On-dna alpha-hydroxy olefinic lead compounds and methods for their synthesis

Abstract

The application provides an On-DNA alpha-hydroxy olefin lead compound and a synthesis method thereof, and belongs to the field of gene coded compound library construction. The application discloses a method for synthesizing an alpha-hydroxy olefin lead compound on DNA. The method has small damage to DNA, is good in universality, simple in operation, mild in condition, and can obtain the On-DNA alpha-hydroxy olefin compound with high conversion rate. Under the reaction condition of the application, the product On-DNA alpha-hydroxy olefin compound has high conversion rate and good DNA integrity. The application enriches the chemical reaction type for synthesizing the coded compound library on DNA, constructs a new alpha-hydroxy olefin skeleton for the gene coded compound library, and has very good application prospect in lead drug development.

Description

Technical Field

[0001] This invention belongs to the field of gene-encoded compound library construction, specifically relating to an on-DNA α-hydroxyolefin lead compound and its synthesis method. Background Technology

[0002] After years of application, development, and improvement, high-throughput screening has established a comprehensive screening process and is an important route for major international drug development companies to obtain lead compounds for target proteins. However, traditional high-throughput screening of single molecule has drawbacks such as limited compound library size, long time consumption, and high cost, and is increasingly unable to meet the needs of new drug development.

[0003] In 1992, Brenner and Lerner creatively proposed a method for screening bioactive compounds using gene-encoded library technology (DELT). The principle of DELT is to label each small molecule compound in the reaction process with gene fragments of different specific sequences. Using a combinatorial chemistry strategy, through splitting and pooling, millions to tens of billions of compounds linked to specific gene sequences can be synthesized in large quantities with limited cost and time. The resulting mixture of compounds is then incubated with a protein target. Physical separation is achieved by washing away compounds that do not bind to the protein target, thus identifying compounds with high affinity. The gene-encoded compound library required for incubating the target protein requires only extremely small doses (micrograms) and can be performed in a very short time (e.g., within one day). Furthermore, DELT technology can easily perform multiple biological screening experiments under different conditions. Currently, screening of gene-encoded compound libraries (DELs) has become a common method for discovering new protein ligands (Goodnow, RA; Dumerin, CE; Keefe, ADDNA-encoded chemistry: Enabling the deeper sampling of chemical space. Nat. Rev. Drug Discov. 2016; Neri, D.; Lerner, RADNA-Encoded Chemical Libraries: A Selection System Based on Endowing Organic Compounds with Amplifiable Information. Annu. Rev. Biochem. 2018, 87, 479-502). Compared with traditional chemistry, DELs offer significant advantages in cost, output, and the construction of larger chemical spaces through high-throughput screening, greatly increasing the number and diversity of compounds. In practical applications, DEL bioscreening has successfully identified affinities for many protein targets (Zimmermann, G.; Neri, D. DNA-encoded chemical libraries: Foundations and applications in lead discovery. Drug Discov. Today 2016, 21, 1828-1834), including small chemical molecules currently in clinical trials (Belyanskaya, SL; Ding, Y.; Callahan, JF; Lazaar, AL; Israel, DIChemBioChem. 2017, 18, 837-842).

[0004] One of the most important areas of research in gene-encoded compound library technology is the development of on-DNA chemical reactions (On-DNA chemical reactions). Several On-DNA chemical reactions have already been reported. For example, Shanghai WuXi AppTec New Drug Development Co., Ltd. (patent application CN201910609569.0) disclosed a method for obtaining On-DNA aromatic compounds via Suzuki coupling reaction: using On-DNA aryl halides as substrates, reacting with potassium organotrifluoroborate reagent in the presence of a Pd catalyst, ligands, and a base to prepare On-DNA aromatic compounds. This method increases the diversity of DNA-encoded compound libraries of On-DNA aryl halides, has high reaction yields, broad substrate versatility, mild conditions, and is easy to operate, making it suitable for the synthesis of DNA-encoded compound libraries in multi-well plates. Chengdu Pioneer Pharmaceuticals Co., Ltd. (patent application CN201910590679.7) disclosed a method for synthesizing On-DNA arylbenzyl-substituted compounds. This method uses On-DNA aldehyde compounds as raw materials, reacting them with indole under alkaline conditions to generate On-DNA indole alcohol compounds. Then, the On-DNA indole alcohol compounds are reduced to indole alkylated compounds by diethyl 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate under acidic conditions. The more diverse and abundant the chemical reactions realized in gene-encoded compound libraries, the more choices are available for the design and synthesis of these libraries, resulting in greater diversity. However, the currently reported types of On-DNA chemical reactions are limited and cannot yet meet the widespread demand for lead compound discovery.

[0005] Alpha-hydroxyolefins are compounds with an α-hydroxyolefin structure that possess various biological activities and are found in many common drug molecules, such as swertiamarin, cinicin, cephalosporin, and hygrophylline. Swertiamarin is an iridoid compound isolated from the gentian plant *Swertia spp.*, which inhibits the autonomous rhythmic activity of isolated rat duodenum, uterus, gallbladder smooth muscle, and bile duct sphincter, and antagonizes the excitatory effects of acetylcholine and norepinephrine. Clinical studies have shown that swertiamarin has significant antispasmodic and analgesic effects on spasmodic pain in the gastrointestinal tract and biliary tract, and also has a certain sedative effect, without causing allergies or irritation. Cnicin, also known as cinicin, mainly has antibacterial, anti-inflammatory, skin-whitening, and antitumor effects, and has bactericidal activity against various bacilli, including *Abortus abortus*.

