Rhein phosphorylation derivative as well as synthesis method and application thereof

Abstract

The invention belongs to the technical field of organic chemical synthesis, and particularly relates to a rhein phosphorylation derivative as well as a synthesis method and application thereof. The preparation method comprises the following steps: taking rhein as a research object, selecting a carboxyl group at a site 3 of the rhein as a modification site, introducing phosphorylation groups containing different substituent groups into a rhein structure on the basis of classical reaction mechanisms of Kabachnic-Fields reaction, esterification and the like and a pharmacophore splicing principle, and designing and synthesizing to obtain 35 rhein phosphorylation derivatives, a novel rhein phosphorylation derivative synthesis method which is mild in reaction condition, environmentally friendly and high in yield is established, the provided novel rhein phosphorylation derivative is low in toxicity to normal hepatocytes and remarkable in anti-proliferation activity to hepatoma carcinoma cells, the action effect is better than that of novel active molecules of rhein, and the novel rhein phosphorylation derivative can be used for preparing the novel rhein phosphorylation derivative. And a new thought is provided for the preparation of antitumor drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of organic chemical synthesis, and specifically relates to a phosphorylated rhein derivative and a synthesis method and application thereof. Background Art

[0002] Malignant tumors are a type of disease that seriously threatens human life. In recent years, the incidence and mortality rates of malignant tumors have been on the rise. At present, the anti-tumor drugs commonly used in clinical practice have serious side effects and are prone to drug resistance, which seriously affects their therapeutic effects. A large number of studies have shown that the anti-tumor active ingredients extracted from natural products not only have diverse chemical structures and mechanisms of action, but also have relatively good safety. Therefore, the optimization of natural anti-tumor active ingredients as lead compounds has attracted the attention of many pharmaceutical workers.

[0003] Rhein is one of the effective components of rhubarb, and has multiple pharmacological activities such as anti-inflammatory, anti-tumor, anti-Alzheimer's disease, antibacterial, antiviral and bone tropism. In particular, rhein has a broad spectrum of anti-tumor effects, and its mechanism of action is diverse. In addition, its low toxicity and high safety provide a good prospect for the development of highly effective, low-toxic and targeted rhein anti-tumor drugs. However, due to its own rigid structure, its clinical application is subject to certain restrictions. Phosphoric acid plays an important physiological role in the cells of organisms. It is a structural component of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It specifically binds to target proteins and receptors in the body and is an important group of protein translation signals and phospholipids.

[0004] Based on this, the present invention uses the C-3 carboxyl group of rhein as the modification site, performs structural modification on rhein, introduces a structural fragment containing a phosphate group, and uses anti-tumor experiments to screen the biological activities of the synthesized rhein phosphoester derivatives, in order to obtain phosphorylated rhein derivatives with excellent anti-tumor activity. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a phosphorylated derivative of rhein and a synthesis method and application thereof. By modifying the No. 3 carboxyl group of rhein, a new phosphorylated derivative of rhein with high yield and significant anti-liver cancer activity is synthesized.

[0006] The technical solution of the present invention is as follows:

[0007] The phosphorylated rhein derivative provided by the present invention is characterized in that the phosphorylated rhein derivative has a structural formula 1:

[0008]

[0009] R1 is methyl or ethyl, and R2 is any one of H, methyl, methoxy, fluoride ion, chloride ion, bromide ion, trifluoromethyl, and trifluoromethoxy.

[0010] The synthesis method of the above-mentioned phosphorylated rhein derivative comprises the following steps:

[0011] (1) Using N-(Boc)-ethylenediamine, benzaldehyde and phosphite as raw materials, condensing to obtain an intermediate;

[0012] (2) removing the protecting group Boc from the intermediate in (1) to obtain compound I;

[0013] (3) subjecting rhein to an esterification reaction with compound I to obtain a phosphorylated rhein derivative having a structure as described in Formula 1;

[0014] The structural formula of compound I is as follows:

[0015]

[0016] Among them, R1 is methyl or ethyl, and R2 is any one of H, methyl, methoxy, fluoride ion, chloride ion, bromide ion, trifluoromethyl, and trifluoromethoxy.

[0017] The synthetic route of phosphorylated derivatives of rhein is as follows:

[0018]

[0019] The synthetic method of the above-mentioned phosphorylated derivative of rhein comprises the following specific steps:

[0020] (1) Using N-(Boc)-ethylenediamine, benzaldehyde and phosphite as raw materials, FeCl3 as catalyst and tetrahydrofuran as solvent, heating to 50-70°C under Ar gas protection for constant temperature reaction for 12-16 hours, then distilling under reduced pressure to recover the solvent, and separating by column chromatography to obtain an intermediate;

[0021] (2) The intermediate in (1) is used as a raw material, trifluoroacetic acid is used as a catalyst, and CH2Cl2 is used as a solvent. The reaction is carried out at 0°C in an ice bath for 30 to 70 minutes. The solvent is then recovered by distillation under reduced pressure, and compound I is obtained by column chromatography.

