CN112587526A

CN112587526A - Application of 1,3, 4-thiadiazole phenyl furan thiocarbamate compound in preparation of alpha-glucosidase inhibitor

Info

Publication number: CN112587526A
Application number: CN202011567038.9A
Authority: CN
Inventors: 崔紫宁; 王鸿; 何敏; 魏斌; 李亚胜
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2021-04-02
Anticipated expiration: 2040-12-25
Also published as: CN112587526B

Abstract

The invention discloses an application of 1,3, 4-thiadiazole phenyl furan thioformate compounds in preparation of alpha-glucosidase inhibitors. The 1,3, 4-thiadiazole phenyl furan thiomethyl ester compound has very obvious inhibition activity on alpha-glucosidase, and is obviously superior to a control drug acarbose and IC₅₀The value range is 0.2-49.6 mu M, and the compound has wide application prospect in the research and development aspect of preparing medicaments for preventing or treating type II diabetes.

Description

Application of 1,3, 4-thiadiazole phenyl furan thiocarbamate compound in preparation of alpha-glucosidase inhibitor

Technical Field

The invention belongs to the technical field of chemical medicine. More particularly, relates to an application of 1,3, 4-thiadiazole phenyl furan thioformate compounds in preparing alpha-glucosidase inhibitors.

Background

Diabetes mellitus is a chronic metabolic disorder syndrome characterized by hyperglycemia resulting from insulin resistance or insufficient insulin secretion. In recent years, the number of diabetes mellitus in the world increases year by year, and according to the latest message issued by the international diabetes association (IDF) in 2019, 2 diabetes mellitus patients exist in every 11 adults, and the number of the diabetes mellitus patients in the middle of 2019 is in the leaderboard and reaches 1.164 hundred million, wherein more than 90 percent of the diabetes mellitus patients are type II diabetes mellitus patients. Type II diabetes mellitus is mainly deficient in insulin secretion and insulin resistance, and in the early stages of onset, hyperglycemia occurs due to increased hepatic glucose production and decreased peripheral glucose uptake. So far, no specific medicine capable of radically curing the disease exists, so that the control of postprandial blood sugar by oral hypoglycemic drugs is the key.

Alpha-glucosidase can hydrolyze 1, 4-alpha-glycosidic bond, is mainly used for digesting maltose and sucrose in food, and the food can be digested and absorbed only by combining with the alpha-glucosidase in small intestine to cause blood sugar rise, so oral administration of a plurality of alpha-glucosidase inhibitors can delay the decomposition of food polysaccharide into glucose, thereby slowing the absorption of glucose by human body and reducing postprandial blood sugar. Therefore, the alpha-glucosidase inhibitor is a specific drug for treating type II diabetes, however, some alpha-glucosidase inhibitors in the market, such as acarbose, voglibose and miglitol, which are 3 hypoglycemic drugs, have certain side effects, such as acarbose and voglibose are absorbed into the body, damage is caused to the liver, liver function needs to be detected regularly, hypoglycemia may occur when other hypoglycemic drugs are used together, and intravenous injection or oral glucose is needed when a patient has hypoglycemia, which brings inconvenience to the patient. Therefore, the screening of the alpha-glucosidase inhibitor and the application of the alpha-glucosidase inhibitor to clinic have great development prospect.

The 1,3, 4-thiadiazole phenyl furan thiomethyl ester compound is a derivative containing a 5-phenyl-2-furan structure, and in terms of the structure, a furan ring is rich in electronic groups and can easily form intermolecular hydrogen bonds with a plurality of biological macromolecules, so that the compound containing the 5-phenyl-2-furan structure has various biological activities, such as antibiosis, antitumor, anti-inflammation, insect resistance and the like, but no relevant research is carried out on whether the compound has alpha-glucosidase inhibitory activity.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art and provides an application of a 1,3, 4-thiadiazole phenyl furan thioformate compound in preparation of an alpha-glucosidase inhibitor.

The above purpose of the invention is realized by the following technical scheme:

the application of 1,3, 4-thiadiazole phenyl furan thio-methyl ester compounds in preparing alpha-glucosidase inhibitors is disclosed in the following formula (I):

in the formula (I), R is₁Is a substituent on a benzene ring, the number of which can be one or more, and R is₁Optionally selected from hydrogen, halogen, nitro, C1-C4 alkyl or C1-C4 alkoxy;

the R is₂Optionally selected from hydrogen, C1-C4 alkyl or C1-C4 alkoxy.

