Deuterated chemical modifications and oligonucleotides comprising same

Abstract

The present disclosure relates to deuterated chemical modifications and oligonucleotides comprising the same. Specifically, the present disclosure relates to an oligonucleotide comprising a chemical modification of formula (I) or a tautomer modification thereof wherein the functional groups are as defined in the disclosure. # imgabs0 #

Description

Deuterated chemical modification and oligonucleotide containing the same

[0001] This disclosure claims priority to Chinese patent application No. 202210448666.8, filed on April 26, 2022, and this disclosure cites the full text of the above-mentioned Chinese patent application. Technical Field

[0002] The present disclosure relates to deuterated chemically modified oligonucleotides, which have improved stability and can be delivered into cells in a targeted manner to exert RNA interference effects. The present disclosure also relates to methods for preparing and using oligonucleotides. Background Art

[0003] RNA interference (RNAi) is an effective method for silencing gene expression. According to statistics, over 80% of disease-related proteins in the human body cannot be targeted by currently available small-molecule drugs and biopharmaceuticals, making them undruggable. Using RNAi technology, we can design appropriate siRNAs based on the mRNA encoding these proteins. These siRNAs specifically target and degrade the target mRNA, thereby inhibiting the production of the associated proteins. Therefore, siRNA holds great promise for drug development.

[0004] However, unmodified small RNAs are unstable in the body (plasma, lysosomes, etc.) and easily degraded by enzymes (endo- and exo-nucleases, etc.). They also have poor pharmacokinetics, are easily filtered by the glomeruli, and are rapidly excreted in the urine after administration. Therefore, siRNA requires specific chemical modifications to enhance its nuclease resistance, improve its in vivo stability, improve tissue distribution, and enhance its pharmacokinetic properties, thereby achieving therapeutic effects on the corresponding diseases.

[0005] CN110072530A discloses a class of 4′-phosphate analogs 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 analogs in vivo, so chemical modification is needed to further improve its in vivo stability, increase the cell's ability to take up RNA, and enhance the activity of the antisense chain on the target, thereby achieving a long-term inhibitory effect on the target mRNA. The present invention further enhances the activity of the antisense chain on the target by deuterating the 5′-terminal nucleotide of the antisense chain of siRNA, improves the stability of siRNA on metabolic enzymes such as nucleases, and further improves its in vivo stability, thereby achieving a more efficient and long-lasting inhibitory effect on the target mRNA and its corresponding protein; this also further reduces the toxicity that may be caused by siRNA targeting mRNA, reduces the frequency of dosing for patients, and further improves the drug-forming properties of siRNA.

[0006] Lipoprotein(a) (Lp(a)), first discovered by Norwegian geneticist Berg in 1963, was identified as a unique lipoprotein (Berg KA, "New serum type system in man—the Lp system." Acta Pathol Microbiol Scand 1963;59:369–82). Lp(a) is composed of two components: lipids and proteins. The lipid component primarily consists of LDL-like particles located in the core; the protein component, located peripherally, is composed of apolipoprotein(a) (apo(a)) and apoB100 linked by disulfide bonds.

[0007] Patients with elevated Lp(a) have a 2-3 times higher risk of cardiovascular events than the normal population, causing cardiovascular diseases including atherosclerotic cardiovascular disease, lower limb artery disease, aortic valve stenosis, etc. (EnAs EA, Varkey B, Dharmarajan TS, 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 contribute to adverse atherosclerotic cardiovascular disease (ASCVD) through two mechanisms: First, because apo(a) has been shown to inhibit fibrinolysis in vitro, it may promote thrombosis at plaque rupture or turbulence at vascular stenosis, leading to vascular obstruction or thrombosis; second, LDL-like particles can promote intimal cholesterol deposition, inflammation, or oxidized phospholipids, leading to atherosclerotic stenosis 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). However, even very high levels of Lp(a) contain cholesterol levels below the traditional LDL cutoff, and therefore, this portion of LDL-like particles may be less pathogenic.

[0008] The 2016 Chinese Guidelines for the Prevention and Treatment of Dyslipidemia in Adults define abnormal Lp(a) levels as >30 mg / dL. Based on this standard, approximately 30% of patients with prior cardiovascular events in China have abnormal Lp(a). In 2019, the US National Lipid Association recommended an elevated Lp(a) level of ≥50 mg / dL. Using this standard, 20% of the global population has elevated Lp(a). Despite the common nature of elevated Lp(a), targeted therapeutics are lacking, and no drugs targeting Lp(a) have been approved for clinical use. The Lp(a) protein, structurally similar to many lipoproteins, is a difficult target for both small and large molecule drugs. However, the mRNA transcribed from the Lp(a) gene is highly specific, allowing for post-transcriptional degradation of the mRNA through siRNA, thereby inhibiting Lp(a) expression. Therefore, siRNA targeting the apo(a) gene (LPA) has been designed to attenuate its expression, thereby reducing serum Lp(a) levels and, consequently, cardiovascular adverse events.

[0009] Summary of the Invention

[0010] The present disclosure provides an oligonucleotide comprising a 5' terminal chemical modification, wherein the 5' terminal chemical modification comprises at least one deuterium.

[0011] The present disclosure provides an oligonucleotide comprising a 5' terminal chemical modification as shown in formula (I), or a tautomer modification thereof, wherein the 5' terminal chemical modification as shown in formula (I) is selected from:

[0012] Among them, R A1 、R A2 Each is independently selected from hydrogen or deuterium; M1 and M2 are each independently selected from -SH or -OH; B is selected from a base, hydrogen, deuterium; R A3 R is selected from hydrogen, deuterium, hydroxyl, halogen, alkyl (e.g., C1, C2, C3, C4, C5, C6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (e.g., C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, C6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), wherein each of the hydroxyl, alkyl, and alkoxy groups is optionally substituted with one or more deuterium groups; A4 is selected from hydrogen, deuterium, and alkyl (e.g., C1, C2, C3, C4, C5, C6 alkyl, including but not limited to methyl, ethyl, isopropyl), each of which is optionally substituted with one or more deuterium; provided that formula (I) contains at least one deuterium.

[0013] In some embodiments, R A3 Selected from hydrogen and deuterium.

[0014] In some embodiments, R A3is selected from halogen (such as fluorine, chlorine, bromine).

[0015] In some embodiments, R A3 Selected from alkyl groups (e.g., C1, C2, C3, C4, C5, C6 alkyl groups, including but not limited to methyl, ethyl, isopropyl), each of which is optionally substituted with one or more deuterium groups.

[0016] In some embodiments, R A3 Selected from alkoxy (e.g., C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, C6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), each of which is optionally substituted with one or more deuterium groups.

[0017] In some embodiments, the 5' terminal chemical modification represented by formula (I) is selected from:

[0018] Among them, R A5 、R A6 、R A7 are each independently selected from hydrogen or deuterium; R A1 、R A2 , M1, M2, and B are as defined in formula (I); provided that formula (I-1) contains at least one deuterium.

[0019] In some embodiments, R A5 、R A6 、R A7 All deuterium; in some embodiments, R A5 Selected from deuterium, R A6 、R A7 is selected from hydrogen; in some embodiments, R A5 、R A6 Selected from deuterium, R A7 Selected from hydrogen.

[0020] In some embodiments, R A1 Selected from hydrogen, R A2 selected from deuterium; in some embodiments, RA1 and R A2 is selected from deuterium; in some embodiments, R A1 and R A2 Selected from hydrogen.

[0021] In some embodiments, the 5' terminal chemical modification represented by formula (I) is selected from:

[0022] B is selected from a base or hydrogen.

[0023] In some embodiments, B is selected from a base; in some specific embodiments, B is selected from adenine, guanine, cytosine, uracil, or thymine.

[0024] In some specific embodiments, B is the base at the corresponding position of the modified nucleotide in the antisense strand.

[0025] In some embodiments, the 5' terminal chemical modification represented by formula (I) is selected from:

[0026]

[0027] and structures in which uracil is replaced by adenine, guanine, cytosine, or thymine.

[0028] In some embodiments, the oligonucleotides described in the present disclosure are selected from 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.

[0029] In some embodiments, the oligonucleotides described herein are single-stranded oligonucleotides.

[0030] In some embodiments, the single stranded oligonucleotide is a single stranded RNAi inhibitor molecule. In some embodiments, the single stranded RNAi inhibitor molecule has 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.

[0031] In some embodiments, the oligonucleotides described herein are selected from double-stranded RNAi inhibitor molecules.

[0032] In some embodiments, the oligonucleotides described herein are selected from siRNA.

[0033] In some embodiments, the oligonucleotides described herein are selected from double-stranded RNAi inhibitor molecules, comprising a sense strand and an antisense strand forming a double-stranded region.

[0034] In some embodiments, the oligonucleotides described herein are selected from siRNAs, comprising a sense strand and an antisense strand forming a double-stranded region.

[0035] In some embodiments, the 5' terminal chemical modification represented by formula (I), or a tautomer modification thereof, is located at the 5' terminus of the antisense strand.

[0036] In some specific embodiments, in the 5'-terminal chemical modification shown in formula (I), B is the base at the corresponding position of the modified nucleotide in the antisense strand.

[0037] In some embodiments, the oligonucleotides described in the present disclosure target the hepatitis B virus (HBV) gene, the apolipoprotein C3 (ApoC3) gene, or the lipoprotein (a) (LPA) gene.

[0038] In some embodiments, the oligonucleotides described herein are siRNAs targeting the hepatitis B virus (HBV) gene.

[0039] In some embodiments, the oligonucleotides described herein are siRNAs targeting HBV-S.

[0040] In some embodiments, the oligonucleotides described herein are siRNAs targeting HBV-X.

[0041] In some embodiments, the oligonucleotides described herein are siRNAs targeting the apolipoprotein C3 (ApoC3) gene.

[0042] In some embodiments, the oligonucleotides described herein are siRNAs targeting the lipoprotein (a) (LPA) gene.

[0043] In some embodiments, the oligonucleotide, double-stranded RNAi inhibitor molecule, or siRNA disclosed herein comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides and differs from the nucleotide sequence of SEQ ID NO: 1 by no more than 2 nucleotides (e.g., 2, 1). In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides and differs from the nucleotide sequence of SEQ ID NO: 2 by no more than 2 nucleotides (e.g., 2, 1).

[0044] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides (e.g., 15, 16, 17, 18, 19) of SEQ ID NO: 1. In some embodiments, the sense strand comprises at least 16 consecutive nucleotides of SEQ ID NO: 1. In some embodiments, the sense strand comprises at least 18 consecutive nucleotides of SEQ ID NO: 1. In some embodiments, the sense strand comprises at least 19 consecutive nucleotides of SEQ ID NO: 1.

[0045] In some embodiments, the antisense strand comprises at least 15 consecutive 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 consecutive nucleotides of SEQ ID NO: 2. In some embodiments, the antisense strand comprises at least 19 consecutive nucleotides of SEQ ID NO: 2. In some embodiments, the antisense strand comprises at least 20 consecutive nucleotides of SEQ ID NO: 2. In some embodiments, the antisense strand comprises at least 21 consecutive nucleotides of SEQ ID NO: 2.

[0046] In some embodiments, the sense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:1.

[0047] In some embodiments, the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:2.

[0048] In some embodiments, the oligonucleotide described in the present disclosure comprises a sense strand as shown in SEQ ID NO: 1 and an antisense strand as shown in SEQ ID NO: 2.

[0049] In some embodiments, in the oligonucleotides disclosed herein, 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.

[0050] In some embodiments, the oligonucleotides disclosed herein are selected from the sense strand set forth in SEQ ID NO: 1 and the antisense strand set forth in SEQ ID NO: 2.

[0051] In the present disclosure, according to the 5'-3' direction,

[0052] SEQ ID NO: 1 is GCUCCUUAUUGUUAUACGA;

[0053] SEQ ID NO: 2 is UCGUAUAACAAUAAGGAGCUG.

[0054] 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, no more than 3, no more than 2, or no more than 1 mismatch between the antisense strand and the target sequence; in some embodiments, the antisense strand is completely reverse complementary to the target sequence.

[0055] 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, no more than 4, no more than 3, no more than 2, or no more than 1 mismatch between the sense strand and the antisense strand; in some embodiments, the sense strand and the antisense strand are completely reverse complementary.

[0056] In some embodiments, the oligonucleotides disclosed herein are selected from siRNAs and comprise one or two blunt ends.

[0057] In some specific embodiments, the siRNA comprises an overhang of 1 to 4 unpaired nucleotides, eg, 1, 2, 3, 4.

[0058] In some embodiments, the oligonucleotide of the present disclosure is selected from siRNA, comprising an overhang located at the 3' end of the antisense strand of the siRNA.

[0059] In some embodiments, the sense strand is 15-35 nucleotides in length and the antisense strand is 15-30 nucleotides in length.

[0060] In some embodiments, the sense and antisense strands each independently have 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides (e.g., 19, 20, 21, 22, 23 nucleotides).

[0061] In some embodiments, the sense strand and the antisense strand are the same or different lengths, with the sense strand being 19-23 nucleotides in length and the antisense strand being 19-26 nucleotides in length. Thus, the disclosure provides that the ratio of the length of the sense and antisense strands 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 to the antisense strand is 19 / 21, 21 / 23, or 23 / 25. In some embodiments, the length ratio of the sense strand to the antisense strand is 19 / 21.

[0062] In some embodiments, the oligonucleotides, double-stranded RNAi inhibitor molecules, or siRNAs described herein do not contain other modified nucleotides.

[0063] In some embodiments, at least one additional nucleotide in the oligonucleotide, double-stranded RNAi inhibitor molecule or siRNA described in the present disclosure is a modified nucleotide; in some embodiments, the oligonucleotide, double-stranded RNAi inhibitor molecule or siRNA comprises a sense strand and an antisense strand forming a double-stranded region, and the sense strand and antisense strand each comprise at least one nucleotide that is a modified nucleotide; in some embodiments, all nucleotides of the sense strand and all nucleotides of the antisense strand described in the present disclosure are modified nucleotides.

[0064] In some embodiments, the modified nucleotides are selected from the group consisting of: 2'-methoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 3'-deoxy-thymine (dT) nucleotides, isonucleotides, LNA, ENA, cET, UNA, GNA.

[0065] In some embodiments, the modified nucleotides are independently selected from the group consisting of: 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides.

