GalNAc monomer containing ribose ring or derivative structure thereof and application of GalNAc monomer in hepatic targeting delivery of small nucleic acid drugs

Abstract

The invention provides a GalNAc monomer containing a ribose ring or a derivative structure of the ribose ring and application of the GalNAc monomer in hepatic targeting delivery of small nucleic acid drugs. A compound of formula (I) or a pharmaceutically acceptable salt thereof is provided. According to the GalNAc-conjugated oligonucleotide prepared by the GalNAc compound provided by the invention, efficient liver targeting delivery can be realized, and the drug curative effect is improved. # imgabs0 #

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and in particular to a GalNAc compound having a ribose ring structure, and a GalNAc-conjugated oligonucleotide prepared therefrom can achieve efficient liver-targeted delivery. Background Art

[0002] Nucleic acid drugs, especially oligonucleotide drugs, are widely used due to their simple synthesis and high activity. Oligonucleotide drugs generally include antisense oligonucleotides (ASO), small interfering RNA (siRNA), micro RNA (miRNA) and nucleic acid aptamers.

[0003] Oligonucleotides are a class of short DNA or RNA molecules, oligomers, that readily bind to their respective complementary oligonucleotides, DNA or RNA in a sequence-specific manner to form duplexes, or less commonly, higher order hybrids. This fundamental property makes oligonucleotides widely used in genetic testing, targeted gene therapy research, and medicine. These small fragments of nucleic acid can be manufactured as single-stranded molecules with any specified sequence. In nature, oligonucleotides are usually small RNA molecules that play a role in the regulation of gene expression, or degradation intermediates derived from the decomposition of larger nucleic acid molecules.

[0004] RNA interference is a natural defense mechanism against foreign genes. siRNA can knock out target genes by recognizing specific sequences and degrading target mRNA.

[0005] N-acetylgalactosamine (GalNAc) is a ligand that binds to the asialoglycoprotein receptor (ASGPR) on the liver surface. The asialoglycoprotein receptor is an endocytic receptor specifically expressed on the surface of hepatocytes. In recent years, some progress has been made in the liver-targeted delivery of nucleic acid drugs using the high-affinity ligand GalNAc of ASGPR as a targeting molecule. For example, Alnylam ( Pharmaceuticals, Inc. reported that siRNA based on GalNAc conjugation technology exerted gene silencing activity in mice (Nair JK, et al. J. Am. Chem. Soc. 2014, 136, 16958). The article reported that the conjugate of GalNAc and siRNA showed good delivery activity in both in vivo and in vitro experiments. In vivo experiments in mice with subcutaneous administration, a single dose of ED 50The dose was determined to be 1 mg / kg, and the single injection dose was less than 1 mL. In long-term dosing experiments, subcutaneous injection once a week can obtain stable interference activity for up to 9 months. The study found that the affinity of tetra-antennary and tri-antennary GalNAc compounds for ASGPR is much higher than that of bi-antennary and mono-antennary GalNAc compounds.

[0006] GalNAc compounds with different structures have very different effects on nucleic acid delivery. In order to improve the delivery effect of liver-targeted drugs, such as AGT inhibitors, anti-chronic hepatitis B infection drugs, and lipid-lowering drugs, there is a need in the art to develop new GalNAc compounds. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a GalNAc monomer containing a ribose ring or its derivative structure and its application in liver-targeted delivery of small nucleic acid drugs. The GalNAc compound provided by the present invention can be used to prepare GalNAc-conjugated oligonucleotides, the delivery efficiency is significantly improved, and the AGT gene expression is significantly inhibited.

[0008] The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0009]

[0010] in,

[0011] R 1 is oxygen or sulfur;

[0012] R 2 For hydrogen, C 1-6 Alkyl, C 1-6 Alkoxy or halogen;

[0013] R 3 Hydrogen, hydroxyl protecting group, phosphorus-containing reactive group, -CO(CH 2 ) x CONH- or -CO(CH 2 ) x COOH, wherein x is an integer from 1 to 10, It is controlled pore glass or polystyrene;

[0014] R 4 is hydrogen or a hydroxyl protecting group;

[0015] A is -(CH 2 ) a -、-(CH 2 CH 2 OCH 2 CH 2 )b -or-(CH 2 OCH 2 ) c -, wherein a is an integer from 1 to 10; b is an integer from 1 to 5; c is an integer from 1 to 7;

[0016] L is -CONH- or -NHCO-;

[0017] G is

[0018] in,

[0019] T is a hydroxyl group fully protected with an acyl group: N-acetyl-galactosamine, galactose, galactosamine, N-formal-galactosamine, N-propionyl-galactosamine, and N-butyryl-galactosamine;

[0020] X 1 For -(CH 2 ) f -or-(CH 2 CH 2 O) f CH 2 -, f is an integer from 1 to 5;

[0021] X 2 For -(CH 2 ) g -, g is an integer from 1 to 6;

[0022] Y 1 is 0 or 1;

[0023] Y 2 is 0, 1, or 2;

[0024] Y 3 is 1, 2, or 3;

[0025] m is an integer from 0 to 4;

[0026] n is an integer from 0 to 4.

[0027] In a certain embodiment, in the compound of formula (I) or a pharmaceutically acceptable salt thereof, the definitions of certain groups may be as described below, and the definitions of other groups may be as described in any of the above embodiments (hereinafter referred to as "a certain embodiment"):

[0028] In one embodiment, the C 1-6 The alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

[0029] In one embodiment, the C 1-6 Alkoxy is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy or tert-butoxy.

[0030] In one embodiment, the halogen is fluorine, chlorine or bromine.

[0031] In one embodiment, A is -CH 2 CH 2 OCH 2 CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -、-(CH 2 ) 4 -or-(CH 2 ) 5 -.

[0032] In one scheme, R 1 For oxygen.

[0033] In one scheme, R 1 It is an α configuration or a β configuration, for example an α configuration.

[0034] In one scheme, R 2 C 1-6 Alkoxy, such as -OCH 3 .

[0035] In one scheme, R 3 In the embodiment, the hydroxyl protecting group is acyl, silyl, trityl, 4-methoxytrityl, 4,4'-bismethoxytrityl, preferably acetyl, 1,1,3,3-tetraisopropyldisiloxane, tert-butyldimethylsilyl, dimethylphenylsilyl, 4,4'-bismethoxytrityl.

[0036] In one scheme, R 3 is a phosphorus-containing reactive group, such as

[0037] In one scheme, R 3 is a phosphorus-containing reactive group, -CO(CH 2 ) x CONH- is controlled pore glass (CPG) or polystyrene, and preferably, x is 2.

[0038] In one scheme, R 3 -CO(CH 2 ) 2 CONH- It is controlled pore glass (CPG).

[0039] In one scheme, R 3-CO(CH 2 ) 2 COOH.

[0040] In one scheme, R 4 It is a hydroxyl protecting group, preferably a trityl group, a 4-methoxytrityl group or a 4,4'-bismethoxytrityl group, and more preferably a 4,4'-bismethoxytrityl group.

[0041] In one embodiment, T is N-acetyl-galactosamine fully protected by acyl groups, and the acyl group may be acetyl or benzoyl, such as acetyl.

[0042] In one scenario, X 1 For -(CH 2 ) f -, f is preferably 1.

[0043] In one embodiment, a is an integer from 2 to 7.

[0044] In one embodiment, b is an integer from 1 to 3.

[0045] In one solution, g is 2.

[0046] In one scenario, Y 1 is 0.

[0047] In one scenario, Y 2 is 0.

[0048] In one scenario, Y 3 is 1.

[0049] In one embodiment, m is 0, 1 or 2, for example 1.

[0050] In one embodiment, n is 0, 1 or 2, for example 0.

[0051] In one embodiment, L is -NHCO-.

[0052] In one scheme, G is

[0053]

[0054] In one embodiment, the compound of formula (I) is a compound represented by the following formula I-1;

[0055]

[0056] Among them, R 2 , R 3 , R 4 , A and X 2 Independently as described in any embodiment of the present invention. In one embodiment, the compound represented by formula (I) is any of the following compounds:

[0057]

[0058]

[0059] In one embodiment, the compound represented by formula (I) is any of the following compounds:

[0060]

[0061]

[0062] It is controlled pore glass (CPG).

[0063] In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof can bind to asialoglycoprotein receptor (ASGPR).

[0064] The present invention provides a use of the compound of formula (I) or a pharmaceutically acceptable salt thereof as an intermediate in the preparation of a GalNAc-conjugated oligonucleotide drug.

[0065] The present invention provides a conjugate comprising an oligonucleotide and a GalNAc moiety; the oligonucleotide and the GalNAc are linked via a phosphate group or a phosphorothioate group (via R 3-1 or R 4-1 The GalNAc portion is one or more GalNAc molecules, which are connected by a phosphate group or a thiophosphate group; wherein the GalNAc molecule is a compound of formula (I) or a pharmaceutically acceptable salt thereof;

[0066]

[0067] Among them, R 3-1 is the connecting key, R 4 is H;

[0068] Or, R 4-1 is the connecting key, R 3-1 is H;

[0069] G is

[0070] T1 is N-acetyl-galactosamine, galactose, galactosamine, N-formal-galactosamine, N-propionyl-galactosamine and N-butyryl-galactosamine, preferably N-acetyl-galactosamine;

[0071] Y 3 , Y 2 , X 2 , Y 1 , X1 , m, n, R 2 , R1, A, and L are as described in any embodiment of the present invention.

[0072] In a certain embodiment, in the conjugate, the oligonucleotide may include a non-thiolated oligonucleotide and a thiolated oligonucleotide.

[0073] In one embodiment, in the conjugate, the non-thio oligonucleotide and the GalNAc moiety may be linked via a phosphate bond.

[0074] In one embodiment, in the conjugate, the phosphorothioate oligonucleotide and the GalNAc moiety may be linked via a phosphorothioate bond.

[0075] In a certain embodiment, the conjugate preferably has the following structure:

[0076]

[0077] Among them, Oligo represents oligonucleotide, X 3 is oxygen or sulfur, q is 1, 2 or 3;

[0078] R 1 , R 2 , R 3 , R 4 , A, L, G, m and n are independently as described in any embodiment of the present invention.

[0079] In a certain embodiment, in the conjugate, the oligonucleotide preferably includes small interfering nucleotides (siRNA), DNA, micro RNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), antisense nucleotides (ASO) or aptamers (Aptamer), preferably antisense nucleotides (ASO) or small interfering nucleotides (siRNA).

[0080] In a certain embodiment, in the conjugate, each nucleotide in the antisense nucleotide (ASO) or small interfering nucleotide (siRNA) is preferably independently a modified or unmodified nucleotide.

[0081] In one embodiment, the conjugate preferably has the following structure:

[0082]

[0083]

[0084]

[0085] In the conjugate, the oligonucleotide regulates the expression of the target gene.

[0086] The present invention provides a pharmaceutical composition comprising the conjugate according to any embodiment of the present invention and at least one pharmaceutically acceptable excipient.

[0087] The present invention provides a use of a conjugate or a pharmaceutically acceptable salt thereof according to any embodiment of the present invention, or a pharmaceutical composition according to any embodiment of the present invention, in the preparation of a drug for treating and / or preventing a pathological condition or disease caused by the expression of a specific gene in liver tissue or in a virus; optionally, the specific gene is selected from hepatitis B (HBV) virus gene, proprotein convertase subtilisin / kexin type 9 (PCSK9) gene, coagulation factor gene, lipoprotein a gene, angiopoietin-like protein 3 gene, angiotensinogen gene or apolipoprotein C3 gene, etc. Preferably, the disease is selected from chronic liver disease, hepatitis, liver fibrosis disease, liver proliferative disease and cardiovascular and cerebrovascular disease; optionally, the cardiovascular and cerebrovascular disease is hypercholesterolemia, hypertriglyceridemia, atherosclerosis or coagulation dysfunction, etc.

[0088] The present invention provides a method for inhibiting the expression of specific genes in hepatocytes, wherein the method comprises administering a therapeutically effective amount of any of the aforementioned conjugates to an individual in need thereof, preferably, the method comprises contacting an effective amount of any of the aforementioned conjugates with the hepatocytes; optionally, the specific gene is selected from the proprotein convertase subtilisin / kexin type 9 gene (PCSK9), hepatitis B virus gene, apolipoprotein a gene, coagulation factor XI gene, angiopoietin-like protein 3 gene, angiotensinogen gene or apolipoprotein C3 gene.

[0089] The present invention provides a kit, which comprises the conjugate described in any embodiment of the present invention.

[0090] Explanation of terms:

[0091] The term "pharmaceutically acceptable" means relatively non-toxic, safe, and suitable for use by patients.

[0092] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. For details, please refer to Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition).

[0093] The term "acyl" refers to R-CO-, where R is C 1-6 Alkyl, C 6-14 Aryl or substituted C 1-6 Alkyl, substituted C 6-14 Aryl, substituted by halogen, cyano, hydroxyl or C 6-14 Aryl.

[0094] All terms therapeutically effective amount refer to an amount of a compound that is sufficient to effectively treat a disease or condition described herein when administered to a subject. Although the amount of a compound that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, and the age of the subject to be treated, it can be determined in a routine manner by those skilled in the art.

[0095] All terms subject, individual, and individual in need refer to any animal that is about to or has received the conjugate or pharmaceutical composition according to the embodiments of the present disclosure, preferably a mammal, and most preferably a human. As used herein, all terms "mammal" include any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., with humans being the most preferred.

[0096] In certain embodiments, treatment or treating refers to the improvement, prevention, or reversal of a disease or condition or at least one discernible symptom thereof. In other embodiments, treatment or treating refers to the improvement, prevention, or reversal of at least one measurable physical parameter of the disease or condition being treated, which disease or condition may not be identified in a mammal. In yet another embodiment, treatment or treating refers to slowing the progression of a disease or condition, either physically, such as stabilization of discernible symptoms, or physiologically, such as stabilization of a physical parameter, or both. In other embodiments, treatment or treating refers to delaying the onset of a disease or condition.

