CN109608415B - Thiazole methanamide compound and synthesis and application thereof - Google Patents

Abstract

The present invention relates to novel thiazole carboxamides, wherein R1, R2, Z, X1, X2, X3, n and m have the meanings defined in the claims. The compound mainly has better inhibition effect on alpha-glucosidase inhibitors, and can be used for preventing or treating diseases related to alpha-glucosidase, especially diabetes. The invention further relates to processes for the preparation of such novel compounds, pharmaceutical compositions containing said compounds as active ingredient and the use of said compounds as medicaments.

Description

Thiazole methanamide compound and synthesis and application thereof

Technical Field

The present invention relates to thiazole carboxamides useful as pharmaceuticals. More particularly, it relates to diseases useful as alpha-glucosidase inhibitors, especially diabetes.

Background

Diabetes Mellitus (DM) is a group of endocrine-metabolic syndromes that are currently prevalent in the world, and its increasing tendency to develop makes it the third major non-infectious disease, second only to cardiovascular diseases and tumors. For a long time, in the treatment of diabetes, attention has been paid to the monitoring of fasting blood glucose rather than Postprandial blood glucose [18 ]. However, recent studies have found that postprandial hyperglycemia and subsequent diabetes often occur during the onset of diabetes [19 ]. For diabetic patients, especially type II patients, postprandial hyperglycemia far outweighs fasting hyperglycemia. Postprandial hyperglycemia is not only very likely to induce complications such as cardiovascular, brain, kidney, eye and nervous system diseases, but also can cause ketoacidosis coma and even threaten life [21 ]. Studies have shown that an independent risk factor for death from cardiovascular and cerebrovascular diseases is postprandial hyperglycemia whose mechanism of action is different from that of sustained hyperglycemia [22 ]. Maintaining postprandial blood glucose levels in the normal range is one of the important ways to prevent diabetes and its complications and reduce mortality. Blood glucose is mainly derived from sugars in food. Carbohydrates are hydrolyzed by alpha-glucosidase catalysis to generate monosaccharides which can be absorbed [23 ]. Therefore, alpha-glucosidase is a key enzyme for regulating blood sugar of food sources and also a target enzyme for regulating postprandial blood sugar [24], and alpha-glucosidase inhibitor (AGI) is also a symptomatic treatment drug for controlling postprandial blood sugar [25 ]. Slowing or inhibiting carbohydrate digestion and absorption is of great importance in regulating postprandial blood glucose in type II diabetes.

Alpha-glucosidase inhibitors (AGI) can reversibly or competitively inhibit the activity of alpha-glucosidase, thereby delaying the conversion of polysaccharides or disaccharides in the body to absorbable glucose and reducing the rate of postprandial blood glucose elevation [41 ]. In addition, normally no carbohydrate component enters the lower small intestine, but after taking the α -glucosidase inhibitor drugs, chyme such as carbohydrate, fat, protein, etc. in the intestine enters the distal ileum, which is the most abundant site of glucagon-like peptide-1 (GLP-1) storage in the small intestine, stimulates the secretion of glucagon-like peptide-1 to increase, interacts with GLP-1 receptor (GLP-1R) in vivo to stimulate β cells to secrete insulin, and at the same time, inhibits the production of glucagon, and finally lowers postprandial blood glucose to maintain a constant level [42,43 ].

At present, alpha-glucosidase inhibitors are widely used for reducing postprandial hyperglycemia, and currently, AGI drugs are on the market: acarbose (Glucobay)^TM,Acarbose)[26]Voglibose (Basen)^TM,Voglibose)[27]Miglitol (Glyset)^TM,Migltol)[28](FIG. 1). Clinical application shows that the three medicines have good curative effect, can be used as a first choice medicine for II type diabetes and an auxiliary medicine for treating insulin for I type diabetes, and have wide application prospect. Because the three medicines do not decompose the carbohydrate, the carbohydrate can generate some gastrointestinal adverse reactions such as abdominal distension, intestinal spasm, abdominal pain and the like when reaching the large intestine. And the preparation process is complex, the production cost is high, and the clinical application is limited to a great extent.

Currently, there are two sources of alpha-glucosidase inhibitors (AGI): i, extracting from animals, plants or microorganisms; and ii, artificial synthesis. The natural alpha-glucosidase inhibitor comprises flavonoid inhibitor, xanthenone inhibitor, cyclitol inhibitor, alkaloid inhibitor, curcumin and curcumin analogue inhibitor, etc. The synthetic alpha-glucosidase inhibitors mainly comprise: chalcones, thiadiazoles and oxadiazoles, benzothiazoles and oxindoles, stilbene urea derivatives, macrolides, imidazoles, and the like.

In the last decades, much interest has been focused on screening antidiabetic drugs with a broad mode of action, thus developing many promising clinics, however, since the marketing of acarbose, voglibose and miglitol, no major progress has been made to date in the field of drug development of α -glucosidase inhibitors despite the fact that hundreds of articles concerning α -glucosidase inhibitors have been published. Therefore, it is necessary to research and develop new α -glucosidase inhibitors with less side effects and better therapeutic effects.

Disclosure of Invention

The invention aims to provide a novel thiazole carboxamide compound which has better alpha-glucosidase inhibitory activity, a preparation method of the compound and application of the compound serving as an alpha-glucosidase inhibitor in treating diseases related to the compound, in particular diabetes.

The thiazole carboxamide compounds have the structure of formula (I):

wherein R1 is independently selected from the group consisting of hydrogen atom, halogen, hydroxy, nitro, amine, carboxy, cyano, alkyl, alkoxy, ester, aryl, heteroaryl, alkyl substituted by halogen or hydroxy or cyano, amine substituted by alkyl, alkoxy substituted by halogen or hydroxy or cyano, aryl substituted by halogen or hydroxy or cyano, heteroaryl substituted by halogen or hydroxy or cyano; or R1 with

Forming a fused ring which may be substituted with halogen, hydroxy, cyano, alkyl, alkoxy, alkyl substituted with halogen, alkoxy substituted with halogen;

r2 is independently selected from hydrogen, halogen, hydroxy, carboxy, nitro, amino, cyano, alkyl, alkoxy, ester, aryl, heteroaryl, alkyl substituted by halogen, hydroxy or cyano, amino substituted by alkyl, alkoxy substituted by halogen, hydroxy or cyano, R2 and

forming a fused ring which may be substituted with halogen, hydroxy, cyano, alkyl, alkoxy, alkyl substituted with halogen, alkoxy substituted with halogen;

n is selected from 0 to 3;

m is selected from 0 to 5;

x1, X2, X3 are independently from each other selected from C, O, S or N, provided that at least one is not C;

z is selected from CO or SO 2.

Further, the compound of formula (1) is preferably a compound of formula (2):

r1, R2, Z, n and m are as defined in formula (1).

