Lipid conjugate for delivering drug to adipose tissue

Abstract

Provided in the present application is a compound or pharmaceutically acceptable salt thereof for delivering an oligonucleotide-based reagent (e.g. a small interfering RNA (siRNA) and an antisense oligonucleotide (ASO)) to adipose tissue and / or adipocytes in vivo, the compound comprising an oligonucleotide, and at least one lipid moiety and at least one polyethylene glycol ether chain-containing moiety. Also provided in the present application are a pharmaceutical composition comprising the compound or pharmaceutically acceptable salt thereof, a preparation method for the compound or the pharmaceutically acceptable salt, and the use thereof for delivering oligonucleotides and / or treating diseases.

Description

Lipid conjugates for drug delivery to adipose tissue Technical Field

[0001] This disclosure pertains to the field of biomedicine, specifically relating to lipid conjugates, their preparation methods and applications. More specifically, this disclosure relates to lipid conjugates for delivering oligonucleotide-based reagents (e.g., small interfering RNA (siRNA) and antisense oligonucleotides (ASO)) to adipose tissue and / or adipocytes in vivo, their preparation methods and applications. Background Technology

[0002] Modified nucleic acids show great potential as a research tool in the laboratory and a therapeutic agent in the clinic in regulating gene expression. Several classes of oligonucleotide or nucleic acid-based therapeutic agents are in clinical trials, including antisense oligonucleotides (ASO), short interfering RNA (siRNA), double-stranded nucleic acids (dsRNA), aptamers, ribozymes, exon skipping and splicing altered oligonucleotides, immunomodulatory oligonucleotides, mRNA, and CRISPR.

[0003] Oligonucleotide (e.g., siRNA and ASO) technologies offer significant advantages in target selection, therapeutic efficiency, and specificity, and have achieved considerable success in drug development, with the technology continuing to evolve. However, delivery technology remains a key bottleneck in siRNA drug development. While consistent delivery to hepatocytes using carbohydrates such as N-acetylgalactosamine has been well-established, significant challenges remain in achieving specific and selective delivery of oligonucleotide-based drugs to extrahepatic cells. For example, nanostructure delivery may trigger immune responses, requiring further research to avoid potential immunogenicity and improve payload release; aptamer delivery, although showing progress in animal disease models, still faces difficulties in clinical translation; and the practical application of peptide-conjugated delivery requires further clinical trials for verification.

[0004] Direct delivery of siRNA to relatively closed or easily accessible tissues such as the central nervous system, eyes, lungs, and skin can enhance siRNA retention in target tissues and ensure adequate cellular uptake. However, existing delivery strategies, such as cholesterol conjugates and lipid nanoparticles (LNPs), have significant limitations. While cholesterol conjugates can promote cellular uptake of siRNA, their non-specific distribution leads to siRNA distribution in non-target tissues and organs, and toxicity issues are frequently reported. Similarly, although LNPs are widely used for nucleic acid delivery, they also suffer from non-specific distribution and potential toxicity problems. Therefore, despite the progress these delivery systems have made in some areas, they still cannot meet the need for efficient and specific delivery of siRNA to non-hepatocytes.

[0005] Therefore, there is still a need for a delivery vector that targets non-hepatic cell types (such as adipose tissue and / or adipocytes) to deliver oligonucleotide-based reagents, especially RNAi reagents, to non-hepatic cell types of tissues and cells. Summary of the Invention

[0006] The problem to be solved

[0007] This disclosure is intended to provide lipid conjugates for delivering therapeutic agents (particularly oligonucleotide-based agents) to target the therapeutic agents to adipose tissue and / or adipocytes.

[0008] Solution for solving the problem

[0009] This disclosure provides compounds (such as those shown in Formula (I)) comprising a lipid group conjugated (or linked) to an oligonucleotide-based reagent for delivering the linked or conjugated oligonucleotide to adipose tissue or certain cell types (e.g., adipocytes, such as subcutaneous adipose tissue cells, brown adipose tissue cells, gonadal adipose tissue cells). The lipid group in the compound (also referred to herein as a lipid PK / PD modulator) facilitates the delivery of the oligonucleotide-based payload to adipocytes or adipose tissue.

[0010] In one aspect, this disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, said compound comprising an oligonucleotide, and at least one lipid group and at least one group containing a polyethylene glycol ether chain.

[0011] [Lipid group]-[Oligo]-[group containing polyethylene glycol ether chain] Formula (I)

[0012] Oligo is an oligonucleotide with a length of about 8 to about 50 nucleotides, wherein each nucleotide may be independently modified or unmodified.

[0013] The lipid group has the structure shown in formula (Ia):

[0014] Wherein, L1 is selected from a bond or a linker connected to the oligonucleotide;

[0015] A1 is selected from chain-like saturated aliphatic hydrocarbon groups optionally substituted with one or more halogens or hydroxyl groups, preferably straight-chain or branched C groups optionally substituted with one or more halogens or hydroxyl groups. 14-28 Chain-like saturated aliphatic hydrocarbon groups;

[0016] R 1 Selected from hydrogen, hydroxyl, C1-C6 alkyl, C3-C6 cycloalkyl, halogen or C1-C6 haloalkyl, wherein the C1-C6 alkyl and C1-C6 haloalkyl are optionally substituted with one or more hydroxyl groups;

[0017] The groups containing polyethylene glycol ether chains have the structure shown in formula (Ib):

[0018] L2 is selected from phosphate ester linking groups or phosphate ester derivative linking groups;

[0019] When present, L3 is selected from a bond or a linker that can connect the PEG molecule to L2;

[0020] Y is selected from bonds, hydroxyl groups, methoxy groups, -O- or -OCH2-;

[0021] When present, A2 is selected from one or more R's. 5 The substituted chain-like saturated aliphatic hydrocarbon group, preferably optionally replaced by one or more R groups. 5 Replacement of straight or branched C 10-22 Chain-like saturated aliphatic hydrocarbon group, the R 5 Each is independently selected from OH or COOH;

[0022] L4 is selected from a key or a connector used to connect A2;

[0023] Each PEG is independently selected from PEGs with a weight average molecular weight of 100 Da to 10000 Da, preferably 100 Da to 6000 Da, or each PEG independently contains n polyethylene glycol repeating units, wherein n is greater than or equal to 2; or each PEG independently contains two or more polyethylene glycol repeating units (i.e., -OCH2CH2-), more preferably containing no more than 150 polyethylene glycol units.

[0024] q is selected from an integer between 1 and 5, and when q is selected from an integer greater than 2, L2-(L3)-PEG may be the same or different from each other.

[0025] L1 is selected from bonds, phosphate ester linking groups, or phosphate ester derivative linking groups, for example, bonds, Preferably, L1 is selected from bonds,

[0026] In a specific implementation, A1 is selected from linear C. 14-28 Chain-like saturated aliphatic hydrocarbon group, the straight-chain C 14-28 The chain-like saturated aliphatic hydrocarbon group is optionally substituted with one or more hydroxyl groups, Cl, or F, preferably with one F. In some embodiments, A1 is selected from straight-chain C. 14 C 15 C 16 C 17 C 18 C 19 C20 C 21 C 22 C 23 C 24 C 25 C 26 C 27 Or C 28 The chain-like saturated aliphatic hydrocarbon group is optionally substituted with one or more Cl or F groups, preferably with one F group. In some embodiments, A1 is selected from straight-chain C. 16 C 18 C 20 Or C 22 Chain-like saturated aliphatic hydrocarbon groups, with straight-chain C being particularly preferred. 16 Or C 22 The chain-like saturated aliphatic hydrocarbon group is optionally substituted with one or more Cl or F, preferably substituted with one F.

[0027] In a specific implementation, A1 is selected from... In a more preferred embodiment, A1 is selected from any of the following structures:

[0028] In some implementations, R 1 Selected from hydrogen, hydroxyl, C1-C6 alkyl, halogen, or C1-C6 haloalkyl, wherein the C1-C6 alkyl and C1-C6 haloalkyl are optionally substituted with one or more hydroxyl or halogens. In some embodiments, R 1 The components are selected from hydrogen, hydroxyl, C1-C3 alkyl, halogen, or C1-C3 haloalkyl, wherein the C1-C3 alkyl and C1-C3 haloalkyl are optionally substituted with one or more hydroxyl or halogens.

[0029] In a preferred embodiment, R 1 Selected from hydrogen, hydroxyl, methyl, ethyl, Cl, F, -CH2OH, -CH2F, -CHF2, or -CF3. In a preferred embodiment, R 1 It is hydrogen.

[0030] In a specific implementation, structural unit Optional In a preferred embodiment, the structural unit for

[0031] In this application, the lipid groups represented by formula (Ia) may be selected from: Where X is O or S; Where X is O or S;

[0032] In a specific embodiment, the lipid group represented by formula (Ia) may be selected from any of the following:

[0033] Where X is O or S;

[0034] Where X is O or S;

[0035] Where X is O or S;

[0036] Where X is O or S;

[0037] Preferably, the lipid group represented by formula (Ia) can be selected from any of the following:

[0038] In some embodiments, the compound of formula (I) of this application or a pharmaceutically acceptable salt thereof contains one, two or three lipid groups of formula (Ia), preferably containing one lipid group of formula (Ia).

[0039] In this application, the lipid group may be attached to the 5' end of the oligonucleotide. For example, the lipid group shown in formula (Ia) may be attached to a nucleotide residue or a 5' modification group at the 5' end of the oligonucleotide, such as a modified or unmodified nucleotide residue. In some embodiments, if the 5' end nucleotide residue does not have a phosphate group, at least one lipid group shown in formula (Ia) may be attached to a hydroxyl group, ribose group, or nucleobase group of a modified or unmodified nucleotide residue at the 5' end of the oligonucleotide; or, if the 5' end nucleotide residue has a phosphate group, at least one lipid group shown in formula (Ia) may be attached to a phosphate group, ribose group, or nucleobase group of a modified or unmodified nucleotide residue at the 5' end of the oligonucleotide.

[0040] In a specific embodiment, the 5' modifying group may be selected from a phosphate derivative (including, without limitation, phosphate ester derivatives, such as thiophosphate groups, EVP), an amino group, or a debased nucleoside (e.g., inverse debased nucleotide residues (invAb)).

[0041] For example, when the lipid group represented by formula (Ia) is attached to the phosphate group of an oligonucleotide, or a phosphate derivative (including, without limitation, phosphate ester derivatives), L1 can be a bond. For example, when the lipid group represented by formula (Ia) is attached to the phosphate group of a modified or unmodified nucleotide residue at the 5' end of an oligonucleotide, the lipid group represented by formula (Ia) can be a bond.

[0042] In this application, for example, when the 5' modifying group of the oligonucleotide is (invAb), the structures of L1 and (invAb) can be as follows: Where X is independently either S or O, * indicates the direction toward the lipid group, and ** indicates the direction toward the oligonucleotide.

[0043] The compound represented by formula (I) of this application or a pharmaceutically acceptable salt thereof may contain 1, 2, 3, 4 and 5 groups containing polyethylene glycol ether chains as shown in formula (Ib), preferably containing 1 or 2 groups containing polyethylene glycol ether chains as shown in formula (Ib).

[0044] For the groups containing polyethylene glycol ether chains shown in formula (Ib) of this application, they preferably contain two or more polyethylene glycol repeating units (i.e., -OCH2CH2-). More preferably, they contain no more than 150 polyethylene glycol units.

[0045] In some embodiments, the PEGs are each independently selected from PEGs with a weight-average molecular weight of 100 Da to 6000 Da; or, the PEGs are preferably PEGs having n repeating units (i.e., -OCH2CH2-), where n is an integer selected from 2 to 120.

[0046] In some embodiments of this application, n can be an integer between 3 and 6, for example, 3, 4, 5, or 6. In specific embodiments, q can be selected from 1, 2, 3, or 4.

[0047] In some implementations, n can be 3 and q can be 1, 2, or 3; or n can be 6 and q can be 1 or 2. In some implementations, q can be 2, wherein n can be independently selected from 3 or 6; or q can be 3 and n can be independently selected from 3 or 6.

[0048] In some embodiments, the groups containing polyethylene glycol ether chains shown in formula (Ib) preferably contain 3-20, more preferably 3-18, and more preferably 3-16 PEG repeating units.

[0049] In some implementations, the PEG may be selected from: or (MW is approximately 1000-5000 Da).

[0050] In a specific embodiment, L2 is selected from phosphate ester linking groups or phosphate ester derivative linking groups, such as phosphate ester linking groups or thiophosphate linking groups.

[0051] L3 can be selected from key, including Linkers, or linkers containing amide groups. In a specific embodiment, it includes... The connector can be Where R 2 and R 3 Each is independently selected from key, C 1-8 Alkylene or C 1-8 alkeneoxy, the C 1-8 Alkylene or C 1-8 The alkoxide may optionally be replaced by one or more halogens, amino groups, or hydroxyl groups.

[0052] In some implementations, R 2 and R 3 Each was independently selected from C 1-6 Alkylene or C 1-6 alkeneoxy, the C 1-6 Alkylene or C 1-6 The alkoxide may optionally be replaced by one or more halogens, amino groups, or hydroxyl groups.

[0053] Preferably, R 2 and R 3 Each is independently selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -C(CH3)2-, -CH2CH(CH3)2-, -CH(CH3)CH2CH3, -CH2CH(CH3)CH2CH3, -CH2C(CH3)2-, -CH2CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH(CH3)CH2CH2CH2CH2CH2-, -CH2CH(CH3)CH2CH2CH2CH2-, -OCH2-, -OCH2CH2 -, -OC(CH3)2-, -OCH2CH2CH2-, -OCH2CH2CH2CH2-, -OCH2CH2CH2CH2CH2-, -OCH2CH2CH2CH2CH2CH2-, -OCH2CH(CH3)2-, -OCH(CH3)CH2CH3, -OCH2CH(CH3)CH2CH3, -OCH2C(CH3)2-, -OCH2CH(CH3)CH2CH2CH2-, -OCH(CH3)CH2CH2CH2CH2-, -OCH(CH3)CH2CH2CH2CH2CH2-, or -OCH2CH(CH3)CH2CH2CH2CH2-, optionally substituted with one or more halogens, amino groups, or hydroxyl groups. For example, R 2 and R 3Each is independently selected from the bond, ethylidene, or hexanediol.

[0054] In some implementations, R 2 For Aziki, R 3 It is a bond or an ethylene.

[0055] In some embodiments, the linker containing the amide group may be selected from -R 4 -NH-C(O)-, where R 4 Selected from C 1-8 Alkylene or C 1-8 alkeneoxy, the C 1-8 Alkylene or C 1-8 The alkoxide may optionally be replaced by one or more halogens, amino groups, or hydroxyl groups.

[0056] For example, R 4 Can be selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -C(CH3)2-, -CH2CH(CH3)2-, -CH(CH3)CH2CH3, -CH2CH (CH3)CH2CH3, -CH2C(CH3)2-, -CH2CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH(CH3)CH2CH2CH2CH2CH2-, -CH2CH(CH3)CH2CH2CH2CH2-, -OCH2-, -OCH2CH2-, -OC(CH3)2-, -OCH2CH2CH2-, -OCH2CH2CH2CH2-, -OCH2CH2CH2CH2CH2-, -OCH2CH2CH2CH2CH2CH2-, -OCH2CH(CH3)2-, -OCH(CH3)CH2CH3, -OCH2CH(CH3)CH2CH3, -OCH2C(CH3)2-, -OCH2CH(CH3)CH2CH2-, -OCH(CH3)CH2CH2CH2-, -OCH(CH3)CH2CH2CH2CH2-, or -OCH2CH(CH3)CH2CH2CH2CH2-, which may optionally be substituted with one or more halogens, amino groups, or hydroxyl groups.

[0057] In some implementations, R 4 Selected from C 1-6 Alkylene or C 1-6 alkeneoxy, the C 1-6 Alkylene or C 1-6 The alkoxide may optionally be replaced by one or more halogens, amino groups, or hydroxyl groups.

[0058] In a specific implementation, L3 may be a key or have a structure selected from the following:

[0059] In a specific implementation, L3 may be a key or have a structure selected from the following:

[0060] In a specific implementation, L2-(L3)-PEG may be selected from the following: Where X is 0 or S, and n is an integer selected from 3 to 10; or, Where X is 0 or S, and n is an integer between 3 and 10.

[0061] In some implementations, L4 may be selected from a key, or have any of the following structures:

[0062] In some implementations, L4 may be selected from a key, or have any of the following structures:

[0063] In some implementations, L4 may be selected from a key, or have any of the following structures:

[0064] In some implementations, A2 can be selected from... Among them, R 5 Selected from OH or COOH;

[0065] Preferred

[0066] In some embodiments, the groups containing polyethylene glycol ether chains represented by formula (Ib) are selected from:

[0067] L2, L3, q, and PEG are defined as above; Y is selected from hydroxyl or methoxy groups.

[0068] When L4-A2 is present, Y is preferably selected from the bond, -O- or -O-CH2-.

[0069] In a specific embodiment, in the group containing the polyethylene glycol ether chain shown in formula (Ib), L2 is selected from phosphate ester linking groups or thiophosphate ester linking groups.

[0070] Alternatively, L3 can be a key or selected from...

[0071] Alternatively, Y can be selected from the key, -O-, or -OCH2-;

[0072] Alternatively, A2 is selected from Among them, R 5 Selected from OH or COOH;

[0073] Alternatively, L4 is selected from

[0074] Alternatively, each PEG independently contains 2 to 8 -OCH2CH2-, preferably selected from the PEG selected from:

[0075] Alternatively, q can be selected from integers between 1 and 5, and when q is selected from integers greater than 2, L2-(L3)-PEG can be the same or different from each other.

[0076] In a specific embodiment, in the group containing the polyethylene glycol ether chain shown in formula (Ib), L2 is selected from phosphate ester linking groups or thiophosphate ester linking groups.

[0077] Alternatively, L3 can be a key or selected from...

[0078] Alternatively, Y can be selected from the key, -O-, or -OCH2-;

[0079] Alternatively, A2 is selected from Among them, R 5 Selected from OH or COOH, preferably

[0080] Alternatively, L4 is selected from

[0081] Alternatively, each PEG independently contains 2 to 8 -OCH2CH2-, preferably selected from the PEG selected from:

[0082] Alternatively, q can be selected from integers between 1 and 3, and when q is selected from integers greater than 2, L2-(L3)-PEG can be the same or different from each other.

[0083] In a specific embodiment, in the group containing the polyethylene glycol ether chain shown in formula (Ib), L2 is selected from phosphate ester linking groups or thiophosphate ester linking groups.

[0084] Alternatively, L3 is selected from

[0085] Alternatively, Y can be selected from the key, -O-, or -OCH2-;

[0086] Alternatively, A2 is selected from Among them, R 5 Selected from OH or COOH, preferably

[0087] Alternatively, L4 is selected from

[0088] Alternatively, each PEG independently contains 3 to 6 -OCH2CH2-, preferably selected from the PEG selected from:

[0089] Alternatively, q can be 1.

[0090] In a specific embodiment, the group containing the polyethylene glycol ether chain shown in formula (Ib) can be selected from any of the following:

[0091] Where X is selected from O or S;

[0092] Where X is selected from O or S;

[0093] Where X is selected from O or S;

[0094] Each of X is independently selected from O or S;

[0095] Each of X is independently selected from O or S;

[0096] Where X is selected from O or S;

[0097] Each of X is independently selected from O or S;

[0098] Each of X is independently selected from O or S;

[0099] In this case, each X is independently selected from either O or S;

[0100] In this case, each X is independently selected from either O or S;

[0101] In this case, each X is independently selected from either O or S;

[0102] In this case, each X is independently selected from either O or S;

[0103] Where X is selected from O or S;

[0104] Where X is selected from O or S;

[0105] Where X is selected from O or S;

[0106] Where X is selected from O or S;

[0107] Where X is selected from O or S;

[0108] Where X is selected from O or S;

[0109] Where X is selected from O or S;

[0110] Where X is selected from O or S;

[0111] Where X is selected from O or S;

[0112] Where X is selected from O or S; or

[0113] Where X is selected from O or S.

[0114] In some preferred embodiments, the groups containing polyethylene glycol ether chains shown in formula (Ib) may be selected from any of the following:

[0115] In this context, X is independently selected from either O or S;

[0116] In this case, each X is independently selected from either O or S;

[0117] Where X is selected from O or S;

[0118] Where X is selected from O or S;

[0119] Where X is selected from O or S;

[0120] Where X is selected from O or S.

[0121] In some preferred embodiments, the groups containing polyethylene glycol ether chains shown in formula (Ib) may be selected from any of the following:

[0122] In this case, each X is independently selected from either O or S;

[0123] In this case, each X is independently selected from either O or S; Where X is selected from O or S;

[0124] Where X is selected from O or S;

[0125] Where X is selected from O or S;

[0126] Where X is selected from O or S;

[0127] Where X is selected from O or S;

[0128] Where X is selected from O or S; or

[0129] Where X is selected from O or S.

[0130] Preferably, the groups containing polyethylene glycol ether chains shown in formula (Ib) are selected from the following structures:

[0131] Preferably, the groups containing polyethylene glycol ether chains shown in formula (Ib) are selected from the following structures:

[0132] Preferably, the groups containing polyethylene glycol ether chains shown in formula (Ib) are selected from the following structures:

[0133] In this application, the group containing the polyethylene glycol ether chain can be attached to the 3' end of the oligonucleotide. For example, the group containing the polyethylene glycol ether chain shown in formula (Ib) can be attached to a nucleotide residue or a 3' modifying group at the 3' end of the oligonucleotide, such as a modified or unmodified nucleotide residue. In some embodiments, at least one group containing the polyethylene glycol ether chain shown in formula (Ib) can be attached to a ribose group or nucleobase group at the 3' end of a modified or unmodified nucleotide residue of the oligonucleotide, such as an OH group at the 3' position of the ribose group.

[0134] In a specific embodiment, the 3' modifying group may be selected from an amino group or a debased nucleoside (e.g., a reverse debased nucleotide residue (invAb)).

[0135] In this application, for example, when the 3' modifying group of the oligonucleotide is (invAb), the structure of one of the L2 groups and (invAb) can be as follows: Where X represents S or O, * indicates the direction toward the oligonucleotide, and ** indicates the direction toward the group containing the polyethylene glycol ether chain.

[0136] In some embodiments, the compound represented by formula (I) of this application or a pharmaceutically acceptable salt thereof is selected from the following structures:

[0137] The double helix structure represents dsRNA.

[0138] In some embodiments, the oligonucleotide in the compound or its pharmaceutically acceptable salt is a single-stranded or double-stranded oligonucleotide.

[0139] In some embodiments, the compound or a pharmaceutically acceptable salt thereof is preferably a double-stranded oligonucleotide, such as siRNA.

[0140] The double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense strand is complementary to the target gene; and the sense strand is complementary to the antisense strand.

[0141] In some implementations, the antisense strand comprises a sequence B whose inverse complementary sequence to at least 15 consecutive nucleotides in the target sequence differs by no more than 3 nucleotides. Specifically, a start nucleotide is selected in the target sequence along the 5' end to the 3' end, and at least 15 nucleotides extending in the 3' direction, including the start nucleotide, serve as the binding region of the siRNA. The antisense strand comprises the inverse complementary sequence of the nucleotide sequence corresponding to the binding region. It should be noted that the start nucleotide can be a nucleotide at any position in the target sequence, as long as extending in the 3' direction from the start nucleotide yields at least 15 consecutive nucleotides (including the nucleotide at the start position).

