Glycosyl protected compounds, processes for their preparation and use

Abstract

The application discloses a glycosyl-protected compound, a preparation method and application thereof, and belongs to the field of organic synthesis. The glycosyl-protected compound is a compound shown in the formula I, or a stereoisomer, a pharmaceutically acceptable salt thereof: the protecting group of the 5' hydroxyl group provided by the compound can be removed by using an oxidizing agent, so that in the synthesis of an oligonucleotide, the oxidation step and the deprotection step are consistent in conditions, one-pot deprotection / oxidation is realized, the cycle step is shortened, and the purpose of fast synthesis of the oligonucleotide, time saving and economic cost saving is achieved.

Description

Technical Field

[0001] This invention relates to the field of nucleotide synthesis technology, and in particular to a glycosyl protected compound, its preparation method, and its application. Background Technology

[0002] The most common DNA synthesis method currently is phosphorus amide solid-phase synthesis. The phosphorus amide method generally involves single-base extension through the following four steps (…). Figure 1 (1) Deprotection: Remove the protecting group on the 5'-OH of the nucleoside molecule to expose the hydroxyl group for the next reaction. (2) Coupling: The phosphoramidite monomer reacts with the free hydroxyl group to obtain a dinucleotide with a phosphite triester bond. (3) Capping: Acetylate the unreacted nucleoside 5'-OH to avoid obtaining a nucleotide chain that does not conform to the preset sequence. (4) Oxidation: Oxidize the unstable P(III) to P(V). Repeat the four steps a certain number of times to obtain the target nucleotide chain.

[0003] In the phosphoramidite cycle, the most commonly used alcohol protecting group is the 4,4'-dimethoxytriphenylmethyl (DMT) group. Currently, the commonly used deprotection methods for the DMT group are (e.g., Figure 2 (As shown): Under acidic conditions, dichloromethane is used as a solvent to react nucleosides with protecting groups with trichloroacetic acid (TCA). This condition is a relatively fast and effective deprotection method, but it still has some problems: (1) the strong reaction conditions can damage the DNA strand; (2) this is a reversible reaction, and the DMT protecting group may recombine with the alcohol hydroxyl group. In addition, in the large-scale synthesis of nucleic acids, dichloromethane, as a chlorinated solvent, causes serious pollution to groundwater, and the post-processing is complex and the cost of harmless treatment is high. Summary of the Invention

[0004] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a glycosyl protected compound, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a compound of formula I, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0007]

[0008] Among them, R1 is selected from C1 to C2. 20 Alkyl, C1-C 20 Alkoxy; wherein the alkyl or alkoxy group is optionally unsubstituted, or is substituted by one or more R groups. a replace;

[0009] Each R2 is independently selected from deuterium, CD3, halogen, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, -C0-C5 alkylene, and -OR. 21 ;

[0010] R4, each R 21 Each of them is independently selected from hydrogen, deuterium, CD3, halogen, C1-C6 alkyl, and halogen-substituted C1-C6 alkyl;

[0011] R3 is selected from C1 to C2. 20 Alkyl, C2-C8 alkenyl, C2-C8 ynyl, 3-20 membered cycloalkyl, 4-20 membered heterocycloalkyl, 6-20 membered aryl, 5-20 membered heteroaryl, 5-20 membered bridged cycloalkyl, 5-20 membered bridged heterocycloalkyl, 5-20 membered spirocycloalkyl, 5-20 membered spiroheterocycloalkyl, 5-20 membered fused cycloalkyl, 5-20 fused heterocycloalkyl; wherein the alkyl, alkenyl, ynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, bridged cycloalkyl, bridged heterocycloalkyl, spirocycloalkyl, spiroheterocycloalkyl, fused cycloalkyl, fused heterocycloalkyl are optionally non-substituted, or are modified by one or more R b replace;

[0012] Each R b Each element is independently selected from deuterium, CD3, halogens, C1-C6 alkyl groups, halogen-substituted C1-C6 alkyl groups, -C0-C5 alkylene groups, and -OR. 21 3-20 membered cycloalkyl, 4-20 membered heterocycloalkyl, 6-20 membered aryl, 5-20 membered heteroaryl;

[0013] Indicates the presence or absence of carbon-carbon single bonds; n is a natural number representing the number of substituents.

[0014] In some embodiments of the present invention, each R2 is independently selected from deuterium, CD3, halogen, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, C1-C6 alkoxy, and halogen-substituted C1-C6 alkoxy.

[0015] In some embodiments of the present invention, R3 is selected from C1 to C2. 10 Alkyl, 3-20 membered cycloalkyl, 4-20 membered heterocycloalkyl, 6-20 membered aryl, 5-20 membered heteroaryl, 5-20 membered bridged cycloalkyl, 5-20 membered bridged heterocycloalkyl, 5-20 membered spirocycloalkyl, 5-20 membered spiroheterocycloalkyl, 5-20 membered fused cycloalkyl, 5-20 fused heterocycloalkyl; wherein, the alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, bridged cycloalkyl, bridged heterocycloalkyl, spirocycloalkyl, spiroheterocycloalkyl, fused cycloalkyl, fused heterocycloalkyl are optionally unsubstituted, or are modified by one or more R b replace.

[0016] In some embodiments of the present invention, R3 is selected from C1 to C2.10 Alkyl, 3-20 membered cycloalkyl, 6-20 membered aryl, 5-20 membered heteroaryl, 5-20 membered fused cycloalkyl, 5-20 membered heterocyclic; wherein the alkyl, cycloalkyl, aryl, heteroaryl, fused cycloalkyl, and fused heterocyclic groups are optionally unsubstituted or substituted with one or more R groups. b replace.

[0017] In some embodiments of the present invention, R3 is selected from C1 to C2. 10 Alkyl, 5-20 membered heteroaryl, 5-20 fused heterocyclic groups; wherein the alkyl, heteroaryl, or fused heterocyclic group is denoted by one or more R groups. b replace.

[0018] In some embodiments of the present invention, each R b Each is independently selected from deuterium, CD3, halogen, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, C1-C6 alkoxy, halogen-substituted C1-C6 alkoxy, 3-20 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-20 membered aryl, and 5-20 membered heteroaryl.

[0019] In some embodiments of the present invention, each R b Each is independently selected from deuterium, CD3, halogen, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, 3-20 membered cycloalkyl, 4-20 membered heterocycloalkyl, 6-20 membered aryl, and 5-20 membered heteroaryl.

[0020] In some embodiments of the present invention, n is a natural number representing the number of substituents, such as a suitable number of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0021] In some embodiments of the present invention, the compound of formula I has the structure of formula Ia, formula Ib or formula Ic:

[0022]

[0023] In some embodiments of the present invention, R3 is selected from R3.

[0024] Wherein, Ar is selected from phenyl or naphthyl; the phenyl or naphthyl group is optionally unsubstituted, or is substituted with one or more R groups. b replace.

[0025] In some embodiments of the present invention, R3 is selected from 5-20 membered heteroaryl groups and 5-20 fused heterocyclic groups; such as acridinel, benzoindolyl, carbazolel, biphenylfuranyl, phenanthrolyl, phenanthidyl, phenpyrazinyl, phenazinyl, phenothiazinyl, phenotoxazinyl, and xanthonyl; wherein the heteroaryl or fused heterocyclic group is surrounded by one or more R3 groups. b Substitution, such as phenyl, methoxyphenyl.

[0026] In some embodiments of the present invention, each R b Selected from methyl, ethyl, isopropyl, butyl, tert-butyl, methoxy, ethoxy, propoxy, methylamino, ethylamino, dimethylamino, diethylamino, benzylamino, aminomethyl, 2-aminoethyl, 3-aminopropyl, and 4-aminobutyl.

[0027] In some embodiments of the present invention, R3 is selected from...

