Galnac monomers containing a ribose ring or a derivative thereof and their use in the liver-targeted delivery of small nucleic acid drugs

Abstract

The application provides a GalNAc monomer containing a ribose ring or a derivative structure and application thereof in liver-targeted delivery of small nucleic acid drugs. Specifically provided is a compound of formula (I) or a pharmaceutically acceptable salt thereof. The GalNAc compound provided by the application can realize efficient liver-targeted delivery of a GalNAc conjugated oligonucleotide, and improve drug efficacy.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a GalNAc compound with a ribocyclic structure, and the GalNAc-conjugated oligonucleotides prepared therefrom can achieve efficient liver-targeted delivery. Background Technology

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

[0003] Oligonucleotides are a class of short DNA or RNA molecules, oligomers, that readily bind in a sequence-specific manner to their complementary oligonucleotides, DNA, or RNA to form double strands, or less commonly, higher-order hybrids. This fundamental characteristic makes oligonucleotides widely applicable in gene sequencing, targeted gene therapy research, and medicine. These small fragments of nucleic acids can be manufactured into single-stranded molecules with any specified sequence. In nature, oligonucleotides are typically small RNA molecules that play a role in gene expression regulation, or degradation intermediates derived from the breakdown 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 breaking down target mRNA.

[0005] N-acetylgalactosamine (GalNAc) is a ligand that binds to the desialization glycoprotein receptor (ASGPR) on the liver surface. The desialization glycoprotein receptor is an endocytic receptor specifically expressed on the surface of hepatocytes. In recent years, the use of GalNAc, a high-affinity ligand of ASGPR, as a targeting molecule has led to some progress in the liver-targeted delivery of nucleic acid drugs. For example, Alnilam Pharmaceuticals (… 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 GalNAc-siRNA conjugate exhibited good delivery activity in both in vivo and in vitro experiments. In in vivo experiments in mice administered subcutaneously, a single dose of ED... 50The optimal dosage was determined to be 1 mg / kg, with a single injection dose of less than 1 mL. In long-term dosing studies, subcutaneous injection once weekly resulted in stable interfering activity for up to 9 months. Studies found that tetrapod and tripod GalNAc compounds exhibited significantly higher affinity for ASGPR than bipod and monopod GalNAc compounds.

[0006] GalNAc compounds with different structures exhibit significantly different nucleic acid delivery effects. To improve the delivery of liver-targeted drugs, such as AGT inhibitors, anti-chronic hepatitis B drugs, and lipid-lowering drugs, there is a need in this field to develop new GalNAc compounds. Summary of the Invention

[0007] To address the aforementioned technical problems, this 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 this invention can be used to prepare GalNAc-conjugated oligonucleotides, significantly improving delivery efficiency and significantly inhibiting AGT gene expression.

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

[0009]

[0010] in,

[0011] R1 is oxygen or sulfur;

[0012] R2 is hydrogen, C 1-6 Alkyl, C 1-6 Alkyl or halogen;

[0013] R3 represents hydrogen, a hydroxyl protecting group, a phosphorus-containing reactive group, or -CO(CH2). x CONH- or -CO(CH2) x COOH, where x is an integer from 1 to 10. It can be made of glass with controllable pores or polystyrene;

[0014] R4 is a hydrogen or hydroxyl protecting group;

[0015] A is -(CH2) a -、-(CH2CH2OCH2CH2) b -or-(CH2OCH2) c - where a is an integer from 1 to 10; b is an integer from 1 to 5; and c is an integer from 1 to 7.

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

[0017] G is

[0018] in,

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

[0020] X1 is -(CH2) f -or-(CH2CH2O) f CH2-, f is an integer from 1 to 5;

[0021] X2 is -(CH2) g - g is an integer from 1 to 6;

[0022] Y1 is either 0 or 1;

[0023] Y2 is 0, 1, or 2;

[0024] Y3 is 1, 2, or 3;

[0025] m is an integer between 0 and 4;

[0026] n is an integer between 0 and 4.

[0027] In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof; certain groups may be defined as follows, and other groups may be defined as in any of the preceding embodiments (hereinafter referred to as "in one embodiment"):

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

[0029] In one scheme, the C 1-6 The alkoxy group can be 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 scheme, A is -CH2CH2OCH2CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, or -(CH2)5-.

[0032] In one particular scheme, R1 represents oxygen.

[0033] In a certain scheme, R1 is either α configuration or β configuration, such as α configuration.

[0034] In one particular scheme, R2 is C 1-6 Alkyl groups, such as -OCH3.

[0035] In one embodiment, in R3, the hydroxyl protecting group is acyl, silyl, triphenylmethyl, 4-methoxytriphenylmethyl, or 4,4'-dimethoxytriphenylmethyl, preferably acetyl, 1,1,3,3-tetraisopropyldisiloxane, tert-butyldimethylsilyl, dimethylphenylsilyl, or 4,4'-dimethoxytriphenylmethyl.

[0036] In one embodiment, R3 is a phosphorus-containing reactive group, such as...

[0037] In one scheme, R3 is a phosphorus-containing reactive group, -CO(CH2). x CONH- It is controlled-pore glass (CPG) or polystyrene, preferably, x is 2.

[0038] In one scheme, R3 is -CO(CH2)2CONH- It is a controllable aperture glass (CPG).

[0039] In one scheme, R3 is -CO(CH2)2COOH.

[0040] In one embodiment, R4 is a hydroxyl protecting group, preferably triphenylmethyl, 4-methoxytriphenylmethyl, or 4,4'-dimethoxytriphenylmethyl, more preferably 4,4'-dimethoxytriphenylmethyl.

[0041] In one embodiment, T is an acyl-protected N-acetyl-galactosamine, and the acyl group can be acetyl or benzoyl, for example, acetyl.

[0042] In one particular scheme, X1 is -(CH2). f -, f is preferably 1.

[0043] In one scheme, a is an integer between 2 and 7.

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

[0045] In one particular scheme, g is 2.

[0046] In one particular scheme, Y1 is 0.

[0047] In one particular scheme, Y2 is 0.

[0048] In one particular scheme, Y3 is 1.

