Binuclear oxygen vanadium (IV) complex as well as preparation method and application thereof

Abstract

The invention belongs to the field of metal complexes, and particularly relates to a binuclear oxygen vanadium (IV) complex as well as a preparation method and application thereof. The simple formula of the structure of the complex is [V2 ([mu] O) 2O2 (H2L) 2], wherein H2L is 2-hydroxy-N '-[1-(pyrazine-2-yl) ethylidene] benzoyl hydrazine. The complex is prepared by taking acetoxyl vanadium and 2-hydroxyl-N '-(1-(pyrazine-2-yl) ethylidene) benzoyl hydrazine as raw materials through a heating method. The complex can effectively inhibit the activity of alpha-amylase and alpha-glucosidase, and the IC50 value of alpha-amylase inhibition is lower than that of a positive reference substance acarbose; the complex shows relatively low cytotoxicity to human liver normal cells (7702). Therefore, the complex is a low-toxicity effective alpha-amylase and alpha-glucosidase inhibitor, and can be used as a potential candidate drug for treating type II diabetes mellitus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of metal complexes, and particularly relates to a binuclear oxovanadium (IV) complex and a preparation method and application thereof. BACKGROUND

[0002] Type 2 diabetes (T2DM) is caused by the resistance of body cells to the normal action of insulin, and has been recognized as one of the most serious public health problems in the world. At present, there are various hypoglycemic drugs, which provide diversified treatment options for diabetic patients. However, these drugs are accompanied by some side effects such as gastrointestinal discomfort, liver and kidney function damage, etc. while alleviating the symptoms of the disease. These problems not only affect the quality of life of patients, but also limit the wide application of drugs. In order to overcome the defects of existing hypoglycemic drugs and minimize the harm to patients, many researchers at home and abroad are actively exploring and developing new anti-diabetic drugs. As a necessary metal element for human body, a large number of studies have shown that vanadium compounds have significant insulin-like activity. Therefore, through reasonable screening and design of ligands, the toxicity of vanadium is reduced and the bioavailability is improved, which has become an important innovative direction of vanadium complexes in the field of diabetes treatment.

[0003] Since the diet of type 2 diabetes patients tends to be high in carbohydrates, the postprandial blood glucose fluctuation is more significant, which is characterized by postprandial hyperglycemia. Therefore, type 2 diabetes can be treated by inhibiting carbohydrate enzymes such as α amylase and α glucosidase to reduce postprandial hyperglycemia (PPHG). According to existing research, α amylase and α glucosidase inhibitors can be divided into three categories: natural plant and animal extracts, microbial secondary metabolites, and chemical synthesis. The former two methods are difficult to obtain α amylase and α glucosidase inhibitors due to the lack of raw materials and difficulty in extraction, etc., so that the obtained α amylase and α glucosidase inhibitors are difficult to be widely applied. Therefore, chemical synthesis has become an important research direction for developing α glucosidase and α amylase inhibitors. Metal complexes have attracted much attention due to their strong designability, easy functionalization and modification, etc. Different structures of ligands combined with metal ions can form complex systems with rich structures and diverse properties, thereby opening up a broad research idea and innovative way for designing new α amylase and α glucosidase inhibitors. SUMMARY

[0004] The present application aims at the above technical status, and provides a binuclear oxovanadium (IV) complex, a preparation method and application thereof, and application of the complex as an α-amylase and α-glucosidase inhibitor in a hypoglycemic drug.

[0005] To achieve the above object, the technical scheme of the present application is as follows. In a first aspect, the present application provides a binuclear oxovanadium (IV) complex, the structural formula of which is [V2(μ-O)2O2(H2L)2], wherein H2L is 2-hydroxy-N'-[1-(pyrazin-2-yl)ethylidene] benzhydrazide, and the structural formula is: 。

[0006] The binuclear oxovanadium (IV) complex crystal is monoclinic, belongs to the P21 / n space group, and has the following cell parameters: a = 11.834 (4) Å, b = 8.000 (3) Å, c = 14.389 (5) Å, α = 90°, β = 98.528 (13)°, and γ = 90°. β From the coordination environment of the complex, it can be seen that the central metal vanadium ion forms a six-coordinated octahedral configuration with the oxygen atom on the carbonyl group, the nitrogen atom of the carbon-nitrogen double bond, the nitrogen atom on the pyridine ring, the terminal oxygen atom and the two bridging oxygen atoms in the ligand H2L.

