CN117482242A

CN117482242A - Conjugate and application thereof

Info

Publication number: CN117482242A
Application number: CN202310957841.0A
Authority: CN
Inventors: 吕佳声; 司杨海啸; 任广威; 叶祥胜; 周天伦; 葛建; 孔宪起; 陈大为
Original assignee: Runjia Shanghai Pharmaceutical Engineering Co ltd; Risen Suzhou Pharma Tech Co Ltd
Current assignee: Runjia Shanghai Pharmaceutical Engineering Co ltd; Risen Suzhou Pharma Tech Co Ltd
Priority date: 2022-08-01
Filing date: 2023-08-01
Publication date: 2024-02-02

Abstract

The invention relates to a conjugate, a pharmaceutical composition comprising the conjugate, and the preparation of the conjugate or the pharmaceutical composition comprising the conjugate for treatment and inhibitionUse in the manufacture or prevention of a PCSK 9-associated disease.

Description

Conjugate and application thereof

Technical Field

The invention relates to a conjugate and application of the conjugate in preparing medicines for treating, inhibiting or preventing PCSK9 related diseases.

Background

There are 4 types of fats in human blood: cholesterol, neutral fat, free fatty acids and phospholipids. Cholesterol is an oil complex, mostly produced in the liver. The total amount of cholesterol in the human body is 100 to 200 grams, two thirds of which are self-synthesized in the body and one third of which are from food. Cholesterol must bind to lipoproteins for transport to the various parts of the body. Lipoproteins are in turn divided into low density lipoproteins and high density lipoproteins. The low density and high density lipoprotein levels in serum are one to two, both of which have important roles: low density lipoproteins transport cholesterol from the liver to systemic tissues and high density lipoproteins transport cholesterol from each tissue back to the liver for metabolism. Low density lipoprotein can be oxidized to oxidized low density lipoprotein, and when low density lipoprotein, especially oxidized modified low density lipoprotein (OX-LDL), is excessive, cholesterol carried by it accumulates on the arterial wall, which is liable to cause arteriosclerosis for a long time.

Studies have shown that PCSK9 (member 9 of the Kexin-like pre-convertase subtilisin family) mediates degradation of low density lipoprotein receptor (Low Density Lipoprotein Receptor, LDLR) on hepatocytes, regulates low density lipoprotein (Low Density Lipoprotein, LDL), increases cholesterol (LDL-C) levels, and renders LDL in the blood non-scavenging, leading to hypercholesterolemia or increasing the risk of coronary heart disease and myocardial infarction. Therefore, how to effectively inhibit PCSK9 expression is a valuable research direction.

Disclosure of Invention

The invention mainly solves the technical problem of providing a novel conjugate and realizing better treatment effect through the novel conjugate.

The conjugate disclosed by the invention has a structure shown in a formula (I):

wherein L is a carrier group;

r and R ^* Side chains independently selected from different or identical natural or unnatural amino acids;

m and m 'are independently selected from integers from 0 to 3, when m or m' is 1, structural fragments Independently selected from different or identical amino acid residues, structural fragments when m or m' is 2 or 3Independently selected from different or identical oligopeptide residues;

n and n 'are independently selected from integers from 0 to 10, and n' are not both 0;

A is selected from one of the following groups:

or methylene (CH) ₂ )，

And when A is methylene, m and m' are not both 0;

R ¹ a bio-ligand group that is a cellular receptor;

R ² as a genetic component, the genetic sequence comprising:

from the nucleotide sequence 5' -cs ^x us ^x a ^x g ^x a ^x c ^x Cf ^x u ^x Gf ^x u ^x dT ^x u ^x u ^x g ^x c ^x u ^x u ^x u ^x u ^x g ^x u-3 'sense strand and 5' -ay from the nucleotide sequence ^x Cfy ^x a ^x Af ^x Af ^x Af ^x g ^x Cf ^x a ^x Af ^x a ^x Af ^x c ^x Af ^x g ^x Gf ^x u ^x Cf ^x u ^x a ^x gy ^x ay ^x an antisense strand consisting of a-3',

wherein a, g, c and U are 2 '-O-methyl (2' -OMe) modified A, G, C and U nucleotides, respectively; af. Gf, cf and Uf are 2' -fluoro modified A, G, C and U nucleotides, respectively; dT is deoxythymine; and, any y independently represents s or absence, wherein s is a phosphorothioate linkage

Any x independently represents H or D (deuterium) in which the hydrogen on the pentamethylene of the adjacent nucleotide on the right is naturally abundant, in particular D (deuterium) may be one or two;

or the five-position oxygen of the adjacent nucleotide on the right side is O or isotopically enriched in natural abundance ¹⁸ O；

Or the oxygen atom on the phosphoester bond or phosphorothioate bond of the adjacent nucleotide to the right thereof is optionally selected from naturally abundant O or isotopically enriched ¹⁸ O, an oxygen atom on a phosphate bond or a phosphorothioate bond refers to any oxygen atom attached to a phosphorus atom, specifically, may be an oxygen atom attached to a phosphorus atom by a single bond, may be an oxygen atom attached to a phosphorus atom by a double bond (e.g., p=o bond), and the isotopically enriched oxygen atoms may be one, two, three or four;

Or the five-position oxygen of the adjacent nucleotide on the right side is replaced by sulfur;

by way of limitation, at least one of the above x indicates that the hydrogen on the pentamethylene of its right-hand adjacent nucleotide is D (deuterium), or that the pentaoxygen of its right-hand adjacent nucleotide is isotopically enriched ¹⁸ The oxygen atom on the phosphoester or phosphorothioate bond of O, or the right-hand adjacent nucleotide thereof, being isotopically enriched ¹⁸ O, or the penta-oxygen of the adjacent nucleotide to the right thereof, is replaced by sulfur;

in some embodiments, when x represents that the penta-oxygen of its right adjacent nucleotide is replaced with sulfur, x adjacent y represents absence, i.e., the linkage of the nucleotides flanking y is a phosphate linkage.

The sense strand is linked to the phosphorus atom represented by formula (I) through an oxygen atom at the 3 'end or the 5' end.

The conjugate disclosed by the invention has good biological activity, and can effectively reduce the expression of PSCK9 protein, reduce the blood lipid level, or reduce the level of one or more of high density lipoprotein cholesterol (HDL-c), low density lipoprotein cholesterol (LDL-c), total Cholesterol (TC) and total Triglyceride (TG).

In some embodiments, the genetic component comprises:

from the nucleotide sequence 5' -cs ^x us ^x a ^x g ^x a ^x c ^x Cf ^x u ^x Gf ^x u ^x dT ^x u ^x u ^x g ^x c ^x u ^x u ^x u ^x u ^x g ^x u-3' sense strand and nucleotide sequence

5'-as ^x Cfs ^x a ^x Af ^x Af ^x Af ^x g ^x Cf ^x a ^x Af ^x a ^x Af ^x c ^x Af ^x g ^x Gf ^x u ^x Cf ^x u ^x a ^x gs ^x as ^x a-3'.

In some embodiments, the genome component comprises:

from the nucleotide sequence 5' -as ^DD An antisense strand consisting of CfsafAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 2); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^DD an antisense strand composed of aAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 4); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -ascfsaaafafafafafgfcfaafafafafafgfgcgffcfucffufuags ^DD an antisense strand consisting of asa-3' (SEQ ID NO: 6); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -ascfsafafafafafgfafafafafafafafafgfgfgfaffucffucffuagsas ^DD a-3' (SEQ ID NO: 8); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD Cfs ^DD an antisense strand consisting of aAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 10); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -ascfsaaafafafafafgfcfaafafafafafgfgcgffcfucffufuags ^DD as ^DD a-3' (SEQ ID NO: 12); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD CfsaAfAfAfgCfaAfaAfcAfgGfuCfuags ^DD an antisense strand consisting of asa-3' (SEQ ID NO: 14); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^DD aAfAfAfgCfaAfaAfcAfgGfuCfuags ^DD an antisense strand consisting of asa-3' (SEQ ID NO: 16); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD CfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsas ^DD a-3' (SEQ ID NO: 18); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^DD aAfAfAfgCfaAfaAfcAfgGfuCfuagsas ^DD a-3' (SEQ ID NO: 20); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD CfsaAfAfAfgCfaAfaAfcAfgGfuCfuags ^DD as ^DD a-3' (SEQ ID NO: 22); or alternatively, the first and second heat exchangers may be,

