WO2023208023A1

WO2023208023A1 - Deuterated chemical modification and oligonucleotide including same

Info

Publication number: WO2023208023A1
Application number: PCT/CN2023/090779
Authority: WO
Inventors: 王艳辉; 李云飞; 林晓燕; 张瑱; 董玉琼; 刘敏; 张建羽; 茅松
Original assignee: 上海拓界生物医药科技有限公司
Priority date: 2022-04-26
Filing date: 2023-04-26
Publication date: 2023-11-02
Also published as: TW202400790A

Abstract

The present disclosure relates to a deuterated chemical modification and an oligonucleotide including same. Specifically, the present disclosure relates to an oligonucleotide including a chemical modification represented by formula (I) or a tautomer modification thereof, wherein the functional groups are as defined in the present disclosure.

Description

Deuterated chemical modifications and oligonucleotides containing the same

This disclosure claims priority from Chinese patent application 202210448666.8 with a filing date of April 26, 2022. This disclosure cites the full text of the above-mentioned Chinese patent application.

Technical field

The present disclosure relates to deuterated chemically modified oligonucleotides, which have improved stability and can be targeted and delivered into cells to exert RNA interference effects. The present disclosure also relates to preparation methods and applications of oligonucleotides.

Background technique

RNA interference (RNAi) is an effective way to silence gene expression. According to statistics, among the disease-related proteins in the human body, more than 80% of them cannot be targeted by current conventional small molecule drugs and biological macromolecule preparations, and are undruggable proteins. Using RNA interference technology, appropriate siRNA can be designed based on the mRNA encoding these proteins, specifically targeting the target mRNA and degrading the target mRNA, thereby achieving the purpose of inhibiting the production of related proteins. Therefore, siRNA has very important drug development prospects.

However, unmodified small nucleic acids are unstable in the body (plasma, lysosomes, etc.) and are easily degraded (endo- and exonucleases, etc.). At the same time, the pharmacokinetics are poor, and it is easily filtered by the glomerulus and is quickly excreted in the urine after administration. Therefore, siRNA requires specific chemical modifications to improve its nuclease resistance, improve its in vivo stability, improve tissue distribution, and improve its pharmacokinetic properties to achieve drug treatment effects on the corresponding diseases.

CN110072530A discloses a class of 4'-phosphate analogues for regulating the properties of target genes in cells. However, nucleases and other substances that may act on nucleic acids will reduce the effect of siRNA modified with 4'-phosphate analogues in vivo. Therefore, further chemical modification is needed to improve its stability in vivo and increase the ability of cells to uptake RNA. , enhance the activity of the antisense strand on the target, thereby achieving a long-term inhibitory effect on the target mRNA. By deuterating the 5'-terminal nucleotide of the antisense chain of siRNA, the present disclosure can further enhance the activity of the antisense chain against the target, improve the stability of siRNA against metabolic enzymes such as nucleases, and further improve its in vivo stability. , thereby achieving a more efficient and longer-lasting inhibitory effect on the target mRNA and its corresponding protein; this also further reduces the possible toxicity caused by siRNA targeting mRNA, reduces the frequency of patient administration, and further improves the drug properties of siRNA.

Lipoprotein (a) (Lp(a)), first discovered by Norwegian geneticist Berg in 1963, was identified as a unique lipoprotein (Berg K.A new serum type system in man-the Lp system. Acta Pathol Microbiol Scand 1963; 59: 369–82.). Lp(a) consists of two parts: lipid and protein: the lipid part is mainly LDL-like particles located in the core; the protein part is located in the periphery and consists of apolipoprotein(a)(apo(a)) and apoB100 through disulfide bonds connected.

The risk of cardiovascular events in patients with elevated Lp(a) is 2-3 times that of the normal population, including atherosclerotic cardiovascular disease, lower extremity arterial disease, aortic stenosis, etc. (EnAs EA, Varkey B, Dharmarajan T S,et al.Lipoprotein(a):An independent,genetic,and causal factor for cardiovascular disease and acute myocardial infarction[J].Indian Heart Journal,2019,71(2).). Lp(a) may cause adverse atherosclerotic cardiovascular disease (ASCVD) through the following two mechanisms: on the one hand, because apo(a) has been shown to inhibit fibrinolysis in vitro, it may promote thrombus at the site of plaque rupture. Formation or turbulence in blood vessel stenosis, leading to vessel obstruction or promoting thrombosis; on the other hand, LDL-like particles can promote intimal cholesterol deposition, inflammation or oxidized phospholipids, leading to atherosclerosis or aortic valve stenosis (Albert Youngwoo Jang, Seung Hwan Han, Il Suk Sohn, et al. Lipoprotein(a)and Cardiovascular Diseases[J]. Circulation Journal, 2020, 84:867–874). But even at very high levels of Lp(a), the cholesterol content is below the traditional LDL cutoff, so this part of the LDL-like particles may be less pathogenic.

The 2016 Chinese Adult Dyslipidemia Prevention and Treatment Guidelines define >30 mg/dl as Lp(a) abnormality. Based on this standard, approximately 30% of patients with past cardiovascular events in China have Lp(a) abnormality. In 2019, the U.S. National Lipid Association recommended that Lp(a) ≥ 50 mg/dl as elevated levels. According to this standard, 20% of the global population has elevated Lp(a) levels. Although elevated Lp(a) levels are common, there is a lack of targeted therapeutic drugs, and so far no drug that targets Lp(a) has been approved for clinical use. The Lp(a) protein is structurally similar to a variety of lipoproteins, making it difficult to become a direct target for small and large molecule drugs. However, the mRNA transcribed by the Lp(a) gene is highly specific and can be specifically degraded through the siRNA post-transcriptional regulation mechanism. Its mRNA, thereby inhibiting Lp(a) expression. Therefore, siRNA targeting the apo(a) gene (LPA) is designed to attenuate its expression, thereby reducing Lp(a) levels in serum, thereby reducing adverse cardiovascular events.

Contents of the invention

The present disclosure provides an oligonucleotide comprising a 5' terminal chemical modification that includes at least one deuterium.

The present disclosure provides an oligonucleotide comprising a 5' end chemical modification represented by formula (I), or a tautomer modification thereof, wherein the 5' end chemical modification represented by formula (I) is selected from :

Wherein, R _A1 and R _A2 are each independently selected from hydrogen or deuterium; M ₁ and M ₂ are each independently selected from -SH or -OH; B is selected from base, hydrogen, deuterium; R _A3 is selected from hydrogen, deuterium, Hydroxy, halogen, alkyl (such as C1, C2, C3, C4, C5, C6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C1 alkoxy, C2 alkoxy base, C3 alkoxy group, C4 alkoxy group, C5 alkoxy group, C6 alkoxy group, including but not limited to Methoxy, ethoxy, propoxy, isopropoxy), each of the hydroxyl, alkyl, and alkoxy groups is optionally substituted by one or more deuteriums; R _A4 is selected from hydrogen, deuterium, alkyl (For example, C1, C2, C3, C4, C5, C6 alkyl, including but not limited to methyl, ethyl, isopropyl), each of the alkyl groups is optionally substituted by one or more deuterium; provided that, Formula (I) contains at least one deuterium.

In some embodiments, R _A3 is selected from hydrogen, deuterium.

In some embodiments, R _A3 is selected from halogen (e.g., fluorine, chlorine, bromine).

In some embodiments, R _A3 is selected from alkyl groups (e.g., C1, C2, C3, C4, C5, C6 alkyl, including but not limited to methyl, ethyl, isopropyl), each of which is optionally Replaced by one or more deuteriums.

In some embodiments, R _A3 is selected from alkoxy (e.g., C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, C6 alkoxy, including but not limited to methyl oxy, ethoxy, propoxy, isopropoxy), each of the alkoxy groups is optionally substituted by one or more deuterium.

In some embodiments, the 5' end chemical modification represented by Formula (I) is selected from:

Among them, _RA5 , _RA6 and _RA7 are each independently selected from hydrogen or deuterium; _RA1 , _RA2 , _M1 , _M2 and B are as defined in formula (I); the condition is that in formula (I-1) Contains at least one deuterium.

In some embodiments, _RA5 , _RA6 , and _RA7 are all deuterium; in some embodiments, _RA5 is selected from deuterium, and _RA6 and _RA7 are selected from hydrogen; in some embodiments, _RA5 , _RA6 Selected from deuterium, R _A7 is selected from hydrogen.

In some embodiments, _RA1 is selected from hydrogen and _RA2 is selected from deuterium; in some embodiments, _RA1 and _RA2 are selected from deuterium; in some embodiments, _RA1 and _RA2 are selected from hydrogen.

B is selected from bases or hydrogen.

In some embodiments, B is selected from a base; in some specific embodiments, B is selected from adenine, guanine, cytosine, uracil, or thymine.

In some specific embodiments, B is the base corresponding to the modified nucleotide of the antisense strand.

and structures in which uracil is replaced by adenine, guanine, cytosine, or thymine.

In some embodiments, the oligonucleotides described in the present disclosure are selected from the group consisting of single-stranded oligonucleotides, double-stranded oligonucleotides, antisense oligonucleotides, small interfering RNA (siRNA), double-stranded RNA (dsRNA) , microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, RNAi inhibitor molecules and Dicer enzyme substrates.

In some embodiments, the oligonucleotides of the present disclosure are single-stranded oligonucleotides.

In some embodiments, the single-stranded oligonucleotide is a single-stranded RNAi inhibitor molecule. In some embodiments, the single-stranded RNAi inhibitor molecules have 16 to 50, 16 to 45, 17 to 45, 17 to 40, 18 to 40, 18 to 35, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides.

In some embodiments, oligonucleotides of the present disclosure are selected from double-stranded RNAi inhibitor molecules.

In some embodiments, oligonucleotides of the present disclosure are selected from siRNA.

In some embodiments, oligonucleotides of the present disclosure are selected from double-stranded RNAi inhibitor molecules, including a sense strand and an antisense strand forming a double-stranded region.

In some embodiments, the oligonucleotides described in the present disclosure are selected from siRNA, comprising a sense strand and an antisense strand forming a double-stranded region.

In some embodiments, the 5' end chemical modification represented by formula (I), or a tautomer modification thereof, is located at the 5' end of the antisense strand.

In some specific embodiments, in the chemical modification of the 5' end shown in formula (I), B is the base corresponding to the modified nucleotide of the antisense strand.

In some embodiments, oligonucleotides of the present disclosure target the hepatitis B virus (HBV) gene, the apolipoprotein C3 (ApoC3) gene, the lipoprotein (a) (LPA) gene.

In some embodiments, the oligonucleotides of the present disclosure are siRNAs targeting hepatitis B virus (HBV) genes.

In some embodiments, the oligonucleotides of the present disclosure are siRNAs targeting HBV-S.

In some embodiments, the oligonucleotides of the present disclosure are siRNAs targeting HBV-X.

In some embodiments, the oligonucleotides of the present disclosure are siRNAs targeting the apolipoprotein C3 (ApoC3) gene.

In some embodiments, the oligonucleotides of the present disclosure are siRNAs targeting the lipoprotein(a) (LPA) gene.

In some embodiments, an oligonucleotide, double-stranded RNAi inhibitor molecule or siRNA of the present disclosure comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 contiguous nucleotides, and The nucleotide sequence differs from the nucleotide sequence of SEQ ID NO: 1 by no more than 2 nucleotides (for example, 2, 1). In some embodiments, the antisense strand comprises at least 15 contiguous nucleotide sequences and differs from the nucleotide sequence of SEQ ID NO: 2 by no more than 2 nucleotides (e.g., 2, 1).

In some embodiments, the sense strand comprises at least 15 contiguous nucleotides (eg, 15, 16, 17, 18, 19) of SEQ ID NO: 1. In some embodiments, the sense strand comprises at least 16 contiguous nucleotides of SEQ ID NO:1. In some embodiments, the sense strand comprises at least 18 contiguous nucleotides of SEQ ID NO:1. In some embodiments, the sense strand comprises at least 19 consecutive nucleotides of SEQ ID NO:1 Nucleotides.

In some embodiments, the antisense strand comprises at least 15 contiguous nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21) of the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the antisense strand comprises at least 18 contiguous nucleotides of SEQ ID NO:2. In some embodiments, the antisense strand comprises at least 19 contiguous nucleotides of SEQ ID NO: 2. In some embodiments, the antisense strand comprises at least 20 contiguous nucleotides of SEQ ID NO: 2. In some embodiments, the antisense strand comprises at least 21 contiguous nucleotides of SEQ ID NO: 2.

In some embodiments, the sense strand comprises or is selected from the nucleotide sequence set forth in SEQ ID NO: 1.

In some embodiments, the antisense strand comprises or is selected from the nucleotide sequence set forth in SEQ ID NO: 2.

In some embodiments, the oligonucleotide of the present disclosure includes the sense strand shown in SEQ ID NO: 1 and the antisense strand shown in SEQ ID NO: 2.

In some embodiments, the oligonucleotide of the present disclosure, the sense strand is selected from the nucleotide sequence shown in SEQ ID NO: 1, and the antisense strand is selected from the nucleotide sequence shown in SEQ ID NO: 2 Nucleotide sequence.

In some embodiments, the oligonucleotide of the present disclosure is selected from the sense strand shown in SEQ ID NO: 1 and the antisense strand shown in SEQ ID NO: 2.

In this disclosure, according to the 5’-3’ direction,

SEQ ID NO:1 is GCUCCUUAUUGUUAUACGA;

SEQ ID NO:2 is UCGUAUAACAAUAAGGAGCUG.

In some embodiments, the antisense strand is at least partially reverse complementary to the target sequence to mediate RNA interference; in some embodiments, there are no more than 5, no more than 4, between the antisense strand and the target sequence. No more than 3, no more than 2, no more than 1 mismatch; in some embodiments, the antisense strand is completely reverse complementary to the target sequence.

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

In some embodiments, oligonucleotides of the present disclosure are selected from siRNA and contain one or two blunt ends.

In some specific embodiments, the siRNA contains overhangs with 1 to 4 unpaired nucleotides, such as 1, 2, 3, 4.

In some embodiments, the oligonucleotides of the present disclosure are selected from siRNA and comprise an overhang located at the 3' end of the antisense strand of the siRNA.

In some embodiments, the sense strand is 15-35 nucleotides in length and the antisense strand is 15-30 nucleotides in length.

In some embodiments, the sense strand and the antisense strand each independently have 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31 , 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 cores nucleotides (e.g. 19, 20, 21, 22, 23 nucleotides).

In some embodiments, the sense strand and antisense strand are the same or different in length, with the sense strand being 19-23 nucleotides in length and the antisense strand being 19-26 nucleotides in length. In this way, the length ratio of the sense strand and the antisense strand provided by the present disclosure can be 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20 /21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21/26 ,22/20,22/21,22/22,22/23,22/24,22/25,22/26,23/20,23/21,23/22,23/23,23/24,23 /25 or 23/26. In some embodiments, the length ratio of the sense strand and antisense strand is 19/21, 21/23, or 23/25. In some embodiments, the length ratio of the sense strand and antisense strand is 19/21.

In some embodiments, the oligonucleotides, double-stranded RNAi inhibitor molecules, or siRNAs described in this disclosure contain no other modified nucleotides.

In some embodiments, at least one additional nucleotide in an oligonucleotide, double-stranded RNAi inhibitor molecule, or siRNA of the present disclosure is a modified nucleotide; in some embodiments, the oligonucleotide Acid, double-stranded RNAi inhibitor molecule or siRNA comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand and the antisense strand respectively comprising at least one nucleotide being a modified nucleotide; in some embodiments , all nucleotides of the sense strand and all nucleotides of the antisense strand of the present disclosure are modified nucleotides.

