Rnai agent and pharmaceutical use thereof

Abstract

The present invention relates to an RNAi agent and pharmaceutical use thereof. Specifically, the present invention relates to an RNAi agent targeting MAPT, a pharmaceutical composition, and pharmaceutical use thereof. The present invention also relates to a pharmaceutical composition, cell, or kit comprising the RNAi agent, and a method for using the RNAi agent to treat and / or prevent a related disorder suffered by a subject.

Description

An RNAi agent and its medicinal uses

[0001] This disclosure claims priority to patent application CN202510106770.2 filed on January 23, 2025 and patent application CN202511226113.8 filed on August 29, 2025. Technical Field

[0002] This disclosure pertains to the field of biomedicine and specifically relates to RNAi agents of the microtubule-associated protein tau gene (MAPT), pharmaceutical compositions containing the same, and their pharmaceutical uses. Background Technology

[0003] The microtubule-associated protein tau (MAPT) gene encodes tau, a microtubule-associated protein located on chromosome 17q21.31. Tau protein is a microtubule-associated protein that, under normal physiological conditions, is primarily located in the axons of adult neurons, regulating the stability of microtubule assembly. Exons 9, 10, 11, and 12 of the MAPT gene encode the microtubule-binding repeat sequence of tau protein. Due to alternative splicing of exon 10, tau protein isoforms are classified as 3R-tau and 4R-tau, where "R" refers to the number of repetitions of the microtubule-binding domain. In the adult brain, the ratio of 3R-tau to 4R-tau is approximately 1. The 3R-tau / 4R-tau ratio is skewed in disease states, and deviations from the normal 3R / 4R tau ratio are characteristic of neurodegenerative tau protein diseases.

[0004] Tau protein has a very high content of proline and lysine residues and more than 80 potential phosphorylation sites, making it easier for tau to cross-link with each other due to hyperphosphorylation under pathological conditions and to aggregate with other tau molecules through condensation reactions. Under pathological conditions, the phosphorylation level of tau is 2-3 times higher than normal. This hyperphosphorylation causes tau protein to detach from microtubules, increasing the cytoplasmic tau content and causing aggregation to form insoluble fiber bundles, ultimately leading to neurofibrillary entanglement, which is an important pathological feature of Alzheimer's disease (AD). Tau pathology is also seen in other neurodegenerative dementias, such as frontotemporal dementia and Parkinson's disease associated with chromosome 17 (FTDP-17), Pick's disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), aerobatic granuloma (AGD), tangled dementia (TOD), and chronic traumatic encephalopathy (CTE). In particular, FTDP-17 patients exhibit many exon and intron mutations in the tau gene, leading to tau accumulation. These findings suggest that abnormalities in tau protein lead to its accumulation and neuronal degeneration.

[0005] Currently, there is a lack of acceptable treatments for these neurodegenerative diseases. Therefore, there is a need for drugs that selectively and effectively inhibit or regulate MAPT gene expression in order to effectively treat subjects with MAPT-related diseases (such as Alzheimer's disease, frontotemporal dementia (FTD), PSP, or other tau protein disorders). The aim of this article is to provide RNAi agents and methods for treating these diseases. Summary of the Invention

[0006] This disclosure provides an RNAi agent targeting MAPT, comprising a sense strand and an antisense strand forming a double-stranded region;

[0007] The positive strand comprises at least 15 consecutive nucleotides that differ from any nucleotide sequence in SEQ ID NO:1 to SEQ ID NO:4 by no more than 3 nucleotides; and

[0008] The antisense strand comprises at least 15 consecutive nucleotides that differ from any of the nucleotide sequences in SEQ ID NO:5 to SEQ ID NO:8 by no more than 3 nucleotides.

[0009] In some implementations, the justice chain and antisense chain are at least partially anticomplementary to form a dual-chain region. In some implementations, there are no more than 5, 4, 3, 2, or 1 mismatches between the justice chain and antisense chain. In some implementations, the justice chain and antisense chain are completely anticomplementary.

[0010] In some embodiments, "difference of no more than 3 nucleotides" means a difference of 0, 1, 2, or 3 nucleotides. In some embodiments, "at least 15 consecutive nucleotides" means at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides. In some embodiments, it is at least 17, 18, 19, 20, or 21 consecutive nucleotides.

[0011] In some implementations, the antisense strand is at least partially complementary to the target sequence to mediate RNA interference. In some implementations, there are no more than 5, 4, 3, 2, or 1 mismatches between the antisense strand and the target sequence. In some implementations, the antisense strand is completely anticomplementary to the target sequence.

[0012] In some embodiments, the RNAi agent disclosed herein comprises one or two blunt ends.

[0013] In some embodiments, the sense and / or antisense strands of the RNAi agent disclosed herein each independently contain one or two unpaired nucleotides. In some embodiments, the 3' end of the antisense strand includes a protrusion formed by unpaired nucleotides.

[0014] In some embodiments, the sense strand and antisense strand are each independently composed of 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. In some embodiments, the sense strand and antisense strand are each independently composed of 18, 19, 20, 21, 22, or 23 nucleotides.

[0015] In some implementations, the sense strand and the antisense strand may be the same length or different, with the sense strand being 19-23 nucleotides long and the antisense strand being 19-26 nucleotides long. Therefore, the length ratio of the sense strand to the antisense strand of the RNAi agent provided in this disclosure can be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 19, 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 length ratio of the sense strand to the antisense strand of the RNAi agent is 19 / 19, 19 / 21, 21 / 21, 21 / 23, 23 / 23, or 23 / 25. In some embodiments, the length ratio of the sense strand to the antisense strand is 19 / 21, 21 / 21, or 21 / 23.

[0016] In some embodiments, the sense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:4; and / or the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:5 to SEQ ID NO:8.

[0017] In some embodiments, the sense strand has a nucleotide sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:4; and / or the antisense strand has a nucleotide sequence as shown in any one of SEQ ID NO:5 to SEQ ID NO:8.

[0018] In some embodiments, at least one nucleotide in the sense strand and / or the antisense strand is a modified nucleotide. In some embodiments, all nucleotides in the sense strand and / or the antisense strand are modified nucleotides.

[0019] In some embodiments, the positive strand contains three consecutive 2'-fluorinated nucleosides. In some embodiments, the nucleosides at positions 7, 8, and 9, or positions 8, 9, and 10, of the positive strand are 2'-fluorinated nucleosides, with the remaining nucleotides being non-fluorinated nucleosides. In some embodiments, the non-fluorinated nucleosides are nucleosides containing a lipophilic group, nucleosides with reverse baseless modification, or nucleosides modified with a 2'-methoxy group. The lipophilic group is as defined in any embodiment within the context of this disclosure.

[0020] In some embodiments, the RNAi agent contains one or more reverse-base-free nucleosides, for example, it may contain 1, 2, 3, 4, or 5 reverse-base-free nucleosides. The reverse-base-free nucleosides may be located at any position in the sense strand and / or antisense strand. In some embodiments, the reverse-base-free nucleosides are located in the sense strand.

[0021] In some embodiments, the positive strand contains at least one reverse-base-free nucleoside. In some embodiments, the first nucleoside at the 3' and / or 5' end of the positive strand is a reverse-base-free nucleoside. In some embodiments, the first nucleoside at the 3' and / or 5' end of the positive strand is a reverse-base-free nucleoside.

[0022] In some embodiments, the antisense strand contains at least five 2'-fluoro-modified nucleosides, for example, five, six, seven, eight, nine, or ten 2'-fluoro-modified nucleosides. In some embodiments, the nucleosides at positions 2, 6, 12, 14, and 16 are 2'-fluoro-modified nucleosides, following the direction from the 5' end to the 3' end. In some embodiments, the antisense strand contains five 2'-fluoro-modified nucleosides, with the nucleosides at positions 2, 6, 12, 14, and 16 being 2'-fluoro-modified nucleosides, and the remaining nucleotides being non-2'-fluoro-modified nucleosides. In some embodiments, the antisense chain contains eight 2'-fluorinated nucleosides, wherein the nucleosides at positions 2, 4, 6, 10, 12, 14, 16, and 18 of the antisense chain are each independently 2'-fluorinated nucleosides, and the nucleosides at the remaining positions are non-2'-fluorinated nucleosides. In some embodiments, the non-fluorinated nucleosides are 2'-methoxylated nucleosides, acyclic nucleosides, or nucleosides with the structure shown in formula (IV).

[0023] In equation (IV),

[0024] X is selected from O or C(R) X )2;

[0025] Each R X Each is independently selected from hydrogen, halogen, and C. 1-6 Alkyl, the C 1-6 Alkyl groups are optionally surrounded by one or more components selected from R A The substituents replaced by R A Selected from halogens, hydroxyl groups, cyano groups, and oxo groups;

[0026] R 1A R 1B Each is independently selected from hydrogen, hydroxyl, and C. 1-6 Alkyl, C 1-6 alkoxy, amino, or halogen, wherein the C 1-6 Alkyl, C 1-6 Alkyl, amino, optionally composed of one or more components selected from R A The substituents replaced by R A Selected from halogens, hydroxyl groups, cyano groups, and oxo groups;

[0027] or R 1A R 1B With and R 1A R 1B The bonded carbon atoms form a 3- to 6-membered heterocyclic group, which is optionally surrounded by one or more atoms selected from R A The substituents replaced by R A Selected from halogens, hydroxyl groups, cyano groups, and oxo groups;

[0028] R2 and R3 are each independently selected from hydrogen or C. 1-6 Alkyl, the C 1-6 Alkyl groups are optionally surrounded by one or more components selected from R A The substituents replaced by R A Selected from halogens, hydroxyl groups, cyano groups, and oxo groups;

[0029] m is selected from 0, 1, 2, or 3;

[0030] In formula (IV), ring A is selected from 5- to 12-membered heteroaryl groups or 3- to 12-membered heterocyclic groups;

[0031] R4 is independently selected from hydrogen, cyano, halogen, hydroxyl, amino, C 1-6 Alkyl, C 2-6 alkynyl group, C 2- 6-Alkenyl, -SC 1-6 Alkyl, C 1-6 Alkoxy, -N(C)1-6 Alkyl, 2-oxo, 3- to 6-membered cycloalkyl, 6- to 10-membered aryl, 5- to 12-membered heteroaryl, 3- to 12-membered heterocyclic, -NH-(C=O)-C 1-6 Alkyl, -NH-(C=O)-C 3-6 cycloalkyl, -NH(C=O)-OC 1-6 Alkyl, -NH(C=O)-OC 3-6 Cycloalkyl, -O(C=O)NHC 1- 6-alkyl, -O(C=O)NH-C 3-6 cycloalkyl, -(C=O)NH-C 1-6 Alkyl group, -(C=O)-NH-C 3-6 cycloalkyl, -(C=O)-C 1-6 Alkyl, -(C=O)-C 3-6 cycloalkyl, -SO2-C 1-6 Alkyl group, -SO2-C 3-6 Cycloalkyl, -SO2-NH2, -SO2-NH-C 1-6 Alkyl group, -SO2-NH-C 3-6 cycloalkyl, -SO2-N(C 1-6 Alkyl)2, -SO2-NH(C 3- 6-cycloalkyl)2、-S(O)(NH)-C 1-6 Alkyl group, -S(O)(NH)-C 3-6 cycloalkyl;

[0032] n is selected from 0, 1, 2, 3, 4 or 5;

[0033] Base represents a base.