Further, the compound of formula (1) is preferably a compound of formula (3):

wherein R2, Z, m are as defined for formula (1);

r3 is selected from hydrogen, halogen, hydroxy, carboxy, cyano, nitro, amino, alkyl, alkoxy, ester, alkyl substituted by halogen or hydroxy or cyano or alkoxy substituted by halogen or hydroxy or cyano, amino substituted by alkyl;

q is selected from 0 to 4;

x4, X5, X6 and X7 are selected from C or N, of which up to two are N.

A compound of formula (1), formula (2) or (3),

further, R1 is preferably independently selected from hydrogen atom, halogen, hydroxyl group, nitro group, amine group, carboxyl group, cyano group, alkyl group, alkoxy group, ester group, alkyl group substituted with halogen or hydroxyl group or cyano group, alkoxy group substituted with halogen or hydroxyl group or cyano group, amine group substituted with alkyl group; or R1 with

Forming a fused aromatic heterocycle which may be substituted with halogen, hydroxy, cyano, alkyl, alkoxy, alkyl substituted with halogen, alkoxy substituted with halogen; further, R1 is preferably selected from the group consisting of hydrogen atom, halogen, hydroxyl group, nitro group, amine group, carboxyl group, cyano group, alkyl group, alkoxy group, ester group, alkyl group substituted with halogen or hydroxyl group or cyano group, alkoxy group substituted with halogen or hydroxyl group or cyano group; further, R1 is preferably independently selected from hydrogen atom, halogen, nitro, alkyl, alkoxy, ester group, substituted by halogen or hydroxy or cyanoAlkyl, alkoxy substituted by halogen or hydroxy or cyano;

further, R2 is preferably independently selected from hydrogen atom, halogen, hydroxy, nitro, amino, carboxy, cyano, alkyl, alkoxy, ester, alkyl substituted by halogen, hydroxy or cyano, alkoxy substituted by halogen, hydroxy or cyano, amino substituted by alkyl, R2 and

to form a fused aromatic ring which may be substituted with halogen, hydroxy, cyano, alkyl, alkoxy, alkyl substituted with halogen, alkoxy substituted with halogen. Further, R2 is preferably independently selected from hydrogen atom, halogen, nitro, amino, alkyl, alkoxy, alkyl substituted by halogen or hydroxy or cyano, alkoxy substituted by halogen or hydroxy or cyano, R2 and

form a naphthalene ring which may be substituted by halogen, hydroxy, cyano, alkyl, alkoxy, alkyl substituted by halogen, alkoxy substituted by halogen.

Further, n is preferably 0,1 or 2; further, n is preferably 0 or 1.

Further, m is preferably 0,1, or 3; further, m is preferably 0,1 or 2.

Further, X1, X2, X3 are preferably independently from each other selected from C, S or N, provided that at least one is not C; further, X1, X2, X3 are preferably one of C, one of N, and one of S;

further, Z is preferably SO 2.

Further, R3 is preferably a hydrogen atom, halogen, nitro, amino, alkyl, alkoxy, alkyl substituted with halogen or hydroxy or cyano, or alkoxy substituted with halogen or hydroxy or cyano; further preferred are a hydrogen atom, halogen, nitro, alkyl, alkoxy;

further, q is preferably 0,1, 2; further, q is preferably 1;

further, X4, X5, X6 and X7 are preferably at most one N; further, X4, X5, X6 and X7 are preferably all C.

Further, the alkyl group is preferably a C1-6 alkyl group, and examples thereof include: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl and the like.

Further, the alkoxy group is preferably a C1-6 alkoxy group, and examples thereof include: methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, isopentyloxy, hexyloxy, and the like.

Further, the aryl group or aromatic ring is preferably a C6-10 aryl group, more preferably a phenyl group or a naphthyl group.

Further, the heteroaryl group is preferably a C6-10 heteroaryl group, more preferably a C6-10 heteroaryl group having 1 to 3 heteroatoms, preferably N, S or O, more preferably O or N. For example: furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuryl, benzothienyl, indolyl, indazolyl.

In another aspect, the present invention provides stereoisomers, tautomers, enantiomers, diastereomers, racemates of the above-mentioned compounds, and pharmaceutically acceptable salts thereof with acids or bases.

The salt-forming acid or base may be an organic acid, an inorganic acid, an organic base or an inorganic base, for example: salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, and carbonic acid; salts with organic acids such as formic acid, acetic acid, propionic acid, trifluoroacetic acid, phthalic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; salts with amino acids such as lysine, arginine, ornithine, glutamic acid, and aspartic acid; salts with alkali metals such as sodium, potassium, and lithium; salts with alkaline earth metals such as calcium and magnesium; salts with metals such as aluminum, zinc, iron, etc.; salts with organic bases such as methylamine, ethylamine, diethylamine, trimethylamine, triethylamine, ethylenediamine, piperidine, piperazine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N-methylglucamine, and N, N' -dibenzylethylenediamine; ammonium salts, and the like.

In another aspect, the compounds of the present invention or pharmaceutically acceptable salts thereof may be converted to solvates as desired. Examples of such a solvent include: water, methanol, ethanol, 1-propanol, 2-propanol, butanol, t-butanol, acetonitrile, acetone, methyl ethyl ketone, chloroform, ethyl acetate, diethyl ether, t-butyl methyl ether, benzene, toluene, N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the like. In particular, there may be mentioned: water, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, acetone, methyl ethyl ketone, and ethyl acetate are preferred solvents.

Further, the present invention provides the following compounds or stereoisomers, tautomers, enantiomers, diastereomers, racemates, pharmaceutically acceptable salts thereof with an acid or a base:

further, the present invention provides the following compounds or stereoisomers, tautomers, enantiomers, diastereomers, racemates, pharmaceutically acceptable salts thereof with an acid or a base:

in another aspect, the present invention provides a process for the preparation of a compound of formula (1), formula (2) or formula (3).

Reacting an acid chloride of formula (4) and an amine of formula (5) or an amide of formula (6) with a compound of formula (7) in an organic solvent in the presence of a base at a temperature of ice bath to room temperature to produce a compound of formula (1), wherein each group is as defined in formula (1) and X is halogen.

The organic solvent is preferably dichloromethane, Tetrahydrofuran (THF), N, N-Dimethylformamide (DMF), N, N-Diethylformamide (DEF), N, N-Dimethylacetamide (DMAC), toluene, dimethyl sulfoxide (DMSO).

The base is preferably triethylamine, triethanolamine, pyridine or 4-dimethylaminopyridine;

the temperature is preferably ice bath;

the solvent is preferably an anhydrous solvent;

the reaction is preferably carried out under the protection of nitrogen;

x is preferably Cl or Br.

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (1), formula (2) or formula (3) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a use of a compound of formula (1), formula (2) or formula (3) or a pharmaceutically acceptable salt thereof as an inhibitor of α -glucosidase in the prevention or treatment of a disease associated with the α -glucosidase inhibitor, wherein the associated disease is diabetes.