In some embodiments, the modified nucleotides are selected from: 2'-methoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleosides Acid, 2'-substituted alkyl modified nucleotide, 2'-amino modified nucleotide, 2'-substituted amino modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxynucleotide, 2'-deoxy-2'-fluoro modified nucleotide, 3'-deoxy-thymine (dT) nucleotide, isonucleotide, LNA, ENA, cET, UNA , GNA.

In some embodiments, the modified nucleotides are independently selected from the group consisting of: 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides.

In some embodiments, in the direction from the 5' end to the 3' end, the three consecutive nucleotides located at positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluorinated modified cores. glycosides. In some specific embodiments, the three consecutive nucleotides at positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoro modified nucleotides, and the remaining positions of the sense strand are non-2 '-fluorinated modified nucleotides; in some specific embodiments, the three consecutive nucleotides located at positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluorinated modified nucleosides acid, and the remaining positions of the sense strand are 2'-methoxy modified nucleotides.

In some embodiments, in the direction from the 5' end to the 3' end, the nucleotide at position 1 of the antisense strand is a 5' end chemical modification represented by formula (I) according to any one of the present disclosure. nucleotides; in some specific implementations In an embodiment, the 5' end chemically modified nucleotide represented by formula (I) is The B is the base corresponding to the first nucleotide at the 5' end of the antisense strand.

In some embodiments, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, and 18 of the antisense strand are each independently 2'- Fluorinated modified nucleotides, the remaining positions of the antisense strand are non-2'-fluorinated modified nucleotides. In some specific embodiments, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 in the antisense strand are each independently 2 '-Fluoro-modified nucleotides, and the remaining positions of the antisense strand are 2'-methoxy-modified nucleotides.

In some embodiments, oligonucleotides of the present disclosure comprise at least one phosphate group that is a phosphate group with a modifying group, and in some embodiments, at least one phosphate group is a phosphorothioate diester group. .

In some embodiments, at least one phosphate group in the sense strand and/or antisense strand is a phosphate group with a modifying group. In some embodiments, at least one phosphate group in the sense strand and/or antisense strand It is a phosphorothioate diester group.

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

Between the first nucleotide and the second nucleotide at the 5' end of the sense strand;

Between the second and third nucleotides at the 5' end of the sense strand;

Between the first nucleotide and the second nucleotide at the 3' end of the sense strand;

The first nucleotide end of the 3' end of the sense strand;

Between the first nucleotide and the second nucleotide at the 5' end of the antisense strand;

Between the 2nd nucleotide and the 3rd nucleotide at the 5' end of the antisense strand;

Between the first nucleotide and the second nucleotide at the 3' end of the antisense strand;

Between the 2nd nucleotide and the 3rd nucleotide at the 3' end of the antisense strand; or,

The first nucleotide end of the 3' end of the antisense strand.

In some embodiments, the phosphorothioate diester group is present in:

Between the 1st and 2nd nucleotide at the 5' end of the sense strand.

In some embodiments, the phosphorothioate diester group is present in:

Between the 1st nucleotide and the 2nd nucleotide at the 5' end of the sense strand, and,

Between the 2nd and 3rd nucleotides at the 5' end of the sense strand.

In some embodiments, the phosphorothioate diester group is present in:

between the 1st and 2nd nucleotides at the 5' end of the sense strand, and

between the 2nd and 3rd nucleotides at the 5' end of the sense strand, and

Between the first and second nucleotides at the 3' end of the sense strand.

In some embodiments, the phosphorothioate diester group is present in:

Between the first nucleotide and the second nucleotide at the 5' end of the sense strand; and,

The first nucleotide end of the 3' end of the sense strand.

In some embodiments, the phosphorothioate diester group is present in:

Between the 2nd and 3rd nucleotides at the 5' end of the sense strand; and,

The first nucleotide end of the 3' end of the sense strand.

In some embodiments, the phosphorothioate diester group is present in:

The first nucleotide end of the 3' end of the sense strand.

In some embodiments, the phosphorothioate diester group is present in:

Between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and,

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

In some specific embodiments, the sense strand comprises the nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:4.

In some specific embodiments, the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:5.

In some specific embodiments, the sense strand is selected from the nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:4.

In some specific embodiments, the antisense strand is selected from the nucleotide sequence shown in SEQ ID NO:5.

In some specific embodiments, the sense strand of an oligonucleotide of the present disclosure includes SEQ ID NO: 3, and the antisense strand includes SEQ ID NO: 5.

In some specific embodiments, the sense strand of an oligonucleotide of the present disclosure includes SEQ ID NO: 4, and the antisense strand includes SEQ ID NO: 5.

In some specific embodiments, the sense strand of the oligonucleotide described in the present disclosure is selected from SEQ ID NO: 3, and the antisense strand is selected from SEQ ID NO: 5.

In some specific embodiments, the sense strand of the oligonucleotide described in the present disclosure is selected from SEQ ID NO: 4, and the antisense strand is selected from SEQ ID NO: 5.

In some specific embodiments, the oligonucleotides described in the present disclosure are selected from any of the following:

The sense strand includes the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand includes the nucleotide sequence shown in SEQ ID NO:5;

The sense strand includes the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand includes the nucleotide sequence shown in SEQ ID NO:5.

The sense strand is the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand is the nucleotide sequence shown in SEQ ID NO:5;

The sense strand is the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand is the nucleotide sequence shown in SEQ ID NO:5.

In this disclosure, according to the 5’-3’ direction,

SEQ ID NO:3 is GmsCmsUmCmCmUmUfAfUfUmGmUmUmAmUmAmCmGmAm;

SEQ ID NO:4 is GmsCmsUmCmCmUmUfAfUfUmGmUmUmAmUmAmCmGmsAm;

SEQ ID NO:5 is NA0127CfsGmUfAmUfAmAmCmAfAmUfAmAfGmGfAmGfCmsUmsGm;

Among them, 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; Gm=guanine 2'-OMe ribonucleoside; Um=uracil 2'-OMe ribose Nucleoside (uracil 2'-OMe ribonucleoside);

s means that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate diester groups;

NA0127 means

In some embodiments, the oligonucleotides of the present disclosure further comprise a targeting ligand; in some embodiments, the targeting ligand targets the liver; in some embodiments, the targeting ligand Binds to the asialoglycoprotein receptor (ASGPR); in some embodiments, the targeting ligand includes a cluster of galactose or a cluster of galactose derivatives selected from N-acetyl-galactose Amine, N-trifluoroacetylgalactosamine, N- Propionylgalactosamine, N-n-butyrylgalactosamine, N-acetyl-galactosamine triacetate or N-isobutyrylgalactosamine.

In some embodiments, the targeting ligand includes 3 galactose clusters or galactose derivative clusters.

In some embodiments, the targeting ligand is covalently or non-covalently linked to the oligonucleotide.

In some embodiments, the targeting ligand is linked to the terminus of an oligonucleotide, double-stranded RNAi inhibitor molecule, or siRNA via a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group.

In some embodiments, the targeting ligand is indirectly linked to the terminus of an oligonucleotide, double-stranded RNAi inhibitor molecule, or siRNA through a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group.

In some embodiments, the targeting ligand is directly linked to the terminus of an oligonucleotide, double-stranded RNAi inhibitor molecule, or siRNA via a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group.

In some embodiments, the targeting ligand is directly linked to the terminus of an oligonucleotide, double-stranded RNAi inhibitor molecule, or siRNA via a phosphodiester group or a phosphorothioate diester group.

In some embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of the oligonucleotide, double-stranded RNAi inhibitor molecule, or siRNA via a phosphodiester group or a phosphorothioate diester group.

In some embodiments, the targeting ligand is selected from the structure represented by formula (II') or a pharmaceutically acceptable salt thereof:

Wherein, L ₁ is a C ₁ -C ₃₀ alkyl chain, or a C ₁ -C ₃₀ alkyl chain interrupted by one or more oxygen, sulfur, nitrogen atoms or C=O;

R ₁ and R ₂ are independently chemical bonds, NR ₆ , C=O or -OC(=O)-;

Q is

is a single bond or a double bond, and when When it is a single bond, R ₃ is independently CR ₇ R ₈ , NR ₆ , O or S. When it is a double bond, R ₃ is independently CR ₉ or N;

R ₄ is independently CR ₉ or N;

Ring A is cycloalkyl, heterocyclyl, aryl or heteroaryl, which may or may not be present, and when Ring A is present, R ₅ is independently CR ₉ or N, when Ring A is absent, R _{5 is} independently The ground is CR ₇ R ₈ , NR ₆ or O;

R ₆ and R ₉ are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, SR', S(=O)R', S(=O) ₂ R', S(=O) ₂ NR'(R”), NR'(R”), C(=O)R’, C(=O)OR’ or C(=O)NR’(R” ), the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl group is optionally substituted by one or more halogens (such as fluorine, chlorine, bromine), hydroxyl, oxo, nitro group, cyano group, C _1-6 alkyl, C _1-6 alkoxy Base, C _3-7 cycloalkyl, 3-12 membered heterocyclyl, 5-12 membered aryl, 5-12 membered heteroaryl, SR', S(=O)R', S(=O) ₂ R', S(=O) ₂ NR'(R"), NR'(R"), C(=O)R', C(=O)OR' and C(=O)NR'(R") Replaced by groups in;

R ₇ and R ₈ are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, SR', S(=O)R', S(=O) ₂ R', S(=O) ₂ NR'(R”), NR'(R”), C(=O)R’, C(=O)OR’ or C(=O)NR’(R” ), the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl group is optionally substituted by one or more halogens (such as fluorine, chlorine, bromine), hydroxyl, oxo, nitro group, cyano group, C _1-6 alkyl group, C _1-6 alkoxy group, C _3-7 cycloalkyl group, 3-12 membered heterocyclyl group, 5-12 membered aryl group, 5-12 membered heteroaryl group , SR', S(=O)R', S(=O) ₂ R', S(=O) ₂ NR'(R”), NR'(R”), C(=O)R', C Substituted by groups in (=O)OR' and C(=O)NR'(R”);

R' and R" are independently hydrogen, deuterium, hydroxyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl, said alkyl, alkoxy, cycloalkyl, hetero The cyclic group, aryl or heteroaryl group is optionally substituted by one or more substituents selected from halogen (such as fluorine, chlorine, bromine), hydroxyl, oxo, nitro and cyano;

m, n, p and q are independently 0, 1, 2, 3 or 4;

B 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)-(CH ₂ ) _z8 -O- or -NHC(=O)-(CH ₂ ) _z9 -O-;

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

L ₂ is a C ₁ -C ₃₀ alkyl chain, or contains a C ₁ -C ₃₀ alkyl chain interrupted by one or more oxygen, sulfur, nitrogen atoms or C=O;

G is the targeting moiety that binds to cell receptors;

r is an integer from 1 to 10.

In some embodiments, certain groups are defined as follows, undefined groups are as described in the previous embodiment (hereinafter referred to as "in some embodiments"), L ₁ can be L ₃ or L ₃ -R ₁₀ - R ₁₁ -L ₃ , where L ₃ is independently a C ₁ -C ₁₂ alkyl chain, -(CH ₂ ) _j1 -C(=O)-(CH ₂ ) _j2 - or -(CH ₂ ) _j3 -( CH ₂ CH ₂ O) _1-4 -(CH ₂ ) _j4 -, R ₁₀ and R ₁₁ are independently chemical bonds, -NR ₁₂ -, -C(=O)- or -OC(=O)-, R ₁₂ is hydrogen or C ₁ -C ₁₂ alkyl, j1, j2, j3 and j4 are independently an integer of 0-10, preferably an integer of 0-2 or 4-10, more preferably 0, 1, 2, 6, 7, 8, 9 or 10.

In some embodiments, L ₁ can be -(CH ₂ ) _j1 -C(=O)-(CH ₂ ) _j2 -, j1 and j2 are as defined in the previous embodiment.

In some embodiments, _L can be The definitions of j1 and j2 are the same as in the previous scheme, in which the a1 end is connected to B and the b1 end is connected to R ₁ .

In some embodiments, _L can be Among them, the a1 end is connected to B, and the b1 end is connected to R ₁ .

In some embodiments, _R1 can be a chemical bond and _R2 can be C=O.

In some embodiments, R ₁ can be a chemical bond and R ₂ can be NR ₆ , and R ₆ is as defined in the previous embodiment.

In some embodiments, R ₁ can be a chemical bond and R ₂ can be -OC(=O)-.

In some embodiments, R ₁ can be NR ₆ and R ₂ can be C=O, and R ₆ is as defined in the previous embodiment.

In some embodiments, R ₁ can be NR ₆ and R ₂ can be -OC(=O)-, and R ₆ is as defined in the previous embodiment.

In some embodiments, R ₂ can be NR ₆ and R ₁ can be C=O, and R ₆ is as defined in the previous embodiment.

In some embodiments, R ₂ can be NR ₆ and R ₁ can be -OC(=O)-, and R ₆ is as defined in the previous embodiment.

In some embodiments, _R6 can be hydrogen or _C1-6 alkyl.

In some embodiments, _R6 can be hydrogen, methyl, ethyl, propyl, or isopropyl.

In some embodiments, _R6 can be hydrogen.

In some embodiments, _R7 and _R8 can be hydrogen.

In some embodiments, R ₉ can be hydrogen.

In some embodiments, Ring A, when present, can be C _1-10 aryl, preferably phenyl.

In some embodiments, m can be 0 or 1.

In some embodiments, m can be 3.

In some embodiments, n can be 0 or 1.

In some embodiments, p and q are independently 0 or 1.

In some embodiments, p=1 and q=1.

In some embodiments, p=1 and q=0.

In some embodiments, p=0 and q=1.

In some embodiments, p=0 and q=0.

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

In some embodiments, B can be R _b1 , R _b2 , R _b3 and R _b4 are independently -C(=O)- or -NHC(=O)-, the N atom is connected to L ₁ , and the definitions of z1, z2, z3 and z4 are the same as in the previous scheme. described.

In some embodiments, B can be R _b1 , R _b2 , R _b3 and R _b4 are independently -C(=O)- or -NHC(=O)-, the N atom is connected to L ₁ , R _b1 , R _b3 and R _b4 are the same, z1 and z2 The definitions of , z3 and z4 are the same as those described in the previous scheme.

In some embodiments, B can be

In some embodiments, B can be R _b5 , R _b6 and R _b7 are independently -C(=O)-(CH ₂ ) _z8 -O- or -NHC(=O)-(CH ₂ ) _z9 -O-, and the N atom is connected to L ₁ , The definitions of z5, z6, z7, z8 and z9 are the same as in the previous scheme.

In some embodiments, B can be R _b5 , R _b6 and R _b7 are independently -C(=O)-(CH ₂ ) _z8 -O- or -NHC(=O)-(CH ₂ ) _z9 -O-, and the N atom is connected to L ₁ , R _b5 , R _b6 and R _b7 are the same, and the definitions of z5, z6, z7, z8 and z9 are the same as in the previous scheme.

In some embodiments, B can be

In some embodiments, L ₂ can be L ₄ or L ₄ -R ₁₃ -R ₁₄ -L ₄ , wherein L ₄ is independently a C ₁ -C ₁₂ alkyl chain or -(CH ₂ ) _{j 5} -(OCH ₂ CH ₂ ) _1-4 -(CH ₂ ) _j6 -, R ₁₃ and R ₁₄ are independently chemical bonds, -NR ₁₅ -, -C(=O)- or -OC(=O)-, R ₁₅ is independently is hydrogen or C ₁ -C ₁₂ alkyl, j5 and j6 are independently an integer from 0 to 10, preferably an integer from 0 to 6, more preferably 0, 1, 2, 3 or 4.

