CN111116452B

CN111116452B - Indolone derivatives with alpha-glucosidase inhibitory activity as well as preparation method and application thereof

Info

Publication number: CN111116452B
Application number: CN202010030849.9A
Authority: CN
Inventors: 徐学涛; 陈洁; 邓旭阳; 柯俊杰; 陈思奇; 刘自强; 骆东; 刘子健; 贾振升; 马于然; 李冬利; 马爱军; 吴盼盼; 张焜
Original assignee: International Healthcare Innovation Institute (jiangmen); Wuyi University
Current assignee: International Healthcare Innovation Institute (jiangmen); Wuyi University
Priority date: 2020-01-13
Filing date: 2020-01-13
Publication date: 2022-05-03
Anticipated expiration: 2040-01-13
Also published as: CN111116452A

Abstract

The invention discloses indolone derivatives with alpha-glucosidase inhibitory activity, a preparation method and application thereof, wherein the indolone derivatives have a structure shown in a formula I:

wherein R is substituted or unsubstituted aryl or heteroaryl. The indolone derivatives can be used for preparing compositions, medicaments and health products for preventing and/or treating diabetes. The invention designs and synthesizes a series of 5-fluoroindolone compounds by taking a 5-fluoroindolone structure as a mother nucleus through a chemical synthesis method; meanwhile, through deep discussion on the related biological activity and structure-activity relationship, a solid theoretical basis is provided for finding high-efficiency and low-toxicity alpha-glucose protease inhibitor medicine lead compounds.

Description

Indolone derivatives with alpha-glucosidase inhibitory activity as well as preparation method and application thereof

Technical Field

The invention relates to the field of medicinal chemistry, in particular to indolone derivatives with alpha-glucosidase inhibitory activity and a preparation method and application thereof.

Background

The global diabetes map (9 th edition) newly released by the international diabetes union (IDF) shows that the global population of diabetics is increasing, the global average growth rate is 51%, 4.63 hundred million diabetics currently exist, and 7 hundred million diabetics globally according to the growth trend to 2045 years. The number of diabetics is the first rank in China, the total number of diabetics is about 1.164 hundred million, China is the country with the most number of elderly diabetics, and currently, the number of diabetics over 65 years in China reaches 3550 ten thousands, which is predicted to increase to 5430 thousands by 2030 and more likely to increase to 7810 thousands by 2045 years.

Diabetes mellitus is a chronic metabolic disorder in which blood glucose levels are elevated due to insufficient insulin secretion or damage to islet beta cells. Diabetes causes prolonged excessive blood glucose levels in patients and may cause a range of complications, for example, an increase in blood glucose levels may lead to many microvascular and macrovascular complications. Wherein, the microvascular complications comprise retinopathy, cataract, nephropathy and neuropathy, while the macrovascular complications comprise stroke, cardiovascular diseases, coronary artery diseases, cerebrovascular diseases and diabetic feet, and amputation can be caused when the diabetic feet are serious. The hypoglycemic drugs clinically applied at present mainly comprise sulfonylureas, biguanides, alpha-glucosidase inhibitors, thiazolidinediones, non-sulfonylureas insulin secretagogues and the like.

The glucosidase inhibitor is an oral hypoglycemic medicament which can reduce the digestion rate of carbohydrates and inhibit postprandial hyperglycemia so as to treat diabetes. The action mechanism is as follows: alpha-glucosidase inhibitors interfere with enzymatic actions in the brush border of the small intestine and may slow the release of D-glucose from oligosaccharides and disaccharides, which in turn leads to a decrease in postprandial plasma glucose levels and delayed glucose absorption. Alpha-glucosidases are a family of enzymes located on the brush border surface of small intestine cells, carbohydrate hydrolases that specifically hydrolyze 1, 4-alpha-glucosidic bonds to release alpha-D-glucose. Alpha-glucosidase plays an important role in the overall systemic circulation of mammals, and inhibitors of this enzyme play an important role in the treatment of diabetes on this basis. The natural product resources of China are rich, the alpha-glucosidase inhibitor prepared by taking the natural product as a parent synthesis derivative has low cost, rich sources, challenge and development value, and becomes the main direction of researchers in various countries.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides an indolone derivative with alpha-glucosidase inhibition activity.

The invention also provides a preparation method of the indolone derivatives.

The invention also provides application of the indolone derivatives.

The invention also provides a medicament for preventing and/or treating diabetes.

The invention also provides a medicinal composition for preventing and/or treating diabetes.

An indolone derivative according to an embodiment of the first aspect of the invention has the structure shown in formula I:

wherein R represents an optionally substituted aromatic or heteroaromatic group.

