Synthesis method of GalNAc derivative

Abstract

The invention provides a synthesis method of a GalNAc derivative, and particularly provides a synthesis method of a GalNAc derivative as shown in a formula (I). The method comprises the following steps: S101, carrying out first substitution reaction on a compound (1) and an amino protective agent to obtain a compound (2); s102, carrying out second substitution reaction on the compound (2) and a hydroxyl protective agent to obtain a compound (3); s103, performing a first amino deprotection reaction on the compound (3) to obtain a compound (4); s104, carrying out condensation reaction on the compound (4) and a compound (5) to obtain a compound (6); and S105, carrying out phosphorylation reaction on the compound (6) and a phosphorylation reagent to obtain the GalNAc derivative as shown in the formula (I). The method disclosed by the invention has the advantages of high safety, high final product purity, high yield and the like, and is easy for industrial production. # imgabs0 #

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine technology, and in particular, to a method for synthesizing a GalNAc derivative. Background Art

[0002] N-acetylgalactosamine (GalNAc) is an amino sugar derivative of galactose. GalNAc derivatives are usually the targeting moieties of conjugates comprising a GalNAc moiety and certain oligonucleotides. The GalNAc moiety can functionally deliver the oligonucleotide conjugate to hepatocytes due to its affinity for the asialoglycoprotein receptors located on hepatocytes. The GalNAc conjugate has the potential to act as a pharmacokinetic modulator and is therefore a compound of therapeutic value, as described in PCT Publication WO2012 / 083046. Although these publications also disclose methods for preparing GalNAc derivatives, it has been found that these methods are further improved in terms of large-scale synthesis.

[0003] Therefore, it is necessary to develop a new method for preparing GalNAc derivatives to be suitable for industrial production. Summary of the invention

[0004] The present disclosure aims to solve at least one of the technical problems existing in the prior art to at least some extent.

[0005] Therefore, in the first aspect of the present disclosure, a method for synthesizing a GalNAc derivative represented by formula (I) is proposed. According to an embodiment of the present disclosure, the synthesis method comprises the following steps:

[0006] S101, subjecting compound (1) to a first substitution reaction with an amino protecting agent to obtain compound (2);

[0007] S102, subjecting the compound (2) to a second substitution reaction with a hydroxyl protecting agent to obtain a compound (3);

[0008] S103, subjecting the compound (3) to a first amino group deprotection reaction to obtain a compound (4);

[0009] S104, subjecting the compound (4) to a condensation reaction with the compound (5) to obtain a compound (6);

[0010] S105, subjecting the compound (6) to a phosphitylation reaction with a phosphitylation agent to obtain a GalNAc derivative represented by formula (I);

[0011]

[0012]

[0013] Wherein, R1 represents an amino protecting group;

[0014] R2 represents a hydroxyl protecting group;

[0015] Each R3 is independently selected from optionally substituted C 1-6 Alkanoyl or optionally substituted C 5-12 Aroyl;

[0016] L is selected from optionally substituted C 1-20 alkyl;

[0017] R4 is selected from Each R 4a Independently selected from or C containing a cyano substituent 1-6 Alkoxy; wherein each R 4a ' is independently selected from optionally substituted C 1-6 Thus, the legal method disclosed in the present invention can improve the total yield of the GalNAc derivative of formula (I), wherein the total yield reaches 25.4% and the purity can also reach 99%.

[0018] According to an embodiment of the present disclosure, the synthesis method may further include at least one of the following additional technical features:

[0019] According to an embodiment of the present disclosure, the L is selected from optionally substituted C 1-20 Straight chain alkyl. For example, the optionally substituted C 1-19 Straight chain alkyl, optionally substituted C 1-18 Straight chain alkyl, optionally substituted C 1-17 Straight chain alkyl, optionally substituted C 1-16 Straight chain alkyl, optionally substituted C 1-15 Straight chain alkyl, optionally substituted C 1-14 Straight chain alkyl, optionally substituted C 1-13 Straight chain alkyl, optionally substituted C 1-12 Straight chain alkyl, optionally substituted C 1-11 Straight chain alkyl, optionally substituted C 1-10 Straight chain alkyl, optionally substituted C 1-9 Straight chain alkyl, optionally substituted C 1-8 Straight chain alkyl, optionally substituted C 1-7 Straight chain alkyl, optionally substituted C 1-6 Straight chain alkyl, optionally substituted C 1-5 Straight chain alkyl, optionally substituted C 1-4 Straight chain alkyl, optionally substituted C 2-20 Straight chain alkyl, optionally substituted C 2-19 Straight chain alkyl, optionally substituted C 2-18Straight chain alkyl, optionally substituted C 2-17 Straight chain alkyl, optionally substituted C 2-16 Straight chain alkyl, optionally substituted C 2-15 Straight chain alkyl, optionally substituted C 2-14 Straight chain alkyl, optionally substituted C 2-13 Straight chain alkyl, optionally substituted C 2-12 Straight chain alkyl, optionally substituted C 2-11 Straight chain alkyl, optionally substituted C 2-10 Straight chain alkyl, optionally substituted C 2-9 Straight chain alkyl, optionally substituted C 2-8 Straight chain alkyl, optionally substituted C 2-7 Straight chain alkyl, optionally substituted C 2-6 Straight chain alkyl, optionally substituted C 2-5 Straight chain alkyl, optionally substituted C 2-4 Straight chain alkyl, optionally substituted C 3-20 Straight chain alkyl, optionally substituted C 3-19 Straight chain alkyl, optionally substituted C 3-18 Straight chain alkyl optionally substituted C 3-17 Straight chain alkyl, optionally substituted C 3-16 Straight chain alkyl, optionally substituted C 3-15 Straight chain alkyl, optionally substituted C 3-14 Straight chain alkyl, optionally substituted C 3-13 Straight chain alkyl, optionally substituted C 3-12 Straight chain alkyl, optionally substituted C 3-11 Straight chain alkyl, optionally substituted C 3-10 Straight chain alkyl, optionally substituted C 3-9 Straight chain alkyl, optionally substituted C 3-8 Straight chain alkyl, optionally substituted C 3-7 Straight chain alkyl, optionally substituted C 3-6 Straight chain alkyl, optionally substituted C 3-5 Straight chain alkyl, optionally substituted C 3-4 Straight chain alkyl and optionally substituted C4 straight chain alkyl.

[0020] According to an embodiment of the present disclosure, the L is selected from C 1-20 Straight chain alkyl. For example, C 1-19 Straight chain alkyl, C 1-18 Straight chain alkyl, C 1-17 Straight chain alkyl, C 1-16 Straight chain alkyl, C 1-15 Straight chain alkyl, C 1-14 Straight chain alkyl, C 1-13 Straight chain alkyl, C 1-12 Straight chain alkyl, C 1-11 Straight chain alkyl, C1-10 Straight chain alkyl, C 1-9 Straight chain alkyl, C 1-8 Straight chain alkyl, C 1-7 Straight chain alkyl, C 1-6 Straight chain alkyl, C 1-5 Straight chain alkyl, C 1-4 Straight chain alkyl, C 2-20 Straight chain alkyl, C 2-19 Straight chain alkyl, C 2-18 Straight chain alkyl C 2-17 Straight chain alkyl, C 2-16 Straight chain alkyl, C 2-15 Straight chain alkyl, C 2-14 Straight chain alkyl, C 2-13 Straight chain alkyl, C 2-12 Straight chain alkyl, C 2-11 Straight chain alkyl, C 2-10 Straight chain alkyl, C 2-9 Straight chain alkyl, C 2-8 Straight chain alkyl, C 2-7 Straight chain alkyl, C 2-6 Straight chain alkyl, C 2-5 Straight chain alkyl, C 2-4 Straight chain alkyl, C 3-20 Straight chain alkyl, C 3-19 Straight chain alkyl, C 3-18 Straight chain alkyl, C 3-17 Straight chain alkyl, C 3-16 Straight chain alkyl, C 3-15 Straight chain alkyl, C 3-14 Straight chain alkyl, C 3-13 Straight chain alkyl, C 3-12 Straight chain alkyl, C 3-11 Straight chain alkyl, C 3-10 Straight chain alkyl, C 3-9 Straight chain alkyl, C 3-8 Straight chain alkyl, C 3-7 Straight chain alkyl, C 3-6 Straight chain alkyl, C 3-5 Straight chain alkyl, C 3-4 Straight chain alkyl and C4 straight chain alkyl.

[0021] According to an embodiment of the present disclosure, L is selected from

[0022] According to an embodiment of the present disclosure, each R3 is independently selected from

[0023] According to an embodiment of the present disclosure, each R3 is selected from

[0024] According to an embodiment of the present disclosure, the R2 is selected from a trityl group (Tr group), a 4-methoxytrityl group (MMTr group), a 4,4'-dimethoxytrityl group (DMTr group) or a 4,4',4"-trimethoxytrityl group (TMTr group).

[0025] According to an embodiment of the present disclosure, the R2 is selected from 4,4'-dimethoxytrityl.