[0028] In some embodiments of the present invention, the compound of formula I has the structure of formula Id: in, Indicates the presence or absence of carbon-carbon single bonds;

[0029] Ar is selected from phenyl or naphthyl; the phenyl or naphthyl group is optionally unsubstituted, or substituted with one or more R groups. b Substitution; n is a natural number representing the number of substituents.

[0030] In some embodiments of the present invention, the compound of formula I has the structure of formula Ie, formula If, or formula Ig:

[0031] In some embodiments of the present invention, the compound of formula I is selected from the following compounds:

[0032]

[0033]

[0034] A second aspect of the present invention provides a method for preparing the above-mentioned compound, comprising the following steps:

[0035] The compound of formula II reacts with the compound of formula III to prepare the compound of formula I;

[0036]

[0037] Where X is a halogen, such as F, Cl, Br, I; the definitions of R1, R2, R3, and R4 are as described above.

[0038] In some embodiments of the present invention, the compound of formula II reacts with the compound of formula IIIa to obtain the compound of formula Id;

[0039]

[0040] Wherein, Ar is selected from phenyl or naphthyl; the phenyl or naphthyl group is optionally unsubstituted, or is substituted with one or more R groups. b Substitution. In some embodiments of the present invention, the compound of formula IIa is reacted with the compound of formula IIIa to obtain the compound of formula Ie;

[0041]

[0042] In some embodiments of the present invention, the reaction is carried out under strongly alkaline conditions; preferably, the strong base includes at least one of sodium hydride (NaH), potassium hydride (KH), calcium hydride (CaH2), sodium amino acid (NaNH2), and potassium tert-butoxide (t-BuOK).

[0043] In this invention, the compound of formula II undergoes a nucleophilic substitution reaction with the compound of formula III to form a tertiary carbon CS bond, which is different from the CS bond containing secondary carbon. The tertiary carbon CS bond can only be synthesized under strong base conditions. In addition, the compound of formula II in this invention is halogenated.

[0044] In some embodiments of the present invention, the solvent used in the reaction includes at least one of tetrahydrofuran (THF), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), acetonitrile (MeCN), tert-butyl methyl ether (MTBE), 1,4-dioxane, ethylene glycol dimethyl ether (DME), toluene, xylene, dichloromethane (DCM), and chloroform; preferably, the solvent used in the reaction is tetrahydrofuran (THF), and its post-treatment removal operation is easy to implement.

[0045] In some embodiments of the present invention, the compound of formula IIa-1 undergoes a halogenation reaction to obtain the compound of formula IIa;

[0046]

[0047] In some embodiments of the present invention, the halogenating reagents used in the halogenation reaction include N-chlorosuccinimide (NCS), N-bromosuccinimide (NBS), N-iodosuccinimide (NIS), and benzyltriphenylphosphonium chloride (Ph3P). + CH2ClCl - At least one of the following.

[0048] In some embodiments of the present invention, the compound of formula IIa-2 undergoes an esterification reaction to obtain the compound of formula IIa-1; wherein, R4 is selected from C1 to C6 alkyl groups;

[0049]

[0050] In some embodiments of the present invention, the esterification reaction is carried out under acidic conditions; preferably, the acid includes at least one of sulfuric acid, hydrochloric acid, p-toluenesulfonic acid, phosphoric acid, and sodium bisulfate.

[0051] In some embodiments of the present invention, the compound of formula IIa-2 undergoes an esterification reaction to obtain the compound of formula IIa-1; the compound of formula IIa-1 undergoes a halogenation reaction to obtain the compound of formula IIa; the compound of formula IIa undergoes a nucleophilic reaction with the compound of formula IIIa to obtain the compound of formula Ie; the compound of formula Ie is hydrolyzed and acidified to obtain the compound of formula Ih. R4 is selected from C1 to C6 alkyl groups.

[0052] In some embodiments of the present invention, the hydrolysis and acidification is alkaline hydrolysis followed by acidification; the alkaline hydrolysis uses at least one of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium alkoxide (such as sodium methoxide, sodium ethoxide), carbonate (such as K2CO3, Na2CO3), and triethylamine; the acidification uses at least one of hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, and acetic acid.

[0053] In some embodiments of the present invention, the compound of formula IIa-2 undergoes an esterification reaction to obtain the compound of formula IIa-3; the compound of formula IIa-3 undergoes a bromination reaction to obtain the compound of formula IIa-4; the compound of formula IIa-4 undergoes a nucleophilic reaction with the compound of formula IIIa to obtain the compound of formula Ie-1; the compound of formula Ie-1 is hydrolyzed and acidified to obtain the compound of formula Ih.

[0054]

[0055] A third aspect of the invention provides a compound of formula 1, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0056]

[0057] The definitions of R1, R2, R3, n, and "" are as described above;

[0058] Base PG Selected from bases, bases with protecting groups, or their tautomers;

[0059] G is selected from hydrogen, alcohol protecting groups, R5 and R6 are each independently selected from C1 to C6 alkyl groups; R7 is a phosphate protecting group.

[0060] In some embodiments of the present invention, the compound of formula 1 has the structures of formula 1-1, formula 1-2, and formula 1-3:

[0061] Wherein, Ar is selected from phenyl or naphthyl; the phenyl or naphthyl group is optionally unsubstituted, or is substituted with one or more R groups. b replace.

[0062] In some embodiments of the present invention, the base with the protecting group is an A base with a phenylpropionyl group (Bz), a C base with an acetyl group (Ac), or a G base with an isobutyryl group (iBu).

[0063] In some embodiments of the present invention, Base PG It is selected from adenine, 7-deadenine, thymine, uracil, cytosine, guanine, 7-deadenine, or their tautomers.

[0064] In some embodiments of the present invention, Base PG Selected from

[0065] In some embodiments of the present invention, the compound of formula 1 has the following structure:

[0066]

[0067] In some embodiments of the present invention, the alcohol protecting group includes any one of acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), dimethoxytriphenylmethyl (DMT), methoxymethyl ether (MOM), methoxytriphenylmethyl (MMT), p-methoxybenzyl ether (PMB), trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl ether (TBDPS), tri-isopropylsilyloxymethyl (TOM), triphenylmethyl (triphenylmethyl, Tr), and neopentanoyl (Piv).

[0068] In some embodiments of the present invention, R5 and R6 are each independently selected from methyl, ethyl, and isopropyl.

[0069] In some embodiments of the present invention, R7 is selected from β-cyanoethyl, methyl, benzyl, allyl, 2-(p-nitrophenyl)-1-ethyl, 4-methylthio-1-butyl, 2-(N-acetyl)-amino-1-ethyl or 2-naphthylcarbamoyloxy-1-ethyl, preferably β-cyanoethyl.

[0070] A fourth aspect of the present invention provides a method for preparing the compound of Formula 1, comprising the following steps:

[0071] Compound of Formula 2 was reacted with compound of Formula Ia to prepare compound of Formula 1;

[0072]

[0073] Among them, R1, R2, R3, R4, n, "", Base PG The definition of G is as described above.

[0074] In some embodiments of the present invention, the reaction is the Mitsunobu reaction.

[0075] In some embodiments of the present invention, the Mitsunobu reaction is carried out under the action of an azo reagent and a phosphine reagent; the azo reagent includes at least one of DIAD (diisopropyl azodicarbonate), DEAD (diethyl azodicarbonate), and ADDP (dipiperidine azodicarbonate); the phosphine reagent includes at least one of triphenylphosphine (PPh3) and PBu3 (tributylphosphine).

[0076] In some embodiments of the present invention, the molar ratio of the compound of formula 2 to the compound of formula Ia is 1 to 2:1, such as 1.1:1.

[0077] In some embodiments of the present invention, the compound of formula 2 has the structure of formula 2-1.

[0078] A fifth aspect of the invention provides a compound of formula I, or a stereoisomer thereof, a pharmaceutically acceptable salt thereof, and / or the use of a compound of formula I, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, in the synthesis of oligonucleotides.

