Preparation method of On-DNA halogen-halogen coupling compound

Abstract

The invention discloses a preparation method of an On-DNA halogen-halogen coupling compound, which comprises the following steps: taking an On-DNA aryl halide as a substrate, reacting with an acid aryl halide compound under the conditions of alkali, a boron-containing compound and a palladium catalyst, realizing in-situ conversion of halogen into a boric acid (ester) bifunctional reagent through a one-pot method, and further obtaining the On-DNA halogen-halogen coupling compound through Suzuki reaction. The reaction method is high in yield, wide in substrate universality, mild in condition, convenient to operate and suitable for synthesis of the DNA coding compound library through a porous plate.

Description

Technical Field

[0001] The present invention belongs to the field of DNA-encoded compound libraries, and in particular relates to a method for preparing halogen-halogen coupling compounds in a DNA-encoded compound library through a one-pot process. Background Art

[0002] DNA-Encoded Library (DEL) technology is an emerging small molecule drug screening technique that combines DNA technology with combinatorial chemistry to efficiently construct compound libraries with billions of compounds. Furthermore, during the screening process, DEL can simultaneously screen multiple targets or multiple conditions for the same target. Compared with traditional high-throughput screening, DEL offers significant advantages in terms of compound library capacity, library construction difficulty, and screening time and cost.

[0003] While DNA-encoded compound libraries offer significant advantages in speed, scale, and cost, they also face several drawbacks, including relatively large molecular weights, a limited range of DNA-compatible chemical reactions, insufficient structural diversity, and limited library construction reagent diversity. This limited range of chemical reactions is a key constraint on the further development of DNA-encoded compound library technology. Currently, mature organic synthesis reactions applicable to DNA-encoded compound library technology are very limited. The most commonly used bonding reactions include amide bond formation, reduction, aromatic nucleophilic substitution, Suzuki coupling, Sonogashira coupling, Heck coupling, Buchwald coupling, and Ullmann coupling. One of the key areas of research in the DEL library field is the development of DNA-on-DNA chemical reactions, often referred to as on-DNA reactions. Because DNA requires a specific aqueous phase, pH, temperature, metal ion concentration, and inorganic salt concentration to maintain stability, on-DNA reactions that minimize DNA damage, offer good recovery rates, and possess broad substrate compatibility are essential for the large-scale synthesis of DNA-encoded compound libraries. Currently, there are hundreds of publicly reported On-DNA chemical reactions, including aqueous, solid-phase, and DNA-templated reactions. The reaction conditions for each type range from one to more than a dozen. It can be said that, under the same circumstances, the more types of On-DNA chemical reactions, the richer the conditions, and the wider the substrate types, the higher the diversity and the better the quality of the DNA-encoded compound library.

[0004] Aryl-coupled compounds are widely found in the structures of natural products and drug molecules and possess important biological activities and medicinal value. However, the commercial availability of diverse bifunctional aryl boronic acid substrates in DNA-encoded compound libraries has limited the introduction of further diversity into DNA-encoded compound libraries using the Suzuki reaction. The preparation of bifunctional aryl boronic acids using conventional small molecule synthesis and purification methods requires large quantities, requires long synthesis cycles, is labor-intensive, and is expensive. Utilizing on-DNA halogen-halogen coupling synthesis methods can significantly reduce small molecule synthesis costs, significantly shorten construction cycles, and expand substrate structural diversity. Furthermore, no synthetic methods for on-DNA halogen-halogen coupling compounds have been reported. Therefore, we aimed to develop a simple and rapid method for synthesizing on-DNA halogen-halogen coupling compounds. This research could enrich the structural diversity of DEL libraries and further enhance the application value of DNA-encoded compound library technology. Summary of the Invention

[0005] To address these issues, we have developed a synthesis method for a DNA-encoded compound library featuring stable raw material storage, mild reaction conditions, good substrate universality, minimal DNA damage, and suitability for batch operations using multi-well plates. This method enables a one-pot preparation of On-DNA halogen-halogen coupling compounds.

[0006] The present invention provides a method for preparing an On-DNA halogen-halogen coupling compound. The method comprises the following steps: using an On-DNA aryl halide as a substrate, reacting the substrate with an acid aryl halide compound, and in situ converting the halogen into a boric acid (ester) bifunctional reagent through a one-pot method using a small molecule bifunctional halogen-containing compound. The On-DNA halogen-halogen coupling compound is then obtained through a Suzuki reaction.