[0049] In a certain scheme, m is 0, 1, or 2, for example, 1.

[0050] In a certain scheme, n is 0, 1, or 2, for example, 0.

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

[0052] In a certain scheme, where G is

[0053]

[0054] In one embodiment, the compound of formula (I) is the compound shown in formula I-1 below;

[0055]

[0056] Wherein, R2, R3, R4, A, and X2 are 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 a controllable aperture glass (CPG).

[0063] In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is capable of binding to the desialyl glycoprotein receptor (ASGPR).

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

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

[0066]

[0067] Among them, R 3-1 For the connection key, R4 is H;

[0068] Or, R 4-1 For connection key, R 3-1 For H;

[0069] G is

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

[0071] Y3, Y2, X2, Y1, X1, m, n, R2, R1, A, L are as described in any embodiment of the present invention.

[0072] In one embodiment, the oligonucleotide in the conjugate may include nonthio oligonucleotides and thio oligonucleotides.

[0073] In one embodiment, the nonthiooligonucleotide and the GalNAc moiety in the conjugate are linked by a phosphate ester bond.

[0074] In one embodiment, the thiooligonucleotide and the GalNAc moiety in the conjugate are linked by a thiophosphate bond.

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

[0076]

[0077] Oligo represents oligonucleotide, X3 represents oxygen or sulfur, and q represents 1, 2, or 3.

[0078] R1, R2, R3, R4, A, L, G, m, and n are independently as described in any embodiment of the present invention.

[0079] In one embodiment, the oligonucleotide in the conjugate preferably includes small interfering nucleotide (siRNA), DNA, microRNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), antisense nucleotide (ASO), or aptamer, preferably antisense nucleotide (ASO) or small interfering nucleotide (siRNA).

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

[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 described in any embodiment of the present invention and at least one pharmaceutically acceptable excipient.

[0087] This invention provides the use of any conjugate or pharmaceutically acceptable salt thereof described in any embodiment of the invention, or the pharmaceutical composition described in any embodiment of the invention, in the preparation of a medicament for treating and / or preventing pathological conditions or diseases caused by the expression of specific genes in liver tissue or viruses; optionally, the specific gene is selected from hepatitis B virus (HBV) gene, proprotein convertase subtilisin 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, liver proliferative disease, and cardiovascular and cerebrovascular diseases; optionally, the cardiovascular and cerebrovascular diseases are hypercholesterolemia, hypertriglyceridemia, atherosclerosis, or coagulation dysfunction, etc.

[0088] This invention provides a method for inhibiting the expression of a specific gene in hepatocytes, wherein the method includes administering a therapeutically effective amount of any of the preceding conjugates to an individual in need; preferably, the method includes contacting the hepatocytes with an effective amount of any of the preceding conjugates; optionally, the specific gene is selected from the proprotein convertase subtilisin 9 gene (PCSK9), hepatitis B virus gene, apolipoprotein a gene, coagulation factor 11 gene, angiopoietin-like protein 3 gene, angiotensinogen gene, or apolipoprotein C3 gene.

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

[0090] Terminology Explanation:

[0091] The term "pharmaceutical acceptable" means that something is relatively non-toxic, safe, and suitable for patient use.

[0092] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When a 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 a 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. See Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition) for details.

[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 The aryl group, and the substituents can be halogen, cyano, hydroxyl, or C. 6-14 Aryl.

[0094] All terms "therapeutic effective amount" refer to the amount of a compound that, when administered to a subject, is sufficient to effectively treat the disease or condition described herein. While the amount of a compound constituting a "therapeutic 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 conventional manner by those skilled in the art.

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

[0096] In some embodiments, treatment or ongoing treatment refers to the improvement, prevention, or reversal of a disease or condition or at least one identifiable symptom thereof. In other embodiments, treatment or ongoing treatment refers to the improvement, prevention, or reversal of at least one measurable bodily parameter of a disease or condition that may not be recognized in mammals. However, in yet another embodiment, treatment or ongoing treatment refers to slowing the progression of a disease or condition, either physically, such as stabilizing identifiable symptoms, or physiologically, such as stabilizing bodily parameters, or both. In still other embodiments, treatment or ongoing treatment refers to delaying the onset of a disease or condition.

[0097] In some embodiments, the compounds of this disclosure may be administered as a preventative measure. As used herein, “prevention” or “being prevented” means reducing the risk of acquiring a given disease or condition. In a preferred mode of the embodiments, the designated compound is given as a preventative measure to a subject, such as a subject with a family history or predisposition to cancer or an autoimmune disease.

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

[0099] The reagents and raw materials used in this invention are all commercially available.

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

[0101] 1. This application designs a series of novel GalNAc compounds with completely different chemical structures compared with existing GalNAc compounds.

[0102] 2. The GalNAc-conjugated oligonucleotides prepared from the GalNAc compounds designed in this application have significantly improved delivery efficiency compared with those prepared from existing GalNAc compounds (e.g., L96 and YK-GAL-325), and significantly improved inhibition rates of AGT protein expression in mouse serum and AGT mRNA levels in the liver. Detailed Implementation

[0103] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the described embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0104] This application may be implemented in other specific forms without departing from its essential attributes. It should be understood that, without conflict, any and all embodiments of this application may be combined with technical features of any or more other embodiments to obtain further embodiments. This application includes such further embodiments obtained through combination.

[0105] All publications and patents mentioned in this application are incorporated herein by reference in their entirety. In the event of any conflict between the use or terminology used in any publications and patents incorporated by reference and the use or terminology used in this application, the use and terminology of this application shall prevail.

[0106] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the subject matter.

[0107] Unless otherwise specified, all technical and scientific terms used herein have their usual meaning in the field to which the claimed subject matter pertains. Where multiple definitions exist for a term, the definition herein shall prevail.

[0108] Unless otherwise indicated in the working embodiments or elsewhere, all figures for quantitative properties such as dosage set forth in the specification and claims should be understood to be modified in all cases by the term "about". It should also be understood that any range of figures listed in this application is intended to include all subranges within that range and any combination of the endpoints of that range or subranges.