[0066] In some embodiments, in the direction from the 5' end to the 3' end, the three consecutive nucleotides at positions 7, 8, and 9 of the 5' end of the sense strand are 2'-fluoro-modified nucleotides. In some specific embodiments, the three consecutive nucleotides at positions 7, 8, and 9 of the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining positions of the sense strand are non-2'-fluoro-modified nucleotides; in some specific embodiments, the three consecutive nucleotides at positions 7, 8, and 9 of the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining positions of the sense strand are 2'-methoxy-modified nucleotides.

[0067] In some embodiments, in the direction from the 5' end to the 3' end, the first nucleotide of the antisense strand is a 5' terminal chemically modified nucleotide as shown in any one of the formula (I) described in the present disclosure; in some specific embodiments, the 5' terminal chemically modified nucleotide as shown in formula (I) is The B is the base corresponding to the first nucleotide at the 5' end of the antisense strand.

[0068] In some embodiments, 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 a 2'-fluoro-modified nucleotide, and the remaining positions of the antisense strand are all non-2'-fluoro-modified nucleotides. In some specific embodiments, 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 a 2'-fluoro-modified nucleotide, and the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides.

[0069] In some embodiments, the oligonucleotides described herein comprise at least one phosphate group that is a phosphate group having a modified group, and in some embodiments, comprise at least one phosphate group that is a phosphorothioate diester group.

[0070] In some embodiments, at least one phosphate group in the sense strand and / or the antisense strand is a phosphate group having a modified group. In some embodiments, at least one phosphate group in the sense strand and / or the antisense strand is a phosphorothioate diester group.

[0071] In some embodiments, the phosphorothioate diester group is present in at least one of the following positions:

[0072] between the first and second nucleotides at the 5' end of the sense strand;

[0073] between the second and third nucleotides at the 5' end of the sense strand;

[0074] between the first and second nucleotides at the 3' end of the sense strand;

[0075] The first nucleotide end of the 3' end of the sense strand;

[0076] between the first and second nucleotides at the 5' end of the antisense strand;

[0077] between the second and third nucleotides at the 5' end of the antisense strand;

[0078] between the first and second nucleotides at the 3' end of the antisense strand;

[0079] between the second and third nucleotides at the 3' end of the antisense strand; or

[0080] The first nucleotide end of the 3' end of the antisense strand.

[0081] In some embodiments, the phosphorothioate diester group is present in:

[0082] between the first and second nucleotides at the 5' end of the sense strand.

[0083] In some embodiments, the phosphorothioate diester group is present in:

[0084] between the first and second nucleotides at the 5' end of the sense strand, and

[0085] between the second and third nucleotides at the 5' end of the sense strand.

[0086] In some embodiments, the phosphorothioate diester group is present in:

[0087] Between the first and second nucleotides at the 5' end of the sense strand, and

[0088] Between the second and third nucleotides at the 5' end of the sense strand, and

[0089] between the first and second nucleotides at the 3' end of the sense strand.

[0090] In some embodiments, the phosphorothioate diester group is present in:

[0091] between the first nucleotide and the second nucleotide at the 5' end of the sense strand; and

[0092] The first nucleotide end of the 3' end of the sense strand.

[0093] In some embodiments, the phosphorothioate diester group is present in:

[0094] between the first and second nucleotides at the 5' end of the sense strand;

[0095] between the second and third nucleotides at the 5' end of the sense strand; and

[0096] The first nucleotide end of the 3' end of the sense strand.

[0097] In some embodiments, the phosphorothioate diester group is present in:

[0098] The first nucleotide end of the 3' end of the sense strand.

[0099] In some embodiments, the phosphorothioate diester group is present in:

[0100] between the first and second nucleotides at the 5' end of the antisense strand;

[0101] between the second and third nucleotides at the 5' end of the antisense strand;

[0102] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0103] between the second and third nucleotides at the 3' end of the antisense strand.

[0104] In some specific embodiments, the sense strand comprises the nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:4.

[0105] In some specific embodiments, the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:5.

[0106] In some specific embodiments, the sense strand is selected from the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0107] In some specific embodiments, the antisense strand is selected from the nucleotide sequence shown in SEQ ID NO:5.

[0108] In some specific embodiments, the sense strand of the oligonucleotide described herein comprises SEQ ID NO:3, and the antisense strand comprises SEQ ID NO:5.

[0109] In some specific embodiments, the sense strand of the oligonucleotide described herein comprises SEQ ID NO:4, and the antisense strand comprises SEQ ID NO:5.

[0110] In some specific embodiments, the sense strand of the oligonucleotide described herein is selected from SEQ ID NO: 3, and the antisense strand is selected from SEQ ID NO: 5.

[0111] In some specific embodiments, the sense strand of the oligonucleotide described herein is selected from SEQ ID NO:4, and the antisense strand is selected from SEQ ID NO:5.

[0112] In some specific embodiments, the oligonucleotides described in the present disclosure are selected from any one of the following:

[0113] The sense strand comprises the nucleotide sequence set forth in SEQ ID NO:3, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:5;

[0114] The sense strand comprises the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:5.

[0115] In some specific embodiments, the oligonucleotides described in the present disclosure are selected from any one of the following:

[0116] 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;

[0117] 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.

[0118] In the present disclosure, according to the 5'-3' direction,

[0119] SEQ ID NO: 3 is GmsCmsUmCmCmUmUfAfUfUmGmUmUmAmUmAmCmGmAm;

[0120] SEQ ID NO:4 is GmsCmsUmCmCmUmUfAfUfUmGmUmUmAmUmAmCmGmsAm;

[0121] SEQ ID NO:5 is NA0127CfsGmUfAmUfAmAmCmAfAmUfAmAfGmGfAmGfCmsUmsGm;

[0122] Wherein, 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;

[0123] s indicates that the two nucleotides adjacent to the letter s are connected by a phosphorothioate diester group;

[0124] NA0127 indicates

[0125] In some embodiments, the oligonucleotides described in 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 comprises a galactose cluster or a galactose derivative cluster selected from N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-acetyl-galactosamine triacetate, or N-isobutyrylgalactosamine.

[0126] In some embodiments, the targeting ligand comprises three clusters of galactose or galactose derivatives.

[0127] In some embodiments, the targeting ligand is covalently or non-covalently linked to the oligonucleotide.

[0128] In some embodiments, the targeting ligand is linked to the end of the oligonucleotide, double-stranded RNAi inhibitor molecule, or siRNA through a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group.

[0129] In some embodiments, the targeting ligand is indirectly linked to the end of the oligonucleotide, double-stranded RNAi inhibitor molecule, or siRNA through a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group.

[0130] In some embodiments, the targeting ligand is directly linked to the end of an oligonucleotide, a double-stranded RNAi inhibitor molecule, or a siRNA via a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group.

[0131] In some embodiments, the targeting ligand is directly linked to the end of an oligonucleotide, double-stranded RNAi inhibitor molecule, or siRNA via a phosphodiester group or a phosphorothioate diester group.

[0132] In some embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of an oligonucleotide, a double-stranded RNAi inhibitor molecule, or an siRNA via a phosphodiester group or a phosphorothioate diester group.

[0133] In some embodiments, the targeting ligand is selected from the structure represented by formula (II') or a pharmaceutically acceptable salt thereof:

[0134] Among them, L1 is C1-C 30 Alkyl chains, or C1-C ... 30 alkyl chain;

[0135] R1 and R2 are independently a chemical bond, NR6, C=O or -OC(=O)-;

[0136] Q is

[0137] is a single bond or a double bond, and when When it is a single bond, R3 is independently CR7R8, NR6, O or S. When it is a double bond, R3 is independently CR9 or N;

[0138] R4 is independently CR9 or N;

[0139] Ring A is a cycloalkyl, heterocyclyl, aryl or heteroaryl group which may be present or absent, and when ring A is present, R5 is independently CR9 or N, and when ring A is absent, R5 is independently CR7R8, NR6 or O;

[0140] R6 and R9 are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR' or C(=O)NR'(R"), wherein the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted by one or more halogen (such as fluorine, chlorine, bromine), hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-7 substituted by a group selected from cycloalkyl, 3-12 membered heterocyclyl, 5-12 membered aryl, 5-12 membered heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR' and C(=O)NR'(R");

[0141] R7 and R8 are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR' or C(=O)NR'(R"), wherein the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted by one or more halogen (such as fluorine, chlorine, bromine), hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-7 substituted by a group selected from cycloalkyl, 3-12 membered heterocyclyl, 5-12 membered aryl, 5-12 membered heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR' and C(=O)NR'(R");

[0142] R' and R" are independently hydrogen, deuterium, hydroxy, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with one or more substituents selected from halogen (such as fluorine, chlorine, bromine), hydroxy, oxo, nitro and cyano;

[0143] m, n, p and q are independently 0, 1, 2, 3 or 4;

[0144] B is

[0145] R b1 、R b2 、R b3 、R b4 、R b5 、R b6 and R b7 are independently -C(=O)-, -NHC(=O)-, -C(=O)O-, -C(=O)-(CH2) z8 -O- or -NHC(=O)-(CH2) z9 -O-;

[0146] z1, z2, z3, z4, z5, z6, z7, z8 and z9 are independently an integer from 0 to 10;

[0147] L2 is C1-C 30 Alkyl chains, or C1-C ... 30 alkyl chain;

[0148] G is a targeting moiety that binds to cell receptors;

[0149] r is an integer from 1 to 10.

[0150] In some embodiments, some groups are defined as follows, and undefined groups are the same as those described in any of the previous schemes (hereinafter referred to as "in some embodiments"), L1 can be L3 or L3-R 10 -R 11 -L3, wherein L3 is independently C1-C 12 Alkyl chain, -(CH2) j1 -C(=O)-(CH2) j2 -or-(CH2) j3 -(CH2CH2O) 1-4 -(CH2) j4 -, R 10 and R 11 independently chemical bonds, -NR 12-, -C(=O)- or -OC(=O)-, R 12 is hydrogen or C1-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.

[0151] In some embodiments, L1 can be -(CH2) j1 -C(=O)-(CH2) j2 -, the definitions of j1 and j2 are the same as those described in any of the previous solutions.

[0152] In some embodiments, L1 may be The definitions of j1 and j2 are the same as those described in any of the previous solutions, wherein the a1 end is connected to B and the b1 end is connected to R1.

[0153] In some embodiments, L1 may be Among them, the a1 end is connected to B, and the b1 end is connected to R1.

[0154] In some embodiments, R1 can be a chemical bond and R2 can be C=O.

[0155] In some embodiments, R1 may be a chemical bond and R2 may be NR6, where R6 is as defined in any of the previous embodiments.

[0156] In some embodiments, R1 may be a chemical bond and R2 may be -OC(=O)-.

[0157] In some embodiments, R1 may be NR6 and R2 may be C=O, and R6 is as defined in any of the previous embodiments.

[0158] In some embodiments, R1 may be NR6 and R2 may be -OC(=O)-, and R6 is as defined in any of the previous embodiments.

[0159] In some embodiments, R2 may be NR6 and R1 may be C=O, and R6 is as defined in any of the previous embodiments.

[0160] In some embodiments, R2 may be NR6 and R1 may be -OC(=O)-, and R6 is as defined in any of the previous embodiments.

[0161] In some embodiments, R6 can be hydrogen or C 1-6 alkyl.

[0162] In some embodiments, R6 can be hydrogen, methyl, ethyl, propyl, or isopropyl.

[0163] In some embodiments, R6 can be hydrogen.

[0164] In some embodiments, R7 and R8 can be hydrogen.

[0165] In some embodiments, R9 can be hydrogen.

[0166] In some embodiments, when Ring A is present, Ring A may be C 1-10 Aryl, preferably phenyl.

[0167] In some embodiments, m can be 0 or 1.

[0168] In some embodiments, m may be 3.

[0169] In some embodiments, n can be 0 or 1.

[0170] In some embodiments, p and q are independently 0 or 1.

[0171] In some embodiments, p=1 and q=1.

[0172] In some embodiments, p=1 and q=0.

[0173] In some embodiments, p=0 and q=1.

[0174] In some embodiments, p=0 and q=0.

[0175] In some embodiments, z1, z2, z3, z4, z5, z6, z7, z8 and z9 can independently be an integer from 0 to 4, preferably 0, 1 or 2.

[0176] In some embodiments, B may be R b1 、R b2 、R b3 and R b4 are independently -C(=O)- or -NHC(=O)-, the N atom is connected to L1, and the definitions of z1, z2, z3 and z4 are the same as those described in any of the previous schemes.

[0177] In some embodiments, B may be R b1 、R b2 、R b3 and R b4 are independently -C(=O)- or -NHC(=O)-, the N atom is connected to L1, R b1 、R b3 and R b4 Similarly, the definitions of z1, z2, z3 and z4 are the same as those described in any of the previous solutions.

[0178] In some embodiments, B may be

[0179] In some embodiments, B may be

[0180] In some embodiments, B may be R b5 、R b6 and R b7 are independently -C(=O)-(CH2) z8 -O- or -NHC(=O)-(CH2) z9 -O-, the N atom is connected to L1, and the definitions of z5, z6, z7, z8 and z9 are the same as those described in any of the previous schemes.

[0181] In some embodiments, B may be R b5 、R b6 and R b7 are independently -C(=O)-(CH2) z8 -O- or -NHC(=O)-(CH2) z9 -O-, N atom is connected to L1, R b5 、R b6 and R b7 Similarly, the definitions of z5, z6, z7, z8 and z9 are the same as those described in any of the previous solutions.

[0182] In some embodiments, B may be

[0183] In some embodiments, L2 may be L4 or L4-R 13 -R 14 -L4, wherein L4 is independently C1-C 12 Alkyl chain or -(CH2) j5 -(OCH2CH2) 1-4 -(CH2) j6 -, R 13 and R 14 independently chemical bonds, -NR 15 -, -C(=O)- or -OC(=O)-, R 15 are independently hydrogen or C1-C 12 Alkyl, j5 and j6 are independently an integer of 0-10, preferably an integer of 0-6, more preferably 0, 1, 2, 3 or 4.

[0184] In some embodiments, L2 can be -(CH2) j5 -(OCH2CH2) 1-4 -(CH2) j6 -, the definitions of j5 and j6 are the same as those described in any of the previous solutions.

[0185] In some embodiments, L2 may be Among them, the O atom is connected to G, the C atom is connected to B, and L2 is preferably

[0186] In some embodiments, L2 may be Wherein, the definition of j6 is the same as that described in any of the previous schemes, the O atom is connected to G, and the C atom is connected to B.

