GalNAc monomers containing a ribose ring or its derivative structure, and their use in liver-targeted delivery of small nucleic acid drugs.

Abstract

This invention provides a GalNAc monomer containing a ribose ring or a derivative thereof, and its use in liver-targeted delivery of small nucleic acid drugs. [Solution] The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof. GalNAc-binding oligonucleotides produced with the GalNAc compound provided by the present invention can achieve efficient liver target delivery and improve the efficacy of drugs. JPEG2026059798000067.jpg37165

Description

[Technical Field]

[0001] This application relates to the field of biomedicine, and more specifically to a GalNAc compound having a ribose ring structure, wherein a GalNAc-binding oligonucleotide produced therefrom can achieve efficient liver target delivery. [Background technology]

[0002] Nucleic acid drugs, particularly oligonucleotide drugs, are widely used due to their ease of synthesis and high activity. Oligonucleotide drugs generally include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), and nucleic acid aptamers.

[0003] Oligonucleotides are short DNA or RNA molecules, a type of oligomer, that readily bind sequence-specifically to their complementary oligonucleotides, DNA, or RNA to form double helixes, and, less commonly, higher-order heterozygotes. This fundamental property makes oligonucleotides widely used in genetic testing, targeted gene therapy research, and the pharmaceutical field. These nucleic acid fragments can be synthesized into single-stranded molecules with any specified sequence. In nature, oligonucleotides are generally small RNA molecules that play a role in regulating gene expression, or degradation intermediates derived from the degradation 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. N-acetylgalactosamine (GalNAc) is a ligand that binds to the asialoglycoprotein receptor (ASGPR) on the surface of the liver. The asialoglycoprotein receptor is an endocytosis receptor specifically expressed on the surface of liver cells. In recent years, the use of GalNAc, a high-affinity ligand for ASGPR, as a target molecule has brought about significant progress in liver-targeted delivery of nucleic acid drugs. For example, Alnylam® Pharmaceuticals, Inc. reported that siRNA based on GalNAc binding technology exhibits gene silencing activity in mice (Nair JK, et al. J. Am. Chem. Soc. 2014, 136, 16958). The paper reported that the binding of GalNAc and siRNA showed excellent delivery activity in both in vivo and in vitro experiments. In in vivo experiments with mice using subcutaneous administration, a single dose of ED showed superior delivery activity. 50 The concentration was determined to be 1 mg / kg, and the single injection dose was less than 1 mL. In long-term administration experiments, stable interference activity was obtained for up to 9 months with weekly subcutaneous injections. The study revealed that tetrabranched and tribranched GalNAc compounds have a much higher affinity for ASGPR than bibranched and monobranched GalNAc compounds.

[0005] GalNAc compounds with different structures exhibit significantly different effects on nucleic acid delivery. In this field, there is a need to develop new GalNAc compounds to improve the delivery efficacy of liver-targeted drugs such as AGT inhibitors, chronic hepatitis B infection treatments, and lipid-lowering drugs. [Overview of the Initiative]

[0006] To solve the above technical problems, the present invention provides GalNAc monomers comprising a ribose ring or a derivative thereof, and their use in liver target delivery of small nucleic acid drugs. The GalNAc compounds provided by the present invention can be used to produce GalNAc-binding oligonucleotides, which significantly improve delivery efficiency and significantly inhibit AGT gene expression.

[0007] The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

[0008] [ka]

[0009] however, R1 is oxygen or sulfur. R2 is hydrogen, C 1-6 Alkyl alkyl group, C 1-6 It is an alkoxy group or halogen, R3 contains hydrogen, a hydroxy protecting group, and a phosphorus-containing active reactive group.

[0010] [ka] or -CO(CH2) x COOH, where x is an integer from 1 to 10.

[0011] [ka] It is a controllable porous glass or polystyrene.

[0012] R4 is a hydrogen or hydroxyl protecting group. A is -(CH2) a -,-(CH2CH2OCH2CH2) b -or-(CH2OCH2) c -where a is an integer between 1 and 10, b is an integer between 1 and 5, and c is an integer between 1 and 7. L is -CONH- or -NHCO-, G is

[0013] [ka] And,

[0014] however, T is N-acetylgalactosamine, galactose, galactosamine, N-formylgalactosamine, N-propionylgalactosamine, and N-butanoylgalactosamine in which the hydroxy group is completely protected by an acyl group, 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.

[0015] In one embodiment, for the compound represented by the formula (I), or a pharmaceutically acceptable salt thereof, the definitions of some groups are as described below, and the definitions of the remaining groups may be as described in any one of the above embodiments (hereinafter abbreviated as "in one embodiment").

[0016] In one embodiment, the C 1-6 alkyl group may be a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group.

[0017] In one embodiment, the C 1-6 alkoxy group may be a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, or a tert-butoxy group.

[0018] In one embodiment, the halogen is fluorine, chlorine or bromine. In one embodiment, A is -CH2CH2OCH2CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4- or -(CH2)5-.

[0019] In one embodiment, R1 is oxygen. In one embodiment, R1 is in an α configuration or a β configuration, for example, in an α configuration. In one embodiment, R2 is C 1-6 It is an alkoxy group, for example, -OCH3.

[0020] In one embodiment, R3, the hydroxy protecting group is an acyl group, a silyl group, a trityl group, a 4-methoxytrityl group, or a 4,4'-dimethoxytrityl group, preferably an acetyl group, a 1,1,3,3-tetraisopropyldisiloxane group, a tert-butyldimethylsilyl group, a dimethylphenylsilyl group, or a 4,4'-dimethoxytrityl group.

[0021] In one embodiment, R3 is a phosphorus-containing active reactive group, and the phosphorus-containing active reactive group is, for example,

[0022] [ka] That is the case.

[0023] In one embodiment, R3 is a phosphorus-containing active reactive group,

[0024] [ka] And,

[0025] [ka] x is a controllable porous glass (CPG) or polystyrene, preferably x is 2.

[0026] In one embodiment, R3 is

[0027] [ka] And,

[0028] [ka] It is a controllable porous glass (CPG).

[0029] In one embodiment, R3 is -CO(CH2)2COOH. In one embodiment, R4 is a hydroxy protecting group, preferably a trityl group, a 4-methoxytrityl group, or a 4,4'-dimethoxytrityl group, and more preferably a 4,4'-dimethoxytrityl group.

[0030] In one embodiment, T is N-acetylgalactosamine completely protected by an acyl group, and the acyl group may be an acetyl group or a benzoyl group, for example, an acetyl group.

[0031] In one embodiment, X1 is -(CH2) f - and f is preferably 1. In one embodiment, a is an integer between 2 and 7. In one embodiment, b is an integer between 1 and 3.

[0032] In one embodiment, g is 2. In one embodiment, Y1 is 0. In one embodiment, Y2 is 0.

[0033] In one embodiment, Y3 is 1. In one embodiment, m is 0, 1, or 2, for example, 1. In one embodiment, n is 0, 1, or 2, for example, 0.

[0034] In one embodiment, where L is -NHCO-, In one embodiment, here G is

[0035] [ka] That is the case.

[0036] In one embodiment, the compound represented by formula (I) is the compound represented by the following formula I-1,

[0037] [ka]

[0038] Here, R2, R3, R4, A, and X2 are independently as described in any one embodiment of the present invention. In one embodiment, the compound represented by formula (I) is one of the following:

[0039] [ka]

[0040] [ka]

[0041] In one embodiment, the compound represented by formula (I) is one of the following compounds:

[0042] [ka]

[0043] [ka]

[0044] [ka] It is a controllable porous glass (CPG).

[0045] In one embodiment, the compound represented by formula (I), or a pharmaceutically acceptable salt thereof, can bind to the asialoglycoprotein receptor (ASGPR). The present invention further provides the use of a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, as an intermediate in the production of a GalNAc-binding oligonucleotide pharmaceutical.

[0046] The present invention provides a conjugate comprising an oligonucleotide and a GalNAc moiety, wherein the oligonucleotide and GalNAc are linked via a phosphate ester group or a phosphorothioate group (R 3-1 or R 4-1 (linked via) the GalNAc portion is one or more GalNAc molecules, which are formed by linking via a phosphate ester group or a phosphorothioate group, where the GalNAc molecule is a compound represented by formula (I), or a pharmaceutically acceptable salt thereof.

[0047] [ka]

[0048] Here, R 3-1 This is a linking bond, and R4 is H. Alternatively, R 4-1 R is a linking bond, 3-1 H is H, G is

[0049] [ka] And,

[0050] T1 is N-acetylgalactosamine, galactose, galactosamine, N-formylgalactosamine, N-propionylgalactosamine, and N-butanoylgalactosamine, preferably N-acetylgalactosamine. Y3, Y2, X2, Y1, X1, m, n, R2, R1, A, and L are as described in any one embodiment of the present invention.

[0051] In one embodiment, the jugate comprises a non-phosphorothioate oligonucleotide and a phosphorothioate oligonucleotide. In one embodiment, the nonphosphorothioate oligonucleotide and the GalNAc moiety are linked in the jugate via a phosphate ester bond.

[0052] In one embodiment, the phosphorothioate oligonucleotide and the GalNAc moiety are linked via a phosphorothioate bond in the jugate. In one embodiment, the djugate preferably has the following structure.

[0053] [ka]

[0054] Here, Oligo represents an oligonucleotide, X3 is oxygen or sulfur, and q is 1, 2, or 3. R1, R2, A, L, G, m, and n are independently as described in any one embodiment of the present invention.

[0055] In one embodiment, the conjugate preferably comprises an oligonucleotide, DNA, microRNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), antisense nucleotide (ASO), or aptamer, and is preferably an antisense nucleotide (ASO) or small interfering nucleotide (siRNA).

[0056] In one embodiment, in the conjugate, each nucleotide in the antisense nucleotide (ASO) or small interfering nucleotide (siRNA) is independently modified or unmodified.

[0057] In one embodiment, the jugate has the following structure.

[0058] [ka]

[0059] [ka]

[0060] [ka]

[0061] In the aforementioned conjugate, the oligonucleotide regulates the expression of the target gene. The present invention provides a pharmaceutical composition comprising a conjugate described in any one embodiment of the present invention and at least one pharmaceutically acceptable excipient.

[0062] The present invention provides the use of a conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, according to any one embodiment of the present invention, in the manufacture of a pharmaceutical for treating and / or preventing a pathological condition or disease caused by the expression of a specific gene in liver tissue or a virus, wherein the specific gene is selectively selected from the hepatitis B (HBV) virus gene, the proprotein-converting enzyme subtilisin / kexin type 9 (PCSK9) gene, a coagulation factor gene, the lipoprotein A gene, the angiopoietin-like protein 3 gene, the angiotensinogen gene, or the apolipoprotein C3 gene. Preferably, the disease is selected from chronic liver disease, hepatitis, hepatic fibrosis, hepatoproliferative disorders, and cardiovascular diseases, and selectively, the cardiovascular disease is hypercholesterolemia, hypertriglyceridemia, atherosclerosis, or a blood coagulation disorder.

[0063] The present invention provides a method for inhibiting the expression of a specific gene in hepatocytes, wherein the method comprises the step of administering a therapeutically effective amount of any one of the aforementioned conjugates to an individual in need, preferably the method comprising the step of contacting the hepatocytes with an effective amount of any one of the aforementioned conjugates, wherein the specific gene is selectively selected from the proprotein convertase subtilisin / kexin 9 gene (PCSK9), the hepatitis B virus gene, the apolipoprotein a gene, the coagulation factor 11 gene, the angiopoietin-like protein 3 gene, the angiotensinogen gene, or the apolipoprotein C3 gene.

[0064] The present invention provides a kit comprising a conjugate described in any one embodiment of the present invention. Explanation of terms: The term "pharmaceutically acceptable" refers to a substance that is relatively non-toxic, safe, and suitable for use in patients.

[0065] 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 relatively acidic functional groups, 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 relatively basic functional groups, 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 specifics, refer to Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition).

