TW202302851A

TW202302851A - Hepatitis b virus sirnas and sirna conjugates

Info

Publication number: TW202302851A
Application number: TW111112868A
Authority: TW
Inventors: 黃金宇; 劉楠; 周雅琴; 羅敏
Original assignee: 大陸商上海拓界生物醫藥科技有限公司
Priority date: 2021-04-02
Filing date: 2022-04-01
Publication date: 2023-01-16
Also published as: WO2022206946A1; CN117241837A

Abstract

The present disclosure relates to hepatitis B virus siRNAs and siRNA conjugates. In particular, the present disclosure relates to siRNAs and siRNA conjugates targeting the hepatitis B virus (HBV) genome, and compositions and medical uses thereof. The present disclosure also relates to pharmaceutical compositions, cells or kits comprising the siRNA, and methods of using the siRNA for treating and/or preventing a subject suffering from HBV infection or HBV-related disease.

Description

Hepatitis B virus siRNA and siRNA conjugates

本申請要求申請日為2021年04月02日的中國專利申請CN202110361273.9的優先權，本申請引用上述中國專利申請的全文。 This application claims the priority of the Chinese patent application CN202110361273.9 with an application date of April 2, 2021, and this application cites the full text of the above-mentioned Chinese patent application.

本揭露關於用於抑制B型肝炎病毒基因表達的小干擾RNA(siRNA)綴合物及其使用方法。 The present disclosure relates to small interfering RNA (siRNA) conjugates and methods of use thereof for inhibiting hepatitis B virus gene expression.

B肝病毒(HBV)是一種嚴格嗜肝性、含有雙股DNA的病毒，是誘發B型肝炎的主要病因。全球大約有2.57億B肝患者，其中約30%以上的B肝患者會發展為慢性肝病，並最終導致肝癌的發生。目前，治療B肝的抗病毒藥物主要包括核苷/核苷酸類似物和干擾素兩類。 Hepatitis B virus (HBV) is a strictly hepatotropic virus containing double-stranded DNA, which is the main cause of hepatitis B. There are about 257 million hepatitis B patients in the world, and more than 30% of them will develop chronic liver disease and eventually lead to liver cancer. Currently, antiviral drugs for the treatment of hepatitis B mainly include nucleoside/nucleotide analogues and interferon.

核苷/核苷酸類似物則藉由抑制DNA聚合酶和逆轉錄酶活性，終止病毒DNA股的延長和生成，從而發揮抗病毒作用。然而，在治療結束後，只有少數患者實現完全和持久的緩解。此外，B型肝炎病毒隨著治療持續時間的增加而產生耐藥性。干擾素的主要作用是阻斷病毒核酸和蛋白質的合成，抑制HBV複製，提高人體免疫系統機能，但此類藥物並不能徹底清除病毒且復發率高。 Nucleoside/nucleotide analogues inhibit the activity of DNA polymerase and reverse transcriptase, and terminate the elongation and generation of viral DNA strands, thereby exerting antiviral effects. However, only a minority of patients achieve complete and durable remission after treatment ends. In addition, HBV develops drug resistance with increasing duration of treatment. The main function of interferon is to block the synthesis of viral nucleic acid and protein, inhibit HBV replication, and improve the function of the human immune system, but these drugs cannot completely eliminate the virus and have a high recurrence rate.

siRNA藉由轉錄後調控機制，可以特異性降解靶基因mRNA。在B肝病毒生命週期中，主要產生五種mRNA轉錄產物，其長度分別為3.5、3.5、2.4、2.1和0.7kb，這些轉錄產物的序列存在相同區域。針對B肝病毒轉錄出的全部mRNA產物相同區域設計siRNA化合物，可以抑制B肝病毒生命週期中的全部mRNA轉錄產物，進而抑制B肝病毒的複製和B肝表面抗原的分泌，達到治療B型肝炎的目的。 siRNA can specifically degrade target gene mRNA through post-transcriptional regulation mechanism. In the life cycle of hepatitis B virus, five kinds of mRNA transcripts are mainly produced, the lengths of which are 3.5, 3.5, 2.4, 2.1 and 0.7kb, and the sequences of these transcripts have the same region. Designing siRNA compounds for the same region of all mRNA products transcribed by hepatitis B virus can inhibit all mRNA transcription products in the life cycle of hepatitis B virus, thereby inhibiting the replication of hepatitis B virus and the secretion of liver B surface antigen, so as to achieve the treatment of hepatitis B the goal of.

本揭露提供一種以HBV為靶向的siRNA。 The present disclosure provides an siRNA targeting HBV.

一些實施方案中，本揭露提供了一種siRNA，其包含形成雙股區的正義股與反義股；該正義股包含至少15個連續核苷酸，與SEQ ID NO：1的核苷酸序列相差不超過3個核苷酸；該反義股包含至少15個連續核苷酸序列，與SEQ ID NO：2的核苷酸序列相差不超過3個核苷酸。 In some embodiments, the present disclosure provides an siRNA comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 No more than 3 nucleotides; the antisense strand contains at least 15 continuous nucleotide sequences, which differ from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides.

一些實施方案中，反義股在其5’區域的第2位至第8位(如第2位、第3位、第4位、第5位、第6位、第7位、第8位)中的至少一個核苷酸位置處包含式(I)所示的化學修飾或其互變異構體修飾： In some embodiments, the antisense strand is at position 2 to position 8 (e.g. position 2, position 3, position 4, position 5, position 6, position 7, position 8) of its 5' region ) at least one nucleotide position comprising chemical modifications shown in formula (I) or tautomer modifications thereof:

其中，Y選自O、NH和S； Wherein, 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 X is independently selected from CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ', R ₅ 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;

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 ; wherein 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； Optionally, R ₁ and R ₂ are directly connected to form a ring;

B是鹼基； B is a base;

其中，該式(I)所示的化學修飾或其互變異構體修飾不是

。 Wherein, the chemical modification shown in the formula (I) or its tautomer modification is not

.

在一些實施方案中，正義股不是5’-CACCUCUGCACGUCGCAUG(SEQ ID NO：108)；反義股不是5’-UAUGCGACGUGCAGAGGUGA-3’(SEQ ID NO：109)。 In some embodiments, the sense strand is not 5'-CACCUCUGCACGUCGCAUG (SEQ ID NO: 108); the antisense strand is not 5'-UAUGCGACGUGCAGAGGUGA-3' (SEQ ID NO: 109).

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, _R is not H.

一些實施方案中，B是鹼基；例如選自嘌呤鹼基、嘧啶鹼基、吲哚、5-硝基吲哚和3-硝基吡咯中的那些。 In some embodiments, B is a base; for example, those selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.

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-dimethylamine Base purine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, Pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidines, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

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.

一些實施方案中，B是該反義股在其5’區域的第2位至第8位(如第2位、第3位、第4位、第5位、第6位、第7位、第8位)中對應位置的鹼基。 In some embodiments, B is the 2nd to 8th positions (such as the 2nd, 3rd, 4th, 5th, 6th, 7th, The base at the corresponding position in position 8).

一些具體的實施方案中，B是該反義股在其5’區域的第2位至第8位(如第2位、第3位、第4位、第5位、第6位、第7位、第8位)中對應位置的天然鹼基。 In some specific embodiments, B is the 2nd to 8th (such as the 2nd, 3rd, 4th, 5th, 6th, 7th position, the natural base at the corresponding position in position 8).

在一些實施方案中，反義股在其5’區域的第2位至第8位(如第2位、第3位、第4位、第5位、第6位、第7位、第8位)中的至少一個核苷酸位置處包含式(I-1)所示的化學修飾或其互變異構體修飾： In some embodiments, the antisense strand is at position 2 to position 8 (e.g., position 2, position 3, position 4, position 5, position 6, position 7, position 8) of its 5' region. At least one nucleotide position in position) comprises the chemical modification shown in formula (I-1) or its tautomer modification:

其中，Y選自O、NH和S； Wherein, 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;

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 ; wherein 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).

在一些實施方案中，反義股在其5’區域的第2位至第8位(如第2位、第3位、第4位、第5位、第6位、第7位、第8位)中的至少一個核苷酸位置處包含式(I-2)所示的化學修飾或其互變異構體修飾： In some embodiments, the antisense strand is at position 2 to position 8 (e.g., position 2, position 3, position 4, position 5, position 6, position 7, position 8) of its 5' region. At least one nucleotide position in position) comprises the chemical modification shown in formula (I-2) or its tautomer modification:

其中Y選自O、NH和S； Wherein 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 ; wherein 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).

在一些實施方案中，上述化學修飾或其互變異構體修飾不是 In some embodiments, the aforementioned chemical modifications or tautomeric modifications thereof are not

在一些實施方案中，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, each X is independently selected from CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ', R ₅ are each independently H or C ₁ -C ₃ alkyl groups;

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 ; wherein 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;

視需要地，R₁和R₂直接相連成環。 Optionally, R ₁ and R ₂ are directly connected to form a ring.

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

在一些實施方案中，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, each X is independently selected from CR ₄ (R ₄ '), S, NR ₅ , and NH-CO, wherein R ₄ , R ₄ ', and R ₅ are each independently H, methyl, ethyl radical, 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 is selected from H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl 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).

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, Y is O or NH; each X is independently selected from NH-CO, _CH 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 of J ₁ and J ₂ is 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;

在一些實施方案中，該式(I)所示的化學修飾或其互變異構體修飾選自： In some embodiments, the chemical modification represented by the formula (I) or its tautomeric modification is selected from:

其中，B是鹼基；例如選自嘌呤鹼基、嘧啶鹼基、吲哚、5-硝基吲哚和3-硝基吡咯中的那些。 wherein, B is a base; for example, those selected from purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.

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-dimethylamine Base purine, 2-amino purine, N6-alkyl adenine, O6-alkyl guanine, 7-deaza purine, Cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole.

本揭露提供了一種siRNA，其包含形成雙股區的正義股與反義股；該正義股包含至少15個連續核苷酸，與SEQ ID NO：1的核苷酸序列相差不超過3個核苷酸；該反義股包含至少15個連續核苷酸序列，與SEQ ID NO：2的核苷酸序列相差不超過3個核苷酸； The present disclosure provides an siRNA comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises at least 15 consecutive nucleotides, and differs from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 cores Nucleotide; the antisense strand comprises at least 15 continuous nucleotide sequences, which differ from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides;

其中，大寫字母C、G、U、A表示核苷酸的鹼基組成； Among them, capital letters C, G, U, and A represent the base composition of nucleotides;

該反義股在其5’區域的第2位至第8位(如第2位、第3位、第4位、第5位、第6位、第7位、第8位)中的至少一個核苷酸位置處包含式(I’)所示的化學修飾或其互變異構體修飾： The antisense strand has at least one of the 2nd to 8th positions (such as 2nd, 3rd, 4th, 5th, 6th, 7th, 8th) in its 5' region A nucleotide position comprises a chemical modification shown in formula (I') or a tautomer modification thereof:

其中，Y選自O、NH和S； Wherein, 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;

B是鹼基； B is a base;

M為O或S； M is O or S;

其中，該式(I’)所示的化學修飾或其互變異構體修飾不是

。 Wherein, the chemical modification shown in the formula (I') or its tautomer modification is not

.

在一些實施方案中，反義股在其5’區域的第2位至第8位(如第2位、第3位、第4位、第5位、第6位、第7位、第8位)中的至少一個核苷酸位置處包含式(I’-1)所示的化學修飾或其互變異構體修飾： In some embodiments, the antisense strand is at position 2 to position 8 (e.g., position 2, position 3, position 4, position 5, position 6, position 7, position 8) of its 5' region. At least one nucleotide position in position) comprises the chemical modification shown in formula (I'-1) or its tautomer modification:

其中，Y選自O、NH和S； Wherein, 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').

在一些實施方案中，反義股在其5’區域的第2位至第8位(如第2位、第3位、第4位、第5位、第6位、第7位、第8位)中的至少一個核苷酸位置處包含式(I’-2)所示的化學修飾或其互變異構體修飾： In some embodiments, the antisense strand is at position 2 to position 8 (e.g., position 2, position 3, position 4, position 5, position 6, position 7, position 8) of its 5' region. At least one nucleotide position in position) comprises the chemical modification shown in formula (I'-2) or its tautomer modification:

其中Y選自O、NH和S； Wherein 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').

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;

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.

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;

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 is selected from H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl and (CH ₂ ) _q R ₇ ; where R ₇ is selected from OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, q=1 or 2;

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

在一些實施方案中，該式(I’)所示的化學修飾或其互變異構體修飾選自： In some embodiments, the chemical modification represented by the formula (I') or its tautomeric modification is selected from:

其中，M為O或S； Wherein, M is O or S;

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

其中，M為O或S； Wherein, M is O or S;

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

其中，M為O或S； Wherein, M is O or S;

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

在一些實施方案中，該式(I’)所示的化學修飾或其互變異構體修飾包括但不限於： In some embodiments, the chemical modification or tautomer modification represented by the formula (I') includes but is not limited to:

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 the adenine in their structure is replaced by guanine, cytosine, uracil or thymine.

在一些實施方案中，反義股在其5’區域的第2位至第8位、第3位至第8位、第4位至第8位、第5位至第8位、第5位至第7位中的至少一個核苷酸位置處包含上述式(I)或式(I’)所示的化學修飾或其互變異構體修飾。 In some embodiments, the antisense strand has positions 2 to 8, 3 to 8, 4 to 8, 5 to 8, 5 At least one nucleotide position to the 7th position contains the chemical modification represented by the above formula (I) or formula (I') or its tautomer modification.

在一些實施方案中，本揭露的siRNA，反義股在其5’區域的第5位、第6位或第7位包含上述式(I)或式(I’)所示的化學修飾或其互變異構體修飾。 In some embodiments, the siRNA of the present disclosure, the antisense strand comprises the chemical modification shown in the above-mentioned formula (I) or formula (I') or its Tautomeric modification.

一些具體的實施方案中，式(I)或式(I’)所示的化學修飾或其互變異構體修飾在其5’區域的第5位，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；在一些實施方案中選自腺嘌呤。 In some specific embodiments, the chemical modification represented by formula (I) or formula (I') or its tautomer modification is at the 5th position of its 5' region, and B is selected from adenine, guanine, 2, 6-Diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole; in some implementations The scheme is selected from adenine.

一些具體的實施方案中，式(I)或式(I’)所示的化學修飾或其互變異構體修飾在其5’區域的第6位，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；在一些實施方案中選自腺嘌呤。 In some specific embodiments, the chemical modification represented by formula (I) or formula (I') or its tautomer modification is at the 6th position of its 5' region, and B is selected from adenine, guanine, 2, 6-Diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole; in some implementations The scheme is selected from adenine.

一些具體的實施方案中，式(I)或式(I’)所示的化學修飾或其互變異構體修飾在其5’區域的第7位，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；在一些實施方案中選自鳥嘌呤。 In some specific embodiments, the chemical modification shown in formula (I) or formula (I') or its tautomer modification is at the 7th position of its 5' region, and B is selected from adenine, guanine, 2, 6-Diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole; in some implementations The scheme is selected from guanine.

在一些實施方案中，正義股和反義股各自獨立地具有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。 In some embodiments, the sense and antisense strands each independently have 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31 , 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24 , or 19 to 23 nucleotides.

在一些實施方案中，正義股和反義股長度相同或不同，該正義股的長度為19-23個核苷酸，反義股的長度為19-26個核苷酸。這樣，本揭露提供的siRNA的正義股和反義股的長度比可以是19/20、19/21、19/22、19/23、19/24、19/25、19/26、20/20、20/21、20/22、20/23、20/24、20/25、20/26、21/20、21/21、21/22、21/23、21/24、21/25、21/26、22/20、22/21、22/22、22/23、22/24、22/25、22/26、23/20、23/21、23/22、23/23、23/24、23/25或23/26。在一些實施方式中，該siRNA的正義股和反義股的長度比為19/21、21/23或23/25。在一些實施方式中，該siRNA的正義股和反義股的長度比為19/21。 In some embodiments, the sense strand and the antisense strand are the same or different in length, the sense strand being 19-23 nucleotides in length and the antisense strand being 19-26 nucleotides in length. In this way, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure can be 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20 , 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21 /26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24 , 23/25 or 23/26. In some embodiments, the sense and antisense strands of the siRNA have a length ratio of 19/21, 21/23, or 23/25. In some embodiments, the length ratio of the sense strand and the antisense strand of the siRNA is 19/21.

在一些實施方案中，反義股與靶序列至少部分地反向互補以介導RNA干擾；在一些實施方案中，反義股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，反義股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 to mediate RNA interference; in some embodiments, there are no more than 5, no more than 4, between the antisense strand and the target sequence , no more than 3, no more than 2, no more than 1 mismatch; in some embodiments, the antisense strand is fully reverse complementary to the target sequence.

在一些實施方案中，正義股與反義股至少部分地反向互補以形成雙股區；在一些實施方案中，正義股與反義股之間存在不多於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，正義股與反義股完全反向互補。 In some embodiments, the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region; in some embodiments, there are no more than 5, no more than 4, between the sense and antisense strands , no more than 3, no more than 2, no more than 1 mismatch; in some embodiments, the sense and antisense strands are fully reverse complementary.

在一些實施方案中，本揭露siRNA包含一個或兩個平端。 In some embodiments, siRNAs of the present disclosure comprise one or two blunt ends.

一些具體的實施方案中，siRNA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 specific embodiments, the siRNA comprises an overhang with 1 to 4 unpaired nucleotides, eg 1, 2, 3, 4.

在一些實施方案中，本揭露siRNA包含位於該siRNA反義股3’端的突出端。 In some embodiments, a siRNA of the present disclosure comprises an overhang located at the 3' end of the antisense strand of the siRNA.

一些實施方案中，本揭露siRNA正義股包含至少15個連續核苷酸，且與SEQ ID NO：3、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：20和SEQ ID NO：22中的任一核苷酸序列相差不超過3個核苷酸。 In some embodiments, the siRNA sense strand of the present disclosure comprises at least 15 consecutive nucleotides and is identical to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID Any nucleus in NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 22 The nucleotide sequences differ by no more than 3 nucleotides.

一些實施方案中，本揭露siRNA反義股包含至少15個連續核苷酸序列，且與SEQ ID NO：4、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21和SEQ ID NO：23中的任一核苷酸序列相差不超過3個核苷酸。 In some embodiments, the siRNA antisense strand of the present disclosure comprises at least 15 contiguous nucleotide sequences, and is identical to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO : 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23 have a difference of no more than 3 nucleotides.

一些實施方案中，本揭露siRNA正義股核苷酸序列包含或為： In some embodiments, the nucleotide sequence of the siRNA sense strand disclosed herein comprises or is:

5’-GUG UGC ACU UCG CUU CAC C-3’(SEQ ID NO：3)；或， 5'-GUG UGC ACU UCG CUU CAC C-3' (SEQ ID NO: 3); or,

5’-CUU UUG UCU UUG GGU AUA U-3’(SEQ ID NO：5)；或， 5'-CUU UUG UCU UUG GGU AUA U-3' (SEQ ID NO: 5); or,

5’-UUA CCA AUU UUC UUU UGU U-3’(SEQ ID NO：7)；或， 5'-UUA CCA AUU UUC UUU UGU U-3' (SEQ ID NO: 7); or,

5’-CGU GUG CAC UUC GCU UCA C-3’(SEQ ID NO：9)；或， 5'-CGU GUG CAC UUC GCU UCA C-3' (SEQ ID NO: 9); or,

5’-UGU CUU UGG GUA UAC AUU U-3’(SEQ ID NO：11)；或， 5'-UGU CUU UGG GUA UAC AUU U-3' (SEQ ID NO: 11); or,

5’-CUU UUG UCU UUG GGU AUA C-3’(SEQ ID NO：13)；或， 5'-CUU UUG UCU UUG GGU AUA C-3' (SEQ ID NO: 13); or,

5’-CAU CUU CUU GUU GGU UCU U-3’(SEQ ID NO：14)；或， 5'-CAU CUU CUU GUU GGU UCU U-3' (SEQ ID NO: 14); or,

5’-UGU CUG CGG CGU UUU AUC A-3’(SEQ ID NO：16)；或， 5'-UGU CUG CGG CGU UUU AUC A-3' (SEQ ID NO: 16); or,

5’-UGC ACU UCG CUU CAC CUC U-3’(SEQ ID NO：18)；或， 5'-UGC ACU UCG CUU CAC CUC U-3' (SEQ ID NO: 18); or,

5’-GCA CUU CGC UUC ACC UCU G-3’(SEQ ID NO：20)；或， 5'-GCA CUU CGC UUC ACC UCU G-3' (SEQ ID NO: 20); or,

5’-GGC GCU GAA UCC UGC GGA C-3’(SEQ ID NO：22)。 5'-GGC GCU GAA UCC UGC GGA C-3' (SEQ ID NO: 22).

一些實施方案中，本揭露siRNA正義股核苷酸序列選自： In some embodiments, the nucleotide sequence of the siRNA sense strand disclosed herein is selected from:

一些實施方案中，本揭露siRNA反義股核苷酸序列包含或為： In some embodiments, the siRNA antisense strand nucleotide sequence disclosed herein comprises or is:

5’-AGU GAA W’CG AAG UGC ACA CGG-3’(SEQ ID NO：110)；或， 5'-AGU GAA W'CG AAG UGC ACA CGG-3' (SEQ ID NO: 110); or,

5’-AUA UAC W’CA AAG ACA AAA GAA-3’(SEQ ID NO：111)；或， 5'-AUA UAC W'CA AAG ACA AAA GAA-3' (SEQ ID NO: 111); or,

5’-AAC AAA W’GA AAA UUG GUA ACA-3’(SEQ ID NO：112)；或， 5'-AAC AAA W'GA AAA UUG GUA ACA-3' (SEQ ID NO: 112); or,

5’-AUG AAG W’GA AGU GCA CAC GGU-3’(SEQ ID NO：113)；或， 5'-AUG AAG W'GA AGU GCA CAC GGU-3' (SEQ ID NO: 113); or,

5’-AAA UGU W’UA CCC AAA GAC AAA-3’(SEQ ID NO：114)；或， 5'-AAA UGU W'UA CCC AAA GAC AAA-3' (SEQ ID NO: 114); or,

5’-AAG AAC W’AA CAA GAA GAU GAG-3’(SEQ ID NO：115)；或， 5'-AAG AAC W'AA CAA GAA GAU GAG-3' (SEQ ID NO: 115); or,

5’-UGA UAA W’AC GCC GCA GAC ACA-3’(SEQ ID NO：116)；或， 5'-UGA UAA W'AC GCC GCA GAC ACA-3' (SEQ ID NO: 116); or,

5’-AGA GGU W’AA GCG AAG UGC ACA-3’(SEQ ID NO：117)；或， 5'-AGA GGU W'AA GCG AAG UGC ACA-3' (SEQ ID NO: 117); or,

5’-UAG AGG W’GA AGC GAA GUG CAC-3’(SEQ ID NO：118)；或， 5'-UAG AGG W'GA AGC GAA GUG CAC-3' (SEQ ID NO: 118); or,

5’-AUC CGC W’GG AUU CAG CGC CGA-3’(SEQ ID NO：119)；其中，W’表示含有本揭露任一式(I)或式(I’)所示的化學修飾或其互變異構體修飾的核苷酸。 5'-AUC CGC W'GG AUU CAG CGC CGA-3' (SEQ ID NO: 119); wherein, W' represents any chemical modification shown in formula (I) or formula (I') of the present disclosure or its mutual Isomerically modified nucleotides.

一些實施方案中，本揭露siRNA反義股核苷酸序列選自： In some embodiments, the siRNA antisense strand nucleotide sequence disclosed herein is selected from:

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AGU GAA W’CG AAG UGC ACA CGG-3’(SEQ ID NO：120)，該W’含有的化學修飾或其互變異構體修飾選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-AGU GAA W'CG AAG UGC ACA CGG-3' (SEQ ID NO: 120), the W' contains chemical modification or Its tautomeric modification is selected from:

、

或

；其中，B為鳥嘌呤。

,

or

; Wherein, B is guanine.

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AUA UAC W’CA AAG ACA AAA GAA-3’(SEQ ID NO：121)，該W’含有的化學修飾或其互變異構體修飾選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-AUA UAC W'CA AAG ACA AAA GAA-3' (SEQ ID NO: 121), the W' contains chemical modification or Its tautomeric modification is selected from:

、

或

；其中，B為胞嘧啶。

,

or

; Wherein, B is cytosine.

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AAC AAA W’GA AAA UUG GUA ACA-3’(SEQ ID NO：122)，該W’含有的化學修飾或其互變異構體修飾選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-AAC AAA W'GA AAA UUG GUA ACA-3' (SEQ ID NO: 122), the W' contains chemical modification or Its tautomeric modification is selected from:

或

；其中，B為腺嘌呤。

or

; Wherein, B is adenine.

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AUG AAG W’GA AGU GCA CAC GGU-3’(SEQ ID NO：123)，該W’含有的化學修飾或其互變異構體修飾選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-AUG AAG W'GA AGU GCA CAC GGU-3' (SEQ ID NO: 123), the W' contains chemical modification or Its tautomeric modification is selected from:

、

或

；其中，B為胞嘧啶。

,

or

; Wherein, B is cytosine.

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AAA UGU W’UA CCC AAA GAC AAA-3’(SEQ ID NO：124)，該W’含有的化學修飾或其互變異構體修飾選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-AAA UGU W'UA CCC AAA GAC AAA-3' (SEQ ID NO: 124), the W' contains chemical modification or Its tautomeric modification is selected from:

、

或

；其中，B為腺嘌呤。

,

or

; Wherein, B is adenine.

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AAG AAC W’AA CAA GAA GAU GAG-3’(SEQ ID NO：125)，該W’含有的化學修飾或其互變異構體修飾選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-AAG AAC W'AA CAA GAA GAU GAG-3' (SEQ ID NO: 125), the W' contains chemical modification or Its tautomeric modification is selected from:

、

或

；其中，B為胞嘧啶。

,

or

; Wherein, B is cytosine.

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-UGA UAA W’AC GCC GCA GAC ACA-3’(SEQ ID NO：126)，該W’含有的化學修飾或其互變異構體修飾選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-UGA UAA W'AC GCC GCA GAC ACA-3' (SEQ ID NO: 126), the W' contains chemical modification or Its tautomeric modification is selected from:

、

或

；其中，B為腺嘌呤。

,

or

; Wherein, B is adenine.

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AGA GGU W’AA GCG AAG UGC ACA-3’(SEQ ID NO：127)，該W’含有的化學修飾或其互變異構體修飾選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-AGA GGU W'AA GCG AAG UGC ACA-3' (SEQ ID NO: 127), the W' contains chemical modification or Its tautomeric modification is selected from:

、

或

；其中，B為鳥嘌呤。

,

or

; Wherein, B is guanine.

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-UAG AGG W’GA AGC GAA GUG CAC-3’(SEQ ID NO：128)，該W’含有的化學修飾或其互變異構體修飾選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-UAG AGG W'GA AGC GAA GUG CAC-3' (SEQ ID NO: 128), the W' contains chemical modification or Its tautomeric modification is selected from:

、

或

；其中，B為尿嘧啶。

,

or

; Wherein, B is uracil.

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AUC CGC W’GG AUU CAG CGC CGA-3’(SEQ ID NO：129)，該W’含有的化學修飾或其互變異構體修飾選自： In some embodiments, the siRNA antisense strand disclosed herein comprises or is a nucleotide sequence: 5'-AUC CGC W'GG AUU CAG CGC CGA-3' (SEQ ID NO: 129), the W' contains chemical modifications or Its tautomeric modification is selected from:

、

或

；其中，B為腺嘌呤。

,

or

; Wherein, B is adenine.

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AGU GAA W’CG AAG UGC ACA CGG-3’(SEQ ID NO：130)，該W’選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-AGU GAA W'CG AAG UGC ACA CGG-3' (SEQ ID NO: 130), the W' is selected from:

、

和

；其中，M為O或S；其中， B為鳥嘌呤。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。

,

and

; Wherein, M is O or S; Wherein, B is guanine. In some specific embodiments, M is S. In some specific embodiments, M is O.

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AUA UAC W’CA AAG ACA AAA GAA-3’(SEQ ID NO：131)，該W’選自： In some embodiments, the disclosed siRNA antisense strand comprises or is a nucleotide sequence: 5'-AUA UAC W'CA AAG ACA AAA GAA-3' (SEQ ID NO: 131), the W' is selected from:

、

和

；其中，M為O或S；其中， B為胞嘧啶。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。

,

and

; Wherein, M is O or S; Wherein, B is cytosine. In some specific embodiments, M is S. In some specific embodiments, M is O.

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AAC AAA W’GA AAA UUG GUA ACA-3’(SEQ ID NO：132)，該W’選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-AAC AAA W'GA AAA UUG GUA ACA-3' (SEQ ID NO: 132), the W' is selected from:

、

和

；其中，M為O或S；其中， B為腺嘌呤。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。

,

and

; Wherein, M is O or S; Wherein, B is adenine. In some specific embodiments, M is S. In some specific embodiments, M is O.

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AUG AAG W’GA AGU GCA CAC GGU-3’(SEQ ID NO：133)，該W’選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-AUG AAG W'GA AGU GCA CAC GGU-3' (SEQ ID NO: 133), the W' is selected from:

、

和

,

and

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AAA UGU W’UA CCC AAA GAC AAA-3’(SEQ ID NO：134)，該W’選自： In some embodiments, the siRNA antisense strand disclosed herein comprises or is a nucleotide sequence: 5'-AAA UGU W'UA CCC AAA GAC AAA-3' (SEQ ID NO: 134), the W' is selected from:

、

和

,

and

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AAG AAC W’AA CAA GAA GAU GAG-3’(SEQ ID NO：135)，該W’選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-AAG AAC W'AA CAA GAA GAU GAG-3' (SEQ ID NO: 135), the W' is selected from:

、

和

,

and

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-UGA UAA W’AC GCC GCA GAC ACA-3’(SEQ ID NO：136)，該W’選自： In some embodiments, the siRNA antisense strand disclosed herein comprises or is a nucleotide sequence: 5'-UGA UAA W'AC GCC GCA GAC ACA-3' (SEQ ID NO: 136), the W' is selected from:

、

和

,

and

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AGA GGU W’AA GCG AAG UGC ACA-3’(SEQ ID NO：137)，該W’選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-AGA GGU W'AA GCG AAG UGC ACA-3' (SEQ ID NO: 137), the W' is selected from:

、

和

,

and

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-UAG AGG W’GA AGC GAA GUG CAC-3’(SEQ ID NO：138)，該W’選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-UAG AGG W'GA AGC GAA GUG CAC-3' (SEQ ID NO: 138), the W' is selected from:

、

和

；其中，M為O或S；其中， B為尿嘧啶。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。

,

and

; Wherein, M is O or S; Wherein, B is uracil. In some specific embodiments, M is S. In some specific embodiments, M is O.

一些實施方案中，本揭露siRNA反義股包含或為核苷酸序列：5’-AUC CGC W’GG AUU CAG CGC CGA-3’(SEQ ID NO：139)，該W’選自： In some embodiments, the siRNA antisense strand of the present disclosure comprises or is a nucleotide sequence: 5'-AUC CGC W'GG AUU CAG CGC CGA-3' (SEQ ID NO: 139), the W' is selected from:

、

和

,

and

本揭露還提供了一種siRNA，其為經修飾的上述siRNA，除含有上述式(I)或式(I’)所示的化學修飾或其互變異構體修飾外，正義股和/或反義股中至少一個另外的核苷酸為修飾的核苷酸。 The present disclosure also provides an siRNA, which is the modified above-mentioned siRNA, except containing the chemical modification shown in the above formula (I) or formula (I') or its tautomer modification, the sense strand and/or the antisense At least one additional nucleotide in the strand is a modified nucleotide.

在一些實施方案中，除含有上述式(I)或式(I’)所示的化學修飾或其互變異構體修飾外，全部另外的核苷酸為修飾的核苷酸。 In some embodiments, all additional nucleotides are modified nucleotides, except those containing the chemical modifications shown in formula (I) or formula (I') above or tautomeric modifications thereof.

在一些實施方案中，修飾的核苷酸相互獨立地選自脫氧-核苷酸、3’-末端脫氧-胸腺嘧啶(dT)核苷酸、2’-O-甲基修飾的核苷酸、2’-氟修飾的核苷酸、2’-脫氧-修飾的核苷酸、鎖核苷酸、非鎖核苷酸、構象限制性核苷酸、限制性乙基核苷酸、無鹼基核苷酸、2’-胺基-修飾的核苷酸、2’-O-烯丙基-修飾的核苷酸、2’-C-烷基-修飾的核苷酸、2’-羥基-修飾的核苷酸、2’-甲氧基乙基修飾的核苷酸、2’-O-烷基-修飾的核苷酸、嗎啉基核苷酸、胺基磷酸酯、包含非天然鹼基的核苷酸、四氫吡喃修飾的核苷酸、1,5-脫水己糖醇修飾的核苷酸、環己烯基修飾的核苷酸、包含硫代磷酸酯基的核苷酸、包含甲基膦酸酯基的核苷酸、包含5’-磷酸酯的核苷酸及包含5’-磷酸酯模擬物的核苷酸。 In some embodiments, the modified nucleotides are independently selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally constrained nucleotides, constrained ethyl nucleotides, abasic Nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy- Modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, containing unnatural bases nucleotides modified with tetrahydropyran, nucleotides modified with 1,5-anhydrohexitol, nucleotides modified with cyclohexenyl, nucleotides containing phosphorothioate groups , a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, and a nucleotide comprising a 5'-phosphate mimetic.

在一些實施方案中，修飾的核苷酸相互獨立地選自：2'-烷氧基修飾的核苷酸、2'-經取代的烷氧基修飾的核苷酸、2'-烷基修飾的核苷酸、2'-經取代的烷基修飾的核苷酸、2'-胺基修飾的核苷酸、2'-經取代的胺基修飾的核苷酸、2'-氟修飾的核苷酸、2'-脫氧核苷酸、2'-脫氧-2'-氟修飾的核苷酸、3'-脫氧-胸腺嘧啶(dT)核苷酸、異核苷酸、LNA、ENA、cET、UNA和GNA； In some embodiments, the modified nucleotides are independently selected from: 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl modified Nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amine-modified nucleotides, 2'-fluoro-modified Nucleotides, 2'-deoxynucleotides, 2'-deoxy-2'-fluoro-modified nucleotides, 3'-deoxy-thymine (dT) nucleotides, isonucleotides, LNA, ENA, cET, UNA and GNA;

在一些實施方案中，修飾的核苷酸相互獨立地選自：2'-甲氧基修飾的核苷酸、2'-氟修飾的核苷酸和2'-脫氧-修飾的核苷酸。 In some embodiments, the modified nucleotides are independently selected from: 2'-methoxy-modified nucleotides, 2'-fluoro-modified nucleotides, and 2'-deoxy-modified nucleotides.

在本揭露的上下文中，2’-氟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。在一些實施方式中，2'-烷氧基修飾的核苷酸為2’-甲氧基修飾的核苷酸(2'-OMe)。在一些實施方式中，2'-經取代的烷氧基修飾的核苷酸，例如可以是2'-O-甲氧基乙基修飾的核苷酸(2'-MOE)或2'-胺基修飾的核苷酸(2'-NH₂)。 In the context of the present disclosure, a 2'-fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine. In some embodiments, the 2'-alkoxy modified nucleotide is a 2'-methoxy modified nucleotide (2'-OMe). In some embodiments, the 2'-substituted alkoxy-modified nucleotide, for example, can be a 2'-O-methoxyethyl-modified nucleotide (2'-MOE) or a 2'-amine base-modified nucleotides (2'-NH ₂ ).

在一些實施方案中，按照5'末端到3'末端的方向，反義股的第2、6、14位的核苷酸各自獨立地為2'-脫氧核苷酸或2'-氟修飾的核苷酸。 In some embodiments, according to the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, and 14 of the antisense strand are each independently 2'-deoxynucleotide or 2'-fluoro modified Nucleotides.

在一些實施方案中，按照5'末端到3'末端的方向，反義股的第2、6、14和16位的核苷酸各自獨立地為2'-脫氧核苷酸或2'-氟修飾的核苷酸。 In some embodiments, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are each independently a 2'-deoxynucleotide or a 2'-fluoronucleotide in the direction from the 5' end to the 3' end Modified nucleotides.

在一些實施方案中，按照5'末端到3'末端的方向，反義股的第2、6、9、12和14位的核苷酸各自獨立地為2'-脫氧核苷酸或2'-氟修飾的核苷酸。 In some embodiments, the nucleotides at positions 2, 6, 9, 12 and 14 of the antisense strand are each independently a 2'-deoxynucleotide or a 2' - Fluorine-modified nucleotides.

在一些實施方案中，按照5'末端到3'末端的方向，反義股的第2、6、10、12和14位的核苷酸各自獨立地為2'-脫氧核苷酸或2'-氟修飾的核苷酸。 In some embodiments, the nucleotides at positions 2, 6, 10, 12 and 14 of the antisense strand are each independently a 2'-deoxynucleotide or a 2' - Fluorine-modified nucleotides.

在一些實施方案中，按照5'末端到3'末端的方向，反義股的第2、4、6、9、12、14和18位的核苷酸各自獨立地為2'-脫氧核苷酸或2'-氟修飾的核苷酸。 In some embodiments, the nucleotides at positions 2, 4, 6, 9, 12, 14, and 18 of the antisense strand are each independently a 2'-deoxynucleoside in the direction from the 5' end to the 3' end Acid or 2'-fluoro modified nucleotides.

在一些實施方案中，按照5'末端到3'末端的方向，反義股的第2、4、6、10、12、14和18位的核苷酸各自獨立地為2'-脫氧核苷酸或2'-氟修飾的核苷酸。 In some embodiments, the nucleotides at positions 2, 4, 6, 10, 12, 14, and 18 of the antisense strand are each independently a 2'-deoxynucleoside in the direction from the 5' end to the 3' end Acid or 2'-fluoro modified nucleotides.

在一些實施方案中，按照5'末端到3'末端的方向，反義股的第2、4、6、9、12、14、16和18位的核苷酸各自獨立地為2'-脫氧核苷酸或2'-氟修飾的核苷酸。 In some embodiments, the nucleotides at positions 2, 4, 6, 9, 12, 14, 16, and 18 of the antisense strand are each independently 2'-deoxy in the direction from the 5' end to the 3' end Nucleotides or 2'-fluoro-modified nucleotides.

在一些實施方案中，按照5'末端到3'末端的方向，反義股的第2、4、6、10、12、14、16和18位的核苷酸各自獨立地為2'-脫氧核苷酸或2'-氟修飾的核苷酸。 In some embodiments, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, and 18 of the antisense strand are each independently 2'-deoxy in the direction from the 5' end to the 3' end Nucleotides or 2'-fluoro-modified nucleotides.

在一些實施方案中，按照5'末端到3'末端的方向，反義股的第2、4、6、9、10、12、14、16和18位的核苷酸各自獨立地為2'-脫氧核苷酸或2'-氟修飾的核苷酸。 In some embodiments, the nucleotides at positions 2, 4, 6, 9, 10, 12, 14, 16, and 18 of the antisense strand are each independently 2' in the direction from the 5' end to the 3' end - deoxynucleotides or 2'-fluoro-modified nucleotides.

在一些實施方案中，本揭露所述的siRNA正義股具有如下式所示的核苷酸序列： In some embodiments, the siRNA sense strand described in the present disclosure has 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，每個N_a和N_b各自獨立地表示修飾的核苷酸或者未修飾的核苷酸，且N_a和N_b是不同的修飾，每個X獨立地為N_a或N_b。 Wherein, each N _a and N _b independently represents a modified nucleotide or an unmodified nucleotide, and Na _and N _b are different modifications, and each X is independently N _a or N _b .

在一些實施方案中，本揭露所述的siRNA反義股具有如下式所示的核苷酸序列： In some embodiments, the siRNA antisense strand described in the present disclosure has a nucleotide sequence as shown in the following formula:

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

其中，每個N_a’和N_b’獨立地表示修飾的核苷酸或者未修飾的核苷酸，其中N_a’和N_b’上的修飾不同；每個X’獨立地為N_a’或N_b’，Y’為N_a’或N_b’，W’表示含有本揭露任一式(I)或式(I’)所示的化學修飾或其互變異構體修飾的核苷酸。 Wherein, each N _a ' and N _b ' independently represents a modified nucleotide or an unmodified nucleotide, wherein the modifications on Na _' and N _b ' are different; each X' is independently N _a ' Or N _b ', Y' is N _a ' or N _b ', W' represents a nucleotide containing any chemical modification shown in formula (I) or formula (I') of the present disclosure or its tautomer modification.

在一些實施方案中，X’和Y’上的修飾不同。 In some embodiments, the modifications on X' and Y' are different.

在一些實施方案中，N_a為2'-甲氧基修飾的核苷酸，N_b為2'-氟修飾的核苷酸或2'-脫氧-修飾的核苷酸。 In some embodiments, _Na is a 2'-methoxy-modified nucleotide and _Nb is a 2'-fluoro-modified or 2'-deoxy-modified nucleotide.

在一些實施方案中，N_a’為2'-甲氧基修飾的核苷酸，N_b’為2'-氟修飾的核苷酸或2'-脫氧-修飾的核苷酸。 In some embodiments, _Na ' is a 2'-methoxy-modified nucleotide and _Nb ' is a 2'-fluoro-modified or 2'-deoxy-modified nucleotide.

在一些具體的實施方案中，N_a為2'-甲氧基修飾的核苷酸，N_b為2'-氟修飾的核苷酸。 In some specific embodiments, N _a is a 2'-methoxy modified nucleotide and N _b is a 2'-fluoro modified nucleotide.

在一些具體的實施方案中，N_a’為2'-甲氧基修飾的核苷酸，N_b’為2'-氟修飾的核苷酸。 In some specific embodiments, N _a ' is a 2'-methoxy-modified nucleotide, and N _b ' is a 2'-fluoro-modified nucleotide.

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

其中，每個X’獨立地為N_a’或N_b’，Y’為N_a’或N_b’，且X’和Y’上的修飾不同；N_a’為2'-甲氧基修飾的核苷酸，N_b’為2'-氟修飾的核苷酸；W’ 表示含有本揭露任一式(I)或式(I’)所示的化學修飾或其互變異構體修飾的核苷酸。 Wherein, each X' is independently Na _' or N _b ', Y' is Na _' or N _b ', and the modifications on X' and Y' are different; _Na ' is 2'-methoxy modification N _b 'is a 2'-fluorine-modified nucleotide; W' represents a chemical modification or a tautomer modification containing any formula (I) or formula (I') of the present disclosure. glycosides.

5’-N_aN_aN_aN_aN_aN_aN_bN_bN_bN_aN_aN_aN_aN_aN_aN_aN_aN_aN_a-3’；或， 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'; or,

5’-N_aN_aN_aN_aN_bN_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 _b 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。 Wherein, N _a is a 2'-methoxy-modified nucleotide, and N _b is a 2'-fluoro-modified nucleotide.

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

5’-N_a’N_b’N_a’N_b’N_a’N_b’W’N_a’N_b’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 _b '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';

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； Wherein, N _a 'is a 2'-methoxy-modified nucleotide, and N _b ' is a 2'-fluoro-modified nucleotide;

W’表示含有本揭露任一式(I)或式(I’)所示的化學修飾或其互變異構體修飾的核苷酸。 W' represents a nucleotide containing any chemical modification shown in formula (I) or formula (I') of the present disclosure or a tautomer modification thereof.

在一些具體的實施方案中，W’表示含有化學修飾或其互變異構體修飾的核苷酸；該化學修飾或其互變異構體修飾選自： In some specific embodiments, W' represents a nucleotide containing a chemical modification or a tautomeric modification thereof; the chemical modification or a tautomeric modification thereof is selected from:

、

或

；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選自反義股在其5’區域的第7位中對應位置的鹼基。

,

or

; Wherein, B is selected from guanine, adenine, cytosine or uracil. In some specific embodiments, B is selected from the base corresponding to the 7th position in the 5' region of the antisense strand.

、

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選自反義股在其5’區域的第7位中對應位置的鹼基。

,

and

; Wherein, M is O or S; Wherein, B is selected from guanine, adenine, cytosine or uracil. In some specific embodiments, B is selected from the base corresponding to the 7th position in the 5' region of the antisense strand.

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_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，每個N_a和N_b各自獨立地表示修飾的核苷酸或者未修飾的核苷酸，且N_a和N_b是不同的修飾，每個X獨立地為N_a或N_b；和/或 Wherein, each N _a and N _b independently represents a modified nucleotide or an unmodified nucleotide, and N _a and N _b are different modifications, and each X is independently N _a or N _b ; and /or

反義股具有如下式所示的核苷酸序列： The antisense strand has the nucleotide sequence shown in the following formula:

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

其中，每個N_a’和N_b’獨立地表示修飾的核苷酸或者未修飾的核苷酸，其中N_a’和N_b’上的修飾不同；每個X’獨立地為N_a’或N_b’；Y’為N_a’或N_b’；W’表示含有本揭露任一式(I)或式(I’)所示的化學修飾或其互變異構體修飾的核苷酸。 Wherein, each N _a ' and N _b ' independently represents a modified nucleotide or an unmodified nucleotide, wherein the modifications on Na _' and N _b ' are different; each X' is independently N _a ' or N _b ';Y' is N _a ' or N _b ';W' represents a nucleotide containing any chemical modification shown in formula (I) or formula (I') of the present disclosure or its tautomer modification.

