Active oxygen activated lipoic acid modified chalcone derivative as well as preparation method and application thereof

Abstract

The invention discloses an active oxygen activated lipoic acid modified chalcone derivative as well as a preparation method and application thereof, and relates to a chalcone derivative as well as a preparation method and application thereof. In order to solve the problems that chalcone lacks tumor selectivity, is insufficient in efficacy and has pharmacokinetics and safety through an ROS anticancer mechanism, the invention comprises three routes: route I: Claisen-Schmidt reaction is combined with Steglich esterification reaction; the route II is as follows: a Claisen-Schmidt reaction is combined with acylating chlorination and Schopper-Baumann esterification; and route 3: arsenic reagent catalytic cyclization is combined with Steglich esterification. The synthesized compound can efficiently and selectively induce cancer cells to generate ROS, exerts excellent anti-cancer activity, reduces oxidative damage to normal tissues to the greatest extent, and provides a new strategy for designing anti-cancer drugs targeting ROS. The invention is applied to the field of medicinal chemistry.

Description

TECHNICAL FIELD

[0001] The present application relates to a reactive oxygen species-activated lipoic acid-modified chalcone derivative, a preparation method and application thereof. BACKGROUND

[0002] Cancer is a major global health burden. Existing treatments such as chemotherapy, radiotherapy, targeted therapy and immunotherapy often have serious side effects, drug resistance and high cost, and there is an urgent need to develop new treatment strategies with greater selectivity and effectiveness. Chalcone (1,3-diphenylpropenone) is a natural flavonoid precursor widely present in plants, which has a simple structure and is easy to modify chemically. A large number of in vitro and in vivo studies have shown that a variety of chalcones and their synthetic derivatives exhibit significant broad-spectrum anticancer activity. Isobavachalcone inhibits mitochondrial electron transport (ETC) NADH dehydrogenase (Complex I), leading to increased electron leakage and superoxide anion production. Derivatives such as hesperetin methyl chalcone can down-regulate the expression of superoxide dismutase (SOD) and catalase (CAT), and weaken the ability of cells to remove reactive oxygen species (ROS). Some chalcones induce lipid peroxidation of cell membranes by consuming glutathione (GSH) or inhibiting glutathione peroxidase 4 (GPX4), thereby triggering ferroptosis in cancer cells.

[0003] Although the ROS-based anticancer mechanism of chalcone has been widely reported, there are still serious challenges in developing safe and effective clinical drugs. The core problems are lack of tumor selectivity, insufficient potency, and pharmacokinetics and safety. Therefore, the key to developing new chalcone derivatives is to design a chalcone derivative that can selectively induce rapid and significant ROS production in cancer cells while minimizing toxicity to normal cells, i.e., to develop a chalcone derivative with high cancer cell selectivity and strong ROS induction capacity. SUMMARY

[0004] In order to solve the problems of lack of tumor selectivity, insufficient potency, and pharmacokinetics and safety in the ROS-based anticancer mechanism of chalcone, the present application provides a reactive oxygen species-activated lipoic acid-modified chalcone derivative, a preparation method and application thereof.

[0005] The active oxygen activated lipoic acid modified chalcone derivative is lipoic acid modified chalcone derivative MA-LA, lipoic acid modified chalcone derivative BA-LA or lipoic acid modified chalcone derivative CA-LA.

[0006] The structural formula of the derivative is as follows:

[0007] ;

[0008] The structural formula of R in the lipoic acid modified chalcone derivative MA-LA is as follows: The structural formula of R in the lipoic acid modified chalcone derivative BA-LA is as follows: The structural formula of R in the lipoic acid modified chalcone derivative CA-LA is as follows: .

[0009] The novel active oxygen activated lipoic acid modified chalcone derivative has three synthesis routes.

[0010] Route one, Claisen-Schmidt reaction combined with Steglich esterification reaction:

[0011] The specific preparation method is as follows:

[0012] I. The raw material A and p-hydroxyacetophenone are dissolved in methanol, NaOH solid is added for reflux reaction, after the reaction is completed, the mixture is poured into ice water, the pH is adjusted to neutral, the crude product is extracted and dried, the crude product is dissolved in dichloromethane, and column chromatography purification is carried out to obtain the chalcone intermediate; wherein the molar ratio of the raw material A, p-hydroxyacetophenone and NaOH solid is 1: (1.1-1.5): (5-6);

[0013] II. Dissolve lipoic acid in dichloromethane, add chalcone intermediate, then cool to 0 ℃, add 4-dimethylaminopyridine DMAP and 1-ethyl-(3-dimethylaminopropyl) carbodiimide EDCI, react at room temperature, then remove the solvent by rotary evaporation, purify by column chromatography to obtain lipoic acid-modified chalcone derivative; wherein the molar ratio of chalcone intermediate, lipoic acid, 4-dimethylaminopyridine, 1-ethyl-(3-dimethylaminopropyl) carbodiimide is 1: (1~1.2): (0.1~0.3): (1.2~1.5); wherein raw material A is benzaldehyde, 2,3-dihydrobenzofuran-5-carboxaldehyde or 2,2-difluoro-1,3-benzodioxolane-5-carboxaldehyde; if raw material A is benzaldehyde, lipoic acid-modified chalcone derivative MA-LA is prepared; if raw material A is 2,3-dihydrobenzofuran-5-carboxaldehyde, lipoic acid-modified chalcone derivative BA-LA is prepared; if raw material A is 2,2-difluoro-1,3-benzodioxolane-5-carboxaldehyde, lipoic acid-modified chalcone derivative CA-LA is prepared.