[0066] The term "acyl group" refers to R-CO-, where R is C. 1-6 Alkyl alkyl group, C 6-14 Aryl group or substituted C 1-6 Alkyl alkyl group, substituted C 6-14 It is an aryl group, and the substituents are halogen, cyano, hydroxyl, or C 6-14 It may also be an aryl group.

[0067] The term "therapeutic dose" as used refers to the amount of a compound sufficient to effectively treat the disease or condition described herein when administered to a subject. The amount of compound constituting the "therapeutic dose" varies depending on the compound, the disease and its severity, and the age of the subject being treated, but can be determined by the routine methods of those skilled in the art.

[0068] The terms "subject," "individual," and "individual required" as used refer to any animal that is intended to be administered, or has already been administered, the conjugate or pharmaceutical composition according to the embodiments of this disclosure, preferably a mammal, and most preferably a human. The term "mammal" as used herein includes all mammals. Mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans, and most preferably humans.

[0069] In some embodiments, treatment or being treated means improvement, prevention, or recovery of a disease or symptom, or at least one identifiable symptom thereof. In some other embodiments, treatment or being treated means improvement, prevention, or recovery of at least one measurable bodily parameter of a disease or symptom being treated, even if the disease or symptom is not specified in the mammal. In yet another embodiment, treatment or being treated means slowing the progression of a disease or symptom, whether physical, such as stabilization of a recognizable symptom, or physiological, such as stabilization of a bodily parameter, or both. In some other embodiments, treatment or being treated means delaying the onset of a disease or symptom.

[0070] In some embodiments, the compounds of this disclosure may be administered as a prophylactic measure. As used herein, “prevention” or “prevention” means reducing the risk of developing a particular disease or condition. In preferred embodiments, the designated compounds are administered as a prophylactic measure to subjects such as subjects with a family history or tendency toward cancer or autoimmune disease.

[0071] The aforementioned preferred conditions can be combined in any way, without violating the ordinary knowledge of the art, to obtain any preferred embodiment of the present invention. The reagents and raw materials used in this invention are commercially available.

[0072] The GalNAc compounds provided in this application and the GalNAc-binding oligonucleotides produced thereby have one or more beneficial effects as described below. 1. This application designs a series of novel GalNAc compounds that have completely different chemical structures compared to conventional GalNAc compounds.

[0073] 2. GalNAc-binding oligonucleotides produced with the GalNAc compound designed in this application exhibit significantly improved delivery efficiency and significantly improved inhibition rates of AGT protein expression in mouse serum and AGT mRNA levels in the liver compared to GalNAc-binding oligonucleotides produced with conventional GalNAc compounds (e.g., L96 and YK-GAL-325). [Modes for carrying out the invention]

[0074] To further clarify the object, technical solution, and advantages of the present invention, the technical solution of the embodiments of this application will be described clearly and completely below. Obviously, the embodiments described are a part of the embodiments of this application, but not all of them. All other embodiments that a person of the ordinary skill of the art could obtain without creative work based on the embodiments described in this application are also within the scope of protection of this application.

[0075] This application may also be implemented in other specific forms without departing from the essential attributes of this application. It should be understood that, to the extent that they do not contradict each other, any embodiment of this application may be combined with the technical features of any other embodiment or more other embodiments to obtain further embodiments. This application also includes additional embodiments obtained from such combinations.

[0076] All publications and patents described herein are incorporated herein by reference in their entirety. In the event of any conflict between the use or terminology used in any of the incorporated publications and patents and the use or terminology used herein, the use and terminology of this application shall prevail.

[0077] The section headings used in this specification are for organizational purposes only and should not be interpreted as limiting the subject matter being described. Unless otherwise defined, all technical and scientific terms used herein have the common meaning in the technical field to which the claimed subject matter belongs. If there are multiple definitions of a term, the definition provided herein shall prevail.

[0078] Except as otherwise provided in the examples, all numerical values ​​indicating quantitative characteristics such as dosages in the specification and claims shall be understood in all cases to be modified by the term "approximately." It should also be understood that any numerical range described herein is intended to include all subranges within that range, and any combination of the various endpoints of that range or subrange.

[0079] As used in this application, words such as “include,” “contain,” or “equip” mean that the element preceding the word includes the element described after the word and their equivalents, but does not exclude any unrecorded elements. As used herein, the terms “contain” or “include” mean open, semi-closed, or closed. In other words, the terms also include “essentially consist of” or “consist of.”

[0080] The present invention will be further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The conditions of use used in the embodiments can be further adjusted according to the specific different requirements used, and the conditions of use not specified are standard conditions in this industry. The raw materials used in the specific embodiments of this application are commercially available. Unless otherwise specified, all temperatures are given in Celsius. The technical features included in each embodiment of this application can be combined with each other, provided that they do not conflict with each other.

[0081] Manufacturing example: Synthesis of GalNAc compound The following abbreviations represent the reagents: TEA: Triethylamine, DCM: Dichloromethane, 3AMS: 3A Molecular Sieves, MeONa: Sodium Methoxydosodium, MeOH: Methanol, imidazole: Imidazole, Pyridine: Pyridine, NaOH: Sodium Hydroxide, DIPEA: Diisopropylethylamine, THF: Tetrahydrofuran, BF3·Et2O: Boron Trifluoride Diethyl Etherate, DMTrCl: 4,4'-Dimethoxytrityl Chloride, HBTU: O-(Benzotriazole-1- (Iyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, DIPEA: N,N-diisopropylethylamine, DMAP: 4-dimethylaminopyridine, TIPDSCl: 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, TEA·3HF: triethylamine hydrofluoric acid, Protonsponge: 1,8-bis(dimethylamino)naphthalene, Me3OBF4: trimethyloxonium tetrafluoroborate, DMF: N,N-dimethylformamide, Ac2O: acetic anhydride.

[0082] 1. Synthesis of GalNAc phosphoramidite compounds (1) Synthesis of YK-GAL-501 The synthesis route is as follows:

[0083] [ka]

[0084] Step 1: Synthesis of G1-3 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). After cooling to 0°C, G1-2 (121.7 g, 865.1 mmol) was slowly added dropwise while controlling the temperature between 0 and 5°C. After the dropwise addition was complete, the temperature was raised to room temperature and the mixture was stirred for 2 hours. After cooling to 0°C, 1 mol / L hydrochloric acid was added dropwise to adjust the pH to 6-7. Saturated sodium chloride aqueous solution (300 mL) was added, and the mixture was separated by shaking. The aqueous phase was extracted with dichloromethane (800 mL x 3) and combined with the organic phase. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent, and a colorless oily crude product G1-3 (105.0 g) was obtained and used directly in the next step. 1 H NMR (400 MHz, DMSO-d6) δ 8.48 (t, J = 5.6 Hz, 1H), 7.84 (dd, J = 7.2, 1.9 Hz, 2H), 7.56 - 7.49 (m, 1H), 7.46 (dd, J = 8.2, 6.5 Hz, 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.

[0085] Step 2: Synthesis of G1-5 Compound G1-4 (87.2 g, 274.0 mmol) was mixed with dichloromethane (450 mL), stirred to dissolve completely, 3A molecular sieve (80.0 g) was added, and the mixture was cooled to 0°C. Then, boron trifluoride diethyl etherate (116.9 g, 823.7 mmol) was added, and the mixture was stirred for 20 minutes after the addition was complete. While controlling the temperature of the reaction system to 0-5°C, a solution of G1-3 (86.0 g, 411.0 mmol) in dichloromethane (180 mL) was added dropwise, and after the addition was complete, the mixture was stirred overnight at 0-5°C. The reaction system was quenched by filtration and dropwise addition of saturated sodium bicarbonate aqueous solution (125 mL) while controlling the temperature to 0-5°C. The organic phase was then separated and washed sequentially with saturated sodium bicarbonate aqueous solution (900 mL x 2) and semi-saturated sodium chloride aqueous solution (900 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent, and a crude yellow oily product G1-5 (150.0 g) was obtained and used directly in the next step. 1 H NMR (400 MHz, DMSO-d6) δ 8.47 (t, J = 5.6 Hz, 1H), 7.84 (dt, J = 7.0, 1.4 Hz, 2H), 7.56 - 7.48 (m, 1H), 7.45 (dd, J = 8.2, 6.6 Hz, 2H), 5.17 (dd, J = 6.7, 4.9 Hz, 1H), 5.12 - 5.05 (m, 2H), 4.28 (dd, J = 11.7, 3.9 Hz, 1H), 4.25 - 4.18 (m, 1H), 4.04 (dd, J = 11.7, 5.6 Hz, 1H), 3.71 (qd, J = 8.6, 7.4, 4.4 Hz, 1H), 3.62 - 3.50 (m, 5H), 3.42 (q, J = 5.9 Hz, 2H), 2.06 (s, 3H), 2.02 (d, J = 2.0 Hz, 6H). MS (ESI) m / z [M + Na] + = 490.2.

[0086] Step 3: Synthesis of G1-6 G1-5 (150.0 g, 320.9 mol) was dissolved in methanol (1200 mL), the reaction system was cooled to 0°C, and methanol solution of sodium methoxide (3.50 g, 64.8 mmol) (300 mL) was added dropwise while controlling the temperature between 0 and 5°C. After the dropwise addition was complete, the temperature was raised to room temperature and the reaction was allowed to proceed for 1 hour. The system was cooled to 0 and 5°C, and 1 mol / L hydrochloric acid was added dropwise while controlling the temperature between 0 and 5°C to adjust the pH to 6 and 7. The reaction 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 x 8). The aqueous phase was collected, concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by column chromatography (dichloromethane / methanol) to obtain a colorless, transparent oily substance G1-6 (69.0 g, 202.1 mmol), and the total yield of the two steps was 73.8%. 1 H NMR (400 MHz, DMSO-d6) δ 8.48 (t, J = 5.6 Hz, 1H), 7.88 - 7.81 (m, 2H), 7.56 - 7.49 (m, 1H), 7.46 (dd, J = 8.1, 6.5 Hz, 2H), 4.89 (d, J = 1.5 Hz, 1H), 4.83 (d, J = 6.9 Hz, 1H), 4.54 (t, J = 5.7 Hz, 1H), 4.30 (dd, J = 6.9, 4.4 Hz, 1H), 3.98 (td, J = 6.6, 4.7 Hz, 1H), 3.77 - 3.67 (m, 2H), 3.56 - 3.46 (m, 6H), 3.35 (s, 4H). MS (ESI) m / z [M - H] - = 340.3.

[0087] Step 4: Synthesis of G1-7 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 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 controlling the temperature between 0 and 5°C. After the dropwise addition was complete, the mixture was stirred for 10 minutes, then the temperature was raised to room temperature and stirring was continued for 2 hours. After the reaction was complete, purified water (700 mL) was slowly added dropwise to the reaction solution. 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, concentrated under reduced pressure to remove the solvent, and the crude product was obtained. n-heptane (700 mL) was added to form a slurry, filtered, the cake was collected, and vacuum-dried to obtain a white solid G1-7 (74.3 g, 127.3 mmol) with a yield of 63.0%. 1 H NMR (400 MHz, DMSO-d6) δ 8.46 (t, J = 5.6 Hz, 1H), 7.87 - 7.80 (m, 2H), 7.56 - 7.41 (m, 3H), 5.06 (d, J = 3.9 Hz, 1H), 4.76 (s, 1H), 4.30 (dd, J = 6.9, 4.4 Hz, 1H), 3.92 - 3.75 (m, 4H), 3.67 - 3.36 (m, 8H), 1.08 - 0.84 (m, 28H). MS (ESI) m / z [M - H] - = 582.5.

[0088] Step 5: Synthesis of G1-8 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 mixture was cooled to 0°C, trimethyloxonium tetrafluoroborate (35.5 g, 240.0 mmol) was added, the mixture was stirred for 10 minutes, and then the temperature was raised to room temperature and stirring continued for 2 hours. After the reaction was complete, purified water (5.4 mL) was added to quench the mixture, and after stirring for 10 minutes, the mixture was filtered, the filtrate was collected and concentrated under reduced pressure to remove the solvent, then n-heptane (700 mL) was added and the mixture was stirred for 20 minutes, filtered again, 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 the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (n-hexane / ethyl acetate) to obtain a wine-red oily substance G1-8 (56.0 g, 93.7 mmol) with a yield of 78.1%. 1 H 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 [M - H] - = 596.5.