[0079] In this invention, when the compound of formula I, or its stereoisomer, or its pharmaceutically acceptable salt is used as a compound providing a nucleoside 5'-OH protecting group in oligonucleotide synthesis, it has high atom economy, simple synthesis operation, and low cost.

[0080] A sixth aspect of the present invention provides a method for preparing a solid-phase support containing an initiating nucleic acid molecule, comprising the following steps:

[0081] CPG-linker-NH2 reacts with the compound of formula 3 to obtain a solid-phase support containing the starting nucleic acid molecule;

[0082]

[0083] The definitions of R1 and R2 are as described above;

[0084] "" indicates the presence or absence of a carbon-carbon single bond;

[0085] Base PG Selected from bases, bases with protecting groups, or their tautomers;

[0086] Ar is selected from phenyl or naphthyl; the phenyl or naphthyl group is optionally unsubstituted, or substituted with one or more R groups. b replace;

[0087] n is a natural number representing the number of substituents;

[0088] The CPG-linker-NH2 is

[0089] In some embodiments of the present invention, CPG is controlled-pore glass (CPG); conventionally, the support used for solid-phase synthesis can also be polystyrene microspheres (PS beads).

[0090] In some embodiments of the invention, the reaction is carried out in the presence of a condensing agent and a base.

[0091] In some embodiments of the present invention, the condensing agent includes dicyclohexylcarbodiimide (DCC), N,N'-dicyclohexylurea-1,3-diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP).

[0092] In some embodiments of the present invention, the base includes at least one of pyridine, triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), and 4-dimethylaminopyridine (DMAP).

[0093] In some embodiments of the present invention, the reaction further includes the following post-processing steps: washing and drying. The solvent used for washing is pyridine, dichloromethane, and acetonitrile.

[0094] In some embodiments of the present invention, the method for preparing the solid-phase support containing the starting nucleic acid molecule further includes a capping step, for example, acetylation using an acetylation reagent; the acetylation reagent includes acetic anhydride, phenoxyacetic anhydride, N-methylimidazole, and dimethylpyridine; preferably a dimethylpyridine acetonitrile solution, a mixed solution of N-methylimidazole and acetic anhydride; or capping using capping reagent A and capping reagent B.

[0095] A seventh aspect of the present invention provides a method for synthesizing oligonucleotides, comprising the following steps:

[0096] Provide the compound of Formula 1, or its stereoisomers, or pharmaceutically acceptable salts thereof.

[0097] In some embodiments of the present invention, the method for synthesizing the oligonucleotide includes the following steps: providing a compound of formula 1-1, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0098] In some embodiments of the present invention, the method for synthesizing the oligonucleotide includes the following steps: providing a compound of formula 1-2, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0099] In some embodiments of the present invention, the method for synthesizing the oligonucleotide includes the following steps: providing a compound of formula 1-3, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0100] In some embodiments of the present invention, the method for synthesizing the oligonucleotide includes the following steps:

[0101] The protecting group at the 5' position of the hydroxyl group on the compound of Formula 1, or its stereoisomer, or a pharmaceutically acceptable salt thereof, is removed by an oxidizing agent. Among them, R1, R2, R3, n, The definition is as described above.

[0102] In some embodiments of the present invention, the method for synthesizing the oligonucleotide includes the following steps: removing the protecting group at the 5' position of the hydroxyl group of the compound of Formula 1, or its stereoisomer, or a pharmaceutically acceptable salt thereof, using an oxidizing agent; preferably, the oxidizing agent includes at least one of iodine (I2), hydrogen peroxide (H2O2), tert-butyl hydroperoxide (t-BuOOH), m-chloroperoxybenzoic acid (mCPBA), and iodoacetamide (NIS, N-iodosuccinimide); if the oxidizing agent is an I2 / pyridine / acetonitrile / water system, preferably a mixed solution of pyridine, acetonitrile, and water at a concentration of 0.01–0.1 mol / L.

[0103] In this invention, the protecting group at the 5' hydroxyl position of a compound of formula I, or its stereoisomers, or its pharmaceutically acceptable salts, can be removed by an oxidizing agent, so that the oxidation and deprotection steps in oligonucleotide synthesis are under the same conditions, achieving one-pot deprotection / oxidation (e.g., Figure 3 As shown in the figure, the cycle steps are shortened, achieving the goal of rapid oligonucleotide synthesis, saving time and economic costs.

[0104] In some embodiments of the present invention, the molar ratio of the compound of Formula 1 to the oxidant is 1:5 to 15, such as 1:10.

[0105] In some embodiments of the present invention, the method for synthesizing the oligonucleotide includes the following steps:

[0106] (1) Under the influence of an oxidizing agent, the solid support prepared by the method described above is subjected to the following process. The deprotection reaction is carried out to obtain

[0107] (2) Under the activator, the above-mentioned With compounds of formula 1-3 The reaction produces a triphosphite with a protecting group.

[0108] (3) Under the presence of an oxidizing agent, the phosphite triester with a protecting group is subjected to an oxidation reaction and a deprotection reaction to obtain the first extended product;

[0109] (4) Optionally, repeat steps (2) and (3) to obtain the extended product;

[0110] (5) The extended product or the first extended product reacts with the cleavage solvent to cleave CPG-linker-NH2.

[0111] In some embodiments of the present invention, in step (2), the activator is 5-ethimercaptotetrazole, 5-(p-nitrophenyl)-1H-tetrazole, trifluoroacetic acid pyridinium salt, tetrazolium, 5-benzylthio-1H-tetrazole, 4,5-dicyanimidazolium, preferably an acetonitrile solution of 0.25 mol / L 5-ethimercaptotetrazole.

[0112] In some embodiments of the present invention, step (2) further includes a capping step, for example, using capping reagent A and capping reagent B for capping.

[0113] In some embodiments of the present invention, the removal solvent is ammonia, preferably 1 mL of concentrated ammonia. Attached Figure Description

[0114] Figure 1 This is a schematic diagram of the phosphoramide method, a common method for synthesizing DNA.

[0115] Figure 2 This is a schematic diagram of the deprotection reaction of DMT as an alcohol hydroxyl protecting group.

[0116] Figure 3 This is a schematic diagram of the optimized DNA phosphorous amide synthesis method of the present invention.

[0117] Figure 4 This is the mass spectrum of compound 6aa from Example 1 of the present invention.

[0118] Figure 5 This is the mass spectrum of compound 6ab from Example 2 of the present invention.

[0119] Figure 6 This is the mass spectrum of compound 6ac from Example 3 of the present invention.

[0120] Figure 7 This is the mass spectrum of compound 6ba from Example 4 of the present invention.

[0121] Figure 8 This is the mass spectrum of compound 9a from Example 5 of the present invention.

[0122] Figure 9 This is the mass spectrum of compound 9b from Example 6 of the present invention. Detailed Implementation

[0123] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0124] Unless otherwise specified, the above groups and substituents have the general meaning in the field of medicinal chemistry.

[0125] In various parts of this specification, the substituents of the compounds disclosed herein are disclosed according to the type or scope of the groups. In particular, this invention includes every independent secondary combination of the members of these types and scopes of groups.

[0126] Additionally, it should be noted that, unless otherwise explicitly stated, the descriptive phrases “each…independently is / is selected from” and “…independently is / is selected from” used throughout this document are interchangeable and should be interpreted broadly. They can mean either that the specific options expressed by the same or different symbols in different groups do not affect each other, or that the specific options expressed by the same or different symbols in the same group do not affect each other.

[0127] "Substitution" refers to the replacement of hydrogen atoms in a molecule with other different atoms or groups.

[0128] "Multiple" refers to two or more groups. Therefore, the substitution by multiple groups described in this invention means substitution by two or more groups. The specific number of substituents is affected by the number of substituted sites and steric hindrance of the substituted group. It usually means substitution by two, three, four, five or six groups, and more preferably substitution by two or three groups.

