Phenoxazine derivative as well as preparation method and application thereof

Abstract

The invention discloses a phenoxazine derivative as well as a preparation method and application thereof, and belongs to the field of biological medicine synthesis. A series of phenoxazine derivatives are synthesized by using a simple, efficient and low-toxicity method, circular dichroism, fluorescence spectrum and ultraviolet absorption spectrum prove that the molecules not only have non-covalent interaction with DNA, but also can be subjected to transamidation reaction with the DNA to covalently modify the DNA, and meanwhile, the phenoxazine derivatives are relatively good in anti-tumor cell activity and relatively good in anti-tumor effect. And a new strategy is provided for the research of DNA targeted drugs.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceutical synthesis, and specifically relates to a phenoxazine derivative and a preparation method and application thereof. Background Art

[0002] Cancer is one of the main causes of death in humans, involving more than 150 diseases, whose common feature is the dysregulation of cell proliferation and apoptosis. The occurrence of cancer is the result of the accumulation of genetic and epigenetic changes. As a carrier of genetic information, DNA mutation, damage and abnormal epigenetic modification may cause dysregulation of cancer-related gene expression, leading to abnormal gene expression and destruction of cellular homeostasis (Nature, 2024. 629, 688-696.). Therefore, DNA plays a vital role in the occurrence and development of cancer, and drugs targeting DNA have also become an important direction for anti-cancer treatment.

[0003] DNA-targeted drugs interfere with the normal function of DNA by affecting or changing its structure, fundamentally inhibiting the proliferation of cancer cells and inducing their apoptosis. According to the mechanism of action, the DNA-targeted drugs that have been discovered so far are mainly divided into the following categories. Intercalation is an important form of interaction between small molecules and DNA. Organic intercalators generally contain planar or nearly planar aromatic ring structures such as quinoline, indole, phenazine, etc., which achieve intercalation by inserting planar aromatic rings between adjacent DNA base pairs, interfering with the replication and transcription process. The intercalating agents that have been marketed include actinomycin D, daunorubicin and doxorubicin (Proceedings of the National Academy of Sciences, 1989. 86, 3968-3972.); the second category is alkylating agents, which can produce highly active alkylating groups that react with DNA, thereby affecting its structure and function. Typical alkylating agents include cisplatin, whose IC50 value for Hela cells is about 263 μM (Current Topics in Medicinal Chemistry, 2019. 19, 691-712.); the third category is DNA cleavage agents that can induce DNA breaks, and representative drugs include bleomycin; in addition, there are some molecules that can target specific DNA structures to prevent the division and proliferation of cancer cells (Nature Reviews Drug Discovery, 2011.10(4), 261-275.). Although existing DNA targeted drugs have achieved certain results in cancer treatment in the past, they still have problems such as high cytotoxicity, increasing drug resistance and insufficient specificity. Therefore, the development of new DNA targeted drugs is of great significance.

[0004] Tricyclic heterocycles are important skeletons of many known drugs. The most common tricyclic is phenothiazine, which is the core skeleton of many antipsychotic drugs represented by chlorpromazine. Further studies have shown that common phenothiazine drugs such as chlorpromazine and levomepromazine can also significantly inhibit the proliferation of leukemia cells, while having no obvious cytotoxicity to normal lymphocytes (Cancer Chemotherapy and Pharmacology, 2004. 53, 267-275.). Actinomycin D entered clinical trials in the 1960s. It was the first DNA intercalator to enter the clinical stage, and its core structure is a phenoxazine tricyclic structure. The discovery of actinomycin D triggered extensive research on the anti-tumor effects of phenoxazines and their derivatives. A study in 2001 investigated the effect of a phenoxazine derivative Phx on the proliferation of human leukemia cell lines and found that the compound could significantly inhibit the proliferation of leukemia cells such as K562 and HL-60, with an IC50 of about 75 μM, and its inhibitory effect on normal cells was less than that on leukemia cells (Clinical Cancer Research, 2001. 7, 704-708.). In a 2004 article, the interaction between some phenoxazine derivatives and DNA, as well as the effect of the hydrophobic effect of the molecule and the specific charge on the DNA structure were investigated by ultraviolet absorption spectroscopy and circular dichroism spectroscopy (Nucleosides Nucleotides & Nucleic Acids, 2004. 23, 1639-1656.). These studies show that phenoxazine derivatives have broad research prospects in the field of DNA intercalators, and their development and application may provide new solutions for cancer treatment. Summary of the invention

[0005] The present invention provides a phenoxazine derivative and a preparation method and application thereof. The present invention uses a simple, efficient and low-toxic method to synthesize a series of phenoxazine derivatives, and proves through circular dichroism, fluorescence spectrum and ultraviolet absorption spectrum that these molecules not only have non-covalent interactions with DNA, but also can undergo transamidation reaction with DNA to covalently modify DNA, and have good anti-tumor proliferation effect.

