Synthesis method of On-DNA isoxazole compound

Abstract

The invention discloses a synthesis method of On-DNA (deoxyribonucleic acid) isoxazole compounds, which comprises the following steps: firstly, mixing 10 to 500 molar equivalents of aldehyde small molecular compounds with 10 to 500 molar equivalents of hydroxylamine hydrochloride solution, and then adding 10 to 500 molar equivalents of chloramine-T to obtain a first reaction solution; adding 10 to 500 molar equivalents of a metal catalyst and 10 to 500 molar equivalents of a reducing agent into the On-DNA alkyne compound mixed solution to obtain a second reaction solution; and mixing the first reaction solution and the second reaction solution, and reacting at 20-50 DEG C for 2-24 hours to obtain the On-DNA isoxazole compound. The reaction method provided by the invention provides a new method for synthesis of On-DNA isoxazole compounds, and the reaction method is high in yield, wide in substrate universality, mild in condition, convenient to operate, low in cost and suitable for synthesis of a DNA coding compound library by using a porous plate.

Description

Technical Field

[0001] The invention belongs to the technical field of DNA encoded compound library, and specifically relates to a method for synthesizing On-DNA isoxazole compounds. Background Art

[0002] Professor Sydney Brenner and Richard Lerner of the Scripps Research Institute in the United States proposed the concept of DNA Encoded Library (DEL) in 1992 (reference: Proc. Natl. Acad. Sci., 1992, 89, 5381, patent: US5573905). This method connects an organic small molecule reagent with a unique sequence of DNA at the molecular level (i.e., DNA labeling of the small molecule reagent), and uses the "combine-split" strategy of combinatorial chemistry to quickly construct a huge number of compound libraries through two or more cycles. Each compound in the compound library is composed of different organic small molecule fragments and is marked by a corresponding unique base sequence of DNA. A very small amount of DNA encoded compound library is affinity screened with the target. The compound library molecules that are not adsorbed to the target will be washed away first, leaving the compound library molecules that are adsorbed to the target and then eluted. At this time, the concentration of the compound library molecules obtained is very low, and it is difficult to analyze and identify them by conventional means. However, through the unique polymerase chain reaction (PCR) of DNA, the molecular structure of the compound library can be determined by DNA. Chain Reaction, PCR for short) can replicate and amplify the DNA part of the compound library molecules that are adsorbed to the target until the amount of DNA obtained can be recognized by the DNA sequencer. The sequenced data is then decoded through the relationship table between the organic small molecule reagents created when constructing the DNA-encoded compound library and each specific DNA base sequence, and then the small molecule fragments corresponding to the specific compounds corresponding to the potential active molecules are found. These small molecule fragments are then combined together through traditional organic synthesis methods to obtain the screened target molecules, and then their physiological activity on the target is detected and confirmed.

[0003] There are three main methods for constructing DNA-encoded compound libraries. The first is the DNA-Templated Chemical Library Synthesis (DTCL) obtained by using DNA template technology mainly by Ensemble, USA. The second is the DNA-Recorded Chemical Library (DRCL) obtained by using DNA labeling technology mainly by GSK, X-Chem and Chengdu Pioneer, China. The third is the Encoded Self-Assembling Chemical Libraries (ESAC) obtained by using fragment-based drug discovery (FBDD) technology mainly by Philogen, Switzerland. Currently, the second method is the main one widely used in industry to construct DNA-encoded compound libraries. This method is simple to operate, has lower cost, and can more quickly use combinatorial chemistry methods to obtain DNA-encoded compound libraries containing a large number of compounds.

[0004] In addition to the DNA starting fragment (refer to invention patents: CN108070009A, CN109868268A), a large number of DNA tags and organic small molecule reagents that can react in a certain order are required. Among them, the encoding of the DNA tag can be obtained through a certain computer program, and then the primer of the specific DNA base sequence is obtained through a DNA synthesizer; the organic small molecule reagent can be obtained by using a certain computer program to screen the obtained reagent list.