[0014] Route II, Claisen-Schmidt reaction combined with acyl chloride and Schotten-Baumann esterification:

[0015]

[0016] The specific preparation method is:

[0017] I. Dissolve raw material A and p-hydroxyacetophenone in methanol, add NaOH solid for reflux reaction, after the reaction is completed, pour into ice water, then adjust to neutral, filter and dry the crude product, dissolve the crude product in dichloromethane, purify by column chromatography to obtain chalcone intermediate; wherein the molar ratio of raw material A, p-hydroxyacetophenone and NaOH solid is 1: (1.1~1.5): (5~6);

[0018] II. Dissolve lipoic acid in benzene, then add dropwise to a solution of thionyl chloride in benzene, stir at 0 ℃, remove the solvent under reduced pressure after the reaction is completed to obtain lipoic acid chloride; dissolve chalcone intermediate in anhydrous dichloromethane, then add lipoic acid chloride, cool to 0 ℃ in an ice bath, add Et3N, DMAP and EDCI in sequence, react at room temperature, remove the solvent by rotary evaporation after the reaction is completed, purify by recrystallization to obtain lipoic acid-modified chalcone derivative; the molar ratio of lipoic acid, thionyl chloride, chalcone intermediate, Et3N, DMAP and EDCI is 1: (1.5~1.8): (0.8~1): (1.1~1.5): (1.2~1.5): (1~1.2);

[0019] Wherein the raw material A is benzaldehyde, 2,3-dihydrobenzofuran-5-formaldehyde or 2,2-difluoro-1,3-benzodioxol-5-formaldehyde; if the raw material A is benzaldehyde, then the thioctic acid modified chalcone derivative MA-LA is prepared; if the raw material A is 2,3-dihydrobenzofuran-5-formaldehyde, then the thioctic acid modified chalcone derivative BA-LA is prepared; if the raw material A is 2,2-difluoro-1,3-benzodioxol-5-formaldehyde, then the thioctic acid modified chalcone derivative CA-LA is prepared.

[0020] Route three, arsenic reagent catalytic cyclization combined with Steglich esterification:

[0021]

[0022] The specific preparation method is:

[0023] I. Triphenylarsine (Ph3As) and 2-bromo-4-hydroxyacetophenone are dissolved in acetonitrile, and the reaction is carried out at 80 DEG C. After the reaction, the raw material A, N, N-diisopropylethylamine DIPEA and formamide are added, and the reaction is carried out at room temperature. After the reaction is completed, the solvent is evaporated under reduced pressure, and the chalcone intermediate is purified by column chromatography. The molar ratio of Ph3As, 2-bromo-4-hydroxyacetophenone, raw material A, DIPEA is 1: (2~2.5): (1~1.2): (1~1.2), and the volume ratio of acetonitrile and formamide is 50: 1.

[0024] II. Thioctic acid is dissolved in dichloromethane, and the chalcone intermediate is added, and the mixture is cooled to 0 DEG C. DMAP, EDCI are added, and the reaction is carried out at room temperature. After the reaction is completed, the solvent is removed by rotary evaporation, and the thioctic acid modified chalcone derivative is purified by column chromatography. The molar ratio of the chalcone intermediate, thioctic acid, DMAP, EDCI is 1: (1~1.2): (0.1~0.3): (1.2~1.5); wherein the raw material A is benzaldehyde, 2,3-dihydrobenzofuran-5-formaldehyde or 2,2-difluoro-1,3-benzodioxol-5-formaldehyde; if the raw material A is benzaldehyde, then the thioctic acid modified chalcone derivative MA-LA is prepared; if the raw material A is 2,3-dihydrobenzofuran-5-formaldehyde, then the thioctic acid modified chalcone derivative BA-LA is prepared; if the raw material A is 2,2-difluoro-1,3-benzodioxol-5-formaldehyde, then the thioctic acid modified chalcone derivative CA-LA is prepared.

[0025] The application of the active oxygen activated thioctic acid modified chalcone derivative in the preparation of antitumor drugs.

[0026] The application provides a novel lipoic acid modified chalcone derivative, which can efficiently and selectively induce ROS production in cancer cells, and has superior anticancer activity, while minimizing oxidative damage to normal tissues.

[0027] The application has the following beneficial effects:

[0028] In various human tumor cells (T24: bladder cancer cells, A549 lung cancer cells, SKOV3 ovarian cancer cells, HeLa cervical cancer cells), the ROS level is increased by more than 5 times within 6 hours, and the active oxygen level is significantly improved, which is obviously higher than the effect of Rosup (a commercially available active oxygen detection kit provided by Biyun Tian, with a use concentration of 50 μg / mL).

[0029] The cytotoxicity experiment results show that the lipoic acid modified chalcone derivative synthesized in the application has higher toxicity to bladder cancer cells (T24) than to embryonic kidney cells (HEK293), indicating that the lipoic acid modified chalcone derivative has potential antitumor effect.