[0097] In certain embodiments, the compounds of the present disclosure may be administered as a preventive measure. As used herein, "prevention" or "preventing" refers to reducing the risk of acquiring a given disease or condition. In a preferred mode of the embodiments, a given compound is administered as a preventive measure to a subject, such as a subject with a family history or predisposition to cancer or an autoimmune disease.

[0098] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0099] The reagents and raw materials used in the present invention are commercially available.

[0100] The GalNAc compound provided in the present application and the GalNAc-conjugated oligonucleotide prepared therefrom have one or more of the following beneficial effects:

[0101] 1. This application designs a series of novel GalNAc compounds, which have completely different chemical structures compared with the prior art GalNAc compounds.

[0102] 2. The GalNAc-conjugated oligonucleotides prepared from the GalNAc compounds designed in the present application have significantly improved delivery efficiency compared with the GalNAc-conjugated oligonucleotides prepared from the GalNAc compounds of the prior art (such as L96 and YK-GAL-325), and significantly improved the inhibition rate of AGT protein expression in mouse serum and the inhibition rate of AGT mRNA level in the liver. DETAILED DESCRIPTION

[0103] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the described embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0104] The present application may be implemented in other specific forms without departing from the basic attributes of the present application. It should be understood that, under the premise of no conflict, any and all embodiments of the present application may be combined with the technical features in any other embodiment or multiple other embodiments to obtain other embodiments. The present application includes other embodiments obtained by such combination.

[0105] All publications and patents mentioned in this application are hereby incorporated into this application by reference in their entirety. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in this application, then the purposes and terms of this application shall prevail.

[0106] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0107] Unless otherwise specified, all technical and scientific terms used herein have the common meaning in the field to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0108] Except in the working examples or otherwise indicated, all numbers of quantitative properties such as dosage stated in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range recited in the application is intended to include all subranges within the range and any combination of the respective endpoints of the range or subrange.

[0109] The words "include", "contain" or "comprises" and the like used in this application mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unrecorded elements. The terms "contain" or "includes" used herein may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0110] The present application is further described below in conjunction with the examples. However, the present application is not limited to the following examples. The implementation conditions adopted in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. In the specific examples in the present application, the raw materials used can be obtained commercially. Unless otherwise stated, all temperatures are given in degrees Celsius. The technical features involved in each embodiment of the present application can be combined with each other as long as they do not conflict with each other.

[0111] Preparation Example: Synthesis of GalNAc Compounds

[0112] The following abbreviations represent the following reagents: TEA: triethylamine; DCM: dichloromethane; 3A MS: 3A molecular sieve; MeONa: sodium methoxide; MeOH: methanol; imidazole: imidazole; Pyridine: pyridine; NaOH: sodium hydroxide; DIPEA: diisopropylethylamine; THF: tetrahydrofuran; BF 3 ·Et 2O: Boron trifluoride etherate; DMTrCl: 4,4'-dimethoxytriphenylmethane; HBTU: O-benzotriazole-tetramethyluronium hexafluorophosphate; DIPEA: N,N-diisopropylethylamine; DMAP: 4-dimethylaminopyridine; TIPDSCl: 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane; TEA·3HF: triethylamine trihydrofluoride; Protonsponge: 1,8-bis(dimethylaminonaphthalene); Me 3 OBF 4 : trimethyloxonium tetrafluoroborate; DMF: N,N-dimethylformamide; Ac 2 O: acetic anhydride.

[0113] 1. Synthesis of GalNAc phosphoramidite compounds

[0114] (1) Synthesis of YK-GAL-501

[0115] The synthetic route is as follows:

[0116]

[0117] Step 1: Synthesis of G1-3

[0118] Add dichloromethane (1500mL) to the dried compound G1-1 (100.0g, 951.2mmol), then add triethylamine (107.3g, 1.06mol), cool to 0℃, and then slowly add G1-2 (121.7g, 865.1mmol) at 0~5℃. After the addition, warm to room temperature and continue stirring for 2h. Cool to 0℃, add 1mol / L hydrochloric acid to adjust the pH to 6~7, add saturated sodium chloride aqueous solution (300mL), shake and separate, extract the aqueous phase with dichloromethane (800mL×3), and combine the organic phases. Dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure to remove the solvent, and obtain a colorless oily crude product G1-3 (105.0g), which is directly used in the next step. 1HNMR(400MHz,DMSO-d6)δ8.48(t,J=5.6Hz,1H),7.84(dd,J=7.2,1.9Hz,2H),7.56–7.49 (m,1H),7.46(dd,J=8.2,6.5Hz,2H),4.42(s,1H),3.57–3.48(m,4H),3.48–3.38(m,4H). MS(ESI)m / z[M+H] + =210.4.

[0119] Step 2: Synthesis of G1-5

[0120] Add dichloromethane (450 mL) to compound G1-4 (87.2 g, 274.0 mmol), stir to dissolve, add 3A molecular sieve (80.0 g), cool to 0 ° C, add boron trifluoride etherate (116.9 g, 823.7 mmol), and stir for 20 min after addition. Add a dichloromethane (180 mL) solution of G1-3 (86.0 g, 411.0 mmol) dropwise to the system at 0-5 ° C, and stir overnight at 0-5 ° C after addition. Filter, control the temperature at 0-5 ° C, add a saturated sodium bicarbonate aqueous solution (125 mL) to the filtrate to quench the reaction, separate the liquids, wash the organic phase with a saturated sodium bicarbonate aqueous solution (900 mL × 2) and a semi-saturated sodium chloride aqueous solution (900 mL) in turn, dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain a crude yellow oil product G1-5 (150.0 g), which is directly used in the next step. 1H NMR(400MHz, DMSO-d6)δ8.47(t,J=5.6Hz,1H),7.84(dt,J=7.0,1.4Hz,2H),7.56–7.48(m, 1H),7.45(dd,J=8.2,6.6Hz,2H),5.17(dd,J=6.7,4.9Hz,1H),5.12–5.05(m,2H),4.28(dd, J=11.7,3.9Hz,1H),4.25–4.18(m,1H),4.04(dd,J=11.7,5.6Hz,1H),3.71(qd,J=8.6,7.4 ,4.4Hz,1H),3.62–3.50(m,5H),3.42(q,J=5.9Hz,2H),2.06(s,3H),2.02(d,J=2.0Hz,6H). MS(ESI)m / z[M+Na] + =490.2.

[0121] Step 3: Synthesis of G1-6

[0122] G1-5 (150.0 g, 320.9 mol) was dissolved in methanol (1200 mL), the system was cooled to 0 ° C, the temperature was controlled at 0-5 ° C, and a methanol solution (300 mL) of sodium methoxide (3.50 g, 64.8 mmol) was added dropwise to the system, and the temperature was raised to room temperature for 1 h. The temperature was lowered to 0-5 ° C, the temperature was controlled at 0-5 ° C, 1 mol / L hydrochloric acid was added dropwise to adjust the pH to 6-7, the system was concentrated under reduced pressure to remove the solvent, purified water (200 mL) was added, and the system was washed with dichloromethane (400 mL × 8), the aqueous phase was collected and concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by column chromatography (dichloromethane / methanol) to obtain a colorless transparent oil G1-6 (69.0 g, 202.1 mmol), and the combined yield of the two steps was 73.8%. 1H NMR (400MHz, DMSO-d6) δ8.48(t,J=5.6Hz,1H),7.88–7.81(m,2H),7.56–7.49(m,1H),7.46(dd,J=8.1,6.5Hz,2H),4.89(d,J=1.5Hz,1H),4.83(d, J=6.9Hz,1H),4.54(t,J=5.7Hz,1H),4.30(dd,J=6.9,4.4Hz,1H),3.98(t d,J=6.6,4.7Hz,1H),3.77–3.67(m,2H),3.56–3.46(m,6H),3.35(s,4H). MS(ESI)m / z[MH] - =340.3.

[0123] Step 4: Synthesis of G1-7

[0124] G1-6 (69.0 g, 202.1 mmol) was dissolved in dichloromethane / tetrahydrofuran (5 / 1, 700 mL), imidazole (34.4 g, 505.3 mmol) was added, the temperature was lowered to 0°C, 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (66.8 g, 211.8 mmol) was added dropwise at a temperature of 0-5°C, stirred for 10 min after the addition, and stirred for 2 h after the addition was heated to room temperature. After the reaction was completed, purified water (700 mL) was slowly added dropwise to the reaction solution, and the mixture was shaken and separated after the addition was completed. The organic phase was washed with a saturated aqueous sodium chloride solution (700 mL), and the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure to obtain a crude product, and n-heptane (700 mL) was added for pulping, filtered, and the filter cake was collected and dried under vacuum to obtain a white solid G1-7 (74.3 g, 127.3 mmol) with a yield of 63.0%. 1H NMR(400MHz, DMSO-d6)δ8.46(t,J=5.6Hz,1H),7.87–7.80(m,2H),7.56–7.41(m,3H),5.06(d,J=3.9Hz,1 H), 4.76 (s, 1H), 4.30 (dd, J = 6.9, 4.4Hz, 1H), 3.92–3.75 (m, 4H), 3.67–3.36 (m, 8H), 1.08–0.84 (m, 28H). MS(ESI)m / z[MH] - =582.5.

[0125] Step 5: Synthesis of G1-8

[0126] G1-7 (70.0 g, 119.9 mmol) was dissolved in dichloromethane (700 mL), 1,8-bis(dimethylamino)naphthalene (64.0 g, 298.6 mmol) was added, the temperature was lowered to 0°C, trimethyloxonium tetrafluoroboric acid (35.5 g, 240.0 mmol) was added, the mixture was stirred for 10 min, the mixture was heated to room temperature and stirred for 2 h. After the reaction was completed, purified water (5.4 mL) was added to quench the mixture, the mixture was stirred for 10 min, filtered, the filtrate was collected and concentrated under reduced pressure to remove the solvent, and then n-heptane (700 mL) was added and stirred for 20 min, filtered again, the filtrate was collected, the filtrate was washed once with a 15 wt% aqueous solution of ammonium chloride (700 mL), and once with a saturated aqueous solution of sodium chloride (700 mL), the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (n-hexane / ethyl acetate) to obtain a wine red oil G1-8 (56.0 g, 93.7 mmol) with a yield of 78.1%. 1H NMR (400 MHz, DMSO-d6) δ8.46 (t, J = 5.6 Hz, 1H), 7.87–7.80 (m, 1H), 7.56–7.28 (m, 4H), 4.86 (s, 1H), 4.40 (dd, J = 6.9, 4.2 Hz, 1H), 3.92–3.84 (m, 1H), 3.81 (dt, J = 7.2, 5.4 Hz, 2H), 3.68–3.34 (m, 11H), 2.99–2.90 (m, 1H), 1.01 (dt, J = 11.0, 5.6 Hz, 28H). MS (ESI) m / z [MH] - =596.5.

[0127] Step 6: Synthesis of G1-9

[0128] G1-8 (56.0 g, 93.7 mmol) was dissolved in tetrahydrofuran (500 mL), cooled to 0 °C, triethylamine trihydrofluoric acid (45.3 g, 281.0 mmol) was slowly added dropwise, and the temperature was raised to room temperature after the addition, and stirred overnight at room temperature. After the reaction was completed, saturated sodium bicarbonate aqueous solution (600 mL) was added, stirred for 5 min, saturated sodium chloride aqueous solution (600 mL) was added, and the liquid was separated by shaking. The aqueous phase was extracted with tetrahydrofuran (600 mL), and the organic phases were combined. The organic phases were washed with saturated sodium chloride aqueous solution (300 mL) and dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was dissolved in acetonitrile (300 mL), washed with n-heptane (600 mL×3), and the acetonitrile phase was concentrated to obtain a light yellow oil G1-9 (35.0 g), which was directly used in the next step. 1H NMR (400MHz, DMSO-d6) δ8.48(t,J=5.6Hz,1H),7.88–7.81(m,2H),7.56–7.49(m,1H),7.46(dd,J=8.1,6.5Hz,2H),4.89(d,J=1.5Hz,1H),4.8 3(d,J=6.9Hz,1H),4.54(t,J=5.7Hz,1H),3.98(td,J=6.6,4.7Hz,1H),3.77–3.67(m,2H),3.56–3.46(m,6H),3.46–3.39(m,3H),3.35(s,4H). MS(ESI)m / z[MH] - =354.2.

[0129] Step 7: Synthesis of G1-10

[0130] Dissolve the dried G1-9 (35.0 g, 98.5 mmol) and dimethylaminopyridine (1.20 g, 9.82 mmol) in pyridine (350 mL), add 4,4'-dimethoxytriphenylmethane (40.1 g, 118.3 mmol) in 4 batches, and stir at 15-20°C for 1 h. After the reaction is completed, cool to 0°C, and add saturated sodium bicarbonate aqueous solution (350 mL) dropwise at 0-5°C to quench the reaction. After the dropwise addition, stir for 10 min, add dichloromethane (350 mL), shake and separate, wash the organic phase once with purified water (350 mL), and then wash once with 10wt% sodium chloride aqueous solution (350 mL), dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain a crude product. Column chromatography purification (n-heptane / ethyl acetate, 0.1% triethylamine) gave a pale yellow foamy solid G1-10 (50.0 g, 76.0 mmol) with a yield of 77.2%. 1H NMR (400 MHz, DMSO-d6) δ8.46 (t, J = 5.6 Hz, 1H), 7.87–7.80 (m, 2H), 7.56–7.48 (m, 1H), 7.48–7.39 (m, 4H), 7.33–7.24 (m, 6H), 7.24–7.15 (m, 1H), 6.91–6.84 (m, 4H), 4.98 (s, 1H), 4.87 (d, J = 7. 3Hz,1H),4.01(td,J=8.4,7.3,5.7Hz,1H),3.91(td,J=7.0,6.6,2.8Hz,1H),3.73(s,6H),3.71 –3.64(m,1H),3.61–3.35(m,11H),3.10(dd,J=10.0,2.7Hz,1H),2.96(dd,J=10.0,6.0Hz,1H). MS(ESI)m / z[MH] - =656.6.