[0142] In this application, the nucleotide sequence of the antisense strand can be completely complementary or substantially complementary to the target sequence. When the nucleotide sequence of the antisense strand is substantially complementary to the target sequence, there are no more than three mismatched bases in the nucleotide sequence of the antisense strand that are different from the target sequence. For example, there may be one, two, or three mismatched bases. When the nucleotide sequence of the antisense strand is completely complementary to the target sequence, there are no mismatched bases in the nucleotide sequence of the antisense strand and the target sequence.

[0143] Furthermore, the antisense strand consists of at least 15 nucleotides. In some embodiments, the antisense strand consists of 15-28 nucleotides. For example, the length of the antisense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides. Preferably, the antisense strand consists of 18-25 nucleotides, more preferably 18-23 nucleotides, and most preferably 19, 21, or 23 nucleotides.

[0144] In some specific embodiments, the nucleotide that differs from the target sequence is preferably located at the end of the antisense strand. For example, in some specific embodiments, the differing nucleotide is located at the 3' end of the antisense strand. In other specific embodiments, the differing nucleotide is located at the 5' end of the antisense strand.

[0145] In some embodiments, the sense strand comprises sequence A, which differs from at least 15 consecutive nucleotides in the target sequence by no more than 3 nucleotides. The sense strand includes regions complementary to the antisense strand, and the nucleotide sequence of the sense strand is identical or substantially identical to the sequence of the antisense strand binding region on the target sequence. Therefore, the nucleotide sequence of the sense strand comprises / is at least 15 consecutive nucleotides in the target sequence that bind the antisense strand; or, the nucleotide sequence of the sense strand differs from at least 15 consecutive nucleotides in the target sequence that bind the antisense strand by 1, 2, or 3 bases.

[0146] In some embodiments, the sense strand comprises a sequence differing from at least 15 consecutive nucleotides in the target sequence by no more than 3 nucleotides. The sense strand includes regions complementary to the antisense strand, and the nucleotide sequence of the sense strand is identical or substantially identical to the sequence of the antisense strand binding region on the target sequence. Therefore, the nucleotide sequence of the sense strand is at least 15 consecutive nucleotides in the target sequence that bind the antisense strand; or, the nucleotide sequence of the sense strand differs from at least 15 consecutive nucleotides in the target sequence that bind the antisense strand by 1, 2, or 3 bases.

[0147] Furthermore, the positive sense strand consists of at least 15 nucleotides. In some embodiments, the positive sense strand consists of 15-28 nucleotides. For example, the length of the positive sense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides. Preferably, the positive sense strand consists of 18-25 nucleotides, more preferably 18-23 nucleotides, and most preferably 19, 21, or 23 nucleotides.

[0148] In some specific embodiments, the nucleotide that differs from the target sequence is preferably located at the end of the sense strand. For example, in some specific embodiments, the differing nucleotide is located at the 3' end of the sense strand. In other specific embodiments, the differing nucleotide is located at the 5' end of the sense strand.

[0149] In this disclosure, the length of the justice chain and the length of the antisense chain may be the same or different.

[0150] In some implementations, the lengths of the justice chain and the antisense chain are the same. Specifically, the ratio of the justice chain to the antisense chain length is 15 / 15, 16 / 16, 17 / 17, 18 / 18, 19 / 19, 20 / 20, 21 / 21, 22 / 22, 23 / 23, 24 / 24, 25 / 25, 26 / 26, 27 / 27, or 28 / 28. Preferably, the ratio of the justice chain to the antisense chain length is 18 / 18, 19 / 19, 20 / 20, 21 / 21, 22 / 22, 23 / 23, 24 / 24, or 25 / 25; more preferably, it is 19 / 19, 20 / 20, 21 / 21, 22 / 22, or 23 / 23; and most preferably, it is 19 / 19, 21 / 21, or 23 / 23.

[0151] In some implementations, the lengths of the justice chain and the antisense chain differ. For example, the justice chain / antisense chain length ratios are 18 / 19, 18 / 20, 18 / 21, 18 / 22, 18 / 23, 18 / 24, 18 / 25, 18 / 26, 19 / 18, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 19, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 18, 21 / 19 21 / 20, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 18, 22 / 19, 22 / 20, 22 / 21, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 19, 23 / 20, 23 / 21, 23 / 22, 23 / 24, 23 / 25, or 23 / 26, etc.; in some preferred embodiments, the length ratio of the justice chain to the antisense chain is 19 / 21 or 21 / 23.

[0152] In this disclosure, the sense strand and the antisense strand can be fully complementary or substantially complementary. When they are substantially complementary, there are no more than three (e.g., one, two, or three) mismatched bases in the double-stranded region formed by the sense strand and the antisense strand.

[0153] In some embodiments, the nucleotide sequence of the sense strand of the double-stranded ribonucleic acid comprises sequence A and optional sequence D, wherein the 3' end of sequence A in the sense strand is connected to sequence D consisting of 1-2 nucleotides; and / or, the nucleotide sequence of the antisense strand of the double-stranded ribonucleic acid comprises sequence B and optional sequence E, wherein the 3' end of sequence B in the antisense strand is connected to sequence E consisting of 1-2 nucleotides.

[0154] For example, 1-5 nucleotides are added to the 3' end of the sense strand sequence A to form sequence D, such as GA, CG, GC, CA, UC, AC, AA, UG, GG, CC, UU, CU, AG, or dTdT as sequence D. Similarly, 1-5 nucleotides are added to the 3' end of the antisense strand sequence B to form sequence E, such as UC, CG, AG, CU, GC, CA, UG, GG, UU, AC, CC, GA, AA, dTdT, CUG, or G. Optionally, the ribonucleotides in sequences D and E may be modified ribonucleic acids, such as 2'-O-CH3 modified ribonucleotides, 2'-deoxy modified ribonucleotides, or 2'-F modified ribonucleotides. Alternatively, the ribonucleotides in sequence E may be 2'-O-CH3 modified ribonucleotides, 2'-deoxy modified ribonucleotides, or 2'-F modified ribonucleotides (e.g., 2'-F modified guanine ribonucleotides).

[0155] In some embodiments, after the sense and antisense strands are at least partially complementary to form a double-stranded region, the sense, antisense, or combination thereof has protruding nucleotides extending out of the double-stranded region. The number of protruding nucleotides can be one or more, for example, one or two. Furthermore, the one or two protruding nucleotides can be located at the 5' end, 3' end, or both ends of any antisense or sense strand, and each protruding nucleotide can be of any type. For example, in the case where the sense strand consists of a 21-unit sequence A+D and the antisense strand consists of a 21-unit sequence B+E, the nucleotides at positions 1-19 of sequence A are completely anticomplementary to those at positions 1-19 of sequence B in the direction from the 5' end to the 3' end, thereby forming protruding nucleotides at the 3' end of sequences D and E, respectively. For example, when the sense strand consists of a 19-position sequence A and the antisense strand consists of a 21-position sequence B+E, the nucleotides 1-19 of sequence A are completely anticomplementary to those 1-19 of sequence B in the direction from the 5' end to the 3' end, thus forming a protruding nucleotide at the 3' end of sequence E on the antisense strand.

[0156] In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and the 3' end of the sense strand has 1-2 protruding nucleotides extending out of the double-stranded region, while the 3' end of the antisense strand is blunt. In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region, while the 3' end of the sense strand is blunt. In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and both the sense strand and the antisense strand have 1-2 protruding nucleotides extending out of the double-stranded region at their 3' ends. In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and both the sense strand and the antisense strand have blunt 3' ends.

[0157] In some embodiments, each nucleotide of the sense strand is independently a modified or unmodified nucleotide. In some embodiments, each nucleotide of the antisense strand is independently a modified or unmodified nucleotide.

[0158] In some embodiments, any two nucleotides linked in the sense strand are connected by a phosphodiester bond or a phosphothiodiester bond. In some embodiments, any two nucleotides linked in the antisense strand are connected by a phosphodiester bond or a phosphothiodiester bond.

[0159] In some embodiments, the ribosome group of the 5' terminal nucleotide of the positive strand is a 5' hydroxyl group.

[0160] In some embodiments, the ribosome of the 5' terminal nucleotide of the sense strand may have a 5' hydroxyl group, a 5' phosphate group, or a 5' phosphate-derived group. In some embodiments, the ribosome of the 5' terminal nucleotide of the antisense strand has a 5' hydroxyl group, a 5' phosphate group, or a 5' phosphate-derived group. The presence of the 5' group at the 5' position of the ribosome of the sense and / or antisense strand depends on the preparation method used, which is known to those skilled in the art based on the corresponding preparation method.

[0161] For example, the structure of the 5' phosphate group is as follows: The structure of the 5' phosphate derivative group includes, but is not limited to: wait.

[0162] Furthermore, the double-stranded oligonucleotide is a siRNA modifier.

[0163] In some embodiments, the siRNA modifier comprises a modification of at least one nucleotide. The nucleotide modification is selected from at least one of ribose group modification and base modification. In some embodiments, "nucleotide modification" refers to a nucleotide or nucleotide derivative formed by replacing the 2' hydroxyl group of the ribose group with another group, or a nucleotide in which the base is a modified base. The nucleotide modification does not result in a significant weakening or loss of the siRNA's ability to suppress gene expression. For example, modified nucleotides disclosed in JKWatts, G.F. Deleavey, and MJDamha, Chemically Modified siRNA: Tools and Applications. Drug Discov Today, 2008, 13(19-20):842-55 can be selected. Nucleotide modification can improve the stability of siRNA and maintain its high repressive efficiency against the SMAD2 gene.

[0164] For example, the modified nucleosides have the following structures:

[0165] Wherein, Base represents a base, such as A, U, G, C, or T. The hydroxyl group at the 2' position of the ribosome is replaced by R. These hydroxyl groups at the 2' position of the ribosome can be replaced by various groups known to those skilled in the art; for example, R can be selected from halogens, alkyl groups, alkoxy groups, substituted alkyl groups, and substituted alkoxy groups. For example, in some specific embodiments, the modified nucleotide includes, but is not limited to, 2'-fluoro(2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, and 2'-deoxyribonucleotides.

[0166] In some implementations, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe, 2'-O-CH3) modified nucleotide, etc.

[0167] In some embodiments, the 2'-substituted alkoxy modified nucleotide is a nucleotide modified with 2'-methoxyethoxy (2'-O-CH2-CH2-O-CH3), a nucleotide modified with 2'-O-CH2-CH=CH2, etc.

[0168] In some embodiments, the 2'-substituted alkyl-modified nucleotide is a 2'-CH2-CH2-CH=CH2 modified nucleotide, etc.

[0169] In some embodiments, the modification of the nucleotide is a modification of the bases. Base modifications can be of various types known to those skilled in the art. For example, base modifications include, but are not limited to, m... 6 A、Ψ、m 1 A、m 5 A, ms 2 i 6 A、i 6 A、m 3 C, m 5 C、ac 4 C, m 7 G, m 2,2 G, m 2 G, m 1 G, Q, m 5 U, mcm 5 U、ncm 5 U、ncm 5 Um, D, mcm 5 s 2 U, Inosine(I), hm 5 C, s 4 U、s 2 U, azobenzene, Cm, Um, Gm, t 6 A, yW, ms 2 t 6 A or its derivatives.

[0170] In some embodiments, a nucleotide derivative refers to a compound that can replace a nucleotide in a nucleic acid but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, the nucleotide derivative can be an isonucleotide, a bridged nucleic acid (BNA), or an acyclic nucleotide. A BNA refers to a restricted or conformationally restricted nucleotide. A BNA can contain a five-, six-, or seven-membered ring with a "fixed" C3'-endoglucan condensation bridging structure. This bridge is typically incorporated into the 2', 4' position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET, etc.

[0171] The nucleosides of LNA are shown in formula (1), the nucleosides of ENA are shown in formula (2), and the nucleosides of cET are shown in formula (3):

[0172] In this context, "Base" refers to a base.

[0173] In some implementations, acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide, such as unopenable nucleic acids (UNA) or glycerol nucleic acids (GNA), where UNA is shown as formula (4) and GNA is shown as formula (5):

[0174] In formulas (4) and (5) above, Base refers to a base, and R is selected from H, OH or alkoxy (-O-alkyl).

[0175] In some implementations, nucleotide derivative modification refers to the replacement of nucleotides in nucleic acids with nucleotide derivatives. Exemplary examples include isonucleotides, LNA, ENA, cET, UNA, or GNA.

[0176] In some embodiments, the nucleotides in the nucleic acid are replaced with isonucleotides, also referred to as isonucleotide modification in the context of this disclosure. In some embodiments, isonucleotide modification includes incorporating an isonucleotide at one or more sites on the sense and / or antisense strands of the siRNA to be modified, in place of the native nucleoside for coupling at the corresponding position.

[0177] In some embodiments, the isonucleoside modification is D-isonucleoside modification. In other embodiments, the isonucleoside modification is L-isonucleoside modification. In still other embodiments, the isonucleoside modification is a combination of D-isonucleoside and L-isonucleoside modification.

[0178] In some embodiments, the double-stranded ribonucleic acid (siRNA) modifier includes modification of a phosphodiester bond at at least one position. In some embodiments, the modification of the phosphodiester bond involves replacing at least one oxygen atom in the phosphodiester bond with a sulfur atom, thereby forming a phosphothiodiester bond. The phosphothiodiester bond can stabilize the double-stranded structure of siRNA and maintain the specificity of base pairing. An exemplary phosphothiodiester bond structure is shown below:

[0179] In some embodiments, the positive strand may contain one or more capping residues or portions, sometimes referred to in the art as a “cap,” “terminal cap,” or “capping residue.” As used herein, a “capping residue” refers to a nonnucleotide compound or other structural unit that can be incorporated into one or more ends of the nucleotide sequence of the siRNA disclosed herein. In some cases, capping residues may provide certain beneficial properties to the siRNA, such as protection against exonuclease degradation. In some embodiments, an inverse debased deoxyribose residue (invAb) is added as a capping residue. In some embodiments, the capping residue appears at the 3' end or both the 5' and 3' ends of the positive strand.

[0180] In some embodiments, one or more inverted debased deoxyribose residues (invAb) are added to the 3' end of the positive strand. In some embodiments, one or more inverted debased deoxyribose residues (invAb) are added to the 5' end of the positive strand. In some embodiments, one or more inverted debased deoxyribose residues (invAb) are added to both the 5' end and the 3' end of the positive strand. In some embodiments, the inverted debased deoxyribose residues may be linked via phosphate ester bonds, phosphothioester bonds, or other nucleoside internucleotide bonds. When describing modification sites in modification methods, (invAb) is not counted as the first site of the nucleotide sequence. In some embodiments, inverted debased deoxyribose residues (invAb) (also referred to in the art as "inverted debasing sites") may be added. The (invAb) may have the structure shown in the following formula:

[0181] Formula B is used when (invAb) is located at the 3' end of the siRNA; Formula A is used when (invAb) is located at the 5' end of the siRNA.

[0182] In formulas A and B, X is O or S, and the phosphate ester bonds in formulas A and B are the bonds toward the 5' (corresponding to formula A) and 3' (corresponding to formula B) ends of the siRNA being linked, respectively.

[0183] In some implementations, the siRNA modifier comprises at least one of the following chemical modifications:

[0184] (1) Modification of at least one nucleotide in the positive strand,

[0185] (2) Modification of the phosphodiester bond at at least one position in the positive chain.

[0186] (3) Modification of at least one nucleotide in the antisense strand,

[0187] (4) Modification of phosphodiester bonds at at least one position in the antisense chain.

[0188] Furthermore, the double-stranded RNA modifier is an siRNA modifier containing at least one of the chemical modifications in (1)-(4).

[0189] In some embodiments, the sense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 7, 9, 10, and 11 of the sense strand are 2'-fluoro-modified ribonucleotides; and the ribonucleotides at other positions of the sense strand are 2'-methoxy-modified ribonucleotides.

[0190] In some embodiments, when neither the 5' end nor the 3' end of the positive chain is connected (invAb), the positive chain contains a phosphothioester bond located at the following positions:

[0191] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0192] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0193] Between the first nucleotide and the second nucleotide starting at the 3' end of the positive strand; and / or

[0194] Between the second and third nucleotides starting at the 3' end of the positive strand;

[0195] or,

[0196] The positive chain contains phosphothiophosphate diester bonds located at the following positions:

[0197] Between the first nucleotide and the second nucleotide starting at the 5' end of the positive strand; and / or

[0198] Between the second and third nucleotides starting at the 5' end of the positive strand.

[0199] In some embodiments, when only one (invAb) is attached to the 5' end of the positive chain, the positive chain contains a phosphothioester bond located at the following positions:

[0200] Between (invAb) at the 5' end of the positive strand and the first nucleotide starting at the 5' end;

[0201] Between the first and second nucleotides starting at the 5' end of the positive strand.

[0202] In some embodiments, when only one (invAb) is attached to the 3' end of the positive chain, the positive chain contains a phosphothioester bond located at the following positions:

[0203] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0204] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0205] Between (invAb) at the 3' end of the positive strand and the first nucleotide starting at the 3' end.

[0206] In some embodiments, when the 5' end and 3' end of the positive chain are each connected to an (invAb), the positive chain contains a phosphothioester bond located at the following positions:

[0207] Between (invAb) at the 5' end of the positive strand and the first nucleotide starting at the 5' end;

[0208] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0209] Between (invAb) at the 3' end of the positive strand and the first nucleotide starting at the 3' end.

[0210] In some implementations, the ribosome of the 5' terminal nucleotide of the positive strand is a 5' hydroxyl group.

[0211] In some embodiments, the nucleotide structure of the ribosome with a 5' hydroxyl group at the 5' end of the positive strand is shown in Formula X:

[0212] Wherein, Base represents a base, such as A, U, G, C or T; R is a hydroxyl group or is substituted by various groups known to those skilled in the art, for example, R can be 2'-fluoro(2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino, 2'-deoxynucleotide.

[0213] In some embodiments, the positive strand of the siRNA modifier includes phosphothioester bonds at the following positions along the 5' end to the 3' end: between the first and second nucleotides starting at the 5' end, between the second and third nucleotides starting at the 5' end, between the first and second nucleotides starting at the 3' end, and between the second and third nucleotides starting at the 3' end.

[0214] In some implementations, the positive strand of the siRNA modifier includes phosphothioester bonds at the following positions along the 5'-to-3' direction: between the first and second nucleotides starting at the 5' end, and between the second and third nucleotides starting at the 5' end.

[0215] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at any odd-numbered positions in the antisense strand are 2'-methoxy modified ribonucleotides, and the ribonucleotides at any even-numbered positions in the antisense strand are 2'-fluoro modified ribonucleotides.

[0216] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: at least four ribonucleotides at positions 2-18 of the antisense strand are 2'-methoxy modified ribonucleotides, and the ribonucleotides at any even number of positions in the antisense strand are 2'-fluoro modified ribonucleotides.

[0217] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0218] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0219] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 6 of the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0220] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 7 of the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0221] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0222] In some embodiments, the antisense strand of the siRNA modifier includes phosphothioester bonds at the following positions along the 5' end to the 3' end: between the first and second nucleotides starting at the 5' end, between the second and third nucleotides starting at the 5' end, between the first and second nucleotides starting at the 3' end, and between the second and third nucleotides starting at the 3' end.

[0223] In embodiments of this application, the ribosome of the 5' terminal nucleotide of the antisense strand may have: a 5' hydroxyl group, a 5' phosphate group, or a 5' phosphate-derived group.

[0224] In the compounds of this application or pharmaceutically acceptable salts thereof, both the lipid group and the group containing the polyethylene glycol ether chain can be attached to the positive strand of a double-stranded ribonucleic acid (e.g., siRNA). For example, in one embodiment, a lipid group may be attached to the 5' end of the positive strand of the siRNA, and a group containing the polyethylene glycol ether chain may be attached to the 3' end of the positive strand of the siRNA.

[0225] In a specific embodiment, this application provides a mixture comprising one or more compounds of formula (I) or pharmaceutically acceptable salts thereof.

[0226] In some embodiments, this application provides a mixture comprising two or more compounds of formula (I) or pharmaceutically acceptable salts thereof.

[0227] In some embodiments, this application provides a mixture comprising the following:

[0228] The double helix structure represents dsRNA.

[0229] In some embodiments, this application provides a mixture comprising the following:

[0230] The double helix structure represents dsRNA.

[0231] In some embodiments, this application provides a mixture comprising the following:

[0232] The double helix structure represents dsRNA.

[0233] In some embodiments, this application provides a mixture comprising the following:

[0234] The double helix structure represents dsRNA.

[0235] In some embodiments, this application provides a mixture comprising the following:

[0236] The double helix structure represents dsRNA.

[0237] In some embodiments, this application provides a mixture comprising the following:

[0238] The double helix structure represents dsRNA.

[0239] In some embodiments, this application provides a mixture comprising the following:

[0240] The double helix structure represents dsRNA.

[0241] In a specific embodiment, the proportions of the compounds represented by each formula (I) or their pharmaceutically acceptable salts in the mixture can be any proportion.

[0242] Preferably, the compound of formula (I) or its pharmaceutically acceptable salt in the mixture comprises the same double-stranded oligonucleotide.

[0243] Preferably, the compound of formula (I) or its pharmaceutically acceptable salt in the mixture contains only the difference in... Two compounds or their pharmaceutically acceptable salts are attached to the triazole ring at different positions.

[0244] In one aspect, this application also provides an intermediate compound or a pharmaceutically acceptable salt thereof for preparing the above-described compounds of this application, having the following structure:

[0245] In one aspect, this application also provides a method for preparing the compound of Formula I as described in this disclosure or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0246] (1) Attach lipid groups and / or groups containing polyethylene glycol ether chains or their precursors to nucleoside phosphoramidide monomers;

[0247] (2) Synthesize the sense chain and antisense chain described in this application by solid-phase synthesis method;

[0248] (3) Anneal the sense and antisense chains to obtain the compound of Formula I as described in this application or a pharmaceutically acceptable salt thereof.

[0249] When using a precursor containing lipid groups and / or groups containing polyethylene glycol ether chains, the method further includes the step of further reacting the positive chain to contain the lipid groups and / or groups containing polyethylene glycol ether chains.

[0250] On the other hand, this application provides a pharmaceutical composition comprising the compounds described above or pharmaceutically acceptable salts thereof. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0251] In some embodiments, the pharmaceutically acceptable excipient may be, for example, a carrier, transporter, diluent, and / or delivery polymer.

[0252] On the other hand, this application provides the use of the above-described compound or its pharmaceutically acceptable salt or the above-described pharmaceutical composition in the preparation of a medicament.

[0253] In some embodiments, the drug is used to prevent and / or treat obesity-related or metabolic diseases; in other embodiments, the drug is used to prevent and / or treat obesity-related diseases.

[0254] In some embodiments, the drug is used to prevent and / or treat tumor-related diseases.

[0255] The diseases / conditions covered by the publicly available information in this article include, but are not limited to: obesity, type 2 diabetes, hyperlipidemia, etc.

[0256] In another aspect, this application further provides a method for delivering the compound of this application or a pharmaceutically acceptable salt thereof to a specific target in a subject via subcutaneous or intravenous administration. This application further provides a method for delivering the compound of this application or a pharmaceutically acceptable salt thereof to a specific target in a subject via subcutaneous or intravenous administration.

[0257] Another aspect of this application relates to a method for reducing the expression of a target gene in cells, the method comprising contacting the cells with a compound of the present application or a pharmaceutically acceptable salt thereof, the compound or a pharmaceutically acceptable salt thereof comprising an antisense strand complementary to the target gene; a sense strand complementary to the antisense strand; and one or more lipid groups and one or more groups containing polyethylene glycol ether chains.