[0030] The above results show that the active oxygen activated lipoic acid modified chalcone derivative synthesized in the application has potential antitumor effect. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the active oxygen activated lipoic acid modified chalcone derivative MA-LA;

[0032] Figure 2 The nuclear magnetic resonance carbon spectrum of the active oxygen activated lipoic acid modified chalcone derivative MA-LA;

[0033] Figure 3 The high-resolution mass spectrum of the active oxygen activated lipoic acid modified chalcone derivative MA-LA;

[0034] Figure 4 The nuclear magnetic resonance hydrogen spectrum of the active oxygen activated lipoic acid modified chalcone derivative BA-LA;

[0035] Figure 5 The nuclear magnetic resonance carbon spectrum of the active oxygen activated lipoic acid modified chalcone derivative BA-LA;

[0036] Figure 6 The high-resolution mass spectrum of the active oxygen activated lipoic acid modified chalcone derivative BA-LA;

[0037] Figure 7 The nuclear magnetic resonance hydrogen spectrum of the active oxygen activated lipoic acid modified chalcone derivative CA-LA;

[0038] Figure 8NMR spectrum of the active oxygen-activated lipoic acid modified chalcone derivative CA-LA;

[0039] Figure 9 High resolution mass spectrum of the active oxygen-activated lipoic acid modified chalcone derivative CA-LA;

[0040] Figure 10 Graph of the results of the T24 cell toxicity detection experiment of the lipoic acid modified chalcone derivative;

[0041] Figure 11 Graph of the results of the HEK293 cell toxicity detection experiment of the lipoic acid modified chalcone derivative;

[0042] Figure 12 Graph of the results of the experiment of the lipoic acid modified chalcone derivative stimulating T24 cells to produce active oxygen;

[0043] Figure 13 Graph of the results of the experiment of the lipoic acid modified chalcone derivative stimulating five kinds of cells to produce active oxygen. DETAILED DESCRIPTION

[0044] The technical scheme of the present application is not limited to the following specific embodiments, and any combination of the specific embodiments is also included.

[0045] Specific embodiment one: the active oxygen-activated lipoic acid modified chalcone derivative in the embodiment is the lipoic acid modified chalcone derivative MA-LA, the lipoic acid modified chalcone derivative BA-LA or the lipoic acid modified chalcone derivative CA-LA.

[0046] The structural formula of the derivative is as follows:

[0047] ;

[0048] The structural formula of R in the lipoic acid modified chalcone derivative MA-LA is as follows: The structural formula of R in the lipoic acid modified chalcone derivative BA-LA is as follows: The structural formula of R in the lipoic acid modified chalcone derivative CA-LA is as follows: .

[0049] The lipoic acid modified chalcone derivative MA-LA is (S)-4-cinnamoylphenyl 5-(1,2-dithiolane-3-yl)valerate, with the chemical formula C 23 H 24 O3S2, and a molecular weight of 412.56.

[0050] Lipoic acid modified chalcone derivative BA-LA: (E)-4-[3-(2,3-dihydrobenzofuran-6-yl)acryloyl]phenyl-(S)-5-(1,2-dithiopentan-3-yl)valerate, chemical formula C 25 H 26 O4S2, molecular weight 454.60.

[0051] Lipoic acid modified chalcone derivative CA-LA: (E)-4-[3-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)acryloyl]phenyl-(S)-5-(1,2-dithiopentan-3-yl)valerate, chemical formula C 24 H 22 F2O5S2, molecular weight 492.55.

[0052] Specific embodiment two: the preparation method of the active oxygen activated lipoic acid modified chalcone derivative in this embodiment is as follows:

[0053] I. Dissolve raw material A and p-hydroxyacetophenone in methanol, add NaOH solid for reflux reaction, after the reaction is completed, pour the mixture into ice water, adjust the pH to neutral, filter and dry the crude product, dissolve the crude product in dichloromethane, purify by column chromatography to obtain a chalcone intermediate; wherein the molar ratio of raw material A, p-hydroxyacetophenone and NaOH solid is 1: (1.1-1.5): (5-6);

[0054] II. Dissolve lipoic acid in dichloromethane, add the chalcone intermediate, then cool to 0 ℃, add DMAP and EDCI, react at room temperature, then remove the solvent by rotary evaporation, and purify by column chromatography to obtain a lipoic acid modified chalcone derivative; wherein the molar ratio of the chalcone intermediate, lipoic acid, DMAP, EDCI is 1: (1-1.2): (0.1-0.3): (1.2-1.5); wherein raw material A is benzaldehyde, 2,3-dihydrobenzofuran-5-carboxaldehyde or 2,2-difluoro-1,3-benzodioxole-5-carboxaldehyde; if raw material A is benzaldehyde, a lipoic acid modified chalcone derivative MA-LA is prepared; if raw material A is 2,3-dihydrobenzofuran-5-carboxaldehyde, a lipoic acid modified chalcone derivative BA-LA is prepared; if raw material A is 2,2-difluoro-1,3-benzodioxole-5-carboxaldehyde, a lipoic acid modified chalcone derivative CA-LA is prepared.

