GalNAc compounds, conjugates, compositions, and uses thereof

Abstract

The present disclosure relates to the field of medicine, in particular to GalNAc compounds, conjugates, compositions and uses thereof. The oligonucleotide conjugates provided by the present disclosure are synthesized by the GalNAc compounds provided by the present disclosure, which can highly effectively target to hepatocytes and effectively inhibit the expression of target genes in hepatocytes, and can be used for treating and / or preventing liver-derived diseases, and have the advantages of high in vivo activity, stable and long-lasting efficacy, excellent safety and low toxicity at animal level.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of medicine, in particular to GalNAc compounds, conjugates, compositions and uses thereof. BACKGROUND

[0002] The asialoglycoprotein receptor (ASGPR), also known as liver lectin, is a transmembrane protein that is expressed significantly and specifically on the cell surface of the liver sinusoid and basolateral side. The ASGPR is mainly composed of two subunits H1 and H2, which have endocytosis function in combination. A normal mature hepatocyte has about 500,000 receptor molecules, about 10% of which are expressed on the cell membrane, which can specifically recognize, bind and endocytose some glycoproteins in the blood circulation with terminal galactosyl and N-acetylglucosamine groups, so as to be metabolized in hepatocytes. Targeting ligand conjugated oligonucleotides are used, and the targeting ligand is combined with the ASGPR to achieve liver-targeted delivery of oligonucleotides.

[0003] However, the affinity of the ligand of the ASGPR is not high at present, and the liver-targeted delivery of drugs or genes cannot be well achieved to achieve the purpose of regulating target gene expression. SUMMARY

[0004] Therefore, the present disclosure provides GalNAc compounds, conjugates, compositions and uses thereof.

[0005] The oligonucleotide conjugate provided by the present disclosure can be highly effectively targeted to the liver and effectively inhibit the expression of liver target genes, and can be used for treating and / or preventing liver diseases, has higher in vivo activity than the control carrier, more stable and persistent drug efficacy, and is expected to have excellent safety and low animal level toxicity.

[0006] In order to achieve the above-mentioned purpose of the application, the present disclosure provides the following technical solutions:

[0007] In a first aspect of the present disclosure, the present disclosure provides a compound having a structure represented by formula (Ia) or a stereoisomer, a pharmaceutically acceptable salt or a prodrug thereof:

[0008]

[0009] In formula (Ia), R1 is selected from H, a hydroxyl protecting group or The * represents a linking site for connecting a pharmaceutically active molecule;

[0010] R2 is selected from H, a reactive phosphorus group or wherein R 2bR selected from a solid support comprising an amino functional group 2a a covalent linker attached to the amino functional group;

[0011] said j is selected from 0, 1, 2, or 3;

[0012] each M is independently selected from hydroxyl or thiol;

[0013] each p is independently selected from 0, 1, 2, 3, or 4;

[0014] each q is independently selected from 0, 1, 2, 3, or 4;

[0015] each Cy is independently selected from wherein said A is selected from a 5-6 membered saturated oxygen-containing heterocycle; said R3 is selected from H or C1-C6 alkoxy; said n is selected from 1 or 2; each X is independently selected from O, S, or NH; each R4 is independently selected from H, C1-C6 alkyl, or C1-C6 alkoxy; each L1 is independently selected from a substituted or unsubstituted triazole group, or an amide group; said m is selected from 1 or 2; each L2 is independently selected from a substituted or unsubstituted C1-C30 alkylene group, or each r is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, each Z is independently selected from O, S, or NH, and each R L2 each independently selected from a substituted or unsubstituted C1-C10 alkylene group; each Y is independently selected from O, S, or NH; and each R5 is independently selected from H, a substituted or unsubstituted C1-C4 alkyl acyl group, or a substituted or unsubstituted C6-C12 aryl acyl group.

[0016] In some optional embodiments of the present disclosure, said A is selected from a 5-6 membered saturated oxygen-containing heterocycle.

[0017] In some optional embodiments of the present disclosure, said A is selected from a 5-6 membered saturated heterocycle comprising one oxygen atom.

[0018] In some optional embodiments of the present disclosure, said A is selected from

[0019] In some specific embodiments of the present disclosure, said A is selected from

[0020] In some specific embodiments of the present disclosure, said A is selected from

[0021] In some specific embodiments of the present disclosure, said m is selected from 1.

[0022] In some specific embodiments of the present disclosure, said X is selected from O.

[0023] In some embodiments of the disclosure, each R4is selected from H.

[0024] In some alternative embodiments of the disclosure, each L1is independently selected from

[0025] In some embodiments of the disclosure, each L1is selected from

[0026] In some embodiments of the disclosure, each L1is selected from

[0027] In some alternative embodiments of the disclosure, the R3is selected from H or C1-C3alkoxy.

[0028] In some embodiments of the disclosure, the R3is selected from H or methoxy.

[0029] In some embodiments of the disclosure, the R3is selected from H.

[0030] In some embodiments of the disclosure, the R3is selected from methoxy.

[0031] In some alternative embodiments of the disclosure, each L2is independently selected from each r is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, each Z is independently selected from O, S, or NH, each R L2 is independently selected from substituted or unsubstituted C1-C10alkylene.

[0032] In some alternative embodiments of the disclosure, each L L2 is independently selected from substituted or unsubstituted C1-C5alkylene.

[0033] In some alternative embodiments of the disclosure, each L L2 is independently selected from substituted or unsubstituted C1-C3alkylene.

[0034] In some alternative embodiments of the disclosure, each L L2 is independently selected from substituted or unsubstituted C2alkylene.

[0035] In some alternative embodiments of the disclosure, each r is independently selected from 2 or 3.

[0036] In some alternative embodiments of the disclosure, each L2is independently selected from the r is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0037] In some embodiments of the disclosure, each L2is independently selected from said r is selected from 2 or 3.

[0038] In some embodiments of the disclosure, each L2is selected from

[0039] In some embodiments of the disclosure, each L2is selected from

[0040]

[0041] In some embodiments of the disclosure, each Y is selected from O.

[0042] In some embodiments of the disclosure, each R5is independently selected from H,

[0043] In some embodiments of the disclosure, each R5is selected from

[0044] In some embodiments of the disclosure, each R5is selected from H.

[0045] In some embodiments of the disclosure, said p and q are each independently selected from 0 or 1.

[0046] In some embodiments of the disclosure, p = 1 and q = 1.

[0047] In some embodiments of the disclosure, p = 1 and q = 0.

[0048] In some embodiments of the disclosure, p = 0 and q = 1.

[0049] In some embodiments of the disclosure, p = 0 and q = 0.

[0050] In the disclosure, the hydroxyl protecting group can be various hydroxyl protecting groups as long as it can protect the hydroxyl group, and the specific type is not limited. In some embodiments, the hydroxyl protecting group is stable under basic conditions, but can be removed under acidic conditions.

[0051] In some alternative embodiments of the disclosure, the hydroxyl protecting groups that can be used in the disclosure include, but are not limited to, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthine-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthine-9-yl (Mox), trityl (Tr), 4-methoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr), and 4,4',4"-trimethoxytrityl (TMTr).

[0052] In some alternative embodiments of the disclosure, the hydroxyl protecting group is selected from trityl (Tr), 4-methoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr), or 4,4',4"-trimethoxytrityl (TMTr).

[0053] In some specific embodiments of the disclosure, the hydroxyl protecting group is selected from 4,4'-dimethoxytrityl (DMTr).

[0054] In the disclosure, a “reactive phosphorus group” refers to a phosphorus-containing group that can be removed by reaction with other compounds.

[0055] In some alternative embodiments of the disclosure, the reactive phosphorus group is selected from

[0056] In some specific embodiments of the disclosure, the reactive phosphorus group is selected from

[0057] In some alternative embodiments of the disclosure, the pharmaceutically active molecule is selected from a small molecule drug, an antibody, or an oligonucleotide.

[0058] In some alternative embodiments of the disclosure, the oligonucleotide is selected from a single-stranded oligonucleotide and a double-stranded oligonucleotide.

[0059] In some alternative embodiments of the disclosure, the pharmaceutically active molecule is selected from a single-stranded oligonucleotide;

[0060] In some alternative embodiments of the disclosure, the single-stranded oligonucleotide is selected from an antisense oligonucleotide (ASO).

[0061] In some specific embodiments of the disclosure, the pharmaceutically active molecule is selected from a double-stranded oligonucleotide.

[0062] In some alternative embodiments of the disclosure, the double-stranded oligonucleotide is selected from a small interfering RNA (siRNA).

[0063] In some specific embodiments of the disclosure, the pharmaceutically active molecule is selected from a siRNA.

[0064] In some embodiments of the disclosure, the siRNA is conjugated to one of the compounds, and one of the compounds is conjugated to the 3' end of the sense strand of the siRNA.

[0065] In some alternative embodiments of the disclosure, the siRNA is conjugated to one of the compounds, and one of the compounds is conjugated to the 5' end of the sense strand of the siRNA.

[0066] In some embodiments of the disclosure, the siRNA is conjugated to two of the compounds, and two of the compounds are conjugated to the 3' end and the 5' end of the sense strand of the siRNA, respectively.

[0067] In some alternative embodiments of the disclosure, the solid support is selected from a resin comprising an amino functional group or a controlled pore glass bead comprising an amino functional group.

[0068] In some embodiments of the disclosure, R 2b is selected from represents a resin (e.g., polystyrene resin PS) or a controlled pore glass bead (CPG).

[0069] In some embodiments of the disclosure, R 2a is selected from

[0070] In some embodiments of the disclosure, is selected from

[0071] In some alternative embodiments of the disclosure, j is selected from 0, 1, or 2.

[0072] In some embodiments of the disclosure, j is selected from 0.

[0073] In some embodiments of the disclosure, j is selected from 1.

[0074] In some embodiments of the disclosure, j is selected from 2.

[0075] In some alternative embodiments of the disclosure, in Formula (la), each Cy is independently selected from any one of the following structures:

[0076]

[0077] In some embodiments of the disclosure, the compound is selected from any one of the following structures:

[0078]

[0079]

[0080] In some embodiments of the present disclosure, the compound is selected from any one of the following compounds:

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] In a second aspect of the present disclosure, the present disclosure also provides a conjugate having a structure shown in formula (Ib) or a stereoisomer, a pharmaceutically acceptable salt or a prodrug thereof:

[0100]

[0101] In formula (Ib), the Nu represents an oligonucleotide;

[0102] The k is selected from 1 or 2;

[0103] The structure of Q is wherein said j' is selected from 1, 2, 3, or 4;

[0104] said j', M, p, q, Cy are as defined in any of the preceding.

[0105] In some optional embodiments of the present disclosure, said j' is selected from 1, 2, or 3.

[0106] In some specific embodiments of the present disclosure, said j' is selected from 1.

[0107] In some specific embodiments of the present disclosure, said j' is selected from 2.

[0108] In some specific embodiments of the present disclosure, said j' is selected from 3.

[0109] In some specific embodiments of the present disclosure, said k is selected from 1.

[0110] In some specific embodiments of the present disclosure, said k is selected from 2.

[0111] In some optional embodiments of the present disclosure, said oligonucleotide is selected from a single-stranded oligonucleotide or a double-stranded oligonucleotide.

[0112] In some optional embodiments of the present disclosure, said oligonucleotide is selected from a single-stranded oligonucleotide. Said single-stranded oligonucleotide includes but is not limited to: an antisense oligonucleotide (ASO), an aptamer, a ribozyme, a deoxyribozyme, a circRNA, and the like.

[0113] In some optional embodiments of the present disclosure, said single-stranded oligonucleotide is selected from an ASO.

[0114] In some specific embodiments of the present disclosure, said oligonucleotide is selected from a double-stranded oligonucleotide. Said double-stranded oligonucleotide includes but is not limited to: a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a microRNA (miRNA), a small guide RNA (sgRNA), a small activating RNA (saRNA), a short hairpin RNA (shRNA), and the like.

[0115] In some specific embodiments of the present disclosure, said double-stranded oligonucleotide is selected from a siRNA.

[0116] In some specific embodiments of the present disclosure, said Nu represents a double-stranded oligonucleotide.

[0117] In some embodiments of the disclosure, the Nu represents an siRNA.

[0118] In some embodiments of the disclosure, the k is selected from 1, and one of the Q is conjugated to the 3' end of the sense strand of the siRNA.

[0119] In some alternative embodiments of the disclosure, the k is selected from 1, and one of the Q is conjugated to the 5' end of the sense strand of the siRNA.

[0120] In some embodiments of the disclosure, the k is selected from 2, and two of the Q are conjugated to the 3' end and the 5' end of the sense strand of the siRNA, respectively. In some alternative embodiments of the disclosure, the conjugate is selected from any one of the following structures:

[0121]

[0122]

[0123] In some embodiments of the disclosure, the conjugate is selected from any one of the following compounds:

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131] In a third aspect of the disclosure, the disclosure also provides a composition comprising the conjugate of the second aspect.

[0132] In some alternative embodiments of the disclosure, the composition further comprises, optionally, one or more pharmaceutically acceptable carriers or excipients.

[0133] In a fourth aspect of the disclosure, the disclosure also provides the use of any of the following in the preparation of a medicament for the prevention and / or treatment of a disease:

[0134] (I) the compound of the first aspect; and / or

[0135] (II) the conjugate of the second aspect; and / or

[0136] (I) the compound of the first aspect; and / or

[0137] In some embodiments of the present disclosure, the disease is selected from a pathological condition or disease caused by abnormal expression of a specific gene in a target cell.

[0138] In some embodiments of the present disclosure, the disease is selected from a liver-derived disease.

[0139] In a fifth aspect of the present disclosure, the present disclosure further provides use of any of the following in the preparation of a medicament for reducing expression or activity of a target gene.

[0140] (I) the compound of the first aspect; and / or

[0141] (II) the conjugate of the second aspect; and / or

[0142] (I) the compound of the first aspect; and / or

[0143] In a sixth aspect of the present disclosure, the present disclosure further provides a medicament comprising any of the following and a pharmaceutically acceptable adjuvant or aid:

[0144] (I) the compound of the first aspect; and / or

[0145] (II) the conjugate of the second aspect; and / or

[0146] (I) the compound of the first aspect; and / or

[0147] In a seventh aspect of the present disclosure, the present disclosure further provides a pharmaceutical combination comprising the medicament of the sixth aspect and other any effective ingredients.

[0148] In an eighth aspect of the present disclosure, the present disclosure further provides a method for treating and / or preventing a disease in a subject, comprising administering to the subject a pharmaceutically acceptable amount of any of the following:

[0149] (I) the compound of the first aspect; and / or

[0150] (I) the compound of the first aspect; and / or

[0151] (I) the compound of the first aspect; and / or

[0152] (I) the compound of the first aspect; and / or

[0153] In some embodiments of the present disclosure, the disease is selected from a pathological condition or disease caused by abnormal expression of a specific gene in a target cell.

[0154] In a ninth aspect of the present disclosure, the present disclosure also provides a method for reducing the expression or activity of a target gene, comprising contacting any of the following with a cell:

[0155] (I) the conjugate of the second aspect; and / or

[0156] (II) the composition of the third aspect; and / or

[0157] (III) the medicament of the sixth aspect; and / or

[0158] (IV) the pharmaceutical combination of the seventh aspect.

[0159] Advantages of the present disclosure:

[0160] The present disclosure provides GalNAc compounds, conjugates, compositions, and their uses. The present disclosure provides oligonucleotide conjugates by conjugating the compounds provided by the present disclosure to specific oligonucleotides (e.g. siRNA), which are able to highly effectively target the oligonucleotide conjugates to liver cells and effectively inhibit the expression of target genes in liver cells, which can be used for treating and / or preventing pathological conditions or diseases caused by abnormal expression of target genes in liver cells, have higher in vivo activity than control vectors, more stable and persistent pharmacodynamic effects, and are expected to have excellent safety and low animal level toxicity. BRIEF DESCRIPTION OF DRAWINGS

[0161] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure or the prior art, the drawings required to be used in the embodiments or prior art description will be briefly introduced below.

