Compound K413 and application thereof in preparation of diabetes medicines

Abstract

The invention relates to an (E)-4-(2-(5-bromo-2-oxoindoline-3-subunit methyl)-4-oxoquinazoline-3 (4H)-yl) benzoic acid compound and an application of the (E)-4-(2-(5-bromo-2-oxoindoline-3-subunit methyl)-4-oxoquinazoline-3 (4H)-yl) benzoic acid compound in a medicine for treating diabetes. The compound as shown in the following formula (1) is obtained through virtual screening in combination with experimental verification, and the compound has remarkable alpha-glucosidase inhibitory activity. In-vitro and in-vivo experiments prove that the compound effectively reduces the generation of glucose and delays the digestion and decomposition of starch by inhibiting the activity of alpha-glucosidase. The invention discloses the compound and the application thereof in the field of preparation of anti-diabetic drugs for the first time, the alpha-glucosidase inhibitory activity of the compound is high, and a new direction is provided for development of diabetes treatment drugs. Formula (1) below

Description

Technical Field

[0001] This invention belongs to the field of medicine, and specifically relates to a compound as shown in formula (1) and its application in the preparation of drugs for treating diabetes. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a serious chronic metabolic disease characterized by long-term hyperglycemia, resulting from insulin resistance and insufficient insulin secretion by pancreatic beta cells. According to the World Health Organization, there are over 420 million people with diabetes worldwide, and this number is projected to exceed 642 million by 2040. Long-term uncontrolled hyperglycemia can lead to a variety of serious complications, including eye, kidney, heart, and vascular diseases. Postprandial blood glucose control is a crucial means of delaying the onset of complications. Changes in blood glucose levels arise from the breakdown of carbohydrates catalyzed by enzymes such as α-glucosidase and α-amylase. α-glucosidase is an important membrane-bound enzyme located on the brush border membrane of the small intestine, which hydrolyzes 1,4-α-glucosidic bonds to produce α-glucose. Therefore, α-glucosidase inhibitors can delay carbohydrate intake, thereby maintaining postprandial blood glucose within the normal range, and are thus widely used to treat patients with T2DM. Currently, some alpha-glucosidase inhibitors (acarbose, vogose, and miglitol) are used to treat type 2 diabetes, but these drugs have a wide range of side effects, including stomach pain, diarrhea, flatulence, and allergic reactions.

[0003] Virtual screening is currently a primary method in new drug development, mainly used to study large compound libraries. It can rapidly screen small molecules with high potential for interaction with target proteins, facilitating subsequent in vitro biochemical evaluation. Therefore, virtual screening technology can quickly screen high-potential α-glucosidase inhibitors from large compound libraries. Combined with experimental validation, this can greatly improve the ability to screen novel compounds that bind and interact with target proteins. Summary of the Invention

[0004] In view of the shortcomings of existing technologies, the present invention aims to propose a new compound with a more efficient inhibitory effect on α-glucosidase, so as to solve the deficiencies of current clinical drugs.

[0005] The first aspect of the present invention is to provide a compound, said compound being a compound of formula (1):

[0006]

[0007] The compounds are named (E)-4-(2-(5-bromo-2-oxoindolin-3-ylidene)-4-oxoquinazolin-3(4H)-yl)benzoic acid; (E)-4-(2-((5-bromo-2-oxoindolin-3-ylidene)methyl)-4-oxoquinazolin-3(4H)-yl)benzoic acid.

[0008] A second aspect of the present invention is to provide a pharmaceutical composition for treating diabetes, said pharmaceutical composition comprising the compound described in the first aspect and a pharmaceutically acceptable carrier.

[0009] Thirdly, the present invention also provides the use of the compounds described in the first aspect in the preparation of α-glucosidase inhibitors.

[0010] Furthermore, the stated use is for non-therapeutic purposes.

[0011] Fourthly, the present invention also provides the use of the compounds described in the first aspect in the preparation of medicaments for treating diabetes.

[0012] Fifthly, the present invention also provides the use of the method described in the second aspect in the preparation of a medicament for treating diabetes.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] The present invention screened a large compound library through virtual screening to obtain compound (1) which can significantly inhibit α-glucosidase activity, thereby inhibiting the conversion of starch into glucose, and has extremely high anti-diabetic potential. Attached Figure Description

[0015] Figure 1 Verification of virtual screening results.

[0016] Figure 2 Determination of the IC50 value of the compound against α-glucosidase.

[0017] Figure 3 The compound quenches the fluorescence of α-glucosidase.

