CN113563265B - Method for synthesizing On-DNA N, N-monosubstituted indazolone compound - Google Patents
Method for synthesizing On-DNA N, N-monosubstituted indazolone compound Download PDFInfo
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Abstract
The invention relates to a method for synthesizing On-DNA N, N-monosubstituted indazolone compounds, which takes On-DNA o-nitroaromatic amide compounds as raw materials and reacts in the presence of alkali and tetrahydroxy diboron to obtain the On-DNA N, N-monosubstituted indazolone compounds. The method for synthesizing the On-DNA N, N-monosubstituted indazolone compound can be carried out in a mixed water phase of an organic solvent/water phase, has simple post-treatment and mild conditions, can obtain high-diversity DNA coding compounds in a short time and high yield, and is suitable for synthesizing DNA coding compound libraries by porous plates.
Description
Technical Field
The invention belongs to the technical field of coding compound libraries, and particularly relates to a method for synthesizing an On-DNAN, N-monosubstituted indazolone compound.
Background
In drug development, especially new drug development, high throughput screening against biological targets is one of the main means to rapidly obtain lead compounds. However, conventional high throughput screening based on single molecules requires long time, huge equipment investment, limited numbers of library compounds (millions), and the build-up of compound libraries requires decades of accumulation, limiting the efficiency and possibilities of discovery of lead compounds. The recent advent of DNA-encoded compound library technology (WO 2005058479, WO2018166532, CN 103882532), combining combinatorial chemistry and molecular biology techniques, tagged each compound with a DNA tag at the molecular level, and capable of synthesizing up to hundred million classes of compound libraries in extremely short time, has become a trend for the next generation of compound library screening technology, and began to be widely used in the pharmaceutical industry, producing a number of positive effects (Accounts of Chemical Research,2014,47,1247-1255).
The DNA encoding compound library rapidly generates a huge compound library by combinatorial chemistry, and can screen the lead compound with high flux, so that the screening of the lead compound becomes unprecedented rapid and efficient. One of the challenges in constructing libraries of DNA-encoding compounds is the need to synthesize small molecules with chemical diversity on DNA in high yields. Since DNA needs to be stable under certain conditions (solvent, pH, temperature, ion concentration), higher yields are also required for the On-DNA reaction constructed from DNA encoding compound libraries. Therefore, the kind of the reagent, the kind of the reaction and the reaction condition of the chemical reaction (called On-DNA reaction for short) performed On the DNA directly influence the richness and the selectivity of the DNA coding compound library. Thus, the development of chemical reactions compatible with DNA is also a long-term research and study direction of the current DNA coding compound library technology, and directly influences the application and commercial value of the DNA coding compound library.
Indazolone compounds are important drug compound skeleton structures, and introduction of indazolone skeletons into a DNA encoding compound library can further expand the diversity of the compound library, thereby being beneficial to improving the probability of screening effective compounds. However, no method for constructing On-DNA N, N-monosubstituted indazolone compounds by On-DNA ortho-nitroarylcarboxylic acid or On-DNA ortho-nitroarylamide compounds has been reported. Therefore, a new method for synthesizing On-DNA N, N-monosubstituted indazolone compounds, which is suitable for the operation of a large number of porous plates, needs to be developed, the diversity of DNA coding compound libraries is obviously increased, and the application value of the DNA coding compound library technology is further improved.
Disclosure of Invention
In order to solve the problems, the invention develops a synthetic method of a DNA coding compound library, which has the advantages of stable storage of raw materials, mild reaction conditions, high yield, good substrate universality, small damage to DNA, and suitability for batch operation by using porous plates, and can quickly convert the DNA coding o-nitroaromatic amide compound library into the DNA coding N, N-monosubstituted indazolone compound library through one-step reaction.
