Method for synthesizing On-DNA N, N-monosubstituted indazolone compound
阅读说明：本技术 一种合成On-DNA N,N-单取代吲唑酮类化合物的方法 (Method for synthesizing On-DNA N, N-monosubstituted indazolone compound ) 是由 李进 包亚鹏 冯静 刘观赛 万金桥 于 2020-04-29 设计创作，主要内容包括：本发明涉及一种合成On-DNA N,N-单取代吲唑酮类化合物的方法,该方法以On-DNA邻硝基芳酰胺化合物为原料,在碱、四羟基二硼存在下反应得到On-DNA N,N-单取代吲唑酮类化合物。本发明提供的On-DNA N,N-单取代吲唑酮类化合物的合成方法能够在有机溶剂/水相的混合水相中进行,后处理简单,条件温和,能够在较短时间内高收率得到高多样性的DNA编码化合物,并且适用于多孔板进行的DNA编码化合物库的合成。(The invention relates to a method for synthesizing an On-DNA N, N-monosubstituted indazolone compound. The synthesis method of the On-DNA N, N-monosubstituted indazolone compound provided by the invention can be carried out in a mixed water phase of an organic solvent/a water phase, has simple post-treatment and mild conditions, can obtain a high-diversity DNA coding compound in a short time and at a high yield, and is suitable for synthesizing a DNA coding compound library by using a porous plate.)
1. A method for synthesizing an On-DNA N, N-monosubstituted indazolone compound is characterized by comprising the following steps: the method takes an On-DNA o-nitro aromatic amide compound as a raw material, and the On-DNA N, N-monosubstituted indazolone compound is obtained by reaction under the reaction condition of alkali and tetrahydroxy diboron; wherein the structural formula of the On-DNA o-nitro aromatic amide compound is shown in the specificationThe structural formula of the On-DNA N, N-monosubstituted indazolone compound is shown as
Wherein, the DNA in the structural formula comprises a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers, and the nucleotide chain is connected with Ar through one or more chemical bonds or groups;
wherein Ar is a monocyclic or bicyclic aromatic ring; r2Selected from hydrogen or a group having a molecular weight of 1000 or less directly attached to the amide nitrogen atom.
2. The method of claim 1, wherein: r2Selected from alkyl, substituted alkyl, aryl or substituted aryl; wherein the alkyl is C1～C20Alkyl or C3～C8A cycloalkyl group; the number of substituents of the substituted alkyl group is one or more; the substituent of the substituted alkyl is one or more independently selected from halogen, nitro, alkoxy, halogenated phenyl, phenyl and alkyl phenyl; the aryl is selected from pyridyl, quinolyl, thiazolyl, thienyl or phenyl; the number of the substituent groups for substituting the aromatic group is one or more, and the substituent groups for substituting the aromatic group are independently selected from halogen, cyano, nitro, alkoxy, aminoacyl, alkanoyl and C1～C20One or more of alkyl groups.
3. The method of claim 1, wherein: ar is selected from the following groups:
4. according to the claimsThe method according to any one of claims 1 to 3, wherein: an On-DNA o-nitroarylamide compound solution with the molar equivalent of 1 and the molar concentration of 0.1-2 mM, 300-500 times of molar equivalent of alkali and 80-150 times of molar equivalent of B2(OH)4Mixing and reacting for 0.5-24 h at room temperature.
5. The method of claim 4, wherein: the molarity of the On-DNA o-nitroarylamide compound solution is 0.5mM, 1.0mM or 2 mM.
6. The method of claim 4, wherein: the alkali is sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, N-diisopropylethylamine or 1,1,3, 3-tetramethylguanidine.
7. The method of claim 4, wherein: the molar equivalent of the On-DNA o-nitroarylamide compound is 1, the molar equivalent of the base is 300 equivalents, 350 equivalents, 400 equivalents or 500 equivalents, and the molar equivalent of the tetrahydroxy diboron is 80 equivalents, 90 equivalents, 100 equivalents, 120 equivalents or 150 equivalents.
