Compound as well as synthesis method and application thereof

Abstract

The invention relates to a compound as well as a synthesis method and application thereof. The invention provides a compound as shown in a formula 9 and a preparation method of a stereoisomer, a tautomer, a hydrate and a solvate of the compound, the structure of the compound has an electron-donating group, the hydrolysis of the compound can be inhibited, and the compound can be used for being combined with dNTP to form a reversible terminator and is used for nucleic acid sequencing. The synthesis method is high in atom economy, simple and convenient to operate, low in cost and easy to operate, greatly enriches the selection range of reaction substrates, can realize higher synthesis efficiency, and is beneficial to further modification and application of methyl-2-(1-(methyldisulfonyl) ethyl) benzoate derivatives. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid sequencing, and in particular, to a compound and a synthesis method and use thereof. Background Art

[0002] Methyl 2-(1-(methyldisulfanyl)ethyl)benzoate derivatives (hereinafter referred to as SSEB) are highly efficient reversible blocking sequencing reagents, which have been widely used in sequencing services. SSEB is used to block the hydroxyl group at the 3' end during the sequencing process. After each round of sequencing, it can be easily removed and the next round of sequencing can be carried out. SSEB is a benzoic acid derivative containing a disulfide bond at the ortho position. The sequencing principle is that SSEB first forms an ester with the hydroxyl group at the 3' end. After the sequencing is completed, an excision reagent such as tris(3-hydroxypropyl)phosphine (THPP) and tris(2-carboxyethyl)phosphine (TCEP) are added. The excision reagent can cut the disulfide bond in SSEB to obtain a thiol-containing intermediate. The thiol group can easily undergo an intramolecular ester exchange reaction to obtain a five-membered ring thioester, thereby releasing the hydroxyl group at the 3' end, and then the next round of sequencing can be carried out. For convenience, under normal circumstances, the sequencing reagent is preferably prepared and used directly during sequencing. Sequencing reagents are all prepared as buffers, which are weakly alkaline. The blocking group is an ester bond, which will be partially degraded under buffer conditions, thus slightly affecting the sequencing results. Therefore, SSEB sequencing reagents are currently diluted and stored with dimethyl sulfoxide, and then prepared in buffer before use, which means that the sequencing results will be more perfect if they are prepared before use.

[0003] In order to solve the problem of poor stability of SSEB, it was found that the ester bond formed by SSEB will undergo a slow hydrolysis reaction under weak alkaline conditions, that is, a small amount of hydroxyl will attack the ester carbonyl nucleophilically. The hydrolysis product will slightly increase the runon value. In order to solve the hydrolysis problem, researchers have proposed many solutions. The main ideas include the following: (1) Increasing the electron cloud density on the benzene ring (introducing electron-donating groups on the benzene ring, such as methoxy, dimethylamino, etc.) can slow down the nucleophilic attack of the hydroxide on the ester bond; (2) Increasing the steric hindrance next to the carboxylic acid on the benzene ring (introducing alkyl, alkoxy, or halogen at this position); (3) Replacing the straight-chain alkane connected to the disulfide with a branched one (for example, replacing methyl with isopropyl or tert-butyl); (4) Increasing the alkyl group connected to the disulfide can also increase the steric hindrance; (5) Replacing the benzene ring with a five-membered heterocyclic ring can theoretically increase the electron cloud density on the ring (replacing the benzene ring with thiophene, furan, pyrrole, benzothiophene, benzofuran, or indole); (6) Introducing electron-donating groups at the ortho and meta positions of the benzene ring (benzopyran, benzo[1,4]dioxane); (7) Replacing the benzene ring with naphthalene; (8) Introducing a methylene group between the oxygen atom and the carbonyl group of the ester bond.

[0004] In the current SSEB synthesis, the main step involved is the benzylic bromination reaction. The benzylic bromination reaction generally includes the following: (1) free radical reaction, usually using 2,2'-azobisisobutyronitrile (AIBN) or benzoyl peroxide (BPO) as an initiator, carbon tetrachloride (CCl 4 ) as solvent, and N-bromosuccinimide (NBS) as bromination reagent under reflux to obtain the target product. This method is more suitable for substrates with few electron-donating substituents on the benzene ring; (2) S N2 reaction, usually a hydroxyl group is first introduced at the benzylic position, then converted into methanesulfonate or p-toluenesulfonate, and then refluxed with a bromine-containing compound (such as lithium bromide) in acetone to obtain the target product. To obtain a hydroxyl group at the benzylic position, generally a ketone or aldehyde is first introduced, and then reduced (sodium borohydride), or the aldehyde and ketone are reacted with a Grignard reagent to obtain a benzyl alcohol derivative. However, these methods are not applicable to SSEB substrates, because the intermediate of the ketone after reduction and the addition of the Grignard reagent to the aldehyde is an alkoxy anion, which is easy to undergo an ester exchange reaction with the adjacent ester group, and it is easy to obtain a lactone by-product. The last step of the SSEB synthesis is to hydrolyze the ester to obtain the final carboxylic acid product. If there are too many electron-donating groups on the benzene ring, the conventional hydrolysis conditions (lithium hydroxide or sodium hydroxide, ethanol aqueous solution, room temperature) will become very difficult and the conditions are very intense. Generally, potassium hydroxide is refluxed in a methanol aqueous solution to hydrolyze the ester bond, but because the SSEB substrate contains a disulfide bond, this functional group will break under such strong conditions and the final target product cannot be obtained. Therefore, theoretically, introducing electron-donating groups on the benzene ring can inhibit its hydrolysis, but when multiple electron-donating groups are introduced, some steps in the current synthetic route are not universal and the target product cannot be obtained. Therefore, it is urgent to design a SSEB compound with high stability and a method for obtaining improved SSEB derivatives with high atom economy, simple operation, low cost and easy operation. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. To this end, the present invention provides a compound with good stability, which is combined with dNTP as a reversible terminator for nucleic acid sequencing, and the present invention also provides a method for preparing the compound with high atom economy, simple operation, low cost and easy operation. The preparation method greatly enriches the selection range of reaction substrates, can synthesize some SSEB derivatives that are difficult to synthesize using existing technologies, and can achieve higher synthesis efficiency, which is conducive to the further modification and application of SSEB derivatives.

[0006] To this end, the first aspect of the present invention provides a compound, which is a compound represented by Formula 9, and its stereoisomers, tautomers, hydrates, and solvates:

[0007]

[0008] in:

[0009] R 1 , R 2 , R 3 , R 4 are independently selected from H, halogen, -OH, C 1 -C 10 Alkyl, C1 -C 10 Alkoxy, C 1 -C 10 Alkylamino, C 3 -C 10 Cycloalkyl;

[0010] R 5 , R 6 are independently selected from H, C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C 6 -C 20 Aryl.

[0011] The current SSEB compounds are slightly unstable. Analysis of the impurity structure produced by them shows that it is caused by the hydrolysis of the SSEB compounds. In order to solve this technical problem, the present invention provides a new SSEB compound with an electron-donating group, thereby improving the stability of the compound.

[0012] According to an embodiment of the present invention, the R 1 , R 2 , R 3 , R 4 are independently selected from H, halogen, -OH, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 1 -C 6 Alkylamino, C 3 -C 6 Cycloalkyl;

[0013] The R 5 , R 6 are independently selected from H, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, C 6 -C 10 Aryl.

[0014] According to an embodiment of the present invention, the R 1 , R 2 , R 3 , R 4 are independently selected from H, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 1 -C 6 Alkylamino, C3 -C 6 Cycloalkyl;

[0015] The R 5 , R 6 are independently selected from H, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, C 6 -C 10 Aryl.

[0016] According to an embodiment of the present invention, the R 1 , R 2 , R 3 , R 4 are independently selected from H, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 1 -C 6 Alkylamino;

[0017] The R 5 , R 6 are independently selected from C 1 -C 6 alkyl.

[0018] According to an embodiment of the present invention, the R 1 , R 2 , R 3 , R 4 are independently selected from H, C 1 -C 6 Alkoxy;

[0019] The R 5 , R 6 are independently selected from C 1 -C 6 alkyl.

[0020] According to an embodiment of the present invention, the R 1 , R 2 , R 3 , R 4 are independently selected from H, C 1 Alkoxy;

[0021] The R 5 , R 6 are independently selected from C 1 -C 2 alkyl.

[0022] According to an embodiment of the present invention, the R 1, R 2 , R 3 , R 4 are independently selected from H, C 1 Alkoxy, and R 1 , R 2 , R 3 , R 4 At least one of them is not H;

[0023] The R 5 , R 6 are independently selected from C 1 -C 2 alkyl.

[0024] According to an embodiment of the present invention, R 1 , R 3 For H, R 2 , R 4 C 1 Alkoxy;

[0025] The R 5 Methyl, R 6 It is ethyl.