[0089] Step 6: Synthesis of G1-9 G1-8 (56.0 g, 93.7 mmol) was dissolved in tetrahydrofuran (500 mL), cooled to 0°C, and triethylamine hydrofluoric acid (45.3 g, 281.0 mmol) was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred overnight at room temperature. After the reaction was complete, saturated sodium bicarbonate aqueous solution (600 mL) was added and stirred for 5 minutes. Saturated sodium chloride aqueous solution (600 mL) was added, and the mixture was separated by shaking. The aqueous phase was extracted with tetrahydrofuran (600 mL), and the organic phase was combined. The organic phase was washed with saturated sodium chloride aqueous solution (300 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was dissolved in acetonitrile (300 mL), washed with n-heptane (600 mL x 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. 1 H NMR (400 MHz, DMSO-d6) δ 8.48 (t, J = 5.6 Hz, 1H), 7.88 - 7.81 (m, 2H), 7.56 - 7.49 (m, 1H), 7.46 (dd, J = 8.1, 6.5 Hz, 2H), 4.89 (d, J = 1.5 Hz, 1H), 4.83 (d, J = 6.9 Hz, 1H), 4.54 (t, J = 5.7 Hz, 1H), 3.98 (td, J = 6.6, 4.7 Hz, 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 [M - H] - = 354.2.

[0090] Step 7: Synthesis of G1-10 Dried G1-9 (35.0 g, 98.5 mmol) and dimethylaminopyridine (1.20 g, 9.82 mmol) were dissolved in pyridine (350 mL), and 4,4'-dimethoxytrityl chloride (40.1 g, 118.3 mmol) was added in four batches. The mixture was stirred at 15-20°C for 1 hour. After the reaction was complete, the mixture was cooled to 0°C, and the reaction system was quenched by adding saturated sodium bicarbonate aqueous solution (350 mL) dropwise while controlling the temperature to 0-5°C. After the dropwise addition was complete, the mixture was stirred for 10 minutes, dichloromethane (350 mL) was added, and the mixture was separated by shaking. The organic phase was washed once with purified water (350 mL), and then once again with 10% by weight sodium chloride aqueous solution (300 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent, and the crude product was obtained. Purification by column chromatography (n-heptane / ethyl acetate, 0.1% triethylamine) yielded a pale yellow, foamy solid G1-10 (50.0 g, 76.0 mmol) with a yield of 77.2%. 1 H 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 = 7.3 Hz, 1H), 4.01 (td, J = 8.4, 7.3, 5.7 Hz, 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, 2.7 Hz, 1H), 2.96 (dd, J = 10.0, 6.0 Hz, 1H). MS (ESI) m / z [M - H] - = 656.6.

[0091] Step 8: Synthesis of G1-11 G1-10 (50.0 g, 76.0 mmol) was dissolved in methanol (500 mL), sodium hydroxide (136.8 g, 3.42 mol) and purified water (27.4 g, 1.52 mol) were added, the mixture was sealed, and the temperature was raised to 130°C for 2 hours. After the reaction was complete, the mixture was cooled to room temperature, dichloromethane (1000 mL) and a 15% by weight aqueous solution of ammonium chloride (1000 mL) were added, the mixture was stirred for 5 minutes, and the mixture was separated. The organic phase was sequentially washed with purified water (1000 mL) and saturated aqueous solution of sodium chloride (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. 1 H NMR (400 MHz, DMSO-d6) δ 7.43 (d, J = 7.5 Hz, 2H), 7.29 (dd, J = 8.9, 2.8 Hz, 6H), 7.21 (t, J = 7.2 Hz, 1H), 6.88 (d, J = 8.5 Hz, 4H), 4.99 (s, 1H), 4.92 (s, 1H), 4.02 (dd, J = 7.4, 4.5 Hz, 1H), 3.93 (dt, J = 7.6, 3.7 Hz, 1H), 3.73 (s, 7H), 3.55 (ddd, J = 10.9, 6.9, 3.9 Hz, 1H), 3.44 (ddd, J = 15.2, 7.1, 4.3 Hz, 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.

[0092] Step 9: Synthesis of G1-13 G1-11 (40.0 g, 72.2 mmol) was dissolved in dichloromethane (400 mL), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (41.1 g, 108.4 mmol) was added, the mixture was cooled to 0°C, diisopropylethylamine (18.7 g, 144.7 mmol) was added, the mixture was stirred for 10 minutes, and a solution of G1-12 (35.6 g, 79.6 mmol) in dichloromethane (400 mL) was slowly added dropwise. After the dropwise addition was complete, the mixture was stirred for 1 hour. After the reaction was complete, purified water (1000 mL) was added dropwise at 0-5°C, and the mixture was separated by shaking. The organic phase was washed with 10% by weight aqueous sodium chloride solution (1000 mL), the organic phase was dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to remove the solvent and obtain the crude product. Purification by column chromatography (dichloromethane / tetrahydrofuran, 0.1% triethylamine) yielded a pale yellow, foamy solid G1-13 (28.9 g, 29.4 mmol). The yield was 40.7%. 1 H 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, 5.6 Hz, 2H), 3.74 (s, 6H), 3.68 (dq, J = 9.5, 4.5 Hz, 2H), 3.58 - 3.32 (m, 10H), 3.19 - 3.06 (m, 3H), 2.95 (dd, J = 10.0, 6.0 Hz, 1H), 2.10 (s, 3H), 2.05 (t, J = 7.1 Hz, 2H), 1.99 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H), 1.47 (d, J = 14.5 Hz, 4H). MS (ESI) m / z [M - H] - = 981.8.

[0093] Step 10: Synthesis of YK-GAL-501 G1-13 (3.0 g, 3.05 mmol) was dissolved in tetrahydrofuran (15 mL), 3A molecular sieve (750 mg) was added, and the mixture was dried while stirring for 30 minutes. This mixture was labeled as Solution A and stored. Trifluoroacetic acid pyridine (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, and the mixture was dried while stirring for 30 minutes. This mixture was labeled as Solution B and stored. Solution B was cooled to 0°C, and the phosphorus reagent diisopropylaminobis(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 hour. After the reaction was complete, the mixture was filtered, and 30 mL of 5% by weight sodium bicarbonate aqueous solution was added to the filtrate. The mixture was stirred for 5 minutes, and 60 mL of dichloromethane was added. The mixture was shaken to separate the organic phase. The organic phase was then washed sequentially with 30 mL of 5% by weight sodium bicarbonate aqueous solution and 30 mL of saturated sodium chloride aqueous solution. The organic phase was dried over anhydrous sodium sulfate, concentrated to remove the solvent, and the residue was obtained. The residue was dissolved in 3 mL of dichloromethane, added dropwise to 60 mL of n-heptane at 10-15°C, stirred for 30 minutes, and the supernatant was removed to obtain a viscous oily solid. This solid was then vacuum-dried to obtain a white foamy solid YK-GAL-501 (3.4 g, 2.87 mmol) with a yield of 94.4%. 1H NMR (400 MHz, DMSO-d6) δ 7.79 (d, J = 9.2 Hz, 1H), 7.74 (t, J = 5.6 Hz, 1H), 7.47 - 7.37 (m, 2H), 7.34 - 7.17 (m, 7H), 6.91 - 6.83 (m, 4H), 5.21 (d, J = 3.4 Hz, 1H), 5.04 (d, J = 5.1 Hz, 1H), 4.97 (dd, J = 11.2, 3.4 Hz, 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 = 2.0 Hz, 8H), 3.61 (dd, J = 14.9, 4.6 Hz, 2H), 3.53 - 3.33 (m, 11H), 3.21 - 3.09 (m, 2H), 2.95 (dd, J = 10.5, 5.2 Hz, 1H), 2.89 (t, J = 5.8 Hz, 2H), 2.75 (t, J = 6.1 Hz, 1H), 2.56 (t, J = 5.9 Hz, 1H), 2.10 (s, 3H), 2.03 (q, J = 6.4 Hz, 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.

[0094] (2)Synthesis of YK-GAL-502 Synthesis of the road to the next note:

[0095]

change

[0096] Synthesis of ステップ1:G2-2 Using G2-1 (30.4g, 497.8mmol) and G1-2 (70.0g, 498.0mmol) as raw materials, crude product G2-2 (70.0g) was obtained according to the synthesis method of G1-3 and used directly in the next step. 1 H 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.

[0097] Step 2: Synthesis of G2-3 Using G1-4 (77.0g, 241.9 mmol) and G2-2 (60.0g, 363.2 mmol) as raw materials, crude product G2-3 (100.0g) was obtained according to the synthesis method of G1-5 and used directly in the next step. 1 H NMR (400 MHz, Chloroform-d) δ 7.84 - 7.77 (m, 2H), 7.54 - 7.47 (m, 1H), 7.43 (dd, J = 8.2, 6.6 Hz, 2H), 6.78 (s, 1H), 5.31 (dd, J = 6.3, 4.9 Hz, 1H), 5.25 (dd, J = 4.9, 1.4 Hz, 1H), 5.05 (d, J = 1.4 Hz, 1H), 4.32 (dt, J = 9.5, 3.6 Hz, 2H), 3.86 (ddd, J = 10.2, 6.4, 3.5 Hz, 1H), 3.79 - 3.57 (m, 3H), 2.08 (d, J = 14.2 Hz, 6H), 2.02 (s, 3H). MS (ESI) m / z [M + Na] + = 446.2.

[0098] Step 3: Synthesis of G2-4 Using G2-3 (100.0 g, 236.2 mol) as the raw material, a colorless, transparent oily substance G2-4 (40.0 g, 135.4 mmol) was obtained according to the synthesis method of G1-6, and the total yield of the two steps was 56.0%. 1 H NMR (400 MHz, DMSO-d6) δ 8.43 (t, J = 5.6 Hz, 1H), 7.86 - 7.80 (m, 2H), 7.52 (t, J = 7.3 Hz, 1H), 7.45 (dd, J = 8.3, 6.6 Hz, 2H), 4.99 (d, J = 4.5 Hz, 1H), 4.78 (d, J = 6.9 Hz, 2H), 4.65 (t, J = 5.6 Hz, 1H), 3.89 (td, J = 6.7, 4.7 Hz, 1H), 3.75 (qd, J = 8.1, 7.1, 3.4 Hz, 2H), 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 [M - H] - = 296.3.

[0099] Step 4: Synthesis of G2-5 Using G2-4 (34.0g, 115.1 mmol) as the raw material, white solid G2-5 (50.0g, 92.6 mmol) was obtained according to the synthesis method of G1-7, with a yield of 80.5%. 1H NMR (400 MHz, DMSO-d6) δ 8.41 (t, J = 5.6 Hz, 1H), 7.86 - 7.79 (m, 2H), 7.52 (t, J = 7.3 Hz, 1H), 7.44 (t, J = 7.5 Hz, 2H), 5.08 (d, J = 4.0 Hz, 1H), 4.78 (s, 1H), 4.30 (dd, J = 7.7, 4.3 Hz, 1H), 3.91 - 3.74 (m, 4H), 3.68 - 3.59 (m, 1H), 3.49 (dt, J = 9.9, 6.0 Hz, 1H), 3.39 (dt, J = 8.7, 5.9 Hz, 2H), 1.06 - 0.86 (m, 28H). MS (ESI) m / z [M - H] - = 538.4.

[0100] Step 5: Synthesis of G2-6 Using G2-5 (43.0g, 79.7mmol) as a raw material, wine-red oil G2-6 (31.0g, 56.0mmol) was obtained according to the synthesis method of G1-8, with a yield of 70.3%. 1 H NMR (400 MHz, DMSO-d6) δ 8.40 (t, J = 5.6 Hz, 1H), 7.87 - 7.80 (m, 2H), 7.52 (t, J = 7.2 Hz, 1H), 7.45 (t, J = 7.5 Hz, 2H), 4.90 (s, 1H), 4.41 (dd, J = 7.6, 4.1 Hz, 1H), 3.90 - 3.75 (m, 3H), 3.71 - 3.58 (m, 2H), 3.53 (dt, J = 10.0, 5.9 Hz, 1H), 3.45 (s, 3H), 3.41 (t, J = 5.9 Hz, 2H), 1.01 (ddt, J = 13.9, 7.1, 3.2 Hz, 28H). MS (ESI) m / z [M - H] - = 552.4.