[0162] Figure 1 Relative expression level of the target gene in mice after administration of the siRNA conjugate of Example 1 in Example 1;

[0163] Figure 2 Relative expression level of the target gene in mice after administration of the siRNA conjugate of Example 2 in Example 2;

[0164] Figure 3 Relative expression level of the target gene in mice after administration of the siRNA conjugate of Example 3 in Example 3;

[0165] Figure 4 Relative expression level of the target gene in mice after administration of the siRNA conjugate of Example 4 in Example 4;

[0166] Figure 5 Relative expression level of the target gene in mice after administration of the siRNA conjugate of Example 5 in Example 5;

[0167] Figure 6 Relative expression levels of the target gene of interest in mice after administration of the siRNA conjugate described in Example 6 in Example 6;

[0168] Figure 7 Relative expression levels of the target gene of interest in mice after administration of the siRNA conjugate described in Example 7 in Example 7;

[0169] Figure 8 Relative expression levels of the target gene of interest in mice after administration of the siRNA conjugate described in Example 8 in Example 8;

[0170] Figure 9 Relative expression levels of the target gene of interest in mice after administration of the siRNA conjugate described in Example 9 in Example 9;

[0171] Figure 10 Relative expression levels of the target gene of interest in mice after administration of the siRNA conjugate described in Example 10 in Example 10;

[0172] Figure 11 Relative expression levels of the target gene of interest in mice after administration of the siRNA conjugate described in Example 11 in Example 11;

[0173] Figure 12 Relative expression levels of the target gene of interest in mice after administration of the siRNA conjugate described in Example 12 in Example 12;

[0174] Figure 13 Relative expression levels of the target gene of interest in mice after administration of the siRNA conjugate described in Example 13 in Example 13;

[0175] Figure 14 Relative expression levels of the target gene of interest in mice after administration of the siRNA conjugate described in Example 14 in Example 14;

[0176] Figure 15 Relative expression levels of the target gene of interest in mice after administration of the siRNA conjugate described in Example 15 in Example 15;

[0177] Figure 16a Detection of AST in the blood of animals in each group after administration of the siRNA conjugate described in Example 16 in Example 16;

[0178] Figure 16b Detection of ALT in the blood of animals in each group after administration of the siRNA conjugate described in Example 16 in Example 16;

[0179] Figure 16cDetection of TBIL in blood of each group of animals after administration of siRNA conjugate described in Example 16;

[0180] Figure 16d Detection of Urea in blood of each group of animals after administration of siRNA conjugate described in Example 16;

[0181] Figure 16e Detection of Crea in blood of each group of animals after administration of siRNA conjugate described in Example 16;

[0182] Figure 16f Detection of TC in blood of each group of animals after administration of siRNA conjugate described in Example 16;

[0183] Figure 16g Detection of TG in blood of each group of animals after administration of siRNA conjugate described in Example 16;

[0184] Figure 16h Detection of WBC in blood of each group of animals after administration of siRNA conjugate described in Example 16;

[0185] Figure 16i Detection of NEUT in blood of each group of animals after administration of siRNA conjugate described in Example 16;

[0186] Figure 16j Detection of LYMPH in blood of each group of animals after administration of siRNA conjugate described in Example 16;

[0187] Figure 16k Histopathological examination of liver tissue of each group of animals after administration of siRNA conjugate described in Example 16. DETAILED DESCRIPTION

[0188] The present disclosure discloses GalNAc compounds, conjugates, compositions and their uses, which can be implemented by those skilled in the art with reference to the content herein, with appropriate modification of process parameters. It is particularly pointed out that all such similar substitutions and modifications are apparent to those skilled in the art, and are considered to be included within the present disclosure. The methods and applications of the present disclosure have been described by preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present disclosure, to realize and apply the present disclosure technology.

[0189] The affinity of ASGPR to ligands is mainly affected by the following factors:

[0190] a. The type of sugar molecule at the end of the ligand. Both ligand molecules with terminal Gal or GalNAc can be recognized by ASGPR, and the affinity of ASGPR binding to GalNAc is 10-50 times higher than that to Gal. Moreover, GalNAc ligand molecules are more likely to escape the recognition of Kupffer cells and target more to hepatocytes.

[0191] b. The substitution site of the sugar molecule: 1-OH, 2-OH (or 2-amino), 3-OH, 4-OH and 5-CH2- of Gal (or GalNAc) are involved in the binding of ASGPR receptor; the affinity to the receptor is dramatically reduced when 5-CH2- is connected to a negatively charged group or 4-OH, 3-OH, 2-OH (or 2-amino) is substituted; when glycosylation substitution occurs at 1-OH, the affinity to the receptor is reduced when the glycosidic bond is in the α conformation, but is not affected when the substitution group is a linear structure and the glycosidic bond is in the β conformation; only 6-OH is not involved in receptor binding and points to the solvent region, and thus is suitable as a connection site to connect to carriers, drugs, etc.

[0192] c. The spatial distance between the end sugar group and the carrier. The sugar group and the carrier are at least 6 CH2units apart, which can be effectively recognized by ASGPR.

[0193] In addition, different connection modes between ligands with different molecular cluster structures and oligonucleotides can significantly affect the in vivo activity of oligonucleotides. Higher activity means better therapeutic effect or lower dosage; lower dosage also means lower toxicity under the same efficacy.

[0194] According to the affinity characteristics of the ASGPR receptor described above, we designed a ligand with high affinity to ASGPR to achieve liver-targeted delivery of drugs or genes and regulate target gene expression.

[0195] Terminology

[0196] The term "aliphatic ring" refers to a structure with a cyclic carbon skeleton. The term "5-8 membered aliphatic ring" refers to a monocyclic structure with 5 to 8 carbon atoms in the cyclic carbon skeleton. The term "saturated aliphatic ring" refers to a cyclic carbon skeleton composed of carbon-carbon single bonds.

[0197] The term "5-8 membered aliphatic heterocycle" refers to one or more carbon atoms in the aliphatic ring being replaced by a heteroatom. The term "saturated aliphatic heterocycle" refers to one or more carbon atoms in the saturated aliphatic ring being replaced by a heteroatom.

[0198] The term "alkyl" refers to an alkyl hydrocarbon group with the general formula The term "C1-C6 alkyl" refers to an alkyl group with 1 to 6 carbon atoms.

[0199] The term "chain alkylene" refers to a chain alkylene radical of the general formula The chain alkylene radical can be a straight chain alkylene radical or a branched chain alkylene radical.

[0200] The term "CH2CH2" refers to a C2 straight chain alkyl radical (i.e., ethylene), of the formula:

[0201] The term "alkyloxy" refers to

[0202] The term "alkylacyl" refers to For example, "C1-C6alkylacyl" can be formyl, acetyl, propionyl, n-butyryl, or isobutyryl, etc.

[0203] The term "NH" refers to an imino radical, of the formula

[0204] The term "CO" refers to a carbonyl radical, of the formula

[0205] The term "CN" refers to a cyano radical, of the formula

[0206] The term "triazole" has the formula

[0207] In the context of the present disclosure, "pharmaceutically acceptable salts" include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0208] In the context of the present disclosure, "pharmaceutically acceptable acid addition salts" refer to salts with inorganic or organic acids that are able to preserve the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalene disulfonate, etc. These salts can be prepared by methods known in the art.

[0209] In the context of this disclosure, a "pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that retains the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts, with sodium salts being the most preferred. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0210] In the context of this disclosure, "oligonucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), typically composed of 10-50 nucleotides. Oligonucleotides can regulate gene expression through a series of processes, including ribonuclease-mediated target degradation, splicing regulation, non-coding RNA inhibition, gene activation, and programmed gene editing.

[0211] the term This indicates the site where groups are connected by covalent bonds.

[0212] In the structural formulas of the compounds or ligands described in this disclosure, the bond “__” indicates that the configuration is not specified. If chiral isomerism exists in the chemical structure, the bond “-” can be… Or simultaneously include and Two configurations. Although all the above structural formulas are shown in some isomer forms for simplicity, this disclosure can include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers.

[0213] In the structural formula of the compound or ligand described in this disclosure, the bond... This indicates that the configuration is not specified. If cis-trans isomerism exists in the chemical structure, the bond... The configuration can be E-type, Z-type, or both E-type and Z-type.

[0214] The following definitions shall apply unless otherwise indicated. For the purposes of this disclosure, chemical elements are identified by their atomic number as provided in the Periodic Table of the Elements, CAS version, and the American Chemical Society. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the entire contents of which are incorporated herein by reference.

[0215] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to "at least one") of the enumerated items, unless otherwise indicated by the context of the description. The term "the" is used herein to refer to one or to more than one (i.e., to "at least one") of the enumerated items unless otherwise indicated by the context of the description. The use of the terms "a" and "an" and "the" are not intended to be limiting in any way.

[0216] The term "comprising" is used herein to mean including, but not limited to, the recited items.

[0217] "Stereoisomers" refer to compounds which have the same chemical constitution, but differ in the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis / trans), atropisomers, and the like.

[0218] "Chiral" refers to a molecule which has the property of having a mirror image that cannot be superimposed upon it; while "achiral" refers to a molecule which has a mirror image that can be superimposed upon it.

[0219] "Enantiomers" refer to two isomers of a compound which are not superimposable mirror images of one another.

[0220] "Diastereomers" refer to stereoisomers which have two or more chiral centers and which are not mirror images of one another. Diastereomers have different physical properties, even if they have the same molecular formula and structure. Mixtures of diastereomers can be separated by standard techniques, including but not limited to, high resolution analysis operations such as electrophoresis and chromatography, e.g., HPLC.

[0221] The stereochemical definitions and rules used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.

[0222] As described herein, the compounds of the present disclosure can optionally be substituted with one or more substituents, such as described herein for the compounds of the general formulae above, or as described in particular examples, subgenera, and classes of compounds encompassed by the present disclosure.

[0223] In general, the term "substituted" means that one or more hydrogen atoms in a given structure are replaced by a particular substituent. Unless otherwise indicated, a substituted group can have a substituent at each substitutable position of the group. When more than one position in the given structure can be substituted with one or more substituents selected from a particular group, the substituents can be the same or different at each position.

[0224] The term "unsubstituted" means that the designated group bears no substituents.

[0225] The term "optionally substituted" can be used interchangeably with the term "unsubstituted or substituted" to mean that the structure is either unsubstituted or substituted with one or more substituents as described herein. Substituents as described herein include, but are not limited to, D, F, Cl, Br, I, N3, CN, NO2, OH, SH, NH2, alkyl, haloalkyl, haloalkoxy, haloalkylamino, alkenyl, alkynyl, alkoxy, alkylamino, cycloalkyl, heterocyclyl, aryl, heteroaryl, and the like.

[0226] Further, it is noted that the descriptive language "each...is independently" and "each...is independently" and "is independently" as used in the present disclosure can be used interchangeably, and should be interpreted broadly, to mean that the specific options expressed by the same symbol in different groups are independent of each other, and that the specific options expressed by the same symbol in the same group are independent of each other. Using R3as an example, the specific options for R3in the structure "C1-C50alkylene optionally substituted with R3" and the structure "-C(O)-NH-C1-50alkylene optionally substituted with R3" are independent of each other.

[0227] The term "small interfering RNA (siRNA)" is a class of double-stranded RNA comprising a sense strand and an antisense strand, each strand being 17 to 30 nucleotides in length. siRNA mediates the RISC pathway of RNA transcript targeting cleavage by forming a silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through a known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and conversion into proteins.

[0228] In the context of the present disclosure, the term "antisense strand (or guide strand)" includes a region that is substantially complementary to a target sequence. The "sense strand (or passenger strand)" refers to an iRNA strand that contains a sequence that is substantially complementary to the antisense strand. The term "substantially complementary" means completely complementary or at least partially complementary, for example, the antisense strand is completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can exist within the interior or terminal regions of the molecule, wherein the most tolerated mismatches exist within the terminal regions, for example, within 5, 4, 3, or 2 nucleotides of the 5'- and / or 3'-end of the iRNA.

[0229] It should be noted that "at least partially substantially complementary" of the antisense strand to the mRNA means that the antisense strand has a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest.

[0230] In the context of the present disclosure, an "oligonucleotide" is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), generally consisting of 10-50 nucleotides. Oligonucleotides can regulate gene expression through a series of processes such as RNA interference, ribozyme-mediated target degradation, splicing regulation, non-coding RNA inhibition, gene activation, and programmed gene editing.

[0231] In the context of the present disclosure, an "antisense oligonucleotide (ASO)" is a single-stranded oligonucleotide molecule, generally consisting of 10-50 nucleotides. After entering the cell, the ASO binds to its complementary target mRNA through base complementary pairing under the action of ribonuclease H1, inhibiting the expression of the target gene.

[0232] In the context of the present disclosure, capital letters A, U, G, C, T represent the base composition of a nucleotide, lower case letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-methoxy modified nucleotide; lower case letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluoro modified nucleotide; lower case letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond.

[0233] In the context of the present disclosure, the term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating material, surfactant, antioxidant, preservative (e.g., antibacterial agent, antifungal agent), isotonic agent, salt, pharmaceutical stabilizer, binder, excipient, dispersing agent, lubricant, sweetener, flavoring agent, coloring agent, or combinations thereof, which are known to those skilled in the art (as described in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical composition is contemplated.

[0234] In the context of the present disclosure, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent or other liquid excipient, and the like, suitable for the particular target dosage form. Except insofar as any conventional excipient is incompatible with the siRNA of the present disclosure, for example, produces any adverse biological effect or interacts in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, their use is contemplated within the scope of the present disclosure.

[0235] In the context of the present disclosure, "subject" refers to any animal, such as a mammal or a marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., monkeys), murines, porcines, equines, bovines, ovines, and any species of poultry.

[0236] In the context of the present disclosure, "treat," "treatment," or "ameliorate" or "amelioration" are used interchangeably herein. These terms refer to methods of obtaining beneficial or desired results, including but not limited to therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Herein, a therapeutic benefit is achieved with regard to the underlying disorder by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder, thereby improving the subject's condition, although the subject can still be afflicted with the underlying disorder.

[0237] In the context of the present disclosure, "prevent" and "prevention" are used interchangeably. These terms refer to methods of obtaining beneficial or desired results, including but not limited to prophylactic benefit. To obtain "prophylactic benefit," a conjugate, RNAi agent, or composition can be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease can not have been made.

[0238] In the context of the present disclosure, the ratios of the agents described in the various embodiments of the present disclosure are calculated as volume ratios (v / v), unless otherwise specified.

[0239] 2. Preparation of the compound

[0240] The reagents and sources of reagents used in the preparation of the compound of the present disclosure are as follows:

[0241] 1) Tributyltin hydride, CAS No. 688-73-3, purchased from Beijing Coupling Technology Co., Ltd;

[0242] 2) Benzaldehyde dimethyl acetal, CAS No. 125-88-8, purchased from Beijing Coupling Technology Co., Ltd;

[0243] 3) DL-10-camphorsulfonic acid, CAS No. 5872-08-2, purchased from Beijing Coupling Technology Co., Ltd;

[0244] 4) 3-bromopropyne, CAS No. 106-96-7, purchased from Beijing Coupling Technology Co., Ltd;

[0245] 5) 4,4'-Dimethoxytrityl chloride (abbreviated as DMTrCl), CAS No. 40615-36-9, purchased from Beijing Coupling Technology Co., Ltd;

[0246] 6) 4,5-Dicyanoimidazole (abbreviated as DCI), CAS No. 1122-28-7, purchased from Beijing Coupling Technology Co., Ltd;

[0247] 7) Bis(diisopropylamino)(2-cyanoethoxy)phosphine, CAS No. 102691-36-1, purchased from Beijing Coupling Technology Co., Ltd;

[0248] 8) 1-O-acetyl-2,3,5-O-tri-tert-butyldimethylsilyl-BETA-L-ribofuranose, CAS No. 3080-30-6, purchased from Beijing Coupling Technology Co., Ltd;

[0249] 9) Triethylsilane, CAS No. 617-86-7, purchased from Beijing Coupling Technology Co., Ltd;

[0250] 10) Trimethylsilyl trifluoromethanesulfonate (abbreviated as TMSOTf), CAS No. 27607-77-8, purchased from Beijing Coupling Technology Co., Ltd;

[0251] 11) Cesium carbonate, CAS No. 534-17-8, purchased from Beijing Coupling Technology Co., Ltd;

[0252] 12) Silver oxide (Ag2O), CAS No. 20667-12-3, purchased from Beijing Coupling Technology Co., Ltd;

[0253] 13) Potassium iodide (KI), CAS No. 7681-11-0, purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd;

[0254] 14) p-Toluenesulfonyl chloride (TsCl), CAS No. 98-59-9, purchased from Beijing Coupling Technology Co., Ltd;

[0255] 15) Azidotrimethylsilane (TMSN3), CAS No. 4648-54-8, purchased from Beijing Coupling Technology Co., Ltd;

[0256] 16) Potassium fluoride (KF), CAS No. 7789-23-3, purchased from Beijing Coupling Technology Co., Ltd;

[0257] 17) N,N-Dimethylformamide (DMF), CAS No. 68-12-2, purchased from Beijing Coupling Technology Co., Ltd;

[0258] 18) D-Galactosamine pentaacetate, CAS No. 76375-60-5, purchased from Beijing Coupling Technology Co., Ltd;

[0259] 19) Scandium triflate, CAS No. 144026-79-9, purchased from Beijing Coupling Technology Co., Ltd;

[0260] 20) (S)-(+)-Glycerol acetonide, CAS No. 22323-82-6, purchased from Beijing Coupling Technology Co., Ltd;

[0261] 21) Sodium hydride (NaH), CAS No. 7646-69-7, purchased from Beijing Coupling Technology Co., Ltd;

[0262] 21) Amberlyst-15 (Chinese name: polymer of vinylbenzenesulfonic acid and divinylbenzene, English name: amberlyst 15 ion-exchange resin), CAS No. 39389-20-3, purchased from Beijing Coupling Technology Co., Ltd;

[0263] 22) Amino CPG (Aminoalkyl-CPG, model C3006-1000), purchased from Beijing Coupling Technology Co., Ltd;

[0264] 23) Succinic anhydride, CAS No. 108-30-5, purchased from Beijing Coupling Technology Co., Ltd.