The 1,3, 4-thiadiazole phenyl furan thiomethyl ester compound has the first reported inhibition activity on alpha-glucosidase, and has very obvious inhibition activity, IC₅₀IC with value much smaller than that of positive control-acarbose₅₀The value is obtained. Has wide application prospect in the research and development aspects of medicaments for preventing or treating related diseases caused by alpha-glucosidase and medicaments thereof.

Preferably, the 1,3, 4-thiadiazole phenyl furan thio methyl ester compound R₁The number may be one or more, R₁Optionally selected from hydrogen, fluorine, chlorine, bromine, nitro, methyl, ethyl, methoxy, ethoxy; wherein, the number of the fluorine, the chlorine and the bromine can be more than one;

R₂optionally selected from hydrogen, methyl, ethyl, methoxy, ethoxy.

Preferably, the 1,3, 4-thiadiazole phenyl furan thio methyl ester compound R₁Optionally selected from hydrogen, 2-chloro, 3-chloro, 4-chloro, 2-fluoro, 3-fluoro, 4-fluoro, 2-nitro, 3-nitro, 4-bromo, 4-methyl, 4-methoxy, 2, 4-difluoro, 2, 6-difluoro;R₂optionally selected from hydrogen and methyl.

More preferably, the 1,3, 4-thiadiazole phenyl furan thio methyl ester compound R₁Selected from hydrogen, 3-chloro, 3-fluoro, 4-methyl, 4-methoxy, 4-nitro; r₂Is hydrogen.

The invention also provides an alpha-glucosidase inhibitor, which comprises 1,3, 4-thiadiazole phenyl furan thio-methyl ester compounds or pharmaceutically acceptable salts thereof, wherein the structure of the 1,3, 4-thiadiazole phenyl furan thio-methyl ester compounds is shown as the formula (I):

the R is₂Optionally selected from hydrogen, C1-C4 alkyl or C1-C4 alkoxy.

Preferably, the inhibitor can be in the dosage form of powder, wettable powder, granules, water dispersible granules, suspending agents, missible oil, microemulsion or aqueous solution.

The invention also protects the application of the 1,3, 4-thiadiazole phenyl furan thio-methyl ester compound or the pharmaceutically acceptable salt thereof in preparing the medicines for preventing or treating the alpha-glucosidase related diseases.

Preferably, the α -glucosidase related disease is type II diabetes.

The invention also protects a pharmaceutical composition which is composed of the 1,3, 4-thiadiazole phenyl furan thiomethyl ester compound or pharmaceutically acceptable salt thereof as an active ingredient and one or more pharmaceutical excipients.

Compared with the prior art, the invention has the following beneficial effects:

the 1,3, 4-thiadiazole phenyl furan thiomethyl ester compound has the first reported alpha-glucosidase inhibitory activity, has very obvious inhibitory activity, is even obviously superior to a control drug acarbose, and is a novel alpha-glucosidase inhibitor. The compounds can be used as alpha-glucosidase inhibitors for preventing or treating related diseases caused by alpha-glucosidase, such as type II diabetes, and have important medicinal value and wide application prospect.

Drawings

FIG. 1 is a graph showing the concentration-dependent inhibition curves of 1,3, 4-thiadiazole phenyl furan thiomethyl ester compounds and acarbose on alpha-glucosidase;

FIG. 2 is a graph showing the inhibition types of acarbose and 1,3, 4-thiadiazole phenyl furan thiomethyl ester derivative 14 and acarbose on alpha-glucosidase;

FIG. 3 is a graph of the mimetic docking of inhibitor 14 with α -glucosidase.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Among them, the alpha-glucosidase of the present invention is a commercial enzyme derived from Saccharomyces cerevisiae and purchased from Sigma-Aldrich (Cat. No. G0660).