[0006] The phenoxazine derivatives of the present invention have a general structural formula as shown in the following formula I:

[0007]

[0008] Formula I

[0009] in:

[0010] R1 is selected from H, CN or CH2OH;

[0011] R2 is selected from H, OH, O(CH2)2OH or O(CH2)2NR2;

[0012] R3 is selected from CH3, C(CH3)2COOR or CH2COOR;

[0013] R is H or CH3.

[0014] One of the preparation routes of the phenoxazine derivatives of the present invention, in the case where R2 = H and R3 = CH3, comprises the following steps:

[0015] (1) Compound 1 undergoes a nucleophilic substitution reaction with compound 2 in the presence of a base to obtain compound 3;

[0016] (2) Compound 3 undergoes oxidation reaction in the presence of a base to obtain compound 5;

[0017] (3) Compound 5 is condensed with phenoxazine or substituted phenoxazine via an acyl chloride intermediate to give product Ⅰ-1.

[0018] During the nucleophilic substitution reaction of compound 1 and compound 2, the base is selected from any one of potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, and potassium hydroxide, preferably sodium hydroxide. The reaction is carried out in a solvent, and the solvent is selected from any one of ethanol, DMF, THF, 2-butanone, and acetonitrile, preferably 2-butanone. The reaction temperature is 35°C to 100°C, preferably 50°C. The molar ratio of compound 1, compound 2, and base is 1: (1.2~2): (3~5), preferably 1: 1.8: 5.

[0019] During the oxidation reaction of compound 3, the base is any one of potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, and potassium hydroxide, preferably sodium hydroxide. The reaction system also includes an oxidant, which is any one of potassium permanganate, barium permanganate or TEMPO, preferably potassium permanganate. The molar ratio of compound 3 to the oxidant is 1: (1~2), preferably 1: 4 / 3. The reaction is carried out in a solvent, which is a mixed solvent of water and acetonitrile in a ratio of 1: (1~4).

[0020] During the reaction of compound 5 with phenoxazine or substituted phenoxazine, the reaction system also includes a condensing agent, which is cyanuric chloride and pyridine. The solvent used is any one of DMF, DCM, and DCE, preferably DMF. Furthermore, the molar ratio of phenoxazine or substituted phenoxazine, compound 5, cyanuric chloride, and pyridine is 1: (1-1.5): (1-1.8): 1.8, preferably 1: 1.2: 1.2: 1.8.

[0021] Synthesis route 1 is as follows:

[0022]

[0023] The second preparation route of the phenoxazine derivatives of the present invention is the case where R2 = H and R3 = C(CH3)2COOR or CH2COOR (R is H or CH3), comprising the following steps:

[0024] (1) Compound 1 undergoes a nucleophilic substitution reaction with compound 2' in the presence of a base to obtain compound 3';

[0025] (2) Compound 3' undergoes esterification reaction with methanol under acid catalysis to obtain compound 4;

[0026] (3) Compound 4 undergoes oxidation reaction in an alkaline environment to obtain compound 5;

[0027] (4) Compound 5 is condensed with phenoxazine or substituted phenoxazine via an acyl chloride intermediate to give product Ⅰ-2.

[0028] R3 in compounds 4, 5 and product Ⅰ-2 is C(CH3)2COOCH3 or CH2COOCH3.

[0029] (5) Product Ⅰ-2 undergoes hydrolysis reaction in an alkaline environment to obtain product Ⅰ-3. R3 in product Ⅰ-3 is C(CH3)2COOH or CH2COOH.

[0030] During the nucleophilic substitution reaction of compound 1 and compound 2', the base is selected from any one of potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, and potassium hydroxide, preferably sodium hydroxide. The reaction is carried out in a solvent, and the solvent is selected from any one of ethanol, DMF, THF, 2-butanone, and acetonitrile, preferably 2-butanone. The reaction temperature is 35°C to 100°C, preferably 50°C. The molar ratio of compound 1, compound 2, and base is 1: (1.2~2): (3~5), preferably 1: 1.8: 5.

[0031] In the process of esterification of compound 3' with methanol under acid catalysis, the acid is selected from any one of hydrochloric acid, sulfuric acid, and p-toluenesulfonic acid, preferably sulfuric acid. The reaction temperature is 30° C. to 70° C., preferably 50° C. Further, the solvent is methanol.

[0032] During the oxidation reaction of compound 4, the base is any one of potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, and potassium hydroxide, preferably sodium hydroxide. The reaction system also includes an oxidant, which is any one of potassium permanganate, barium permanganate or TEMPO, preferably potassium permanganate. The molar ratio of compound 4 to the oxidant is 1: (1~2), preferably 1: 4 / 3. The reaction is carried out in a solvent, which is a mixed solvent of water and acetonitrile in a ratio of 1: (1~4).