[0258] In one embodiment, the cells are cells from adipose tissue.

[0259] In one implementation, the cell is not a liver cell.

[0260] Another aspect of this application relates to a method for reducing the expression of a target gene in a subject, the method comprising administering a compound or a pharmaceutically acceptable salt thereof to the subject, including contacting the cells with the compound or a pharmaceutically acceptable salt thereof, the compound or a pharmaceutically acceptable salt thereof comprising an antisense strand complementary to the target gene; a sense strand complementary to the antisense strand; and one or more lipid groups and one or more groups containing polyethylene glycol ether chains.

[0261] In some embodiments, the compound or its pharmaceutically acceptable saline is administered extrahepatically.

[0262] In some embodiments, exemplary target genes are SOD1, ALK7, CB1, and adiponectin.

[0263] Another aspect of this application relates to a method for treating a subject suffering from obesity or a subject with a tumor, the method comprising administering to the subject a therapeutically effective amount of the compound of this application or a pharmaceutically acceptable salt thereof, thereby treating the subject. The compound of this application or a pharmaceutically acceptable salt thereof comprises an oligonucleotide (e.g., siRNA), said oligonucleotide comprising an antisense strand complementary to a target gene; a sense strand complementary to said antisense strand; and one or more lipid groups and one or more groups containing polyethylene glycol ether chains. The compound of this application or a pharmaceutically acceptable salt thereof exhibits good inhibitory activity against the target gene in vivo, and can reduce the expression of the target gene for a prolonged period, thus possessing long-lasting effects. The types of diseases / conditions addressed in this disclosure include, but are not limited to, obesity, type 2 diabetes, and hyperlipidemia.

[0264] definition

[0265] Unless otherwise stated, the following terms as used in this application shall have the following meanings. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0266] In this art, “G,” “C,” “A,” “T,” and “U” typically represent the bases of guanine, cytosine, adenine, thymine, and uracil, respectively. However, it is also generally known in the art that each of “G,” “C,” “A,” “T,” and “U” typically also represents a nucleotide containing guanine, cytosine, adenine, thymine, and uracil as a base, respectively. This is a common practice in representing deoxyribonucleic acid (DNA) sequences and / or ribonucleic acid (RNA) sequences. Therefore, in the context of this disclosure, the meanings of “G,” “C,” “A,” “T,” and “U” include all of the above-mentioned possible cases. However, it should be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide (as further detailed below) or one with an alternative substitution. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be substituted with other parts without substantially altering the base-pairing properties of an oligonucleotide (including a nucleotide with such a substitution). For example, without limitation, a nucleotide including inosine as its base can be base-paired with a nucleotide including adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be replaced in the nucleotide sequence of the dsRNA characterized in this disclosure by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively, to form a GU swing base pairing with the target mRNA. Sequences containing such replacement portions are suitable for the compositions and methods characterized in this disclosure. The terms “double-stranded oligonucleotide,” “double-stranded RNA (dsRNA) molecule,” and “dsRNA” as used in the context of this disclosure are used interchangeably. 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, described as having “sense” and “antisense” orientation relative to a target gene, such as the SMAD2 gene. In some embodiments, double-stranded ribonucleic acid (dsRNA) triggers the degradation of target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).

[0267] Typically, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides; however, as detailed herein, each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Additionally, as used herein, “double-stranded ribonucleic acid” can include chemically modified ribonucleotides, phosphate backbones, etc. These modifications can include all types of modifications disclosed herein or known in the art.

[0268] As used in the context of this disclosure, the term "isonucleotide" refers to a compound formed by altering the position of a base on the ribose ring, for example, a compound formed by attaching a base to the 2' or 3' position of the ribose ring instead of the 1' position.

[0269] In some embodiments, the double-stranded RNA disclosed herein is siRNA, which interacts with the mRNA sequence transcribed from the target gene (e.g., the mRNA sequence transcribed from the SMAD2 gene) to guide the cleavage of the target RNA. Not wishing to be bound by theory, long double-stranded RNA introduced into the cell is broken down into siRNA by a type III nuclease called Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer (a type III ribonuclease-like enzyme) processes dsRNA into short 19–23 base pairs of interfering RNA with a characteristic dibase 3' overhang (Bernstein et al., (2001) Nature 409:363). These siRNAs are then incorporated into the RNA-inducible silencing complex (RISC), where one or more helicases unwind the siRNA duplex, enabling complementary antisense strands to guide target recognition (Nykanen et al., (2001) Cell 107:309). Once bound to a suitable target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188).

[0270] As used in the context of this disclosure, the term "protruding nucleotide" refers to one or more unpaired nucleotides that protrude from the double-stranded structure of a dsRNA when one 3' end of one strand extends beyond the 5' end of the other strand, or vice versa. "Flat-ended" or "blunt-terminated" means that there are no unpaired nucleotides at the said end of the double-stranded ribonucleic acid, i.e., no nucleotide protrusions. A "flat-terminated" double-stranded ribonucleic acid is a dsRNA that is double-stranded throughout its entire length, i.e., without nucleotide protrusions at either end of the molecule.

[0271] As used herein, the term "antisense strand" refers to a strand of double-stranded RNA that has a region substantially complementary to a target sequence (e.g., derived from human SMAD2 mRNA). Mismatches at the terminal regions are generally tolerable, provided that the complementary region is not perfectly complementary to the target sequence, and if mismatches do occur, they are typically within one or more terminal regions, such as 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends.

[0272] The term "sense chain" as used in this article refers to a double-stranded RNA strand containing a region that is substantially complementary to the antisense chain region.

[0273] The term “inhibition” as used in this article may be used interchangeably with “reduction,” “silence,” “downregulation,” “suppression,” and other similar terms, and includes any level of inhibition.

[0274] As used herein, the term "suppressing target gene expression" includes suppressing the expression of any target gene (e.g., mouse target gene, rat target gene, monkey target gene, or human target gene) as well as variants (e.g., naturally occurring variants) or mutants of the target gene. Therefore, the target gene can be a wild-type target gene, a mutant target gene, or, in the case of genetically manipulated cells, cell groups, or organisms, a transgenic target gene.

[0275] As used herein, the term "suppressing target gene expression" includes suppression of any level of target gene expression, such as at least partial suppression of target gene expression, including suppression of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0276] As used in this article, the term "chemical modification" or "modification" refers to all changes that nucleic acids undergo through chemical means, such as the addition or removal of chemical parts, or the replacement of one chemical part with another.

[0277] Unless otherwise stated, the compounds described in this application or their pharmaceutically acceptable salts encompass all their isomer forms, including stereoisomers. Unless otherwise specified, the single compounds described in this application encompass racemic mixtures, diastereomer mixtures, and individual isomer forms.

[0278] The term "stereoisomer" as used in this article refers to the phenomenon of isomers with the same molecular formula and functional groups, but different spatial arrangements of atoms or functional groups in the molecule. Stereoisomers include cis-trans isomers and chiral isomers. Chiral isomers are further divided into two main categories: enantiomers and diastereomers.

[0279] As used in this article, "enantiomers" refers to compounds with the same molecular formula and functional groups that are isomers due to differences in the spatial arrangement of atoms or functional groups, and the compounds form mirror images of each other and are not superimposed.

[0280] The term "diastereomer" as used in this article refers to compounds with the same molecular formula and functional groups that are isomers due to different spatial arrangements of atoms, and these compounds are stereoisomers that are not mirror images of each other.

[0281] Involving structural units The term "positional isomers" mentioned here refers to compounds that have the same molecular formula and the same functional groups, but the functional groups or substituents are linked at different positions on the triazole ring of the parent structure. For example, when mentioning... When it comes to its positional isomers, including

[0282] In this application, in the structural formulas of "compound", "chemical group", "nucleoside", "nucleotide", and "nucleotide derivative", a straight covalent bond "—" can indicate that it is coplanar with the paper. In this application, when the atoms connected by the straight covalent bond have stereoisomers, the arrangement of the atoms connected by the straight covalent bond can include being coplanar with the paper, facing outwards from the paper, facing inwards from the paper, or a mixture of various arrangements.

[0283] Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration of the center of a solid is represented by a straight solid line key. and straight dashed key The relative configuration of the center of a solid is indicated by a wavy line. Indicates wedge-shaped solid line key or wedge-shaped dashed key Or use wavy lines Indicates a straight solid line key and straight dashed key

[0284] As defined herein, polyethylene glycol (PEG) is a linear or branched polymer composed of repeating ethylene glycol units (-CH2CH2O-), with the general formula, for example, HO-(CH2CH2O)nH, where n represents the degree of polymerization. Its molecular weight ranges from 200 Da to 50,000 Da, and it is a widely used polyether compound in industrial production and medicine. The terminal hydroxyl groups (-OH) of PEG can be selectively modified with amino (-NH2), carboxyl (-COOH), mercapto (-SH), methoxy (-OCH3), or other reactive functional groups to suit specific chemical coupling requirements. PEG is prepared by polymerizing ethylene oxide and is commercially available in a wide molecular weight range from 300 g / mol to 10,000,000 g / mol. Although PEGs with different molecular weights are used for different applications and have different physical properties, such as viscosity, due to chain length effects, their chemical properties are essentially the same. Depending on the initiator used in the polymerization method, different forms of PEG are also available, the most common being monofunctional methyl ether PEG (methoxy polyethylene glycol), abbreviated as mPEG. PEGs with different geometries are also available. For example, branched or star-shaped PEGs have about 3 to 100 PEG chains (arms) radiating from a central core group. Comb-shaped PEGs have multiple PEG chains typically grafted to the polymer backbone. Any of these PEG polymers can be used in the compounds of this application as defined above or in pharmaceutically acceptable salts thereof. In the context of this application, the term "PEG" preferably includes polymers defined in more than one of these ways.

[0285] The weight-average molecular weight (Mw) of PEG (polyethylene glycol) is a weighted average value obtained through statistical analysis of the polymer's molecular weight distribution. It is commonly used to describe the overall molecular weight characteristics of polydisperse polymers. The weight-average molecular weight is a weighted average calculated based on the product of the molecular weight of each molecule in the molecular weight distribution and its probability of occurrence. It reflects the central position of the polymer's molecular weight distribution and can effectively characterize the overall properties of polydisperse polymers.

[0286] In this document, when referring to modifications of nucleotides, unless otherwise stated, "2'-... modification" refers to a modification present at the 2' position of the ribosome in the nucleotide. Therefore, in this document, when referring to modifications of nucleotides, unless otherwise stated, "2'-... modified nucleotide" refers to a nucleotide formed by a modification present at the 2' position of the ribosome in the nucleotide.

[0287] The terms "substituted" and "substituted" are used interchangeably to mean that any one or more hydrogen atoms on a specific atom in a given structure are replaced by a substituent, provided that the valence state of the specific atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are replaced, and oxoing generally does not occur on the aromatic group. Unless otherwise stated, a substituted group may have one substituent at each substituted position of the group. When more than one position in a given structural formula can be replaced by one or more substituents selected from a specific group, then the substituents may be substituted at the same or different substituted positions.

[0288] The terms “optional” or “optionally” mean that the event or condition subsequently described may or may not occur, including both the occurrence and non-occurrence of said event or condition. For example, the ethyl group “optionally” being halogenated means that the ethyl group can be unsubstituted (-CH2CH3), monosubstituted (e.g., -CH2CH2F), polysubstituted (e.g., -CHFCH2F, -CH2CHF2, etc.), or fully substituted (-CF2CF3). Those skilled in the art will understand that for any group containing one or more substituents, no substitution or substitution pattern that is spatially impossible and / or cannot be synthesized is introduced.

[0289] In this article, the terms “each…independently selected”, “…independently selected”, and “…independently selected” are interchangeable and should be interpreted broadly. They can mean that the specific options expressed by the same symbols in different groups do not affect each other, or that the specific options expressed by the same symbols in the same group do not affect each other.

[0290] C in this article m-n This means that the part has an integer number of carbon atoms within a given range. For example, "C 1-6 "" means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.

[0291] In this application, "unsaturated bond" refers to carbon-carbon double and triple bonds. That is, a carbon atom with one or two unsaturated bonds... 2-4 A hydrocarbon group refers to a hydrocarbon group containing an alkenyl or alkynyl group.

[0292] The term "alkenyl" refers to an unsaturated aliphatic hydrocarbon group consisting of a straight or branched chain of carbon and hydrogen atoms, having at least one double bond. Non-limiting examples of alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, etc.

[0293] The term "alkynyl" refers to a straight-chain or branched unsaturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms and having at least one triple bond. Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), 1-propynyl (-C≡C-CH3), 2-propynyl (-CH2-C≡CH), and 1,3-butyrynyl (-C≡CC≡CH).

[0294] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Therefore, for example, if a group is substituted by two Rs, each R has an independent option.

[0295] As used herein, the terms "bond" or "linker" refer to groups or bonds that are typically formed due to chemical reactions and are generally covalent bonds. The term "hydrolytically stable bond" refers to a bond that is substantially stable in water and does not react with water at an acceptable pH, including (but not limited to) bonds that have a relatively long reaction time under physiological conditions, and possibly even an infinitely long reaction time. "Hydrolytically unstable or degradable bond" means that the bond is degradable in water or aqueous solutions (e.g., blood). "Enzyme-unstable or degradable bond" means that the bond can be degraded by one or more enzymes. As understood in the art, PEG and related polymers may include degradable bonds in the polymer backbone or linker groups, and between the polymer backbone and one or more terminal functional groups of the polymer molecule. For example, ester bonds formed by the reaction of PEG carboxylic acid or active PEG carboxylic acid with an alcohol group on a bioactive agent are typically hydrolyzed under physiological conditions to release the agent. In some embodiments, branched linkers may be used in the compounds of this application or their pharmaceutically acceptable salts.

[0296] When the number of a linking group is 0, for example -(CH2)0-, it indicates that the linking group is a covalent bond.

[0297] When one of the variables is selected as a covalent bond, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a covalent bond, it means that the structure is actually AZ.

[0298] When a substituent is linked to two atoms on a ring structure via a cross bond, the substituent can form bonds with any atom on that ring structure. For example, structural units. This indicates that it can be substituted at any position on the cyclohexyl group or cyclohexadiene.

[0299] In this document, the symbol “﹏” indicates a site where a group is covalently linked. For example, “﹏” can represent a site where the lipophilic monomer is covalently linked to a nucleotide of the double-stranded oligonucleotide. For instance, ﹏ represents a site where the structure is linked to an H, or phosphate group, nucleoside, or other chemical group.

[0300] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of that group can be connected to other groups by chemical bonds. When the chemical bond connection is non-directional and the connectable site contains H atoms, the number of H atoms at that site will decrease accordingly with the number of chemical bonds connected, resulting in a group with a corresponding valence. The chemical bonds connecting the site to other groups can be straight solid line bonds. Straight dashed key or wavy line express.

[0301] In this application, the term "reactive phosphorus group" refers to a phosphorus-containing group that can react with other compounds and be substituted or removed during the reaction. In this application, the reactive phosphorus group can be selected from...

[0302] The term "hydroxyl protecting group" refers to a group that temporarily alters the chemical properties of a hydroxyl group by introducing a protecting group onto it. Typically, the introduction of a hydroxyl protecting group can enhance the stability of a compound, prevent side reactions during the reaction process, and help improve the selectivity of the reaction. Common hydroxyl protecting groups include, but are not limited to, ester protecting groups, aryl protecting groups, alkyl protecting groups, alkoxymethyl protecting groups, and silane protecting groups. Exemplarily, the hydroxyl protecting group may be methyl ether (ROMe), tert-butyl ether (ROtBu), benzyl ether (ROBn), p-methoxybenzyl ether (ROPMB), triphenylmethyl ether (ROTr); trimethylsilyl (TMS), triethylsilyl (TES), dimethyl tert-butylsilyl (TBDMS), triisopropylsilyl (TIPS), M... OM (methoxymethyl ether), MEM (methoxyethoxymethyl ether), BOM (benzyloxymethyl ether), SEM (trimethylsilylethoxymethyl ether), THP (tetrahydropyran ether), t-BuCO (Piv, neopentanoyl), PhCO (Bz, benzoyl), MeCO (Ac, acetyl), ClCH2CO (chloroacetyl, and other haloacetyl), benzylidene (also known as benzenemethyl), acetoneide (isopropylidene), alicyclic ketoneide, DMT (4,4'-dimethoxytriphenylmethyl), etc.

[0303] The term "phosphate derivatives" refers to a class of compounds derived from phosphoric acid (H3PO4) or its anions (such as PO4). 3-Compounds derived from phosphates include phosphates, phosphate esters, and phosphates. For example, these compounds form compounds with different chemical properties by removing or replacing one or more atoms (such as hydrogen atoms) in a phosphate molecule.

[0304] In this text, the term "key" refers to a single bond unless otherwise specified.

[0305] The terms “halogen,” “halogenated,” or “halogen” refer to fluorine, chlorine, bromine, or iodine.

[0306] The term "hydroxyl group" refers to the -OH group.

[0307] The term "alkyl" refers to a compound with the general formula C1. n H 2n+1 A fully saturated (i.e., free of double or triple bonds) chain hydrocarbon group. The alkyl group can be straight-chain or branched. For example, the term "C 1-6 "Alkyl" refers to an alkyl group containing 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc.). Similarly, the alkyl portion (i.e., alkyl) of alkoxy, alkylamino, dialkylamino, alkylsulfonyl, and alkylthio groups has the same definition as above.

[0308] The term "alkylene" refers to a compound with the general formula -C n H 2n - A saturated divalent hydrocarbon group. The alkylene group can be straight-chain or branched. For example, the term "C1-C 10 "Alkylene" refers to alkylene groups having 1 to 10 carbon atoms.

[0309] The term "alkoxy" refers to -O-alkyl. Alkyl groups are defined as described above.

[0310] The term "alkenyl" refers to an unsaturated aliphatic hydrocarbon group consisting of a straight or branched chain of carbon and hydrogen atoms, having at least one double bond. Non-limiting examples of alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, etc.

[0311] The term "heterocyclic group" refers to a non-aromatic ring that is fully saturated or partially unsaturated (but excluding fully unsaturated heteroaromatics) and can exist in monocyclic, bridged, or spirocyclic form. Unless otherwise indicated, the heterocycle is typically a 3- to 12-membered ring containing 1 to 3 heteroatoms independently selected from sulfur, oxygen, and / or nitrogen (preferably 1 or 2 heteroatoms). Non-limiting examples of heterocyclic groups include, but are not limited to, ethylene oxide, tetrahydrofuranyl, dihydrofuranyl, pyrrolyl, N-methylpyrrolyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothiophene, etc.

[0312] The term "heteroatom" refers to any atom other than a carbon atom that can covalently bond with a carbon atom, for example, 1 to 6, such as 1, 2, 3, 4, 5, or 6. Common heteroatoms include, but are not limited to, O, S, N, P, Si, etc., for example, 1, 2, 3, 4, 5, or 6 O, S, N, or P atoms.

[0313] The term "heteroaryl" refers to a monocyclic or fused polycyclic system containing at least one ring atom selected from N, O, and S, with the remaining ring atoms being C, and having at least one aromatic ring. Preferred heteroaryl groups have a single 5- to 6-membered ring. Non-limiting examples of heteroaryl groups include, but are not limited to, pyrroleyl, furanyl, thiopheneyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, and triazineyl.

[0314] In this article, when a letter refers to an object or quantity, it is case-sensitive.

[0315] In this paper, in the general formulas (Ib) and (Ib-1), the symbol “()” indicates the presence or absence of a group. For example, “(L3)” indicates the presence or absence of L3; or “(L4-A2)” indicates the presence or absence of L4-A2.

[0316] The compounds of this application, or their pharmaceutically acceptable salts and intermediates, may exist in various tautomer forms, and all such forms are included within the scope of this application. The terms "tautomer" or "tautomer form" refer to structural isomers of different energies that can interconvert via low energy barriers. For example, proton tautomers (also known as proton transfer tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerization. A specific example of a proton tautomer is the imidazole moiety, in which a proton can migrate between two ring nitrogens. Valence tautomers include interconversions via the recombination of some bonding electrons.

[0317] This application also includes compounds of this application or pharmaceutically acceptable salts thereof that are identical to those described herein, but with one or more atoms replaced by isotopes of atomic weights or mass numbers different from those commonly found in nature. Examples of isotopes that can bind to compounds of this application or pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0318] Certain isotopically labeled compounds of this application or their pharmaceutically acceptable salts (e.g., using...) 3 H and 14 Those labeled with C can be used in the analysis of compound and / or substrate tissue distribution. Tritiumization (i.e. 3 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred due to their ease of preparation and detectability. Positron-emitting isotopes, such as... 15 O、 13 N、 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of this application or pharmaceutically acceptable salts thereof can typically be prepared by replacing unlabeled reagents with isotopically labeled reagents using a similar procedure to those disclosed in the schemes and / or examples below.

[0319] In addition, heavier isotopes (such as deuterium) are used. 2 H)) substitution can provide certain therapeutic advantages resulting from higher metabolic stability (e.g., increased in vivo half-life or reduced dose requirement), and may therefore be preferred in certain situations, where deuterium substitution can be partial or complete, with partial deuterium substitution referring to at least one hydrogen being replaced by at least one deuterium.

[0320] The compounds of this application, or their pharmaceutically acceptable salts, may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, for example, enantiomers and diastereomers. The compounds of this application containing asymmetric carbon atoms can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from a racemic mixture or synthesized using chiral starting materials or chiral reagents.

[0321] In this application, unless otherwise stated, "conjugation" and "linkage" are used interchangeably, referring to the connection of two or more chemical parts, each with a specific function, to each other in a non-covalent or covalent manner; correspondingly, "conjugated compound" refers to a compound formed by the chemical parts through non-covalent or covalent linkage. In this application, conjugated compounds linked to each other in a covalent manner are preferred.

[0322] The terms “subject” or “patient” are used interchangeably herein and generally refer to any human or non-human animal (including mammals) being examined, studied, or treated for the diagnosis, improvement, prognosis, prevention, and / or treatment of a disease, and are not intended to limit this application to any particular type of subject. Examples of subjects include, but are not limited to, humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), livestock (dogs and cats), farm animals (horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents, and adult subjects. Subjects include animal disease models. In some embodiments of this application, humans are preferred subjects, while in other embodiments, non-human animals are preferred subjects, including, but not limited to, mice, monkeys, ferrets, cattle, sheep, goats, pigs, chickens, turkeys, dogs, cats, horses, and reptiles.

[0323] The term "treatment" means administering the compound or preparation described in this application to prevent, improve, or eliminate a disease or one or more symptoms related to said disease, and includes:

[0324] (i) To prevent the occurrence of disease or disease state in mammals, especially when such mammals are susceptible to the disease state but have not yet been diagnosed with the disease state;

[0325] (ii) To suppress the disease or disease state, that is, to curb its development;

[0326] (iii) Alleviate the disease or disease state, even if the disease or disease state subsides.

[0327] The term "therapeutic effective amount" means (i) the amount of the compound of this application or a pharmaceutically acceptable salt thereof used to treat or prevent a particular disease, condition, or disorder; (ii) to reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) to prevent or delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the compound of this application or a pharmaceutically acceptable salt thereof constituting a "therapeutic effective amount" depends on factors such as the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and in conjunction with this disclosure.

