Compound having alpha-glucosidase inhibitory activity and application thereof

Abstract

To provide a compound having α - glucosidase inhibitory activity and its application.SOLUTION: The 5-hydroxymethyl-8-methyl-2 - (N-arylamino) - pyrano [2, 3-c] pyridine-3 - (N-aryl) - carboxamide compound has a structure represented by general formula (I). The compounds according to the invention have alpha-glucosidase inhibitory activity and are of use in the treatment of diabetes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention belongs to the field of chemical medicine technology, and specifically relates to compounds having α-glucosidase inhibitory activity and their applications. [Background technology]

[0002] In the treatment of obesity and diabetes, the control of postprandial blood glucose levels has received widespread attention due to the close relationship between postprandial blood glucose levels and diabetic complications. The control of postprandial blood glucose levels is primarily related to the regulation of starch-digesting enzymes, including salivary amylase, an amylase secreted by the pancreas, and α-glucosidase. Currently, commonly used clinical drugs (including acarbose, voglibose, and miglitol) primarily inhibit α-amylase and α-glucosidase. However, the strong inhibition of these two enzymes results in the inability to digest large amounts of starch. The majority of undigested starch can cause a wide range of gastrointestinal side effects, including intestinal distension due to microbial fermentation in the colon. Therefore, to enhance the application of starch-digesting enzyme inhibitors and improve patient experience, new inhibitors must be developed to address the serious shortcomings of current clinical drugs.

[0003] Currently, the literature has reported that compounds with benzopyran, benzimidazole, quinoline, and acetamide structures have α-glucosidase inhibitory activity. For example, compound b, which is based on quinoline and acetamide structures, has an IC50 value of 30.2 μM against α-glucosidase, demonstrating extremely high inhibitory activity.

[0004] [ka] Summary of the Invention [Problem to be solved by the invention]

[0005] Based on the above research, the present invention synthesized 5-hydroxymethyl-8-methyl-2-(N-arylamino)-pyrano[2,3-c]pyridine-3-(N-aryl)-carboxamide series compounds, and the biological activity measurement results showed that the synthesized compounds have good α-glucosidase inhibitory activity. [Means for solving the problem]

[0006] A first aspect of the present invention provides 5-hydroxymethyl-8-methyl-2-(N-arylamino)-pyrano[2,3-c]pyridine-3-(N-aryl)-carboxamide compounds, which have the structure of the following general formula (I):

[0007] [ka]

[0008] In one specific embodiment, R1 is halogen and R2 is a C1-C2 alcoholic hydroxyl group, more preferably R1 is chlorine and R2 is ethanol, and most preferably R1 is 4-Cl and R2 is a 3-carbinol group.

[0009] The present invention provides a method for preparing 5-hydroxymethyl-8-methyl-2-(N-arylamino)-pyrano[2,3-c]pyridine-3-(N-aryl)-carboxamide compounds according to the first aspect, wherein the compounds are synthesized according to the following route:

[0010] [ka]

[0011] The synthesis method is as follows.

[0012] (1) Using pyridoxal hydrochloride and 2-cyano-N-arylacetamide as raw materials, they are reacted in the presence of piperidine and anhydrous methanol at 40-45°C for 15-25 minutes to self-assemble the 2-H-pyrano[2,3-c]pyridine heterocyclic ring system of the intermediate 2-cyanoacrylamide, and then 2-imino-5-hydroxymethyl-8-methyl-2H-pyrano[3,2-c]pyridine-3-carboxamide is precipitated from the reaction mixture. (2) 2-Imino-5-hydroxymethyl-8-methyl-2H-pyrano[3,2-c]pyridine-3-carboxamide and aniline are reacted in boiling acetic acid for 20 to 30 minutes to produce the compound of general formula (I).

[0013] Furthermore, in step 2), when the mixture is cooled to room temperature, it crystallizes and compound (1) is separated from the reaction mixture by filtration.

[0014] In one specific embodiment, the molar ratio of pyridoxal hydrochloride to 2-cyano-N-arylacetamide in step (1) is 1-1.5:1, preferably 1.2:1.

[0015] The molar ratio of piperidine, pyridoxal hydrochloride, and 2-cyano-N-arylacetamide added in step (1) is 1-3:1-1.5:1, preferably 2:1.2:1.

[0016] A third aspect of the present invention provides a pharmaceutical composition for treating diabetes, the pharmaceutical composition comprising a compound of formula (1) according to the first aspect and a pharmaceutically acceptable carrier.

[0017] A fourth aspect of the present invention provides the use of a compound of formula (1) according to the first aspect in the manufacture of a medicament for treating diabetes.