Therapeutic or prophylactic agents comprising the compound of the present invention or a pharmaceutically acceptable salt thereof, or a solvate of either as an active ingredient can be prepared by using carriers or excipients and other additives which are generally used for formulation. The carrier or excipient for the preparation may be solid or liquid, and examples thereof include: lactose, magnesium stearate, starch, talc, gelatin, agar, pectin, acacia, olive oil, sesame oil, cacao butter, and ethylene glycol. Administration may be in any of the following forms: oral administration in the form of tablets, pills, capsules, granules, powders, liquids, etc., or parenteral administration in the form of injections such as intravenous injection and intramuscular injection, suppositories, transdermal administration, etc.

The effective amount of the active ingredient in the α -glucosidase inhibitor, the therapeutic agent or the prophylactic agent of the present invention varies depending on the administration route, the symptoms of the patient, the age, sex, body weight, and the type of disease, but is usually in the range of 0.01 to 1000mg per day for an adult, and the administration frequency is generally 1 to 3 times per day or 1 to 7 times per week. However, since the dose varies depending on various conditions, an amount smaller than the above dose may be sufficient, and an amount exceeding the above range may be necessary.

Drawings

FIG. 1 is a graph showing the results of MTT experiments.

FIG. 2 shows the interaction of compound W29 with 3L4T (A, B) and 3TOP (C, D).

Detailed Description

The following examples are intended to illustrate embodiments of the present invention in more detail, but the present invention is not limited thereto. The structure of the isolated novel compounds is confirmed by 1H-NMR and/or using mass spectrometry.

Example 1

Synthesis of ethyl benzothiazole-2-formate

1.25g (9.91mmol) of 2-aminobenzenethiol was weighed into a 50mL round-bottomed flask, 2.98g (19.98mmol) of diethyl oxalate was added, and the reaction was stirred at 140 ℃ for 6 hours. TLC monitored the reaction was complete (PE: EA ═ 5: 1). Pouring the reaction system into concentrated hydrochloric acid: water: in an ice-cooled solution of ethanol (5ml:15ml:5ml), a pale yellow solid was precipitated, filtered, and dried to obtain a crude product. After column chromatography separation, 1.55g (7.48mmol) of white solid is obtained, and the yield is 75.51%.

1H-NMR(300MHz,CDCl3)8.27(m,1H),7.99(m,1H),7.57(pd,J＝7.2,1.5Hz,2H),4.57(q,J＝7.1Hz,2H),1.50(t,J＝7.1Hz,3H)；13C-NMR(75MHz,CDCl3)160.68,158.57,153.21,136.78,127.54,127.09,125.52,122.08,63.14,14.30.ESI-HRMSm/z208.0425[M+H]+(calcd for C10H9NO2S,208.0427).

Synthesis of benzothiazole-2-formic acid

1.40g (6.76mmol) of benzothiazole-2-carboxylic acid ethyl ester are weighed into a 100mL round-bottomed flask, 15mL of tetrahydrofuran is added, 4.05mL (8.11mmol) of aqueous sodium hydroxide solution (2M) is added under stirring at room temperature, and after stirring for 2 hours, the reaction is monitored by TLC to be complete (PE: EA: 5: 1). Adjusting the pH value to 2 by using 2M hydrochloric acid, adding water for dilution, and performing suction filtration to obtain a white solid. After drying, 1.06g (5.92mmol) of the product is obtained, and the yield is 87.57%.

Synthesis of theirobenzothiazole-2-formyl chloride

0.80g (4.46mmol) of benzothiazole-2-carboxylic acid is weighed into a 50mL double-neck flask, 10mL of redistilled dichloromethane is added, argon is introduced for protection, and 7.56mL of oxalyl chloride is slowly added dropwise under ice bath. After the addition, one drop of DMF was added, the reaction was gradually warmed to room temperature for 2h, and the completion of the reaction was monitored by TLC (PE: EA: 5: 1). After concentration under reduced pressure, 0.84g (4.25mmol) of pale yellow solid was obtained, with a yield of 95.20%.

Synthesis of N- ((4-bromophenyl) sulfonyl) benzo [ d ] thiazole-2-carboxamide

0.62g (2.61mmol) of p-bromobenzenesulfonamide is weighed into a 50mL three-necked flask, 10mL of ultra-dry THF is added, after stirring and dissolving at 0 ℃, 0.72mL of triethylamine is added, and the reaction is carried out for 15min under the protection of argon. Then, an acid chloride solution (0.62 g (3.13mmol) of benzothiazole-2-carbonyl chloride is weighed out and dissolved in 10mL of ultra-dry THF, and then the mixture is transferred to a 50mL constant pressure dropping funnel) is slowly added dropwise, and the reaction is continuously stirred for 5 hours after the dropwise addition is finished. TLC monitored the reaction was complete (PE: EA ═ 2: 1). Quenching with 2M hydrochloric acid, extracting with ethyl acetate, sequentially washing with saturated sodium bicarbonate water solution, saturated salt water, drying with anhydrous sodium sulfate, vacuum filtering, evaporating solvent, and separating by column chromatography to obtain white solid 0.54g (1.53 mmol). The yield thereof was found to be 58.66%.

m.p:177.2-180.5℃；1H-NMR(400MHz,DMSO-d6)8.21(m,2H),8.02(m,2H),7.73(m,2H),7.65(m,2H)；13C-NMR(101MHz,DMSO-d6)167.54,160.21,152.98,139.79,138.98,136.97,130.16,129.72,130.16,129.72,128.14,127.90,125.07,123.59；ESI-HRMSm/z 352.9816[M+H]+(calcd for C14H9ClN2O3S2,352.9816).

Example 2

By a similar preparation method to that of example 1, compound W2 was obtained in the form of a white solid with a yield of 54.67%, m.p193.8-195.9 ℃; 1H-NMR (400MHz, DMSO-d6)8.23(m,2H),7.92(dd, J ═ 32.2,8.6Hz,4H),7.63(m, 2H); 13C-NMR (101MHz, DMSO-d6)162.08,160.00,152.91,139.18,136.99,132.75,130.24,132.75,130.24,128.32,128.25,127.98,125.12,123.63; ESI-HRMSm/z396.9309[ M + H ] + (calcd for C14H9BrN2O3S2, 396.9311).

Example 3

Compound W3 was prepared in a yellow solid with yield 59.46%, m.p195.8-199.1 ℃ using a similar preparation method to that of example 1; 1H-NMR (400MHz, DMSO-d6)8.32(m,2H),8.22(ddd, J ═ 9.9,5.2,4.5Hz,2H),8.13(m,1H),7.91(t, J ═ 7.9Hz,1H),7.63(tdd, J ═ 14.8,7.2,1.4Hz, 2H); 13C-NMR (101MHz, DMSO-d6)162.47,160.36,152.93,141.45,136.99,132.26,131.25,131.10,130.79,128.16,127.90,125.20,125.06,124.86,123.57; ESI-HRMSm/z387.0076[ M + H ] + (calcd for C15H9F3N2O3S2,387.0079).

