CN117304158A - Epigallocatechin gallate derivative and preparation method and application thereof - Google Patents

Epigallocatechin gallate derivative and preparation method and application thereof Download PDF

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CN117304158A
CN117304158A CN202210709564.7A CN202210709564A CN117304158A CN 117304158 A CN117304158 A CN 117304158A CN 202210709564 A CN202210709564 A CN 202210709564A CN 117304158 A CN117304158 A CN 117304158A
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egcg
fmoc
epigallocatechin gallate
derivative
integers
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高钟镐
刘超
黄伟
金明姬
王启明
陈丽青
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Abstract

The present invention belongs to the field of medicine chemistry and medicine preparation technology. An epigallocatechin gallate derivative is prepared from epigallocatechin gallate (EGCG) and fluorenylmethoxycarbonyl chloride (Fmoc-Cl) through reaction in the presence of acid-binding agent. The derivative can effectively improve the fat solubility, stability and pharmacological activity of EGCG. The EGCG derivative provided by the invention can be self-assembled in water to form nano particles with the particle size of about 100nm, and the property can improve the cell uptake efficiency of EGCG in vivo and increase the capability of the EGCG in vivo for targeting tumor focus positions. The invention can solve the clinical application problems of poor EGCG stability, low bioavailability and the like, has simple preparation method, is easy for industrial production and amplification, and has better application prospect in promoting EGCG clinical application transformation.

Description

Epigallocatechin gallate derivative and preparation method and application thereof
Technical Field
The invention relates to an epigallocatechin gallate derivative, a preparation method and application thereof, belonging to the technical fields of pharmaceutical chemistry and pharmaceutical preparations.
Background
Tea polyphenols are mainly present in tea leaves, which can be classified into six major classes, such as green tea (unfermented), white tea, yellow tea, oolong tea (semi-fermented), black tea, and black tea (fully fermented), depending on the degree of fermentation. Green tea is one of the most consumed beverages in the world, and is mainly extracted from the leaves of evergreen shrubs camellia, and the most complete content of catechin stored in green tea is also the most abundant because green tea is not fermented. Catechin is a subgroup of flavonoids, and mainly comprises Epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), epigallocatechin-3-gallate (EGCG), and the like. The most abundant EGCG content accounts for about 50-80% of the total content of tea polyphenols, so that many health benefits of green tea are related to EGCG, which is also the most widely studied tea polyphenols in current medical research. EGCG has unique antioxidant and free radical quenching characteristics due to its special polyphenol structure, and can reduce or eliminate oxygen free radicals at focus positions, thereby reducing oxidative stress reaction at disease positions and avoiding further occurrence and development of diseases. EGCG has been widely studied for its therapeutic and prophylactic effects on a range of diseases related to oxygen radicals, such as cancer, cardiovascular diseases, antiviral, diabetes, ischemic diseases, metabolic syndrome, inflammation, aging resistance, renal diseases, neurodegenerative diseases, and the like.
EGCG has various beneficial pharmacological activities and has great application potential in the aspects of disease treatment and the like. However, EGCG has some drawbacks in specific applications, such as poor lipid solubility, rapid dissolution, low bioavailability, short half-life, etc. The structural characteristics of EGCG make it susceptible to oxidation, thereby reducing its stability. Furthermore, its absorption and bioavailability in clinical applications may be limited due to its physical and chemical properties at physiological pH values. Studies on EGCG pharmacokinetics have shown that EGCG has low oral bioavailability in rodents, approximately between 2% and 13%. The analysis of the cause may be related to EGCG degradation in gastrointestinal fluids, poor intestinal tract transport caused by EGCG passive diffusion and active excretion, first pass effect of liver and the like.
