TWI866590B - Tetravalent chelated group containing seven-membered heterocyclic ring and its application - Google Patents

Abstract

The present invention discloses a tetravalent conjugated group containing a seven-membered heterocyclic ring and its application, and specifically discloses the structure of the conjugated group in formula (V) and its application. In formula (V), the definition of each substituent is the same as that in the embodiment.

Description

Tetravalent chelated group containing seven-membered heterocyclic ring and its application

This application claims priority over the following Chinese patent applications filed with the National Intellectual Property Administration of China, the full text of which is incorporated herein for reference: Application number: CN202211389523.0, filing date: November 8, 2022; Application number: CN202310166298.2, filing date: February 24, 2023; Application number: CN202310900468.5, filing date: July 20, 2023; Application number: CN202311425671.8, filing date: October 30, 2023.

The present invention relates to a tetravalent conjugated group containing a seven-membered heterocyclic ring and its application, and specifically to the structure of the conjugated group represented by formula (V) and its application.

Tissue-specific delivery of nucleic acid molecules is one of the key technologies for developing nucleic acid drugs. The technology of conjugating nucleic acid molecules to ligands and using ligands to deliver nucleic acid molecules to specific tissues has been widely used. Among them, conjugating ligands containing terminal N-acetylgalactose (GalNAc) and its derivatives to nucleic acid molecules and using the binding of N-acetylgalactose to asialoglycoprotein receptor (ASGPR) to deliver nucleic acid molecules to liver cells is a more common delivery method. Common GalNAc-based ligands usually contain three terminal GalNAc molecules, that is, trivalent GalNAc ligands.

Public reports and literature indicate that, compared with trivalent GalNAc ligand molecules, tetravalent GalNAc ligands containing four terminal GalNAc molecules have better delivery efficiency and tissue specificity. However, the conformation of the four GalNAc molecules is not easy to control, which reduces the binding efficiency of the ligand containing four terminal GalNAc groups to the ASGPR protein, thereby affecting the delivery efficiency of the ligand. Therefore, the present invention provides a novel way to control the conformation of four GalNAc molecules and obtain a tetravalent GalNAc conjugate group with higher delivery efficiency.

The first aspect of the present invention provides a conjugated group represented by formula (V), Among them, L ₁ is selected from and ; L ₂ selected from and ; Selected from , and ; n is selected from 0 and 1; t is selected from 2, 3, 4, 5, 6 and 7.

The present invention also provides a conjugated group represented by formula (I), Among them, L ₁ is selected from ; L ₂ selected from ; n is selected from 0 and 1; t is selected from 2, 3, 4, 5, 6 and 7.

In some technical solutions of the present invention, the above-mentioned conjugated group is selected from (D02): .

In some technical solutions of the present invention, the above-mentioned conjugated group is selected from (D02-1): Where * represents a chiral carbon atom.

In some technical solutions of the present invention, the above-mentioned conjugated group is selected from (D02-1-A), (D02-1-B), (D02-1-C), (D03), (D12) and (D13): , , , , , .

The second aspect of the present invention provides a conjugate or a pharmaceutically acceptable salt thereof, wherein the conjugate is selected from a compound formed by linking a conjugate group defined in any of the above technical solutions to an oligonucleotide via a phosphodiester bond or a thiophosphodiester bond.

In some technical solutions of the present invention, the above-mentioned conjugated group is connected to the oligonucleotide via a phosphodiester bond or a thiophosphodiester bond, which refers to the following connection method: , wherein X is selected from S- or O-.

In some technical solutions of the present invention, the above-mentioned conjugated group is connected to the oligonucleotide via a phosphodiester bond or a thiophosphodiester bond, which refers to the following connection method: , wherein X is selected from S- or O-.

In some technical solutions of the present invention, the above-mentioned conjugated group is connected to the oligonucleotide via a phosphodiester bond or a thiophosphodiester bond, which refers to the following connection method: , wherein X is selected from S- or O-.

In some technical embodiments of the present invention, the above oligonucleotides are selected from RNAi reagents and ASO reagents, and other variables are as defined in the present invention.

In some embodiments of the present invention, the RNAi reagent is selected from single-stranded oligonucleotides and double-stranded oligonucleotides, and other variables are as defined in the present invention.

In some technical solutions of the present invention, the above-mentioned single-stranded oligonucleotide is selected from single-stranded antisense oligonucleotide, and other variables are as defined in the present invention.

In some technical solutions of the present invention, the double-stranded oligonucleotide is selected from double-stranded siRNA, and other variables are as defined in the present invention.

In some technical embodiments of the present invention, the nucleotides of the above oligonucleotides are optionally modified, and other variables are as defined in the present invention.

In some technical solutions of the present invention, the above-mentioned complex can inhibit or block the expression of the gene.

A third aspect of the present invention provides the use of the conjugated group defined in any of the above technical solutions as a delivery platform, wherein the delivery platform is used to enhance the binding of a therapeutic agent to a specific target location.

The fourth aspect of the present invention provides an intermediate compound for preparing a conjugated group represented by formula (V), and its structure is shown in formula (IM), (V-M12) and (V-M13): , , , L ₁ , L ₂ , t, and n are as defined in the present invention.

In some technical solutions of the present invention, the intermediate structure is as shown in formula (D02-M): .

In some technical solutions of the present invention, the intermediate structure is as shown in formula (D02-1-M): Here, * represents a chiral carbon atom.

In some technical solutions of the present invention, the intermediate structures are shown in formulas (D02-1-AM), (D02-1-BM), (D02-1-CM), (D03-M), (D12-M) and (D13-M): , , , , , .

The present invention also provides the conjugates shown in Table 1:

Table 1 List of compounds of the present invention Compound No. Positive-sense strand sequence (5'-3') (the binding group is attached to the 3' end of the positive-sense strand) Antisense chain sequence (5'-3') Compound 1 gs us cauc Cf a Cf Af Af ugagaguaca – D02 (SEQ ID NO: 1) us Gfs uac Tgn cucauug Uf g Gf auga cs gs a (SEQ ID NO: 2) Compound 2 gs us cauc Cf a Cf Af Af ugagaguaca – D02-1-A (SEQ ID NO: 3) us Gfs uac Tgn cucauug Uf g Gf auga cs gs a (SEQ ID NO: 2) Compound 3 gs us cauc Cf a Cf Af Af ugagaguaca – D02-1-B (SEQ ID NO: 4) us Gfs uac Tgn cucauug Uf g Gf auga cs gs a (SEQ ID NO: 2) Compound 4 gs us cauc Cf a Cf Af Af ugagaguaca – D02-1-C (SEQ ID NO: 5) us Gfs uac Tgn cucauug Uf g Gf auga cs gs a (SEQ ID NO: 2) Compound 5 gs us cauc Cf a Cf Af Af ugagaguaca – D03 (SEQ ID NO: 6) us Gfs uac Tgn cucauug Uf g Gf auga cs gs a (SEQ ID NO: 2) Compound 6 gs us cauc Cf a Cf Af Af ugagaguaca – D12 (SEQ ID NO: 7) us Gfs uac Tgn cucauug Uf g Gf auga cs gs a (SEQ ID NO: 2) Compound 7 gs us cauc Cf a Cf Af Af ugagaguaca – D13 (SEQ ID NO: 8) us Gfs uac Tgn cucauug Uf g Gf auga cs gs a (SEQ ID NO: 2) Compound 8 as as Gf c Af a Gf a Uf Af Uf u Uf uu Af u Af aua – D12 (SEQ ID NO: 9) us Afs Uf u Af ua Af a Af aua Uf c Uf u Gf cu us us u dT dT (SEQ ID NO: 10) Compound 9 as as Gf c Af a Gf a Uf Af Uf u Uf uu Af u Af aua – D13 (SEQ ID NO: 11) us Afs Uf u Af ua Af a Af aua Uf c Uf u Gf cu us us u dT dT (SEQ ID NO: 10) Compound 10 gs cs ucaaca Uf Af Uf uugaucagua – D03 (SEQ ID NO: 12) c Gfs a Gf u Uf g Uf a Uf a Af a Cf u Af g Ufs cs Afs u (SEQ ID NO: 13) Compound 11 as as Gf c Af a Gf a Uf Af Uf u Uf uu Af u Af aua – D03 (SEQ ID NO: 14) us Afs Uf u Af ua Af a Af aua Uf c Uf u Gf cu us us u dT dT (SEQ ID NO: 10) .

The present invention also provides the following test method

1. In vitro activity test

Experimental process: The complex of the present invention was incubated with freshly isolated primary mouse hepatocytes (PMH) at room temperature for 30 minutes to allow the complex to enter the PMH cells freely. After 24 hours of cell culture, the cells were lysed, RNA was extracted and purified, and the down-regulation of the target gene was detected by rt-PCR.

Experimental conclusion: The complex of the present invention performed well in the in vitro activity test experiment.

2. In vivo activity test

Objective: To evaluate the target gene in vivo and the inhibitory effect on the target gene in the test sample by using a mouse model with high-pressure tail vein injection of 2-pcDNA-CMV-AGT plasmid.

Experimental process:

1. Experimental materials: 2-pcDNA-CMV-AGT plasmid, BALB/c mice, PBS (phosphate buffered saline), and the conjugate of the present invention.

2. Experimental methods:

Order 6-8 week old female BALB/c mice and acclimate them to quarantine for one week after arrival at the animal facility.

On day 0, mice were randomly divided into groups according to body weight data, with 4 mice in each group. After grouping, all mice were subcutaneously injected with drugs, with a single dose of 10 mL/kg. Mice in group 1 were given PBS; mice in group 2 were given the complex.

On the third day after drug administration, all mice were injected with 2-pcDNA-CMV-AGT plasmid at a volume of 8% of their body weight through the tail vein within 5 seconds (injection volume (mL) = mouse body weight (g) × 8%), and the mass of the plasmid injected into each mouse was 10 µg.

On the 4th day after drug administration, mice in all groups were euthanized by CO2 inhalation, and two liver samples were collected from each mouse. The liver samples were treated with RNAlater at 4°C overnight, then RNAlater was removed and stored at -80°C for detection of AGT gene expression.

Experimental conclusion: The complex of the present invention performed well in the in vivo activity test experiment.

3: Free uptake experiment of human primary hepatocytes

1. Experimental principle:

The test samples were incubated with human primary hepatocytes to evaluate the degree of downregulation of AGT mRNA by the test samples.

2. Experimental materials:

Primary human hepatocytes (PHH), RNeasy® 96 Kit (12) (QIAGEN-74182), FastKing RT Kit (With gDNase) (Tiangen- KR116-02), TaqMan Gene Expression Assay (GAPDH, Thermo, Assay ID-Hs027866666666 ), TaqMan Gene Expression Assay (AGT, Thermo, Assay ID- Hs01586213_m1).

3. Experimental methods:

The complex of the present invention was diluted to 10 times the concentration to be tested with PBS solution. 10uL siRNA was transferred to a 96-well plate. PHH cells were thawed and transplanted to a 96-well plate, and the final cell density was 5.4×105 cells/well. The complex of the present invention was tested at 10 concentration points, with the highest concentration being 500nM, and 4-fold dilution.

The cells were cultured at 37°C, 5% CO ₂ for 48 hours, and the cell status was examined under a microscope.

After incubation, cells were lysed and all RNA was extracted using the lysate and RNeasy® 96 Kit (QIAGEN-74182). cDNA was reverse transcribed using FastKing RT Kit (With gDNase) (Tiangen- KR116-02). AGT cDNA was detected by qPCR.

4. Experimental conclusion: The complex of the present invention can significantly down-regulate the level of AGT mRNA in PHH cells.

The present invention also provides a method for preparing the complex:

Synthesis of single-chain oligoribonucleotides containing conjugates: Oligoribonucleotides were synthesized using the phosphoramidite solid phase synthesis technique. The synthesis was carried out on a solid support made by covalently linking the intermediate compounds corresponding to the conjugate groups with controlled pore glass (amino CPG, 500 Å or 1000 Å). All 2'-modified RNA phosphoramidites and auxiliary reagents were commercially available reagents. All amides were dissolved in anhydrous acetonitrile and added to a molecular sieve (3 Å), and the coupling time was 5 min using 5-ethylthio-1H-tetrazolium (ETT) as an activator, followed by 3 min in 50 mM I ₂ -water/pyridine (volume ratio 1/9) to generate phosphate bonds, or 3 min in 50 mM 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile/pyridine (v/v=1/1) to generate phosphorothioate bonds. All sequences were synthesized after the final removal of the DMTr group.