According to a first aspect of the inventionThe indolone derivatives of the examples have at least the following beneficial effects: the invention designs and synthesizes a series of 5-fluoroindolone compounds by taking a 5-fluoroindolone structure as a mother nucleus, wherein

derivatives

3d, 3f and 3i show the strongest alpha-glucosidase inhibition effect, IC₅₀The values are respectively 56.87 mu M, 49.89 mu M and 35.83 mu M, are 10-15 times of acarbose, and can be used as an alpha-glucosidase inhibitor for treating or preventing diabetes.

According to some embodiments of the invention, R represents optionally substituted phenyl, pyridyl, furyl or thienyl, wherein the substituted substituent is selected from halogen, C₁-C₃Alkyl radical, C₁-C₃Alkoxy groups, thioether groups, and trifluoromethyl groups.

According to some embodiments of the invention, R is selected from the group consisting of:

a method of making an embodiment according to the second aspect of the invention comprises the steps of: 5-fluoro-2-oxindole is taken as an initial raw material and is subjected to condensation reaction with an aromatic aldehyde compound in the presence of alkali to obtain the compound; the reaction equation is as follows:

the preparation method provided by the embodiment of the second aspect of the invention has at least the following beneficial effects: the preparation method is efficient and convenient, and can be used for preparing a series of indolone derivatives taking a 5-fluoroindolone structure as a parent nucleus.

According to some embodiments of the invention, the base is an inorganic base; preferably, the base is at least one of KOH, NaOH, and LiOH; further preferably, the base is KOH.

According to the application of the embodiment of the third aspect of the invention, the indolone derivatives are applied to the preparation of products for preventing and/or treating diabetes.

The application of the embodiment of the third aspect of the invention has at least the following beneficial effects: the invention applies a computer-aided drug design method, combines an in-vitro pharmacological activity evaluation experiment, takes the alpha-glucosidase as a target protein, deeply discusses the related biological activity and structure-activity relationship, and provides a solid theoretical basis for finding the high-efficiency and low-toxicity alpha-glucosidase inhibitor drug lead compound.

According to some embodiments of the invention, the product comprises at least one of a pharmaceutical and a nutraceutical.

The medicament according to the fourth aspect of the embodiment of the invention comprises the indolone derivative and/or pharmaceutically acceptable salt thereof.

According to some embodiments of the invention, the pharmaceutical dosage form is a tablet, a capsule, an oral liquid, or an injection.

According to a fifth aspect of the present invention, an embodiment of the pharmaceutical composition comprises the indolone derivative and/or the pharmaceutically acceptable salt thereof.

As used herein, "optionally substituted" means that the group may or may not be further substituted with one or more groups selected from: alkyl, alkenyl, alkynyl, aryl, halo (halo), haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, amino, alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino, phenylamino, diphenylamino, benzylamino, dibenzylamino, hydrazino, acyl, acylamino, diamido, acyloxy, heterocyclyl, heterocyclyloxy, heterocyclylamino, haloheterocyclyl, carboxyl ester, carboxyl, carboxamido, mercapto, alkylthio, benzylthio, acylthio (acylthio), and phosphorus-containing groups.

As used herein, the term "heteroaryl" represents an aromatic group containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Heteroaryl groups within the scope of this definition include, but are not limited to: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a graph showing the half inhibitory concentration of indolone derivatives as alpha-glucosidase inhibitors in vitro, according to example 2 of the present invention; (a) median inhibitory concentration profile at 3 d; (b) a plot of half maximal inhibitory concentration of 3 f; (c) a plot of half maximal inhibitory concentration of 3 i;

FIG. 2 is a graph showing the in vitro kinetics of indolone derivatives (3d, 3f and 3i) as α -glucosidase inhibitors against α -glucosidase in example 3 of the present invention; (a) enzyme kinetic profile at 3 d; (b) enzyme kinetic profile at 3 f; (c) enzyme kinetic profile at 3 i;

FIG. 3 is a substrate kinetic diagram of indolone derivative 3d as an α -glucosidase inhibitor in vitro against α -glucosidase in example 4 of the present invention; (a) a substrate kinetic map is made for a double reciprocal mapping method; (b) is a slope diagram; (c) is an intercept map;

FIG. 4 is a substrate kinetic diagram of indolone derivatives 3f as alpha-glucosidase inhibitors in vitro in example 4 of the present invention for alpha-glucosidase; (a) a substrate kinetic map is made for a double reciprocal mapping method; (b) is a slope diagram; (c) is an intercept map;

FIG. 5 is a substrate kinetic diagram of indolone derivatives 3i as alpha-glucosidase inhibitors in vitro in example 4 of the present invention for alpha-glucosidase; (a) a substrate kinetic map is made for a double reciprocal mapping method; (b) is a slope diagram; (c) is an intercept map;

FIG. 6 is a molecular docking diagram providing the interaction rule of

compounds

3d, 3f, 3i with α -glucosidase in example 5 of the present invention.

Detailed Description

In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.