The invention provides a method for synthesizing an On-DNA N, N-monosubstituted indazolone compound, which takes an On-DNA o-nitroaromatic amide compound as a raw material and reacts in the presence of alkali and tetrahydroxy diboron to obtain the On-DNA N, N-monosubstituted indazolone compound; wherein the structural formula of the On-DNA o-nitroaromatic amide compoundThe structural formula of the On-DNA N, N-monosubstituted indazolone compound is +.>
Wherein the DNA in the formula comprises a single-stranded or double-stranded nucleotide chain polymerized from artificially modified and/or unmodified nucleotide monomers, the nucleotide chain being linked to Ar by one or more chemical bonds or groups;
wherein the DNA in the structural formula and Ar are connected through one chemical bond or a plurality of chemical bonds or groups. When a chemical bond is formed, the DNA in the structural formula is directly connected with Ar; in the case of multiple chemical bonds or groups, the DNA in the structural formula is linked to Ar by multiple chemical bonds, for example, a methylene group (-CH) is used between DNA and Ar 2 (-) are connected, namely through two chemical bonds; or the DNA and Ar are connected through a carbonyl (-CO-) and also connected through two chemical bonds; or DNA and Ar through a methylenecarbonyl (-CH) 2 CO-) is linked to DNA, also via three chemical bonds.
Wherein Ar is a monocyclic or bicyclic aromatic ring; preferably, ar is a substituted aromatic ring or aromatic heterocyclic ring having a molecular weight of 1000 or less; r is R 2 Selected from hydrogen or a group having a molecular weight of 1000 or less which is directly bonded to the amide nitrogen atom.
R 2 Selected from alkyl, substituted alkyl, aryl or substituted aryl; wherein the alkyl group is C 1 ～C 20 Alkyl or C 3 ～C 8 Cycloalkyl; the number of the substituent groups of the substituted alkyl is one or more, and the substituent groups of the substituted alkyl are one or more selected from halogen, nitro, alkoxy, halogenated phenyl, phenyl and alkylphenyl independently;
the aryl is selected from pyridyl, quinolyl, thiazolyl, thienyl or phenyl; the number of substituents of the substituted aryl is one or more, and the substituents of the substituted aryl are independently selected from halogen, cyano, nitro, alkoxy, alkanoyl, C 1 ～C 20 One or more of the alkyl groups.
Preferably, said R 2 Selected from straight-chain or branched C 1 ～C 12 An alkyl group; further, said R 2 Selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl.
Preferably, said R 2 Selected from C 3 ～C 6 Saturated cycloalkyl is specifically selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.
Preferably, said R 2 Selected from substituted alkyl groups selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, and hexane groups, the substituents of the substituted alkyl groups being phenyl, alkylphenyl, or halophenyl; further, the alkylphenyl is selected from the group consisting of methylphenyl, ethylphenyl, n-propylphenyl, isopropylphenyl, n-butylphenyl, isobutylphenyl, t-butylphenyl, pentanylphenyl, and hexanes-based phenyl; the halogenated phenyl is selected from fluorinated phenyl, chlorinated phenyl, brominated phenyl and iodinated phenyl.
Preferably, said R 2 Selected from the group consisting of substituted aromatic groups, wherein the aromatic groups are phenyl groups, and the substituents of the substituted aromatic groups are C 1 ～C 6 Alkyl, C 1 ～C 6 Alkoxy, alkanoyl, C 1 ～C 6 Alkanoyl or halogen; further; the C is 1 ～C 6 Alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, and hexane; the C is 1 ～C 6 Alkoxy is selected from methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy; the alkanoyl is selected from the group consisting of aminoacyl, methylaminoyl, ethylaminoyl, n-alanyl, isopropylaminoacyl, n-butylaminoyl, isobutylaminoyl, t-butylaminoyl, pentylaminoyl, hexylaminoacyl; the C is 1 ～C 6 Alkanoyl is selected from the group consisting of methanoyl, ethaneacyl, n-propanoyl, isopropanoyl, n-butanoyl, isobutane acyl, t-butanoyl, pentanoyl, hexanoyl; the halogen is fluorine, chlorine, bromine or iodine.
Ar is selected from the following groups:
preferably Ar is
The On-DNA o-nitroaromatic amide compound in the invention can be prepared from an On-DNA o-nitroaromatic carboxylic acid compound, and the reaction route is as follows:
step 1, condensing an On-DNA o-nitroaromatic carboxylic acid compound serving as a substrate with amine under the reaction condition of 20-100 ℃ to obtain an On-DNA o-nitroaromatic amide compound; the reaction is that amide condensation occurs in the presence of N-hydroxy-7-aza-benzotriazol and N, N' -diisopropylcarbodiimide; the reaction is carried out in a solvent, wherein the solvent is one or a mixture of a plurality of aqueous solvents selected from acetonitrile, methanol, ethanol, DMF, DMA, DMSO, THF, water, inorganic salt buffer solution, organic acid buffer solution and organic alkali buffer solution.