在一些實施方案中，N_a為2'-甲氧基修飾的核苷酸，N_b為2'-氟修飾的核苷酸或2'-脫氧-修飾的核苷酸；和/或，N_a’為2'-甲氧基修飾的核苷酸，N_b’為2'-氟修飾的核苷酸或2'-脫氧-修飾的核苷酸； In some embodiments, N _a is a 2'-methoxy-modified nucleotide, N _b is a 2'-fluoro-modified nucleotide or a 2'-deoxy-modified nucleotide; and/or, N _a 'is a 2'-methoxy-modified nucleotide, N _b ' is a 2'-fluoro-modified nucleotide or a 2'-deoxy-modified nucleotide;

在一些具體的實施方案中，N_a為2'-甲氧基修飾的核苷酸，N_b為2'-氟修飾的核苷酸；和/或，N_a’為2'-甲氧基修飾的核苷酸，N_b’為2'-氟修飾的核苷酸。 In some specific embodiments, N _a is a 2'-methoxy modified nucleotide, N _b is a 2'-fluoro modified nucleotide; and/or, N _a ' is a 2'-methoxy Modified nucleotides, N _b ' 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’；和/或 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'; and/or

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

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；和/或，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, N _a is a 2'-methoxy-modified nucleotide, N _b is a 2'-fluoro-modified nucleotide; and/or, N _a ' is a 2'-methoxy-modified nucleotide, N _b 'is a 2'-fluoro-modified nucleotide;

、

或

；其中，B選自鳥嘌呤、腺嘌呤、胞嘧啶或尿嘧啶。在一些實施方案中，B選自反義股在其5’區域的第7位中對應位置的鹼基。

,

or

; Wherein, B is selected from guanine, adenine, cytosine or uracil. In some embodiments, B is selected from the base corresponding to position 7 in the 5' region of the antisense strand.

、

和

,

and

5’-N_aN_aN_aN_aN_bN_aN_bN_bN_bN_aN_aN_aN_aN_aN_aN_aN_aN_aN_a-3’；和/或 5'-N _a N _a N _a N _a N _b 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'; and/or

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；和/或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, N _a is a 2'-methoxy modified nucleotide, N _b is a 2'-fluoro modified nucleotide; and/or N _a ' is a 2'-methoxy modified nucleotide, N _b ' is a 2'-fluoro-modified nucleotide;

、

或

,

or

、

和

,

and

在一些實施方案中，正義股具有如下式所示的核苷酸序列： In some embodiments, the sense strand has a nucleotide sequence represented by the following formula:

5’-N_aN_aN_aN_aN_bN_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 _b 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N_a和N_b各自獨立地表示修飾的核苷酸或者未修飾的核苷酸，且N_a和N_b是不同的修飾；和/或 Wherein, each N _a and N _b independently represent modified nucleotides or unmodified nucleotides, and N _a and N _b are different modifications; and/or

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

其中，每個N_a’和N_b’獨立地表示修飾的核苷酸或者未修飾的核苷酸，其中N_a’和N_b’上的修飾不同；每個X’獨立地為N_a’或N_b’；Y’為N_a’或 N_b’；W’表示含有本揭露任一式(I)或式(I’)所示的化學修飾或其互變異構體修飾的核苷酸。 Wherein, each N _a ' and N _b ' independently represents a modified nucleotide or an unmodified nucleotide, wherein the modifications on Na _' and N _b ' are different; each X' is independently N _a ' or N _b ';Y' is N _a ' or N _b ';W' represents a nucleotide containing any chemical modification shown in formula (I) or formula (I') of the present disclosure or its tautomer modification.

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

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

在一些實施方案中，正義股和/或反義股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siRNA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。具體地，硫代磷酸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。 In some embodiments, at least one phosphate group in the sense and/or antisense strand is a phosphate group with a modification group that confers increased stability to the siRNA in a biological sample or environment. In some embodiments, the phosphate group having a modifying group is a phosphorothioate group. Specifically, a phosphorothioate group refers to a phosphodiester group modified by replacing a non-bridging oxygen atom with a sulfur atom.

在一些實施方案中，硫代磷酸酯基存在於選自以下的位置中的至少一處： In some embodiments, the phosphorothioate group is present in at least one position selected from:

該正義股的5'末端端部第1個核苷酸和第2個核苷酸之間； between the 1st and 2nd nucleotides of the 5' end of the sense strand;

該正義股的5'末端端部第2個核苷酸和第3個核苷酸之間； between the 2nd and 3rd nucleotides of the 5' end of the sense strand;

該正義股的3'末端端部第1個核苷酸末端； the first nucleotide end of the 3' end of the sense strand;

該正義股的3'末端端部第1個核苷酸和第2個核苷酸之間； between the 1st and 2nd nucleotides of the 3' end of the sense strand;

該正義股的3'末端端部第2個核苷酸和第3個核苷酸之間； between the 2nd and 3rd nucleotides of the 3' end of the sense strand;

該反義股的5'末端端部第1個核苷酸和第2個核苷酸之間； Between the 1st nucleotide and the 2nd nucleotide at the end of the 5' end of the antisense strand;

該反義股的5'末端端部第2個核苷酸和第3個核苷酸之間； Between the 2nd nucleotide and the 3rd nucleotide at the end of the 5' end of the antisense strand;

該反義股的3'末端端部第1個核苷酸末端； The first nucleotide end of the 3' end of the antisense strand;

該反義股的3'末端端部第1個核苷酸和第2個核苷酸之間；以及 between the 1st and 2nd nucleotides of the 3' end of the antisense strand; and

該反義股的3'末端端部第2個核苷酸和第3個核苷酸之間。 The 3' end of the antisense strand is between the 2nd and 3rd nucleotides.

在一些實施方案中，正義股選自如下式所示的核苷酸序列： In some embodiments, the sense strand is selected from a nucleotide sequence represented by the following formula:

5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmsNm-3’，或， 5'-NmsNmsNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmsNm-3', or,

5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmsNms-3’，或， 5'-NmsNmsNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmsNms-3', or,

5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmsNm-3’，或， 5'-NmsNmsNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmsNm-3', or,

5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmsNms-3’ 5'-NmsNmsNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmsNms-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、T；Nf表示2’-氟修飾的任意核苷酸，例如2’-氟修飾的C、G、U、A、T； Wherein, Nm represents any nucleotide modified by 2'-methoxy, such as C, G, U, A, T modified by 2'-methoxy; Nf represents any nucleotide modified by 2'-fluoro, such as 2'-fluoro modified C, G, U, A, T;

小寫字母s在大寫字母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酯基。 When the lowercase letter s is in the middle of the uppercase letter, it means that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate groups. The adjacent one nucleotide terminus is a phosphorothioate group.

在一些實施方案中，反義股具有如下式所示的核苷酸序列： In some embodiments, the antisense strand has a nucleotide sequence represented by the following formula:

5’-Nms’Nfs’Nm’Nf’Nm’Nf’W’Nm’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nf’Nms’Nms’Nm’-3’ 5'-Nms'Nfs'Nm'Nf'Nm'Nf'W'Nm'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nf'Nms'Nms'Nm'-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、T；Nf’表示2’-氟修飾的任意核苷酸，例如2’-氟修飾的C、G、U、A、T； Among them, Nm' represents any nucleotide modified by 2'-methoxy, such as C, G, U, A, T modified by 2'-methoxy; Nf' represents any nucleotide modified by 2'-fluoro , such as 2'-fluoro modified C, G, U, A, T;

小寫字母s在大寫字母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酯基； When the lowercase letter s is in the middle of the uppercase letter, it means that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate groups. One adjacent nucleotide terminal is a phosphorothioate group;

W’表示含有化學修飾或其互變異構體修飾的核苷酸；該化學修飾或其互變異構體修飾選自： W' represents a nucleotide containing a chemical modification or a tautomeric modification thereof; the chemical modification or a tautomeric modification thereof is selected from:

、

或

；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，選自反義股在其5’區域的第7位中對應位置的鹼基。

,

or

; Wherein, B is selected from guanine, adenine, cytosine or uracil. In some specific embodiments, the base selected from the corresponding position in the 7th position of the antisense strand in its 5' region.

、

，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選自反義股在其5’區域的第7位中對應位置的鹼基。

,

,and

在一些實施方案中，本揭露siRNA，其中正義股和反義股選自： In some embodiments, the present disclosure discloses siRNAs wherein the sense strand and the antisense strand are selected from:

正義股：5’-GmsUmsGm UmGfCm AfCfUf UmCmGm CmUmUm CmAmCms Cm-3’(SEQ ID NO：140) Sense strand: 5'-GmsUmsGm UmGfCm AfCfUf UmCmGm CmUmUm CmAmCms Cm-3' (SEQ ID NO: 140)

反義股：5’-AmsGfsUm GfAmAf W’CmGm AfAmGf UmGfCm AfCmAf CmsGmsGm-3’(SEQ ID NO：141) Antisense strand: 5'-AmsGfsUm GfAmAf W'CmGm AfAmGf UmGfCm AfCmAf CmsGmsGm-3' (SEQ ID NO: 141)

或， or,

正義股：5’-GmsUmsGm UmGfCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-3’(SEQ ID NO：142) Sense strand: 5'-GmsUmsGm UmGfCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-3' (SEQ ID NO: 142)

或， or,

正義股：5’-GmsUmsGm UmGmCm AfCfUf UmCmGm CmUmUm CmAmCms Cm-3’(SEQ ID NO：143) Sense strand: 5'-GmsUmsGm UmGmCm AfCfUf UmCmGm CmUmUm CmAmCms Cm-3' (SEQ ID NO: 143)

或， or,

正義股：5’-GmsUmsGm UmGmCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-3’(SEQ ID NO：144) Sense strand: 5'-GmsUmsGm UmGmCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-3' (SEQ ID NO: 144)

或， or,

正義股：5’-UmsGmsCm AmCfUm UfCfGf CmUmUm CmAmCm CmUmCms Um-3’(SEQ ID NO：145) Sense strand: 5'-UmsGmsCm AmCfUm UfCfGf CmUmUm CmAmCm CmUmCms Um-3' (SEQ ID NO: 145)

反義股：5’-AmsGfsAm GfGmUf W’AmAm GfCmGf AmAfGm UfGmCf AmsCmsAm-3’(SEQ ID NO：146) Antisense strand: 5'-AmsGfsAm GfGmUf W'AmAm GfCmGf AmAfGm UfGmCf AmsCmsAm-3' (SEQ ID NO: 146)

或， or,

正義股：5’-UmsGmsCm AmCfUm UfCfGf CmUmUm CmAmCm CmUmCms Ums-3’(SEQ ID NO：147) Sense strand: 5'-UmsGmsCm AmCfUm UfCfGf CmUmUm CmAmCm CmUmCms Ums-3' (SEQ ID NO: 147)

或， or,

正義股：5’-UmsGmsCm AmCmUm UfCfGf CmUmUm CmAmCm CmUmCms Um-3’(SEQ ID NO：148) Sense strand: 5'-UmsGmsCm AmCmUm UfCfGf CmUmUm CmAmCm CmUmCms Um-3' (SEQ ID NO: 148)

或， or,

正義股：5’-UmsGmsCm AmCmUm UfCfGf CmUmUm CmAmCm CmUmCms Ums-3’(SEQ ID NO：149) Sense strand: 5'-UmsGmsCm AmCmUm UfCfGf CmUmUm CmAmCm CmUmCms Ums-3' (SEQ ID NO: 149)

其中，小寫字母m表示該字母m左側相鄰的一個核苷酸為2’-甲氧基修飾的核苷酸；小寫字母f表示該字母f左側相鄰的一個核苷酸為2’-氟修飾的核苷酸； Among them, the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is 2'-fluoro Modified nucleotides;

小寫字母s在大寫字母中間時表示與該字母s左右相鄰的兩個核苷酸之間的連接為硫代磷酸酯基連接； When the lowercase letter s is in the middle of the uppercase letter, it means that the connection between the two nucleotides adjacent to the left and right of the letter s is a phosphorothioate group connection;

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酯基； When the lowercase letter s is the first at the 3' end, it means that a nucleotide terminal adjacent to the left side of the letter s is a phosphorothioate group;

W’表示含有化學修飾或其互變異構體修飾的核苷酸，該化學修飾或其互變異構體修飾選自： W' represents a nucleotide containing a chemical modification or a tautomeric modification thereof selected from:

、

或

，中：B為鳥嘌呤。

,

or

, Middle: B is guanine.

、

和

；其中，M為O或S。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。

,

and

; Wherein, M is O or S. In some specific embodiments, M is S. In some specific embodiments, M is O.

正義股：5’-CmsUmsUm UmUfGm UfCfUf UmUmGm GmGmUm AmUmAms Um-3’(SEQ ID NO：150) Sense strand: 5'-CmsUmsUm UmUfGm UfCfUf UmUmGm GmGmUm AmUmAms Um-3' (SEQ ID NO: 150)

反義股：5’-AmsUfsAm UfAmCf W’CmAm AfAmGf AmCfAm AfAmAf GmsAmsAm-3’(SEQ ID NO：151) Antisense strand: 5'-AmsUfsAm UfAmCf W'CmAm AfAmGf AmCfAm AfAmAf GmsAmsAm-3' (SEQ ID NO: 151)

或， or,

正義股：5’-CmsUmsUm UmUfGm UfCfUf UmUmGm GmGmUm AmUmAms Ums-3’(SEQ ID NO：152) Sense strand: 5'-CmsUmsUm UmUfGm UfCfUf UmUmGm GmGmUm AmUmAms Ums-3' (SEQ ID NO: 152)

或， or,

正義股：5’-CmsUmsUm UmUmGm UfCfUf UmUmGm GmGmUm AmUmAms Um-3’(SEQ ID NO：153) Sense strand: 5'-CmsUmsUm UmUmGm UfCfUf UmUmGm GmGmUm AmUmAms Um-3' (SEQ ID NO: 153)

或， or,

正義股：5’-CmsUmsUm UmUmGm UfCfUf UmUmGm GmGmUm AmUmAms Ums-3’(SEQ ID NO：154) Sense strand: 5'-CmsUmsUm UmUmGm UfCfUf UmUmGm GmGmUm AmUmAms Ums-3' (SEQ ID NO: 154)

或， or,

正義股：5’-CmsGmsUm GmUfGm CfAfCf UmUmCm GmCmUm UmCmAms Cm-3’(SEQ ID NO：155) Sense strand: 5'-CmsGmsUm GmUfGm CfAfCf UmUmCm GmCmUm UmCmAms Cm-3' (SEQ ID NO: 155)

反義股：5’-AmsUfsGm AfAmGf W’GmAm AfGmUf GmCfAm CfAmCf GmsGmsUm-3’(SEQ ID NO：156) Antisense strand: 5'-AmsUfsGm AfAmGf W'GmAm AfGmUf GmCfAm CfAmCf GmsGmsUm-3' (SEQ ID NO: 156)

或， or,

正義股：5’-CmsGmsUm GmUfGm CfAfCf UmUmCm GmCmUm UmCmAms Cms-3’(SEQ ID NO：157) Sense strand: 5'-CmsGmsUm GmUfGm CfAfCf UmUmCm GmCmUm UmCmAms Cms-3' (SEQ ID NO: 157)

或， or,

正義股：5’-CmsGmsUm GmUmGm CfAfCf UmUmCm GmCmUm UmCmAms Cm-3’(SEQ ID NO：158) Sense strand: 5'-CmsGmsUm GmUmGm CfAfCf UmUmCm GmCmUm UmCmAms Cm-3' (SEQ ID NO: 158)

或， or,

正義股：5’-CmsGmsUm GmUmGm CfAfCf UmUmCm GmCmUm UmCmAms Cms-3’(SEQ ID NO：159) Sense strand: 5'-CmsGmsUm GmUmGm CfAfCf UmUmCm GmCmUm UmCmAms Cms-3' (SEQ ID NO: 159)

或， or,

正義股：5’-CmsUmsUm UmUfGm UfCfUf UmUmGm GmGmUm AmUmAms Cm-3’(SEQ ID NO：160) Sense strand: 5'-CmsUmsUm UmUfGm UfCfUf UmUmGm GmGmUm AmUmAms Cm-3' (SEQ ID NO: 160)

或， or,

正義股：5’-CmsUmsUm UmUfGm UfCfUf UmUmGm GmGmUm AmUmAms Cms-3’(SEQ ID NO：161) Sense strand: 5'-CmsUmsUm UmUfGm UfCfUf UmUmGm GmGmUm AmUmAms Cms-3' (SEQ ID NO: 161)

或， or,

正義股：5’-CmsUmsUm UmUmGm UfCfUf UmUmGm GmGmUm AmUmAms Cm-3’(SEQ ID NO：162) Sense strand: 5'-CmsUmsUm UmUmGm UfCfUf UmUmGm GmGmUm AmUmAms Cm-3' (SEQ ID NO: 162)

或， or,

正義股：5’-CmsUmsUm UmUmGm UfCfUf UmUmGm GmGmUm AmUmAms Cms-3’(SEQ ID NO：163) Sense strand: 5'-CmsUmsUm UmUmGm UfCfUf UmUmGm GmGmUm AmUmAms Cms-3' (SEQ ID NO: 163)

或， or,

正義股：5’-CmsAmsUm CmUfUm CfUfUf GmUmUm GmGmUm UmCmUms Um-3’(SEQ ID NO：164) Sense strand: 5'-CmsAmsUm CmUfUm CfUfUf GmUmUm GmGmUm UmCmUms Um-3' (SEQ ID NO: 164)

反義股：5’-AmsAfsGm AfAmCf W’AmAm CfAmAf GmAfAm GfAmUf GmsAmsGm-3’(SEQ ID NO：165) Antisense strand: 5'-AmsAfsGm AfAmCf W'AmAm CfAmAf GmAfAm GfAmUf GmsAmsGm-3' (SEQ ID NO: 165)

或， or,

正義股：5’-CmsAmsUm CmUfUm CfUfUf GmUmUm GmGmUm UmCmUms Ums-3’(SEQ ID NO：166) Sense strand: 5'-CmsAmsUm CmUfUm CfUfUf GmUmUm GmGmUm UmCmUms Ums-3' (SEQ ID NO: 166)

或， or,

正義股：5’-CmsAmsUm CmUmUm CfUfUf GmUmUm GmGmUm UmCmUms Um-3’(SEQ ID NO：167) Sense strand: 5'-CmsAmsUm CmUmUm CfUfUf GmUmUm GmGmUm UmCmUms Um-3' (SEQ ID NO: 167)

或， or,

正義股：5’-CmsAmsUm CmUmUm CfUfUf GmUmUm GmGmUm UmCmUms Ums-3’(SEQ ID NO：168) Sense strand: 5'-CmsAmsUm CmUmUm CfUfUf GmUmUm GmGmUm UmCmUms Ums-3' (SEQ ID NO: 168)

、

或

，中：B為胞嘧啶。

,

or

, Middle: B is cytosine.

、

和

,

and

正義股：5’-UmsUmsAm CmCfAm AfUfUf UmUmCm UmUmUm UmGmUms Um-3’(SEQ ID NO：169) Sense strand: 5'-UmsUmsAm CmCfAm AfUfUf UmUmCm UmUmUm UmGmUms Um-3' (SEQ ID NO: 169)

反義股：5’-AmsAfsCm AfAmAf W’GmAm AfAmAf UmUfGm GfUmAf AmsCmsAm-3’(SEQ ID NO：170) Antisense strand: 5'-AmsAfsCm AfAmAf W'GmAm AfAmAf UmUfGm GfUmAf AmsCmsAm-3' (SEQ ID NO: 170)

或， or,

正義股：5-UmsUmsAm CmCfAm AfUfUf UmUmCm UmUmUm UmGmUms Ums-3’(SEQ ID NO：171) Justice strand: 5-UmsUmsAm CmCfAm AfUfUf UmUmCm UmUmUm UmGmUms Ums-3' (SEQ ID NO: 171)

或， or,

正義股：5’-UmsUmsAm CmCmAm AfUfUf UmUmCm UmUmUm UmGmUms Um-3’(SEQ ID NO：172) Sense strand: 5'-UmsUmsAm CmCmAm AfUfUf UmUmCm UmUmUm UmGmUms Um-3' (SEQ ID NO: 172)

或， or,

正義股：5’-UmsUmsAm CmCmAm AfUfUf UmUmCm UmUmUm UmGmUms Ums-3’(SEQ ID NO：173) Sense strand: 5'-UmsUmsAm CmCmAm AfUfUf UmUmCm UmUmUm UmGmUms Ums-3' (SEQ ID NO: 173)

或， or,

正義股：5’-UmsGmsUm CmUfUm UfGfGf GmUmAm UmAmCm AmUmUms Um-3’(SEQ ID NO：174) Sense strand: 5'-UmsGmsUm CmUfUm UfGfGf GmUmAm UmAmCm AmUmUms Um-3' (SEQ ID NO: 174)

反義股：5’-AmsAfsAm UfGmUf W’UmAm CfCmCf AmAfAm GfAmCf AmsAmsAm-3’(SEQ ID NO：175) Antisense strand: 5'-AmsAfsAm UfGmUf W'UmAm CfCmCf AmAfAm GfAmCf AmsAmsAm-3' (SEQ ID NO: 175)

或， or,

正義股：5’-UmsGmsUm CmUfUm UfGfGf GmUmAm UmAmCm AmUmUms Ums-3’(SEQ ID NO：176) Sense strand: 5'-UmsGmsUm CmUfUm UfGfGf GmUmAm UmAmCm AmUmUms Ums-3' (SEQ ID NO: 176)

或， or,

正義股：5’-UmsGmsUm CmUmUm UfGfGf GmUmAm UmAmCm AmUmUms Um-3’(SEQ ID NO：177) Sense strand: 5'-UmsGmsUm CmUmUm UfGfGf GmUmAm UmAmCm AmUmUms Um-3' (SEQ ID NO: 177)

或， or,

正義股：5’-UmsGmsUm CmUmUm UfGfGf GmUmAm UmAmCm AmUmUms Ums-3’(SEQ ID NO：178) Sense strand: 5'-UmsGmsUm CmUmUm UfGfGf GmUmAm UmAmCm AmUmUms Ums-3' (SEQ ID NO: 178)

或， or,

正義股：5’-UmsGmsUm CmUfGm CfGfGf CmGmUm UmUmUm AmUmCms Am-3’(SEQ ID NO：179) Sense strand: 5'-UmsGmsUm CmUfGm CfGfGf CmGmUm UmUmUm AmUmCms Am-3' (SEQ ID NO: 179)

反義股：5’-UmsGfsAm UfAmAf W’AmCm GfCmCf GmCfAm GfAmCf AmsCmsAm-3’(SEQ ID NO：180) Antisense strand: 5'-UmsGfsAm UfAmAf W'AmCm GfCmCf GmCfAm GfAmCf AmsCmsAm-3' (SEQ ID NO: 180)

或， or,

正義股：5’-UmsGmsUm CmUfGm CfGfGf CmGmUm UmUmUm AmUmCms Ams-3’(SEQ ID NO：181) Sense strand: 5'-UmsGmsUm CmUfGm CfGfGf CmGmUm UmUmUm AmUmCms Ams-3' (SEQ ID NO: 181)

或， or,

正義股：5’-UmsGmsUm CmUmGm CfGfGf CmGmUm UmUmUm AmUmCms Am-3’(SEQ ID NO：182) Sense strand: 5'-UmsGmsUm CmUmGm CfGfGf CmGmUm UmUmUm AmUmCms Am-3' (SEQ ID NO: 182)

或， or,

正義股：5’-UmsGmsUm CmUmGm CfGfGf CmGmUm UmUmUm AmUmCms Ams-3’(SEQ ID NO：183) Sense strand: 5'-UmsGmsUm CmUmGm CfGfGf CmGmUm UmUmUm AmUmCms Ams-3' (SEQ ID NO: 183)

或， or,

正義股：5’-GmsGmsCm GmCfUm GfAfAf UmCmCm UmGmCm GmGmAms Cm-3’(SEQ ID NO：184) Sense strand: 5'-GmsGmsCm GmCfUm GfAfAf UmCmCm UmGmCm GmGmAms Cm-3' (SEQ ID NO: 184)

反義股：5’-AmsUfsCm CfGmCf W’GmGm AfUmUf CmAfGm CfGmCf CmsGmsAm-3’(SEQ ID NO：185) Antisense strand: 5'-AmsUfsCm CfGmCf W'GmGm AfUmUf CmAfGm CfGmCf CmsGmsAm-3' (SEQ ID NO: 185)

或， or,

正義股：5’-GmsGmsCm GmCfUm GfAfAf UmCmCm UmGmCm GmGmAms Cms-3’(SEQ ID NO：186) Sense strand: 5'-GmsGmsCm GmCfUm GfAfAf UmCmCm UmGmCm GmGmAms Cms-3' (SEQ ID NO: 186)

或， or,

正義股：5’-GmsGmsCm GmCmUm GfAfAf UmCmCm UmGmCm GmGmAms Cm-3’(SEQ ID NO：187) Sense strand: 5'-GmsGmsCm GmCmUm GfAfAf UmCmCm UmGmCm GmGmAms Cm-3' (SEQ ID NO: 187)

或， or,

正義股：5’-GmsGmsCm GmCmUm GfAfAf UmCmCm UmGmCm GmGmAms Cms-3’(SEQ ID NO：188) Sense strand: 5'-GmsGmsCm GmCmUm GfAfAf UmCmCm UmGmCm GmGmAms Cms-3' (SEQ ID NO: 188)

、

或

，中：B為腺嘌呤。

,

or

, Middle: B is adenine.

、

和

,

and

正義股：5’-GmsCmsAm CmUfUm CfGfCf UmUmCm AmCmCm UmCmUms Gm-3’(SEQ ID NO：189) Sense strand: 5'-GmsCmsAm CmUfUm CfGfCf UmUmCm AmCmCm UmCmUms Gm-3' (SEQ ID NO: 189)

反義股：5’-UmsAfsGm AfGmGf W’GmAm AfGmCf GmAfAm GfUmGf CmsAmsCm-3’(SEQ ID NO：190) Antisense strand: 5'-UmsAfsGm AfGmGf W'GmAm AfGmCf GmAfAm GfUmGf CmsAmsCm-3' (SEQ ID NO: 190)

或， or,

正義股：5’-GmsCmsAm CmUfUm CfGfCf UmUmCm AmCmCm UmCmUms Gms-3’(SEQ ID NO：191) Sense strand: 5'-GmsCmsAm CmUfUm CfGfCf UmUmCm AmCmCm UmCmUms Gms-3' (SEQ ID NO: 191)

或， or,

正義股：5’-GmsCmsAm CmUmUm CfGfCf UmUmCm AmCmCm UmCmUms Gm-3’(SEQ ID NO：192) Sense strand: 5'-GmsCmsAm CmUmUm CfGfCf UmUmCm AmCmCm UmCmUms Gm-3' (SEQ ID NO: 192)

或， or,

正義股：5’-GmsCmsAm CmUmUm CfGfCf UmUmCm AmCmCm UmCmUms Gms-3’(SEQ ID NO：193) Sense strand: 5'-GmsCmsAm CmUmUm CfGfCf UmUmCm AmCmCm UmCmUms Gms-3' (SEQ ID NO: 193)

、

或

，中：B為尿嘧啶。

,

or

, Middle: B is uracil.

、

和

,

and

在一些實施方案中，上述siRNA當接觸到表達靶基因的細胞時，由例如：psiCHECK活性篩選和螢光素酶報告基因檢測法，其他如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，例如Western Blot或流式細胞術測定的，上述siRNA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 some embodiments, the aforementioned siRNAs, when contacted with cells expressing the target gene, are detected by, for example, psiCHECK activity screening and luciferase reporter assays, other methods such as PCR or branched DNA (bDNA)-based, or protein-based The method, such as immunofluorescence analysis, such as Western Blot or flow cytometry, said siRNA can inhibit the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

在一些實施方案中，上述siRNA當接觸到表達靶基因的細胞時，由例如：psiCHECK活性篩選和螢光素酶報告基因檢測法，其他如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，例如Western Blot或流式細胞術測定的，上述siRNA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 some embodiments, the aforementioned siRNAs, when contacted with cells expressing the target gene, are detected by, for example, psiCHECK activity screening and luciferase reporter assays, other methods such as PCR or branched DNA (bDNA)-based, or protein-based method, such as immunofluorescence analysis, such as Western Blot or flow cytometry, the remaining expression percentage of the target gene mRNA caused by the above siRNA 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 higher than 50%, not higher At 45%, not higher than 40%, not higher than 35%, not higher than 30%, not higher than 25%, not higher than 20%, not higher than 15%, or not higher than 10%.

在一些實施方案中，包含本揭露化學修飾的siRNA當接觸到表達靶基因的細胞時，由例如：psiCHECK活性篩選和螢光素酶報告基因檢測法，其他如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，例如Western Blot、或流式細胞術測定的，包含本揭露化學修飾，例如式(I)或式(II)所示化學修飾的siRNA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 some embodiments, siRNAs comprising chemical modifications of the present disclosure, when exposed to cells expressing a target gene, are detected by, for example, psiCHECK activity screening and luciferase reporter gene assays, other assays such as PCR or branched DNA (bDNA)-based assays. method, or a protein-based method, such as immunofluorescence analysis, such as Western Blot, or flow cytometry assay, comprising chemical modifications of the present disclosure, such as chemically modified siRNA shown in formula (I) or formula (II) in 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%, while maintaining on-target activity %, at least 70%, or at least 75%.

在一些實施方案中，包含本揭露化學修飾的siRNA當接觸到表達靶基因的細胞時，由例如：psiCHECK活性篩選和螢光素酶報告基因檢測法，其他如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，例如Western Blot、或流式細胞術測定的，包含本揭露化學修飾，例如式(I)或式(II)所示化學修飾的siRNA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 some embodiments, siRNAs comprising chemical modifications of the present disclosure, when exposed to cells expressing a target gene, are detected by, for example, psiCHECK activity screening and luciferase reporter gene assays, other assays such as PCR or branched DNA (bDNA)-based assays. method, or a protein-based method, such as immunofluorescence analysis, such as Western Blot, or flow cytometry assay, comprising a chemical modification of the present disclosure, such as chemically modified siRNA shown in formula (I) or formula (II) Reduce off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, while reducing on-target activity by at most 20%, at most 19%, at most 15%, at most 10%, at most 5%, or more than 1% , 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%.

在一些實施方案中，包含本揭露化學修飾的siRNA當接觸到表達靶基因的細胞時，由例如：psiCHECK活性篩選和螢光素酶報告基因檢測法，其他如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，例如Western Blot、或流式細胞術測定的，包含本揭露化學修飾，例如式(I)或式(II)所示化學修飾的siRNA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 some embodiments, siRNAs comprising chemical modifications of the present disclosure, when exposed to cells expressing a target gene, are detected by, for example, psiCHECK activity screening and luciferase reporter gene assays, other assays such as PCR or branched DNA (bDNA)-based assays. method, or a protein-based method, such as immunofluorescence analysis, such as Western Blot, or flow cytometry assay, comprising a chemical modification of the present disclosure, such as chemically modified siRNA shown in formula (I) or formula (II) 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% increase in target activity %,At least 60%, at least 65%, at least 70%, at least 75%, or at least 80% 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%.

本揭露還提供了一種siRNA綴合物，其包含上述siRNA中的任意一種和連接至該siRNA的靶向配體。 The present disclosure also provides an siRNA conjugate comprising any one of the above siRNAs and a targeting ligand linked to the siRNA.

在一些實施方案中，siRNA和該靶向配體共價或非共價連接。 In some embodiments, the siRNA and the targeting ligand are covalently or non-covalently linked.

在一些實施方案中，該靶向配體連接至該siRNA的正義股3’末端。 In some embodiments, the targeting ligand is attached to the 3' end of the sense strand of the siRNA.

在一些實施方案中，為了促進siRNA進入細胞，可以在siRNA正義股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小干擾核酸結合，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，siRNA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 promote the entry of siRNA into cells, a lipophilic group such as cholesterol can be introduced at the end of the siRNA sense strand. protein, vitamin E, etc., in order to facilitate the interaction with the mRNA in the cell through the cell membrane composed of lipid bilayer. At the same time, siRNA can also be modified by non-covalent bonds, such as binding phospholipid molecules, polypeptides, and cationic polymers through hydrophobic bonds or ionic bonds to increase stability and biological activity.

在一些實施方案中，靶向配體藉由磷酸酯基團、硫代磷酸酯基團或膦酸基團與siRNA末端連接。 In some embodiments, the targeting ligand is attached to the end of the siRNA via a phosphate group, phosphorothioate group, or phosphonate group.

在一些實施方案中，靶向配體藉由磷酸酯基團、硫代磷酸酯基團或膦酸基團與siRNA末端間接連接。 In some embodiments, the targeting ligand is indirectly attached to the end of the siRNA via a phosphate group, phosphorothioate group, or phosphonate group.

在一些實施方案中，靶向配體藉由磷酸酯基團、硫代磷酸酯基團或膦酸基團與siRNA末端直接連接。 In some embodiments, the targeting ligand is directly attached to the end of the siRNA via a phosphate group, phosphorothioate group, or phosphonate group.

在一些實施方案中，靶向配體藉由硫代磷酸酯基團與siRNA末端直接連接。 In some embodiments, the targeting ligand is directly attached to the end of the siRNA via a phosphorothioate group.

在一些實施方案中，靶向配體藉由硫代磷酸酯基團與siRNA正義股3’末端直接連接。 In some embodiments, the targeting ligand is directly attached to the 3' end of the sense strand of the siRNA via a phosphorothioate group.

在一些實施方案中，靶向配體該靶向配體包含靶向部分T，T選自半乳糖、半乳糖胺、N-甲醯基-半乳糖胺、N-乙醯基-半乳糖胺、N-丙醯基-半乳糖胺、N-正丁醯基-半乳糖胺和N-異丁醯基-半乳糖胺。在一些實施方案中，T選自N-乙醯基-半乳糖胺。 In some embodiments, the targeting ligand comprises a targeting moiety T selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine , N-propionyl-galactosamine, N-n-butyryl-galactosamine and N-isobutyryl-galactosamine. In some embodiments, T is selected from N-acetyl-galactosamine.

在一些實施方案中，靶向配體結構如下式(II)所示， In some embodiments, the structure of the targeting ligand is shown in the following formula (II),

其中T為靶向部分，E為分支基團，L₁為接頭部分，L₂為靶向部分與分支基團間的栓系部分，其中i選自1到10的整數，例如1、2、3、4、5、6、7、8、9、10。 Wherein T is a targeting moiety, E is a branching group, _L1 is a linker part, _L2 is a tethering part between the targeting moiety and the branching group, wherein i is selected from integers from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

在一些實施方案中，i選自2到8的整數。 In some embodiments, i is selected from integers from 2 to 8.

在一些實施方案中，i選自3到5的整數。 In some embodiments, i is selected from an integer from 3 to 5.

在一些實施方案中，L₁為 In some embodiments, L _is

R⁹及R¹⁰各自獨立的選自為-S-、-NH-、-O-、-C(O)-、-OC(O)-、-C(O)O-、-NHC(O)-、-C(O)NH-、-CH₂-、-CH₂NH-、-CH₂O-、-NH-C(O)-CH₂-、-C(O)-CH₂-NH-、-NH(CO)NH-、3-12員雜環基，該-CH₂-視需要被選自鹵素、烷基、烷氧基、烷胺基的取代基取代，該烷基視需要進一步被選自羥基、胺基、鹵素的取代基取代； R ⁹ and R ¹⁰ are each independently selected from -S-, -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O) -, -C(O)NH-, -CH ₂ -, -CH ₂ NH-, -CH ₂ O-, -NH-C(O)-CH ₂ -, -C(O)-CH ₂ -NH- , -NH(CO)NH-, 3-12 membered heterocyclic group, the -CH ₂ - is optionally substituted by a substituent selected from halogen, alkyl, alkoxy, alkylamino, and the alkyl is further Substituted by a substituent selected from hydroxyl, amino, halogen;

R¹¹選自氘、鹵素、烷基、胺基、氰基、硝基、烯基、炔基、羧基、羥基、巰基、烷巰基、烷氧基、烷胺基、-C(O)-烷基、-C(O)-O-烷基、-CONH₂、-CONH-烷基、-OC(O)-烷基、-NH-C(O)-烷基、-S(O)O-烷基、-S(O)ONH₂、-S(O)ONH-烷基，該烷基、烯基、炔基、烷基巰基、烷基氧基、-C(O)-烷基、-C(O)-O-烷基、-CONH-烷基、-OC(O)-烷基、-NH-C(O)-烷基、-S(O)O-烷基、-S(O)ONH-烷基，視需要進一步被選自鹵素、羥基、胺基、巰基的取代基取代； ^R is selected from deuterium, halogen, alkyl, amine, cyano, nitro, alkenyl, alkynyl, carboxyl, hydroxyl, mercapto, alkylmercapto, alkoxy, alkylamino, -C(O)-alk radical, -C(O)-O-alkyl, -CONH ₂ , -CONH-alkyl, -OC(O)-alkyl, -NH-C(O)-alkyl, -S(O)O- Alkyl, -S(O)ONH ₂ , -S(O)ONH-alkyl, the alkyl, alkenyl, alkynyl, alkylmercapto, alkyloxy, -C(O)-alkyl, - C(O)-O-alkyl, -CONH-alkyl, -OC(O)-alkyl, -NH-C(O)-alkyl, -S(O)O-alkyl, -S(O ) ONH-alkyl, optionally further substituted by a substituent selected from halogen, hydroxyl, amino, mercapto;

該k選自0，1，2，3，4； The k is selected from 0, 1, 2, 3, 4;

該j選自1到20的整數(例如0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20)。 The j is selected from integers from 1 to 20 (for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20).

在一些實施方案中，L₁為 In some embodiments, L _is

，其中R¹¹選自氘、鹵素、烷基、胺基、氰基、硝基、烯基、炔基、羧基、羥基、巰基、烷巰基、烷氧基、烷胺基、-C(O)-烷基、-C(O)-O-烷基、-CONH₂、-CONH-烷基、-OC(O)-烷基、-NH-C(O)-烷基、-S(O)O-烷基、-S(O)ONH₂、-S(O)ONH-烷基，該烷基、烯基、炔基、羧基、烷基巰基、烷基氧基、-C(O)-烷基、-C(O)-O-烷基、-CONH-烷基、-OC(O)-烷基、-NH-C(O)-烷基、-S(O)O-烷基、-S(O)ONH-烷基，選進一步被選自鹵素、羥基、胺基、巰基的取代基取代；

, wherein R ¹¹ is selected from deuterium, halogen, alkyl, amino, cyano, nitro, alkenyl, alkynyl, carboxyl, hydroxyl, mercapto, alkylmercapto, alkoxyl, alkylamino, -C(O) -Alkyl, -C(O)-O-Alkyl, -CONH ₂ , -CONH-Alkyl, -OC(O)-Alkyl, -NH-C(O)-Alkyl, -S(O) O-alkyl, -S(O)ONH ₂ , -S(O)ONH-alkyl, the alkyl, alkenyl, alkynyl, carboxyl, alkylmercapto, alkyloxy, -C(O)- Alkyl, -C(O)-O-Alkyl, -CONH-Alkyl, -OC(O)-Alkyl, -NH-C(O)-Alkyl, -S(O)O-Alkyl, -S(O)ONH-alkyl, selected to be further substituted by a substituent selected from halogen, hydroxyl, amino, mercapto;

該k選自0，1，2，3，4。 The k is selected from 0,1,2,3,4.

在一些實施方案中，L₁為 In some embodiments, L _is

或

or

該k選自0、1、2、3、4。 The k is selected from 0,1,2,3,4.

在一些實施方案中，L₁為 In some embodiments, L _is

在一些實施方案中，L₁為 In some embodiments, L _is

在一些實施方案中，L₁為 In some embodiments, L _is

在一些實施方案中，L₁為 In some embodiments, L _is

在一些實施方案中，L₁為 In some embodiments, L _is

在一些實施方案中，靶向配體中E為 In some embodiments, E in the targeting ligand is

該R¹²、R¹³、R¹⁴及R¹⁵各自獨立的選自-C(O)NH-、-C(O)-，該羰基視需要進一步被烷基取代，該烷基視需要進一步被選自烷基，羥基，-C(O)O-，-C(O)O-烷基-，-C(O)NH-取代； The R ¹² , R ¹³ , R ¹⁴ and R ¹⁵ are each independently selected from -C(O)NH-, -C(O)-, the carbonyl group is further substituted by an alkyl group, and the alkyl group is further selected from Substituted from alkyl, hydroxyl, -C(O)O-, -C(O)O-alkyl-, -C(O)NH-;

該X²、X³、X⁴及X⁵各自獨立的選自0到10的整數(例如0、1、2、3、4、5、6、7、8、9、10)。 The X ² , X ³ , X ⁴ and X ⁵ are each independently selected from integers from 0 to 10 (eg 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

該R¹²、R¹³、R¹⁴及R¹⁵各自獨立的選自-C(O)NH-、-C(O)-，該-C(O)NH-、-C(O)-視需要進一步被烷基取代，該烷基視需要進一步被選自烷基、羥基、-C(O)O-、-C(O)O-烷基-、-C(O)NH-取代； The R ¹² , R ¹³ , R ¹⁴ and R ¹⁵ are each independently selected from -C(O)NH-, -C(O)-, and the -C(O)NH-, -C(O)- may be further Substituted by an alkyl group, the alkyl group is optionally further substituted by an alkyl group, a hydroxyl group, -C(O)O-, -C(O)O-alkyl-, -C(O)NH-;

該R¹²、R¹³、R¹⁴及R¹⁵各自獨立的選自-C(O)NH-、-C(O)-，該-C(O)NH-，-C(O)-進一步被選自 The R ¹² , R ¹³ , R ¹⁴ and R ¹⁵ are each independently selected from -C(O)NH-, -C(O)-, the -C(O)NH-, -C(O)- are further selected from since

，

，

，

，

，

的取代基取代，該X²、X³、X⁴及X⁵各自獨立的選自0到10的整數(例如0、1、2、3、4、5、6、7、8、9、10)。

,

The substituents are substituted, the X ² , X ³ , X ⁴ and X ⁵ are each independently selected from integers from 0 to 10 (such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ).

在一些實施方案中，靶向配體中E選自 In some embodiments, E in the targeting ligand is selected from

在一些實施方案中，靶向配體中E選自

。 In some embodiments, E in the targeting ligand is selected from

.

在一些實施方案中，靶向配體中E選自

。 In some embodiments, E in the targeting ligand is selected from

.

，L₁選自如下結構：

, L ₁ is selected from the following structures:

或者

，

or

,

R⁹及R¹⁰各自獨立的選自為-S-、-NH-、-O-、-S-、-C(O)-、-OC(O)-、-C(O)O-、-NHC(O)-、-C(O)NH-、-CH₂-、-CH₂NH-、-CH₂O-、-NH-C(O)-CH₂-、-C(O)-CH₂-NH-、-NH(CO)NH-、3-12員雜環基，該-CH₂-視需要被選自鹵素、烷基、烷氧基、烷胺基的取代基取代，該烷基視需要進一步被選自羥基、胺基、鹵素的取代基取代； R ⁹ and R ¹⁰ are each independently selected from -S-, -NH-, -O-, -S-, -C(O)-, -OC(O)-, -C(O)O-, - NHC(O)-, -C(O)NH-, -CH ₂ -, -CH ₂ NH-, -CH ₂ O-, -NH-C(O)-CH ₂ -, -C(O)-CH ₂ -NH-, -NH(CO)NH-, 3-12-membered heterocyclic group, the -CH ₂ - is optionally substituted by a substituent selected from halogen, alkyl, alkoxy, and alkylamino, the alkyl The group is optionally further substituted by a substituent selected from hydroxyl, amino, halogen;

該k選自0、1、2、3、4； The k is selected from 0, 1, 2, 3, 4;

該j選自0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20。 The j is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

在一些實施方案中，靶向配體中E選自： In some embodiments, E in the targeting ligand is selected from:

或

，L₁選自：

，或

。

or

, _L1 is selected from:

,or

.

或

，L₁選自：

、

或

。

or

, _L1 is selected from:

,

or

.

，L₁選自：

，即E-L₁為

。

, _L1 is selected from:

, ie EL ₁ is

.

，L₁選自：

，即E-L₁為

。

, _L1 is selected from:

, ie EL ₁ is

.

，L₁選自：

，即E-L₁為

。

, _L1 is selected from:

, ie EL ₁ is

.

在一些實施方案中，靶向配體中E選自

，L₁選自

，即E-L₁為

。 In some embodiments, E in the targeting ligand is selected from

, L ₁ selected from

, ie EL ₁ is

.

在一些實施方案中，靶向配體中E選自

，L₁選自

，即E-L₁為

。 In some embodiments, E in the targeting ligand is selected from

, L ₁ selected from

, ie EL ₁ is

.

在一些實施方案中，靶向配體中E選自

，L₁選自

，即E-L₁為

。 In some embodiments, E in the targeting ligand is selected from

, L ₁ selected from

, ie EL ₁ is

.

本揭露中L₂為靶向部分與分支基團間的栓系部分，L₂在分支基團和各靶向部分之間發揮連接、間隔作用。 In the present disclosure, L ₂ is the tethering part between the targeting moiety and the branching group, and L ₂ plays the role of connection and spacer between the branching group and each targeting moiety.

在一些實施方式中，L₂的一端直接連接至靶向配體，而另一端直接連接至分支基團E。 In some embodiments, one _end of L is directly attached to the targeting ligand, and the other end is directly attached to the branching group E.

在一些實施方式中，L₂的一端直接連接至靶向配體，而另一端間接地連接至分支基團E。 In some embodiments, one end of _L2 is directly connected to the targeting ligand and the other end is indirectly connected to the branching group E.

在一些實施方式中，L₂的一端間接地連接至靶向配體，而另一端間接地連接至分支基團E。 In some embodiments, one end of _L is indirectly linked to the targeting ligand and the other end is indirectly linked to the branching group E.

在一些實施方式中，本文公開的靶向配體包括2個L₂和2個靶向部分。 In some embodiments, a targeting ligand disclosed herein includes 2 _L2 and 2 targeting moieties.

在一些實施方式中，本文公開的靶向配體包括3個L₂和3個靶向部分。 In some embodiments, a targeting ligand disclosed herein includes 3 _L2 and 3 targeting moieties.

在一些實施方式中，本文公開的靶向配體包括4個L₂和4個靶向部分。 In some embodiments, a targeting ligand disclosed herein includes 4 _L2 and 4 targeting moieties.

在一些實施方式中，本文公開的靶向配體包括多個L₂和多個靶向部分。 In some embodiments, a targeting ligand disclosed herein includes multiple _L2 and multiple targeting moieties.