[0328] The therapeutic dose of the compound or its pharmaceutically acceptable salt thereof may be determined based on a variety of factors, such as the specific purpose of treatment, the manner of administration of the compound, the health and condition of the subject or patient, and the judgment of the prescribing physician. The proportion or concentration of the compound in the pharmaceutical composition may not be fixed, depending on a variety of factors, including the dose, the chemical properties (e.g., hydrophobicity) of the compound, and the route of administration. For example, the compound may be provided parenterally via a physiologically buffered saline solution containing about 0.1 to 10% w / v of the compound. In some embodiments, certain typical dose ranges are from about 1 μg / kg to about 2 g / kg body weight / day. In some embodiments, the dose range is from about 0.01 mg / kg to about 500 mg / kg body weight / day. The dose of the compound may depend on variables such as the type and severity of the disease or condition, the general health condition of the specific subject or patient, the relative biological potency of the selected compound, the excipient formulation used, and the route of administration. The dose may be obtained by extrapolation from dose-response curves derived from in vitro or animal model testing systems.

[0329] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0330] The term "pharmaceutically acceptable salt" refers to a compound obtained by forming a salt with an acid or a base, such as, but not limited to, metal salts, ammonium salts, salts formed with organic bases, salts formed with inorganic acids, salts formed with organic acids, and salts formed with basic or acidic amino acids.

[0331] The term "pharmaceutical composition" refers to a mixture of one or more compounds of this application or their salts with a pharmaceutically acceptable carrier, excipient, or excipient. The purpose of a pharmaceutical composition is to facilitate the administration of the compound of this application to an organism; it may refer to a composition containing an active ingredient for the treatment of a disease or for use in in vitro cell culture experiments. In this document, the terms "pharmaceutical composition" and "formulation" have the same meaning and are used interchangeably. Carriers, excipients, or excipients as used herein include any and all solvents, diluents or other liquid excipients, dispersants or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., suitable for the desired specific dosage form. Various carriers, excipients, or excipients used to formulate pharmaceutically acceptable compositions and methods for their preparation may be employed using known techniques. The use of any conventional carrier media, except those incompatible with the compounds of this application (e.g., producing any adverse biological effects or otherwise interacting harmfully with any other component of the pharmaceutically acceptable composition), is covered within the scope of this application.

[0332] The term "pharmaceuticalally acceptable excipient" refers to excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0333] The pharmaceutical compositions of this application can be prepared by combining the compounds of this application or their pharmaceutically acceptable salts with suitable pharmaceutically acceptable excipients, for example, in solid, semi-solid, liquid, or gaseous formulations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalers, gels, microspheres, and aerosols. In some embodiments of this application, when used for the prevention and / or treatment of disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry.

[0334] Typical routes of administration for the compounds described in this application, their isomers, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0335] The pharmaceutical composition of this application can be manufactured using methods well known in the art, such as conventional mixing, dissolving, granulation, sugar-coated pill making, grinding, emulsification, freeze drying, etc.

[0336] In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of this application to be formulated into tablets, pills, lozenges, sugar-coated tablets, capsules, liquids, gels, pastes, suspensions, etc., for oral administration to patients.

[0337] In some embodiments, solid oral pharmaceutical compositions can be prepared by conventional mixing, filling, or tableting methods. For example, the active compound can be obtained by mixing it with solid excipients, optionally milling the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain a tablet or sugar-coated core. Suitable excipients include, but are not limited to, binders, diluents, disintegrants, lubricants, glidants, sweeteners, or flavoring agents.

[0338] In some embodiments, the pharmaceutical compositions described herein may also be suitable for parenteral administration, such as in suitable unit dosage forms of sterile solutions, suspensions or lyophilized products.

[0339] Unless otherwise specified, singular terms encompass plural terms in this document, and vice versa. Unless otherwise specified, the words “a” or “an” mean “at least one” or “at least one”. Unless otherwise specified, the word “or” means “and / or”.

[0340] In this application, the terms "comprise" or "comprising" and their English variations such as "comprises" or "comprising" should be understood in an open, non-exclusive sense, meaning "including but not limited to," implying that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be included. The term "composed of" generally indicates that no other components can exist (or similarly, features, integers, steps, etc.). Unless the context clearly specifies otherwise, nouns without a defined quantity also include plural indicators.

[0341] Unless otherwise stated, in this document, parameter values ​​representing the amount of an ingredient, physicochemical properties, or reaction conditions, etc., should be understood to be modified by the term "about" in all cases. When the term "about" is used herein to describe a parameter value of this application, the term "about" indicates that the parameter value is allowed to have a certain range of error, for example, a variation within ±5%, ±1%, or ±0.1% of a specific value.

[0342] For purposes of description and disclosure, all patents, patent applications, and other identified publications mentioned herein are expressly incorporated herein by reference. Such publications are mentioned only because their publication predates the filing date of this application. Any representations regarding the dates or contents of these publications are based on information currently available to the applicant and do not constitute an acknowledgment of the accuracy of their contents. Any reference in this document to the aforementioned publications also does not constitute an endorsement that such publications are part of common general knowledge in the art.

[0343] The compounds of this application or their pharmaceutically acceptable salts can be prepared by a variety of synthetic methods known to those skilled in the art, including but not limited to the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this application.

[0344] The chemical reactions in the specific embodiments of this application are carried out and completed in a suitable solvent, which must be suitable for the chemical changes involved in this application and the reagents and materials required therefor. In order to obtain the compounds of this application or their pharmaceutically acceptable salts, in some cases, those skilled in the art may make appropriate modifications or selections to the synthetic steps or reaction processes based on existing embodiments; such modifications or selections are conventional techniques used by those skilled in the art. The solvents, reagents, and materials used in this application are available from commercial sources.

[0345] An important consideration in synthetic route planning in this field is the selection of appropriate protecting groups for reactive functional groups (such as amino groups in this application). For example, see Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in this application are incorporated herein by reference in their entirety.

[0346] Technical effect

[0347] The compound of this application or its pharmaceutically acceptable salt can deliver siRNA to adipose tissue and effectively knock down target mRNA, which can be used to treat obesity and metabolic-related diseases and has broad application prospects. Attached Figure Description

[0348] Figure 1 shows a schematic diagram of the siRNA conjugate according to the present invention used in the experimental examples of this application. In the figure, black bases are methoxy, green bases are fluorinated, orange vertical lines represent thio, and VP indicates that the base is modified with EVP.

[0349] Figure 2 shows the inhibitory effect of the siRNA conjugate of this application on the target protein adiponectin in mice. Detailed Implementation

[0350] The present application is described in detail below through examples, but this does not imply any adverse limitation thereof. The compounds of this application can be prepared by various synthetic methods well known to those skilled in the art, including but not limited to the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this application. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of this application without departing from the spirit and scope of this application, and all such changes should fall within the protection scope of this application.

[0351] This application uses the following abbreviations:

[0352] PE represents petroleum ether; EA represents ethyl acetate; DCM represents dichloromethane; THF represents tetrahydrofuran; DMF represents N,N-dimethylformamide; MeOH represents methanol; TBDPSCl represents tert-butyldiphenylchlorosilane; TBDPSO represents TBDPS bonded to oxygen; TBAF represents tetrabutylammonium fluoride; DIPEA represents N,N-diisopropylethylamine; TCEP represents tris(2-carboxyethyl)phosphine; PBS represents phosphate buffer; TEAA represents triethylamine acetate; DMSO represents dimethyl sulfoxide; TBTU represents 2-(1H-benzotriazo-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate; TFA represents trifluoroacetic acid; HATU represents 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate.

[0353] Unless otherwise stated, the compounds described in this application are artificially or... Software naming conventions and names of commercially available compounds are used from the supplier's catalog. Unless otherwise specified in the examples, conditions generally applicable in the art or as recommended by the manufacturer are followed. Unless otherwise stated, all reagents and consumables used in this application, unless otherwise specified, are commercially available products.

[0354] Example

[0355] The present application will now be described in further detail through embodiments, but the present application is not limited to these embodiments.

[0356] All reagents used in this application are commercially available and can be used without further purification.

[0357] Synthesis of structural units modified with phosphorus amide monomers or said conjugates:

[0358] Preparation Example 1: Synthesis of compound D-ER-FY003100-P1

[0359] Synthesis of compound D-ER-FY003100-P1-A2: At room temperature, compound D-ER-FY003100-P1-A1 ((S)-3-pyrrolidone, 10.00 g, 114.78 mmol, 1.0 eq.), imidazole (11.72 g, 172.15 mmol, 1.5 eq.), and dichloromethane (200 mL) were added sequentially to a reaction flask. After the addition was complete, the mixture was moved to 0 °C and stirred for 15 min. Then, tert-butyldiphenylchlorosilane (37.86 g, 137.74 mmol, 1.2 eq.) was added, and the mixture was moved to room temperature and reacted overnight. The reaction mixture was poured into water (200 mL), extracted three times with dichloromethane (200 mL), and the combined dichloromethane phases were dried over anhydrous sodium sulfate with stirring. The mixture was filtered, concentrated under vacuum, and purified by column chromatography (dichloromethane / methanol = 15 / 1, v / v) to give compound D-ER-FY003100-P1-A2 (30.0 g, yield 80.32%); MS: m / z 326.2 [M+H] + .

[0360] Synthesis of compound D-ER-FY003100-P1-A3: Compound D-ER-FY003100-P1-A2 (10.00 g, 30.75 mmol, 1 eq.) was weighed at room temperature and added to a reaction flask. DMF (100 mL) was added, and saturated sodium hydroxide aqueous solution (20 mL) was added dropwise with stirring at room temperature. After the addition was complete, the mixture was stirred for 30 min. Then, hexadecane bromo (18.80 g, 61.57 mmol, 2 eq.) was added, and the mixture was stirred overnight at room temperature. The reaction mixture was poured into water and extracted three times with ethyl acetate (150 mL). The combined ethyl acetate phases were washed three times with saturated sodium chloride aqueous solution (300 mL). The ethyl acetate phase was dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and purified by column chromatography (petroleum ether / ethyl acetate = 5 / 1, v / v) to obtain compound D-ER-FY003100-P1-A3 (12.0 g, yield: 71.01%). MS:m / z 550.6 [M+H] + .

[0361] Synthesis of compound D-ER-FY003100-P1-A4: At room temperature, 12.00 g (21.84 mmol, 1 eq.) of compound D-ER-FY003100-P1-A3 was weighed, and 150 mL of THF was added. Tetrabutylammonium fluoride (11.42 g, 43.68 mmol, 2 eq.) was added with stirring, and the reaction was allowed to proceed overnight. The reaction mixture was evaporated to dryness, and 100 mL of water was added. The mixture was extracted three times with 150 mL of ethyl acetate. The combined ethyl acetate phases were dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and purified by column chromatography (dichloromethane / methanol = 10 / 1, v / v) to give compound D-ER-FY003100-P1-A4 (4.1 g, yield: 60.481%). MS: m / z 312.4 [M+H] + .

[0362] Synthesis of compound D-ER-FY003100-P1: Compound D-ER-FY003100-P1-A4 (500 mg, 1.61 mmol, 1 eq.), triethylamine (487.55 mg, 4.82 mmol, 3 eq.), and DCM (10 mL) were added sequentially to a reaction flask to dissolve the contents. Nitrogen gas was then introduced, followed by dropwise addition of 2-cyanoethyl N,N-diisopropylphosphoramide (760.25 mg, 3.21 mmol, 2.0 eq.). After the addition was complete, the reaction mixture was allowed to react at room temperature for 4 h. Post-treatment: The reaction mixture was concentrated and subjected to wet chromatography for purification (eluent: PE / EA = 1 / 1, v / v) to obtain compound D-ER-FY003100-P1 (540.00 mg, yield: 65.74%). MS: m / z 429.4 [M-83+H] + .

[0363] Preparation Example 2: Synthesis of compound D-ER-FY003120-P1

[0364] Except for replacing hexadecane bromide with docosane bromide in Preparation Example 1, D-ER-FY003120-P1 (500.00 mg) was prepared according to the same steps and methods as in Preparation Example 1; MS: m / z 513.6 [M-83+1] + .

[0365] Synthesis of compound D-ER-FY003301-P1 (Preparation Example 3)

[0366] Synthesis of compound D-ER-FY003301-P1-A2: Compound D-ER-FY003301-P1-A1 (12.5 g, 36.49 mmol) was added to a reaction flask, and cyclohexane (150 mL) was added as a solvent. After the system became a white suspension, 48% hydrobromic acid aqueous solution (170 mL) was added to the system, and the reaction was refluxed at 81 °C for 18 h. The reaction was monitored by TLC until completion. 150 mL of water and 150 mL of... The system was filtered with DCM, and the filtrate was allowed to stand and separate into layers. The organic phase was separated, and the aqueous phase was extracted three times with DCM. The filter cake was washed with DCM / MeOH = 10 / 1 (v / v) solution. The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography (eluent: PE:EA = 25:1 to 8:1, v:v) to obtain compound D-ER-FY003301-P1-A2 (12.1 g, yield 82.0%).

[0367] Synthesis of compound D-ER-FY003301-P1-A3: Compound D-ER-FY003301-P1-A2 (900 mg, 2.23 mmol, 1 eq.), anhydrous pyridine (264 mg, 3.34 mmol, 1.5 eq.), and anhydrous DCM as solvent (15 mL) were added to a reaction flask. After dissolution, the mixture was purged with nitrogen three times. After cooling at 0 °C, trifluoromethanesulfonic anhydride (942 mg, 3.34 mmol, 1.5 eq.) was added dropwise. The reaction was carried out at 0 °C for 2 h. The system was washed with 1 M hydrochloric acid (15 mL), and the aqueous phase was extracted once with DCM after separation. The organic phases were combined and dried over anhydrous sodium sulfate until the solution was clear. The solution was filtered and evaporated to dryness to obtain crude compound D-ER-FY003301-P1-A3 (1.4 g).

[0368] Synthesis of compound D-ER-FY003301-P1-A4: Compound D-ER-FY003301-P1-A3 (1.4 g, 2.61 mmol, 1 eq.) was added to a reaction flask, with DCM (20 mL) as solvent, followed by the addition of tetrabutylamine hydrofluoride (882 mg, 3.13 mmol, 1.2 eq.). The reaction was carried out overnight at room temperature. The reaction was monitored by TLC until completion. The system was evaporated to dryness to obtain the crude product, which was purified by column chromatography (eluent PE) to obtain compound D-ER-FY003301-P1-A4 (760 mg, yield 71.6%).

[0369] Synthesis of compound D-ER-FY003301-P1-A5: Compound D-ER-FY003100-P1-A2 (608 mg, 1.87 mmol, 1 eq.) was added to a reaction flask, along with ground potassium carbonate (776 mg, 5.61 mmol, 3 eq.). Acetonitrile (15 mL) was used as the solvent. After stirring at room temperature for 5 min, compound D-ER-FY003301-P1-A4 (760 mg, 1.87 mmol, 1 eq.) was added, and the reaction was carried out at 60 °C for 8 h. The system was filtered, the filter cake was washed with DCM, and the filtrate was evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography (eluent: PE:EA = 5:1, v:v) to obtain compound D-ER-FY003301-P1-A5 (970 mg, yield 79.6%). MS: m / z 652.52 [M+1] + .

[0370] Synthesis of compound D-ER-FY003301-P1-A6: D-ER-FY003301-P1-A5 (970 mg, 1.49 mmol, 1 eq.) was added to a reaction flask, with THF (10 mL) as the solvent. TBAF (1 M, in THF) (3.72 mL, 3.72 mmol, 2.5 eq.) was added to the system. After the addition was complete, the reaction was carried out at room temperature for 4 h, and the reaction was monitored by TLC to indicate completion. After concentrating most of the solvent from the system, 10 mL of water was added, and the aqueous phase was extracted five times with EA. The combined organic phases were washed twice with saturated ammonium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography (eluent: first PE:EA = 2:1, v:v; then DCM:MeOH = 1:0~20:1, v:v) to obtain compound D-ER-FY003301-P1-A6 (585 mg, yield 95.1%). MS: m / z 414.40 [M+1] + .

[0371] Synthesis of compound D-ER-FY003301-P1: Compound D-ER-FY003301-P1-A6 (250 mg, 0.605 mmol, 1 eq.) and dry DIPEA (313 mg, 2.42 mmol, 4 eq.) were added to a reaction flask. Anhydrous DCM (6 mL) was used as the solvent, and the mixture was purged with nitrogen three times. After stirring at room temperature for 10 min, anhydrous DCM solution of 2-cyanoethyl N,N-diisopropylchlorophosphine (286 mg, 1.21 mmol, 2 eq., 0.5 mL) was added dropwise. After the addition was complete, the reaction was allowed to proceed at room temperature for 1.5 h. The reaction was monitored by TLC to indicate completion. The system was concentrated and directly loaded onto a wet plate, then purified rapidly by column chromatography under nitrogen pressure (eluent:PE:EA:TEA = 1:0:0.01–1:2:0.01, v:v) to give compound D-ER-FY003301-P1 (280 mg, yield 75.5%). MS: m / z 531.40 [M-82] + .

[0372] Synthesis of compound D-ER-FY003305M31-P1 (i.e., SM2) in Preparation Example 4

[0373] Synthesis of compound D-ER-FY003305M31-P1-A1: Hexadecanoic acid monotert-butyl ester (3.0 g, 8.76 mmol, 1.0 eq.), DMF (50 mL), TBTU (3.1 g, 9.64 mmol, 1.1 eq.), and DIPEA (3.4 g, 26.28 mmol, 3.0 eq.) were added to a reaction flask and stirred at room temperature for 10 minutes. Then, 2-[2-(propynyloxy)ethoxy]ethylamine (1.38 g, 9.64 mmol, 1.1 eq.) was added, and the reaction was carried out at room temperature for 1 hour. After the reaction was complete, the reaction system was transferred to a 300 mL container. The EA was diluted and washed with 3% citric acid solution (60 mL × 3), water (60 mL × 2), and saturated sodium chloride solution (60 mL × 1). The organic phase was dried over anhydrous sodium sulfate and then evaporated to dryness. The crude product was purified by column chromatography (PE:EA = 1:1, v:v) to give compound D-ER-FY003305M31-P1-A1 (3.5 g, yield 85.4%). MS: m / z 468.2 [M+1] + .

[0374] 1H NMR (400MHz, DMSO-d6) δ7.81(t,J=5.7Hz,1H),4.14(d,J=2.3Hz,2H),3.55(tt,J=10.6,5.3Hz,4H),3.43–3.35(m,3H ),3.18(q,J=5.8Hz,2H),2.16(t,J=7.3Hz,2H),2.04(t,J=7.4Hz,2H),1.57–1.42(m,4H),1.39(s,9H),1.23(s,20H).

[0375] 13 C NMR(101MHz,DMSO-d6)δ172.18,172.13,80.19,79.20,76.99,69.28,69.18,68.43,57.49,3 8.39,35.28,34.73,29.04,29.01,28.95,28.86,28.81,28.64,28.35,27.69,25.25,24.57.

[0376] Synthesis of compound D-ER-FY003305M31-P1: Compound D-ER-FY003305M31-P1-A1 (1.0 g, 2.14 mmol, 1.0 eq.), DCM (5 mL), and TFA (5 mL) were added to a reaction flask. The reaction was carried out at room temperature for 1 h. After the reaction was complete, the reaction system was concentrated to dryness, and the crude product was purified by column chromatography (DCM:MeOH = 60:1, v:v) to obtain compound D-ER-FY003305M31-P1 (718 mg, yield 81.6%). MS: m / z 412.2 [M+1] + .

[0377] 1 H NMR (400MHz, DMSO-d6) δ11.86(s,1H),7.81(t,J=5.7Hz,1H),4.14(d,J=2.4Hz,2H),3.54(qd,J=5.1,2.3Hz,4H),3.44– 3.35 (m, 3H), 3.18 (q, J = 5.8Hz, 2H), 2.18 (t, J = 7.4Hz, 2H), 2.04 (t, J = 7.4Hz, 2H), 1.46 (p, J = 7.0Hz, 4H), 1.23 (s, 20H).

[0378] 13C NMR(101MHz,DMSO-d6)δ174.46,172.18,77.06,69.28,69.17,68.44,57.49,38.39,3 5.28,33.64,29.03,28.99,28.94,28.91,28.79,28.73,28.62,28.54,25.25,24.47.

[0379] Synthesis of compound D-ER-FY003305M29-P1 (i.e., SM3) in Example 5

[0380] 100 mg (0.23 mmol, 1 eq) of D-ER-FY003305M29-P1-A1 (CAS No. 843666-21-7) was dissolved in 3 mL of DMF. HATU (174.9 mg, 0.46 mmol, 2.0 eq.) and DIPEA (89.2 mg, 0.69 mmol, 3.0 eq.) were added to the reaction system, followed by 2-[2-(2-azidoethoxy)ethoxy]ethylamine (80.1 mg, 0.46 mmol, 2.0 eq.). The mixture was stirred overnight at room temperature until the reaction was complete. The reaction system was concentrated to dryness, and the crude product was purified by column chromatography (petroleum ether: ethyl acetate = 1:1, v / v) to give compound D-ER-FY003305M29-P1 (100 mg, yield: 72.6%). MS: m / z 600.2 [M+1] + .

[0381] Preparation Example 6: Synthesis of Compound SM4

[0382] Add tert-butyl eicosanoate (3.00 g, 7.53 mmol, 1.0 eq.), DMF (50 mL), TBTU (2.66 g, 8.28 mmol, 1.1 eq.), and DIPEA (2.92 g, 22.59 mmol, 3.0 eq.) to the reaction flask, stir at room temperature for 10 minutes, then add 2-[2-(2-azidoethoxy)ethoxy]ethylamine (1.44 g, 8.28 mmol, 1.1 eq.). The reaction mixture was reacted at room temperature for 1 hour until complete. The reaction system was diluted with 300 mL of ethyl acetate, washed with 3% citric acid aqueous solution (60 mL × 3), washed with water (60 mL × 2), and washed with saturated sodium chloride aqueous solution (60 mL × 1). The organic phase was then dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (PE:EA = 1:1, v / v) to give compound SM4-A1 (3.90 g, yield 93.4%). MS: m / z 555.2 [M+1]+.

[0383] Compound SM4-A1 (1.00 g, 1.80 mmol, 1.0 eq.), DCM (5 mL), and 4 M HCl ethyl acetate solution (5 mL) were added to a reaction flask. The reaction was carried out at room temperature for 1 h. After the reaction was completed, the reaction system was concentrated to dryness. The crude product was slurried with cyclohexane (10 mL), filtered, and the solid was collected to give compound SM4 (0.71 g, yield 79.0%). MS: m / z 499.1 [M+1]+.

[0384] The preparation of the siRNA conjugates described in this application will be further illustrated below through more examples, but these examples do not constitute any limitation on this application. The compounds, sequences, and structures related to the preparation of the siRNA conjugates are listed in Tables 1 to 5.

[0385] Table 1. Compounds involved in the synthesis of siRNA conjugates

[0386] The relevant sequences and structures of the siRNA conjugates in the examples are shown in Tables 2, 3, and 4.

[0387] Table 2. siRNA sequences targeting SOD1

[0388] Table 3 Sequences targeting ALK7

[0389] Table 4 Sequences of adiponectin target sites

[0390] In Tables 2, 3, and 4, A, U, G, and C represent native adenine ribonucleotides, uracil ribonucleotides, guanine ribonucleotides, and cytosine ribonucleotides, respectively; lowercase letters a, u, c, and g represent 2'-methoxy-modified adenosine, uridine, cytosine, and guanosine; Af, Uf, Cf, and Gf represent 2'-F-modified adenosine, uridine, cytosine, and guanosine; lowercase letter s indicates that the two nucleotides to the left and right of s are linked by a phosphothioester bond; EVP indicates that the nucleotide adjacent to its right is modified by EVP; the letter combination (invAb) represents an inverted debased deoxyribose residue; (invAb)s represents an inverted debased deoxyribose residue -5'-phosphothioester, and s(invAb) represents a phosphothioester -3'-inverted debased deoxyribose residue.

