Compound, its preparation method and use

Abstract

The present disclosure relates to the field of nucleic acid drugs, and specifically discloses a compound represented by formula (I), a preparation method thereof, and uses thereof. The targeting ligand and RNAi drug prepared by using the compound provided by the present disclosure can highly effectively target hepatocytes, and effectively inhibit the expression of target genes in hepatocytes, and can be used for treating and / or preventing liver-derived diseases, and has the advantages of high in vivo activity, stable and lasting drug effect, excellent safety, and low toxicity.

Description

Technical Field

[0001] The present disclosure relates to the field of synthesis, specifically to the field of nucleic acid drugs, and more specifically provides compounds, their preparation methods and uses. Background Art

[0002] Asialoglycoprotein receptor (ASGPR), also known as liver lectin, is a transmembrane protein that is significantly and specifically expressed on the cell surface of liver sinusoids and basolateral. ASGPR is mainly composed of two subunits, H1 and H2, and the combination of the two has an endocytic effect. A normal mature hepatocyte has approximately 500,000 receptor molecules, and about 10% of ASGPR is expressed on the cell membrane, which specifically recognizes, binds and internalizes some glycoproteins with galactosyl and N-acetylgalactosaminyl groups at the end in the blood circulation, enabling their metabolism in hepatocytes. By conjugating oligonucleotides with a targeting ligand and using the targeting ligand to bind to ASGPR, hepatic targeting delivery of oligonucleotides can be achieved.

[0003] However, the current ligands of ASGPR have low affinity and cannot achieve good hepatic targeting delivery of drugs or genes to achieve the purpose of regulating the expression of target genes. Summary of the Invention

[0004] In view of this, the present disclosure provides compounds, their preparation methods and uses.

[0005] The targeting ligand and RNAi drug prepared using the compounds provided by the present disclosure can highly effectively target hepatocytes and effectively inhibit the expression of target genes in hepatocytes, and can be used for the treatment and / or prevention of liver diseases, and has the advantages of high in vivo activity, stable and lasting drug effect, excellent safety and low toxicity.

[0006] To achieve the above-mentioned invention purposes, the present disclosure provides the following technical solutions:

[0007] In the first aspect of the present disclosure, the present disclosure provides a compound of formula (I) or its stereoisomer:

[0008]

[0009] Wherein, p and q each independently selected from 0, 1, 2 or 3;

[0010] Each n independently selected from 1, 2, 3, 4 or 5;

[0011] R1 is selected from H or R1', and the R1' is selected from a hydroxyl protecting group;

[0012] R2 is selected from H or R2', and the R2' is selected from Wherein, R 2a is selected from secondary amino groups, and R 2b is selected from secondary amino groups or cyano-substituted C1-C3 alkoxy groups;

[0013] R3 is selected from H or C1-C3 alkoxy groups;

[0014] Each R4 is independently selected from

[0015] In some specific embodiments of the present disclosure, each R4 is selected from

[0016] In some specific embodiments of the present disclosure, each R4 is selected from

[0017] In some alternative embodiments of the present disclosure, p and q are independently selected from 0 or 1.

[0018] In some alternative embodiments of the present disclosure, p = 1 and q = 1.

[0019] In some specific embodiments of the present disclosure, p = 1 and q = 0.

[0020] In some alternative embodiments of the present disclosure, or p = 0 and q = 1.

[0021] In some alternative embodiments of the present disclosure, p = 0 and q = 0.

[0022] In some specific embodiments of the present disclosure, each n is selected from 1.

[0023] In some alternative embodiments of the present disclosure, each n is selected from 2.

[0024] In some alternative embodiments of the present disclosure, each n is selected from 3.

[0025] In some alternative embodiments of the present disclosure, each n is selected from 4.

[0026] In some alternative embodiments of the present disclosure, each n is selected from 5.

[0027] Exemplarily, when p = 1, q = 0, and n = 1, the structural formula of the compound of formula (I) is

[0028] In some alternative embodiments of the present disclosure, the structural formula of the hydroxyl protecting group is Wherein, each R 1a is independently selected from H, C1-C3 alkyl groups, or C1-C3 alkoxy groups.

[0029] In some alternative embodiments of the present disclosure, each R 1a is independently selected from H or a C1-C3 alkoxy group;

[0030] In some alternative embodiments of the present disclosure, each R 1a is independently selected from H or a methoxy group.

[0031] In some alternative embodiments of the present disclosure, the hydroxyl protecting group is selected from trityl (Tr group), 4-methoxytrityl (MMTr group), 4,4'-dimethoxytrityl (DMTr group), or 4,4',4''-trimethoxytrityl (TMTr group).

[0032] In some specific embodiments of the present disclosure, the hydroxyl protecting group is selected from 4,4'-dimethoxytrityl (DMTr group).

[0033] In some specific embodiments of the present disclosure, R 2a is selected from

[0034] In some alternative embodiments of the present disclosure, R 2b is selected from

[0035] In some specific embodiments of the present disclosure, R 2b is selected from selected from

[0036] In some alternative embodiments of the present disclosure, is selected from

[0037] In some specific embodiments of the present disclosure, is selected from

[0038] In some alternative embodiments of the present disclosure, R2 is selected from H,

[0039] In some alternative embodiments of the present disclosure, R2 is selected from H or

[0040] In some specific embodiments of the present disclosure, R2 is selected from H.

[0041] In some specific embodiments of the present disclosure, R2 is selected from

[0042] In some alternative embodiments of the present disclosure, the compound is selected from any of the following structures:

[0043] In some alternative embodiments of the present disclosure, the compound is selected from any of the following structures:

[0044]

[0045] In a second aspect of the present disclosure, there is provided a method for preparing a compound, wherein the compound is represented by formula (I-1a) or formula (I-1b) or formula (I-1c):

[0046]

[0047] wherein, R1', R2', R3, p, q, n are as defined above;

[0048] The preparation method includes the following steps:

[0049]

[0050] S1-1: The compound of formula (1) and the compound of formula (2) undergo an acetalization reaction in the presence of an acidic catalyst to obtain a compound of formula (3); wherein, R5 is selected from a substituted or unsubstituted C6-C12 aryl group;

[0051] S1-2: The compound of formula (3), a hydrogenated salt, and the compound of formula (4) undergo an electrophilic substitution reaction to obtain a compound of formula (5); wherein, X1 represents a halogen;

[0052] S1-3: The compound of formula (5) undergoes a dehydroxy protection reaction under acidic conditions to obtain a compound of formula (I-1a);

[0053] S1-4: The compound of formula (I-1a) and the compound of formula (6) undergo a nucleophilic substitution reaction to obtain a compound of formula (I-1b); wherein, X2 represents a halogen;

[0054] S1-5: The compound of formula (I-1b) and the compound of formula (7) undergo a condensation reaction to obtain a compound of formula (I-1c); wherein, R 2c is selected from a halogen or a secondary amino group.

[0055] In some alternative embodiments of the present disclosure, in step S1-1, the molar ratio of the compound of formula (1), the compound of formula (2), and the acidic catalyst is 1:(2-4):(0.01-0.2). For example, 1:(2.5-3.5):(0.01-0.2), 1:(2.8-3.2):(0.01-0.2), 1:3:(0.01-0.2), 1:(2-4):(0.05-0.15), 1:(2-4):(0.08-0.12), 1:(2-4):0.1, 1:3:0.1, etc.

[0056] In some specific embodiments of the present disclosure, in step S1-1, the molar ratio of the compound of formula (1), the compound of formula (2), and the acidic catalyst is 1:3:0.1.

[0057] In some specific embodiments of the present disclosure, in step S1-1, the acidic catalyst is selected from sulfonic acid catalysts.

[0058] In some specific embodiments of the present disclosure, the sulfonic acid catalyst is selected from DL-10-camphorsulfonic acid (CAS No. 5872-08-2).

[0059] In some alternative embodiments of the present disclosure, in step S1-1, the reaction temperature of the acetalization reaction is selected from 20 to 40 °C; for example, 25 °C, 30 °C, 35 °C, etc.

[0060] In some alternative embodiments of the present disclosure, in step S1-1, the reaction time of the acetalization reaction is 4 to 10 h, for example, 4.5 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h, 8 h, 8.5 h, 9 h, 9.5 h, etc.

[0061] In some alternative embodiments of the present disclosure, in step S1-1, the reaction solvent of the acetalization reaction is a polar aprotic solvent, such as acetonitrile.

[0062] In some specific embodiments of the present disclosure, in step S1-1, R5 is selected from phenyl (Ph group).

[0063] In some alternative embodiments of the present disclosure, in step S1-2, the molar ratio of the compound of formula (3), the compound of formula (4), and the catalyst is 1:(3 to 5):(3 to 5); for example, 1:(3.5 to 4.5):(3 to 5), 1:(3.8 to 4.2):(3 to 5), 1:4:(3 to 5), 1:(3 to 5):(3.5 to 4.5), 1:(3 to 5):(3.8 to 4.2), 1:(3 to 5):4, 1:4:4.

[0064] In some specific embodiments of the present disclosure, in step S1-2, the molar ratio of the compound of formula (3), the compound of formula (4), and the catalyst is 1:4:4.

[0065] In some alternative embodiments of the present disclosure, in step S1-2, the hydride salt is selected from sodium hydride, potassium hydride, or magnesium hydride.

[0066] In some specific embodiments of the present disclosure, in step S1-2, the hydride salt is selected from sodium hydride.

[0067] In some alternative embodiments of the present disclosure, in step S1-2, the conditions for the electrophilic substitution reaction are as follows: The compound of formula (3) and the hydrogenated salt are first reacted at -10 to 0 °C (such as -9 °C, -8 °C, -7 °C, -6 °C, -5 °C, -4 °C, -3 °C, -2 °C, -1 °C, etc.) for 25 to 35 min (such as 30 min, etc.), and then the compound of formula (4) is added and reacted at 20 to 40 °C (such as 25 °C, 30 °C, 35 °C, etc.) for 1 to 5 h (such as 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, etc.).

[0068] In some alternative embodiments of the present disclosure, in step S1-2, the reaction solvent for the electrophilic substitution reaction is a polar aprotic solvent. For example, N,N-dimethylformamide (DMF).

[0069] In some specific embodiments of the present disclosure, in step S1-2, X1 is selected from bromine (Br).

[0070] In some alternative embodiments of the present disclosure, in step S1-3, the acidic condition is: an organic acid. Among them, the organic acid can be selected from formic acid (CAS No. 64-18-6), acetic acid (CAS No. 64-19-7), propionic acid (CAS No. 79-09-4), butyric acid (CAS No. 107-92-6), and valeric acid (CAS No. 109-52-4).

[0071] In some specific embodiments of the present disclosure, in step S1-3, the acidic condition is: acetic acid. Specifically, it is a 50 to 80 mass% aqueous acetic acid solution, and more specifically, it is a 70 mass% aqueous acetic acid solution.

[0072] In some alternative embodiments of the present disclosure, in step S1-3, the reaction temperature for the dehydroxy protection reaction is selected from 60 to 80 °C; such as 65 °C, 70 °C, 75 °C, etc.

[0073] In some alternative embodiments of the present disclosure, in step S1-3, the reaction solvent for the deprotection reaction is a polar aprotic solvent. For example, dichloromethane (DCM).

[0074] In some alternative embodiments of the present disclosure, in step S1-4, the molar ratio of the compound of formula (I-1a) to the compound of formula (6) is 1:(1.1 to 1.5); such as 1:1.15, 1:1.2, 1:1.25, 1:1.3, 1:1.35, 1:1.4, 1:1.45, etc.

[0075] In some alternative embodiments of the present disclosure, in step S1-4, the compound of formula (6) is selected from trityl chloride (also known as triphenylchloromethane, abbreviated as Trt-Cl, CAS No. 76-83-5), trityl bromide (CAS No. 596-43-0), 4-methyltrityl chloride (also known as 4-methyltriphenylchloromethane, CAS No. 23429-44-9), 4-methoxytrityl chloride (also known as 4-methoxytriphenylchloromethane, MMT-Cl, CAS No. 14470-28-1), 4,4'-dimethoxytrityl chloride (also known as 4,4'-dimethoxytriphenylchloromethane, abbreviated as DMT-Cl, CAS No. 40615-36-9), and 4,4',4''-trimethoxytrityl chloride (CAS No. 49757-42-8).

[0076] In some specific embodiments of the present disclosure, in step S1-4, the compound of formula (6) is selected from 4,4'-dimethoxytrityl chloride.

[0077] In some specific embodiments of the present disclosure, in step S1-4, the nucleophilic substitution reaction is carried out in an inert atmosphere, such as nitrogen, helium, etc.

[0078] In some alternative embodiments of the present disclosure, in step S1-4, the reaction temperature of the nucleophilic substitution reaction is selected from 20 to 40 °C; for example, 25 °C, 30 °C, 35 °C, etc.

[0079] In some alternative embodiments of the present disclosure, in step S1-4, the reaction time of the nucleophilic substitution reaction is 2 to 5 h, for example: 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, etc.

[0080] In some specific embodiments of the present disclosure, in step S1-4, X2 is selected from bromine (Br).