[0022] (3) Using 1-ethyl-3 (3-dimethylpropylamine) carbodiimide and 4-dimethylaminopyridine as catalysts and tetrahydrofuran as solvent, rhein and compound I are subjected to an esterification reaction to finally obtain a phosphorylated rhein derivative having a structure described in formula 1.

[0023] Preferably, in (1), the benzaldehyde is selected from any one of p-tolualdehyde, p-methoxybenzaldehyde, 4-chlorobenzaldehyde, 4-bromobenzaldehyde, p-fluorobenzaldehyde, 2-fluorobenzaldehyde, 3,4,5-trimethoxybenzaldehyde, 4-chloro-3-fluorobenzaldehyde, 3,4-dimethylbenzaldehyde, 2,4-difluorobenzaldehyde, 2-bromo-4-fluorobenzaldehyde, 3-bromo-4-fluorobenzaldehyde, 5-bromo-2-fluorobenzaldehyde, 2,4-dichlorobenzaldehyde, p-trifluoromethylbenzaldehyde, p-trifluoromethoxybenzaldehyde, 2-trifluoromethylbenzaldehyde, 2-fluoro-4-methoxybenzaldehyde, 3-fluoro-4-methoxybenzaldehyde, 3-fluorobenzaldehyde, and 4-(difluoromethoxy)benzaldehyde;

[0024] The phosphite is selected from any one of Formula 1 to Formula 2:

[0025]

[0026] The application of the above-mentioned phosphorylated rhein derivatives in the preparation of anti-tumor drugs is the key content to be protected by the present invention; the above-mentioned anti-tumor drugs refer to: any one of drugs for cervical cancer, gastric cancer, liver cancer, and non-small cell lung cancer.

[0027] The beneficial effects of the present invention are:

[0028] (1) The present invention uses rhein as the research object, selects its No. 3 carboxyl group as the modification site, introduces phosphoryl groups containing different substituents into the structure of rhein based on the classic Kabachnic-Fields reaction, esterification and other reaction mechanisms and the principle of pharmacophore splicing, designs and synthesizes 35 phosphorylated derivatives of rhein, and establishes a new method for synthesizing phosphorylated derivatives of rhein with mild reaction conditions, environmental friendliness and high yield;

[0029] (2) The new phosphorylated rhein derivatives provided by the present invention have low toxicity to normal liver cells and significant anti-proliferation activity against a variety of cancer cells, providing a new idea for the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Effects of phosphorylated rhein derivatives C9 and D9 on the migration ability of HepG2 cells;

[0031] Figure 2 The effects of phosphorylated derivatives of rhein, C9 and D9, on apoptosis of HepG2 cells;

[0032] Figure 3 This is the effect of phosphorylated rhein derivatives C9 and D9 on the cell cycle distribution of HepG2 cells. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0034] Example 1

[0035] A phosphorylated rhein derivative having a structure of general formula 1:

[0036]

[0037] The phosphorylated rhein derivative having the general formula 1 is prepared by the following method:

[0038] (1) N-(Boc)-ethylenediamine (2 mmol, 320 μL), benzaldehyde (2 mmol, 247 μL) and phosphite (2 mmol, 183 μL) are used as raw materials in a molar ratio of 1:1:1, ferric chloride is used as a catalyst in an amount of 25 mg, and tetrahydrofuran is used as a solvent in an amount of 5 mL. The mixture is heated to 50-70° C. under Ar gas protection for constant temperature reaction for 12-16 hours. After the reaction is completed, the mixture is cooled to room temperature, and the solvent is recovered by vacuum distillation. The mixture is separated by silica gel column chromatography, and the eluent used is dichloromethane:acetone = 80:1-50:1 (volume ratio, the elution ratios hereinafter refer to volume ratios unless otherwise specified) by gradient elution to obtain an intermediate;

[0039] (2) The intermediate in (1) was used as a raw material (70 mg), trifluoroacetic acid was used as a catalyst (2 mL), and dichloromethane was used as a solvent (4 mL). The mixture was reacted in an ice bath at 0°C for 30 to 70 min, and then the solvent was recovered by distillation under reduced pressure. The mixture was separated by silica gel column chromatography, and the eluent volume ratio was dichloromethane:methane = 30:1 to 5:1 gradient elution to obtain compound I.