[0026] The second aspect of the present invention provides a method for synthesizing the compound described in the first aspect, the method comprising:

[0027] (1) subjecting the compound represented by Formula 4 to an esterification reaction to obtain the compound represented by Formula 5;

[0028] (2) subjecting the compound represented by Formula 5 to benzyl bromination reaction to obtain the compound represented by Formula 6;

[0029] (3) contacting the compound represented by Formula 6 with thiomethanesulfonate to obtain the compound represented by Formula 7;

[0030] (4) subjecting the compound represented by Formula 7 to a reduction reaction to obtain the compound represented by Formula 8;

[0031] (5) subjecting the compound represented by Formula 8 to a hydrolysis reaction to obtain a compound represented by Formula 9;

[0032]

[0033]

[0034] The current SSEB compounds are somewhat unstable. After analyzing the impurity structure produced, it was found that it was caused by the hydrolysis of SSEB. In theory, the introduction of electron-donating groups on the benzene ring can inhibit its hydrolysis. When multiple electron-donating groups are introduced, some steps in the current synthesis route are not universal and the target product cannot be obtained. For example, it is impossible to obtain a benzyl bromination product (the current reaction conditions are to use 2,2'-azobisisobutyronitrile / N-bromosuccinimide dissolved in carbon tetrachloride and refluxed). Under this condition, the electron-rich benzene ring (such as the benzene ring has two or more methoxy groups) has a bromination reaction that occurs directly on the benzyl ring, not at the benzyl position. The present invention has found a new method that can construct a benzyl bromide product in an electron-rich system. The carboxyl group is replaced with other protecting groups (2-trimethylsilylethanol) instead of the current methyl ester. The new protecting group is easily removed in the presence of fluoride ions to obtain the target product.

[0035] According to an embodiment of the present invention, the esterification reaction in step (1) is carried out in a first solvent, wherein the compound represented by formula 4 is contacted with a first catalyst, oxalyl chloride, and 2-trimethylsilylethanol to obtain a compound represented by formula 5.

[0036] According to an embodiment of the present invention, the first solvent includes at least one selected from dichloromethane, chloroform, 1,2-dichloroethane, and 1,1-dichloroethane.

[0037] According to an embodiment of the present invention, the first catalyst includes catalyst A, catalyst B and catalyst C.

[0038] According to an embodiment of the present invention, the catalyst A comprises at least one selected from N,N-dimethylformamide, N,N-dimethylaniline, N,N-diethylformamide, and pyridine;

[0039] Catalyst B comprises at least one selected from 4-dimethylaminopyridine, pyridine, 4-pyrrolidinylpyridine, and 4-piperidinylpyridine;

[0040] The catalyst C includes at least one selected from triethylamine, tripropylamine, tributylamine, N,N-diisopropylethylamine and pyridine.

[0041] According to an embodiment of the present invention, the esterification reaction further comprises:

[0042] S1, in a first solvent, contacting the compound represented by formula 4 with a catalyst A and oxalyl chloride to obtain a mixed solution a;

[0043] S2, contacting the mixed solution a with catalyst B, catalyst C and 2-trimethylsilylethanol to obtain a compound represented by formula 5.

[0044] According to an embodiment of the present invention, step S1 is to add the compound shown in formula 4, catalyst A and oxalyl chloride to the first solvent at -5-0°C, and adjust the reaction temperature to 24-28°C to obtain the mixed solution a.

[0045] According to an embodiment of the present invention, in step S2, catalyst B, catalyst C and 2-trimethylsilyl alcohol are added to the mixed solution a at -5-3°C, and the reaction temperature is adjusted to 24-28°C to obtain the compound represented by formula 5.

[0046] According to an embodiment of the present invention, the molar ratio of the compound represented by Formula 4, oxalyl chloride and 2-trimethylsilylethanol is (1-2): (1-3): (1-3), preferably 1:1.02:1.02.

[0047] According to an embodiment of the present invention, the molar ratio of the catalyst A, the catalyst B and the catalyst C is 0.05:0.1:2.6.

[0048] According to an embodiment of the present invention, the benzylic bromination reaction in step (2) is carried out by contacting the compound represented by formula 5 with a mixed solution b and a bromine reagent in a second solvent at -80--75°C to obtain a compound represented by formula 6;

[0049] Wherein, the mixed solution b is a solution obtained by the reaction of n-butyl lithium and an organic base.

[0050] According to an embodiment of the present invention, the second solvent includes at least one selected from tetrahydrofuran, diethyl ether, and hexamethylphosphoramide.

[0051] According to an embodiment of the present invention, the organic base includes at least one selected from 2,2,6,6-tetramethylpiperidine, diisopropylamine, bis(trimethylsilyl)amine, piperidine, and tetrahydropyrrole.

[0052] According to an embodiment of the present invention, the bromine reagent includes at least one selected from 1,2-dibromotetrafluoroethane, N-bromosuccinimide, liquid bromine, and tetrabromomethane.

[0053] According to an embodiment of the present invention, the molar ratio of n-butyl lithium to the organic base is (1-2):(1-3), preferably 1.2:1.3.

[0054] According to an embodiment of the present invention, the molar ratio of the compound represented by Formula 5 to the bromine reagent is 1.0:1.3.

[0055] According to an embodiment of the present invention, step (3) further comprises: contacting the compound represented by formula 6 with a second catalyst and thiomethanesulfonate in a third solvent to obtain a compound represented by formula 7.

[0056] According to an embodiment of the present invention, the third solvent includes at least one selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, methanol, and ethanol.

[0057] According to an embodiment of the present invention, the second catalyst includes at least one selected from tetrabutylammonium bromide, tetrabutylammonium iodide, benzyltrimethylammonium bromide, octadecyltrimethylammonium bromide, and dodecyltrimethylammonium bromide.

[0058] According to an embodiment of the present invention, the thiomethanesulfonate is selected from sodium thiomethanesulfonate or potassium thiomethanesulfonate.

[0059] According to an embodiment of the present invention, the molar ratio of the compound represented by Formula 6, the second catalyst and the thiomethanesulfonate is 1.0:(0.3-0.9):(1.0-10), preferably 1.0:0.3:3.0.

[0060] According to an embodiment of the present invention, the reaction temperature of step (3) is -20-25°C, preferably -10°C.

[0061] According to an embodiment of the present invention, the reduction reaction in step (4) is carried out in a fourth solvent, wherein the compound represented by formula 7 is contacted with a third catalyst and a thiol compound to obtain a compound represented by formula 8.

[0062] According to an embodiment of the present invention, the fourth solvent includes at least one selected from the group consisting of dichloromethane, 1,2-dichloroethane, chlorobenzene, 1,1-dichloroethane, and carbon tetrachloride.

[0063] According to an embodiment of the present invention, the third catalyst includes at least one selected from triethylamine, tripropylamine, tributylamine, N,N-diisopropylethylamine, and pyridine.

[0064] According to an embodiment of the present invention, the thiol compound includes at least one selected from ethyl mercaptan, methyl mercaptan, propyl mercaptan, tert-butyl mercaptan, and thiophenol.

[0065] According to an embodiment of the present invention, the molar ratio of the compound represented by Formula 7, the third catalyst and the thiol compound is (1.0-2.0):(1.5-3.0):(1.2-2.5), preferably 1.0:1.5:1.2.

[0066] According to an embodiment of the present invention, the reaction temperature of step (4) is -20-5°C, preferably -10°C.

[0067] According to an embodiment of the present invention, the hydrolysis reaction in step (5) is carried out in a fifth solvent, and the compound represented by formula 8 is contacted with a fourth catalyst to obtain a compound represented by formula 9.

[0068] According to an embodiment of the present invention, the fifth solvent includes at least one selected from tetrahydrofuran, acetonitrile, and ethyl acetate.

[0069] According to an embodiment of the present invention, the fourth catalyst includes at least one selected from tetrabutylammonium fluoride, triethylamine hydrogen fluoride, hydrogen fluoride, and pyridine hydrogen fluoride.

[0070] According to an embodiment of the present invention, the molar ratio of the compound represented by Formula 8 to the fourth catalyst is 1:(30-60), preferably 1:30.

[0071] According to an embodiment of the present invention, the reaction temperature of step (5) is 20-70°C.

[0072] According to an embodiment of the present invention, the synthesis method further comprises: subjecting the compound represented by Formula 3 to an oxidation reaction to obtain

[0073] The compound shown in formula 4;

[0074]

[0075] According to an embodiment of the present invention, the oxidation reaction is carried out in a sixth solvent by contacting the compound represented by Formula 3 with a buffer and an oxidant to obtain the compound represented by Formula 4.

[0076] According to an embodiment of the present invention, the sixth solvent includes at least one selected from dimethyl sulfoxide, N,N-dimethylformamide, and N-methylpyrrolidone.

[0077] According to an embodiment of the present invention, the buffer is selected from sodium dihydrogen phosphate or potassium dihydrogen phosphate.

[0078] According to an embodiment of the present invention, the oxidant includes at least one selected from sodium hypochlorite, calcium hypochlorite, potassium hypochlorite, and sodium periodate.