[0081] In some alternative embodiments of the present disclosure, in step S1-5, the molar ratio of the compound of formula (I-1b) to the compound of formula (7) is 1:(0.6 to 0.9); for example, 1:0.65, 1:0.7, 1:0.71, 1:0.72, 1:0.73, 1:0.74, 1:0.75, 1:0.76, 1:0.77, 1:0.78, 1:0.79, 1:0.8, 1:0.85, etc.

[0082] In some specific embodiments of the present disclosure, in step S1-5, the molar ratio of the compound of formula (I-1b) to the compound of formula (7) is 1.5:1.1 (about 1:0.73).

[0083] In some alternative embodiments of the present disclosure, in step S1-5, the compound of formula (7) is selected from 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite (CAS No. 89992-70-1), bis(diisopropylamino)chlorophosphine, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (CAS No. 102691-36-1).

[0084] Compared with bis(diisopropylamino)(2-cyanoethoxy)phosphine, 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite is not only expensive, but also has high activity and is prone to deterioration; due to the inconvenient use and storage of 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite, it is also more likely to introduce other impurities during the reaction, resulting in unsatisfactory purity and yield. Therefore, those skilled in the art tend to choose bis(diisopropylamino)(2-cyanoethoxy)phosphine, and it is well known in the art that bis(diisopropylamino)(2-cyanoethoxy)phosphine must undergo a condensation reaction with an alcohol under the catalysis of a weak acid reagent (such as tetrazole, 4,5-dicyanoimidazole, etc.). In some specific embodiments of the present disclosure, in step S1-5, the compound of formula (7) is selected from 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite.

[0085] In some alternative embodiments of the present disclosure, in step S1-5, R 2c is selected from halogen.

[0086] In some specific embodiments of the present disclosure, in step S1-5, R 2c is selected from chlorine (Cl).

[0087] In some alternative embodiments of the present disclosure, in step S1-5, the condensation reaction is carried out in an inert atmosphere, such as nitrogen, helium, etc.

[0088] In some alternative embodiments of the present disclosure, in step S1-5, the reaction solvent for the condensation reaction is a polar aprotic solvent, such as dichloromethane.

[0089] When R3 is selected from H, the structural formula of the compound of formula (1) is Among them, the compound of formula (1) (R3 selected from H) can be obtained by market purchase, but the price is relatively high. In order to save costs, the compound of formula (1) (R3 selected from H) can be obtained by the following preparation method.

[0090] In some specific embodiments of the present disclosure, the preparation method of the compound of formula (1) (R3 selected from H) includes the following steps:

[0091]

[0092] Step 1: The compound of formula (1-a) undergoes a hydrodehalogenation reaction in the presence of a reducing agent and a radical initiator to obtain the compound of formula (1b); wherein, X4 represents a halogen (such as F, Cl, Br, I).

[0093] Step 2: The compound of formula (1b) undergoes a hydrolysis reaction to obtain the compound of formula (1).

[0094] In some alternative embodiments of the present disclosure, in Step 1, the molar ratio of the compound of formula (1-a), the reducing agent, and the radical initiator is 1:(0.9 - 1.2):(0.01 - 0.2), such as 1:(0.95 - 1.2):(0.01 - 0.2), 1:(0.95 - 1.1):(0.01 - 0.2), 1:(0.98 - 1.05):(0.01 - 0.2), 1:(0.98 - 1.02):(0.01 - 0.2), 1:1:(0.01 - 0.2), 1:(0.9 - 1.2):(0.05 - 0.15), 1:(0.9 - 1.2):(0.08 - 0.12), 1:(0.9 - 1.2):0.1, 1:1:0.1, etc.

[0095] In some specific embodiments of the present disclosure, in Step 1, the molar ratio of the compound of formula (1-a), the reducing agent, and the radical initiator is 1:1:0.1.

[0096] In some specific embodiments of the present disclosure, in Step 1, the reducing agent is tributyltin hydride.

[0097] In some alternative embodiments of the present disclosure, in Step 1, the radical initiator is an azo initiator. Among them, the azo initiator can be azobisisobutyronitrile.

[0098] In some alternative embodiments of the present disclosure, in Step 1, the reaction temperature of the hydrodehalogenation reaction is 100 - 120 °C, such as: 105 °C, 110 °C, 115 °C, etc.

[0099] In some alternative embodiments of the present disclosure, in Step 1, the reaction solvent of the hydrodehalogenation reaction is an aprotic solvent, such as: toluene.

[0100] In some alternative embodiments of the present disclosure, in Step 1, the reaction time of the hydrodehalogenation reaction is 1 - 5 h, such as 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, etc.

[0101] In some alternative embodiments of the present disclosure, in Step 2, the hydrolysis reaction is an alkaline hydrolysis reaction.

[0102] In some alternative embodiments of the present disclosure, in step two, the conditions for the alkaline hydrolysis reaction are: C1-C3 alkoxide and C1-C3 alcohol.

[0103] In some alternative embodiments of the present disclosure, the C1-C3 alkoxide is selected from lithium methoxide, sodium methoxide, potassium methoxide, rubidium methoxide, cesium methoxide, lithium ethoxide, sodium ethoxide, potassium ethoxide, sodium n-propoxide, potassium n-propoxide, sodium isopropoxide, and potassium isopropoxide.

[0104] In some specific embodiments of the present disclosure, the C1-C3 alkoxide is selected from lithium methoxide.

[0105] In some alternative embodiments of the present disclosure, the C1-C3 alcohol is selected from methanol, ethanol, n-propanol, and isopropanol.

[0106] In some specific embodiments of the present disclosure, the C1-C3 alcohol is selected from methanol.

[0107] In some alternative embodiments of the present disclosure, in step two, the molar ratio of the compound of formula (1-b) to the alkoxide is 1:(0.05 - 0.2). For example, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1, 1:0.11, 1:0.12, 1:0.13, 1:0.14, 1:0.15, 1:0.16, 1:0.17, 1:0.18, 1:0.19.

[0108] In a second aspect of the present disclosure, the present disclosure provides a method for preparing a compound, the compound being represented by formula (I-2):

[0109]

[0110] wherein R1', R3, p, q, n are as defined above;

[0111] The preparation method includes the following steps:

[0112]

[0113] S2-1. The compound of formula (3), the hydrogenated salt, the compound of formula (8), and the iodide salt undergo an electrophilic substitution reaction to obtain the compound of formula (9); wherein, R5 is selected from a substituted or unsubstituted C6-C12 aryl group, X3 represents a halogen, and R6 is selected from a substituted or unsubstituted C1-C5 alkyl group;

[0114] S2-2. The compound of formula (9) undergoes a dehydroxy protection reaction under acidic conditions to obtain the compound of formula (10);

[0115] S2-3. The compound of formula (10) and the compound of formula (6) undergo a nucleophilic substitution reaction to obtain the compound of formula (11);

[0116] S2-4. The compound shown in formula (11) undergoes a hydrolysis reaction to obtain the compound of formula (I-2).

[0117] In some alternative embodiments of the present disclosure, in step S2-1, the molar ratio of the compound of formula (3), the hydrogenated salt, the compound of formula (8), and the iodide salt is 1:(3-5):(1.5-2.5):(1-2); for example, 1:(3.5-4.5):(1.5-2.5):(1-2), 1:(3.8-4.2):(1.5-2.5):(1-2), 1:4:(1.5-2.5):(1-2), 1:(3-5):(1.8-2.2):(1-2), 1:(3-5):(1.9-2.1):(1-2), 1:(3-5):2:(1-2), 1:(3-5):(1.5-2.5):(1.3-1.7), 1:(3-5):(1.5-2.5):(1.4-1.6), 1:(3-5):(1.5-2.5):1.5, 1:4:2:1.5, etc.

[0118] In some specific embodiments of the present disclosure, in step S2-1, the molar ratio of the compound of formula (3), the hydrogenated salt, the compound of formula (8), and the iodide salt is 1:4:2:1.5.

[0119] In some alternative embodiments of the present disclosure, in step S2-1, the conditions for the electrophilic substitution reaction are as follows: the compound of formula (3) and the hydrogenated salt first react at -10 to 0 °C (for example, -9 °C, -8 °C, -7 °C, -6 °C, -5 °C, -4 °C, -3 °C, -2 °C, -1 °C, etc.) for 0.5 to 1.5 h (for example, 0.75 h, 1 h, 1.25 h, etc.), then the compound of formula (8) and the iodide salt are added and the reaction is carried out at 20 to 40 °C (for example, 25 °C, 30 °C, 35 °C, etc.) for 3 to 5 h (for example, 3.5 h, 4 h, 4.5 h, etc.).

[0120] In some specific embodiments of the present disclosure, in step S2-1, R5 is selected from phenyl (Ph group);

[0121] In some alternative embodiments of the present disclosure, in step S2-1, R6 is selected from substituted or unsubstituted C1-C3 alkyl.

[0122] In some specific embodiments of the present disclosure, in step S2-1, R6 is selected from ethyl.

[0123] In some alternative embodiments of the present disclosure, in step S2-2, the acidic condition is: an organic acid; wherein, the organic acid can be selected from formic acid (CAS No. 64-18-6), acetic acid (CAS No. 64-19-7), propionic acid (CAS No. 79-09-4), butyric acid (CAS No. 107-92-6), and valeric acid (CAS No. 109-52-4).

[0124] In some specific embodiments of the present disclosure, in step S2-2, the acidic condition is: acetic acid. Specifically: a 50-80% by mass aqueous acetic acid solution, more specifically: a 70% by mass aqueous acetic acid solution.

[0125] In some alternative embodiments of the present disclosure, in step S2-2, the reaction temperature of the dehydroxy protection reaction is selected from 40-60 °C; for example, 45 °C, 50 °C, 55 °C, etc.

[0126] In some alternative embodiments of the present disclosure, in step S2-3, the molar ratio of the compound of formula (10) to the compound of formula (6) is 1:(1.1-1.5), for example, 1:1.15, 1:1.2, 1:1.25, 1:1.3, 1:1.35, 1:1.4, 1:1.45, etc.

[0127] In some specific embodiments of the present disclosure, in step S2-3, the molar ratio of the compound of formula (10) to the compound of formula (6) is 1:1.3.

[0128] In some alternative embodiments of the present disclosure, in step S2-3, the reaction temperature of the nucleophilic substitution reaction is selected from 20-40 °C; for example, 25 °C, 30 °C, 35 °C, etc.

[0129] In some alternative embodiments of the present disclosure, in step S2-4, the hydrolysis reaction is selected from basic hydrolysis reactions.

[0130] In some alternative embodiments of the present disclosure, in step S2-4, the hydrolysis reaction conditions are selected from a 0.5-1.5 mol / L aqueous NaOH solution, for example, a 0.8 mol / L aqueous NaOH solution, a 1 mol / L aqueous NaOH solution, a 1.2 mol / L aqueous NaOH solution, etc.

[0131] In some alternative embodiments of the present disclosure, in step S2-4, the temperature of the hydrolysis reaction is 20-40 °C; for example, 25 °C, 30 °C, 35 °C, etc.

[0132] In the fourth aspect of the present disclosure, the present disclosure provides the use of the compound described in the first aspect in the preparation of a targeting ligand capable of binding to a cell surface receptor.

[0133] In some specific embodiments of the present disclosure, the receptor is selected from asialoglycoprotein receptors.

[0134] Exemplarily, the targeting ligand is selected from any one of the following compounds:

[0135]

[0136]

[0137]

[0138]

[0139] In the fourth aspect of the present disclosure, the present disclosure provides the use of the compound of the first aspect in the preparation of a drug for reducing the expression or activity of a target gene in hepatocytes.

[0140] In some specific embodiments of the present disclosure, the target cells are selected from hepatocytes.

[0141] In some specific embodiments of the present disclosure, the drug is selected from RNAi drugs;

[0142] In some specific embodiments of the present disclosure, the drug is selected from siRNA drugs.

[0143] Exemplarily, the siRNA drug is selected from any one of the following compounds:

[0144]

[0145]

[0146]

[0147] Wherein, represents siRNA. Wherein, when there is one targeting ligand in the siRNA drug, one targeting ligand is conjugated to the 3'-end of the sense strand. When there are two targeting ligands in the siRNA drug, the two targeting ligands are respectively conjugated to the 3'-end and 5'-end of the sense strand.