From the nucleotide sequence 5' -asCfs ^DD aAfAfAfgCfaAfaAfcAfgGfuCfuags ^DD as ^DD a-3' (SEQ ID NO: 24); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD Cfs ^DD aAfAfAfgCfaAfaAfcAfgGfuCfuagsas ^DD a-3' (SEQ ID NO: 26); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD Cfs ^DD aAfAfAfgCfaAfaAfcAfgGfuCfuags ^DD an antisense strand consisting of asa-3' (SEQ ID NO: 28); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD Cfs ^DD aAfAfAfgCfaAfaAfcAfgGfuCfuags ^DD as ^DD a-3' (SEQ ID NO: 30); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -ascfsaaafafafafafgfgcfaaf ^DD an antisense strand consisting of aAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 122); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD CfsaAfAfAfgCfaAf ^DD aAfcAfgGfuCfuagsas ^DD a-3' (SEQ ID NO: 124); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD Cfs ^DD aAfAfAfgCfaAf ^DD aAfcAfgGfuCfuagsas ^DD a-3' (SEQ ID NO: 126); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD CfsaAfAfAfgCfaAf ^DD aAfcAfgGfuCfuags ^DD as ^DD a-3' (SEQ ID NO: 128); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD Cfs ^DD aAfAfAfgCfaAf ^DD aAfcAfgGfuCfuags ^DD as ^DD a-3' (SEQ ID NO: 130); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -ascafsaafafafafafgfgcfaafa ^DD An antisense strand consisting of AfcAfgGfuCfuagsasa-3' (SEQ ID NO: 132); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD CfsaAfAfAfgCfaAfa ^DD AfcAfgGfuCfuagsas ^DD a-3' (SEQ ID NO: 134); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD Cfs ^DD aAfAfAfgCfaAfa ^DD AfcAfgGfuCfuagsas ^DD a-3' (SEQ ID NO: 136); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD CfsaAfAfAfgCfaAfa ^DD AfcAfgGfuCfuags ^DD as ^DD a-3' (SEQ ID NO: 138); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^DD Cfs ^DD aAfAfAfgCfaAfa ^DD AfcAfgGfuCfuags ^DD as ^DD a-3' (SEQ ID NO: 140); or alternatively, the first and second heat exchangers may be,

From the nucleotide sequence 5' -ascfsaaafafafafafgfgcfaaf ^DD a ^DD An antisense strand consisting of AfcAfgGfuCfuagsasa-3' (SEQ ID NO: 142); or (b)

From the nucleotide sequence 5' -ascafsaafafafafafgfgcfaafa ^DD AfcAfgGfuCfuagsas ^DD a-3' (SEQ ID NO: 144); or (b)

From the nucleotide sequence 5' -ascfsaaafafafafafgfgcfaaf ^DD aAfcAfgGfuCfuagsas ^DD a-3' (SEQ ID NO: 146); or (b)

From the nucleotide sequence 5' -ascafsaafafafafafgfgcfaafa ^DD AfcAfgGfuCfuags ^DD an antisense strand consisting of asa-3' (SEQ ID NO: 148); or (b)

From the nucleotide sequence 5' -ascfsaaafafafafafgfgcfaaf ^DD aAfcAfgGfuCfuags ^DD an antisense strand consisting of asa-3' (SEQ ID NO: 150); or (b)

From the nucleotide sequence 5' -ascafsaafafafafafgfgcfaafa ^DD AfcAfgGfuCfuags ^DD as ^DD a-3' (SEQ ID NO: 152); or (b)

From the nucleotide sequence 5' -ascfsaaafafafafafgfgcfaaf ^DD aAfcAfgGfuCfuags ^DD as ^DD a-3' (SEQ ID NO: 154) of the sequence set forth belowA chain; or (b)

From the nucleotide sequence 5' -as ^DD CfsaAfAfAfgCfaAfa ^DD An antisense strand consisting of AfcAfgGfuCfuagsasa-3' (SEQ ID NO: 156); or (b)

From the nucleotide sequence 5' -as ^DD CfsaAfAfAfgCfaAf ^DD an antisense strand consisting of aAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 158); or (b)

From the nucleotide sequence 5' -asCfs ^DD aAfAfAfgCfaAfa ^DD An antisense strand consisting of AfcAfgGfuCfuagsasa-3' (SEQ ID NO: 160); or (b)

From the nucleotide sequence 5' -asCfs ^DD aAfAfAfgCfaAf ^DD an antisense strand consisting of aAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 162); or (b)

From the nucleotide sequence 5' -as ^DD Cfs ^DD aAfAfAfgCfaAfa ^DD An antisense strand consisting of AfcAfgGfuCfuagsasa-3' (SEQ ID NO: 164); or (b)

From the nucleotide sequence 5' -as ^DD Cfs ^DD aAfAfAfgCfaAf ^DD an antisense strand consisting of aAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 166);

wherein the superscript DD indicates that the penta-methylene group of the adjacent nucleotide on the right side is a dideuteric methylene group, i.e., -CD ₂ -。

In some embodiments, the genome component comprises:

from the nucleotide sequence 5' -as ^oa An antisense strand consisting of CfsafAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 32); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^oa an antisense strand consisting of aAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 34); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -ascfsaaafafafafafgfcfaafafafafafgfgcgffcfucffufuags ^oa an antisense strand consisting of asa-3' (SEQ ID NO: 36); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -ascfsafafafafafgfafafafafafafafafgfgfgfaffucffucffuagsas ^oa a-3' (SEQ ID NO: 38); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^oa Cfs ^oa an antisense strand consisting of aAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 40); or alternatively, the first and second heat exchangers may be,

by the coreNucleotide sequence 5' -ascfsaaafafafafafgfgcafafafgfgfgcgfaffucfaags ^oa as ^oa a-3' (SEQ ID NO: 42); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^oa CfsaAfAfAfgCfaAfaAfcAfgGfuCfuags ^oa an antisense strand consisting of asa-3' (SEQ ID NO: 44); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^oa aAfAfAfgCfaAfaAfcAfgGfuCfuags ^oa an antisense strand consisting of asa-3' (SEQ ID NO: 46); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^oa CfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsas ^oa a-3' (SEQ ID NO: 48); or alternatively, the first and second heat exchangers may be,

From the nucleotide sequence 5' -asCfs ^oa aAfAfAfgCfaAfaAfcAfgGfuCfuagsas ^oa a-3' (SEQ ID NO: 50); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^oa CfsaAfAfAfgCfaAfaAfcAfgGfuCfuags ^oa as ^oa a-3' (SEQ ID NO: 52); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^oa aAfAfAfgCfaAfaAfcAfgGfuCfuags ^oa as ^oa a-3' (SEQ ID NO: 54); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^oa Cfs ^oa aAfAfAfgCfaAfaAfcAfgGfuCfuagsas ^oa a-3' (SEQ ID NO: 56); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^oa Cfs ^oa aAfAfAfgCfaAfaAfcAfgGfuCfuags ^oa an antisense strand consisting of asa-3' (SEQ ID NO: 58); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^oa Cfs ^oa aAfAfAfgCfaAfaAfcAfgGfuCfuags ^oa as ^oa a-3' (SEQ ID NO: 60);

wherein the superscript oa indicates that the five-position oxygen of the adjacent nucleotide on the right side is ¹⁸ O。

In some embodiments, the genome component comprises:

from the nucleotide sequence 5' -as ^ob An antisense strand consisting of CfsafAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 62); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^ob an antisense strand consisting of aAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 64); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -ascfsaaafafafafafgfcfaafafafafafgfgcgffcfucffufuags ^ob an antisense strand consisting of asa-3' (SEQ ID NO: 66); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -ascfsafafafafafgfafafafafafafafafgfgfgfaffucffucffuagsas ^ob a-3' (SEQ ID NO: 68); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^ob Cfs ^ob an antisense strand consisting of aAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 70); or alternatively, the first and second heat exchangers may be,

From the nucleotide sequence 5' -ascfsaaafafafafafgfcfaafafafafafgfgcgffcfucffufuags ^ob as ^ob a-3' (SEQ ID NO: 72); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^ob CfsaAfAfAfgCfaAfaAfcAfgGfuCfuags ^ob an antisense strand consisting of asa-3' (SEQ ID NO: 74); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^ob aAfAfAfgCfaAfaAfcAfgGfuCfuags ^ob an antisense strand consisting of asa-3' (SEQ ID NO: 76); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^ob CfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsas ^ob a-3' (SEQ ID NO: 78); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^ob aAfAfAfgCfaAfaAfcAfgGfuCfuagsas ^ob a-3' (SEQ ID NO: 80); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^ob CfsaAfAfAfgCfaAfaAfcAfgGfuCfuags ^ob as ^ob a-3' (SEQ ID NO: 82); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^ob aAfAfAfgCfaAfaAfcAfgGfuCfuags ^ob as ^ob a-3' (SEQ ID NO: 84) of the antisense strand; or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^ob Cfs ^ob aAfAfAfgCfaAfaAfcAfgGfuCfuagsas ^ob a-3' (SEQ ID NO: 86); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^ob Cfs ^ob aAfAfAfgCfaAfaAfcAfgGfuCfuags ^ob an antisense strand consisting of asa-3' (SEQ ID NO: 88); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^ob Cfs ^ob aAfAfAfgCfaAfaAfcAfgGfuCfuags ^ob as ^ob a-3' (SEQ ID NO: 90);

wherein the superscript ob denotes that the oxygen atom on the phosphoester bond or phosphorothioate bond of the adjacent nucleotide on the right side thereof is ¹⁸ O。

In some embodiments, the genome component comprises:

from the nucleotide sequence 5' -as ^os An antisense strand consisting of CfsafAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 92); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^os an antisense strand consisting of aAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 94); or alternatively, the first and second heat exchangers may be,