[0187] In some embodiments, L2 may be Among them, the O atom is connected to G, and the C atom is connected to B.

[0188] In some embodiments, G can be an asialoglycoprotein receptor targeting moiety.

[0189] In some embodiments, G can be galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, or N-isobutyrylgalactosamine.

[0190] In some embodiments, r may be 3, 4, 5, or 6, for example, 3.

[0191] In some embodiments, Q may be Preferred Wherein, the definitions of R3, R4, R5 and n are the same as those described in any of the previous schemes.

[0192] In some embodiments, Can Wherein, the definitions of R3, R4, R5, p and q are the same as those described in any of the previous schemes.

[0193] In some embodiments, Can Wherein, the definitions of R3, R4, R5, p and q are the same as those described in any of the previous schemes.

[0194] In some embodiments, Can Preferred More preferred The definitions of p and q are the same as those described in any of the previous schemes.

[0195] In some embodiments, Can Preferred More preferred The definitions of p and q are the same as those described in any of the previous schemes.

[0196] In some embodiments, Can Wherein, the definitions of R3, R4, n, p and q are the same as those described in any of the previous schemes.

[0197] In some embodiments, Can Wherein, the definitions of R3, R4, R5, n, p and q are the same as those described in any of the previous schemes.

[0198] In some embodiments, Can The definitions of n, p and q are the same as those described in any of the previous schemes.

[0199] In some embodiments, Can The definitions of n, p and q are the same as those described in any of the previous schemes.

[0200] In some embodiments, the targeting ligand is selected from the structure shown in formula (II), or a pharmaceutically acceptable salt thereof:

[0201] Among them, L1 is C1-C 30 Alkyl chains, or C1-C ... 30 alkyl chain;

[0202] R1 and R2 are independently a chemical bond, NR6, C=O or -OC(=O)-;

[0203] Q is

[0204] is a single bond or a double bond, and when When it is a single bond, R3 is independently CR7R8, NR6, O or S. When it is a double bond, R3 is independently CR9 or N;

[0205] R4 is independently CR9 or N;

[0206] Ring A is a cycloalkyl, heterocyclyl, aryl or heteroaryl group which may be present or absent, and when ring A is present, R5 is independently CR9 or N, and when ring A is absent, R5 is independently CR7R8, NR6 or O;

[0207] R6 and R9 are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR' or C(=O)NR'(R"), wherein the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted by one or more halogen (such as fluorine, chlorine, bromine), hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-7 substituted by a group selected from cycloalkyl, 3-12 membered heterocyclyl, 5-12 membered aryl, 5-12 membered heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR' and C(=O)NR'(R");

[0208] R7 and R8 are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR' or C(=O)NR'(R"), wherein the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted by one or more halogen (such as fluorine, chlorine, bromine), hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-7 substituted by a group selected from cycloalkyl, 3-12 membered heterocyclyl, 5-12 membered aryl, 5-12 membered heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR' and C(=O)NR'(R");

[0209] R' and R" are independently hydrogen, deuterium, hydroxy, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with one or more substituents selected from halogen (such as fluorine, chlorine, bromine), hydroxy, oxo, nitro and cyano;

[0210] m, n, p and q are independently 0, 1, 2, 3 or 4;

[0211] B is

[0212] R b1 、R b2 、R b3 、R b4 、R b5 、Rb6 and R b7 are independently -C(=O)-, -NHC(=O)-, -C(=O)O-, -C(=O)-(CH2) z8 -O- or -NHC(=O)-(CH2) z9 -O-;

[0213] z1, z2, z3, z4, z5, z6, z7, z8 and z9 are independently an integer from 0 to 10;

[0214] L2 is C1-C 30 Alkyl chains, or C1-C ... 30 alkyl chain;

[0215] r is an integer from 1 to 10.

[0216] In some embodiments, L1 may be L3 or L3-R 10 -R 11 -L3, wherein L3 is independently C1-C 12 Alkyl chain, -(CH2) j1 -C(=O)-(CH2) j2 -or-(CH2) j3 -(CH2CH2) 1-4 -(CH2) j4 -, R 10 and R 11 independently chemical bonds, -NR 12 -, -C(=O)- or -OC(=O)-, R 12 is hydrogen or C1-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.

[0217] In some embodiments, L1 can be -(CH2) j1 -C(=O)-(CH2) j2 -, the definitions of j1 and j2 are the same as those described in any of the previous solutions.

[0218] In some embodiments, L1 may be The definitions of j1 and j2 are the same as those described in any of the previous solutions, wherein the a1 end is connected to B and the b1 end is connected to R1.

[0219] In some embodiments, L1 may be Among them, the a1 end is connected to B, and the b1 end is connected to R1.

[0220] In some embodiments, R1 can be a chemical bond and R2 can be C=O.

[0221] In some embodiments, R1 may be a chemical bond and R2 may be NR6, where R6 is as defined in any of the previous embodiments.

[0222] In some embodiments, R1 may be a chemical bond and R2 may be -OC(=O)-.

[0223] In some embodiments, R1 may be NR6 and R2 may be C=O, and R6 is as defined in any of the previous embodiments.

[0224] In some embodiments, R1 may be NR6 and R2 may be -OC(=O)-, and R6 is as defined in any of the previous embodiments.

[0225] In some embodiments, R2 may be NR6 and R1 may be C=O, and R6 is as defined in any of the previous embodiments.

[0226] In some embodiments, R2 may be NR6 and R1 may be -OC(=O)-, and R6 is as defined in any of the previous embodiments.

[0227] In some embodiments, R6 can be hydrogen or C 1-6 alkyl.

[0228] In some embodiments, R6 can be hydrogen, methyl, ethyl, propyl, or isopropyl.

[0229] In some embodiments, R6 can be hydrogen.

[0230] In some embodiments, R7 and R8 can be hydrogen.

[0231] In some embodiments, R9 can be hydrogen.

[0232] In some embodiments, when Ring A is present, Ring A may be C 1-10 Aryl, preferably phenyl.

[0233] In some embodiments, m can be 0 or 1.

[0234] In some embodiments, m may be 3.

[0235] In some embodiments, n can be 0 or 1.

[0236] In some embodiments, p and q are independently 0 or 1.

[0237] In some embodiments, p=1 and q=1.

[0238] In some embodiments, p=1 and q=0.

[0239] In some embodiments, p=0 and q=1.

[0240] In some embodiments, p=0 and q=0.

[0241] In some embodiments, z1, z2, z3, z4, z5, z6, z7, z8 and z9 can independently be an integer from 0 to 4, preferably 0, 1 or 2.

[0242] In some embodiments, B may be R b1 、R b2 、R b3 and R b4 are independently -C(=O)- or -NHC(=O)-, the N atom is connected to L1, and the definitions of z1, z2, z3 and z4 are the same as those described in any of the previous schemes.

[0243] In some embodiments, B may be R b1 、R b2 、R b3 and R b4 are independently -C(=O)- or -NHC(=O)-, the N atom is connected to L1, R b1 、R b3 and R b4 Similarly, the definitions of z1, z2, z3 and z4 are the same as those described in any of the previous solutions.

[0244] In some embodiments, B may be

[0245] In some embodiments, B may be

[0246] In some embodiments, B may be R b5 、R b6 and R b7 are independently -C(=O)-(CH2) z8 -O- or -NHC(=O)-(CH2) z9 -O-, the N atom is connected to L1, and the definitions of z5, z6, z7, z8 and z9 are the same as those described in any of the previous schemes.

[0247] In some embodiments, B may be R b5 、R b6 and R b7 are independently -C(=O)-(CH2) z8 -O- or -NHC(=O)-(CH2) z9 -O-, N atom is connected to L1, Rb5 、R b6 and R b7 Similarly, the definitions of z5, z6, z7, z8 and z9 are the same as those described in any of the previous solutions.

[0248] In some embodiments, B may be

[0249] In some embodiments, L2 may be L4 or L4-R 13 -R 14 -L4, wherein L4 is independently C1-C 12 Alkyl chain or -(CH2) j5 -(OCH2CH2) 1-4 -(CH2) j6 -, R 13 and R 14 independently chemical bonds, -NR 15 -, -C(=O)- or -OC(=O)-, R 15 are independently hydrogen or C1-C 12 Alkyl, j5 and j6 are independently an integer of 0-10, preferably an integer of 0-6, more preferably 0, 1, 2, 3 or 4.

[0250] In some embodiments, L2 can be -(CH2) j5 -(OCH2CH2) 1-4 (CH2) j6 -, the definitions of j5 and j6 are the same as those described in any of the previous solutions.

[0251] In some embodiments, L2 may be Preferred Among them, the O atom is connected to G, and the C atom is connected to B.

[0252] In some embodiments, L2 may be C1-C 12 Alkyl chain.

[0253] In some embodiments, L2 may be

[0254] In some embodiments, L2 may be Preferred More preferred Among them, the a3 end is connected to G, and the b3 end is connected to B.

[0255] In some embodiments, L2 may be Among them, the a3 end is connected to G, and the b3 end is connected to B.

[0256] In some embodiments, r may be 3, 4, 5, or 6, preferably 3.

[0257] In some embodiments, Q may be Preferred Wherein, the definitions of R3, R4, R5 and n are the same as those described in any of the previous schemes.

[0258] In some embodiments, Can Wherein, the definitions of R3, R4, R5, p and q are the same as those described in any of the previous schemes.

[0259] In some embodiments, Can Wherein, the definitions of R3, R4, R5, p and q are the same as those described in any of the previous schemes.

[0260] In some embodiments, Can Preferred More preferred The definitions of p and q are the same as those described in any of the previous schemes.

[0261] In some embodiments, Can Preferred More preferred The definitions of p and q are the same as those described in any of the previous schemes.

[0262] In some embodiments, Can Wherein, the definitions of R3, R4, n, p and q are the same as those described in any of the previous schemes.

[0263] In some embodiments, Can Wherein, the definitions of R3, R4, R5, n, p and q are the same as those described in any of the previous schemes.

[0264] In some embodiments, Can Preferred The definitions of n, p and q are the same as those described in any of the previous schemes.

[0265] In some embodiments, Can The definitions of n, p and q are the same as those described in any of the previous schemes.

[0266] In some embodiments, the targeting ligand is any of the following structures, or a pharmaceutically acceptable salt thereof:

[0267] In some embodiments, the targeting ligand is selected from any of the following structures, or a pharmaceutically acceptable salt thereof:

[0268] In some embodiments, the N-acetyl-galactosamine moiety in the above ligands can be replaced with N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, or N-isobutyrylgalactosamine.

[0269] In some embodiments, the N-acetyl-galactosamine triacetate portion of the above-mentioned targeting ligands is replaced with N-trifluoroacetylgalactosamine triacetate, N-propionylgalactosamine triacetate, N-n-butyrylgalactosamine triacetate, or N-isobutyrylgalactosamine triacetate.

[0270] In some specific embodiments, the sense strand comprises the nucleotide sequence shown in SEQ ID NO:6 or SEQ ID NO:7.

[0271] In some specific embodiments, the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:5.

[0272] In some specific embodiments, the sense strand is selected from the nucleotide sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7;

[0273] In some specific embodiments, the antisense strand is selected from the nucleotide sequence shown in SEQ ID NO:5.

[0274] In some specific embodiments, the sense strand of the oligonucleotide described herein comprises SEQ ID NO:6, and the antisense strand comprises SEQ ID NO:5.

[0275] In some specific embodiments, the sense strand of the oligonucleotide described herein comprises SEQ ID NO:7, and the antisense strand comprises SEQ ID NO:5.

[0276] In some specific embodiments, the sense strand of the oligonucleotide described herein is selected from SEQ ID NO:6, and the antisense strand is selected from SEQ ID NO:5.

[0277] In some specific embodiments, the sense strand of the oligonucleotide described herein is selected from SEQ ID NO: 7, and the antisense strand is selected from SEQ ID NO: 5.

[0278] In some specific embodiments, the oligonucleotides described in the present disclosure are selected from any one of the following:

[0279] The sense strand comprises the nucleotide sequence set forth in SEQ ID NO:6, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:5;

[0280] The sense strand comprises the nucleotide sequence shown in SEQ ID NO:7, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:5.

[0281] In some specific embodiments, the oligonucleotides described in the present disclosure are selected from any one of the following:

[0282] 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;

[0283] 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.

[0284] In the present disclosure, according to the 5'-3' direction,

[0285] SEQ ID NO:6 is GmsCmsUmCmCmUmUfAfUfUmGmUmUmAmUmAmCmGmAm-NAG0052';

[0286] SEQ ID NO:7 is GmsCmsUmCmCmUmUfAfUfUmGmUmUmAmUmAmCmGmsAm-NAG0052';

[0287] SEQ ID NO:5 is NA0127CfsGmUfAmUfAmAmCmAfAmUfAmAfGmGfAmGfCmsUmsGm;

[0288] Wherein, 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;

[0289] s indicates that the two nucleotides adjacent to the letter s are connected by a phosphorothioate diester group;

[0290] NA0127 indicates

[0291] NAG0052' indicates

[0292] In some embodiments, the oligonucleotide is in an anionic form as shown in the following structure or a pharmaceutically acceptable salt thereof:

[0293] in,

[0294] Af = adenine 2'-F ribonucleoside;

[0295] Cf = cytosine 2'-F ribonucleoside;

[0296] Uf = uracil 2'-F ribonucleoside;

[0297] Gf = guanine 2'-F ribonucleoside;

[0298] Am = adenine 2'-OMe ribonucleoside;

[0299] Cm = cytosine 2'-OMe ribonucleoside;

[0300] Gm = guanine 2'-OMe ribonucleoside;

[0301] Um = uracil 2'-OMe ribonucleoside;

[0302] represents a phosphorothioate group, represents a phosphodiester group;

[0303] NAG0052' indicates

[0304] NA0127' means

[0305] Pharmaceutically acceptable salts may be conventional salts in the art, including but not limited to sodium salts, potassium salts, ammonium salts, amine salts, and the like.