[0026] According to an embodiment of the present disclosure, the hydroxyl protecting agent is selected from trityl chloride (also known as triphenylmethane chloride, abbreviated as Tr-Cl, CAS No. 76-83-5), trityl bromide (CAS No. 596-43-0), 4-methyltriphenylmethyl chloride (also known as 4-methyltriphenylmethane chloride, CAS No. 23429-44-9), 4-methoxytrityl chloride (also known as 4-methoxytriphenylmethane chloride, MMT-Cl, CAS No. 14470-28-1), 4,4'-dimethoxytrityl chloride (also known as 4,4'-dimethoxytriphenylmethane chloride, abbreviated as DMT-Cl, CAS No. 40615-36-9) and 4,4',4"-trimethoxytrityl chloride (CAS No. 49757-42-8).

[0027] According to an embodiment of the present disclosure, the hydroxyl protecting agent is selected from 4,4'-dimethoxytrityl chloride.

[0028] According to an embodiment of the present disclosure, the phosphitylation agent is selected from 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (CAS No. 89992-70-1), bis(diisopropylamino)chlorophosphine (CAS No. 56183-63-2) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (CAS No. 102691-36-1).

[0029] According to an embodiment of the present disclosure, the phosphitylation agent is selected from bis(diisopropylamino)(2-cyanoethoxy)phosphine.

[0030] According to an embodiment of the present disclosure, R4 is selected from

[0031] According to an embodiment of the present disclosure, R4 is selected from

[0032] According to an embodiment of the present disclosure, the amino protecting agent is selected from an alkoxycarbonyl amino protecting agent, an acyl amino protecting agent and an alkyl amino protecting agent.

[0033] According to an embodiment of the present disclosure, the amino protecting agent is selected from alkoxycarbonyl amino protecting agents.

[0034] According to an embodiment of the present disclosure, the structural formula of the alkoxycarbonyl amino protecting agent is Wherein R5 is selected from optionally substituted C 1-10 Alkyl, if R5 contains substituents, the substituents are independently selected from halogen, optionally substituted C 5-20 Aryl, optionally substituted C 1-6 an alkene group or an optionally substituted silane group.

[0035] According to the embodiments of the present disclosure, the alkoxycarbonyl amino protecting agent includes but is not limited to: benzyl chloroformate (Cbz-Cl, CAS No. 501-53-1), tert-butyl chloroformate (Boc-Cl, CAS No. 24608-52-4), 9-fluorenylmethyl chloroformate (Fmoc-Cl, CAS No. 28920-43-6), allyl chloroformate (Alloc-Cl, CAS No. 2937-50-0), 2-(trimethylsilyl)ethyl chloroformate (Teoc-Cl, CAS No. 2937-50-0), 2-(trimethylsilyl)ethyl chloroformate (Teoc-Cl, CAS No. 2937-50-0), decyl chloroformate (CAS No. 2937-50-0), decyl chloroformate (CAS No. 2937-50-0), 2-(trimethylsilyl)ethyl chloroformate (Teoc-Cl, ... 488-51-2), nonyl chloroformate (CAS No. 57045-82-6), octyl chloroformate (CAS No. 7452-59-7), 2-octyl chloroformate (CAS No. 15586-11-5), heptyl chloroformate (CAS No. 33758-34-8), hexyl chloroformate (CAS No. 6092-54-2), 3-hexyl chloroformate (CAS No. 58906-62-0), cyclohexyl chloroformate (CAS No. 13248-54-9), (-)-menthyl chloroformate (CAS No. 14602-86-9), chloroformic acid n-Amyl chloroformate (CAS No. 638-41-5), isoamyl chloroformate (CAS No. 628-50-2), cyclopentyl chloroformate (CAS No. 50715-28-1), n-butyl chloroformate (CAS No. 592-34-7), isobutyl chloroformate (CAS No. 543-27-1), cyclobutyl chloroformate (CAS No. 81228-87-7), sec-butyl chloroformate (CAS No. 17462-58-7), n-propyl chloroformate (CAS No. 109-61-5), 3-chloro-n-propyl chloroformate (CAS No. 628-11-5), isopropyl chloroformate ( CAS No. 108-23-6), 2,2-dimethylpropyl chloroformate (CAS No. 20412-38-8), ethyl chloroformate (CAS No. 541-41-3), 2-methoxyethyl chloroformate (CAS No. 628-12-6), 2,2,2-trichloroethyl chloroformate (CAS No. 17341-93-4), chloroethyl chloroformate (CAS No. 627-11-2), methyl chloroformate (CAS No. 79-22-1), chloromethyl chloroformate (CAS No. 22128-62-7) and trichloromethyl chloroformate (CAS No. 503-38-8).

[0036] According to an embodiment of the present disclosure, the alkoxycarbonyl amino protecting agent is selected from benzyl chloroformate, tert-butyl chloroformate, 9-fluorenylmethyl chloroformate, allyl chloroformate, 2-(trimethylsilyl)ethyl chloroformate, ethyl chloroformate and methyl chloroformate.

[0037] According to an embodiment of the present disclosure, the alkoxycarbonyl amino protecting agent is selected from 9-fluorenylmethyl chloroformate.

[0038] According to an embodiment of the present disclosure, the R5 is selected from

[0039] According to an embodiment of the present disclosure, the R1 is selected from an alkoxycarbonyl-type amino protecting group, an acyl-type amino protecting group and an alkyl-type amino protecting group.

[0040] According to an embodiment of the present disclosure, the R1 is selected from an alkoxycarbonyl-type amino protecting group.

[0041] According to the embodiments of the present disclosure, the alkoxycarbonyl amino protecting groups include but are not limited to: benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), n-decyloxycarbonyl, n-nonyloxycarbonyl, n-octyloxycarbonyl, 2-octyloxycarbonyl, n-heptyloxycarbonyl, n-hexyloxycarbonyl, 3-hexyloxycarbonyl, 4-Butyloxycarbonyl, cyclohexyloxycarbonyl, n-pentyloxycarbonyl, isopentyloxycarbonyl, cyclopentyloxycarbonyl, n-butoxycarbonyl, isobutoxycarbonyl, cyclobutyloxycarbonyl, sec-butoxycarbonyl, n-propyloxycarbonyl, 3-chloro-n-propyloxycarbonyl, isopropyloxycarbonyl, 2,2-dimethyl-n-propyloxycarbonyl, ethoxycarbonyl, 2-methoxyethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-chloroethoxycarbonyl, methoxycarbonyl, monochloromethoxycarbonyl and trichloromethoxycarbonyl.

[0042] According to an embodiment of the present disclosure, the alkoxycarbonyl amino protecting group is selected from benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), ethoxycarbonyl and methoxycarbonyl.

[0043] According to an embodiment of the present disclosure, the alkoxycarbonyl-type amino protecting group is selected from fluorenylmethoxycarbonyl (Fmoc).

[0044] According to an embodiment of the present disclosure, the GalNAc derivative shown in formula (I) has a structure of

[0045] According to an embodiment of the present disclosure, the structure of the compound (6) is

[0046] According to an embodiment of the present disclosure, the structure of the compound (5) is

[0047] According to an embodiment of the present disclosure, the structure of the compound (4) is

[0048] According to an embodiment of the present disclosure, the structure of the compound (3) is

[0049] According to an embodiment of the present disclosure, the structure of the compound (2) is

[0050] According to an embodiment of the present disclosure, in step S101, the molar ratio of the compound (1) to the amino protecting agent is 1:(1.2-1.7). For example, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, etc. The inventors have found through a large number of experiments that when the molar ratio of the compound (1) to the amino protecting agent is within this range, the yield of the GalNAc derivative represented by formula (I) can be improved.

[0051] According to an embodiment of the present disclosure, in step S101, the reaction solvent of the first substitution reaction is selected from 1,4-dioxane.

[0052] According to an embodiment of the present disclosure, in step S101, the amino protecting agent is added at a temperature of -5 to 5°C, such as 0°C (ice bath).

[0053] According to an embodiment of the present disclosure, in step S101, the first substitution reaction is performed under stirring conditions.

[0054] According to an embodiment of the present disclosure, in step S101, the reaction temperature of the first substitution reaction is 20-30° C., for example, 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C. or 30° C.

[0055] It should be noted that, in step S101, a saturated aqueous solution of sodium bicarbonate may be added to the reaction system of the first substitution reaction, wherein the sodium bicarbonate may neutralize the HCl in compound (1).

[0056] According to an embodiment of the present disclosure, in step S101, after the first substitution reaction is completed, the compound (2) is further subjected to a first purification treatment to obtain the compound (2).

[0057] According to an embodiment of the present disclosure, in step S101, the first purification treatment includes a first extraction treatment and / or a first column chromatography purification treatment.

[0058] According to an embodiment of the present disclosure, in step S101, the extractant of the first extraction treatment includes ethyl acetate and water, wherein the volume ratio of ethyl acetate to water is (3-5):(3-5), for example, 5:3.

[0059] According to an embodiment of the present disclosure, in step S101, the first column chromatography purification treatment is selected from column chromatography normal phase purification.

[0060] According to an embodiment of the present disclosure, in step S101, the eluent of the first column chromatography purification treatment includes methanol and dichloromethane, wherein the volume ratio of methanol to dichloromethane is (3-10):(90-97), for example, 6:94.