8. The method of claim 4, wherein: the reaction time is 1h, 2h, 3h, 4h, 6h, 8h or 24 h.
9. The method of claim 1, wherein: the reaction is carried out in a solvent, wherein the solvent is one or a mixture of water in acetonitrile, methanol, ethanol, DMF, DMA, DMSO, THF, water, inorganic salt buffer solution, organic acid buffer solution and organic base buffer solution.
10. The method of claim 9, wherein: the solvent contains ethanol.
11. The method of claim 10, wherein: the adding amount of the ethanol is 5-20% of the total volume of the reaction system.
12. The method of claim 10, wherein: the feeding sequence of the reaction is as follows: sequentially adding the On-DNA o-nitro aromatic amide compound solution, alkali and ethanol, and finally adding tetrahydroxy diboron.
13. The method according to any one of claims 1-12, wherein: the method is used for batch multi-well plate operations.
14. The method according to any one of claims 1-12, wherein: the method is used for the synthesis of libraries of DNA-encoding compounds for multi-well plates.
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 for biological targets is one of the main means for rapidly obtaining lead compounds. However, traditional high throughput screening based on single molecules requires long time, large equipment investment, limited number of library compounds (millions), and the building of compound libraries requires decades of accumulation, limiting the efficiency and possibility of discovery of lead compounds. The recent DNA-encoded compound library technologies (WO2005058479, WO2018166532, CN103882532) combine the technologies of combinatorial chemistry and molecular biology, add a DNA tag to each compound on the molecular level, and synthesize up to hundred million levels of compound libraries in a very short time, which is a trend of the next generation compound library screening technology, and begin to be widely applied in the pharmaceutical industry, resulting in many positive effects (Accounts of Chemical Research,2014,47, 1247-.
The DNA coding compound library can rapidly generate a giant compound library through combinatorial chemistry, and can screen out a lead compound with high flux, so that the screening of the lead compound becomes unprecedented rapidness and high efficiency. One of the challenges in constructing libraries of DNA-encoding compounds is the need to synthesize chemically diverse small molecules on DNA in high yields. Because DNA can be kept stable under certain conditions (solvent, pH, temperature and ion concentration), the On-DNA reaction applied to the construction of the DNA coding compound library also needs higher yield. Therefore, the reagent type, reaction type and reaction condition of the chemical reaction (On-DNA reaction for short) carried out On DNA directly influence the richness and selectivity of the DNA coding compound library. Therefore, the development of chemical reactions compatible with DNA is also a long-term research and research direction of the current DNA coding compound library technology, and the application and commercial value of the DNA coding compound library are directly influenced.
The indazolone compound is an important medicine compound framework structure, and the indazolone framework is introduced into a DNA coding compound library, so that the diversity of the compound library can be further expanded, and the probability of screening effective compounds is favorably improved. However, no method for constructing an On-DNA N, N-monosubstituted indazolone compound by using an On-DNA o-nitroarylcarboxylic acid or an On-DNA o-nitroarylamide compound is reported at present. Therefore, a new method for synthesizing the On-DNA N, N-monosubstituted indazolone compound, which is suitable for large-scale multi-well plate operation, needs to be developed, the diversity of a DNA coding compound library 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 synthesis 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 and small damage to DNA and is suitable for batch operation by using a porous plate, and can quickly convert the DNA coding o-nitroarylamide 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 comprises the steps of reacting an On-DNA o-nitro aromatic amide compound serving as a raw material 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-nitro aromatic amide compoundThe structural formula of the On-DNA N, N-monosubstituted indazolone compound is shown as
Wherein, the DNA in the structural formula comprises a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers, and the nucleotide chain is connected with Ar through one or more chemical bonds or groups;
wherein, the DNA in the structural formula is connected with Ar through one chemical bond or a plurality of chemical bonds or groups. When the chemical bond is one, the DNA in the structural formula is directly connected with Ar; multiple chemical bonds or groups are defined as multiple bonds spaced between the DNA and Ar in the structural formula, e.g., the DNA and Ar are linked by a methylene group (-CH)2-) are linked, i.e. linked by two chemical bonds; or the DNA is connected with the Ar through a carbonyl (-CO-) and also through two chemical bonds; or DNA and Ar through a methylene carbonyl group (-CH)2CO-) to DNA, also by three chemical bonds.