[0109] As used herein, the terms “comprising,” “containing,” or “including” mean that the element preceding the word encompasses the elements listed following the word and their equivalents, without excluding elements not described herein. The terms “containing” or “comprising (including)” as used herein can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently composed of” or “composed of”.

[0110] The present application will be further described below with reference to embodiments. However, the present application is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. In the specific embodiments of the present application, the raw materials used can all be obtained commercially. Unless otherwise stated, all temperatures are given in degrees Celsius. The technical features involved in the various embodiments 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; BF3·Et2O: boron trifluoride ether; DMTrCl: 4,4'-dimethoxytriphenylchloromethane; HBTU: O-benzotriazole-tetramethylurea 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); Me3OBF4: trimethyloxonium tetrafluoroboric acid; DMF: N,N-dimethylformamide; Ac2O: acetic anhydride.

[0113] 1. Synthesis of GalNAc phosphorous amide compounds

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

[0115] The synthesis route is as follows:

[0116]

[0117] Step 1: Synthesis of G1-3

[0118] Dichloromethane (1500 mL) was added to dried compound G1-1 (100.0 g, 951.2 mmol), followed by triethylamine (107.3 g, 1.06 mol). The mixture was cooled to 0 °C, and then G1-2 (121.7 g, 865.1 mmol) was slowly added dropwise while maintaining the temperature between 0 and 5 °C. After the addition was complete, the mixture was heated to room temperature and stirred for 2 h. The pH was adjusted to 6–7 by adding 1 mol / L hydrochloric acid after cooling to 0 °C. A saturated sodium chloride aqueous solution (300 mL) was added, and the mixture was shaken and separated. The aqueous phase was extracted with dichloromethane (800 mL × 3), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure to obtain a colorless oily crude product G1-3 (105.0 g), which was used directly 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] Dichloromethane (450 mL) was added to compound G1-4 (87.2 g, 274.0 mmol), and the mixture was stirred until dissolved. 3A molecular sieve (80.0 g) was added, and the mixture was cooled to 0 °C. Boron trifluoride diethyl ether (116.9 g, 823.7 mmol) was then added, followed by stirring for 20 min. A dichloromethane solution of G1-3 (86.0 g, 411.0 mmol) in 180 mL was added dropwise to the system at 0–5 °C, and the mixture was stirred overnight at 0–5 °C. The mixture was filtered, and the reaction was quenched by adding saturated sodium bicarbonate aqueous solution (125 mL) dropwise to the filtrate at 0–5 °C. The mixture was separated, and the organic phase was washed successively with saturated sodium bicarbonate aqueous solution (900 mL × 2) and semi-saturated sodium chloride aqueous solution (900 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure to obtain a yellow oily crude product, G1-5 (150.0 g), which was used directly 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, and a methanol solution (300 mL) of sodium methoxide (3.50 g, 64.8 mmol) was added dropwise to the system at 0–5 °C. After the addition was complete, the temperature was raised to room temperature and reacted for 1 h. The temperature was then lowered to 0–5 °C, and 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, and purified water (200 mL) was added. The mixture was washed with dichloromethane (400 mL × 8), and the aqueous phase was collected and concentrated under reduced pressure to remove the solvent, yielding the crude product. The crude product was purified by column chromatography (dichloromethane / methanol) to give a colorless, transparent oily substance G1-6 (69.0 g, 202.1 mmol). 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), and imidazole (34.4 g, 505.3 mmol) was added. The mixture was cooled to 0 °C, and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (66.8 g, 211.8 mmol) was added dropwise while maintaining the temperature at 0–5 °C. After the addition was complete, the mixture was stirred for 10 min, and then heated to room temperature and stirred for 2 h. After the reaction was complete, purified water (700 mL) was slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was shaken and separated. The organic phase was washed with saturated sodium chloride aqueous solution (700 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent, yielding a crude product. This crude product was slurried with n-heptane (700 mL), filtered, and the filter cake was collected and dried under vacuum to give 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), and 1,8-bis(dimethylamino)naphthalene (64.0 g, 298.6 mmol) was added. The mixture was cooled to 0 °C, and trimethyloxonium tetrafluoroboric acid (35.5 g, 240.0 mmol) was added. The mixture was stirred for 10 min, then heated to room temperature and stirred for 2 h. After the reaction was complete, purified water (5.4 mL) was added to quench the reaction, and the mixture was stirred for 10 min. The mixture was filtered, and the filtrate was collected and concentrated under reduced pressure to remove the solvent. Then, n-heptane (700 mL) was added and stirred for 20 min. The mixture was filtered again, and the filtrate was collected. The filtrate was washed once with 15 wt% ammonium chloride aqueous solution (700 mL) and once with saturated sodium chloride aqueous solution (700 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent, yielding the crude product. The crude product was purified by column chromatography (n-hexane / ethyl acetate) to give a wine-red oil, G1-8 (56.0 g, 93.7 mmol), in 78.1% yield. ¹H NMR (400 MHz, DMSO-d6) δ 8.46 (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, and triethylamine trihydrofluoric acid (45.3 g, 281.0 mmol) was slowly added dropwise. After the addition was complete, the temperature was raised to room temperature and stirred overnight at room temperature. After the reaction was complete, saturated sodium bicarbonate aqueous solution (600 mL) was added, and the mixture was stirred for 5 min. Saturated sodium chloride aqueous solution (600 mL) was then added, and the mixture was shaken and separated. The aqueous phase was extracted with tetrahydrofuran (600 mL), and the organic phases were combined. The organic phase was washed with saturated sodium chloride aqueous solution (300 mL) and dried over anhydrous sodium sulfate. The solvent was removed by concentration under reduced pressure to obtain the 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 pale yellow oily substance G1-9 (35.0 g), which was used directly 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] Dry G1-9 (35.0 g, 98.5 mmol) and dimethylaminopyridine (1.20 g, 9.82 mmol) were dissolved in pyridine (350 mL). 4,4'-dimethoxytriphenylchloromethane (40.1 g, 118.3 mmol) was added in four portions, and the mixture was stirred at 15–20 °C for 1 h. After the reaction was complete, the temperature was lowered to 0 °C, and the reaction was quenched by adding saturated sodium bicarbonate aqueous solution (350 mL) dropwise at 0–5 °C. After the addition was complete, the mixture was stirred for 10 min, and then dichloromethane (350 mL) was added. The mixture was shaken and separated. The organic phase was washed once with purified water (350 mL), and then once with 10 wt% sodium chloride aqueous solution (350 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure to obtain the crude product. Column chromatography purification (n-heptane / ethyl acetate, 0.1% triethylamine) yielded a pale yellow, foamy solid G1-10 (50.0 g, 76.0 mmol), 77.2%. ¹H NMR (400 MHz, DMSO-d6) δ 8.46 (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 = 7.0 mmol). 