CN114478670B - A method for synthesizing On-DNA β-substituted ketone compounds - Google Patents

Abstract

The invention relates to a method for synthesizing On-DNA beta substituted ketone compound, which takes On-DNA alpha, beta unsaturated carbonyl compound as raw material, reacts with aromatic boric acid or alkenyl boric acid compound in the presence of palladium catalyst and alkali to obtain On-DNA beta substituted ketone compound. The method can be carried out in a mixed water phase of an organic solvent/water phase, has simple post-treatment and mild conditions, can obtain a DNA coding compound library with high diversity in a short time and high yield, and is suitable for synthesizing the DNA coding compound by a porous plate.

Description

A method for synthesizing On-DNA β-substituted ketone compounds

Technical field

The invention belongs to the technical field of encoded compound libraries, and specifically relates to a method for constructing On-DNA β-substituted ketone compounds in a DNA-encoded compound library.

Background technique

In drug research and development, especially new drug research and development, high-throughput screening of biological targets is one of the main means to quickly obtain lead compounds. However, traditional high-throughput screening based on single molecules requires a long time, huge equipment investment, limited number of library compounds (millions), and the construction of compound libraries requires decades of accumulation, which limits the efficiency and efficiency of lead compound discovery. possibility. The DNA-encoded compound library technology that has emerged in recent years (WO2005058479, WO2018166532, CN103882532) combines combinatorial chemistry and molecular biology technology to add a DNA tag to each compound at the molecular level, and can be synthesized in a very short time Compound libraries of up to 100 million levels have become the trend of next-generation compound library screening technology, and have begun to be widely used in the pharmaceutical industry, producing many positive effects (Accounts of Chemical Research, 2014, 47, 1247-1255).

DNA-encoded compound libraries quickly generate giant compound libraries through combinatorial chemistry, and can screen lead compounds in high-throughput, making the screening of lead compounds faster and more efficient than ever before. One of the challenges in constructing DNA-encoded compound libraries is the need to synthesize chemically diverse small molecules on DNA with high yields. Since DNA needs to be stable under certain conditions (solvent, pH, temperature, ion concentration), On-DNA reactions used in the construction of DNA-encoded compound libraries also need to have a higher yield. Therefore, the types of reagents, reaction types, and reaction conditions of chemical reactions performed on DNA (referred to as On-DNA reactions) directly affect the richness and selectivity of the DNA-encoded compound library. Therefore, the development of chemical reactions compatible with DNA has become a long-term exploration and research direction of current DNA-encoded compound library technology, which also directly affects the application and commercial value of DNA-encoded compound libraries.

α,β unsaturated carbonyl compounds and boronic acid compounds form sp ² hybridized carbon atom connection sites at the β position of the carbonyl group through conjugate addition, which can enrich the topological structure of pharmaceutical compounds and introduce such β-substituted ketone compounds into The DNA-encoded compound library can further expand the diversity of the compound library, which is helpful to increase the probability of screening effective compounds. However, there is currently no report on the synthesis of On-DNAβ-substituted ketones through On-DNAα,β-unsaturated carbonyl compounds. Therefore, it is hoped to develop a new synthesis method for On-DNAβ-substituted ketone compounds suitable for large-scale multi-well plate operation to increase the diversity of DNA-encoded compound libraries and further enhance the application value of DNA-encoded compound library technology.

Contents of the invention

In order to solve the above problems, we have developed a method for synthesizing a DNA-encoded compound library with stable storage of raw materials, mild reaction conditions, good substrate versatility, little damage to DNA, and suitable for batch operation using multi-well plates, which can quickly On-DNAα,β-unsaturated carbonyl compounds are converted into On-DNAβ substituted ketones through a one-step reaction.

The technical solutions adopted by the present invention are as follows:

A method for synthesizing On-DNAβ-substituted ketone compounds: the method uses On-DNAα,β unsaturated carbonyl compounds as raw materials, reacts with boric acid compounds in the presence of a palladium catalyst and a base to obtain On-DNAβ-substituted ketone compounds; wherein , the structural formula of On-DNAα,β unsaturated carbonyl compound is The structural formula of boric acid compound is:/> The structural formula of On-DNAβ substituted ketone compounds is:/>

Wherein, the DNA in the structural formula includes a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers, and the nucleotide chain is connected to the nucleotide chain through one or more chemical bonds or groups. The remaining parts of the compound are connected; the length of the DNA is 10 to 200 bp.