[0006]

[0007] However, because DNA must maintain stability under specific aqueous phase, pH, temperature, metal ion concentration, and inorganic salt concentration conditions, and because reactions used for constructing DNA-encoded compound libraries require high conversion rates, there are currently no reports on methods for constructing α-hydroxyolefin gene-encoded compounds on DNA. Therefore, developing a method for synthesizing On-DNA α-hydroxyolefin compounds with minimal DNA damage and high conversion rates is of great significance. Summary of the Invention

[0008] The purpose of this invention is to provide an On-DNA α-hydroxyolefin lead compound and its synthesis method, as well as the use of the synthesis method in constructing a gene-encoded compound library.

[0009] This invention provides an On-DNA α-hydroxyolefin lead compound of Formula I:

[0010]

[0011] DNA is a single-stranded or double-stranded nucleotide chain;

[0012] M represents the connection unit;

[0013] R2 is selected from nitro, cyano, and COR. 2a NHR 2a CONHR 2a , NHCOR 2a ;

[0014] R3 and R4 are each independently selected from hydrogen, halogen, carboxyl, nitro, cyano, hydroxyl, mercapto, and COR. 2a NHR 2a CONHR 2a , NHCOR 2a Not replaced or R 3a The following groups are substituted: C1- 12 Alkyl, C 1-12 Alkoxy, C 2-8 alkenyl, C 2-8 alkynyl, 3-8 membered cycloalkyl, 5-6 membered aryl, 5-6 membered heteroaryl, R 3a Selected from C 1-12 Alkyl, C 1-12 Alkyl groups; or, R3 and R4 linked together to form a ring;

[0015] R 2a Selected from C 1-12 Alkyl, C 1-12 Alkoxy, LR 2b OLR 2b L is C 1-6 Alkylene, R 2bSelected from C 1-12 Alkyl, unsubstituted or R 2c Substituted phenyl, R 2c Selected from C 1-12 Alkyl, C 1-12 Alkoxy;

[0016] Alternatively, R4 is hydrogen, and R2 and R3 are linked together to form a ring.

[0017] Further, M is X1-Y1-P-Y2-X2-Q, where X1, Y1, P, Y2, X2, and Q are each independently selected from the following groups: unsubstituted, unsubstituted, or substituted by R1: NHCO, CONH, NH, 5-6 aryl, 5-6 heteroaryl, 3-8 saturated cycloalkyl, 3-8 saturated heterocyclic, C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group;

[0018] m is an integer from 1 to 10;

[0019] R1 is selected from hydrogen, halogen, nitro, cyano, mercapto, carboxyl, hydroxyl, halogenated or unhalogenated C. 1-6 Alkyl, halogenated or unhalogenated C 1-6 Alkoxy;

[0020] X1, Y1, P, Y2, X2, and Q are not all zero at the same time.

[0021] Furthermore, the structure of the On-DNA α-hydroxyolefin lead compound is shown in Formula II-1:

[0022]

[0023] Among them, DNA is a single-stranded or double-stranded oligonucleotide chain;

[0024] X1 is NHCO or CONH, X2 is NHCO or CONH;

[0025] Y1 is either none or C 1-4 Alkylene, Y2 is absent or C 1-4 Alkylene;

[0026] Q is selected from the following groups that are either unsubstituted or substituted by R1: 5-6 aryl, 5-6 heteroaryl, 3-6 saturated cycloalkyl, 3-6 saturated heterocyclic, C 1-4 Alkylene;

[0027] m is an integer from 1 to 8;

[0028] R1 is selected from hydrogen, halogen, nitro, cyano, C 1-6 Alkyl, C1-6 Alkoxy;

[0029] R2 is selected from nitro, cyano, and COR. 2a NHR 2a CONHR 2a , NHCOR 2a ;

[0030] R3 and R4 are each independently selected from hydrogen, halogen, carboxyl, nitro, cyano, hydroxyl, mercapto, and COR. 2a NHR 2a CONHR 2a , NHCOR 2a Not replaced or R 3a The following groups are substituted: C1-6 alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, 3-8 membered cycloalkyl, 5-6 membered aryl, 5-6 membered heteroaryl, R 3a Selected from C 1-6 Alkyl, C 1-6 Alkyl groups; or, R3 and R4 linked together to form a ring;

[0031] R 2a Selected from C 1-6 Alkyl, C 1-6 Alkoxy, LR 2b OLR 2b L is C 1-4 Alkylene, R 2b Selected from C 1-6 Alkyl, unsubstituted or R 2c Substituted phenyl, R 2c Selected from C 1-6 Alkyl, C 1-6 Alkoxy;

[0032] Alternatively, R4 is hydrogen, and R2 and R3 are connected to form a ring;

[0033] Preferably, the structure of the On-DNA α-hydroxyolefin lead compound is shown in Formula II-2:

[0034]

[0035] Furthermore, the structures of the On-DNA α-hydroxyolefin lead compounds are shown in formulas III-a, III-b, III-c, III-d, III-e, or III-f:

[0036]

[0037]

[0038] Among them, DNA is a single-stranded or double-stranded oligonucleotide chain;

[0039] R1 is selected from hydrogen, halogens, and C. 1-3 Alkyl, C 1-3 Alkoxy;

[0040] n is selected from 1, 2, and 3;