Synthesis of single-chain oligoribonucleotides without binding groups: Oligoribonucleotides were synthesized using the phosphoramidite solid phase synthesis technique. The synthesis was performed on a universal controlled pore glass (CPG) (500 Å or 1000 Å). All 2'-modified RNA phosphoramidites and auxiliary reagents were commercially available reagents. All amides were dissolved in anhydrous acetonitrile and added to a molecular sieve (3 Å). The coupling time was 5 min using 5-ethylthio-1H-tetrazolium (ETT) as an activator, followed by 3 min in 50 mM I ₂ -water/pyridine (volume ratio 1/9) to generate phosphate bonds, or 3 min in 50 mM 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile/pyridine (v/v=1/1) to generate phosphorothioate bonds. All sequences were synthesized after the final removal of the DMT group.

Cleavage and deprotection of oligomers bound to CPG: After the solid phase synthesis was terminated, the protecting groups were removed by treatment with acetonitrile solution containing 20% diethylamine for 30 minutes without cleaving the oligonucleotide from CPG. Subsequently, the dried CPG was treated with concentrated ammonia for 18 hours at 40 degrees Celsius. After centrifugation, the supernatant was transferred to a new tube and the CPG was washed with ammonia. The combined solution was concentrated to obtain a solid mixture.

Purification of single-stranded oligoribonucleotides: Oligomers were purified by HPLC using NanoQ anion exchange. Buffer A was 10 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile, and buffer B was 500 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile. The desired product was isolated and desalted using a reverse phase C18 column.

Annealing of single-stranded oligoribonucleotides to produce siRNA: Prepare the single-stranded oligoribonucleotides to be annealed to 200 μM with sterile RNase Free H ₂ O (RNA hydrolase-free). Set up the annealing reaction system as follows: Place 10 nmol of the total volume of 100 μL of the mixture in a 95°C water bath for 10 minutes (≥100 nmol requires high temperature for 20 minutes) → quickly place in a 60°C water bath and cool naturally → The solution after annealing is not stored at high temperature. Complementary chains are formed by combining equal molar single-stranded oligoribonucleotide solutions.

Technical Effects

The binding group described in the present invention, after being bound to a nucleic acid molecule, can efficiently and specifically deliver the nucleic acid molecule to liver tissue. Oligonucleotides using this binding group can better bind to the ASGPR protein, thereby allowing the oligonucleotides to enter liver cells more efficiently. At the same time, this binding group enables the nucleic acid molecule bound to it to have good tissue specificity, that is, it reduces the enrichment of nucleic acid molecules in extrahepatic tissues. At the same time, this binding group has low toxicity in the body, so that when it is used as an oligonucleotide delivery platform, the toxicity of the oligonucleotide is also low. More importantly, the siRNA obtained by using the binding group to connect double-stranded RNA sequences of different target genes shows excellent efficacy and long-term effect in the in vivo model. In addition, the synthesis of the conjugated group of the present application has a concise route, simple synthesis operation, and is easy for post-processing operation.

Definition and description

Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be ambiguous or unclear in the absence of a specific definition, but should be understood according to the meaning understood by a person of ordinary skill in the art. When a trade name appears in this article, it is intended to refer to the corresponding product or its active ingredient.

Unless otherwise specified, the terms "comprising, including and containing" or equivalents are open-ended expressions, meaning that in addition to the listed elements, components or steps, other unspecified elements, components or steps may also be included.

The "therapeutic agent" mentioned in the present invention refers to a drug used to treat a disease or improve symptoms, and the drug includes but is not limited to chemical therapeutic agents and biological therapeutic agents.

The conjugated groups of the present invention can enhance the delivery of therapeutic agents to specific target locations (e.g., specific organs or tissues) in subjects such as humans or animals. In some embodiments of the present invention, the conjugated groups can enhance the target delivery of expression inhibitory oligonucleotides. In some embodiments of the present invention, the conjugated groups can enhance the delivery of expression inhibitory oligonucleotides to the liver.

The conjugate groups described herein can be directly or indirectly linked to a compound, such as a therapeutic agent, for example, a performance inhibitory oligonucleotide, for example, the 3' or 5' end of the performance inhibitory oligonucleotide. In some embodiments of the present invention, the performance inhibitory oligonucleotide comprises one or more modified nucleotides. In some embodiments of the present invention, the performance inhibitory oligonucleotide is an RNAi reagent, such as a double-stranded RNAi reagent comprising a sense strand and an antisense strand. In some embodiments of the present invention, the conjugate groups disclosed herein are linked to the 5' end of the sense strand of the double-stranded RNAi reagent. In some embodiments, the conjugate group disclosed herein is linked to the expression inhibitory oligonucleotide reagent at the 5' end of the sense strand of the double-stranded RNAi reagent via a phosphate, phosphorothioate or phosphonate group.

The term "linked" as used herein, when referring to the connection between two molecules, means that the two molecules are linked via a covalent bond or the two molecules are associated via a non-covalent bond (eg, a hydrogen bond or an ionic bond).

The "oligonucleotide" of the present invention is a nucleotide sequence containing 10 to 80 nucleotides or nucleotide base pairs. In some embodiments of the present invention, the oligonucleotide has a nucleotide base sequence that is at least partially complementary to the coding sequence in the target nucleic acid or target gene expressed in the cell. The nucleotide can be optionally modified. In some embodiments of the present invention, after the oligonucleotide is delivered to the cell expressing the gene, the oligonucleotide can inhibit the expression of the potential gene, and is referred to as an "expression inhibitory oligonucleotide" in the present invention, which can inhibit gene expression in vitro or in vivo. “Oligonucleotide” includes but is not limited to: single-stranded oligonucleotide, single-stranded antisense oligonucleotide, short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro RNA (miRNA), short hairpin RNA (shRNA), ribozyme, interfering RNA molecule and Dicer enzyme substrate.

The "RNAi reagent" described in the present invention refers to a reagent containing RNA or RNA-like (such as chemically modified RNA) oligonucleotide molecules that can degrade or inhibit the translation of messenger RNA (mRNA) transcripts of target mRNA in a sequence-specific manner. The RNAi reagent described in the present invention can operate through an RNA interference mechanism (i.e., inducing RNA interference by interacting with components of the RNA interference pathway of mammalian cells (RNA-induced silencing complex or RISC)), or through any other mechanism or pathway. Although the RNAi reagent described in the present invention mainly operates through an RNA interference mechanism, the disclosed RNAi reagent is not limited or constrained to any specific pathway or mechanism of action. RNAi reagents include but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and Dicer substrates. The RNAi reagents described in the present invention include oligonucleotides having a strand that is at least partially complementary to the target mRNA. In some embodiments of the present invention, the RNAi reagents described herein are double-stranded and include an antisense strand and a sense strand that is at least partially complementary to the antisense strand. The RNAi reagent may include modified nucleotides and/or one or more non-phosphodiester linkages. In some embodiments, the RNAi reagents described herein are single-stranded.

The term "single-stranded oligonucleotide" used in the present invention refers to a single-stranded oligonucleotide having a sequence that is at least partially complementary to a target mRNA, which can hybridize with the target mRNA through hydrogen bonds under mammalian physiological conditions (or an equivalent in vitro environment). In some embodiments of the present invention, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.

The term "double-stranded oligonucleotide" in the present invention refers to a duplex structure comprising two antiparallel and substantially complementary nucleotide chains, one of which is a sense chain and the other is an antisense chain, wherein the antisense chain refers to a chain that is substantially complementary to the corresponding region of the target sequence (e.g., AGT mRNA), and can hybridize with the target mRNA through hydrogen bonds under mammalian physiological conditions (or an equivalent in vitro environment). The "substantially complementary" means that the corresponding positions of the two sequences can be completely complementary, or there can be one or more mismatches, and when there is a mismatch, there are usually no more than 3, 2 or 1 mismatched base pairs. In a double-stranded nucleic acid molecule, the bases of one strand are paired with the bases of the other strand in a complementary manner. The purine base adenine (A) always matches the pyrimidine base uracil (U); the purine base guanine (C) always matches the pyrimidine base cytosine (G). In some embodiments of the present invention, the double-stranded oligonucleotide is a double-stranded siRNA.

The "short interfering RNA (siRNA)" described in the present invention is a type of RNA molecule with a double-stranded region length of 17 to 25 base pairs, similar to miRNA, and operates within the RNA interference (RNAi) pathway, which interferes with the translation of mRNA of a specific gene that complements the nucleotide sequence, resulting in mRNA degradation. The short interfering RNA (siRNA) described in the present invention includes double-stranded siRNA (including positive-sense and anti-sense chains) and single-stranded siRNA (anti-sense chain only).

As used herein, "silencing", "reducing", "inhibiting", "down-regulating" or "knockdown", when referring to the expression of a given gene, means that the expression of the gene is reduced when the cell, cell population, or tissue is treated with an oligonucleotide linked to a binding group as described herein, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein or protein subunit translated from mRNA in the cell, cell population, tissue or subject that transcribes the gene, compared to a second cell, cell population or tissue that has not been so treated.

The "sequence" or "nucleotide sequence" of the present invention refers to the order or sequence of nucleobases or nucleotides described by a sequence of letters using standard nucleotide nomenclature.

Unless otherwise specified, the "optionally modified nucleotides" mentioned in the present invention means that the nucleotides can be unmodified nucleotides or modified nucleotides, and the "unmodified nucleotides" refer to nucleotides composed of natural nucleobases, sugar rings and phosphates. The "modified nucleotides" refer to nucleotides composed of modified nucleobases, and/or modified sugar rings, and/or modified phosphates. In some embodiments of the present invention, the "modified nucleotide" consists of a modified nucleobase, a modified sugar ring and a natural phosphate; in some embodiments of the present invention, the "modified nucleotide" consists of a modified nucleobase, a modified phosphate and a natural sugar ring; in some embodiments of the present invention, the "modified nucleotide" consists of a natural nucleobase, a modified sugar ring and a modified phosphate; in some embodiments of the present invention, the "modified nucleotide" consists of a modified nucleobase, a natural sugar ring and a natural phosphate; in some embodiments of the present invention, the "modified nucleotide" consists of a natural nucleobase, a modified sugar ring and a natural phosphate; in some embodiments of the present invention, the "modified nucleotide" consists of a natural nucleobase, a modified sugar ring and a natural phosphate; In some embodiments of the present invention, the "modified nucleotide" consists of a modified nucleobase, a modified sugar ring and a modified phosphate.

Unless otherwise specified, the "natural sugar ring" of the present invention is a five-membered sugar ring selected from 2'-OH.

Unless otherwise specified, the "natural base" of the present invention is selected from the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

Unless otherwise specified, the "modified nucleobase" of the present invention refers to a 5-12 membered saturated, partially unsaturated or aromatic heterocyclic ring other than a natural base, including a monocyclic or condensed ring, and specific examples thereof include but are not limited to thiophene, thianthrene, furan, pyran, isobenzofuran, benzothiazine, pyrrole, imidazole, substituted or unsubstituted triazole, pyrazole, isothiazole, isoxazole, oxazine, indolizine, indole, isoindole, isoquinoline, quinoline, naphthopyridine, quinazoline, carbazole, phenanthridine, piperidine, phenazine, phenazine, phenothiazine, furanane, Phenoxazine, pyrrolidine, pyrroline, imidazolidine, imidazoline, pyrazolidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 2-aminoadenine, 2-aminoguanine, 2-propyl adenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halogenated uracil and cytosine, 5-propynyl uracil and cytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-hydrosulfanyl, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogen, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine, , , , and .

