TW202339773A

TW202339773A - Dsrna, the application and preparation method thereof

Info

Publication number: TW202339773A
Application number: TW112102810A
Authority: TW
Inventors: 林曉燕; 董玉瓊; 張瑱; 李云飛; 王艶輝; 茅松; 周雅琴; 黃龍飛; 黃燕芬; 黃敏印
Original assignee: 大陸商上海拓界生物醫藥科技有限公司
Priority date: 2022-01-20
Filing date: 2023-01-19
Publication date: 2023-10-16
Also published as: TW202340470A; WO2023138650A1; WO2023138663A1

Abstract

The present disclosure relates to dsRNA, the application and preparation method thereof. The present disclosure also relates to a pharmaceutical composition, cell or kit containing the dsRNA. The disclosed dsRNA can interfere with the expression of HSD17B13, prevent and/or treat related diseases.

Description

A kind of dsRNA, its application and preparation method

本揭露要求申請日為2022年01月20日的中國專利申請202210064314.2的優先權，本揭露引用上述中國專利申請的全文。 This disclosure claims the priority of Chinese patent application 202210064314.2 with a filing date of January 20, 2022. This disclosure cites the full text of the above-mentioned Chinese patent application.

本揭露關於一種dsRNA、其應用及製備方法。 The present disclosure relates to a dsRNA, its applications and preparation methods.

RNA干擾(RNAi)是一種有效的沉默基因表達的方式。據統計，在人體內的疾病相關蛋白中，超過80%的蛋白質不能被目前常規的小分子藥物以及生物大分子製劑所靶向，屬於不可成藥蛋白。利用RNA干擾技術，可以根據編碼這些蛋白的mRNA，設計合適的dsRNA，特異性靶向目標mRNA並降解目標mRNA，從而達到抑制相關的蛋白生成。因此dsRNA具有非常重要的藥物開發前景。然而要實現體內的治療目的RNA干擾效應，需要向體內特定的細胞遞送dsRNA分子。 RNA interference (RNAi) is an effective way to silence gene expression. According to statistics, more than 80% of disease-related proteins in the human body cannot be targeted by current conventional small molecule drugs and biological macromolecule preparations, and are undruggable proteins. Using RNA interference technology, appropriate dsRNA can be designed based on the mRNA encoding these proteins, specifically targeting the target mRNA and degrading the target mRNA, thereby inhibiting the production of related proteins. Therefore, dsRNA has very important drug development prospects. However, to achieve therapeutic RNA interference effects in vivo, dsRNA molecules need to be delivered to specific cells in the body.

採用靶向配體納入dsRNA，利用靶向配體與細胞膜表面的受體分子結構，從而內吞進入到細胞內，是一種有效的藥物遞送方式。例如，去唾液酸糖蛋白受體(ASGPR)是肝細胞特異性表達的受體，在肝細胞表面具有高豐度，胞內外轉換快速的特點。半乳糖、半乳糖胺、N-乙醯半乳糖胺等單糖和多糖分子對ASGPR有高親和性。文獻報導(黃淵餘等人，去唾液酸糖蛋白受體及其在藥物肝靶向遞送中的應用；生物化學與生物物理進展，2015,42(6)：10)使用胺基半乳糖分子簇(GalNAc)可以有效遞送RNA到肝細胞，GalNAc分子被設計成三價或四價的分子簇可以顯著提高單價或二價的GalNAc分子靶向肝細胞的能力。 It is an effective drug delivery method to incorporate targeting ligands into dsRNA and utilize the targeting ligands to interact with the receptor molecular structure on the cell membrane surface to be endocytosed into cells. For example, asialoglycoprotein receptor (ASGPR) is a receptor specifically expressed on hepatocytes and is highly abundant on the surface of hepatocytes. Characterized by rapid intracellular and intracellular conversion. Monosaccharide and polysaccharide molecules such as galactose, galactosamine, and N-acetylgalactosamine have high affinity for ASGPR. Literature reports (Huang Yuanyu et al., Asialoglycoprotein receptor and its application in liver-targeted drug delivery; Progress in Biochemistry and Biophysics, 2015, 42(6): 10) used aminogalactose molecular clusters ( GalNAc) can effectively deliver RNA to liver cells. GalNAc molecules designed into trivalent or tetravalent molecular clusters can significantly improve the ability of monovalent or bivalent GalNAc molecules to target liver cells.

HSD17B13是一類固醇類脫氫酶，屬於17β-羥基類固醇脫氫酶(17β-HSDs)家族的一員。目前在哺乳動物中，17β-HSDs家族已報導有14名成員。這14個成員有著不同的組織分佈、亞細胞定位和功能，作為與類固醇激素、前列腺素、脂質、異生物素和類維生素A代謝有關的酶，主要參與機體的生殖、發育、肥胖等一系列生理與病生理過程。 HSD17B13 is a steroid dehydrogenase, a member of the 17β-hydroxysteroid dehydrogenase (17β-HSDs) family. Currently, 14 members of the 17β-HSDs family have been reported in mammals. These 14 members have different tissue distribution, subcellular localization and functions. As enzymes related to the metabolism of steroid hormones, prostaglandins, lipids, xenobiotics and retinoids, they are mainly involved in a series of reproduction, development, obesity and other aspects of the body. Physiological and pathophysiological processes.

研究發現，在患有單純性脂肪變性的人類受試者中，HSD17B13蛋白表達在脂肪肝脂質液滴(LDs)中明顯上調，並確定其主要位於LDs的表面。當HSD17B13蛋白過表達時，會導致肝細胞中LDs數量和大小的增加，並且會顯著增加肝臟的脂肪生成和甘油三酯(TG)含量(Su W et al.Comparative proteomic study reveals 17β-HSD13 as a pathogenic protein in nonalcoholic fatty liver disease[J].Proceedings of the National Academy of Sciences of the United States of America,2014,111(31)：11437)。 The study found that in human subjects with simple steatosis, HSD17B13 protein expression was significantly upregulated in fatty liver lipid droplets (LDs) and was determined to be mainly located on the surface of LDs. When HSD17B13 protein is overexpressed, it leads to an increase in the number and size of LDs in hepatocytes, and significantly increases liver lipogenesis and triglyceride (TG) content (Su W et al. Comparative proteomic study reveals 17β-HSD13 as a Pathogenic protein in nonalcoholic fatty liver disease[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(31): 11437).

最新研究進一步表明，HSD17B13基因表達在非酒精性脂肪肝疾病(NAFLD)與非酒精性脂肪肝炎(NASH)的發病機制中起著重要作用。HSD17B13(rs72613567：TA)功能缺失型突變體降低了穀丙轉胺酶(ALT)和穀草轉胺酶(AST)的水平，同時減少了脂肪肝患者的炎症和肝損傷(Abul-Husn N S et al.A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease.[J].N Engl J Med,2018,378(12)：1096-1106)。 The latest research further shows that HSD17B13 gene expression plays an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). HSD17B13 (rs72613567: TA) loss-of-function mutant reduces alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels while reducing inflammation and liver damage in patients with fatty liver disease (Abul-Husn N S et al. A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease. [J]. N Engl J Med, 2018, 378(12): 1096-1106).

NAFLD是與肥胖、胰島素抵抗、2型糖尿病、高血壓、高脂血症和代謝綜合症相關的肝病。NAFLD患者有近四分之一的人會進展為以肝臟炎症、肝細胞損傷和肝臟纖維化為特徵的NASH。在HSD17B13(rs72613567：TA)突變體攜帶者中發現，幾種關鍵炎症因子表達水平減少，尤其白細胞介素(IL-6)血漿濃度顯著低於非攜帶者，同時發現患者血清ALT和小兒NAFLD纖維化指數均降低，這揭示了HSD17B13功能缺失的變異體可降低NASH和進行性肝損傷的風險(Luukkonen P K et al.Hydroxysteroid 17-β dehydrogenase 13 variant increases phospholipids and protects against fibrosis in nonalcoholic fatty liver disease[J].JCI Insight,2020,5(5))。 NAFLD is a liver disease associated with obesity, insulin resistance, type 2 diabetes, hypertension, hyperlipidemia, and metabolic syndrome. Nearly one-quarter of NAFLD patients will progress to NASH, which is characterized by liver inflammation, hepatocellular damage, and liver fibrosis. It was found that the expression levels of several key inflammatory factors were reduced in HSD17B13 (rs72613567: TA) mutant carriers, especially the plasma concentration of interleukin (IL-6) was significantly lower than that of non-carriers. It was also found that patients' serum ALT and pediatric NAFLD fiber Hydroxysteroid 17-β dehydrogenase 13 variant increases phospholipids and protects against fibrosis in nonalcoholic fatty liver disease[J ].JCI Insight,2020,5(5)).

因此，本領域仍需要提供一種有效靶向HSD17B13的藥物。 Therefore, there is still a need in the art to provide a drug that effectively targets HSD17B13.

第一方面，本揭露提供了一種雙鏈核糖核酸(dsRNA)，其包含有義鏈和反義鏈，按照5'端到3'端的方向： In a first aspect, the present disclosure provides a double-stranded ribonucleic acid (dsRNA), which includes a sense strand and an antisense strand, in the direction from the 5' end to the 3' end:

該有義鏈第7、8和9位的核苷酸為2'-氟修飾的核苷酸，第5位的核苷酸獨立地為2'-甲氧基修飾的核苷酸或2'-氟修飾的核苷酸，其餘位置的核苷酸為2'-甲氧基修飾的核苷酸； The nucleotides at positions 7, 8 and 9 of the sense strand are 2'-fluoro modified nucleotides, and the nucleotide at position 5 is independently a 2'-methoxy modified nucleotide or 2' -Fluorine modified nucleotides, the remaining nucleotides are 2'-methoxy modified nucleotides;

該反義鏈第2和14位的核苷酸為2'-氟修飾的核苷酸，第4、6、8、9、10、12、16和18位的核苷酸獨立地為2'-甲氧基或2'-氟修飾的核苷酸，其餘位置的核苷酸為2'-甲氧基修飾的核苷酸；該反義鏈中2'-氟修飾的核苷酸的個數為2-7個； The nucleotides at positions 2 and 14 of the antisense strand are 2'-fluoro modified nucleotides, and the nucleotides at positions 4, 6, 8, 9, 10, 12, 16 and 18 are independently 2' -methoxy or 2'-fluoro modified nucleotides, The nucleotides at the remaining positions are 2'-methoxy modified nucleotides; the number of 2'-fluoro modified nucleotides in the antisense strand is 2-7;

該反義鏈第2位至第8位中的至少一個核苷酸位置處包含式(I)所示的化學修飾、其互變異構體或其藥學上可接受的鹽： At least one nucleotide position in positions 2 to 8 of the antisense strand includes a chemical modification represented by formula (I), its tautomer or a pharmaceutically acceptable salt thereof:

其中，Y選自O、NH和S； Among them, Y is selected from O, NH and S;

每個X獨立地選自CR₄(R₄’)、S、NR₅和NH-CO，其中R₄、R₄’、R₅分別獨立地為H或C₁-C₆烷基； _Each _{_} _{_} _{_} _{_} _{_} _{_} _{_}

J₂為H或C₁-C₆烷基； J ₂ is H or C ₁ -C ₆ alkyl;

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

R₃選自H、OH、鹵素、NH₂、C₁-C₆烷基、C₁-C₆烷氧基、C₂-C₆烯基、C₂-C₆炔基、S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH₃、-O-烷基胺基和(CH₂)_pR₆；其中R₆選自OH、鹵素、甲氧基、乙氧基、N₃、C₂-C₆烯基和C₂-C₆炔基，p=1、2或3； R ₃ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ ; wherein R ₆ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, p=1, 2 or 3;

Q₁為

，Q₂為R₂；或者Q₁為R₂，Q₂為

； Q ₁ is

, Q ₂ is R ₂ ; or Q ₁ is R ₂ , Q ₂ is

;

其中， in,

R₁選自H、C₁-C₆烷基、C₁-C₆烷氧基、C₂-C₆烯基、C₂-C₆炔基和(CH₂)_qR₇；其中R₇選自OH、鹵素、甲氧基、乙氧基、N₃、C₂-C₆烯基和C₂-C₆炔基，q=1、2或3； R ₁ is selected from H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ ; wherein R ₇ Selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, q=1, 2 or 3;

J₁為H或C₁-C₆烷基； J ₁ is H or C ₁ -C ₆ alkyl;

R₂選自H、OH、鹵素、NH₂、C₁-C₆烷基、C₁-C₆烷氧基、C₂-C₆烯基、C₂-C₆炔基、S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH₃、-O-烷基胺基和(CH₂)_rR₈；其中R₈選自OH、鹵素、甲氧基、乙氧基、N₃、C₂-C₆烯基和C₂-C₆炔基，r=1、2或3； R ₂ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ ; where R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, r=1, 2 or 3;

視需要地，R₁和R₂直接相連成環； If necessary, R ₁ and R ₂ are directly connected to form a ring;

B是鹼基； B is the base;

該式(I)所示的化學修飾、其互變異構體或其藥學上可接受的鹽修飾不是

。 The chemical modification represented by formula (I), its tautomer or its pharmaceutically acceptable salt modification is not

.

在一些實施方案中，當X為NH-CO時，R₁不是H。 In some embodiments, when X is NH-CO, _R1 is other than H.

在另一些實施方案中，以2’-甲氧基修飾替換式(I)所示的化學修飾、其互變異構體或其藥學上可接受的鹽。 In other embodiments, the chemical modification represented by Formula (I), its tautomer, or its pharmaceutically acceptable salt is replaced with a 2'-methoxy modification.

在一些實施方案中，該反義鏈第2位至第8位中的至少一個核苷酸為2’-甲氧基修飾的核苷酸。 In some embodiments, at least one nucleotide in positions 2 to 8 of the antisense strand is a 2'-methoxy modified nucleotide.

在一些具體的實施方案中，該反義鏈5’端起第2、4、6、10、12、14和16位為2'-氟修飾的核苷酸。 In some specific embodiments, positions 2, 4, 6, 10, 12, 14 and 16 from the 5' end of the antisense strand are 2'-fluoro modified nucleotides.

在一些具體的實施方案中，該反義鏈5’端起第2、4、6、10、14和16位為2'-氟修飾的核苷酸。 In some specific embodiments, positions 2, 4, 6, 10, 14 and 16 from the 5' end of the antisense strand are 2'-fluoro modified nucleotides.

在一些具體的實施方案中，該反義鏈5’端起第2、4、6、12、14和16位為2'-氟修飾的核苷酸。 In some specific embodiments, positions 2, 4, 6, 12, 14 and 16 from the 5' end of the antisense strand are 2'-fluoro modified nucleotides.

在一些具體的實施方案中，該反義鏈5’端起第2、6、10、12、14和16位為2'-氟修飾的核苷酸。 In some specific embodiments, positions 2, 6, 10, 12, 14 and 16 from the 5' end of the antisense strand are 2'-fluoro modified nucleotides.

在一些具體的實施方案中，該反義鏈5’端起第2、6、14和16位為2'-氟修飾的核苷酸。 In some specific embodiments, positions 2, 6, 14 and 16 from the 5' end of the antisense strand are 2'-fluoro modified nucleotides.

在一些具體的實施方案中，該反義鏈5’端起第2、4、6、14和16位為2'-氟修飾的核苷酸。 In some specific embodiments, positions 2, 4, 6, 14 and 16 from the 5' end of the antisense strand are 2'-fluoro modified nucleotides.

在一些具體的實施方案中，該反義鏈5’端起第2、6、10、14和16位為2'-氟修飾的核苷酸。 In some specific embodiments, positions 2, 6, 10, 14 and 16 from the 5' end of the antisense strand are 2'-fluoro modified nucleotides.

在一些具體的實施方案中，該反義鏈5’端起第2、6、12、14和16位為2'-氟修飾的核苷酸。 In some specific embodiments, positions 2, 6, 12, 14 and 16 from the 5' end of the antisense strand are 2'-fluoro modified nucleotides.

在一些具體的實施方案中，該反義鏈5’端起第6、14和16位為2'-氟修飾的核苷酸。 In some specific embodiments, positions 6, 14 and 16 from the 5' end of the antisense strand are 2'-fluoro modified nucleotides.

在一些具體的實施方案中，該反義鏈5’端起第2、14和16位為2'-氟修飾的核苷酸。 In some specific embodiments, positions 2, 14 and 16 from the 5' end of the antisense strand are 2'-fluoro modified nucleotides.

在一些具體的實施方案中，該反義鏈5’端起第2、6和14位為2'-氟修飾的核苷酸。 In some specific embodiments, positions 2, 6 and 14 from the 5' end of the antisense strand are 2'-fluoro modified nucleotides.

在一些實施方案中，式(I)所示的化學修飾選自式(I-1)所示的化學修飾： In some embodiments, the chemical modification represented by Formula (I) is selected from the group consisting of chemical modifications represented by Formula (I-1):

其中，Y選自O、NH和S； Among them, Y is selected from O, NH and S;

每個J₁、J₂分別獨立地為H或C₁-C₆烷基； Each J ₁ and J ₂ are independently H or C ₁ -C ₆ alkyl;

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

B如式(I)中所定義。 B is as defined in formula (I).

在式(I-1)的一些實施方案中，B選自嘌呤鹼基、嘧啶鹼基、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments of Formula (I-1), B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.

在式(I-1)的一些實施方案中，B選自腺嘌呤、鳥嘌呤、異鳥嘌呤、次黃嘌呤、黃嘌呤、C2修飾的嘌呤、N8修飾的嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、N6-烷基腺嘌呤、O6-烷基鳥嘌呤、7-脫氮嘌呤、胞嘧啶、5-甲基胞嘧啶、異胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修飾的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments of formula (I-1), B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diamino group Purine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine , pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole.

在式(I-1)的一些實施方案中，B選自腺嘌呤、鳥嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments of formula (I-1), B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, urea Pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole.

在式(I-1)的一些實施方案中，B與反義鏈該位置處核苷酸未被修飾時的鹼基相同。 In some embodiments of formula (I-1), B is the same base as the nucleotide at that position in the antisense strand when the nucleotide is unmodified.

在一些實施方案中，式(I)所示的化學修飾選自式(I-2)所示的化學修飾： In some embodiments, the chemical modification represented by formula (I) is selected from the chemical modification represented by formula (I-2):

其中Y選自O、NH和S； Where Y is selected from O, NH and S;

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

R₂選自H、C₁-C₆烷基、C₁-C₆烷氧基、S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH₃、-O-烷基胺基和(CH₂)_rR₈；其中R₈選自OH、鹵素、甲氧基、乙氧基、N₃、C₂-C₆烯基和C₂-C₆炔基；r=1、2或3； R ₂ is selected from H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ ; where R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl; r=1, 2 or 3;

B如式(I)中所定義。 B is as defined in formula (I).

在式(I-2)的一些實施方案中，B選自嘌呤鹼基、嘧啶鹼基、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments of Formula (I-2), B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.

在式(I-2)的一些實施方案中，B選自腺嘌呤、鳥嘌呤、異鳥嘌呤、次黃嘌呤、黃嘌呤、C2修飾的嘌呤、N8修飾的嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、N6-烷基腺嘌呤、O6-烷基鳥嘌呤、7-脫氮嘌呤、胞嘧啶、5-甲基胞嘧啶、異胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修飾的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments of formula (I-2), B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diamino group Purine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine , pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole.

在式(I-2)的一些實施方案中，B選自腺嘌呤、鳥嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments of formula (I-2), B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, urea Pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole.

在式(I-2)的一些實施方案中，B與反義鏈該位置處核苷酸未被修飾時的鹼基相同。 In some embodiments of formula (I-2), B is the same base as the nucleotide at that position on the antisense strand when it is not modified.

在式(I)的一些實施方案中，每個X獨立地選自CR₄(R₄’)、S、NR₅和NH-CO，其中R₄、R₄’、R₅分別獨立地為H或C₁-C₃烷基； In _some embodiments _of formula ( _I ₎ _, _each or C ₁ -C ₃ alkyl;

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

每個J₁、J₂分別獨立地為H或C₁-C₃烷基； Each J ₁ and J ₂ are independently H or C ₁ -C ₃ alkyl;

R₃選自H、OH、鹵素、NH₂、C₁-C₃烷基、C₁-C₃烷氧基、C₂-C₄烯基、C₂-C₄炔基、S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH₃、-O-烷基胺基和(CH₂)_pR₆；其中R₆選自OH、鹵素、甲氧基、乙氧基、N₃、C₂-C₆烯基和C₂-C₆炔基，p=1、2或3； R ₃ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ ; wherein R ₆ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, p=1, 2 or 3;

R₁選自H、C₁-C₃烷基、C₁-C₃烷氧基、C₂-C₄烯基、C₂-C₄炔基和(CH₂)_qR₇；其中R₇選自OH、鹵素、甲氧基、乙氧基、N₃、C₂-C₄烯基和C₂-C₄炔基，q=1、2或3； R ₁ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl and (CH ₂ ) _q R ₇ ; wherein R ₇ Selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₄ alkenyl and C ₂ -C ₄ alkynyl, q=1, 2 or 3;

R₂選自H、OH、鹵素、NH₂、C₁-C₃烷基、C₁-C₃烷氧基、C₂-C₄烯基、C₂-C₄炔基、S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH₃、-O-烷基胺基和(CH₂)_rR₈；其中R₈選自OH、鹵素、甲氧基、乙氧基、N₃、C₂-C₄烯基和C₂-C₄炔基，r=1、2或3； R ₂ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ ; where R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₄ alkenyl and C ₂ -C ₄ alkynyl, r=1, 2 or 3;

B如式(I)中所定義。 B is as defined in formula (I).

在一些實施方案中，B選自嘌呤鹼基、嘧啶鹼基、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments, B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.

在一些實施方案中，B選自腺嘌呤、鳥嘌呤、異鳥嘌呤、次黃嘌呤、黃嘌呤、C2修飾的嘌呤、N8修飾的嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、N6-烷基腺嘌呤、O6-烷基鳥嘌呤、7-脫氮嘌呤、胞嘧啶、5-甲基胞嘧啶、異胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修飾的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethyl Aminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil , pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole.

在一些實施方案中，B選自腺嘌呤、鳥嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole , 5-nitroindole and 3-nitropyrrole.

在一些實施方案中，B與反義鏈該位置處核苷酸未被修飾時的鹼基相同。 In some embodiments, B is the same base as the unmodified nucleotide at that position on the antisense strand.

在式(I)的一些實施方案中，每個X獨立地選自CR₄(R₄’)、S、NR₅和NH-CO，其中R₄、R₄’、R₅分別獨立地為H、甲基、乙基、正丙基或異丙基； In _some embodiments _of formula ( _I ₎ _, _each , methyl, ethyl, n-propyl or isopropyl;

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

每個J₁、J₂分別獨立地為H或甲基； Each J ₁ and J ₂ are independently H or methyl;

R₃選自H、OH、F、Cl、NH₂、甲基、乙基、正丙基、異丙基、甲氧基、乙氧基、正丙氧基、異丙氧基、乙烯基、烯丙基、乙炔基、炔丙基、S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH₃、-O-甲基胺基、-O-乙基胺基和(CH₂)_pR₆；其中R₆選自OH、F、Cl、甲氧基、乙氧基、N₃、乙烯基、烯丙基、乙炔基和炔丙基，p=1或2； R ₃ is selected from H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, Allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _p R ₆ ; wherein R ₆ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, p=1 or 2;

R₁選自H、甲基、乙基、正丙基、異丙基、甲氧基、乙氧基、正丙氧基、異丙氧基、乙烯基、烯丙基、乙炔基、炔丙基和(CH₂)_qR₇；其中R₇選自OH、F、Cl、甲氧基、乙氧基、N₃、乙烯基、烯丙基、乙炔基和炔丙基，q=1或2； R ₁ is selected from H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl group and (CH ₂ ) _q R ₇ ; wherein R ₇ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, q=1 or 2;

R₂選自H、OH、F、Cl、NH₂、甲基、乙基、正丙基、異丙基、甲氧基、乙氧基、正丙氧基、異丙氧基、乙烯基、烯丙基、乙炔基、炔丙基、S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH₃、-O-甲基胺基、-O-乙基胺基和(CH₂)_rR₈；其中R₈選自OH、F、Cl、甲氧基、乙氧基、N₃、乙烯基、烯丙基、乙炔基和炔丙基，r=1或2； R ₂ is selected from H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, Allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _r R ₈ ; wherein R ₈ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, r=1 or 2;

B如式(I)中所定義。 B is as defined in formula (I).

在式(I)的一些實施方案中，Y為O或NH；每個X獨立地選自NH-CO、CH₂和NH； In some embodiments of Formula (I), Y is O or NH; each X is independently selected from NH-CO, _CH2 , and NH;

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

每個J₁、J₂分別獨立地為H； Each J ₁ and J ₂ are independently H;

R₁選自H、甲基和CH₂OH； R ₁ is selected from H, methyl and CH ₂ OH;

R₂選自H、OH、NH₂、甲基和CH₂OH； R ₂ is selected from H, OH, NH ₂ , methyl and CH ₂ OH;

R₃選自H、OH、NH₂、甲基和CH₂OH； R ₃ is selected from H, OH, NH ₂ , methyl and CH ₂ OH;

B如式(I)中所定義。 B is as defined in formula (I).

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

R₂選自H、甲基和CH₂OH； R ₂ is selected from H, methyl and CH ₂ OH;

B如式(I)中所定義。 B is as defined in formula (I).

在式(I)的一些實施方案中，Y為O或NH； In some embodiments of formula (I), Y is O or NH;

每個X獨立地選自CR₄(R₄’)、NR₅和NH-CO，R₄、R₄’、R₅分別獨立地為H或C₁-C₆烷基； _Each _{_} _{_} _{_} _{_} _{_} _{_} _{_}

J₂為H或C₁-C₆烷基； J ₂ is H or C ₁ -C ₆ alkyl;

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

R₃選自H、OH、NH₂、C₁-C₆烷基、C₁-C₆烷氧基和(CH₂)_pR₆；R₆選自OH、甲氧基和乙氧基，p=1、2或3； R ₃ is selected from H, OH, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy and (CH ₂ ) _p R ₆ ; R ₆ is selected from OH, methoxy and ethoxy, p=1, 2 or 3;

Q₁為

，Q₂為R₂；或者Q₁為R₂，Q₂為

； Q ₁ is

, Q ₂ is R ₂ ; or Q ₁ is R ₂ , Q ₂ is

;

R₁選自H、OH、C₁-C₆烷基、C₁-C₆烷氧基和(CH₂)_qR₇；R₇選自OH、甲氧基和乙氧基，q=1、2或3； R ₁ is selected from H, OH, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy and (CH ₂ ) _q R ₇ ; R ₇ is selected from OH, methoxy and ethoxy, q=1 , 2 or 3;

J₁為H或C₁-C₆烷基； J ₁ is H or C ₁ -C ₆ alkyl;

R₂選自H、OH、C₁-C₆烷基、C₁-C₆烷氧基和(CH₂)_rR₈；R₈選自OH、甲氧基和乙氧基，r=1、2或3； R ₂ is selected from H, OH, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy and (CH ₂ ) _r R ₈ ; R ₈ is selected from OH, methoxy and ethoxy, r=1 , 2 or 3;

視需要地，R₁和R₂直接相連成3-6員環； If necessary, R ₁ and R ₂ are directly connected to form a 3-6 member ring;

B是鹼基； B is the base;

.

在式(I)的一些實施方案中，X獨立地選自CR₄(R₄’)和NH-CO。 In some embodiments of Formula (I), X is independently selected from _CR4 ( _R4 ') and NH-CO.

在式(I)的一些實施方案中，X獨立地選自CR₄(R₄’)。 In some embodiments of Formula (I), X is independently selected from _CR4 ( _R4 ').

在式(I)的一些實施方案中，R₃選自H、C₁-C₆烷基和(CH₂)_pR₆。 In some embodiments of Formula (I), R ₃ is selected from H, C ₁ -C ₆ alkyl, and (CH ₂ ) _p R ₆ .

在式(I)的一些實施方案中，R₃選自H和C₁-C₆烷基。 In some embodiments of formula (I), _R3 is selected from H and _C1 - _C6 alkyl.

在式(I)的一些實施方案中，R₁選自H、C₁-C₆烷基和(CH₂)_qR₇。 In some embodiments of Formula (I), R ₁ is selected from H, C ₁ -C ₆ alkyl, and (CH ₂ ) _q R ₇ .

在式(I)的一些實施方案中，R₁選自H和C₁-C₆烷基。 In some embodiments of Formula (I), _R1 is selected from H and _C1 - _C6 alkyl.

在式(I)的一些實施方案中，R₂選自H、OH、C₁-C₆烷基和(CH₂)_rR₈。 In some embodiments of Formula (I), _R2 is selected from H, OH, _C1 - _C6 alkyl, and ( _CH2 ) _rR8 _.

在式(I)的一些實施方案中，R₂選自H、C₁-C₆烷基和(CH₂)_rR₈。 In some embodiments of _Formula (I), _R2 is selected from H, _C1 - _C6 alkyl, and ( _CH2 ) _rR8 .

在式(I)的一些實施方案中，Y為O； In some embodiments of formula (I), Y is O;

每個X獨立地選自CR₄(R₄’)和NH-CO，R₄和R₄’分別獨立地為H或C₁-C₆烷基； Each X is independently selected from CR ₄ (R ₄ ') and NH-CO, R ₄ and R ₄ ' are independently H or C ₁ -C ₆ alkyl;

J₂為H或C₁-C₆烷基； J ₂ is H or C ₁ -C ₆ alkyl;

R₃選自H、C₁-C₆烷基和(CH₂)_pR₆；R₆是OH，p=1、2或3； R ₃ is selected from H, C ₁ -C ₆ alkyl and (CH ₂ ) _p R ₆ ; R ₆ is OH, p=1, 2 or 3;

Q₁為

，Q₂為R₂；或者Q₁為R₂，Q₂為

； Q ₁ is

, Q ₂ is R ₂ ; or Q ₁ is R ₂ , Q ₂ is

;

R₁選自H、C₁-C₆烷基和(CH₂)_qR₇；R₇選自OH，q=1、2或3； R ₁ is selected from H, C ₁ -C ₆ alkyl and (CH ₂ ) _q R ₇ ; R ₇ is selected from OH, q=1, 2 or 3;

J₁為H或C₁-C₆烷基； J ₁ is H or C ₁ -C ₆ alkyl;

R₂選自H、OH、C₁-C₆烷基和(CH₂)_rR₈；R₈選自OH，r=1、2或3； R ₂ is selected from H, OH, C ₁ -C ₆ alkyl and (CH ₂ ) _r R ₈ ; R ₈ is selected from OH, r=1, 2 or 3;

視需要地，R₁和R₂直接相連成5-6員環； If necessary, R ₁ and R ₂ are directly connected to form a 5-6 member ring;

B是鹼基。 B is a base.

每個X獨立地選自CR₄(R₄’)，R₄和R₄’分別獨立地為H或C₁-C₆烷基； Each X is independently selected from CR ₄ (R ₄ '), R ₄ and R ₄ ' are independently H or C ₁ -C ₆ alkyl;

J₂為H； J ₂ is H;

R₃選自H和C₁-C₆烷基； R ₃ is selected from H and C ₁ -C ₆ alkyl;

Q₁為

，Q₂為R₂；或者Q₁為R₂，Q₂為

； Q ₁ is

, Q ₂ is R ₂ ; or Q ₁ is R ₂ , Q ₂ is

;

R₁選自H和C₁-C₆烷基； R ₁ is selected from H and C ₁ -C ₆ alkyl;

J₁為H或C₁-C₆烷基； J ₁ is H or C ₁ -C ₆ alkyl;

R₂選自H、C₁-C₆烷基和(CH₂)_rR₈；R₈選自OH，r=1、2或3； R ₂ is selected from H, C ₁ -C ₆ alkyl and (CH ₂ ) _r R ₈ ; R ₈ is selected from OH, r=1, 2 or 3;

B是鹼基。 B is a base.

在一些實施方案中，B選自腺嘌呤、鳥嘌呤、異鳥嘌呤、次黃嘌呤、黃嘌呤、C2修飾的嘌呤、N8修飾的嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、N6-烷基腺嘌呤、O6-烷基鳥嘌呤、7-脫氮嘌呤、胞嘧啶、5- 甲基胞嘧啶、異胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修飾的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethyl Aminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5- Methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, thymine, indole, 5-nitroindole Indoles and 3-nitropyrrole.

在式(I)的一些實施方案中，Y為O。 In some embodiments of Formula (I), Y is O.

在一些實施方案中，X獨立地選自CR₄(R₄’)、NR₅和NH-CO，R₄、R₄’、R₅分別獨立地為H、甲基、乙基、正丙基或異丙基。在一些實施方案中，X獨立地選自NH-CO、CH₂和NH。在一些實施方案中，X獨立地選自NH-CO和CH₂。在一些實施方案中，X為CH₂。 _In _some _embodiments _, _{_} _{_} Or isopropyl. In some embodiments, X is independently selected from NH-CO, _CH2 , and NH. In some embodiments, X is independently selected from NH-CO and _CH2 . In some embodiments, X is _CH2 .

在一些實施方案中，J₂為H或甲基。在一些實施方案中，J₂為H。 In some embodiments, _J2 is H or methyl. In some embodiments, _J2 is H.