[0148] Beneficial effects:

[0149] The targeting ligand and RNAi drug (such as siRNA drug) prepared by using the compound provided by the present disclosure can highly effectively target hepatocytes and effectively inhibit the expression of the target gene in hepatocytes, and can be used for treating and / or preventing pathological conditions or diseases caused by abnormal expression of the target gene in hepatocytes, and has the advantages of high in vivo activity, stable and lasting drug effect, excellent safety and low toxicity. Description of the Drawings

[0150] Figure 1 It is the graph of the inhibitory activity results of RZ802010 and RZ502031 in Example 1 of the present disclosure on the target gene CC3 in mice;

[0151] Figure 2 It is the graph of the inhibitory activity results of RZ899009, RZ899011 and RZ599001 in Example 2 of the present disclosure on the target gene SOD1 in mice;

[0152] Figure 3 It is the graph of the inhibitory activity results of RZ899011, RZ899040 and RZ599001 in Example 3 of the present disclosure on the target gene SOD1 in mice;

[0153] Figure 4 It is the graph of the inhibitory activity results of RZ802011 and RZ502031 in Example 4 of the present disclosure on the target gene CC3 in mice;

[0154] Figure 5 It is the graph of the inhibitory activity results of RZ899048, RZ899011 and RZ599001 in Example 5 of the present disclosure on the target gene SOD1 in mice;

[0155] Figure 6 It is the graph of the inhibitory activity results of RZ899038, RZ899011 and RZ599001 in Example 6 of the present disclosure on the target gene SOD1 in mice;

[0156] Figure 7 It is the graph of the inhibitory activity results of RZ899041, RZ899042, RZ899038 and RZ599001 in Example 7 of the present disclosure on the target gene SOD1 in mice;

[0157] Figure 8a It is the graph of the AST results in the blood of mice after injecting RZ899011 and RZ899038 in Example 8 of the present disclosure;

[0158] Figure 8b It is the graph of the ALT results in the blood of mice after injecting RZ899011 and RZ899038 in Example 8 of the present disclosure;

[0159] Figure 8c It is the graph of the TBIL results in the blood of mice after injecting RZ899011 and RZ899038 in Example 8 of the present disclosure;

[0160] Figure 8dIt is the graph of Urea results in the blood after injecting RZ899011 and RZ899038 into mice in Example 8 of the present disclosure;

[0161] Figure 8e It is the graph of Crea results in the blood after injecting RZ899011 and RZ899038 into mice in Example 8 of the present disclosure;

[0162] Figure 8f It is the graph of TC results in the blood after injecting RZ899011 and RZ899038 into mice in Example 8 of the present disclosure;

[0163] Figure 8g It is the graph of TG results in the blood after injecting RZ899011 and RZ899038 into mice in Example 8 of the present disclosure;

[0164] Figure 8h It is the graph of WBC results in the blood after injecting RZ899011 and RZ899038 into mice in Example 8 of the present disclosure;

[0165] Figure 8i It is the graph of NEUT results in the blood after injecting RZ899011 and RZ899038 into mice in Example 8 of the present disclosure;

[0166] Figure 8j It is the graph of LYMPH results in the blood after injecting RZ899011 and RZ899038 into mice in Example 8 of the present disclosure;

[0167] Figure 8k It is the graph of the histopathological staining results of liver tissues after injecting RZ899011 and RZ899038 into mice in Example 8 of the present disclosure. Detailed implementation manners

[0168] The present disclosure discloses compounds, their preparation methods and uses. Those skilled in the art can draw on the content of this article and appropriately improve process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present disclosure. The methods and applications of the present disclosure have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate alterations and combinations to the methods and uses described herein without departing from the content, spirit and scope of the present disclosure to implement and apply the technology of the present disclosure.

[0169] The affinity between ASGPR and ligands is mainly affected by the following factors:

[0170] a. Type of the ligand terminal sugar molecule. Ligand molecules with Gal or GalNAc at the terminal can be recognized by ASGPR. The affinity of GalNAc for binding to ASGPR is 10 - 50 times higher than that of Gal, and GalNAc ligand molecules are more likely to escape the recognition of Kupffer cells and target hepatocytes more.

[0171] b. Sugar molecule substitution site: 1-OH, 2-OH (or 2-amino), 3-OH, 4-OH, and 5-CH2- on Gal (or GalNAc) are all involved in the binding of the ASGPR receptor; when 5-CH2- is linked with an electronegative group or 4-OH, 3-OH, 2-OH (or 2-amino) are substituted, the affinity with the receptor drops sharply; when 1-OH undergoes glycosylation substitution, when the glycosidic bond is in the α conformation, the affinity with the receptor weakens, and when it is in the β conformation and the substituent is a straight-chain structure, the affinity is not affected; only 6-OH is not involved in the receptor binding and points to the solvent region, so it is suitable as a linking site to connect with carriers, drugs, etc.

[0172] c. Spatial distance between the terminal glycosyl and the carrier. The glycosyl can be effectively recognized by ASGPR when there is at least a 6-CH2 unit interval between the glycosyl and the carrier.

[0173] In addition, Ligands with different molecular cluster structures , different connection methods between the oligonucleotide and the ligand will significantly affect the activity of the oligonucleotide in vivo. Higher activity means better therapeutic effects or lower dosing doses; at the same drug efficacy, lower dosing doses also mean lower toxicity reactions.

[0174] According to the above affinity characteristics of the ASGPR receptor, we designed a ligand with high affinity for ASGPR to achieve liver-targeted delivery of drugs or genes and achieve the purpose of regulating the expression of target genes.

[0175] Glossary of terms

[0176] The term represents the site where the group is covalently linked.

[0177] In the structural formula of the compound or the ligand described in the present disclosure, the bond "-" represents an unspecified configuration. If there are chiral isomers in the chemical structure, the bond "-" can be or simultaneously contain both configurations. Although all the above structural formulas are drawn in the form of certain isomers for simplicity, the present disclosure can include all isomers, such as: tautomers, rotamers, geometric isomers, diastereomers, racemates, and enantiomers.

[0178] Unless otherwise indicated, the following definitions apply to the terms used herein. For the purposes of this disclosure, chemical elements are in accordance with the CAS version of the Periodic Table of the Elements and the Handbook of Chemistry and Physics, 75th Edition, 1994. In addition, general principles of organic chemistry can be referred to the descriptions 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.

[0179] Unless otherwise specified or there is an obvious conflict in the context, the articles "a", "an", and "the" as used herein are intended to include "at least one" or "one or more". Thus, these articles as used herein refer to articles for one or more (i.e., at least one) objects. For example, "a component" refers to one or more components, that is, there may be more than one component considered to be adopted or used in the implementation of the described embodiment.

[0180] The term "comprising" is an open-ended expression, that is, it includes the content specified in this disclosure, but does not exclude other aspects.

[0181] "Stereoisomers" refer to compounds having the same chemical structure but different spatial arrangements of atoms or groups. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans isomers), atropisomers, and so on.

[0182] "Chiral" is a molecule with the property that it cannot be superimposed on its mirror image; while "achiral" refers to a molecule that can be superimposed on its mirror image.

[0183] "Enantiomers" refer to two isomers of a compound that cannot be superimposed but are mirror images of each other.

[0184] "Diastereomers" refer to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. Mixtures of diastereomers can be separated by high-resolution analytical operations such as electrophoresis and chromatography, such as HPLC.

[0185] The stereochemical definitions and rules used in this disclosure 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.

[0186] In the context of this disclosure, unless otherwise specified, “conjugation” means that two or more chemical moieties each having a specific function are connected to each other in a covalent linkage; correspondingly, a “conjugate” means a compound formed by covalent linkage between the respective chemical moieties. Further, a “drug conjugate” means a compound formed by covalently linking one or more chemical moieties with specific functions to an active drug. Hereinafter, sometimes, especially in the examples, the drug conjugates of this disclosure are also simply referred to as “conjugates”. A drug conjugate should be understood, according to the context, as the general term for drug conjugates or a specific drug conjugate represented by a specific structural formula.

[0187] Generally, the term “substituted” means that one or more hydrogen atoms in the given structure are replaced by specific substituents. 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 structural formula can be substituted by one or more substituents selected from a specific group, then the substituents can be the same or different at each substitutable position.

[0188] The term “unsubstituted” means that the designated group bears no substituent.

[0189] The term “optionally substituted by...” can be used interchangeably with the term “unsubstituted or substituted by...”, that is, the structure is unsubstituted or substituted by one or more substituents described in this disclosure. The substituents described in this disclosure 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, heterocyclic group, aryl, heteroaryl, and the like.

[0190] In addition, it should be noted that, unless otherwise explicitly specified, the description methods "each... independently is", "... each independently is", and "... independently is" adopted in this disclosure can be interchanged and should be understood in a broad sense. It can either mean that among different groups, the specific options expressed between the same symbols do not affect each other, or it can mean that within the same group, the specific options expressed between the same symbols do not affect each other. Taking R3 as an example, the specific options of R3 between the structural formula "optionally substituted C1-C50 alkylene group by R3" and the structural formula "optionally substituted -C(O)-NH-C1-50 alkylene group by R3" do not affect each other.

[0191] The term "Small interfering RNA (siRNA)" refers to a class of double-stranded RNA, which contains a sense strand and an antisense strand, and the length of each strand is 17 to 30 nucleotides. siRNA mediates the targeted cleavage of RNA transcripts in the RISC pathway by forming the RNA-induced silencing complex (RISC). Specifically, siRNA guides the specific degradation of mRNA sequences through the known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and the conversion into proteins.

[0192] In the context of this disclosure, "treat", "alleviate", or "improve" can be used interchangeably here. These terms refer to methods of obtaining beneficial or desired results, including but not limited to therapeutic benefits. "Therapeutic benefit" means eradicating or improving the underlying disorder being treated. Here, the therapeutic benefit is obtained by eradicating or improving one or more physiological symptoms associated with the underlying disorder, thereby observing an improvement in the subject, although the subject may still be suffering from the underlying disorder.

[0193] In the context of this disclosure, "prevent" and "prevent from" can be used interchangeably. These terms refer to methods of obtaining beneficial or desired results, including but not limited to preventive benefits. To obtain "preventive benefit", the conjugate, RNAi reagent, or composition can be administered to a subject at risk of developing a specific disease, or to a subject reporting one or more physiological symptoms of the disease, even if the diagnosis of the disease may not have been made.

[0194] The raw materials and reagents used in the compounds, their preparation methods, and uses provided in this disclosure are all commercially available.

[0195] The reagents and their sources used in this disclosure are as follows:

[0196] 1) Tin hydride tributyl, CAS No. 688-73-3, purchased from Beijing Coupling Technology Co., Ltd.;

[0197] 2) DL-10-Camphorsulfonic acid, CAS No. 5872-08-2, purchased from Beijing Coupling Technology Co., Ltd.;

[0198] 3) 3-Bromopropyne, CAS No. 106-96-7, purchased from Beijing Coupling Technology Co., Ltd.;

[0199] 4) 4,4'-Dimethoxytrityl chloride (abbreviated as DMTrCl), CAS No. 40615-36-9, purchased from Beijing Coupling Technology Co., Ltd.;

[0200] 5) 4,5-Dicyanoimidazole (abbreviated as DCI), CAS No. 1122-28-7, purchased from Beijing Coupling Technology Co., Ltd.;

[0201] 6) Bis(diisopropylamino)(2-cyanoethoxy)phosphine, CAS No. 102691-36-1, purchased from Beijing Coupling Technology Co., Ltd.;

[0202] 7) Potassium iodide (KI), CAS No. 7681-11-0, purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.;

[0203] 8) Trimethylsilyl azide (TMSN3), CAS No. 4648-54-8, purchased from Beijing Coupling Technology Co., Ltd.;

[0204] 9) Potassium fluoride (KF), CAS No. 7789-23-3, purchased from Beijing Coupling Technology Co., Ltd.;

[0205] 10) N,N-Dimethylformamide (DMF), CAS No. 68-12-2, purchased from Beijing Coupling Technology Co., Ltd.;

[0206] 11) D-Galactosamine pentaacetate, CAS No. 76375-60-5, purchased from Beijing Coupling Technology Co., Ltd.;

[0207] 12) Scandium trifluoromethanesulfonate, CAS No. 144026-79-9, purchased from Beijing Coupling Technology Co., Ltd.;

[0208] 13) Sodium hydride (NaH), CAS No. 7646-69-7, purchased from Beijing Coupling Technology Co., Ltd.;

[0209] 14) 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.;

[0210] 15) Aminoalkyl-CPG (model C3006-1000), purchased from Beijing Coupling Technology Co., Ltd.;

[0211] 16) Succinic anhydride, CAS No. 108-30-5, purchased from Beijing Coupling Technology Co., Ltd.

[0212] Unless otherwise specified, the reagents, consumables (Table 1) and instruments and equipment (Table 2) used in this disclosure are all commercially available products from the following manufacturers.

[0213] Table 1 Main reagents and consumables

[0214] Name Manufacturer 1×PBS Zhongke Maichen (Beijing) Technology Co., Ltd. Hanwei RNA Extraction Kit Zhejiang Hanwei Technology Co., Ltd. Reverse Transcription System Promega Corporation SYBR Select Master Mix ABI RNALater Thermo Fisher Scientific

[0215] Table 2 Main instruments and equipment

[0216]

[0217]

[0218] This disclosure provides a compound of formula (I) or its isomer:

[0219]

[0220] When R4 is selected from The synthetic routes of the compound of formula (I-1a), the compound of formula (I-1b) or the compound of formula (I-1c) are as follows:

[0221]

[0222] Using the compound of formula (1) as the starting material, through acetalization reaction, electrophilic substitution reaction and dehydroxy protection reaction in sequence, the compound of formula (I-1a) is obtained; the compound of formula (I-1a) undergoes nucleophilic substitution reaction to obtain the compound of formula (I-1b); the compound of formula (I-1a) undergoes condensation reaction to obtain the compound of formula (I-1c).