Unless otherwise specified, the "modified sugar ring" of the present invention may include, but is not limited to, one of the following modifications at the 2' position: H; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH ₂ ) _n O] _m CH ₃ , O(CH ₂ ) _n OCH ₃ , O(CH ₂ ) _n NH ₂ , O(CH ₂ ) _n CH ₃ , O(CH ₂ ) _n ONH ₂ , and O(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ )] ₂ , wherein n and m are from 1 to 10. In other embodiments, the modification at the 2' position includes but is not limited to one of the following: substituted or unsubstituted C1 to C10 lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA, or group for improving the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification includes, but is not limited to, 2'-methoxyethoxy (2'-O-CH ₂ CH ₂ OCH ₃ , also known as 2'-O-(2-methoxyethyl) or 2'-MOE).

Unless otherwise specified, the "modified phosphate" of the present invention includes but is not limited to: thiophosphate modification, and the "thiophosphate" includes (R)- and (S)-isomers and/or mixtures thereof.

In some embodiments of the invention, the modified nucleotide may comprise one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide with a modified ribose moiety, wherein the ribose moiety comprises an additional bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in a 3'-endo conformation.

In some embodiments of the invention, the modified nucleotides comprise one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which any sugar bonds have been removed, thereby forming an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the bond between C1'-C4' has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., a covalent carbon-carbon bond between the C2' and C3' carbons) has been removed.

In some embodiments of the present invention, the modified nucleotide comprises one or more monomers of GNA (glycerol nucleic acid) nucleotides. GNA includes GNA-A, GNA-T, GNA-C, GNA-G and GNA-U. The structure of GNA-A is ; GNA-T or Tgn structure is ; The structure of GNA-C is ; The structure of GNA-G is ; The structure of GNA-U is .

In some example sequences of the present invention, the modified nucleotides contain one or more dX (deoxynucleotide) nucleotide monomers. dX includes dA, dT, dC, dG and dU. The structure of dA is ; dT structure is ; dC structure is ; The structure of dG is ; dU structure is .

In some embodiments of the invention, the modified nucleotides may also include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified via a two-atom bridge. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring.

The "plurality" mentioned in the present invention refers to an integer greater than or equal to 2, including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, up to the theoretical upper limit of the siRNA analogs.

Unless otherwise specified, the "overhang" described in the present invention refers to at least one unpaired nucleotide protruding from the double-stranded region structure of the double-stranded compound. For example, the 3'-end of one chain extends beyond the 5'-end of the other chain, or the 5'-end of one chain extends beyond the 3'-end of the other chain. The overhang may contain at least one nucleotide; or the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five or more nucleotides. The nucleotides at the nucleotide overhang are optionally modified nucleotides. The overhang may be located on the positive chain, the antisense chain, or any combination thereof. In addition, the overhang may be present at the 5'-end, 3'-end, or both ends of the antisense or positive chain of the double-stranded compound. In some embodiments of the present invention, the antisense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 3'-end and/or the 5'-end. In some embodiments of the present invention, the positive sense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 3'-end and/or the 5'-end. In some embodiments of the present invention, the antisense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 3'-end and the positive sense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 3'-end. In some embodiments of the invention, the antisense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 5'-end and the sense strand has an overhang of 1 to 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) at the 5'-end.

In the present invention, the conjugate of the double-stranded RNAi analog (also referred to as "conjugate") is a compound formed by linking the double-stranded RNAi analog and a pharmaceutically acceptable conjugating group, and the double-stranded RNAi analog and the pharmaceutically acceptable conjugating group are covalently linked.

In the present invention, a pharmaceutically acceptable conjugate group can be linked to the 3' end and/or 5' end of the sense chain and/or antisense chain of the double-stranded RNAi analog.

In the present invention, the number of pharmaceutically acceptable conjugated groups is 1, 2, 3, 4 or 5, and the pharmaceutically acceptable conjugated groups can be independently linked to the 3' end and/or 5' end of the sense chain and/or antisense chain of the double-stranded RNAi.

In the context of the present invention, unless otherwise specified, "conjugation" refers to the connection between two or more chemical moieties, each having a specific function, by covalent linkage; correspondingly, "conjugate" refers to a compound formed by covalent linkage between the chemical moieties.

In the present invention, unless otherwise specified, "linker" refers to an organic moiety group that connects two parts of a compound, for example, covalently attaches two parts of a compound. This bond usually includes a direct bond or an atom (such as oxygen or sulfur), an atom group (such as NRR, C(O), C(O)NH, SO, SO ₂ , SO ₂ NH), a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, wherein any one or more C atoms in the substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl can be replaced by a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl.

The cleavable linker is fully stable outside the cell, but once inside the target cell it will cleave to release the two moieties that the linker co-fixes.

The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and their mixtures are included within the scope of the present invention.

Unless otherwise indicated, the term "enantiomers" or "optical isomers" refers to stereoisomers that are mirror images of each other.

Unless otherwise indicated, the term "diastereomers" refers to stereoisomers of a molecule having two or more chiral centers that are in a non-mirror image relationship.

Unless otherwise noted, the solid wedge key ( ) and the Dashed Wedge Key ( ) represents the absolute configuration of a stereocenter, with a straight solid line bond ( ) and the straight dashed line key ( ) indicates the relative configuration of the stereocenter, with a wavy line ( ) indicates a solid wedge key ( ) or a dashed wedge key ( ), or a wavy line ( ) indicates a straight solid line key ( ) or the dashed straight line key ( ).

The present invention, structural fragment The straight solid key in ) indicates that the two substituents connected in the structure are in the same direction.

Unless otherwise indicated, the terms "enriched in one isomer", "isomer enrichment", "enriched in one enantiomer" or "enantiomeric enrichment" mean that the content of one isomer or enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

Unless otherwise indicated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.

Optically active (R)- and (S)-isomers as well as D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereoisomer salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, separation of enantiomers and diastereomers is often accomplished by the use of chromatographic methods employing chiral stationary phases optionally coupled with chemical derivatization (e.g. carbamate formation from amines).

The compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more atoms constituting the compound. For example, the compound may be labeled with a radioactive isotope, such as tritium ( ^3H ), iodine-125 ( ^125I ) or C-14 ( ^14C ). For another example, deuterated drugs may be formed by replacing hydrogen with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have the advantages of reducing side effects, increasing drug stability, enhancing therapeutic efficacy, and extending the biological half-life of drugs. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.

Unless otherwise indicated, the terms "phosphothioate" and "phosphothioate" refer to , its protonated form (e.g. , ) and its tautomers (e.g. ) compounds.

Unless otherwise indicated, the term "phosphate" is used in its ordinary sense as understood by those skilled in the art and includes its protonated form (e.g., and ).

The term "salt" refers to a salt of the compounds of the present invention, prepared from the compounds with specific substituents discovered in the present invention and a relatively non-toxic acid or base. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrosulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid, etc.; also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and can therefore be converted into either base or acid addition salts.

The salts of the present invention can be synthesized from parent compounds containing acid or alkaline groups by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or alkaline form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two.

The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitution methods known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention.

The compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more atoms constituting the compound. For example, the compound may be labeled with a radioactive isotope, such as tritium ( ^3H ), iodine-125 ( ^125I ) or C-14 ( ^14C ). For another example, deuterated drugs may be formed by replacing hydrogen with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have the advantages of reducing side effects, increasing drug stability, enhancing therapeutic efficacy, and extending the biological half-life of drugs. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.

When a linker group is listed without specifying its direction of attachment, the direction of attachment is arbitrary, e.g. The connecting group L is -MW-. In this case, -MW- can connect ring A and ring B in the same direction as the reading order from left to right. , or by connecting Ring A and Ring B in the opposite direction of the reading order from left to right. Combinations of linking groups, substituents and/or variants thereof are permitted only if such combinations result in stable compounds.

The term "optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where said event or circumstance does not occur.

The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the compound of the substituent is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may be substituted or unsubstituted. Unless otherwise specified, the type and number of substituents can be any on a chemically feasible basis.

When any variable (e.g., R) occurs more than once in the composition or structure of a compound, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may optionally be substituted with up to two Rs, and each occurrence of R is an independent choice. Furthermore, combinations of substituents and/or variants thereof are permitted only if such combinations result in stable compounds.

When the number of a linking group is 0, for example -(CRR) ₀ -, it means that the linking group is a single bond.

When a substituent is vacant, it means that the substituent does not exist. For example, when X is vacant in A-X, it means that the structure is actually A. When the substituent is listed without specifying which atom it is connected to the substituted group through, this substituent can be bonded through any atom of it. For example, pyridyl as a substituent can be connected to the substituted group through any carbon atom on the pyridine ring.

The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitution methods known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention.

The structure of the compound of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the field. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data of the cultured single crystal using a Bruker D8 venture diffractometer, the light source is CuKα radiation, and the scanning method is φ/ω scanning. After collecting relevant data, the direct method (Shelxs97) is further used to analyze the crystal structure to confirm the absolute configuration.

The solvent used in the present invention can be obtained from commercial sources.

Unless otherwise specified, the solvent ratios used in the column chromatography and the preparation of thin layer silica gel chromatography of the present invention are all volume ratios.

The present invention uses the following abbreviations: DMSO represents dimethyl sulfoxide; CBz represents benzyloxycarbonyl, which is an amine protecting group; Boc represents tert-butyloxycarbonyl, which is an amine protecting group; _Boc2O represents di-tert-butyl dicarbonate; DMTr represents dimethoxytrityl; Fmoc represents 9-fluorenylmethoxycarbonyl; ANGPTL3 represents angiopoietin-like 3; AGT represents angiotensinogen; and complement C5 represents complement component 5.

The abbreviations of nucleotide monomers used in the description of nucleic acid sequences are specifically shown in Table 2:

Table 2 Abbreviations of nucleotide monomers Abbreviation Nucleotides A Adenosine 3'-phosphate U Uridine-3'-phosphate G Guanosine-3'-phosphate C Cytidine-3'-phosphate dT Thymidine deoxynucleotide gs 2'-O-methylguanosine-3'-phosphorothioate us 2'-O-methyluridine-3'-phosphorothioate as 2'-O-methyladenosine-3'-phosphorothioate cs 2'-O-methylcytidine-3'-phosphorothioate c 2'-O-methylcytidine-3'-phosphate a 2'-O-methyladenosine-3'-phosphate u 2'-O-methyluridine-3'-phosphate g 2'-O-methylguanosine-3'-phosphate Cf 2'-Fluorocytidine-3'-phosphate A 2'-Fluoroadenosine-3'-phosphate Uf 2'-Fluorouridine-3'-phosphate Gf 2'-Fluoroguanosine-3'-phosphate Cfs 2'-Fluorocytidine-3'-phosphorothioate Af 2'-Fluoroadenosine-3'-phosphorothioate Ufs 2'-Fluorouridine-3'-phosphorothioate Gf 2'-Fluoroguanosine-3'-phosphorothioate GNA-C Cytidine Glycerol Nucleic Acid GNA-A Adenosine Glycerol Nucleic Acid GNA-T or Tgn TgRNA GNA-G GGNA GNA-U UGN

Compounds were named according to conventional nomenclature in this field or using ChemDraw® software. Commercially available compounds were named according to the supplier's catalog name.

Specific implementation method

The present invention is described in detail below through examples, but it does not mean any adverse limitation to the present invention. The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitution methods known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention. It will be obvious to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

Embodiment 1

Step A:

Under nitrogen protection, sodium hydrogen (2.75 g, 68.68 mmol, 60% purity) was added gradually to a solution of diethyl malonate (10 g, 62.43 mmol, 9.43 ml) in tetrahydrofuran (100 ml) at 0 degrees Celsius. After the addition, the mixture was stirred for 0.5 hours, and then a solution of compound 1-1 (17.86 g, 62.43 mmol) in tetrahydrofuran (50 ml) was added dropwise at 0 degrees Celsius. The mixture was stirred at 25 degrees Celsius for 15.5 hours. At 10-25 degrees Celsius, add saturated ammonium chloride (20 ml) to quench the reaction, dilute with water (20 ml), extract with ethyl acetate (50 ml*2), wash with saturated brine (30 ml*2), dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain a crude product. The crude product is purified by passing through a silica gel column (petroleum ether/ethyl acetate = 1/0 to 9/1) to obtain compound 1-2 .