[0131] Step 8: Synthesis of G1-11

[0132] Dissolve G1-10 (50.0 g, 76.0 mmol) in methanol (500 mL), add sodium hydroxide (136.8 g, 3.42 mol) and purified water (27.4 g, 1.52 mol), seal and heat to 130 ° C, and react for 2 hours. After the reaction is completed, cool to room temperature, add dichloromethane (1000 mL) and 15 wt% ammonium chloride aqueous solution (1000 mL), stir for 5 minutes, separate the liquids, wash the organic phase with purified water (1000 mL) and saturated sodium chloride aqueous solution (500 mL) in turn, dry the organic phase with anhydrous sodium sulfate, and then concentrate under reduced pressure to remove the solvent to obtain a pale yellow solid crude product G1-11 (40.0 g), which is directly used in the next step. 1H NMR(400MHz, DMSO-d6)δ7.43(d,J=7.5Hz,2H),7.29(dd,J=8.9,2.8Hz,6H),7.21(t,J=7.2Hz,1H), 6.88(d,J=8.5Hz,4H),4.99(s,1H),4.92(s,1H),4.02(dd,J=7.4,4.5Hz,1H),3.93(dt,J=7.6,3.7 Hz,1H),3.73(s,7H),3.55(ddd,J=10.9,6.9,3.9Hz,1H),3.44(ddd,J=15.2,7.1,4.3Hz,6H),3.29 (t, J = 5.8 Hz, 2H), 3.11 (dd, J = 9.9, 2.8 Hz, 1H), 2.98 (dd, J = 10.0, 6.0 Hz, 1H), 2.59 (t, J = 5.8 Hz, 2H). MS(ESI)m / z[M+Na] + =576.4.

[0133] Step 9: Synthesis of G1-13

[0134] Dissolve G1-11 (40.0 g, 72.2 mmol) in dichloromethane (400 mL), add O-benzotriazole-tetramethyluronium hexafluorophosphate (41.1 g, 108.4 mmol), cool to 0°C, add diisopropylethylamine (18.7 g, 144.7 mmol), stir for 10 min, slowly drop G1-12 (35.6 g, 79.6 mmol) in dichloromethane (400 mL), and stir for 1 h after the dropwise addition. After the reaction is completed, add purified water (1000 mL) dropwise at 0-5°C, shake and separate, wash the organic phase with 10 wt% sodium chloride aqueous solution (1000 mL), dry the organic phase with anhydrous sodium sulfate, and then concentrate under reduced pressure to remove the solvent to obtain a crude product. Column chromatography purification (dichloromethane / tetrahydrofuran, 0.1% triethylamine) gave a pale yellow foamy solid G1-13 (28.9 g, 29.4 mmol). Yield 40.7%. 1H NMR (400 MHz, DMSO-d6) δ7.83–7.72 (m, 2H), 7.42 (d, J = 7.2 Hz, 2H), 7.34–7.17 (m, 7H), 6.88 (d, J = 8.6 Hz, 4H), 5.21 (d, J = 3.4 Hz, 1H), 4.98 (s, 2H), 4.88 (d, J = 7.3 Hz, 1H), 4.48 (d, J = 8.5 Hz, 1H), 4.02 (q, J = 4.2 Hz, 4H), 3.89 (ddt, J = 19.9 ,11.0,5.6Hz,2H),3.74(s,6H),3.68(dq,J=9.5,4.5Hz,2H),3.58–3.32(m,10H),3.19–3.06(m,3H),2.95(dd,J=10 .0, 6.0Hz, 1H), 2.10 (s, 3H), 2.05 (t, J = 7.1Hz, 2H), 1.99 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H), 1.47 (d, J = 14.5Hz, 4H). MS(ESI)m / z[MH] - =981.8.

[0135] Step 10: Synthesis of YK-GAL-501

[0136] G1-13 (3.0 g, 3.05 mmol) was dissolved in tetrahydrofuran (15 mL), 3A molecular sieve (750 mg) was added, stirred and dried for 30 min, recorded as solution A, and set aside; pyridine trifluoroacetate (1.18 g, 6.11 mmol) and triphenylphosphine (80 mg, 0.31 mmol) were dissolved in tetrahydrofuran (15 mL), 3A molecular sieve (750 mg) was added, stirred and dried for 30 min, recorded as solution B, and set aside. Solution B was cooled to 0°C, phosphorus reagent: bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.30 g, 7.63 mmol) was added, and then solution A was added dropwise to solution B and stirred for 1 h. After the reaction is completed, filter, add 5wt% sodium bicarbonate aqueous solution (30mL) to the filtrate, stir for 5min, add dichloromethane (60mL), shake and separate, wash the organic phase with 5wt% sodium bicarbonate aqueous solution (30mL) and saturated sodium chloride aqueous solution (30mL) in sequence, dry the organic phase with anhydrous sodium sulfate, and concentrate to remove the solvent to obtain a residue. Dissolve the residue in dichloromethane (3mL), add dropwise to n-heptane (60mL) at 10-15°C, stir for 30min, remove the supernatant, and obtain a viscous oily solid. Vacuum dry to obtain a white foamy solid YK-GAL-501 (3.4g, 2.87mmol), with a yield of 94.4%. 1H NMR (400MHz, DMSO-d6) δ7.79(d,J=9.2Hz,1H),7.74(t,J=5.6Hz,1H),7.47–7.37(m,2H),7.34–7.17(m,7H),6.91–6.83(m,4H),5.21(d,J=3.4Hz, 1H),5.04(d,J=5.1Hz,1H),4.97(dd,J=11.2,3.4Hz,1H),4.49(d,J=8.5 Hz,1H),4.13–3.96(m,5H),3.92–3.82(m,1H),3.73(d,J=2.0Hz,8H),3.6 1(dd,J=14.9,4.6Hz,2H),3.53–3.33(m,11H),3.21–3.09(m,2H),2.95(dd,J=10.5,5.2Hz,1H),2.89(t,J=5.8Hz,2H),2.75(t,J=6.1Hz,1H),2.5 6(t,J=5.9Hz,1H),2.10(s,3H),2.03(q,J=6.4Hz,2H),1.99(s,3H),1.89 (s,3H),1.77(s,3H),1.45(s,4H),1.26–1.04(m,12H).MS(ESI)m / z[M+H] + =1184.2.

[0137] (2) Synthesis of YK-GAL-502

[0138] The synthetic route is as follows:

[0139]

[0140] Step 1: Synthesis of G2-2

[0141] Using G2-1 (30.4 g, 497.8 mmol) and G1-2 (70.0 g, 498.0 mmol) as raw materials, according to the synthesis method of G1-3, the crude product G2-2 (70.0 g) was obtained and used directly in the next step. 1H NMR (400 MHz, DMSO-d6) δ 8.45 (t, J = 5.5 Hz, 1H), 7.90–7.83 (m, 2H), 7.56–7.41 (m, 3H), 4.75 (t, J = 5.6 Hz, 1H), 3.52 (q, J = 6.0 Hz, 2H), 3.35 (t, J = 6.0 Hz, 2H). MS (ESI) m / z [M+H] + =166.3.

[0142] Step 2: Synthesis of G2-3

[0143] Using G1-4 (77.0 g, 241.9 mmol) and G2-2 (60.0 g, 363.2 mmol) as raw materials, according to the synthesis method of G1-5, the crude product G2-3 (100.0 g) was obtained and used directly in the next step. 1H NMR(400MHz,Chloroform-d)δ7.84–7.77(m,2H),7.54–7.47(m,1H),7.43(dd,J=8.2,6.6Hz,2H),6.78(s,1H),5.31(dd,J=6.3,4.9Hz,1H),5.25(dd,J=4.9, 1.4Hz,1H),5.05(d,J=1.4Hz,1H),4.32(dt,J=9.5,3.6Hz,2H),3.86(ddd,J=1 0.2, 6.4, 3.5Hz, 1H), 3.79–3.57 (m, 3H), 2.08 (d, J = 14.2Hz, 6H), 2.02 (s, 3H). MS(ESI)m / z[M+Na] + =446.2

[0144] Step 3: Synthesis of G2-4

[0145] Using G2-3 (100.0 g, 236.2 mol) as raw material, according to the synthesis method of G1-6, a colorless transparent oil G2-4 (40.0 g, 135.4 mmol) was obtained, and the combined yield of the two steps was 56.0%. NMR(400MHz, DMSO-d6)δ8.43(t,J=5.6Hz,1H),7.86–7.80(m,2H),7.52(t,J=7.3H z,1H),7.45(dd,J=8.3,6.6Hz,2H),4.99(d,J=4.5Hz,1H),4.78(d,J=6.9Hz,2H), 4.65(t,J=5.6Hz,1H),3.89(td,J=6.7,4.7Hz,1H),3.75(qd,J=8.1,7.1,3.4Hz,2 H), 3.68 (dd, J = 11.0, 5.0 Hz, 1H), 3.52 (tt, J = 10.0, 4.6 Hz, 2H), 3.45–3.33 (m, 3H). MS (ESI) m / z [MH] - =296.3.

[0146] Step 4: Synthesis of G2-5

[0147] Using G2-4 (34.0 g, 115.1 mmol) as raw material and following the synthesis method of G1-7, a white solid G2-5 (50.0 g, 92.6 mmol) was obtained with a yield of 80.5%. 1H NMR (400MHz, DMSO-d6) δ8.41(t,J=5.6Hz,1H),7.86–7.79(m,2H),7.52(t,J=7.3Hz,1H),7.44(t,J=7.5Hz,2H),5.08(d,J=4.0Hz,1H),4.78(s,1H) ,4.30(dd,J=7.7,4.3Hz,1H),3.91–3.74(m,4H),3.68–3.59(m,1H),3.49 (dt,J=9.9,6.0Hz,1H),3.39(dt,J=8.7,5.9Hz,2H),1.06–0.86(m,28H). MS(ESI)m / z[MH] - =538.4.

[0148] Step 5: Synthesis of G2-6

[0149] Using G2-5 (43.0 g, 79.7 mmol) as raw material, according to the synthesis method of G1-8, wine red oil G2-6 (31.0 g, 56.0 mmol) was obtained with a yield of 70.3%. 1H NMR (400MHz, DMSO-d6) δ8.40(t,J=5.6Hz,1H),7.87–7.80(m,2H),7.52(t,J=7.2Hz,1H),7.45(t,J=7.5Hz,2H),4.90(s,1H),4.41(dd,J=7.6,4.1Hz ,1H),3.90–3.75(m,3H),3.71–3.58(m,2H),3.53(dt,J=10.0,5.9Hz,1H) ,3.45(s,3H),3.41(t,J=5.9Hz,2H),1.01(ddt,J=13.9,7.1,3.2Hz,28H). MS(ESI)m / z[MH] - =552.4.

[0150] Step 6: Synthesis of G2-7

[0151] G2-6 (31.0 g, 56.0 mmol) was used as the raw material, and the synthetic method of G1-9 was followed to obtain a light yellow oil G2-7 (17.5 g), which was used directly in the next step. 1H NMR (400 MHz, Chloroform-d) δ7.83–7.76 (m, 2H), 7.54–7.46 (m, 1H), 7.43 (dd, J = 8.2, 6.6 Hz, 2H), 5.04 (d, J = 1.4 Hz, 1H), 4.34 (t, J = 5.5 Hz, 1H), 4.00 (dt, J = 6.2, 3.3 Hz, 1H), 3.82 (ddt, J = 17.2, 10.6, 3.2 Hz, 3H), 3.77–3.58 (m, 4H), 3.51 (s, 3H). MS (ESI) m / z [MH] - =310.2.

[0152] Step 7: Synthesis of G2-8

[0153] Using dried G2-7 (16.8 g, 54.0 mmol) as raw material, according to the synthesis method of G1-10, a light yellow foam solid G2-8 (19.0 g, 31.0 mmol) was obtained with a yield of 57.4%. 1H NMR (400 MHz, DMSO-d6) δ8.42 (t, J = 5.6 Hz, 1H), 7.84–7.78 (m, 2H), 7.56–7.48 (m, 1H), 7.48–7.39 (m, 4H), 7.34–7.24 (m, 6H), 7.19 (t, J = 7.3 Hz, 1H), 6.87 (d, J = 8.4 Hz, 4H), 5.01 (s, 1H), 4.88 (d, J = 7.2 Hz, 1H), 4.01(td,J=7.2,4.4Hz,1H),3.92(td,J=7.4,6.7,2.7Hz,1H),3.72(d,J=2.6Hz,7H),3.66–3.58(m,1H) ,3.47(d,J=4.6Hz,1H),3.46–3.33(m,5H),3.10(dd,J=10.1,2.7Hz,1H),2.98(dd,J=10.0,6.0Hz,1H). MS(ESI)m / z[MH] - =612.5.

[0154] Step 8: Synthesis of G2-9

[0155] G2-8 (18.0 g, 29.3 mmol) was used as the raw material, and the synthetic method of G1-11 was followed to obtain a pale yellow solid crude product G2-9 (15.0 g), which was used directly in the next step. 1H NMR (400 MHz, DMSO-d6) δ7.46–7.39 (m, 2H), 7.34–7.25 (m, 6H), 7.24–7.18 (m, 1H), 6.92–6.83 (m, 4H), 4.94 (d, J = 1.2 Hz, 1H), 4.87 (d, J = 6.9 Hz, 1H), 4.00 (s, 1H), 3.91 (td, J = 6.7, 2.7 Hz,1H),3.73(s,6H),3.58(dt,J=9.7,5.6Hz,1H),3.51–3.45(m,1H),3.35(dd,J=9.6,6.2 Hz, 4H), 3.09 (dd, J = 10.0, 2.8 Hz, 1H), 2.96 ( dd, J = 10.0, 5.9 Hz, 1H), 2.59 ( t, J = 5.9 Hz, 2H). MS(ESI)m / z[M+Na] + =532.4.