[0007] The technical solutions of the present invention are as follows:

[0008] A method for preparing an On-DNA halogen-halogen coupling compound, characterized in that: the method comprises using an On-DNA aryl halide as a substrate and reacting it with an acid aryl halide compound under the conditions of a base, a boron-containing compound, and a palladium catalyst to prepare the On-DNA halogen-halogen coupling compound;

[0009] Wherein, the structure of the On-DNA aryl halide is:

[0010] The structural formula of the acid aromatic halide compound is:

[0011] The structural formula of the On-DNA halogen-halogen coupling compound is:

[0012] Wherein, the DNA in the structural formula is a single-stranded or double-stranded nucleotide chain obtained by polymerization of artificially modified and / or unmodified nucleotide monomers, and the nucleotide chain is connected to the rest of the compound through one or more chemical bonds or groups;

[0013] The length of the DNA is 10 to 200 bp;

[0014] Wherein, the DNA and L in the structural formula are connected by one or more chemical bonds. When there is one chemical bond, it means that the DNA and L in the structural formula are directly connected; when there are multiple chemical bonds, it means that the DNA and L in the structural formula are connected by multiple chemical bonds. For example, DNA and L are connected to the amino group of DNA through a methylene (-CH2-), that is, they are connected through two chemical bonds; or DNA and L are connected to the amino group of DNA through a carbonyl (-CO-), also through two chemical bonds; or DNA and L are connected to the amino group of DNA through a methylene carbonyl (-CH2CO-), also through three consecutive chemical bonds.

[0015] Wherein, the L is selected from -(L Y ) q - or L does not exist; q is an integer from 1 to 30; each L Y are independently selected from CH2, C(O), O, S, S(O), S(O)2, NH, 3-10 membered cycloalkyl, 3-10 membered heterocycloalkyl, 6-10 membered aromatic ring, 5-10 membered aromatic heterocycle; the methylene, cycloalkyl, heterocycloalkyl, aromatic ring, aromatic heterocycle are optionally replaced by one, two or three independent R L replace;

[0016] Each R L are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1～6 Alkyl, halogen-substituted -C 1～6 alkyl;

[0017] The Ar 1 Selected from 6-10 membered aromatic rings, 5-10 membered aromatic heterocycles; the aromatic rings, aromatic heterocycles are optionally replaced by one, two or three independent R 1a replace;

[0018] Each R 1a are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1～6 Alkyl, halogen-substituted -C 1～6 Alkyl, -C 0～2 Alkylene-OH, -C 0～2 Alkylene-O(C 1～6 alkyl), 3-10 membered cycloalkyl, 3-10 membered heterocycloalkyl, 6-10 membered aromatic ring, 5-10 membered aromatic heterocycle, C 1～6Alkyl-substituted 5- to 10-membered aromatic heterocycle;

[0019] The Ar 2 Selected from 6-10 membered aromatic rings, 5-10 membered aromatic heterocycles; the aromatic rings, aromatic heterocycles are optionally replaced by one, two or three independent R 1b replace;

[0020] Each R 1b are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1～6 Alkyl, halogen-substituted -C 1～6 Alkyl, -C 0～2 Alkylene-OH, -C 0～2 Alkylene-O(C 1～6 alkyl), -NH2, -NH(C 1～6 Alkyl), -N(C 1～6 Alkyl)(C 1～6 alkyl), 3-10 membered cycloalkyl, 3-10 membered heterocycloalkyl, 6-10 membered aromatic ring, 5-10 membered aromatic heterocycle, -C(O)NH(C 1～6 alkyl), -NHC(O)(C 1～6 alkyl);

[0021] The R is selected from the group consisting of 1～6 alkyl, 3-10 membered cycloalkyl, 3-10 membered heterocycloalkyl, 6-10 membered aryl, 5-10 membered aromatic heterocyclic group or absent; the alkylene, cycloalkyl, heterocycloalkyl, aryl, aromatic heterocyclic group is optionally replaced by one, two or three independent R 1c replace;

[0022] Each R 1c are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1～6 Alkyl, halogen-substituted -C 1～6 Alkyl, -C 0～2 Alkylene-OH, -C 0～2 Alkylene-O(C 1～6 alkyl), -NH2, -NH(C 1～6 Alkyl), -N(C 1～6 Alkyl)(C 1～6 alkyl);

[0023] X1 and X2 are independently selected from halogen.

[0024] As preferred: said L is selected from CH2, NH, C(O), Or it does not exist. The non-existence refers to being directly connected by a covalent bond.

[0025] The Ar 1Selected from 6-membered aromatic ring, 5-membered aromatic heterocycle, 6-membered aromatic heterocycle, 9-membered aromatic heterocycle, 10-membered aromatic heterocycle; the aromatic ring, aromatic heterocycle is optionally replaced by one, two or three independent R 1a replace;

[0026] Each R 1a Each of the following is independently selected from hydrogen, methyl, ethyl, propyl, methoxy, and a 5-membered aromatic heterocycle substituted with methyl.

[0027] The Ar 1 Selected from

[0028] The On-DNA aryl halide is selected from:

[0029] As preferred: 2 Selected from 6-membered aromatic ring, 10-membered aromatic ring; the aromatic ring is optionally replaced by one, two or three independent R 1b replace;

[0030] Each R 1b Each is independently selected from hydrogen, methyl, ethyl, propyl, fluorine, -NHC(O)CH3.

