CN112661803A

CN112661803A - Synthetic method of On-DNA 4-amino quinazoline compound in construction of DNA coding compound library

Info

Publication number: CN112661803A
Application number: CN202011494835.9A
Authority: CN
Inventors: 文华楠; 葛蕊; 曲毅; 吴阿亮; 安玉龙; 苏文姬; 蒯乐天; 杨洪芳; 彭宣嘉
Original assignee: Wuxi Apptec Co Ltd
Current assignee: Wuxi Apptec Co Ltd
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2021-04-16

Abstract

The invention discloses a synthetic method of an On-DNA 4-amino quinazoline compound in the construction of a DNA coding compound library, which comprises the following steps: the method takes an On-DNA amino compound as a raw material, uses a condensing agent as an activating agent, does not need to add any metal catalyst in the presence of organic alkali, and can realize the high-efficiency amination reaction of the 4-hydroxyquinazoline compound after reacting for a period of time at a certain temperature. The preparation method of the On-DNA 4-amino quinazoline compound provided by the invention has the advantages of mild reaction conditions, no use of any metal catalyst, low cost, simple post-treatment, environmental friendliness and high yield, and is suitable for synthesizing a DNA coding compound library by using a porous plate.

Description

Synthetic method of On-DNA 4-amino quinazoline compound in construction of DNA coding compound library

Technical Field

The invention belongs to the technical field of DNA coding compound libraries, and particularly relates to a method for synthesizing an On-DNA 4-amino quinazoline compound by directly aminating a 4-hydroxy quinazoline compound to realize the reaction of the On-DNA amino compound and the 4-hydroxy quinazoline compound.

Background

The concept of libraries of DNA-Encoded Library (DEL) was proposed in 1992 by the teaching of Sydney Brenner and Richard Lerner of the Scripps institute of America (reference: Proc. Natl.Acad.Sci.,1992,89,5381) by linking an organic small molecule reagent to a unique sequence of DNA at the molecular level, rapidly constructing a huge Library of compounds each consisting of different organic small molecule reagent residues and identified by DNA of a corresponding unique base sequence through two to many cycles using a combinatorial chemistry "combinatorial-resolution" strategy, affinity-screening a small Library of DNA-Encoded compounds to a target, washing away Library molecules that are not adsorbed to the target, leaving Library molecules adsorbed to the target and then washing away, where the resulting Library molecule concentrations are low and difficult to analyze and identify by conventional means, Polymerase Chain Reaction of DNA Polymerase (Polymerase Chain Reaction, PCR for short) can be carried out to copy and amplify the DNA part in the obtained library molecule adsorbed with the target until the obtained DNA quantity can be identified by a DNA sequencer, the sequenced data is decoded by a relation table between small molecule reagents and DNA base sequences established when a DNA coding compound library is constructed, then the small molecule reagents corresponding to specific compounds corresponding to potential active molecules are found, then the small molecule reagents are combined together by a traditional organic synthesis method to obtain the screened target molecules, and the biological activity of the target molecules on the target is detected and confirmed.

The method for constructing DNA coding compound Library mainly includes three kinds, the first kind is DNA-guided Chemical Library Synthesis (DTCL) mainly obtained by using DNA template technology from Ensemble corporation in America, the second kind is DNA-Recorded Chemical Library (DRCL) mainly obtained by using DNA marking technology from GSK corporation in America, X-Chem corporation and domestic leaders, the third kind is coding Self-assembly molecule Library (ESAC) mainly obtained by Fragment-based drug design (Fragment-based drug discovery, FBDD) technology from Philogen corporation in Switzerland, the method for constructing DNA coding compound Library which is industrially applied in large quantity at present is mainly the second kind, the method is simple to operate and lower in cost, and can quickly obtain a DNA coding compound library containing massive compounds by using a combinatorial chemistry method.

Besides the DNA starting fragment (see the invention patents of the present company: CN108070009A, CN109868268A), a large number of DNA tags and small organic molecule reagents which can be reacted in a certain order are required. The DNA tag code can be obtained by a computer program (see the present invention: CN107958139A), and a primer of a specific DNA base sequence can be obtained by a DNA synthesizer. The small organic molecule reagent can be obtained by screening the obtained reagent list by using a certain computer program (see the invention patent of the company: CN 108959855A).