Preferably, the solvent is a borate buffer containing DMA; further, the pH of the buffer=9.4.
Preferably, the reaction temperature is 20 ℃,30 ℃, 40 ℃, 50 ℃,60 ℃, 70 ℃, 80 ℃ or 90 ℃.
The molar equivalent of the On-DNA o-nitroaromatic carboxylic acid compound in the reaction is 1, the molar equivalent of the amine is 150 to 300 equivalents, preferably the molar equivalent of the amine is 150 equivalents, 200 equivalents, 250 equivalents or 300 equivalents.
Step 2, taking an On-DNA o-nitroaromatic amide compound as a raw material, and reacting under the reaction conditions of alkali and tetrahydroxy diboron to obtain an On-DNA N, N-monosubstituted indazolone compound. The reaction steps are as follows:
the molar equivalent is 1, the molar concentration is 0.1 to 2mM of the solution of the On-DNA o-nitroaromatic amide compound, 300 to 500 times of the molar concentrationEquivalent of base, 80 to 150 times mole equivalent of B 2 (OH) 4 Mixing and reacting for 0.5-24 h at room temperature.
Further, the molar concentration of the On-DNA o-nitroaromatic amide compound solution is 0.5mM, 1.0mM or 2mM.
Preferably, the base is sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, N-diisopropylethylamine or 1, 3-tetramethylguanidine.
Preferably, when the molar equivalent of the On-DNA ortho-nitroaromatic amide compound is 1, the molar equivalent of the base is 300 equivalents, 350 equivalents, 400 equivalents, or 500 equivalents; the molar equivalent of tetrahydroxydiboron is 80 equivalents, 90 equivalents, 100 equivalents, 120 equivalents or 150 equivalents.
Further, the reaction time is 1h, 2h, 3h, 4h, 6h, 8h or 24h.
Further, the reaction is carried out in a solvent, wherein the solvent is one or a mixture of a plurality of aqueous solvents selected from acetonitrile, methanol, ethanol, DMF, DMA, DMSO, THF, water, inorganic salt buffer solution, organic acid buffer solution and organic base buffer solution.
Preferably, the solvent contains ethanol, and the addition amount of the ethanol is 5-20% of the total volume of the reaction system.
Further, the addition amount of the ethanol is 10 to 20 percent of the total volume of the On-DNA o-nitroaromatic amide compound solution and the alkali solution.
Further, the reaction is carried out in the following feeding sequence: sequentially adding On-DNA o-nitroaromatic amide compound solution, alkali and ethanol, and finally adding tetrahydroxy diboron.
Further, the above method is used for batch multi-well plate operations.
Further, the above method is used for the synthesis of DNA encoding compound libraries in multiwell plates.
The method can realize the synthesis of the On-DNA N, N-monosubstituted indazolone compound in the DNA coding compound library through the On-DNA o-nitroaromatic amide compound, and can be widely applied to various On-DNA o-nitroaromatic amide substrates. The On-DNA o-nitroaromatic amide compound used in the invention can be obtained by condensing On-DNA o-nitroaromatic acid and amino compound through amide, and can be introduced into amino compound On a large scale as a synthesis module. The method has the advantages of high yield, single product, simple operation and environmental protection, and is suitable for synthesizing the DNA coding compound library by using a porous plate.
In a preferred embodiment of the present invention, by adding ethanol to the reaction system, the production of by-products can be suppressed, and the accuracy of library construction applied to DNA encoding compounds can be improved.
Definition of terms used in connection with the present invention: unless otherwise indicated, the initial definitions provided for groups or terms herein apply to the groups or terms throughout the specification; for terms not specifically defined herein, the meanings that one skilled in the art can impart based on the disclosure and the context.
"substituted" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.