Example 4

By a similar preparation method to that of example 1, compound W4 was obtained as a white solid in 53.17% yield, a white solid; m.p174.3-178.4 ℃; 1H-NMR (400MHz, DMSO-d6)8.39(d, J ═ 7.6Hz,1H),8.21(dt, J ═ 17.4,7.6Hz,2H),7.95(m,3H),7.64(m, 2H); 13C-NMR (101MHz, DMSO-d6)162.91,160.54,153.05,139.14,136.94,134.21,133.50,132.97,128.66,128.60,128.03,127.79,126.52,125.04,123.51; ESI-HRMSm/z387.0079[ M + H ] + (calcd for C15H9F3N2O3S2,387.0079).

Example 5

By a similar preparation method to that of example 1, compound W6 was obtained in the form of a white solid with a yield of 28.07%, m.p. 251.1-254.3 ℃; 1H-NMR (400MHz, DMSO-d6)8.71(dt, J ═ 4.6,1.2Hz,1H),8.20(dd, J ═ 9.9,8.3Hz,2H),8.15(m,2H),7.70(ddd, J ═ 6.2,4.7,2.7Hz,1H),7.63(m, 2H); 13C-NMR (101MHz, DMSO-d6)161.04,157.49,153.08,150.30,150.30,139.10,136.90,128.00,128.00,127.77,125.04,123.49,123.49; ESI-HRMSm/z320.0156[ M + H ] + (calcd for C13H9N3O3S2,320.0158).

Example 6

By a similar preparation method to that of example 1, compound W7 was obtained in the form of a white solid with a yield of 39.06%, m.p. 195.8-204.3 ℃; 1H-NMR (400MHz, DMSO-d6)8.22(m,2H),8.03(td, J ═ 7.9,1.7Hz,1H),7.78(m,1H),7.64(m,2H),7.46(m, 2H); 13C-NMR (101MHz, DMSO-d6)162.37,160.37,157.55,152.98,136.94,136.81,131.65,128.16,127.90,125.25,125.11,123.57,117.85,117.64; ESI-HRMSm/z337.0108[ M + H ] + (calcd for C14H9FN2O3S2,337.0111).

Example 7

By a similar preparation method to that of example 1, compound W8 was obtained in the form of a white solid with a yield of 40.79%, a white solid, m.p297.6-300.2 ℃; 1H-NMR (400MHz, DMSO-d6)8.06(dd, J ═ 16.1,7.8Hz,2H),7.80(t, J ═ 8.0Hz,1H),7.53(m, 4H); 13C-NMR (101MHz, DMSO-d6)169.33,163.57,153.73,136.67,132.47,132.45,127.36,127.32,126.66,126.60,124.38,122.86,120.15,119.90; ESI-HRMSm/z414.9205[ M + H ] + (calcd for C14H8BrFN2O3S2,414.9217).

Example 8

By a similar preparation method to that of example 1, compound W9 was obtained as a white solid in 58.88% yield, m.p212.4-218.1 ℃; 1H-NMR (400MHz, DMSO-d6)8.20(m,2H),7.67(m,3H),7.61(m, 2H); 13C-NMR (101MHz, DMSO-d6)163.61,163.49,161.12,161.00,160.90,153.02,136.95,127.95,127.73,124.96,123.47,111.89,111.61,109.40; ESI-HRMSm/z355.0018[ M + H ] + (calcd for C14H8F2N2O3S2,355.0017).

Example 9

By a similar preparation method to that of example 1, compound W10 was obtained as a white solid in a yield of 32.97%, m.p165.0-168.6 ℃; 1H-NMR (300MHz, DMSO-d6)8.23(m,2H),7.93(d, J ═ 8.3Hz,2H),7.65(pd, J ═ 7.2,1.5Hz,2H),7.46(d, J ═ 8.1Hz,2H),2.40(s, 3H); 13C-NMR (75MHz, CDCl3)166.57,164.37,157.64,149.81,141.71,141.54,134.84,133.07,134.84,133.07,133.03,132.76,129.89,128.40, 26.33; ESI-HRMSm/z333.0352[ M + H ] + (calcd for C15H12N2O3S2,333.0362).

Example 10

By a similar preparation method to that of example 1, compound W11 was prepared as a white solid in 51.36% yield, m.p289.0-292.4 ℃; 1H-NMR (300MHz, DMSO-d6)11.44(s,1H),8.25(t, J ═ 8.6Hz,2H),7.98(m,4H),7.65(m,2H),7.36(s, 2H); 13C-NMR (75MHz, DMSO-d6)164.64,159.05,153.08,141.28,140.07,136.94,127.80,127.74,127.03,127.03,124.72,123.59,121.04,121.04.

Example 11

By a similar preparation method to that of example 1, compound W12 was prepared as a white solid with a yield of 49.97% m.p179.2-182.4 ℃; 1H-NMR (300MHz, DMSO-d6)8.22(m,3H),7.67(m, 5H); 13C-NMR (75MHz, DMSO-d6)161.83,159.98,152.95,137.22,136.95,135.64,132.69,132.23,131.37,128.26,128.15,127.97,125.17,123.60; ESI-HRMSm/z352.9813[ M + H ] + (calcd for C14H9ClN2O3S2, 352.9816).

Example 12

By a similar preparation method to that of example 1, compound W13 was obtained in the form of a white solid with a yield of 46.49% m.p. 230.5-232.9 ℃; 1H-NMR (300MHz, DMSO-d6)8.76(s,1H),8.48(dd, J ═ 27.1,7.8Hz,2H),8.20(d, J ═ 1.6Hz,2H),7.95(t, J ═ 8.0Hz,1H),7.62(dd, J ═ 7.3,6.0Hz, 2H); 13C-NMR (75MHz, DMSO-d6)162.47,160.44,152.90,148.12,141.76,136.96,134.22,131.56,128.59,128.17,127.90,125.05,123.55,123.19; ESI-HRMSm/z364.0057[ M + H ] + (calcd for C14H9N3O5S2,364.0056).

Example 13

By a production method similar to that of example 1, compound W14 was obtained as a white solid in a yield of 30.43% in m.p211.3-213.9 ℃; 1H-NMR (300MHz, DMSO-d6)8.05(dd, J ═ 16.8,8.6Hz,3H),7.77(dd, J ═ 47.8,7.8Hz,2H),7.49(m, 3H); 13C-NMR (75MHz, DMSO-d6)172.01,163.69,153.61,147.57,136.65,133.73,130.83,130.01,128.65,126.70,126.36,124.40,122.88,121.32; ESI-HRMS M/z396.9312[ M + H ] + (calcd for C14H9BrN2O3S2, 396.9311).

Example 14

By a similar preparation method to that of example 1, compound W15 was obtained as a white solid with a yield of 62.95%, m.p209.7-212.5 ℃; 1H-NMR (400MHz, DMSO-d6)12.02(s,1H),8.23(m,4H),8.04(d, J ═ 8.4Hz,2H),7.64(m, 2H); 13C-NMR (101MHz, DMSO-d6)161.84,159.84,152.47,143.55,136.54,133.29,132.84,128.77,127.76,127.49,126.42,124.77,124.64,123.15,122.06; ESI-HRMS M/z387.0079[ M + H ] + (calcd for C15H9F3N2O3S2,387.0079).