One of the major tasks in the DEL library field at present is the development of chemical reactions On DNA, called On-DNA chemistry for short. Because DNA must be kept stable in a certain aqueous phase, pH, temperature, metal ion concentration and inorganic salt concentration, the On-DNA chemical reaction which has small DNA damage, better recovery rate and wide substrate adaptability is required for the synthesis of a DNA coding compound library in a large scale. There are nearly 120 types of On-DNA chemistry reported in the prior publications, including aqueous, solid-phase and DNA template reactions (see in detail On-line database DEL ChemFinder,https://delopen.org/ reactions) The reaction conditions are as few as one, and as many as a dozen, and in the same manner as the other cases, the On-DNAThe more the types of chemical reactions are, the more the conditions are, the better the universality is, the more the selectivity is in the design of the DNA coding compound library, the higher the synthesis success rate of the final DNA coding compound library is, and the more the diversity of the obtained DNA coding compound library is.

4-aminoquinazoline compounds are widely present in the structure of natural products and drug molecules, and their construction has attracted extensive research interest to chemists. For this reason, many custom synthetic methods have been developed, particularly the construction of cyclic systems. However, no synthetic method of On-DNA 4-aminoquinazoline compounds has been reported in the literature and patents for the construction of libraries of DNA-encoding compounds. In order to solve the above problems, it is desirable to develop a simple and rapid method for synthesizing On-DNA 4-aminoquinazoline compounds, so as to meet the requirement of mass production of libraries of DNA-encoding compounds in multi-well plates.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for quickly constructing an On-DNA 4-amino quinazoline compound in the construction of a DNA coding compound library.

In order to solve the technical problems, the invention provides a method for synthesizing an On-DNA 4-amino quinazoline compound in the construction of a DNA coding compound library, which synthesizes the On-DNA 4-amino quinazoline compound by directly ammoniating the 4-hydroxy quinazoline compound by adopting an On-DNA amine compound, and comprises the following steps:

mixing an On-DNA amine compound with the molar concentration of 0.1-2.0 mM with 10-500 molar equivalent of a 4-hydroxyquinazoline compound solution, 10-500 molar equivalent of an organic base reagent and 10-500 molar equivalent of a condensing agent solution, and reacting at 20-80 ℃ for 1-24 hours to react the On-DNA amine compound with the 4-hydroxyquinazoline compound to obtain an On-DNA 4-aminoquinazoline compound;

wherein, the On-DNA amino compound is an On-DNA compound obtained by connecting a chemical group containing primary amine or secondary amine with amino or carboxyl of a DNA sequence at the molecular level through a covalent bond;

the DNA is a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers;

the 4-hydroxyquinazoline compound is a compound which contains a 4-hydroxyquinazoline heterocyclic structure in a molecular structure, has a molecular weight of less than 1000 and does not contain other functional groups capable of directly carrying out chemical reaction with primary amine or secondary amine.

Specifically, the general structural formula of the On-DNA amino compound can be DNA-R-NH₂The compound includes an On-DNA arylamine compound and an On-DNA alkylamine compound. On-DNA amine-based compounds include, but are not limited to, the following structures:

wherein R is₁Functional groups, R, directly attached to the DNA moiety₁Is a functional group which complementarily reacts with a functional group on DNA and is selected from any one of amino, carboxyl, aldehyde group and aromatic halide; r₁Directly connected with the aromatic ring or the alkyl, or connected with the aromatic ring or the alkyl at intervals of a plurality of chemical bonds; r₂Independently selected from hydrogen, aryl, alkyl, halogen, amino, nitro, cyano, hydroxy, mercapto, aryl ketone, alkyl ketone, C₁-C₁₂Alkyl radical, C₁-C₆Alkylene radical, C₁-C₆Alkynyl radical, C₃-C₈Cycloalkyl radical, C₁-C₆Alkyloxy, C₁-C₆Any one of alkylamino, R₂The number on the ring is one or more.

Specifically, the structural general formula of the 4-hydroxyquinazoline compound is

Wherein R' is independently selected from hydrogen, aryl, alkyl, halogen, nitro, cyano, C₁-C₁₂Alkyl radical, C₁-C₆Alkylene radical, C₁-C₆Alkynyl radical, C₃-C₈Cycloalkyl radical, C₁-C₆Alkyloxy, C₁-C₆Any one of alkylamino groups.