[0391] The structure of PC16s is shown as number 1 in Table 5;

[0392] The structure of PC22s is shown as number 2 in Table 5;

[0393] The structure of PFC22s is shown as number 3 in Table 5;

[0394] The structure of Q1 is shown as number 4 in Table 5;

[0395] The structure of Q2 is shown as number 5 in Table 5;

[0396] The structure of Q3 is shown as number 6 in Table 5;

[0397] The structure of Q4 is shown as number 7 in Table 5;

[0398] The structure of Q5 is shown as number 8 in Table 5;

[0399] The structure of Q6 is shown as number 9 in Table 5;

[0400] The structure of Q7 is shown as number 10 in Table 5;

[0401] The structure of Q8 is shown as number 11 in Table 5;

[0402] The structure of (invAb)s is shown as number 12 in Table 5;

[0403] The structure of s(invAb) is shown as number 13 in Table 5;

[0404] The structure of EVPu is shown as number 14 in Table 5;

[0405] The structure of EVPa is shown as number 15 in Table 5;

[0406] The structure of Q9 is shown as number 16 in Table 5;

[0407] The structure of Q10 is shown as number 17 in Table 5;

[0408] The structure of Q11 is shown as number 18 in Table 5;

[0409] The structure of Q12 is shown as number 19 in Table 5;

[0410] The structure of Q13 is shown as number 20 in Table 5;

[0411] The structure of Q14 is shown as number 21 in Table 5;

[0412] The structure of Q15 is shown as number 22 in Table 5;

[0413] The structure of Q16 is shown as number 23 in Table 5;

[0414] The structure of Q17 is shown as number 24 in Table 5;

[0415] The structure of Q18 is shown as number 25 in Table 5;

[0416] The structure of Q19 is shown as number 26 in Table 5;

[0417] The structure of Q20 is shown as number 27 in Table 5;

[0418] The structure of Q21 is shown as number 28 in Table 5;

[0419] The structure of Q22 is shown as number 29 in Table 5.

[0420] Table 5. Structure of lipid PK / PD regulators, modified monomers, and linkers

[0421] Example 1: Synthesis of siRNA conjugate D-ER-FY003303

[0422] (1-1) Preparation of siRNA

[0423] 1.1 Synthesis of the Justice Chain (SS Chain)

[0424] The oligonucleotide was synthesized using a solid-phase phosphoramide method, employing a CPG solid-phase support (Suzhou NewSys Biotechnology Co., Ltd.) as the starting cycle. Nucleoside monomers were sequentially linked from the 3'-5' direction according to the nucleotide arrangement of the positive strand. Each linkage of a nucleoside monomer involved four steps: deprotection, coupling, oxidation or thiolation, and capping. The synthesis scale was 1 μmol of oligonucleotides. The synthetic conditions are as follows:

[0425] The nucleoside monomers were provided in 1.0 g / 10 mL acetonitrile solution. The reaction conditions were the same for each step, i.e., the temperature was 25 °C. Deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution.

[0426] The activator used in the coupling reaction was a 0.25 mol / L acetonitrile solution of 5-ethylthiotetrazole, and the coupling reaction was carried out for 8 min.

[0427] Oxidation was performed using 0.05 mol / L iodine in tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v) for 0.5 min; or thiolation was performed using 0.05 mol / L N,N-dimethyl-N'-(3-thio-3H-1,2,4-dithiazo-5-yl)formamidin in acetonitrile / pyridine (2 / 3, v / v) for 8 min.

[0428] Use a 20% acetic anhydride-acetonitrile solution and 15% N-methylimidazole / acetonitrile to cap the product, and cap it twice.

[0429] 1.2 Synthesis of the antisense chain (AS chain)

[0430] The solid-phase phosphoramide synthesis method utilizes a CPG solid-phase support as the starting cycle, linking nucleoside monomers sequentially from the 3'-5' direction according to the nucleotide arrangement sequence of the antisense strand. Each linking of a nucleoside monomer involves four steps: deprotection, coupling, oxidation or thiolation, and capping. The synthesis conditions for 1 μmol of oligonucleotides in the antisense strand are the same as those for the sense strand.

[0431] 1.3 Purification and Annealing of Oligonucleotides

[0432] 1.3.1 Ammonolysis

[0433] The synthesized solid-phase support (sense or antisense chain) was added to a 1.5 mL centrifuge tube, and 1.0 mL of 50% methylamine / ammonia water (v / v) was added. The mixture was reacted in a metal bath shaker at 25 °C for 10 hours. The supernatant was collected, and the solid-phase support was washed twice with enzyme-free water, 200 μL each time. The supernatants were combined, centrifuged and concentrated, and the crude product was purified.

[0434] 1.3.2 Purification

[0435] Purification was performed using a reverse chromatography system with a Shim-pack GIS C18 column (SHIMADZU) and gradient elution with a hexylamine acetate-acetonitrile system. The prepared liquid was collected and lyophilized to obtain the single-chain product.

[0436] 1.3.3 Annealing

[0437] The sense chain (SS chain) and the antisense chain (AS chain) were mixed in a molar ratio (SS chain / AS chain = 1 / 1), reacted in a metal bath shaker at 37°C for 4 hours, naturally cooled to room temperature, and the system was freeze-dried to obtain the product.

[0438] (1-2) Synthesis of D-ER-FY003303-A1

[0439] The positive strand sequence of the siRNA conjugate D-ER-FY003303 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, N,N-bis(1-methylethyl)aminophosphonic acid 6-[[6-[bis(4-methoxyphenyl)phenylmethoxy]hexyl]dithio]hexyl 2-cyanoethyl ester (CAS: 148254-21-1) was coupled to the first nucleotide at the 3' end, and the synthesized phosphoramidite monomer D-ER-FY003100-P1 was coupled to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the crude oligonucleotide single strand D-ER-FY003303-A1 as shown in the following formula.

[0440] (1-3) Synthesis of D-ER-FY003303-A2

[0441] The crude D-ER-FY003303-A1 (2.78 μmol) obtained in steps (1-2) of Example 1 was dissolved in 750 μL of purified water, vortexed, centrifuged at high speed for 5 min, and the supernatant (680 μL, 15.60 mg / mL) was collected. 15.91 mg of TCEP was dissolved in 680 μL of purified water and added to the supernatant. The mixture was shaken at 37 °C for 2 h. The reaction process was monitored using a Waters mass spectrometer. After the reaction was complete, 136 μL of 3M sodium acetate (pH = 5.2) was added to the reaction system, vortexed, and 12.24 mL of anhydrous ethanol was added. The mixture was then vortexed and allowed to settle overnight at -20 °C.

[0442] Centrifuge the sedimentation system for 30 minutes, discard the supernatant, add 1.5 mL of 10% methanol aqueous solution (volume ratio) to dissolve, ultrafilter 8 times with 10% methanol aqueous solution (volume ratio), and then ultrafilter 8 times with 1×PBS to obtain D-ER-FY003303-A2 (1.24 μmol, yield: 44.60%) (calculated molecular weight: 7403.08; measured molecular weight: 7402.26).

[0443] (1-4) Synthesis of D-ER-FY003303-A3

[0444] The D-ER-FY003303-A2 (1.24 μmol) obtained in steps (1-3) was dissolved in 3.0 mL of 1×PBS solution and vortexed to mix. Separately, 12.4 mg of methoxy polyethylene glycol maleimide (CAS: 99126-64-4) was dissolved in 3.0 mL of DMF solution and added to the above system. The reaction was carried out at 25 °C with shaking for 2 h. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, purification was performed using an Agilent 1260 HPLC system. The mobile phase conditions used were: Phase A: 50 mM TEAA (pH 1.5). 7.0-7.4); Phase B: methanol; gradient conditions: 0-30 min, Phase B: 5%-75%; collect product peaks, and replace the obtained fraction with 10% methanol aqueous solution by ultrafiltration 8 times, and then replace with purified water by ultrafiltration 8 times to obtain D-ER-FY003303-A3 (0.78 μmol, yield: 62.90%) (calculated molecular weight: 12381.08; measured molecular weight: 12384.17).

[0445] (1-5) Synthesis of D-ER-FY003303

[0446] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003303 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003303-A3 obtained in step (1-4) was dissolved in enzyme-free water in an equimolar amount with the antisense strand prepared by solid-phase synthesis. The two were then annealed to obtain the conjugate D-ER-FY003303 in Table 2.

[0447] Example 2: Synthesis of siRNA conjugate D-ER-FY003304

[0448] (2-1) Synthesis of D-ER-FY003304-A1

[0449] D-ER-FY003303-A2 was dissolved in 3.0 mL of 1×PBS solution and vortexed. Separately, 0.68 mg of maleimide-ethylene glycol (CAS: 1421933-37-0) was dissolved in 3.0 mL of DMF solution and added to the above system. The reaction was carried out at 25 °C with shaking for 2 h. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, purification was performed using an Agilent 1260 HPLC system with the following mobile phase conditions: Phase A: 50 mM TEAA (pH = 7.0–7.4); Phase B: methanol; gradient conditions: 0–30 min, Phase B: 5%–75%; collect product peaks, and replace the obtained fraction with 10% (v / v) methanol aqueous solution by ultrafiltration 8 times, and then replace with purified water by ultrafiltration 8 times to obtain D-ER-FY003304-A1 (0.89 μmol, yield: 71.77%) (calculated molecular weight: 7676.35; measured molecular weight: 7675.49).

[0450] (2-2) Synthesis of D-ER-FY003304

[0451] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003304 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003304-A1 obtained in step (2-1) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003304 in Table 2.

[0452] The siRNA conjugates D-ER-FY003304M1 and D-ER-FY003304M2 were obtained using a similar synthesis method as in Example 2.

[0453] Example 3 Synthesis of siRNA conjugate D-ER-FY003304M3

[0454] (3-1) Synthesis of D-ER-FY003304M3-A1

[0455] The positive strand sequence of the siRNA conjugate D-ER-FY003304M3 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, N,N-bis(1-methylethyl)aminophosphonic acid 6-[[6-[bis(4-methoxyphenyl)phenylmethoxy]hexyl]dithio]hexyl 2-cyanoethyl ester (CAS: 148254-21-1) was coupled to the first nucleotide at the 3' end, and the synthesized phosphoramidite monomer D-ER-FY003120-P1 was linked to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the crude oligonucleotide single strand D-ER-FY003304M3-A1 as shown in the following formula.

[0456] (3-2) Synthesis of D-ER-FY003304M3-A2

[0457] The crude D-ER-FY003304M3-A1 (3.25 μmol) obtained in step (3-1) of Example 3 was dissolved in 800 μL of purified water, vortexed, centrifuged at high speed for 5 min, and the supernatant (705 μL, 16.38 mg / mL) was collected. 17.32 mg of TCEP was dissolved in 705 μL of purified water and added to the supernatant. The mixture was shaken at 37 °C for 2 h. The reaction process was monitored using a Waters mass spectrometer. After the reaction was complete, 141 μL of 3M sodium acetate (pH = 5.2) was added to the reaction system, vortexed, and 12.69 mL of anhydrous ethanol was added, vortexed, and the mixture was allowed to settle overnight at -20 °C.

[0458] Centrifuge the sedimentation system for 30 minutes, discard the supernatant, add 1.5 mL of 10% methanol aqueous solution (volume ratio) to dissolve, ultrafilter 8 times with 10% methanol aqueous solution (volume ratio), and then ultrafilter 8 times with 1*PBS to obtain D-ER-FY003304M3-A2 (1.45 μmol, yield: 44.61%) (calculated molecular weight: 7503.30; measured molecular weight: 7502.44).

[0459] (3-3) Synthesis of D-ER-FY003304M3-A3

[0460] 1.45 μmol of D-ER-FY003304M3-A2 was dissolved in 3.0 mL of 1×PBS solution and vortexed. Separately, 0.79 mg of maleimide-ethylene glycol (CAS: 1421933-37-0) was dissolved in 3.0 mL of DMF solution and added to the above system. The reaction was carried out at 25 °C with shaking for 2 h. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, purification was performed using an Agilent 1260 HPLC system with the following mobile phase conditions: Phase A: 50 mM TEAA (pH = 7.0–7.4); Phase B: methanol; gradient conditions: 0–30 min, Phase B: 5%–75%; collect product peaks, and replace the obtained fraction with 10% methanol aqueous solution (volume ratio) by ultrafiltration 8 times, and then replace with purified water by ultrafiltration 8 times to obtain D-ER-FY003304M3-A3 (0.85 μmol, yield: 58.62%) (calculated molecular weight: 7776.58; measured molecular weight: 7775.55).

[0461] (3-4) Synthesis of D-ER-FY003304M3

[0462] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY003304M3 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003304M3-A3 obtained in step (3-3) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003304M3 in Table 2.

[0463] Example 4: Synthesis of siRNA conjugate D-ER-FY003305

[0464] (4-1) Synthesis of D-ER-FY003305-A1

[0465] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, Spacers-9 phosphorus amide monomer (CAS: 146668-73-7) was used to couple to the first nucleotide at the 3' end, and the synthesized phosphorus amide monomer D-ER-FY003100-P1 was linked to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support and purified by high performance liquid chromatography to obtain the oligonucleotide single strand D-ER-FY003305-A1 as shown in the following formula.

[0466] (4-2) Synthesis of D-ER-FY003305

[0467] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003305 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305-A1 obtained in step (4-1) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305 in Table 2.

[0468] Example 5 Synthesis of siRNA conjugate D-ER-FY003305M1

[0469] (5-1) Synthesis of D-ER-FY003305M1-A1

[0470] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M1 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, three nucleotides prior to the first nucleotide at the 3' end were sequentially coupled using Spacer 9 phosphoramide monomer (CAS: 146668-73-7), and the synthesized phosphoramide monomer D-ER-FY003100-P1 was linked to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support and purified by high-performance liquid chromatography to obtain the oligonucleotide single strand D-ER-FY003305M1-A1 as shown in the following formula.

[0471] (5-2) Synthesis of D-ER-FY003305M1

[0472] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY003305M1 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M1-A1 obtained in step (5-1) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M1 in Table 2.

[0473] Example 6 Synthesis of siRNA conjugate D-ER-FY003305M2

[0474] (6-1) Synthesis of D-ER-FY003305M2-A1

[0475] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M2 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, Spacer 18 phosphorus amide (CAS: 125607-09-2) and Spacer 9 phosphorus amide monomer (CAS: 146668-73-7) were sequentially coupled before the first nucleotide at the 3' end, and the synthesized phosphorus amide monomer D-ER-FY003100-P1 was linked to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support and purified by high performance liquid chromatography to obtain the oligonucleotide single strand D-ER-FY003305M2-A1 as shown in the following formula.

[0476] (6-2) Synthesis of D-ER-FY003305M2

[0477] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY003305M2 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M2-A1 obtained in step (6-1) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M2 in Table 2.

[0478] Example 7 Synthesis of siRNA conjugate D-ER-FY003305M3

[0479] (7-1) Synthesis of D-ER-FY003305M3-A1

[0480] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M3 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, Spacer 18 phosphorus amide (CAS: 125607-09-2) was used to couple the synthesized phosphorus amide monomer D-ER-FY003100-P1 to the 5' end of the nucleic acid sequence before the first nucleotide at the 3' end. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support and purified by high performance liquid chromatography to obtain the oligonucleotide single strand D-ER-FY003305M3-A1 as shown in the following formula.

[0481] (7-2) Synthesis of D-ER-FY003305M3

[0482] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY003305M3 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M3-A1 obtained in step (7-1) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M3 in Table 2.

[0483] Example 8 Synthesis of siRNA conjugate D-ER-FY003305M4

[0484] (8-1) Synthesis of D-ER-FY003305M4-A1

[0485] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M4 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, two nucleotides before the first nucleotide at the 3' end were continuously coupled using Spacer 18 phosphorus amide (CAS: 125607-09-2), and the synthesized phosphorus amide monomer D-ER-FY003100-P1 was linked to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support and purified by high-performance liquid chromatography to obtain the oligonucleotide single strand D-ER-FY003305M4-A1 as shown in the following formula.

[0486] (8-2) Synthesis of D-ER-FY003305M4

[0487] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY003305M4 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M4-A1 obtained in step (8-1) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M4 in Table 2.

[0488] Example 9 Synthesis of siRNA conjugate D-ER-FY003305M5

[0489] (9-1) Synthesis of D-ER-FY003305M5-A1

[0490] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M5 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, Spacer 18 phosphorus amide (CAS: 125607-09-2) was used to couple the synthesized phosphorus amide monomer D-ER-FY003100-P1 to the 5' end of the nucleic acid sequence before the first nucleotide at the 3' end. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support and purified by high performance liquid chromatography to obtain the oligonucleotide single strand D-ER-FY003305M5-A1 as shown in the following formula.

[0491] (9-2) Synthesis of D-ER-FY003305M5

[0492] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY003305M5 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M5-A1 obtained in step (9-1) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M5 in Table 2.

[0493] Example 10 Synthesis of siRNA conjugate D-ER-FY003305M6

[0494] Synthesis of (10-1)D-ER-FY003305M6-A1

[0495] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M6 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, Spacer 18 phosphorus amide (CAS: 125607-09-2) was used to couple the synthesized phosphorus amide monomer D-ER-FY003120-P1 to the 5' end of the nucleic acid sequence before the first nucleotide at the 3' end. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support and purified by high performance liquid chromatography to obtain the oligonucleotide single strand D-ER-FY003305M6-A1 as shown in the following formula.

[0496] Synthesis of (10-2)D-ER-FY003305M6

[0497] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY003305M6 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M6-A1 obtained in step (10-1) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M6 in Table 2.

[0498] Example 11 Synthesis of siRNA conjugate D-ER-FY003305M8

[0499] Synthesis of (11-1)D-ER-FY003305M8-A1

[0500] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M8 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, Spacer 18 phosphorus amide (CAS: 125607-09-2) and Spacer 9 phosphorus amide monomer (CAS: 146668-73-7) were sequentially coupled before the first nucleotide at the 3' end, and the synthesized phosphorus amide monomer D-ER-FY003301-P1 was linked to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support and purified by high performance liquid chromatography to obtain the oligonucleotide single strand D-ER-FY003305M8-A1 as shown in the following formula.

[0501] (11-2) Synthesis of D-ER-FY003305M8

[0502] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY003305M8 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M8-A1 obtained in step (11-1) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M8 in Table 2.

[0503] Example 12 Synthesis of siRNA conjugate N-ER-FY059305M2

[0504] Synthesis of (12-1) N-ER-FY059305M2-A1

[0505] The siRNA positive strand sequence of the siRNA conjugate N-ER-FY059305M2 in Table 3 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, Spacer 18 phosphorous amide (CAS: 125607-09-2) and Spacer were used. Nine phosphorus amide monomers (CAS: 146668-73-7) and ((2R,3S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)tetrahydrofuran-2-yl)methyl(2-cyanoethyl)diisopropylphosphorus amide (CAS: 401813-16-9) were sequentially coupled before the first nucleotide at the 3' end. Then, ((2R,3S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)tetrahydrofuran-2-yl)methyl(2-cyanoethyl)diisopropylphosphorus amide (CAS: 401813-16-9) and the synthesized phosphorus amide monomer D-ER-FY003100-P1 were sequentially linked to the 5' end of the nucleic acid sequence. After solid-phase synthesis, the oligonucleotide single strand was cleaved from the solid-phase support and purified by high-performance liquid chromatography to obtain the oligonucleotide single strand N-ER-FY059305M2-A1 as shown in the following formula.

[0506] Synthesis of (12-2) N-ER-FY059305M2

[0507] Following the method described in Example 1 (1-1), the antisense strand of siRNA in N-ER-FY059305M2 in Table 3 was synthesized by solid-phase synthesis. The sense strand N-ER-FY059305M2-A1 obtained in step (12-1) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate N-ER-FY059305M2 in Table 3.

[0508] Example 13 Synthesis of siRNA conjugate D-ER-FY003305M9

[0509] (13-1) Synthesis of D-ER-FY003305M9-A1

[0510] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M9 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, Spacer 18 phosphorus amide (CAS: 125607-09-2) and Spacer 9 phosphorus amide monomer (CAS: 146668-73-7) were sequentially coupled before the first nucleotide at the 3' end, and the synthesized phosphorus amide monomer D-ER-FY003120-P1 was linked to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support and purified by high-performance liquid chromatography to obtain the oligonucleotide single strand D-ER-FY003305M9-A1 as shown in the following formula.

[0511] (13-2) Synthesis of D-ER-FY003305M9

[0512] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY003305M9 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M9-A1 obtained in step (13-1) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M9 in Table 2.

[0513] Example 14 Synthesis of siRNA conjugate D-ER-FY003AC002873-305M2

[0514] Synthesis of (14-1)D-ER-FY003AC002873-305M2-A1

[0515] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003AC002873-305M2 in Table 4 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, Spacer 18 phosphoramidite (CAS: 125607-09-2), Spacer 9 phosphoramidite monomer (CAS: 146668-73-7), and ((2R,3S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)tetrahydrofuran-2-yl)methyl(2-cyanoethyl)diisopropylphosphoramidite (CAS: 401813-16-9) were sequentially coupled before the first nucleotide at the 3' end. 2-(2-yl)methyl(2-cyanoethyl)diisopropylphosphonamide (CAS: 401813-16-9) and the synthesized phosphoramide monomer D-ER-FY003100-P1 were linked to the 5' end of the nucleic acid sequence. After solid-phase synthesis, the oligonucleotide single strand was cleaved from the solid-phase support and purified by high performance liquid chromatography to obtain the oligonucleotide single strand D-ER-FY003AC002873-305M2-A1 as shown in the following formula.

[0516] Synthesis of (14-2)D-ER-FY003AC002873-305M2

[0517] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003AC002873-305M2 in Table 4 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003AC002873-305M2-A1 obtained in step (14-1) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003AC002873-305M2 in Table 4.

[0518] Example 15 Synthesis of siRNA conjugate D-ER-FY003305M10

[0519] (15-1) Synthesis of D-ER-FY003305M10-A1

[0520] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M10 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, 3'-Amino-Modifier C7 CPG 1000 (Hebei Dinaxingke) was used as the starting cycle, and Spacer 18 phosphorus amide (CAS: 125607-09-2) and Spacer 9 phosphorus amide (CAS: 146668-73-7) were sequentially coupled before the first nucleotide at the 3' end. The synthesized phosphorus amide monomer D-ER-FY003100-P1 was then linked to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support and purified by high-performance liquid chromatography to obtain the oligonucleotide single strand D-ER-FY003305M10-A1 as shown in the following formula.

[0521] (15-2) Synthesis of D-ER-FY003305M10-A2

[0522] Dissolve D-ER-FY003305M10-A1 (398 nmol) prepared in step (15-1) in 1160 μL of 1×PBS and vortex to mix. Separately, dissolve 1.29 mg of dibenzocyclooctynyl-tetraethylene glycol-active ester (CAS: 1427004-19-0) in 1160 μL of DMSO and add it to the D-ER-FY003305M10-A1 solution. Incubate at 37°C. The reaction was allowed to proceed overnight with shaking. The reaction process was monitored using a Waters mass spectrometer. After the reaction was completed, the reaction system was replaced 5 times with 10% methanol aqueous solution (volume ratio) by ultrafiltration, and then replaced 5 times with 1×PBS by ultrafiltration. The solution in the ultrafiltration tube was taken out to obtain D-ER-FY003305M10-A2 (78 nmol, 350 μL) (calculated molecular weight: 8538.74; measured molecular weight: 8537.56).