[0031] Specifically: the Ar 2 Selected from The aa end is connected to X2;

[0032] Preferably, the R is selected from cyclopentyl, cyclohexyl, methylene, ethylene, phenyl, 5-membered aromatic heterocyclic group; the methylene, ethylene, phenyl, aromatic heterocyclic group is optionally replaced by one, two or three independent R 1c substituted or R is absent;

[0033] Each R 1c Each is independently selected from hydrogen, halogen, hydroxy, methyl, ethyl, methoxy, and -O(isopropyl).

[0034] More specifically: said R is selected from or or it doesn’t exist;

[0035] More specifically: the bifunctional acid aromatic halide compound is selected from:

[0036] Preferably, X1 and X2 are independently selected from fluorine, chlorine, bromine and iodine.

[0037] At the same time, the present invention provides a method for preparing an On-DNA halogen-halogen coupling compound, the method comprising the following steps:

[0038] Step 1, adding 50-1000 times the molar equivalent of a boron-containing compound, 50-2000 times the molar equivalent of a base and 1-15 times the molar equivalent of a palladium catalyst (molar equivalent relative to the molar amount of DNA) to a 50-1000 times molar equivalent of an acid aryl halide solution, and reacting at 10° C. to 100° C. for 0.1 to 24 hours;

[0039] Step 2: Adding a molar equivalent of 1 and a molar concentration of 0.1-5 mM On-DNA aryl halide solution to the system after the reaction in step 1, 50-2000 times the molar equivalent of a base and 1-15 times the molar equivalent of a palladium catalyst, reacting at 10° C. to 100° C. for 0.1 to 24 hours until the reaction is completed, and performing alcohol precipitation to obtain a reaction product.

[0040] Preferably, the boron-containing compound in step 1 is one or both of tetrahydroxydiboron and biboronic acid pinacol ester.

[0041] Furthermore, in step 1 and step 2, the base is selected from sodium borate, lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, potassium acetate, sodium carbonate, potassium carbonate, cesium carbonate, sodium phosphate, potassium phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate, N-methylmorpholine, triethylamine, diisopropylethylamine, DBU (1,8-diazabicycloundec-7-ene), 4-dimethylaminopyridine, 2,6-lutidine or N-methylimidazole; preferably, the base in step 1 is potassium acetate; the base in step 2 is cesium hydroxide.

[0042] Further, in step 1 and step 2, the palladium catalyst is selected from palladium acetate, palladium dichloride, palladium hydroxide, tetrakis(triphenylphosphine)palladium, tris(dibenzylideneacetone)palladium, bis(dibenzylideneacetone)palladium, bis(acetonitrile)palladium dichloride (II), bis(triphenylphosphine)palladium chloride, 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride, bis(benzonitrile)palladium dichloride, 1,4-bis(diphenylphosphino)butane-palladium chloride, [di-tert-butyl(chloro)phosphine]palladium dichloride (II) dimer, bis(methyldiphenylphosphine)palladium dichloride (II), benzylbis(triphenylphosphine)palladium chloride (II), di Dichlorobis(di-tert-butylphosphinyl-KP) palladium (2-yl), chloro(sodium-2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl-3'-sulfonate)[2-(2'-amino-1,1'-biphenyl)]palladium(II), chloro(2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II), methanesulfonic acid(2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium (II), methanesulfonic acid (2-dicyclohexylphosphino-2',6'-diisopropyloxy-1,1'-biphenyl) (2-amino-1,1'-biphenyl-2-yl) palladium (II), chloro (2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl) [2-(2'-amino-1,1'-biphenyl)] palladium (II), chloro (2-dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl) [2-(2-aminoethylphenyl)] palladium (II), methanesulfonic acid (9,9-dimethylamino) One or more of: palladium (II) selected from the group consisting of (4-(N,N-dimethylamino)phenyl)di-tert-butylphosphine (2-amino-1,1'-biphenyl-2-yl) palladium (II) and (2-amino-1,1'-biphenyl-2-yl) palladium (II) methanesulfonate.

[0043] Preferably, the palladium catalyst in step 1 is 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride, and the palladium catalyst in step 2 is chloro(2-dicyclohexylphosphine-2',6'-dimethoxy-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II).

[0044] Furthermore, the reaction is carried out in a solvent, which is any one or a mixed solvent of water, methanol, ethanol, acetonitrile, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, an inorganic salt buffer, an organic acid buffer, and an organic base buffer; preferably, the reaction solvent in step 1 is dimethylacetamide, water, and dimethyl sulfoxide; the reaction solvent in step 1 is sodium borate / boric acid buffer solution and dimethylacetamide.