Example 1: preparation of indolone derivatives

Indole is natural product of jasmine oil or narcissus oil, and has blood sugar lowering activity. Indolones are important derivatives of indole, and are the lead structures of many natural alkaloids. Therefore, the invention prepares a series of indolone derivatives from the mother nucleus structure of indolone to search for the lead compound with hypoglycemic activity.

5-fluoro-2-oxindole is used as an initial raw material and is subjected to condensation reaction with aromatic aldehyde compounds (2 a-2 v) in the presence of KOH to directly obtain target products (3 a-3 v), and the chemical reaction formula is shown as follows:

the specific operation steps are as follows:

to 5-fluoroindol-2-one (1mmol) and

KOH (6mmol) was added to a 10mL solution of aldehydes in anhydrous ethanol, and the mixture was stirred at room temperature and checked by TLC until the reaction was complete. After adjusting the pH to 2-3 and evaporating the ethanol, the mixture was extracted with ethyl acetate. With saturated NaHCO₃And the ethyl acetate layer was washed with brine, and then concentrated to give a crude product. Recrystallizing the obtained crude product with ethanol to respectively obtain the compounds

The yield of 3a was 65%.

Preparation of the resulting product

Comprises the following steps:

wherein R of 3a to 3v is shown in Table 1 below:

TABLE 1 structural formula of R group

The structures of 3a to 3v were characterized by NMR, MS and melting point, as follows are the properties, yield, nuclear magnetic and mass spectrometry results for each compound:

(Z)-5-fluoro-3-(2-fluorobenzylidene)indolin-2-one(3a).Orange-yellow crystal；yield 65.0％；mp:228.3-230.2℃；¹H NMR(500MHz,DMSO-d₆)δ10.73(s,1H),7.77(td,J＝7.7,1.7Hz,1H),7.65-7.55(m,2H),7.47-7.36(m,2H),7.13(td,J＝9.0,2.6Hz,1H),6.94(dd,J＝9.0,2.6Hz,1H),6.88(dd,J＝8.5,4.6Hz,1H)；HRMS(ESI)calcd for C₁₅H₉F₂NO[M-H]^-:m/z＝256.24,found 255.85。

(Z)-5-fluoro-3-(3-fluorobenzylidene)indolin-2-one(3b).Orange-yellow crystal；yield 41.2％；mp:191.4-192.2℃；¹H NMR(500MHz,Chloroform-d)δ7.81(s,1H),7.49(td,J＝8.0,5.7Hz,1H),7.42(dp,J＝7.6,0.9Hz,1H),7.30(ddd,J＝8.9,6.8,2.4Hz,2H),7.20-7.15(m,1H),6.97(td,J＝8.7,2.5Hz,1H),6.82(dd,J＝8.6,4.4Hz,1H)；HRMS(ESI)calcd for C₁₅H₉F₂NO[M-H]^-:m/z＝256.24,found 256.09。

(Z)-5-fluoro-3-(4-fluorobenzylidene)indolin-2-one(3c).Orange-yellow crystal；yield 55.7％；mp:217.0-219.6℃；¹H NMR(500MHz,Chloroform-d)δ8.10(s,1H),7.82(s,1H),7.64(dd,J＝8.5,5.4Hz,2H),7.32(dd,J＝9.1,2.5Hz,1H),7.20(t,J＝8.4Hz,2H),6.96(td,J＝8.8,2.6Hz,1H),6.83(dd,J＝8.5,4.4Hz,1H)；HRMS(ESI)calcd for C₁₅H₉F₂NO[M-H]^-:m/z＝256.24,found 256.17。

(Z)-3-(2-chlorobenzylidene)-5-fluoroindolin-2-one(3d).Orange-yellow crystal；yield 82.4％；mp:219.7-221.9℃；¹H NMR(500MHz,Chloroform-d)δ8.12(s,1H),7.92(s,1H),7.68(dd,J＝7.5,1.8Hz,1H),7.54(dd,J＝7.9,1.4Hz,1H),7.40(dtd,J＝20.5,7.5,1.6Hz,2H),7.04(dd,J＝8.9,2.6Hz,1H),6.94(td,J＝8.8,2.6Hz,1H),6.82(dd,J＝8.5,4.3Hz,1H)；HRMS(ESI)calcd for C₁₅H₉ClFNO[M-H]^-:m/z＝272.69,found 271.9。

(Z)-3-(3-chlorobenzylidene)-5-fluoroindolin-2-one(3e).Orange-yellow crystal；yield 47.8％；mp:237.4-238.9℃；¹H NMR(500MHz,Chloroform-d)δ7.97(s,1H),7.79(s,1H),7.58(dd,J＝2.1,1.1Hz,1H),7.54-7.50(m,1H),7.46-7.43(m,2H),7.25(d,J＝2.5Hz,1H),6.97(td,J＝8.7,2.6Hz,1H),6.82(dd,J＝8.5,4.4Hz,1H)；HRMS(ESI)calcd for C₁₅H₉ClFNO[M-H]^-:m/z＝272.69,found 272.10。