[0265] 24) Reference compound L96-PS

[0266] The compound L96-PS was purchased from KELAIYING PHARMACEUTICAL GROUP (TIANJIN) CO., LTD. with a loading of 120 ± 12 μmol / g (detection method: UV / HPLC).

[0267] The structural formula of the reference compound L96-PS is as follows:

[0268]

[0269] In the formula, PS represents a polystyrene resin solid-phase carrier.

[0270] Unless otherwise specified, the reagents and consumables (Table 1) and instrument equipment (Table 2) used in the present disclosure are all commercially available products from the following manufacturers.

[0271] Table 1 Main reagents and consumables

[0272]

[0273] Table 2 Main instrument equipment

[0274]

[0275] The raw materials and reagents used in the GalNAc compounds, conjugates, compositions and their uses provided by the present disclosure can be commercially available.

[0276] The present disclosure is further described below in conjunction with examples:

[0277] Preparation Example 1: Synthesis of compound NM041

[0278] In the present preparation example, the synthesis process of the compound NM041 is as shown below:

[0279]

[0280] (1-1) Synthesis of compound NM041-2

[0281] Compound NM041-1 (25.0 g, 1.0 eq) was dissolved in 250 ml of toluene, and the temperature was raised to 110°C. Tributyltin hydride (17.7 g, 1.0 eq) and azobisisobutyronitrile (1 g, 0.1 eq) were added, and the reaction was refluxed at 110°C for 2 hours. After the reaction was completed, the reaction solution was cooled to 25°C. Ethyl acetate (100 ml) and potassium fluoride (10.59 g in 30 ml of water, 3.0 eq) were added, and the mixture was stirred at 25°C for 2 hours. The mixture was filtered, and the organic phase was separated. The aqueous phase was washed twice with ethyl acetate (50 ml x 2), and the organic phases were combined. The organic phase was washed once with 50 ml of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain compound NM041-2 (18.7 g, 87.9% yield) as a light yellow oil. MS ESI (m / z) = 333.1 [M+H] + .

[0282] (1-2) Synthesis of compound NM041-3

[0283] Compound NM041-2 (18.7 g, 1 eq) was dissolved in 50 ml of anhydrous methanol, and sodium methoxide (0.288 g, 0.1 eq) was added. The mixture was stirred at 25°C for 1 hour. After the reaction was completed, the pH of the reaction solution was adjusted to 6-7 with 4 mol / L hydrogen chloride in 1,4-dioxane under ice bath. The reaction solution was concentrated, and acetonitrile was added twice. The mixture was dried under vacuum to obtain compound NM041-3 (9.4 g, 100% yield) as a white solid.

[0284] (1-3) Synthesis of compound NM041-4

[0285] Compound NM041-3 (9.4 g, 1.0 eq) was dissolved in 150 ml of acetonitrile, and benzaldehyde dimethyl acetal (30 ml, 3 eq) and DL-10-camphorsulfonic acid (1.5 g, 0.1 eq) were added, respectively. The mixture was stirred at 25°C for 5 hours, and 3 ml of triethylamine was added. The mixture was stirred at 25°C for 30 minutes. After the reaction was completed, the reaction solution was concentrated. 100 ml of water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate (100 ml x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and purified by column chromatography (normal phase) (eluent: ethyl acetate / petroleum ether = 57 / 43, v / v) to obtain compound NM041-4 (8.6 g, 59.5% yield) as a white solid. MS ESI (m / z) = 252.2 [M+H] + .

[0286] (1-4) Synthesis of compound NM041-5

[0287] Compound NM041-4 (4.3 g, 1.0 eq) was dissolved in 40 ml of N,N- dimethylformamide (DMF), and sodium hydride (2.7 g, 68 mmol, 4 eq) was added under ice bath, and the reaction was performed for 30 minutes under ice bath, 3-bromopropynyl (8.1 g, 4 eq) was added, and the reaction was performed for 2 hours at 25°C, and 20 ml of water was added for quenching. After the reaction was completed, the reaction solution was extracted with ethyl acetate three times (50 ml x 3), the combined organic phase was washed with saturated sodium chloride solution five times (20 ml x 5), the organic phase was dried over anhydrous sodium sulfate and filtered, and concentrated to obtain compound NM041-5 (yield 100%) as brown oil. MS ESI (m / z) = 329.2 [M+H] + .

[0288] (1-5) Synthesis of compound NM041-6

[0289] Compound NM041-5 (5.6 g, 1.0 eq) was dissolved in 30 ml of dichloromethane (DCM), and 300 ml of 70 mass% aqueous acetic acid solution was added, and the reaction was performed for 1 hour at 70°C. After the reaction was completed, the reaction solution was directly concentrated to obtain compound NM041-6 (yield 100%) as yellow oil. MS ESI (m / z) = 241 [M+H] + .

[0290] (1-6) Synthesis of compound NM041-7

[0291] Compound NM041-6 (5.6 g, 1.0 eq) was dissolved in 50 ml of pyridine, 4,4'- dimethoxytrityl chloride (10.2 g, 1.3 eq, abbreviated as DMTrCl) was added under ice bath, nitrogen was replaced three times, and the reaction was performed for 3 hours at 25°C, and 50 ml of methanol was added for quenching. After the reaction was completed, the reaction solution was concentrated, 50 ml of water was added, and the reaction was extracted with ethyl acetate three times (50 ml x 3), the combined organic phase was dried over anhydrous sodium sulfate and filtered, and concentrated, and purified by column chromatography (eluent: ethyl acetate / petroleum ether = 16 / 84, v / v) to obtain compound NM041-7 (5 g, yield 39.6%) as yellowish solid. MS ESI (m / z) = 543.1 [M+H] + .

[0292] (1-7) Synthesis of compound NM041

[0293] Compound NM041-7 (2.0 g, 1.5 eq) was dissolved in 20 ml of anhydrous dichloromethane, and DCI (347.4 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.22 g, 1.1 eq) were added, respectively, and replaced with nitrogen for 3 times, and stirred at 25 °C for 2 hours. After the reaction was completed, 20 ml of saturated sodium bicarbonate solution was added to the reaction solution, extracted with dichloromethane 3 times (20 ml x 3), and the organic phase was combined, dried with anhydrous sodium sulfate, and filtered, concentrated, and purified by column chromatography in reverse phase (C18 column, eluent: acetonitrile / water = 72 / 28, v / v), and vacuum dried for 12 hours to obtain compound NM041 (2 g, yield 73.09%) as a white powder. MS ESI (m / z) = 743.2 [M+H] + .

[0294] 1 HNMR (400 MHz, Acetonitrile-d3) δ 7.53 - 7.47 (m, 2H), 7.36 (tt, J = 9.6, 3.4 Hz, 6H), 7.28 - 7.22 (m, 1H), 6.94 - 6.86 (m, 4H), 4.45 (d, J = 2.4 Hz, 1H), 4.35 (dt, J = 6.1, 1.9 Hz, 2H), 4.18 (ddd, J = 11.2, 5.4, 2.6 Hz, 1H), 3.81 (s, 7H), 3.79 - 3.20 (m, 8H), 3.07 (dt, J = 10.5, 7.0 Hz, 1H), 2.79 (q, J = 2.6 Hz, 1H), 2.72 (dt, J = 5.1, 2.4 Hz, 1H), 2.69 - 2.63 (m, 1H), 2.48 - 2.34 (m, 1H), 2.17 (d, J = 1.0 Hz, 2H), 1.08 (dd, J = 6.8, 4.1 Hz, 10H), 0.90 (d, J = 6.8 Hz, 2H).

[0295] 2.34 (m, 1H), 2.17 (d, J = 1.0 Hz, 2H), 1.08 (dd, J = 6.8, 4.1 Hz, 10H), 0.90 (d, J = 6.8 Hz, 2H). Preparation Example 2: Synthesis of Reference Compound NM042

[0296] In the present preparation example, the synthesis process of compound NM042 is as shown below:

[0297]

[0298] (2-1) Synthesis of compound NM042-2

[0299] Compound NM042-1 (5.0 g, 1.0 eq.) and sodium hydride (3.78 g, 2.5 eq.) were added to 60 ml of DMF, and the temperature was lowered to 0 °C. The mixture was stirred at 0 °C for 0.5 h, and bromopropargyl (9.0 g, 2.0 eq.) was slowly added during stirring. The mixture was stirred at 25 °C for 4 h. After the reaction was completed, 100 ml of water was added to the reaction solution, and the mixture was extracted with ethyl acetate three times (3 x 100 ml). The organic phase was combined and washed with 100 ml of saturated brine once, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound NM042-2 (9.0 g) as an oil. ESI-MS (m / z) = 171 [M+H] + .

[0300] (2-2) Synthesis of compound NM042-3

[0301] Compound NM042-2 (9.0 g, 37.8 mmol) and Amberlyst-15 resin (13.5 g, 150% w / w) were added to 180 ml of methanol, and the mixture was replaced with nitrogen three times and stirred at 25 °C for 6 h. After the reaction was completed, the reaction solution was filtered and concentrated to obtain compound NM042-3 (9.1 g) as an oil. ESI-MS (m / z) = 131.2 [M+H] + .

[0302] (2-3) Synthesis of compound NM042-4

[0303] Compound NM042-3 (5 g, 1.0 eq.) was added to 75 ml of a pyridine solution, and the temperature was lowered to 0 °C. DMTrCl (16.9 g, 1.69 eq.) was added portionwise at 0 °C, and the mixture was stirred at 25 °C for 3 h. After the reaction was completed, the reaction solution was concentrated, and the product was purified by column chromatography in a reverse phase (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound NM042-4 (15 g, yield 90.4%). ESI-MS (m / z) = 433.2 [M+H] + .

[0304] (2-4) Synthesis of compound NM042

[0305] Compound NM042-4 (2.5 g, 1.0 eq.), bis(diisopropylamino)(2-cyanoethoxy) phosphine (3.5 g, 2.0 eq.), DCl (0.567 g, 0.8 eq.) were added into 50 ml of DCM respectively, and stirred for 3 hours under nitrogen protection. After the reaction was completed, 50 ml of saturated sodium bicarbonate solution was added to the reaction solution for washing once, and the organic phase was separated. The organic phase was washed with 50 ml of saturated brine once, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by reverse phase HPLC to obtain compound NM042 (2.0 g, yield 56%) in white oil. ESI-MS (m / z): 619.3 [M+H] + .

[0306] 1 HNMR (400 MHz, DMSO-d6) δ 0.97-1.04 (d, J = 6.7 Hz, 3H), 1.06-1.20 (m, 9H), 3.05-3.14 (dd, J = 5.5, 10.7 Hz, 2H), 3.46-3.69 (m, 6H), 3.70-3.75 (d, J = 2.5 Hz, 7H), 3.98-4.09 (dt, J = 4.9, 10.3 Hz, 1H), 4.09-4.17 (m, 2H), 6.81-6.93 (m, 4H), 7.15-7.34 (m, 7H), 7.35-7.46 (m, 2H).

[0307] Preparation Example 3: Synthesis of compound NM014 and compound NM014A

[0308] In this preparation example, the synthesis process of compound NM014 and compound NM014A is as shown below:

[0309]

[0310] (3-1) Synthesis of compound NM014-2

[0311] Compound NM014-1 (50 g, 1.0 eq.) was added into 100 ml of ACN, and replaced with nitrogen, the temperature was reduced to 0°C, TMSOTf (44.0 g, 2.0 eq.) was added dropwise at 0°C, followed by the addition of triethylsilane (34.5 g, 3.0 eq.), and slowly warmed to 25°C, and reacted at 25°C for 12 hours. After the reaction was completed, 200 ml of purified water was added to the reaction solution, extracted with 200 ml of ethyl acetate twice, the organic phase was combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: n-heptane / ethyl acetate = 4 / 1, v / v) to obtain compound NM014-2 (41 g, yield 93.1%). ESI-MS (m / z) = 445.3 [M+H] +.

[0312] Synthesis of compound NM014-3

[0313] Compound NM014-2 (41 g, 1.0 eq.) was added to 410 ml of monomethylamine methanol, and the reaction was carried out at 25°C for 12 hours. After the reaction was completed, the reaction solution was directly concentrated, and column chromatography was performed for normal phase purification (eluent: dichloromethane / methanol = 10 / 1 was used first to remove impurities; then dichloromethane / methanol = 5 / 1 was used for elution, v / v) to obtain compound NM014-3 (7.5 g, yield 60.6%). ESI-MS (m / z) = 135.2 [M+H] + .

[0314] Synthesis of compound NM014-4

[0315] Compound NM014-3 (5.16 g, 1.0 eq.) was added to 30 ml of pyridine, and the temperature was lowered to 0°C. DMTrCl (14.32 g, 1.1 eq.) was added in batches at 0°C, and the reaction was carried out at 25°C for 6 hours. Then 10 ml of methanol was added for quenching. After the reaction was completed, the reaction solution was directly concentrated, diluted with 50 ml of ethyl acetate, washed once with 30 ml of saturated sodium bicarbonate solution and twice with 30 ml of purified water. The organic phase was separated, concentrated, and column chromatography was performed for normal phase purification (normal heptane / ethyl acetate = 5 / 1 to normal heptane / ethyl acetate = 1 / 1 gradient elution, v / v) to obtain compound NM014-4 (9.0 g, yield 53.6%). ESI-MS (m / z) = 437.3 [M+H] + .

[0316] Synthesis of compound NM014-5 and compound NM014-5A

[0317] Compound NM014-4 (9.0 g, 1.0 eq.) was dissolved in 90 ml of DMF, and cesium carbonate (7.4 g, 1.1 eq.) was added. The reaction was carried out at 25°C for 10 min, and the temperature was lowered to 0°C. Bromopropargyl (2.5 g, 1.0 eq.) was added dropwise at 0°C, and the temperature was slowly raised to 25°C. The reaction was carried out at 25°C for 16 hours. After the reaction was completed, 100 ml of ethyl acetate was added to the reaction solution, and the mixture was washed three times with 10 ml of purified water (10 ml x 3). The organic phase was separated, dried with anhydrous sodium sulfate, and filtered. The organic phase was concentrated, and column chromatography was performed for normal phase purification (eluent: normal heptane / ethyl acetate = 5 / 1, v / v, first to remove impurities, then to maintain the polarity unchanged) to obtain the front peak compound NM014-5 (2.7 g, yield 27.6%) and the rear peak compound NM014-5A (2.3 g, yield 23.7%). The ESI-MS (m / z) of the front peak product compound NM014-5 was 475.4 [M+H]+ ESI-MS (m / z) = 475.4 [M+H] of the later peak compound NM014-5A + .

[0318] Synthesis of compound NM014

[0319] The former peak compound NM014-5 (2.57 g, 1.0 eq.) was added to 26 ml of dichloromethane, stirring was continued, DCI (0.83 g, 1.3 eq.) was added, and nitrogen was replaced, at which time the reaction system was white turbidity, and the temperature was lowered to 0°C. Bis (diisopropylamino) (2-cyanoethoxy) phosphine (2.44 g, 1.5 eq.) was added dropwise at 0°C, and after the dropwise addition was completed, the temperature was slowly raised to 25°C, and stirring was continued at 25°C for 3 hours. After the reaction was completed, the reaction system was diluted with 50 ml of dichloromethane, the organic phase was washed once with 20 ml of saturated sodium bicarbonate, dried over anhydrous sodium sulfate and filtered, and the organic phase was concentrated and purified by column chromatography (eluent: n-heptane / 0.1 volume% triethylamine in ethyl acetate solution = 4 / 1, v / v) to obtain compound NM014 (907 mg). ESI-MS (m / z) = 698.2 [M+Na] + .

[0320] 1 HNMR (400 MHz, DMSO-d6) δ 0.88-0.94 (d, J = 6.8 Hz, 4H), 1.05-1.14 (m, 8H), 1.11-1.22 (m, 1H), 1.18-1.26 (m, 1H), 2.52-2.60 (t, J = 5.9 Hz, 1H), 2.72-2.80 (m, 1H), 2.89-3.00 (td, J = 5.1, 10.7 Hz, 1H), 3.15-3.28 (ddd, J = 2.8, 10.2, 23.1 Hz, 1H), 3.41-3.59 (m, 4H), 3.71-3.78 (d, J = 2.0 Hz, 6H), 3.75-3.84 (m, 1H), 3.88-3.99 (dq, J = 2.7, 18.2 Hz, 1H), 3.99-4.07 (m, 1H), 4.14-4.21 (m, 1H), 4.18-4.30 (m, 1H), 4.27-4.38 (m, 1H), 6.84-6.93 (m, 4H), 7.17-7.35 (m, 7H), 7.36-7.44 (ddd, J = 1.3, 4.0, 8.2 Hz, 2H).