[0055] Specific embodiment three: the difference between this embodiment and specific embodiment two is that the eluent for column chromatography purification in step I is a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 10:1. Other steps are the same as specific embodiment two.

[0056] Specific embodiment four: the difference between this embodiment and specific embodiment two or three is that: in step two, the eluent for column chromatography purification is a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 4:1. The others are the same as specific embodiment two or three.

[0057] Specific embodiment five: a method for synthesizing an active oxygen activated lipoic acid modified chalcone derivative is as follows:

[0058] I. The raw material A and p-hydroxyacetophenone are dissolved in methanol, and NaOH solid is added for reflux reaction. After the reaction is completed, it is poured into ice water, and then neutralized. After filtration and drying, the crude product is dissolved in dichloromethane and purified by column chromatography to obtain a chalcone intermediate. The molar ratio of raw material A, p-hydroxyacetophenone and NaOH solid is 1: (1.1-1.5): (5-6);

[0059] II. Lipoic acid is dissolved in benzene, and then added dropwise into a benzene solution of thionyl chloride. Stirring is carried out at 0 ℃, and after the reaction is completed, the solvent is removed under reduced pressure to obtain lipoic acid chloride. The chalcone intermediate and anhydrous dichloromethane are mixed, and then lipoic acid chloride is added. The mixture is cooled to 0 ℃ in an ice bath, and then Et3N, DMAP and EDCI are added in sequence. Reaction is carried out at room temperature. After the reaction is completed, the solvent is removed by rotary evaporation, and the product is purified by recrystallization to obtain a lipoic acid modified chalcone derivative. The molar ratio of lipoic acid, thionyl chloride, chalcone intermediate, Et3N, DMAP and EDCI is 1: (1.5-1.8): (0.8-1): (1.1-1.5): (1.2-1.5): (1-1.2);

[0060] Wherein, the raw material A is benzaldehyde, 2,3-dihydrobenzofuran-5-carboxaldehyde or 2,2-difluoro-1,3-benzodioxol-5-carboxaldehyde. If the raw material A is benzaldehyde, a lipoic acid modified chalcone derivative MA-LA is prepared. If the raw material A is 2,3-dihydrobenzofuran-5-carboxaldehyde, a lipoic acid modified chalcone derivative BA-LA is prepared. If the raw material A is 2,2-difluoro-1,3-benzodioxol-5-carboxaldehyde, a lipoic acid modified chalcone derivative CA-LA is prepared.

[0061] Specific embodiment six: the difference between this embodiment and specific embodiment five is that: in step two, the reagent for recrystallization is a mixture of n-hexane and ethyl acetate, and the volume ratio of n-hexane to ethyl acetate is 1:1. The others are the same as specific embodiment five.

[0062] Specific embodiment seven: a method for preparing an active oxygen activated lipoic acid modified chalcone derivative is as follows:

[0063] I. triphenylarsine (Ph3As) and 2-bromo-4-hydroxyacetophenone are dissolved in acetonitrile, and the reaction is carried out at 80 ℃. After the reaction, raw material A, DIPEA and formamide are added, and the reaction is carried out at room temperature. After the reaction is completed, the solvent is evaporated under reduced pressure, and the chalcone intermediate is purified by column chromatography. The molar ratio of Ph3As, 2-bromo-4-hydroxyacetophenone, raw material A, DIPEA is 1: (2~2.5): (1~1.2): (1~1.2), and the volume ratio of acetonitrile and formamide is 50: 1;

[0064] II. lipoic acid is dissolved in dichloromethane, and the chalcone intermediate is added. The mixture is cooled to 0 ℃, and DMAP and EDCI are added. The reaction is carried out at room temperature. After the reaction is completed, the solvent is removed by rotary evaporation, and the lipoic acid-modified chalcone derivative is purified by column chromatography. The molar ratio of the chalcone intermediate, lipoic acid, DMAP and EDCI is 1: (1~1.2): (0.1~0.3): (1.2~1.5). Raw material A is benzaldehyde, 2,3-dihydrobenzofuran-5-carboxaldehyde or 2,2-difluoro-1,3-benzodioxol-5-carboxaldehyde. If raw material A is benzaldehyde, lipoic acid-modified chalcone derivative MA-LA is prepared. If raw material A is 2,3-dihydrobenzofuran-5-carboxaldehyde, lipoic acid-modified chalcone derivative BA-LA is prepared. If raw material A is 2,2-difluoro-1,3-benzodioxol-5-carboxaldehyde, lipoic acid-modified chalcone derivative CA-LA is prepared.

[0065] Specific embodiment eight: this embodiment is different from specific embodiment seven in that the reaction time in step one is 30 min. The others are the same as specific embodiment seven.

[0066] Specific embodiment nine: the application of the active oxygen-activated lipoic acid-modified chalcone derivative in the preparation of an antitumor drug.

[0067] Specific embodiment ten: this embodiment is different from specific embodiment nine in that the active oxygen-activated lipoic acid-modified chalcone derivative stimulates the production of active oxygen by tumor cells. The others are the same as specific embodiment nine.