Example 15

By a similar preparation method to that of example 1, compound W16 was obtained as a white solid in 31.17% yield, a white solid; m.p104.1-106.8 ℃; 1H-NMR (400MHz, DMSO-d6)8.19(dt, J ═ 36.8,14.2Hz,1H),8.03(t, J ═ 20.2Hz,1H),7.78(m,2H),7.13(m,4H),3.83(d, J ═ 11.1Hz, 3H); 13C-NMR (101MHz, DMSO-d6)163.84,162.10,159.57,152.90,136.94,136.65,131.01,130.73,128.15,125.12,123.62,114.46,56.03.

Example 16

Synthesis of thiazole-4-formyl chloride

0.25g (1.94mmol) of thiazole-4-carboxylic acid was weighed into a 50mL round-bottomed flask, and 2.81mL of thionyl chloride was added thereto, followed by stirring under reflux for 2 hours. The reaction was monitored by TLC for completion (methanol: dichloro ═ 1: 2), and the reaction solution was concentrated under reduced pressure to give 0.23g (1.56mmol) of a pale yellow solid in 80.50% yield.

Synthesis of N- ((4-nitro) benzenesulfonyl) thiazole-4-formamide

0.50g (2.47mmol) of p-nitrobenzenesulfonamide is weighed into a 50mL three-necked flask, 10mL of ultra-dry THF is added, stirring is carried out at 0 ℃ to dissolve the mixture, 0.41mL of triethylamine is added, and the reaction is carried out for 15min under the protection of argon. Then, an acyl chloride solution (0.38 g (2.60mmol) of thiazole-4-formyl chloride is weighed and added with 10mL of ultra-dry THF for dissolution and then transferred to a 50mL constant pressure dropping funnel) is slowly dropped, and the stirring reaction is continued for 5 hours after the dropping is finished. TLC monitored the reaction was complete (PE: EA ═ 2: 1). Column chromatography gave 0.33g (1.05mmol) of a white solid. The yield thereof was found to be 42.59%.

m.p 151.1-155.4℃；1H-NMR(400MHz,DMSO-d6)9.23(d,J＝1.5Hz,1H),8.77(s,1H),8.63(d,J＝1.6Hz,1H),8.55(dd,J＝8.1,1.1Hz,1H),8.44(d,J＝7.8Hz,1H),7.96(t,J＝8.1Hz,1H)；13C-NMR(101MHz,DMSO-d6)159.85,156.20,148.21,148.15,141.32,134.23,131.66,129.75,128.83,123.22；ESI-HRMSm/z313.9900[M+H]+(calcd for C10H7N3O5S2,313.9900)。

Example 17

By a similar preparation method to that of example 16, compound W18 was obtained in the form of a white solid with a yield of 45.17%; m.p169.8-171.1 ℃; 1H-NMR (400MHz, DMSO-d6)8.61(t, J ═ 2.0Hz,1H),8.45(ddd, J ═ 8.2,2.2,0.8Hz,1H),8.25(m,1H),7.90(t, J ═ 8.0Hz,1H),7.74(s, 2H); 13C-NMR (101MHz, DMSO-d6)148.19,146.03,132.19,132.19,131.59,131.59,127.01,127.01,121.03,121.03.

Example 18

By a similar preparation method to that of example 16, compound W19 was obtained as a yellow solid in 41.84% yield, m.p130.6-132.5 ℃; 1H-NMR (400MHz, DMSO-d6)12.20(s,1H),9.21(m,1H),8.59(d, J ═ 1.9Hz,1H),7.96(m,2H),7.15(m,2H),3.85(d, J ═ 4.9Hz, 3H); 13C-NMR (101MHz, DMSO-d6)163.66,159.44,156.02,148.57,131.29,130.64,130.64,128.91,114.68,114.68, 56.24; ESI-HRMSm/z299.0152[ M + H ] + (calcd for C11H10N2O4S2,299.0155).

Example 19

By a production method similar to that of example 16, compound W20 was obtained in the form of a white solid with a yield of 46.27%, m.p170.8-173.5 ℃; 1H-NMR (400MHz, DMSO-d6)9.23(d, J ═ 1.9Hz,1H),8.64(dd, J ═ 4.2,2.1Hz,2H),8.26(dd, J ═ 8.5,2.2Hz,1H),8.07(m, 1H); 13C-NMR (101MHz, DMSO-d6)160.16,156.13,148.48,147.59,140.09,133.48,133.00,131.17,129.63,125.80; ESI-HRMSm/z347.9510[ M + H ] + (calcd for C10H6ClN3O5S2,347.9510).

Example 20

By a similar preparation method to that of example 16, compound W21 was obtained in the form of a white solid with a yield of 69.29% m.p275.4-278.7 ℃; 1H-NMR (400MHz, DMSO-d6)10.68(s,1H),9.29(d, J ═ 2.0Hz,1H),8.57(d, J ═ 2.0Hz,1H),7.93(m,4H),7.30(s, 2H); 13C-NMR (101MHz, DMSO-d6)159.95,155.71,150.71,141.97,139.39,126.93,126.93,126.80,120.54,120.54.

Example 21

By a production method similar to that of example 16, compound W22 was obtained in the form of a white solid with a yield of 44.31%, m.p. 176.8-181.4 ℃; 1H-NMR (400MHz, DMSO-d6)9.22(t, J ═ 3.5Hz,1H),8.61(d, J ═ 1.9Hz,1H),8.02(m,2H),7.72(d, J ═ 8.6Hz, 2H); 13C-NMR (101MHz, DMSO-d6)159.77,156.11,148.45,139.15,138.77,130.17,129.74,130.17,129.74,129.32; ESI-HRMSm/z302.9657[ M + H ] + (calcd for C10H7ClN2O3S2, 302.9659).

Example 22

By a production method similar to that of example 16, compound W23 was obtained in the form of a white solid with a yield of 31.82% m.p192.7-195.4 ℃; 1H-NMR (400MHz, DMSO-d6)9.00(d, J ═ 1.1Hz,1H),8.79(d, J ═ 1.4Hz,1H),8.11(m,2H),7.51(dt, J ═ 17.0,8.6Hz, 2H); 13C-NMR (101MHz, DMSO-d6)164.56,159.89,155.79,149.50,135.34,132.72,132.62,132.35,117.06,116.83; ESI-HRMSm/z286.9953[ M + H ] + (calcd for C10H7FN2O3S2,286.9955).

Example 23

By a similar preparation method to that of example 16, compound W24 was obtained in the form of a white solid with a yield of 50.59%, m.p187.9-188.4 ℃; (ii) a 1H-NMR (300MHz, DMSO-d6)11.22(s,1H),8.81(dd, J ═ 4.6,1.5Hz,1H),8.62(dd, J ═ 8.4,1.5Hz,1H),7.73(m,1H),2.70(t, J ═ 7.3Hz,2H),1.57(m,2H),128(m,6H),0.89(dt, J ═ 9.5,5.9Hz, 3H); 13C-NMR (75MHz, DMSO-d6)173.50,163.41,158.98,158.75,150.50,145.97,133.03,123.46,37.30,31.49,28.62,24.30,22.45, 14.38; ESI-HRMSm/z292.1123[ M + H ] + (calcd for C14H17N3O2S, 292.1114).