[0034] In some implementations, n in equation (IV) is selected from 0 or 1.

[0035] In some embodiments, one or more nucleosides in the antisense chain are nucleosides as shown in formula (IV). In some embodiments, one nucleoside in the antisense chain is a nucleoside as shown in formula (IV).

[0036] In some embodiments, the nucleoside represented by formula (IV) has a structure represented by formula (IV-a):

[0037] In equation (IV-a), R 1A R2, R3, R4, m, n, ring A, and Base are defined as in equation (IV).

[0038] In some embodiments, the nucleoside represented by formula (IV) has a structure represented by formula (IV-b):

[0039] In equation (IV-b), R 1A R4, m, n, ring A, and Base are defined as in equation (IV).

[0040] In some embodiments, the nucleoside represented by formula (IV) has a structure represented by formula (IV-c):

[0041] In formula (IV-c), R 1A R2, R3, R4, n, ring A, and Base are defined as in equation (IV).

[0042] In some embodiments, the nucleoside represented by formula (IV) has a structure represented by formula (IV-d):

[0043] In equation (IV-d), R 1A R4, n, ring A, and Base are defined as in equation (IV).

[0044] In some implementations, R in formula (IV) 1A Selected from hydroxyl, C 1-6 Alkyl groups (e.g., methoxy, ethoxy, propoxy, butoxy), the C 1-6 Alkyl groups are optionally surrounded by one or more components selected from R A The substituents replaced by R A Selected from halogens, hydroxyl groups, cyano groups, and oxo groups.

[0045] In some implementations, R in formula (IV) 1A Selected from C 1-6 Alkoxy, the C 1-6 Alkyl groups are optionally surrounded by one or more R groups. A The substituents replaced by R A Selected from halogens, hydroxyl groups, cyano groups, and oxo groups.

[0046] In an optional implementation, R in formula (IV) 1A Selected from methoxy groups.

[0047] In some implementations, R as described in formula (IV) 1A Selected from halogens (e.g., fluorine, chlorine, bromine, iodine).

[0048] In an optional implementation, R in formula (IV) 1A Selected from fluorine.

[0049] In some embodiments, the ring A in formula (IV) is selected from 5 to 12 heteroaryl groups (e.g., 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered, 11-membered, 12-membered).

[0050] In some embodiments, the ring A in formula (IV) is selected from 5- to 6-membered heteroaryl groups.

[0051] In some embodiments, ring A in formula (IV) is selected from 1,2,4-triazole ring.

[0052] In some specific implementations, the ring A in formula (IV) is selected from...

[0053] In some embodiments, the ring A in formula (IV) is selected from 3 to 12-membered heterocyclic groups (e.g., 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered, 11-membered, 12-membered).

[0054] In some implementations, the ring A in formula (IV) is selected from 3- to 6-membered heterocyclic groups.

[0055] In some embodiments, ring A in formula (IV) is selected from morpholine ring or azacyclic butane.

[0056] In some specific implementations, the ring A in formula (IV) is selected from...

[0057] In some embodiments, R4 in formula (IV) is independently selected from hydrogen, cyano, halogen (e.g., fluorine, chlorine, bromine, iodine), hydroxyl, amino, C 1-6 Alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl), C 2-6 Alkynyl (e.g., ethynyl, propynyl, butynyl, pentynyl, pentyn-4-ynyl, and pentyn-1,4-diynyl), C 2-6 Alkenyl (e.g., vinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl), -SC 1-6 Alkyl groups (e.g., methylthio, ethylthio, propylthio, butylthio), C 1-6 Alkyl groups (e.g., methoxy, ethoxy, propoxy, butoxy), -N(C) 1-6 Alkyl)2 (e.g., -NMe2, -NEt2, -N(nPr)2, -N(iPr)2).

[0058] In some implementations, R4 in formula (IV) is independently selected from hydrogen, C 1-6 Alkyl, -SC 1-6 Alkyl, C 1-6 Alkoxy, -N(C) 1-6 Alkyl)2.

[0059] In some specific implementations, R4 in formula (IV) is independently selected from hydrogen, -N(C 1-6 Alkyl)2.

[0060] In some embodiments, the structure shown in formula (IV) is the structure shown in formula (IV-1):

[0061] Where Base represents a base.

[0062] The nucleoside represented by formula (IV) can be located at any position in the sense or antisense strand. In some embodiments, the nucleoside represented by formula (IV) is located at any position in the antisense strand. In some embodiments, the nucleoside represented by formula (IV) is located at the 9th position from the 5' end.

[0063] In some embodiments, the acyclic nucleotide has the structure shown in PCT application WO2022028462A1. In some embodiments, the nucleoside in the acyclic nucleotide has the structure shown in formula (III):

[0064] Where Base represents a base, following the direction from the 5' end to the 3' end in the sequence, "*" indicates the site where the nucleoside is connected to the nucleoside adjacent to its 5' end, and "#" indicates the site where it is connected to the nucleoside adjacent to its 3' end. In some embodiments, the nucleoside at position 7 of the antisense strand has the structure shown in Formula (III).

[0065] In some embodiments, the 5' position of the sugar ring of the first nucleotide at the 5' end of the sense or antisense chain is a 5'-vinyl phosphate diester group (VP). In some embodiments, the 5' position of the sugar ring of the first nucleotide at the 5' end of the antisense chain is a 5'-vinyl phosphate diester group (VP). In some embodiments, the 5'-vinyl phosphate diester group is a 5'-trans-vinyl phosphate diester group (E-VP).

[0066] In some embodiments, at least one phosphodiester group in the sense strand and / or antisense strand is a phosphodiester group with a modifying group. The modifying group increases the stability of the RNAi agent in a biological sample or environment. In some embodiments, the sense strand and / or antisense strand includes multiple phosphodiester groups with modifying groups. In some embodiments, both the sense strand and the antisense strand contain multiple phosphodiester groups with modifying groups. In some embodiments, the sense strand contains four phosphodiester groups with modifying groups, and the antisense strand contains four phosphodiester groups with modifying groups. In some embodiments, the sense strand contains two phosphodiester groups with modifying groups, and the antisense strand contains four phosphodiester groups with modifying groups.

[0067] In some embodiments, the phosphodiester group with the modifying group is present at one or more locations selected from: between any two adjacent nucleotides from the first to the fourth nucleotide at the 5' end and / or the 3' end of the sense chain, and / or between any two adjacent nucleotides from the first to the fourth nucleotide at the 5' end and / or the 3' end of the antisense chain. For example, it may be located between the first and second nucleotides at the 5' end of the sense chain;

[0068] Between the first and second nucleotides at the 5' end of the positive chain;

[0069] Between the second and third nucleotides at the 5' end of the positive chain;

[0070] Between the first and second nucleotides at the 3' end of the positive chain;

[0071] Between the second and third nucleotides at the 3' end of the positive chain;

[0072] Between the first and second nucleotides at the 5' end of the antisense strand;

[0073] Between the second and third nucleotides at the 5' end of the antisense strand;

[0074] Between the first and second nucleotides at the 3' end of the antisense strand; and

[0075] Between the second and third nucleotides at the 3' end of the antisense strand.

[0076] In some embodiments, the phosphate diester group having the modifying group is a thiophosphate diester group.

[0077] In some embodiments, the justice chain includes or is selected from the justice chain shown in any one of SEQ ID NO:9 to SEQ ID NO:42 and / or the antisense chain includes or is selected from the antisense chain shown in any one of SEQ ID NO:43 to SEQ ID NO:76.

[0078] In some embodiments, the sense strand comprises or is selected from nucleotide sequences such as those shown in SEQ ID NO:13, 10, or 42; and the antisense strand comprises or is selected from nucleotide sequences such as those shown in SEQ ID NO:47, 44, or 76.

[0079] In some embodiments, the RNAi agent comprises or is selected from the group consisting of a sense strand and an antisense strand:

[0080] (1) The positive chain as shown in SEQ ID NO:13 and the negative chain as shown in SEQ ID NO:47;

[0081] (2) The positive chain as shown in SEQ ID NO:10 and the negative chain as shown in SEQ ID NO:44;

[0082] (2) The justice chain as shown in SEQ ID NO:42 and the antisense chain as shown in SEQ ID NO:76.

[0083] In some embodiments, the RNAi agent further comprises one or more delivery groups. In some embodiments, the delivery group is a lipophilic group or an antibody. The delivery group is covalently or non-covalently linked to any position in the sense and / or antisense strands. The delivery group is capable of delivering the RNAi agent of this disclosure to a site where MAPT gene expression is present.

[0084] In some embodiments, the delivery group is an antibody that can bind to the transferrin receptor (TfR).

[0085] In some embodiments, the antibody and the RNAi agent are covalently linked to form a conjugate. In some embodiments, any one nucleotide of the antibody and the RNAi agent is linked by a covalent bond or a linker group. In some embodiments, the antibody is linked to the sense strand. In some embodiments, the antibody is linked to the first nucleotide at the 5' or 3' end of the sense strand.

[0086] In some embodiments, the delivery group is a lipophilic group. The lipophilic group can be attached to any position in the positive and / or negative strands. In some embodiments, the lipophilic group is attached to the 5' and / or 3' ends of the positive and / or negative strands. In some embodiments, the lipophilic group is attached to the 5' end of the positive strand.

