CN118355121A

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Publication number: CN118355121A
Application number: CN202280080982.2A
Authority: CN
Inventors: 李云飞; 张瑱; 林晓燕; 侯哲; 张建羽; 耿俊; 黄龙飞; 周雅琴; 吕珍珍
Original assignee: Shanghai Tuojie Biomedical Technology Co ltd
Current assignee: Shanghai Tuojie Biomedical Technology Co ltd
Priority date: 2021-12-16
Filing date: 2022-12-16
Publication date: 2024-07-16
Also published as: WO2023109935A1; WO2023109945A1; TW202340466A; CN118541482A; TW202333751A

Abstract

DsRNA, preparation method and application thereof, and pharmaceutical composition, cell or kit containing dsRNA, wherein the dsRNA can interfere with the expression of FXI gene and prevent and/or treat related diseases.

Description

DsRNA, preparation method and application thereof

The present disclosure claims priority to chinese patent application 202111542323.X, date 2021, 12, 16, the present disclosure incorporates the entirety of the above-mentioned chinese patent application.

Technical Field

The present disclosure relates to a dsRNA that can be targeted for delivery into a cell, exerting the effects of RNA interference. The disclosure also relates to methods of making and using the dsrnas.

Background

RNA interference (RNAi) is an effective way to silence gene expression. It is counted that, of the disease-associated proteins in humans, approximately more than 80% of proteins cannot be targeted by currently conventional small molecule drugs and biomacromolecule preparations, which are non-patentable proteins. By utilizing the RNA interference technology, proper siRNA can be designed according to mRNA encoding the proteins, target mRNA is specifically targeted and degraded, and thus, the generation of related proteins is inhibited. Therefore, siRNA has very important drug development prospect. However, to achieve the therapeutic objective RNA interference effect in vivo, it is necessary to deliver siRNA molecules to specific cells in vivo.

The targeting ligand is adopted to conjugate siRNA, and the targeting ligand is used for combining with a receptor on the surface of a cell membrane, so that endocytosis enters the cell, and the targeting ligand is an effective drug delivery mode. For example, asialoglycoprotein receptor (ASGPR) is a receptor specifically expressed by liver cells, and has the characteristics of high abundance on the surface of liver cells and rapid intracellular and extracellular conversion. Mono-and polysaccharide molecules such as galactose, galactosamine, N-acetylgalactosamine and the like have high affinity for ASGPR. Literature reports (10.16476/j. Pibb.2015.0028) that the use of a cluster of galactosamine molecules (GalNAc) can effectively deliver siRNA to hepatocytes, and that GalNAc molecules designed as a trivalent or tetravalent cluster of molecules can significantly enhance the ability of monovalent or divalent GalNAc molecules to target hepatocytes.

Different molecular cluster structures and different connection modes between the molecular cluster structures and the siRNA can obviously influence the activity of the siRNA in vivo, higher activity means better treatment effect or lower administration dosage, and under the same drug effect, lower administration dosage means lower toxic reaction.

Disclosure of Invention

In a first aspect, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) comprising an siRNA comprising a sense strand and an antisense strand comprising a chemical modification represented by formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof, at least one nucleotide position from position 2 to position 8 from the 5' end thereof, and one or more ligands conjugated thereto:

Wherein: y is selected from O, NH and S;

Each X is independently selected from CR₄(R₄')、S、NR₅ and NH-CO, wherein R₄、R₄'、R₅ is each independently H or C₁-C₆ alkyl;

J₂ is H or C₁-C₆ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

R₃ is selected from H, OH, halogen, NH₂、C₁-C₆ alkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyl, C₂-C₆ alkynyl, S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH₃, -O-alkylamino, and (CH₂)_pR₆; wherein R₆ is selected from OH, halogen, methoxy, ethoxy, N₃、C₂-C₆ alkenyl, and C₂-C₆ alkynyl, p=1, 2, or 3;

Q₁ isQ₂ is R₂; or alternatively

Q₁ is R₂,Q₂

Wherein:

R₁ is selected from H, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyl, C₂-C₆ alkynyl and (CH₂)_qR₇; wherein R₇ is selected from OH, halogen, methoxy, ethoxy, N₃、C₂-C₆ alkenyl and C₂-C₆ alkynyl, q=1, 2 or 3;

J₁ is H or C₁-C₆ alkyl;

r₂ is selected from H, OH, halogen, NH₂、C₁-C₆ alkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyl, C₂-C₆ alkynyl, S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH₃, -O-alkylamino, and (CH₂)_rR₈; wherein R₈ is selected from OH, halogen, methoxy, ethoxy, N₃、C₂-C₆ alkenyl, and C₂-C₆ alkynyl, R = 1,2, or 3;

Optionally, R₁ and R₂ are directly linked to form a ring;

B is a base;

the chemical modification shown in the formula (I), the tautomer or the pharmaceutically acceptable salt modification thereof is not

The ligand is a compound shown as a formula (II) or pharmaceutically acceptable salt thereof,

Wherein L₁ is a C₁-C₃₀ alkyl chain, or a C₁-C₃₀ alkyl chain comprising a break with one or more oxygen, sulfur, nitrogen atoms, or c=o;

R₁₁ and R₁₂ are independently a bond, NR₁₆, c=o or-OC (=o) -;

Q₃ is

Is a single bond or a double bond, and whenR₁₃ is independently CR₁₇R₁₈、NR₁₆, O or S when a single bond is presentWhen a double bond, R₁₃ is independently CR₁₉ or N;

R₁₄ is independently CR₁₉ or N;

Ring a is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, with or without ring a present, and R₁₅ is independently CR₁₉ or N when ring a is not present, and R₁₅ is independently CR₁₇R₁₈、NR₁₆ or O when ring a is not present;

R₁₆ and R₁₉ are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, SR ', S (=o) R', S (=o)₂R'、S(＝O)₂ NR '(R "), C (=o) R', C (=o) OR 'OR C (=o) NR' (R"), said alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl OR heteroaryl being optionally substituted with one OR more groups selected from halogen, hydroxy, oxo, nitro, cyano, C_1-6 alkyl, C_1-6 alkoxy, C_3-7 cycloalkyl, 3-12 membered heterocycloalkyl, 6-12 membered aryl, 5-12 membered heteroaryl, SR ', S (=o) R', S (=o)₂R'、S(＝O)₂ NR '(R "), C (=o) R', C (=o) OR 'and C (=o) NR' (R").

R₁₇ and R₁₈ are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, SR ', S (=o) R', S (=o)₂R'、S(＝O)₂ NR '(R "), C (=o) R', C (=o) OR 'OR C (=o) NR' (R"), said alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl OR heteroaryl being optionally substituted with one OR more groups selected from halogen, hydroxy, oxo, nitro, cyano, C_1-6 alkyl, C_1-6 alkoxy, C_3-7 cycloalkyl, 3-12 membered heterocycloalkyl, 6-12 membered aryl, 5-12 membered heteroaryl, SR ', S (=o) R', S (=o)₂R'、S(＝O)₂ NR '(R "), C (=o) R', C (=o) OR 'and C (=o) NR' (R").

R' and R "are independently hydrogen, deuterium, hydroxy, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, said alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted with one or more substituents selected from halogen, hydroxy, oxo, nitro and cyano;

m1, n1, p1 and q1 are independently 0, 1, 2, 3 or 4;

B1 is

R_b1、R_b2、R_b3、R_b4、R_b5、R_b6 and R_b7 are independently-C (=o) -, -NHC (=o) -, -C (=o) O-, -C (=o) - (CH₂)_z8 -O-, or-NHC (=o) - (CH₂)_z9 -O-;

z1, z2, z3, z4, z5, z6, z7, z8 and z9 are independently integers from 0 to 10;

L₂ is a C₁-C₃₀ alkyl chain, or a C₁-C₃₀ alkyl chain comprising a break with one or more oxygen, sulfur, nitrogen atoms, or c=o;

r1 is an integer of 1 to 10.

In some embodiments, when X is nh—co, R₁ is not H.

In some embodiments, the chemical modification shown in formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof is replaced with a 2' -methoxy modification.

In some embodiments, the chemical modification of formula (I) is a chemical modification of formula (I-1):

Wherein: y is selected from O, NH and S;

each J₁、J₂ is independently H or C₁-C₆ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Optionally, R₁ and R₂ are directly linked to form a ring;

B is as defined in formula (I).

In some embodiments of formula (I-1), B is selected from the group consisting of a purine base, a pyrimidine base, an indole, a 5-nitroindole, and a 3-nitropyrrole.

In some embodiments of formula (I-1), B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments of formula (I-1), B is selected from adenine, guanine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments of formula (I-1), B is the same as the base at which the nucleotide at that position of the antisense strand was not modified.

In some embodiments, the chemical modification of formula (I) is a chemical modification of formula (I-2):

Wherein Y is selected from O, NH and S;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

each J₁、J₂ is independently H or C₁-C₆ alkyl;

R₂ is selected from H, C₁-C₆ alkyl, C₁-C₆ alkoxy, S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH₃, -O-alkylamino, and (CH₂)_rR₈; wherein R₈ is selected from OH, halogen, methoxy, ethoxy, N₃、C₂-C₆ alkenyl, and C₂-C₆ alkynyl; R = 1, 2, or 3;

Optionally, R₁ and R₂ are directly linked to form a ring;

B is as defined in formula (I).

In some embodiments of formula (I-2), B is selected from the group consisting of a purine base, a pyrimidine base, an indole, a 5-nitroindole, and a 3-nitropyrrole.

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

In some embodiments of formula (I-2), B is selected from adenine, guanine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments of formula (I-2), B is the same as the base at which the nucleotide at that position of the antisense strand was not modified.

In some embodiments, each X is independently selected from CR₄(R₄')、S、NR₅ and NH-CO, wherein R₄、R₄'、R₅ is each independently H or C₁-C₃ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J₁、J₂ is independently H or C₁-C₃ alkyl;

R₃ is selected from H, OH, halogen, NH₂、C₁-C₃ alkyl, C₁-C₃ alkoxy, C₂-C₄ alkenyl, C₂-C₄ alkynyl, S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH₃, -O-alkylamino, and (CH₂)_pR₆; wherein R₆ is selected from OH, halogen, methoxy, ethoxy, N₃、C₂-C₆ alkenyl, and C₂-C₆ alkynyl, p=1, 2, or 3;

R₁ is selected from H, C₁-C₃ alkyl, C₁-C₃ alkoxy, C₂-C₄ alkenyl, C₂-C₄ alkynyl and (CH₂)_qR₇; wherein R₇ is selected from OH, halogen, methoxy, ethoxy, N₃、C₂-C₄ alkenyl and C₂-C₄ alkynyl, q=1, 2 or 3;

r₂ is selected from H, OH, halogen, NH₂、C₁-C₃ alkyl, C₁-C₃ alkoxy, C₂-C₄ alkenyl, C₂-C₄ alkynyl, S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH₃, -O-alkylamino, and (CH₂)_rR₈; wherein R₈ is selected from OH, halogen, methoxy, ethoxy, N₃、C₂-C₄ alkenyl, and C₂-C₄ alkynyl, R = 1,2, or 3;

Optionally, R₁ and R₂ are directly linked to form a ring;

B is as defined in formula (I).

In some embodiments, B is selected from the group consisting of a purine base, a pyrimidine base, an indole, a 5-nitroindole, and a 3-nitropyrrole.

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

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

In some embodiments, B is the same base as when the nucleotide at that position of the antisense strand is not modified.

In some embodiments, each X is independently selected from CR₄(R₄')、S、NR₅ and NH-CO, wherein R₄、R₄'、R₅ is each independently H, methyl, ethyl, n-propyl, or isopropyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

each J₁、J₂ is independently H or methyl;

R₃ is selected from H, OH, F, cl, NH₂, methyl, ethyl, N-propyl, isopropyl, methoxy, ethoxy, N-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH₃, -O-methylamino, -O-ethylamino, and (CH₂)_pR₆; wherein R₆ is selected from OH, F, cl, methoxy, ethoxy, N₃, vinyl, allyl, ethynyl, and propargyl, p = 1 or 2;

R₁ is selected from H, methyl, ethyl, N-propyl, isopropyl, methoxy, ethoxy, N-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, and (CH₂)_qR₇; wherein R₇ is selected from OH, F, cl, methoxy, ethoxy, N₃, vinyl, allyl, ethynyl, and propargyl, q=1 or 2;

R₂ is selected from H, OH, F, cl, NH₂, methyl, ethyl, N-propyl, isopropyl, methoxy, ethoxy, N-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH₃, -O-methylamino, -O-ethylamino, and (CH₂)_rR₈; wherein R₈ is selected from OH, F, cl, methoxy, ethoxy, N₃, vinyl, allyl, ethynyl, and propargyl, R = 1 or 2;

Optionally, R₁ and R₂ are directly linked to form a ring;

B is as defined in formula (I).

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

In some embodiments, Y is O or NH; each X is independently selected from NH-CO, CH₂, and NH;

n=0 or 1; m=0 or 1; s=0 or 1;

Each J₁、J₂ is independently H;

R₁ is selected from H, methyl and CH₂ OH;

r₂ is selected from H, OH, NH₂, methyl and CH₂ OH;

R₃ is selected from H, OH, NH₂, methyl and CH₂ OH;

Optionally, R₁ and R₂ are directly linked to form a ring;

B is as defined in formula (I).

n=0 or 1; m=0 or 1; s=0 or 1;

Each J₁、J₂ is independently H;

R₁ is selected from H, methyl and CH₂ OH;

R₂ is selected from H, methyl and CH₂ OH;

R₃ is selected from H, OH, NH₂, methyl and CH₂ OH;

Optionally, R₁ and R₂ are directly linked to form a ring;

B is as defined in formula (I).

In some embodiments, Y is O or NH;

Each X is independently selected from CR₄(R₄')、NR₅ and NH-CO, R₄、R₄'、R₅ is each independently H or C₁-C₆ alkyl;

J₂ is H or C₁-C₆ alkyl;

n=0 or 1; m=0 or 1; s=0 or 1;

R₃ is selected from H, OH, NH₂、C₁-C₆ alkyl, C₁-C₆ alkoxy and (CH₂)_pR₆;R₆ is selected from OH, methoxy and ethoxy, p=1, 2 or 3;

Q₁ isQ₂ is R₂; or Q₁ is R₂,Q₂ is

R₁ is selected from H, OH, C₁-C₆ alkyl, C₁-C₆ alkoxy and (CH₂)_qR₇;R₇ is selected from OH, methoxy and ethoxy, q=1, 2 or 3;

J₁ is H or C₁-C₆ alkyl;

R₂ is selected from H, OH, C₁-C₆ alkyl, C₁-C₆ alkoxy and (CH₂)_rR₈;R₈ is selected from OH, methoxy and ethoxy, r=1, 2 or 3;

Optionally, R₁ and R₂ are directly linked to form a 3-6 membered ring;

B is a base;

In some embodiments, X is independently selected from CR₄(R₄') and NH-CO.

In some embodiments, X is independently selected from CR₄(R₄').

In some embodiments, R₃ is selected from H, C₁-C₆ alkyl and (CH₂)_pR₆).

In some embodiments, R₃ is selected from H and C₁-C₆ alkyl.

In some embodiments, R₁ is selected from H, C₁-C₆ alkyl and (CH₂)_qR₇).

In some embodiments, R₁ is selected from H and C₁-C₆ alkyl.

In some embodiments, R₂ is selected from H, OH, C₁-C₆ alkyl, and (CH₂)_rR₈).

In some embodiments, R₂ is selected from H, C₁-C₆ alkyl and (CH₂)_rR₈).

In some embodiments, Y is O;

Each X is independently selected from CR₄(R₄ ') and NH-CO, R₄ and R₄' are each independently H or C₁-C₆ alkyl;

J₂ is H or C₁-C₆ alkyl;

R₃ is selected from H, C₁-C₆ alkyl and (CH₂)_pR₆;R₆ is selected from OH, p=1, 2 or 3;

Q₁ isQ₂ is R₂; or Q₁ is R₂,Q₂ is

R₁ is selected from H, C₁-C₆ alkyl and (CH₂)_qR₇;R₇ is selected from OH, q=1, 2 or 3;

J₁ is H or C₁-C₆ alkyl;

R₂ is selected from H, OH, C₁-C₆ alkyl and (CH₂)_rR₈;R₈ is selected from OH, r=1, 2 or 3;

Optionally, R₁ and R₂ are directly linked to form a 5-6 membered ring;

B is a base.

