Method for directly synthesizing unprotected alpha-thioglycoside without catalyst under open condition and medicine for preventing or treating diabetes mellitus

Abstract

The invention provides a method for directly synthesizing unprotected alpha-thioglycoside without a catalyst under an open condition and a medicine for preventing or treating diabetes mellitus, which comprises the following steps: adding a sugar donor, disulfide and zinc powder into a reaction test tube, dissolving trimethylchlorosilane into a solvent, adding into the reaction test tube, reacting at room temperature, detecting the reaction process by TLC (Thin Layer Chromatography), and determining the content of alpha-thioglycoside in the reaction test tube. After the glucose raw material completely disappears, filtering reaction liquid, removing solid impurities, quenching filtrate by adding water, extracting by using DCM, carrying out reduced pressure distillation on an organic phase to obtain an oily crude product, and then carrying out column chromatography by using a petroleum ether / ethyl acetate / dichloromethane / methanol solution as a mobile phase to obtain the unprotected alpha-thioglycoside. The method is characterized in that a catalyst is not needed, water and oxygen isolation operation is not needed, the method has high stereoselectivity, a C-OH bond can be effectively cracked, an anomeric free radical is generated, high efficiency and stereoselectivity of the reaction are ensured, and the alpha-configuration unprotected thioglycoside can be obtained.

Description

TECHNICAL FIELD

[0001] The application provides a method for directly synthesizing unprotected alpha-thio glycoside under open conditions without a catalyst and application thereof in pharmacy, and belongs to the technical field of organic synthesis. BACKGROUND

[0002] Carbohydrate compounds are not only the energy source required for human life activities, but also play an important role in various physiological and pathological processes. As a special class of glycoside compounds, thio glycosides have the structural feature that the oxygen atom in the glycoside molecule is replaced by a sulfur atom to form a general structure of sugar-S-acceptor. Such compounds have wide significance in the analysis of life activity mechanisms and medical research. Because the C-S bond is more stable than the O-glycoside bond, it can resist acid hydrolysis and enzymatic hydrolysis, and thio glycosides are often used as tool molecules in glycosylation research to simulate the function of glycoproteins or glycolipids in cell recognition and signal transduction. In addition, thio glycosides can interfere with the glycometabolism process of pathogens (such as malaria parasites) or affect the interaction between the host and the pathogen by competitively inhibiting glycosidases (such as β-glucosidase). In the aspects of antiviral and antitumor research, thio glycosides also show important potential: some thio glycoside analogs can inhibit the synthesis of bacterial peptidoglycan (such as Mycobacterium tuberculosis) or the activity of viral glycosidase (such as the neuraminidase of HIV and influenza virus); and certain thio glycoside derivatives (such as sulforaphane) can exert antioxidant and anticancer effects by activating the Nrf2 / ARE pathway, or induce apoptosis of tumor cells (such as colorectal cancer and prostate cancer cells). Due to its unique chemical properties and diverse biological activities, thio glycosides have important value in the functional research of natural products, drug development (covering the fields of anticancer, anti-infection and metabolic diseases), and tool molecules of glycochemistry.

