A phosphorylated derivative of rhein and its synthesis method and application

Abstract

The present invention belongs to the technical field of organic chemical synthesis, and specifically relates to a phosphorylated derivative of rhein, and a synthesis method and application thereof. The present invention takes rhein as the research object, selects its No. 3 carboxyl group as the modification site, and introduces phosphorylation groups containing different substituents into the rhein structure based on the classic Kabachnic-Fields reaction, esterification and other reaction mechanisms and the pharmacophore splicing principle. 35 phosphorylated derivatives of rhein are designed and synthesized, and a synthesis method of novel phosphorylated derivatives of rhein with mild reaction conditions, environmental friendliness and high yield is established. The provided novel phosphorylated derivatives of rhein have low toxicity to normal liver cells, significant anti-proliferative activity against liver cancer cells, and are novel active molecules with better effects than rhein, providing a new idea for the preparation of anti-tumor drugs.

Description

Technical Field

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

[0002] Malignant tumors are a serious threat to human life, with morbidity and mortality rates increasing in recent years. Currently, commonly used anti-tumor drugs are associated with significant side effects and the development of drug resistance, severely impacting their therapeutic efficacy. Numerous studies have demonstrated that active anti-tumor compounds extracted from natural products possess diverse chemical structures and mechanisms of action, while also exhibiting relatively good safety profiles. Consequently, the optimization of lead compounds using natural anti-tumor active ingredients has attracted considerable attention in pharmaceutical research.

[0003] Rhein, one of the active ingredients in rhubarb, possesses a variety of pharmacological activities, including anti-inflammatory, anti-tumor, anti-Alzheimer's, antibacterial, antiviral, and bone-tropism activities. Rhein, in particular, exhibits broad-spectrum antitumor effects with diverse mechanisms of action. Its low toxicity and high safety profile, combined with its low safety profile, offer promising prospects for the development of highly effective, low-toxic, and targeted rhein-based antitumor drugs. However, its rigid structure limits its clinical application. Phosphate plays an important physiological role in cells. It is a structural component of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), specifically binding to target proteins and receptors in vivo, and serves as a key group in protein translation signals and phospholipids.

[0004] Based on this, the present invention uses the C-3 carboxyl group of rhein as the modification site, modifies the structure of 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 rhein phosphorylated 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 its synthesis method and application. 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 solutions of the present invention are 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, chloride, bromide, 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) esterifying rhein with compound I to obtain a phosphorylated rhein derivative having the structure of formula 1;

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

[0015]

[0016] Wherein, 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 synthesis method of the above-mentioned phosphorylated rhein derivative comprises the following specific steps:

[0020] (1) Using N-(Boc)-ethylenediamine, benzaldehyde and phosphite as raw materials, FeCl3 as catalyst and tetrahydrofuran as solvent, the mixture is heated to 50-70°C under Ar gas protection and reacted at a constant temperature for 12-16 hours. The solvent is then recovered by vacuum distillation and the intermediate is separated by column chromatography;

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

[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 obtain a phosphorylated rhein derivative having the structure described in formula 1.

[0023] Preferably, in (1), the benzaldehyde is selected from any one of p-methylbenzaldehyde, 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, drugs for gastric cancer, drugs for liver cancer, and drugs for 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, and introduces phosphoryl groups containing different substituents into the structure of rhein based on the classic Kabachnic-Fields reaction, esterification and pharmacophore splicing principles. 35 phosphorylated derivatives of rhein were designed and synthesized, and a new method for synthesizing phosphorylated derivatives of rhein with mild reaction conditions, environmental friendliness and high yield was established;

[0029] (2) The new phosphorylated derivatives of rhein provided by the present invention have low toxicity to normal liver cells and significant anti-proliferative activity against various 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 effect of phosphorylated derivatives of rhein C9 and D9 on apoptosis of HepG2 cells;

[0032] Figure 3 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 will be further explained in conjunction with specific embodiments.

[0034] Example 1

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

[0036]

[0037] The phosphorylated rhein derivative having the general structure of 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) were used as raw materials in a molar ratio of 1:1:1, ferric chloride was used as a catalyst in an amount of 25 mg, and tetrahydrofuran was used as a solvent in an amount of 5 mL. The mixture was heated to 50-70°C under Ar gas protection and reacted at this temperature for 12-16 h. After the reaction was completed, the mixture was cooled to room temperature, and the solvent was recovered by vacuum distillation. The mixture was separated by silica gel column chromatography using a gradient elution of dichloromethane:acetone = 80:1-50:1 (volume ratio, the elution ratios below are all volume ratios unless otherwise specified) to obtain an intermediate.