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] G1-10 (50.0 g, 76.0 mmol) was dissolved in methanol (500 mL), and sodium hydroxide (136.8 g, 3.42 mol) and purified water (27.4 g, 1.52 mol) were added. The mixture was sealed and heated to 130 °C, and reacted for 2 h. After the reaction was complete, the mixture was cooled to room temperature, and dichloromethane (1000 mL) and a 15 wt% ammonium chloride aqueous solution (1000 mL) were added. The mixture was stirred for 5 min, and the liquid was separated. The organic phase was washed successively with purified water (1000 mL) and saturated sodium chloride aqueous solution (500 mL). The organic phase was dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to remove the solvent, yielding a pale yellow solid crude product G1-11 (40.0 g), which was used directly 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] G1-11 (40.0 g, 72.2 mmol) was dissolved in dichloromethane (400 mL), and O-benzotriazole-tetramethylurea hexafluorophosphate (41.1 g, 108.4 mmol) was added. The mixture was cooled to 0 °C, and diisopropylethylamine (18.7 g, 144.7 mmol) was added. The mixture was stirred for 10 min, and a dichloromethane (400 mL) solution of G1-12 (35.6 g, 79.6 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred for 1 h. After the reaction was complete, purified water (1000 mL) was added dropwise at 0–5 °C, and the mixture was shaken and separated. The organic phase was washed with a 10 wt% sodium chloride aqueous solution (1000 mL), dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to remove the solvent, yielding the crude product. Column chromatography purification (dichloromethane / tetrahydrofuran, 0.1% triethylamine) yielded a pale yellow, frothy solid, G1-13 (28.9 g, 29.4 mmol). Yield: 40.7%. ¹H NMR (400 MHz, DMSO-d6) δ 7.83–7.72 (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), and 3A molecular sieve (750 mg) was added. The mixture was stirred and dried for 30 min, and this solution was designated as solution A. Pyridine trifluoroacetate (1.18 g, 6.11 mmol) and triphenylphosphine (80 mg, 0.31 mmol) were dissolved in tetrahydrofuran (15 mL), and 3A molecular sieve (750 mg) was added. The mixture was stirred and dried for 30 min, and this solution was designated as solution B. Solution B was cooled to 0 °C, and a phosphorus reagent, bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.30 g, 7.63 mmol), was added. Then, solution A was added dropwise to solution B, and the mixture was stirred for 1 h. After the reaction was complete, the mixture was filtered. A 5 wt% sodium bicarbonate aqueous solution (30 mL) was added to the filtrate, and the mixture was stirred for 5 min. Dichloromethane (60 mL) was then added, and the mixture was shaken and separated. The organic phase was washed successively with a 5 wt% sodium bicarbonate aqueous solution (30 mL) and a saturated sodium chloride aqueous solution (30 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to remove the solvent, yielding a residue. The residue was dissolved in dichloromethane (3 mL) and added dropwise to n-heptane (60 mL) at 10–15 °C, and stirred for 30 min. The supernatant was removed, yielding a viscous, oily solid. This solid was dried under vacuum to obtain a white, foamy solid, YK-GAL-501 (3.4 g, 2.87 mmol), 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 synthesis 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 starting materials, crude product G2-2 (70.0 g) was obtained following the synthesis method of G1-3 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, crude product G2-3 (100.0 g) was obtained by following the synthesis method of G1-5, and was directly used 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 a starting material, a colorless, transparent oil G2-4 (40.0 g, 135.4 mmol) was obtained by following the synthesis method of G1-6. The combined yield of the two steps was 56.0%. 1H 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, 5.0Hz, 1H), 3.52 (tt, J=10.0, 4.6Hz, 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 the starting material, G2-5 (50.0 g, 92.6 mmol) was synthesized according to the method of G1-7, yielding a white solid G2-5 (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 the starting material, G2-6 (31.0 g, 56.0 mmol) was synthesized according to the method of G1-8, 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] Using G2-6 (31.0 g, 56.0 mmol) as a starting material, G2-7 (17.5 g) was synthesized according to the method for G1-9, yielding a pale yellow oil, which was directly used in the next step. ¹H NMR (400 MHz, Chloroform-d) δ 7.83–7.76 (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 a starting material, G2-8 (19.0 g, 31.0 mmol) was synthesized according to the method for G1-10, yielding a pale yellow, foamy solid, with a yield of 57.4%. ¹H NMR (400 MHz, DMSO-d6) δ 8.42 (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] Using G2-8 (18.0 g, 29.3 mmol) as the starting material, and following the synthesis method of G1-11, a pale yellow solid crude product G2-9 (15.0 g) was obtained and directly used in the next step. ¹H NMR (400 MHz, DMSO-d6) δ 7.46–7.39 (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 mmol). 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 starting materials, G2-10 (18.6 g, 19.8 mmol) was synthesized according to the method for G1-13, yielding a pale yellow, foamy solid, 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 a starting material, a white, foamy solid YK-GAL-502 (2.0 g, 1.76 mmol) was obtained according to the synthesis method of YK-GAL-501, with a yield of 82.3%. 1H 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 synthesis 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 starting materials, and following the synthesis method of G1-3, a colorless oily crude product G3-2 (63.0 g) was obtained and directly used in the next step. ¹H NMR (400 MHz, DMSO-d6) δ 8.42 (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 starting materials, and following the synthesis method of G1-5, a yellow oily crude product G3-3 (135.0 g) was obtained and directly used in the next step. ¹H NMR (400 MHz, DMSO-d6) δ 8.40 (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] Using G3-3 (135 g, 308.6 mol) as the starting material, a colorless, transparent oil G3-4 (31.9 g, 102.5 mmol) was obtained by following the synthesis method of G1-6. The combined yield of the two steps was 33.2%. ¹H NMR (400 MHz, DMSO-d6) δ 8.42 (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). 