在一些實施方式中，本揭露中L₂選自以下基團中的1個或2-20個共價連接的組合： In some embodiments, L in the present disclosure is selected from ₁ or a combination of 2-20 covalent linkages in the following groups:

、取代的或未取代的環烷基 (例如環己基、環丙基、環丁基、環戊基、環庚基、環辛基等)，取代的或未取代的環烯基(例如環己烯基、環丁烯基、環戊烯基、環庚烯基、環辛烯基、環己二烯基、環戊二烯基、環庚二烯基、環辛二烯基等)、取代的或未取代的芳基(例如苯基、萘基、聯萘基、蒽基等)、取代的或未取代的雜芳基(例如吡啶基、嘧啶基、吡咯、咪唑、呋喃、苯并呋喃、吲哚等)和取代的或未取代的雜環基(例如四氫呋喃、四氫吡喃、哌啶、吡咯烷等)。

, substituted or unsubstituted cycloalkyl (such as cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclooctyl, etc.), substituted or unsubstituted cycloalkenyl (such as cyclohexyl alkenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), substituted substituted or unsubstituted aryl (such as phenyl, naphthyl, binaphthyl, anthracenyl, etc.), substituted or unsubstituted heteroaryl (such as pyridyl, pyrimidyl, pyrrole, imidazole, furan, benzofuran , indole, etc.) and substituted or unsubstituted heterocyclic groups (such as tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, etc.).

在一些實施方式中，本揭露中L₂選自以下基團中的1個或2-20個(例如，2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)共價連接的組合： In some embodiments, _L in the present disclosure is selected from 1 or 2-20 of the following groups (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) Combinations of covalent linkages:

的基團的共價組合。

A covalent combination of groups.

在一些實施方式中，靶向配體包括具有如下所示結構的L₂，

，其中x⁶是從1至20的整數(例如，1、2、3、4、5、6、7、 8、9、10、11、12、13、14、15、16、17、18、19或20)。 In some embodiments, the targeting ligand comprises _L2 having the structure shown below,

, where ^x6 is an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

在一些實施方式中，靶向配體包括具有如下所示結構的L₂， In some embodiments, the targeting ligand comprises _L2 having the structure shown below,

，其中，x⁷是從1至20的整數(例如，1、2、3、4、5、6、 7、8、9、10、11、12、13、14、15、16、17、18、19或20)，並且Z是

, wherein x ⁷ is an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19 or 20), and Z is

在一些實施方式中，靶向配體具有如下所示結構的L₂， In some embodiments, the targeting ligand has _L2 of the structure shown below,

其中，x⁸是從1至20的整數(例如，1、2、3、4、5、6、7、8、9、10、 11、12、13、14、15、16、17、18、19或20)，並且Z是

。 where ^x8 is an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and Z is

.

，其中x⁹和X¹⁰各自獨立選自從1至20的整數(例如， 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、 19或20)，並且Z是

。

, wherein x ⁹ and X ¹⁰ are each independently selected from an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19 or 20), and Z is

.

其中，x⁷和X⁸各自獨立選自從1至20的整數(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)，並且Z Wherein, x ⁷ and X ⁸ are each independently selected from integers from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19 or 20), and Z

在一些具體的實施方式中，靶向配體具有以下結構： In some specific embodiments, the targeting ligand has the following structure:

一些實施方案中，靶向配體的靶向部分是由一個或多個靶向基團或靶向部分組成，靶向配體協助引導將與其連接的治療性試劑遞送至所需靶位置。在一些情況中，靶向部分可以結合細胞或細胞受體，並且啟動內吞作用以促進治療性試劑進入細胞。靶向部分可以包括對細胞受體或細胞表面分子或抗體具有親和性的化合物。含有靶向部分的各種靶向配體可以與治療性試劑和其它化合物連接以將試劑靶向細胞和特定細胞受體。 In some embodiments, the targeting moiety of the targeting ligand is comprised of one or more targeting groups or targeting moieties that assist in directing the delivery of the therapeutic agent attached thereto to the desired target location. In some instances, a targeting moiety can bind a cell or a cell receptor and initiate endocytosis to facilitate entry of a therapeutic agent into the cell. Targeting moieties may include compounds that have affinity for cell receptors or cell surface molecules or antibodies. Various targeting ligands containing targeting moieties can be linked to therapeutic agents and other compounds to target the agent to cells and specific cellular receptors.

一些實施方案中，靶向部分的類型包括碳水化合物、膽固醇和膽甾醇基團或類固醇。可以結合細胞受體的靶向部分包括糖類，諸如半乳糖、半乳糖衍生物(如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、N-異丁醯基半乳糖胺)、甘露糖和甘露糖衍生物。 In some embodiments, the types of targeting moieties include carbohydrates, cholesterol, and cholesteryl groups or steroids. Targeting moieties that can bind to cellular receptors include carbohydrates such as galactose, galactose derivatives (e.g. N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N- Acrylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine), mannose and mannose derivatives.

已知結合脫唾液酸糖蛋白受體(ASGPR)的靶向部分可特別用於引導遞送寡聚化合物至肝臟。脫唾液酸糖蛋白受體在肝臟細胞(肝細胞)上大量表達。靶向ASCPR的細胞受體靶向部分包括半乳糖和半乳糖衍生物。具體而言，半乳糖衍生物的簇，包括由2、3、4或超過4個N-乙醯基-半乳糖胺(GalNAc或NAG)組成的簇可以促進肝細胞中某些化合物的攝取。偶聯寡聚化合物的GalNAc簇用於引導組成物至肝臟，在這裡N-乙醯基-半乳糖胺糖能夠結合肝臟細胞表面的脫唾液酸糖蛋白受體。脫唾液酸糖蛋白受體的結合被認為將啟動受體介導的內吞作用，從而促進化合物進入細胞內部。 Targeting moieties known to bind the asialoglycoprotein receptor (ASGPR) are particularly useful for directing the delivery of oligomeric compounds to the liver. Asialoglycoprotein receptors are abundantly expressed on liver cells (hepatocytes). Cellular receptor targeting moieties that target ASCPR include galactose and galactose derivatives. Specifically, clusters of galactose derivatives, including clusters consisting of 2, 3, 4 or more than 4 N-acetyl-galactosamines (GalNAc or NAG), can enhance the uptake of certain compounds in hepatocytes. GalNAc clusters coupled to oligomeric compounds are used to direct the constituents to the liver, where the N-acetyl-galactosamine sugar can bind to asialoglycoprotein receptors on the liver cell surface. Binding of the asialoglycoprotein receptor is thought to initiate receptor-mediated endocytosis, thereby facilitating the entry of compounds into the cell interior.

一些實施方案中，靶向配體可以包括2、3、4或超過4個靶向部分。在一些實施方式中，本文所公開的靶向配體可以包括1、2、3、4或超過4個藉由L₂連接至分支基團的靶向部分。 In some embodiments, a targeting ligand may comprise 2, 3, 4 or more than 4 targeting moieties. In some embodiments, a targeting ligand disclosed herein can include 1, 2, 3, 4, or more than 4 targeting moieties linked to a branching group via _L2 .

在一些實施方式中，靶向配體是半乳糖簇的形式。 In some embodiments, the targeting ligand is in the form of a galactose cluster.

在一些實施方式中，各靶向部分包括半乳糖胺衍生物，其為N-乙醯基-半乳糖胺。可用作靶向部分且對脫唾液酸糖蛋白受體具有親和性的其他糖可選自半乳糖、半乳糖胺、N-甲醯基-半乳糖胺、N-乙醯基-半乳糖胺、N-丙醯基-半乳糖胺、N-正丁醯基-半乳糖胺和N-異丁醯基-半乳糖胺等。 In some embodiments, each targeting moiety includes a galactosamine derivative which is N-acetyl-galactosamine. Other sugars useful as targeting moieties and having an affinity for the asialoglycoprotein receptor may be selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine , N-propionyl-galactosamine, N-n-butyryl-galactosamine and N-isobutyryl-galactosamine, etc.

在一些實施方式中，本揭露中的靶向配體包括N-乙醯半乳糖胺做為靶向部分， In some embodiments, the targeting ligands of the present disclosure include N-acetylgalactosamine as a targeting moiety,

在一些實施方式中，靶向配體包括三個末端半乳糖胺或半乳糖胺衍生物(諸如N-乙醯基-半乳糖胺)，其各自對唾液酸糖蛋白受體均具有親和性。在一些實施方式中，靶向配體包括三個末端N-乙醯基-半乳糖胺(GalNAc或NAG)作為靶向部分。 In some embodiments, the targeting ligand comprises three terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has an affinity for the sialoglycoprotein receptor. In some embodiments, the targeting ligand includes three terminal N-acetyl-galactosamine (GalNAc or NAG) as the targeting moiety.

在一些實施方式中，靶向配體包括四個末端半乳糖胺或半乳糖胺衍生物(諸如N-乙醯基-半乳糖胺)，其各自對脫唾液酸糖蛋白受體均具有親和性。在一些實施方式中，靶向配體包括四個末端N-乙醯基-半乳糖胺(GalNAc或NAG)作為靶向部分。 In some embodiments, the targeting ligand comprises four terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for the asialoglycoprotein receptor . In some embodiments, the targeting ligand includes four terminal N-acetyl-galactosamine (GalNAc or NAG) as the targeting moiety.

當述及三個末端N-乙醯基-半乳糖胺時本領域常用的術語包括三觸角(tri-antennary)、三價物(tri-valent)和三聚體。 Terms commonly used in the art when referring to the three terminal N-acetyl-galactosamines include tri-antennary, tri-valent and trimer.

當述及四個末端N-乙醯基-半乳糖胺時本領域常用的術語包括四觸角(tetra-antennary)、四價物(tetra-valent)和四聚體。 Terms commonly used in the art when referring to the four terminal N-acetyl-galactosamines include tetra-antennary, tetra-valent and tetramer.

一些具體實施方案中，本揭露提供的靶向配體具有如下結構， In some specific embodiments, the targeting ligand provided by the present disclosure has the following structure,

在一些實施方式中，本揭露siRNA連接至本揭露靶向配體，形成具有如下所示的siRNA綴合物， In some embodiments, an siRNA of the present disclosure is linked to a targeting ligand of the present disclosure to form a siRNA conjugate having the following,

其中T為靶向部分，E為分支基團，L₁為接頭部分，L₂為靶向部分與分支基團間的栓系部分，其中x選自1到10的整數，D為靶向HBV的siRNA。 Wherein T is the targeting moiety, E is the branching group, _L1 is the linker part, _L2 is the tethering part between the targeting moiety and the branching group, wherein x is an integer selected from 1 to 10, and D is the targeting HBV siRNA.

在一些實施方式中，D為靶向HBV-X的siRNA。 In some embodiments, D is an siRNA targeting HBV-X.

在一些實施方式中，D為本揭露任一siRNA。在一些實施方案中，該L₁連接至該siRNA的正義股3’末端。 In some embodiments, D is any siRNA disclosed herein. In some embodiments, the _L1 is linked to the 3' end of the sense strand of the siRNA.

在一些具體的實施方案中，本揭露提供一種siRNA綴合物， In some specific embodiments, the present disclosure provides an siRNA conjugate,

，其中，D為靶向HBV的siRNA。

, wherein, D is the siRNA targeting HBV.

在一些實施方式中，D為靶向HBV-X的siRNA。在一些實施方式中，D為本揭露任一siRNA。在一些具體的實施方案中，靶向配體藉由硫代磷酸酯基團與siRNA正義股3’末端直接連接。 In some embodiments, D is an siRNA targeting HBV-X. In some embodiments, D is any siRNA disclosed herein. In some specific embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of the siRNA via a phosphorothioate group.

，其中，D為靶向HBV的siRNA。在一些實施方式中，D為靶向HBV-X的siRNA。在一些實施方式中，D為本揭露任一siRNA。

, wherein, D is the siRNA targeting HBV. In some embodiments, D is an siRNA targeting HBV-X. In some embodiments, D is any siRNA disclosed herein.

在一些具體的實施方案中，靶向配體藉由硫代磷酸酯基團與siRNA正義股3’末端直接連接。 In some specific embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of the siRNA via a phosphorothioate group.

，其中，D為靶向HBV的siRNA。在一些實施方式中，D為靶向HBV-X的siRNA。在一些實施方式中，D為本揭露任一siRNA。在一些具體的實施方案中，靶向配體藉由硫代磷酸酯基團與siRNA正義股3’末端直接連接。

, wherein, D is the siRNA targeting HBV. In some embodiments, D is an siRNA targeting HBV-X. In some embodiments, D is any siRNA disclosed herein. In some specific embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of the siRNA via a phosphorothioate group.

在一些具體的實施方案中，該L₁與D藉由磷酸酯基團、硫代磷酸酯基團或膦酸基團連接。 In some specific embodiments, the _L and D are linked via a phosphate, phosphorothioate, or phosphonic acid group.

在一些具體的實施方案中，該L₁與D正義股3’末端藉由磷酸酯基團、硫代磷酸酯基團或膦酸基團連接。 In some specific embodiments, the L ₁ is linked to the 3' end of the D sense strand via a phosphate group, phosphorothioate group or phosphonate group.

在一些具體的實施方案中，該L₁與D正義股3’末端藉由硫代磷酸酯基團直接連接。 In some specific embodiments, the _L1 is directly linked to the 3' end of the D sense strand via a phosphorothioate group.

在一些具體的實施方案中，該L₁與D正義股3’末端藉由硫代磷酸酯基團間接連接。 In some specific embodiments, the _L1 is indirectly linked to the 3' end of the D sense strand via a phosphorothioate group.

在一些具體的實施方案中，siRNA綴合物選自： In some specific embodiments, the siRNA conjugate is selected from:

正義股(5’-3’)：GmsUmsGm UmGfCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG1(SEQ ID NO：24) Sense strand (5'-3'): GmsUmsGm UmGfCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG1 (SEQ ID NO: 24)

反義股(5’-3’)：AmsGfsUm GfAmAf W’CmGm AfAmGf UmGfCm AfCmAf CmsGmsGm(SEQ ID NO：194) Antisense strand (5'-3'): AmsGfsUm GfAmAf W'CmGm AfAmGf UmGfCm AfCmAf CmsGmsGm (SEQ ID NO: 194)

或， or,

正義股(5’-3’)：GmsUmsGm UmGmCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG1(SEQ ID NO：26) Sense strand (5'-3'): GmsUmsGm UmGmCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG1 (SEQ ID NO: 26)

其中，大寫字母C、G、U、A表示核苷酸的鹼基組成；小寫字母m表示該字母m左側相鄰的一個核苷酸為2’-甲氧基修飾的核苷酸；小寫字母f表示該字母f左側相鄰的一個核苷酸為2’-氟修飾的核苷酸； Among them, the uppercase letters C, G, U, and A indicate the base composition of nucleotides; the lowercase letter m indicates that the adjacent nucleotide on the left side of the letter m is a 2'-methoxy modified nucleotide; the lowercase letter f means that the nucleotide adjacent to the left side of the letter f is a 2'-fluoro modified nucleotide;

NAG1結構為：

； The structure of NAG1 is:

;

W’結構為：

、

或

，其中M為O， B為鳥嘌呤。 The structure of W' is:

,

or

, wherein M is O, and B is guanine.

正義股(5’-3’)：GmsUmsGm UmGfCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG2(SEQ ID NO：195) Sense strand (5'-3'): GmsUmsGm UmGfCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG2 (SEQ ID NO: 195)

反義股(5’-3’)：AmsGfsUm GfAmAf W’CmGm AfAmGf UmGfCm AfCmAf CmsGmsGm(SEQ ID NO：196) Antisense strand (5'-3'): AmsGfsUm GfAmAf W'CmGm AfAmGf UmGfCm AfCmAf CmsGmsGm (SEQ ID NO: 196)

或， or,

正義股(5’-3’)：GmsUmsGm UmGmCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG2(SEQ ID NO：197) Sense strand (5'-3'): GmsUmsGm UmGmCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG2 (SEQ ID NO: 197)

NAG2構為：

； NAG2 is structured as:

;

W’結構為：

、

或

，其中M為O， B為鳥嘌呤。 The structure of W' is:

,

or

, wherein M is O, and B is guanine.

正義股(5’-3’)：GmsUmsGm UmGfCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG3(SEQ ID NO：198) Sense strand (5'-3'): GmsUmsGm UmGfCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG3 (SEQ ID NO: 198)

反義股(5’-3’)：AmsGfsUm GfAmAf W’CmGm AfAmGf UmGfCm AfCmAf CmsGmsGm(SEQ ID NO：199) Antisense strand (5'-3'): AmsGfsUm GfAmAf W'CmGm AfAmGf UmGfCm AfCmAf CmsGmsGm (SEQ ID NO: 199)

或， or,

正義股(5’-3’)：GmsUmsGm UmGmCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG3(SEQ ID NO：200) Sense strand (5'-3'): GmsUmsGm UmGmCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG3 (SEQ ID NO: 200)

NAG3結構為：

； The structure of NAG3 is:

;

W’結構為：

、

或

，其中M為O， B為鳥嘌呤。 The structure of W' is:

,

or

, wherein M is O, and B is guanine.

正義股(5’-3’)：GmsUmsGm UmGfCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG4(SEQ ID NO：201) Sense strand (5'-3'): GmsUmsGm UmGfCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG4 (SEQ ID NO: 201)

反義股(5’-3’)：AmsGfsUm GfAmAf W’CmGm AfAmGf UmGfCm AfCmAf CmsGmsGm(SEQ ID NO：202) Antisense strand (5'-3'): AmsGfsUm GfAmAf W'CmGm AfAmGf UmGfCm AfCmAf CmsGmsGm (SEQ ID NO: 202)

或， or,

正義股(5’-3’)：GmsUmsGm UmGmCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG4(SEQ ID NO：203) Sense strand (5'-3'): GmsUmsGm UmGmCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG4 (SEQ ID NO: 203)

NAG4結構為：

； The structure of NAG4 is:

;

W’結構為：

、

或

，其中M為O， B為鳥嘌呤。 The structure of W' is:

,

or

, wherein M is O, and B is guanine.

正義股：5’-GmsUmsGm UmGfCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG1-3’(SEQ ID NO：24) Sense strand: 5'-GmsUmsGm UmGfCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG1-3' (SEQ ID NO: 24)

反義股：5’-AmsGfsUm GfAmAf W’CmGm AfAmGf UmGfCm AfCmAf CmsGmsGm-3’(SEQ ID NO：204) Antisense strand: 5'-AmsGfsUm GfAmAf W'CmGm AfAmGf UmGfCm AfCmAf CmsGmsGm-3' (SEQ ID NO: 204)

或， or,

正義股：5’-GmsUmsGm UmGmCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG1-3’(SEQ ID NO：26) Sense strand: 5'-GmsUmsGm UmGmCm AfCfUf UmCmGm CmUmUm CmAmCms Cms-NAG1-3' (SEQ ID NO: 26)

或， or,

正義股：5’-UmsGmsCm AmCfUm UfCfGf CmUmUm CmAmCm CmUmCms Ums-NAG1-3’(SEQ ID NO：47) Sense strand: 5'-UmsGmsCm AmCfUm UfCfGf CmUmUm CmAmCm CmUmCms Ums-NAG1-3' (SEQ ID NO: 47)

反義股：5’-AmsGfsAm GfGmUf W’AmAm GfCmGf AmAfGm UfGmCf AmsCmsAm-3’(SEQ ID NO：205) Antisense strand: 5'-AmsGfsAm GfGmUf W'AmAm GfCmGf AmAfGm UfGmCf AmsCmsAm-3' (SEQ ID NO: 205)

或， or,

正義股：5’-UmsGmsCm AmCmUm UfCfGf CmUmUm CmAmCm CmUmCms Ums-NAG1-3’(SEQ ID NO：49) Sense strand: 5'-UmsGmsCm AmCmUm UfCfGf CmUmUm CmAmCm CmUmCms Ums-NAG1-3' (SEQ ID NO: 49)

、

或

，其中，B為鳥嘌呤。

,

or

, wherein, B is guanine.

該NAG1結構為： The NAG1 structure is:

一些具體的實施方案中，W’結構為：

、

或

，其中M為O，B為鳥嘌呤。 In some specific embodiments, the structure of W' is:

,

or

, wherein M is O, and B is guanine.

正義股：5’-CmsUmsUm UmUfGm UfCfUf UmUmGm GmGmUm AmUmAms Ums-NAG1-3’(SEQ ID NO：27) Sense strand: 5'-CmsUmsUm UmUfGm UfCfUf UmUmGm GmGmUm AmUmAms Ums-NAG1-3' (SEQ ID NO: 27)

反義股：5’-AmsUfsAm UfAmCf W’CmAm AfAmGf AmCfAm AfAmAf GmsAmsAm-3’(SEQ ID NO：206) Antisense strand: 5'-AmsUfsAm UfAmCf W'CmAm AfAmGf AmCfAm AfAmAf GmsAmsAm-3' (SEQ ID NO: 206)

或， or,

正義股：5’-CmsUmsUm UmUmGm UfCfUf UmUmGm GmGmUm AmUmAms Ums-NAG1-3’(SEQ ID NO：29) Sense strand: 5'-CmsUmsUm UmUmGm UfCfUf UmUmGm GmGmUm AmUmAms Ums-NAG1-3' (SEQ ID NO: 29)

或， or,

正義股：5’-CmsGmsUm GmUfGm CfAfCf UmUmGm GmCmUm UmCmAms Cms-NAG1-3’(SEQ ID NO：33) Sense strand: 5'-CmsGmsUm GmUfGm CfAfCf UmUmGm GmCmUm UmCmAms Cms-NAG1-3' (SEQ ID NO: 33)

反義股：5’-AmsUfsGm AfAmGf W’GmAm AfGmUf GmCfAm CfAmCf GmsGmsUm-3’(SEQ ID NO：207) Antisense strand: 5'-AmsUfsGm AfAmGf W'GmAm AfGmUf GmCfAm CfAmCf GmsGmsUm-3' (SEQ ID NO: 207)

或， or,

正義股：5’-CmsGmsUm GmUmGm CfAfCf UmUmCm GmCmUm UmCmAms Cms-NAG1-3’(SEQ ID NO：35) Sense strand: 5'-CmsGmsUm GmUmGm CfAfCf UmUmCm GmCmUm UmCmAms Cms-NAG1-3' (SEQ ID NO: 35)

或， or,

正義股：5’-CmsUmsUm UmUfGm UfCfUf UmUmGm GmGmUm AmUmAms Cms-NAG1-3’(SEQ ID NO：39) Sense strand: 5'-CmsUmsUm UmUfGm UfCfUf UmUmGm GmGmUm AmUmAms Cms-NAG1-3' (SEQ ID NO: 39)

反義股：5’-AmsUfsAm UfAmCf W’CmAm AfAmGf AmCfAm AfAmAf GmsAmsAm-3’(SEQ ID NO：208) Antisense strand: 5'-AmsUfsAm UfAmCf W'CmAm AfAmGf AmCfAm AfAmAf GmsAmsAm-3' (SEQ ID NO: 208)

或， or,

正義股：5’-CmsUmsUm UmUmGm UfCfUf UmUmGm GmGmUm AmUmAms Cms-NAG1-3’(SEQ ID NO：40) Sense strand: 5'-CmsUmsUm UmUmGm UfCfUf UmUmGm GmGmUm AmUmAms Cms-NAG1-3' (SEQ ID NO: 40)

或， or,

正義股：5’-CmsAmsUm CmUfUm CfUfUf GmUmUm GmGmUm UmCmUms Ums-NAG1-3’(SEQ ID NO：41) Sense strand: 5'-CmsAmsUm CmUfUm CfUfUf GmUmUm GmGmUm UmCmUms Ums-NAG1-3' (SEQ ID NO: 41)

反義股：5’-AmsAfsGm AfAmCf W’AmAm CfAmAf GmAfAm GfAmUf GmsAmsGm-3’(SEQ ID NO：209) Antisense strand: 5'-AmsAfsGm AfAmCf W'AmAm CfAmAf GmAfAm GfAmUf GmsAmsGm-3' (SEQ ID NO: 209)

或， or,

正義股：5’-CmsAmsUm CmUmUm CfUfUf GmUmUm GmGmUm UmCmUms Ums-NAG1-3’(SEQ ID NO：43) Sense strand: 5'-CmsAmsUm CmUmUm CfUfUf GmUmUm GmGmUm UmCmUms Ums-NAG1-3' (SEQ ID NO: 43)

、

或

，中：B為胞嘧啶。

,

or

, Middle: B is cytosine.

一些具體的實施方案中，W’結構為：

、

或

，其中M為O，B為胞嘧啶。 In some specific embodiments, the structure of W' is:

,

or

, where M is O and B is cytosine.

正義股：5’-UmsUmsAm CmCfAm AfUfUf UmUmCm UmUmUm UmGmUms Ums-NAG1-3’(SEQ ID NO：30) Sense strand: 5'-UmsUmsAm CmCfAm AfUfUf UmUmCm UmUmUm UmGmUms Ums-NAG1-3' (SEQ ID NO: 30)

反義股：5’-AmsAfsCm AfAmAf W’GmAm AfAmAf UmUfGm GfUmAf AmsCmsAm-3’(SEQ ID NO：210) Antisense strand: 5'-AmsAfsCm AfAmAf W'GmAm AfAmAf UmUfGm GfUmAf AmsCmsAm-3' (SEQ ID NO: 210)

或， or,

正義股：5’-UmsUmsAm CmCmAm AfUfUf UmUmCm UmUmUm UmGmUms Ums-NAG1-3’(SEQ ID NO：32) Sense strand: 5'-UmsUmsAm CmCmAm AfUfUf UmUmCm UmUmUm UmGmUms Ums-NAG1-3' (SEQ ID NO: 32)

或， or,

正義股：5’-UmsGmsUm CmUfUm UfGfGf GmUmAm UmAmCm AmUmUms Ums-NAG1-3’(SEQ ID NO：36) Sense strand: 5'-UmsGmsUm CmUfUm UfGfGf GmUmAm UmAmCm AmUmUms Ums-NAG1-3' (SEQ ID NO: 36)

反義股：5’-AmsAfsAm UfGmUf W’UmAm CfCmCf AmAfAm GfAmCf AmsAmsAm-3’(SEQ ID NO：211) Antisense strand: 5'-AmsAfsAm UfGmUf W'UmAm CfCmCf AmAfAm GfAmCf AmsAmsAm-3' (SEQ ID NO: 211)

或， or,

正義股：5’-UmsGmsUm CmUmUm UfGfGf GmUmAm UmAmCm AmUmUms Ums-NAG1-3’(SEQ ID NO：38) Sense strand: 5'-UmsGmsUm CmUmUm UfGfGf GmUmAm UmAmCm AmUmUms Ums-NAG1-3' (SEQ ID NO: 38)

或， or,

正義股：5’-UmsGmsUm CmUfGm CfGfGf CmGmUm UmUmUm AmUmCms Ams-NAG1-3’(SEQ ID NO：44) Sense strand: 5'-UmsGmsUm CmUfGm CfGfGf CmGmUm UmUmUm AmUmCms Ams-NAG1-3' (SEQ ID NO: 44)

反義股：5’-UmsGfsAm UfAmAf W’AmCm GfCmCf GmCfAm GfAmCf AmsCmsAm-3’(SEQ ID NO：212) Antisense strand: 5'-UmsGfsAm UfAmAf W'AmCm GfCmCf GmCfAm GfAmCf AmsCmsAm-3' (SEQ ID NO: 212)

或， or,

正義股：5’-UmsGmsUm CmUmGm CfGfGf CmGmUm UmUmUm AmUmCms Ams-NAG1-3’(SEQ ID NO：46) Sense strand: 5'-UmsGmsUm CmUmGm CfGfGf CmGmUm UmUmUm AmUmCms Ams-NAG1-3' (SEQ ID NO: 46)

或，正義股：5’-GmsGmsCm GmCfUm GfAfAf UmCmCm UmGmCm GmGmAms Cms-NAG1-3’(SEQ ID NO：53) Or, sense strand: 5'-GmsGmsCm GmCfUm GfAfAf UmCmCm UmGmCm GmGmAms Cms-NAG1-3' (SEQ ID NO: 53)

反義股：5’-AmsUfsCm CfGmCf W’GmGm AfUmUf CmAfGm CfGmCf CmsGmsAm-3’(SEQ ID NO：213) Antisense strand: 5'-AmsUfsCm CfGmCf W'GmGm AfUmUf CmAfGm CfGmCf CmsGmsAm-3' (SEQ ID NO: 213)

或， or,

正義股：5’-GmsGmsCm GmCmUm GfAfAf UmCmCm UmGmCm GmGmAms Cms-NAG1-3’(SEQ ID NO：55) Sense strand: 5'-GmsGmsCm GmCmUm GfAfAf UmCmCm UmGmCm GmGmAms Cms-NAG1-3' (SEQ ID NO: 55)

、

或

，中：B為腺嘌呤。

,

or

, Middle: B is adenine.

一些具體的實施方案中，W’結構為：

、

或

，其中M為O，B為腺嘌呤。 In some specific embodiments, the structure of W' is:

,

or

, where M is O and B is adenine.

正義股：5’-GmsCmsAm CmUfUm CfGfCf UmUmCm AmCmCm UmCmUms Gms-NAG1-3’(SEQ ID NO：50) Sense strand: 5'-GmsCmsAm CmUfUm CfGfCf UmUmCm AmCmCm UmCmUms Gms-NAG1-3' (SEQ ID NO: 50)

反義股：5’-UmsAfsGm AfGmGf W’GmAm AfGmCf GmAfAm GfUmGf CmsAmsCm-3’(SEQ ID NO：214) Antisense strand: 5'-UmsAfsGm AfGmGf W'GmAm AfGmCf GmAfAm GfUmGf CmsAmsCm-3' (SEQ ID NO: 214)

或， or,

正義股：5’-GmsCmsAm CmUmUm CfGfCf UmUmCm AmCmCm UmCmUms Gms-NAG1-3’(SEQ ID NO：52) Sense strand: 5'-GmsCmsAm CmUmUm CfGfCf UmUmCm AmCmCm UmCmUms Gms-NAG1-3' (SEQ ID NO: 52)

、

或

，中：B為尿嘧啶。

,

or

, Middle: B is uracil.

一些具體的實施方案中，W’結構為：

、

或

，其中M為O，B為尿嘧啶。 In some specific embodiments, the structure of W' is:

,

or

, where M is O and B is uracil.

本揭露還提供了一種醫藥組成物，其包含本揭露的siRNA或siRNA綴合物。 The present disclosure also provides a pharmaceutical composition comprising the siRNA or siRNA conjugate of the present disclosure.

在一些實施方案中，該醫藥組成物中還可以包含藥學上可接受的輔料和/或佐劑，該輔料可以為一種或多種本領域常規採用的各種製劑或化合物。例如，該藥學上可接受的輔料可以包括pH緩衝劑、保護劑和滲透壓調節劑中的至少一種。 In some embodiments, the pharmaceutical composition may also contain pharmaceutically acceptable excipients and/or adjuvants, and the excipients may be one or more various preparations or compounds conventionally used in the art. For example, the pharmaceutically acceptable excipient may include at least one of pH buffering agent, protective agent and osmotic pressure regulator.

在一些實施方案中，上述siRNA綴合物或醫藥組成物當接觸到表達靶基因的細胞時，由例如：psiCHECK活性篩選和螢光素酶報告基因檢測法，其他如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，例如Western Blot或流式細胞術測定的，上述siRNA綴合物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 some embodiments, when the above-mentioned siRNA conjugates or pharmaceutical compositions are exposed to cells expressing the target gene, they are screened by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, other methods such as PCR or branched DNA (bDNA)-based ) method, or protein-based method, such as immunofluorescence analysis, such as Western Blot or flow cytometry, the above-mentioned siRNA conjugate 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%, to 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%.

在一些實施方案中，上述siRNA綴合物或醫藥組成物當接觸到表達靶基因的細胞時，由例如：psiCHECK活性篩選和螢光素酶報告基因檢測法，其他如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，例如Western Blot或流式細胞術測定的，上述siRNA綴合物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 some embodiments, when the above-mentioned siRNA conjugates or pharmaceutical compositions are exposed to cells expressing the target gene, they are screened by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, other methods such as PCR or branched DNA (bDNA)-based ) method, or a protein-based method, such as immunofluorescence analysis, such as Western Blot or flow cytometry, the remaining expression percentage of the target gene mRNA caused by the above-mentioned siRNA conjugate 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 higher than 50%, not higher than 45%, not higher than 40%, not higher than 35%, not higher than 30%, not higher than 25%, not higher than 20%, not higher less than 15%, or not higher than 10%.

在一些實施方案中，siRNA綴合物或醫藥組成物當接觸到表達靶基因的細胞時，由例如：psiCHECK活性篩選和螢光素酶報告基因檢測法，其他如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，例如Western Blot、或流式細胞術測定的，siRNA綴合物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 some embodiments, siRNA conjugates or pharmaceutical compositions, when exposed to cells expressing a target gene, are screened by, for example, psiCHECK activity and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based siRNA conjugates, while maintaining on-target activity, reduce off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

在一些實施方案中，siRNA綴合物或醫藥組成物當接觸到表達靶基因的細胞時，由例如：psiCHECK活性篩選和螢光素酶報告基因檢測法，其他如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，例如Western Blot、或流式細胞術測定的，siRNA綴合物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 some embodiments, siRNA conjugates or pharmaceutical compositions, when exposed to cells expressing a target gene, are screened by, for example, psiCHECK activity and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based siRNA conjugates reduce on-target activity by at most 20%, at most 19%, at most 15%, at most 10%, up to 5% or more 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%.

在一些實施方案中，siRNA綴合物或醫藥組成物當接觸到表達靶基因的細胞時，由例如：psiCHECK活性篩選和螢光素酶報告基因檢測法，其他如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，例如Western Blot、或流式細胞術測定的，siRNA綴合物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 some embodiments, siRNA conjugates or pharmaceutical compositions, when exposed to cells expressing a target gene, are screened by, for example, psiCHECK activity and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based siRNA conjugates increase 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% 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%.

本揭露還提供了一種細胞，其包含本揭露的siRNA或siRNA綴合物。 The present disclosure also provides a cell comprising the siRNA or siRNA conjugate of the present disclosure.

本揭露還提供了一種試劑盒，其包含本揭露的siRNA或siRNA綴合物。 The present disclosure also provides a kit comprising the siRNA or siRNA conjugate of the present disclosure.

本揭露還提供了一種用於沉默細胞中靶基因或靶基因的mRNA的方法，該方法包括將根據本揭露的siRNA、siRNA綴合物和/或醫藥組成物引入該細胞中的步驟。 The present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell, the method comprising the step of introducing the siRNA, siRNA conjugate and/or pharmaceutical composition according to the present disclosure into the cell.

本揭露還提供了一種用於在體內或在體外沉默細胞中靶基因或靶基因的mRNA的方法，該方法包括將根據本揭露的siRNA、siRNA綴合物和/或醫藥組成物引入該細胞中的步驟。 The present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell in vivo or in vitro, the method comprising introducing an siRNA, siRNA conjugate and/or pharmaceutical composition according to the present disclosure into the cell A step of.

本揭露還提供了一種用於抑制靶基因或靶基因的mRNA表達的方法，該方法包括向有其需要的受試者施用有效量或有效劑量的根據本揭露的siRNA、siRNA綴合物和/或醫藥組成物。 The present disclosure also provides a method for inhibiting a target gene or mRNA expression of a target gene, the method comprising administering to a subject in need thereof an effective amount or an effective dose of siRNA, siRNA conjugate and/or siRNA according to the present disclosure or pharmaceutical compositions.

在一些實施方案中，施用藉由包括肌肉內、支氣管內、胸膜內、腹膜內、動脈內、淋巴、靜脈內、皮下、腦脊髓、或其組合的給藥方式進行。 In some embodiments, administration is by means of administration including intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, cerebrospinal, or combinations thereof.

在一些實施方案中，siRNA、siRNA綴合物和/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 effective dosage of siRNA, siRNA conjugate and/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.

在一些實施方案中，靶基因是B型肝炎病毒(HBV)基因。 In some embodiments, the target gene is a hepatitis B virus (HBV) gene.

本揭露提供了前述siRNA和/或醫藥組成物和/或siRNA綴合物，用於治療和/或預防受試者的B型肝炎病毒(HBV)感染。 The present disclosure provides the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate for treating and/or preventing hepatitis B virus (HBV) infection in a subject.

本揭露提供了前述siRNA和/或醫藥組成物和/或siRNA綴合物，用於治療和/或預防受試者的與B型肝炎病毒(HBV)相關疾病。在一些實施方案中，該與HBV相關的疾病是慢性肝炎。在一些實施方案中，該受試者是HBeAg陽性或HBeAg陰性。 The present disclosure provides the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate for treating and/or preventing hepatitis B virus (HBV)-related diseases in a subject. In some embodiments, the HBV-related disease is chronic hepatitis. In some embodiments, the subject is HBeAg positive or HBeAg negative.

本揭露提供了前述siRNA和/或醫藥組成物和/或siRNA綴合物在製備用於治療和/或預防受試者的B型肝炎病毒(HBV)感染的藥物中的用途。 The present disclosure provides the use of the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate in the preparation of a medicament for treating and/or preventing hepatitis B virus (HBV) infection in a subject.

本揭露提供了前述siRNA和/或醫藥組成物和/或siRNA綴合物在製備用於治療和/或預防受試者的與B型肝炎病毒(HBV)相關疾病的藥物中的用途。在一些實施方案中，該與HBV相關的疾病是慢性肝炎。在一些實施方案中，該受試者是HBeAg陽性或HBeAg陰性。 The present disclosure provides the use of the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate in the preparation of a medicament for treating and/or preventing hepatitis B virus (HBV)-related diseases in a subject. In some embodiments, the HBV-related disease is chronic hepatitis. In some embodiments, the subject is HBeAg positive or HBeAg negative.

本揭露提供了一種治療和/或預防患有B型肝炎病毒(HBV)感染的受試者的方法，包括將本揭露的siRNA和/或醫藥組成物和/或siRNA綴合物給予有需要的受試者。 The disclosure provides a method for treating and/or preventing a subject suffering from hepatitis B virus (HBV) infection, comprising administering the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the disclosure to a patient in need subject.

本揭露提供了一種治療和/或預防患有B型肝炎病毒(HBV)相關疾病的受試者的方法，包括將本揭露的siRNA和/或醫藥組成物和/或siRNA綴合物給予有需要的受試者。在一些實施方案中，該與HBV相關的疾病是慢性肝炎。在一些實施方案中，該受試者是HBeAg陽性或HBeAg陰性。 The present disclosure provides a method for treating and/or preventing a subject suffering from hepatitis B virus (HBV)-related diseases, comprising administering the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure to those in need of subjects. In some embodiments, the HBV-related disease is chronic hepatitis. In some embodiments, the subject is HBeAg positive or HBeAg negative.

本揭露提供了一種體內遞送抑制B型肝炎病毒(HBV)基因表達和/或複製的siRNA至肝臟的方法，包括將本揭露的siRNA和/或醫藥組成物和/或siRNA綴合物給予有需要的受試者。 The present disclosure provides a method for in vivo delivery of siRNA that inhibits hepatitis B virus (HBV) gene expression and/or replication to the liver, comprising administering the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure to the liver in need of subjects.

遞送可以是藉由局部給藥(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。 Delivery can be by topical administration (e.g., direct injection or implantation) or systemic administration, and administration can also be by oral, rectal, or parenteral routes 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, intranasal administration), intracranial administration, etc.

可選的實施方案中，本揭露提供的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 alternative embodiments, the pharmaceutical compositions provided in the present disclosure may be administered by injection, eg, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal or intraperitoneal injection.

各種遞藥系統是已知的並且可以用於本揭露的siRNA、siRNA綴合物或醫藥組成物，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。 Various delivery systems are known and can be used for the siRNAs, siRNA conjugates or pharmaceutical compositions of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of nucleic acids as part of retroviral or other vectors.

本揭露還提供了抑制細胞中B型肝炎病毒(HBV)複製的方法，該方法包括使細胞與本揭露的siRNA和/或醫藥組成物和/或siRNA綴合物接觸，從而抑制細胞中HBV的複製。在一些實施方案中，細胞在受試者體內。在一些實施方案中，細胞是體外的。 The present disclosure also provides a method for inhibiting the replication of hepatitis B virus (HBV) in a cell, the method comprising conjugating the cell with the siRNA and/or the pharmaceutical composition and/or the siRNA of the present disclosure Compound contact, thereby inhibiting the replication of HBV in cells. In some embodiments, the cells are in a subject. In some embodiments, the cells are in vitro.

本揭露還提供了降低感染了HBV的受試者中B型肝炎病毒(HBV)抗原水平的方法，其包括向受試者施用治療有效量的本揭露的siRNA和/或醫藥組成物和/或siRNA綴合物，從而降低受試者中HBV抗原的水平。在一些實施方案中，HBV抗原是HBsAg。在一些實施方案中，HBV抗原是HBeAg。在一些實施方案中，該受試者是HBeAg陽性，在一些實施方案中，該受試者是HBeAg陰性。 The disclosure also provides a method for reducing hepatitis B virus (HBV) antigen levels in a subject infected with HBV, comprising administering to the subject a therapeutically effective amount of the siRNA and/or the pharmaceutical composition and/or siRNA conjugates, thereby reducing the level of HBV antigen in the subject. In some embodiments, the HBV antigen is HBsAg. In some embodiments, the HBV antigen is HBeAg. In some embodiments, the subject is HBeAg positive, in some embodiments, the subject is HBeAg negative.

本揭露還提供了前述siRNA和/或醫藥組成物和/或siRNA綴合物在製備用於預防和/或治療由B肝病毒所引起的病理學病症和疾病的藥物中的用途。 The present disclosure also provides the use of the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate in the preparation of a medicament for preventing and/or treating pathological conditions and diseases caused by hepatitis B virus.

在一些實施方案中，與HBV相關的疾病選自急性B型肝炎、慢性B型肝炎、D型肝炎病毒感染、D型肝炎、肝纖維化、晚期肝病和肝細胞癌。 In some embodiments, the HBV-related disease is selected from acute hepatitis B, chronic hepatitis B, hepatitis D virus infection, hepatitis D, liver fibrosis, advanced liver disease, and hepatocellular carcinoma.

在一些實施方案中，前述siRNA和/或醫藥組成物和/或siRNA綴合物在調節肝臟中表達的基因，或者在治療肝細胞中基因異常表達而引起的病理狀況或疾病中，表現出優異的在靶活性和降低的脫靶活性。在一些實施方式中，該基因選自B型肝炎病毒基因。相應地，該疾病選自慢性肝病、肝炎、肝纖維化疾病、肝增生性疾病和血脂異常。在一些實施方案中，該血脂異常為高膽固醇血症、高甘油三酯血症或動脈粥樣硬化。 In some embodiments, the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate are excellent in regulating genes expressed in the liver, or in treating pathological conditions or diseases caused by abnormal expression of genes in liver cells On-target activity and reduced off-target activity. In some embodiments, the gene is selected from hepatitis B virus genes. Accordingly, the disease is selected from chronic liver disease, hepatitis, liver fibrotic disease, liver proliferative disease and dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

在一些實施方案中，前述siRNA、siRNA綴合物和/或醫藥組成物也可以用於治療其他肝臟疾病，包括以不需要的細胞增殖為特徵的疾病、血液疾病、代謝疾病和以炎症為特徵的疾病。肝臟的增殖疾病可以是良性或惡性疾病，例如癌症、肝細胞癌(HCC)、肝轉移或肝母細胞瘤。肝臟血液學或炎症疾病可以是關於凝血因子、補體介導的炎症或纖維化的疾病。肝臟的代謝疾病包括血脂異常和葡萄糖調節的不規則性。 In some embodiments, the foregoing siRNAs, siRNA conjugates, and/or pharmaceutical compositions may also be used to treat other liver diseases, including diseases characterized by unwanted cell proliferation, blood diseases, metabolic diseases and inflammation characterized disease. A proliferative disease of the liver may be a benign or malignant disease, such as cancer, hepatocellular carcinoma (HCC), liver metastases, or hepatoblastoma. liver The hematological or inflammatory disease may be a disease involving coagulation factors, complement-mediated inflammation or fibrosis. Metabolic disorders of the liver include dyslipidemia and irregularities in glucose regulation.