[0306] In some embodiments, the oligonucleotides disclosed herein are of the following structure or a pharmaceutically acceptable salt thereof:

[0307] in,

[0308] Af = adenine 2'-F ribonucleoside;

[0309] Cf = cytosine 2'-F ribonucleoside;

[0310] Uf = uracil 2'-F ribonucleoside;

[0311] Gf = guanine 2'-F ribonucleoside;

[0312] Am = adenine 2'-OMe ribonucleoside;

[0313] Cm = cytosine 2'-OMe ribonucleoside;

[0314] Gm = guanine 2'-OMe ribonucleoside;

[0315] Um = uracil 2'-OMe ribonucleoside;

[0316] represents a phosphorothioate group, represents a phosphodiester group;

[0317] NAG0052' indicates:

[0318] NA0127 indicates

[0319] Pharmaceutically acceptable salts may be conventional salts in the art, including but not limited to sodium salts, potassium salts, ammonium salts, amine salts, and the like.

[0320] In some embodiments, the oligonucleotide is selected from TJR100747, TJR100848.

[0321] In some embodiments, the oligonucleotide is selected from the following structures:

[0322] In some embodiments, the oligonucleotide is selected from the following structures:

[0323] in,

[0324] Af = adenine 2'-F ribonucleoside;

[0325] Cf = cytosine 2'-F ribonucleoside;

[0326] Uf = uracil 2'-F ribonucleoside;

[0327] Gf = guanine 2'-F ribonucleoside;

[0328] Am = adenine 2'-OMe ribonucleoside;

[0329] Cm = cytosine 2'-OMe ribonucleoside;

[0330] Gm = guanine 2'-OMe ribonucleoside;

[0331] Um = uracil 2'-OMe ribonucleoside;

[0332] represents a phosphorothioate group, represents a phosphodiester group;

[0333] NAG0052' indicates:

[0334] NA0127' means

[0335] The present disclosure also provides a compound represented by formula (IV) or a tautomer thereof,

[0336] Among them, R A8 、R A9 Each is independently selected from -CH3, -CH2CH3, -CH2CH2CN, -CH2OPiv (ie, -CH2OCOC(CH3)3), CH2OCH2CH2Si(CH3)3 or a protecting group; R A10 Selected from phosphorus-containing reactive groups; R A1 、R A2 Each is independently selected from hydrogen or deuterium; M4 is selected from O or S; B is selected from base, hydrogen, deuterium; R A3 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), the hydroxyl, alkyl, alkoxy are each optionally substituted with one or more deuterium; B is selected from base, hydrogen, deuterium; with the proviso that formula (IV) contains at least one deuterium.

[0337] In some embodiments, Formula (IV) is selected from Formula (IV-1),

[0338] Among them, R A5 、R A6 、R A7 are each independently selected from hydrogen or deuterium; R A1 、R A2 、R A8 、RA9 、R A10 , B is as defined in formula (IV), provided that formula (IV-1) contains at least one deuterium.

[0339] In some embodiments, the compound represented by formula (IV) is selected from:

[0340] B is selected from bases and hydrogen.

[0341] The present disclosure also provides a method for preparing the oligonucleotide of the present disclosure, comprising the following steps:

[0342] (1) synthesizing the compound represented by formula (IV) or its tautomer described in the present disclosure;

[0343] (2) Synthesizing the oligonucleotide described in the present disclosure using the compound represented by formula (IV) or its tautomer in step (1).

[0344] The present disclosure also provides a method for preparing the compound represented by formula (IV) or its tautomer according to the present disclosure, comprising:

[0345] Dissolve compound (IV-2) in dichloromethane, add 2-cyanoethyl N, N-diisopropylchlorophosphoramidite and diisopropylethylamine at room temperature, and stir until the reaction is complete to obtain product (IV-1).

[0346] Among them, R A5 、R A6 、R A7 are each independently selected from hydrogen or deuterium; R A1 、R A2 、R A8 、R A9 、R A10 , B is as defined in formula (IV), provided that formula (IV-1) and (IV-2) contain at least one deuterium.

[0347] On the other hand, the present disclosure further 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:

[0348] Wherein, M3 is selected from -S-, -NH-, -NR A11 -, R A11 Selected from C1-C6 alkylene, R A1 、R A2 , M1, M2, B, R A3 、R A4As defined in formula (I), with the proviso that formula (V) contains at least one deuterium.

[0349] In some embodiments of the present disclosure, the compound represented by formula (I), (I-1), (IV) or (IV-1), wherein the abundance of deuterium in the deuterium atom is at least 2000 times greater than the natural abundance of deuterium, and the natural abundance of deuterium is 0.015%.

[0350] 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 abundance of deuterium in the deuterium atom is at least 3000 times greater than the natural abundance of deuterium, and the natural abundance of deuterium is 0.015%.

[0351] 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 abundance of deuterium in the deuterium atom is at least 3340 times greater than the natural abundance of deuterium, and the natural abundance of deuterium is 0.015%.

[0352] 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 abundance of deuterium in the deuterium atom is at least 3500 times greater than the natural abundance of deuterium, and the natural abundance of deuterium is 0.015%.

[0353] 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 abundance of deuterium in the deuterium atom is at least 4000 times greater than the natural abundance of deuterium, and the natural abundance of deuterium is 0.015%.

[0354] 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 abundance of deuterium in the deuterium atom is at least 4500 times greater than the natural abundance of deuterium, and the natural abundance of deuterium is 0.015%.

[0355] 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 abundance of deuterium in the deuterium atom is at least 5000 times greater than the natural abundance of deuterium, and the natural abundance of deuterium is 0.015%.

[0356] 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 abundance of deuterium in the deuterium atom is at least 5500 times greater than the natural abundance of deuterium, and the natural abundance of deuterium is 0.015%.

[0357] 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 abundance of deuterium in the deuterium atom is at least 6000 times greater than the natural abundance of deuterium, and the natural abundance of deuterium is 0.015%.

[0358] 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 abundance of deuterium in the deuterium atom is at least 6333.3 times greater than the natural abundance of deuterium, and the natural abundance of deuterium is 0.015%.

[0359] 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 abundance of deuterium in the deuterium atom is at least 6466.7 times greater than the natural abundance of deuterium, and the natural abundance of deuterium is 0.015%.

[0360] 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 abundance of deuterium in the deuterium atom is at least 6600 times greater than the natural abundance of deuterium, and the natural abundance of deuterium is 0.015%.

[0361] 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 abundance of deuterium in the deuterium atom is at least 6633.3 times greater than the natural abundance of deuterium, and the natural abundance of deuterium is 0.015%.

[0362] In another aspect, the present disclosure provides a pharmaceutical composition comprising the oligonucleotide of the present disclosure.

[0363] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0364] In some embodiments, the pharmaceutically acceptable excipient can be, for example, a carrier, a vehicle, a diluent, and / or a delivery polymer.

[0365] In some embodiments, the oligonucleotide may be in a therapeutically effective amount.

[0366] In some embodiments, the unit dose of the oligonucleotide may be 0.001 mg-1000 mg.

[0367] In some embodiments, based on the total weight of the pharmaceutical composition, the content of the oligonucleotide may be 0.01-99.99%, further 0.1-99.9%, further 0.5%-99.5%, further 1%-99%, and further 2%-98%.

[0368] In some embodiments, based on the total weight of the pharmaceutical composition, the content of the pharmaceutically acceptable excipient may be 0.01-99.99%, further 0.1-99.9%, further 0.5%-99.5%, further 1%-99%, and further 2%-98%.

[0369] In some embodiments, the effective amount or effective dose 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.

[0370] Various delivery systems are known and can be used with the oligonucleotides or pharmaceutical compositions of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, constructing the nucleic acid as part of a retroviral or other vector.

[0371] In some embodiments, the oligonucleotides or pharmaceutical compositions of the present invention are administered in a conventional manner, and may be administered topically (e.g., by direct injection or implantation) or systemically, or may be administered orally, rectally, or parenterally, including but not limited to subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, transdermal administration, inhalation administration (e.g., aerosol), mucosal administration (e.g., sublingual, intranasal administration), intracranial administration, and the like.

[0372] In some embodiments, the oligonucleotides or pharmaceutical compositions provided by the present disclosure can be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal injection.

[0373] In some embodiments, the oligonucleotides or pharmaceutical compositions provided by the present disclosure can be packaged in a kit.

[0374] In another aspect, the present disclosure provides a use of the aforementioned oligonucleotide or the aforementioned pharmaceutical composition in preparing a drug.

[0375] In some embodiments, the drug can 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 ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower limb artery disease or aortic valve stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, and heart failure. In some embodiments, the drug is used to inhibit the expression of LPA.

[0376] In some embodiments, the drug can be used to prevent and / or treat cardiovascular disease. In some embodiments, the cardiovascular disease is selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower limb artery disease or aortic valve stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, heart failure.

[0377] In some embodiments, the medicament can be used to prevent and / or treat thromboembolic complications. In some embodiments, the thromboembolic complications can be deep vein thrombosis, pulmonary embolism, myocardial infarction or stroke.

[0378] In some embodiments, the medicament can be used to prevent and / or treat a disease associated with the hepatitis B virus. In some embodiments, the disease associated with the 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.

[0379] In some embodiments, the medicament can be used to prevent and / or treat a disease associated with APOC3 gene expression. In some embodiments, the disease is selected from hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, or familial partial lipid dystrophy.

[0380] In some embodiments, the medicament can 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 hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, or familial partial lipid dystrophy.

[0381] In another aspect, the present disclosure provides a method for preventing and / or treating a disease, comprising administering an effective amount or effective dose of the aforementioned oligonucleotide or the aforementioned pharmaceutical composition to a subject.

[0382] 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 ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower limb artery disease or aortic valve stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, and heart failure.

[0383] In some embodiments, the disease may be a cardiovascular disease. In some embodiments, the cardiovascular disease is selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower limb artery disease or aortic valve stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, heart failure.

[0384] In some embodiments, the disease may be a disease associated with the hepatitis B virus. In some embodiments, the disease associated with the 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.

[0385] In some embodiments, the disease may be a disease associated with APOC3 gene expression. In some embodiments, the disease is selected from hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, or familial partial lipid dystrophy.

[0386] 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 hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, or familial partial lipid dystrophy.

[0387] 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.

[0388] In another aspect, the present disclosure provides a method for reducing low-density lipoprotein levels in a subject, comprising administering to the subject an effective amount or effective dose of the aforementioned oligonucleotide or the aforementioned pharmaceutical composition.

[0389] In some embodiments, the effective amount or effective dose 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.

[0390] In another aspect, the present disclosure provides a method for reducing apolipoprotein(a) and / or apolipoprotein(a) levels in a subject, comprising administering to the subject an effective amount or effective dose of the above oligonucleotide or the above pharmaceutical composition.

[0391] In some embodiments, the effective amount or effective dose 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.

[0392] In another aspect, the present disclosure provides a method for silencing a target gene or its mRNA in a cell in vivo or in vitro, comprising the step of introducing the aforementioned oligonucleotide or the aforementioned pharmaceutical composition into the cell.

[0393] In some embodiments, the oligonucleotides or pharmaceutical compositions disclosed herein can reduce the expression level of a target gene or its mRNA in a subject such as a cell, a cell population, a tissue, or a subject, comprising: administering a therapeutically effective amount of the oligonucleotides or pharmaceutical compositions described herein to the subject, thereby inhibiting the expression of the target gene or its mRNA in the subject.

[0394] 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.

[0395] In another aspect, the present disclosure provides a method for delivering an oligonucleotide to the liver, comprising administering to a subject an effective amount or an effective dose of the oligonucleotide or the pharmaceutical composition.

[0396] In another aspect, the present disclosure further provides a cell comprising the aforementioned oligonucleotide or the aforementioned pharmaceutical composition.

[0397] On the other hand, the present disclosure also provides a kit comprising the aforementioned oligonucleotide or the aforementioned pharmaceutical composition.

[0398] In the present disclosure, when the above-mentioned oligonucleotides or pharmaceutical compositions are contacted with cells expressing the target gene, the above-mentioned oligonucleotides or pharmaceutical compositions can 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% when measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, 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.

[0399] In the present disclosure, when the above-mentioned oligonucleotides or pharmaceutical compositions are contacted with cells expressing the target gene, the percentage of residual target gene mRNA expression caused by the above-mentioned oligonucleotides or pharmaceutical compositions is no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, 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%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, 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.

[0400] In the present disclosure, when the above-mentioned oligonucleotides or pharmaceutical compositions are contacted with cells expressing the target gene, the oligonucleotides reduce off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75% while maintaining target activity, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assays, 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.

[0401] In the present disclosure, when the above-mentioned oligonucleotides or pharmaceutical compositions are contacted with cells expressing the target gene, the oligonucleotides reduce the target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75%, while reducing the off-target activity by at least 20%, at least 19%, at most 15%, at most 10%, at most 5% or more than 1%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, 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.

[0402] The present disclosure also provides a method for preparing an oligonucleotide or a pharmaceutical composition, which comprises: synthesizing the oligonucleotide or the pharmaceutical composition described in the present disclosure.

[0403] Unless otherwise specified, the "nucleic acid", "oligonucleotide", "single-stranded oligonucleotide", "double-stranded oligonucleotide", "antisense oligonucleotide", "small interfering RNA (siRNA)", "double-stranded RNA (dsRNA)", "microRNA (miRNA)", "short hairpin RNA (shRNA)", "ribozyme", "RNAi inhibitor molecule", "double-stranded RNAi inhibitor molecule" and "Dicer enzyme substrate", "compound" of the present disclosure can independently exist in the form of salt, mixed salt or non-salt (such as free acid or free base). When it exists in the form of a salt or a mixed salt, it can be a pharmaceutically acceptable salt.

[0404] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0405] 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, and phosphates; organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, and naphthalene disulfonate. These salts can be prepared by methods known in the art.

[0406] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain 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 salts, sodium salts, potassium salts, calcium salts, and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, following salts: primary amines, secondary amines and tertiary amines, substituted amines, including natural substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the art.

[0407] Without specifying the configuration, the compounds of the present disclosure (e.g., oligonucleotides, siRNA, chemical modifications shown in formula (I), targeting ligands, compounds shown in formula (IV), etc.) may exist in specific geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present disclosure. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure form or in racemic form. Optically pure forms can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0408] Optically active (R)- and (S)-isomers, as well as 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 a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary groups are cleaved to provide the 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), diastereomeric salts are formed with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and then the pure enantiomers are recovered. In addition, separation of enantiomers and diastereoisomers is typically accomplished using chromatography using a chiral stationary phase, optionally combined with chemical derivatization (e.g., to form carbamates from amines).

[0409] In the chemical structures of the compounds disclosed herein (e.g., oligonucleotides, siRNA, chemical modifications represented by formula (I), targeting ligands, compounds represented by formula (IV), etc.), without specifying the configuration, the bonds Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or include both Two configurations. In the chemical structure of the compound disclosed in the present invention, the bond No configuration is specified, i.e. the bond The configuration can be E-type or Z-type, or include both E and Z configurations.