[0156] Step 9: Synthesis of G2-10

[0157] Using G2-9 (13.5 g, 26.5 mmol) and G1-12 (13.0 g, 29.1 mmol) as raw materials, according to the synthesis method of G1-13, a light yellow foam solid G2-10 (18.6 g, 19.8 mmol) was obtained with a yield of 74.7%. NMR (400MHz, DMSO-d6) δ7.80(d,J=9.3Hz,1H),7.71(t,J=5.6Hz,1H),7.42(d,J=7.3Hz,2H),7.34–7.25(m,6H),7.21(t,J=7.3Hz,1H),6.88( d,J=8.6Hz,4H),5.21(d,J=3.4Hz,1H),5.01–4.94(m,2H),4.88(d,J=7.3Hz,1H),4.48(d,J=8.5Hz,1H),4.02(d,J=5.5Hz,4H),3.95–3.83(m, 2H),3.74(s,6H),3.71–3.64(m,1H),3.64–3.54(m,1H),3.46(d,J=4.6Hz,1H),3.45–3.35(m,5H),3.21(dd,J=13.5,5.8Hz,1H),3.16–3.06(m ,2H),2.96(dd,J=10.0,6.0Hz,1H),2.10(s,3H),2.02(d,J=6.8Hz,2H),1.99(s,3H),1.89(s,3H),1.77(s,3H),1.46(dt,J=13.0,7.4Hz,4H). MS(ESI)m / z[MH] - =937.7.

[0158] Step 10: Synthesis of YK-GAL-502

[0159] Using G2-10 (2.0 g, 2.13 mmol) as raw material, according to the synthesis method of YK-GAL-501, white foam solid YK-GAL-502 (2.0 g, 1.76 mmol) was obtained with a yield of 82.3%. NMR(400MHz,DMSO-d6)δ7.79(d,J=9.2Hz,1H),7.73–7.63(m,1H),7.46–7.39 (m,2H),7.28(t,J=8.7Hz,6H),7.22(t,J=5.5Hz,1H),6.87(dd,J=8.7,4.8Hz ,4H),5.21(d,J=3.4Hz,1H),5.02(d,J=4.2Hz,1H),4.97(dd,J=11.3,3.5Hz, 1H),4.48(d,J=8.5Hz,1H),4.02(s,5H),3.93–3.81(m,1H),3.73(d,J=1.9Hz ,6H),3.71–3.65(m,1H),3.65–3.56(m,2H),3.52–3.42(m,4H),3.39(d,J=13 .9Hz,4H),3.17(d,J=40.4Hz,3H),2.95(s,1H),2.89(t,J=5.9Hz,1H),2.75( t,J=5.9Hz,1H),2.56(t,J=5.9Hz,1H),2.10(s,3H),2.02(d,J=6.8Hz,2H),1 .99(s,3H),1.89(s,3H),1.76(s,3H),1.52–1.38(m,4H),1.22–1.03(m,12H). MS (ESI) m / z [M+H] + =1140.1.

[0160] 1) Synthesis of YK-GAL-503

[0161] (3) Synthesis of YK-GAL-503

[0162] The synthetic route is as follows:

[0163]

[0164] Step 1: Synthesis of G3-2

[0165] Using G3-1 (22.5 g, 300.0 mmol) and G1-2 (42.2 g, 300.0 mmol) as raw materials, according to the synthesis method of G1-3, a colorless oily crude product G3-2 (63.0 g) was obtained, which was used directly in the next step. 1H NMR (400 MHz, DMSO-d6) δ8.42 (s, 1H), 7.87–7.79 (m, 2H), 7.55–7.48 (m, 1H), 7.45 (dd, J = 8.2, 6.4 Hz, 2H), 4.46 (t, J = 5.2 Hz, 1H), 3.31 (td, J = 6.9, 5.7 Hz, 4H), 1.67 (p, J = 6.5 Hz, 2H). MS (ESI) m / z [M+H] + =180.3.

[0166] Step 2: Synthesis of G3-3

[0167] Using G1-4 (70.0 g, 219.9 mmol) and G3-2 (51.2 g, 285.9 mmol) as raw materials, according to the synthesis method of G1-5, a yellow oily crude product G3-3 (135.0 g) was obtained, which was directly used in the next step. 1H NMR (400 MHz, DMSO-d6) δ8.40 (t, J = 5.7 Hz, 1H), 7.89–7.80 (m, 2H), 7.49–7.45 (m, 1H), 7.43 (dd, J = 8.1, 6.4 Hz, 2H), 5.15 (dd, J = 6.6, 4.9 Hz, 1H), 5.10 (dd, J = 4.9, 1.4 Hz, 1H), 5.04 (d, J = 1. 3Hz,1H),4.30–4.21(m,2H),4.04(ddt,J=10.9,7.2,3.2Hz,1H),3.66–3.58(m,1H),3.44–3. 38(m,1H),3.28(t,J=6.1Hz,2H),2.06(s,3H),2.08(d,J=3.6Hz,6H),1.62(p,J=3.1Hz,2H). MS(ESI)m / z[M+Na] + =460.2.

[0168] Step 3: Synthesis of G3-4

[0169] G3-3 (135 g, 308.6 mol) was used as the raw material, and the synthesis method of G1-6 was followed to obtain a colorless transparent oil G3-4 (31.9 g, 102.5 mmol), with a combined yield of 33.2% in two steps. 1H NMR (400 MHz, DMSO-d6) δ8.42 (t, J = 5.7 Hz, 1H), 7.86–7.79 (m, 2H), 7.56–7.48 (m, 1H), 7.45 (dd, J = 8.2, 6.5 Hz, 2H), 4.97 (d, J = 4.5 Hz, 1H), 4.78 (d, J = 6.7 Hz, 1H), 4.74 (d, J = 1.2 Hz, 1H), 4.65 (t, J = 5.7 Hz,1H),3.87(td,J=6.7,4.8Hz,1H),3.80–3.71(m,2H),3.67(dt,J=9.7,6.4Hz,1H),3.53(ddd,J= 11.5, 5.7, 3.8Hz, 1H), 3.41–3.34 (m, 2H), 3.29 (td, J = 6.9, 6.4, 2.4Hz, 2H), 1.74 (p, J = 6.6Hz, 2H). MS(ESI)m / z[MH] - =310.3.

[0170] Step 4: Synthesis of G3-5

[0171] Using G3-4 (30.0 g, 96.4 mmol) as raw material, according to the synthesis method of G1-7, a white solid G3-5 (43.0 g, 77.6 mmol) was obtained with a yield of 80.6%. 1H NMR (400MHz, DMSO-d6) δ8.38(t,J=5.7Hz,1H),7.85–7.78(m,2H),7.53–7.40(m,3H),5.06(d,J=4.0Hz,1H),4.74(s,1H),4.30(dd,J=7.1, 4.4Hz,1H),3.92–3.74(m,4H),3.64–3.57(m,1H),3.41–3.34(m,1H),3.26(dd,J=10.0,4.3Hz,2H),1.77–1.68(m,2H),1.07–0.85(m,28H). MS(ESI)m / z[MH] - =552.6.

[0172] Step 5: Synthesis of G3-6

[0173] Using G3-5 (40.0 g, 72.2 mmol) as raw material, according to the synthesis method of G1-8, a wine red oil G3-6 (31.2 g, 54.9 mmol) was obtained with a yield of 76.0%. 1H NMR (400 MHz, DMSO-d6) δ8.39 (t, J = 5.6 Hz, 1H), 7.86–7.79 (m, 2H), 7.51 (t, J = 7.3 Hz, 1H), 7.45 (dd, J = 8.2, 6.5 Hz, 2H), 4.85 (s, 1H), 4.42 (dd, J = 7.1, 4.3 Hz, 1H), 3.91–3.78 (m,3H),3.63(dd,J=9.7,6.4Hz,1H),3.59(d,J=4.3Hz,1H),3.46(s,3H),3.40(dt,J= 9.7, 6.4Hz, 1H), 3.33–3.25 (m, 2H), 1.74 (pd, J=6.8, 2.0Hz, 2H), 1.07–0.95 (m, 28H). MS(ESI)m / z[MH] - =566.6.

[0174] Step 6: Synthesis of G3-7

[0175] G3-6 (30.0 g, 52.8 mmol) was used as the raw material, and the synthetic method of G1-9 was followed to obtain a light yellow oil G3-7 (19.1 g), which was used directly in the next step. 1H NMR (400 MHz, DMSO-d6) δ 8.42 (t, J = 5.6 Hz, 1H), 7.87–7.79 (m, 2H), 7.56–7.48 (m, 1H), 7.48–7.41 (m, 2H), 4.87 (d, J = 1.7 Hz, 1H), 4.82 (d, J = 6.9 Hz, 1H), 4.67 (t, J = 5.7 Hz, 1H) ,3.98(td,J=6.5,4.6Hz,1H),3.78–3.63(m,2H),3.56–3.47(m,1H),3.45(dd,J=4.8, 1.7Hz, 1H), 3.44–3.39 (m, 1H), 3.36 (s, 4H), 3.34–3.25 (m, 2H), 1.75 (p, J = 6.7Hz, 2H). MS(ESI)m / z[MH] - =324.4.

[0176] Step 7: Synthesis of G3-8

[0177] G3-7 (18.0 g, 55.3 mmol) was used as the raw material, and the synthetic method of G1-10 was followed to obtain a pale yellow foam solid G3-8 (24.5 g, 39.0 mmol), with a yield of 70.6%. 1H NMR (400 MHz, DMSO-d6) δ8.39 (t, J = 5.8 Hz, 1H), 7.81 (d, J = 7.6 Hz, 2H), 7.51 (dd, J = 8.3, 6.2 Hz, 1H), 7.44 (t, J = 8.2 Hz, 4H), 7.33–7.24 (m, 6H), 7.18 (t, J = 7.3 Hz, 1H), 6.91–6.84 (m, 4H), 4.96 (s, 1H), 4.87 (d, J = 7.1 Hz, 1H) ,4.08–3.98(m,1H),3.91(t,J=7.5Hz,1H),3.71(d,J=1.9Hz,7H),3.47(d,J=4.4Hz,2H),3.39(d,J=1.7Hz,3 H), 3.25 (p, J = 6.7Hz, 2H), 3.10 (dd, J = 10.1, 2.7Hz, 1H), 2.95 (dd, J = 10.1, 5.8Hz, 1H), 1.71 (p, J = 6.8Hz, 2H). MS(ESI)m / z[MH] - =626.4.

[0178] Step 8: Synthesis of G3-9

[0179] G3-8 (23.0 g, 36.6 mmol) was used as the raw material, and the synthetic method of G1-11 was followed to obtain a pale yellow solid crude product G3-9 (18.5 g), which was used directly in the next step. 1H NMR (400 MHz, DMSO-d6) δ7.47–7.40 (m, 2H), 7.29 (dd, J = 8.7, 3.0 Hz, 6H), 7.25–7.17 (m, 1H), 6.92–6.84 (m, 4H), 4.92 (d, J = 1.2 Hz, 1H), 4.90–4.78 (m, 1H), 3.99 (dd, J = 7.2, 4.5 Hz, 1H), 3.91 ( td,J=7.6,6.7,2.7Hz,1H),3.73(s,6H),3.67(dt,J=9.5,6.5Hz,1H),3.48–3.35(m,5H),3.09 (dd,J=9.9,2.8Hz,1H),2.95(dd,J=9.9,5.9Hz,1H),2.53–2.43(m,2H),1.50(p,J=6.7Hz,2H). MS(ESI)m / z[M+Na] + =546.4.

[0180] Step 9: Synthesis of G3-10

[0181] Using G3-9 (18.0 g, 34.4 mmol) and G1-12 (16.9 g, 37.8 mmol) as raw materials, according to the synthesis method of G1-13, a light yellow foam solid G3-10 (23.0 g, 24.1 mmol) was obtained with a yield of 70.1%. NMR (400MHz, DMSO-d6) δ7.80(d,J=9.2Hz,1H),7.67(t,J=5.6Hz,1H),7.46–7.39(m,2H),7.34–7.24(m,6H),7.21(t,J=7.3Hz,1H),6.88(d,J=8 .5Hz,4H),5.22(d,J=3.4Hz,1H),4.97(dd,J=11.3,3.4Hz,1H),4.93(s,1H),4.86(d,J=7.2Hz,1H),4.49(d,J=8.5Hz,1H),4.02(d,J=5.8Hz,4H) ,3.95–3.77(m,2H),3.73(s,6H),3.72–3.56(m,2H),3.46(d,J=4.5Hz,1 H),3.41(s,5H),3.10(dd,J=9.9,2.7Hz,1H),3.01(p,J=6.6Hz,2H),2.9 4(dd,J=10.1,5.8Hz,1H),2.10(s,3H),2.00(d,J=8.7Hz,5H),1.89(s,3H),1.76(d,J=3.8Hz,3H),1.56(p,J=6.8Hz,2H),1.47(p,J=7.1Hz,4H). MS(ESI)m / z[MH] - =951.8.

[0182] Step 10: Synthesis of YK-GAL-503

[0183] Using G3-10 (5.0 g, 5.25 mmol) as raw material, according to the synthesis method of YK-GAL-501, white foam solid YK-GAL-503 (5.1 g, 4.42 mmol) was obtained with a yield of 84.3%. NMR(400MHz,DMSO-d6)δ7.80(d,J=9.2Hz,1H),7.66(d,J=6.1Hz,1H),7.43(dd ,J=7.7,5.1Hz,2H),7.29(td,J=8.9,8.1,3.6Hz,6H),7.21(dd,J=8.4,5.9Hz, 1H),6.87(dd,J=8.7,4.2Hz,4H),5.21(d,J=3.4Hz,1H),4.97(dd,J=11.6,3.6 Hz,2H),4.49(d,J=8.4Hz,1H),4.13–3.95(m,5H),3.93–3.80(m,1H),3.73(d,J =2.0Hz,9H),3.61(dd,J=16.2,4.5Hz,1H),3.56–3.35(m,7H),3.21(dd,J=26. 4,10.1Hz,1H),3.02(d,J=7.3Hz,2H),2.89(t,J=5.9Hz,2H),2.75(t,J=5.9Hz, 1H),2.56(t,J=5.9Hz,1H),2.10(s,3H),2.00(d,J=8.2Hz,5H),1.89(s,3H),1 .77(s,3H),1.58(q,J=6.3Hz,2H),1.46(d,J=12.8Hz,4H),1.21–1.02(m,12H). MS (ESI) m / z [M+H] + =1154.3.