[0039] (2) The intermediate in (1) was used as a raw material (70 mg), trifluoroacetic acid as a catalyst (2 mL), and dichloromethane 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 using a gradient elution method with a volume ratio of dichloromethane to methane of 30:1 to 5:1 to obtain compound I.

[0040] (3) With the 3-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 using a gradient elution of dichloromethane:methanol = 120:1 to 80:1 as the eluent, to finally obtain a phosphorylated rhein derivative having the structure described in formula 1.

[0041] Example 1-1

[0042] The only difference from Example 1 is:

[0043] In (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] In (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] In (1), the selected benzaldehyde is 4-bromobenzaldehyde and the phosphite is diethyl phosphite;

[0051] As can be seen from the above, the purpose of synthesizing the target product can be achieved by changing the benzaldehyde in Examples 1-1, 1-2, and 1-3 and selecting dimethyl phosphite or diethyl phosphite. 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, etc. aldehyde, 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, 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 obtained phosphorylated rhein derivatives 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 on 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 assay. The results are shown in Tables 2 and 3:

[0060] Table 2 Antiproliferative activity of rhein and its phosphorylated derivatives C1-C23 against four cancer cells and cytotoxicity against normal cell 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 exhibited different antiproliferative activities against the selected tumor cell lines and also showed different cytotoxicity against normal liver cells. Among them, the target compounds C9 and D9 had significant antiproliferative activity against HepG2 cells, with IC50 values ​​of 11.88±0.93μM and 15.87±1.76μM, respectively, which were superior to 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 against liver cancer cells HepG2 was at least 8 times that of their cytotoxicity against normal liver cells L02, indicating that compounds C9 and D9 had certain selectivity in antiproliferative activity against normal cells and tumor cells. Therefore, compounds C9 and D9 were selected as representative compounds and their inhibitory activity and mechanism of action against 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-dependency. 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. Figures 1 to 3 shown.

Claims

1. A phosphorylated derivative of rhein, characterized in that: The phosphorylated rhein derivative has a structural formula 1: R1 is methyl or ethyl, and R2 is any one of H, methyl, methoxy, fluoride, chloride, bromide, and trifluoromethoxy.

2. The method for synthesizing the phosphorylated derivative of rhein as claimed in claim 1, comprising the following steps: (1) Using N-(Boc)-ethylenediamine, benzaldehyde or substituted benzaldehyde and phosphite as raw materials, condensing to obtain an intermediate; The substituted benzaldehyde is selected from any one of p-tolualdehyde, p-methoxybenzaldehyde, 4-chlorobenzaldehyde, 4-bromobenzaldehyde, p-fluorobenzaldehyde, 2-fluorobenzaldehyde, 3-fluorobenzaldehyde, and p-trifluoromethoxybenzaldehyde; (2) removing the protecting group Boc from the intermediate in (1) to obtain compound I; (3) esterifying rhein with compound I to obtain a phosphorylated rhein derivative having the structure of formula 1; The structural formula of the compound I is as follows: Wherein, R1 is methyl or ethyl, and R2 is any one of H, methyl, methoxy, fluoride ion, chloride ion, bromide ion, and trifluoromethoxy.

3. The synthesis method according to claim 2, wherein the specific steps are as follows: (1) Using N-(Boc)-ethylenediamine, benzaldehyde or substituted benzaldehyde and phosphite as raw materials, FeCl3 as catalyst and tetrahydrofuran as solvent, heating to 50-70°C under Ar gas protection and reacting at this temperature for 12-16 hours, then recovering the solvent by vacuum distillation and separating the intermediate by column chromatography; (2) The intermediate in (1) was used as a raw material, trifluoroacetic acid as a catalyst, and CH2Cl2 as a solvent. The reaction was carried out in an ice bath at 0°C for 30 to 70 minutes. The solvent was then recovered by distillation under reduced pressure and separated 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 obtain a phosphorylated rhein derivative having the structure described in formula 1.

4. The synthesis method according to claim 2, wherein In (1), the phosphite is selected from any one of Formulas 1 to 2:

5. The synthesis method according to claim 3, wherein In (1), the molar ratios of N-(Boc)-ethylenediamine, benzaldehyde / substituted benzaldehyde, and phosphite are 1:1:1; The amount of FeCl3 used accounts for 55% to 65% of the total weight of the reaction system consisting of N-(Boc)-ethylenediamine, benzaldehyde / substituted benzaldehyde and phosphite; The amount of tetrahydrofuran used accounts for 30% to 40% of the volume of the reaction system.

6. The synthesis method according to claim 3, wherein In (2), the volume mass ratio of trifluoroacetic acid to the intermediate is 1 mL:30 mg~40 mg, and the volume ratio of trifluoroacetic acid to CH2Cl2 is 1:1.5~4.

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