[0007] In a second aspect, the present application provides a preparation method of the binuclear oxovanadium (IV) complex of the first aspect, which comprises the following steps: adding acetoxyvanadium and 2-hydroxy-N'-(1-(pyrazin-2-yl)ethylidene) benzhydrazide into an alcohol solvent, and obtaining the binuclear oxovanadium (IV) complex through heating reaction.

[0008] Further, the molar ratio of the acetoxyvanadium to the 2-hydroxy-N'-(1-(pyrazin-2-yl)ethylidene) benzhydrazide is 1:1.

[0009] Further, the alcohol solvent is at least one of methanol, ethanol, a mixed solvent of methanol and water, and a mixed solvent of ethanol and water.

[0010] Further, the heating reaction is performed at a temperature of 40°C for 10 h.

[0011] In a third aspect, the present application provides an α-amylase inhibitor comprising the binuclear oxovanadium (IV) complex of the first aspect. The binuclear oxovanadium (IV) complex provided by the present application can effectively inhibit α the activity of amylase, with an IC 50 value of 88.14 ± 0.16 μg / mL, which is less than that of the positive control acarbose (IC50 = 309.6 ± 0.23 μg / mL); for α - The inhibition mechanism of amylase is mainly characterized by non-competitive inhibition.

[0012] Fourthly, the present invention provides an α-glucosidase inhibitor comprising the binuclear vanadium(IV) complex described in the first aspect. The binuclear vanadium(IV) complex provided by the present invention inhibits... α -IC50 of glucosidase 50 The value was 311.88 ± 0.17 μg / mL; for α The inhibition mechanism of glucosidase is mainly characterized by competitive inhibition.

[0013] Fifthly, the present invention provides the use of the binuclear vanadium(IV) complex described in the first aspect, or the α-amylase inhibitor described in the third aspect, or the α-glucosidase inhibitor described in the fourth aspect in the preparation of a drug for treating diabetes.

[0014] Furthermore, the diabetes mentioned is type II diabetes.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The binuclear vanadium(IV) complex of this invention is obtained under conventional heating reaction conditions. The preparation process is simple, with high yield and purity, and its structure is novel, exhibiting excellent biological properties. The complex effectively inhibits the activities of α-amylase and α-glucosidase, and its IC50 inhibition of α-amylase is [not specified in the original text]. 50 Its value is lower than that of the positive control acarbose, which can be used as an inhibitor of α-amylase and α-glucosidase targets, and has potential application value in the field of diabetes treatment. Attached Figure Description

[0016] Figure 1 Synthetic route diagram of the binuclear vanadium(IV) complex of the present invention.

[0017] Figure 2 Crystal structure diagram of the binuclear vanadium(IV) complex of the present invention.

[0018] Figure 3 The electron paramagnetic resonance spectrum of the binuclear vanadium(IV) complex of this invention.

[0019] Figure 4 Electrospray mass spectra of the binuclear vanadium(IV) complex of this invention.

[0020] Figure 5 The binuclear vanadium(IV) complex of this invention inhibits α - Lineweaver-Burk, Dixon, and Cornish-Bowden curves of amylase.

[0021] Figure 6 The dinuclear oxovanadium (IV) complex of the present application inhibits α Lineweaver-Burk, Dixon, Cornish-Bowden plots of -glucosidase.

[0022] Figure 7 The dinuclear oxovanadium (IV) complex of the present application inhibits α Molecular docking diagram of -amylase.

[0023] Figure 8 The dinuclear oxovanadium (IV) complex of the present application inhibits α Molecular docking diagram of -glucosidase.

[0024] Figure 9 MTT diagram of the dinuclear oxovanadium (IV) complex of the present application inhibiting 7702 cell proliferation. DETAILED DESCRIPTION

[0025] For the purpose of promoting an understanding of the principles of the application, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same. It will, nevertheless, be understood that no limitation of the scope of the application is thereby intended, any alterations and further modifications in the illustrated embodiments, and any further applications of the principles of the application as illustrated therein are contemplated as falling within the scope of the application.

[0026] Example 1: Preparation of the dinuclear oxovanadium (IV) complex 25 mL H2L (0.5 mmol, 0.1280 g) methanol solution was mixed with 15 mL vanadyl acetate methanol solution (0.5 mmol, 0.1326 g), heated at 40 °C for 10 h, suction filtered, washed with methanol for several times, the powder was yellow, the yield was 0.1023 g, and the yield was 60.34%. The mother liquor was left at room temperature for 2 weeks to obtain red-brown block crystals. The synthetic route is shown in Figure 1 .

