Dsrna molecule, pharmaceutical composition, and use of dsrna molecule and pharmaceutical composition extrahepatic delivery

Abstract

A dsRNA molecule containing a bulge structure and a preparation method therefor, a pharmaceutical composition containing the dsRNA molecule, and a use of the dsRNA molecule and the pharmaceutical composition in extrahepatic delivery.

Description

dsRNA molecules, pharmaceutical compositions and their application in extrahepatic delivery Technical Field

[0001] This disclosure relates to dsRNA molecules containing a bulging structure and methods for preparing the same, pharmaceutical compositions containing the dsRNA molecules, and the use of the dsRNA molecules and pharmaceutical compositions in extrahepatic delivery. Background Technology

[0002] In mammals, microRNAs (miRNAs) and small interfering RNAs (siRNAs) mediate gene repression through Argonaute (Ago) proteins. These two small RNA species differ in their intrinsic properties and modes of action. Typically, siRNAs consist of two perfectly complementary strands called the "guide strand" and the "passenger strand." When the siRNA double strand is loaded onto the Ago protein, the passenger strand is removed, while the guide strand remains associated with the Ago protein via its phosphate-sugar backbone; ultimately, the guide strand binds to its mRNA target with complete or near-complete base complementarity. Of the four human Argonaute proteins, only Ago2 possesses endonuclease activity. Ago2 exerts this activity by acting as a protein component of the RNA-induced silencing complex (RISC). Extensive complementarity with the siRNA guide strand is crucial for cleaving the mRNA target within the RISC.

[0003] Compared to traditional siRNAs, mature miRNA duplexes are characterized by incomplete sequence homology, containing various mismatches and bulges. It is speculated that these mismatches and bulges represent an alternative strategy for achieving thermodynamically unstable structures.

[0004] The small RNA mechanism in mammals cannot distinguish between siRNA and miRNA. Since endogenous miRNAs may contain protrusions, RISC must be able to tolerate these structures to some extent. This can occur during double-strand loading and unwinding. siRNAs containing protrusions, especially ribotropically modified siRNAs, may benefit from thermodynamic instability during RISC maturation, ultimately accelerating passenger strand removal.

[0005] Dua et al. (US9,260,470B2) investigated the activity of siRNAs with bulging guide strands. They designed bulging siRNAs by inserting additional nucleotides into the guide strand, and these siRNAs were used to reduce off-target effects. To date, there have been no reports on exploring the use of bulging structures of siRNAs for other purposes, such as siRNA delivery.

[0006] Existing siRNA drug delivery technologies mainly include liver-targeted delivery mediated by LNP and GalNAc, antibody-mediated muscle-targeted delivery, integrin ligand-mediated lung delivery, lipid-mediated CNS delivery, peptide nanoparticle-mediated tumor delivery, AAV delivery, and exosome delivery. Currently, GalNAc-conjugated delivery is the most widely studied, and 5 out of the 6 marketed siRNA drugs utilize this technology. Extrahepatic delivery remains in a very early stage of development.

[0007] Because free siRNA in the blood cannot cross the blood-brain barrier (BBB), delivery of siRNA to the central nervous system (CNS) is challenging. One effective method for delivering siRNA to the CNS is local administration. However, this does not solve the problem of internalization in CNS target cells. Therefore, further development of efficient in vivo delivery methods is needed to enable siRNA drugs to exert their effects in extrahepatic tissues such as the CNS.

[0008] Invention Overview

[0009] One aspect of this disclosure provides a dsRNA molecule comprising an antisense strand and a sense strand complementary to the antisense strand, the sense strand including at least one monomeric structure connected in a bulging manner between any two nucleotides, the monomeric structure including a lipophilic portion, and the monomeric structure not affecting the inherent nucleotide base pairing of the dsRNA.

[0010] In some embodiments, the sense strand of the dsRNA molecule contains at least one structural unit 1 as shown in formula a, and the antisense strand contains at least one structural unit 2 as shown in formula b, which is complementary to structural unit 1.

[0011] in:

[0012] In formula a, Q1 and Q2 are the T-th and T+1-th nucleotides on the sense strand starting from the 5' end, respectively, where T is a natural number. M is a monomeric structure that is connected between Q1 and Q2 in a bulging form. It does not pair complementaryly with structural unit 2. The monomeric structure contains a lipophilic part, and Q1, M and Q2 are connected in sequence.

[0013] In formula b, Q1' and Q2' are two nucleotides sequentially linked on the antisense strand and are complementary to Q1 and Q2 on the sense strand, respectively.

[0014] When Q1 and Q2 in structural unit 1 are paired with Q1' and Q2' in structural unit 2 respectively, structural unit 3 is formed as shown in equation c below:

[0015] Where: Q1, Q2, M, Q1', and Q2' are as defined above.

[0016] The dsRNA molecule contains at least one structural unit 3.

[0017] In some embodiments, Q1 is connected to M, and Q2 is connected to M by a phosphodiester bond, a thiophosphate diester bond, a hydrocarbon phosphate diester bond, or a thiohydrocarbon phosphate diester bond, and Q1' and Q2' are connected by a phosphodiester bond or a thiophosphate diester bond. The structural formulas of the hydrocarbon phosphate diester bond and the thiohydrocarbon phosphate diester bond are shown below:

[0018] Wherein, R is a hydrocarbon group, preferably C. 10-30 Aliphatic hydrocarbon group, further preferably C 16-22 Alkyl, more preferably C 16 straight-chain alkyl, C 22 Straight-chain alkyl groups.

[0019] In some implementations, T is a natural number not greater than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15.

[0020] In some embodiments, the lipophilic portion comprises a lipid group selected from C 10-30 Aliphatic hydrocarbon group, C 9-29 Aliphatic hydrocarbon group -C(O)-, preferably C 16-22 Alkyl, C 15-21 Alkyl-C(O)-, more preferably C 16 straight-chain alkyl, C 15 Straight-chain alkyl-C(O)-, C 22 straight-chain alkyl, C 21 Straight-chain alkyl-C(O)-.

[0021] In some implementations, the monomer structure contains the structure shown in Formula I:

[0022] -R1-R2(I),

[0023] in:

[0024] R1 is selected from -O-, -NH-, and -S-.

[0025] R2 is selected from C 10-30 Aliphatic hydrocarbon group, C 9-29 Aliphatic hydrocarbon group -C(O)-, preferably C 16-22 Alkyl, C 15-21 Alkyl-C(O)-, more preferably C 16 straight-chain alkyl, C 15 Straight-chain alkyl-C(O)-, C 22 straight-chain alkyl, C 21Straight-chain alkyl-C(O)-.

[0026] In some embodiments, the monomer structure has any one or more of the following formulas: I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9 and I-10, or their pharmaceutically acceptable salt forms:

[0027] in:

[0028] R1 is selected from -O-, -NH-, and -S-, and R2 is selected from C. 10-30 Aliphatic hydrocarbon group, C 9-29 Aliphatic hydrocarbon group -C(O)-, preferably C 16-22 Alkyl, C 15-21 Alkyl-C(O)-, more preferably C 16 straight-chain alkyl, C 15 Straight-chain alkyl-C(O)-, C 22 straight-chain alkyl, C 21 Straight-chain alkyl-C(O)-;

[0029] R3 is selected from -O-, -CH2-, -S-, and -NH-, with -O- being the preferred choice;

[0030] R4 is selected from -P(=O)(OH)- -P(=S)(OH)- R5 does not exist in the form of its salts; or R5 is selected from -P(=O)(OH)- -P(=S)(OH)- R4 does not exist in its salt form;

[0031] R6 is selected from H, natural or modified bases;

[0032] n is an integer selected from 0 to 2, preferably 0 or 1;

[0033] X is selected from -O- and -S-, with -O- being preferred;

[0034] R7 is selected from C 10-30 Aliphatic hydrocarbon group, preferably C 16-22 Alkyl, more preferably C 16 straight-chain alkyl, C 22 Straight-chain alkyl;

[0035] R8 is selected from H, OH, halogen, unsubstituted or C-substituted. 1-6 Alkoxy, C 1-6 Alkylamine, OH, amino, (C 1-6 (alkyl)2N-C 1-6 alkoxy-substituted C1-6 Alkoxy, C 2-6 alkenyl-O-,C 1-6 Alkyl-NH-C 1-6 Alkyl-C(=O)-O-; preferably H, OH, F, OMe, methoxyethoxy (MOE-O-), N-methyl-acetoxy, N,N-dimethylethoxyethoxy, allyl-O-, N-methyl-acetoxy; further preferably H, OH, F, OMe, methoxyethoxy (MOE-O-); more preferably H, OMe, F.

[0036] In some embodiments, the monomer structure has any one or more of the following general formulas or pharmaceutically acceptable salt forms:

[0037] In some implementations, the monomer structure does not contain a base structure.

[0038] In some embodiments, the monomer structure has any one or more of the following general formulas or pharmaceutically acceptable salt forms:

[0039] In some embodiments, the monomer structure is any one or more of the following formula or its pharmaceutically acceptable salt form:

[0040] When the monomer structures disclosed herein exist in the form of pharmaceutically acceptable salts, they can be acidic or basic salts.

[0041] In this disclosure, the addition of the monomeric structure to the dsRNA molecule does not affect the inherent double-stranded base pairing. The effect refers to increasing or decreasing the number of Watson-Crick or Wobble base pairs between the bases in the double-stranded sequence.

[0042] In some embodiments, the sense strand of the dsRNA molecule described in this disclosure includes one of the monomeric structures.

[0043] In some embodiments, the monomeric structure is located between any two nucleotides of the sense strand in the dsRNA molecule, preferably between any two nucleotides from position 1 to 20 from the 5' end, more preferably between any two nucleotides from position 4 to 18, and even more preferably between any two nucleotides from position 4 to 8 and position 15 to 18 from the 5' end, such as between nucleotides from position 4 to 5, position 5 to 6, position 6 to 7, position 7 to 8, position 15 to 16, position 16 to 17, or position 17 to 18 from the 5' end.

[0044] The nucleotides in the dsRNA molecules disclosed herein are unmodified or modified, and at least one modified nucleotide includes: 2'-O-methyl nucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, locked nucleotide, open-ring nucleotide (UNA), glycol nucleotide (GNA), 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, baseless nucleotide, ribitol, reverse nucleotide, reverse baseless nucleotide, reverse 2'-OMe nucleotide, reverse 2'-deoxynucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholinonucleotide and 3'-OMe nucleotide, nucleotide containing a 5'-thiophosphate group, terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bis(decanoic acid) group, 2'-amino-modified nucleotide, aminophosphate, or non-natural bases containing nucleotides. In some embodiments, it also includes an E-vinylphosphonate nucleotide at the 5' end of the sense strand and / or antisense strand; or a linking between at least one thiophosphate nucleotide of the sense strand and / or antisense strand.

[0045] In some implementations, the length of the double-stranded region of the dsRNA molecule can be 19 to 30 nucleotide pairs; or 19 to 25 nucleotide pairs; or 19 to 23 nucleotide pairs; or 19 to 21 nucleotide pairs; or 21 to 23 nucleotide pairs; or the length of each strand independently is no more than 30 nucleotides; or the length of each strand independently is no more than 25 nucleotides; or the length of each strand independently is no more than 23 nucleotides; or the length of each strand independently is no more than 21 nucleotides; or the length of each strand independently is no more than 19 nucleotides.

[0046] In some implementations, the dsRNA molecule has two blunt ends; or at least one strand contains a 3' overhang of at least one nucleotide; or at least one strand contains a 3' overhang of at least two nucleotides.

[0047] The sense strand and antisense strand contain 21 and 23 nucleotides respectively, and the lipophilic portion of the monomer structure is C. 16 Taking a straight-chain alkyl group as an example, the structural diagram of the dsRNA molecule is as follows:

[0048] Another aspect of this disclosure provides an intermediate compound or a salt thereof for forming the above-described monomer structure, the intermediate compound having the following structural formula:

[0049] in:

[0050] R1 is selected from -O-, -NH-, and -S-.