在一些實施方案中，R₃選自H、OH、NH₂、甲基、乙基、正丙基、異丙基、甲氧基、乙氧基、正丙氧基、異丙氧基和(CH₂)_pR₆，R₆選自OH、甲氧基和乙氧基，p=1或2。在一些實施方案中，R₃選自H、甲基、乙基、正丙基、異丙基和(CH₂)_pR₆，R₆選自OH，p=1或2。在一些實施方案中，R₃選自H和甲基。 In some embodiments, _R3 is selected from H, OH, _NH2 , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, and ( CH ₂ ) _p R ₆ , R ₆ is selected from OH, methoxy and ethoxy, p=1 or 2. In some embodiments, R ₃ is selected from H, methyl, ethyl, n-propyl, isopropyl, and (CH ₂ ) _p R ₆ , R ₆ is selected from OH, and p=1 or 2. In some embodiments, _R3 is selected from H and methyl.

在一些實施方案中，R₁選自H、OH、甲基、乙基、正丙基、異丙基、甲氧基、乙氧基、正丙氧基、異丙氧基和(CH₂)_qR₇，R₇選自OH，q=1或2。在一些實施方案中，R₁選自H、甲基、乙基、正丙基、異丙基和(CH₂)_qR₇，R₇選自OH，q=1或2。在一些實施方案中，R₁選自H和甲基。 In some embodiments, R ₁ is selected from H, OH, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, and (CH ₂ ) _q R ₇ , R ₇ is selected from OH, q=1 or 2. In some embodiments, R ₁ is selected from H, methyl, ethyl, n-propyl, isopropyl, and (CH ₂ ) _q R ₇ , R ₇ is selected from OH, q=1 or 2. In some embodiments, _R1 is selected from H and methyl.

在一些實施方案中，R₂選自H、OH、甲基、乙基、正丙基、異丙基、甲氧基、乙氧基、正丙氧基、異丙氧基和(CH₂)_rR₈，R₈選自OH，r=1或2。在一些實施方案中，R₂選自H、OH、甲基、乙基、正丙基、異丙基和(CH₂)_rR₈，R₈選自OH，r=1或2。在一些實施方案中，R₂選自H、甲基和CH₂OH。 In some embodiments, _R2 is selected from H, OH, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, and ( _CH2 ) _r R ₈ , R ₈ is selected from OH, r=1 or 2. In some embodiments, R ₂ is selected from H, OH, methyl, ethyl, n-propyl, isopropyl, and (CH ₂ ) _r R ₈ , R ₈ is selected from OH, and r=1 or 2. In some embodiments, _R2 is selected from H, methyl, and _CH2OH .

在一些實施方案中，R₁和R₂直接相連成5-6員環。在一些實施方案中，R₁和R₂直接相連形成3-6員環烷基。在一些實施方案中，R₁和R₂直接相連形成環戊基或環己基。 In some embodiments, R ₁ and R ₂ are directly connected to form a 5-6 membered ring. In some embodiments, R ₁ and R ₂ are directly linked to form a 3-6 membered cycloalkyl group. In some embodiments, R ₁ and R ₂ are directly connected to form cyclopentyl or cyclohexyl.

在一些實施方案中，該式(I)所示的化學修飾選自以下任一結構： In some embodiments, the chemical modification represented by formula (I) is selected from any of the following structures:

其中，B選自嘌呤鹼基、嘧啶鹼基、吲哚、5-硝基吲哚和3-硝基吡咯。 Among them, B is selected from purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.

在一些實施方案中，該包含式(I)所示的化學修飾、其互變異構體或其藥學上可接受的鹽的核苷酸選自包含式(I’)所示的化學修飾、其互變異構體或其藥學上可接受的鹽的核苷酸， In some embodiments, the nucleotide comprising a chemical modification represented by Formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof is selected from the group consisting of a chemical modification represented by Formula (I'), a tautomer thereof, or a pharmaceutically acceptable salt thereof. Nucleotides that are tautomers or pharmaceutically acceptable salts thereof,

其中，Y選自O、NH和S； Among them, Y is selected from O, NH and S;

J₂為H或C₁-C₆烷基； J ₂ is H or C ₁ -C ₆ alkyl;

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

Q_1’為

，Q_2’為R₂；或者Q_1’為R₂，Q_2’為

； Q _1' is

, Q _2' is R ₂ ; or Q _1' is R ₂ , Q _2' is

;

其中， in,

J₁為H或C₁-C₆烷基； J ₁ is H or C ₁ -C ₆ alkyl;

R₂選自H、OH、鹵素、NH₂、C₁-C₆烷基、C₁-C₆烷氧基、C₂-C₆烯基、C₂-C₆炔基、S-CH₃、NCH₃(CH₃)、OCH₂CH₂OCH3、-O-烷基胺基和(CH₂)_rR₈；其中R₈選自OH、鹵素、甲氧基、乙氧基、N₃、C₂-C₆烯基和C₂-C₆炔基，r=1、2或3； R ₂ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH3, -O-alkylamino and (CH ₂ ) _r R ₈ ; where R ₈ is selected from OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, r=1, 2 or 3;

B是鹼基； B is the base;

M為O或S； M is O or S;

該式(I’)所示的化學修飾、其互變異構體或其藥學上可接受的鹽不是

。 The chemical modification represented by the formula (I'), its tautomers or its pharmaceutically acceptable salts are not

.

在式(I’)的一些實施方案中，當X為NH-CO時，R₁不是H。 In some embodiments of Formula (I'), when X is NH-CO, _R1 is other than H.

在式(I’)的一些實施方案中，以2’-甲氧基修飾替換式(I’)所示的化學修飾、其互變異構體或其藥學上可接受的鹽。 In some embodiments of Formula (I'), the chemical modification represented by Formula (I'), its tautomer, or its pharmaceutically acceptable salt is replaced with a 2'-methoxy modification.

在一些實施方案中，該式(I’)所示的化學修飾選自式(I’-1)所示的化學修飾： In some embodiments, the chemical modification represented by formula (I') is selected from the chemical modification represented by formula (I'-1):

其中，Y選自O、NH和S； Among them, Y is selected from O, NH and S;

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

M為O或S； M is O or S;

B如式(I’)中所定義。 B is as defined in formula (I').

在式(I’-1)的一些實施方案中，B選自嘌呤鹼基、嘧啶鹼基、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments of Formula (I'-1), B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.

在式(I’-1)的一些實施方案中，B選自腺嘌呤、鳥嘌呤、異鳥嘌呤、次黃嘌呤、黃嘌呤、C2修飾的嘌呤、N8修飾的嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、N6-烷基腺嘌呤、O6-烷基鳥嘌呤、7-脫氮嘌呤、胞嘧啶、5-甲基胞嘧啶、異胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修飾的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments of formula (I'-1), B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diamine Purine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, heterocytosine Pyrimidine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole .

在式(I’-1)的一些實施方案中，B選自腺嘌呤、鳥嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments of formula (I'-1), B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, Uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.

在式(I’-1)的一些實施方案中，B與反義鏈該位置處核苷酸未被修飾時的鹼基相同。 In some embodiments of formula (I'-1), B is the same base as the nucleotide at that position in the antisense strand when it is not modified.

在一些實施方案中，式(I’)所示的化學修飾選自式(I’-2)所示的化學修飾： In some embodiments, the chemical modification represented by formula (I') is selected from the chemical modification represented by formula (I'-2):

其中，Y選自O、NH和S； Among them, Y is selected from O, NH and S;

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

M為O或S； M is O or S;

B如式(I’)中所定義。 B is as defined in formula (I').

在式(I’-2)的一些實施方案中，B選自嘌呤鹼基、嘧啶鹼基、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments of Formula (I'-2), B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.

在式(I’-2)的一些實施方案中，B選自腺嘌呤、鳥嘌呤、異鳥嘌呤、次黃嘌呤、黃嘌呤、C2修飾的嘌呤、N8修飾的嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、N6-烷基腺嘌呤、O6-烷基鳥嘌呤、7-脫氮嘌呤、胞嘧啶、5-甲基胞嘧啶、異胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修飾的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments of formula (I'-2), B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diamine Purine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, heterocytosine Pyrimidine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole .

在式(I’-2)的一些實施方案中，B選自腺嘌呤、鳥嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments of formula (I'-2), B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, Uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.

在式(I’-2)的一些實施方案中，B與反義鏈該位置處核苷酸未被修飾時的鹼基相同。 In some embodiments of formula (I'-2), B is the same base as the nucleotide at that position in the antisense strand when it is not modified.

在式(I’)、式(I’-1)或式(I’-2)的一些實施方案中，每個X獨立地選自CR₄(R₄’)、S、NR₅和NH-CO，其中R₄、R₄’、R₅分別獨立地為H或C₁-C₃烷基； In some embodiments of Formula (I'), Formula (I'-1), or Formula (I'-2), each X is independently selected from CR ₄ (R ₄ '), S, NR ₅ , and NH- CO, where R ₄ , R ₄ ', and R ₅ are each independently H or C ₁ -C ₃ alkyl;

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

B如式(I’)中所定義。 B is as defined in formula (I').

在式(I’)、式(I’-1)或式(I’-2)的一些實施方案中，每個X獨立地選自CR₄(R₄’)、S、NR₅和NH-CO，其中R₄、R₄’、R₅分別獨立地為H、甲基、乙基、正丙基或異丙基； In some embodiments of Formula (I'), Formula (I'-1), or Formula (I'-2), each X is independently selected from CR ₄ (R ₄ '), S, NR ₅ , and NH- CO, where R ₄ , R ₄ ', and R ₅ are independently H, methyl, ethyl, n-propyl or isopropyl;

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

R₂選自H、OH、F、Cl、NH₂、甲基、乙基、正丙基、異丙基、甲氧基、乙氧基、正丙氧基、異丙氧基、乙烯基、烯丙基、乙炔基、炔丙基、S-CH₃、NCH₃(CH₃)、-OCH₂CH₂OCH₃、-O-甲基胺基、-O-乙基胺基和(CH₂)_rR₈；其中R₈選自OH、F、Cl、甲氧基、乙氧基、N₃、乙烯基、烯丙基、乙炔基和炔丙基，r=1或2； R ₂ is selected from H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, Allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), -OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _r R ₈ ; wherein R ₈ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, r=1 or 2;

B如式(I’)中所定義。 B is as defined in formula (I').

在一些實施方案中，B選自腺嘌呤、鳥嘌呤、異鳥嘌呤、次黃嘌呤、黃嘌呤、C2修飾的嘌呤、N8修飾的嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、N6-烷基腺嘌呤、O6-烷基鳥嘌呤、7-脫氮嘌呤、胞嘧啶、5-甲基胞嘧啶、異胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修飾的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethyl Amino group Purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudocytosine Uracil, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole.

在式(I’)、式(I’-1)或式(I’-2)的一些實施方案中，Y為O或NH；每個X獨立地選自NH-CO、CH₂和NH； In some embodiments of Formula (I'), Formula (I'-1), or Formula (I'-2), Y is O or NH; each X is independently selected from NH-CO, _CH2, and NH;

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

B如式(I’)中所定義。 B is as defined in formula (I').

在一些實施方案中，B選自腺嘌呤、鳥嘌呤、異鳥嘌呤、次黃嘌呤、黃嘌呤、C2修飾的嘌呤、N8修飾的嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、N6-烷基腺嘌呤、O6-烷基鳥嘌呤、7-脫氮嘌呤、胞嘧啶、5- 甲基胞嘧啶、異胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修飾的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2 modified purine, N8 modified purine, 2,6-diaminopurine, 6-dimethyl Aminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5- Methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, thymine, indole, 5-nitroindole Indole and 3-nitropyrrole.

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

B如式(I’)中所定義。 B is as defined in formula (I').

在式(I’)的一些實施方案中，Y為O或NH； In some embodiments of formula (I'), Y is O or NH;

J₂為H或C₁-C₆烷基； J ₂ is H or C ₁ -C ₆ alkyl;

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

Q_1’為

，Q_2’為R₂；或者Q_1’為R₂，Q_2’為

； Q _1' is

, Q _2' is R ₂ ; or Q _1' is R ₂ , Q _2' is

;

J₁為H或C₁-C₆烷基； J ₁ is H or C ₁ -C ₆ alkyl;

M為O或S； M is O or S;

B是鹼基； B is the base;

.

在式(I’)的一些實施方案中，X獨立地選自CR₄(R₄’)和NH-CO。 In some embodiments of Formula (I'), X is independently selected from _CR4 ( _R4 ') and NH-CO.

在式(I’)的一些實施方案中，X獨立地選自CR₄(R₄’)。 In some embodiments of Formula (I'), X is independently selected from CR ₄ (R ₄ ').

在式(I’)的一些實施方案中，R₃選自H、C₁-C₆烷基和(CH₂)_pR₆。 In some embodiments of formula (I'), R ₃ is selected from H, C ₁ -C ₆ alkyl, and (CH ₂ ) _p R ₆ .

在式(I’)的一些實施方案中，R₃選自H和C₁-C₆烷基。 In some embodiments of formula (I'), R ₃ is selected from H and C ₁ -C ₆ alkyl.

在式(I’)的一些實施方案中，R₁選自H、C₁-C₆烷基和(CH₂)_qR₇。 In some embodiments of Formula (I'), R ₁ is selected from H, C ₁ -C ₆ alkyl, and (CH ₂ ) _q R ₇ .

在式(I’)的一些實施方案中，R₁選自H和C₁-C₆烷基。 In some embodiments of formula (I'), R ₁ is selected from H and C ₁ -C ₆ alkyl.

在式(I’)的一些實施方案中，R₂選自H、OH、C₁-C₆烷基和(CH₂)_rR₈。 In some embodiments of Formula (I'), _R2 is selected from H, OH, _C1 - _C6 alkyl, and ( _CH2 ) _rR8 _.

在式(I’)的一些實施方案中，R₂選自H、C₁-C₆烷基和(CH₂)_rR₈。 In some embodiments of formula (I'), R ₂ is selected from H, C ₁ -C ₆ alkyl, and (CH ₂ ) _r R ₈ .

在式(I’)的一些實施方案中，Y為O； In some embodiments of formula (I'), Y is O;

J₂為H或C₁-C₆烷基； J ₂ is H or C ₁ -C ₆ alkyl;

R₃選自H、C₁-C₆烷基和(CH₂)_pR₆；R₆選自OH，p=1、2或3； R ₃ is selected from H, C ₁ -C ₆ alkyl and (CH ₂ ) _p R ₆ ; R ₆ is selected from OH, p=1, 2 or 3;

Q_1’為

，Q_2’為R₂；或者Q_1’為R₂，Q_2’為

； Q _1' is

, Q _2' is R ₂ ; or Q _1' is R ₂ , Q _2' is

;

J₁為H或C₁-C₆烷基； J ₁ is H or C ₁ -C ₆ alkyl;

M為O或S； M is O or S;

B是鹼基。 B is a base.

J₂為H； J ₂ is H;

Q_1’為

，Q_2’為R₂；或者Q_1’為R₂，Q_2’為

； Q _1' is

, Q _2' is R ₂ ; or Q _1' is R ₂ , Q _2' is

;

J₁為H或C₁-C₆烷基； J ₁ is H or C ₁ -C ₆ alkyl;

M為O或S； M is O or S;

B是鹼基。 B is a base.

在式(I’)的一些實施方案中，Y為O。 In some embodiments of Formula (I'), Y is O.

在式(I’)的一些實施方案中，X獨立地選自CR₄(R₄’)、NR₅和NH-CO，R₄、R₄’、R₅分別獨立地為H、甲基、乙基、正丙基或異丙基。在一些實施方案中，X獨立地選自NH-CO、CH₂和NH。在一些實施方案中，X獨立地選自NH-CO和CH₂。在一些實施方案中，X為CH₂。 In _some embodiments _of formula ₍ _I _' ) _, Ethyl, n-propyl or isopropyl. In some embodiments, X is independently selected from NH-CO, _CH2 , and NH. In some embodiments, X is independently selected from NH-CO and _CH2 . In some embodiments, X is _CH2 .

在式(I’)的一些實施方案中，J₂為H或甲基。在一些實施方案中，J₂為H。 In some embodiments of Formula (I'), _J2 is H or methyl. In some embodiments, _J2 is H.

在式(I’)的一些實施方案中，R₃選自H、OH、NH₂、甲基、乙基、正丙基、異丙基、甲氧基、乙氧基、正丙氧基、異丙氧基和(CH₂)_pR₆，R₆選自OH、甲氧基和乙氧基，p=1或2。在一些實施方案中，R₃選自H、甲基、乙基、正丙基、異丙基和(CH₂)_pR₆，R₆選自OH，p=1或2。在一些實施方案中，R₃選自H和甲基。 In some embodiments of formula (I'), _R3 is selected from H, OH, _NH2 , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, Isopropoxy and (CH ₂ ) _p R ₆ , R ₆ is selected from OH, methoxy and ethoxy, p=1 or 2. In some embodiments, R ₃ is selected from H, methyl, ethyl, n-propyl, isopropyl, and (CH ₂ ) _p R ₆ , R ₆ is selected from OH, and p=1 or 2. In some embodiments, _R3 is selected from H and methyl.

在式(I’)的一些實施方案中，R₁選自H、OH、甲基、乙基、正丙基、異丙基、甲氧基、乙氧基、正丙氧基、異丙氧基和(CH₂)_qR₇，R₇選自OH，q=1或2。在一些實施方案中，R₁選自H、甲基、乙基、正丙基、異丙基和(CH₂)_qR₇，R₇選自OH，q=1或2。在一些實施方案中，R₁選自H和甲基。 In some embodiments of formula (I'), _R1 is selected from H, OH, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy The radicals are (CH ₂ ) _q R ₇ , R ₇ is selected from OH, q=1 or 2. In some embodiments, R ₁ is selected from H, methyl, ethyl, n-propyl, isopropyl, and (CH ₂ ) _q R ₇ , R ₇ is selected from OH, q=1 or 2. In some embodiments, _R1 is selected from H and methyl.

在式(I’)的一些實施方案中，R₂選自H、OH、甲基、乙基、正丙基、異丙基、甲氧基、乙氧基、正丙氧基、異丙氧基和(CH₂)_rR₈，R₈選自OH，r=1或2。在一些實施方案中，R₂選自H、OH、甲基、乙基、正丙基、異丙基和(CH₂)_rR₈，R₈選自OH，r=1或2。在一些實施方案中，R₂選自H、甲基和CH₂OH。 In some embodiments of formula (I'), R _is selected from H, OH, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy The radicals are (CH ₂ ) _r R ₈ , R ₈ is selected from OH, r=1 or 2. In some embodiments, R ₂ is selected from H, OH, methyl, ethyl, n-propyl, isopropyl, and (CH ₂ ) _r R ₈ , R ₈ is selected from OH, and r=1 or 2. In some embodiments, _R2 is selected from H, methyl, and _CH2OH .

在式(I’)的一些實施方案中，R₁和R₂直接相連成5-6員環。在一些實施方案中，R₁和R₂直接相連形成3-6員環烷基。在一些實施方案中，R₁和R₂直接相連形成環戊基或環己基。 In some embodiments of formula (I'), R ₁ and R ₂ are directly connected to form a 5-6 membered ring. In some embodiments, R ₁ and R ₂ are directly linked to form a 3-6 membered cycloalkyl group. In some embodiments, R ₁ and R ₂ are directly connected to form cyclopentyl or cyclohexyl.

在一些實施方案中，該式(I’)所示的化學修飾選自以下任一結構： In some embodiments, the chemical modification represented by formula (I') is selected from any of the following structures:

其中，M為O或S； Among them, M is O or S;

B選自嘌呤鹼基、嘧啶鹼基、吲哚、5-硝基吲哚和3-硝基吡咯。 B is selected from purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.

其中，M為O或S； Among them, M is O or S;

其中，M為O或S； Among them, M is O or S;

以及它們結構中的腺嘌呤被置換為鳥嘌呤、胞嘧啶、尿嘧啶或胸腺嘧啶的那些。 and those in which adenine in their structure is replaced by guanine, cytosine, uracil, or thymine.

在一些實施方案中，B與該反義鏈該位置處核苷酸未被修飾時的鹼基相同。 In some embodiments, B is the same base as the unmodified nucleotide at that position on the antisense strand.

在一些實施方案中，該式(I)所示的化學修飾為： In some embodiments, the chemical modification represented by formula (I) is:

再一方面，本揭露提供了一種dsRNA，其包含有義鏈和反義鏈： In yet another aspect, the present disclosure provides a dsRNA comprising a sense strand and an antisense strand:

在一些實施方案中，該反義鏈與靶序列至少部分地反向互補。在一些實施方案中，該反義鏈與靶序列之間存在不多於5個、不多於4個、不多於 3個、不多於2個、不多於1個錯配。在一些實施方案中，該反義鏈與靶序列完全反向互補。 In some embodiments, the antisense strand is at least partially reverse complementary to the target sequence. In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 mismatch. In some embodiments, the antisense strand is completely reverse complementary to the target sequence.

在一些實施方案中，該有義鏈與反義鏈至少部分地反向互補以形成雙鏈區。在一些實施方案中，該有義鏈與反義鏈之間存在不多於5個、不多於4個、不多於3個、不多於2個、不多於1個錯配。在一些實施方案中，該有義鏈與反義鏈完全反向互補。 In some embodiments, the sense strand and 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, and no more than 1 mismatches between the sense strand and the antisense strand. In some embodiments, the sense strand is completely reverse complementary to the antisense strand.

在一些實施方案中，該有義鏈和反義鏈各自獨立地具有16至35個、16至34個、17至34個、17至33個、18至33個、18至32個、18至31個、18至30個、18至29個、18至28個、18至27個、18至26個、18至25個、18至24個、18至23個、19至25個、19至24個、或19至23個核苷酸(例如19、20、21、22、23個核苷酸)。 In some embodiments, the sense strand and the antisense strand each independently have 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides (eg 19, 20, 21, 22, 23 nucleotides).

在一些實施方案中，該有義鏈和反義鏈長度相同或不同，該有義鏈的長度為19-23個核苷酸，該反義鏈的長度為19-26個核苷酸。在一些實施方案中，該有義鏈和反義鏈的長度比可以是19/19、19/20、19/21、19/22、19/23、19/24、19/25、19/26、20/19、20/20、20/21、20/22、20/23、20/24、20/25、20/26、21/20、21/21、21/22、21/23、21/24、21/25、21/26、22/20、22/21、22/22、22/23、22/24、22/25、22/26、23/20、23/21、23/22、23/23、23/24、23/25或23/26。在一些實施方案中，該有義鏈和反義鏈的長度比為19/21、21/23或23/25。在一些實施方案中，該有義鏈和反義鏈的長度比為19/21。 In some embodiments, the sense strand and the antisense strand are the same or different in length, the sense strand is 19-23 nucleotides in length, and the antisense strand is 19-26 nucleotides in length. In some embodiments, the length ratio of the sense strand and the antisense strand can be 19/19, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26 , 20/19, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21 /24, 21/25, 21/26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22 , 23/23, 23/24, 23/25 or 23/26. In some embodiments, the length ratio of the sense strand and antisense strand is 19/21, 21/23, or 23/25. In some embodiments, the length ratio of the sense strand and antisense strand is 19/21.

在一些實施方案中，該dsRNA包含一個或兩個平端。 In some embodiments, the dsRNA contains one or two blunt ends.

在一些實施方案中，該dsRNA包含具有1至4個未配對核苷酸的突出端，例如1個、2個、3個、4個。 In some embodiments, the dsRNA includes an overhang with 1 to 4 unpaired nucleotides, such as 1, 2, 3, 4.

在一些實施方案中，該dsRNA包含位於該反義鏈3’端的突出端。 In some embodiments, the dsRNA includes an overhang located at the 3' end of the antisense strand.

在一些實施方案中，包含式(I)或式(I’)所示的化學修飾、其互變異構體或其藥學上可接受的鹽的核苷酸位於反義鏈5’端起第5位、第6位或第7位。 In some embodiments, a nucleotide comprising a chemical modification represented by Formula (I) or Formula (I'), a tautomer thereof, or a pharmaceutically acceptable salt thereof is located at the 5' end of the antisense strand. position, 6th or 7th position.

在一些實施方案中，包含式(I)或式(I’)所示的化學修飾、其互變異構體或其藥學上可接受的鹽的核苷酸位於反義鏈5’端起第7位。 In some embodiments, the nucleotide comprising the chemical modification represented by Formula (I) or Formula (I'), a tautomer thereof, or a pharmaceutically acceptable salt thereof is located at the 7th position from the 5' end of the antisense strand. Bit.

在一些實施方案中，式(I)或式(I’)所示的化學修飾、其互變異構體或其藥學上可接受的鹽修飾在反義鏈5’端起第5位時，B選自腺嘌呤、鳥嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments, when the chemical modification represented by Formula (I) or Formula (I'), its tautomer or its pharmaceutically acceptable salt modification is at position 5 from the 5' end of the antisense strand, B Selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.

在一些實施方案中，式(I)或式(I’)所示的化學修飾、其互變異構體或其藥學上可接受的鹽修飾在反義鏈5’端起第6位時，B選自腺嘌呤、鳥嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments, when the chemical modification represented by Formula (I) or Formula (I'), its tautomer or its pharmaceutically acceptable salt modification is at position 6 from the 5' end of the antisense strand, B Selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.

在一些實施方案中，式(I)或式(I’)所示的化學修飾、其互變異構體或其藥學上可接受的鹽修飾在反義鏈5’端起第7位時，B選自腺嘌呤、鳥嘌呤、2,6-二胺基嘌呤、6-二甲基胺基嘌呤、2-胺基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。 In some embodiments, when the chemical modification represented by Formula (I) or Formula (I'), its tautomer or its pharmaceutically acceptable salt modification is at position 7 from the 5' end of the antisense strand, B Selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.

在一些實施方案中，B與該反義鏈在其5’端起第5位核苷酸未被修飾時的鹼基相同。 In some embodiments, B is the same base as the 5th nucleotide from the 5' end of the antisense strand when it is not modified.

在一些實施方案中，B與該反義鏈在其5’端起第6位核苷酸未被修飾時的鹼基相同。 In some embodiments, B is the same base as the 6th nucleotide from the 5' end of the antisense strand when it is not modified.

在一些實施方案中，B與該反義鏈在其5’端起第7位核苷酸未被修飾時的鹼基相同。 In some embodiments, B is the same base as the 7th nucleotide from the 5' end of the antisense strand when it is not modified.

在一些實施方案中，該有義鏈第5位的核苷酸為2'-甲氧基修飾的核苷酸。 In some embodiments, the nucleotide at position 5 of the sense strand is a 2'-methoxy modified nucleotide.

在一些實施方案中，該反義鏈第7位的核苷酸為2'-甲氧基修飾的核苷酸。 In some embodiments, the nucleotide at position 7 of the antisense strand is a 2'-methoxy modified nucleotide.

在一些實施方案中，該有義鏈和反義鏈包含或為如下式所示的核苷酸序列： In some embodiments, the sense strand and antisense strand comprise or are a nucleotide sequence represented by the following formula:

有義鏈：5’-N_aN_aN_aN_aXN_aN_bN_bN_bN_aN_aN_aN_aN_aN_aN_aN_aN_aN_a-3’； Sense strand: 5'-N _a N _a N _a N _a XN _a N _b N _b N _b N _a N _a N _a N _a N _a N _a N _a N _a N _a N _a -3';

反義鏈：5’-N_a’N_b’N_a’X’N_a’X’W’X’X’X’N_a’X’N_a’N_b’N_a’X’N_a’X’N_a’N_a’N_a’-3’； Antisense strand: 5'-N _a 'N _b 'N _a 'X'N _a 'X'W'X'X'X'N _a 'X'N _a 'N _b 'N _a 'X'N _a 'X'N _a 'N _a 'N _a '-3';

其中，每個X獨立地為N_a或N_b，每個X’獨立地為N_a’或N_b’；N_a和N_a’為2'-甲氧基修飾的核苷酸，N_b和N_b’為2'-氟修飾的核苷酸； Wherein _, each _X is independently _Na _or _N _b , and _each and N _b ' is a 2'-fluorine modified nucleotide;

W’表示2'-甲氧基修飾的核苷酸或包含式(I)或式(I’)所示的化學修飾、其互變異構體或其藥學上可接受的鹽的核苷酸。 W' represents a 2'-methoxy modified nucleotide or a nucleotide comprising a chemical modification represented by formula (I) or formula (I'), a tautomer thereof or a pharmaceutically acceptable salt thereof.

在一些實施方案中，W’表示2'-甲氧基修飾的核苷酸。 In some embodiments, W' represents a 2'-methoxy modified nucleotide.

在一些實施方案中，W’表示2'-甲氧基修飾的核苷酸，該dsRNA靶向LPA基因。 In some embodiments, W' represents a 2'-methoxy modified nucleotide and the dsRNA targets the LPA gene.

在另一些實施方案中，該有義鏈和反義鏈包含或為如下式所示的核苷酸序列： In other embodiments, the sense strand and antisense strand comprise or are a nucleotide sequence represented by the following formula:

有義鏈：5’-N_aN_aN_aN_aN_aN_aN_bN_bN_bN_aN_aN_aN_aN_aN_aN_aN_aN_aN_a-3’； Sense strand: 5'-N _a N _a N _a N _a N _a N _a N _b N _b N _b N _a N _a N _a N _a N _a N _a N _a N _a N _a N _a -3';

其中，每個X’獨立地為N_a’或N_b’；N_a和N_a’為2'-甲氧基修飾的核苷酸，N_b和N_b’為2'-氟修飾的核苷酸； _Wherein _, _each _{_} _{_} _{_} glycolic acid;

在一些實施方案中，該有義鏈包含或為如下式所示的核苷酸序列： In some embodiments, the sense strand comprises or is a nucleotide sequence represented by the following formula:

5’-N_aN_aN_aN_aXN_aN_bN_bN_bN_aN_aN_aN_aN_aN_aN_aN_aN_aN_a-3’； 5'-N _a N _a N _a N _a XN _a N _b N _b N _b N _a N _a N _a N _a N _a N _a N _a N _a N _a N _a -3';

其中，X為N_a或N_b；N_a為2'-甲氧基修飾的核苷酸，N_b為2'-氟修飾的核苷酸。 Wherein, X is _Na or _Nb ; _Na is a 2'-methoxy modified nucleotide, and _Nb is a 2'-fluoro modified nucleotide.

5’-N_aN_aN_aN_aN_aN_aN_bN_bN_bN_aN_aN_aN_aN_aN_aN_aN_aN_aN_a-3’； 5'-N _a N _a N _a N _a N _a N _a N _b N _b N _b N _a N _a N _a N _a N _a N _a N _a N _a N _a N _a -3';

其中，N_a為2'-甲氧基修飾的核苷酸，N_b為2'-氟修飾的核苷酸。 Among them, _Na is a 2'-methoxy modified nucleotide, and N _b is a 2'-fluoro modified nucleotide.

在一些實施方案中，該反義鏈包含或為如下式所示的核苷酸序列： In some embodiments, the antisense strand comprises or is a nucleotide sequence represented by the following formula:

5’-N_a’N_b’N_a’X’N_a’X’W’X’X’X’N_a’X’N_a’N_b’N_a’X’N_a’X’N_a’N_a’N_a’-3’； 5'-N _a 'N _b 'N _a 'X'N _a 'X'W'X'X'X'N _a 'X'N _a 'N _b 'N _a 'X'N _a 'X'N _a 'N _a 'N _a '-3';

其中，每個X’獨立地為N_a’或N_b’；N_a’為2'-甲氧基修飾的核苷酸，N_b’為2'-氟修飾的核苷酸； _Wherein _, _each _{_}

在一些實施方案中，W’表示包含式(I)或式(I’)所示的化學修飾、其互變異構體或其藥學上可接受的鹽的核苷酸。 In some embodiments, W' represents a nucleotide comprising a chemical modification represented by Formula (I) or Formula (I'), a tautomer thereof, or a pharmaceutically acceptable salt thereof.

在一些實施方案中，式(I)所示的化學修飾選自： In some embodiments, the chemical modification represented by Formula (I) is selected from:

、

和

；其中，B選自鳥嘌呤、腺嘌呤、胞嘧啶和尿嘧啶；在一些具體的實施方案中，B與該反義鏈在其5’端起第7位核苷酸未被修飾時的鹼基相同。

,

and

; Wherein, B is selected from guanine, adenine, cytosine and uracil; in some specific embodiments, B and the base of the 7th nucleotide from the 5' end of the antisense strand are not modified The basis is the same.

、

和

；其中，M為O或S；其中，B選自鳥嘌呤、腺嘌呤、胞嘧啶或尿嘧啶；在一些具體的實施方案中，B與該反義鏈在其5’端起第7位核苷酸未被修飾時的鹼基相同。

,

and

; Wherein, M is O or S; Wherein, B is selected from guanine, adenine, cytosine or uracil; In some specific embodiments, B and the antisense strand are located at the 7th position from its 5' end. The bases of the unmodified nucleotides are the same.

在一些具體的實施方案中，M為S。一些具體的實施方案中，M為O。 In some specific embodiments, M is S. In some specific embodiments, M is O.