From the nucleotide sequence 5' -ascfsaaafafafafafgfcfaafafafafafgfgcgffcfucffufuags ^os an antisense strand consisting of asa-3' (SEQ ID NO: 96); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -ascfsafafafafafgfafafafafafafafafgfgfgfaffucffucffuagsas ^os a-3' (SEQ ID NO: 98); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^os Cfs ^os an antisense strand consisting of aAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 100); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -ascfsaaafafafafafgfcfaafafafafafgfgcgffcfucffufuags ^os as ^os a-3' (SEQ ID NO: 102); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^os CfsaAfAfAfgCfaAfaAfcAfgGfuCfuags ^os an antisense strand consisting of asa-3' (SEQ ID NO: 104); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^os aAfAfAfgCfaAfaAfcAfgGfuCfuags ^os an antisense strand consisting of asa-3' (SEQ ID NO: 106); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^os CfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsas ^os a-3' (SEQ ID NO: 108); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^os aAfAfAfgCfaAfaAfcAfgGfuCfuagsas ^os a-3'(SEQ ID NO110) an antisense strand; or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^os CfsaAfAfAfgCfaAfaAfcAfgGfuCfuags ^os as ^os a-3' (SEQ ID NO: 112); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^os aAfAfAfgCfaAfaAfcAfgGfuCfuags ^os as ^os a-3' (SEQ ID NO: 114); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^os Cfs ^os aAfAfAfgCfaAfaAfcAfgGfuCfuagsas ^os a-3' (SEQ ID NO: 116); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^os Cfs ^os aAfAfAfgCfaAfaAfcAfgGfuCfuags ^os an antisense strand consisting of asa-3' (SEQ ID NO: 118); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^os Cfs ^os aAfAfAfgCfaAfaAfcAfgGfuCfuags ^os as ^os a-3' (SEQ ID NO: 120); or (b)

From the nucleotide sequence 5' -a ^os An antisense strand consisting of CfsafAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 168); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCf ^os an antisense strand consisting of aAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 170); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -ascfsaaafafafafafgfcfaafafafafafgfgcgfaffucffufumag ^os an antisense strand consisting of asa-3' (SEQ ID NO: 172); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -ascfsafafafafafgfafafafafafafafgfgfgfafgcfucffuagagsa ^os a-3' (SEQ ID NO: 174); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -a ^os Cf ^os an antisense strand consisting of aAfAfgCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID NO: 176); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -ascfsaaafafafafafgfcfaafafafafafgfgcgfaffucffufumag ^os a ^os a-3' (SEQ ID NO: 178); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -a ^os CfsaAfAfAfgCfaAfaAfcAfgGfuCfuag ^os an antisense strand consisting of asa-3' (SEQ ID NO: 180); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCf ^os aAfAfAfgCfaAfaAfcAfgGfuCfuag ^os an antisense strand consisting of asa-3' (SEQ ID NO: 182); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -as ^os CfsaAfAfAfgCfaAfaAfcAfgGfuCfuagsa ^os a-3' (SEQ ID NO: 184); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCfs ^os aAfAfAfgCfaAfaAfcAfgGfuCfuagsa ^os a-3' (SEQ ID NO: 186) of the antisense strand; or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -a ^os CfsaAfAfAfgCfaAfaAfcAfgGfuCfuag ^os a ^os a-3' (SEQ ID NO: 188); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -asCf ^os aAfAfAfgCfaAfaAfcAfgGfuCfuag ^os a ^os a-3' (SEQ ID NO: 190); or alternatively, the first and second heat exchangers may be,

From the nucleotide sequence 5' -a ^os Cf ^os aAfAfAfgCfaAfaAfcAfgGfuCfuagsa ^os a-3' (SEQ ID NO: 192); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -a ^os Cf ^os aAfAfAfgCfaAfaAfcAfgGfuCfuag ^os an antisense strand consisting of asa-3' (SEQ ID NO: 194); or alternatively, the first and second heat exchangers may be,

from the nucleotide sequence 5' -a ^os Cf ^os aAfAfAfgCfaAfaAfcAfgGfuCfuag ^os a ^os a-3' (SEQ ID NO: 196);

wherein the superscript os indicates that the five O positions of the adjacent nucleotide on the right side thereof are replaced by sulfur.

In some embodiments, a is methylene and the conjugate may be a compound of formula (II):

in one aspect, the amino acid of formula (I) or formula (II) is selected from the group consisting of, but not limited to, citrulline, homocysteine, lysine, homolysine, asparagine, glutamine, arginine, glycine, methionine, phenylalanine, albiziamine, valine, and any combination thereof.

In another aspect, the invention discloses a conjugate of formula (III) and formula (IV):

wherein Y is independently selected from oxygen (O) and Nitrogen (NH). When Y is oxygen, the amino acid residue in the molecule is from citrulline; when Y is N, the amino acid residue in the molecule is from arginine.

In one aspect, the amino acid residues in the conjugate are derived from natural amino acids, unnatural amino acids, and any combination thereof.

In another aspect, the amino acid residues in the conjugate are derived from L-amino acids, D-amino acids, DL-amino acids, and any combination thereof.

Alternatively, the amino acid is an L-amino acid.

In some embodiments, the amino acid is citrulline. Alternatively, citrulline has the L-configuration.

In one aspect, the carrier group is a nitrogen-containing heterocycle-derived group.

Alternatively, the nitrogen-containing heterocycle may be a four-, five-, or six-membered ring.

Further, the carrier group L may be selected from one of the following groups:

wherein Z is selected from oxygen (O), sulfur (S), and Nitrogen (NH); r is R ³ And R is ⁴ Independently selected from hydrogen (H), hydroxy (-OH), OR-OR ⁵ Wherein R is ⁵ Is a substituent or protecting group on a hydroxy group, and R ⁵ Can be selected from aliphatic hydrocarbon groups, aromatic hydrocarbon groups, acyl groups, phosphono groups and the like.

In some embodiments, the carrier group contains a five-membered nitrogen containing heterocyclic structure.

In other embodiments, the nitrogen-containing five-membered heterocyclic carrier group is of the structure:

optionally, the nitrogen-containing five-membered heterocyclic carrier group has the following steric structure:

in some embodiments, the present disclosure discloses that the bioregand group contained in the conjugate contains a lipophilic body selected from the group consisting of cholesteryl, cholic acid, adamantaneacetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyloxyhexyl, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl, palmitic acid, myristic acid, O-3- (oleoyl) lithocholic acid, O-3- (oleoyl) cholanic acid, dimethoxy tribenzyl, and phenoxazine.

Further, the bio-ligand group contains a carbohydrate selected from the group consisting of allose, altrose, arabinose, cladinose, erythrose, fructose, D-fucose alcohol, L-fucose alcohol, fucose amine, fucose, fucoidan, galactosamine, D-galactosamine alcohol, N-acetyl-galactosamine (GalNAc), galactose, glucosamine, N-acetyl-glucosamine, glucitol, glucose-6-phosphate, guloglycol, L-glycerol-D-mannose-heptose, glycerol, glyceron, gulose, idose, lyxose, mannosamine, mannose-6-phosphate, allose, quinolone, quiniose, quini-furfuryl amine, murine Li Tangchun, rhamnose amine, murine, ribose, ribulose, heptulose, sorbose, tagatose, talose, tartaric acid, and xylulose. Specifically, the bio-ligand group is a ligand group containing N-acetyl-galactosamine (GalNAc).

In other embodiments, the presently disclosed conjugates specifically bind to specific receptors of a particular tissue, thereby achieving tissue-specific targeting. In some embodiments, the conjugates of the invention specifically target to hepatocyte surface receptors, thereby specifically targeting to liver tissue. In some embodiments, the conjugates of the invention specifically target asialoglycoprotein receptors (asialoglycoprotein receptors, ASGPR) on the surface of hepatocytes. In some embodiments, the biological ligand group is a ligand group containing N-acetyl-galactosamine (GalNAc).

Further, the bioregand group comprises a carbohydrate, specifically including the following structures;

specifically, the foregoing conjugates include, but are not limited to, examples of structures of compounds in table 1, in which the sense strand of all conjugates in table 1 are linked to the phosphorus atom through the 3' -terminal oxygen atom.

TABLE 1 examples of conjugates

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The conjugates or compounds coupled to conjugates of the present invention include, but are not limited to, optical isomers, racemic compounds and other mixtures thereof. All conjugates of the invention can also be considered as a single compound entity, i.e., all compounds of the invention include both the components used to couple the conjugate and the entity of the conjugate obtained after coupling the components. In these cases, a single enantiomer or diastereoisomer, i.e. an optically active configuration, may be obtained by asymmetric synthesis or chiral resolution. Resolution of the racemate may be accomplished, for example, by conventional means, such as recrystallization in the presence of a resolving agent, or using, for example, chiral High Pressure Liquid Chromatography (HPLC) column chromatography. In addition, some compounds containing carbon-carbon double bonds have Z-and E-configurations (or cis-and trans-configurations). When a compound described herein is tautomeric, the term "compound" (including conjugates or conjugates) includes all tautomeric forms of the compound. Such compounds also include crystals and chelates. Similarly, the term "salt" includes all tautomeric forms of the compounds and crystalline forms of the compounds.

In some embodiments, the presently disclosed conjugates have a genome that is a double-stranded oligonucleotide comprising a sense strand and an antisense strand, the nucleotide sequence of the conjugate being linked to a phosphorus atom via the 3 'or 5' end. The sense or antisense strand described herein is linked to the phosphorus atom by a 3 'or 5' terminus, i.e., the 3 'or 5' terminus of the sense or antisense strand is linked to the carrier group by a phosphoester linkage. In some embodiments, the double-stranded oligonucleotide is attached to the phosphorus atom through the 3' end of the sense strand. In some embodiments, the double-stranded oligonucleotide is attached to the phosphorus atom through the 5' end of the sense strand. In some embodiments, the double-stranded oligonucleotide is attached to the phosphorus atom through the 3' end of the antisense strand. In some embodiments, the double-stranded oligonucleotide is attached to the phosphorus atom through the 5' end of the antisense strand.

