CN113307800A

CN113307800A - EGCG derivative and preparation method thereof

Info

Publication number: CN113307800A
Application number: CN202110700379.7A
Authority: CN
Inventors: 陈志亮; 朱海亮
Original assignee: Wuyishan Chenglong Tianchuang Tea Technology Co ltd
Current assignee: Wuyishan Chenglong Tianchuang Tea Technology Co ltd
Priority date: 2021-06-23
Filing date: 2021-06-23
Publication date: 2021-08-27

Abstract

The invention relates to the fields of dietetics, molecular biology, medicine and the like, and provides an EGCG derivative containing a pyrazole skeleton and a preparation method thereof by adopting EGCG extracted from tea leaves for site modification.

Description

EGCG derivative and preparation method thereof

Technical Field

Background

Tea is a traditional drink in China. In recent years, extensive studies on tea leaves have revealed that the active ingredient having a broad spectrum disease-preventing effect in tea leaves is mainly polyphenols in tea leaves, and typical examples thereof are catechins.

Catechin (Catechin) is also called tea tannin and Catechin. Catechin is the general term of flavonol substances in tea, and accounts for 60% -80% of the content of tea polyphenol. The first catechin was extracted from the catechu, a traditional Chinese medicine, and is relatively rich in green tea. Catechin is the main active substance in tea, and has effects of resisting oxidation, resisting tumor, and preventing cardiovascular disease etc. A plurality of catechin monomers are separated, purified and identified from tea leaves at present. For example, theaflavin digallate may inhibit the formation of endogenous carcinogens and thus inhibit tumor formation and development by inducing a decrease in the synthesis of NO Synthase (iNOs). The tea pigment has effects of remarkably enhancing T lymphocyte transformation and natural killer cell activity, and effectively inhibiting cell mutation, gene and chromosome mutation caused by carcinogens such as aflatoxin and benzopyrene, and cell canceration induced by radioactive rays.

A large number of reports indicate that COX-2 is expressed and enhanced in various solid tumors such as colorectal cancer, esophageal cancer, lung cancer, skin cancer, breast cancer and the like, and is closely related to various processes such as tumorigenesis, angiogenesis, cell proliferation and apoptosis. Over the past several decades, a number of selective COX-2 inhibitors of the coxib class, such as celecoxib, rofecoxib, parecoxib, and valdecoxib, have been discovered that are effective in reducing the side effects of traditional tnnsaids and that exhibit some activity in many oncology studies. Unfortunately, many clinical studies have shown that chronic and high dose administration of COX-2 inhibitors increases the risk of myocardial infarction, and even threatens the life of the patient in the severe cases. In addition, the conventional COX-2 drugs are difficult to prepare due to the problem of water solubility.

Therefore, the invention uses epigallocatechin gallate (EGCG) as a framework, combines with a part of the structure of celecoxib which is a classical COX-2 inhibitor, discovers the EGCG derivative containing a pyrazole skeleton, and researches the contents of COX-2 inhibition, antitumor activity, toxicity and the like.

Disclosure of Invention

The invention relates to the fields of food science, molecular biology, medicine and the like, and provides an EGCG derivative containing a pyrazole skeleton and a preparation method thereof by adopting EGCG extracted from tea leaves for site modification.

In order to achieve the purpose, the invention provides the following technical scheme:

an EGCG derivative having a structure of formula I and pharmaceutically acceptable salts thereof, wherein the structure is as follows:

a process for preparing EGCG derivatives having the structure of formula I and pharmaceutically acceptable salts thereof, comprising the steps of:

s1: EGCG, ethyl bromoacetate and anhydrous potassium carbonate are dissolved in anhydrous acetone, and the mixture is heated and stirred for reaction. After the reaction is finished, cooling to room temperature, and filtering to obtain a crude product; the crude product is dissolved in methanol solution again, and strong base is added for reflux under the stirring condition. After the reaction is finished, pouring the reaction liquid into ice water, adjusting the pH value to be strong acid by using 1M dilute hydrochloric acid, and precipitating and filtering to obtain a compound 1.

S2: dissolving dimethyl butynedioate in ether, and stirring at low temperature. And (2) weighing monomethylhydrazine, dissolving in diethyl ether, adding a strong base solution to adjust the pH to be alkaline, pouring the mixed solution into a constant-pressure dropping funnel, dropwise adding a low-temperature reaction dimethyl butynedioate diethyl ether solution, performing suction filtration, leaching and drying after reaction to obtain a compound 2.