[0003] Generally, the synthesis of unprotected thio glycosides usually requires the protection of the hydroxyl group on the sugar donor, the reaction with the thio-substituted sugar acceptor to form the thio glycoside, and then the removal of the protection, and various metal catalysts or photosensitizers are required for the reaction conditions, and even water-free and oxygen-free operations are required, and the process is relatively complicated. Therefore, the synthesis of unprotected thio glycosides (i.e., directly using unprotected sugar as a raw material) has attracted widespread attention due to its high efficiency, atom economy and green chemistry advantages. For example, in 2009, Tokyo University reported a method for directly converting unprotected sugar into beta configuration unprotected thio glycoside in aqueous medium using 2-chloro-1,3-dimethylimidazoline chloride (Tetrahedron Lett. 2009, 50, 3078-3080). Chemistry Letters .2009, 38, 458–459), but its yield is generally low and the substrate is not practical for the synthesis of α-thio glycosides; in 2022, Francesca Paradisi's research group abroad reported a new type of microbial glycoside hydrolase engineering, which was endowed with the ability to synthesize thio glycosides efficiently through triple mutation (M299R / E166A / E354G), and realized the efficient and selective synthesis of unprotected thio glycosides (https: / / doi.org / 10.1002 / anie.202207593) Chem. Commun. 2022, 58 , 12118 ), but the cost of the enzyme is high because the specificity of the enzyme leads to poor substrate applicability; in 2024, Ming Joo Koh's research group at the National University of Singapore and Benjamin G. Davis's research group at the University of Oxford pre-synthesized 2,3,5,6-tetrafluoropyridine-4-thio glycoside as an intermediate by generating unprotected sugar (in situ), and then realized the stereoselective synthesis of five unprotected α-thio glycosides under light induction (https: / / doi.org / 10.1002 / anie.202410230) Nature ,2024, 631 , 319–327 ), with a two-step separation yield of 17%-82%, which belongs to a non-direct synthesis method and requires blue light conditions. Therefore, the synthesis of unprotected α-thio glycosides is a frontier topic in sugar chemistry, but due to the complicated synthesis steps, low atomic economic efficiency, and the need for anhydrous and anaerobic synthesis operation, stereoselectivity and other problems, how to simplify the synthesis route, improve the green chemistry index, and better simulate the synthesis of natural thio glycosides is still a very difficult and challenging work. SUMMARY

[0004] To solve the above technical problems, the present application provides a method for directly synthesizing unprotected α-thio glycosides under open conditions without catalyst, comprising the following steps: The sugar donor, disulfide and zinc powder are added to the reaction container, and then the chlorosilane additive is dissolved in the green solvent and added to the reaction system, and the reaction is stirred at room temperature, and the reaction process is monitored by TLC, and after the reaction is completed, the reaction liquid is filtered, quenched, extracted and concentrated to obtain the crude product, which is purified by column chromatography to obtain the unprotected α-thio glycoside.

[0005] The reaction formula is as follows: .

[0006] It can be understood that the preparation process in the present application also includes post-treatment, specifically, after the glucose raw material is completely reacted and disappears, the reaction liquid is filtered to remove solid impurities, the filtrate is quenched with water and then extracted with DCM, and the organic phase is distilled under reduced pressure to obtain an oily crude product, and then petroleum ether / ethyl acetate / dichloromethane / methanol solution is used as the mobile phase for column chromatography to obtain the unprotected α-thio glycoside.

[0007] The chlorosilane additives include any one of Et3ClSi, Me2HClSi, Me3ClSi, and HSiCl3.

[0008] The solvent includes any one of THF, 1,4-Dioxane, 2-MeTHF, and MeCN.

[0009] The amount of the chlorosilane additive is 5–11 equivalents, preferably 9 equivalents.

[0010] The reaction temperature ranges from room temperature to 70°C, and the reaction time is 0.5–6 hours.

[0011] The unprotected sugar donors include any one of D-glucose, D-galactose, D-mannose, L-rhamnose, and L-arabinose.

[0012] The structure of R in the sugar acceptor disulfide is a benzene ring and a substituted benzene ring. The substituents of the substituted benzene ring include any one of 4-methyl substituent, 4-ethyl substituent, 4-trifluoromethyl substituent, ortho, meta, and para halogen substituents.

[0013] The molar ratio of the sugar donor, zinc powder, additives, and sugar acceptor is 1.0 : 3.0 : 9.0 : 1.0.

[0014] The use of an unprotected α-glucosinolate synthesized by the method described above in the preparation of α-glucosidase inhibitor drugs.

[0015] A medicine for the prevention or treatment of diabetes, comprising an unprotected α-glucosinolate synthesized by the method described above.

[0016] A pharmaceutical composition comprising an unprotected α-thioglycoside synthesized by the method as an active ingredient, and a pharmaceutically acceptable carrier or excipient.

[0017] This invention utilizes relatively mild conditions, requiring no anhydrous or oxygen-free operation and no expensive catalysts, demonstrating significant innovation and high stereoselectivity. Furthermore, the elimination of hydroxyl group protection greatly shortens the reaction process, allowing for a one-step reaction that significantly improves production efficiency and reduces costs. Finally, the synthesized unprotected α-glucosinolate exhibits good stability, making it highly valuable in drug synthesis.