Example 1 microplate method for screening alpha-glucosidase inhibitors

(1) Preparation of enzyme: 2mg of purchased alpha-glucosidase is precisely weighed and prepared into 2mg/mL (22.10U/mL) mother solution by Phosphate Buffer Solution (PBS), and the mother solution is preserved at the temperature of minus 20 ℃ for standby; for each experiment, the enzyme solution was prepared at 0.15U/mL (e.g., 6. mu.L of a 2mg/mL stock solution was added to 878. mu.L of PBS).

(2) Preparation of a p-nitrophenol (PNP) standard: preparing 1mM PNP solution (PBS is dissolved), operating in a 96-well plate, adding 0, 10, 20, 40, 60 and 80 mu L of 1mM PNP solution in each group of 3 parallel plates, respectively, supplementing to 100 mu L with PBS, measuring absorbance at 405nM wavelength for 0min and 30min respectively by using a microplate reader (the reaction is incubated for 30min at 37 ℃), then making a scatter diagram by using Excel to obtain a relation formula of the absorbance and the PNP concentration, namely y 3.2617x +0.0547, wherein y is the absorbance, x is the PNP concentration, converting and removing blank interference by unit, and the relation formula of the absorbance and the PNP concentration at the mu M concentration is y 0.003262 x.

(3) Screening of alpha-glucosidase inhibitors (final concentration of inhibitor 10. mu.M):

inhibitor (B): 1,3, 4-thiadiazole phenyl furan thiomethyl ester compounds (compounds 1-30 in Table 1) are respectively prepared into a solution with the concentration of 0.1mM by using dimethyl sulfoxide (DMSO) to be used as an inhibitor for standby.

Positive control: acarbose (Acarbose, ACAR, available from Sigma-Aldrich) was prepared as a 0.1mM solution in dimethyl sulfoxide (DMSO) as a positive control for future use.

Substrate: 4-Nitrophenyl- α -D-glucopyranoside (α -PNPG, available from Sigma-Aldrich) was made up into 2.5mM stock solution with PBS for use.

Enzyme: and (2) taking the alpha-glucosidase prepared in the step (1) at 0.15U/mL as a reaction enzyme solution.

IC₅₀Determination of the value: determination of IC of 30 1,3, 4-thiadiazole phenyl furan thio-methyl ester compounds₅₀The value is that a series of inhibitor concentration points (such as 0.001, 0.01, 0.1, 0.3, 0.5, 1,3, 5, 10 and 30 mu M) are arranged in a final concentration of 0.001-100 mu M, and the reaction is carried out in a 96-well plate, wherein the reaction system is as follows: blank group: enzyme 10 μ L + PBS 70 μ L + volume fraction 1% DMSO 10 μ L +2.5mM substrate 10 μ L; experimental groups: enzyme 10. mu.L + PBS 70. mu.L + inhibitor at different concentrations 10. mu.L +2.5mM substrate 10. mu.L; setting 3 parallel groups in each group, loading samples according to the sequence of enzyme, PBS, inhibitor/positive control and substrate, respectively measuring OD values of 0min and 30min under the wavelength of 405nM of an enzyme labeling instrument (incubation at 37 ℃), obtaining the relative activity value of each inhibitor to alpha-glucosidase under different solubility conditions through calculation, finally converting the unit of mu M of the inhibitor concentration point into nM, taking a derivative with 10 as the bottom to obtain an lg value, taking the lg value as a horizontal coordinate and the relative activity as a vertical coordinate, and drawing an IC by utilizing Graphad Prism 6.0 software₅₀The graphs (see FIG. 1) were obtained and analyzed by the software to obtain the IC of each inhibitor for alpha-glucosidase₅₀Value, each compoundIC₅₀The values are shown in Table 1.

The specific calculation process is as follows:

ΔOD＝OD_30min–OD_0min；

ΔC_PNPΔ OD/0.003262(0.003262 is a correlation coefficient between the absorbance obtained in step (2) and the PNP concentration)

Relative activity (%) (Experimental group. DELTA.C)_PNPBlank group Δ C_PNP)×100％；

TABLE 11 IC of thiomethyl 3, 4-thiadiazole phenyl furancarboxylates₅₀Value of

As can be seen from Table 1, the IC of 1,3, 4-thiadiazole phenyl furan thiomethyl ester compound for alpha-glucosidase₅₀Are all less than the positive control compound ACAR, IC₅₀The value range is 0.2-49.6. mu.M, and the alpha-glucosidase inhibitor has obvious inhibition effect.