By increasing the fat solubility of the water-soluble EGCG, the capacity of penetrating cells can be improved, so that the bioavailability of the EGCG in clinical application is effectively improved. Therefore, many researchers are devoting to the modification research of tea polyphenols such as EGCG, for example, they improve the solubility and stability of tea polyphenols such as EGCG in lipid medium by physical method, chemical modification method, enzyme catalysis method and the like, thereby increasing the bioavailability of tea polyphenols in vivo. The chemical modification method is to modify some lipophilic substituent groups to the molecular structure of tea polyphenol such as EGCG, so as to improve the physical and chemical properties of the tea polyphenol, increase the fat solubility and stability of the tea polyphenol, and retain or even enhance the pharmacological activities such as oxidation resistance and the like of the tea polyphenol. For example, it is reported in the literature that peracetic acid modified EGCG derivatives (Lambert JD, sang S, hong J, et al Drug MetabDispos.2006;34 (12): 2111-2116) have higher growth inhibitory activity on human esophageal cancer cells and human colon cancer cells than EGCG, which can be rapidly converted to EGCG by HCT116 cells, and that the EGCG concentration in HCT116 cells is 2.8-30 times that in the EGCG treated group. In addition, the bioavailability, stability and protease inhibition and anticancer activity of the acetylated protected EGCG in human breast cancer cells and tumors are higher (Landris-Piwowar KR, huo C, chen D, et al cancer Res.2007; 67 (9): 4303-4310). And, tea polyphenol palmitate is taken as a novel grease antioxidant, and is recorded in GB2760-2014 'national food safety Standard food additive use Standard' by national guard and Committee in month 6 of 2014.
According to the invention, the fat-soluble 9-fluorenylmethoxycarbonyl (Fmoc) is modified on the water-soluble EGCG to obtain the fat-soluble epigallocatechin gallate 9-fluorenylmethoxycarbonyl derivative (EGCG-Fmoc), and the derivative can effectively improve the fat-solubility, stability and pharmacological activity of EGCG. In addition, the derivative can self-assemble in water to form micelle-like nano particles with the particle diameter of about 100nm, and the property can improve the cell uptake efficiency of EGCG in vivo and increase the capability of targeting tumor focus in vivo. The invention provides a novel fat-soluble EGCG derivative and a preparation method thereof for the first time, and the invention can solve the clinical application problem of EGCG and has better application potential in promoting the conversion of the clinical application of EGCG. In addition, the preparation method of the derivative is simple, is beneficial to industrial production and amplification, and has wide prospect in the aspects of rapid conversion and clinical application.
Disclosure of Invention
The invention aims to solve the technical difficulties of low stability, poor fat solubility and difficult delivery in the clinical application process of water-soluble epigallocatechin gallate, and provides a self-assembled fat-soluble epigallocatechin gallate derivative EGCG-Fmoc.
The second object of the invention is to provide a preparation method of epigallocatechin gallate derivative EGCG-Fmoc which can be self-assembled.
The third object of the invention is to provide a self-assembled epigallocatechin gallate derivative EGCG-Fmoc application in targeted delivery of EGCG.
In order to solve the technical problems of the invention, the invention provides the following technical scheme:
in a first aspect, the invention provides an epigallocatechin gallate derivative EGCG-Fmoc or pharmaceutically acceptable salt thereof, which is characterized by having a structural formula shown in formula (I):
wherein a and b are independently selected from integers of 0, 1, 2 or 3 and c is selected from integers of 0, 1 or 2. The dotted circular line indicates that any of the phenol groups in the epigallocatechin gallate structure may be modified by an ester linkage.
Further, the epigallocatechin gallate derivative EGCG-Fmoc or the pharmaceutically acceptable salt thereof is characterized by comprising a structure shown in a general formula (II):
wherein a and b are independently selected from integers of 0, 1, 2 or 3 and c is selected from integers of 0 or 1.
Further, the epigallocatechin gallate derivative EGCG-Fmoc or the pharmaceutically acceptable salt thereof is characterized by comprising a structure shown in a general formula (III):
wherein a and c are independently selected from integers of 0, 1 or 2 and b is selected from integers of 0, 1, 2 or 3.