In some embodiments, Y is O;

Each X is independently selected from CR₄(R₄'),R₄ and R₄' is each independently H or C₁-C₆ alkyl;

J₂ is H;

r₃ is selected from H and C₁-C₆ alkyl;

Q₁ isQ₂ is R₂; or Q₁ is R₂,Q₂ is

R₁ is selected from H and C₁-C₆ alkyl;

J₁ is H or C₁-C₆ alkyl;

R₂ is selected from H, C₁-C₆ alkyl and (CH₂)_rR₈;R₈ is selected from OH, r=1, 2 or 3;

Optionally, R₁ and R₂ are directly linked to form a 5-6 membered ring;

B is a base.

In some embodiments, Y is O.

In some embodiments, X is independently selected from CR₄(R₄')、NR₅ and NH-CO, and R₄、R₄'、R₅ is each independently H, methyl, ethyl, n-propyl, or isopropyl. In some embodiments, X is independently selected from NH-CO, CH₂, and NH. In some embodiments, X is independently selected from NH-CO and CH₂. In some embodiments, X is CH₂.

In some embodiments, J₂ is H or methyl. In some embodiments, J₂ is H.

In some embodiments, R₃ is selected from H, OH, NH₂, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, and (CH₂)_pR₆,R₆ is selected from OH, methoxy, and ethoxy, p=1 or 2. In some embodiments, R₃ is selected from H, methyl, ethyl, n-propyl, isopropyl, and (CH₂)_pR₆,R₆ is selected from OH, p=1 or 2. In some embodiments, R₃ is selected from H and methyl.

In some embodiments, R₁ is selected from H, OH, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, and (CH₂)_qR₇,R₇ is selected from OH, q=1 or 2. In some embodiments, R₁ is selected from H, methyl, ethyl, n-propyl, isopropyl, and (CH₂)_qR₇,R₇ is selected from OH, q=1 or 2. In some embodiments, R₁ is selected from H and methyl.

In some embodiments, R₂ is selected from H, OH, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, and (CH₂)_rR₈,R₈ is selected from OH, r=1 or 2. In some embodiments, R₂ is selected from H, OH, methyl, ethyl, n-propyl, isopropyl, and (CH₂)_rR₈,R₈ is selected from OH, r=1 or 2. In some embodiments, R₂ is selected from H, methyl, and CH₂ OH.

In some embodiments, R₁ and R₂ are directly linked to form a 5-6 membered ring. In some embodiments, R₁ and R₂ are directly linked to form a 3-6 membered cycloalkyl. In some embodiments, R₁ and R₂ are directly linked to form cyclopentyl or cyclohexyl.

In some embodiments, the chemical modification of formula (I) is selected from any one of the following structures:

wherein: b is selected from purine bases, pyrimidine bases, indoles, 5-nitroindoles, and 3-nitropyrroles.

Wherein: b is selected from purine bases, pyrimidine bases, indoles, 5-nitroindoles and 3-nitropyrroles.

In some embodiments, the nucleotide comprising the chemical modification of formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof is a nucleotide comprising the chemical modification of formula (I'), a tautomer thereof, or a pharmaceutically acceptable salt thereof,

Wherein: y is selected from O, NH and S;

J₂ is H or C₁-C₆ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Q_1' isQ_2' is R₂; or Q_1' is R₂,Q_2' is

Wherein:

J₁ is H or C₁-C₆ alkyl;

Optionally, R₁ and R₂ are directly linked to form a ring;

B is a base;

M is O or S;

the chemical modification represented by the formula (I'), a tautomer thereof or a pharmaceutically acceptable salt thereof is not

In some embodiments, when X is nh—co, R₁ is not H.

In some embodiments, the chemical modification of formula (I ') is a chemical modification of formula (I' -1):

Wherein: y is selected from O, NH and S;

each J₁、J₂ is independently H or C₁-C₆ alkyl;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

M is O or S;

Optionally, R₁ and R₂ are directly linked to form a ring;

B is as defined in formula (I').

In some embodiments of formula (I' -1), B is selected from the group consisting of a purine base, a pyrimidine base, an indole, a 5-nitroindole, and a 3-nitropyrrole.

In some embodiments of formula (I' -1), B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole.

In some embodiments of formula (I' -1), B is selected from adenine, guanine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments of formula (I' -1), B is the same as the base when the nucleotide at that position of the antisense strand is not modified.

In some embodiments, the chemical modification of formula (I ') is a chemical modification of formula (I' -2):

Wherein Y is selected from O, NH and S;

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

each J₁、J₂ is independently H or C₁-C₆ alkyl;

Optionally, R₁ and R₂ are directly linked to form a ring;

M is O or S;

B is as defined in formula (I').

In some embodiments of formula (I' -2), B is selected from the group consisting of a purine base, a pyrimidine base, an indole, a 5-nitroindole, and a 3-nitropyrrole.

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

In some embodiments of formula (I' -2), B is selected from adenine, guanine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments of formula (I' -2), B is the same as the base when the nucleotide at that position of the antisense strand is not modified.

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

Each J₁、J₂ is independently H or C₁-C₃ alkyl;

Optionally, R₁ and R₂ are directly linked to form a ring;

B is as defined in formula (I').

n=0, 1 or 2; m=0, 1 or 2; s=0 or 1;

each J₁、J₂ is independently H or methyl;

Optionally, R₁ and R₂ are directly linked to form a ring;

B is as defined in formula (I').

n=0 or 1; m=0 or 1; s=0 or 1;

Each J₁、J₂ is independently H;

R₁ is selected from H, methyl and CH₂ OH;

r₂ is selected from H, OH, NH₂, methyl and CH₂ OH;

R₃ is selected from H, OH, NH₂, methyl and CH₂ OH;

Optionally, R₁ and R₂ are directly linked to form a ring;

B is as defined in formula (I').

n=0 or 1; m=0 or 1; s=0 or 1;

Each J₁、J₂ is independently H;

R₁ is selected from H, methyl and CH₂ OH;

R₂ is selected from H, methyl and CH₂ OH;

R₃ is selected from H, OH, NH₂, methyl and CH₂ OH;

Optionally, R₁ and R₂ are directly linked to form a ring;

B is as defined in formula (I').

In some embodiments, Y is O or NH;

J₂ is H or C₁-C₆ alkyl;

n=0 or 1; m=0 or 1; s=0 or 1;

Q_1' isQ_2' is R₂; or Q_1' is R₂,Q_2' is

J₁ is H or C₁-C₆ alkyl;

Optionally, R₁ and R₂ are directly linked to form a 3-6 membered ring;

M is O or S;

B is a base;

In some embodiments, X is independently selected from CR₄(R₄') and NH-CO.

In some embodiments, X is independently selected from CR₄(R₄').

In some embodiments, R₃ is selected from H and C₁-C₆ alkyl.

In some embodiments, R₁ is selected from H and C₁-C₆ alkyl.

In some embodiments, Y is O;

J₂ is H or C₁-C₆ alkyl;

Q_1' isQ_2' is R₂; or Q_1' is R₂,Q_2' is

J₁ is H or C₁-C₆ alkyl;

Optionally, R₁ and R₂ are directly linked to form a 5-6 membered ring;

M is O or S;

B is a base.

In some embodiments, Y is O;

J₂ is H;

r₃ is selected from H and C₁-C₆ alkyl;

Q_1' isQ_2' is R₂; or Q_1' is R₂,Q_2' is

R₁ is selected from H and C₁-C₆ alkyl;

J₁ is H or C₁-C₆ alkyl;

Optionally, R₁ and R₂ are directly linked to form a 5-6 membered ring;

M is O or S;

B is a base.

In some embodiments, Y is O.

In some embodiments, J₂ is H or methyl. In some embodiments, J₂ is H.

In some embodiments, the chemical modification represented by formula (I') is selected from any one of the following structures:

Wherein: m is O or S;

b is selected from purine bases, pyrimidine bases, indoles, 5-nitroindoles, and 3-nitropyrroles.

Wherein: m is O or S;

And they are

Adenine in the structure is replaced with guanine, cytosine, uracil or thymine.

In some embodiments, the ligand is a compound of formula (II) or a pharmaceutically acceptable salt thereof,

R₁₁ and R₁₂ are independently a bond, NR₁₆ or c=o;

Q₃ is

R₁₃ is CR₁₇R₁₈、NR₁₆, O or S;

R₁₄ is CR₁₉;

R₁₅ is independently CR₁₇R₁₈、NR₁₆ or O;

R₁₆ to R₁₉ are independently hydrogen, deuterium or alkyl;

m1, p1 and q1 are independently 0, 1, 2, 3 or 4;

B1 is

R_b5、R_b6 and R_b7 are independently-C (=o) -, -NHC (=o) -, -C (=o) O-, -C (=o) - (CH₂)_z8 -O-, or-NHC (=o) - (CH₂)_z9 -O-;

z5, z6, z7, z8 and z9 are independently integers from 0 to 10;

r1 is an integer of 1 to 10.

In some embodiments, L₁ is- (CH₂)_j11-C(＝O)-(CH₂)_j12 -;

R₁₁ and R₁₂ are independently a bond, NR₁₆ or c=o;

Q₃ is

R₁₃ is CR₁₇R₁₈ or O;

R₁₄ is CR₁₉;

R₁₅ is independently CR₁₇R₁₈ or O;

R₁₆ to R₁₉ are independently hydrogen or alkyl;

m1, p1 and q1 are independently 0 or 1;

B1 is

R_b5、R_b6 and R_b7 are independently-C (=O) - (CH₂)_z8 -O-or-NHC (=O) - (CH₂)_z9 -O-;

z8 and z9 are independently integers from 0 to 10;

L₂ is- (CH₂)_j15-(OCH₂CH₂)_1-4-(CH₂)_j16) -or

J15 and j16 are independently integers from 0 to 4;

r1 is 3, 4, 5 or 6.

In some embodiments, L₁ can be L₃ or L₃-R₁₁₀-R₁₁₁-L₃, where L₃ is independently a C₁-C₁₂ alkyl chain, - (CH₂)_j11-C(＝O)-(CH₂)_j12 -or -(CH₂)_j13-(CH₂CH₂O)_1-4-(CH₂)_j14-,R₁₁₀) and R₁₁₁ is independently a bond, -NR₁₁₂ -, -C (=O) -or-OC (=O) -, R₁₁₂ is hydrogen or C₁-C₁₂ alkyl, j11, j12, j13 and j14 are independently integers from 0 to 10, in some embodiments, j11, j12, j13 and j14 are independently integers from 0 to 2 or 4 to 10, in some embodiments, j11, j12, j13 and j14 are independently 0, 1,2, 6, 7, 8, 9 or 10.

In some embodiments, L₁ may be- (CH₂)_j11-C(＝O)-(CH₂)_j12 -, with j11 and j12 being as defined in any of the previous schemes.

In some embodiments, L₁ may beThe definition of j12 is the same as any one of the previous schemes, wherein the a1 end is connected with B, and the B1 end is connected with R₁.

In some embodiments, L₁ may beWherein, the a1 end is connected with B₁, and the B1 end is connected with R₁₁.

In some embodiments, R₁₁ can be a bond and R₁₂ can be c=o.

In some embodiments, R₁₁ can be a bond and R₁₂ can be NR₁₆,R₁₆ as defined in any of the previous schemes.

In some embodiments, R₁₁ can be a bond and R₁₂ can be-OC (=o) -.

In some embodiments, R₁₁ can be NR₁₆ and R₁₂ can be c=o, and R₁₆ is as defined in any of the previous schemes.

In some embodiments, R₁ can be NR₁₆ and R₁₂ can be-OC (=o) -, and R₁₆ is as defined in any of the previous embodiments.

In some embodiments, R₁₂ can be NR₁₆ and R₁₁ can be c=o, and R₁₆ is as defined in any of the previous schemes.

In some embodiments, R₁₂ can be NR₁₆ and R₁₁ can be-OC (=o) -, and R₁₆ is as defined in any of the previous embodiments.

In some embodiments, R₁₁ can be NH and R₁₂ can be c=o.

In some embodiments, R₁₂ can be NH and R₁₁ can be c=o.

In some embodiments, R₁₆ can be hydrogen or C_1-6 alkyl.

In some embodiments, R₁₆ can be hydrogen, methyl, ethyl, propyl, or isopropyl.

In some embodiments, R₁₆ can be hydrogen.

In some embodiments, R₁₇ and R₁₈ may be hydrogen.

In some embodiments, R₁₉ can be hydrogen.

In some embodiments, ring a, when present, may be a C_6-12 aryl group.

In some embodiments, ring a may be phenyl.

In some embodiments, m1 may be 0 or 1.

In some embodiments, m1 may be 3.

In some embodiments, n1 may be 0 or 1.

In some embodiments, p1 and q1 are independently 0 or 1.

In some embodiments, p1=1 and q1=1.

In some embodiments, p1=1 and q1=0.

In some embodiments, p1=0 and q1=1.

In some embodiments, p1=0 and q1=0.

In some embodiments, z1, z2, z3, z4, z5, z6, z7, z8, and z9 may independently be integers from 0 to 4. In some embodiments, z1, z2, z3, z4, z5, z6, z7, z8, and z9 can independently be 0, 1, or 2.

In some embodiments, B₁ may beR_b1、R_b2、R_b3 and R_b4 are independently-C (=O) -or-NHC (=O) -, the N atom is attached to L₁, and z1, z2, z3 and z4 are as defined in any of the previous schemes.

In some embodiments, B₁ may beR_b1、R_b2、R_b3 and R_b4 are independently-C (=O) -or-NHC (=O) -, the N atom is attached to L₁, R_b1、R_b3 and R_b4 are the same, and z1, z2, z3 and z4 are as defined in any of the previous schemes.

In some embodiments, B₁ may be

In some embodiments, B₁ may beR_b5、R_b6 and R_b7 are independently-C (=O) - (CH₂)_z8 -O-or-NHC (=O) - (CH₂)_z9 -O-, the N atom is attached to L₁ and z5, z6, z7, z8 and z9 are as defined in any of the previous schemes.

In some embodiments, B₁ may beR_b5、R_b6 and R_b7 are independently-C (=O) - (CH₂)_z8 -O-or-NHC (=O) - (CH₂)_z9 -O-, the N atom is attached to L₁, R_b5、R_b6 and R_b7 are the same, and z5, z6, z7, z8 and z9 are as defined in any of the previous schemes.

In some embodiments, B₁ may be

In some embodiments, L₂ can be L₄ or L₄-R₁₃-R₁₄-L₄, wherein L₄ is independently a C₁-C₁₂ alkyl chain or -(CH₂)_j15-(OCH₂CH₂)_1-4-(CH₂)_j16-,R₁₁₃ and R₁₁₄ are independently a bond, -NR₁₁₅ -, -C (=o) -or-OC (=o) -, R₁₁₅ is independently hydrogen or C₁-C₁₂ alkyl, and j15 and j16 are independently integers from 0 to 10. In some embodiments, j15 and j16 are independently integers from 0 to 6. In some embodiments, j15 and j16 are independently 0, 1,2, 3, or 4.

In some embodiments, L₂ may be- (CH₂)_j15-(OCH₂CH₂)_1-4-(CH₂)_j16 -, with j15 and j16 being as defined in any of the preceding schemes.

In some embodiments, L₂ may beIn some embodiments, L₂ may beWherein the left side is connected with an O atom, and the right side is connected with B₁.

In some embodiments, L₂ can be a C₁-C₁₂ alkyl chain.

In some embodiments, L₂ may be

In some embodiments, L₂ may beIn some embodiments, L₂ may beIn some embodiments, L₂ may beIn some embodiments, L₂ may beWherein, the a3 end is connected with the O atom, and the B3 end is connected with the B₁.