[0184] (4) Synthesis of YK-GAL-504

[0185] The synthetic route is as follows:

[0186]

[0187] Step 1: Synthesis of G4-2

[0188] Using G4-1 (60.0 g, 673.1 mmol) and G1-2 (94.6 g, 673.0 mmol) as raw materials, according to the synthesis method of G1-3, a colorless oily crude product G4-2 (135.0 g) was obtained, which was used directly in the next step. 1H NMR (400 MHz, Chloroform-d) δ7.77 (dt, J = 7.0, 1.4 Hz, 2H), 7.54–7.46 (m, 1H), 7.43 (dd, J = 8.2, 6.5 Hz, 2H), 6.48 (s, 1H), 3.73 (t, J = 5.9 Hz, 2H), 3.51 (t, J = 6.6 Hz, 2H), 1.78–1.65 (m, 4H). MS (ESI) m / z [MH] - =194.4.

[0189] Step 2: Synthesis of G4-3

[0190] Using G1-4 (120.0 g, 377.0 mmol) and G4-2 (110 g, 569.2 mmol) as raw materials, according to the synthesis method of G1-5, a yellow oily crude product G4-3 (194.0 g) was obtained, which was directly used in the next step. 1H NMR (400 MHz, DMSO-d6) δ8.43 (t, J = 5.7 Hz, 1H), 7.87–7.80 (m, 2H), 7.55–7.48 (m, 1H), 7.45 (dd, J = 8.2, 6.5 Hz, 2H), 5.17 (dd, J = 6.5, 4.9 Hz, 1H), 5.09 (dd, J = 4.9, 1.2 Hz, 1H), 5.03 (d, J = 1. 3Hz,1H),4.32–4.19(m,2H),4.04(ddt,J=10.8,7.1,3.2Hz,1H),3.69–3.59(m,1H),3.46–3. 39 (m, 1H), 3.28 (t, J = 6.0Hz, 2H), 2.07 (s, 3H), 2.02 (d, J = 3.7Hz, 6H), 1.56 (p, J = 3.1Hz, 4H). MS(ESI)m / z[M+Na] + =474.4.

[0191] Step 3: Synthesis of G4-4

[0192] G4-3 (194.0 g, 429.7 mol) was used as the raw material and the synthesis method of G1-6 was followed to obtain a colorless transparent oil G4-4 (51.2 g, 157.4 mmol), with a combined yield of 41.7% in two steps. 1H NMR (400 MHz, DMSO-d6) δ8.44 (t, J = 5.7 Hz, 1H), 7.87–7.80 (m, 2H), 7.56–7.40 (m, 3H), 4.96 (d, J = 4.4 Hz, 1H), 4.78 (d, J = 6.6 Hz, 1H), 4.73 (d, J = 1.2 Hz, 1H), 4.59 (t, J = 5.7 Hz, 1H), 3.84 (td, J = 6 .5,4.7Hz,1H),3.74(ddd,J=17.5,7.7,4.2Hz,2H),3.63(dt,J=9.0,5.7Hz,1H),3.52(ddd,J=1 1.5, 5.7, 3.9Hz, 1H), 3.35 (q, J = 5.7Hz, 2H), 3.26 ( q, J = 6.4Hz, 2H), 1.54 ( dd, J = 6.7, 3.6Hz, 4H). MS(ESI)m / z[MH] - =324.4.

[0193] Step 4: Synthesis of G4-5

[0194] Using G4-4 (45.0 g, 138.3 mmol) as raw material and following the synthesis method of G1-7, a white solid G4-5 (59.0 g, 103.9 mmol) was obtained with a yield of 75.2%. 1H NMR(400MHz, DMSO-d6)δ8.42(t,J=5.7Hz,1H),7.87–7.80(m,2H),7.50(dd,J=8 .4,6.0Hz,1H),7.44(t,J=7.4Hz,2H),5.04(d,J=3.9Hz,1H),4.72(s,1H),4.30( dd,J=7.3,4.4Hz,1H),3.93–3.73(m,4H),3.56(dt,J=9.3,6.0Hz,1H),3.33(d, J=6.6Hz, 1H), 3.25 (d, J=5.9Hz, 2H), 1.53 (p, J=6.6Hz, 4H), 1.06–0.82 (m, 28H). MS (ESI) m / z [MH] - =566.6.

[0195] Step 5: Synthesis of G4-6

[0196] Using G4-5 (59.0 g, 103.9 mmol) as raw material, according to the synthesis method of G1-8, a wine red oil G4-6 (41.0 g, 70.5 mmol) was obtained with a yield of 67.8%. 1H NMR (400 MHz, DMSO-d6) δ8.42 (t, J = 5.7 Hz, 1H), 7.86–7.79 (m, 2H), 7.51 (t, J = 7.3 Hz, 1H), 7.44 (dd, J = 8.2, 6.5 Hz, 2H), 4.82 (s, 1H), 4.41 (dd, J = 7.5, 4.3 Hz, 1H), 3.91– 3.83(m,1H),3.83–3.74(m,2H),3.57(dd,J=8.4,5.3Hz,2H),3.45(s,3H),3.40–3. 32(m,1H),3.24(t,J=6.1Hz,2H),1.52(dt,J=10.1,5.1Hz,4H),1.09–0.86(m,28H). MS(ESI)m / z[MH] - =580.5.

[0197] Step 6: Synthesis of G4-7

[0198] G4-6 (34.0 g, 58.4 mmol) was used as the raw material and the synthetic method of G1-9 was followed to obtain a light yellow oil G4-7 (22.0 g), which was used directly in the next step. 1H NMR (400 MHz, DMSO-d6) δ8.44 (t, J = 5.7 Hz, 1H), 7.84 (dt, J = 7.0, 1.5 Hz, 2H), 7.56–7.48 (m, 1H), 7.45 (dd, J = 8.1, 6.5 Hz, 2H), 4.86 (d, J = 1.7 Hz, 1H), 4.82 (d, J = 6.8 Hz, 1H), 4.62 (s, 1H), 3.96 (t d,J=6.5,4.8Hz,1H),3.83–3.70(m,1H),3.70–3.56(m,2H),3.50(ddd,J=11.5,5.7,4.2Hz,1H) ,3.44(dd,J=4.8,1.8Hz,1H),3.41–3.33(m,4H),3.33–3.22(m,2H),1.54(h,J=4.7,3.8Hz,4H). MS(ESI)m / z[MH] - =338.3.

[0199] Step 7: Synthesis of G4-8

[0200] G4-7 (20.0 g, 58.9 mmol) was used as the raw material and the synthetic method of G1-10 was followed to obtain a pale yellow foam solid G4-8 (23.1 g, 36.0 mmol) with a yield of 61.0%. 1H NMR (400 MHz, DMSO-d6) δ8.40 (t, J = 5.7 Hz, 1H), 7.86–7.79 (m, 2H), 7.55–7.48 (m, 1H), 7.48–7.40 (m, 4H), 7.33–7.24 (m, 6H), 7.18 (t, J = 7.3 Hz, 1H), 6.87 (d, J = 8.3 Hz, 4H), 4.94 (s, 1H), 4.86 (d, J = 7.3 Hz, 1H), 4.02 (td, J = 7.1, 4.2 Hz, 1H),3.91(td,J=7.2,6.6,2.7Hz,1H),3.72(s,6H),3.70–3.63(m,1H),3.45(d,J=4.5Hz,1H),3.43–3.35(m,4H),3. 22(dd,J=7.9,4.5Hz,2H), 3.10(dd,J=10.0,2.8Hz,1H), 2.95(dd,J=10.0,5.8Hz,1H), 1.48(dd,J=6.6,3.4Hz,4H). MS(ESI)m / z[MH] - =640.6.

[0201] Step 8: Synthesis of G4-9

[0202] Using G4-8 (21.0 g, 32.7 mmol) as the raw material, according to the synthetic method of G1-11, the pale yellow solid crude product G4-9 (16.5 g) was obtained and used directly in the next step. 1H NMR (400 MHz, DMSO-d6) δ7.47–7.40 (m, 2H), 7.34–7.25 (m, 6H), 7.21 (t, J = 7.1 Hz, 1H), 6.88 (d, J = 8.7 Hz, 4H), 4.93 (s, 1H), 4.88 (s, 1H), 4.00 (dd, J = 7.3, 4.6 Hz, 1H), 3.95–3.88 (m, 1H), 3.73 (s, 6H), 3. 65–3.58(m,1H),3.44(d,J=4.5Hz,1H),3.41–3.32(m,4H),3.10(dd,J=10.0,2.7Hz,1H),2.94(dd,J =9.9, 5.9Hz, 1H), 2.45 (t, J = 6.9Hz, 2H), 1.43 (q, J = 7.0Hz, 2H), 1.26 (ddd, J = 11.1, 8.7, 5.4Hz, 2H). MS(ESI)m / z[M+Na] + =560.5.

[0203] Step 9: Synthesis of G4-10

[0204] Using G4-9 (15.0 g, 27.9 mmol) and G1-12 (13.7 g, 30.6 mmol) as raw materials, according to the synthesis method of G1-13, a light yellow foam solid G4-10 (19.4 g, 20.1 mmol) was obtained with a yield of 71.9%. NMR(400MHz, DMSO-d6)δ7.80(d,J=9.2Hz,1H),7.66(t,J=5.6Hz,1H),7.46–7.39(m,2H),7.34–7.25(m,6H),7.25–7.17(m,1H),6.91–6.84 (m,4H),5.22(d,J=3.4Hz,1H),4.97(dd,J=11.2,3.4Hz,1H),4.93(d,J=1.1Hz,1H),4.86(d,J=7.3Hz,1H),4.49(d,J=8.6Hz,1H),4.07–3. 96(m,4H),3.95–3.84(m,2H),3.74(s,6H),3.67–3.56(m,2H),3.47–3.33(m,6H),3.10(dd,J=10.0,2.8Hz,1H),2.96(td,J=11.7,10.2,6. 1Hz, 3H), 2.10 (s, 3H), 2.03 (t, J = 7.1Hz, 2H), 1.99 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H), 1.45 (tq, J = 13.1, 6.6Hz, 6H), 1.34 (d, J = 14.9Hz, 2H). MS(ESI)m / z[MH] - =965.8.

[0205] Step 10: Synthesis of YK-GAL-504

[0206] Using G4-10 (3.0 g, 3.10 mmol) as raw material, according to the synthesis method of YK-GAL-501, white foam solid YK-GAL-504 (3.2 g, 2.74 mmol) was obtained with a yield of 88.4%. NMR(400MHz,DMSO-d6)δ7.80(d,J=9.2Hz,1H),7.70–7.61(m,1H),7.43(dd,J =7.7,5.3Hz,2H),7.29(td,J=9.0,8.2,2.8Hz,6H),7.21(dt,J=8.2,3.8Hz,1H ),6.87(dd,J=8.7,4.1Hz,4H),5.21(d,J=3.4Hz,1H),4.97(dd,J=11.4,3.6Hz ,2H),4.49(d,J=8.5Hz,1H),4.03(q,J=4.9,3.9Hz,5H),3.87(dt,J=11.2,8.9 Hz,1H),3.73(d,J=2.1Hz,6H),3.70–3.56(m,3H),3.56–3.35(m,8H),3.21(dd d,J=27.3,10.4,2.8Hz,1H),3.02–2.85(m,4H),2.75(t,J=6.0Hz,1H),2.56(t ,J=5.9Hz,1H),2.10(s,3H),2.01(d,J=12.3Hz,5H),1.89(s,3H),1.77(s,3H) ,1.45(hept,J=7.0Hz,6H),1.33(dt,J=10.2,4.9Hz,2H),1.22–1.03(m,12H). MS (ESI) m / z [M+H] + =1168.3.

[0207] (5) Synthesis of YK-GAL-505

[0208]

[0209] Step 1: Synthesis of G5-2

[0210] Using G5-1 (60.0 g, 581.6 mmol) and G1-2 (81.8 g, 581.9 mmol) as raw materials, according to the synthesis method of G1-3, a colorless oily crude product G5-2 (123.0 g) was obtained, which was used directly in the next step. 1H NMR (400 MHz, DMSO-d6) δ8.41 (s, 1H), 7.88–7.79 (m, 2H), 7.56–7.49 (m, 1H), 7.42 (dd, J = 8.1, 6.5 Hz, 2H), 4.56 (t, J = 5.2 Hz, 1H), 3.34 (td, J = 6.7, 5.6 Hz, 4H), 1.67-1.53 ​​(m, 4H), 1.34 (m, 2H). MS (ESI) m / z [MH] - =208.4.

[0211] Step 2: Synthesis of G5-3

[0212] Using G1-4 (80.0 g, 251.4 mmol) and G5-2 (78.9 g, 377.1 mmol) as raw materials, according to the synthesis method of G1-5, a yellow oily crude product G5-3 (124.0 g) was obtained, which was directly used in the next step. 1H NMR (400 MHz, DMSO-d6) δ8.43 (t, J = 5.7 Hz, 1H), 7.88–7.81 (m, 2H), 7.55–7.49 (m, 1H), 7.48 (dd, J = 8.1, 6.5 Hz, 2H), 5.16 (dd, J = 6.6, 5.0 Hz, 1H), 5.07 (dd, J = 4.8, 1.2 Hz, 1H), 5.01 (d, J = 1.3 Hz, 1H), 4 .32–4.17(m,2H),4.05(ddt,J=11.0,7.0,3.4Hz,1H),3.70–3.59(m,1H),3.41–3.32(m,1H),3.26( t,J=6.0Hz,2H),2.11(s,3H),2.01(d,J=3.5Hz,6H),1.54(p,J=3.0Hz,4H),1.34(p,J=2.0Hz,2H). MS(ESI)m / z[M+Na] + =488.3.

