Synthetic methods for on-DNA 2-thioquinazolinone compounds

Abstract

The present application relates to a synthesis method of On-DNA 2-thioxoquinazolinone compounds, and belongs to the technical field of DNA coded compound library. The synthesis method comprises the following steps: firstly, On-DNA amine compounds are converted into On-DNA isothiocyanate compounds under the action of 1,1'-thiocarbonyldiimidazole; then the obtained On-DNA isothiocyanate compounds are subjected to oxidative addition reaction with isatoic anhydride compounds and tert-butyl hydroperoxide in an aqueous solvent system, so that On-DNA 2-thioxoquinazolinone compounds are obtained. The synthesis method has small damage to DNA, good substrate universality, high yield and low cost, is suitable for the construction of DNA coded compound library, and is conducive to promoting the development of On-DNA 2-thioxoquinazolinone drugs.

Description

Technical Field

[0001] This invention belongs to the technical field of DNA-encoded compound libraries and relates to a method for synthesizing On-DNA 2-thioquinoazolinone compounds. Background Technology

[0002] In the field of drug development, the rapid, efficient, and low-cost discovery of new lead compounds has long been a goal pursued by researchers and large pharmaceutical companies. Research shows that DNA-encoded library (DEL) technology can rapidly select novel, potentially druggable compounds from tens of millions to billions of molecules, thereby significantly shortening drug research cycles and reducing development costs. To date, DEL technology has discovered a large number of high-quality lead compounds targeting various therapeutically relevant sites, such as the epoxidase inhibitor GSK2256294 and the death domain receptor-associated receptor kinase RIP1 inhibitor GSK2982772. These compounds have entered clinical trials, demonstrating the important role of DEL in new drug development.

[0003] Currently, the main limiting factor in DEL technology is how to construct more high-quality compound libraries with structural diversity. Since DNA must remain stable in a specific aqueous phase within a certain pH range, temperature range, metal ion concentration, and inorganic salt concentration to maintain stability, developing on-DNA chemical reactions that minimize DNA damage, achieve high conversion rates, have good DNA recovery rates, and broad substrate adaptability is urgently needed in the synthesis of DNA-encoded compound libraries.

[0004] 2-Thioquinazolinones are an important class of fused heterocyclic compounds that can act as denitrifying flavins (F... 420These compounds are inhibitors of various important proteases, including nitroreductase (Ddn) inhibitors (Y. Jian, HE Forbes, et al., J. Med. Chem., 2021, 64, 440) and type II NADH dehydrogenase inhibitors (D. Murugesan, PCRay, et al., ACS Infect. Dis. 2018, 4, 954). Furthermore, some of these compounds can also act as antagonists or agonists of receptor proteins such as estrogen receptor β (ER) and G protein-coupled receptors (GPCRs) (AK Knox, C. Kalchschmid, et al., J. Med. Chem., 2021, 64, 5766), thus playing a crucial role in medicinal chemistry. In particular, the cyclic thiourea moiety in these compounds is a potential derivatization site. Therefore, 2-thioquinazolinone compounds are commonly used as intermediates for various quinazolinone-containing molecules and are widely used in the synthesis of drugs including fluconazole. However, no synthetic methods for On-DNA 2-thioquinazolinone compounds have been reported in the construction of DNA-encoded compound libraries. Therefore, we aim to develop a simple and rapid On-DNA synthetic method to prepare DNA-encoded compound libraries containing this important structure. On the one hand, this research can enrich the types of chemical reactions for DEL library construction; on the other hand, it is beneficial for discovering other novel targets based on this structure. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide a method for synthesizing On-DNA 2-thioquinazolinone compounds; another objective of the present invention is to provide a library of DNA-encoded 2-thioquinazolinone compounds.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] 1. A method for synthesizing On-DNA 2-thioquinazolinone compounds, the method comprising the following steps:

[0008] Provide On-DNA amine compounds as shown in general formula (I), On-DNA isothiocyanate compounds as shown in general formula (II), and indigo anhydride compounds as shown in general formula (III):

[0009]

[0010] The On-DNA amine compound is first converted into the On-DNA isothiocyanate compound under the action of 1,1'-thiocarbonyl diimidazole. Then, the On-DNA isothiocyanate compound, the indigo anhydride compound, and hydrogen peroxide tert-butanol are reacted in an aqueous solvent system to obtain the On-DNA 2-thioquinoazolinone compound as shown in general formula (IV).