[0068] The following examples are used to verify the beneficial effects of the present application:

[0069] Example 1

[0070] An active oxygen-activated lipoic acid-modified chalcone derivative, the synthesis route of which is as follows:

[0071]

[0072] The preparation method of the above-mentioned active oxygen-activated lipoic acid-modified chalcone derivative comprises the following steps:

[0073] (1) In a 250 mL round-bottom flask, add benzaldehyde (11.5 mmol) and p-hydroxyacetophenone (12.7 mmol), add 100 mL of methanol, magnetically stir until the solid is completely dissolved, weigh NaOH solid (2.3 g, 5 eq), slowly add to the flask in batches, install a reflux condenser, heat to reflux in an oil bath, continue to react for 6 h, cool the reaction solution to room temperature, slowly pour into a beaker containing 100 g of crushed ice, adjust the solution to neutral pH with dilute hydrochloric acid, vacuum filter, wash the filter cake with ice water (20 mL x 2), place the solid on the filter cake in a petri dish, and dry in a vacuum drying oven at 50°C for 2 h, obtain the crude product, purify the crude product by column chromatography (silica gel 200-300 mesh), eluent is petroleum ether (PE), ethyl acetate (EA) = 10: 1 (v / v), remove the solvent by rotary evaporation, to obtain compound MA (light yellow solid), which is (E)-1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one, chemical formula C 15 H 12 O2, molecular weight 224.26.

[0074] (2) In a dry 250 mL round-bottom flask, add lipoic acid (11 mmol, 1.1 eq) and 80 mL of anhydrous dichloromethane, add compound MA (10 mmol), magnetically stir to dissolve, cool the reaction system to 0°C in an ice bath, add DMAP (4-dimethylaminopyridine, 0.24 g, 0.2 eq), EDCI (1-ethyl-(3-dimethylaminopropyl) carbodiimide, 2.88 g, 1.5 eq) in turn, keep stirring at 0°C for 30 min, remove the ice bath, and raise to room temperature, continue to stir for 5 h, after the reaction is completed, directly rotary evaporate the reaction solution to remove dichloromethane, purify by column chromatography (silica gel 200-300 mesh), eluent gradient is PE:EA=4: 1 (v / v), rotary evaporate the solvent to dryness, to obtain lipoic acid modified chalcone MA-LA.

[0075] Test the nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum, and high-resolution mass spectrum of MA-LA prepared in this embodiment, and the test results are shown in FIGS. 1-3. Figures 1-3 As can be seen from the figures:

[0076] Example 2

[0077] A lipoic acid modified chalcone derivative activated by active oxygen, the synthesis route is as follows:

[0078]

[0079] The preparation method of the active oxygen-activated lipoic acid modified chalcone derivative includes the following steps:

[0080] (1) In a 250 mL round-bottom flask, add 2,3-dihydrobenzofuran-5-carboxaldehyde (11.5 mmol) and p-hydroxyacetophenone (12.7 mmol), add 100 mL of methanol, and stir magnetically until the solid is completely dissolved. Weigh NaOH solid (2.3 g, 5 eq) and slowly add it to the flask. Install a reflux condenser and heat to reflux in an oil bath. Continue the reaction for 6 h. Cool the reaction solution to room temperature, slowly pour it into a beaker containing 100 g of crushed ice, and adjust the solution pH to neutral with dilute hydrochloric acid. Vacuum filter, wash the filter cake with ice water (20 mL x 2), and dry the solid on the filter cake in a surface dish in a vacuum drying oven at 50°C for 2 h. The crude product is obtained. Purify the crude product by column chromatography (silica gel 200-300 mesh) with PE:EA=10:1 (v / v) as the eluent. Remove the solvent by rotary evaporation to obtain compound BA (light yellow solid). Specifically, (E)-1-(4-hydroxyphenyl)-3-(2,3-dihydrobenzofuran-6-yl)prop-2-en-1-one, chemical formula C 17 H 14 O3, molecular weight 266.30.

[0081] (2) In a dry 50 mL flask, add thionyl chloride (SOCl2, 0.89 g, 7.5 mmol) and anhydrous benzene (10 mL), and cool to 0°C. In another flask, dissolve lipoic acid (1.03 g, 5 mmol) in anhydrous benzene (10 mL). Add the lipoic acid benzene solution dropwise to the SOCl2 solution. Continue stirring at 0°C for 3 h, then remove the benzene and excess SOCl2 by rotary evaporation under reduced pressure to obtain lipoic acid chloride. In a dry 250 mL round-bottom flask, add compound BA (5 mmol) and anhydrous dichloromethane (50 mL), and add the prepared lipoic acid chloride. Cool to 0°C in an ice bath. Add Et3N (0.77 mL, 1.1 eq), DMAP (0.73 g, 1.2 eq), and EDCI (0.96 g, 1 eq) in sequence. Remove the ice bath and stir at room temperature for 5 h. After the reaction is complete, remove the dichloromethane by rotary evaporation. Recrystallize with a mixture of n-hexane:ethyl acetate=1:1 (v / v). Dissolve the crude product by heating, filter while hot, cool in an ice bath, vacuum filter, and wash with cold n-hexane (5 mL) to obtain yellow solid product BA-LA.