[0321] Synthesis of compound NM014A

[0322] The post-peak compound NM014-5A (2.3 g, 1.0 eq.) was added to 23 ml of dichloromethane, stirring was continued, DCI (0.74 g, 1.3 eq.) was added, and nitrogen was replaced, at which time the reaction system was white turbidity, and the temperature was lowered to 0°C. Bis (diisopropylamino) (2-cyanoethoxy) phosphine (2.19 g, 1.5 eq.) was added dropwise at 0°C, and after the dropwise addition was completed, the temperature was slowly raised to 25°C, and stirring was continued at 25°C for 3 hours. After the reaction was completed, the reaction system was diluted with 50 ml of dichloromethane, the organic phase was washed once with 20 ml of saturated sodium bicarbonate, dried over anhydrous sodium sulfate, and filtered, and the organic phase was concentrated and purified by column chromatography (eluent: n-heptane / 0.1 volume% triethylamine in ethyl acetate solution = 4 / 1, v / v) to obtain compound NM014A (520 mg). ESI-MS (m / z) = 698.2 [M+Na] + .

[0323] 1 HNMR (400 MHz, DMSO-d6) δ 1.10-1.20 (dd, J = 6.7, 8.6 Hz, 12H), 2.74-2.82 (m, 2H), 2.96-3.04 (dd, J = 4.4, 10.2 Hz, 1H), 3.11-3.19 (dd, J = 3.3, 10.2 Hz, 1H), 3.38-3.43 (q, J = 2.3 Hz, 1H), 3.55-3.66 (m, 2H), 3.71-3.75 (s, 6H), 3.75-3.85 (m, 3H), 3.85-3.94 (m, 1H), 4.01-4.07 (dd, J = 4.4, 9.4 Hz, 1H), 4.09-4.20 (m, 2H), 4.22-4.31 (m, 1H), 4.43-4.52 (dq, J = 4.1, 12.0 Hz, 1H), 6.86-6.93 (m, 4H), 7.19-7.35 (m, 7H), 7.38-7.43 (m, 2H). Preparation Example 4: Synthesis of compound NM043

[0324] In the present preparation example, the synthesis process of compound NM043 is as follows:

[0325]

[0326] (4-1) Synthesis of compound NM043-2

[0327] Compound NM043-1 (10 g, 1.0 eq.), silver oxide (23.16 g, 1.5 eq.), potassium iodide (12.16 g, 1.1 eq.) were added into 100 ml dichloromethane, and the solution was cooled to 0 °C. A solution of TsCl (12.7 g, 1.0 eq.) in 100 ml dichloromethane was added slowly, and the mixture was stirred at 25 °C for 2 h. After the reaction was completed, the reaction solution was filtered, and the organic phase was washed twice with 100 ml water, once with 100 ml saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to give compound NM043-2 (12.0 g) as an oil. ESI-MS (m / z) = 305.1 [M+H] + .

[0328] (4-2) Synthesis of compound NM043-3

[0329] Compound NM043-2 (12.0 g, 1.0 eq.), TMSN3 (azidotrimethylsilane, CAS No. 4648-54-8, 13.62 g, 3.0 eq.), potassium fluoride (6.9 g, 3.0 eq.) were added into 72 ml DMF, and the solution was replaced with nitrogen for 3 times. The mixture was warmed to 60 °C and stirred at 60 °C for 1.5 h. After the reaction was completed, the solution was cooled to 25 °C, 100 ml water was added to the reaction solution, and the mixture was extracted with 60 ml ethyl acetate for 3 times. The organic phase was combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to give compound NM043-3 (3 g) as an oil. ESI-MS (m / z) = 176.3 [M+H] + .

[0330] (4-3) Synthesis of compound NM043

[0331] Compound NM043-3 (3.0 g, 1.01 eq.), D-galactosamine pentaacetate (6.0 g, 1.0 eq.) were added into 75 ml dichloroethane, and the solution was warmed to 85 °C. Scandium triflate (0.38 g, 0.05 eq.) was added in portions at 85 °C, and the mixture was stirred at 85 °C for 3 h. 16 ml triethylamine was added to quench the reaction. After the reaction was completed, the reaction system was cooled to 25 °C, 60 ml water and 60 ml dichloromethane were added to the reaction solution, and the mixture was filtered. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to give compound NM043 (4.5 g) as an oil. ESI-MS (m / z) = 505.3 [M+H] + .

[0332] Preparation Example 5: Synthesis of compound NM015

[0333] In this preparation example, the synthesis process of compound NM015 is as follows:

[0334]

[0335] Compound NM015-1 (5 g, 1.1 eq.), D-galactosamine pentaacetate (8.07 g, 1.0 eq.) were added into 96 ml dichloroethane, and the temperature was raised to 85 °C. Scandium triflate (0.38 g, 0.77 mmol) was added in batches at 85 °C, and stirred at 85 °C for 3 hours. 16 ml triethylamine was added to quench the reaction. After the reaction was completed, the reaction system was cooled to 25 °C, 100 ml water and 100 ml dichloromethane were added to the reaction solution, filtered, and the organic phase was separated, dried with anhydrous sodium sulfate and filtered, concentrated, and purified by column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound NM015 (6.6 g) in the form of oil. ESI-MS (m / z) = 549 [M+H] + .

[0336] Preparation Example 6: Synthesis of compound NM064

[0337] In this preparation example, the synthesis process of compound NM064 is as follows:

[0338]

[0339] (6-1) Synthesis of compound NM064-2

[0340] Compound NM064-1 (13 g, 1.0 eq.) was dissolved in 150 ml acetonitrile, and benzaldehyde dimethyl acetal (30 ml, 3.0 eq.) and DL-10-camphorsulfonic acid (1.5 g, 0.1 eq.) were added respectively, and stirred at 25 °C for 5 hours. 3 ml of triethylamine was added, and stirred at 25 °C for 30 min. After the reaction was completed, the reaction solution was concentrated, 100 ml water was added, extracted with ethyl acetate twice (100 ml x 2), the organic phase was combined, dried with anhydrous sodium sulfate and filtered, concentrated, and purified by column chromatography (eluent: ethyl acetate / petroleum ether = 57 / 43, v / v) to obtain compound NM064-2 (11.5 g, yield 60.8%) in the form of white solid. MS ESI (m / z) = 283 [M+H] + .

[0341] (6-2) Synthesis of compound NM064-3

[0342] Compound NM064-2 (5 g, 1.0 eq) was dissolved in 40 ml of DMF, sodium hydride (2.7 g, 4 eq) was added under ice bath, and the reaction was carried out for 30 minutes under ice bath. 3-Bromopropynyl (8.1 g, 4 eq) was added, and the reaction was stirred at 25°C for 2 hours. The reaction was quenched by adding 20 ml of water. After the reaction was completed, the reaction solution was extracted with 50 ml of ethyl acetate three times (50 ml x 3), and the combined organic phase was washed with 20 ml of saturated sodium chloride solution five times (20 ml x 5). The organic phase was dried over anhydrous sodium sulfate and filtered, and concentrated to obtain brown oil of NM064-3 (6.3 g, yield 100%). MS ESI (m / z) = 359 [M+H] + .

[0343] Synthesis of compound NM064-4

[0344] Compound NM064-3 (6.3 g, 1.0 eq) was dissolved in 30 ml of DCM, and 300 ml of 70 mass% aqueous acetic acid solution was added, and the reaction was carried out at 70°C for 1 hour. After the reaction was completed, the reaction solution was directly concentrated to obtain yellow oil of compound NM064-4 (4.78 g, yield 100%). MS ESI (m / z) = 271 [M+H] + .

[0345] Synthesis of compound NM064-5

[0346] Compound NM064-4 (4.78 g, 1.0 eq) was dissolved in 50 ml of pyridine, and DMTrCl (7.8 g, 1.3 eq) was added under ice bath, and the reaction was carried out under nitrogen substitution three times, and the reaction was stirred at 25°C for 3 hours. The reaction was quenched by adding 50 ml of methanol. After the reaction was completed, the reaction solution was concentrated, 50 ml of water was added, and the reaction was extracted with 50 ml of ethyl acetate three times (50 ml x 3), and the combined organic phase was dried over anhydrous sodium sulfate and filtered, and concentrated, and purified by column chromatography (eluent: ethyl acetate / petroleum ether = 16 / 84, v / v) to obtain yellowish solid of compound NM064-5 (6.7 g, yield 66.3%). MS ESI (m / z) = 573 [M+H] + .

[0347] Synthesis of compound NM064

[0348] Compound NM064-5 (2.0 g, 1.0 eq) was dissolved in 20 mL of anhydrous dichloromethane. DCI (330.4 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.16 g, 1.1 eq) were added separately. The mixture was purged with nitrogen three times and stirred at 25 °C for 2 hours. After the reaction was complete, 20 mL of saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the mixture was extracted three times with 20 mL of dichloromethane (20 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse-phase column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v). The solution was dried under vacuum for 12 hours to obtain a white powder, compound NM064 (2 g, yield 74.07%). MS ESI (m / z) = 774 [M+H] + .

[0349] 1 H NMR(400MHz, DMSO-d6)δ7.45–7.39(d,J=7.8Hz,2H),7.36–7.18(tt,J=14.5,8.5 Hz,7H),6.95–6.84(d,J=7.5Hz,4H),5.01–4.96(s,1H),4.43–4.28(s,4H),3.81–

[0350] 3.70(s,8H),3.65–3.35(m,12H),3.28–3.18(dt,J=14.5,7.2Hz,1H),3.06–2.97(t,J=9.5 Hz,1H),2.75–2.68(m,1H),1.07–0.95(q,J=7.4,6.8Hz,10H),0.84–0.78(d,J=6.6Hz,2H).

[0351] Preparation Example 7: Synthesis of compound CR01022 and compound CR01022Z (a cluster of CR01022, denoted as (CR01022)×1)

[0352] In this preparation example, the synthesis process of compounds CR01022 and CR01022Z is ​​as follows:

[0353]

[0354] (7-1) Synthesis of compound CR01022-1

[0355] Compound NM015 (1114 mg, 2.03 mmol, 2.2 eq.), compound NM041-7 (500 mg, 0.922 mmol, 1.0 eq.), cuprous iodide (701 mg, 3.69 mmol, 4.0 eq.), N,N-diisopropyl ethylamine (476 mg, 3.69 mmol, 4.0 eq.) were added into 3 ml of N,N-dimethylformamide, stirred at 25 °C for 1.5 hours. After the reaction was completed, 5 ml of ethyl acetate and 5 ml of water were added to the reaction solution, filtered, and the organic phase was separated, dried with anhydrous sodium sulfate and filtered, concentrated, and purified by column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound CR01022-1 (1 g) as a foam. ESI-MS (m / z) = 1639 [M+H] + .

[0356] (7-2) Synthesis of compound CR01022

[0357] Compound CR01022-1 (850 mg, 0.523 mmol, 1.0 eq.) was added to 17 ml of dichloromethane, and then bis(diisopropylamino)(2-cyanoethoxy) phosphine (313 mg, 1.04 mmol, 2.0 eq.) was added in portions, followed by the addition of 4,5-dicyanoimidazole (49 mg, 0.415 mmol, 0.8 eq.). After nitrogen replacement for 3 times, it was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was washed once with saturated aqueous sodium bicarbonate solution, and the organic phase was separated, concentrated, and purified by column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01022 (560 mg). MS ESI (m / z) = 1839 [M+H] + .

[0358] (7-3) Synthesis of compound CR01022Z

[0359] Compound CR01022-1 (160 mg, 0.1 mmol, 1.0 eq.) was added to 3.2 ml of dichloromethane, and then succinic anhydride (18 mg, 0.18 mmol, 1.8 eq.), 4-dimethylaminopyridine (1.2 mg, 0.01 mmol, 0.01 eq.) and triethylamine (19.7 mg, 0.2 mmol, 2.0 eq.) were added. After nitrogen replacement for 3 times, it was stirred at 25 °C for 16 hours. After the reaction was completed, it was purified by column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain 100 mg of the product.

[0360] The above product (100 mg, 0.058 mmol, 1.0 eq.), amino CPG (1.44 g, 0.115 mmol, 80 umol / g), benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (32.67 mg, 0.086 mmol, 1.5 eq.) and N, N-diisopropyl ethylamine (14.8 mg, 0.115 mmol, 2.0 eq.) were mixed, and the reaction was shaken for 16 hours. The reaction liquid was filtered, the solid was washed once with 10 ml of acetonitrile and dried under vacuum. The solid was mixed with 4-dimethylaminopyridine (2 mg, 0.017 mmol), Cap1 (15 ml) and Cap2 (1.5 ml), shaken for 6 hours, the reaction liquid was filtered, the filter cake was washed once with 10 ml of acetonitrile and dried under vacuum to obtain compound CR01022Z (1.366 g, loading 20-30 umol / g).

[0361] Cap1 and Cap2 are cap reagent solutions, Cap1 is a 20% (by volume) N-methylimidazole solution in pyridine / acetonitrile, and the volume ratio of pyridine to acetonitrile is 3:5; Cap2 is a 20% (by volume) acetic anhydride solution in acetonitrile.

[0362] Preparation Example 8: Synthesis of compound CR01027 and compound CR01027Z (a cluster of CR01027, denoted as (CR01027) x1)

[0363] In this preparation example, the synthesis process of compound CR01027 and compound CR01027Z is as shown below:

[0364]

[0365] (8-1) Synthesis of compound CR01027-1

[0366] Compound NM015 (1125 mg, 2.03 mmol, 2.2 eq.), compound NM064-5 (500 mg, 0.922 mmol, 1.0 eq.), cuprous iodide (701 mg, 3.69 mmol, 4.0 eq.), N, N-diisopropyl ethylamine (476 mg, 3.69 mmol, 4.0 eq.) were added to 3 ml of N, N-dimethylformamide, and stirred at 25°C for 1.5 hours. After the reaction was completed, 5 ml of ethyl acetate and 5 ml of water were added to the reaction liquid, filtered, and the organic phase was separated, dried with anhydrous sodium sulfate, and concentrated. Purification by column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01027-1 (1 g, yield 68.5%) in the form of a foam. ESI-MS (m / z): 1669.7 [M+H] + .

[0367] Synthesis of compound CR01027

[0368] Compound CR01027-1 (870 mg, 0.530 mmol, 1.0 eq.) was added to 20 ml of dichloromethane, bis(diisopropylamino)(2-cyanoethoxy)phosphine (330 mg, 1.04 mmol, 2.0 eq.) was added in portions, followed by 4,5-dicyanoimidazole (52 mg, 0.415 mmol, 0.8 eq.), nitrogen was replaced for 3 times, stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was washed with sodium bicarbonate aqueous solution once, the organic phase was separated, concentrated, and purified by column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01027 (560 mg, yield 60.8%). MS ESI (m / z) = 1869.8 [M+H] + .

[0369] Synthesis of compound CR01027Z

[0370] Compound CR01027Z was prepared according to the method for synthesizing compound CR01022Z in (7-3) of Preparation Example 7.

[0371] In this preparation example, the synthesis of compound CR01020 and compound CR01020Z is as shown below:

[0372]

[0373] Synthesis of compound CR01020-1

[0374] Compound NM015 (2.55 g, 4.64 mmol, 2.2 eq.), compound NM014-5 (1.0 g, 2.11 mmol, 1.0 eq.), cuprous iodide (1.61 g, 8.44 mmol, 4.0 eq.), N,N-diisopropylethylamine (1.09 g, 8.44 mmol, 4.0 eq.) were added to 10 ml of N,N-dimethylformamide, and stirred at 25 °C for 1.5 hours. After the reaction was completed, 15 ml of ethyl acetate and 15 ml of water were added to the reaction solution, filtered, and the organic phase was separated, dried with anhydrous sodium sulfate and filtered, concentrated, and purified by column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01020-1 (1.6 g, yield 74.1%) as a foam. ESI-MS (m / z) = 1023.2 [M+H] + .

[0375] Synthesis of compound CR01020

[0376] Compound CR01020-1 (1.0 g, 0.98 mmol, 1.0 eq.) was added to 20 ml of dichloromethane, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.59 g, 1.96 mmol, 2.0 eq.) was added in portions, followed by 4,5-dicyanoimidazole (92 mg, 0.78 mmol, 0.8 eq.), and the reaction was stirred at room temperature for 1 hour under nitrogen. After the reaction was completed, the reaction solution was washed once with an aqueous sodium bicarbonate solution, and the organic phase was separated, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated and purified by column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01020 (780 mg, yield 65.0%). MS ESI (m / z) = 1223.5 [M+H] + .

[0377] Synthesis of compound CR01020Z

[0378] Compound CR01020Z was prepared according to the synthesis method of compound CR01022Z (7-3) in Preparation Example 7.

[0379] In this preparation example, the synthesis of compound CR01021 and compound CR01021Z is as shown below:

[0380]

[0381] Synthesis of compound CR01021-1

[0382] Compound NM015 (2.55 g, 4.64 mmol, 2.2 eq.), compound NM014-5A (1.0 g, 2.11 mmol, 1.0 eq.), cuprous iodide (1.61 g, 8.44 mmol, 4.0 eq.), and N,N-diisopropylethylamine (1.09 g, 8.44 mmol, 4.0 eq.) were added to 10 ml of N,N-dimethylformamide, and the mixture was stirred at 25°C for 1.5 hours. After the reaction was completed, 15 ml of ethyl acetate and 15 ml of water were added to the reaction solution, and the mixture was filtered. The organic phase was separated, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated and purified by column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01021-1 (1.7 g, yield 78.7%) as a foam. ESI-MS (m / z): 1023.2 [M+H] + .