[0018] A fifth aspect of the present invention provides the use of a compound of formula (1) according to the first aspect in the manufacture of an α-glucosidase inhibitor. [Effects of the Invention]

[0019] The beneficial effects of the present invention are as follows:

[0020] The present inventors first discovered that many GFY analogs have potential antidiabetic effects, but the level of their activity is significantly affected by the different combinations of substituents selected. Even with the same core structure, different types of substituents can significantly change the activity. Next, the inventors limited the structure of the compound to be protected to that of formula [I], and very strictly restricted the substituents therein. Compared with compounds with general antidiabetic activity, the compounds of the present invention with extremely strong antidiabetic activity have more obvious and important application value. [Brief explanation of the drawings]

[0021] [Figure 1] Fluorescence quenching values ​​of GFY-based compounds for α-glucosidase. [Figure 2] Activity of GFY compounds against α-glucosidase. DETAILED DESCRIPTION OF THE INVENTION

[0022] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments, which are not intended to limit the present invention.

[0023] The experimental methods described in each of the following examples are conventional unless otherwise specified. The reagents and materials designated are commercially available unless otherwise specified.

[0024] Example 1 Synthesis of Compounds For the preparation of 5-hydroxymethyl-8-methyl-2-(N-arylamino)-pyrano[2,3-c]pyridine-3-(N-aryl)-carboxamide shown in formula (I), the compound number is indicated as GFY-14 (R1 is selected from 4-Cl-, and R2 is selected from 3-CH2OH-), and the specific preparation process is as follows:

[0025] [ka]

[0026] The specific manufacturing process is as follows: 488.68 mg (2.4 mMol) of pyridoxal hydrochloride and 356.32 mg (2 mMol) of N-4-chloro-2-cyanoacetamide were added to a 10 ml round-bottom flask, 4 ml of methanol was pipetted into the flask, the flask was placed in an oil bath, stirred, and heated to 40-45°C, after which 395 μl (4 mMol) of piperidine was slowly added. The reaction solution was initially a yellow, cloudy liquid that gradually became clear as the reaction progressed, and then a precipitate was formed. The resulting mixture was allowed to stand at 40-45°C for 20 minutes, and the reaction product formed a precipitate. The precipitate was filtered, washed with methanol, and recrystallized from ethanol to obtain a yellow solid (N-(4-chlorophenyl)-5-(hydroxymethyl)-2-imino-8-methyl-2hydro-pyrano[2,3-c]pyridine-3-carboxamide) in a 63% yield.

[0027] A 10 mL round-bottom flask was charged with 343.77 mg (1 mMol) of N-(4-chlorophenyl)-5-(hydroxymethyl)-2-imino-8-methyl-2-hydro-pyrano[2,3-c]pyridine-3-carboxamide and 147.78 mg (1.2 mMol) of 3-aminobenzyl alcohol. 5 mL of glacial acetic acid was then pipetted into the round-bottom flask. The flask was placed in an oil bath, stirred, and heated to 115°C for 20-30 min. The reaction mixture was then cooled to room temperature, and the precipitated product was filtered and recrystallized from ethanol, DMF, or ethanol-DMF to give a yellow solid in 62% yield.

[0028] Example 2 Synthesis of a series of compounds The other series of compounds GFY-1 to GFY-13 and GFY-15 to GFY-31 were prepared using the same preparation method as in Example 1, with the only difference being that the selected N-4-chloro-2-cyanoacetamide and 3-aminobenzyl alcohol were replaced with the N-2-cyanoacetamide derivatives and aniline derivatives shown in the process flow chart above, where the R substituent selection was the same as the definition of R in the Summary of the Invention.

[0029] The physicochemical data of the compounds GFY-1 to GFY-31 produced in Examples 1 and 2 are shown in Table 1 below, and their 1H-NMR, 13C-NMR, and HRMS data are shown in Table 2.

[0030] Table 1. Physicochemical data of compounds GFY-1 to GFY-31

[0031] [Table 1]

[0032] Table 2 Compounds GFY-1 to GFY-31 1 H-NMR, 13 C-NMR and HRMS data

[0033] [Table 2] TIFF2026025949000007.tif250170TIFF2026025949000008.tif250170TIFF2026025949000009.tif130170

[0034] Example 3: Verification of α-glucosidase inhibitory activity of compounds GFY-1 to GFY-29 1. Measurement of GFY-1 to GFY-31 inhibitory activity at a concentration of 250 μmol / L Activity measurements were performed using an in vitro α-glucosidase activity assay.

[0035] Each compound was weighed and dissolved in DMSO to prepare a 250 μmol / L reagent for use in subsequent experiments. Briefly, α-glucosidase solution (final concentration: 0.5 U / mL) was reacted with 250 μmol / L of compound and 0.6 mM pNαGP solution in 0.1 mM phosphate buffer (pH 6.8) to measure the inhibitory effect of the compound on the substrate converted by α-glucosidase. At the same time, acarbose and DMSO were used as the standard inhibitor and control inhibitor, respectively. 250 μmol / L compound (10 μL), enzyme solution (40 μL), and potassium phosphate buffer (100 μL) were preincubated in a 96-well plate at 37°C for 10 min. Subsequently, 50 μL of substrate (pNαGP, 0.6 mM) was added to each microwell, and the mixture was incubated at 37° C. for 20 minutes. The absorbance was measured at 405 nm to examine the change in enzyme activity.