[0051] R2 is selected from C 10-30 Aliphatic hydrocarbon group, C 9-29 Aliphatic hydrocarbon group -C(O)-, preferably C 16-22 Alkyl, C 15-21 Alkyl-C(O)-, more preferably C 16 straight-chain alkyl, C 15 Straight-chain alkyl-C(O)-, C 22 straight-chain alkyl, C 21 Straight-chain alkyl-C(O)-;

[0052] R3 is selected from -O-, -CH2-, -S-, and -NH-, with -O- being the preferred choice;

[0053] R6 is selected from H, natural or modified bases;

[0054] n is an integer selected from 0 to 2, preferably 0 or 1;

[0055] R7 is selected from C 10-30 Aliphatic hydrocarbon group, preferably C 16-22 Alkyl, more preferably C 16 straight-chain alkyl, C 22 Straight-chain alkyl;

[0056] R8 is selected from H, OH, halogen, unsubstituted or C-substituted. 1-6 Alkoxy, C 1-6 Alkylamine, OH, amino, (C 1-6 (alkyl)2N-C 1-6 alkoxy-substituted C 1-6 Alkoxy, C 2-6 alkenyl-O-,C 1-6 Alkyl-NH-C 1-6 Alkyl-C(=O)-O-; preferably H, OH, F, OMe, methoxyethoxy (MOE-O-), N-methyl-acetoxy, N,N-dimethylethoxyethoxy, allyl-O-, N-methyl-acetoxy; more preferably H, OH, F, OMe, methoxyethoxy (MOE-O-); more preferably H, OMe, F;

[0057] R9 is selected from hydroxyl protecting groups, preferably benzyl, benzoyl, 2,6-dichlorobenzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, methanesulfonyl, toluenesulfonyl, dimethoxytriphenylmethyl (DMTr), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX), more preferably DMTr;

[0058] R 10 Selected from cyano-substituted C1-C6 alkyl groups, preferably cyanoethyl, cyanopropyl, or cyanobutyl, and more preferably cyanoethyl (CN-(CH2)2-);

[0059] R 11 R 12 Each independently is C1-C 10 Alkyl group, preferably C1-C6 alkyl group, more preferably isopropyl group.

[0060] In some embodiments, the intermediate compound is selected from:

[0061] In some embodiments, the intermediate compound is selected from:

[0062] In some embodiments, the intermediate compound is selected from:

[0063] When the intermediate compounds disclosed herein exist in the form of salts, they may be acidic or basic salts, preferably pharmaceutically acceptable salts.

[0064] Without specifying the configuration, the dsRNA molecules, compounds, and monomer structures of this disclosure may exist in specific geometric or stereoisomeric forms. All such dsRNA molecules, compounds, and monomer structures of this disclosure, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, and their racemic mixtures and other mixtures, such as mixtures enriched with enantiomers or diastereomers, are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in alkyl or other substituents. All such isomers and mixtures thereof are included within the scope of this disclosure.

[0065] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthetic methods, or using chiral reagents or other techniques conventional in the art. To obtain an enantiomer of a dsRNA molecule or compound disclosed herein, preparation can be achieved by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated, and the auxiliary group is cleaved to provide a pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished using chromatography employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., from amines to carbamates).

[0066] This disclosure also includes compounds identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, and fluorine, 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 and 18 F, etc.

[0067] Another aspect of this disclosure provides a pharmaceutical composition comprising any one or more of the above-described dsRNA molecules and a pharmaceutically acceptable carrier.

[0068] In some embodiments, the pharmaceutical composition is an injectable dosage form, comprising a sterile aqueous solution or dispersion and a sterile powder. In some embodiments, the sterile solution may include a diluent such as water; a physiological saline solution; a non-volatile oil, polyethylene glycol, glycerin, or propylene glycol, etc.

[0069] In some embodiments, the pharmaceutical composition is an oral dosage form. In some embodiments, the oral dosage form includes excipients, such as polyethylene glycol, glyceryl stearate, etc. In some embodiments, the oral dosage form includes plasticizers, such as diethyl phthalate, glyceryl triacetate, dibutyl sebacate, dibutyl phthalate, or triethyl citrate, etc. In some embodiments, the oral dosage form includes a permeation enhancer, which can be bile salts or fatty acids. Bile salts can be ursodeoxycholic acid, chenodeoxycholic acid, and their salts. Fatty acids can be decanoic acid, lauric acid, and their salts, etc.

[0070] In some embodiments, the pharmaceutical composition is a pulmonary or nasal dosage form.

[0071] In some embodiments, the pharmaceutical composition includes a local delivery agent, which may be a plurality of microvesicles, and the microvesicles may be liposomes.

[0072] Another aspect of this disclosure provides the use of the above-described dsRNA molecule or the above-described pharmaceutical composition in extrahepatic delivery (e.g., central nervous system, adipose tissue, muscle tissue, lung, eye, heart, groin, etc.), or in the preparation of a drug delivered extrahepatically.

[0073] Another aspect of this disclosure provides a method for reducing the expression of target genes in cells, or provides the use of the above-mentioned dsRNA molecule or the above-mentioned pharmaceutical composition in the preparation of a drug that reduces the expression of target genes in cells, the method or application comprising contacting the cells with the dsRNA molecule of this disclosure or the dsRNA molecule in the above-mentioned pharmaceutical composition; the target gene is selected from SOD1, APP, SNCA, GPR75, SCN9A, PMP22, MAPT, Leptin, MSTN1, MALAT1, Adiponectin, ACTR2, ACVR1C, ACVR2A, ACVR2B, etc., and the cells are extrahepatic cells. Furthermore, the method involves extrahepatic application to reduce the expression of target genes in cells such as central nervous system cells, adipose tissue cells, muscle tissue cells, lung tissue cells, eye tissue cells, heart tissue cells, and groin tissue cells. The cells are derived from one or more of the following groups: cerebellum, brainstem, thoracic vertebrae, thoracic spinal cord, thoracic DRG, striatum, cortex, lumbar vertebrae, hippocampus, heart, quadriceps femoris muscle, inguinal fat (iWAT), epididymal fat (eWAT), mesenteric fat (vAT), and pre-abdominal fat (BAT).

[0074] In some embodiments, the above-described dsRNA molecules or the above-described pharmaceutical compositions are used to prevent and / or treat diseases associated with lung, fat, muscle, eye, heart, groin, and central nervous system (CNS) disorders, or to prepare a medicament for the prevention and / or treatment of diseases associated with lung, fat, muscle, eye, heart, groin, and central nervous system (CNS) disorders.

[0075] This disclosure connects at least one monomeric structure containing a lipophilic moiety to any two nucleotides of the sense strand in a bulging manner, enabling the dsRNA molecule to achieve efficient extrahepatic delivery and demonstrating good knockdown effect on target genes in cells of different extrahepatic tissues such as the central nervous system, fat, muscle, lung, eye, heart, and groin, with minimal toxic side effects. Attached Figure Description

[0076] Figure 1 shows the SOD1 expression levels of dsRNA molecules QLAD-0002028, QLAD-002029, and QLAD-002030, the negative control molecule QLAD-000270, and the blank group in rats after 14 days of ICM.

[0077] Figure 2 shows the SOD1 expression levels of dsRNA molecules QLAD-0002028, QLAD-002029, and QLAD-002030, the negative control molecule QLAD-000270, and the blank group in rats after 42 days of ICM.

[0078] Figure 3 shows the IC50 of the dsRNA molecule in this disclosure and the negative control molecule QLAD-000270 in CT26 cells.

[0079] Figure 4 shows the SOD1 expression levels of the dsRNA molecules QLAD-000249, QLAD-002536, QLAD-002574, QLAD-002656, QLAD-002657, QLAD-002658, QLAD-002659, QLAD-002545, QLAD-002660, QLAD-002662, QLAD-002663, QLAD-002664, and QLAD-002665 in rats after 42 days of ICM in the control group.

[0080] Figure 5 shows the SOD1 expression levels of dsRNA molecules QLAD-002586, QLAD-002587, and QLAD-002588 in rats compared with those in the control group after 28 days of intravenous administration. Detailed Implementation

[0081] the term

[0082] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if specifically and individually indicated that each individual publication, patent or patent application is incorporated by reference.

[0083] Before this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0084] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for the purposes of brevity and convenience only, and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. For example, a description of a range from 1 to 30 should be considered to specifically disclose subranges from 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 10, 2 to 4, 2 to 6, 3 to 6, 10 to 30, 12 to 25, 15 to 29, 16 to 22, etc., and specific numerical points within these ranges, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30. The above principles apply equally regardless of the breadth of the stated numerical values. When a range description is used, the range includes the endpoints of the range.

[0085] When referring to measurable values ​​such as quantities, temporary durations, etc., the term “about” means a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0086] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the relevant gene, including mRNA that is a primary transcription product of RNA processing. The target portion of the sequence is at least long enough that the nucleotide sequence portion of the mRNA molecule formed during the transcription of the relevant gene, or in its vicinity, serves as a substrate for iRNA-guided cleavage.

[0087] The target sequence can be approximately 18 to 35 nucleotides in length, for example, approximately 18 to 30 nucleotides. For example, the target sequence can be approximately 19 to 30 nucleotides, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides in length. In some embodiments, the target sequence is approximately 19 to approximately 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 19 to about 21 nucleotides in length. Ranges and lengths between those listed above are also considered part of the invention.

[0088] As used interchangeably herein, the terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent” refer to an agent containing RNA as defined herein, which mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNA guides the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). For example, “iRNA” used in the compositions, uses, and methods of this disclosure can be double-stranded RNA and is referred to herein as a “double-stranded RNA agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA reagent,” “dsRNA,” “double-stranded RNA (dsRNA) conjugate,” or “double-stranded RNA (dsRNA) conjugate molecule.” The term “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, meaning having a “sense” orientation and an “antisense” orientation relative to the target RNA.

[0089] The terms “silence,” “reduction,” “inhibition,” “knockdown,” “block,” or “downregulation,” and other similar terms are used interchangeably and include any level of inhibition.

[0090] The term "chain containing a sequence" refers to an oligonucleotide containing a nucleotide chain, described by reference to a sequence using standard nucleotide nomenclature.

[0091] Typically, "G", "C", "A", "T", and "U" each represent a nucleotide containing guanine, cytosine, adenine, thymine, and uracil as bases, respectively. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be replaced by other parts without substantially altering the base-pairing properties of the oligonucleotide containing the nucleotide with this replacement part. For example, but not limited to, a nucleotide containing inosine as its base can base-pair with a nucleotide containing adenine, cytosine, or guanine. Therefore, nucleotides containing uracil, guanine, or adenine in the nucleotide sequence of the dsRNA characterized in this invention can be replaced with nucleotides containing, for example, inosine. In another example, adenine and cytosine at any position in the oligonucleotide can be replaced with guanine and uracil, respectively, to form a GU wobble base pairing with the target mRNA. Sequences containing such replacement parts are suitable for the compositions and methods characterized in this invention.

[0092] The terms "sequence" and "nucleotide sequence" refer to a continuous sequence of nucleobases or nucleotides, described using standard nomenclature with consecutive letters. Nucleic acid molecules may contain unmodified and / or modified nucleotides. Nucleotide sequences may contain unmodified and / or modified nucleotides.

[0093] The terms "base," "nucleotide base," or "nucleobase" refer to heterocyclic pyrimidine or purine compounds that are components of nucleotides, including primary purine bases adenine and guanine, and primary pyrimidine bases cytosine, thymine, and uracil. Nucleobases can be further modified to include, but are not limited to, universal bases, hydrophobic bases, hybrid bases, size-enlarged bases, and fluorinated bases. The synthesis of such modified nucleobases (including phosphoramidite compounds containing modified nucleobases) is known in the art.

[0094] The term "nucleotide" has the same meaning as commonly understood in the art. Therefore, as used herein, "nucleotide" refers to a glycoside comprising a sugar moiety, a base moiety, and a covalently linked group (linking group) such as a phosphate or thiophosphate internucleotide linking group, and encompasses naturally occurring nucleotides, such as DNA or RNA, as well as non-naturally occurring nucleotides comprising modified sugar and / or base moieties, which are also referred to herein as nucleotide analogs. In this document, a single nucleotide may be referred to as a monomer or unit.