Wherein Ar is a monocyclic or bicyclic aromatic ring; preferably, Ar is a substituted aromatic ring or aromatic heterocycle having a molecular weight of 1000 or less; r2Selected from hydrogen or a group having a molecular weight of 1000 or less directly attached to the amide nitrogen atom.
R2Selected from alkyl, substituted alkyl, aryl or substituted aryl; wherein the alkyl is C1～C20Alkyl or C3～C8A cycloalkyl group; 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 independently selected from halogen, nitro, alkoxy, halogenated phenyl, phenyl and alkyl phenyl;
the aryl is selected from pyridyl, quinolyl, thiazolyl, thienyl or phenyl; the number of the substituent groups of the substituted aromatic group is one or more, and the substituent groups of the substituted aromatic group are independently selected from halogen, cyano, nitro, alkoxy, alkanamido, alkanoyl and C1～C20One or more of alkyl groups.
Preferably, R is2Selected from straight or branched C1～C12An alkyl group; further, said R2Selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and hexyl.
Preferably, R is2Is selected from C3～C6Saturated cycloalkyl, in particular selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.
Preferably, R is2The alkyl is selected from substituted alkyl, the alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and hexyl, and the substituent of the substituted alkyl is phenyl, alkylphenyl or halogenated phenyl; still further, the alkylphenyl group is selected from the group consisting of methylphenyl group, ethylphenyl group, n-propylphenyl group, isopropylphenyl group, n-butylphenyl group, isobutylphenyl group, tert-butylphenyl group, pentylphenyl group, hexylphenyl group; the halogenated phenyl is selected from the group consisting of fluorinated phenyl, chlorinated phenyl, brominated phenyl and iodophenyl.
Preferably, R is2Is selected from substituted aryl, the aryl is phenyl, and the substituent of the substituted aryl is C1～C6Alkyl radical, C1～C6Alkoxy, alkylaminoacyl, C1～C6Alkanoyl orHalogen; further on; said C is1～C6The alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and hexyl; said C is1～C6Alkoxy is selected from methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy; the alkylaminoacyl group is selected from aminoacyl, methionyl, ethylacyl, n-alaninyl, isopropylacyl, n-butylacyl, isobutylyl, tert-butylacyl, pentylyl, hexylyl; said C is1～C6The alkanoyl is selected from the group consisting of methanoyl, ethanoyl, n-propanoyl, isopropanoyl, n-butanoyl, iso-butanoyl, tert-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-nitro aromatic amide compound can be prepared from an On-DNA o-nitro aromatic carboxylic acid compound, and the reaction route is as follows:
step 1, condensing an On-DNA o-nitro aromatic carboxylic acid compound serving as a substrate with amine at the temperature of 20-100 ℃ to obtain an On-DNA o-nitro aromatic amide compound; the reaction is that amide condensation occurs in the presence of N-hydroxy-7-azabenzotriazole and N, N' -diisopropylcarbodiimide; the reaction is carried out in a solvent, wherein the solvent is one or a mixture of water in acetonitrile, methanol, ethanol, DMF, DMA, DMSO, THF, water, inorganic salt buffer solution, organic acid buffer solution and organic base buffer solution.
Preferably, the solvent is a borate buffer containing DMA; further, the pH of the buffer was 9.4.
Preferably, the reaction temperature is 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ or 90 ℃.
In the reaction, the molar equivalent of the On-DNA o-nitro aromatic carboxylic acid compound is 1, the molar equivalent of the amine is 150-300 equivalents, and preferably, the molar equivalent of the amine is 150 equivalents, 200 equivalents, 250 equivalents or 300 equivalents.
And 2, reacting the On-DNA o-nitro aromatic amide compound serving as a raw material under the reaction condition of alkali and tetrahydroxy diboron to obtain the On-DNA N, N-monosubstituted indazolone compound. The reaction steps are as follows:
an On-DNA o-nitroarylamide compound solution with the molar equivalent of 1 and the molar concentration of 0.1-2 mM, 300-500 times of molar equivalent of alkali and 80-150 times of molar equivalent of B2(OH)4Mixing and reacting for 0.5-24 h at room temperature.