[0018] This invention utilizes a system of chlorosilane, zinc powder, and ether solvent (2-MeTHF) to synergistically activate the hydroxyl groups of sugar donors. Due to the higher stability of the anodic radicals of the sugar ring, the anodic hydroxyl groups are selectively activated. The in-situ generated anodic radicals couple with disulfide ethers to form α-selective thioglycosides due to the unique anodic effect of sugars.

[0019] The product yield obtained by using the technical solution of the present invention is mostly greater than 50%, preferably more than 60%, and even more preferably more than 70%, and the stereoselectivity is mostly 10:1 or more, and can reach more than 20:1. Attached Figure Description

[0020] Figure 1 This is the 1H NMR spectrum of compound 3b.

[0021] Figure 2 This is the carbon NMR spectrum of compound 3b. Detailed Implementation

[0022] Experimental reagents 2-Methyltetrahydrofuran (Shanghai Haohong Biomedical Technology Co., Ltd.), Trimethylchlorosilane (Shanghai Haohong Biomedical Technology Co., Ltd.), Petroleum ether (boiling range 60-90 ℃, Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.), Dichloromethane (analytical grade, Tianjin Kemio Chemical Reagent Co., Ltd.), Ethyl acetate (analytical grade, Tianjin Kemio Chemical Reagent Co., Ltd.), Anhydrous sodium sulfate (analytical grade, Sinopharm Chemical Reagent Co., Ltd.), Deuterated acetone (99.9% deuterium content, 0.03% TMS content, 10g / bottle, Shanghai Maclean Biochemical Technology Co., Ltd.), Deuterated methanol (99.8% deuterium content, 0.03% TMS content, 25mL / bottle, Beijing Bailingwei Technology Co., Ltd.); NMR tube (5mm 100 / pk2 ST500-8, Norell, USA).

[0023] Experimental instruments ZXZ-4 Rotary Vane Vacuum Pump (Linhai Tanshi Vacuum Equipment Co., Ltd.), DZF-6020 Vacuum Drying Oven (Shanghai Xinmiao Medical Instrument Manufacturing Co., Ltd.), SHB-IIIA Circulating Water Multipurpose Vacuum Pump (Shanghai Yukang Science and Education Instrument Equipment Co., Ltd.), CL-4 Flat Plate Magnetic Stirrer (Zhengzhou Changcheng Science and Industry Trade Co., Ltd.), EYELA SB-1100 Rotary Evaporator (Shanghai Ailang Instrument Co., Ltd.), FA2104B Analytical Balance (Shanghai Yueping Science and Technology Instrument Co., Ltd.), XRC-1 Micro Melting Point Tester (Sichuan University Scientific Instrument Factory), DF-101S Heat Collector-type Constant Temperature Heating Magnetic Stirrer (Gongyi Yingyu Yuhua Instrument Factory), GZX-9240MBE Digital Display Blower Drying Oven (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), ZF-6 Three-Purpose Ultraviolet Analyzer (Shanghai Jiapeng Technology Co., Ltd.), Ultrashied 400 MHz Plus Nuclear Magnetic Resonance Spectrometer (Bruker AG, Switzerland), API 4000 LC-MS / MS mass spectrometer (Bruckdorf Dalton GmbH, Germany) Example 1 D-glucose (36.32 mg, 0.2 mmol), diphenyl disulfide (43.7 mg, 0.2 mmol), and zinc powder (39.2 mg, 0.6 mmol) were added to a reaction tube. Triethylchlorosilane (211.0 mg, 1.4 mmol) was dissolved in 1 mL of THF and added to the reaction tube. The reaction was carried out at room temperature, and the reaction progress was monitored by TLC. After the glucose starting material was completely eliminated, the reaction solution was filtered to remove solid impurities. The filtrate was quenched with water and extracted with DCM. The organic phase was distilled under reduced pressure to obtain an oily crude product. Then, column chromatography was performed using petroleum ether / ethyl acetate / methanol / dichloromethane solution as the mobile phase to obtain (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-(phenylthio)tetrahydro-2H-pyran-3,4,5-triol (yield 54%).