[0027] Example 2: Crystal structure determination of the dinuclear oxovanadium (IV) complex X-ray single crystal diffraction data of the zinc-coordinated polymer was collected by a Bruker Smart Apex II diffractometer, and a molybdenum target (λ = 0.71073 Å) was used as the radiation source in the test, and the crystal structure was analyzed by using SHELXTL-97 and Olex2 software. The detailed crystal determination data is shown in Table 1, and the crystal structure is shown in Lambda . Figure 2

[0028] Table 1 Crystallographic data of the dinuclear oxovanadium (IV) complex of the present application ​

[0029] Example 3: Electron paramagnetic resonance spectrum test of the binuclear oxovanadium (IV) complex The binuclear oxovanadium (IV) complex in the mixed solvent of N,N-dimethylformamide : toluene = 1 : 9 was subjected to electron spin resonance (EPR) test under the condition of 60 K low temperature.

[0030] According to the spectrum characteristics and g value size, the results show that the complex is a tetravalent vanadium complex, and the electronic configuration of the vanadium ion is d 1 . See Figure 3 and Table 2.

[0031] Table 2 EPR data of the binuclear oxovanadium (IV) complex of the application

[0032] Example 4: Electrospray mass spectrum test of the binuclear oxovanadium (IV) complex The methanol solution of the complex was prepared and analyzed by using an electrospray mass spectrometer. The positive ion mass spectrum of the complex was obtained. Figure 4 ).

[0033] The results show that m / z = 415.20 belongs to [M / 2 + OH + Na + NH4+ H2O] + ion peak, m / z = 415.20 belongs to [M / 2 - 2H + 3Na + CH3O + NH4+ H2O] + ion peak.

[0034] Example 5: Inhibition of the binuclear oxovanadium (IV) complex on α - amylase activity 50 μL of sample solution with different concentrations (0.1, 0.3, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 mM) and 50 μL of 2 U / mL α - amylase solution were added to a 96-well plate, mixed, incubated at 37°C for 15 min, 100 μL of 1 mg / mL starch solution was added, and then incubated for another 30 min, immediately 100 μL of DNS (3,5-dinitrosalicylic acid) was added, reacted in a boiling water bath for 5 min, and then cooled, the absorbance value was determined at 540 nm wavelength, acarbose was used as a positive control, each group was repeated for 3 times, and the inhibition rate was calculated by using formula (1).

[0035] α - amylase inhibition rate = 1- (A3-A2) / (A1-A0) × 100% (1) A3: sample, α- A mixture of amylase and starch solution; A2: A mixture of sample, PBS (phosphate buffered solution), and starch solution; A1: Solvent, α - A mixture of amylase and starch solution; A0: A mixture of solvent, PBS and starch solution.

[0036] right α -IC50 of amylase inhibition 50 The results are shown in Table 3. Coordination pairs α -Amylase inhibitory activity is significantly enhanced compared to the corresponding ligands and metal salts, and IC50 is significantly higher. 50 The values ​​were all lower than those of the positive control, acarbose.

[0037] Table 3. IC50 values ​​of the binuclear vanadium(IV) complex of the present invention for the inhibition of α-amylase and α-glucosidase. 50 value Sample name IC 50 (μg / mL) Complex 88.14 ± 0.16 Vanadyl acetoacetonate ＞ 930 Ligand 501.76 ± 0.26 Acarbose 309.6 ± 0.23 Example 6: Binuclear vanadium(IV) complex pair α -Inhibition of glucosidase activity Add 25 µL of sample solutions of different concentrations (0.1, 0.3, 0.5, 0.7, 1.0, 1.2, 1.5 mM) and 25 µL of 1 U / mL [unclear] to a 96-well plate. α - Glucosidase solution, mix well, incubate at 37°C for 15 min, add 80 µL of 3 mM pNPG (p-nitrobenzene- α After reacting with a carbose solution for 25 min, 80 µL of 0.1 M Na2CO3 solution was added to terminate the reaction. The absorbance at 405 nm was measured, with acarbose as a positive control. Each group was repeated 3 times, and the inhibition rate was calculated using formula (2).