Further, the molarity of the On-DNA o-nitroarylamide compound solution was 0.5mM, 1.0mM, or 2 mM.
Preferably, the base is sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, N-diisopropylethylamine or 1,1,3, 3-tetramethylguanidine.
Preferably, when the molar equivalent of the On-DNA o-nitroarylamide 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 24 h.
Further, the reaction is carried out in a solvent, wherein the solvent is one or a mixture of water in 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.
Furthermore, the adding amount of the ethanol is 10-20% of the total volume of the On-DNA o-nitroarylamide compound solution and the alkali solution.
Further, the feeding sequence of the reaction is as follows: sequentially adding the On-DNA o-nitro aromatic 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 libraries of DNA-encoding compounds for multi-well plates.
The method can realize the synthesis of the On-DNA N, N-monosubstituted indazolone compound by the On-DNA o-nitro aromatic amide compound in a DNA coding compound library, and can be widely applied to various On-DNA o-nitro aromatic amide substrates. The On-DNA o-nitro aromatic amide compound used by the invention can be obtained by carrying out amide condensation On the On-DNA o-nitro aromatic formic acid and an amino compound, and the amino compound can be introduced in 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 the preferred embodiment of the invention, by adding ethanol into the reaction system, the generation of byproducts can be inhibited, and the accuracy of library construction applied to the DNA coding compounds can be improved.
Definitions of terms used in connection with the present invention: the initial definitions provided herein for a group or term apply to that group or term throughout the specification unless otherwise indicated; for terms not specifically defined herein, the meanings that would be given to them by a person skilled in the art are to be given in light of 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 indicated by a prefix, e.g. prefix (Ca-C)b) Alkyl means any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C1～C20Alkyl radicalThe alkyl group is a straight chain or branched chain alkyl group having 1 to 20 carbon atoms.
Alkyl is a straight or branched chain hydrocarbon radical derived from an alkane molecule by the removal of one hydrogen atom, e.g. methyl-CH3ethyl-CH2CH3. The alkyl group may also be part of another group, such as C1～C6An 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.
The aryl group means a group obtained by substituting a part of H on an aromatic ring, such as pyridyl, quinolyl, thiazolyl or phenyl.
Alkoxy means that the alkyl radical is linked to an oxygen atom to form a substituent, e.g. methoxy is-OCH3。
The halophenyl group means a group in which H on a phenyl group is substituted with halogen.
Alkanoyl means an alkyl group linked to a C atom in a carbonyl group to form a substituent, e.g. acetyl is-C (O) CH3。
Aminoacyl means that the N atom in the amino group is linked to the C atom in the carbonyl group to form a substituent, e.g. aminoacyl is-C (O) NH2And methinyl is-C (O) NHCH3。
Aromatic heterocyclic ring means an aromatic unsaturated ring containing at least one heteroatom; wherein the hetero atom means a nitrogen atom, an oxygen atom, a sulfur atom.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples, but it should not be construed that the scope of the above subject matter is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1: the conversion rate distribution corresponding to the 22 On-DNAN, N-monosubstituted indazolone compounds obtained in example 5 of the invention is shown.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
In the invention, the term "room temperature" means 20-25 ℃.
HOAT: n-hydroxy-7-azabenzotriazole; DIC: n, N' -diisopropylcarbodiimide;
DMA: dimethylacetamide; DMF: dimethylformamide (DMF).
HATU: 2- (7-azabenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate;
DIPEA: n, N-diisopropylethylamine.
DMSO, DMSO: dimethyl sulfoxide; THF: tetrahydrofuran.
DNA-NH in the present invention2Is formed by single-stranded or double-stranded DNA and a linker group and has-NH2DNA constructs for linkers, e.g. DNA-NH of "compound 1" in WO20050584792And (5) structure.