[0223] Preparation Example 1

[0224] When R4 is selected from p is selected from 1, q is selected from 0, and R3 is selected from H, the compound of formula (I-1a) NM041 compound, the compound of formula (I-1b) NM041 compound and the compound of formula (I-1c) NM041 compound are as follows:

[0225]

[0226] The compound of formula (I-1a) NM041 compound, the compound of formula (I-1b)NM041 Compound and formula (I-1c) NM041 The synthetic route of the compound is as follows:

[0227]

[0228] (1-1) Formula (1) NM041 Synthesis of the compound

[0229] The synthetic route of the compound of formula (1) is as follows:

[0230]

[0231] Using the compound of formula (1-a) as the starting material, through hydrodehalogenation reaction and hydrolysis reaction in sequence, the compound of formula (1) is obtained NM041 Compound

[0232] (1-1-1) Synthesis of the compound of formula (1-b)

[0233] Dissolve the compound of formula (1-a) (25.0 g, 1.0 eq) in 250 ml of toluene, heat up to 110 °C, add tributyltin hydride (17.7 g, 1.0 eq) and azobisisobutyronitrile (1 g, 0.1 eq), and reflux the reaction at 110 °C for 2 hours. After the reaction is completed, cool the reaction solution to 25 °C, add ethyl acetate (100 ml) and potassium fluoride (10.59 g in 30 ml of water, 3.0 eq), stir at 25 °C for 2 hours, filter the reaction solution and separate the organic phase. Wash the aqueous phase with 50 ml of ethyl acetate twice (50 ml × 2), combine the organic phases, wash the combined organic phase with 50 ml of saturated sodium chloride aqueous solution once (50 ml × 1), dry with anhydrous sodium sulfate and filter by suction, concentrate to obtain the compound of formula (1-b) as a pale yellow oil (18.7 g, yield 87.9%). MS ESI (m / z) = 333.1 [M+H] + .

[0234] (1-1-2) Formula (1) NM041 Synthesis of the compound

[0235] Dissolve the compound of formula (1-b) (18.7 g, 1 eq) in anhydrous methanol (50 ml), add sodium methoxide (0.288 g, 0.1 eq), and stir the reaction at 25 °C for 1 hour. After the reaction is completed, adjust the pH of the reaction solution to 6-7 with a 1,4-dioxane solution of 4 mol / L hydrogen chloride in an ice bath, concentrate, azeotrope with acetonitrile twice, and then dry in vacuo to obtain the compound of formula (1) NM041 Compound (9.4 g, yield 100%).

[0236] (1-2) Formula (3) NM041Synthesis of Compounds

[0237] Dissolve the compound of formula (1) NM041 The compound (9.4 g, 1.0 eq) was dissolved in 150 ml of acetonitrile. The compound of formula (2) (30 ml, 3 eq, benzaldehyde dimethyl acetal) and DL-10-camphorsulfonic acid (1.5 g, 0.1 eq) were added respectively. The mixture was stirred at 25 °C for 5 hours, 3 ml of triethylamine was added, and then stirred at 25 °C for 30 min. After the reaction was completed, the reaction solution was concentrated. 100 ml of water was added to the reaction solution, and then extracted twice with 100 ml of ethyl acetate (100 ml × 2). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated and purified by normal-phase column chromatography (eluent: ethyl acetate / petroleum ether = 57 / 43, v / v) to obtain the compound of formula (3) in the form of a white solid NM041 compound (8.6 g, yield 59.5%). MS ESI (m / z) = 252.2 [M+H] + .

[0238] (1 - 3) Formula (5) NM041 Synthesis of Compounds

[0239] Dissolve the compound of formula (3) (4.3 g, 1.0 eq) in 40 ml of DMF. Sodium hydride (2.7 g, 4 eq) was added under an ice bath and reacted for 30 minutes under the ice bath. The compound of formula (4) (8.1 g, 4 eq, 3-bromopropyne) was added, and the mixture was stirred at 25 °C for 2 hours. 20 ml of water was added to quench the reaction. After the reaction was completed, the reaction solution was extracted three times with 50 ml of ethyl acetate (50 ml × 3). The organic phases were combined, washed five times with 20 ml of saturated sodium chloride solution (20 ml × 5), dried over anhydrous sodium sulfate and filtered, and then concentrated to obtain the compound of formula (5) in the form of a brown oil NM041 compound (yield 100%). MS ESI (m / z) = 329.2 [M+H] + .

[0240] (1 - 4) Formula (I - 1a) NM041 Synthesis of Compounds

[0241] Dissolve the compound of formula (5) NM041 compound (5.6 g, 1.0 eq) in 30 ml of DCM. 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 the compound of formula (I - 1a) in the form of a yellow oil NM041 compound (yield 100%). MS ESI (m / z) = 241 [M+H] + .

[0242] (1 - 5) Formula (I - 1b) NM041 Synthesis of Compounds

[0243] Compound of formula (I-1a) NM041 The compound (5.6 g, 1.0 eq) was dissolved in 50 ml of pyridine. Under an ice bath, the compound of formula (6) (10.2 g, 30.16 mmol, 1.3 eq, 4,4'-dimethoxytriphenyl chloromethane, abbreviated as DMTrCl) was added. The reaction system was purged with nitrogen three times and stirred at 25 °C for 3 hours. Then 50 ml of methanol was added to quench the reaction. After the reaction was completed, the reaction solution was concentrated, 50 ml of water was added, and the mixture was extracted with 50 ml of ethyl acetate three times (50 ml×3). The organic phases were combined, dried over anhydrous sodium sulfate and filtered by suction, concentrated, and purified by normal-phase column chromatography (eluent: ethyl acetate / ether = 16 / 84, v / v) to obtain the compound of formula (I-1b) as a pale yellow solid NM041 Compound (5 g, yield 39.6%). MS ESI (m / z) = 543.1 [M+H] + .

[0244] (1-6) Compound of formula (I-1c) NM041 Synthesis of the compound

[0245] The compound of formula (I-1b) NM041 The compound (2.0 g, 1.5 eq) was dissolved in 20 ml of anhydrous dichloromethane. 4,5-Dicyanoimidazole (347.4 mg, 0.8 eq) and the compound of formula (7) (1.22 g, 1.1 eq, bis(diisopropylamino)(2-cyanoethoxy)phosphine) were added respectively. The reaction system was purged with nitrogen three 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, and the mixture was extracted with 20 ml of dichloromethane three times (20 ml×3). The organic phases were combined, dried over anhydrous sodium sulfate and filtered, concentrated, and purified by reverse-phase column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v), and dried under vacuum for 12 hours to obtain the compound of formula (I-1c) as a white powder NM041 Compound (2 g, yield 73.09%). MS ESI (m / z): 743.2 [M+H] + .

[0246] 11H NMR (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).

[0247] Preparation Example 2

[0248] When R4 is selected from in the case where p is 1, q is 0, and R3 is methoxy, formula (I-1a) NM064 compound, formula (I-1b) NM064 compound and formula (I-1c) NM064 compounds are as follows:

[0249]

[0250] Formula (I-1a) NM064 compound, formula (I-1b) NM064 compound and formula (I-1c) NM064 The synthetic routes of the compounds are as follows:

[0251]

[0252] (2-1) Formula (3) NM064 Synthesis of the compound

[0253] For formula (1) NM064Compound (13 g, 1.0 eq) was dissolved in 150 ml of acetonitrile. Compound of formula (2) (30 ml, 3.0 eq, benzaldehyde dimethyl acetal) and DL-10-camphorsulfonic acid (1.5 g, 0.1 eq) were added respectively. The mixture was stirred at 25 °C for 5 hours, 3 ml of triethylamine was added, and the mixture was stirred at 25 °C for 30 min. After the reaction was completed, the reaction solution was concentrated, 100 ml of water was added, and the mixture was extracted twice with 100 ml of ethyl acetate (100 ml × 2). The organic phases were combined, dried over anhydrous sodium sulfate and filtered, concentrated, and purified by normal-phase purification on a purification column (eluent: ethyl acetate / petroleum ether = 57 / 43, v / v) to obtain the compound of formula (3) in the form of a white solid NM064 Compound (11.5 g, yield 60.8%). MS ESI (m / z) = 283 [M+H] + .

[0254] (2-2) Formula (5) NM064 Synthesis of the compound

[0255] Compound of formula (3) NM064 (5 g, 1.0 eq) was dissolved in 40 ml of N,N-dimethylformamide. Sodium hydride (2.7 g, 4 eq) was added under ice bath, and the reaction was carried out under ice bath for 30 minutes. Compound of formula (4) (8.1 g, 4 eq, 3-bromopropyne) was added, and the mixture was stirred at 25 °C for 2 hours. 20 ml of water was added to quench the reaction. After the reaction was completed, the reaction solution was extracted three times with 50 ml of ethyl acetate (50 ml × 3). The organic phases were combined, washed five times with 20 ml of saturated sodium chloride solution (20 ml × 5), dried over anhydrous sodium sulfate and filtered, concentrated, to obtain the compound of formula (5) in the form of a brown oil NM064 Compound (6.3 g, yield 100%). MS ESI (m / z) = 359 [M+H] + .

[0256] (2-3) Formula (I-1a) NM064 Synthesis of the compound

[0257] Compound of formula (5) NM064 (6.3 g, 17 mmol, 1.0 eq) was dissolved in 30 ml of dichloromethane. 300 ml of 70% by 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 the compound of formula (I-1a) in the form of a yellow oil NM064 Compound (4.78 g, yield 100%). MS ESI (m / z) = 271 [M+H] + .

[0258] (2-4) Formula (I-1b) NM064 Synthesis of the compound

[0259] Compound of formula (I-1a)NM064 The compound (4.78 g, 17.7 mmol, 1.0 eq) was dissolved in 50 ml of pyridine. The compound of formula (6) (7.8 g, 23.0 mmol, 1.3 eq, DMTrCl) was added under an ice bath. The reaction system was purged with nitrogen three times and stirred at 25 °C for 3 hours, then quenched with 50 ml of methanol. After the reaction was completed, the reaction solution was concentrated, 50 ml of water was added, and the mixture was extracted three times with 50 ml of ethyl acetate (50 ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (eluent: ethyl acetate / petroleum ether = 16 / 84, v / v) to obtain the compound of formula (I-1b) as a pale yellow solid NM064 Compound (6.7 g, yield 66.3%). MS ESI (m / z) = 573 [M+H] + .

[0260] (2 - 5) Formula (I-1c) NM064 Synthesis of the compound

[0261] The compound of formula (I-1b) NM064 The compound (2.0 g, 1.0 eq) was dissolved in 20 ml of anhydrous dichloromethane. DCI (330.4 mg, 0.8 eq, 4,5-dicyanoimidazole) and the compound of formula (7) (1.16 g, 1.1 eq, bis(diisopropylamino)(2-cyanoethoxy)phosphine) were added respectively. The reaction system was purged with nitrogen three times and stirred at 25 °C for 2 hours. After the reaction was completed, 20 ml of saturated aqueous sodium bicarbonate 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 (eluent: acetonitrile / water = 72 / 28, v / v), and dried in vacuo for 12 hours to obtain the compound of formula (I-1c) as a white powder NM064 Compound (2 g, yield 74.07%). MS ESI (m / z) = 774 [M+H] + .

[0262] 11H NMR (400 MHz, DMSO-d6) δ 7.45 - 7.39 (d, J = 7.8 Hz, 2H), 7.36 - 7.18 (tt, J = 14.5, 8.5 Hz, 7H), 6.95 - 6.84 (d, J = 7.5 Hz, 4H), 5.01 - 4.96 (s, 1H), 4.43 - 4.28 (s, 4H), 3.81 - 3.70 (s, 8H), 3.65 - 3.35 (m, 12H), 3.28 - 3.18 (dt, J = 14.5, 7.2 Hz, 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, 6.8 Hz, 10H), 0.84 - 0.78 (d, J = 6.6 Hz, 2H).

[0263] When R4 is selected from the synthetic route of the compound of formula (I-2) is as follows:

[0264]

[0265] Starting from the compound of formula (3), through electrophilic substitution reaction, dehydroxy protection reaction, nucleophilic substitution reaction and hydrolysis reaction in sequence, the compound of formula (I-2) is obtained.

[0266] Preparation Example 3

[0267] When R4 is selected from p is selected from 1, q is selected from 0, and R3 is selected from H, the synthetic route of the compound of formula (I-2) is as follows:

[0268]

[0269] (3-1) Synthesis of the compound of formula (9)

[0270] Using the compound of formula (3) NM041The compound (4.3 g, 17.0 mmol, 1.0 eq) was dissolved in 50 ml of ultra-dry DMF. NaH (2.7 g, 68 mmol, 4.0 eq) was added under an 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 the reaction was carried out at 25 °C for 3 hours. The reaction was quenched by adding saturated ammonium chloride aqueous solution. After the reaction was completed, 20 ml of water was added to the reaction solution, and it was extracted with 50 ml of ethyl acetate three times (50 ml × 3). The organic phases were combined, washed with 20 ml of saturated sodium chloride aqueous solution ten times (20 ml × 10), dried over anhydrous sodium sulfate and filtered, concentrated to obtain the compound of formula (9) as a dark brown oil, which was directly used for the next step. MS ESI (m / z) = 425.2 [M + H] + .

[0271] (3 - 2) Synthesis of the compound of formula (10)

[0272] The compound of formula (9) (5 g, 1.0 eq) was dissolved in 30 ml of dichloromethane, and 300 ml of 70 mass% aqueous acetic acid solution was added. The mixture was stirred at 50 °C for 2 hours. After the reaction was completed, the reaction solution was directly concentrated to obtain the compound of formula (10) as a dark brown oil, which was directly used for the next step. MS ESI (m / z) = 337 [M + H] + .