EXAMPLE 2 Positive control ACAR and Studies of the type of inhibition of alpha-glucosidase by Compound 14

Select IC₅₀Minimum value of Compound 14(R is 4-Me, IC)₅₀0.2 μ M) and positive control ACAR (IC)₅₀516.9 μ M) were performed. The substrate alpha-PNPG is firstly prepared into solutions with the concentrations of 2, 3, 5 and 10mM by using PBS, namely the final concentrations of the reaction system are 200, 300, 500 and 1000 mu M, and then the inhibitor is also prepared into a series of solutions with concentration gradients by using PBS. For example, the positive control ACAR is prepared into a solution with the concentration of 0, 3000, 6000 or 9000 μ M, namely the final concentration of the reaction system is 0, 300, 600 or 900 μ M; compound 14 was formulated in 0, 0.2, 0.5, 1. mu.M solutions, i.e., final concentrations of 0, 0.02, 0.05, 0.1. mu.M. A table of substrate and inhibitor solubility combinations was prepared for compound 14 as an example, as shown in Table 2.

TABLE 2 permutation and combination of different concentration points of substrate and Compound 14

Note that: c_PNPGDenotes the final substrate concentration, C_InThe final concentration of inhibitor (compound 14) is indicated, and a circle indicates one experimental group (corresponding to one well of a 96-well plate), and 3 groups are made for each concentration combination in parallel.

Then according to the reaction system: performing reaction on 10 mu L of enzyme, 70 mu L of PBS, 10 mu L of inhibitor with different concentrations and 10 mu L of substrate with different concentrations (the combination of the inhibitor and the substrate with different concentrations is shown in Table 2), setting 3 combinations in parallel in a 96-well plate, loading the enzyme, the PBS, the inhibitor/positive control and the substrate in sequence, measuring the absorbance of 0min and 30min at the wavelength of 405nm by using a microplate reader (incubation period 37 ℃), calculating the concentration difference of PNP corresponding to different solubility combinations according to the calculation process of the step (3) in the example 1, and finally calculating the values of 1/V (mu mol/min/mg) and 1/PNPG (mu mol/min/mg), wherein V (mu mol/min/mg) is the catalysis speed of the enzyme and represents the molar amount of the product catalytically produced per milligram of enzyme per minute under the conditions of certain temperature, pH value and substrate concentration;

the calculation process is as follows:

1/V(μmol/min/mg)＝1/(ΔC_PNP*100/10/30/1)；1/PNPG＝1/ΔC_PNP；

wherein Δ C_PNPThe difference in PNP concentration between the 0min and 30min systems was shown, 100 was 100. mu.L for the reaction system, 10 was 10. mu.L for the enzyme, 30 was 30min for the reaction time, and 1 was 1. mu.g/mL for the enzyme preparation solubility.

Finally, a graph of double reciprocal inhibition is drawn by utilizing Graphad Prism 6.0 software linear regression, the result is shown in figure 3, the ACAR and the compound 14 which are positive controls correspond to A, B in figure 3 respectively, and the inhibition type is judged according to the intersection point of the curves.

As can be seen from FIG. 2, the positive control ACAR and the compound 14 images both have an intersection point with the y-axis, indicating that the inhibition of alpha-glucosidase by the compound 14 and the positive control ACAR are similar and competitive inhibition. The compound 14 can rob the binding site of an alpha-PNPG substrate and alpha-glucosidase in the enzyme activity reaction process, so that the degradation activity of the alpha-glucosidase is inhibited.

Example 3 molecular docking simulation

The rule of the interaction of the compound 14 with the best inhibitory activity and the alpha-glucosidase is analyzed by a Molecular docking method.