[0410] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can 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 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0411] Compared to non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and prolonged biological half-life. All isotopic variations of the compounds disclosed herein, whether radioactive or not, are encompassed by the present disclosure. Each available hydrogen atom attached to a carbon atom can be independently replaced with a deuterium atom, where the deuterium replacement can be partial or complete. Partial deuterium replacement refers to the replacement of at least one hydrogen atom with at least one deuterium atom.

[0412] Unless otherwise indicated, compounds of the present disclosure, when a position is specifically designated as "deuterium" or "D," are understood to have an abundance of deuterium at that position that is at least 1000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 15% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 1000 times greater than the natural abundance of deuterium (i.e., at least 15% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 2000 times greater than the natural abundance of deuterium (i.e., at least 30% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 3000 times greater than the natural abundance of deuterium (i.e., at least 45% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 3340 times greater than the natural abundance of deuterium (i.e., at least 50.1% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 3500 times greater than the natural abundance of deuterium (i.e., at least 52.5% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 4000 times greater than the natural abundance of deuterium (i.e., at least 60% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 4500 times greater than the natural abundance of deuterium (i.e., at least 67.5% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 5000 times greater than the natural abundance of deuterium (i.e., at least 75% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 5500 times greater than the natural abundance of deuterium (i.e., at least 82.5% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6000 times greater than the natural abundance of deuterium (i.e., at least 90% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6333.3 times greater than the natural abundance of deuterium (i.e., at least 95% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6466.7 times greater than the natural abundance of deuterium (i.e., at least 97% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6600 times greater than the natural abundance of deuterium (i.e., at least 99% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6633.3 times greater than the natural abundance of deuterium (i.e., at least 99.5% deuterium incorporation). Those skilled in the art will be able to synthesize deuterated compounds with reference to the relevant literature. Commercially available deuterated starting materials can be used in the preparation of deuterated compounds, or they can be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane.

[0413] In addition, without specifying the configuration, the compounds and intermediates of the present disclosure (e.g., oligonucleotides, siRNA, chemical modifications shown in formula (I), targeting ligands, compounds shown in formula (IV), etc.) may also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also known as prototransfer tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine, lactam-lactim isomerizations. An example of a lactam-lactim equilibrium is between A and B as shown below,

[0414] All tautomeric forms are within the scope of the present disclosure. The naming of compounds does not exclude any tautomers.

[0415] This disclosure incorporates the full text of WO2023274395A and PCT / CN2022 / 139500.

[0416] Explanation of terms

[0417] In order to make the present disclosure more easily understood, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0418] Unless otherwise specified, 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 monkey LPA, mouse LPA, and rat LPA. Their amino acid and complete coding sequences, as well as their mRNA sequences, are readily available using publicly available databases, such as GenBank, UniProt, OMIM, and the Macaca mulatta genome project website.

[0419] The term "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during LPA transcription, including mRNAs that are processed as the primary transcription product. The targeted portion of the target sequence should be sufficiently long to serve as a substrate for iRNA-directed cleavage. In one embodiment, the target sequence is within the protein-coding region of LPA.

[0420] As used herein, in the context of RNA-mediated gene silencing, the positive strand (also known as SS, SS strand or sense strand) refers to the strand comprising a sequence identical or substantially identical to the target mRNA sequence; the antisense strand (also known as AS or AS strand) refers to the strand having a sequence complementary to the target mRNA sequence.

[0421] In the context of describing the sense strand described herein, the term "the sense strand comprises 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, optionally, the sense strand described herein comprises at least 16 consecutive nucleotides of SEQ ID NO: 1, the sense strand described herein comprises at least 18 consecutive nucleotides of SEQ ID NO: 1, or the sense strand described herein comprises at least 19 consecutive nucleotides of SEQ ID NO: 1.

[0422] In the context of describing the antisense strand described herein, the term "the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 2" is intended to mean that the antisense strand described herein comprises at least 15 consecutive nucleotides of SEQ ID NO: 2, optionally, the antisense strand described herein comprises at least 16 consecutive nucleotides of SEQ ID NO: 2, the antisense strand described herein comprises at least 18 consecutive nucleotides of SEQ ID NO: 2, the antisense strand described herein comprises at least 19 consecutive nucleotides of SEQ ID NO: 2, the antisense strand described herein comprises at least 20 consecutive nucleotides of SEQ ID NO: 2, or the antisense strand described herein comprises at least 21 consecutive nucleotides of SEQ ID NO: 2.

[0423] As used herein, in the context of RNA-mediated gene silencing, the positive strand (also known as SS, SS strand or sense strand) refers to the strand comprising a sequence identical or substantially identical to the target mRNA sequence; the antisense strand (also known as AS or AS strand) refers to the strand having a sequence complementary to the target mRNA sequence.

[0424] Unless otherwise specified, in the context of this disclosure, "G," "C," "A," "T," and "U" represent nucleotides, including guanine, cytosine, adenine, thymidine, and uracil, respectively. A lowercase letter "m" indicates that the nucleotide immediately upstream of the letter "m" is a methoxy-modified nucleotide; a lowercase letter "f" indicates that the nucleotide immediately upstream of the letter "f" is a fluorinated nucleotide; and a lowercase letter "s" indicates that the two nucleotides immediately to the left and right of the letter "s" are linked by a phosphorothioate diester linkage.

[0425] In the present disclosure, unless otherwise specified, the phosphate groups between natural nucleotides are phosphodiester groups.

[0426] In the present disclosure, a thiophosphate diester group refers to a phosphodiester group modified by replacing a non-bridging oxygen atom with a sulfur atom. (M is an S atom) are used interchangeably.

[0427] As used in the present disclosure, the term "2'-fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and "non-2'-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorine group.

[0428] As used in the present disclosure, the term "2'-methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0429] In the context of the present disclosure, "a nucleotide difference" or "a difference of no more than n nucleotides" between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position in the former is changed compared to the latter. For example, when a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, the two nucleotide sequences are considered to have a nucleotide difference at that position, or to differ by one nucleotide. In some embodiments, when a nucleotide at a position that is not chemically modified or modified with a tautomer as shown in formula (I) is replaced by an abasic nucleotide or its equivalent, a nucleotide difference can also be considered to have occurred at that position.

[0430] As used herein, the terms "complementary" and "reverse complement" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one chain are paired with bases on the other chain in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair consists of a purine and a pyrimidine. When adenine on one chain is always paired with thymine (or uracil) on the other chain, and guanine is always paired with cytosine, the two chains are considered to be complementary to each other, and the sequence of the chain can be inferred from the sequence of its complementary chain. Accordingly, "mismatch" means in the art that the bases at corresponding positions in a double-stranded nucleic acid are not paired in a complementary manner.

[0431] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "repress," and other similar terms, and includes any level of inhibition.

[0432] "Effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the onset of a condition, including the biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes that present during the progression of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various conditions associated with the disclosed target genes, target mRNAs, or target proteins, or ameliorating one or more symptoms of the condition, reducing the dose of another agent required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of a condition associated with the disclosed target genes, target mRNAs, or target proteins in a patient.

[0433] In some embodiments, the effective amount or effective dose of an oligonucleotide, double-stranded RNAi inhibitor molecule, or siRNA 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.

[0434] A "pharmaceutical composition" comprises an oligonucleotide, a double-stranded RNAi inhibitor molecule or siRNA disclosed herein, and a pharmaceutically acceptable excipient and / or adjuvant. The excipient may be one or more of various formulations or compounds conventionally used in the art. For example, the pharmaceutically acceptable excipient may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0435] As used herein, "subject," "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as mammals, eg, humans or monkeys.

[0436] The oligonucleotides, double-stranded RNAi inhibitor molecules or siRNA provided by the present disclosure can be obtained by conventional preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis already has commercial customization services. Modified nucleotide groups can be introduced into the oligonucleotides, double-stranded RNAi inhibitor molecules or siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into oligonucleotides, double-stranded RNAi inhibitor molecules or siRNA are also well known to those skilled in the art.

[0437] The term "chemically modified" or "modification" includes all changes in a nucleotide by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0438] The term "base" includes any known DNA and RNA base, base analogs, such as purine or pyrimidine, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogs. Base analogs can also be universal bases.

[0439] The terms "blunt end" or "blunt end" are used interchangeably to refer to a given end of an siRNA having no unpaired nucleotides or nucleotide analogs, i.e., no nucleotide overhangs. In most cases, an siRNA having both ends blunt-ended will be double-stranded throughout its entire length.

[0440] The terms "about" and "approximately" refer to values ​​that are within an acceptable error range for a specific value as determined by one of ordinary skill in the art, which depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean a standard deviation within or exceeding 1. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, or between 0.5% and 1%. In this disclosure, each case in which a number or numerical range is preceded by the term "about" also includes embodiments of the given number. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0441] The term "optionally" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "C 1-6 The term "alkyl" means that halogen or cyano may but need not be present, and the description includes both the case where the alkyl is substituted by halogen or cyano and the case where the alkyl is not substituted by halogen and cyano.

[0442] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing from 1 to 20 carbon atoms, and in some embodiments is selected from alkyl groups containing from 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-dimethylpentyl, 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-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. In some embodiments, selected from the group consisting of alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which 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, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment and, in some embodiments, is selected from one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylate.

[0443] The term "alkoxy" refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from halogen, hydroxy, oxo, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, 3- to 7-membered cycloalkyl or 3- to 7-membered heterocycloalkyl, wherein the alkyl, alkoxy, cycloalkyl or heterocycloalkyl is optionally substituted by halogen, hydroxy, nitro, cyano or amino.

[0444] The term "alkylthio" refers to -S-(alkyl), wherein alkyl is as defined above. Non-limiting examples of alkylthio include: methylthio, ethylthio, propylthio, butylthio. Alkylthio may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from C 1-6 Alkoxy, 3 to 6 membered cycloalkyl, 3 to 6 membered heterocycloalkyl, 3 to 6 membered cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 1-6 Alkylthio, 3- to 6-membered cycloalkylthio, 3- to 6-membered heterocycloalkylthio, wherein the alkoxy, cycloalkyl, heterocycloalkyl, cycloalkyloxy, heterocyclooxy, alkylthio, cycloalkylthio, heterocycloalkylthio are optionally substituted with halogen, hydroxy, cyano or amino.

[0445] The term "alkenyl" refers to a straight or branched non-aromatic hydrocarbon group containing at least one carbon-carbon double bond and having 2 to 10 carbon atoms. Up to 5 carbon-carbon double bonds may be present in such a group. For example, "C 2- C6" alkenyl is defined as an alkenyl group having 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, and cyclohexenyl. The straight, branched, or cyclic portion of the alkenyl group may contain double bonds and may be optionally mono-, di-, tri-, tetra-, or penta-substituted at any position permitted by normal valence.

[0446] The term "alkynyl" refers to a straight or branched hydrocarbon group containing 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Up to 5 carbon-carbon triple bonds may be present. Thus, "C2-C6 alkynyl" refers to 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 straight and branched portions of the alkynyl group may contain triple bonds as allowed by normal valence and may be optionally mono-, di-, tri-, tetra-, or penta-substituted at any position allowed by normal valence.

[0447] The term "cycloalkyl" or "carbocycle" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, in some embodiments selected from 3 to 7 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls. Cycloalkyls may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, in some embodiments selected from one or more of the following groups, independently selected from halogen, deuterium, hydroxy, 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 heterocycloalkoxy, C 3-8 Cycloalkenyloxy, 5 to 6 membered aryl 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 The cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl group is optionally substituted by one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, and cyano.

[0448] The cycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a cycloalkyl, non-limiting examples of which include indanyl, tetrahydronaphthyl, benzocycloheptanyl, etc. The cycloalkyl 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, hydroxy, 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 heterocycloalkoxy, C 3-8 Cycloalkenyloxy, 5 to 6 membered aryl 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 The cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl group is optionally substituted by one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, and cyano.

[0449] The term "heterocycloalkyl" or "heterocycle" or "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, but excluding the ring portion 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, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocycloalkyl groups include spirocyclic, fused ring and bridged heterocycloalkyl groups. Non-limiting examples of "heterocycloalkyl" include:

[0450]

[0451] etc.

[0452] The heterocycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a heterocycloalkyl, non-limiting examples of which include:

[0453] wait.

[0454] Heterocycloalkyl may be optionally substituted or unsubstituted. When substituted, the substituents are, in some embodiments, selected from one or more of the following groups, which are independently selected from halogen, deuterium, hydroxy, 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 heterocycloalkoxy, C 3-8 Cycloalkenyloxy, 5 to 6 membered aryl 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 The cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl group is optionally substituted by one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, and cyano.

[0455] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated π 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, wherein the ring attached to the parent structure is the aryl ring, non-limiting examples of which include:

[0456] Aryl groups may be substituted or unsubstituted. When substituted, the substituents are, in some embodiments, selected from one or more of the following groups, which are independently selected from halogen, deuterium, hydroxy, 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 heterocycloalkoxy, C 3-8 Cycloalkenyloxy, 5 to 6 membered aryl 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 The cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl group is optionally substituted by one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, and cyano.

[0457] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. In some embodiments, the heteroaryl group is selected from 6 to 12 members, and in some embodiments, is selected from 5 or 6 members. For example, non-limiting examples include: imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, triazolyl, indazolyl, benzimidazolyl, wait.

[0458] The heteroaryl ring may be fused to an aryl, heterocycloalkyl or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring, non-limiting examples of which include:

[0459] Heteroaryl groups may be optionally substituted or unsubstituted. When substituted, the substituents are, in some embodiments, selected from one or more of the following groups, which are independently selected from halogen, deuterium, hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C2-6 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, 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 The cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl group is optionally substituted by one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, and cyano.

[0460] The term "alkylamino" refers to a group having the structure -NH(C1-C12 alkyl).

[0461] The term "hydroxyalkyl" refers to an alkyl group substituted with one or more hydroxy groups, wherein alkyl is as defined above.

[0462] The term "hydroxy" refers to an -OH group.

[0463] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0464] The term "haloalkyl" refers to an alkyl group substituted with a halogen, wherein alkyl is as defined above.

[0465] The term "haloalkoxy" refers to an alkoxy group substituted with a halogen, wherein alkoxy is as defined above.

[0466] The term "cyano" refers to -CN.