S3: dissolving compound 1, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC. HCl), 1-Hydroxybenzotriazole (HOBT) and 4-Dimethylaminopyridine (DMAP) in dichloromethane, stirring under heating for reaction, adding compound 2, and continuing to stir for reaction. After the reaction is finished, washing the reaction product for three times by using a leaching solution, and purifying the reaction product by column chromatography to obtain the target compound shown in the formula I.

Wherein, in reaction step S1: the addition ratio of the EGCG, the ethyl bromoacetate, the anhydrous potassium carbonate and the strong base is as follows: (1-3): (1-2): (1-3): (1-3); heating and stirring reaction conditions are 60-90 ℃; the strong base is at least one of potassium hydroxide and sodium hydroxide; the strongly acidic pH is 1-5.

Wherein, in reaction step S2: the addition ratio of dimethyl butynedioate to monomethylhydrazine is as follows: (1-3): (1-3); the strong alkali solution is at least one of potassium hydroxide solution and sodium hydroxide solution; the pH adjusting range is 7-10.

Wherein, in reaction step S3: the adding proportion of the compound 1, EDC & HCl, HOBT, DMAP and the compound 2 is as follows: (1-3): (1-3): (1-2): (1-2): (1-3); the heating temperature is 30-65 ℃; the ratio of the saturated sodium chloride solution to the dichloromethane of the leacheate is as follows: (1-2): (1-2).

A pharmaceutical composition comprising an EGCG derivative having the structure of formula i as claimed in claim 1 and pharmaceutically acceptable salts thereof.

The EGCG derivative with the structure of formula I and the pharmaceutically acceptable salt thereof or the pharmaceutical composition containing the structure are used as COX-2 inhibitors in the preparation of drugs for treating tumors.

Further, the tumor is selected from: esophageal cancer, gastric cancer, pancreas, liver cancer, lung cancer, colon cancer, rectal cancer, cervical cancer, uterine body cancer, skin cancer, and breast cancer.

The invention has the following beneficial effects:

compared with the prior art, the invention has the beneficial effects of good antitumor activity, low toxic and side effects and excellent water solubility:

the EGCG derivative containing pyrazole skeleton has the advantages of formula I:

formula I is an organic derivative with a main framework structure from epigallocatechin gallate (EGCG), is derived from tea, and can obviously reduce the risks of myocardial infarction, cardiovascular diseases, hemorrhage, ulcer and perforation of stomach or intestinal tracts and reduce the toxic and side effects on patients compared with COX-2 inhibitor medicaments chemically synthesized by celecoxib, rofecoxib, parecoxib, valdecoxib and the like;

secondly, the formula I has an inhibition effect on the proliferation of tumor cells, and has better anti-tumor activity compared with the mother framework structure EGCG of the formula I;

formula I is used as EGCG derivative with anti-tumor activity, on one hand, formula I has specific COX-2 inhibiting effect, and can play analgesic and anti-inflammatory roles besides anti-tumor; on the other hand, the inhibitor has no obvious inhibition effect on COX-1, and the targeted COX-2 inhibition effect can reduce the adverse reaction caused by the inhibition of COX-1 by the traditional or less specific COX inhibitor;

and the formula I has more hydrophilic groups such as polyphenol hydroxyl and the like, and has excellent water solubility compared with COX-2 inhibitor medicaments such as celecoxib, rofecoxib and the like.

The preparation method of the EGCG derivative containing pyrazole skeleton disclosed by the invention has the advantages that:

firstly, the extraction of EGCG is not easy, and the yield is high and the raw materials are saved by using the preparation method;

secondly, the reaction rate and the precipitation efficiency can be improved by regulating and controlling the pH of S1 and S2, and the preparation process is simplified;

and thirdly, redundant impurities can be removed through leaching in each step, and the purity and the yield of the obtained target compound are improved.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1:

s1: EGCG (1mmol), ethyl bromoacetate (1mmol) and anhydrous potassium carbonate (1mmol) were dissolved in 10mL of anhydrous acetone and reacted at 60 ℃ with stirring for 9 h. After the reaction is finished, cooling the reaction liquid to room temperature, and filtering to obtain a crude product; the crude product was redissolved in 10mL of methanol and potassium hydroxide (1.5mmol) was added with stirring and the mixture refluxed for 3 h. After the reaction is finished, pouring the reaction solution into 80mL of ice water, adjusting the pH value to 1-2 by using 1M dilute hydrochloric acid, and performing suction filtration to obtain a compound 1.