[0087] In some embodiments, the lipophilic group is directly linked to the first nucleotide at the 5' end of the positive strand. In some embodiments, the lipophilic group and the first nucleotide at the 5' end of the positive strand are linked by a linker bond, which is a phosphodiester group or a phosphodiester group with a modifying group. In some embodiments, the phosphodiester group with the modifying group is a thiophosphate diester group.

[0088] In some embodiments, the lipophilic group is linked to a nucleoside at position 1, 2, 7, 20, or 21 of the 5' end of the positive chain.

[0089] In some embodiments, the lipophilic group is linked to a base of the nucleotide. In some embodiments, the lipophilic group is linked to a sugar ring of the nucleotide. In some embodiments, the lipophilic group is linked to a nucleoside between two adjacent nucleotides via a linker group.

[0090] In some embodiments, the lipophilic group comprises saturated or unsaturated C. 4-30 A hydrocarbon chain, and optionally a functional group selected from halogens, alkoxy groups, hydroxyl groups, amines, carboxylic acids, sulfonates, phosphates, thiols, azides, and alkynes.

[0091] In some embodiments, the lipophilic group comprises saturated or unsaturated C. 6-18 Hydrocarbon chain. In some embodiments, the lipophilic group comprises saturated or unsaturated C atoms. 16 Hydrocarbon chain. In some embodiments, the lipophilic group is n-hexadecyl.

[0092] In some embodiments, the RNAi agent contains a lipophilic group, wherein the lipophilic group is n-hexadecyl.

[0093] In some embodiments, the lipophilic group is covalently linked to the 2' position of the nucleoside sugar ring in the nucleotide, in which case the nucleoside with the lipophilic group has a structure as shown in formula (I):

[0094] in,

[0095] X1 is selected from O, S, N, and C atoms;

[0096] X2 is an O or S atom;

[0097] R1 is selected from C 10 -C 30 Straight-chain alkyl groups, or C groups truncated by one or more O or S atoms. 10 -C 30 Straight-chain alkyl; optionally, the C 10 -C 30 Straight-chain alkyl groups can be generated by one or more R groups. a Instead, or optionally, the C 10 -C 30 A C10 bond is formed between two adjacent carbon atoms of a straight-chain alkyl group. 3-6 cycloalkyl;

[0098] R a Each is independently selected from hydrogen, deuterium, halogen, hydroxyl, cyano, alkyl, haloalkyl, alkoxy, cycloalkyl, heterocyclic alkyl;

[0099] n is 1;

[0100] B represents a base.

[0101] In some embodiments, the nucleoside represented by formula (I) has the structure of the nucleoside moiety in a compound represented by formula (I) as described in PCT application WO2024125556A1 or a pharmaceutically acceptable salt thereof, the entire contents of which are incorporated herein by reference.

[0102] In some embodiments, X1 in formula (I) is selected from O or S atoms. In some embodiments, it is an O atom.

[0103] In some embodiments, X2 in formula (I) is selected from O or S atoms. In some embodiments, it is an O atom.

[0104] In some implementations, R1 in formula (I) is selected from C 14 -C 24 Straight-chain alkyl groups (e.g., C14) 14 C 15 C 16 C 17 C 18 C 19 C 20 C 21 C 22 C 23 C 24 Straight-chain alkyl groups, or C atoms truncated by one or more O or S atoms. 14 -C 24 Straight-chain alkyl groups (e.g., C14) 14 C 15 C 16 C17 C 18 C 19 C 20 C 21 C 22 C 23 C 24 (linear alkyl groups).

[0105] In some implementations, R in equation (I) a Each is independently selected from hydrogen, deuterium, halogens (e.g., fluorine, chlorine, bromine), and C. 1-6 Alkyl groups (e.g., C1, C2, C3, C4, C5, C6 alkyl groups, including but not limited to methyl, ethyl, and isopropyl), C 1-6 Alkoxy groups (e.g., C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, C6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy).

[0106] In some implementations, R in equation (I) a Each is independently selected from hydrogen, deuterium, fluorine, methyl, and methoxy.

[0107] In some implementations, R1 in formula (I) is selected from: End a is connected to X2.

[0108] In some implementations, B is selected from adenine, guanine, cytosine, uracil, and thymine.

[0109] In some embodiments, the nucleoside represented by formula (I) is selected from:

[0110] In some embodiments, the nucleoside represented by formula (I) is selected from:

[0111] In some embodiments, the nucleoside represented by formula (I) is:

[0112] In this context, B represents a base.

[0113] In some embodiments, the nucleoside represented by formula (I) can be located at any position in the sense strand and / or antisense strand of the RNAi agent. In some embodiments, the nucleoside represented by formula (I) is located in the sense strand. In some embodiments, the nucleoside of formula (I) is located at one or more positions from position 1 to position 8 starting from the 5' end or 3' end of the sense strand, for example, it can be located at position 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the nucleoside represented by formula (I) is located at one or more positions from position 1, 2, 6, 7, 20, or 21 starting from the 5' end of the sense strand.

[0114] In some embodiments, the RNAi agent contains one or more nucleosides as shown in formula (I), for example, it may contain 1, 2, 3, 4 or 5 nucleosides as shown in formula (I). In some embodiments, the RNAi agent contains 1 nucleoside as shown in formula (I).

[0115] In some embodiments, the positive strand is selected from or comprises a nucleotide sequence as shown in any one of SEQ ID NO: 77 to SEQ ID NO: 125.

[0116] In some embodiments, the antisense strand is selected from or comprises the nucleotide sequence shown in any one of SEQ ID NO: 126 to SEQ ID NO: 174.

[0117] In some implementations, the justice chain is selected from or includes a sequence as shown in any one of the justice chains in Tables 1a, 1b, and 1c, and the antisense chain is selected from or includes a sequence as shown in any one of the antisense chains in Tables 1a, 1b, and 1c.

[0118] In some embodiments, the RNAi agent is selected from any one of the RNAi agents shown in Tables 1a, 1b, and 1c.

[0119] In some embodiments, the RNAi agent is selected from TJR104439, TJR106045, TJR104441, TJR104468, TJR104470, TJR106560, TJR106558, TJR105324, TJR106545, TJR106546, or TJR104440.

[0120] On the other hand, this disclosure provides a pharmaceutical composition comprising the RNAi agent described herein, and one or more pharmaceutically acceptable excipients, such as a vehicle, carrier, diluent, and / or delivery polymer. Various drug delivery systems are known and can be used with the RNAi agent of this disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the RNAi agent, receptor-mediated endocytosis, and constructing nucleic acids as part of a retrovirus or other vector.

[0121] In some embodiments, the pharmaceutical composition may further comprise pharmaceutically acceptable excipients and / or adjuvants, which may be one or more formulations or compounds conventionally used in the art. For example, the pharmaceutically acceptable excipients may include at least one of pH buffers, protectants, and osmotic pressure regulators.

[0122] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg to 1000 mg.

[0123] In some embodiments, based on the total weight of the composition, the pharmaceutical composition contains 0.01-99.99% of the aforementioned RNAi agent or a pharmaceutically acceptable salt or isotopic substitution thereof. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned RNAi agent or a pharmaceutically acceptable salt or isotopic substitution thereof. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned RNAi agent or a pharmaceutically acceptable salt or isotopic substitution thereof. In some embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned RNAi agent or a pharmaceutically acceptable salt or isotopic substitution thereof. In some embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned RNAi agent or a pharmaceutically acceptable salt or isotopic substitution thereof.

[0124] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% pharmaceutically acceptable excipients based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 1% to 99% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 2% to 98% pharmaceutically acceptable excipients.

[0125] In some embodiments, when the RNAi agent or pharmaceutical composition of this disclosure comes into contact with cells expressing a target gene, it is determined by, for example, psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA) based methods, or protein-based methods such as immunofluorescence assays, such as Western blotting or flow cytometry, that the RNAi agent or pharmaceutical composition of this disclosure inhibits 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%.

[0126] In some embodiments, when the RNAi agent or pharmaceutical composition of this disclosure comes into contact with cells expressing the target gene, the percentage of residual expression of the target gene mRNA induced by the RNAi agent or pharmaceutical composition of this disclosure is determined by, for example, psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA) based methods, or protein-based methods such as immunofluorescence analysis, such as Western blotting or flow cytometry, to be no higher than 99%, no higher than 95%, no higher than 90%, no higher than 85%, no higher than 80%, no higher than 75%, no higher than 70%, no higher than 65%, no higher than 60%, no higher than 55%, no higher than 50%, no higher than 45%, no higher than 40%, no higher than 35%, no higher than 30%, no higher than 25%, no higher than 20%, no higher than 15%, or no higher than 10%.

[0127] In some embodiments, when the RNAi agent or pharmaceutical composition described herein comes into contact with cells expressing a target gene, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA) based methods, or protein-based methods such as immunofluorescence assays, such as Western blotting, or flow cytometry, the RNAi agent 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% while maintaining target activity.

[0128] In some embodiments, when the RNAi agent or pharmaceutical composition described herein comes into contact with cells expressing a target gene, the RNAi agent, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA) based methods, or protein-based methods such as immunofluorescence assays, such as Western blotting, or flow cytometry, 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% while reducing target activity by at least 20%, at least 19%, at least 15%, at most 10%, at most 5%, or more than 1%.

[0129] In some embodiments, when the RNAi agent or pharmaceutical composition described herein comes into contact with cells expressing a target gene, the RNAi agent, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA) based methods, or protein-based methods such as immunofluorescence assays, such as Western blotting, or flow cytometry, 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%, at least 75%, or at least 80%, while increasing 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%, or at least 75%.

[0130] On the other hand, this disclosure also provides a cell containing the RNAi agent of this disclosure.

[0131] On the other hand, this disclosure also provides a kit containing the RNAi agent or pharmaceutical composition of this disclosure.

[0132] On the other hand, this disclosure also provides a method for reducing the expression of the microtubule-associated protein tau gene (MAPT), the method comprising administering to a subject an effective amount of the RNAi agent of this disclosure, or a pharmaceutical composition, or a kit.

[0133] On the other hand, this disclosure provides an RNAi agent, pharmaceutical composition, or kit for treating and / or preventing diseases.

[0134] On the other hand, this disclosure provides a RAi agent, pharmaceutical composition, and kit for use as a medicament.

[0135] On the other hand, this disclosure also provides a method for treating and / or preventing disease, the method comprising administering to a subject an effective amount of the RNAi agent of this disclosure, or a pharmaceutical composition, or a kit.