[0273] (3 - 3) Synthesis of the compound of formula (11)

[0274] The compound of formula (10) (3.2 g, 9.52 mmol, 1.0 eq) was dissolved in 70 ml of pyridine. The compound of formula (6) (4.19 g, 12.4 mmol, 1.3 eq, DMTrCl) was added under an ice bath, and the mixture was stirred at 25 °C for 3 hours. The reaction was quenched by adding 30 ml of methanol. After the reaction was completed, the reaction solution was concentrated, 20 ml of water was added, and it was extracted with 50 ml of ethyl acetate three times (50 ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate and filtered, concentrated, and purified by reverse-phase column chromatography (C18 chromatographic column, eluent: acetonitrile / water = 52 / 48, v / v) to obtain the compound of formula (11) as a yellow solid (5 g, yield 83%). MS ESI (m / z) = 639 [M + H] + .

[0275] (3 - 4) Synthesis of the compound of formula (I - 2)

[0276] Dissolve the compound of formula (11) (2g, 3.16 mmol, 1.0 eq) in 10 ml of methanol, add an aqueous NaOH solution (6.3 ml, 1 mol / L), and stir at 25 °C for 1 hour. After the reaction is completed, adjust the pH of the reaction solution to neutral with hydrochloric acid, filter the reaction solution, concentrate the filtrate, and purify it by reverse-phase column chromatography (C18 chromatographic column, eluent: acetonitrile / water = 26 / 74, v / v) to obtain the compound of formula (I-2) as a pale yellow solid (920 mg, yield 51%). MS ESI (m / z) = 583 [M+H] + .

[0277] Preparation Example 4: Synthesis of Compounds CR01022 and CR01022Z (a cluster of CR01022, denoted as (CR01022)×1)

[0278] In this preparation example, the synthetic routes of compounds CR01022 and CR01022Z are as follows:

[0279]

[0280] (4-1) Synthesis of compound NM015

[0281]

[0282] Add compound NM015-1 (5g, 1.1 eq.) and D-galactosamine pentaacetate (8.07g, 1.0 eq.) to 96 ml of dichloroethane, heat to 85 °C, add scandium trifluoromethanesulfonate (0.38g, 0.77 mmol) in portions at 85 °C, stir at 85 °C for 3 hours, and add 16 ml of triethylamine to quench. After the reaction is completed, cool the reaction system to 25 °C, add 100 ml of water and 100 ml of dichloromethane to the reaction solution, filter, separate the organic phase, dry the organic phase with anhydrous sodium sulfate and filter, concentrate, and purify it by reverse-phase column chromatography (C18 chromatographic column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound NM015 as an oil (6.6g). ESI-MS (m / z) = 549 [M+H] + .

[0283] (4-2) Synthesis of compound CR01022-1

[0284] Add compound NM015 (1114 mg, 2.03 mmol, 2.2 eq.) and the compound of formula (I-1b) NM041Compound (500 mg, 0.922 mmol, 1.0 eq.), copper(I) iodide (701 mg, 3.69 mmol, 4.0 eq.), N,N-diisopropylethylamine (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 h. After the reaction was completed, 5 ml of ethyl acetate and 5 ml of water were added to the reaction solution. The mixture was filtered and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse-phase column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain the compound CR01022-1 as a foam (1 g). ESI-MS (m / z) = 1639 [M+H] + .

[0285] (4-3) Synthesis of compound CR01022

[0286] Compound CR01022-1 (850 mg, 0.523 mmol, 1.0 eq.) was added to 17 ml of dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (313 mg, 1.04 mmol, 2.0 eq.) was added in portions first, and then 4,5-dicyanoimidazole (49 mg, 0.415 mmol, 0.8 eq.) was added. The reaction system was purged with nitrogen three times and stirred at 25 °C for 1 h. After the reaction was completed, the reaction solution was washed once with saturated aqueous sodium bicarbonate solution. The organic phase was separated, concentrated, and purified by reverse-phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01022 (560 mg). MS ESI (m / z) = 1839 [M+H] + .

[0287] (4-4) Synthesis of compound CR01022Z

[0288] Compound CR01022-1 (160 mg, 0.1 mmol, 1.0 eq.) was added to 3.2 ml of dichloromethane in a 25-ml reaction kettle. 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. The reaction system was purged with nitrogen three times and stirred at 25 °C for 16 h. After the reaction was completed, it was purified by reverse-phase column chromatography (C18 column, acetonitrile / water = 72 / 28) to obtain the intermediate product (100 mg).

[0289] The above intermediate product (100 mg, 0.058 mmol, 1.0 eq.), amino CPG (1.44 g, 0.115 mmol, 80 μmol / g), benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate (32.67 mg, 0.086 mmol, 1.5 eq.), and N,N-diisopropylethylamine (14.8 mg, 0.115 mmol, 2.0 eq.) were mixed and shaken on a shaker for 16 hours. The reaction solution was filtered, and 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 on a shaker for 6 hours, the reaction solution was filtered, and 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 μmol / g).

[0290] Among them, Cap1 and Cap2 are capping reagent solutions. Cap1 is a pyridine / acetonitrile mixed solution of 20% N-methylimidazole, and the volume ratio of pyridine to acetonitrile is 3:5; Cap2 is an acetonitrile solution of 20% acetic anhydride.

[0291] Preparation Example 5: Synthesis of Compound CR01027 and Compound CR01027Z (a cluster of CR01027, denoted as (CR01027)×1) Synthesis

[0292] In this preparation example, the synthesis processes of compound CR01027 and compound CR01027Z are as follows:

[0293]

[0294] (5-1) Synthesis of compound CR01027-1

[0295] Compound NM015 (1125 mg, 2.03 mmol, 2.2 eq.), the compound of formula (I-1b) NM064 compound (500 mg, 0.922 mmol, 1.0 eq.), copper(I) iodide (701 mg, 3.69 mmol, 4.0 eq.), and N,N-diisopropylethylamine (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 solution, filtered, the organic phase was separated, the organic phase was dried with anhydrous sodium sulfate and concentrated, and purified by reverse-phase column chromatography (C18 chromatographic column, acetonitrile / water = 72 / 28, v / v) to obtain a foamy compound CR01027-1 (1 g, yield 68.5%). ESI-MS (m / z): 1669.7 [M+H] + .

[0296] (Synthesis of Compound CR01027)

[0297] 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 first, and then 4,5-dicyanoimidazole (52 mg, 0.415 mmol, 0.8 eq.) was added. The mixture was purged with nitrogen three times and stirred at 25 °C for 1 hour. After the reaction was completed, the reaction mixture was washed once with an aqueous sodium bicarbonate solution, the organic phase was separated, concentrated, and purified by reverse-phase 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] + .

[0298] (Synthesis of Compound CR01027Z)

[0299] Compound CR01027Z was prepared according to the synthesis method of compound CR01022Z in Preparation Example 4.

[0300] Preparation Example 6: Synthesis of Compound CR01017 and Compound CR01017Z (a cluster of CR01017, denoted as (CR01027)×1) Synthesis

[0301] In this preparation example, the synthesis processes of compound CR01017 and compound CR01017Z are as follows:

[0302]

[0303] (Synthesis of Compound CR01017-1)

[0304] The compound of formula (I-2) (900 mg, 1.55 mmol, 1 eq.), (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-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. DIEA (800 mg, 6.2 mmol, 4.0 eq) was added under an ice bath, and the mixture was stirred at 25 °C for 12 hours. After the reaction was completed, it was purified by reverse-phase column chromatography (C18 column, eluent: acetonitrile / water = 20 / 80, v / v) to obtain compound CR01017-1 in the form of a white solid (700 mg, yield 28.6%). MS ESI (m / z) = 1591.2 [M+H] + .

[0305] (6-2) Synthesis of Compound CR01017

[0306] Dissolve compound CR01017 (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) in 10 ml of ultradry dichloromethane, and react at 25 °C for 2 hours under a nitrogen atmosphere. After the reaction is completed, wash the reaction solution twice with 10 ml of saturated sodium bicarbonate (10 ml×2), concentrate the organic phase, and purify by reverse-phase column chromatography (C18 column, eluent: acetonitrile / water = 80 / 20, v / v) to obtain compound CR01017 in the form of a white solid (480 mg, yield 83.1%). MS ESI (m / z) = 1791.2 [M+H] + .

[0307] (6-3) Synthesis of Compound CR01017-2

[0308] Dissolve compound CR01017-1 (200 mg, 0.13 mmol, 1.0 eq) in 10 ml of dichloromethane, add succinic anhydride (14 mg, 0.14 mmol, 1.1 eq) and triethylamine (26 mg, 0.26 mmol, 2.0 eq), and stir at 25 °C for 12 hours. After the reaction is completed, concentrate the reaction solution, and purify by reverse-phase column chromatography (C18 column, eluent: acetonitrile / water = 36 / 64, v / v) to obtain compound CR01017-2 in the form of a white solid (150 mg, yield 71.4%). MS ESI (m / z) = 1691.2 [M+H] + .

[0309] (6-4) Synthesis of Compound CR01017Z

[0310] Dissolve compound CR01017-2 (70 mg, 0.041 mmol, 1.0 eq), benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate (22 mg, 0.062 mmol, 1.5 eq, abbreviated as HBTU), and N,N-diisopropylethylamine (10 mg, 0.082 mmol, 2.0 eq) in 10 ml of acetonitrile, stir at 25 °C for 5 minutes, add amino CPG (970 mg, 80 umol / g), react on a shaker at 25 °C for 18 hours, filter, wash the filter cake twice with 10 ml of dichloromethane (10 ml × 2) and then twice with 10 ml of acetonitrile (10 ml × 2). Mix the filter cake, DMAP (3 mg, 0.024 mmol), Cap1 (10 ml), and Cap2 (1 ml), react on a shaker at 25 °C for 5 hours, filter, rinse the filter cake 3 times with 10 ml of acetonitrile, and dry to obtain compound CR01017Z (720 mg).

[0311] Among them, Cap1 and Cap2 are capping reagent solutions. Cap1 is a pyridine / acetonitrile mixed solution containing 20 vol% N-methylimidazole, and the volume ratio of pyridine to acetonitrile is 3:5; Cap2 is an acetonitrile solution containing 20 vol% acetic anhydride.

[0312] Preparation Example 9: Synthesis of Reference Compound NM042

[0313] In this preparation example, the synthesis process of compound NM042 is as follows:

[0314]

[0315] (7-1) Synthesis of compound NM042-2

[0316] Add compound NM042-1 (5.0 g, 1.0 eq.) and sodium hydride (3.78 g, 2.5 eq.) to 60 ml of DMF, cool down to 0 °C, stir at 0 °C for 0.5 hour, slowly add propargyl bromide (9.0 g, 2.0 eq.) during stirring, and stir at 25 °C for 4 hours. After the reaction is completed, add 100 ml of water to the reaction solution, extract three times with ethyl acetate (3 × 100 ml), combine the organic phases, wash the organic phase once with 100 ml of saturated brine, dry with anhydrous sodium sulfate and filter, and concentrate to obtain oily compound NM042-2 (9.0 g). ESI-MS (m / z) = 171 [M + H] + .

[0317] (7-2) Synthesis of compound NM042-3

[0318] 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. The mixture was purged with nitrogen three times and stirred at 25 °C for 6 hours. After the reaction was completed, the reaction solution was filtered and concentrated to obtain oily compound NM042-3 (9.1 g). ESI-MS (m / z) = 131.2 [M+H] + .

[0319] (7-3) Synthesis of compound NM042-4

[0320] Compound NM042-3 (5 g, 1.0 eq.) was added to 75 ml of pyridine solution. The temperature was lowered to 0 °C, and DMTrCl (16.9 g, 1.69 eq.) was added portionwise at 0 °C. The mixture was stirred at 25 °C for 3 hours. After the reaction was completed, the reaction solution was concentrated and purified by reverse-phase column chromatography (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] + .

[0321] (7-4) Synthesis of compound NM042

[0322] Compound NM042-4 (2.5 g, 1.0 eq.), bis(diisopropylamino)(2-cyanoethoxy)phosphine (3.5 g, 2.0 eq.), and DCI (0.567 g, 0.8 eq.) were respectively added to 50 ml of DCM. The mixture was stirred under nitrogen protection for 3 hours. After the reaction was completed, the reaction solution was washed once with 50 ml of saturated sodium bicarbonate solution. The organic phase was separated, washed once with 50 ml of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by reverse-phase HPLC to obtain white oily compound NM042 (2.0 g, yield 56%). ESI-MS (m / z): 619.3 [M+H] + .

[0323] 1 H NMR (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).

[0324] Preparation Example 10: Synthesis of Reference Compound CR01023 and Compound CR01023Z

[0325] In this preparation example, the synthesis processes of compound CR01023 and compound CR01023Z are as follows:

[0326]

[0327] (8-1) Synthesis of compound CR01023-1

[0328] Compound NM015 (2.79 g, 5.08 mmol, 2.2 eq.), compound NM042-4 (1.0 g, 2.31 mmol, 1.0 eq.), copper(I) iodide (1.76 g, 9.24 mmol, 4.0 eq.), and N,N-diisopropylethylamine (1.19 g, 9.24 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. Then, 15 ml of ethyl acetate and 15 ml of water were added. The mixture was filtered, and the layers were separated. The organic layer was dried over anhydrous sodium sulfate and filtered, concentrated, and purified by reverse-phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain a foamy compound CR01023-1 (1.6 g, yield 70.5%). ESI-MS (m / z) = 980.2 [M+H] + .