[0213] Step 3: Synthesis of G5-4

[0214] G5-3 (88.0 g, 189.0 mol) was used as the raw material, and the synthesis method of G1-6 was followed to obtain a colorless transparent oil G5-4 (23.0 g, 67.8 mmol), with a combined yield of 35.8% in two steps. 1H NMR (400 MHz, DMSO-d6) δ8.44 (t, J = 5.7 Hz, 1H), 7.88–7.81 (m, 2H), 7.55–7.48 (m, 3H), 4.97 (d, J = 4.6 Hz, 1H), 4.78 (d, J = 6.5 Hz, 1H), 4.75 (d, J = 1.3 Hz, 1H), 4.58 (t, J = 5.7 Hz, 1H), 3.80 (td, J = 6.9, 4.8H z,1H),3.71(ddd,J=17.9,7.6,4.2Hz,2H),3.65(dt,J=9.0,5.9Hz,1H),3.51(ddd,J=11.2,5.9,3.5 Hz, 1H), 3.36 (q, J = 5.7 Hz, 2H), 3.28 ( q, J = 6.5 Hz, 2H), 1.54 ( p, J = 3.1 Hz, 4H), 1.34 ( p, J = 2.1 Hz, 2H). MS(ESI)m / z[MH] - =338.4.

[0215] Step 4: Synthesis of G5-5

[0216] G5-4 (20.0 g, 58.9 mmol) was used as the raw material, and the synthesis method of G1-7 was followed to obtain white solid G5-5 (25.1 g, 43.1 mmol), with a yield of 73.2%. 1H NMR (400 MHz, DMSO-d6) δ8.43 (t, J = 5.7 Hz, 1H), 7.88–7.81 (m, 2H), 7.52 (dd, J = 8.4, 6.1 Hz, 1H), 7.43 (t, J = 7.2 Hz, 2H), 5.07 (d, J = 3.9 Hz, 1H), 4.79 (s, 1H), 4.26 (dd, J = 7.4, 4.6Hz,1H),3.90–3.77(m,4H),3.58(dt,J=9.2,6.3Hz,1H),3.36(d,J=6.5Hz,1H),3. 21(d,J=5.7Hz,2H),1.53(p,J=3.0Hz,4H),1.35(p,J=2.0Hz,2H),1.04–0.87(m,28H). MS(ESI)m / z[MH] - =580.5.

[0217] Step 5: Synthesis of G5-6

[0218] Using G5-5 (24.0 g, 41.2 mmol) as raw material, according to the synthesis method of G1-8, a wine red oil G5-6 (19.4 g, 32.6 mmol) was obtained with a yield of 78.9%. 1H NMR (400 MHz, DMSO-d6) δ8.44 (t, J = 5.7 Hz, 1H), 7.87–7.79 (m, 2H), 7.50 (t, J = 7.4 Hz, 1H), 7.47 (dd, J = 8.2, 6.5 Hz, 2H), 4.82 (s, 1H), 4.43 (dd, J = 7.4, 4.5 Hz, 1H), 3.90–3.85 (m, 1H) ,3.86–3.73(m,2H),3.56(dd,J=8.4,5.3Hz,2H),3.47(s,3H),3.41–3.34(m,1H),3.25(t ,J=6.5Hz,2H),1.54(dt,J=10.2,5.1Hz,4H),1.35(p,J=2.0Hz,2H),1.09–0.86(m,28H). 1 HNMR(400MHz,)δppm. MS(ESI)m / z[MH] - =594.5.

[0219] Step 6: Synthesis of G5-7

[0220] G5-6 (19.0 g, 31.9 mmol) was used as the raw material, and the synthetic method of G1-9 was followed to obtain a light yellow oil G5-7 (12.3 g), which was used directly in the next step. 1H NMR (400 MHz, DMSO-d6) δ8.44 (t, J = 5.7 Hz, 1H), 7.85 (dt, J = 7.1, 1.5 Hz, 2H), 7.57–7.47 (m, 1H), 7.47 (dd, J = 8.2, 6.5 Hz, 2H), 4.83 (d, J = 1.8 Hz, 1H), 4.82 (d, J = 6.5 Hz, 1H), 4.65 (s, 1H), 3.97 (td, J = 6.5, 4. 8Hz,1H),3.88–3.71(m,1H),3.69–3.55(m,2H),3.51(ddd,J=11.2,5.5,4.3Hz,1H),3.42(dd,J=4.9, 1.8Hz, 1H), 3.40–3.33 (m, 4H), 3.33–3.21 (m, 2H), 1.54 (h, J = 4.9, 3.7Hz, 4H), 1.35 (p, J = 2.1Hz, 2H). MS(ESI)m / z[MH] - =352.3.

[0221] Step 7: Synthesis of G5-8

[0222] Using G5-7 (12.0 g, 34.0 mmol) as raw material, according to the synthesis method of G1-10, a light yellow foam solid G5-8 (15.3 g, 23.3 mmol) was obtained with a yield of 68.6%. NMR(400MHz, DMSO-d6)δ8.42(t,J=5.7Hz,1H),7.86–7.77(m,2H),7.57–7.49(m,1H),7.48–7.41(m,4H),7.32–7.26(m,6 H),7.19(t,J=7.3Hz,1H),6.87(d,J=8.3Hz,4H),4.95(s,1H),4.89(d,J=7.3Hz,1H),4.04(td,J=7.1,4.2Hz,1H),3.90(t d,J=7.2,6.6,2.7Hz,1H),3.72(s,6H),3.71–3.64(m,1H),3.45(d,J=4.5Hz,1H),3.42–3.34(m,4H),3.22(dd,J=7.9,4. 5Hz, 2H), 3.11 (dd, J = 10.0, 2.8Hz, 1H), 2.96 (dd, J = 10.0, 5.8Hz, 1H), 1.50 (dd, J = 6.6, 3.4Hz, 4H), 1.31 (p, J = 2.1Hz, 2H). MS(ESI)m / z[MH] - =654.6.

[0223] Step 8: Synthesis of G5-9

[0224] G5-8 (13.0 g, 19.8 mmol) was used as the raw material, and the synthetic method of G1-11 was followed to obtain a pale yellow solid crude product G3-9 (10.4 g), which was used directly in the next step. 1H NMR (400 MHz, DMSO-d6) δ7.48–7.40 (m, 2H), 7.33–7.22 (m, 6H), 7.20 (t, J = 7.1 Hz, 1H), 6.85 (d, J = 8.7 Hz, 4H), 4.93 (s, 1H), 4.86 (s, 1H), 4.01 (dd, J = 7.3, 4.6 Hz, 1H), 3.96–3.88 (m, 1H), 3.73 (s ,6H),3.64–3.59(m,1H),3.45(d,J=4.5Hz,1H),3.41–3.33(m,4H),3.11(dd,J=10.0,2.7Hz,1H ), 2.95 (dd, J = 9.9, 5.9 Hz, 1H), 2.43 (t, J = 6.9 Hz, 2H), 1.43 (q, J = 7.0 Hz, 2H), 1.39-1.26 (m, 4H). MS(ESI)m / z[M+Na] +=574.5.

[0225] Step 9: Synthesis of G5-10

[0226] Using G5-9 (10.0 g, 18.1 mmol) and G1-12 (8.9 g, 19.9 mmol) as raw materials, according to the synthesis method of G1-13, a light yellow foam solid G5-10 (11.9 g, 12.1 mmol) was obtained with a yield of 66.9%. NMR(400MHz,DMSO-d6)δ7.80(d,J=9.2Hz,1H),7.66(t,J=5.6Hz,1H),7.46– 7.39(m,2H),7.32–7.25(m,6H),7.21(t,J=7.3Hz,1H),6.87(d,J=8.6Hz,4H ),5.21(d,J=3.4Hz,1H),4.97(dd,J=11.2,3.4Hz,1H),4.92(s,1H),4.86(d ,J=7.3Hz,1H),4.48(d,J=8.5Hz,1H),4.05–3.95(m,4H),3.95–3.83(m,2H) ,3.73(s,6H),3.72–3.66(m,1H),3.60(d,J=9.0Hz,1H),3.44(d,J=4.6Hz,1 H),3.38(s,4H),3.34(d,J=8.8Hz,1H),3.09(dd,J=9.9,2.8Hz,1H),2.95(d q,J=12.1,5.8Hz,3H),2.10(s,3H),2.01(d,J=12.5Hz,5H),1.89(s,3H),1. 77(s,3H),1.44(td,J=14.3,7.1Hz,6H),1.30(q,J=7.2Hz,2H),1.18(s,2H). MS(ESI)m / z[MH] - =979.8.

[0227] Step 10: Synthesis of YK-GAL-505

[0228] Using G5-10 (5.0 g, 5.10 mmol) as raw material, according to the synthesis method of YK-GAL-501, white foam solid YK-GAL-505 (5.0 g, 4.23 mmol) was obtained with a yield of 83.3%. NMR(400MHz, DMSO-d6)δ7.80(d,J=9.2Hz,1H),7.70–7.63(m,1H),7.46–7.40(m,2H),7.32–7.26(m,6H),7.21(dt,J=8.2,3.8Hz,1H),6.87(dd,J= 8.7,4.1Hz,4H),5.21(d,J=3.4Hz,1H),4.97(dd,J=11.4,3.6Hz,2H),4.5 0(d,J=8.5Hz,1H),4.05–3.95(m,4H),3.95–3.84(m,2H),3.73(d,J=2.1H z,6H),3.69–3.51(m,3H),3.51–3.30(m,8H),3.22(ddd,J=27.3,10.4,2 .8Hz,1H),3.09–2.87(m,4H),2.74(t,J=6.0Hz,1H),2.57(t,J=5.9Hz,1H ),2.10(s,3H),2.01(d,J=12.5Hz,5H),1.89(s,3H),1.77(s,3H),1.44(t d,J=14.3,7.1Hz,6H),1.31(dt,J=10.2,4.9Hz,4H),1.23–1.03(m,12H). MS(ESI)m / z[M+H] + =1182.2.

[0229] (6) Synthesis of YK-GAL-325

[0230]

[0231] According to the synthesis method of YK-GAL-325 in CN116854754B, page 62, 417.6 mg of product was obtained, MS (ESI) m / z [M+H] + =1452.2.

[0232] 2. Synthesis of GalNAc solid phase support compounds

[0233] (1) Synthesis of YK-GAL-501-SP

[0234] The synthetic route is as follows:

[0235]

[0236] Step 1: Synthesis of G1-14

[0237] G1-13 (200 mg, 204 μmol), 4-dimethylaminopyridine (24.86 mg, 204 μmol), diisopropylethylamine (105.2 mg, 814 μmol), and succinic anhydride (102.1 mg, 1.02 mmol) were dissolved in N,N-dimethylformamide (2.0 mL) and stirred at 30 ° C for 16 h under nitrogen protection. Purification by preparative chromatography gave a white solid G1-14 (115.4 mg, 106 μmol) with a yield of 52.5%.

[0238] Step 2: Synthesis of YK-GAL-501-SP

[0239] G1-14 (50.0 mg, 46.2 μmol), 4-dimethylaminopyridine (5.64 mg, 46.2 μmol), diisopropylethylamine (47.8 mg, 370 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (87.6 mg, 231 μmol) were dissolved in N,N-dimethylformamide (10.0 mL), followed by the addition of CPG-NH 2 (800 mg), stirred at 40 ° C for 16 hours. The reaction solution was filtered, and the filtrate was washed with methanol and dichloromethane in turn and dried in vacuum. The filtrate was then added to 10 mL of acetic anhydride / pyridine (1:4) solution and stirred at 40 ° C for 0.5 hours. Filtered, washed with dichloromethane and methanol in turn, and the filtrate was dried in vacuum for 12 hours to obtain a white solid compound YK-GAL-501-SP (749 mg, loading 32.7 μmol / g).

[0240] (2) Synthesis of other solid phase carrier compounds

[0241] Using G2-10, G3-10, G4-10 and G5-10 as starting materials, respectively, according to the method for synthesizing YK-GAL-501-SP, other GalNAc solid phase carrier compounds YK-GAL-502-SP, YK-GAL-503-SP, YK-GAL-504-SP and YK-GAL-505-SP listed in Table 1 were synthesized.

[0242] Table 1 GalNAc solid phase carrier compounds

[0243]

[0244] Example 2: Coupling of GalNAc compounds to oligonucleotides

[0245] In this example, the same oligonucleotide sequence was used for synthesis, and the GalNAc compound was coupled with the oligonucleotide to obtain a GalNAc-conjugated oligonucleotide, wherein the conjugated oligonucleotide sequence is a sequence numbered D579-DV25P, and the sequence of D579-DV25P is as follows:

[0246] Sense strand (D579-DV25P-SS): 5'-Cms-Cms-Um-Um-Um-Um-Cf-Um-Uf-Cf-Uf-Am-Am-Um-Gm-Am-Gf-Um-Cm-Gm-Am-3' (SEQ ID NO: 1),

[0247] Antisense strand (D579-DV25P-AS): 5′-UmsEVP-Cfs-Gm-Am-Cm-Uf-Cm-Am-Um-Um-Am-Gm-Am-Af-Gm-Af-Am-Am-Am-Gm-Gms-Ums-Gm-3′ (SEQ ID NO: 2).

[0248] Among them, A, U, C, and G represent the base composition of the nucleotide; m represents that the nucleotide adjacent to the left of m is modified with 2'-OMe; f represents that the nucleotide adjacent to the left of f is modified with 2'-F; s represents that the two nucleotides adjacent to the left and right of s are connected by thiophosphate groups; EVP represents that the 5' end is modified with vinylphosphonic acid.