[0082] Test the BA-LA prepared in this embodiment for nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum, and high-resolution mass spectrum. The test results are shown in Figures 4-6 The figure shows that:

[0083] Example 3

[0084] A lipoic acid-modified chalcone derivative activated by active oxygen, and a synthetic route thereof is as follows:

[0085]

[0086] The preparation method of the lipoic acid-modified chalcone derivative activated by active oxygen comprises the following steps:

[0087] (1) A dry 250 mL round-bottom flask was added with triphenylarsine (122.4 mg, 0.4 mmol), 2-bromo-4-hydroxyacetophenone (199 mg, 0.925 mmol), anhydrous acetonitrile (100 mL) was added, and the solution was stirred magnetically and dissolved, and heated to 80 ℃ in an oil bath, and stirred at reflux for 30 min. The temperature was lowered to room temperature, and 2,2-difluoro-1,3-benzodioxol-5-formaldehyde (74.4 mg, 0.4 mmol), DIPEA (N,N-diisopropylethylamine, 70 μL, 0.4 mmol), formamide (2 mL) were added in sequence, and stirred at room temperature for 30 min. After the reaction was completed, the acetonitrile and formamide were removed by rotary evaporation under reduced pressure, and purified by column chromatography (silica gel 200-300 mesh) with eluent gradient PE: EA = 10: 1 (v / v), and the solvent was rotary evaporated to dryness to obtain the compound CA (yellow solid). Specifically, (E)-1-(4-hydroxyphenyl)-3-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)prop-2-en-1-one, chemical formula C 16 H 10 F2O4, molecular weight 304.25.

[0088] (2) In a dry 250 mL round-bottom flask, lipoic acid (11 mmol, 1.1 eq) and 80 mL of anhydrous dichloromethane were added, and compound CA (10 mmol) was added, and the solution was stirred magnetically and dissolved, and the reaction system was cooled to 0 ℃ in an ice bath, and DMAP (4-dimethylaminopyridine, 0.24 g, 0.2 eq), EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide, 2.88 g, 1.5 eq) were added in sequence, and stirred at 0 ℃ for 30 min, and the ice bath was removed, and the temperature was raised to room temperature, and stirring was continued for 5 h. The reaction liquid was directly rotary evaporated to remove dichloromethane, and purified by column chromatography (silica gel 200-300 mesh) with eluent gradient PE: EA = 4: 1 (v / v), and the solvent was rotary evaporated to dryness to obtain the lipoic acid-modified chalcone CA-LA.

[0089] The CA-LA prepared in this example was tested for nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum, and high-resolution mass spectrum, and the test results are as follows:Figures 7-9 As shown in the figure:

[0090] Example 4

[0091] The in vitro cytotoxicity evaluation of lipoic acid modified chalcone derivatives includes the following steps:

[0092] (1) Take the logarithmic growth period of bladder cancer cells (T24) and embryonic kidney cells (HEK293) to make cell suspension, and inoculate 100 μL of cells (density 5 x 10 4 cells / mL) per well in a 96-well plate, and place it in a cell incubator at 37 ℃, 5% CO2.

[0093] (2) Under the inverted microscope, when the cell confluence rate reaches 60%, remove the original culture medium, wash 2 to 3 times with DPBS, and add complete culture medium containing different concentrations (0, 2, 10, 50, 100 μmol / L) of MA-LA, BA-LA or CA-LA in turn (avoiding bubbles). Set different culture times (24, 48 h), and set 3 biological replicates for each concentration.

[0094] (3) After drug treatment, centrifuge the cell culture plate at 400 g for 5 min using a multi-well plate centrifuge. Transfer 90 μL of supernatant from each well to the corresponding well of a new 96-well plate, immediately add 45 μL of LDH detection working solution (lactic acid solution: INT solution: enzyme solution in a volume ratio of 1: 1: 1) to each well, incubate at room temperature for 30 min, and use an enzyme label instrument to measure the absorbance (OD) of each well at 490 nm, using 600 nm wavelength as the reference wavelength.

[0095] (4) Calculate the cell survival rate:

[0096]

[0097] The experimental results are shown in Figure 10 As shown in the figure, the activity detection results of cells treated with lipoic acid modified chalcone derivatives for 24 h show that as the concentration of lipoic acid modified chalcone derivatives increases, the toxicity to bladder cancer cells T24 increases, the reciprocal of the logarithm of the drug concentration is taken, and the nonlinear regression curve fitting is calculated to calculate the half-inhibition rate IC 50 of cells, respectively, BA-LA: 96.62 μmol / L, MA-LA: 63.73 μmol / L, CA-LA: 55.16 μmol / L; Similarly, the activity detection of cells treated with lipoic acid modified chalcone for 48 h, the half-inhibition rate IC 50BA-LA: 63.67 μmol / L, MA-LA: 76.28% at 100 μM, thus >100 μmol / L, CA-LA: 76.28% at 100 μM, thus >100 μmol / L, respectively.