[0041] R 2a Selected from C 1-3 Alkyl, C 1-3 Alkoxy, LR 2b OLR 2b L is C 1-2 Alkylene, R 2b Selected from C 1-3 Alkyl, unsubstituted or R 2c Substituted phenyl, R 2c Selected from C 1-3 Alkyl, C 1-3 Alkoxy;

[0042] R3 is selected from hydrogen, C 1-3 Alkyl, C 1-3 Alkoxy, unsubstituted or R 3a The substituted phenyl group, R4 is selected from hydrogen, C 1-3 Alkyl, C 1-3 Alkoxy, unsubstituted or R 3a Substituted phenyl groups, or R3 and R4 linkages, form unsubstituted or R-substituted phenyl groups. 3b Substituted 4-membered saturated nitrogen heterocycle; R 3a Selected from C 1-3 Alkyl, C 1-3 Alkoxy, R 3b Selected from Boc, C 1-3 Alkyl, C 1-3 Alkoxy;

[0043] Y3 is C 1-4 Alkylene;

[0044] Y4 is C 1-4 Alkylene.

[0045] Furthermore, the On-DNA α-hydroxyolefin lead compound is selected from one of the following compounds:

[0046]

[0047] This invention also provides a method for synthesizing the above-mentioned On-DNA α-hydroxy olefin lead compound, the method comprising the following steps: using the compound shown in formula Ia and the compound shown in formula Ib as raw materials, reacting them in a solvent in the presence of a base to obtain the On-DNA α-hydroxy olefin lead compound shown in formula I:

[0048]

[0049] Furthermore, the equivalent ratio of the compound shown in formula Ia, the compound shown in formula Ib, and the base is 1:(500-2000):(500-2000);

[0050] The reaction is carried out at a temperature of 0-90℃ for 1-24 hours.

[0051] The solvent is selected from water, methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2,2-difluoroethanol, 2,2,2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, diethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1,4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, etc. One or a mixture of two or more of the following: dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 4-methoxypyridine, toluene, and xylene;

[0052] The base is selected from one or a mixture of two or more of the following: triethylenediamine, triethylamine, n-butylamine, isobutylamine, 4-dimethylaminopyridine, pyridine, N,N-diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N,N',N'-tetramethylethylenediamine, 1,1,3,3-tetramethylguanidine, N,N-dicyclohexylmethylamine, dicyclohexylamine, tetrahydropyrrole, carbonates, phosphates, borates, triethylammonium acetate, and tris(hydroxymethyl)aminomethane.

[0053] Furthermore, the equivalent ratio of the compound shown in formula Ia, the compound shown in formula Ib, and the base is 1:1000:1000;

[0054] The reaction was carried out at a temperature of 25°C for 16 hours.

[0055] The solvent is selected from a mixture of water and acetonitrile, a mixture of water and N,N-dimethylformamide, and a mixture of water and ethanol;

[0056] The base is triethylenediamine.

[0057] Furthermore, in the mixture of water and acetonitrile, the volume ratio of water to acetonitrile is (1-2):(1-2);

[0058] In the mixture of water and N,N-dimethylformamide, the volume ratio of water to N,N-dimethylformamide is (1-2):(1-2);

[0059] In the mixture of water and ethanol, the volume ratio of water to ethanol is (1-2):(1-2).

[0060] Furthermore, the compound shown in formula Ia is The preparation method of the compound shown in Formula Ia includes the following steps:

[0061]

[0062] (1) Compound HP reacts with compound A1 to give compound A2;

[0063] (2) Compound A2 reacts with a deprotecting agent to give compound A3;

[0064] (3) Compound A3 reacts with compound A4 to obtain the compound shown in formula Ia.

[0065] Further, in step (1), the reaction is carried out in the presence of sodium tetraborate and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, with the equivalent ratio of compound HP, compound A1, sodium tetraborate, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride being 1:(30-50):(200-300):(30-50), and the solvent for the reaction is selected from... Water, methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2,2-difluoroethanol, 2,2,2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, ethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1,4-dioxane, anisole, dimethyl sulfide, diethyl sulfide. The reaction mixture comprises one or more of the following: ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 4-methoxypyridine, toluene, and xylene. The reaction temperature is 0-30°C, and the reaction time is 0.5-4 hours. Preferably, the equivalent ratio of compound HP, compound Al, sodium tetraborate, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is 1:40:250:40. The solvent for the reaction is a mixed solution of water and dimethyl sulfoxide. The reaction temperature is 4°C, and the reaction time is 1 hour.

[0066] In step (2), the deprotecting agent is piperidine;

[0067] In step (3), the reaction is carried out in the presence of sodium tetraborate and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, with the equivalent ratio of compound A3, compound A4, sodium tetraborate, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride being 1:(30-50):(200-300):(30-50). The solvent for the reaction is selected from water and methanol. Ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2,2-difluoroethanol, 2,2,2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, ethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1,4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol The reaction mixture comprises one or more of the following: dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 4-methoxypyridine, toluene, and xylene. The reaction temperature is 0-30°C, and the reaction time is 0.5-4 hours. Preferably, the equivalent ratio of compound A3, compound A4, sodium tetraborate, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is 1:40:250:40. The solvent for the reaction is a mixed solution of water and dimethyl sulfoxide. The reaction temperature is room temperature, and the reaction time is 2 hours.

[0068] The present invention also provides the use of the above-described synthesis method in constructing gene-encoded compound libraries.