[0033] During the reaction of compound 5 with phenoxazine or substituted phenoxazine, the reaction system also includes a condensing agent, which is cyanuric chloride and pyridine. The solvent used is any one of DMF, DCM, and DCE, preferably DMF. Furthermore, the molar ratio of phenoxazine or substituted phenoxazine, compound 5, cyanuric chloride, and pyridine is 1: (1-1.5): (1-1.8): 1.8, preferably 1: 1.2: 1.2: 1.8.

[0034] During the hydrolysis reaction of product Ⅰ-2, the base is any one of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium bromide, and lithium chloride, preferably lithium bromide; further, the solvent is a mixed solvent of water and acetonitrile, the ratio is 1: (20~50), preferably 1:50; further, the reaction temperature is 25°C~70°C, preferably 40°C.

[0035] Synthesis route 2 is as follows:

[0036]

[0037] The third preparation route of the phenoxazine derivatives of the present invention, when R2≠H and R3 = CH3, comprises the following steps:

[0038] (1) Compound 7 and compound 8 undergo nucleophilic substitution reaction in the presence of a base to obtain compound 9;

[0039] (2) Compound 9 is condensed with phenoxazine or substituted phenoxazine via an acyl chloride intermediate to obtain compound 10;

[0040] (3) Compound 10 undergoes hydrolysis in the presence of a base to obtain product Ⅰ-4;

[0041] (4) Product Ⅰ-4 undergoes nucleophilic substitution reaction with 2-bromoethanol or ethanolamine or N-methyl-2-hydroxyethylamine or N,N-dimethylethanolamine in the presence of a base to obtain product Ⅰ-5.

[0042] During the reaction of compound 7 and compound 8, the base is any one of pyridine, sodium hydroxide, and potassium hydroxide, preferably sodium hydroxide; further, the solvent is water; further, the molar ratio of compound 7, compound 8 and the base is 1: (1-3): 2, preferably 1: 2.3: 2.

[0043] In the reaction process of compound 9 and phenoxazine or substituted phenoxazine, the reaction system also includes a condensing agent, and the condensing agent is cyanuric chloride and pyridine. Further, the solvent used is any one of DMF, DCM, and DCE, preferably DMF; further, the molar ratio of phenoxazine or substituted phenoxazine, compound 9, cyanuric chloride, and pyridine is 1: (1-1.5): (1-1.8): 1.8, preferably 1: 1.2: 1.2: 1.8.

[0044] During the hydrolysis reaction of compound 10, the base is any one of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium bromide, and lithium chloride, preferably lithium bromide; further, the solvent is a mixed solvent of water and acetonitrile, the ratio of which is 1:(20~50), preferably 1:50; further, the reaction temperature is 25°C~70°C, preferably 40°C.

[0045] During the reaction of product Ⅰ-4, the base is any one of potassium carbonate, sodium carbonate, sodium hydroxide, and potassium hydroxide, preferably potassium carbonate; further, the solvent is any one of DMF, acetonitrile, and acetone, preferably DMF; further, the molar ratio of product Ⅰ-4, another reactant, and base is 1:1:1.2; further, the reaction temperature is 60°C~100°C, preferably 70°C.

[0046] Synthesis route three is as follows:

[0047]

[0048] The fourth preparation route of the phenoxazine derivative of the present invention is the case where R2≠H and R3 = C(CH3)2COOR or CH2COOR (R is H or CH3), comprising the following steps:

[0049] (1) Compound 11 undergoes a nucleophilic substitution reaction with compound 2' in the presence of a base to obtain compound 12;

[0050] (2) Compound 12 reacts with methanol under acid catalysis to obtain compound 13;

[0051] (3) Compound 13 reacts with phenoxazine or substituted phenoxazine via an acyl chloride intermediate to obtain compound 14;

[0052] (4) Compound 14 reacts with 48% hydrobromic acid in the presence of a phase transfer catalyst to obtain compound 15;

[0053] (5) Compound 15 undergoes nucleophilic substitution reaction with 2-bromoethanol or ethanolamine or N-methyl-2-hydroxyethylamine or N,N-dimethylethanolamine in the presence of a base to give product Ⅰ-6. In product Ⅰ-6, R3 is C(CH3)2COOCH3 or CH2COOCH3.

[0054] (6) Product Ⅰ-6 undergoes hydrolysis reaction in an alkaline environment to obtain product Ⅰ-7. In product Ⅰ-7, R3 is C(CH3)2COOH or CH2COOH.

[0055] During the reaction of compound 11 and compound 2', the base is selected from any one of potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, and potassium hydroxide, preferably sodium hydroxide. The reaction is carried out in a solvent, and the solvent is selected from any one of ethanol, DMF, THF, 2-butanone, and acetonitrile, preferably 2-butanone. The reaction temperature is 35°C to 100°C, preferably 50°C. The molar ratio of compound 11, compound 2', and base is 1: (1.2~2): (3~5), preferably 1: 1.8: 5.

[0056] In the process of esterification of compound 12 with methanol under acid catalysis, the acid is selected from any one of hydrochloric acid, sulfuric acid, and p-toluenesulfonic acid, preferably sulfuric acid. The reaction temperature is 30° C. to 70° C., preferably 50° C. Further, the solvent is methanol.