[0101] Step 6: Synthesis of G2-7 Using G2-6 (31.0g, 56.0mmol) as a raw material, a pale yellow oily substance G2-7 (17.5g) was obtained according to the synthesis method of G1-9 and used directly in the next step. 1 H 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 [M - H] - = 310.2.

[0102] Step 7: Synthesis of G2-8 Using G2-7 (16.8g, 54.0 mmol) as a raw material, a pale yellow foamy solid G2-8 (19.0g, 31.0 mmol) was obtained according to the synthesis method of G1-10, 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, 4.4 Hz, 1H), 3.92 (td, J = 7.4, 6.7, 2.7 Hz, 1H), 3.72 (d, J = 2.6 Hz, 7H), 3.66 - 3.58 (m, 1H), 3.47 (d, J = 4.6 Hz, 1H), 3.46 - 3.33 (m, 5H), 3.10 (dd, J = 10.1, 2.7 Hz, 1H), 2.98 (dd, J = 10.0, 6.0 Hz, 1H). MS (ESI) m / z [M - H] - = 612.5.

[0103] Step 8: Synthesis of G2-9 Using G2-8 (18.0 g, 29.3 mmol) as a raw material, a pale yellow solid crude product G2-9 (15.0 g) was obtained according to the synthesis method of G1-11 and used directly 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 Hz, 1H), 3.73 (s, 6H), 3.58 (dt, J = 9.7, 5.6 Hz, 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.

[0104] Step 9: Synthesis of G2-10 Using G2-9 (13.5g, 26.5mmol) and G1-12 (13.0g, 29.1mmol) as raw materials, a pale yellow foamy solid G2-10 (18.6g, 19.8mmol) was obtained according to the synthesis method of G1-13, with a yield of 74.7%. 1H NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 9.3 Hz, 1H), 7.71 (t, J = 5.6 Hz, 1H), 7.42 (d, J = 7.3 Hz, 2H), 7.34 - 7.25 (m, 6H), 7.21 (t, J = 7.3 Hz, 1H), 6.88 (d, J = 8.6 Hz, 4H), 5.21 (d, J = 3.4 Hz, 1H), 5.01 - 4.94 (m, 2H), 4.88 (d, J = 7.3 Hz, 1H), 4.48 (d, J = 8.5 Hz, 1H), 4.02 (d, J = 5.5 Hz, 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 = 4.6 Hz, 1H), 3.45 - 3.35 (m, 5H), 3.21 (dd, J = 13.5, 5.8 Hz, 1H), 3.16 - 3.06 (m, 2H), 2.96 (dd, J = 10.0, 6.0 Hz, 1H), 2.10 (s, 3H), 2.02 (d, J = 6.8 Hz, 2H), 1.99 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H), 1.46 (dt, J = 13.0, 7.4 Hz, 4H). MS (ESI) m / z [M - H] - =937.7.

[0105] Step 10: Synthesis of YK-GAL-502 Using G2-10 (2.0 g, 2.13 mmol) as a raw 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 (400 MHz, DMSO-d6) δ 7.79 (d, J = 9.2 Hz, 1H), 7.73 - 7.63 (m, 1H), 7.46 - 7.39 (m, 2H), 7.28 (t, J = 8.7 Hz, 6H), 7.22 (t, J = 5.5 Hz, 1H), 6.87 (dd, J = 8.7, 4.8 Hz, 4H), 5.21 (d, J = 3.4 Hz, 1H), 5.02 (d, J = 4.2 Hz, 1H), 4.97 (dd, J = 11.3, 3.5 Hz, 1H), 4.48 (d, J = 8.5 Hz, 1H), 4.02 (s, 5H), 3.93 - 3.81 (m, 1H), 3.73 (d, J = 1.9 Hz, 6H), 3.71 - 3.65 (m, 1H), 3.65 - 3.56 (m, 2H), 3.52 - 3.42 (m, 4H), 3.39 (d, J = 13.9 Hz, 4H), 3.17 (d, J = 40.4 Hz, 3H), 2.95 (s, 1H), 2.89 (t, J = 5.9 Hz, 1H), 2.75 (t, J = 5.9 Hz, 1H), 2.56 (t, J = 5.9 Hz, 1H), 2.10 (s, 3H), 2.02 (d, J = 6.8 Hz, 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.

[0106] Synthesis of YK-GAL-503 (3) Synthesis of YK-GAL-503 The synthesis route is as follows:

[0107] [ka]

[0108] Step 1: Synthesis of G3-2 Using G3-1 (22.5g, 300.0mmol) and G1-2 (42.2g, 300.0mmol) as raw materials, a crude colorless oily product G3-2 (63.0g) was obtained according to the synthesis method of G1-3 and used directly in the next step. 1 H 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.

[0109] Step 2: Synthesis of G3-3 Using G1-4 (70.0g, 219.9mmol) and G3-2 (51.2g, 285.9mmol) as raw materials, the crude yellow oily product G3-3 (135.0g) was obtained according to the synthesis method of G1-5 and used directly in the next step. 1 H 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 (d, J = 6.6, 4.9 Hz, 1H), 5.10 (d, J = 4.9, 1.4 Hz, 1H), 5.04 (t, J = 1.3 Hz, 1H), 4.04 (td, J = 10.9, 7.2, 3.2 Hz, 1H), 3.66 - 3.58 (m, 2H), 3.44 - 3.46 (m, 1H), 3.46 - 2.06 (m, 3H), 1.62 (s, 2H). MS (ESI) m / z [M + Na] + = 460.2.

[0110] Step 3: Synthesis of G3-4 Using G3-3 (135 g, 308.6 mol) as the raw material, a colorless, transparent oily substance G3-4 (31.9 g, 102.5 mmol) was obtained according to the synthesis method of G1-6, and the total yield of the two steps was 33.2%. 1 H 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), 3.87 (td, J = 6.7, 4.8 Hz, 1H), 3.80 - 3.71 (m, 2H), 3.67 (dt, J = 9.7, 6.4 Hz, 1H), 3.53 (ddd, J = 11.5, 5.7, 3.8 Hz, 1H), 3.41 - 3.34 (m, 2H), 3.29 (td, J = 6.9, 6.4, 2.4 Hz, 2H), 1.74 (p, J = 6.6 Hz, 2H). MS (ESI) m / z [M - H] - = 310.3.

[0111] Step 4: Synthesis of G3-5 Using G3-4 (30.0 g, 96.4 mmol) as the raw material, white solid G3-5 (43.0 g, 77.6 mmol) was obtained according to the synthesis method of G1-7, with a yield of 80.6%. 1H NMR (400 MHz, DMSO-d6) δ 8.38 (t, J = 5.7 Hz, 1H), 7.85 - 7.78 (m, 2H), 7.53 - 7.40 (m, 3H), 5.06 (d, J = 4.0 Hz, 1H), 4.74 (s, 1H), 4.30 (dd, J = 7.1, 4.4 Hz, 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, 4.3 Hz, 2H), 1.77 - 1.68 (m, 2H), 1.07 - 0.85 (m, 28H). MS (ESI) m / z [M - H] - = 552.6.

[0112] Step 5: Synthesis of G3-6 Using G3-5 (40.0g, 72.2 mmol) as a raw material, wine-red oil G3-6 (31.2g, 54.9 mmol) was obtained according to the synthesis method of G1-8, with a yield of 76.0%. 1 H 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, 6.4 Hz, 1H), 3.59 (d, J = 4.3 Hz, 1H), 3.46 (s, 3H), 3.40 (dt, J = 9.7, 6.4 Hz, 1H), 3.33 - 3.25 (m, 2H), 1.74 (pd, J = 6.8, 2.0 Hz, 2H), 1.07 - 0.95 (m, 28H). MS (ESI) m / z [M - H] - = 566.6.

[0113] Step 6: Synthesis of G3-7 Using G3-6 (30.0g, 52.8mmol) as a raw material, a pale yellow oily substance G3-7 (19.1g) was obtained according to the synthesis method of G1-9 and used directly in the next step. 1 H NMR (400 MHz, DMSO-d6) δ 8.42 (t, J = 5.7 Hz, 1H), 7.87 - 7.79 (m, 2H), 7.56 - 7.48 (m, 1H), 7.48 (dd, J = 8.2, 6.5 Hz, 2H), 7.41 (d, J = 4.5 Hz, 1H), 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, 4.6 Hz, 1H), 3.78 - 3.63 (m, 2H), 3.56 (dt, J = 9.7, 6.4 Hz, 1H), 3.45 (ddd, J = 11.5, 4.8, 1.7 Hz, 1H), 3.44 - 3.39 (m, 2H), 3.36 (td, J = 3.34, 3.25, 2.4 Hz, 2H), 1.75 (p, J = 6.7Hz, 2H). MS (ESI) m / z [M - H] - = 324.4.

[0114] Step 7: Synthesis of G3-8 Using G3-7 (18.0 g, 55.3 mmol) as a raw material, a pale yellow foamy solid G3-8 (24.5 g, 39.0 mmol) was obtained according to the synthesis method of G1-10, 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 = 7.5 Hz, 1H), 3.71 (d, J = 1.9 Hz, 7H), 3.47 (d, J = 4.4 Hz, 2H), 3.39 (d, J = 1.7 Hz, 3H), 3.25 (p, J = 6.7 Hz, 2H), 3.10 (dd, J = 10.1, 2.7 Hz, 1H), 2.95 (dd, J = 10.1, 5.8 Hz, 1H), 1.71 (p, J = 6.8 Hz, 2H). MS (ESI) m / z [M - H] - = 626.4.

[0115] Step 8: Synthesis of G3-9 Using G3-8 (23.0 g, 36.6 mmol) as a raw material, a pale yellow solid crude product G3-9 (18.5 g) was obtained according to the synthesis method of G1-11 and used directly in the next step. 1H NMR (400 MHz, DMSO-d6) δ 7.47 - 7.40 (m, 2H), 7.29 - 7.25 (m, 6H), 7.25 - 7.17 (m, 1H), 6.92 - 6.84 (m, 4H), 4.92 (d, J = 1.2 Hz, 1H), 4.90-4.90 (d, J = 6.9 Hz, 1H), 4.00 (s, 1H), 3.91 (td, J = 7.6, 6.7, 2.7 Hz, 1H), 3.73 (s, 6H), 3.67 (dt, J = 9.5, 6.5 Hz, 1H), 3.48 - 3.35 (m, 1H), 3.09 (dd, J = 9.9, 2.8 Hz, 4H), 2.95 (dd, J = 9.9, 5.9 Hz, 1H), 2.53 (dd, J = 10.0, 5.9 Hz, 1H), 1.50 (t, J = 6.7 Hz, 2H). (ESI) m / z [M + Na] + = 546.4.

[0116] Step 9: Synthesis of G3-10 Using G3-9 (18.0g, 34.4 mmol) and G1-12 (16.9g, 37.8 mmol) as raw materials, a pale yellow foamy solid G3-10 (23.0g, 24.1 mmol) was obtained according to the synthesis method of G1-13, with a yield of 70.1%. 1H NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 9.2 Hz, 1H), 7.67 (t, J = 5.6 Hz, 1H), 7.46 - 7.39 (m, 2H), 7.34 - 7.24 (m, 6H), 7.21 (t, J = 7.3 Hz, 1H), 6.88 (d, J = 8.5 Hz, 4H), 5.22 (d, J = 3.4 Hz, 1H), 4.97 (dd, J = 11.3, 3.4 Hz, 1H), 4.93 (s, 1H), 4.86 (d, J = 7.2 Hz, 1H), 4.49 (d, J = 8.5 Hz, 1H), 4.02 (d, J = 5.8 Hz, 4H), 3.95 - 3.77 (m, 2H), 3.73 (s, 6H), 3.72 - 3.56 (m, 2H), 3.46 (d, J = 4.5 Hz, 1H), 3.41 (s, 5H), 3.10 (dd, J = 9.9, 2.7 Hz, 1H), 3.01 (p, J = 6.6 Hz, 2H), 2.94 (dd, J = 10.1, 5.8 Hz, 1H), 2.10 (s, 3H), 2.00 (d, J = 8.7 Hz, 5H), 1.89 (s, 3H), 1.76 (d, J = 3.8 Hz, 3H), 1.56 (p, J = 6.8 Hz, 2H), 1.47 (p, J = 7.1 Hz, 4H). MS (ESI) m / z [M - H] - = 951.8.