[0079] According to an embodiment of the present invention, the molar ratio of the compound represented by Formula 3, the buffer and the oxidant is 1.0:(1.0-5.0):(1.0-6.0), preferably 1.0:2.5:2.4.

[0080] According to an embodiment of the present invention, the reaction temperature of the oxidation reaction is 0-30°C.

[0081] According to an embodiment of the present invention, the synthesis method further comprises: subjecting the compound of Formula 2 to a Vilsmeier reaction to obtain a compound of Formula 3;

[0082]

[0083] According to an embodiment of the present invention, the Vilsmeier reaction is carried out in a seventh solvent, wherein the compound represented by Formula 2 is contacted with phosphorus oxychloride to obtain the compound represented by Formula 3.

[0084] According to an embodiment of the present invention, the seventh solvent includes N,N-dimethylformamide or N-dimethylformamide dimethyl acetal.

[0085] According to an embodiment of the present invention, the Vilsmeier reaction further comprises contacting the compound of formula 2 dissolved in a seventh solvent with phosphorus oxychloride dissolved in a seventh solvent, and adjusting the reaction temperature to room temperature to obtain the compound of formula 3.

[0086] According to an embodiment of the present invention, the molar ratio of the compound represented by Formula 2 to phosphorus oxychloride is 1.0:1.3.

[0087] According to an embodiment of the present invention, the synthesis method further comprises: subjecting the compound represented by Formula 1 to a Grignard reaction to obtain

[0088] The compound shown in formula 2;

[0089]

[0090] The third aspect of the present invention provides use of the compound described in the first aspect as a reversible blocking sequencing reagent.

[0091] The compound described in the first aspect is a benzoic acid derivative containing a disulfide bond at the ortho position, which can form an ester with the hydroxyl group at the 3' end. After sequencing is completed, an excision reagent is added, such as tri(3-hydroxypropyl)phosphine, tri(2-carboxyethyl)phosphine, etc. The excision reagent can cut the disulfide bond in the compound to obtain a thiol-containing intermediate. The thiol group can easily undergo an intramolecular ester exchange reaction to obtain a five-membered ring thioester, thereby releasing the hydroxyl group at the 3' end, and then proceeding to the next round of sequencing.

[0092] The fourth aspect of the present invention provides a kit, which comprises the compound described in the first aspect.

[0093] The fifth aspect of the present invention provides use of the compound described in the first aspect or the kit described in the fourth aspect in nucleic acid sequencing.

[0094] A sixth aspect of the present invention provides a nucleic acid sequencing method, comprising:

[0095] The compound described in the first aspect is used to undergo an esterification reaction with the 3'-hydroxyl group of dNTP to obtain a reactant, and the reactant is used for sequencing.

[0096] According to an embodiment of the present invention, the nucleic acid sequencing method further comprises: removing the compound using an excision reagent to release the hydroxyl group at the 3' position of the dNTP for subsequent sequencing reaction.

[0097] According to an embodiment of the present invention, the cleavage reagent is a reagent capable of cleaving a disulfide bond contained in the compound according to any one of claims 1 to 4.

[0098] According to an embodiment of the present invention, the excision reagent includes at least one selected from tris(3-hydroxypropyl)phosphine, tris(2-carboxyethyl)phosphine, triphenylphosphine, tributylphosphine, and glutathione.

[0099] Compared with the prior art, the present invention has the following beneficial effects:

[0100] The present invention aims at the deficiencies of the prior art and provides a new compound that can solve the technical problem that the existing SSEB derivatives are easily hydrolyzed. At the same time, the present invention also provides a synthetic route for the compound, which can conveniently synthesize SSEB derivatives and has the advantages of high total yield, simple reaction conditions, convenient operation, wide range of substrate selection, etc. This method can improve the synthesis efficiency, realize the synthesis of various types of SSEB derivatives, greatly enrich the selection range of substrates, and synthesize some SSEB derivatives that are difficult to synthesize using the previous method, which is conducive to the further modification and application of SSEB derivatives.

[0101] Additional aspects and advantages of the present invention 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 invention. DETAILED DESCRIPTION

[0102] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0103] 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 invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0104] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0105] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0106] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0107] 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.

[0108] Unless otherwise stated, conventional methods within the technical scope of the art, such as mass spectrometry, NMR, IR and UV / Vis spectroscopy and pharmacological methods, are used. Unless specifically defined, the terms used herein in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and drugs and medicinal chemistry are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and in the treatment of patients. For example, the manufacturer's instructions for the use of the kit can be used, or the reaction and purification can be carried out in a manner known in the art or in accordance with the description of this application. The above techniques and methods can usually be implemented according to conventional methods well known in the art, according to the descriptions in the multiple general and more specific documents cited and discussed in this specification. In this specification, groups and substituents thereof can be selected by those skilled in the art to provide stable structural parts and compounds. When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes a chemically equivalent substituent obtained when the structural formula is written from right to left. For example, CH 2 O is equivalent to OCH 2 .

[0109] The term "stereoisomer" refers to isomers produced by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, diastereomers and conformers. The stereochemical definitions and conventions used in the present invention are generally defined in accordance with SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.

[0110] According to the selection of raw materials and methods, the compounds of the present invention can exist in the form of one of the possible isomers or their mixture, for example as a pure optical isomer, or as an isomer mixture, such as as a racemic and diastereomeric mixture, depending on the number of asymmetric carbon atoms. When describing optically active compounds, prefixes D and L or R and S are used to represent the absolute configuration of the molecule with respect to the chiral center (or multiple chiral centers) in the molecule. The prefixes D and L or (+) and (-) are symbols for the rotation of plane polarized light caused by the specified compound, wherein (-) or L represent that the compound is left-handed. The compound prefixed with (+) or D is right-handed. With respect to a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers may also be referred to as enantiomers, and the mixture of the isomers is usually referred to as a mixture of enantiomers. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process. Many geometric isomers of olefins, C=N double bonds, etc. may also exist in the compounds described herein, and all such stable isomers are contemplated in the present invention. When the compounds described herein contain olefinic double bonds, unless otherwise specified, such double bonds include both E and Z geometric isomers. If the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituents may be in the cis- or trans- configuration.

[0111] When the bonds to the chiral carbon in the formula of the present invention are depicted as straight lines, it should be understood that both the (R) and (S) configurations of the chiral carbon and the enantiomerically pure compounds and mixtures thereof produced therefrom are included within the scope of the general formula. The graphic representation of racemates or enantiomerically pure compounds herein is from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, the absolute configuration of a stereocenter is indicated by a wedge-shaped bond and a dashed bond.

[0112] Optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral preparations, or resolved using conventional techniques. Compounds of the invention containing asymmetrically substituted carbon atoms can be separated in optically active form or racemic form. Resolution of a racemic mixture of a compound can be carried out by any of a number of methods known in the art. Exemplary methods include fractional recrystallization using a chiral resolution acid that is an optically active salified organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or various optically active camphorsulfonic acids such as the D and L forms of β-camphorsulfonic acid. Other resolving agents suitable for fractional crystallization methods include α-methyl-benzylamine (e.g., S and R forms or diastereomeric pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, etc. The resolution of the racemic mixture can also be carried out by eluting on a column filled with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). High performance liquid chromatography (HPLC) can also be used to carry out supercritical fluid chromatography (SFC). The selection of specific methods and elution conditions, the selection of chromatographic columns can be selected by those skilled in the art according to the structure of the compound and the test results. Further, optically pure starting materials or reagents of known configurations can also be used to obtain any enantiomer or diastereomer of the compounds described in the present invention through stereo organic synthesis.

[0113] The term "tautomer" refers to functional group isomers resulting from the rapid movement of an atom in a molecule between two positions. The compounds of the present invention may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Prototropic tautomers arise from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium, and attempts to separate a single tautomer usually produce a mixture whose physicochemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical characteristics within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates; while in phenols, the enol form predominates. The present invention encompasses all tautomeric forms of the compounds.

[0114] The compounds of the invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound. For example, the compounds may be labeled with radioactive isotopes, such as deuterium ( 2 H), tritium ( 3 H), iodine-125( 125 I) or C-14( 14C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

[0115] The term "halogen" is to be understood as fluorine, chlorine, bromine or iodine.

[0116] The term "C 1 -C 10 “Alkyl” is understood to mean a linear or branched, saturated, monovalent hydrocarbon radical having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The alkyl radical is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc. or their isomers. In particular, the radical has 1, 2, 3, 4, 5, 6 carbon atoms (“C 1 -C 6 alkyl), for example methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, tert-butyl, more particularly, the radical having 1, 2 or 3 carbon atoms (“C 1 -C 3 alkyl"), for example methyl, ethyl, n-propyl or isopropyl.

[0117] The term "C 1 -C 10 "Alkoxy" is understood to mean -O-(C 1 -C 10 alkyl), where "C 1 -C 10 "Alkyl" has the above definition.