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 the starting material, G3-5 (43.0 g, 77.6 mmol) was synthesized according to the method of G1-7, yielding a white solid G3-5 (43.0 g, 77.6 mmol) in 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 the starting material, a wine-red oily compound G3-6 (31.2 g, 54.9 mmol) was synthesized according to the method for G1-8, with a yield of 76.0%. ¹H NMR (400 MHz, DMSO-d6) δ 8.39 (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] Using G3-6 (30.0 g, 52.8 mmol) as a starting material, G3-7 (19.1 g) was synthesized according to the method for G1-9, and was directly used 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] Using G3-7 (18.0 g, 55.3 mmol) as a starting material, G3-8 (24.5 g, 39.0 mmol) was synthesized according to the method for G1-10, yielding a pale yellow, foamy solid, with a yield of 70.6%. ¹H NMR (400 MHz, DMSO-d6) δ 8.39 (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] Using G3-8 (23.0 g, 36.6 mmol) as the starting material, and following the synthesis method of G1-11, a pale yellow solid crude product G3-9 (18.5 g) was obtained and directly used in the next step. ¹H NMR (400 MHz, DMSO-d6) δ 7.47–7.40 (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 starting materials, G3-10 (23.0 g, 24.1 mmol) was synthesized following the same method as G1-13, yielding a pale yellow, foamy solid, G3-10, in 70.1% yield. 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 a starting material, a white, foamy solid YK-GAL-503 (5.1 g, 4.42 mmol) was synthesized according to the method for YK-GAL-501, with a yield of 84.3%. 1H 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 synthesis 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 starting materials, and following the synthesis method of G1-3, a colorless oily crude product G4-2 (135.0 g) was obtained and directly used in the next step. ¹H NMR (400 MHz, Chloroform-d) δ 7.77 (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 starting materials, and following the synthesis method of G1-5, a yellow oily crude product G4-3 (194.0 g) was obtained and directly used in the next step. ¹H NMR (400 MHz, DMSO-d6) δ 8.43 (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] Using G4-3 (194.0 g, 429.7 mol) as the starting material, a colorless, transparent oil G4-4 (51.2 g, 157.4 mmol) was obtained by following the synthesis method of G1-6, with a combined yield of 41.7%. ¹H NMR (400 MHz, DMSO-d6) δ 8.44 (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 = 6.6 Hz, 1H). .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 the starting material, G4-5 (59.0 g, 103.9 mmol) was synthesized according to the method of G1-7, with a white solid 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 the starting material, a wine-red oily compound G4-6 (41.0 g, 70.5 mmol) was synthesized according to the method for G1-8, with a yield of 67.8%. ¹H NMR (400 MHz, DMSO-d6) δ 8.42 (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] Using G4-6 (34.0 g, 58.4 mmol) as a starting material, G4-7 (22.0 g) was synthesized according to the method for G1-9, and was directly used in the next step. ¹H NMR (400 MHz, DMSO-d6) δ 8.44 (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] Using G4-7 (20.0 g, 58.9 mmol) as a starting material, G4-8 (23.1 g, 36.0 mmol) was synthesized according to the method for G1-10, yielding a pale yellow, foamy solid, G4-8 (23.1 g, 36.0 mmol), with a yield of 61.0%. ¹H NMR (400 MHz, DMSO-d6) δ 8.40 (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). 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 starting material, and following the synthesis method of G1-11, a pale yellow solid crude product G4-9 (16.5 g) was obtained and directly used in the next step. ¹H NMR (400 MHz, DMSO-d6) δ 7.47–7.40 (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 starting materials, G4-10 (19.4 g, 20.1 mmol) was synthesized according to the method for G1-13, yielding a pale yellow, foamy solid, G4-10, in 71.9% yield. 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 a starting material, a white, foamy solid YK-GAL-504 (3.2 g, 2.74 mmol) was synthesized according to the method for YK-GAL-501, with a yield of 88.4%. 1H 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 starting materials, and following the synthesis method of G1-3, a colorless oily crude product G5-2 (123.0 g) was obtained and directly used in the next step. ¹H NMR (400 MHz, DMSO-d6) δ 8.41 (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, the crude yellow oily product G5-3 (124.0 g) was obtained according to the synthesis method of G1-5 and used directly in the next step. ¹H NMR (400 MHz, DMSO-d6) δ 8.43 (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] Using G5-3 (88.0 g, 189.0 mol) as the starting material, a colorless, transparent oil G5-4 (23.0 g, 67.8 mmol) was obtained by following the synthesis method of G1-6, with a combined yield of 35.8%. ¹H NMR (400 MHz, DMSO-d6) δ 8.44 (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, 4.8 Hz). 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] Using G5-4 (20.0 g, 58.9 mmol) as the starting material, G5-5 (25.1 g, 43.1 mmol) was synthesized according to the method for G1-7, yielding a white solid G5-5 (25.1 g, 43.1 mmol), with a yield of 73.2%. ¹H NMR (400 MHz, DMSO-d6) δ 8.43 (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, 1H). 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 the starting material, a wine-red oily compound G5-6 (19.4 g, 32.6 mmol) was synthesized according to the method for G1-8, with a yield of 78.9%. ¹H NMR (400 MHz, DMSO-d6) δ 8.44 (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] Using G5-6 (19.0 g, 31.9 mmol) as a starting material, G5-7 (12.3 g) was synthesized according to the method for G1-9, and was directly used in the next step. ¹H NMR (400 MHz, DMSO-d6) δ 8.44 (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, 4.5 Hz, 2H). 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 a starting material, G5-8 (15.3 g, 23.3 mmol) was synthesized according to the method for G1-10, yielding a pale yellow, foamy solid, with a yield of 68.6%. 1H 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] Using G5-8 (13.0 g, 19.8 mmol) as the starting material, and following the synthesis method of G1-11, a pale yellow solid crude product G3-9 (10.4 g) was obtained and directly used in the next step. ¹H NMR (400 MHz, DMSO-d6) δ 7.48–7.40 (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 starting materials, G5-10 (11.9 g, 12.1 mmol) was synthesized according to the method for G1-13, yielding a pale yellow, foamy solid, G5-10, in 66.9% yield. 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 a starting material, a white, foamy solid YK-GAL-505 (5.0 g, 4.23 mmol) was obtained according to the synthesis method of YK-GAL-501, with a yield of 83.3%. 1H 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] Following the synthesis method for YK-GAL-325 described on page 62 of CN116854754B, 417.6 mg of the product was obtained, with 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 synthesis 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. Preparative chromatographic purification yielded 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-tetramethylurea hexafluorophosphate (87.6 mg, 231 μmol) were dissolved in N,N-dimethylformamide (10.0 mL), followed by the addition of CPG-NH2 (800 mg), and the mixture was stirred at 40 °C for 16 h. The reaction solution was filtered, and the filtrate was washed successively with methanol and dichloromethane, and dried under vacuum. The filtrate was then added to 10 mL of acetic anhydride / pyridine (1:4) solution, and stirred at 40 °C for 0.5 h. After filtration, the filtrate was washed successively with dichloromethane and methanol, and dried under vacuum for 12 h to obtain a white solid compound YK-GAL-501-SP (749 mg, loading 32.7 μmol / g).