[0069] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0070] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a-b Alkyl groups refer to any alkyl group containing one to two carbon atoms ("a" to "b"). For example, C 1-6 Alkyl groups are straight-chain or branched alkyl groups containing 1 to 6 carbon atoms.

[0071] "Aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group with a conjugated π-electron system, such as phenyl and naphthyl. The aryl ring can be fused to other cyclic groups (including saturated and unsaturated rings), but cannot contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of attachment to the parent group must be on a carbon atom of a ring with a conjugated π-electron system. The aryl group can be substituted or unsubstituted.

[0072] "Heteroaryl" refers to a heteroaryl group containing one or more heteroatoms. Heteratoms include oxygen, sulfur, and nitrogen. Examples include furanyl, thiophene, pyridinyl, pyrazolyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0073] "Connecting units" include chemical bonds and groups that can act as connectors.

[0074] Halogens include fluorine, chlorine, bromine, and iodine.

[0075] This invention discloses for the first time a method for synthesizing α-hydroxyolefin lead compounds on DNA. This method causes little damage to DNA, has good versatility, is simple to operate, operates under mild conditions, and can produce On-DNA α-hydroxyolefin compounds with high conversion rates.

[0076] As is well known in the art, DNA must remain stable under certain conditions, and reactions used for constructing DNA-encoded compound libraries require high conversion rates. Under the reaction conditions of this invention, the resulting On-DNA α-hydroxyolefin compounds not only exhibit high conversion rates but also good DNA integrity. The integrity of the On-DNA α-hydroxyolefin compounds obtained in this invention can be confirmed not only by liquid chromatography-mass spectrometry but also by further verification through DNase-catalyzed coupling reactions. This invention enriches the types of chemical reactions for synthesizing DNA-encoded compound libraries, provides a novel α-hydroxyolefin backbone for constructing gene-encoded compound libraries, and has excellent application prospects in lead drug development.

[0077] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0078] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0079] Figure 1 The structure of raw material HP.

[0080] Figure 2 : Liquid chromatography-mass spectrometry detection chromatogram of S2.

[0081] Figure 3 : Liquid chromatography-mass spectrometry detection chromatogram of S3.

[0082] Figure 4 :S 4-1 The liquid chromatography-mass spectrometry detection chromatogram.

[0083] Figure 5 :S 4-2 The liquid chromatography-mass spectrometry detection chromatogram.

[0084] Figure 6 :S 4-3 The liquid chromatography-mass spectrometry detection chromatogram.

[0085] Figure 7 :S 4-4 The liquid chromatography-mass spectrometry detection chromatogram.

[0086] Figure 8 :S 4-5 The liquid chromatography-mass spectrometry detection chromatogram.

[0087] Figure 9 :S 4-6 The liquid chromatography-mass spectrometry detection chromatogram.

[0088] Figure 10 :S 4-7 The liquid chromatography-mass spectrometry detection chromatogram.

[0089] Figure 11 :S 4-8 The liquid chromatography-mass spectrometry detection chromatogram.

[0090] Figure 12 :S 4-9 The liquid chromatography-mass spectrometry detection chromatogram.

[0091] Figure 13 :S 4-10 The liquid chromatography-mass spectrometry detection chromatogram.

[0092] Figure 14 :S 4-11 The liquid chromatography-mass spectrometry detection chromatogram.

[0093] Figure 15 :S 4-12 The liquid chromatography-mass spectrometry detection chromatogram.

[0094] Figure 16: Liquid chromatography-mass spectrometry detection chromatogram of P1.

[0095] Figure 17 : Liquid chromatography-mass spectrometry detection chromatogram of P2.

[0096] Figure 18 : Liquid chromatography-mass spectrometry detection chromatogram of P3.

[0097] Figure 19 : Liquid chromatography-mass spectrometry detection chromatogram of P4.

[0098] Figure 20 : Liquid chromatography-mass spectrometry detection chromatogram of P5.

[0099] Figure 21 : Liquid chromatography-mass spectrometry detection chromatogram of P6.

[0100] Figure 22 : Liquid chromatography-mass spectrometry detection chromatogram of P7.

[0101] Figure 23 : Liquid chromatography-mass spectrometry detection chromatogram of P8.

[0102] Figure 24 : Liquid chromatography-mass spectrometry detection chromatogram of P9.

[0103] Figure 25 :P 10 The liquid chromatography-mass spectrometry detection chromatogram.

[0104] Figure 26 :P 11 The liquid chromatography-mass spectrometry detection chromatogram.

[0105] Figure 27 :P 12 The liquid chromatography-mass spectrometry detection chromatogram.

[0106] Figure 28 :P 13 The liquid chromatography-mass spectrometry detection chromatogram.

[0107] Figure 29 :P 14 The liquid chromatography-mass spectrometry detection chromatogram.

[0108] Figure 30 :P 15 The liquid chromatography-mass spectrometry detection chromatogram.

[0109] Figure 31 :P 16 The liquid chromatography-mass spectrometry detection chromatogram.

[0110] Figure 32 :P 17 The liquid chromatography-mass spectrometry detection chromatogram.

[0111] Figure 33 :P 18 The liquid chromatography-mass spectrometry detection chromatogram.

[0112] Figure 34 :P 19 The liquid chromatography-mass spectrometry detection chromatogram.

[0113] Figure 35 :P 1- P 19 Summary of structure and conversion rate.

[0114] Figure 36 Liquid chromatography-mass spectrometry (LC-MS) chromatogram of Tag A-P7.