[0057] In the reaction process of compound 13 and phenoxazine or substituted phenoxazine, the reaction system also includes a condensing agent, and the condensing agent is cyanuric chloride and pyridine. Further, the solvent used is any one of DMF, DCM, and DCE, preferably DMF; further, the molar ratio of phenoxazine or substituted phenoxazine, compound 13, cyanuric chloride, and pyridine is 1: (1-1.5): (1-1.8): 1.8, preferably 1: 1.2: 1.2: 1.8.

[0058] In the reaction process of compound 14 with 48% hydrobromic acid in the presence of a phase transfer catalyst, the phase transfer catalyst is selected from any one of tetrabutylammonium bromide, hexadecyl tributylphosphonium bromide, tetraoctylammonium bromide, and trioctylmethylammonium bromide, preferably tetrabutylammonium bromide. The reaction temperature is 115°C. Furthermore, the molar ratio of compound 14, hydrogen bromide, and phase transfer catalyst is 1:(5-10):(0.05-0.2), preferably 1:5:0.1.

[0059] During the reaction of compound 15, the base is any one of potassium carbonate, sodium carbonate, sodium hydroxide, and potassium hydroxide, preferably potassium carbonate; further, the solvent is any one of DMF, acetonitrile, and acetone, preferably DMF; further, the molar ratio of compound 15, another reactant, and the base is 1:1:1.2; further, the reaction temperature is 60°C~100°C, preferably 70°C.

[0060] During the hydrolysis reaction of product Ⅰ-6, the base is any one of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium bromide, and lithium chloride, preferably lithium bromide; further, the solvent is a mixed solvent of water and acetonitrile, the ratio is 1: (20~50), preferably 1:50; further, the reaction temperature is 25°C~70°C, preferably 40°C.

[0061] Synthesis route 4 is as follows:

[0062]

[0063] The invention discloses an application of the phenoxazine derivative in preparing a DNA intercalating agent.

[0064] The invention discloses an application of the phenoxazine derivative in preparing a drug for inhibiting the growth of cancer cells.

[0065] The invention discloses an application of the phenoxazine derivative in the preparation of anti-tumor drugs.

[0066] The present invention also explores the interaction mechanism between the synthesized phenoxazine derivatives and DNA, comprising the following steps:

[0067] Phenoxazine or substituted phenoxazine, DNA, product, phenoxazine or substituted phenoxazine and DNA, product and DNA are dissolved in appropriate solutions at appropriate concentrations to prepare samples to be tested.

[0068] The circular dichroism spectra of DNA, product and mixed samples of product and DNA were tested respectively.

[0069] The fluorescence spectra of all samples to be tested were tested separately.

[0070] The UV absorption spectra of DNA, product, and mixed samples of product and DNA were tested respectively.

[0071] The invention determines the cell activity of the product through MTT experiment.

[0072] The preparation methods, mechanism of action and activities of two of the products are provided in the examples.

[0073] The occurrence of cancer is the result of the accumulation of genetic and epigenetic changes. As a carrier of genetic information, DNA mutation, damage and abnormal epigenetic modification may cause dysregulated expression of cancer-related genes, playing a vital role in the occurrence and development of cancer. Existing DNA intercalators such as actinomycin D have achieved certain results in cancer treatment in the past, but they are difficult to synthesize artificially, drug resistance is increasing, and most DNA intercalators only have non-covalent interactions with DNA.

[0074] The present invention uses a simple, efficient and low-toxic method to synthesize a series of phenoxazine derivatives, and proves through circular dichroism, fluorescence spectrum and ultraviolet absorption spectrum that these molecules not only have non-covalent interactions with DNA, but also can undergo transamidation reaction with DNA to covalently modify DNA, and at the same time have good anti-tumor cell activity, which is better than the anticancer drug cisplatin that also has a covalent effect with DNA, and provides a new strategy for the research of DNA targeted drugs.

[0075] The main advantages of the present invention are: (1) this type of phenoxazine derivative can undergo a transamidation reaction with DNA to covalently modify DNA, which is different from existing DNA intercalators and can provide a new strategy for DNA-targeted cancer treatment; (2) the several phenoxazine derivatives provided by the present invention have high anti-tumor cell activity and are superior to cisplatin, an anticancer drug that also has a covalent effect with DNA; (3) the synthesis method provided by the present invention is simple and efficient, can be synthesized in large quantities, and is low in cost; (4) in response to the drug resistance problem of existing drugs, the phenoxazine derivatives provided by the present invention provide a new treatment plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 : The specific synthetic route of compound A in Example 1.

[0077] Figure 2 : H NMR spectrum (400 MHz, DMSO-d6) of compound A in Example 1.

[0078] Figure 3 : Carbon NMR spectrum (400 MHz, DMSO-d6) of compound A in Example 1.