Step B:

Under nitrogen protection, sodium borohydride (15.2 g, 401.77 mmol) was added gradually to a solution of compound 1-2 (15 g, 41.06 mmol) in methanol (150 ml) at 60 °C over 0.5 hours, and the mixture was stirred for 0.5 hours. The mixture was stirred at 65 °C for 1 hour. At 0-25 degrees Celsius, water was added to quench the reaction, stirred for 0.5 hours, and water (50 ml) was added to dilute. The reaction solution was concentrated under reduced pressure at 30-35 degrees Celsius to remove methanol, extracted with ethyl acetate (100 ml*2), washed with saturated brine (50 ml*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 1-3 .

Step C:

Compound 1-3 (8.5 g, 30.22 mmol) and potassium phthalimide (14.00 g, 75.56 mmol) were mixed in N,N-dimethylformamide (90 ml) solution, replaced with nitrogen three times, and the reaction solution was reacted at 100 degrees Celsius for 16 hours under nitrogen protection. The reaction solution was diluted with water (300 ml), and a white solid precipitated. The filter cake was collected by filtration, and the filter cake was passed through a silica gel column (petroleum ether/ethyl acetate = 1/0 to 1/1) to purify compound 1-4 .

Step D:

Compound 1-4 (6.1 g, 17.56 mmol), 4,4-dimethoxytrityl chloride (7.14 g, 21.07 mmol) and triethylenediamine (2.95 g, 26.33 mmol, 2.90 ml) were mixed in a dichloromethane (60 ml) solution, replaced with nitrogen three times, and the reaction solution was reacted at 25 degrees Celsius for 16 hours under nitrogen protection. The reaction solution was diluted with water (30 ml), extracted with dichloromethane (50 ml), and the organic phase was washed with saturated brine (30 ml*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by passing through a silica gel column (petroleum ether/ethyl acetate = 1/0 to 4/1, adding 0.1% triethylamine) to obtain compound 1-5 .

Step E:

Compound 1-5 (7.1 g, 10.93 mmol) and hydrazine monohydrate (1.52 g, 29.76 mmol, 1.48 ml, 98% purity) were mixed in an ethanol (80 ml) solution and replaced with nitrogen three times. Under nitrogen protection, the reaction solution was reacted at 70 degrees Celsius for 16 hours. At 35-40 degrees Celsius, the ethanol was removed by reduced pressure and concentration to obtain compound 1-6 .

Step F:

Compound 1-7A (15 g, 62.41 mmol, 14.71 ml), compound 1-7 (15.35 g, 62.41 mmol) and triethylamine (20.21 g, 199.71 mmol, 27.80 ml) were mixed in a toluene (150 ml) and methanol (15 ml) solution and replaced with nitrogen three times. Under nitrogen protection, the reaction solution was reacted at 70 degrees Celsius for 16 hours. The toluene was removed by reducing pressure and concentration to obtain a crude product. The crude product was diluted with 0.5 mol hydrochloric acid (50 ml), extracted with ethyl acetate (50 ml * 2), and the aqueous phase was retained. The aqueous phase was adjusted to pH 13 with sodium carbonate, extracted with ethyl acetate (50 ml * 2), and the aqueous phase was retained. The aqueous phase was concentrated under reduced pressure to obtain a crude product, which was slurried with methanol and ethyl acetate in a ratio of 1:4 (50 ml), filtered, the mother liquor was collected, and concentrated under reduced pressure to obtain a crude product. The crude product was then purified by preparative chromatography (formic acid conditions; chromatography column: Phenomenex luna C18 250*80 mm*10 μm; mobile phase: [water (hydrochloric acid)-acetonitrile]; acetonitrile%: 5%-35%, 20 minutes) to obtain compound 1-7 .

Step G:

Compound 1-6 (5.68 g, 10.93 mmol), compound 1-7 (5.13 g, 14.21 mmol, HCl), triethylamine (4.42 g, 43.72 mmol, 6.08 ml) and tri-n-propyl cyclophosphoric anhydride (14.47 g, 22.74 mmol, 13.52 ml, 50% purity) were mixed in a dichloromethane (50 ml) solution, replaced with nitrogen three times, and the reaction solution was reacted at 25 degrees Celsius for 16 hours under nitrogen protection. The reaction solution was diluted with water (30 ml) and extracted with dichloromethane (100 ml*2). The organic phase was washed with saturated brine (30 ml*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative chromatography (neutral conditions; chromatographic column: Kromasil Eternity XT 250*80 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 70%-100%, 20 minutes) to obtain compound 1-8 .

Step H:

Compound 1-8 (4.9 g, 5.93 mmol) and palladium carbon (10%) were mixed in ethyl acetate (100 ml) solution, and the atmosphere was replaced with hydrogen three times. The reaction solution was stirred at 25 degrees Celsius for 100 hours under hydrogen pressure (15 psi). The filtrate was filtered, collected, and concentrated under reduced pressure to obtain compound 1-9 .

Step I:

Compound 1-10 (5 g, 16.43 mmol) was added to a solution of ethyl acetate hydrochloride (4 mol/L, 50.00 ml) and reacted at 25 degrees Celsius for 1 hour. The reaction solution was concentrated under reduced pressure to obtain compound 1-11 .

Step J:

To a solution of compound 1-11 (2.91 g, 16.44 mmol) in sodium hydroxide (2 M, 24.66 ml), benzyl chloroformate (6.17 g, 36.16 mmol, 5.14 ml) was added at 0 degrees Celsius within 2 minutes. After the addition, sodium hydroxide (2 M, 18.08 ml) solution was added at 0 degrees Celsius. The reaction solution was reacted at 25 degrees Celsius for 0.5 hours. The reaction solution was diluted with water (50 ml), extracted with ethyl acetate (100 ml*2), the aqueous phase was retained, 2 mol/L hydrochloric acid was added to the aqueous phase to adjust the pH to 4-5, and extracted with ethyl acetate (100 ml*2). The organic phase was washed with saturated brine (50 ml), dried over anhydrous sodium sulfate, and filtered. The product was concentrated under reduced pressure to obtain compound 1-12 .

Step K:

Compound 1-12 (1.2 g, 1.86 mmol), compound 1-9 (1.52 g, 4.09 mmol), triethylamine (752.02 mg, 7.43 mmol, 1.03 ml) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (1.55 g, 4.09 mmol) were mixed in N,N-dimethylformamide (10 ml) solution and replaced with nitrogen three times. Under nitrogen protection, the reaction solution was reacted at 25 degrees Celsius for 2 hours. The reaction solution was diluted with water (50 ml), extracted with ethyl acetate (50 ml*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative chromatography (neutral conditions; chromatographic column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 64%-94%, 10 minutes) to obtain compound 1-13.

Step L:

Compound 1-13 (1.5 g, 1.11 mmol) and palladium carbon (200 mg, 10% purity) were mixed in a solution of methanol (10 ml) and tetrahydrofuran (5 ml), and the hydrogen atmosphere was replaced three times. The mixture was reacted at 25 degrees Celsius for 40 hours under hydrogen pressure (15 psi). The mother liquor was collected by filtration and concentrated under reduced pressure to obtain compound 1-14 .

Step M:

Compound 1-14 (465 mg, 568.42 μmol), compound 1-15 (1.51 g, 2.84 mmol), triethylamine (575.19 mg, 5.68 mmol, 791.18 μl) and benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate (1.51 g, 3.98 mmol) were mixed in N,N-dimethylformamide (5 ml) solution and replaced with nitrogen three times. The reaction was carried out at 25 degrees Celsius for 16 hours under nitrogen protection. The reaction solution was filtered, the mother liquor was collected, and purified by preparative chromatography (neutral conditions; chromatographic column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 48%-78%, 10 minutes) to obtain compound 1-16 .

Step N:

Compound 1-16 (402 mg, 139.77 μmol), compound 1-15 (1.51 g, 2.84 mmol), succinic anhydride (47.84 mg, 419.31 μmol), 4-dimethylaminopyridine (3.429 mg, 27.95 μmol) and triethylamine (42.43 mg, 419.31 μmol, 58.36 μl) were mixed in a dichloromethane (10 ml) solution and replaced with nitrogen three times. Under nitrogen protection, the reaction solution was stirred at 25 degrees Celsius for 16 hours. The dichloromethane was removed by reducing the pressure and concentrating to obtain a crude product. The crude product was purified by preparative chromatography (neutral conditions; chromatographic column: Waters Xbridge 150*25 mm*5 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 28%-58%, 8 minutes) to obtain compound D02-M .

¹ H NMR (400 MHz, DMSO-d6) δ = 8.55-7.59 (m, 10H), 7.40-7.27 (m, 4H), 7.23 (br d, J=8.3Hz, 5H), 6.89 (br d, J =8.6 Hz, 4H), 5.21 (d, J=2.8 Hz, 3H), 4.97 (br dd, J=2.9, 11.1 Hz, 4H), 4.87-4.62 (m, 1H), 4.49 (br d, J=8.4 Hz, 3H), 4.24-3.96 (m, 13H) , 3.93-3.81 (m, 5H), 3.79-3.60 (m, 12H), 3.56-3.35 (m, 13H), 3.13-2.87 (m, 15H), 2.47-2.34 (m, 6H), 2.16-2.01 (m, 27H), 2.00 (s, 12H), 1.89 (s, 11H), 1.77 (s, 14H), 1.68-1.33 (m, 28H), 1.32-0.98 (m, 16H).

Embodiment 2

Step A:

Compound 2-1 (10 g, 41.61 mmol, 9.80 ml) and triethylamine (8.42 g, 83.21 mmol, 11.58 ml) in toluene (100 ml) were added dropwise to a toluene solution (100 ml) of compound 2-2 (11.03 g, 42.44 mmol) at 25 degrees Celsius. The reaction was stirred at 100 degrees Celsius for 12 hours. Saturated sodium carbonate aqueous solution (100 ml) was added to the reaction solution and then extracted with ethyl acetate (100 ml*2). The combined organic phases were washed with saturated brine (100 ml*2), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated and separated by silica gel column chromatography to obtain compound 2-3 .

Step B:

Palladium carbon (5 g, 26.21 mmol, 10% content) and Boc ₂ O (12.59 g, 57.67 mmol, 13.25 ml) were added to a methanol solution (95 ml) of compound 2-3 (9.5 g, 26.21 mmol). After replacing the atmosphere with argon three times, the atmosphere was replaced with hydrogen three times, and the reaction was carried out at 25 degrees Celsius under normal pressure hydrogen atmosphere for 3 hours. The reaction solution was filtered through diatomaceous earth and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound 2-4 , which was used directly in the next step without purification.

Step C:

A tetrahydrofuran solution (25 ml) of compound 2-4 (7.3 g, 20.37 mmol) was added to a mixed solution of sodium hydroxide (3.24 g, 81.06 mmol) in methanol (25 ml) and water (25 ml). The reaction solution was stirred at 25 degrees Celsius for 2 hours. After the reaction solution was concentrated to remove methanol, 100 ml of water was added and the aqueous phase was washed with ethyl acetate (100 ml*2), and the ethyl acetate phase was discarded. The pH of the aqueous phase was adjusted to 2-3 with 1 mol/L hydrochloric acid, and the aqueous phase was extracted with dichloromethane (100 ml*2). The combined dichloromethane phases were washed with saturated brine (100 ml) and dried over anhydrous sodium sulfate. The filtrate was concentrated after filtration to obtain compound 2-5 , which was used directly in the next step.

Step D:

Potassium carbonate (4.09 g, 29.62 mmol) and benzyl bromide (3.04 g, 17.77 mmol, 2.11 ml) were added to a solution of compound 2-5 (5.1 g, 14.81 mmol) in N,N-dimethylformamide (50 ml). The reaction solution was stirred at 25 degrees Celsius for 12 hours. After adding 200 ml of water to the reaction solution, it was extracted with ethyl acetate (100 ml*2). The combined organic phases were washed with 100 ml of saturated brine and dried over anhydrous sodium sulfate. The filtered solution was concentrated and separated by silica gel column chromatography to obtain compound 2-6 .

Step E:

To a solution of compound 2-6 in ethyl acetate (10 ml) was added ethyl acetate hydrochloride solution (4 M, 48 ml) at 25 degrees Celsius. The reaction mixture was stirred at 25 degrees Celsius for 0.5 hours. The mixture was concentrated under reduced pressure to obtain compound 2-7 , which was used directly in the next step.