In some embodiments, L ₂ can be -(CH ₂ ) _j5 -(OCH ₂ CH ₂ ) _1-4 -(CH ₂ ) _j6 -, j5 and j6 are as defined in the previous embodiment.

In some embodiments, _L2 can be Among them, the O atom is connected to G, the C atom is connected to B, and preferably L ₂ is

In some embodiments, _L2 can be Among them, the definition of j6 is the same as described in the previous scheme, O atom is connected to G, and C atom is connected to B.

In some embodiments, _L2 can be Among them, the O atom is connected to G, and the C atom is connected to B.

In some embodiments, G can be an asialoglycoprotein receptor targeting moiety.

In some embodiments, G can be galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, or N -Isobutyrylgalactosamine.

In some embodiments, r can be 3, 4, 5 or 6, such as 3.

In some embodiments, Q can be preferred Among them, the definitions of R ₃ , R ₄ , R ₅ and n are the same as those described in the previous solution.

In some embodiments, can be Among them, the definitions of R ₃ , R ₄ , R ₅ , p and q are the same as those described in the previous solution.

In some embodiments, can be preferred More preferred The definitions of p and q are the same as described in the previous scheme.

In some embodiments, can be Among them, the definitions of R ₃ , R ₄ , n, p and q are the same as those described in the previous solution.

In some embodiments, can be Among them, the definitions of R ₃ , R ₄ , R ₅ , n, p and q are the same as those described in the previous solution.

In some embodiments, can be The definitions of n, p and q are the same as in the previous scheme.

In some embodiments, the targeting ligand is selected from the structure shown in formula (II), or a pharmaceutically acceptable salt thereof:

R ₁ and R ₂ are independently chemical bonds, NR ₆ , C=O or -OC(=O)-;

Q is

R ₄ is independently CR ₉ or N;

R ₆ and R ₉ are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, SR', S(=O)R', S(=O) ₂ R', S(=O) ₂ NR'(R”), NR'(R”), C(=O)R’, C(=O)OR’ or C(=O)NR’(R” ), the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl group is optionally substituted by one or more halogens (such as fluorine, chlorine, bromine), hydroxyl, oxo, nitro group, cyano group, C _1-6 alkyl group, C _1-6 alkoxy group, C _3-7 cycloalkyl group, 3-12 membered heterocyclyl group, 5-12 membered aryl group, 5-12 membered heteroaryl group , SR', S(=O)R', S(=O) ₂ R', S(=O) ₂ NR'(R”), NR'(R”), C(=O)R', C Substituted by groups in (=O)OR' and C(=O)NR'(R”);

R ₇ and R ₈ are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, SR', S(=O)R', S(=O) ₂ R', S(=O) ₂ NR'(R”), NR'(R”), C(=O)R’, C(=O)OR’ or C(=O)NR’(R” ), the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl group is optionally substituted by one or more halogens (such as fluorine, chlorine, bromine), hydroxyl, oxo, nitro group, cyano group, C _1-6 alkyl group, C _1-6 alkoxy group, C _3-7 cycloalkyl group, 3-12 membered heterocyclyl group, 5-12 membered aryl group, 5-12 membered heteroaryl group ,SR',S(=O)R',S(=O) ₂ R', S(=O) ₂ NR'(R"), NR'(R"), C(=O)R', C(=O)OR' and C(=O)NR'(R") Replaced by regiment;

m, n, p and q are independently 0, 1, 2, 3 or 4;

B is

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

r is an integer from 1 to 10.

In some embodiments, L ₁ can be L ₃ or L ₃ -R ₁₀ -R ₁₁ -L ₃ , where L ₃ is independently a C ₁ -C ₁₂ alkyl chain, -(CH ₂ ) _j1 -C( =O)-(CH ₂ ) _j2 - or -(CH ₂ ) _j3 -(CH ₂ CH ₂ ) _1-4 -(CH ₂ ) _j4 -, R ₁₀ and R ₁₁ are independently chemical bonds, -NR ₁₂ -, -C(=O)- or -OC(=O)-, R ₁₂ is hydrogen or C ₁ -C ₁₂ alkyl, j1, j2, j3 and j4 are independently integers from 0 to 10, preferably 0 to 2 or An integer of 4-10, more preferably 0, 1, 2, 6, 7, 8, 9 or 10.

In some embodiments, _R1 can be a chemical bond and _R2 can be C=O.

In some embodiments, R ₁ can be a chemical bond and R ₂ can be -OC(=O)-.

In some embodiments, R ₁ can be NR ₆ and R ₂ can be C=O, and R ₆ is as defined in the previous embodiment. narrate.

In some embodiments, _R6 can be hydrogen or _C1-6 alkyl.

In some embodiments, _R6 can be hydrogen, methyl, ethyl, propyl, or isopropyl.

In some embodiments, _R6 can be hydrogen.

In some embodiments, _R7 and _R8 can be hydrogen.

In some embodiments, R ₉ can be hydrogen.

In some embodiments, m can be 0 or 1.

In some embodiments, m can be 3.

In some embodiments, n can be 0 or 1.

In some embodiments, p and q are independently 0 or 1.

In some embodiments, p=1 and q=1.

In some embodiments, p=1 and q=0.

In some embodiments, p=0 and q=1.

In some embodiments, p=0 and q=0.

In some embodiments, B can be R _b1 , R _b2 , R _b3 and R _b4 are independently -C(=O)- or -NHC(=O)-, the N atom is connected to L ₁ , R _b1 , R _b3 and R _b4 are the same, z1 and z2 , The definitions of z3 and z4 are the same as described in the previous scheme.

In some embodiments, B can be

In some embodiments, L ₂ can be L ₄ or L ₄ -R ₁₃ -R ₁₄ -L ₄ , wherein L ₄ is independently a C ₁ -C ₁₂ alkyl chain or -(CH ₂ ) _{j 5} -(OCH ₂ CH ₂ ) _1-4 -(CH ₂ ) _j6 -, R ₁₃ and R ₁₄ are independently chemical bonds, -NR ₁₅ -, -C(=O)- or -OC(=O)-, R ₁₅ is independently hydrogen or C ₁ -C ₁₂ alkyl, j5 and j6 are independently an integer from 0 to 10, preferably an integer from 0 to 6, more preferably 0, 1, 2 , 3 or 4.

In some embodiments, L ₂ can be -(CH ₂ ) _j5 -(OCH ₂ CH ₂ ) _1-4 (CH ₂ ) _j6 -, j5 and j6 are as defined in the previous embodiment.

In some embodiments, _L2 can be preferred Among them, the O atom is connected to G, and the C atom is connected to B.

In some embodiments, _L2 can be a _C1 - _C12 alkyl chain.

In some embodiments, _L2 can be

In some embodiments, _L2 can be preferred More preferred Among them, the a3 end is connected to G, and the b3 end is connected to B.

In some embodiments, _L2 can be Among them, the a3 end is connected to G, and the b3 end is connected to B.

In some embodiments, r can be 3, 4, 5 or 6, with 3 being preferred.

In some embodiments, can be preferred The definitions of n, p and q are the same as in the previous scheme.

In some embodiments, the targeting ligand is any of the following structures, or a pharmaceutically acceptable salt thereof:

In some embodiments, the targeting ligand is selected from any of the following structures, or a pharmaceutically acceptable salt thereof:

In some embodiments, N- in the above ligands can be replaced by N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine. Acetyl-galactosamine moiety.

In some embodiments, N-trifluoroacetylgalactosamine triacetate, N-propionylgalactosamine triacetate, N-n-butyrylgalactosamine triacetate, or N-isobutyl Acylgalactosamine triacetate replaces the N-acetyl-galactosamine triacetate moiety in the above targeting ligand.

In some specific embodiments, the sense strand comprises the nucleotide sequence shown in SEQ ID NO:6 or SEQ ID NO:7.

In some specific embodiments, the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:5.

In some specific embodiments, the sense strand is selected from the nucleotide sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7;

In some specific embodiments, the sense strand of an oligonucleotide of the present disclosure includes SEQ ID NO: 6, and the antisense strand includes SEQ ID NO: 5.

In some specific embodiments, the sense strand of an oligonucleotide of the present disclosure includes SEQ ID NO: 7, and the antisense strand includes SEQ ID NO: 5.

In some specific embodiments, the sense strand of an oligonucleotide of the present disclosure is selected from SEQ ID NO: 6, and the antisense strand is selected from SEQ ID NO: 5.

In some specific embodiments, the sense strand of the oligonucleotide of the present disclosure is selected from SEQ ID NO: 7, and the antisense strand is selected from SEQ ID NO: 5.

The sense strand includes the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand includes the nucleotide sequence shown in SEQ ID NO:5;

The sense strand includes the nucleotide sequence shown in SEQ ID NO:7, and the antisense strand includes the nucleotide sequence shown in SEQ ID NO:5.

The sense strand is the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand is the nucleotide sequence shown in SEQ ID NO:5;

The sense strand is the nucleotide sequence shown in SEQ ID NO:7, and the antisense strand is the nucleotide sequence shown in SEQ ID NO:5.

In this disclosure, according to the 5’-3’ direction,

SEQ ID NO:6 is GmsCmsUmCmCmUmUfAfUfUmGmUmUmAmUmAmCmGmAm-NAG0052’;

SEQ ID NO:7 is GmsCmsUmCmCmUmUfAfUfUmGmUmUmAmUmAmCmGmsAm-NAG0052’;

SEQ ID NO:5 is NA0127CfsGmUfAmUfAmAmCmAfAmUfAmAfGmGfAmGfCmsUmsGm;

Among them, Af = adenine 2'-F ribonucleoside; Cf = 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; Gm=guanine 2'-OMe ribonucleoside; Um=uracil 2'-OMe ribose Nucleoside (uracil 2'-OMe ribonucleoside);

NA0127 means

NAG0052' means

In some embodiments, the oligonucleotide is an anionic form represented by the following structure or a pharmaceutically acceptable salt thereof:

in,

Af=adenine 2'-F ribonucleoside (adenine 2'-F ribonucleoside);

Cf=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);

Represents phosphorothioate group, Represents phosphodiester group;

NAG0052' means

NA0127' means

Pharmaceutically acceptable salts can be conventional salts in the art, including but not limited to: sodium salt, potassium salt, ammonium salt, amine salt, etc.

In some embodiments, the oligonucleotides described in the present disclosure are the following structures or pharmaceutically acceptable salts thereof:

in,

Af=adenine 2'-F ribonucleoside (adenine 2'-F ribonucleoside);

Cf=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);

Represents phosphorothioate group, Represents phosphodiester group;

NAG0052' means:

NA0127 means

In some embodiments, the oligonucleotide is selected from TJR100747, TJR100848.

In some embodiments, the oligonucleotide is selected from the following structures:

in,

Af=adenine 2'-F ribonucleoside (adenine 2'-F ribonucleoside);

Cf=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);

Represents phosphorothioate group, Represents phosphodiester group;

NAG0052' means:

NA0127' means

The present disclosure also provides a compound represented by formula (IV) or a tautomer thereof,

Among them, _RA8 and _RA9 are each independently selected from -CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₂ CN, -CH ₂ OPiv (i.e. -CH ₂ OCOC(CH ₃ ) ₃ ), CH ₂ OCH ₂ CH ₂ Si(CH ₃ ) ₃ or protecting group; R _A10 is selected from phosphorus-containing reactive groups; R _A1 and R _A2 are each independently selected from hydrogen or deuterium; M ₄ is selected from O or S; B is selected from bases , hydrogen, deuterium; R _A3 is selected from hydrogen, deuterium, hydroxyl, halogen (such as fluorine, chlorine, bromine), alkyl (such as C1, C2, C3, C4, C5, C6 alkyl, including but not limited to methyl, Ethyl, isopropyl), alkoxy (such as C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, C6 alkoxy, including but not limited to methoxy , ethoxy, propoxy, isopropoxy), each of the hydroxyl, alkyl, and alkoxy groups is optionally substituted by one or more deuteriums; B is selected from base, hydrogen, and deuterium; the condition is, Formula (IV) contains at least one deuterium.

In some embodiments, Formula (IV) is selected from Formula (IV-1),

Among them, _RA5 , _RA6 and _RA7 are each independently selected from hydrogen or deuterium; _RA1 , _RA2 , _RA8 , _RA9 , _RA10 and B are as defined by formula (IV), with the condition that formula (IV- 1) contains at least one deuterium.

In some embodiments, the compound of formula (IV) is selected from:

B is selected from base and hydrogen.

The present disclosure also provides a method for preparing the oligonucleotide of the present disclosure, which includes the following steps:

(1) Synthesize the compound represented by formula (IV) or its tautomer described in this disclosure;

(2) Use the compound represented by formula (IV) in step (1) or its tautomer to synthesize the oligonucleotide described in the present disclosure.

The present disclosure also provides a method for preparing the compound represented by formula (IV) or its tautomer according to the present disclosure, including:

Dissolve compound (IV-2) in dichloromethane and add 2-cyanoethyl N,N-diisopropyl chloride at room temperature. Phosphoramidite and diisopropylethylamine are stirred until the reaction is complete to obtain product (IV-1),

Among them, _RA5 , _RA6 and _RA7 are each independently selected from hydrogen or deuterium; _RA1 , _RA2 , _RA8 , _RA9 , _RA10 and B are as defined by formula (IV), with the condition that formula (IV- 1), (IV-2) contains at least one deuterium.

On the other hand, the present disclosure also provides an oligonucleotide comprising at least one chemical modification represented by formula (V) or a tautomer modification thereof, wherein the chemical modification represented by formula (V) is selected from since:

Among them, M ₃ is selected from -S-, -NH-, -NR _A11 -, R _A11 is selected from C ₁ -C ₆ alkylene, R _A1 , R _A2 , M ₁ , M ₂ , B, R _A3 , R _A4 is as defined by formula (I), with the proviso that formula (V) contains at least one deuterium.

In some embodiments of the present disclosure, the compound represented by formula (I), (I-1), (IV) or (IV-1), wherein the deuterium atom has an abundance greater than that of deuterium The natural abundance of deuterium is at least 2000 times greater, and the natural abundance of deuterium is 0.015%.

In some embodiments of the present disclosure, the compound represented by formula (I), (I-1), (IV) or (IV-1) or a pharmaceutically acceptable salt thereof, wherein the deuterium atom The abundance of deuterium is at least 3000 times greater than the natural abundance of deuterium, which is 0.015%.

In some embodiments of the present disclosure, the compound represented by formula (I), (I-1), (IV) or (IV-1) or a pharmaceutically acceptable salt thereof, wherein the deuterium atom The abundance of deuterium is at least 3340 times greater than the natural abundance of deuterium, which is 0.015%.

In some embodiments of the present disclosure, the compound represented by formula ((I), (I-1), (IV) or (IV-1) or a pharmaceutically acceptable salt thereof, wherein the deuterium The atomic abundance of deuterium is at least 3500 times greater than the natural abundance of deuterium, which is 0.015%.

In some embodiments of the present disclosure, the compound represented by formula (I), (I-1), (IV) or (IV-1) or a pharmaceutically acceptable salt thereof, wherein the deuterium atom The abundance of deuterium is at least 4000 times greater than the natural abundance of deuterium, which is 0.015%.