[0038] α - Glucosidase inhibition rate = 1 - (A3 - A2) / (A1 - A0) × 100% (2) A3: Sample α A1: A mixture of glucosidase and pNPG; A2: A mixture of sample, PBS, and pNPG solution; A1: Solvent, α - A mixture of glucosidase and pNPG solution; A0: A mixture of solvent, PBS and pNPG solution.

[0039] right α IC50 of glucosidase inhibition 50 The results are shown in Table 4. Coordination pairs α - The inhibitory capacity for glucosidase is significantly enhanced compared to the corresponding ligands and metal salts, but IC50 is still significantly reduced. 50 The value is greater than that of the positive control acarbose.

[0040] Table 4. Binuclear vanadium(IV) complexes of the present invention α -Amylase and α -ICP-glucosidase inhibition 50 value Sample name IC 50 (μg / mL) Complex 311.88 ± 0.17 Vanadyl acetoacetonate ＞ 400 Ligand ＞ 390 Acarbose 225.75 ± 0.33 Example 7: Binuclear vanadium(IV) complex pair α -Amylase and α -Inhibition kinetics of glucosidase activity (1) α -Amylase activity inhibition kinetics: α A mixture of 50 μL amylase solution (2 U / mL) and 50 μL of complex solutions at different concentrations (0, 0.5, 1.0, 1.5 mmol / L) was incubated at 37 °C for 10 min. 100 μL of starch solutions at different concentrations (1.0, 2.0, 2.5, 4.0, 5.0 mg / mL) was added to initiate the reaction. At 0, 10, 20, and 30 min of reaction time, 100 μL of DNS was added, and the mixture was incubated in a boiling water bath for 5 min. The absorbance at 540 nm was measured. The data were analyzed using the Lineweaver-Burk double reciprocal equation to determine the complex pair. α - Types of amylase inhibition.

[0041] (3) According to the Dixon equation, the competitive inhibition equation can be expressed in the following form: (4) The mixed suppression equation can be written as: (5) in, v The initial reaction rate, V max is the maximum reaction rate. K m It is the Michaelis constant, [ S [] represents the substrate concentration, and i represents the inhibitor concentration. K ic It represents the competitive inhibition constant. K iu This is a non-competitive inhibition constant.

[0042] When calculating the non-competitive inhibition constant, equation (5) can be transformed into the Cornish-Bowden equation, which takes the following form: (6) According to 1 / v From the i-curve, we can obtain K ic= -i, according to [S] v and i curves, it can be concluded that K iu = -i. (2) α - Glucosidase activity inhibition kinetics: α - Glucosidase (25 μL, 1 U / mL) and different concentrations (0, 0.5, 1.0, 1.5 mmol / L) of complex solution were mixed, and after incubation at 37°C for 10 min, 80 μL of pNPG solution with concentrations of 2.0, 2.5, 4.0, and 5.0 mg / mL was added, respectively, and after 0, 5, 10, and 15 min of reaction, 80 μL of 0.1 M Na2CO3 solution was added to terminate the reaction. The absorbance value at 405 nm was measured, and the data were processed according to the above method to determine the inhibition type of the complex on α - glucosidase.

[0043] As shown in Figure 5 and Figure 6 , through Lineweaver-Burk curve analysis, the complex intersects at a point in the third quadrant, indicating that its inhibition mechanism does not belong to typical competitive or non-competitive inhibition. Again, through Dixon and Cornish-Bowden curve analysis, the Dixon and Cornish-Bowden curves of the complex both show single-point intersection, proving that the inhibition type of the complex on α - amylase and α - glucosidase is mixed-type inhibition. By calculating the corresponding K ic (competitive inhibition constant) and K iu (non-competitive inhibition constant) values of the complex (as shown in Table 5), the results show that in the inhibition mechanism of α - amylase, the K ic value of the complex is greater than the K iu value, indicating that this mixed-type inhibitor mainly exhibits non-competitive inhibition characteristics; in the inhibition mechanism of α - glucosidase, the K ic value of the complex is less than the K iu value, indicating that this mixed-type inhibitor mainly exhibits competitive inhibition characteristics.