In another aspect, the double stranded oligonucleotide is an siRNA, each nucleotide in the siRNA being independently a modified or unmodified nucleotide.

Further, the target point corresponding to the siRNA may be: apoB, apoC, ANGPTL3, PCSK9, SCD1, FVII, p53, HBV, HCV. Specifically, the siRNA is PCSK9-siRNA.

Alternatively, the sequence of the siRNA includes any one of the sirnas in table 2.

TABLE 2 siRNA sequence listing in some embodiments

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Note that: s, sense strand; AS, antisense strand; wherein a, g, c and U are 2 '-O-methyl (2' -OMe) modified A, G, C and U nucleotides, respectively; C. g, U, A the cytidine-3 '-phosphate, guanosine-3' -phosphate, uridine-3 '-phosphate, and adenosine-3' -phosphate, respectively; af. Gf, cf and Uf are 2' -fluoro modified A, G, C and U nucleotides, respectively; dT is deoxythymine; s represents that the linkage between two adjacent nucleotides around the letter s is a phosphorothioate linkage; the superscript ob indicates that the phosphoester bond of the adjacent nucleotide on the right side is ¹⁸ O-phosphoester bonds, i.e. the oxygen atom in the phosphorus-oxygen bond (p=o) on the phosphoester bond is ¹⁸ O，s ^ob Representing the letter s ^ob The linkage between two adjacent nucleotides is thio- ¹⁸ O-phosphorothioate bonds, i.e. having oxygen atoms in phosphorothioate bonds ¹⁸ O; the superscript DD indicates that one nucleotide adjacent to the right side of the superscript DD is a 5' -dideuterogenic nucleotide, namely, the penta-methylene is dideuterogenic methylene; the superscript oa indicates that the adjacent nucleotide on the right side of the superscript oa is 5' -) ¹⁸ O-substituted nucleotides, i.e. having the five-position oxygen of the adjacent nucleotide to the right side thereof as ¹⁸ O，s ^oa Representing the letter combination s ^oa The linkage between two adjacent nucleotides is phosphorothioate bond and one adjacent nucleotide on the right side is 5' -) ¹⁸ O-substituted nucleotides; the superscript os indicates that the adjacent nucleotide on the right side of the superscript os is a 5' -sulfur-substituted nucleotide, i.e., a nucleotide in which the five-position O is replaced with sulfur, s ^os Representing the letter combination s ^os The linkage between two adjacent nucleotides on the left and right is a phosphorothioate linkage and one adjacent nucleotide on the right is a 5' -thio modified nucleotide. The nucleotide or phosphoester bond in which the above-mentioned moiety is modified or substituted can be referred to the structure shown in Table 3.

Table 3 partially modified or substituted nucleoside or phosphoester linkages

Above-mentioned

Further, the genomic component of the conjugate is PCSK9-siRNA. The conjugate can be used for preparing medicines for treating or preventing PSCK9 related diseases, disorders or conditions. PSCK 9-related diseases include atherosclerosis, hypercholesterolemia, hypertriglyceridemia, acute coronary syndrome, dyslipidemia, myocardial infarction, coronary artery disease, stroke, coronary artery disease, cardiovascular disease, diabetes, hyperlipidemia, type II diabetes, kidney disease.

The invention also provides a pharmaceutical composition comprising any of the conjugates described above, or pharmaceutically acceptable salts and esters thereof, and a pharmaceutically acceptable carrier, useful for the practical use of the conjugates of the invention in the prevention and/or treatment of a variety of corresponding diseases or conditions.

In some embodiments, the pharmaceutically acceptable carrier comprises a cream, emulsion, gel, liposome, or nanoparticle.

The pharmaceutical composition provided by the invention adopts the conjugate which is arbitrarily connected with the gene component and the biological ligand group, and can effectively improve the delivery efficiency of the gene component, thereby improving the treatment effect of the conjugate and the pharmaceutical composition thereof.

Drawings

Fig. 1: schematic representation of human PCSK9 protein levels: group I, blank group (saline group); group II, positive control group (Inclisiran group); conjugate 8.

Fig. 2: schematic representation of human PCSK9 protein levels: group I, blank group (saline group); group II, positive control group (Inclisiran group); conjugate 105.

Detailed Description

In order to provide a clear and consistent understanding of the terms used in the description of the present invention, some definitions are provided below. Furthermore, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The use of the word "a" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "an" but it is also known to the meaning of "one or more", "at least one" and "one or more". Similarly, the word "another" may mean at least a second or more.

The word "comprising" (and any form of comprising, such as "comprising" and "comprises"), "having" (and any form of having, "having", "including" and "containing") as used in this specification and claims is inclusive and open-ended and does not exclude additional unrecited elements or process steps. The terms "about" or "approximately" are used to indicate that the value includes errors in the instruments and methods used in determining the value.

The term "derivative" as used herein is understood to be another compound that is structurally similar and differs in some minor structures.

The term "conjugate" (sometimes also referred to as conjugate, coupler, sometimes also referred to as conjugate in the literature) as used herein corresponds to "conjugate" or "conjugates" in the english language. A conjugate refers to a novel compound formed by covalently linking (coupling) two or more molecules of a compound through a bivalent or multivalent compound molecule having a linking function. Conjugates can also be formed from two molecules directly coupled or condensed. A common anti-drug conjugate (ADC) is a conjugate, also known as an antibody drug conjugate. In the present invention, the product of the siRNA molecule coupled to the bio-ligand group via the linking group and the attachment of the bivalent compound is also a conjugate.

The term "bivalent compound" (bivalent compound) as used herein refers to an organic compound or organic molecule having two other compound groups or residues linked (also referred to as coupled) at two sites in the molecule that can be derivatized, respectively, to form a new compound or conjugate. For example, 1, 12-dodecanedioic acid is a typical bivalent compound, which can link or couple two other molecules with two carboxyl groups in the 1-and twelve positions, respectively, as amides or esters, to form a new compound, i.e., a conjugate. The two sites from which the bivalent compounds may be derived may be different or the same.

The term "coupling" as used herein refers to the chemical process by which two or more molecules of a compound undergo a reaction to form a new chemical bond and a new molecule. In certain contexts, "coupled" may be used interchangeably with "connected" or in lieu of "connected".

Isotopically enriched one is a process whereby one particular isotope is enriched (i.e., increased) and the corresponding other isotope is reduced or depleted by changing the relative abundance of its isotopes of a given element. The term "isotopically enriched" compound or derivative as used herein means that one or more specific isotopes in the compound are increased (i.e., one or more specific isotopic elements are enriched or augmented). Typically, in an isotopically enriched compound or derivative, the particular isotopic element at a particular position of the compound is enriched or augmented. However, it is understood that the compounds may have two or more isotopic elements enriched or augmented, including different isotopes of the same element and respective isotopes of different elements. Furthermore, isotopically enriched compounds can be isotopically enriched mixed forms, i.e., containing a plurality of specific isotopes or elements, or both.

Typically, deuterium (D or ² H) (stable isotope thereof with mass about twice that of hydrogen), nitrogen-15 # ¹⁵ N), C-13% ¹³ C) Oxygen-18% ¹⁸ O) and oxygen-17% ¹⁷ O) is 0.016%,0.37%,1.11%,0.204% and 0.037%, respectively. The "isotopically enriched" compounds or derivatives used in the present invention have isotopic levels higher than the natural abundance. The level of isotopic enrichment depends on the natural abundance of the particular isotope itself. In some embodiments, the compound or the isotopically enriched level of the element in the compound may be about 1 to about 100 molesPercent (%), e.g., about 2%, about 5%, about 17%, about 30%, about 51%, about 83%, about 90%, about 95%, about 96%, about 97%, about 98%, and greater than about 98%, about 99%, or 100%. In one embodiment, the isotopically enriched compounds of the present invention have an isotopic enrichment level of about 5% or greater, or greater than about 10%. In another embodiment, the isotopically enriched compounds of the present invention have an isotopic enrichment level of about 20% or greater, or greater than about 50%. In another embodiment, the isotopically enriched compounds of the present invention have an isotopic enrichment level of about 75% or greater, or about 90% or greater. In another embodiment, the isotopically enriched compounds of the present invention have an isotopic enrichment level of about 95% or more, or 100%. In another embodiment, the isotopically enriched compounds of the present invention are isotopically enriched at a level of about 98% to 100%. It is noted that the level of isotopic enrichment of a particular compound or of a particular element of a compound will depend on several properties of the compound, including chemistry, pharmacokinetics and therapeutic effect, for the purpose of improving the therapeutic efficacy, therapeutic biodistribution, bioavailability, metabolism, stability and/or pharmacokinetics of the compound, among others.

The term "natural abundance element" or "natural abundance element" as used herein refers to the element of atomic mass that is most abundant in nature. For example, the natural abundance element of hydrogen is ¹ The natural abundance elements of H and nitrogen are ¹⁴ N; the natural abundance of oxygen is ¹⁶ The natural abundance elements of O and carbon are ¹² C, etc. A "non-isotopically enriched" compound is a compound in which all atoms or elements in the compound are isotopes of natural abundance, i.e., the atomic mass of all atoms or elements is the most abundant in nature. Whereas isotopically enriched compounds refer to compounds in which one or more specific elements are enriched in the form of isotopes that are not naturally abundant. Non-isotopically enriched compounds are excluded from the compounds provided herein.