[0005] One of the most important tasks in the field of DEL libraries is the development of chemical reactions on DNA, referred to as On-DNA chemical reactions. Because DNA must remain stable under certain water phases, pH, temperature, metal ion concentrations, and inorganic salt concentrations, On-DNA chemical reactions that cause little DNA damage, have a good recovery rate, and have a wide substrate adaptability are what is needed for large-scale application in the synthesis of DNA-encoded compound libraries. Currently, there are nearly 120 types of On-DNA chemical reactions publicly reported, including aqueous, solid-phase, and DNA template reactions, with each type having a reaction condition ranging from one to more than a dozen. It can be said that, under the same other conditions, the more types of On-DNA chemical reactions, the richer the conditions, the better the universality, the more selectivity in the design of DNA-encoded compound libraries, the higher the success rate of DNA-encoded compound library synthesis, and the richer the diversity of the resulting DNA-encoded compound library.

[0006] In the construction of DNA-encoded compound libraries, isoxazole is an important azole compound containing adjacent N and O atoms and is widely used in organic synthesis. This compound has many biological activities and also has good pharmacological properties. It has a great help to humans in reducing human blood sugar, eliminating human pain, resisting human inflammation, killing harmful bacteria, and controlling and reducing the harm of HIV. At present, the method of applying isoxazole to On-DNA synthesis is very limited. Rongfeng Wu et al. (Chem. Asian J., 2020, 15 (23), 4033-4037) reported a method for obtaining On-DNA isoxazole compounds by on DNA-α, β-unsaturated ketone and hydroxylamine. However, the raw material on DNA-α, β-unsaturated ketone of this scheme is not easy to prepare, and because it can only form a ring with hydroxylamine, it limits the chemical diversity of the isoxazole DEL library, while increasing the cost, which is not conducive to large-scale industrial production.

[0007] In addition to these conventional chemical reactions reported in the literature, due to the diverse characteristics of library synthesis, functional group transformation is often required. However, there are currently no literature or patents reporting methods for synthesizing On-DNA isoxazole compounds through metal-catalyzed strategies.

[0008] In order to solve the limitations of the synthesis method of On-DNA isoxazole compounds in the construction of DNA-encoded compound libraries, it is urgent to develop a synthesis process for DNA-encoded compound libraries with easily available raw materials, mild reaction conditions, high yield, good substrate universality and suitable for the use of multi-well plates, so as to increase the diversity of DNA-encoded compound libraries. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a method for synthesizing an On-DNA isoxazole compound, comprising the following steps:

[0010] 1) First, 10 to 500 molar equivalents of an aldehyde small molecule compound and 10 to 500 molar equivalents of a hydroxylamine hydrochloride solution are mixed, and then 10 to 500 molar equivalents of chloramine-T are added to obtain a first reaction solution, wherein the aldehyde small molecule compound is selected from the following compounds:

[0011]

[0012] 2) adding 10 to 500 molar equivalents of a metal catalyst and 10 to 500 molar equivalents of a reducing agent to the mixed solution of the On-DNA alkyne compound to obtain a second reaction solution, wherein the On-DNA alkyne compound has a structural formula of DNA-NHCOCH2CH2-C≡CH, and the DNA in the structural formula is a single-stranded or double-stranded nucleotide chain obtained by polymerization of artificially modified and / or unmodified nucleotide monomers;

[0013] 3) The first reaction solution and the second reaction solution are mixed and reacted at 20 to 50° C. for 2 to 24 hours to obtain a reaction product.

[0014] The reaction chemical formula is as follows:

[0015]

[0016] in, Represents aldehyde small molecule compounds.

[0017] Since DEL screening is a rapidly developing and efficient screening method for early drug discovery, the lead compounds obtained after decoding the screening data need to be synthesized off-DNA to obtain relevant lead compounds with specific conformations, and then verify their activity against the target. The development of On-DNA chemical reactions pays more attention to the novelty of chemical space, and the doubts about the specific isomer configuration and conformation of the products produced by the reaction can be confirmed in the verification stage of the lead compounds screened later. Therefore, in the reaction of the present invention, the product has two isomers, the ratio of which is not limited, and both isomers are On-DNA isoxazole compounds in the synthesis of DNA-encoded compound libraries.

[0018] Specifically, the concentration of the On-DNA alkyne compound mixed solution is 0.1-2.0 mM.

[0019] Specifically, the On-DNA alkyne compound mixed solution comprises any one or more aqueous mixed solvents selected from acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butanol, isopropanol, tetrahydrofuran, inorganic salt buffer, organic acid buffer, and organic base buffer.