[0117] Step 10: Synthesis of YK-GAL-503 Using G3-10 (5.0 g, 5.25 mmol) as a raw material, a white foamy solid YK-GAL-503 (5.1 g, 4.42 mmol) was obtained according to the synthesis method for YK-GAL-501, with a yield of 84.3%. 1H NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 9.2 Hz, 1H), 7.66 (d, J = 6.1 Hz, 1H), 7.43 (dd, J = 7.7, 5.1 Hz, 2H), 7.29 (td, J = 8.9, 8.1, 3.6 Hz, 6H), 7.21 (dd, J = 8.4, 5.9 Hz, 1H), 6.87 (dd, J = 8.7, 4.2 Hz, 4H), 5.21 (d, J = 3.4 Hz, 1H), 4.97 (dd, J = 11.6, 3.6 Hz, 2H), 4.49 (d, J = 8.4 Hz, 1H), 4.13 - 3.95 (m, 5H), 3.93 - 3.80 (m, 1H), 3.73 (d, J = 2.0 Hz, 9H), 3.61 (dd, J = 16.2, 4.5 Hz, 1H), 3.56 - 3.35 (m, 7H), 3.21 (dd, J = 26.4, 10.1 Hz, 1H), 3.02 (d, J = 7.3 Hz, 2H), 2.89 (t, J = 5.9 Hz, 2H), 2.75 (t, J = 5.9 Hz, 1H), 2.56 (t, J = 5.9 Hz, 1H), 2.10 (s, 3H), 2.00 (d, J = 8.2 Hz, 5H), 1.89 (s, 3H), 1.77 (s, 3H), 1.58 (q, J = 6.3 Hz, 2H), 1.46 (d, J = 12.8 Hz, 4H), 1.21 - 1.02 (m, 12H). MS (ESI) m / z [M + H] + =1154.3.

[0118] (4)Synthesis of YK-GAL-504 Synthesis of the road to the next note:

[0119]

change

[0120] Synthesis of ステップ1:G4-2 Using G4-1 (60.0g, 673.1mmol) and G1-2 (94.6g, 673.0mmol) as raw materials, a crude colorless oily product G4-2 (135.0g) was obtained according to the synthesis method of G1-3 and used directly in the next step. 1 H 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 [M - H] - = 194.4.

[0121] Step 2: Synthesis of G4-3 Using G1-4 (120.0g, 377.0mmol) and G4-2 (110g, 569.2mmol) as raw materials, a crude yellow oily product G4-3 (194.0g) was obtained according to the synthesis method of G1-5 and used directly in the next step. 1 H 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 = 1.3 Hz, 1H), 4.32 - 4.19 (m, 2H), 4.04 (ddt, J = 10.8, 7.1, 3.2 Hz, 1H), 3.69 - 3.59 (m, 1H), 3.46 - 3.39 (m, 1H), 3.28 (t, J = 6.0 Hz, 2H), 2.07 (s, 3H), 2.02 (d, J = 3.7 Hz, 6H), 1.56 (p, J = 3.1 Hz, 4H). MS (ESI) m / z [M + Na] + = 474.4.

[0122] Step 3: Synthesis of G4-4 Using G4-3 (194.0 g, 429.7 mmol) as the raw material, following the synthesis method of G1-6, a colorless and transparent oily substance G4-4 (51.2 g, 157.4 mmol) was obtained, and the total yield of the two steps was 41.7%. 1 H 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 = 6.5, 4.7 Hz, 1H), 3.74 (ddd, J = 17.5, 7.7, 4.2 Hz, 2H), 3.63 (dt, J = 9.0, 5.7 Hz, 1H), 3.52 (ddd, J = 11.5, 5.7, 3.9 Hz, 1H), 3.35 (q, J = 5.7 Hz, 2H), 3.26 (q, J = 6.4 Hz, 2H), 1.54 (dd, J = 6.7, 3.6 Hz, 4H). MS (ESI) m / z [M - H] - = 324.4。

[0123] Step 4: Synthesis of G4-5 Using G4-4 (45.0 g, 138.3 mmol) as the raw material, following the synthesis method of G1-7, a white solid G4-5 (59.0 g, 103.9 mmol) was obtained, and the yield was 75.2%. 1H NMR (400 MHz, DMSO-d6) δ 8.42 (t, J = 5.7 Hz, 1H), 7.87 - 7.80 (m, 2H), 7.50 (dd, J = 8.4, 6.0 Hz, 1H), 7.44 (t, J = 7.4 Hz, 2H), 5.04 (d, J = 3.9 Hz, 1H), 4.72 (s, 1H), 4.30 (dd, J = 7.3, 4.4 Hz, 1H), 3.93 - 3.73 (m, 4H), 3.56 (dt, J = 9.3, 6.0 Hz, 1H), 3.33 (d, J = 6.6 Hz, 1H), 3.25 (d, J = 5.9 Hz, 2H), 1.53 (p, J = 6.6 Hz, 4H), 1.06 - 0.82 (m, 28H). MS (ESI) m / z [M - H] - = 566.6.

[0124] Step 5: Synthesis of G4-6 Using G4-5 (59.0g, 103.9mmol) as a raw material, a wine-red oily substance G4-6 (41.0g, 70.5mmol) was obtained according to the synthesis method of G1-8, with a yield of 67.8%. 1 H 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, 5.3 Hz, 2H), 3.45 (s, 3H), 3.40 - 3.32 (m, 1H), 3.24 (t, J = 6.1 Hz, 2H), 1.52 (dt, J = 10.1, 5.1 Hz, 4H), 1.09 - 0.86 (m, 28H). MS (ESI) m / z [M - H] - = 580.5.

[0125] Step 6: Synthesis of G4-7 Using G4-6 (34.0 g, 58.4 mmol) as the raw material, a pale yellow oily substance G4-7 (22.0 g) was obtained according to the synthesis method of G1-9 and directly used in the next step. 1 H 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 (td, J = 6.5, 4.8 Hz, 1H), 3.83 - 3.70 (m, 1H), 3.70 - 3.56 (m, 2H), 3.50 (ddd, J = 11.5, 5.7, 4.2 Hz, 1H), 3.44 (dd, J = 4.8, 1.8 Hz, 1H), 3.41 - 3.33 (m, 4H), 3.33 - 3.22 (m, 2H), 1.54 (h, J = 4.7, 3.8 Hz, 4H). MS (ESI) m / z [M - H] - = 338.3.

[0126] Step 7: Synthesis of G4-8 Using G4-7 (20.0 g, 58.9 mmol) as the raw material, a pale yellow foamy solid G4-8 (23.1 g, 36.0 mmol) was obtained according to the synthesis method of G1-10, and the yield was 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), 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 = 4.5 Hz, 1H), 3.43 - 3.35 (m, 4H), 3.22 (dd, J = 7.9, 4.5 Hz, 2H), 3.10 (dd, J = 10.0, 2.8 Hz, 1H), 2.95 (dd, J = 10.0, 5.8 Hz, 1H), 1.48 (dd, J = 6.6, 3.4 Hz, 4H). MS (ESI) m / z [M - H] - = 640.6.

[0127] Step 8: Synthesis of G4-9 Using G4-8 (21.0 g, 32.7 mmol) as a raw material, a pale yellow solid crude product G4-9 (16.5 g) was obtained according to the synthesis method of G1-11 and used directly 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 = 4.5 Hz, 1H), 3.41 - 3.32 (m, 4H), 3.10 (dd, J = 10.0, 2.7 Hz, 1H), 2.94 (dd, J = 9.9, 5.9 Hz, 1H), 2.45 (t, J = 6.9 Hz, 2H), 1.43 (q, J = 7.0 Hz, 2H), 1.26 (ddd, J = 11.1, 8.7, 5.4 Hz, 2H). MS (ESI) m / z [M + Na] + = 560.5.

[0128] Step 9: Synthesis of G4-10 Using G4-9 (15.0 g, 27.9 mmol) and G1-12 (13.7 g, 30.6 mmol) as raw materials, a pale yellow foamy solid G4-10 (19.4 g, 20.1 mmol) was obtained according to the synthesis method of G1-13, with a yield of 71.9%. 1H NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 9.2 Hz, 1H), 7.66 (t, J = 5.6 Hz, 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 = 3.4 Hz, 1H), 4.97 (dd, J = 11.2, 3.4 Hz, 1H), 4.93 (d, J = 1.1 Hz, 1H), 4.86 (d, J = 7.3 Hz, 1H), 4.49 (d, J = 8.6 Hz, 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, 2.8 Hz, 1H), 2.96 (td, J = 11.7, 10.2, 6.1 Hz, 3H), 2.10 (s, 3H), 2.03 (t, J = 7.1 Hz, 2H), 1.99 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H), 1.45 (tq, J = 13.1, 6.6 Hz, 6H), 1.34 (d, J = 14.9 Hz, 2H). MS (ESI) m / z [M - H] - =965.8.

[0129] Step 10: Synthesis of YK-GAL-504 Using G4-10 (3.0 g, 3.10 mmol) as a raw material, a white foamy solid YK-GAL-504 (3.2 g, 2.74 mmol) was obtained according to the synthesis method of YK-GAL-501, with a yield of 88.4%. 1H NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 9.2 Hz, 1H), 7.70 - 7.61 (m, 1H), 7.43 (dd, J = 7.7, 5.3 Hz, 2H), 7.29 (td, J = 9.0, 8.2, 2.8 Hz, 6H), 7.21 (dt, J = 8.2, 3.8 Hz, 1H), 6.87 (dd, J = 8.7, 4.1 Hz, 4H), 5.21 (d, J = 3.4 Hz, 1H), 4.97 (dd, J = 11.4, 3.6 Hz, 2H), 4.49 (d, J = 8.5 Hz, 1H), 4.03 (q, J = 4.9, 3.9 Hz, 5H), 3.87 (dt, J = 11.2, 8.9 Hz, 1H), 3.73 (d, J = 2.1 Hz, 6H), 3.70 - 3.56 (m, 3H), 3.56 - 3.35 (m, 8H), 3.21 (ddd, J = 27.3, 10.4, 2.8 Hz, 1H), 3.02 - 2.85 (m, 4H), 2.75 (t, J = 6.0 Hz, 1H), 2.56 (t, J = 5.9 Hz, 1H), 2.10 (s, 3H), 2.01 (d, J = 12.3 Hz, 5H), 1.89 (s, 3H), 1.77 (s, 3H), 1.45 (hept, J = 7.0 Hz, 6H), 1.33 (dt, J = 10.2, 4.9 Hz, 2H), 1.22 - 1.03 (m, 12H). MS (ESI) m / z [M + H] + =1168.3.

[0130] (5)Synthesis of YK-GAL-505

[0131]

change

[0132] Synthesis of ステップ1:G5-2 Using G5-1 (60.0g, 581.6mmol) and G1-2 (81.8g, 581.9mmol) as raw materials, a crude colorless oily product G5-2 (123.0g) was obtained according to the synthesis method of G1-3 and used directly in the next step. 1 H 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 [M - H] - = 208.4.

[0133] Step 2: Synthesis of G5-3 Using G1-4 (80.0g, 251.4 mmol) and G5-2 (78.9g, 377.1 mmol) as raw materials, a crude yellow oily product G5-3 (124.0g) was obtained according to the synthesis method of G1-5 and used directly in the next step. 1 H 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, 3.4 Hz, 1H), 3.70 - 3.59 (m, 1H), 3.41 - 3.32 (m, 1H), 3.26 (t, J = 6.0 Hz, 2H), 2.11 (s, 3H), 2.01 (d, J = 3.5 Hz, 6H), 1.54 (p, J = 3.0 Hz, 4H), 1.34 (p, J = 2.0 Hz, 2H). MS (ESI) m / z [M + Na]+ = 488.3.