S2: dimethylbutynedioate (3mmol) was dissolved in 10ml of diethyl ether and poured into a round-bottomed flask, which was placed in a low-temperature reaction vessel and stirred at-20 ℃ for 30 min. Weighing monomethylhydrazine (9mmol), dissolving in 10ml of diethyl ether, adding a sodium hydroxide solution into a beaker, adjusting the pH to 7-8, pouring the prepared mixed solution into a constant-pressure dropping funnel, adding the mixed solution into a reaction system at a speed of about 1 drop per second, and continuing stirring for 1 hour after the addition is finished. And (4) carrying out suction filtration, leaching with diethyl ether, and drying to obtain a compound 2.

S3: compound 1(1mmol), EDC. HCl (2mmol), HOBT (1mmol) and DMAP (1mmol) were dissolved in 10mL of dichloromethane and reacted at 30 ℃ with stirring for 60min, then compound 2(1.5mmol) was added and reacted with stirring overnight. After completion of the reaction, the reaction mixture was washed three times with saturated brine (50mL) and methylene chloride (100mL), and purified by column chromatography to obtain the objective compound of formula I in a yield of about 39%.

Example 2

S1: EGCG (2mmol), ethyl bromoacetate (1mmol) and anhydrous potassium carbonate (2mmol) were dissolved in 10mL of anhydrous acetone and the reaction was stirred at 70 ℃ for 6 h. After the reaction is finished, cooling the reaction liquid to room temperature, and filtering to obtain a crude product; the crude product was redissolved in 10mL of methanol and potassium hydroxide (2mmol) was added with stirring and the mixture refluxed for 2 h. After the reaction is finished, pouring the reaction solution into 100mL of ice water, adjusting the pH value to 3-4 by using 1M dilute hydrochloric acid, and performing suction filtration to obtain a compound 1.

S2: dimethylbutynedioate (5mmol) was dissolved in 10ml of diethyl ether and poured into a round-bottomed flask, which was placed in a low-temperature reaction vessel and stirred at-5 ℃ for 30 min. Weighing monomethylhydrazine (5mmol) and dissolving in 10ml of diethyl ether, adding a sodium hydroxide solution into a beaker, adjusting the pH to 8-9, then pouring the prepared mixed solution into a constant-pressure dropping funnel, adding the mixed solution into a reaction system at a speed of about 1 drop per second, and continuing stirring for 1h after the addition is finished. And (4) carrying out suction filtration, leaching with diethyl ether, and drying to obtain a compound 2.

S3: compound 1(1mmol), EDC. HCl (1.2mmol), HOBT (0.48mmol) and DMAP (0.5mmol) were dissolved in 10mL of dichloromethane and reacted at 45 ℃ for 30min with stirring, then Compound 2(1.1mmol) was added and reacted with stirring overnight. After the reaction was completed, the reaction mixture was washed three times with saturated saline (50mL) and dichloromethane (50mL), and purified by column chromatography to obtain the objective compound of formula I in a yield of about 40%.

Example 3

S1: EGCG (2mmol), ethyl bromoacetate (2mmol) and anhydrous potassium carbonate (3.5mmol) were dissolved in 10mL of anhydrous acetone and the reaction was stirred at 90 ℃ for 8 h. After the reaction is finished, cooling the reaction liquid to room temperature, and filtering to obtain a crude product; the crude product was redissolved in 10mL of methanol and sodium hydroxide (3mmol) was added with stirring and the mixture refluxed for 3 h. After the reaction is finished, pouring the reaction solution into 120mL of ice water, adjusting the pH value to 4-5 by using 1M dilute hydrochloric acid, and performing suction filtration to obtain a compound 1.

S2: dimethylbutynedioate (7mmol) was dissolved in 10ml of diethyl ether and poured into a round-bottomed flask, which was placed in a low-temperature reaction vessel and stirred at-10 ℃ for 45 min. Weighing monomethylhydrazine (4mmol) and dissolving in 10ml of diethyl ether, adding a potassium hydroxide solution into a beaker, adjusting the pH to 9-10, then pouring the prepared mixed solution into a constant-pressure dropping funnel, adding the mixed solution into a reaction system at a speed of about 1 drop per second, and continuing stirring for 2 hours after the addition is finished. And (4) carrying out suction filtration, leaching with diethyl ether, and drying to obtain a compound 2.