[0115] Figure 37 : Validation roadmap for real-time quantitative polynucleotide chain reaction.

[0116] Figure 38 qPCR product PS 4-1 The amplification curve.

[0117] Figure 39 Amplification curve of qPCR product TP-P7.

[0118] Figure 40 Standard curve of qPCR product TP-P7.

[0119] Figure 41 qPCR product PS 4-1 Melting curves of TP-P7. Detailed Implementation

[0120] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0121] The α-EWG-olefin reagent used in the embodiments of this invention is one of the following compounds, all of which are commercially available products:

[0122]

[0123] The oligonucleotide-aldehyde compound used in the embodiments of this invention is S. 4-1 -S 4-12 One of them, S 4-1 -S 4-12 The structure is as follows:

[0124]

[0125] The following is S 4-1 -S 4-12 Synthesis method.

[0126] (I) Synthesis of oligonucleotide-aldehyde compounds S4-1

[0127] Step 1: Synthesize DNA-NHFmoc (S2)

[0128] DNA-NHFmoc(S2) is synthesized according to the following reaction formula:

[0129]

[0130] 100 nanomolar HP (HP structure as shown in...) Figure 1 As shown, a commercially available product was dissolved in deionized water to prepare a 1 mmol / L solution (100 μL, 1 equivalent). 40 equivalents of a DMSO solution (20 μL, 200 mmol / L) of the starting head fragment compound (commercially available product), 250 equivalents of a sodium tetraborate (Na₂B₄O₇) buffer solution (pH 9.5, 100 μL, 250 mmol / L), and 40 equivalents of an aqueous solution of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) (20 μL, 200 mmol / L) were mixed and thoroughly vortexed. This mixture was then added to the HP solution, mixed thoroughly, and reacted at 4°C for 1 hour. After the reaction was complete, 10% (by total volume) of a 5 mol / L sodium chloride solution was added to the reaction solution. Then, three times the total volume of anhydrous ethanol was added, shaken thoroughly, and the reaction solution was placed in a -80°C freezer for 2 hours. Next, centrifuge at 4000 rpm for half an hour, and discard the supernatant. The remaining precipitate, after drying, yields the oligonucleotide-NHFmoc, abbreviated as DNA-NHFmoc(S2). The chromatogram of DNA-NHFmoc(S2) was detected using liquid chromatography-mass spectrometry, as shown below. Figure 2 As shown, its molecular weight is 5406.

[0131] Step 2: Synthesize DNA-NH2(S3)

[0132] DNA-NH2(S3) is synthesized according to the following reaction formula:

[0133]

[0134] 100 nanomolars of DNA-NHFmoc(S2) were dissolved in deionized water to prepare a 1 mmol / L (100 μL, 1 equivalent) solution. 56 μL (659 equivalents) of a 10% piperidine aqueous solution was added, and the mixture was thoroughly mixed and reacted at room temperature for 1 hour. After the reaction, 10% (5 mol / L) sodium chloride solution was added to the reaction solution. Then, three times the total volume of anhydrous ethanol was added, and the mixture was shaken thoroughly. The reaction mixture was then frozen at -80°C for 2 hours. Afterward, the mixture was centrifuged at 4000 rpm for half an hour, and the supernatant was discarded. The remaining precipitate was dried to obtain the oligonucleotide-NH2(S3), abbreviated as DNA-NH2. The chromatogram of DNA-NH2(S3) was detected using liquid chromatography-mass spectrometry (LC-MS / MS), as shown below. Figure 3 As shown, its molecular weight is 5184.

[0135] Step 3: Synthesize oligonucleotide-aldehyde compound S 4-1

[0136] Oligonucleotide-aldehyde compound S was synthesized according to the following reaction formula. 4-1 :

[0137]

[0138] Dissolve 10 nanomoles of DNA-NH2(S3) in deionized water to prepare a 1 mmol / L (10 μL, 1 equivalent) solution. Mix 40 equivalents of a DMSO solution of 3-fluoro-5-formylbenzoic acid (commercially available product) (2 μL, 200 mmol / L), 250 equivalents of a sodium tetraborate (Na2B4O7) buffer solution at pH 9.5 (10 μL, 250 mmol / L), and 40 equivalents of an aqueous solution of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) (2 μL, 200 mmol / L). Vortex thoroughly to mix the mixture. Add the mixture to the DNA-NH2 solution, mix well, and react at room temperature for 2 hours. After the reaction is complete, add 10% by volume of a 5 mol / L sodium chloride solution to the reaction solution. Then, add three times the total volume of anhydrous ethanol, shake well, and freeze the reaction solution at -80°C for 2 hours. Afterward, centrifuge at 4000 rpm for half an hour and discard the supernatant. The remaining precipitate, after drying, yields the oligonucleotide-aldehyde compound S. 4-1 S was detected using liquid chromatography-mass spectrometry. 4-1 The spectrum, such as Figure 4 As shown, its molecular weight is 5334 and the yield is 90%.

[0139] (II) Synthesis of oligonucleotide-aldehyde compound S 4-2 -S 4-12

[0140] Following the same method described above, the only difference is that 3-fluoro-5-formyl-benzoic acid in step 3 is replaced with the corresponding raw material to synthesize oligonucleotide-aldehyde compounds S. 4-2 -S 4-12 S was detected using liquid chromatography-mass spectrometry. 4-2 -S 4-12 The spectrum, such as Figures 5-15 As shown.