[0129] The term "alkyl" refers to a straight-chain saturated hydrocarbon group having 1 to the specified number of carbon atoms, or a branched or cyclic saturated hydrocarbon group (such as C1-C6 alkyl) having 3 to the specified number of carbon atoms. Specific examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, tert-butyl, cyclopropylmethyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, neohexyl, and similar structures.

[0130] The term "alkylene" refers to a divalent saturated aliphatic hydrocarbon group having a specified number of carbon atoms. For example, "C a ~C b "alkylene" refers to an alkylene group having a to b carbon atoms. Alkylene groups can be straight-chain or branched. For example, "C1 to C6 alkylene" is intended to include methylene, ethylene, propylene, 2-methylpropylene, dimethyl ethylene, pentylene, etc.

[0131] The term "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group having a specified number of carbon atoms and at least one carbon-carbon double bond (>C=C<). For example, C a ~C b Alkenyl refers to an unsaturated hydrocarbon group containing an alkenyl group that has a to b carbon atoms. Specific examples of alkenyl groups include vinyl, propenyl, isopropenyl, 1,3-butadienyl, etc.

[0132] The term "alkynyl" refers to a straight-chain or branched monovalent hydrocarbon group containing at least one carbon-carbon triple bond. The term "alkynyl" is also intended to include hydrocarbon groups having one triple bond and one double bond. Specific examples of C2–C6 alkynyl groups include ethynyl and propynyl.

[0133] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0134] The term "halogen substitution" refers to the substitution of a group by one or more halogen atoms, including partial substitution and complete substitution. For example, halogen-substituted C1 to C4 alkyl groups refer to alkyl groups containing 1 to 4 carbon atoms that are substituted by one or more halogen atoms, such as trifluoromethyl, difluoromethyl, monofluoromethyl, etc.

[0135] The term "alkoxy" refers to any of the aforementioned alkyl groups (e.g., C1 to C6 alkyl groups) that are attached to the rest of the molecule by an oxygen atom (-O-).

[0136] The term "cycloalkyl" refers to a saturated or partially saturated cyclic group having a plurality of carbon atoms and no heterocyclic atoms, and having a single ring or multiple rings (including fused rings). Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclooctyl, cyclopentenyl, cyclohexenyl, and polycyclic alkyl rings, such as dicyclopropyl, dicyclohexyl, dicyclopentyl, dicyclooctyl, etc.

[0137] The term "bridged ring" refers to a saturated or partially saturated ring structure formed by two or more ring structures sharing two non-adjacent carbon atoms. Therefore, the "5-12 quinary bridged ring base" described in this invention includes both "5-12 quinary saturated bridged ring bases" and "5-12 quinary partially saturated bridged ring bases," preferably 5-10 quinary bridged ring bases. Specific examples of "bridged ring bases" include: etc. The term "bridged ring" also includes adamantane, including but not limited to the following structures.

[0138] The term "spirocyclic" refers to a saturated or partially saturated cyclic structure formed by two or more cyclic structures sharing a single ring atom and lacking cyclic heteroatoms. The "5-12 membered spirocyclic group" described in this invention includes both "5-12 membered saturated spirocyclic groups" and "5-12 membered partially saturated spirocyclic groups," with specific examples including: wait.

[0139] The term "fused-ring group" refers to a fully carbon polycyclic group in which each ring in a system shares an adjacent pair of carbon atoms with the other rings in the system, wherein one or more rings may contain one or more double bonds, but no ring has a fully conjugated π-electron system. In a particular embodiment, the fused-ring group comprises 5 to 20 (C 5-20 ), 6 to 14 (C 6-14 ) or 7 to 10 (C 7-10 (For example, 7, 8, 9, 10) carbon atoms. Depending on the number of rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused ring groups, further in one embodiment being bicyclic or tricyclic, further in another embodiment being 3-membered / 5-membered, 4-membered / 5-membered, 5-membered / 5-membered, or 5-membered / 6-membered bicyclic alkyl. In one embodiment, the fused ring group is an optionally substituted fused ring group described elsewhere herein or an fused ring group optionally fused with a heterocyclic group, aryl group, or heteroaryl group, further in one embodiment being bicyclic or tricyclic, further in another embodiment being a C3-C8 cycloalkyl 3-8-membered heterocyclic group, a C3-C8 cycloalkyl phenyl group, or a C3-C8 cycloalkyl 5-8-membered heteroaryl group, and even further in another embodiment being a C3-C6 cycloalkyl 3-6-membered heterocyclic group, a C3-C6 cycloalkyl phenyl group, or a C3-C6 cycloalkyl 5-6-membered heteroaryl group.

[0140] The term "bridged heterocycle" refers to a saturated or partially saturated cyclic structure formed by two or more ring structures sharing two non-adjacent carbon atoms, containing at least one heteroatom in the ring. The bridged heterocycle generally contains CO, N, O, S, NO, SO, or S(O)₂, preferably independently containing 1 to 3 CO, N, and / or O atoms, and more preferably independently containing 1 O and / or 1 N atom. The "5-12 membered bridged heterocycle group" of this invention includes "5-12 membered saturated bridged heterocycle group" and "5-12 membered partially saturated bridged heterocycle group," preferably 5-10 membered bridged heterocycle group, 6-8 membered bridged heterocycle group, 7-8 membered bridged heterocycle group, 6-8 membered saturated bridged heterocycle group, 6-8 membered nitrogen-containing bridged heterocycle group, 6-8 membered saturated nitrogen-containing bridged heterocycle group, etc. Specific examples of bridged heterocycles include: wait.

[0141] The term "spiroheterocycle" refers to a saturated or partially saturated cyclic structure formed by two or more cyclic structures sharing a single ring atom, containing at least one heteroatom. The heteroatom is generally selected from CO, N, O, S, NO, SO, and S(O)₂, preferably independently containing 1 to 3 CO, N, and / or O atoms, and more preferably independently containing 1 CO and 1 N atom. The "5-12 membered spiroheterocyclic group" of this invention includes "5-12 membered saturated spiroheterocyclic group" and "5-12 membered partially saturated spiroheterocyclic group," preferably 7-12 membered spiroheterocyclic group, 7-11 membered spiroheterocyclic group, 8-11 membered spiroheterocyclic group, 8-11 membered saturated spiroheterocyclic group, 9-11 membered saturated spiroheterocyclic group, 9-11 membered nitrogen-containing spiroheterocyclic group, and 9-11 membered nitrogen-containing saturated spiroheterocyclic group. Specific examples of spiroheterocycles include, but are not limited to:

[0142] wait.

[0143] The term "fused heterocyclic group" refers to a polycyclic heterocyclic group in which each ring in a system shares an adjacent pair of atoms with other rings in the system. One or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are heteroatoms selected from nitrogen, oxygen, boron, phosphorus, or sulfur, and the remaining ring atoms are carbon. In a particular embodiment, the fused heterocyclic group comprises 5 to 20 or 6 to 14 ring atoms, and in one embodiment, 7 to 10 (e.g., 7, 8, 9, 10) ring atoms. Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic groups; bicyclic or tricyclic is preferred; in one embodiment, it is a 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic group; in one embodiment, the fused heterocyclic group is optionally substituted as described elsewhere herein, or may be associated with cycloalkyl, heterocyclic, aryl, or heterocyclic groups. The fused heterocyclic group, preferably bicyclic or tricyclic, is in one embodiment a 3-8 membered heterocyclic group with a C3-C8 cycloalkyl group, a 3-8 membered heterocyclic group with a 3-8 membered heterocyclic group, a 3-8 membered heterocyclic group with a phenyl group, or a 3-8 membered heterocyclic group with a 5-8 membered heteroaryl group. In a further embodiment, it is a 3-6 membered heterocyclic group with a C3-C6 cycloalkyl group, a 3-6 membered heterocyclic group with a 3-6 membered heterocyclic group, a 3-6 membered heterocyclic group with a phenyl group, or a 3-6 membered heterocyclic group with a 5-6 membered heteroaryl group. Non-limiting examples of the fused heterocyclic group include:

[0144] The terms "aromatic ring" and "aryl" refer to an aromatic hydrocarbon group having multiple carbon atoms. Aryl groups typically comprise monocyclic, bicyclic, or tricyclic aryl groups. Furthermore, as used herein, the term "aryl" refers to an aromatic substituent that can be a single aromatic ring or a combination of multiple aromatic rings fused together. Non-limiting examples include phenyl, naphthyl, or tetrahydronaphthyl.