5’-N_a’N_b’N_a’N_b’N_a’N_b’W’N_a’N_a’N_b’N_a’N_b’N_a’N_b’N_a’N_b’N_a’N_a’N_a’N_a’N_a’-3’； 5'-N _a 'N _b 'N _a 'N _b _' N a 'N _b 'W'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b ' N _a 'N _a 'N _a 'N _a 'N _a '-3';

5’-N_a’N_b’N_a’N_b’N_a’N_b’W’N_a’N_a’N_b’N_a’N_a’N_a’N_b’N_a’N_b’N_a’N_b’N_a’N_a’N_a’-3’； 5'-N _a 'N _b 'N _a 'N _b _' N a 'N _b 'W'N _a 'N _a 'N _b 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b ' N _a 'N _b 'N _a 'N _a 'N _a '-3';

5’-N_a’N_b’N_a’N_b’N_a’N_b’W’N_a’N_a’N_a’N_a’N_b’N_a’N_b’N_a’N_b’N_a’N_b’N_a’N_a’N_a’-3’； 5'-N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'W'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b ' N _a 'N _b 'N _a 'N _a 'N _a '-3';

5’-N_a’N_b’N_a’N_a’N_a’N_b’W’N_a’N_a’N_b’N_a’N_b’N_a’N_b’N_a’N_b’N_a’N_b’N_a’N_a’N_a’-3’； 5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _b 'W'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b ' N _a 'N _b 'N _a 'N _a 'N _a '-3';

5’-N_a’N_b’N_a’N_b’N_a’N_b’W’N_a’N_a’N_a’N_a’N_a’N_a’N_b’N_a’N_b’N_a’N_a’N_a’N_a’N_a’-3’； 5'-N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'W'N _a 'N _a 'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b ' N _a 'N _a 'N _a 'N _a 'N _a '-3';

5’-N_a’N_b’N_a’N_a’N_a’N_b’W’N_a’N_a’N_b’N_a’N_a’N_a’N_b’N_a’N_b’N_a’N_a’N_a’N_a’N_a’-3’； 5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _b 'W'N _a 'N _a 'N _b 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b ' N _a 'N _a 'N _a 'N _a 'N _a '-3';

5’-N_a’N_b’N_a’N_a’N_a’N_b’W’N_a’N_a’N_a’N_a’N_b’N_a’N_b’N_a’N_b’N_a’N_a’N_a’N_a’N_a’-3’； 5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _b 'W'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b ' N _a 'N _a 'N _a 'N _a 'N _a '-3';

5’-N_a’N_b’N_a’N_a’N_a’N_a’W’N_b’N_b’N_a’N_a’N_a’N_a’N_b’N_a’N_b’N_a’N_a’N_a’N_a’N_a’-3’； 5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _a 'W'N _b 'N _b 'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b ' N _a 'N _a 'N _a 'N _a 'N _a '-3';

5’-N_a’N_b’N_a’N_a’N_a’N_b’W’N_a’N_a’N_a’N_a’N_a’N_a’N_b’N_a’N_b’N_a’N_a’N_a’N_a’N_a’-3’； 5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _b 'W'N _a 'N _a 'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b ' N _a 'N _a 'N _a 'N _a 'N _a '-3';

5’-N_a’N_b’N_a’N_a’N_a’N_b’W’N_a’N_a’N_a’N_a’N_a’N_a’N_b’N_a’N_a’N_a’N_a’N_a’N_a’N_a’-3’； 5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _b 'W'N _a 'N _a 'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _a ' N _a 'N _a 'N _a 'N _a 'N _a '-3';

5’-N_a’N_b’N_a’N_a’N_a’N_a’W’N_a’N_a’N_a’N_a’N_a’N_a’N_b’N_a’N_b’N_a’N_a’N_a’N_a’N_a’-3’；或， 5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _a 'W'N _a 'N _a 'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b ' N _a 'N _a 'N _a 'N _a 'N _a '-3'; or,

5’-N_a’N_b’N_a’N_a’N_a’N_a’W’N_a’N_a’N_a’N_a’N_a’N_a’N_b’N_a’N_a’N_a’N_a’N_a’N_a’N_a’-3’； 5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _a 'W'N _a 'N _a 'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _a ' N _a 'N _a 'N _a 'N _a 'N _a '-3';

其中，N_a’為2'-甲氧基修飾的核苷酸，N_b’為2'-氟修飾的核苷酸； Among them, N _a ' is a 2'-methoxy modified nucleotide, and N _b ' is a 2'-fluorine modified nucleotide;

、

和

,

and

在一些實施方案中，式(I’)所示的化學修飾選自： In some embodiments, the chemical modification represented by formula (I') is selected from:

和

and

在一些實施方案中，該有義鏈和/或反義鏈中至少一個磷酸酯基為具有修飾基團的磷酸酯基，該修飾基團使得該dsRNA在生物樣品或環境中具有增加的穩定性；在一些實施方案中，該具有修飾基團的磷酸酯基為硫代磷酸酯基。在一些實施方案中，該具有修飾基團的磷酸酯基為硫代磷酸二酯基。 In some embodiments, at least one phosphate group in the sense strand and/or antisense strand is a phosphate group with a modifying group that allows the dsRNA to have increased stability in biological samples or environments. ; In some embodiments, the phosphate group having a modifying group is a phosphorothioate group. In some embodiments, the phosphate group having a modifying group is a phosphorothioate diester group.

在一些實施方案中，該硫代磷酸二酯基存在於以下位置中的至少一處： In some embodiments, the phosphorothioate diester group is present at at least one of the following positions:

該有義鏈的5'端第1個核苷酸和第2個核苷酸之間； Between the first and second nucleotides at the 5' end of the sense strand;

該有義鏈的5'端第2個核苷酸和第3個核苷酸之間； Between the second and third nucleotides at the 5' end of the sense strand;

該反義鏈的5'端第1個核苷酸和第2個核苷酸之間； Between the first and second nucleotides at the 5' end of the antisense strand;

該反義鏈的5'端第2個核苷酸和第3個核苷酸之間； Between the second and third nucleotides at the 5' end of the antisense strand;

該反義鏈的3'端第1個核苷酸和第2個核苷酸之間；以及 Between the first and second nucleotides at the 3' end of the antisense strand; and

該反義鏈的3'端第2個核苷酸和第3個核苷酸之間。 Between the 2nd and 3rd nucleotides at the 3' end of the antisense strand.

在一些實施方案中，該有義鏈和/或反義鏈中包括多個硫代磷酸二酯基，該硫代磷酸二酯基存在於： In some embodiments, the sense strand and/or antisense strand includes multiple phosphorothioate diester groups, the phosphorothioate diester groups being present in:

該有義鏈的5'端第1個核苷酸和第2個核苷酸之間；和， Between the first and second nucleotides at the 5' end of the sense strand; and,

該有義鏈的5'端第2個核苷酸和第3個核苷酸之間；和， Between the 2nd and 3rd nucleotides at the 5' end of the sense strand; and,

該反義鏈的5'端第1個核苷酸和第2個核苷酸之間；和， Between the first and second nucleotides at the 5' end of the antisense strand; and,

該反義鏈的5'端第2個核苷酸和第3個核苷酸之間；和， Between the 2nd and 3rd nucleotides at the 5' end of the antisense strand; and,

該反義鏈的3'端第1個核苷酸和第2個核苷酸之間；和， Between the first and second nucleotides at the 3' end of the antisense strand; and,

5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’，或， 5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm-3’, or,

5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’， 5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm-3’,

其中，Nm表示2'-甲氧基修飾的任意核苷酸，例如2'-甲氧基修飾的C、G、U、A；Nf表示2'-氟修飾的任意核苷酸，例如2'-氟修飾的C、G、U、A； Among them, Nm represents any nucleotide modified by 2'-methoxy group, such as 2'-methoxy modified C, G, U, A; Nf represents any nucleotide modified by 2'-fluorine, such as 2' -Fluorine modified C, G, U, A;

小寫字母s在中間時表示與該字母s左右相鄰的兩個核苷酸之間為硫代磷酸二酯基連接；小寫字母s在3’端第一個時表示與該字母s左側相鄰的一個核苷酸末端為硫代磷酸二酯基。 When the lowercase letter s is in the middle, it means that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate diester groups; when the lowercase letter s is the first one at the 3' end, it means it is adjacent to the left side of the letter s. One of the nucleotide ends is a phosphorothioate diester group.

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

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

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

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

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

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

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

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

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

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

5’-Nm’sNf’sNm’Nm’Nm’Nm’W’Nm’Nm’Nm’Nm’Nm’Nm’Nf’Nm’Nf’Nm’Nm’Nm’sNm’sNm’-3’；或， 5'-Nm'sNf'sNm'Nm'Nm'Nm'W'Nm'Nm'Nm'Nm'Nm'Nm'Nf'Nm'Nf'Nm'Nm'Nm'sNm'sNm'-3'; or ,

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

其中，Nm’表示2'-甲氧基修飾的任意核苷酸，例如2'-甲氧基修飾的C、G、U、A；Nf’表示2'-氟修飾的任意核苷酸，例如2'-氟代修飾的C、G、U、A； Among them, Nm' represents any nucleotide modified by 2'-methoxy, such as 2'-methoxy modified C, G, U, A; Nf' represents any nucleotide modified by 2'-fluorine, such as 2'-fluorinated modified C, G, U, A;

小寫字母s在中間時表示與該字母s左右相鄰的兩個核苷酸之間為硫代磷酸二酯基連接，小寫字母s在3’端第一個時表示與該字母s左側相鄰的一個核苷酸末端為硫代磷酸二酯基； When the lowercase letter s is in the middle, it means that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate diester groups. When the lowercase letter s is the first one at the 3' end, it means it is adjacent to the left side of the letter s. One of the nucleotide ends is a phosphorothioate diester group;

W’表示2'-甲氧基修飾的核苷酸或包含式(I)所示的化學修飾、其互變異構體或其藥學上可接受的鹽修飾的核苷酸。 W' represents a 2'-methoxy modified nucleotide or a modified nucleotide containing a chemical modification represented by formula (I), its tautomer or a pharmaceutically acceptable salt thereof.

、

和

；其中，B選自鳥嘌呤、腺嘌呤、胞嘧啶和尿嘧啶；在一些實施方案中，B與該反義鏈在其5’端起第7位核苷酸未被修飾時的鹼基相同。

,

and

; Wherein, B is selected from guanine, adenine, cytosine and uracil; in some embodiments, B is the same base as the 7th nucleotide from the 5' end of the antisense strand when it is not modified .

一些實施方案中，或式(I’)所示的化學修飾選自： In some embodiments, the chemical modification represented by formula (I') is selected from:

、

和

,

and

在一些實施方案中，M為S。在一些具體的實施方案中，M為O。 In some embodiments, M is S. In some specific embodiments, M is O.

在一些實施方案中，該dsRNA抑制第13型17β-羥基類固醇脫氫酶(HSD17B13)的表達和/或活性。 In some embodiments, the dsRNA inhibits the expression and/or activity of 17β-hydroxysteroid dehydrogenase type 13 (HSD17B13).

在一些實施方案中，該有義鏈包含與SEQ ID NO：1的核苷酸序列相差不超過3個核苷酸，且包含至少15個連續核苷酸，和/或， In some embodiments, the sense strand contains no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1 and contains at least 15 contiguous nucleotides, and/or,

該反義鏈包含與SEQ ID NO：2的核苷酸序列相差不超過3個核苷酸，且包含至少19個連續核苷酸。 The antisense strand contains no more than 3 nucleotides different from the nucleotide sequence of SEQ ID NO: 2 and contains at least 19 consecutive nucleotides.

在一些實施方案中，該有義鏈包含與SEQ ID NO：1的核苷酸序列相差不超過3個核苷酸，且包含至少19個連續核苷酸，和/或， In some embodiments, the sense strand contains no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1 and contains at least 19 contiguous nucleotides, and/or,

該反義鏈包含與SEQ ID NO：2的核苷酸序列相差不超過3個核苷酸，且包含至少21個連續核苷酸。 The antisense strand contains no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 2 and contains at least 21 consecutive nucleotides.

在一些實施方案中，該有義鏈包含或選自SEQ ID NO：1的核苷酸序列，和/或，該反義鏈包含或選自SEQ ID NO：2的核苷酸序列。 In some embodiments, the sense strand comprises or is selected from the nucleotide sequence of SEQ ID NO: 1, and/or the antisense strand comprises or is selected from the nucleotide sequence of SEQ ID NO: 2.

在一些實施方案中，該有義鏈和反義鏈為以下任一組： In some embodiments, the sense strand and antisense strand are any of the following:

在一些實施方案中，該dsRNA的有義鏈的核苷酸序列包含或選自SEQ ID NO：3，反義鏈的核苷酸序列包含或選自SEQ ID NO：5至SEQ ID NO：14中任一的核苷酸序列。 In some embodiments, the nucleotide sequence of the sense strand of the dsRNA includes or is selected from SEQ ID NO: 3, and the nucleotide sequence of the antisense strand includes or is selected from SEQ ID NO: 5 to SEQ ID NO: 14 any nucleotide sequence.

在一些實施方案中，該dsRNA為以下任一組： In some embodiments, the dsRNA is any of the following:

有義鏈的核苷酸序列包含SEQ ID NO：3，反義鏈的核苷酸序列包含SEQ ID NO：5； The nucleotide sequence of the sense strand includes SEQ ID NO: 3, and the nucleotide sequence of the antisense strand includes SEQ ID NO: 5;

有義鏈的核苷酸序列包含SEQ ID NO：3，反義鏈的核苷酸序列包含SEQ ID NO：6； The nucleotide sequence of the sense strand includes SEQ ID NO: 3, and the nucleotide sequence of the antisense strand includes SEQ ID NO: 6;

有義鏈的核苷酸序列包含SEQ ID NO：3，反義鏈的核苷酸序列包含SEQ ID NO：7； The nucleotide sequence of the sense strand includes SEQ ID NO: 3, and the nucleotide sequence of the antisense strand includes SEQ ID NO: 7;

有義鏈的核苷酸序列包含SEQ ID NO：3，反義鏈的核苷酸序列包含SEQ ID NO：8； The nucleotide sequence of the sense strand includes SEQ ID NO: 3, and the nucleotide sequence of the antisense strand includes SEQ ID NO: 8;

有義鏈的核苷酸序列包含SEQ ID NO：3，反義鏈的核苷酸序列包含SEQ ID NO：9； The nucleotide sequence of the sense strand includes SEQ ID NO: 3, and the nucleotide sequence of the antisense strand includes SEQ ID NO: 9;

有義鏈的核苷酸序列包含SEQ ID NO：3，反義鏈的核苷酸序列包含SEQ ID NO：10； The nucleotide sequence of the sense strand contains SEQ ID NO: 3, and the nucleotide sequence of the antisense strand contains SEQ ID NO: 10;

有義鏈的核苷酸序列包含SEQ ID NO：3，反義鏈的核苷酸序列包含SEQ ID NO：11； The nucleotide sequence of the sense strand contains SEQ ID NO: 3, and the nucleotide sequence of the antisense strand contains SEQ ID NO: 11;

有義鏈的核苷酸序列包含SEQ ID NO：3，反義鏈的核苷酸序列包含SEQ ID NO：12； The nucleotide sequence of the sense strand contains SEQ ID NO: 3, and the nucleotide sequence of the antisense strand contains SEQ ID NO: 12;

有義鏈的核苷酸序列包含SEQ ID NO：3，反義鏈的核苷酸序列包含SEQ ID NO：13； The nucleotide sequence of the sense strand contains SEQ ID NO: 3, and the nucleotide sequence of the antisense strand contains SEQ ID NO: 13;

有義鏈的核苷酸序列包含SEQ ID NO：3，反義鏈的核苷酸序列包含SEQ ID NO：14。 The nucleotide sequence of the sense strand contains SEQ ID NO:3, and the nucleotide sequence of the antisense strand contains SEQ ID NO:14.

有義鏈的核苷酸序列選自SEQ ID NO：3，反義鏈的核苷酸序列選自SEQ ID NO：5； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 3, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 5;

有義鏈的核苷酸序列選自SEQ ID NO：3，反義鏈的核苷酸序列選自SEQ ID NO：6； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 3, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 6;

有義鏈的核苷酸序列選自SEQ ID NO：3，反義鏈的核苷酸序列選自SEQ ID NO：7； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 3, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 7;

有義鏈的核苷酸序列選自SEQ ID NO：3，反義鏈的核苷酸序列選自SEQ ID NO：8； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 3, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 8;

有義鏈的核苷酸序列選自SEQ ID NO：3，反義鏈的核苷酸序列選自SEQ ID NO：9； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 3, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 9;

有義鏈的核苷酸序列選自SEQ ID NO：3，反義鏈的核苷酸序列選自SEQ ID NO：10； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 3, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 10;

有義鏈的核苷酸序列選自SEQ ID NO：3，反義鏈的核苷酸序列選自SEQ ID NO：11； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 3, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 11;

有義鏈的核苷酸序列選自SEQ ID NO：3，反義鏈的核苷酸序列選自SEQ ID NO：12； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 3, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 12;

有義鏈的核苷酸序列選自SEQ ID NO：3，反義鏈的核苷酸序列選自SEQ ID NO：13； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 3, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 13;

有義鏈的核苷酸序列選自SEQ ID NO：3，反義鏈的核苷酸序列選自SEQ ID NO：14。 The nucleotide sequence of the sense strand is selected from SEQ ID NO:3, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO:14.

包含SEQ ID NO：3所示的有義鏈和SEQ ID NO：5至SEQ ID NO：14中任一項所示的反義鏈。 It contains the sense strand shown in SEQ ID NO: 3 and the antisense strand shown in any one of SEQ ID NO: 5 to SEQ ID NO: 14.

選自SEQ ID NO：3所示的有義鏈和SEQ ID NO：5至SEQ ID NO：14中任一項所示的反義鏈。 Selected from the sense strand shown in SEQ ID NO: 3 and the antisense strand shown in any one of SEQ ID NO: 5 to SEQ ID NO: 14.

本揭露中，按照5’-3’方向， In this disclosure, according to the 5’-3’ direction,

SEQ ID NO：3是 AmsAmsAmUmGmAmAfAfUfGmAmAmUmAmAmAmUmAmAm； SEQ ID NO:3 is AmsAmsAmUmGmAmAfAfUfGmAmAmUmAmAmAmUmAmAm;

SEQ ID NO：5是UmsUfsAmUfUmUf(-)hmpNA(A)UmUmCfAmUfUmUfCmAfUmUmUmsUmsGm； SEQ ID NO: 5 is UmsUfsAmUfUmUf(-)hmpNA(A)UmUmCfAmUfUmUfCmAfUmUmUmsUmsGm;

SEQ ID NO：6是UmsUfsAmUfUmUf(-)hmpNA(A)UmUmCfAmUmUmUfCmAfUmUfUmsUmsGm； SEQ ID NO: 6 is UmsUfsAmUfUmUf(-)hmpNA(A)UmUmCfAmUmUmUfCmAfUmUfUmsUmsGm;

SEQ ID NO：7是UmsUfsAmUfUmUf(-)hmpNA(A)UmUmCmAmUfUmUfCmAfUmUfUmsUmsGm； SEQ ID NO: 7 is UmsUfsAmUfUmUf(-)hmpNA(A)UmUmCmAmUfUmUfCmAfUmUfUmsUmsGm;

SEQ ID NO：8是UmsUfsAmUmUmUf(-)hmpNA(A)UmUmCfAmUfUmUfCmAfUmUfUmsUmsGm； SEQ ID NO: 8 is UmsUfsAmUmUmUf(-)hmpNA(A)UmUmCfAmUfUmUfCmAfUmUfUmsUmsGm;

SEQ ID NO：9是UmsUfsAmUmUmUf(-)hmpNA(A)UmUmCmAmUmUmUfCmAfUmUmUmsUmsGm； SEQ ID NO: 9 is UmsUfsAmUmUmUf(-)hmpNA(A)UmUmCmAmUmUmUfCmAfUmUmUmsUmsGm;

SEQ ID NO：10是UmsUfsAmUfUmUf(-)hmpNA(A)UmUmCmAmUmUmUfCmAfUmUmUmsUmsGm； SEQ ID NO: 10 is UmsUfsAmUfUmUf(-)hmpNA(A)UmUmCmAmUmUmUfCmAfUmUmUmsUmsGm;

SEQ ID NO：11是UmsUfsAmUmUmUf(-)hmpNA(A)UmUmCfAmUmUmUfCmAfUmUmUmsUmsGm； SEQ ID NO: 11 is UmsUfsAmUmUmUf(-)hmpNA(A)UmUmCfAmUmUmUfCmAfUmUmUmsUmsGm;

SEQ ID NO：12是UmsUfsAmUmUmUf(-)hmpNA(A)UmUmCmAmUfUmUfCmAfUmUmUmsUmsGm； SEQ ID NO: 12 is UmsUfsAmUmUmUf(-)hmpNA(A)UmUmCmAmUfUmUfCmAfUmUmUmsUmsGm;

SEQ ID NO：13是 UmsUfsAmUmUmUm(-)hmpNA(A)UmUmCmAmUmUmUfCmAfUmUmUmsUmsGm； SEQ ID NO:13 is UmsUfsAmUmUmUm(-)hmpNA(A)UmUmCmAmUmUmUfCmAfUmUmUmsUmsGm;

SEQ ID NO：14是UmsUfsAmUmUmUf(-)hmpNA(A)UmUmCmAmUmUmUfCmAmUmUmUmsUmsGm； SEQ ID NO: 14 is UmsUfsAmUmUmUf(-)hmpNA(A)UmUmCmAmUmUmUfCmAmUmUmUmsUmsGm;

其中，Af=腺嘌呤2'-F核糖核苷(adenine 2'-F ribonucleoside)；Cf=胞嘧啶2'-F核糖核苷(cytosine 2'-F ribonucleoside)；Uf=尿嘧啶2'-F核糖核苷(uracil 2'-F ribonucleoside)；Gf=鳥嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；Am=腺嘌呤2'-OMe核糖核苷(adenine 2'-OMe ribonucleoside)；Cm=胞嘧啶2'-OMe核糖核苷(cytosine 2'-OMe ribonucleoside)；Gm=鳥嘌呤2'-OMe核糖核苷(guanine 2'-OMe ribonucleoside)；Um=尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)； Among them, Af=adenine 2'-F ribonucleoside; Cf=cytosine 2'-F ribonucleoside; Uf=uracil 2'-F Ribonucleoside (uracil 2'-F ribonucleoside); Gf=guanine 2'-F ribonucleoside (guanine 2'-F ribonucleoside); Am=adenine 2'-OMe ribonucleoside (adenine 2'-OMe ribonucleoside ); Cm=cytosine 2'-OMe ribonucleoside; Gm=guanine 2'-OMe ribonucleoside; Um=uracil 2'-OMe Ribonucleoside (uracil 2'-OMe ribonucleoside);

s表示與該字母s左右相鄰的兩個核苷酸之間為硫代磷酸二酯基連接； s means that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate diester groups;

(-)hmpNA(A)表示

。 (-)hmpNA(A) means

.

在一些實施方案中，該dsRNA還包括綴合的配體，該配體選自靶向肝臟的靶向配體。在一些實施方案中，該配體結合脫唾液酸糖蛋白受體(ASGPR)。在一些實施方案中，該配體包括半乳糖簇或半乳糖衍生物簇，該半乳糖衍生物選自N-乙醯基-半乳糖胺、N-三氟乙醯基半乳糖胺、N-丙醯基半乳糖胺、N-正丁醯基半乳糖胺、異丁醯基半乳糖胺、或其組合。 In some embodiments, the dsRNA further includes a conjugated ligand selected from the group consisting of liver-targeting targeting ligands. In some embodiments, the ligand binds asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand includes a cluster of galactose or a cluster of galactose derivatives selected from the group consisting of N-acetyl-galactosamine, N-trifluoroacetyl-galactosamine, N- Propionyl galactosamine, N-n-butyl galactosamine, isobutyl galactosamine, or combinations thereof.

在一些實施方案中，該dsRNA還包括綴合的配體，該配體為如式(II)所示化合物或其藥學上可接受的鹽， In some embodiments, the dsRNA also includes a conjugated ligand, which is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof,

其中，L₁為C₁-C₃₀烷基鏈、或包含被一個或多個氧、硫、氮原子或C=O間斷的C₁-C₃₀烷基鏈； Wherein, L ₁ is a C ₁ -C ₃₀ alkyl chain, or a C ₁ -C ₃₀ alkyl chain interrupted by one or more oxygen, sulfur, nitrogen atoms or C=O;

R₁₁和R₁₂獨立地為化學鍵、NR₁₆、C=O或-OC(=O)-； R ₁₁ and R ₁₂ are independently chemical bonds, NR ₁₆ , C=O or -OC(=O)-;

Q₃為

或

； Q ₃ is

or

;

為單鍵或雙鍵，且當

為單鍵時，R₁₃獨立地為CR₁₇R₁₈、NR₁₆、O或S，當

為雙鍵時，R₁₃獨立地為CR₁₉或N；

is a single bond or a double bond, and when

When it is a single bond, R ₁₃ is independently CR ₁₇ R ₁₈ , NR ₁₆ , O or S, when

When it is a double bond, R ₁₃ is independently CR ₁₉ or N;

R₁₄獨立地為CR₁₉或N； R ₁₄ is independently CR ₁₉ or N;

環A為存在或不存在的環烷基、雜環烷基、芳基或雜芳基，且當環A存在時，R₁₅獨立地為CR₁₉或N，當環A不存在時，R₁₅獨立地為CR₁₇R₁₈、NR₁₆或O； Ring A is cycloalkyl, heterocycloalkyl, aryl or heteroaryl present or absent, and when Ring A is present, R ₁₅ is independently CR ₁₉ or N, when Ring A is absent, R ₁₅ Independently CR ₁₇ R ₁₈ , NR ₁₆ or O;

R₁₆和R₁₉獨立地為氫、氘、烷基、烷氧基、環烷基、雜環烷基、芳基、雜芳基、SR'、S(=O)R'、S(=O)₂R'、S(=O)₂NR'(R")、NR'(R")、C(=O)R'、C(=O)OR'或C(=O)NR'(R")，該烷基、烷氧基、環烷基、雜環烷基、芳基或雜芳基視需要被一個或多個選自鹵素、羥基、側氧、硝基、氰基、C_1-6烷基、C_1-6烷氧基、C_3-7環烷基、3-12員雜環烷基、6-12員芳基、5-12員雜芳基、SR'、S(=O)R'、 S(=O)₂R'、S(=O)₂NR'(R")、NR'(R")、C(=O)R'、C(=O)OR'和C(=O)NR'(R")中的基團所取代； R ₁₆ and R ₁₉ are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, SR', S(=O)R', S(=O ) ₂ R', S(=O) ₂ NR'(R"), NR'(R"), C(=O)R', C(=O)OR' or C(=O)NR'(R "), the alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group is optionally replaced by one or more selected from the group consisting of halogen, hydroxyl, pendant oxygen, nitro, cyano, C _{1 -6} alkyl, C _1-6 alkoxy, C _3-7 cycloalkyl, 3-12 membered heterocycloalkyl, 6-12 membered aryl, 5-12 membered heteroaryl, SR', S ( =O)R', S(=O) ₂ R', S(=O) ₂ NR'(R"), NR'(R"), C(=O)R', C(=O)OR' And substituted by the groups in C(=O)NR'(R");

R₁₇和R₁₈獨立地為氫、氘、烷基、烷氧基、環烷基、雜環烷基、芳基、雜芳基、SR'、S(=O)R'、S(=O)₂R'、S(=O)₂NR'(R")、NR'(R")、C(=O)R'、C(=O)OR'或C(=O)NR'(R")，該烷基、烷氧基、環烷基、雜環烷基、芳基或雜芳基視需要被一個或多個選自鹵素、羥基、側氧、硝基、氰基、C_1-6烷基、C_1-6烷氧基、C_3-7環烷基、3-12員雜環烷基、6-12員芳基、5-12員雜芳基、SR'、S(=O)R'、S(=O)₂R'、S(=O)₂NR'(R")、NR'(R")、C(=O)R'、C(=O)OR'和C(=O)NR'(R")中的基團所取代； R ₁₇ and R ₁₈ are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, SR', S(=O)R', S(=O ) ₂ R', S(=O) ₂ NR'(R"), NR'(R"), C(=O)R', C(=O)OR' or C(=O)NR'(R "), the alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group is optionally replaced by one or more selected from the group consisting of halogen, hydroxyl, pendant oxygen, nitro, cyano, C _{1 -6} alkyl, C _1-6 alkoxy, C _3-7 cycloalkyl, 3-12 membered heterocycloalkyl, 6-12 membered aryl, 5-12 membered heteroaryl, SR', S( =O)R', S(=O) ₂ R', S(=O) ₂ NR'(R"), NR'(R"), C(=O)R', C(=O)OR' And substituted by the groups in C(=O)NR'(R");

R'和R"獨立地為氫、氘、羥基、烷基、烷氧基、環烷基、雜環烷基、芳基或雜芳基，該烷基、烷氧基、環烷基、雜環烷基、芳基或雜芳基視需要被一個或多個選自鹵素、羥基、側氧、硝基和氰基中的取代基所取代； R' and R" are independently hydrogen, deuterium, hydroxyl, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, and the alkyl, alkoxy, cycloalkyl, heteroaryl The cycloalkyl, aryl or heteroaryl group is optionally substituted with one or more substituents selected from halogen, hydroxyl, pendant oxygen, nitro and cyano;

m1、n1、p1和q1獨立地為0、1、2、3或4； m1, n1, p1 and q1 are independently 0, 1, 2, 3 or 4;

B1為

或

； B1 is

or

;

R_b1、R_b2、R_b3、R_b4、R_b5、R_b6和R_b7獨立地為-C(=O)-、-NHC(=O)-、-C(=O)O-、-C(=O)-(CH₂)_z8-O-或-NHC(=O)-(CH₂)_z9-O-； R _b1 , R _b2 , R _b3 , R _b4 , R _b5 , R _b6 and R _b7 are independently -C(=O)-, -NHC(=O)-, -C(=O)O-, -C (=O)-(CH ₂ ) _z8 -O- or -NHC(=O)-(CH ₂ ) _z9 -O-;

z1、z2、z3、z4、z5、z6、z7、z8和z9獨立地為0-10的整數； z1, z2, z3, z4, z5, z6, z7, z8 and z9 are independently integers from 0 to 10;

L₂為C₁-C₃₀烷基鏈、或包含被一個或多個氧、硫、氮原子或C=O間斷的C₁-C₃₀烷基鏈； L ₂ is a C ₁ -C ₃₀ alkyl chain, or contains a C ₁ -C ₃₀ alkyl chain interrupted by one or more oxygen, sulfur, nitrogen atoms or C=O;

r1為1-10的整數。 r1 is an integer from 1 to 10.

在一些實施方案中，L₁為C₁-C₃₀烷基鏈、或包含被一個或多個氧、硫、氮原子或C=O間斷的C₁-C₃₀烷基鏈； In some embodiments, L ₁ is a C ₁ -C ₃₀ alkyl chain, or includes a C ₁ -C ₃₀ alkyl chain interrupted by one or more oxygen, sulfur, nitrogen atoms, or C=O;

R₁₁和R₁₂獨立地為化學鍵、NR₁₆或C=O； R ₁₁ and R ₁₂ are independently chemical bonds, NR ₁₆ or C=O;

R₁₆為氫或C_1-6烷基； R ₁₆ is hydrogen or C _1-6 alkyl;

Q₃為

Q ₃ is

R₁₃為CR₁₇R₁₈、NR₁₆、O或S； R ₁₃ is CR ₁₇ R ₁₈ , NR ₁₆ , O or S;

R₁₄為CR₁₉； R ₁₄ is CR ₁₉ ;

R₁₅獨立地為CR₁₇R₁₈、NR₁₆或O； R ₁₅ is independently CR ₁₇ R ₁₈ , NR ₁₆ or O;

R₁₇至R₁₉獨立地為氫、氘或烷基； R ₁₇ to R ₁₉ are independently hydrogen, deuterium or alkyl;

m1、p1和q1獨立地為0、1、2、3或4； m1, p1 and q1 are independently 0, 1, 2, 3 or 4;

B1為

； B1 is

;

R_b5、R_b6和R_b7獨立地為-C(=O)-、-NHC(=O)-、-C(=O)O-、-C(=O)-(CH₂)_z8-O-或-NHC(=O)-(CH₂)_z9-O-； R _b5 , R _b6 and R _b7 are independently -C(=O)-, -NHC(=O)-, -C(=O)O-, -C(=O)-(CH ₂ ) _z8 -O -or-NHC(=O)-(CH ₂ ) _z9 -O-;

z5、z6、z7、z8和z9獨立地為0-10的整數； z5, z6, z7, z8 and z9 are independently integers from 0 to 10;

r1為1-10的整數。 r1 is an integer from 1 to 10.