本揭露還提供了一種siRNA，其包含或為以下任一組的正義股和反義股： The present disclosure also provides an siRNA comprising or being a sense strand and an antisense strand of any of the following groups:

組1，正義股：5’-GUG UGC ACU UCG CUU CAC C-3’(SEQ ID NO：3)；反義股：5’-AGU GAA GCG AAG UGC ACA CGG(SEQ ID NO：4)； Group 1, sense strand: 5'-GUG UGC ACU UCG CUU CAC C-3' (SEQ ID NO: 3); antisense strand: 5'-AGU GAA GCG AAG UGC ACA CGG (SEQ ID NO: 4);

組2，正義股：5’-CUU UUG UCU UUG GGU AUA U-3’(SEQ ID NO：5)；反義股：5’-AUA UAC CCA AAG ACA AAA GAA-3’(SEQ ID NO：6)； Group 2, sense strand: 5'-CUU UUG UCU UUG GGU AUA U-3' (SEQ ID NO: 5); antisense strand: 5'-AUA UAC CCA AAG ACA AAA GAA-3' (SEQ ID NO: 6 );

組3，正義股：5’-UUA CCA AUU UUC UUU UGU U-3’(SEQ ID NO：7)；反義股：5’-AAC AAA AGA AAA UUG GUA ACA-3’(SEQ ID NO：8)； Group 3, sense strand: 5'-UUA CCA AUU UUC UUU UGU U-3' (SEQ ID NO: 7); antisense strand: 5'-AAC AAA AGA AAA UUG GUA ACA-3' (SEQ ID NO: 8 );

組4，正義股：5’-CGU GUG CAC UUC GCU UCA C-3’(SEQ ID NO：9)；反義股：5’-AUG AAG CGA AGU GCA CAC GGU-3’(SEQ ID NO：10)； Group 4, sense strand: 5'-CGU GUG CAC UUC GCU UCA C-3' (SEQ ID NO: 9); antisense strand: 5'-AUG AAG CGA AGU GCA CAC GGU-3' (SEQ ID NO: 10 );

組5，正義股：5’-UGU CUU UGG GUA UAC AUU U-3’(SEQ ID NO：11)；反義股：5’-AAA UGU AUA CCC AAA GAC AAA-3’(SEQ ID NO：12)； Group 5, sense strand: 5'-UGU CUU UGG GUA UAC AUU U-3' (SEQ ID NO: 11); antisense strand: 5'-AAA UGU AUA CCC AAA GAC AAA-3' (SEQ ID NO: 12 );

組6，正義股：5’-CUU UUG UCU UUG GGU AUAC-3’(SEQ ID NO：13)；反義股：5’-AUA UAC CCA AAG ACA AAA GAA-3’(SEQ ID NO：6)； Group 6, sense strand: 5'-CUU UUG UCU UUG GGU AUAC-3' (SEQ ID NO: 13); antisense strand: 5'-AUA UAC CCA AAG ACA AAA GAA-3' (SEQ ID NO: 6) ;

組7，正義股：5’-CAU CUU CUU GUU GGU UCU U-3’(SEQ ID NO：14)；反義股：5’-AAG AAC CAA CAA GAA GAU GAG-3’(SEQ ID NO：15)； Group 7, sense strand: 5'-CAU CUU CUU GUU GGU UCU U-3' (SEQ ID NO: 14); antisense strand: 5'-AAG AAC CAA CAA GAA GAU GAG-3' (SEQ ID NO: 15 );

組8，正義股：5’-UGU CUG CGG CGU UUU AUC A-3’(SEQ ID NO：16)；反義股：5’-UGA UAA AAC GCC GCA GAC ACA-3’(SEQ ID NO：17)； Group 8, sense strand: 5'-UGU CUG CGG CGU UUU AUC A-3' (SEQ ID NO: 16); antisense strand: 5'-UGA UAA AAC GCC GCA GAC ACA-3' (SEQ ID NO: 17 );

組9，正義股：5’-UGC ACU UCG CUU CAC CUC U-3’(SEQ ID NO：18)；反義股：5’-AGA GGU GAA GCG AAG UGC ACA-3’(SEQ ID NO：19)； Group 9, sense strand: 5'-UGC ACU UCG CUU CAC CUC U-3' (SEQ ID NO: 18); antisense strand: 5'-AGA GGU GAA GCG AAG UGC ACA-3' (SEQ ID NO: 19 );

組10，正義股：5’-GCA CUU CGC UUC ACC UCU G-3’(SEQ ID NO：20)；反義股：5’-UAG AGG UGA AGC GAA GUG CAC-3’(SEQ ID NO：21)； Group 10, sense strand: 5'-GCA CUU CGC UUC ACC UCU G-3' (SEQ ID NO: 20); antisense strand: 5'-UAG AGG UGA AGC GAA GUG CAC-3' (SEQ ID NO: 21 );

組11，正義股：5’-GGC GCU GAA UCC UGC GGA C-3’(SEQ ID NO：22)；反義股：5’-AUC CGC AGG AUU CAG CGC CGA-3’(SEQ ID NO：23)。 Group 11, sense strand: 5'-GGC GCU GAA UCC UGC GGA C-3' (SEQ ID NO: 22); antisense strand: 5'-AUC CGC AGG AUU CAG CGC CGA-3' (SEQ ID NO: 23 ).

本揭露提供了一種siRNA，其包含形成雙股區的正義股與反義股；該反義股包含至少15個連續核苷酸，與X04615.1(HBV序列，可在GenBank中查詢到)互補且相差不超過3個核苷酸；該正義股與該反義股互補。 The disclosure provides an siRNA comprising a sense strand and an antisense strand forming a double-stranded region; the antisense strand comprises at least 15 consecutive nucleotides, complementary to X04615.1 (HBV sequence, available in GenBank) And the difference is no more than 3 nucleotides; the sense strand is complementary to the antisense strand.

本揭露還提供了一種式(V)所示的化合物或其互變異構體形式， The present disclosure also provides a compound represented by formula (V) or its tautomeric form,

其中，Y選自O、NH和S； Wherein, 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

，Q₂為R₂；或者Q₁為R₂，Q’₂為

； Q' ₁ for

, Q ₂ is R ₂ ; or Q ₁ is R ₂ , Q' ₂ is

;

其中，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； Wherein, 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 is 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;

B是鹼基；和 B is a base; and

W是離去基團，Z是含磷活性反應基團。 W is a leaving group, and Z is a phosphorus-containing active reactive group.

在一些實施方案中，W是MMTr或DMTr。 In some embodiments, W is MMTr or DMTr.

在一些實施方案中，Z是

。 In some embodiments, Z is

.

在一些實施方案中，上述化合物或其互變異構體形式不是

，其中W，B，Z如上所定義。 In some embodiments, the above-mentioned compounds or tautomeric forms thereof are not

, where W, B, Z are as defined above.

在一些實施方案中，上述化合物或其互變異構體形式不是： In some embodiments, the compound described above or a tautomeric form thereof is not:

和

，其中Z如上所定義。

and

, where Z is as defined above.

在一些實施方案中，式(V)所示的化合物或其互變異構體形式具體為：式(V-1)所示的化合物或其互變異構體形式， In some embodiments, the compound represented by formula (V) or its tautomeric form is specifically: the compound represented by formula (V-1) or its tautomeric form,

其中，X、Y、W、Z、J₁、J₂、B、R₁、R₂、m、n和s如上式(V)中所定義。 Wherein, X, Y, W, Z, J ₁ , J ₂ , B, R ₁ , R ₂ , m, n and s are as defined in the above formula (V).

在一些實施方案中，式(V)所示的化合物或其互變異構體形式具體為：式(V-2)所示的化合物或其互變異構體形式， In some embodiments, the compound represented by formula (V) or its tautomeric form is specifically: the compound represented by formula (V-2) or its tautomeric form,

在一些實施方案中，上述化合物或其互變異構體形式包括但不限於： In some embodiments, the aforementioned compounds or tautomeric forms thereof include, but are not limited to:

以及它們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化合物。 and those compounds in which adenine is replaced by guanine, cytosine, uracil or thymine.

本揭露還提供了一種式(VI)所示的化合物或其互變異構體形式， The present disclosure also provides a compound represented by formula (VI) or its tautomeric form,

其中，Y選自O、NH和S； Wherein, 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

，Q₂為R₂；或者Q₁為R₂，Q”₂為

； Q” ₁ for

, 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;

B是鹼基；和 B is a base; and

W是離去基團。 W is a leaving group.

在一些實施方案中，上述化合物或其互變異構體形式不是 In some embodiments, the above-mentioned compounds or tautomeric forms thereof are not

，其中W和B如上所定義。

, where W and B are as defined above.

在一些實施方案中，上述化合物或其互變異構體形式不是以下的化合物中的一種或多種： In some embodiments, the aforementioned compound or tautomeric form thereof is not one or more of the following compounds:

在一些實施方案中，式(VI)所示的化合物或其互變異構體形式具體為：式(VI-1)所示的化合物或其互變異構體形式， In some embodiments, the compound represented by formula (VI) or its tautomeric form is specifically: the compound represented by formula (VI-1) or its tautomeric form,

其中，X、Y、W、J₁、J₂、B、R₁、R₂、m、n和s如上式(VI)中所定義。 Wherein, X, Y, W, J ₁ , J ₂ , B, R ₁ , R ₂ , m, n and s are as defined in the above formula (VI).

在一些實施方案中，式(VI)所示的化合物或其互變異構體形式具體為：式(VI-2)所示的化合物或其互變異構體、形式， In some embodiments, the compound represented by formula (VI) or its tautomeric form is specifically: the compound represented by formula (VI-2) or its tautomeric form,

以及它們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化合物，和 and those compounds in which adenine is replaced by guanine, cytosine, uracil or thymine, and

，

以及它們中的鹼基被置換為嘌呤、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-硝基吲哚或3-硝基吡咯的那些化合物。

,

And the bases in them are replaced by purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, Those compounds of indole, 5-nitroindole or 3-nitropyrrole.

本揭露還提供了一種siRNA或siRNA綴合物，其特徵在於以2’-甲氧基修飾，替換本揭露任一siRNA或siRNA綴合物的反義股式(I)或(I’)所示的化學修飾或其互變異構體修飾。 The present disclosure also provides an siRNA or siRNA conjugate, which is characterized in that the antisense strand of any siRNA or siRNA conjugate of the present disclosure is modified by 2'-methoxy group, represented by formula (I) or (I'). The indicated chemical modifications or their tautomeric modifications.

本揭露還提供了一種siRNA或siRNA綴合物，其特徵在於本揭露任一siRNA或siRNA綴合物的反義股式(I)或(I’)所示的化學修飾為2’-甲氧基修飾。 The present disclosure also provides an siRNA or siRNA conjugate, characterized in that the antisense strand formula (I) or (I') of any siRNA or siRNA conjugate of the present disclosure is chemically modified as 2'-methoxy base modification.

本揭露還提供了一種siRNA或siRNA綴合物，該反義股在其5’區域的第2位至第8位中的至少一個核苷酸包含修飾，該修飾為式(I)所示的化學修飾或互變異構體修飾。 The present disclosure also provides an siRNA or siRNA conjugate, the antisense strand contains a modification in at least one nucleotide from the 2nd to the 8th position of the 5' region, and the modification is represented by formula (I) Chemical modification or tautomeric modification.

術語定義 Definition of Terms

如無特殊說明，本揭露的“siRNA”、“siRNA綴合物”、“化學修飾”和“靶向配體”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，其可為可藥用鹽。 Unless otherwise specified, the "siRNA", "siRNA conjugate", "chemical modification" and "targeting ligand" of the present disclosure can be independently salt, mixed salt or non-salt (such as free acid or free base) form exists. When present in the form of a salt or mixed salt, it may be a pharmaceutically acceptable salt.

本揭露中所述化合物可藥用鹽選自無機鹽或有機鹽，本揭露所述化合物可與酸性或鹼性物質反應成相應鹽。 The pharmaceutically acceptable salts of the compounds described in this disclosure are selected from inorganic salts or organic salts, and the compounds described in this disclosure can react with acidic or basic substances to form corresponding salts.

“與酸性物質反應成相應鹽”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。 "Reacting with an acidic substance to form a corresponding salt" refers to a salt formed with an inorganic acid or an organic acid that can retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include but not limited to hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; organic acid salts include but not limited to formate, acetate, 2,2-dichloroacetate, tris Fluoroacetate, Propionate, Caproate, Caprylate, Caprate, Undecylenate, Glycolate, Gluconate, Lactate, Sebacate, Adipate, Pentadiol salt, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, Laurate, Malate, Glutamate, Pyroglutamate, Aspartate, Benzoate, Methanesulfonate, Benzenesulfonate, p-Toluenesulfonate, Alginate, Ascorbate, salicylate, 4-aminosalicylate, naphthalene disulfonate, etc. These salts can be prepared by methods known in the art.

“與鹼性物質反應成相應鹽”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。 "Reacting with a basic substance to form a corresponding salt" refers to a salt formed with an inorganic base or an organic base 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, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts and magnesium salts, preferably sodium salts. Salts derived from organic bases include, but are not limited to, those of the following: primary, secondary, 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-diethylaminoethanol Ethanol, Dicyclohexylamine, Lysine, Arginine, Histidine, Caffeine, Procaine, Choline, Betaine, B Diamine, glucosamine, methylglucamine, 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.

雖然為簡便起見將全部上述結構式畫成某些異構體形式，但是本發明可以包括所有的異構體，如互變異構體、旋轉異構體、幾何異構體、非對映異構體、外消旋體和對映異構體。 Although all of the above formulas are drawn as certain isomeric forms for simplicity, the present invention may include all isomers such as tautomers, rotamers, geometric isomers, diastereoisomers isomers, racemates and enantiomers.

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於本揭露的範圍之內。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。 Compounds of the present disclosure may exist in particular geometric or stereoisomeric forms. This disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and their racemic and other mixtures, such as enantiomerically 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 the present disclosure.

另外，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。 In addition, 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 prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine, lactam-lactimine isomerizations. An example of a lactam-lactimine equilibrium is between A and B shown below.

本發明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發明的範圍內。化合物的命名不排除任何互變異構體。 All compounds in the present invention can be drawn as Form A or Form B. All tautomeric forms are within the scope of the invention. The naming of compounds does not exclude any tautomers.

本揭露化合物可以是不對稱的，例如，具有一個或多個立體異構體。除非另有說明，所有立體異構體都包括，如對映異構體和非對映異構體。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 be asymmetric, eg, possess one or more stereoisomers. Unless otherwise stated, all stereoisomers are included, such as enantiomers and diastereomers. Compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or 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)。 Optically active (R)- and (S)-isomers as well as D and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, wherein 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), a diastereoisomeric salt is formed with an appropriate optically active acid or base, and then by conventional methods known in the art Methods The diastereoisomers were resolved and the pure enantiomers were recovered. 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. amines to amines formate).

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 disclosure also includes isotopically labeled compounds of the disclosure that are identical to those described herein, but wherein one or more atoms are replaced by an atom of an atomic mass or mass number different from that normally found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ¹²³ I, ¹²⁵ I and ³⁶ Cl, 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化合物。在製備氘代形式的式I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), the position is understood to have an abundance of deuterium (i.e., at least 10 % deuterium incorporation). Exemplary compounds having a natural abundance greater than deuterium can be at least 1000 times more abundant deuterium, at least 2000 times more abundant deuterium, at least 3000 times more abundant deuterium, at least 4000 times more abundant deuterium, at least 5000 times more abundant deuterium, at least 6000 times more abundant deuterium deuterium or higher abundance deuterium. The present disclosure also includes various deuterated forms of compounds of formula I. Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can refer to the relevant literature to synthesize the deuterated form of the compound of formula I. Commercially available deuterated starting materials can be used in the preparation of deuterated forms of compounds of formula I, or they can be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane Tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide and deuterated methyl iodide, etc.

“視需要地”或“視需要”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“視需要的被鹵素或者氰基取代的C_1-6烷基”是指鹵素或者氰基可以但不必須存在，該說明包括烷基被鹵素或者氰基取代的情形和烷基不被鹵素和氰基取代的情形。 "Optionally" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "C _1-6 alkyl optionally substituted by halogen or cyano" means that halogen or cyano may but not necessarily exist, and this description includes the case where the alkyl is substituted by halogen or cyano and the alkyl is not substituted by halogen. And the case of cyano substitution.

本發明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。雖然為簡便起見將全部上述結構式畫成某些異構體形式，但是本發明可以包括所有的異構體，如互變異構體、旋轉異構體、幾何異構體、非對映異構體、外消旋體和對映異構體。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 compound described in the present invention, the bond "

"indicates that no configuration is specified, i.e. if chiral isomers exist in the chemical structure, the bond"

"can be"

"or"

, or both "

"and"

"Two configurations. Although all of the above structural formulas are drawn as certain isomer forms for simplicity, the present invention may include all isomers, such as tautomers, rotamers, geometric isomers Body, diastereoisomer, racemate and enantiomer. In the chemical structure of the compound described in this disclosure, the bond "

"No configuration specified, i.e. key"

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

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，否則當具體值在本揭露中出現時，“約”或“基本上包含”的含義應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 determined by one of ordinary skill in the art, and the value depends in part on how it is measured or determined (ie, the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "comprising essentially" may mean a range of up to 20%, such as between 1% and 15%, Varying between 1% and 10%, between 1% and 5%, between 0.5% and 5%, between 0.5% and 1%, in this disclosure, numbers or numerical ranges are preceded by the term "about Each instance of " also includes the given number of embodiments. Unless otherwise stated, when a specific value appears in this disclosure, the meaning of "about" or "comprising essentially" shall be within an acceptable error range for the specific value.

公開中數值為儀器測量值或儀器測量後計算值，存在一定程度的誤差，一般而言，正負10%均屬於合理誤差範圍內。當然需要考慮該數值所用之處的上下文，例如，總雜質的含量，該數值為測量後誤差變化不超過正負10%，可以為正負9%、正負8%、正負7%、正負6%、正負5%、正負4%、正負3%、正負2%或正負1%，較佳正負5%。 The disclosed values are measured by instruments or calculated by instruments, and there is a certain degree of error. Generally speaking, plus or minus 10% is within a reasonable error range. Of course, the context where the value is used needs to be considered, for example, the content of total impurities. The value is that the error after measurement does not change by more than plus or minus 10%, which can be plus or minus 9%, plus or minus 8%, plus or minus 7%, plus or minus 6%, plus or minus 5%, plus or minus 4%, plus or minus 3%, plus or minus 2% or plus or minus 1%, preferably plus or minus 5%.

如本文所使用的，在RNA介導的基因沉默的情形中，siRNA的正義股(又稱SS、SS股或有義股)是指包含與靶mRNA序列相同或基本上相同的序列的股；siRNA的反義股(又稱AS或AS股)是指具有與靶mRNA序列互補的序列的股。 As used herein, in the context of RNA-mediated gene silencing, the sense strand (also known as SS, SS strand or sense strand) of an siRNA refers to a strand comprising a sequence that is identical or substantially identical to the target mRNA sequence; The antisense strand (also known as AS or AS strand) of an siRNA refers to a strand that has a sequence that is complementary to the target mRNA sequence.

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，在雙股核酸分子中，一條股的鹼基與另一條股上的鹼基以互補的方式相配對。在DNA中，嘌呤鹼基腺嘌呤(A)始終與嘧啶鹼基胸腺嘧啶(T)(或者在RNA中為尿嘧啶(U))相配對；嘌呤鹼基鳥嘌呤(C)始終與嘧啶鹼基胞嘧啶(G)相配對。每個鹼基對都包括一個嘌呤和一個嘧啶。當一條股上的腺嘌呤始終與另一條股上的胸腺嘧啶(或尿嘧啶)配對，以及鳥嘌呤始終與胞嘧啶配對時，兩條股被認為是彼此相互補的，以及從其互補股的序列中可以推斷出該股的序列。與此相應地，“錯配”在本領域中意指在雙股核酸中，對應位置上的鹼基並未以互補的形式配對存在。 The terms "complementary" or "reverse complementary" are used interchangeably and have the meaning known to those of ordinary skill in the art, i.e., in a double-stranded nucleic acid molecule, a base on one strand interacts with a base on the other strand. The bases are paired in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base Cytosine (G) is paired. 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 the sequence of their complementary strands The sequence of the strand can be inferred. Correspondingly, "mismatch" in the art means that in a double-stranded nucleic acid, the bases at the corresponding positions are not paired in a complementary form.

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。 The term "base" includes any known DNA and RNA base, base analogs such as purine or pyrimidine, which also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and Natural analogs.

術語“鹼基類似物”是指位於可併入核酸雙股體中的經過修飾的核苷酸中核苷酸糖部分的1’位(或可併入核酸雙股體中的核苷酸糖部分取代物的等效位置)的雜環部分。在本揭露中，鹼基類似物一般為嘌呤或嘧啶鹼基，不包括常見鹼基：鳥嘌呤(G)、胞嘧啶(C)、腺嘌呤(A)、胸腺嘧啶(T)和尿嘧啶(U)。鹼基的非限制性實例包括次黃嘌呤(I)、黃嘌呤(X)、3β-D-呋喃核糖基-(2,6-二胺基嘧啶)(K)、3-β-D-呋喃核糖基-(1-甲基-吡唑并[4,3-d]嘧啶-5,7(4H,6H)-二酮)(P)、異胞嘧啶(iso-C)、異鳥嘌呤(iso-G)、1-β-D-呋喃核糖基-(5-硝基吲哚)、1-β-D-呋喃核糖基-(3-硝基吡咯)、5-溴尿嘧啶、2-胺基嘌呤、4-硫代-dT、7-(2-噻吩基)-咪唑并[4，5-b]吡啶(Ds)和吡咯-2-甲醛(Pa)、2-胺基-6-(2-噻吩基)嘌呤(S)、2-側氧吡啶(Y)、二氟甲苯基、4-氟-6-甲基苯并咪唑、4-甲基苯并咪唑、3-甲基羥基異喹啉基、5-甲基羥基異喹啉基和3-甲基-7-丙炔基羥基異喹啉基、7-氮雜吲哚基、6-甲基-7-氮雜吲哚基、咪唑并吡啶基、9-甲基-咪唑并吡啶基、吡咯并吡嗪基、羥基異喹啉基、7-丙炔基羥基異喹啉基、丙炔基-7-氮雜吲哚基、2,4,5-三甲基苯基、4-甲基吲哚基、4,6-二甲基吲哚基、苯基、萘基、蒽基、菲基、芘基、茋基(stilbenzyl)、并四苯基、并五苯基和其結構衍生物。鹼基類似物還可以是通用鹼基。 The term "base analogue" refers to the 1' position of the nucleotide sugar moiety in a modified nucleotide that can be incorporated into a nucleic acid duplex (or the nucleotide sugar moiety that can be incorporated into a nucleic acid duplex) The equivalent position of the substituent) of the heterocyclic moiety. In this disclosure, base analogs are generally purine or pyrimidine bases, excluding common bases: guanine (G), cytosine (C), adenine (A), thymine (T) and uracil ( U). Non-limiting examples of bases include hypoxanthine (I), xanthine (X), 3β-D-ribofuranosyl-(2,6-diaminopyrimidine) (K), 3-β-D-furan Ribosyl-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione) (P), isocytosine (iso-C), isoguanine ( iso-G), 1-β-D-ribofuranosyl-(5-nitroindole), 1-β-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2- Aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa), 2-amino-6- (2-thienyl)purine (S), 2-oxopyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylhydroxy Isoquinolyl, 5-methylhydroxyisoquinolyl and 3-methyl-7-propynylhydroxyisoquinolyl, 7-azaindole, 6-methyl-7-azaindole Base, imidazopyridyl, 9-methyl-imidazopyridyl, pyrrolopyrazinyl, hydroxyisoquinolyl, 7-propynyl hydroxyisoquinolyl, propynyl-7-azaindole Base, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, stilbene (stilbenzyl), naphthacene, pentacene and their structural derivatives. Base analogs can also be universal bases.

術語“通用鹼基”是指位於經過修飾的核苷酸中核苷酸糖部分的1'位或核苷酸糖部分取代物中等效位置的雜環部分，該雜環部分當存在於核酸雙股體中時，可與一種以上鹼基相對定位，同時不改變雙螺旋結構(例如，磷酸骨架的結構)。此外，通用鹼基不會破壞其所駐留的單股核酸與靶核酸形成雙股體的能力。含有通用鹼基的單股核酸與靶核酸形成雙股體的能力可藉由所屬技術領域具有通常知識者顯而易見的方法測定(例如，UV吸光度、圓二色性、凝膠遷移法、單股核酸酶敏感性等)。另外，可以改變能觀察到雙股體形成的條件，以確定雙股體穩定性或形成，例如，溫度，如解股溫度(Tm)，與核酸雙股體穩定性相關。相較於與靶核酸精確互補的參考單股核酸，含有通用鹼基的單股核酸與靶核酸形成的雙股體的Tm低於與互補核酸形成的雙股體。然而，與其中通用鹼基被鹼基置換而產生單一錯配的參考單股核酸相比較，含有通用鹼基的單股核酸與靶核酸形成的雙股體的Tm高於用具有錯配鹼基的核酸形成的雙股體。 The term "universal base" refers to a heterocyclic moiety located at the 1 ' position of a nucleotide sugar moiety in a modified nucleotide or an equivalent position in a nucleotide sugar moiety substitute, which heterocyclic moiety when present in a nucleic acid double-stranded In vivo, it can be positioned relative to more than one base without changing the double helix structure (eg, the structure of the phosphate backbone). In addition, universal bases do not disrupt the ability of the single-stranded nucleic acid on which they reside to form double-stranded forms with the target nucleic acid. The ability of a single-stranded nucleic acid containing a universal base to form a duplex with a target nucleic acid can be determined by methods apparent to those of ordinary skill in the art (e.g., UV absorbance, circular dichroism, gel shift, single-stranded nucleic acid enzyme sensitivity, etc.). In addition, the conditions under which duplex formation can be observed can be varied to determine duplex stability or formation, eg, temperature, such as the dissolving temperature (Tm), correlates with nucleic acid duplex stability. Compared to a reference single-stranded nucleic acid that is precisely complementary to a target nucleic acid, a duplex formed by a single-stranded nucleic acid containing a universal base and a target nucleic acid has a lower Tm than a duplex formed with a complementary nucleic acid. However, compared to a reference single-stranded nucleic acid in which a universal base is replaced by a base to generate a single mismatch, the Tm of a duplex formed by a single-stranded nucleic acid containing a universal base and a target nucleic acid is higher than that obtained with a base having a mismatch. nucleic acid to form double-stranded bodies.

一些通用鹼基能夠藉由在通用鹼基與所有鹼基鳥嘌呤(G)、胞嘧啶(C)、腺嘌呤(A)、胸腺嘧啶(T)和尿嘧啶(U)在鹼基配對條件下形成氫鍵，來實現鹼基配對。通用鹼基不是只與單一互補鹼基形成鹼基對的鹼基。在雙股體中，通用鹼基可與雙股體相對股上與之相對的G、C、A、T和U中每一者不形成氫鍵、形成一個氫鍵或形成一個以上氫鍵。在一些實施方案中，通用鹼基不與雙股體相對股上與之相對的鹼基相互作用。在雙股體中，與通用鹼基之間發生的鹼基配對不會改變磷酸骨架的雙螺旋結構。通用鹼基也可以借助堆疊相互作用與相同核酸股上的相鄰核苷酸中的鹼基相互作用。這種堆疊相互作用可使雙股體穩定，尤其是在通用鹼基不與雙股體相對股上與之相對定位的鹼基形成任何氫鍵的情形中。通用結合核苷酸的非限制性實例包括肌苷、1-β-D-呋喃核糖基-5-硝基吲哚和/或1-β-D-呋喃核糖基-3-硝基吡咯。 Some universal bases can be formed by base pairing conditions between the universal base and all bases guanine (G), cytosine (C), adenine (A), thymine (T) and uracil (U). Form hydrogen bonds to achieve base pairing. A universal base is not a base that only forms a base pair with a single complementary base. In a duplex, the universal base may form no hydrogen bonds, form one hydrogen bond, or form more than one hydrogen bond with each of the opposing G, C, A, T, and U on the opposite strand of the duplex. In some embodiments, the universal base does not interact with the opposing base on the opposite strand of the duplex. In duplexes, base pairing with universal bases does not alter the double helix structure of the phosphate backbone. Universal bases can also interact with bases in adjacent nucleotides on the same nucleic acid strand via stacking interactions. This stacking interaction can stabilize the duplex, especially if the universal base does not form any hydrogen bonds with the bases positioned opposite it on the opposite strand of the duplex. Non-limiting examples of universal binding nucleotides include inosine, 1-β-D-ribofuranosyl-5-nitroindole, and/or 1-β-D-ribofuranosyl-3-nitropyrrole.

“G”、“C”、“A”、“T”和“U”分別一般代表核苷酸，其分別包含鳥苷、胞苷、腺苷、胸苷和尿苷的鹼基。 "G," "C," "A," "T," and "U," respectively, generally represent nucleotides that include the bases of guanosine, cytidine, adenosine, thymidine, and uridine, respectively.

術語“平端”或“平末端”可互換使用，是指在siRNA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siRNA將在其整個長度範圍內是雙股的。 The terms "blunt end" or "blunt end" are used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of an siRNA, ie, no nucleotide overhangs. In most cases, siRNAs with both blunt-ended ends will be double-stranded throughout their entire length.

術語“突出端”是指至少一個未配對的核苷酸從siRNA雙螺旋結構中突出。例如：當siRNA中一股的3’端超過另一股5’端，或反之亦然時，即有一個核苷酸突出端。siRNA可包含含有至少一個核苷酸的突出端；可替代地，該突出端可包含至少兩個核苷酸、至少三個核苷酸、至少四個核苷酸、至少五個核苷酸或更多個。核苷酸突出端可包含或其組成可為核苷酸/核苷類似物，包括脫氧核苷酸/核苷。該(等)突出端可位於正義股、反義股或其任何組合。此外，突出端的核苷酸(群)可出現在siRNA的反義或正義股的5’-端、3’-端或兩個末端。 The term "overhang" refers to at least one unpaired nucleotide protruding from the siRNA duplex structure. For example, there is a nucleotide overhang when the 3' end of one strand of siRNA exceeds the 5' end of the other strand, or vice versa. The siRNA can comprise an overhang comprising at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. Nucleotide overhangs may comprise or consist of nucleotide/nucleoside analogs, including deoxynucleotides/nucleosides. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotide(s) of the overhang can occur at the 5'-end, 3'-end, or both ends of the antisense or sense strand of the siRNA.

本揭露中，正義股或反義股的“5’區域”也即“5’端”，兩者可替換使用。例如反義股5’區域的第2位至第8位的核苷酸，也可替換為反義股5’端的第2位至第8位的核苷酸。同理，正義股或反義股的“3’區域”和“3’端”也可替換使用。 In this disclosure, the "5' region" of the sense strand or the antisense strand is also the "5' end", and the two can be used interchangeably. For example, the 2nd to 8th nucleotides in the 5' region of the antisense strand can also be replaced with the 2nd to 8th nucleotides at the 5' end of the antisense strand. Similarly, the "3' region" and "3' end" of the sense strand or the antisense strand can also be used interchangeably.

本揭露上下文中，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。 In the context of this disclosure, the term "chemical modification" or "modification" includes all alterations of nucleotides by chemical means, such as the addition or removal of chemical moieties, or the substitution of one chemical moiety for another.

術語“2’-氟修飾的核苷酸”指核苷酸的核糖基2'位的羥基被氟取代形成的核苷酸，“非氟修飾的核苷酸”指核苷酸的核糖基2'位的羥基被非氟基團取代形成的核苷酸或核苷酸類似物，“核苷酸類似物”指能夠在核酸中代替核苷酸，但結構不同於腺嘌呤核糖核苷酸、鳥嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脫氧核糖核苷酸的基團。如異核苷酸、橋聯的核苷酸(bridged nucleic acid，簡稱BNA)或無環核苷酸。該甲氧基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。異核苷酸是指核苷酸中鹼基在核糖環上的位置發生改變而形成的化合物。在一些實施方式中，異核苷酸可以是鹼基從核糖環的1'-位移動至2'-位或3'-位而形成的化合物。BNA是指受約束的或不能接近的核苷酸。BNA可以含有五員環、六員環、或七員環的具有“固定的”C3'-內切糖縮攏的橋聯結構。通常將該橋摻入到該核糖的2'-、4'-位處以提供一個2',4'-BNA核苷酸。在一些實施方式中，BNA可以是LNA、ENA、cET BNA等。無環核苷酸是核苷酸的糖環被打開形成的一類核苷酸。在一些實施方式中，無環核苷酸可以是解鎖核酸(UNA)或甘油核酸(GNA)。 The term "2'-fluorine-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and "non-fluorine-modified nucleotide" refers to the ribose group 2 of the nucleotide. Nucleotides or nucleotide analogues formed by replacing the hydroxyl group at the ' position with non-fluorine groups. "Nucleotide analogues" refer to nucleotides that can replace nucleotides in nucleic acids, but are different in structure from adenine ribonucleotides, adenine ribonucleotides, A group of guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxyribonucleotides. Such as isonucleotides, bridged nucleic acid (BNA) or acyclic nucleosides acid. The methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is substituted with a methoxy group. Isonucleotide refers to a compound formed by changing the position of the base in the nucleotide on the ribose ring. In some embodiments, the isonucleotide may be a compound formed by moving a base from the 1'-position to the 2'-position or the 3'-position of the ribose ring. BNA refers to constrained or inaccessible nucleotides. BNAs may contain five-, six-, or seven-membered bridging structures with "fixed" C3'-endosugar constrictions. Typically the bridge is incorporated at the 2'-,4'-position of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, the BNA can be LNA, ENA, cET BNA, and the like. Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of the nucleotide. In some embodiments, the acyclic nucleotide may be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA).

術語“抑制”，可以與“減少”、“沉默”、“下調”、“阻抑”和其他類似術語交替使用，並且包括任何水平的抑制。抑制可藉由這些變量中的一個或多個與對照水平相比的絕對或相對水平的減少來評估。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siRNA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+siRNA/Ratioreporter only)*100%；本揭露中，剩餘mRNA表達量比例(或剩餘活性%)=100%-抑制率(%)。 The term "inhibit", is used interchangeably with "reduce", "silence", "downregulate", "suppress" and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to control levels. The control level can be any type of control level used in the art, such as a pre-dose baseline level or from a similar untreated or control (eg buffer only or inert control) treated subject, cell , or sample-determined levels. For example, the residual expression of mRNA can be used to characterize the inhibition degree of siRNA on target gene expression, such as the remaining expression of mRNA is not higher than 99%, not higher than 95%, not higher than 90%, not higher than 85%, not higher than More than 80%, not more than 75%, not more than 70%, not more than 65%, not more than 60%, not more 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 more than 10%. The inhibition rate of target gene expression can be detected by Dual-Glo® Luciferase Assay System, and the firefly (Firefly) chemiluminescence value and Renilla (Renilla) chemiluminescence value are read respectively, and the relative value Ratio=Ren/Fir is calculated, and the inhibition rate (% )=1-(Ratio+siRNA/Ratioreporter only)*100%; in this disclosure, the remaining mRNA expression ratio (or remaining activity %)=100%-inhibition rate (%).

術語“有效量”或“有效劑量”指指獲得任一種或多種有益的或所需的治療結果所必需的藥物、化合物或醫藥組成物的量。對於預防用途，有益的或所需的結果包括消除或降低風險、減輕嚴重性或延遲病症的發作，包括病症、其併發症和在病症的發展過程中呈現的中間病理表型的生物化學、組織學和/或行為症狀。對於治療應用，有益的或所需的結果包括臨床結果，諸如減少各種本揭露靶基因、靶mRNA或靶蛋白相關病症的發病率或改善該病症的一個或更多個症狀，減少治療病症所需的其它藥劑的劑量，增強另一種藥劑的療效，和/或延緩患者的本揭露靶基因、靶mRNA或靶蛋白相關病症的進展。 The terms "effective amount" or "effective dose" refer to the amount of drug, compound or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired results include elimination or reduction of risk, lessening of severity, or delay of onset of the disorder, including the biochemical, histological Physical and/or behavioral symptoms. For therapeutic applications, beneficial or desired outcomes include clinical outcomes, such as reducing the incidence of or improving one or more symptoms of a disorder associated with various target genes, target mRNAs, or target proteins of the present disclosure, reducing the need to treat the disorder The dosage of other medicaments can enhance the curative effect of another medicament, and/or delay the progress of the disease related to the target gene, target mRNA or target protein of the present disclosure in patients.

術語“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。 The terms "subject", "patient", "subject" or "individual" are used interchangeably and include humans or non-human animals such as mammals such as humans or monkeys.

本揭露提供的siRNA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制服務。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siRNA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siRNA的方法也是所屬技術領域具有通常知識者所熟知的。 The siRNA provided in this disclosure can be obtained by conventional preparation methods in the art (such as solid-phase synthesis and liquid-phase synthesis). Among them, solid-phase synthesis has commercialized customized services. The modified nucleotide group can be introduced into the siRNA described in the present disclosure by using the correspondingly modified nucleoside monomer, the method for preparing the correspondingly modified nucleoside monomer and the modified nucleotide group Methods for introducing siRNA are also well known to those skilled in the art.

術語“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。 The term "pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically acceptable salt or prodrug thereof and other chemical components, as well as other components such as a physiologically acceptable carrier and excipient. The purpose of the pharmaceutical composition is to promote the administration to the living body, facilitate the absorption of the active ingredient and thus exert the biological activity.

術語“可藥用賦形劑”或“藥學上可接受的賦形劑”包括但不限於任何已經被美國食品和藥物管理局批准對於人類或家畜動物使用可接受的任何助劑、載體、賦形劑、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。 The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient that has been approved by the U.S. Food and Drug Administration for use in humans or livestock animals. excipients, glidants, sweeteners, diluents, preservatives, dyes/coloring Coloring agent, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent or emulsifying agent.

術語“有效量”或“有效治療量”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、患者的總體健康情況、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。 The term "effective amount" or "therapeutically effective amount" encompasses an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. Effective amounts for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the method, route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

術語“B型肝炎病毒相關疾病”或“HBV相關疾病”是指由HBV感染和/或複製引起或與其相關的疾病或病症。本文所採用術語“HBV相關疾病”包括可因降低HBV基因表達和/或複製而受益的疾病、病症或症狀。HBV相關疾病的非限制性實例包括例如：D型肝炎病毒感染、δ型(delta)肝炎、急性B型肝炎；急性暴發性B型肝炎；慢性B型肝炎；肝纖維化；末期肝病；肝細胞癌。 The term "hepatitis B virus-associated disease" or "HBV-associated disease" refers to a disease or condition caused by or associated with HBV infection and/or replication. As used herein, the term "HBV-associated disease" includes diseases, disorders or conditions that would benefit from reduced HBV gene expression and/or replication. Non-limiting examples of HBV-related diseases include, for example: hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; cancer.

術語“烷基”指飽和脂肪族烴基團，其為包含1至20個碳原子的直鏈或支鏈基團，在一些實施方案中選自含有1至12個碳原子的烷基。非限制性實例包括甲基、乙基、正丙基、異丙基、正丁基、異丁基、第三丁基、第二丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各種支鏈異構體等。在一些實施方案中選自含有1至6個碳原子的烷基，非限制性實施例包括甲基、乙基、正丙基、異丙基、正丁基、異丁基、第三丁基、第二丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，當被取代時，取代基可以在任何可使用的連接點上被取代，該取代基在一些實施方案中選自一個或多個以下基團，其獨立地選自烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、鹵素、巰基、羥基、硝基、氰基、環烷基、雜環烷基、芳基、雜芳基、環烷氧基、雜環烷氧基、環烷硫基、雜環烷硫基、側氧基、羧基或羧酸酯基。 The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, selected in some embodiments from alkyl groups containing 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, second-butyl, n-pentyl, 1,1-dimethylpropyl , 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl- 2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1 ,3-Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl , 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-di Methylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl , 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl Base, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethyl Diethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branches chain isomers, etc. In some embodiments, selected from alkyl groups containing 1 to 6 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl , second butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methyl butyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1, 2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4 -methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, the substituents being in some embodiments selected from one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, hetero Aryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, pendant oxy, carboxyl or carboxylate.

術語“烷氧基”指-O-(烷基)和-O-(非取代的環烷基)，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、環丙氧基、環丁氧基、環戊氧基、環己氧基。烷氧基可以是視需要取代的或非取代的，當被取代時，取代基在一些實施方案中選自一個或多個以下基團，其獨立地選自鹵素、氘、羥基、側氧、硝基、氰基、C_1-6烷基、C_1-6烷氧基、C_2-6烯氧基、C_2-6炔氧基、C_3-6環烷氧基、3至6員雜環烷氧基、C_3-8環烯氧基、5至6員芳基或雜芳基，該C_1-6烷基、C_1-6烷氧基、C_2-6烯氧基、C_2-6炔氧基、C_3-6環烷氧基、3至6員雜環烷氧基、C_3-8環烯氧基、5至6員芳基或雜芳基視需要被一個或多個選自鹵素、氘、羥基、側氧、硝基、氰基所取代。同理，“炔氧基”、“烯氧基”、“環烷氧基”、“雜環烷氧基”、“環烯氧基”的定義如上述“烷氧基”定義。 The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. Alkoxy groups may be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups independently selected from halogen, deuterium, hydroxyl, pendant oxygen, Nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 members Heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl are optionally replaced by one Or more selected from halogen, deuterium, hydroxyl, side oxygen, nitro, cyano substituted. Similarly, the definitions of "alkynyloxy", "alkenyloxy", "cycloalkoxy", "heterocycloalkoxy", and "cycloalkenyloxy" are as defined above for "alkoxy".

術語“烯基”指直鏈或支鏈的非芳香族烴基，其含有至少一個碳-碳雙鍵，並且具有2-10個碳原子。在這樣的基團中可以存在多達5個碳-碳雙鍵。例如，“C_2-C₆”烯基被定義為具有2-6個碳原子的烯基。烯基的示例包括但不限於：乙烯基、丙烯基、丁烯基和環己烯基。烯基的直鏈、支鏈或環狀部分可以含有雙鍵，並且在正常化合價所允許的任何位置視需要地被單-、二-、三-、四-或五-取代。 The term "alkenyl" refers to a straight or branched non-aromatic hydrocarbon group containing at least one carbon-carbon double bond and having 2 to 10 carbon atoms. There may be as many as 5 carbon-carbon double bonds present in such groups. For example, "C2 _- _C6 "alkenyl is defined as an alkenyl group having 2-6 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, and cyclohexenyl. Straight chain, branched chain or cyclic moieties of alkenyl groups may contain double bonds and be optionally mono-, di-, tri-, tetra- or penta-substituted at any position permitted by normal valence.

術語“環烯基”表示具有特定數量的碳原子和至少一個碳-碳雙鍵的單環烴基。 The term "cycloalkenyl" denotes a monocyclic hydrocarbon group having the specified number of carbon atoms and at least one carbon-carbon double bond.

術語“炔基”指直鏈或支鏈的烴基，其含有2-10個碳原子並且含有至少一個碳-碳三鍵。可以存在多達5個碳-碳三鍵。因此，“C₂-C₆炔基”表示具有2-6個碳原子的烯基。炔基的示例包括但不限於：乙炔基、2-丙炔基和2-丁炔基。炔基的直鏈、支鏈部分可以含有正常化合價所允許的三鍵，並且在正常化合價所允許的任何位置視需要地被單-、二-、三-、四-或五-取代。 The term "alkynyl" refers to a straight or branched chain hydrocarbon group containing 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. There can be as many as 5 carbon-carbon triple bonds. Thus, " _C2 - _C6alkynyl " means an alkenyl group having 2-6 carbon atoms. Examples of alkynyl include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl. The straight chain, branched moieties of the alkynyl groups may contain triple bonds as permitted by normal valences and may be optionally mono-, di-, tri-, tetra- or penta-substituted at any position permitted by normal valences.

術語“酮”指代本文所述藉由羰基橋連接的任何烷基、烯基、炔基、環烷基、環烯基、雜環基、雜芳基或芳基。酮基的示例包括但不限於：烷醯基(例如，乙醯基、丙醯基、丁醯基、戊醯基、己醯基)、烯醯基(例如，丙烯醯基)炔醯基(例如，乙炔醯基、丙炔醯基、丁炔醯基、戊炔醯基、己炔醯基)、芳醯基(例如，苯甲醯基)、雜芳醯基(例如，吡咯醯基、咪唑醯基、喹啉醯基、吡啶醯基)。 The term "keto" refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or aryl group described herein attached through a carbonyl bridge. Examples of keto groups include, but are not limited to: alkyl (e.g., acetyl, propionyl, butyryl, pentyl, hexyl), alkenoyl (e.g., acryl) alkynyl (e.g., Ethynyl, propynyl, butynyl, pentynyl, hexynyl), arayl (e.g., benzoyl), heteroaryl (e.g., pyrroyl, imidazolyl) base, quinolinyl, pyridinyl).

術語“烷氧基羰基”指藉由羰基橋連接的上述定義的任何烷氧基(即，-C(O)O-烷基)。烷氧基羰基的示例包括但不限於：甲氧基羰基、乙氧基羰基、異丙氧基羰基、正丙氧基羰基、第三丁氧基羰基、苄氧基羰基或正戊氧基羰基。 The term "alkoxycarbonyl" refers to any alkoxy group defined above attached through a carbonyl bridge (ie, -C(O)O-alkyl). Examples of alkoxycarbonyl include, but are not limited to: methoxycarbonyl, ethyl Oxycarbonyl, isopropoxycarbonyl, n-propoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl or n-pentyloxycarbonyl.

術語“芳氧基羰基”指藉由氧羰基橋連接的上述定義的任何芳基(即，-C(O)O-芳基)。芳氧基羰基的例子包括但不限於：苯氧基羰基和萘氧基羰基。 The term "aryloxycarbonyl" refers to any aryl group as defined above attached through an oxycarbonyl bridge (ie, -C(O)O-aryl). Examples of aryloxycarbonyl include, but are not limited to, phenoxycarbonyl and naphthoxycarbonyl.

術語“雜芳氧基羰基”指藉由氧基羰基橋連接的上述定義的任何雜芳基(即，-C(O)O-雜芳基)。雜芳基氧基羰基的示例包括但不限於：2-吡啶氧基羰基、2-噁唑基氧基羰基、4-噻唑基氧基羰基或嘧啶基氧基羰基。 The term "heteroaryloxycarbonyl" refers to any heteroaryl group defined above attached through an oxycarbonyl bridge (ie, -C(O)O-heteroaryl). Examples of heteroaryloxycarbonyl include, but are not limited to, 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

術語“環烷基”或“碳環”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，在一些實施方案中包含3至7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、稠環和橋環的環烷基。環烷基可以是取代的或未取代的，當被取代時，取代基可以在任何可使用的連接點上被取代，在一些實施方案中選自一個或多個以下基團，獨立地選自鹵素、氘、羥基、側氧、硝基、氰基、C_1-6烷基、C_1-6烷氧基、C_2-6烯氧基、C_2-6炔氧基、C_3-6環烷氧基、3至6員雜環烷氧基、C_3-8環烯氧基、5至6員芳基或雜芳基，該C_1-6烷基、C_1-6烷氧基、C_2-6烯氧基、C_2-6炔氧基、C_3-6環烷氧基、3至6員雜環烷氧基、C_3-8環烯氧基、5至6員芳基或雜芳基視需要被一個或多個選自鹵素、氘、羥基、側氧、硝基、氰基所取代。 The term "cycloalkyl" or "carbocycle" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, and in some embodiments from 3 to 7 carbon atom. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, etc.; multicyclic cycloalkyls include spiro Cycloalkyl rings, fused rings and bridged rings. Cycloalkyl groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, and in some embodiments are selected from one or more of the following groups, independently selected from Halogen, deuterium, hydroxyl, side oxygen, nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy , C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3-6 membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5-6 membered aromatic The radical or heteroaryl is optionally substituted by one or more selected from halogen, deuterium, hydroxyl, pendant oxygen, nitro, cyano.