[0036] The formula for calculating the enzyme inhibitory activity of the measured compound is as follows:

[0037] Inhibition % = (blank absorbance value - sample absorbance value) ÷ blank absorbance value × 100% 2. Measurement of IC50 values ​​of GFY-1 to GFY-29 against α-glucosidase Activity measurements were performed using an in vitro α-glucosidase activity assay.

[0038] Each compound was weighed and dissolved in DMSO to prepare a 1 mMol / L stock solution, which was then gradient-diluted to 500 μmol / L, 250 μmol / L, 125 μmol / L, 62.5 μmol / L, 31.25 μmol / L, 15.625 μmol / L, and 7.8125 μmol / L. Various concentrations of compound (10 μL), 0.5 U / mL enzyme solution (40 μL), and 0.1 mM, pH 6.8 phosphate buffer (100 μL) were preincubated in a 96-well plate at 37°C for 10 min. Subsequently, 50 μL of substrate (pNαGP, 0.6 mM) was added to each microwell and incubated at 37°C for 20 min. The absorbance at 405 nm was measured to assess changes in enzyme activity. Acarbose and DMSO were used as standard and control inhibitors, respectively.

[0039] Table 3. Inhibitory activity and IC50 value of GFY-1 to GFY-29 against α-glucosidase at a concentration of 250 μmol / L

[0040] [Table 3]

[0041] As can be seen from Table 3, all of the GFY series compounds had a certain level of inhibitory effect on α-glucosidase, demonstrating broad spectrum activity. However, although the inhibitory effect on α-glucosidase varied greatly depending on the compound, the skeletal compounds generally exhibited good α-glucosidase inhibitory activity.

[0042] Example 4: Fluorescence quenching experiment of compounds against α-glucosidase α-Glucosidase (1.0 mL, 2 U / mL) was titrated with inhibitor solutions of different concentrations (1.0 mL, 0–1000 mM) and equilibrated for 5 min before fluorescence measurement. The fluorescence intensity of the reaction solution was measured at different temperatures (305.15, 310.15, and 315.15 K) using a fluorescence spectrometer (F-7100, Tokyo, Japan). The excitation and emission slit widths were 5.0 nm, the excitation wavelength was 280 nm, and the emission wavelength range was 290–500 nm.

[0043] The results are shown in Figure 1. α-glucosidase fluorescence quenching was used to characterize the affinity of the compounds with the enzyme. α-glucosides have intrinsic fluorescence at approximately 340 nm due to Trp and Tyr residues. Therefore, the degree of binding can be determined by measuring the effect of the compounds on the emission spectra of the two enzymes. As can be seen from Figure 1, the fluorescence intensity of the enzyme significantly decreased with increasing compound concentration, indicating that all five compounds were able to quench the intrinsic fluorescence of the enzyme and bind to it. However, when comparing the effects of the compounds on the enzyme fluorescence at the same concentration, GFY-based compounds had a greater effect on the fluorescence quenching of α-glucosidase, indicating a stronger affinity and interaction strength between GFY-based compounds and α-glucosidase, thereby exerting a stronger inhibitory effect on the enzyme.

[0044] Example 5 In vitro starch digestion inhibition experiment The effect of inhibitors on in vitro starch digestibility was measured using a slightly modified Englyst method. Cornstarch (300 mg) and guar gum (25 mg) were added to a 50 mL centrifuge tube and dissolved in 7.5 mL of distilled water. The tube was boiled in a boiling water bath for 10 min, cooled to room temperature, and sodium acetate buffer (2.5 mL, 0.4 M, pH 5.2, containing 0.18% (w / v) CaCl2) was added. After equilibrating the tube at 37 °C for 15 min, fresh porcine trypsin extract, amyloglucosidase, and inhibitor mixture (5.5 mL) was added to hydrolyze starch. A control group without inhibitor and acarbose group served as blank and positive controls. At 20, 60, 120, 240, and 360 min, 250 μL of starch hydrolysate was removed from the centrifuge tube and added to 10.0 mL of 66% (v / v) ethanol. Glucose production was measured using a d-glucose assay kit (GOPOD).

[0045] The results are shown in Figure 2. The results show that GFY-based compounds can significantly inhibit α-glucosidase activity, thereby inhibiting the conversion of starch to glucose, and have extremely high anti-diabetic potential.

[0046] The above embodiments are merely preferred embodiments given to fully explain the present invention, and the scope of protection is not limited thereto. Any equivalent replacement or conversion made by those skilled in the art based on the present invention falls within the scope of protection of the present invention.

Claims

1. A compound having α-glucosidase inhibitory activity, having the structure of the following general formula (I): 【Chemistry 1】 A compound having α-glucosidase inhibitory activity, wherein R1 is a halogen and R2 is a C1-C2 alcoholic hydroxyl group.

2. A pharmaceutical composition for treating diabetes, comprising the compound of claim 1 and a pharmaceutically acceptable carrier.

3. 10. Use of the compound of claim 1 in the manufacture of a medicament for treating diabetes.

4. Use of the compound according to claim 1 in the production of an α-glucosidase inhibitor.

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