Stemona alkaloid skeleton-based small-molecule probe and preparation method and application thereof
阅读说明：本技术 一种基于百部生物碱骨架的小分子探针及制备方法和应用 (Stemona alkaloid skeleton-based small-molecule probe and preparation method and application thereof ) 是由 马开庆 张梦晨 武兴康 阴彩霞 于 2020-04-07 设计创作，主要内容包括：本发明具体涉及一种基于百部生物碱骨架的小分子探针及制备方法和应用,属有机化物及其制备的技术领域。本发明完成了基于化合物SA-11结构的小分子探针(阳性探针)的设计与合成,该小分子探针可以与生物正交反应联用,发现基于百部生物碱骨架的活性小分子的作用靶点,为后续小分子结构优化奠定基础,还可对结肠癌细胞HCT-116抑制。(The invention particularly relates to a small molecular probe based on a stemona alkaloid skeleton, a preparation method and application thereof, belonging to the technical field of organic compounds and preparation thereof. The invention completes the design and synthesis of a small molecular probe (positive probe) based on the compound SA-11 structure, the small molecular probe can be used together with a bioorthogonal reaction, the effect target of an active small molecule based on the stemona alkaloid skeleton is found, the foundation is laid for the subsequent optimization of the small molecular structure, and the small molecular probe can also inhibit colon cancer cells HCT-116.)
1. The small molecule probe based on the stemona alkaloid skeleton is characterized in that the small molecule probe is (3aR,10bR) -1-methylene-2-oxy-N- (prop-2-yne-1-amido) -1,3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrole [1,2-a ] azepine-8-formamide, and the structural formula of the small molecule probe is as follows:
2. a preparation method of a stemona alkaloid skeleton-based small molecule probe is characterized by comprising the following steps:
step 1, oxalyl chloride was added dropwise to dry dimethylformamide under argon protection, the reaction was continued under these conditions after white crystals were produced, and this was mixedDichloromethane is added to the compound, then (3aR,10bR) -1-methylene-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] is added]Pyrrole [1,2-a ]]Continuing the reaction of the solution of azepine-2-ketone in dichloromethane, adding aqueous solution of sodium acetate, stirring, stopping the reaction, extracting with dichloromethane, and adding anhydrous Na2SO4Drying, and separating by column chromatography to obtain (3aR,10bR) -1-methylene-2-oxo-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3, 2-c)]Pyrrole [1,2-a ]]Azepine-8-carbaldehyde;
step 2, adding sodium dihydrogen phosphate dihydrate, sodium hypochlorite and 2-methyl-2-butene into (3aR,10bR) -1-methylene-2-oxo-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] in sequence at room temperature under the protection of argon]Pyrrole [1,2-a ]]Adding 1, 4-dioxane solution of azepine-8-formaldehyde, stirring overnight, adding glacial acetic acid into the reaction mixture, extracting with mixed solvent, and adding anhydrous Na2SO4Drying and separating by column chromatography to obtain (3aR,10bR) -1-methylene-2-oxygen-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3, 2-c)]Pyrrole [1,2-a ]]Azepine-8-carboxylic acid;
step 3, under the protection of argon, propargylamine, triethanolamine, 1-hydroxybenzotriazole and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride are sequentially added into (3aR,10bR) -1-methylene-2-oxo-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] at room temperature]Pyrrole [1,2-a ]]The azepine-8-formic acid is put into dichloromethane solution and reacts at room temperature overnight, and then dichloromethane is used for extraction, anhydrous Na is used2SO4Drying and separating by column chromatography to obtain (3aR,10bR) -1-methylene-2-oxygen-N- (prop-2-alkyne-1-amido) -1,3a,4,5,6,10 b-hexahydro-2H-furo [3, 2-c)]Pyrrole [1,2-a ]]Azepine-8-carboxamide.
3. The preparation method of the stemona alkaloid skeleton-based small molecule probe as claimed in claim 2, wherein the reaction temperature of dropwise adding oxalyl chloride into dry dimethylformamide in step 1 is 0-10 ℃, the reaction time after white crystals are generated is 15min, the reaction time after adding (3aR,10bR) -1-methylene-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrole [1,2-a ] azepine-2-one is 0.5H, the stirring time is 0.5H, and the extraction times are 3 times.
4. The preparation method of the stemona alkaloid skeleton-based small molecule probe as claimed in claim 2, wherein the molar ratio of dimethylformamide to oxalyl chloride in step 1 is 2-5: 2-5, the volume of dichloromethane is 8-16 m L, the concentration of dichloromethane solution of (3aR,10bR) -1-methylene-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrolo [1,2-a ] azepine-2-one is 0.2-0.8 mol/L, the volume is 2-8 m L, the concentration of sodium acetate aqueous solution is 1-3 mol/L, and the volume is 2-10 m L.