Example 24

By a production method similar to that of example 16, compound W25 was obtained in the form of a white solid with a yield of 40.17% m.p192.7-195.4 ℃; 1H-NMR (400MHz, DMSO-d6)8.27(s,1H),8.06(m,2H),7.48(m, 2H); 13C-NMR (101MHz, DMSO-d6)166.56,164.05,162.33,160.50,160.24,158.34,131.25,127.17,125.80,116.89; ESI-HRMSm/z364.9058[ M + H ] + (calcd for C10H6BrFN2O3S2,364.9060).

Example 25

By a production method similar to that of example 16, compound W26 was obtained in the form of a white solid with a yield of 38.49% m.p109.9-112.5 ℃; 1H-NMR (400MHz, DMSO-d6)9.23(t, J ═ 3.7Hz,1H),8.65(d, J ═ 1.9Hz,1H),8.31(m,2H),8.14(d, J ═ 7.9Hz,1H),7.92(t, J ═ 7.9Hz, 1H); 13C-NMR (101MHz, DMSO-d6)159.81,156.22,148.21,140.99,132.27,131.36,131.04,130.27,129.72,124.94,122.43; ESI-HRMSm/z336.9920[ M + H ] + (calcd for C11H7F3N2O3S2,336.9923).

Example 26

By a similar preparation method to that of example 16, compound W27 was obtained in the form of a white solid with a yield of 42.50% m.p187.9-188.4 ℃; 1H-NMR (400MHz, DMSO-d6)9.23(d, J ═ 1.7Hz,1H),8.62(d, J ═ 1.7Hz,1H),8.23(d, J ═ 8.3Hz,2H),8.05(d, J ═ 8.4Hz, 2H); 13C-NMR (101MHz, DMSO-d6)159.91,156.16,148.35,143.82,133.48,129.55,129.20,126.88,126.84,125.19,122.48; ESI-HRMSm/z336.9920[ M + H ] + (calcd for C11H7FN2O3S2,336.9923).

Example 27

By a similar preparation method to that of example 16, compound W28 was obtained as a yellow solid with a yield of m.p109.9-112.5 ℃; 1H-NMR (400MHz, DMSO-d6)12.63(s,1H),9.22(d, J ═ 1.9Hz,1H),8.60(dd, J ═ 7.1,2.0Hz,1H),8.07(dd, J ═ 7.9,1.1Hz,1H),7.57(td, J ═ 7.5,1.1Hz,1H),7.45(t, J ═ 7.5Hz,1H),7.39(d, J ═ 7.5Hz, 1H); 13C-NMR (101MHz, DMSO-d6)159.50,156.07,148.42,137.97,137.58,134.08,132.86,130.93,129.03,126.66,126.53.

Example 28

Compound W29 was prepared in a similar manner to example 16 in a white solid with a yield of 32.49%, 1H-NMR (400MHz, DMSO-d6)9.29(t, J ═ 5.2Hz,1H),9.24(dd, J ═ 7.1,1.9Hz,1H),8.94(d, J ═ 1.9Hz,1H),8.64(d, J ═ 1.9Hz,1H),8.11(m,2H),7.60(m, 2H); 13C-NMR (101MHz, DMSO-d6)159.14,156.66,156.13,154.49,145.75,137.47,132.42,130.20,129.36,123.25.

Example 29

Compound W30 was prepared in a similar manner to example 16 in 46.36% yield as a white solid, 1H-NMR (400MHz, DMSO-d6)9.20(m,1H),8.62(d, J ═ 1.9Hz,1H),7.91(m, 4H); 13C-NMR (101MHz, DMSO-d6)159.69,156.14,148.33,139.10,132.72,130.22,132.72,130.22,129.42,128.29; ESI-HRMSm/z346.9150[ M + H ] + (calcd for C10H7BrN2O3S2,346.9154).

Example 30

By a similar preparation method to that of example 16, compound W31 was obtained in the form of a white solid with a yield of 60.96% m.p296.9-302.6 ℃; 1H-NMR (400MHz, DMSO-d6)10.68(s,1H),8.03(d, J ═ 8.5Hz,2H),7.97(d, J ═ 8.8Hz,2H),7.82(d, J ═ 8.7Hz,2H),7.63(d, J ═ 8.5Hz,2H),7.31(s, 2H); 13C-NMR (101MHz, DMSO-d6)165.30,142.44,139.35,137.25,133.61,130.28,129.01,127.00,120.41; ESI-HRMSm/z311.0246[ M + H ] + (calcd for C13H11ClN2O3S,311.0252).

Example 31

By a similar preparation method to that of example 16, Compound W32 was obtained in a white solid with a yield of 37.58%, 1H-NMR (400MHz, DMSO-d6)7.91(m,1H),7.78(m,4H),7.59(m, 2H); 13C-NMR (101MHz, DMSO-d6)159.65,157.09,133.16,131.59,131.44,130.92,130.32,129.19,128.35,126.86,126.77,121.08,120.83; ESI-HRMSm/z391.9157[ M + H ] + (calcd for C13H8BrClFNO3S,391.9154).

Example 32

Compound W33 was prepared in a similar manner to example 16 in a white solid with a yield of 40.79%, 1H-NMR (400MHz, DMSO-d6)7.80(m,4H),7.45(d, J ═ 8.6Hz,2H),7.11(d, J ═ 7.9Hz,2H),2.30(s, 3H); 13C-NMR (101MHz, DMSO-d6)170.40,145.75,139.91,136.80,134.79,129.33,128.86,128.46,128.05,129.33,128.86,128.46,128.05, 21.42; ESI-HRMSm/z310.0297[ M + H ] + (calcd for C14H12ClNO3S,310.0299).

Determination of Compound Activity

Activity assay for alpha-glucosidase (AG)

1. Preparation of the solution

Preparing 0.1mol/L solution of potassium dihydrogen phosphate (6.805g) and disodium hydrogen phosphate dodecahydrate (17.905g) by using ultrapure water (500mL), and mixing the solution with a pH meter to prepare Phosphate Buffer Solution (PBS) with pH of 6.8 and concentration of 0.1 mol/L;

preparing a Na2CO3 solution with the concentration of 1mol/L by using ultrapure water (10mL) and anhydrous Na2CO3(1.0584 g);

preparing an alpha-p-nitrophenol glucoside (p-NPG) solution with the final concentration of 2mmol/L by using PBS (10mL) and alpha-p-nitrophenol glucoside (p-NPG, 6.025 mg).

Measurement procedure for alpha-glucosidase Activity

The total activity of the alpha-glucosidase lyophilized powder was 750UN (Saccharomyces cerevisiae, sigma), and the powder was dissolved by adding 7.5mL of LPBS, and then dispensed into 10 EP tubes (750. mu.L per tube, enzyme activity was 100U/mL). 2.4 mu L of the split charging liquid is taken from 100U/mL and added into 97.6 mu LPBS, the enzyme activity is diluted to 3.2U/mL, and the gradient is set to 2.4U/mL in sequence; 1.2U/mL; 0.6U/mL; 0.3U/mL; 0.15U/mL; 0U/mL.