In some embodiments, L₂ may beWherein, the a3 end is connected with the O atom, and the B3 end is connected with the B₁.

In some embodiments, r1 may be 3,4, 5, or 6. In some embodiments, r1 may be 3.

In some embodiments, Q₃ may beIn some embodiments, Q₃ may beWherein R₁₃、R₁₄、R₁₅ and n1 are as defined in any one of the preceding schemes.

In some embodiments of the present invention, in some embodiments,Can beWherein R₁₃、R₁₄、R₁₅, p1 and q1 are as defined in any one of the preceding schemes.

In some embodiments of the present invention, in some embodiments,Can beIn some embodiments of the present invention, in some embodiments,Can beIn some embodiments of the present invention, in some embodiments,Can beThe definition of p1 and q1 is as in any one of the previous schemes.

In some embodiments of the present invention, in some embodiments,Can beIn some embodiments of the present invention, in some embodiments,Can be

In some embodiments of the present invention, in some embodiments,Can beThe definition of p1 and q1 is as in any one of the previous schemes.

In some embodiments of the present invention, in some embodiments,Can beWherein R₁₃、R₁₄, n1, p1 and q1 are as defined in any one of the preceding schemes.

In some embodiments of the present invention, in some embodiments,Can beIn some embodiments of the present invention, in some embodiments,Can beN1, p1 and q1 are as defined in any one of the preceding schemes.

In some embodiments of the present invention, in some embodiments,Can beN1, p1 and q1 are as defined in any one of the preceding schemes.

In some embodiments, the ligand may be any one of the following structures or a pharmaceutically acceptable salt thereof,

In some embodiments, the ligand may be of the structure or a pharmaceutically acceptable salt thereof,

In some embodiments, the chemical modification of formula (I) isB is selected from guanine, adenine, cytosine and uracil; and the ligand is any one of the following structures or pharmaceutically acceptable salts thereof,

In some embodiments, the chemical modification of formula (I) isB is selected from guanine, adenine, cytosine and uracil, and the ligand is any one of the following structures or pharmaceutically acceptable salts thereof,

In some embodiments, the chemical modification of formula (I) isB is selected from guanine, adenine, cytosine and uracil; and the ligand is of the following structure or pharmaceutically acceptable salt thereof,

In some embodiments, the N-acetyl-galactosamine moiety in the above ligand may be replaced with N-trifluoroacetyl galactosamine, N-propionyl galactosamine, N-N-butyryl galactosamine, or N-isobutyryl galactosamine.

In some embodiments, the siRNA and the ligand are covalently or non-covalently linked.

In some embodiments, the 3 'and/or 5' end of the sense strand may be conjugated to the ligand.

In some embodiments, the 3' end of the sense strand may be conjugated to the ligand.

In some embodiments, the ligand is linked to the siRNA terminus via a phosphate group or a phosphorothioate group.

In some embodiments, the ligand is linked to the siRNA terminal via a phosphodiester group or a phosphorothioate diester group.

In some embodiments, the ligand is linked to the siRNA end via a phosphodiester group.

In some embodiments, the ligand is indirectly linked to the siRNA end via a phosphate group or a phosphorothioate group.

In some embodiments, the ligand is directly linked to the siRNA end via a phosphate group or a phosphorothioate group.

In some embodiments, the ligand is directly linked to the 3' end of the sense strand of the siRNA through a phosphate group or a phosphorothioate group.

In some embodiments, the phosphate group is a phosphate monoester group or a phosphate diester group. In some embodiments, the phosphate group is a phosphodiester group.

In some embodiments, the phosphorothioate group is a phosphorothioate monoester group or a phosphorothioate diester group. In some embodiments, the phosphorothioate group is a phosphorothioate diester group.

In some alternative embodiments, to facilitate entry of the siRNA into the cell, a lipophilic group such as cholesterol may be introduced at the end of the sense strand of the siRNA, including covalent bond to the siRNA, such as end-introduced cholesterol, lipoproteins, vitamin E, etc., to facilitate interaction with mRNA within the cell through the cell membrane comprised of the lipid bilayer. Meanwhile, siRNA can also be modified by non-covalent bonds, such as through hydrophobic bonds or ionic bonds to phospholipid molecules, polypeptides, cationic polymers and the like, so as to increase stability and biological activity.

In some embodiments, a nucleotide comprising a chemical modification of formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof is located at position 5, 6, or 7 from the 5' end of the antisense strand.

In some embodiments, a nucleotide comprising a chemical modification of formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof is located at position 7 from the 5' end of the antisense strand.

In some embodiments, the chemical modification of formula (I), tautomer thereof, or pharmaceutically acceptable salt thereof modifies at position 5 from the 5' end thereof and B is selected from adenine, guanine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, the chemical modification of formula (I), tautomer thereof, or pharmaceutically acceptable salt thereof modifies at position 6 from the 5' end thereof and B is selected from adenine, guanine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, the chemical modification of formula (I), tautomer thereof, or pharmaceutically acceptable salt thereof modifies at position 7 from the 5' end thereof and B is selected from adenine, guanine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, the chemical modification of formula (I), tautomer thereof, or pharmaceutically acceptable salt thereof modifies at position 8 from the 5' end thereof and B is selected from adenine, guanine, 2, 6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is the same base as the antisense strand when the 5 th nucleotide is not modified from its 5' end.

In some embodiments, B is the same base as the antisense strand when the nucleotide at position 6 from its 5' end is not modified.

In some embodiments, B is the same base as the antisense strand when the nucleotide at position 7 from its 5' end is not modified.

In some embodiments, B is the same base as the antisense strand when the 8 th nucleotide is not modified from its 5' end.

In some embodiments, the sense strand contains three consecutive nucleotides with the same modification. In some embodiments, the three nucleotides with the same modification are 2' -fluoro modified nucleotides.

In some embodiments, the antisense strand is 2' -fluoro modified nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18, each independently, in a 5' to 3' orientation.

In some embodiments, the antisense strand is at least partially reverse-complementary to a target sequence. In some embodiments, no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 mismatches exist between the antisense strand and the target sequence; in some embodiments, the antisense strand is fully reverse-complementary to the target sequence.

In some embodiments, the sense strand is at least partially reverse-complementary to the antisense strand to form a double-stranded region. In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 mismatches between the sense strand and the antisense strand. In some embodiments, the sense strand is fully reverse-complementary to the antisense strand.

In some embodiments, the sense strand and the antisense strand each independently have 16 to 35, 16 to 34, 17 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 (e.g., 19, 20, 21, 22, 23).

In some embodiments, the sense strand and the antisense strand are the same or different in length, the sense strand is 19-23 nucleotides in length, and the antisense strand is 19-26 nucleotides in length. The length ratio of the sense strand and the antisense strand in the dsRNA provided by the present disclosure may 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 ratio of the length of the sense strand to the antisense strand is 19/21, 21/23, or 23/25. In some embodiments, the ratio of the length of the sense strand to the antisense strand is 19/21.

In some embodiments, the siRNA comprises one or two blunt ends.

In some specific embodiments, each strand of the siRNA independently comprises an overhang formed from 1 to 2 unpaired nucleotides.

In some embodiments, the siRNA comprises an overhang at the 3' end of the antisense strand.

In some embodiments, the sense strand comprises a nucleotide sequence (5 '-3') as shown in the following formula:

N_aN_aN_aN_aXN_aN_bN_bN_bN_aN_aN_aN_aN_aN_aN_aN_aN_aN_a

Wherein each X is independently N_a or N_b;N_a is a 2 '-methoxy modified nucleotide and N_b is a 2' -fluoro modified nucleotide.

In some embodiments, the sense strand comprises a nucleotide sequence represented by the formula:

5'-N_aN_aN_aN_aN_aN_aN_bN_bN_bN_aN_aN_aN_aN_aN_aN_aN_aN_aN_a-3'; Or alternatively, the first and second heat exchangers may be,

5'-N_aN_aN_aN_aN_bN_aN_bN_bN_bN_aN_aN_aN_aN_aN_aN_aN_aN_aN_a-3';

Wherein N_a is a 2 '-methoxy modified nucleotide and N_b is a 2' -fluoro modified nucleotide.

In some embodiments, the antisense strand comprises a nucleotide sequence represented by the formula:

5'-N_a'N_b'N_a'N_b'N_a'N_b'W'N_a'N_a'N_b'N_a'N_b'N_a'N_b'N_a'N_b'N_a'N_b'N_a'N_a'N_a'-3';

Wherein N_a 'is a 2' -methoxy modified nucleotide and N_b 'is a 2' -fluoro modified nucleotide; w' represents a nucleotide comprising a chemical modification represented by formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof.

In some specific embodiments, W represents a nucleotide comprising a chemical modification represented by formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof.

In some specific embodiments, the chemical modification represented by formula (I) is selected from:

Wherein: b is selected from guanine, adenine, cytosine and uracil. In some specific embodiments, B is the same base as the antisense strand when the nucleotide at position 7 from its 5' end is not modified.

Wherein: m is O or S; wherein: b is selected from guanine, adenine, cytosine or uracil. In some specific embodiments, B is the same base as the antisense strand when the nucleotide at position 7 from its 5' end is not modified.

In some specific embodiments, M is S. In some specific embodiments, M is O.

In some embodiments, at least one phosphate group in the sense strand and/or the antisense strand is a phosphate group having a modification group that allows for increased stability of the siRNA in a biological sample or environment; in some embodiments, the phosphate group having a modifying group is a phosphorothioate group. In some embodiments, the phosphate group having a modifying group is a phosphorothioate diester group.

In some embodiments, the phosphorothioate diester group is present in at least one of the following positions:

Between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand;

between nucleotide 2 and nucleotide 3 from the 5' end of the sense strand;

the 5' end of the antisense strand is between nucleotide 1 and nucleotide 2;

the 5' end of the antisense strand is between nucleotide 2 and nucleotide 3;

the 3' end of the antisense strand is between nucleotide 1 and nucleotide 2; and

The antisense strand is between nucleotide 2 and nucleotide 3 from the 3' end.

In some embodiments, the sense strand and/or antisense strand includes a plurality of phosphorothioate diester groups therein, the phosphorothioate diester groups being present in:

Between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand; and, a step of, in the first embodiment,

Between nucleotide 2 and nucleotide 3 from the 5' end of the sense strand; and, a step of, in the first embodiment,

The 5' end of the antisense strand is between nucleotide 1 and nucleotide 2; and, a step of, in the first embodiment,

The 5' end of the antisense strand is between nucleotide 2 and nucleotide 3; and, a step of, in the first embodiment,

The 3' end of the antisense strand is between nucleotide 1 and nucleotide 2; and, a step of, in the first embodiment,

The antisense strand is between nucleotide 2 and nucleotide 3 from the 3' end.

5'-NMSNMSNMNMNFNMNFNFNFNMNMNMNMNMNMNMNMNMNM-3', or,

5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’，

Where Nm represents any 2 '-methoxy modified nucleotide, e.g. 2' -methoxy modified C, G, U, A; nf represents any 2 '-fluoro modified nucleotide, for example, 2' -fluoro modified C, G, U, A;

lower case letter s in the middle means that there is a phosphorothioate diester linkage between two nucleotides adjacent to the letter s.

5’-Nm’sNf’sNm’Nf’Nm’Nf’W’Nm’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nf’Nm’sNm’sNm’-3’；

Where Nm ' represents any 2' -methoxy modified nucleotide, e.g. 2' -methoxy modified C, G, U, A; nf ' represents any 2' -fluoro modified nucleotide, for example, 2' -fluoro modified C, G, U, A;

Lower case letter s in the middle means that there is a phosphorothioate diester linkage between two nucleotides adjacent to the letter s;

W 'represents a 2' -methoxy modified nucleotide or a nucleotide comprising a chemical modification represented by formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof.

In some embodiments, W represents a 2' -methoxy modified nucleotide.

In some embodiments, the chemical modification represented by formula (I) is selected from:

Wherein: b is selected from guanine, adenine, cytosine and uracil; in some embodiments, B is the same base as the antisense strand when the nucleotide at position 7 from its 5' end is not modified.

Wherein: m is O or S; wherein: b is selected from guanine, adenine, cytosine or uracil; in some specific embodiments, B is the same base as the antisense strand when the nucleotide at position 7 from its 5' end is not modified.

In some embodiments, M is S. In some specific embodiments, M is O.

DsRNA targeting coagulation Factor XI (FXI) gene

In some embodiments, the siRNA is an siRNA targeting a coagulation Factor XI (FXI) gene.

In some embodiments, the nucleotide sequence of the sense strand of the siRNA comprises a sequence that differs by NO more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs 1 to SEQ ID NOs 3, and comprises at least 15 consecutive nucleotides (in some embodiments, at least 19), and/or,

The nucleotide sequence of the antisense strand comprises NO more than 3 nucleotides from the nucleotide sequence of either SEQ ID NO. 4 or SEQ ID NO. 5, and comprises at least 19 consecutive nucleotides (in some embodiments, at least 21).

In some embodiments, the nucleotide sequence of the sense strand of the siRNA comprises any one of the nucleotide sequences of SEQ ID NO. 1 through SEQ ID NO. 3 and/or the nucleotide sequence of the antisense strand comprises any one of the nucleotide sequences of SEQ ID NO. 4 or SEQ ID NO. 5.

In some embodiments, the sense strand and the antisense strand of the siRNA are selected from any one of the following sets of protocols:

The nucleotide sequence of the sense strand comprises the nucleotide sequence of SEQ ID NO. 3 and the nucleotide sequence of the antisense strand comprises the nucleotide sequence of SEQ ID NO. 5;

The nucleotide sequence of the sense strand comprises the nucleotide sequence of SEQ ID NO. 2 and the nucleotide sequence of the antisense strand comprises the nucleotide sequence of SEQ ID NO. 5;

The nucleotide sequence of the sense strand comprises the nucleotide sequence of SEQ ID NO. 1 and the nucleotide sequence of the antisense strand comprises the nucleotide sequence of SEQ ID NO. 4.

In some embodiments, the sense strand and the antisense strand of the siRNA are selected from any one of the following groups:

The nucleotide sequence of the sense strand is SEQ ID NO. 3, and the nucleotide sequence of the antisense strand is SEQ ID NO. 5;

The nucleotide sequence of the sense strand is SEQ ID NO. 2, and the nucleotide sequence of the antisense strand is SEQ ID NO. 5;

the nucleotide sequence of the sense strand is SEQ ID NO. 1 and the nucleotide sequence of the antisense strand is SEQ ID NO. 4.

In the present disclosure, according to the 5'-3' direction,

SEQ ID NO. 1 is CUUGCAACAAAGACAUUUA;

SEQ ID NO. 2 is UCAGGAUGAUUUUCUUAUU;

SEQ ID NO. 3 is UAAAUGUCUUUGUUGCAAGCG;

SEQ ID NO. 4 is UAUAAGAAAAUCAUCCUGAAA.

In some embodiments, the sense strand of the dsRNA comprises any one of SEQ ID No. 6 to SEQ ID No. 10 and/or the antisense strand comprises any one of SEQ ID No. 14 or SEQ ID No. 15.

In some embodiments, the sense strand and the antisense strand of the dsRNA are selected from any one of the following groups:

Group 1): the sense strand comprises SEQ ID NO. 10 and the antisense strand comprises SEQ ID NO. 15;

Group 2): the sense strand comprises SEQ ID NO. 9 and the antisense strand comprises SEQ ID NO. 15;

Group 3): the sense strand comprises SEQ ID NO. 7 and the antisense strand comprises SEQ ID NO. 14;

Group 4): the sense strand comprises SEQ ID NO. 8 and the antisense strand comprises SEQ ID NO. 15;

group 5): the sense strand comprises SEQ ID NO. 6 and the antisense strand comprises SEQ ID NO. 14.

Group 1): the sense strand is shown as SEQ ID NO. 10, and the antisense strand is shown as SEQ ID NO. 15;

Group 2): the sense strand is shown as SEQ ID NO. 9, and the antisense strand is shown as SEQ ID NO. 15;

group 3): the sense strand is shown as SEQ ID NO. 7, and the antisense strand is shown as SEQ ID NO. 14;

group 4): the sense strand is shown as SEQ ID NO. 8, and the antisense strand is shown as SEQ ID NO. 15;

group 5): the sense strand is shown in SEQ ID NO. 6 and the antisense strand is shown in SEQ ID NO. 14.