[0467] The term "nitro" refers to -NO2.

[0468] The term "oxo" refers to a =0 group, eg, a carbon atom connected to an oxygen atom via a double bond, where a ketone or aldehyde group is formed.

[0469] The term "amino" refers to -NH2.

[0470] The term "carboxy" refers to -C(O)OH.

[0471] In the chemical structural formula disclosed herein, One or more groups according to the scope of the invention described herein may be attached; an asterisk "*" indicates a chiral center.

[0472] The term "linked" when referring to a connection between two molecules means that the two molecules are connected by a covalent bond or the two molecules are associated via a non-covalent bond (eg, hydrogen bond or ionic bond), including direct connection and indirect connection.

[0473] The term "directly linked" refers to a first compound or group being linked to a second compound or group without any intervening atoms or groups of atoms.

[0474] The term "indirectly linked" means that a first compound or group is linked to a second compound or group through an intermediate group, compound or molecule (eg, a linking group).

[0475] The term "substituted" means that any one or more hydrogen atoms on a designated atom (typically a carbon, oxygen, and nitrogen atom) are replaced by any group as defined herein, provided that the normal valence of the designated atom is not exceeded and the substitution produces a stable compound. Non-limiting examples of substituents include C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cyano, hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, aryl, ketone, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halogen (e.g., F, Cl, Br, I). When a substituent is ketone or oxo (i.e., =O), then two (2) hydrogen atoms on the atom are replaced.

[0476] "Substituted by one or more..." means that the compound may be substituted by a single or multiple substituents. When substituted by multiple substituents, the substituents may be multiple identical substituents or a combination of one or more different substituents.

[0477] 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 under conditions that will not degrade a significant proportion of the molecule being synthesized.

[0478] Some abbreviations used in this disclosure are defined as follows: DCE: 1,2-dichloroethane; Sc(OTf)3: scandium trifluoromethanesulfonate; THF: tetrahydrofuran; Pd / C: palladium on 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'-tetramethyluronium hexafluorophosphate; LiOH: lithium hydroxide; DMAP: 4-dimethylaminopyridine; HBTU: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate; CF3SO3H: trifluoromethanesulfonic acid; BnBr: benzyl bromide; DEPBT: 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4-one; Bz: benzoyl protecting group; MMTr: methoxyphenyldiphenylmethyl; DMTr: dimethoxytrityl protecting group; Piv: pivaloyl. BRIEF DESCRIPTION OF THE DRAWINGS

[0479] Figure 1 shows the mRNA expression levels of TRD002218 and TRD007205 in TTR 7 days after administration.

[0480] Figure 2 shows the mRNA expression levels of TRD002218 and TRD007205 in TTR 28 days after administration.

[0481] Figure 3 shows the serum Lp(a) levels of TJR100747, TJR100848, and TJR100553 mice on day 21. ** indicates P < 0.01 compared with the TJR100553 control group.

[0482] Figure 4 shows the serum Lp(a) levels of TJR100747, TJR100848 and TJR100553 mice on day 28. ** indicates that the difference between each group and the TJR100553 control group was P<0.01; * indicates that the difference between each group and the TJR100553 control group was P<0.1. DETAILED DESCRIPTION

[0483] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples disclosed herein, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the raw material or commercial manufacturer. Reagents whose sources are not specified can be obtained from any molecular biology reagent supplier of a quality / purity suitable for molecular biology applications.

[0484] Example

[0485] Example 1. Synthesis of targeting ligands

[0486] Example 1.1 Preparation of NAG0052 and L96

[0487] Compounds NAG0024 and NAG0026 were purchased from Tianjin WuXi AppTec Pharmaceutical Development Co., Ltd. Unless otherwise specified, all reagents used in the following examples were commercially available products.

[0488] (1) Synthesis of compound NAG0052

[0489] The starting material, compound 1, was purchased from Jiangsu Beida Pharmaceutical Technology Co., Ltd.

[0490] The specific preparation process of NAG0052 is as follows:

[0491] Compound 2

[0492] To a solution of compound 1 (12.3 mL, 101 mmol) in THF (300 mL) at 0°C under nitrogen was added portionwise NaH (12.2 g, 304 mmol, 60% purity). The mixture was stirred at 20°C for 1 hour, then cooled to 0°C again. Benzyl bromide (36.3 mL, 304 mmol) was then added dropwise, and stirred at 20°C for 12 hours. The reaction mixture was quenched with H₂O (100 mL) and extracted with EtOAc (200 mL x 2). The combined organic phases were washed with saturated brine (100 mL), dried over Na₂SO₄, filtered, and concentrated. The resulting residue was separated by silica gel column chromatography to yield the target compound 2 (20.0 g, 51.8 mmol, 51% yield).

[0493] 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] + .

[0494] 1 H NMR: (400MHz, CDCl3) δppm 7.35-7.12(m,10H),5.06-4.95(m,1H), 4.51-4.39(m,4H),4.24-3.87(m,2H),3.50-3.40(m,2H),3.38-3.20(m,3H),2.20-1.91(m,2H).

[0495] Compound 3 and Compound 4

[0496] To a solution of compound 2 (13.0 g, 33.6 mmol) in DCM (300 mL) at 20°C under nitrogen was added TMSCN (13.5 mL, 101 mmol) in one portion, followed by the dropwise addition of a solution of TMSOTf (9.14 mL, 50.5 mmol) in DCM (30 mL). The reaction was stirred at 20°C for 15 hours. After completion of the reaction, the system was quenched with saturated aqueous NaHCO₃ (80 mL) and extracted with DCM (150 mL x 2). The combined organic phases were washed with saturated brine (80 mL), dried over Na₂SO₄, filtered, concentrated, and then separated by silica gel column chromatography to afford the target compound 3 (3.30 g, 9.18 mmol, 27% yield) and compound 4 (8.50 g, 9.18 mmol, 70% yield) as a pale yellow oily liquid.

[0497] Compound 3

[0498] 1 H NMR: (400MHz, CDCl3) δppm 7.42-7.29(m,10H),4.81(t,J=7.8Hz,1H),4.65-4.49(m,4H),4.30-4.21(m,2H),3.65-3.57(m,1H),3.57-3.49(m,1H),2.49-2.40(m,2H).

[0499] Compound 4

[0500] 1 H NMR: (400MHz, CDCl3) δppm 7.42-7.26(m,10H),4.93-4.87(m,1H),4.65-4.48(m,4H),4.43-4.38(m,1H),4 .21-4.17(m,1H),3.79-3.70(m,1H),3.54(d,J=4.0Hz,1H),2.45-2.37(m,2H).

[0501] Compound 5

[0502] A solution of compound 4 (3.00 g, 9.28 mmol) in THF (15 mL) was added dropwise to a solution of LiAlH4 (0.79 g, 20.9 mmol) in THF (15 mL) at 0°C under nitrogen. After complete addition, the system was allowed to react at 0°C for 1 hour. TLC (PE:EtOAc = 3:1) confirmed the complete disappearance of the starting material. Sodium sulfate decahydrate was slowly added to the reaction solution until bubbling ceased. The reaction mixture was then filtered, and the filter cake was washed three times with dichloromethane (60 mL). The filtrate was collected and dried to afford the title compound 5 (3.00 g, 90% yield).

[0503] 1 H NMR: (400MHz, DMSO-d6) δppm 7.40-7.14(m,10H),4.54-4.38(m,4H),4.06-3.99(m,2H),3.91(q,J=6.4Hz,1H ),3.48-3.37(m,2H),2.67-2.52(m,2H),2.21-2.18(m,1H),1.77-1.73(m,1H).

[0504] Compound 6

[0505] Under nitrogen, compound 5 (3.00 g, 8.25 mmol) was dissolved in DCM (30 mL), and TEA (3.44 mL, 24.7 mmol) and CbzCl (1.76 mL, 12.4 mmol) were added. The mixture was allowed to react at 20°C for 2 hours. LCMS indicated the reaction was complete. The reaction solution was extracted with dichloromethane (30 mL) and water (60 mL). The organic phase was washed three times with water (60 mL x 3), dried over anhydrous sodium sulfate, concentrated, and purified using a forward column chromatography (PE:EtOAc = 1:1) to afford the target compound 6 (2.5 g, 90% yield).

[0506] LCMS:t R =0.810min in 5-95AB_1min,MS(ESI)m / z=462.2[M+H] + .

[0507] 1H NMR: (400MHz, CDCl3) δppm 7.39-7.29(m,15H),5.35(s,1H),5.15-5.01(m,2H),4.72(d,J=6.0Hz,1H),4.54-4.40(m,3H),4.26(s,1H),4.23-4.18(m,1H), 4.11-4.04(m,1H),3.54-3.41(m,3H),3.37-3.25(m,1H),2.34-2.23(m,1H),1.85-1.79(m,1H).

[0508] Compound 7

[0509] Under nitrogen, compound 6 (2.00 g, 3.90 mmol) was dissolved in DCM (5 mL) and a 1 M solution of BCl in THF (27.3 mL) was added at -78°C. The reaction was allowed to react for 1 hour. TLC (DCM:MeOH = 10:1) indicated complete disappearance of the starting material. The reaction mixture was quenched by methanol (20 mL) at -78°C, concentrated, and purified using a forward column chromatography (DCM:MeOH = 10:1) to afford the target compound 7 (2.00 g, 60% yield).

[0510] 1 H NMR: (400MHz, CD3OD) δppm 7.41-7.23(m,5H),5.08(s,2H),4.25-4.07(m,2H),3.85-3.75(m,1H),3.63-3.56( m,1H),3.54-3.48(m,1H),3.30-3.27(m,2H),2.34-2.21(m,1H),1.71-1.64(m,1H).

[0511] Compound 8

[0512] Under nitrogen, compound 7 (0.50 g, 1.78 mmol) was dissolved in pyridine (5 mL). 4A molecular sieves (500 mg) and DMTrCl (0.66 mL, 2.13 mmol) were added at 0°C, and the mixture was heated to 20°C for 1.5 hours. TLC (PE:EtOAc = 2:1) indicated 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 x 3), dried over anhydrous sodium sulfate, concentrated, and purified using a forward phase column (PE:EtOAc = 1:1) to obtain the target compound 8 (800 mg, 90% yield).

[0513] 1H NMR: (400MHz, CDCl3) δppm 7.44(d,J=7.6Hz,2H),7.37-7.23(m,11H),7.22-7.15(m,1H),6.84(d,J=8.8Hz,4H),5.09(s,2H),4.31-4.17(m,2H),4. 02-3.91(m,1H),3.84-3.73(m,6H),3.33(s,1H),3.28(s,1H),3.19-3.01(m,2H),2.34-2.25(m,1H),1.70-1.62(m,1H).

[0514] Compound 9

[0515] Compound 8 (800 mg, 1.234 mmol) was dissolved in EtOAc (5 mL), and 10% Pd / C (800 mg, 7.517 mmol) was added. The reaction was allowed to proceed under H₂ conditions (15 psi) at 20°C for 1 hour. LCMS indicated the reaction was complete. The reaction mixture 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 afford compound 9 (300 mg, 54%).

[0516] LCMS:t R =2.586min in 10-80CD_3min MS(ESI)m / z=450.2[M+H] + .

[0517] Compound 11

[0518] Compound 10 (435 mg, 1.780 mmol) was dissolved in DCM (10 mL), and DIEA (0.441 mL, 2.67 mmol) and HATU (677 mg, 1.78 mmol) were added. Compound 9 (400 mg, 0.890 mmol) was then added and allowed to react at 20°C for 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 x 3), dried over anhydrous sodium sulfate, concentrated, and purified using a normal phase column chromatography (PE:EtOAc = 0:1, product peak at 100%) to obtain the target compound 11 (600 mg, 90% yield).

[0519] LCMS:t R =2.745min in 30-90CD_3min,MS(ESI)m / z=698.4[M+Na] + .

[0520] 1 H NMR: (400MHz, CD3OD) δppm 7.46-7.38(m,2H),7.35-7.24(m,6H),7.22-7.16(m,1H),6.90-6.78(m,4H),4.29-4.21(m,2H),4.02-3.95(m,1H),3.77(s, 6H),3.66-3.62(m,3H),3.41(s,1H),3.18-3.04(m,2H),2.36-2.17(m,5H),1.71-1.50(m,5H),1.39-1.25(m,14H).

[0521] Compound 12

[0522] Compound 11 (600 mg, 0.799 mmol) was dissolved in THF (3 mL) and H₂O (1 mL). LiOH.H₂O (134 mg, 3.20 mmol) was added and allowed to react at 20°C for 12 hours. TLC (DCM:MeOH = 10:1) indicated the reaction was complete. The reaction solution was spin-dried, dissolved in water (5 mL) and methanol (5 mL), and purified using a reverse-phase column chromatography (H₂O:CH₃CN = 1:1, peak at approximately 35%) to obtain the target compound 12 (460 mg, 100% yield, lithium salt).

[0523] LCMS:t R =1.346min in 10-80CD_3min,MS(ESI)m / z=684.3[M+Na] + .

[0524] HPLC:t R =1.879min in 10-80CD_6min.

[0525] 1 H NMR: (400MHz, CD3OD) δppm 7.47-7.39(m,2H),7.35-7.24(m,6H),7.22-7.15(m,1H),6.91-6.79(m,4H),4.31-4.18(m,2H),4.02-3.95(m,1H),3.78(s ,6H),3.44-3.33(m,2H),3.18-3.04(m,2H),2.35-2.27(m,1H),2.24-2.10(m,4H),1.70-1.51(m,5H),1.31-1.23(m,12H).

[0526] Compound 13

[0527] At room temperature and under nitrogen, compound NAG0024 (271 mg, 0.151 mmol) was dissolved in anhydrous THF (2 mL) and anhydrous DMF (4 mL). 3A molecular sieves were added, followed by compound 12 (100 mg, 0.151 mmol), HOBt (25 mg, 0.181 mmol), DCC (38 mg, 0.181 mmol), and DIEA (39 mg, 0.302 mmol). The reaction mixture was allowed to react at 45°C for 16 h. After LC-MS showed completion of the reaction, the mixture was quenched with water and filtered. The filtrate was concentrated and purified by C18 reverse-phase column chromatography (HO / MeCN) to afford compound 13 (210 mg, 57% yield).