In some embodiments of the present disclosure, the compound represented by formula (I), (I-1), (IV) or (IV-1) or a pharmaceutically acceptable salt thereof, wherein the deuterium atom The abundance of deuterium is at least 4500 times greater than the natural abundance of deuterium, which is 0.015%.

In some embodiments of the present disclosure, the compound represented by formula (I), (I-1), (IV) or (IV-1) or a pharmaceutically acceptable salt thereof, wherein the deuterium atom The abundance of deuterium is at least 5,000 times greater than the natural abundance of deuterium, which is 0.015%.

In some embodiments of the present disclosure, the compound represented by formula (I), (I-1), (IV) or (IV-1) or a pharmaceutically acceptable salt thereof, wherein the deuterium atom The abundance of deuterium is at least 5500 times greater than the natural abundance of deuterium, which is 0.015%.

In some embodiments of the present disclosure, the compound represented by formula (I), (I-1), (IV) or (IV-1) or a pharmaceutically acceptable salt thereof, wherein the deuterium atom The abundance of deuterium is at least 6000 times greater than the natural abundance of deuterium, which is 0.015%.

In some embodiments of the present disclosure, the compound represented by formula (I), (I-1), (IV) or (IV-1) or a pharmaceutically acceptable salt thereof, wherein the deuterium atom The abundance of deuterium is at least 6333.3 times greater than the natural abundance of deuterium, which is 0.015%.

In some embodiments of the present disclosure, the compound represented by formula (I), (I-1), (IV) or (IV-1) or a pharmaceutically acceptable salt thereof, wherein the deuterium atom The abundance of deuterium is at least 6466.7 times greater than the natural abundance of deuterium, which is 0.015%.

In some embodiments of the present disclosure, the compound represented by formula (I), (I-1), (IV) or (IV-1) or a pharmaceutically acceptable salt thereof, wherein the deuterium atom The abundance of deuterium is at least 6600 times greater than the natural abundance of deuterium, which is 0.015%.

In some embodiments of the present disclosure, the compound represented by formula (I), (I-1), (IV) or (IV-1) or a pharmaceutically acceptable salt thereof, wherein the deuterium atom The abundance of deuterium is at least 6633.3 times greater than the natural abundance of deuterium, which is 0.015%.

In another aspect, the present disclosure provides a pharmaceutical composition comprising an oligonucleotide of the present disclosure.

In some embodiments, the pharmaceutical composition further includes one or more pharmaceutically acceptable excipients.

In some embodiments, the pharmaceutically acceptable excipients may be, for example, carriers, carriers, diluents, and/or delivery polymers.

In some embodiments, the oligonucleotide can be in a therapeutically effective amount.

In some embodiments, the unit dosage of the oligonucleotide may be 0.001 mg-1000 mg.

In some embodiments, based on the total weight of the pharmaceutical composition, the content of the oligonucleotide may be 0.01-99.99%, or 0.1-99.9%, or 0.5%-99.5%, or further 1 %-99%, further can be 2%-98%.

In some embodiments, the content of the pharmaceutically acceptable excipients may be 0.01-99.99%, 0.1-99.9%, or 0.5%-99.5%, based on the total weight of the pharmaceutical composition. It may further be 1%-99%, and further it may be 2%-98%.

In some embodiments, the effective amount or dosage of the oligonucleotide or pharmaceutical composition is about 0.001 mg/kg body weight to about 200 mg/kg body weight, about 0.01 mg/kg body weight to about 100 mg/kg body weight, or About 0.5 mg/kg body weight to about 50 mg/kg body weight.

Various drug delivery systems are known and can be used in the oligonucleotides or pharmaceutical compositions of the present disclosure, e.g. Encapsulation in liposomes, microparticles, microvesicles, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, constructed nucleic acid as part of a retroviral or other vector.

In some embodiments, the oligonucleotides or pharmaceutical compositions of the present disclosure are administered in a conventional manner, either locally (e.g., direct injection or implantation) or systemically, orally or rectally. Or administration by parenteral route, which includes but is not limited to subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, transdermal administration, inhalation administration (such as aerosol), mucosal administration (such as tongue intranasal administration), intracranial administration, etc.

In some embodiments, the oligonucleotides or pharmaceutical compositions provided by the present disclosure can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally, or intraperitoneally.

In some embodiments, oligonucleotides or pharmaceutical compositions provided by the present disclosure can be packaged in kits.

On the other hand, the present disclosure provides a use of the above-mentioned oligonucleotide or the above-mentioned pharmaceutical composition in the preparation of medicines.

In some embodiments, the medicaments may be used to prevent and/or treat diseases associated with elevated levels of lipoprotein(a) and/or apolipoprotein(a). In some embodiments, the disease associated with elevated levels of lipoprotein(a) and/or apolipoprotein(a) is selected from cardiovascular disease. In some embodiments, the cardiovascular disease is selected from the group consisting of ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity arterial disease or aortic stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral stenosis, Arterial narrowing, heart failure. In some embodiments, the drug is used to inhibit the expression of LPA.

In some embodiments, the medicaments may be used to prevent and/or treat cardiovascular disease. In some embodiments, the cardiovascular disease is selected from the group consisting of ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity arterial disease or aortic stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral stenosis, Arterial narrowing, heart failure.

In some embodiments, the medicaments may be used to prevent and/or treat thromboembolic complications. In some embodiments, the thromboembolic complication may be deep vein thrombosis, pulmonary embolism, myocardial infarction, or stroke.

In some embodiments, the medicaments may be used to prevent and/or treat diseases associated with hepatitis B virus. In some embodiments, the disease associated with hepatitis B virus is chronic hepatitis and the subject is HBeAg positive or HBeAg negative. In some embodiments, the disease associated with the hepatitis B virus is hepatitis B; in some embodiments, the hepatitis B is selected from chronic hepatitis B or acute hepatitis B.

In some embodiments, the medicaments can be used to prevent and/or treat diseases associated with APOC3 gene expression. In some embodiments, the disease is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, abnormalities of lipid and/or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hyperglycemia Triesteremia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome Symptoms or familial partial lipodystrophy.

In some embodiments, the medicaments may be used to prevent and/or treat diseases mediated by elevated triglyceride levels or elevated cholesterol levels. In some embodiments, the disease is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, abnormalities of lipid and/or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hyperglycemia Triesteremia-induced pancreatitis, metabolic syndrome, type 2 diabetes, familial chylomicronemia syndrome, or familial partial lipodystrophy.

On the other hand, the present disclosure provides a method for preventing and/or treating diseases, which includes administering an effective amount or an effective dose of the above-mentioned oligonucleotide or the above-mentioned pharmaceutical composition to a subject.

In some embodiments, the disease may be a disease associated with elevated levels of lipoprotein(a) and/or apolipoprotein(a). In some embodiments, the disease associated with elevated levels of lipoprotein(a) and/or apolipoprotein(a) is selected from cardiovascular disease. In some embodiments, the cardiovascular disease is selected from the group consisting of ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity arterial disease or aortic stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral stenosis, Arterial narrowing, heart failure.

In some embodiments, the disease may be cardiovascular disease. In some embodiments, the cardiovascular disease is selected from the group consisting of ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity arterial disease or aortic stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral stenosis, Arterial narrowing, heart failure.

In some embodiments, the disease may be a disease associated with hepatitis B virus. In some embodiments, the disease associated with hepatitis B virus is chronic hepatitis and the subject is HBeAg positive or HBeAg negative. In some embodiments, the disease associated with the hepatitis B virus is hepatitis B; in some embodiments, the hepatitis B is selected from chronic hepatitis B or acute hepatitis B.

In some embodiments, the disease may be a disease associated with APOC3 gene expression. In some embodiments, the disease is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, abnormalities of lipid and/or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hyperglycemia Triesteremia-induced pancreatitis, metabolic syndrome, type 2 diabetes, familial chylomicronemia syndrome, or familial partial lipodystrophy.

In some embodiments, the disease may be a disease mediated by elevated triglyceride levels or elevated cholesterol levels. In some embodiments, the disease is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, abnormalities of lipid and/or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hyperglycemia Triesteremia-induced pancreatitis, metabolic syndrome, type 2 diabetes, familial chylomicronemia syndrome, or familial partial lipodystrophy.

In some embodiments, the disease may be a thromboembolic complication. In some embodiments, the thromboembolic complication may be deep vein thrombosis, pulmonary embolism, myocardial infarction, or stroke.

On the other hand, the present disclosure provides a method for reducing low-density lipoprotein levels in a subject, which includes administering to the subject an effective amount or an effective dose of the above-mentioned oligonucleotide or the above-mentioned pharmaceutical composition.

In another aspect, the present disclosure provides a method of reducing apolipoprotein(a) and/or apolipoprotein(a) levels in a subject, which includes administering to the subject an effective amount or an effective dose of the above. Oligonucleotides or pharmaceutical compositions as described above.

On the other hand, the present disclosure provides a method for silencing a target gene or its mRNA in a cell in vivo or in vitro, which includes the step of introducing the above-mentioned oligonucleotide or the above-mentioned pharmaceutical composition into the cell.

In some embodiments, an oligonucleotide or pharmaceutical composition of the present disclosure can reduce the expression level of a target gene or its mRNA in a cell, cell population, tissue, or subject, including: administering a therapeutically effective amount to the subject An oligonucleotide or pharmaceutical composition described herein, thereby inhibiting the expression of a target gene or its mRNA in a subject.

In some embodiments, the subject has been previously identified as having pathological upregulation of the target gene or its mRNA in the targeted cell, cell population, tissue, or subject.

On the other hand, the present disclosure provides a method of delivering an oligonucleotide to the liver, which includes administering an effective amount or an effective dosage of the above-mentioned oligonucleotide or the above-mentioned pharmaceutical composition to a subject.

On the other hand, the present disclosure also provides a cell comprising the above-mentioned oligonucleotide or the above-mentioned pharmaceutical composition.

On the other hand, the present disclosure also provides a kit comprising the above-mentioned oligonucleotide or the above-mentioned pharmaceutical composition.

In the present disclosure, when the above-mentioned oligonucleotide or pharmaceutical composition comes into contact with cells expressing the target gene, it is detected by, for example: psiCHECK activity screening and luciferase reporter gene detection methods, other methods such as PCR or branched DNA (bDNA)-based methods. , or protein-based methods, such as immunofluorescence analysis, such as Western Blot or flow cytometry, the above oligonucleotide or pharmaceutical composition will inhibit the expression of the target gene by at least 5%, at least 10%, at least 15% , at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

In the present disclosure, when the above-mentioned oligonucleotide or pharmaceutical composition comes into contact with cells expressing the target gene, it is detected by, for example: psiCHECK activity screening and luciferase reporter gene detection methods, other methods such as PCR or branched DNA (bDNA)-based methods. , or protein-based methods such as immunofluorescence assays, such as Western Blot Or measured by flow cytometry, the remaining expression percentage of the target gene mRNA caused by the above-mentioned oligonucleotide or pharmaceutical composition is no more than 99%, no more than 95%, no more than 90%, no more than 85%, Not higher than 80%, not higher than 75%, not higher than 70%, not higher than 65%, not higher than 60%, not higher than 55%, not higher than 50%, not higher than 45%, not higher No more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%.

In the present disclosure, when the above-mentioned oligonucleotide or pharmaceutical composition comes into contact with cells expressing the target gene, it is detected by, for example: psiCHECK activity screening and luciferase reporter gene detection methods, other methods such as PCR or branched DNA (bDNA)-based methods. , or protein-based methods, such as immunofluorescence assays, such as Western Blot, or flow cytometry, the oligonucleotide reduces off-target activity by at least 20%, at least 25%, while maintaining target activity. 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%.

In the present disclosure, when the above-mentioned oligonucleotide or pharmaceutical composition comes into contact with cells expressing the target gene, it is detected by, for example: psiCHECK activity screening and luciferase reporter gene detection methods, other methods such as PCR or branched DNA (bDNA)-based methods. , or protein-based methods such as immunofluorescence assays, such as Western Blot, or flow cytometry, oligonucleotides reduce on-target activity by up to 20%, up to 19%, up to 15%, up to 10%, Up to 5% or more than 1% while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60% , at least 65%, at least 70%, or at least 75%.

The present disclosure also provides a method of preparing an oligonucleotide or pharmaceutical composition, which includes: synthesizing the oligonucleotide or pharmaceutical composition described in the present disclosure.

Unless otherwise specified, the terms “nucleic acid”, “oligonucleotide”, “single-stranded oligonucleotide”, “double-stranded oligonucleotide”, “antisense oligonucleotide” and “small interfering RNA” in this disclosure (siRNA)", "double-stranded RNA (dsRNA)", "microRNA (miRNA)", "short hairpin RNA (shRNA)", "ribozyme", "RNAi inhibitor molecule", "double-stranded RNAi inhibitor" "Molecular" and "Dicer enzyme substrate" and "compound" may independently exist in the form of salts, mixed salts, or non-salts (eg, free acids or free bases). When present in the form of a salt or mixed salts, they may be pharmaceutically acceptable salts.

The term "pharmaceutically acceptable salts" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

In some embodiments, "pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include but are not limited to hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, etc.; organic acid salts include but are not limited to formates, acetates, and 2,2-dichloroacetates. , trifluoroacetate, propionate, caproate, caprylate, decanoate, undecenoate, glycolate, gluconate, lactate, sebacate, adipate Acid, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate , cinnamate, laurate, malate, glutamate, coke Amate, aspartate, benzoate, mesylate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate , naphthalene disulfonate, etc. These salts can be prepared by methods known in the art.

"Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that can maintain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salt, sodium salt, potassium salt, calcium salt and magnesium salt, with sodium salt being preferred. Salts derived from organic bases include, but are not limited to, the following salts: 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, bicyclic Hexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperazine Biridine, N-ethylpiperidine, polyamine resin, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the art.

Without specifying the configuration, the compounds of the present disclosure (such as oligonucleotides, siRNA, chemical modifications represented by formula (I), targeting ligands, compounds represented by formula (IV), etc.) may exist in specific Geometric or stereoisomeric forms. This disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof and other mixtures, such as enantiomeric or diastereomerically enriched mixtures, all of which are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of this disclosure. Compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically active pure form or racemic form. Optically active pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

The optically active (R)- and (S)-isomers as well as the 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 can be prepared by asymmetric synthesis or derivatization with chiral auxiliaries, where the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with a suitable optically active acid or base, and then the salt is formed by conventional methods known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally combined with chemical derivatization methods (e.g., generation of amino groups from amines). formate).

Without specifying the configuration, the chemistry of the compounds described in the present disclosure (such as oligonucleotides, siRNA, chemical modifications represented by formula (I), targeting ligands, compounds represented by formula (IV), etc.) structure, key Indicates that the configuration is not specified, i.e. if there are chiral isomers in the chemical structure, the bond can be or both Two configurations. In the chemical structure of the compounds described in this disclosure, the bond No configuration is specified, i.e. the key The configuration can be E-type or Z-type, or contain both E and Z configurations.

The present disclosure also includes some isotopically labeled compounds that are the same as those described herein, but in which one or more atoms are replaced by an atom having an atomic weight or mass number different from that typically found in nature. Examples of isotopes that may be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹³ respectively N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ¹²³ I, ¹²⁵ I and ³⁶ Cl, etc.

Compared with non-deuterated drugs, deuterated drugs have the advantages of reducing side effects, increasing drug stability, enhancing efficacy, and extending the biological half-life of the drug. All variations in the isotopic composition of the compounds of the present disclosure, whether radioactive or not, are included within the scope of the present disclosure. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom, where the replacement of deuterium can be partial or complete. The replacement of partial deuterium means that at least one hydrogen is replaced by at least one deuterium.