[0118] The term "C 1 -C 10 "Alkylamino" is understood to mean -N-(C 1 -C 10 alkyl), where "C 1 -C 10 "Alkyl" has the above definition.

[0119] The term "C 6 -C 20 "Aryl" is understood to mean a monovalent aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring having 6 to 20 carbon atoms, in particular a ring having 6 carbon atoms ("C 6aryl), such as phenyl; or biphenyl, or a ring having 9 carbon atoms ("C 9 aryl), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C 10 "aryl"), such as tetrahydronaphthyl, dihydronaphthyl or naphthyl. When the C 6 -C 20 When the aryl group is substituted, it may be monosubstituted or polysubstituted. Also, there is no limitation on the substitution position, for example, it may be substituted at the ortho position, para position or meta position.

[0120] The term "C 3 -C 10 "Cycloalkyl" is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 10 carbon atoms, including fused or bridged polycyclic ring systems, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or a bicyclic hydrocarbon ring such as a decalin ring.

[0121] The term "reversible terminator" refers to a sequencing reagent whose 3' hydroxyl end contains a group that can be cleaved by a chemical reagent. Each sequencing cycle allows only one base to be incorporated. After laser scanning, the type of polymerized base can be obtained. Subsequently, a cutting reagent is added to release the 3' sticky end and continue to polymerize nucleotides.

[0122] According to a specific embodiment of the present invention, the present invention provides a compound, which is a compound represented by Formula 9, and its stereoisomers, tautomers, hydrates, and solvates:

[0123]

[0124] in:

[0125] R 1 , R 2 , R 3 , R 4 are independently selected from H, halogen, -OH, C 1 -C 10 Alkyl, C 1 -C 10 Alkoxy, C 1 -C 10 Alkylamino, C 3 -C 10 Cycloalkyl;

[0126] R 5 , R 6 are independently selected from H, C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C6 -C 20 Aryl.

[0127] According to an embodiment of the present invention, the present invention also provides a method for preparing the aforementioned compound, specifically, a bromobenzene derivative (1) can be used as a raw material, and sequentially coupled with an alkyl derivative (step 1), an aldehyde group is added (step 2), the aldehyde is oxidized to a carboxylic acid (step 3), the carboxylic acid is protected as an ester (step 4), benzyl bromination (step 5), S of sodium methanesulfonate is added, and the reaction mixture is stirred for 2 hours. N 2 substitution (step 6), disulfide bond formation (step 7), ester hydrolysis (step 8) and post-treatment and purification to obtain the target product 9.

[0128]

[0129] Step 1: Synthesis of a key intermediate having a structure shown in Formula 2 by reacting a bromobenzene derivative (compound shown in Formula 1) with a halogenated alkane

[0130] The specific operation method is preferably as follows: 1.0 mol of bromobenzene derivative is dissolved in anhydrous tetrahydrofuran, 1.1 mol of magnesium bars and a catalytic amount of 1,2-dibromoethane are added, and the mixture is heated to reflux. After most of the magnesium bars disappear, the temperature of the reaction solution is lowered to 0°C, and 1.4 mol of iodoalkane and 0.05 mol of Li are added. 2 CuCl 4 Sequentially add to the reaction solution, keep at zero temperature for 1.5 hours. Warm to room temperature overnight. After the reaction is completed, slowly add saturated ammonium chloride to the reaction system to quench the reaction, extract with dichloromethane, evaporate the solvent, separate by silica gel column chromatography, and use a mixed solvent of dichloromethane and n-hexane as the eluent, with dichloromethane:n-hexane = 1:20 by volume, to obtain the key intermediate 2.

[0131] Step 2: Convert the Vilsmeier intermediate (compound shown in Formula 2) into the key aldehyde intermediate (compound shown in Formula 3)

[0132] The specific operation method is preferably: at 0°C, 1.3 mol of POCl 3 (phosphorus oxychloride) is added to 1 / 3-1 / 2 of the total volume of DMF (N,N-dimethylformamide) required and stirred for half an hour. The key intermediate 2 is dissolved in the remaining N,N-dimethylformamide and added to the reaction bottle, and then warmed to room temperature overnight. At 0°C, water is used to quench the reaction. 10% sodium hydroxide is used to adjust the pH to 10, stirred for 0.5h, and then concentrated hydrochloric acid is used to adjust the pH to 3. After stirring for 0.5h, ethyl acetate is extracted. After concentration, the solvent is evaporated and separated by silica gel column chromatography. The eluent is a mixed solvent of ethyl acetate and n-hexane. By volume, ethyl acetate: n-hexane = 1:20 to obtain the key intermediate 3.

[0133] Step 3: Convert the aldehyde intermediate (compound shown in Formula 3) into the key carboxylic acid intermediate (compound shown in Formula 4) by Pinnick oxidation

[0134] The specific operation method is preferably as follows: at 0°C, 1.0 mole of the compound shown in formula 3 is dissolved in DMSO (dimethyl sulfoxide), and 2.5 moles of NaH 2 PO 4 (sodium dihydrogen phosphate) (3.28 M dissolved in water) and 2.4 moles of NaClO 2 (sodium chlorite) (3.13M dissolved in water) was sequentially added to the above solution, warmed to room temperature, and the reaction was detected by TLC. Water was added to quench the reaction, extracted with ethyl acetate, concentrated, and the solvent was evaporated. The eluent was a mixed solvent of ethyl acetate and n-hexane, by volume, ethyl acetate: n-hexane = 1:2 (1% acetic acid was added), and the key intermediate 4 was obtained.

[0135] Step 4: Convert the carboxylic acid intermediate (compound shown in Formula 4) into the key ester (compound shown in Formula 5) via an acyl chloride

[0136] The specific operation method is preferably as follows: 1.0 mol of the compound shown in formula 4 is dissolved in dichloromethane, and a catalytic amount of N,N-dimethylformamide is added at 0°C, and then 1.01 mol of oxalyl chloride is slowly added to the above solution. The mixture is stirred at room temperature for 1 hour, and then the reaction solution is cooled to 0°C, and 0.5 mol of 4-dimethylaminopyridine, 2.6 mol of triethylamine, and 1.01 mol of 2-trimethylsilylethanol are added to the above reaction solution in sequence. The mixture is heated to room temperature for 1 hour, and the reaction is quenched with saturated ammonium chloride. The mixture is extracted with ethyl acetate, the solvent is evaporated, and the mixture is separated by silica gel column chromatography. The eluent is a mixed solvent of dichloromethane and n-hexane, and the volume ratio of dichloromethane: n-hexane is 1:2, to obtain the key intermediate 5.

[0137] Step 5: The key ester (compound shown in Formula 5) is converted into the key intermediate benzyl bromide (compound shown in Formula 6) by extracting hydrogen with a strong base.

[0138] The specific operation method is preferred: 1.3 mol of 2,2,6,6-tetramethylpiperidine is dissolved in tetrahydrofuran at 0°C and under argon protection, and then 1.2 mol of n-butyl lithium is added dropwise to the above solution and kept warm for half an hour. Take another reaction bottle, dissolve 1.0 mol of the key ester (the compound shown in Formula 5) in tetrahydrofuran, and cool the solution to -78°C. Under argon protection, add the above lithium reagent dropwise to the solution containing the key ester (the compound shown in Formula 5), ​​keep warm at -78°C for one hour, and then quickly add 1.3 mol of 1,2-dibromotetrafluoroethane to the reaction bottle. After the solution changes from dark red to light yellow, stir for 1.5 minutes, and immediately quench the reaction with saturated ammonium chloride. Extract with ethyl acetate, evaporate the solvent, separate by silica gel column chromatography, and the eluent is a mixed solvent of ethyl acetate and n-hexane. By volume, n-hexane: ethyl acetate = 1:20 to obtain the key benzyl bromide intermediate 6.

[0139] Step 6: Through S N 2 reaction, converting the benzyl bromide intermediate (compound shown in formula 6) into thiomethanesulfonate (compound shown in formula 7)

[0140] The specific operation method is preferably as follows: 1.0 mol of benzyl bromide intermediate (compound shown in Formula 6) is dissolved in anhydrous N,N-dimethylformamide, 0.3 mol of tetrabutylammonium bromide and 3.0 mol of sodium thiomethanesulfonate are added in sequence, and the reaction solution is heated to 50°C and stirred overnight. The reaction is quenched with water. The mixture is extracted with ethyl acetate, the solvent is evaporated, and the mixture is separated by silica gel column chromatography. The eluent is a mixed solvent of ethyl acetate and n-hexane, and n-hexane: ethyl acetate = 1:5 by volume, to obtain the key intermediate 7.

[0141] Step 7: Through S N 2 reaction, converting the mesylate intermediate (compound shown in Formula 7) into a disulfide intermediate (compound shown in Formula 8)

[0142] The specific operation method is preferably as follows: 1.0 mol of the mesylate intermediate (compound shown in Formula 7) is dissolved in dichloromethane, the solution is cooled to -10°C, 1.5 mol of triethylamine and 1.2 mol of thiol are sequentially added, and the mixture is stirred for 1 hour. The reaction is quenched with water. The mixture is extracted with ethyl acetate, the solvent is evaporated, and the mixture is separated by silica gel column chromatography, the eluent is a mixed solvent of ethyl acetate and n-hexane, and the volume ratio of n-hexane: ethyl acetate = 1:20, to obtain the key intermediate 8.