The term "genetic component" is a component comprising a modified or unmodified nucleotide sequence, and the genetic component may be a single-stranded nucleotide sequence or a paired double-stranded nucleotide sequence, the paired double-stranded nucleotide sequences may be completely paired or partially paired, and the two strands of the double-stranded nucleotide sequence may be aligned in whole or in part. In some embodiments, the genomic component is a polynucleide such as ribonucleic acid (RNA) or a derivative thereof. For example, and without limitation, the genomic component may be an RNA therapeutic molecule, such as a small interfering RNA (siRNA), RNA aptamer, or antisense RNA. In some embodiments, the genome is a modified or unmodified double-stranded RNA comprising a sense strand and an antisense strand, wherein the sense strand is coupled via an oxygen atom at the five position on the 3 'or 5' end, in particular, the oxygen atom is linked to a phosphorus atom represented by formula I. In some embodiments, the double stranded RNA is two at least partially paired oligonucleotide sequences, each oligonucleotide sequence having 10-30 modified or unmodified nucleotides.

In some embodiments, the genetic component of the conjugate is selected from the group consisting of SEQ ID NOs 1/2 shown in table 2, wherein the sense strand SEQ ID NO 1 is linked to the phosphorus atom shown in formula (I) through the 3' terminal oxygen atom; in some embodiments, the genetic component of the conjugate is selected from the group consisting of SEQ ID NOs 9/10 shown in table 2, wherein the sense strand SEQ ID NO 9 is linked to the phosphorus atom shown in formula (I) through the 3' terminal oxygen atom; in some embodiments, the genetic component of the conjugate is selected from the group consisting of SEQ ID NOs 121/122 shown in table 2, wherein the sense strand SEQ ID NO 121 is linked to the phosphorus atom shown in formula (I) through the 3' terminal oxygen atom; in some embodiments, the genetic component of the conjugate is selected from the group consisting of SEQ ID NO 131/132 shown in Table 2, wherein the sense strand SEQ ID NO 131 is linked to the phosphorus atom shown in formula (I) through an oxygen atom at the 3' end. All "/" of the present invention, when they occur between two gene sequence numbers, denote the relationship of "and", e.g., SEQ ID NO 1/2 denotes the double stranded nucleotides formed by the combination of SEQ ID NO 1 and SEQ ID NO 2.

The term "amino acid" generally refers to an organic compound that contains both carboxylic acid groups and amine groups. The term "amino acid" includes "natural" and "unnatural" amino acids. In addition, the term amino acid includes O-alkylated or N-alkylated amino acids, as well as amino acids having a side chain containing nitrogen, sulfur or oxygen (e.g., lys, cys or Ser), where the nitrogen, sulfur or oxygen atom may or may not be acylated or alkylated. The amino acid may be an L-amino acid, a D-amino acid, or a mixture of L-and D-amino acids, including but not limited to a racemic mixture.

The term "natural amino acid" and equivalent expression as used herein refers to the L-amino acid typically found in naturally occurring proteins. Examples of natural amino acids include, but are not limited to, citrulline (Citn), alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (gin), arginine (Arg), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), beta-alanine (beta-Ala), gamma-aminobutyric acid (GABA), and the like.

The term "unnatural amino acid" as used herein refers to any derivative of a natural amino acid, including D-amino acids and derivatives thereof, as well as alpha-and beta-amino acid derivatives. It should be noted that certain unnatural amino acids (e.g., hydroxyproline) in the present invention may occur in nature in certain biological tissues or in certain proteins. Amino acids having many different protecting groups suitable for direct use in solid phase peptide synthesis are commercially available. In addition to the twenty most common natural amino acids, the following examples of unnatural amino acids and amino acid derivatives (common abbreviations in brackets) may be used in accordance with the invention: 2-aminoadipic acid (Aad), 3-aminoadipic acid (β -Aad), 2-aminobutyric acid (2-Abu), α, β -dehydro-2-aminobutyric acid (8-AU), 1-aminocyclopropane-1-carboxylic Acid (ACPC), aminoisobutyric acid (Aib), 3-aminoisobutyric acid (β -Aib), 2-aminothiazoline-4-carboxylic acid, 5-aminopentanoic acid (5-Ava), 6-aminocaproic acid (6-Ahx), 2-aminoheptanoic acid (Ahe), 8-aminocaprylic acid (8-Aoc), 11-aminoundecanoic acid (11-Aun), 12-aminododecanoic acid (12-Ado), 2-aminobenzoic acid (2-Abz), 3-aminobenzoic acid (3-Abz), 4-aminobenzoic acid (4-Abz), 4-amino-3-hydroxy-6-methylheptanoic acid (Statine, stata), aminooxyacetic acid (Aoa), 2-aminotetralin-2-carboxylic Acid (ATC), 4-aminohexyl-5-aminopentanamic acid (2-Acp-Ala), 2-dihydroxyphenylalanine (2-Ala), p-bromophenylalanine (4-Br-Phe), o-chlorophenylalanine (2-Cl-Phe), m-chlorophenylalanine (3-Cl-Phe), p-chlorophenylalanine (3-Cl-Phe), m-chlorotyrosine (3-Cl-Tyr), p-benzoylphenylalanine (Bpa), t-butylglycine (TLG), cyclohexylalanine (Cha), cyclohexylglycine (Chg), desmin (Des), 2-diaminopimelic acid (Dpm), 2, 3-diaminopropionic acid (Dpr), 2, 4-diaminobutyric acid (Dbu), 3, 4-dichlorophenylalanine (3, 4-Cl 2-Phe), 3, 4-difluorophenylalanine (3, 4-F2-Phe), 3, 5-diiodotyrosine (3, 5-I2-Tyr), N-ethylglycine (EtG), N-ethylasparamide (Easn), o-fluorophenylalanine (2-F-Phe), m-fluorophenylalanine (3-F-Phe), p-fluorophenylalanine (OH), homolysine (Trp-5-hydroxy-Tyr), homolysine (OH) and homolysine (Trp-OH), 3-or 4-hydroxyproline (3-or 4-Hyp), p-iodophenylalanine-iso-tyrosine (3-I-Tyr), indoline-2-carboxylic acid (Idc), iso Ai Dumei (Ide), isoleucine (alpha-Ile), isopiperidinic acid (Inp), N-methylisoleucine (MeLys), m-methyltyrosine (3-Me-Tyr), N-methylvaline (MeVal), 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), p-nitrophenylalanine (4-NO 2-Phe), 3-nitrotyrosine (3-NO 2-Tyr), norleucine (Nle), norvaline (Nva), ornithine (Orn), orthophosphoric tyrosine (H2 PO 3-Tyr), octahydroindole-2-carboxylic acid (Penicillamine), pentafluorophenylalanine (F5-Phe), phenylglycine (Phg), piperidinic acid (Pip), propargyl glycine (PGa), pyroquinoline (PrLU), tetrahydroisoquinoline (Sar), tetrahydroisoquinoline (3-proline (Tic), thiazolidine-carboxylic acid (Tth-4-thiocarboxylic acid).

The term "side chain of an amino acid" as used herein refers to the side chains of the above-mentioned natural and unnatural amino acids.

The term "amino acid residue" as used herein refers to an incomplete amino acid, i.e., a structural fragment that remains after at least a portion of an amino acid molecule has been lost. For example, a polypeptide is formed by a plurality of amino acids linked to one another by peptide bonds; amino acids in a polypeptide chain are referred to as amino acid residues in the remaining structural portion, since some of their groups are involved in the formation of peptide bonds. Amino acid residues are not limited to peptide molecules, and are collectively referred to as amino acid residues when the amino acid participates in an incomplete amino acid moiety formed upon attachment to other molecules. Also, the term "oligopeptide residue" refers to an incomplete oligopeptide.

The term "carbohydrate" refers to a monosaccharide, disaccharide, trisaccharide or polysaccharide.

The term "monosaccharide" includes the group of allose, maltose, arabinose, cladinose, brown sugar, erythrose, fructose, D-fucose, L-fucose, fucose amine, fucose, galactosamine, D-galactosamine, N-acetyl-galactosamine, galactose, glucosamine, N-acetyl-glucosamine, glucitol, glucose-6-phosphate, glyceraldehyde, L-glycerol-D-mannose-heptose, glycerol, iodine, lyxose, mannosamine, mannose-6-phosphate, allose, isorhamnose, quini-furfuryl amine, murine Li Tangchun, rhamnose, ribose, ribulose, heptose, sorbose, tagatose, talose, tartaric acid, threose and xylose. The monosaccharides may be in the D-or L-configuration. The monosaccharides may also be deoxy sugars (alcoholic hydroxyl groups substituted with hydrogen), amino sugars (alcoholic hydroxyl groups substituted with amino groups), thio sugars (alcoholic hydroxyl groups substituted with thiol), or cyclic epoxy groups substituted with CS or with sulfur), seleno sugars, telluroses, aza sugars (cyclic carbon substituted with nitrogen), imino sugars (epoxy substituted with nitrogen), phospho sugars (epoxy substituted with phosphorus), phospho sugars (cyclic carbon substituted with phosphorus), C-substituted monosaccharides (hydrogen on non-terminal carbon atoms substituted with carbon), unsaturated monosaccharides, sugar alcohols (carbonyl groups substituted with CHOH groups), aldonic acids (aldehyde groups substituted with carboxyl groups), ketoaldonic acids, uronic acids, aldonic acids, and the like. The amino sugar includes amino monosaccharides, preferably galactosamine, glucosamine, mannosamine, fucosylamine, quinitol, neuraminic acid, muramic acid, lactosamine, acosamine, bacillus glycosylamine, daunorubimine, desugarized amine, fu Luo Shaming, carbamamide, canola amine, mannosamine, trehalose, mycolamine, peroxidase amine, pneumonamine, purine ribomine, rhodamine amine. It should be understood that monosaccharides and the like may be further substituted.