[0383] Synthesis of compound CR01021

[0384] Compound CR01020-1 (1.0 g, 0.98 mmol, 1.0 eq.) was added to 20 ml of dichloromethane, bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.59 g, 1.96 mmol, 2.0 eq.) was added in portions, followed by 4,5-dicyanoimidazole (92 mg, 0.78 mmol, 0.8 eq.), nitrogen was replaced for 3 times, and stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was washed with sodium bicarbonate aqueous solution once, the organic phase was separated, concentrated, and purified by column chromatography (C18 column, acetonitrile / water = 72 / 28) to obtain compound CR01021 (880 mg, yield 73.3%). MS ESI (m / z) = 1223.5 [M+H] + .

[0385] Synthesis of compound CR01021Z

[0386] Compound CR01021Z was prepared according to the method for synthesizing compound CR01022Z in Preparation Example 7 (7-3).

[0387] In this preparation example, the synthesis of compound CR01023 and compound CR01023Z is as shown below:

[0388]

[0389] Synthesis of compound CR01023-1

[0390] Compound NM015 (2.79 g, 5.08 mmol, 2.2 eq.), compound NM042-4 (1.0 g, 2.31 mmol, 1.0 eq.), cuprous iodide (1.76 g, 9.24 mmol, 4.0 eq.), N,N-diisopropylethylamine (1.19 g, 9.24 mmol, 4.0 eq.) were added to 10 ml of N,N-dimethylformamide, and stirred at 25 °C for 1.5 hours, 15 ml of ethyl acetate and 15 ml of water were added, filtered, separated, the organic phase was dried with anhydrous sodium sulfate and filtered, concentrated, and purified by column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01023-1 (1.6 g, yield 70.5%) in the form of foam. ESI-MS (m / z) = 980.2 [M+H] + .

[0391] Synthesis of compound CR01023

[0392] Compound CR01023-1 (1.0 g, 1.02 mmol, 1.0 eq.) was added to 20 ml of dichloromethane, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.61 g, 2.04 mmol, 2.0 eq.) was added in portions, followed by the addition of 4,5-dicyanoimidazole (96 mg, 0.82 mmol, 0.8 eq.), and the reaction was stirred at 25 °C for 1 hour under nitrogen. After the reaction was completed, the reaction solution was washed once with an aqueous sodium bicarbonate solution, and the organic phase was separated, concentrated, and purified by column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01023 (880 mg, yield 73.3%). MS ESI (m / z) = 1181.5 [M+H] + .

[0393] Synthesis of compound CR01023Z

[0394] Compound CR01023Z was prepared according to the synthesis method of compound CR01022Z (7-3) in Preparation Example 7.

[0395] In this preparation example, the synthesis of compound CR01024 and compound CR01024Z is as shown below:

[0396]

[0397] Synthesis of compound CR01024-1

[0398] Compound NM043 (2.34 g, 4.64 mmol, 2.2 eq.), compound NM014-5 (1.0 g, 2.11 mmol, 1.0 eq.), cuprous iodide (1.61 g, 8.44 mmol, 4.0 eq.), and N,N-diisopropylethylamine (1.09 g, 8.44 mmol, 4.0 eq.) were added to 10 ml of N,N-dimethylformamide, and the mixture was stirred at 25 °C for 1.5 hours. After the reaction was completed, 15 ml of ethyl acetate and 15 ml of water were added to the reaction solution, which was filtered, and the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound CR01024-1 (1.7 g, yield 80%) as a foam. ESI-MS (m / z): 979.2 [M+H] + .

[0399] (12-2) Synthesis of compound CR01024

[0400] Into a 25-ml reaction kettle, compound CR01024-1 (1.0 g, 1.02 mmol, 1.0 eq.), dichloromethane (20 ml), bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.61 g, 2.04 mmol, 2.0 eq.) and 4,5-dicyanoimidazole (96 mg, 0.82 mmol, 0.8 eq.) were added in batches, replaced with nitrogen for 3 times, stirred at room temperature for 1 hour, washed once with sodium bicarbonate aqueous solution, separated, concentrated the organic phase, purified by column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v), to obtain compound CR01024 (770 mg, yield 64.2%). MS ESI (m / z) = 1179.5 [M+H] + .

[0401] (12-3) Synthesis of compound CR01024Z

[0402] Compound CR01024Z was prepared according to the method for synthesizing compound CR01022Z (7-3) in Preparation Example 7.

[0403] In this preparation example, the synthesis process of compound CR01025 and compound CR01025Z is as shown below:

[0404]

[0405] (13-1) Synthesis of compound CR01025-1

[0406] Into a 10-ml N,N-dimethylformamide, compound NM043 (2.34 g, 4.64 mmol, 2.2 eq.), compound NM14-5A (1.0 g, 2.11 mmol, 1.0 eq.), cuprous iodide (1.61 g, 8.44 mmol, 4.0 eq.), N,N-diisopropylethylamine (1.09 g, 8.44 mmol, 4.0 eq.) were added, stirred at 25°C for 1.5 hours. After the reaction was completed, 15 ml of ethyl acetate and 15 ml of water were added to the reaction solution, filtered, separated the organic phase, dried and filtered by adding anhydrous sodium sulfate to the organic phase, concentrated, purified by column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v), to obtain compound CR01025-1 (1.7 g, yield 80%) in the form of foam. ESI-MS (m / z) = 979.2 [M+H] + .

[0407] Synthesis of compound CR01025

[0408] Compound CR01025-1 (1.0 g, 1.02 mmol, 1.0 eq.) was added to 20 ml of dichloromethane, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.61 g, 2.04 mmol, 2.0 eq.) was added in portions, followed by the addition of 4,5-dicyanoimidazole (96 mg, 0.82 mmol, 0.8 eq.), and the reaction was stirred at room temperature for 1 hour under nitrogen. After the reaction was completed, the reaction solution was washed once with an aqueous sodium bicarbonate solution, and the organic phase was separated and concentrated. The resulting product was purified by column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01025 (775 mg, yield 64.3%). MS ESI (m / z) = 1179.5 [M+H] + .

[0409] Synthesis of compound CR01025Z

[0410] Compound CR01025Z was prepared according to the synthesis method of compound CR01022Z (7-3) in Preparation Example 7. Preparation Example 14: Synthesis of compound CR01026 and compound CR01026Z (a cluster of CR01026, denoted as (CR01026)×1)

[0411] In this preparation example, the synthesis of compound CR01026 and compound CR01026Z was carried out as shown below:

[0412]

[0413] Synthesis of compound CR01026-1

[0414] Compound NM043 (4.08 g, 8.10 mmol, 4.4 eq.), compound NM041-7 (1.0 g, 1.84 mmol, 1.0 eq.), cuprous iodide (2.10 g, 11.04 mmol, 6.0 eq.), and N,N-diisopropylethylamine (1.43 g, 11.04 mmol, 6.0 eq.) were added to 20 ml of N,N-dimethylformamide, and the mixture was stirred at 25°C for 1.5 hours. After the reaction was completed, 25 ml of ethyl acetate and 25 ml of water were added to the reaction solution, and the mixture was filtered. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting product was purified by column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01026-1 (2.5 g, yield 87.4%) as a foam. ESI-MS (m / z) = 1551.2 [M+H] + .

[0415] Synthesis of compound CR01026

[0416] Compound CR01026-1 (1.0 g, 0.64 mmol, 1.0 eq.) was added to 20 ml of dichloromethane, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.39 g, 1.28 mmol, 2.0 eq.) was added in portions, followed by the addition of 4,5-dicyanoimidazole (60 mg, 0.51 mmol, 0.8 eq.), and the reaction was stirred at 25 °C for 1 hour under nitrogen. After the reaction was completed, the reaction solution was washed once with an aqueous sodium bicarbonate solution, and the organic phase was separated and concentrated. The resulting product was purified by column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01026 (670 mg, yield 55.8%). MS ESI (m / z) = 1751.5 [M+H] + .

[0417] Synthesis of compound CR01026Z

[0418] Compound CR01026Z was prepared according to the synthesis method of compound CR01022Z in Preparation Example 7 (7-3).

[0419] In this preparation example, the synthesis of compound CR01017 and compound CR01017Z is as shown below:

[0420]

[0421] Synthesis of compound CR01017-1

[0422] Compound NM041-4 (4.3 g, 17.0 mmol, 1.0 eq) was dissolved in 50 ml of super dry DMF, NaH (2.7 g, 68 mmol, 4.0 eq) was added under ice bath and stirred for 1 hour; then ethyl bromoacetate (5.7 g, 34 mmol, 2.0 eq, CAS No. 105-36-2) and KI (4.3 g, 25.9 mmol, 1.5 eq) were added and reacted at 25 °C for 3 hours, and then saturated aqueous ammonium chloride solution was added for quenching. After the reaction was completed, 20 ml of water was added, and 50 ml of ethyl acetate was extracted for 3 times (50 ml x 3), the organic phase was combined, and the organic phase was washed with 20 ml of saturated aqueous sodium chloride solution for 10 times (20 ml x 10), dried over anhydrous sodium sulfate and filtered, and concentrated to obtain compound CR01017-1 in dark brown oil, which was directly used in the next step. MS ESI (m / z) = 425.2 [M+H] + .

[0423] Synthesis of compound CR01017-2

[0424] CR01017-1 (5 g, 1.0 eq) was dissolved in 30 ml of dichloromethane (DCM), and 300 ml of 70% by mass aqueous acetic acid was added, and stirred at 50 °C for 2 hours. After the reaction was completed, the reaction solution was directly concentrated to obtain compound CR01017-2 in dark brown oil, which was directly used in the next step. MS ESI (m / z) = 337 [M+H] + .

[0425] Synthesis of compound CR01017-3

[0426] Compound CR01017-2 (3.2 g, 9.52 mmol, 1.0 eq) was dissolved in 70 ml of pyridine, DMTrCl (4.19 g, 12.4 mmol, 1.3 eq) was added under ice bath, and stirred at 25 °C for 3 hours, and then 30 ml of methanol was added for quenching. After the reaction was completed, the reaction solution was concentrated, 20 ml of water was added, and 50 ml of ethyl acetate was extracted for 3 times (50 ml x 3), the organic phase was combined, and the organic phase was dried over anhydrous sodium sulfate and filtered, and concentrated, and then purified by column chromatography (C18 column, eluent: acetonitrile / water = 52 / 48, v / v) to obtain compound CR01017-3 (5 g, yield 83%) in yellow solid. MS ESI (m / z) = 639 [M+H] + .

[0427] Synthesis of compound CR01017-4

[0428] Compound CR01017-3 (2 g, 3.16 mmol, 1.0 eq) was dissolved in 10 ml of methanol, and an aqueous NaOH solution (6.3 ml, 1 mol / L) was added, and stirred at 25°C for 1 hour. After the reaction was completed, the pH of the reaction solution was adjusted to neutral with hydrochloric acid, the reaction solution was filtered, and the filtrate was concentrated and purified by column chromatography (C18 column, eluent: acetonitrile / water = 26 / 74, v / v) to obtain compound CR01017-4 (920 mg, yield 51%) as a light yellow solid. MS ESI (m / z) = 583 [M+H] + .

[0429] (15-5) Synthesis of compound CR01017-5

[0430] Compound CR01017-4 (900 mg, 1.55 mmol, 1 eq), (2R,3R,4R,5R,6R)-5-acetamido-2- (acetyloxymethyl)-6-(2-(2-(aminoethoxy)ethoxy (2.02 g, 3.88 mmol, 2.5 eq), and HATU (1.35 g, 3.56 mmol, 2.3 eq) were dissolved in 25 ml of N,N-dimethylformamide, and DIEA (800 mg, 6.2 mmol, 4.0 eq) was added under ice bath, and stirred at 25°C for 12 hours. After the reaction was completed, it was purified by column chromatography (C18 column, eluent: acetonitrile / water = 20 / 80, v / v) to obtain compound CR01017-5 (700 mg, yield 28.6%) as a white solid. MS ESI (m / z) = 1591.2 [M+H] + .

[0431] (15-6) Synthesis of compound CR01017

[0432] Compound CR01017-5 (500 mg, 0.31 mmol, 1.0 eq), 4,5-dicyanoimidazole (30 mg, 0.25 mmol, 0.8 eq), and bis(diisopropylamino)(2-cyanoethoxy)phosphine (138 mg, 0.46 mmol, 1.5 eq) were dissolved in 10 ml of super dry dichloromethane, and reacted at 25°C for 2 hours under nitrogen atmosphere. After the reaction was completed, the reaction solution was washed twice with 10 ml of saturated sodium bicarbonate (10 ml x 2), the organic phase was concentrated, and purified by column chromatography (C18 column, eluent: acetonitrile / water = 80 / 20, v / v) to obtain compound CR01017 (480 mg, yield 83.1%) as a white solid. MS ESI (m / z) = 1791.2 [M+H] + .

[0433] (15-7) Synthesis of compound CR01017-6

[0434] Compound CR01017-5 (200 mg, 0.13 mmol, 1.0 eq) was dissolved in 10 ml of dichloromethane, succinic anhydride (14 mg, 0.14 mmol, 1.1 eq) and triethylamine (26 mg, 0.26 mmol, 2.0 eq) were added, and stirred at 25 °C for 12 hours. After the reaction was completed, the reaction solution was concentrated, and purified by column chromatography (C18 column, eluent: acetonitrile / water = 36 / 64, v / v) to obtain compound CR01017-6 (150 mg, yield 71.4%) in the form of a white solid. MS ESI (m / z) = 1691.2 [M+H] + .

[0435] (15-8) Synthesis of compound CR01017Z

[0436] Compound CR01017-6 (70 mg, 0.041 mmol, 1.0 eq), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (22 mg, 0.062 mmol, 1.5 eq, abbreviated as HBTU), N,N-diisopropyl ethylamine (10 mg, 0.082 mmol, 2.0 eq) were dissolved in 10 ml of acetonitrile, stirred at 25 °C for 5 minutes, and then amino CPG (970 mg, 80 umol / g) was added. The reaction was carried out on a shaking table at 25 °C for 18 hours, and then filtered. The filter cake was washed twice with 10 ml of dichloromethane (10 ml x 2) and then twice with 10 ml of acetonitrile (10 ml x 2). The filter cake, DMAP (3 mg, 0.024 mmol), Cap1 (10 ml), and Cap2 (1 ml) were mixed, and the reaction was carried out on a shaking table at 25 °C for 5 hours. The reaction mixture was filtered, the filter cake was washed with 10 ml of acetonitrile three times, and then dried to obtain compound CR01017Z (720 mg).

[0437] wherein Cap1 and Cap2 are cap reagent solutions, Cap1 is a 20% (by volume) N-methylimidazole solution in pyridine / acetonitrile, and the volume ratio of pyridine to acetonitrile is 3:5; and Cap2 is a 20% (by volume) acetic anhydride solution in acetonitrile.

[0438] Preparation Example 16: Synthesis method one of siRNA conjugate

[0439] (16-1) Synthesis of sense strand SS

[0440] By the method of phosphoramidite nucleic acid solid phase synthesis, using the above compound (i.e. CR01017Z, CR01020Z, CR01021Z, CR01022Z, CR01023Z, CR01024Z, CR01025Z, CR01026Z, CR01027Z, L96-PS) attached to the solid support as the starting material, the nucleoside monomers are sequentially linked in the order of 3'-5' according to the nucleotide sequence (the compounds CR01017, CR01020, CR01021, CR01022, CR01023, CR01024, CR01025 and CR01026 can be regarded as a nucleoside monomer, respectively).

[0441] Each nucleoside monomer linkage includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows:

[0442] The nucleoside monomers are prepared into an acetonitrile solution of nucleoside monomers with a concentration of 0.1 M.

[0443] The conditions of deprotection reaction in each step are the same. The conditions of deprotection reaction are as follows: the temperature is 25°C, the reaction time is 70 seconds, the deprotection reagent is dichloroacetic acid in dichloromethane (3% by volume), and the molar ratio of dichloroacetic acid to 4,4'-dimethoxytrityl protecting group on the solid support is 5:1.

[0444] The conditions of coupling reaction in each step are the same. The conditions of coupling reaction are as follows: the temperature is 25°C, the molar ratio of the nucleic acid sequence attached to the solid support to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence attached to the solid support to the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is 5-ethylthio-1H-tetrazole in acetonitrile with a concentration of 0.5 M, and the thio reagent is a mixture of acetonitrile / pyridine with a volume ratio of 1:1, in which the concentration of hydrogenated xanthine is 0.2 M.