Among them, the DNA in the structural formula and R ₁ or R ₃ are connected through one chemical bond or multiple chemical bonds. When there is one chemical bond, it means that the DNA in the structural formula is directly connected to R ₁ or R ₃ ; when there are multiple chemical bonds, it means that there are multiple chemical bonds between the DNA and R ₁ or R ₃ in the structural formula. For example, DNA is connected to R ₁ or R 3. R ₃ are connected through a methylene group (-CH ₂ -), that is, connected through two chemical bonds; or DNA and R ₁ or R ₃ are connected through a carbonyl group (-CO-) to the amino group of DNA, also through two chemical bonds. Connected by a chemical bond; or DNA and R ₁ or R ₃ are connected to the amino group of DNA through a methylene carbonyl group (-CH ₂ CO-), which is also connected through three consecutive chemical bonds.

R ₁ is selected from a group with a molecular weight of less than 1000 that is directly connected to DNA and carbonyl carbon atoms or does not exist;

R ₂ is selected from a group with a molecular weight of less than 1000 directly connected to an alkenyl carbon atom;

R ₃ is selected from a group with a molecular weight of less than 1000 that is directly connected to DNA and alkenyl carbon atoms or does not exist;

R ₄ is selected from a group with a molecular weight of less than 1000 that is directly connected to the carbonyl carbon atom;

R ₅ is selected from hydrogen or a group with a molecular weight of less than 1000 directly connected to the alkenyl carbon atom;

R ₆ is selected from a group with a molecular weight of less than 1000 that is directly connected to the boron atom in the boric acid compound;

Or R ₅ forms a ring with R ₁ or R ₄ respectively.

Preferably: R ₁ , R ₂ , R ₃ and R ₄ are respectively selected from alkyl, substituted alkyl, aryl or substituted aryl; wherein, the alkyl is C ₁ to C ₂₀ alkyl or C ₃ ~C ₈ cycloalkyl; the number of substituents of the substituted alkyl group is one or more; the substituents of the substituted alkyl group are independently selected from halogen, nitro, alkoxy, halophenyl, phenyl, One or more of alkylphenyl and heterocyclyl; the aromatic group is selected from pyridyl, quinolyl, thiazolyl, thienyl or phenyl; the number of substituents of the substituted aromatic group is one or more Each, the substituents of the substituted aromatic group are independently selected from one or more types of halogen, cyano, nitro, alkoxy, C ₁ to C ₂₀ alkyl, and trifluoromethyl;

The R ₅ is selected from hydrogen and C ₁ to C ₂₀ alkyl;

The R ₆ is selected from alkenyl, substituted alkenyl, aryl or substituted aryl; the alkenyl is selected from C ₂ to C ₂₀ alkenyl or C ₃ to C ₈ cycloalkenyl; the substituents of the substituted alkenyl are The number is one or more, and the substituents of the substituted alkenyl group are independently selected from one or more of halogen, cyano, nitro, alkoxy, C ₁ to C ₂₀ alkyl, and trifluoromethyl. ; The aryl group is selected from pyridyl, quinolyl, thiazolyl, thienyl or phenyl; the number of substituents of the substituted aryl group is one or more, and the substituents of the substituted aryl group are independently selected from halogen , one or more of cyano group, nitro group, alkoxy group, C ₁ to C ₂₀ alkyl group, and trifluoromethyl group.

further:

The R ₁ is selected from phenyl and thienyl; the R ₂ is selected from phenyl, C ₁ to C ₆ alkyl; the C ₁ to C ₆ alkyl is specifically selected from methyl, ethyl, n- Propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl;

The R ₃ is selected from phenyl and thienyl; the R ₄ is selected from phenyl, C ₁ to C ₆ alkyl; the C ₁ to C ₆ alkyl is specifically selected from methyl, ethyl, n- Propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl;

The R ₅ is selected from hydrogen, C ₁ to C ₆ alkyl; or R ₅ forms a ring with R ₁ or R ₄ respectively; the C ₁ to C ₆ alkyl is specifically selected from methyl, ethyl, n-propyl , isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl;

The R ₆ is selected from phenyl, trifluoromethyl-substituted phenyl, C ₁ to C ₆ alkoxy-substituted phenyl, C ₃ to C ₈ unsaturated cycloalkyl; the C ₁ to C ₆ The alkoxy group is specifically selected from methoxy, ethoxy, propoxy, butoxy, pentyloxy, and hexyloxy;

Preferably: the On-DNAα,β-unsaturated carbonyl compound is specifically selected from:

The boric acid compound is specifically selected from:

A method for synthesizing On-DNAβ-substituted ketone compounds. The method includes the following steps: adding 10 to 1000 times to an On-DNAα,β-unsaturated carbonyl compound solution with a molar equivalent of 1 and a molar concentration of 0.5-5mM. Molar equivalents of a boric acid compound, 10 to 1000 molar equivalents of a base, and then 0.1 to 100 molar equivalents of a palladium catalyst are added, and the reaction is carried out at 10°C to 100°C for 0.1 to 24 hours.