Step F:

N,N-diisopropylethylamine (4.75 g, 36.72 mmol, 6.40 ml), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.35 g, 11.47 mmol), and compound 2-7 (1.41 g, 4.59 mmol) were added to a solution of compound 2-8 (3.07 g, 10.10 mmol) in N,N-dimethylformamide (25 ml). The reaction was stirred at 25 degrees Celsius for 12 hours. The reaction solution was diluted with water (150 ml) and extracted with ethyl acetate (100 ml * 2). The combined organic phases were washed with saturated brine (100 ml), dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated and separated by preparative HPLC (column: Phenomenex luna C18 250*80 mm*10 μm; mobile phase: [water (formic acid)-acetonitrile]; acetonitrile%: 65%-95%, 20 minutes) to obtain compound 2-9 .

Step G:

Compound 2-9 (3.4 g, 4.21 mmol) was dissolved in ethyl acetate hydrochloride solution (4 M, 50 ml) and stirred at 25 degrees Celsius for 2 hours. The reaction solution was concentrated and dried under vacuum to obtain compound 2-10 .

Step H:

Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.92 g, 7.69 mmol), N,N-diisopropylethylamine (3.18 g, 24.62 mmol, 4.29 ml), and compound 2-10 (0.85 g, 1.54 mmol) were added to a solution of compound 2-11 in N,N-dimethylformamide (30 ml). The reaction solution was stirred at 25 degrees Celsius for 12 hours. The reaction solution was added to water (150 ml) and extracted with dichloromethane/isopropanol = 3/1 (200 ml * 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative HPLC (column: Phenomenex luna C18 (250*70 mm, 10 μm); mobile phase: [water (trifluoroacetic acid)-acetonitrile]; acetonitrile%: 25%-50%, 19 minutes) to obtain compound 2-12 .

Step I:

Palladium carbon (0.4 g, 750.64 μmol, 10% content) was added to a methanol solution (30 ml) of compound 2-12 (1.85 g, 750.64 μmol), and the atmosphere was replaced with nitrogen three times and then replaced with hydrogen three times. The mixture was stirred at 25 degrees Celsius for 12 hours under a hydrogen atmosphere at normal pressure. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to obtain compound 2-13 .

Step J:

Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (191.66 mg, 505.38 μmol), N,N-diisopropylethylamine (174.18 mg, 1.35 mmol) and compound 2-14 (175.10 mg, 336.92 μmol) were added to a solution of compound 2-13 (0.8 g, 336.92 μmol) in N,N-dimethylformamide (15 ml). The reaction solution was stirred at 25 degrees Celsius for 12 hours. Water (100 ml) was added to the reaction solution, followed by extraction with dichloromethane (100 ml * 2). The combined organic phases were dried over anhydrous sodium sulfate and purified by preparative HPLC to obtain compound 2-15 (column: Waters XbridgeC18 150 * 50 mm * 10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 36%-66%, 10 minutes).

Step K:

4-Dimethylaminopyridine (25.49 mg, 208.61 μmol), triethylamine (42.22 mg, 417.23 μmol), and compound 2-16 (125.26 mg, 1.25 mmol) were added to a dichloromethane solution (15 ml) of compound 2-15 (600 mg, 208.61 μmol). The reaction solution was stirred at 25 degrees Celsius for 12 hours, concentrated under reduced pressure, and purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 24%-54%, 10 minutes) to obtain compound D02-1-AM . LCMS ([(M-2)/2], m/z: 1487), ¹ H NMR (400 MHz, DMSO-d6) δ ppm 0.99 - 1.33 (m, 19 H) 1.35 - 1.67 (m, 28 H) 1.77 (s, 12 H) 1.89 (s, 12 H) 1.99 (s, 12 H) 2.01 - 2.14 (m, 28 H) 2.42 (s, 4 H) 2.89 - 3.13 (m, 14 H) 3.35 - 3.44 (m, 6 H) 3.62 - 3.79 (m, 12 H) 3.82 - 3.93 (m, 5 H) 3.94 - 4.23 (m, 16 H)4.48(d, J=8.44 Hz, 4 H) 4.97 (dd, J=11.19, 3.24 Hz, 4 H) 5.21 (d, J=3.30 Hz, 4 H) 6.88 (d, J=8.68 Hz, 4 H) 7.18 - 7.26 (m, 5 H) 7.27 - 7.39 (m, 4 H) 7.72 - 7.89 (m, 9 H) 7.91 - 8.25 (m, 3 H).

Embodiment 3

Step A:

Compound 2-7 (1.41 g, 4.59 mmol) and N,N-diisopropylethylamine (3.56 g, 27.54 mmol, 4.80 ml) were added to a solution of compound 3-1 (3.07 g, 10.10 mmol), N,N-diisopropylethylamine (1.19 g, 9.18 mmol, 1.60 ml) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.35 g, 11.47 mmol) in N,N-dimethylformamide (20 ml). The reaction solution was stirred at 25 degrees Celsius for 12 hours. After the reaction was completed, 80 ml of water was added and extracted with ethyl acetate (80 ml*2). After the organic phases were combined, they were washed with saturated brine (80 ml * 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by preparative HPLC to obtain compound 3-2 (column: Phenomenex luna C18 (250 * 70 mm, 10 μm); mobile phase: [water (formic acid) - acetonitrile]; acetonitrile%: 50% - 80%, 26 minutes).

Step B:

Compound 3-2 (3.79 g, 4.52 mmol) was dissolved in ethyl acetate hydrochloride (4 M, 50 ml). The mixture was stirred at 25 degrees Celsius for 2 hours. The mixture was concentrated under reduced pressure to obtain compound 3-3 , which was used directly in the next step without further purification.

Step C:

N,N-diisopropylethylamine (4.90 g, 37.95 mmol, 6.61 ml), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.50 g, 11.86 mmol) and compound 3-3 (1.31 g, 2.37 mmol) were added to a solution of compound 2-11 (5.43 g, 10.20 mmol) in N,N-dimethylformamide (60 ml). The reaction was stirred at 25 degrees Celsius for 12 hours. After adding 200 ml of water, the aqueous phase (100 ml * 4) was extracted with dichloromethane/isopropanol = 3/1. The organic phases were combined and washed with saturated brine (200 ml * 2), dried and filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by preparative HPLC (column: Phenomenex Synergi Max-RP 250*50 mm*10 μm; mobile phase: [water (trifluoroacetic acid)-acetonitrile]; acetonitrile%: 25%-50%, 17 min) to obtain compound 3-4 .

Step D:

A methanol solution (10 ml) of compound 3-4 (380 mg, 151.95 μmol) was added to palladium carbon (50 mg, 151.95 μmol, 10% content). After three replacements with argon and three replacements with hydrogen, the reaction was carried out at 25 degrees Celsius under normal pressure and hydrogen atmosphere for 12 hours. The reaction solution was filtered through diatomaceous earth and concentrated to obtain compound 3-5 , which was directly used in the next step.

Step E:

N,N-diisopropylethylamine (67.49 mg, 522.23 μmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (74.27 mg, 195.84 μmol) and compound 2-14 (61.07 mg, 117.50 μmol) were added to a solution of compound 3-5 (310 mg, 130.56 μmol) in N,N-dimethylformamide (10 ml). The reaction solution was stirred at 25 degrees Celsius for 12 hours. After the reaction was completed, 200 ml of water was added and extracted with ethyl acetate (100 ml*2). The organic phase was washed with 100 ml of saturated brine, dried over anhydrous sodium sulfate, and filtered. The obtained filtrate was concentrated and purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 37%-67%, 10 minutes) to obtain compound 3-6 .

Step F:

A dichloromethane solution (5 ml) of compound 3-6 (142 mg, 46.38 μmol) was added with triethylamine (4.69 mg, 46.38 μmol), 4-dimethylaminopyridine (5.67 mg, 46.38 μmol), and compound 2-16 (11.60 mg, 115.94 μmol). The reaction solution was stirred at 25 degrees Celsius for 12 hours. After concentration under reduced pressure, the product was purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 mm; mobile phase: [water (ammonium bicarbonate-acetonitrile)]; acetonitrile%: 25%-55%, 10 minutes) to obtain compound D02-1-BM . LCMS ([(M-2)/2], m/z: 1487), ¹ H NMR (400 MHz, DMSO-d6) δ = 7.95 - 7.68(m, 9H), 7.40 - 7.28(m, 4H), 7.23(d, J = 8.6 Hz, 5H), 6.89(d, J = 8.4 Hz, 4H), 5.22(d, J = 3.3 Hz, 4H), 4.98(dd, J = 3.1, 11.3 Hz, 4H), 4.90 - 4.76(m, 1H), 4.49(d, J = 8.4 Hz, 4H), 4.15 - 3.97(m, 15H), 3.94 - 3.83(m, 5H), 3.77 - 3.66(m, 11H), 3.40(br s, 7H), 3.13 - 2.89(m, 14H), 2.69 - 2.66(m, 1H), 2.44 - 2.30(m, 6H), 2.10(s, 14H), 2.08 - 2.01(m, 13H), 2.01 - 1.98(m, 12H), 1.89(s, 12H), 1.78(s, 12H), 1.58(br d, J = 2.9 Hz, 8H), 1.46(br s, 18H), 1.29 - 1.02(m, 15H).

Embodiment 4

Step A:

Compound 4-1 (20 g, 76.95 mmol) was dissolved in 200 ml of chloroform and the temperature was controlled between 0-5 degrees Celsius. Benzene bromide (20.61 g, 192.36 mmol, 20.97 ml, 2.5 eq) was added dropwise over 15 minutes under stirring and the temperature was kept between 0-5 degrees Celsius. N,N-diisopropylethylamine (19.89 g, 153.89 mmol, 26.81 ml, 2 eq) was added slowly at 25 degrees Celsius and the temperature was raised to 70 degrees Celsius for two hours. After the reaction was cooled to 25 degrees Celsius naturally, the organic phase was washed with water (2*200 ml) and saturated brine (2*200 ml) in sequence and dried over anhydrous sodium sulfate. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/ethyl acetate, gradient elution) to obtain compound 4-2 .

Step B:

Palladium carbon (2 g, 10% content) was added to a mixed solution of compound 4-2 (13 g, 41.61 mmol) in methanol (80 ml) and acetic acid (160 ml). After replacing the atmosphere with argon three times and hydrogen three times, the reaction was carried out at 38 degrees Celsius under a hydrogen atmosphere with a pressure of 50 PSI for 15 hours. The reaction solution was cooled and filtered, and the filtrate was concentrated by reducing the pressure. After adding 200 ml of acetonitrile, it was re-concentrated. After repeating twice, the obtained product was dissolved in methanol (125 ml) and cooled to 15 degrees Celsius. 23 ml of concentrated hydrochloric acid was slowly added, and the temperature was kept no higher than 26 degrees Celsius. After concentrating the solution, it was repeated with the same volume of methanol and concentrated hydrochloric acid. The obtained product was slurried with methanol (25 ml) and methyl tert-butyl ether (500 ml) at 25 degrees Celsius for 20 minutes. After filtration, compound 4-3 was obtained. The product was directly used in the next step.

Step C:

Sodium bicarbonate (1.60 g, 19.02 mmol) and Boc ₂ O (2.84 g, 12.99 mmol, 2.99 ml) were added to a suspension of compound 4-3 (1.3 g, 6.34 mmol) in ethanol (13 ml). The reaction solution was stirred at 45 degrees Celsius for 3 hours. After reduced pressure concentration, 50 ml of water was added and extracted with ethyl acetate (50 ml * 2). The combined organic phase was washed with 100 ml of saturated brine and dried over anhydrous sodium sulfate. The filtered filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether/ethyl acetate, gradient elution) to obtain compound 4-4 .

Step D:

Sodium hydroxide (469.29 mg, 11.73 mmol) was added to a mixed solution of compound 4-4 (1.95 g, 5.87 mmol) in tetrahydrofuran (6 ml), methanol (6 ml) and water (6 ml). The reaction solution was stirred at 25 degrees Celsius for 2 hours. After reduced pressure concentration, 20 ml of water was added, and the pH was adjusted to 3-4 with saturated citric acid solution. The solution was extracted with ethyl acetate (100 ml), and the organic phase was washed with 100 ml of saturated brine and concentrated under reduced pressure to obtain compound 4-5 .