[0528] Compound NAG0052

[0529] At room temperature, compound 13 (230 mg, 0.094 mmol) was dissolved in pyridine (5 mL), molecular sieves were added, and DMAP (12 mg, 0.283 mmol) and succinic anhydride (28 mg, 0.283 mmol) were added. Under nitrogen, the mixture was stirred at 50°C for 16 hours. LCMS confirmed the reaction was complete, and the product was filtered and dried. After purification by C18 reverse-phase column chromatography, it was repurified by preparative HPLC (acetonitrile / water, gradient elution from 0% acetonitrile to 100% acetonitrile) to obtain the target compound NAG0052 (123 mg, 0.048 mmol, 51% yield).

[0530] MS (ESI) m / z = 2535.3 [M-1] - , theoretical: 2536.2.

[0531] 1H NMR (400 MHz, acetonitrile-d3) δ 7.48-7.43 (m, 2H), 7.37-7.12 (m, 11H), 7.00-6.85 (m, 10H), 6.66 (s, 1H), 5.31 (dd, J = 3.4, 1.1 Hz, 3H), 5.20-5.13 (m, 1H), 5.05 (dd, J = 11.3, 3.4 Hz, 3H), 4.56 (d, J = 8.5 Hz, 3H), 4.30 (dd, J = 7.7, 5.3 Hz, 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),2.1 7(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).

[0532] Synthesis of L96

[0533] L96 shown in the above structural formula was prepared according to the method described in patent application WO2014025805A1.

[0534] Example 1.2 Synthesis of siRNA conjugates containing NAG0052

[0535] 1. Make your own resin with a carrier

[0536] The carboxylic acid-containing compound NAG0052 (157 mg, 0.062 mmol) was dissolved in anhydrous DMF (3 mL). After complete dissolution, anhydrous acetonitrile (4 mL), DIEA (0.03 mL, 0.154 mmol, 2.5 eq), and HBTU (35 mg, 0.093 mmol, 1.5 eq) were added sequentially. After the reaction mixture was thoroughly mixed, macroporous amine methyl resin (476 mg, blank loading 0.41 mmol / g, target loading 0.1 mmol / g) was added. The reaction mixture was shaken overnight on a shaker (temperature: 25°C, speed: 200 rpm). The reaction mixture was filtered, and the filter cake was washed with DCM and anhydrous acetonitrile. The solid was collected and dried under vacuum overnight.

[0537] The solid from the previous step was dispersed in anhydrous acetonitrile (5 mL), and pyridine (0.18 mL), DMAP (3 mg), NMI (0.12 mL), and CapB1 (2.68 mL) were added sequentially. The reaction mixture was shaken on a shaker (temperature: 25°C, speed: 200 rpm) for 2 hours. The reaction mixture was filtered, and the filter cake was washed with anhydrous acetonitrile. The solid was collected and vacuum dried overnight to obtain a resin with a support. The loading capacity was determined to be 0.1 mmol / g.

[0538] 2. Using NAG0052 already attached to the resin as a starting point, nucleoside monomers are attached one by one from the 3' to the 5' direction according to the nucleotide sequence. Each attachment step involves four steps: deprotection, coupling, capping, and oxidation or sulfurization, all performed according to conventional procedures in the art. Compound NAG0052 is attached to the sequence via solid-phase synthesis. After aminolysis, some functional groups in NAG0052 are removed, resulting in NAG0052'.

[0539] The prepared siRNA conjugates had the sense and antisense strands shown in Tables 1 and 2.

[0540] Table 1. List of siRNA conjugates

[0541] Table 2. Nucleic acid sequences of sense and antisense strands

[0542] Table 3. Structure of the conjugates

[0543] Among them, the conjugate TRD002218 is used as a reference positive compound, and Z represents siRNA.

[0544] Example 2: Inhibition of target gene mRNA expression by siRNA conjugates in vivo

[0545] This experiment investigated the inhibitory efficiency of the disclosed siRNA conjugates with different structures on the target gene mRNA expression in vivo.

[0546] 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 of the present disclosure, the reference positive nucleic acid ligand conjugate TRD002218 and PBS were administered to each group of mice.

[0547] All animals were dosed according to body weight. After dissolving the drug in saline, the drug was administered subcutaneously as a single dose. The siRNA conjugate dosage (based on the amount of siRNA) was 1 mg / kg, and the administration volume was 5 mL / kg. Mice were killed 7 and 28 days after administration, and the livers were collected and preserved with RNA later (Sigma Aldrich). The liver tissue was then homogenized with a tissue homogenizer, and total RNA from the liver tissue was extracted using a tissue RNA extraction kit (Fanzhi Medical Technology, FG0412) according to the operating instructions. The total RNA was reverse transcribed into cDNA and the expression of TTR mRNA in the liver tissue was detected by real-time fluorescence quantitative PCR. In this fluorescence quantitative PCR method, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference gene, and Taqman probe primers for TTR and GAPDH were used to detect the mRNA expression levels of TTR and GAPDH, respectively.

[0548] Table 4 Grouping information of compounds tested in mice

[0549] The TTR mRNA expression level was calculated according to the following formula: TTR mRNA expression level = [(TTR mRNA expression level in the test group / GAPDH mRNA expression level in the test group) / (TTR mRNA expression level in the control group / GAPDH mRNA expression level in the control group)] x 100%.

[0550] The inhibitory efficiency of the siRNA conjugates with different structures on the target gene mRNA expression in vivo after 7 and 28 days of administration is shown in FIG1 and FIG2 , respectively.

[0551] As shown in Figure 1, the conjugate TRD007205 exhibited a significant inhibitory effect on TTR mRNA expression 7 days after administration, demonstrating its ability to mediate more efficient delivery. Figure 2 shows that after 28 days of administration, TRD007205 exhibited a superior inhibitory effect on target gene mRNA expression compared to TRD002218. Therefore, the ligand NAG0052 exhibited a significant advantage.

[0552] Example 3, human LPA siRNA

[0553] 1) siRNA Design: Human LPA (NM_005577.3) was used as the target gene, and 19 / 21 nt siRNAs were designed to meet the general rules for active siRNAs. The unmodified sense and antisense strand sequences are detailed in Tables 5 and 6.

[0554] Table 5. Unmodified sense and antisense strands of human LPA siRNA

[0555] Table 6. Unmodified sense and antisense strands of human LPA siRNA (control group)

[0556] Example 4: siRNA psiCHECK Target Activity at 9 Concentrations

[0557] In HEK293A cells, siRNA conjugates were screened for on-target activity at the in vitro molecular level using nine concentration gradients.

[0558] HEK293A cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a seeding density of 8 × 10 cells per well. 3 cells, 100 μL culture medium per well.

[0559] According to the instructions, cells were co-transfected with siRNA and the corresponding plasmid using Lipofectamine 2000 (ThermoFisher, 11668019), using 0.3 μL Lipofectamine 2000 per well. The plasmid transfection amount was 40 ng per well. For the target sequence plasmid, siRNA was set at 9 concentration points, with the highest concentration point having a final concentration of 20 nM. The 3-fold gradient dilution had 9 concentration points of 20 nM, 6.666666667 nM, 2.222222222 nM, 0.740740741 nM, 0.24691358 nM, 0.082304527 nM, 0.027434842 nM, 0.009144947 nM, and 0.003048316 nM. 24 h after transfection, the target level was detected using the Dual-Luciferase Reporter Assay System (Promega, E2940).

[0560] In this example, TJR100373 (comprising the sense chain shown in SEQ ID NO: 1 and the antisense chain shown in SEQ ID NO: 2) whose sense chain and antisense chain were not modified was used for testing.

[0561] The results showed that the unmodified siRNA (naked sequence) TJR100373 had significant activity compared with similar unmodified siRNA sequences (TJR100374-TJR100380).

[0562] Table 7. siRNA activity results at 9 concentration points in the psi-Check system

[0563] Example 5. Inhibition of human LPA in primary human hepatocytes (PHH) by siRNA - Inhibitory activity at 7 concentrations

[0564] siRNA sequences were screened for PHH activity in primary human hepatocytes (PHH) using a 7-point concentration gradient. Each siRNA sample was transfected at a concentration of 20 nM, with 5-fold gradient dilution and 7 concentration points.

[0565] PHH were frozen in liquid nitrogen. 24 h before transfection, primary human hepatocytes (PHH) were revived and seeded into 96-well plates at a seeding density of 3 × 10 cells per well. 4 cells, 80 μL of culture medium per well.

[0566] siRNA was transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150) according to the product instructions. The final concentration of siRNA transfection was 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, and 0.00128 nM. After 24 hours of treatment, total RNA was extracted from cells using a high-throughput RNA extraction kit, followed by reverse transcription and quantitative real-time PCR. Human LPA mRNA levels were measured and corrected for GAPDH levels.

[0567] Among them, in the quantitative real-time PCR detection, a probe Q-PCR detection experiment was used, and its primer information is shown in Tables 8 and 9.

[0568] After the Taqman probe Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct value. Comparative Ct refers to calculating the difference in gene expression by the difference between the Ct value of the internal reference gene, also known as 2 -△△Ct , ΔΔCt=[(target gene in Ct experimental group - internal reference in Ct experimental group)-(target gene in Ct control group - internal reference in Ct control group)]. Inhibition rate (%)=(1-remaining amount of target gene expression)×100%.

[0569] The results are expressed as the percentage of human LPA mRNA expression remaining relative to that in cells treated with control siRNA. 50 The results are shown in Table 10.

[0570] The results showed that TJR100373 had significantly superior activity.

[0571] Table 8. Taqman LPA probe primer information

[0572] Table 9. Taqman GAPDH probe primer information

[0573] Table 10. Multiple-dose inhibitory activity of siRNA on human LPA in primary human hepatocytes (PHH)

[0574] Example 6: Synthesis of Deuterated 5'-Terminal Chemically Modified Phosphoramidites

[0575] first step

[0576] 1-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilaneoctan-8-yl)pyrimidine-2,4(1H,3H)-dione A1-2

[0577] Under a nitrogen atmosphere, compound A1-1 (100 g, 409 mmol) was dissolved in pyridine (1000 mL). 1,3-Dichloro-1,1,3,3-tetraisopropyldimethylsilyl ether (TIPDSiCl2) (135.6 g, 430.0 mmol) was added at 0°C, and the mixture was reacted at 20°C for 18 hours. The reaction solution was extracted with ethyl acetate (1500 mL) and water (1000 mL). The organic phase was washed three times with saturated brine (1000 mL x 3), dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain compound A1-2 (185 g, 380 mmol, 92.8% yield).

[0578] Step 2

[0579] 3-((Benzyloxy)methyl)-1-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-][1,3,5,2,4]trioxadisilocan-8-yl)pyrimidine-2,4(1H,3H)-dione A1-3

[0580] Compound A1-2 (185 g, 380.1 mmol) was dissolved in N,N-dimethylformamide (1850 mL), and benzyl chloromethyl ether (BOMCl) (89.29 g, 570.1 mmol) and 1,8-diazabicycloundec-7-ene (DBU) (115.7 g, 760.2 mmol) were added. The mixture was reacted at 20°C for 3 hours. The reaction solution was extracted with ethyl acetate (3000 mL) and water (3000 mL). The organic phase was washed three times with saturated brine (2000 mL x 3), dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain compound A1-3 (the target compound, 172 g, 275.6 mmol, 72.5% yield).

[0581] Step 3

[0582] 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]trioxadisilocan-8-yl)pyrimidine-2,4(1H,3H)-dione A1-4

[0583] Compound A1-3 (150 g, 247.2 mmol) was dissolved in acetonitrile (300 mL), and CD3I (107.5 g, 741.5 mmol) and silver oxide (114.5 g, 494.3 mmol) were added. The mixture was reacted at 55°C for 24 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain crude compound A1-4 (the target compound, 125 g, 200.3 mmol, 81% yield).

[0584] Step 4

[0585] 3-((Benzyloxy)methyl)-1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-(methoxy-d3)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione A1-5

[0586] Compound A1-4 (125 g, 200 mmol) was dissolved in tetrahydrofuran (1250 mL), and pyridine hydrofluoride (158.8 g, 1.60 mol) was added. The mixture was allowed to react 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 (the target compound, 59 g, 154.7 mmol, 77% yield).

[0587] Step 5

[0588] (2R,3R,4R,5R)-5-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-(methoxy-d3)tetrahydrofuran-3-yl benzoate A1-6

[0589] Compound A1-5 (59 g, 154.7 mmol) was dissolved in pyridine (590 mL), and tert-butyldimethylsilyl chloride (TBSCl) (93.2 g, 618 mmol) was added. The reaction was allowed to proceed at 25°C for 18 hours. Benzoyl chloride (32.62 g, 232.0 mmol) was then added, and the reaction was continued at 25°C for 3 hours. The reaction solution was extracted with ethyl acetate (1000 mL) and water (1000 mL). The organic phase was washed twice with saturated brine (1000 mL x 2), dried over anhydrous magnesium sulfate, concentrated, and purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain compound A1-6 (the target compound, 60.0 g, 100.3 mmol, 64.6% yield).

[0590] Step 6

[0591] (2R,3R,4R,5R)-5-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-hydroxymethyl-4-(methoxy-d3)tetrahydrofuran-3-yl benzoate A1-7

[0592] Compound A1-6 (60.0 g, 100.3 mmol) was dissolved in methanol (600 mL) at 0°C, and acetyl chloride (10.21 g, 130.1 mmol) was added. The mixture was allowed to react at 20°C for 2 hours. Silver carbonate was then added, and the mixture was stirred at 20°C for 1 hour. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound A1-7 (46.0 g, 94.7 mmol, 94.7% yield).

[0593] Step 7

[0594] (2S,3S,4R,5R)-3-(Benzyloxy)-5-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(methoxy-d3)tetrahydrofuran-2-carboxylic acid A1-8

[0595] Compound A1-7 (46.0 g, 94.75 mmol) and 4-carbonyl-tetramethylpiperidinyl oxide (TEMPO) (6.95 g, 44.49 mmol) were dissolved in CH3CN (230 mL) and H2O (230 mL), and 5(6)-amino-1-(4-aminophenyl)-1,3,3-trimethylindane (PIDA) (67.14 g, 208.4 mmol) was added. The mixture was reacted 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 over anhydrous sodium sulfate and concentrated. It was purified by column chromatography (dichloromethane:methanol = 10:1) and concentrated under reduced pressure to obtain compound A1-8 (target compound, 43.0 g, 86 mmol, yield 90%).