[0095] As used herein, unless otherwise stated, the term "complementary" is used to describe a first nucleobase or nucleotide sequence (e.g., sense strand or targeting mRNA of an RNAi reagent) or an antisense strand or single-stranded antisense oligonucleotide associated with a second nucleobase or nucleotide sequence (e.g., RNAi reagent antisense strand or targeting mRNA), referring to the hybridization of an oligonucleotide or polynucleotide containing the first nucleotide sequence (forming base pair hydrogen bonds under mammalian physiological conditions (or other suitable in vivo or in vitro conditions) and forming a double-stranded or double-helix structure with an oligonucleotide containing the second nucleotide sequence under certain standard conditions. Those skilled in the art will be able to select the set of conditions most suitable for the hybridization test. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include native or modified nucleotides or nucleotide mimics to at least the extent required to satisfy the hybridization requirements described above. Sequence identity or complementarity is independent of modification. For example, a and Af, as defined herein, are complementary to U (or T) and identical to A for the purpose of determining identity or complementarity.

[0096] As used herein, “perfect complementarity” means that in a pair of hybridized nucleobase or nucleotide sequences, all (100%) bases in the first oligonucleotide sequence will hybridize with the same number of bases in the second oligonucleotide sequence. The sequence may contain all or part of the first or second nucleotide sequence.

[0097] As used herein, when referring to a connection between two compounds or molecules, the terms “connection” or “combination” mean that the two compounds or molecules are connected by a covalent bond. Unless otherwise stated, the terms “connection” and “combination” as used herein may refer to a connection between a first compound and a second compound, with or without any inserted atoms or groups.

[0098] As used in this article, "extrahepatic delivery" refers to the delivery of drugs to tissues outside the liver, including but not limited to the central nervous system, adipose tissue, muscle tissue, lung tissue, eye tissue, heart tissue, groin tissue, etc. However, considering toxicity issues, it does not include kidney tissue.

[0099] As used herein, the terms “individual” or “subject” refer to any animal, such as a mammal or marsupial. Individuals covered by this disclosure include, but are not limited to, humans, non-human primates (such as cynomolgus monkeys or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.

[0100] As used herein, the terms “disease,” “symptom,” or “disorder,” etc., refer to any alteration or dysregulation that impairs or interferes with the normal function of cells, tissues, or organs. For example, “disease” includes, but is not limited to: tumors, pathogen infections, autoimmune diseases, T-cell dysfunction disorders, or deficiencies in immune tolerance (such as transplant rejection).

[0101] As used in this article, the term "treatment" refers to a clinical intervention in the process of attempting to alter an individual's or treat a disease caused by cells, which can be preventative or intervention in the clinicopathological process. Therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and alleviating or improving prognosis.

[0102] As used herein, the term "prevention" means that, prior to the onset of a disease, the symptoms of the disease are reduced compared to those without exposure by exposing the subject to (e.g., administering medication) the dsRNA molecule disclosed herein or a pharmaceutical composition containing the dsRNA molecule. It does not imply that the disease must be completely suppressed.

[0103] The article uses Indicates the bonding site.

[0104] The term "lipophilic moiety" as used in this article refers broadly to any compound or chemical moiety that has an affinity for lipids.

[0105] As used in this article, the term "aliphatic hydrocarbon group" refers to the residue formed after an aliphatic hydrocarbon loses at least one hydrogen atom. Aliphatic hydrocarbon groups include saturated and unsaturated aliphatic hydrocarbon groups, and can be straight-chain or branched. For example: C 1-3 Aliphatic hydrocarbon group, C 1-6 Aliphatic hydrocarbon group, C 9-29 Aliphatic hydrocarbon group, C 10-30 Aliphatic hydrocarbon group, C 12-25 Aliphatic hydrocarbon group, C 15-29 Aliphatic hydrocarbon group, C 16-22 Aliphatic hydrocarbon groups, etc. Representative aliphatic hydrocarbon groups include alkyl, alkenyl, and alkynyl groups.

[0106] As used herein, the term "alkyl" can refer to a straight-chain or branched alkyl group, for example: C 1-3 Alkyl, C 1-6 Alkyl, C 9-29 Alkyl, C 10-30 Alkyl, C 12-25 Alkyl, C 15-29 Alkyl, C 16-22 Alkyl groups, etc. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, eicosyl or docosyl and similar groups.

[0107] As used herein, the term "alkenyl" refers to an alkyl group as defined above that contains at least one double bond between adjacent carbon atoms, and can be straight-chain or branched, for example: C 2-6 alkenyl, C 9-29 alkenyl, C 10-30 alkenyl, C 12-25 alkenyl, C 15-29 alkenyl, C 16-22 Alkenyl groups, etc. Alkenyl groups include both cis and trans isomers. Representative alkenyl groups include, but are not limited to, vinyl, propenyl, allyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, pentadecenyl, hexadecenyl, dodecenyl, dodecenyl, dodecenyl, dodecenyl, dodecenyl, tridecenyl, and similar groups.

[0108] As used herein, the term "alkynyl" refers to an alkyl group as defined above that contains at least one triple bond between adjacent carbon atoms, and can be straight-chain or branched, for example: C 2-6 alkynyl group, C 9-29 alkynyl group, C 10-30 alkynyl group, C 12-25 alkynyl group, C 15-29 alkynyl group, C 16-22 Alkynyl groups, etc. Representative alkenyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentyynyl, hexynyl, heptyynyl, octyynyl, nonynyl, decyynyl, undecynyl, dodecynyl, pentadecynyl, hexadecynyl, 22decynyl, 29decynyl, 30decynyl, and similar groups. As used herein, the term "pharmaceutically acceptable salt" or "salt" refers to acidic and / or basic salts formed by a monomeric structure or compound with inorganic and / or organic acids and bases, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkylammonium salts. Pharmaceutically acceptable salts and the salts mentioned herein include, but are not limited to, hydrochlorides, sulfates, citrates, benzenesulfonates, hydrobromides, hydrofluoric acids, phosphates, formates, acetates, propionates, succinates, oxalates, malates, succinates, fumarates, maleates, tartrates, trifluoroacetates, methanesulfonates, and toluenesulfonates.

[0109] Example

[0110] Example 1: Synthesis of a phosphorusamide monomer with a bulging structure containing lipid groups

[0111] 1.1 Synthesis of compound QLR-2

[0112] Compounds 1-2

[0113] 1,2,4-Butanetriol (compound 1-1, 20 g, 1.0 eq.) was dissolved in DCM. Benzaldehyde (28 g, 1.4 eq.) and trimethoxymethane (28 g, 1.4 eq.) were added separately under nitrogen protection. The mixture was cooled to 0-5 °C, and 2,2,2-trifluoroacetic acid (13.97 g, 0.65 eq.) was added dropwise to the reaction solution. After the addition was complete, the mixture was warmed back to 25 °C and reacted for 48 h. After the reaction was complete, sodium methoxide (0.65 eq.) was added to the reaction system, followed by tert-butyl methyl ether. The mixture was cooled to 0-5 °C and stirred for 1 h. The mixture was filtered through diatomaceous earth. The filtrate was concentrated to dryness at 30-35 °C. The crude product was subjected to silica gel chromatography to obtain 26 g of a pale yellow oily compound 1-2 (UPLC purity: 98.8%).

[0114] 1 H NMR(500MHz,d6-DMSO)δ7.46-7.41(m,2H),7.41-7.31(m,3H),5.52(s,1H),5.03-4.49(m,1H),4.17-4.12(m,1H),3.96-3.83(m, 2H),3.48(dd,J=11.4,5.9Hz,1H),3.41(dd,J=11.4,4.7Hz,1H),1.69-1.57(m,1H),1.55-1.48(m,1H).MS(ESI)m / z=195.24[MH] - Theory: 195.10.

[0115] Compounds 1-3

[0116] Compounds 1-2 (25 g, 1.0 eq.) were dissolved in THF under nitrogen protection, followed by the addition of KOH (36.11 g, 5.0 eq.) and TBAI (4.75 g, 0.1 eq.). The mixture was heated to 65 °C, and then 1-bromohexadecane (117.91 g, 3.0 eq.) was added dropwise. After the addition was complete, the reaction was carried out at 65 ± 5 °C for 16 h. After the reaction was complete, the system was cooled to 10 ± 5 °C, and then extracted once with deionized water and tert-butyl methyl ether. The organic phase was washed once with 20% NH4Cl aqueous solution and then once with 10% NaCl aqueous solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The concentrate was crystallized in methanol and tert-butyl methyl ether, and after filtration, 52 g of white solid compound 1-3 (UPLC: 96.7%) was obtained.

[0117] 1H NMR(500MHz,d6-DMSO)δ7.42-7.31(m,5H),5.52(s,1H),4.16(dd,J=11.3,4.1Hz,1H),4.07-3.99(m,1H),3.92(td,J=12.1,2.6Hz,1H),3.45(dd,J =10.7,6.1Hz,1H),3.42-3.37(m,3H),1.67-1.62(m,1H),1.49-1.40(m,3 H),1.31-1.16(m,26H),0.85(t,J=6.9Hz,3H).MS(ESI)m / z=419.28[M+H] + Theoretical value: 419.35.

[0118] Compounds 1-4

[0119] Compounds 1-3 (50 g, 1.0 eq.) were dissolved in 25 mL of methanol, and 100 g of cation exchange resin and 100 mL of deionized water were added. The mixture was then heated to 60 °C and reacted for 13 h. After the reaction was complete, the temperature was lowered to 25 °C, 300 mL of THF was added, and the mixture was stirred until the system was clear. The mixture was filtered, and the pH of the filtrate was adjusted to 7–8 with 1 N NaOH solution, followed by concentration. The concentrate was washed once with ethyl acetate and 10% NaCl aqueous solution. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was then subjected to silica gel chromatography to obtain 24 g of product compound 1-4.

[0120] 1 H NMR(500MHz,d6-DMSO)δ4.43(d,J=5.1Hz,1H),4.30(t,J=5.1Hz,1H),3.71-3.63(m,1H),3.49(dd,J=12.2,5.9Hz,2H),3.34(dd,J=11.6,4 .9Hz,2H),3.22-3.16(m,2H),1.61-1.53(m,1H),1.49-1.36(m,3H),1.30-1.05(m,26H),0.84(t,J=6.9Hz,3H).MS(ESI)m / z=331.35[M+H] + Theoretical: 331.32.

[0121] Compounds 1-5

[0122] Compounds 1-4 (15 g, 1.0 eq.) were dissolved in 54 g of pyridine, and DMTrCl (18.45 g, 1.2 eq.) was added. The reaction was carried out at 5-10 °C for 13 h. After the reaction was completed, ethanol was added dropwise to quench the reaction, and the mixture was stirred at 20-25 °C for 1-2 h. The mixture was then concentrated to dryness. The concentrate was extracted with ethyl acetate and 5% NaHCO3 aqueous solution. The organic phase was washed once with 10% NaCl aqueous solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by reverse-phase chromatography to obtain 10 g of product compound 1-5 (UPLC purity: 99.6%).

[0123] 1 H NMR(500MHz,d6-DMSO)δ7.42-4.36(m,2H),7.33-7.22(m,6H),7.16(t,J=7.3Hz,1H),6.84 (d,J=9.0Hz,4H),4.45(d,J=5.1Hz,1H),3.82-3.64(m,7H),3.31(t,J=6.6Hz,2H),3.25(d d,J=9.5,5.7Hz,1H),3.19(dd,J=9.5,5.5Hz,1H),3.16-3.06(m,2H),1.83-1.72(m,1H),1 .59-1.50(m,1H),1.45(dd,J=13.0,6.4Hz,2H),1.32-1.14(m,26H),0.83(t,J=6.9Hz,3H).

[0124] Compound QLR-2

[0125] Compounds 1-5 (6 g, 1.0 eq.) were dissolved in 60 mL of DCM, followed by the addition of bis(diisopropylamino)(2-cyanoethoxy)phosphine (3.71 g, 1.3 eq.) and 1H-tetrazole (0.66 g, 1.0 eq.). The mixture was then heated to 30 °C and reacted for 5 h. After the reaction and post-treatment were completed, the crude product was purified by silica gel chromatography to yield 4.5 g of product compound QLR-2. UPLC purity: 99.8%. 31 P-NMR purity: 99.7%.