[0045] Furthermore, the reaction temperature of the reaction is 10°C to 100°C; preferably, the reaction temperature is 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, or 100°C.

[0046] Furthermore, the reaction time is 0.5 to 24 hours; preferably, the reaction time is 0.5 hours, 1 hour, 2 hours, 2.5 hours, 4 hours, 8 hours, 10 hours, 16 hours, 18 hours, or 20 hours.

[0047] Furthermore, in step 1, the molar equivalent of the acid aryl halide compound is 50 to 1000, the molar equivalent of the boron-containing compound is 50 to 1000, the molar equivalent of the base is 50 to 2000; and the molar equivalent of the palladium catalyst is 1 to 15;

[0048] Preferably, the molar equivalent of the acid aromatic halide compound in step 1 is 50, 100, 200, 300, 400, 500, 600, 800, 1000, the molar equivalent of the boron-containing compound is 50, 100, 200, 300, 400, 500, 600, 800, 1000, the molar equivalent of the base is 50, 100, 200, 300, 400, 500, 600, 800, 1000, 1500, 2000; the molar equivalent of palladium is 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 10, 12, 14, 15.

[0049] Most preferably, in step 1, the molar equivalent of the acid aryl halide is 100, the molar equivalent of the boron-containing compound is 200, the molar equivalent of the base is 100, and the molar equivalent of the palladium catalyst is 2.5.

[0050] In step 2, the molar equivalent of the On-DNA aryl halide in the method is 1, the molar equivalent of the base is 50 to 2000, and the molar equivalent of the palladium catalyst is 1-15; preferably, the molar equivalent of the base is 50, 100, 200, 300, 400, 450, 500, 600, 800, 1000, 1500, 2000; the molar equivalent of palladium is 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 10, 12, 14, 15; most preferably, the molar equivalent of the base is 450, and the molar equivalent of the palladium catalyst is 2.5.

[0051] Furthermore, the method is used for batch multi-well plate operations.

[0052] Furthermore, the method is used for the synthesis of DNA-encoded compound libraries in multi-well plates.

[0053] The method of the present invention enables a one-pot preparation of On-DNA halogen-halogen coupling compounds from a DNA-encoded compound library. It is widely applicable to various On-DNA aryl halides and can introduce a variety of substituted aryl halides and bifunctional halogen-containing compounds as synthetic modules on a large scale. The method has high yield, produces a single product, can be carried out in an organic solvent / aqueous mixed aqueous phase, is simple to operate, is environmentally friendly, and is suitable for synthesizing DNA-encoded compound libraries using multiwell plates.

[0054] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined herein, they should be given the meaning that a person skilled in the art would give them based on the disclosure and context.

[0055] "Substitution" means that the hydrogen atoms in a molecule are replaced by other different atoms or groups; or the lone pair of electrons in the atoms in the molecule are replaced by other atoms or groups. For example, the lone pair of electrons on the S atom can be replaced by an O atom to form

[0056] "Optionally substituted" means that "substitution" may but need not occur, and the description includes instances where it occurs and instances where it does not occur.

[0057] The minimum and maximum carbon atom content in the hydrocarbon group is indicated by a prefix, for example, the prefix (Ca~C b )alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C1-C 20 The alkyl group refers to a straight-chain or branched alkyl group containing 1 to 20 carbon atoms.

[0058] "Alkyl" refers to a saturated hydrocarbon chain having a specified number of member atoms. Alkyl groups can be straight or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl groups may optionally be substituted with one or more substituents as defined herein. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and tert-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl. Alkyl groups may also be part of other groups, such as -O(C 1～6 alkyl).

[0059] "Alkylene" refers to a divalent saturated aliphatic hydrocarbon radical having the specified number of member atoms. a ~ b Alkylene refers to an alkylene group having a to b carbon atoms. Alkylene groups include branched and straight chain hydrocarbon groups. For example, the term "propylene" can be exemplified by the following structure: Likewise, the term "dimethylbutylene" can be exemplified, for example, by any of the following structures:

[0060] The -C0~4 alkylene group of the present invention can be a C0 alkylene group, a C1 alkylene group (e.g., -CH2-), a C2 alkylene group (e.g., -CH2CH2-, etc.), a C3 alkylene group or a C4 alkylene group; the C0 alkylene group means that the group here does not exist and is connected in the form of a chemical bond, such as A-C0 alkylene-B means AB, that is, the A group and the B group are directly connected by a chemical bond.

[0061] The unsaturated group mentioned in the present invention refers to a group or molecule containing a carbon-carbon double bond, a carbon-carbon triple bond, a carbon-oxygen double bond, a carbon-sulfur double bond, a carbon-nitrogen triple bond, etc.

[0062] "Alkenyl" refers to a straight or branched chain hydrocarbon group having at least one site of vinyl unsaturation (>C=C<). For example, C a-b Alkenyl refers to an alkenyl group having a to b carbon atoms and is intended to include, for example, ethenyl, propenyl, isopropenyl, 1,3-butadienyl, and the like.

