A natural product photoaffinity probe reactant based on phenylhydrazine and its preparation method and application

Abstract

The invention discloses a natural product photoaffinity probe reactant based on phenylhydrazine and its preparation method and application. This photoaffinity probe reactant based on phenylhydrazine has three functional groups of phenylhydrazine structure, photocrosslinking group and bioorthogonal group alkynyl. Its preparation method includes using 4-tert-butyloxycarbonylhydrazine benzoic acid and 3-aminoethyl-3-(butyl-3-alkynyl) diaziridine as raw materials, performing addition reaction at room temperature, and then removing the tert-butyloxycarbonyl to obtain the target product. The photoaffinity probe reactant designed in the present application can be used for chemical small molecule modification with ketone or aldehyde functional groups. Since the chemical selectivity of the reaction is high, functional group-specific modification can be achieved, and the reaction conditions are mild, the process is simple, and a photoaffinity probe with biological activity can be quickly and efficiently prepared, and further used in target discovery and drug development.

Description

Technical Field

[0001] The invention relates to the technical field of chemical proteomics, and in particular to a natural product photoaffinity probe reactant based on phenylhydrazine and a preparation method and application thereof. Background Art

[0002] With the continuous development of science and technology, the development methods of drugs have gradually diversified, such as separation from plants and other natural sources, synthetic chemistry, combinatorial chemistry and molecular modeling. Although pharmaceutical companies have used the above technical means for the development of new drugs, natural products are still an important source of new drugs. In addition to participating in the physiological activities of plants as endogenous small molecules, these natural products also have excellent efficacy on some human diseases. Natural products are generally low in content in nature, complex in structure, difficult to synthesize, and difficult to achieve mass production, but natural products also have highly selective and specific pharmacological activities. According to statistics in 2020, more than 45% of drugs are derived from natural products and their derivatives, and the diseases treated include cancer, AIDS and multiple sclerosis. It can be seen that innovative drug research and development based on natural products has an irreplaceable position in the field of new drug research and development, but natural products also face many challenges in the development process, including the revelation of the active mechanism of natural product drugs and the discovery of their targets.

[0003] As one of the hot research directions in chemical biology, chemical proteomics combines active molecular probes and quantitative mass spectrometry methods to discover target proteins that interact with specific active compound molecules. Currently, chemical proteomics technology has played an important role in drug mechanism revelation and new drug development. In 2018, Wang Chu's research group at Peking University used chemical proteomics to reveal the molecular mechanism of baicalin, a traditional Chinese medicine active molecule, in treating obesity, fatty liver and related metabolic diseases (Proc Natl Acad Sci USA. 2018; 115 (26): E5896-E5905). By chemically derivatizing baicalin molecules, they prepared photoaffinity probes with similar drug activity to natural baicalin, thereby finding the pharmacodynamic targets of baicalin directly acting in cells.

[0004] Patent CN113061126A discloses a preparation method and application of a Sorafenib photoaffinity probe molecule based on a halogen intermediate. The preparation method comprises reacting a halogen intermediate with sodium azide to generate an azide, reacting the azide with triphenylphosphine to obtain a photoaffinity linker, reacting Sorafenib with sodium hydroxide in anhydrous ethanol to obtain an intermediate product with a monocarboxylic acid, and condensing the photoaffinity linker and the intermediate product with a monocarboxylic acid under the action of EDC·HCl to generate a Sorafenib photoaffinity probe molecule based on a halogen intermediate, wherein the halogen intermediate contains a photoactive group of diaziridine and an iodine element. The preparation method is simple, easy to implement, and has a high yield.