[0126] 1H NMR (500MHz, CDCl3) δ7.45-7.40(m,2H),7.33-7.27(m,4H),7.24(dd,J=8.4,1.5Hz,2H),7.18(dd,J=13. 4,7.2Hz,1H),6.84-6.76(m,4H),4.18-4.04(m,1H),3.88-3.66(m,7H),3.58-3.32(m,7H),3.26-3.09(m, 2H),2.62-2.50(m,1H),2.43-2.29(m,1H),2.09-1.81(m,3H),1.59-1.45(m,2H),1.39-1.18(m,26H),1.1 3(dd,J=12.2,6.7Hz,9H),1.01(dd,J=19.0,4.2Hz,2H),0.87(t,J=6.9Hz,3H).MS(ESI)m / z=833.58[M+H] + Theoretical: 833.56.

[0127] 1.2 Synthesis of compound QLR-3

[0128] Compound 2-2

[0129] Palmitic acid (compound 2-1, 10 g, 1.0 eq.) was dissolved in 100 mL of DCM, followed by the addition of DIPEA (10.08 g, 2.0 eq.), 3-amino-1,2-propanediol (4.62 g, 1.3 eq.), EDCI (8.97 g, 1.2 eq.), and HOBT (1.05 g, 0.2 eq.). The reaction was carried out at 20-25 °C for 15 h, resulting in the precipitation of a large amount of solid. After the reaction was complete, deionized water was added and stirred. The mixture was filtered, and the solid was dried at 40 °C for 5 h to obtain 12 g of white solid compound 2-2 with a purity of 69.3%. MS (ESI) m / z = 388.32 [M + CH3COO] - Theoretical: 388.31.

[0130] Compounds 2-3

[0131] Compound 2-2 (12 g, 1.0 eq.) was dissolved in 120 mL of pyridine, and DMTr-Cl (13.6 g, 1.1 eq.) was added. The reaction was carried out at 0-5 °C for 16 h. After the reaction was completed, deionized water and NaHCO3 were added to quench the reaction. The mixture was concentrated to dryness at 40 °C, and then extracted with DCM and deionized water. The mixture was then dried and filtered. The crude product was concentrated to dryness and subjected to silica gel chromatography to obtain 13.5 g of product compound 2-3 with a purity of 98.8%.

[0132] 1H NMR(500MHz,d6-DMSO)δ7.62(t,J=5.6Hz,1H),7.40(d,J=7.4Hz,2H),7.33-7.24(m,6H ),7.20(t,J=7.3Hz,1H),6.87(d,J=8.3Hz,4H),4.95(d,J=5.3Hz,1H),3.76-3.67(m,7 H),3.26-3.20(m,1H),3.04-2.95(m,1H),2.89-2.83(m,2H),2.05-1.95(m,2H),1.46- 1.36(m,2H),1.25(d,J=15.2Hz,24H),0.85(t,J=6.9Hz,3H).MS(ESI)m / z=632.43[M+H] + Theoretical value: 632.39.

[0133] compound QLR-3

[0134] Compound 2-3 (8 g, 1.0 eq.) was dissolved in 80 mL of LCM, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (4.58 g, 1.2 eq.) and 1H-tetrazole (0.71 g, 0.8 eq.) were added. The mixture was heated to 25 °C and reacted for 1.5 h. The crude product after reaction was purified by silica gel chromatography to give 6.5 g of an oily compound QLR-3 with a purity of 97.3%. 31 P-NMR purity: 97.5%.

[0135] 1 H NMR(500MHz,d6-DMSO)δ7.58-7.50(m,1H),7.43-7.36(m,2H),7.33-7.23(m,6H),7.19-7.12(m,1H ),6.86(t,J=9.3Hz,4H),4.11-3.98(m,1H),3.87-3.66(m,8H),3.65-3.48(m,2H),3.24-3.16(m,1H ),3.18-2.92(m,2H),2.83-2.68(m,1H),2.67-2.56(m,1H),2.04-1.87(m,3H),1.46-1.35(m,2H), 1.30-1.09(m,34H),1.01(t,J=16.9Hz,2H),0.85(t,J=6.9Hz,3H).MS(ESI)m / z=890.55[M+CH3COO] - Theoretical value: 890.55.

[0136] 1.3 Synthesis of compound QLR-4

[0137] Compound 3-2

[0138] Compound 3-1 (17.8 g, 1.0 eq.) was dissolved in 180 mL of acetonitrile. After cooling to 0-5 °C, Et3SiH (9.94 g, 3.0 eq.) and TMSOTf (12.66 g, 2.0 eq.) were added sequentially, and the mixture was then heated to 20 °C and reacted for 10 h. After the reaction was complete, ethyl acetate was added to the reaction solution, and the mixture was washed twice with 5% NaHCO3 aqueous solution, followed by one wash with 10% NaCl aqueous solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 20 g of oily compound 3-2 with a purity of 76.8%. MS (ESI) m / z = 584.38 [M+NH4] + Theoretical value: 584.39.

[0139] Compound 3-3

[0140] Compound 3-2 (16.15 g, 1.0 eq.) was dissolved in 80 mL of acetonitrile, then NaOH aqueous solution (8.0 eq.) and deionized water (20 mL) were added, and the reaction was carried out at 40 °C for 16 h. Extraction was performed using DCM, and the organic phase was washed once each with 5% NaHCO3 aqueous solution and 10% NaCl aqueous solution. The mixture was dried over anhydrous sodium sulfate, filtered, concentrated to dryness, and dried again with DCM. After drying, 8.5 g of white solid product compound 3-3 with a purity of 77.0% was obtained.

[0141] 1 H NMR(500MHz,d6-DMSO)δ4.65-4.50(m,2H),3.91-3.73(m,3H),3.63-3.51(m,3H),3.46-3 .38(m,1H),3.41-3.32(m,2H),1.55-1.44(m,2H),1.24-1.16(m,26H),0.93-0.82(m,3H).

[0142] Compounds 3-4

[0143] Compound 3-3 (8.5 g, 1.0 eq.) was dissolved in 85 mL of pyridine, and DMT-Cl (8.43 g, 1.05 eq.) was added. The reaction was allowed to proceed for 15 h. After the reaction was complete, ethanol was added, and the mixture was heated to 25 °C and stirred for 1 h before being concentrated to dryness. The crude product was dissolved in DCM and deionized water, and the organic phase was separated by standing. The organic phase was then washed once each with 5% NaHCO3 aqueous solution and 10% NaCl aqueous solution. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain a yellow oily crude product. After purification by reverse phase preparation, 6.0 g of yellow oily compound 3-4 with a purity of 99.1% was obtained.

[0144] 1 H NMR(600MHz,d6-DMSO)δ7.39(d,J=7.4Hz,2H),7.30(t,J=7.7Hz,2H),7.25(d,J=8.5Hz,4H),7.21(t,J=7.3Hz ,1H),6.88(d,J=8.9Hz,4H),4.65(d,J=6.7Hz,1H),3.92-3.86(m,1H),3.85-3.83(m,1H),3.82-3.76(m,2H),3 .73(s,6H),3.65-3.60(m,1H),3.54-3.48(m,1H),3.40-3.34(m,1H),3.32-3.28(m,2H),3.08-3.01(m,1H),2. 94-2.85(m,1H),1.54-1.46(m,2H),1.28-1.21(m,24H),0.85(t,J=7.0Hz,3H).MS(ESI)m / z=1338.22[2M+H2O] + Theoretical: 1338.89.

[0145] Compound QLR-4

[0146] Compounds 3-4 (5 g, 1.0 eq.) were dissolved in 50 mL of DCM, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (3.19 g, 1.4 eq.) and 1H-tetrazole (0.42 g, 0.8 eq.) were added sequentially. The mixture was then heated to 25 °C and reacted for 8 h. After the reaction and post-treatment were completed, the crude product was purified by silica gel chromatography to obtain 3 g of oily product with a purity of 99.7% by UPLC. 31 P-NMR: 100%.

[0147] 1H NMR (500MHz, CDCl3) δ7.46-7.41(m,2H),7.39-7.31(m,4H),7.28-7.22(m,2H),7.19-7.10(m,1H),6 .85-6.76(m,4H),4.32-3.95(m,4H),3.94-3.79(m,2H),3.76(d,J=3.8Hz,6H),3.66-3.25(m,6H),3. 06-2.82(m,1H),2.68-2.52(m,1H),2.38-2.23(m,1H),1.92-1.87(m,1H),1.65-1.53(m,2H),1.42-1 .21(m,26H),1.15-1.02(m,9H),1.00-0.92(m,2H),0.88(t,J=6.9Hz,3H).MS(ESI)m / z=861.60[M+H] + Theoretical value: 861.55.

[0148] 1.4 Synthesis of compound QLR-27

[0149] C in the above compounds 16 H 33 C 16 Straight-chain alkyl groups.

[0150] Compound 4-3

[0151] Compound 4-1 (20 g, 1.0 eq) was dissolved in acetonitrile (200 mL), and then Et3SiH (1.6 g, 3.0 eq) and TMSOTf (14.2 g, 2.0 eq) were added to the system. The mixture was stirred for 16 hours, and after post-treatment, it was concentrated to dryness to obtain an oily crude product. The crude product was dissolved in acetonitrile (100 mL), and NaOH aqueous solution (8.0 eq) was added. The mixture was stirred for 16 hours, and after post-treatment, it was concentrated to dryness to obtain 11.6 g of a pale yellow solid product, which was directly fed into the next step.

[0152] Compound 4-4

[0153] Compound 4-3 (5 g, 1.0 eq) was dissolved in pyridine (50 mL), and TBSCl (2.5 g, 1.2 eq) was added to the system. The mixture was stirred for 4 hours. After post-treatment, the product was concentrated to dryness to obtain 6.4 g of a pale yellow oily product, which was directly fed into the next step.

[0154] Compounds 4-6

[0155] Compound 4-4 (6.4 g, 1.0 eq) was dissolved in DCM (640 mL). 2,4,6-Trimethylpyridine (8.2 g, 5.0 eq) and DMTCl (9.2 g, 2.0 eq) were added to the system, and the mixture was stirred for 22 hours. Methanol (6.2 mL) was then added to the reaction mixture, followed by triethylamine (8.2 g, 6.0 eq) and triethylamine hydrogen fluoride (6.5 g, 3.0 eq). The mixture was stirred for 46 hours after the addition was complete. The product was then purified by column chromatography after post-treatment, yielding 4.8 g of product with a purity of 99.11%.

[0156] Compound QLR-27

[0157] Compound 4-6 (4 g, 1.0 eq) was weighed and dissolved in 10 mL of dichloromethane. Then, bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.6 g, 1.4 eq) and 1H-tetrazole (0.3 g, 0.8 eq) were added, and the mixture was stirred for 4 hours. The crude product after reaction and post-treatment was purified by silica gel column chromatography to obtain 0.8 g of a colorless oily product. The purity was 99.55%, and the phosphorus spectrum purity was 99.56%.

[0158] 1 H NMR (500MHz, CDCl3) δ7.58-7.52(m,2H),7.48-7.40(m,4H),7.31-7.24(m,2H),7.23-7.17(m,1H),6 .84-6.77(m,4H),4.04-3.96(m,1H),3.97-3.83(m,2H),3.79(s,6H);3.77-3.63(m,3H),3.57-3.44( m,3H),3.37-3.13(m,2H),3.11-2.90(m,2H),2.60-2.47(m,2H),1.66-1.55(m,2H),1.42-1.21(m,26 H),1.14(dd,J=6.5,1.5Hz,6H),1.11-1.04(m,6H),0.88(t,J=7.0Hz,3H).MS(ESI)m / z=861.63[M+H] + Theoretical value: 861.55.