Step E:

N,N-diisopropylethylamine (1.85 g, 14.32 mmol, 2.49 ml), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.70 g, 4.48 mmol) and compound 4-6 (550 mg, 1.79 mmol) were added to a DMF solution (6 ml) of compound 4-5 (1.20 g, 3.76 mmol). The reaction solution was reacted at 25 degrees Celsius for 3 hours. Ethyl acetate (100 ml) and water (50 ml) were added to the reaction solution. After separation, the organic phase was dried over anhydrous sodium sulfate, and the filtrate obtained after filtration was concentrated under reduced pressure and purified by silica gel chromatography (petroleum ether/ethyl acetate, gradient elution) to obtain compound 4-7 .

Step F:

Compound 4-7 (1.5 g, 1.80 mmol) was dissolved in ethyl acetate hydrochloride (4 M, 20 ml). The reaction solution was stirred at 25 degrees Celsius for 5 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product of compound 4-8 . The crude product was directly used in the next step without purification.

Step G:

To a solution of compound 2-11 (3.63 g, 6.82 mmol) in N,N-dimethylformamide (30 ml) were added N,N-diisopropylethylamine (3.28 g, 25.36 mmol, 4.42 ml), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.71 g, 7.13 mmol) and compound 4-8 (920 mg, 1.59 mmol). The reaction was stirred at 25 degrees Celsius for 12 hours. After concentration under reduced pressure, the crude product was separated by preparative HPLC to obtain compound 4-9 (column: Kromasil Eternity XT 250*80 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 20%-50%, 20 minutes).

Step H:

Compound 4-9 (1.92 g, 770.27 μmol) was dissolved in 20 ml of methanol, and palladium carbon (300 mg, 10% content) was added. The reaction was replaced with hydrogen three times and then replaced with hydrogen three times, and stirred at 25 degrees Celsius for 12 hours under normal pressure hydrogen atmosphere. After the reaction was completed, it was filtered with diatomaceous earth, and the filtrate was concentrated to obtain compound 4-10 .

Step I:

Compound 4-10 (1.7 g, 707.60 μmol) in N,N-dimethylformamide solution (10 ml) was added with N,N-diisopropylethylamine (365.81 mg, 2.83 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (402.53 mg, 1.06 mmol), and compound 2-14 (330.97 mg, 636.84 μmol). The reaction was stirred at 25 degrees Celsius for 12 hours. After the reaction solution was filtered, the filtrate was spin-dried and purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 46%-76%, 10 min) to obtain compound 4-11 .

Step J:

Triethylamine (112.05 mg, 1.11 mmol), 4-dimethylaminopyridine (33.82 mg, 276.84 mmol) and compound 2-16 (110.82 mg, 1.11 mmol) were added to a dichloromethane solution (8 ml) of compound 4-11 (804 mg, 276.84 mmol). The reaction solution was stirred at 25 degrees Celsius for 12 hours. The reaction solution was filtered and concentrated under reduced pressure, and purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 24%-54%, 10 minutes) to obtain compound D03-M . LCMS ([(M-2)/2], m/z: 1501), ¹ H NMR(DMSO-d6)δ = 9.94 - 9.65(m, 1H), 8.72(br s, 1H), 8.22 - 8.19(m, 1H), 8.15(s, 2H), 8.05(br d, J = 8.0 Hz, 2H), 7.66(s, 1H), 7.58(s, 2H), 5.09 - 4.92(m, 1H), 4.11 - 3.88(m, 3H), 3.53(br s, 3H), 3.48 - 3.32(m, 2H), 3.23(br d, J = 2.1 Hz, 3H), 2.25 - 2.06(m, 3H), 1.91-1.65(m, 3H), 1.64-1.55(m, 1H), 1.34(s, 6H).

Embodiment 5

Step A:

Compound 2-8 (3.68 g, 12.08 mmol), triethylamine (6.11 g, 60.38 mmol, 8.40 ml) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (10.08 g, 26.57 mmol) were dissolved in N,N-dimethylformamide (50 ml) and stirred at 25 degrees Celsius for half an hour. The reaction solution was slowly added dropwise to a solution of compound 2-7 (7.42 g, 24.15 mmol) in N,N-dimethylformamide (50 ml). The reaction solution was stirred at 25 degrees Celsius for half an hour. After the reaction was completed, 500 ml of water was added and extracted with dichloromethane (500 ml*2). The combined organic phases were washed with saturated brine (500 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by silica gel column chromatography to obtain compound 5-1 (gradient elution, petroleum ether/ethyl acetate: 1/1, 1/2, 1/3, 0/1, all containing 0.1% triethylamine).

Step B:

Compound 3-1 (2.86 g, 9.41 mmol), N,N-diisopropylethylamine (3.65 g, 28.24 mmol, 4.92 ml) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.35 g, 14.12 mmol) were dissolved in N,N-dimethylformamide (30 ml), and slowly added to a solution of compound 5-1 (4.9 g, 9.41 mmol) in N,N-dimethylformamide (30 ml). The reaction solution was stirred at 25 degrees Celsius for 12 hours. After the reaction was completed, 400 ml of water was added and extracted with ethyl acetate (400 ml*2). The combined organic phases were washed with saturated brine (500 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by preparative HPLC to obtain compound 5-2 (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; acetonitrile%: 24%-54%, 10 min).

Step C:

Compound 5-2 (6 g, 7.44 mmol) was dissolved in ethyl acetate hydrochloride (4 M, 64.62 ml). The mixture was stirred at 25 degrees Celsius for 3 hours. The mixture was concentrated under reduced pressure to obtain compound 5-3 , which was used directly in the next step without further purification.

Step D:

N,N-diisopropylethylamine (5.62 g, 43.45 mmol, 7.57 ml), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.15 g, 13.58 mmol) and compound 5-3 (1.50 g, 2.72 mmol) were added to a solution of compound 2-11 (6.51 g, 12.22 mmol) in N,N-dimethylformamide (60 ml). The reaction was stirred at 25 degrees Celsius for 12 hours. After adding 400 ml of saturated sodium bicarbonate solution, the aqueous phase (300 ml * 3) was extracted with dichloromethane/isopropanol = 3/1, and the organic phases were combined, dried and filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by preparative HPLC (column: Phenomenex luna C18 150*40 mm*15 μm; mobile phase: [water(trifluoroacetic acid)-acetonitrile]; gradient: 30%-60% acetonitrile, 10 min) to obtain compound 5-4 .

Step E:

A methanol solution (30 ml) of compound 5-4 (2.6 g, 1.05 mmol) was added to palladium carbon (200 mg, 1.05 mmol, 10% content). The reaction was replaced with argon three times and hydrogen three times, and then reacted at 25 degrees Celsius under normal pressure and hydrogen atmosphere for 16 hours. The reaction solution was filtered through diatomaceous earth and concentrated to obtain compound 5-5 , which was directly used in the next step.

Step F:

N,N-diisopropylethylamine (478.99 mg, 3.71 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (478.99 mg, 3.71 mmol) and compound 2-14 (433.99 mg, 833.88 mmol) were added to a solution of compound 5-5 (2.2 g, 926.53 μmol) in N,N-dimethylformamide (20 ml). The reaction solution was stirred at 25 degrees Celsius for 4 hours. After the reaction was completed, the mixture was filtered. The obtained filtrate was concentrated and purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water(ammonium bicarbonate)-acetonitrile]; gradient: 35%-65% acetonitrile, 10 min) to obtain compound 5-6 .

Step G:

A dichloromethane solution (5 ml) of compound 5-6 (500 mg, 46.38 μmol) was added to triethylamine (70.36 mg, 695.38 μmol), 4-dimethylaminopyridine (21.24 mg, 173.84 μmol), 4-dimethylaminopyridine (21.24 mg, 173.84 μmol), and compound 2-16 (69.59 mg, 695.38 μmol). The reaction solution was stirred at 25 degrees Celsius for 12 hours. After concentration under reduced pressure, the product was purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 mm; mobile phase: [water (ammonium bicarbonate-acetonitrile)]; acetonitrile%: 25%-55%, 10 minutes) to obtain compound D02-1-CM . LCMS ([(M-2)/2], m/z: 1487), ¹ H NMR (400 MHz, DMSO-d6) δ = 8.33 - 7.70 (m, 12H), 7.38 - 7.16 (m, 9H), 6.88 (d, J = 8.7 Hz, 4H), 5.21 (d, J = 3.3 Hz, 4H), 4.97 (dd, J = 3.5, 11.2 Hz, 4H), 4.48 (d, J = 8.4 Hz, 4H), 4.09 - 3.97 (m, 14H), 3.92 - 3.81 (m, 5H), 3.76 - 3.66 (m, 11H), 3.47 - 3.36 (m, 5H), 3.16 - 2.88 (m, 14H), 2.68 - 2.65 (m, 4H), 2.42 (s, 7H), 2.34 - 2.31 (m, 4H), 2.10 (s, 15H), 2.04 (br d, J = 3.7 Hz, 12H), 1.99 (s, 13H), 1.89 (s, 12H), 1.77 (s, 12H), 1.63 - 1.37 (m, 27H), 1.30 - 1.11 (m, 14H).

Embodiment 6

Step A:

Compound 6-1 (2 g, 10.68 mmol) was dissolved in a mixed solution of acetonitrile (20 ml) and water (20 ml), and sodium bicarbonate (1.79 g, 21.36 mmol, 831.06 μL) and N-(9-fluorenylmethoxycarbonyloxy)succinimide (4.32 g, 12.82 mmol) were added in sequence. The reaction solution was reacted at 25°C for 12 hours. The reaction solution was diluted with 1 mol/L hydrochloric acid aqueous solution (200 ml), extracted with ethyl acetate (100 ml*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was added to a mixed solution of petroleum ether (10 ml) and ethyl acetate (10 ml), and slurried for 12 hours to obtain compound 6-2 .

Step B:

Compound 6-2 (3.2 g, 7.81 mmol) was dissolved in dichloromethane (50 ml) solution, and N,N-diisopropylethylamine (23.44 mmol, 4.08 ml), 1-hydroxy-7-azabenzotriazole (1.60 g, 11.72 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.25 g, 11.72 mmol) and compound 6-3 (3.11 g, 7.42 mmol) were added in sequence. The reaction solution was reacted at 25°C for 12 hours. The reaction solution was diluted with water (200 ml), extracted with dichloromethane (100 ml*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column to obtain compound 6-4 .

Step C:

Compound 6-4 (3.8 g, 4.69 mmol) and diethylamine (187.42 mmol, 19.31 ml) were dissolved in dichloromethane (30 ml) solution, and the reaction solution was reacted at 25°C for 12 hours. The mixture was concentrated under reduced pressure to obtain compound 6-5 .

Step D:

Under nitrogen protection, compound 4-10 (1.7 g, 707.60 μmol) was dissolved in dichloromethane solution (17 ml), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (203.47 mg, 1.06 mmol), 1-hydroxy-7-azabenzotriazole (144.47 mg, 1.06 mmol), N,N-diisopropylethylamine were added in sequence at 25-30°C. Amine (2.12 mmol, 369.75 μl), the mixed solution was stirred at 25-30°C for half an hour, and then compound 6-5 (603.79 mg, 707.60 μmol) was added, and then stirred at 25-30°C for 12 hours. After the reaction was completed, dichloromethane was removed by concentration at 40-45°C, and purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 30%-60% acetonitrile, 10 minutes) to obtain compound 6-6 .