[0005] However, many natural products with excellent drug activity, such as glycyrrhizin flavonoids, shafotoside, and neohesperidin, contain a large number of hydroxyl groups in their molecular structures, which makes site-selective derivatization reactions very difficult, making it impossible to carry out the photoaffinity probe-assisted target discovery strategy. Therefore, designing a reactant that can combine with the above natural products to synthesize photoaffinity probes is a technical problem that has not yet been solved. Summary of the invention

[0006] For many natural products with excellent pharmaceutical activity, such as glycyrrhizin flavonoids, shafotoside, neohesperidin, silybin A, silybin B, myricetin, vorseloto, quercetin or silybin, their molecular structures contain a large number of hydroxyl groups, which makes site-selective derivatization reactions very difficult, resulting in the inability to carry out photoaffinity probe-assisted target discovery strategies. However, it is worth noting that the above-mentioned types of natural products all have a ketone or aldehyde functional group, so the scheme of the present application is based on the above ideas and provides a phenylhydrazine-based photoaffinity probe reactant that can bind to a wide range of natural products containing ketone or aldehyde functional groups, as well as its preparation method and application.

[0007] First, the present application provides a phenylhydrazine-based natural product photoaffinity probe reactant PDAA, which has the following structure:

[0008]

[0009] Secondly, the present application provides a method for preparing a natural product photoaffinity probe reactant based on phenylhydrazine, which is used to prepare the above-mentioned natural product photoaffinity probe reactant based on phenylhydrazine, comprising the following steps:

[0010] 1) dissolving compound II and compound III in an organic solvent, adding triethylamine, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and reacting at room temperature to obtain a reaction solution containing compound IV;

[0011] 2) adding water to the reaction solution containing compound IV, adding ethyl acetate, extracting, washing the organic layer with saturated brine, removing water with anhydrous sodium sulfate, and rotary evaporating to obtain a crude compound IV, and purifying the crude compound IV by column chromatography to obtain compound IV;

[0012] 3) Compound IV was dissolved in methanol, and the tert-butyloxycarbonyl protection of compound IV was removed in 1,4-epoxyhexahydrochloride at room temperature, and the reaction mixture was rotary evaporated to obtain compound I.

[0013]

[0014] Furthermore, the molar ratio of the compound II to the compound III is 1:1, and the molar ratio of the compound II to triethylamine, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:2.4:1.2:1.2.

[0015] Furthermore, the organic solvent is dichloromethane or dichloroethane; the reaction is carried out at room temperature to obtain a reaction solution containing compound IV for 3 hours.

[0016] Furthermore, the reaction time for removing the tert-butyloxycarbonyl protection of compound IV is 12-36 hours.

[0017] Finally, the present application also provides an application of a phenylhydrazine-based natural product photoaffinity probe reactant, which is used to combine with a natural product containing a ketone or aldehyde functional group to prepare a natural product photoaffinity probe, wherein the natural product containing a ketone or aldehyde functional group can be glycyrrhizin flavonoids, shafotoside, neohesperidin, silybin A, silybin B, myricetin, vorseloto, quercetin or quercetin, etc.

[0018] Furthermore, the natural product containing a ketone or aldehyde functional group is neohesperidin or silybin A.

[0019] Furthermore, it is characterized in that the structure of the natural product photoaffinity probe is:

[0020]

[0021] Furthermore, the step of preparing the natural product photoaffinity probe comprises:

[0022] 1) adding ethanol to dissolve compound I and a natural product containing an aldehyde or ketone, and adding sodium acetate to react to obtain a reaction solution containing a natural product photoaffinity probe;

[0023] 2) Cooling the reaction solution containing the natural product photoaffinity probe to room temperature, and purifying the natural product photoaffinity probe by liquid chromatography.

[0024] Furthermore, the molar ratio of the compound I, the natural product containing aldehyde or ketone and sodium acetate is 1:0.5-0.7; the reaction temperature of adding sodium acetate for the reaction is 70°C, and the reaction time is 12h.