[0024] Regarding the above process, this invention takes D-glucose as an example and optimizes the additives, solvents, additive equivalents, and temperature. The screening conditions are shown in Table 1:

[0025] Note: All experiments were conducted using 0.2 mmol D-glucose with 0.2 mmol diphenyl disulfide, 0.6 mmol zinc powder, and 1.8 mmol trimethylchlorosilane in 1 mL 2-MeTHF solvent under stirring at room temperature; the yields and stereoselectivity were determined by 1H NMR spectroscopy.

[0026] The reaction condition screening experiments showed that using D-glucose as the glycosyl donor and diphenyl disulfide as the acceptor was optimal (Table 1). First, Et3SiCl was used as an additive and THF as the solvent to verify the experimental hypothesis. Fortunately, the reaction proceeded smoothly, and unprotected glucosinolate was successfully synthesized in 54% yield at an isomer ratio of 6.3:1 (Item 1). Then, other chlorosilane additives, including Me2HSiCl, Me3SiCl, and HSiCl3, were screened, and Me3SiCl was selected as the best additive (Items 2-4). To further optimize the study, a series of solvents, such as 1,4-Dioxane, 2-MeTHF, and MeCN, were explored, further improving the yield and selectivity (Items 5-8). Subsequently, the additive equivalents and reaction temperature were screened (Items 8-12). Finally, the optimal reaction conditions were determined to be Me3SiCl as the additive, 2-MeTHF as the solvent, 9.0 equivalents of additive, and room temperature as the reaction temperature (Item 9).

[0027] The experimental results show that the optimal reaction conditions for unprotected glucosinolates are: Me3SiCl as additive, 2-MeTHF as solvent, 9.0 equivalents of additive, and room temperature as reaction temperature.

[0028] Example 2 This invention uses D-glucose as a raw material and p-tolyl disulfide as a sugar acceptor to prepare (2) R ,3 S 4 R 5 R 6 R )-2-(hydroxymethyl)-5-methyl-6-( p -tolylthio)tetrahydro-2 H -pyran-3,4-diol, the technical route is as follows:

[0029] D-glucose (36.32 mg, 0.2 mmol), p-tolyl disulfide (49.28 mg, 0.2 mmol), and zinc powder (39.2 mg, 0.6 mmol) were added to a reaction tube. Trimethylchlorosilane (195.6 mg, 1.8 mmol) was dissolved in 1 mL of 2-MeTHF and added to the reaction tube. The reaction was carried out at room temperature, and the reaction progress was monitored by TLC. After the glucose starting material was completely eliminated, the reaction solution was filtered to remove solid impurities. The filtrate was quenched with water and extracted with DCM. The organic phase was distilled under reduced pressure to obtain an oily crude product. Then, column chromatography was performed using a petroleum ether / ethyl acetate / methanol / dichloromethane solution as the mobile phase to obtain (2R,3S,4R,5R,6R)-2-(hydroxymethyl)-5-methyl-6-(p-tolylthio)tetrahydro-2H-pyran-3,4-diol (yield 76%).

[0030] The (2) prepared in Example 2 above R ,3 S 4 R 5 R 6 R )-2-(hydroxymethyl)-5-methyl-6-( p -tolylthio)tetrahydro-2 H 1H and 1C spectral data of pyran-3,4-diol 1H NMR (400MHz, Acetone-d6) δ 7.45 – 7.39 (m, 2H), 7.13 (d, J = 8.0 Hz, 2H), 5.48 (d, J= 5.6 Hz, 1H), 4.56 (m, 2H), 4.09 (m, 1H) 3.78 – 3.70 (m, 2H, 3H), 3.62 –3.48 (m, 2H), 3.48 – 3.38 (m, 1H), 2.29 (s, 3H). 13 C NMR (100 MHz, Acetone-d6)δ 137.7, 133.1, 132.6, 130.4, 90.9, 75.7, 74.3, 73.1, 71.6, 62.6, 21.0. HRMS(ESI) m / z : calcd for C 13 H 18 O5S + (M + H) + 287.0948, found, 287.0939; = 15.052 ( c = 1.0, CHCl3). The following products were also prepared under these reaction conditions: .