Specifically, the molar concentration of the On-DNA amino compound dissolved in the inorganic salt buffer solution is 0.1-2.0 mM; preferably, the On-DNA aldehyde based compound is dissolved in the inorganic salt buffer solution at a molar concentration of 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM, 1.1mM, 1.2mM, 1.3mM, 1.4mM, 1.5mM, 1.6mM, 1.7mM, 1.8mM, 1.9mM, or 2.0 mM; more preferably, the On-DNA aldehyde compound has a molar concentration of 1.0mM after being dissolved in an inorganic salt buffer solution.

Specifically, the solvent of the On-DNA amino compound solution is a water-containing mixed solvent containing any one or more of acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butyl alcohol, isopropanol, tetrahydrofuran, an inorganic salt buffer solution, an organic acid buffer solution and an organic base buffer solution; preferably, the On-DNA carbonyl compound is dissolved in an inorganic salt buffer.

Specifically, the molar equivalent of the 4-hydroxyquinazoline compound is 10-500 equivalents; preferably, the molar equivalents of the amino acid or amino acid derivative are 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 60 equivalents, 70 equivalents, 80 equivalents, 90 equivalents, 100 equivalents, 150 equivalents, 200 equivalents, 250 equivalents, 300 equivalents; more preferably, the molar equivalent of the amino acid derivative is 200 equivalents.

Specifically, the organic base reagent is N, N-Diisopropylethylamine (DIPEA), 1,8 diazabicycloundecen-7-ene (DBU), triethylene diamine, triethylamine, trimethylamine, pyridine, piperidine; preferably, the organic base reagent is 1,8 diazabicycloundec-7-ene (DBU).

Specifically, the molar equivalent of the organic base reagent is 10-500 equivalents; preferably, the molar equivalents of base are 10 equivalents, 20 equivalents, 40 equivalents, 80 equivalents, 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, 500 equivalents; more preferably, the molar equivalent of the base is 200 equivalents.

Specifically, the condensing agent is 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU, CAS 148893-10-1), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, CAS 128625-52-5), benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP, CAS 56602-33-6), (3H-1,2, 3-triazolo [4,5-b ] pyridin-3-yloxy) tris-1-pyrrolidinylphosphonium hexafluorophosphate (PyAOP, CAS 156311-83-0), tripyrrolidinylphosphonium hexafluorophosphate (PyBrOP, CAS 132705-51-2), 6-chlorobenzotriazole-1, 1,3, 3-tetramethyluronium hexafluorophosphate (HCTU, CAS 330645-87-9); preferably, the condensing agent is benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP, CAS 56602-33-6).

Specifically, the molar equivalent of the condensing agent is 10 to 500 equivalents; preferably, the molar equivalents of the condensing agent are 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 60 equivalents, 70 equivalents, 80 equivalents, 90 equivalents, 100 equivalents, 150 equivalents, 200 equivalents, 250 equivalents, 300 equivalents; more preferably, the molar equivalent of the condensing agent is 10 to 500 equivalents.

Specifically, the reaction temperature of the reaction is 20-80 ℃; preferably, the reaction temperature of the reaction is 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃ or 80 ℃; more preferably, the temperature of the reaction is 60 ℃.

Specifically, the reaction time of the reaction is 0.1-24 hours; preferably, the reaction time of the reaction is 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 16 hours, 24 hours; more preferably, the reaction time of the reaction is 3 hours.

In particular, the method is used for batch multi-well plate operations; preferably, the method is used for the synthesis of libraries of DNA-encoding compounds for multiwell plates.

The invention provides a method for directly aminating a 4-hydroxyquinazoline compound, which realizes the high-efficiency reaction of an On-DNA amino compound and the 4-hydroxyquinazoline compound in the construction of a DNA coding compound library, can be used for synthesizing the On-DNA 4-aminoquinazoline compound, enlarges the diversity of the DNA coding compound library of the company, and ensures that library molecules of the obtained DNA coding compound library can better meet the market requirements. The method has the advantages of good universality to reaction substrates, mild conditions, low cost, convenient operation and high yield, and is suitable for synthesizing the DNA coding compound library by using a porous plate.

Drawings

FIG. 1 is a representative structural formula of the On-DNA 4-aminoquinazoline 5 obtained by the reaction between the On-DNA amino compound 3 and the 4-hydroxyquinazoline compound 4.