Further, the epigallocatechin gallate derivative EGCG-Fmoc or the pharmaceutically acceptable salt thereof is characterized by comprising a structure shown in a general formula (IV):
wherein b and c are independently selected from integers of 0, 1, or 2, and a is selected from integers of 0, 1, 2, or 3.
Further, the epigallocatechin gallate derivative EGCG-Fmoc or the pharmaceutically acceptable salt thereof is characterized by comprising a structure shown in the following formula V:
in a second aspect, the invention provides a preparation method of epigallocatechin gallate derivative EGCG-Fmoc, which is characterized by comprising the following steps:
and (3) carrying out esterification reaction on the EGCG and Fmoc-Cl in the presence of an acid binding agent to obtain the self-assembled fat-soluble epigallocatechin gallate derivative EGCG-Fmoc.
Wherein the acid binding agent is one or more of sodium acetate, sodium carbonate, sodium bicarbonate, triethylamine and N, N-diisopropylethylamine; the esterification reaction temperature is 0-50 ℃, the reaction time is 1-12h, and the reaction solvent is one or more of acetone, ethyl acetate and tetrahydrofuran.
In a third aspect, the epigallocatechin gallate derivative EGCG-Fmoc or pharmaceutically acceptable salt thereof provided by the invention has self-assembly property, can improve the stability and pharmacological activity of EGCG, and provides an application in targeted delivery of EGCG.
The beneficial technical effects are as follows:
1. the EGCG-Fmoc of the epigallocatechin gallate derivative provided by the invention can improve the stability, the fat solubility and the pharmacological activity of EGCG and can improve the bioavailability of EGCG applied in vivo.
2. The epigallocatechin gallate derivative EGCG-Fmoc provided by the invention can be self-assembled into nano particles with the particle size of about 100nm in water, so that the cell uptake efficiency of EGCG can be increased, and the disease targeting of EGCG in-vivo application can be improved.
3. The preparation process of the epigallocatechin gallate derivative EGCG-Fmoc provided by the invention is simple, is beneficial to the process amplification production and is beneficial to the promotion of the clinical transformation.
Drawings
Fig. 1: the embodiment 2 of the invention provides an EGCG-Fmoc synthesis route pattern of an EGCG derivative.
Fig. 2: the embodiment 2 of the invention provides an EGCG-Fmoc nuclear magnetic resonance hydrogen spectrogram of an EGCG derivative.
Fig. 3: the epigallocatechin gallate derivative EGCG-Fmoc mass spectrum provided by the embodiment 2 of the invention.
Fig. 4: the epigallocatechin gallate derivative EGCG-Fmoc infrared spectrogram provided by the embodiment 2 of the invention.
Fig. 5: the epigallocatechin gallate derivative EGCG-Fmoc micelle particle size diagram provided by the embodiment 3 of the invention.
Fig. 6: the epigallocatechin gallate derivative EGCG-Fmoc micelle zeta potential map provided in the embodiment 3 of the invention.
Fig. 7: the epigallocatechin gallate derivative EGCG-Fmoc micelle proliferation inhibition chart provided by the embodiment 3 of the invention.
Fig. 8: the epigallocatechin gallate derivative EGCG-Fmoc micelle provided by the embodiment 3 of the invention is compared with EGCG proliferation inhibition.
Fig. 9: the Cou-6 marked epigallocatechin gallate derivative EGCG-Fmoc micelle cell uptake concentration dependence experiment provided in the embodiment 6 of the invention.
Fig. 10: the Cou-6 marked epigallocatechin gallate derivative EGCG-Fmoc micelle cell uptake time dependence experiment provided in the embodiment 6 of the invention.
Fig. 11: the Cou-6 marked epigallocatechin gallate derivative EGCG-Fmoc micelle provided in the embodiment 6 of the invention is compared with free Cou-6.
Fig. 12: the DiR marked epigallocatechin gallate derivative EGCG-Fmoc micelle and free DiR provided in the embodiment 9 of the invention have distribution patterns in the internal organs of tumor-bearing mice.