在一些實施方案中，L₁為-(CH₂)_j11-C(=O)-(CH₂)_j12-； In some embodiments, L ₁ is -(CH ₂ ) _j11 -C(=O)-(CH ₂ ) _j12 -;

R₁₆為氫或C_1-6烷基； R ₁₆ is hydrogen or C _1-6 alkyl;

Q₃為

； Q ₃ is

;

R₁₃為CR₁₇R₁₈或O； R ₁₃ is CR ₁₇ R ₁₈ or O;

R₁₄為CR₁₉； R ₁₄ is CR ₁₉ ;

R₁₅獨立地為CR₁₇R₁₈或O； R ₁₅ is independently CR ₁₇ R ₁₈ or O;

R₁₇至R₁₉獨立地為氫或烷基； R ₁₇ to R ₁₉ are independently hydrogen or alkyl;

m1、p1和q1獨立地為0或1； m1, p1 and q1 are independently 0 or 1;

B1為

； B1 is

;

R_b5、R_b6和R_b7獨立地為-C(=O)-(CH₂)_z8-O-或-NHC(=O)-(CH₂)_z9-O-； R _b5 , R _b6 and R _b7 are independently -C(=O)-(CH ₂ ) _z8 -O- or -NHC(=O)-(CH ₂ ) _z9 -O-;

z8和z9獨立地為0-10的整數； z8 and z9 are independently integers from 0 to 10;

L₂為-(CH₂)_j15-(OCH₂CH₂)_1-4-(CH₂)_j16-或

； L ₂ is -(CH ₂ ) _j15 -(OCH ₂ CH ₂ ) _1-4 -(CH ₂ ) _j16 -or

;

j15和j16獨立地為0-4的整數； j15 and j16 are independently integers from 0 to 4;

r1為3、4、5或6。 r1 is 3, 4, 5 or 6.

在一些實施方案中，L₁可為L₃或L₃-R₁₁₀-R₁₁₁-L₃，其中，L₃獨立地為C₁-C₁₂烷基鏈、-(CH₂)_j11-C(=O)-(CH₂)_j12-或-(CH₂)_j13-(CH₂CH₂O)_1-4-(CH₂)_j14-，R₁₁₀和R₁₁₁獨立地為化學鍵、-NR₁₁₂-、-C(=O)-或-OC(=O)-，R₁₁₂為氫或C₁-C₁₂烷基，j11、j12、j13和j14獨立地為0-10的整數。在一些實施方案中，j11、j12、j13和j14獨立地為0-2或4-10的整數。在一些實施方案中，j11、j12、j13和j14獨立地為0、1、2、6、7、8、9或10。 In some embodiments, L ₁ can be L ₃ or L ₃ -R ₁₁₀ -R ₁₁₁ -L ₃ , where L ₃ is independently a C ₁ -C ₁₂ alkyl chain, -(CH ₂ ) _j11 -C( =O)-(CH ₂ ) _j12 -or-(CH ₂ ) _j13 -(CH ₂ CH ₂ O) _1-4 -(CH ₂ ) _j14 -, R ₁₁₀ and R ₁₁₁ are independently chemical bonds, -NR ₁₁₂ - , -C(=O)- or -OC(=O)-, R ₁₁₂ is hydrogen or C ₁ -C ₁₂ alkyl, j11, j12, j13 and j14 are independently integers from 0 to 10. In some embodiments, j11, j12, j13, and j14 are independently integers from 0 to 2 or from 4 to 10. In some embodiments, j11, j12, j13, and j14 are independently 0, 1, 2, 6, 7, 8, 9, or 10.

在一些實施方案中，L₁可為-(CH₂)_j11-C(=O)-(CH₂)_j12-，j11和j12的定義同前任一方案所述。 In some embodiments, L ₁ can be -(CH ₂ ) _j11 -C(=O)-(CH ₂ ) _j12 -, j11 and j12 are defined as in the previous embodiment.

在一些實施方案中，L₁可為

，j12的定義同前任一方案所述，其中，a1端與B₁相連，b1端與R₁₁相連。 In some embodiments, _L can be

, the definition of j12 is the same as described in the previous scheme, in which the a1 end is connected to B ₁ and the b1 end is connected to R ₁₁ .

在一些實施方案中，L₁可為

、

、

、

或

，其中，a1端與B₁相連，b1端與R₁₁相連。 In some embodiments, _L can be

,

or

, where the a1 end is connected to B ₁ , and the b1 end is connected to R ₁₁ .

在一些實施方案中，R₁₁可為化學鍵且R₁₂可為C=O。 In some embodiments, R ₁₁ can be a chemical bond and R ₁₂ can be C=O.

在一些實施方案中，R₁₁可為化學鍵且R₁₂可為NR₁₆，R₁₆的定義同前任一方案所述。 In some embodiments, R ₁₁ can be a chemical bond and R ₁₂ can be NR ₁₆ , and R ₁₆ is as defined in the previous embodiment.

在一些實施方案中，R₁₁可為化學鍵且R₁₂可為-OC(=O)-。 In some embodiments, R ₁₁ can be a chemical bond and R ₁₂ can be -OC(=O)-.

在一些實施方案中，R₁₁可為NR₁₆且R₁₂可為C=O，R₁₆的定義同前任一方案所述。 In some embodiments, R ₁₁ can be NR ₁₆ and R ₁₂ can be C=O, with R ₁₆ being as defined in either embodiment.

在一些實施方案中，R₁可為NR₁₆且R₁₂可為-OC(=O)-，R₁₆的定義同前任一方案所述。 In some embodiments, R ₁ can be NR ₁₆ and R ₁₂ can be -OC(=O)-, with R ₁₆ being as defined in either embodiment.

在一些實施方案中，R₁₂可為NR₁₆且R₁₁可為C=O，R₁₆的定義同前任一方案所述。 In some embodiments, R ₁₂ can be NR ₁₆ and R ₁₁ can be C=O, with R ₁₆ being as defined in either embodiment.

在一些實施方案中，R₁₂可為NR₁₆且R₁₁可為-OC(=O)-，R₁₆的定義同前任一方案所述。 In some embodiments, R ₁₂ can be NR ₁₆ and R ₁₁ can be -OC(=O)-, with R ₁₆ being as defined in the previous embodiment.

在一些實施方案中，R₁₁可為NH且R₁₂可為C=O。 In some embodiments, R ₁₁ can be NH and R ₁₂ can be C=O.

在一些實施方案中，R₁₂可為NH且R₁₁可為C=O。 In some embodiments, R ₁₂ can be NH and R ₁₁ can be C=O.

在一些實施方案中，R₁₆可為氫或C_1-6烷基。 In some embodiments, R ₁₆ can be hydrogen or C _1-6 alkyl.

在一些實施方案中，R₁₆可為氫、甲基、乙基、丙基或異丙基。 In some embodiments, R ₁₆ can be hydrogen, methyl, ethyl, propyl, or isopropyl.

在一些實施方案中，R₁₆可為氫。 In some embodiments, R ₁₆ can be hydrogen.

在一些實施方案中，R₁₇和R₁₈可為氫。 In some embodiments, R ₁₇ and R ₁₈ can be hydrogen.

在一些實施方案中，R₁₉可為氫。 In some embodiments, R ₁₉ can be hydrogen.

在一些實施方案中，環A存在時，環A可為C_6-12芳基。 In some embodiments, Ring A, when present, can be C _6-12 aryl.

在一些實施方案中，環A可為苯基。 In some embodiments, Ring A can be phenyl.

在一些實施方案中，m1可為0或1。 In some embodiments, m1 can be 0 or 1.

在一些實施方案中，m1可為3。 In some embodiments, m1 can be 3.

在一些實施方案中，n1可為0或1。 In some embodiments, n1 can be 0 or 1.

在一些實施方案中，p1和q1獨立地為0或1。 In some embodiments, pi and q1 are independently 0 or 1.

在一些實施方案中，p1=1且q1=1。 In some embodiments, p1=1 and q1=1.

在一些實施方案中，p1=1且q1=0。 In some embodiments, p1=1 and q1=0.

在一些實施方案中，p1=0且q1=1。 In some embodiments, p1=0 and q1=1.

在一些實施方案中，p1=0且q1=0。 In some embodiments, p1=0 and q1=0.

在一些實施方案中，z1、z2、z3、z4、z5、z6、z7、z8和z9可獨立地為0-4的整數。在一些實施方案中，z1、z2、z3、z4、z5、z6、z7、z8和z9可獨立地為0、1或2。 In some embodiments, z1, z2, z3, z4, z5, z6, z7, z8, and z9 may independently be an integer from 0 to 4. In some embodiments, z1, z2, z3, z4, z5, z6, z7, z8, and z9 can independently be 0, 1, or 2.

在一些實施方案中，B₁可為

，R_b1、R_b2、R_b3 和R_b4獨立地為-C(=O)-或-NHC(=O)-，N原子與L₁相連，z1、z2、z3和z4的定義同前任一方案所述。 In some embodiments, B ₁ can be

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

在一些實施方案中，B₁可為

，R_b1、R_b2、R_b3 和R_b4獨立地為-C(=O)-或-NHC(=O)-，N原子與L₁相連，R_b1、R_b3和R_b4相同，z1、z2、z3和z4的定義同前任一方案所述。 In some embodiments, B ₁ can be

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

在一些實施方案中，B₁可為

。 In some embodiments, B ₁ can be

.

在一些實施方案中，B₁可為

。 In some embodiments, B ₁ can be

.

在一些實施方案中，B₁可為

，R_b5、R_b6和R_b7獨立地為-C(=O)-(CH₂)_z8-O-或-NHC(=O)-(CH₂)_z9-O-，N原子與L₁相連，z5、z6、z7、z8和z9的定義同前任一方案所述。 In some embodiments, B ₁ can be

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

在一些實施方案中，B₁可為

，R_b5、R_b6和R_b7獨立地為-C(=O)-(CH₂)_z8-O-或-NHC(=O)-(CH₂)_z9-O-，N原子與L₁相連，R_b5、R_b6和R_b7相同，z5、z6、z7、z8和z9的定義同前任一方案所述。 In some embodiments, B ₁ can be

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

在一些實施方案中，B₁可為

。 In some embodiments, B ₁ can be

.

在一些實施方案中，L₂可為L₄或L₄-R₁₃-R₁₄-L₄，其中，L₄獨立地為C₁-C₁₂烷基鏈或-(CH₂)_j15-(OCH₂CH₂)_1-4-(CH₂)_j16-，R₁₃和R₁₄獨立地為化學鍵、-NR₁₁₅-、-C(=O)-或-OC(=O)-，R₁₁₅獨立地為氫或C₁-C₁₂烷基，j15和j16獨立地為0-10的整數。在一些實施方案中，j15和j16獨立地為0-6的整數。在一些實施方案中，j15和j16獨立地為0、1、2、3或4。 In some embodiments, L ₂ can be L ₄ or L ₄ -R ₁₃ -R ₁₄ -L ₄ , wherein L ₄ is independently a C ₁ -C ₁₂ alkyl chain or -(CH ₂ ) _{j 15} -(OCH ₂ CH ₂ ) _1-4 -(CH ₂ ) _j16 -, R ₁₃ and R ₁₄ are independently chemical bonds, -NR ₁₁₅ -, -C(=O)- or -OC(=O)-, R ₁₁₅ is independently is hydrogen or C ₁ -C ₁₂ alkyl, j15 and j16 are independently an integer from 0 to 10. In some embodiments, j15 and j16 are independently integers from 0-6. In some embodiments, j15 and j16 are independently 0, 1, 2, 3, or 4.

在一些實施方案中，L₂可為-(CH₂)_j15-(OCH₂CH₂)_1-4-(CH₂)_j16-，j15和j16的定義同前任一方案所述。 In some embodiments, L ₂ can be -(CH ₂ ) _j15 -(OCH ₂ CH ₂ ) _1-4 -(CH ₂ ) _j16 -, j15 and j16 being as defined in the previous embodiment.

在一些實施方案中，L₂可為

或

。在一些實施方案中，L₂可為

，其中，一側與O原子相連，另一側與 B₁相連。 In some embodiments, _L2 can be

or

. In some embodiments, _L2 can be

, where one side is connected to the O atom and the other side is connected to B ₁ .

在一些實施方案中，L₂可為C₁-C₁₂烷基鏈。 In some embodiments, _L2 can be a _C1 - _C12 alkyl chain.

在一些實施方案中，L₂可為

、

、 In some embodiments, _L2 can be

,

在一些實施方案中，L₂可為

。在一些實施方案中，L₂ 可為

。在一些實施方案中，L₂可為

。在一些實施方案中，L₂可為

。其中，a3端與O原子相連，b3端與B₁相連。 In some embodiments, _L2 can be

. In some embodiments, _L2 can be

. Among them, the a3 end is connected to the O atom, and the b3 end is connected to B ₁ .

在一些實施方案中，L₂可為

，其中，a3端與O原子相連，b3端與B₁相連。 In some embodiments, _L2 can be

, where the a3 end is connected to the O atom, and the b3 end is connected to B ₁ .

在一些實施方案中，r1可為3、4、5或6。在一些實施方案中，r1可為3。 In some embodiments, r1 can be 3, 4, 5, or 6. In some embodiments, r1 can be 3.

在一些實施方案中，Q₃可為

或

。在一些實施方案中，Q₃可為

。其中，R₁₃、R₁₄、R₁₅和n1的定義同前任一方案所述。 In some embodiments, _Q3 can be

or

. In some embodiments, _Q3 can be

. Among them, the definitions of R ₁₃ , R ₁₄ , R ₁₅ and n1 are the same as those described in the previous solution.

在一些實施方案中，

可為

，其中，R₁₃、R₁₄、 R₁₅、p1和q1的定義同前任一方案所述。 In some embodiments,

can be

, where the definitions of R ₁₃ , R ₁₄ , R ₁₅ , p1 and q1 are the same as those described in the previous solution.

在一些實施方案中，

可為

、

、

或

，其中，R₁₃、R₁₄、R₁₅、p1和q1的定義同前任一方案所述。 In some embodiments,

can be

,

or

在一些實施方案中，

可為

、

或

。在一些實施方案中，

可為

或

。在一些實施方案中，

可為

。p1和q1的定義同前任一方案所述。 In some embodiments,

can be

,

or

. In some embodiments,

can be

or

. In some embodiments,

can be

. The definitions of p1 and q1 are the same as described in the previous scheme.

在一些實施方案中，

可為

、

、

、 In some embodiments,

can be

,

或

。在一些實施方案中，

可為

、

、

、

、

、

、

或

。在一些實施方案中，

可為

、

、

或

。p1和q1的定義同前任一方案所述。

or

. In some embodiments,

can be

,

or

. In some embodiments,

can be

,

or

在一些實施方案中，

可為

，其中，R₁₃、R₁₄、 n1、p1和q1的定義同前任一方案所述。 In some embodiments,

can be

, where the definitions of R ₁₃ , R ₁₄ , n1, p1 and q1 are the same as those described in the previous solution.

在一些實施方案中，

可為

或

，其中，R₁₃、R₁₄、n1、p1和q1的定義同前任一方案所述。 In some embodiments,

can be

or

在一些實施方案中，

可為

或

在一些實施方案中，

可為

。n1、p1和q1的定義同前任一方案所述。 In some embodiments,

can be

or

In some embodiments,

can be

. The definitions of n1, p1 and q1 are the same as described in the previous scheme.

在一些實施方案中，

可為

或

， n1、p1和q1的定義同前任一方案所述。 In some embodiments,

can be

or

, the definitions of n1, p1 and q1 are the same as those described in the previous scheme.

在一些實施方案中，該配體可為以下任一結構或其藥學上可接受的鹽， In some embodiments, the ligand can be any of the following structures or a pharmaceutically acceptable salt thereof,

在一些實施方案中，該配體可為以下結構或其藥學上可接受的鹽， In some embodiments, the ligand can be the following structure or a pharmaceutically acceptable salt thereof,

在一些實施方案中，可以用N-三氟乙醯基半乳糖胺、N-丙醯基半乳糖胺、N-正丁醯基半乳糖胺或N-異丁醯基半乳糖胺替換以上配體中的N-乙醯基-半乳糖胺部分。 In some embodiments, N in the above ligands can be replaced with N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butylgalactosamine or N-isobutylgalactosamine. - Acetyl-galactosamine moiety.

在一些實施方案中，該式(I)所示的化學修飾為

、

或

，B選自鳥嘌呤、腺嘌呤、胞嘧啶和尿嘧啶；且該配體為如下任一結構或其藥學上可接受的鹽， In some embodiments, the chemical modification represented by formula (I) is

,

or

, B is selected from guanine, adenine, cytosine and uracil; and the ligand is any of the following structures or a pharmaceutically acceptable salt thereof,

。在一些實施方案中，該有義鏈和反義鏈的定義同前任一方案所述。

. In some embodiments, the sense strand and antisense strand are as defined in the previous embodiment.

在一些實施方案中，該式(I)所示的化學修飾為

、

或

，B選自鳥嘌呤、腺嘌呤、胞嘧啶和尿嘧啶；且 In some embodiments, the chemical modification represented by formula (I) is

,

or

, B is selected from guanine, adenine, cytosine and uracil; and

該配體為如下任一結構或其藥學上可接受的鹽， The ligand is any of the following structures or a pharmaceutically acceptable salt thereof,

在一些實施方案中，該式(I)所示的化學修飾為

、

或

,

or

, B is selected from guanine, adenine, cytosine and uracil; and

該配體為如下結構或其藥學上可接受的鹽， The ligand has the following structure or a pharmaceutically acceptable salt thereof,

在一些實施方案中，該有義鏈和反義鏈的定義同前任一方案所述。 In some embodiments, the sense strand and antisense strand are as defined in the previous embodiment.

在一些實施方案中，該有義鏈和/或反義鏈和該配體共價或非共價連接。 In some embodiments, the sense strand and/or antisense strand and the ligand are covalently or non-covalently linked.

在一些實施方案中，該有義鏈的3’端和/或5’端與該配體綴合。 In some embodiments, the 3' end and/or 5' end of the sense strand is conjugated to the ligand.

在一些實施方案中，該有義鏈的3’端與該配體綴合。 In some embodiments, the 3' end of the sense strand is conjugated to the ligand.

在一些實施方案中，該配體藉由磷酸酯基團或硫代磷酸酯基團與該有義鏈末端連接。 In some embodiments, the ligand is attached to the sense strand end via a phosphate group or a phosphorothioate group.

在一些實施方案中，該配體藉由磷酸二酯基團或硫代磷酸二酯基團與該有義鏈末端連接。 In some embodiments, the ligand is attached to the sense strand end via a phosphodiester group or a phosphorothioate diester group.

在一些實施方案中，該配體藉由磷酸二酯基團與該有義鏈末端連接。 In some embodiments, the ligand is attached to the sense strand end via a phosphodiester group.

在一些實施方案中，該配體藉由磷酸酯基團或硫代磷酸酯基團與該有義鏈末端間接連接。 In some embodiments, the ligand is indirectly linked to the sense strand end via a phosphate group or phosphorothioate group.

在一些實施方案中，該配體藉由磷酸酯基團或硫代磷酸酯基團與該有義鏈末端直接連接。 In some embodiments, the ligand is directly linked to the sense strand end via a phosphate group or phosphorothioate group.

在一些實施方案中，該配體藉由磷酸酯基團或硫代磷酸酯基團與該有義鏈3’末端直接連接。 In some embodiments, the ligand is directly linked to the 3' end of the sense strand via a phosphate group or phosphorothioate group.

在一些實施方案中，該磷酸酯基團為磷酸一酯基團或磷酸二酯基團。在一些實施方案中，該磷酸酯基團為磷酸二酯基團。 In some embodiments, the phosphate ester group is a phosphate monoester group or a phosphate diester group. In some embodiments, the phosphate ester group is a phosphodiester group.

在一些實施方案中，該硫代磷酸酯基團為硫代磷酸一酯基團或硫代磷酸二酯基團。在一些實施方案中，該硫代磷酸酯基團為硫代磷酸二酯基團。 In some embodiments, the phosphorothioate group is a phosphorothioate monoester group or a phosphorothioate diester group. In some embodiments, the phosphorothioate group is a phosphorothioate diester group.

在一些實施方案中，為了促進進入細胞，可以在有義鏈的末端引入膽固醇等親脂性的基團，親脂性的基團包括以共價鍵與dsRNA結合，如末端引入膽固醇、脂蛋白、維生素E等，以利於藉由由脂質雙分子層構成的細胞膜與細胞內的mRNA發生作用。同時，dsRNA也可以進行非共價鍵修飾，如藉由疏水鍵或離子鍵結合磷脂分子、多肽、陽離子聚合物等增加穩定性和生物學活性。 In some embodiments, in order to facilitate entry into cells, lipophilic groups such as cholesterol can be introduced at the end of the sense strand. The lipophilic groups include binding to dsRNA through covalent bonds, such as introducing cholesterol, lipoproteins, and vitamins at the end. E, etc., to facilitate the interaction between the cell membrane composed of lipid bilayers and The mRNA in the cell acts. At the same time, dsRNA can also undergo non-covalent modification, such as binding to phospholipid molecules, polypeptides, cationic polymers, etc. through hydrophobic bonds or ionic bonds to increase stability and biological activity.

在一些實施方案中，該配體的個數包括但不限於：1、2、3或4個。在一些實施方案中，該配體的個數為1個。 In some embodiments, the number of ligands includes, but is not limited to: 1, 2, 3 or 4. In some embodiments, the number of ligands is one.

在一些實施方案中，該dsRNA的有義鏈的核苷酸序列包含或選自SEQ ID NO：4，反義鏈的核苷酸序列包含或選自SEQ ID NO：5至SEQ ID NO：14中任一的核苷酸序列； In some embodiments, the nucleotide sequence of the sense strand of the dsRNA includes or is selected from SEQ ID NO: 4, and the nucleotide sequence of the antisense strand includes or is selected from SEQ ID NO: 5 to SEQ ID NO: 14 The nucleotide sequence of any one;

有義鏈的核苷酸序列包含SEQ ID NO：4，反義鏈的核苷酸序列包含SEQ ID NO：5； The nucleotide sequence of the sense strand includes SEQ ID NO: 4, and the nucleotide sequence of the antisense strand includes SEQ ID NO: 5;

有義鏈的核苷酸序列包含SEQ ID NO：4，反義鏈的核苷酸序列包含SEQ ID NO：6； The nucleotide sequence of the sense strand includes SEQ ID NO: 4, and the nucleotide sequence of the antisense strand includes SEQ ID NO: 6;

有義鏈的核苷酸序列包含SEQ ID NO：4，反義鏈的核苷酸序列包含SEQ ID NO：7； The nucleotide sequence of the sense strand includes SEQ ID NO: 4, and the nucleotide sequence of the antisense strand includes SEQ ID NO: 7;

有義鏈的核苷酸序列包含SEQ ID NO：4，反義鏈的核苷酸序列包含SEQ ID NO：8； The nucleotide sequence of the sense strand includes SEQ ID NO: 4, and the nucleotide sequence of the antisense strand includes SEQ ID NO: 8;

有義鏈的核苷酸序列包含SEQ ID NO：4，反義鏈的核苷酸序列包含SEQ ID NO：9； The nucleotide sequence of the sense strand includes SEQ ID NO: 4, and the nucleotide sequence of the antisense strand includes SEQ ID NO: 9;

有義鏈的核苷酸序列包含SEQ ID NO：4，反義鏈的核苷酸序列包含SEQ ID NO：10； The nucleotide sequence of the sense strand contains SEQ ID NO: 4, and the nucleotide sequence of the antisense strand contains SEQ ID NO: 10;

有義鏈的核苷酸序列包含SEQ ID NO：4，反義鏈的核苷酸序列包含SEQ ID NO：11； The nucleotide sequence of the sense strand contains SEQ ID NO: 4, and the nucleotide sequence of the antisense strand contains SEQ ID NO: 11;

有義鏈的核苷酸序列包含SEQ ID NO：4，反義鏈的核苷酸序列包含SEQ ID NO：12； The nucleotide sequence of the sense strand contains SEQ ID NO: 4, and the nucleotide sequence of the antisense strand contains SEQ ID NO: 12;

有義鏈的核苷酸序列包含SEQ ID NO：4，反義鏈的核苷酸序列包含SEQ ID NO：13； The nucleotide sequence of the sense strand contains SEQ ID NO: 4, and the nucleotide sequence of the antisense strand contains SEQ ID NO: 13;

有義鏈的核苷酸序列包含SEQ ID NO：4，反義鏈的核苷酸序列包含SEQ ID NO：14。 The nucleotide sequence of the sense strand contains SEQ ID NO: 4, and the nucleotide sequence of the antisense strand contains SEQ ID NO: 14.

有義鏈的核苷酸序列選自SEQ ID NO：4，反義鏈的核苷酸序列選自SEQ ID NO：5； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 4, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 5;

有義鏈的核苷酸序列選自SEQ ID NO：4，反義鏈的核苷酸序列選自SEQ ID NO：6； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 4, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 6;

有義鏈的核苷酸序列選自SEQ ID NO：4，反義鏈的核苷酸序列選自SEQ ID NO：7； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 4, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 7;

有義鏈的核苷酸序列選自SEQ ID NO：4，反義鏈的核苷酸序列選自SEQ ID NO：8； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 4, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 8;

有義鏈的核苷酸序列選自SEQ ID NO：4，反義鏈的核苷酸序列選自SEQ ID NO：9； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 4, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 9;

有義鏈的核苷酸序列選自SEQ ID NO：4，反義鏈的核苷酸序列選自SEQ ID NO：10； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 4, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 10;

有義鏈的核苷酸序列選自SEQ ID NO：4，反義鏈的核苷酸序列選自SEQ ID NO：11； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 4, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 11;

有義鏈的核苷酸序列選自SEQ ID NO：4，反義鏈的核苷酸序列選自SEQ ID NO：12； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 4, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 12;

有義鏈的核苷酸序列選自SEQ ID NO：4，反義鏈的核苷酸序列選自SEQ ID NO：13； The nucleotide sequence of the sense strand is selected from SEQ ID NO: 4, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO: 13;

有義鏈的核苷酸序列選自SEQ ID NO：4，反義鏈的核苷酸序列選自SEQ ID NO：14。 The nucleotide sequence of the sense strand is selected from SEQ ID NO:4, and the nucleotide sequence of the antisense strand is selected from SEQ ID NO:14.

包含SEQ ID NO：4所示的有義鏈和SEQ ID NO：5至SEQ ID NO：14中任一項所示的反義鏈。 It contains the sense strand shown in SEQ ID NO:4 and the antisense strand shown in any one of SEQ ID NO:5 to SEQ ID NO:14.

選自SEQ ID NO：4所示的有義鏈和SEQ ID NO：5至SEQ ID NO：14中任一項所示的反義鏈。 Selected from the sense strand shown in SEQ ID NO: 4 and the antisense strand shown in any one of SEQ ID NO: 5 to SEQ ID NO: 14.

SEQ ID NO：4是AmsAmsAmUmGmAmAfAfUfGmAmAmUmAmAmAmUmAmAm-NAG0052’； SEQ ID NO: 4 is AmsAmsAmUmGmAmAfAfUfGmAmAmUmAmAmAmUmAmAm -NAG0052';

SEQ ID NO：8是 UmsUfsAmUmUmUf(-)hmpNA(A)UmUmCfAmUfUmUfCmAfUmUfUmsUmsGm； SEQ ID NO:8 is UmsUfsAmUmUmUf(-)hmpNA(A)UmUmCfAmUfUmUfCmAfUmUfUmsUmsGm;

SEQ ID NO：11是UmsUfsAmUmUmUf-)hmpNA(A)UmUmCfAmUmUmUfCmAfUmUmUmsUmsGm； SEQ ID NO: 11 is UmsUfsAmUmUmUf-)hmpNA(A)UmUmCfAmUmUmUfCmAfUmUmUmsUmsGm;

SEQ ID NO：13是UmsUfsAmUmUmUm(-)hmpNA(A)UmUmCmAmUmUmUfCmAfUmUmUmsUmsGm； SEQ ID NO: 13 is UmsUfsAmUmUmUm(-)hmpNA(A)UmUmCmAmUmUmUfCmAfUmUmUmsUmsGm;

SEQ ID NO：14是UmsUfsAmUmUmUm(-)hmpNA(A)UmUmCmAmUmUmUfCmAfUmUmUmsUmsGm； SEQ ID NO: 14 is UmsUfsAmUmUmUm(-)hmpNA(A)UmUmCmAmUmUmUfCmAfUmUmUmsUmsGm;

其中，Af=腺嘌呤2'-F核糖核苷(adenine 2'-F ribonucleoside)；Cf=胞嘧啶2'-F核糖核苷(cytosine 2'-F ribonucleoside)；Uf=尿嘧啶2'-F核糖核苷(uracil 2'-F ribonucleoside)；Gf=鳥嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；Am=腺嘌呤2'-OMe核糖核苷(adenine 2'-OMe ribonucleoside)；Cm=胞嘧啶2'-OMe核糖核苷(cytosine 2'-OMe ribonucleoside)；Gm=鳥嘌呤2'-OMe核糖核苷(guanine 2'-OMe ribonucleoside)；Um=尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)； Among them, Af=adenine 2'-F ribonucleoside; Cf=cytosine 2'-F ribonucleoside; Uf=uracil 2'-F Ribonucleoside (uracil 2'-F ribonucleoside); Gf=guanine 2'-F ribonucleoside (guanine 2'-F ribonucleoside); Am=adenine 2'-OMe ribonucleoside; Cm=cytosine 2'-OMe ribonucleoside; Gm=guanine 2'- OMe ribonucleoside (guanine 2'-OMe ribonucleoside); Um=uracil 2'-OMe ribonucleoside (uracil 2'-OMe ribonucleoside);

NAG0052’表示

， NAG0052' means

,

(-)hmpNA(A)表示

。 (-)hmpNA(A) means

.

在一些實施方案中，該dsRNA選自如下結構或其藥學上可接受的鹽： In some embodiments, the dsRNA is selected from the following structures or pharmaceutically acceptable salts thereof:

其中，Af=腺嘌呤2'-F核糖核苷(adenine 2'-F ribonucleoside)；Cf=胞嘧啶2'-F核糖核苷(cytosine 2'-F ribonucleoside)；Uf=尿嘧啶2'-F核糖核苷(uracil 2'-F ribonucleoside)；Am=腺嘌呤2'-OMe核糖核苷(adenine 2'-OMe ribonucleoside)；Cm=胞嘧啶2'-OMe核糖核苷(cytosine 2'-OMe ribonucleoside)； Gf=鳥嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；Gm=鳥嘌呤2'-OMe核糖核苷(guanine 2'-OMe ribonucleoside)；Um=尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)。 Among them, Af=adenine 2'-F ribonucleoside; Cf=cytosine 2'-F ribonucleoside; Uf=uracil 2'-F Ribonucleoside (uracil 2'-F ribonucleoside); Am=adenine 2'-OMe ribonucleoside (adenine 2'-OMe ribonucleoside); Cm=cytosine 2'-OMe ribonucleoside (cytosine 2'-OMe ribonucleoside) ); Gf=guanine 2'-F ribonucleoside; Gm=guanine 2'-OMe ribonucleoside; Um=uracil 2'-OMe ribonucleoside Glycoside (uracil 2'-OMe ribonucleoside).

表示硫代磷酸二酯基，

表示磷酸二酯基，

Represents phosphorothioate diester group,

Represents phosphodiester group,

NAG0052’表示

， NAG0052' means

,

(-)hmpNA(A)表示

。 (-)hmpNA(A) means

.

在一些實施方案中，該藥學上可接受的鹽可為本領域常規的鹽，包括但不限於：鈉鹽、鉀鹽、銨鹽、胺鹽等。 In some embodiments, the pharmaceutically acceptable salt can be a conventional salt in the art, including but not limited to: sodium salt, potassium salt, ammonium salt, amine salt, etc.

在一些實施方案中，該dsRNA選自TRD008225、TRD008218、TRD008219、TRD008220、TRD008221、TRD008222、TRD008223、TRD008224、TRD008227、TRD008228中的任一項。 In some embodiments, the dsRNA is selected from any one of TRD008225, TRD008218, TRD008219, TRD008220, TRD008221, TRD008222, TRD008223, TRD008224, TRD008227, TRD008228.

在一些實施方案中，該dsRNA為TRD008225，其為如下結構 In some embodiments, the dsRNA is TRD008225, which has the following structure

在一些實施方案中，該dsRNA為TRD008218，其為如下結構 In some embodiments, the dsRNA is TRD008218, which has the following structure

在一些實施方案中，該dsRNA為TRD008219，其為如下結構 In some embodiments, the dsRNA is TRD008219, which has the following structure

在一些實施方案中，該dsRNA為TRD008220，其為如下結構 In some embodiments, the dsRNA is TRD008220, which has the following structure

在一些實施方案中，該dsRNA為TRD008221，其為如下結構 In some embodiments, the dsRNA is TRD008221, which has the following structure

在一些實施方案中，該dsRNA為TRD008222，其為如下結構 In some embodiments, the dsRNA is TRD008222, which has the following structure

在一些實施方案中，該dsRNA為TRD008223，其為如下結構 In some embodiments, the dsRNA is TRD008223, which has the following structure

在一些實施方案中，該dsRNA為TRD008224，其為如下結構 In some embodiments, the dsRNA is TRD008224, which has the following structure

在一些實施方案中，該dsRNA為TRD008227，其為如下結構 In some embodiments, the dsRNA is TRD008227, which has the following structure

在一些實施方案中，該dsRNA為TRD008228，其為如下結構 In some embodiments, the dsRNA is TRD008228, which has the following structure

其中，Af=腺嘌呤2'-F核糖核苷(adenine 2'-F ribonucleoside)；Cf=胞嘧啶2'-F核糖核苷(cytosine 2'-F ribonucleoside)；Uf=尿嘧啶2'-F核糖核苷(uracil 2'-F ribonucleoside)；Am=腺嘌呤2'-OMe核糖核苷(adenine 2'-OMe ribonucleoside)；Cm=胞嘧啶2'-OMe核糖核苷(cytosine 2'-OMe ribonucleoside)；Gf=鳥嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；Gm=鳥嘌呤2'-OMe核糖核苷(guanine 2'-OMe ribonucleoside)；Um=尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)。 Among them, Af=adenine 2'-F ribonucleoside; Cf=cytosine 2'-F ribonucleoside; Uf=uracil 2'-F Ribonucleoside (uracil 2'-F ribonucleoside); Am=adenine 2'-OMe ribonucleoside (adenine 2'-OMe ribonucleoside); Cm=cytosine 2'-OMe ribonucleoside (cytosine 2'-OMe ribonucleoside) ); Gf=guanine 2'-F ribonucleoside; Gm=guanine 2'-OMe ribonucleoside; Um=uracil 2'-OMe Ribonucleoside (uracil 2'-OMe ribonucleoside).