[0063] "Alkynyl" refers to a straight chain monovalent hydrocarbon radical or a branched monovalent hydrocarbon radical containing at least one triple bond. The term "alkynyl" is also intended to include those hydrocarbon radicals having one triple bond and one double bond. For example, C 2-6 Alkynyl is meant to include ethynyl, propynyl, and the like.

[0064] "Cycloalkyl" and "cycloalkane" refer to saturated or partially saturated cyclic groups having carbon atoms and no ring heteroatoms and having a single ring or multiple rings (including fused and combined). For polycyclic ring systems with aromatic and non-aromatic rings without ring heteroatoms, the term "cycloalkyl" is applicable when the point of attachment is at a non-aromatic carbon atom (e.g., 5,6,7,8,-tetrahydronaphthalene-5-yl). The term "cycloalkyl" includes cycloalkenyl groups such as cyclohexenyl. Examples of cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclooctyl, cyclopentenyl, and cyclohexenyl. Examples of cycloalkyl groups including polybicycloalkyl ring systems are bicyclohexyl, bicyclopentyl, bicyclooctyl, etc. For example

[0065] "Heterocycle", "heterocycloalkyl" and "heterocycloalkane" refer to a saturated ring or non-aromatic unsaturated cyclic group having a single ring or multiple rings (including fused and combined) containing at least one heteroatom; wherein the heteroatom refers to a nitrogen atom, an oxygen atom, or a sulfur atom;

[0066] "Aryl" and "aromatic ring" are used interchangeably to refer to an all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group with a conjugated π electron system, such as "C 6-10 The term "aryl" refers to a monocyclic or bicyclic aromatic group having 6 to 10 carbon atoms. Non-limiting examples of aryl include phenyl, naphthyl, and the like.

[0067] The "aromatic heterocycle" described in the present invention refers to an aromatic unsaturated ring containing at least one heteroatom; wherein the heteroatom refers to a nitrogen atom, an oxygen atom, a sulfur atom, etc. It is usually an aromatic monocyclic or bicyclic hydrocarbon containing multiple ring atoms, wherein one or more ring atoms are selected from O, N, and S heteroatoms. Preferably, there are one to three heteroatoms. Heterocyclic aromatic groups are represented by pyridyl, indolyl, quinoxalinyl, quinolyl, isoquinolyl, benzothiophenyl, benzofuranyl, benzothiophenyl, benzopyranyl, benzothiapyranyl, furyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, oxadiazolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl

[0068] The "aromatic heterocycle" mentioned in the invention also includes aromatic rings, aromatic heterocycles and saturated heterocycles or saturated rings, for example:

[0069] The halogen is fluorine, chlorine, bromine or iodine.

[0070] The "halogen-substituted alkyl" mentioned in the present invention refers to an alkyl group in which one or more hydrogen atoms are replaced by halogen; for example, a halogen-substituted C 1～4 The alkyl group refers to an alkyl group containing 1 to 4 carbon atoms in which hydrogen atoms are substituted by one or more halogen atoms; examples include monofluoromethyl, difluoromethyl, and trifluoromethyl.

[0071] Alkoxy: refers to an alkyl group connected to an oxygen atom to form a substituent, for example, methoxy is -OCH3.

[0072] The "-OR", "-NRR" and the like described in the present invention refer to that the R group is connected to the oxygen atom or nitrogen atom via a single bond.

[0073] The oxygen atom in "-C(O)R", "-S(O)2R" and the like described in the present invention is connected to the carbon atom or the sulfur atom via a double bond.

[0074] In the present invention, the oxygen atom in "-C(O)R", "-S(O)2R", etc. is connected to the carbon atom or sulfur atom by a double bond, and the R group is connected to the oxygen atom or sulfur atom by a single bond. For example, "-S(O)(NH)R" means that the oxygen atom and the nitrogen atom are connected to the sulfur atom by a double bond, and the R group is connected to the sulfur atom by a single bond.

[0075] The “---” in the description of the group of the present invention, It is used to describe the position of the substitution group. For example It means that the tetrahydropyrrole ring is fused with other rings in the structure through the position of "---".

[0076] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0077] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 : Structures and conversion rates of 12 On-DNA halogen-halogen coupling compounds in Example 2 of the present invention. DETAILED DESCRIPTION

[0079] The following is a detailed description of the above contents of the present invention by way of specific embodiments in the form of examples, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0080] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0081] In the present invention, DNA-NH2 is a DNA structure with an -NH2 linker formed by single-stranded or double-stranded DNA and a linker group, such as the DNA-NH2 structure of "compound 1" in WO2005058479. For example, the following DNA structure:

[0082]

[0083] Among them, A is adenine, T is thymine, C is cytosine, and G is guanine.