[0098] The experimental results are shown in Table 1. Figure 11 The activity of HEK293 cells treated with lipoic acid-modified chalcone derivatives for 24 h was detected, and the reciprocal of the logarithm of the drug concentration on the abscissa was calculated. Nonlinear regression curve fitting was performed to calculate the cell half-inhibition rate IC 50 BA-LA: 63.67 μmol / L, MA-LA: 76.28% at 100 μM, thus >100 μmol / L, CA-LA: 76.28% at 100 μM, thus >100 μmol / L, respectively. The activity of cells treated with lipoic acid-modified chalcone derivatives for 48 h was detected, and the cell half-inhibition rate IC 50 BA-LA: 63.67 μmol / L, MA-LA: 76.28% at 100 μM, thus >100 μmol / L, CA-LA: 76.28% at 100 μM, thus >100 μmol / L, respectively. The activity of cells treated with lipoic acid-modified chalcone derivatives for 48 h was detected, and the cell half-inhibition rate IC

[0099] Example 5

[0100] The evaluation of intracellular reactive oxygen species (ROS) induction ability of lipoic acid-modified chalcone derivatives includes the following steps:

[0101] (1) Select cells in good growth state and in logarithmic growth phase, and inoculate 400 μL (density 5 x 10 4 cells / mL) per well in a 48-well culture plate, and place it in a cell incubator for culture. When the cell growth state and morphology are good, and the density reaches 70%-80%, replace it with complete culture medium containing different concentrations (0, 12.5, 25, 50, 100 μmol / L) of MA-LA, BA-LA or CA-LA, and treat the cells for 6 h. After treatment, the culture medium in the 48-well plate is aspirated, and the cells are washed 3 times with sterile DPBS. Add 400 μL of DCFH-DA (5 μmol / L) containing medium per well, and incubate it in a 37 °C cell incubator for 20 min. Wash the cells 3 times with serum-free cell culture medium to thoroughly remove the DCFH-DA probe that has not entered the cells.

[0102] (2) The detection was performed by flow cytometry within 1 h, the excitation wavelength was 488 nm, and the emission wavelength was 525 nm. The data were processed using Flowjo software.

[0103] (3) DCFH-DA working solution: DCFH-DA was diluted with serum-free DMEM at a ratio of 1:2000 to prepare a working solution, and the concentration used was 5 μmol / L. The reagent used in the ROS positive control group was Rosup (50 mg / mL) provided by the Biyun Tian active oxygen detection kit, which needed to be diluted with serum-free DMEM at a ratio of 1:1000 before use, and the concentration used was 50 μg / mL.

[0104] The experimental results showed that the ROS level changed after treatment with lipoic acid-modified chalcone derivatives. The ROS level was detected after treating the cells with different concentrations of drugs (0, 12.5, 25, 50, 100 μmol / L) for 6 h. The results showed that the ROS level in the treated cells (T24, A549, HEK293, HeLa, SKOV3) increased significantly, and reached a peak at 100 μmol / L drug treatment. As shown in Figure 12 , the ability of lipoic acid-modified chalcone derivatives to stimulate T24 cells to produce reactive oxygen species was stronger than that of Rosup reagent, as shown in Figure 13 , by comparing the ability of lipoic acid-modified chalcone derivatives to produce ROS in different cells in 5, the ability to stimulate T24 cells to produce ROS was significantly stronger than that in other cells, indicating that lipoic acid-modified chalcone derivatives had the potential to specifically kill bladder cancer cells and reduce oxidative damage to normal tissues.

Claims

1. An active oxygen-activated thioctic acid-modified chalcone derivative, characterized by, The lipoic acid modified chalcone derivative is lipoic acid modified chalcone derivative MA-LA, lipoic acid modified chalcone derivative BA-LA or lipoic acid modified chalcone derivative CA-LA. The structural formula of the derivative is as follows: ； The structure of R in the lipoic acid modified chalcone derivative MA-LA is: The structure of R in the lipoic acid modified chalcone derivative BA-LA is: The structure of R in the lipoic acid modified chalcone derivative CA-LA is: .

2. A process for the preparation of active oxygen-activated thioctic acid-modified chalcone derivatives as claimed in claim 1, wherein, The preparation method is as follows: I. raw material A and p-hydroxyacetophenone are dissolved in methanol, NaOH solid is added for reflux reaction, after the reaction is completed, the mixture is poured into ice water, the pH is adjusted to neutral, the crude product is extracted and dried, the crude product is dissolved in dichloromethane, and column chromatography purification is performed to obtain a chalcone intermediate; wherein the molar ratio of raw material A, p-hydroxyacetophenone and NaOH solid is 1: (1.1-1.5): (5-6); II. lipoic acid is dissolved in dichloromethane, the chalcone intermediate is added, then cooled to 0 ℃, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added, and the temperature is raised to room temperature for reaction, then the solvent is removed by rotary evaporation, and column chromatography purification is performed to obtain a lipoic acid modified chalcone derivative; wherein the molar ratio of the chalcone intermediate, lipoic acid, 4-dimethylaminopyridine, 1-ethyl-(3-dimethylaminopropyl) carbodiimide is 1: (1-1.2): (0.1-0.3): (1.2-1.5); wherein raw material A is benzaldehyde, 2,3-dihydrobenzofuran-5-carboxaldehyde or 2,2-difluoro-1,3-benzodioxolane-5-carboxaldehyde; if raw material A is benzaldehyde, lipoic acid modified chalcone derivative MA-LA is prepared; if raw material A is 2,3-dihydrobenzofuran-5-carboxaldehyde, lipoic acid modified chalcone derivative BA-LA is prepared; if raw material A is 2,2-difluoro-1,3-benzodioxolane-5-carboxaldehyde, lipoic acid modified chalcone derivative CA-LA is prepared.