Further, the base is selected from the group consisting of sodium borate, lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, 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- Dimethylpyridine, N-methylimidazole; preferably, the base is cesium hydroxide.

Further, the reaction is carried out in a solvent, and the solvent is any one or aqueous mixture of several of water, methanol, ethanol, acetonitrile, dimethyl sulfoxide, inorganic salt buffer, organic acid buffer, and organic base buffer. Solvent; Preferably, the reaction solvent contains water and dimethyl sulfoxide.

Further, the palladium catalyst used in the reaction is Pd ₂ (dba) ₃ , palladium tetrakis triphenylphosphine, palladium acetate, palladium trifluoroacetate, elemental palladium (palladium black), sSPhosPd-G2; preferably, the palladium catalyst It is sSPhos-Pd-G2, palladium acetate or tetrakis triphenylphosphine palladium.

Further, the reaction also requires the addition of ligands, the amount of ligands being added is 0.1-100 molar equivalents, and the ligands are selected from the group consisting of triphenylphosphine, triaryl-substituted phosphine, tricyclohexylphosphine, and trialkyl-substituted phosphine. or monoaryldialkylphosphine. Preferably, the molar equivalent of the added ligand is 0.5 equivalent, 2 equivalent, 5 equivalent, 10 equivalent, 15 equivalent, 20 equivalent, 30 equivalent, 50 equivalent, or 80 equivalent.

Further, 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, 100°C.

Further, the reaction time of the reaction is 0.5 to 24 hours; preferably, the reaction time is 1 hour, 2 hours, 4 hours, 8 hours, 10 hours, 16 hours, 18 hours, or 20 hours.

Further, in the method, the molar equivalent of On-DNAα,β-unsaturated carbonyl compound is 1, the molar equivalent of the boric acid compound is 10-1000, and the molar equivalent of the base is 10-1000; preferably, the molar equivalent of the boric acid compound They are 50 equivalents, 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, 500 equivalents, 600 equivalents, 800 equivalents, and 1000 equivalents; the molar equivalents of bases are 50 equivalents, 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, and 500 equivalents. , 600 equivalents, 800 equivalents, 1000 equivalents; the molar equivalents of the palladium catalyst are 0.5 equivalents, 1.5 equivalents, 2.5 equivalents, 3.5 equivalents, 5 equivalents, 10 equivalents, 20 equivalents, 40 equivalents, 80 equivalents; most preferably, the boric acid compound The molar equivalents are 100, the molar equivalents of the base are 100, and the molar equivalents of the palladium catalyst are 2.5.

Further, the method is used for batch multi-well plate operations.

Further, the method is used for the synthesis of DNA-encoded compound libraries in multi-well plates.

The method of the invention can obtain On-DNAβ-substituted ketone compounds through On-DNAα,β-unsaturated carbonyl compounds in the DNA-encoded compound library, and can be widely used in various On-DNAα,β-unsaturated carbonyl substrates, and Various substituted boronic acid compounds can be introduced on a large scale as synthetic modules. This method has a single product, can be carried out in a mixed aqueous phase of organic solvent/water phase, is simple to operate, is environmentally friendly, and is suitable for the synthesis of DNA-encoded compound libraries using multi-well plates.

Definitions of terms used in the present invention: Unless otherwise stated, the initial definition provided for a group or term in this article applies to the group or term in the entire specification; for terms that are not specifically defined in this article, it should be based on the disclosure content and context. , giving the meanings that those skilled in the art can give them.

"Substitution" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.

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

Alkyl refers to a linear or branched hydrocarbon group formed by substitution of H in an alkane molecule, such as methyl CH ₃ -, ethyl CH ₃ CH ₂ -, and methylene -CH ₂ -.

Cycloalkyl: refers to a saturated or unsaturated cycloalkyl group formed by H being substituted;

Halogen: is fluorine, chlorine, bromine or iodine.

Aryl group: refers to a group in which part of the H on the aromatic ring is substituted, such as pyridyl, quinolyl, thiazolyl or phenyl.