[0084] DIPEA: N,N-diisopropylethylamine; DIC: N,N'-diisopropylcarbodiimide; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DMA: dimethylacetamide; DMSO: dimethyl sulfoxide; DDTC: sodium diethyldithiocarbamate. Pd(dppf)Cl2: 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride sSPhosPd G2: Chloro(sodium-2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl-3'-sulfonate)[2-(2'-amino-1,1'-biphenyl)]palladium(II)

[0085] Example 1: Method for constructing On-DNA halogen-halogen coupling compounds

[0086] Step 1. Synthesis of On-DNA Aryl Halogen Compounds

[0087]

[0088] DNA (HP-AOP-NH2) was dissolved in 250 mM borate buffer (pH 9.4) to a 1 mM solution (20 μL, 20 nmol). m-Bromobenzoic acid (50 equivalents, 1000 nmol, 200 mM dissolved in di-DMA), HATU (50 equivalents, 1000 nmol, 400 mM dissolved in DMA), and DIPEA (50 equivalents, 1000 nmol, 400 mM dissolved in DMA) were mixed sequentially at 0°C. The mixture was thoroughly mixed by vortexing and then stored at 0°C for 5 minutes. The activation solution was then added to the DNA solution, mixed thoroughly, and reacted at 25°C for 1 hour.

[0089] After the reaction, ethanol precipitation was performed: 10% of the total volume of 5M sodium chloride aqueous solution was added to the reaction solution, and then 3 times the total volume of anhydrous ethanol was added. After shaking evenly, the reaction was placed in dry ice and frozen for 0.5 hours. Then, it was centrifuged at a speed of 12000 rpm and low temperature (4°C) for half an hour. The supernatant was poured out, and the remaining precipitate was freeze-dried and dissolved in deionized water to obtain an aqueous solution of On-DNA aryl halogen compound (1). After OD quantification, LC-MS was sent to confirm that the conversion rate of compound 1 was 90%.

[0090] Step 2: Synthesis of On-DNA halogen-halogen coupling compounds

[0091]

[0092] (1) 1-(3-Bromophenyl)cyclohexanecarboxylic acid (2) (100 equivalents, 2000 nanomoles, 200 mM dissolved in di-DMA), pinacol diboron (3) (200 equivalents, 4000 nanomoles, 200 mM dissolved in di-DMA), potassium acetate (100 equivalents, 2000 nanomoles, 200 mM dissolved in di-H2O), and Pd(dppf)Cl2 (2.5 equivalents, 50 nanomoles, 10 mM dissolved in di-DMSO) were mixed uniformly and activated at 90°C for 2.5 hours. The reaction was completed without additional treatment and the reaction solution was placed at room temperature;

[0093] (2) The On-DNA aryl halide compound (1) was prepared into a 1 mM solution (20 μL, 20 nmol) with deionized water and added to the reaction solution cooled at room temperature in step 1. Subsequently, cesium hydroxide (450 equivalents, 9000 nmol, 1 M dissolved in H2O) and sSPhos-Pd-G2 (2.5 equivalents, 50 nmol, 10 mM dissolved in DMSO) were added in sequence, mixed well, and reacted at 90°C for 30 minutes.

[0094] After the reaction, ethanol precipitation was performed: 10% of the total volume of 5M sodium chloride aqueous solution was added to the reaction solution, and then 3 times the total volume of anhydrous ethanol was added. After shaking evenly, the reaction was placed in dry ice and frozen for 0.5 hours. Then, it was centrifuged at a speed of 12000 rpm and low temperature (4°C) for half an hour. The supernatant was poured out, and the remaining precipitate was freeze-dried and dissolved in deionized water to obtain an aqueous solution of On-DNA halo-halogen coupling compound (4). After quantification by OD, LC-MS was sent to confirm that the conversion rate of compound (4) was 86%.

[0095] Example 2: Method for constructing On-DNA halogen-halogen coupling compounds

[0096]

[0097] (1) 100 equivalents of aryl halide (6) (0.2 M, dissolved in DMA), 200 equivalents of bis(pinacolato) borate (3) (0.2 M, dissolved in DMA), 100 equivalents of potassium acetate (0.2 M, dissolved in H2O), and 2.5 equivalents of Pd(dppf)Cl2 (10 mM, dissolved in DMSO) were mixed and reacted at 90°C for 2.5 hours. After the reaction was completed, the reaction solution was allowed to stand at room temperature.

[0098] (2) Dissolve the On-DNA aryl halide (5) in water to prepare a 1 mM solution, and add it to the reaction solution in step 1. Then, add 450 equivalents of cesium hydroxide (1 M concentration, dissolved in H2O) and 2.5 equivalents of sSPhos-Pd-G2 (10 mM concentration, dissolved in DMA) in sequence, mix well, and react at 90°C for 2 hours.