[0011]

[0012] in, R1 is the DNA; R1 is the functional group that links the DNA portion, specifically a functional group that can undergo a complementary reaction with functional groups on the DNA, selected from any one of amide, amino, carboxyl, aldehyde, aryl, or alkyne groups; R2 is selected from -(R4). m -(R5) n - where m and n are integers and not both 0; R3 is selected from hydrogen, halogen, trifluoromethyl, carboxyl, amino, nitro, cyano, hydroxyl, phenyl, ester, amide, aryl ketone, alkyl ketone, alkyl with 1 to 12 carbon atoms, olefin with 2 to 6 carbon atoms, alkyne with 2 to 6 carbon atoms, cycloalkyl with 3 to 8 carbon atoms, alkyloxy with 1 to 6 carbon atoms, or alkylamino with 1 to 6 carbon atoms; R4 is selected from alkyl, alkoxy, or cycloalkyl; R5 is selected from aryl, aromatic heteroyl, substituted aryl, or substituted heteroaryl.

[0013] The DNA is a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and / or unmodified nucleotide monomers.

[0014] Preferably, the substituents in the substituted aryl group and the substituents in the substituted heteroaryl group are independently selected from any one or more of alkyl, alkoxy, cycloalkyl, halogen, hydroxy, trifluoromethyl, amino, ester, amide, nitro, cyano or phenyl.

[0015] Preferably, the specific steps for converting the On-DNA amine compound into the On-DNA isothiocyanate compound under the action of 1,1'-thiocarbonyldiimidazole include: adding 100 to 4000 molar equivalents of 1,1'-thiocarbonyldiimidazole solution to the solution of the On-DNA amine compound, and then reacting at 5 to 40°C for 0.5 to 4 hours.

[0016] Preferably, the solvent in the solution of the On-DNA amine compound is any one of water, an inorganic salt buffer solution, an inorganic alkaline aqueous solution, an organic solvent, or a mixture of an organic solvent and water; the organic solvent is any one or more of acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butanol, isopropanol, tetrahydrofuran, or 1,4-dioxane; the volume ratio of the organic solvent to water in the mixture is 1:1; and the solvent in the 1,1'-thiocarbazide diimidazole solution is N,N'-dimethylacetamide.

[0017] Preferably, the specific steps for reacting the On-DNA isothiocyanate compound, the indocyanine anhydride compound, and tert-butanol peroxide in an aqueous solvent system include: mixing 200–4000 molar equivalents of the indocyanine anhydride compound, 100–2000 molar equivalents of tert-butanol peroxide, dimethyl sulfoxide, and a phosphate buffer solution with pH = 5.5 and a concentration of 200 mmol / L, and then reacting at 4–60°C for 0.5–4 h; after the reaction is completed, adding a solution of 1 molar equivalent of the On-DNA isothiocyanate compound, and then reacting at 4–40°C for 2–12 h.

[0018] Preferably, the solvent in the solution of the On-DNA isothiocyanate compound is any one of water, inorganic salt buffer, inorganic base aqueous solution, organic acid buffer, or organic base buffer.

[0019] Preferably, the concentration of the On-DNA isothiocyanate compound solution is 0.001 to 0.05 mmol / L.

[0020] 2. A DNA-encoded compound library comprising On-DNA 2-thioquinazolinone compounds prepared by the synthetic method described above.

[0021] The beneficial effects of this invention are as follows: This invention provides a method for synthesizing On-DNA 2-thioquinazolinone compounds, which offers a new technical means for constructing On-DNA 2-thioquinazolinone compounds. The synthesis process uses inexpensive, readily available, and stable On-DNA amine compounds, 1,1'-thiocarbonyldiimidazole, and On-DNA isothiocyanate compounds as raw materials. The reaction conditions are mild (most reactions can be carried out at room temperature), the operation is convenient, and it is suitable for the synthesis of large-scale DNA-encoded compound libraries in multi-well plates.