[0141] Example 1: Synthesis of oligonucleotide-α-hydroxyolefin compound P1

[0142]

[0143] At 1.0 nanomolar oligonucleotide-aldehyde compound S 4-1 1000 equivalents of DABCO (triethylenediamine, commercially available, 1.0 μL, 1.0 mol / L acetonitrile solution) and 1000 equivalents of 2-cyclopentenone (commercially available, 1.0 μL, 1.0 mol / L acetonitrile solution) were added to an aqueous solution of 1.0 mmol / L (1.0 μL, 1.0 mol / L acetonitrile solution). The mixture was thoroughly mixed by vortexing and reacted at 25°C for 16 hours. After the reaction was complete, 10% of the total volume of 5 mol / L sodium chloride solution and 3 times the total volume of anhydrous ethanol were added to the reaction solution. After shaking thoroughly, the reaction solution was frozen at -80°C for 2 hours, followed by high-speed refrigerated centrifugation (4°C, 12000 rpm, 15 minutes). The supernatant was discarded, and the remaining precipitate was dried to obtain the product oligonucleotide-α-hydroxyolefin compound P1. P1 was detected by liquid chromatography-mass spectrometry. The detection results are shown in the figure. Figure 16 The molecular weight is 5416, and the conversion rate is 78%. This invention utilizes liquid chromatography-mass spectrometry to accurately detect the conversion rate of the target product.

[0144] Example 2: Synthesis of oligonucleotide-α-hydroxyolefin compound P2-P 19

[0145] The synthesis method is the same as in Example 1, except that the oligonucleotide-aldehyde compound S is used instead. 4-1 Replace with S 4-1 -S 4-12 One of the methods involves replacing 2-cyclopentenone with the corresponding α-EWG-olefin reagent to synthesize oligonucleotide-α-hydroxyolefin compounds P2-P. 19 P2-P was detected using liquid chromatography-mass spectrometry. 19 The test results are shown below. Figures 17-34 .

[0146] Oligomeric nucleic acid-α-hydroxyolefin compound P1-P19 The structure and conversion rate are summarized as follows: Figure 35 As shown.

[0147] The following experimental examples demonstrate the beneficial effects of the present invention.

[0148] Experiment Example 1: Screening Experiment for Synthesizing Oligonucleotide-α-hydroxyolefin Compounds

[0149] Referring to Example 2, oligonucleotide-α-hydroxyolefin compound P was synthesized. 19 The method differs only in that the following parameters are controlled according to Table 1: type of base, equivalent amount of base, equivalent amount of α-EWG-olefin reagent (i.e., acrylonitrile), reaction solvent, and ratio. Calculate P under different parameters. 19 The product conversion rate was calculated. The results are shown in Table 1.

[0150]

[0151] Table 1. Synthesis of oligonucleotide-α-hydroxyolefin compound P under different conditions 19 Product conversion rate

[0152]

[0153] It can be seen that under reaction conditions numbered 1 and 3-5, the product conversion rate of the obtained oligonucleotide-α-hydroxyolefin compound was 0; under reaction conditions numbered 9-10 and 12-15, the product conversion rate of the obtained oligonucleotide-α-hydroxyolefin compound was as high as 49% or more; and under reaction condition numbered 10, the product conversion rate of the obtained oligonucleotide-α-hydroxyolefin compound was the highest. This indicates that the method using the specific parameters of the embodiments of the present invention yields the oligonucleotide-α-hydroxyolefin compound with the highest product conversion rate.

[0154] Experimental Example 2: Verification of the integrity of oligonucleotides in the oligonucleotide-α-hydroxyolefin compound of the present invention

[0155] 1) Enzyme ligation reaction verification

[0156] The integrity of the oligonucleotide was verified by linking oligonucleotide-α-hydroxyolefin compound P7 with Tag A (a short-chain oligonucleotide with molecular weights of 4064 and 5884, respectively):

[0157]

[0158] Procedure: Dissolve 1.0 nanomolar P7 in deionized water to prepare a 1.0 mmol / L solution (1.0 μL, 1.0 equivalent). Add 1.2 equivalents of Tag A (1.0 mmol / L aqueous solution, 1.2 μL), 1.0 μL of 10×T4 DNA ligation buffer, and 0.5 μL of T4 DNA ligase. Mix the solutions thoroughly and react at room temperature for 1 hour. After the reaction, add 10% of the total volume of 5.0 mol / L sodium chloride solution to the reaction solution, followed by 3.0 times the total volume of anhydrous ethanol. After shaking thoroughly, freeze the reaction mixture at -80°C for 2.0 hours. Centrifuge at 4000 rpm for half an hour and discard the supernatant. Collect the remaining precipitate and name it Tag A-P7. Dissolve Tag A-P7 in deionized water and confirm the molecular weight of the product by liquid chromatography-mass spectrometry (LC-MS / MS). The molecular weight is 15346. The mass spectrometry results are shown in the figure. Figure 36 .

[0159] LCMS analysis showed that the oligonucleotide-α-hydroxyolefin compound P7 could be successfully coupled with Tag A. This indicates that the DNA strand integrity of the oligonucleotide-α-hydroxyolefin compound obtained according to the synthesis method of this invention is good. Further LCMS mass spectrometry accurately displayed its molecular weight, confirming that its nucleotides were not damaged. Therefore, the reaction method in this invention does not damage the basic structure and activity of DNA.