S3: compound 1(1mmol), EDC. HCl (1.5mmol), HOBT (0.8mmol) and DMAP (0.8mmol) were dissolved in 10mL of dichloromethane and reacted at 65 ℃ for 45min with stirring, then Compound 2(1.2mmol) was added and reacted with stirring overnight. After completion of the reaction, the reaction mixture was washed three times with saturated brine (100mL) and dichloromethane (50mL), and purified by column chromatography to obtain the objective compound of formula I in about 39% yield.

The target compounds obtained in the above examples 1-3 have the same formula I as that obtained in the above examples, and one of the data is as follows:

1H NMR(600MHz,DMSO-d6)δ:12.83(s,1H),8.09(d,J＝7.4Hz,2H),7.66-7.60(m,1H),7.58(t,J＝7.5Hz,3H),7.43-7.35(m,3H),7.35-7.28(m,3H),7.27-7.22(m,2H),7.21-7.16(m,2H),7.06(s,2H),6.89(d,J＝2.3Hz,1H),6.65(s,2H),6.48(d,J＝2.3Hz,1H),5.09(s,4H).,1.56(s,3H).13C NMR(150MHz,DMSO-d6)δ:182.58,168.47,164.04,161.70,157.66,147.97,144.12,139.92,132.65,131.00,130.11,129.60,129.56,129.06,128.96,128.81,126.93,125.69,108.09,105.88,105.81,99.07,94.15,65.47,60.68.MS(ESI):654.58[M+H]+.Anal.

thus, the structure of the target compound shown as the formula I is confirmed to be shown as the structural diagram of the formula I, and the structure are consistent.

The accompanying drawings, which are included to provide an understanding of the principles of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

In the drawings:

FIG. 1 is a model of the binding of a target compound of the present invention to COX-2;

FIG. 2 shows the results of the apoptosis test of HeLa cells with the objective compound of the present invention of formula I;

FIG. 3 shows the results of water solubility experiments for the target compounds of formula I and EGCG of the present invention.

Evaluation of biological Activity:

1. COX inhibition assay of formula i:

we examined the COX-1 and COX-2 inhibitory potency of the synthesized target compound of formula i by a COX-1/COX-2ELISA kit. The results show that formula I shows certain inhibitory activity against COX-2, IC₅₀The value was 0.31. mu.M, but showed relatively weak inhibitory activity against COX-1, IC against COX-1₅₀The value was 49.64. mu.M. Positive control, IC of positive control drug celecoxib on COX-2₅₀The value of 0.36. mu.M, it is believed that the COX-2 inhibitory activity of formula I is similar to, or even slightly superior to, that of the positive control celecoxib, indicating that formula I is effective in selectively inhibiting COX-2.

2. The COX-2 binding model of formula I:

as shown in figure 1, the target compound formula I is tightly combined with COX-2, and preliminary evaluation of biological activity shows that the medicine has a remarkable COX-2 inhibition effect.

3. Apoptosis assay of formula i:

in view of the excellent COX-2 inhibitory activity of the target compound of formula I, we further evaluated the effect of formula I on HeLa apoptosis by flow cytometry. The percentage of apoptotic cells increased from 4.01% to 58.30% after 24h treatment of HeLa cells with formula I at concentrations of 0, 1.25, 2.5, 5 and 10 μ M; these results demonstrate that formula i can inhibit the growth of HeLa cells by inducing apoptosis in a dose and time dependent manner. The apoptosis pattern is shown in figure 2.

4. Antitumor activity assay of formula i:

CaES-17, N87, Pan02, HepG2, A549, MC-38, SW-480, HeLa, A431 and MCF-7 cells in logarithmic growth phase were inoculated into a 96-well plate at a density of 7X 104 cells/mL and cultured for 12 hours. Then adding the tested compound with different concentrations for incubation for 48 h. Subsequently, 10. mu.L of MTT solution (5mg/mL) was added to each well and the incubation was continued for 4 h. The stock culture in the wells was removed and 150. mu.L DMSO was added and shaken for 15 min. Finally, detecting the absorbance at 570nm by using a microplate reader, and calculating the corresponding cytotoxicity according to the following equation:

wherein OD_positiveIndicates the absorbance, OD, of the blank control group_blankIndicates the absorbance, OD, of the negative zero-adjusted group_sampleThe absorbance of the sample treatment group is indicated.