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

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

[0242] Table 1. GalNAc solid-phase support compounds

[0243]

[0244] Example 2: Conjugation of GalNAc compounds with oligonucleotides

[0245] In this embodiment, the same oligonucleotide sequence was used for synthesis. The conjugation of the GalNAc compound with the oligonucleotide yielded GalNAc-conjugated oligonucleotides, wherein the conjugated oligonucleotide sequence is the sequence numbered D579-DV25P, and the D579-DV25P sequence is as follows:

[0246] Chain of Justice (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 chain (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] In this context, A, U, C, and G represent the base composition of the nucleotide; m indicates that the nucleotide to the left of m is modified with 2'-OMe; f indicates that the nucleotide to the left of f is modified with 2'-F; s indicates that the two nucleotides to the left and right of s are linked by a thiophosphate group; and EVP indicates that the 5' end is modified with vinylphosphonic acid.

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

[0250] Chain of Justice: 5'-CCUUUUCUUCUAAUGAGUCGA-3'(SEQ ID NO:3)

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

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

[0253] GalNAc-conjugated oligonucleotides (hereinafter referred to as conjugates) were synthesized on a solid support using a phosphorus amide chemical method.

[0254] When synthesizing conjugates 1-5 (sequence numbers D579-DV25PG501, D579-DV25PG502, D579-DV25PG503, D579-DV25PG504, and D579-DV25PG505), a general-purpose CPG solid support or the GalNAc solid support compounds 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) were used. c-phosphorous amide compounds (YK-GAL-501, YK-GAL-502, YK-GAL-503, YK-GAL-504, and YK-GAL-505) were used as monomers; when synthesizing conjugate 6 (sequence number D579-DV25PG325), a general-purpose CPG solid-phase support was used, with the GalNAc phosphorous amide compound YK-GAL-325 synthesized in Example 1 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 support, wherein the solid-phase support synthesis scale was 1 μmol. All these GalNAc compounds were conjugated to the 3' end of the oligonucleotide.

[0255] (1) Preparation of reagents and monomers

[0256] The following reagents were used: a monomeric acetonitrile solution (1 / 20, w / v), a 0.25M 5-benzylthiotetrazole acetonitrile solution as an activator, a 0.2M hydroflavin acetonitrile / pyridine solution (1 / 4, v / v) as a thioating agent, a 0.05M iodine-water / pyridine solution (1 / 9, v / v) as an oxidizing agent, 20% acetic anhydride in acetonitrile (v / v) as capping agent A, 20 / 30 / 50 (1-methylimidazolium / pyridine / acetonitrile, v / v / v) as capping agent B, 20% diethylamine in acetonitrile (v / v) as a decyanoethylating agent, and 3% dichloroacetic acid in toluene (v / v) as a DMT-removing agent. These were loaded into the designated reagent positions on a 192P model DNA / RNA automated synthesizer.

[0257] (2) Crude product synthesis

[0258] Input the specified oligonucleotide sequence and set the synthesis program. After verifying that everything is correct, begin the oligonucleotide synthesis cycle. The monomer coupling time is approximately 1 minute, with an oxygenation time of approximately 30-45 seconds and a thiolation time of approximately 2 minutes. After the cycle is complete, the solid-phase synthesis of the oligonucleotide is finished.

[0259] (3) Deprotection

[0260] After synthesis, the solid support was transferred to a reactor, and the oligonucleotides were lysed from the solid support with concentrated ammonia (25-28%) at 50-60°C for 16-24 hours. The system was then cooled to room temperature, filtered, and washed with a mixture of purified water and ethanol. The filtrates were combined and concentrated at low temperature to obtain the crude residue.

[0261] (4) Purification

[0262] The crude residue after deprotection was dissolved in purified water, purified by HPLC, the product peak solution was collected and the content was measured by an enzyme-linked immunosorbent assay (ELISA) reader, and the molecular weight was confirmed by ESIMS.

[0263] This step involves conjugating the GalNAc phosphoridamide compound synthesized in Example 1 to the 3' end of the positive strand of the siRNA.

[0264] When synthesizing conjugates 1-6, the GalNAc phosphorous amide 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 general CPG solid support was used, the GalNAc phosphorous amide compound was repeated 3 times in the synthesis sequence; when the GalNAc solid support synthesized in Example 1 was used, the GalNAc phosphorous amide compound was repeated 2 times in the synthesis sequence, so that all conjugates 1-6 were conjugated with 3 GalNAc compounds.

[0265] 2. Preparation of GalNAc-free siRNA antisense strands

[0266] The antisense strand of siRNA was synthesized using the same method as for synthesizing the sense strand of siRNA, with a universal CPG solid-phase carrier. The synthesis scale of each antisense strand complementary to the sense strand was 1 μmol.