5. The preparation method of the stemona alkaloid skeleton-based small molecule probe as claimed in claim 2, wherein the molar ratio of sodium dihydrogen phosphate dihydrate, sodium hypochlorite and 2-methyl-2-butene in the step 2 is 4-10: 4-10, (3aR,10bR) -1-methylene-2-oxo-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrolo [1,2-a ] azepine-8-carbaldehyde 1, 4-dioxane solution with the concentration of 0.01-1 mol/L and the volume of 1-5 m L.
6. The preparation method of the stemona alkaloid skeleton-based small molecule probe as claimed in claim 2, wherein the stirring temperature in step 2 is 28 ℃, the amount of glacial acetic acid is 2-5 m L, and the volume ratio of dichloromethane to methanol is 10: 1-20: 1.
7. The preparation method of the stemona alkaloid skeleton-based small molecule probe as claimed in claim 2, wherein the molar ratio of propargylamine, triethanolamine, 1-hydroxybenzotriazole and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the step 3 is 1.5-5: 10-15: 1.5-5: 5, (3aR,10bR) -1-methylene-2-oxo-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrolo [1,2-a ] azepine-8-carboxylic acid in dichloromethane is 0.02-1 mol/L, the volume is 0.5-2 m L, and the number of extractions is 3 times.
8. The application of the small molecular probe based on the stemona alkaloid skeleton is characterized in that the small molecular probe can be used together with bioorthogonal reaction to perform action target fishing of a stemona alkaloid skeleton compound; can inhibit proliferation of colon cancer cell HCT-116.
Technical Field
The invention particularly relates to a small molecular probe based on a stemona alkaloid skeleton, a preparation method and application thereof, belonging to the technical field of organic compounds and preparation thereof.
Background
The traditional Chinese medicine and the natural medicine are treasures of medicine discovery, and the ever anti-malarial artemisinin and the newly discovered tetrandrine capable of resisting the Ebola virus are both derived from the traditional Chinese medicine. Under the background of high research cost, long time and increasing difficulty of innovative drugs, the search of lead compounds from traditional Chinese medicines and natural medicines is again a hot spot in recent years. Natural products which can be separated from traditional Chinese medicines and natural medicines have remarkable biological activity, but the action targets of the natural products are unknown, and the small yield of the natural products also limits further pharmacological mechanism research. The research on the effective new research method has important significance.
Currently, various strategies are developed and applied to target search of active natural products, wherein the strategy based on active-based Protein Profiling (ABPP) is widely applied to target search of active small molecules. The application of multiple natural product star molecules has completed the confirmation of target points, such as adenitin, triptolide, HEP14, artemisinin, baicalin, etc. Whereas the ABPP strategy first step is to construct probes based on active small molecule structures. In the previous research, we found that small molecules based on stemona alkaloid skeleton have significant activity of inhibiting colon cancer, but the action mechanism is not clear. Therefore, on the basis of accumulation of the former-stage synthesis method and structure-activity relationship, design and synthesis of a positive probe based on the structure of the compound SA-11 are completed, and in order to exclude non-specific protein binding in the target fishing process, design and synthesis of a negative probe corresponding to the positive probe are completed at the same time, and activity verification is completed. In the pull-down experiment, the target proteins were separated by SDS-PAGE electrophoresis, and several distinct protein bands were found compared to the negative probe.
The structural formula of the compound SA-11 is as follows:
disclosure of Invention
The invention aims to provide a small molecular probe based on a stemona alkaloid skeleton, a synthesis method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a small molecule probe (positive probe) based on stemona alkaloid skeleton is (3aR,10bR) -1-methylene-2-oxygen-N- (prop-2-alkyne-1-amido) -1,3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrole [1,2-a ] azepine-8-formamide, and the structural formula is as follows:
the preparation method of the molecular probe comprises the following steps:
(3aR,10bR) -1-methylene-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrolo [1,2-a ] azepine-2-one is reacted with oxalyl chloride and dimethylformamide to form (3aR,10bR) -1-methylene-2-oxo-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrolo [1,2-a ] azepine-8-carbaldehyde, (3aR,10bR) -1-methylene-2-oxo-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrolo [1,2-a ] azepine-8-carbaldehyde Oxidizing with the following to generate (3aR,10bR) -1-methylene-2-oxo-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrole [1,2-a ] azepine-8-formic acid, and carrying out condensation reaction on the (3aR,10bR) -1-methylene-2-oxo-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrole [1,2-a ] azepine-8-formic acid and propargylamine to generate (3aR,10bR) -1-methylene-2-oxo-N- (prop-2-yne-1-amino) -1,3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrolo [1,2-a ] azepine-8-carboxamide.