[0134] Step 3: Synthesis of G5-4 Using G5-3 (88.0 g, 189.0 mol) as a raw material, a colorless, transparent oily substance G5-4 (23.0 g, 67.8 mmol) was obtained according to the synthesis method of G1-6, and the total yield of the two steps was 35.8%. 1 H 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, 1H), 3.71 (ddd, J = 17.9, 7.6, 4.2 Hz, 2H), 3.65 (dt, J = 9.0, 5.9 Hz, 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 = 2.1Hz, 2H). MS (ESI) m / z [M - H] - = 338.4.

[0135] Step 4: Synthesis of G5-5 Using G5-4 (20.0 g, 58.9 mmol) as the raw material, white solid G5-5 (25.1 g, 43.1 mmol) was obtained according to the synthesis method of G1-7, 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, 4.6 Hz, 1H), 3.90 - 3.77 (m, 4H), 3.58 (dt, J = 9.2, 6.3 Hz, 1H), 3.36 (d, J = 6.5 Hz, 1H), 3.21 (d, J = 5.7 Hz, 2H), 1.53 (p, J = 3.0 Hz, 4H), 1.35 (p, J = 2.0 Hz, 2H), 1.04 - 0.87 (m, 28H). MS (ESI) m / z [M - H] - = 580.5.

[0136] Step 5: Synthesis of G5-6 Using G5-5 (24.0g, 41.2mmol) as a raw material, wine-red oil G5-6 (19.4g, 32.6mmol) was obtained according to the synthesis method of G1-8, with a yield of 78.9%. 1 H 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, 5.3 Hz, 2H), 3.47 (s, 3H), 3.41 - 3.34 (m, 1H), 3.25 (t, J = 6.5 Hz, 2H), 1.54 (dt, J = 10.2, 5.1 Hz, 4H), 1.35 (p, J = 2.0 Hz, 2H), 1.09 - 0.86 (m, 28H). 1HNMR (400 MHz,) δ ppm. MS (ESI) m / z [M - H] - = 594.5.

[0137] Step 6: Synthesis of G5-7 Using G5-6 (19.0g, 31.9mmol) as a raw material, a pale yellow oily substance G5-7 (12.3g) was obtained according to the synthesis method of G1-9 and used directly in the next step. 1 H 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, 4.8 Hz, 1H), 3.88 - 3.71 (m, 1H), 3.69 - 3.55 (m, 2H), 3.51 (ddd, J = 11.2, 5.5, 4.3 Hz, 1H), 3.42 (dd, J = 4.9, 1.8 Hz, 1H), 3.40 - 3.33 (m, 4H), 3.33 - 3.21 (m, 2H), 1.54 (h, J = 4.9, 3.7 Hz, 4H), 1.35 (p, J = 2.1 Hz, 2H). MS (ESI) m / z [M - H] - = 352.3.

[0138] Step 7: Synthesis of G5-8 Using G5-7 (12.0 g, 34.0 mmol) as a raw material, a pale yellow foamy solid G5-8 (15.3 g, 23.3 mmol) was obtained according to the synthesis method of G1-10, with a yield of 68.6%. 1H NMR (400 MHz, DMSO-d6) δ 8.42 (t, J = 5.7 Hz, 1H), 7.86 - 7.77 (m, 2H), 7.57 - 7.49 (m, 1H), 7.48 - 7.41 (m, 4H), 7.32 - 7.26 (m, 6H), 7.19 (t, J = 7.3 Hz, 1H), 6.87 (d, J = 8.3 Hz, 4H), 4.95 (s, 1H), 4.89 (d, J = 7.3 Hz, 1H), 4.04 (td, J = 7.1, 4.2 Hz, 1H), 3.90 (td, J = 7.2, 6.6, 2.7Hz, 1H), 3.72 (s, 6H), 3.71 - 3.64 (m, 1H), 3.45 (d, J = 4.5 Hz, 1H), 3.42 - 3.34 (m, 4H), 3.22 (dd, J = 7.9, 4.5 Hz, 2H), 3.11 (dd, J = 10.0, 2.8 Hz, 1H), 2.96 (dd, J = 10.0, 5.8 Hz, 1H), 1.50 (dd, J = 6.6, 3.4 Hz, 4H), 1.31 (p, J = 2.1 Hz, 2H). MS (ESI) m / z [M - H] - = 654.6.

[0139] Step 8: Synthesis of G5-9 Using G5-8 (13.0 g, 19.8 mmol) as a raw material, a pale yellow solid crude product G3-9 (10.4 g) was obtained according to the synthesis method of G1-11 and used directly 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 = 4.5 Hz, 1H), 3.41 - 3.33 (m, 4H), 3.11 (dd, J = 10.0, 2.7 Hz, 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.

[0140] Step 9: Synthesis of G5-10 Using G5-9 (10.0g, 18.1mmol) and G1-12 (8.9g, 19.9mmol) as raw materials, a pale yellow foamy solid G5-10 (11.9g, 12.1mmol) was obtained according to the synthesis method of G1-13, with a yield of 66.9%. 1H NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 9.2 Hz, 1H), 7.66 (t, J = 5.6 Hz, 1H), 7.46 - 7.39 (m, 2H), 7.32 - 7.25 (m, 6H), 7.21 (t, J = 7.3 Hz, 1H), 6.87 (d, J = 8.6 Hz, 4H), 5.21 (d, J = 3.4 Hz, 1H), 4.97 (dd, J = 11.2, 3.4 Hz, 1H), 4.92 (s, 1H), 4.86 (d, J = 7.3 Hz, 1H), 4.48 (d, J = 8.5 Hz, 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 = 9.0 Hz, 1H), 3.44 (d, J = 4.6 Hz, 1H), 3.38 (s, 4H), 3.34 (d, J = 8.8 Hz, 1H), 3.09 (dd, J = 9.9, 2.8 Hz, 1H), 2.95 (dq, J = 12.1, 5.8 Hz, 3H), 2.10 (s, 3H), 2.01 (d, J = 12.5 Hz, 5H), 1.89 (s, 3H), 1.77 (s, 3H), 1.44 (td, J = 14.3, 7.1 Hz, 6H), 1.30 (q, J = 7.2 Hz, 2H), 1.18 (s, 2H). MS (ESI) m / z [M - H] - = 979.8.

[0141] Step 10: Synthesis of YK-GAL-505 Using G5-10 (5.0 g, 5.10 mmol) as a raw 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 (400 MHz, DMSO-d6) δ 7.80 (d, J = 9.2 Hz, 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, 3.8 Hz, 1H), 6.87 (dd, J = 8.7, 4.1 Hz, 4H), 5.21 (d, J = 3.4 Hz, 1H), 4.97 (dd, J = 11.4, 3.6 Hz, 2H), 4.50 (d, J = 8.5 Hz, 1H), 4.05 - 3.95 (m, 4H), 3.95 - 3.84 (m, 2H), 3.73 (d, J = 2.1 Hz, 6H), 3.69 - 3.51 (m, 3H), 3.51 - 3.30 (m, 8H), 3.22 (ddd, J = 27.3, 10.4, 2.8 Hz, 1H), 3.09 - 2.87 (m, 4H), 2.74 (t, J = 6.0 Hz, 1H), 2.57 (t, J = 5.9 Hz, 1H), 2.10 (s, 3H), 2.01 (d, J = 12.5 Hz, 5H), 1.89 (s, 3H), 1.77 (s, 3H), 1.44 (td, J = 14.3, 7.1 Hz, 6H), 1.31 (dt, J = 10.2, 4.9 Hz, 4H), 1.23 - 1.03 (m, 12H). MS (ESI) m / z [M + H] + = 1182.2.

[0142] (6) Synthesis of YK-GAL-325

[0143] [ka]

[0144] Following the synthesis method for YK-GAL-325 described on page 62 of CN116854754B, 417.6 mg of the product was obtained, and the MS (ESI) m / z [M + H] + = 1452.2.

[0145] 2. Synthesis of GalNAc Solid-Phase Support Compounds (1) Synthesis of YK-GAL-501-SP The synthesis route is as follows:

[0146] [ka]

[0147] Step 1: Synthesis of G1-14 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 hours under the protection of nitrogen gas. Purification by preparative chromatography yielded a white solid G1-14 (115.4 mg, 106 μmol) with a yield of 52.5%.

[0148] Step 2: Synthesis of YK-GAL-501-SP G1-14 (50.0 mg, 46.2 μmol), 4-dimethylaminopyridine (5.64 mg, 46.2 μmol), diisopropylethylamine (47.8 mg, 370 μmol), and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (87.6 mg, 231 μmol) were dissolved in N,N-dimethylformamide (10.0 mL), and then CPG-NH2 (800 mg) was added and the mixture was stirred at 40°C for 16 hours. The reaction solution was filtered, and the filtrate was successively washed with methanol and dichloromethane and vacuum-dried. The filtrate was then added to 10 mL of anhydrous acetic acid / pyridine (1:4) solution and stirred at 40°C for 0.5 hours. The mixture was filtered, washed sequentially with dichloromethane and methanol, and the filtrate was dried under vacuum for 12 hours to obtain the white solid compound YK-GAL-501-SP (749 mg, loading amount: 32.7 μmol / g).

[0149] (2) Synthesis of other solid support compounds Using G2-10, G3-10, G4-10, and G5-10 as starting materials, these compounds were synthesized according to the synthesis method for YK-GAL-501-SP to obtain the other GalNAc solid support compounds YK-GAL-502-SP, YK-GAL-503-SP, YK-GAL-504-SP, and YK-GAL-505-SP listed in Table 1.

[0150] [Table 1]

[0151] Example 2: Coupling of GalNAc compound with oligonucleotide In this example, the same oligonucleotide sequence was used for synthesis, and the GalNAc compound was coupled with the oligonucleotide to obtain a GalNAc-binding oligonucleotide. Here, the binding oligonucleotide sequence is the sequence numbered D579-DV25P, and the sequence D579-DV25P is as follows.

[0152] Sense 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' (Sequence ID 1), 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' (Sequence ID 2).

[0153] Here, A, U, C, and G represent the base composition of the nucleotide; m indicates that the nucleotide adjacent to the left of m is 2'-OMe modified; f indicates that the nucleotide adjacent to the left of f is 2'-F modified; s indicates that there is a thiophosphate ester group bond between the two nucleotides adjacent to the left and right of s; and EVP indicates that the 5' end is vinylphosphonic acid modified.

[0154] The basic sequence of the double-stranded oligonucleotide with the number D579-DV25P is as follows: Sense chain: 5'-CCUUUUCUUCUAAUGAGUCGA-3' (Sequence ID 3), Antisense chain: 5'-UCGACUCAUUAGAAGAAAAGGUG-3' (SEQ ID NO: 4).

[0155] 1. Production of GalNAc-conjugated siRNA sense strands GalNAc-linked oligonucleotides were synthesized on a solid support according to the phosphoramidite chemistry method, and these will be abbreviated as conjugates below.

[0156] When synthesizing conjugates 1-5 (SEQ ID NOs: D579-DV25PG501, D579-DV25PG502, D579-DV25PG503, D579-DV25PG504, and D579-DV25PG505), a general 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, and the GalNAc phosphoramidite compounds synthesized in Example 1 (YK-GAL-501, YK-GAL-502, YK-GAL-503, YK-GAL-504, and YK-GAL-505) were used as monomers. When synthesizing conjugate 6 (SEQ ID NO: D579-DV25PG325), a general CPG solid support was used, and the GalNAc phosphoramidite compound YK-GAL-325 synthesized in Example 1 was used as the monomer. When synthesizing conjugate 7 (SEQ ID NO: D579-DV25PL96), purchased CPG-L96 (Tianjin WuXi AppTec New Drug Development Co., Ltd; L96 is referred to in claim 10 of US10465194B2) was used as the solid support, and the solid support was synthesized on a 1 μmol scale. All of these GalNAc compounds were bonded to the 3' end of oligonucleotides.