Unless otherwise stated, in a compound of the present disclosure, when a position is specifically designated as "deuterium" or "D", that position is understood to have an abundance of deuterium greater than the natural abundance of deuterium, which is 0.015%. At least 1000 times (i.e. at least 15% deuterium incorporation). In some embodiments, the abundance of deuterium per designated deuterium atom is at least 1000 times greater than the natural abundance of deuterium (ie, at least 15% deuterium incorporation). In some embodiments, the abundance of deuterium per designated deuterium atom is at least 2000 times greater than the natural abundance of deuterium (ie, at least 30% deuterium incorporation). In some embodiments, the abundance of deuterium per designated deuterium atom is at least 3000 times greater than the natural abundance of deuterium (ie, at least 45% deuterium incorporation). In some embodiments, the abundance of deuterium per designated deuterium atom is at least 3340 times greater than the natural abundance of deuterium (ie, at least 50.1% deuterium incorporation). In some embodiments, the abundance of deuterium per designated deuterium atom is at least 3500 times greater than the natural abundance of deuterium (ie, at least 52.5% deuterium incorporation). In some embodiments, the abundance of deuterium per designated deuterium atom is at least 4000 times greater than the natural abundance of deuterium (ie, at least 60% deuterium incorporation). In some embodiments, the abundance of deuterium per designated deuterium atom is at least 4500 times greater than the natural abundance of deuterium (ie, at least 67.5% deuterium incorporation). In some embodiments, the abundance of deuterium per designated deuterium atom is at least 5000 times greater than the natural abundance of deuterium (ie, at least 75% deuterium incorporation). In some embodiments, the abundance of deuterium per designated deuterium atom is at least 5500 times greater than the natural abundance of deuterium (ie, at least 82.5% deuterium incorporation). In some embodiments, the abundance of deuterium per designated deuterium atom is at least 6000 times greater than the natural abundance of deuterium (ie, at least 90% deuterium incorporation). In some embodiments, the abundance of deuterium per designated deuterium atom is at least 6333.3 times greater than the natural abundance of deuterium (ie, at least 95% deuterium incorporation). In some embodiments, the abundance of deuterium per designated deuterium atom is at least 6466.7 times greater than the natural abundance of deuterium (ie, at least 97% deuterium incorporation). In some embodiments, the abundance of deuterium per designated deuterium atom is at least 6600 times greater than the natural abundance of deuterium (ie, at least 99% deuterium incorporation). In some embodiments, the abundance of deuterium per designated deuterium atom is at least 6633.3 times greater than the natural abundance of deuterium (ie, at least 99.5% deuterium incorporation). Those skilled in the art can refer to relevant literature to synthesize the deuterated form of compound. Commercially available deuterated starting materials may be used in the preparation of deuterated forms of the compounds, or they may be synthesized using conventional techniques using deuterated reagents including, but not limited to, deuterated borane, trideuterated borane in tetrahydrofuran. , Deuterated lithium aluminum hydride, deuterated ethyl iodide and deuterated methyl iodide, etc.

In addition, without specifying the configuration, the compounds and intermediates of the present disclosure (such as oligonucleotides, siRNA, chemical modifications represented by formula (I), targeting ligands, compounds represented by formula (IV) etc.) may also exist in different tautomeric forms, and all such forms are included within the scope of this 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 called proton transfer tautomers) include tautomers via proton migration, such as keto-enol and imine-enamine, lactam-lactam isomerizations . An example of a lactam-lactam equilibrium is between A and B as shown below,

All tautomeric forms are within the scope of this disclosure. The naming of the compounds does not exclude any tautomers.

This disclosure incorporates the full text of WO2023274395A and PCT/CN2022/139500.

Terminology explanation

In order to make the present disclosure easier to understand, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined 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.

Unless otherwise specified, in the context of this disclosure, the terms "apolipoprotein(a) gene", "Apo(a) gene", and "LPA" are used interchangeably in this disclosure. LPA includes but is not limited to human LPA, cynomolgus LPA, mouse LPA, and rat LPA. Its amino acid, complete coding sequence, and mRNA sequence are easily obtained using published databases, such as GenBank, UniProt, OMIM, and macaque (Macaca). Genome Project website.

The term "target sequence" refers to the contiguous portion of the nucleotide sequence of the mRNA molecule formed during the transcription of LPA, including the mRNA as the primary transcript product of RNA processing. The targeted portion of the target sequence should be long enough to serve as a substrate for iRNA-directed cleavage. In one embodiment, the target sequence is within the protein coding region of LPA.

As used herein, in the context of RNA-mediated gene silencing, the sense strand (also known as SS, SS strand, or sense strand) refers to the strand that contains the same or substantially the same sequence as the target mRNA sequence; antisense A strand (also known as AS or AS strand) refers to a strand that has a sequence complementary to the target mRNA sequence.

In the context of describing the sense strand described herein, the term "sense strand comprising at least 15 consecutive nucleotides of SEQ ID NO:1" is intended to mean that the sense strand described herein comprises at least 15 consecutive nucleotides of SEQ ID NO:1 consecutive nucleotides, optionally, the sense strand described herein includes at least 16 consecutive nucleotides of SEQ ID NO:1 Nucleotides, the sense strand described herein includes at least 18 consecutive nucleotides of SEQ ID NO: 1, and the sense strand described herein includes at least 19 consecutive nucleotides of SEQ ID NO: 1.

In the context of describing an antisense strand as described herein, the term "antisense strand comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 2" is intended to mean that the antisense strand as described herein comprises SEQ ID NO:2 At least 15 consecutive nucleotides of SEQ ID NO: 2. Optionally, the antisense strand described herein includes at least 16 consecutive nucleotides of SEQ ID NO: 2. The antisense strand described herein includes SEQ ID NO. : at least 18 consecutive nucleotides of SEQ ID NO:2, the antisense strand described herein includes at least 19 consecutive nucleotides of SEQ ID NO:2, the antisense strand described herein includes at least 20 of SEQ ID NO:2 Contiguous nucleotides, the antisense strand described herein includes at least 21 contiguous nucleotides of SEQ ID NO: 2.

Unless otherwise specified, in the context of this disclosure, "G", "C", "A", "T" and "U" respectively represent nucleotides, which respectively include guanine, cytosine, adenine, thymidine and The base of uracil. The lowercase letter m indicates that the nucleotide adjacent to the upstream of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the upstream of the letter f is a fluoro-modified nucleotide; lowercase letter The letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate diester groups.

In the present disclosure, unless otherwise specified, the phosphate group between natural nucleotides is a phosphodiester group.

In the present disclosure, a phosphorothioate diester group refers to a phosphodiester group in which a non-bridging oxygen atom is replaced by a sulfur atom. (M is S atom) are used interchangeably.

As used in this disclosure, the term "2'-fluoromodified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by fluorine. "Non-2'-fluoromodified nucleotide" "Nucleotide" refers to a nucleotide or nucleotide analogue formed by replacing the 2' hydroxyl group of the ribose group with a non-fluorine group.

As used in this disclosure, the term "2'-methoxy modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group is replaced by a methoxy group.

In the context of this disclosure, "nucleotide differences" and "no more than n nucleotide differences" between one nucleotide sequence and another nucleotide sequence refer to the fact that the former has the same position as the latter compared to the latter. The base type of the nucleotide has changed. For example, when one nucleotide base is A in the latter, the corresponding nucleotide base at the same position in the former is U, C, G or T. , it is considered that there is a nucleotide difference at this position between the two nucleotide sequences, or a difference of one nucleotide. In some embodiments, when the nucleotide at the original position is replaced by an abasic nucleotide or its equivalent at a position that is not a chemical modification represented by formula (I) or a tautomer modification thereof, it can also be It is believed that a nucleotide difference occurs at this position.

As used herein, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are in contact with the bases of the other strand. The bases in the strand are paired in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base Pairs with cytosine (G). Each base pair consists of 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 said to be complementary to each other, and from their complementary strands The sequence of the chain can be inferred from the sequence. Correspondingly, "mismatch" in this field means that in double-stranded nucleic acids, the bases at corresponding positions do not pair in a complementary manner.

As used herein, the term "inhibition," may be used interchangeably with "reduction," "silencing," "downregulation," "repression" and other similar terms and includes any level of inhibition.

"Effective amount" or "effective dosage" refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results. For prophylactic uses, beneficial or desired results include elimination or reduction of risk, reduction of severity, or delay of onset of a condition, including biochemical, histological, and biological aspects of the condition, its complications, and intermediate pathological phenotypes present during the development of the condition. academic and/or behavioral symptoms. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of, or ameliorating one or more symptoms of, various target gene, target mRNA, or target protein-related disorders of the present disclosure, reducing the treated disorder The dosage of the other agent required to enhance the efficacy of the other agent and/or delay the progression of a disorder associated with the target gene, target mRNA or target protein of the present disclosure in a patient.

In some embodiments, the effective amount or dosage of the oligonucleotide, double-stranded RNAi inhibitor molecule or siRNA is from about 0.001 mg/kg body weight to about 200 mg/kg body weight, from about 0.01 mg/kg body weight to about 100 mg/kg Body weight or about 0.5 mg/kg body weight to about 50 mg/kg body weight.

"Pharmaceutical composition" includes the oligonucleotide, double-stranded RNAi inhibitor molecule or siRNA of the present disclosure and pharmaceutically acceptable excipients and/or adjuvants. The excipients can be one or more various substances commonly used in the art. a preparation or compound. For example, the pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

As used herein, "subject," "patient," "subject" or "individual" are used interchangeably and include humans or non-human animals, such as mammals, such as humans or monkeys.

The oligonucleotides, double-stranded RNAi inhibitor molecules or siRNA provided by the present disclosure can be obtained by conventional preparation methods in the art (such as solid phase synthesis and liquid phase synthesis methods). Among them, solid-phase synthesis already has commercial customization services. Nucleoside monomers with corresponding modifications can be prepared by using nucleoside monomers with corresponding modifications to introduce modified nucleotide groups into oligonucleotides, double-stranded RNAi inhibitor molecules, or siRNAs described in the present disclosure. Methods and methods of introducing modified nucleotide groups into oligonucleotides, double-stranded RNAi inhibitor molecules or siRNA are also well known to those skilled in the art.

The term "chemical modification" or "modification" includes all changes in nucleotides by chemical means, e.g. The addition or removal of moieties, or the substitution of one chemical moiety for another.

The term "base" includes any known DNA and RNA base, base analogues, such as purine or pyrimidine, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and Natural analogues. Base analogs can also be universal bases.

The terms "blunt end" or "blunt end" are used interchangeably and mean that there are no unpaired nucleotides or nucleotide analogs at a given end of the siRNA, ie, no nucleotide overhangs. In most cases, an siRNA that is blunt-ended at both ends will be double-stranded throughout its length.

The terms "about" and "approximately" mean that a value is within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "consisting essentially of" may mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% It varies between 0.5% and 1%. In this disclosure, every instance in which a number or numerical range is preceded by the term "about" also includes the embodiment of the given number. Unless stated otherwise, when a specific value appears in this application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

The terms "optionally" or "optionally" are intended to mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance does or does not occur. For example, "C _1-6 alkyl optionally substituted by halogen or cyano group" means that halogen or cyano group may but does not have to be present. This description includes the case where the alkyl group is substituted by halogen or cyano group and the alkyl group is not substituted by halogen. and the case of cyano substitution.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group that is a straight or branched chain group containing 1 to 20 carbon atoms, and in some embodiments is selected from alkyl groups containing 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 -Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2 -Methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethyl Pentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2 ,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethyl Hexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethyl Hexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers, etc. In some embodiments selected from alkyl groups containing 1 to 6 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl base, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methyl Butyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethyl Propyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, which in some embodiments are selected from one or more of the following groups, which Independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, hetero Aryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate group.

The term "alkoxy" refers to -O-(alkyl), where alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy. The alkoxy group may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from halogen, hydroxyl, oxo, cyano, amino, C _{1 -6} alkyl, C _1-6 alkoxy, 3 to 7-membered cycloalkyl or 3 to 7-membered heterocycloalkyl, the alkyl, alkoxy, cycloalkyl or heterocycloalkyl is optionally Substituted by halogen, hydroxyl, nitro, cyano or amino.

The term "alkylthio" refers to -S-(alkyl), where alkyl is as defined above. Non-limiting examples of alkylthio groups include: methylthio, ethylthio, propylthio, butylthio. The alkylthio group may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from C _1-6 alkoxy, 3 to 6 membered cycloalkyl base, 3 to 6-membered heterocycloalkyl group, 3 to 6-membered cycloalkoxy group, 3 to 6-membered heterocycloalkoxy group, C _1-6 alkylthio group, 3 to 6-membered cycloalkylthio group, 3 to 6 One-membered heterocycloalkylthio group, the alkoxy group, cycloalkyl group, heterocycloalkyl group, cycloalkoxy group, heterocyclic oxy group, alkylthio group, cycloalkylthio group and heterocycloalkylthio group are optionally replaced by halogen , hydroxyl, cyano or amino substituted.

The term "alkenyl" refers to a straight or branched chain non-aromatic hydrocarbon radical containing at least one carbon-carbon double bond and having 2 to 10 carbon atoms. Up to 5 carbon-carbon double bonds can be present in such groups. For example, "C _2- C ₆ "alkenyl is defined as an alkenyl group having 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to: vinyl, propenyl, butenyl, and cyclohexenyl. The linear, branched or cyclic portion of the alkenyl group may contain double bonds and is optionally mono-, di-, tri-, tetra- or penta-substituted at any position permitted by normal valency.

The term "alkynyl" refers to a straight or branched chain hydrocarbon group containing 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Up to 5 carbon-carbon triple bonds can be present. Thus, "C ₂ -C ₆ alkynyl" means an alkenyl group having 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to: ethynyl, 2-propynyl, and 2-butynyl. The linear, branched portion of the alkynyl group may contain as many triple bonds as normal valency allows, and may be optionally mono-, di-, tri-, tetra- or penta-substituted at any position permitted by normal valency.

The term "cycloalkyl" or "carbocycle" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent. The cycloalkyl ring contains 3 to 20 carbon atoms, and in some embodiments is selected from the group consisting of 3 to 20 carbon atoms. 7 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, etc.; polycyclic cycloalkyl groups include spiro Cycloalkyl groups of rings, fused rings and bridged rings. Cycloalkyl groups may be substituted or unsubstituted, and when substituted, the substituents may be taken at any available point of attachment. Generation, in some embodiments selected from one or more of the following groups, independently selected from halogen, deuterium, hydroxyl, oxo, nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy , C _2-6 alkenyloxy group, C _2-6 alkynyloxy group, C _3-6 cycloalkoxy group, 3 to 6 membered heterocycloalkoxy group, C _3-8 cycloalkenyloxy group, 5 to 6 membered aromatic group base or heteroaryl, the C _1-6 alkyl, C 1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy _, 3 to 6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5 to 6-membered aryl or heteroaryl are optionally substituted by one or more members selected from halogen, deuterium, hydroxyl, oxo, nitro, and cyano. replaced.