[0020] Preferably, the molar equivalent of the aldehyde small molecule compound is 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 100 equivalents, 150 equivalents, 200 equivalents, 250 equivalents, 300 equivalents, 350 equivalents, 400 equivalents, 450 equivalents or 500 equivalents.

[0021] Preferably, the molar equivalent of the aldehyde small molecule compound is 100 equivalents.

[0022] Preferably, the molar equivalent of the hydroxylamine hydrochloride solution is 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 100 equivalents, 150 equivalents, 200 equivalents, 250 equivalents, 300 equivalents, 350 equivalents, 400 equivalents, 450 equivalents or 500 equivalents.

[0023] Preferably, the molar equivalent of the hydroxylamine hydrochloride solution is 100 equivalents.

[0024] Preferably, the molar equivalent of the chloramine-T is 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 100 equivalents, 150 equivalents, 200 equivalents, 250 equivalents, 300 equivalents, 350 equivalents, 400 equivalents, 450 equivalents or 500 equivalents.

[0025] Preferably, the molar equivalent of the chloramine-T is 20 equivalents.

[0026] Specifically, the metal catalyst is one or a mixture of palladium chloride, platinum chloride, copper chloride, silver chloride, zinc chloride, nickel chloride and cobalt chloride.

[0027] Preferably, the metal catalyst is cupric chloride.

[0028] Preferably, the molar equivalent of the metal catalyst is 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 100 equivalents, 150 equivalents, 200 equivalents, 250 equivalents, 300 equivalents, 350 equivalents, 400 equivalents, 450 equivalents or 500 equivalents.

[0029] Preferably, the molar equivalent of the metal catalyst is 30 equivalents.

[0030] Specifically, the reducing agent is one or a mixture of sodium ascorbate, sodium bisulfite, glucose, mercaptoethanol, and sodium hydroxide.

[0031] Preferably, the reducing agent is sodium ascorbate.

[0032] Preferably, the molar equivalent of the reducing agent is 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 100 equivalents, 150 equivalents, 200 equivalents, 250 equivalents, 300 equivalents, 350 equivalents, 400 equivalents, 450 equivalents or 500 equivalents.

[0033] Preferably, the molar equivalent of the reducing agent is 100 equivalents.

[0034] Specifically, in step 3), the reaction temperature is 20°C.

[0035] Specifically, in step 3), the reaction time is 2 hours.

[0036] In particular, the method is used for batch operations of multiwell plates.

[0037] The present invention realizes the cycloaddition reaction of On-DNA alkyne compounds and in situ generated oxime compounds, which is used to synthesize On-DNA isoxazole compounds. The method provided by the present invention expands the chemical space diversity of DNA-encoded compound libraries. The reaction disclosed by the present invention has good universality of reaction substrates, mild conditions, low cost, convenient operation, high yield, and is suitable for the synthesis of DNA-encoded compound libraries in multi-well plates. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The present invention discloses a chemical reaction formula for preparing On-DNA isoxazole compounds. DETAILED DESCRIPTION

[0039] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental materials involved in the present invention are all commercial reagents unless otherwise specified and can be obtained from commercial channels.

[0040] Example 1

[0041]

[0042] according to Figure 1 The chemical formula shown in Figure 1 shows the synthesis steps of On-DNA isoxazole compounds as follows:

[0043] (1) Aldehyde small molecule compounds (50 mM dimethylacetamide solution, 100 molar equivalents) and hydroxylamine hydrochloride (100 mM dimethylacetamide and water 1:1 mixed solution, 100 molar equivalents) were added to a 96-well plate, mixed and reacted in a shaker at 20°C for 1 hour, and then chloramine-T (50 mM dimethylacetamide and water 1:1 mixed solution, 20 molar equivalents) was added to the reaction solution, mixed and reacted in a shaker at 20°C for 1 hour.

[0044] (2) At the same time, the On-DNA alkyne compound in the above formula Dissolved in 50mM, pH=8.2 Mops buffer, prepared into a 1mM concentration of On-DNA alkyne compound mixed solution, dispensed into 96-well plates, added with copper chloride (75mM aqueous solution, 30 molar equivalents) and sodium ascorbate (250mM aqueous solution, 100 molar equivalents), respectively, replaced with nitrogen three times after mixing, and reacted in a shaker at 20°C for 1 hour.