[0143] Step 8: Convert the disulfide intermediate (the compound shown in Formula 8) into a carboxylic acid product (the compound shown in Formula 9) by deprotection

[0144] The specific operation method is preferably as follows: 1.0 mole of the disulfide intermediate (the compound shown in Formula 8) is dissolved in tetrahydrofuran, 30 moles of tetrabutylammonium fluoride (1M dissolved in tetrahydrofuran) is added to the above solution, the temperature is raised to 50°C, and the mixture is stirred overnight. The pH of the reaction solution is adjusted to 2 with 1M hydrochloric acid, extracted with ethyl acetate, concentrated, and separated by silica gel column chromatography, the eluent being a mixed solvent of n-hexane and ethyl acetate, calculated by volume, n-hexane:ethyl acetate=1:4 (add 1% acetic acid), to obtain SSEB derivative 9.

[0145] The reaction solvent used in each reaction step described in the present invention is not particularly limited, and any solvent that can dissolve the starting material to a certain extent and does not inhibit the reaction is included in the present invention. In addition, many similar modifications, equivalent replacements, or solvents, solvent combinations, and different ratios of solvent combinations that are equivalent to those described in the present invention are all considered to be included in the scope of the present invention.

[0146] Example 1 Synthesis of Compound 9a

[0147]

[0148] The specific steps are as follows:

[0149] Step 1:

[0150]

[0151] In a 100mL round-bottom flask equipped with a magnetic stirrer, add anhydrous tetrahydrofuran, 3,5-dimethoxybromobenzene (5.0g, 23.0mmol), 1,2-dibromoethane (0.2mL, catalytic amount), and magnesium stick (615mg, 25.3mmol). Heat to reflux for 6 hours under argon protection. Lower the reaction temperature to 0℃, then add iodoethane (2.59mL, 32.25mmol) and Li 2 CuCl 4 (11.5mL, 1.15mmol) were sequentially added to the reaction solution and kept at zero temperature for 1.5h. The temperature was raised to room temperature overnight. After the reaction was completed, saturated ammonium chloride was slowly added to the reaction system to quench the reaction, extracted with dichloromethane, the solvent was evaporated, and separated by silica gel column chromatography. The eluent was a mixed solvent of dichloromethane and n-hexane, with a volume ratio of dichloromethane: n-hexane = 1:20, to obtain 3.16g of the target product with a yield of 82.6%.

[0152] Step 2:

[0153]

[0154] Under argon protection, add anhydrous N,N-dimethylformamide (5mL) to a 50mL round-bottom flask equipped with a magnetic stirrer. At 0°C, phosphorus oxychloride (2.3mL, 24.7mmol) was slowly added dropwise to the reaction solution. After the addition was complete, the reaction was continued at the same temperature for 0.5 hours. Intermediate 2a (3.16g, 19mmol) was dissolved in 5.0mL anhydrous N,N-dimethylformamide and slowly added dropwise to the above reaction solution. After the addition was complete, the reaction slowly warmed to room temperature and stirred overnight at room temperature. Cool to 0°C and add 10mL of ice water to the reaction flask to quench the reaction. After adjusting the pH to 8-9 with 1M sodium hydroxide, 15mL of ethyl acetate was added, the organic layer was separated, the aqueous layer was extracted three times with ethyl acetate, the organic layers were combined, washed once with 20mL of saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation, and the residue was separated by silica gel column chromatography. The eluent was a mixed solvent of ethyl acetate and n-hexane, with ethyl acetate:n-hexane = 1:20 by volume, to obtain 3.09 g of the target product with a yield of 84%.

[0155] Step 3:

[0156]

[0157] Under argon protection, DMSO and intermediate 3a (2.71 g, 13.99 mmol) were added to a 100 mL round-bottom flask equipped with a magnetic stirrer. 2 PO 4 (sodium dihydrogen phosphate) (4.2 g, 35 mmol, dissolved in 10 mL water) and NaClO 2 (Sodium chlorite) (3.8 g, 33.58 mmol, dissolved in 10 mL of water) was slowly added dropwise to the reaction solution. After the addition was completed, the reaction was continued at the same temperature for 0.5 hours. The mixture was warmed to room temperature and the reaction was detected by TLC. Water was added to quench the reaction, and the mixture was extracted with ethyl acetate. After concentration, the solvent was evaporated and separated by silica gel column chromatography. The eluent was a mixed solvent of ethyl acetate and n-hexane, and by volume, ethyl acetate: n-hexane = 1:2 (1% acetic acid was added), and 2.73 g of the key intermediate 4a was obtained with a yield of 92.9%.

[0158] Step 4:

[0159]

[0160] Under argon protection, intermediate 4a (3.35 g, 13.99 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stirrer, and anhydrous dichloromethane (50 mL) and anhydrous N,N-dimethylformamide (5 mL) were added to dissolve. The reaction flask was cooled to 0 °C and (COCl) 2(Oxalyl chloride) (1.44mL, 16.79mmol) was slowly added dropwise to the reaction solution. After the addition was completed, the mixture was heated to room temperature and the reaction was continued for 1 hour. The reaction solution was cooled to 0°C, and DMAP (4-dimethylaminopyridine) (342mg, 2.8mmol), triethylamine (5.1mL, 36.4mmol) and 2-trimethylsilylethanol (2.1mL, 14.68mmol) were added in sequence, and the mixture was heated to room temperature and reacted for 1 hour. Saturated ammonium chloride was added to quench the reaction, and the mixture was extracted with dichloromethane. After concentration, the solvent was evaporated and separated by silica gel column chromatography. The eluent was a mixed solvent of dichloromethane and n-hexane. By volume, dichloromethane: n-hexane = 1:2, and 4.7g of the key intermediate 5a was obtained with a yield of 87.3%.

[0161] Step 5:

[0162]

[0163] Under argon protection, 2,2,6,6-tetramethylpiperidine (2.17 mL, 12.75 mmol) and anhydrous tetrahydrofuran (75 mL) were added to a 100 mL round-bottom flask equipped with a magnetic stirrer. The reaction flask was cooled to 0°C, and n-BuLi (n-butyllithium) (4.5 mL, 11.25 mmol) was slowly added dropwise to the reaction solution. After the addition was completed, the reaction was continued for 0.5 hours while keeping warm. In another reaction bottle, the intermediate 5a (2.32 g, 7.5 mmol) was dissolved in anhydrous tetrahydrofuran (16 mL), and the solution was cooled to -78 ° C. Under Ar protection, the above lithium reagent was added dropwise to the solution containing the intermediate 5a. After keeping at -78 ° C for one hour, 1,2-dibromotetrafluoroethane (1.88 mL, 15.75 mmol) was quickly added to the reaction bottle. After the solution changed from dark red to light yellow, it was stirred for 1.5 minutes and immediately quenched with saturated ammonium chloride (40 mL). Extracted with ethyl acetate, the solvent was evaporated, and separated by silica gel column chromatography. The eluent was a mixed solvent of ethyl acetate and n-hexane. By volume, n-hexane: ethyl acetate = 1:20, and the key benzyl bromide intermediate 6a 1.94 g was obtained with a yield of 66.5%.

[0164] Step 6:

[0165]

[0166] Add benzyl bromide intermediate 6a (1.58 g, 4.06 mmol) and N,N-dimethylformamide (20 mL) to a 50 mL round-bottom flask equipped with a magnetic stirrer, add tetrabutylammonium bromide (262 mg, 0.81 mmol) and sodium thiomethanesulfonate (1.63 g, 12.1 mmol) in sequence, and heat the reaction solution to 50 ° C and stir overnight. Quench the reaction with 40 mL of water. Extract with ethyl acetate, evaporate the solvent, separate by silica gel column chromatography, the eluent is a mixed solvent of ethyl acetate and n-hexane, calculated by volume, n-hexane: ethyl acetate = 1:5, and obtain 866 mg of the key intermediate 7a, with a yield of 50.8%.

[0167] Step 7:

[0168]

[0169] Add intermediate 7a (696 mg, 1.65 mmol) and anhydrous dichloromethane (8 mL) to a 25 mL round-bottom flask equipped with a magnetic stirrer, cool to -10 °C, add triethylamine (0.34 mL, 2.47 mmol) and ethanethiol (0.15 mL, 2.15 mmol) in sequence, and stir the reaction solution for 1 hour. Quench the reaction with 10 mL of water. Extract with ethyl acetate, evaporate the solvent, separate by silica gel column chromatography, the eluent is a mixed solvent of ethyl acetate and n-hexane, calculated by volume, n-hexane: ethyl acetate = 1:20, and obtain 632 mg of the key intermediate 8a, with a yield of 95%.