In some embodiments, the dsRNA is selected from any one of the following groups:

Group 1): comprising or selected from SEQ ID NO:10 and the sense strand shown in SEQ ID NO:15, and an antisense strand shown in seq id no;

Group 2): comprising or selected from SEQ ID NO:9 and the sense strand shown in SEQ ID NO:15, and an antisense strand shown in seq id no;

group 3): comprising or selected from SEQ ID NO:7 and the sense strand shown in SEQ ID NO:14, an antisense strand shown in seq id no;

Group 4): comprising or selected from SEQ ID NO:8 and the sense strand shown in SEQ ID NO:15, and an antisense strand shown in seq id no;

group 5): comprising or selected from SEQ ID NO:6 and the sense strand shown in SEQ ID NO: 14.

In the present disclosure, according to the 5'-3' direction,

SEQ ID NO. 6 is

CmsUmsUmGmCfAmAfCfAfAmAmGmAmCmAmUmUmUmAm-NAG0052’；

SEQ ID NO. 7 is

CmsUmsUmGmCmAmAfCfAfAmAmGmAmCmAmUmUmUmAm-NAG0052’；

SEQ ID NO. 8 is

UmsCmsAmGmGfAmUfGfAfUmUmUmUmCmUmUmAmUmUm-NAG0052’；

SEQ ID NO. 9 is

UmsCmsAmGmGmAmUfGfAfUmUmUmUmCmUmUmAmUmUm-NAG0052’；

SEQ ID NO. 10 is

UmsCmsAmGmGmAmUfGfAfUmUmUmUmCmUmUmAmUmAm-NAG0052’

SEQ ID NO. 14 is

UmsAfsAmAfUmGf(-)hmpNA(U)CmUmUfUmGfUmUfGmCfAmAfGmsCmsGm；

SEQ ID NO. 15 is

UmsAfsUmAfAmGf(-)hmpNA(A)AmAmAfUmCfAmUfCmCfUmGfAmsAmsAm；

Wherein af=adenine 2'-F ribonucleoside (adenine 2' -F ribonucleoside); cf=cytosine 2'-F ribonucleoside (cytosine 2' -F ribonucleoside); uf=uracil 2'-F ribonucleoside (uracil 2' -F ribonucleoside); gf=guanine 2'-F ribonucleoside (guanine 2' -F ribonucleoside); am = adenine 2'-OMe ribonucleoside (adenine 2' -OMe ribonucleoside); cm = cytosine 2'-OMe ribonucleoside (cytosine 2' -OMe ribonucleoside); gm=guanine 2'-OMe ribonucleoside (guanine 2' -OMe ribonucleoside); um = uracil 2'-OMe ribonucleoside (uracil 2' -OMe ribonucleoside);

s represents a phosphorothioate diester linkage between two nucleotides adjacent to the letter s;

NAG0052' represents

(-) HmpNA (A) represents(-) HmpNA (U) represents

In some embodiments, the dsRNA is selected from the following structures or pharmaceutically acceptable salts thereof:

Wherein af=adenine 2'-F ribonucleoside (adenine 2' -F ribonucleoside); cf=cytosine 2'-F ribonucleoside (cytosine 2' -F ribonucleoside); uf=uracil 2'-F ribonucleoside (uracil 2' -F ribonucleoside); am = adenine 2'-OMe ribonucleoside (adenine 2' -OMe ribonucleoside); cm = cytosine 2'-OMe ribonucleoside (cytosine 2' -OMe ribonucleoside); gf=guanine 2'-F ribonucleoside (guanine 2' -F ribonucleoside); gm=guanine 2'-OMe ribonucleoside (guanine 2' -OMe ribonucleoside); um = uracil 2'-OMe ribonucleoside (uracil 2' -OMe ribonucleoside); Represents a phosphorothioate diester group, and,Represents a group of a phosphoric acid diester,

NAG0052' represents

(-) HmpNA (U) represents

(-) HmpNA (A) represents

In some embodiments, the pharmaceutically acceptable salt may be a salt conventional in the art, including but not limited to: sodium salt, potassium salt, ammonium salt, amine salt, and the like.

In some embodiments, the dsRNA is selected from TJR100407, TRD008003-1, TRD008002-1, TRD008003, or TRD008002.

In some embodiments, the dsRNA is TRD008002, which is of the structure:

in some embodiments, the dsRNA is TRD008002-1, which is of the structure:

in some embodiments, the dsRNA is TRD008003, which is of the structure:

In some embodiments, the dsRNA is TRD008003-1, which is of the structure:

in some embodiments, the dsRNA is TJR100407, which is of the structure:

wherein af=adenine 2'-F ribonucleoside (adenine 2' -F ribonucleoside); cf=cytosine 2'-F ribonucleoside (cytosine 2' -F ribonucleoside); uf=uracil 2'-F ribonucleoside (uracil 2' -F ribonucleoside); am = adenine 2'-OMe ribonucleoside (adenine 2' -OMe ribonucleoside); cm = cytosine 2'-OMe ribonucleoside (cytosine 2' -OMe ribonucleoside); gf=guanine 2'-F ribonucleoside (guanine 2' -F ribonucleoside); gm=guanine 2'-OMe ribonucleoside (guanine 2' -OMe ribonucleoside); um=uracil 2'-OMe ribonucleoside (uracil 2' -OMe ribonucleoside).

Represents a phosphorothioate diester group, and,Represents a group of a phosphoric acid diester,

NAG0052' represents

(-) HmpNA (U) represents

(-) HmpNA (A) represents

Pharmaceutical composition

In another aspect, the present disclosure provides a pharmaceutical composition comprising the dsRNA described above.

In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients. Various drug delivery systems are known and can be used in the dsRNA or pharmaceutical compositions of the present disclosure, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of nucleic acids as part of a retrovirus or other vector.

In some embodiments, the dsRNA or pharmaceutical compositions of the present disclosure are administered in a conventional manner, either topically (e.g., directly injected or implanted) or systemically, and also by oral, rectal, or parenteral routes including, but not limited to, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, transdermal administration, inhaled administration (e.g., aerosol), mucosal administration (e.g., sublingual, intranasal administration), intracranial administration, and the like.

In some embodiments, the dsRNA or pharmaceutical compositions provided by the present disclosure can be administered by injection, e.g., intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal injection.

In some embodiments, the dsRNA or pharmaceutical compositions provided by the present disclosure can be packaged in a kit.

In the present disclosure, the effective amount or dose of the dsRNA or pharmaceutical composition is about 0.001mg/kg body weight to about 200mg/kg body weight, about 0.01mg/kg body weight to about 100mg/kg body weight, or about 0.5mg/kg body weight to about 50mg/kg body weight.

In the present disclosure, the dsRNA, pharmaceutical composition is an effective amount or an effective dose.

Use and method of treatment

In another aspect, the present disclosure provides use of the dsRNA described above or the pharmaceutical composition described above in the preparation of a medicament.

In some embodiments, the medicament may be used to prevent and/or treat thromboembolic complications. In some embodiments, the thromboembolic complication is selected from any one or a combination of the following: deep vein thrombosis, pulmonary embolism, myocardial infarction or stroke.

In some embodiments, the medicament may be used to prevent and/or treat diseases associated with blood coagulation factor XI gene expression. In some embodiments, the disorder associated with the expression of the factor XI gene may be a thromboembolic complication. In some embodiments, the thromboembolic complication is selected from any one or a combination of the following: deep vein thrombosis, pulmonary embolism, myocardial infarction or stroke.

In another aspect, the present disclosure provides a method of preventing and/or treating a disease comprising administering to a subject an effective amount or effective dose of the dsRNA described above or the pharmaceutical composition described above.

In some embodiments, the disease may be a thromboembolic complication. In some embodiments, the thromboembolic complication is selected from any one or a combination of the following: deep vein thrombosis, pulmonary embolism, myocardial infarction or stroke.

In some embodiments, the disease may be a disease associated with blood coagulation factor XI gene expression. In some embodiments, the disorder associated with the expression of the factor XI gene may be a thromboembolic complication. In some embodiments, the thromboembolic complication is selected from any one or a combination of the following: deep vein thrombosis, pulmonary embolism, myocardial infarction or stroke.

Methods of modulating expression of a target gene

In another aspect, the present disclosure provides a method for silencing a target gene FXI or mRNA thereof in a cell in vivo or in vitro comprising the step of introducing into the cell the dsRNA described above or the pharmaceutical composition described above.

In another aspect, the present disclosure provides a method of inhibiting expression of a target gene FXI or mRNA thereof, comprising administering to a subject an effective amount or effective dose of the dsRNA described above or the pharmaceutical composition described above.

The dsRNA or pharmaceutical composition of the present disclosure can reduce the expression level of a target gene or mRNA thereof in a cell, cell population, tissue, or subject, or the like, comprising: administering to a subject a therapeutically effective amount of a dsRNA or pharmaceutical composition described herein, thereby inhibiting expression of a target gene or mRNA thereof in the subject.

In some embodiments, the subject has been previously identified as having a pathological upregulation of the target gene FXI or mRNA thereof in a targeted cell, cell population, tissue or subject. In another aspect, the present disclosure provides a method of delivering an oligonucleotide to the liver comprising administering to a subject an effective amount or effective dose of the dsRNA described above or the pharmaceutical composition described above.

In another aspect, the present disclosure provides an RNAi (RNA interference) agent comprising the dsRNA described above or the pharmaceutical composition described above.

In another aspect, the present disclosure also provides a cell comprising the dsRNA described above or the pharmaceutical composition described above.

In another aspect, the present disclosure also provides a kit or kit comprising the dsRNA described above or the pharmaceutical composition described above.

In the present disclosure, the dsRNA or pharmaceutical composition as described above inhibits 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% when contacted with a cell expressing the target gene, as determined (e.g., by psiCHECK activity screening, luciferase reporter gene assay, PCR or branched DNA-based method, or protein-based method, such as immunofluorescence assay, western Blot, or flow cytometry).

In the present disclosure, the dsRNA or pharmaceutical composition described above 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% when contacted with a cell expressing a target gene, as determined (e.g., by psiCHECK activity screening, luciferase reporter gene assay, PCR or branched DNA-based methods, or protein-based methods such as immunofluorescence analysis, western Blot, or flow cytometry).

In the present disclosure, the dsRNA or pharmaceutical composition described above 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% when contacted with a cell expressing a target gene, as determined (e.g., by psiCHECK activity screening, luciferase reporter assay, PCR or a branched DNA-based method, or a protein-based method, such as immunofluorescence assay, western Blot, or flow cytometry), while reducing target activity by at most 20%, at most 19%, at most 15%, at most 10%, at most 5%, or more than 1%.

In the present disclosure, the above-described dsRNA or pharmaceutical compositions, when contacted with a cell expressing a target gene, are as determined (e.g., by psiCHECK activity screening, luciferase reporter gene assay, PCR or branched DNA-based methods, or protein-based methods, such as immunofluorescence analysis, western Blot, or flow cytometry), the dsRNA results in a reduction in off-target activity by at least 20%, at least 25%, at least 30%, at least 50%, 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%.

The present disclosure also provides a method of making a dsRNA or pharmaceutical composition comprising: the ligand, siRNA and dsRNA disclosed in the disclosure are synthesized.

The disclosure is incorporated in full into WO2022028457 A1.

The compounds of the present disclosure may exist in particular geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (-) -and (+) -pairs of enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the 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 the present disclosure. The asymmetric carbon atom containing compounds of the present disclosure may be isolated in optically active pure or racemic forms. Optically pure forms can be resolved from the racemic mixture or synthesized by using chiral starting materials or chiral reagents.

Optically active (R) -and (S) -isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it may be prepared by asymmetric synthesis or derivatization with chiral auxiliary wherein the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomer. Or when the molecule contains a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl), forms a diastereomeric salt with an appropriate optically active acid or base, and then undergoes diastereomeric resolution by conventional methods well known in the art, followed by recovery of the pure enantiomer. Furthermore, separation of enantiomers and diastereomers is typically accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (e.g., carbamate formation from amine).

In the chemical structure of the compounds of the present disclosure, the bondIndicating unspecified configuration, i.e. bonds if chiral isomers are present in the chemical structureMay beOr at the same time containTwo configurations. In the chemical structure of the compounds of the present disclosure, the bondNot specifying configuration, i.e. keysThe form of (C) may be E-form or Z-form, or both E and Z-forms may be included.

In the chemical structural formula of the present disclosure,One or more of any group may be attached according to the scope of the invention described herein; asterisks indicate chiral centers.

The compounds and intermediates of the present disclosure may also exist in different tautomeric forms without specifying the configuration, and all such forms are included within the scope of the present disclosure.

The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also known as proton transfer tautomers) include tautomers via proton transfer, such as keto-enol and imine-enamine, lactam-lactam isomerization. Examples of lactam-lactam balances are between a and B as shown below.

All compounds in the present disclosure may be drawn as form a or form B. All tautomeric forms are within the scope of the invention. The naming of the compounds does not exclude any tautomers.

The present disclosure also includes some isotopically-labeled compounds of the present disclosure which are identical to those recited herein, but for the replacement of one or more atoms by an atom having an atomic weight or mass number different from the atomic weight or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as²H、³H、¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O、¹⁸O、³¹P、³²P、³⁵S、¹⁸F、¹²³I、¹²⁵I and³⁶ Cl, respectively, and the like.

Unless otherwise indicated, "optionally," "optional," or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally, R₁ and R₂ are directly linked in a ring" means that R₁ and R₂ are directly linked in a ring may occur, but need not be, and the description includes the case where R₁ and R₂ are directly linked in a ring and the case where R₁ and R₂ are not.

Interpretation of the terms

For easier understanding of the present disclosure, some technical and scientific terms are specifically defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Target refers to the guest against which the dsRNA (or conjugate thereof) of the present disclosure is directed; the target may be a nucleic acid (gene, mRNA, etc.), or a protein (precursor, mature protein, isoform, variant, etc.). In the present disclosure, target refers in particular to the factor XI gene or its expression product.

Factor XI is to be construed broadly in this disclosure to mean the factor XI gene itself and its expression products in various forms during various stages, such as, but not limited to, molecules produced by the gene during amplification, replication, transcription, splicing, processing, translation, modification, such as cDNA, mRNA, precursor proteins, mature proteins, natural variants, modified forms, and fragments thereof.

In the context of the present disclosure, the terms "factor XI", "factor 11", "coagulation factor XI", "coagulation factor 11", "FXI" and "F11" are used interchangeably. "factor XI nucleic acid" refers to any nucleic acid encoding factor XI. For example, in some embodiments, a factor XI nucleic acid includes a DNA sequence encoding factor XI, e.g. "factor XI gene", an RNA sequence transcribed from DNA encoding factor XI (including genomic DNA containing introns and exons), and an mRNA sequence encoding factor XI. The "factor XI gene" is any one of the following sequences: GENBANK accession number nm_000128.4; GENBANK accession number nt_022792.17, 19598000 to 19624000 deleted; GENBANK accession No. nm_028066.3, exons 1-15; GENBANK accession number xm_006253144.4, xm_006253145.3; GENBANK accession numbers xm_015139652.2, xm_015139653.2; GENBANK accession number xm_005556483.3, xm_005556484.3; GENBANK accession number nw_001118167.1. "factor XI mRNA" refers to mRNA encoding factor XI protein.

The skilled person will understand that although a specific accession number is given in a specific embodiment, factor XI is not limited to numbers in a specific database, but is intended to cover any literature, books, equivalent references in databases in the prior art. When the dsRNA targets a specific target of factor XI or its expression product, the skilled person is able to determine the equivalent location of the target in different databases. As used herein, the sense strand (also known as the SS, or sense strand) refers to a strand comprising a sequence that is identical or substantially identical to a target mRNA sequence; the antisense strand (also known AS an AS or AS strand) refers to a strand having a sequence complementary or partially complementary to a target mRNA sequence.