(Z)-3-(4-chlorobenzylidene)-5-fluoroindolin-2-one(3f).Orange-yellow crystal；yield 44.2％；mp:200.6-202.5℃；¹H NMR(500MHz,Chloroform-d)δ7.80(s,1H),7.58(d,J＝8.3Hz,2H),7.51-7.45(m,2H),7.30(dd,J＝8.9,2.5Hz,1H),6.96(td,J＝8.7,2.6Hz,1H),6.82(dd,J＝8.5,4.4Hz,1H)；HRMS(ESI)calcd for C₁₅H₉ClFNO[M-H]^-:m/z＝272.69,found 272.39。

(Z)-3-(2-bromobenzylidene)-5-fluoroindolin-2-one(3g).Orange-yellow crystal；yield 69.7％；mp:194.9-197.1℃；¹H NMR(500MHz,Chloroform-d)δ7.90-7.82(m,1H),7.73(dd,J＝8.1,1.1Hz,1H),7.66(dt,J＝7.7,1.9Hz,1H),7.44(td,J＝7.4,1.1Hz,1H),7.34(td,J＝7.7,1.6Hz,1H),7.00(dd,J＝8.9,2.6Hz,1H),6.94(td,J＝8.7,2.6Hz,1H)；HRMS(ESI)calcd for C₁₅H₉BrFNO[M+Na]⁺:m/z＝340.15,found 340.59。

(Z)-3-(3-bromobenzylidene)-5-fluoroindolin-2-one(3h).Orange-yellow crystal；yield 70.4％；mp:222.6-223.8℃；¹H NMR(500MHz,Chloroform-d)δ8.31(s,1H),7.86-7.67(m,2H),7.66-7.48(m,2H),7.38(t,J＝7.9Hz,1H),7.24(d,J＝2.6Hz,1H),6.97(td,J＝8.7,2.6Hz,1H),6.84(dd,J＝8.5,4.4Hz,1H)；HRMS(ESI)calcd for C₁₅H₉BrFNO[M+Na]⁺:m/z＝357.15,found 357.97。

(Z)-3-(4-bromobenzylidene)-5-fluoroindolin-2-one(3i).Orange-yellow crystal；yield 65.7％；mp:249.3-251.6℃；¹H NMR(500MHz,Chloroform-d)δ7.77(s,1H),7.67-7.61(m,2H),7.51(d,J＝8.4Hz,2H),7.30(dd,J＝9.0,2.6Hz,1H),6.96(td,J＝8.7,2.6Hz,1H),6.81(dd,J＝8.5,4.4Hz,1H)；HRMS(ESI)calcd for C₁₅H₉BrFNO[M+Na]⁺:m/z＝341.15,found 340.97。

(Z)-5-fluoro-3-(2-methoxybenzylidene)indolin-2-one(3j).Orange-yellow crystal；yield 52.7％；mp:227.1-227.8℃；¹H NMR(500MHz,Chloroform-d)δ8.02(s,1H),7.97(s,1H),7.67(dd,J＝7.7,1.7Hz,1H),7.49-7.42(m,1H),7.28(dd,J＝9.2,2.7Hz,1H),7.06(td,J＝7.5,1.0Hz,1H),7.00(dd,J＝8.3,1.0Hz,1H),6.91(td,J＝8.8,2.6Hz,1H),6.80(dd,J＝8.5,4.4Hz,1H),3.89(s,3H)；HRMS(ESI)calcd for C₁₆H₁₂FNO₂[M-H]^-:m/z＝268.28,found 268.19。

(Z)-5-fluoro-3-(3-methoxybenzylidene)indolin-2-one(3k).Orange-yellow crystal；yield 73.3％；mp:200.1-202.1℃；¹H NMR(500MHz,Chloroform-d)δ7.86(s,1H),7.45-7.38(m,2H),7.23(d,J＝7.5Hz,1H),7.14(t,J＝2.0Hz,1H),7.04-6.90(m,2H),6.82(dd,J＝8.6,4.4Hz,1H),3.86(s,3H)；HRMS(ESI)calcd for C₁₆H₁₂FNO₂[M-H]^-:m/z＝268.28,found 268.01。

(Z)-5-fluoro-3-(4-methoxybenzylidene)indolin-2-one(3l).Orange-yellow crystal；yield 42.8％；mp:358.8-359.9℃；¹H NMR(500MHz,Chloroform-d)δ7.84(s,1H),7.68-7.62(m,2H),7.49(dd,J＝9.3,2.6Hz,1H),7.04-7.00(m,2H),6.93(dd,J＝8.8,2.6Hz,1H),6.83(dt,J＝9.0,3.6Hz,1H),3.91(s,3H)；HRMS(ESI)calcd for C₁₆H₁₂FNO₂[M-H]^-:m/z＝268.28,found 268.11。