[0045] (3) Then, the reaction solution of the above-mentioned aldehyde small molecule compound was added to the mixed solution of the On-DNA alkyne compound, and after mixing, the mixture was replaced with nitrogen three times and reacted at 20° C. for 2 hours in a shaker. Finally, sodium diethyldithiocarbamate trihydrate (100 mM aqueous solution, 100 molar equivalents) was added to the reaction solution, and the mixture was reacted at 20° C. for 1 hour to remove metallic copper.

[0046] Ethanol precipitation: add 5M sodium chloride solution of 10% of the total reaction solution volume to the reaction wells of the 96-well plate, seal the film, shake and mix, add 3 times the total volume of cold anhydrous ethanol stored at -20°C, freeze in a -80°C refrigerator for 2 hours, then take out and centrifuge at 4°C with a centrifugal force of 4000G for 30 minutes, remove the supernatant, dissolve the precipitate with deionized water and freeze-dry at -40°C in a vacuum to obtain the product, detect OD by NanoDrop to confirm the recovery rate, and detect LC-MS to confirm the conversion rate of each small molecule. The results are shown in Table 1 below. The conversion rate is the total conversion rate of the two isomers of On-DNA isoxazole compounds. The ratio of the two isomers is not fixed, and the yield is based on the TIC peak area on LCMS-LTQ.

[0047] Table 1

[0048]

[0049]

[0050]

[0051] To sum up, the above embodiments and drawings are only some representative embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for synthesizing an On-DNA isoxazole compound, characterized in that: The following steps are involved: 1) First, 10 to 500 molar equivalents of an aldehyde small molecule compound and 10 to 500 molar equivalents of a hydroxylamine hydrochloride solution are mixed, and then 10 to 500 molar equivalents of chloramine-T are added to obtain a first reaction solution, wherein the aldehyde small molecule compound is selected from the following compounds: 2) adding 10 to 500 molar equivalents of a metal catalyst and 10 to 500 molar equivalents of a reducing agent to the mixed solution of the On-DNA alkyne compound to obtain a second reaction solution, wherein the On-DNA alkyne compound has a structural formula of DNA-NHCOCH2CH2-C≡CH, and the DNA in the structural formula is a single-stranded or double-stranded nucleotide chain obtained by polymerization of artificially modified and / or unmodified nucleotide monomers; 3) The first reaction solution and the second reaction solution are mixed and reacted at 20 to 50° C. for 2 to 24 hours to obtain a reaction product.

2. The method for synthesizing an On-DNA isoxazole compound according to claim 1, characterized in that: The concentration of the On-DNA alkyne compound mixed solution is 0.1-2.0 mM.

3. The method for synthesizing an On-DNA isoxazole compound according to claim 1, characterized in that: The On-DNA alkyne compound mixed solution comprises any one or more aqueous mixed solvents selected from acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butanol, isopropanol, tetrahydrofuran, inorganic salt buffer, organic acid buffer, and organic base buffer.

4. The method for synthesizing an On-DNA isoxazole compound according to claim 1, characterized in that: The molar equivalent of the aldehyde small molecule compound is 100 equivalents.

5. The method for synthesizing an On-DNA isoxazole compound according to claim 1, characterized in that: The metal catalyst is one or a mixture of palladium chloride, platinum chloride, copper chloride, silver chloride, zinc chloride, nickel chloride and cobalt chloride.

6. The method for synthesizing an On-DNA isoxazole compound according to claim 1, characterized in that: The molar equivalent of the metal catalyst is 30 equivalents.

7. The method for synthesizing an On-DNA isoxazole compound according to claim 1, characterized in that: The reducing agent is one or a mixture of sodium ascorbate, sodium bisulfite, glucose, mercaptoethanol and sodium hydroxide.

8. The method for synthesizing On-DNA isoxazole compounds according to claim 1, characterized in that: The molar equivalent of the reducing agent is 100 equivalents.

9. The method for synthesizing an On-DNA isoxazole compound according to claim 1, characterized in that: In step 3), the reaction temperature is 20°C.

10. The method for synthesizing On-DNA isoxazole compounds according to claim 1, characterized in that: In step 3), the reaction time is 2 hours.

11. The method for synthesizing On-DNA isoxazole compounds according to claim 1, characterized in that: The method is used for batch operation of multiwell plates.

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