[0099] After the reaction is completed, 100 equivalents of sodium diethyldithiocarbamate (concentration 0.4M, dissolved in H2O) are added to the reaction solution, mixed evenly, and reacted at 90°C for 10 minutes. After the reaction is completed, ethanol precipitation is performed: 10% of the total volume of 5M sodium chloride solution is added to the reaction solution, and then 3 times the total volume of anhydrous ethanol is added. After shaking evenly, the reaction is placed in dry ice and frozen for 0.5 hours, and then centrifuged at a speed of 12000 rpm for half an hour, the supernatant is poured out, and the remaining precipitate is dissolved in deionized water to obtain a solution of On-DNA product. After quantification by OD of an enzyme marker, LC-MS is detected at the same time to confirm the conversion rate of each target compound (7). The results are shown in the attached manual. Figure 1 .

[0100] This example illustrates that the method of the present invention is an innovative method for preparing On-DNA halogen-halogen coupling compounds.

[0101] In summary, the above embodiments and drawings are only some representative embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0102] This method is applicable to a variety of different On-DNA aryl halides and bifunctional halogen-containing compounds, showing good universality. In summary, the present invention successfully constructs halogen-halogen coupling compounds by controlling the solvent, temperature, pH and other conditions during the reaction in the presence of a palladium catalyst, a base, and a boron-containing compound. The substrate of this method has a wide range of applications and can be carried out in a mixed aqueous phase of an organic solvent / aqueous phase. It is simple to operate, environmentally friendly, and suitable for the synthesis of DNA-encoded compound libraries using porous plates.

Claims

1. A method for preparing an On-DNA halogen-halogen coupling compound, characterized in that: The method is to use an On-DNA aryl halide as a substrate, react it with an acid aryl halide compound under the conditions of a base, a boron-containing compound and a palladium catalyst to prepare an On-DNA halogen-halogen coupling compound; Wherein, the structure of the On-DNA aryl halide is: The structural formula of the acid aromatic halide compound is: The structural formula of the On-DNA halogen-halogen coupling compound is: Wherein, the DNA in the structural formula is a single-stranded or double-stranded nucleotide chain obtained by polymerization of artificially modified and / or unmodified nucleotide monomers, and the nucleotide chain is connected to the rest of the compound through one or more chemical bonds or groups; Wherein, the L is selected from -(L Y ) q - or does not exist; q is an integer from 1 to 30; each L Y are independently selected from CH2, C(O), O, S, S(O), S(O)2, NH, 3-10 membered cycloalkyl, 3-10 membered heterocycloalkyl, 6-10 membered aromatic ring, 5-10 membered aromatic heterocycle; the methylene, cycloalkyl, heterocycloalkyl, aromatic ring, aromatic heterocycle are optionally replaced by one, two or three independent R L replace; Each R L are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1～6 Alkyl, halogen-substituted -C 1～6 alkyl; The Ar 1 Selected from 6-10 membered aromatic rings, 5-10 membered aromatic heterocycles; the aromatic rings, aromatic heterocycles are optionally replaced by one, two or three independent R 1a replace; Each R 1a are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1～6 Alkyl, halogen-substituted -C 1～6 Alkyl, -C 0～2 Alkylene-OH, -C 0～2 Alkylene-O(C 1～6 alkyl), 3-10 membered cycloalkyl, 3-10 membered heterocycloalkyl, 6-10 membered aromatic ring, 5-10 membered aromatic heterocycle, C 1～6 Alkyl-substituted 5- to 10-membered aromatic heterocycle; The Ar 2 Selected from 6-10 membered aromatic rings, 5-10 membered aromatic heterocycles; the aromatic rings, aromatic heterocycles are optionally replaced by one, two or three independent R 1b replace; Each R 1b are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1～6 Alkyl, halogen-substituted -C 1～6 Alkyl, -C 0～2 Alkylene-OH, -C 0～2 Alkylene-O(C 1～6 alkyl), -NH2, -NH(C 1～6 Alkyl), -N(C 1～6 Alkyl)(C 1～6 alkyl), 3-10 membered cycloalkyl, 3-10 membered heterocycloalkyl, 6-10 membered aromatic ring, 5-10 membered aromatic heterocycle, -C(O)NH(C 1～6 alkyl), -NHC(O)(C 1～6 alkyl); The R is selected from the group consisting of 1～6 alkyl, 3-10 membered cycloalkyl, 3-10 membered heterocycloalkyl, 6-10 membered aryl, 5-10 membered aromatic heterocyclic group or absent; the alkylene, cycloalkyl, heterocycloalkyl, aryl, aromatic heterocyclic group is optionally replaced by one, two or three independent R 1c replace; Each R 1c are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1～6 Alkyl, halogen-substituted -C 1～6 Alkyl, -C 0～2 Alkylene-OH, -C 0～2 Alkylene-O(C 1～6 alkyl), -NH2, -NH(C 1～6 Alkyl), -N(C 1～6 Alkyl)(C 1～6 alkyl); X1 and X2 are independently selected from halogen.