[0159] 1.5 Synthesis of compound QLR-29

[0160] Compound 5-1 (4 g, 1.0 eq) was weighed and dissolved in 40 mL of dichloromethane. Then, compound 5-2 (5 g, 1.24 eq) and 4,5-dicyanimidazole (1 g, 1.0 eq) were added, and the mixture was stirred for 3 hours. The crude product after reaction and post-treatment was purified by silica gel column chromatography to obtain 1.2 g of product with a purity of 98.95% and a phosphorus spectrum purity of 98.95%.

[0161] 1 H NMR (500MHz, CDCl3) δ7.51-7.41(m,2H),7.40-7.29(m,4H),7.26-7.22(m,2H),7.21-7.14(m,1H),6.80-6.7 0(m,4H),4.29-4.12(m,1H),4.09-4.07(m,1H),4.06-3.99(m,1H),3.99-3.80(m,2H),3.77(s,6H),3.41(d, J=14.8Hz,4H),3.29-3.24(m,1H),3.03-3.00(m,1H),1.72-1.62(m,1H),1.58-1.53(m,1H),1.51-1.18(m,2 9H),1.16-1.17(m,2H),1.04-1.00(m,6H),0.94-0.93(m,4H),0.89-0.86(m,3H).MS(ESI)m / z=806.64[M+H] + Theoretical value: 806.55.

[0162] 1.6 Synthesis of compound QLR-28

[0163] Compound 6-2

[0164] Compound 6-1 (28 g, 1.0 eq.) was dissolved in acetonitrile. Triethylsilane (15.6 g, 3.0 eq.) and trimethylsilyl trifluoromethanesulfonate (19.9 g, 2.0 eq.) were added to the solution at 15–25 °C, and the mixture was stirred overnight at this temperature. After the reaction was complete, ethyl acetate was added to the reaction solution, and the mixture was extracted. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain 25 g of crude product 6-2. The purity was 98.0%.

[0165] Compound 6-3

[0166] Compound 6-2 (25 g, 1.0 eq.) was dissolved in methanol, and 50 mL of 2 N sodium hydroxide solution was added. After the addition was complete, the reaction was carried out at 20–30 °C for 3 h. After the reaction was completed, the reaction solution was concentrated to remove methanol, and the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain 14 g of crude product 6-3, which was directly fed into the next step.

[0167] Compound 6-4

[0168] Compound 6-3 (14 g, 1.0 eq.) was dissolved in pyridine (140 mL), cooled to 0–10 °C, and then DMT-Cl (15.6 g, 1.2 eq.) was added. The mixture was stirred overnight at 0–10 °C. After the reaction was complete, the organic phase was extracted, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain the crude product. Purification by column chromatography yielded 14 g of product 6-4 with a purity of 97.3%.

[0169] 1 H NMR (500MHz, DMSO-d6) δ7.42-7.36(m,2H),7.33-7.23(m,6H),7.21(t,J=7.3Hz,1H),6.87(d,J= 8.6Hz,4H),4.65(d,J=5.0Hz,1H),4.22-4.09(m,1H),3.92-3.89(m,1H),3.85-3.78(m,1H),3.73 (s,6H),3.66-3.64(m,1H),3.62-3.60(m,1H),3.54-3.50(m,1H),3.13-3.10(m,1H),2.94-2.91 (m,1H),1.48-1.37(m,2H),1.25-1.13(m,18H),0.84(t,J=6.9Hz,3H).MS(ESI)m / z=661.48[M+H] + Theoretical value: 661.45.

[0170] Compound QLR-28

[0171] Compound 6-4 (8.4 g, 1.0 eq.) was dissolved in dichloromethane (80 mL). Phosphoramide (5.7 g, 1.5 eq.) and 1H-tetrazole (0.71 g, 0.8 eq.) were added to the solution at 0–10 °C. After the addition was complete, the temperature was raised to 20–30 °C and the mixture was stirred for 5 h. After the reaction was complete, the crude product was post-processed and, after chromatography, yielded 6 g of product with a UPLC purity of 96.8%. 31 The purity of the β-NMR spectrum is 96.6%.

[0172] 1H NMR (500MHz, CDCl3) δ7.46 (d, J = 7.5Hz, 2H), 7.38-7.30 (m, 3H), 7.27-7.24 (m, 2H), 7.19-7.17 (m, 1H ),6.81-6.78(m,4H),4.39-4.36(m,1H),4.12-4.10(m,1H),3.98-3.96(m,1H),3.94-3.80(m,3H),3 .77(s,6H),3.75-3.69(m,1H),3.68-3.54(m,3H),3.33-3.30(m,2H),3.09-3.07(m,1H),2.67-2.56 (m,2H),1.49(d,J=6.0Hz,2H),1.31-1.13(m,38H),0.88(t,J=6.9Hz,3H).MS(ESI)m / z=861.70[M+H] + Theoretical value: 861.55.

[0173] 1.7 Synthesis of compound QLR-63

[0174] Compound 7-2

[0175] Compound 7-1 (34 g, 1.0 eq.) was dissolved in methanol, and 5 N NaOH was added. The reaction was carried out at 20–25 °C. After the reaction was complete, the reaction solution was concentrated to dryness, extracted with DCM, and concentrated to obtain 22 g of crude product 7-2, which was directly used in the next step.

[0176] Compound 7-3

[0177] Compound 7-2 (24 g, 1.0 eq.) was dissolved in pyridine, and TBDPS-Cl (23.9 g, 1.3 eq.) was added dropwise. The reaction was carried out at 0–5 °C. After the reaction was complete, deionized water and ethanol (6.2 g, 2.0 eq.) were added to the reaction solution to quench the reaction, followed by DCM extraction. The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain 44 g of crude product 7-3, which was used directly in the next step. MS (ESI) m / z = 636.22 [M+K] + Theoretical value: 635.39

[0178] Compound 7-4

[0179] Compound 7-3 (44 g, crude product, 1.0 eq.) was dissolved in DCM. 2,4,6-Trimethylpyridine (40.58 g, 5.0 eq.) and DMTr-Cl (45.3 g, 2.0 eq.) were added to the system, and the reaction was carried out at 35 °C. After the reaction was complete, methanol was added to quench the reaction solution. After extraction and washing, the organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain 93 g of crude product 7-4, which proceeded directly to the next step. MS (ESI) m / z = 1816.08 [2M + H + H2O] + Theory: 1816.13.

[0180] Compound 7-5

[0181] Compound 7-4 (93 g, crude product) was dissolved in THF, and then TBAF (35 g, 2.0 eq.) was added. The reaction was carried out at 35 °C. After the reaction was complete, the solution was concentrated to dryness to obtain the crude product. The crude product was purified by column chromatography to obtain 18 g of product 7-5, with a UPLC purity of 98.61%. MS (ESI) m / z = 1339.07 [2M + H + H₂O] + Theoretical value: 1339.90.

[0182] 1 H NMR(600MHz,DMSO)δ7.45(d,J=7.8Hz,2H),7.35-7.26(m,6H),7.22(t,J=7.3Hz,1H), 6.88-6.80(m,4H),4.64(t,J=5.5Hz,1H),3.88-3.84(m,1H),3.73(s,7H),3.47-3.37( m,2H),3.33-3.30(m,1H),3.27-3.18(m,2H),3.04(t,J=7.9Hz,1H),2.87(t,J=7.1Hz, 1H),1.61-1.50(m,2H),1.44-1.34(m,2H),1.31-1.18(m,24H),0.84(t,J=6.8Hz,3H).

[0183] Compound QLR-63

[0184] Compound 7-5 (10 g, 1.0 eq.) was dissolved in DCM. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (5.5 g, 1.2 eq.) and tetrazolium (0.85 g, 0.8 eq.) were added to the system, and the reaction was carried out at 20–25 °C. After the reaction was complete, the reaction solution was extracted and washed. The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain a crude product. After purification by column chromatography, 6 g of the product was obtained with a purity of 99.32% (UPLC). 31P-NMR: 99.78%. MS(ESI)m / z=861.49[M+H] + Theoretical value: 861.55.

[0185] 1 H NMR (500MHz, CDCl3) δ7.53(d,J=7.5Hz,2H),7.40(t,J=7.4Hz,4H),7.27(t,J=6.6Hz,2H),7 .20(d,J=4.7Hz,1H),6.82(d,J=8.1Hz,4H),4.00(d,J=17.9Hz,2H),3.73(d,J=26.7Hz,6H) ,3.66(d,J=6.5Hz,1H),3.55-3.44(m,4H),3.43-3.21(m,3H),2.46-2.43(m,2H),1.72-1.5 6(m,2H),1.48-1.38(m,2H),1.26-1.21(m,24H),1.20-1.01(m,12H),0.88(t,J=6.5Hz,3H).

[0186] 1.8 Synthesis of compound QLR-65

[0187] Compound 8-2

[0188] Compound 8-1 (45 g, 1.0 eq.) was dissolved in 2-methyltetrahydrofuran, and palmitic acid (26.9 g, 0.95 eq.) was added. The mixture was stirred until completely dissolved, followed by the sequential addition of HATU (46.2 g, 1.1 eq.) and DMAP (27 g, 2.0 eq.). After the addition was complete, the reaction was carried out at 25 °C. The reaction solution was filtered, washed, and the organic phase was dried and concentrated to obtain the crude product. The crude product was purified by chromatography to give 63.8 g of product with a purity of 98.82%. MS (ESI) m / z = 644.38 [MH] - Theoretical value: 644.43.

[0189] 1H NMR(600MHz,DMSO)δ7.45(d,J=9.1Hz,1H),7.39(d,J=7.5Hz,2H),7.30-7.22(m,6H) ,7.19(t,J=7.3Hz,1H),6.86(d,J=8.5Hz,4H),4.51(d,J=5.2Hz,1H),3.96-3.86(m, 2H),3.73(s,6H),3.07-3.04(m,1H),2.88-2.84(m,1H),2.14(t,J=7.3Hz,2H),1.54 -1.46(m,2H),1.21(d,J=17.7Hz,24H),0.96(d,J=6.3Hz,3H),0.85(t,J=6.8Hz,3H).

[0190] Compound QLR-65

[0191] Compound 8-2 (23 g, 1.0 eq.), bis(diisopropylamino)(2-cyanoethoxy)phosphine (12.9 g, 1.2 eq.), and tetrazolium (2 g, 0.8 eq.) were added to the system. After the addition was complete, the reaction was carried out at 25 °C. After the reaction was complete, the reaction solution was extracted and washed. The organic phase was dried over anhydrous Na₂SO₄ and concentrated to dryness to obtain the crude product. The crude product was purified by column chromatography to obtain 21 g of product with a purity of 99.22%. MS (ESI) m / z = 884.51 [M+K] + Theoretical: 884.51.

[0192] 1 H NMR (500MHz, CDCl3) δ7.44-7.40(m,2H),7.33-7.25(m,6H),7.19-7.14(m,1H),6.86- 6.79(m,4H),5.70-5.67(m,1H),4.40-4.13(m,2H),3.77(d,J=3.2Hz,6H),3.58-3.43 (m,3H),3.16-3.12(m,2H),2.58-2.29(m,2H),2.20-2.15(m,2H),1.67-1.58(m,2H), 1.34-1.19(m,28H),1.18-1.08(m,10H),0.99(d,J=6.8Hz,2H),0.88(t,J=7.0Hz,3H).

[0193] 1.9 Synthesis of compound QLR-66

[0194] Compound 9-2

[0195] Bis(diisopropylamino)phosphorus chloride (30.8 g, 1.05 eq.) was dissolved in toluene. Then, toluene solutions of triethylamine (41.6 g, 5.0 eq.) and 1-hexadecanoic acid (compound 9-1, 20 g, 1.0 eq.) were added sequentially to the system, and the mixture was stirred to react. The reaction solution was filtered and concentrated to dryness to obtain a crude product. After purification by silica gel column chromatography, 12.7 g of product 9-2 was obtained, with a phosphorus spectrum purity of 94.60%.

[0196] Compound QLR-66

[0197] Compound 2a (6 g, 1.0 eq.) was dissolved in DCM, and compound 9-2 (11 g, 1.5 eq.) and DCI (1.5 g, 0.8 eq.) were added sequentially to the system, followed by stirring. After post-treatment, the product was purified by column chromatography to obtain 6 g of product with a UPLC purity of 98.8% and a phosphorus spectrum purity of 97.9%.