Detailed Description
The following examples are intended to illustrate the invention and are not intended to be limiting. The present invention will be further illustrated in detail with reference to examples, but the present invention is not limited to these examples and the preparation methods used. Moreover, the present invention may be equivalently replaced, combined, improved, or modified by those skilled in the art in light of the description of the present invention, but are included in the scope of the present invention.
Example 1: the epigallocatechin gallate derivative EGCG-Fmoc provided by the invention is mainly obtained by reacting one of phenolic hydroxyl groups in an EGCG structure with Fmoc-Cl to form ester, and the structure is shown in a formula V:
example 2: the embodiment is a preparation method of epigallocatechin gallate derivative EGCG-Fmoc shown in embodiment 1, the synthetic route is shown in figure 1, and the preparation method comprises the following steps:
in a reaction flask, 1mmol of EGCG was first dissolved in 10mL of acetone and the solution was stirred. Then 5mmol of anhydrous sodium acetate was added, and stirring was continued and the temperature was raised to 40 ℃. 1mmmol of Fmoc-Cl was dissolved in 5mL of acetone, and then added dropwise to the reaction system, and after the addition was completed, the reaction was continued with stirring for 6 hours. After the reaction, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a pale yellow foamy solid. After washing the foamy solid with dichloromethane and water, respectively, it was purified by column chromatography on silica gel (ethyl acetate: petroleum ether: acetic acid=20:10:1) to finally give a white foamy solid. The overall yield of the reaction was 45.72%. The reaction products were characterized by nuclear magnetic resonance hydrogen spectroscopy, mass spectrometry and infrared spectroscopy, respectively, and EGCG-Fmoc was successfully synthesized as can be seen in FIGS. 2,3 and 4.
Example 3: and (3) researching self-assembly performance of EGCG-Fmoc of the epigallocatechin gallate derivative. EGCG-Fmoc 10mg was weighed accurately, 200. Mu.L of absolute ethanol was added for dissolution, and added dropwise to 5mL of mildly stirred deionized water, during which the aqueous phase was transparent and milky white with opalescence. The particle size and zeta potential were measured by dynamic light scattering as shown in FIGS. 5 and 6. The epigallocatechin gallate derivative EGCG-Fmoc can be self-assembled in water to form nano particles with the particle size of about 100nm, the potential of the nano particles is negative, and the stability and in-vivo uptake of EGCG can be effectively improved.
Example 4: EGCG-Fmoc inhibition ability investigation of 4T1 cell proliferation
Log-grown 4T1 cells were seeded into 96-well plates at a density of 5000 cells/well and were allowed to stand at 37 ℃ under 5% CO2 for 24h. After the completion of the culture, the old medium was aspirated, and EGCG-Fmoc micelle solutions were added at concentrations of 1, 5, 10, 50, 100, 150, 250, 500. Mu.g/mL, respectively. Simultaneously setting a zeroing group (without adding cells and without adding drugs) and a control group (with cells and without adding drugs), and continuously placing the cells into a CO2 incubator for culturing for 24 hours, 48 hours and 72 hours respectively. After the end of the incubation, the medium was aspirated, 200 μl of serum-free medium containing 10% cck-8 was added to each well, incubation was continued for 2h, and OD values at λ=450 nm were measured with a microplate reader, and 6 replicate wells were set per group. Wherein the cell viability was calculated using the following formula.
As shown in FIG. 7, EGCG-Fmoc has no toxicity to cells at the concentration of 1-10 mug/mL, has better biocompatibility, starts to show toxicity when the concentration reaches 50 mug/mL, and shows proliferation inhibition effect on 4T1 at the concentration of 100-500 mug/mL.