[0249] The basic sequence of the double-stranded oligonucleotide numbered D579-DV25P is:

[0250] Sense strand: 5'-CCUUUUCUUCUAAUGAGUCGA-3' (SEQ ID NO: 3)

[0251] Antisense strand: 5'-UCGACUCAUUAGAAGAAAAGGUG-3' (SEQ ID NO: 4).

[0252] 1. Preparation of GalNAc-conjugated siRNA sense strand

[0253] GalNAc-conjugated oligonucleotides, hereinafter referred to as conjugates, were synthesized on a solid support according to phosphoramidite chemistry.

[0254] When synthesizing conjugates 1-5 (sequence numbers D579-DV25PG501, D579-DV25PG502, D579-DV25PG503, D579-DV25PG504 and D579-DV25PG505), a general CPG solid phase carrier or the GalNAc solid phase carrier compound synthesized in Example 1 (YK-GAL-501-SP, YK-GAL-502-SP, YK-GAL-503-SP, YK-GAL-504-SP and YK-GAL-505-SP) was used, and the GalNAc synthesized in Example 1 was added to the solid phase carrier. c phosphoramidite compounds (YK-GAL-501, YK-GAL-502, YK-GAL-503, YK-GAL-504 and YK-GAL-505) as monomers; when synthesizing conjugate 6 (sequence number D579-DV25PG325), a universal CPG solid phase carrier was used, and the GalNAc phosphoramidite compound YK-GAL-325 synthesized in Example 1 was used as a monomer; when synthesizing conjugate 7 (sequence number D579-DV25PL96), purchased CPG-L96 (Tianjin WuXi AppTec New Drug Development Co., Ltd., L96 see US 10465194B2 claim 10) was used as a solid phase carrier, wherein the solid phase carrier synthesis scale was 1 μmol. These GalNAc compounds were conjugated to the 3' end of the oligonucleotide.

[0255] (1) Preparation of reagents and monomers

[0256] By using acetonitrile solution of monomer (1 / 20, w / v), 0.25M 5-benzylthiotetrazolyl acetonitrile solution as activator, 0.2M hydrogenated xanthan in acetonitrile / pyridine (1 / 4, v / v) solution as thio reagent, 0.05M iodine in water / pyridine (1 / 9, v / v) solution as oxidizing reagent, 20% acetic anhydride in acetonitrile (v / v) as capping agent A, 20 / 30 / 50 (1-methylimidazole / pyridine / acetonitrile, v / v / v) as capping agent B, 20% diethylamine in acetonitrile (v / v) as decyanoethyl reagent, 3% dichloroacetic acid in toluene (v / v) as de-DMT reagent. And loaded into the designated reagent position in the 192P model DNA / RNA automatic synthesizer.

[0257] (2) Crude product synthesis

[0258] Enter the designated oligonucleotide sequence and set the synthesis program. After checking that everything is correct, start the cyclic oligonucleotide synthesis. The monomer coupling time is about 1 minute, of which the oxygen generation time is about 30-45 seconds and the thio generation time is about 2 minutes. After the cycle is completed, the solid phase synthesis of the oligonucleotide is completed.

[0259] (3) Deprotection

[0260] After the synthesis is completed, the solid phase carrier is transferred to a reactor, and the oligonucleotide is cleaved from the solid phase carrier with concentrated ammonia (25-28%) at 50-60°C for 16-24 hours. The system is cooled to room temperature and then filtered, rinsed with a mixed solution of purified water and ethanol, the filtrates are combined, and the filtrates are concentrated at low temperature to obtain a crude residue.

[0261] (4) Purification

[0262] The crude residue after deprotection was dissolved in purified water and purified by HPLC. The product peak solution was collected and the content was measured by an ELISA instrument, and the molecular weight was confirmed by ESIMS.

[0263] In this step, the GalNAc phosphoramidite compound synthesized in Example 1 is conjugated to the 3' end of the sense strand of the siRNA.

[0264] When synthesizing conjugates 1-6, the GalNAc phosphoramidite compounds (YK-GAL-501, YK-GAL-502, YK-GAL-503, YK-GAL-504, YK-GAL-505 and YK-GAL-325) synthesized in Example 1 were used as monomers. When a universal CPG solid phase support was used, the GalNAc phosphoramidite compound was repeated 3 times in the synthesis sequence; when the GalNAc solid phase support synthesized in Example 1 was used, the GalNAc phosphoramidite compound was repeated 2 times in the synthesis sequence, and ultimately the synthesized conjugates 1-6 were conjugated with 3 GalNAc compounds.

[0265] 2. Preparation of siRNA antisense strand without GalNAc conjugation

[0266] The siRNA antisense chain was synthesized according to the method for synthesizing the siRNA sense chain, wherein a universal CPG solid phase carrier was used, and the synthesis scale of each antisense chain complementary to the sense chain was 1 μmol.

[0267] 3. Preparation of Conjugated Oligonucleotides

[0268] The GalNAc-conjugated siRNA sense strand and complementary antisense strand were mixed in a ratio of 1:1 according to the UV absorption content, heated to 95°C, and cooled to room temperature after 3 minutes to form a double strand. The obtained double-stranded solution was characterized by HPLC, and the content was determined by an ELISA instrument after the product purity was qualified, and lyophilized to obtain a solid powder for storage. The sequence and molecular weight of the obtained GalNAc-conjugated oligonucleotide are shown in Table 2.

[0269] Table 2 GalNAc-conjugated double-stranded siRNA

[0270]

[0271]

[0272] Wherein: SS is the sense strand, AS is the antisense strand, and the resulting GalNAc-conjugated oligonucleotide structure is as follows:

[0273]

[0274]

[0275]

[0276]

[0277] The GalNAc compound in D579-DV25PG325 is YK-GAL-325 (CN116854754B, page 62),

[0278]

[0279] The GalNAc compound in D579-DV25PL96 is L96 (US10465194B2, compound according to claim 10).

[0280] Example 3: Inhibitory effect of GalNAc-conjugated oligonucleotides on AGT protein expression in mouse serum and liver and effect on liver AGT mRNA levels

[0281] Angiotensinogen (AGT) protein is a secretory protein that is mainly expressed in the liver in the human body. As an upstream protein of the renin-angiotensin-aldosterone system (RAAS), inhibiting its expression will fundamentally inhibit the blood pressure-raising effect of the RAAS system, thereby lowering blood pressure.

[0282] In this example, transgenic mice expressing the human AGT gene were used to detect the inhibitory effect of the GalNAc-conjugated double-stranded siRNA prepared in Example 2 on the AGT protein in serum at different time points by ELISA, and the inhibitory effect on the AGT mRNA level in the liver was detected.

[0283] 1. Experimental Materials

[0284] Tested drugs:

[0285] GalNAc conjugated oligonucleotides: D579-DV25PL96, D579-DV25PG325, D579-DV25PG501, D579-DV25PG502, D579-DV25PG503, D579-DV25PG504, and D579-DV25PG505.

[0286] Experimental Animal Information:

[0287] Species / strain: hAGT transgenic mice

[0288] Rating: SPF

[0289] Gender: Male

[0290] Quantity: 64

[0291] Age: 6 to 8 weeks

[0292] Weight: 18-28g

[0293] Source: Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0294] Production license number: SCXK (Su) 2018-0008

[0295] Feeding and management:

[0296] Feeding conditions: The experimental animals were received and kept by Beijing Boruishian Technology Co., Ltd., with a license number of SYXK (Beijing) 2022-0025. The animals were strictly reviewed in accordance with the requirements of the Institutional Animal Care and Use Committee (IACUC) to ensure animal welfare. The cage specifications were length × width × height = 29.0 cm × 18.5 cm × 13.0 cm; the set temperature range was 20-26 ° C, the set humidity range was 40%-70%, the air change frequency was no less than 15 times of fresh air / h, and the artificial lighting was alternating between 12h light and 12h dark.

[0297] The feeding environment conditions are in accordance with the National Standard of the People's Republic of China GB14925-2010, and the environment is controlled by a modular air conditioning unit. Animals have free access to food and water.

[0298] 2. Experimental Methods

[0299] Dosage design and grouping

[0300] Definition of experimental day: The day when animals were administered with vehicle or test drug was defined as day 0.

[0301] Grouping and administration: After 3 days of adaptive feeding, the experimental animals were randomly divided into a negative control group and a test drug group according to the serum AGT protein content, with 8 animals in each group. The drug was administered by a single subcutaneous injection, with a dose of 1 mg / kg, a volume of 5 mL / kg, and a concentration of 0.2 mg / mL. The day of administration was recorded as day 0.

[0302] The animal grouping information is shown in Table 3:

[0303] Table 3 Animal grouping information

[0304] Group drug Dosage (mg / kg) Dosage route / frequency Cycle (days) Number of animals Negative control group solvent / / 35 8 Test drug group siRNA 1 Subcutaneous injection / 1 time 35 8

[0305] Detection indicators

[0306] (1) General observation

[0307] The subjects were observed once a day from one week before administration to the end of the experiment.

[0308] Observation content: Observe the animal's death or dying, mental state, behavioral activities, feces characteristics, feed and water supply, etc. beside the cage.

[0309] Test animals: all animals in the negative control group and the test drug group.

[0310] (2) Expression of AGT protein in serum

[0311] Testing time: before dosing on day 3 (day-3), 1 week after dosing on day 7 (1w), 2 weeks after dosing on day 14 (2w), 3 weeks after dosing on day 21 (3w), 4 weeks after dosing on day 28 (4w), and 5 weeks after dosing on day 35 (5w).

[0312] AGT protein level detection method: ELISA kit was used for detection.

[0313] Test animals: all animals in the negative control group and the test drug group.

[0314] (3) Detection of liver mRNA levels

[0315] Liver tissue collection and homogenate preparation: On Day 35, appropriate amounts of fresh liver tissue were collected from all animals and quickly placed in an EP tube containing 1 mL of RNA lysis solution (TRIzol) according to tissue weight: RNA lysis solution (TRIzol) = 100 mg: 1 mL to prepare liver homogenate. The homogenate was immediately tested or placed at -80°C. The remaining liver tissue was quickly frozen and stored at -80°C.

[0316] RNA extraction:

[0317] a. Place the tissue in a 1.5 mL RNase-free EP tube, add 1 mL TRIzol reagent to every 100 mg of tissue, and grind by shaking to prepare a tissue homogenate.

[0318] b. Centrifuge at 4℃, 12000g for 3min, transfer 400μL of tissue homogenate supernatant to a 1.5mL RNase-free EP tube and place on ice. Add 80μL of chloroform to each tube, shake vigorously for 15sec, and let stand at room temperature for 5min. Centrifuge at 4℃, 12000g for 15min, and take 150μL of supernatant to a new EP tube.

[0319] c. Add an equal volume of isopropanol, gently mix the liquid in the tube by turning it upside down, let it stand at -20℃ for 10 min, centrifuge at 4℃ (white precipitate can be seen), 12000g for 15 min, and discard the supernatant.

[0320] d. Add 1 mL of 75% ethanol, gently wash the RNA precipitate, centrifuge at 4°C, 7500 g for 5 min, and remove the supernatant. Repeat the rinse once, 4°C, 7500 g for 5 min, and remove the residual ethanol with a micropipette.

[0321] e. Dry the residual ethanol at room temperature for 10 min, add 100 μL RNase-free ddH 2 O, dissolve.

[0322] RNA concentration detection and reverse transcription: The RNA concentration was detected using a UV-visible spectrophotometer. 2 μL RNase-free ddH 2 O was used as a blank control, and 2 μL RNA samples were used for testing each time. The sample concentration was recorded. Then, cDNA was synthesized using PrimeScript RT Master Mix according to the instructions. The reverse transcription reaction system was prepared in 0.2 mL eight-well strips according to Table 4. The volume of RNA and sterile enzyme-free water can be adjusted according to the RNA concentration. A 10 μL system can process up to 500 ng RNA, and the system can be expanded proportionally according to the required RNA amount.

[0323] Table 4 Reverse transcription system

[0324] Components volume Master Mix 2.0μL RNA 5.0μL Sterile enzyme-free water 3.0μL

[0325] Flick the eight-well strip to mix the system. Centrifuge briefly using the Short function of the centrifuge, and then use a PCR instrument to perform the reverse transcription reaction. The program is as follows: 37℃ for 15min, 85℃ for 5s, and 4℃ for maintenance.

[0326] Realtime-qPCR: Using TB Premix Ex Taq TM(Tli RNaseH Plus), Bulk for qPCR. According to Table 4, 10 μL reaction system was prepared in a 96-well plate, and the cDNA of GAPDH and AGT was detected by qPCR, with GAPDH as the internal reference gene. qPCR was performed in a 96-well plate, with each well containing 10 μL reaction system, 3 wells of GAPDH primers for each sample, and 3 wells of each pair of target gene primers. When there are too many samples and the samples of one biological replicate need to be dispersed on multiple 96-well plates for detection, ensure that each group of samples is amplified simultaneously on the same 96-well plate, and a control group sample is included on each 96-well plate. The qPCR reaction program is: heating at 95℃ for 30s, then entering the cycle mode, heating at 95℃ for 5s, then heating at 60℃ for 34s, for a total of 40 cycles; heating the template at 95℃ for 15s, heating at 60℃ for 1min, then heating at 95℃ for 15s, and performing melting curve analysis.

[0327] Table 5 qPCR reaction system

[0328] Element Volume / μL 2×Master Mix 5.00 Upstream primer (10 μM) 0.20 Downstream primer (10 μM) 0.20 cDNA 1.00 ROX 0.04 <![CDATA[ddH 2 The]]> 3.56

[0329] (4) Data processing and statistical analysis

[0330] The experimental data were expressed as mean ± standard deviation (Mean ± SD), and the data were analyzed using GraphPad Prism 8.3 analysis software. Two-way ANOVA and post hoc test were used for statistical analysis. The LSD test was used for homogeneity of variance, and the inequality of variance was tested by

[0331] 3. Experimental results

[0332] Specific experimental results are shown in Table 6-1, Table 6-2 and Table 7.