[0329] (8-2) Synthesis of compound CR01023

[0330] Compound CR01023-1 (1.0 g, 1.02 mmol, 1.0 eq.) was added to 20 ml of dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.61 g, 2.04 mmol, 2.0 eq.) was added in portions first, and then 4,5-dicyanoimidazole (96 mg, 0.82 mmol, 0.8 eq.) was added. The reaction system was purged with nitrogen three times and stirred at 25 °C for 1 hour. After the reaction was completed, the reaction mixture was washed once with an aqueous sodium bicarbonate solution. The organic layer was separated, concentrated, and purified by reverse-phase 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] + .

[0331] (8-3) Synthesis of compound CR01023Z

[0332] Compound CR01023Z was prepared according to the synthesis method of compound CR01022Z in Preparation Example 4.

[0333] Reference Compound L96-PS

[0334] Compound L96-PS was purchased from CITIC Pharmaceutical Group (Tianjin) Co., Ltd., with a loading of 120 ± 12 μmol / g (the detection method is UV / HPLC).

[0335] The structural formula of the reference compound L96-PS is as follows:

[0336]

[0337] Among them, PS represents the polystyrene resin solid-phase support.

[0338] Preparation Example 9: Method 1 for the Synthesis of siRNA Conjugates

[0339] (9-1) Synthesis of the sense strand (SS)

[0340] By the method of phosphoramidite nucleic acid solid-phase synthesis, using the above compounds (i.e., CR01022Z, CR01023Z, CR01027Z, L96-PS) linked to the solid-phase support to initiate the cycle, nucleotide monomers are sequentially linked one by one in the 3'-5' direction according to the nucleotide sequence (compounds CR01022, CR01023, and CR01027 can be regarded as one nucleotide monomer respectively). Each linkage of a nucleotide monomer includes four steps of deprotection, coupling, capping, and oxidation or sulfidation. The synthesis conditions are given as follows:

[0341] The nucleotide monomer is formulated into an acetonitrile solution of the nucleotide monomer with a concentration of 0.1M.

[0342] The conditions for each deprotection reaction are the same. The conditions for the deprotection reaction: the temperature is 25°C, the reaction time is 70 seconds, the deprotection reagent is a dichloromethane solution of dichloroacetic acid (3 vol%), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid-phase support is 5:1.

[0343] The conditions for each coupling reaction are the same. The conditions for the coupling reaction are: the temperature is 25°C, the molar ratio of the nucleic acid sequence linked to the solid-phase support to the nucleotide monomer is 1:10, the molar ratio of the nucleic acid sequence linked to the solid-phase support and the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is an acetonitrile solution of 5-ethylthio-1H-tetrazole with a concentration of 0.5M, and the sulfurizing reagent is an acetonitrile / pyridine mixed solution (the volume ratio of acetonitrile and pyridine is 1:1) of hydrogencyanate with a concentration of 0.2M.

[0344] The conditions for each capping reaction are the same. The conditions for the capping reaction are as follows: the temperature is 25 °C; the reaction time is 2 minutes; the capping reagent solution is a mixed solution of Cap1 and Cap2 with a molar ratio of 1:1. Cap1 is a pyridine / acetonitrile mixed solution of N-methylimidazole with a concentration of 20% by volume, and the volume ratio of pyridine to acetonitrile is 3:5. Cap2 is an acetonitrile solution of acetic anhydride with a concentration of 20% by volume. The molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent to the nucleic acid sequence linked to the solid support is 1:1:1.

[0345] The conditions for each oxidation reaction are the same. The conditions for the oxidation reaction are as follows: the temperature is 25 °C; the reaction time is 3 seconds; the concentration of the oxidation reagent is 0.05 M iodine water, and the molar ratio of iodine to the nucleic acid sequence linked 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 as follows: the temperature is 25 °C; the reaction time is 360 seconds; the concentration of the thio reagent is a pyridine solution of 0.2 M hydrogen xanthate, and the molar ratio of the thio reagent to the nucleic acid sequence linked 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).

[0346] After the connection of the last nucleoside monomer is completed, the nucleic acid sequence linked to the solid support is successively subjected to cleavage, deprotection, purification, and desalting, and then freeze-dried to obtain the sense strand, where:

[0347] The cleavage and deprotection conditions are as follows: The synthesized nucleotide sequence linked to the solid support is added to ammonia water with a concentration of 25% by mass, and the amount of ammonia water used is 0.5 ml / μmol. The reaction is carried out at 55 °C for 16 hours, the solvent is removed, and it is concentrated to dryness under vacuum. After the ammonia treatment, relative to the amount of single-stranded nucleic acid, the product is dissolved in 0.4 ml / μmol N-methylpyrrolidone, and then 0.3 ml / μmol triethylamine and 0.6 ml / μmol triethylamine trihydrofluoride are added to remove the 2'-O-TBDMS protection on the ribose.

[0348] Purification and desalting conditions: The nucleic acid was purified by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, Eluent 1 was 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (volume ratio of water to acetonitrile was 9:1); Eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (volume ratio of water to acetonitrile was 9:1); the elution gradient was Eluent 1:Eluent 2 = (100:0)-(50:50). After collecting and combining the product eluate, reverse chromatography purification column was used for desalting. The desalting conditions included using a Sephadex column for desalting, with the packing material being Sephadex G25, and eluting with deionized water.

[0349] Detection: Ion exchange chromatography (IEX-HPLC) was used for purity detection; a liquid chromatography-mass spectrometry instrument (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier) was used for molecular weight detection. The measured molecular weight was compared with the theoretical value. If the measured value was consistent with the theoretical value, it indicated that the compound was conjugated to the 3'-end of the sense strand of siRNA.

[0350] During the synthesis of the sense strand, the following targeting ligands (vectors) were synthesized respectively:

[0351] The structural formula of two clusters of CR01017 (denoted as (CR01017)×2) is:

[0352]

[0353] The structural formula of two clusters of CR01022 (denoted as (CR01022)×2) is:

[0354]

[0355] The structural formula of three clusters of CR01022 (denoted as (CR01022)×3) is:

[0356]

[0357] The structural formula of three clusters of CR01023 (denoted as (CR01023)×3) is:

[0358]

[0359] The structural formula of two clusters of CR01027 (denoted as (CR01027)×2) is:

[0360]

[0361] Synthesis of Antisense Strand (AS)

[0362] The antisense strand was synthesized using a universal solid support. The conditions and steps for deprotection, coupling, capping, oxidation or sulfidation reactions, cleavage and deprotection, purification and desalting in the solid-phase synthesis method of the antisense strand were the same as those for synthesizing the sense strand in step (9-1).

[0363] Detection: Purity detection was performed using ion exchange chromatography (IEX-HPLC); molecular weight detection was performed using liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). The measured value of the molecular weight was compared with the theoretical value. If the measured value and the theoretical value were consistent, it indicated that the siRNA antisense strand was obtained.

[0364] (9-3) Synthesis of siRNA Conjugate

[0365] The sense strand synthesized in step (9-1) and the antisense strand synthesized in step (9-2) were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. Then, it was slowly cooled to room temperature and maintained at room temperature for 10 minutes to allow the sense strand and the antisense strand to form a double-stranded structure through hydrogen bonds, thereby obtaining the target-designed siRNA conjugate.

[0366] After diluting each siRNA conjugate to a concentration of 0.2 mg / ml (calculated based on siRNA) using ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2 MΩ·cm (25°C)), molecular weight detection was performed using liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). If the measured value was consistent with the theoretical value, it indicated that the synthesized conjugate was the target-designed double-stranded nucleic acid sequence.

[0367] Preparation Example 10: Method 2 for the Synthesis of siRNA Conjugates

[0368] In this preparation example, the siRNA, linker groups (IC-NM041 and NM042), and ligand group (NM015) were sequentially connected to form the siRNA conjugate.

[0369] Taking the linker group IC-NM041 and the ligand group NM015 as examples, the synthesis route of the siRNA conjugate is shown as follows:

[0370]

[0371] (10-1) Synthesis of Sense Strand (SS)

[0372] Specifically, the preparation method of the sense strand includes the following process:

[0373] S1. Regarding the compound of formula (I-1c) NM041 as a nucleoside monomer, and connecting the compound of formula (I-1c) NM041 to a solid-phase carrier by the phosphoramidite solid-phase synthesis method, and then connecting nucleoside monomers one by one in the 3' to 5' direction according to the nucleotide types and sequences of the sense strand of the above siRNA. The connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The reaction conditions of deprotection, coupling, capping, oxidation or sulfurization in the synthesis of the sense strand are the same as those in step (9-1) of Preparation Example 9 for synthesizing the sense strand.

[0374] Isolate the sense strand semi-conjugate containing the linking group (denoted as SS-(NM041)×2). Among them, the linking group NM041 is connected to the 3' end of the siRNA sense strand.

[0375] Specifically, the preparation method of the sense strand includes the following process:

[0376] Step 1. Take 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) and mix them to obtain a mixed solvent; dissolve SS-(NM041)×2 in this mixed solvent to obtain a SS-(NM041)×2 solution with a concentration of 1.0 eq.

[0377] Step 2. Take 6.0 eq of ligand compound NM015 and dissolve it with 70 μL of DMF to obtain an NM015 solution

[0378] Step 3. Mix the SS-(NM041)×2 solution obtained in Step 1 and the NM015 solution obtained in Step 2 to obtain a reaction mixture.

[0379] Step 4. Take 10.0 eq of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and 3.0 eq of CuSO4·5H2O and mix them according to the volume ratio THPTA:CuSO4·5H2O = 10:3. After shaking at 40 °C for 5 min, take 37 μL and add it to the reaction mixture obtained in Step 3 above, and perform vortex shaking to obtain an intermediate mixture. Measure the pH of this intermediate mixture to be 8.

[0380] Step 5. Quickly add 25.0 eq of sodium ascorbate to the intermediate mixture obtained in Step 4 and perform vortex shaking treatment. React at 40 °C for 1 h to obtain a product mixture.

[0381] Step 6. Purification: Take 3 μL of the product mixture obtained in Step S3, dilute the product mixture with a mixed solution of DMF and H2O (the volume ratio of DMF to H2O is 1:5), and then perform separation and purification on it by HPLC treatment. Among them, the chromatographic column used in the HPLC treatment process is a C18 chromatographic column, the mobile phase is an ammonium bicarbonate buffer solution, and gradient elution is used. The product after HPLC treatment and purification is subjected to freeze-drying treatment.

[0382] (10-2) Synthesis of antisense strand (AS):

[0383] It is prepared according to the synthesis method of the antisense strand (AS) in (9-2) of Preparation Example 9.

[0384] (10-3) Synthesis of siRNA conjugate:

[0385] It is prepared according to the synthesis method of the siRNA conjugate in (9-3) of Preparation Example 9.

[0386] In the two methods for synthesizing siRNA conjugates provided by the present disclosure, in the first synthesis method of siRNA conjugates, the vector is synthesized first, then the sense strand conjugate is synthesized, and finally the siRNA conjugate is synthesized; in the second synthesis method of siRNA conjugates, the sense strand semi-conjugate is synthesized first, then the sense strand conjugate is synthesized, and finally the siRNA conjugate is synthesized.

[0387] Among them, when synthesizing the same amount of siRNA conjugate, compared with the second synthesis method, the first synthesis method uses less raw materials when synthesizing the vector, but takes a longer time to synthesize the siRNA conjugate; compared with the first synthesis method, the second synthesis method takes a shorter time for the siRNA conjugate, but uses more raw materials when synthesizing the vector.

[0388] When the vector is a cluster of CR01017, the structural formula of the siRNA conjugate is as follows:

[0389]

[0390]

[0391] When the vector is two clusters of CR01017, the structural formula of the siRNA conjugate is as follows:

[0392]

[0393] When the vector is a cluster of CR01022, the structural formula of the siRNA conjugate is:

[0394] When the vector is two clusters of CR01022, the structural formula of the siRNA conjugate is:

[0395]

[0396] When the carrier is three clusters of CR01022, the structural formula of the siRNA conjugate is:

[0397]

[0398] When the carrier is three clusters of CR01023, the structural formula of the siRNA conjugate is:

[0399]

[0400] When the carrier is two clusters of CR01027, the structural formula of the siRNA conjugate is:

[0401]

[0402] When the carrier is L96, the structural formula of the siRNA conjugate is:

[0403]

[0404] Wherein, represents siRNA.

[0405] The present disclosure synthesized the siRNA conjugates in Table 3 according to the methods of Preparation Example 9 and Preparation Example 10 respectively.

[0406] In the present disclosure, the conjugates RZ599001 and RZ502031 can only be synthesized by Synthesis Method 1 of the siRNA conjugate described in Preparation Example 9.