表示硫代磷酸二酯基，

表示磷酸二酯基，

Represents phosphorothioate diester group,

Represents phosphodiester group,

NAG0052’表示

， NAG0052' means

,

(-)hmpNA(A)表示

。 (-)hmpNA(A) means

.

另一方面，本揭露提供了一種醫藥組成物，其包含上述任一dsRNA。 On the other hand, the present disclosure provides a pharmaceutical composition comprising any of the above-mentioned dsRNA.

在一些實施方案中，該醫藥組成物還包含一種或多種藥學上可接受的賦形劑。在非限制示例中，在遞藥系統中提供本揭露的醫藥組成物。各種遞藥系統是已知的並且可以用於本揭露的dsRNA或醫藥組成物，例如封裝在脂質體中、微粒、微囊、能夠表達該化合物的重組細胞、受體介導的細胞內吞作用、構建核酸作為逆轉錄病毒或其他載體的一部分。 In some embodiments, the pharmaceutical composition further includes one or more pharmaceutically acceptable excipients. In a non-limiting example, pharmaceutical compositions of the present disclosure are provided in a drug delivery system. Various drug delivery systems are known and can be used with the dsRNA or pharmaceutical compositions of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis , construct nucleic acids as part of retroviruses or other vectors.

在一些實施方案中，本揭露的dsRNA或醫藥組成物的給藥方式是常規的，可藉由局部給藥(例如，直接注射或植入)或全身給藥，也可藉由口服、直腸或胃腸外途徑進行給藥，該腸胃外途徑包括但不限於皮下注射、靜脈注射、肌肉注射、腹腔注射、透皮給藥、吸入給藥(如氣溶膠)、黏膜給藥(如舌下、鼻內給藥)、顱內給藥等。 In some embodiments, the mode of administration of the dsRNA or pharmaceutical composition of the present disclosure is conventional, and may be by local administration (e.g., direct injection or implantation) or systemic administration, or by oral, rectal or Administration is by parenteral route, including but not limited to subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, transdermal administration, inhalation administration (such as aerosol), mucosal administration (such as sublingual, nasal intracranial administration), intracranial administration, etc.

在一些實施方案中，本揭露提供的dsRNA或醫藥組成物可以藉由注射給予，例如，靜脈內、肌內、皮內、皮下、十二指腸內或腹膜內注射。 In some embodiments, the dsRNA or pharmaceutical composition provided by the present disclosure can be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal or intraperitoneal injection.

在一些實施方案中，本揭露提供的dsRNA或醫藥組成物可被包裝在試劑盒中。 In some embodiments, the dsRNA or pharmaceutical composition provided by the present disclosure can be packaged in a kit.

另一方面，本揭露提供了一種上述的dsRNA或上述的醫藥組成物在製備藥物中的應用。 On the other hand, the present disclosure provides a use of the above-mentioned dsRNA or the above-mentioned pharmaceutical composition in the preparation of medicines.

在一些實施方案中，該藥物可用於預防和/或治療肝炎、肝纖維化、非酒精性脂肪性肝炎(NASH)、非酒精性脂肪性肝病(NAFLD)、肝硬化、酒精性脂肪性肝炎(ASH)、酒精性脂肪肝病(ALD)、HCV-相關的硬化、藥物引起的肝損傷、或肝細胞壞死、慢性纖維炎性肝病。 In some embodiments, the drug can be used to prevent and/or treat hepatitis, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis ( ASH), alcoholic fatty liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury or hepatocellular necrosis, chronic fibroinflammatory liver disease.

在一些實施方案中，該藥物可用於預防和/或治療與HSD17B13基因表達相關的疾病。在一些實施方案中，該與HSD17B13基因表達相關的疾病可為肝炎、肝纖維化、非酒精性脂肪性肝炎(NASH)、非酒精性脂肪性肝病(NAFLD)、肝硬化、酒精性脂肪性肝炎(ASH)、酒精性脂肪肝病(ALD)、HCV-相關的硬化、藥物引起的肝損傷、肝細胞壞死、慢性纖維炎性肝病。在一些實施方案中，該與HSD17B13基因表達相關的疾病可為慢性纖維炎性肝病。在一些實施方案中，該慢性纖維炎性肝病與脂質液滴在肝臟中的蓄積及/或擴張有關。 In some embodiments, the medicament can be used to prevent and/or treat diseases associated with HSD17B13 gene expression. In some embodiments, the disease associated with HSD17B13 gene expression can be hepatitis, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic fatty liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury, hepatocellular necrosis, chronic fibroinflammatory liver disease. In some embodiments, the disease associated with HSD17B13 gene expression can be chronic fibroinflammatory liver disease. In some embodiments, the chronic fibroinflammatory liver disease is associated with the accumulation and/or expansion of lipid droplets in the liver.

在一些實施方案中，該dsRNA或醫藥組成物的有效量或有效劑量為約0.001mg/kg體重至約200mg/kg體重、約0.01mg/kg體重至約100mg/kg體重或約0.5mg/kg體重至約50mg/kg體重。 In some embodiments, the effective amount or dosage of the dsRNA 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.

另一方面，本揭露提供了一種預防和/或治療疾病的方法，其包括向受試者給予有效量或有效劑量的上述的dsRNA或上述的醫藥組成物。 On the other hand, the present disclosure provides a method for preventing and/or treating diseases, which includes administering an effective amount or an effective dose of the above-mentioned dsRNA or the above-mentioned pharmaceutical composition to a subject.

在一些實施方案中，該疾病可為肝炎、肝纖維化、非酒精性脂肪性肝炎(NASH)、非酒精性脂肪性肝病(NAFLD)、肝硬化、酒精性脂肪性肝炎(ASH)、酒精性脂肪肝病(ALD)、HCV-相關的硬化、藥物引起的肝損傷或肝細胞壞死、慢性纖維炎性肝病。 In some embodiments, the disease may be hepatitis, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic Fatty liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury or hepatocellular necrosis, chronic fibroinflammatory liver disease.

在一些實施方案中，該疾病可為與HSD17B13基因表達相關的疾病。在一些實施方案中，該與HSD17B13基因表達相關的疾病可為肝炎、肝纖維化、非酒精性脂肪性肝炎(NASH)、非酒精性脂肪性肝病(NAFLD)、肝硬化、酒精性脂肪性肝炎(ASH)、酒精性脂肪肝病(ALD)、HCV-相關的硬化、藥物引起的肝損傷或肝細胞壞死、慢性纖維炎性肝病。在一些實施方案中，該與HSD17B13基因表達相關的疾病可為慢性纖維炎性肝病。在一些實施方案中，該慢性纖維炎性肝病與脂質液滴在肝臟中的蓄積及/或擴張有關。 In some embodiments, the disease may be a disease associated with HSD17B13 gene expression. In some embodiments, the disease associated with HSD17B13 gene expression can be hepatitis, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic fatty liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury or hepatocellular necrosis, chronic fibroinflammatory liver disease. In some embodiments, the disease associated with HSD17B13 gene expression can be chronic fibroinflammatory liver disease. In some embodiments, the chronic fibroinflammatory liver disease is associated with the accumulation and/or expansion of lipid droplets in the liver.

另一方面，本揭露提供了一種用於在體內或在體外沉默細胞中靶基因或其mRNA的方法，其包括將上述的dsRNA或上述的醫藥組成物引入該細胞中的步驟。 On the other hand, the present disclosure provides a method for silencing a target gene or its mRNA in a cell in vivo or in vitro, which includes the step of introducing the above-mentioned dsRNA or the above-mentioned pharmaceutical composition into the cell.

在一些實施方案中，該靶基因為HSD17B13。 In some embodiments, the target gene is HSD17B13.

另一方面，本揭露提供了一種抑制靶基因或其mRNA表達的方法，其包括向受試者給予有效量或有效劑量的上述的dsRNA或上述的醫藥組成物。 On the other hand, the present disclosure provides a method for inhibiting the expression of a target gene or its mRNA, which includes administering an effective amount or an effective dose of the above-mentioned dsRNA or the above-mentioned pharmaceutical composition to a subject.

在一些實施方案中，該靶基因可為HSD17B13。 In some embodiments, the target gene can be HSD17B13.

本揭露的dsRNA或醫藥組成物可以在細胞、細胞群、組織或受試者等對象中降低靶基因或其mRNA的表達水平或抑制其活性，包括：向對象給予治療有效量的本文該dsRNA或醫藥組成物，從而抑制靶基因或其mRNA在對象中的表達。 The dsRNA or pharmaceutical composition of the present disclosure can reduce the expression level or inhibit the activity of a target gene or its mRNA in cells, cell groups, tissues or subjects, including: administering to the subject A therapeutically effective amount of the dsRNA or pharmaceutical composition herein is administered, thereby inhibiting the expression of the target gene or its mRNA in the subject.

在一些實施方案中，該受試者已在先前被鑑定為在靶向的細胞、細胞群、組織或受試者中具有靶基因或其mRNA的病理性上調。 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.

另一方面，本揭露提供了一種遞送寡核苷酸至肝臟的方法，其包括向受試者給予有效量或有效劑量的上述的dsRNA或上述的醫藥組成物。 On the other hand, the present disclosure provides a method of delivering oligonucleotides to the liver, which includes administering an effective amount or an effective dose of the above-mentioned dsRNA or the above-mentioned pharmaceutical composition to a subject.

另一方面，本揭露提供了一種RNA干擾(RNAi)試劑，其包含上述的dsRNA或上述的醫藥組成物。 On the other hand, the present disclosure provides an RNA interference (RNAi) reagent, which includes the above-mentioned dsRNA or the above-mentioned pharmaceutical composition.

另一方面，本揭露還提供了一種細胞，其包含上述的dsRNA或上述的醫藥組成物。 On the other hand, the present disclosure also provides a cell comprising the above-mentioned dsRNA or the above-mentioned pharmaceutical composition.

在本揭露中，該細胞不能發育成動物或植物個體。 In the present disclosure, the cells are unable to develop into animal or plant individuals.

另一方面，本揭露還提供了一種試劑盒，其包含上述的dsRNA或上述的醫藥組成物。 On the other hand, the present disclosure also provides a kit comprising the above-mentioned dsRNA or the above-mentioned pharmaceutical composition.

一些實施方案中，本揭露的dsRNA是siRNA。 In some embodiments, the dsRNA of the present disclosure is siRNA.

本揭露中，上述dsRNA或醫藥組成物當接觸到表達靶基因的細胞時，如所測定的(例如藉由psiCHECK活性篩選和螢光素酶報告基因檢測法、PCR或基於分支DNA(bDNA)的方法、或基於蛋白質的方法，如免疫螢光分析法、Western Blot、或流式細胞術)，上述dsRNA或醫藥組成物會抑制靶基因的表達至少5%、至少10%、至少15%、至少20%、至少25%、至少30%、至少35%、至少40%、至少45%、至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、或至少99%。 In the present disclosure, when the above-mentioned dsRNA or pharmaceutical composition comes into contact with cells expressing the target gene, as measured (for example, by psiCHECK activity screening and luciferase reporter gene detection, PCR or branched DNA (bDNA)-based method, or protein-based method, such as immunofluorescence analysis, Western Blot, or flow cytometry), the above dsRNA or pharmaceutical composition will inhibit the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

本揭露中，上述dsRNA或醫藥組成物當接觸到表達靶基因的細胞時，如所測定的(例如藉由psiCHECK活性篩選和螢光素酶報告基因檢測法、PCR或基於分支DNA(bDNA)的方法、或基於蛋白質的方法，如免疫螢光分析法、Western Blot、或流式細胞術)，上述dsRNA或醫藥組成物引起的靶基因mRNA剩餘表達百分比為不高於99%、不高於95%、不高於90%、不高於85%、不高於80%、不高於75%、不高於70%、不高於65%、不高於60%、不高於55%、不高於50%、不高於45%、不高於40%、不高於35%、不高於30%、不高於25%、不高於20%、不高於15%、或不高於10%。 In the present disclosure, when the above-mentioned dsRNA or pharmaceutical composition comes into contact with cells expressing the target gene, as measured (for example, by psiCHECK activity screening and luciferase reporter gene detection, PCR or branched DNA (bDNA)-based method, or protein-based method, such as immunofluorescence analysis, Western Blot, or flow cytometry), the remaining expression percentage of the target gene mRNA caused by the above dsRNA or pharmaceutical composition is not higher than 99%, not higher than 95 %, not higher than 90%, not higher than 85%, not higher than 80%, not higher than 75%, not higher than 70%, not higher than 65%, not higher than 60%, not higher than 55%, Not more than 50%, not more than 45%, not more than 40%, not more than 35%, not more than 30%, not more than 25%, not more than 20%, not more than 15%, or not higher than 10%.

本揭露中，上述dsRNA或醫藥組成物當接觸到表達靶基因的細胞時，如所測定的(例如藉由psiCHECK活性篩選和螢光素酶報告基因檢測法、PCR或基於分支DNA(bDNA)的方法、或基於蛋白質的方法，如免疫螢光分析法、Western Blot、或流式細胞術)，dsRNA在保持在靶活性的同時，將脫靶活性減少了至少20%、至少25%、至少30%、至少35%、至少40%、至少45%、至少50%、至少55%、至少60%、至少65%、至少70%或至少75%。 In the present disclosure, when the above-mentioned dsRNA or pharmaceutical composition comes into contact with cells expressing the target gene, as measured (for example, by psiCHECK activity screening and luciferase reporter gene detection, PCR or branched DNA (bDNA)-based method, or protein-based method, such as immunofluorescence assay, Western Blot, or flow cytometry), dsRNA reduces off-target activity by at least 20%, at least 25%, or at least 30% while maintaining on-target activity. , 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%.

本揭露中，上述dsRNA或醫藥組成物當接觸到表達靶基因的細胞時，如所測定的(例如藉由psiCHECK活性篩選和螢光素酶報告基因檢測法、PCR或基於分支DNA(bDNA)的方法、或基於蛋白質的方法，如免疫螢光分析法、Western Blot、或流式細胞術)，dsRNA使在靶活性降低至多20%、至多19%、至多15%、至多10%、至多5%或超過1%的同時，將脫靶活性減少了至少20%、至少25%、至少30%、至少35%、至少40%、至少45%、至少50%、至少55%、至少60%、至少65%、至少70%或至少75%。 In the present disclosure, when the above-mentioned dsRNA or pharmaceutical composition comes into contact with cells expressing the target gene, as measured (for example, by psiCHECK activity screening and luciferase reporter gene detection, PCR or branched DNA (bDNA)-based method, or protein-based method such as immunofluorescence assay, Western Blot, or flow cytometry), dsRNA reduces on-target activity by up to 20%, up to 19%, up to 15%, up to 10%, up to 5% Or more than 1%, while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% %, at least 70% or at least 75%.

本揭露中，上述dsRNA或醫藥組成物當接觸到表達靶基因的細胞時，如所測定的(例如藉由psiCHECK活性篩選和螢光素酶報告基因檢測法、PCR或基於分支DNA(bDNA)的方法、或基於蛋白質的方法，如免疫螢光分析法、Western Blot、或流式細胞術)，dsRNA使在靶活性提高至少1%、至少5%、至少10%、至少15%、至少20%、至少25%、至少30%、至少35%、至少40%、至少45%、至少50%、至少55%、至少60%、至少65%、至少70%、至少75%或至少80%的同時，將脫靶活性減少了至少20%、至少25%、至少30%、至少35%、至少40%、至少45%、至少50%、至少55%、至少60%、至少65%、至少70%或至少75%。 In the present disclosure, when the above-mentioned dsRNA or pharmaceutical composition comes into contact with cells expressing the target gene, as measured (for example, by psiCHECK activity screening and luciferase reporter gene detection, PCR or branched DNA (bDNA)-based method, or protein-based method, such as immunofluorescence assay, Western Blot, or flow cytometry), dsRNA increases on-target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20% , at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% at the same time , reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or At least 75%.

本揭露化合物可以存在特定的幾何或立體異構體形式。本揭露設想所有的這類化合物，包括順式和反式異構體、(-)-和(+)-對對映體、(R)-和(S)-對映體、非對映異構體、(D)-異構體、(L)-異構體，及其外消旋混合物和其他混合物，例如對映異構體或非對映體富集的混合物，所有這些混合物都屬本揭露的範圍之內。烷基等取代基中可存在另外的不對稱碳原子。所有這些異構體以及它們的混合物，均包括在本揭露的範圍之內。本揭露的含有不對稱碳原子的化合物可以以光學活性純的形式或外消旋形式被分離出來。光學活性純的形式可以從外消旋混合物拆分，或藉由使用手性原料或手性試劑合成。 Compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. This disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, ( R )- and ( S )-enantiomers, diastereomers isomer, the ( D )-isomer, the ( L )-isomer, and their racemic mixtures and other mixtures, such as enantiomeric or diastereomerically enriched mixtures, all of which are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of this disclosure. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically active pure form or racemic form. Optically active pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

可以藉由的手性合成或手性試劑或者其他常規技術製備光學活性的(R)-和(S)-異構體以及D和L異構體。如果想得到本揭露某化合物的一種對映體，可以藉由不對稱合成或者具有手性助劑的衍生作用來製備，其中將所得非對映體混合物分離，並且輔助基團裂開以提供純的所需對映異構體。或者，當分子中含有鹼性官能團(如胺基)或酸性官能團(如羧基)時，與適當的光學活性的酸或鹼形成非對映異構體的鹽，然後藉由本領域所公知的常規方法進行非對映異構體拆分，然後回收得到純的對映體。此外，對映異構體和非對映異構體的分離通常是藉由使用色譜法完成的，該色譜法採用手性固定相，並視需要地與化學衍生法相結合(例如由胺生成胺基甲酸鹽)。 The optically active ( R )- and ( S )-isomers as well as the D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If an enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliaries, where the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amine group) or an acidic functional group (such as a carboxyl group), it can form a diastereomeric salt with an appropriate optically active acid or base, and then use conventional methods known in the art to The method performs diastereoisomer resolution and then recovers the pure enantiomers. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using a chiral stationary phase, optionally combined with chemical derivatization methods (e.g., generation of amines from amines). formate).

本揭露所述化合物的化學結構中，鍵“

”表示未指定構型，即如果化學結構中存在手性異構體，鍵“

”可以為“

”或“

”，或者同時包含“

”和“

”兩種構型。本揭露所述化合物的化學結構中，鍵“

”並未指定構型，即鍵“

”的構型可以為E型或Z型，或者同時包含E和Z兩種構型。 In the chemical structure of the compounds described in this disclosure, the bond "

"Indicates that the configuration is not specified, i.e. if there are chiral isomers in the chemical structure, the bond"

"can be"

"or"

”, or both “

"and"

"Two configurations. In the chemical structures of the compounds described in this disclosure, bonds"

"The configuration, i.e. the key, is not specified"

"The configuration can be E -type or Z -type, or contain both E and Z configurations.

在本揭露的化學結構式中，“

”、“

”、“

”可以根據本文所述發明範圍連接一個或多個任何基團；星號“*”表示手性中心。 In the chemical structural formula of the present disclosure, "

","

"One or more of any group may be attached in accordance with the scope of the invention described herein; an asterisk "*" indicates a chiral center.

在不指明構型的情況下，本揭露的化合物和中間體還可以以不同的互變異構體形式存在，並且所有這樣的形式包含於本揭露的範圍內。術語“互變異構體”或“互變異構體形式”是指可經由低能壘互變的不同能量的結構異構體。例如，質子互變異構體(也稱為質子轉移互變異構體)包括經由質子遷移的互變，如酮-烯醇及亞胺-烯胺、內醯胺-內醯亞胺異構化。內醯胺-內醯亞胺平衡實例是在如下所示的A和B之間。 Without specifying a configuration, the compounds and intermediates of the present disclosure 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 proton transfer tautomers) include tautomers via proton migration, such as keto-enol and imine-enamine, lactam-lactam imine isomerizations. An example of a lactam-lactamimine equilibrium is between A and B as shown below.

本揭露中的所有化合物可以被畫成A型或B型。所有的互變異構形式在本揭露的範圍內。化合物的命名不排除任何互變異構體。 All compounds in this disclosure can be drawn as Form A or Form B. All tautomeric forms are within the scope of the present disclosure. The naming of the compounds does not exclude any tautomers.

本揭露還包括一些與本文中記載的那些相同的，但一個或多個原子被原子量或質量數不同於自然中通常發現的原子量或質量數的原子置換的同位素標記的本揭露化合物。可結合到本揭露化合物的同位素的實例包括氫、碳、氮、氧、磷、硫、氟、碘和氯的同位素，諸如分別為²H、³H、¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O、¹⁸O、³¹P、³²P、³⁵S、¹⁸F、¹²³I、¹²⁵I和³⁶Cl等。 The present disclosure also includes some isotopically labeled compounds that are the same as those described herein, but in which one or more atoms are replaced by an atom having an atomic weight or mass number different from that typically found in nature. Examples of isotopes that may be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹³ respectively N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ¹²³ I, ¹²⁵ I and ³⁶ Cl, etc.

除另有說明，當一個位置被特別地指定為氘(D)時，該位置應理解為具有大於氘的天然豐度(其為0.015%)至少1000倍的豐度的氘(即，至少10%的氘摻入)。示例中化合物的具有大於氘的天然豐度可以是至少1000倍的豐度的氘、至少2000倍的豐度的氘、至少3000倍的豐度的氘、至少4000倍的豐度的氘、至少5000倍的豐度的氘、至少6000倍的豐度的氘或更高豐度的氘。本揭露還包括各種氘化形式的式(I)、式(I’)、式(II)化合物。與碳原子連接的各個可用的氫原子可獨立地被氘原子替換。所屬技術領域具有通常知識者能夠參考相關文獻合成氘化形式的式(I)、式(I’)、式(II)化合物。在製備氘代形式的式(I)、式(I’)、式(II)化合物時可使用市售的氘代起始物質，或它們可使用常規技術採用氘代試劑合成，氘代試劑包括但不限於氘代硼烷、三氘代硼烷四氫呋喃溶液、氘代氫化鋰鋁、氘代碘乙烷和氘代碘甲烷等。 Unless otherwise stated, when a position is specifically designated as deuterium (D), that position is understood to have an abundance of deuterium that is at least 1000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10 % deuterium incorporation). Examples of compounds having a natural abundance greater than deuterium may be at least 1000 times the abundance of deuterium, at least 2000 times the abundance of deuterium, at least 3000 times the abundance of deuterium, at least 4000 times the abundance of deuterium, at least 5000 times more abundant deuterium, at least 6000 times more abundant deuterium, or more abundant deuterium. The present disclosure also includes various deuterated forms of compounds of Formula (I), Formula (I'), and Formula (II). Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. A person with ordinary knowledge in the art can refer to relevant literature to synthesize deuterated forms of compounds of formula (I), formula (I'), and formula (II). Commercially available deuterated starting materials may be used in the preparation of deuterated forms of compounds of formula (I), formula (I'), and formula (II), or they may be synthesized using conventional techniques using deuterated reagents, including deuterated reagents. But it is not limited to deuterated borane, trideuterated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide, deuterated methyl iodide, etc.

本揭露還提供了一種製備dsRNA或醫藥組成物的方法，其包括：合成本揭露所述的dsRNA或醫藥組成物。 The disclosure also provides a method for preparing dsRNA or a pharmaceutical composition, which includes: synthesizing the dsRNA or pharmaceutical composition described in the disclosure.

本揭露一些實施方案中，“包含”可替換為“由......組成”。 In some embodiments of the present disclosure, "comprising" may be replaced by "composed of."

本揭露引入WO2022028462A1、WO2022223015A1、WO2023274395A1、WO2022223015A1、PCT/CN2022/139481全文。 This disclosure introduces the full text of WO2022028462A1, WO2022223015A1, WO2023274395A1, WO2022223015A1, and PCT/CN2022/139481.

術語解釋Terminology explanation

為了更容易理解本揭露，以下具體定義了一些技術和科學術語。除非在本文中另有明確定義，本文使用的所有其它技術和科學術語都具有本揭露所屬技術領域具有通常知識者通常理解的含義。 To make this disclosure easier to understand, some technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

靶標是指本揭露的dsRNA所針對的客體；靶標可以是核酸(基因、mRNA等)，也可以是蛋白(前體、成熟蛋白、同種型、變體等)。在本揭露中，靶標尤其是指HSD17B13基因或其表達產物。 The target refers to the object targeted by the dsRNA of the present disclosure; the target can be a nucleic acid (gene, mRNA, etc.) or a protein (precursor, mature protein, isotype, variant, etc.). In the present disclosure, the target specifically refers to the HSD17B13 gene or its expression product.

術語“HSD17B13”是指第13型17β羥基類固醇脫氫酶，屬17β羥基類固醇脫氫酶(hsd17b)家族的一員，本揭露中HSD17B13應作做廣泛的解讀，是指HSD17B13基因本身及其在各階段中各種形式的表達產物，例如但不限於基因在擴增、複製、轉錄、剪接、加工、翻譯、修飾過程中所產生的分子，例如cDNA、mRNA、前體蛋白、成熟蛋白、天然變體、修飾形式、及其片段。HSD17B13序列包括：GENBANK登錄號NM_001136230.3，GENBANK登錄號NM_001136230.3。所屬技術領域具有通常知識者應當理解，儘管在具體實施方案中給出了具體的登錄號，但是HSD17B13不限於特定數據庫中的編號，還意圖涵蓋現有技術中任何文獻、書籍、數據庫中的等同指代物。當dsRNA靶向HSD17B13或其表達產物的特定靶點時，所屬技術領域具有通常知識者能夠確定該靶點在不同數據庫中的等同位置。 The term "HSD17B13" refers to type 13 17β-hydroxysteroid dehydrogenase, a member of the 17β-hydroxysteroid dehydrogenase (hsd17b) family. In this disclosure, HSD17B13 should be interpreted broadly and refers to the HSD17B13 gene itself and its functions in various Various forms of expression products in stages, such as but not limited to molecules produced during gene amplification, replication, transcription, splicing, processing, translation, and modification, such as cDNA, mRNA, precursor proteins, mature proteins, and natural variants , modified forms, and fragments thereof. The HSD17B13 sequence includes: GENBANK accession number NM_001136230.3, GENBANK accession number NM_001136230.3. Those with ordinary knowledge in the technical field should understand that although specific registration numbers are given in specific embodiments, HSD17B13 is not limited to numbers in specific databases, and is also intended to cover equivalent references in any documents, books, and databases in the prior art. Substitute things. When dsRNA targets a specific target of HSD17B13 or its expression product, a person with ordinary skill in the art can determine the equivalent position of the target in different databases.

除另有說明，“視需要地”、“視需要”、“可選的”或“可選”是指意味著隨後所描述的事件或環境可以但不必發生，該說明包括該事件或環境發生或不發生的場合。例如“視需要地，R₁和R₂直接相連成環”是指R₁和R₂直接相連成環可以發生但不必須存在，該說明包括R₁和R₂直接相連成環的情形和R₁和R₂不成環的情形。 Unless otherwise stated, "optionally,""optionally,""optional" or "optional" means that the subsequently described event or circumstances may but need not occur, and that the description includes the occurrence of that event or circumstances or does not occur. For example, "if necessary, R ₁ and R ₂ are directly connected to form a ring" means that R ₁ and R ₂ are directly connected to form a ring, which can occur but does not necessarily exist. This description includes the situation where R ₁ and R ₂ are directly connected to form a ring and R ₁ and R ₂ do not form a ring.

術語“約”、“大約”是指數值在由所屬技術領域具有通常知識者所測定的具體值的可接受誤差範圍內，該數值部分取決於怎樣測量或測定(即測量體系的限度)。例如，“約”可意味著在1內或超過1的標準差。或者，“約”或“基本上包含”可意味著至多±20%的範圍，例如±1%至15%之間、在±1%至10%之間、在±1%至5%之間、在±0.5%至5%之間、在±0.5%至1%之間變化。本揭露中，數字或數值範圍之前有術語“約”的每種情況也包括給定數的實施方案。除非另外說明，否則當具體值在本申請和請求項中出現時，“約”或“基本上包含”的含義應該假定為在該具體值的可接受誤差範圍內。 The terms "about" and "approximately" mean that the value is within an acceptable error range of the specific value as determined by a person of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "substantially includes" may mean a range of up to ±20%, such as between ±1% and 15%, between ±1% and 10%, between ±1% and 5% , varies between ±0.5% and 5%, and varies between ±0.5% and 1%. In this disclosure, every instance in which a number or numerical range is preceded by the term "about" also includes the embodiment of the given number. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about" or "substantially including" should be assumed to be within an acceptable error range for that specific value.

如無特殊說明，本揭露的“化合物”、“化學修飾”、“配體”、“dsRNA”、“核酸”均可獨立地以鹽、混合鹽或非鹽(例如游離酸或游離鹼)的形式存在。當以鹽或混合鹽的形式存在時，其可為藥學上可接受的鹽。 Unless otherwise specified, the "compounds", "chemical modifications", "ligands", "dsRNA" and "nucleic acids" of the present disclosure can be independently expressed as salts, mixed salts or non-salts (such as free acids or free bases). Form exists. When present in the form of a salt or mixed salts, they may be pharmaceutically acceptable salts.

“藥學上可接受的鹽”可選自無機鹽或有機鹽，也可包括藥學上可接受的酸加成鹽和藥學上可接受的鹼加成鹽。 "Pharmaceutically acceptable salts" may be selected from inorganic salts or organic salts, and may also include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

“藥學上可接受的酸加成鹽”是指能夠保留游離鹼的生物有效性而無其它副作用的，與無機酸或有機酸所形成的鹽。無機酸鹽包括但不限於鹽酸鹽、氫溴酸鹽、硫酸鹽、硝酸鹽、磷酸鹽等；有機酸鹽包括但不限於甲酸鹽、乙酸鹽、2,2-二氯乙酸鹽、三氟乙酸鹽、丙酸鹽、己酸鹽、辛酸鹽、癸酸鹽、十一碳烯酸鹽、乙醇酸鹽、葡糖酸鹽、乳酸鹽、癸二酸鹽、己二酸鹽、戊二酸鹽、丙二酸鹽、草酸鹽、馬來酸鹽、琥珀酸鹽、富馬酸鹽、酒石酸鹽、檸檬酸鹽、棕櫚酸鹽、硬脂酸鹽、油酸鹽、肉桂酸鹽、月桂酸鹽、蘋果酸鹽、谷胺酸鹽、焦谷胺酸鹽、天冬胺酸鹽、苯甲酸鹽、甲磺酸鹽、苯磺酸鹽、對甲苯磺酸鹽、海藻酸鹽、抗壞血酸鹽、水楊酸鹽、4-胺基水楊酸鹽、萘二磺酸鹽等。這些鹽可藉由本領域已知的方法製備。 "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 hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; organic acid salts include but are not limited to formate, acetate, 2,2-dichloroacetate, trichloroacetate, etc. Fluoroacetate, propionate, caproate, caprylate, caprate, undecenoate, glycolate, gluconate, lactate, sebacate, adipate, glutamate acid salt, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, Laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, mesylate, benzenesulfonate, p-toluenesulfonate, alginate, Ascorbate, salicylate, 4-aminosalicylate, naphthalene disulfonate, etc. These salts can be prepared by methods known in the art.