3. A process for the preparation of active oxygen-activated thioctic acid-modified chalcone derivatives according to claim 2, characterized by, The eluent for column chromatography purification in step one is a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 10:

1.

4. A process for the preparation of active oxygen-activated thioic acid-modified chalcone derivative according to claim 2, characterized by, The eluent for column chromatography purification in step two is a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 4:

1.

5. The method of synthesis of active oxygen-activated thioctic acid-modified chalcone derivative according to claim 1, characterized in that, The preparation method is as follows: I. raw material A and p-hydroxyacetophenone are dissolved in methanol, NaOH solid is added for reflux reaction, after the reaction is completed, the mixture is poured into ice water, the pH is adjusted to neutral, the crude product is extracted and dried, the crude product is dissolved in dichloromethane, and column chromatography purification is performed to obtain a chalcone intermediate; wherein the molar ratio of raw material A, p-hydroxyacetophenone and NaOH solid is 1: (1.1-1.5): (5-6) II. lipoic acid is dissolved in benzene, then added dropwise to a solution of thionyl chloride in benzene, stirred at 0℃, after the reaction is completed, the solvent is removed under reduced pressure, to obtain lipoic acid chloride; chalcone intermediate is dissolved in anhydrous dichloromethane, then lipoic acid chloride is added, cooled to 0℃ in an ice bath, Et3N, 4-dimethylaminopyridine, 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added in turn, the reaction is carried out at room temperature, after the reaction is completed, the solvent is removed by rotary evaporation, and the lipoic acid modified chalcone derivative is obtained by recrystallization purification; the molar ratio of lipoic acid, thionyl chloride, chalcone intermediate, Et3N, 4-dimethylaminopyridine, 1-ethyl-(3-dimethylaminopropyl) carbodiimide is 1: (1.5~1.8): (0.8~1): (1.1~1.5): (1.2~1.5): (1~1.2); Wherein the raw material A is benzaldehyde, 2,3-dihydrobenzofuran-5-carboxaldehyde or 2,2-difluoro-1,3-benzodioxolane-5-carboxaldehyde; if the raw material A is benzaldehyde, the lipoic acid modified chalcone derivative MA-LA is prepared; if the raw material A is 2,3-dihydrobenzofuran-5-carboxaldehyde, the lipoic acid modified chalcone derivative BA-LA is prepared; if the raw material A is 2,2-difluoro-1,3-benzodioxolane-5-carboxaldehyde, the lipoic acid modified chalcone derivative CA-LA is prepared.

6. The method of synthesis of active oxygen-activated thioctic acid-modified chalcone derivative according to claim 5, characterized by, The reagent used for recrystallization in step two is a mixture of n-hexane and ethyl acetate, wherein the volume ratio of n-hexane to ethyl acetate is 1:

1.

7. A method of preparing an active oxygen-activated thioctic acid-modified chalcone derivative according to claim 1, characterized by, The preparation method is: I. triphenylarsine and 2-bromo-4-hydroxyacetophenone are dissolved in acetonitrile, and the reaction is carried out at 80℃, then the raw material A, N,N-diisopropylethylamine and formamide are added, and the reaction is carried out at room temperature, after the reaction is completed, the solvent is evaporated under reduced pressure, and the chalcone intermediate is purified by column chromatography, wherein the molar ratio of triphenylarsine, 2-bromo-4-hydroxyacetophenone, raw material A, DIPEA is 1: (2~2.5): (1~1.2): (1~1.2), and the volume ratio of acetonitrile to formamide is 50:1; II. Dissolve lipoic acid in dichloromethane, add chalcone intermediate, and cool the mixture to 0 ℃, add 4-dimethylaminopyridine, 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and react at room temperature. After the reaction is completed, remove the solvent by rotary evaporation, purify by column chromatography, and obtain lipoic acid-modified chalcone derivatives; wherein the molar ratio of chalcone intermediate, lipoic acid, 4-dimethylaminopyridine, and EDCI is 1: (1-1.2): (0.1-0.3): (1.2-1.5); wherein the raw material A is benzaldehyde, 2,3-dihydrobenzofuran-5-carboxaldehyde, or 2,2-difluoro-1,3-benzodioxolane-5-carboxaldehyde; if the raw material A is benzaldehyde, lipoic acid-modified chalcone derivatives MA-LA are prepared; if the raw material A is 2,3-dihydrobenzofuran-5-carboxaldehyde, lipoic acid-modified chalcone derivatives BA-LA are prepared; if the raw material A is 2,2-difluoro-1,3-benzodioxolane-5-carboxaldehyde, lipoic acid-modified chalcone derivatives CA-LA are prepared.

8. A process for the preparation of an active oxygen-activated thioctic acid-modified chalcone derivative according to claim 7, characterized by, The reaction time in step one is 30 min.

9. Use of the active oxygen-activated lipoic acid-modified chalcone derivative of claim 1 for preparing an antitumor drug.

10. Use according to claim 9, characterized in that, The active oxygen-activated lipoic acid-modified chalcone derivative stimulates tumor cells to produce active oxygen.

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