In this disclosure, the "5 'region" of the sense strand or antisense strand, i.e., "5' end", is used interchangeably. For example, the nucleotides at positions 2 to 8 from the 5 '-end of the antisense strand may be replaced with the nucleotides at positions 2 to 8 from the 5' -end of the antisense strand. Similarly, the "3' region", "3' end" and "3' end" of the sense strand or antisense strand may be used interchangeably.

In the context of describing the sense strand described in the present disclosure, the term "differs by NO more than 3 nucleotide sequences from the nucleotide sequence of any one of SEQ ID NO. 1 to SEQ ID NO. 3, and comprises at least 15 consecutive nucleotides" is intended to mean that the siRNA sense strand described herein comprises at least 15 consecutive nucleotides of any one of SEQ ID NO. 1 to SEQ ID NO. 3, or differs by NO more than 3 nucleotide sequences from at least 15 consecutive nucleotides of the sense strand of any one of SEQ ID NO. 1 to SEQ ID NO. 3 (optionally, by NO more than 2 nucleotide sequences; optionally, by 1 nucleotide sequence). Optionally, the siRNA sense strands described herein comprise at least 16 consecutive nucleotides of any one of SEQ ID NO. 1 to SEQ ID NO. 3, or differ by NO more than 3 nucleotide sequences (optionally, by NO more than 2 nucleotide sequences, optionally, by 1 nucleotide sequence) from at least 16 consecutive nucleotides of any one of SEQ ID NO. 1 to SEQ ID NO. 3.

In the context of describing the antisense strands described in this disclosure, the term "differ from either of SEQ ID NO. 4 or SEQ ID NO. 5 by NO more than 3 nucleotide sequences and comprises at least 15 consecutive nucleotides" is intended to mean that the siRNA antisense strand described herein comprises at least 15 consecutive nucleotides of either of SEQ ID NO. 4 or SEQ ID NO. 5 or differs from at least 15 consecutive nucleotides of either of SEQ ID NO. 4 or SEQ ID NO. 5 by NO more than 3 nucleotide sequences (optionally, by NO more than 2 nucleotide sequences, optionally, by 1 nucleotide sequence).

The term "dsRNA" refers to a double-stranded RNA molecule capable of RNA interference, comprising a sense strand and an antisense strand.

In the context of the present disclosure, "G", "C", "a", "T" and "U" represent nucleotides, respectively, comprising bases of guanine, cytosine, adenine, thymidine and uracil, respectively, unless otherwise specified; the lowercase letter d indicates that the adjacent nucleotide downstream of the letter d is a deoxyribonucleotide; the lower case letter m indicates that the adjacent nucleotide upstream of the letter m is a methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide upstream of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that there is a phosphorothioate diester linkage between two nucleotides adjacent to and about the letter s.

As used in this disclosure, the term "2 '-fluoro (2' -F) modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide is substituted with fluorine, and "non-fluoro modified nucleotide" refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorine group.

As used in this disclosure, the term "2' -methoxy (2 ' -OMe) -modified nucleotide" refers to a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with a methoxy group.

In the context of the present disclosure, the presence of a "nucleotide difference" in one nucleotide sequence from another means that the base type of the nucleotide at the same position is changed in the former as compared with the latter, for example, in the case where one nucleotide base is A, the corresponding nucleotide base at the same position in the former is U, C, G or T, it is determined that there is a nucleotide difference between the two nucleotide sequences at that position. In some embodiments, a nucleotide difference is also considered to occur at an original position when the nucleotide is replaced with an abasic nucleotide or its equivalent.

As used herein, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art that in a double stranded nucleic acid molecule, the bases of one strand pair with the bases on the other strand in a complementary manner. In DNA, the purine base adenine is always paired with the pyrimidine base thymine (or uracil in RNA); the purine base guanine always pairs with the pyrimidine base cytosine (G). Each base pair includes a purine and a 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 to each other, and the sequence of the strand can be deduced from the sequence of its complementary strand. Accordingly, "mismatch" means in the art that bases at corresponding positions do not exist in complementary pairs in a double-stranded nucleic acid.

The term "chemical modification" or "modification" includes all changes in the nucleotide through chemical means, such as the addition or removal of chemical moieties, or substitution of one chemical moiety for another.

The term "base" encompasses any known DNA and RNA base, base analogue, such as purine or pyrimidine, which also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogues. The base analogue may also be a universal base.

The terms "blunt end" or "blunt end" are used interchangeably to refer to the absence of unpaired nucleotides or nucleotide analogs, i.e., no nucleotide overhang, at a given end of the siRNA. In most cases, an siRNA that is blunt-ended at both ends will be double-stranded throughout its length.

The terms "about" and "approximately" refer to values that are within acceptable tolerances for the particular values being determined by one of ordinary skill in the art, which values depend in part on how the measurement or determination is made (i.e., the limits of the measurement system). For example, "about" may mean within 1 or exceeding a standard deviation of 1. Or "about" or "substantially comprising" may mean a range of up to 20%, for example, between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 1%, in this disclosure, each instance preceded by the term "about" by the numerical value or numerical range also includes the given number of embodiments. Unless otherwise indicated, when a particular value is found in the present disclosure and claims, the meaning of "about" or "consisting essentially of" should be assumed to be within the acceptable error range for that particular value.

In this disclosure, the term "comprising" may be replaced by "consisting of … …".

Unless otherwise indicated, the "compounds", "chemical modifications", "ligands", "dsRNA", "nucleic acids" and "RNAi" of the present disclosure may each independently exist in the form of salts, mixed salts or non-salts (e.g., free acids or free bases). When present in salt or mixed salt form, it may be a pharmaceutically acceptable salt.

The "pharmaceutically acceptable salt" may be selected from inorganic salts or organic salts, and may also include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

By "pharmaceutically acceptable acid addition salt" is meant a salt with an inorganic or organic acid that retains the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like; organic acid salts include, but are not limited to, formate, acetate, 2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalenedisulfonate, and the like. These salts can be prepared by methods known in the art.

By "pharmaceutically acceptable base addition salt" is meant a salt formed with an inorganic or organic base that is capable of maintaining 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, aluminum, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts, preferably sodium salts. Salts derived from organic bases include, but are not limited to, the following: primary, secondary and tertiary amines, substituted amines including natural 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, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

"Alkyl" refers to saturated aliphatic hydrocarbon groups, such as straight and branched chain groups (C₁-C₃₀ alkyl) comprising 1 to 30 carbon atoms, and further such as alkyl groups containing 1 to 6 carbon atoms (C₁-C₆ alkyl), and further such as alkyl groups containing 1 to 3 carbon atoms (C₁-C₃ alkyl). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, and various branched isomers thereof, and the like.

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

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

The term "alkoxy" refers to-O- (alkyl) wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy.

"Cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, preferably from 3 to 6 carbon atoms, more preferably 5-6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.

"Heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S (O)_m (where m is an integer from 0 to 2), but excluding the ring portion of-O-O-, -O-S-, or-S-S-, and the remaining ring atoms are carbon. Preferably containing 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably from 3 to 7 ring atoms. Non-limiting examples of "heterocycloalkyl" include:

Etc.

The heterocycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a heterocycloalkyl group, non-limiting examples of which include:

Etc.

"Aryl" refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated pi-electron system, preferably 6 to 12 membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocycloalkyl, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples of which include:

"heteroaryl" refers to a heteroaromatic system containing from 1 to 4 heteroatoms, from 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. Heteroaryl is preferably 6 to 12 membered, more preferably 5 or 6 membered. For example. Non-limiting examples of which include: imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl (oxazolyl), isoxazolyl (isoxazolyl), pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, triazolyl, indazolyl, benzimidazolyl,Etc.

The heteroaryl ring may be fused to an aryl, heterocycloalkyl, or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring, non-limiting examples of which include:

the term "hydroxy" refers to an-OH group.

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

The term "cyano" refers to-CN.

The term "amino" refers to-NH₂.

The term "nitro" refers to-NO₂.

The term "oxo" refers to an =o substituent.

In the present disclosure, the "phosphate group" may be a phosphomonoester group, a phosphodiester group, or a phosphotriester group, with a phosphodiester group being preferred.

In the present disclosure, phosphorothioate diester refers to a modified phosphodiester group having one non-bridging oxygen atom replaced with a sulfur atom, which may be used(M is an S atom) are used interchangeably.

"Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents. When the substituent is ketone or oxo (i.e., =o), then two (2) hydrogens on the atom are replaced.

In the context of the present disclosure, a groupIn (a) and (b)The moiety may be replaced by any group capable of achieving ligation to an adjacent nucleotide.

The term "linked," when referring to a connection between two molecules, refers to the connection of the two molecules by covalent bonds or the association of the two molecules via non-covalent bonds (e.g., hydrogen or ionic bonds), including direct connection, indirect connection.

The term "directly linked" means that a first compound or group is linked to a second compound or group without any intervening atoms or groups of atoms.

The term "indirectly attached" refers to a first compound or group being attached to a second compound or group through an intervening group, compound or molecule (e.g., a linking group).

"Pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically acceptable salt or prodrug thereof, and other chemical components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

"Pharmaceutically acceptable excipients" include, but are not limited to, any auxiliary, carrier, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent or emulsifying agent that has been approved for use in humans or livestock animals.

An "effective amount" or "effective dose" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the route of administration and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

As used herein, "subject," "patient," "subject," or "individual" are used interchangeably and include a human or non-human animal, such as a mammal, e.g., a human or monkey.

Dsrnas or sirnas provided by the present disclosure can be obtained by methods of preparation conventional in the art (e.g., methods of solid phase synthesis and liquid phase synthesis). Among them, solid-phase synthesis already has commercial subscription services. Methods of preparing nucleoside monomers having corresponding modifications and methods of introducing modified nucleotide groups into dsRNA or siRNA described in the present disclosure can be also well known to those of skill in the art by using nucleoside monomers having corresponding modifications to introduce modified nucleotide groups into dsRNA or siRNA.

Drawings

FIG. 1 shows the residual expression levels of mRNA in TTR at 7 days after administration of TRD002218 and TRD 007205.

FIG. 2 shows the residual expression levels of mRNA in TTR at day 28 after administration of TRD002218 and TRD 007205.

FIG. 3 is an inhibition of the expression of the humanized mouse (hF 11) serum FXI protein, # indicates that TRD0008003-1 has a P <0.05 compared to TJR 100362; TRD0008002-1 shows P <0.05 compared with TJR 100364; * Each group was represented as P <0.05 compared to the blank; * P <0.0001 for each group compared to the placebo; ns indicates that each group was not statistically different from the blank.

Detailed Description

The present disclosure is further described below in connection with the examples, which are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not noted in the examples of the present disclosure are generally performed under conventional conditions or under conditions suggested by the manufacturer of the raw materials or goods. Reagents of particular origin are not noted, but are available from any supplier of molecular biological reagents in quality/purity for molecular biological applications.

Example 1: preparation of chemical modifications

1.1 Synthesis of Compounds 1-1a and 1-1b

Compound 1 (500 mg,3.42 mmol) and triethylamine (Et₃ N,692mg,6.84mmol,0.95 mL) were dissolved in dichloromethane (DCM, 10 mL), a solution of 4-toluenesulfonyl chloride (TsCl, 719 mg,3.76 mmol) in dichloromethane (10 mL) was added dropwise under ice, after the addition was completed, the reaction was stirred at room temperature overnight, after the reaction was completed, quenched with water, the aqueous phase was extracted three times with dichloromethane (15 mL), the combined organic phases were washed with saturated aqueous sodium bicarbonate (10 mL) first, then with saturated brine (20 mL), and then the solvent was evaporated under reduced pressure to give crude 2 (820 mg, 80%) which was used directly in the next reaction. MS m/z: c₁₄H₂₁O₅S,[M+H]⁺ theory: 301.10 actual measurement: 301.2.

Compound 3 (239 mg,1.22 mmol) was dissolved in dimethylformamide (DMF, 10 mL), naH (60% in mineral oil, 93mg,2.33 mmol) was added under ice-bath, stirring was carried out for 30min under this reaction, then compound 2 (350 mg,1.16 mmol) was added dropwise, the reaction was stirred at 60℃for 5H after the addition was completed, after the reaction was completed, water was added to quench the reaction, the aqueous phase was extracted three times with ethyl acetate (15 mL), the combined organic phases were washed three times with water (10 mL) first, then with saturated brine (10 mL), followed by evaporation of the solvent under reduced pressure, and reversed phase preparative HPLC (C¹⁸, condition: 5-50% (A: H₂O,B:CH₃ CN), flow rate: 70 mL/min) was carried out to give 220mg of compound 4 after lyophilization. MS m/z: c₁₉H₂₁N₅O₃Na,[M+Na]⁺ theory: 390.16, actual measurement: 390.3.

Compound 4 (1.50 g,4.08 mmol) was dissolved in a mixture of 20mL of acetic acid and water (4:1) at room temperature, stirred at 60℃for 30 min, after the reaction was completed, the solvent was evaporated under reduced pressure, and 1.10g of Compound 5 was obtained by lyophilization through reverse phase preparative HPLC (C¹⁸, condition: 5-25% (A: H₂O,B:CH₃ CN), flow rate: 70 mL/min). MS m/z: c₁₆H₁₈N₅O₃,[M+H]⁺ theory: 328.13, actual measurement: 328.4.

Compound 5 (1.00 g,3.05 mmol) was dissolved in pyridine (Py, 10 mL), a solution of 4,4' -dimethoxytrityl chloride (DMTrCl, 1.50g,4.58 mmol) in pyridine (5 mL) was added dropwise in ice, the reaction was stirred at room temperature overnight after the addition, quenched with water after the reaction was completed, the solvent was evaporated under reduced pressure, and 1.00g of Compound 6 was obtained after lyophilization by reverse phase preparative HPLC (C¹⁸, condition: 5-80% (A: H₂O,B:CH₃ CN), flow rate: 70 mL/min). MS m/z: c₃₇H₃₆N₅O₅,[M-H]⁺ theory: 630.26, actual measurement: 630.5. racemate compound 6 was subjected to chiral column (DaicelIE 250.6mm, 5 μm, A: n-hexane, B: ethanol) gives 410mg 6A (-) and 435mg 6B (+).

Compound 6A (-) (200 mg,0.32 mmol), tetrazole (11 mg,0.16 mmol), N-methylimidazole (5 mg,0.06 mmol), 3A molecular sieves (500 mg) were dissolved in 10mL of acetonitrile, compound 7 (144 mg,0.48 mmol) was added at room temperature, and stirred at room temperature overnight. After the reaction was completed, the molecular sieve was filtered off, dichloromethane (30 mL) was added, and the mixture was washed three times with saturated aqueous sodium bicarbonate (10 mL) and then with saturated brine (20 mL), and the filtrate was dried by spin-drying and subjected to reverse phase preparative HPLC (C¹⁸, condition: 5-100% (A: water, B: CH₃ CN), flow rate: 70 mL/min) to obtain 200mg of compound 1-1a after lyophilization. MS m/z: c₄₀H₃₉N₆O₇ P, [ M-diisopropyl+OH ]⁺ theory: 747.26, actual measurement: 747.6.1H NMR (400 MHz, acetonitrile -d₃)δ7.56,7.54(2s,1H),7.36-7.27(m,2H),7.24-7.21(m,7H),6.83-6.80(m,4H),4.12-4.10(m,2H),3.75-3.68(m,10H),3.20-2.80(m,2H),2.68-2.54(m,4H),1.22-1.04(m,18H).

Compound 6B (+) (200 mg,0.32 mmol), tetrazole (11 mg,0.16 mmol), N-methylimidazole (5 mg,0.06 mmol), 3A molecular sieves (500 mg) were dissolved in 10mL of acetonitrile, compound 7 (144 mg,0.48 mmol) was added at room temperature, and stirred at room temperature overnight. After the reaction was completed, the molecular sieve was filtered off, dichloromethane (30 mL) was added, and the mixture was washed three times with saturated aqueous sodium bicarbonate (10 mL) and then with saturated brine (20 mL), and the filtrate was dried by spin-drying and subjected to reverse phase preparative HPLC (C¹⁸, conditions: 5-100% (A: water, B: CH₃ CN), flow rate: 70 mL/min) to obtain 200mg of compound 1-1B after lyophilization. MS m/z: c₄₀H₃₉N6O₇ P, [ M-diisopropyl+OH ]⁺ theory: 747.26, actual measurement: 747.5.