[0145] The term "heteroaryl" refers to an optionally substituted monocyclic, polycyclic group or ring system comprising at least one aromatic ring having one or more heteroatoms independently selected from O, S, and N. In certain embodiments, a heteroaryl comprises 5 to 20, 5 to 14, or 5 to 10 ring atoms, of which 1 to 4 are heteroatoms; in one embodiment, a heteroaryl comprises 5 or 6 ring atoms; in certain embodiments, a heteroaryl may further refer to a bicyclic, tricyclic, or tetracyclic ring, wherein at least one ring is an aromatic ring having one or more heteroatoms independently selected from O, S, and N, and the other rings may be saturated, partially unsaturated carbocyclic rings, or rings comprising one or more heteroatoms independently selected from O, S, and N. In one embodiment, the heteroaryl group is selected from heteroaryl-6-10 aryl, heteroaryl-3-10 cycloalkyl, or heteroaryl-3-10 heterocyclic groups; in a further embodiment, the heteroaryl group is selected from 5- or 6-membered heteroaryl-6-10 aryl, 5- or 6-membered heteroaryl-3-6 cycloalkyl, or 5- or 6-membered heteroaryl-3-6 heterocyclic groups, wherein the heterocyclic group is a heterocyclic group containing 1 to 3 nitrogen atoms, oxygen atoms, or sulfur atoms. Non-limiting examples include: furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrroloyl, thiadiazolyl, thiazolyl, thiophene, tetrazolyl, triazinyl, triazolyl, benzofuranyl, benzimidazolyl, benziisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiaphenyl, benzobenzenethio, benzothiaphenyl, benzotriazolyl, imidazopyridyl, imidazothiazolyl Indazinyl, indolyl, inzolyl, isobenzofuranyl, isobenzothiophenyl, isoindolyl, isoquinolinyl, naphridinyl, oxazolopyridyl, phthalazinyl, pteridinyl, purine, pyridopyridyl, pyrrolopyridyl, quinolinyl, quinoxolinyl, quinazolinyl, thiadiazopyrimidinyl, thienenopyridyl, acridineyl, benzoindolyl, carbazole, biphenylfuranyl, phenanthrololinyl, phenanthidyl, phenpyrazinyl, phenazinyl, phenthiazinyl, phenoxazinyl, xanthonyl,

[0146] The term "stereoisomer" includes enantiomers and diastereomers, as well as cis-trans isomers and tautomers.

[0147] The term "pharmaceutical acceptable" means that a drug is chemically or physically compatible with other components that make up a drug dosage form and is physiologically compatible with receptors.

[0148] The term "salt" refers to an acidic or basic salt formed by a compound or its stereoisomer with an inorganic acid, organic acid, or base, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained through separation and purification during compound preparation, or by mixing the aforementioned compound or its stereoisomer with an appropriate amount (e.g., equimolar amounts) of an acid or base. These salts may precipitate in solution and be collected by filtration, or be recovered after solvent evaporation, or be prepared by freeze-drying after reaction in an aqueous medium. The salts described in this invention can be hydrochlorides, sulfates, citrates, benzenesulfonates, hydrobromides, hydrofluoric acids, phosphates, acetates, propionates, succinates, oxalates, malates, succinates, fumarates, maleates, tartrates, or trifluoroacetates of the compound.

[0149] In the substituent structure, This indicates that the atom is a bonded atom.

[0150] In this specification, "nucleoside," as a building block of oligonucleotides, refers to a compound formed by N-glycosidization of a nucleic acid base to the 1-position of a sugar (e.g., 2-deoxyribose, ribose, 2-deoxyribose formed by the combination of the 2-carbon and 4-carbon atoms with a divalent organic group). "Sugar" in this specification also includes amino sugars with a hydroxyl group replaced by an amino group, and ribose with a 2-hydroxyl group replaced by a halogen atom. Nucleosides can be naturally occurring nucleosides or modified nucleosides such as adenosine, thymidine, guanosine, cytidine, and uridine. Modified nucleosides are not limited to these; for example, modified nucleosides in which the 3' or 5' hydroxyl groups are replaced by thiol or amino groups can be cited. Nucleoside bases can be naturally occurring bases or modified nucleoside bases such as adenine, guanine, cytosine, thymine, and uracil. The sugar moiety of a nucleoside can be naturally occurring deoxyribose or ribose, and can be in the D or L configuration.

[0151] In this specification, "nucleotide" refers to a compound obtained by combining a nucleoside with a phosphate group. It can be naturally occurring nucleotides such as adenosine triphosphate, thymidine triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine triphosphate, or modified nucleotides. The nucleoside base portion of the nucleotide can be adenine, guanine, cytosine, thymine, and uracil, or modified nucleoside bases. The sugar portion of the nucleoside can be naturally occurring deoxyribose or ribose, and can be in the D or L configuration. The phosphate portion can be, for example, thiophosphate, dithiophosphate, methylphosphonate, and methyl phosphate.

[0152] In this invention, oligonucleotides refer to compounds having a structure in which nucleoside bases, sugars, and phosphates are linked by phosphodiester bonds, including naturally occurring oligonucleotides such as 2'-deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as nucleic acids containing modified sugar moieties, modified phosphate moieties, or modified nucleoside bases. Modification of the sugar moieties includes replacing the ribose ring with a hexose, cyclopentyl, or cyclohexyl ring. Alternatively, the D-ribose ring of a naturally occurring nucleic acid may be replaced with an L-ribose ring, or the β-terminal isomer of a naturally occurring nucleic acid may be replaced with an α-terminal isomer. Oligonucleotides may also contain more than one non-basic moieties. Modified phosphate moieties include thiophosphates, dithiophosphates, methylphosphonates, and methyl phosphates. Such nucleic acid analogs are known to those skilled in the art. Oligonucleotides containing two or more of the above-mentioned compounds can be manufactured, for example, from oligonucleotides containing mixtures of deoxyribose and ribonucleosides, particularly mixtures of deoxyribonucleosides with 2'-O-substituted ribonucleosides such as 2'-O-methyl or 2'-O-methoxyethyl ribonucleosides. Examples of oligonucleotides containing mixtures of nucleosides include ribozymes.

[0153] Protecting group: As used herein, the term "protecting group" refers to a temporary substituent that prevents potentially reactive functional groups from undergoing undesirable chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and the corresponding acetals and ketals of aldehydes and ketals.

[0154] Protected hydroxyl groups are those well known in the art and included in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Watts, 3rd Edition, John Wiley & Sons, 1999, the entire contents of which are incorporated herein by reference. Examples of suitable protected hydroxyl groups also include, but are not limited to, ethers, carbonates, sulfonates, allyl ethers, silyl ethers, alkyl ethers, aralkyl ethers, and alkoxyalkyl ethers. Examples of suitable esters include formates, acetates, propionates, valerates, crotonates, and benzoates. Specific examples of suitable esters include formate, benzoylcarbamate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, methyl 4-oxovalerate, methyl 4,4-(dithioethylidene)valerate, neovalerate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate, p-benzylbenzoate, and 2,4,6-trimethylbenzoate. Examples of suitable carbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(benzenesulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonates. Examples of suitable silyl ethers include trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl ether, and other trialkylsilyl ethers. Examples of suitable alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, triphenylmethyl, tert-butyl, and allyl ethers or derivatives thereof. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, β-(trimethylsilyl)ethoxymethyl, and tetrahydropyran-2-yl ether. Examples of suitable arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2-pyridinemethyl, and 4-pyridinemethyl ethers.