“藥學上可接受的鹼加成鹽”是指能夠保持游離酸的生物有效性而無其它副作用的、與無機鹼或有機鹼所形成的鹽。衍生自無機鹼的鹽包括但不限於鈉鹽、鉀鹽、鋰鹽、銨鹽、鈣鹽、鎂鹽、鐵鹽、鋅鹽、銅鹽、錳鹽、鋁鹽等。較佳的無機鹽為銨鹽、鈉鹽、鉀鹽、鈣鹽及鎂鹽，較佳鈉鹽。衍生自有機鹼的鹽包括但不限於以下的鹽：一級胺類、二級胺類及三級胺類，被取代的胺類，包括天然的被取代胺類、環狀胺類及鹼性離子交換樹脂，例如胺、異丙胺、三甲胺、二乙胺、三乙胺、三丙胺、乙醇胺、二乙醇胺、三乙醇胺、二甲基乙醇胺、2-二甲胺基乙醇、2-二乙胺基乙醇、二環己胺、賴胺酸、精胺酸、組胺酸、咖啡因、普魯卡因、膽鹼、甜菜鹼、乙二胺、葡萄糖胺、甲基葡萄糖胺、可可鹼、嘌呤、哌嗪、哌啶、N-乙基哌啶、聚胺樹脂等。較佳的有機鹼包括異丙胺、二乙胺、乙醇胺、三甲胺、二環己基胺、膽鹼及咖啡因。這些鹽可藉由本領域已知的方法製備。 "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that can maintain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salt, sodium salt, potassium salt, calcium salt and magnesium salt, with sodium salt being preferred. Salts derived from organic bases include, but are not limited to, the following salts: primary amines, secondary amines and tertiary amines, substituted amines, including natural substituted amines, cyclic amines and basic ions Exchange resins such as amines, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylamine Ethanol, dicyclohexylamine, lysine, arginine, histine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, Piperazine, piperidine, N-ethylpiperidine, polyamine resin, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the art.

“烷基”指飽和的脂族烴基團，例如包括1至30個碳原子的直鏈和支鏈基團(C₁-C₃₀烷基)，又例如含有1至6個碳原子的烷基(C₁-C₆烷基)，又例如1至3個碳原子的烷基(C₁-C₃烷基)。非限制性實例包括甲基、乙基、正丙基、異丙基、正丁基、異丁基、三級丁基、二級丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基及其各種支鏈異構體等。 "Alkyl" refers to a saturated aliphatic hydrocarbon group, such as linear and branched chain groups (C ₁ -C ₃₀ alkyl) of 1 to 30 carbon atoms, and alkyl groups of 1 to 6 carbon atoms. (C ₁ -C ₆ alkyl), another example is an alkyl group of 1 to 3 carbon atoms (C ₁ -C ₃ alkyl). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tertiary butyl, tertiary butyl, n-pentyl, 1,1-dimethylpropyl , 1,2-dimethylpropyl, 2,2-dimethylpropyl and various branched isomers, etc.

術語“烯基”是指含有至少一個雙鍵的烴基。烯基的非限制性實例包括但不限於：乙烯基、1-丙烯基、2-丙烯基、1-丁烯基或2-丁烯基及其各種支鏈異構體。 The term "alkenyl" refers to a hydrocarbon group containing at least one double bond. Non-limiting examples of alkenyl groups include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl or 2-butenyl and various branched isomers thereof.

術語“炔基”指含有至少一個三鍵的烴基。炔基的非限制性實例包括但不限於：乙炔基、1-丙炔基、2-丙炔基、1-丁炔基或2-丁炔基及其各種支鏈異構體。 The term "alkynyl" refers to a hydrocarbon group containing at least one triple bond. Non-limiting examples of alkynyl groups include, but are not limited to: ethynyl, 1-propynyl, 2-propynyl, 1-butynyl or 2-butynyl and various branched isomers thereof.

術語“烷氧基”指-O-(烷基)，其中烷基的定義如上所述。烷氧基的非限制性實例包括：甲氧基、乙氧基、丙氧基、丁氧基。 The term "alkoxy" refers to -O-(alkyl), where alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy.

“環烷基”指飽和或部分不飽和單環或多環環狀烴取代基，環烷基環包含3至20個碳原子，較佳包含3至6個碳原子，更佳包含5-6個碳原子。單環環烷基的非限制性實例包括環丙基、環丁基、環戊基、環戊烯基、環己基、環己烯基、環己二烯基等；多環環烷基包括螺環、並環和橋環的環烷基。 "Cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent. The cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 6 carbon atoms, and more preferably 5-6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, etc.; polycyclic cycloalkyl groups include spiro Cycloalkyl groups of rings, bridged rings and bridged rings.

“雜環烷基”指飽和或部分不飽和單環或多環環狀烴取代基，其包含3至20個環原子，其中一個或多個環原子為選自氮、氧或S(O)_m(其中m是整數0至2)的雜原子，但不包括-O-O-、-O-S-或-S-S-的環部分，其餘環原子為碳。較佳包含3至12個環原子，其中1至4個是雜原子；更佳包含3至7個環原子。“雜環烷基”非限制性實例包括： "Heterocycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O) _m (where m is an integer from 0 to 2), excluding the ring portion of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. Preferably it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably it contains 3 to 7 ring atoms. Non-limiting examples of "heterocycloalkyl" include:

和

，等等。

and

,etc.

該雜環烷基環可以稠合於芳基或雜芳基環上，其中與母體結構連接在一起的環為雜環烷基，其非限制性實例包括： The heterocycloalkyl ring can be fused to an aryl or heteroaryl ring, where the ring connected to the parent structure is a heterocycloalkyl group. Non-limiting examples include:

和

等。

and

wait.

“芳基”指具有共軛的π電子體系的6至14員全碳單環或稠合多環(也就是共享毗鄰碳原子對的環)基團，較佳為6至12員，例如苯基和萘基。該芳基環可以稠合於雜芳基、雜環烷基或環烷基環上，其中與母體結構連接在一起的環為芳基環，其非限制性實例包括： "Aryl" refers to a 6 to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6 to 12 members, such as benzene base and naphthyl. The aryl ring can be fused to a heteroaryl, heterocycloalkyl or cycloalkyl ring, where the ring attached to the parent structure is an aryl ring, non-limiting examples of which include:

“雜芳基”指包含1至4個雜原子、5至14個環原子的雜芳族體系，其中雜原子選自氧、硫和氮。雜芳基較佳為6至12員，更佳為5員或6員。例如。其非限制性實例包括：咪唑基、呋喃基、噻吩基、噻唑基、吡唑基、噁唑基(oxazolyl)、異噁唑基(isoxazolyl)、吡咯基、四唑基、吡啶基、嘧啶基、噻二唑、吡嗪基、三唑基、吲唑基、苯并咪唑基、

，

、

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

,

wait.

該雜芳基環可以稠合於芳基、雜環烷基或環烷基環上，其中與母體結構連接在一起的環為雜芳基環，其非限制性實例包括： The heteroaryl ring can be fused to an aryl, heterocycloalkyl or cycloalkyl ring, where the ring attached to the parent structure is a heteroaryl ring, non-limiting examples of which include:

術語“羥基”指-OH基團。 The term "hydroxy" refers to the -OH group.

術語“鹵素”指氟、氯、溴或碘。 The term "halogen" refers to fluorine, chlorine, bromine or iodine.

術語“氰基”指-CN。 The term "cyano" refers to -CN.

術語“胺基”指-NH₂。 The term "amine" refers to _-NH2 .

術語“硝基”指-NO₂。 The term "nitro" refers to _-NO2 .

術語“側氧”指=O取代基。 The term "pendant oxygen" refers to an =O substituent.

本揭露中，“磷酸酯基團”可為磷酸一酯基團、磷酸二酯基團或磷酸三酯基團，較佳磷酸二酯基團。 In the present disclosure, the "phosphate ester group" may be a phosphate monoester group, a phosphate diester group or a phosphate triester group, preferably a phosphate diester group.

本揭露中，硫代磷酸二酯基是指一個非橋接氧原子被硫原子替代而修飾的磷酸二酯基，可用

，

(M為S原子)互換使用。 In this disclosure, a phosphorothioate diester group refers to a phosphodiester group in which a non-bridging oxygen atom is replaced by a sulfur atom.

,

(M is S atom) are used interchangeably.

“取代”指基團中的一個或多個氫原子，較佳為最多5個，更佳為1至3個氫原子彼此獨立地被相應數目的取代基取代。當取代基是酮或側氧(即，=O)時，則原子上有兩個(2個)氫被替代。 "Substituted" means that one or more hydrogen atoms in a group, preferably up to 5, more preferably from 1 to 3 hydrogen atoms, are independently substituted with a corresponding number of substituents. When the substituent is a ketone or a pendant oxygen (ie, =O), then two (2) hydrogens on the atom are replaced.

本揭露上下文中，基團

中的

部分可以替換為能夠與相鄰核苷酸實現連接的任意基團。 In the context of this disclosure, the group

middle

The moiety may be replaced by any group capable of linking to adjacent nucleotides.

術語“連接”，當表示兩個分子之間的聯繫時，指兩個分子藉由共價鍵連接或者兩個分子經由非共價鍵(例如，氫鍵或離子鍵)關聯，包括直接連接、間接連接。 The term "connection", when referring to the connection between two molecules, means that the two molecules are connected by a covalent bond or that the two molecules are related by a non-covalent bond (for example, a hydrogen bond or an ionic bond), including direct connection, indirect connection.

術語“直接連接”指第一化合物或基團與第二化合物或基團在沒有任何間插原子或原子基團的情況下連接。 The term "directly linked" means that a first compound or group is linked to a second compound or group without any intervening atoms or groups of atoms.

術語“間接連接”指第一化合物或基團與第二化合物或基團藉由中間基團、化合物或分子(例如，連接基團)連接。 The term "indirectly linked" means that a first compound or group and a second compound or group are connected through an intervening group, compound or molecule (eg, a linking group).

“醫藥組成物”表示含有一種或多種本文所述化合物或其生理學上可藥用的鹽或前體藥物與其他化學組分的混合物，以及其他組分例如生理學可藥用的載體和賦形劑。醫藥組成物的目的是促進對生物體的給藥，利於活性成分的吸收進而發揮生物活性。 "Pharmaceutical composition" means a mixture containing one or more compounds described herein, or physiologically acceptable salts or prodrugs thereof, and other chemical components, together with other ingredients such as physiologically acceptable carriers and excipients. form agent. The purpose of pharmaceutical compositions is to facilitate administration to living organisms and facilitate the absorption of active ingredients to exert biological activity.

“藥學上可接受的賦形劑”包括但不限於任何已經被批准對於人類或家畜動物使用可接受的任何助劑、載體、助流劑、甜味劑、稀釋劑、防腐劑、染料/著色劑、增香劑、表面活性劑、潤濕劑、分散劑、助懸劑、穩定劑、等滲劑、溶劑或乳化劑。 "Pharmaceutically acceptable excipients" include, but are not limited to, any adjuvants, carriers, glidants, sweeteners, diluents, preservatives, dyes/colorants that have been approved for use by humans or livestock animals Agent, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier.

如本文所使用的，術語“抑制”，可以與“減少”、“沉默”、“下調”、“阻抑”、“阻斷”、“減弱”、“剔除”和其他類似術語交替使用，並且包括任何水平或活性的抑制。應當理解的是，dsRNA干擾或抑制靶標基因的表達或活性是指：相對於未經處理的對照而言，靶標基因的表達或活性被抑制(或等同術語)。可藉由變量與對照相比的絕對或相對水平的減少，來評估抑制。該對照水平可以是本領域中使用的任何類型的對照水平，例如給藥前基線水平或從類似的未經處理或經對照(例如僅緩衝液對照或惰性劑對照)處理的受試者、細胞、或樣品確定的水平。例如，可以採用mRNA剩餘表達量來表徵dsRNA對靶基因表達的抑制程度，如mRNA剩餘表達量為不高於99%、不高於95%、不高於90%、不高於85%、不高於80%、不高於75%、不高於70%、不高於65%、不高於60%、不高於55%、不高於50%、不高於45%、不高於40%、不高於35%、不高於30%、不高於25%、不高於20%、不高於15%、或不高於10%。靶基因表達的抑制率可以採用Dual-Glo® Luciferase Assay System檢測，分別讀取螢火蟲(Firefly)化學發光值和海腎(Renilla)化學發光值，計算相對值Ratio=Ren/Fir，抑制率(%)=1-(Ratio+dsRNA/僅報告基因)*100%；本揭露中，剩餘mRNA表達量比例(或剩餘活性%)=100%-抑制率(%)。 As used herein, the term "inhibition," may be used interchangeably with "reduce," "silence," "downregulate," "repress," "block," "attenuate," "knock out" and other similar terms, and Includes any level or inhibition of activity. It should be understood that interference or inhibition of the expression or activity of a target gene by dsRNA means that the expression or activity of the target gene is inhibited relative to an untreated control (or equivalent terms). Inhibition can be assessed by reduction in absolute or relative levels of a variable compared to a control. The control level may be any type of control level used in the art, such as a pre-dose baseline level or cells from a similar untreated or treated subject, such as a buffer only control or an inert control. , or the level determined by the sample. For example, the remaining expression level of mRNA can be used to characterize the degree of inhibition of target gene expression by dsRNA. For example, the remaining expression level of mRNA 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%. The inhibition rate of target gene expression can be detected using the Dual-Glo® Luciferase Assay System. The firefly (Firefly) chemiluminescence value and the Renilla (Renilla) chemiluminescence value are respectively read, and the relative value Ratio=Ren/Fir is calculated. The inhibition rate (% )=1-(Ratio+dsRNA/reporter gene only)*100%; in this disclosure, the remaining mRNA expression ratio (or remaining activity %)=100%-inhibition rate (%).

“有效量”或“有效劑量”包含足以改善或預防醫學病症的症狀或病症的量。有效量還意指足以允許或促進診斷的量。用於特定患者或獸醫學受試者的有效量可依據以下因素而變化：如待治療的病症、受試者的總體健康情況、給藥的方法途徑和劑量以及副作用嚴重性。有效量可以是避免顯著副作用或毒性作用的最大劑量或給藥方案。 An "effective amount" or "effective dosage" includes an amount sufficient to ameliorate or prevent symptoms or symptoms of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the general health of the subject, the method, route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dosage or dosage regimen that avoids significant side effects or toxic effects.

如本文所使用的，“對象”、“患者”、“受試者”或“個體”可互換使用，包括人類或者非人類動物，例如哺乳動物，例如人或猴。 As used herein, "subject," "patient," "subject" or "individual" are used interchangeably and include humans or non-human animals, such as mammals, such as humans or monkeys.

如本文所使用的，有義鏈(又稱SS、SS鏈或正義鏈)是指包含與靶mRNA序列相同或基本上相同的序列的鏈；反義鏈(又稱AS或AS鏈)是指具有與靶mRNA序列互補或部分互補的序列的鏈。 As used herein, the sense strand (also known as SS, SS strand or sense strand) refers to the strand that contains the same or substantially the same sequence as the target mRNA sequence; the antisense strand (also known as AS or AS strand) refers to A strand having a sequence that is complementary or partially complementary to the target mRNA sequence.

在描述本文所述的有義鏈的上下文中，術語“SEQ ID NO：1的核苷酸序列相差不超過3個核苷酸序列，且包含至少15個連續核苷酸”旨在表示本文所述的有義鏈包含SEQ ID NO：1的至少15個連續核苷酸，或與SEQ ID NO：1的至少15個連續核苷酸相差不超過3個核苷酸序列，視需要地，相差不超過2個核苷酸序列，視需要地，相差1個核苷酸序列。視需要地，本文所述的有義鏈包含SEQ ID NO：1的至少16個連續核苷酸，或與SEQ ID NO：1的至少16個連續核苷酸相差不超過3個核苷酸序列，視需要地，相差不超過2個核苷酸序列，視需要地，相差1個核苷酸序列； In the context of describing the sense strand as described herein, the term "the nucleotide sequences of SEQ ID NO: 1 differ by no more than 3 nucleotide sequences and comprise at least 15 contiguous nucleotides" is intended to mean that the nucleotide sequences of SEQ ID NO:1 are Said sense strand contains at least 15 consecutive nucleotides of SEQ ID NO: 1, or differs from at least 15 consecutive nucleotides of SEQ ID NO: 1 by no more than 3 nucleotide sequences, as appropriate. No more than 2 nucleotide sequences differ, and optionally 1 nucleotide sequence. Optionally, the sense strand described herein comprises at least 16 contiguous nucleotides of SEQ ID NO: 1, or differs from at least 16 contiguous nucleotides of SEQ ID NO: 1 by no more than 3 nucleotide sequences. , optionally, differing by no more than 2 nucleotide sequences, optionally differing by 1 nucleotide sequence;

在描述本文所述的反義鏈的上下文中，術語“與SEQ ID NO：2相差不超過3個核苷酸序列，且包含至少15個連續核苷酸”旨在表示本文所述的SEQ ID NO：2的至少15個連續核苷酸，或與SEQ ID NO：2的至少15個連續核苷酸相差不超過3個核苷酸序列，視需要地，相差不超過2個核苷酸序列，視需要地，相差1個核苷酸序列。 In the context of describing the antisense strand described herein, the term "different from SEQ ID NO:2 by no more than 3 nucleotide sequences and containing at least 15 contiguous nucleotides" is intended to mean SEQ ID NO:2 described herein At least 15 consecutive nucleotides of NO:2, or no more than 3 nucleotide sequences, and optionally no more than 2 nucleotide sequences different from at least 15 consecutive nucleotides of SEQ ID NO:2 , optionally differing by 1 nucleotide sequence.

如無特別說明，在本揭露上下文中，“G”、“C”、“A”、“T”與“U”分別代表核苷酸，其分別包含鳥嘌呤、胞嘧啶、腺嘌呤、胸苷與尿嘧啶的鹼基。小寫字母m表示該字母m上游相鄰的一個核苷酸為甲氧基修飾的核苷酸；小寫字母f表示該字母f上游相鄰的一個核苷酸為氟修飾的核苷酸；小寫字母s表示與該字母s左右相鄰的兩個核苷酸之間為硫代磷酸二酯基連接。 Unless otherwise specified, in the context of this disclosure, "G", "C", "A", "T" and "U" respectively represent nucleotides, which respectively include guanine, cytosine, adenine and thymidine. with the base of uracil. The lowercase letter m indicates that the nucleotide adjacent to the upstream of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the upstream of the letter f is a fluorine-modified nucleotide; the lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate diester groups.

如本揭露所使用的，術語“2'-氟(2’-F)修飾的核苷酸”指核苷酸的核糖基2'位的羥基被氟取代形成的核苷酸。 As used in this disclosure, the term "2'-fluoro (2'-F) modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by fluorine.

如本揭露所使用的，術語“2'-甲氧基(2’-OMe)修飾的核苷酸”指核糖基的2'-羥基被甲氧基取代而形成的核苷酸。 As used in this disclosure, the term "2'-methoxy (2'-OMe) modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group.

在本揭露的上下文中，一個核苷酸序列與另外一個核苷酸序列存在“核苷酸差異”，是指前者與後者相比，相同位置的核苷酸的鹼基種類發生了改變，例如，在後者中一個核苷酸鹼基為A時，在前者的相同位置處的對應核苷酸鹼基為U、C、G或者T的情況下，認定為兩個核苷酸序列之間在該位置處存在核苷酸差異。在一些實施方案中，以無鹼基核苷酸或其等同物代替原位置的核苷酸時，也可認為在該位置處產生了核苷酸差異。 In the context of this disclosure, a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence refers to a change in the base type of the nucleotide at the same position in the former compared to the latter, e.g. , when one nucleotide base in the latter is A, and when the corresponding nucleotide base at the same position in the former is U, C, G or T, it is deemed that there is a gap between the two nucleotide sequences. There is a nucleotide difference at this position. In some embodiments, when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, a nucleotide difference at that position may also be considered to have occurred.

如本文所使用的，術語“互補”或“反向互補”一詞可互相替代使用，並具有所屬技術領域具有通常知識者周知的含義，即，在雙鏈核酸分子中，一條鏈的鹼基與另一條鏈上的鹼基以互補的方式相配對。在DNA中，嘌呤鹼基腺嘌呤始終與嘧啶鹼基胸腺嘧啶(或者在RNA中為尿嘧啶)相配對；嘌呤鹼基鳥嘌呤始終與嘧啶鹼基胞嘧啶相配對。每個鹼基對都包括一個嘌呤和一個嘧啶。當一條鏈上的腺嘌呤始終與另一條鏈上的胸腺嘧啶(或尿嘧啶)配對，以及鳥嘌呤始終與胞嘧啶配對時，兩條鏈被認為是彼此相互補的，以及從其互補鏈的序列中可以推斷出該鏈的序列。與此相應地，“錯配”在本領域中意指在雙鏈核酸中，對應位置上的鹼基並未以互補的形式配對存在。 As used herein, the terms "complementary" or "reverse complement" are used interchangeably and have the meaning well known to those of ordinary skill in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand Pairs complementary to bases on the other strand. In DNA, the purine base adenine always pairs with the pyrimidine base thymine (or uracil in RNA); the purine base guanine always pairs with the pyrimidine base cytosine. Each base pair consists of a purine and a pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are said to be complementary to each other, and from their complementary strands The sequence of the chain can be inferred from the sequence. Correspondingly, "mismatch" in this field means that in double-stranded nucleic acids, the bases at corresponding positions do not pair in a complementary manner.

術語“dsRNA”是指能夠進行RNA干擾的雙鏈RNA分子，包含有義鏈和反義鏈。雙鏈核糖核酸(dsRNA)是指由兩條多核苷酸鏈藉由完全互補或部分互補而形成的RNA分子。在干擾領域中，dsRNA能夠調節(本揭露中尤其用於干擾、抑制、沉默、或失活)靶標基因(或其表達產物)的表達或活性。 The term "dsRNA" refers to a double-stranded RNA molecule capable of performing RNA interference, including a sense strand and an antisense strand. Double-stranded ribonucleic acid (dsRNA) refers to two polynucleotide strands that are completely complementary or RNA molecules formed by partial complementarity. In the field of interference, dsRNA is capable of modulating (especially used in this disclosure to interfere, inhibit, silence, or inactivate) the expression or activity of a target gene (or its expression product).

術語“化學修飾”或“修飾”包括核苷酸經化學手段的所有改變，例如化學部分的添加或去除、或以一個化學部分取代另一個化學部分。 The term "chemical modification" or "modification" includes all changes in a nucleotide by chemical means, such as the addition or removal of chemical moieties, or the substitution of one chemical moiety for another.

術語“鹼基”包含任何已知的DNA和RNA鹼基、鹼基類似物，例如嘌呤或嘧啶，其還包括天然化合物腺嘌呤、胸腺嘧啶、鳥嘌呤、胞嘧啶、尿嘧啶、次黃苷和天然類似物。鹼基類似物還可以是通用鹼基。 The term "base" includes any known DNA and RNA base, base analogues, such as purine or pyrimidine, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and Natural analogues. Base analogs can also be universal bases.

術語“平端”或“平末端”可互換使用，是指在dsRNA的給定的末端沒有非配對的核苷酸或核苷酸類似物，即，沒有核苷酸突出。大多數情況下，兩個末端都是平末端的dsRNA將在其整個長度範圍內呈現雙鏈結構。 The terms "blunt end" or "blunt end" are used interchangeably and mean that there are no unpaired nucleotides or nucleotide analogs at a given end of a dsRNA, ie, no nucleotide overhangs. In most cases, a dsRNA with both ends blunted will exhibit a double-stranded structure throughout its length.

本揭露提供的dsRNA可以藉由本領域常規的製備方法(例如固相合成和液相合成的方法)得到。其中，固相合成已經有商業化訂製服務。可以藉由使用具有相應修飾的核苷單體來將修飾的核苷酸基團引入本揭露所述的dsRNA中，製備具有相應修飾的核苷單體的方法及將修飾的核苷酸基團引入dsRNA的方法也是所屬技術領域具有通常知識者所熟知的。 The dsRNA provided by the present disclosure can be obtained by conventional preparation methods in the art (such as solid phase synthesis and liquid phase synthesis methods). Among them, solid-phase synthesis already has commercial customization services. Modified nucleotide groups can be introduced into the dsRNA described in the present disclosure by using nucleoside monomers with corresponding modifications, methods of preparing nucleoside monomers with corresponding modifications, and modifying nucleotide groups. Methods for introducing dsRNA are also well known to those of ordinary skill in the art.

圖1為TRD002218和TRD007205在給藥後第7天TTR中mRNA的剩餘表達量。 Figure 1 shows the remaining expression of mRNA in the TTR of TRD002218 and TRD007205 on day 7 after administration.

圖2為TRD002218和TRD007205在給藥後第28天TTR中mRNA的剩餘表達量。 Figure 2 shows the remaining expression of mRNA in the TTR of TRD002218 and TRD007205 on day 28 after administration.

以下結合實施例進一步描述本揭露，但這些實施例並非限制著本揭露的範圍。本揭露實施例中未註明具體條件的實驗方法，通常按照常規條件或按照原料或商品製造廠商所建議的條件。未註明具體來源的試劑，則該試劑可自任意分子生物學試劑的供應商以用於分子生物學應用的質量/純度而獲得。 The present disclosure is further described below with reference to examples, but these examples do not limit the scope of the present disclosure. Experimental methods without specifying specific conditions in the examples of this disclosure usually follow conventional conditions or conditions recommended by raw material or product manufacturers. If a specific source of reagent is not stated, the reagent can be obtained from any supplier of molecular biology reagents in a quality/purity suitable for molecular biology applications.

實施例1：化學修飾的製備Example 1: Preparation of chemical modifications

1.1 合成化合物1-1a和化合物1-1b1.1 Synthesis of compound 1-1a and compound 1-1b

將化合物1(500mg,3.42mmol)和三乙胺(Et₃N，692mg,6.84mmol,0.95mL)溶於二氯甲烷(DCM，10mL)中，冰浴下滴加4-甲苯磺醯氯(TsCl,717mg,3.76mmol)的二氯甲烷(10mL)溶液，滴加完畢後反應在室溫下攪拌過夜，待反應完畢後，用水淬滅，水相用二氯甲烷(15mL)提取三次，合併的有機相先用飽和碳酸氫鈉水溶液(10mL)洗滌，再用飽和食鹽水(20mL)洗滌，隨後減壓蒸乾溶劑得到粗品2(820mg,80%)，直接用於下一步反應。MS m/z：C₁₄H₂₁O₅S，[M+H]⁺理論：301.10實測：301.2。 Compound 1 (500 mg, 3.42 mmol) and triethylamine (Et ₃ N, 692 mg, 6.84 mmol, 0.95 mL) were dissolved in dichloromethane (DCM, 10 mL), and 4-toluenesulfonyl chloride ( TsCl, 717 mg, 3.76 mmol) in dichloromethane (10 mL). After the dropwise addition, the reaction was stirred at room temperature overnight. After the reaction was completed, quenched with water, the aqueous phase was extracted three times with dichloromethane (15 mL), and combined The organic phase was first washed with saturated aqueous sodium bicarbonate solution (10 mL) and then with saturated brine (20 mL), and then the solvent was evaporated to dryness under reduced pressure to obtain crude product 2 (820 mg, 80%), which was directly used in the next reaction. MS m/z: C ₁₄ H ₂₁ O ₅ S, [M+H] ⁺ Theory: 301.10 Measured: 301.2.

將化合物3(239mg,1.22mmol)溶解於二甲基甲醯胺(DMF，10mL)中，冰浴下加入NaH(60%溶解在礦物油中，93mg,2.33mmol)溶液，該反應下攪拌30分鐘，然後滴加化合物2(350mg,1.16mmol)，滴加完畢後反應在60℃下攪拌5小時，反應完畢後，加水淬滅，水相用乙酸乙酯(15mL)提取三次，合併的有機相先用水(10mL)洗滌三次，再用飽和食鹽水(10mL)洗滌，隨後減壓蒸乾溶劑，經反相製備HPLC(C¹⁸，條件：5-50%(A：H₂O,B：CH₃CN)，流速：70mL/min)，凍乾後得到220mg化合物4。MS m/z：C₁₉H₂₁N₅O₃Na，[M+Na]⁺理論：390.16，實測：390.3。 Compound 3 (239 mg, 1.22 mmol) was dissolved in dimethylformamide (DMF, 10 mL), NaH (60% dissolved in mineral oil, 93 mg, 2.33 mmol) solution was added under ice bath, and the reaction was stirred for 30 minutes, then compound 2 (350 mg, 1.16 mmol) was added dropwise. After the addition was completed, the reaction was stirred at 60°C for 5 hours. After the reaction was completed, water was added to quench the water phase. The aqueous phase was extracted three times with ethyl acetate (15 mL). The combined organic The phase was first washed three times with water (10 mL) and then with saturated brine (10 mL). The solvent was then evaporated to dryness under reduced pressure and subjected to reversed-phase preparative HPLC (C ¹⁸ , conditions: 5-50% (A: H ₂ O, B: CH ₃ CN), flow rate: 70 mL/min), and 220 mg of compound 4 was obtained after lyophilization. MS m/z: C ₁₉ H ₂₁ N ₅ O ₃ Na, [M+Na] ⁺ theory: 390.16, measured: 390.3.

室溫下將化合物4(1.50g,4.08mmol)溶解於20mL的醋酸和水(4：1)的混合溶液中，60℃下攪拌30分鐘，待反應完畢後減壓蒸乾溶劑，經反相製備HPLC(C¹⁸，條件：5-25%(A：H₂O,B：CH₃CN)，流速：70mL/min)，凍乾後得到1.10g化合物5。MSm/z：C₁₆H₁₈N₅O₃，[M+H]⁺理論：328.13，實測：328.4。 Compound 4 (1.50g, 4.08mmol) was dissolved in 20 mL of a mixed solution of acetic acid and water (4:1) at room temperature, and stirred at 60°C for 30 minutes. After the reaction was completed, the solvent was evaporated to dryness under reduced pressure, and the solution was reversed phase. Preparative HPLC (C ¹⁸ , conditions: 5-25% (A: H ₂ O, B: CH ₃ CN), flow rate: 70 mL/min), and 1.10 g of compound 5 was obtained after lyophilization. MSm/z: C ₁₆ H ₁₈ N ₅ O ₃ , [M+H] ⁺ theory: 328.13, measured: 328.4.

將化合物5(1.00g,3.05mmol)溶於吡啶(Py，10mL)中，冰浴下滴4,4'-雙甲氧基三苯甲基氯(DMTrCl,1.50g,4.58mmol)的吡啶(5mL)溶液，滴加完畢後反應在室溫下攪拌過夜，待反應完畢後，用水淬滅，減壓蒸乾溶劑，經反相製備HPLC(C¹⁸，條件：5-80%(A：H₂O,B：CH₃CN)，流速：70mL/min)，凍乾後得到1.00g化合物6。MS m/z：C₃₇H₃₆N₅O₅，[M-H]⁺理論：630.26，實測：630.5。消旋體化合物6經手性管柱(Daicel CHIRALPAK® IE 250*4.6mm,5μm,A：正己烷，B：乙醇)拆分得410mg 6A(-)和435mg 6B(+)。 Compound 5 (1.00g, 3.05mmol) was dissolved in pyridine (Py, 10mL), and 4,4'-bismethoxytrityl chloride (DMTrCl, 1.50g, 4.58mmol) was added dropwise in an ice bath. 5mL) solution, after the dropwise addition is completed, the reaction is stirred at room temperature overnight. After the reaction is completed, quench with water, evaporate the solvent to dryness under reduced pressure, and perform reverse-phase preparative HPLC (C ¹⁸ , conditions: 5-80% (A: H ₂ O, B: CH ₃ CN), flow rate: 70 mL/min), and 1.00 g of compound 6 was obtained after lyophilization. MS m/z: C ₃₇ H ₃₆ N ₅ O ₅ , [MH] ⁺ theory: 630.26, measured: 630.5. The racemic compound 6 was separated by a chiral column (Daicel CHIRALPAK® IE 250*4.6mm, 5μm, A: n-hexane, B: ethanol) to obtain 410mg 6A(-) and 435mg 6B(+).

將化合物6A(-)(200mg,0.32mmol)，四唑(11mg,0.16mmol)，N-甲基咪唑(5mg,0.06mmol)，3A分子篩(500mg)溶於10mL的乙腈中，室溫下加入化合物7(144mg,0.48mmol)，在室溫下攪拌過夜。反應完畢後，將分子篩過濾掉，加入二氯甲烷(30mL)，飽和碳酸氫鈉水溶液(10mL)洗滌三次，再用飽和食鹽水(20mL)洗滌，濾液旋乾並經反相製備HPLC(C¹⁸，條件：5-100%(A：水，B：CH₃CN)，流速：70mL/min)，凍乾後得到200mg化合物1-1a。MS m/z：C₄₀H₃₉N₆O₇P，[M-二異丙基+OH]⁺理論：747.26，實測：747.6。1H NMR(400MHz,乙腈-d ₃)δ 7.56,7.54(2s,1H),7.36-7.27(m,2H),7.24-7.21(m,7H),6.83-6.80(m,4H),4.12-4.10(m,2H),3.75-3.68(m,10H),3.20-2.80(m,2H),2.68-2.54(m,4H),1.22-1.04(m,18H)。 Dissolve compound 6A(-) (200mg, 0.32mmol), tetrazole (11mg, 0.16mmol), N-methylimidazole (5mg, 0.06mmol), 3A molecular sieve (500mg) in 10mL of acetonitrile, and add at room temperature Compound 7 (144 mg, 0.48 mmol), stirred at room temperature overnight. After the reaction is completed, filter the molecular sieve, add dichloromethane (30 mL), wash three times with saturated aqueous sodium bicarbonate solution (10 mL), and then wash with saturated brine (20 mL). The filtrate is spin-dried and subjected to reversed-phase preparative HPLC (C ¹⁸ , conditions: 5-100% (A: water, B: CH ₃ CN), flow rate: 70 mL/min), and 200 mg of compound 1-1a was obtained after freeze-drying. MS m/z: C ₄₀ H ₃₉ N ₆ O ₇ P, [M-diisopropyl+OH] ⁺ theory: 747.26, measured: 747.6. 1H NMR (400MHz, acetonitrile- d ₃ ) δ 7.56, 7.54 (2s ,1H),7.36-7.27(m,2H),7.24-7.21(m,7H),6.83-6.80(m,4H),4.12-4.10(m,2H),3.75-3.68(m,10H),3.20 -2.80(m,2H),2.68-2.54(m,4H),1.22-1.04(m,18H).