Example 5: EGCG-Fmoc and EGCG have comparison investigation on 4T1 cell proliferation inhibition capability
Log-grown 4T1 cells were seeded into 96-well plates at a density of 5000 cells/well and were allowed to stand at 37 ℃ under 5% CO2 for 24h. After the completion of the culture, the old medium was aspirated, and EGCG aqueous solution and EGCG-Fmoc micelle solution having EGCG concentration of 0.5, 2.5, 5, 25, 50, 150, 250, 500. Mu.g/mL were added, respectively. Simultaneously setting a zeroing group (without adding cells and without adding drugs) and a control group (with cells and without adding drugs), and continuously placing the cells into a CO2 incubator for culturing for 24 hours. After the end of the incubation, the medium was aspirated, 200 μl of serum-free medium containing 10% cck-8 was added to each well, incubation was continued for 2h, and OD values at λ=450 nm were measured with a microplate reader, and 6 replicate wells were set per group. Wherein the cell viability was calculated using the following formula.
As shown in FIG. 8, under the condition of the same EGCG concentration, the inhibition capacity of EGCG-Fmoc on 4T1 cell proliferation is obviously stronger than that of EGCG on 4T1 cells, and the analysis reasons probably relate to higher cell uptake efficiency and stronger pharmacological activity of EGCG-Fmoc micelle.
Example 6: preparation of coumarin 6 (Cou-6) -labeled EGCG-Fmoc micelle
1mg of Cou-6 was weighed and dissolved in 1mL of absolute ethanol to prepare a Cou-6 ethanol solution having a concentration of 1 mg/mL. Accurately weighing 10mg of the fat-soluble epigallocatechin gallate derivative EGCG-Fmoc, adding 150 mu L of absolute ethyl alcohol and 50 mu L of 1mg/mL of Cou-6 ethanol solution for dissolution, and dropwise adding into 5mL of gently stirred deionized water. After the completion of the dropwise addition, stirring was continued for 10 minutes, and excess ethanol was removed by depressurization, whereby Cou-6 labeled EGCG-Fmoc micelle, namely Cou-6@EGCG-Fmoc, was finally obtained, wherein the concentration of Cou-6 was 10. Mu.g/mL.
Example 7: EGCG-Fmoc cell uptake efficiency investigation
And quantitatively examining the uptake efficiency of the EGCG-Fmoc nano micelle by adopting a flow cytometer. Logarithmic growth of 4T1 cells was taken at 15X 10 4 Is inoculated in 12-well plate at 37℃with 5% CO 2 And (5) standing and culturing for 24 hours under the condition. After the end of the culture, the old medium was aspirated. Cou-6 solution (Cou-6 concentration is 1 mug/mL) diluted by serum-free culture medium and Cou-6@EGCG-Fmoc solution (Cou-6 concentration is 0.5, 1 and 2 mug/mL) diluted by serum-free culture medium are respectively added, 3 compound holes are arranged in each group, and the whole experiment is protected from light. Setting serum-free culture solution as blank control group, culturing for 15min, 60min, and 120min respectively, and sucking off culture medium. The cells were rinsed 3 times with cold PBS, digested 5-10min with 0.25% pancreatin, collected and centrifuged at 500g for 5min. Cells were resuspended with 0.5ml pbs, filtered through a screen, and the filtrate was loaded and examined by flow cytometry for uptake of Cou-6 labeled EGCG-Fmoc nanomicelle by 4T1 cells.
As shown in fig. 9, 10, 11, 4T1 cells had time-and concentration-dependence on uptake of EGCG-Fmoc micelles and were more efficient in uptake relative to the free coumarin 6 solution.
Example 8: establishment of in-situ lotus 4T1 breast cancer mouse model
Female BALB/c mice were selected for 1×10 breast cancer cells in log phase 5 Seed at 0.2 mL/min in BALB/cAnd constructing a tumor-bearing mouse model under the fourth mammary gland cushion of the mouse. Tumor mass volume was measured dynamically with vernier calipers. Tumor volume calculation formula: v=0.5×l×d 2 (wherein V is tumor volume, L is tumor major diameter, and D is tumor minor diameter).