該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環烷基，非限制性實例包括茚滿基、四氫萘基、苯并環庚烷基等。環烷基可以是視需要取代的或非取代的，當被取代時，取代基在一些實施方案中選自一個或多個以下基團，其獨立地選自鹵素、氘、羥基、側氧、硝基、氰基、C_1-6烷基、C_1-6烷氧基、C_2-6烯氧基、C_2-6炔氧基、C_3-6環烷氧基、3至6員雜環烷氧基、C_3-8環烯氧基、5至6員芳基或雜芳基，該C_1-6烷基、C_1-6烷氧基、C_2-6烯氧基、C_2-6炔氧基、C_3-6環烷氧基、3至6員雜環烷氧基、C_3-8環烯氧基、5至6員芳基或雜芳基視需要被一個或多個選自鹵素、氘、羥基、側氧、硝基、氰基所取代。 The cycloalkyl ring may be fused to an aryl or heteroaryl ring where the ring bonded to the parent structure is a cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl Alkyl etc. Cycloalkyl groups can be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups independently selected from halogen, deuterium, hydroxyl, pendant oxygen, Nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 members Heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl are optionally replaced by one Or more selected from halogen, deuterium, hydroxyl, side oxygen, nitro, cyano substituted.

術語“雜環烷基”或“雜環”或“雜環基”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。在一些實施方案中選自包含3至12個環原子，其中1~4個是雜原子；在一些實施方案中包含3至7個環原子。單環雜環烷基的非限制性實例包括吡咯烷基、咪唑烷基、四氫呋喃基、四氫噻吩基、二氫咪唑基、二氫呋喃基、二氫吡唑基、二氫吡咯基、哌啶基、哌嗪基、嗎啉基、硫代嗎啉基、高哌嗪基等。多環雜環烷基包括螺環、稠環和橋環的雜環烷基。“雜環烷基”非限制性實例包括： The term "heterocycloalkyl" or "heterocycle" or "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms in which one or more ring Atoms are heteroatoms selected from nitrogen, oxygen, or S(O) _m (where m is an integer from 0 to 2), excluding ring portions of -OO-, -OS- or -SS-, the remaining ring atoms being carbon. In some embodiments is selected from comprising 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; in some embodiments comprising 3 to 7 ring atoms. Non-limiting examples of monocyclic heterocycloalkyl include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperrolyl, Pyridyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Multicyclic heterocycloalkyls include spiro, fused and bridged heterocycloalkyls. Non-limiting examples of "heterocycloalkyl" include:

，等等。

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

和

等。

and

wait.

雜環烷基可以是視需要取代的或非取代的，當被取代時，取代基在一些實施方案中選自一個或多個以下基團，其獨立地選自鹵素、氘、羥基、側氧、硝基、氰基、C_1-6烷基、C_1-6烷氧基、C_2-6烯氧基、C_2-6炔氧基、C_3-6環烷氧基、3至6員雜環烷氧基、C_3-8環烯氧基、5至6員芳基或雜芳基，該C_1-6烷基、C_1-6烷氧基、C_2-6烯氧基、C_2-6炔氧基、C_3-6環烷氧基、3至6員雜環烷氧基、C_3-8環烯氧基、5至6員芳基或雜芳基視需要被一個或多個選自鹵素、氘、羥基、側氧、硝基、氰基所取代。 Heterocycloalkyl groups can be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups independently selected from halogen, deuterium, hydroxyl, pendant oxygen , nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy , C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl are optionally One or more selected from halogen, deuterium, hydroxyl, side oxygen, nitro, cyano are substituted.

術語“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，在一些實施方案中選自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： The term "aryl" refers to a 6 to 14 membered full carbon monocyclic or fused polycyclic (that is, rings sharing adjacent pairs of carbon atoms) group with a conjugated π-electron system, selected in some embodiments from 6 to 12 members, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocycloalkyl or cycloalkyl ring, where the ring bonded to the parent structure is an aryl ring, non-limiting examples of which include:

芳基可以是取代的或非取代的，當被取代時，取代基在一些實施方案中選自一個或多個以下基團，其獨立地選自鹵素、氘、羥基、側氧、硝基、氰基、C_1-6烷基、C_1-6烷氧基、C_2-6烯氧基、C_2-6炔氧基、C_3-6環烷氧基、3至6員雜環烷氧基、C_3-8環烯氧基、5至6員芳基或雜芳基，該C_1-6烷基、C_1-6烷氧基、C_2-6烯氧基、C_2-6炔氧基、C_3-6環烷氧基、3至6員雜環烷氧基、C_3-8環烯氧基、5至6員芳基或雜芳基視需要被一個或多個選自鹵素、氘、羥基、側氧、硝基、氰基所取代。 Aryl groups may be substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups independently selected from halogen, deuterium, hydroxyl, pendant oxygen, nitro, Cyano, C _1-6 alkyl, C 1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to ₆ membered heterocycloalkane Oxygen, C _3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _{2- 6} alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl are optionally replaced by one or more Substituted by halogen, deuterium, hydroxyl, side oxygen, nitro, cyano.

術語“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。雜芳基在一些實施方案中選自6至12員，在一些實施方案中選自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。 The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is in some embodiments selected from 6 to 12 members, in some embodiments from 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 may be fused to an aryl, heterocycloalkyl, or cycloalkyl ring where the ring bonded to the parent structure is a heteroaryl ring, non-limiting examples of which include:

雜芳基可以是視需要取代的或非取代的，當被取代時，取代基在一些實施方案中選自一個或多個以下基團，其獨立地選自鹵素、氘、羥基、側氧、硝基、氰基、C_1-6烷基、C_1-6烷氧基、C_2-6烯氧基、C_2-6炔氧基、C_3-6環烷氧基、3至6員雜環烷氧基、C_3-8環烯氧基、5至6員芳基或雜芳基，該C_1-6烷基、C_1-6烷氧基、C_2-6烯氧基、C_2-6炔氧基、C_3-6環烷氧基、3至6員雜環烷氧基、C_3-8環烯氧基、5至6員芳基或雜芳基視需要被一個或多個選自鹵素、氘、羥基、側氧、硝基、氰基所取代。 Heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups independently selected from halogen, deuterium, hydroxyl, pendant oxygen, Nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 members Heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl are optionally replaced by one Or more selected from halogen, deuterium, hydroxyl, side oxygen, nitro, cyano substituted.

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

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

術語“鹵烷基”指被鹵素取代的烷基，其中烷基如上所定義。 The term "haloalkyl" refers to an alkyl group substituted by halogen, wherein alkyl is as defined above.

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

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

術語“側氧”指=O基團，例如，碳原子與氧原子藉由雙鍵連接，其中形成酮或醛基。 The term "side oxygen" refers to an =O group, for example, a carbon atom and an oxygen atom connected by a double bond, wherein a ketone or aldehyde group is formed.

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

術語“羧基”指-C(O)OH。 The term "carboxy" refers to -C(O)OH.

術語“醛基”指-CHO。 The term "aldehyde" refers to -CHO.

在本揭露的化學結構式中，“

”或

其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, "

"or

It may be attached to one or more of any group according to the scope of the invention described herein; an asterisk "*" indicates a chiral center.

本揭露中，術語“包含”可替換為“選自”。 In this disclosure, the term "comprising" can be replaced with "selected from".

本揭露中，“磷酸酯基”和“磷酸酯基團”可替換使用，其可為磷酸一酯基、磷酸二酯基或磷酸三酯基；若無特別說明，天然核苷間鍵磷酸酯基為磷酸二酯基。 In this disclosure, "phosphate group" and "phosphate group" can be used interchangeably, and it can be a phosphoric acid monoester group, a phosphoric acid diester group or a phosphoric acid triester group; The group is a phosphodiester group.

本揭露中，“硫代磷酸酯基”和“硫代磷酸酯基團”可替換使用，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

或

(M為S原子)表示，兩者可替換使用。 In this disclosure, "phosphorothioate group" and "phosphorothioate group" can be used interchangeably, referring to a phosphodiester group modified by replacing a non-bridging oxygen atom with a sulfur atom, which can be

or

(M is an S atom) indicates that the two can be used interchangeably.

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

A moiety can be replaced by any group that enables linkage 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。 The term "linked", when referring to an association between two molecules, means that the two molecules are connected by a covalent bond or that the two molecules are associated by a non-covalent bond (eg, a hydrogen bond or an ionic bond).

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 is linked to a second compound or group via an intervening group, compound or molecule (eg, a linking group).

術語“取代的”表示指定原子(通常是碳、氧和氮原子)上的任何一個或多個氫原子被本文所限定的任何基團所替代，條件是不超過該指定原子的正常化合價並且取代生成穩定化合物。取代基的非限制性示例包括C1-C6烷基、C2-C6烯基、C2-C6炔基、氰基、羥基、側氧基、羧基、環烷基、環烯基、雜環基、雜芳基、芳基、酮、烷氧基羰基、芳氧基羰基、雜芳氧基羰基或鹵素(例如，F、Cl、Br、I)。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。 The term "substituted" means that any one or more hydrogen atoms on the designated atom (usually carbon, oxygen and nitrogen atoms) are replaced by any group as defined herein, provided that the designated atom's normal valence is not exceeded and the replacement Forms stable compounds. Non-limiting examples of substituents include C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cyano, hydroxyl, pendant oxy, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, hetero Aryl, aryl, ketone, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halogen (eg, F, Cl, Br, I). When the substituent is a ketone or pendant oxygen (ie, =0), then two (2) hydrogens are replaced on the atom.

“被一個或多個......取代”是指可以被單個或多個取代基取代。當被多個取代基取代時，可以是複數個相同取代基，也可以是一個或複數個不同取代基的組合。 "Substituted by one or more" means that it can be substituted by single or multiple substituents. When substituted by a plurality of substituents, it may be a plurality of the same substituents, or one or a combination of a plurality of different substituents.

本揭露中，SEQ ID NO：1序列為(5’-3’)： In this disclosure, the sequence of SEQ ID NO: 1 is (5'-3'):

本揭露中，SEQ ID NO：2序列為(5’-3’)： In this disclosure, the sequence of SEQ ID NO: 2 is (5'-3'):

圖1顯示GalNAc綴合siRNA對於鼠原代肝細胞mTTR基因抑制活性； Figure 1 shows the inhibitory activity of GalNAc-conjugated siRNA on the mTTR gene of primary mouse hepatocytes;

圖2顯示GalNAc綴合siRNA對於小鼠mTTR基因的體內抑制活性。 Figure 2 shows the in vivo inhibitory activity of GalNAc-conjugated siRNA on mouse mTTR gene.

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

本揭露中的一些縮略語定義如下： Some abbreviations used in this disclosure are defined as follows:

DCE：二氯乙烷； DCE: Dichloroethane;

Sc(OTf)₃：三氟甲烷磺酸鈧； Sc(OTf) ₃ : scandium trifluoromethanesulfonate;

TFH：四氫呋喃； TFH: Tetrahydrofuran;

Pd/C：鈀-碳； Pd/C: palladium-carbon;

TFA：三氟乙酸； TFA: trifluoroacetic acid;

DMF：二甲基甲醯胺； DMF: Dimethylformamide;

DIPEA：N-乙基二異丙基胺； DIPEA: N-ethyldiisopropylamine;

HoBt：1-羥基苯并三唑； HoBt: 1-hydroxybenzotriazole;

EDCI：1-乙基-(3-二甲基胺基丙基)碳醯二亞胺鹽酸鹽； EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

DMTrCl：4,4'-雙甲氧基三苯甲基氯； DMTrCl: 4,4'-bismethoxytrityl chloride;

DIEA：N,N-二異丙基乙胺； DIEA: N,N-Diisopropylethylamine;

HATU：2-(7-氮雜苯并三唑)-N,N,N',N'-四甲基脲六氟磷酸鹽； HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate;

LiOH：氫氧化鋰； LiOH: lithium hydroxide;

DMAP：4-二甲胺基吡啶； DMAP: 4-dimethylaminopyridine;

HBTU：苯并三唑-N,N,N',N'-四甲基脲六氟磷酸鹽； HBTU: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate;

DMTrCl：1-[氯(4-甲氧基苯基)苄基]-4-甲氧基苯 DMTrCl: 1-[Chloro(4-methoxyphenyl)benzyl]-4-methoxybenzene

CF₃SO₃H：三氟甲磺酸； CF ₃ SO ₃ H: Trifluoromethanesulfonic acid;

BnBr：溴化苄； BnBr: benzyl bromide;

DEPBT：3-(二乙氧基磷醯氧基)-1,2,3-苯并三嗪-4-酮； DEPBT: 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4-one;

Bz：苯甲醯基保護基； Bz: benzoyl protecting group;

MMTr：甲氧基苯基二苯甲基； MMTr: methoxyphenylbenzhydryl;

DMTr：二甲氧三苯甲基保護基。 DMTr: Dimethoxytrityl protecting group.

實施例Example

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，如冷泉港的抗體技術實驗手冊，分子選殖手冊；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，為市場購買的常規試劑。 The present disclosure is further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure. The experimental methods in the examples of the present disclosure that do not indicate specific conditions are usually in accordance with conventional conditions, such as Cold Spring Harbor Antibody Technology Experiment Manual, Molecular Breeding Manual; or in accordance with the conditions suggested by raw material or commodity manufacturers. Reagents without specific sources indicated are conventional reagents purchased in the market.

一、式(I)化學修飾的製備和活性評價1. Preparation and activity evaluation of chemical modification of formula (I)

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

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, 717mg, 3.76mmol) in dichloromethane (10mL) solution, 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 (15mL), and combined The organic phase was washed with saturated aqueous sodium bicarbonate (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: _C14H21O5S , [M+H] ⁺ theoretical: 301.10 found _: _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 (239mg, 1.22mmol) was dissolved in dimethylformamide (DMF, 10mL), NaH (60% dissolved in mineral oil, 93mg, 2.33mmol) solution was added under ice-cooling, and the reaction was stirred for 30 Minutes, then dropwise added compound 2 (350mg, 1.16mmol), after the dropwise addition, the reaction was stirred at 60°C for 5 hours, after the reaction was completed, quenched with water, the aqueous phase was extracted three times with ethyl acetate (15mL), and the combined organic The phase was washed three times with water (10 mL), then washed with saturated brine (10 mL), then the solvent was evaporated to dryness under reduced pressure, and the reverse phase preparative HPLC (C ¹⁸ , condition: 5-50% (A: H ₂ O, B: CH ₃ CN), flow rate: 70 mL/min), and 220 mg of compound 4 were obtained after lyophilization. MS m/z: _C19H21N5O3Na , [M+Na] ⁺ _theoretical : _390.16 , _found : 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。MS m/z：C₁₆H₁₈N₅O₃，[M+H]⁺理論：328.13，實測：328.4。 Compound 4 (1.50g, 4.08mmol) was dissolved in 20mL 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. Preparative HPLC (C ¹⁸ , condition: 5-25% (A: H ₂ O, B: CH ₃ CN), flow rate: 70 mL/min) obtained 1.10 g of compound 5 after lyophilization. MS m/z: _C16H18N5O3 , [M+H] ⁺ theoretical: _328.13 _, _found : 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 pyridine ( 5mL) solution, after the dropwise addition, the reaction was stirred overnight at room temperature. After the reaction was completed, it was quenched with water, and the solvent was evaporated to dryness under reduced pressure. Preparative HPLC (C ¹⁸ , condition: 5-80% (A:H ₂ O, B: CH ₃ CN), flow rate: 70 mL/min), and lyophilized to obtain 1.00 g of compound 6 . MS m/z _: _C37H36N5O5 , [MH] ⁺ theoretical: _630.26 _, found: 630.5. Racemate compound 6 was resolved by 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). Compound 6A(-) (200mg, 0.32mmol), tetrazole (11mg, 0.16mmol), N-methylimidazole (5mg, 0.06mmol), 3A molecular sieve (500mg) were dissolved in 10mL of acetonitrile, and added at room temperature Compound 7 (144mg, 0.48mmol), stirred overnight at room temperature. After the reaction was completed, the molecular sieves were filtered off, dichloromethane (30 mL) was added, washed three times with saturated aqueous sodium bicarbonate (10 mL), and then washed with saturated brine (20 mL), the filtrate was spin-dried and subjected to reverse-phase preparative HPLC (C ¹⁸ , condition: 5-100% (A: water, B: CH ₃ CN), flow rate: 70 mL/min), and 200 mg of compound 1-1a was obtained after lyophilization. MS m/z: C ₄₀ H ₃₉ N ₆ O ₇ P, [M-Diisopropyl+OH] ⁺ Theory: 747.26, Found: 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。 Compound 6B(+) (200mg, 0.32mmol), tetrazole (11mg, 0.16mmol), N-methylimidazole (5mg, 0.06mmol), 3A molecular sieves (500mg) were dissolved in 10mL of acetonitrile, and added at room temperature Compound 7 (144mg, 0.48mmol), stirred overnight at room temperature. After the reaction was completed, the molecular sieves were filtered off, dichloromethane (30 mL) was added, washed three times with saturated aqueous sodium bicarbonate (10 mL), and then washed with saturated brine (20 mL), the filtrate was spin-dried and subjected to reverse-phase preparative HPLC (C ¹⁸ , condition: 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: _C40H39N6O7P , [M-diisopropyl+OH] ⁺ _theory : _747.26 , found: 747.5.

1.2 合成化合物1-21.2 Synthesis of compound 1-2

將化合物1(2g,8.36mmol)溶解在DMF(20mL)中，室溫條件下，氬氣保護下緩慢加入NaH(0.37g,9.2mmol,60%溶解在礦物油中)。室溫攪拌2個小時後，將化合物2(3.3g,16.72mmol)加入到反應液中。室溫攪拌12h後，將反應液濃縮後，殘留物用乙醇(EtOH，50mL)再結晶得到目標產物3A(1.3g，收率44.0%)(二氯甲烷：乙酸乙酯=2：1,Rf =0.2)和目標產物3B(0.6g，化合物1的混合物)(二氯甲烷：乙酸乙酯=2：1,Rf=0.18)。 Compound 1 (2 g, 8.36 mmol) was dissolved in DMF (20 mL), and NaH (0.37 g, 9.2 mmol, 60% dissolved in mineral oil) was slowly added under the protection of argon at room temperature. After stirring at room temperature for 2 hours, compound 2 (3.3 g, 16.72 mmol) was added to the reaction solution. After stirring at room temperature for 12 h, the reaction solution was concentrated, and the residue was recrystallized with ethanol (EtOH, 50 mL) to obtain the target product 3A (1.3 g, yield 44.0%) (dichloromethane:ethyl acetate=2:1, Rf =0.2) and target product 3B (0.6g, mixture of compound 1) (dichloromethane:ethyl acetate=2:1, Rf=0.18).

將化合物3A(1.3g,3.68mmol)溶解在混合液三氟乙酸(TFA，4mL)和DCM(20mL)後，室溫攪拌12h，將反應液濃縮，得到的殘留物用反向管柱純化(C¹⁸,H₂O+乙腈)得到目標產物4(1g，收率91.44%)。MS m/z：C₃₉H₃₈N₆O₆，[M+H]⁺：687.5. Compound 3A (1.3g, 3.68mmol) was dissolved in a mixture of trifluoroacetic acid (TFA, 4mL) and DCM (20mL), stirred at room temperature for 12h, the reaction solution was concentrated, and the obtained residue was purified by reverse column ( C ¹⁸ , H ₂ O+acetonitrile) to obtain the target product 4 (1 g, yield 91.44%). _MS m/z: _C39H38N6O6 , [M+H] ⁺ : _687.5 _.

將化合物(D-Threoninol 5，1.2g,11.4mmol)溶解在吡啶(10mL)然後緩慢加入DMTrCl(4.64g,13.70mmol)的吡啶(15mL)溶液。室溫下攪拌16h後，將反應液用H₂O(10mL)淬滅並濃縮。將反應液濃縮，得到的殘留物用反向管柱純化(C¹⁸,H₂O+乙腈)得到目標產物6(4.0g，收率86.0%)。MS m/z：C₂₅H₂₉NO₄，[M+Na]⁺：430.4. The compound (D-Threoninol 5 , 1.2 g, 11.4 mmol) was dissolved in pyridine (10 mL) and then a solution of DMTrCl (4.64 g, 13.70 mmol) in pyridine (15 mL) was added slowly. After stirring at room temperature for 16 h, the reaction was quenched with H ₂ O (10 mL) and concentrated. The reaction solution was concentrated, and the obtained residue was purified by a reverse column (C ¹⁸ , H ₂ O+acetonitrile) to obtain the target product 6 (4.0 g, yield 86.0%). _MS m/z: _C25H29NO4 , [M+Na] ⁺ : 430.4 _.

將化合物6(600mg,2.02mmol)、化合物4(822.5mg,2.02mmol)和二氫喹啉(EEDQ，998.2mg,4.04mmol)溶解在DCM(10mL)和甲醇(MeOH，5mL)中。該混合液室溫攪拌16h後，將固體濾掉，濾液用DCM(100mL)稀釋。有機相用H₂O(30mL)洗滌三次，用無水Na₂SO₄乾燥，過濾再濃縮。得到的殘留物用反向管柱純化(C¹⁸,H₂O+乙腈)得到目標產物7(780mg，收率56.3%)。MS m/z：C₃₉H₃₈N₆O₆，[M+H]⁺：687.5. Compound 6 (600 mg, 2.02 mmol), compound 4 (822.5 mg, 2.02 mmol) and dihydroquinoline (EEDQ, 998.2 mg, 4.04 mmol) were dissolved in DCM (10 mL) and methanol (MeOH, 5 mL). After the mixture was stirred at room temperature for 16 h, the solid was filtered off and the filtrate was diluted with DCM (100 mL). The organic phase was washed three _times with _H2O (30 mL), dried over anhydrous _Na2SO4 , filtered and concentrated. The obtained residue was purified by reverse column (C ¹⁸ , H ₂ O+acetonitrile) to obtain the target product 7 (780 mg, yield 56.3%). _MS m/z: _C39H38N6O6 , [M+H] ⁺ : _687.5 _.

將化合物7(780mg,1.13mmol)、四唑(39.8mg,0.57mmol)、N-甲基咪唑(18.7mg,0.23mmol)溶解在CH₃CN(10mL)中，加入3A分子篩(700mg)。氬氣保護下室溫攪拌5min後，加入化合物8(513.5mg,1.70mmol)。室溫攪拌1h後，將分子篩過濾掉，固體用DCM(30mL)淋洗三次。濾液分別用飽和的NaHCO₃水溶液(30mL×4)和H₂O(30mL×4)洗滌；有機相在30℃下濃縮。得到殘留物用反向管柱純化(C¹⁸,H₂O+乙腈，乙腈90%)，凍乾後得到目標化合物1-2(700mg，收率69.5%)。MS m/z：C₄₈H₅₅N₈O₇P，[M-氰乙基-二異丙基+OH]^-：749.3. Compound 7 (780 mg, 1.13 mmol), tetrazole (39.8 mg, 0.57 mmol), N-methylimidazole (18.7 mg, 0.23 mmol) were dissolved in CH ₃ CN (10 mL), and 3A molecular sieves (700 mg) were added. After stirring at room temperature for 5 min under the protection of argon, compound 8 (513.5 mg, 1.70 mmol) was added. After stirring at room temperature for 1 h, the molecular sieves were filtered off, and the solid was rinsed three times with DCM (30 mL). The filtrate was washed with saturated aqueous NaHCO ₃ (30 mL×4) and H ₂ O (30 mL×4), respectively; the organic phase was concentrated at 30°C. The obtained residue was purified by reverse column (C ¹⁸ , H ₂ O+acetonitrile, acetonitrile 90%), and the target compound 1-2 (700 mg, yield 69.5%) was obtained after lyophilization. MS m/z: C ₄₈ H ₅₅ N ₈ O ₇ P, [M-cyanoethyl-diisopropyl+OH] ^- : 749.3.

1.3 合成化合物1-31.3 Synthesis of compounds 1-3

將化合物1(2g,8.36mmol)溶解在DMF(20mL)中，室溫條件下，氬氣保護下緩慢加入NaH(0.37g,9.2mmol,60%溶解在礦物油中)。室溫攪拌2個小時後，將化合物2(3.3g,16.72mmol)加入到反應液中。室溫攪拌12h後，將反應也濃縮後，殘留物用EtOH(50mL)再結晶得到目標產物3A(1.3g，收率44.0%)(二氯甲烷：乙酸乙酯=2：1,Rf= 0.2)和目標產物3B(0.6g，化合物1的混合物)(二氯甲烷：乙酸乙酯=2：1,Rf=0.18)。 Compound 1 (2 g, 8.36 mmol) was dissolved in DMF (20 mL), and NaH (0.37 g, 9.2 mmol, 60% dissolved in mineral oil) was slowly added under the protection of argon at room temperature. After stirring at room temperature for 2 hours, compound 2 (3.3 g, 16.72 mmol) was added to the reaction solution. After stirring at room temperature for 12 h, the reaction was also concentrated, and the residue was recrystallized with EtOH (50 mL) to obtain the target product 3A (1.3 g, yield 44.0%) (dichloromethane: ethyl acetate=2:1, Rf=0.2 ) and the target product 3B (0.6 g, a mixture of compound 1) (dichloromethane: ethyl acetate = 2: 1, Rf = 0.18).

將化合物3A(1.3g,3.68mmol)溶解在混合液TFA(4mL)和DCM(20mL)後，室溫攪拌12h，將反應液濃縮，得到的殘留物用反向管柱純化(C¹⁸,H₂O+乙腈)得到目標產物4(1g，收率91.44%)。MS m/z：C₃₉H₃₈N₆O₆，[M+H]⁺：687.5. Compound 3A (1.3g, 3.68mmol) was dissolved in a mixture of TFA (4mL) and DCM (20mL), stirred at room temperature for 12h, the reaction solution was concentrated, and the obtained residue was purified by reverse column (C ¹⁸ , H ₂ O+acetonitrile) to obtain the target product 4 (1 g, yield 91.44%). _MS m/z: _C39H38N6O6 , [M+H] ⁺ : _687.5 _.

將化合物L-蘇胺醇(L-Threoninol 5，1.2g,11.4mmol)溶解在吡啶(10mL)，然後緩慢加入DMTrCl(4.64g,13.70mmol)的吡啶(15mL)溶液。室溫下攪拌16h後，將反應液用H₂O(10mL)淬滅並濃縮。將反應液濃縮，得到的殘留物用反向管柱純化(C¹⁸,H₂O+乙腈)得到目標產物6(4.0g，收率86.0%)。MS m/z：C₂₅H₂₉NO₄，[M+Na]⁺：430.4. The compound L-threoninol 5 (L-Threoninol 5 , 1.2 g, 11.4 mmol) was dissolved in pyridine (10 mL), and then a solution of DMTrCl (4.64 g, 13.70 mmol) in pyridine (15 mL) was added slowly. After stirring at room temperature for 16 h, the reaction was quenched with H ₂ O (10 mL) and concentrated. The reaction solution was concentrated, and the obtained residue was purified by a reverse column (C ¹⁸ , H ₂ O+acetonitrile) to obtain the target product 6 (4.0 g, yield 86.0%). _MS m/z: _C25H29NO4 , [M+Na] ⁺ : 430.4 _.

將化合物6(600mg,2.02mmol)、化合物4(822.5mg,2.02mmol)、四甲基脲六氟磷酸鹽(HATU，1.15g,3.03mmol)和二異丙基乙胺(DIEA，1mL,6.05mmol)溶解在DMF(10mL)中。室溫攪拌16h後，反應液過濾，濾液用DCM(100mL)稀釋。有機相用H₂O(30mL)洗三次，用無水Na₂SO₄乾燥，過濾，濃縮。得到的殘留物用反向管柱純化(C¹⁸,H₂O+乙腈，乙腈60%)，凍乾後得到目標化合物7(1.0g，收率72.1%)。MS m/z：C₃₉H₃₈N₆O₆，[M+H]⁺：687.5. Compound 6 (600mg, 2.02mmol), compound 4 (822.5mg, 2.02mmol), tetramethyluronium hexafluorophosphate (HATU, 1.15g, 3.03mmol) and diisopropylethylamine (DIEA, 1mL, 6.05 mmol) was dissolved in DMF (10 mL). After stirring at room temperature for 16 h, the reaction solution was filtered, and the filtrate was diluted with DCM (100 mL). The organic phase was washed three _times with _H2O (30 mL), dried over anhydrous _Na2SO4 , filtered and concentrated. The obtained residue was purified by reverse column (C ¹⁸ , H ₂ O+acetonitrile, acetonitrile 60%), and the target compound 7 was obtained after lyophilization (1.0 g, yield 72.1%). _MS m/z: _C39H38N6O6 , [M+H] ⁺ : _687.5 _.

將化合物7(1.2g,1.75mmol)、四唑(61.2mg,0.87mmol),N-甲基咪唑(28.7mg,0.35mmol)溶解在CH₃CN(10mL)中，加入3A分子篩(700mg)。氬氣保護下室溫攪拌5min後，加入化合物8(0.79g,2.62mmol)。室溫攪拌1h後，將分子篩過濾掉，固體用DCM(30mL)淋洗三次。濾液分別用飽和的NaHCO₃水溶液(30mL×4)和H₂O(30mL×4)洗。有機相在30℃下濃縮。得到殘留物用反向管柱純化(C¹⁸,H₂O+乙腈，乙腈90%)，凍乾後得到目標化合物1-3(1.2g，收率77.4%)。MS m/z：C₄₈H₅₅N₈O₇P，[M-氰乙基-二異丙基+OH]^-：749.3. Compound 7 (1.2 g, 1.75 mmol), tetrazole (61.2 mg, 0.87 mmol), N-methylimidazole (28.7 mg, 0.35 mmol) were dissolved in CH ₃ CN (10 mL), and 3A molecular sieves (700 mg) were added. After stirring at room temperature for 5 min under the protection of argon, compound 8 (0.79 g, 2.62 mmol) was added. After stirring at room temperature for 1 h, the molecular sieves were filtered off, and the solid was rinsed three times with DCM (30 mL). The filtrate was washed with saturated aqueous NaHCO ₃ (30 mL×4) and H ₂ O (30 mL×4), respectively. The organic phase was concentrated at 30°C. The obtained residue was purified by reverse column (C ¹⁸ , H ₂ O+acetonitrile, acetonitrile 90%), and the target compound 1-3 (1.2 g, yield 77.4%) was obtained after lyophilization. MS m/z: C ₄₈ H ₅₅ N ₈ O ₇ P, [M-cyanoethyl-diisopropyl+OH] ^- : 749.3.

1.4 合成化合物1-4a和化合物1-4b1.4 Synthesis of compound 1-4a and compound 1-4b

將化合物1A(6.73g,28.14mmol)溶解在乾燥的DMF(80mL)中，氬氣保護下緩慢加入NaH(60%,1.24g,30.95mmol)。該混合液室溫攪拌30min後，將該反應液加到溶有四(三苯基膦)鈀(Pd(PPh₃)₄，1.95g,1.69mmol)，三苯基膦(PPh₃，0.74g,2.81mmol)和化合物1(4.0g,28.14mmol)的四氫呋喃(THF，60mL)溶液中。該反應液在55℃攪拌16 h後，將固體濾掉並用DCM(60mL)洗滌三次。將濾液濃縮，得到的殘留物用正向管柱純化(先用乙酸乙酯，再用乙酸乙酯：甲醇(12：1沖洗管柱)得目標產物2(7g，粗品)。 Compound 1A (6.73g, 28.14mmol) was dissolved in dry DMF (80mL), and NaH (60%, 1.24g, 30.95mmol) was added slowly under the protection of argon. After the mixture was stirred at room temperature for 30 min, the reaction solution was added to a solution of tetrakis(triphenylphosphine)palladium (Pd(PPh ₃ ) ₄ , 1.95g, 1.69mmol), triphenylphosphine (PPh ₃ , 0.74g , 2.81mmol) and compound 1 (4.0g, 28.14mmol) in tetrahydrofuran (THF, 60mL) solution. After the reaction was stirred at 55 °C for 16 h, the solid was filtered off and washed three times with DCM (60 mL). The filtrate was concentrated, and the obtained residue was purified by a forward column (washing the column with ethyl acetate first, then ethyl acetate:methanol (12:1)) to obtain the target product 2 (7 g, crude product).

將化合物2(8g，粗品)和DMTrCl(12.65g,37.34mmol)溶解在吡啶(10mL)。該混合物室溫攪拌16h後，用水(80mL)淬滅並濃縮。得到的殘留物用反向管柱純化(C¹⁸，水+乙腈)凍乾後，得到目標化合物3(13g，收率83.7%)。 Compound 2 (8 g, crude) and DMTrCl (12.65 g, 37.34 mmol) were dissolved in pyridine (10 mL). After stirring the mixture at room temperature for 16 h, it was quenched with water (80 mL) and concentrated. The obtained residue was purified by reverse column (C ¹⁸ , water+acetonitrile) and lyophilized to obtain the target compound 3 (13 g, yield 83.7%).

將化合物3(5g,8.02mmol)溶解在甲醇(MeOH，20mL)和氨水(6mL)中，該混合液室溫攪拌16h後，將反應液濃縮。得到的殘留物用正向管柱(DCM：MeOH=20：1)純化得到目標化合物4(4g，收率96.0%)。 Compound 3 (5 g, 8.02 mmol) was dissolved in methanol (MeOH, 20 mL) and ammonia water (6 mL), and the mixture was stirred at room temperature for 16 h, and then the reaction solution was concentrated. The obtained residue was purified with a forward column (DCM:MeOH=20:1) to obtain the target compound 4 (4 g, yield 96.0%).

氬氣保護下，0℃下將硼烷(BH₃)四氫呋喃溶液(1.0M溶解於THF中，38.54mL,38.54mmol)逐滴滴加到溶有化合物4(4.00g,7.71mmol)的THF(12mL)溶液中。該化合物在氬氣保護下0℃攪拌6h後，逐滴滴加H₂O(27mL)。然後，將3M的NaOH水溶液(52mL,156mmol) 0℃下逐滴加入反應液中後，再將30%的H₂O₂(106mL)的水溶液逐滴滴加反應液中，再加入EtOH(10mL)。該反應液室溫攪拌48h後，0℃下用飽和的Na₂S₂O₃緩慢加入，直到無氣泡產生。將H₂O(300mL)加到反應液中，用DCM(4×200mL)萃取。有機相用無水Na₂SO₄乾燥，過濾並濃縮。得到的殘留物用反向管柱純化(C¹⁸，乙腈+H2O,50%)，凍乾後得到目標產物5a(730mg，收率17.6%)和目標產物5b(1.1g,26.6%)。 Under the protection of argon, borane (BH ₃ ) tetrahydrofuran solution (1.0M dissolved in THF, 38.54mL, 38.54mmol) was added dropwise to THF ( 12mL) solution. After the compound was stirred at 0° C. for 6 h under the protection of argon, H ₂ O (27 mL) was added dropwise. Then, 3M NaOH aqueous solution (52mL, 156mmol) was added dropwise to the reaction solution at 0°C, then 30% H ₂ O ₂ (106mL) aqueous solution was added dropwise to the reaction solution, and then EtOH (10mL ). After the reaction solution was stirred at room temperature for 48 h, saturated Na ₂ S ₂ O ₃ was added slowly at 0°C until no bubbles were generated. H ₂ O (300 mL) was added to the reaction solution, extracted with DCM (4×200 mL). The organic phase was dried over _anhydrous _Na2SO4 , filtered and concentrated. The obtained residue was purified by reverse column (C ¹⁸ , acetonitrile+H2O, 50%), and the target product 5a (730 mg, yield 17.6%) and target product 5b (1.1 g, 26.6%) were obtained after lyophilization.

將化合物5a(730mg,1.36mmol)溶解在吡啶(8mL)中，氬氣保護和室溫下加入TMSCl(0.67g,6.14mmol)。室溫攪拌1h後，BzCl(0.29mL,2.46mmol)加到反應液中。室溫攪拌16h後，反應液用H₂O(10mL)淬滅並濃縮。得到的殘留物溶解在THF(30mL)中，加入四丁基氟化銨(TBAF，1mL)。室溫攪拌1h後，將氨水(0.5mL)加入，室溫攪拌5h後。反應液用乙醇(EA，100mL)稀釋並用飽和食鹽水(30mL)洗五次。將有機相濃縮，得到殘留物用反相管柱純化(C18,H₂O+乙腈，乙腈60%)凍乾得到目標產物6a(480mg，收率74.8%)。MS m/z：C₃₈H₃₅N₅O₅，[M+H]⁺：642.6. Compound 5a (730 mg, 1.36 mmol) was dissolved in pyridine (8 mL), and TMSCl (0.67 g, 6.14 mmol) was added under argon protection at room temperature. After stirring at room temperature for 1 h, BzCl (0.29 mL, 2.46 mmol) was added to the reaction solution. After stirring at room temperature for 16 h, the reaction was quenched with H ₂ O (10 mL) and concentrated. The obtained residue was dissolved in THF (30 mL), and tetrabutylammonium fluoride (TBAF, 1 mL) was added. After stirring at room temperature for 1 h, aqueous ammonia (0.5 mL) was added and stirred at room temperature for 5 h. The reaction solution was diluted with ethanol (EA, 100 mL) and washed five times with saturated brine (30 mL). The organic phase was concentrated, and the residue obtained was purified by a reverse-phase column (C18, H ₂ O+acetonitrile, acetonitrile 60%) and lyophilized to obtain the target product 6a (480 mg, yield 74.8%). _MS m/z: _C38H35N5O5 , [M+H] ⁺ : _642.6 _.

將化合物5b(1.1g,2.05mmol)溶解在吡啶(20mL)中，氬氣保護和室溫下加入TMSCl(1.34g,1.28mmol)。室溫攪拌1h後，苯甲醯氯(BzCl，0.59mL,5.92mmol)加到反應液中。室溫攪拌16h後，反應液用H₂O(10mL)淬滅並濃縮。得到的殘留物溶解在THF(30mL)中，加入TBAF(2mL)。室溫攪拌1h後，將氨水(0.5mL)加入，室溫攪拌5h後。反應液用EA(100mL)稀釋並用飽和食鹽水(30mL)洗五次。將有機相濃縮，得到殘留物用反相管柱純化(C18,H₂O+乙腈，乙腈60%)凍乾得到目標產物6b(1.4g，收率82.1%)。MS m/z：C₃₈H₃₅N₅O₅，[M+H]⁺：642.5. Compound 5b (1.1 g, 2.05 mmol) was dissolved in pyridine (20 mL), and TMSCl (1.34 g, 1.28 mmol) was added under argon protection at room temperature. After stirring at room temperature for 1 h, benzoyl chloride (BzCl, 0.59 mL, 5.92 mmol) was added to the reaction solution. After stirring at room temperature for 16 h, the reaction was quenched with H ₂ O (10 mL) and concentrated. The resulting residue was dissolved in THF (30 mL), and TBAF (2 mL) was added. After stirring at room temperature for 1 h, aqueous ammonia (0.5 mL) was added and stirred at room temperature for 5 h. The reaction solution was diluted with EA (100 mL) and washed five times with saturated brine (30 mL). The organic phase was concentrated, and the residue obtained was purified by a reverse-phase column (C18, H ₂ O+acetonitrile, acetonitrile 60%) and lyophilized to obtain the target product 6b (1.4 g, yield 82.1%). _MS m/z: _C38H35N5O5 , [M+H] ⁺ : _642.5 _.

將化合物6a(700mg,1.04mmol)、四唑(26.2mg,0.37mmol)、N-甲基咪唑溶解在CH₃CN(10mL)中，加入3A分子篩(500mg)。氬氣保護，室溫攪拌5min後，加入化合物7(470.4mg,1.56mmol)。室溫攪拌1h後，將分子篩過濾掉，固體用DCM(50mL)淋洗三次。濾液分別用飽和的NaHCO₃水溶液(50mL×4)和H₂O(50mL×4)洗。有機相在30℃下濃縮。得到殘留物用反向管柱純化(C¹⁸,H₂O+乙腈，乙腈90%)，凍乾後得到目標化合物1-4A(600mg，收率66.1%)。MS m/z：C₄₇H₅₂N₇O₆P，[M-氰乙基-二異丙基+OH]^-：704.3. Compound 6a (700 mg, 1.04 mmol), tetrazole (26.2 mg, 0.37 mmol), N-methylimidazole were dissolved in CH ₃ CN (10 mL), and 3A molecular sieves (500 mg) were added. Under argon protection, after stirring at room temperature for 5 min, compound 7 (470.4 mg, 1.56 mmol) was added. After stirring at room temperature for 1 h, the molecular sieves were filtered off, and the solid was rinsed three times with DCM (50 mL). The filtrate was washed with saturated aqueous NaHCO ₃ (50 mL×4) and H ₂ O (50 mL×4), respectively. The organic phase was concentrated at 30°C. The obtained residue was purified by reverse column (C ¹⁸ , H ₂ O+acetonitrile, acetonitrile 90%), and the target compound 1-4A (600 mg, yield 66.1%) was obtained after lyophilization. MS m/z: C ₄₇ H ₅₂ N ₇ O ₆ P, [M-cyanoethyl-diisopropyl+OH] ^- : 704.3.

將化合物6b(1.3g,2.03mmol)、四唑(71.0mg,1.01mmol)、N-甲基咪唑(33.3mg,0.41mmol)溶解在CH₃CN(20mL)中，加入3A分子篩(700mg)。氬氣保護，室溫攪拌5min後，加入化合物7(0.92g,3.04mmol)。室溫攪拌1h後，將分子篩過濾掉，固體用DCM(50mL)淋洗三次。濾液分別用飽和的NaHCO₃水溶液(50mL×4)和H₂O(50mL×4)洗。有機相在30℃下濃縮。得到殘留物用反向管柱純化(C¹⁸,H₂O+乙腈，乙腈90%)，凍乾後得到目標化合物1-4b(1.4g，收率82.1%)。MS m/z：C₄₇H₅₂N₇O₆P，[M-氰乙基-二異丙基]^-：704.3. Compound 6b (1.3 g, 2.03 mmol), tetrazole (71.0 mg, 1.01 mmol), N-methylimidazole (33.3 mg, 0.41 mmol) were dissolved in CH ₃ CN (20 mL), and 3A molecular sieves (700 mg) were added. Under argon protection, after stirring at room temperature for 5 min, compound 7 (0.92 g, 3.04 mmol) was added. After stirring at room temperature for 1 h, the molecular sieves were filtered off, and the solid was rinsed three times with DCM (50 mL). The filtrate was washed with saturated aqueous NaHCO ₃ (50 mL×4) and H ₂ O (50 mL×4), respectively. The organic phase was concentrated at 30°C. The obtained residue was purified by reverse column (C ¹⁸ , H ₂ O+acetonitrile, acetonitrile 90%), and the target compound 1-4b (1.4 g, yield 82.1%) was obtained after lyophilization. MS m/z: C ₄₇ H ₅₂ N ₇ O ₆ P, [M-cyanoethyl-diisopropyl] ^- : 704.3.

1.5 合成化合物1-51.5 Synthesis of compounds 1-5

將化合物1A(6.73g,28.14mmol)溶解在乾燥的DMF(80mL)中，氬氣保護下緩慢加入NaH(60%,1.24g,30.95mmol)。該混合液室溫攪拌30min後，將該反應液加到溶有Pd(PPh₃)₄(1.95g,1.69mmol)、PPh₃(0.74g,2.81mmol)和化合物1(4.0g,28.14mmol)的THF(60mL)溶液中。該反應液在55℃攪拌16h後，將固體濾掉並用DCM(60mL)洗滌三次。將濾液濃縮，得到的殘留物用正向管柱純化(先用乙酸乙酯，再用乙酸乙酯：甲醇(12：1沖洗管柱)得目標固體2(7g，粗品)。 Compound 1A (6.73g, 28.14mmol) was dissolved in dry DMF (80mL), and NaH (60%, 1.24g, 30.95mmol) was added slowly under the protection of argon. After the mixture was stirred at room temperature for 30min, the reaction solution was added to a solution of Pd(PPh ₃ ) ₄ (1.95g, 1.69mmol), PPh ₃ (0.74g, 2.81mmol) and compound 1 (4.0g, 28.14mmol) in THF (60 mL) solution. After the reaction was stirred at 55 °C for 16 h, the solid was filtered off and washed three times with DCM (60 mL). The filtrate was concentrated, and the obtained residue was purified by a forward column (washing the column with ethyl acetate first, then ethyl acetate:methanol (12:1) to obtain the target solid 2 (7 g, crude product).

將化合物3(1g,1.60mmol)、KHCO₃(0.48g,4.81mmol)和乙二醇(0.40g,6.41mmol)溶解在丙酮(50mL)中，-30℃下緩慢加入KMnO₄(40%溶解於水中，0.67g,1.68mmol)。-30℃下攪1h後，用飽和的硫代硫酸鈉水溶液(30mL)將反應淬滅。該混合液用DCM(30mL)萃取四次。有機相用無水Na₂SO₄乾燥，過濾並濃縮。殘留物用反向管柱純化(C18,H₂O+乙腈，乙腈60%)凍乾得到目標產物4(600mg，收率56.9%)。MS m/z：C₃₈H₃₅N₅O₆，[M+H]⁺：658.5. Compound 3 (1g, 1.60mmol), KHCO ₃ (0.48g, 4.81mmol) and ethylene glycol (0.40g, 6.41mmol) were dissolved in acetone (50mL), and KMnO ₄ (40% dissolved In water, 0.67g, 1.68mmol). After stirring at -30°C for 1 h, the reaction was quenched with saturated aqueous sodium thiosulfate (30 mL). The mixture was extracted four times with DCM (30 mL). The organic phase was dried over _anhydrous _Na2SO4 , filtered and concentrated. The residue was purified by reverse column (C18, H ₂ O+acetonitrile, acetonitrile 60%) and lyophilized to obtain the target product 4 (600 mg, yield 56.9%). MS m/z _: _C38H35N5O6 , [M+H] ⁺ : _658.5 _.