將化合物6B(+)(200mg,0.32mmol)，四唑(11mg,0.16mmol)，N-甲基咪唑(5mg,0.06mmol)，3A分子篩(500mg)溶於10mL的乙腈中，室溫下加入化合物7(144mg,0.48mmol)，在室溫下攪拌過夜。反應完畢後，將分子篩過濾掉，加入二氯甲烷(30mL)，飽和碳酸氫鈉水溶液(10mL)洗滌三次，再用飽和食鹽水(20mL)洗滌，濾液旋乾並經反相製備HPLC(C¹⁸，條件：5-100%(A：水，B：CH₃CN)，流速：70mL/min)，凍乾後得到200mg化合物1-1b。MS m/z：C₄₀H₃₉N6O₇P，[M-二異丙基+OH]⁺理論：747.26，實測：747.5。 Dissolve compound 6B ( + ) (200 mg, 0.32 mmol), tetrazole (11 mg, 0.16 mmol), N-methylimidazole (5 mg, 0.06 mmol), and 3A molecular sieve (500 mg) in 10 mL of acetonitrile, and add at room temperature Compound 7 (144 mg, 0.48 mmol), stirred at room temperature overnight. After the reaction is completed, filter the molecular sieve, add dichloromethane (30 mL), wash three times with saturated aqueous sodium bicarbonate solution (10 mL), and then wash with saturated brine (20 mL). The filtrate is spin-dried and subjected to reversed-phase preparative HPLC (C ¹⁸ , conditions: 5-100% (A: water, B: CH ₃ CN), flow rate: 70 mL/min), and 200 mg of compound 1-1b was obtained after lyophilization. MS m/z: C ₄₀ H ₃₉ N6O ₇ P, [M-diisopropyl+OH] ⁺ theory: 747.26, measured: 747.5.

1.2 合成化合物1-6a1.2 Synthesis of compound 1-6a

將化合物1(10g,68.404mmol)、化合物2(15g,62.186mmol)和三苯基膦(32.62g,124.371mmol)溶於無水THF(30mL)，於0℃下緩慢滴加DIAD(24.656mL，124.371mmol)。該反應液在25℃下反應12h。LCMS顯示反應完成。將該反應液用乙酸乙酯(200mL)和水(200mL)萃取，有機相乾燥將濾液濃縮，得到的殘留物用正相管柱純化(DCM/MeOH=10/1)得目標產物3(20g)。 Compound 1 (10g, 68.404mmol), compound 2 (15g, 62.186mmol) and triphenylphosphine (32.62g, 124.371mmol) were dissolved in anhydrous THF (30mL), and DIAD (24.656mL) was slowly added dropwise at 0°C. 124.371mmol). The reaction solution was reacted at 25°C for 12 hours. LCMS showed the reaction was complete. The reaction solution was extracted with ethyl acetate (200 mL) and water (200 mL). The organic phase was dried and the filtrate was concentrated. The obtained residue was purified with a normal phase column (DCM/MeOH=10/1) to obtain the target product 3 (20 g ).

將化合物3(20g,28.585mmol)溶於醋酸(24mL,426.016mmol)和H₂O(12mL)中，60℃攪拌1小時。之後將反應液旋乾加入THF(12mL)和H₂O(12mL)，80℃攪拌7小時。LCMS顯示反應完成。將反應液加入乙酸乙酯(200mL)和水(100mL)萃取，水相加入碳酸鈉固體直到水相有大量固體析出。將固體過濾，用水洗滌，將濾餅用油泵拉乾，得到目標化合物5(9g)。 Compound 3 (20g, 28.585mmol) was dissolved in acetic acid (24mL, 426.016mmol) and H ₂ O (12mL), and stirred at 60°C for 1 hour. Afterwards, the reaction solution was spun to dryness, THF (12 mL) and H ₂ O (12 mL) were added, and the mixture was stirred at 80° C. for 7 hours. LCMS showed the reaction was complete. The reaction solution was added to ethyl acetate (200 mL) and water (100 mL) for extraction, and sodium carbonate solid was added to the water phase until a large amount of solid precipitated out of the water phase. The solid was filtered, washed with water, and the filter cake was dried with an oil pump to obtain target compound 5 (9g).

在氮氣保護下，將化合物5(6.8g,18.581mmol)溶於吡啶(80mL)中，於0℃下緩慢加入TMSCl(14.250mL,111.489mmol)，攪拌2h。之後在0℃下加入異丁醯氯(2.044mL,19.511mmol)，於25℃下攪拌1h。LCMS顯示反應完成。用二氯甲烷(200mL)和水(200mL)萃取，有機相乾燥旋乾後拌樣，用正相管柱純化(DCM：MeOH=10：1)過管柱，在4.8%處出峰)，得到黃色油狀化合物6(12g). Under nitrogen protection, compound 5 (6.8g, 18.581mmol) was dissolved in pyridine (80mL), TMSCl (14.250mL, 111.489mmol) was slowly added at 0°C, and stirred for 2 hours. Then, isobutyl chloride (2.044 mL, 19.511 mmol) was added at 0°C, and stirred at 25°C for 1 hour. LCMS showed the reaction was complete. Extract with dichloromethane (200mL) and water (200mL), dry the organic phase and spin it to dryness, mix the sample, and purify with a normal phase column (DCM: MeOH=10:1) passes through the column and peaks at 4.8%), Compound 6 (12g) was obtained as a yellow oil.

在氮氣保護下，將化合物6(5.5g,12.392mmol)溶於吡啶(30mL)，加入MOLECULAR SIEVE 4A 1/16(7g,12.392mmol)，然後在0℃下分批加入DMTrCl(5.04g,14.870mmol)固體，25℃反應2h。TLC(PE：EtOAc=1：1,Rf= 0.69)顯示反應已經完成。該反應液和TJN200879-040-P1合併一起處理。將反應液用乙酸乙酯(200mL)和水(200mL)萃取，有機相乾燥旋乾後拌樣用正相管柱純化(PE：EtOAc過管柱，在84%處出峰)，得到黃色油狀化合物7(12g)。 Under nitrogen protection, compound 6 (5.5g, 12.392mmol) was dissolved in pyridine (30mL), MOLECULAR SIEVE 4A 1/16 (7g, 12.392mmol) was added, and then DMTrCl (5.04g, 14.870) was added in batches at 0°C. mmol) solid, react at 25°C for 2 hours. TLC(PE:EtOAc=1:1,Rf= 0.69) indicates that the reaction is complete. The reaction solution and TJN200879-040-P1 were combined and processed together. The reaction solution was extracted with ethyl acetate (200 mL) and water (200 mL). The organic phase was dried and spin-dried, and the mixed sample was purified by a normal phase column (PE: EtOAc passed through the column and peaked at 84%) to obtain a yellow oil. Compound 7 (12g).

將化合物7(12g,15.389mmol)溶於EtOAc(140mL)，加入濕鈀碳Pd/C(7g,15.389mmol)該反應液在25℃，氫氣(15Psi)下反應2小時。TLC(PE：EtOAc=0：1,Rf=0.09)顯示反應已經完成。將反應液過濾，濾餅用乙酸乙酯*30mL)沖洗三遍後，收集濾液。濾液旋乾後加入50mL二氯甲烷和2mL三乙胺拌樣用正相管柱純化(DCM：MeOH=10：1過管柱，在0.5%處出峰)，得到9g(黃色泡沫狀固體)。將所得消旋化合物SFC拆分，得到產品目標化合物7A(-)(3.9g)和目標化合物7B(+)(3.8g)。 Compound 7 (12g, 15.389mmol) was dissolved in EtOAc (140mL), and wet palladium on carbon Pd/C (7g, 15.389mmol) was added. The reaction solution was reacted at 25°C under hydrogen gas (15Psi) for 2 hours. TLC (PE:EtOAc=0:1, Rf=0.09) showed that the reaction was complete. Filter the reaction solution, rinse the filter cake three times with ethyl acetate*30 mL), and collect the filtrate. After the filtrate was dried, 50 mL of dichloromethane and 2 mL of triethylamine were added to the mixed sample and purified by a normal phase column (DCM: MeOH = 10:1 passed through the column, peaking at 0.5%) to obtain 9g (yellow foamy solid) . The obtained racemic compound was separated by SFC to obtain target compound 7A(-) (3.9g) and target compound 7B(+) (3.8g).

將化合物7A(-)(3.30g,5.40mmol)，四唑(190mg,2.70mmol)，1-甲基咪唑(90mg,1.10mmol)，3A分子篩(500mg)溶於30mL的乙腈中，室溫下加入化合物8(2.50g,8.10mmol)，在室溫下攪拌2h。反應完畢後，將分子篩過濾掉，加入DCM(150mL)，飽和碳酸氫鈉水溶液洗滌(30mL*3)，再用飽和食鹽水(30mL)洗滌，濾液旋乾並經反相製備HPLC(C18，條件：5-100%(A：水，B：CH3CN)，流速：70mL/min)，凍乾後得到1-6a(2.9g,66%)。MS m/z：C43H55N7O7P[M+H]+，理論：812.38，實測：812.5。1H NMR(400MHz，乙腈-d3)δ 7.56,7.54(2s,1H),7.36-7.27(m,2H),7.24-7.21(m,7H),6.83-6.80(m,4H),4.12-4.10(m,2H),3.75-3.68(m,10H),3.20-2.80(m,2H),2.68-2.54(m,4H),1.22-1.04(m,18H)。 Dissolve compound 7A(-) (3.30g, 5.40mmol), tetrazole (190mg, 2.70mmol), 1-methylimidazole (90mg, 1.10mmol), and 3A molecular sieve (500mg) in 30 mL of acetonitrile at room temperature. Compound 8 (2.50g, 8.10mmol) was added and stirred at room temperature for 2h. After the reaction is completed, filter out the molecular sieve, add DCM (150mL), wash with saturated aqueous sodium bicarbonate solution (30mL*3), and then wash with saturated brine (30mL). The filtrate is spin-dried and subjected to reversed-phase preparative HPLC (C18, conditions : 5-100% (A: water, B: CH3CN), flow rate: 70mL/min), 1-6a (2.9g, 66%) was obtained after freeze-drying. MS m/z: C43H55N7O7P[M+H]+, theory: 812.38, measured: 812.5. 1H NMR (400MHz, acetonitrile-d3) δ 7.56, 7.54 (2s, 1H), 7.36-7.27 (m, 2H), 7.24 -7.21(m,7H),6.83-6.80(m,4H),4.12-4.10(m,2H),3.75-3.68(m,10H),3.20-2.80(m,2H),2.68-2.54(m, 4H),1.22-1.04(m,18H).

1.3 合成化合物1-7a1.3 Synthesis of compound 1-7a

在氮氣保護下，將化合物1(5g,23.1272mmol)、化合物2(6.76g,46.254mmol)和三苯基磷(7.28g,27.753mmol)溶於30mL二噁烷中，於0℃緩慢滴加入DEAD(5.502mL,27.753mmol)。滴加完成後，反應緩慢升溫至25℃繼續反應1h。在反應液裡加入100mL H₂O和100mL EtOAc萃取，有機相合併乾燥過濾濃縮後拌樣過管柱，用正相管柱純化(PE：EtOAc=1：1過管柱得目標產物(4g)。 Under nitrogen protection, compound 1 (5g, 23.1272mmol), compound 2 (6.76g, 46.254mmol) and triphenylphosphorus (7.28g, 27.753mmol) were dissolved in 30mL dioxane, and slowly added dropwise at 0°C. DEAD (5.502mL, 27.753mmol). After the dropwise addition was completed, the reaction temperature was slowly raised to 25°C and the reaction was continued for 1 h. Add 100 mL H ₂ O and 100 mL EtOAc to the reaction solution for extraction. The organic phases are combined, dried, filtered and concentrated, then mixed and passed through the column. Purify with a normal phase column (PE: EtOAc=1:1 and pass through the column to obtain the target product (4g). .

將化合物3(3.3g)溶於HOAc(16mL)和H₂O(4mL)，油浴60℃加熱0.5h。將反應液旋乾得到的殘留物用正相管柱純化(PE：EtOAc=0：1過管柱)，得到目標產物4(3g)。 Compound 3 (3.3g) was dissolved in HOAc (16mL) and H ₂ O (4mL), and heated in an oil bath at 60°C for 0.5h. The reaction solution was spin-dried and the residue obtained was purified by a normal phase column (PE:EtOAc=0:1 through the column) to obtain the target product 4 (3g).

將化合物4(3g,8.873mmol)溶於5mL吡啶中，在氮氣保護下於0℃緩慢滴加DMTrCl(3.91g,11.535mmol)的10mL吡啶的溶液。滴加完畢後反應升溫至25℃並繼續反應1h。在反應液中加入50mL水和100mL乙酸乙酯萃取。水相再用100mL乙酸乙酯萃取三次，有機相合併乾燥過濾濃縮用正相管柱純化(用PE：EtOAc=2：1)。得到目標產物5(4g). Compound 4 (3g, 8.873mmol) was dissolved in 5mL of pyridine, and a solution of DMTrCl (3.91g, 11.535mmol) in 10mL of pyridine was slowly added dropwise at 0°C under nitrogen protection. After the dropwise addition was completed, the reaction temperature was raised to 25°C and the reaction was continued for 1 h. Add 50 mL of water and 100 mL of ethyl acetate to the reaction solution and extract. The aqueous phase was extracted three times with 100 mL of ethyl acetate. The organic phases were combined, dried, filtered, concentrated and purified by a normal phase column (using PE:EtOAc=2:1). The target product 5 (4g) was obtained.

將化合物5(4g,5.769mmol)溶於甲醇(10mL)，加入飽和的NH3甲醇溶液(40mL)，0℃反應6h。將反應液旋乾用正相管柱純化(用PE：EtOAc=0：1)得消旋化合物2.4g SFC拆分，得到目標產物6A(750mg,100%純度)和目標產物6B(400mg,99.16%純度)。 Compound 5 (4g, 5.769mmol) was dissolved in methanol (10mL), saturated NH3 methanol solution (40mL) was added, and the reaction was carried out at 0°C for 6h. Spin the reaction solution to dryness and purify it with a normal phase column (using PE:EtOAc=0:1) to obtain 2.4g of the racemic compound. SFC separation yields the target product 6A (750mg, 100% purity) and the target product 6B (400mg, 99.16 % purity).

將化合物6A(-)(700mg,1.40mmol)，四唑(50mg,0.70mmol)，1-甲基咪唑(23mg,0.28mmol)，3A分子篩(500mg)溶於10mL的乙腈中，室溫下加入化合物7(630mg,2.10mmol)，在室溫下攪拌2h。反應完畢後，將分子篩過濾掉，加入DCM(50mL)，飽和碳酸氫鈉水溶液洗滌(10mL*3)，再用飽和食鹽水(20mL)洗滌，濾液旋乾並經反相製備HPLC(C18，條件：5-100%(A：水B：CH₃CN)，流速：70mL/min)，凍乾後得到1-7a(700mg,72%)。MS m/z：C38H47N4O7PNa[M+Na]+，理論：725.32，實測：725.5。 Dissolve compound 6A(-) (700mg, 1.40mmol), tetrazole (50mg, 0.70mmol), 1-methylimidazole (23mg, 0.28mmol), 3A molecular sieve (500mg) in 10mL of acetonitrile, and add at room temperature Compound 7 (630 mg, 2.10 mmol), stirred at room temperature for 2 h. After the reaction is completed, filter out the molecular sieve, add DCM (50mL), wash with saturated sodium bicarbonate aqueous solution (10mL*3), and then wash with saturated brine (20mL). The filtrate is spin-dried and subjected to reversed-phase preparative HPLC (C18, conditions : 5-100% (A: water B: CH ₃ CN), flow rate: 70mL/min), 1-7a (700mg, 72%) was obtained after lyophilization. MS m/z: C38H47N4O7PNa[M+Na]+, theory: 725.32, measured: 725.5.

1.4 合成化合物1-8a1.4 Synthesis of compound 1-8a

將化合物1(8.5g,76.508mmol)，化合物2(30.64g,91.809mmol)溶於DMF(150mL)，加入CS2CO3(29.91g,91.809mmol)，反應於氮氣保護下，90℃反應12h。LCMS檢測反應完成。將反應液過濾，油泵旋乾，正相管柱分離純化(80g,DCM/MeOH=10/1至5/1)得到目標產物3(13.5g,80%純度)。 Compound 1 (8.5g, 76.508mmol) and compound 2 (30.64g, 91.809mmol) were dissolved in DMF (150mL), CS2CO3 (29.91g, 91.809mmol) was added, and the reaction was carried out under nitrogen protection at 90°C for 12h. LCMS detects that the reaction is complete. The reaction solution was filtered, and the oil pump was rotated to dryness. The normal phase column was separated and purified (80g, DCM/MeOH=10/1 to 5/1) to obtain the target product 3 (13.5g, 80% purity).

將化合物3(10.5g,35.105mmol)溶於吡啶(65mL)和CH₃CN(65mL)，向溶液中滴加BzCl(4.894mL,42.126mmol)，於25℃反應2h。LCMS檢測大部分原料反應完成，加H₂O(100mL)淬滅，EtOAc(100mL X 3)萃取，乾燥旋乾，管柱分離(合併TJN200872-101)純化(80g,PE/EtOAc=10/1~0/1,DCM/MeOH=10/1)得到目標產物4(14g,90%純度)。 Compound 3 (10.5g, 35.105mmol) was dissolved in pyridine (65mL) and CH ₃ CN (65mL), BzCl (4.894mL, 42.126mmol) was added dropwise to the solution, and the reaction was carried out at 25°C for 2h. LCMS detects that most of the raw material reactions are completed, add H ₂ O (100 mL) to quench, extract with EtOAc (100 mL ~0/1, DCM/MeOH=10/1) to obtain the target product 4 (14g, 90% purity).

將化合物4(14g,36.694mmol)溶於HOAc(56mL,314.796mmol)和H₂O(14mL)，於60℃反應2h,LCMS顯示反應完成。油泵濃縮，正相管柱分離(40g,DCM/MeOH=1/0至5/1)得到目標產物5(8.4g,90%純度& 2.4g,80%純度)。 Compound 4 (14g, 36.694mmol) was dissolved in HOAc (56mL, 314.796mmol) and H ₂ O (14mL), and the reaction was carried out at 60°C for 2h. LCMS showed that the reaction was complete. Concentrate with oil pump and separate with normal phase column (40g, DCM/MeOH=1/0 to 5/1) to obtain the target product 5 (8.4g, 90% purity & 2.4g, 80% purity).

將化合物5(7.4g,21.957mmol)，DMAP(0.54g,4.391mmol)，MOLECULAR SIEVE 4A(11.1g,2.967mmol)溶於吡啶(60mL)，冰浴下攪拌10min，然後加入DMTrCl(8.93g,26.348mmol)，反應攪拌1.8h。LCMS檢測約19%原料剩餘，約60%目標MS。合併(TJN200872-105&106)一起純化。向反應液中加入H₂O(50mL)，經DCM(50mL×3)萃取，乾燥，旋乾，管柱分離(120g,PE/(EA：DCM：TEA=1：1：0.05)=1/0至0/1至DCM/MeOH=10/1)得到目標化合物6(11g,89%純度，TJN200872-105&106&107)，回收原料(3.0g,70%純度)。 Compound 5 (7.4g, 21.957mmol), DMAP (0.54g, 4.391mmol), MOLECULAR SIEVE 4A (11.1g, 2.967mmol) were dissolved in pyridine (60mL), stirred in an ice bath for 10min, and then added DMTrCl (8.93g, 26.348mmol), the reaction was stirred for 1.8h. LCMS detected about 19% raw material remaining and about 60% target MS. Combine (TJN200872-105&106) and purify together. Add H ₂ O (50 mL) to the reaction solution, extract with DCM (50 mL × 3), dry, spin dry, column separation (120g, PE/(EA:DCM:TEA=1:1:0.05)=1/ 0 to 0/1 to DCM/MeOH=10/1) to obtain the target compound 6 (11g, 89% purity, TJN200872-105&106&107), and the raw material (3.0g, 70% purity) was recovered.

化合物6(15g,22.041mmol)經SFC(DAICEL CHIRALPAK AD(250mm*50mm,10μm)；0.1% NH₃ H₂O EtOH，B：45%-45%；200ml/min)分離得到目標產物6A(5.33g,94.29%純度)，目標產物6B(6.14g,97.91%純度)，化合物6回收1.0g。 Compound 6 (15g, 22.041mmol) was separated by SFC (DAICEL CHIRALPAK AD (250mm*50mm, 10μm); 0.1% NH ₃ H ₂ O EtOH, B: 45%-45%; 200ml/min) to obtain the target product 6A (5.33 g, 94.29% purity), target product 6B (6.14g, 97.91% purity), and 1.0g of compound 6 was recovered.

將化合物6B(-)(5.4g,8.92mmol)，四氮唑(312mg,4.46mmol)，1-甲基咪唑(146mg,1.78mmol)，3A分子篩(500mg)溶於40mL的乙腈中，室溫下加入化合物7(4g,13.4mmol)，在室溫下攪拌2h。反應完畢後，將分子篩過濾掉，加入DCM(200mL)，飽和碳酸氫鈉水溶液洗滌(30mL*3)，再用飽和食鹽水(50mL)洗滌，濾液旋乾並經反相製備HPLC(C18，條件：5-100%(A：水，B：CH₃CN)，流速：70mL/min)，凍乾後得到1-8a(5.8g,80%)。MS m/z：C45H51N5O7P，[M+H]+，理論：804.36，實測：804.4。 Dissolve compound 6B(-) (5.4g, 8.92mmol), tetrazole (312mg, 4.46mmol), 1-methylimidazole (146mg, 1.78mmol), and 3A molecular sieve (500mg) in 40 mL of acetonitrile at room temperature. Compound 7 (4g, 13.4mmol) was added at room temperature and stirred for 2h at room temperature. After the reaction is completed, filter out the molecular sieve, add DCM (200mL), wash with saturated sodium bicarbonate aqueous solution (30mL*3), and then wash with saturated brine (50mL). The filtrate is spin-dried and subjected to reversed-phase preparative HPLC (C18, conditions : 5-100% (A: water, B: CH ₃ CN), flow rate: 70mL/min), 1-8a (5.8g, 80%) was obtained after freeze-drying. MS m/z: C45H51N5O7P, [M+H]+, theory: 804.36, measured: 804.4.

實施例2：不同化學修飾表徵Example 2: Characterization of different chemical modifications

其中，我們將由2-羥甲基-1,3-丙二醇為起始原料合成的核苷酸定義hmpNA； Among them, we define hmpNA from nucleotides synthesized from 2-hydroxymethyl-1,3-propanediol as the starting material;

(+)hmpNA(A)為實施例1.1節中核苷亞磷醯胺單體1-1b藉由固相合成獲得，絕對構型為(S)-hmpNA(A)； (+)hmpNA(A) is obtained by solid-phase synthesis of nucleoside phosphoramidite monomer 1-1b in Section 1.1 of Example, and its absolute configuration is ( S )-hmpNA(A);

(-)hmpNA(A)為實施例1.1節中核苷亞磷醯胺單體1-1a藉由固相合成獲得，絕對構型為(R)-hmpNA(A)； (-)hmpNA(A) is obtained by solid-phase synthesis of nucleoside phosphoramidite monomer 1-1a in Section 1.1 of Example, and its absolute configuration is ( R )-hmpNA(A);

類似的，替換hmpNA的鹼基種類，藉由固相合成獲得以下結構並確認絕對構型： Similarly, by replacing the base species of hmpNA, the following structure was obtained by solid-phase synthesis and the absolute configuration was confirmed:

(+)hmpNA(G)，絕對構型為(S)-hmpNA(G)； (+)hmpNA(G), the absolute configuration is ( S )-hmpNA(G);

(-)hmpNA(G)，絕對構型為(R)-hmpNA(G)； (-)hmpNA(G), the absolute configuration is ( R )-hmpNA(G);

(+)hmpNA(C)，絕對構型為(S)-hmpNA(C)； (+)hmpNA(C), the absolute configuration is ( S )-hmpNA(C);

(-)hmpNA(C)，絕對構型為(R)-hmpNA(C)； (-)hmpNA(C), the absolute configuration is ( R )-hmpNA(C);

(+)hmpNA(U)，絕對構型為(R)-hmpNA(U)； (+)hmpNA(U), the absolute configuration is ( R )-hmpNA(U);

(-)hmpNA(U)，絕對構型為(S)-hmpNA(U)。 (-)hmpNA(U), the absolute configuration is ( S )-hmpNA(U).

(S)-hmpNA(G)、(R)-hmpNA(G)、(S)-hmpNA(C)、(R)-hmpNA(C)、(S)-hmpNA(U)和(R)-hmpNA(U)的絕對構型由其中間體或衍生物經X-Ray衍射而確認。 ( S )-hmpNA(G), ( R )-hmpNA(G), ( S )-hmpNA(C), ( R )-hmpNA(C), ( S )-hmpNA(U), and ( R )-hmpNA The absolute configuration of (U) is confirmed by X-Ray diffraction of its intermediates or derivatives.

中間體或衍生物的結構為： The structure of the intermediate or derivative is:

TJ-NA067：檢測晶體為無色塊狀(0.30×0.10×0.04mm3)，屬單斜晶系P21空間群。晶胞參數a=16.0496(5)Å，b=4.86260(10)Å，c=16.4686(5)Å，α=90°，β=118.015(4)°，γ=90°，V=1134.65(7)Å3，Z=4。計算密度Dc=1.389g/cm3，單胞中電子數F(000)=504.0，單胞的線性吸收係數μ(Cu Kα)=0.840mm-1，衍射實驗溫度T=150.00(11)K。 TJ-NA067: The detection crystal is a colorless block (0.30×0.10×0.04mm3) and belongs to the monoclinic P21 space group. Unit cell parameters a=16.0496(5)Å, b=4.86260(10)Å, c=16.4686(5)Å, α =90°, β =118.015(4)°, γ =90°, V=1134.65(7 )Å3, Z=4. The calculated density Dc=1.389g/cm3, the number of electrons in the unit cell F (000)=504.0, the linear absorption coefficient μ (Cu Kα)=0.840mm-1 of the unit cell, and the diffraction experiment temperature T=150.00(11)K.

6A(+)：檢測晶體為無色塊狀(0.30×0.20×0.10mm3)，屬單斜晶系P21空間群。晶胞參數a=22.6688(7)Å，b=8.5595(2)Å，c=23.3578(5)Å， α=90°，β=113.876(3)°，γ=90°，V=4144.3(2)Å3，Z=2。計算密度Dc=0.999g/cm3，單胞中電子數F(000)=1318.0，單胞的線性吸收係數μ(Cu Kα)=0.570mm-1，衍射實驗溫度T=100.01(18)K。 6A( + ): The detected crystal is a colorless block (0.30×0.20×0.10mm3) and belongs to the monoclinic P21 space group. Unit cell parameters a=22.6688(7)Å, b=8.5595(2)Å, c=23.3578(5)Å, α =90°, β =113.876(3)°, γ =90°, V=4144.3(2 )Å3, Z=2. The calculated density Dc=0.999g/cm3, the number of electrons in the unit cell F (000)=1318.0, the linear absorption coefficient μ (Cu Kα)=0.570mm-1 of the unit cell, and the diffraction experiment temperature T=100.01(18)K.

TJ-NA048：檢測晶體為無色針狀(0.30×0.04×0.04mm3)，屬單斜晶系P1空間群。晶胞參數a=7.6165(4)Å，b=11.3423(5)Å，c=17.3991(8)Å，α=85.007(4)°，β=88.052(4)°，γ=70.532(4)°，V=1411.75(12)Å3，Z=2。計算密度Dc=1.366g/cm3，單胞中電子數F(000)=620.0，單胞的線性吸收係數μ(Cu Kα)=0.856mm-1，衍射實驗溫度T=150.00(13)K。 TJ-NA048: The detection crystal is colorless needle-shaped (0.30×0.04×0.04mm3) and belongs to the monoclinic P1 space group. Unit cell parameters a=7.6165(4)Å, b=11.3423(5)Å, c=17.3991(8)Å, α =85.007(4)°, β =88.052(4)°, γ =70.532(4)° , V=1411.75(12)Å3, Z=2. The calculated density Dc=1.366g/cm3, the number of electrons in the unit cell F (000)=620.0, the linear absorption coefficient μ (Cu Kα) of the unit cell=0.856mm-1, and the diffraction experiment temperature T=150.00(13)K.

TJ-NA092：檢測晶體為無色棱柱狀(0.30×0.10×0.10mm3)，屬三斜晶系P1空間群。晶胞參數a=5.17960(10)Å，b=8.0667(2)Å，c=12.4077(2)Å，α=93.146(2)°，β=101.266(2)°，γ=96.134(2)°，V=503.993(18)Å3，Z=2。計算密度Dc=1.412g/cm3，單胞中電子數F(000)=228.0，單胞的線性吸收係數μ(Cu Kα)=0.945mm-1，衍射實驗溫度T=100.00(10)K。 TJ-NA092: The detection crystal is colorless prismatic (0.30×0.10×0.10mm3) and belongs to the triclinic P1 space group. Unit cell parameters a=5.17960(10)Å, b=8.0667(2)Å, c=12.4077(2)Å, α =93.146(2)°, β =101.266(2)°, γ =96.134(2)° , V=503.993(18)Å3, Z=2. The calculated density Dc=1.412g/cm3, the number of electrons in the unit cell F (000)=228.0, the linear absorption coefficient μ (Cu Kα)=0.945mm-1 of the unit cell, and the diffraction experiment temperature T=100.00(10)K.

實施例3：製備NAG0052、L96Example 3: Preparation of NAG0052, L96

化合物NAG0024、NAG0026購買自天津藥明康德新藥開發有限公司。除非特別說明，以下實施例中所用的試劑均為市售商品。 Compounds NAG0024 and NAG0026 were purchased from Tianjin WuXi AppTec New Drug Development Co., Ltd. Unless otherwise stated, the reagents used in the following examples are all commercially available products.

化合物NAG0052的合成Synthesis of compound NAG0052

起始原料化合物1採購自江蘇倍達醫藥科技有限公司。化合物NAG0052的合成路線如下所示： The starting material compound 1 was purchased from Jiangsu Beida Pharmaceutical Technology Co., Ltd. The synthetic route of compound NAG0052 is as follows:

上述路線中涉及的具體中間產物以及終產物的合成以及鑑定如下所述： The synthesis and identification of specific intermediate products and final products involved in the above route are as follows:

化合物2Compound 2

在0℃以及氮氣保護下，往化合物1(12.3mL,101mmol)的THF(300mL)溶液中分批加入NaH(12.2g,304mmol，純度60%)。該混合物在20℃下攪拌1小時之後再次冷卻到0℃，接著往體系中逐滴加入苄溴(36.3mL,304mmol)，並且在20℃攪拌12小時。將該反應液用H₂O(100mL)淬滅後，用EtOAc(200mL x 2)萃取。合併後的有機相用飽和食鹽水(100mL)洗滌，Na₂SO₄乾燥，過濾，濃縮得到的殘留物經過矽膠管柱層析分離後得到目標化合物2(20.0g,51.8mmol，產率51%)。 At 0°C and under nitrogen protection, NaH (12.2g, 304mmol, purity 60%) was added in batches to a solution of compound 1 (12.3mL, 101mmol) in THF (300mL). The mixture was stirred at 20°C for 1 hour and then cooled to 0°C again, then benzyl bromide (36.3 mL, 304 mmol) was added dropwise to the system and stirred at 20°C for 12 hours. The reaction solution was quenched with H ₂ O (100 mL), and then extracted with EtOAc (200 mL x 2). The combined organic phases were washed with saturated brine (100 mL), dried over Na ₂ SO ₄ , filtered, and concentrated. The residue obtained was separated by silica gel column chromatography to obtain target compound 2 (20.0 g, 51.8 mmol, yield 51%). ).

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

¹ H NMR：(400MHz,CDCl₃)δ ppm 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)。 ¹ H NMR: (400MHz, CDCl ₃ ) δ ppm 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).

化合物3和4Compounds 3 and 4

在20℃以及氮氣保護下，往化合物2(13.0g,33.6mmol)的DCM(300mL)溶液中一次性加入TMSCN(13.5mL,101mmol)，接著逐滴加入TMSOTf(9.14mL,50.5mmol)的DCM(30mL)溶液。該反應液在20℃下攪拌15小時。反應結束之後用飽和NaHCO₃水溶液(80mL)淬滅該體系，並且用DCM(150mL x 2)萃取，合併後的有機相用飽和食鹽水(80mL)洗滌，Na₂SO₄乾燥，過濾以及濃縮後藉由矽膠管柱層析分離後得到目標化合物3(3.30g,9.18mmol，產率27%)以及淡黃色油狀液體化合物4(8.50g,9.18mmol，產率70%)。 At 20°C and under nitrogen protection, add TMSCN (13.5 mL, 101 mmol) in one go to a solution of compound 2 (13.0 g, 33.6 mmol) in DCM (300 mL), and then add TMSOTf (9.14 mL, 50.5 mmol) in DCM dropwise. (30mL) solution. The reaction solution was stirred at 20°C for 15 hours. After the reaction, the system was quenched with saturated NaHCO ₃ aqueous solution (80 mL), and extracted with DCM (150 mL x 2). The combined organic phases were washed with saturated brine (80 mL), dried over Na ₂ SO ₄ , filtered and concentrated. After separation by silica gel column chromatography, target compound 3 (3.30g, 9.18mmol, yield 27%) and light yellow oily liquid compound 4 (8.50g, 9.18mmol, yield 70%) were obtained.