[0018] Figure 4 Compound docking with α-glucosidase molecules

[0019] Figure 5 The compound inhibits starch digestion. Detailed Implementation

[0020] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0021] Example 1: Compound Screening

[0022] The Autodock Vina was used to perform virtual screening of 100K+ compounds in a representative diversity library in the ChemDiv database. Additionally, 2QMJ and 1CXW were selected from a protein database as crystal structures for docking with α-glucosidase and α-amylase, respectively. The compounds were docked with α-glucosidase and α-amylase, respectively, and scoring results were obtained. Compounds with docking scores <-9.0 with α-glucosidase and >-5.0 with α-amylase were selected, synthesized by ChemDiv, and their activity was verified. The synthesis method of compound (1) is as follows:

[0023]

[0024] The inhibitory effects of the screened compounds on the two digestive enzymes at a concentration of 250 μM were then determined, and the results are as follows: Figure 1 As shown in Table 1, among the five screened compounds, compound (1) showed a significant α-glucosidase-specific inhibitory effect, with an inhibition effect of 94.2% on α-glucosidase at a concentration of 250 μM.

[0025] Table 1. Compound structures and inhibition rates of α-glucosidase and α-amylase.

[0026]

[0027]

[0028] Example 2: Determination of IC50 value of compound (1) against α-glucosidase

[0029] In vitro α-glucosidase activity was determined spectrophotometrically. A 0.5 U / mL solution of α-glucosidase and 0.6 mM p-nitrophenyl-diglucoside (pNαGP) was prepared using phosphate buffer (pH 6.8). All test chemicals, including the standard drug acarbose, were dissolved in DMSO to form a 10 mM stock solution. Serial dilutions with phosphate buffer were performed to obtain sample solutions of different concentrations. First, different concentrations of the compound (10 μL), enzyme solution (40 μL), and potassium phosphate buffer (100 μL) were pre-incubated in 96-well plates at 37°C for 10 min. Then, 50 μL of substrate (pNαGP, 0.6 mM) was added to each well, and the plates were incubated at 37°C for 20 min. The absorbance was measured at 405 nm to detect changes in enzyme activity. Acarbose and DMSO were used as the standard inhibitor and control inhibitor, respectively.

[0030] The formula for calculating the enzyme inhibitory activity of the tested compounds is as follows:

[0031]

[0032] The IC50 value of the test compound was calculated using nonlinear fitting (logit method).

[0033] The results are as follows Figure 2 As shown, by Figure 2 It can be seen that compound (1) (66.30 μM) has a much higher inhibitory activity against α-glucosidase than pharmaceutical acarbose (562.22 μM).

[0034] Example 3: Fluorescence quenching experiment of compound (1) on α-glucosidase

[0035] α-glucosidase (1.0 mL, 2 U / mL) was titrated with inhibitor solutions of different concentrations (1.0 mL, 0–1000 mM), and fluorescence was measured after equilibration for 5 min. The fluorescence intensity of the reaction solution was measured using a fluorescence spectrometer (Tokyo F-7100, Japan) at different temperatures (305.15, 310.15, and 315.15 K). The excitation and emission slit widths were set to 5.0 nm, the excitation wavelength to 280 nm, and the emission wavelength range to 290 nm–500 nm.

[0036] The results are as follows Figure 3 As shown, fluorescence quenching of α-glucosidase can be used to characterize the affinity between a compound and the enzyme. α-glucosides exhibit a strong intrinsic fluorescence around 340 nm, induced by Trp and Tyr residues. Therefore, the degree of binding can be determined by measuring the effect of the compound on the emission spectra of the two enzymes. Figure 3As can be seen from the table, the fluorescence intensity of the enzyme decreases significantly with increasing compound concentration, indicating that the compound can quench the enzyme's intrinsic fluorescence and bind to it. However, by comparing the effects of the compound on the enzyme fluorescence at the same concentration, it can be seen that compound (1) has a greater effect on the fluorescence quenching value of α-glucosidase, which also indicates that compound (1) has a stronger affinity and interaction with α-glucosidase, thus exerting a stronger inhibitory effect on the enzyme. Since Ksv can reflect the interaction (binding) between the inhibitor and α-glucosidase, the higher the Ksv value, the higher the binding performance of the inhibitor. Table 2 shows that the Ksv value of compound (1) at different temperatures is much higher than that of acarbose, further indicating that compound (1) has a stronger affinity for α-glucosidase.

[0037] Table 2. Affinity determination of compounds with α-glucosidase at different temperatures.