[0523] (15-3) Synthesis of D-ER-FY003305M10-A3

[0524] 0.17 mg of the azide compound SM1 (CAS: 3049664-45-8) was dissolved in 350 μL of DMSO, vortexed, and added to a 1×PBS solution of D-ER-FY003305M10-A2 (78 nmol, 350 μL). The mixture was allowed to react overnight at 37°C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, the product was purified by Agilent 1260 HPLC. The mobile phase conditions were: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min, Phase B: 5%-75%. The product peaks were collected, and the fractions were combined and lyophilized to obtain D-ER-FY003305M10-A3 (D-ER-FY003305M10-A3 contains positional isomers isomer 1 and isomer 2). 2)(44.5 nmol, yield: 57%))(Calculated molecular weight: 8980.74; Measured molecular weight: 8979.56).

[0525] (15-4) Synthesis of D-ER-FY003305M10

[0526] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003305M10 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M10-A3 obtained in step (15-3) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M10 in Table 2 (D-ER-FY003305M10 contains positional isomers isomer 1 and isomer 2).

[0527] Example 16 Synthesis of siRNA conjugate D-ER-FY003305M11

[0528] Synthesis of (16-1)D-ER-FY003305M11-A1

[0529] Dissolve D-ER-FY003305M10-A1 (398 nmol) prepared in step 15-1 of Example 15 in 1350 μL of 1×PBS, vortex to mix. Separately, dissolve 0.78 mg of propynyl-tetraethylene glycol-succinimide acrylate (CAS: 1393330-40-9) in 1350 μL of DMSO, and add it to the D-ER-FY003305M10-A1 solution. The reaction was carried out overnight with shaking at 5°C. The reaction process was monitored using a Waters mass spectrometer. After the reaction was completed, the reaction system was replaced 5 times with 10% methanol aqueous solution (volume ratio) by ultrafiltration, and then replaced 5 times with 1×PBS by ultrafiltration. The solution in the ultrafiltration tube was taken out to obtain D-ER-FY003305M11-A1 (228 nmol, 620 μL) (calculated molecular weight: 8289.19; measured molecular weight: 8288.64).

[0530] (16-2) Synthesis of D-ER-FY003305M11-A2

[0531] Under nitrogen protection, anhydrous acetonitrile (74.4 μL), 20 mmol / L copper sulfate aqueous solution (123 μL), and 200 mmol / L sodium bicarbonate aqueous solution (247 μL) were added sequentially to D-ER-FY003305M11-A1 (228 nmol, 620 μL). 0.41 mg of the azide compound SM1 (CAS: 3049664-45-8) was dissolved in 106 μL of DMF and added to the system. 20 mmol / L TCEP aqueous solution (310 μL) was also added. The mixture was vortexed and reacted overnight at 25°C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, purification was performed using an Agilent 1260 HPLC system with the following mobile phase conditions: Phase A: 50 mM TEAA (pH = 7.0–7.4); Phase B: acetonitrile; gradient conditions: 0–30 min, Phase B: 5%–75%; collect product peaks, combine fractions and freeze-dry to obtain D-ER-FY003305M11-A2 (38 nmol, yield: 38%) (calculated molecular weight: 8292.19; measured molecular weight: 8291.38).

[0532] (16-3) Synthesis of D-ER-FY003305M11

[0533] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003305M11 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M11-A2 obtained in step (16-2) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M11 in Table 2.

[0534] Example 17 Synthesis of siRNA conjugate D-ER-FY003305M12

[0535] Synthesis of (17-1)D-ER-FY003305M12-A1

[0536] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M12 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, 3'-Amino-Modifier C7 CPG 1000 (Hebei Dinaxingke) was used as the starting cycle, and Spacer 18 phosphorus amide (CAS: 125607-09-2) was used to couple to the first nucleotide at the 3' end. The synthesized phosphorus amide monomer D-ER-FY003100-P1 was then linked to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support and prepared by high-performance liquid chromatography to obtain the oligonucleotide single strand D-ER-FY003305M12-A1 as shown in the following formula.

[0537] (17-2) Synthesis of D-ER-FY003305M12-A2

[0538] The D-ER-FY003305M12-A1 (420 nmol) prepared in step 17-1 was dissolved in 1210 μL of 1×PBS and vortexed. Separately, 0.84 mg of propynyl-tetraethylene glycol-succinimide acrylate (CAS: 1393330-40-9) was dissolved in 1210 μL of DMSO and added to the D-ER-FY003305M12-A1 solution. The mixture was shaken and reacted overnight at 25 °C. The reaction process was monitored using a Waters mass spectrometer. After the reaction was completed, the reaction system was replaced 5 times with 10% methanol aqueous solution (volume ratio) by ultrafiltration, and then replaced 5 times with 1×PBS by ultrafiltration. The solution in the ultrafiltration tube was removed to obtain D-ER-FY003305M12-A2 (334 nmol, 1000 μL). (Calculated molecular weight: 8063.22; Measured molecular weight: 8061.82).

[0539] Synthesis of (17-3)D-ER-FY003305M12-A3

[0540] Under nitrogen protection, anhydrous acetonitrile (120 μL), 20 mmol / L copper sulfate aqueous solution (200 μL), and sodium bicarbonate aqueous solution (400 μL) were added sequentially to D-ER-FY003305M12-A2 (334 nmol, 1000 μL). 0.59 mg of the azide compound SM1 (CAS: 3049664-45-8) was dissolved in 172 μL of DMF and added to the system. Separately, 20 mmol / L TCEP aqueous solution (500 μL) was added to the system. The mixture was vortexed and reacted overnight at 25°C with shaking. After the reaction was complete, purification was performed using an Agilent 1260 HPLC system with the following mobile phase conditions: Phase A: 50 mM TEAA (pH = 7.0–7.4); Phase B: acetonitrile; gradient conditions: 0–30 min, Phase B: 5%–75%; collect product peaks, combine fractions and freeze-dry to obtain D-ER-FY003305M12-A3 (38 nmol, yield: 38%) (calculated molecular weight: 8505.34; measured molecular weight: 8504.96).

[0541] Synthesis of (17-4)D-ER-FY003305M12

[0542] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003305M12 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M12-A3 obtained in step (17-3) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water, and then the two were annealed to obtain the conjugate D-ER-FY003305M12 in Table 2.

[0543] Example 18 Synthesis of siRNA conjugate D-ER-FY003305M13

[0544] Synthesis of (18-1)D-ER-FY003305M13-A1

[0545] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M13-A1 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, a 3'-Amino-Modifier C7 CPG 1000 (Hebei Dinaxingke) was used as the starting cycle to link the synthesized phosphoramide monomer D-ER-FY003100-P1 to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY003305M13-A1 as shown in the following formula.

[0546] (18-2) Synthesis of D-ER-FY003305M13-A2

[0547] Dissolve 320 nmol of D-ER-FY003305M13-A1 prepared in step 18-1 in 1000 μL of 1×PBS and vortex to mix. Separately, dissolve 0.64 mg of propynyl-tetraethylene glycol-succinimide acrylate (CAS: 1393330-40-9) in 1000 μL of DMSO and add it to the D-ER-FY003305M13-A1 solution. Incubate at 25°C. The reaction was allowed to proceed overnight with shaking. The reaction process was monitored using a Waters mass spectrometer. After the reaction was completed, the reaction system was replaced 5 times with 10% methanol aqueous solution (volume ratio) by ultrafiltration, and then replaced 5 times with 1×PBS by ultrafiltration. The solution in the inner tube of the ultrafiltration tube was removed to obtain D-ER-FY003305M13-A2 (265 nmol, 1000 μL) (calculated molecular weight: 7703.19; measured molecular weight: 7702.88).

[0548] Synthesis of (18-3)D-ER-FY003305M13-A3

[0549] Under nitrogen protection, anhydrous acetonitrile (120 μL), 20 mmol / L copper sulfate aqueous solution (200 μL), and sodium bicarbonate aqueous solution (400 μL) were added to D-ER-FY003305M13-A2 (265 nmol, 1000 μL). 0.47 mg of azide compound SM1 (CAS: 3049664-45-8) was dissolved in 172 μL of DMF and added to the system. 20 mmol / L TCEP aqueous solution (500 μL) was also added to the system. The mixture was vortexed and reacted overnight at 25 °C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, the mixture was purified by HPLC to obtain D-ER-FY003305M13-A3 (106 nmol, yield: 40%) (calculated molecular weight: 8145.19; measured molecular weight: 8144.55).

[0550] Synthesis of (18-4)D-ER-FY003305M13

[0551] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY003305M13 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M13-A3 obtained in step (18-3) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water, and then the two were annealed to obtain the conjugate D-ER-FY003305M13 in Table 2.

[0552] Example 19 Synthesis of siRNA conjugate D-ER-FY003305M14

[0553] Synthesis of (19-1)D-ER-FY003305M14-A1

[0554] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M14 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, a 3'-Amino-Modifier C7 CPG 1000 (Hebei Dinaxingke) was used as the starting cycle to link the synthesized phosphoramide monomer D-ER-FY003301-P1 to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY003305M14-A1 as shown in the following formula.

[0555] Synthesis of (19-2)D-ER-FY003305M14-A2

[0556] Dissolve 360 ​​nmol of D-ER-FY003305M14-A1 prepared in step (19-1) in 1200 μL of 1×PBS and vortex to mix. Separately, dissolve 0.72 mg of propynyl-tetraethylene glycol-succinimide acrylate (CAS: 1393330-40-9) in 1200 μL of DMSO and add it to the D-ER-FY003305M14-A1 solution. Incubate at 25°C. The reaction was allowed to proceed overnight under shaking. The reaction process was monitored using a Waters mass spectrometer. After the reaction was completed, the reaction system was replaced 5 times with 10% methanol aqueous solution (volume ratio) by ultrafiltration, and then replaced 5 times with 1×PBS by ultrafiltration. The solution in the ultrafiltration tube was taken out to obtain D-ER-FY003305M14-A2 (244 nmol, 1000 μL) (calculated molecular weight: 7805.34; measured molecular weight: 7804.47).

[0557] Synthesis of (19-3)D-ER-FY003305M14-A3

[0558] Under nitrogen protection, anhydrous acetonitrile (120 μL), 20 mmol / L copper sulfate aqueous solution (200 μL), and sodium bicarbonate aqueous solution (400 μL) were added sequentially to D-ER-FY003305M14-A2 (224 nmol, 1000 μL). 0.39 mg of azide compound SM1 (CAS: 3049664-45-8) was dissolved in 172 μL of DMF and added to the system. 20 mmol / L TCEP aqueous solution (500 μL) was also added to the system. The mixture was vortexed and reacted overnight at 25°C with shaking. After the reaction was complete, the mixture was purified by HPLC to obtain D-ER-FY003305M14-A3 (100 nmol, yield: 44%) (calculated molecular weight: 8247.34; measured molecular weight: 8246.91).

[0559] Synthesis of (19-4)D-ER-FY003305M14

[0560] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003305M14 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M14-A3 obtained in step (19-3) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water, and then the two were annealed to obtain the conjugate D-ER-FY003305M14 in Table 2.

[0561] Example 20 Synthesis of siRNA conjugate D-ER-FY003305M15

[0562] Synthesis of (20-1)D-ER-FY003305M15-A1

[0563] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M15 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, 3'-PT-Amino-Modifier C6 CPG (Hebei Dinaxingke) was used as the starting cycle to link the synthesized phosphoramide monomer D-ER-FY003301-P1 to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY003305M15-A1 as shown in the following formula.

[0564] Synthesis of (20-2)D-ER-FY003305M15-A2

[0565] Dissolve 305 nmol of D-ER-FY003305M15-A1 prepared in step (20-1) in 720 μL of 1×PBS and vortex to mix. Separately, dissolve 0.61 mg of propynyl-tetraethylene glycol-succinimide acrylate (CAS: 1393330-40-9) in 720 μL of DMSO and add it to the D-ER-FY003305M15-A1 solution. Incubate at 25°C. The reaction was allowed to proceed overnight with shaking. The reaction process was monitored using a Waters mass spectrometer. After the reaction was completed, the reaction system was replaced 5 times with 10% methanol aqueous solution (volume ratio) by ultrafiltration, and then replaced 5 times with 1×PBS by ultrafiltration. The solution in the ultrafiltration tube was taken out to obtain D-ER-FY003305M15-A2 (270 nmol, 780 μL) (calculated molecular weight: 7774.32; measured molecular weight: 7774.15).

[0566] Synthesis of (20-3)D-ER-FY003305M15-A3

[0567] Under nitrogen protection, anhydrous acetonitrile (93 μL), 20 mmol / L copper sulfate aqueous solution (156 μL), and 200 mmol / L sodium bicarbonate aqueous solution (312 μL) were added sequentially to D-ER-FY003305M15-A2 (270 nmol, 780 μL). 0.39 mg of azide compound SM1 (CAS: 3049664-45-8) was dissolved in 134 μL of DMF and added to the system. 20 mmol / L TCEP aqueous solution (156 μL) was also added to the system. The mixture was vortexed and reacted overnight at 25°C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, the mixture was purified by HPLC to obtain D-ER-FY003305M15-A3 (137 nmol, yield: 50%) (calculated molecular weight: 8216.34; measured molecular weight: 8215.91).

[0568] Synthesis of (20-4)D-ER-FY003305M15

[0569] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY00305M15 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M15-A3 obtained in step (20-3) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M15 in Table 2.

[0570] Example 21 Synthesis of siRNA conjugate D-ER-FY003305M18

[0571] (21-1) Synthesis of D-ER-FY003305M18-A1

[0572] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M18 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, 3'-PT-Amino-Modifier C6 CPG (Hebei Dinaxingke) was used as the starting cycle to link the synthesized phosphoramide monomer D-ER-FY003100-P1 to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY003305M18-A1 as shown in the following formula.

[0573] (21-2) Synthesis of D-ER-FY003305M18-A2

[0574] Dissolve 332 nmol of D-ER-FY003305M18-A1 prepared in step (21-1) in 740 μL of 1×PBS and vortex to mix. Separately, dissolve 0.66 mg of propynyl-tetraethylene glycol-succinimide acrylate (CAS: 1393330-40-9) in 740 μL of DMSO and add it to the D-ER-FY003305M18-A1 solution. Shake and mix at 25°C. The reaction should be allowed to stand overnight. The reaction process was monitored using a Waters mass spectrometer. After the reaction was completed, the reaction system was replaced 5 times with 10% methanol aqueous solution (volume ratio) by ultrafiltration, and then replaced 5 times with 1×PBS by ultrafiltration. The solution in the inner tube of the ultrafiltration tube was taken out to obtain D-ER-FY003305M18-A2 (277 nmol, 780 μL, yield: 83%) (calculated molecular weight: 7672.17; measured molecular weight: 7671.89).

[0575] (21-3) Synthesis of D-ER-FY003305M18-A3

[0576] Under nitrogen protection, anhydrous acetonitrile (93 μL), 20 mmol / L copper sulfate aqueous solution (156 μL), and 200 mmol / L sodium bicarbonate aqueous solution (312 μL) were added sequentially to D-ER-FY003305M18-A2 (277 nmol, 780 μL). 0.49 mg of azide compound SM1 (CAS: 3049664-45-8) was dissolved in 134 μL of DMF and added to the system. 20 mmol / L TCEP aqueous solution (156 μL) was also added to the system. The mixture was vortexed and reacted overnight at 25°C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, the mixture was purified by HPLC to obtain D-ER-FY003305M18-A3 (130 nmol, yield: 47%) (calculated molecular weight: 8114.17; measured molecular weight: 8113.91).

[0577] (21-4) Synthesis of D-ER-FY003305M18

[0578] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY003305M18 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M18-A3 obtained in step (21-3) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water, and then the two were annealed to obtain the conjugate D-ER-FY003305M18 in Table 2.

[0579] Example 22 Synthesis of siRNA conjugate D-ER-FY003305M19

[0580] (22-1) Synthesis of D-ER-FY003305M19-A1

[0581] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003305M19 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, 3'-PT-Amino-Modifier C6 CPG (Hebei Dinaxingke) was used as the starting cycle to link the synthesized phosphoramide monomer D-ER-FY003120-P1 to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY003305M19-A1 as shown in the following formula.

[0582] (22-2) Synthesis of D-ER-FY003305M19-A2

[0583] Dissolve 320 nmol of D-ER-FY003305M19-A1 prepared in step (22-1) in 800 μL of 1×PBS and vortex to mix. Separately, dissolve 0.64 mg of propynyl-tetraethylene glycol-succinimide acrylate (CAS: 1393330-40-9) in 800 μL of DMSO and add it to the D-ER-FY003305M19-A1 solution. Incubate at 25°C. The reaction was allowed to proceed overnight under shaking. The reaction process was monitored using a Waters mass spectrometer. After the reaction was completed, the reaction system was replaced 5 times with 10% methanol aqueous solution (volume ratio) by ultrafiltration, and then replaced 5 times with 1×PBS by ultrafiltration. The solution in the ultrafiltration tube was taken out to obtain D-ER-FY003305M19-A2 (300 nmol, 1000 μL) (calculated molecular weight: 7756.33; measured molecular weight: 7755.97).

[0584] (22-3) Synthesis of D-ER-FY003305M19-A3

[0585] Under nitrogen protection, anhydrous acetonitrile (93 μL), 20 mmol / L copper sulfate aqueous solution (156 μL), and 200 mmol / L sodium bicarbonate aqueous solution (312 μL) were added sequentially to D-ER-FY003305M19-A2 (300 nmol, 1000 μL). 0.53 mg of azide compound SM1 (CAS: 3049664-45-8) was dissolved in 134 μL of DMF and added to the system. 20 mmol / L TCEP aqueous solution (156 μL) was also added to the system. The mixture was vortexed and reacted overnight at 25 °C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, the mixture was purified by HPLC to obtain D-ER-FY003305M19-A3 (145 nmol, yield: 48%) (calculated molecular weight: 8198.33; measured molecular weight: 8197.89).

[0586] (22-4) Synthesis of D-ER-FY003305M19

[0587] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY003305M19 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M19-A3 obtained in step (22-3) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water, and then the two were annealed to obtain the conjugate D-ER-FY003305M19 in Table 2.

[0588] Example 23 Synthesis of siRNA conjugate D-ER-FY003305M28

[0589] (23-1) Synthesis of D-ER-FY003305M28-A1

[0590] The D-ER-FY003305M18-A1 (4.4 mg) prepared in step (21-1) was dissolved in 1600 μL of 1×PBS and vortexed. Separately, 2.63 mg of dibenzocyclooctynyl tetraethylene glycol-active ester (CAS: 1427004-19-0) was dissolved in 1600 μL of DMSO and added to the D-ER-FY003305M18-A1 solution. The reaction was carried out overnight at 25 °C with shaking. The reaction process was monitored using a Waters mass spectrometer. After the reaction was completed, the reaction system was replaced 5 times with 10% methanol aqueous solution (volume ratio) by ultrafiltration, and then replaced 5 times with 1×PBS by ultrafiltration. The solution in the ultrafiltration tube was taken out to obtain D-ER-FY003305M28-A1 (3.04 mg, yield 69%) (calculated molecular weight: 7921.7; measured molecular weight: 7922.0).

[0591] (23-2) Synthesis of D-ER-FY003305M28-A2

[0592] 2.6 mg of the azide compound SM1 (CAS: 3049664-45-8) was dissolved in 1000 μL of DMSO, vortexed, and added to a 1×PBS solution of D-ER-FY003305M28-A1 (3.04 mg, 1000 μL). The mixture was allowed to react overnight at 37°C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, the product was purified by Agilent 1260 HPLC. The mobile phase conditions were: Phase A: 50 mM TEAA (pH = 7.0–7.4); Phase B: acetonitrile; gradient conditions: 0–30 min, Phase B: 5%–75%. The product peaks were collected, and the fractions were combined and lyophilized to obtain D-ER-FY003305M28-A2 (D-ER-FY003305M28-A2 contains positional isomers isomer 1 and isomer 2). 2)(1.2 mg, yield: 39%) (calculated molecular weight: 8363.7; measured molecular weight: 8365.0).

[0593] (23-3) Synthesis of D-ER-FY003305M28

[0594] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003305M28 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M28-A2 obtained in step (23-2) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M28 in Table 2 (D-ER-FY003305M28 contains positional isomers isomer 1 and isomer 2).

[0595] Example 24 Synthesis of siRNA conjugate D-ER-FY003305M31

[0596] (24-1) Synthesis of D-ER-FY003305M31-A1

[0597] Dissolve 3.5 mg of D-ER-FY003305M18-A1 prepared in step (21-1) in 458 μL of 1×PBS and vortex to mix. Separately, dissolve 1.85 mg of azide-polyethylene glycol-succinimide acrylate (CAS: 1245718-89-1) in 458 μL of DMSO and add it to the D-ER-FY003305M18-A1 solution. Shake and react overnight at 25°C. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, replace the reaction system with 10% methanol aqueous solution (volume ratio) five times by ultrafiltration, and then replace it with 1×PBS five times by ultrafiltration. Take out the solution in the ultrafiltration tube to obtain D-ER-FY003305M31-A1 (3.02 mg, yield: 86%) (calculated molecular weight: 7615.0; measured molecular weight: 7616.0).

[0598] (24-2) Synthesis of D-ER-FY003305M31-A2

[0599] Under nitrogen protection, anhydrous acetonitrile (68 μL), 20 mmol / L copper sulfate aqueous solution (114 μL), and 200 mmol / L sodium bicarbonate aqueous solution (228 μL) were added sequentially to D-ER-FY003305M31-A1 (3.02 mg, 570 μL). 10.5 mg of the azide compound SM2 was dissolved in 2.82 mL of DMF, and 98 μL of this solution was added to the system. 20 mmol / L TCEP aqueous solution (114 μL) was also added to the system. The mixture was vortexed and reacted overnight at 25 °C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, the mixture was purified by HPLC to obtain D-ER-FY003305M31-A2 (0.8 mg, yield: 26%) (calculated molecular weight: 8026.3; measured molecular weight: 8026.7).

[0600] (24-3) Synthesis of D-ER-FY003305M31

[0601] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003305M31 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M31-A2 obtained in step (24-2) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M31 in Table 2.

[0602] Example 25 Synthesis of siRNA conjugate D-ER-FY003305M32

[0603] (25-1) Synthesis of D-ER-FY003305M32-A1

[0604] Dissolve 3.5 mg of D-ER-FY003305M18-A1 prepared in step (21-1) in 458 μL of 1×PBS and vortex to mix. Separately, dissolve 2.08 mg of 15-azido-4,7,10,13-tetraoxopentadecanoic acid-N-succinimide ester (CAS: 944251-24-5) in 458 μL of DMSO and add it to the D-ER-FY003305M18-A1 solution. The reaction was carried out overnight at 25°C with shaking. The reaction process was monitored using a Waters mass spectrometer. After the reaction was completed, the reaction system was replaced 5 times with 10% methanol aqueous solution (volume ratio) by ultrafiltration, and then replaced 5 times with 1×PBS by ultrafiltration. The solution in the ultrafiltration tube was taken out to obtain D-ER-FY003305M32-A1 (3.15 mg, yield 90%) (calculated molecular weight: 7659.0; measured molecular weight: 7660.0).