FIG. 2 is an example of the reaction equation for the synthesis of the On-DNA 4-aminoquinazoline compound according to the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

EXAMPLE 1 Synthesis of On-DNA 4-hydroxyquinazoline Compound 5

1) Synthesis of On-DNA arylamine Compound 3a

DNA-NH₂Compound 1 (e.g., the starting headpiece mentioned in patent CN 108070009A) was dissolved in 250mM boric acid buffer at pH 9.5 to prepare a 1mM concentration solution, and reacted with acid 2a (200mM solution in dimethylacetamide, 60 molar equivalents) using EDCI (200mM solution in dimethylsulfoxide, 50 molar equivalents) as a condensing agent and s-NHS (200mM solution in dimethylsulfoxide and water at a volume ratio of 1:1, 30 molar equivalents) as a condensation activator at 20 ℃ for 16 hours to give the corresponding on-DNA arylnitro compound intermediate, which was subjected to ethanol precipitation only after completion of the reaction and concentrated and dried for direct use in the next reaction.

The on-DNA arylnitro compound intermediate was dissolved in 250mM boric acid buffer (pH 9.5) to prepare a 1mM solution, and B was added₂(OH)₄Reacting the water solution (200mM, 200 mol equivalent) at 80 ℃ for 4 hours to obtain the On-DNA arylamine compound 3a, only carrying out ethanol precipitation treatment after the reaction is finished, concentrating and drying the product, and directly using the product for the next reaction.

2) Synthesis of On-DNA alkylamine Compound 3b

DNA-NH₂Compound 1 (e.g. the starting headpiece mentioned in patent CN 108070009A) was dissolved in 250mM, pH 9.5 boric acid buffer to make a 1mM concentration solution, reacted with acid 2b (200mM solution of dimethylacetamide, 60 molar equivalents) using EDCI (200mM solution of dimethylsulfoxide, 50 molar equivalents) as a condensing agent and s-NHS (200mM solution of dimethylsulfoxide and water in a volume ratio of 1:1, 30 molar equivalents) as a condensation activator at 20 ℃ for 16 hours to give the corresponding on-DNA compound intermediate, which was subjected to ethanol precipitation only after completion of the reaction, concentrated and dried and then used directly in the next step.

Dissolving the on-DNA compound intermediate in H₂And O, preparing a 1mM concentration solution, adding an equal volume of 20% piperidine aqueous solution, reacting at 25 ℃ for 2 hours to obtain an On-DNA alkylamine compound 3b, only carrying out ethanol precipitation treatment after the reaction is finished, and concentrating and drying the product to be directly used for the next reaction.

It is noted that the On-DNA arylamine compound 3a and the On-DNA alkylamine compound 3b will jointly form the On-DNA arylamine compound 3 (DNA-R-NH)₂)。

3) Synthesis of On-DNA 4-aminoquinazoline compound 5

The On-DNA amino compound 3 was dissolved in water to prepare a 1mM solution. 4-hydroxyquinazoline compound 4(200mM in N-methylpyrrolidone, 200 molar equivalents) and benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP, CAS 56602-33-6,200mM in N-methylpyrrolidone, 200 molar equivalents) and 1,8 diazabicycloundecen-7-ene (DBU,200mM in N-methylpyrrolidone, 200 molar equivalents) were mixed, vortexed, dispensed into a 96-well plate, reacted at 60 ℃ for 5 minutes in PCR, then 1mM in On-DNA amino compound 3 was added to the reaction solution, mixed, vortexed, centrifuged, and reacted at 60 ℃ for 3 hours in PCR.

Adding ethanol for precipitation:

adding 5M sodium chloride solution with 10% of total reaction solution volume into reaction holes of a 96-well plate, sealing a membrane, oscillating and uniformly mixing, adding cold absolute ethyl alcohol with 3 times of total volume and stored at the temperature of 20 ℃ below zero, freezing for 2 hours in a refrigerator at the temperature of 80 ℃ below zero, taking out and centrifuging for 30 minutes at the temperature of 4 ℃ under the centrifugal force of 4000G, sucking supernatant, dissolving precipitates with deionized water, performing vacuum freeze-drying at the temperature of 40 ℃ below zero to obtain a product, detecting OD (optical density) by using an enzyme labeling instrument to confirm recovery rate, and simultaneously detecting LC-MS (liquid chromatography-mass spectrometry) to confirm the conversion rate of each small molecule (see figure 1). As shown in FIG. 1, the conversion rates of the On DNA amino compounds were all 50% or more, and the conversion rates of half or more of the On DNA amino compounds were 80% or more.