1.2 Synthesis of Compounds 1-6a

Compound 1 (10 g,68.404 mmol), compound 2 (15 g,62.186 mmol) and triphenylphosphine (32.62 g,124.371 mmol) were dissolved in anhydrous THF (30 mL) and DIAD (24.650 mL,124.371 mmol) was slowly added dropwise at 0deg.C. The reaction was reacted at 25℃for 12h.LCMS showed completion. The reaction was extracted with ethyl acetate (200 mL) and water (200 mL), the organic phase was dried and the filtrate was concentrated, and the resulting residue was purified by forward column (DCM/meoh=10/1) to give the desired product 3 (20 g).

Compound 3 (20 g,28.585 mmol) was dissolved in acetic acid (24 mL,426.016 mmol) and H₂ O (12 mL) and stirred at 60℃for 1 hour. The reaction mixture was then dried by spin-drying, and THF (12 mL) and H₂ O (12 mL) were added thereto, followed by stirring at 80℃for 7 hours. LCMS showed the reaction was complete. The reaction was extracted with ethyl acetate (200 mL) and water (100 mL), and the aqueous phase was added with sodium carbonate solids until a significant amount of the solids had precipitated in the aqueous phase. The solid was filtered, washed with water, and the filter cake was pulled up with an oil pump to give the objective compound 5 (9 g).

Compound 5 (6.8 g,18.581 mmol) was dissolved in pyridine (80 mL) under nitrogen, TMSCL (14.250 mL,111.489 mmol) was slowly added at 0deg.C and stirred for 2h. Isobutyryl chloride (2.044 mL,19.511 mmol) was then added at 0deg.C and stirred at 25deg.C for 1h. Extraction with dichloromethane (200 mL) and water (200 mL) and drying of the organic phase followed by stirring and purification on a forward column (DCM: meoh=10:1) peak at 4.8%) afforded compound 6 (12 g) as a yellow oil.

Compound 6 (5.5 g, 12.390 mmol) was dissolved in pyridine (30 mL) under nitrogen, MOLECULAR SIEVE 4A 1/16 (7 g, 12.390 mmol) was added, then DMTrCl (5.04 g, 14.87mmol) was added in portions at 0deg.C as a solid and reacted at 25deg.C for 2h.TLC (PE: etOAc=1:1, rf=0.69) indicating the reaction was complete. The reaction mixture was treated with TJN200879,879-040-P1 in combination. The reaction was extracted with ethyl acetate (200 mL) and water (200 mL), and the organic phase was dried and spun-dried and purified on a forward column (PE: etOAc, peak 84%) to give compound 7 (12 g) as a yellow oil.

Compound 7 (12 g,15.389 mmol) was dissolved in EtOAc (140 mL) and wet Pd/C on carbon (7 g,15.389 mmol) was added and the reaction was allowed to react for 2 hours under hydrogen (15 Psi) at 25 ℃. TLC (PE: etoac=0:1, rf=0.09) showed the reaction was complete. The reaction mixture was filtered, and the cake was washed three times with ethyl acetate (30 mL), and the filtrate was collected. After spin-drying the filtrate was added 50mL of dichloromethane and 2mL of triethylamine and purified by forward column (DCM: meoh=10:1, peak at 0.5%) to give 9g (yellow foamy solid) the resulting racemic compound SFC was resolved to give the product title compound 7A (-) (3.9 g) and the title compound 7B (+) (3.8 g).

Compound 7A (-) (3.30 g,5.40 mmol), tetrazole (190 mg,2.70 mmol), 1-methylimidazole (90 mg,1.10 mmol), 3A molecular sieves (500 mg) were dissolved in 30mL of acetonitrile, compound 8 (2.50 g,8.10 mmol) was added at room temperature, and stirred at room temperature for 2h. After completion of the reaction, the molecular sieve was filtered off, DCM (150 mL) was added, washed with saturated aqueous sodium bicarbonate (30 mL x 3), then with saturated brine (30 mL), the filtrate was dried by spinning and lyophilized to give 1-6a (2.9 g, 66%) by reverse phase preparative HPLC (C18, conditions: 5-100% (a: water, B: CH3 CN), flow rate: 70 mL/min). MS m/z: c43H55N7O7P [ m+h ] +, theory: 812.38, actual measurement: 812.5.1H NMR (400 MHz, acetonitrile -d3)δ7.56,7.54(2s,1H),7.36-7.27(m,2H),7.24-7.21(m,7H),6.83-6.80(m,4H),4.12-4.10(m,2H),3.75-3.68(m,10H),3.20-2.80(m,2H),2.68-2.54(m,4H),1.22-1.04(m,18H).

1.3 Synthesis of Compounds 1-7a

Compound 1 (5 g,23.1272 mmol), compound 2 (6.76 g,46.254 mmol) and triphenylphosphine (7.28 g,27.753 mmol) were dissolved in 30mL dioxane under nitrogen and DEAD (5.502 mL,27.753 mmol) was slowly added dropwise at 0deg.C. After the completion of the dropwise addition, the reaction was slowly warmed to 25℃and continued for 1 hour. To the reaction mixture was added 100mL of H₂ O and 100mL of EtOAc, and the organic phase was combined, dried, filtered, concentrated, and purified by column chromatography (PE: etoac=1:1) to give the desired product (4 g).

Compound 3 (3.3 g) was dissolved in HOAc (16 mL) and H₂ O (4 mL), heated in an oil bath at 60℃for 0.5H, and the residue obtained by spin-drying the reaction was purified on a forward column (PE: etOAc=0:1 passage through a column) to give the desired product 4 (3 g).

Compound 4 (3 g,8.873 mmol) was dissolved in 5mL of pyridine and a solution of DMTrCl (3.91 g,11.535 mmol) in 10mL of pyridine was slowly added dropwise under nitrogen at 0deg.C. After the completion of the dropwise addition, the reaction was warmed to 25℃and continued for 1 hour. To the reaction mixture was added 50mL of water and 100mL of ethyl acetate for extraction. The aqueous phase was extracted three more times with 100mL ethyl acetate and the organic phases were combined, dried, filtered and concentrated and purified on a forward column (with PE: etoac=2:1). Target product 5 (4 g) was obtained.

Compound 5 (4 g,5.769 mmol) was dissolved in methanol (10 mL), saturated NH3 methanol solution (40 mL) was added and reacted for 6h at 0deg.C the reaction solution was spin dried and purified with a forward column (PE: etOAc=0:1) to give 2.4g of SFC as the racemic compound which was resolved to give the desired product 6A (750 mg,100% purity) and the desired product 6B (400 mg,99.16% purity).

Compound 6A (-) (700 mg,1.40 mmol), tetrazole (50 mg,0.70 mmol), 1-methylimidazole (23 mg,0.28 mmol), 3A molecular sieves (500 mg) were dissolved in 10mL of acetonitrile, compound 7 (630 mg,2.10 mmol) was added at room temperature, and stirred at room temperature for 2h. After completion of the reaction, the molecular sieve was filtered off, DCM (50 mL) was added, washed with saturated aqueous sodium bicarbonate (10 mL x 3), then with saturated brine (20 mL), the filtrate was dried by spinning and lyophilized to give 1-7a (700 mg, 72%) by reverse phase preparative HPLC (C18, conditions: 5-100% (a: water B: CH₃ CN), flow rate: 70 mL/min). MS m/z: C38H47N4O7PNa [ m+na ] +, theory: 725.32, actual measurement: 725.5.

1.4 Synthesis of Compounds 1-8a

Compound 1 (8.5 g,76.508 mmol), compound 2 (30.64 g,91.809 mmol) was dissolved in DMF (150 mL), CS2CO3 (29.91 g,91.809 mmol) was added and reacted under nitrogen for 12h at 90 ℃. LCMS detects completion of the reaction. The reaction was filtered, oil pump dried, and purified by column chromatography (80 g, dcm/meoh=10/1 to 5/1) to give the desired product 3 (13.5 g,80% purity).

Compound 3 (10.5 g,35.105 mmol) was dissolved in pyridine (65 mL) and CH₃ CN (65 mL), bzCl (4.894 mL,42.126 mmol) was added dropwise to the solution and reacted at 25℃for 2h. LCMS detected completion of most starting material reaction, quench with H₂ O (100 mL), extract with EtOAc (100 mL X3), dry spin-dry, purify by column separation (combination TJN 200872-101) (80 g, pe/etoac=10/1 to 0/1, dcm/meoh=10/1) to afford the desired product 4 (14 g,90% purity).

Compound 4 (14 g,36.694 mmol) was dissolved in HOAc (56 mL,314.796 mmol) and H₂ O (14 mL) and reacted at 60℃for 2H, LCMS indicated the reaction was complete. Oil pump concentration and forward column separation (40 g, dcm/meoh=1/0 to 5/1) gave the desired product 5 (8.4 g,90% purity &2.4g,80% purity).

Compound 5 (7.4 g,21.957 mmol), DMAP (0.54 g, 4.399 mmol), MOLECULAR SIEVE 4A (11.1 g,2.967 mmol) was dissolved in pyridine (60 mL), stirred for 10min under ice, then DMTrCl (8.93 g,26.348 mmol) was added and the reaction stirred for 1.8h.LCMS detected about 19% starting material remaining, about 60% target MS. The fractions were combined (TJN 200872-105& 106) and purified together. H₂ O (50 mL) was added to the reaction solution, extracted with DCM (50 mL. Times.3), dried, spin-dried, and column separated (120 g, PE/(EA: DCM: TEA=1:1:0.05) =1/0 to 0/1 to DCM/MeOH=10/1) to give the target compound 6 (11 g,89% purity, TJN200872-105&106& 107), and the starting material (3.0 g,70% purity) was recovered.

Compound 6 (15 g,22.041 mmol) was isolated by SFC (DAICEL CHIRALPAK AD (250 mm. Times.50 mm,10 um); 0.1% NH₃H₂ O EtOH, B:45% -45%;200 ml/min) to give target product 6A (5.33 g,94.29% purity), target product 6B (6.14 g,97.91% purity), and compound 6 was recovered at 1.0g.

Compound 6B (-) (5.4 g,8.92 mmol), tetrazole (312 mg,4.46 mmol), 1-methylimidazole (146 mg,1.78 mmol), 3A molecular sieves (500 mg) were dissolved in 40mL of acetonitrile, compound 7 (4 g,13.4 mmol) was added at room temperature, and stirred at room temperature for 2h. After completion of the reaction, the molecular sieve was filtered off, DCM (200 mL) was added, washed with saturated aqueous sodium bicarbonate (30 mL x 3), then with saturated brine (50 mL), the filtrate was dried by spinning and subjected to reverse phase preparative HPLC (C18, conditions: 5-100% (a: water, B: CH₃ CN), flow rate: 70 mL/min) to give 1-8a (5.8 g, 80%) after lyophilization. MS m/z: c45H51N5O7P, [ m+h ] +, theory: 804.36, actual measurement: 804.4.

Example 2: synthesis of siRNA

The synthesis of siRNA is not different from the common solid-phase synthesis method of phosphoramidite, and when synthesizing nucleotide modified at the 5' 7 th position of AS chain, the synthesized phosphoramidite monomer is used for replacing parent sequence original nucleotide. The synthesis process is briefly described as follows: nucleoside phosphoramidite monomers were individually linked according to the synthetic procedure starting with the Universal CPG vector on a Dr.Oligo48 synthesizer (Biolytic). In addition to the above described nucleoside phosphoramidite monomers at position 7 of the AS-strand 5', the remaining nucleoside monomer starting materials, 2' -F RNA, 2' -O-methyl RNA, and other nucleoside phosphoramidite monomers, were purchased from Shanghai megadimension or Stuzhou Ji Ma. 5-ethylthio-1H-tetrazole (ETT) was used as the activator (0.6M acetonitrile solution), 0.22M PADS was dissolved in a 1:1 volume ratio of acetonitrile and trimethylpyridine (Ke Lema, suzhou) solution as the sulfiding agent, and iodopyridine/water solution (Ke Lema) as the oxidizing agent.

After the solid phase synthesis was completed, the oligoribonucleotides were cleaved from the solid support and immersed in a 3:1 solution of 28% ammonia and ethanol at 50℃for 16 hours. The supernatant was then transferred to another centrifuge tube, concentrated to dryness, purified using C18 reverse chromatography, mobile phase 0.1M TEAA and acetonitrile, and DMTr removed using 3% trifluoroacetic acid solution. The target oligonucleotides were collected, lyophilized, and identified as target products by LC-MS, and then quantified by UV (260 nm).

The obtained single-stranded oligonucleotide is annealed according to complementary pairing according to the equimolar ratio, and finally the obtained dsRNA is dissolved in 1 XPBS and adjusted to the concentration required by the experiment for standby.

Example 3: psiCHECK activity screening assay

DsRNA Synthesis see above, plasmids were derived from the division of biological engineering (Shanghai). The psiCHECK experimental consumables are shown in table 1.

TABLE 1 psicheck assay consumables and reagents

The experimental steps are as follows: cell plating, cell transfection, wherein specific formulation amounts of the transfection complexes are shown in table 2.

TABLE 2 amount of transfection complex required per well of 96 well plates

Note that: lipo:0.2 μl/well; plasmid: 0.05 μl/well; opti-MEM: 10. Mu.L/well.

According to Table 3, the working fluid was diluted to different concentrations according to different experimental requirements and prepared as it was. After 24h of transfection, the method is carried out according toThe test protocol of the Luciferase ASSAY SYSTEM test kit was followed. Calculating a relative value ratio=ren/Fir (Renilla/firefly Ratio); calculating inhibition Ratio 1- (Ratio + siRNA/Ratio when reporter gene alone) ×100% = inhibition Ratio (%); in the present disclosure, residual activity% (also referred to as mRNA residual expression amount% or mRNA residual expression ratio) =100% -inhibition (%).

TABLE 3 Multi-concentration Point siRNA dilution protocol

Final concentration (nM)	Adding water and dsRNA
40	4μL siRNA(20μM)+96μL H₂O
13.33333333	30μL dsRNA+60μL H₂O
4.444444444	30μL dsRNA+60μL H₂O
1.481481481	30μL dsRNA+60μL H₂O
0.49382716	30μL dsRNA+60μL H₂O
0.164609053	30μL dsRNA+60μL H₂O
0.054869684	30μL dsRNA+60μL H₂O
0.018289895	30μL dsRNA+60μL H₂O
0.006096632	30μL dsRNA+60μL H₂O
0.002032211	30μL dsRNA+60μL H₂O
0.000677404	30μL dsRNA+60μL H₂O

Example 4: characterization of different chemical modifications

Wherein: we define hmpNA the nucleotide synthesized from 2-hydroxymethyl-1, 3-propanediol as starting material;

(+) hmpNA (a) is obtained by solid phase synthesis of nucleoside phosphoramidite monomer 1-1b of section 1.1 of example, absolute configuration is (S) -hmpNA (a);

(-) hmpNA (a) is obtained by solid phase synthesis of nucleoside phosphoramidite monomer 1-1a of section 1.1 of example, absolute configuration (R) -hmpNA (a);

(+) hmpNA (G), absolute configuration (S) -hmpNA (G);

(-) hmpNA (G), absolute configuration (R) -hmpNA (G);

(+) hmpNA (C), absolute configuration (S) -hmpNA (C);

(-) hmpNA (C), absolute configuration (R) -hmpNA (C);

(+) hmpNA (U), absolute configuration (R) -hmpNA (U);

(-) hmpNA (U), absolute configuration (S) -hmpNA (U).

The absolute configuration of (S) -hmpNA (G), (R) -hmpNA (G), (S) -hmpNA (C), (R) -hmpNA (C), (S) -hmpNA (U) and (R) -hmpNA (U) is confirmed by X-Ray diffraction of the intermediate or derivative thereof.