[0022] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0023] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0024] Figure 1 The spectrum of On-DNA 2-thioquinazolinone compound with a 4-1 structure is shown, where A is the chromatogram and B is the mass spectrum.

[0025] Figure 2 The spectrum of the On-DNA 2-thioquinazolinone compound with a 4-2 structure is shown, where A is the chromatogram and B is the mass spectrum.

[0026] Figure 3 The spectrum of the On-DNA 2-thioquinazolinone compound with a 4-3 structure is shown, where A is the chromatogram and B is the mass spectrum.

[0027] Figure 4 The spectrum of On-DNA 2-thioquinazolinone compound with a 4-4 structure is shown, where A is the chromatogram and B is the mass spectrum.

[0028] Figure 5 The spectrum of On-DNA 2-thioquinazolinone compound with a 4-5 structure is shown, where A is the chromatogram and B is the mass spectrum.

[0029] Figure 6 The spectrum of On-DNA 2-thioquinazolinone compound with a 4-6 structure is shown, where A is the chromatogram and B is the mass spectrum.

[0030] Figure 7 The spectrum of On-DNA 2-thioquinazolinone compound with a 4-7 structure is shown, where A is the chromatogram and B is the mass spectrum.

[0031] Figure 8 The spectrum of On-DNA 2-thioquinazolinone compound with a 4-8 structure is shown, where A is the chromatogram and B is the mass spectrum.

[0032] Figure 9 The spectrum of On-DNA 2-thioquinazolinone compound with a 4-9 structure is shown, where A is the chromatogram and B is the mass spectrum.

[0033] Figure 10The spectrum is shown for an On-DNA 2-thioquinazolinone compound with a 4-10 structure, where A is a chromatogram and B is a mass spectrum. Detailed Implementation

[0034] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0035] The abbreviations used in the following examples are as follows:

[0036] DMA stands for N,N'-dimethylacetamide; HATU stands for 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; DIPEA stands for N,N-diisopropylethylamine; DMSO stands for dimethyl sulfoxide.

[0037] The DNA in each of the following representative 2-thioquinazolinone compounds 4 is a commercially available headpiece with a molecular weight of 4937 and the following structural formula:

[0038]

[0039] Example 1

[0040] On-DNA amine compounds 2 were prepared according to the following reaction formula.

[0041]

[0042] (1) Dissolve DNA-NH2 in sodium borate buffer solution with a concentration of 250 mmol / L and pH = 9.4 to prepare a solution with a concentration of 100 μmol / L for later use;

[0043] (2) Add HATU solution, DIPEA solution and aminocarboxylic acid small molecule solution with a concentration of 200 mmol / L, a volume of 3.5 μL and a solvent of DMA to the EP tube in sequence, mix thoroughly and activate for 20 min;

[0044] The structural formula of the small aminocarboxylic acid molecule is:

[0045] (3) Take another EP tube, add 10 μL of DNA-NH2 solution with a concentration of 100 μmol / L in step (1) and 10 μL of activation solution in step (2), mix well and react at room temperature for 2 h. After the reaction is completed, perform ethanol precipitation to obtain On-DNA amine compound 2. Confirm the recovery rate by microplate reader and confirm the conversion rate of each small molecule by LC-MS.

[0046] The specific procedure for ethanol precipitation is as follows: After the reaction is complete, add 80 μL of primary water, 10 μL of 5 mol / L sodium chloride aqueous solution, and 330 μL of anhydrous ethanol stored at -20℃ to the EP tube in sequence. After mixing, freeze at -80℃ overnight. After taking it out, centrifuge at 13500 rpm for 30 min in a centrifuge at 4℃. Remove the supernatant, dissolve the precipitate in primary water, and freeze-dry under vacuum at -80℃ to obtain On-DNA amine compound 2.

[0047] Example 2

[0048] On-DNA isothiocyanate compounds were prepared according to the following reaction formula.