[0061] According to an embodiment of the present disclosure, in step S102, the molar ratio of the compound (2) to the hydroxyl protecting agent is 1:(1.1-2), such as 1:1.1, 1:1.2, 1:13, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, etc. The inventors have found through a large number of experiments that the molar ratio of the compound (2) to the hydroxyl protecting agent within this range can make the isomers produced in this step unnecessary to undergo a purification and separation step, thus saving time and simplifying the operation process. In addition, the use of a hydroxyl protecting agent can also help to improve the yield of the GalNAc derivative shown in formula (I) finally prepared.

[0062] According to an embodiment of the present disclosure, in step S102, the reaction solvent of the second substitution reaction is selected from pyridine.

[0063] According to an embodiment of the present disclosure, in step S102, the hydroxyl protective agent is added at a temperature of -5 to 5°C, such as 0°C (ice bath). The inventors have found through experiments that when the hydroxyl protective agent is added under ice bath conditions, the yield of the GalNAc derivative represented by formula (I) can be improved.

[0064] According to an embodiment of the present disclosure, in step S102, the reaction temperature of the second substitution reaction is 20-30° C. For example, 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., etc.

[0065] According to an embodiment of the present disclosure, in step S102, after the second substitution reaction is completed, the compound (3) is further subjected to a second purification treatment.

[0066] According to an embodiment of the present disclosure, in step S102, the second purification treatment includes a second extraction treatment and / or a second column chromatography purification treatment.

[0067] According to an embodiment of the present disclosure, in step S102, the organic solvent of the second extraction treatment is selected from ethyl acetate.

[0068] According to an embodiment of the present disclosure, in step S102, the second column chromatography purification treatment is selected from column chromatography normal phase purification.

[0069] According to an embodiment of the present disclosure, in step S102, the eluent of the second column chromatography purification treatment includes ethyl acetate and petroleum ether, wherein the volume ratio of ethyl acetate to petroleum ether is (20-35):(65-80), for example, 28:72.

[0070] According to an embodiment of the present disclosure, in step S103, the reaction solvent of the first amino deprotection reaction is selected from acetonitrile.

[0071] According to an embodiment of the present disclosure, in step S103, the first amino group deprotection reaction is performed under alkaline conditions.

[0072] According to an embodiment of the present disclosure, in step S103, the first amino group deprotection reaction is performed in the presence of a basic deprotecting agent.

[0073] According to an embodiment of the present disclosure, the basic deprotecting agent is selected from tetrahydropyrrole, triethylamine and piperidine.

[0074] According to an embodiment of the present disclosure, the alkaline deprotecting agent is selected from tetrahydropyrrole.

[0075] According to an embodiment of the present disclosure, in step S103, the reaction temperature of the first amino group deprotection reaction is 22-27° C., for example, 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., etc.

[0076] According to an embodiment of the present disclosure, in step S103, after the first amino deprotection reaction is completed, the compound (6) is further purified through a third purification process.

[0077] According to an embodiment of the present disclosure, the third purification treatment includes a third column chromatography purification treatment.

[0078] According to an embodiment of the present disclosure, the third column chromatography purification treatment is selected from column chromatography normal phase purification.

[0079] According to an embodiment of the present disclosure, the eluent of the third column chromatography purification treatment includes methanol and dichloromethane containing (0.5-2) volume % ammonia water, for example, dichloromethane containing 1 volume % ammonia water. Wherein, the volume ratio of methanol to dichloromethane containing (0.5-2) volume % ammonia water is (4-6):(94-96), for example, 5:95.

[0080] According to an embodiment of the present disclosure, in step S104, the molar ratio of the compound (4) to the compound (5) is (1.2-1.8):1, for example, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, etc.

[0081] According to an embodiment of the present disclosure, in step S104, the condensation reaction is performed in the presence of a condensation agent.

[0082] According to an embodiment of the present disclosure, the molar ratio of the condensing agent to the compound (5) is (1.2-1.8): 1. Further, the molar ratio is (1.3-1.7): 1. Further, the molar ratio is 1.5:1.

[0083] According to an embodiment of the present disclosure, the condensing agent is selected from 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0084] According to an embodiment of the present disclosure, the condensing agent is selected from 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0085] According to an embodiment of the present disclosure, the condensation reaction is carried out in the presence of an organic base.

[0086] According to an embodiment of the present disclosure, the molar ratio of the organic base to the compound (5) is (2.5-3.5):1; for example, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1. Further, the molar ratio of the organic base to the compound (5) is (2.8-3.2):1. Further, the molar ratio of the organic base to the compound (5) is 3:1.

[0087] According to an embodiment of the present disclosure, the organic base is selected from triethylamine, N-methylmorpholine and diisopropylethylamine.

[0088] According to an embodiment of the present disclosure, the organic base is selected from diisopropylethylamine.

[0089] According to an embodiment of the present disclosure, the organic base is added at a temperature of -5 to 5°C, such as 0°C (ice bath).

[0090] According to an embodiment of the present disclosure, in step S104, the reaction temperature of the condensation reaction is 22-27°C, for example, 22°C, 23°C, 24°C, 25°C, 26°C or 27°C.

[0091] According to an embodiment of the present disclosure, the reaction solvent of the condensation reaction is selected from N,N-dimethylformamide and dichloromethane.

[0092] According to an embodiment of the present disclosure, the reaction solvent of the condensation reaction is selected from N,N-dimethylformamide.

[0093] According to a specific embodiment of the present disclosure, in step S104, after the condensation reaction is completed, the compound (6) is further obtained by undergoing a post-treatment reaction and a fourth purification treatment.

[0094] According to an embodiment of the present disclosure, the fourth purification treatment includes a fourth column chromatography purification treatment.

[0095] According to an embodiment of the present disclosure, the fourth column chromatography purification treatment is selected from column chromatography reverse phase purification or column chromatography normal phase purification.

[0096] According to an embodiment of the present disclosure, when the fourth column chromatography purification treatment is selected from column chromatography reverse phase purification, the eluent of the fourth column chromatography purification treatment includes acetonitrile and water in a volume ratio of (30-40):(60-70), for example, 36:64, etc.

[0097] According to an embodiment of the present disclosure, in step S105, the molar ratio of the compound (6) to the phosphitylation agent is (1.2-2): 1. For example, 1.2: 1, 1.3: 1, 1.4: 1, 1.5: 1, 1.6: 1, 1.7: 1, 1.8: 1, 1.9: 1, 2: 1, etc.

[0098] According to an embodiment of the present disclosure, in step S105, the reaction solvent of the phosphonylation reaction is selected from dichloromethane.

[0099] According to an embodiment of the present disclosure, in step S105, the phosphonylation reaction is performed in an inert atmosphere, such as nitrogen, argon, etc.

[0100] According to an embodiment of the present disclosure, in step S105, the phosphonylation reaction is carried out in nitrogen.

[0101] According to an embodiment of the present disclosure, in step S105, the phosphonylation reaction is carried out under stirring conditions.

[0102] According to an embodiment of the present disclosure, in step S105, the reaction temperature of the phosphonylation reaction is 20-30°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, etc.

[0103] According to an embodiment of the present disclosure, in step S105, after the phosphonylation reaction is completed, a fifth purification treatment is further performed to obtain the GalNAc derivative represented by formula (I).

[0104] According to an embodiment of the present disclosure, the fifth purification treatment includes a fifth extraction treatment and / or a fifth column chromatography purification treatment.

[0105] According to an embodiment of the present disclosure, the organic solvent of the extraction treatment is selected from dichloromethane.

[0106] According to an embodiment of the present disclosure, the fifth column chromatography purification treatment is selected from column chromatography reverse phase purification.

[0107] According to an embodiment of the present disclosure, the eluent of the fifth column chromatography purification treatment includes acetonitrile and water in a volume ratio of (60-75):(25-40), for example, 68:32.

[0108] The compound (1) (CAS No. 2408968-41-0) used in the present disclosure can be obtained from the market, but it is expensive. In order to further control the production cost, the present disclosure provides a new synthesis method of compound (1).

[0109] According to an embodiment of the present disclosure, the preparation method of the compound (1) comprises the following steps:

[0110]

[0111] S201, mixing compound (1-1) and formaldehyde, and subjecting the reaction system to an aldol condensation reaction and then a Cannizzaro reaction to obtain compound (1-2);

[0112] S202. The compound (1-2) undergoes a second amino deprotection reaction to obtain compound (1).

[0113] According to an embodiment of the present disclosure, in step S201, the molar ratio of the compound (1-1) to the formaldehyde is 1:(2-3); for example, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, etc.

[0114] According to an embodiment of the present disclosure, in step S201, the reaction system is alkaline. Further, the alkalinity is selected from an aqueous solution of an alkali metal hydroxide (e.g., an aqueous solution of KOH, an aqueous solution of NaOH), with a concentration of 40 to 50% by mass (e.g., 45.3% by mass). Further, the alkalinity is selected from an aqueous solution of KOH with a concentration of 45.3% by mass.

[0115] According to an embodiment of the present disclosure, in step S201, the solvent in the reaction system is selected from alcohol organic solvents, and the alcohol organic solvent is selected from methanol and ethanol.