[0170] Step 8:

[0171]

[0172] Add intermediate 8a (632 mg, 1.57 mmol) and tetrahydrofuran to a 100 mL round-bottom flask equipped with a magnetic stirrer. Under argon protection, add tetrabutylammonium fluoride (62 mL, 62 mmol, 1 M dissolved in tetrahydrofuran) to the reaction flask, heat to 50 ° C, and stir overnight. Remove most of the volatile substances by rotary evaporation, and quench the reaction with 50 mL of water. Adjust the pH of the solution to 2-3 with 1 M hydrochloric acid, extract with ethyl acetate, evaporate the solvent, and separate by silica gel column chromatography. The eluent is a mixed solvent of ethyl acetate and n-hexane. By volume, n-hexane: ethyl acetate = 1:4 (add 1% acetic acid), and obtain 335 mg of the target product 9a with a yield of 70.6%.

[0173] Example 2 Synthesis of Compound 9b

[0174]

[0175] The specific steps are as follows:

[0176] Step 1:

[0177]

[0178] In a 100mL round-bottom flask equipped with a magnetic stirrer, add anhydrous tetrahydrofuran, 3-methoxybromobenzene (5.61g, 30.0mmol), 1,2-dibromoethane (0.2mL, catalytic amount), and magnesium stick (792mg, 33mmol). Heat to reflux for 6 hours under argon protection. Lower the reaction temperature to 0℃, then add iodoethane (3.38mL, 42mmol) and Li 2 CuCl 4 (15mL, 1.5mmol) were sequentially added to the reaction solution and kept at zero temperature for 1.5h. The temperature was raised to room temperature overnight. After the reaction was completed, saturated ammonium chloride was slowly added to the reaction system to quench the reaction, extracted with dichloromethane, and the solvent was evaporated. The mixture was separated by silica gel column chromatography. The eluent was a mixed solvent of dichloromethane and n-hexane. By volume, dichloromethane:n-hexane = 1:20, and 3.31g of the target product 2b was obtained with a yield of 81.2%.

[0179] Step 2:

[0180]

[0181] Under argon protection, add anhydrous N,N-dimethylformamide (5mL) to a 50mL round-bottom flask equipped with a magnetic stirrer. At 0°C, phosphorus oxychloride (2.42mL, 26mmol) was slowly added dropwise to the reaction solution. After the addition was complete, the reaction was continued at the same temperature for 0.5 hours. Intermediate 2b (2.72g, 20mmol) was dissolved in 5.2mL anhydrous N,N-dimethylformamide and slowly added dropwise to the above reaction solution. After the addition was complete, the reaction slowly warmed to room temperature and stirred overnight at room temperature. Cool to 0°C and add 10mL of ice water to the reaction flask to quench the reaction. After adjusting the pH to 8-9 with 1M sodium hydroxide, 15mL of ethyl acetate was added, the organic layer was separated, the aqueous layer was extracted three times with ethyl acetate, the organic layers were combined, washed once with 20mL of saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation, and the residue was separated by silica gel column chromatography. The eluent was a mixed solvent of ethyl acetate and n-hexane, with ethyl acetate:n-hexane = 1:20 by volume, to obtain 2.78 g of the target product with a yield of 85%.

[0182] Step 3:

[0183]

[0184] Under argon protection, DMSO and intermediate 3b (2.46 g, 15 mmol) were added to a 100 mL round-bottom flask equipped with a magnetic stirrer. 2 PO 4(sodium dihydrogen phosphate) (4.5 g, 37.5 mmol, dissolved in 12 mL water) and NaClO 2 (Sodium chlorite) (3.26 g, 36 mmol, dissolved in 12 mL of water) was slowly added dropwise to the reaction solution. After the addition was completed, the reaction was continued at the same temperature for 0.5 hours. The mixture was warmed to room temperature and the reaction was detected by TLC. Water was added to quench the reaction, and the mixture was extracted with ethyl acetate. After concentration, the solvent was evaporated and separated by silica gel column chromatography. The eluent was a mixed solvent of ethyl acetate and n-hexane, and by volume, ethyl acetate: n-hexane = 1:2 (1% acetic acid was added), and 2.52 g of the key intermediate 4b was obtained with a yield of 93.2%.

[0185] Step 4:

[0186]

[0187] Under argon protection, intermediate 4a (2.34 g, 13 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stirrer, and anhydrous dichloromethane (48 mL) and anhydrous N,N-dimethylformamide (4.8 mL) were added to dissolve. The reaction flask was cooled to 0 °C and (COCl) 2 (Oxalyl chloride) (1.13mL, 13.13mmol) was slowly added dropwise to the reaction solution. After the addition was completed, the mixture was heated to room temperature and the reaction was continued for 1 hour. The reaction solution was cooled to 0°C, and DMAP (4-dimethylaminopyridine) (158.8mg, 1.3mmol), triethylamine (4.71mL, 33.8mmol) and 2-trimethylsilylethanol (1.88mL, 13.13mmol) were added in sequence, and the mixture was heated to room temperature and reacted for 1 hour. Saturated ammonium chloride was added to quench the reaction, and the mixture was extracted with dichloromethane. After concentration, the solvent was evaporated and separated by silica gel column chromatography. The eluent was a mixed solvent of dichloromethane and n-hexane. By volume, dichloromethane: n-hexane = 1:2, and 3.15g of the key intermediate 5a was obtained with a yield of 86.5%.

[0188] Step 5:

[0189]

[0190] Under argon protection, 2,2,6,6-tetramethylpiperidine (2.43 mL, 14.3 mmol) and anhydrous tetrahydrofuran (80 mL) were added to a 150 mL round-bottom flask equipped with a magnetic stirrer. The reaction flask was cooled to 0°C, and n-BuLi (n-butyllithium) (5.3 mL, 13.2 mmol) was slowly added dropwise to the reaction solution. After the addition was completed, the reaction was continued for 0.5 hours while keeping warm. In another reaction bottle, the intermediate 5b (3.08 g, 11 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL), and the solution was cooled to -78 ° C. Under Ar protection, the above lithium reagent was added dropwise to the solution containing the intermediate 5b. After keeping at -78 ° C for one hour, 1,2-dibromotetrafluoroethane (1.71 mL, 14.3 mmol) was quickly added to the reaction bottle. After the solution changed from dark red to light yellow, it was stirred for 1.5 minutes and immediately quenched with saturated ammonium chloride (40 mL). Extracted with ethyl acetate, the solvent was evaporated, and separated by silica gel column chromatography. The eluent was a mixed solvent of ethyl acetate and n-hexane. By volume, n-hexane: ethyl acetate = 1:20, and the key benzyl bromide intermediate 6b 2.54 g was obtained with a yield of 64.3%.

[0191] Step 6:

[0192]

[0193] Add benzyl bromide intermediate 6b (2.96 g, 8.23 ​​mmol) and N,N-dimethylformamide (30 mL) to a 50 mL round-bottom flask equipped with a magnetic stirrer, add tetrabutylammonium bromide (796 mg, 2.47 mmol) and sodium thiomethanesulfonate (3.35 g, 24.96 mmol) in sequence, and heat the reaction solution to 50 ° C and stir overnight. Quench the reaction with 40 mL of water. Extract with ethyl acetate, evaporate the solvent, separate by silica gel column chromatography, the eluent is a mixed solvent of ethyl acetate and n-hexane, calculated by volume, n-hexane: ethyl acetate = 1:5, and obtain 1.68 g of the key intermediate 7b, with a yield of 52.4%.

[0194] Step 7:

[0195]

[0196] Add intermediate 7b (1.68 g, 4.3 mmol) and anhydrous dichloromethane (20 mL) to a 25 mL round-bottom flask equipped with a magnetic stirrer, cool to -10 °C, add triethylamine (0.9 mL, 6.45 mmol) and ethanethiol (0.37 mL, 5.16 mmol) in sequence, and stir the reaction solution for 1 hour. Quench the reaction with 20 mL of water. Extract with ethyl acetate, evaporate the solvent, separate by silica gel column chromatography, and use a mixed solvent of ethyl acetate and n-hexane as the eluent. By volume, n-hexane: ethyl acetate = 1:20 to obtain 1.47 g of the key intermediate 8b with a yield of 92%.

[0197] Step 8:

[0198]

[0199] Add intermediate 8b (1.47 g, 3.9 mmol) and tetrahydrofuran to a 100 mL round-bottom flask equipped with a magnetic stirrer. Under argon protection, add tetrabutylammonium fluoride (117 mL, 117 mmol, 1M dissolved in tetrahydrofuran) to the reaction flask, heat to 50 ° C, and stir overnight. Remove most of the volatile substances by rotary evaporation, and quench the reaction with 70 mL of water. Adjust the pH of the solution to 2-3 with 1 M hydrochloric acid, extract with ethyl acetate, evaporate the solvent, and separate by silica gel column chromatography. The eluent is a mixed solvent of ethyl acetate and n-hexane. By volume, n-hexane: ethyl acetate = 1:4 (add 1% acetic acid), and obtain 657.7 mg of the target product 9b with a yield of 62%.