化合物3 Compound 3

¹ H NMR：(400MHz,CDCl₃)δ ppm 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)。 ¹ H NMR: (400MHz, CDCl ₃ ) δ ppm 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).

化合物4 Compound 4

¹ H NMR：(400MHz,CDCl₃)δ ppm 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)。 ¹ H NMR: (400MHz, CDCl ₃ ) δ ppm 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).

化合物5Compound 5

在0℃及氮氣保護下將化合物4(3.00g,9.28mmol)的THF(15mL)溶液，滴加到LiAlH₄(0.79g,20.9mmol)的THF(15mL)溶液中，滴加完後體系在0℃反應1小時。TLC(PE：EtOAc=3：1)監測到原料完全消失。向反應液中緩慢加入十水硫酸鈉，加至不冒泡為止。之後將反應液過濾，濾餅用二氯甲烷(60mL)洗滌三次後，收集濾液旋乾，得目標化合物5(3.00g，產率90%). A solution of compound 4 (3.00g, 9.28mmol) in THF (15mL) was added dropwise to a solution of LiAlH ₄ (0.79g, 20.9mmol) in THF (15mL) at 0°C under nitrogen protection. After the dropwise addition, the system was React at 0°C for 1 hour. TLC (PE:EtOAc=3:1) detected the complete disappearance of the raw material. Slowly add sodium sulfate decahydrate to the reaction solution until bubbling stops. Afterwards, the reaction solution was filtered, and the filter cake was washed three times with dichloromethane (60 mL). The filtrate was collected and spin-dried to obtain target compound 5 (3.00 g, yield 90%).

¹ H NMR：(400MHz,DMSO-d ₆)δ ppm 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)。 ¹ H NMR: (400MHz, DMSO- d ₆ ) δ ppm 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).

化合物6Compound 6

在氮氣保護下，將化合物5(3.00g,8.25mmol)溶於DCM(30mL)，加入TEA(3.44mL,24.7mmol)和CbzCl(1.76mL,12.4mmol)，20℃反應2小時。LCMS顯示反應完成。將反應液加入二氯甲烷(30mL)和水(60mL)萃取。有機相用水(60mL x 3)洗滌三次，無水硫酸鈉乾燥，濃縮用正相管柱純化(PE：EtOAc=1：1)，得到目標化合物6(2.5g，產率90%)。 Under nitrogen protection, compound 5 (3.00g, 8.25mmol) was dissolved in DCM (30mL), TEA (3.44mL, 24.7mmol) and CbzCl (1.76mL, 12.4mmol) were added, and the reaction was carried out at 20°C for 2 hours. LCMS showed the reaction was complete. Dichloromethane (30 mL) and water (60 mL) were added to the reaction liquid and extracted. The organic phase was washed three times with water (60mL

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

¹ H NMR：(400MHz,CDCl₃)δ ppm 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)。 ¹ H NMR: (400MHz, CDCl ₃ ) δ ppm 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).

化合物7Compound 7

在氮氣保護下，將化合物6(2.00g,3.90mmol)溶於DCM(5mL)，在-78℃下加入BCl₃的THF溶液(1M,27.3mL)，反應1小時。TLC(DCM：MeOH=10：1)監測到原料完全消失。將反應液在-78℃下加入甲醇(20mL)淬滅，濃縮，用正相管柱純化(DCM：MeOH=10：1)，得到目標化合物7(2.00g，產率60%)。 Under nitrogen protection, compound 6 (2.00g, 3.90mmol) was dissolved in DCM (5mL), BCl ₃ in THF solution (1M, 27.3mL) was added at -78°C, and the reaction was carried out for 1 hour. TLC (DCM: MeOH=10:1) detected the complete disappearance of the raw material. The reaction solution was quenched by adding methanol (20 mL) at -78°C, concentrated, and purified with a normal phase column (DCM: MeOH=10:1) to obtain target compound 7 (2.00 g, yield 60%).

¹ H NMR：(400MHz,CD₃OD)δ ppm 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)。 ¹ H NMR: (400MHz, CD ₃ OD) δ ppm 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).

化合物8Compound 8

在氮氣保護下，將化合物7(0.50g,1.78mmol)溶於吡啶(5mL)中，在0℃下加入4A分子篩(500mg)和DMTrCl(0.66mL,2.13mmol)，之後升溫至20℃反應1.5小時。TLC(PE：EtOAc=2：1)監測到原料完全消失。將反應液加入乙酸乙酯(60mL)和水(60mL)萃取，有機相用水(60mL x 3)洗滌三次後用無水硫酸鈉乾燥，濃縮，用正相管柱純化(PE：EtOAc=1：1)，得到目標化合物8(800mg，產率90%)。 Under nitrogen protection, compound 7 (0.50g, 1.78mmol) was dissolved in pyridine (5mL), 4A molecular sieve (500mg) and DMTrCl (0.66mL, 2.13mmol) were added at 0°C, and then the temperature was raised to 20°C for reaction 1.5 hours. TLC (PE:EtOAc=2:1) detected the complete disappearance of the raw 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 with a normal phase column (PE: EtOAc=1:1 ) to obtain target compound 8 (800 mg, yield 90%).

¹ H NMR：(400MHz,CDCl₃)δ ppm 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)。 ¹ H NMR: (400MHz, CDCl ₃ ) δ ppm 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).

化合物9Compound 9

將化合物8(800mg,1.234mmol)溶於EtOAc(5mL)，加入Pd/C 10%(800mg,7.517mmol)，反應在H₂條件(15Psi)，20℃下反應1小時。LCMS顯示反應已經完成。反應液過濾，濾餅用二氯甲烷(100mL)和甲醇(100mL)洗滌三次，濃縮，經過反相管柱分離得到化合物9(300mg,54%)。 Compound 8 (800mg, 1.234mmol) was dissolved in EtOAc (5mL), Pd/C 10% (800mg, 7.517mmol) was added, and the reaction was carried out under H ₂ conditions (15Psi) and 20°C for 1 hour. LCMS showed the reaction was complete. The reaction solution was filtered, and the filter cake was washed three times with dichloromethane (100 mL) and methanol (100 mL), concentrated, and separated through a reversed-phase column to obtain compound 9 (300 mg, 54%).

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

化合物11Compound 11

將化合物10(435mg,1.780mmol)溶於DCM(10mL)，加入DIEA(0.441mL,2.67mmol)和HATU(677mg,1.78mmol)後，再加入化合物9(400mg,0.890mmol)，20℃反應1小時。TLC(DCM：MeOH=10：1)監測反應完成。將反應液加入二氯甲烷(60mL)和水(60mL)萃取，有機相用水(60mL x 3)洗滌三次，無水硫酸鈉乾燥，濃縮用正相管柱純化(PE：EtOAc=0：1過管柱，在100%處出產品峰)，得到目標化合物11(600mg，產率90%)。 Dissolve compound 10 (435mg, 1.780mmol) in DCM (10mL), add DIEA (0.441mL, 2.67mmol) and HATU (677mg, 1.78mmol), then add compound 9 (400mg, 0.890mmol), and react at 20°C 1 hours. TLC (DCM:MeOH=10:1) monitored the completion of the reaction. 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 by a normal phase column (PE: EtOAc=0:1). column, the product peak appeared at 100%), and the target compound 11 (600 mg, yield 90%) was obtained.

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

¹ H NMR：(400MHz,CD₃OD)δ ppm 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)。 ¹ H NMR: (400MHz, CD ₃ OD) δ ppm 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).

化合物12Compound 12

將化合物11(600mg,0.799mmol)溶於THF(3mL)和H₂O(1mL)，加入LiOH.H₂O(134mg,3.20mmol)，20℃反應12小時。TLC(DCM：MeOH=10：1)顯示反應完成。將反應液旋乾，用水(5mL)和甲醇(5mL)溶解，用反相管柱純化(H₂O：CH₃CN=1：1，在35%左右出峰)，得到目標化合物12(460mg，產率100%，鋰鹽)。 Compound 11 (600 mg, 0.799 mmol) was dissolved in THF (3 mL) and H ₂ O (1 mL), LiOH.H ₂ O (134 mg, 3.20 mmol) was added, and the reaction was carried out at 20°C for 12 hours. TLC (DCM:MeOH=10:1) showed the reaction was complete. Spin the reaction solution to dryness, dissolve it in water (5 mL) and methanol (5 mL), and purify it with a reverse-phase column (H ₂ O: CH ₃ CN = 1: 1, peak at about 35%) to obtain target compound 12 (460 mg , yield 100%, lithium salt).

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

HPLC：t_R=1.879min in 10-80CD_6min。 HPLC: t _R =1.879min in 10-80CD_6min.

¹ H NMR：(400MHz,CD₃OD)δ ppm 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)。 ¹ H NMR: (400MHz, CD ₃ OD) δ ppm 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).

化合物13Compound 13

室溫環境，氮氣保護下，將化合物NAG0024(271mg,0.151mmol)溶解於無水THF(2mL)和無水DMF(4mL)，加入3A分子篩，再依次加入化合物12(100mg,0.151mmol)、HOBt(25mg,0.181mmol)、DCC(38mg,0.181mmol)和DIEA(39mg,0.302mmol)。反應液45℃反應16h。LC-MS顯示反應完全後，加水淬滅，過濾。濾液濃縮後，經C18反相管柱純化(H₂O/MeCN)，得到化合物13(210mg，產率57%)。 At room temperature, under nitrogen protection, dissolve compound NAG0024 (271 mg, 0.151 mmol) in anhydrous THF (2 mL) and anhydrous DMF (4 mL), add 3A molecular sieve, and then add compound 12 (100 mg, 0.151 mmol), HOBt (25 mg) in sequence ,0.181mmol), DCC (38mg, 0.181mmol) and DIEA (39mg, 0.302mmol). The reaction solution was reacted at 45°C for 16 hours. After LC-MS showed that the reaction was complete, add water to quench and filter. After the filtrate was concentrated, it was purified through a C18 reverse-phase column (H ₂ O/MeCN) to obtain compound 13 (210 mg, yield 57%).

化合物NAG0052Compound NAG0052

室溫環境下，化合物13(230mg，0.094mmol)溶於吡啶(5mL)，加入分子篩，加入DMAP(12mg，0.283mmol)，丁二酸酐(28mg，0.283mmol)。氮氣保護，50℃攪拌16小時。LCMS檢測反應完全，過濾旋乾。過C18反相管柱純化後，由製備HPLC二次純化，得到目標化合物NAG0052(123mg，0.048mmol，產率51%)。 At room temperature, compound 13 (230 mg, 0.094 mmol) was dissolved in pyridine (5 mL), molecular sieves were added, DMAP (12 mg, 0.283 mmol), and succinic anhydride (28 mg, 0.283 mmol) were added. Under nitrogen protection, stir at 50°C for 16 hours. LCMS detects that the reaction is complete, filter and spin dry. After purification through a C18 reverse-phase column and secondary purification by preparative HPLC, the target compound NAG0052 (123 mg, 0.048 mmol, yield 51%) was obtained.

MS(ESI)m/z=2535.3[M-1]^-.理論：2536.2。 MS(ESI)m/z=2535.3[M-1] ^- .Theory: 2536.2.

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

NAG0052’NAG0052’

化合物NAG0052經固相合成連接到序列上，再經過胺解後，NAG0052結構脫去一部分官能團成為前述NAG0052’。 Compound NAG0052 is connected to the sequence through solid-phase synthesis, and after aminolysis, part of the functional groups of the NAG0052 structure is removed to become the aforementioned NAG0052'.

L96的合成Synthesis of L96

按照專利申請WO2014025805A1記載的方法製備獲得如上結構式所示的L96。 L96 shown in the above structural formula was prepared according to the method described in patent application WO2014025805A1.

實施例4：dsRNA的合成Example 4: Synthesis of dsRNA

1.自製帶有載體的樹脂 1. Homemade resin with carrier

將含有羧酸基團的化合物NAG0052(157mg,0.062mmol)溶於無水DMF(3mL)，待底物完全溶解後，依次加入無水乙腈(4mL)，DIEA(0.03mL,0.154mmol，2.5eq)和HBTU(35mg,0.093mmol，1.5eq)。反應液混合均勻後，再加入大孔胺甲基樹脂(476mg，空白載量為0.41mmol/g，目標載量為0.1mmol/g)。將反應液放入搖床上(溫度：25℃，轉速：200rpm)振搖過夜。反應液過濾，濾餅依次分別用DCM，無水乙腈洗滌，收集固體，真空乾燥過夜。 The compound NAG0052 (157mg, 0.062mmol) containing a carboxylic acid group was dissolved in anhydrous DMF (3mL). After the substrate was completely dissolved, anhydrous acetonitrile (4mL), DIEA (0.03mL, 0.154mmol, 2.5eq) and HBTU (35mg, 0.093mmol, 1.5eq). After the reaction solution is mixed evenly, macroporous amine methyl resin (476 mg, blank loading is 0.41 mmol/g, target loading is 0.1 mmol/g) is added. Place the reaction solution on a shaker (temperature: 25°C, rotation speed: 200 rpm) and shake overnight. The reaction solution was filtered, and the filter cake was washed with DCM and anhydrous acetonitrile in sequence. The solids were collected and dried under vacuum overnight.

將上步固體分散於無水乙腈(5mL)，依次加入吡啶(0.18mL)，DMAP(3mg)，NMI(0.12mL)和CapB1(2.68mL)。將反應液放入搖床上(溫度：25 ℃，轉速：200rpm)振搖2h。反應液過濾，濾餅用無水乙腈洗滌，收集固體，真空乾燥過夜，得到帶有載體的樹脂。載量經過測定為0.1mmol/g。 Disperse the solid from the previous step in anhydrous acetonitrile (5 mL), and add pyridine (0.18 mL), DMAP (3 mg), NMI (0.12 mL) and CapB1 (2.68 mL) in sequence. Place the reaction solution on a shaker (temperature: 25 ℃, rotation speed: 200rpm) and shake for 2h. The reaction solution was filtered, and the filter cake was washed with anhydrous acetonitrile. The solid was collected and dried under vacuum overnight to obtain a resin with a carrier. The loading capacity was determined to be 0.1mmol/g.

2.對於已經連接在樹脂上的NAG0052，使用該樹脂作為起始，按照核苷酸排布順序自3’-5’方向逐一連接核苷單體。每連接一個核苷單體都包括脫保護、偶聯、蓋帽、氧化或硫化四步反應。操作為本領域常規。 2. For NAG0052 that has been connected to the resin, use the resin as a starting point and connect the nucleoside monomers one by one from the 3’-5’ direction in the order of the nucleotide arrangement. Each connection of a nucleoside monomer involves four steps of deprotection, coupling, capping, oxidation or sulfation. The operation is routine in the art.

化合物NAG0052經過固相合成連接到序列上，再經過胺解後，NAG0052結構脫去一部分官能團成為NAG0052’。 Compound NAG0052 is connected to the sequence through solid-phase synthesis, and after aminolysis, part of the functional groups of the NAG0052 structure is removed to become NAG0052’.

製得的dsRNA具有表1和表2中所示的有義鏈和反義鏈。 The prepared dsRNA had the sense strand and antisense strand shown in Table 1 and Table 2.

表1. dsRNA列表

Table 1. dsRNA list

表2. 有義鏈和反義鏈的核酸序列

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

在上表中，大寫字母G、A、C、U分別表示包含鳥嘌呤、腺嘌呤、胞嘧啶和尿嘧啶的核苷酸，小寫字母m表示2'-甲氧基修飾，小寫字母f表示2'-氟修飾，小寫字母s表示與該字母s左右相鄰的兩個核苷酸之間為硫代磷酸二酯基連接；以下同。 In the above table, the capital letters G, A, C, and U represent nucleotides containing guanine, adenine, cytosine, and uracil respectively, the small letter m represents 2'-methoxy modification, and the small letter f represents 2 '-Fluorine modification, the lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate diester groups; the same below.

實施例5：dsRNA在體內對靶基因mRNA表達量的抑制Example 5: Inhibition of target gene mRNA expression by dsRNA in vivo

本實驗考察本揭露的綴合不同的dsRNA在體內對靶基因mRNA表達量的抑制效率。 This experiment examines the inhibitory efficiency of the disclosed conjugated different dsRNAs on target gene mRNA expression in vivo.

將雄性6-8週齡C57BL/6小鼠隨機分組，每組共6隻，每個時間點各3隻，分別向每組小鼠給予TRD007205、參比陽性TRD002218以及PBS。 Male 6-8 week old C57BL/6 mice were randomly divided into 6 groups, 3 mice at each time point. TRD007205, reference positive TRD002218, and PBS were administered to each group of mice.

所有動物依據體總計算給藥量，採用皮下注射方式單次給藥，dsRNA給藥劑量(以無配體核苷酸的量計)為1mg/kg，給藥體積為5mL/kg。給藥7天、28天後處死小鼠，收集肝臟，用RNA later(Sigma Aldrich公司)保存；隨後用組織勻漿儀勻漿肝組織，再用組織RNA提取試劑盒(凡知醫療科技，FG0412)根據操作說明書標註的操作步驟提取得到肝組織總RNA。將總RNA反轉錄成cDNA並採用實時螢光定量PCR方法檢測肝組織中的TTR mRNA的表達量。在該螢光定量PCR法中，以甘油醛3-磷酸脫氫酶(GAPDH)基因作為內參基因，使用針對TTR和GAPDH的Taqman探針引子分別檢測TTR和GAPDH的mRNA表達量。 The dosage of all animals was calculated based on the total body weight, and a single dose was administered by subcutaneous injection. The dsRNA dosage (based on the amount of ligand-free nucleotides) was 1 mg/kg, and the dosage volume was 5 mL/kg. The mice were killed 7 days and 28 days after administration, and the livers were collected and preserved with RNA later (Sigma Aldrich Company); then the liver tissue was homogenized with a tissue homogenizer, and then tissue RNA extraction kit (Fanzhi Medical Technology, FG0412 ) Extract total RNA from liver tissue according to the operating steps marked in the operating instructions. Total RNA was reverse transcribed into cDNA and real-time fluorescence quantitative PCR method was used to detect the expression of TTR mRNA in liver tissue. In this fluorescent quantitative PCR method, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as the internal reference gene, and Taqman probe primers for TTR and GAPDH were used to detect the mRNA expression levels of TTR and GAPDH respectively.

表3. 小鼠體內實驗分組信息：

Table 3. Mouse in vivo experimental grouping information:

表4. 檢測引子的序列參見如下：

Table 4. The sequence of the detection primer is as follows:

TTR mRNA表達量按照如下等式計算： TTR mRNA expression is calculated according to the following equation:

TTR mRNA表達量=【(測試組TTR mRNA表達量/測試組GAPDH mRNA表達量)/(對照組TTR mRNA表達量/對照組GAPDH mRNA表達量)】x 100%。 TTR mRNA expression = [(TTR mRNA expression in the test group/GAPDH mRNA expression in the test group)/(TTR mRNA expression in the control group/GAPDH mRNA expression in the control group)] x 100%.

給藥7天、28天後，本揭露的綴合不同結構的dsRNA的在體內對靶基因mRNA表達量的抑制效率分別見圖1和圖2。由圖1的結果可知，TRD007205在給藥後7天對於TTR mRNA的表達抑制均具有良好的效果。由圖2可知，給藥28天後，TRD007205對靶基因mRNA表達量的抑制作用均優於TRD002218。 After 7 days and 28 days of administration, the inhibitory efficiency of the dsRNA conjugated with different structures of the present disclosure on the expression of target gene mRNA in vivo is shown in Figure 1 and Figure 2 respectively. It can be seen from the results in Figure 1 that TRD007205 has a good effect on inhibiting TTR mRNA expression 7 days after administration. As can be seen from Figure 2, after 28 days of administration, TRD007205 has a better inhibitory effect on target gene mRNA expression than TRD002218.

實施例6：合成dsRNAExample 6: Synthesis of dsRNA

1.自製帶有載體的樹脂 1. Homemade resin with carrier

2.使用帶有NAG0052的樹脂作為起始，按照核苷酸排布順序自3’-5’方向逐一連接核苷單體。每連接一個核苷單體都包括脫保護、偶聯、蓋帽、氧化或硫化四步反應。 2. Use the resin with NAG0052 as the starting point, and connect the nucleoside monomers one by one from the 3’-5’ direction in the order of nucleotide arrangement. Each connection of a nucleoside monomer involves four steps of deprotection, coupling, capping, oxidation or sulfation.

製得的dsRNA具有表5和表6中所示的有義鏈和反義鏈。 The prepared dsRNA had the sense strand and antisense strand shown in Table 5 and Table 6.

表5. dsRNA列表

Table 5. dsRNA list

表6. dsRNA的有義鏈和反義鏈

Table 6. Sense strand and antisense strand of dsRNA

表7. dsRNA的有義鏈和反義鏈對應的裸序列

Table 7. Naked sequences corresponding to the sense strand and antisense strand of dsRNA

其中，(-)hmpNA(A)表示

； Among them, (-)hmpNA(A) means

;

NAG0052’的結構為： The structure of NAG0052’ is:

實施例7：dsRNA序列psiCHECK在靶活性Example 7: On-target activity of dsRNA sequence psiCHECK

在HEK293A細胞中採用9個濃度梯度對dsRNA進行體外分子水平模擬在靶活性篩選。 In HEK293A cells, 9 concentration gradients were used to screen dsRNA for target activity through in vitro molecular level simulation.

以人HSD17B13基因構建dsRNA對應的在靶序列，插入到psiCHECK-2質粒中。該質粒包含海腎螢光素酶基因及螢火蟲螢光素酶基因。作為雙報告基因系統，dsRNA的靶序列插入到海腎螢光素酶基因的3’UTR區域，dsRNA對於靶標序列的活性可以藉由經螢火蟲螢光素酶校準後的海腎螢光素酶表達情況的檢測來反映，檢測使用Dual-Luciferase Reporter Assay System(Promega，E2940)。 The target sequence corresponding to dsRNA was constructed using the human HSD17B13 gene and inserted into the psiCHECK-2 plasmid. The plasmid contains the Renilla luciferase gene and the firefly luciferase gene. As a dual reporter gene system, the target sequence of dsRNA is inserted into the 3'UTR region of the Renilla luciferase gene. The activity of dsRNA on the target sequence can be expressed by Renilla luciferase calibrated with firefly luciferase. The situation is reflected by the detection, and the Dual-Luciferase Reporter Assay System (Promega, E2940) is used for detection.

HEK293A細胞培養於含10%胎牛血清的DMEM高糖培養基中，在37℃，5% CO₂條件下培養。轉染前24h，將HEK293A細胞接種於96孔板，接種密度為每孔8×10³個細胞，每孔100μL培養基。 HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal calf serum at 37°C and 5% _CO2 . 24 h before transfection, HEK293A cells were seeded in a 96-well plate at a seeding density of 8 × 10 ³ cells per well and 100 μL culture medium per well.

按照說明書，使用Lipofectamine2000(ThermoFisher，11668019)對細胞共轉染dsRNA及對應質粒，Lipofectamine2000每孔使用0.2μL，質粒轉染量為20ng每孔。對於在靶序列質粒，dsRNA共設置9個濃度點，最高濃度點終濃度為20nM，4倍梯度稀釋，20nM、5nM、1.25nM、0.3125nM、0.0781nM、0.0195nM、0.0049nM、0.0012nM和0.0003nM。轉染後24h，採用Dual-Luciferase Reporter Assay System(Promega，E2940)檢測在靶水平。檢測序列的在靶活性如表8所示。結果表明，所有dsRNA在psiCHECK實驗中對在靶質粒的表達具有高水平的抑制活性，均顯著優於對照TJR100347。 According to the instructions, cells were co-transfected with dsRNA and the corresponding plasmid using Lipofectamine2000 (ThermoFisher, 11668019). Lipofectamine2000 was used at 0.2 μL per well, and the plasmid transfection amount was 20 ng per well. For the target sequence plasmid, a total of 9 concentration points are set for dsRNA. The final concentration of the highest concentration point is 20nM, 4-fold gradient dilution, 20nM, 5nM, 1.25nM, 0.3125nM, 0.0781nM, 0.0195nM, 0.0049nM, 0.0012nM and 0.0003 nM. 24h after transfection, the Dual-Luciferase Reporter Assay System (Promega, E2940) was used to detect the target level. The on-target activity of the detection sequences is shown in Table 8. The results showed that all dsRNAs had a high level of inhibitory activity on the expression of the target plasmid in the psiCHECK experiment, and were significantly better than the control TJR100347.

表8. dsRNA psiCHECK在靶活性篩選結果

Table 8. Screening results of dsRNA psiCHECK on-target activity

TW202339773A_112102810_SEQL.xmlTW202339773A_112102810_SEQL.xml

Claims

A double-stranded ribonucleic acid (dsRNA), which contains a sense strand and an antisense strand, in the direction from the 5' end to the 3' end,

The nucleotides at positions 7, 8 and 9 of the sense strand are 2'-fluoro modified nucleotides, and the nucleotide at position 5 is independently a 2'-methoxy modified nucleotide or 2' -Fluorine modified nucleotides, the remaining nucleotides are 2'-methoxy modified nucleotides;

The nucleotides at positions 2 and 14 of the antisense strand are 2'-fluoro modified nucleotides, and the nucleotides at positions 4, 6, 8, 9, 10, 12, 16 and 18 are independently 2' -Methoxy or 2'-fluoro modified nucleotides, the remaining nucleotides are 2'-methoxy modified nucleotides;

The number of 2'-fluorine modified nucleotides in the antisense strand is 2-7;

The 7th nucleotide position of the antisense strand contains a chemical modification represented by formula (I), its tautomer or its pharmaceutically acceptable salt:

The chemical modification represented by formula (I) is selected from any of the following structures:

,

, B is the base of the antisense strand when the 7th nucleotide from the 5' end is not modified;

The dsRNA inhibits the expression and/or activity of 17β-hydroxysteroid dehydrogenase type 13 (HSD17B13).

dsRNA as described in claim 1, wherein,

The sense strand contains the nucleotide sequence represented by the following formula:

5'-N _a N _a N _a N _a N _a N _a N _b N _b N _b N _a N _a N _a N _a N _a N _a N _a N _a N _a N _a -3';

Among them, _Na is a 2'-methoxy modified nucleotide, and N _b is a 2'-fluoro modified nucleotide.

dsRNA as described in claim 1 or 2, wherein,

The antisense strand contains the nucleotide sequence represented by the following formula:

5'-N _a 'N _b 'N _a 'X'N _a 'X'W'X'X'X'N _a 'X'N _a 'N _b 'N _a 'X'N _a 'X'N _a 'N _a 'N _a '-3';

Preferably, the antisense strand includes a nucleotide sequence represented by the following formula:

5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _b 'W'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b ' N _a 'N _a 'N _a 'N _a 'N _a '-3';

5'-N _a 'N _b 'N _a 'N _b _' N a 'N _b 'W'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b ' N _a 'N _a 'N _a 'N _a 'N _a '-3';

5'-N _a 'N _b 'N _a 'N _b _' N a 'N _b 'W'N _a 'N _a 'N _b 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b ' N _a 'N _b 'N _a 'N _a 'N _a '-3';

5'-N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'W'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b ' N _a 'N _b 'N _a 'N _a 'N _a '-3';

5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _b 'W'N _a 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b ' N _a 'N _b 'N _a 'N _a 'N _a '-3';

5'-N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'W'N _a 'N _a 'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b ' N _a 'N _a 'N _a 'N _a 'N _a '-3';

5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _b 'W'N _a 'N _a 'N _b 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b ' N _a 'N _a 'N _a 'N _a 'N _a '-3';5'-

N _a 'N _b 'N _a 'N _a _' N _a 'N a 'W'N _b 'N _b 'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b 'N _a ' N _a 'N _a 'N _a 'N _a '-3';

5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _b 'W'N _a 'N _a 'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b ' N _a 'N _a 'N _a 'N _a 'N _a '-3';

5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _b 'W'N _a 'N _a 'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _a ' N _a 'N _a 'N _a 'N _a 'N _a '-3';

5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _a 'W'N _a 'N _a 'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _b ' N _a 'N _a 'N _a 'N _a 'N _a '-3'; or,

5'-N _a 'N _b 'N _a 'N _a 'N _a 'N _a 'W'N _a 'N _a 'N _a 'N _a 'N _a 'N _a 'N _b 'N _a 'N _a ' N _a 'N _a 'N _a 'N _a 'N _a '-3';

in,

Each X' is independently N _a ' or N _b ';

N _a ' is a 2'-methoxy modified nucleotide,

N _b ' is a 2'-fluorine modified nucleotide;

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

The dsRNA according to any one of claims 1 to 3, wherein,

At least one phosphate group in the sense strand and/or antisense strand is a phosphate group with a modifying group, preferably a phosphorothioate group, more preferably a phosphorothioate diester group.

The dsRNA as described in claim 4, wherein the phosphorothioate diester group is present at at least one of the following positions:

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

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

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

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

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

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

Preferably, the sense strand and/or antisense strand includes multiple phosphorothioate diester groups, and the phosphorothioate diester groups are present in:

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

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

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

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

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

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

The dsRNA according to any one of claims 1 to 5, wherein,

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

The naked sequence of the antisense strand contains no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 2, and contains at least 19 consecutive nucleotides;

Preferably,

The naked sequence of the sense strand includes the nucleotide sequence shown in SEQ ID NO: 1, and

The naked sequence of the antisense strand includes the nucleotide sequence shown in SEQ ID NO:2.

The dsRNA according to any one of claims 1 to 6, wherein,

The antisense strand includes SEQ ID NO: 12, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO : 11. The nucleotide sequence shown in any one of SEQ ID NO: 13 and SEQ ID NO: 14;

Preferably, the sense strand includes the nucleotide sequence shown in SEQ ID NO: 3.

The dsRNA according to any one of claims 1 to 7, wherein the dsRNA further comprises a conjugated ligand, the ligand comprising at least one N-acetyl-galactosamine.

dsRNA as described in claim 8, wherein,

The ligand is as shown in the following structure or a pharmaceutically acceptable salt thereof,

The dsRNA as described in any one of claim 8 or 9, wherein the 3' end of the sense strand is conjugated to the ligand.

The dsRNA as described in any one of claims 8 to 10, wherein the ligand is connected to the 3' end of the sense strand of the dsRNA through a phosphate group or a phosphorothioate group; preferably through A phosphodiester group or a phosphorothioate diester group is attached, preferably via a phosphodiester group.

The dsRNA according to any one of claims 8 to 11, wherein,

Preferably, the sense strand includes the nucleotide sequence shown in SEQ ID NO: 4.

The dsRNA according to any one of claims 8 to 12, wherein,

The dsRNA is shown in the following structure or a pharmaceutically acceptable salt thereof:

in,

Af = adenine 2'-F ribonucleoside;

Cf=cytosine 2'-F ribonucleoside;

Uf=uracil 2'-F ribonucleoside;

Gf=guanine 2'-F ribonucleoside;

Am = adenine 2'-OMe ribonucleoside;

Cm=cytosine 2'-OMe ribonucleoside;

Gm=guanine 2'-OMe ribonucleoside;

Um=uracil 2'-OMe ribonucleoside;

Represents phosphorothioate diester group,

Represents phosphodiester group;

NAG0052’ means

(-)hmpNA(A) means

.

A pharmaceutical composition containing:

dsRNA as described in any one of claims 1 to 13;

Optionally, the pharmaceutical composition further contains one or more pharmaceutically acceptable excipients.

The use of a dsRNA as described in any one of claims 1 to 13 or a pharmaceutical composition as described in claim 14 in the preparation of medicines;

This medicine is used to prevent and/or treat any of the following:

Non-alcoholic fatty liver disease, cirrhosis, alcoholic steatohepatitis, alcoholic fatty liver disease, HCV-related cirrhosis, liver injury, hepatocellular necrosis, chronic fibroinflammatory liver disease, drug-induced liver injury.

A method for inhibiting the expression of target gene HSD17B13 or its mRNA, which includes:

An effective amount or dose of the dsRNA according to any one of claims 1 to 13 or the pharmaceutical composition according to claim 14 is administered to the subject.

A method of delivering oligonucleotides to the liver, comprising:

An effective amount or dose of the dsRNA according to any one of claims 8 to 13 or the pharmaceutical composition according to claim 14 is administered to the subject.

A cell comprising the dsRNA according to any one of claims 1 to 13.

A vector comprising the dsRNA as described in any one of claims 1 to 13.

A kit comprising the dsRNA as described in any one of claims 1 to 13 or the pharmaceutical composition as described in any one of claim 14.

A method of preparing dsRNA or pharmaceutical composition, which includes:

Synthesize dsRNA as described in any one of claims 1 to 13.