(Z)-5-fluoro-3-(2-(trifluoromethyl)benzylidene)indolin-2-one(3m).Orange-yellow crystal；yield 53.1％；mp:235.2-236.4℃；¹H NMR(500MHz,Chloroform-d)δ8.33-8.22(m,1H),8.01(q,J＝2.5Hz,1H),7.83(d,J＝7.8Hz,1H),7.70-7.65(m,2H),7.63-7.55(m,1H),6.93(td,J＝8.8,2.6Hz,1H),6.83(dd,J＝8.5,4.3Hz,1H),6.66(dd,J＝8.7,2.6Hz,1H)；HRMS(ESI)calcd for C₁₆H₉F₄NO[M-H]^-:m/z＝306.25,found 305.91。

(Z)-5-fluoro-3-(3-(trifluoromethyl)benzylidene)indolin-2-one(3n).Orange-yellow crystal；yield 42.9％；mp:199.7-200.5℃；¹H NMR(500MHz,Chloroform-d)δ7.87(s,1H),7.85(s,1H),7.81(d,J＝7.7Hz,1H),7.73(d,J＝7.8Hz,1H),7.64(t,J＝7.8Hz,1H),7.18(dd,J＝8.9,2.6Hz,1H),6.97(td,J＝8.7,2.6Hz,1H),6.83(dd,J＝8.6,4.3Hz,1H)；HRMS(ESI)calcd for C₁₆H₉F₄NO[M-H]^-:m/z＝306.25,found 306.01。

(Z)-3-(2,4-difluorobenzylidene)-5-fluoroindolin-2-one(3o).Orange-yellow crystal；yield 55.3％；mp:221.1-224.3℃；¹H NMR(500MHz,Chloroform-d)δ8.31-8.21(s,1H),8.01(s,1H),7.83(d,J＝7.8Hz,1H),7.68-7.65(m,2H),7.62-7.58(m,1H),6.93(td,J＝8.8,2.6Hz,1H),6.83(dd,J＝8.5,4.3Hz,1H),6.66(dd,J＝8.7,2.6Hz,1H)；HRMS(ESI)calcd for C₁₆H₉F₄NO[M+H]⁺:m/z＝276.23,found 278.01。

(Z)-3-(3,4-difluorobenzylidene)-5-fluoroindolin-2-one(3p).Orange-yellow crystal；yield 44.6％；mp:189.2-190.2℃；¹H NMR(500MHz,Chloroform-d)δ8.99(dd,J＝10.1,2.7Hz,1H),8.91(ddd,J＝4.6,2.0,0.8Hz,1H),7.83(td,J＝7.7,1.9Hz,1H),7.73(s,1H),7.63(dd,J＝7.7,1.1Hz,1H),7.36(ddd,J＝7.7,4.7,1.1Hz,1H),7.01(td,J＝8.6,2.7Hz,1H),6.80(dd,J＝8.5,4.4Hz,1H)；HRMS(ESI)calcd for C₁₅H₈F₃NO[M-H]^-:m/z＝274.23,found 274.02。

(Z)-3-benzylidene-5-fluoroindolin-2-one(3q).Orange-yellow crystal；yield 76.3％；mp:198.9-199.7℃；¹H NMR(500MHz,Chloroform-d)δ8.93(s,1H),7.90(s,1H),7.67-7.62(m,2H),7.53-7.46(m,3H),7.36(dd,J＝9.1,2.6Hz,1H),6.94(td,J＝8.7,2.5Hz,1H),6.86(dd,J＝8.5,4.5Hz,1H)；HRMS(ESI)calcd for C₁₅H₈F₃NO[M-H]^-:m/z＝238.25,found 237.99。

(Z)-5-fluoro-3-(4-(methylthio)benzylidene)indolin-2-one(3r).Orange-yellow crystal；yield 41.8％；mp:246.1-247.1℃；¹H NMR(500MHz,Chloroform-d)δ8.13(s,1H),7.81(s,1H),7.61-7.55(m,2H),7.44(dd,J＝9.0,2.6Hz,1H),7.36-7.31(m,2H),6.94(td,J＝8.7,2.5Hz,1H),6.82(dd,J＝8.5,4.4Hz,1H),2.56(s,3H)；HRMS(ESI)calcd for C₁₆H₁₂FNOS[M-H]^-:m/z＝284.34,found 284.13。

(Z)-5-fluoro-3-(furan-2-ylmethylene)indolin-2-one(3s).Orange-yellow crystal；yield 72.5％；mp:237.9-238.8℃；¹H NMR(500MHz,Chloroform-d)δ7.76(s,1H),7.41(s,1H),7.24-7.16(m,2H),6.88(td,J＝8.8,2.5Hz,1H),6.82(dd,J＝8.5,4.2Hz,2H),6.42(dt,J＝4.0,2.1Hz,1H)；HRMS(ESI)calcd for C₁₆H₁₂FNOS[M-H]^-:m/z＝228.21,found 227.95。