[0160] 2) Validation by real-time quantitative polynucleotide chain reaction (qPCR)

[0161] according to Figure 37 The route shown describes the synthesis of the on-DNA α-hydroxyolefin lead compound P-P7 using the method of this invention. This lead compound, along with Tag E and a reverse primer, was then enzymatically linked to yield the full-length DNA target fragment TP-P7. The molecular weight of TP-P7 was accurately displayed using LCMS mass spectrometry. Subsequently, qPCR experiments were performed on TP-P7 to verify the integrity of the DNA.

[0162] TP-P7 was serially diluted 10-fold each time, for a total of 8 dilutions, and a standard curve was obtained through qPCR experiments. The first 7 dilutions were used as templates for qPCR. A fluorescent dye kit (SYBR GreenMaster Mix kit, Thermo Fisher Scientific) was added to the reaction solution, and the final volume was controlled to 20 μL. Amplification was performed using a real-time quantitative PCR instrument (Quant Studio 3). All samples were repeated in triplicate. The qPCR cycling program was set as follows: heating at 50°C for 2 minutes, thermal activation at 95°C for 10 minutes; then 40 cycles of denaturation at 95°C for 15 seconds, annealing and extension at 60°C for 1 minute. Melting curve stage: 95°C for 15 seconds, 60°C for 1 minute, 95°C for 15 seconds.

[0163] qPCR results showed that PS 4-1 No significant CT displacement was observed in either serially diluted TP-P7 or other solutions. Figure 38 (39), indicating that the synthesis method of the present invention did not cause DNA damage. No multiple peaks were observed in the qPCR melting curve ( Figure 41 This indicates that there was no significant change in the types of DNA after the reaction.

[0164] The qPCR method further demonstrates that the reaction method of the present invention does not damage the basic structure and activity of DNA.

[0165] In summary, this invention discloses for the first time a method for synthesizing α-hydroxyolefin lead compounds on DNA. This method causes minimal DNA damage, has good versatility, is simple to operate, operates under mild conditions, and can produce On-DNA α-hydroxyolefin compounds with high conversion rates. Under the reaction conditions of this invention, the produced On-DNA α-hydroxyolefin compounds not only have high conversion rates but also exhibit good DNA integrity. This invention enriches the types of chemical reactions for synthesizing DNA-encoded compound libraries, constructs novel α-hydroxyolefin backbones for gene-encoded compound libraries, and has excellent application prospects in lead drug development.

Claims

1. A method for synthesizing On-DNA α-hydroxyolefin lead compounds, characterized in that: The method includes the following steps: using the compounds shown in Formula Ia and Formula Ib as raw materials, reacting them in a solvent in the presence of a base to obtain the On-DNA α-hydroxy olefin lead compound shown in Formula I: ； The equivalent ratio of the compound shown in formula Ia, the compound shown in formula Ib, and the base is 1:(500-1000):(500-1000). The reaction is carried out at a temperature of 0-90℃ for 1-24 hours. The solvent is selected from a mixture of water and acetonitrile, a mixture of water and N,N-dimethylformamide, a mixture of water and ethanol, and a mixture of water and tetrahydrofuran; The alkali is triethylenediamine; The On-DNA α-hydroxyolefin lead compound is shown in Formula I: Formula I DNA is a single-stranded or double-stranded nucleotide chain; M is the connection unit; R2 is selected from nitro, cyano, and COR. 2a NHR 2a CONHR 2a , NHCOR 2a ; R3 and R4 are each independently selected from hydrogen, halogen, carboxyl, nitro, cyano, hydroxyl, mercapto, and COR. 2a NHR 2a CONHR 2a , NHCOR 2a Not replaced or R 3a The following groups are substituted: C1- 12 Alkyl, C 1-12 Alkoxy, C 2-8 alkenyl, C 2-8 alkynyl, 3-8 membered cycloalkyl, 5-6 membered aryl, 5-6 membered heteroaryl, R 3a Selected from C 1-12 Alkyl, C 1-12 Alkyl groups; or, R3 and R4 linked together to form a ring; R 2a Selected from C 1-12 Alkyl, C 1-12 Alkoxy, LR 2b OLR 2b L is C 1-6 Alkylene, R 2b Selected from C 1-12 Alkyl, unsubstituted or R 2c Substituted phenyl, R 2c Selected from C 1-12 Alkyl, C 1-12 Alkoxy; Alternatively, R4 is hydrogen, and R2 and R3 are linked together to form a ring.

2. The method according to claim 1, characterized in that: In Formula I, M is X1-Y1-P-Y2-X2-Q, and X1, Y1, P, Y2, X2, and Q are each independently selected from the following groups: unsubstituted, unsubstituted, or substituted by R1: NHCO, CONH, NH, 5-6 aryl, 5-6 heteroaryl, 3-8 saturated cycloalkyl, 3-8 saturated heterocyclic, C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group; m is an integer from 1 to 10; R1 is selected from hydrogen, halogen, nitro, cyano, mercapto, carboxyl, hydroxyl, halogenated or unhalogenated C. 1-6 Alkyl, halogenated or unhalogenated C 1-6 Alkoxy; X1, Y1, P, Y2, X2, and Q are not all zero at the same time.