[0200] Example 3 Synthesis of Compound 9c

[0201]

[0202] The specific steps are as follows:

[0203] Step 1:

[0204]

[0205] In a 100mL round-bottom flask equipped with a magnetic stirrer, add anhydrous tetrahydrofuran, 3,4,5-trimethoxybromobenzene (6.17g, 25.0mmol), 1,2-dibromoethane (0.2mL, catalytic amount), and magnesium stick (660mg, 27.5mmol). Heat to reflux for 6 hours under argon protection. Lower the reaction temperature to 0℃, then add iodoethane (2.89mL, 35.0mmol) and Li 2 CuCl 4(12.5mL, 1.25mmol) were sequentially added to the reaction solution and kept at zero temperature for 1.5h. The temperature was raised to room temperature overnight. After the reaction was completed, saturated ammonium chloride was slowly added to the reaction system to quench the reaction, extracted with dichloromethane, the solvent was evaporated, and separated by silica gel column chromatography. The eluent was a mixed solvent of dichloromethane and n-hexane, with dichloromethane: n-hexane = 1:20 by volume, to obtain 3.93g of the target product 2c, with a yield of 80.3%.

[0206] Step 2:

[0207]

[0208] Under argon protection, add anhydrous N,N-dimethylformamide (9 mL) to a 50 mL round-bottom flask equipped with a magnetic stirrer. At 0°C, phosphorus oxychloride (2.43 mL, 26.1 mmol) was slowly added dropwise to the reaction solution. After the addition was complete, the reaction was continued at the same temperature for 0.5 hours. Intermediate 2c (3.93 g, 20.0 mmol) was dissolved in 9.0 mL of anhydrous N,N-dimethylformamide and slowly added dropwise to the above reaction solution. After the addition was complete, the reaction slowly warmed to room temperature and stirred overnight at room temperature. Cool to 0°C and add 16 mL of ice water to the reaction flask to quench the reaction. After adjusting the pH to 8-9 with 1 M sodium hydroxide, add 20 mL of ethyl acetate, separate the organic layer, extract the aqueous layer three times with ethyl acetate, combine the organic layers, wash once with 30 mL of saturated brine, and dry over anhydrous sodium sulfate. The solvent was removed by rotary evaporation, and the residue was separated by silica gel column chromatography. The eluent was a mixed solvent of ethyl acetate and n-hexane, with ethyl acetate:n-hexane = 1:20 by volume, to obtain 3.85 g of the target product with a yield of 86%.

[0209] Step 3:

[0210]

[0211] Under argon protection, DMSO (17 mL) and intermediate 3c (3.85 g, 17.2 mmol) were added to a 100 mL round-bottom flask equipped with a magnetic stirrer. 2 PO 4 (sodium dihydrogen phosphate) (5.16 g, 43.0 mmol, dissolved in 14 mL water) and NaClO 2(Sodium chlorite) (3.73 g, 41.28 mmol, dissolved in 14 mL of water) was slowly added dropwise to the reaction solution. After the addition was completed, the reaction was continued at the same temperature for 0.5 hours. The mixture was warmed to room temperature and the reaction was detected by TLC. Water was added to quench the reaction, and the mixture was extracted with ethyl acetate. After concentration, the solvent was evaporated and separated by silica gel column chromatography. The eluent was a mixed solvent of ethyl acetate and n-hexane, and by volume, ethyl acetate: n-hexane = 1:2 (1% acetic acid was added), and 3.78 g of the key intermediate 4c was obtained with a yield of 91.5%.

[0212] Step 4:

[0213]

[0214] Under argon protection, intermediate 4c (3.78 g, 15.73 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stirrer, and anhydrous dichloromethane (60 mL) and anhydrous N,N-dimethylformamide (6 mL) were added to dissolve. The reaction flask was cooled to 0 °C and (COCl) 2 (Oxalyl chloride) (1.36mL, 15.89mmol) was slowly added dropwise to the reaction solution. After the addition was completed, the mixture was heated to room temperature and the reaction was continued for 1 hour. The reaction solution was cooled to 0°C, and DMAP (4-dimethylaminopyridine) (191.8mg, 1.57mmol), triethylamine (5.7mL, 40.9mmol) and 2-trimethylsilylethanol (2.28mL, 15.89mmol) were added in sequence, and the mixture was heated to room temperature and reacted for 1 hour. Saturated ammonium chloride was added to quench the reaction, and the mixture was extracted with dichloromethane. After concentration, the solvent was evaporated and separated by silica gel column chromatography. The eluent was a mixed solvent of dichloromethane and n-hexane. By volume, dichloromethane: n-hexane = 1:2, and 4.56g of the key intermediate 5c was obtained with a yield of 85.2%.

[0215] Step 5:

[0216]

[0217] Under argon protection, 2,2,6,6-tetramethylpiperidine (2.92 mL, 17.42 mmol) and anhydrous tetrahydrofuran (80 mL) were added to a 100 mL round-bottom flask equipped with a magnetic stirrer. The reaction flask was cooled to 0°C, and n-BuLi (n-butyllithium) (6.43 mL, 16.08 mmol) was slowly added dropwise to the reaction solution. After the addition was completed, the reaction was continued for 0.5 hours while keeping warm. In another reaction bottle, the intermediate 5c (4.56 g, 13.4 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL), and the solution was cooled to -78 ° C. Under Ar protection, the above lithium reagent was added dropwise to the solution containing the intermediate 5c. After keeping at -78 ° C for one hour, 1,2-dibromotetrafluoroethane (1.91 mL, 17.42 mmol) was quickly added to the reaction bottle. After the solution changed from dark red to light yellow, it was stirred for 1.5 minutes and immediately quenched with saturated ammonium chloride (40 mL). Extracted with ethyl acetate, the solvent was evaporated, and separated by silica gel column chromatography. The eluent was a mixed solvent of ethyl acetate and n-hexane. By volume, n-hexane: ethyl acetate = 1:20, and the key benzyl bromide intermediate 6c 3.49 g was obtained with a yield of 62.2%.

[0218] Step 6:

[0219]

[0220] Add benzyl bromide intermediate 6c (3.49 g, 8.33 mmol) and N,N-dimethylformamide (40 mL) to a 50 mL round-bottom flask equipped with a magnetic stirrer, add tetrabutylammonium bromide (805 mg, 2.5 mmol) and sodium thiomethanesulfonate (3.35 g, 24.99 mmol) in sequence, and heat the reaction solution to 50 ° C and stir overnight. Quench the reaction with 40 mL of water. Extract with ethyl acetate, evaporate the solvent, separate by silica gel column chromatography, the eluent is a mixed solvent of ethyl acetate and n-hexane, calculated by volume, n-hexane: ethyl acetate = 1:5, and obtain 1.83 g of the key intermediate 7c, with a yield of 48.9%.

[0221] Step 7:

[0222]

[0223] Add intermediate 7c (1.83 g, 4.07 mmol) and anhydrous dichloromethane (8 mL) to a 25 mL round-bottom flask equipped with a magnetic stirrer, cool to -10 °C, add triethylamine (0.74 mL, 5.29 mmol) and ethanethiol (0.35 mL, 4.88 mmol) in sequence, and stir the reaction solution for 1 hour. Quench the reaction with 20 mL of water. Extract with ethyl acetate, evaporate the solvent, separate by silica gel column chromatography, and use a mixed solvent of ethyl acetate and n-hexane as the eluent. By volume, n-hexane: ethyl acetate = 1:20 to obtain 1.6 g of the key intermediate 8c with a yield of 91.3%.

[0224] Step 8:

[0225]

[0226] Add intermediate 8c (1.6 g, 3.71 mmol) and tetrahydrofuran to a 100 mL round-bottom flask equipped with a magnetic stirrer. Under argon protection, add tetrabutylammonium fluoride (110 mL, 110 mmol, 1 M dissolved in tetrahydrofuran) to the reaction flask, heat to 50 ° C, and stir overnight. Remove most of the volatile substances by rotary evaporation, and quench the reaction with 50 mL of water. Adjust the pH of the solution to 2-3 with 1 M hydrochloric acid, extract with ethyl acetate, evaporate the solvent, and separate by silica gel column chromatography. The eluent is a mixed solvent of ethyl acetate and n-hexane. By volume, n-hexane: ethyl acetate = 1:4 (add 1% acetic acid), and obtain 827 mg of the target product 9c with a yield of 67.2%.

[0227] Although the embodiments of the present invention 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 invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A compound, the compound being a compound represented by Formula 9, and its stereoisomers, tautomers, hydrates, and solvates: in: R 1 , R 2 , R 3 , R 4 are independently selected from H, halogen, -OH, C 1 -C 10 Alkyl, C 1 -C 10 Alkoxy, C 1 -C 10 Alkylamino, C 3 -C 10 Cycloalkyl; R 5 , R 6 are independently selected from H, C 1 -C 10 Alkyl, C 3 -C 10 Cycloalkyl, C 6 -C 20 Aryl.