[0267] 3. Preparation of conjugated oligonucleotides

[0268] The GalNAc-conjugated siRNA sense and antisense strands were mixed at a 1:1 ratio according to their UV absorption content, heated to 95℃ for 3 minutes, and then cooled to room temperature to form double strands. The resulting double-stranded solution was characterized by HPLC to ensure product purity. After passing the purity test, the content was determined using a microplate reader, and the product was lyophilized to obtain a solid powder for later storage. The sequences and molecular weights of the obtained GalNAc-conjugated oligonucleotides are shown in Table 2.

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

[0270]

[0271]

[0272] Where SS is the sense strand and AS is the antisense strand, 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 of claim 10).

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

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

[0282] In this embodiment, 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 serum AGT protein at different time points using the ELISA method, and the inhibitory effect on AGT mRNA levels in the liver was also detected.

[0283] 1. Experimental Materials

[0284] Test drug:

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

[0286] Laboratory animal information:

[0287] Species / Strain: hAGT transgenic mice

[0288] Grade: SPF

[0289] Sex: Male

[0290] Quantity: 64

[0291] Age: 6 - 8 weeks

[0292] Body weight: 18 - 28 g

[0293] Source: Jiangsu Genscript Biotech Co., Ltd.

[0294] Production License Number: SCXK(Su)2018 - 0008

[0295] Feeding and Management:

[0296] Feeding Conditions: After receiving the experimental animals, they were raised in Beijing Boshi An Technology Co., Ltd., with the use license number: SYXK(Jing)2022 - 0025. It was carried out strictly in accordance with the requirements of the Institutional Animal Care and Use Committee (IACUC) to ensure animal welfare. The specifications of the breeding cages 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 rate was not less than 15 times of fresh air per hour, and the artificial lighting was alternated with 12 hours of light and 12 hours of darkness.

[0297] The standards of the feeding environmental conditions were referred to the national standard of the People's Republic of China GB14925 - 2010, and the environment was controlled by a combined air conditioning unit. The animals were fed freely and had free access to water.

[0298] 2. Experimental Methods

[0299] Dose Design and Grouping

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

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

[0302] Details of the animal grouping information are shown in Table 3:

[0303] Table 3 Animal Grouping Information

[0304] Group drug Dosage (mg / kg) Dosage method / 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] Observe once a day from one week before administration until the end of the trial.

[0308] Observation content: Observe the animal's death or near death, mental state, behavior, fecal characteristics, and the supply of feed and water at the cage.

[0309] Animals tested: All animals in the negative control group and the test drug group.

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

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

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

[0313] Animals tested: All animals in the negative control group and the test drug group.

[0314] (3) Liver mRNA level detection

[0315] Liver tissue collection and homogenate preparation: On Day 35, appropriate amounts of fresh liver tissue were collected from all animals. The tissue weight was adjusted to 100 mg of RNA lysis buffer (TRIzol) per 1 mL, and the mixture was quickly placed into EP tubes containing 1 mL of TRIzol to prepare a liver homogenate. The homogenate was immediately tested or stored at -80°C. The remaining liver tissue was flash-frozen and stored at -80°C.

[0316] RNA extraction:

[0317] a. Take tissue and place it into a 1.5 mL RNase-free EP tube. Add 1 mL TRIzol reagent to every 100 mg of tissue and vortex to prepare a tissue homogenate.

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

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

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

[0321] e. Allow the residual ethanol to air dry at room temperature for 10 minutes, then add 100 μL of RNase-free ddH2O to dissolve.

[0322] RNA Concentration Detection and Reverse Transcription: RNA concentration was detected using a UV-Vis spectrophotometer. 2 μL of RNA-free ddH2O was used as a blank control, and 2 μL of RNA sample was used for each test. The sample concentration was recorded. cDNA was then synthesized using PrimeScript RT Master Mix according to the manufacturer's instructions. The reverse transcription reaction system was prepared in 0.2 mL octet in Table 4. The volumes 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 of RNA, and the system can be scaled up proportionally according to the required amount of RNA.

[0323] Table 4 Reverse Transcription System

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

[0325] Gently tap the eight-pack to mix the system. Briefly centrifuge using the Short function of a centrifuge, then perform a reverse transcription reaction using a PCR instrument with the following program: 37°C for 15 min, 85°C for 5 s, and maintain at 4°C.

[0326] Realtime-qPCR: Using TB Premix Ex Taq TM(Tli RNaseH Plus), Bulk for qPCR. Prepare 10 μL reaction mixtures in 96-well plates according to Table 4. qPCR was used to detect GAPDH and AGT cDNA, with GAPDH serving as an internal control gene. qPCR was performed in 96-well plates, with 10 μL reaction mixture per well. Three wells were used for each sample's GAPDH primers, and three wells were used for each target gene primer pair. When there were too many samples and it was necessary to distribute samples for a single biological replicate across multiple 96-well plates, ensure that each group of samples was amplified simultaneously on the same 96-well plate, and that each 96-well plate included a control sample. The qPCR reaction program was as follows: 95°C for 30 s, then cycling at 95°C for 5 s, followed by 60°C for 34 s, for a total of 40 cycles; template was heated at 95°C for 15 s, then at 60°C for 1 min, followed by 95°C for 15 s, and then melt curve analysis was performed.

[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[ddH2O]]> 3.56

[0329] (4) Data processing and statistical analysis

[0330] Experimental data are expressed as mean ± standard deviation (Mean ± SD). Data analysis was performed using GraphPad Prism 8.3 software. Statistical analysis was conducted using two-way ANOVA and post-hoc tests. Homogeneity of variance was assessed using the LSD test, and unequal variances were analyzed using...

[0331] 3. Experimental Results

[0332] The specific experimental results are shown in Tables 6-1, 6-2 and 7.