[0198] 1 H NMR (500MHz, CDCl3) δ7.46-7.40(m,2H),7.35-7.29(m,4H),7.25(t,J=7.6Hz,2 H),7.17(t,J=7.3Hz,1H),6.83-6.77(m,4H),3.81-3.79(m,1H),3.75(s,6H),3 .74-3.66(m,1H),3.63-3.46(m,4H),3.23-3.09(m,2H),1.93-1.91(m,2H),1.6 2-1.52(m,2H),1.35-1.22(m,26H),1.14-1.01(m,12H),0.88(t,J=6.9Hz,3H). MS(ESI)m / z = 750.47 [M+H] + Theoretical value: 750.52.

[0199] 1.10 Synthesis of compound QLR-48

[0200] Compound 10⁻³

[0201] Compound 10-1 (73.3 g, 1.0 eq.) was dissolved in ACN (600 mL) and THF (100 mL) by stirring. The system was cooled to 0-5 °C, and triethylsilane (36.1 g, 3 eq.) was added. After the addition was complete, trimethylsilyl trifluoromethanesulfonate (46 g, 2 eq.) was added dropwise. After the addition was complete, the temperature was raised to 20 °C and the reaction was carried out for 13 h. After extraction, the organic phase was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 70 g of a brownish-yellow oil with a purity of 97.4%. THF was added to the crude product and stirred to dissolve. Then, sodium hydroxide solution (350 mL) was added. After the addition was complete, the temperature was raised to 40 °C and the reaction was carried out for 19 h. After extraction, the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain 43 g of compound 10-3.

[0202] 1 H NMR (500MHz, CDCl3) δ4.11-4.03(m,2H),3.93-3.90(m,1H),3.89-3.83(m,2H),3.82-3.77(m,1H),3.69-3.67(m,1H),3.58-3.55 (m,1H),3.50-3.46(m,1H),2.78(d,J=5.3Hz,1H),1.67-1.58(m,2H),1.34-1.23(m,36H),1.02-0.84(m,5H),0.57-0.54(m,1H).

[0203] Compound 10⁻⁴

[0204] Compound 10⁻³ (43 g, 1 eq.) was dissolved in dried pyridine (380 mL) with stirring. DMTCl (34.55 g, 1.05 eq.) was added, and the reaction was carried out at 0–5 °C. After the reaction was complete, the mixture was extracted, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated until no dripping occurred. The crude product was purified by column chromatography, resuspended twice in ACN, filtered, and dried to obtain 30 g of product 10⁻⁴ with a purity of 96.3%.

[0205] Compound QLR-48

[0206] Compound 10-4 (25 g, 1 eq.) was dissolved in DCM (230 mL), and phosphoramidite (30.3 g, 3 eq.) and 4,5-dicyanimidazole (5.94 g, 1.5 eq.) were added sequentially. After the addition was complete, the temperature was raised to 20–25 °C for reaction. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a crude product. After chromatography, 22 g of product QLR-48 was obtained with a purity of 94.48% (UPLC). 31 P-NMR: 95.9%.

[0207] 1H NMR (500MHz, CDCl3) δ7.49-7.42(m,2H),7.37-7.30(m,4H),7.27-7.24(m,2H),7.19-7.17(m,1H),6.8 5-6.78(m,4H),4.32-3.95(m,4H),3.94-3.80(m,3H),3.77(d,J=3.8Hz,6H),3.65-3.39(m,5H),3.32- 3.28(m,1H),3.06-3.03(m,1H),2.64-2.53(m,1H),2.31-2.28(m,1H),1.65-1.52(m,2H),1.34-1.21( m,39H),1.15(t,J=6.7Hz,9H),0.99(d,J=6.8Hz,3H),0.88(t,J=6.9Hz,3H).MS(ESI)m / z=945.95[M+H] + Theoretical value: 945.65.

[0208] 1.11 Synthesis of compound QLR-49

[0209] Compound 11-2

[0210] THF was added to compound 11-1 (100.3 g, 1.0 eq.) and stirred to dissolve. Then, 4.5 N NaOH solution was added, and the mixture was heated to 40 °C to react. After post-treatment of the reaction solution, the organic phase was dried, filtered, and concentrated to obtain 55 g of product 11-2.

[0211] 1 H NMR (500MHz, CDCl3) δ4.31-4.22(m,1H),4.03-4.00(m,1H),3.91-3.81(m,4H),3.66-3.50 (m,3H),2.75-2.72(m,1H),1.61-1.57(m,3H),1.25-1.17(m,38H),0.88(t,J=7.0Hz,3H).

[0212] Compound 11-3

[0213] Compound 11-2 (32 g, 1 eq.) was dissolved in pyridine. DMT-Cl (25 g, 1.02 eq.) was added in three equal portions at 2 ± 3 °C, with each addition spaced 10–20 minutes apart. The reaction was maintained at 2 ± 3 °C after each addition. After the reaction was complete, NaHCO3 aqueous solution was slowly added dropwise to quench the reaction. The mixture was then concentrated to dryness after stirring. The solution was extracted with DCM, and the organic phase was dried and filtered for concentration. The crude product was purified by column chromatography to give 29 g of product 11-3 with a purity of 98.6%. MS (ESI) m / z = 745.41 [M+H] + Theoretical value: 745.54.

[0214] Compound QLR-49

[0215] Compound 11-3 (21.7 g, 1 eq.) was dissolved in DCM, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (26.4 g, 3 eq.) and DCI (5.16 g, 1.5 eq.) were added sequentially. The reaction was carried out at 20–25 °C after the addition was complete. After the reaction was complete, the reaction solution was post-treated by extraction and washing. The organic phase was dried and concentrated to give 26 g of product QLR-49 with a purity of 95.9% by UPLC. 31 P-NMR: 97.1%. MS(ESI)m / z=945.95[M+H] + Theoretical value: 945.65.

[0216] 1 H NMR (500MHz, CDCl3) δ7.46 (d, J = 7.4Hz, 2H), 7.38-7.31 (m, 4H), 7.27-7.24 (m, 2H), 7 .21-7.15(m,1H),6.84-6.78(m,4H),4.45-4.34(m,1H),4.12-4.10(m,1H),4.03-3.8 0(m,5H),3.77-3.74(m,6H),3.67-3.55(m,3H),3.41-3.25(m,2H),3.09-3.04(m,1H ),2.62-2.58(m,2H),1.33-1.22(m,38H),1.22-1.14(m,13H),0.88(t,J=6.9Hz,3H).

[0217] 1.12 Synthesis of compound QLR-64

[0218] Compound 12-2

[0219] Under nitrogen protection, compound 12-1 (13.5 g, 1 eq.) was dissolved in pyridine. TBDPS-Cl (7.5 g, 1.0 eq.) was added to the system, and after stirring to dissolve, the mixture was reacted at 20-25 °C. After the reaction was complete, ethanol (2.0 eq.) and solid NaHCO3 (6.9 g, 3.0 eq.) were added to the reaction solution, and the mixture was concentrated to dryness. The concentrate was post-treated to yield 18.2 g of product 12-2, with a UPLC purity of 85.6%. MS (ESI) m / z = 698.57 [M+NH4]. + Theoretical value: 698.55.

[0220] Compound 12-3

[0221] Compound 12-2 (21.2 g, 1.0 eq.), 2,4,6-trimethylpyridine (18.8 g, 5 eq.), and DMT-Cl (21.1 g, 2.0 eq.) were added to the system under nitrogen protection, and the reaction was carried out at 35–37 °C. After the reaction was completed, ethanol (2.9 g, 2 eq.) and solid NaHCO3 (7.8 g, 3.0 eq.) were added to the reaction solution. The reaction solution was then post-treated and condensed to obtain crude product 12-3, which was directly used in the next step. MS (ESI) m / z = 1984.12 [2M + H + H2O] + Theory: 1984.32.

[0222] Compound 12-4

[0223] Under nitrogen protection, compound 12-3 (16.13 g, 1.0 eq.) was dissolved in THF, and TBAF (8.6 g, 2 eq.) was added. The reaction was carried out at 40–45 °C. After the reaction was completed, the reaction solution was concentrated at 30–35 °C to obtain a crude product. The crude product was purified by column chromatography to obtain 7.6 g of product 12-4, with a UPLC purity of 96.8%. MS (ESI) m / z = 1507.16 [2M + NH4] + Theory: 1507.10.

[0224] 1H NMR(600MHz,DMSO)δ7.45(d,J=7.7Hz,2H),7.34-7.26(m,6H),7.21(t,J=7.3Hz,1H),6 .87-6.84(m,4H),4.61(t,J=5.6Hz,1H),3.88-3.84(m,1H),3.72-3.66(m,7H),3.40-3 .38(m,2H),3.31-3.28(m,1H),3.26-3.17(m,2H),3.05(t,J=7.9Hz,1H),2.88-2.84(m ,1H),1.58-1.51(m,2H),1.37-1.33(m,2H),1.22-1.12(m,36H),0.84(t,J=6.9Hz,3H).

[0225] Compound QLR-64

[0226] Compound 12-4 (7.6 g, 1 eq.) was dissolved in DCM, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (9.2 g, 3 eq.) and DCI (1.8 g, 1.5 eq.) were added sequentially. The reaction was carried out at 20–25 °C. After extraction and washing of the reaction solution, the organic phase was dried over anhydrous Na₂SO₄ and concentrated to obtain the crude product. The crude product was purified by chromatography to obtain 6.1 g of product with a UPLC purity of 94%. MS (ESI) m / z = 946.18 [M+H] + Theoretical value: 945.65.

[0227] 1 H NMR (500MHz, CDCl3) δ7.52 (d, J = 8.3Hz, 2H), 7.44-7.35 (m, 4H), 7.28-7.24 (m, 2H), 7 .21-7.18(m,1H),6.82-6.78(m,4H),4.05-3.94(m,2H),3.79-3.74(m,6H),3.74-3. 41(m,8H),3.30-3.25(m,3H),2.50-2.44(m,2H),1.69-1.59(m,2H),1.44-1.36(m,2 H),1.31-1.23(m,36H),1.16-1.10(m,6H),1.09-1.05(m,6H),0.88(t,J=6.9Hz,3H).

[0228] Example 2: Synthesis of dsRNA molecules with monomeric structures linked between two nucleotides in a bulging manner as disclosed herein, and negative control dsRNA molecules.

[0229] The synthesis of dsRNA molecules was similar to that of conventional phosphoramide solid-phase synthesis. Using a Mermade 12 synthesizer (LGC), a universal CPG carrier was used as the starting material, and nucleoside phosphoramide monomers were linked one by one according to the synthesis program. Nucleoside monomer raw materials, such as 2'-F RNA and 2'-O-methyl RNA, were purchased from Shanghai Zhaowei. 5-Ethylthio-1H-tetrazole (ETT) was used as the activator (0.6M acetonitrile solution), 0.22M PADS dissolved in a 1:1 volume ratio of acetonitrile and trimethylpyridine was used as the sulfidation agent, and iodopyridine / aqueous solution was used as the oxidizing agent. After solid-phase synthesis, the oligonucleotides were cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. The mixture was then centrifuged, and the supernatant was transferred to another centrifuge tube. After concentration and evaporation to dryness, it was purified by C18 reversed-phase chromatography with 0.1M TEAA and acetonitrile as the mobile phase. DMTr was removed using 3% trifluoroacetic acid solution. The target oligonucleotide was collected, lyophilized, identified as the target product by LC-MS, and then quantified by UV (260nm).

[0230] The obtained single-stranded oligonucleotides were annealed according to complementary pairing in equimolar ratios to finally obtain the double-stranded dsRNA molecules shown in Table 1. The double-stranded dsRNA molecules were dissolved in 1×PBS and adjusted to the required concentration for the experiment.

[0231] Table 1. Nucleotide sequences of unmodified SOD1 sense and antisense strands.

[0232] Table 2 lists the abbreviations for one or more nucleotides used in nucleic acid sequence representation. It is understood that these monomers, when present in oligonucleotides, are linked together by 5'-3'-phosphodiester bonds.