The cycloalkyl ring can be fused to an aryl or heteroaryl ring, where the ring attached to the parent structure is a cycloalkyl ring, non-limiting examples include indanyl, tetrahydronaphthyl, benzo ring Heptyl etc. Cycloalkyl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups, which are independently selected from halogen, deuterium, hydroxyl, oxo, Nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenoxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 yuan Heterocycloalkoxy, C _3-8 cycloalkenoxy, 5 to 6-membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenoxy , C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5 to 6-membered aryl or heteroaryl are optionally One or more substitutes selected from halogen, deuterium, hydroxyl, oxo, nitro, and cyano.

The term "heterocycloalkyl" or "heterocycle" or "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms, one or more of which The atoms are heteroatoms selected from nitrogen, oxygen or S(O) _m (where m is an integer from 0 to 2), but excluding the ring part of -OO-, -OS- or -SS-, the remaining ring atoms are carbon. In some embodiments, it is selected from 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; in some embodiments, it is selected from 3 to 7 ring atoms. Non-limiting examples of monocyclic heterocycloalkyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuryl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuryl, dihydropyrazolyl, dihydropyrrolyl, piperazoyl Aldyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocycloalkyl groups include spiro, fused and bridged heterocycloalkyl groups. Non-limiting examples of "heterocycloalkyl" include:

etc.

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

wait.

Heterocycloalkyl may be optionally substituted or unsubstituted, and when substituted, the substituent in some embodiments is selected from one or more of the following groups, which are independently selected from halogen, deuterium, hydroxyl, oxo , nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenoxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 1-membered heterocycloalkoxy, C _3-8 cycloalkenoxy, 5 to 6-membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyl base, C _2-6 alkynyloxy group, C _3-6 cycloalkoxy group, 3 to 6 membered heterocycloalkoxy group, C _3-8 cycloalkenyloxy group, 5 to 6 membered aryl group or heteroaryl group optional Substituted by one or more selected from halogen, deuterium, hydroxyl, oxo, nitro, and cyano.

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

Aryl groups may be substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups, which are independently selected from halogen, deuterium, hydroxyl, oxo, nitro, Cyano, C _1-6 alkyl, C 1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to ₆ -membered heterocycloalkane Oxy group, C _3-8 cycloalkenyloxy group, 5 to 6-membered aryl group or heteroaryl group, the C _1-6 alkyl group, C _1-6 alkoxy group, C _2-6 alkenyloxy group, C _{2 -6} alkynyloxy, C _3-6 cycloalkoxy, 3 to 6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5 to 6-membered aryl or heteroaryl are optionally substituted by one or more Each is selected from the group consisting of halogen, deuterium, hydroxyl, oxo, nitro, and cyano.

The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is selected from 6- to 12-membered in some embodiments, and in some embodiments is selected from 5- or 6-membered. For example. Non-limiting examples thereof include: imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl , Thiadiazole, pyrazinyl, triazolyl, indazolyl, benzimidazolyl, wait.

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

Heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups, which are independently selected from halogen, deuterium, hydroxyl, oxo, Nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenoxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 yuan Heterocycloalkoxy, C _3-8 cycloalkenoxy, 5 to 6-membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenoxy , C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5 to 6-membered aryl or heteroaryl are optionally One or more substitutes selected from halogen, deuterium, hydroxyl, oxo, nitro, and cyano.

The term "alkylamino" refers to a group having the structure -NH (C1-C12 alkyl).

The term "hydroxyalkyl" refers to an alkyl group substituted with one or more hydroxyl groups, where alkyl is as defined above.

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

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

The term "haloalkyl" refers to an alkyl group substituted by halogen, wherein alkyl is as defined above.

The term "haloalkoxy" refers to an alkoxy group substituted by halogen, wherein alkoxy is as defined above.

The term "cyano" refers to -CN.

The term "nitro" refers to _-NO2 .

The term "oxo" refers to an =O group, for example, a carbon atom linked to an oxygen atom by a double bond, whereby a ketone or aldehyde group is formed.

The term "amino" refers to _-NH2 .

The term "carboxy" refers to -C(O)OH.

In the chemical structural formula of the present disclosure, One or more groups according to the scope of the invention described herein may be attached; an asterisk "*" indicates a chiral center.

The term "connection", when referring to a connection between two molecules, means that the two molecules are connected by a covalent bond or that the two molecules are associated via a non-covalent bond (e.g., hydrogen bonding or ionic bonding), including direct connection, indirect connection, connect.

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 linked" means that a first compound or group is connected to a second compound or group through an intervening group, compound or molecule (eg, a linking group).

The term "substituted" means that any one or more hydrogen atoms on a designated atom (generally carbon, oxygen, and nitrogen atoms) are replaced by any group defined herein, provided that the normal valency of the designated atom is not exceeded and Substitution produces stable compounds. Non-limiting examples of substituents include C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cyano, hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, heterocyclic Aryl, aryl, ketone, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl or halogen (eg, F, Cl, Br, I). When the substituent is a ketone or oxo (ie, =O), then two (2) hydrogens on the atom are replaced.

"Substituted by one or more" means that it may be substituted by single or multiple substituents. When substituted by multiple substituents, it may be a plurality of the same substituent, or it may be one or a combination of multiple different substituents.

The term "protecting group" is used in the conventional chemical sense as a group that reversibly renders a functional group unreactive under certain conditions of the desired reaction. After the desired reaction, the protecting group can be removed to deprotect the protected functional group. All protecting groups should be removable without degrading a significant proportion of the molecule being synthesized.

Some abbreviations used in this disclosure are defined as follows:
DCE: 1,2-dichloroethane;
Sc(OTf) ₃ : scandium trifluoromethanesulfonate;
THF: tetrahydrofuran;
Pd/C: palladium-carbon;
TFA: trifluoroacetic acid;
DMF: dimethylformamide;
DIPEA: N-ethyldiisopropylamine;
HoBt: 1-hydroxybenzotriazole;
EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;
DMTrCl: 4,4'-bismethoxytrityl chloride;
DIEA: N,N-diisopropylethylamine;
HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate;
LiOH: lithium hydroxide;
DMAP: 4-dimethylaminopyridine;
HBTU: benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate;
CF ₃ SO ₃ H: trifluoromethanesulfonic acid;
BnBr: benzyl bromide;
DEPBT: 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4-one;
Bz: benzoyl protecting group;
MMTr: methoxyphenyl diphenylmethyl;
DMTr: dimethoxytrityl protecting group;
Piv: pivaloyl.

Description of the drawings

Figure 1 shows the expression of mRNA in TTR of TRD002218 and TRD007205 7 days after administration.

Figure 2 shows the expression of mRNA in TTR of TRD002218 and TRD007205 28 days after administration.

Figure 3 shows the serum Lp(a) content of TJR100747, TJR100848 and TJR100553 mice on day 21. ** indicates the comparison between each group and the TJR100553 control, P<0.01.

Figure 4 shows the serum Lp(a) content of TJR100747, TJR100848 and TJR100553 mice on day 28. ** indicates that each group is compared with the TJR100553 control, P<0.01; * indicates that each group is compared with the TJR100553 control, P<0.1.

Detailed ways

The present disclosure is further described below in conjunction with examples, but these examples do not limit the scope of the disclosure. Experimental methods without specifying specific conditions in the examples of this disclosure usually follow conventional conditions or conditions recommended by raw material or product manufacturers. If a specific source of reagent is not stated, the reagent can be obtained from any supplier of molecular biology reagents in a quality/purity suitable for molecular biology applications.

Example

Example 1. Synthesis of targeting ligands

Example 1.1 Preparation of NAG0052, L96

Compounds NAG0024 and NAG0026 were purchased from Tianjin WuXi AppTec New Drug Development Co., Ltd. Unless otherwise stated, the reagents used in the following examples are all commercially available products.

(1) Synthesis of compound NAG0052

The starting material compound 1 was purchased from Jiangsu Beida Pharmaceutical Technology Co., Ltd.

The specific preparation process of NAG0052 is as follows:

Compound 2

To a solution of compound 1 (12.3 mL, 101 mmol) in THF (300 mL) at 0° C. and nitrogen protection, NaH (12.2 g, 304 mmol, purity 60%) was added in portions. After the mixture was stirred at 20°C for 1 hour, it was cooled to 0°C again, then benzyl bromide (36.3 mL, 304 mmol) was added dropwise to the system, and stirred at 20°C for 12 hours. The reaction solution was quenched with H ₂ O (100 mL), and then extracted with EtOAc (200 mL×2). The combined organic phases were washed with saturated brine (100 mL), dried over Na ₂ SO ₄ , filtered, and concentrated. The residue obtained was separated by silica gel column chromatography to obtain the target compound 2 (20.0 g, 51.8 mmol, yield 51%).

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] ⁺ .

¹ H NMR: (400MHz, CDCl ₃ )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).

Compound 3 and Compound 4

At 20°C and under nitrogen protection, TMSCN (13.5 mL, 101 mmol) was added in one go to a solution of compound 2 (13.0 g, 33.6 mmol) in DCM (300 mL), and then TMSOTf (9.14 mL, 50.5 mmol) in DCM was added dropwise. (30mL) solution. The reaction solution was stirred at 20°C for 15 hours. After the reaction, the system was quenched with saturated NaHCO ₃ aqueous solution (80 mL), and extracted with DCM (150 mL × 2). The combined organic phases were washed with saturated brine (80 mL), dried over Na ₂ SO ₄ , filtered and concentrated. , separated by silica gel column chromatography, and obtained target compound 3 (3.30g, 9.18mmol, yield 27%) and light yellow oily liquid compound 4 (8.50g, 9.18mmol, yield 70%).

Compound 3

¹ H NMR: (400MHz, CDCl ₃ ) 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

¹ H NMR: (400MHz, CDCl ₃ ) 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 (3.00g, 9.28mmol) in THF (15mL) was added dropwise to a solution of LiAlH ₄ (0.79g, 20.9mmol) in THF (15mL) at 0°C under nitrogen protection. After the dropwise addition, the system was React at 0°C for 1 hour. TLC (PE:EtOAc=3:1) detected the complete disappearance of the starting material. Slowly add sodium sulfate decahydrate to the reaction solution until bubbling stops. Afterwards, the reaction solution was filtered, and the filter cake was washed three times with dichloromethane (60 mL). The filtrate was collected and spin-dried to obtain target compound 5 (3.00 g, yield 90%).

¹ H NMR: (400MHz, DMSO-d ₆ ) 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

Under nitrogen protection, compound 5 (3.00g, 8.25mmol) was dissolved in DCM (30mL), TEA (3.44mL, 24.7mmol) and CbzCl (1.76mL, 12.4mmol) were added, and the reaction was carried out at 20°C for 2 hours. LCMS showed the reaction was complete. Dichloromethane (30 mL) and water (60 mL) were added to the reaction liquid and extracted. The organic phase was washed three times with water (60 mL×3), dried over anhydrous sodium sulfate, concentrated and purified by forward column (PE:EtOAc=1:1) to obtain target compound 6 (2.5 g, yield 90%).

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

¹ H NMR: (400MHz, CDCl ₃ ) 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

Under nitrogen protection, compound 6 (2.00g, 3.90mmol) was dissolved in DCM (5mL), BCl ₃ in THF solution (1M, 27.3mL) was added at -78°C, and the reaction was carried out for 1 hour. TLC (DCM:MeOH=10:1) detected the complete disappearance of the starting material. The reaction solution was quenched by adding methanol (20 mL) at -78°C, concentrated, and purified with a forward column (DCM:MeOH=10:1) to obtain target compound 7 (2.00 g, yield 60%).

¹ H NMR: (400MHz, CD ₃ OD) 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

Under nitrogen protection, compound 7 (0.50g, 1.78mmol) was dissolved in pyridine (5mL), 4A molecular sieve (500mg) and DMTrCl (0.66mL, 2.13mmol) were added at 0°C, and then the temperature was raised to 20°C for reaction 1.5 Hour. TLC (PE:EtOAc=2:1) detected the complete disappearance of the starting material. The reaction solution was extracted with ethyl acetate (60 mL) and water (60 mL). The organic phase was washed three times with water (60 mL × 3), dried over anhydrous sodium sulfate, concentrated, and purified with a forward column (PE:EtOAc=1:1 ) to obtain target compound 8 (800 mg, yield 90%).

¹ H NMR: (400MHz, CDCl ₃ ) 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), Pd/C 10% (800 mg, 7.517 mmol) was added, and the reaction was carried out under H ₂ conditions (15 Psi) and 20°C for 1 hour. LCMS showed the reaction was complete. The reaction solution was filtered, and the filter cake was washed three times with dichloromethane (100 mL) and methanol (100 mL), concentrated, and separated by reverse-phase column chromatography to obtain compound 9 (300 mg, 54%).

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

Compound 11

Dissolve compound 10 (435mg, 1.780mmol) in DCM (10mL), add DIEA (0.441mL, 2.67mmol) and HATU (677mg, 1.78mmol), then add compound 9 (400mg, 0.890mmol), and react at 20°C 1 Hour. TLC (DCM:MeOH=10:1) monitored the reaction completion. The reaction solution was extracted with dichloromethane (60 mL) and water (60 mL). The organic phase was washed three times with water (60 mL × 3), dried over anhydrous sodium sulfate, concentrated and purified by forward column (PE:EtOAc=0:1). , the product peak appeared at 100%), and the target compound 11 (600 mg, yield 90%) was obtained.

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

¹ H NMR: (400MHz, CD ₃ OD) 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 H ₂ O (1 mL), LiOH.H ₂ O (134 mg, 3.20 mmol) was added, and the reaction was carried out at 20°C for 12 hours. TLC (DCM:MeOH=10:1) showed the reaction was complete. Spin the reaction solution to dryness, dissolve it in water (5 mL) and methanol (5 mL), and purify it with a reverse column (H ₂ O: CH ₃ CN = 1:1, peak at about 35%) to obtain target 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.

¹ H NMR: (400MHz, CD ₃ OD) 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

Dissolve compound NAG0024 (271 mg, 0.151 mmol) in anhydrous THF (2 mL) and anhydrous DMF (4 mL) at room temperature under nitrogen protection, add 3A molecular sieve, and then add compound 12 (100 mg, 0.151 mmol), HOBt ( 25mg, 0.181mmol), DCC (38mg, 0.181mmol) and DIEA (39mg, 0.302mmol). The reaction solution was reacted at 45°C for 16 hours. After LC-MS showed that the reaction was complete, water was added to quench and filtered. After the filtrate was concentrated, it was purified by C18 reverse-phase column chromatography (H ₂ O/MeCN) to obtain compound 13 (210 mg, yield 57%).

Compound NAG0052

At room temperature, compound 13 (230 mg, 0.094 mmol) was dissolved in pyridine (5 mL), molecular sieves were added, DMAP (12 mg, 0.283 mmol), and succinic anhydride (28 mg, 0.283 mmol) were added. Under nitrogen protection, stir at 50°C for 16 hours. LCMS detects that the reaction is complete, filter and spin dry. After purification by C18 reversed-phase column chromatography, and secondary purification by preparative HPLC (acetonitrile/water, gradient elution ratio from 0% acetonitrile to 100% acetonitrile), the target compound NAG0052 (123 mg, 0.048 mmol, yield 51%) was obtained. .

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

¹ H NMR (400MHz, acetonitrile-d ₃ ) 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).

Synthesis of L96

L96 shown in the above structural formula was prepared according to the method described in patent application WO2014025805A1.