[0079] Figure 4 : Circular dichroism spectrum in Example 2.

[0080] Figure 5 : Fluorescence emission spectrum in Example 2.

[0081] Figure 6 : Ultraviolet absorption spectrum in Example 2.

[0082] Figure 7 : The possible mechanism of action of compound A and DNA in Example 2.

[0083] Figure 8 : Relationship diagram between Hela cell activity and compound A concentration in Example 3.

[0084] Fig. 9 : The specific synthetic route of compound B in Example 4.

[0085] Fig.10: H NMR spectrum of compound B in Example 4 (400 MHz, DMSO-d6).

[0086] Fig.11 : Carbon NMR spectrum (400 MHz, DMSO-d6) of compound B in Example 4.

[0087] Fig.12 : Circular dichroism spectrum in Example 5.

[0088] Fig.13 : Relationship diagram between Hela cell activity and compound B concentration in Example 6. DETAILED DESCRIPTION

[0089] The technical solution of the present invention is further analyzed and explained below through specific embodiments.

[0090] Embodiment 1:

[0091] A method for preparing a DNA-targeting phenoxazine derivative A, the implementation route is as follows Figure 1 As shown, the specific steps are as follows:

[0092] Step a: Dissolve p-hydroxybenzyl alcohol (5 mmol, 620.7 mg) in 10 mL 2-butanone, add finely ground sodium hydroxide solid (25 mmol, 1 g), and stir at 50 °C for 2 h. Add 2-bromoisobutyric acid (9 mmol, 1.5030 g) to the reaction solution and react for 3 h. Add 5 mL of water and stir at room temperature overnight. After removing 2-butanone by rotary evaporation, add 15 mL of ethyl acetate, adjust the pH to 6.5 with 3 M hydrochloric acid, and then add sodium bisulfite (10 mmol, 1.0406 g), and stir at room temperature overnight. Adjust the pH to 1.6 with 3 M hydrochloric acid, extract with ethyl acetate, wash the organic phase with saturated brine and dry with anhydrous sodium sulfate, and rotary evaporate to obtain 836.6 mg of yellow powdered solid with a yield of 80%.

[0093] Step b: The product of the previous step was dissolved in 4 mL of methanol, 2 drops of concentrated sulfuric acid were added, and the reaction was stirred at 55 °C for 3 h. The methanol was removed by rotary evaporation, 1 M sodium bicarbonate solution was added to adjust the pH to 8, extracted with DCM, dried over anhydrous sodium sulfate, and rotary evaporated to obtain 662 mg of brown oily product, which was directly carried out to the next step without purification.

[0094] Step c: The product of the previous step was dissolved in 3 mL of acetonitrile, potassium permanganate (3.94 mmol, 622 mg) was dissolved in as little water as possible, and then dripped into the reaction solution, and 2 drops of 6 M sodium hydroxide solution were added, and the reaction was stirred at room temperature for 1 h. 4 mL of water was added to the reaction solution, and the pH was adjusted to 1 with 3 M hydrochloric acid. The brown-black solid was removed by suction filtration, the filtrate was extracted with ethyl acetate, the organic phase was washed with sodium bicarbonate solution, the aqueous phase was separated, the pH was adjusted to 1 with 3 M hydrochloric acid, and then extracted with ethyl acetate, the organic phase was collected, dried with anhydrous sodium sulfate, and rotary evaporated to obtain 351.7 mg of a brown-yellow oily product with almost no impurities, with a yield of 50%.

[0095] Step d: The product of the previous step was dissolved in 5 mL of anhydrous DMF, and phenoxazine (1.48 mmol, 270.4 mg), pyridine (2.21 mmol, 179 μL) and cyanuric chloride (1.48 mmol, 272.2 mg) were added, and stirred at room temperature overnight. Diluted with water, adjusted to pH 2 with 1 M hydrochloric acid, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and rotary evaporated to obtain 339.5 mg of brown oil, which was directly carried out to the next step without purification.

[0096] Step e: The product of the previous step was dissolved in a mixed solvent of 3.4 mL acetonitrile and 68 μL water, and 350 μL triethylamine and lithium bromide (8.42 mmol, 730.9 mg) were added. The reaction mixture was stirred at 40°C for 40 min. Water was added to dilute the reaction solution, the pH was adjusted to 2 with 3 M hydrochloric acid, and extracted with ethyl acetate. The organic phase was washed with sodium bicarbonate solution, the aqueous phase was collected, the pH was adjusted to 2 with 3 M hydrochloric acid, and then extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and rotary evaporated, and purified by column chromatography (DCM: MeOH = 10: 1) to obtain product A (65.5 mg, yield 20%). 1 H NMR(400 MHz, DMSO-d6, ppm) δ 7.40(dd, J =8, 4 Hz, 2H), 7.28-7.18(m, 6H), 7.02(td, 2H), 6.70(d, 2H), 1.45(s, 6H). 13 CNMR(101 MHz, DMSO-d6) δ 174.68, 167.32, 158.19, 149.63, 130.46, 130.17,126.74, 126.42, 124.35, 123.71, 117.03, 116.71, 79.64, 25.48 ppm. MS(ESI)calculated for C23H19NO5 [M - H]- 388.41; found 388.22.