[0157] (1) Production of reagents and monomers A monomer acetonitrile solution (1 / 20, w / v), a 0.25 M 5-benzylthio-1H-tetrazole acetonitrile solution as an activator, a 0.2 M xanthan hydride acetonitrile / pyridine (1 / 4, v / v) solution as a thiolation reagent, a 0.05 M iodine water / pyridine (1 / 9, v / v) solution as an oxidation reagent, a 20% anhydrous acetic acid (v / v) acetonitrile as capping agent A, a 20 / 30 / 50 (1-methylimidazole / pyridine / acetonitrile, v / v / v) capping agent B, a 20% diethylamine acetonitrile (v / v) decyanoethylation reagent, and a 3% dichloroacetic acid toluene (v / v) DMT reagent were used. These were loaded into the designated reagent positions of a 192P model DNA / RNA automated synthesizer.

[0158] (2) Synthesis of crude products After inputting the specified oligonucleotide sequence, setting the synthesis program, and verifying that everything was correct, the cyclic oligonucleotide synthesis cycle was started. The monomer coupling time was approximately 1 minute, of which the oxygenation time was approximately 30-45 seconds and the thiolation time was approximately 2 minutes. After the cycle was completed, the solid-phase synthesis of the oligonucleotide was finished.

[0159] (3) Deprotection After the synthesis was complete, the solid support was transferred to a reactor, and oligonucleotides were cleaved from the solid support using concentrated aqueous ammonia (25-28%) over 16-24 hours under conditions of 50-60°C. The reaction system was then cooled to room temperature, filtered, rinsed with a mixed solution of purified water and ethanol, the filtrates were combined, and the filtrates were concentrated at a low temperature to obtain the residue of the crude product.

[0160] (4) Purification The residue of the deprotected crude product was dissolved in purified water, purified by HPLC, the peak solution of the product was collected, the content was measured with an enzyme reader, and the molecular weight was confirmed by ESI MS.

[0161] In this step, the GalNAc phosphoramidite compound synthesized in Example 1 was attached to the 3' end of the sense strand of the siRNA. When synthesizing conjugates 1-6, the GalNAc phosphoramidite compounds synthesized in Example 1 (YK-GAL-501, YK-GAL-502, YK-GAL-503, YK-GAL-504, YK-GAL-505, and YK-GAL-325) were used as monomers. When using a general CPG solid support, the GalNAc phosphoramidite compound was repeated three times in the synthesis sequence. When using the GalNAc solid support synthesized in Example 1, the GalNAc phosphoramidite compound was repeated twice in the synthesis sequence. Finally, all of the synthesized conjugates 1-6 were bound to three GalNAc compounds.

[0162] 2. Production of GalNAc-conjugated siRNA antisense strands siRNA antisense strands were synthesized according to the method for synthesizing siRNA sense strands. Here, a common CPG solid support was used, and the synthesis scale for each antisense strand complementary to the sense strand was 1 μmol.

[0163] 3. Production of bound oligonucleotides GalNAc-binding siRNA sense strands and complementary antisense strands were mixed in a 1:1 ratio according to their UV absorption levels, heated to 95°C, and cooled to room temperature after 3 minutes to form double helixes. The resulting double-stranded solution was characterized by HPLC to ensure product purity, and after measuring the content using a microplate reader, it was freeze-dried to obtain a solid powder, which was stored for later use. The sequences and molecular weights of the obtained GalNAc-binding oligonucleotides are shown in Table 2.

[0164] [Table 2] TIFF2026059798000032.tif39170

[0165] Here, SS is the sense strand and AS is the antisense strand, and the structure of the resulting GalNAc-binding oligonucleotide is as follows.

[0166] [Chemical]

[0167] [Chemical]

[0168] [Chemical]

[0169] [Chemical]

[0170] [[ID=3l]] The GalNAc compound of D579-DV25PG325 is YK-GAL-325 (CN116854754B, page 62),

[0171] [Chemical]

[0172] The GalNAc compound of D579-DV25PL96 is L96 (US10465194B2, the compound of claim 10). Example 3: Inhibitory effect of GalNAc-conjugated oligonucleotides on AGT protein expression in mouse serum and liver, and effect on liver AGT mRNA level Angiotensinogen (AGT) protein is a secreted protein and is mainly expressed in the human liver. As an upstream protein of the renin-angiotensin-aldosterone system (RAAS), by inhibiting its expression, the blood pressure-raising effect of the RAAS system is fundamentally inhibited and blood pressure decreases.

[0173] In this example, gene-modified mice expressing the human-derived AGT gene were used to detect the inhibitory effect of the GalNAc-conjugated double-stranded siRNA produced in Example 2 on AGT protein in serum at different time points by ELISA, and the inhibitory effect on AGT mRNA levels in the liver was detected.

[0174] 1. Experimental materials Test drug: GalNAc-conjugated oligonucleotides: D579-DV25PL96, D579-DV25PG325, D579-DV25PG501, D579-DV25PG502, D579-DV25PG503, D579-DV25PG504, and D579-DV25PG505.

[0175] Experimental animal information: Species / strain: hAGT gene-modified mice Grade: SPF Gender: Male Number of animals: 64 Age: 6 - 8 weeks old Weight: 18 - 28 g Origin: GemPharmatech Production license number: SCXK (Su) 2018 - 0008 Husbandry and management: Husbandry conditions: After the experimental animals were received, they were housed at GemPharmatech, with the use permission number: SYXK (Jing) 2022 - 0025. The experiment was carried out strictly in accordance with the requirements of the Institutional Animal Care and Use Committee (IACUC) to ensure animal welfare. The cage dimensions were length × width × height = 29.0 cm × 18.5 cm × 13.0 cm, the set temperature range was 20 -​​​The rearing environment conditions conformed to the National Standard GB14925-2010 of the People's Republic of China, and the environment was controlled by a modular air conditioning unit. The animals had free access to food and water. 2. Experimental Method Dosage design and group classification Definition of experimental day: The day on which the solvent or test drug was administered to the animal was defined as day 0.

[0177] Grouping and Administration: Experimental animals were reared adaptively for 3 days, then randomly divided into a negative control group and a test drug group based on serum AGT protein content, with 8 animals in each group. The drug was administered by a single subcutaneous injection at 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 designated as day 0.

[0178] The grouping information for the animals is shown in Table 3.

[0179] [Table 3]

[0180] Detection indicators (1) General observations The subjects were observed once a day from one week before administration until the end of the experiment.

[0181] Observations: The animals were observed next to their cages for death or near-death status, mental state, behavior, fecal characteristics, and feeding and watering conditions. Test animals: All animals in the negative control group and the test drug group.

[0182] (2) Expression of AGT protein in serum Detection timing: Day 3 before administration, Day 7 one week after administration, Day 14 two weeks after administration, Day 21 three weeks after administration, Day 28 four weeks after administration, and Day 35 five weeks after administration.

[0183] AGT protein level detection method: Detection was performed using an ELISA kit. Detected animals: All animals in the negative control group and the test drug group. (3) Detection of liver mRNA level Collection of liver tissue and preparation of homogenate: On the 35th day, an appropriate amount of fresh liver tissue was collected from all animals. According to the ratio of tissue weight: RNA lysis solution (Trizol) = 100 mg: 1 mL, it was quickly transferred to an EP tube containing 1 mL of RNA lysis solution (TRIzol) to prepare liver homogenate. The homogenate was immediately examined or stored at -80°C. The remaining liver tissue was quickly frozen and stored at -80°C.

[0184] RNA extraction: a. Take the tissue and put it into a 1.5 mL RNase-free EP tube. Add 1 mL of TRIzol reagent for every 100 mg of tissue, shake and grind to prepare tissue homogenate.

[0185] b. Centrifuge at 12,000 g for 3 minutes at 4°C, transfer 400 μL of the tissue homogenate supernatant to a 1.5 mL RNase-free EP tube, and place it on ice. Add 80 μL of chloroform to each tube, shake vigorously for 15 seconds, and leave it at room temperature for 5 minutes. Centrifuge at 12,000 g for 15 minutes at 4°C, and transfer 150 μL of the supernatant to a new EP tube.

[0186] c. Add the same volume of isopropanol, invert the liquid in the tube and gently mix, leave it at -20°C for 10 minutes, centrifuge at 12,000 g for 15 minutes at 4°C (a white precipitate can be seen), and discard the supernatant.

[0187] d. Add 1 mL of 75% ethanol to gently wash the RNA pellet, centrifuge at 7,500 g for 5 minutes at 4°C, and aspirate and remove the supernatant. Repeat the rinsing once at 7,500 g for 5 minutes at 4°C, and use a micropipette to remove the residual ethanol.

[0188] e. Dry the residual ethanol at room temperature for 10 minutes, and add 100 μL of RNase-free ddH2O to dissolve it. RNA concentration detection and reverse transcription: RNA concentration was detected using a UV-Vis spectrophotometer. 2 μL of RNase-free ddH2O was used as a blank control, and 2 μL of RNA sample was used for detection each time. Sample concentrations were recorded. cDNA was then synthesized using PrimeScript RT Master Mix according to the instructions. A reverse transcription reaction system was prepared in 0.2 mL 8-well strips according to Table 4 below. The volumes of RNA and sterile enzyme-free water can be adjusted according to the RNA concentration; a 10 μL reaction system can process up to 500 ng of RNA, and can be scaled up proportionally according to the required amount of RNA.

[0189] [Table 4]

[0190] The 8-well strip was gently flicked to homogeneously mix the reaction system. After short-term centrifugation using the Short function of a centrifuge, the reverse transcription reaction was carried out using a PCR instrument. The program was to react at 37°C for 15 minutes, then at 85°C for 5 seconds, and then maintain the temperature at 4°C.

[0191] Realtime-qPCR:TB Green(registered trademark) Premix Ex Taq TMqPCR was performed using (Tli RNaseH Plus), Bulk. A 10 μL reaction system was prepared in a 96-well plate according to Table 4, and qPCR was used to detect cDNA of GAPDH and AGT, with GAPDH used as the internal reference gene. qPCR was performed in a 96-well plate, with each well containing 10 μL of reaction system, using three wells for GAPDH primers and three wells for each pair of target gene primers for each sample. If the number of samples was too large to disperse and test biologically replicated samples across multiple 96-well plates, each sample group was amplified simultaneously in the same 96-well plate, ensuring that a control sample was included in each 96-well plate. qPCR reaction program: Heat at 95°C for 30 seconds, then enter cycling mode, heat at 95°C for 5 seconds, then heat at 60°C for 34 seconds, for a total of 40 cycles. Heat the template at 95°C for 15 seconds, then at 60°C for 1 minute, then heat at 95°C for 15 seconds, and perform melting curve analysis.

[0192] [Table 5]

[0193] (4) Data processing and statistical analysis Experimental data were expressed as mean ± standard deviation (Mean ± SD), and the data were analyzed using GraphPad Prism 8.3 analysis software. Statistical analysis was performed using two-way ANOVA and post-hoc tests, and heterogeneity of variances was evaluated using the Dunnett T3 test. A p-value < 0.05 was considered statistically significant.

[0194] 3. Experimental Results The specific experimental results are shown in Tables 6-1, 6-2, and 7. (1) Inhibition rate of AGT protein levels in serum

[0195] [Table 6] TIFF2026059798000042.tif121161

[0196] [Table 7] TIFF2026059798000044.tif32161

[0197] As can be seen from Table 6, these GalNAc-binding oligonucleotides can significantly and continuously 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. The experimental results showed that coupling the GalNAc compound of this application with the oligonucleotide sequence D579-DV25P allowed for efficient delivery to the animal liver and significant inhibition of AGT gene expression.

[0198] Compared to siRNA conjugates produced with the conventional GalNAc compounds YK-GAL-325 and L96, GalNAc-binding oligonucleotides produced with the GalNAc compound of this application significantly improved the inhibition rate of AGT protein levels in serum. For example, the inhibition rate at day 7 in the D579-DV25PG502 group was improved by 12.22% compared to the inhibition rate in the D579-DV25PL96 group, and the inhibition rate at day 35 in the D579-DV25PG504 group was improved by 14.48% compared to the inhibition rate in the D579-DV25PL96 group.