[0040] (3) Taking the 3-position carboxyl group of rhein as the modification site, 120 mg of rhein and 160 mg of compound I were weighed, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (EDCI, 240 mg) and 4-dimethylaminopyridine (DMAP, 200 mg) were used as catalysts, and tetrahydrofuran (10 mL) was used as solvent to carry out esterification reaction between rhein and compound I. After the reaction was completed, the solvent was recovered by vacuum distillation, and the mixture was separated by silica gel column chromatography. The eluent used was dichloromethane:methanol = 120:1 to 80:1 gradient elution, and finally a phosphorylated rhein derivative having the structure described in Formula 1 was obtained.

[0041] Example 1-1

[0042] The only difference from Example 1 is:

[0043] (1), the selected benzaldehyde is p-methoxybenzaldehyde, and the phosphite is dimethyl phosphite;

[0044] The other steps are the same as those in Example 1.

[0045] Example 1-2

[0046] The only difference from Example 1 is:

[0047] (1), the selected benzaldehyde is 4-chlorobenzaldehyde, and the phosphite is diethyl phosphite;

[0048] Examples 1-3

[0049] The only difference from Example 1 is:

[0050] (1), the selected benzaldehyde is 4-bromobenzaldehyde, and the phosphite is diethyl phosphite;

[0051] As can be seen from the above, changing the benzaldehyde in Examples 1-1, 1-2, and 1-3 and selecting dimethyl phosphite or diethyl phosphite can achieve the purpose of synthesizing the target product. Therefore, the benzaldehyde in Example 1 can be p-methoxybenzaldehyde, 4-chlorobenzaldehyde, 4-bromobenzaldehyde, p-fluorobenzaldehyde, 2-fluorobenzaldehyde, 3,4,5-trimethoxybenzaldehyde, 4-chloro-3-fluorobenzaldehyde, 3,4-dimethylbenzaldehyde, in addition to p-methylbenzaldehyde. The phosphite may be any one of methyl phosphite, 2,4-difluorobenzaldehyde, 2-bromo-4-fluorobenzaldehyde, 3-bromo-4-fluorobenzaldehyde, 5-bromo-2-fluorobenzaldehyde, 2,4-dichlorobenzaldehyde, p-trifluoromethylbenzaldehyde, p-trifluoromethoxybenzaldehyde, 2-trifluoromethylbenzaldehyde, 2-fluoro-4-methoxybenzaldehyde, 3-fluoro-4-methoxybenzaldehyde, 3-fluorobenzaldehyde, and 4-(difluoromethoxy)benzaldehyde, and the phosphite may be dimethyl phosphite or diethyl phosphite.

[0052] The phosphorylated rhein derivatives in C1-C23 and D1-D22 were prepared by the method in Example 1. The specific structures of the phosphorylated rhein derivatives obtained are shown in Table 1:

[0053] Table 1 Structures of phosphorylated derivatives of rhein

[0054]

[0055]

[0056]

[0057]

[0058] Experimental Example 1 Evaluation of the Anti-liver Cancer Activity of Phosphorylated Rhein Derivatives

[0059] The cytotoxicity of rhein and its phosphorylated derivatives in Example 1 to human cervical cancer cells HeLa, human liver cancer cells HepG-2, human gastric cancer cells MGC803, human non-small cell lung cancer cells A549 and normal cells L02 was determined by MTT method. The results are shown in Table 2 and Table 3:

[0060] Table 2 Antiproliferative activity of rhein and its phosphorylated derivatives C1-C23 against four cancer cells and cytotoxicity against normal cells L02

[0061]

[0062]

[0063] Table 3 Antiproliferative activity of rhein and its phosphorylated derivatives D1-D22 against four cancer cells and cytotoxicity against normal cells L02

[0064]

[0065] As shown in Tables 2 and 3, the target compounds showed different antiproliferative activities on the selected tumor cell lines, and also showed different cytotoxicity to normal liver cells. Among them, the target compounds C9 and D9 had significant antiproliferative activity on HepG2 cells, with IC50 values ​​of 11.88±0.93μM and 15.87±1.76μM, respectively, which were better than the positive control drugs cisplatin and 5-fluorouracil (IC50 values ​​of 83.28±5.91μM and 20.26±0.99μM, respectively). In addition, the antitumor activity of compounds C9 and D9 on liver cancer cells HepG2 was at least 8 times that of their cytotoxicity on normal liver cells L02, which indicated that compounds C9 and D9 had certain selectivity in antiproliferative activity on normal cells and tumor cells. Therefore, compounds C9 and D9 were selected as representative compounds and their inhibitory activity and mechanism of action on HepG2 cells were further studied.

[0066] The results of cell wound healing experiments showed that the phosphorylated derivatives of rhein C9 and D9 had a certain inhibitory effect on the migration and repair of liver cancer cells HepG2, and showed a dose-dependent effect. The results of cell cycle experiments showed that the phosphorylated derivatives of rhein C9 and D9 could induce apoptosis of HepG2 cells and had a certain concentration dependence. The results of cell cycle experiments showed that the phosphorylated derivatives of rhein C9 and D9 could block HepG2 cells in the G2 / M phase in a dose-dependent manner, thereby exerting anti-tumor activity. Figure 1 to Figure 3 shown.