在250mL圓底瓶中加入反應物4(5.0g,7.601mmol)，NaIO₄和1,4-二噁烷/水(50mL/5mL)，室溫反應2小時後，減壓除去溶劑，得到白色固體(6.0g)，然後溶於甲醇(50mL)，加入硼氫化鈉(1.62g,38mmol)，室溫攪拌2小時後，加入10%氯化銨溶液(10mL)，減壓除去溶劑後，C18管柱層析(水/乙腈：5%-95%)得到產品P1，目標產物5(2.0g,3.0315mmol,39%)。LCMS,MS+,[M+H]+：660. Add reactant 4 (5.0g, 7.601mmol), NaIO ₄ and 1,4-dioxane/water (50mL/5mL) in a 250mL round-bottom flask, react at room temperature for 2 hours, and remove the solvent under reduced pressure to obtain a white Solid (6.0g), then dissolved in methanol (50mL), added sodium borohydride (1.62g, 38mmol), stirred at room temperature for 2 hours, added 10% ammonium chloride solution (10mL), after removing the solvent under reduced pressure, C18 Column chromatography (water/acetonitrile: 5%-95%) gave product P1, the target product 5 (2.0 g, 3.0315 mmol, 39%). LCMS, MS+, [M+H]+: 660.

將化合物5(1.7g,2.58mmol)和DBU(0.77mL,5.15mmol)溶解在DCM(20mL)，氬氣保護-70℃下將BzCl(0.5M in DCM,0.8mL)逐滴滴加到反應中。該反應液置於-70℃下1h，該反應液用乙醇(5mL)淬滅。淬滅的反應液用DCM(100mL)稀釋並用水(30mL)洗滌三次，有機相用無水Na₂SO₄乾燥，過濾並濃縮。得到的殘留物用正向管柱純化(DCM：EA=1：1)得到目標產物6(80mg，收率4.14%)。MS m/z：C₄₅H₄₁N₅O₇，[M+H]⁺：764.5. Compound 5 (1.7g, 2.58mmol) and DBU (0.77mL, 5.15mmol) were dissolved in DCM (20mL), and BzCl (0.5M in DCM, 0.8mL) was added dropwise to the reaction at -70°C under argon protection. middle. The reaction solution was placed at -70°C for 1 h, and the reaction solution was quenched with ethanol (5 mL). The quenched reaction was diluted with DCM (100 mL) and washed three _times with water (30 mL), the organic phase was dried over anhydrous _Na2SO4 , filtered and concentrated. The obtained residue was purified by a forward column (DCM:EA=1:1) to obtain the target product 6 (80 mg, yield 4.14%). MS _m /z _: _C45H41N5O7 , [M+H] ⁺ : 764.5 _.

將化合物4(380mg,0.50mmol)、四唑(17.43mg,0.25mmol)、N-甲基咪唑(8.17mg,0.10mmol)溶解在CH₃CN(10mL)中，加入3A分子篩(500mg)。氬氣保護，室溫攪拌5min後，加入化合物7(224.95mg,0.75mmol)。室溫攪拌1h後，將分子篩過濾掉，固體用DCM(50mL)淋洗三次。濾液分別用飽和的NaHCO₃水溶液(50mL×4)和H₂O(50mL×4)洗。有機相在30℃下濃縮。得到殘留物用反向管柱純化(C¹⁸,H₂O+乙腈，乙腈90%)，凍乾後得到目標產物1-5(330mg，收率68.8%)。MS m/z：C₅₄H₅₈N₇O₈P，[M-氰乙基-二異丙基]^-：826.3. Compound 4 (380 mg, 0.50 mmol), tetrazole (17.43 mg, 0.25 mmol), N-methylimidazole (8.17 mg, 0.10 mmol) were dissolved in CH ₃ CN (10 mL), and 3A molecular sieves (500 mg) were added. Under argon protection, after stirring at room temperature for 5 min, compound 7 (224.95 mg, 0.75 mmol) was added. After stirring at room temperature for 1 h, the molecular sieves were filtered off, and the solid was rinsed three times with DCM (50 mL). The filtrate was washed with saturated aqueous NaHCO ₃ (50 mL×4) and H ₂ O (50 mL×4), respectively. The organic phase was concentrated at 30°C. The obtained residue was purified by reverse column (C ¹⁸ , H ₂ O+acetonitrile, acetonitrile 90%), and the target product 1-5 (330 mg, yield 68.8%) was obtained after lyophilization. MS m/z: C ₅₄ H ₅₈ N ₇ O ₈ P, [M-cyanoethyl-diisopropyl] ^- : 826.3.

1.6 合成化合物1-6a1.6 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度下反應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，得到的殘留物用正向管柱純化(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, 124.371 mmol). The reaction solution was reacted at 25 degrees for 12h. LCMS showed that the reaction was complete. The reaction solution was extracted with ethyl acetate (200mL) and water (200mL), the organic phase was dried and the filtrate was concentrated, and the residue obtained was purified by a forward column (DCM/MeOH=10/1) to obtain the target product 3 (20g ).

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 (20 g, 28.585 mmol) was dissolved in acetic acid (24 mL, 426.016 mmol) and H2O (12 mL), and stirred at 60° C. for 1 hour. Then the reaction solution was spin-dried, THF (12 mL) and H2O (12 mL) were added, and 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 solid sodium carbonate was added to the aqueous phase until a large amount of solids precipitated out of the aqueous phase. The solid was filtered, washed with water, and the filter cake was pulled dry with an oil pump to obtain the target compound 5 (9 g).

在氮氣保護下，將化合物5(6.8g,18.581mmol)溶於吡啶(80mL)中，於0度下緩慢加入TMSCl(14.250mL,111.489mmol)，攪拌2h。之後在0度下加入Isobutyryl chloride(2.044mL,19.511mmol)，於25度下攪拌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，用正向管柱純化(DCM：MeOH=10：1)過管柱，在4.8%處出峰)，得到目標產物6(12g)。 Under nitrogen protection, compound 5 (6.8g, 18.581mmol) was dissolved in pyridine (80mL), and TMSCl (14.250mL, 111.489mmol) was slowly added at 0 degrees, and stirred Stir for 2h. Then Isobutyryl chloride (2.044 mL, 19.511 mmol) was added at 0°C and stirred at 25°C for 1 h. LCMS showed that the reaction was complete. Extracted with dichloromethane (200mL) and water (200mL), the organic phase was dried and spin-dried, and the sample was mixed, and purified by a forward column (DCM:MeOH=10:1) through the column, and the peak was at 4.8%), The target product 6 (12 g) was obtained.

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向管柱純化(PE：EtOAc過管柱，在84%處出峰)，得到目標產物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 mmol) solid, reacted at 25°C for 2h. TLC (PE:EtOAc=1:1, Rf=0.69) showed that the reaction had been completed. The reaction liquid and TJN200879-040-P1 are combined and processed together. The reaction solution was extracted with ethyl acetate (200mL) and water (200mL), the organic phase was dried and spin-dried, and the mixed sample was purified with a forward column (PE: EtOAc passed through the column, and the peak was at 84%) to obtain the target product 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度，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三乙胺拌樣用正向管柱純化(DCM：MeOH=10： 1過管柱，在0.5%處出峰)，得到9g(粗品).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), wet palladium carbon Pd/C (7g, 15.389mmol) was added, and the reaction solution was reacted at 25°C under hydrogen (15Psi) for 2 hours. TLC (PE:EtOAc=0:1, Rf=0.09) showed that the reaction was complete. The reaction solution was filtered, and the filter cake was washed three times with ethyl acetate (30 mL), and the filtrate was collected. After the filtrate was spin-dried, add 50 mL of dichloromethane and 2 mL of triethylamine and mix the sample with a forward column for purification (DCM: MeOH=10: 1 passes through the column, and goes out at 0.5% place), obtains 9g (crude product). Gained racemic compound SFC is resolved, obtains product 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,Condition：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). Compound 7A(-) (3.30g, 5.40mmol), tetrazole (190mg, 2.70mmol), 1-methylimidazole (90mg, 1.10mmol), 3A molecular sieve (500mg) were dissolved in 30mL of acetonitrile, at room temperature Compound 8 (2.50 g, 8.10 mmol) was added and stirred at room temperature for 2 h. After the reaction was completed, molecular sieves were filtered off, DCM (150mL) was added, washed with saturated aqueous sodium bicarbonate (30mL*3), and then washed with saturated brine (30mL), the filtrate was spin-dried and subjected to reverse-phase preparative HPLC (C18, Condition : 5-100% (A: water, B: CH3CN), flow rate: 70mL/min), 1-6a (2.9g, 66%) was obtained after lyophilization. MS m/z: C43H55N7O7P[M+H]+, Theory: 812.38, Found: 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).

1.7 合成化合物1-7a1.7 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，用正向管柱純化(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 triphenylphosphine (7.28g, 27.753mmol) were dissolved in 30mL of dioxin In alkanes, DEAD (5.502 mL, 27.753 mmol) was slowly added dropwise at 0°C. After the dropwise addition was completed, the temperature of the reaction was slowly raised to 25° C. to continue the reaction for 1 h. 100mL H2O and 100mL EtOAc were added to the reaction solution for extraction, the organic phases were combined, dried, filtered and concentrated, then the sample was mixed and passed through the column, and purified with a forward column (PE:EtOAc=1:1) 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向管柱純化(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 H2O (4mL), and heated in an oil bath at 60°C for 0.5h. The residue obtained by spinning the reaction solution to dryness was purified by a forward column (PE:EtOAc=0:1 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向管柱純化(用PE：EtOAc=2：1)。得到目標產物5(4g). Compound 4 (3 g, 8.873 mmol) was dissolved in 5 mL of pyridine, and a solution of DMTrCl (3.91 g, 11.535 mmol) in 10 mL of pyridine was slowly added dropwise at 0° C. under nitrogen protection. After the dropwise addition, the temperature of the reaction was raised to 25° C. and the reaction was continued for 1 h. 50 mL of water and 100 mL of ethyl acetate were added to the reaction solution for extraction. The aqueous phase was extracted three times with 100 mL of ethyl acetate, and the organic phases were combined, dried, filtered, concentrated, and purified with a forward 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。將反應液旋乾用正向管柱純化(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 (4 g, 5.769 mmol) was dissolved in methanol (10 mL), added saturated NH3 methanol solution (40 mL), and reacted at 0° C. for 6 h. The reaction solution was spin-dried and purified with a forward column (PE:EtOAc=0:1) to obtain 2.4 g of the racemic compound. SFC resolution gave the target product 6A (750 mg, 100% purity) and target product 6B (400 mg, 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。 Compound 6A(-) (700mg, 1.40mmol), tetrazole (50mg, 0.70mmol), 1-methylimidazole (23mg, 0.28mmol), 3A molecular sieve (500mg) were dissolved in 10mL of acetonitrile, and added at room temperature Compound 7 (630mg, 2.10mmol), stirred at room temperature for 2h. After the reaction was completed, the molecular sieves were filtered off, DCM (50 mL) was added, washed with saturated aqueous sodium bicarbonate (10 mL*3), and then washed with saturated brine (20 mL), the filtrate was spin-dried and subjected to reverse-phase preparative HPLC (C18, condition : 5-100% (A: water, B: CH3CN), flow rate: 70mL/min), 1-7a (700mg, 72%) was obtained after lyophilization. MS m/z: C38H47N4O7PNa[M+Na]+, theory: 725.32, found: 725.5.

1.8 合成化合物1-8a1.8 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，正向管柱分離純化(80g,DCM/MeOH=10/1~5/1)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 reacted under nitrogen protection at 90°C for 12h. LCMS detected that the reaction was complete. The reaction solution was filtered, spin-dried with an oil pump, and separated and purified by forward column (80g, DCM/MeOH=10/1~5/1) to obtain the target product 3 (13.5g, 80% purity).

將化合物3(10.5g,35.105mmol)溶於pyridine(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 CH3CN (65mL), and BzCl (4.894mL, 42.126mmol) was added dropwise to the solution, and reacted at 25°C for 2h. LCMS detected that most of the raw materials were reacted, quenched by adding H2O (100mL), extracted with EtOAc (100mL X 3), dried and spin-dried, and purified by column separation (merging TJN200872-101) (80g, PE/EtOAc=10/1~0 /1, DCM/MeOH=10/1) to obtain the target product 4 (14 g, 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，正向管柱分離(40g,DCM/MeOH=1/0~5/1)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 H2O (14mL), reacted at 60°C for 2h, LCMS showed that the reaction was complete. The oil pump was concentrated, and the forward column separation (40g, DCM/MeOH=1/0~5/1) gave 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pyridine(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 X 3)萃取，乾燥，旋乾，管柱分離(120g,PE/(EA：DCM：TEA=1：1：0.05)=1/0~0/1至DCM/MeOH=10/1)得到目標產物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 for 10min under ice bath, then DMTrCl (8.93g, 26.348mmol), the reaction was stirred for 1.8h. LCMS detected that about 19% of the raw material remained, and about 60% of the target MS. Combined (TJN200872-105&106) were purified together. Add H2O (50mL) to the reaction solution, extract with DCM (50mL X 3), dry, spin dry, column separation (120g, PE/(EA:DCM:TEA=1:1:0.05)=1/0~ 0/1 to DCM/MeOH=10/1) to obtain the target product 6 (11 g, 89% purity, TJN200872-105&106&107), and recover the starting material (3.0 g, 70% purity).

化合物6(15g,22.041mmol)經SFC(DAICEL CHIRALPAK AD(250mm*50mm,10um)；0.1% NH3H2O 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, 10um); 0.1% NH3H2O EtOH, B: 45%-45%; 200ml/min) to obtain the target product 6A (5.33g, 94.29% Purity), target product 6B (6.14g, 97.91% purity), compound 6 recovered 1.0g.

將化合物6B(-)(5.4g,8.92mmol)，四唑(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。 Compound 6B(-) (5.4g, 8.92mmol), tetrazole (312mg, 4.46mmol), 1-methylimidazole (146mg, 1.78mmol), 3A molecular sieve (500mg) were dissolved in 40mL of acetonitrile, at room temperature Compound 7 (4g, 13.4mmol) was added and stirred at room temperature for 2h. After the reaction was completed, the molecular sieves were filtered off, DCM (200 mL) was added, washed with saturated aqueous sodium bicarbonate (30 mL*3), and then washed with saturated brine (50 mL), the filtrate was spin-dried and subjected to reverse-phase preparative HPLC (C18, condition : 5-100% (A: water, B: CH3CN), flow rate: 70mL/min), and 1-8a (5.8g, 80%) was obtained after lyophilization. MS m/z: C45H51N5O7P, [M+H]+, Theoretical: 804.36, Found: 804.4.

實施例2 siRNA的合成Example 2 Synthesis of siRNA

siRNA的合成與通常的亞磷醯胺固相合成法無異，在合成AS股5’第7位修飾的核苷酸時，使用上述合成的亞磷醯胺單體替換母序列原核苷酸。合成過程簡要描述如下：於Dr.Oligo48合成器(Biolytic)上，以Universal CPG載體為起始，根據合成程序逐個連接核苷亞磷醯胺單體。除上述描述的AS股5’第7位的核苷亞磷醯胺單體外，其餘核苷單體原料2’-F RNA、2’-O-甲基RNA等核苷亞磷醯胺單體購自上海兆維或蘇州吉瑪。採用5-乙基硫-1H-四唑(ETT)作為活化劑(0.6M乙腈溶液)，使用0.22M的PADS溶於1：1體積比的乙腈和三甲基吡啶(蘇州柯樂瑪)溶液作為硫化試劑，使用碘吡啶/水溶液(柯樂瑪)作為氧化劑。 The synthesis of siRNA is the same as the usual phosphoramidite solid-phase synthesis method. When synthesizing the nucleotide modified at the 5'-7th position of the AS strand, the phosphoramidite monomer synthesized above is used to replace the original nucleotide of the mother sequence. The synthesis process is briefly described as follows: On Dr. Oligo48 synthesizer (Biolytic), starting with Universal CPG carrier, nucleoside phosphoramidite monomers are connected one by one according to the synthesis procedure. Except for the nucleoside phosphoramidite monomer at the 7th position of AS strand 5' described above, other nucleoside monomer raw materials such as 2'-F RNA, 2'-O-methyl RNA and other nucleoside phosphoramidite monomers The body was purchased from Shanghai Zhaowei or Suzhou Jima. Using 5-ethylthio-1H-tetrazole (ETT) as the activator (0.6M acetonitrile solution), using 0.22M PADS dissolved in acetonitrile and collidine (Suzhou Kelema) solution with a volume ratio of 1:1 As a vulcanizing agent, iodopyridine/water solution (Koroma) was used as an oxidizing agent.

固相合成完成後，寡核糖核苷酸自該固體支撐物裂解，採用3：1的28%氨水和乙醇溶液在50℃條件下浸泡16小時。然後離心，將上清液轉移到另一個離心管中，濃縮蒸發乾後，使用C18反向色譜純化，流動相為0.1MTEAA和乙腈，並使用3%三氟乙酸溶液脫除DMTr。目標寡核苷酸收集後凍乾，並經LC-MS鑑定為目標產物，再經過UV(260nm)定量。 After the solid-phase synthesis was completed, the oligoribonucleotides were cleaved from the solid support, and soaked in a 3:1 solution of 28% ammonia water and ethanol at 50°C for 16 hours. Then centrifuge, transfer the supernatant to another centrifuge tube, concentrate and evaporate to dryness, then use C18 reverse chromatography to purify, the mobile phase is 0.1MTEAA and acetonitrile, and use 3% trifluoroacetic acid solution to remove DMTr. The target oligonucleotides were collected and freeze-dried, identified as the target product by LC-MS, and then quantified by UV (260nm).

所得到的單股寡核苷酸，根據等莫耳比，按照互補配對，退火，最後所得到的雙股siRNA溶於1×PBS中，並調整至實驗所需濃度備用。 The obtained single-stranded oligonucleotides were annealed according to the equimolar ratio and complementary pairing, and finally the obtained double-stranded siRNA was dissolved in 1×PBS and adjusted to the concentration required for the experiment for later use.

實施例3 psiCHECK活性篩選實驗Example 3 psiCHECK activity screening experiment

Huh7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18h，將Huh7細胞接種於96孔板，接種密度為每孔1萬個細胞，每孔100μL培養基。 Huh7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO ₂ . Eighteen hours before transfection, Huh7 cells were seeded in 96-well plates at a seeding density of 10,000 cells per well and 100 μL of medium per well.

轉染前將孔中的含10%胎牛血清的DMEM高糖培養基吸棄，換成80μL Opti-MEM，饑餓處理1.5h。然後按照說明書，使用Lipofectamine2000(ThermoFisher，11668019)對細胞共轉染siRNA及對應質粒。在96孔板的每孔中添加含有0.2μL Lipofectamine2000，20ng質粒，以及2.2uL siRNA(最高濃度40nM，3倍梯度稀釋，共設置11個濃度點，每個濃度雙複孔)的20uL Opti-MEM。37度培養箱溫育4小時後，補充含20%胎牛血清的DMEM高糖培養基。繼續培養24h後，按照Dual-Glo® Luciferase Assay System(Promega Cat.# E2940)檢測試劑盒的實驗操作方案進行螢光素酶活性檢測。根據檢測信號計算相對值Ratio=Ren/Fir(海腎/螢火蟲比值)，以及抑制率1-(Ratio+siRNA/Ratio reporter only)*100%=抑制率(%)。本揭露中，剩餘活性%(也稱為mRNA剩餘表達量%或mRNA剩餘表達比例)=100%-抑制率(%)。應用Graphpad Prism軟體分析(four parameter logistic equations)計算IC₅₀值。 Before transfection, the DMEM high-glucose medium containing 10% fetal bovine serum in the well was discarded, replaced with 80 μL Opti-MEM, and starved for 1.5 h. Then, according to the instructions, cells were co-transfected with siRNA and corresponding plasmids using Lipofectamine2000 (ThermoFisher, 11668019). Add 20uL Opti-MEM containing 0.2μL Lipofectamine2000, 20ng plasmid, and 2.2uL siRNA (maximum concentration 40nM, 3-fold serial dilution, a total of 11 concentration points, double wells for each concentration) to each well of a 96-well plate . After incubating in a 37 degree incubator for 4 hours, DMEM high glucose medium containing 20% fetal bovine serum was supplemented. After continuing to culture for 24 hours, detect luciferase activity according to the experimental protocol of the Dual-Glo® Luciferase Assay System (Promega Cat.# E2940) detection kit. Calculate the relative value Ratio=Ren/Fir (renilla/firefly ratio) according to the detection signal, and the inhibition rate 1-(Ratio+siRNA/Ratio reporter only)*100%=inhibition rate (%). In the present disclosure, remaining activity % (also referred to as remaining mRNA expression % or remaining mRNA expression ratio)=100%-inhibition rate (%). _IC50 values were calculated using Graphpad Prism software analysis (four parameter logistic equations).

實施例4 包含不同化學修飾的siRNA的在靶活性和脫靶活性實驗Example 4 On-target activity and off-target activity experiments of siRNAs comprising different chemical modifications

利用實施例1的化合物、採用實施例2的方法合成表5的siRNA，並藉由實施例3的方法驗證各siRNA的在靶活性和脫靶活性，其中各siRNA採用相同的正義股，且反義股5’端第7位分別為以下修飾核苷酸/化學修飾， Using the compound of Example 1, the siRNAs in Table 5 were synthesized by the method of Example 2, and the on-target activity and off-target activity of each siRNA were verified by the method of Example 3, wherein each siRNA used the same sense strand, and the antisense The 7th position of the 5' end of the strand is the following modified nucleotides/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 will define hmpNA from nucleotides synthesized from 2-hydroxymethyl-1,3-propanediol as the starting material;

TJ-NA019(A)為實施例1.1節中核苷亞磷醯胺單體1-2藉由固相合成獲得； TJ-NA019(A) is obtained by solid-phase synthesis of nucleoside phosphoramidite monomer 1-2 in Example 1.1;

TJ-NA020(A)為實施例1.1節中核苷亞磷醯胺單體1-3藉由固相合成獲得； TJ-NA020(A) is obtained by solid-phase synthesis of nucleoside phosphoramidite monomer 1-3 in Example 1.1;

TJ-NA026(A)為實施例1.1節中核苷亞磷醯胺單體1-4a藉由固相合成獲得； TJ-NA026(A) is obtained by solid-phase synthesis of the nucleoside phosphoramidite monomer 1-4a in Example 1.1;

TJ-NA027(A)為實施例1.1節中核苷亞磷醯胺單體1-4b藉由固相合成獲得； TJ-NA027(A) is obtained by solid-phase synthesis of the nucleoside phosphoramidite monomer 1-4b in Example 1.1;

(+)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； (+) hmpNA(A) is obtained by solid-phase synthesis of the nucleoside phosphoramidite monomer 1-1b in Example 1.1;

(-)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； (-)hmpNA(A) is obtained by solid-phase synthesis of the nucleoside phosphoramidite monomer 1-1a in Example 1.1;

TJ-NA038(A)為實施例1.1節中核苷亞磷醯胺單體1-5藉由固相合成獲得。 TJ-NA038(A) was obtained by solid-phase synthesis of nucleoside phosphoramidite monomers 1-5 in Example 1.1.

(+)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 Example 1.1, 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 the nucleoside phosphoramidite monomer 1-1a in Example 1.1 obtained by solid-phase synthesis, and the 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, 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) was 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)，屬於單斜晶系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), which belongs to the monoclinic crystal system 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 of the unit cell μ (Cu Kα)=0.840mm-1, 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)，屬於單斜晶系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 detection crystal is a colorless block (0.30×0.20×0.10mm3), belonging to the monoclinic crystal system 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 of the unit cell μ (Cu Kα)=0.570mm-1, 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)，屬於單斜晶系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 detected crystal is colorless needle-shaped (0.30×0.04×0.04mm3), belonging 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 of the unit cell μ (Cu Kα)=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)，屬於三斜晶系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 a colorless prism (0.30×0.10×0.10mm3), belonging to the P1 space group of the triclinic crystal system. 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 of the unit cell μ (Cu Kα)=0.945mm-1, and the diffraction experiment temperature T=100.00(10)K.

其中，大寫字母C、G、U、A表示核苷酸的鹼基組成；小寫字母m表示該字母m左側相鄰的一個核苷酸為2’-甲氧基修飾的核苷酸；小寫字母f表示該字母f左側相鄰的一個核苷酸為2’-氟修飾的核苷酸； Among them, the uppercase letters C, G, U, and A indicate the base composition of nucleotides; the lowercase letter m indicates that the adjacent nucleotide on the left side of the letter m is a 2'-methoxy modified nucleotide; the lowercase letter f indicates that the adjacent nucleotide to the left of the letter f is a 2'-fluoro-modified nucleotide;

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酯基；以下相同。 When the lowercase letter s is the first at the 3' end, it means that the nucleotide terminal adjacent to the left side of the letter s is a phosphorothioate group; the same below.

在靶活性實驗結果參見表6，脫靶活性實驗結果參見表7，目前實驗的化合物，在測試序列上均顯示了與母序列相當或略優的活性，說明修飾沒有影響在靶活性，其中活性最優的是分別包含GNA(A)/Abasic/Id、TJ-NA019(A)、TJ-NA020(A)、TJ-NA026(A)、(+)hmpNA(A)和(-)hmpNA(A)的siRNA。另外，母序列具有明顯的脫靶活性，所有修飾均顯示了明顯的抑制脫靶活性的效果，特別是對於分別包含TJ-NA027(A)、(+)hmpNA(A)和(-)hmpNA(A)的siRNA，並未觀察到脫靶活性。 See Table 6 for the results of on-target activity experiments, and Table 7 for the results of off-target activity experiments. The compounds tested so far have shown activities comparable to or slightly superior to those of the parent sequences on the test sequences, indicating that the modification does not affect the on-target activity. It is preferred to contain GNA (A) /Abasic/Id, TJ-NA019 (A) , TJ-NA020 (A) , TJ-NA026 (A) , (+)hmpNA(A) and (-)hmpNA(A) respectively siRNA. In addition, the parent sequence has obvious off-target activity, and all modifications showed obvious effects of inhibiting off-target activity, especially for TJ-NA027 (A) , (+)hmpNA(A) and (-)hmpNA(A) respectively. siRNA, no off-target activity was observed.

實施例5 包含不同化學修飾的siRNA的序列依賴性實驗Example 5 Sequence-dependent experiments comprising siRNAs of different chemical modifications

已知Abasic修飾具有siRNA序列依賴性，因此發明人在多條不同序列上測試了本揭露的實驗化合物。使用了靶向不同基因(HBV-S、HBV-X)mRNA的siRNA(序列如表8所示)，使用實施例1的化合物TJ-NA020(A)、TJ-NA027(A)、(+)hmpNA(A)、(-)hmpNA(A)和作為對照的GNA(A)、Id化合物修飾AS股5’端第7位(序列如表9所示)，再與母序列比較在靶活性和脫靶活性。 It is known that Abasic modification is siRNA sequence dependent, so the inventors tested the experimental compounds of the present disclosure on multiple different sequences. The siRNA (sequence shown in Table 8) targeting different gene (HBV-S, HBV-X) mRNA was used, and the compounds TJ-NA020 (A) , TJ-NA027 (A) , (+) of Example 1 were used hmpNA (A), (-) hmpNA (A) and GNA (A) as a control, the Id compound modifies the 7th position of the 5' end of the AS strand (sequence is shown in Table 9), and then compared with the parent sequence in the target activity and off-target activity.

在靶活性實驗結果參見表10，GNA顯現出明顯的序列依賴性，不同序列的在靶活性差異明顯。本揭露的實驗化合物沒有顯示出明顯的序列依賴性，普遍適用性更強。 The results of the on-target activity experiment are shown in Table 10. GNA shows obvious sequence dependence, and the on-target activity of different sequences is significantly different. The experimental compounds disclosed in the present disclosure do not show obvious sequence dependence, and have stronger general applicability.

二、靶向配體的製備和活性評價2. Preparation and activity evaluation of targeting ligands

實施例6 連接到固相載體上的胺基半乳糖化合物1-tExample 6 Galactosamine compound 1-t linked to a solid support

合成路線如下： The synthetic route is as follows:

1)化合物1-g的合成路線 1) Synthetic route of compound 1-g

2)化合物1-h的合成路線 2) Synthetic route of compound 1-h

3)化合物1-l的合成路線 3) The synthetic route of compound 1-1

4)化合物1-q的合成 4) Synthesis of compound 1-q

5)連接到固相載體上的胺基半乳糖化合物1-t的合成 5) Synthesis of galactosamine compound 1-t linked to a solid support

步驟一 step one

將原料1-a(297g,763mmol)和原料1-b(160g,636mmol)溶解於960mlDCE，在15℃條件下，加入Sc(OTf)₃(15.6g,31.8mmol)，然後升高反應溫度到85℃，攪拌反應2h，反應結束後加入1.5L飽和NaHCO₃中止反應，分出有機相，並再用1.5升飽和食鹽水洗滌，有機相無水Na₂SO₄乾燥，過濾後的溶液減壓蒸餾後矽膠管柱層析純化(石油醚：乙酸乙酯5：1-0：1)，的到目標產物1-c(328g，544mmol，收率為85.5%，純度為96.4%)。 Dissolve raw material 1-a (297g, 763mmol) and raw material 1-b (160g, 636mmol) in 960ml DCE, add Sc(OTf) ₃ (15.6g, 31.8mmol) at 15°C, then increase the reaction temperature to Stir the reaction at 85°C for 2 hours, add 1.5L saturated NaHCO ₃ to stop the reaction after the reaction, separate the organic phase and wash it with 1.5 liter saturated brine, dry the organic phase with anhydrous Na ₂ SO ₄ , and distill the filtered solution under reduced pressure After purification by silica gel column chromatography (petroleum ether:ethyl acetate 5:1-0:1), the target product 1-c (328g, 544mmol, yield 85.5%, purity 96.4%) was obtained.

¹HNMR：(400MHz,CDCl₃)δ 7.44-7.29(m,5H),5.83(d,J=8.8Hz,1H),5.40-5.23(m,2H),5.18-5.06(m,2H),4.86(s,1H),4.66(d,J=8.4Hz,1H),4.21-4.07(m,2H),4.04-3.77(m,3H),3.51-3.45(m,1H),3.31-3.11(m,2H),2.18(d,J=2.0Hz,1H),2.14(s,3H),2.06(s,3H),2.03-1.99(m,3H),1.95(s,3H),1.64-1.46(m,4H),1.43-1.29(m,4H). ¹ HNMR: (400MHz, CDCl ₃ )δ 7.44-7.29(m,5H),5.83(d,J=8.8Hz,1H),5.40-5.23(m,2H),5.18-5.06(m,2H),4.86 (s,1H),4.66(d,J=8.4Hz,1H),4.21-4.07(m,2H),4.04-3.77(m,3H),3.51-3.45(m,1H),3.31-3.11(m ,2H),2.18(d,J=2.0Hz,1H),2.14(s,3H),2.06(s,3H),2.03-1.99(m,3H),1.95(s,3H),1.64-1.46( m,4H),1.43-1.29(m,4H).

MS，C₂₈H₄₀N₂O¹¹，實測M⁺ 581.3。 MS, C ₂₈ H ₄₀ N ₂ O ¹¹ , found M ⁺ 581.3.

步驟二 step two

將步驟一所得化合物平行分成兩份進行：每個反應包含化合物1-c(72.0g,124mmol)加入432mL THF中，在氬氣保護下加入Pd/C(20.0g,10%純度)，再加入TFA(14.1g,124mmol,9.18mL)，在反應溶液中通入氫氣，保持氣體壓力在30Psi，加熱至30℃並攪拌反應16h。反應完成後，合併兩個平行進行的反應，過濾，並減壓濃縮濾液。殘餘物使用二氯甲烷稀釋並重複減壓濃縮，重複三次。減壓抽乾後得到目標化合物1-d(139g)。 The compound obtained in step 1 was divided into two parts in parallel: each reaction contained compound 1-c (72.0g, 124mmol) was added to 432mL THF, Pd/C (20.0g, 10% purity) was added under the protection of argon, and then TFA (14.1g, 124mmol, 9.18mL), hydrogen was passed into the reaction solution, the gas pressure was kept at 30Psi, heated to 30°C and stirred for 16h. After completion of the reaction, two parallel reactions were combined, filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure three times. The target compound 1-d (139 g) was obtained after pumping to dryness under reduced pressure.

¹HNMR(400MHz,DMSO-d ₆)δ 7.85(d,J=9.2Hz,1H),7.74(s,3H),5.21(d,J=3.6Hz,1H),4.97(dd,J=2.8,10.8Hz,1H),4.48(d,J=8.8Hz,1H),4.06-3.98(m,3H),3.93-3.82(m,1H),3.73-3.68(m,1H),3.63-3.56(m,1H),3.43-3.38(m,1H),2.82-2.71(m,2H),2.13-2.09(m,3H),2.01-1.97(m,3H),1.91-1.87(m,3H),1.77(s,3H),1.76-1.73(m,1H),1.52-1.44(m,4H),1.28(s,4H). ¹ HNMR(400MHz,DMSO- d ₆ )δ 7.85(d, J =9.2Hz,1H),7.74(s,3H),5.21(d, J =3.6Hz,1H),4.97(dd, J =2.8, 10.8Hz,1H),4.48(d, J =8.8Hz,1H),4.06-3.98(m,3H),3.93-3.82(m,1H),3.73-3.68(m,1H),3.63-3.56(m ,1H),3.43-3.38(m,1H),2.82-2.71(m,2H),2.13-2.09(m,3H),2.01-1.97(m,3H),1.91-1.87(m,3H),1.77 (s,3H),1.76-1.73(m,1H),1.52-1.44(m,4H),1.28(s,4H).

步驟三 step three

將化合物1-d(139g,247mmol)和化合物1-e(75.3g,223mmol)加入DMF溶液(834mL)，在0℃下再加入DIPEA(41.6g,322mmol,56.1mL)、HOBt(36.8g,272mmol)和EDCI(52.2g,272mmol)，保持15℃攪拌反應16h，反應完成後將，反應液用二氯甲烷((400mL)稀釋，然後用飽和氯化銨溶液(1L)、飽和NaHCO₃(1.00L)、飽和食鹽水依次洗滌，分出有機相用無水硫酸鈉乾燥，過濾後減壓蒸餾除去溶劑，殘餘物矽膠管柱層析純化(石油醚：乙酸乙酯=5：1-0：1)，得到目標化合物1-f(108g，收率為56.8%)。 Compound 1-d (139g, 247mmol) and compound 1-e (75.3g, 223mmol) were added to DMF solution (834mL), and DIPEA (41.6g, 322mmol, 56.1mL), HOBt (36.8g, 272mmol) and EDCI (52.2g, 272mmol), kept stirring at 15°C for 16h, after the reaction was completed, the reaction solution was diluted with dichloromethane ((400mL), then saturated ammonium chloride solution (1L), saturated NaHCO ₃ ( 1.00L), washed with saturated saline in sequence, the organic phase was separated and dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1-0: 1), obtain target compound 1-f (108g, yield is 56.8%).

¹HNMR(40(400MHz,DMSO-d ₆)δ 7.89-7.78(m,2H),7.41-7.27(m,6H),5.21(d,J=3.2Hz,1H),5.08-4.92(m,3H),4.48(d,J=8.4Hz,1H),4.07-3.99(m,3H),3.97-3.81(m,2H),3.75-3.64(m,1H),3.42-3.37(m,1H),3.13-2.93(m,2H),2.20(t,J=8.0Hz,2H),2.10(s,3H),1.99(s,3H),1.89(s,3H),1.87-1.79(m,1H),1.76(s,3H),1.74-1.64(m,1H),1.48-1.41(m,2H),1.38(s,12H),1.29-1.20(m,4H),1.19-1.14(m,1H). ¹ HNMR (40(400MHz,DMSO- d ₆ )δ 7.89-7.78(m,2H),7.41-7.27(m,6H),5.21(d, J =3.2Hz,1H),5.08-4.92(m,3H ),4.48(d, J =8.4Hz,1H),4.07-3.99(m,3H),3.97-3.81(m,2H),3.75-3.64(m,1H),3.42-3.37(m,1H), 3.13-2.93(m,2H),2.20(t, J =8.0Hz,2H),2.10(s,3H),1.99(s,3H),1.89(s,3H),1.87-1.79(m,1H) ,1.76(s,3H),1.74-1.64(m,1H),1.48-1.41(m,2H),1.38(s,12H),1.29-1.20(m,4H),1.19-1.14(m,1H) .

MS，C₃₇H₅₅N₃O₁₄，實測值M⁺766.4。 MS, _C37H55N3O14 , found _M ₊ ^766.4 _.

步驟四 step four

將上述所得化合物1-f平行分成兩份進行：每個反應包含化合物6(47.0g,61.3mmol)加入280mL THF中，在氬氣保護下加入Pd/C(15.0g,10%純度)，再加入TFA(7.00g,61.3mmol,4.54mL)，在反應溶液中通入氫氣，保持氣體壓力在30Psi，加熱至30℃並攪拌反應16h。反應完成後，合併兩個平行進行的反應，過濾，並減壓濃縮濾液。殘餘物使用二氯甲烷稀釋並重複減壓濃縮，重複三次。減壓抽乾後得到目標化合物1-g(94.0g,crude)。 The compound 1-f obtained above was divided into two parts in parallel: each reaction contained compound 6 (47.0g, 61.3mmol) was added to 280mL THF, and Pd/C (15.0g, 10% purity) was added under the protection of argon, and then TFA (7.00g, 61.3mmol, 4.54mL) was added, hydrogen gas was passed through the reaction solution, and the gas pressure was kept at 30Psi, heated to 30°C and stirred for 16h. After completion of the reaction, two parallel reactions were combined, filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure three times. The target compound 1-g (94.0 g, crude) was obtained after pumping to dryness under reduced pressure.

¹HNMR(400MHz,DMSO-d6)δ 8.38(s,1H),8.10(s,3H),7.83(d,J=9.2Hz,1H),5.21(d,J=3.2Hz,1H),4.96(dd,J=3.6,11.2Hz,1H),4.47(d,J=8.4Hz,1H),4.06-3.98(m,3H),3.92-3.82(m,1H),3.75-3.67(m,2H),3.60(s,1H),3.43-3.37(m,1H),3.18-3.04(m,2H),2.30-2.24(m,2H),2.10(s,3H),2.00(s,3H),1.95-1.90(m,2H),1.89(s,3H),1.78-1.75(m,3H),1.49-1.41(m,3H),1.40(s,9H),1.26(s,4H). ¹ HNMR(400MHz,DMSO-d6)δ 8.38(s,1H),8.10(s,3H),7.83(d,J=9.2Hz,1H),5.21(d,J=3.2Hz,1H),4.96( dd,J=3.6,11.2Hz,1H),4.47(d,J=8.4Hz,1H),4.06-3.98(m,3H),3.92-3.82(m,1H),3.75-3.67(m,2H) ,3.60(s,1H),3.43-3.37(m,1H),3.18-3.04(m,2H),2.30-2.24(m,2H),2.10(s,3H),2.00(s,3H),1.95 -1.90(m,2H),1.89(s,3H),1.78-1.75(m,3H),1.49-1.41(m,3H),1.40(s,9H),1.26(s,4H).

步驟五 step five

將上述所得化合物1-f平行分成兩份進行：每個反應包含化合物1-f(46.0g,60mmol)加入HCl-EtOAc(2.00M,276mL)中，在15℃ 條件下攪拌反應16h。反應完成後合併兩個反應溶液，減壓蒸餾濃縮，殘餘物使用二氯甲烷稀釋並重複減壓濃縮，重複三次。減壓抽乾後得到目標化合物1-h(91.0g，粗品)。 Compound 1-f obtained above was divided into two parts in parallel: each reaction contained compound 1-f (46.0 g, 60 mmol) added to HCl-EtOAc (2.00 M, 276 mL), and stirred at 15°C for 16 h. After the reaction was completed, the two reaction solutions were combined and concentrated by distillation under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure three times. The target compound 1-h (91.0 g, crude product) was obtained after vacuum drying.

¹HNMR(400MHz,DMSO-d ₆)δ 7.91-7.80(m,2H),7.42-7.26(m,6H),5.21(d,J=3.2Hz,1H),5.07-4.92(m,4H),4.48(d,J=8.4Hz,1H),4.06-3.98(m,3H),3.98-3.82(m,3H),3.73-3.65(m,1H),3.44-3.35(m,1H),3.12-2.94(m,2H),2.22(t,J=8.0Hz,2H),2.10(s,3H),2.01-1.97(m,4H),1.94-1.90(m,1H),1.89(s,3H),1.87-1.79(m,2H),1.76(s,3H),1.74-1.67(m,1H),1.49-1.40(m,2H),1.40-1.32(m,2H),1.24(d,J=4.0Hz,4H),1.19-1.13(m,1H). ¹ HNMR(400MHz,DMSO- d ₆ )δ 7.91-7.80(m,2H),7.42-7.26(m,6H),5.21(d, J =3.2Hz,1H),5.07-4.92(m,4H), 4.48(d, J =8.4Hz,1H),4.06-3.98(m,3H),3.98-3.82(m,3H),3.73-3.65(m,1H),3.44-3.35(m,1H),3.12- 2.94(m,2H),2.22(t,J=8.0Hz,2H),2.10(s,3H),2.01-1.97(m,4H),1.94-1.90(m,1H),1.89(s,3H) ,1.87-1.79(m,2H),1.76(s,3H),1.74-1.67(m,1H),1.49-1.40(m,2H),1.40-1.32(m,2H),1.24(d, J = 4.0Hz,4H),1.19-1.13(m,1H).

MS，C₃₃H₄₇N₃O₁₄，實測M⁺710.3。 MS, C ₃₃ H ₄₇ N ₃ O ₁₄ , found M ⁺ 710.3.

步驟六 step six

平行進行兩個反應：每個反應包含化合物1-g(45.0g,60.3mmol)和化合物1-h(38.5g,54.3mmol)加入到270mL DMF，在0℃下再加入DIPEA(10.1g,78.4mmol,13.6mL)，再加入HOBt(8.97g,66.3mmol)和EDCI(12.7g,66.3mmol)。在15℃條件下攪拌反應16h。反應完成後合併兩個反應溶液，並加入300mL DCM稀釋，依次使用飽和氯化銨(800mL)、飽和NaHCO₃(800mL)和飽和食鹽水(800mL)洗滌，有機相用無水Na₂SO₄乾燥。過濾後，加壓蒸發濃縮，殘餘物使用矽膠管柱層析純化(石油醚：乙酸乙酯=5：1-0：1)，得到目標化合物1-i(66.0g,47.4mmol，收率為39.3%，純度為95.1%)。 Two reactions were carried out in parallel: each reaction contained compound 1-g (45.0 g, 60.3 mmol) and compound 1-h (38.5 g, 54.3 mmol) into 270 mL DMF, and DIPEA (10.1 g, 78.4 mmol, 13.6 mL), HOBt (8.97 g, 66.3 mmol) and EDCI (12.7 g, 66.3 mmol) were added. The reaction was stirred at 15°C for 16h. After the reaction was complete, the two reaction solutions were combined and diluted with 300 mL of DCM, washed sequentially with saturated ammonium chloride (800 mL), saturated NaHCO ₃ (800 mL) and saturated brine (800 mL), and the organic phase was dried over anhydrous Na ₂ SO ₄ . After filtration, concentrated by evaporation under pressure, the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1-0:1) to obtain the target compound 1-i (66.0g, 47.4mmol, the yield was 39.3%, with a purity of 95.1%).

¹HNMR(400MHz,DMSO-d ₆)δ 7.96-7.78(m,5H),7.41-7.25(m,6H),5.21(d,J=3.6Hz,2H),5.05-4.92(m,4H),4.48(d,J=8.8Hz,2H),4.22-4.12(m,1H),4.02(s,6H),3.94-3.80(m,3H),3.74-3.64(m,2H), 3.45-3.35(m,2H),3.11-2.92(m,4H),2.20-2.12(m,4H),2.10(s,6H),1.99(s,6H),1.89(s,6H),1.82-1.79(m,2H),1.76(s,6H),1.74-1.63(m,2H),1.44(d,J=6.0Hz,4H),1.37(s,12H),1.24(s,9H). ¹ HNMR(400MHz,DMSO- d ₆ )δ 7.96-7.78(m,5H),7.41-7.25(m,6H),5.21(d, J =3.6Hz,2H),5.05-4.92(m,4H), 4.48(d, J =8.8Hz,2H),4.22-4.12(m,1H),4.02(s,6H),3.94-3.80(m,3H),3.74-3.64(m,2H), 3.45-3.35( m,2H),3.11-2.92(m,4H),2.20-2.12(m,4H),2.10(s,6H),1.99(s,6H),1.89(s,6H),1.82-1.79(m, 2H),1.76(s,6H),1.74-1.63(m,2H),1.44(d, J =6.0Hz,4H),1.37(s,12H),1.24(s,9H).

MS：C₆₂H₉₄N₆O₂₅，實測值m/z 1323.8。 MS: _C62H94N6O25 _, found m _/ z 1323.8 _.

步驟七 step seven

分成11個反應進行：在每個反應中加入化合物1-i(5.00g,3.78mmol)和甲苯(300mL)，加入矽膠(45.0g)。在100℃下攪拌反應40h，反應完成後合併11個反應混合物。減壓蒸餾除去溶劑後，殘餘物加入異丙醇和二氯甲烷，並攪拌20min。過濾除去不溶物，並使用異丙醇洗滌濾餅至無產物溶出，得到的溶液除去溶劑並抽乾後得到目標化合物1-j(43.2g,34.0mmol，收率為82.0%)。 It was carried out in 11 reactions: compound 1-i (5.00 g, 3.78 mmol) and toluene (300 mL) were added to each reaction, and silica gel (45.0 g) was added. The reaction was stirred at 100° C. for 40 h, and 11 reaction mixtures were combined after completion of the reaction. After the solvent was distilled off under reduced pressure, isopropanol and dichloromethane were added to the residue, and stirred for 20 min. The insoluble matter was removed by filtration, and the filter cake was washed with isopropanol until no product was dissolved. The resulting solution was desolventized and dried to obtain the target compound 1-j (43.2 g, 34.0 mmol, yield 82.0%).