[0199] (2) Inhibition rate of AGT mRNA levels in the liver

[0200] [Table 8]

[0201] As can be seen from Table 7, these GalNAc-binding oligonucleotides were able to significantly and continuously inhibit AGT mRNA levels in mouse liver, with the D579-DV25PG504 group showing the highest inhibition rate at 74.64%. The experimental results demonstrate that GalNAc compounds coupled with the oligonucleotide sequence D579-DV25P of this application can be efficiently delivered to the liver of animals and can significantly inhibit AGT mRNA levels in the liver.

[0202] Compared to siRNA conjugates produced with the conventional GalNAc compounds YK-GAL-325 and L96, GalNAc-binding oligonucleotides produced with the GalNAc compounds of this application significantly improved the inhibition rate of AGT mRNA levels in mouse liver. For example, the inhibition rate in the D579-DV25PG504 group was improved by 8.39% and 13.70% compared to the D579-DV25PG325 group and the D579-DV25PL96 group, respectively.

[0203] Comparing the GalNAc compounds of this application with the conventional GalNAc compounds YK-GAL-325 and L96, coupling the oligonucleotide sequence D579-DV25P with the GalNAc compound of this application significantly improved the delivery efficiency of the oligonucleotide to the liver compared to coupling with YK-GAL-325 and L96, demonstrating that the delivery efficiency of the oligonucleotide cannot be simply predicted based on the chemical structure of the GalNAc compound.

[0204] This application describes the design of a series of novel GalNAc compounds, YK-GAL-501, YK-GAL-502, YK-GAL-503, YK-GAL-504, and YK-GAL-505. The GalNAc-binding oligonucleotides produced using these compounds achieve efficient liver target delivery, which is significantly improved compared to representative GalNAc compounds in the prior art.

[0205] 1. The GalNAc compound of this application has a completely different chemical structure from the GalNAc compound of the prior art and is an entirely new GalNAc compound. The GalNAc compound designed in this application has a ribose ring structure introduced into the linker arm, and an oxygen or sulfur atom introduced at the 1' position of the ribose ring.

[0206] 2. The GalNAc compounds designed in this application are bound to the oligonucleotide sequence D579-DV25P, enabling efficient delivery of the oligonucleotide to the animal's liver and significant and sustained inhibition of serum AGT protein expression and hepatic mRNA levels. For example, D579-DV25PG501 achieved serum AGT protein inhibition rates of 66.21%, 75.63%, and 75.24% on days 7, 14, and 28, respectively, and hepatic AGT mRNA level inhibition rate reached 71.29% after 35 days.

[0207] 3. Compared with conventional GalNAc compounds (e.g., L96 and YK-GAL-325), GalNAc-binding oligonucleotides produced with the GalNAc compound designed in this application significantly improved the inhibition rate of AGT protein expression in mouse serum and significantly improved the inhibition rate of AGT mRNA levels in mouse liver.

[0208] For example, compared to the L96-conjugated oligonucleotide D579-DV25PL96, the YK-GAL-503-conjugated oligonucleotide D579-DV25PG503 improved the inhibition rate of AGT protein expression in mouse serum by 10.89% on day 7 and improved the inhibition rate of AGT mRNA levels in mouse liver by 12.25% on day 35. This indicates that the GalNAc compound designed in this application significantly improves delivery efficiency, enhances drug bioavailability, improves drug pharmacokinetic properties, and thereby enables the drug to exert superior efficacy.

[0209] 4. In this application, we found that GalNAc-binding oligonucleotides prepared with various structurally similar GalNAc compounds 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 levels in mouse liver, indicating that it is difficult to accurately predict oligonucleotide delivery efficiency based on the chemical structure of GalNAc compounds.

[0210] For example, compared to L96, YK-GAL-503 changes the prolinol structure of the L96 backbone to a ribose ring structure, while the rest of the structure is exactly the same. However, compared to the L96-binding oligonucleotide D579-DV25PL96, the YK-GAL-503-binding oligonucleotide D579-DV25PG503 improved the inhibition rate of AGT protein expression in mouse serum by 10.89% on day 7 and improved the inhibition rate of AGT mRNA levels in mouse liver by 12.25% on day 35. Compared to YK-GAL-325, YK-GAL-503 has only a shorter bond between the ribose ring and GalNAc compared to YK-GAL-325, while the rest of the structure is exactly the same. However, compared to YK-GAL-325-conjugated oligonucleotides, YK-GAL-503-conjugated oligonucleotides improved the inhibition rate of AGT protein expression in mouse serum by 7.85% on day 7 of administration and improved the inhibition rate of AGT mRNA levels in mouse liver by 6.94% on day 35 of administration.

[0211] Although the present invention has been described with reference to the specific examples described above, it is not limited thereto. Rather, the present invention should be interpreted as encompassing the general embodiments disclosed herein and allowing for various modifications and embodiments without departing from the spirit and scope of the invention.

[0212] Sequence ID 1 Sense 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'.

[0213] Sequence ID 2 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'.

[0214] Sequence ID 3: Sense chain: 5'-CCUUUUCUUCUAAUGAGUCGA-3'. Sequence ID 4 Antisense chain: 5'-UCGACUCAUUAGAAGAAAAGGUG-3'.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof. 【Chemistry 1】 (R １ It is oxygen or sulfur, R ２ is hydrogen, C １－６ Alkyl alkyl group, C １－６ It is an alkoxy group or halogen, R ３ It contains hydrogen, a hydroxy protecting group, and a phosphorus-containing active reactive group. 【Chemistry 2】 or -CO(CH ２ ) ｘ COOH, where x is an integer from 1 to 10. 【Transformation 3】 It is a controllable porous glass or polystyrene. R ４ is a hydrogen or hydroxy protecting group, 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, L is -CONH- or -NHCO-, G is 【Chemistry 4】 And, Here, T is N-acetylgalactosamine, galactose, galactosamine, N-formylgalactosamine, N-propionylgalactosamine, and N-butanoylgalactosamine, in which the hydroxyl group is completely protected by an acyl group. X １ ha- (CH ２ ) ｆ - or - (CH ２ CH ２ O) ｆ CH ２ - and f is an integer from 1 to 5, X ２ ha- (CH ２ ) ｇ - and g is an integer from 1 to 6, Y １ is 0 or 1, Y ２ is 0, 1, or 2, Y ３ is 1, 2, or 3, m is an integer between 0 and 4. n is an integer between 0 and 4.

2. R ３ The hydroxy protecting group is preferably an acyl group, a silyl group, a trityl group, a 4-methoxytrityl group, or a 4,4'-dimethoxytrityl group, and more preferably an acetyl group, a 1,1,3,3-tetraisopropyldisiloxane group, a tert-butyldimethylsilyl group, a dimethylphenylsilyl group, or a 4,4'-dimethoxytrityl group. Or, R ３ is a phosphorus-containing active reactive group, and the phosphorus-containing active reactive group is, for example, 【Transformation 5】 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that it is such.

3. R ４ The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that is a hydroxy protecting group, preferably a trityl group, a 4-methoxytrityl group, or a 4,4'-dimethoxytrityl group.

4. (1) The condition is that a is an integer between 2 and 7. (2) b is an integer between 1 and 3, (3) The acyl group is either an acetyl group or a benzoyl group, for example, under the condition that it is an acetyl group, (4) The condition that m is 0, 1 or 2, (5) The condition that n is 0, 1 or 2, (6) R ３ is a phosphorus-containing active reactive group, 【Transformation 6】 And, 【Transformation 7】 x is a controllable porous glass or polystyrene, preferably under the condition that x is 2. A compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized by satisfying one or more of the following conditions.

5. (1) A is -CH ２ CH ２ OCH ２ CH ２ -, - (CH ２ ) ２ -, - (CH ２ ) ３ -, - (CH ２ ) ４ - or - (CH ２ ) ５ - condition, (2) R １ The condition is that it is oxygen. (3) Caution １ The conditions are that it is an α configuration or a β configuration. (4) R ２ ha-OCH ３ The condition is, (5) Caution ３ teeth 【Transformation 8】 or -CO(CH ２ ) ２ COOH, 【Chemistry 9】 The condition is that it is a controllable porous glass. (6) R ４ The condition is that it is a 4,4'-dimethoxytrityl group, (7) T is N-acetylgalactosamine that is completely protected by an acyl group, (8) X １ ha- (CH ２ ) ｆ - and the condition that f is 1, (9) The condition that g is 2, (10) Y １ The condition is that it is 0. (11) Y ２ The condition is that it is 0. (12) Condition that m is 1, (13) Condition that n is 0, (14) The condition that L is -NHCO-, (15) Y ３ The condition is that it is 1. A compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized by satisfying one or more of the following conditions.

6. G is 【Chemistry 10】 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that it is such.

7. The aforementioned compound is one of the following compounds: 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that it is a controllable porous glass.

8. A compound represented by formula (I) according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, characterized by being able to bind to an asialoglycoprotein receptor.

9. It comprises an oligonucleotide and a GalNAc moiety, the oligonucleotide and GalNAc linked via a phosphate ester group or a phosphorothioate group, and the GalNAc moiety is one or more GalNAc molecules, which are formed by linking via a phosphate ester group or a phosphorothioate group. Here, the GalNAc molecule is a compound represented by formula I-2 or a pharmaceutically acceptable salt thereof. 【Chemistry 15】 Here, R ３－１ This is a linked connection, R ４ H is, Or, R ４－１ This is a linked connection, R ３－１ H is, G is 【Chemistry 16】 And, T1 is N-acetylgalactosamine, galactose, galactosamine, N-formylgalactosamine, N-propionylgalactosamine, and N-butanoylgalactosamine, preferably N-acetylgalactosamine. Y ３ , Y ２ , X ２ , Y １ , X １ , m, n, R ２ , R １ A and L are as described in any one of claims 1 to 7, conjugate.

10. The conjugate according to claim 9, characterized in that the oligonucleotide comprises a non-phosphorothioate oligonucleotide and a phosphorothioate oligonucleotide.

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

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

13. It has the following structure, 【Chemistry 17】 or [Chemistry 18] Here, Oligo represents an oligonucleotide, X ３ is oxygen or sulfur, q is 1, 2, or 3, R １ , R ２ The conjugate according to claim 9, characterized in that A, L, G, m, and n are as described in claim 9.

14. The conjugate according to claim 9, wherein the oligonucleotide comprises a small interfering nucleotide, DNA, microRNA, small activating RNA, small guide RNA, transfer RNA, antisense nucleotide, or aptamer, and is preferably an antisense nucleotide or a small interfering nucleotide.

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

16. The conjugate according to claim 9, characterized by having the following structure. 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】

17. The conjugate according to claim 9, characterized in that the oligonucleotide regulates the expression of a target gene.

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

19. The use of a conjugate according to any one of claims 9 to 17, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 18, in the manufacture of a pharmaceutical for treating and / or preventing a pathological condition or disease caused by the expression of a specific gene in liver tissue or a virus, Selectively, the specified gene used is selected from the hepatitis B virus gene, the proprotein-converting enzyme subtilisin / kexin type 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.

20. The use according to claim 19, wherein the disease is selected from chronic liver disease, hepatitis, hepatic fibrosis, hepatic proliferative disorders, and cardiovascular diseases, and selectively, the cardiovascular disease is hypercholesterolemia, hypertriglyceridemia, atherosclerosis, or blood coagulation disorders.

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

22. A method for inhibiting the expression of a specific gene in hepatocytes, Herein, the method includes the step of bringing the hepatocytes into contact with an effective amount of the conjugate according to any one of claims 9 to 17, or the pharmaceutical composition according to claim 18. A method wherein the specific gene is selectively selected from the proprotein convertase subtilisin / kexin type 9 gene, hepatitis B virus gene, apolipoprotein a gene, coagulation factor 11 gene, angiopoietin-like protein 3 gene, angiotensinogen gene, or apolipoprotein C3 gene.

Images