[0155] The oligonucleotide synthesis method of the present invention is applicable to either liquid-phase methods (including liquid-phase methods using pseudo-solid-phase protecting groups) or solid-phase methods, preferably to liquid-phase methods (including liquid-phase methods using pseudo-solid-phase protecting groups). The synthesis method of the present invention includes both a form in which the oligonucleotide chain is extended in the direction from the 3' end to the 5' end (hereinafter sometimes simply referred to as "3'-5' synthesis") and a form in which the oligonucleotide chain is extended in the direction from the 5' end to the 3' end (hereinafter sometimes simply referred to as "5'-3' synthesis"). First, the synthesis method of the present invention as a 3'-5' synthesis will be described. The oligonucleotide may be chiral.

[0156] Example 1

[0157] In this embodiment, compound 6aa was prepared, and the specific process is as follows:

[0158]

[0159] Step 1:

[0160]

[0161] Compound 1a (18.5 g) was dissolved in 40 mL of methanol, and 3.9 mL of sulfuric acid was added. The mixture was heated to reflux for 3.5 h, after which heating was stopped and the mixture was cooled to room temperature. Ice (100 g) was added to the reaction mixture, and the mixture was extracted three times with 50 mL of diethyl ether. The organic layer was washed with water, sodium bicarbonate solution, water, and brine, and dried over sodium sulfate. The solvent was evaporated to give 20.5 g of the target product, a brown oily liquid, and intermediate 2a. The yield was greater than 99%.

[0162] Step 2:

[0163]

[0164] The crude intermediate 2a (20.5 g) obtained in step 1 was dissolved in 200 mL of ethyl acetate. 500 mg of benzoyl peroxide (BPO) was added as a free radical initiator, and the mixture was reacted with 22 g of N-bromosuccinimide (NBS) under reflux. After cooling to room temperature, the system was concentrated to 100 mL under reduced pressure. A certain proportion of ethyl acetate and n-hexane (1 / 9, 100 mL) was added, and the mixture was filtered, retaining the filtrate. The solid was washed with a mixed solvent of ethyl acetate / n-hexane, and the filtrate and washings were combined. The mother liquor was concentrated under reduced pressure, and the crude product was separated by thin-layer chromatography (TLC) with n-hexane as the mobile phase, yielding 24.6 g of a colorless oily liquid 3a. The yield was 81%.

[0165] Step 3:

[0166]

[0167] 4.6 g of compound 4a was weighed and dissolved in 70 mL of tetrahydrofuran. Sodium hydride (60%, 800 mg) was added at 0 °C, and the mixture was allowed to return to room temperature. The mixture was stirred for 30 min. Compound 3a (2.4 g) was added to the system, and the mixture was stirred for 2 h to obtain compound 5aa. 10 mL of 8 mol / L potassium hydroxide solution and 20 mL of ethanol were added to the system, and the mixture was heated to 90 °C and stirred for 24 h. The mixture was cooled to room temperature, acidified with 4 mol / L hydrochloric acid, and extracted three times with 50 mL of dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, concentrated under reduced pressure, and precipitated with toluene / n-hexane to obtain crude compound 6aa. Separation was performed by silica gel column chromatography using a mixed solvent of n-hexane, dichloromethane, and methanol as the eluent. The ratio was changed from n-hexane:dichloromethane:methanol = 3:7:0 to n-hexane:dichloromethane:methanol = 0:100:3 (by volume) to give 2.81 g of compound 6aa. The yield was 66%, and the purity was identified as 99.5% by HPLC.

[0168] The results of the proton NMR spectrum identification of compound 6aa are as follows:

[0169] 1 H NMR (400MHz, CDCl3): 1.41 (d, J = 7.2Hz, 3H), 4.73 (q, J = 7.1Hz, 1H), 7.16 (m, 8H), 7.24 (t, J = 7.6 Hz, 1H), 7.33 (d, J = 7.5Hz, 6H), 7.49 (t, J = 7.6Hz), 7.70 (d, J = 7.9Hz, 1H), 7.77 (d, J = 7.8Hz, 1H).

[0170] The mass spectrum of compound 6aa is shown below. Figure 4 As shown.

[0171] Comparative Example

[0172] In this comparative example, compound 3a and compound 4a were reacted in the presence of potassium carbonate. The specific process was as follows:

[0173]

[0174] Compound 3a (2.4 mg) and K2CO3 (2.8 mg) were added to a THF (0.5 mL) solution, followed by compound 4a (4.1 mg) at room temperature. After stirring overnight, the reaction mixture was analyzed by HPLC, which showed that no target product was formed and that compounds 3a and 4a were recovered.

[0175] Example 2

[0176] In this embodiment, a compound 6ab was prepared, and the specific process is as follows:

[0177]

[0178] The preparation method of compound 6ab is the same as that of compound 6aa in Example 1, except that in step 3, 4.6 g of compound 4b was weighed and dissolved in 70 mL of tetrahydrofuran. Sodium hydride (60%, 800 mg) was added at 0 °C, and the mixture was allowed to return to room temperature and stirred for 30 min. Compound 3a (2.4 g) was added to the system, and stirring was continued for 2 h to obtain compound 5ab. 10 mL of 8 mol / L potassium hydroxide solution and 20 mL of ethanol were added to the system, and the mixture was heated to 90 °C and stirred for 24 h. The mixture was cooled to room temperature, acidified with 4 mol / L hydrochloric acid, and extracted three times with 50 mL of dichloromethane. The organic layer was washed with brine, dried with sodium sulfate, concentrated under reduced pressure, and precipitated with toluene / n-hexane to obtain crude compound 6ab. Separation was performed by silica gel column chromatography using a mixed solvent of n-hexane, dichloromethane, and methanol as the eluent. The ratio was changed from n-hexane:dichloromethane:methanol = 3:7:0 to n-hexane:dichloromethane:methanol = 0:100:3 (by volume) to yield 2.9 g of compound 6ab. The yield was 64%, and the purity was determined to be 94% by HPLC.

[0179] The results of the proton NMR spectrum identification of compound 6ab are as follows:

[0180] 1 H NMR (400MHz, CDCl3): δ1.45(d,J=7.2Hz,3H),3.75(s,3H),4.77(q,J=7.1Hz,1H),6.66(app.d,J=3.0Hz,2H),7.11-7.21( m,J=2.6Hz,8H),7.23-7.27(m,1H),7.30-7.37(m,4H),7.47-7.52(m,1H),7.68-7.74(m,1H),7.79(dd,J=1.2,7.9Hz,1H)

[0181] The mass spectrum of compound 6ab is shown below. Figure 5 As shown.

[0182] Example 3

[0183] In this embodiment, a compound 6ac was prepared, and the specific process is as follows:

[0184]

[0185] The preparation method of compound 6ac is the same as that of compound 6aa in Example 1, except that in step 3, 4.5 g of compound 4c was weighed and dissolved in 70 mL of tetrahydrofuran. Sodium hydride (60%, 800 mg) was added at 0 °C, and the mixture was allowed to return to room temperature and stirred for 30 min. Compound 3a (2.4 g) was added to the system, and stirring was continued for 2 h to obtain compound 5ac. 10 mL of 8 mol / L potassium hydroxide solution and 20 mL of ethanol were added to the system, and the mixture was heated to 90 °C and stirred for 24 h. After cooling to room temperature, the mixture was acidified with 4 mol / L hydrochloric acid and extracted three times with 50 mL of dichloromethane. The organic layer was washed with brine, dried with sodium sulfate, concentrated under reduced pressure, and precipitated with toluene / n-hexane to obtain crude compound 6ac. Separation was performed by silica gel column chromatography using a mixed solvent of n-hexane, dichloromethane, and methanol as the eluent. The ratio was changed from n-hexane:dichloromethane:methanol = 3:7:0 to n-hexane:dichloromethane:methanol = 0:100:3 (by volume) to give 4.95 g of a yellow solid compound, 6ac. The yield was 91%, and the purity was determined to be 97% by HPLC.

