CN112521408B - Bisindole alkaloid compound and preparation method and application thereof - Google Patents

Abstract

The invention discloses a bisindole alkaloid compound and a preparation method and application thereof. The compounds have the common characteristic of having a vincristine main body structure but having no acetoxyl group at the 17-position. The preparation of the compounds generally comprises the following steps: (a) protecting hydroxyl of raw material alkaloid according to the requirement; (b) ester hydrolysis is carried out at the 17-position; (c) reacting the 17-position hydroxyl with p-toluenesulfonyl chloride to generate sulfonic ester which is easy to remove; (d) deprotection and sulfonate; (e) derivatizing the product; (f) and carrying out preparation and separation on the reaction in the last step. Compared with vincristine, the bisindole alkaloid compound disclosed by the invention has lower cytotoxicity, but maintains or enhances the antitumor activity of vincristine.

Description

Bisindole alkaloid compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of pharmaceutical chemistry, and particularly relates to a bisindole alkaloid compound, and a preparation method and application thereof.

Background

Catharanthus roseus (Catharanthus roseus) is a herbaceous plant of the Apocynaceae family and is widely used in folk medicine for hemostasis, analgesia, wound cleansing, and treatment of scurvy and diabetes.

With the 60 s of the 20 th century, researchers discovered that vinblastine and vincristine had anti-tumor activity, and subsequently many similar compounds were found to be useful in the treatment of cancer, such as: vinorelbine, vindesine, and the like. Although they have similar mother nuclei and all stop mitosis in the metaphase by acting on cellular tubulin, there is a great difference between the pharmacological effects of different compounds. In addition, although the vinca alkaloids have long application time, they have toxicity of different degrees, such as the neurotoxicity of vincristine, which limits their clinical application. Therefore, the discovery of new low-toxicity vinca alkaloids with antitumor activity is of great significance in improving the quality of life of patients.

Disclosure of Invention

The invention aims to provide a bisindole alkaloid compound or a pharmaceutically acceptable salt thereof, which has the common characteristics that the bisindole alkaloid compound has a vincristine main structure but has no acetoxyl group at the 17-position, has antitumor activity similar to or better than that of vincristine, and has lower cytotoxicity.

In order to solve the technical problems, the invention adopts the following technical scheme:

a bisindole alkaloid compound having a structure represented by formula 1:

wherein R1 and R3 are each independently H, substituted or unsubstituted acyl, and R2 and R4 are each independently hydroxy, substituted or unsubstituted alkoxy.

Further, the compound has one of the structures shown in 1a, 1b and 1 c:

an application of the bisindole alkaloid compound or the pharmaceutically acceptable salt thereof in preparing a medicament for inhibiting tumor cells.

Further, the tumor cells comprise one or more of cervical cancer cells, lung cancer cells, breast cancer cells, nasopharyngeal cancer cells, leukemia cells and neuroma cells.

Compared with vincristine, the vincristine has lower neuron cytotoxicity, and has higher inhibition rate on tumor cells such as cervical cancer cell (HeLa), lung cancer (A549), breast cancer (MCF-7), nasopharyngeal carcinoma (CNE-2Z), leukemia (K562), neuroma cell (PC-12), etc.

A preparation method of the bisindole alkaloid compound or the pharmaceutically acceptable salt thereof comprises the following steps:

wherein R1 and R3 are each independently H, substituted or unsubstituted acyl, and R2 and R4 are each independently hydroxy, substituted or unsubstituted alkoxy.

Further, the method comprises the following steps:

a. reacting the hydroxyl group of vincristine according to the synthesis requirement to obtain the intermediate 1;

b. selecting a reagent of potassium hydroxide, reacting the intermediate 1 and the potassium hydroxide at a mass ratio of 1:1.1 at room temperature for 5-6 hours, and extracting, concentrating and drying a reaction product to obtain an intermediate 2;

c. selecting a reagent as p-toluenesulfonyl chloride, reacting the intermediate 2 and the p-toluenesulfonyl chloride at a molar ratio of 1: 0.9-1.1 at 50-60 ℃ for 8-9 hours, and concentrating a reaction product to obtain an intermediate 3;

d. selecting a liquid ammonia solution with a reagent of calcium, wherein the intermediate 3: calcium: reacting liquid ammonia for 1-2 hours at room temperature according to the mass-volume ratio of 1g to 1.5-3 g to 60-80 ml, then adding bromobenzene until the solution turns blue, evaporating the liquid ammonia, and concentrating to obtain a crude product;

f. and (2) performing preparative separation on the crude product by selecting chromatography, mixing methanol and chloroform according to the volume ratio of 1:99 to obtain a mobile phase reagent, and combining concentrated collecting solutions to obtain the formula 1.

Further, between step d and step f, the method further comprises the following steps:

e. according to the requirements, the R of the crude product is added₁、R₂、R₃、R₄Any one of which is further derivatized.

Further, in step b, the intermediate 1 is reacted in the following solvent system: mixing methanol, chloroform and water according to a volume ratio of 20-30: 1: 1; in step c, the intermediate 2 is reacted in the following solvent system: mixing pyridine and chloroform according to the volume ratio of 2.5-3: 1; in step d, the intermediate 3 is reacted in the following solvent system: and mixing toluene and chloroform according to the volume ratio of 2-5: 1.

Further, the room temperature is 20-30 ℃.