The minimum and maximum values of the carbon atom content in the hydrocarbon group are represented by prefixes, for example, prefixes (Ca to C b ) Alkyl indicates any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C 1 ～C 20 Alkyl refers to straight or branched chain alkyl groups containing 1 to 20 carbon atoms.
Alkyl refers to a straight or branched hydrocarbon radical containing at least one hydrogen atom in the alkane molecule, e.g. methyl-CH 3 ethyl-CH 2 CH 3 . The alkyl group may also be part of another group, such as C 1 ～C 6 An alkoxy group.
"cycloalkyl" refers to a saturated or partially saturated cyclic group having multiple carbon atoms and no ring heteroatoms and having a single ring or multiple rings (including fused, bridged and spiro ring systems).
Halogen is fluorine, chlorine, bromine or iodine.
Aryl refers to a group in which part H on the aromatic ring is substituted, such as pyridyl, quinolinyl, thiazolyl or phenyl.
Alkoxy means that the alkyl group is attached to an oxygen atom to form a substituent, examplesFor example methoxy is-OCH 3 。
Halo phenyl refers to a group formed by substitution of H on phenyl with halogen.
Alkanoyl means that the alkyl group is attached to the C atom of the carbonyl group to form a substituent, e.g. acetyl is-C (O) CH 3 。
Aminoacyl means that the N atom of the amino group is linked to the C atom of the carbonyl group to form a substituent, e.g. aminoacyl is-C (O) NH 2 The methylamyl group being-C (O) NHCH 3 。
Aromatic heterocyclic ring refers to an aromatic unsaturated ring comprising at least one heteroatom; wherein the hetero atom refers to nitrogen atom, oxygen atom and sulfur atom.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail by way of the following embodiments, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
Fig. 1: the corresponding conversion profile of the 22 On-DNAN, N-monosubstituted indazolone compounds obtained in example 5 of the present invention.
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
In the present invention, "room temperature" means 20 to 25 ℃.
HOAT: n-hydroxy-7-azabenzotriazoles; DIC: n, N' -diisopropylcarbodiimide;
DMA: dimethylacetamide; DMF: dimethylformamide.
HATU:2- (7-azabenzotriazol) -N, N' -tetramethylurea hexafluorophosphate;
DIPEA: n, N-diisopropylethylamine.
DMSO: dimethyl sulfoxide; THF: tetrahydrofuran.
DNA-NH in the present invention 2 Is formed by single-stranded or double-stranded DNA and a linker group and carries-NH 2 DNA structure of linker, e.g. DNA-NH of "component 1" in WO2005058479 2 Structure is as follows.
Example 1: synthesis of On-DNA ortho-nitro aromatic carboxylic acid compound
100 times molar equivalent of 4-nitro-3-methoxycarbonylbenzoic acid or 2-nitroterephthalic acid monomethyl ester solution (concentration: 200mM, dissolved in DMA) and 100 times molar equivalent of HATU (concentration: 400mM, dissolved in DMA) and 100 times equivalent of DIPEA (concentration: 400mM, dissolved in DMA) were placed in a-20deg.C refrigerator, cooled for 5min, mixed, thoroughly mixed with vortex vibration, then placed in a 4deg.C refrigerator, and stored for 5min, and the above mixture was added to DNA-NH 2 In the solution (1 mM concentration; borate buffer solution, pH=9.4, 250 mM solution), the reaction solution is fully and uniformly mixed by vortex oscillation, the reaction is carried out for 2 hours at room temperature, ethanol precipitation is carried out after the reaction is finished, the freeze-drying reagent is dissolved in double distilled water to be 0.5mM concentration, 300 times molar equivalent of 1M sodium hydroxide aqueous solution is added into the solution, and the mixture is fully and uniformly mixed by vortex oscillation and then reacted for 1 hour at 60 ℃;
ethanol precipitation is carried out after the reaction is finished: adding 5M sodium chloride solution with the total volume of 10% into the solution, continuously adding absolute ethanol with the total volume being 3 times of the total volume, shaking uniformly, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000 rpm for half an hour, pouring out the supernatant, and directly using the supernatant for the next reaction after freeze-drying.