The terms "disaccharide", "trisaccharide" and "polysaccharide" include groups of abbe quinone saccharide, aclatose, glucosamine, pullulan, amylose, apiose, glucosamine, ascose, ascorbic acid, flavone sugar, cellobiose, cellotriose, cellulose, chalcotriose, thioether, chitin, collagen, cyclodextrin, melamine, dextrin, 2-deoxyribose, 2-deoxyglucose, diglucose, maltose, digital ketose, evalcose, evodia, fructo-oligosaccharide, galactooligosaccharide, gentian, gentiobiose, dextran, glycogen, hamametose, heparin, inulin, isoevotigenin, isomaltose, isomaltotriose, curcin, lactose, lactosamine, lactose diamine, layered arabinose, levoglucosan, mannooligosaccharide, mannite, disaccharide, muramic acid, neuraminic acid, blackose, nualamycin, sucrose, and so on. Furthermore, it should be understood that "disaccharides", "trisaccharides" and "polysaccharides" and the like may be further substituted. Disaccharides also include amino sugars and derivatives thereof, in particular mycoaminosugars derived at the C-4 'position or 4-deoxy-3-amino-glucose derived at the C-6' position.

"pharmaceutically acceptable salt" of a compound refers to a salt of a pharmaceutically acceptable compound. Salts of desirable compounds (basic, acidic or charged functional groups) may retain or improve the biological activity and properties of the parent compound as defined herein and are not biologically undesirable.

The term "ester" means an ester derived from the compounds of the general formulae herein, including physiologically hydrolyzable esters (compounds of the present invention that hydrolyze under physiological conditions to release the free acid or alcohol form). The compounds of the invention may themselves be esters.

The genome is coupled to the carrier group via a phosphate linkage. The carrier group is generally of a preferred cyclic structure. The cyclic structure may be a carbocyclic ring system, i.e. all ring atoms are carbon atoms, or a heterocyclic ring system, i.e. one or more ring atoms may be heteroatoms, such as nitrogen, oxygen, sulfur. The cyclic structure may be a single ring system or may comprise two or more rings, such as fused rings. The cyclic structure may be a fully saturated ring system or it may contain one or more double bonds.

In one embodiment, the carrier group of the genetic component is a nitrogen-containing heterocycle, preferably a four-, five-or six-membered ring, having at least one substituent containing a reactive functional group thereon, the nitrogen heterocycle being coupled to the genetic component via the reactive functional group on the substituent.

In some embodiments, the genetic component is a functional oligonucleotide, optionally, the functional oligonucleotide is selected from one of a small interfering RNA, a microrna, an anti-microrna, a microrna antagonist, a microrna mimetic, a decoy oligonucleotide, an immunostimulatory substance, a G-quadrapole, an alternative splice, a single stranded RNA, an antisense nucleic acid, a nucleic acid aptamer, a stem loop RNA, an mRNA fragment, an activating RNA, or DNA. In some embodiments, the functional oligonucleotide is a single stranded oligonucleotide, the carrier is attached to the end of the single stranded oligonucleotide, the end of the single stranded oligonucleotide refers to the first 4 nucleotides from one end of the single stranded oligonucleotide; alternatively, the vector is ligated to the end of a single stranded oligonucleotide; alternatively, the bivalent compound is attached to the 3 'end or the 5' end of the single stranded oligonucleotide.

In some embodiments, the functional oligonucleotide is a double-stranded oligonucleotide comprising a sense strand and an antisense strand, and the vector is attached to the ends of the double-stranded oligonucleotide. In some embodiments, the double stranded oligonucleotide is an siRNA, each nucleotide in the siRNA being independently a modified or unmodified nucleotide.

In some embodiments, the conjugate comprises a sense strand directly attached to the conjugate and an antisense strand complementary to the sense strand, the conjugate producing a therapeutic effect upon entry of the active molecule as a whole into the body.

Various hydroxyl protecting groups may be used in the present invention. In general, the protecting group renders the chemical functional group insensitive to specific reaction conditions and can be added and removed from the functional group in the molecule without substantially damaging the remainder of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al, tetrahedron 1992,48,2223-2311, and Greeneand Wuts, protective Groups in Organic Synthesis, chapter 2,2d ed,John Wiley&Sons,New York,1991, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under alkaline conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used in the present invention include Dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), and 9- (p-methoxyphenyl) xanthen-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include trityl (Tr), 4-methoxytrityl (MMTr), 4 '-dimethoxytrityl (DMTr), and 4,4',4 "-trimethoxytrityl (TMTr).

In some embodiments, the bioregand groups of the invention bind to a cell surface receptor. For this purpose, any cell surface receptor or biomarker or portion thereof is considered suitable. In some embodiments, the bio-ligand groups of the invention specifically bind to specific receptors of a particular tissue, thereby achieving tissue-specific targeting. In some embodiments, the bio-ligand groups of the invention specifically target to hepatocyte surface receptors, thereby specifically targeting to liver tissue. In some embodiments, the bio-ligand groups of the invention specifically target cell surface receptors specific for hepatocytes. In some embodiments, the bioregand group of the invention comprises GalNAc, an asialoglycoprotein receptor (asialoglycoprotein receptors, ASGPR) that specifically targets the surface of hepatocytes.

In some embodiments, the oligonucleotide conjugates of the invention have excellent liver targeting specificity, and are capable of efficiently delivering the conjugated functional oligonucleotides to the liver, thereby effectively modulating the expression of specific genes within hepatocytes. In some embodiments, the oligonucleotide conjugates of the invention have excellent biological activity, thereby effectively treating or preventing diseases associated with the targeted site. The oligonucleotide conjugate of the invention has wide application prospect.

In some embodiments, the target of the siRNA is selected from ApoB, apoC, ANGPTL3, PCSK9, SCD1, FVII, p53, HBV, and HCV.

In some embodiments, the genomic component is a double stranded siRNA against a target gene. In one embodiment, the target gene is PCSK9. In some embodiments, the genomic component is an siRNA targeting the PCSK9 gene.

In some embodiments, the subject has a disorder mediated by PCSK9 expression or a disease or disorder associated with PCSK9.

In some embodiments, there is provided a method of treating a subject suffering from a disease mediated by PCSK9 expression comprising administering to the subject a therapeutically effective amount of a compound described herein or a pharmaceutical composition thereof, such that the subject is treated. In some embodiments, the subject has hypercholesterolemia, dyslipidemia, or hyperlipidemia. In some embodiments, the subject's serum cholesterol level is reduced following administration of a compound or composition described herein.

The invention also provides a pharmaceutical composition comprising any of the conjugates described above, or pharmaceutically acceptable salts and esters thereof, and a pharmaceutically acceptable carrier, and the active molecule thereof is PSCK9-siRNA. In some embodiments, the above pharmaceutical compositions are useful in the preparation of a medicament for treating or preventing a PSCK 9-related disease, disorder or condition. Specifically, PSCK 9-related diseases include atherosclerosis, hypercholesterolemia, acute coronary syndrome, dyslipidemia, myocardial infarction, coronary artery disease, stroke, coronary artery disease, cardiovascular disease, diabetes, hyperlipidemia, type II diabetes, kidney disease, and the like.

The invention provides a method of inhibiting expression of a target gene in a cell, the method comprising contacting the cell with a conjugate or pharmaceutical composition thereof disclosed herein, and allowing the cell to stand for a time sufficient to obtain degradation of an mRNA transcript of the target gene, thereby inhibiting expression of the target gene in the cell. In some embodiments, expression of the gene of interest is inhibited by at least 30%, at least about 40%, at least about 50%, at least about 60%, or at least 70%.

The invention also provides a method for treating and preventing high cholesterol. In some embodiments, the methods further comprise the step of determining serum cholesterol levels in the subject. Serum cholesterol levels may be determined before, during and/or after such administration.

In some embodiments of the methods of treatment and prophylaxis provided herein, the subject is a mammal, such as a primate, rodent or human.

In some embodiments of the therapeutic and prophylactic methods provided herein, the conjugates are administered at a dose of about 0.01mg/kg to about 10mg/kg, about 0.5mg/kg to about 50mg/kg, or about 10mg/kg to about 30 mg/kg.

In some embodiments of the therapeutic and prophylactic methods provided herein, administration of the conjugates or pharmaceutical compositions of the invention results in a reduction in serum cholesterol in a subject.

In some embodiments of the therapeutic and prophylactic methods provided herein, the compounds or compositions are administered parenterally (e.g., subcutaneously, intramuscularly, or intravenously) in the form of injection or infusion solutions. In one embodiment, the compound or composition is administered subcutaneously.

It is clear to a person skilled in the art that the method of preparing the corresponding modified nucleoside monomer and the method of introducing the modified nucleotide group into the siRNA described in the present invention can be introduced by using the corresponding modified nucleoside monomer are also well known to a person skilled in the art. All modified nucleoside monomers are commercially available or can be prepared using known methods.

The carrier groups of certain of the genome components provided by the present invention are some of the carrier groups derived from nitrogen heterocycles. It will be appreciated that the carrier groups are not limited to nitrogen heterocycle containing groups, but include a variety of carrier groups that can be used as carriers for the genome component.