[0097] In addition, the condensation agent used in step d is cyanuric chloride, but it can also be achieved by using a combination of oxalyl chloride and DMF, so oxalyl chloride and DMF are alternatives. The specific method is: dissolve the product of the previous step in anhydrous dichloromethane, add 2 drops of DMF, and slowly add oxalyl chloride to the reaction solution. After reacting at room temperature for a period of time, the reaction solution is spun dry, anhydrous dichloromethane is added to dissolve, phenoxazine or substituted phenoxazine and TEA are mixed and dissolved in anhydrous dichloromethane, slowly dripped into the reaction solution, DMAP is added, and heated to react. TLC detects that phenoxazine or substituted phenoxazine is basically consumed, water is added to dilute the reaction solution, the pH is adjusted to 1-2, EA is extracted, the organic phase is washed with 1 M sodium bicarbonate solution, dried with anhydrous sodium sulfate and spun dry. Column chromatography purification (PE: EA = 5: 1). Furthermore, the molar ratio of the product obtained in step c to phenoxazine or substituted phenoxazine, oxalyl chloride, TEA, and DMAP is 1:(1-1.5):(1-2):(1-2):(0.02-0.1), preferably 1:1:1.3:1.3:0.05; further, the final reaction temperature is room temperature to 60°C, preferably 30°C.

[0098] Embodiment 2:

[0099] Investigation of the interaction mechanism between compound A synthesized by the present invention and DNA:

[0100] 1. Prepare the solution: Weigh 0.05 mmol EDTA disodium salt, 0.31 mmol disodium hydrogen phosphate and 0.19 mmol sodium dihydrogen phosphate and dissolve them in ultrapure water to prepare 50 mL of pH=7 buffer solution. Mix the buffer solution with ethanol in a volume ratio of 3:0.06 as the solvent for all subsequent samples. Prepare phenoxazine into a 35 μM solution, compound A into a 35 μM solution, and DNA from salmon into a 0.021 mg / mL solution. Phenoxazine is mixed with DNA, and compound A is mixed with DNA. The concentrations of phenoxazine and compound A are both 35 μM, and the concentration of DNA is 0.021 mg / mL. The solvent volume of each sample is 3.06 mL. All samples are treated at 4 °C for 24 hours.

[0101] 2. Using a mixed solvent of buffer solution and ethanol as the background solution, scan the circular dichroism spectra of DNA, compound A, and the mixed sample of compound A and DNA in sequence with a step size of 0.1 nm. Each sample is scanned 6 times for superposition. Figure 4 As shown, after compound A was mixed with DNA, the intensities of the two peaks of DNA at 275 nm and 250 nm weakened, and the peak shape around 220 nm was obviously destroyed, which indicates that compound A can interact with DNA and thus affect or even destroy the double helix structure of DNA.

[0102] 3. Use an ELISA reader to test the maximum fluorescence excitation wavelength of the five samples, and test the emission spectra of phenoxazine (312 nm excitation), phenoxazine and DNA mixture (312 nm excitation), DNA (282 nm excitation), compound A (374 nm excitation), and compound A and DNA mixture (364 nm excitation) at their respective maximum excitation wavelengths. Figure 5 As shown, after compound A is mixed with DNA, it emits the maximum emission wavelength of phenoxazine at 418 nm, while the maximum emission wavelength of compound A or DNA alone is somewhat different from 418 nm, which indicates that compound A can react with DNA to generate phenoxazine with strong fluorescence.

[0103] 4. Using a mixed solvent of buffer solution and ethanol as the background solution, scan the UV absorption spectra of DNA, compound A, and a mixed sample of compound A and DNA between 200 nm and 450 nm in a step size of 5 nm, and then simply add the spectral data of the two samples of DNA and compound A to obtain the corresponding curve. Figure 6 As shown, the curve obtained by simply adding the data of two separate samples of compound A and DNA cannot completely overlap with the curve measured by the compound A and DNA complex sample, and there is a large difference, indicating that compound A interacts with DNA.

[0104] The above results can prove that compound A has a strong non-covalent interaction with DNA, affecting the structure of DNA. In addition, some reactions generate phenoxazines with strong fluorescence. The possible mechanism is that the phenoxazine chromophore on compound A is embedded between DNA base pairs in a similar action mode to actinomycin D. The lone pair of electrons of the amino or imine on the DNA base pair attacks the carbonyl carbon atom of the amide on compound A, causing a transamidation reaction, covalently modifying DNA, resulting in significant changes in the DNA structure and the release of phenoxazines, such as Figure 7 shown.