Example 1.2 Synthesis of siRNA conjugates containing NAG0052

1. Homemade resin with carrier

Dissolve the compound NAG0052 (157mg, 0.062mmol) containing a carboxylic acid group in anhydrous DMF (3mL). After the substrate is completely dissolved, add anhydrous acetonitrile (4mL) and DIEA (0.03mL, 0.154mmol, 2.5eq) in sequence. ) and HBTU (35mg, 0.093mmol, 1.5eq). After the reaction solution is mixed evenly, macroporous amine methyl resin (476 mg, blank loading is 0.41 mmol/g, target loading is 0.1 mmol/g) is added. Place the reaction solution on a shaker (temperature: 25°C, rotation speed: 200 rpm) and shake overnight. The reaction solution was filtered, and the filter cake was washed with DCM and anhydrous acetonitrile in sequence. The solids were collected and dried under vacuum overnight.

Disperse the solid from the previous step in anhydrous acetonitrile (5 mL), and add pyridine (0.18 mL), DMAP (3 mg), NMI (0.12 mL) and CapB1 (2.68 mL) in sequence. Place the reaction solution on a shaker (temperature: 25°C, rotation speed: 200 rpm) and shake for 2 hours. The reaction solution was filtered, and the filter cake was washed with anhydrous acetonitrile. The solid was collected and dried under vacuum overnight to obtain a resin with a carrier. The loading capacity was determined to be 0.1mmol/g.

2. For NAG0052 that has been connected to the resin, use the resin as a starting point and connect the nucleoside monomers one by one from the 3’-5’ direction in the order of nucleotide arrangement. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfation, and the operations are routine in the art. Compound NAG0052 is connected to the sequence through solid-phase synthesis, and after aminolysis, part of the functional groups of the NAG0052 structure is removed to become NAG0052’.

The prepared siRNA conjugates had the sense strand and antisense strand shown in Table 1 and Table 2.

Table 1. List of siRNA conjugates

Table 2. Nucleic acid sequences of sense strand and antisense strand

Table 3. Structure of conjugates

Among them, conjugate TRD002218 is used as the reference positive compound, and Z represents siRNA.

Example 2. Inhibition of target gene mRNA expression by siRNA conjugates in vivo

This experiment examines the inhibition efficiency of the siRNA conjugates of the present disclosure conjugated with different structures on the expression of target gene mRNA in vivo.

Male 6-8 week old C57BL/6 mice were randomly divided into groups, with 6 mice in each group and 3 mice at each time point. The conjugate TRD007205 and the reference positive nucleic acid ligand of the present disclosure were administered to each group of mice respectively. Conjugate TRD002218 and PBS.

The dosage of all animals was calculated based on body weight. After dissolving the drug in physiological saline, a single dose was administered by subcutaneous injection. The dosage of siRNA conjugate (based on the amount of siRNA) was 1 mg/kg, and the dosage volume was 5 mL/kg. . The mice were sacrificed 7 days and 28 days after administration, and the livers were collected and preserved with RNA later (Sigma Aldrich Company); then the liver tissue was homogenized with a tissue homogenizer, and then tissue RNA extraction kit (Fanzhi Medical Technology, FG0412) Total RNA from liver tissue was extracted according to the operating procedures marked in the operating instructions. Total RNA was reverse transcribed into cDNA and real-time fluorescence quantitative PCR method was used to detect the expression of TTR mRNA in liver tissue. In this fluorescence quantitative PCR method, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as the internal reference gene, and Taqman probe primers for TTR and GAPDH were used to detect the mRNA expression levels of TTR and GAPDH respectively.

Table 4 Grouping information of experimental compounds in mice

TTR mRNA expression is calculated according to the following formula:
TTR mRNA expression amount = [(TTR mRNA expression amount in test group/GAPDH mRNA expression amount in test group)/(TTR mRNA expression amount in control group/GAPDH mRNA expression amount in control group)] x 100%.

After 7 days and 28 days of administration, the inhibition efficiency of the siRNA conjugates of the present disclosure conjugated with different structures on target gene mRNA expression in vivo is shown in Figure 1 and Figure 2 respectively.

As can be seen from the results in Figure 1, the conjugate TRD007205 has a good effect on inhibiting the expression of TTR mRNA 7 days after administration, indicating that it can mediate more efficient delivery efficiency. As can be seen from Figure 2, after 28 days of administration, TRD007205 has a better inhibitory effect on target gene mRNA expression than TRD002218. Therefore, the ligand NAG0052 has significant advantageous effects.

Example 3, human LPA siRNA

1) siRNA design, using human LPA (NM_005577.3) as the target gene to design 19/21nt siRNA to meet the general rules of active siRNA. The unmodified sense strand and antisense strand sequences are detailed in Table 5 and Table 6.

Table 5. Unmodified sense strand and antisense strand of human LPA siRNA

Table 6. Unmodified sense strand and antisense strand of human LPA siRNA (control group)

Example 4. Target activity of siRNA psiCHECK at 9 concentration points

In HEK293A cells, siRNA conjugates were screened for in vitro molecular level simulation and target activity using 9 concentration gradients.

HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal calf serum at 37°C and 5% _CO2 . 24 h before transfection, HEK293A cells were seeded in a 96-well plate at a seeding density of 8 × 10 ³ cells per well and 100 μL culture medium per well.

According to the instructions, use Lipofectamine2000 (ThermoFisher, 11668019) to co-transfect cells with siRNA and corresponding plasmids, using 0.3 μL Lipofectamine2000 in each well. The transfection amount of plasmid is 40ng per well. For the plasmid in the target sequence, SiRNA has a total of 9 concentration points, the maximum concentration point concentration is 20nm, 3 times gradient dilution, 9 concentration points are 20nm, 6.666666667nm, 2.2222222222nm, 0.740740741Nm, 0.24691358nm, 0.08230444 527nm, 0.027434842 nM, 0.009144947nM, 0.003048316nM. 24h after transfection, the Dual-Luciferase Reporter Assay System (Promega, E2940) was used to detect the target level.

In this example, TJR100373 (including the sense strand shown in SEQ ID NO: 1 and the antisense strand shown in SEQ ID NO: 2) in which both the sense and antisense strands were unmodified was used for testing.

The results showed that unmodified siRNA (naked sequence) TJR100373 had significant activity compared with similar unmodified siRNA sequences (TJR100374-TJR100380).

Table 7. siRNA activity results at 9 concentration points of psi-Check system

Example 5. Inhibitory activity of siRNA on human LPA in primary human hepatocytes (PHH) at 7 concentration points

The siRNA sequences were screened for PHH activity in primary human hepatocytes (PHH) using 7 concentration gradients. Each siRNA sample was always at a concentration of 20 nM from the time of transfection, with 5-fold gradient dilution and 7 concentration points.

PHH was cryopreserved in liquid nitrogen. 24 hours before transfection, primary human hepatocytes (PHH) were recovered and seeded in a 96-well plate at a seeding density of 3 × 10 ⁴ cells per well and 80 μL culture medium per well.

According to the product instruction manual, Lipofectamine RNAi MAX (ThermoFisher, 13778150) was used to transfect siRNA. The final gradient concentrations of siRNA transfection were 20nM, 4nM, 0.8nM, 0.16nM, 0.032nM, 0.0064nM and 0.00128nM. After 24 hours of treatment, a high-throughput cell RNA extraction kit was used to extract total cellular RNA, RNA reverse transcription experiments and quantitative real-time PCR detection to determine the mRNA level of human LPA. The mRNA level of human LPA was measured based on the GAPDH internal reference gene level. Correction.

Among them, in the quantitative real-time PCR detection, the probe Q-PCR detection experiment was used, and the primer information is shown in Tables 8 and 9.

After the Taqman probe Q-PCR detection experiment is completed, the corresponding Ct value is obtained according to the threshold automatically set by the system. The expression of a certain gene can be relatively quantified by comparing the Ct value. Comparing Ct refers to calculating the gene expression difference through the difference between the Ct value of the internal reference gene, also known as 2- ^△△Ct , △△Ct=[(Ct experimental group target gene-Ct experimental group internal reference)- (Ct control group target gene-Ct control group internal reference)]. Inhibition rate (%) = (1 - remaining amount of target gene expression) × 100%.

Results are expressed as percent remaining human LPA mRNA expression relative to control siRNA-treated cells. The IC ₅₀ results of inhibition rates are shown in Table 10.

The results show that TJR100373 has significant advantageous activity.

Table 8. Taqman LPA probe primer information table

Table 9. Taqman GAPDH probe primer information table

Table 10. Multiple-dose inhibitory activity of siRNA on human LPA in primary human hepatocytes (PHH)

Example 6. Synthesis of deuterated 5' terminal chemically modified phosphoramidite

first step

1-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5, 2,4]trioxadisilacyclooctan-8-yl)pyrimidine-2,4(1H,3H)-dione A1-2

Under a nitrogen atmosphere, compound A1-1 (100g, 409mmol) was dissolved in pyridine (1000mL), and 1,3-dichloro-1,1,3,3-tetraisopropyldimethylsiloxane was added at 0°C. Ether (TIPDSiCl ₂ ) (135.6g, 430.0mmol), reacted at 20°C for 18 hours. The reaction solution was added to ethyl acetate (1500mL) and water (1000mL) for extraction. The organic phase was washed three times with saturated brine (1000mL x 3), dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether). :ethyl acetate=2:1) to obtain compound A1-2 (185g, 380mmol, yield 92.8%).

Step 2

3-((benzyloxy)methyl)-1-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3 ,2-][1,3,5,2,4]trioxadisilacyclooctane-8-yl)pyrimidine-2,4(1H,3H)-dione A1-3

After compound A1-2 (185g, 380.1mmol) was dissolved in N,N-dimethylformamide (1850mL), benzyl chloromethyl ether (BOMCl) (89.29g, 570.1mmol) and 1,8-dimethylformamide were added. Azabicycloundec-7-ene (DBU) (115.7g, 760.2mmol), reacted at 20°C for 3 hours. The reaction solution was extracted with ethyl acetate (3000mL) and water (3000mL). The organic phase was washed three times with saturated brine (2000mL x 3), dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether). : ethyl acetate = 3:1) to obtain compound A1-3 (target compound, 172 g, 275.6 mmol, yield 72.5%).

third step

3-((benzyloxy)methyl)-1-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-(methoxy-d3)tetrahydro- 6H-Furo[3,2-][1,3,5,2,4]trioxadisilacyclooctan-8-yl)pyrimidine-2,4(1H,3H)-dione A1- 4

Compound A1-3 (150g, 247.2mmol) was dissolved in acetonitrile (300mL), CD ₃ I (107.5g, 741.5mmol) and silver oxide (114.5g, 494.3mmol) were added, and the reaction was carried out at 55°C for 24 hours. The reaction solution was filtered and concentrated under reduced pressure to obtain crude compound A1-4 (target compound, 125 g, 200.3 mmol, yield 81%).

the fourth step

3-((benzyloxy)methyl)-1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-(methoxy-d3)tetrahydrofuran-2- base)pyrimidine-2,4(1H,3H)-dione A1-5

After compound A1-4 (125g, 200mmol) was dissolved in tetrahydrofuran (1250mL), pyridine hydrogen fluoride (158.8g, 1.60mol) was added and the reaction was carried out at 25°C for 18 hours. The reaction solution was extracted with ethyl acetate (1000 mL) and water (1000 mL). The organic phase was washed once with saturated brine (1000 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography ( Dichloromethane: methanol = 20:1) to obtain compound A1-5 (target compound, 59 g, 154.7 mmol, yield 77%).

the fifth step

(2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-2 -(((tert-butyldimethylsilyl)oxy)methyl)-4-(methoxy-d3)tetrahydrofuran-3-yl benzoate A1-6

After compound A1-5 (59g, 154.7mmol) was dissolved in pyridine (590mL), tert-butyldimethylsilyl chloride (TBSCl) (93.2g, 618mmol) was added, and the reaction was carried out at 25°C for 18 hours. Then add benzoyl Chlorine (32.62g, 232.0mmol), react at 25°C for 3 hours. The reaction solution was extracted with ethyl acetate (1000mL) and water (1000mL). The organic phase was washed twice with saturated brine (1000mL x 2), dried over anhydrous magnesium sulfate, concentrated, and purified by column chromatography (petroleum ether). : ethyl acetate = 5:1) to obtain compound A1-6 (target compound, 60.0 g, 100.3 mmol, yield 64.6%).

Step 6

(2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-2 -Hydroxymethyl-4-(methoxy-d3)tetrahydrofuran-3-yl benzoate A1-7

After dissolving compound A1-6 (60.0g, 100.3mmol) in methanol (600mL) at 0°C, acetyl chloride (10.21g, 130.1mmol) was added and the reaction was carried out at 20°C for 2 hours. Then add silver carbonate and stir at 20°C for 1 hour. The reaction liquid was filtered, and the filtrate was concentrated under reduced pressure to obtain compound A1-7 (46.0 g, 94.7 mmol, yield 94.7%).

Step 7

(2S,3S,4R,5R)-3-(benzyloxy)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl)-4-(methoxy-d3)tetrahydrofuran-2-carboxylic acid A1-8

Compound A1-7 (46.0g, 94.75mmol) and 4-carbonyl-tetramethylpiperidine oxide TEMPO) (6.95g, 44.49mmol) were dissolved in CH ₃ CN (230 mL) and H ₂ O (230 mL), 5(6)-amino-1-(4-aminophenyl)-1,3,3-trimethylindane (PIDA) (67.14g, 208.4mmol) was added, and the reaction was carried out at 25°C for 18 hours. Ethyl acetate (500 mL) and water (300 mL) were added to the reaction solution for extraction. The organic phase was dried with anhydrous sodium sulfate and concentrated. It was purified by column chromatography (dichloromethane:methanol=10:1), and the pressure was reduced. Concentrate to obtain compound A1-8 (target compound, 43.0 g, 86 mmol, yield 90%).

Step 8

(3S,4R,5R)-2-acetoxy-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1(2H)- methyl)-4-(methoxy-d3)tetrahydrofuran-3-yl benzoate A1-9

Compound A1-8 (5 g, 10.0 mmol) was added to a dry flask, purged with argon, and then N,N-dimethylformamide (50 mL) was added. Then add pyridine (8mL, 100mmol) and lead acetate (9.7mL, 50mmol) in sequence. The reaction was protected from light and stirred at room temperature for 48 hours. The reaction was quenched with water (200 mL) and diluted with ethyl acetate (200 mL). The resulting suspension was filtered through a pad of Celite. The solid was rinsed with ethyl acetate. The organic layer was separated and concentrated in vacuo. The crude product was purified on a C18 reverse column to give the title product A1-9 as an α/β mixture (1.5 g, yield: 29%).

MS (ESI): m/z=514.2[M+H] ⁺ .

Step 9

(3S,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-2-( (Diethoxyphosphoryl)methoxy)-4-(methoxy-d3)tetrahydrofuran-3ylbenzoate A1-10

Under an argon atmosphere, (hydroxymethyl)phosphonate diethyl ester (2.5g, 156.7mmol) and boron trifluoride diethyl ether complex (5.7mL, 43.8mmol) were added to compound A1-9 (1.5g, 2.9 mmol) in anhydrous dichloromethane (15 mL). The reaction was stirred at room temperature for 16 hours. The reaction was quenched with water and extracted with ethyl acetate. The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified on a C18 reverse column to obtain the title compound A1-10 (1 g, yield 55%).

MS (ESI): m/z=622.3[M+H] ⁺ .

Step 10

(3S,4R,5R)-2-((diethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl) -4-(methoxy-d3)tetrahydrofuran-3-yl benzoate A1-11

A solution of compound Al-10 (1 g, 14.6 mmol) in TFA (5 mL) was stirred at 80°C for 30 min and then concentrated in vacuo. The title compound A1-11 (1 g, crude product) was obtained and used directly in the next step.