Example 9: preparation of Dir-labeled EGCG-Fmoc micelle
1mg of 1, 1-octacosyl-3, 3-tetramethylindocyanine iodide (Dir) was weighed and dissolved in 1mL of absolute ethanol to prepare a Cou-6 ethanol solution having a concentration of 1 mg/mL. Accurately weighing EGCG-Fmoc 10mg, adding 150 mu L absolute ethyl alcohol and 50 mu L1 mg/mL Dir ethanol solution for dissolution, and dropwise adding into 5mL mildly stirred deionized water. After the dropwise addition, stirring is continued for 10 minutes, and excess ethanol is removed by decompression, so that the DIR-marked EGCG-Fmoc micelle, namely Dir@EGCG-Fmoc, can be finally obtained, wherein the concentration of Dir is 10 mug/mL.
Example 10: in vivo imaging method for small animals to detect tissue distribution of DiR@EGCG-Fmoc:
BALB/c tumor-bearing mice were randomly divided into 2 groups of 3 groups of free DiR group and Dir@EGCG-Fmoc group. Until the tumor grows to 500mm 3 At this time, a single tail vein injection was performed with a DiR administration concentration of 10. Mu.g/kg. The fluorescence signal of DiR in mice was detected using in vivo imaging techniques 2, 4, 6, 8, 24 hours post-dose and recorded by photography. As shown in FIG. 12, the distribution of DiR-labeled EGCG-Fmoc micelle at the tumor site of the mice is more, which indicates that the EGCG-Fmoc micelle has the targeting distribution characteristic in vivo.

Claims (9)

1. An epigallocatechin gallate derivative EGCG-Fmoc or pharmaceutically acceptable salt thereof, which is characterized by having a structural formula (I):
wherein a and b are independently selected from integers of 0, 1, 2 or 3 and c is selected from integers of 0, 1 or 2; the dotted circular line indicates that any of the phenol groups in the epigallocatechin gallate structure may be modified by an ester linkage.
2. The epigallocatechin gallate derivative EGCG-Fmoc or a pharmaceutically acceptable salt thereof according to claim 1, comprising a structure of formula (ii):
wherein a and b are independently selected from integers of 0, 1, 2 or 3 and c is selected from integers of 0 or 1.
3. The epigallocatechin gallate derivative EGCG-Fmoc or a pharmaceutically acceptable salt thereof according to claim 1, comprising a structure of formula (iii):
wherein a and c are independently selected from integers of 0, 1 or 2 and b is selected from integers of 0, 1, 2 or 3.
4. The epigallocatechin gallate derivative EGCG-Fmoc or a pharmaceutically acceptable salt thereof according to claim 1, comprising a structure of formula (iv):
wherein b and c are independently selected from integers of 0, 1, or 2, and a is selected from integers of 0, 1, 2, or 3.
5. An epigallocatechin gallate derivative EGCG-Fmoc or a pharmaceutically acceptable salt thereof according to claim 1, comprising the structure of formula v:
6. the method for preparing epigallocatechin gallate derivative EGCG-Fmoc according to any one of claims 1 to 5, comprising the steps of:
and (3) carrying out esterification reaction on the EGCG and Fmoc-Cl in the presence of an acid binding agent to obtain the self-assembled fat-soluble epigallocatechin gallate derivative EGCG-Fmoc.
7. The preparation method according to claim 6, wherein the acid binding agent is one or more of sodium acetate, sodium carbonate, sodium bicarbonate, triethylamine, and N, N-diisopropylethylamine; the esterification reaction temperature is 0-50 ℃, the reaction time is 1-12h, and the reaction solvent is one or more of acetone, ethyl acetate and tetrahydrofuran.
8. The epigallocatechin gallate derivative EGCG-Fmoc or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, wherein the compound has self-assembly properties, and can improve the stability and pharmacological activity of EGCG.
9. Use of an epigallocatechin gallate derivative EGCG-Fmoc, or a pharmaceutically acceptable salt thereof, according to any of claims 1-5, in the preparation of targeted delivery of EGCG.
CN202210709564.7A 2022-06-22 2022-06-22 Epigallocatechin gallate derivative and preparation method and application thereof Pending CN117304158A (en)

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