The MOE2014.09 software is mainly used for molecular docking research, and the specific implementation steps are as follows: since the actual protein crystal structure of alpha-glucosidase has not been successfully resolved, homology modeling was performed to find a protein crystal structure with high homology to alpha-glucosidase. The amino acid sequence of alpha-glucosidase (accession number P53341) was found from the UniProt protein resource database. A similarity search was performed on the PDB database in MOE-2014.0901 using default parameters, selecting the crystal structure of Saccharomyces cerevisiae isomaltase (PDB ID: 3AJ7,

resolution) as template, which has higher target sequence homology (72.4%), an α -glucosidase homology model was established based on target template alignment. After the homologous model is established, preparing a protein model structure by using MOE-2014.0901 (the specific steps are: structure preparation-structure correction-protonation-energy minimization) to obtain a butt-jointed protein, then preparing a small molecule, drawing a structural formula of the small molecule by using a drawing function carried by MOE-2014.0901, and quickly preparing to obtain the butt-jointed small molecule. And finally, carrying out molecular docking, selecting a docking pocket of the Site Finder automatic recognition protein, then selecting a triangular docking method for docking, generating 30 conformations of ligand-protein complexes by software according to the scoring condition, and selecting the conformation with the best score, namely the optimal molecular conformation for docking with the protein pocket.

Fig. 3 (a) and (B) are graphs showing docking simulations of compound 14 with α -glucosidase homology model protein. From the graph (A), it can be seen that compound 14 can well enter the active pocket cavity of the α -glucosidase homologous model protein. From (B) in the figureIt was found that the sulfur atom of thiadiazole of compound 14 forms a hydrogen bonding force with Glu304 of the magnitude

In addition, compound 14 in turn forms a p- π conjugated interaction with amino acid residue Arg 312. In conclusion, the molecular docking simulation well shows the interaction of the compound 14 and the alpha-glucosidase.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

The application of 1,3, 4-thiadiazole phenyl furan thio-methyl ester compounds in preparing alpha-glucosidase inhibitors is disclosed in the following formula (I):

in the formula (I), R is₁Is a substituent on a benzene ring, the number of which can be one or more, and R is₁Optionally selected from hydrogen, halogen, nitro, C1-C4 alkyl or C1-C4 alkoxy;

the R is₂Optionally selected from hydrogen, C1-C4 alkyl or C1-C4 alkoxy.
2. The use according to claim 1, wherein the 1,3, 4-thiadiazole phenyl furan thio-methyl ester compound R₁The number may be one or more, R₁Optionally selected from hydrogen, fluorine, chlorine, bromine, nitro, methyl, ethyl, methoxy, ethoxy;

R₂optionally selected from hydrogen, methyl, ethyl, methoxy, ethoxy.
3. The use according to claim 2, wherein the 1,3, 4-thiadiazole phenyl furan thio-methyl ester compound R₁Optionally selected from hydrogen, 2-chloro, 3-chloro, 4-chloro, 2-fluoro, 3-fluoro, 4-fluoro, 2-nitro, 3-nitro, 4-bromo, 4-methyl, 4-methoxy, 2, 4-difluoro, 2, 6-difluoro;

R₂optionally selected from hydrogen and methyl.
4. The use according to claim 3, wherein the 1,3, 4-thiadiazole phenyl furan thio-methyl ester compound R₁Optionally selected from hydrogen, 3-chloro, 3-fluoro, 4-methyl, 4-methoxy, 4-nitro; r₂Is hydrogen.
5. An alpha-glucosidase inhibitor is characterized by comprising a 1,3, 4-thiadiazole phenyl furan thio methyl ester compound or pharmaceutically acceptable salt thereof, wherein the structure of the 1,3, 4-thiadiazole phenyl furan thio methyl ester compound is shown as a formula (I):

in the formula (I), R is₁Is a substituent on a benzene ring, the number of which can be one or more, and R is₁Optionally selected from hydrogen, halogen, nitro, C1-C4 alkyl or C1-C4 alkoxy;

the R is₂Optionally selected from hydrogen, C1-C4 alkyl or C1-C4 alkoxy.
6. The alpha-glucosidase inhibitor as claimed in claim 5, wherein the inhibitor is in the form of powder, wettable powder, granule, water dispersible granule, suspension, emulsifiable concentrate, microemulsion or aqueous solution.
7. The alpha-glucosidase inhibitor as defined in claim 5, wherein the inhibitor is used for preparing a medicament for preventing or treating alpha-glucosidase related diseases.
8. The α -glucosidase inhibitor as defined in claim 7, wherein the α -glucosidase related disease is type II diabetes.
9. A pharmaceutical composition comprising the α -glucosidase inhibitor of claim 5 as an active ingredient in combination with one or more pharmaceutically acceptable excipients.