In some embodiments, the invention employs a phosphono group to link the genome to the carrier moiety. It will be appreciated that the manner of attachment or coupling of such bivalent compounds to the genome component is varied and includes, but is not limited to, direct coupling and indirect coupling, and that indirect coupling may be via a variety of carrier groups and attachment means suitable for coupling.

Examples

The invention will be more readily understood by reference to the following examples, which are provided to illustrate the invention and should not be construed to limit the scope of the invention in any way.

Unless defined otherwise or the context clearly indicates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. Materials and instruments used in the present invention are conventionally commercially available unless otherwise indicated.

Preparation example: synthesis of conjugates

Conjugates were prepared according to the following reaction scheme.

Step 1, preparation of intermediate M1

(S) -2- (((benzyloxy) carbonyl) amino) -5-carbamidinvalproic acid (0.8 g,2.59mmol,1 eq.) EDCI (991.60 mg,5.17mmol,2 eq.) N, N-diisopropylethylamine (1.34 g,10.35mmol,1.80mL,4 eq.) HOBt (349.47 mg,2.59mmol,1 eq.) was mixed in 20mL dichloromethane and S2A (1.09 g,2.60mmol,1 eq.) was added. The resulting mixture was stirred under nitrogen at 25℃for 16h. LC-MS monitors new product formation. After the reaction mixture was diluted with 20mL of methylene chloride, 20mL of water was added thereto. Standing and layering to obtain an organic phase. The organic phase was dried over anhydrous sodium sulfate and the solvent was dried to give the crude product. The crude product was chromatographed on silica gel to give M1 (1.2 g, yield 65.0%).

Step 2, preparation of intermediate M2

M1 (1.2 g,1.69mmol,1 eq.) was dissolved in 10mL of methanol and Pd/C (102.52 mg) was added. The mixture obtained is at H ₂ Stirring was carried out at 25℃for 16h under an atmosphere. LC-MS monitors target product formation. The reaction mixture was filtered to obtain a solution. After spin-drying the solvent, M2 (860 mg) was obtained and used directly in the next reaction.

Step 3, preparation of intermediate M3

Monomethyl azelate (301.61 mg,1.49mmol,1 eq.) HBTU (1.13 g,2.98mmol,2 eq.) and N, N-diisopropylethylamine (770.94 mg,5.97mmol,1.04mL,4 eq.) were mixed in 10mL dichloromethane and M2 (862mg, 1.49mmol,1 eq.) was added. The resulting mixture was stirred under nitrogen at 25℃for 16h. LC-MS monitors target product formation. After the reaction mixture was diluted with 20mL of methylene chloride, 20mL of water was added thereto. Standing and layering to obtain an organic phase. The organic phase was dried over anhydrous sodium sulfate and the solvent was dried by spin-drying to give the crude product. The crude product was chromatographed on silica gel (methanol/dichloromethane=0-6%) to give M3 (680 mg, 59.9% yield).

Step 4, preparation of intermediate M4

M3 (600 mg, 788.53. Mu. Mol,1 eq.) was dissolved in 10mL of methanol and 10mL of water, followed by addition of lithium hydroxide (188.85 mg,7.89mmol,10 eq.). The resulting mixture was stirred at 80℃for 3h. LC-MS monitors target product formation. The solvent in the reaction mixture was dried to give M4 (590 mg) which was used directly in the next reaction.

Step 5, preparation of intermediate M5

M4 (201.43 mg, 267.57. Mu. Mol,1.5 eq) was dissolved in 5mL DMF, and HOBT (1 eq.) was added, EDCI (2 eq.) and DIPEA (2 eq.). After stirring for 0.5h, M13 (320 mg, 178.38. Mu. Mol,1 eq.) was added. The resulting mixture was stirred under nitrogen at 25℃for 16h. LC-MS monitors target product formation. After 15mL of water was added to the reaction system, the mixture was extracted with dichloromethane (15 mL. Times.3). The organic phases are combined and the solvent is dried by spin to obtain the crude product. The crude product was separated by column chromatography on silica gel (dichloromethane (0.1% triethylamine)/methanol=90:10, 80:20, 70:30) to give M5 (230 mg).

Step 6, preparing intermediate M6

M5%230mg, 91.17. Mu. Mol,1 eq.) in 10mL of dichloromethane, DMAP (22.28 mg, 182.34. Mu. Mol,2 eq.) and triethylamine (415.14 mg,4.10mmol, 570.25. Mu.L, 45 eq.) were added. After cooling in an ice bath, succinic anhydride (136.85 mg,1.37mmol,15 eq.) was added to the reaction system. The resulting mixture was gradually warmed to 25℃and stirred under this condition for 16h. LC-MS monitors target product formation. After the reaction mixture was concentrated, the crude product was separated by HPLC (carbon-18 column, acetonitrile/0.01% ammonia) to give M6 (33.8 mg). HPLC purity: 99.83%. LCMS (ESI): cal.for C124H184N14O47:2622.89, found [ M-H ] ] ^- :2621.7

Step 7, preparation of intermediate M7

(I) M6 (33.8 mg, 12.89. Mu. Mol,1 eq.) and HBTU (9.77 mg, 25.77. Mu. Mol,2 eq.) were dissolved in 5mL acetonitrile and N, N-diisopropylethylamine (6.66 mg, 51.55. Mu. Mol, 8.98. Mu.L, 4 eq.) was added. After shaking for 3-4min, CPG-amino resin (386.7 mg,50 umol/g) was added. The resulting mixture was shaken on a shaker at room temperature for 48h. The reaction mixture was filtered and the filter cake was washed twice with acetonitrile (25 mL). The resulting solid was dried at 35℃for 2h to give a white solid (382.6 mg).

(II) the white solid obtained above was mixed with pyridine (4.54 mg, 57.39. Mu. Mol, 4.62. Mu.L, 3eq. Relative to acetic anhydride) in 10mL of acetonitrile, and acetic anhydride (1.95 mg, 19.13. Mu. Mol, 1.81. Mu.L, 1eq. Relative to free amino groups on CPG) was added under ice bath conditions. The resulting mixture was shaken on a shaker at room temperature for 0.5h. The mixture was filtered and the filter cake was washed twice with acetonitrile (10 mL). The resulting solid was dried at 40℃for 2h to give M7 (261.6 mg) as a white solid. The loading was 20.5. Mu. Mol/g.

Step 8, preparing the conjugate

The RNA sequences used in this application were all synthesized by Suzhou Bei Xin Biotechnology Co.

R ² SEQ ID NO 1/2 is selected, and the solid phase supported product M7 is subjected to solid phase synthesis and deprotection (see M.J.Damha, K.K.Ogilvie, methods mol. Biol.1993,20,81-114. The same applies below) to obtain the conjugate 2.

R ² SEQ ID NO 7/8 is selected, and the solid phase loaded product M7 is subjected to solid phase synthesis and deprotection to obtain the conjugate 8.

R ² SEQ ID NO 9/10 is selected, and the solid phase loaded product M7 is subjected to solid phase synthesis and deprotection to obtain the conjugate 9.

R ² SEQ ID NO 121/122 is selected, and the solid phase loaded product M7 is subjected to solid phase synthesis and deprotection to obtain the conjugate 91.

R ² SEQ ID NO 131/132 is selected, and the solid phase loaded product M7 is subjected to solid phase synthesis and deprotection to obtain the conjugate 96.

R ² SEQ ID NO 145/146 is selected, and the solid phase loaded product M7 is subjected to solid phase synthesis and deprotection to obtain the conjugate 105.

Positive control

The positive control adopts Incisiiran, and the synthesis steps refer to CN104854242B, and the specific structure is as follows:

biological assays

1. Free uptake of cynomolgus monkey primary hepatocytes

Cynomolgus monkey primary hepatocytes (cryopreservation) were obtained from Miaotong (Shanghai) Biotechnology Co., ltd.) and incubated at 37℃and 5% CO ₂ The culture is carried out in a resuscitating culture medium in a humidifying incubator under atmosphere. After resuscitation, hepatocytes were treated at 5x 10 ⁵ Cell/well density was seeded into 96-well plates coated with coating medium. After 24 hours of adherence, the supernatant was aspirated, siRNA (starting at 500nM, 10-fold dilution, 2 times total) was added and the culture was performed with the maintenance medium.

After a co-culture of 72 hours, primary hepatocytes were lysed and Dynabeads were used according to the protocol ^TM mRNA Purification Kit mRNA was extracted, cDNA was obtained by reverse transcription and PCSK9 and GAPDH mRNA levels were detected using the SYBR Green method. Normalized PCSK9/GAPDH ratios were used as a plot of relative levels of PCSK9 mRNA.

Detection of PCSK9 knock-down level in PCSK9 humanized mice

PCSK9 humanized mice were divided into four groups, group A subcutaneous injection of conjugate 8 diluted with normal saline at the designed dose (1 mg/kg); group B subcutaneously injected conjugate 105 diluted with normal saline at the designed dose (1 mg/kg); group I is blank control group, and the same volume of physiological saline is injected subcutaneously; group II is a positive control group, and the selected positive control drug, incisiiran, was diluted with physiological saline by subcutaneous injection at a designed dose (1 mg/kg). Blood was collected by orbital procedures at corresponding time points in 100 μl, and blood plasma was obtained by centrifugation after anticoagulation with EDTA and frozen at-80 ℃. After the experiment is finished, the protein level and the blood lipid level of PCSK9 in serum of each group of mice are detected by using an ELISA method or a biochemical analyzer.