3. The method according to claim 2, characterized in that: The structure of the On-DNA α-hydroxyolefin lead compound is shown in Formula II-1: Formula II-1 Among them, DNA is a single-stranded or double-stranded oligonucleotide chain; X1 is NHCO or CONH, X2 is NHCO or CONH; Y1 is either none or C 1-4 Alkylene, Y2 is absent or C 1-4 Alkylene; Q is selected from the following groups that are either unsubstituted or substituted by R1: 5-6 aryl, 5-6 heteroaryl, 3-6 saturated cycloalkyl, 3-6 saturated heterocyclic, C 1-4 Alkylene; m is an integer from 1 to 8; R1 is selected from hydrogen, halogen, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy; R2 is selected from nitro, cyano, and COR. 2a NHR 2a CONHR 2a , NHCOR 2a ; R3 and R4 are each independently selected from hydrogen, halogen, carboxyl, nitro, cyano, hydroxyl, mercapto, and COR. 2a NHR 2a CONHR 2a , NHCOR 2a Not replaced or R 3a The following groups are substituted: C1-6 alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, 3-8 membered cycloalkyl, 5-6 membered aryl, 5-6 membered heteroaryl, R 3a Selected from C 1-6 Alkyl, C 1-6 Alkyl groups; or, R3 and R4 linked together to form a ring; R 2a Selected from C 1-6 Alkyl, C 1-6 Alkoxy, LR 2b OLR 2b L is C 1-4 Alkylene, R 2b Selected from C 1-6 Alkyl, unsubstituted or R 2c Substituted phenyl, R 2c Selected from C 1-6 Alkyl, C 1-6 Alkoxy; Alternatively, R4 is hydrogen, and R2 and R3 are linked together to form a ring.

4. The method according to claim 3, characterized in that: The structure of the On-DNA α-hydroxyolefin lead compound is shown in Formula II-2: Formula II-2.

5. The method according to claim 3, characterized in that: The structures of the On-DNA α-hydroxyolefin lead compounds are shown in formulas III-a, III-b, III-c, III-d, III-e, or III-f: Formula III-a Formula III-b Formula III-c Formula III-d Formula III-e Formula III-f Among them, DNA is a single-stranded or double-stranded oligonucleotide chain; R1 is selected from hydrogen, halogen, C 1-3 Alkyl, C 1-3 Alkoxy; n is selected from 1, 2, and 3; R 2a Selected from C 1-3 Alkyl, C 1-3 Alkoxy, LR 2b OLR 2b L is C 1-2 Alkylene, R 2b Selected from C 1-3 Alkyl, unsubstituted or R 2c Substituted phenyl, R 2c Selected from C 1-3 Alkyl, C 1-3 Alkoxy; R3 is selected from hydrogen, C 1-3 Alkyl, C 1-3 Alkoxy, unsubstituted or R 3a The substituted phenyl group, R4 is selected from hydrogen, C 1-3 Alkyl, C 1-3 Alkoxy, unsubstituted or R 3a Substituted phenyl groups, or R3 and R4 linkages, form unsubstituted or R-substituted phenyl groups. 3b Substituted 4-membered saturated nitrogen heterocycle; R 3a Selected from C 1-3 Alkyl, C 1-3 Alkoxy, R 3b Selected from Boc, C 1-3 Alkyl, C 1-3 Alkoxy; Y3 is C 1-4 Alkylene; Y4 is C 1-4 Alkylene.

6. The method according to claim 1, characterized in that: The On-DNA α-hydroxyolefin lead compound is selected from one of the following compounds: 。 7. The method according to claim 1, characterized in that: The equivalent ratio of the compound shown in Formula Ia, the compound shown in Formula Ib, and the base is 1:1000:1000; The reaction was carried out at a temperature of 25°C for 16 hours.

8. The method according to any one of claims 1-7, characterized in that: The compound shown in formula Ia is The preparation method of the compound shown in Formula Ia includes the following steps: (1) Compound HP reacts with compound A1 to give compound A2; (2) Compound A2 reacts with a deprotecting agent to give compound A3; (3) Compound A3 reacts with compound A4 to obtain the compound shown in formula Ia.

9. The method according to claim 8, characterized in that: In step (1), the reaction is carried out in the presence of sodium tetraborate and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, with an equivalence ratio of compound HP, compound A1, sodium tetraborate, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride of 1:(30-50):(200-300):(30-50). The solvent for the reaction is selected from water, methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2,2-difluoroethanol, 2,2,2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, Glycerol, diethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1,4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 4-methoxypyridine, toluene, xylene, or a mixture of two or more of these, reacted at a temperature of 0-30°C. o C, the reaction time is 0.5-4 hours.

10. The method according to claim 9, characterized in that: The equivalent ratio of compound HP, compound A1, sodium tetraborate, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is 1:40:250:

40. The solvent for the reaction is a mixed solution of water and dimethyl sulfoxide, and the reaction temperature is 4... o C, the reaction time is 1 hour; In step (2), the deprotecting agent is piperidine; In step (3), the reaction is carried out in the presence of sodium tetraborate and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, with an equivalence ratio of compound A3, compound A4, sodium tetraborate, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride of 1:(30-50):(200-300):(30-50). The solvent for the reaction is selected from water, methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2,2-difluoroethanol, 2,2,2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, Glycerol, diethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1,4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 4-methoxypyridine, toluene, xylene, or a mixture of two or more of these, reacted at a temperature of 0-30°C. o C, the reaction time is 0.5-4 hours.

11. The method according to claim 10, characterized in that: The equivalent ratio of compound A3, compound A4, sodium tetraborate, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is 1:40:250:

40. The solvent for the reaction is a mixed solution of water and dimethyl sulfoxide. The reaction temperature is room temperature, and the reaction time is 2 hours.

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