¹HNMR：(400MHz,DMSO-d6)δ 8.01(d,J=7.6Hz,1H),7.93-7.79(m,2H),7.39-7.27(m,3H),5.21(d,J=3.2Hz,1H),5.06-4.91(m,2H),4.48(d,J=8.0Hz,1H),4.07-3.97(m,3H),3.94-3.82(m,2H),3.73-3.65(m,1H),3.45-3.36(m,2H),3.10-2.94(m,2H),2.15(d,J=7.6Hz,2H),2.10(s,3H),1.99(s,3H),1.89(s,3H),1.86-1.79(m,1H),1.77(s,3H),1.74-1.65(m,1H),1.44(s,2H),1.37(d,J=5.2Hz,2H),1.24(s,4H)。 ¹ HNMR: (400MHz,DMSO-d6)δ 8.01(d,J=7.6Hz,1H),7.93-7.79(m,2H),7.39-7.27(m,3H),5.21(d,J=3.2Hz, 1H),5.06-4.91(m,2H),4.48(d,J=8.0Hz,1H),4.07-3.97(m,3H),3.94-3.82(m,2H),3.73-3.65(m,1H) ,3.45-3.36(m,2H),3.10-2.94(m,2H),2.15(d,J=7.6Hz,2H),2.10(s,3H),1.99(s,3H),1.89(s,3H ),1.86-1.79(m,1H),1.77(s,3H),1.74-1.65(m,1H),1.44(s,2H),1.37(d,J=5.2Hz,2H),1.24(s, 4H).

MS：C₅₈H₈₆N₆O₂₅，實測m/z=1267.8. MS: C ₅₈ H ₈₆ N ₆ O ₂₅ , found m/z=1267.8.

步驟八 step eight

此步平行分成兩個反應進行：每個反應包含化合物1-d(11.8g,21.0mmol)和化合物1-j(21.3g,16.8mmol)加入到70mL DMF，在0℃下再加入DIPEA(3.54g,27.3mmol,4.77mL)，再加入HOBt(3.13g,23.1mmol)和EDCI(4.44g,23.1mmol)。在15℃條件下攪拌反應16h。反應完成後合併兩個反應溶液，並加入500mL DCM稀釋，依次使用飽和氯化銨(1.5L)、飽和NaHCO₃(1.5mL)和飽和食鹽水(1.5mL)洗滌，有機相用無水Na₂SO₄乾燥。過濾後，加壓蒸發濃縮，殘餘物使用矽膠管柱層析純化(二氯甲烷：甲醇=50：1-10：1)，得到目標化合物1-k(54.0g,31.8mmol，收率為75.6%)。 This step was divided into two reactions in parallel: each reaction contained compound 1-d (11.8g, 21.0mmol) and compound 1-j (21.3g, 16.8mmol) into 70mL DMF, then added DIPEA (3.54 g, 27.3 mmol, 4.77 mL), HOBt (3.13 g, 23.1 mmol) and EDCI (4.44 g, 23.1 mmol) were added. The reaction was stirred at 15°C for 16h. After the reaction was completed, the two reaction solutions were combined, diluted with 500 mL of DCM, washed sequentially with saturated ammonium chloride (1.5 L), saturated NaHCO ₃ (1.5 mL) and saturated brine (1.5 mL), and the organic phase was washed with anhydrous Na ₂ SO ₄ dry. After filtration, concentrated by evaporation under pressure, the residue was purified by silica gel column chromatography (dichloromethane:methanol=50:1-10:1) to obtain the target compound 1-k (54.0g, 31.8mmol, yield 75.6 %).

¹HNMR(400MHz,DMSO-d ₆)δ 7.91(d,J=7.6Hz,1H),7.87-7.78(m,5H),7.73(t,J=5.2Hz,1H),7.42-7.24(m,6H),5.21(d,J=3.6Hz,3H),5.06-4.92(m,5H),4.48(d,J=8.4Hz,3H),4.19-4.09(m,2H),4.07-3.97(m,10H),3.94-3.80(m,4H),3.76-3.64(m,3H),3.42-3.37(m,4H),3.08-2.94(m,6H),2.20-2.12(m,2H),2.10(s,9H),2.08-2.01(m,2H),1.99(s,9H),1.89(s,9H),1.87-1.79(m,2H),1.77(s,9H),1.74-1.63(m,2H),1.44(d,J=5.6Hz,6H),1.40-1.31(m,6H),1.24(s,13H). ¹ HNMR(400MHz,DMSO- d ₆ )δ 7.91(d,J=7.6Hz,1H),7.87-7.78(m,5H),7.73(t,J=5.2Hz,1H),7.42-7.24(m, 6H), 5.21(d, J=3.6Hz, 3H), 5.06-4.92(m, 5H), 4.48(d, J=8.4Hz, 3H), 4.19-4.09(m, 2H), 4.07-3.97(m ,10H),3.94-3.80(m,4H),3.76-3.64(m,3H),3.42-3.37(m,4H),3.08-2.94(m,6H),2.20-2.12(m,2H),2.10 (s,9H),2.08-2.01(m,2H),1.99(s,9H),1.89(s,9H),1.87-1.79(m,2H),1.77(s,9H),1.74-1.63(m ,2H),1.44(d,J=5.6Hz,6H),1.40-1.31(m,6H),1.24(s,13H).

MS：C₇₈H₁₁₈N₈O₃₃，實測值m/z=1696.1。 MS: C ₇₈ H ₁₁₈ N ₈ O ₃₃ , found m/z = 1696.1.

步驟九 Step Nine

此步平行分成3個反應進行：在每個反應中加入化合物1-k(17.0g,10.0mmol)和THF(100mL)，在氬氣保護下加入Pd/C(5.0g,10%純度)，再加入TFA(1.14g,10.0mmol,742uL)，在反應溶液中通入氫氣，保持氣體壓力在15Psi，加熱至30℃並攪拌反應4h。反應完成後，合併3個平行進行的反應，過濾，並減壓濃縮濾液。殘餘物使用二氯甲烷稀釋並重複減壓濃縮，重複三次。殘餘物使用製備液相色譜(C18，流動相A 0.1%TFA-水，流動相B：10-40% CAN，20min)純化得到目標化合物1-l(17.3g,10.2mmol，收率為34.0%)。 This step is divided into 3 reactions in parallel: add compound 1-k (17.0g, 10.0mmol) and THF (100mL) to each reaction, add Pd/C (5.0g, 10% purity) under argon protection, Then TFA (1.14g, 10.0mmol, 742uL) was added, hydrogen gas was passed into the reaction solution, and the gas pressure was kept at 15Psi, heated to 30°C and stirred for 4h. After the reaction was complete, 3 parallel reactions were combined, filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure three times. The residue was purified by preparative liquid chromatography (C18, mobile phase A 0.1%TFA-water, mobile phase B: 10-40% CAN, 20min) to obtain the target compound 1-l (17.3g, 10.2mmol, yield 34.0% ).

¹HNMR：(400MHz,DMSO-d ₆)δ 8.45(t,J=5.2Hz,1H),8.14(d,J=5.2Hz,3H),7.97(t,J=5.2Hz,1H),7.90-7.77(m,4H),5.21(d,J=2.8Hz,3H),4.96(dd,J=3.2,11.6Hz,3H),4.47(d,J=8.4Hz,3H), 4.20-4.10(m,1H),4.02(s,8H),3.87(q,J=9.6Hz,3H),3.75-3.61(m,4H),3.46-3.34(m,3H),3.21-2.93(m,6H),2.21(s,2H),2.14-2.02(m,11H),1.99(s,9H),1.96-1.82(m,12H),1.80-1.65(m,10H),1.44(d,J=5.6Hz,8H),1.36(d,J=6.4Hz,4H),1.30-1.17(m,12H) ¹ HNMR: (400MHz, DMSO- d ₆ )δ 8.45(t, J =5.2Hz, 1H), 8.14(d, J =5.2Hz, 3H), 7.97(t, J =5.2Hz, 1H), 7.90- 7.77(m,4H),5.21(d, J =2.8Hz,3H),4.96(dd, J =3.2,11.6Hz,3H),4.47(d, J =8.4Hz,3H), 4.20-4.10(m ,1H),4.02(s,8H),3.87(q, J =9.6Hz,3H),3.75-3.61(m,4H),3.46-3.34(m,3H),3.21-2.93(m,6H), 2.21(s,2H),2.14-2.02(m,11H),1.99(s,9H),1.96-1.82(m,12H),1.80-1.65(m,10H),1.44(d, J =5.6Hz, 8H), 1.36(d, J =6.4Hz, 4H), 1.30-1.17(m, 12H)

MS：C₇₀H₁₁₂N₈O₃₁，實測值m/2z=781.8 MS: C ₇₀ H ₁₁₂ N ₈ O ₃₁ , found m/2z=781.8

步驟十 step ten

將化合物1-m(2g,12.64mmol)溶於吡啶(10mL)中，室溫下滴加DMTrCl(4.71g,13.90mmol)的吡啶(10mL)溶液，反應在室溫下攪拌5小時，待反應完畢後，用甲醇淬滅，減壓濃縮得到粗品，用矽膠純化(石油醚：乙酸乙酯=10：1沖提)，收集產物沖提液，減壓蒸乾溶劑得到4g的化合物1-n。 Compound 1-m (2g, 12.64mmol) was dissolved in pyridine (10mL), and a solution of DMTrCl (4.71g, 13.90mmol) in pyridine (10mL) was added dropwise at room temperature, and the reaction was stirred at room temperature for 5 hours. After completion, it was quenched with methanol, concentrated under reduced pressure to obtain the crude product, purified with silica gel (petroleum ether: ethyl acetate = 10:1 eluting), collected the product eluate, evaporated the solvent under reduced pressure to obtain 4 g of compound 1-n .

MS m/z：C₂₉H₃₂O₅，[M+H]⁺實測：461.3。 MS m/z _: _C29H32O5 , [M+H] ⁺ found: _461.3 .

步驟十一 step eleven

將化合物1-n(2g,4.34mmol)，N,N-二異丙基乙胺(DIEA，1.43mL,8.68mmol)和HATU(2.47g,6.51mmol)溶解於DMF(10mL)中，室溫下加入化合物1-o的DMF(5mL)溶液，該反應在室溫下攪拌8小時。反應完畢後，加水淬滅，水相用乙酸乙酯提取，合併的有機相先用水洗滌，再用飽和食鹽水(20mL)洗滌，隨後減壓蒸乾溶劑，經反相製備HPLC(管柱：Boston Green ODS 150*30mm*5um，條件：25-80%(A：水0.075% NH₃．H₂O,B：CH₃CN)，流速：55mL/min)，凍乾後得到2.4g化合物1-p。 Compound 1-n (2g, 4.34mmol), N,N-diisopropylethylamine (DIEA, 1.43mL, 8.68mmol) and HATU (2.47g, 6.51mmol) were dissolved in DMF (10mL) at room temperature A solution of compound 1-o in DMF (5 mL) was added at low temperature, and the reaction was stirred at room temperature for 8 hours. After the reaction was completed, quenched with water, the aqueous phase was extracted with ethyl acetate, the combined organic phase was washed with water first, and then washed with saturated brine (20mL), then the solvent was evaporated to dryness under reduced pressure, and the HPLC was prepared by reverse phase (column: Boston Green ODS 150*30mm*5um, condition: 25-80% (A: water 0.075% NH ₃ ．H ₂ O, B: CH ₃ CN), flow rate: 55mL/min), after lyophilization, 2.4g of compound 1 was obtained -p .

MS m/z：C₃₃H₃₉NO₇，[M+H]⁺實測：562.4。 MS m/z: _C33H39NO7 , [M+H ^]+ _found _: 562.4.

步驟十二 step twelve

將化合物1-p(2.4g,4.27mmol)溶解於15mL的甲醇和水(2：1)的混合溶液中，室溫下加入LiOH(0.36g,8.54mmol)並攪拌過夜。待反應完畢後減壓蒸乾溶劑，經反相製備HPLC(管柱：Boston Green ODS 150*30mm*5um，條件：25-75%(A：水0.075% NH₃．H₂O,B：CH₃CN)，流速：55mL/min)，凍乾後得到2g化合物1-q。 Compound 1-p (2.4g, 4.27mmol) was dissolved in 15mL of a mixed solution of methanol and water (2:1), and LiOH (0.36g, 8.54mmol) was added at room temperature and stirred overnight. After the reaction was completed, the solvent was evaporated to dryness under reduced pressure, and HPLC was prepared by reverse phase (column: Boston Green ODS 150*30mm*5um, condition: 25-75% (A: water 0.075% NH ₃ .H ₂ O, B: CH ₃ CN), flow rate: 55mL/min), and 2 g of compound 1-q were obtained after lyophilization.

MS m/z：C₃₂H₃₇NO₇，[M+H]⁺實測：548.6。 MS m/z: _C32H37NO7 , [M+H] ₊ found: ^548.6 _.

步驟十三 Step Thirteen

將化合物1-q(0.37g,0.69mmol)，DIEA(0.19mL,1.15mmol)和HATU(0.32g,0.86mmol)溶於2mL的DMF中，室溫下加入化合物1-l(0.9g,0.69mmol)的DMF(2mL)溶液，在室溫下攪拌過夜。反應完畢後，經二氯甲烷(10mL)稀釋反應液，並依次用飽和NaHCO₃(20mL)和飽和食鹽水(20mL)洗滌，有機相無水Na₂SO₄乾燥，過濾後減壓濃縮。經反相製備HPLC(管柱：Boston Green ODS 150*30mm*5um，條件：25-65%(A：水0.075% NH₃．H₂O,B：CH₃CN)，流速：45mL/min)純化，凍乾後得到0.5g化合物1-r。 Compound 1-q (0.37g, 0.69mmol), DIEA (0.19mL, 1.15mmol) and HATU (0.32g, 0.86mmol) were dissolved in 2mL of DMF, and compound 1-l (0.9g, 0.69 mmol) in DMF (2 mL), stirred overnight at room temperature. After the reaction was complete, the reaction solution was diluted with dichloromethane (10 mL), washed successively with saturated NaHCO ₃ (20 mL) and saturated brine (20 mL), and the organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Preparative HPLC by reverse phase (column: Boston Green ODS 150*30mm*5um, condition: 25-65% (A: water 0.075% NH ₃ .H ₂ O, B: CH ₃ CN), flow rate: 45mL/min) After purification and lyophilization, 0.5 g of compound 1-r was obtained.

MS m/z：C₁₀₂H₁₄₇N₉O₃₇，[M-H]⁺實測：2088.5。 MS m/z _: _{C102H147N9O37} , [MH] ⁺ found: _2088.5 _.

步驟十四 Step Fourteen

將化合物1-r(300mg,0.14mmol)和丁二酸酐(28.70mg,0.28mmol)溶解於四氫呋喃中，向反應液中加入DMAP(3.50mg,0.028mmol)並在40℃下攪拌過夜。待反應完畢後，待反應完畢後，加入甲醇(18.8mg)，並攪拌反應10min，然後將反應液用二氯甲烷(3mL)稀釋反應液，並用飽和NaHCO3(5mL)洗滌2次。將有機相減壓濃縮至乾，經反相製備HPLC(管柱：Boston Green ODS 150*30mm*5um，條件：25-65%(A：水0.075% NH₃．H₂O,B：CH₃CN)，流速：35mL/min)純化，凍乾後得到140mg化合物1-s。 Compound 1-r (300mg, 0.14mmol) and succinic anhydride (28.70mg, 0.28mmol) were dissolved in tetrahydrofuran, DMAP (3.50mg, 0.028mmol) was added to the reaction solution and stirred overnight at 40°C. After the reaction was completed, methanol (18.8 mg) was added, and the reaction was stirred for 10 min, then the reaction solution was diluted with dichloromethane (3 mL), and washed twice with saturated NaHCO3 (5 mL). Concentrate the organic phase to dryness under reduced pressure, and perform reverse-phase preparative HPLC (column: Boston Green ODS 150*30mm*5um, condition: 25-65% (A: water 0.075% NH ₃ .H ₂ O, B: CH ₃ CN), flow rate: 35mL/min), and lyophilized to obtain 140mg of compound 1-s .

MS m/z：C₁₀₆H₁₅₁N₉O₄₀，[M-H]⁺實測：2189.4。 MS _m /z: _{C106H151N9O40} , [MH] ⁺ found: _2189.4 _.

步驟十五 Step fifteen

將上步得到的化合物1-r(140mg，64ummol)加入乙腈(5mL)，再加入HBTU(48.7mg，128umol)，加入表面胺基修飾的固相支撐物(CPG-NH2，2.3g)，加入DIEA(41.5mg,320umol,55uL)，保持30℃震盪反應16h。反應完成後，過濾，並依次用甲醇(8mL x 4)、二氯甲烷(8mL x 4)洗滌。固體繼續加入吡啶：乙酸酐(v：v=4：1,10.0mL)中，繼續保持30℃震盪反應16h。反應完成後，過濾，並依次用甲醇(8mL x 4)、二氯甲烷(8mL x 4)洗滌。得到連接在固相載體上的化合物1-t 2.1g。 Add the compound 1-r (140mg, 64ummol) obtained in the previous step into acetonitrile (5mL), then add HBTU (48.7mg, 128umol), add the solid phase support (CPG-NH2, 2.3g) modified by the surface amino group, add DIEA (41.5mg, 320umol, 55uL), keep shaking at 30°C for 16h. After the reaction was completed, it was filtered and washed with methanol (8 mL x 4), dichloromethane (8 mL x 4) successively. The solid was continued to be added to pyridine:acetic anhydride (v:v=4:1, 10.0 mL), and the reaction was kept at 30°C for 16 hours with shaking. After the reaction was completed, it was filtered and washed with methanol (8 mL x 4), dichloromethane (8 mL x 4) successively. Obtained 2.1 g of compound 1-t linked on the solid phase support.

實施例7 連接到固相載體上的胺基半乳糖化合物2-eExample 7 Galactosamine compound 2-e linked to a solid support

合成路線如下： The synthetic route is as follows:

1)化合物2-b的合成 1) Synthesis of compound 2-b

2)化合物2-e的合成 2) Synthesis of compound 2-e

步驟一 step one

將化合物2-a(1.00g,2.37mmol)加入THF(7.5mL)和H₂O(7.5mL)中，再加入LiOH.H₂O(109mg,2.60mmol)，保持16℃攪拌反應16h。反應完成後，減壓蒸發除去溶劑，殘餘物繼續冷凍乾燥，得到目標化合物2-b(960mg,2.32mmol，收率為97.8%)。 Compound 2-a (1.00g, 2.37mmol) was added to THF (7.5mL) and H ₂ O (7.5mL), then LiOH.H ₂ O (109mg, 2.60mmol) was added, and the reaction was stirred at 16°C for 16h. After the reaction was completed, the solvent was evaporated under reduced pressure, and the residue was further freeze-dried to obtain the target compound 2-b (960 mg, 2.32 mmol, yield 97.8%).

¹HNMR：(400MHz,DMSO-d6)δ 7.44(d,J=8.4Hz,2H),7.34-7.23(m,6H),7.22-7.15(m,1H),6.86(d,J=8.0Hz,4H),3.73(s,6H),3.66(d,J=6.4Hz,1H),3.32(d,J=12.0Hz,1H),3.11(dd,J=2.0,9.2Hz,1H),2.85(t,J=8.8Hz,1H). ¹ HNMR: (400MHz,DMSO-d6)δ 7.44(d,J=8.4Hz,2H),7.34-7.23(m,6H),7.22-7.15(m,1H),6.86(d,J=8.0Hz, 4H),3.73(s,6H),3.66(d,J=6.4Hz,1H),3.32(d,J=12.0Hz,1H),3.11(dd,J=2.0,9.2Hz,1H),2.85( t,J=8.8Hz,1H).

MS m/z：C₂₄H₂₄O₆，實測m/z：407.2。 MS m/z: _C24H24O6 , found m/z _: 407.2 _.

步驟二 step two

將化合物1-l(500mg,0.30mmol)加入二氯甲烷(3mL)中，15℃下，再將化合物2-b(0.14g,0.34mmol)加入反應中，在0℃下再加入HBTU(142mg,375umol)和DIEA((115mg,895umol)，保持15℃反應16h。反應完畢後，經二氯甲烷(10mL)稀釋反應液，並依次用飽和NaHCO₃(20mL)和飽和食鹽水(20mL)洗滌，有機相無水Na₂SO4乾燥，過濾後減壓濃縮，殘餘物經過製備液相純化(管柱：Welch Xtimate C18 250*70mm#10um；流動相：[水-ACN]；B%：40%-66%,18min)，得到化合物2-c。 Compound 1-l (500mg, 0.30mmol) was added in dichloromethane (3mL), at 15°C, compound 2-b (0.14g, 0.34mmol) was added to the reaction, and HBTU (142mg , 375umol) and DIEA ((115mg, 895umol), kept at 15°C for 16h. After the reaction was completed, the reaction solution was diluted with dichloromethane (10mL), and washed successively with saturated NaHCO ₃ (20mL) and saturated brine (20mL) , the organic phase was dried over anhydrous Na ₂ SO4 , filtered and concentrated under reduced pressure, and the residue was purified by preparative liquid phase (column: Welch Xtimate C18 250*70mm#10um; mobile phase: [water-ACN]; B%: 40%- 66%, 18min), to obtain compound 2-c .

MS m/z：C₉₄H₁₃₄N₈O₃₆，[M-H]+實測：1952.1。 MS m _/ z: _C94H134N8O36 , [MH]+ found: _1952.1 _.

步驟三 step three

將化合物2-c(230mg,0.12mmol)和丁二酸酐(23.5mg,0.26mmol)溶解於二氯甲烷溶液(2mL)中，向反應液中加入DMAP(43.1mg,0.35mmol)，保持在15oC下攪拌16h。待反應完畢後，加入甲醇(18.8mg)，並攪拌反應10min，然後將反應液用二氯甲烷(3mL)稀釋反應液，並用飽和NaHCO₃洗滌2次。將反應液減壓濃縮抽乾得到化合物2-d(240mg，粗品)。 Compound 2-c (230mg, 0.12mmol) and succinic anhydride (23.5mg, 0.26mmol) were dissolved in dichloromethane solution (2mL), DMAP (43.1mg, 0.35mmol) was added to the reaction solution and kept at 15oC Under stirring for 16h. After the reaction was complete, methanol (18.8 mg) was added, and the reaction was stirred for 10 min, then the reaction solution was diluted with dichloromethane (3 mL), and washed twice with saturated NaHCO ₃ . The reaction solution was concentrated under reduced pressure and sucked to dryness to obtain compound 2-d (240 mg, crude product).

MS m/z：C106H151N9O40，[M-H]+實測：m/2z：2070.2 MS m/z: C106H151N9O40, [M-H]+ measured: m/2z: 2070.2

步驟四 step four

將上步得到的化合物2-d(240mg，116ummol)加入乙腈(8mL)，再加入HBTU(88.7mg，233umol)，加入表面胺基修飾的固相支撐物(CPG-NH2，4g)，加入DIEA(75.5mg,584umol,101uL)，保持30℃震盪反應16h。反應完成後，過濾，並依次用甲醇(8mL x 4)、二氯甲烷(8mL x 4)洗滌。固體繼續加入吡啶：乙酸酐(v：v=4：1,10.0mL)中，繼續保持30℃震盪反應16h。反應完成後，過濾，並依次用甲醇(8mL x 4)、二氯甲烷(8mL x 4)洗滌。得到目標產物連接在固相載體上的化合物2-e 3.7g。 Add the compound 2-d (240mg, 116ummol) obtained in the previous step into acetonitrile (8mL), then add HBTU (88.7mg, 233umol), add the surface amine-modified solid phase support (CPG-NH2, 4g), add DIEA (75.5mg, 584umol, 101uL), keep shaking at 30°C for 16h. After the reaction was completed, it was filtered and washed with methanol (8 mL x 4), dichloromethane (8 mL x 4) successively. The solid was continued to be added to pyridine:acetic anhydride (v:v=4:1, 10.0 mL), and the reaction was kept at 30°C for 16 hours with shaking. After the reaction was completed, it was filtered and washed with methanol (8 mL x 4), dichloromethane (8 mL x 4) successively. Obtained 3.7g of compound 2 -e in which the target product was linked to a solid phase carrier.

實施例8 連接到固相載體上的胺基半乳糖化合物3-nExample 8 Galactosamine compound 3-n linked to a solid support

合成路線如下： The synthetic route is as follows:

1)化合物3-d的合成 1) Synthesis of compound 3-d

2)化合物3-g的合成 2) Synthesis of compound 3-g

3)化合物3-n的合成 3) Synthesis of compound 3-n

步驟一 step one

將原料3-a(78.8g,202mmol)和原料3-b(40g,168mmol)溶解於DCE(250mL)，在15℃條件下，加入CF₃SO₃H(4.15g,8.43mmol,)，然後升高反應溫度到75℃，攪拌反應2h，反應結束後加入1L飽和NaHCO₃中止反應，分出有機相，並再用1L飽和食鹽水洗滌，有機相無水Na₂SO₄乾燥，過濾後的溶液減壓蒸餾後矽膠管柱層析純化(石油醚：乙酸乙酯5：1-0：1)，的到目標產物3-c(63.2g,107mmol，收率為63.5%)。 Material 3-a (78.8g, 202mmol) and material 3-b (40g, 168mmol) were dissolved in DCE (250mL), and CF ₃ SO ₃ H (4.15g, 8.43mmol,) was added at 15°C, and then Raise the reaction temperature to 75°C, stir the reaction for 2 hours, add 1L saturated NaHCO ₃ after the reaction to stop the reaction, separate the organic phase, and then wash with 1L saturated brine, dry the organic phase with anhydrous Na ₂ SO ₄ , filter the solution After vacuum distillation, silica gel column chromatography purification (petroleum ether: ethyl acetate 5:1-0:1), the target product 3-c (63.2g, 107mmol, yield 63.5%) was obtained.

¹HNMR：(400MHz,CDCl₃)δ 7.35-7.26(m,5H),5.88(s,1H),5.34-5.25(m,2H),4.65(d,J=8.4Hz,1H),4.16-4.13(m,2H),3.92-3.87(m,3H),3.18-3.17(m,1H),3.15-3.14(m,2H),2.16-1.91(m,15H),1.58-1.50(m,5H),1.49-1.36(m,2H). ¹ HNMR: (400MHz, CDCl ₃ )δ 7.35-7.26(m,5H),5.88(s,1H),5.34-5.25(m,2H),4.65(d, J =8.4Hz,1H),4.16-4.13 (m,2H),3.92-3.87(m,3H),3.18-3.17(m,1H),3.15-3.14(m,2H),2.16-1.91(m,15H),1.58-1.50(m,5H) ,1.49-1.36(m,2H).

MS m/z：C₂₄H₄₀N₂O₁₁，實測m/z：567.4。 MS m/z: _C24H40N2O11 , found _m /z _: 567.4 _.

步驟二 step two

將上述所得化合物3-c(60.0g,106mmol)加入360mL THF中，在氬氣保護下加入Pd/C(15.0g,10%純度)，再加入TFA(12.1g,106mmol,7.84mL)，在反應溶液中通入氫氣，保持氣體壓力在30Psi，加熱至30℃並攪拌反應16h。反應完成後，過濾，並減壓濃縮濾液。殘餘物使用二氯甲烷稀釋並重複減壓濃縮，重複三次(500mL x 3)。減壓抽乾後得到目標化合物3-d(44g,102mmol，收率為96.1%)。 The compound 3-c (60.0g, 106mmol) obtained above was added in 360mL THF, and Pd/C (15.0g, 10% purity) was added under the protection of argon, and then TFA (12.1g, 106mmol, 7.84mL) was added. Hydrogen gas was introduced into the reaction solution to keep the gas pressure at 30Psi, heated to 30°C and stirred for 16h. After the reaction was completed, it was filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure repeatedly three times (500 mL x 3). The target compound 3-d (44 g, 102 mmol, yield 96.1%) was obtained after vacuum drying.

步驟三 step three

將化合物3-e(60.0g，447mmol)溶解於DMF(300mL)，加入K₂CO₃(92.7g，671mmol)，在0℃條件下滴加入BnBr(115g，671mmol，79.7mL)。保持25℃攪拌反應6h。將反應液倒入碎冰中，然後使用乙酸乙酯(100mL*6)萃取，有機相再依次用水洗滌(100ml*2)，飽和食鹽水洗滌(100ml*3)。有機相無水硫酸鈉乾燥後減壓蒸餾除去溶劑，殘餘物使用矽膠管柱層析純化(石油醚：乙酸乙酯=2：1-0：1)，得到目標固體化合物3-f(60.3g，269mmol，收率60.1%)。 Compound 3-e (60.0 g, 447 mmol) was dissolved in DMF (300 mL), K ₂ CO ₃ (92.7 g, 671 mmol) was added, and BnBr (115 g, 671 mmol, 79.7 mL) was added dropwise at 0°C. Keep stirring at 25°C for 6h. The reaction solution was poured into crushed ice, then extracted with ethyl acetate (100mL*6), and the organic phase was washed with water (100ml*2) and saturated brine (100ml*3) successively. The organic phase was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1-0:1) to obtain the target solid compound 3-f (60.3g, 269mmol, yield 60.1%).

¹HNMR：(400MHz,CDCl₃)δ 7.37-7.26(m,5H),5.18(d,J=4.4Hz,2H),3.95-3.90(m,2H),3.75-3.71(m,2H),1.08(s,1H). ¹ HNMR: (400MHz, CDCl ₃ )δ 7.37-7.26(m,5H),5.18(d,J=4.4Hz,2H),3.95-3.90(m,2H),3.75-3.71(m,2H),1.08 (s,1H).

MS m/z：C₁₂H₁₆O₄，實測m/z：223.5。 MS m/z: _C12H16O4 , found m/z _: 223.5 _.

步驟四 step four

將化合物3-f(50.0g，223mmol)溶解在二氯甲烷中(300mL)，加入吡啶(73.5g，929mmol，75mL)和溶解於二氯甲烷(50mL)的對硝基苯基氯甲酸酯(180g，892mmol)，氮氣保護下保持25℃攪拌反應24h。反應完成後，加入二氯甲烷(250mL)稀釋，然後依次使用NaHSO₄溶液(30mL*3)和飽和食鹽水(30mL*2)洗滌，有機相使用MgSO₄乾燥後，過濾，減壓蒸發掉溶劑。所得到的粗品使用矽膠管柱層析純化(石油醚：乙酸乙酯=3：1)，得到目標化合物3-g(37.0g，66.7mmol，收率為29.9%) Compound 3-f (50.0 g, 223 mmol) was dissolved in dichloromethane (300 mL), pyridine (73.5 g, 929 mmol, 75 mL) and p-nitrophenyl chloroformate dissolved in dichloromethane (50 mL) were added (180g, 892mmol), stirred and reacted at 25°C for 24h under the protection of nitrogen. After the reaction is complete, add dichloromethane (250mL) to dilute, then wash with NaHSO ₄ solution (30mL*3) and saturated brine (30mL*2) successively, the organic phase is dried with MgSO ₄ , filtered, and the solvent is evaporated under reduced pressure . The obtained crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate=3:1) to obtain the target compound 3-g (37.0g, 66.7mmol, yield 29.9%)

MS m/z：C₂₆H₂₂N₂O₁₂，實測m/z：553.4 MS m/z: C ₂₆ H ₂₂ N ₂ O ₁₂ , found m/z: 553.4

步驟五 step five

將化合物3-g(22.0g，39.7mmol)加入乙腈(120mL)中，在氮氣保護條件下，再加入三乙胺(24.1g，238mmol，33.1mL)，將反應液冷卻到0℃，滴加入溶解在乙腈(120mL)中的化合物3-d(42.1g，40mmol)，升高溫度到25℃，並攪拌反應1h。反應完成後，減壓濃縮除去溶劑，然後使用矽膠管柱層析純化(石油醚：乙酸乙酯=2：1)得到目標化合物3-h(37.0g，12.0mmol，收率為30.2%)。 Compound 3-g (22.0g, 39.7mmol) was added to acetonitrile (120mL), under nitrogen protection, triethylamine (24.1g, 238mmol, 33.1mL) was added, the reaction solution was cooled to 0°C, and added dropwise Compound 3-d (42.1 g, 40 mmol) was dissolved in acetonitrile (120 mL), the temperature was raised to 25° C., and the reaction was stirred for 1 h. After the reaction was completed, the solvent was concentrated under reduced pressure to remove the solvent, and then purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain the target compound 3-h (37.0 g, 12.0 mmol, yield 30.2%).

MS m/z：C₅₂H₇₆N₄O₂₄，實測m/z：1141.8。 MS _m _/ z: _C52H76N4O24 , found m/z: 1141.8 _.

步驟六 step six

將化合物3-h(11.0g，9.64mmol)溶解於乙酸乙酯(60mL)中，加入Pd/C(2.00g,10%純度)，反應溶液中通入氫氣，保持氣體壓力在40Psi，溫度25℃攪拌反應8h。反應完成後，過濾，並減壓蒸發至乾燥，得到目標化合物3-i(10.0g,9.42mmol，收率為97.7%)。 Compound 3-h (11.0g, 9.64mmol) was dissolved in ethyl acetate (60mL), Pd/C (2.00g, 10% purity) was added, and hydrogen gas was introduced into the reaction solution to keep the gas pressure at 40Psi and the temperature at 25 The reaction was stirred at ℃ for 8h. After the reaction was completed, it was filtered and evaporated to dryness under reduced pressure to obtain the target compound 3-i (10.0 g, 9.42 mmol, yield 97.7%).

¹HNMR：(400MHz,DMSO-d ₆)δ 7.79(d,J=9.2Hz,2H),7.10(s,2H),5.74(t,J=1.6Hz,2H),5.21(d,J=3.6Hz,2H),4.98-4.95(m,2H),4.48(d,J=8.4Hz,2H),4.02(d,J=4.8Hz,11H),3.87-3.84(m,2H),3.69-3.67(m,2H),3.41-3.39(m,2H),2.94-2.90(m,4H),2.10(s,5H),1.99(s,7H),1.89(s,6H),1.77(s,6H),1.47-1.35(m,8H),1.26-1.24(m,4H),1.23-1.08(m,3H). ¹ HNMR: (400MHz,DMSO- d ₆ )δ 7.79(d,J=9.2Hz,2H),7.10(s,2H),5.74(t,J=1.6Hz,2H),5.21(d,J=3.6 Hz,2H),4.98-4.95(m,2H),4.48(d,J=8.4Hz,2H),4.02(d,J=4.8Hz,11H),3.87-3.84(m,2H),3.69-3.67 (m,2H),3.41-3.39(m,2H),2.94-2.90(m,4H),2.10(s,5H),1.99(s,7H),1.89(s,6H),1.77(s,6H ),1.47-1.35(m,8H),1.26-1.24(m,4H),1.23-1.08(m,3H).

MS m/z：C₄₅H₇₀N₄O₂₄，實測m/z：1051.4 MS m/z: C ₄₅ H ₇₀ N ₄ O ₂₄ , Found m/z: 1051.4

步驟七 step seven

將化合物3-i(5.00g,4.76mmol)加入到二氯甲烷(30mL)和DMF(30mL)的混合溶劑中，再加入化合物33(312mg,2.38mmol)，加入HBTU(1.80g,4.76mmol)和DIEA(615mg,4.76mmol)，保持25℃攪拌反應12h。反應完成後，將反應液倒入到乙酸乙酯(100mL)中，然後使用飽和食鹽水洗滌，無水Na₂SO₄乾燥後，過濾並減壓蒸發除去溶劑，殘餘物使用製備HPLC純化得到目標化合物3-k(2.1g,956umol，收率為20.1%)。 Compound 3-i (5.00g, 4.76mmol) was added to a mixed solvent of dichloromethane (30mL) and DMF (30mL), then compound 33 (312mg, 2.38mmol) was added, and HBTU (1.80g, 4.76mmol) was added And DIEA (615mg, 4.76mmol), kept stirring at 25°C for 12h. After the reaction was completed, the reaction solution was poured into ethyl acetate (100 mL), washed with saturated brine, dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to remove the solvent, and the residue was purified by preparative HPLC to obtain the target compound 3-k (2.1 g, 956 umol, yield 20.1%).

¹HNMR：(400MHz,DMSO-d ₆)δ 7.84-7.81(m,5H),7.12-7.07(m,3H),5.21(d,J=3.6Hz,4H),4.99-4.96(m,4H),4.49(d,J=8.4Hz,4H),4.06-4.00(m,24H),3.88-3.86(m,4H),3.55-3.52(m,4H),3.49-3.43(m,4H),3.25-3.05(m,4H),2.94-2.93(m,8H),2.11(s,12H),2.00(s,16H),1.90(s,12H),1.78(s,12H),1.46-1.44(m,8H),1.38-1.35(m,8H),1.26-1.24(m,8H),1.18-1.16(m,6H),1.09-0.99(m,2H). ¹ HNMR: (400MHz,DMSO- d ₆ )δ 7.84-7.81(m,5H),7.12-7.07(m,3H),5.21(d,J=3.6Hz,4H),4.99-4.96(m,4H) ,4.49(d,J=8.4Hz,4H),4.06-4.00(m,24H),3.88-3.86(m,4H),3.55-3.52(m,4H),3.49-3.43(m,4H),3.25 -3.05(m,4H),2.94-2.93(m,8H),2.11(s,12H),2.00(s,16H),1.90(s,12H),1.78(s,12H),1.46-1.44(m ,8H),1.38-1.35(m,8H),1.26-1.24(m,8H),1.18-1.16(m,6H),1.09-0.99(m,2H).

MS m/z：C₉₆H₁₅₃N₁₁O₄₆，實測m/z：2197.5。 MS m _/ z: _{C96H153N11O46} _, _found m/z: 2197.5.

步驟八 step eight

將化合物3-k(100mg,45.5umol)加入DMF(1mL)中，再將化合物2-b(21.1mg,54umol)加入反應中，再加入HBTU(21.8mg,57.3umol)和DIEA((17.7mg,136umol)，保持15℃反應16h。反應完畢後，經二氯甲烷(10mL)稀釋反應液，並依次用飽和NaHCO₃和飽和食鹽水洗滌，有機相無水Na₂SO₄乾燥，過濾後減壓濃縮，殘餘物經過製備液相純化(管柱：Phenomenex Gemini-NX 150*30mm*5um；流動相：[水-ACN]；B%：35%-75%,12min)，得到化合物3-l。MS m/z：C₁₂₀H₁₇₅N₁₁O₅₁，實測：2586.9。 Compound 3-k (100mg, 45.5umol) was added in DMF (1mL), then compound 2-b (21.1mg, 54umol) was added to the reaction, then HBTU (21.8mg, 57.3umol) and DIEA ((17.7mg , 136umol), kept at 15°C for 16h. After the reaction was completed, the reaction solution was diluted with dichloromethane (10mL), washed with saturated NaHCO ₃ and saturated brine in turn, and the organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and depressurized After concentration, the residue was purified by preparative liquid phase (column: Phenomenex Gemini-NX 150*30mm*5um; mobile phase: [water-ACN]; B%: 35%-75%, 12min) to obtain compound 3-1 . MS _m /z: _{C120H175N11O51} , found: _2586.9 _.

步驟八 step eight

將化合物3-l(14mg,5.4umol)和丁二酸酐(1.08mg,10.8umol)溶解於二氯甲烷溶液(1mL)中，向反應液中加入DMAP(2.0mg, 16umol)和TEA(1.1mg,10.8umol,1.5uL)，保持在15℃下攪拌16h。待反應完畢後，加入甲醇(0.9mg)，並攪拌反應10min，然後將反應液用二氯甲烷稀釋反應液，並用飽和NaHCO₃洗滌2次。將反應液減壓濃縮抽乾得到化合物3-m(18mg)。 Compound 3-l (14mg, 5.4umol) and succinic anhydride (1.08mg, 10.8umol) were dissolved in dichloromethane solution (1mL), DMAP (2.0mg, 16umol) and TEA (1.1mg , 10.8umol, 1.5uL), kept stirring at 15°C for 16h. After the reaction was completed, methanol (0.9 mg) was added, and the reaction was stirred for 10 min, then the reaction solution was diluted with dichloromethane and washed twice with saturated NaHCO ₃ . The reaction solution was concentrated under reduced pressure and sucked to dryness to obtain compound 3-m (18 mg).

MS m/z：C₁₂₄H₁₇₉N₁₁O⁵⁴，實測：2687.2。 MS _m /z: _{C124H179N11O54} ^, found: 2687.2 _.

步驟九 Step Nine

將上步得到的化合物3-m(18mg，6.7ummol)加入乙腈(3mL)，再加入HBTU(5.1mg，13.4umol)，加入表面胺基修飾的固相支撐物(CPG-NH2，200mg)，加入DIEA(4.3mg,33.5umol,5.8uL)，保持30℃震盪反應16h。反應完成後，過濾，並依次用甲醇(2mL x 4)、二氯甲烷(2mL x 4)洗滌。固體繼續加入吡啶：乙酸酐(v：v=4：1,2mL)中，繼續保持30℃震盪反應16h。反應完成後，過濾，並依次用甲醇、二氯甲烷洗滌。得到目標產物連接在固相載體上的化合物3-n 200mg。 Compound 3-m (18mg, 6.7ummol) obtained in the previous step was added to acetonitrile (3mL), then HBTU (5.1mg, 13.4ummol) was added, and the surface amine-modified solid phase support (CPG-NH2, 200mg) was added, Add DIEA (4.3mg, 33.5umol, 5.8uL) and keep shaking at 30°C for 16h. After the reaction was completed, it was filtered, and washed successively with methanol (2 mL x 4), dichloromethane (2 mL x 4). The solid continued to be added to pyridine:acetic anhydride (v:v=4:1, 2mL), and the reaction was kept at 30°C for 16h with shaking. After the reaction was completed, it was filtered and washed successively with methanol and dichloromethane. Obtained 200 mg of compound 3-n in which the target product was linked to a solid phase carrier.

實施例9 連接到固相載體上的胺基半乳糖化合物4-cExample 9 Galactosamine compound 4-c linked to a solid support

合成路線如下： The synthetic route is as follows:

1)化合物4-c的合成 1) Synthesis of compound 4-c

步驟一 step one

將化合物3-k(149.5mg,68ummol)、DIEA(141.0mg,1.09mmol)、3A分子篩(500mg)和DEPBT(163.4mg,0.55mmol)溶於5mL的DCM中，室溫下加入化合物1-q(400mg,0.18mmol)，在室溫下攪拌過夜。反應完畢後，將分子篩過濾掉，濾液旋乾並經反相製備HPLC(管柱：Boston Green ODS 150*30mm*5um，條件：5-50%(A：水，B：CH3CN)，流速：45mL/min)，凍乾後得到化合4-a(118mg，32ummol，收率為62.6%)。 Compound 3-k (149.5mg, 68ummol), DIEA (141.0mg, 1.09mmol), 3A molecular sieves (500mg) and DEPBT (163.4mg, 0.55mmol) were dissolved in 5mL of DCM, and compound 1-q was added at room temperature (400mg, 0.18mmol), stirred overnight at room temperature. After the reaction is completed, the molecular sieves are filtered off, and the filtrate is spin-dried and subjected to reverse-phase preparative HPLC (column: Boston Green ODS 150*30mm*5um, condition: 5-50% (A: water, B: CH3CN), flow rate: 45mL /min), to obtain compound 4-a (118mg, 32ummol, yield 62.6%) after lyophilization.

MS m/z：C₁₂₈H₁₈₈N₁₂O₅₂，實測[M+HCOO^-]=2770.6。 MS m/z: C ₁₂₈ H ₁₈₈ N ₁₂ O ₅₂ , found [M+HCOO ⁻ ] = 2770.6.

步驟二 step two

將化合物4-a(110mg,4.0umol)，DMAP(7.4mg,40umol),3A分子篩(100mg)和丁二酸酐(11.9mg,120umol)溶於5mL的THF中，氬氣保護，40℃下攪拌4h。反應完畢後，將分子篩過濾掉，濾液旋乾並經反相製備HPLC純化(管柱：Boston Green ODS 150*30mm*5um，條件：5-50%(A：水，B：CH₃CN)，流速：45mL/min)，凍乾後得到化合物4-b(80mg,28.3ummol，收率為70.8%)。 Compound 4-a (110mg, 4.0umol), DMAP (7.4mg, 40umol), 3A molecular sieves (100mg) and succinic anhydride (11.9mg, 120umol) were dissolved in 5mL of THF, protected by argon, and stirred at 40°C 4h. After the reaction, the molecular sieves were filtered off, the filtrate was spin-dried and purified by reverse-phase preparative HPLC (column: Boston Green ODS 150*30mm*5um, condition: 5-50% (A: water, B: CH ₃ CN), Flow rate: 45mL/min), compound 4-b (80mg, 28.3ummol, yield 70.8%) was obtained after lyophilization.

MS m/z：C₁₃₂H₁₉₂N₁₂O₅₅，[M-H]⁺實測：2824.6。 _MS m/z: _{C132H192N12O55} _, [MH] ⁺ found: _2824.6 .

步驟三 step three

將上步得到的化合物38(71mg，25ummol)加入乙腈(5mL)，再加入HBTU(19.0mg，50umol)，加入表面胺基修飾的固相支撐物(CPG-NH2，0.86g)，加入DIEA(16.2mg,125umol,21.6uL)，保持30℃震盪反應16h。反應完成後，過濾，並依次用甲醇(5mL x 4)、二氯甲烷(5mL x 4)洗滌。固體繼續加入吡啶：乙酸酐(v：v=4：1,6.0mL)中，繼續保持30℃震盪反應16h。反應完成後，過濾，並依次用甲醇、二氯甲烷洗滌。得到連接在固相載體上的化合物4-c 0.74g。 The compound 38 (71mg, 25ummol) obtained in the previous step was added to acetonitrile (5mL), then HBTU (19.0mg, 50umol) was added, and the surface amine-modified solid phase support (CPG-NH2, 0.86g) was added, and DIEA ( 16.2mg, 125umol, 21.6uL), keep shaking at 30°C for 16h. After the reaction was completed, it was filtered and washed with methanol (5 mL x 4), dichloromethane (5 mL x 4) successively. The solid was continued to be added to pyridine:acetic anhydride (v:v=4:1, 6.0mL), and the reaction was kept at 30°C for 16h with shaking. After the reaction was completed, it was filtered and washed successively with methanol and dichloromethane. Obtained 0.74 g of compound 4-c attached to the solid support.