Alkoxy group: refers to an alkyl group connected to an oxygen atom to form a substituent, for example, methoxy group is -OCH ₃ .

Halophenyl: refers to a group formed by replacing H on the phenyl group with halogen.

Alkylphenyl: refers to a group formed by replacing H on the phenyl group with an alkyl group.

Heterocyclyl: a saturated or unsaturated monocyclic or polycyclic hydrocarbon group with at least one 3 to 8 atoms selected from O, S, and N.

Obviously, according to the above content of the present invention, according to the common technical knowledge and common means in the field, without departing from the above basic technical idea of the present invention, various other forms of modifications, replacements or changes can also be made.

The above contents of the present invention will be further described in detail below through specific implementation methods in the form of examples. However, this should not be understood to mean 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.

Description of the drawings

Figures 1 and 2 are conversion rate distribution diagrams of 32 On-DNA β-aryl substituted ketone compounds obtained in Example 2 of the present invention.

Detailed ways

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

In the present invention, "room temperature" means 20 to 25°C.

DMA: Dimethylacetamide;

DMSO: dimethyl sulfoxide.

In the present invention, DNA-NH ₂ is a DNA structure with an -NH ₂ linker formed by single-stranded or double-stranded DNA and a linker group, such as the DNA-NH ₂ structure of "compound1" in WO2005058479. For example, the following DNA structure:

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

Example 1. Synthesis of On-DNA β-aryl substituted ketone compounds

Step 1. Synthesis of On-DNAα,β-unsaturated carbonyl compounds

Dissolve On-DNA aryl ethyl ketone compound (A) into 250mM, pH=9.4 boric acid buffer, prepare a 1mM concentration solution (20μL, 20nmol), and add benzaldehyde (4000nmol, 200 equivalents, 200 equivalents) to the solution in sequence. 200mM DMSO solution), potassium hydroxide (10000nmol, 500 equivalents, 1000mM H ₂ O solution), mix evenly, and react at 30°C for 1 hour.

Dissolve On-DNA aryl aldehyde compound (B) into 250mM, pH=9.4 boric acid buffer, prepare a 1mM concentration solution (20μL, 20nmol), and add acetophenone (2000nmol, 100 equivalent, 200mM DMSO solution), potassium hydroxide (6000nmol, 300 equivalents, 1000mM H ₂ O solution), mix evenly, and react at 30°C for 1 hour.

After the reaction, perform ethanol precipitation: add 10% of the total volume of 5M sodium chloride solution to the reacted solution, then continue to add 3 times the total volume of absolute ethanol, shake evenly, and freeze the reaction in dry ice. 0.5 hours, and then centrifuge at low temperature (-4°C) at 12000 rpm for half an hour, pour off the supernatant, and dissolve the remaining precipitate with deionized water to obtain On-DNA α, β-unsaturated carbonyl compounds (1, 2 ) solution was quantified by OD using a microplate reader and then sent to LCMS to confirm that the conversion rates of compounds 1 and 2 were 95% and 83% respectively.

Step 2. Synthesis of On-DNAβ-aryl substituted ketones

Prepare On-DNAα,β-unsaturated carbonyl compounds (1, 2) with deionized water to prepare a 1mM concentration solution (20μL, 20nmol), and add phenylboronic acid (2000nmol, 100 equivalents, 200mM DMA solution) to the solution in sequence. Cesium hydroxide (2000nmol, 100 equivalents, 500mM H ₂ O solution), Pd(OAc) ₂ (50nmol, 2.5 equivalents, 10mM DMA solution), PPh ₃ (200nmol, 10 equivalents, 50mM DMA solution); mix well , 90°C, reaction for 2 hours.

After the reaction is completed, add sodium diethyldithiocarbamate (DDTC) (2000 nmol, 100 equivalents, 400 mM H ₂ O solution) to the reaction system, mix evenly, and react at 80°C for 30 minutes. After the reaction is completed, add 10% of the total volume of 5M sodium chloride solution to the reacted solution, then continue to add 3 times the total volume of absolute ethanol, shake evenly, and freeze the reaction in dry ice for 0.5 hours. Then centrifuge at low temperature (-4°C) for half an hour at 12,000 rpm, discard the supernatant, and dissolve the remaining precipitate with deionized water to obtain a solution of On-DNA β-aryl substituted ketone compounds (1a, 2a). , after quantification by OD of a microplate reader, sent to LCMS to confirm that the conversion rates of compounds 1a and 2a were 84% and 81% respectively.