2. The compound according to claim 1, It is characterized in that The R 1 , R 2 , R 3 , R 4 are independently selected from H, halogen, -OH, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 1 -C 6 Alkylamino, C 3 -C 6 Cycloalkyl; The R 5 , R 6 are independently selected from H, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, C 6 -C 10 Aryl; Optionally, the R 1 , R 2 , R 3 , R 4 are independently selected from H, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 1 -C 6 Alkylamino, C 3 -C 6 Cycloalkyl; The R 5 , R 6 are independently selected from H, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, C 6 -C 10 Aryl.

3. The compound according to claim 1, It is characterized in that The R 1 , R 2 , R 3 , R 4 are independently selected from H, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 1 -C 6 Alkylamino; The R 5 , R 6 are independently selected from C 1 -C 6 alkyl; Optionally, the R 1 , R 2 , R 3 , R 4 are independently selected from H, C 1 -C 6 Alkoxy; The R 5 , R 6 are independently selected from C 1 -C 6 alkyl; Optionally, the R 1 , R 2 , R 3 , R 4 are independently selected from H, C 1 Alkoxy; The R 5 , R 6 are independently selected from C 1 -C 2 alkyl; Optionally, the R 1 , R 2 , R 3 , R 4 are independently selected from H, C 1 Alkoxy, and R 1 , R 2 , R 3 , R 4 At least one of them is not H; The R 5 , R 6 are independently selected from C 1 -C 2 alkyl; Optionally, R 1 , R 3 For H, R 2 , R 4 C 1 Alkoxy; The R 5 Methyl, R 6 It is ethyl.

4. A method for synthesizing the compound according to any one of claims 1 to 3, It is characterized in that The synthesis method comprises: (1) subjecting the compound represented by Formula 4 to an esterification reaction to obtain the compound represented by Formula 5; (2) subjecting the compound represented by Formula 5 to benzyl bromination reaction to obtain the compound represented by Formula 6; (3) contacting the compound represented by Formula 6 with thiomethanesulfonate to obtain the compound represented by Formula 7; (4) subjecting the compound represented by Formula 7 to a reduction reaction to obtain the compound represented by Formula 8; (5) subjecting the compound represented by Formula 8 to a hydrolysis reaction to obtain a compound represented by Formula 9; 5. The synthesis method according to claim 4, It is characterized in that The esterification reaction in step (1) is carried out by contacting the compound represented by formula 4 with a first catalyst, oxalyl chloride and 2-trimethylsilyl alcohol in a first solvent to obtain a compound represented by formula 5; Optionally, the first solvent comprises at least one selected from dichloromethane, chloroform, 1,2-dichloroethane, and 1,1-dichloroethane; Optionally, the first catalyst includes catalyst A, catalyst B and catalyst C; Wherein, the catalyst A comprises at least one selected from N,N-dimethylformamide, N,N-dimethylaniline, N,N-diethylformamide, and pyridine; Catalyst B comprises at least one selected from 4-dimethylaminopyridine, pyridine, 4-pyrrolidinylpyridine, and 4-piperidinylpyridine; Catalyst C comprises at least one selected from triethylamine, tripropylamine, tributylamine, N,N-diisopropylethylamine, and pyridine; Optionally, the esterification reaction further comprises: S1, in a first solvent, contacting the compound represented by formula 4 with a catalyst A and oxalyl chloride to obtain a mixed solution a; S2, contacting the mixed solution a with catalyst B, catalyst C and 2-trimethylsilylethanol to obtain a compound represented by formula 5; Optionally, step S1 is to add the compound of formula 4, catalyst A and oxalyl chloride to the first solvent at -5-0°C, and adjust the reaction temperature to 24-28°C to obtain the mixed solution a; Optionally, in step S2, catalyst B, catalyst C and 2-trimethylsilyl alcohol are added to the mixed solution a at -5-3°C, and the reaction temperature is adjusted to 24-28°C to obtain the compound represented by formula 5.

6. The synthesis method according to claim 4, It is characterized in that The benzyl bromination reaction in step (2) is carried out by contacting the compound represented by formula 5 with a mixed solution b and a bromine reagent in a second solvent at -80-75°C to obtain a compound represented by formula 6; Wherein, the mixed solution b is a solution obtained by the reaction of n-butyl lithium and an organic base; Optionally, the second solvent comprises at least one selected from tetrahydrofuran, diethyl ether, and hexamethylphosphoramide; Optionally, the organic base comprises at least one selected from 2,2,6,6-tetramethylpiperidine, diisopropylamine, bis(trimethylsilyl)amine, piperidine, and tetrahydropyrrole; Optionally, the bromine reagent includes at least one selected from 1,2-dibromotetrafluoroethane, N-bromosuccinimide, liquid bromine, and tetrabromomethane.

7. The synthesis method according to claim 4, It is characterized in that Step (3) further comprises: contacting the compound represented by Formula 6 with a second catalyst and thiomethanesulfonate in a third solvent to obtain a compound represented by Formula 7; Optionally, the third solvent includes at least one selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, methanol, and ethanol; Optionally, the second catalyst comprises at least one selected from tetrabutylammonium bromide, tetrabutylammonium iodide, benzyltrimethylammonium bromide, octadecyltrimethylammonium bromide, and dodecyltrimethylammonium bromide; Optionally, the thiomethanesulfonate salt comprises a salt selected from sodium thiomethanesulfonate or potassium thiomethanesulfonate.

8. The synthesis method according to claim 4, It is characterized in that The reduction reaction in step (4) is carried out in a fourth solvent, wherein the compound represented by formula 7 is contacted with a third catalyst and a thiol compound to obtain a compound represented by formula 8; Optionally, the fourth solvent comprises at least one selected from dichloromethane, 1,2-dichloroethane, chlorobenzene, 1,1-dichloroethane, and carbon tetrachloride; Optionally, the third catalyst comprises at least one selected from triethylamine, tripropylamine, tributylamine, N,N-diisopropylethylamine, and pyridine; Optionally, the thiol compound includes at least one selected from ethanethiol, methyl mercaptan, propyl mercaptan, tert-butyl mercaptan, and thiophenol.

9. The synthesis method according to claim 4, It is characterized in that The hydrolysis reaction in step (5) is carried out in a fifth solvent, wherein the compound represented by formula 8 is contacted with a fourth catalyst to obtain a compound represented by formula 9; Optionally, the fifth solvent comprises at least one selected from tetrahydrofuran, acetonitrile, and ethyl acetate; Optionally, the fourth catalyst comprises at least one selected from tetrabutylammonium fluoride, triethylamine hydrogen fluoride, hydrogen fluoride, and pyridine hydrogen fluoride.

10. The synthesis method according to claim 4, It is characterized in that The synthesis method further comprises: subjecting the compound of Formula 3 to an oxidation reaction to obtain a compound of Formula 4; Optionally, the oxidation reaction is carried out in a sixth solvent, wherein the compound represented by Formula 3 is contacted with a buffer and an oxidant to obtain a compound represented by Formula 4; Optionally, the sixth solvent comprises at least one selected from dimethyl sulfoxide, N,N-dimethylformamide, and N-methylpyrrolidone; Optionally, the buffer comprises a member selected from sodium dihydrogen phosphate or potassium dihydrogen phosphate; Optionally, the oxidant comprises at least one selected from sodium hypochlorite, calcium hypochlorite, potassium hypochlorite, and sodium periodate.

11. The synthesis method according to claim 10, It is characterized in that The synthesis method further comprises: subjecting the compound of Formula 2 to a Vilsmeier reaction to obtain a compound of Formula 3; Optionally, the synthesis method further comprises: subjecting the compound of Formula 1 to a Grignard reaction to obtain a compound of Formula 2; 12. Use of the compound according to any one of claims 1 to 3 as a reversible blocking sequencing agent.

13. A kit, It is characterized in that The kit comprises the compound according to any one of claims 1 to 3.

14. Use of the compound according to any one of claims 1 to 3 or the kit according to claim 13 in nucleic acid sequencing.

15. A nucleic acid sequencing method, It is characterized in that The nucleic acid sequencing method comprises: Using the compound according to any one of claims 1 to 3 to undergo an esterification reaction with the 3'-hydroxyl group of dNTP to obtain a reactant, and using the reactant to perform sequencing; Optionally, the nucleic acid sequencing method further comprises: removing the compound using an excision reagent to release the hydroxyl group at the 3' position of the dNTP for subsequent sequencing reaction; Optionally, the excision agent is a reagent capable of cleaving a disulfide bond contained in the compound according to any one of claims 1 to 3; Optionally, the cleavage agent comprises at least one selected from tris(3-hydroxypropyl)phosphine, tris(2-carboxyethyl)phosphine, triphenylphosphine, tributylphosphine, and glutathione.

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