[0136] In some embodiments, the disease is a disease associated with MAPT gene expression. In some embodiments, the disease is selected from tau proteinopathy, Alzheimer's disease (AD), frontotemporal dementia and Parkinson's disease associated with chromosome 17 (FTDP-17), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), epilepsy, Dravet syndrome (DS), Pick's disease (PiD), aerophilic granulomatosis (AGD), and glial globular inclusion body tau proteinopathy (GGT).

[0137] On the other hand, this disclosure provides the use of the RNAi agents, pharmaceutical compositions, and kits described herein in pharmaceuticals for treating and / or preventing diseases in subjects. In some embodiments, the disease is a disease associated with the MAPT gene. In some embodiments, the disease is selected from tau proteinopathy, Alzheimer's disease (AD), frontotemporal dementia and Parkinson's disease associated with chromosome 17 (FTDP-17), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), epilepsy, Dravet syndrome (DS), Pick's disease (PiD), aerophilic granulomatosis (AGD), and glial cell globular inclusion body tau proteinopathy (GGT).

[0138] On the other hand, this disclosure also provides a cell containing the RNAi agent of this disclosure. The cell does not develop into a complete plant or animal.

[0139] On the other hand, this disclosure also provides a method for in vivo delivery of an RNAi agent that inhibits MAPT expression and / or replication, the method comprising administering to a subject an effective amount of the RNAi agent of this disclosure, or a pharmaceutical composition, or a kit.

[0140] The RNAi agents or pharmaceutical compositions and methods disclosed herein can reduce the level of target mRNA in cells, cell populations, tissues, or subjects, including: administering the RNAi agent or pharmaceutical composition of this disclosure to a subject, wherein the RNAi agent is linked to the delivery group, thereby inhibiting the expression of the target mRNA in the subject. The target mRNA is mRNA expressed by the MAPT gene.

[0141] In some embodiments, the subject has been identified as having pathological upregulation of the target gene in the targeted cells or tissues prior to administration of the RNAi agent and / or pharmaceutical composition of this disclosure.

[0142] The subjects mentioned in this disclosure refer to subjects who are diagnosed with (or suspected of having, or are susceptible to) a disease or condition that would benefit from a reduction or inhibition of target mRNA expression.

[0143] The RNAi agents and / or pharmaceutical compositions disclosed herein can be administered via local (e.g., direct injection, implantation, or topical administration) or systemic administration. This includes administration via subcutaneous, intravenous, intraperitoneal, or parenteral routes, such as intracranial (e.g., intraventricular, intraparenchymal, and intrasheathal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, buccal, and sublingual administration, or any suitable route of administration commonly found in the art. Alternatively, the pharmaceutical compositions provided herein can be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, duodenal, or intraperitoneal injection.

[0144] This disclosure also provides a method for silencing mRNA of a target gene in cells, the method comprising the step of introducing an RNAi agent and / or pharmaceutical composition of the present disclosure into the cell.

[0145] This disclosure also provides a method for silencing a target gene or its mRNA in cells in vivo or in vitro, the method comprising the step of introducing an RNAi agent and / or pharmaceutical composition according to this disclosure into the cell.

[0146] This disclosure also provides a method for inhibiting the expression of a target gene or its mRNA, the method comprising administering to a subject in need an effective amount or effective dose of an RNAi agent and / or pharmaceutical composition according to this disclosure.

[0147] In some embodiments, the effective amount or dose of the RNAi agent 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.

[0148] In some implementations, the target gene is the MAPT gene, and the target mRNA is the mRNA expressed by the target gene.

[0149] This disclosure also provides a method for preparing an RNAi agent or pharmaceutical composition, comprising: synthesizing the RNAi agent or pharmaceutical composition described in this disclosure.

[0150] The compounds described in this disclosure include the RNAi agents of this disclosure. Pharmaceutically acceptable salts of the compounds described in this disclosure are selected from inorganic or organic salts, and the compounds described in this disclosure are reactable with acidic or basic substances to form the corresponding salts. In some embodiments, the compounds of this disclosure are present in the form of sodium salts. In some embodiments, one or more phosphodiester groups or phosphodiester groups with modifying groups in the RNAi agents of this disclosure form sodium phosphodiester salts. In some specific embodiments, all phosphodiester groups or phosphodiester groups with modifying groups in the RNAi agents of this disclosure form sodium phosphodiester salts.

[0151] On the other hand, without specifying the configuration, the compounds disclosed herein, including the RNAi agents disclosed herein, may exist in specific geometric or stereoisomeric forms. This disclosure contemplates all such compounds, including cis and 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 enriched with enantiomers or diastereomers, all of which are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this disclosure.

[0152] Furthermore, without specifying the configuration, the compounds of this disclosure, including the RNAi agents and intermediates of this disclosure, may also exist in different tautomer forms, and all such forms are included within the scope of this disclosure. The terms "tautomer" or "tautomer form" refer to structural isomers with different energies that can interconvert via low energy barriers.

[0153] The compounds disclosed herein, including the RNAi agents disclosed herein, may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds containing asymmetric carbon atoms of this disclosure can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from a racemic mixture or synthesized using chiral starting materials or chiral reagents.

[0154] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. If an enantiomer of a compound of this disclosure comprising the RNAi agent of this disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide a pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and then recovery of the pure enantiomer. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., from amines to carbamates).

[0155] This disclosure also includes compounds of this disclosure that are identical to those described herein, but in which one or more atoms are replaced by isotopes with atomic weights or mass numbers different from those commonly found in nature, including the RNAi agents of this disclosure. Examples of isotopes that can bind to the compounds of this disclosure, including the RNAi agents of this disclosure, include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 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 Cl, etc.

[0156] Without specifying the configuration, in the chemical structure of this disclosure, the bonds are... This indicates that the configuration is not specified; that is, if chiral isomers exist in the chemical structure, the bond... It can be Or simultaneously include Two configurations. Although all structural formulas described herein are represented in certain isomer forms for simplicity, this disclosure can include all isomers, such as tautomers, rotatimers, geometric isomers, diastereomers, racemates, and enantiomers. In the chemical structure of the compounds described herein, bonds... No configuration was specified, i.e., key The configuration can be E-type or Z-type, or it can contain both E-type and Z-type configurations.

[0157] This disclosure introduces the full text of PCT applications WO2022028462A1, WO2024125556A1, WO2025026390A1, and WO2025252152A1.

[0158] Terminology Explanation

[0159] To facilitate understanding of this disclosure, some technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0160] As used herein, “RNAi agent” refers to an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of degrading, inactivating, or inhibiting (e.g., under appropriate conditions) the translation of a target mRNA transcript in a sequence-specific manner. RNAi agents used herein may act via RNA interference mechanisms (i.e., by interacting with RNA interference pathway mechanisms in mammalian cells, such as RNA-induced silencing complexes or RISC) or through any alternative mechanism or pathway. Although the term RNAi agent as used herein is considered to act primarily through RNA interference mechanisms, the disclosed RNAi agents are not bound to or limited by any particular pathway or mechanism of action. The disclosed RNAi agents comprise, or consist of, a sense strand and an antisense strand, or are composed of a sense strand and an antisense strand; for example, the RNAi agents disclosed herein may be presented in the form of short (or small) interfering RNA (siRNA). The antisense strand of the RNAi agent described herein is at least partially complementary to the targeted mRNA. RNAi agents may contain one or more modified nucleotides and / or one or more phosphodiester groups having modifying groups.

[0161] Unless otherwise specified, the term “RNAi agent” in this disclosure should be understood to encompass the range of pharmaceutically acceptable salts.

[0162] Unless otherwise specified, in the context of this disclosure, the terms "microtubule-associated protein tau gene," "MAPT," and "Microtubule-associated protein tau" are used interchangeably. MAPT includes, but is not limited to, human MAPT, cynomolgus monkey MAPT, mouse MAPT, and rat MAPT, the amino acid and complete coding sequences of which, as well as mRNA sequences, are readily available from publicly available databases, such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0163] The term "MAPT" also refers to naturally occurring DNA sequence variations in the MAPT gene, such as single nucleotide polymorphisms (SNPs) in the MAPT gene. Exemplary SNPs can be found in the dbSNP database.

[0164] The term "target sequence" refers to a continuous portion of the nucleotide sequence of the mRNA molecule formed during MAPT transcription, including mRNA processed from the major transcription product. The targeted portion of the target sequence should be long enough to serve as a substrate for iRNA-directed cleavage. In one embodiment, the target sequence is located within the protein-coding region of MAPT.

[0165] The term "MAPT gene-related diseases" includes any disease or condition associated with decreased MAPT expression and / or activity. Symptoms or signs typically include cognitive decline, memory impairment, loss of comprehension or verbal expression, behavioral abnormalities, impaired motor function, and an increase in the number or volume of neurofibrillary inclusions. Typical "MAPT gene-related neurodegenerative diseases" include tau proteinopathy, Alzheimer's disease (AD), frontotemporal dementia and Parkinson's disease associated with chromosome 17 (FTDP-17), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), epilepsy, Dravet syndrome (DS), Pick's disease (PiD), aerophilic granulomatosis (AGD), and glial globular inclusion body tau proteinopathy (GGT).

[0166] As used in this article, in the case of RNA-mediated gene silencing, the sense strand (also known as SS, SS chain, or sense strand) is a strand that contains a sequence that is identical or substantially identical to the target mRNA sequence; the antisense strand (also known as AS, AS chain) is a strand that has a sequence that is complementary to the target mRNA sequence.

[0167] In the context of describing the sense strand of the RNAi agent described herein, the term "at least 15 consecutive nucleotides differing by no more than 3 nucleotides from any of the nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:4" is intended to mean that the sense strand of the RNAi agent described herein comprises at least 15 consecutive nucleotides of the nucleotide sequences shown in any of SEQ ID NO:1 to SEQ ID NO:4, or differs from at least 15 consecutive nucleotides of the nucleotide sequences shown in any of SEQ ID NO:1 to SEQ ID NO:4 by no more than 3 nucleotides (optionally, by no more than 2 nucleotides; optionally, by 1 nucleotide; optionally, by 0 nucleotides). Other similar descriptions in the context of this disclosure should also be understood similarly, and "difference" as used herein does not include nucleotides containing different modifications; that is, nucleotides containing the same bases but different modifications are not considered differing nucleotides as described in this disclosure. Other similar descriptions of the sense strand and / or antisense strand in the context of this disclosure should also be understood similarly.