[0105] Embodiment 3:

[0106] The inhibitory activity of compound A on Hela cells was detected by MTT assay. Its IC50 on Hela cells was 99 μM, which was superior to the widely used anticancer drugs cisplatin and paclitaxel. The IC50 of cisplatin on Hela cells was 263 μM, and the IC50 of paclitaxel on Hela cells was about 132 μM.

[0107] Embodiment 4:

[0108] A method for preparing a phenoxazine derivative B, the implementation route is as follows Fig. 9 As shown, the specific steps are as follows:

[0109] Step f: Dissolve 3 mmol NaOH in 1.7 mL water, add 1.5 mmol 3-hydroxy-4-methoxybenzoic acid to the solution under an ice-water bath, then add 3.45 mmol acetic anhydride, and stir at room temperature for 30 minutes. TLC shows that 3-hydroxy-4-methoxybenzoic acid is almost consumed, filter, wash the filter cake with ice water, collect the filter cake, dissolve the filter cake with a mixed solvent of ethyl acetate and dichloromethane, dry it with anhydrous sodium sulfate, and spin dry it to obtain 294 mg of pink powder with a yield of 93%.

[0110] Step g: 150 mg of the product from the previous step was dissolved in 2 mL of DMF, and 1 equivalent of phenoxazine, 1.5 equivalents of pyridine and 1 equivalent of cyanuric chloride were added in sequence, and the reaction was allowed to proceed overnight at room temperature. The mixture was diluted with water, the pH was adjusted to 2 with 1 M hydrochloric acid, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and rotary evaporated to obtain 107 mg of a brown-yellow oily product.

[0111] Step h: The product of the previous step was dissolved in a mixed solvent of 1.07 mL acetonitrile and 22 μL water, and 1 equivalent of lithium bromide and 3 equivalents of triethylamine were added, and the reaction was stirred at room temperature. After about 1.5 h, TLC showed that the raw material was almost exhausted. Dilute with water, adjust the pH to 2 with 1 M hydrochloric acid, extract with EA, dry the organic phase with anhydrous sodium sulfate, spin dry, and separate by column chromatography (PE: EA = 3:1) to obtain product B (55 mg, light yellow powder, yield 58%). 1 H NMR(400 MHz, DMSO-d6, ppm) δ 9.29(s, 1H), 7.40(dd,J1 = 8 Hz, J2 = 4 Hz, 2H), 7.25-7.17(m, 4H), 7.02(td, 2H), 6.87(d, 1H), 6.84-6.78(m, 2H), 3.75(s, 3H). 13 C NMR(101 MHz, DMSO-d6) δ 167.47, 150.01, 149.55,146.02, 130.27, 126.88, 126.68, 124.18, 123.75, 120.90, 116.69, 116.12,111.15, 55.64 ppm. MS(ESI) calculated for C23H19NO5 [M - H]- 332.34; found332.19.

[0112] In addition, the condensation agent used in step g is cyanuric chloride, but it can also be achieved by using a combination of oxalyl chloride and DMF, so oxalyl chloride and DMF are alternatives. The specific method is: dissolve the product of the previous step in anhydrous dichloromethane, add 2 drops of DMF, and slowly add oxalyl chloride to the reaction solution. After reacting at room temperature for a period of time, the reaction solution is spun dry, anhydrous dichloromethane is added to dissolve, phenoxazine or substituted phenoxazine and TEA are mixed and dissolved in anhydrous dichloromethane, slowly dripped into the reaction solution, DMAP is added, and heated to react. TLC detects that phenoxazine or substituted phenoxazine is basically consumed, dilute with water to the reaction solution, adjust the pH to 1-2, extract with EA, wash the organic phase with 1 M sodium bicarbonate solution, dry with anhydrous sodium sulfate and spun dry. Purify by column chromatography (PE: EA = 5: 1). Furthermore, the molar ratio of the product obtained in step f to phenoxazine or substituted phenoxazine, oxalyl chloride, TEA, and DMAP is 1:(1-1.5):(1-2):(1-2):(0.02-0.1), preferably 1:1:1.3:1.3:0.05; further, the final reaction temperature is room temperature to 60°C, preferably 30°C.

[0113] Embodiment 5:

[0114] Investigation of the interaction mechanism between compound B synthesized by the present invention and DNA:

[0115] 1. Prepare the solution: Weigh 0.05 mmol EDTA disodium salt, 0.31 mmol disodium hydrogen phosphate and 0.19 mmol sodium dihydrogen phosphate and dissolve them in ultrapure water to prepare 50 mL of pH=7 buffer solution. Mix the buffer solution with ethanol in a volume ratio of 3:0.06 as the solvent for all subsequent samples. Prepare phenoxazine into a 35 μM solution, compound B into a 30 μM solution, and DNA from salmon into a 0.021 mg / mL solution. Phenoxazine is mixed with DNA, and compound B is mixed with DNA. The concentrations of phenoxazine and compound B are 35 μM and 30 μM, respectively, and the concentration of DNA is 0.021 mg / mL. The solvent volume of each sample is 3.06 mL. All samples are treated at 4°C for 24 hours.