[0049]

[0050] The On-DNA amine compound 2 prepared in Example 1 was dissolved in primary water to prepare a solution with a concentration of 100 μmol / L. Then, 10 μL of this solution was placed in an EP tube, and 4 μL of a 1,1'-thiocarbazide diimidazole solution with a concentration of 200 mmol / L and DMA as the solvent was added sequentially. Then, 6 μL of DMA was added to make the total volume of the system 20 μL. The reaction was then carried out at 5 °C for 2 h. After the reaction was completed, ethanol precipitation was performed in the same manner as in Example 1 to obtain the On-DNA isothiocyanate compound 3.

[0051] Example 3

[0052] Prepare On-DNA 2-thioquinazolinone compounds according to the following reaction formula 4

[0053]

[0054] (1) Dissolve the On-DNA isothiocyanate compound 3 obtained in Example 2 in primary water to prepare a solution with a concentration of 0.05 mmol / L for later use;

[0055] (2) Take an EP tube and add 800 nmol of indigo anhydride, 800 nmol of hydrogen peroxide tert-butanol, 16 μL of phosphate buffer with a concentration of 200 mmol / L and pH = 5.5, and 24 μL of DMSO to the tube to make the total volume of the system 40 μL. Mix well and react at 25 °C for 30 min.

[0056] (3) After the reaction in step (2) is completed, add 2 μL of the solution in step (1), and then react at 25°C for 12 h. Finally, ethanol precipitation is performed in the same way as in Example 1 to obtain On-DNA 2-thioquinazolinone compound 4. LC-MS confirms the conversion rate and molecular weight data of each small molecule.

[0057] A series of representative 2-thioquinazolinone compounds were synthesized using the method described above, as shown in Table 1.

[0058] Table 1 shows the chemical structure, conversion rate, and theoretical molecular weight data of a representative 2-thioquinazolinone compound 4.

[0059]

[0060]

[0061]

[0062] The above-mentioned representative 2-thioquinazolinone compounds were analyzed using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), and the following conclusions can be drawn: On-DNA 2-thioquinazolinone compounds with a 4-1 structure have high purity (e.g., ...). Figure 1 As shown in Figure A), the measured molecular weight is 5216.99, which basically matches the theoretical molecular weight (as shown in Figure A). Figure 1 (As shown in B); On-DNA 2-thioquinazolinone compounds with a 4-2 structure have high purity (e.g., ... Figure 2 As shown in Figure A), the measured molecular weight is 5234.99, which basically matches the theoretical molecular weight (as shown in Figure A). Figure 2 (As shown in B); On-DNA 2-thioquinazolinone compounds with a 4-3 structure have high purity (e.g., ... Figure 3 As shown in Figure A), the measured molecular weight is 5231.01, which basically matches the theoretical molecular weight (as shown in Figure A). Figure 3 (As shown in B); On-DNA 2-thioquinazolinone compounds with a 4-4 structure have high purity (e.g., Figure 4 As shown in Figure A), the measured molecular weight is 5231.01, which basically matches the theoretical molecular weight (as shown in Figure A). Figure 4 (As shown in B); On-DNA 2-thioquinazolinone compounds with 4-5 structures have high purity (e.g., shown in B). Figure 5 As shown in Figure A), the measured molecular weight is 5309.92, which basically matches the theoretical molecular weight (as shown in Figure A). Figure 5 (As shown in B); On-DNA 2-thioquinazolinone compounds with a 4-6 structure have high purity (e.g., ... Figure 6 As shown in Figure A), the measured molecular weight is 5231.00, which matches the theoretical molecular weight (as shown in Figure A). Figure 6 (As shown in B); On-DNA 2-thioquinazolinone compounds with a 4-7 structure have high purity (e.g., Figure 7 As shown in Figure A), the measured molecular weight is 5234.97, which basically matches the theoretical molecular weight (as shown in Figure A). Figure 7 (As shown in B); On-DNA 2-thioquinazolinone compounds with a 4-8 structure have high purity (e.g., Figure 8 As shown in Figure A), the measured molecular weight is 5295.87, which basically matches the theoretical molecular weight (as shown in Figure A). Figure 8 (As shown in B); On-DNA 2-thioquinazolinone compounds with a 4-9 structure have high purity (e.g., Figure 9 As shown in Figure A), the measured molecular weight is 5251.94, which basically matches the theoretical molecular weight (as shown in Figure A). Figure 9 (As shown in B); On-DNA 2-thioquinazolinone compounds with a 4-10 structure have high purity (e.g., shown in B). Figure 10 As shown in Figure A), the measured molecular weight is 5231.00, which matches the theoretical molecular weight (as shown in Figure A). Figure 10 (As shown in B).