Human PCSK9 ELISA method detection

Human PCSK9 protein levels were tested (R & D) according to the protocol provided by the supplier. After the sample was sufficiently dissolved, diluted 10-fold with PBS, added to the ELISA plate coated with the capture antibody, incubated at room temperature for 2 hours, washed and added with biotinylated detection antibody and SA-HRP mixture, and incubated at room temperature for 1 hour. After the washing was completed, color development was performed using TMB, and light absorption at 450nm was detected using an m5e multifunctional microplate reader. Standard curves were fitted with four parameters and used for sample human PCSK9 protein concentration conversion.

The results of the test for conjugate 8 and conjugate 105 are shown in figures 1 and 2.

Blood lipid level detection

Animal blood samples were collected on days 3, 7, 14, 21, 28, 35, 42, and 49, respectively, serum samples were sufficiently dissolved, diluted with an equal volume of physiological saline, and high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), total Cholesterol (TC), and total Triglyceride (TG) levels were measured using a corresponding assay kit, and Chemray 800 was produced for Shenzhen Leidum life technologies using a fully automatic biochemical analyzer. All assays were performed by Shanghai Biyun biotechnology Co.

Although the present invention has been described in detail with reference to the embodiments thereof, these embodiments are provided for the purpose of illustration and not limitation of the invention. Other embodiments that can be obtained according to the principles of the present invention fall within the scope of the invention as defined in the claims.

Claims

1. A conjugate, or a pharmaceutically acceptable salt or ester thereof, having the structure of formula (I):

wherein,

l is a carrier group;

a is selected from one of the following groups:

or methylene (CH) ₂ )，

When A is methylene, m and m' are not both 0;

R ¹ a bio-ligand group that is a cellular receptor;

R ² as a genome component, the genome component comprising:

wherein a, g, c and U are 2' -O-methyl modified A, G, C and U nucleotides, respectively; af. Gf, cf and Uf are 2' -fluoro modified A, G, C and U nucleotides, respectively; dT is deoxythymine; and, any y independently represents s or is absent, wherein s is a phosphorothioate linkage;

any x independently represents H or D (deuterium) in which the hydrogen on the pentamethylene of the right-hand adjacent nucleotide is naturally abundant, or O or isotopically enriched in which the pentaoxygen of the right-hand adjacent nucleotide is naturally abundant ¹⁸ O, or the oxygen atom on the phosphoester bond or phosphorothioate bond of the adjacent nucleotide to the right thereof, is optionally selected from naturally abundant O or isotopically enriched ¹⁸ O, or the penta-oxygen of its right-adjacent nucleotide, is replaced by sulfur, and at least one of said x's represents that the hydrogen on the penta-methylene of its right-adjacent nucleotide is D (deuterium), or that the penta-oxygen of its right-adjacent nucleotide is isotopically enriched ¹⁸ The oxygen atom on the phosphoester or phosphorothioate bond of O, or the right-hand adjacent nucleotide thereof, being isotopically enriched ¹⁸ O, or the penta-oxygen of the adjacent nucleotide to the right thereof, is replaced by sulfur;

the sense strand is linked to a phosphorus atom represented by formula (I) through an oxygen atom at the 3 'end or the 5' end.

2. The conjugate of claim 1, wherein the genetic component comprises: from the nucleotide sequence 5' -cs ^x us ^x a ^x g ^x a ^x c ^x Cf ^x u ^x Gf ^x u ^x dT ^x u ^x u ^x g ^x c ^x u ^x u ^x u ^x u ^x g ^x u-3 'sense strand and nucleotide sequence 5' -as ^x Cfs ^x a ^x Af ^x Af ^x Af ^x g ^x Cf ^x a ^x Af ^x a ^x Af ^x c ^x Af ^x g ^x Gf ^x u ^x Cf ^x u ^x a ^x gs ^x as ^x a-3'.

3. The conjugate of claim 1, wherein the genetic component comprises:

from the nucleotide sequence 5' -ascafsaafafafafafgfgcfaafa ^DD AfcAfgGfuCfuagsasa-3'(SEQ IDNO: 132) an antisense strand; or alternatively, the first and second heat exchangers may be,

From the nucleotide sequence 5' -ascfsaaafafafafafgfgcfaaf ^DD aAfcAfgGfuCfuags ^DD as ^DD a-3' (SEQ ID NO: 154); or (b)

From the nucleotide sequence 5' -as ^DD CfsaAfAfAfgCfaAfa ^DD AfcAfgGfuCfAn antisense strand consisting of uagsa-3' (SEQ ID NO: 156); or (b)

wherein the superscript DD indicates that the pentamethylene group of the adjacent nucleotide on the right side is a dideuteric methylene group.

4. The conjugate of claim 1, wherein the genetic component comprises:

from the nucleotide sequence 5' -ascfsaaafafafafafgfcfaafafafafafgfgcgffcfucffufuags ^oa as ^oa a-3'(SEQ ID NO.42) an antisense strand; or alternatively, the first and second heat exchangers may be,

5. The conjugate of claim 1, wherein the genetic component comprises:

from the nucleotide sequence 5' -as ^ob Cfs ^ob aAfAfAfgCfaAfaAfcAfgGfuCfuags ^ob antisense strand composed of asa-3' (SEQ ID NO: 88)The method comprises the steps of carrying out a first treatment on the surface of the Or alternatively, the first and second heat exchangers may be,

6. The conjugate of claim 1, wherein the genetic component comprises:

from the nucleotide sequence 5' -asCfs ^os aAfAfAfgCfaAfaAfcAfgGfuCfuagsas ^os a-3'(SEQ ID NO:110 An antisense strand consisting of; or alternatively, the first and second heat exchangers may be,

from nucleotide sequencesColumn 5' -asCf ^os aAfAfAfgCfaAfaAfcAfgGfuCfuag ^os an antisense strand consisting of asa-3' (SEQ ID NO: 182); or alternatively, the first and second heat exchangers may be,

wherein, the superscript os indicates that the five-position O of the adjacent nucleotide on the right side is thio-substituted.

7. The conjugate of claim 1, wherein the natural or unnatural amino acid is selected from the group consisting of citrulline, homocysteine, lysine, homolysine, asparagine, glutamine, arginine, glycine, methionine, phenylalanine, albiziamine, valine, and combinations thereof.

8. The conjugate according to claim 1, wherein the carrier group L is a nitrogen-containing heterocycle, preferably a quaternary nitrogen-containing heterocycle, a five-membered nitrogen-containing heterocycle or a six-membered nitrogen-containing heterocycle.

9. The conjugate of claim 7, wherein the carrier group L is selected from:

wherein Z is selected from oxygen (O), sulfur (S), and Nitrogen (NH);

R ³ and R is ⁴ Independently selected from hydrogen (H), hydroxy (-OH), OR-OR ⁵ Wherein R is ⁵ Is a substituent or protecting group on a hydroxy group, and R ⁵ Selected from aliphatic hydrocarbon groups, aromatic hydrocarbon groups, acyl groups and phosphono groups.

10. The conjugate of any one of claims 1 to 9, wherein the cellular receptor is an asialoglycoprotein cellular receptor (ASGPR).

11. The conjugate according to any one of claims 1 to 9, wherein the bio-ligand group contains a lipophilic body selected from the group consisting of cholesterol, cholic acid, adamantaneacetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyloxyhexyl, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl, palmitic acid, myristic acid, O-3- (oleoyl) lithocholic acid, O-3- (oleoyl) cholanic acid, dimethoxytribenzyl and phenoxazine.

12. The conjugate of any one of claims 1 to 9, wherein the biological ligand group contains a carbohydrate selected from allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fucose, L-fucose, fucose amine, fucose, fuco-fucose, galactosamine, D-galactosamine, N-acetyl-galactosamine (GalNAc), galactose, glucosamine, N-acetyl-glucosamine, glucitol, glucose-6-phosphate, guloglycol, L-glycerol-D-mannose-heptose, glycerol, glyceron, gulose, idose, threose, mannosamine, mannose-6-phosphate, psicose, quiniose, quinionamine, murine Li Tangchun, rhamnose amine, rhamnose, rhodiola, ribose, ketose, xylose, threose, ketose, tartaric acid, and tartaric acid; preferably, wherein the bio-ligand group is a ligand group containing N-acetyl-galactosamine (GalNAc).

13. The conjugate according to any one of claims 1 to 9, wherein said R ¹ Is one of the following structures:

14. The conjugate of any one of claims 1 to 13, wherein the sense strand is linked to the phosphorus atom of formula (I) by an oxygen atom at the 3' end.

15. The conjugate according to any one of claims 1 to 14, wherein the conjugate is selected from the group consisting of:

/>

or pharmaceutically acceptable salts and esters thereof, the sense strand of the conjugate being linked to the phosphorus atom of formula (I) by an oxygen atom at the 3' end.

16. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 15, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier.

17. Use of a conjugate according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 16 in the manufacture of a medicament for the treatment or prophylaxis of a PCSK 9-related disease, disorder or condition.

18. The use according to claim 17, wherein the PCSK 9-related disease comprises atherosclerosis, high gall

Sterolaemia, hypercholesteraemia, acute coronary syndrome, dyslipidaemia, myocardial infarction, coronary lesions, stroke,

coronary artery disease, cardiovascular disease, diabetes, hyperlipidemia, type II diabetes, and kidney disease.