[0116] According to an embodiment of the present disclosure, in step S201, the reaction temperature of the aldol condensation reaction is 20-30°C; for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C, etc.

[0117] According to an embodiment of the present disclosure, in step S201, the reaction time of the aldol condensation reaction is 20 to 40 minutes; for example, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes or 40 minutes, etc.

[0118] According to an embodiment of the present disclosure, in step S201, the reaction temperature of the Cannizzaro reaction is 55-65°C; for example, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, etc. Wherein, Cannizzaro Reaction is translated as Cannizzaro Reaction, Cannizzaro Reaction, and Cannizzaro Reaction.

[0119] According to an embodiment of the present disclosure, in step S201, the reaction time of the Cannizzaro reaction is 1 to 3 hours, for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours or 3 hours.

[0120] According to an embodiment of the present disclosure, in step S202, the second amino group deprotection reaction is performed in the presence of an acidic deprotecting agent.

[0121] According to an embodiment of the present disclosure, the acidic deprotecting agent is selected from inorganic strong acids.

[0122] According to an embodiment of the present disclosure, the inorganic strong acid is selected from HCl and HBr.

[0123] According to an embodiment of the present disclosure, the inorganic strong acid is selected from HCl.

[0124] According to an embodiment of the present disclosure, the reaction solvent of the second amino deprotection reaction is selected from 1,4-dioxane.

[0125] According to an embodiment of the present disclosure, the reaction temperature of the second amino deprotection reaction is 20-30°C; for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C, etc.

[0126] According to an embodiment of the present disclosure, the reaction time of the second amino group deprotection reaction is 0.5 to 3 hours; for example, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours or 3 hours. It should be noted that in each reaction step of the present disclosure, the reaction time is not fixed, and the reaction progress only needs to be monitored by monitoring whether the reaction raw materials are completely reacted at a suitable reaction temperature, and the reaction process can be terminated when there is no reaction raw material in the reaction solution.

[0127] Additional aspects and advantages of the present disclosure will be given in part in the following description and in part will be obvious from the following description or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] The above and / or additional aspects and advantages of the present disclosure will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0129] Figure 1 is a liquid chromatogram of compound (4) before normal phase purification by column chromatography according to an embodiment of the present disclosure;

[0130] Figure 2 is a liquid chromatogram of compound (4) after normal phase purification by column chromatography according to an embodiment of the present disclosure;

[0131] Figure 3 is a liquid chromatogram of compound (6) before normal phase purification by column chromatography according to an embodiment of the present disclosure;

[0132] Figure 4 is a liquid chromatogram of compound (6) after normal phase purification by column chromatography according to an embodiment of the present disclosure;

[0133] Figure 5 is a GalNAc derivative (I) according to an embodiment of the present disclosure CR01008 's H NMR spectrum;

[0134] Figure 6 is a GalNAc derivative (I) according to an embodiment of the present disclosure CR01008 The nuclear magnetic phosphorus spectrum of

[0135] Figure 7 The GalNAc derivative (I) after column chromatography normal phase purification according to the embodiment of the present disclosure CR01008 Liquid chromatogram of . DETAILED DESCRIPTION

[0136] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure.

[0137] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present disclosure, unless otherwise specified, the meaning of "plurality" is two or more.

[0138] Herein, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present disclosure but not excluding other contents.

[0139] In this document, the term "selected from... and..." can be used interchangeably with the term "selected from.... or...", which means that one or more of them can be selected.

[0140] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0141] In general, the term "substituted" means that one or more hydrogen atoms in a given structure are replaced by a specified substituent. Unless otherwise indicated, a substituted group may have a substituent at each substitutable position of the group. When more than one position in a given structure can be substituted by one or more substituents selected from a specified group, then the substituents may be the same or different at each substitutable position.

[0142] The term "unsubstituted" means that the designated group bears no substituents.

[0143] The term "optionally substituted ..." can be used interchangeably with the term "optionally substituted by..." or "unsubstituted or substituted by...", i.e., the structure is unsubstituted or substituted by one or more substituents described in the present disclosure. The substituents described in the present disclosure include, but are not limited to, D, F, =O, Cl, Br, I, N3, CN, NO2, OH, SH, NH2, alkyl, haloalkyl, haloalkoxy, haloalkylamino, alkenyl, alkynyl, alkoxy, alkylamino, cycloalkyl, heterocyclyl, aryl, heteroaryl, and the like.

[0144] Unless otherwise indicated, the following definitions used herein shall apply. For purposes of this disclosure, chemical elements are consistent with the Periodic Table of the Elements, CAS version, and Handbook of Chemistry and Physics, 75th edition, 1994. In addition, general principles of organic chemistry can be found in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the entire contents of which are incorporated herein by reference.

[0145] The term "alkyl" refers to a saturated linear or branched monovalent hydrocarbon group of 1-50 carbon atoms, or 1-40 carbon atoms, or 1-20 carbon atoms, or 1-10 carbon atoms, or 1-6 carbon atoms, or 1-4 carbon atoms, wherein the alkyl group may be independently and optionally substituted with one or more substituents described in the present disclosure, including but not limited to deuterium, amino, hydroxyl, cyano, F, Cl, Br, I, mercapto, nitro, oxo (=O), etc. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (n-Bu, -CH2CH2CH2CH3), isobutyl (i-Bu, -CH2CH(CH3)2), sec-butyl (s-Bu, -CH(CH3)CH2CH3), tert-butyl (t-Bu, -CH(CH3)CH2CH3), -C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1- Butyl (-CH2CH(CH3)CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3 ), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), n-heptyl, n-octyl, and the like. The term "alkyl" and its prefix "alkane" as used herein include both straight and branched saturated carbon chains.

[0146] The term "alkoxy" refers to the group -O-RQ, wherein RQ is an "alkyl" as defined above.

[0147] The terms "cycloalkyl" and "heterocycloalkyl" by themselves or in combination with other terms refer to cyclic forms of "alkyl" and "heteroalkyl", respectively. In addition, for heterocycloalkyl, a heteroatom may occupy the position where the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophene-2-yl, tetrahydrothiophene-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. The terms "cycloalkenyl" and "heterocycloalkenyl" refer to divalent derivatives of cycloalkyl and heterocycloalkyl, respectively.

[0148] The term "alkenyl" or "alkene group" refers to a linear or branched monovalent hydrocarbon group of two to forty carbon atoms having at least one carbon-carbon sp2 double bond (e.g., C2-C6 alkenyl, and C2-C4 alkenyl), and includes groups with "cis" and "trans" orientations or "E" and "Z" orientations. Examples of alkenyl include, but are not limited to, vinyl and allyl.

[0149] The term "aryl" may be used alone or as a large part of "aralkyl", "aralkyloxy" or "aryloxyalkyl" to refer to monocyclic, bicyclic and tricyclic carbon ring systems containing a total of 6-14 ring members, wherein at least one ring system is aromatic, wherein each ring system contains 3-7 ring members and has one or more points of attachment to the rest of the molecule. The term "aryl" may be used interchangeably with the term "aromatic ring", for example, aromatic rings may include phenyl, naphthyl and anthracenyl. And the aryl group may be substituted or unsubstituted, wherein the substituent may be, but is not limited to, deuterium, hydroxyl, amino, halogen, cyano, aryl, heteroaryl, alkoxy, alkylamino, alkyl, alkenyl, alkynyl, heterocyclic, thiol, nitro, aryloxy, hydroxy-substituted alkoxy, hydroxy-substituted alkyl-C(=O)-, alkyl-C(=O)-, alkyl-S(=O)-, alkyl-S(=O)2-, hydroxy-substituted alkyl-S(=O)-, hydroxy-substituted alkyl-S(=O)2-, carboxyl-substituted alkoxy, and the like.

[0150] The term "heteroaryl" may be used alone or as a large part of "heteroarylalkyl" or "heteroarylalkoxy" to refer to monocyclic, bicyclic and tricyclic ring systems containing a total of 5-14 ring members, wherein at least one ring system is aromatic and at least one ring system contains one or more heteroatoms, wherein the heteroatoms have the meanings described in the present disclosure, wherein each ring system contains 3-7 ring members and has one or more points of attachment to the rest of the molecule.

[0151] The term "heteroaryl" can be used interchangeably with the term "aromatic heterocycle" or "heteroaromatic compound". And the heteroaryl group can be substituted or unsubstituted, wherein the substituent can be, but is not limited to, deuterium, hydroxyl, amino, halogen, cyano, aryl, heteroaryl, alkoxy, alkylamino, alkyl, alkenyl, alkynyl, heterocyclyl, mercapto, nitro, aryloxy, hydroxy-substituted alkoxy, hydroxy-substituted alkyl-C(=O)-, alkyl-C(=O)-, alkyl-S(=O)-, alkyl-S(=O)2-, hydroxy-substituted alkyl-S(=O)-, hydroxy-substituted alkyl-S(=O)2-, carboxyl-substituted alkoxy, and the like.