[0605] (25-2) Synthesis of D-ER-FY003305M32-A2

[0606] Under nitrogen protection, anhydrous acetonitrile (69 μL), 20 mmol / L copper sulfate aqueous solution (116 μL), and 200 mmol / L sodium bicarbonate aqueous solution (232 μL) were added sequentially to D-ER-FY003305M32-A1 (3.15 mg, 580 μL). 0.76 mg of the azide compound SM2 was dissolved in 102 μL of DMF and added to the system. 20 mmol / L TCEP aqueous solution (116 μL) was also added to the system. The mixture was vortexed and reacted overnight at 25 °C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, the mixture was purified by HPLC to obtain D-ER-FY003305M32-A2 (0.75 mg, yield: 24%) (calculated molecular weight: 8070.3; measured molecular weight: 8069.7).

[0607] (25-3) Synthesis of D-ER-FY003305M32

[0608] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY003305M32 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M32-A2 obtained in step (25-2) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M32 in Table 2.

[0609] Example 26 Synthesis of siRNA conjugate D-ER-FY003305M33

[0610] (26-1) Synthesis of D-ER-FY003305M33-A1

[0611] Dissolve 3.21 mg of D-ER-FY003305M18-A1 prepared in step (21-1) in 1100 μL of 1×PBS and vortex to mix. Separately, dissolve 1.8 mg of azido-tetraethylene glycol-vinyl succinimide (CAS: 1807534-82-2) in 1100 μL of DMSO and add it to the D-ER-FY003305M18-A1 solution. Shake and react overnight at 25°C. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, replace the reaction system with 10% methanol aqueous solution (volume ratio) five times by ultrafiltration, and then replace it with 1×PBS five times by ultrafiltration. Take out the solution in the ultrafiltration tube to obtain D-ER-FY003305M33-A1 (3.05 mg, yield: 95%) (calculated molecular weight: 7646.3; measured molecular weight: 7645.0).

[0612] (26-2) Synthesis of D-ER-FY003305M33-A2

[0613] Under nitrogen protection, anhydrous acetonitrile (199 μL), 20 mmol / L copper sulfate aqueous solution (332 μL), and 200 mmol / L sodium bicarbonate aqueous solution (664 μL) were added sequentially to D-ER-FY003305M33-A1 (3.05 mg, 1661 μL). 0.84 mg of the azide compound SM2 was dissolved in 286 μL of DMF and added to the system. 20 mmol / L TCEP aqueous solution (332 μL) was also added to the system. The mixture was vortexed and reacted overnight at 25°C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, the mixture was purified by HPLC to obtain D-ER-FY003305M33-A2 (0.75 mg, yield: 25%) (calculated molecular weight: 8057.6; measured molecular weight: 8056.7).

[0614] (26-3) Synthesis of D-ER-FY003305M33

[0615] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003305M33 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M33-A2 obtained in step (26-2) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M33 in Table 2.

[0616] Example 27 Synthesis of siRNA conjugate D-ER-FY003305M34

[0617] (27-1) Synthesis of D-ER-FY003305M34-A1

[0618] Dissolve 3.5 mg of D-ER-FY003305M18-A1 prepared in step (21-1) in 458 μL of 1×PBS and vortex to mix. Separately, dissolve 2.24 mg of AZIDO-PEG5-CH2CO2-NHS (CAS: 2144777-77-3) in 458 μL of DMSO and add it to the D-ER-FY003305M18-A1 solution. Shake and react overnight at 25 °C. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, replace the reaction system with 10% methanol aqueous solution (volume ratio) five times by ultrafiltration, and then replace it with 1×PBS five times by ultrafiltration. Take out the solution in the ultrafiltration tube to obtain D-ER-FY003305M34-A1 (3.02 mg, yield: 86%) (calculated molecular weight: 7689.0; measured molecular weight: 7690.0).

[0619] (27-2) Synthesis of D-ER-FY003305M34-A2

[0620] Under nitrogen protection, anhydrous acetonitrile (99 μL), 20 mmol / L copper sulfate aqueous solution (166 μL), and 200 mmol / L sodium bicarbonate aqueous solution (332 μL) were added sequentially to D-ER-FY003305M34-A1 (3.02 mg, 830 μL). 0.73 mg of the azide compound SM2 was dissolved in 98 μL of DMF and added to the system. 20 mmol / L TCEP aqueous solution (166 μL) was also added to the system. The mixture was vortexed and reacted overnight at 25°C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, the mixture was purified by HPLC to obtain D-ER-FY003305M34-A2 (0.75 mg, yield: 25%) (calculated molecular weight: 8100.3; measured molecular weight: 8100.7).

[0621] (27-3) Synthesis of D-ER-FY003305M34

[0622] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003305M34 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M34-A2 obtained in step (27-2) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M34 in Table 2.

[0623] Example 28 Synthesis of siRNA conjugate D-ER-FY003305M29

[0624] Synthesis of (28-1)D-ER-FY003305M29-A1

[0625] Under nitrogen protection, anhydrous acetonitrile (216 μL), 20 mmol / L copper sulfate aqueous solution (359 μL), and 200 mmol / L sodium bicarbonate aqueous solution (718 μL) were added sequentially to the D-ER-FY003305M18-A2 (550.8 nmol, 1796 μL) prepared in step 21-2 of Example 21. 1.32 mg of the azide compound SM3 was dissolved in 309 μL of DMF and added to the system. Separately, 20 mmol / L... TCEP aqueous solution (359 μL) was added to the system, vortexed and mixed, and reacted overnight at 25 °C with shaking. The reaction system was replaced 5 times with 10% methanol aqueous solution (volume ratio) by ultrafiltration, and then replaced 5 times with 1×PBS by ultrafiltration. The solution in the inner tube of the ultrafiltration tube was removed to obtain crude product D-ER-FY003305M29-A1 (550 nmol) (calculated molecular weight: 8272.6; measured molecular weight: 8272.0).

[0626] (28-2) Synthesis of D-ER-FY003305M29-A2

[0627] 550 nmol of D-ER-FY003305M29-A1 was dissolved in 1100 μL of 10% piperidine aqueous solution (v / v), and the reaction was carried out at 40 °C with shaking for 16 h. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was completed, the product was purified by IEX chromatography to obtain D-ER-FY003305M29-A2 (25.8 nmol, yield: 4.7%) (calculated molecular weight: 8244.6; measured molecular weight: 8242.8).

[0628] Synthesis of (28-3)D-ER-FY003305M29

[0629] Following the method described in Example 1 (1-1), the antisense strand of siRNA in D-ER-FY003305M29 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M29-A2 obtained in step (28-2) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M29 in Table 2.

[0630] Example 29 Synthesis of siRNA conjugate D-ER-FY003305M35

[0631] (29-1) Synthesis of D-ER-FY003305M35-A1

[0632] The D-ER-FY003305M19-A1 (4.4 mg) prepared in step (22-1) was dissolved in 1600 μL of 1×PBS and vortexed. Separately, 2.63 mg of dibenzocyclooctynyl tetraethylene glycol-active ester (CAS: 1427004-19-0) was dissolved in 1600 μL of DMSO and added to the D-ER-FY003305M19-A1 solution. The reaction was carried out overnight at 25 °C with shaking. The reaction process was monitored using a Waters mass spectrometer. After the reaction was completed, the reaction system was replaced 5 times with 10% methanol aqueous solution (volume ratio) by ultrafiltration, and then replaced 5 times with 1×PBS by ultrafiltration. The solution in the ultrafiltration tube was taken out to obtain D-ER-FY003305M35-A1 (3.1 mg, yield: 70%) (calculated molecular weight: 8005.9; measured molecular weight: 8005.0).

[0633] (29-2) Synthesis of D-ER-FY003305M35-A2

[0634] 2.6 mg of the azide compound SM1 (CAS: 3049664-45-8) was dissolved in 1000 μL of DMSO, vortexed, and added to a 1*PBS solution of D-ER-FY003305M35-A1 (3.1 mg, 1000 μL). The mixture was allowed to react overnight at 37°C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, the product was purified by Agilent 1260 HPLC. The mobile phase conditions were: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min, Phase B: 5%-75%. The product peaks were collected, and the fractions were combined and lyophilized to obtain D-ER-FY003305M35-A2 (D-ER-FY003305M35-A2 contains positional isomers isomer 1 and isomer 2). 2)(1.3 mg, yield: 42%) (calculated molecular weight: 8447.9; measured molecular weight: 8446.0).

[0635] (29-3) Synthesis of D-ER-FY003305M35

[0636] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003305M35 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M35-A2 obtained in step (29-2) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M35 in Table 2 (D-ER-FY003305M35 contains positional isomers isomer 1 and isomer 2).

[0637] Example 30 Synthesis of siRNA conjugate D-ER-FY003305M40

[0638] Synthesis of (30-1)D-ER-FY003305M40-A1

[0639] 2.9 mg of the azide compound SM4 was dissolved in 1000 μL of DMSO, vortexed, and added to a 1×PBS solution of D-ER-FY003305M28-A1 (3.1 mg, 1000 μL) obtained in step 23-1 of Example 23. The mixture was reacted overnight at 37°C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was completed, the product was purified by Agilent 1260 HPLC. The mobile phase conditions were: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min, Phase B: 5%-75%. The product peaks were collected, and the fractions were combined and lyophilized to obtain D-ER-FY003305M40-A1 (D-ER-FY003305M40-A1 contains positional isomers isomer 1 and isomer 2). 2)(1.3 mg, yield: 41%))(Calculated molecular weight: 8419.7; Measured molecular weight: 8418.0).

[0640] Synthesis of (30-2)D-ER-FY003305M40

[0641] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003305M40 in Table 2 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003305M40-A1 obtained in step (30-1) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003305M40 in Table 2 (D-ER-FY003305M40 contains positional isomers isomer 1 and isomer 2).

[0642] Example 31 Synthesis of siRNA conjugate D-ER-FY003AC002873-305M35

[0643] Synthesis of (31-1)D-ER-FY003AC002873-305M35-A1

[0644] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003AC002873-305M35 in Table 4 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, 3'-PT-Amino-Modifier C6 CPG (Hebei Dinaxingke) was used as the starting cycle to link the synthesized phosphoramide monomer D-ER-FY003120-P1 to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY003AC002873-305M35-A1 as shown in the following formula.

[0645] (31-2) Synthesis of D-ER-FY003AC002873-305M35-A2

[0646] Dissolve 4.6 mg of D-ER-FY003AC002873-305M35-A1 prepared in step (31-1) in 1600 μL of 1×PBS and vortex to mix. Separately, dissolve 2.6 mg of dibenzocyclooctynyl tetraethylene glycol-active ester (CAS: 1427004-19-0) in 1600 μL of DMSO and add it to the D-ER-FY003AC002873-305M35-A1 solution. The reaction was carried out overnight at 25°C with shaking. The reaction process was monitored using a Waters mass spectrometer. After the reaction was completed, the reaction system was replaced 5 times with 10% methanol aqueous solution by ultrafiltration, and then replaced 5 times with 1*PBS by ultrafiltration. The solution in the ultrafiltration tube was taken out to obtain D-ER-FY003AC002873-305M35-A2 (2.9 mg, yield: 63%) (calculated molecular weight: 8417.1; measured molecular weight: 8415.7).

[0647] Synthesis of (31-3)D-ER-FY003AC002873-305M35-A3

[0648] 2.9 mg of the azide compound SM1 (CAS: 3049664-45-8) was dissolved in 1000 μL of DMSO, vortexed, and added to 1*PBS solution of D-ER-FY003AC002873-305M35-A2 (3.4 mg, 1000 μL). The mixture was allowed to react overnight at 37°C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was completed, the mixture was purified by Agilent 1260 HPLC. The mobile phase conditions were: Phase A: 50 mM TEAA (pH 1.5). 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min, Phase B: 5%-75%; collect product peaks, combine fractions and freeze-dry to obtain D-ER-FY003AC002873-305M35-A3 (D-ER-FY003AC002873-305M35-A3 contains positional isomers isomer 1 and isomer 2) (1.4 mg, yield: 48%) (calculated molecular weight: 8859.1; measured molecular weight: 8859.0).

[0649] (31-4) Synthesis of D-ER-FY003AC002873-305M35

[0650] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003AC002873-305M35 in Table 4 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003AC002873-305M35-A3 obtained in step (31-3) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003AC002873-305M35 in Table 4 (D-ER-FY003AC002873-305M35 contains positional isomers isomer 1 and isomer 2).

[0651] Example 32 Synthesis of siRNA conjugate D-ER-FY003AC002873-305M40

[0652] Synthesis of (32-1)D-ER-FY003AC002873-305M40-A1

[0653] The siRNA positive strand sequence of the siRNA conjugate D-ER-FY003AC002873-305M40 in Table 4 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, 3'-PT-Amino-Modifier C6 CPG (Hebei Dinaxingke) was used as the starting cycle to link the synthesized phosphoramide monomer D-ER-FY003100-P1 to the 5' end of the nucleic acid sequence. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY003AC002873-305M40-A1 as shown in the following formula.

[0654] (32-2) Synthesis of D-ER-FY003AC002873-305M40-A2

[0655] Dissolve 2.3 mg of D-ER-FY003AC002873-305M40-A1 prepared in step (32-1) in 1600 μL of 1*PBS and vortex to mix. Separately, dissolve 1.3 mg of dibenzocyclooctynyl-tetraethylene glycol-active ester (CAS: 1427004-19-0) in 1600 μL of DMSO and add it to the D-ER-FY003AC002873-305M40-A1 solution. The reaction was carried out overnight with shaking at 25°C. The reaction process was monitored using a Waters mass spectrometer. After the reaction was completed, the reaction system was replaced 5 times with 10% methanol aqueous solution by ultrafiltration, and then replaced 5 times with 1*PBS by ultrafiltration. The solution in the inner tube of the ultrafiltration tube (volume ratio) was taken out to obtain D-ER-FY003AC002873-305M40-A2 (1.5mg, yield: 65%) (calculated molecular weight: 8332.9; measured molecular weight: 8334.0).

[0656] Synthesis of (32-3)D-ER-FY003AC002873-305M40-A3

[0657] 1.6 mg of the azide compound SM4 was dissolved in 1000 μL of DMSO, vortexed, and added to a 1*PBS solution of D-ER-FY003AC002873-305M40-A2 (1.5 mg, 1000 μL). The mixture was allowed to react overnight at 37°C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, the product was purified by Agilent 1260 HPLC. The mobile phase conditions were: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min, Phase B: 5%-75%. The product peaks were collected, and the fractions were combined and lyophilized to obtain D-ER-FY003AC002873-305M40-A3 (D-ER-FY003AC002873-305M40-A3 contains positional isomers isomer 1 and isomer 2). 2)(0.7 mg, yield: 47%)(calculated molecular weight: 8830.9; measured molecular weight: 8833.0).

[0658] Synthesis of (32-4)D-ER-FY003AC002873-305M40

[0659] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003AC002873-305M40 in Table 4 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003AC002873-305M40-A3 obtained in step (32-3) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003AC002873-305M40 in Table 4 (D-ER-FY003AC002873-305M40 contains positional isomers isomer 1 and isomer 2).

[0660] Example 33 Synthesis of siRNA conjugate D-ER-FY003AC002873-305M28

[0661] Synthesis of (33-1)D-ER-FY003AC002873-305M28-A1

[0662] Dissolve 1.6 mg of the azide compound SM1 in 1000 μL of DMSO, vortex to mix, and add to a 1*PBS solution of D-ER-FY003AC002873-305M40-A2 (1.8 mg, 1000 μL) obtained in step 32-2 of Example 32. The reaction was allowed to proceed overnight at 37°C with shaking. The reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, purification was performed using an Agilent 1260 HPLC system. The mobile phase conditions were: Phase A: 50 mM TEAA (pH 1.5). 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min, Phase B: 5%-75%; collect product peaks, combine fractions and freeze-dry to obtain D-ER-FY003AC002873-305M28-A1 (D-ER-FY003AC002873-305M28-A1 contains positional isomers isomer 1 and isomer 2) (0.65 mg, yield: 41%) (calculated molecular weight: 8774.9; measured molecular weight: 8776.0).

[0663] (33-2) Synthesis of D-ER-FY003AC002873-305M28

[0664] Following the method described in Example 1 (1-1), the antisense strand of the siRNA in D-ER-FY003AC002873-305M28 in Table 4 was synthesized by solid-phase synthesis. The sense strand D-ER-FY003AC002873-305M28-A1 obtained in step (33-1) was dissolved in an equimolar amount with the antisense strand prepared by solid-phase synthesis using enzyme-free water. The two were then annealed to obtain the conjugate D-ER-FY003AC002873-305M28 in Table 4 (D-ER-FY003AC002873-305M28 contains positional isomers isomer 1 and isomer 2).

[0665] Example 34 Synthesis of siRNA conjugate N-ER-FY059Y01-305M28

[0666] The synthesis steps for siRNA conjugate N-ER-FY059Y01-305M28 are the same as those for conjugate D-ER-FY003AC002873-305M28, except that the sequences of the sense and antisense strands are replaced with the sequences corresponding to N-ER-FY059Y01-305M28 in Table 3.

[0667] Example 35 Synthesis of siRNA conjugate N-ER-FY059Y01-305M35

[0668] The synthesis steps for siRNA conjugate N-ER-FY059Y01-305M35 are the same as those for conjugate D-ER-FY003AC002873-305M35, except that the sequences of the sense and antisense strands are replaced with the sequences corresponding to N-ER-FY059Y01-305M35 in Table 3.

[0669] Experimental Example 1: Inhibitory activity of the disclosed compound on the target gene SOD1 in mice.

[0670] This experiment used 6-8 week old male C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.). After 3 days of acclimatization, the mice were randomly divided into groups of 5 mice each based on body weight. The mice were administered subcutaneously with a single dose of 5 mg / kg siRNA conjugate (as shown by the siRNA ID in Table 6 below) and PBS (purchased from Gibco, catalog number 10010-023). The injection volume was 5 μL / g, and the day of administration was recorded as day 0 (D0). One week after administration, the mice were euthanized, and adipose tissue (gonadal fat, subcutaneous fat, and brown adipose tissue) and kidneys were collected. Homogenized samples were prepared, and RT-qPCR was used to analyze and detect SOD1 mRNA in different tissues. The inhibition rate relative to the PBS group was calculated. Inhibition rate % = (1 - mean of the administered group / mean of the control group) * 100.

[0671] The results are shown in Table 6.

[0672] Table 6

[0673] Experimental results show that the siRNA conjugate sequence of this application has a good inhibition rate on SOD1 mRNA in adipose tissue, especially the best inhibition on subcutaneous fat.

[0674] Experimental Example 2: Inhibitory activity of the disclosed compound on the target gene SOD1 in mice.

[0675] Six- to eight-week-old male C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 3 days and then randomly divided into groups of 5 mice each based on body weight. The mice were administered subcutaneously with a single dose of 5 mg / kg of siRNA conjugate (as shown by the siRNA ID in Table 7 below) and PBS (purchased from Gibco, catalog number 10010-023), with an injection volume of 5 μL / g. One week after administration, the mice were euthanized, and adipose tissue (gonadal fat, subcutaneous fat, and brown adipose tissue) and kidneys were collected. Homogenized samples were prepared, and SOD1 mRNA in different tissues was detected using the method described in Experiment Example 1. The inhibition rate relative to the PBS group was calculated, and the results are shown in Table 7.

[0676] Table 7

[0677] Experimental results show that the siRNA conjugate sequence of this application has a good inhibition rate against SOD1 mRNA in adipose tissue.

[0678] In addition, at the same dose (5 mg / kg), the siRNA conjugate D-ER-FY003109 inhibited the mRNA of SOD1 in subcutaneous fat by only 43.64%.

[0679] Experimental Example 3: Inhibitory activity of the disclosed compound on the target gene SOD1 in mice.

[0680] Six- to eight-week-old male C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for three days and then randomly divided into groups of five mice each based on body weight. The mice were administered subcutaneously with different doses of siRNA conjugates (as shown by the siRNA IDs in Table 8 below) and PBS (purchased from Gibco, catalog number 10010-023) at an injection volume of 5 μL / g. Two weeks after administration, the mice were euthanized, and adipose tissue (gonadal fat, subcutaneous fat, and brown adipose tissue) was collected and homogenized. SOD1 mRNA in different tissues was detected using the method described in Experiment Example 1, and its inhibition rate relative to the PBS group was calculated. The results are shown in Table 8.

[0681] Table 8

[0682] Experimental results show that the siRNA conjugate sequence of this application has a good inhibition rate against SOD1 mRNA in adipose tissue.

[0683] Experimental Example 4: Inhibitory activity of the disclosed compound on the target gene ALK7 in mice.

[0684] Six- to eight-week-old male C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 3 days and then randomly divided into groups of 5 mice each based on body weight. The mice were administered subcutaneously with a single dose of 5 mg / kg of the siRNA conjugate N-ER-FY059305M2 and PBS (purchased from Gibco, catalog number 10010-023), with an injection volume of 5 μL / g. Two weeks after administration, the mice were euthanized. Gonadal adipose tissue, subcutaneous adipose tissue, and brown adipose tissue were homogenized. ALK7 mRNA in different tissues was detected using the method described in Example 1, and its inhibition rate relative to the PBS group was calculated. The results are shown in Table 9.

[0685] Table 9

[0686] Experimental results show that the siRNA conjugate sequence of this application has a good inhibition rate against ALK7 mRNA in adipose tissue.

[0687] Experimental Example 5: Inhibition of the target protein adiponectin by the disclosed compound in mice.

[0688] Six- to eight-week-old male C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for three days and then randomly divided into groups of five mice each based on body weight. Each group received a subcutaneous injection of either 2 mg / kg or 5 mg / kg of a single dose of the siRNA conjugate D-ER-FY003AC002873-305M2, or PBS (purchased from Gibco, catalog number 10010-023), with an injection volume of 5 μL / g. Serum samples were collected weekly before (day 0) and after administration for subsequent adiponectin ELISA detection. The adiponectin detection experiment in serum was performed using an adiponectin ELISA kit (purchased from RD, catalog number: MRP300). The collected serum samples were processed according to the kit instructions, and then the light absorption at 450 nm was detected using a multi-functional microplate reader (purchased from PE, model: Ensight). The adiponectin content in each sample was calculated by comparing with the standard curve. The adiponectin content in the serum collected after drug administration was compared with the adiponectin content in the serum of the PBS group to observe the adiponectin inhibition in each group of mice. The results are shown in Table 10.

[0689] Table 10

[0690] Experimental results show that the siRNA conjugate sequence of this application has a good inhibition rate against adiponectin in serum.

[0691] Experimental Example 6: Inhibitory activity of the disclosed compound on the target gene SOD1 in mice.

[0692] Six- to eight-week-old male C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 3 days and then randomly divided into groups of 5 mice each based on body weight. The mice were administered subcutaneously with a single dose of 5 mg / kg of siRNA conjugate (as shown in Table 11) and PBS (purchased from Gibco, catalog number 10010-023), with an injection volume of 5 μL / g. One week after administration, the mice were euthanized, and subcutaneous adipose tissue was collected and homogenized. Following the method described in Example 1, SOD1 mRNA in the subcutaneous tissue was detected, and its inhibition rate relative to the PBS group was calculated. The results are shown in Table 11.

[0693] Table 11

[0694] Experimental results show that the siRNA conjugate sequence of this application has a good inhibition rate against SOD1 mRNA in adipose tissue.

[0695] Experimental Example 7: Inhibitory activity of the disclosed compound on the target gene SOD1 in mice.