[0025] The natural product photoaffinity probe reactant PDAA provided in the present application is a trifunctional reactant having a phenylhydrazine structure, a photocrosslinking group and a bioorthogonal group alkynyl, and is composed of three elements. The phenylhydrazine structure is a reactive group, which is used for addition condensation with ketones or aldehydes in natural products to generate a stable phenylhydrazone structure; the photocrosslinking group, i.e., the diazirine structure, can generate free radicals under the excitation of ultraviolet light, and undergo insertion crosslinking reactions with adjacent CH or CC bonds, thereby covalently capturing the protein bound by the photoaffinity probe; the bioorthogonal group, i.e., the alkynyl, can be used for subsequent coupling with biotin or fluorescein to separate, enrich or fluorescently image the protein labeled with the photoaffinity probe, and due to its small size, the synthesis is very simple.

[0026] Natural products with excellent pharmaceutical activity, such as neohesperidin and silybin A, have complex structures, contain multiple active reactive groups, and have important biological activities. Neohesperidin, as a flavonoid glycoside with neuroprotective and antioxidant abilities, can not only reduce blood sugar in diabetic mice and regulate insulin sensitivity, but also induce apoptosis in human breast cancer MDA-MB-231 cells. Silybin A has been recommended as a drug in multiple guidelines, including drug-induced liver injury. In addition to protecting the liver, it also has the effects of anti-HCV replication, lowering blood lipids, protecting the myocardium, inhibiting cancer cell proliferation, protecting nerves, and anti-depression and anxiety.

[0027] PDAA reactants can be used to modify small chemical molecules with ketone or aldehyde functional groups. Due to the high chemical selectivity of the reaction, functional group-specific modification can be achieved. In addition, the reaction conditions are mild and the process is simple. It can quickly and efficiently prepare biologically active photoaffinity probes, which can be further used in target discovery and drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following is a brief description of the contents expressed in the drawings of the specification:

[0029] Figure 1 The natural product photoaffinity probe reactant PDAA prepared in Example 1 of the present invention 1 H NMR spectrum;

[0030] Figure 2 The new hesperidin-derived photoaffinity probe prepared in Example 2 of the present invention 1 H NMR spectrum;

[0031] Figure 3 The new hesperidin-derived photoaffinity probe prepared in Example 2 of the present invention 13 C NMR spectrum;

[0032] Figure 4 is a mass spectrum of the new hesperidin-derived photoaffinity probe prepared in Example 2 of the present invention;

[0033] Figure 5 is the silybin A-derived photoaffinity probe prepared in Example 3 of the present invention 1 H NMR spectrum;

[0034] Figure 6 is the silybin A-derived photoaffinity probe prepared in Example 3 of the present invention 13 C NMR spectrum;

[0035] Figure 7 It is the mass spectrum of the silybin A-derived photoaffinity probe prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1

[0038] Synthesis of trifunctional reactant PDAA:

[0039] Dissolve 4-tert-butyloxycarbonylhydrazine benzoic acid (50 mg, 0.2 mmol), 3-aminoethyl-3-(but-3-ynyl)diaziridine (27.2 mg, 0.2 mmol) and triethylamine (0.062 ml, 0.48 mmol) in a reaction flask with 3 ml of dichloromethane. Add 1-hydroxybenzotriazole (32.4 mg, 0.24 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (46.1 mg, 0.24 mmol) thereto, and react at room temperature for 3 hours to obtain a reaction solution containing compound IV. Then add 80 ml of water to the reaction solution, extract the reaction solution with ethyl acetate, then wash the organic layer with saturated brine, remove water with anhydrous sodium sulfate, and then rotary evaporate to obtain a crude product of compound IV. The crude product of compound IV is purified by column chromatography, and the eluent is dichloromethane: methanol = 150:1 to obtain a yellow solid product, i.e. compound IV.

[0040] Compound IV (200 mg, 0.53 mmol) was dissolved in 2 ml of methanol, 3.5 ml of 1,4-epoxyhexacyclopentane hydrochloride was added to the solution, and the mixture was reacted at room temperature for 12 h overnight. The solvent was removed by rotary evaporation to obtain a dark yellow solid product, PDAA (75 mg, 0.24 mmol). The obtained product was tested and the obtained product was 1 H NMR spectrum Figure 1 shown.