[0152] In other embodiments, the aromatic heterocycle includes the following monocyclic rings, but is not limited to these monocyclic rings: 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 4-methylisoxazol-5-yl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, pyrimidin-5-yl, pyridazinyl (such as 3-pyridazinyl), 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl (such as 5-tetrazolyl), triazolyl (such as 2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl, pyrazolyl (such as 2-pyrazolyl ), isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiodiazolyl, 1,3,4-thiodiazolyl, 1,2,5-thiodiazolyl, 1,3,4-thiadiazol-2-yl, pyrazinyl, pyrazin-2-yl, 1,3,5-triazinyl, benzo[d]thiazolyl oxazol-2-yl, imidazo[1,5-a]pyridin-6-yl; also includes the following bicyclic rings, but is in no way limited to these bicyclic rings: benzimidazolyl, benzofuranyl, benzothiophenyl, indolyl (such as 2-indolyl), purinyl, quinolyl (such as 2-quinolyl, 3-quinolyl, 4-quinolyl) and isoquinolyl (such as 1-isoquinolyl, 3-isoquinolyl or 4-isoquinolyl).

[0153] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be construed as limiting the present disclosure. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0154] Unless otherwise specified, the reagents used in the preparation of the compounds disclosed herein were purchased from Beijing Coupling Technology Co., Ltd. The information of the main reagents is shown in Table 1.

[0155] Table 1 Main reagents

[0156]

[0157]

[0158] Preparation of compound (1)

[0159]

[0160] The compound (1) used in the present disclosure can be obtained from the market, but it is expensive. In order to further control the production cost, the present disclosure provides a new synthesis method of compound (1).

[0161] The synthesis route of compound (1) provided in the present disclosure is as follows:

[0162]

[0163] Compound (1-1) (trans-4-(Boc-amino)cyclohexylcarboxaldehyde) is used as a starting material, and undergoes aldol condensation reaction, Cannizzaro reaction and second amino deprotection reaction in sequence to obtain compound (1).

[0164] Preparation Example 1

[0165] (1-1) Synthesis of compound (1-2)

[0166] Compound (1-1) (10.0 g, 1.0 eq) and formaldehyde aqueous solution (8.9 g, 37% by mass, 2.4 eq) were dissolved in 33 ml of methanol, and 13 ml of 45.3% by mass KOH aqueous solution was added dropwise. After the addition was completed, the reaction was stirred at 25°C for 30 minutes, and the temperature was raised to 60°C and refluxed at 60°C for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness under reduced pressure to obtain a crude white solid. A small amount of water was added to the crude product for pulping, and filtered to obtain a white solid compound (1-2) (9 g, yield 78.9%). MS-ESI (m / z) = 260 [M+H] +.

[0167] (1-2) Synthesis of Compound (1)

[0168] Compound (1-2) (9 g, 1 eq) was dissolved in 70 ml of 1,4-dioxane, and a solution of hydrogen chloride in 1,4-dioxane (45 ml, 4 M) was added, and the mixture was stirred at 25°C for 1 hour. After the reaction, the reaction solution was evaporated under reduced pressure to obtain compound (1) (6.8 g, yield 100%) as a white solid.

[0169] Therefore, in the preparation of the GalNAc derivative represented by the following formula (I) in the present disclosure, both Preparation Example 2 and Comparative Preparation Example 3 use the compound (1) prepared in Preparation Example 1.

[0170] Preparation of GalNAc derivatives represented by formula (I)

[0171] The present disclosure provides a method for synthesizing a GalNAc derivative represented by formula (I), and the specific synthesis route is as follows:

[0172]

[0173] Compound (1) (CAS No. 2408968-41-0) is used as the starting compound, and a first substitution reaction (amino protection reaction), a second substitution reaction (hydroxyl protection reaction), a first amino deprotection reaction, a condensation reaction and a phosphonylation reaction are sequentially performed to obtain a GalNAc derivative represented by formula (I).

[0174] GalNAc derivatives (I) CR01008 Preparation

[0175]

[0176] Preparation Example 2

[0177] In this preparation example, the GalNAc derivative (I) CR01008 The synthetic route is as follows:

[0178]

[0179] (2-1) Synthesis of compound (2)

[0180] Compound (1) (5.5 g, 28.1 mmol, 1.0 eq) and saturated sodium bicarbonate aqueous solution (15 ml) were added to a 1,4-dioxane (50 ml) solution, and a 1,4-dioxane solution (10 ml) of Fmoc-Cl (7.9 g, 42.1 mmol, 1.5 eq) was added dropwise under an ice bath. After the addition was complete, the mixture was stirred at 25°C for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and 50 ml of ethyl acetate and 30 ml of water were added for extraction. The organic phase was separated and dried over anhydrous sodium sulfate and filtered. The organic phase was purified by column chromatography (eluent: methanol / dichloromethane = 6 / 94, v / v) to obtain a light yellow solid compound (2) (6.7 g, yield 65%), which was used directly in the next step without purification. MS ESI (m / z) = 382 [M+H] + .

[0181] (2-2) Synthesis of compound (3)

[0182] The compound (2) (6.7 g, 17.5 mmol, 1.0 eq) prepared in step (2-1) was dissolved in 40 ml of pyridine, and DMTrCl (6.5 g, 26.25 mmol, 1.5 eq) was added under ice bath, and the mixture was reacted at 25°C for 12 hours, and quenched by adding 20 ml of methanol. After the reaction, the reaction solution was concentrated under reduced pressure, extracted with ethyl acetate three times (20 ml each time), and purified by column chromatography (eluent: ethyl acetate / petroleum ether = 28 / 72, v / v) to obtain a light yellow solid compound (3) (7.6 g, yield 65%). MS ESI (m / z) = 683 [M+H] + .

[0183] (2-3) Synthesis of compound (4)

[0184] The compound (3) (3.7 g, 5.42 mmol, 1.0 eq) prepared in step (2-2) was dissolved in 15 ml of acetonitrile, and 10 ml of tetrahydropyrrole was added, and stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by column chromatography (eluent: methanol / dichloromethane containing 1% by volume of ammonia water = 5 / 95, v / v) to obtain a white solid compound (4) (1.8 g, yield 90%). MS ESI (m / z) = 462 [M+H] + .

[0185] The liquid chromatogram of compound (4) before column chromatography normal phase purification is as follows: Figure 1 As shown, the liquid chromatogram of the purified compound (4) is as follows Figure 2 As shown in the figure, it can be seen that the purity of compound (4) changes from 70% to 95% after purification.

[0186] (2-4) Synthesis of Compound (6)

[0187] Compound (4) (3.1 g, 6.7 mmol, 1.5 eq) prepared in step (2-3), compound (5) (2 g, 4.46 mmol, 1.0 eq), and HATU (2.5 g, 6.7 mmol, 1.5 eq) were dissolved in 50 ml of N,N-dimethylformamide, and DIEA (1.7 g, 13.4 mmol, 3.0 eq) was added dropwise under ice bath, and stirred at 25°C for 3 hours. After the reaction, the reaction solution was directly purified by column chromatography (C18 column, eluent: acetonitrile / water = 36 / 64, v / v) to obtain compound (6) (3.5 g, yield 87%) as a white solid. MS ESI (m / z) = 589 [M+H] + .

[0188] The liquid chromatogram of compound (6) before column chromatography normal phase purification is as follows: Figure 3As shown, the liquid chromatogram of the purified compound (6) is as follows Figure 4 shown.

[0189] (2-5) GalNAc derivatives (I) CR01008 Synthesis

[0190] The compound (6) (3.1 g, 3.47 mmol, 1 eq) prepared in step (2-4) was dissolved in 30 ml of anhydrous dichloromethane, and then DCI (362 mg, 2.77 mmol, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.28 g, 2.42 mmol, 1.1 eq) were added respectively, and the atmosphere was replaced with nitrogen three times, and stirred at 25° C. for 3 hours. After the reaction was completed, 20 ml of saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the solution was extracted with dichloromethane three times (20 ml each time), and the organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (C18 column, eluent: acetonitrile / water=68 / 32, v / v). The fraction was vacuum dried for 12 hours to obtain a white powdery GalNAc derivative (I). CR01008 (2.73 g, yield 76.9%, purity 99%). MS ESI (m / z) = 1113 [M+Na] + .

[0191] GalNAc derivative (1) obtained in Preparation Example 2 CR01008 The H NMR spectrum is as follows Figure 5 As shown, the nuclear magnetic phosphorus spectrum is as follows Figure 6 As shown. Combined Figure 5 and Figure 6 It can be seen that the GalNAc derivative (1) was prepared in Preparation Example 2 CR01008 .

[0192] GalNAc derivative (1) obtained in Preparation Example 2 CR01008 The liquid chromatogram is as follows Figure 7 As shown. Figure 7 It can be seen that the GalNAc derivative (1) obtained in Preparation Example 2 CR01008 The purity is 99%.