TABLE 1

According to the results in table 1, it is shown that the target compound of formula i has significantly superior tumor cell proliferation inhibition effect on 10 cancer cells (esophageal cancer, gastric cancer, pancreas cancer, liver cancer, lung cancer, colon cancer, rectal cancer, cervical cancer, skin cancer and breast cancer) compared to EGCG in the positive control group, i.e., formula i has more superior anti-tumor activity.

5. Water solubility test of formula I

The target compounds of formula I and EGCG with the same mass are taken, a certain volume of pure water is added dropwise, and the comparison graph shows that the formula I has more excellent water solubility:

as shown in FIG. 3, the left EP tube contains the compound of formula I, the right EP tube contains EGCG, the EP tube of formula I is clear and transparent, but the right EP tube is slightly turbid, and the excellent water solubility of formula I relative to EGCG can be seen.

Although the preferred embodiments of the present invention and the evaluation of biological activity have been described in detail, the present invention is not limited to the details of the above embodiments, and various equivalent changes can be made to the technical solution of the present invention within the technical spirit of the present invention, and these equivalent changes are within the scope of the present invention. It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition. In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Claims

1. An EGCG derivative having a structure of formula I and pharmaceutically acceptable salts thereof, wherein the structure is as follows:

2. a process for the preparation of EGCG derivatives having the structure of formula i as claimed in claim 1 and pharmaceutically acceptable salts thereof comprising the steps of:

s1: dissolving EGCG, ethyl bromoacetate and anhydrous potassium carbonate in anhydrous acetone, and heating and stirring for reaction; after the reaction is finished, cooling to room temperature, and filtering to obtain a crude product; re-dissolving the crude product in methanol solution, adding strong base under stirring condition, and refluxing; after the reaction is finished, pouring the reaction liquid into ice water, adjusting the pH value to be strong acid by using 1M dilute hydrochloric acid, and precipitating and filtering to obtain a compound 1;

s2: dissolving dimethyl butynedioate in ether, and stirring at low temperature. Weighing monomethylhydrazine, dissolving in diethyl ether, adding a strong base solution to adjust the pH to be alkaline, pouring the mixed solution into a constant-pressure dropping funnel, dropwise adding a low-temperature reaction dimethyl butynedioate diethyl ether solution, performing suction filtration, leaching and drying after reaction to obtain a compound 2;

s3: dissolving compound 1, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC. HCl), 1-Hydroxybenzotriazole (HOBT) and 4-Dimethylaminopyridine (DMAP) in dichloromethane, stirring under heating for reaction, adding compound 2, and continuing stirring for reaction; after the reaction is finished, washing the reaction product for three times by using a leaching solution, and purifying the reaction product by column chromatography to obtain the target compound shown in the formula I.

3. The method of claim 2, wherein: in reaction step S1:

the addition ratio of the EGCG, the ethyl bromoacetate, the anhydrous potassium carbonate and the strong base is as follows: (1-3): (1-2): (1-3): (1-3);

and/or the heating and stirring reaction condition is 60-90 ℃;

and/or the strong base is at least one of potassium hydroxide and sodium hydroxide;

and/or the pH value of the strong acid is 1-5.

4. The method of claim 2, wherein: in reaction step S2:

the addition ratio of dimethyl butynedioate to monomethylhydrazine is as follows: (1-3): (1-3);

and/or the strong alkali solution is at least one of a potassium hydroxide solution and a sodium hydroxide solution;

and/or the pH adjusting range is 7-10.

5. The method of claim 2, wherein: in reaction step S3:

the adding proportion of the compound 1, EDC & HCl, HOBT, DMAP and the compound 2 is as follows: (1-3): (1-3): (1-2): (1-2): (1-3);

and/or the temperature of the heating condition is 30-65 ℃;

and/or the leacheate is a saturated sodium chloride solution and dichloromethane in the following proportion: (1-2): (1-2).

6. A pharmaceutical composition characterized by: an EGCG derivative having the structure of formula i as claimed in claim 1 and pharmaceutically acceptable salts thereof.

7. The use of EGCG derivatives having the structure of formula i and pharmaceutically acceptable salts thereof as claimed in claim 1 as COX-2 inhibitors for the preparation of medicaments for the treatment of tumors.

8. Use of a pharmaceutical composition according to claim 6 as a COX-2 inhibitor in the manufacture of a medicament for the treatment of a tumour.

9. Use according to claim 7 or 8, wherein the tumour is selected from: esophageal cancer, gastric cancer, pancreas cancer, liver cancer, lung cancer, colon cancer, rectal cancer, cervical cancer, skin cancer or breast cancer.