[0445] The conditions of capping reaction in each step are the same. The conditions of capping reaction are as follows: the temperature is 25°C, the reaction time is 2 minutes, the capping reagent solution is a mixture of Cap1 and Cap2 with a molar ratio of 1:1, Cap1 is N-methylimidazole in pyridine / acetonitrile with a concentration of 20% by volume, the volume ratio of pyridine to acetonitrile is 3:5, and Cap2 is acetic anhydride in acetonitrile with a concentration of 20% by volume; the molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent, and the nucleic acid sequence attached to the solid support is 1:1:1.

[0446] The conditions for each oxidation reaction are the same. The conditions for the oxidation reaction are: temperature is 25°C; reaction time is 3 seconds; the concentration of the oxidizing reagent is 0.05 M iodine water, the molar ratio of iodine to the nucleic acid sequence attached to the solid support in the coupling reaction is 30:1; the oxidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9). The conditions for the sulfurization reaction are: temperature is 25°C; reaction time is 360 seconds; the concentration of the sulfurizing reagent is 0.2 M pyridine solution of hydrogen xanthate, the molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid support in the coupling reaction is 4:1; the sulfurization reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9).

[0447] After the last nucleoside monomer is attached, the nucleic acid sequence attached to the solid support is sequentially subjected to cleavage, deprotection, purification, desalting, and then lyophilization to obtain the sense strand, wherein:

[0448] The cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence attached to the solid support is added to 25% ammonia water by mass, the amount of ammonia water is 0.5 ml / μmol, the reaction is carried out at 55°C for 16 hours, the solvent is removed, and vacuum concentration is carried out to dryness. After ammonia water treatment, the product is dissolved in 0.4 ml / μmol N-methylpyrrolidine, followed by the addition of 0.3 ml / μmol triethylamine and 0.6 ml / μmol triethylamine trifluoromethanesulfonate, and the 2'-O-TBDMS protection on the ribose is removed.

[0449] The purification and desalting conditions are as follows: the nucleic acid is purified by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent 1 is 20 mM sodium phosphate (pH=8.1), the solvent is a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile is 9:1); eluent 2 is 1.5 M sodium chloride, 20 mM sodium phosphate (pH=8.1), the solvent is a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile is 9:1); the elution liquid is eluent 1:eluent 2=(100:0)-(50:50). After the product elution liquid is collected, desalting is carried out using a reverse phase chromatography purification column, and the desalting conditions include desalting using a dextran gel column, the filler is dextran gel G25, and elution is carried out using deionized water.

[0450] Detection: purity detection is carried out using ion exchange chromatography (IEX-HPLC); molecular weight detection is carried out using a liquid chromatograph-mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters Company, model: LCT Premier); the actual value of the molecular weight is compared with the theoretical value; if the actual value and the theoretical value are consistent, it is indicated that the compound is conjugated to the 3' end of the sense strand of the siRNA.

[0451] During the synthesis of the sense strand, the following ligands (carriers) were synthesized separately:

[0452] The structural formula of the doublet CR01017 (denoted as (CR01017) x 2) is:

[0453]

[0454] The structural formula of the triad CR01020 (denoted as (CR01020) x 3) is:

[0455]

[0456] The structural formula of the triad CR01021 (denoted as (CR01021) x 3) is:

[0457]

[0458] The structural formula of the doublet CR01022 (denoted as (CR01022) x 2) is:

[0459]

[0460] The structural formula of the triad CR01022 (denoted as (CR01022) x 3) is:

[0461]

[0462] The structural formula of the triad CR01023 (denoted as (CR01023) x 3) is:

[0463]

[0464] The structural formula of the triad CR01024 (denoted as (CR01024) x 3) is:

[0465]

[0466] The structural formula of the triad CR01025 (denoted as (CR01025) x 3) is:

[0467]

[0468] The structural formula of the doublet CR01026 (denoted as (CR01026) x 2) is:

[0469]

[0470] The structural formula of the doublet CR01027 (denoted as (CR01027) x 2) is:

[0471]

[0472] (16-2) Synthesis of antisense strand (AS)

[0473] The antisense strand was synthesized using a general solid support. The deprotection, coupling, capping, oxidation or sulfuration reaction conditions, cleavage and deprotection conditions, purification and desalination conditions and steps in the solid phase synthesis method of the antisense strand are the same as those in the synthesis of the sense strand in step (16-1).

[0474] Detection: Purity was detected using ion exchange chromatography (IEX-HPLC); molecular weight was detected using liquid chromatography-mass spectrometry (LC-MS, Liquid ChromatogRLhy-Mass SP1ectrometry, purchased from Waters Corporation, model: LCT Premier), and the measured value was compared with the theoretical value. If the measured value and the theoretical value are consistent, it is indicated that the siRNA antisense strand is obtained.

[0475] (16-3) Synthesis of siRNA conjugate

[0476] The sense strand synthesized in step (16-1) and the antisense strand synthesized in step (16-2) were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. Slowly cool to room temperature and keep at room temperature for 10 minutes, so that the sense strand and the antisense strand form a double-stranded structure through hydrogen bonding, thereby obtaining the target siRNA conjugate.

[0477] Detection: After each conjugate was diluted to a concentration of 0.2 mg / ml (calculated as siRNA) using ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2 MΩ*cm (25°C)), the molecular weight was detected using liquid chromatography-mass spectrometry (LC-MS, Liquid ChromatogRLhy-Mass SP1ectrometry, purchased from Waters Corporation, model: LCT Premier). The measured value is consistent with the theoretical value, indicating that the synthesized conjugate is the double-stranded nucleic acid sequence designed.

[0478] Preparation Example 17: Synthesis method two of siRNA conjugate

[0479] In this preparation example, the linker group for connecting the siRNA and the ligand group in the siRNA conjugate provided by the present disclosure is as shown in formula A (NM014, NM014A, NM041 and NM042):

[0480]

[0481] The ligand group is as shown in formula B (NM015 and NM043):

[0482]

[0483] For example, the synthesis of siRNA with the linker group NM041 and the ligand group NM015 comprises the following steps:

[0484] (17-1) Synthesis of the sense strand

[0485] The synthesis route of the sense strand is shown as follows:

[0486]

[0487] Specifically, the preparation method of the sense strand comprises the following process:

[0488] S1, taking the compound NM041 as a nucleoside monomer, the compound NM041 is connected to the solid phase carrier by the phosphoramidite solid phase synthesis method, and then the nucleoside monomers are connected one by one in the order of 3' to 5' according to the nucleotide type and sequence of the siRNA sequence sense strand, and the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions in the synthesis of the sense strand are the same as those in step (16-1) for synthesizing the sense strand.

[0489] The sense strand half-conjugate containing the linker group (denoted as SS-(NM041)×2) is separated. Among them, the linker groups NM041 are connected to the 3' terminal of the siRNA sense strand.

[0490] S2, the conjugation connection between the terminal alkyne group in SS-(NM041)×2 and the ligand group NM015 is connected by click reaction to obtain the sense strand conjugate.

[0491] S2-1, 150 μL of H2O, 70 μL of 0.2 mol / L carbonate buffer solution (pH = 9.2) and 70 μL of N,N-dimethylformamide (DMF) are mixed to obtain a mixed solvent; the SS-(NM041)×2 is dissolved in the mixed solvent to obtain a SS-(NM041)×2 solution with a concentration of 1.0 eq.

[0492] S2-2, 6.0 eq of ligand compound NM015 is dissolved in 70 μL of DMF to obtain an NM015 solution

[0493] S2-3, the SS-(NM041)×2 solution obtained in step S2-1 and the NM015 solution obtained in step S2-2 are mixed to obtain a reactant mixture.

[0494] S2-4, 10.0 eq of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and 3.0 eq of CuSO4·5H2O were mixed in a volume ratio of THPTA:CuSO4·5H2O = 10:3, and after shaking at 40°C for 5 min, 37 μL was added to the reaction mixture obtained in step S2-3, and vortexed to obtain an intermediate mixture, and the pH of the intermediate mixture was measured to be 8.

[0495] S3, 25.0 eq of sodium ascorbate was quickly added to the intermediate mixture obtained in step 2-4, and vortexed to obtain a product mixture, which was reacted at 40°C for 1 h.

[0496] S4, purification: 3 μL of the product mixture obtained in step S3 was diluted with a mixed solution of DMF and H2O (volume ratio of DMF:H2O = 1:5), and then separated and purified by HPLC treatment. In the HPLC treatment, a C18 column was used, and a gradient elution method was used with an ammonium bicarbonate buffer solution as the mobile phase. The product after HPLC treatment was freeze-dried.

[0497] (17-2) Synthesis of antisense strand

[0498] Prepared according to the synthesis method of the antisense strand (AS) in Preparation Example 16 (16-2).

[0499] (17-3) Synthesis of siRNA conjugate

[0500] Prepared according to the synthesis method of the siRNA conjugate in Preparation Example 16 (16-3).

[0501] In the two methods for synthesizing siRNA conjugates provided in the present disclosure, the first method for synthesizing siRNA conjugates is to synthesize a carrier first, then synthesize a sense strand conjugate, and finally synthesize an siRNA conjugate; the second method for synthesizing siRNA conjugates is to synthesize a sense strand half-conjugate first, then synthesize a sense strand conjugate, and finally synthesize an siRNA conjugate.

[0502] In the case of synthesizing the same amount of siRNA conjugate, compared with the second method, the first method uses less raw material in the synthesis of the carrier, but takes longer to synthesize the siRNA conjugate; compared with the first method, the second method takes less time to synthesize the siRNA conjugate, but uses more raw material in the synthesis of the carrier.

[0503] When the carrier is a cluster of CR01017, the structural formula of the siRNA conjugate is as follows:

[0504]

[0505] In the case of the carrier being the two-cluster CR01017, the structure of the siRNA conjugate is as follows:

[0506]

[0507] In the case of the carrier being the three-cluster CR01020, the structure of the siRNA conjugate is as follows:

[0508]

[0509] In the case of the carrier being the three-cluster CR01021, the structure of the siRNA conjugate is as follows:

[0510]

[0511] In the case of the carrier being the one-cluster CR01022, the structure of the siRNA conjugate is as follows:

[0512]

[0513] In the case of the carrier being the two-cluster CR01022, the structure of the siRNA conjugate is as follows:

[0514]

[0515] In the case of the carrier being the three-cluster CR01022, the structure of the siRNA conjugate is as follows:

[0516]

[0517] In the case of the carrier being the three-cluster CR01023, the structure of the siRNA conjugate is as follows:

[0518]

[0519] In the case of the carrier being the three-cluster CR01024, the structure of the siRNA conjugate is as follows:

[0520]

[0521] In the case of the carrier being the three-cluster CR01025, the structure of the siRNA conjugate is as follows:

[0522]

[0523] In the case of the carrier being the two-cluster CR01026, the structure of the siRNA conjugate is as follows:

[0524]

[0525] In the case of the carrier being CR01027, the structural formula of the siRNA conjugate is:

[0526]

[0527] In the case of the carrier being L96, the structural formula of the siRNA conjugate is:

[0528]

[0529] The siRNA conjugates in Table 3 were synthesized according to the methods of Preparation Example 16 and Preparation Example 17, respectively, in the present disclosure.

[0530] In the present disclosure, the conjugates RZ502031, RZ599001, RZ597002, RZ899038, RZ899041, RZ899042 can only be synthesized by the synthesis method one of the siRNA conjugate described in Preparation Example 16.

[0531] The conjugates RZ802008, RZ802009, RZ899007, RZ802014, RZ899022, RZ899023, RZ899024, RZ802010, RZ899009, RZ899011, RZ899040, RZ899025, RZ802011, RZ899010, RZ899048, RZ897013 can be synthesized by either the synthesis method one of the siRNA conjugate described in Preparation Example 16 or the synthesis method two of the siRNA conjugate described in Preparation Example 17. Specifically, in the present disclosure, the conjugates RZ802010, RZ899009, RZ899011, RZ899040 are synthesized by the synthesis method one of the siRNA conjugate described in Preparation Example 16; the conjugates RZ802008, RZ802009, RZ899007, RZ802014, RZ899022, RZ899023, RZ899024, RZ899025, RZ802011, RZ899010, RZ899048, RZ897013 are synthesized by the synthesis method two of the siRNA conjugate described in Preparation Example 17.

[0532] UmsUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmAmAm, as shown in SEQ ID No. 1;

[0533] UmsUfsUmAmGmAfGmUmGmAmGmGmAmUfUmAfAmAmAmsUmsGm, as shown in SEQ ID No. 2;

[0534] CmsAmsGmAmCmAmGfAfCfAmAmGmAmCmCmAmUmCmUm, as set forth in SEQ ID No. 5;

[0535] AmsGfsAmUmGmGfUmCmUmUmGmUmCmUfGmUfCmUmGmsGmsAm, as set forth in SEQ ID No. 4;

[0536] CmsAmsGmAmCmAmGfAfCfAmAmGmAmCmCmAmUmCmUm, as set forth in SEQ ID No. 5;

[0537] CmsCmsAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm, as set forth in SEQ ID No. 6;

[0538] UmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmsUmsUm, as set forth in SEQ ID No. 7.

[0539] Table 3 siRNA conjugates

[0540]

[0541]

[0542]

[0543]

[0544] Unless otherwise specified, the base composition and modification meaning described in each embodiment of the present disclosure are as follows: capital letters A, U, G, C, T represent the base composition of nucleotides, and lowercase letter m represents that the nucleotide represented by the previous letter is a methoxy-modified nucleotide; lowercase letter f represents that the nucleotide represented by the previous letter is a fluorine-modified nucleotide; and lowercase letter s represents that the nucleotides represented by the two letters before and after it are connected by a phosphorothioate bond.

[0545] Unless otherwise specified, the siRNA sequences used in the present disclosure are synthesized by Suzhou Bexin Biotechnology Co., Ltd.; the PCR primers used in the present disclosure are synthesized by Beijing Qikang Biotechnology Co., Ltd.; and the experimental animals C57BL / 6J mice used in the present disclosure are purchased from Spafas (Beijing) Biotechnology Co., Ltd.

[0546] Table 4 Detection results of siRNA conjugates

[0547]

[0548]

[0549] From the data in Table 4, it can be seen that the sense strand (SS) and antisense strand (AS) can be well and highly-purely connected to the ligand.

[0550] General experiment

[0551] Method for evaluating target gene inhibition activity in mice in vivo

[0552] 6-8 week old C57BL / 6J mice were randomly grouped by weight (all female). The mice in each group were calculated according to the weight of the drug dose, and the abdominal subcutaneous injection method was used for single dose, and the siRNA conjugate was respectively configured into a solution of corresponding concentration (calculated by siRNA) for administration, and the administration volume was 5 ml / kg (calculated by mouse weight). The PBS control group was given a PBS solution (without drug conjugate), and the administration volume was 5 ml / kg (calculated by mouse weight). The day of administration was recorded as day 1 (recorded as D1), and at the preset time after administration, 5 mice were sacrificed in each group. The necropsy of the sacrificed mice was performed and the liver tissue of each sacrificed mouse was collected, and the liver tissue was cut into about 2 mm 3 Small pieces, preserved with RNA Later.

[0553] The liver tissue samples at different time points in different experimental groups were taken from the above RNA later, and the liver tissue samples were crushed in a Tissuelyser II type automatic tissue homogenizer for 60 s, and then a full-automatic nucleic acid extractor (purchased from Zhejiang Hanwei Science and Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Science and Technology Co., Ltd.) were used to extract total RNA according to the standard operation procedure for total RNA extraction.

[0554] Take the above 1 μg total RNA, use the reverse transcription kit (Promega Company, Reverse Transcription System, A3500) and select Oligo(dT)15 reverse transcription primer, configure 20 μL reverse transcription system according to the method recorded in the reverse transcription kit instruction book and complete the reverse transcription reaction. After the reaction, 80 μL RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. Then the real-time fluorescent quantitative PCR kit (ABI Company, SYBR TMSelect Master Mix, Catalog number: 4472908) to detect the expression of target gene mRNA in liver tissue. In the real-time fluorescent quantitative PCR method, the target gene and the internal reference gene are detected by using primers for the target gene and primers for the internal reference gene, respectively. According to the method recorded in the real-time fluorescent quantitative PCR kit instruction book, 20 μL of real-time PCR reaction system is configured for each PCR detection well, and each reaction system contains 5 μL of cDNA solution obtained by reverse transcription reaction, 10 μL of SYBR TM Select Master Mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 4 μL of RNase-Free H2O. The prepared reaction system is placed on a real-time fluorescent quantitative PCR instrument (ABI Company, StepOnePlus TM ) to perform Real-time PCR amplification by using a three-step method, and the amplification program is 95°C pre-denaturation for 10 min, then 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, and repeating the denaturation, annealing and extension process for 40 cycles. In the real-time fluorescent quantitative PCR method, the ΔΔCt method is used to perform relative quantitative calculation on the expression level and inhibition rate of target gene mRNA in each test group, and the calculation method is as follows:

[0555] ΔCt (test group) = Ct (test group target gene) - Ct (test group internal reference gene)

[0556] ΔCt (control group) = Ct (control group target gene) - Ct (control group internal reference gene)

[0557] ΔΔCt (test group) = ΔCt (test group) - ΔCt (control group average)

[0558] ΔΔCt (control group) = ΔCt (control group) - ΔCt (control group average)

[0559] Wherein, ΔCt (control group average) is the arithmetic mean of ΔCt (control group) of each of the 5 mice in the control group at the same time point. Therefore, each mouse in the test group and the control group corresponds to a ΔΔCt value.