(Z)-5-fluoro-3-(thiophen-2-ylmethylene)indolin-2-one(3t).Orange-yellow crystal；yield 44.6％；mp:219.0-223.4℃；¹H NMR(500MHz,Chloroform-d)δ8.01(dd,J＝9.4,2.5Hz,1H),7.97(s,1H),7.85(s,1H),7.65(dd,J＝23.4,4.4Hz,2H),7.23(dd,J＝5.1,3.7Hz,1H),6.99(td,J＝8.7,2.5Hz,1H),6.84(dd,J＝8.5,4.5Hz,1H)；HRMS(ESI)calcd for C₁₆H₁₂FNOS[M-H]^-:m/z＝244.27,found 244.14。

(Z)-3-((1H-pyrrol-2-yl)methylene)-5-fluoroindolin-2-one(3u).Orange-yellow crystal；yield 54.4％；mp:198.9-201.6℃；¹H NMR(500MHz,Chloroform-d)δ13.30(s,1H),7.76(s,1H),7.41(s,1H),7.22(s,1H),7.19(dd,J＝8.7,2.5Hz,1H),6.88(td,J＝8.8,2.5Hz,1H),6.82(d,J＝4.2Hz,2H),6.42(dt,J＝4.0,2.1Hz,1H)；HRMS(ESI)calcd for C₁₃H₉FN₂O[M-H]^-:m/z＝227.23,found 227.02。

(Z)-5-fluoro-3-(pyridin-2-ylmethylene)indolin-2-one(3v).Orange-yellow crystal；yield 47.9％；mp:248.5-251.2℃；¹H NMR(500MHz,Chloroform-d)δ8.99(dd,J＝10.1,2.7Hz,1H),8.91(ddd,J＝4.6,2.0,0.8Hz,1H),7.83(td,J＝7.7,1.9Hz,1H),7.73(s,1H),7.63(dd,J＝7.7,1.1Hz,1H),7.36(ddd,J＝7.7,4.7,1.1Hz,1H),7.01(td,J＝8.6,2.7Hz,1H),6.80(dd,J＝8.5,4.4Hz,1H)；HRMS(ESI)calcd for C₁₃H₉FN₂O[M-H]^-:m/z＝239.24,found 238.93。

the method can successfully prepare a series of indolone derivatives taking the 5-fluoroindolone structure as the parent nucleus, and has the advantages of high yield and simple operation.

Example 2: test for alpha-glucosidase inhibitory Activity of indolone derivatives

1. Preparation of reagent and standard solution

(1)100mM phosphate buffer (PBS, pH 6.8): weighing a certain mass of potassium dihydrogen phosphate and disodium hydrogen phosphate, and dissolving with ultrapure water for dissolving and diluting the reagent.

(2) Preparing an alpha-glucosidase solution: adding a proper amount of 100mM PBS into the enzyme with the enzyme activity of 100U to prepare the working concentration of 0.05U/mL, and subpackaging and freezing.

(3) Preparing a substrate: accurately weighing a proper amount of 4-nitrophenyl-D-glucopyranoside (PNPG), adding 100mM PBS solution for dissolving, preparing a substrate working solution with the concentration of 0.25mM, uniformly mixing by vortex, and preparing freshly before each experiment.

The preparation of the test medicine comprises accurately weighing appropriate amount of the medicine to be tested, dissolving with DMSO to prepare 10mM stock solution, and storing at-20 deg.C in dark place. The samples were diluted with DMSO to different desired concentrations (0-200. mu.M) before the experiment, with DMSO content equal to 5%.

2. Experimental procedure

(1)10 μ L of alpha-glucosidase with a working concentration of 0.05U/mL, 130 μ L (pH 6.8) of phosphate buffer with a concentration of 100mM, 10 μ L of compounds with different concentrations (indolone derivatives 3a to 3v prepared in example 1) were sequentially added to a 96-well plate, 10 μ L of DMSO with a content of 5% was used in place of 10 μ L of compounds in the blank control group, acarbose was used as a positive control, 4 multiple wells were arranged in parallel in each group, and the enzyme reaction system was incubated on a microplate reader at 37 ℃ for 10 min.

(2) Subsequently, 50 μ L of substrate PNPG was added to the enzyme reaction system to start the enzyme reaction, the microplate was placed on a microplate reader and incubated at 37 ℃ for a further 15min, 3 times of incubation were equally divided, each time was read once at a wavelength of 405nm, and the reading was recorded as OD₁、OD₂、OD₃。

(3) The α -glucosidase inhibitory activity of the test compound was calculated according to the following formula:

inhibition ratio (%) [ (OD)₃-OD)-(OD₁-OD)]/OD₃-OD×100％

Wherein OD represents the absorbance value of the blank control, data processing: data were processed using MS Excel analysis and half maximal Inhibitory Concentration (IC) was calculated using Origin 9.1₅₀)，IC₅₀Represents the concentration of test compound required to inhibit the activity of alpha-glucosidase by 50% under the experimental conditions.