[0233] Table 2 List of Abbreviations

[0234] Table 3. Nucleotide sequences of modified SOD1 sense and antisense strands.

[0235] Example 3: Evaluation of dsRNA molecules comprising monomeric structures of the present disclosure linked in a bulging manner between two nucleotides in vivo in rats for 14 days.

[0236] To assess the in vivo activity of SOD1 double-stranded nucleotides (also known as dsRNA molecules), rats were administered ICM.

[0237] Male rats were randomly divided into four groups: PBS group (solvent control group, n=4), QLAD-000270 (n=4), QLAD-0002028 (n=4), QLAD-002029 (n=4), and QLAD-002030 (n=4). For each test compound, it was dissolved and diluted with PBS to 7.5 μg / μL (0.3 mg injection dose) and 45 μg / μL (1.8 mg injection dose), and administered at a volume of 40 μL per rat. The specific steps were as follows: After anesthetizing the SD rats, the head was flexed as far towards the chest as possible. The depression above the first cervical vertebra (foramen magnum) was located with the left hand and fixed in position. A scalp needle was taken with the right hand (the tip of the needle was blunted), and the needle was carefully inserted vertically into the cerebellomedullary cistern along the midline of this depression. When the needle is correctly inserted into the cerebellomedullary cistern, the person administering the injection will feel no resistance as the needle moves forward, and will hear a very soft "click" sound. This indicates that the needle has passed through the dura mater and entered the cerebellomedullary cistern, and clear cerebrospinal fluid can be extracted. At this point, the medication can be injected.

[0238] Beforehand, use a microsyringe to measure 30 μL of PBS and 40 μL of the test sample, respectively. Then, using a 1 mL syringe, sequentially draw 30 μL of PBS, 5 μL of air, and 40 μL of the drug solution into a scalp vein needle, for a total of 75 μL. Inject over 40 seconds. After administration, return the animal to its cage.

[0239] On day 14 post-injection, each rat was sacrificed by CO2. Cerebellum, brainstem, thoracic spinal cord, striatum, cortex, and hippocampus tissues were then rapidly extracted and flash-frozen in liquid nitrogen. Subsequently, mRNA was extracted using Thermofisher Kingfisher reagent and its accompanying reagents (Thermofisher, 5400930) and analyzed using TaqMan Faster. TM SOD1 mRNA levels were quantified using a one-step premix (Applied Biosystems, 4444434), human GAPDH, and a rat SOD1 TaqMan probe. SOD1 mRNA expression was normalized using GAPDH mRNA expression. Results were normalized to those from the PBS group, and relative expression levels of the target gene were calculated using a 2-1... - ΔΔCT The calculation formula is as follows:

[0240] ΔCT = Average Ct value of target gene (SOD1) - Average Ct value of internal reference gene (GAPDH);

[0241] ΔCT = ΔCT (drug-treated group) - ΔCT (PBS control group);

[0242] relative mRNA expression level = 2 -ΔΔCT The results are shown in Table 4 and Figure 1.

[0243] Table 4 shows the in vivo evaluation of dsRNA molecules containing the monomeric structures of this disclosure in rats over 14 days.

[0244] The results show that, compared with the negative control molecule (QLAD-000270) without lipid delivery and the blank group, the dsRNA molecules disclosed in this study (QLAD-0002028, QLAD-002029, QLAD-002030) showed good mRNA knockdown effects in different CNS regions in rats during the 14-day experiment.

[0245] Example 4: Evaluation of dsRNA molecules comprising monomeric structures of the present disclosure linked between two nucleotides in a bulging manner in 42-day rat in vivo.

[0246] To assess the in vivo activity of SOD1 double-stranded nucleotides (also known as dsRNA molecules), rats were administered ICM.

[0247] Male rats were randomly divided into four groups: PBS group (solvent control group, n=4), QLAD-000270 (n=4), QLAD-0002028 (n=4), QLAD-002029 (n=4), and QLAD-002030 (n=4). For each test compound, it was dissolved and diluted with PBS to 7.5 μg / μL (0.3 mg injection dose) and 45 μg / μL (1.8 mg injection dose), and administered at a volume of 40 μL per rat. The specific experimental method was the same as in Example 3. Forty-two days after injection, each rat was sacrificed by CO2. The cerebellum, brainstem, thoracic spinal cord, cortex, and hippocampus tissues were then rapidly extracted and flash-frozen in liquid nitrogen. Data processing was the same as in Example 3. The results are shown in Table 5 and Figure 2.

[0248] Table 5 shows the in vivo evaluation of dsRNA molecules containing the monomeric structures of this disclosure in rats over 42 days.

[0249] The results show that, compared with the negative control molecule (QLAD-000270) without lipid delivery and the blank group, the dsRNA molecules disclosed in this study (QLAD-0002028, QLAD-002029, QLAD-002030) showed good mRNA knockdown effects in different CNS regions in rats during the 42-day experiment.

[0250] Example 5: Study on the positional effect of the disclosed monomer structure in the sense chain

[0251] To explore the effect of different positions of the monomer structure in the sense chain on activity, a position-by-position stepwise approach was used for in vitro evaluation. The list of tested compounds is as follows:

[0252] Table 6 shows the modified SOD1 sense and antisense nucleotide sequences.

[0253] To test the knockdown effect of siRNA on SOD1 mRNA, we transfected CT26 cells using Lipofectamine 2000 (Invitrogen, 1166801) as described. TM siRNA conjugate transfection was performed using RNAiMAX (Invitrogen, 13778500) transfection reagent (50 nM, diluted 1:3, 9 concentrations). The siRNA conjugate was transfected at a concentration of 1.0 x 10⁻⁶. 4 Cells were seeded at a density per well in a 96-well plate.

[0254] Forty-eight hours after transfection, mRNA was extracted using Thermofisher Kingfisher and its matching reagents (Thermofisher, 5400930) and TaqMan Fast. TM SOD1 mRNA levels were quantified using a one-step premix (Applied Biosystems, 4444434), mouse GAPDH, and a mouse SOD1 TaqMan probe. SOD1 mRNA expression was homogenized using GAPDH mRNA expression. Untransfected siRNA cells were used as a blank control. Results are shown in Table 7 and Figure 3.

[0255] Table 7 contains the in vitro evaluation of dsRNA molecules with monomeric structures disclosed herein.

[0256] The results showed that the siRNA monomer structure knocked down SOD1 at different positions in the sense strand in CT26 cells.

[0257] Example 6: Evaluation of dsRNA molecules comprising monomeric structures of the present disclosure linked in a bulging manner between two nucleotides in vivo in rats for 42 days.

[0258] Table 8. Nucleotide sequences of modified SOD1 sense and antisense strands

[0259] To assess the in vivo activity of SOD1 double-stranded nucleotide (also known as dsRNA molecule), rats were administered the drug via ICM, following the administration method described in Example 3.

[0260] On day 42 post-injection, each rat was sacrificed by CO2. Cortical, hippocampal, and brainstem samples were then rapidly extracted and flash-frozen in liquid nitrogen. Subsequently, mRNA was extracted using Thermofisher Kingfisher reagent and its accompanying reagents (Thermofisher, 5400930) and analyzed using TaqMan Faster. TM SOD1 mRNA levels were quantified using a one-step premix (Applied Biosystems, 4444434), human GAPDH, and the rat SOD1 TaqMan probe. SOD1 mRNA expression was normalized using GAPDH mRNA expression. Results were normalized to those from the PBS group, and relative expression levels of the target gene were calculated using a 2-1... -ΔΔCT The calculation formula is as follows:

[0261] ΔCT = Average Ct value of target gene (SOD1) - Average Ct value of internal reference gene (GAPDH);

[0262] ΔCT = ΔCT (drug-treated group) - ΔCT (PBS control group);

[0263] relative mRNA expression level = 2 -ΔΔCT The results are shown in Table 9 and Figure 4.

[0264] Table 9 shows the in vivo evaluation of dsRNA molecules containing the monomeric structures of this disclosure in rats over 42 days.

[0265] The results showed that, at 42 days, the monomeric dsRNA molecules with a bulging structure linked between two nucleotides all exhibited good inhibitory activity.

[0266] Example 7 evaluates the efficacy of dsRNA molecules with monomeric structures linked between two nucleotides in a bulging manner, as disclosed in this disclosure, in mouse adipose tissue and other tissues.

[0267] Table 10 shows the modified SOD1 sense and antisense nucleotide sequences.

[0268] The Uda structure is as follows:

[0269] To assess the in vivo activity of SOD1 double-stranded nucleotides (also known as dsRNA molecules), mice were administered the drug intravenously.

[0270] On day 28 post-injection, each mouse was sacrificed by CO2. Liver, heart, kidney, quadriceps femoris muscle, inguinal fat (iWAT), epididymal fat (eWAT), and pre-abdominal fat (BAT) tissues were then rapidly extracted and flash-frozen in liquid nitrogen. Subsequently, mRNA was extracted using Thermofisher Kingfisher and its accompanying reagents (Thermofisher, 5400930) and analyzed using TaqMan Fast. TM SOD1 mRNA levels were quantified using a one-step premix (Applied Biosystems, 4444434), human GAPDH, and a mouse SOD1 TaqMan probe. SOD1 mRNA expression was normalized using GAPDH mRNA expression assays. Results were normalized to those from the PBS group, and relative expression levels of the target gene were calculated using a 2-1... -ΔΔCT The calculation formula is as follows:

[0271] ΔCT = Average Ct value of target gene (SOD1) - Average Ct value of internal reference gene (GAPDH);

[0272] ΔCT = ΔCT (drug-treated group) - ΔCT (PBS control group);

[0273] relative mRNA expression level = 2 -ΔΔCT The results are shown in Table 11 and Figure 5.

[0274] Table 11 In vivo evaluation of SOD1 expression levels of dsRNA molecules containing monomeric structures of this disclosure after 28 days.

[0275] The results showed that at 28 days, the monomeric dsRNA molecule with a bulging structure linked between two nucleotides had an inhibitory effect on various tissues comparable to the control QLAD-002586.

Claims

1. A dsRNA molecule comprising an antisense strand and a sense strand complementary to the antisense strand, the sense strand including at least one monomeric structure connected in a bulging manner between any two nucleotides, the monomeric structure including a lipophilic portion, and the monomeric structure not affecting the inherent nucleotide base pairing of the dsRNA.

2. The dsRNA molecule as described in claim 1, characterized in that: The meaningful chain includes at least one structural unit 1 as shown in formula a, and the antisense chain includes at least one structural unit 2 as shown in formula b, which is complementary to structural unit 1. in: In formula a, Q1 and Q2 are the T-th and T+1-th nucleotides on the sense strand starting from the 5' end, respectively, where T is a natural number not greater than 30, and M is a monomeric structure connected between Q1 and Q2 in a bulging form. It does not pair complementaryly with structural unit 2. The monomeric structure contains a lipophilic portion, and Q1, M and Q2 are connected sequentially. In formula b, Q1' and Q2' are two nucleotides sequentially linked on the antisense strand and are complementary to Q1 and Q2 on the sense strand, respectively.

3. The dsRNA molecule according to any one of claims 1-2, characterized in that: Q1 is connected to M, and Q2 is connected to M by a phosphodiester bond, a thiophosphate diester bond, a hydrocarbon phosphate diester bond, or a thiohydrocarbon phosphate diester bond. Q1' and Q2' are connected by a phosphodiester bond or a thiophosphate diester bond. The structural formulas of the hydrocarbon phosphate diester bond and the thiohydrocarbon phosphate diester bond are shown below: Wherein, R is a hydrocarbon group, preferably C. 10-30 Aliphatic hydrocarbon group, further preferably C 16-22 Alkyl, more preferably C 16 straight-chain alkyl, C 22 Straight-chain alkyl groups.

4. The dsRNA molecule according to any one of claims 1-3, characterized in that: The lipophilic portion comprises a lipid group, which is selected from C 10-30 Aliphatic hydrocarbon group, C 9-29 Aliphatic hydrocarbon group -C(O)-, preferably C 16-22 Alkyl, C 15-21 Alkyl-C(O)-, more preferably C 16 straight-chain alkyl, C 15 Straight-chain alkyl-C(O)-, C 22 straight-chain alkyl, C 21 Straight-chain alkyl-C(O)-.