[0116] 2. Using a mixed solvent of buffer solution and ethanol as the background solution, scan the circular dichroism spectra of DNA, compound B, and the mixed sample of compound B and DNA in sequence with a step size of 0.1 nm. Each sample was scanned 6 times for superposition. Fig.10 As shown, after compound B was mixed with DNA, the peak shape of DNA at around 220 nm was obviously destroyed, which indicates that compound B can interact with DNA and thus affect or even destroy the double helix structure of DNA.

[0117] Embodiment 6:

[0118] The inhibitory activity of compound B on Hela cells was detected by MTT assay, and its IC50 on Hela cells was 15 μM, and its anticancer activity was much higher than that of cisplatin and paclitaxel.

[0119] The above is a specific description of a feasible embodiment of the present invention, but the embodiment is not intended to limit the protection scope of the present invention. Any equivalent implementation or modification that does not deviate from the technical essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A phenoxazine derivative, characterized in that Its general structural formula is shown in the following formula I: ； in: R1 is selected from H, CN or CH2OH; R2 is selected from H, OH, O(CH2)2OH or O(CH2)2NR2; R3 is selected from CH3, C(CH3)2COOR or CH2COOR; R is H or CH3.

2. The method for preparing the phenoxazine derivative according to claim 1, wherein R2 = H, and R3 = CH3, characterized in that The steps include: (1) Compound 1 undergoes a nucleophilic substitution reaction with compound 2 in the presence of a base to obtain compound 3; (2) Compound 3 undergoes oxidation reaction in the presence of a base to obtain compound 5; (3) Compound 5 reacts with phenoxazine or substituted phenoxazine via an acyl chloride intermediate to obtain product Ⅰ-1; The synthetic route is as follows: 。 3. The method for preparing the phenoxazine derivative according to claim 1, wherein R2 = H and R3 = C(CH3)2COOR or CH2COOR, R is H or CH3, characterized in that The steps include: (1) Compound 1 undergoes a nucleophilic substitution reaction with compound 2' in the presence of a base to obtain compound 3'; (2) Compound 3' undergoes esterification reaction with methanol under acid catalysis to obtain compound 4; (3) Compound 4 undergoes oxidation reaction in an alkaline environment to obtain compound 5; (4) Compound 5 is condensed with phenoxazine or substituted phenoxazine via an acyl chloride intermediate to obtain product Ⅰ-2; R3 in compounds 4, 5 and product Ⅰ-2 is C(CH3)2COOCH3 or CH2COOCH3; (5) Product Ⅰ-2 undergoes hydrolysis reaction under alkaline conditions to obtain product Ⅰ-3; R3 in product Ⅰ-3 is C(CH3)2COOH or CH2COOH; The synthetic route is as follows: 。 4. The method for preparing a phenoxazine derivative according to claim 1, wherein R2≠H, and R3 = CH3, characterized in that The steps include: (1) Compound 7 and compound 8 undergo nucleophilic substitution reaction in the presence of a base to obtain compound 9; (2) Compound 9 is condensed with phenoxazine or substituted phenoxazine via an acyl chloride intermediate to obtain compound 10; (3) Compound 10 undergoes hydrolysis in the presence of a base to obtain product Ⅰ-4; (4) Product Ⅰ-4 undergoes nucleophilic substitution reaction with 2-bromoethanol or ethanolamine or N-methyl-2-hydroxyethylamine or N,N-dimethylethanolamine in the presence of a base to obtain product Ⅰ-5; The synthetic route is as follows: 。 5. The method for preparing a phenoxazine derivative according to claim 1, wherein R2≠H, and R3 = C(CH3)2COOR or CH2COOR, R is H or CH3, characterized in that The steps include: (1) Compound 11 undergoes a nucleophilic substitution reaction with compound 2' in the presence of a base to obtain compound 12; (2) Compound 12 reacts with methanol under acid catalysis to obtain compound 13; (3) Compound 13 reacts with phenoxazine or substituted phenoxazine via an acyl chloride intermediate to obtain compound 14; (4) Compound 14 reacts with hydrobromic acid in the presence of a phase transfer catalyst to obtain compound 15; (5) Compound 15 undergoes nucleophilic substitution reaction with 2-bromoethanol or ethanolamine or N-methyl-2-hydroxyethylamine or N,N-dimethylethanolamine in the presence of a base to obtain product Ⅰ-6; in which R3 is C(CH3)2COOCH3 or CH2COOCH3; (6) Product Ⅰ-6 undergoes hydrolysis reaction in an alkaline environment to obtain product Ⅰ-7; in which R3 is C(CH3)2COOH or CH2COOH; Synthesis route 4 is as follows: 。 6. Use of the phenoxazine derivative according to claim 1 in the preparation of DNA intercalators.

7. Use of the phenoxazine derivative according to claim 1 in the preparation of a drug for inhibiting the growth of cancer cells.

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

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