實施例10 對照化合物L96的製備Example 10 Preparation of Reference Compound L96

按照專利WO2014025805A1所述的方法製備了對照化合物。按照上述的連接固相載體同樣的方法，連接到CPG固相載體上，得到化合物L96。 A reference compound was prepared according to the method described in patent WO2014025805A1. According to the same method as above for connecting the solid phase support, it was connected to the CPG solid phase support to obtain compound L96.

實施例11 合成胺基半乳糖分子簇綴合的siRNAExample 11 Synthesis of siRNA conjugated to galactosamine clusters

用於測試的siRNA，靶向小鼠TTR基因mRNA的siRNA(Molecular Therapy Vol.26 No 3 March 2018)如下，在SS股的3’末端藉由共價鍵連接胺基半乳糖分子簇M， The siRNA used for the test, the siRNA targeting mouse TTR gene mRNA (Molecular Therapy Vol.26 No 3 March 2018) is as follows, the 3' end of the SS strand is covalently linked to the galactosamine cluster M,

SS股(5'-3‘)：CmsAmsGm UmGfUm UfCfUf UmGmCm UmCmUm AmUmAm Am-M(SEQ ID NO：88) SS strand (5'-3'): CmsAmsGm UmGfUm UfCfUf UmGmCm UmCmUm AmUmAm Am-M (SEQ ID NO: 88)

AS股(5'-3’)：UmsUfsAm UmAmGf AmGmCm AmAmGm AmAfCm AfCmUm GmsUmsUm(SEQ ID NO：89) AS strand (5'-3'): UmsUfsAm UmAmGf AmGmCm AmAmGm AmAfCm AfCmUm GmsUmsUm (SEQ ID NO: 89)

siRNA的合成與通常的亞磷醯胺固相合成法無異，所不同的是在合成siRNA的SS股時，使用上述所合成的連接有胺基半乳糖簇的CPG載體代替通常的Universal-CPG載體。簡要描述如下：於Dr.Oligo48合成器(Biolytic)上，以上述合成的胺基半乳糖連接的CPG載體為起始，根據合成程序逐個連接核苷亞磷醯胺單體。核苷單體原料2’-F RNA、2’-O-甲基RNA等核苷亞磷醯胺單體購自上海兆維或蘇州吉瑪。採用5-乙基硫-1H-四唑(ETT)作為活化劑(0.6M乙腈溶液)，使用0.22M的PADS溶於1：1體積比的乙腈和三甲基吡啶(蘇州柯樂瑪)溶液作為硫化試劑，使用碘吡啶/水溶液(柯樂瑪)作為氧化劑。 The synthesis of siRNA is the same as the usual phosphoramidite solid-phase synthesis method, the difference is that when synthesizing the SS strand of siRNA, the CPG carrier synthesized above is used instead of the usual Universal-CPG carrier. A brief description is as follows: On the Dr. Oligo48 synthesizer (Biolytic), starting from the galactosamine-linked CPG carrier synthesized above, the nucleoside phosphoramidite monomers were linked one by one according to the synthesis procedure. Nucleoside monomer raw materials 2'-F RNA, 2'-O-methyl RNA and other nucleoside phosphoramidite monomers were purchased from Shanghai Zhaowei or Suzhou Jima. Using 5-ethylthio-1H-tetrazole (ETT) as the activator (0.6M acetonitrile solution), using 0.22M PADS dissolved in acetonitrile and collidine (Suzhou Kelema) solution with a volume ratio of 1:1 As a vulcanizing agent, iodopyridine/water solution (Koroma) was used as an oxidizing agent.

所得到的單股寡核苷酸，根據等莫耳比，按照互補配對，與AS股退火，最後所得到的雙股siRNA溶於1X PBS中，並調整至實驗所需濃度。 The obtained single-stranded oligonucleotides were annealed with the AS strand according to the equimolar ratio and complementary pairing, and finally the obtained double-stranded siRNA was dissolved in 1X PBS and adjusted to the concentration required for the experiment.

合成胺基半乳糖簇綴合的siRNA，實驗所用siRNA靶向小鼠TTR mRNA。 Synthesis of galactosamine cluster-conjugated siRNA targeting mouse TTR mRNA.

M= M =

實施例12 胺基半乳糖分子簇綴合的siRNA在肝原代細胞對mRNA表達的抑制Example 12 Inhibition of mRNA expression by siRNA conjugated to galactosamine clusters in primary liver cells

參照Severgini等報導的方法(Cytotechnology.2012；64(2)：187-195.)分離獲得新鮮小鼠原代肝細胞。 According to the method reported by Severgini et al. (Cytotechnology. 2012; 64(2): 187-195.), fresh mouse primary hepatocytes were isolated and obtained.

原代肝細胞分離後，按照10萬每孔接種於24孔板中，按照終濃度為50nM，10nM，2nM，0.4nM，0.08nM，0.016nM，0.0032nM，0.00064nM分別加入待測siRNA。隨後，將原代肝細胞置於37℃，5% CO₂的環境中培養24h。24h後，採用qPCR方法檢測mTTR的mRNA表達水平。 After the primary hepatocytes were isolated, they were inoculated in 24-well plates according to 100,000 per well, and the siRNA to be tested was added at the final concentrations of 50nM, 10nM, 2nM, 0.4nM, 0.08nM, 0.016nM, 0.0032nM, and 0.00064nM. Subsequently, the primary hepatocytes were cultured at 37°C and 5% CO ₂ for 24 hours. After 24 hours, the mRNA expression level of mTTR was detected by qPCR method.

如圖1所示，S-1、S-2、S-3、S-4均表現了優秀的mTTR基因表達抑制效率。S-1及S-4的IC50值低於其他兩組，與對照組S-L96的IC50值0.280nM相比，S-1的IC50值為0.131nM，S-4的IC50值為0.135nM，表明綴合S-1及S-4化合物的siRNA體外被原代肝細胞自由攝取的效率優於對照組，S-1及S-4化合物能更高效介導siRNA進入原代肝細胞。 As shown in Figure 1, S-1, S-2, S-3, and S-4 all showed excellent mTTR gene expression inhibition efficiency. The IC50 values of S-1 and S-4 were lower than those of the other two groups. Compared with the IC50 value of S-L96 in the control group of 0.280nM, the IC50 value of S-1 was 0.131nM, and the IC50 value of S-4 was 0.135nM. It shows that the siRNA conjugated with S-1 and S-4 compounds is more efficiently taken up by primary hepatocytes in vitro than the control group, and S-1 and S-4 compounds can mediate siRNA into primary hepatocytes more efficiently.

實施例13 胺基半乳糖分子簇綴合的siRNA在體內對mRNA表達的抑制Example 13 Inhibition of mRNA expression by siRNA conjugated to galactosamine clusters in vivo

使用8週齡的C57BL/6小鼠(昭衍生物、SPF級，雌性)，採用上述的siRNA經皮下注射遞送。在第1天，在小鼠肩頸部的鬆弛皮膚上，給予100μl溶液的皮下注射，其含有PBS或PBS配置的1mg/kg(mpk)， 0.2mpk劑量的對應的siRNA(S-L96、S-3、S-2、S-4或S-1)。各個組別注射6隻小鼠。 8-week-old C57BL/6 mice (Zhao derivative, SPF grade, female) were used to deliver the above-mentioned siRNA by subcutaneous injection. On day 1, a subcutaneous injection of 100 μl of a solution containing 1 mg/kg (mpk) in PBS or in PBS was given on the loose skin of the shoulder and neck of the mouse, The corresponding siRNA (S-L96, S-3, S-2, S-4 or S-1) at a dose of 0.2 mpk. Each group injected 6 mice.

在給藥3天後，脫頸處死小鼠，使用qPCR檢測小鼠肝組織mTTR的mRNA表達水平。 After 3 days of administration, the mice were killed by dislocation, and the mRNA expression level of mTTR in mouse liver tissue was detected by qPCR.

如圖2所示，S-1、S-2、S-3、S-4均表現了優秀的mTTR基因表達抑制效率。其中，S-2、S-3、S-4與對照組S-L96相比，在1mpk及0.2mpk的活性相近。S-1在1mpk及0.2mpk的活性水平優於對照組S-L96。 As shown in Figure 2, S-1, S-2, S-3, and S-4 all exhibited excellent mTTR gene expression inhibition efficiency. Among them, compared with the control group S-L96, S-2, S-3 and S-4 had similar activities at 1mpk and 0.2mpk. The activity level of S-1 at 1mpk and 0.2mpk was better than that of the control group S-L96.

三、siRNA、siRNA綴合物的製備和活性評價3. Preparation and activity evaluation of siRNA and siRNA conjugates

實施例14 合成siRNA綴合物Example 14 Synthesis of siRNA conjugates

藉由固相亞磷醯胺法，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。正義股和反義股採用相同的合成條件。 By the solid-phase phosphoramidite method, the nucleoside monomers are connected one by one from the 3'-5' direction according to the nucleotide arrangement sequence. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. Sense and antisense shares were subjected to the same synthesis conditions.

寡核酸合成儀器設備型號：Biolytic Dr.Oligo 48寡核酸固相合成儀，GE oligo pilot100寡核酸固相合成儀。 Oligo nucleic acid synthesis instrument equipment model: Biolytic Dr.Oligo 48 oligo nucleic acid solid phase synthesizer, GE oligo pilot100 oligo nucleic acid solid phase synthesizer.

以Biolytic Dr.Oligo 48寡核酸固相合成儀為例，合成條件給定如下： Taking the Biolytic Dr.Oligo 48 oligonucleotide solid-phase synthesizer as an example, the synthesis conditions are given as follows:

核苷單體以0.05M濃度的乙腈溶液提供，每一步的脫保護反應的條件相同，即溫度為25度，反應時間為3分鐘，脫保護試劑為DCA，進樣體積180μL。 Nucleoside monomers were provided in acetonitrile solution with a concentration of 0.05M. The deprotection reaction conditions of each step were the same, that is, the temperature was 25 degrees, the reaction time was 3 minutes, the deprotection reagent was DCA, and the injection volume was 180 μL.

每一步偶聯反應條件均相同，包括溫度為25度，反應時間為3分鐘。核苷單體進樣體積90μL，催化劑ACT進樣體積110μL。 The coupling reaction conditions of each step are the same, including a temperature of 25 degrees and a reaction time of 3 minutes. The injection volume of nucleoside monomer is 90 μL, and the injection volume of catalyst ACT is 110 μL.

每一步蓋帽條件均相同，包括溫度為25度，反應時間為2分鐘。蓋帽試劑溶液的莫耳比為1：1的CapA和CapB(CapB1：CapB2=1：1)的混合溶液。蓋帽試劑進樣體積180μL。 The capping conditions in each step are the same, including a temperature of 25 degrees and a reaction time of 2 minutes. The capping reagent solution is a mixed solution of CapA and CapB (CapB1:CapB2=1:1) with a molar ratio of 1:1. The injection volume of capping reagent is 180 μL.

每一步氧化反應條件均相同，包括溫度為25度，反應時間為3分鐘，氧化試劑OXD進樣體積為180μL。 The oxidation reaction conditions for each step were the same, including a temperature of 25 degrees, a reaction time of 3 minutes, and an injection volume of oxidizing reagent OXD of 180 μL.

每一步硫化反應條件均相同，包括溫度為25度，反應時間為4分鐘，硫化試劑為0.05M PADS的吡啶乙腈溶液。硫化試劑進樣體積180μL。 The vulcanization reaction conditions of each step are the same, including a temperature of 25 degrees, a reaction time of 4 minutes, and a 0.05M PADS solution of pyridine in acetonitrile as the vulcanization reagent. The injection volume of the sulfide reagent was 180 μL.

2.待最後一個核苷單體連接完成後，依次對固相載體上連接的核酸序列進行切割、脫保護、純化、脫鹽，隨後凍乾獲得正義股和反義股，其中 2. After the connection of the last nucleoside monomer is completed, the nucleic acid sequences connected on the solid phase carrier are sequentially cut, deprotected, purified, and desalted, and then freeze-dried to obtain the sense strand and the antisense strand, wherein

2-1 切割和脫保護條件如下：將合成的連接有載體的核苷酸序列加入氨水：乙醇=3：1的混合溶液至體積為0.8mL。在50度反應15h，過濾除去剩餘載體，將上清液真空濃縮至乾。 2-1 The cleavage and deprotection conditions are as follows: Add the synthesized nucleotide sequence linked to the carrier to a mixed solution of ammonia:ethanol=3:1 to a volume of 0.8 mL. React at 50°C for 15 h, remove the remaining carrier by filtration, and concentrate the supernatant to dryness in vacuo.

2-2 純化和脫鹽條件如下：利用C18反相色譜管柱進行脫鹽。具體條件包括： 2-2 Purification and desalting conditions are as follows: use C18 reverse phase chromatographic column for desalting. Specific conditions include:

2-2-1 樣品的準備 2-2-1 Sample preparation

向寡核苷酸樣品中加入0.1M的TEAA(三乙胺醋酸鹽)至體積為0.8mL。 0.1 M TEAA (triethylamine acetate) was added to the oligonucleotide samples to a volume of 0.8 mL.

2-2-2 96孔板的活化 2-2-2 Activation of 96-well plate

活化：0.8mL乙腈藉由96孔板的每個孔中進行活化 Activation: 0.8mL acetonitrile is activated in each well of a 96-well plate

平衡：用0.8mL TEAA(pH 7.0)溶液進行96孔板的平衡 Equilibration: Equilibrate the 96-well plate with 0.8mL TEAA (pH 7.0) solution

2-2-3 純化過程依次按照如下操作 2-2-3 Purification process in sequence as follows

將0.8mL包含寡核苷酸的溶液藉由脫鹽管柱 Pass 0.8mL of the solution containing oligonucleotide through the desalting column

用0.8mL 6.5%氨水洗滌96孔板2次，去除失敗的序列 Wash the 96-well plate twice with 0.8mL 6.5% ammonia water to remove the failed sequences

用0.8mL去離子水沖洗96孔板2次，去除鹽分 Rinse the 96-well plate twice with 0.8mL deionized water to remove salt

用0.8mL 3%三氟乙酸沖洗96孔板3次，去除DMT，觀察到吸附層變橙紅色 Rinse the 96-well plate 3 times with 0.8mL 3% trifluoroacetic acid to remove DMT, and observe that the adsorption layer turns orange-red

用0.8mL 0.1M TEAA沖洗96孔板 Rinse the 96-well plate with 0.8mL 0.1M TEAA

用0.8mL去離子水沖洗96孔板2次，去除三氟乙酸和殘餘的鹽分 Rinse the 96-well plate twice with 0.8mL deionized water to remove trifluoroacetic acid and residual salt

用0.6mL 20%乙腈進行沖提，並收集凍乾 Elute with 0.6mL 20% acetonitrile, and collect and lyophilize

檢測方法如下：使用Waters Acquity UPLC-SQD2 LCMS(column：ACQUITY UPLC BEH C18)檢測上述正義股和反義股純度並分析分子量。實測值與理論值相符，表明所合成的是3’末端綴合了綴合分子的正義股以及反義股。 The detection method is as follows: use Waters Acquity UPLC-SQD2 LCMS (column: ACQUITY UPLC BEH C18) to detect the purity of the above-mentioned sense strand and antisense strand and analyze the molecular weight. The measured value is consistent with the theoretical value, indicating that the synthesized is the sense strand and the antisense strand conjugated with the conjugate molecule at the 3' end.

退火操作如下：將步驟2合成獲得的正義股和反義股分別溶於注射用水中，得到1000ng/μL的溶液，在QPCR儀上以等莫耳比混合，90度加熱10分鐘，每降5度保持3分鐘，最後25度保持10分鐘，使它們藉由氫鍵形成雙股結構。實測值與理論值相符，說明所合成的siRNA綴合物是目標設計的帶有綴合分子的雙股核酸序列。該siRNA具有表16中所示的正義股和反義股。 The annealing operation is as follows: Dissolve the sense strand and antisense strand synthesized in Step 2 in water for injection to obtain a 1000 ng/μL solution, mix them in an equimolar ratio on a QPCR instrument, heat at 90°C for 10 minutes, and drop by 5 ℃ for 3 minutes, and finally 25 degrees for 10 minutes, so that they form a double-strand structure through hydrogen bonding. The measured value is consistent with the theoretical value, indicating that the synthesized siRNA conjugate is a double-stranded nucleic acid sequence with a conjugate molecule designed in the target. This siRNA has the sense and antisense strands shown in Table 16.

(-)hmpNA(G)為實施例1.6節中核苷亞磷醯胺單體1-6a藉由固相合成獲得； (-)hmpNA(G) is obtained by solid-phase synthesis of the nucleoside phosphoramidite monomer 1-6a in Example 1.6;

(-)hmpNA(C)為實施例1.8節中核苷亞磷醯胺單體1-8a藉由固相合成獲得； (-)hmpNA(C) is obtained by solid-phase synthesis of the nucleoside phosphoramidite monomer 1-8a in Example 1.8;

(-)hmpNA(U)為實施例1.7節中核苷亞磷醯胺單體1-7a藉由固相合成獲得； (-)hmpNA(U) is obtained by solid-phase synthesis of the nucleoside phosphoramidite monomer 1-7a in Example 1.7;

表16中，NAG1結構為(靶向部分為N-乙醯基-半乳糖胺)： In Table 16, the structure of NAG1 is (the targeting part is N-acetyl-galactosamine):

實施例15 siRNA序列活性及脫靶水平驗證Example 15 Verification of siRNA sequence activity and off-target level

在HEK293A細胞中對本揭露化合物進行體外分子水平模擬在靶及脫靶水平篩選。 In HEK293A cells, the compounds of the present disclosure were screened at the on-target and off-target levels by in vitro molecular simulation.

分別構建siRNA序列對應的在靶序列和脫靶序列，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，siRNA的靶序列插入到海腎螢光素酶基因的3’UTR區域，siRNA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 on-target sequence and off-target sequence corresponding to the siRNA sequence were respectively constructed and inserted into the psiCHECK-2 plasmid. The plasmid contains Renilla luciferase gene and Firefly luciferase gene. As a dual reporter gene system, the target sequence of siRNA is inserted into the 3'UTR region of Renilla luciferase gene, and the activity of siRNA to the target sequence can be expressed by Renilla luciferase calibrated by firefly luciferase To reflect the detection of the situation, the detection uses the Dual-Luciferase Reporter Assay System (Promega, E2940).

siRNA序列對應的靶標質粒構建規則如下： The target plasmid construction rules corresponding to the siRNA sequence are as follows:

針對siRNA的反義股，分別構建與AS股完全互補的在靶質粒；構建與反義股5’端1-8位完全互補，而其它位置的鹼基完全不匹配的脫靶質粒，鹼基錯配對應規則為A與C互配、G與T互配。 For the antisense strands of siRNA, construct on-target plasmids that are completely complementary to the AS strands; construct off-target plasmids that are completely complementary to the 1-8 positions of the 5' end of the antisense strands, but the bases at other positions are completely mismatched, and the bases are wrong. The pairing rules are A and C, and G and T.

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1萬個細胞，每孔100μL培養基。 HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO ₂ . 24 hours before transfection, HEK293A cells were seeded in a 96-well plate at a seeding density of 10,000 cells per well and 100 μL of medium per well.

按照說明書，使用Lipofectamine2000(ThermoFisher，11668019)對細胞共轉染siRNA及對應質粒，每孔使用0.2μL Lipofectamine2000。質粒轉染量為10ng每孔。對於在靶序列質粒和脫靶序列質粒，siRNA共設置5個濃度點或11個濃度點。5個濃度點條件下，轉染時最高濃度點為10nM，10倍梯度稀釋；11個濃度點條件下，轉染時最高濃度點終濃度為20nM，3倍梯度稀釋。轉染後24h，採用Dual-Luciferase Reporter Assay System(Promega，E2940)檢測脫靶水平。 According to the instructions, Lipofectamine2000 (ThermoFisher, 11668019) was used to co-transfect the cells with siRNA and the corresponding plasmid, using 0.2 μL Lipofectamine2000 per well. The amount of plasmid transfection was 10ng per well. For the on-target sequence plasmid and the off-target sequence plasmid, siRNA was set at 5 concentration points or 11 concentration points. Under the condition of 5 concentration points, the highest concentration point during transfection is 10nM, 10-fold gradient dilution; under the condition of 11 concentration points, the final concentration of the highest concentration point during transfection is 20nM, 3-fold gradient dilution. 24h after transfection, the off-target level was detected by Dual-Luciferase Reporter Assay System (Promega, E2940).

表17-表20結果表明，本揭露的化合物TRD006890和TRD006924具有高的在靶活性的同時，還具有低脫靶性，均顯著優於陽性對照AD81890。 The results in Table 17-Table 20 show that the disclosed compounds TRD006890 and TRD006924 have high on-target activity and low off-target activity, both of which are significantly better than the positive control AD81890.

表21結果表明，GNA修飾具有明顯的序列位點依賴性。當GNA修飾位點在AS股5’第7位(TRD006912)或第6位時(AD81890)，脫靶活性均較高，預示著毒性較大。此外，TRD006912相對於AD81890，在靶活性進一步降低(0.68降低為0.96)。 The results in Table 21 show that GNA modification has obvious sequence-site dependence. When the GNA modification site was at position 7 (TRD006912) or position 6 (AD81890) of AS strand 5', the off-target activity was higher, indicating greater toxicity. In addition, compared with AD81890, the on-target activity of TRD006912 was further reduced (from 0.68 to 0.96).

實施例16 應用HepG2.2.15細胞評價siRNA化合物體外抗HBV活性Example 16 Application of HepG2.2.15 cells to evaluate the anti-HBV activity of siRNA compounds in vitro

第1天種HepG2.2.15細胞到96孔板，每孔2萬個細胞。種細胞同時用RNAiMax將不同濃度的siRNA轉入HepG2.2.15細胞；第4天收集細胞培養上清，ELISA檢測HBsAg(剩餘上清凍存備用)。最後收集細胞，提取細胞內RNA，RT-PCR分別檢測總HBV RNA(包括3.5kb+2.4kb+2.1kb+0.7kb RNA)和3.5kb HBV RNA(包括pgRNA+preCore RNA)，同時檢測GAPDH基因RNA作為內參。待測化合物為5個濃度點，平行測定2複孔。培養液中DMSO的終濃度為0.5%。 On day 1, HepG2.2.15 cells were seeded into 96-well plates with 20,000 cells per well. At the same time, RNAiMax was used to transfer different concentrations of siRNA into HepG2.2.15 cells; the cell culture supernatant was collected on the 4th day, and HBsAg was detected by ELISA (the remaining supernatant was frozen for future use). Finally, cells were collected, intracellular RNA was extracted, and total HBV RNA (including 3.5kb+2.4kb+2.1kb+0.7kb RNA) and 3.5kb HBV RNA (including pgRNA+preCore RNA) were detected by RT-PCR, while GAPDH gene RNA was detected as an internal reference. The compounds to be tested are 5 concentration points, and 2 replicate wells are measured in parallel. The final concentration of DMSO in the culture medium was 0.5%.

抑制百分比計算公式如下： The formula for calculating percentage inhibition is as follows:

% HBsAg抑制率=(1-樣品中HBsAg含量/DMSO對照組中HBsAg含量)×100 % HBsAg inhibition rate=(1-HBsAg content in sample/HBsAg content in DMSO control group)×100

% HBV RNA抑制率=(1-樣品中HBV的RNA含量/DMSO對照組中HBV的RNA含量)×100 % HBV RNA inhibition rate=(1-HBV RNA content in sample/HBV RNA content in DMSO control group)×100

%細胞活力=(樣品吸光值-培養液對照的吸光值)/(DMSO對照的吸光值-培養液對照的吸光值)×100。 % cell viability=(absorbance value of sample-absorbance value of culture medium control)/(absorbance value of DMSO control-absorbance value of culture medium control)×100.

應用Graphpad Prism軟體分析(four parameter logistic equations)計算EC50值。 EC50 values were calculated using Graphpad Prism software analysis (four parameter logistic equations).

如表22所示，參比對照化合物AD66810及AD81890，綜合抗病毒活性檢測指標，測試化合物TRD006890、TRD006894、TRD006895、TRD006896、TRD006897、TRD006905、TRD006906、TRD006907和TRD006908在HepG2.2.15細胞上展現出優秀的抗病毒活性。 As shown in Table 22, with reference to the control compounds AD66810 and AD81890, comprehensive antiviral activity detection indicators, the test compounds TRD006890, TRD006894, TRD006895, TRD006896, TRD006897, TRD006905, TRD006906, TRD006907 and TRD006908 showed excellent performance on HepG2.2.15 cells Antiviral activity.

實施例17 應用人原代肝細胞評價siRNA化合物體外抗HBV感染活性Example 17 Application of human primary hepatocytes to evaluate the in vitro anti-HBV infection activity of siRNA compounds

第0天種人原代肝細胞到48孔板，每孔細胞數量為12萬個。種細胞同時加入待測化合物，採用自由攝取的方式將siRNA化合物轉入人原代肝細胞，siRNA化合物起始濃度為200nM，5倍稀釋7個濃度。第1天，加入D型HBV感染人原代肝細胞。第2、4和6天，更換新鮮培養基，培養液中DMSO的終濃度為2%。第8天，收集細胞培養上清，qPCR檢測HBV DNA，ELISA檢測HBeAg和HBsAg。待測化合物和對照化合物均7個濃度點，平行測定2複孔。 On day 0, primary human hepatocytes were seeded into 48-well plates, and the number of cells per well was 120,000. The test compound was added to the cells at the same time, and the siRNA compound was transferred into primary human hepatocytes by free uptake. The initial concentration of the siRNA compound was 200 nM, and 7 concentrations were diluted 5 times. On day 1, primary human hepatocytes were infected with type D HBV. On the 2nd, 4th and 6th day, the fresh medium was replaced, and the final concentration of DMSO in the culture medium was 2%. On day 8, the cell culture supernatant was collected, and HBV DNA was detected by qPCR, and HBeAg and HBsAg were detected by ELISA. The compounds to be tested and the control compounds were tested at 7 concentration points, and 2 replicate wells were measured in parallel.

如表23所示，參比對照化合物AD81890，綜合抗病毒活性檢測指標，測試化合物TRD006924在人原代肝細胞上展現出顯著性更優的抗病毒活性。 As shown in Table 23, the test compound TRD006924 exhibited significantly better antiviral activity on primary human hepatocytes compared with the control compound AD81890 and comprehensive antiviral activity detection indicators.

實施例18 siRNA化合物體內抗HBV活性Example 18 siRNA compound anti-HBV activity in vivo

第28天，小鼠(C57BL/6，雄性)經尾靜脈注射rAAV8-1.3HBV。病毒注射後第14天和第21天，所有實驗小鼠藉由頜下靜脈採血用於收集血漿。血漿用於測試HBV DNA、HBeAg和HBsAg含量。 On day 28, mice (C57BL/6, male) were injected with rAAV8-1.3HBV via tail vein. On day 14 and day 21 after virus injection, all experimental mice were blood-collected through the submandibular vein for plasma collection. Plasma was used to test HBV DNA, HBeAg and HBsAg levels.

病毒注射後第28天，根據病毒注射後第14天和第21天血漿樣品的測試結果，將小鼠隨機分組。 On day 28 after virus injection, mice were randomly divided into groups according to the test results of plasma samples on days 14 and 21 after virus injection.

所有小鼠在病毒注射後第28天開始皮下給藥，給藥1次，給藥劑量為3mg/kg。給藥前所有小鼠頜下採血收集血漿，用於檢測HBV DNA、HBeAg，HBsAg和ALT。給藥當天為第0天。給藥後第7天、14天、21天所有小鼠頜下採血用於收集血漿，用於檢測。 All mice were administered subcutaneously on the 28th day after virus injection, and the administration dose was 3 mg/kg once. Before the administration, the submandibular blood of all mice was collected to collect plasma for the detection of HBV DNA, HBeAg, HBsAg and ALT. The day of administration was defined as day 0. On the 7th, 14th, and 21st days after administration, submandibular blood was collected from all mice for plasma collection and detection.

應用qPCR定量血漿HBV DNA。應用ELISA定量血漿中HBeAg和HBsAg。 Plasma HBV DNA was quantified by qPCR. HBeAg and HBsAg in plasma were quantified by ELISA.

“抑制率”與“表達剩餘百分比”之間的計算轉換公式：表達剩餘百分比=100%-抑制率。 The calculation conversion formula between "inhibition rate" and "expression remaining percentage": expression remaining percentage=100%-inhibition rate.

第7天，參比對照化合物AD81890，測試化合物TRD006924在小鼠體內展現出優秀的抗病毒活性。測試化合物TRD006924在體內能夠維持較長時間的活性，在第14天、第21天均能夠有效抑制病毒活性。 On day 7, compared with the control compound AD81890, the test compound TRD006924 exhibited excellent antiviral activity in mice. The test compound TRD006924 can maintain the activity for a long time in vivo, and can effectively inhibit the virus activity on the 14th day and the 21st day.

實施例19. 評估AS股9位和10位不同修飾Example 19. Evaluation of different modifications at positions 9 and 10 of the AS strand

本實驗考察本揭露的不同位點2’-氟修飾的siRNA綴合物在體內對靶基因mRNA表達量的抑制效率。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測試綴合物(2個，TRD007047和TRD006870)、對比綴合物(TRD002218)以及PBS。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，siRNA綴合物給藥劑量(以siRNA的量計)為1mg/kg，給藥體積為5mL/kg。給藥7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。化合物信息參見表25，小鼠體內實驗化合物分組信息參見表26，引子參見表27。 This experiment investigates the inhibitory efficiency of siRNA conjugates modified with 2'-fluorine at different positions disclosed herein on the expression of target gene mRNA in vivo. Male 6-8 week-old C57BL/6 mice were randomly divided into 6 groups, 3 at each time point, and the test conjugates (2, TRD007047 and TRD006870) were administered to each group of mice, compared with Conjugate (TRD002218) and PBS. All animals were dosed according to their total body weight, and were administered once by subcutaneous injection. The dose of siRNA conjugate (based on the amount of siRNA) was 1 mg/kg, and the volume of administration was 5 mL/kg. After 7 days of administration, the mice were sacrificed, the liver was collected, and preserved with RNA later (Sigma Aldrich Company); then the liver tissue was homogenized with a tissue homogenizer, and then the tissue RNA extraction kit (Fanzhi Medical Technology, FG0412) was used according to the operation. The operation steps marked in the instruction manual were used to extract the total RNA of the liver tissue. The total RNA was reverse-transcribed into cDNA, and the expression of TTR mRNA in liver tissue was detected by real-time fluorescent quantitative PCR method. In the fluorescent quantitative PCR method, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference gene, and Taqman probe primers for TTR and GAPDH were used to detect the mRNA expression levels of TTR and GAPDH, respectively. See Table 25 for the compound information, see Table 26 for the grouping information of the experimental compounds in mice, and see Table 27 for the primer.

其中，NAG1的結構為

。 Among them, the structure of NAG1 is

.

給藥28天後，本揭露的不同位點氟修飾的siRNA綴合物的在體內對靶基因mRNA表達量的抑制效率見表28。參比陽性對照化合物TRD002218，不同位點氟修飾的siRNA化合物在給藥後28天對於TTR mRNA的表達抑制高於參比陽性化合物，兩種修飾方法均表現出高抑制效率且無顯著性差異，說明兩種修飾方法能夠介導更高效的siRNA抑制效率。 After 28 days of administration, the in vivo inhibition efficiency of the siRNA conjugates modified with fluorine at different positions on the target gene mRNA expression is shown in Table 28. With reference to the positive control compound TRD002218, the siRNA compounds modified with fluorine at different sites inhibited the expression of TTR mRNA 28 days after administration was higher than that of the reference positive compound. Both modification methods showed high inhibition efficiency without significant difference. It shows that the two modification methods can mediate more efficient siRNA inhibition efficiency.

TTR mRNA表達量按照如下等式計算： TTR mRNA expression was 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 test group/GAPDH mRNA expression in test group)/(TTR mRNA expression in control group/GAPDH mRNA expression in control group)] x 100%

雖然為了清楚的理解，已經借助於附圖和實例詳細描述了上述發明，但是描述和實例不應當解釋為限制本揭露的範圍。本文中引用的所有專利和科學文獻的公開內容藉由引用完整地清楚結合。 Although the foregoing invention has been described in detail by means of drawings and examples for clarity of understanding, the description and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

An siRNA comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises at least 15 contiguous nucleotides and is identical to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 7, SEQ ID NO: Any nucleotide sequence in ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 22 differs by no more than 3 nucleotides ; The antisense strand comprises at least 15 consecutive nucleotide sequences, and is identical to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO : 19. Any nucleotide sequence in SEQ ID NO: 21 and SEQ ID NO: 23 differs by no more than 3 nucleotides;

At least one nucleotide in the 2nd to 8th positions of the antisense strand comprises a chemical modification or a tautomer modification shown in formula (I):

Among them, _Q1 is

, Q ₂ is R ₂ ; or Q ₁ is R ₂ , Q ₂ is

;

Y is selected from O, NH and S;

Each X is independently selected from CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein R ₄ , R ₄ ', R ₅ are independently H, methyl, ethyl, n-propyl or Isopropyl;

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

Each J ₁ and J ₂ are independently H or methyl;

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;

_R is selected from H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl 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;

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;

Optionally, R ₁ and R ₂ are directly connected to form a ring;

B is a base;

Wherein, the chemical modification shown in the formula (I) or its tautomer modification is not

;

Preferably, when X is NH-CO, R ₁ is not H.

The siRNA as claimed in item 1, wherein the antisense strand comprises formula (I-1) or formula (I-2) at least one nucleotide position in the 2nd to 8th positions of its 5' region The indicated chemical modifications or their tautomeric modifications:

siRNA as described in claim item 2, wherein, in formula (I-1) and formula (I-2),

Y is O or NH; each X is independently selected from NH-CO, _CH and NH;

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

Each of J ₁ and J ₂ is independently H;

R ₁ is selected from H, methyl and CH ₂ OH;

R ₂ is selected from H, OH, NH ₂ , methyl and CH ₂ OH;

R ₃ is selected from H, OH, NH ₂ , methyl and CH ₂ OH;

Optionally, R ₁ and R ₂ are directly connected to form a ring;

B is a base;

Preferably, B is the base corresponding to the position from the 2nd to the 8th position of the antisense strand in its 5' region.

The siRNA as described in any one of claims 1 to 3, wherein the chemical modification shown in the formula (I) or its tautomer modification is selected from:

Preferably, the chemical modification represented by the formula (I) or its tautomeric modification is selected from:

More preferably, the chemical modification represented by the formula (I) or its tautomeric modification is selected from:

Further preferably, the chemical modification represented by the formula (I) or its tautomeric modification is selected from:

Wherein, B is a base;

The siRNA as described in any one of claims 1 to 4, wherein the antisense strand comprises the 5th, 6th or 7th position in its 5' region as defined in any one of claims 1 to 4 The chemical modification shown in formula (I) or its tautomer modification;

When the chemical modification shown in formula (I) or its tautomer modification is at the 5th position of its 5' region, B is the base corresponding to the position of the antisense strand at the 5th position of its 5' region;

When the chemical modification shown in formula (I) or its tautomer is modified at the 6th position of its 5' region, B is the base corresponding to the position of the antisense strand at the 6th position of its 5' region; or

When the chemical modification shown in formula (I) or its tautomer modification is at the 7th position of its 5' region, B is the base corresponding to the position of the antisense strand at the 7th position of its 5' region.

The siRNA as described in any one of claim items 1 to 5, wherein the sense strand nucleotide sequence comprises:

5'-GUG UGC ACU UCG CUU CAC C-3' (SEQ ID NO: 3); or,

5'-CUU UUG UCU UUG GGU AUA U-3' (SEQ ID NO: 5); or,

5'-UUA CCA AUU UUC UUU UGU U-3' (SEQ ID NO: 7); or,

5'-CGU GUG CAC UUC GCU UCA C-3' (SEQ ID NO: 9); or,

5'-UGU CUU UGG GUA UAC AUU U-3' (SEQ ID NO: 11); or,

5'-CUU UUG UCU UUG GGU AUA C-3' (SEQ ID NO: 13); or,

5'-UGC ACU UCG CUU CAC CUC U-3' (SEQ ID NO: 18); or,

5'-GCA CUU CGC UUC ACC UCU G-3' (SEQ ID NO: 20); or,

5'-GGC GCU GAA UCC UGC GGA C-3' (SEQ ID NO: 22).

The siRNA as described in any one of claim items 1 to 6, wherein the antisense strand nucleotide sequence comprises:

5'-AGU GAA W'CG AAG UGC ACA CGG-3' (SEQ ID NO: 215); or,

5'-AUA UAC W'CA AAG ACA AAA GAA-3' (SEQ ID NO: 216); or,

5'-AAC AAA W'GA AAA UUG GUA ACA-3' (SEQ ID NO: 217); or,

5'-AUG AAG W'GA AGU GCA CAC GGU-3' (SEQ ID NO: 218); or,

5'-AAA UGU W'UA CCC AAA GAC AAA-3' (SEQ ID NO: 219); or,

5'-AGA GGU W'AA GCG AAG UGC ACA-3' (SEQ ID NO: 220); or,

5'-UAG AGG W'GA AGC GAA GUG CAC-3' (SEQ ID NO: 221); or,

5'-AUC CGC W'GG AUU CAG CGC CGA-3'(SEQ ID NO: 222); Wherein, W' means containing the chemical compound shown in formula (I) as defined in any one of claims 1 to 4 Modified or tautomer-modified nucleotides.

siRNA as described in claim item 7, wherein

The W' contains a chemical modification or a tautomeric modification thereof selected from:

,

or

;in;

When the antisense strand nucleotide sequence comprises: 5'-AGU GAA W'CG AAG UGC ACA CGG-3' (SEQ ID NO: 120), the B is guanine; or,

When the antisense strand nucleotide sequence comprises: 5'-AUA UAC W'CA AAG ACA AAA GAA-3' (SEQ ID NO: 121), the B is cytosine; or,

When the antisense strand nucleotide sequence comprises: 5'-AAC AAA W'GA AAA UUG GUA ACA-3' (SEQ ID NO: 122), the B is adenine; or,

When the antisense strand nucleotide sequence comprises: 5'-AUG AAG W'GA AGU GCA CAC GGU-3' (SEQ ID NO: 123), the B is cytosine; or,

When the antisense strand nucleotide sequence comprises: 5'-AAA UGU W'UA CCC AAA GAC AAA-3' (SEQ ID NO: 124), the B is adenine; or,

When the antisense strand nucleotide sequence comprises: 5'-AGA GGU W'AA GCG AAG UGC ACA-3' (SEQ ID NO: 127), the B is guanine; or,

When the antisense strand nucleotide sequence comprises: 5'-UAG AGG W'GA AGC GAA GUG CAC-3' (SEQ ID NO: 128), the B is uracil; or,

When the antisense strand nucleotide sequence comprises: 5'-AUC CGC W'GG AUU CAG CGC CGA-3' (SEQ ID NO: 129), the B is adenine.

The siRNA as described in any one of claim items 1 to 8, wherein in addition to the chemical modification shown in formula (I) or its tautomer modification as defined in any one of claim items 1 to 5, the siRNA At least one additional nucleotide in the sense strand and/or the antisense strand is a modified nucleotide;

Preferably, the modified nucleotides are independently selected from: 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified Nucleotides, 2'-Substituted Alkyl Modified Nucleotides, 2'-Amine Modified Nucleotides, 2'-Substituted Amine Modified Nucleotides, 2'-Fluoro Modified Nucleotides nucleotides, 2'-deoxynucleotides, 2'-deoxy-2'-fluoro-modified nucleotides, 3'-deoxy-thymidine nucleotides, isonucleotides, LNA, ENA, cET, UNA and GNA;

More preferably, the modified nucleotides are independently selected from: 2'-methoxy-modified nucleotides and 2'-fluoro-modified nucleotides.

The siRNA as described in claim item 9, wherein according to the direction from the 5' end to the 3' end, the nucleotides at positions 2, 4, 6, 9, 12, 14, 16 and 18 of the antisense strand are each independently 2'-fluoro-modified nucleotides;

Alternatively, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 of the antisense strand are each independently a 2'-fluoro-modified nucleoside according to the direction from the 5' end to the 3' end acid.

The siRNA as described in any one of claim items 1 to 10, wherein the sense strand has a nucleotide sequence as shown in 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'; and/or

The antisense strand has the nucleotide sequence shown in the following formula:

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

Wherein, N _a is a 2'-methoxy modified nucleotide, N _b is a 2'-fluoro modified nucleotide; and/or N _a ' is a 2'-methoxy modified nucleotide, N _b ' is a 2'-fluoro-modified nucleotide;

W' represents the nucleotide containing the chemical modification shown in formula (I) as defined in any one of claim items 1 to 4 or its tautomer modification;

Preferably, the chemical modification or tautomer modification contained in W' is selected from:

,

or

; Wherein, B is selected from guanine, adenine, cytosine or uracil, preferably selected from the base corresponding to the 7th position of the antisense strand in its 5' region.

5'-N _a N _a N _a N _a N _b 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'; and/or

,

or

The siRNA according to any one of claims 1 to 12, wherein at least one phosphate group in the sense strand and/or antisense strand is a phosphate group with a modification group, preferably a phosphorothioate group.

A siRNA conjugate comprising the siRNA as described in any one of claims 1 to 13 and a targeting ligand connected to the end of the siRNA;

Preferably, the targeting ligand is attached to the 3' end of the sense strand of the siRNA.

The siRNA conjugate as claimed in claim 14, wherein the targeting ligand comprises a targeting moiety T, and T is selected from galactose, galactosamine, N-formyl-galactosamine, N-acetyl- Galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine and N-isobutyryl-galactosamine, preferably, T is selected from N-acetyl-galactosamine.

A pharmaceutical composition comprising the siRNA according to any one of claims 1 to 13 or the siRNA conjugate according to claim 14 or 15, and a pharmaceutically acceptable carrier.

A siRNA as described in any one of claims 1 to 13 or an siRNA conjugate as described in claim 14 or 15, or a pharmaceutical composition as described in claim 16 during preparation Use in a medicament for treating and/or preventing hepatitis B virus infection or hepatitis B virus-related diseases in a subject;

Preferably, the hepatitis B virus-related diseases are acute hepatitis B, chronic hepatitis B, hepatitis D virus infection, hepatitis D, liver fibrosis, advanced liver disease and hepatocellular carcinoma, and the subject is HBeAg positive or HBeAg negative.

A method for inhibiting hepatitis B virus gene expression and/or replication, the method comprising administering an effective amount or an effective dose of the siRNA as described in any one of claim items 1 to 13 or as described in claim item 14 or 15 siRNA conjugate, or the pharmaceutical composition as described in Claim 16; preferably, the hepatitis B virus gene is HBV-X and/or HBV-S.

A method for in vivo delivery of siRNA that inhibits hepatitis B virus gene expression and/or replication to the liver, the method comprising administering to a subject the siRNA conjugate as described in Claim 14 or 15, or as described in Claim 16 The pharmaceutical composition; preferably, the hepatitis B virus gene is HBV-X and/or HBV-S.

A method for preparing siRNA or siRNA conjugates, comprising: synthesizing siRNA as described in any one of claims 1 to 13 or siRNA conjugates as described in claim 14 or 15; and purifying the siRNA or the siRNA conjugates.

An siRNA comprising a sense strand and an antisense strand selected from any of the following groups:

Group 1, sense strand: 5'-GUG UGC ACU UCG CUU CAC C-3' (SEQ ID NO: 3); antisense strand: 5'-AGU GAA GCG AAG UGC ACA CGG (SEQ ID NO: 4);

Group 2, sense strand: 5'-CUU UUG UCU UUG GGU AUA U-3' (SEQ ID NO: 5); antisense strand: 5'-AUA UAC CCA AAG ACA AAA GAA-3' (SEQ ID NO: 6 );

Group 3, sense strand: 5'-UUA CCA AUU UUC UUU UGU U-3' (SEQ ID NO: 7); antisense strand: 5'-AAC AAA AGA AAA UUG GUA ACA-3' (SEQ ID NO: 8 );

Group 4, sense strand: 5'-CGU GUG CAC UUC GCU UCA C-3' (SEQ ID NO: 9); antisense strand: 5'-AUG AAG CGA AGU GCA CAC GGU-3' (SEQ ID NO: 10 );

Group 5, sense strand: 5'-UGU CUU UGG GUA UAC AUU U-3' (SEQ ID NO: 11); antisense strand: 5'-AAA UGU AUA CCC AAA GAC AAA-3' (SEQ ID NO: 12 );

Group 6, sense strand: 5'-CUU UUG UCU UUG GGU AUA C-3' (SEQ ID NO: 13); antisense strand: 5'-AUA UAC CCA AAG ACA AAA GAA-3' (SEQ ID NO: 6 );

Group 7, sense strand: 5'-UGC ACU UCG CUU CAC CUC U-3' (SEQ ID NO: 18); antisense strand: 5'-AGA GGU GAA GCG AAG UGC ACA-3' (SEQ ID NO: 19 );

Group 8, sense strand: 5'-GCA CUU CGC UUC ACC UCU G-3' (SEQ ID NO: 20); antisense strand: 5'-UAG AGG UGA AGC GAA GUG CAC-3' (SEQ ID NO: 21 );

Group 9, sense strand: 5'-GGC GCU GAA UCC UGC GGA C-3' (SEQ ID NO: 22); antisense strand: 5'-AUC CGC AGG AUU CAG CGC CGA-3' (SEQ ID NO: 23 ).