[0041] Example 2

[0042] Synthesis of Neohesperidin-derived photoaffinity probe (Neohesperidin-PDAA):

[0043]

[0044] First, PDAA (120 mg, 0.39 mmol) and neohesperidin (166 mg, 0.273 mmol) were dissolved in 45% ethanol, and then sodium acetate (35 mg, 0.429 mmol) was added to the above solution, and the reaction was carried out at 70°C for 12 hours. After the reaction, the reaction solution was cooled to room temperature and purified by preparative high performance liquid chromatography to obtain the neohesperidin-derived photoaffinity probe Neohesperidin-PDAA (50 mg, 0.058 mmol). Figure 2 and Figure 3 Neohesperidin-PDAA 1 H NMR and 13 C NMR spectrum, Figure 4 This is the mass spectrum of Neohesperidin-PDAA.

[0045] Example 3

[0046] Synthesis of Silybin A-PDAA-derived photoaffinity probe:

[0047]

[0048] First, PDAA (120 mg, 0.39 mmol) and silybin A (125 mg, 0.259 mmol) were dissolved in 45% ethanol, and finally sodium acetate (35 mg, 0.429 mmol) was added to the above solution, and the mixture was reacted at 70°C for 12 h. After the reaction, the reaction solution was cooled to room temperature and purified by preparative high performance liquid chromatography to obtain silybin A-PDAA (52 mg, 0.071 mmol), a photoaffinity probe derived from silybin A. Figure 5 and Figure 6 They are Silybin A-PDAA 1 H NMR and13 CNMR spectrum. Figure 7 This is the mass spectrum of Silybin A-PDAA.

[0049] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A natural product photoaffinity probe has the structure:

2. A natural product photoaffinity probe according to claim 1, characterized in that: The preparation steps of the natural product photoaffinity probe include: 1) Compound I and a natural product containing an aldehyde or ketone are dissolved in ethanol, and sodium acetate is added for reaction to obtain a reaction solution containing a natural product photoaffinity probe, wherein the structure of Compound I is: 2) Cooling the reaction solution containing the natural product photoaffinity probe to room temperature, and purifying the natural product photoaffinity probe by liquid chromatography.

3. The natural product photoaffinity probe according to claim 2, characterized in that: The molar ratio of the compound I, the natural product containing aldehyde or ketone and sodium acetate is 1:0.5-0.7; the reaction temperature of adding sodium acetate to react is 70°C, and the reaction time is 12h.

4. A use of a reactant for preparing the natural product photoaffinity probe according to claim 1, characterized in that: The reaction substructure is:

5. The use of a reactant according to claim 4 for preparing the natural product photoaffinity probe, characterized in that: The preparation step of the reactant comprises: 1) dissolving compound II and compound III in an organic solvent, adding triethylamine, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and reacting at room temperature to obtain a reaction solution containing compound IV; 2) adding water to the reaction solution containing compound IV, adding ethyl acetate, extracting, washing the organic layer with saturated brine, removing water with anhydrous sodium sulfate, and rotary evaporating to obtain a crude compound IV, and purifying the crude compound IV by column chromatography to obtain compound IV; 3) Compound IV was dissolved in methanol, and the tert-butyloxycarbonyl protection of Compound IV was removed in 1,4-epoxyhexacyclopentane hydrochloride at room temperature, and rotary evaporation was performed to obtain Compound I; The molar ratio of the compound II to the compound III is 1:1, and the molar ratio of the compound II to triethylamine, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:2.4:1.2:1.2; The organic solvent is dichloromethane or dichloroethane; the reaction is carried out at room temperature to obtain a reaction solution containing compound IV for 3 hours; 6. The use of the reactant according to claim 5, characterized in that: The reaction time for removing the tert-butyloxycarbonyl protection of compound IV is 12-36 hours.

7. The use of a reactant according to claim 4 for preparing the natural product photoaffinity probe, characterized in that: The natural product is neohesperidin or silybin A.

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