[0696] Six- to eight-week-old male C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 3 days and then randomly divided into groups of 5 mice each based on body weight. Different doses of siRNA conjugates (1 mg / kg and 3 mg / kg) and PBS (purchased from Gibco, catalog number 10010-023) were administered subcutaneously at an injection volume of 5 μL / g. One week after administration, the mice were euthanized, and subcutaneous adipose tissue was collected, homogenized, and the SOD1 mRNA in the subcutaneous tissue was detected. The inhibition rate relative to day 0 was calculated, and the results are shown in Table 12.

[0697] Table 12

[0698] Experimental results show that the siRNA conjugate sequence of this application has a good inhibition rate against SOD1 mRNA in adipose tissue.

[0699] Experimental Example 8: Inhibition of the target protein adiponectin by the disclosed compound in mice.

[0700] Six- to eight-week-old male C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into groups of five based on body weight after a three-day acclimatization period. Each group received a single subcutaneous dose of one of the following siRNA conjugates: D-ER-FY003AC002873-305M28 (1 mg / kg, 3 mg / kg), D-ER-FY003AC002873-305M35 (1 mg / kg, 3 mg / kg), or D-ER-FY003AC002873-305M40 (1 mg / kg, 3 mg / kg), or PBS (Gibco, catalog number 10010-023). Serum samples were collected weekly before (day 0) and after administration for subsequent ELISA detection of adiponectin levels in the serum. The adiponectin detection experiment in serum was performed using an adiponectin ELISA kit (purchased from RD, catalog number: MRP300). The collected serum samples were processed according to the kit instructions, and then the light absorption at 450 nm was detected using a multi-functional microplate reader (purchased from PE, model: Ensight). The adiponectin content in each sample was calculated by comparing with the standard curve. The adiponectin content in the serum collected after drug administration was compared with the adiponectin content in the serum of the PBS group to observe the adiponectin inhibition in each group of mice. The results are shown in Table 13 and Figure 2.

[0701] Table 13

[0702] Experimental results show that the siRNA conjugate sequence of this application has a good inhibition rate against adiponectin in serum.

[0703] Experimental Example 9: Inhibitory activity of the disclosed compound on the target gene SOD1 in mice.

[0704] Six- to eight-week-old male C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 3 days and then randomly divided into groups of 5 mice each based on body weight. The mice were subcutaneously administered siRNA conjugate (1 mg / kg) and PBS (purchased from Gibco, catalog number 10010-023) at an injection volume of 5 μL / g. One week after administration, the mice were euthanized, and subcutaneous adipose tissue, liver, quadriceps femoris muscle, myocardium, and kidneys were collected. Homogenized samples were prepared, and SOD1 mRNA in each tissue was detected. The inhibition rate relative to day 0 was calculated using the method described in Example 1. The results are shown in Table 14.

[0705] Table 14

[0706] Experimental results show that the siRNA conjugate of this application has a good adipose tissue-specific knockdown effect on target genes.

[0707] Experimental Example 10: Target gene inhibitory activity of the disclosed compounds in ALK7 humanized mice. 6-8 week old ALK7 humanized mice (Jiangsu Jicui, catalog number: T066492) were acclimatized for 3 days and then randomly divided into groups of 5 mice per group based on body weight. They were subcutaneously administered a single dose of 5 mg / kg of the siRNA conjugates N-ER-FY059Y01D1, N-ER-FY059Y01-305M28, and N-ER-FY059Y01-305M28. 5M35 and PBS (purchased from Gibco, catalog number 10010-023), injected at a volume of 5 μL / g, were administered to mice. Two weeks after administration, the mice were euthanized, and subcutaneous fat, gonadal fat, and brown adipose tissue were collected and homogenized. Following the method described in Example 1, ALK7 mRNA in different tissues was detected, and its inhibition rate relative to day 0 was calculated. The results are shown in Table 15. N-ER-FY059Y01D1 (Arrowhead) served as a positive control, and its structure is as follows:

[0708] Table 15

[0709] Experimental results show that the siRNA conjugate sequence of this application has a good inhibition rate against ALK7 mRNA in adipose tissue.

[0710] Example 11: Repeated-dose toxicity test of subcutaneous injection of siRNA conjugate in SD rats

[0711] This experiment consisted of three groups. The main experimental group comprised three male and three female SD rats (from Beijing Vital River Laboratory Animal Science and Technology Co., Ltd., Hebei Province). After a three-day acclimatization period, the rats were subcutaneously injected with the solvent control (0.9% sodium chloride injection), 100 or 300 mg / kg of N-ER-FY059Y01D1 (positive control), N-ER-FY059Y01-305M28, and N-ER-FY059Y01-305M35, respectively. Administration was repeated every two weeks for a total of three times (D0, D14, and D28), with a dosage of 5 mL / kg. A planned dissection was performed on D30 for each group at the end of the administration period. A satellite group (with the same dosage and administration method as the main experimental group) was established, with three male and three female rats per group for blood collection to study cytokines (including IFN-γ, TNF-α, IL-6, IL-10, etc.).

[0712] The toxicity parameters in this study included: general observation, body weight, food intake, clinicopathological parameters (hematological and coagulation parameters, serum biochemical parameters), cytokines, toxicokinetics, tissue distribution, gross necropsy, and histopathology. Blood samples were collected from satellite group animals at the adaptation period, on D1, and 4 and 24 hours after drug administration for cytokine analysis.

[0713] Under the conditions of this experiment, SD rats were subcutaneously injected with N-ER-FY059Y01D1 at doses of 100 and 300 mg / kg for 4 consecutive weeks (once every 2 weeks, for a total of 3 times). The no-observed-adverse-effect level (NOAEL) was 100 mg / kg. SD rats were subcutaneously injected with N-ER-FY059Y01-305M28 at doses of 100 and 300 mg / kg for 4 consecutive weeks (once every 2 weeks, for a total of 3 times). The NOAEL was 300 mg / kg. SD rats were subcutaneously injected with N-ER-FY059Y01-305M35 at doses of 100 and 300 mg / kg for 4 consecutive weeks (once every 2 weeks, for a total of 3 times). The NOAEL was 300 mg / kg.

[0714] In summary, the siRNA conjugates N-ER-FY059Y01-305M28 and N-ER-FY059Y01-305M35 involved in this application have higher NOAEL values ​​than the positive control N-ER-FY059Y01D1, indicating lower toxicity.

[0715] The above specific embodiments are merely illustrative of the content of this application and do not constitute a limitation thereof. For those skilled in the art, any modifications, alterations, or equivalent substitutions made without departing from the spirit and substance of this application should be considered to fall within the protection scope of this application.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, said compound comprising an oligonucleotide, and at least one lipid group and at least one group containing a polyethylene glycol ether chain. [Lipid group]-[Oligo]-[group containing polyethylene glycol ether chain] Formula (I) Oligo is an oligonucleotide, wherein the length of the oligonucleotide is 8 to 50 nucleotides, in which... Each nucleotide can be independently modified or unmodified. The lipid group has the structure shown in formula (Ia): Wherein, L1 is selected from a bond or a linker connected to the oligonucleotide; A1 is selected from chain-like saturated aliphatic hydrocarbon groups optionally substituted with one or more halogens or hydroxyl groups, preferably straight-chain or branched C groups optionally substituted with one or more halogens or hydroxyl groups. 14-28 Chain-like saturated aliphatic hydrocarbon groups; R 1 Selected from hydrogen, hydroxyl, C1-C6 alkyl, C3-C6 cycloalkyl, halogen or C1-C6 haloalkyl, wherein the C1-C6 alkyl and C1-C6 haloalkyl are optionally substituted with one or more hydroxyl groups; The groups containing polyethylene glycol ether chains have the structure shown in formula (Ib): L2 is selected from phosphate ester linking groups or phosphate ester derivative linking groups; When present, L3 is selected from a bond or a linker that can connect the PEG molecule to L2; Y is selected from bonds, hydroxyl groups, methoxy groups, -O- or -OCH2-; When present, A2 is selected from one or more R's. 5 The substituted chain-like saturated aliphatic hydrocarbon group, preferably optionally replaced by one or more R groups. 5 Replacement of straight or branched C 10-22 Chain-like saturated aliphatic hydrocarbon groups, the R 5 Each is independently selected from OH or COOH; L4 is selected from a key or a connector used to connect A2; Each PEG unit is independently selected from PEGs with a weight-average molecular weight between 100 Da and 10000 Da, or each PEG unit independently contains n polyethylene glycol repeating units, where n is greater than or equal to 2. q is selected from integers between 1 and 5, and when q is selected from integers above 2, L2-(L3)-PEG are either the same or different from each other.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, L1 is selected from bonds, phosphate ester linking groups, or phosphate ester derivative linking groups.

3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein, L1 is selected from key, 4. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein, L1 is selected from key, 5. The compound of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein, A1 is selected from the straight chain C. 14-28 Chain-like saturated aliphatic hydrocarbon group, the straight-chain C 14-28 The chain-like saturated aliphatic hydrocarbon group is optionally substituted with one or more hydroxyl groups, Cl or F, preferably with one F.

6. The compound of any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein, A1 is selected from the straight chain C. 14 C 15 C 16 C 17 C 18 C 19 C 20 C 21 C 22 C 23 C 24 C 25 C 26 C 27 Or C 28 A chain of saturated aliphatic hydrocarbon groups, which may optionally be substituted with one or more Cl or F groups.

7. The compound of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein, A1 is selected from the straight chain C. 16 C 18 C 20 Or C 22 A chain of saturated aliphatic hydrocarbon groups, which may optionally be substituted with one or more Cl or F groups.

8. The compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, wherein, A1 is selected from the straight chain C. 16 Or C 22 A chain-like saturated aliphatic hydrocarbon group, which is optionally substituted with one F.

9. The compound of any one of claims 1-8 or a pharmaceutically acceptable salt thereof, wherein, A1 is selected from 10. The compound of any one of claims 1-9, or a pharmaceutically acceptable salt thereof, wherein, A1 is selected from any of the following structures:

11. The compound of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein, R 1 It is selected from hydrogen, hydroxyl, C1-C6 alkyl, halogen or C1-C6 haloalkyl, wherein the C1-C6 alkyl and C1-C6 haloalkyl are optionally substituted with one or more hydroxyl or halogens.

12. The compound of any one of claims 1-11, or a pharmaceutically acceptable salt thereof, wherein, R 1 It is selected from hydrogen, hydroxyl, C1-C3 alkyl, halogen or C1-C3 haloalkyl, wherein the C1-C3 alkyl and C1-C3 haloalkyl are optionally substituted with one or more hydroxyl or halogens.

13. The compound of any one of claims 1-12, or a pharmaceutically acceptable salt thereof, wherein, R 1 It is selected from hydrogen, hydroxyl, methyl, ethyl, Cl, F, -CH2OH, -CH2F, -CHF2 or -CF3.

14. The compound of any one of claims 1-13, or a pharmaceutically acceptable salt thereof, wherein, R 1 It is hydrogen.

15. The compound of any one of claims 1-14, or a pharmaceutically acceptable salt thereof, wherein, Structural unit Selected from 16. The compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, wherein, Structural unit for 17. The compound of any one of claims 1-16, or a pharmaceutically acceptable salt thereof, wherein, The lipid groups represented by formula (Ia) are selected from: Where X is O or S; Where X is O or S; 18. The compound of any one of claims 1-17 or a pharmaceutically acceptable salt thereof, wherein, The lipid group represented by formula (Ia) is selected from any of the following: Where X is O or S; Where X is O or S; Where X is O or S; Where X is O or S; 19. The compound of any one of claims 1-18, or a pharmaceutically acceptable salt thereof, wherein, The lipid group represented by formula (Ia) is selected from any of the following:

20. The compound of any one of claims 1-19, or a pharmaceutically acceptable salt thereof, wherein, The compound represented by formula (I) contains one, two, or three lipid groups represented by formula (Ia).

21. The compound of any one of claims 1-20, or a pharmaceutically acceptable salt thereof, wherein, When the lipid group shown in formula (Ia) is attached to the phosphate group of a modified or unmodified nucleotide residue at the 5' end of an oligonucleotide, the lipid group shown in formula (Ia) is Alternatively, when the 5' modifying group of the oligonucleotide is (invAb), the structures of L1 and (invAb) are as follows: Where X is independently either S or O, * indicates the direction toward the lipid group, and ** indicates the direction toward the oligonucleotide.

22. The compound of any one of claims 1-21, or a pharmaceutically acceptable salt thereof, wherein, The compound represented by formula (I) contains 1, 2, 3, 4 or 5 groups containing polyethylene glycol ether chains as shown in formula (Ib).

23. The compound of any one of claims 1-22, or a pharmaceutically acceptable salt thereof, wherein, The group containing a polyethylene glycol ether chain shown in formula (Ib) comprises two or more polyethylene glycol repeating units and contains no more than 150 polyethylene glycol repeating units.

24. The compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-23, wherein, Each of the PEGs is independently selected from PEGs with a weight-average molecular weight between 100 Da and 6000 Da.

25. The compound of any one of claims 1-24, or a pharmaceutically acceptable salt thereof, wherein, n is an integer between 3 and 6; or q is selected from 1, 2, 3 or 4.

26. The compound of claim 25 or a pharmaceutically acceptable salt thereof, wherein, n is 3, and q is 1, 2, or 3; or n is 6, and q is 1 or 2.

27. The compound of claim 25 or a pharmaceutically acceptable salt thereof, wherein, q is 2, where n is independently selected from 3 or 6; or q is 3, where n is independently selected from 3 or 6.

28. The compound of any one of claims 1-25, or a pharmaceutically acceptable salt thereof, wherein, The PEG is selected from: or (MW is approximately 1000-5000 Da).

29. The compound of any one of claims 1-28 or a pharmaceutically acceptable salt thereof, wherein, L2 is selected from phosphate ester linking groups or phosphate ester derivative linking groups.

30. The compound of claim 29 or a pharmaceutically acceptable salt thereof, wherein, L2 is selected from phosphate ester linking groups or thiophosphate ester linking groups.

31. The compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-30, wherein, L3 is selected from key, containing Linkers, or linkers containing amide groups.

32. The compound of claim 31 or a pharmaceutically acceptable salt thereof, wherein, The inclusion The connector is Where R 2 and R 3 Each is independently selected from key, C 1-8 Alkylene or C 1-8 alkeneoxy, the C 1-8 Alkylene or C 1-8 The alkoxide may optionally be replaced by one or more halogens, amino groups, or hydroxyl groups.

33. The compound of claim 32 or a pharmaceutically acceptable salt thereof, wherein, R 2 and R 3 Each is independently selected from C 1-6 Alkylene or C 1-6 alkeneoxy, the C 1-6 Alkylene or C 1-6 The alkoxide may optionally be replaced by one or more halogens, amino groups, or hydroxyl groups.

34. The compound of claim 33 or a pharmaceutically acceptable salt thereof, wherein, R 2 and R 3 Each is independently selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -C(CH3)2-, -CH2CH(CH3)2-, -CH(CH3)CH2CH3, -CH2CH(CH3)CH2CH3, -CH2C(CH3)2-, -CH2CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH(CH3)CH2CH2CH2CH2CH2-, -CH2CH(CH3)CH2CH2CH2CH2-, -OCH2-, -OCH2CH2 -, -OC(CH3)2-, -OCH2CH2CH2-, -OCH2CH2CH2CH2-, -OCH2CH2CH2CH2CH2-, -OCH2CH2CH2CH2CH2CH2-, -OCH2CH(CH3)2-, -OCH(CH3)CH2CH3, -OCH2CH(CH3)CH2CH3, -OCH2C(CH3)2-, -OCH2CH(CH3)CH2CH2CH2-, -OCH(CH3)CH2CH2CH2CH2-, -OCH(CH3)CH2CH2CH2CH2CH2-, or -OCH2CH(CH3)CH2CH2CH2CH2-, which may optionally be substituted with one or more halogens, amino groups or hydroxyl groups.

35. The compound of claim 34 or a pharmaceutically acceptable salt thereof, wherein, R 2 and R 3 Each is independently selected from the group, ethylidene, or hexanede; or, R 2 For Aztec, R 3 It is a bond or an ethylene.

36. The compound of claim 31 or a pharmaceutically acceptable salt thereof, wherein, The linker containing the amide group is selected from -R 4 -NH-C(O)-, where R 4 Selected from C 1-8 Alkylene or C 1-8 alkeneoxy, the C 1-8 Alkylene or C 1-8 The alkoxide may optionally be replaced by one or more halogens, amino groups, or hydroxyl groups.

37. The compound of claim 36 or a pharmaceutically acceptable salt thereof, wherein, R 4 Selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -C(CH3)2-, -CH2CH(CH3)2-, -CH(CH3)CH2CH3, -CH2CH( CH3)CH2CH3, -CH2C(CH3)2-, -CH2CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH(CH3)CH2CH2CH2CH2CH2-, -CH2CH(CH3)CH2CH2CH2CH2-, -OCH2-, -OCH2CH2-, -OC(CH3)2-, -OCH2CH2CH2-, -OCH2CH2CH2CH2-, -OCH2CH2CH2CH2CH2-, -OCH2CH2CH2CH2CH2CH2-, -OCH2CH(CH3)2-, -OCH(CH3)CH2CH3, -OCH2CH(CH3)CH2CH3, -OCH2C(CH3)2-, -OCH2CH(CH3)CH2CH2-, -OCH(CH3)CH2CH2CH2-, -OCH(CH3)CH2CH2CH2CH2-, or -OCH2CH(CH3)CH2CH2CH2CH2-, which may optionally be substituted with one or more halogens, amino groups, or hydroxyl groups.

38. The compound of claim 36 or a pharmaceutically acceptable salt thereof, wherein, R 4 Selected from C 1-6 Alkylene or C 1-6 alkeneoxy, the C 1-6 Alkylene or C 1-6 The alkoxide may optionally be replaced by one or more halogens, amino groups, or hydroxyl groups.

39. The compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-38, wherein, L3 is a bond or has a structure selected from the following:

40. The compound of any one of claims 1-39, or a pharmaceutically acceptable salt thereof, wherein, L2-(L3)-PEG is selected from any of the following: Where X is 0 or S, and n is an integer selected from 3 to 10; or, Where X is 0 or S, and n is an integer between 3 and 10.

41. The compound of any one of claims 1-40, or a pharmaceutically acceptable salt thereof, wherein, L4 is selected from a bond, or has any of the following structures:

42. The compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-41, wherein, A2 is selected from Among them, R 5 Selected from OH or COOH; Preferably, A2 is selected from the following structures:

43. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, The groups containing polyethylene glycol ether chains shown in formula (Ib) are selected from: Wherein, L2, L3, q and PEG are as defined in claim 1; wherein Y is selected from hydroxyl or methoxy.

44. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, When L4-A2 is present, Y is selected from the bond, -O-, or -O-CH2-.

45. The compound of any one of claims 1-42, or a pharmaceutically acceptable salt thereof, wherein, L2 is selected from phosphate ester linking groups or thiophosphate ester linking groups. Alternatively, L3 can be a key or selected from... Alternatively, Y can be selected from the key, -O-, or -OCH2-; Alternatively, A2 is selected from Among them, R 5 Selected from OH or COOH; Alternatively, L4 is selected from Alternatively, each PEG independently contains 2 to 8 -OCH2CH2-, preferably selected from the PEG selected from: Alternatively, q can be selected from integers between 1 and 5, and when q is selected from integers greater than 2, L2-(L3)-PEG can be the same or different from each other.

46. ​​The compound of claim 44 or a pharmaceutically acceptable salt thereof, wherein, L2 is selected from phosphate ester linking groups or thiophosphate ester linking groups. Alternatively, L3 can be a key or selected from... Alternatively, Y can be selected from the key, -O-, or -OCH2-; Alternatively, A2 is selected from Among them, R 5 Selected from OH or COOH, preferably Alternatively, L4 is selected from Alternatively, each PEG independently contains 2 to 8 -OCH2CH2-, preferably selected from the PEG selected from: Alternatively, q can be selected from integers between 1 and 3, and when q is selected from integers greater than 2, L2-(L3)-PEG can be the same or different from each other.

47. The compound of claim 44 or a pharmaceutically acceptable salt thereof, wherein, L2 is selected from phosphate ester linking groups or thiophosphate ester linking groups. Alternatively, L3 is selected from Alternatively, Y can be selected from the key, -O-, or -OCH2-; Alternatively, A2 is selected from Among them, R 5 Selected from OH or COOH, preferably Alternatively, L4 is selected from Alternatively, each PEG independently contains 3 to 6 -OCH2CH2-, preferably selected from the PEG selected from: Alternatively, q can be 1.

48. The compound of claims 1-47 or a pharmaceutically acceptable salt thereof, wherein, The groups containing polyethylene glycol ether chains shown in formula (Ib) are selected from any of the following: Where X is selected from O or S; Where X is selected from O or S; Where X is selected from O or S; Each of X is independently selected from O or S; Each of X is independently selected from O or S; Where X is selected from O or S; Each of X is independently selected from O or S; Each of X is independently selected from O or S; Each of X is independently selected from O or S; In this case, each X is independently selected from either O or S; In this case, each X is independently selected from either O or S; In this case, each X is independently selected from either O or S; Where X is selected from O or S; Where X is selected from O or S; Where X is selected from O or S; Where X is selected from O or S; Where X is selected from O or S; Where X is selected from O or S; Where X is selected from O or S; Where X is selected from O or S; Where X is selected from O or S; Where X is selected from O or S; or Where X is selected from O or S.

49. The compound of claim 48 or a pharmaceutically acceptable salt thereof, wherein, The groups containing polyethylene glycol ether chains shown in formula (Ib) are selected from any of the following:

50. The compound of any one of claims 1-49, or a pharmaceutically acceptable salt thereof, wherein, When the 3' modifying group of the oligonucleotide is (invAb), the structure of one of the L2 groups and (invAb) can be as follows: Where X represents S or O, * indicates the direction toward the oligonucleotide, and ** indicates the direction toward the group containing the polyethylene glycol ether chain.

51. A compound or a pharmaceutically acceptable salt thereof, selected from the following: in, The double helix structure represents dsRNA.

52. The compound of any one of claims 1-51 or a pharmaceutically acceptable salt thereof, wherein, The oligonucleotides in the compound are single-stranded or double-stranded oligonucleotides.

53. The compound of claim 52 or a pharmaceutically acceptable salt thereof, wherein, The oligonucleotide is siRNA.

54. The compound of claim 52 or a pharmaceutically acceptable salt thereof, wherein, The double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense strand is complementary to the target gene; the sense strand is complementary to the antisense strand.

55. The compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-54, wherein it is a mixture comprising one or more compounds of formula (I) or pharmaceutically acceptable salts thereof; Optionally, the mixture comprises two or more compounds of formula (I) or pharmaceutically acceptable salts thereof; Optionally, the mixture is a mixture comprising: Optionally, the mixture is a mixture comprising: Optionally, the mixture is a mixture comprising: Optionally, the mixture is a mixture comprising: Optionally, the mixture is a mixture comprising: Optionally, the mixture is a mixture comprising: Optionally, the mixture is a mixture comprising:

56. A pharmaceutical composition comprising the compound of any one of claims 1-55 or a pharmaceutically acceptable salt thereof.

57. The pharmaceutical composition of claim 56, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

58. The use of the compound of any one of claims 1-55 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 56 or 57 in the preparation of a medicament.

59. The application as described in claim 58, wherein, The drug is used for the prevention and / or treatment of metabolic-related diseases / conditions; or... The drug is used to prevent and / or treat obesity-related diseases; or, The drug is used to prevent and / or treat tumor-related diseases.

60. The application as described in claim 58 or 59, wherein, The diseases / conditions targeted by the drug include: obesity, type 2 diabetes, and hyperlipidemia.

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