Step E:

Under nitrogen protection, compound 6-6 (350 mg, 105.94 μmol), succinic anhydride (53.01 mg, 529.69 μmol), 4-dimethylaminopyridine (12.94 mg, 105.94 μmol) and triethylamine (635.63 μmol, 88.47 μl) were mixed in a dichloromethane (3 ml) solution, and the nitrogen was replaced 3 times. Under nitrogen protection, the reaction solution was stirred at 25°C for 16 hours. The reaction was detected to be complete. The dichloromethane was removed by reducing the pressure and concentrating to obtain a crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water(ammonium bicarbonate)-acetonitrile]; gradient: 30%-60% acetonitrile, 10 min) to obtain compound D12-M . ¹ H NMR(400 MHz, DMSO-d6) δ ppm: 7.55 - 8.38 (m, 13 H) 7.11 - 7.40 (m, 9 H) 6.78 - 6.97 (m, 4 H) 5.21 (d, J=3.18 Hz, 4 H) 4.97 (dd, J=11.25, 3.18 Hz, 4 H) 4.49 (d, J=8.44 Hz, 4 H) 4.02 (s, 12 H) 3.81 - 3.93 (m, 7 H) 3.65 - 3.77 (m, 12 H) 2.84 - 3.25 (m, 33 H) 2.49 - 2.50 (m, 3 H) 2.44 (br d, J=19.20 Hz, 6 H) 2.17 - 2.30 (m, 3 H) 2.10 (s, 12 H) 2.01 - 2.08 (m, 14 H) 1.99 (s, 12 H) 1.89 (s, 12 H) 1.77 (s, 12 H) 1.54 - 1.66 (m, 8 H) 1.46 (br s, 20 H) 1.25 (br s, 10 H).

Embodiment 7

Step A:

Compound 6-2 (3 g, 7.33 mmol) was dissolved in tetrahydrofuran (30 ml) solution, and N, N-diisopropylethylamine (1.14 g, 8.79 mmol, 1.53 ml) and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (2.43 g, 8.06 mmol) were added in sequence. The reaction solution was reacted at 25°C for 12 hours. The reaction solution was diluted with water (100 ml) and filtered to obtain a crude product. The crude product was purified by silica gel column (eluent: petroleum ether: ethyl acetate = 2:1) to obtain compound 7-1 .

Step B:

Compound 7-1 (2.86 g, 5.65 mmol) was dissolved in dichloromethane (30 ml) solution, and compound 7-2 (1.44 g, 9.03 mmol) was added. The reaction solution was reacted at 25°C for 12 hours. The reaction solution was diluted with water (200 ml), extracted with dichloromethane (100 ml*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 40%-70% acetonitrile, 10 minutes) to obtain compound 7-3 .

Step C:

Compound 7-3 (2 g, 3.63 mmol) was dissolved in dichloromethane (20 ml) solution, and 4A molecular sieve (2 g), triethylenediamine (488.84 mg, 4.36 mmol) and 4,4-dimethoxytrityl chloride (1.35 g, 3.99 mmol) were added in sequence. The reaction solution was reacted at 25°C for 16 hours. The reaction solution was diluted with water (200 ml), extracted with dichloromethane (100 ml*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by preparative HPLC (column: Kromasil Eternity XT 250*80 mm*10 μm; mobile phase: [water (ammonia water)-acetonitrile]; gradient: 60%-90% acetonitrile, 25 minutes) to obtain compound 7-4 .

Step D:

Compound 7-4 (1.78 g, 2.09 mmol) and diethylamine (7.90 g, 108.00 mmol, 11.13 ml) were dissolved in dichloromethane (15 ml) solution, and the reaction solution was reacted at 25°C for 12 hours. The reaction was detected to be complete. The mixture was concentrated under reduced pressure to obtain compound 7-5 .

Step E:

Under nitrogen protection, compound 4-10 (1.5 g, 624.35 μmol, 1 equivalent) was dissolved in dichloromethane solution (17 ml), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (179.53 mg, 936.53 μmol), 1-hydroxy-7-azabenzotriazole (127.47 mg, 936.53 μmol), N,N-diisopropylethylamine (1.87 mmol, 326.25 μL) were added in sequence at 25-30 degrees Celsius. The mixed solution was stirred at 25-30 degrees Celsius for half an hour, and then compound 7-5 (472.65 mg, 749.22 The mixture was stirred at 25-30°C for 16 hours. After the reaction, the dichloromethane was concentrated and removed at 40-45°C, and the mixture was purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; gradient: 34%-64% acetonitrile, 10 minutes) to obtain compound 7-6 .

Step F:

Under nitrogen protection, compound 7-6 (570 mg, 184.84 μmol), succinic anhydride (83.24 mg, 831.77 μmol), 4-dimethylaminopyridine (22.58 mg, 184.84 μmol) and triethylamine (924.19 μmol, 128.63 μl) were mixed in a dichloromethane (5 ml) solution and replaced with nitrogen three times. Under nitrogen protection, the reaction solution was stirred at 25 degrees Celsius for 16 hours. The reaction was detected to be complete. The dichloromethane was removed by reducing the pressure and concentrating to obtain a crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 μm; mobile phase: [water(ammonium bicarbonate)-acetonitrile]; gradient: 23%-53% acetonitrile, 10 min) to obtain compound D13-M . ¹ H NMR (400 MHz, DMSO-d6) δ ppm 7.65 - 8.35 (m, 13 H) 7.15 - 7.42 (m, 9 H) 6.82 - 6.94 (m, 4 H) 5.21 (d, J=3.18 Hz, 4 H) 4.97 (dd, J=11.25, 3.30 Hz, 4 H) 4.49 (d, J=8.44 Hz, 4 H) 4.02 (s, 12 H) 3.81 - 3.95 (m, 7 H) 3.67 - 3.77 (m, 12 H) 2.89 - 3.25 (m, 33 H) 2.49 - 2.50 (m, 4 H) 2.29 - 2.44 (m, 6 H) 2.10 (s, 12 H) 2.06 (br d, J=13.20 Hz, 14 H) 1.99 (s, 12 H) 1.89 (s, 12 H) 1.77 (s, 12 H) 1.54 - 1.69 (m, 9 H) 1.46 (br s, 21 H) 1.24 (br d, J=6.60 Hz, 10 H) 1.09 (br d, J=10.15 Hz, 3 H) 0.97 (br d, J=2.93 Hz, 3 H).

Experimental Example 1 : Free uptake experiment of human primary hepatocytes - target gene AGT

1. Experimental Principle:

GalNAc can be recognized by the specific ASGPR on the surface of hepatocytes and take up the siRNA conjugated to it into hepatocytes through endocytosis, thereby achieving downregulation of the mRNA level of the target gene by GalNAc-siRNA.

2. Experimental Materials:

Human primary hepatocytes (BioIVT), RNeasy® 96 Kit (12) (QIAGEN-74182), FastKing RT Kit (With gDNase) (Tiangen- KR116-02), AceQ Universal U+ Probe Master Mix V2 (Vazyme-Q513-03 ), TaqMan Gene Expression Assay (GAPDH, Thermo, Assay ID-Hs02786624_g1), TaqMan Gene Expression Assay (AGT, Thermo, Assay ID- Hs01586213_m1).

3. Experimental methods:

The complex of the present invention was diluted to 10 times the concentration to be tested with PBS solution. 10 μL of siRNA was transferred to a 96-well plate. Human primary hepatocytes were thawed and transferred to a collagen-coated 96-well plate, with a final cell density of 5.4×10 ⁵ cells/100 μL/well. The complex of the present invention was tested at 10 concentration points, with the highest concentration being 500 nM, 4-fold dilution, and 2 replicates.

The cells were incubated with the complex of the present invention for 48 hours at 37 degrees Celsius and 5% CO _2. After the incubation, the cells were lysed, all RNA was extracted using RNeasy® 96 Kit (QIAGEN-74182), and cDNA was reverse transcribed using FastKing RT Kit (With gDNase) (Tiangen- KR116-02). The expression level of AGT mRNA was detected by qPCR.

4. Experimental results: The experimental results are shown in Table 3, Table 4, and Table 5.

Table 3 Results of the free uptake experiment of the complex of the present invention in human primary hepatocytes Compound No. Absolute maximum half effective concentration (nM) Target Gene Compound 1 0.101 AGT

Table 4 Results of the free uptake experiment of the complex of the present invention in human primary hepatocytes Compound No. Absolute maximum half effective concentration (nM) Target Gene Compound 2 0.226 AGT Compound 3 0.207 Compound 4 0.220 Compound 5 0.208

Table 5 Results of the free uptake experiment of the complex of the present invention in human primary hepatocytes Compound No. Absolute maximum half effective concentration (nM) Target Gene Compound 6 0.040 AGT Compound 7 0.040

Note: Tables 3, 4 and 5 are from different batches of tests. The activity of human primary hepatocytes is greatly affected by different sources.

Conclusion: The complexes of the present invention exhibited high inhibitory activity against AGT mRNA in human primary hepatocytes, demonstrating that the GalNAc delivery system of the present invention has good liver target delivery ability for siRNA sequences.

Experimental example 2 ：Free uptake experiment of human primary hepatocytes Targeted gene complement C5

1. Experimental Principle:

GalNAc can be recognized by the specific ASGPR on the surface of hepatocytes and take up the siRNA conjugated to it into hepatocytes through endocytosis, thereby achieving downregulation of the mRNA level of the target gene by GalNAc-siRNA.

2. Experimental Materials:

Human primary hepatocytes (BioIVT). The main reagents used in this experiment include HiScript III RT SuperMix for qPCR (Vazyme, Cat. No. R323-01), RNA extraction kit (Qiagen, Cat. No. 74182), AceQ Universal U+ Probe Master Mix V2 (Vazyme, Cat. No. Q513-03), TaqMan Gene Expression Assay (GAPDH, Thermo, Assay ID- Hs02786624_g1) and TaqMan Gene Expression Assay (C5, Thermo, Assay ID- Hs00156197_m1).

3. Experimental methods:

On the first day, siRNA was diluted to 5000 nM with Nuclease-free water as the starting point, and then diluted 4-fold gradient, with a total of 10 concentration points, and then 10 μL was taken to the collagen-coated 96-well cell plate. One human primary hepatocyte was transferred to the preheated InvitroGRO CP Medium complete culture medium, and inoculated into the 96-well cell plate at a density of 54,000 cells per well (90 μL/well), and the final culture medium per well was 100 μL. The complex of the present invention was tested at 10 concentration points, with the highest concentration of 500 nM, 4-fold dilution, and 2 replicates. The cells were placed in a 5% CO ₂ , 37 degrees Celsius incubator for 48 hours. After incubation, the cells were lysed and the obtained lysis buffer was used to extract RNA according to the instructions of the RNA extraction kit (Qiagen, 74182). The RNA was reverse transcribed into cDNA according to the instructions of HiScript III RT SuperMix for qPCR (Vazyme, catalog number R323-01), and then the expression level of C5 mRNA was detected by qPCR.

4. Experimental results: The experimental results are shown in Table 6

Table 6 Results of the free uptake experiment of the complex of the present invention in human primary hepatocytes Compound No. Absolute maximum half effective concentration (nM) Target Gene Compound 8 1.020 C5 Compound 9 1.000

Conclusion: The conjugates of the present invention exhibited high inhibitory activity against C5 mRNA in human primary hepatocytes, demonstrating that the delivery system has good liver target delivery capabilities for siRNA sequences.

Experimental example 3 ：Free uptake experiment of human primary hepatocytes Target gene ANGPTL3

1. Experimental Principle:

GalNAc can be recognized by the specific ASGPR on the surface of hepatocytes and take up the siRNA conjugated to it into hepatocytes through endocytosis, thereby achieving downregulation of the mRNA level of the target gene by GalNAc-siRNA.

2. Experimental Materials:

The main reagents used in this experiment include FastQuant RT Kit (with gDNase) (TianGen, Cat. No. KR106-02), RNA Extraction Kit (Qiagen, Cat. No. 74182), FastStart Universal Probe Master (Rox) (Roche, Cat. No. 04914058001), TaqMan Gene Expression Assay (GAPDH, Thermo, Assay ID- Hs02786624_g1) and TaqMan Gene Expression Assay (ANGPTL3, Thermo, Assay ID- Hs00205581_m1).

3. Experimental methods:

On the first day, siRNA was diluted to 5000 nM with Nuclease-free water as the starting point, and then diluted 4-fold gradient, with a total of 10 concentration points, and then 10 μL was taken to the collagen-coated 96-well cell plate. One human primary hepatocyte was transferred to the preheated InvitroGRO CP Medium complete culture medium, and inoculated into the 96-well plate at a density of 54,000 cells per well (90 μL/well), and the final culture medium per well was 100 μL. The complex of the present invention was tested at 10 concentration points, with the highest concentration being 500 nM, 4-fold dilution, and 2 replicates. The cells were placed in a 5% CO ₂ , 37 degrees Celsius incubator for 48 hours.