[0407] The conjugates RZ899040, RZ899011, RZ899009, RZ899048, RZ802010, RZ802011, RZ899041, RZ899042, and RZ899038 can be synthesized either by Synthesis Method 1 of the siRNA conjugate described in Preparation Example 9 or by Synthesis Method 2 of the siRNA conjugate described in Preparation Example 10. Specifically, the conjugates RZ899040, RZ899011, RZ899009, and RZ802010 are synthesized by Synthesis Method 1 of the siRNA conjugate described in Preparation Example 9; the conjugates RZ899048, RZ802011, RZ899041, RZ899042, and RZ899038 are synthesized by Synthesis Method 2 of the siRNA conjugate described in Preparation Example 10.

[0408] UmsUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmAmAm, as shown in SEQ ID No.1;

[0409] UmsUfsUmAmGmAfGmUmGmAmGmGmAmUfUmAfAmAmAmsUmsGm, as shown in SEQ ID No.2;

[0410] CmsAmsGmAmCmAmGfAfCfAmAmGmAmCmCmAmUmCmUm, as shown in SEQ ID No.3;

[0411] AmsGfsAmUmGmGfUmCmUmUmGmUmCmUfGmUfCmUmGmsGmsAm, as shown in SEQ ID No.4 SEQ ID No.3.

[0412] Table 3 siRNA Conjugates

[0413]

[0414]

[0415] Unless otherwise specified, the base composition and modification meanings described in the embodiments of the present disclosure are as follows: The capital letters A, U, G, C, and T represent the base composition of nucleotides, the lowercase letter m represents that the nucleotide represented by the previous letter is a methoxy-modified nucleotide; the lowercase letter f represents that the nucleotide represented by the previous letter is a fluoro-modified nucleotide; the lowercase letter s represents that there is a phosphorothioate bond connection between the nucleotides represented by the two letters before and after it.

[0416] In the context of this application and the present disclosure, unless otherwise stated, the siRNA sequences used in this application and the present disclosure are all synthesized by Suzhou Beixin Biotechnology Co., Ltd.; the synthesis of PCR primers used in this application and the present disclosure is all completed by Beijing Tsingke Biotechnology Co., Ltd.; the experimental animals C57BL / 6J mice used in this application and the present disclosure are all purchased from Spf (Beijing) Biotechnology Co., Ltd.

[0417] Table 4 Detection Results of siRNA Conjugates

[0418]

[0419]

[0420] It can be seen from the data in Table 4 that the sense strand (SS) and the antisense strand (AS) can be connected to the ligand well and maintain a relatively high purity.

[0421] General Experiments

[0422] Method for Evaluating the Inhibitory Activity of Target Genes in Mice

[0423] 6 - 8 week-old C57BL / 6J female mice were randomly grouped by body weight. The drug dosage for each mouse in the group was calculated based on body weight, and a single dose was administered by abdominal subcutaneous injection. Each siRNA conjugate was prepared into a corresponding concentration (calculated by siRNA) solution with PBS for administration, and the administration volume was 5 ml / kg mouse body weight (calculated by siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate). The day of administration was recorded as day 1 (denoted as D1). At the preset time after administration, 5 mice in each group were sacrificed. The sacrificed mice were dissected grossly, and the liver tissues of each sacrificed mouse were collected. The liver tissues were cut into small pieces of about 2 mm3 and stored with RNA Later.

[0424] Liver tissue samples at different time points in different experimental groups were taken from the above RNA later. The liver tissue samples were disrupted in a Tissuelyser II type automatic tissue homogenizer for 60 s, and then total RNA was extracted using an automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd.) according to the standard operating procedures for total RNA extraction.

[0425] Taking 1 μg of the above total RNA, using a reverse transcription kit (Promega, Reverse Transcription System, A3500) and selecting Oligo(dT)15 reverse transcription primer, a 20 μL reverse transcription system was prepared and the reverse transcription reaction was completed according to the method described in the reverse transcription kit instructions. After the reaction, 80 μL of RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. Then, a real-time fluorescence quantitative PCR kit (ABI, SYBR TM Select MasterMix, Catalog number: 4472908) was used to detect the expression level of the target gene mRNA in the liver tissue. In this real-time fluorescence quantitative PCR method, primers specific to the target gene and primers specific to the internal reference gene were used to detect the target gene and the internal reference gene respectively. A 20 μL Real-time PCR reaction system for each PCR detection well was prepared according to the method described in the real-time fluorescence quantitative PCR kit instructions. Each reaction system contained 5 μL of the cDNA solution obtained from the above reverse transcription reaction, 10 μL SYBR TM Select Master Mix, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, and 4 μL of RNase-Free H2O. The prepared reaction system was placed in a real-time fluorescence quantitative PCR instrument (ABI, StepOnePlus TM)For Real-time PCR amplification, the three-step method was used. The amplification program was pre-denaturation at 95°C for 10 min, followed by denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. The process of denaturation, annealing, and extension was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the ΔΔCt method was used to relatively quantify the expression level and inhibition rate of the target gene mRNA in each test group. The calculation method was as follows:

[0426] ΔCt (test group) = Ct (target gene in test group) - Ct (reference gene in test group)

[0427] ΔCt (control group) = Ct (target gene in control group) - Ct (reference gene in control group)

[0428] ΔΔCt (test group) = ΔCt (test group) - ΔCt (average of control group)

[0429] ΔΔCt (control group) = ΔCt (control group) - ΔCt (average of control group)

[0430] Among them, ΔCt (average of control group) is the arithmetic mean of ΔCt (control group) of 5 mice sacrificed at the same time point in the control group. Therefore, each mouse in the test group and the control group corresponds to a ΔΔCt value.

[0431] Based on the control group, the expression level of the target gene mRNA in the test group was normalized, and the expression level of the target gene mRNA in the control group was defined as 100%.

[0432] Relative expression level of target gene mRNA in test group = 2-ΔΔCt (test group) × 100%

[0433] Inhibition rate of target gene mRNA expression in test group = (1 - relative expression level of target gene mRNA in test group) × 100%

[0434] Unless otherwise specified, the in vivo activity experimental data were expressed as X±SD, and the experimental data were plotted and analyzed using GraphPad Prism 8.0 software.

[0435] Example 1: In vivo activity evaluation of trimeric CR01022 vector conjugated with CC3 target siRNA

[0436] In this example, the in vivo target gene inhibition activity evaluation method for mice was used to evaluate the inhibitory activity of the siRNA conjugate RZ802010 with trimeric CR01022 conjugated to the 3' end of the sense strand of the same CC3 target siRNA and the L96 conjugate RZ502031 on the target gene CC3 in mice.

[0437] C57BL / 6j mice at 6 - 8 weeks of age were randomly grouped by body weight, with 15 mice in each group and a total of 3 groups. Each group of mice was given a PBS solution and the siRNA conjugate mentioned in this example by abdominal subcutaneous administration. Among them, the dosage for each mouse in the PBS control group was 5 ml / kg, and the dosage for each mouse in the siRNA conjugate experimental group was 3 mg / kg (calculated based on siRNA), with a dosing volume of 5 ml / kg. The day of administration was recorded as the first day (D1). On the 8th day (D8) after administration, 5 mice in each group were sacrificed; on the 15th day (D15), 5 mice in each group were sacrificed; and on the 29th day (D29), 5 mice in each group were sacrificed. Liver tissues were collected for RNA extraction, reverse transcription reaction, and Real - time PCR detection, and relative quantification of the target gene mRNA in each test group was calculated according to the aforementioned ΔΔCt method.

[0438] The primer sequences of this example are shown in Table 5.

[0439] Table 5 Primer sequence list of this example

[0440]

[0441] The results of Example 1 showed that the conjugate RZ802010 with three clusters of CR01022 conjugated to the 3' end of the siRNA sense strand had comparable maximum inhibitory activity and better pharmacodynamic persistence compared with the L96 conjugate RZ502031 ( Figure 1 , Table 6).

[0442] Table 6 Inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in this example

[0443]

[0444] Example 2: In - vivo activity evaluation of siRNA targeting SOD1 conjugated with two - cluster and three - cluster CR01022 vectors

[0445] In this example, the in - vivo target gene inhibitory activity evaluation method for mice was used to evaluate the inhibitory activity of the siRNA sequences RZ899009 with three clusters of CR01022 conjugated to the 3' end of the sense strand of the same SOD1 - targeting siRNA, the siRNA sequence RZ899011 with two clusters of CR01022 conjugated, and the L96 conjugate RZ599001 on the target gene SOD1 in mice.

[0446] C57BL / 6j mice at 6 - 8 weeks of age were randomly grouped by body weight, with 20 mice in each group and a total of 4 groups. Each group of mice was given PBS solution and the siRNA conjugate mentioned in this example by abdominal subcutaneous administration. The dosage for each mouse in the PBS control group was 5 ml / kg, and the dosage for each mouse in the siRNA conjugate experimental group was 1 mg / kg (calculated based on siRNA), with a dosing volume of 5 ml / kg. The day of administration was recorded as the first day (D1). On the 8th day (D8), 5 mice were sacrificed from each group; on the 15th day (D15), 5 mice were sacrificed from each group; on the 29th day (D29), 5 mice were sacrificed from each group; on the 43rd day (D43), 5 mice were sacrificed from each group. 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.

[0447] The primer sequences of this example are shown in Table 7.

[0448] Table 7 Primer Sequence Table

[0449]

[0450] The results of Example 2 showed that the siRNA sequences RZ899009 with three clusters of CR01022 conjugated to the 3' end of the siRNA sense strand and RZ899011 with two clusters of CR01022 conjugated to the 3' end of the siRNA sense strand had basically equivalent highest inhibitory activities and sustained drug effects on the target gene SOD1, and both were superior to the L96 conjugate sequence RZ599001 ( Figure 2 , Table 8).

[0451] Table 8 Inhibitory Activities of Target Genes in Mice after Administration of the siRNA Conjugates Described in this Example

[0452]

[0453] Example 3: In - vivo Activity Evaluation of SOD1 - target siRNA Conjugated with One Cluster of CR01022 Vector and Two Clusters of CR01022 Vector

[0454] In this example, the inhibitory activities of the siRNA sequences RZ899011 with two clusters of CR01022 vector conjugated to the 3' end of the siRNA sense strand of the same SOD1 - target siRNA, the siRNA sequence RZ899040 with one cluster of CR01022 conjugated, and the L96 conjugate RZ599001 on the target gene SOD1 in mice were evaluated by the method of evaluating the inhibitory activity of target genes in mice in vivo.

[0455] 6 - 8 - week - old C57BL / 6j mice were randomly grouped by body weight, with 5 mice in each group and a total of 4 groups. Each group of mice was given PBS solution and the siRNA conjugate mentioned in this example by abdominal subcutaneous administration. The dosage for each mouse in the PBS control group was 5 ml / kg, and the dosage for each mouse in the siRNA conjugate experimental group was 1 mg / kg (calculated based on siRNA), with a dosing volume of 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. Liver tissues were collected for RNA extraction, reverse transcription reaction, and Real - time PCR detection, and relative quantification of the target gene mRNA in each test group was calculated according to the aforementioned ΔΔCt method.

[0456] The primer sequences of this example are shown in Table 7 of Example 2.

[0457] The results of Example 3 showed that the siRNA sequences RZ899011 and RZ899040, which conjugated two clusters of CR01022 at the 3' end of the siRNA sense strand and one cluster of CR01022 respectively, were both able to produce a highly efficient inhibitory effect on the target gene SOD1 at D8, with basically equivalent inhibitory activities, and both were slightly superior to the L96 conjugate RZ599001 ( Figure 3 , Table 9).

[0458] Table 9 Inhibitory activities of the target genes in mice after administration of the siRNA conjugates described in this example

[0459]

[0460] Example 4: In - vivo activity evaluation of siRNA conjugated with three clusters of CR01023 vectors targeting the CC3 target

[0461] In this example, the in - vivo target gene inhibitory activity evaluation method for mice was used to evaluate the inhibitory activities of the siRNA sequence RZ802011, which conjugated three clusters of CR01023 vectors at the 3' end of the siRNA sense strand, and the L96 conjugate RZ502031 on the target gene CC3 in mice.

[0462] 6 - 8 week - old C57BL / 6j mice were randomly grouped by body weight, with 15 mice in each group and a total of 3 groups. Each group of mice was given a PBS solution and the siRNA conjugate mentioned in this example by abdominal subcutaneous administration. Among them, each mouse in the PBS control group was given a dose of 5 ml / kg of the administration volume, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (calculated based on siRNA), with an administration volume of 5 ml / kg. The day of administration was recorded as the first day (D1). On D8, D15, and D29 after administration, 5 mice were sacrificed from each group respectively. 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.

[0463] The primer sequences of this example are shown in Table 5 of Example 1.

[0464] The results of Example 4 showed that compared with the conjugate RZ502031 of L96, the conjugate RZ802011 of three clusters of CR01023 had comparable overall activities in both groups on D8 and D15, but the conjugate RZ502031 of L96 had a relatively higher inhibitory effect on the target gene at D29. ( Figure 4 , Table 10).