MS (ESI): m/z=502.3[M+H] ⁺ .

Step 11

((((3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-3-hydroxy-4-(methoxy-d3 )tetrahydrofuran-2-yl)oxy)methyl)phosphonate diethyl ester A1-12

A solution of compound A1-11 (1 g, 2.0 mmol) in ammonia methanol solution (7N, 10 mL) was stirred at room temperature for 16 hours. The reaction mixture was concentrated in vacuo. The crude product was purified on a C18 reverse column to obtain the title compound A1-12 (300 mg, yield: 38%).

MS (ESI): m/z=398.2[M+H] ⁺ .

Step 12

2-cyanoethyl((2R,3S,4R,5R)-2-((diethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidine -1(2H)-yl)-4-(methoxy-d3)tetrahydrofuran-3-yl)diisopropylphosphoramidite A1

DIPEA (58 mg, 0.45 mmol) was added to a solution of compound A1-12 (100 mg, 0.252 mmol) in anhydrous dichloromethane (2 mL), followed by 3-[chloro-(diisopropylamino)phosphane methyl]oxypropionitrile (83 mg, 0.35 mmol). The reaction mixture was stirred at room temperature for 2 hours and then quenched with MeOH. The reaction mixture was diluted with ethyl acetate and washed with saturated sodium bicarbonate, water and brine. The organic layer was concentrated in vacuo. The crude product was purified on a C18 reverse column to obtain phosphoramidite monomer A1 (80 mg, yield: 53%).

MS(ESI): m/z=596.0[MH] ⁺ .

¹ H NMR (400MHz, CD ₃ OD) δ7.74-7.70(m,1H),6.33-6.30(m,1H),5.84-5.82(m,1H),5.25-5.18(m,1H),4.51- 4.44(m,1H),4.27-3.67(m,11H),2.82-2.79(m,2H),1.41-1.36(m,6H),1.29-1.23(m,12H).

³¹ P NMR (400 MHz, CD ₃ OD) δ 151.95, 150.57, 21.29.

Example 7. Synthesis of siRNA containing deuterated chemical modification at the 5’ end

The synthesis of siRNA is no different from the usual phosphoramidite solid-phase synthesis method. When synthesizing the nucleotide modified at the end of the antisense chain, the phosphoramidite monomer A1 synthesized in Example 6 is used.

The synthesis process is briefly described as follows: On the Dr.Oligo48 synthesizer (Biolytic), starting from the Universal CPG vector, the nucleoside phosphoramidite monomers are connected one by one according to the synthesis procedure. Except for the nucleoside phosphoramidite monomer A1 described in Example 6, the remaining nucleoside monomer raw materials 2'-F RNA, 2'-O-methyl RNA and other nucleoside phosphoramidite monomers were purchased from Shanghai Zhaowei or Suzhou Jima. Use 5-ethylthio-1H-tetrazole (ETT) as the activator (0.6M acetonitrile solution), and use 0.22M PADS dissolved in a 1:1 volume ratio of acetonitrile and trimethylpyridine (Suzhou Croma) solution As a sulfiding reagent, phosphorothioate can be introduced at a designated position; using iodopyridine/aqueous solution (Croma) as an oxidizing agent, phosphorothioate can be introduced at a designated position.

After the solid-phase synthesis is completed, in some embodiments, TMSI needs to be used to remove the 5'-end protecting group. For example, after solid-phase synthesis using phosphoramidite monomer A1, the crude oligoribonucleotide is suspended in anhydrous acetonitrile. , add TMSI at room temperature, stir thoroughly and remove the ethyl group (Et) in the oligoribonucleotide to obtain the oligoribonucleotide with NA0127. Then, the oligoribonucleotide is further cleaved from the solid support. The cleaving conditions are: soaking in a 3:1 solution of 28% ammonia and ethanol at 50° C. for 16 hours. Then centrifuge, transfer the supernatant to another centrifuge tube, concentrate and evaporate to dryness, use C18 reverse chromatography to purify, the mobile phase is 0.1M TEAA and acetonitrile, and use 3% trifluoroacetic acid solution to remove DMTr. Target oligonucleotides were collected, lyophilized, and subjected to LC-MS The target product was identified and then quantified by UV (260nm).

The obtained single-stranded oligonucleotides were complementary paired and annealed based on equimolar ratios. Finally, the obtained double-stranded siRNA was dissolved in 1×PBS and adjusted to the concentration required for the experiment.

The sequences of the sense strand and antisense strand that contain deuterated chemical modifications at the 5’ end, 2’-fluoro, 2’-methoxy and other modifications are shown in Table 11.

Table 11. Modified sense strand and antisense strand of human LPA siRNA

The lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is 2'-fluoro modified nucleotides;

When the lowercase letter s is in the middle, it means that the connection between the two nucleotides adjacent to the left and right of the letter s is a phosphorothioate group connection;

NAG0052' structure is:

NA0127 and NA0149 respectively represent:

Among them, NA0127 is as described in Example 7, and the synthesis method of NA0149 refers to US20190177729A.

Example 8. Target activity of siRNA conjugate psiCHECK at 9 concentration points

In HEK293A cells, 9 concentration gradients were used for in vitro molecular level simulation to screen the target activity.

According to the instructions, use Lipofectamine2000 (ThermoFisher, 11668019) to co-transfect cells with siRNA conjugates and corresponding plasmids, using 0.3 μL Lipofectamine2000 in each well. The transfection amount of plasmid is 40ng per well. For the target sequence plasmid, a total of 9 concentration points are set for the siRNA/siRNA conjugate. The final concentration of the highest concentration point is 20nM, 3-fold gradient dilution, and the 9 concentration points are: 20nM, 6.666666667nM, 2.222222222nM, 0.740740741nM, 0.24691358 nM, 0.082304527nM, 0.027434842nM, 0.009144947nM, 0.003048316nM. 24h after transfection, the Dual-Luciferase Reporter Assay System (Promega, E2940) was used to detect the target level.

The results are shown in Table 12. The results show that compared with TJR100437, TJR100747 and TJR100848 have a high level of on-target inhibitory activity against the LPA gene in the psiCHECK system.

Table 12. Screening results of psi-CHECK on-target activity

Example 9. Determination of dsRNA activity in humanized mice (hu-Lp(a))

The double-transgenic humanized mouse (hu-Lp(a)) used in this example, in which the humanized Apo(a) construct contains 6 kringle copies, ApoB100 was introduced from Taconic Company's B6.SJL-Tg (APOB )1102Sgy N20+? Use a biochemical analyzer to detect Lp(a) concentration. Biochemical analyzer model: Ci4100 fully automatic biochemical immune all-in-one machine.

According to the serum Lp(a) protein content, they were evenly divided into groups, 6 (male) in each group, 3 at each time point. Each group was given the drugs TJR100553, TJR100747, and TJR100848 (dissolved in normal saline) by subcutaneous injection, and the dosage was Both are 3 mg/kg, and the compound administration volume is 10 μl/g. 40 μl of serum was collected before administration. The mice were euthanized on the 21st and 28th days after administration, and 40 μl of serum were collected from each group. Use a biochemical analyzer to detect the remaining lipoprotein Lp(a) in the serum. One-way ANOVA was used to perform statistics using the measured serum Lp(a) content of each mouse. The experimental results are shown in Table 13, Figure 3 and Figure 4.

According to the results in Table 13 and Figure 3, compared with TJR100747 and TJR100848, there was a significant difference between TJR100553 and TJR100747 and TJR100848 on day 21, P<0.001.

According to the results in Table 13 and Figure 4, compared with TJR100747 and TJR100848, there was a significant difference between TJR100553 and TJR100747 on day 28, P<0.01, and there was a significant difference between TJR100553 and TJR100848, P<0.001.

Table 13. Serum content of hu-Lp(a) mice

The results show that deuterated chemical modification at the 5' end can significantly extend the action time of siRNA and improve biological activity.

Claims

An oligonucleotide comprising a 5' end chemical modification shown in Formula (I), or a tautomer modification thereof, wherein the 5' end chemical modification shown in Formula (I) is selected from:

Wherein, R A1 and R A2 are each independently selected from hydrogen or deuterium; M 1 and M 2 are each independently selected from -SH or -OH; B is selected from base, hydrogen, deuterium; R A3 is selected from hydrogen, deuterium, Hydroxy, halogen, alkyl, alkoxy, each of the hydroxyl, alkyl, alkoxy is optionally substituted by one or more deuterium; R A4 is selected from hydrogen, deuterium, alkyl, alkoxy, the Each of the alkyl group and alkoxy group is optionally substituted by one or more deuterium; the condition is that at least one deuterium is included in formula (I).
The oligonucleotide according to claim 1, wherein said formula (I) is selected from formula (I-1),

Among them, RA5 , RA6 and RA7 are each independently selected from hydrogen or deuterium; RA1 , RA2 , M1 , M2 and B are defined according to claim 1, provided that the formula (I-1) contains At least one deuterium.
The oligonucleotide according to claim 1 or 2, wherein said formula (I) is selected from:

B is selected from bases or hydrogen.
The oligonucleotide according to any one of claims 1 to 3, which is a double-stranded RNAi inhibitor molecule, comprising a sense strand and an antisense strand forming a double-stranded region; the formula (I ), or the tautomer modification thereof, is located on the antisense strand; preferably, the oligonucleotide is siRNA.
The oligonucleotide according to any one of claims 1 to 4, wherein at least one nucleotide other than the chemical modification of the 5' end represented by formula (I) is a modified nucleotide.
The oligonucleotide according to claim 5, wherein in the direction from the 5' end to the 3' end,

The three consecutive nucleotides at positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluorinated modified nucleotides, and the remaining positions of the sense strand are non-2'-fluorinated modified Nucleotide;

The nucleotide located at the 1st position at the 5' end of the antisense strand is a 5' end chemically modified nucleotide represented by formula (I) according to any one of claims 1 to 3, The nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 at the 5' end of the antisense strand are independently 2'-fluorinated modified nucleotides, and the remaining positions of the antisense strand are non- 2'-Fluoromodified nucleotides.
The oligonucleotide of claim 6, wherein the non-2'-fluoro-modified nucleotide is a 2'-methoxy-modified nucleotide.
The oligonucleotide according to any one of claims 4 to 7, wherein at least one phosphate group in the sense strand and/or the antisense strand is a phosphate group with a modifying group, preferably a phosphorothioate diester base.
The oligonucleotide of any one of claims 1 to 8, wherein the oligonucleotide targets the lipoprotein (a) (LPA) gene.
The oligonucleotide according to any one of claims 1 to 9, said oligonucleotide comprising a sense strand and an antisense strand forming a double-stranded region;

The sense strand contains at least 15 consecutive nucleotides and differs from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides;

The antisense strand contains at least 15 consecutive nucleotides and differs from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides.
The oligonucleotide according to claim 10, which includes the sense strand shown in SEQ ID NO: 1 and the antisense strand shown in SEQ ID NO: 2.
The oligonucleotide according to any one of claims 1 to 11, wherein,

The oligonucleotide includes the sense strand shown in SEQ ID NO:3 and the antisense strand shown in SEQ ID NO:5; or,

The oligonucleotide includes the sense strand shown in SEQ ID NO:4 and the antisense strand shown in SEQ ID NO:5.
The oligonucleotide according to any one of claims 1 to 12, wherein the oligonucleotide further comprises a targeting ligand, preferably, the targeting ligand targets the liver; more preferably, The targeting ligand binds to the asialoglycoprotein receptor (ASGPR); more preferably, the targeting ligand includes a galactose cluster or a galactose derivative cluster, and the galactose derivative is selected from N- Acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine.
The oligonucleotide of claim 13, wherein the targeting ligand is attached to the 3' end of the sense strand.
The oligonucleotide according to claim 13 or 14, wherein the targeting ligand is:
The oligonucleotide according to any one of claims 1 to 15, wherein,

The oligonucleotide includes the sense strand shown in SEQ ID NO:6 and the antisense strand shown in SEQ ID NO:5; or,

The oligonucleotide includes the sense strand shown in SEQ ID NO:7 and the antisense strand shown in SEQ ID NO:5.
The oligonucleotide according to any one of claims 1 to 16, wherein the oligonucleotide is selected from the following structure or a pharmaceutically acceptable salt thereof:

Among them, Af = adenine 2'-F ribonucleoside; Cf = cytosine 2'-F ribonucleoside; Uf = uracil 2'-F ribonucleoside; Gf = guanine 2'-F ribonucleoside; Am = adenine 2'-OMe ribonucleoside; Cm = cytosine 2'-OMe ribonucleoside; Gm = guanine 2'-OMe ribonucleoside; Um = uracil 2'-OMe ribonucleoside; Represents phosphorothioate diester group, Indicates phosphodiester group, NAG0052' indicates NA0127' means
The oligonucleotide according to any one of claims 1 to 16, wherein the oligonucleotide is selected from the following structure or a pharmaceutically acceptable salt thereof:

Among them, Af = adenine 2'-F ribonucleoside; Cf = cytosine 2'-F ribonucleoside; Uf = uracil 2'-F ribonucleoside; Gf = guanine 2'-F ribonucleoside; Am = adenine 2'-OMe ribonucleoside; Cm = cytosine 2'-OMe ribonucleoside; Gm = guanine 2'-OMe ribonucleoside; Um = uracil 2'-OMe ribonucleoside; Represents phosphorothioate diester group, Indicates phosphodiester group, NAG0052' indicates NA0127 means
A compound represented by formula (IV) or its tautomer,

Among them, R A8 and R A9 are each independently selected from -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CN, -CH 2 OPiv, CH 2 OCH 2 CH 2 Si(CH 3 ) 3 or protecting group; R A10 is selected from phosphorus-containing active reactive groups; M 4 is selected from O or S;

R A1 , R A2 , R A3 and B are defined according to claim 1, provided that the formula (IV) contains at least one deuterium;

Preferably, formula (IV) is selected from formula (IV-1),

Wherein, RA5 , RA6 and RA7 are each independently selected from hydrogen or deuterium. The condition defined by RA1 , RA2 and B according to claim 1 is that the formula (IV-1) contains at least one deuterium;

RA8 and RA9 are each independently selected from -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CN, -CH 2 OPiv, CH 2 OCH 2 CH 2 Si(CH 3 ) 3 or protecting group; R A10 is selected from phosphorus-containing active reactive groups; R A10 is selected from phosphorus-containing active reactive groups.
The compound represented by formula (IV) or its tautomer according to claim 19, wherein the compound represented by formula (IV) is selected from:

B is selected from bases.
A method for preparing the oligonucleotide according to any one of claims 1 to 18, comprising the following steps:

The oligonucleotide according to any one of claims 1 to 18 is synthesized using the compound represented by formula (IV) according to claim 19 or 20 or its tautomer.
A pharmaceutical composition comprising the oligonucleotide according to any one of claims 1 to 18; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients agent.
An application of the oligonucleotide according to any one of claims 1 to 18 or the pharmaceutical composition according to claim 22 in the preparation of medicines for preventing and/or treating cardiovascular diseases , or, the medicament is used to prevent and/or treat diseases associated with elevated levels of lipoprotein (a) and/or apolipoprotein (a); Diseases associated with elevated apolipoprotein(a) levels are selected from cardiovascular diseases; the cardiovascular diseases are selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity arterial disease or aortic stenosis, Myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, heart failure.
A method of delivering an oligonucleotide to the liver, comprising administering to a subject an effective amount or an effective dose of an oligonucleotide according to any one of claims 1 to 18 or an oligonucleotide according to claim 22 Pharmaceutical compositions.