The structure of the intermediate or derivative is as follows:

TJ-NA067, the detection crystal is colorless block (0.30X0.10X0.04 mm 3), belonging to monoclinic system P21 space group. Unit cell parametersα＝90°,β＝118.015(4)°,γ＝90°,Z=4. The density dc=1.389 g/cm3, the electron number F (000) =504.0 in the unit cell, the linear absorption coefficient μ (Cu kα) =0.840 mm-1 in the unit cell, and the diffraction experiment temperature t=150.00 (11) K were calculated.

6A (+) (0.30X0.20X0.10mm3) of colorless block-shaped detection crystal belongs to monoclinic system P21 space group. Unit cell parametersα＝90°,β＝113.876(3)°,γ＝90°,Z=2. The density dc=0.999 g/cm3, the electron number F (000) = 1318.0 in the unit cell, the linear absorption coefficient μ (Cu kα) =0.570 mm-1 of the unit cell, and the diffraction experiment temperature t=100.01 (18) K were calculated.

TJ-NA048, the detection crystal is colorless needle-shaped (0.30X0.04 X0.04 mm 3), belonging to the monoclinic system P1 space group. Unit cell parametersα＝85.007(4)°,β＝88.052(4)°,γ＝70.532(4)°,Z=2. The density dc=1.366 g/cm3, the electron number F (000) =620.0 in the unit cell, the linear absorption coefficient μ (Cu kα) =0.856 mm-1 in the unit cell, and the diffraction experiment temperature t=150.00 (13) K were calculated.

TJ-NA092, the detection crystal is colorless prism (0.30X0.10X0.10mm3), belonging to the triclinic system P1 space group. Unit cell parametersα＝93.146(2)°,β＝101.266(2)°,γ＝96.134(2)°,Z=2. The density dc=1.412 g/cm3, the electron number F (000) =228.0 in the unit cell, the linear absorption coefficient μ (Cu kα) =0.945 mm-1 of the unit cell, and the diffraction experiment temperature t=100.00 (10) K were calculated.

Example 5: sequence dependent experiments with siRNAs comprising different chemical modifications

GNA modifications are known to have siRNA sequence dependence, and thus the inventors tested the experimental compounds of the present disclosure on a number of different sequences. siRNA targeting mRNA of different genes (ANGPTL 3, HBV-S, HBV-X) (sequences shown in Table 4) were used, the 5' 7 th position of AS strand was modified with the compounds (+) hmpNA (A), (-) hmpNA (A) of example 1 and the GNA (A) compound AS a control (sequences shown in Table 5), and the targeting and off-targeting activities were compared with the parent sequences. Experimental procedure reference is made to example 3.

TABLE 4 siRNA sequences targeting different genes

TABLE 5 siRNA sequences comprising chemical modifications targeting different genes

As a result of the target activity experiment, see Table 6, GNA (A) showed a clear sequence dependence, and the difference of the target activities of different sequences was clear. The experimental compounds of the present disclosure do not show significant sequence dependence and are more universally applicable.

As can be seen from the results of the off-target activity experiments referring to table 7, the modification of the present disclosure significantly reduced the off-target activity of the siRNA relative to the parent sequence.

TABLE 6 on-target Activity results of siRNAs against different target sequences

TABLE 7 off-target Activity results of siRNA against different target sequences

Example 6: preparation of ligands (NAG 0052, L96)

Compounds NAG0024 and NAG0026 were purchased from Tianjin Ming Kangde New drug development Co. Unless otherwise indicated, the reagents used in the examples below are all commercially available.

Synthesis of Compound NAG0052

Starting material compound 1 was purchased from Jiangsu-times pharmaceutical technology Co., ltd.

Compound 2

To a solution of Compound 1 (12.3 mL,101 mmol) in THF (300 mL) was added NaH (12.2 g,304mmol, 60% purity) in portions at 0deg.C under nitrogen. The mixture was stirred at 20℃for 1 hour and then cooled to 0℃again, then benzyl bromide (36.3 mL,304 mmol) was added dropwise to the system, and stirred at 20℃for 12 hours. The reaction mixture was quenched with H₂ O (100 mL) and extracted with EtOAc (200 mL. Times.2). The combined organic phases were washed with saturated brine (100 mL), dried over Na₂SO₄, filtered, and the residue obtained by concentration was separated by silica gel column chromatography to give the title compound 2 (20.0 g,51.8mmol, 51% yield).

LCMS:t_R＝2.615and 2.820min in 30-90AB_7min_220&254_Shimadzu.lcm(Xtimate C18,3um,2.1*30mm),MS(ESI)m/z＝351.2[M+Na]⁺.

¹H NMR:(400MHz,CDCl₃)δppm 7.35-7.12(m,10H),5.06-4.95(m,1H),4.51-4.39(m,4H),4.24-3.87(m,2H),3.50-3.40(m,2H),3.38-3.20(m,3H),2.20-1.91 (m,2H).

Compounds 3 and 4

To a solution of compound 2 (13.0 g,33.6 mmol) in DCM (300 mL) at 20deg.C under nitrogen is added TMSCN (13.5 mL,101 mmol) in one portion followed by dropwise addition of a solution of TMSOTF (9.14 mL,50.5 mmol) in DCM (30 mL). The reaction solution was stirred at 20℃for 15 hours. After the reaction was completed, the system was quenched with saturated aqueous NaHCO₃ (80 mL) and extracted with DCM (150 ml×2), the combined organic phases were washed with saturated brine (80 mL), dried over Na₂SO₄, filtered and concentrated to give the title compound 3 (3.30 g,9.18mmol, 27% yield) as a pale yellow oil, compound 4 (8.50 g,9.18mmol, 70% yield) after separation by silica gel column chromatography.

Compound 3

¹H NMR:(400MHz,CDCl₃)δppm 7.42-7.29(m,10H),4.81(t,J＝7.8Hz,1H),4.65-4.49(m,4H),4.30-4.21(m,2H),3.65-3.57(m,1H),3.57-3.49(m,1H),2.49-2.40(m,2H).

Compound 4

¹H NMR:(400MHz,CDCl₃)δppm 7.42-7.26(m,10H),4.93-4.87(m,1H),4.65-4.48(m,4H),4.43-4.38(m,1H),4.21-4.17(m,1H),3.79-3.70(m,1H),3.54(d,J＝4.0Hz,1H),2.45-2.37(m,2H).

Compound 5

A solution of Compound 4 (3.00 g,9.28 mmol) in THF (15 mL) was added dropwise to a solution of LiAlH₄ (0.79 g,20.9 mmol) in THF (15 mL) at 0deg.C under nitrogen, and the system was reacted at 0deg.C for 1 hour after the dropwise addition. TLC (PE: etoac=3:1) monitored complete disappearance of starting material. Sodium sulfate decahydrate was slowly added to the reaction solution until bubbling did not occur. The reaction mixture was then filtered, and the cake was washed three times with methylene chloride (60 mL), and the filtrate was collected and dried to give the objective compound 5 (3.00 g, yield 90%).

¹H NMR:(400MHz,DMSO-d₆)δppm 7.40-7.14(m,10H),4.54-4.38(m,4H),4.06-3.99(m,2H),3.91(q,J＝6.4Hz,1H),3.48-3.37(m,2H),2.67-2.52(m,2H),2.21-2.18(m,1H),1.77-1.73(m,1H).

Compound 6

Compound 5 (3.00 g,8.25 mmol) was dissolved in DCM (30 mL) under nitrogen and TEA (3.44 mL,24.7 mmol) and CbzCl (1.76 mL,12.4 mmol) were added and reacted at 20℃for 2 h. LCMS showed the reaction was complete. The reaction mixture was extracted with dichloromethane (30 mL) and water (60 mL). The organic phase was washed three times with water (60 ml×3), dried over anhydrous sodium sulfate, and concentrated to dryness on a forward column (PE: etoac=1:1) to give the title compound 6 (2.5 g, yield 90%).

LCMS:t_R＝0.810min in 5-95AB_1min,MS(ESI)m/z＝462.2[M+H]⁺。

¹H NMR:(400MHz,CDCl₃)δppm 7.39-7.29(m,15H),5.35(s,1H),5.15-5.01(m,2H),4.72(d,J＝6.0Hz,1H),4.54-4.40(m,3H),4.26(s,1H),4.23-4.18(m,1H),4.11-4.04(m,1H),3.54-3.41(m,3H),3.37-3.25(m,1H),2.34-2.23(m,1H),1.85-1.79(m,1H).

Compound 7

Compound 6 (2.00 g,3.90 mmol) was dissolved in DCM (5 mL) under nitrogen and BCl₃ in THF (1M, 27.3 mL) was added at-78deg.C and reacted for 1 hour. TLC (DCM: meoh=10:1) monitored complete disappearance of starting material. The reaction was quenched by the addition of methanol (20 mL) at-78 ℃, concentrated and purified by forward column (DCM: meoh=10:1) to give the title compound 7 (2.00 g, 60% yield).

¹H NMR:(400MHz,CD₃OD)δppm 7.41-7.23(m,5H),5.08(s,2H),4.25-4.07(m,2H),3.85-3.75(m,1H),3.63-3.56(m,1H),3.54-3.48(m,1H),3.30-3.27(m,2H),2.34-2.21(m,1H),1.71-1.64(m,1H).

Compound 8

Compound 7 (0.50 g,1.78 mmol) was dissolved in pyridine (5 mL) under nitrogen, 4A molecular sieve (500 mg) and DMTrCl (0.66 mL,2.13 mmol) were added at 0deg.C, and then warmed to 20deg.C for 1.5 hours. TLC (PE: etoac=2:1) monitored complete disappearance of starting material. The reaction solution was extracted with ethyl acetate (60 mL) and water (60 mL), and the organic phase was washed three times with water (60 ml×3), dried over anhydrous sodium sulfate, concentrated, and purified by a forward column (PE: etoac=1:1) to give the objective compound 8 (800 mg, yield 90%).

¹H NMR:(400MHz,CDCl₃)δppm 7.44(d,J＝7.6Hz,2H),7.37-7.23(m,11H),7.22-7.15(m,1H),6.84(d,J＝8.8Hz,4H),5.09(s,2H),4.31-4.17(m,2H),4.02-3.91(m,1H),3.84-3.73(m,6H),3.33(s,1H),3.28(s,1H),3.19-3.01(m,2H),2.34-2.25(m,1H),1.70-1.62(m,1H).

Compound 9

Compound 8 (800 mg,1.234 mmol) was dissolved in EtOAc (5 mL) and Pd/C10% (800 mg,7.517 mmol) was added and the reaction was carried out under H₂ conditions (15 Psi) at 20℃for 1 hour. LCMS showed the reaction was complete. The reaction solution was filtered, and the cake was washed three times with dichloromethane (100 mL) and methanol (100 mL), concentrated, and separated by reverse phase column to give compound 9 (300 mg, 54%).

LCMS:t_R＝2.586min in 10-80CD_3min MS(ESI)m/z＝450.2[M+H]⁺。

Compound 11

Compound 10 (435 mg,1.780 mmol) was dissolved in DCM (10 mL), DIEA (0.4471 mL,2.67 mmol) and HATU (677 mg,1.78 mmol) were added and then Compound 9 (400 mg,0.890 mmol) was added and reacted at 20℃for 1 hour. TLC (DCM: meoh=10:1) monitored the reaction was complete. The reaction was extracted with dichloromethane (60 mL) and water (60 mL), the organic phase was washed three times with water (60 mL x 3), dried over anhydrous sodium sulfate, concentrated and purified on a forward column (PE: etoac=0:1 column, product peak at 100%) to give the title compound 11 (600 mg, yield 90%).

LCMS:t_R＝2.745min in 30-90CD_3min,MS(ESI)m/z＝698.4[M+Na]⁺。

¹H NMR:(400MHz,CD₃OD)δppm 7.46-7.38(m,2H),7.35-7.24(m,6H),7.22-7.16(m,1H),6.90-6.78(m,4H),4.29-4.21(m,2H),4.02-3.95(m,1H),3.77(s,6H),3.66-3.62(m,3H),3.41(s,1H),3.18-3.04(m,2H),2.36-2.17(m,5H),1.71-1.50(m,5H),1.39-1.25(m,14H).

Compound 12

Compound 11 (600 mg,0.799 mmol) was dissolved in THF (3 mL) and H₂ O (1 mL), liOH.H2₂ O (134 mg,3.20 mmol) was added and reacted at 20℃for 12 hours. TLC (DCM: meoh=10:1) showed the reaction was complete. The reaction solution was dried by spin-drying, dissolved in water (5 mL) and methanol (5 mL), and purified by reverse column (H₂O:CH₃ cn=1:1, peak around 35%) to give the objective compound 12 (460 mg, yield 100%, lithium salt).

LCMS:t_R＝1.346min in 10-80CD_3min,MS(ESI)m/z＝684.3[M+Na]⁺。

HPLC:t_R＝1.879min in 10-80CD_6min。

¹H NMR:(400MHz,CD₃OD)δppm 7.47-7.39(m,2H),7.35-7.24(m,6H),7.22-7.15(m,1H),6.91-6.79(m,4H),4.31-4.18(m,2H),4.02-3.95(m,1H),3.78(s,6H),3.44-3.33(m,2H),3.18-3.04(m,2H),2.35-2.27(m,1H),2.24-2.10(m,4H),1.70-1.51(m,5H),1.31-1.23(m,12H).

Compound 13

Compound NAG0024 (271mg, 0.151 mmol) was dissolved in anhydrous THF (2 mL) and anhydrous DMF (4 mL) at room temperature under nitrogen, 3A molecular sieve was added followed by compound 12 (100 mg,0.151 mmol), HOBt (25 mg,0.181 mmol), DCC (38 mg,0.181 mmol) and DIEA (39 mg,0.302 mmol) in sequence. After the reaction was completed by LC-MS at 45℃for 16 hours, the reaction mixture was quenched with water and filtered. After concentration of the filtrate, purification by C18 reverse phase column (H₂ O/MeCN) afforded compound 13 (210 mg, 57% yield).

Compound NAG0052

Compound 13 (230 mg,0.094 mmol) was dissolved in pyridine (5 mL), molecular sieves were added, DMAP (12 mg,0.283 mmol) was added, and succinic anhydride (28 mg,0.283 mmol) was added at room temperature. The mixture was stirred at 50℃for 16 hours under nitrogen. LCMS detected complete reaction and filtered spin-dry. After purification on a C18 reverse phase column, purification by preparative HPLC gave the title compound NAG0052 (123 mg,0.048mmol, 51% yield).

MS (ESI) m/z=2535.3 [ m-1]^- theory: 2536.2.

¹ H NMR (400 MHz, acetonitrile -d₃)δ7.48-7.43(m,2H),7.37-7.12(m,11H),7.00-6.85(m,10H),6.66(s,1H),5.31(dd,J＝3.4,1.1Hz,3H),5.20-5.13(m,1H),5.05(dd,J＝11.3,3.4Hz,3H),4.56(d,J＝8.5Hz,3H),4.30(dd,J＝7.7,5.3Hz,1H),4.18-3.93(m,14H),3.79(s,10H),3.65(q,J＝4.7,3.6Hz,13H),3.56-3.07(m,24H),2.56(s,6H),2.37(t,J＝5.8Hz,10H),2.17(t,J＝7.5Hz,9H),2.02-1.96(m,20H),1.88(s,8H),1.82-1.73(m,2H),1.60(dt,J＝15.0,7.3Hz,16H),1.27(s,13H). )

NAG0052’

The compound NAG0052 is connected to the sequence through solid phase synthesis, and after aminolysis, a part of functional groups of NAG0052 structure are removed to form NAG0052'.

Synthesis of L96

Is prepared according to the method described in patent application WO2014025805A 1.