[0465] Table 10 Inhibitory activities of the target genes in mice after administration of the siRNA conjugate described in this example

[0466]

[0467] Example 5: In - vivo activity evaluation of siRNA conjugated with two clusters of CR01027 vectors and two clusters of CR01022 vectors targeting the SOD1

[0468] In this example, the in - vivo target gene inhibitory activity evaluation method of mice was used to evaluate the inhibitory activities of the siRNA sequence RZ899048 conjugated with two clusters of CR01027 vectors at the 3' end of the sense strand of siRNA, the siRNA sequence RZ899011 conjugated with two clusters of CR01022 vectors, and the conjugate RZ599001 conjugated with L96 on the target gene SOD1 in mice. The difference is that CR01027 has a methoxy group added at the 1 - position of the CR01022 parent nucleus structure.

[0469] C57BL / 6j mice at 6 - 8 weeks of age were randomly grouped by body weight, with 5 mice in each group and a total of 4 groups. Each group of mice was given the siRNA conjugate mentioned in this example by subcutaneous administration in the abdomen. Among them, each mouse in the PBS control group was given a dose of 5 ml / kg of the administration volume, and each administration dose in the siRNA conjugate experimental group was 3 mg / kg, with an 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 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.

[0470] The primer sequences of this example are shown in Table 7 of Example 2.

[0471] The results of Example 5 showed that the conjugate RZ899048 of the two - cluster CR01027 had a higher inhibitory effect on the target gene at D8 compared with the conjugate RZ599001 of L96, and the effect was comparable to that of the conjugate (RZ899011) of the two - cluster CR01022 ( Figure 5 , Table 11).

[0472] Table 11 Inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in this example

[0473]

[0474] Example 6: In - vivo activity evaluation of the conjugates of the two - cluster CR01017 vector and the two - cluster CR01022 vector conjugated with SOD1 - target siRNA

[0475] In this example, the inhibitory activities of the siRNA sequences RZ899038 conjugated with two - cluster CR01017 vectors at the 3' end of the sense strand of SOD1 - target siRNA, the siRNA sequence RZ899011 conjugated with two - cluster CR01022 vectors, and the L96 conjugate RZ599001 on the target gene SOD1 in mice were evaluated by the method of evaluating the inhibitory activity of the target gene in mice in vivo. The main difference between the two - cluster CR01017 vector and the two - cluster CR01022 vector is that the linker part of the CR01017 vector is an amide - bond structure, and the linker part of the CR01022 vector is a triazole structure.

[0476] C57BL / 6j mice at 6 - 8 weeks of age were randomly grouped by body weight, with 15 mice in each group and a total of 4 groups. Each group of mice was given a PBS solution and the siRNA conjugate mentioned in this example by abdominal subcutaneous administration. Among them, each mouse in the PBS control group was given a dose of 5 ml / kg of the administration volume, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (calculated based on siRNA), with an administration volume of 5 ml / kg. The day of administration was recorded as the first day (D1). On the 8th day (D8), 5 mice in each group were sacrificed, on the 29th day (D29), 5 mice in each group were sacrificed, and on the 54th day (D54), 5 mice in each group were sacrificed. 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.

[0477] The primer sequences of this example are shown in Table 7 of Example 2.

[0478] The results of Example 6 showed that the in vivo activities of the siRNA sequences RZ899038 conjugated with two clusters of CR01017 vectors at the 3' end of the siRNA sense strand and RZ899011 conjugated with two clusters of CR01022 vectors were both superior to the L96 conjugated sequence RZ599001. The linker having a triazole structure (CR01022) or an amide structure (CR01017) had little effect on the vector activity ( Figure 6 , Table 12).

[0479] Table 12 Inhibitory activities of target genes in mice after administration of the siRNA conjugates described in this example

[0480]

[0481] Example 7: Evaluation of the in vivo activities of siRNA targeting SOD1 conjugated with different numbers and positions of CR01017

[0482] In this example, the in vivo target gene inhibitory activity evaluation method for mice was used to evaluate the inhibitory activities of the siRNA sequence RZ899041 conjugated with one cluster of CR01017 vectors at the 3' end of the siRNA sense strand, the siRNA sequence RZ899042 conjugated with one cluster of CR01017 vectors at both the 5' end and 3' end of the siRNA sense strand, the siRNA sequence RZ899038 conjugated with two clusters of CR01017 vectors at the 3' end of the siRNA sense strand, and the L96 conjugate RZ599001 on the target gene SOD1 in mice.

[0483] C57BL / 6j mice at 6 - 8 weeks of age were randomly grouped by body weight, with 15 mice in each group and a total of 5 groups. Each group of mice was given a PBS solution or the siRNA conjugate mentioned in this example by subcutaneous administration in the abdomen. Among them, each mouse in the PBS control group was given a dose of 5 ml / kg of the administration volume, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (calculated based on siRNA), with an administration volume of 5 ml / kg. The day of administration was recorded as the first day (D1). On the 8th day (D8), 5 mice in each group were sacrificed; on the 29th day (D29), 5 mice in each group were sacrificed; on the 54th day (D54), 5 mice in each group were sacrificed.

[0484] The primer sequences of this example are shown in Table 7 of Example 2.

[0485] The results of Example 7 showed that the siRNA conjugate RZ899038 with a two - cluster CR01017 vector conjugated to the 3' end of the siRNA sense strand, the siRNA conjugate RZ899041 with a one - cluster CR01017 vector conjugated to the 3' end of the siRNA sense strand, and the siRNA conjugate RZ899042 with a one - cluster CR01017 vector conjugated to both the 5' and 3' ends of the siRNA sense strand had an inhibitory effect equivalent to that of the L96 conjugate RZ599001 at D8 and D29, and had a higher inhibitory effect on the target gene at D59 compared to the L96 conjugate RZ599001. This indicates that the CR01017 conjugate has a longer pharmacodynamic action time ( Figure 7 , Table 13).

[0486] Table 13 Inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in this example

[0487]

[0488] Example 8: The present disclosure selected the sequence RZ899011 of two - cluster CR01022 conjugated to the SOD1 - target siRNA and the sequence RZ899038 of two - cluster CR01017 vector conjugated to the SOD1 - target siRNA for in - vivo toxicological evaluation in mice

[0489] In this example, the nature and degree of the toxic reaction of the siRNA sequence RZ899011 conjugated with two - cluster CR01022 and the siRNA sequence RZ899038 conjugated with two - cluster CR01017 vectors were evaluated by subcutaneous injection in ICR mice.

[0490] ICR mice at 6 - 8 weeks of age were randomly grouped by body weight, with 10 mice in each group (5 males and 5 females), for a total of 3 groups. Each group of mice was given a PBS solution (without siRNA conjugate) or the siRNA conjugate mentioned in this example by abdominal subcutaneous administration. Each mouse in the PBS control group was given a dose of 10 ml / kg of mouse body weight in terms of administration volume, and each mouse in the siRNA conjugate administration group was given a dose of 300 mg / kg of mouse body weight (calculated based on siRNA), with an administration volume of 10 ml / kg.

[0491] The day of administration was recorded as the first day (D1). During the experiment, clinical observations were made at least once a day after the first administration. On the 15th day of the experiment (recorded as D15), blood was collected from all animals (mice were fasted for at least 12 hours but not water - deprived before sampling for detection), and hematological and blood biochemical tests were performed. Blood was collected using EDTA - K2 anticoagulation for routine blood tests using an automated modular hematology and body fluid analyzer (Sysmex XN - 10B1); blood was collected using sodium citrate anticoagulation, centrifuged at room temperature for 10 min (centrifugal force approximately 2000g), and plasma was separated for coagulation index tests using a fully automated coagulation analyzer (Mindray C3510); blood samples (without anticoagulation) were placed at room temperature for approximately 30 min, and after blood coagulation, centrifuged at 2000g for 10 min at 4°C, and blood biochemical tests were performed using a fully automated biochemical analyzer (Sysmex BX - 4000). On the 15th day of the experiment (recorded as D15), all animals were dissected. Before dissection, the mice to be dissected were fasted but not water - deprived for at least 12 h, anesthetized with Zoletil 50 (Virbac S.A., 8G4VA), euthanized by exsanguination from the abdominal aorta after blood collection, and gross anatomical observations were made; histopathological examinations (using hematoxylin - eosin staining) were performed on the livers of all animals, and when necessary, histopathological examinations were also performed on organs with obvious abnormal changes in size, shape, color, texture, etc.

[0492] The primer sequences in this example are shown in Table 7 of Example 2.

[0493] The results of Example 8 showed that after the siRNA conjugate shown in this example, no abnormalities were observed in the clinical observations of the PBS group, the RZ899011 administration group, and the RZ899038 administration group during the experiment; at the experimental endpoint D15, no obvious abnormalities were found in the gross anatomy; no abnormalities were found in the hematological and blood biochemical tests performed on the animals in each group ( Figure 8a -j, Table 14); no abnormalities were found in the histopathological staining of the liver tissues of the animals in each group ( Figure 8k ); indicating that the siRNA conjugate and its carrier molecule of the present disclosure have good safety and low toxicity at the animal level.

[0494] Table 14 Hematological and blood biochemical test results of mice after administration of the siRNA conjugate described in this example

[0495]

[0496] It can be understood that the above embodiments are merely exemplary embodiments adopted to illustrate the principles of the present disclosure. However, the present disclosure is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and essence of the present disclosure, and these modifications and improvements are also regarded as the protection scope of the present disclosure.

Claims

1. A compound represented by formula (I): In formula (I), p and q each independently selected from 0, 1, 2 or 3; Each n is independently selected from 1, 2, 3, 4 or 5; R1 is selected from R1', and said R1' is selected from hydroxyl protecting groups; said hydroxyl protecting groups are selected from 4,4'-dimethoxytrityl (DMTr group); R2 is selected from R2', and R2' is selected from selected from R3 is selected from H or C1-C3 alkoxy; Each R4 is independently selected from 2. The compound according to claim 1, wherein Each R4 is selected from 3. The compound according to claim 1, wherein Each R4 is selected from 4. The compound according to claim 1, wherein p and q each independently selected from 0 or 1; Each n is selected from 1.

5. The compound according to claim 1, wherein The compound is selected from any of the following structures:

6. The compound according to claim 1, characterized in that, The compound is selected from any of the following structures:

7. A method for preparing a compound, characterized in that, The compound is represented by formula (I-1c): Wherein, R1', R2', R3, p, q, n are defined as in any one of claims 1-6; The preparation method includes the following steps: S1-1. The compound of formula (1) and the compound of formula (2) undergo an acetal reaction in the presence of an acidic catalyst to obtain the compound of formula (3); wherein, R5 is selected from phenyl (Ph group); S1-2. The compound of formula (3), a hydrogenated salt and the compound of formula (4) undergo an electrophilic substitution reaction to obtain the compound of formula (5); wherein, X1 represents a halogen; S1-3. The compound of formula (5) undergoes a dehydroxy protection reaction under acidic conditions to obtain the compound of formula (I-1a); S1-4. The compound of formula (I-1a) and the compound of formula (6) undergo a nucleophilic substitution reaction to obtain the compound of formula (I-1b); S1-5. The compound of formula (I-1b) and the compound of formula (7) undergo a condensation reaction to obtain the compound of formula (I-1c); In step S1-1, the molar ratio of the compound of formula (1), the compound of formula (2) and the acidic catalyst is 1:(2-4):(0.01-0.2); In step S1-1, the acidic catalyst is selected from DL-10-camphorsulfonic acid; In step S1-1, the reaction temperature of the acetal reaction is selected from 20-40°C; In step S1-2, the molar ratio of the compound of formula (3), the compound of formula (4) and the hydrogenated salt is 1:(3-5):(3-5); In step S1-2, the hydrogenated salt is selected from sodium hydride, potassium hydride or magnesium hydride; In step S1-2, the conditions for the electrophilic substitution reaction are: the compound of formula (3) and the hydrogenated salt are first reacted at -10-0°C for 25-35 min, then the compound of formula (4) is added and reacted at 20-40°C for 1-5 h; In step S1-3, the acidic condition is: acetic acid; In step S1-3, the reaction temperature of the dehydroxy protection reaction is selected from 60-80°C; In step S1-4, the molar ratio of the compound of formula (I-1a) and the compound of formula (6) is 1:(1.1-1.5); In step S1-4, the compound of formula (6) is selected from 4,4'-dimethoxytrityl chloride; In step S1-4, the nucleophilic substitution reaction is carried out in an inert atmosphere; In step S1-4, the reaction temperature of the nucleophilic substitution reaction is selected from 20-40°C; In step S1-5, the molar ratio of the compound of formula (I-1b) and the compound of formula (7) is 1:(0.6-0.9); In step S1-5, the compound of formula (7) is selected from bis(diisopropylamino)(2-cyanoethoxy)phosphine; In step S1-5, the condensation reaction is carried out in an inert atmosphere.

8. The preparation method according to claim 7, characterized in that, In step S1-1, the molar ratio of the compound of formula (1), the compound of formula (2) and the acidic catalyst is 1:3:0.

1.

9. The preparation method according to claim 7, characterized in that In step S1-2, the molar ratio of the compound of formula (3), the compound of formula (4) and the hydrogenated salt is 1:4:

4.

10. Use of a compound according to any one of claims 1 to 6 in the preparation of a targeting ligand capable of binding to a cell surface receptor; The receptor is selected from asialoglycoprotein receptor.

11. Use of a compound according to any one of claims 1 to 6 in the preparation of a drug for reducing the expression or activity of a specific gene in a target cell, wherein the specific gene is at least one of CC3 and SOD1; The target cell is selected from hepatocytes; The drug is selected from siRNA drugs.

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