In summary, the above embodiments and drawings are only preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. The method is characterized in that an On-DNA amine compound with the molar concentration of 0.1-2.0 mM is mixed with a 4-hydroxyquinazoline compound solution with 10-500 molar equivalents, an organic base reagent with 10-500 molar equivalents and a condensing agent solution with 10-500 molar equivalents, and the mixture is reacted for 1-24 hours at the temperature of 20-80 ℃ to ensure that the On-DNA amine compound and the 4-hydroxyquinazoline compound are subjected to direct ammoniation reaction to obtain an On-DNA 4-aminoquinazoline compound;

wherein, the On-DNA amino compound is an On-DNA compound obtained by connecting a chemical group containing primary amine or secondary amine with amino or carboxyl of a DNA sequence at a molecular level through a covalent bond; the DNA is a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers;

2. The method of claim 1, wherein the On-DNA amine-based compound is selected from the following structures:

wherein R is₁R1 is a functional group which reacts complementarily with a functional group on DNA and is a functional group directly linked to a DNA moiety, and is selected from any one of amino groups, carboxyl groups, aldehyde groups and aromatic halides; r₁Directly connected with the aromatic ring or the alkyl, or connected with the aromatic ring or the alkyl at intervals of a plurality of chemical bonds; r₂Independently selected from hydrogen, aryl, alkyl, halogen, amino, nitro, cyano, hydroxy, mercapto, aryl ketone, alkyl ketone, C₁-C₁₂Alkyl radical, C₁-C₆Alkylene radical, C₁-C₆Alkynyl radical, C₃-C₈Cycloalkyl radical, C₁-C₆Alkyloxy, C₁-C₆Any one of alkylamino, R₂The number on the ring is one or more.

3. The method of claim 1, wherein the molar concentration of the On-DNA amine-based compound after dissolution in an aqueous solution is 0.1-2.0 mM; preferably, the molar concentration of the On-DNA amino compound aqueous solution is 1.0 mM.

4. The method according to claim 1, wherein the solution is an aqueous mixed solvent containing any one or more of acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, methanol, ethanol, t-butanol, isopropanol, tetrahydrofuran, an inorganic salt buffer, an organic acid buffer, and an organic base buffer; preferably, the On-DNA amine compound is soluble in water.

5. The method of claim 1, wherein the molar equivalent of the 4-hydroxyquinazoline is 10 to 500 equivalents; preferably, the molar equivalent of the small molecule 4-hydroxyquinazoline is 200 equivalents.

6. The method of claim 1, wherein the organic base reagent is N, N-Diisopropylethylamine (DIPEA), 1,8 diazabicycloundecen-7-ene (DBU), triethylenediamine, triethylamine, trimethylamine, pyridine, piperidine; preferably, the organic base reagent is 1,8 diazabicycloundec-7-ene (DBU).

7. The method of claim 1, wherein the molar equivalents of the organic base reagent are 10 to 500 equivalents; preferably, the molar equivalent of the organic base reagent is 200 equivalents.

8. The method of claim 1, wherein the condensing agent is 2- (7-azobenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate, (3H-1,2, 3-triazolo [4,5-b ] pyridin-3-yloxy) tris-1-pyrrolidinylphosphonium hexafluorophosphate, tripyrrolidinylphosphonium hexafluorophosphate, 6-chlorobenzotriazol-1, 1,3, 3-tetramethyluronium hexafluorophosphate; preferably, the condensing agent is benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate.

9. The method of claim 1, wherein the molar equivalent of the condensing agent is 10 to 500 equivalents; preferably, the molar equivalent of the small molecule amine is 200 equivalents.

10. The method of claim 1, wherein the reaction temperature of the reaction is 20 to 80 ℃; preferably, the reaction temperature of the reaction is 60 ℃.

11. The method of claim 1, wherein the reaction time is 1 to 24 hours; preferably, the reaction time of the reaction is 3 hours.