3. Analysis of results

The synthesized compound was evaluated for α -glucosidase inhibitory activity using in vitro enzymology experiments, and the results are shown in table 2:

TABLE 2 evaluation of in vitro inhibitory Activity of the screened Compounds with alpha-glucosidase

^aThe inhibition rate of the test compound is lower than 50% at 100 mu M;

^bvalues are mean ± S of the results of three independent experiments.

IC of positive control drug Acarbose (Acarbose) therein₅₀569.43 μ M. Three derivatives (3d, 3f and 3i) with better alpha-glucosidase inhibitory activity are screened out, the inhibition rates of the 3d, 3f and 3i are respectively 56.87 mu M, 49.89 mu M and 35.83 mu M, and the inhibition rates are 10-15 of acarboseAnd (4) doubling. The results show that the small molecule compounds show stronger binding affinity when interacting with alpha-glucosidase. Therefore, the indole skeleton structure plays an important role in better alpha-glucosidase inhibition activity of the compounds, and the

compounds

3d, 3f and 3i can be used as alpha-glucosidase inhibitors for treating or preventing diabetes.

Example 3 enzyme kinetics experiments

And (3) performing dynamic evaluation on the alpha-glucosidase inhibitory activity of the synthesized active compound by adopting an in-vitro enzyme kinetic experiment:

1. preparation of reagent and standard solution

(2) Preparing an alpha-glucosidase solution: adding 100U enzyme with enzyme activity into appropriate amount of 100mM PBS to obtain working concentrations of 0.0375U/mL, 0.05U/mL, 0.0625U/mL, and 0.075U/mL, and subpackaging and freezing.

The preparation of the test medicine comprises accurately weighing appropriate amount of the medicine to be tested, dissolving with DMSO to prepare 10mM stock solution, and storing at-20 deg.C in dark place. Before the experiment, the samples were diluted with DMSO to different required concentrations (0-200. mu.M), with a DMSO content equal to 5%.

2. Experimental procedure

(1) mu.L of alpha-glucosidase at a concentration of 0.0375U/mL, 0.05U/mL, 0.0625U/mL, 0.075U/mL, 130. mu.L (pH 6.8) of phosphate buffer at a concentration of 100mM, 10. mu.L of compounds at different concentrations (

indolone compounds

3d, 3f, and 3i prepared in example 1) were sequentially added to a 96-well plate, 10. mu.L of DMSO at a concentration of 5% was used in place of 10. mu.L of compound in the blank control group, acarbose was used as a positive control, 4 duplicate wells were placed in parallel in each group, and the enzyme reaction was incubated on a microplate reader at 37 ℃ for 10 min.

(2) Subsequently, 50 μ L of substrate PNPG with concentration of 0.25mM was added to the enzyme reaction system to start the enzyme reaction, the microplate was placed on a microplate reader and incubated at 37 ℃ for another 15min, 3 times of incubation were equally divided, each time was read at a wavelength of 405nm, and the reading was recorded as OD₁、OD₂、OD₃。

(3) Data processing: the data were analyzed and processed by MS Excel analysis, and the reaction rate of the enzyme reaction system was Δ OD/min.

3. Analysis of results

The results of the enzyme kinetic inhibition type evaluation experiments are shown in FIG. 2. As can be seen from FIG. 2, the inhibitor binds to the enzyme non-covalently to repress the activity of the enzyme, a reversible inhibition.

Example 4: substrate kinetics experiments

Performing alpha-glucosidase inhibition activity kinetic evaluation on the synthesized active compound by adopting an in vitro substrate kinetic experiment:

1. preparation of reagent and standard solution

(3) Preparing a substrate: an appropriate amount of 4-nitrophenyl-D-glucopyranoside (PNPG) was weighed out accurately, dissolved in 100mM PBS, and prepared into substrate working solutions (0.25mM, 0.5mM, 0.75mM, 1mM) in concentration, mixed by vortexing, and prepared freshly before each experiment.

2. Experimental procedure

(1) mu.L of alpha-glucosidase at a concentration of 0.5U/mL, 130. mu.L (pH 6.8) of phosphate buffer at a concentration of 100mM, and 10. mu.L of compounds at different concentrations (

indolone compounds

3d, 3f, and 3i prepared in example 1) were sequentially added to a 96-well plate, 10. mu.L of DMSO at a concentration of 5% was used in place of 10. mu.L of the compound in the blank control group, acarbose was used as a positive control, 4 multiple wells were provided in parallel for each group, and the enzyme reaction system was incubated on a microplate reader at 37 ℃ for 10 min.