[0044] Table 5 Inhibition type K ic (mmol / L) ​ K iu (mmol / L)]]> ​ 1 / K ic (L / mmol) 1 / K iu (L / mmol) Amylase Mixed inhibition 1.04±0.23c 0.54±0.06c 0.96±0.29b 1.87±0.13b Glucosidase Mixed inhibition 0.75±0.09b 1.69±0.18b 1.37±1.70c 0.60±0.06c Example 8: Dinuclear oxovanadium (IV) complex and α - amylase and αMolecular docking of glucosidase Molecular docking technique is an effective method to visualize the interaction of small molecule ligand with macromolecular receptor, to determine the possible binding site and spatial conformation of ligand, so the present experiment studies the binding site of the complex with amylase and glucosidase by using molecular docking technique. α - Amylase and α - Glucosidase. The molecular docking results of the complex with α - Amylase and α - Glucosidase (as shown in Figure 7 and Figure 8 ), the binding energy of the complex with α - Amylase is -7.81 Kcal / mol, and the binding energy of the complex with α - Glucosidase is -6.01 Kcal / mol, indicating that the complex can form a complex with α - Amylase and α - Glucosidase. From Figure 7 and Figure 8 , it can be seen that the specific docking position and binding mode of the complex with α - Amylase and α - Glucosidase, the complex binds with amino acid residues in α - Amylase through hydrogen bond, hydrophobic force, salt bridge and GLN63, ARG195, ASP197, HIS305, TRP59, TYR62, LEU165, GLU233, ASP300, etc. α - Glucosidase through hydrogen bond, hydrophobic force and LYS373, ASN489, TYR566, LYS568, PHE563, LYS568, etc. Through these weak interactions, the complex forms a new complex with the enzyme, thereby inhibiting the competitive binding of the active site with the substrate, or affecting the structure of some allosteric sites of the enzyme by forming a complex, thereby changing the spatial structure of the enzyme and affecting its activity, so the weak interaction of the complex with amino acid residues has important influence on its inhibitory activity.

[0045] Example 9: Effect of binuclear oxovanadium (IV) complex on the proliferation of normal liver cells 7702 As shown in Figure 9 , from these data, it can be seen that after 48 h, the cell survival rate decreases from about 97% to 70% when the complex concentration increases from 1 μM to 50 μM, showing low toxicity in this concentration range.

[0046] The above only serves to better explain the embodiments of the present application, and is not a limitation thereof, any modification or equivalent replacement without departing from the spirit and scope of the present application shall fall within the scope of the present application.

Claims

1. A binuclear vanadium(IV) complex, characterized in that, The structural formula of the binuclear vanadium(IV) complex is: [V2(μ-O)2O2(H2L)2], where H2L is 2-hydroxy-N'-[1-(pyrazin-2-yl)ethylidene]benzoylhydrazine, with the following structural formula: 。 2. The binuclear vanadium(IV) complex according to claim 1, characterized in that, The aforementioned binuclear vanadium(IV) complex crystal is a monoclinic crystal system. P2 1 / n Space group, cell parameters are: a = 11.834 (4) Å, b = 8.000 (3) Å, c = 14.389 (5) Å, α = 90°, β = 98.528 (13)°, γ = 90°.

3. A method for preparing the binuclear vanadium(IV) complex as described in claim 1 or 2, characterized in that, The process includes the following steps: adding acetylvanadium oxyhydrazine and 2-hydroxy-N'-(1-(pyrazin-2-yl)ethylene)benzoylhydrazine to an alcohol solvent, and reacting by heating to obtain the binuclear vanadium(IV) complex.

4. The method for preparing the binuclear vanadium(IV) complex according to claim 3, characterized in that, The molar ratio of the acetylvanadium to 2-hydroxy-N'-(1-(pyrazin-2-yl)ethylidene)benzoylhydrazine is 1:

1.

5. The method for preparing the binuclear vanadium(IV) complex according to claim 3, characterized in that, The alcohol solvent is at least one of methanol, ethanol, a mixture of methanol and water, or a mixture of ethanol and water.

6. The method for preparing the binuclear vanadium(IV) complex according to claim 3, characterized in that, The heating reaction was carried out at a temperature of 40°C for 10 hours.

7. An α-amylase inhibitor, characterized in that, It comprises the binuclear vanadium(IV) complex as described in claim 1 or 2.

8. An α-glucosidase inhibitor, characterized in that, It comprises the binuclear vanadium(IV) complex as described in claim 1 or 2.

9. The use of the binuclear vanadium(IV) complex of claim 1 or 2, or the α-amylase inhibitor of claim 7, or the α-glucosidase inhibitor of claim 8 in the preparation of a medicament for the treatment of diabetes.

10. The application according to claim 9, characterized in that, The diabetes mentioned is type II diabetes.

Images