[0193] Preparation Comparative Example 3

[0194] In this comparative preparation example, the GalNAc derivative (I) CR01008 The synthetic route is as follows:

[0195]

[0196] (3-1) Synthesis of Compound (7)

[0197] Compound (1) (1.8 g, 2.0 eq), compound (5) (5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq) and DIEA (N,N-diisopropylethylamine, 3.5 g, 6.0 eq) were dissolved in 15 ml of DMF, HBTU (1.9 g, 1.1 eq) was added, and the mixture was stirred at 25° C. for 3 hours in a nitrogen atmosphere. After the reaction was completed, the reaction solution was evaporated to dryness under reduced pressure and purified by reverse phase column chromatography (C18 column, eluent: acetonitrile / water=22 / 78, v / v) to obtain compound (7) (1.78 g, yield 64.4%) as a white solid. MS-ESI (m / z)=589[M+H] + .

[0198] (3-2) Synthesis of Compound (6)

[0199] The compound (7) (1.54 g, 1.0 eq) prepared in step (3-1) was dissolved in 15 ml of pyridine, the reaction system was cooled to 0°C with an ice-water bath, and DMTrCl (1.32 g, 1.5 eq) was added at 0°C, and the reaction was carried out at 25°C for 3 hours. 15 ml of methanol was added to the reaction solution to quench the reaction. After the reaction was completed, the reaction solution was evaporated under reduced pressure and purified by column chromatography (C18 column, eluent: acetonitrile / water = 60 / 40, v / v) to obtain a yellow solid compound (6) (1 g, yield 42.7%). MS-ESI (m / z) = 891 [M+H] + .

[0200] (3-3) GalNAc derivatives (I) CR01008 Synthesis

[0201] The compound (6) (1.08 g, 1.0 eq) prepared in step (3-2) was dissolved in 20 ml of anhydrous dichloromethane, and DCI (115 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (732 mg, 2.1 eq) were added respectively, and the atmosphere was replaced with nitrogen three times. The mixture was stirred and reacted at 25° C. for 2 hours. After the reaction was completed, 20 ml of saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the mixture was extracted three times with 20 ml of dichloromethane (20 ml each time). The organic phases were combined, evaporated to dryness under reduced pressure, and purified by reverse phase column chromatography (C18 column, eluent: acetonitrile / water=72 / 28, v / v). The fraction was vacuum dried for 12 hours to obtain a white powdery GalNAc derivative (I). CR01008 (1 g, yield 76.0%, purity 95%). MS-ESI (m / z) = 1091 [M+Na] + .

[0202] 1 H NMR(400MHz, DMSO-d6)δ1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,6H),1.37-1.17(m,5H),1.60-1.40(m,6H),1.68-1.62(m,1H),1.80(s,3H), 1.80(s,3H),1.92(s,3H),2.02(s,5H),2.13(s,3H),2.71(t,J=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,1H),3.36(s,1H),3.58- 3.39(m,3H),3.69-3.60(m,2H),3.75(s,7H),3.90(dt,J=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H) ,5.24(d,J=3.4Hz,1H),5.78(s,1H),6.93-6.87(m,4H),7.35-7.21(m,7H),7.44-7.37(m,2H),7.66(d,J=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0203] Combining Preparation Example 2 and Preparation Comparative Example 3, it can be seen that both Preparation Example 2 and Preparation Comparative Example 3 use Compound (1) as the starting material to synthesize GalNAc derivative (I) CR01008 , wherein, there are five steps of reaction in Preparation Example 2, and the total yield is 25.4%, and the purity is 99%; there are three steps of reaction in Preparation Comparative Example 3, and the total yield is 20.1%, and the purity is 95%. It can be seen that compared with Preparation Comparative Example 3, although the synthetic route of Preparation Example 2 is extended, the total yield and purity of Preparation Example 2 are improved.

[0204] Moreover, compared with the preparation of comparative example 3, the process route of preparation example 2 is simple and the synthesis cost is more economical.

[0205] Preparation of siRNA conjugates

[0206] Unless otherwise specified, the base composition and modification meanings disclosed herein are as follows: capital letters A, U, G, C, and T represent the base composition of the nucleotide, respectively; a lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-O-methyl-modified (2'-methoxy-modified) nucleotide; a lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; a lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond.

[0207] Preparation Example 4

[0208] (4-1) Formula (I) CR01008Z Preparation of the indicated compounds

[0209]

[0210] Formula (I) CR01008Z The synthetic route of the compound shown is as follows:

[0211]

[0212] (4-1-1) Preparation of compound (8)

[0213] Compound (6) (500 mg) was dissolved in 10 ml of dichloromethane, and succinic anhydride (112 mg), DMAP (6.8 mg) and triethylamine (226.2 mg) were added. The atmosphere was replaced with nitrogen three times and stirred at 25°C for 16 hours. After the reaction, column chromatography was performed for reverse phase purification (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound (8) (300 mg, yield 53.6%). MS-ESI (m / z) = 1013 [M+Na] + .

[0214] (4-1-2) Compound (I) CR01008Z Preparation

[0215] Compound (8) (50 mg), aminoCPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) were added to a 20 ml sample bottle and shaken for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was first washed once with 10 ml of acetonitrile (1×10 ml) and then vacuum dried. The dried filter cake, DMAP (3 mg), Cap1 (10 ml) and Cap2 (1 ml) were added to a 20 ml sample bottle and shaken for 6 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was first washed once with 10 ml of acetonitrile (1×10 ml) and then vacuum dried to obtain compound (I) CR01008Z (1.03 g, loading 20-30 μmol / g).

[0216] Wherein, Cap1 and Cap2 are capping reagents, Cap1 is a 20 volume % N-methylimidazole mixed solution in pyridine / acetonitrile, and the volume ratio of pyridine to acetonitrile is 3:5; Cap2 is a 20 volume % acetic anhydride solution in acetonitrile.

[0217] (4-2) Preparation of ligands The ligands were prepared by phosphoramidite nucleic acid solid phase synthesis using the compound (I) attached to a solid phase support.CR01008Z Initial cycle: connecting GalNAc derivatives one by one (I) CR01008 .

[0218] Each GalNAc derivative (I) is linked CR01008 All of them include four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows:

[0219] The GalNAc derivative (I) was reacted with acetonitrile. CR01008 Prepare a solution with a concentration of 0.1M.

[0220] The conditions of the deprotection reaction in each step are the same. The conditions of the deprotection reaction are: temperature of 25° C., reaction time of 70 seconds, deprotection reagent of dichloroacetic acid in dichloromethane solution (3% by volume), and the molar ratio of dichloroacetic acid to 4,4'-dimethoxytrityl protecting group on the solid phase carrier is 5:1.

[0221] The conditions of each coupling reaction are the same. The coupling reaction conditions are: temperature is 25°C, the molar ratio of the compound connected to the solid phase carrier to the compound not connected to the solid phase carrier is 1:10, the molar ratio of the compound connected to the solid phase carrier and the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is a 0.5M 5-ethylthio-1H-tetrazole acetonitrile solution, and the thio reagent is a 0.2M acetonitrile / pyridine mixed solution of hydrogenated xanthanone (the volume ratio of acetonitrile and pyridine is 1:1).

[0222] The conditions of each step of the capping reaction are the same. The conditions of the capping reaction are: temperature of 25°C; reaction time of 2 minutes; the capping reagent solution is a mixed solution of Cap1 and Cap2 at a molar ratio of 1:1, Cap1 is a pyridine / acetonitrile mixed solution of N-methylimidazole at a concentration of 20 volume%, the volume ratio of pyridine to acetonitrile is 3:5, Cap2 is an acetonitrile solution of acetic anhydride at a concentration of 20 volume%; the molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent to the compound connected to the solid phase carrier is 1:1:1.

[0223] The conditions for each step of oxidation or sulfidation reaction are the same. The conditions for the oxidation reaction are: temperature of 25°C; reaction time of 3 seconds; oxidation reagent concentration of 0.05M iodine water, the molar ratio of iodine to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 30:1; oxidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9). The conditions for the sulfidation reaction are: temperature of 25°C; reaction time of 360 seconds; sulfidation reagent concentration of 0.2M hydrogenated xanthan gum pyridine solution, the molar ratio of sulfidation reagent to the compound connected to the solid phase carrier in the coupling reaction is 4:1; sulfidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9).

[0224] The three clusters of GalNAc derivatives (I) were obtained by the above method. CR01008 (denoted as: ((I) CR01008 ×3)), whose structural formula is as follows:

[0225]

[0226] in, Stands for Controlled Porous Glass (CPG) carrier.

[0227] (4-3) Conjugation of the 3' end of the positive strand (I) CR01008 ×3) Preparation of vector-siRNA conjugates

[0228] (4-3-1) Synthesis of the positive chain (SS)

[0229] SS:5'-UmsUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmAmAm-3' (SEQ ID NO: 1)

[0230] By the method of phosphoramidite nucleic acid solid phase synthesis, using ((I) CR01008 ×3) The carrier starts the cycle, and the above nucleotide sequence is connected to the nucleoside monomers one by one in the 3'-5' direction. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows:

[0231] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0232] The conditions of the deprotection reaction in each step are the same. The conditions of the deprotection reaction are: temperature of 25° C., reaction time of 70 seconds, deprotection reagent of dichloroacetic acid in dichloromethane solution (3% by volume), and the molar ratio of dichloroacetic acid to 4,4'-dimethoxytrityl protecting group on the solid phase carrier is 5:1.