2. The method according to claim 1, wherein: The method comprises the following steps: Step 1: Add 50-1000 times the molar equivalent of a boron-containing compound, 50-2000 times the molar equivalent of a base, and 1-15 times the molar equivalent of a palladium catalyst to a 50-1000 times molar equivalent of an acid aryl halide solution, and react at 10° C. to 100° C. for 0.1 to 24 hours; Step 2: Add an On-DNA aryl halide solution with a molar equivalent of 1 and a molar concentration of 0.1-5 mM to the system after the reaction in step 1, then add 50-2000 times the molar equivalent of a base and 1-15 times the molar equivalent of a palladium catalyst, react at 10° C. to 100° C. for 0.1 to 24 hours, until the reaction is completed, and precipitate the reaction with alcohol to obtain a reaction product.

3. The method according to claim 2, wherein: The boron-containing compound in step 1 is one or both of tetrahydroxydiboron and biboric acid pinacol ester.

4. The method according to claim 2, wherein: In step 1 and step 2, the base is selected from sodium borate, lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, potassium acetate, sodium carbonate, potassium carbonate, cesium carbonate, sodium phosphate, potassium phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate, N-methylmorpholine, triethylamine, diisopropylethylamine, DBU (1,8-diazabicycloundec-7-ene), 4-dimethylaminopyridine, 2,6-lutidine or N-methylimidazole.

5. The method according to claim 2, wherein: In step 1 and step 2, the palladium catalyst is selected from palladium acetate, palladium dichloride, palladium hydroxide, tetrakis(triphenylphosphine)palladium, tris(dibenzylideneacetone)palladium, bis(dibenzylideneacetone)palladium, bis(acetonitrile)palladium dichloride (II), bis(triphenylphosphine)palladium chloride, 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride, bis(benzonitrile)palladium dichloride, 1,4-bis(diphenylphosphino)butane-palladium chloride, [di-tert-butyl(chloro)phosphine]palladium dichloride (II) dimer, bis(methyldiphenylphosphine)palladium dichloride (II), benzylbis(triphenylphosphine)palladium dichloride Phosphine) palladium(II) chloride, dihydrogen dichlorobis(di-tert-butylphosphinyl-KP) palladium acid(2-), chloro(sodium-2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl-3'-sulfonate)[2-(2'-amino-1,1'-biphenyl)]palladium(II), methanesulfonic acid(2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II), methanesulfonic acid(2-dicyclohexylphosphino-2',6'-diisopropyloxy- Palladium(II) chloride, 2-(2-amino-1,1'-biphenyl)-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II), chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II), chloro(2-dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethylphenyl)]palladium(II), methanesulfonic acid (9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene) One or more of (2'-amino-1,1'-biphenyl-2-yl) palladium (II), methanesulfonate (2-dicyclohexylphosphino-N,N-dimethylamino-1,1'-biphenyl) (2'-amino-1,1'-biphenyl-2-yl) palladium (II), methanesulfonate [(4-(N,N-dimethylamino)phenyl]di-tert-butylphosphine (2-amino-1,1'-biphenyl-2-yl) palladium (II), and methanesulfonate-1,1'-bis(diphenylphosphino)ferrocene (2-amino-1,1'-biphenyl-2-yl) palladium (II).

6. The method according to claim 2, wherein: In step 1 and step 2, the reaction is carried out in a solvent, which is any one or a mixed solvent of water, methanol, ethanol, acetonitrile, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, inorganic salt buffer, organic acid buffer, and organic base buffer.

7. The method according to claim 2, wherein: In step 1 and step 2, the reaction temperature is 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C.

8. The method according to claim 2, wherein: In step 1 and step 2, the reaction time is 0.5 hour, 1 hour, 2 hours, 2.5 hours, 4 hours, 8 hours, 10 hours, 16 hours, 18 hours, and 20 hours.

9. The method according to claim 2, wherein: In step 1 and step 2, the molar equivalent of the On-DNA aryl halide in the method is 1, the molar equivalent of the acid aryl halide compound is 50, 100, 200, 300, 400, 500, 600, 800, 1000; the molar equivalent of the boron-containing compound is 50, 100, 200, 300, 400, 500, 600, 800, 1000; the molar equivalent of the base is 50, 100, 200, 300, 400, 450, 500, 600, 800, 1000, 1500, 2000; and the molar equivalent of palladium is 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 10, 12, 14, 15.

10. The method according to any one of claims 1 to 9, characterized in that: The method is used for batch multiwell plate operations.

11. The method according to any one of claims 1 to 9, characterized in that: The method is used for the synthesis of DNA-encoded compound libraries in multi-well plates.

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