[0560] Taking the control group as the reference, the target gene mRNA expression level of the test group is normalized, and the target gene mRNA expression level of the control group is defined as 100%.

[0561] Test group target gene mRNA relative expression level = 2-ΔΔCt (test group) x 100%

[0562] Test group target gene mRNA inhibition rate = (1 - test group target gene mRNA relative expression level) x 100%

[0563] Unless otherwise specified, the in vivo activity experiment data are expressed as The experimental data are plotted and analyzed using GraphPad prism 8.0 software.

[0564] Example 1: In vivo activity evaluation of trispecific CR01020 carrier conjugated complement component 3 (CC3) siRNA

[0565] In this example, the siRNA conjugates RZ802008 and RZ802009, which are siRNA conjugates of trispecific CR01020 carrier conjugated to the 3' end of the sense strand of the CC3 target siRNA, and the siRNA conjugate RZ502031, which is a siRNA conjugate of L96 carrier conjugated to the 3' end of the sense strand of the CC3 target siRNA, were evaluated for their in vivo inhibitory activity on the target gene CC3 in mice.

[0566] Six to eight-week-old C57BL / 6j mice were randomly divided into groups by body weight, with 15 mice in each of the four groups. Each group of mice was administered PBS solution and the siRNA conjugates mentioned in this example by subcutaneous injection in the abdomen, wherein each mouse in the PBS control group was administered a dose of 5 ml / kg; each mouse in the siRNA conjugate experimental group was administered a dose of 3 mg / kg (calculated as siRNA) and a volume of 5 ml / kg. The day of administration was designated as day 1 (D1), and 5 mice from each group were sacrificed on day 8 (D8), 5 mice from each group were sacrificed on day 15 (D15), and 5 mice from each group were sacrificed on day 29 (D29) after administration. Liver tissue was collected for RNA extraction, reverse transcription reaction, and Real-time PCR detection, and the relative quantification of the target gene mRNA in each test group was calculated according to the aforementioned ΔΔCt method.

[0567] The primer sequences of this example are shown in Table 5.

[0568] Table 5 Primer sequence list

[0569]

[0570] The results of Example 1 show that the in vivo activity and duration of the conjugate RZ802008, which is a conjugate of trispecific CR01020 conjugated to the 3' end of the sense strand of the siRNA, are basically equivalent to those of the conjugate RZ502031, which is a conjugate of L96 conjugated to the 3' end of the sense strand of the siRNA; the trispecific CR01020 conjugate RZ802009 has better in vivo activity and comparable pharmacodynamic duration than the L96 conjugate RZ502031 (Table 6). Figure 1 ​

[0571] Table 6 Inhibition activity of target gene in mice after administration of siRNA conjugates described in this example

[0572]

[0573] Example 2: In vivo activity evaluation of siRNA conjugate of trispecific CR01020 carrier conjugated Superoxide Dismutase 1 (SOD1) siRNA

[0574] This example evaluated the inhibition activity of siRNA conjugate RZ899007 of trispecific CR01020 carrier conjugated to the 3' end of the sense strand of the same SOD1 target siRNA, and conjugate RZ599001 of L96 carrier conjugated to the 3' end of the sense strand of the same SOD1 target siRNA, on the target gene SOD1 in mice, using the method of evaluating the inhibition activity of target gene in mice.

[0575] Six to eight-week-old C57BL / 6j mice were randomly divided into groups by weight, with 20 mice in each group, and a total of 3 groups. Each group of mice was administered PBS solution and the siRNA conjugates mentioned in this example by subcutaneous injection in the abdomen, wherein the dose administered to each mouse in the PBS control group was 5 ml / kg, and the dose administered to each mouse in the siRNA conjugate test group was 1 mg / kg (calculated as siRNA) with a dose volume of 5 ml / kg. The day of administration was designated as day 1 (D1), and 5 mice from each group were sacrificed on day 8 (D8), 5 mice from each group were sacrificed on day 15 (D15), 5 mice from each group were sacrificed on day 29 (D29), and 5 mice from each group were sacrificed on day 43 (D43). Liver tissue was collected for RNA extraction, reverse transcription reaction, and Real-time PCR detection, and the relative quantification of the target gene mRNA in each test group was calculated according to the aforementioned ΔΔCt method.

[0576] The primer sequences of this example are shown in Table 7.

[0577] Table 7 Primer sequence list

[0578]

[0579] The results of Example 2 show that the conjugate RZ899007 of trispecific CR01020 conjugated to the 3' end of the sense strand of siRNA has comparable maximum inhibition activity and better pharmacodynamic duration of action than the conjugate RZ599001 of L96 conjugated to the 3' end of the sense strand of siRNA (Table 8). Figure 2

[0580] Table 8 Inhibition activity of target gene in mice after administration of siRNA conjugates described in this example ​

[0581]

[0582]

[0583] Example 3: In vivo activity evaluation of tri-cluster CR01021 vector and tri-cluster CR01020 vector conjugated CC3 target siRNA

[0584] This example used the mouse in vivo target gene inhibition activity evaluation method to evaluate the inhibition activity of siRNA conjugates RZ802014 conjugated with tri-cluster CR01021 vector, siRNA conjugate RZ802009 conjugated with tri-cluster CR01020 vector, and conjugate RZ502031 conjugated with L96 vector on the target gene CC3 in mice. The difference between the tri-cluster CR01021 and the tri-cluster CR01020 vector is that the cyclic parent nucleus part is an isomer.

[0585] 6-8 week old C57BL / 6j mice were randomly divided into groups according to body weight, 10 mice in each group, a total of 4 groups. Each group of mice was given PBS solution and the siRNA conjugates mentioned in this example by subcutaneous injection in the abdomen, wherein each mouse in the PBS control group was given a dose of 5 ml / kg, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (calculated as siRNA) and a volume of 5 ml / kg. The day of administration was recorded as the first day (D1), and 5 mice were sacrificed in each group on the 8th day (D8) and 5 mice were sacrificed in each group on the 22nd day (D22) after administration. The liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was quantitatively calculated according to the aforementioned ΔΔCt method.

[0586] The primer sequences of this example are shown in Table 5 in Example 1.

[0587] The results of Example 3 show that the in vivo activity of tri-cluster CR01021 conjugate RZ802014 is better than that of L96 conjugate RZ502031; the in vivo activity of tri-cluster CR01020 conjugate RZ802009 is basically the same as that of L96 conjugate RZ502031 (Table 9). Figure 3

[0588] Table 9 shows the inhibition activity of the target gene in mice after administration of the siRNA conjugates described in this example

[0589]

[0590] ​Example 4: In vivo activity evaluation of siRNA conjugates of tris-CR01021 carrier and tris-CR01020 carrier conjugating SOD1 target siRNA

[0591] This example evaluated the inhibitory activity of siRNA conjugates RZ899022 of tris-CR01021 carrier conjugating siRNA at the 3' end of the same SOD1 target siRNA, siRNA conjugate RZ899007 of tris-CR01020 carrier conjugating siRNA, and conjugate RZ599001 of L96 carrier conjugating siRNA on the target SOD1 gene in mice in vivo using the method of evaluating the inhibitory activity of target genes in mice in vivo. The tris-CR01021 and tris-CR01020 carriers differ in that the cyclic parent nucleus moieties are isomers of each other.

[0592] Six to eight-week-old C57BL / 6j mice were randomly divided into groups by weight, with 5 mice in each of 4 groups. Each group of mice was administered PBS solution and the siRNA conjugates mentioned in this example by subcutaneous administration in the abdomen, wherein each mouse in the PBS control group was administered a dose of 5 ml / kg of the administration volume, and each mouse in the siRNA conjugate test group was administered a dose of 3 mg / kg (calculated as siRNA) and a volume of 5 ml / kg. The day of administration was designated as the first day (D1), and 5 mice from each group were sacrificed on the 8th day (D8) after administration. The liver tissue was collected for RNA extraction, reverse transcription reaction, and Real-time PCR detection, and the target gene mRNA in each test group was quantitatively calculated according to the aforementioned ΔΔCt method.

[0593] The primer sequences of this example are shown in Table 7 in Example 2.

[0594] The results of Example 4 show that the siRNA conjugate RZ899022 of tris-CR01021 conjugating the 3' end of the sense strand of siRNA and the conjugate RZ899007 of tris-CR01020 conjugating siRNA have better in vivo activity than the L96 conjugate RZ599001 Figure 4 , Table 10).

[0595] Table 10 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugates described in this example

[0596]

[0597]

[0598] Example 5: In vivo activity evaluation of siRNA conjugates of tris-CR01024 carrier and tris-CR01025 carrier conjugating SOD1 target siRNA

[0599] This embodiment uses a mouse in vivo target gene inhibitory activity assessment method to evaluate the inhibitory activity of siRNA conjugates RZ899023 (conjugated to the 3' end of the CR01024 vector), RZ899024 (conjugated to the CR01025 vector), and RZ599001 (conjugated to L96) on the target gene SOD1 in mice. The CR01024 and CR01020 conjugates have the same circular nucleus structure but use linkers of different lengths, while the CR01025 and CR01021 conjugates have the same circular nucleus structure but use linkers of different lengths.

[0600] Six- to eight-week-old C57BL / 6j mice were randomly divided into four groups of five mice each, based on body weight. Each group was administered PBS solution or the siRNA conjugate mentioned in this example via subcutaneous abdominal administration. The PBS control group received 5 ml / kg of PBS solution per mouse, while the siRNA conjugate experimental group received 3 mg / kg of siRNA solution per mouse, administered in a volume of 5 ml / kg. The day of administration was designated as day 1 (D1), and mice were sacrificed on day 8 (D8). Liver tissue was collected for RNA extraction, reverse transcription, and real-time PCR detection. The relative quantification of target gene mRNA in each test group was performed using the aforementioned ΔΔCt method.

[0601] The primer sequences for this embodiment are shown in Table 7 of Embodiment 2.

[0602] The results of Example 5 showed that the in vivo activities of the siRNA conjugates RZ899023 (containing three clusters of CR01024) and RZ899024 (containing three clusters of CR01025) were both superior to those of the L96 conjugate RZ599001. Figure 5 (Table 11).

[0603] The results from Examples 4 and 5 indicate that different linker lengths have little effect on the activity of the carrier.

[0604] Table 11 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in this embodiment.

[0605]

[0606] Example 6: In vivo activity assessment of CC3 target siRNA conjugated to the tri-cluster CR01022 vector

[0607] The same CC3 target siRNA with 3' end of sense strand conjugated with tri-cluster CR01022 siRNA conjugate RZ802010 and L96 conjugate RZ502031 were evaluated for their inhibitory activity on target gene CC3 in mice in vivo by the method of evaluating inhibitory activity of target gene in mice in vivo.

[0608] The 6-8 week old C57BL / 6j mice were randomly divided into groups by weight, 15 mice in each group, and 3 groups in total. The mice in each group were administered with PBS solution and the siRNA conjugates mentioned in this example by subcutaneous administration in the abdomen, wherein the administration dose of each mouse in the PBS control group was 5 ml / kg, and the administration dose of each mouse in the siRNA conjugate experimental group was 3 mg / kg (calculated based on siRNA), and the administration volume was 5 ml / kg. The day of administration was recorded as the first day (D1), and 5 mice in each group were sacrificed on the 8th day (D8), 5 mice in each group were sacrificed on the 15th day (D15), and 5 mice in each group were sacrificed on the 29th day (D29). The liver tissues were collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the relative quantification of the target gene mRNA in each test group was calculated according to the aforementioned ΔΔCt method.

[0609] The primer sequences of this example are shown in Table 5 in Example 1.

[0610] The results of Example 6 show that the conjugate RZ802010 of siRNA sense strand 3' end conjugated with tri-cluster CR01022 has comparable highest inhibitory activity and better pharmacodynamic duration of action compared with L96 conjugate RZ502031 (Table 12). Figure 6

[0611] Table 12 Inhibitory activity of target gene in mice after administration of siRNA conjugates described in this example

[0612]

[0613] Example 7: In vivo activity evaluation of SOD1 target siRNA conjugated with di-cluster CR01022 carrier and tri-cluster CR01022 carrier

[0614] The same SOD1 target siRNA with 3' end of sense strand conjugated with tri-cluster CR01022 siRNA sequence RZ899009, conjugated with di-cluster CR01022 siRNA sequence RZ899011, and L96 conjugate RZ599001 were evaluated for their inhibitory activity on target gene SOD1 in mice in vivo by the method of evaluating inhibitory activity of target gene in mice in vivo.

[0615] ​6-8 weeks old C57BL / 6j mice were randomly grouped by weight, 20 mice in each group, a total of 4 groups. Each group of mice was given PBS solution and siRNA conjugate mentioned in this example by abdominal subcutaneous administration, each mouse in the PBS control group was given a dose of 5 ml / kg, each mouse in the siRNA conjugate experimental group was given a dose of 1 mg / kg (calculated as siRNA), and the administration volume was 5 ml / kg. The day of administration was recorded as the first day (D1), and 5 mice were sacrificed in each group on the 8th day (D8), 5 mice were sacrificed in each group on the 15th day (D15), 5 mice were sacrificed in each group on the 29th day (D29), and 5 mice were sacrificed in each group on the 43rd day (D43). The liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the relative quantitative calculation of the target gene mRNA in each test group was carried out according to the foregoing ΔΔCt method.

[0616] The primer sequences of this example are shown in Table 7 in Example 2.

[0617] The results of Example 7 show that the siRNA sequence RZ899009 conjugated with three clusters of CR01022 and the siRNA sequence RZ899011 conjugated with two clusters of CR01022 on the 3' end of the sense strand of siRNA have basically equivalent highest inhibitory activity and pharmacodynamic sustained effect on the target gene SOD1, and are both superior to the L96 conjugated sequence RZ599001 (Table 13). Figure 7

[0618] Table 13 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in this example

[0619]

[0620] Example 8: In vivo activity evaluation of siRNA conjugated with one cluster of CR01022 carrier and two clusters of CR01022 carrier for SOD1 target

[0621] In this example, the inhibitory activity of the same SOD1 target siRNA sequence RZ899011 conjugated with two clusters of CR01022 carrier on the 3' end of the sense strand, the siRNA sequence RZ899040 conjugated with one cluster of CR01022, and the L96 conjugate RZ599001 in mice was evaluated by the method of target gene inhibition activity evaluation in mice.

[0622] ​6-8 weeks old C57BL / 6j mice were randomly divided into groups according to body weight, 5 mice in each group, a total of 4 groups. Each group of mice was given PBS solution and siRNA conjugate mentioned in this example by subcutaneous injection in the abdomen, the dose of each mouse in the PBS control group was 5 ml / kg, the dose of each mouse in the siRNA conjugate experimental group was 1 mg / kg (calculated by siRNA), and the volume was 5 ml / kg. The day of administration was recorded as the first day (D1), and the mice were sacrificed on the 8th day (D8) after administration. The liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the relative quantification of the target gene mRNA in each test group was calculated according to the aforementioned ΔΔCt method.

[0623] The primer sequences of this example are shown in Table 7 in Example 2.

[0624] The results of Example 8 show that siRNA sense strand 3' end conjugated with two clusters of CR01022 siRNA sequence RZ899011 and one cluster of CR01022 siRNA sequence RZ899040 can produce high-efficiency inhibition of target gene SOD1 on D8, and the inhibition activities are basically equivalent, and both are slightly better than L96 conjugate RZ599001 (Table 14). Figure 8

[0625] Table 14 Inhibition activity of target genes in mice after administration of siRNA conjugates described in this example

[0626]

[0627] Example 9: In vivo activity evaluation of two clusters of CR01026 carrier and two clusters of CR01022 conjugated SOD1 target siRNA

[0628] This example uses the mouse in vivo target gene inhibition activity evaluation method to evaluate the inhibition activity of siRNA sequence RZ899025 conjugated with two clusters of CR01026 carrier, siRNA sequence RZ899011 conjugated with two clusters of CR01022 carrier, and L96 conjugate RZ599001 on target gene SOD1 in mice. The same SOD1 target siRNA sense strand 3' end. The two clusters of CR01026 and the two clusters of CR01022 have the same cyclic parent nucleus structure, but use different lengths of linkers.

[0629] ​6-8 weeks old C57BL / 6j mice were randomly divided into groups according to body weight, 20 mice in each group, and 4 groups in total. Each group of mice was given PBS solution and siRNA conjugate mentioned in this example by abdominal subcutaneous administration, wherein each mouse in the PBS control group was given a dose of 5 ml / kg of administration volume, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (calculated as siRNA) of administration volume of 5 ml / kg. The day of administration was recorded as the first day (D1), and 5 mice were sacrificed in each group on the 8th day (D8) after administration, 5 mice were sacrificed in each group on the 29th day (D29), 5 mice were sacrificed in each group on the 43rd day (D43), and 5 mice were sacrificed in each group on the 57th day (D57). Liver tissues were collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the relative quantitative calculation of the target gene mRNA in each test group was carried out according to the aforementioned ΔΔCt method.