[0186] The results of the proton NMR spectrum identification of compound 6ac are as follows:

[0187] 1 H NMR (400MHz, CDCl3): δ1.48 (d, J=7.2Hz, 3H), 3.75 (s, 6H), 4.77 (q, J=7.1Hz, 1H), 6.65-6.69 (m, J=3.1Hz, 4H), 7.1 1-7.23(m,J=4.7Hz,8H),7.32-7.36(m,2H),7.46-7.52(m,1H),7.71(d,J=7.8Hz,1H),7.79(dd,J=1.2,7.9Hz,1H)

[0188] The mass spectrum of compound 6ac is shown below. Figure 6 As shown.

[0189] Example 4

[0190] In this embodiment, a compound 6ba was prepared, and the specific process is as follows:

[0191]

[0192] The preparation method of compound 6ba is the same as that of compound 6aa in Example 1, except that in step 3, 4.6 g of compound 4a was weighed and dissolved in 70 mL of tetrahydrofuran. Sodium hydride (60%, 800 mg) was added at 0 °C, and the mixture was allowed to return to room temperature and stirred for 30 min. Commercially available compound 3b (2.3 g) was added to the system, and stirring was continued for 2 h to obtain compound 5ba. An 8 mol / L potassium hydroxide solution (10 mL) and ethanol (20 mL) were added to the system, and the mixture was heated to 90 °C and stirred for 24 h. After cooling to room temperature, the mixture was acidified with 4 mol / L hydrochloric acid and extracted three times with 50 mL of dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, concentrated under reduced pressure, and precipitated with toluene / n-hexane to obtain crude compound 6ba. Separation was performed by silica gel column chromatography using a mixed solvent of n-hexane, dichloromethane, and methanol as the eluent. The ratio was changed from n-hexane:dichloromethane:methanol = 3:7:0 to n-hexane:dichloromethane:methanol = 0:100:3 (by volume) to give 2.9 g of a white solid compound 6ba. The yield was 71%, and the purity was determined to be 98% by HPLC.

[0193] The results of the proton NMR spectrum identification of compound 6ba are as follows:

[0194] 1 H NMR (400MHz, CDCl3): δ3.86 (s, 2H), 6.99 (dd, J = 1.0, 7, 6Hz, 1H), 7.26-7.22 (m, 3H), 7.35-7 .31(m,7H),7.41(dt,J=1.5,7.5Hz,1H),7.53(d,J=7.3Hz,6H),8.06(dd,J=1.4,7.8Hz,1H).

[0195] The mass spectrum of compound 6ba is shown below. Figure 7 As shown.

[0196] Example 5

[0197] In this embodiment, compound 9a was prepared using compound 6aa as a protecting group. The specific process is as follows:

[0198]

[0199] Weigh 1.1 g of compound 6aa, 1.3 g of compound 7a, and 2.6 g of triphenylphosphine into a round-bottom flask, dissolve in 25 mL of tetrahydrofuran, add 2.0 g of diisopropyl azodicarbonate (DIAD) at 0 °C, and stir at room temperature for 3 h. After concentration under reduced pressure, separate by silica gel column chromatography using a mixed solvent of n-hexane, dichloromethane, and methanol, with a volume ratio of n-hexane:dichloromethane:ethyl acetate = 3:7:3. Collect the fraction containing the product and concentrate under reduced pressure. Add 5 mL of a tetrahydrofuran solution containing 10 mL of tetrahydrofuran and 1 mol / L tetrabutylammonium fluoride (TBAF), and stir at room temperature for 16 h. Concentrate the reaction mixture under reduced pressure. Separate by silica gel column chromatography using a mixed solvent of n-hexane, dichloromethane, and methanol, with the volume ratio changing from n-hexane:dichloromethane:ethyl acetate = 1:1:1 to 0:2:1. The product is 712 mg of a white solid. The yield was 44%, and the purity was determined to be 99% by HPLC.

[0200] The results of the proton NMR spectrum identification of compound 9a are as follows:

[0201] 1 H NMR (400MHz, CDCl3): δ1.37-1.42(m,3H),1.78-1.82(m,3H),2.05-2.15(m,1H),2.30-2.40(m,1H),4.07-4 .55(m,5H),6,.24-6.30(m,1H),7.10-7.20(m,10H),7.28-7.34(m,6H),7.38-7.64(m,4H),8.34(brs,1H);

[0202] LRMS(ESI):Calcd.for C 38 H 36 N₂O₆SNa(M+Na) + 671.22, found 671.21

[0203] The mass spectrum of compound 9a is shown below. Figure 8 As shown.

[0204]

[0205] 6.5 mg of compound 9a was dissolved in a mixed solvent of acetonitrile (450 μL), water (50 μL), and pyridine (500 μL). 25.4 mg of iodine was added, and the mixture was stirred at room temperature for 5 min. The reaction was monitored by HPLC, using biphenyl as an internal standard. The yield was 24%, demonstrating that this method can rapidly remove the protecting group in the phosphorusamide process.

[0206] Example 6

[0207] In this embodiment, compound 9b was prepared using compound 6ba as a protecting group compound. The specific process is as follows:

[0208]

[0209] Weigh 1.0 g of compound 6ba, 1.3 g of compound 7a, and 30 mg of DMAP into a round-bottom flask, dissolve in 25 mL of DCM, add 730 mg of EDCI at 0 °C, and stir at room temperature for 20 h. Add water to the reaction mixture and separate the organic layer. Extract the aqueous phase three times with 20 mL of dichloromethane. Wash the combined aqueous phases with saturated brine, dry with anhydrous sodium sulfate, and then concentrate under reduced pressure. Add 5 mL of a tetrahydrofuran solution of 10 mL tetrahydrofuran and 1 mol / L tetrabutylammonium fluoride (TBAF) to the resulting mixture, stir at room temperature for 16 h, and concentrate the reaction mixture under reduced pressure. Separate by silica gel column chromatography, using a mixed solvent of n-hexane, dichloromethane, and methanol, with the eluent changing from a volume ratio of n-hexane:dichloromethane:ethyl acetate = 1:1:1 to 0:2:1. The product is 560 mg of white solid. The yield is 35%, and the purity is 95% as determined by HPLC.

[0210] The results of the proton NMR spectrum identification of compound 9b are as follows:

[0211] 1 H NMR (400MHz, CDCl3): δ1.57(s,3H),1.98-2.07(m,1H),2.28-2.35(m,1H),3 .65(d,J=11.6Hz,1H),3.91(d,J=11.6Hz,1H),4.17(q,J=3.6Hz,1H),4.42-4 .49(m,2H),4.73(dd,J=3.2,12.2Hz,1H),6.28(t,J=6.4Hz,1H),6.78(d,J=7 .0Hz,1H),7.23-7.31(m,12H),7.39(d,J=7.3Hz,6H),7.75(dd,J=1.2,7.7Hz Hz,1H),9.02(brs,1H)

[0212] LRMS(ESI):Calcd.for C 37 H 34 N₂O₆SNa(M+Na) + 657.20, found 657.31

[0213] The mass spectrum of compound 9b is as follows: Figure 9 As shown.