[0168] In this disclosure, the "5' region," also known as the "5' end" or "5' terminus," of the sense or antisense strand can be used interchangeably. For example, the nucleotides at positions 2 to 8 of the 5' region of the antisense strand can be replaced with the nucleotides at positions 2 to 8 of the 5' terminus of the antisense strand. Similarly, the "3' region," "3' terminus," and "3' terminus" of the sense or antisense strand can also be used interchangeably.

[0169] Unless otherwise specified, in the context of this disclosure, "G", "C", "A", "T" and "U" represent nucleotides, each containing the bases of guanine, cytosine, adenine, thymine, and uracil, respectively. It is well known to those skilled in the art that substitutions of bases T and U do not significantly affect the properties of the RNAi agent sequence. In the sequences disclosed herein, U can be arbitrarily replaced with T, and the resulting sequences are also within the scope of protection of this disclosure. In the sequences disclosed herein, for the same nucleic acid chain, the direction from the 5' end to the 3' end is defined as left to right. The lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy modified nucleoside; the lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro modified nucleoside; the lowercase letter s indicates that the two nucleosides adjacent to the letter s are connected by a thiophosphate diester group. Unless otherwise specified, the two nucleosides are connected by a phosphate diester group; VP indicates that the 5' end of the nucleoside adjacent to the left / right of the letter is a 5'-vinyl phosphate diester group. The 5'-vinyl phosphate diester group can be cis or trans, that is, it can be the 5'-E-VP isomer (i.e., trans-vinyl phosphate diester group), the 5'-Z-VP isomer (i.e., cis-vinyl phosphate diester group), or a mixture thereof; hmpNA indicates that the nucleoside in parentheses adjacent to its right is a nucleoside modified with hmpNA.

[0170] Unless otherwise specified, the terms "RNAi agent," "nucleotide," "compound," "chemical modification," "oligonucleotide," "double-stranded RNAi inhibitor molecule," "siRNA," "dsRNA," "nucleic acid," and "RNAi" in this disclosure can exist independently as salts, mixed salts, or non-salts (e.g., free acids or free bases). When present as salts or mixed salts, they are pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. When present as salts, some groups may ionize to form anions / cations; for example, phosphodiester groups and thiophosphonate diester groups can exist in anionic form. Unless otherwise specified, the salt forms of the following structures are also within the scope of this disclosure. The "compounds of this disclosure" include the RNAi agents of this disclosure.

[0171] Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each strand is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each strand is a hydroxyl group.

[0172] The above-mentioned modifications and linking groups have the structures shown in Table 2, where Base represents the base at the corresponding position, 5' represents the bond towards the 5' end of the nucleotide sequence, and 3' represents the bond towards the 3' end of the nucleotide sequence.

[0173] The terms "lipophilic group" or "lipophilic moiety" broadly refer to any compound or chemical part that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is through the octanol-water partition coefficient logK. ow K ow This represents the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the aqueous phase at equilibrium in a two-phase system. In principle, logK... ow When the value exceeds 0, the chemical substance exhibits lipophilicity. Typically, the logK of the lipophilic portion... ow Values ​​exceeding 1, 1.5, 2, 3, 4, 5, or 10, such as the logK of 6-aminohexanol. ow The logK of cholesterol-based N-(hexyl-6-ol)carbamate is approximately 0.7. ow It is 10.7.

[0174] The lipophilicity of a molecule can be altered relative to the functional groups it carries. For example, adding a hydroxyl or amino group to the end of the lipophilic moiety can increase or decrease the partition coefficient (e.g., logK) of the lipophilic moiety. owThe lipophilic moiety can be aliphatic, cyclic (e.g., alicyclic), or polycyclic (e.g., polycyclic alicyclic compounds), such as steroids (e.g., sterols) or straight-chain or branched aliphatic hydrocarbons. The lipophilic moiety may generally comprise a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, such as oxygen or sulfur atoms. Such lipophilic aliphatic moieties include, but are not limited to, saturated or unsaturated C4-C... 30 Hydrocarbons (e.g., C) 10 -C 30 Hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric esters of fatty acids and fatty diamides), terpenes (e.g., C464 ... 10 Terpenes, C 15 Sesquiterpenes, C 20 Diterpenes, C 30 Triterpenes and C 40 Tetraterpenes and other polycyclic hydrocarbons; for example, the lipophilic moiety may be optionally substituted C 10-30 Straight-chain alkyl; for example, the lipophilic moiety may be an optionally substituted C. 14-24 Straight-chain alkyl groups.

[0175] As used herein, the terms “complementary” or “reverse complementary” are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired in a complementary manner with the bases of the other strand. In DNA, the purine base adenine always pairs with the pyrimidine base thymine (or uracil in RNA); the purine base guanine always pairs with the pyrimidine base cytosine. Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. Correspondingly, “mismatch” in the art means, in the case of a double-stranded nucleic acid, that the bases at corresponding positions are not paired in a complementary manner.

[0176] As used herein, the term “inhibition” may be used interchangeably with “reduction,” “silencing,” “downregulation,” “blocking,” and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in one or more of these variables at an absolute or relative level compared to a control level. This control level can be any type of control level used in the art, such as a baseline level before administration or a level determined from a subject, cell, or sample that has been treated untreated or with a control (e.g., a buffer-only control or an inert control). For example, the degree of inhibition of target gene expression by an RNAi agent can be characterized by residual mRNA expression levels such as not exceeding 99%, not exceeding 95%, not exceeding 90%, not exceeding 85%, not exceeding 80%, not exceeding 75%, not exceeding 70%, not exceeding 65%, not exceeding 60%, not exceeding 55%, not exceeding 50%, not exceeding 45%, not exceeding 40%, not exceeding 35%, not exceeding 30%, not exceeding 25%, not exceeding 20%, not exceeding 15%, or not exceeding 10%. The inhibition rate of target gene expression can be measured using... The Luciferase Assay System was used to detect the chemiluminescence values ​​of fireflies (Fir) and sea urchins (Ren), and the relative value Ratio = Ren / Fir was calculated. In this disclosure, the proportion of remaining mRNA expression (or remaining activity %) = Ratio (RNAi agent treatment group) / Ratio (no RNAi agent control group), and the inhibition rate (%) = 100% - remaining mRNA expression (%).

[0177] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0178] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylic acid salts, and naphthalene disulfonates. These salts can be prepared by methods known in the art.

[0179] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. In some implementations, the inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring 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, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Some embodiments include organic bases such as isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0180] "Effective amount" or "effective dose" refers to the amount of RNAi agent, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcomes. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of symptoms, including the symptoms, their complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes presented during the development of the symptoms. For therapeutic applications, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various symptoms associated with the target genes, target mRNAs, or target proteins of this disclosure, or improving one or more symptoms of said symptoms, reducing the dosage of other agents required to treat the symptoms, enhancing the efficacy of another agent, and / or delaying the progression of the symptoms associated with the target genes, target mRNAs, or target proteins of this disclosure in patients.

[0181] As used herein, the terms "patient," "subject," or "individual" are used interchangeably and include human or non-human animals, such as mammals, such as humans or monkeys. In some specific implementations, "subject" may even include test cells, test models, or tissues, depending on the context.

[0182] The RNAi agents provided in this disclosure can be obtained using conventional preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis). Solid-phase synthesis is already available as a commercially available custom service. Modified nucleotide groups can be introduced into the RNAi agents described in this disclosure using appropriately modified nucleoside monomers. Methods for preparing appropriately modified nucleoside monomers and for introducing modified nucleotide groups into RNAi agents are also well known to those skilled in the art.

[0183] The term “chemical modification” or “modification” includes all alterations to nucleotides by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0184] The term "base" includes any known DNA and RNA base, base analogues such as purines or pyrimidines, and also includes natural compounds such as adenine, thymine, guanine, cytosine, uracil, hypoxanthoside, and natural analogues.

[0185] The terms "blunt-ended" or "knuckle-ended" are used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogues at a given end of an RNAi agent; that is, no nucleotide protrusions. In most cases, RNAi agents with blunt ends will be double-stranded throughout their entire length.

[0186] The terms “about” and “approximately” mean that a numerical value is within an acceptable margin of error for a specific value as determined by a person skilled in the art, the numerical value depending in part on how it is measured or measured (i.e., the limits of the measurement system). For example, “about” may mean within or above 1 standard deviation. Alternatively, “about” or “substantially includes” may mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, or between 0.5% and 1%. In this disclosure, each instance of a number or range of values ​​preceded by the term “about” also includes embodiments of a given number. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of “about” or “substantially includes” should be assumed to be within an acceptable margin of error for that specific value.

[0187] Unless otherwise stated, "optionally," "optionally," "optional," or "optional" means that the event or situation described below may but does not have to occur, and this description includes the possibility that the event or situation may or may not occur. For example, "optionally, R1 and R2 are directly connected to form a loop" means that R1 and R2 being directly connected to form a loop may occur but is not required to exist, and this description includes both the case where R1 and R2 are directly connected to form a loop and the case where R1 and R2 are not connected to form a loop.

[0188] In the chemical structural formula disclosed herein, It can be connected with one or more groups according to the scope of the invention described herein.

[0189] The term "connection" refers to the link between two molecules, either through a covalent bond or through a non-covalent bond (e.g., hydrogen bond or ionic bond), including direct and indirect connections.

[0190] The term "direct link" refers to the connection between a first compound or group and a second compound or group without any intercalating atoms or atomic groups.

[0191] The term "indirect link" refers to the connection between a first compound or group and a second compound or group through an intermediate group, compound, or molecule (e.g., a linking group).

[0192] The term "substituted" means that any one or more hydrogen atoms on a specified atom (typically carbon, oxygen, or nitrogen atoms) are replaced by any group defined herein, provided that the substitution does not exceed the normal valence of the specified atom and yields a stable compound. Non-limiting examples of substituents include C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cyano, hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclic, heteroaryl, aryl, ketone, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halogens (e.g., F, Cl, Br, I). When the substituent is a ketone or oxo (i.e., =O), two (2) hydrogen atoms on the atom are substituted.

[0193] "Being replaced by one or more..." means that it can be replaced by a single or multiple substituents. When replaced by multiple substituents, it can be a plurality of identical substituents or a combination of one or a plurality of different substituents. Detailed Implementation

[0194] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents whose specific source is not specified are available from any molecular biology reagent supplier at the quality / purity required for molecular biology applications. Unless otherwise stated, all reagents used in the following embodiments are commercially available products.