EXAMPLE 2 Synthesis of On-DNAN, N-monosubstituted indazolone Compounds
Step 1, synthesis of On-DNA o-nitroanilide Compound
On-DNA o-nitrobenzoic acid 1 was dissolved in borate buffer (0.5M, pH=9.4) to prepare a 1mM concentration solution (10. Mu.L, 10 nmol), to which aniline (300 molar equivalents, 300mM MeCN/H) was added in sequence 2 O=1:1 solution), HOAt (600 molar equivalents, 600mM DMSO solution), DIC (600 molar equivalents, 600mM DMSO solution), 1h at room temperature;
ethanol precipitation is carried out after the reaction is finished: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethanol with the total volume being 3 times of the total volume, shaking uniformly, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000 rpm for half an hour, pouring out supernatant, and freeze-drying to obtain an On-DNA compound 2;
step 2 Synthesis of On-DNA N, N-monosubstituted indazolone Compounds
The On-DNA compound 2 was dissolved in water to prepare a 1mM concentration solution (10. Mu.L, 10 nmol), and NaOH (300 molar equivalents, 1M H) was added to the solution in this order 2 O solution), etOH (10% of the total volume of On-DNA compound 2 solution and NaOH solution), B 2 (OH) 4 (80 molar equivalents, 100mM H) 2 O solution), room temperature reaction 2h;
ethanol precipitation is carried out after the reaction is finished: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethanol with the total volume of 3 times, shaking uniformly, then placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000 rpm for half an hour, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain a solution of an On-DNA product, and sending LCMS to confirm that the reaction conversion rate is 90%.
EXAMPLE 3 Synthesis of On-DNAN, N-monosubstituted indazolone Compounds
R 2 ＝C 6 H 5 -or C 6 H 5 -CH 2 -CH 2 -。
Step 1: synthesis of On-DNA o-nitroanilide compound
On-DNA o-nitrobenzoic acid 1 was dissolved in borate buffer (0.5M, pH=9.4) to prepare 1mM concentration solution (10. Mu.L, 10 nmol), and aniline or phenethylamine (300 molar equivalents, 300mM MeCN/H) was added to the solution, respectively 2 O=1:1 solution), HOAt (600 molar equivalents, 600mM DMSO solution), DIC (600 molar equivalents, 600mM DMSO solution), 1h at room temperature;
ethanol precipitation is carried out after the reaction is finished: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethanol with the total volume being 3 times of the total volume, shaking uniformly, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000 rpm for half an hour, pouring out supernatant, and freeze-drying to obtain two On-DNA compounds 2;
step 2 Synthesis of On-DNA N, N-monosubstituted indazolone Compounds
Two On-DNA compounds 2 were dissolved in water to prepare 1mM concentration solutions (10. Mu.L, 10 nmol), and NaOH (X molar equivalents, 1M H) was added to the solutions in this order 2 O solution), etOH (Y% V/V), B 2 (OH) 4 (Z molar equivalent, 100mM H) 2 O solution), room temperature reaction for 24 hours;
ethanol precipitation is carried out after the reaction is finished: adding 5M sodium chloride solution with the total volume of 10% to the solution, continuously adding absolute ethanol with the total volume of 3 times, shaking uniformly, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000 rpm for half an hour, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain a solution of an On-DNA product 3, and sending the solution to LCMS to confirm the reaction conversion rate, wherein the specific reaction conditions and the product conversion rate are shown in the following table 1:
y% represents the ratio of the volume of EtOH addition to the total volume of the On-DNA compound 2 solution and NaOH solution.