[0214]

[0215] 6.5 mg of compound 9b was dissolved in a mixed solvent of acetonitrile (450 μL), water (50 μL), and pyridine (500 μL), and 25.4 mg of iodine was added. The mixture was stirred at room temperature for 5 min. The reaction was monitored by HPLC, using biphenyl as an internal standard. The yield was 4%. This demonstrates that this method can rapidly remove the protecting group in the phosphorusamide process.

[0216] Comparing Examples 5 and 6, it can be seen that the presence of methyl groups in the structure of the alcohol hydroxyl protecting agent compound affects the deprotection efficiency.

[0217] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A compound of formula I, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: in, R1 is selected from C1 to C1. 20 Alkyl, C1-C 20 Alkoxy; wherein the alkyl or alkoxy group is optionally unsubstituted, or is substituted by one or more R groups. a replace; Each R a Each is independently selected from deuterium, CD3, halogen, C1-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl; Each R2 is independently selected from deuterium, CD3, halogen, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, -C0-C5 alkylene, and -OR. 21 ; R4, each R 21 Each of them is independently selected from hydrogen, deuterium, CD3, halogen, C1-C6 alkyl, and halogen-substituted C1-C6 alkyl; R3 is selected from C1 to C2. 20 Alkyl, C2-C8 alkenyl, C2-C8 ynyl, 3-20 membered cycloalkyl, 4-20 membered heterocycloalkyl, 6-20 membered aryl, 5-20 membered heteroaryl, 5-20 membered bridged cycloalkyl, 5-20 membered bridged heterocycloalkyl, 5-20 membered spirocycloalkyl, 5-20 membered spiroheterocycloalkyl, 5-20 membered fused cycloalkyl, 5-20 fused heterocycloalkyl; wherein the alkyl, alkenyl, ynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, bridged cycloalkyl, bridged heterocycloalkyl, spirocycloalkyl, spiroheterocycloalkyl, fused cycloalkyl, fused heterocycloalkyl are optionally non-substituted, or are modified by one or more R b replace; Each R b Each element is independently selected from deuterium, CD3, halogens, C1-C6 alkyl groups, halogen-substituted C1-C6 alkyl groups, -C0-C5 alkylene groups, and -OR. 21 3-20 membered cycloalkyl, 4-20 membered heterocycloalkyl, 6-20 membered aryl, 5-20 membered heteroaryl; "" indicates the presence or absence of a carbon-carbon single bond; n is a natural number representing the number of substituents.

2. The compound of formula I according to claim 1, characterized in that: Each R2 is independently selected from deuterium, CD3, halogen, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, C1-C6 alkoxy, halogen-substituted C1-C6 alkoxy; and / or, R3 is selected from C1-C6 alkyl, CD3, halogen, C1-C6 alkyl, .... 10 Alkyl, 3-20 membered cycloalkyl, 4-20 membered heterocycloalkyl, 6-20 membered aryl, 5-20 membered heteroaryl, 5-20 membered bridged cycloalkyl, 5-20 membered bridged heterocycloalkyl, 5-20 membered spirocycloalkyl, 5-20 membered spiroheterocycloalkyl, 5-20 membered fused cycloalkyl, 5-20 fused heterocycloalkyl; wherein, the alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, bridged cycloalkyl, bridged heterocycloalkyl, spirocycloalkyl, spiroheterocycloalkyl, fused cycloalkyl, fused heterocycloalkyl are optionally unsubstituted, or are modified by one or more R b Replace; and / or, each R b Each is independently selected from deuterium, CD3, halogen, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, 3-20 membered cycloalkyl, 4-20 membered heterocycloalkyl, 6-20 membered aryl, and 5-20 membered heteroaryl.

3. The compound of formula I according to claim 1, characterized in that: Compounds of formula I have the structure of formula Ia, formula Ib, or formula Ic:

4. The compound of formula I according to claim 2, characterized in that: R3 is selected from Wherein, Ar is selected from phenyl or naphthyl; the phenyl or naphthyl group is optionally unsubstituted, or is substituted with one or more R groups. b replace.

5. The compound of formula I according to claim 4, characterized in that: R3 is selected from 6. The compound of formula I according to claim 1, characterized in that: Compounds of formula I have the structures of formula Id, formula Ie, formula If, or formula Ig: Where "" represents a carbon-carbon single bond that is present or absent; Ar is selected from phenyl or naphthyl; the phenyl or naphthyl group is optionally unsubstituted, or substituted with one or more R groups. b replace; n is a natural number representing the number of substituents.

7. The compound of formula I according to claim 1, characterized in that: Compounds of Formula I are selected from the following compounds:

8. A method for preparing a compound of formula I according to any one of claims 1 to 7, characterized in that, Includes the following steps: The compound of formula II reacts with the compound of formula III to prepare the compound of formula I; Wherein, X is a halogen; the definitions of R1, R2, R3, R4, "", and n are as described in any one of claims 1 to 7.

9. The method for preparing the compound of formula I according to claim 8, characterized in that: The compound of formula II reacts with the compound of formula IIIa to prepare the compound of formula Id; Ar is selected from phenyl or naphthyl; the phenyl or naphthyl group is optionally unsubstituted, or substituted with one or more R groups. b replace.

10. The method for preparing the compound of formula I according to claim 8 or 9, characterized in that: The reaction is carried out under strongly alkaline conditions; preferably, the strong base includes at least one of sodium hydride, potassium hydride, calcium hydride, sodium aminohydride, and potassium tert-butoxide.

11. The method for preparing the compound of formula I according to claim 8 or 9, characterized in that: The reaction is carried out in a solvent, which includes at least one of tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone, acetonitrile, tert-butyl methyl ether, 1,4-dioxane, ethylene glycol dimethyl ether, toluene, xylene, dichloromethane, and chloroform; preferably, the solvent used in the reaction is tetrahydrofuran.

12. The use of a compound of formula I according to any one of claims 1 to 7, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, in the synthesis of oligonucleotides.

13. A method for preparing a solid-phase support containing an initiating nucleic acid molecule, characterized in that, Includes the following steps: CPG-linker-NH2 reacts with the compound of formula 3 to obtain a solid-phase support containing the starting nucleic acid molecule; The definitions of R1, R2, "", and n are as described in any one of claims 1 to 7; Base PG Selected from bases, bases with protecting groups, or their tautomers; Ar is selected from phenyl or naphthyl; the phenyl or naphthyl group is optionally unsubstituted, or substituted with one or more R groups. b replace; The CPG-linker-NH2 is 14. A method for synthesizing oligonucleotides, characterized in that, Includes the following steps: The oxidizing agent is used to remove the protecting group at the 5' position of the hydroxyl group on the compound of formula 1, or its stereoisomer, or a pharmaceutically acceptable salt thereof; the oxidizing agent includes at least one of iodine, hydrogen peroxide, tert-butylhydrogen peroxide, m-chloroperoxybenzoic acid, and iodoacetamide. The definitions of R1, R2, "", and n are as described in any one of claims 1 to 7; Base PG Selected from bases, bases with protecting groups, or their tautomers; G is selected from hydrogen, alcohol protecting groups, R5 and R6 are each independently selected from C1 to C6 alkyl groups; R7 is a phosphate protecting group.

15. The method for synthesizing oligonucleotides according to claim 14, characterized in that: The method for synthesizing the oligonucleotides includes the following steps: (1) The solid-phase support prepared by the method for preparing the solid-phase support containing the starting nucleic acid molecule as described in claim 13 under an oxidizing agent. The deprotection reaction is carried out to obtain (2) Under the activator, the above-mentioned With compounds of formula 1-3 The reaction produces a phosphite triester with a protecting group; (3) Under the presence of an oxidizing agent, the phosphite triester with a protecting group is subjected to an oxidation reaction and a deprotection reaction to obtain the first extended product; (4) Optionally, repeat steps (2) and (3) to obtain the extended product; (5) The extended product or the first extended product is reacted with the cleavage solvent to cleave CPG-linker-NH2; Preferably, the abrasion solvent is ammonia.

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