[0195] Abbreviations: NMM: N-methylmorpholine; DCM: dichloromethane; MeOH: methanol; EtOH: ethanol; AcOH: acetic acid; DMF: N,N-dimethylformamide; DMTrCl: 4,4'-bismethoxytriphenylmethyl chloride; NMI: N-methylimidazolium; DCE: 1,2-dichloroethane; BSA: N,O-bistrimethylsilylacetamide; TMSOTf: trimethylsilyl trifluoromethanesulfonate; BzCl: benzoyl chloride; TMSCl: trimethylchlorosilane; LCMS: liquid chromatography-mass spectrometry; NMR: nuclear magnetic resonance.

[0196] Example 1. Design of MAPT RNAi Agent

[0197] The sequences of the sense and antisense strands in the RNAi agents disclosed herein are shown in Tables 1a, 1b, and 1c below. Tables 1a, 1b, and 1c use the human MAPT gene (NM_016841.4) as the target gene to design RNAi agent sequences in accordance with the general rules for active dsRNA.

[0198] Table 1a. RNAi agent sequences targeting the MAPT gene

[0199] Table 1b. RNAi agent sequences targeting the MAPT gene

[0200] Table 1c. RNAi agent sequences targeting the MAPT gene

[0201] In Tables 1a, 1b, and 1c, within the same nucleic acid sequence, G, C, A, and U represent nucleosides containing guanine, cytosine, adenine, and uracil bases, respectively. The direction from the 5' end to the 3' end is considered left-to-right. A lowercase letter m indicates that the nucleoside adjacent to the left of m is 2'-methoxy modified; a lowercase letter f indicates that the nucleoside adjacent to the left of f is 2'-fluoro modified; and an uppercase letter VP indicates that the 5' position of the sugar ring of the nucleoside adjacent to its right is E-. Vinyl phosphate group modification; lowercase s indicates that the two nucleosides adjacent to the letter s are linked by a thiophosphate diester group; NA0133' indicates that the nucleoside adjacent to its right is a nucleoside with the structure shown in formula (I), and its structure is shown in Table 2 below; IB indicates a nucleoside without reverse base modification; hmpNA indicates that the nucleoside adjacent to its right is a nucleoside modified with hmpNA, and NA0218 indicates that the nucleoside adjacent to its right is a nucleoside containing the nucleoside trimming of formula (IV-1).

[0202] Unless otherwise specified, two adjacent or linked nucleosides are connected to the delivery group via a phosphodiester group. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each chain is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each chain is a hydroxyl group.

[0203] The structures of the 2'-methoxy modified nucleosides, 2'-fluoro modified nucleosides, thiophosphate diester groups, phosphodiester groups, 5'-E-VP, NA0133', nucleosides with structures as shown in formula (IV-1), and reverse abase-free nucleosides are shown in Table 2 below. When the RNAi agent of this disclosure exists in salt form, such as in sodium salt form, the salt form structures corresponding to the structures in Table 2 below are also within the protection scope of this disclosure. In the formula, Base represents a base, 5' represents a bond towards the 5' end of the nucleotide sequence, and 3' represents a bond towards the 3' end of the nucleotide sequence.

[0204] Table 2.

[0205] The RNAi agent provided in this disclosure can be obtained by conventional preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis). Solid-phase synthesis is already available as a commercially available custom service. Modified nucleotide groups can be introduced into the RNAi agent described in this disclosure using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into RNAi agents are well known to those skilled in the art. In the RNAi agent of this disclosure, 2'-methoxy modified, 2'-fluorinated modified, E-vinyl phosphate-containing, and reverse-base-free nucleosides of phosphoramidite monomers were purchased from Jiangsu Shenji Company or Zhaowei Technology Co., Ltd. The phosphoramidite monomer NA0133' having U, A, and G bases was prepared according to the method described in PCT application WO2024125556A1 and the methods described in Examples 2 and 3 below. A phosphoramidite monomer containing the structure of formula (IV-1) was prepared according to the method described in WO2025252152A1.

[0206] RNAi agents containing hmpNA-modified nucleosides were prepared according to the method described in PCT application WO2022028462A1.

[0207] Example 2 Synthesis of phosphoramide monomer NA0133(A)

[0208] The phosphoramide monomer NA0133(A) was prepared according to the path shown in the figure below:

[0209] 2.1 Synthesis of compounds 2-4

[0210] N6-benzoyladenine 2-3 (9.05 g, 37.9 mmol) and N,O-bis(trimethylsilylacetamide) (15.6 mL, 63.0 mmol) were dissolved in anhydrous 1,2-dichloroethane (200 mL). Under nitrogen protection, the reaction was carried out at 50°C in an oil bath for 30 minutes. The resulting reaction solution was cooled to room temperature, and under nitrogen protection, a 1,2-dichloroethane solution of compound 2-2 (6.7 g, 12.6 mmol, prepared according to the method described in PCT application WO2024125556A1) and trimethyl trifluoromethanesulfonate were added sequentially. Silicon ester (6.86 mL, 50.8 mmol) was used. The resulting reaction solution was heated to 50°C and stirred for 60 minutes, then heated to 105°C and stirred for 2 hours. LC-MS monitoring showed the reaction was complete. The reaction was cooled to room temperature, quenched with saturated sodium bicarbonate aqueous solution (150 mL), and extracted three times with dichloromethane solution (150 mL). The combined organic phases were washed once with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. Purification was performed by normal-phase silica gel column chromatography to give compound 2-4 (5.2 g, 56% yield) as a white solid. MS (ESI) m / z = 739.0 [M+H] + .

[0211] 2.2 Synthesis of compounds 2-5

[0212] Compound 2-4 (10.9 g, 14.8 mmol) was dissolved in 7 M ammonia-methanol solution (210 mL), stirred at 60°C for 48 hours, concentrated, and purified by normal-phase column chromatography to give compound 2-5 (6 g, 77% yield) as a white solid. MS (ESI) m / z = 530.7 [M+H] + .

[0213] 2.3 Synthesis of compounds 2-6

[0214] Compound 2-5 (6.00 g, 11.3 mmol) was dissolved in anhydrous pyridine (56 mL). TMSCl (7.23 mL, 56.6 mmol) was added at 0°C, and the mixture was stirred at 0°C for 1 hour under nitrogen protection. Benzoyl chloride (6.57 mL, 56.6 mmol) was then added dropwise. After the addition was complete, the mixture was brought to room temperature and stirred for 3 hours. The reaction mixture was cooled to 0°C, water (15 mL) was added, and the mixture was stirred for 30 minutes. Then, concentrated ammonia (30 mL, 28% purity) was added, and the mixture was stirred at room temperature for 2 hours. LC-MS monitoring showed the reaction was complete. The mixture was carefully poured into water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and filtered. The solvent was evaporated, and the mixture was purified by normal-phase silica gel column chromatography to give compound 2-6 (4.6 g, 64% yield) as a white solid. MS (ESI) m / z = 634.8 [M+H] + .

[0215] 2.4 Synthesis of compounds 2-7

[0216] Under nitrogen atmosphere, NaH (0.95 g, 23.6 mmol, 60% mineral oil) was dissolved in N,N-dimethylformamide (50 mL), and stirred in an ice bath for 15 minutes. Then, a solution of compound 2-6 (3.00 g, 4.73 mmol) in N,N-dimethylformamide (30 mL) was added, and the mixture was stirred in an ice bath for 15 minutes. Next, a solution of hexadecane (4.01 mL, 12.8 mmol) in N,N-dimethylformamide (5 mL) was added dropwise. The mixture was slowly heated to room temperature and reacted at 30°C for 4 hours. LC-MS monitoring showed that the reaction was incomplete, with a conversion rate of approximately 70%. The reaction solution was quenched in a saturated ammonium chloride aqueous solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and purified by normal-phase column chromatography to obtain compound 2-7 (2.8 g, 69% yield) as a yellow oily product. MS (ESI) m / z = 857.5 [MH]. + .

[0217] 2.5 Synthesis of compounds 2-8

[0218] Compound 2-7 (3.00 g, 3.49 mmol) was dissolved in dichloromethane (60 mL), purged with nitrogen, and cooled to -78°C. Boron trichloride (23.7 mL, 23.7 mmol) was slowly added dropwise to the reaction solution, with the internal temperature controlled not to exceed -60°C. After the addition was complete, the mixture was stirred at -78°C for 1 hour, then slowly heated for 1.5 hours to -20°C. LC-MS monitoring showed the starting material had disappeared. The reaction solution was cooled to -78°C, and methanol (20 mL) and triethylamine (12 mL) were added sequentially to quench the reaction, with the internal temperature controlled not to exceed -60°C. The mixture was then slowly heated for 0.5 hours to 0°C. The reaction solution was concentrated and purified by normal-phase column chromatography to give compound 2-8 (1.6 g, 75% yield) as a pale yellow solid. MS (ESI) m / z = 609.1 [MH] + .

[0219] 2.6 Synthesis of compounds 2-9

[0220] Compound 2-8 (1.60 g, 2.62 mmol) was dissolved in dichloromethane (32 mL), and 3A molecular sieves were added. Under ice bath conditions, N-methylmorpholine (0.865 mL, 7.87 mmol) and 4,4'-dimethoxytriphenylmethylchloro (936 mg, 2.76 mmol) were added sequentially. The mixture was stirred at room temperature for half an hour, and the reaction was monitored for completeness by LC-MS. The reaction solution was concentrated and purified directly by normal-phase column chromatography (3% MeOH in 0.1% TEA / DCM) to give compound 2-9 (1.35 g, 56% yield) as a yellow solid. MS (ESI) m / z = 911.6 [MH] + .

[0221] 2.7 Synthesis of NA0133(A) phosphorous amide monomer

[0222] Under nitrogen protection, 3A molecular sieves were added to a dichloromethane (30 mL) solution of compound 2-9 (1.59 g, 1.74 mmol). After stirring for 5 minutes at room temperature, compound 2-10 (1.58 g, 5.23 mmol) was added in an ice bath, followed by a pre-prepared dry acetonitrile solution (4 mL) of 1H-tetrazole (0.45 M) and N-methylimidazole (0.18 M). The reaction was carried out in an oil bath at 35°C for 4 hours. The reaction solution was quenched in a saturated sodium bicarbonate aqueous solution in an ice bath, extracted with dichloromethane, washed with brine, dried and concentrated with anhydrous sodium sulfate, purified by reverse-phase column chromatography (100% MeCN), and lyophilized to give compound NA0133(A) (1.53 g, 79% yield) as a white solid. MS (ESI) m / z = 1112.4 [MH] + .