Table 1: specific reaction conditions and product conversion of example 3
EXAMPLE 4 Synthesis of On-DNAN, N-monosubstituted indazolone Compounds
R 2 ＝C 6 H 5 -or C 6 H 5 -CH 2 -CH 2 -。
Step 1: synthesis of On-DNA o-nitroanilide compound
On-DNA o-nitrobenzoic acid 1 was dissolved in borate buffer (0.5M, pH=9.4) to prepare 1mM concentration solution (10. Mu.L, 10 nmol), and aniline or phenethylamine (300 molar equivalents, 300mM MeCN/H) was added to the solution in this order, respectively 2 O=1:1 solution), HOAt (600 molar equivalents, 600mM DMSO solution), DIC (600 molar equivalents, 600mM DMSO solution), 1h at room temperature;
ethanol precipitation is carried out after the reaction is finished: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethanol with the total volume being 3 times of the total volume, shaking uniformly, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000 rpm for half an hour, pouring out supernatant, and freeze-drying to obtain two On-DNA compounds 2;
step 2 Synthesis of On-DNA N, N-monosubstituted indazolone Compounds
Two On-DNA compounds 2 were dissolved in water to prepare 1mM concentration solutions (10. Mu.L, 10 nmol), and NaOH (X molar equivalents, 1M H) was added to the solutions in this order 2 O solution), etOH (Y% V/V), B 2 (OH) 4 (Z molar equivalent, 100mM H) 2 O solution), for 2h at room temperature;
ethanol precipitation is carried out after the reaction is finished: adding 5M sodium chloride solution with the total volume of 10% to the solution, continuously adding absolute ethanol with the total volume of 3 times, shaking uniformly, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000 rpm for half an hour, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain a solution of an On-DNA product 3, and sending the solution to LCMS to confirm the reaction conversion rate, wherein the specific reaction conditions and the product conversion rate are shown in the following table 2:
y% represents the ratio of the volume of EtOH addition to the total volume of the On-DNA compound 2 solution and NaOH solution.
Table 2: specific reaction conditions and product conversion of example 4
The experimental result shows that the ethanol is added into the reaction system to inhibit the generation of byproducts and effectively improve the yield of the reaction. The yield of the target product prepared under the reaction condition 2 is highest.
EXAMPLE 5 Synthesis of On-DNAN, N-monosubstituted indazolone Compounds of different substrates
On-DNA o-nitrobenzoic acid 1 was dissolved in borate buffer (0.5M, pH=9.4) to prepare 1mM concentration solution (10. Mu.L, 10 nmol), and 22 kinds of primary amine molecules (300 molar equivalents, 300mM MeCN/H) were sequentially added to each of the solutions 2 O=1:1 solution), HOAt (600 molar equivalents, 600mM DMSO solution), DIC (600 molar equivalents, 600mM DMSO solution), 1h at room temperature;
ethanol precipitation is carried out after the reaction is finished: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethanol with the total volume being 3 times of the total volume, shaking uniformly, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000 rpm for half an hour, pouring out supernatant, and freeze-drying to obtain 22 On-DNA compounds 2;
22 kinds of On-DNA compounds 2 were dissolved in water to prepare 1mM concentration solutions (10. Mu.L, 10 nmol), and NaOH (300 molar equivalents, 1M H) was added sequentially to the solutions 2 O solution), etOH (10% of the total volume of the reaction solution), B 2 (OH) 4 (80 molar equivalents, 100mM H) 2 O solution), for 2h at room temperature;
ethanol precipitation is carried out after the reaction is finished: adding 5M sodium chloride solution with the total volume of 10% into the solution, continuously adding absolute ethanol with the total volume being 3 times of the total volume, shaking uniformly, then placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000 rpm for half an hour, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain 22 solutions of On-DNA products 3, and sending LCMS to confirm the conversion rate of the reaction.
In summary, the present invention can obtain On-DNA N, N-monosubstituted indazolone compound from On-DNA o-nitroaniline compound in the presence of tetrahydroxy diboron by controlling the conditions of solvent, temperature, pH and the like during the reaction. The method can be carried out in a mixed water phase of an organic solvent/water phase, has single product, simple operation and environmental protection, and is suitable for synthesizing the DNA coding compound library by using a porous plate.