5. The dsRNA molecule according to any one of claims 1-4, characterized in that: The monomer structure contains the structure shown in Formula I: -R1-R2(I), in: R1 is selected from -O-, -NH-, and -S-; R2 is selected from C 10-30 Aliphatic hydrocarbon group, C 9-29 Aliphatic hydrocarbon group -C(O)-, preferably C 16-22 Alkyl, C 15-21 Alkyl-C(O)-, more preferably C 16 straight-chain alkyl, C 15 Straight-chain alkyl-C(O)-, C 22 straight-chain alkyl, C 21 Straight-chain alkyl-C(O)-.

6. The dsRNA molecule according to any one of claims 1-5, characterized in that: The monomer structure has any one or more of the following formulas: I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9 and I-10, or their pharmaceutically acceptable salt forms: in: R1 is selected from -O-, -NH-, and -S-, and R2 is selected from C. 10-30 Aliphatic hydrocarbon group, C 9-29 Aliphatic hydrocarbon group -C(O)-, preferably C 16-22 Alkyl, C 15-21 Alkyl-C(O)-, more preferably C 16 straight-chain alkyl, C 15 Straight-chain alkyl-C(O)-, C 22 straight-chain alkyl, C 21 Straight-chain alkyl-C(O)-; R3 is selected from -O-, -CH2-, -S-, and -NH-, with -O- being the preferred choice; R4 is selected from -P(=O)(OH)-, -P(=S)(OH)- and their salts, and R5 does not exist; or, R5 is selected from -P(=O)(OH)-, -P(=S)(OH)- and their salts, and R4 does not exist; R6 is selected from H, natural or modified bases; n is an integer selected from 0 to 2, preferably 0 or 1; X is selected from -O- and -S-, with -O- being preferred; R7 is selected from C 10-30 Aliphatic hydrocarbon group, preferably C 16-22 Alkyl, more preferably C 16 straight-chain alkyl, C 22 Straight-chain alkyl; R8 is selected from H, OH, halogen, unsubstituted or C-substituted. 1-6 Alkoxy, C 1-6 Alkylamine, OH, amino, (C 1-6 Alkyl)2N-C 1-6 alkoxy-substituted C 1-6 Alkoxy, C 2-6 alkenyl-O-,C 1-6 Alkyl-NH-C 1-6 Alkyl-C(=O)-O-; preferably H, OH, F, OMe, methoxyethoxy (MOE-O-), N-methyl-acetoxy, N,N-dimethylethoxyethoxy, allyl-O-, N-methyl-acetoxy; further preferably H, OH, F, OMe, methoxyethoxy (MOE-O-); more preferably H, OMe, F.

7. The dsRNA molecule according to any one of claims 1-6, characterized in that: The monomer structure has any one or more of the following general formulas or pharmaceutically acceptable salt forms: in: R3 is selected from -O-, -CH2-, -S-, and -NH-, with -O- being the preferred choice; R4 is selected from -P(O)(OH)-, -P(S)(OH)- and their salts, and R5 does not exist; or, R5 is selected from -P(O)(OH)-, -P(S)(OH)- and their salts, and R4 does not exist; R6 is selected from H, natural or modified bases; n is an integer selected from 0 to 2, preferably 0 or 1; X is selected from -O- and -S-, with -O- being preferred; R8 is selected from H, OH, halogen, unsubstituted or C-substituted. 1-6 Alkoxy, C 1-6 Alkylamine, OH, amino, (C 1-6 Alkyl)2N-C 1-6 alkoxy-substituted C 1-6 Alkoxy, C 2-6 alkenyl-O-,C 1-6 Alkyl-NH-C 1-6 Alkyl-C(=O)-O-; preferably H, OH, F, OMe, methoxyethoxy (MOE-O-), N-methyl-acetoxy, N,N-dimethylethoxyethoxy, allyl-O-, N-methyl-acetoxy; further preferably H, OH, F, OMe, methoxyethoxy (MOE-O-); more preferably H, OMe, F.

8. The dsRNA molecule according to any one of claims 1-7, characterized in that: The monomer structure has any one or more of the following general formulas or pharmaceutically acceptable salt forms: in: R4 is selected from -P(O)(OH)-, -P(S)(OH)- and their salts, and R5 does not exist; or, R5 is selected from -P(O)(OH)-, -P(S)(OH)- and their salts, and R4 does not exist; X is selected from -O- and -S-, with -O- being preferred; R8 is selected from H, OH, halogen, unsubstituted or C-substituted. 1-6 Alkoxy, C 1-6 Alkylamine, OH, amino, (C 1-6 Alkyl)2N-C 1-6 alkoxy-substituted C 1-6 Alkoxy, C 2-6 alkenyl-O-,C 1-6 Alkyl-NH-C 1-6 Alkyl-C(=O)-O-; preferably H, OH, F, OMe, methoxyethoxy (MOE-O-), N-methyl-acetoxy, N,N-dimethylethoxyethoxy, allyl-O-, N-methyl-acetoxy; further preferably H, OH, F, OMe, methoxyethoxy (MOE-O-); more preferably H, OMe, F.

9. The dsRNA molecule according to any one of claims 1-8, characterized in that: The monomer structure is any one or more of the following formula or its pharmaceutically acceptable salt forms:

10. The dsRNA molecule according to any one of claims 1-9, characterized in that: The sense strand of the dsRNA molecule contains one of the monomeric structures.

11. The dsRNA molecule according to any one of claims 1-10, characterized in that: The monomeric structure is located in the dsRNA molecule between any two nucleotides from position 1 to 20 of the sense strand starting from the 5' end, preferably between any two nucleotides from position 4 to 18, more preferably between any two nucleotides from position 4 to 8 and position 15 to 18 of the sense strand starting from the 5' end, such as between nucleotides from position 4 to 5, position 5 to 6, position 6 to 7, position 7 to 8, position 15 to 16, position 16 to 17, or position 17 to 18 of the sense strand starting from the 5' end.

12. The dsRNA molecule according to any one of claims 1-11, characterized in that: The nucleotides in the dsRNA molecule are unmodified or modified, and at least one modified nucleotide includes: 2'-O-methyl nucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, locked nucleotide, open-ring nucleotide (UNA), glycol nucleotide (GNA), 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, baseless nucleotide, ribitol, reverse nucleotide, reverse baseless nucleotide, reverse 2'-OMe nucleotide, reverse 2'-deoxynucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholinonucleotide and 3'-OMe nucleotide, nucleotide containing a 5'-thiophosphate group, terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bis(decyl)amide group, 2'-amino modified nucleotide, aminophosphate, or non-natural bases containing nucleotides, containing E-vinylphosphonate nucleotides at the 5' end of the sense strand and / or antisense strand, or linked between at least one thiophosphate nucleotide in the sense strand and / or antisense strand.

13. The dsRNA molecule according to any one of claims 1-12, characterized in that: The length of the double-stranded region of the dsRNA molecule can be 19 to 30 nucleotide pairs; or 19 to 25 nucleotide pairs; or 19 to 23 nucleotide pairs; or 19 to 21 nucleotide pairs; or 21 to 23 nucleotide pairs; or the length of each strand independently does not exceed 30 nucleotides; or the length of each strand independently does not exceed 25 nucleotides; or the length of each strand independently does not exceed 23 nucleotides; or the length of each strand independently does not exceed 21 nucleotides; or the length of each strand independently does not exceed 19 nucleotides.

14. The dsRNA molecule according to any one of claims 1-13, characterized in that: The dsRNA molecule has two blunt ends; or at least one strand contains a 3' overhang of at least one nucleotide; or at least one strand contains a 3' overhang of at least two nucleotides.

15. The following compound or its salt: in: R1 is selected from -O-, -NH-, and -S-; R2 is selected from C 10-30 Aliphatic hydrocarbon group, C 9-29 Aliphatic hydrocarbon group -C(O)-, preferably C 16-22 Alkyl, C 15-21 Alkyl-C(O)-, more preferably C 16 straight-chain alkyl, C 15 Straight-chain alkyl-C(O)-, C 22 straight-chain alkyl, C 21 Straight-chain alkyl-C(O)-; R3 is selected from -O-, -CH2-, -S-, and -NH-, with -O- being the preferred choice; R6 is selected from H, natural or modified bases; n is an integer selected from 0 to 2, preferably 0 or 1; R7 is selected from C 10-30 Aliphatic hydrocarbon group, preferably C 16-22 Alkyl, more preferably C 16 straight-chain alkyl, C 22 Straight-chain alkyl; R8 is selected from H, OH, halogen, unsubstituted or C-substituted. 1-6 Alkoxy, C 1-6 Alkylamine, OH, amino, (C 1-6 Alkyl)2N-C 1-6 alkoxy-substituted C 1-6 Alkoxy, C 2-6 alkenyl-O-,C 1-6 Alkyl-NH-C 1-6 Alkyl-C(=O)-O-; preferably H, OH, F, OMe, methoxyethoxy (MOE-O-), N-methyl-acetoxy, N,N-dimethylethoxyethoxy, allyl-O-, N-methyl-acetoxy; more preferably H, OH, F, OMe, methoxyethoxy (MOE-O-); more preferably H, OMe, F; R9 is selected from hydroxyl protecting groups, preferably benzyl, benzoyl, 2,6-dichlorobenzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, methanesulfonyl, toluenesulfonyl, dimethoxytriphenylmethyl (DMTr), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX), more preferably DMTr; R 10 Selected from cyano-substituted C1-C6 alkyl groups, preferably cyanoethyl, cyanopropyl, cyanobutyl, and more preferably cyanoethyl (CN-(CH2)2-); R 11 R 12 Each independently is C1-C 10 Alkyl group, preferably C1-C6 alkyl group, more preferably isopropyl group.

16. The compound or a salt thereof as claimed in claim 15, characterized in that, The compound is selected from: Among them, R1, R3, R6, R8, R9, R 10 R 11 R 12 n is as defined in claim 15.

17. The compound or a salt thereof as described in any one of claims 15-16, characterized in that, The compound is selected from: Among them, R8, R9, R 10 R 11 R 12 n is as defined in any one of claims 15-16.

18. The compound or a salt thereof as claimed in any one of claims 15-17, characterized in that, The compound is selected from:

19. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises any one or more dsRNA molecules as described in any one of claims 1-14 and a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 19, characterized in that: The pharmaceutical composition is an injectable dosage form, an oral dosage form, a pulmonary or nasal dosage form, or a locally delivered dosage form.

21. The use of the dsRNA molecule as described in any one of claims 1-14 or the pharmaceutical composition as described in any one of claims 19-20 in extrahepatic delivery (e.g., central nervous system, adipose tissue, muscle tissue, lung, eye, heart, groin, etc.).

22. A method for reducing the expression of a target gene in a cell, the method comprising contacting the cell with a dsRNA molecule as described in any one of claims 1-14 or a dsRNA molecule in a pharmaceutical composition as described in any one of claims 19-20, wherein the cell is an extrahepatic cell.

23. The method as described in claim 22, characterized in that: The target genes are selected from SOD1, APP, SNCA, GPR75, SCN9A, PMP22, MAPT, Leptin, MSTN1, MALAT1, Adiponectin, ACTR2, ACVR1C, ACVR2A, ACVR2B, etc.

24. The method according to any one of claims 22-23, characterized in that: The method reduces the expression of target genes in cells of the central nervous system, which are derived from one or more of the following groups: cerebellum, brainstem, thoracic vertebrae, thoracic spinal cord, thoracic DRG, striatum, cortex, lumbar vertebrae, hippocampus, heart, quadriceps femoris muscle, inguinal fat (iWAT), epididymal fat (eWAT), mesenteric fat (vAT), and pre-abdominal fat (BAT).

25. The use of the dsRNA molecule as described in any one of claims 1-14 or the pharmaceutical composition as described in any one of claims 19-20 in the prevention and / or treatment of diseases associated with disorders of the lungs, fat, muscles, eyes, heart, groin, and central nervous system (CNS).

Images