[0223] Example 3. Synthesis of phosphoramide monomer NA0133(G)

[0224] The phosphoramid monomer NA0133(G) was prepared according to the path shown in the figure below:

[0225] 3.1 Synthesis of Compound 3-3

[0226] 2,6-Diaminopurine 3-2 (5.6 g, 37.6 mmol) and N,O-bis(trimethylsilylacetamide) (18.6 mL, 75.2 mmol) were dissolved in anhydrous 1,2-dichloroethane (400 mL). The reaction was carried out under nitrogen protection at 70°C in an oil bath for 60 minutes, yielding a clear reaction solution. The solution was cooled to room temperature, and under nitrogen protection, 1,2-dichloroethane of compound 2-2 (10 g, 18.8 mmol, prepared according to the method described in PCT application WO2024125556A1) was added sequentially. Ethane solution (50 mL) and trimethylsilyl trifluoromethanesulfonate (7 mL, 37.6 mmol) were reacted. The reaction solution was heated to 105 °C and stirred for 16 hours. The reaction was monitored by LC-MS until complete. The reaction solution was cooled to room temperature and poured into a saturated sodium bicarbonate aqueous solution (200 mL) under ice bath conditions. The mixture was extracted three times with dichloromethane solution (200 mL). The combined organic phases were dried over anhydrous sodium sulfate and filtered. The solvent was evaporated, and the mixture was subjected to normal-phase silica gel column chromatography to give compound 3-3 (6.9 g, 57% yield) as a pale yellow solid. MS (ESI) m / z = 649.5 [M+H] + .

[0227] 3.2 Synthesis of compounds 3-4

[0228] Compound 3-3 (13.8 g, 21 mmol) was dissolved in 7 M ammonia-methanol solution (200 mL), stirred at 60°C for 48 hours, concentrated, and purified by normal-phase column chromatography to give compound 3-4 (8.5 g, 75% yield) as a pale yellow solid. MS (ESI) m / z = 545.2 [M+H] + .

[0229] 3.3 Synthesis of compounds 3-5

[0230] Compounds 3-4 (8.5 g, 15.6 mmol) were dissolved in anhydrous pyridine (85 mL). TMSCl (10 mL, 78 mmol) was added at 0°C, and the mixture was stirred at 0°C for 1 hour under nitrogen protection. Benzoyl chloride (9 mL, 78 mmol) was then added dropwise. After the addition was complete, the mixture was brought to room temperature and stirred for 3 hours. The reaction mixture was cooled to 0°C, water (20 mL) was added, and the mixture was stirred for 30 minutes. Concentrated ammonia (45 mL, 28% purity) was then added, and the mixture was stirred at room temperature for 2 hours. LC-MS monitoring showed the reaction was complete. The mixture was carefully poured into water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and filtered. The solvent was evaporated, and the mixture was purified by normal-phase silica gel column chromatography to give compound 3-5 (11 g, 93% yield) as a pale yellow solid. MS (ESI) m / z = 751.0 [MH] - .

[0231] 3.4 Synthesis of compounds 3-6

[0232] Under nitrogen atmosphere, NaH (2.34 g, 58.4 mmol, 60% mineral oil) was dissolved in N,N-dimethylformamide (80 mL), and stirred in an ice bath for 10 minutes. Then, a solution of compound 3-5 (11 g, 14.6 mmol) in N,N-dimethylformamide (25 mL) was added, and the mixture was stirred in an ice bath for 15 minutes. Next, a solution of hexadecane (12.8 g, 36.5 mmol) in N,N-dimethylformamide (5 mL) was added dropwise. The reaction mixture was then reacted in an oil bath at 30°C for 4 hours. The reaction solution was quenched in a saturated ammonium chloride aqueous solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and purified by normal-phase column chromatography to give compound 3-6 (10 g, 70% yield) as a pale yellow oil. MS (ESI) m / z = 975.6 [MH] - .

[0233] 3.5 Synthesis of compounds 3-7

[0234] Compound 3-6 (10 g, 10.3 mmol) was dissolved in 7 M ammonia-methanol solution (150 mL), stirred at 50 °C for 24 hours, concentrated, and purified by normal-phase column chromatography to give compound 3-7 (8 g, 89% yield) as a pale yellow solid. MS (ESI) m / z = 871.5 [MH] - .

[0235] 3.6 Synthesis of compounds 3-8

[0236] Compound 3-7 (2.5 g, 2.86 mmol) was dissolved in ethanol (60 mL), followed by the addition of 10% Pd / C (1 g). The mixture was purged three times under hydrogen atmosphere, and the reaction solution was stirred at 50°C for 2 hours. Then, 10% Pd / C (1 g) was added again, followed by three more purgings under hydrogen atmosphere and stirring at 50°C. Monitoring was performed every 1.5 hours. After three repeated additions of palladium on carbon, LC-MS showed complete reaction of the starting material. After diatomaceous earth filtration, the filtrate was concentrated and purified directly by normal silica gel column chromatography to obtain compound 3-8 (660 mg, 37% yield) as a pale yellow solid. MS (ESI) m / z = 623.5 [MH] - .

[0237] 3.7 Synthesis of compounds 3-9

[0238] Compound 3-8 (660 mg, 1.06 mmol) was dissolved in acetic acid (45 mL), and the solution was heated to 60°C to ensure complete dissolution. An aqueous solution of sodium nitrite (875 mg, 12.7 mmol) (10 mL) was slowly added, and the reaction was carried out at 60°C for 2 hours. After the reaction was complete as monitored by LC-MS, the reaction mixture was concentrated under vacuum and poured into water. The mixture was extracted with dichloromethane, and the combined organic phases were washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, concentrated, and purified by normal-phase silica gel column chromatography to give compound 3-9 (500 mg, 80% yield) as a pale yellow solid. MS (ESI) m / z = 624.4 [MH] - .

[0239] 3.8 Synthesis of Compounds 3-10

[0240] Compound 3-9 (500 mg, 0.8 mmol) was dissolved in dichloromethane (20 mL), and 3A molecular sieve was added. N-methylmorpholine (0.3 mL, 2.4 mmol) and 4,4'-dimethoxytriphenylmethylchloro (330 mg, 0.96 mmol) were added sequentially under ice bath conditions. The mixture was stirred at room temperature for 1 hour. The reaction was monitored by LC-MS until complete. The reaction was quenched with methanol (2 mL), and the mixture was directly concentrated under vacuum after adding basic silica gel. Normal-phase silica gel column chromatography yielded compound 3-10 (700 mg, 94% yield) as a pale yellow solid. MS (ESI) m / z = 926.7 [MH] - .

[0241] 3.9 Synthesis of NA0133(G) phosphorous amide monomer

[0242] Under nitrogen protection, 3A molecular sieves were added to a 24 mL solution of compound 3-10 (1.20 g, 1.29 mmol) in dichloromethane. After stirring at room temperature for 5 minutes, compound 3-11 (1.17 g, 3.88 mmol) was added, followed by a 4.31 mL solution of 1H-tetrazole (0.45 M) and N-methylimidazole (0.18 M) in dry acetonitrile. The reaction was carried out at 30°C for two hours. The reaction was monitored by LC-MS until complete. The reaction solution was quenched with saturated sodium bicarbonate solution, extracted with dichloromethane, washed with brine, dried over anhydrous sodium sulfate, concentrated, filtered, and purified by phenyl reverse-phase column chromatography to obtain compound NA0133(G) (970 mg, yield: 66%) as a white solid. MS (ESI) m / z = 1126.9 [M⁻¹] + . 1 H NMR (400MHz, DMSO) δ12.30(s,1H),11.76(s,1H),8.15(d,J=3.2Hz,1H),8.00(d,J=8.0Hz,2H),7.70(t ,J=7.6Hz,1H),7.57(t,J=7.6Hz,2H),7.35(t,J=8.4Hz,2H),7.26–7.17(m,7H),6.65-6.88(m,4H),6.0 9(dd,J=24.4,8.0Hz,1H),4.63–4.37(m,1H),4.17(dd,J=23.6,20.4Hz,1H),3.78–3.41(m,14H),3.30 –3.13(m,3H),2.77(t,J=6.0Hz,1H),2.58(t,J=6.0Hz,1H),1.10-1.45(m,40H),0.85(t,J=6.8Hz,3H). 31 P NMR (162MHz, DMSO) δ149.02 (d, J=16.8Hz).

[0243] Example 4. Inhibitory activity of RNAi agents against human MAPT in A172 cells.

[0244] In vitro molecular-level activity screening of RNAi agents was performed in A172 cells (human glioblastoma cell line). Each RNAi agent sample was initially transfected at a concentration of 10 nM, with 5-fold serial dilutions and 7 concentration points.

[0245] A172 cells were cultured in Dulbecco's modified eagle medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 hours before transfection, A172 cells were seeded into 96-well plates at a density of 1 × 10⁶ cells per well. 4100 μL of culture medium per well for each cell.

[0246] Referring to the product manual, Lipofectamine RNAi MAX (ThermoFisher, 13778150) was used to transfect RNAi agents. Seven or nine concentration points were set for the RNAi agent, with an initial concentration of 10 nM, and each concentration was tested in duplicate. Forty-eight hours after treatment, total RNA was extracted from cells using a high-throughput cell RNA extraction kit FG0417-L / FG0418-XL (ZhiFan Medical, magnetic bead method). RNA reverse transcription experiments (Takara, RR037A) and quantitative real-time PCR (Thermo, 4444557) were performed to detect the mRNA level of human MAPT. The mRNA level of human MAPT was corrected based on the level of the GAPDH internal reference gene.

[0247] In the real-time quantitative PCR detection, probe Q-PCR detection experiment was used, and its primer information is shown in Table 3 below.

[0248] Table 3. Taqman Primer Information

[0249] Results analysis method:

[0250] After the Q-PCR assay is completed, the corresponding Ct value is obtained according to the threshold automatically set by the system. The expression of a specific gene can be relatively quantified by comparing Ct values: comparing Ct refers to calculating the difference in gene expression by comparing the Ct value with that of the internal reference gene, also known as 2-1. -△△Ct △△Ct=[(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)]. Inhibition rate (%)=(1-remaining amount of target gene expression)*100%.