[0233] The conditions of each coupling reaction are the same. The coupling reaction conditions are: temperature is 25°C, the molar ratio of the nucleic acid sequence connected to the solid phase support to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence connected to the solid phase support to the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is an acetonitrile solution of 5-ethylthio-1H-tetrazole with a concentration of 0.5M, and the thiolation reagent is an acetonitrile / pyridine mixed solution of hydrogenated xanthanol with a concentration of 0.2M (the volume ratio of acetonitrile and pyridine is 1:1).

[0234] The conditions of each capping reaction are the same. The conditions of the capping reaction are: temperature is 25°C; reaction time is 2 minutes; the capping reagent solution is a mixed solution of Cap1 and Cap2 with a molar ratio of 1:1, Cap1 is a pyridine / acetonitrile mixed solution of N-methylimidazole with a concentration of 20 volume%, the volume ratio of pyridine to acetonitrile is 3:5, Cap2 is an acetonitrile solution of acetic anhydride with a concentration of 20 volume%; the molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent to the nucleic acid sequence connected to the solid phase carrier is 1:1:1.

[0235] The conditions for each step of the oxidation reaction are the same. The conditions for the oxidation reaction are: temperature of 25°C; reaction time of 3 seconds; oxidation reagent concentration of 0.05M iodine water, the molar ratio of iodine to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 30:1; oxidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9). The conditions for the sulfidation reaction are: temperature of 25°C; reaction time of 360 seconds; thiolation reagent concentration of 0.2M hydrogenated xanthan gum pyridine solution, the molar ratio of thiolation reagent to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 4:1; thiolation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9).

[0236] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase carrier is cut, deprotected, purified, desalted, and then freeze-dried to obtain the positive chain, wherein:

[0237] The cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence connected to the solid phase carrier is added to 25% by mass ammonia water, the amount of ammonia water is 0.5 ml / μmol, the reaction is carried out at 55°C for 16 hours, the solvent is removed, and the product is concentrated to dryness in vacuo. After the ammonia water treatment, the product is dissolved with 0.4 ml / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, and then 0.3 ml / μmol triethylamine and 0.6 ml / μmol triethylamine trihydrofluoride are added to remove the 2'-O-TBDMS protection on the ribose.

[0238] Purification and desalting conditions: The nucleic acid was purified by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent 1 was 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1); eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1); the elution gradient was eluent 1: eluent 2 = (100:0)-(50:50). The product eluates were collected and combined, and desalted using a reverse chromatography purification column. The desalting conditions included desalting using a dextran gel column, the filler was dextran gel G25, and eluted with deionized water.

[0239] Detection: Use ion exchange chromatography (IEX-HPLC) for purity detection; use liquid chromatography-mass spectrometry (LC-MS) for molecular weight detection, compare the measured value of molecular weight with the theoretical value, if the measured value ≈ the theoretical value, it indicates that ((I) CR01008 ×3) The vector is conjugated to the 3' end of the siRNA sense strand.

[0240] (4-3-2) Synthesis of antisense strand (AS)

[0241] AS:5'-UmsUfsUmAmGmAfGmUmGmAmGmGmAmUfUmAfAmAmAmsUmsGm-3' (SEQ ID NO: 2)

[0242] The antisense chain was synthesized using a universal solid phase carrier. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification and desalting conditions in the solid phase synthesis method of the antisense chain are the same as those in step (4-3-1) for synthesizing the sense chain.

[0243] Detection: Use ion exchange chromatography (IEX-HPLC) for purity detection; use liquid chromatography-mass spectrometry (LC-MS) for molecular weight detection, and compare the measured value of the molecular weight with the theoretical value. If the measured value is consistent with the theoretical value, it indicates that the siRNA antisense chain is obtained.

[0244] (4-3-3) Synthesis of siRNA conjugates

[0245] The sense chain synthesized in step (4-3-1) and the antisense chain synthesized in step (4-3-2) are mixed in an equimolar ratio, dissolved in water for injection and heated to 95°C, slowly cooled to room temperature and kept at room temperature for 10 minutes, so that the sense chain and the antisense chain form a double-stranded structure through hydrogen bonds, thereby obtaining a siRNA conjugate (conjugate RIZ) having a sense chain nucleotide sequence of SEQ ID NO: 1 and an antisense chain nucleotide sequence of SEQ ID NO: 2.

[0246] Among them, the 3' end of the positive strand is conjugated with ((I) CR01008 ×3) The structural formula of the siRNA conjugated to the vector is as follows:

[0247]

[0248] in, Represents siRNA. ((I) CR01008 ×3) The vector is conjugated to the 3' end of the siRNA sense strand via a phosphodiester bond.

[0249] Table 2 Detection results of conjugate RIZ

[0250]

[0251]

[0252] It can be seen from the data in Table 2 that the sense strand (SS) and the antisense strand (AS) can be well connected to the ligand with a high purity.

[0253] Biological detection of conjugate RIZ experiment

[0254] Example 5 Inhibitory activity of conjugate RIZ on target gene superoxide dismutase in mice

[0255] 6-8 week old C57BL / 6J mice were randomly divided into 2 groups according to their body weight, namely, a PBS control group and a conjugate RIZ experimental group, with 5 mice in each group. Each group of mice was given a PBS solution without the conjugate and a PBS solution containing the conjugate RIZ by subcutaneous administration in the abdomen. Among them, each mouse in the conjugate RIZ experimental group was given a dose of 1 mg (in terms of siRNA) / kg (mouse body weight), and the administration volume was 5 mL / kg (mouse body weight); the administration volume for each mouse in the PBS control group was 5 mL / kg (mouse body weight). The day of administration was recorded as day 1 (D1), and the mice were killed on the 8th day (D8) after administration. The animals were grossly dissected, the liver tissue was collected, and cut into several 2 mm 3 Small pieces were stored with RNA Later.

[0256] RNA extraction: Take out an appropriate amount of liver tissue samples from RNALater and break them in Tissuelyser II fully automatic tissue homogenizer for 60 seconds. Use Zhejiang Hanwei Technology Co., Ltd.'s fully automatic nucleic acid extraction instrument and nucleic acid extraction kit to extract total RNA from each liver tissue sample according to the method described in the instructions.

[0257] Reverse transcription reaction: 1 μg of total RNA from the extracted liver tissue samples was taken, and the Promega reverse transcription kit (Reverse Transcription System, A3500) was used. Oligo (dT) 15 reverse transcription primer was selected, and 20 μL reverse transcription system was prepared according to the method described in the kit manual to complete the reverse transcription reaction. After the reaction was completed, 80 μL RNase-Free water was added to the reverse transcription system to obtain the cDNA solution for Real-time PCR detection.

[0258] Real-time PCR detection: using ABI SYBR TM Select Master Mix (Catalog number: 4472908) reagent, and prepare 20 μL Real-time PCR reaction system for each PCR detection well according to the method described in the kit manual. Each detection system contains 5 μL cDNA template obtained by the above reverse transcription reaction, 10 μL SYBR TM SelectMaster Mix, 0.5μL 10μM upstream primer, 0.5μL 10μM downstream primer (primer information see Table 3), 4μL RNase-FreeH2O. The configured reaction system was placed on the ABI StepOnePlus PCR instrument, and Real-time PCR amplification was performed using the three-step method. The amplification program was 95℃ pre-denaturation for 10min, then 95℃ denaturation for 30s, 60℃ annealing for 30s, and 72℃ extension for 30s. The denaturation, annealing, and extension process were repeated for 40 cycles. After the program was completed, the gene expression difference was calculated by the ΔΔCt method.

[0259] The real-time PCR detection data of the in vivo activity experiments involved in the present disclosure all use the ΔΔCt method to perform relative quantitative calculation of the target gene mRNA in each test group. The calculation method is summarized as follows:

[0260] ΔCt(test group) = Ct(test group target gene) - Ct(test group reference gene)

[0261] ΔCt(control group) = Ct(control group target gene) - Ct(control group internal reference gene)

[0262] ΔΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0263] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0264] The mRNA expression level of the target gene in the test group was normalized based on the control group, and the remaining expression level of the target gene mRNA in the control group was defined as 100%.

[0265] Relative residual expression level of target gene mRNA in the test group = 2 -ΔΔCt (Test group) × 100%

[0266] Test group target gene mRNA inhibition rate = 100% - test group target gene mRNA relative expression level

[0267] The experimental results are shown in Table 4, indicating that on day 8, the conjugate RIZ had good activity in mice.

[0268] Table 3 Primer sequence list

[0269]

[0270] Table 4 Inhibitory activity of target genes in mice

[0271]

[0272] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0273] Although the embodiments of the present disclosure have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A method for synthesizing a GalNAc derivative represented by formula (I), characterized in that: The synthesis method comprises the following steps: S101, compound (1) and an amino protecting agent undergo a first substitution reaction to obtain compound (2); S102, the compound (2) undergoes a second substitution reaction with a hydroxyl protecting agent to obtain a compound (3); S103, the compound (3) undergoes a first amino group deprotection reaction to obtain a compound (4); S104, the compound (4) and the compound (5) undergo condensation reaction to obtain a compound (6); S105, the compound (6) and a phosphitylation agent undergo a phosphonylation reaction to obtain a GalNAc derivative represented by formula (I); Wherein, R1 represents an amino protecting group; R2 represents a hydroxyl protecting group; Each R3 is independently selected from optionally substituted C 1-6 Alkanoyl or optionally substituted C 5-12 Aroyl; L is selected from optionally substituted C 1-20 alkyl; R4 is selected from Each R 4a Independently selected from or C containing a cyano substituent 1-6 Alkoxy; wherein each R 4a ' is independently selected from optionally substituted C 1-6 alkyl.