Example 2

According to the method of Example 1, keeping other conditions unchanged, 8 kinds of α, β-unsaturated carbonyl compounds (1-8, as shown in the accompanying drawing) and 4 kinds of boric acids (a-d, as shown in the accompanying drawing) are reacted to obtain 32 kinds of On -DNAβ-aryl substituted ketone compounds, the specific reaction products are shown in the attached figure.

In summary, the present invention can react On-DNAα,β-unsaturated carbonyl compounds and boric acid compounds to obtain On-DNAβ-substituted ketone compounds by controlling the solvent, temperature, pH and other conditions during the reaction. This method has a wide range of substrates and can be carried out in a mixed aqueous phase of organic solvent/water phase. It is simple to operate and environmentally friendly. It is suitable for the synthesis of DNA-encoded compound libraries using multi-well plates.

Claims (7)

1. A method for synthesizing On-DNA β-substituted ketone compounds, characterized in that: the method uses On-DNA α, β unsaturated carbonyl compounds as raw materials, and reacts with boric acid compounds in the presence of palladium acetate and cesium hydroxide. On-DNA β-substituted ketone compounds are obtained; among them, the structural formula of On-DNA α, β unsaturated carbonyl compounds is: The structural formula of boric acid compound is:/> The structural formula of On-DNAβ substituted ketone compounds is: Wherein, the DNA in the structural formula includes a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers, and the nucleotide chain is connected to the nucleotide chain through one or more chemical bonds or groups. The remaining parts of the compound are connected; The method includes the following steps: adding 10 to 1000 times molar equivalents of a boric acid compound and 10 to 1000 times molar equivalents to an On-DNA α, β-unsaturated carbonyl compound solution with a molar equivalent of 1 and a molar concentration of 0.5-5mM. of cesium hydroxide, then add 0.1-100 molar equivalents of palladium acetate, then add 0.1-100 molar equivalents of triphenylphosphine, and react at 10°C to 100°C for 0.1 to 24 hours; The R ₁ is selected from phenyl, thienyl or does not exist; The R ₂ is selected from phenyl, C ₁ to C ₆ alkyl; the C ₁ to C ₆ alkyl is specifically selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl base, tert-butyl, pentyl, hexyl; The R ₃ is selected from phenyl and thienyl; The R ₄ is selected from phenyl, C ₁ to C ₆ alkyl; the C ₁ to C ₆ alkyl is specifically selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl base, tert-butyl, pentyl, hexyl; The R ₅ is selected from hydrogen, C ₁ to C ₆ alkyl; or R ₅ forms a ring with R ₁ or R ₄ respectively; the C ₁ to C ₆ alkyl is specifically selected from methyl, ethyl, n-propyl , isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl; The R ₆ is selected from phenyl, trifluoromethyl-substituted phenyl, C ₁ to C ₆ alkoxy-substituted phenyl, C ₃ to C ₈ unsaturated cycloalkyl; the C ₁ to C ₆ The alkoxy group is specifically selected from methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy. 2. The method according to claim 1, characterized in that: the reaction is carried out in a solvent, and the solvent is water, methanol, ethanol, acetonitrile, dimethyl sulfoxide, inorganic salt buffer, organic acid buffer, organic base Any one or several aqueous mixed solvents in the buffer solution. 3. The method according to claim 1, characterized in that: the reaction temperature of the reaction is 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 100°C. 4. The method according to claim 1, characterized in that: the reaction time of the reaction is 1 hour, 2 hours, 4 hours, 8 hours, 10 hours, 16 hours, 18 hours, or 20 hours. 5. The method according to claim 1, characterized in that: in the method, the molar equivalent of On-DNA α, β-unsaturated carbonyl compound is 1, and the molar equivalent of the boric acid compound is 50 equivalents, 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, 500 equivalents, 600 equivalents, 800 equivalents, 1000 equivalents; the molar equivalents of cesium hydroxide are 50 equivalents, 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, 500 equivalents, 600 equivalents, 800 equivalents, 1000 Equivalents; the molar equivalents of palladium acetate are 0.5 equivalents, 1.5 equivalents, 2.5 equivalents, 3.5 equivalents, 5 equivalents, 10 equivalents, 20 equivalents, 40 equivalents, and 80 equivalents. 6. The method according to any one of claims 1 to 5, characterized in that the method is used for batch multi-well plate operations. 7. The method according to any one of claims 1 to 5, characterized in that the method is used for the synthesis of a DNA-encoded compound library in a multi-well plate.