[0251] In vitro molecular-level activity screening of RNAi agent sequences was performed in A172 cells using seven concentration gradients. Results are expressed as the percentage of residual human MAPT mRNA expression relative to cells treated with the control RNAi agent. The IC50 of the inhibition rate is also considered. 50 The results are shown in Tables 4 and 5 below.

[0252] Table 4. RNAi agent activity at 7 concentration sites in A172 cells (IC50) 50 )

[0253] Table 5. RNAi agent activity at 7 concentration sites in A172 cells (IC50) 50 )

[0254] Example 5. RNAi agent at psiCHECK target activity

[0255] The RNAi agent disclosed herein was screened for in vitro molecular-level target activity simulation using nine concentration gradients in HEK293A cells.

[0256] The target sequence of the RNAi agent was constructed using the target plasmid GSCM and inserted into the psiCHECK-2 plasmid, which contains the Renida luciferase gene and the firefly luciferase gene. As a dual reporter gene system, the target sequence of the dsRNA was inserted into the 3'UTR region of the Renida luciferase gene. The activity of the RNAi agent against the target sequence can be reflected by detecting the expression of Renida luciferase after calibration with firefly luciferase.

[0257] HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 hours before transfection, HEK293A cells were seeded into 96-well plates at a density of 8 × 10⁶ cells per well. 3 100 μL of culture medium per well for each cell.

[0258] Following the instructions, Lipofectamine 2000 (ThermoFisher, 11668019) was used to co-transfect cells with the RNAi agent and the corresponding GSCM plasmid, with 0.2 μL of Lipofectamine 2000 per well. The plasmid transfection volume was 20 ng per well. For the target sequence plasmid, nine concentrations of the RNAi agent were set, starting at 20 nM and serially diluted 3-fold, with each concentration in duplicate. Target levels were detected 24 h post-transfection using the Dual-Luciferase Reporter Assay System (Promega, E2940). The results are shown in Table 6.

[0259] Table 6. Activity results of RNAi agents at 9 concentration points in the psi-Check system (IC50) 50 )

[0260] Example 6. IC50 of RNAi agents inhibiting MAPT expression in glioblastoma cells (A172) 50 active

[0261] A172 cells were cultured in DMEM medium (Gibco) (containing 10% FBS) at 37°C and 5% CO2.

[0262] 24 hours before transfection, A172 cells were inoculated at 1.0 × 10⁶ cells per cell line. 4Cells were seeded at a density of 100 μL / well in 96-well plates. Following the manufacturer's instructions, cells were transfected with RNAi MAX transfection reagent (ThermoFisher, 13778150). Seven concentration points were set for the RNAi reagent, with the highest concentration at 10 nM (7-fold dilution) and the lowest concentration at 0.00008 nM. Forty-eight hours after transfection, total RNA was extracted using a high-throughput cell RNA extraction kit (ThermoFisher, A27828), RNA reverse transcription was performed (Takara, RR037B), and quantitative real-time PCR was conducted (ThermoFisher, 4444557) to determine the mRNA level of human MAPT. The mRNA level of human MAPT was corrected for the level of the GAPDH internal reference gene.

[0263] In the real-time quantitative PCR detection, probe Q-PCR detection experiment was used, and its primer information is shown in Table 3 above.

[0264] Results Analysis Methods

[0265] After the Q-PCR assay is completed, the corresponding Ct value is obtained according to the threshold automatically set by the system. The expression of a specific gene can be relatively quantified by comparing Ct values: comparing Ct refers to calculating the difference in gene expression based on the difference between the Ct value and that of the internal reference gene, also known as 2-1. -△△Ct △△Ct=[(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)]. Inhibition rate (%)=(1-remaining amount of target gene expression)*100%.

[0266] Results are expressed as the percentage of remaining human MAPT mRNA expression relative to cells treated with the control RNAi agent. IC50 of inhibition rate. 50 The results are shown in Table 7 below.

[0267] The results in Table 7 show that the RNAi agent disclosed herein has a high level of inhibitory activity against the MAPT gene in A172 cells.

[0268] Table 7. RNAi agent activity at 7 concentration sites in A172 cells (IC50) 50 )

[0269] Example 7. Inhibitory activity of RNAi agents against human MAPT in A172 cells.

[0270] In vitro molecular-level activity screening of RNAi agents was performed in A172 cells. Each RNAi agent sample was initially transfected at a concentration of 10 nM, with 5-fold serial dilutions and 7 concentration points.

[0271] A172 cells were cultured in Dulbecco's modified eagle medium containing 10% fetal bovine serum at 37°C and 5% CO2. On the day of transfection, A172 cells were seeded into 96-well plates at a density of 1.5 × 10⁶ cells per well. 4 100 μL of culture medium per well for each cell.

[0272] Following the product manual, dsRNA was transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150). Seven or nine concentration points were set for dsRNA, with an initial concentration of 10 nM, and each concentration was tested in duplicate. Twenty-four hours after treatment, total RNA was extracted from cells using a high-throughput cell RNA extraction kit FG0417-L / FG0418-XL (ZhiFan Medical, magnetic bead method). RNA reverse transcription experiments (Takara, RR037A) and quantitative real-time PCR (Thermo, 4444557) were performed to determine the mRNA level of human MAPT. The mRNA level of human MAPT was corrected based on the level of the GAPDH internal reference gene.

[0273] In the real-time quantitative PCR detection, probe Q-PCR detection experiment was used, and the primer information and data processing method were the same as in Example 6.

[0274] The results are shown in Table 8 below. The results in Table 8 show that the RNAi agent disclosed herein has a high level of inhibitory activity against the MAPT gene in A172 cells.

[0275] Table 8. IC50 of RNAi agents in A172 cells 50

[0276] Example 8. Endogenous cellular activity of RNAi agents

[0277] In A172 cells, RNAi agents were screened at seven concentration gradients to simulate endogenous cell activity at the in vitro molecular level.

[0278] The RNAi agent disclosed herein was serially diluted 6-fold at an initial concentration of 10 nM using RNAi MAX transfection reagent (ThermoFisher, 13778150) to obtain seven concentration spots (10 nM, 1.6667 nM, 0.2778 nM, 0.0463 nM, 0.00772 nM, 0.00129 nM, and 0.00021 nM). A172 cells were cultured in Dulbecco's modified eagle medium (Gibco) containing 10% FBS at 37°C and 5% CO2. After adding the RNAi agent to 96-well plates, the seeding density was 1.5 × 10⁶ cells / well. 4 A172 cells per well (90 μL of culture medium per well). Total RNA was extracted 24 hours after transfection using a high-throughput cell RNA extraction kit (Zhiang Biotechnology, MNTR / FX96). The mRNA level of human MAPT was measured by reverse transcription assay (Takara, RR037A) and quantitative real-time PCR (ThermoFisher, 4444557). The expression level was corrected using GAPDH as an internal reference gene.

[0279] Results analysis method:

[0280] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold automatically set by the system. The expression of a certain gene can be relatively quantified by comparing the Ct values: comparing Ct refers to calculating the difference in gene expression by comparing the Ct value with that of the internal reference gene, also known as 2-△△Ct, △△Ct=[(Ct target gene in experimental group - Ct internal reference in experimental group) - (Ct target gene in control group - Ct internal reference in control group)].

[0281] Inhibition rate (%) = (1 - remaining amount of target gene expression) * 100%.

[0282] Results are expressed as the percentage of residual human MAPT expression relative to cells treated with the RNAi agent. IC50 of inhibition rate. 50 The results are shown in Table 9 below. Table 9 shows that the RNAi agent disclosed herein has excellent inhibitory activity in A172 cells.

[0283] Table 9. Endogenous activity (IC50) of the RNAi agent of this disclosure in A172 cells. 50 )

[0284] Example 9. Endogenous cellular activity of RNAi agents

[0285] In A172 cells, RNAi agents were screened at an in vitro molecular level to mimic the activity of endogenous cells using eight concentration gradients.

[0286] A172 cells were cultured in DMEM medium (Gibco, 11965092) (containing 10% FBS) at 37°C and 5% CO2. Following the manufacturer's instructions, RNAi MAX transfection reagent (ThermoFisher, 13778150) was used to transfect the cells with RNAi, 0.3 μL per well. The RNAi reagent was serially diluted 6-fold at an initial concentration of 10 nM to create eight concentration spots (10 nM, 1.67 nM, 0.278 nM, 0.0463 nM, 0.00771 nM, 0.00129 nM, 0.000214 nM, 0.000036 nM). After adding the RNAi reagent to the 96-well plates, A172 cells were seeded at a density of 1.5 × 10⁶ cells / well. 4 Cells / well (90 μL of culture medium per well). Total RNA was extracted 24 hours after transfection using a high-throughput cell RNA extraction kit (Zhiang Bio, MNTR / FX96). The mRNA level of human MAPT was measured by reverse transcription assay (Takara, RR037A) and quantitative real-time PCR (ThermoFisher, 4444557). The expression level was corrected using GAPDH as an internal reference gene.

[0287] The instruments involved in this experiment are shown in Table 10.

[0288] Table 10. Experimental Apparatus

[0289] In the real-time quantitative PCR detection, probe Q-PCR detection experiment was used, and its primer information is shown in Table 3 above.

[0290] Results analysis method:

[0291] After the Q-PCR assay is completed, the corresponding Ct value is obtained according to the threshold automatically set by the system. The expression of a specific gene can be relatively quantified by comparing Ct values: comparing Ct refers to calculating the difference in gene expression by comparing the Ct value with that of the internal reference gene, also known as 2-1. -△△Ct △△Ct=[(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)]. Inhibition rate (%)=(1-remaining amount of target gene expression)*100%.

[0292] IC was calculated based on the inhibition rate of human MAPT expression in cells treated with RNAi agents. 50The results are shown in Tables 11a and 11b below. Table 11a shows that the RNAi agents of this disclosure, after different modifications, all exhibited good MAPT inhibitory activity. Table 11b shows that, compared to the positive control RNAi agents MAPTsi005, MAPTsi006, and MAPTsi007, the RNAi agent MAPTsi001 of this disclosure exhibits significantly superior MAPT inhibitory activity.

[0293] Table 11a. Activity results of RNAi agents in A172 cells (IC50) 50 )

[0294] Table 11b. Activity results of RNAi agents in A172 cells (IC50) 50 )

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