2. The synthesis method according to claim 1, characterized in that The L is selected from optionally substituted C 1-20 Straight chain alkyl; Optionally, said L is selected from optionally substituted C 2-10 Straight chain alkyl; Optionally, the L is selected from C 1-20 Straight chain alkyl; Optionally, the L is selected from C 2-10 Straight chain alkyl; Optionally, the L is selected from 3. The synthesis method according to claim 1, characterized in that Each R3 is independently selected from Optionally, each R3 is selected from 4. The synthesis method according to claim 1, characterized in that The R2 is selected from trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytrityl; Optionally, the R2 is selected from 4,4'-dimethoxytrityl; Optionally, the hydroxy protecting agent is selected from trityl chloride, trityl bromide, 4-methyltrityl chloride, 4-methoxytrityl chloride, 4,4'-dimethoxytrityl chloride and 4,4',4"-trimethoxytrityl chloride; Optionally, the hydroxyl protecting agent is selected from 4,4'-dimethoxytrityl chloride.

5. The synthesis method according to claim 1, characterized in that The phosphitylation agent is selected from 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, bis(diisopropylamino)chlorophosphine and bis(diisopropylamino)(2-cyanoethoxy)phosphine; Optionally, the phosphitylating agent is selected from bis(diisopropylamino)(2-cyanoethoxy)phosphine; Optionally, R4 is selected from Optionally, R4 is selected from 6. The synthesis method according to claim 1, characterized in that The amino protecting agent is selected from alkoxycarbonyl amino protecting agents, acyl amino protecting agents and alkyl amino protecting agents; Optionally, the amino protecting agent is selected from alkoxycarbonyl amino protecting agents; Optionally, the structural formula of the alkoxycarbonyl amino protecting agent is Wherein R5 is selected from optionally substituted C 1-10 Alkyl, if R5 contains substituents, the substituents are independently selected from halogen, optionally substituted C 5-20 Aryl, optionally substituted C 1-6 an alkene group or an optionally substituted silane group; Optionally, the alkoxycarbonyl amino protecting agent is selected from benzyl chloroformate, tert-butyl chloroformate, 9-fluorenylmethyl chloroformate, allyl chloroformate, 2-(trimethylsilyl)ethyl chloroformate, n-decyl chloroformate, n-nonyl chloroformate, n-octyl chloroformate, 2-octyl chloroformate, n-heptyl chloroformate, n-hexyl chloroformate, 3-hexyl chloroformate, cyclohexyl chloroformate, (-)-menthyl chloroformate, n-(trimethylsilyl)ethyl chloroformate, amyl chloroformate, isoamyl chloroformate, cyclopentyl chloroformate, n-butyl chloroformate, isobutyl chloroformate, cyclobutyl chloroformate, sec-butyl chloroformate, n-propyl chloroformate, 3-chloro-n-propyl chloroformate, isopropyl chloroformate, 2,2-dimethyl n-propyl chloroformate, ethyl chloroformate, 2-methoxyethyl chloroformate, 2,2,2-trichloroethyl chloroformate, chloroethyl chloroformate, methyl chloroformate, chloromethyl chloroformate and trichloromethyl chloroformate; Optionally, the alkoxycarbonyl amino protecting agent is selected from benzyl chloroformate, tert-butyl chloroformate, 9-fluorenylmethyl chloroformate, allyl chloroformate, 2-(trimethylsilyl)ethyl chloroformate, ethyl chloroformate and methyl chloroformate; Optionally, the alkoxycarbonyl amino protecting agent is selected from 9-fluorenylmethyl chloroformate; Optionally, the R1 is selected from an alkoxycarbonyl amino protecting group, an acyl amino protecting group and an alkyl amino protecting group; Optionally, the R1 is selected from an alkoxycarbonyl-type amino protecting group; Optionally, the alkoxycarbonyl-type amino protecting group is selected from benzyloxycarbonyl, tert-butyloxycarbonyl, fluorenylmethoxycarbonyl, allyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, methoxycarbonyl and ethoxycarbonyl; Optionally, the alkoxycarbonyl-type amino protecting group is selected from fluorenylmethoxycarbonyl.

7. The synthesis method according to claim 1, characterized in that The compound (2), the compound (3), the compound (4), the compound (5), the compound (6) and the GalNAc derivative represented by formula (I) respectively have the following structures:

8. The synthesis method according to any one of claims 1 to 7, characterized in that In step S101, the molar ratio of the compound (1) to the amino protecting agent is 1:(1.2-1.7); Optionally, the amino protecting agent is added at a temperature of -5 to 5°C; Optionally, the reaction temperature of the first substitution reaction is 20 to 30°C; Optionally, the reaction solvent of the first substitution reaction is selected from 1,4-dioxane; Optionally, the first substitution reaction is carried out under stirring conditions.

9. The synthesis method according to any one of claims 1 to 7, characterized in that In step S102, the molar ratio of the compound (2) to the hydroxyl protecting agent is 1:(1.1-2); Optionally, the hydroxyl protecting agent is added at a temperature of -5 to 5°C; Optionally, the reaction temperature of the second substitution reaction is 20 to 30°C; Optionally, the reaction solvent of the second substitution reaction is selected from pyridine.

10. The synthesis method according to any one of claims 1 to 7, characterized in that: In step S103, the first amino group deprotection reaction is performed in the presence of a basic deprotecting agent; Optionally, the basic deprotecting agent is selected from tetrahydropyrrole, triethylamine and piperidine; Optionally, the basic deprotecting agent is selected from tetrahydropyrrole; Optionally, the reaction temperature of the first amino group deprotection reaction is 22 to 27°C; Optionally, the reaction solvent of the first amino group deprotection reaction is selected from acetonitrile.

11. The synthesis method according to any one of claims 1 to 7, characterized in that: In step S104, the molar ratio of the compound (4) to the compound (5) is (1.2-1.8):1; Optionally, the condensation reaction is carried out in the presence of a condensation agent; Optionally, the molar ratio of the condensing agent to the compound (5) is (1.2-1.8):1; Optionally, the condensing agent is selected from 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; Optionally, the condensing agent is selected from 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; Optionally, the condensation reaction is carried out in the presence of an organic base; Optionally, the molar ratio of the organic base to the compound (5) is (2.5-3.5):1; Optionally, the organic base is selected from triethylamine, N-methylmorpholine and diisopropylethylamine; Optionally, the organic base is selected from diisopropylethylamine; Optionally, the organic base is added at a temperature of -5 to 5°C; Optionally, the reaction temperature of the condensation reaction is 22 to 27°C; Optionally, the reaction solvent of the condensation reaction is selected from N,N-dimethylformamide and dichloromethane.

12. The synthesis method according to any one of claims 1 to 7, characterized in that: In step S105, the molar ratio of the compound (6) to the phosphitylation agent is (1.2-2):1; Optionally, the reaction temperature of the phosphonylation reaction is 20 to 30°C; Optionally, the reaction solvent of the phosphonylation reaction is selected from dichloromethane; Optionally, the phosphonylation reaction is carried out in an inert atmosphere; Optionally, the phosphonylation reaction is carried out under stirring conditions.

13. The synthesis method according to any one of claims 1 to 7, characterized in that: The preparation method of the compound (1) comprises the following steps: S201, mixing compound (1-1) and formaldehyde, and subjecting the reaction system to an aldol condensation reaction and then a Cannizzaro reaction to obtain compound (1-2); S202. The compound (1-2) undergoes a second amino deprotection reaction to obtain compound (1).

14. The synthesis method according to claim 13, characterized in that: In step S201, the molar ratio of the compound (1-1) to the formaldehyde is 1:(2-3); In step S201, the reaction system is alkaline; In step S201, the solvent in the reaction system is selected from an alcohol organic solvent; In step S201, the reaction temperature of the aldol condensation reaction is 20-30°C; In step S201, the reaction time of the aldol condensation reaction is 20 to 40 minutes; In step S201, the reaction temperature of the Cannizzaro reaction is 55-65° C.; In step S201, the reaction time of the Cannizzaro reaction is 1 to 3 hours.

15. The synthesis method according to claim 13, characterized in that: In step S202, the second amino group deprotection reaction is performed in the presence of an acidic deprotecting agent; Optionally, the acidic deprotecting agent is selected from a strong inorganic acid; Optionally, the strong inorganic acid is selected from HCl and HBr; Optionally, the reaction solvent for the second amino group deprotection reaction is selected from 1,4-dioxane; Optionally, the reaction temperature of the second amino group deprotection reaction is 20 to 30°C; Optionally, the reaction time of the second amino group deprotection reaction is 0.5 to 3 hours.

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