[0333] (1) Inhibition rate of AGT protein level in serum

[0334] Table 6-1 Inhibition rate of different GalNAc-conjugated oligonucleotides on AGT protein level in serum

[0335]

[0336]

[0337] Table 6-2 Inhibition rate of AGT protein level in serum by different GalNAc-conjugated oligonucleotides

[0338]

[0339] As can be seen from Table 6, these GalNAc-conjugated oligonucleotides can significantly and continuously inhibit the AGT protein level in mouse serum. For example, the inhibition rates of D579-DV25PG503 on the 7th, 14th, 21st, 28th and 35th days were 67.91%, 77.15%, 73.25%, 74.12% and 69.50%, respectively. The experimental results show that the use of oligonucleotide sequence D579-DV25P coupled with the GalNAc compound of the present application can be efficiently delivered to the animal liver and significantly inhibit AGT gene expression.

[0340] Compared with the siRNA conjugates prepared from the prior art GalNAc compounds YK-GAL-325 and L96, the GalNAc-conjugated oligonucleotides prepared from the GalNAc compounds of the present application significantly improved the inhibition rate of AGT protein levels in serum. For example, the inhibition rate of the D579-DV25PG502 group on the 7th day was 12.22% higher than that of the D579-DV25PL96 group, and the inhibition rate of the D579-DV25PG504 group on the 35th day was 14.48% higher than that of the D579-DV25PL96 group.

[0341] (2) Inhibition rate of AGT mRNA level in liver

[0342] Table 7 Inhibition rate of liver AGT mRNA level in hAGT transgenic mice after administration for 35 days

[0343]

[0344] As shown in Table 7, these GalNAc-conjugated oligonucleotides can significantly and continuously inhibit the AGT mRNA level in mouse liver, and the D579-DV25PG504 group has the highest inhibition rate, reaching 74.64%. The experimental results show that the oligonucleotide sequence D579-DV25P coupled with the GalNAc compound of the present application can be efficiently delivered to the animal liver and significantly inhibit the AGT mRNA level in the liver.

[0345] Compared with the siRNA conjugates prepared from the prior art GalNAc compounds YK-GAL-325 and L96, the GalNAc-conjugated oligonucleotides prepared from the GalNAc compounds of the present application significantly improved the inhibition rate of mouse liver AGT mRNA levels. For example, the inhibition rate of the D579-DV25PG504 group was 8.39% and 13.70% higher than that of the D579-DV25PG325 group and the D579-DV25PL96 group, respectively.

[0346] Compared with the prior art GalNAc compounds YK-GAL-325 and L96, the efficiency of delivering oligonucleotides to the liver by coupling the oligonucleotide sequence D579-DV25P to the GalNAc compound of the present application is significantly improved compared with coupling YK-GAL-325 and L96, indicating that the efficiency of delivering oligonucleotides cannot be simply inferred based on the chemical structure of the GalNAc compound.

[0347] The present application designs a series of novel GalNAc compounds, such as YK-GAL-501, YK-GAL-502, YK-GAL-503, YK-GAL-504 and YK-GAL-505. The GalNAc-conjugated oligonucleotides prepared therefrom can achieve efficient liver-targeted delivery, and their activity is significantly improved compared to the representative GalNAc compounds in the prior art.

[0348] 1. The GalNAc compound of the present application has a completely different chemical structure from the prior art GalNAc compound and is a completely new GalNAc compound. The GalNAc compound designed in the present application introduces a ribose ring structure in the linker and introduces an oxygen or sulfur atom at the 1' position of the ribose ring.

[0349] 2. The GalNAc compound designed in this application is conjugated with the oligonucleotide sequence D579-DV25P, which can efficiently deliver the oligonucleotide to the liver of animals and can significantly and continuously inhibit the expression of AGT protein in serum and the mRNA level in the liver. For example, D579-DV25PG501 inhibited AGT protein in serum by 66.21%, 75.63% and 75.24% on the 7th, 14th and 28th days, respectively; after 35 days of administration, the inhibition rate of AGT mRNA level in the liver reached 71.29%.

[0350] 3. Compared with the GalNAc compounds of the prior art (such as L96 and YK-GAL-325), the GalNAc-conjugated oligonucleotides prepared from the GalNAc compounds designed in the present application significantly improved the inhibition rate of AGT protein expression in mouse serum and the inhibition rate of AGT mRNA levels in mouse liver.

[0351] For example, compared with the oligonucleotide D579-DV25PL96 conjugated with L96, the oligonucleotide D579-DV25PG503 conjugated with YK-GAL-503 increased the inhibition rate of AGT protein expression in mouse serum by 10.89% on day 7, and increased the inhibition rate of AGT mRNA level in mouse liver by 12.25% on day 35. This indicates that the GalNAc compound designed in this application can significantly improve the delivery efficiency, increase the bioavailability of the drug, improve the pharmacokinetic properties of the drug, and exert a better drug effect.

[0352] 4. The present application found that the GalNAc-conjugated oligonucleotides prepared from different GalNAc compounds with similar structures are very likely to have significant differences in the inhibition rate of AGT protein expression in mouse serum and the inhibition rate of AGT mRNA level in mouse liver, indicating that it is difficult to accurately infer the oligonucleotide delivery efficiency through the chemical structure of the GalNAc compound.

[0353] For example, compared with L96, YK-GAL-503 only changes the prolinol structure in the main chain of L96 to a ribose ring structure, and the other structures are exactly the same. However, compared with the oligonucleotide D579-DV25PL96 conjugated with L96, the oligonucleotide D579-DV25PG503 conjugated with YK-GAL-503 increased the inhibition rate of AGT protein expression in mouse serum by 10.89% after 7 days of administration, and increased the inhibition rate of AGT mRNA level in mouse liver by 12.25% after 35 days. Compared with YK-GAL-325, YK-GAL-503 only shortens the connection chain between the ribose ring and GalNAc in YK-GAL-325, and the other structures are exactly the same. However, compared with the YK-GAL-325 conjugated oligonucleotide, the YK-GAL-503 conjugated oligonucleotide increased the inhibition rate of AGT protein expression in mouse serum by 7.85% after 7 days of administration and increased the inhibition rate of AGT mRNA level in mouse liver by 6.94% after 35 days.

[0354] Although the present application has been described by way of specific examples above, it should not be construed as being limited thereto, but rather the present application encompasses the general aspects previously disclosed and may be modified and have various embodiments without departing from the spirit and scope of the present application.

[0355] SEQ ID NO:1

[0356] Sense strand (D579-DV25P-SS): 5'-Cms-Cms-Um-Um-Um-Um-Cf-Um-Uf-Cf-Uf-Am-Am-Um-Gm-Am-Gf-Um-Cm-Gm-Am-3'.

[0357] SEQ ID NO:2

[0358] Antisense strand (D579-DV25P-AS): 5′-UmsEVP-Cfs-Gm-Am-Cm-Uf-Cm-Am-Um-Um-Am-Gm-Am-Af-Gm-Af-Am-Am-Am-Gm-Gms-Ums-Gm-3′.

[0359] SEQ ID NO:3:

[0360] Sense strand: 5′-CCUUUUCUUCUAAUGAGUCGA-3′.

[0361] SEQ ID NO:4

[0362] Antisense strand: 5′-UCGACUCAUUAGAAGAAAAGGUG-3′.

Claims

1. The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof: R1 is oxygen or sulfur; R2 is hydrogen, C 1-6 Alkyl, C 1-6 Alkoxy or halogen; R3 is hydrogen, hydroxyl protecting group, phosphorus-containing reactive group, -CO(CH2) x CONH- or -CO(CH2) x COOH, wherein x is an integer from 1 to 10, It is controlled pore glass or polystyrene; R4 is hydrogen or a hydroxyl protecting group; A is -(CH2) a -、-(CH2CH2OCH2CH2) b -or-(CH2OCH2) c -,in, a is an integer from 1 to 10; b is an integer from 1 to 5; c is an integer from 1 to 7; L is -CONH- or -NHCO-; G is in, T is a hydroxyl group fully protected with an acyl group: N-acetyl-galactosamine, galactose, galactosamine, N-formal-galactosamine, N-propionyl-galactosamine, and N-butyryl-galactosamine; X1 is -(CH2) f -or-(CH2CH2O) f CH2-, f is an integer from 1 to 5; X2 is -(CH2) g -, g is an integer from 1 to 6; Y1 is 0 or 1; Y2 is 0, 1 or 2; Y3 is 1, 2 or 3; m is an integer from 0 to 4; n is an integer from 0 to 4.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: R3 is a hydroxyl protecting group, preferably an acyl group, a silyl group, a trityl group, a 4-methoxytrityl group, a 4,4'-bismethoxytrityl group, and more preferably an acetyl group, a 1,1,3,3-tetraisopropyldisiloxanyl group, a tert-butyldimethylsilyl group, a dimethylphenylsilyl group, a 4,4'-bismethoxytrityl group; Alternatively, R3 is a phosphorus-containing reactive group, such as 3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: R4 is a hydroxyl protecting group, preferably a trityl group, a 4-methoxytrityl group or a 4,4'-bismethoxytrityl group.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: It meets one or more of the following conditions: (1) a is an integer from 2 to 7; (2) b is, for example, an integer from 1 to 3; (3) The acyl group may be acetyl or benzoyl, for example, acetyl; (4) m is 0, 1 or 2; (5) n is 0, 1 or 2; (6) R3 is a phosphorus-containing reactive group, -CO(CH2) x CONH- is controlled pore glass or polystyrene, and preferably, x is 2.

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: It meets one or more of the following conditions: (1) A is -CH2CH2OCH2CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4- or -(CH2)5-; (2) R1 is oxygen; (3) R1 is α- or β-configuration; (4) R2 is -OCH3; (5) R3 is -CO(CH2)2CONH- or -CO(CH2)2COOH, It is a controlled pore glass; (6) R4 is 4,4'-bismethoxytrityl; (7) T is N-acetyl-galactosamine with fully protected acyl groups; (8) X1 is -(CH2) f -, f is 1; (9) g is 2; (10) Y1 is 0; (11) Y2 is 0; (12) m is 1; (13)n is 0; (14) L is -NHCO-; (15)Y3 is 1.

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: G is 7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound is any of the following compounds: It is controlled pore glass.

8. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, characterized in that: It is able to bind to the asialoglycoprotein receptor.

9. A conjugate, characterized in that It comprises an oligonucleotide and a GalNAc portion, wherein the oligonucleotide and the GalNAc are connected via a phosphate group or a thiophosphate group; the GalNAc portion is one or more GalNAc molecules, which are connected via a phosphate group or a thiophosphate group; Wherein, the GalNAc molecule is a compound of formula I-2 or a pharmaceutically acceptable salt thereof; Among them, R 3-1 is a connecting bond, R4 is H; Or, R 4-1 is the connecting key, R 3-1 is H; G is T1 is N-acetyl-galactosamine, galactose, galactosamine, N-formal-galactosamine, N-propionyl-galactosamine and N-butyryl-galactosamine, preferably N-acetyl-galactosamine; Y3, Y2, X2, Y1, X1, m, n, R2, R1, A, and L are as described in any one of claims 1 to 7.

10. The conjugate according to claim 9, characterized in that The oligonucleotides include non-thiolated oligonucleotides and thiolated oligonucleotides.

11. The conjugate according to claim 9 or 10, characterized in that wherein the non-thio oligonucleotide and the GalNAc moiety are linked via a phosphate bond.

12. The conjugate according to claim 9 or 10, characterized in that wherein the thio-oligonucleotide and the GalNAc moiety are linked via a phosphorothioate bond.

13. The conjugate according to claim 9, characterized in that: It has the following structure: wherein Oligo represents oligonucleotide, X3 is oxygen or sulfur, q is 1, 2 or 3, and R1, R2, R3, R4, A, L, G, m and n are as described in claim 9.

14. The conjugate according to claim 9, characterized in that The oligonucleotides include small interfering nucleotides, DNA, microRNA, small activating RNA, small guide RNA, transfer RNA, antisense nucleotides or aptamers, preferably antisense nucleotides or small interfering nucleotides.

15. The conjugate according to claim 14, characterized in that Each nucleotide in the antisense nucleotide or small interfering nucleotide is independently a modified or unmodified nucleotide.

16. The conjugate according to claim 9, characterized in that It has the following structure:

17. The conjugate of claim 9, wherein the oligonucleotide modulates expression of a target gene.

18. A pharmaceutical composition comprising the conjugate of any one of claims 9 to 17 and at least one pharmaceutically acceptable excipient.

19. The conjugate according to any one of claims 9 to 17 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 18, for use in the preparation of a composition for treating and / or preventing a pathological condition or disease caused by the expression of a specific gene in liver tissue or in a virus. The use of the specific gene in a drug for treating a disease; optionally, the specific gene is selected from the group consisting of hepatitis B virus gene, pro Protein convertase subtilisin 9 gene, Coagulation factor gene, lipoprotein a gene or angiopoietin-like protein 3 gene, angiotensinogen gene or apolipoprotein C3 gene.

20. The use according to claim 19, wherein The disease is selected from chronic liver disease, hepatitis, liver fibrosis, liver proliferative disease and cardiovascular and cerebrovascular diseases; optionally, the cardiovascular and cerebrovascular diseases are hypercholesterolemia, hypertriglyceridemia, atherosclerosis or coagulation dysfunction, etc.

21. A kit comprising the conjugate of any one of claims 9 to 17.

22. A method for inhibiting the expression of a specific gene in hepatocytes, wherein: The method comprises contacting the hepatocyte with an effective amount of the conjugate of any one of claims 9 to 17 or the pharmaceutical composition of claim 18; optionally, the specific gene is selected from the proprotein convertase subtilisin 9 gene, the hepatitis B virus gene, the apolipoprotein a gene, the coagulation factor XI gene, the angiopoietin-like protein 3 gene, the angiotensinogen gene or the apolipoprotein C3 gene.

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