Briefly, 8 μ L2.4U/mL of enzyme solution was added to 62 μ LPBS (pH 6.8) and added to 96-well plates (2 plus 3 wells per set). Starting from the second group, 70. mu.L of LPBS was added to each group, diluted sequentially with a line gun, mixed well and added to a mixed solution of PBS and DMSO (58. mu.L of PBS + 12. mu.L of DMSO (0.8%)). Immediately putting the mixture into a multifunctional enzyme-linked immunosorbent assay, vibrating the plate for 30s, and incubating the plate for 10min at the constant temperature of 37 ℃;

after incubation is finished, immediately adding 20 mu L of alpha-p-nitrophenol glucoside (p-NPG) with the concentration of 2mmol/L into each hole by using an eight-channel pipettor, immediately putting into a multifunctional enzyme labeling instrument after the addition is finished, and carrying out incubation at the constant temperature of 37 ℃ for 30min, wherein the circular shaking is carried out for 30 s;

and after the incubation is finished, taking out the 96 micro-porous plate, immediately adding 80 mu L of sodium carbonate solution with the concentration of 1mol/L into each hole by using an eight-channel pipettor, and finishing the reaction. After the addition, the plate was placed in a multifunctional microplate reader, vibrated for 30 seconds, and its OD value was measured at a wavelength of 405 nm. The enzyme concentration was selected in an appropriate absorbance range (OD ═ 0.9 to 1.2), and finally determined to be 0.6U/mL.

Determination of acarbose (positive control) inhibitory Activity

The preparation method comprises the steps of weighing 5.2mg of acarbose and dissolving the acarbose in 80.5 mu of LDMSO (dimethyl sulfoxide) to obtain a solution with the concentration of 100 mmol/L. After mixing, 80. mu.L of the mixture was added to 420. mu.L of LPBS to prepare a solution having a concentration of 16 mmol/L. The prepared solution was added to a 96-well plate and two sets were added (3 wells per set, 12. mu.L of Acarbose (16mmol/L) + 58. mu.L of PBS per well). From the second group, 70. mu.L of PBS and DMSO mixed solution (58. mu.L of PBS + 12. mu.L of DMSO (0.8%)) were added in sequence. Sequentially diluting to obtain final concentration gradient of 800 μ M; 400 mu M; 200 mu M; 100 mu M; 50 mu M; 25 mu M; 0 μ M.

The prepared enzyme solution (8. mu.L of enzyme solution (0.6U/mL) + 62. mu.L of LPBS (pH 6.8)) was added to each well of a 96-well microplate. Immediately putting the mixture into a multifunctional enzyme-linked immunosorbent assay, and incubating the mixture for 10min at a constant temperature of 37 ℃, wherein the mixture is circularly shaken for 30 s;

thirdly, after the incubation is finished, taking out the 96 microporous plate, immediately adding 20 mu L of alpha-p-nitrophenol glucoside solution (p-NPG,2mmol/L) into each hole by using a discharging gun, immediately putting into a multifunctional enzyme labeling instrument, vibrating the plate for 30s, and incubating at the constant temperature of 37 ℃ for 30 min;

after incubation is finished, taking out the 96 micro-porous plate, immediately adding 80 mu L of 1mol/L sodium carbonate aqueous solution into each hole by using a line gun to stop reaction, immediately putting the micro-porous plate into a multifunctional microplate reader, circularly shaking for 30s, measuring the OD value of each hole under the wavelength of 405nm, calculating the average value of each group of samples, and calculating the alpha-glucosidase inhibition rate (%) under different concentrations of acarbose according to a formula.

Note: a. the_DMSOThe OD value of the blank control group; a. the_AcarboseIs the OD value of the acarbose group; a. the_BlankOD values for the background group.

The above experiment was repeated for 2 times. The inhibition rate is used as an ordinate, the Log value (Log) of the sample concentration is used as an abscissa, the inhibition curve of the sample is drawn by Graph Pad Prism software, and the half inhibition concentration IC50 value of the acarbose on the alpha-glucosidase (IC50 is 228.3 +/-9.2 mu M) is calculated from the curve.

Determination of alpha-glucosidase inhibitory Activity of Compounds

The first compound was prepared into solutions of 100mmol/L concentration using DMSO (dimethyl sulfoxide), and 80 μ L of the solutions was added to 420 μ LPBS to prepare 16mmol/L solutions. The prepared solution was added in two sets of 3 duplicate wells (12 μ L compound +58 μ L PBS per well) in 96 microwell plates. Dilution sequentially with DMSO solution (12 μ L DMSO (0.8%) +58 μ L PBS) to final concentration gradient: 800 mu M; 400 mu M; 200 mu M; 100 mu M; 50 mu M; 25 mu M; 12.5. mu.M. The Blank control group was supplemented with an equal amount of DMSO (12. mu. LDMSO + 58. mu. LPBS), and the background control group (Blank) was supplemented directly with 70. mu. LPBS. 70. mu.L PBS was added to the Untreated group (UT), and Acarbose solution (12. mu.L Acarbose (4mM) + 58. mu.L PBS) was added to the positive control group.

And after uniform mixing, adding an alpha-glucosidase solution (8 mu L of enzyme solution +62 mu L of LPBS) with the concentration of 0.6U/mL into each hole. Blank group was added 70. mu.LPBS. Immediately transferring the 96 micro-porous plate into a multifunctional microplate reader, and incubating at constant temperature of 37 ℃ for 10min, wherein the plate is circularly shaken for 30 s;

thirdly, after the incubation at the constant temperature of 37 ℃, taking out the 96 microporous plate, immediately adding 20 mu L of alpha-p-nitrophenol glucoside (p-NPG,2mmol/L) into each hole by using an eight-channel pipettor, immediately putting the hole into a multifunctional enzyme labeling instrument, vibrating the plate for 30s, and then incubating at the constant temperature of 37 ℃ for 30 min;

after incubation is finished, the 96 micro-porous plate is taken out, 80 mu L of sodium carbonate solution with the concentration of 1mol/L is immediately added into each hole by an eight-channel pipette to stop reaction, the micro-porous plate is immediately placed into a multifunctional microplate reader, the circular shaking is carried out for 30s, and the OD value of each hole is measured at the wavelength of 405 nm. The average value of each group of samples is calculated, and the inhibition rate (%) of the alpha-glucosidase at different concentrations of each compound is calculated according to the formula.

Note: a. the_DMSOThe OD value of the blank control group; (ii) a A. the_compoundIs the OD value of the compound group; a. the_BlankOD values for the background group.

(5) The above experiment was repeated 2 times. And (3) drawing an inhibition curve of the sample by using Graph Pad Prism software with the inhibition rate as an ordinate and a sample concentration Log as an abscissa, and calculating a half inhibition concentration IC50 value of the compound to the alpha-glucosidase from the curve. And (3) judging whether the compound has an inhibiting effect or not and the effect relative to a positive control (acarbose).