[0063] In summary, this invention provides a method for synthesizing On-DNA 2-thioquinazolinone compounds. The raw materials used in the synthesis are inexpensive and readily available, the reaction conditions are mild, and the operation is convenient, providing a new technical means for constructing On-DNA 2-thioquinazolinone compounds.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for synthesizing on-DNA 2-thioquinazolinone compounds, characterized in that: The synthesis method includes the following steps: Provide On-DNA amine compounds as shown in general formula (I), On-DNA isothiocyanate compounds as shown in general formula (II), and indomethacin anhydride compounds as shown in general formula (III): The On-DNA amine compound is first converted into the On-DNA isothiocyanate compound under the action of 1,1'-thiocarbonyldiimidazole, which includes the following steps: adding 100 to 4000 molar equivalents of 1,1'-thiocarbonyldiimidazole solution to the aqueous solution of the On-DNA amine compound, and then reacting at 5 to 40°C for 0.5 to 4 hours. Then, the On-DNA isothiocyanate compound, 200-4000 molar equivalents of the indomethacin anhydride compound, and 100-2000 molar equivalents of tert-butanol peroxide are reacted in an aqueous solvent system, including the following steps: the indomethacin anhydride compound, tert-butanol peroxide, dimethyl sulfoxide, and phosphate buffer with pH=5.5 and a concentration of 200 mmol / L are mixed and reacted at 4-60°C for 0.5-4 h. After the reaction is completed, the solution of the On-DNA isothiocyanate compound is added, and the reaction is carried out at 4-40°C for 2-12 h to obtain the On-DNA 2-thioquinazolinone compound as shown in general formula (IV); in, R1 is the DNA; R1 is the functional group that links the DNA portion, specifically a functional group that can undergo a complementary reaction with functional groups on the DNA, selected from any one of amide, amino, carboxyl, aldehyde, aryl, or alkyne groups; R2 is selected from -(R4). m -(R5) n - where m and n are integers and not both 0; R3 is selected from hydrogen, halogen, trifluoromethyl, carboxyl, amino, nitro, cyano, hydroxyl, phenyl, ester, amide, aryl ketone, alkyl ketone, alkyl with 1-12 carbon atoms, olefin with 2-6 carbon atoms, alkyne with 2-6 carbon atoms, cycloalkyl with 3-8 carbon atoms, alkyloxy with 1-6 carbon atoms, or alkylamino with 1-6 carbon atoms; R4 is selected from alkyl, alkoxy, or cycloalkyl; R5 is selected from aryl, aromatic heteroyl, substituted aryl, or substituted heteroaryl; the substituents in the substituted aryl and the substituted heteroaryl are independently selected from alkyl, alkoxy, cycloalkyl, halogen, hydroxyl, trifluoromethyl, amino, ester, amide, nitro, cyano, or phenyl; The DNA is a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and / or unmodified nucleotide monomers.

2. The synthesis method according to claim 1, characterized in that: The solvent in the solution of the On-DNA amine compound is any one of water, inorganic salt buffer solution, inorganic alkaline aqueous solution, organic solvent, or a mixture of organic solvent and water; the organic solvent is any one or more of acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butanol, isopropanol, tetrahydrofuran, or 1,4-dioxane; the volume ratio of organic solvent to water in the mixture is 1:1; the solvent in the 1,1'-thiocarbazide diimidazole solution is N,N'-dimethylacetamide.

3. The synthesis method according to claim 1, characterized in that: The solvent in the solution of the On-DNA isothiocyanate compound is any one of water, inorganic salt buffer, inorganic base aqueous solution, organic acid buffer, or organic base buffer.

4. The synthesis method according to claim 1, characterized in that: The concentration of the solution of the On-DNA isothiocyanate compound is 0.001~0.05 mmol / L.

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