RNA was extracted according to the instructions of the RNA extraction kit (Qiagen, 74182), and the RNA was reverse transcribed into cDNA according to the instructions of the FastQuant RT Kit (with gDNase) (TianGen, cat. no. KR106-02), and the level of ANGPTL3 mRNA was detected by qPCR.

4. Experimental results: The experimental results are shown in Table 7

Table 7 Results of the free uptake experiment of the complex of the present invention in human primary hepatocytes Compound No. Absolute maximum half effective concentration (nM) Target Gene Compound 10 1.571 ANGPTL3

Conclusion: The complexes of the present invention exhibited high inhibitory activity against ANGPTL3 mRNA in human primary hepatocytes, demonstrating that the GalNAc delivery system of the present invention has good liver-targeted delivery capabilities for siRNA sequences.

Experimental example 4 ：In vivo activity test in mice Target gene AGT

1. Experimental Principle:

By rapidly injecting physiological saline containing the target gene recombinant plasmid 2-pcDNA-CMV-AGT through the caudal vein at high pressure, the target gene AGT was efficiently expressed in the mouse liver, thereby evaluating the knockdown effect of the test complex on the target gene after entering the mouse body.

2. Experimental Materials:

2-pcDNA-CMV-AGT plasmid, BALB/c mice, PBS (phosphate buffered saline), conjugate of the present invention.

3. Experimental methods:

Order 6-8 week old female BALB/c mice and acclimate them to quarantine for one week after arrival at the animal facility.

On day 0, mice were randomly divided into groups according to body weight data. After grouping, all mice were given subcutaneous injection of drugs. The drug volume was 10 mL/kg for a single dose. The mice in group 1 were given PBS; the mice in other groups were given the complex.

On the third day after drug administration, all mice were injected with physiological saline containing 2-pcDNA-CMV-AGT plasmid via the tail vein within 5 seconds. The injection volume (mL) = mouse weight (g) × 8%, and the mass of the injected plasmid for each mouse was 10 µg.

On the 4th day after drug administration, mice in all groups were euthanized by CO ₂ inhalation, and two liver samples were collected from each mouse. The liver samples were treated with RNAlater at 4°C overnight, then RNAlater was removed and stored at -80°C for detection of AGT gene expression.

4. Experimental results: The experimental results are shown in Tables 8 and 9.

Table 8 Results of in vivo activity test of the conjugate of the present invention (4 mice per group) Compound No. Dosage (mg/kg) AGT mRNA down-regulation percentage Standard Deviation % (SD) Compound 2 1 42.26 4.66 Compound 3 1 48.44 8.05 Compound 4 1 57.24 8.37 Compound 5 1 49.98 4.18

Note: "AGT mRNA downregulation percentage" refers to the downregulation percentage of AGT-mRNA in the liver of mice in the drug group relative to the PBS blank group.

Table 9 Results of in vivo activity test of the conjugate of the present invention (3 mice per group) Compound No. Dosage (mg/kg) AGT mRNA down-regulation percentage Standard Deviation % (SD) Compound 5 0.1 8.56 32.92 Compound 5 0.3 10.39 30.60 Compound 5 1 28.55 15.21 Compound 5 3 74.61 3.94

Note: "AGT mRNA downregulation percentage" refers to the downregulation percentage of AGT-mRNA in the liver of mice in the drug group relative to the PBS blank group.

Conclusion: The conjugates of the present invention can down-regulate AGT-mRNA in the liver in the AGT-HDI mouse model, and the down-regulation activity shows dose dependence. Since this test is the mRNA level in the liver, it can be proved that the GalNAc delivery system of the present invention can effectively carry out sequential liver target delivery.

Experimental example 5 ：In vivo activity test in mice Target gene AGT

1. Experimental Principle:

By rapidly injecting physiological saline containing the target gene recombinant plasmid 2-pcDNA-CMV-AGT through the caudal vein at high pressure, the target gene AGT was efficiently expressed in the mouse liver, thereby evaluating the knockdown effect of the test complex on the target gene after entering the mouse body.

2. Experimental Materials:

2-pcDNA-CMV-AGT plasmid, BALB/c mice, PBS (phosphate buffered saline), conjugate of the present invention.

3. Experimental methods:

Order 6-8 week old BALB/c female mice. After the mice arrive at the animal house, they are quarantined for one week. The day of the high pressure tail vein of the mouse is defined as day 0 of the experiment, the day before is day -1, the day after is day 1, and so on.

On day -14, mice were randomly divided into groups according to body weight data, with 4 mice in each group. After grouping, 3 groups of mice were given drugs by subcutaneous injection, with a single dose of 10 mL/kg. The first group of mice was given PBS; the other two groups of mice were given the mixture, and the remaining groups of mice were raised normally without drug administration.

On day -3, the remaining groups of mice were given the complex.

On day 0, all mice were injected with 2-pcDNA-CMV-AGT plasmid in saline solution via the tail vein within 5 seconds (injection volume (mL) = mouse weight (g) × 8%), and the mass of the injected plasmid was 10 µg per mouse.

On day 1, mice in all groups were euthanized by CO ₂ inhalation, and two liver samples were collected from each mouse. The liver samples were treated with RNAlater at 4°C overnight, then RNAlater was removed and stored at -80°C for detection of AGT gene expression.

4. Experimental results: The experimental results are shown in Table 10

Table 10 Results of in vivo activity test of the conjugate of the present invention Compound No. Dosage (mg/kg) Time of medication AGT mRNA downregulation percentage % (nM) Standard Deviation % (SD) Compound 6 3 -3 days 76.10 10.27 Compound 6 3 -14 days 62.26 16.85 Compound 7 3 -3 days 73.40 11.65 Compound 7 3 -14 days 65.38 6.14

Note: "AGT mRNA downregulation percentage" refers to the downregulation percentage of AGT-mRNA in the liver of the drug-treated mice relative to the PBS blank group.

Conclusion: Within the test time, all the complexes down-regulated AGT mRNA in the AGT-HDI mouse model. Since this test is the mRNA level in the liver, it can be proved that the GalNAc delivery system of the present invention can effectively carry out sequential liver target delivery.

Experimental example 6 ：In vivo activity test in mice Targeted gene complement C5

1. Experimental Principle:

C57BL/6 mice express complement C5. The test sample can reach the liver after subcutaneous injection into the mouse body, thereby inhibiting the expression of the target gene C5 in liver cells. By measuring the concentration of target protein C5 in mouse plasma at different time points after administration, the in vivo activity and long-term effect of the test sample can be evaluated.

2. Experimental Materials:

C57 BL/6 mice, PBS (phosphate buffered saline), conjugate of the present invention.

3. Experimental methods:

Two days before siRNA administration (Day -2), plasma samples were collected from C57BL/6 (female, 7 weeks old) mice, and the C5 protein level in mouse plasma was measured by ELISA (Abcam). Mice were selected according to the test results and randomly divided into groups, with 5 mice in each group.

All animals were dosed according to their body volume. On Day 0, a single subcutaneous injection was given, and the siRNA administration volume was 10 mL/kg. Plasma was collected from mice on Days 7, 14, 21, and 28 after administration, and the concentration of C5 protein in mouse plasma was determined by ELISA. The relative expression of C5 protein in the plasma of mice in each group was used to evaluate the in vivo effectiveness of different siRNAs. The relative expression of C5 protein was calculated according to the following formula: C5 relative expression = C5 concentration of the administration group at different time points / C5 concentration in the plasma of the administration group on Day-2 × 100%.

4. Experimental results: The experimental results are shown in Table 11 and Table 12.

Table 11 Results of in vivo activity test of the conjugates of the present invention Compound No. Dosage (mg/kg) C5 protein downregulation percentage (Day 7) Standard Deviation % (SD) C5 protein downregulation percentage (Day 14) Standard Deviation % (SD) C5 protein downregulation percentage (day 21) Standard Deviation % (SD) C5 protein downregulation percentage (day 28) Standard Deviation % (SD) Compound 11 1 89.56 3.27 83.52 4.70 77.29 4.72 67.29 7.97 Compound 11 5 97.99 0.24 97.99 0.24 96.47 2.27 92.44 3.08

Note: "C5 protein downregulation percentage" refers to the percentage of C5 protein concentration in the plasma of mice in each drug group at different time points relative to the plasma C5 protein of each group of mice before drug administration (Day-2)

Table 12 Results of in vivo activity test of the conjugates of the present invention Compound No. Dosage (mg/kg) C5 protein relative expression % (Day 7) Standard Deviation % (SD) Relative expression of C5 protein (day 14) Standard Deviation % (SD) Relative expression of C5 protein (day 21) Standard Deviation % (SD) Relative expression of C5 protein (day 28) Standard Deviation % (SD) Compound 8 0.5 15.92 2.42 12.93 2.73 23.55 5.23 24.76 6.18 Compound 8 1 6.47 1.65 7.85 0.89 10.78 1.20 12.75 1.83 Compound 9 0.5 14.08 3.95 10.86 2.06 24.71 3.86 19.01 3.59 Compound 9 1 6.72 1.50 6.19 0.88 9.89 1.87 11.58 1.86

Note: "Relative expression of C5 protein" refers to the percentage of plasma C5 protein concentration of mice in each group at different time points after drug administration relative to the concentration on the second day before drug administration (Day-2) of each group.

Conclusion: This experiment shows that the complex of the present invention has a sustained inhibition on C5 protein and the dose-dependency of the inhibitory effect, and has excellent effectiveness and long-term effect. At the same time, it shows good sustained inhibition on C5 protein and the dose-dependency of the inhibitory effect. Since the complement C5 mRNA has the characteristics of liver origin, the results of this experiment show the good liver-targeted delivery ability of the GalNAc delivery system.

without

without

TWI866590B_112142933_SEQL.xml

Claims (13)

A conjugated group represented by formula (V),

Among them, L ₁ is selected from

and

; L ₂ selected from

and

;

Selected from

,

and

; n is selected from 0 and 1; t is selected from 2, 3, 4, 5, 6 and 7. The conjugated group as described in claim 1, wherein the conjugated group is (D02):

The conjugated group as claimed in claim 1, wherein the conjugated group is selected from (D02-1-A), (D02-1-B), (D02-1-C), (D03), (D12) and (D13):

A conjugate or a pharmaceutically acceptable salt thereof, wherein the conjugate is selected from a compound formed by linking a conjugate group as described in any one of claims 1 to 3 to an oligonucleotide via a phosphodiester bond or a phosphorothioate diester bond. The conjugate or a pharmaceutically acceptable salt thereof as described in claim 4, wherein the oligonucleotide is selected from RNAi reagents and ASO reagents. The conjugate or a pharmaceutically acceptable salt thereof as described in claim 5, wherein the RNAi agent is selected from single-stranded oligonucleotides and double-stranded oligonucleotides. The conjugate or a pharmaceutically acceptable salt thereof as described in claim 6, wherein the single-stranded oligonucleotide is selected from single-stranded antisense oligonucleotides. The conjugate or a pharmaceutically acceptable salt thereof as described in claim 6, wherein the double-stranded oligonucleotide is selected from double-stranded siRNA. A conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 4 to 8, wherein the nucleotides of the oligonucleotide are optionally modified. A use of a conjugated group as described in any one of claims 1 to 3 in the preparation of a drug for enhancing the binding of a therapeutic agent to a specific target site. An intermediate compound for preparing the conjugated group as described in claim 1, whose structure is shown in formula (IM), (V-M12) and (V-M13):

DMTr represents dimethoxytrityl; L ₁ , L ₂ , t and n are as defined in claim 1. An intermediate compound for preparing the conjugated group as described in claim 2, whose structure is shown in formula (D02-M):

DMTr stands for dimethoxytrityl. An intermediate compound for preparing the conjugated group as described in claim 3, wherein the structures thereof are respectively shown in formulas (D02-1-AM), (D02-1-BM), (D02-1-CM), (D03-M), (D12-M) and (D13-M):

DMTr stands for dimethoxytrityl.