[0630] The primer sequences of this example are shown in Table 7 in Example 2.

[0631] The results of Example 8 show that the siRNA sequence RZ899025 conjugated with duster CR01026 at the 3' end of the sense strand of siRNA and the siRNA sequence RZ899011 conjugated with duster CR01022 have in vivo activity comparable to that of L96 conjugated sequence RZ599001 at D8 and D29, and have in vivo activity significantly better than that of L96 conjugated sequence RZ599001 at D43, indicating that the duster CR01026 conjugate RZ899025 and the duster CR01022 conjugate RZ899011 have a longer duration of drug efficacy than the L96 conjugate RZ599001, and different lengths of linkers have little effect on the activity of CR01026 and CR01022 vectors. Figure 9 Table 15 shows the inhibitory activity of target genes in mice after administration of siRNA conjugates described in this example.

[0632] Table 15 shows the inhibitory activity of target genes in mice after administration of siRNA conjugates described in this example.

[0633]

[0634] Example 10: In vivo activity evaluation of siRNA conjugated with tri-cluster CR01023 vector targeting CC3

[0635] In this example, the inhibitory activity of siRNA conjugated with tri-cluster CR01023 vector targeting CC3 in mice was evaluated using the method of target gene inhibition activity evaluation in mice.

[0636] 6-8 weeks old C57BL / 6j mice were randomly divided into groups by weight, 15 mice in each group, a total of 3 groups. Each group of mice was given PBS solution and siRNA conjugate mentioned in this example by abdominal subcutaneous administration, each mouse in the PBS control group was given a dose of 5 ml / kg of administration volume, each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (calculated as siRNA) of administration volume 5 ml / kg. The day of administration is recorded as the first day (D1), and 5 mice were sacrificed in each group on D8, 5 mice were sacrificed in each group on D15, and 5 mice were sacrificed in each group on D29. Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the relative quantification of target gene mRNA in each test group was calculated according to the aforementioned ΔΔCt method.

[0637] The primer sequences of this example are shown in Table 5 in Example 1.

[0638] The results of Example 10 show that the conjugate RZ802011 of the triple CR01023 has comparable overall activity to the L96 conjugate RZ502031 in both groups of test subjects on D8 and D15, but the L96 conjugate RZ502031 has relatively higher target gene inhibition on D29. Figure 10 , Table 16).

[0639] Table 16 gives the inhibition activity of the target gene in mice after administration of the siRNA conjugate described in this example

[0640]

[0641] Example 11: In vivo activity evaluation of triple CR01023 carrier conjugated SOD1 target siRNA

[0642] This example uses the mouse in vivo target gene inhibition activity evaluation method to evaluate the inhibition activity of siRNA sense strand 3' end conjugated triple CR01023 siRNA sequence RZ899010 and L96 conjugate RZ599001 on the target gene SOD1 in mice.

[0643] 6-8 weeks old C57BL / 6j mice were randomly divided into groups by weight, 15 mice in each group, a total of 3 groups. Each group of mice was given PBS solution and siRNA conjugate mentioned in this example by abdominal subcutaneous administration, each mouse in the PBS control group was given a dose of 5 ml / kg of administration volume, each mouse in the siRNA conjugate experimental group was given a dose of 1 mg / kg (calculated as siRNA) of administration volume 5 ml / kg. The day of administration is recorded as the first day (D1), and 5 mice were sacrificed in each group on the 8th day (D8), 5 mice were sacrificed in each group on the 15th day (D15), and 5 mice were sacrificed in each group on the 29th day (D29). Collect liver tissue for RNA extraction, reverse transcription reaction and Real-time PCR detection, and calculate the relative quantification of the target gene mRNA in each test group according to the aforementioned ΔΔCt method.

[0644] The primer sequences of this example are shown in Table 7 in Example 2.

[0645] The results of Example 11 show that the three-cluster CR01023 conjugate RZ899010 has comparable overall activity to the L96 conjugate RZ599001 in both groups of test subjects on D8 and D15, but the L96 conjugate RZ599001 has a relatively higher target gene inhibition effect on D29 (Table 17). Figure 11

[0646] Table 17 Inhibition activity of target genes in mice after administration of siRNA conjugates described in this example

[0647]

[0648] Example 12: In vivo activity evaluation of two-cluster CR01027 carrier and two-cluster CR01022 carrier conjugated SOD1 target siRNA

[0649] This example uses the mouse in vivo target gene inhibition activity evaluation method to evaluate the inhibition activity of siRNA on the target gene SOD1 in mice of siRNA sequence RZ899048 conjugated with two-cluster CR01027 carrier at the 3' end of the sense strand, siRNA sequence RZ899011 conjugated with two-cluster CR01022 carrier, and conjugate RZ599001 conjugated with L96. The difference is that CR01027 increases the methoxy group at position 1 of the CR01022 parent nucleus structure.

[0650] ​6-8 weeks old C57BL / 6j mice were randomly divided into groups according to body weight, 5 mice in each group, a total of 4 groups. Each group of mice was given siRNA conjugate mentioned in this example by abdominal subcutaneous administration, wherein each mouse in the PBS control group was given a dose of 5 ml / kg of administration volume, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg of administration volume of 5 ml / kg. The day of administration was recorded as the first day (D1), and 5 mice in each group were sacrificed on the 8th day (D8) after administration. Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was quantitatively calculated according to the aforementioned ΔΔCt method.

[0651] The primer sequences of this example are shown in Table 7 in Example 2.

[0652] The results of Example 12 show that the conjugate RZ899048 of the two-cluster CR01027 has a higher target gene inhibition effect on D8 than the L96 conjugate RZ599001, and the effect is comparable to that of the conjugate (RZ899011) of the two-cluster CR01022. Figure 12 , Table 18).

[0653] Table 18 gives the inhibition activity of the target target gene in mice after administration of the siRNA conjugate described in this example

[0654]

[0655] Example 13: In vivo activity evaluation of two-cluster CR01027 carrier conjugated angiopoietin-like 3 (ANGPTL3) target siRNA

[0656] This example uses the mouse in vivo target gene inhibition activity evaluation method to evaluate the siRNA sequence RZ897013 of the siRNA conjugated with the 3' end of the sense strand of the two-cluster CR01027 carrier, and the inhibition activity of the conjugate RZ597002 conjugated with L96 on the target gene ANGPTL3 in mice.

[0657] 6-8 weeks old C57BL / 6j mice were randomly divided into groups according to body weight, 5 mice in each group, a total of 4 groups. Each group of mice was given siRNA conjugate mentioned in this example by abdominal subcutaneous administration, each mouse in the PBS control group was given a dose of 5ml / kg of administration volume, each mouse in the siRNA conjugate experimental group was given a dose of 3mg / kg, and the administration volume was 5ml / kg. The day of administration was recorded as the first day (D1), and 5 mice in each group were sacrificed on the 8th day (D8) after administration. Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the relative quantification of target gene mRNA in each test group was calculated according to the aforementioned ΔΔCt method.

[0658] The primer sequences of this example are shown in Table 19.

[0659] Table 19 Primer sequence list

[0660]

[0661] The results of Example 13 show that the di-cluster CR01027 conjugate RZ897013 has comparable inhibitory effect compared with the L96 conjugate RZ597002 (Table 20). Figure 13

[0662] Table 20 Inhibitory activity of target genes in mice after administration of siRNA conjugates described in this example

[0663]

[0664] Example 14: In vivo activity evaluation of di-cluster CR01017 carrier and di-cluster CR01022 carrier conjugated SOD1 target siRNA

[0665] This example uses the mouse in vivo target gene inhibitory activity evaluation method to evaluate the inhibitory activity of SOD1 target siRNA on the target gene SOD1 in mice. The siRNA sequence RZ899038 conjugated with the di-cluster CR01017 carrier, the siRNA sequence RZ899011 conjugated with the di-cluster CR01022 carrier, and the L96 conjugate RZ599001. The main difference between the di-cluster CR01017 carrier and the di-cluster CR01022 carrier is that the CR01017 carrier linker part is an amide bond structure, and the CR01022 carrier linker part is a triazole structure.

[0666] ​6-8 weeks old C57BL / 6j mice were randomly divided into groups by weight, 15 mice in each group, a total of 4 groups. Each group of mice was given PBS solution and siRNA conjugate mentioned in this example by abdominal subcutaneous administration, each mouse in the PBS control group was given a dose of 5 ml / kg of administration volume, each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (calculated as siRNA) of administration volume of 5 ml / kg. The day of administration was recorded as the first day (D1), and 5 mice were sacrificed in each group on the 8th day (D8), 5 mice were sacrificed in each group on the 29th day (D29), and 5 mice were sacrificed in each group on the 54th day (D54). The liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the relative quantitative calculation of the target gene mRNA in each test group was carried out according to the aforementioned ΔΔCt method.

[0667] The primer sequences of this example are shown in Table 7 in Example 2.

[0668] The results of Example 14 show that the siRNA sequence RZ899038 conjugated with the double-cluster CR01017 carrier at the 3' end of the siRNA sense strand and the siRNA sequence RZ899011 conjugated with the double-cluster CR01022 carrier have better in vivo activity than the L96 conjugated sequence RZ599001, and the use of triazole structure (CR01022) or amide structure (CR01017) for the linker has little effect on the activity of the carrier (Table 21). Figure 14

[0669] Table 21 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in this example

[0670]

[0671] Example 15: In vivo activity evaluation of CR01017 conjugated SOD1 target siRNA with different numbers and different positions

[0672] In this example, the inhibitory activity of the target gene in mice was evaluated by evaluating the inhibitory activity of the siRNA sequence RZ897041 conjugated with a single-cluster CR01017 carrier at the 3' end of the siRNA sense strand, the siRNA sequence RZ897042 conjugated with a single-cluster CR01017 carrier at the 5' end and the 3' end of the siRNA sense strand, the siRNA sequence RZ897038 conjugated with a double-cluster CR01017 carrier at the 3' end of the siRNA sense strand, and the L96 conjugate RZ599001 in mice.

[0673] ​6-8 weeks old C57BL / 6j mice were randomly divided into groups according to body weight, 15 mice in each group, a total of 5 groups. Each group of mice was given PBS solution or siRNA conjugate mentioned in this example by subcutaneous injection, and each mouse in the PBS control group was given a dose of 5 ml / kg, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (calculated as siRNA) and a dose of 5 ml / kg. The day of administration was recorded as the first day (D1), and 5 mice were sacrificed in each group on the 8th day (D8), 5 mice were sacrificed in each group on the 29th day (D29), and 5 mice were sacrificed in each group on the 54th day (D54).

[0674] The primer sequences of this example are shown in Table 7 in Example 2.

[0675] The results of Example 15 show that siRNA conjugate RZ899038 with two clusters of CR01017 conjugated to the 3' end of the sense strand of siRNA, siRNA conjugate RZ899041 with one cluster of CR01017 conjugated to the 3' end of the sense strand of siRNA, and siRNA conjugate RZ899042 with one cluster of CR01017 conjugated to the 5' end and 3' end of the sense strand of siRNA, respectively, have an inhibitory effect comparable to L96 conjugate RZ599001 at D8 and D29, and have a higher inhibitory effect on the target gene than L96 conjugate RZ599001 at D59. This indicates that CR01017 conjugates have a longer pharmacodynamic effect time (Table 22). Figure 15

[0676] Table 22 Inhibitory activity of target genes in mice after administration of siRNA conjugates described in this example

[0677]

[0678] Example 16: The disclosure selects siRNA sequence RZ899011 conjugated with two clusters of CR01022 and siRNA sequence RZ899038 conjugated with two clusters of CR01017 targeting SOD1 to evaluate the toxicity in mice

[0679] In this example, the toxicity of siRNA sequence RZ899011 conjugated with two clusters of CR01022 and siRNA sequence RZ899038 conjugated with two clusters of CR01017 was evaluated in ICR mice by subcutaneous injection.

[0680] ​Six- to eight-week-old ICR mice were randomly divided into three groups of 10 mice each (half male and half female) according to their body weight. Each group of mice was administered either PBS solution (without siRNA conjugate) or the siRNA conjugate mentioned in this example via subcutaneous abdominal administration. In the PBS control group, each mouse was given a dose of 10 ml / kg body weight, while in the siRNA conjugate group, each mouse was given a dose of 300 mg / kg body weight (based on siRNA) at a dose of 10 ml / kg.

[0681] The day of administration was designated as day 1 (D1). Clinical observation was conducted at least once daily after the first administration during the experiment. On day 15 (D15), blood was collected from all animals (mice were fasted for at least 12 hours before sampling, but water was allowed) for hematological and blood biochemical tests. Blood samples were anticoagulated with EDTA-K2 and subjected to routine blood tests using an automated modular blood and body fluid analyzer (Sysmex XN-10B1). Blood samples were also anticoagulated with sodium citrate, centrifuged at room temperature for 10 minutes (approximately 2000g), and plasma was separated and tested for coagulation parameters using an automated coagulation analyzer (Mindray C3510). Blood samples (without anticoagulation) were placed at room temperature for approximately 30 minutes until coagulation, then centrifuged at 2000g for 10 minutes at 4°C and subjected to blood biochemical tests using an automated biochemical analyzer (Sysmex BX-4000). On day 15 of the experiment (D15), all animals were dissected. Before dissection, mice were fasted for at least 12 hours but allowed free access to water. They were anesthetized with 50 ozonol (Victal, France, 8G4VA), and euthanized by exsanguination of the abdominal aorta after blood collection. Gross anatomical observation was then performed. The livers of all animals were subjected to histopathological examination (using hematoxylin-eosin staining). If necessary, organs showing obvious abnormalities in size, shape, color, or texture were also subjected to histopathological examination.

[0682] The primer sequences for this embodiment are shown in Table 7 of Embodiment 2.

[0683] The results of Example 16 showed that, based on the siRNA conjugate shown in this example, no abnormalities were observed in clinical observation during the experiment in the PBS group, RZ899011 administration group, and RZ899038 administration group; at the experimental endpoint D15, no obvious abnormalities were found in gross anatomy; and no abnormalities were found in hematological and blood biochemical tests performed on the animals in each group. Figure 16a -j, Table 23); no abnormalities were found in the histopathological staining of liver tissue from each group of animals ( Figure 16k This indicates that the siRNA conjugates and their vector molecules disclosed herein have good safety and low animal-level toxicity.

[0684] Table 23. Hematology and blood biochemistry results in mice after administration of siRNA conjugates described in this Example

[0685]

[0686] The above merely illustrates the exemplary embodiments of the present disclosure, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present disclosure, and these improvements and refinements should also be considered as the protection scope of the present disclosure.

Claims

1. A compound or a stereoisomer, a pharmaceutically acceptable salt thereof, characterized by, said compound is selected from any one of the following structures: said R1is selected from H, a hydroxyl protecting group or ; said represents a linking site for attachment of a pharmaceutically active molecule; said hydroxyl protecting group is selected from 4,4'-dimethoxybenzhydryl; said R2is selected from H, a reactive phosphorus group or ; wherein said reactive phosphorus group is selected from ; selected from ; each M is independently selected from hydroxyl or thiol; P is 1; q is 0; j is selected from 0, 1 or 2; Y is O; L2 is ; said r is selected from 1 to 3; R3 is selected from H or C1-C3 alkoxy; each R5is independently selected from H or .

2. The compound of claim 1, or a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, said compound is selected from any one of the following compounds: 。 3. A conjugate having the structure of Formula (lb) or a stereoisomer, a pharmaceutically acceptable salt thereof: In Formula (lb), the Nu represents an oligonucleotide; said k is selected from 1 or 2; The structural formula of the Q is wherein, said j' is selected from 1, 2, 3 or 4; said , M, p, q, Cy are as defined in claim 1 or 2.

4. The conjugate according to claim 3, characterized in that, said j' is selected from 1, 2 or 3.

5. The conjugate of claim 3, wherein, said conjugate has any one of the following structures: 。 6. The conjugate of claim 3, wherein, said conjugate is selected from any one of the following compounds: 。 7. Composition, characterized in that said composition comprises the conjugate of any one of claims 3 to 6; said composition further comprises one or more pharmaceutically acceptable carriers or excipients.

8. Use of any of the following in the manufacture of a medicament for the prevention and / or treatment of a disease: (I) the compound of claim 1 or 2 or a stereoisomer, a pharmaceutically acceptable salt thereof; and / or (II) the conjugate of any one of claims 3 to 6; and / or (III) the composition of claim 7.

9. Use of any of the following in the manufacture of a medicament for reducing the expression or activity of a target gene: (I) the compound of claim 1 or 2; and / or (II) the conjugate of any one of claims 3 to 6; and / or (III) the composition of claim 7.

10. A medicament, characterized by, comprising any of the following and a pharmaceutically acceptable excipient or adjuvant: (I) the compound of claim 1 or 2; and / or (II) the conjugate of any one of claims 3 to 6; and / or (III) the composition of claim 7.

11. A pharmaceutical combination, characterized in that comprising the medicament of claim 10.

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