Example 1: synthesis of On-DNA o-nitro aromatic carboxylic acid compound
Respectively placing 100 times molar equivalent of 4-nitro-3-methoxycarbonylbenzoic acid or 2-nitro terephthalic acid monomethyl ester solution (with concentration of 200mM dissolved in DMA) and 100 times molar equivalent of HATU (with concentration of 400mM dissolved in DMA) and 100 times equivalent of DIPEA (with concentration of 400mM dissolved in DMA) in a refrigerator at-20 deg.C, cooling for 5min, mixing, fully mixing the mixture by vortex oscillation, storing in a refrigerator at 4 deg.C for 5min, and adding the mixture into DNA-NH2To the solution (1 mM; borate buffer, pH 9.4, 250 mM solution), the reaction mixture was vortexed and mixed thoroughly, and reacted at room temperature for 2 hoursAfter the completion of the reaction, ethanol precipitation is carried out, a freeze-drying reagent is dissolved in double distilled water to be set to the concentration of 0.5mM, 1M sodium hydroxide aqueous solution with the molar equivalent of 300 times is added into the solution, and the solution is reacted for 1h at 60 ℃ after being fully and uniformly mixed by vortex oscillation;
and (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, putting the reaction into dry ice for freezing for 2h, then centrifuging for half an hour at the rotating speed of 12000 rpm, pouring out a 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 μ L, 10nmol), and aniline (300 molar equivalents, 300mM MeCN/H) was added to the solution in sequence2O1: 1 solution), HOAt (600 molar equivalents, 600mM in DMSO), DIC (600 molar equivalents, 600mM in DMSO), reaction at room temperature for 1 h;
and (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the volume of 3 times of the total volume, uniformly oscillating, placing the reaction in dry ice for freezing for 2h, centrifuging at the rotating speed of 12000 rpm for half an hour, pouring out a supernatant, and freeze-drying to obtain an On-DNA compound 2;
step 2, synthesis of On-DNA N, N-monosubstituted indazolone compound
On-DNA Compound 2 was dissolved in water to prepare a 1mM concentration solution (10. mu.L, 10nmol), and NaOH (300 molar equivalent, 1M H) was sequentially added to the solution2O solution), EtOH (10% of the total volume of the On-DNA compound 2 solution and the NaOH solution), B2(OH)4(80 molar equivalents, 100mM H2O solution), room temperature reaction2 h；
And (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2h, then centrifuging for half an hour at the rotating speed of 12000 rpm, pouring out a supernatant, dissolving the rest precipitate with deionized water to obtain a solution of an On-DNA product, and sending LCMS to confirm that the conversion rate of the reaction is 90%.
Example 3 Synthesis of On-DNAN, N-monosubstituted indazolone Compounds
R2＝C6H5-or C6H5-CH2-CH2-。
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 μ L, 10nmol), to which aniline or phenethylamine (300 molar equivalents, 300mM MeCN/H) was added, respectively2O1: 1 solution), HOAt (600 molar equivalents, 600mM in DMSO), DIC (600 molar equivalents, 600mM in DMSO), reaction at room temperature for 1 h;
and (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the volume of 3 times of the total volume, after uniformly oscillating, placing the reaction in dry ice for freezing for 2h, then centrifuging for half an hour at the rotating speed of 12000 rpm, pouring out a supernatant, and freeze-drying to obtain two On-DNA compounds 2;
step 2, synthesis of On-DNA N, N-monosubstituted indazolone compound
Two On-DNA compounds 2 were dissolved in water to prepare a 1mM solution (10. mu.L, 10nmol), and NaOH (X molar equivalent, 1M H) was sequentially added to the solution2O solution), EtOH (Y% V/V), B2(OH)4(Z molar equivalents, 100mM H2O solution), reacting for 24 hours at room temperature;
and (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the total volume of 3 times, after uniformly oscillating, placing the reaction in dry ice for freezing for 2h, then centrifuging at 12000 rpm for half an hour, pouring out the supernatant, dissolving the rest precipitate with deionized water to obtain a solution of an On-DNA product 3, sending LCMS to confirm the reaction conversion rate, wherein the specific reaction conditions and the product conversion rate are as shown in the following table 1:
y% represents the volume of EtOH added in relation to the total volume of the On-DNA compound 2 solution and NaOH solution.