[0596] Step 8

[0597] (3S,4R,5R)-2-Acetoxy-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(methoxy-d3)tetrahydrofuran-3-ylbenzoate A1-9

[0598] 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. Pyridine (8 mL, 100 mmol) and lead acetate (9.7 mL, 50 mmol) were added in sequence. The reactants were 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 celite pad. 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 obtain the title product A1-9 (1.5 g, yield: 29%) as an α / β mixture.

[0599] MS (ESI): m / z = 514.2 [M+H] + .

[0600] Step 9

[0601] (3S,4R,5R)-5-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((diethoxyphosphoryl)methoxy)-4-(methoxy-d3)tetrahydrofuran-3-ylbenzoate A1-10

[0602] Under an argon atmosphere, diethyl (hydroxymethyl) phosphonate (2.5 g, 156.7 mmol) and boron trifluoride diethyl etherate (5.7 mL, 43.8 mmol) were added to a solution of compound A1-9 (1.5 g, 2.9 mmol) in anhydrous dichloromethane (15 mL). The reactant was stirred at room temperature for 16 hours. The reactant 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 title compound A1-10 (1 g, 55% yield).

[0603] MS (ESI): m / z = 622.3 [M+H] + .

[0604] Step 10

[0605] (3S,4R,5R)-2-((diethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(methoxy-d3)tetrahydrofuran-3-ylbenzoate A1-11

[0606] A solution of compound A1-10 (1 g, 14.6 mmol) in TFA (5 mL) was stirred at 80° C. for 30 minutes and then concentrated in vacuo to afford the title compound A1-11 (1 g, crude product), which was used directly in the next step.

[0607] MS (ESI): m / z = 502.3 [M+H] + .

[0608] Step 11

[0609] Diethyl ((((3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-(methoxy-d3)tetrahydrofuran-2-yl)oxy)methyl)phosphonate A1-12

[0610] A solution of compound A1-11 (1 g, 2.0 mmol) in ammonia methanol solution (7 N, 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 give the title compound A1-12 (300 mg, yield: 38%).

[0611] MS (ESI): m / z = 398.2 [M+H] + .

[0612] Step 12

[0613] 2-Cyanoethyl((2R,3S,4R,5R)-2-((diethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(methoxy-d3)tetrahydrofuran-3-yl)diisopropylphosphoramidite A1

[0614] 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 the addition of 3-[chloro-(diisopropylamino)phosphoalkyl]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 give phosphoramidite monomer A1 (80 mg, yield: 53%).

[0615] MS (ESI): m / z = 596.0 [MH] + .

[0616] 1 H NMR(400MHz,CD3OD)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).

[0617] 31 P NMR (400MHz, CD3OD) δ151.95, 150.57, 21.29.

[0618] Example 7: Synthesis of siRNA with Deuterated Chemical Modification at the 5' End

[0619] The synthesis of siRNA is the same as the conventional phosphoramidite solid phase synthesis method. When synthesizing the terminal-modified nucleotide of the antisense chain, the phosphoramidite monomer A1 synthesized in Example 6 is used.

[0620] The synthesis process is briefly described as follows: Nucleoside phosphoramidite monomers were ligated one by one according to the synthesis protocol, starting with the Universal CPG vector on a Dr. Oligo 48 synthesizer (Biolytic). With the exception of nucleoside phosphoramidite monomer A1 described in Example 6, the remaining nucleoside monomer starting materials, including 2'-F RNA and 2'-O-methyl RNA, were purchased from Shanghai Zhaowei or Suzhou Jima. 5-Ethylthio-1H-tetrazole (ETT) was used as an activator (0.6 M in acetonitrile). A 0.22 M solution of PADS in a 1:1 volume ratio of acetonitrile and collidine (Suzhou Kelema) was used as a sulfurization reagent to introduce phosphorothioates at specific positions. An iodine pyridine / water solution (Kelema) was used as an oxidant to introduce phosphoroxoates at specific positions.

[0621] After solid-phase synthesis, some embodiments require the use of TMSI to remove the 5'-terminal protecting group. For example, after solid-phase synthesis using phosphoramidite monomer A1, the crude oligoribonucleotide is suspended in anhydrous acetonitrile. TMSI is added at room temperature and stirred thoroughly to remove the ethyl group (Et) from the oligoribonucleotide, yielding an oligoribonucleotide bearing NA0127. The oligoribonucleotide is then cleaved from the solid support using a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. The mixture is then centrifuged, and the supernatant is transferred to another centrifuge tube, concentrated and evaporated to dryness. The resulting product is then purified using C18 reversed-phase chromatography using a mobile phase of 0.1 M TEAA and acetonitrile, with 3% trifluoroacetic acid to remove DMTr. The target oligonucleotide is then lyophilized and identified as the desired product by LC-MS and quantified by UV spectroscopy (260 nm).

[0622] The obtained single-stranded oligonucleotides were annealed according to the complementary pairing in an equal molar ratio, and the resulting double-stranded siRNA was dissolved in 1× PBS and adjusted to the required concentration for the experiment.

[0623] The sequences of the sense and antisense chains with 5'-terminal chemical modifications containing deuteration, 2'-fluoro, 2'-methoxy, etc. are shown in Table 11.

[0624] Table 11. Modified sense and antisense strands of human LPA siRNA

[0625] A lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; a lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide;

[0626] When the lowercase letter s is in the middle, it means that the connection between the two nucleotides adjacent to the letter s is a phosphorothioate connection;

[0627] The structure of NAG0052 is:

[0628] NA0127 and NA0149 respectively indicate:

[0629] Among them, NA0127 is as described in Example 7, and the synthesis method of NA0149 is referred to US20190177729A.

[0630] Example 8. Target activity of siRNA conjugate psiCHECK at nine concentrations

[0631] In vitro molecular level simulation on-target activity screening was performed using 9 concentration gradients in HEK293A cells.

[0632] HEK293A cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a seeding density of 8 × 10 cells per well. 3 cells, 100 μL culture medium per well.

[0633] According to the instructions, cells were co-transfected with siRNA conjugates and corresponding plasmids using Lipofectamine 2000 (ThermoFisher, 11668019), using 0.3 μL Lipofectamine 2000 per well. The plasmid transfection amount was 40 ng per well. For the target sequence plasmid, 9 concentration points were set for siRNA / siRNA conjugates, with the highest concentration point having a final concentration of 20 nM. The 9 concentration points were diluted 3-fold and were: 20 nM, 6.666666667 nM, 2.222222222 nM, 0.740740741 nM, 0.24691358 nM, 0.082304527 nM, 0.027434842 nM, 0.009144947 nM, and 0.003048316 nM. 24 h after transfection, the target level was detected using the Dual-Luciferase Reporter Assay System (Promega, E2940).

[0634] The results are shown in Table 12, which indicate that, compared with TJR100437, TJR100747 and TJR100848 have higher levels of on-target inhibitory activity against the LPA gene in the psiCHECK system.

[0635] Table 12. Psi-CHECK on-target activity screening results

[0636] Example 9: Determination of dsRNA activity in humanized mice (hu-Lp(a))

[0637] The double-transgenic humanized mice (hu-Lp(a)) used in this example contained six copies of the humanized Apo(a) construct, and ApoB100 was introduced from Taconic's B6.SJL-Tg(APOB)1102Sgy N20+?. Lp(a) concentrations were measured using a biochemical analyzer (Ci4100 fully automatic biochemical and immunoassay instrument).

[0638] The mice were evenly divided into groups according to the serum Lp(a) protein content, with 6 mice (male) in each group and 3 mice at each time point. Each group was given drugs TJR100553, TJR100747, and TJR100848 (dissolved in saline) by subcutaneous injection. The dosage was 3 mg / kg and the compound administration volume was 10 μl / g. 40 μl of serum was collected before administration. Mice were euthanized on the 21st and 28th days after administration, and 40 μl of serum was collected from each group. The remaining lipoprotein Lp(a) in the serum was detected using a biochemical analyzer. 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, Figures 3 and 4.

[0639] According to the results in Table 13 and Figure 3, TJR100553 showed significant differences from TJR100747 and TJR100848 on day 21 (P<0.001).

[0640] According to the results in Table 13 and Figure 4, compared with TJR100747 and TJR100848, on day 28, TJR100553 had significant differences with TJR100747, P<0.01, and TJR100553 had significant differences with TJR100848, P<0.001.

[0641] Table 13. hu-Lp(a) content in mouse serum

[0642] The results showed that deuterated chemical modification at the 5' end could significantly prolong the action time of siRNA and enhance its biological activity.

Claims

1. An oligonucleotide comprising a 5' terminal chemical modification represented by formula (I), or a tautomer modification thereof, wherein the 5' terminal chemical modification represented by formula (I) is selected from: in, R A1 、R A2 Each is independently selected from hydrogen or deuterium; M1 and M2 are each independently selected from -SH or -OH; B is selected from a base, hydrogen, deuterium; R A3 R is selected from hydrogen, deuterium, hydroxy, halogen, alkyl, and alkoxy, wherein each of the hydroxy, alkyl, and alkoxy groups is optionally substituted with one or more deuterium groups; A4 is selected from hydrogen, deuterium, alkyl, and alkoxy, wherein the alkyl and alkoxy groups are each optionally substituted with one or more deuterium groups; provided that formula (I) contains at least one deuterium group.

2. The oligonucleotide according to claim 1, wherein the formula (I) is selected from formula (I-1), in, R A5 、R A6 、R A7 are each independently selected from hydrogen or deuterium; R A1 、R A2 , M1, M2, and B are as defined in claim 1, with the proviso that formula (I-1) contains at least one deuterium.

3. The oligonucleotide according to claim 1 or 2, wherein the formula (I) is selected from: B is selected from a base or hydrogen.

4. The oligonucleotide according to any one of claims 1 to 3, wherein the oligonucleotide is a double-stranded RNAi inhibitor molecule, comprising a sense strand and an antisense strand forming a double-stranded region; the 5'-terminal chemical modification represented by formula (I), or a tautomer modification thereof, is located on the antisense strand; preferably, the oligonucleotide is siRNA.

5. The oligonucleotide according to any one of claims 1 to 4, wherein At least one nucleotide other than the 5'-terminal chemically modified nucleotide represented by formula (I) is a modified nucleotide.

6. 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 of the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining positions of the sense strand are all non-2'-fluoro-modified nucleotides; The nucleotide at the 1st position at the 5' end of the antisense chain is a 5'-terminal chemically modified nucleotide represented by formula (I) according to any one of claims 1 to 3, and the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 at the 5' end of the antisense chain are each independently a 2'-fluoro-modified nucleotide, and the remaining positions of the antisense chain are all non-2'-fluoro-modified nucleotides.

7. The oligonucleotide according to claim 6, wherein The non-2'-fluoro modified nucleotide is a 2'-methoxy modified nucleotide.

8. 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 having a modified group, preferably a phosphorothioate diester group.

9. The oligonucleotide according to any one of claims 1 to 8, wherein the oligonucleotide targets the lipoprotein (a) (LPA) gene.

10. The oligonucleotide according to any one of claims 1 to 9, comprising a sense strand and an antisense strand forming a double-stranded region; The sense strand comprises 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 comprises 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 , comprising the sense strand shown in SEQ ID NO: 1 and the antisense strand shown in SEQ ID NO:

2.

12. The oligonucleotide according to any one of claims 1 to 11, wherein The oligonucleotide comprises the sense strand shown in SEQ ID NO: 3 and the antisense strand shown in SEQ ID NO: 5; or, The oligonucleotide comprises a sense strand shown in SEQ ID NO:4 and an antisense strand shown in SEQ ID NO:

5.

13. 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); even more preferably, the targeting ligand comprises 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.

14. The oligonucleotide according to claim 13, wherein The targeting ligand is linked to the 3' end of the sense strand.

15. The oligonucleotide according to claim 13 or 14, wherein The targeting ligand is:

16. The oligonucleotide according to any one of claims 1 to 15, wherein The oligonucleotide comprises the sense strand shown in SEQ ID NO: 6 and the antisense strand shown in SEQ ID NO: 5; or, The oligonucleotide comprises a sense strand shown in SEQ ID NO:7 and an antisense strand shown in SEQ ID NO:

5.

17. The oligonucleotide according to any one of claims 1 to 16, wherein The oligonucleotide is selected from the following structures or pharmaceutically acceptable salts thereof: Wherein, 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 a phosphorothioate diester group, represents a phosphodiester group, NAG0052' represents NA0127' means 18. The oligonucleotide according to any one of claims 1 to 16, wherein The oligonucleotide is selected from the following structures or pharmaceutically acceptable salts thereof: Wherein, 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 a phosphorothioate diester group, represents a phosphodiester group, NAG0052' represents NA0127 indicates 19. A compound represented by formula (IV) or a tautomer thereof, in, R A8 、R A9 Each is independently selected from -CH3, -CH2CH3, -CH2CH2CN, -CH2OPiv, CH2OCH2CH2Si(CH3)3 or a protecting group; R A10 is selected from phosphorus-containing reactive groups; M4 is selected from O or S; R A1 、R A2 、R A3 , B as defined in claim 1, provided that formula (IV) contains at least one deuterium; Preferably, formula (IV) is selected from formula (IV-1), Among them, R A5 、R A6 、R A7 are each independently selected from hydrogen or deuterium, R A1 、R A2 , B as defined in claim 1, provided that formula (IV-1) contains at least one deuterium; R A8 、R A9 Each independently selected from -CH3, -CH2CH3, -CH2CH2CN, -CH2OPiv, CH2OCH2CH2Si(CH3)3 or protecting group; R A10 Selected from phosphorus-containing reactive groups; R A10 Selected from phosphorus-containing reactive groups.

20. The compound of formula (IV) or its tautomer according to claim 19, wherein the compound of formula (IV) is selected from: B is selected from bases.

21. A method for preparing an 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.

22. 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.

23. Use of an oligonucleotide according to any one of claims 1 to 18 or a pharmaceutical composition according to claim 22 in the preparation of a medicament for preventing and / or treating cardiovascular diseases, or for preventing and / or treating diseases associated with elevated levels of lipoprotein (a) and / or apolipoprotein (a); preferably, the diseases associated with elevated levels of lipoprotein (a) and / or apolipoprotein (a) are selected from cardiovascular diseases; the cardiovascular diseases are selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower limb artery disease or aortic valve stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, and heart failure.

24. A method of delivering an oligonucleotide to the liver, comprising administering to a subject an effective amount or effective dose of the oligonucleotide according to any one of claims 1 to 18 or the pharmaceutical composition according to claim 22.

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