[0031] Activity evaluation The in vitro inhibitory activity of the enzyme α-glucosidase was tested, and the inhibitory activity of multiple compounds against α-glucosidase was detected. Acarbose was used as a positive control. For reaction solution preparation, the samples and positive control were dissolved in DMSO at concentrations of 90, 30, and 10 μM.

[0032] In vitro inhibitory activity of α-glucosidase was investigated, and the inhibitory activity of multiple compounds on α-glucosidase was detected. The experiment was divided into four groups: enzyme activity group (α-glucosidase solution and buffer solution), enzyme blank group (buffer solution and sample), positive group (α-glucosidase solution and positive drug solution), positive blank group (buffer solution and positive drug solution), sample group (α-glucosidase solution and sample), and sample blank group (buffer solution and sample). For each group, we conducted the corresponding research. First, 1-2 mg of sample and positive drug (acarbose) were accurately weighed and dissolved separately in dimethyl sulfoxide (DMSO), and then the sample solution was diluted to the corresponding concentration with PBS buffer (0.1 mol / mL, pH = 6.8). Second, 15 μL of sample and 45 μL of α-glucosidase solution (0.3 μL / mL) were accurately pipetteted into a 96-well plate and mixed by shaking for 4 minutes to ensure complete mixing. The mixture was then preheated at 37 °C, and 20 μl of substrate (PNPG) solution was added. The mixture was shaken to mix thoroughly and reacted at 37 °C for half an hour. Third, 100 μl of Na₂CO₃ solution was added to terminate the reaction. Finally, the inhibitory activity of the sample was determined by measuring the absorbance (OD) at 405 nm.

[0033] Table 1 shows the determination results of the compounds.

[0034] Table 1: Results of compound determination .

Claims

1. A method for the direct synthesis of unprotected α-glucosinolates under open conditions without a catalyst, characterized in that, Includes the following steps: The sugar donor, disulfide, and zinc powder were added to the reaction vessel. Then, a chlorosilane additive was dissolved in a solvent and added to the reaction system. The mixture was stirred at room temperature, and the reaction progress was monitored by TLC. After the reaction was complete, the mixture was filtered, quenched, extracted, and concentrated to obtain the crude product. The crude product was then purified by column chromatography to obtain unprotected α-glucosinolate. The reaction formula is as follows: 。 2. The method according to claim 1, characterized in that, The additives include any one of Et3ClSi, Me2HClSi, Me3ClSi, and HSiCl3, and the solvents include any one of THF, 1,4-Dioxane, 2-MeTHF, and MeCN.

3. The method according to claim 1, characterized in that, The amount of the chlorosilane additive is 5–11 equivalents, preferably 9 equivalents.

4. The method according to claim 1, characterized in that, The reaction temperature ranges from room temperature to 70°C, and the reaction time is 0.5–6 hours.

5. The method according to claim 1, characterized in that, The unprotected sugar donors include any one of D-glucose, D-galactose, D-mannose, L-rhamnose, and L-arabinose.

6. The method according to claim 1, characterized in that, The structure of R in the sugar acceptor disulfide is a benzene ring and a substituted benzene ring. The substituents of the substituted benzene ring include any one of 4-methyl substituent, 4-ethyl substituent, 4-trifluoromethyl substituent, ortho, meta, and para halogen substituents.

7. The method according to claim 1, characterized in that, The molar ratio of the sugar donor, zinc powder, additives, and sugar acceptor is 1.0 : 3.0 : 9.0 : 1.

0.

8. The use of an unprotected α-glucosinolate synthesized by any one of claims 1–7 in the preparation of an α-glucosidase inhibitor drug.

9. A drug for the prevention or treatment of diabetes, characterized in that, Including unprotected α-thioglycosides synthesized by the method according to any one of claims 1 to 7.

10. A pharmaceutical composition, characterized in that, It comprises an unprotected α-thioglycoside synthesized by any one of claims 1 to 7 as an active ingredient, and a pharmaceutically acceptable carrier or excipient.

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