Table 1: specific reaction conditions and product conversion for example 3
Example 4 Synthesis of On-DNAN, N-monosubstituted indazolone Compounds
R2＝C6H5-or C6H5-CH2-CH2-。
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 solution (10. mu.L, 10nmol), and aniline or phenethylamine (300 molar equivalents, 300mM MeCN/H) was added to the solution in this order2O1: 1 solution), HOAt (600 molar equivalents, 600mM in DMSO), DIC (600 molar equivalents, 600mM in DMSO), reaction at room temperature for 1 h;
and (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the volume of 3 times of the total volume, after uniformly oscillating, placing the reaction in dry ice for freezing for 2h, then centrifuging for half an hour at the rotating speed of 12000 rpm, pouring out a supernatant, and freeze-drying to obtain two On-DNA compounds 2;
step 2, synthesis of On-DNA N, N-monosubstituted indazolone compound
Two On-DNA compounds 2 were dissolved in water to prepare a 1mM solution (10. mu.L, 10nmol), and NaOH (X molar equivalent, 1M H) was sequentially added to the solution2O solution), EtOH (Y% V/V), B2(OH)4(Z molar equivalents, 100mM H2O solution), reacting for 2h at room temperature;
and (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the total volume of 3 times, after uniformly oscillating, placing the reaction in dry ice for freezing for 2h, then centrifuging at 12000 rpm for half an hour, pouring out the supernatant, dissolving the rest precipitate with deionized water to obtain a solution of an On-DNA product 3, and sending LCMS to confirm the reaction conversion rate, wherein the specific reaction conditions and the product conversion rate are as shown in the following table 2:
y% represents the volume of EtOH added in relation to the total volume of the On-DNA compound 2 solution and NaOH solution.
Table 2: specific reaction conditions and product conversion for example 4
The experimental results show that the addition of ethanol into the reaction system can inhibit the generation of byproducts and effectively improve the yield of the reaction. The reaction condition 2 produces the highest yield of the target product.
Example 5 Synthesis of On-DNAN, N-monosubstituted indazolone Compounds from different substrates
On-DNA o-nitrobenzoic acid 1 was dissolved in borate buffer (0.5M, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), and 22 primary amine molecules (300 molar equivalents) were added to the solution in sequence300mM MeCN/H2O1: 1 solution), HOAt (600 molar equivalents, 600mM in DMSO), DIC (600 molar equivalents, 600mM in DMSO), reaction at room temperature for 1 h;
and (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the volume of 3 times of the total volume, after uniformly oscillating, placing the reaction in dry ice for freezing for 2h, then centrifuging for half an hour at the rotating speed of 12000 rpm, pouring out the supernatant, and freeze-drying to obtain 22 On-DNA compounds 2;
22 On-DNA compounds 2 were dissolved in water to prepare 1mM solutions (10. mu.L, 10nmol), and NaOH (300 molar equivalents, 1M H) was sequentially added to the solutions2O solution), EtOH (10% of the total volume of the reaction solution), B2(OH)4(80 molar equivalents, 100mM H2O solution), reacting for 2h at room temperature;
and (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the total volume of 3 times of the solution, after uniformly oscillating, putting the reaction into dry ice for freezing for 2h, then centrifuging for half an hour at the rotating speed of 12000 rpm, pouring out a supernatant, dissolving the rest precipitate with deionized water to obtain 22 solutions of the On-DNA products 3, and sending the solution to LCMS to confirm the conversion rate of the reaction.
In conclusion, the invention can obtain the On-DNA N, N-monosubstituted indazolone compound from the On-DNA o-nitroanilide compound in the presence of tetrahydroxydiboron by controlling the conditions of solvent, temperature, pH and the like during the reaction. The method can be carried out in a mixed aqueous phase of an organic solvent and an aqueous phase, has the advantages of single product, simple operation and environmental friendliness, and is suitable for synthesizing a DNA coding compound library by using a porous plate.
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