(2) Subsequently, 50 μ L of substrate PNPG with different concentrations is added into the enzyme reaction system to start the enzyme reaction, the microplate is placed on an enzyme-linked immunosorbent assay (ELISA) instrument to continue incubation for 15min at 37 ℃, the time is averagely distributed for 3 times in the incubation process, the reading is carried out once at the wavelength of 405nm in each period of time, and the reading is recorded as OD₁、OD₂、OD₃。

(3) Data processing: the data were analyzed and processed using MS Excel, and the reaction rate of the enzyme reaction system was Δ OD/min.

3. Analysis of results

The results of the substrate kinetic inhibition type evaluation experiments are shown in fig. 3 to 5, fig. 3 to 5 correspond to the substrate kinetic profiles of

indolone derivatives

3d, 3f and 3i for α -glucosidase in vitro, respectively, in fig. 3 to 5, (a) is a substrate kinetic profile by the double reciprocal plot, (b) is a slope profile, and (c) is an intercept profile. As can be seen from fig. 3 to 5, the inhibitors obtained by screening were mixed inhibitors. Indicating that it can bind not only to alpha-glucosidase but also to the alpha-glucosidase-substrate complex. Meanwhile, as can be seen from fig. 3, the inhibition constants KI of the

compounds

3d, 3f and 3i are 14.96, 33.85 and 22.72, respectively.

Example 5: activity analysis of indolone derivatives

The interaction law of the

compounds

3d, 3f, 3i prepared in activity example 1 and alpha-glucosidase was analyzed by Molecular docking (Molecular docking):

in order to clarify the possible mutual binding mode and action site of the screened

inhibitors

3d, 3f and 3i and the alpha-glucosidase, a molecular docking method is adopted to investigate the interaction rule of the three and the alpha-glucosidase. Alpha-glucosidase consists of one subunit. In the docking experiment, the molecular docking result is analyzed, the conformation with the highest docking score of the compound and the alpha-glucosidase is determined according to the docking score, the binding free energy is the lowest, namely the optimal conformation, and the compound is further analyzed. Among them, as a result of molecular docking analysis, as shown in fig. 6,

inhibitors

3d, 3f and 3i showed similar binding patterns, all bound to the active pocket site of α -glucosidase (fig. 6 (a)), and the carbonyl groups of

compounds

3d, 3f and 3i formed hydrogen bonds with the amino acid sequence of GLN330 to increase the affinity with α -glucosidase (fig. 6 (b)). 3d, 3f and 3i with the active pocket. The outer active pocket is more lipophilic than the inner one (fig. 6 (c)). As seen in fig. 6 (d), fluorophenyl groups as lipophilic moieties of 3d, 3f and 3i are blocked in the lipophilic potential region, while pyrrole rings as hydrophilic moieties are close to the hydrophilic region. From this, it was demonstrated that the presence of an acting force such as a hydrogen bond or van der waals force formed between the small molecule inhibitor and α -glucosidase plays a very important role in stabilizing the conformation of the binding protein receptor-ligand complex.

In conclusion, the indolone derivatives 3 a-3 v provided by the invention are determined to have activity on alpha-glucosidase. The results show that most of the synthesized derivatives show significant inhibitory activity on alpha-glucosidase, especially compounds 3d, 3f and 3i show the strongest alpha-glucosidase inhibition, IC₅₀The values are respectively 56.87 mu M, 49.89 mu M and 35.83 mu M, are 10-15 times of acarbose, and can be used as an alpha-glucosidase inhibitor for treating or preventing diabetes.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

Claims

1. An indolone derivative having an α -glucosidase inhibitory activity, characterized in that: the indolone derivatives have a structure shown in a formula I:

wherein R is selected from the group consisting of:

2. the method for producing an indolone derivative according to claim 1, wherein: the method comprises the following steps: 5-fluoro-2-oxindole is taken as an initial raw material and is subjected to condensation reaction with an aromatic aldehyde compound in the presence of alkali to obtain the compound; the reaction equation is as follows:

wherein the base is an inorganic base.

3. The method of claim 2, wherein: the alkali is at least one of KOH, NaOH and LiOH.

4. The production method according to claim 3, characterized in that: the base is KOH.

5. The use of the indolone derivative of claim 1 in the preparation of a medicament for the prevention and/or treatment of diabetes.

6. A medicament for preventing and/or treating diabetes, which is characterized in that: comprising the indolone derivative according to claim 1 and/or a pharmaceutically acceptable salt thereof.

7. The medicament of claim 6, wherein: the dosage form of the medicine is tablets, capsules, oral liquid or injection.

8. A pharmaceutical composition for preventing and/or treating diabetes, characterized in that: comprising the indolone derivative according to claim 1 and/or a pharmaceutically acceptable salt thereof.