[0038]

[0039] Example 4: Molecular docking of the compound with α-glucosidase

[0040] This experiment used AutoDock Vina for molecular docking. The first step was to prepare the protein crystal structure. 2QMJ was selected from a protein database as the crystal structure for α-glucosidase docking, and the protein crystal structure was modified by removing water molecules and adding hydrogen atoms. The second step was small molecule preparation. The three-dimensional structure of the inhibitor was plotted using ChemBiodraw Ultra 14.0 (WalthamPerkin Elmer Instruments Co., MA, USA), and the energy of the small molecule was minimized. The third step involved generating a docking cassette centered on the inhibitor and performing docking using AutoDock Vina. Ten docking positions were generated during the docking process; the position with the highest Glide score was selected to study the interaction between the inhibitor and α-glucosidase.

[0041] The results are as follows Figure 4 As shown, the binding of the clinical drug acarbose to the active site mainly involves multiple hydrogen bonds, especially the binding to the acarbose ring. The interaction mainly occupies the -1 and +1 binding sites, and the same phenomenon was found in the docking of the screened compounds. Compound (1) mainly forms a strong binding by forming hydrophobic, salt bridge or π-π interactions with PHE450, PHE575, TYR299, ASP542 and TRP406 in the α-glucosidase active pocket.

[0042] Example 4: In vitro starch digestion inhibition experiment

[0043] The effect of the inhibitor on the in vitro digestibility of starch was determined using a slightly modified Englyst method. Corn starch (300 mg) and guar gum (25 mg) were added to 50 mL centrifuge tubes and dissolved in 7.5 mL of distilled water. The mixture was boiled in a water bath for 10 min, cooled to room temperature, and then infused with sodium acetate buffer (2.5 mL, 0.4 M, pH 5.2, containing 0.18% (w / v) CaCl2). After equilibration at 37°C for 15 min, a mixture of fresh porcine trypsin extract, amylase, and the inhibitor (5.5 mL) was added to hydrolyze the starch. A control group (no inhibitor and acarbose) served as blank and positive controls, respectively. At 20, 60, 120, and 240 min, 250 μL of the starch hydrolysate was collected from the centrifuge tubes and 10.0 mL of 66% (v / v) ethanol was added. Glucose production was measured using a d-glucose assay kit (GOPOD).

[0044] The results are shown in Table 3. The results indicate that compound (1) significantly reduced α-glucosidase activity. At 20 min, 60 min, 120 min, and 240 min, compound (1) reduced glucose production by 12.21%, 11.90%, 18.12%, and 20.92% compared to acarbose, respectively. This suggests that compound (1) is more effective at inhibiting starch conversion to glucose and possesses extremely high anti-diabetic potential.

[0045] Table 3. GOPOD Detection and Determination of Glucose Production

[0046]

[0047] Example 5: In vivo starch digestion inhibition experiment

[0048] Intervention experiments on normal mice ( Figure 5 Pre-administered compound (1) and acarbose effectively reduced the rapid spike in blood glucose levels within 30 minutes prior to starch suspension consumption (P<0.01). Figure 5A), and also revealed some other interesting findings. First, we observed that compound (1) had a similar effect to acarbose in controlling postprandial blood glucose and in alleviating the side effects of acarbose. This suggests that compound (1) is a promising alternative treatment option for those who have difficulty with starch digestion and postprandial blood glucose control. AUC represents the overall blood glucose response over a period of time, and a lower AUC curve value indicates less postprandial blood glucose fluctuation and less harm to diabetic patients. When comparing the area under the curve (AUC) of the control group with that of the compound (1) or acarbose treatment group, the results showed a statistically significant difference (P<0.001). At the same time, the area under the curve of the AUC of the compound (1) group was significantly lower than that of the acarbose group, and the blood glucose fluctuation was only 86.6% of that of the acarbose group. In this case, it clearly shows that both compound (1) and acarbose are more effective in reducing postprandial blood glucose levels than no treatment, with compound (1) being more effective.

[0049] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A compound, said compound having the formula (1):

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound of claim 1 and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, characterized in that, It also contains excipients.

4. The pharmaceutical composition according to claim 3, characterized in that, The excipient is at least one of the following: sustained-release agent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, or lubricant.

5. The pharmaceutical composition according to claim 3 or 4, characterized in that, The dosage form of the pharmaceutical composition is tablets, pills, capsules, oral liquids, or injections.

6. Use of the compound of claim 1 or the pharmaceutical composition of any one of claims 2-5 in the preparation of an α-glucosidase inhibitor.

7. The use of the compound of claim 1 or the pharmaceutical composition of any one of claims 2-5 in the preparation of a medicament for treating diabetes.

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