Claims (8)
1. A method for synthesizing On-DNA N, N-monosubstituted indazolone compounds is characterized in that: the method takes an On-DNA o-nitroaromatic amide compound as a raw material, and the On-DNA N, N-monosubstituted indazolone compound is obtained by reaction under the reaction conditions of alkali and tetrahydroxy diboron; wherein the structural formula of the On-DNA o-nitroaromatic amide compound isThe structural formula of the On-DNA N, N-monosubstituted indazolone compound is +.>
Wherein the DNA in the formula comprises a single-stranded or double-stranded nucleotide chain polymerized from artificially modified and/or unmodified nucleotide monomers, the nucleotide chain being linked to Ar by one or more chemical bonds or groups;
wherein Ar is selected from the following groups:
R 2 selected from alkyl, substituted alkyl, aryl orA substituted aromatic group; wherein the number of substituents of the substituted alkyl group is one or more; the substituent of the substituted alkyl is one or more of halogen, nitro, alkoxy, halogenated phenyl, phenyl and alkylphenyl which are independent of each other; the alkyl group being C 1 ～C 20 Alkyl or C 3 ～C 8 Cycloalkyl;
the aryl is selected from pyridyl, quinolyl, thiazolyl, thienyl or phenyl; the number of substituents of the substituted aryl is one or more, and the substituents of the substituted aryl are independently selected from halogen, cyano, nitro and C 1 ～C 6 Alkoxy, aminoacyl, C 1 ～C 6 Alkanoyl, C 1 ～C 20 One or more of alkyl groups;
the method comprises the following steps: the molar equivalent is 1, the molar concentration is 0.1 to 2mM of the On-DNA o-nitroaromatic amide compound solution, 300 to 500 times of the molar equivalent of the alkali, 80 to 150 times of the molar equivalent of the B 2 (OH) 4 Mixing and reacting for 0.5-24 h at room temperature;
the alkali is sodium hydroxide; the reaction is carried out in a solvent, wherein the solvent is a mixed solvent of ethanol and water.
2. The method according to claim 1, characterized in that: the molar concentration of the On-DNA o-nitroaromatic amide compound solution is 0.5mM, 1.0mM or 2mM.
3. The method according to claim 1, characterized in that: the molar equivalent of the On-DNA o-nitroaromatic amide compound is 1, the molar equivalent of the base is 300, 350, 400 or 500, and the molar equivalent of the tetrahydroxydiboron is 80, 90, 100, 120 or 150.
4. The method according to claim 1, characterized in that: the reaction time is 1h, 2h, 3h, 4h, 6h, 8h or 24h.
5. The method according to claim 1, characterized in that: the addition amount of the ethanol is 5-20% of the total volume of the reaction system.
6. The method according to claim 1, characterized in that: the reaction is carried out in the following feeding sequence: sequentially adding On-DNA o-nitroaromatic amide compound solution, alkali and ethanol, and finally adding tetrahydroxy diboron.
7. The method according to any one of claims 1-6, wherein: the method is used for batch multi-well plate operation.
8. The method according to any one of claims 1-6, wherein: the method is used for the synthesis of DNA encoding compound libraries in multiwell plates.
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| CN107082763A (en) * | 2016-02-15 | 2017-08-22 | 中山大学 | A kind of method for synthesizing indazole ketone compounds |
| CN109680342A (en) * | 2018-12-18 | 2019-04-26 | 上海药明康德新药开发有限公司 | The method that On-DNA virtue nitro compound is reduced into On-DNA aromatic amine compound in DNA encoding compound library |
| CN110066308A (en) * | 2019-04-26 | 2019-07-30 | 上海药明康德新药开发有限公司 | Synthetic method for the On-DNA sulfamide compound in the building of DNA encoding compound library |
| CN110818749A (en) * | 2019-10-30 | 2020-02-21 | 上海药明康德新药开发有限公司 | Method for synthesizing On-DNA aryl sulfonamide compounds in construction of DNA coding compound library |
| WO2020047095A1 (en) * | 2018-08-28 | 2020-03-05 | The Scripps Research Institute | Use of non-covalent immobilization in dna encoded libraries |
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| CN107082763A (en) * | 2016-02-15 | 2017-08-22 | 中山大学 | A kind of method for synthesizing indazole ketone compounds |
| WO2020047095A1 (en) * | 2018-08-28 | 2020-03-05 | The Scripps Research Institute | Use of non-covalent immobilization in dna encoded libraries |
| CN109680342A (en) * | 2018-12-18 | 2019-04-26 | 上海药明康德新药开发有限公司 | The method that On-DNA virtue nitro compound is reduced into On-DNA aromatic amine compound in DNA encoding compound library |
| CN110066308A (en) * | 2019-04-26 | 2019-07-30 | 上海药明康德新药开发有限公司 | Synthetic method for the On-DNA sulfamide compound in the building of DNA encoding compound library |
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