A preparation method of a stemona alkaloid skeleton-based small molecule probe comprises the following steps:
step 1, oxalyl chloride is added dropwise to dry dimethylformamide under argon protection, the reaction is continued under these conditions after white crystals have formed, anTo this mixture was added dichloromethane followed by (3aR,10bR) -1-methylene-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ]]Pyrrole [1,2-a ]]Adding sodium acetate aqueous solution after continuing the reaction of the dichloromethane solution of the azepine-2-ketone (compound 1), stirring, stopping the reaction, extracting with dichloromethane, adding anhydrous Na2SO4Drying, and separating by column chromatography to obtain (3aR,10bR) -1-methylene-2-oxo-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3, 2-c)]Pyrrole [1,2-a ]]Azepine-8-carbaldehyde (compound 3);
step 2, adding sodium dihydrogen phosphate dihydrate, sodium hypochlorite and 2-methyl-2-butene into (3aR,10bR) -1-methylene-2-oxo-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] in sequence at room temperature under the protection of argon]Pyrrole [1,2-a ]]Adding 1, 4-dioxane solution of azepine-8-formaldehyde, stirring overnight, adding glacial acetic acid into the reaction mixture, extracting with mixed solvent, and adding anhydrous Na2SO4Drying and separating by column chromatography to obtain (3aR,10bR) -1-methylene-2-oxygen-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3, 2-c)]Pyrrole [1,2-a ]]Azepine-8-carboxylic acid (compound 4);
step 3, under the protection of argon, propargylamine, triethanolamine, 1-hydroxybenzotriazole and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride are sequentially added into (3aR,10bR) -1-methylene-2-oxo-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] at room temperature]Pyrrole [1,2-a ]]The azepine-8-formic acid is put into dichloromethane solution and reacts at room temperature overnight, and then dichloromethane is used for extraction, anhydrous Na is used2SO4Drying and separating by column chromatography to obtain (3aR,10bR) -1-methylene-2-oxygen-N- (prop-2-alkyne-1-amido) -1,3a,4,5,6,10 b-hexahydro-2H-furo [3, 2-c)]Pyrrole [1,2-a ]]Azepine-8-carboxamide (Compound 2).
The route realizes the efficient synthesis of the stemona alkaloid skeleton-based active small molecular probe.
Further, in the step 1, oxalyl chloride is dropwise added into dry dimethylformamide at a reaction temperature of 0-10 ℃, the time for continuing the reaction after white crystals are generated is 15min, the time for continuing the reaction after (3aR,10bR) -1-methylene-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrole [1,2-a ] azepine-2-one is added for 0.5H, the stirring time is 0.5H, and the extraction times are 3 times.
Further, in the step 1, the molar ratio of dimethylformamide to oxalyl chloride is 2-5: 2-5, the volume of dichloromethane is 8-16 m L, the concentration of a dichloromethane solution of (3aR,10bR) -1-methylene-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrole [1,2-a ] azepine-2-one is 0.2-0.8 mol/L, the volume is 2-8 m L, the concentration of a sodium acetate aqueous solution is 1-3 mol/L, and the volume is 2-10 m L.
Further, in the step 2, the molar ratio of the sodium dihydrogen phosphate dihydrate to the sodium hypochlorite to the 2-methyl-2-butene is 4-10: 4-10, and the concentration of the 1, 4-dioxane solution of the (3aR,10bR) -1-methylene-2-oxo-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrolo [1,2-a ] azepine-8-formaldehyde is 0.01-1 mol/L, and the volume is 1-5 m L.
Further, the stirring temperature in the step 2 is 28 ℃, the amount of glacial acetic acid is 2-5 m L, and the volume ratio of dichloromethane to methanol is 10: 1-20: 1.
In the step 3, the molar ratio of propargylamine, triethanolamine, 1-hydroxybenzotriazole and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1.5-5: 10-15: 1.5-5, and the dichloromethane solution of (3aR,10bR) -1-methylene-2-oxo-1, 3a,4,5,6,10 b-hexahydro-2H-furo [3,2-c ] pyrrolo [1,2-a ] azepine-8-carboxylic acid has the concentration of 0.02-1 mol/L, the volume of 0.5-2 m L and the extraction times of 3 times.
An application of a small molecular probe based on radix Stemonae alkaloid skeleton can be used together with bioorthogonal reaction to perform effect target fishing of radix Stemonae alkaloid skeleton compound; can inhibit HCT-116 of colon cancer cells.
Compared with the existing fluorescent probe for detecting the micromolecule mercaptan, the fluorescent probe has the following advantages:
1. the invention provides a simple synthesis route of a small molecular probe based on a stemona alkaloid skeleton, and the probe is easy to obtain raw materials and low in cost;
2. the invention provides application of a small molecular probe, which can be used together with bioorthogonal reaction to perform action target fishing of stemona alkaloid skeleton compounds.
Drawings
FIG. 1 is a hydrogen spectrum of nuclear magnetic resonance of a small molecule probe;
FIG. 2 is a carbon spectrum of nuclear magnetic resonance of a small molecule probe;
FIG. 3 is data of inhibitory activity of small molecule probes on colon cancer cells HCT-116;
FIG. 4 is polyacrylamide gel electrophoresis of a target protein of a small molecule probe.
Detailed Description