Claims

1. A phosphorylated derivative of rhein, It is characterized in that The phosphorylated rhein derivative has a structural formula 1: R 1 is methyl or ethyl, R 2 It is any one of H, methyl, methoxy, fluoride, chloride, bromide, trifluoromethyl and trifluoromethoxy.

2. The method for synthesizing the phosphorylated derivative of rhein as claimed in claim 1, The following steps are involved: (1) Using N-(Boc)-ethylenediamine, benzaldehyde and phosphite as raw materials, condensing to obtain an intermediate; (2) removing the protecting group Boc from the intermediate in (1) to obtain compound I; (3) subjecting rhein to an esterification reaction with compound I to obtain a phosphorylated rhein derivative having a structure as described in Formula 1; The structural formula of the compound I is as follows: Among them, R 1 is methyl or ethyl, R 2 It is any one of H, methyl, methoxy, fluoride, chloride, bromide, trifluoromethyl and trifluoromethoxy.

3. The synthesis method according to claim 2, wherein the specific steps are as follows: (1) Using N-(Boc)-ethylenediamine, benzaldehyde and phosphite as raw materials, FeCl 3 As catalyst, tetrahydrofuran is used as solvent, and the mixture is heated to 50-70°C under the protection of Ar gas for constant temperature reaction for 12-16 hours, and then the solvent is recovered by vacuum distillation, and the intermediate is obtained by column chromatography separation; (2) Using the intermediate in (1) as raw material and trifluoroacetic acid as catalyst, CH 2 Cl 2 As solvent, react at 0℃ in an ice bath for 30-70min, then recover the solvent by distillation under reduced pressure, and separate by column chromatography to obtain compound Ⅰ; (3) Using 1-ethyl-3 (3-dimethylpropylamine) carbodiimide and 4-dimethylaminopyridine as catalysts and tetrahydrofuran as solvent, rhein and compound I are subjected to an esterification reaction to finally obtain a phosphorylated rhein derivative having a structure described in formula 1.

4. The synthesis method according to claim 2, It is characterized in that In (1), the benzaldehyde is selected from any one of p-tolualdehyde, p-methoxybenzaldehyde, 4-chlorobenzaldehyde, 4-bromobenzaldehyde, p-fluorobenzaldehyde, 2-fluorobenzaldehyde, 3,4,5-trimethoxybenzaldehyde, 4-chloro-3-fluorobenzaldehyde, 3,4-dimethylbenzaldehyde, 2,4-difluorobenzaldehyde, 2-bromo-4-fluorobenzaldehyde, 3-bromo-4-fluorobenzaldehyde, 5-bromo-2-fluorobenzaldehyde, 2,4-dichlorobenzaldehyde, p-trifluoromethylbenzaldehyde, p-trifluoromethoxybenzaldehyde, 2-trifluoromethylbenzaldehyde, 2-fluoro-4-methoxybenzaldehyde, 3-fluoro-4-methoxybenzaldehyde, 3-fluorobenzaldehyde, and 4-(difluoromethoxy)benzaldehyde; The phosphite is selected from any one of Formula 1 to Formula 2:

5. The synthesis method according to claim 3, It is characterized in that In (1), the molar ratios of N-(Boc)-ethylenediamine, benzaldehyde and phosphite are 1:1:1; FeCl 3 The amount of the reaction system composed of N-(Boc)-ethylenediamine, benzaldehyde and phosphite is 55% to 65% by weight. The amount of tetrahydrofuran used accounts for 30% to 40% of the volume of the above reaction system.

6. The synthesis method according to claim 3, It is characterized in that (2), the volume mass ratio of trifluoroacetic acid to the intermediate is 1 mL: 30 mg to 40 mg, trifluoroacetic acid, CH 2 Cl 2 The volume ratio = 1:1.5~4.

7. The synthesis method according to claim 3, It is characterized in that (3), the mass ratios of compound I to 1-ethyl-3 (3-dimethylpropylamine) carbodiimide and 4-dimethylaminopyridine are 4:6:5, respectively; The molar ratio of rhein to compound I = 1:1 to 3:5; The tetrahydrofuran accounts for 25% to 35% of the volume of the reaction system composed of compound I, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide and 4-dimethylaminopyridine.

8. Use of the phosphorylated rhein derivative according to claim 1 in the preparation of anti-tumor drugs.

9. The use according to claim 8, It is characterized in that The anti-tumor drug refers to any one of: drugs for cervical cancer, drugs for gastric cancer, drugs for liver cancer, and drugs for non-small cell lung cancer.

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