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

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

[0335]

[0336]

[0337] Table 6-2 Inhibition rate of different GalNAc-conjugated oligonucleotides on serum AGT protein levels

[0338]

[0339] As shown in Table 6, these GalNAc-conjugated oligonucleotides can consistently and significantly inhibit AGT protein levels in mouse serum. For example, D579-DV25PG503 achieved inhibition rates of 67.91%, 77.15%, 73.25%, 74.12%, and 69.50% on days 7, 14, 21, 28, and 35, respectively. These experimental results demonstrate that conjugating the GalNAc compound of this application with the oligonucleotide sequence D579-DV25P can efficiently deliver it to the animal liver and significantly inhibit AGT gene expression.

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

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

[0342] Table 7 shows the inhibition rate of hepatic AGT mRNA levels in hAGT transgenic mice after 35 days of administration.

[0343]

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

[0345] Compared with siRNA conjugates prepared from existing GalNAc compounds YK-GAL-325 and L96, the GalNAc-conjugated oligonucleotides prepared from the GalNAc compounds of this application significantly enhanced the inhibition rate of AGT mRNA levels in mouse liver. 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 GalNAc compound of this application, when coupled with the oligonucleotide sequence D579-DV25P, significantly improves the efficiency of oligonucleotide delivery to the liver compared with coupling with YK-GAL-325 and L96. This indicates that the efficiency of oligonucleotide delivery cannot be simply inferred from the chemical structure of the GalNAc compound.

[0347] This 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 from these compounds can achieve highly efficient liver-targeted delivery, with significantly improved activity compared to representative GalNAc compounds in the prior art.

[0348] 1. The GalNAc compound of this application has a completely different chemical structure compared with the prior art GalNAc compounds, and is a novel GalNAc compound. The GalNAc compound designed in this application introduces a ribose ring structure in the linker arm and introduces an oxygen or sulfur atom at the 1' position of the ribose ring.

[0349] 2. The GalNAc compound designed in this application, conjugated with the oligonucleotide sequence D579-DV25P, can efficiently deliver the oligonucleotide to the animal liver and sustainably and significantly inhibit serum AGT protein expression and liver mRNA levels. For example, D579-DV25PG501 achieved an inhibition rate of 66.21%, 75.63%, and 75.24% on serum AGT protein at days 7, 14, and 28, respectively; and an inhibition rate of 71.29% on liver AGT mRNA levels after 35 days of administration.

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

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

[0352] 4. This application found that the oligonucleotides conjugated with GalNAc prepared from different GalNAc compounds with similar structures are 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 the delivery efficiency of oligonucleotides is difficult to accurately infer from the chemical structure of GalNAc compounds.

[0353] For example, compared to L96, YK-GAL-503 differs only in that the proline structure in the L96 backbone is replaced with a ribocyclic structure; all other structures are identical. However, compared to the L96-conjugated oligonucleotide D579-DV25PL96, the YK-GAL-503-conjugated oligonucleotide D579-DV25PG503 showed a 10.89% increase in the inhibition rate of AGT protein expression in mouse serum after 7 days of administration, and a 12.25% increase in the inhibition rate of AGT mRNA levels in mouse liver after 35 days. Compared to YK-GAL-325, YK-GAL-503 differs only in that the linker chain between the ribocyclic ring and GalNAc in YK-GAL-325 is shortened; all other structures are identical. However, compared with the oligonucleotide conjugated with YK-GAL-325, the oligonucleotide conjugated with YK-GAL-503 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 this application has been described through specific embodiments, it should not be construed as being limited thereto. Rather, this application covers the general aspects previously disclosed and may be modified and implemented in various ways without departing from the spirit and scope of this application.

[0355] SEQ ID NO:1

[0356] Justice Chain (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 chain (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] Chain of Justice: 5'-CCUUUUCUUCUAAUGAGUCGA-3'.

[0361] SEQ ID NO:4

[0362] Antonym chain: 5'-UCGACUCAUUAGAAGAAAAGGUG-3'.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound is any one of the following compounds: ， ， ， , ， ， ， ； It is a glass with controllable aperture.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, It can bind to the desialyl glycoprotein receptor.

3. A conjugate, characterized in that, It has the following structure: ， ， ， 。 4. The conjugate according to claim 3, wherein the oligonucleotide regulates the expression of the target gene.

5. The conjugate according to claim 3, characterized in that, Oligonucleotides include small interfering nucleotides, DNA, microRNA, small activating RNA, small guide RNA, transfer RNA, antisense nucleotides, or aptamers.

6. The conjugate according to claim 3, characterized in that, The oligonucleotides are either antisense nucleotides or small interfering nucleotides.

7. The conjugate according to claim 5, characterized in that, Each of the antisense nucleotides or small interfering nucleotides therein is independently a modified or unmodified nucleotide.

8. A pharmaceutical composition comprising the conjugate of any one of claims 3-7 and at least one pharmaceutically acceptable excipient.

9. The use of the conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 3-7, or the pharmaceutical composition according to claim 8, in the preparation of a medicament for treating and / or preventing pathological conditions or diseases caused by the expression of a specific gene in liver tissue or a virus.

10. The use according to claim 9, characterized in that, The specific gene is selected from the hepatitis B virus gene, the proprotein convertase subtilisin-9 gene, the coagulation factor gene, the lipoprotein a gene or the angiopoietin-like protein 3 gene, the angiotensinogen gene or the apolipoprotein C3 gene.

11. The use according to claim 9, wherein, The diseases mentioned are selected from chronic liver disease, hepatitis, liver fibrosis, liver proliferative diseases, and cardiovascular and cerebrovascular diseases.

12. The use according to claim 11, characterized in that, The cardiovascular and cerebrovascular diseases mentioned are hypercholesterolemia, hypertriglyceridemia, atherosclerosis, or coagulation dysfunction.

13. A kit comprising the conjugate according to any one of claims 3-7.

14. Use of a conjugate as described in any one of claims 3-7 or the pharmaceutical composition as described in claim 8 in the preparation of an inhibitor of specific gene expression in hepatocytes.

15. The application as described in claim 14, characterized in that, The specific gene is selected from the proprotein convertase subtilisin-9 gene, hepatitis B virus gene, apolipoprotein a gene, coagulation factor 11 gene, angiopoietin-like protein 3 gene, angiotensinogen gene, or apolipoprotein C3 gene.

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