The results are shown in Table 1

MTT assay

1. Preparation of the solution

Configuring a PBS: weighing 8g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate and 0.24g of potassium dihydrogen phosphate dodecahydrate, dissolving with ultrapure water, adjusting the pH to 7.4 by using a pH meter, and fixing the volume of the solution to 1L;

secondly, 50mg of MTT is weighed and added into 10mL of PBS, filtered by a 0.22 mu m filter membrane and wrapped by tinfoil paper, and stored in a refrigerator at 4 ℃ in a dark place, wherein the concentration of the MTT is 5 mg/mL.

2. Cell culture

Firstly, resuscitating cells: the cryopreserved cells (RAW264.7) were taken out from the liquid nitrogen tank, placed in a 37 ℃ ultrasonic instrument for rapid thawing (completed about 1-1.5 min), sterilized by spraying alcohol, and placed on a clean bench.

The cell suspension (1mL) is sucked into a 15mL centrifuge tube containing 9mL DMEME culture medium (containing 1% double antibody and 10% BI serum), and centrifuged for 3min at 800 rpm.

Thirdly, sucking the supernatant, adding 1mL of culture medium to suspend the cells, sucking the cells into a 10cm culture dish containing 5mL of the EME culture medium, and slightly shaking the culture dish up, down, left and right to uniformly disperse the cells.

The cells were cultured in a constant temperature incubator at 37 ℃ and 5% CO 2.

And fifthly, absorbing the culture medium when the growth is over 70-80%, washing with PBS, adding 2ml of the culture medium for blowing, subculturing, and continuously adding 5ml of the culture medium for culturing. After 3 passages, the cell suspension can be used for cytotoxicity experiments.

Cytotoxicity test

Carrying out counting: after the cell culture fluid in the logarithmic growth phase is diluted by 50 times, 20 mu L of the cell culture fluid is added into a cell counting plate, the cell count plate is used for counting under an inverted microscope, the number of cells in each milliliter of culture medium is calculated, and the cell culture fluid is diluted to 30 ten thousand/mL.

Two seed boards: the prepared cells were inoculated into 96-well plates, 30000 cells (100. mu.L per well) were inoculated into each well, and the peripheral wells were filled with 100. mu.L of sterile PBS and cultured for 12 hours.

The medicine preparation: the compound (stock concentration 100mM) was diluted in medium to different concentration gradients, slightly above half the inhibitory concentration for primary screening. W10 (260. mu.M), W24 (150. mu.M), W25 (60. mu.M), W27 (260. mu.M), W28 (25. mu.M), W29 (110. mu.M), acarbose (250. mu.M), doxorubicin (10. mu.M).

Fourth, dosing: the medium in 96 microwell plates was aspirated, 100. mu.L of drug-containing medium was added, and the culture was carried out for 24 hours. A blank control group (DMSO), acarbose as a negative control group, adriamycin as a positive control group, and a UT group (only 100. mu.L of the culture medium) were also set. A background control group of 100. mu.L PBS alone was added to the periphery. Each set is provided with 5 multiple holes.

After 24 hours of culture, 20 μ L of 0.5% MTT was added to each well, and the incubator was kept at constant temperature for 4 hours.

Sixthly, sucking the supernate dry by using a syringe, carefully not touching bottom cells, adding 150 mu LDMSO, and shaking for 1min to fully dissolve formazan. And (3) measuring the light absorption value at the wavelength of 570nm by using a multifunctional microplate reader.

Calculation of viability of RAW264.7 cells based on measured OD value (%)

Note: a. the_DMSOThe OD value of the blank control group; (ii) a A. the_compoundIs the OD value of the compound group; a. the_BlankOD values for the background group.

MTT test results

The alpha-glucosidase activity inhibition experiment shows that the compounds W10, W11, W12, W19, W29 and W32 have better inhibition activity. Therefore, we performed MTT experiments at slightly higher concentrations than their IC50 to test for cytotoxicity. A three-group parallel experiment is carried out by taking adriamycin (10 mu M) as a positive control and DMSO as a blank control, and the cell survival rate is calculated. The experimental results show that (figure 1), the cell survival rate of the adriamycin is 5.4%, while the cell survival rates of the compounds W19 (180. mu.M), W32 (150. mu.M), W29 (60. mu.M) and W12 (110. mu.M) are all more than 90%, and the compounds are considered to have no toxic or side effect on normal cells. The cell survival rates of the compounds W10(260 mu M) and W27(25 mu M) are 81.6% and 66.4% respectively, and the compounds have certain toxic effects on normal cells. It was preliminarily speculated that the inclusion of amino groups in the structure of the compound enhances cytotoxicity.

Computer simulation results

After experimental studies of enzyme activity and cytotoxicity, we also performed in silico molecular docking of the best active compound W29 (as shown in figure 2). From FIG. 2, it is clear that W29 hydrogen-interacts with the ASP542 and ARG202 residues of the 3L4T active site of the protein structure of α -glucosidase, hydrogen-interacts with the ARG1510, ASP1157 and TRP1369 residues of the 3TOP active site, and thiazole rings interact with TRP1369 and also with aryl groups. Indicating that compound W29 binds well to the active pocket of alpha-glucosidase.

Claims (9)

1. A compound represented by the following formula (3) or a pharmaceutically acceptable salt thereof, characterized in that the compound is a compound of the formula (3):

wherein the content of the first and second substances,

r2 are independently from each other selected from the group consisting of hydrogen, halogen, hydroxy, nitro, cyano, alkyl, alkoxy, alkyl substituted by halogen or hydroxy or cyano, alkoxy substituted by halogen or hydroxy or cyano;

z is selected from SO₂；

R3 is selected from hydrogen, halogen, hydroxy, cyano, nitro, alkyl, alkoxy, alkyl substituted by halogen or hydroxy or cyano or alkoxy substituted by halogen or hydroxy or cyano;

m is selected from 0 to 5;

q is selected from 0 to 4;

x4, X5, X6 and X7 are C;

the alkyl is C1-C6 alkyl;

the alkoxy is C1-C6 alkoxy.

2. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein m is 0,1 or 3 and q is 0,1, 2.

3. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein m is 0,1 or 2 and q is 1.

4. Selected from the group consisting of the following compounds or pharmaceutically acceptable salts thereof,

。

5. selected from the group consisting of the following compounds or pharmaceutically acceptable salts thereof,

。

6. a process for the preparation of a compound according to any one of claims 1 to 5, characterized in that:

reacting an acid chloride of formula (4) and an amine of formula (5) or an amide of formula (6) with a compound of formula (7) in an organic solvent in the presence of a base at a temperature in an ice bath to room temperature to form a compound of any one of claims 1 to 5, wherein each group is as defined in claims 1 to 5 and X is Cl.

7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

8. Use of a compound according to any one of claims 1 to 5 or a composition according to claim 7 for the manufacture of a medicament for the prophylaxis or treatment of a disease associated with an α -glucosidase inhibitor.

9. Use according to claim 8, characterized in that the disease is diabetes.

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