Example 7: synthesis of dsRNA

1. Homemade resin with carrier

Compound NAG0052 (157 mg,0.062 mmol) containing carboxylic acid groups was dissolved in anhydrous DMF (3 mL), and after complete dissolution of the substrate, anhydrous acetonitrile (4 mL), DIEA (0.03 mL,0.154mmol,2.5 eq) and HBTU (35 mg,0.093mmol,1.5 eq) were added sequentially. After the reaction mixture was mixed uniformly, a large Kong An methyl resin (476 mg, blank load 0.41mmol/g, target load 0.1 mmol/g) was added. The reaction solution was placed on a shaking table (temperature: 25 ℃ C., rotation speed: 200 rpm) and shaken overnight. The reaction was filtered and the filter cake was washed with DCM, anhydrous acetonitrile, respectively, and the solids were collected and dried overnight in vacuo.

The above solid was dispersed in anhydrous acetonitrile (5 mL), pyridine (0.18 mL), DMAP (3 mg), NMI (0.12 mL) and CapB1 (2.68 mL) were added sequentially. The reaction solution was placed on a shaking table (temperature: 25 ℃ C., rotation speed: 200 rpm) and shaken for 2 hours. The reaction solution was filtered, and the filter cake was washed with anhydrous acetonitrile, and the solid was collected and dried under vacuum overnight to give a supported resin. The loading was determined to be 0.1mmol/g.

2. For NAG0052 which has been attached to the resin, nucleoside monomers were attached one by one in the 3'-5' direction in the nucleotide arrangement order, using the resin as a starting point. Each nucleoside monomer attached includes four steps of deprotection, coupling, capping, oxidation or vulcanization. The operation is conventional in the art.

The resulting dsRNA had sense and antisense strands shown in tables 8 and 9.

TABLE 8 list of dsRNAs

DsRNA numbering	Sense strand numbering	Antisense strand numbering
TRD002218	TJR4373-SS	TJR0414-AS
TRD007205	TJR013485S	TJR0414-AS

TABLE 9 nucleic acid listing of sense and antisense strands

TABLE 10 Structure of dsRNA

Wherein TRD002218 is used as a reference positive compound, and Z represents siRNA.

Example 8: inhibition of mRNA expression of target genes by dsRNA in vivo

This experiment examined the inhibition efficiency of dsRNA conjugated with different structures of the present disclosure on the expression level of target gene mRNA in vivo.

Male 6-8 week old C57BL/6 mice were randomly grouped, 6 in each group, 3 at each time point, and each group of mice was given TRD007205, reference positive TRD002218, and PBS, respectively, of the present disclosure.

All animals calculated the dose in total on a per-body basis, and were given in a single dose by subcutaneous injection, with dsRNA (in terms of siRNA) doses of 1mg/kg and 5mL/kg of dose volume. Mice were sacrificed 7 days and 28 days after dosing, livers were collected and saved with RNA later (SIGMA ALDRICH Co.); subsequently, liver tissues are homogenized by a tissue homogenizer, and total RNA of the liver tissues is extracted by using a tissue RNA extraction kit (known medical science and technology, FG 0412) according to the operation steps marked by the operation instructions. The total RNA is reversely transcribed into cDNA and the expression quantity of TTR mRNA in liver tissue is detected by adopting a real-time fluorescence quantitative PCR method. In the fluorescent quantitative PCR method, the mRNA expression levels of TTR and GAPDH are detected using Taqman probe primers for TTR and GAPDH, respectively, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as an internal reference gene.

Table 11. Grouping information of experimental compounds in mice:

Table 12. Sequences of detection primers are as follows:

TTR mRNA expression amount was calculated according to the following equation:

TTR mRNA expression level= [ test set TTR mRNA expression level/test set GAPDH MRNA expression level)/(control set TTR mRNA expression level/control set GAPDH MRNA expression level) ]x100%.

The inhibition efficiency of dsRNA conjugated with different structures of the present disclosure on the target gene mRNA expression level in vivo 7 days and 28 days after administration is shown in fig. 1 and 2, respectively. From the results of fig. 1, TRD007205 had a good effect on inhibition of TTR mRNA expression 7 days after administration. As can be seen from FIG. 2, the TRD007205 had better effect of inhibiting the expression level of mRNA of the target gene than TRD002218 after 28 days of administration.

Example 9: synthesis of dsRNA

1. Homemade resin with carrier

The specific operation was the same as in example 7.

2. Using a resin with NAG0052 as a starting material, nucleoside monomers were linked one by one in the nucleotide arrangement order from the 3'-5' direction. Each nucleoside monomer attached includes four steps of deprotection, coupling, capping, oxidation or vulcanization. Reference is made specifically to the synthetic method of example 2.

The dsRNA produced had sense and antisense strands shown in table 13 and 14. The naked sequences corresponding to dsRNA are shown in Table 15.

TABLE 13 list of dsRNAs

TABLE 14 sequence of dsRNA

TABLE 15 naked sequences corresponding to sense and antisense strands of dsRNA

Wherein, (-) hmpNA (a), (-) hmpNA (G), (-) hmpNA (C), (-) hmpNA (U) are structured as described in example 4.

NAG0052' has the structure:

NAG1 has the structure of

Example 10: on-target Activity of dsRNA

In vitro molecular level simulation of dsRNA was screened for target activity in HEK293A cells using 11 concentration gradients.

The human FXI gene is used to construct the target sequence corresponding to siRNA and inserted into psiCHECK-2 plasmid. The plasmid contains a Renilla luciferase gene and a firefly luciferase gene. As a Dual reporter system, the target sequence of dsRNA was inserted into the 3' UTR region of Renilla luciferase gene, and the activity of dsRNA on the target sequence was reflected by detection of Renilla luciferase expression after firefly luciferase calibration, using Dual-Luciferase Reporter ASSAY SYSTEM (Promega, E2940).

HEK293A cells were cultured in DMEM high sugar medium containing 10% fetal bovine serum at 37 ℃ under 5% co₂. 24h before transfection, HEK293A cells were seeded in 96-well plates at a density of 8X 10³ cells per well and 100. Mu.L of medium per well.

According to the instructions, the cells were co-transfected with dsRNA and the corresponding plasmid using Lipofectamine2000 (ThermoFisher, 11668019), 0.2. Mu.L per well of Lipofectamine2000, 20ng per well of plasmid. For the plasmid in the target sequence, the dsRNA was set to 11 concentration points in total, with the highest concentration points having final concentrations of 20nM, 3-fold gradient dilutions, 20nM,6.6667nM,2.2222nM,0.7407nM,0.2469nM,0.0823nM,0.0274nM,0.0091nM,0.0030nM,0.0010nM and 0.0003nM. 24h after transfection, the target level was detected using Dual-Luciferase Reporter ASSAY SYSTEM (Promega, E2940). The on-target activity of the detected sequences is shown in Table 16.

TABLE 16 psi-CHECK screening results for dsRNA on target Activity

N/A represents: inapplicable;

The above results indicate that TJR100407, TRD008003-1, TRD008002-1, TRD008003, and TRD008002 compounds have high levels of on-target inhibitory activity against FXI gene in psiCHECK system.

Example 11: inhibition of human FXI in human primary hepatocytes (PHH) by dsRNA

PHH activity screening was performed on dsRNA in human primary hepatocytes (PHH) using 7 concentration gradients. The final transfection concentration for each dsRNA sample was 20nM, 5-fold gradient dilution and 7 concentration points.

PHH cells (Novabiosis, nHPHepatocytes) were frozen in liquid nitrogen, and after 24h before transfection, PHH cells were resuscitated and plated in 96 well plates at a plating density of 3X 10⁴ cells per well, 80. Mu.L of medium per well.

Referring to the product instruction manual, lipofectamine RNAi MAX (thermo Fisher, 13778150) was used to transfect dsRNA, the final gradient of which was 10nM,2nM,0.4nM,0.08nM,0.016nM,0.0032nM and 0.00064nM. After 24 hours of treatment, total cellular RNA extraction, RNA reverse transcription experiments (Takara, 6210B) and quantitative real-time PCR detection (thermo fisher, 4444557) were performed using a high throughput cellular RNA extraction kit (known as FG 0417), the mRNA levels of human FXI were determined and corrected according to GAPDH reference gene levels.

In the quantitative real-time PCR detection, a probe Q-PCR detection experiment was used, and the primer information is shown in Table 17.

TABLE 17 Taqman primer information Table

Primer name	SEQ ID NO	Primer sequences
hFXI-PF	40	TTTGCTGGGAGAGGGTGTTG
hFXI-PR	41	TACAAACACCAAGCCCCTTCA
hFXI-P	42	CCAGCATGCTTCCTCCACAGTAACACG
hGAPDH-PF1-MGB	43	GACCCCTTCATTGACCTCAACTAC
hGAPDH-PR1-MGB	44	TTGACGGTGCCATGGAATTT
hGAPDH-P1-MGB	45	TTACATGTTCCAATATGATTCC

Result analysis method

After the Q-PCR detection experiment is finished, corresponding Ct values are obtained according to the threshold value automatically set by the system, and the expression of a certain gene can be relatively quantified through Ct value comparison: comparing Ct refers to calculating the difference in gene expression by the difference between the Ct values of the reference genes, also referred to as 2^-△△Ct, ΔΔct= [ (Ct experimental group gene of interest-Ct experimental group reference) - (Ct control group gene of interest-Ct control group reference) ]. Inhibition ratio (%) = (residual amount of 1-target gene expression) ×100%.

Results are expressed as the remaining percentage of mRNA expression of human FXI relative to control dsRNA treated cells. The results of the inhibition IC₅₀ are shown in Table 18.

The results indicate that TRD008002, TRD008003 compounds have high levels of on-target inhibitory activity against FXI gene in PHH cells.

TABLE 18 inhibitory Activity of dsRNA in PHH cells

Example 12: inhibition of FXI in cynomolgus primary hepatocytes (PCH) by dsRNA

Reverse transfection activity screening was performed in PCH cells (Miaoshun, CCH100 CY-V09201) using 7 concentration gradients of FXI dsRNA. The final transfection concentration for each dsRNA sample was 10nM, 5-fold gradient dilution and 7 concentration spots.

PCH cells were frozen in liquid nitrogen, and after resuscitating PCH cells prior to transfection, they were plated in 96-well plates at a plating density of 3X 10⁴ cells per well, 90uL medium per well (Miaoshun, HEP044, HEP024, HEP054, HEP 064).

Referring to the product instruction manual, lipofectamine RNAi MAX (thermo Fisher, 13778150) was used to transfect dsRNA, the final gradient of which was 10nM,2nM,0.4nM,0.08nM,0.016nM,0.0032nM and 0.00064nM. After 24 hours of treatment, total cellular RNA extraction, RNA reverse transcription experiments and quantitative real-time PCR detection were performed using a high throughput cellular RNA extraction kit, the mRNA level of monkey FXI was determined and corrected according to GAPDH reference gene levels.

TABLE 19 monkey Taqman probe primer information Table

Primer name	SEQ ID NO	Primer sequences
mkFXI-V1-PF1	46	CTGGATATTGTTGCTGTGAAAGGT
mkFXI-V1-PR1	47	CCTTCGTTGCAAGATGCTTGA
mkFXI-V1-P1	48	CTGTGCACCAATGCCGTCCGC
mkGAPDH-PF1-MGB	49	AGTCAGCCGCATTTTCTCTTG
mkGAPDH-PR1-MGB	50	AAATCCGTTGACTCCGACCTT
mkGAPDH-P1-MGB	51	ATCGCCAGCGCATC

Results are expressed as the remaining percentage of monkey FXI mRNA expression relative to control treated cells. The results of the inhibition IC₅₀ are shown in Table 20. The results show that TRD008002 and TRD008003 have good inhibiting activity against FXI in PCH cells.

TABLE 20 Multi-dose inhibitory Activity of dsRNA in PCH

Example 13: determination of the Activity of dsRNA in vivo (in vivo) in humanized mice (hF 11)

The humanized mice (hF 11) used in this example were purchased from biotechnology limited of the racing industry (su state), product number: c001272 serum 40ul was collected, anticoagulated with EDTAK and assayed for FXI protein content in serum from each of the above groups using a Human Coagulation Factor XI ELISA kit (Sigma Co., lot 0309J2350, cat# RAB1385-1 KT).

The serum FXI proteins were uniformly distributed, 6 groups (2 males, 4 females) were 5 groups each, and five groups were each administered to a control group (physiological saline) by subcutaneous injection, with dsRNA administration volumes of 10. Mu.l/g, TJR100362, TRD008003-1, TJR100364, and TRD008002-1 each at a dose of 10mg/kg. Serum was collected at 40 μl on the day of dosing, at 15 days, plasma FXI protein content was measured using Human Coagulation Factor XI ELISA kit, and inhibition of serum FXI protein expression in humanized mice (hF 11) by dsRNA was calculated. The experimental results are shown in table 21 and fig. 3.

On the day of administration, the average serum FXI content of blood collected before administration of a blank control group (physiological saline) was used as a baseline. On day 8, 15, 22, the ratio of serum FXI content to baseline was calculated for each group and statistics were performed using Two-way ANOVA (day 8, 15)/One-way ANOVA (day 22), giving fig. 3.

As can be seen from the results in fig. 3, TRD008003-1 was significantly different from the placebo group (normal saline) on day 22 (ns in fig. 3), whereas TRD008003-1 was P <0.0001 (x in fig. 3) compared to the placebo group (normal saline), indicating that no therapeutic effect was given on day 22, TJR100362, and TRD008003-1 remained therapeutic.

No significant difference in TJR100364 compared to the placebo group (normal saline) on day 15 (ns in fig. 3) for TRD008002-1 compared to TJR100364, whereas P <0.0001 (x in fig. 3) for TRD008002-1 compared to the placebo group (normal saline), indicating that on day 15, TJR100364 has no therapeutic effect, while TRD008002-1 remains therapeutic.

Meanwhile, on days 15 and 22, the TRD008003-1 inhibition rate is significantly better than TJR100362 (denoted by # in fig. 3, #, P < 0.05); on day 15, the TRD008002-1 inhibition rate was significantly better than TJR100364 (shown in figure 3 as &, & P < 0.05).

ELISA detection

The serum FXI protein content of each group of mice was tested using Human Coagulation Factor XI ELISA kit (Sigma Co., cat. RAB1385-1 KT).

And diluting the samples in the kit according to instructions to obtain the diluents A, B, C, D for later use, and diluting the sample diluent A for the mouse serum 5000 times to serve as a sample to be detected for later use.

The reaction was terminated by following the instruction, and the optical density value was immediately read at a wavelength of 450nm using a full-automatic microplate reader (PERKIN ELMER, envision 2015).

Fitting a standard curve according to the optical density value measured on the standard concentration gradient solution, calculating the protein concentration in the serum to be measured according to the standard curve, wherein the standard curve obtained by fitting accords with the following calculation formula:

Y＝slope X+intercept

Wherein: y is the corresponding optical density value read at 450 nm; x is the concentration value (pg/mL) in the standard curve, intercept is the intercept of the standard curve, and slope is the slope of the curve.

From the obtained standard curve, the optical density value measured for each serum sample was substituted to obtain a concentration value X corresponding to each sample, and serum FXI protein concentration value=5000X (pg/mL) for each given different sample was calculated.

From the plasma serum FXI protein concentration values, FXI protein inhibition rate = (control histone concentration-test histone concentration)/control histone concentration x 100% was calculated relative to the protein concentration of the control group. The inhibition ratios are shown in table 21.

TABLE 21 inhibition of Activity of dsRNA in humanized mice (hF 11)

Example 14: determination of the Activity of dsRNA in vivo (in vivo) in humanized mice (hF 11)

The groups were evenly divided according to the serum FXI protein content, 6 groups (2 males, 4 females) were administered to the control group (physiological saline) by subcutaneous injection, the TJR100407 administration volume was 10. Mu.l/g, and the TJR100407 administration dose was reduced to 3mg/kg. Serum was collected at 40 μl on the day of administration, at 40 μl on the 8 th day after administration, plasma FXI protein content was measured using Human Coagulation Factor XI ELISA kit, and inhibition of serum FXI protein expression by dsRNA on humanized mice (hF 11) was calculated. ELISA detection and inhibition calculation, as in example 13, the experimental results are shown in Table 22.

The results in Table 22 show that TJR100407 still has a high inhibition of human FXI protein expression at lower doses when the dose is reduced to 3 mg/kg.

TABLE 22 inhibition of Activity of dsRNA in humanized mice (hF 11)