A pharmaceutical composition, which comprises the bisindole alkaloid compound or pharmaceutically acceptable salt thereof.

Compared with the prior art, the implementation of the invention has the following beneficial effects:

1. the bisindole alkaloid compound or the pharmaceutically acceptable salt thereof is confirmed to have the antitumor activity similar to or better than that of vincristine and lower cytotoxicity;

2. the application provides a preparation method of the bisindole alkaloid compound or the pharmaceutically acceptable salt thereof, and provides a stable source for the application and research of the bisindole alkaloid compound in medicines.

Drawings

FIG. 1 is a comparison of neuronal cytotoxicity assays for vincristine and 1a samples;

FIG. 2 shows the IC of vincristine and each tumor cell of the 1a sample₅₀The results are compared with the figures.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

Example 1

Synthetic route for methyl (3aR, 3a1S, 5R, 5aR, 10bR) -3 a-ethyl-9- ((3S, 5S, 7S, 9S) -5-ethyl-5-hydroxy-9- (methoxycarbonyl) -1,4,5,6,7,8,9, 10-octahydro-2H-3, 7-methyl [1] azacycloundec [5,4-b ] indol-9-yl) -6-formyl-5-hydroxy-8-methoxy-3 a, 3a1,4,5,5a, 6,11, 12-octahydro-1H-indolo [8,1-cd ] carbazole-5-carboxylate:

step a: dissolving 10g of commercially available vincristine (824.96g/mol) in a dry mixed solvent of N, N-Dimethylformamide (DMF) and acetone, wherein the volumes of the solvents are 250ml and 50ml respectively, sequentially adding 0.64g of sodium hydride (1.1equiv, 24.00g/mol), 3.2ml of benzyl bromide (1.1equiv, 171.04g/mol) and 0.18g of sodium iodide (149.89g/mol), reacting at room temperature for 5 hours, adding water to generate a precipitate after the reaction is finished, filtering and drying to obtain an intermediate 1(11.5g), and obtaining the yield of 94.4%;

step b: dissolving 10g of the intermediate 1 in a mixed solvent of methanol, chloroform and water, wherein the volumes of the solvents are respectively 300ml, 10ml and 10ml, adding 11g of potassium hydroxide, stirring at room temperature for reaction for 6 hours, concentrating the solvent in vacuum after the reaction is finished, and adding chloroform: extracting the reaction product with 50ml of water for three times, and then combining and concentrating organic phases to obtain an intermediate 2(9.1g), wherein the yield is 95.0%;

step c: dissolving the intermediate 2(9g) in a mixed solvent of pyridine and chloroform, wherein the volume of the solvent is 90ml and 30ml respectively, adding 1.96g of paratoluensulfonyl chloride (190.65g/mol), stirring at 50 ℃ for reacting for 8 hours, and after the reaction is finished, concentrating the reaction solution in vacuum to obtain an intermediate 3(10.3g), wherein the yield is 98.6%;

step d: 10g of intermediate 3 was dissolved in a mixed solvent of dry toluene and chloroform in a volume of 150ml and 30ml, respectively, and a solution of calcium in liquid ammonia (15g of calcium in 600ml of liquid ammonia) was added dropwise over 10 minutes, followed by vigorous stirring for 1 hour. Adding bromobenzene until the solution turns blue, evaporating liquid ammonia at room temperature, and concentrating to obtain a crude product;

step f: and d, carrying out preparative separation on the crude product in the step d, wherein the mobile phase reagents are methanol and chloroform, the volume ratio is 1:99, combining and concentrating collected liquid to obtain an intermediate 4(6.1g), and the yield is 88.9%.

Example 2

Synthetic route for methyl (3aR, 3a1S, 5R, 5aR, 10bR) -5-acetoxy-9- (((3S, 5S, 7S, 9S) -5-acetoxy-5-ethyl-9- (methoxycarbonyl) -1,4,5,6,7,8,9, 10-octahydro-2H-3, 7-methyl [1] azacycloundec [5,4-b ] indol-9-yl) -3 a-ethyl-6-formyl-8-methoxy-3 a, 3a1,4,5,5a, 6,11, 12-octahydro-1H-indolo [8,1-cd ] carbazole-5-carboxylate:

step e: dissolving 0.5ml acetyl chloride in a mixed solvent of DMF and chloroform, wherein the volumes of the solvents are 80ml and 10ml respectively, adding 0.93g N, N' -Carbonyldiimidazole (CDI) and stirring for reaction at room temperature for 30 minutes, adding 2g of the compound intermediate 4 obtained in the example 1 and reacting for 8 hours at 50 ℃, adding 20ml water to precipitate a product after the reaction is finished, and filtering and drying to obtain a crude product;

step f: and e, performing preparative separation on the crude product in the step e, wherein the mobile phase reagents are methanol and chloroform, the volume ratio is 1:99, and combining concentrated collected liquid to obtain the yield of the formula 1a (2.1g) of 95.4%.

Example 3

Synthetic route of (3aR, 3a1S, 5R, 5aR, 10bR) -9- (((3S, 5S, 7S, 9S) -9-carboxy-5-ethyl-5-hydroxy-1, 4,5,6,7,8,9, 10-octahydro-2H-3, 7-methyl [1] azacycloundec [5,4-b ] indol-9-yl) -3 a-ethyl-6-formyl-5-hydroxy-8-methoxy-3 a, 3a1,4,5,5a, 6,11, 12-octahydro-1H-indolo [8,1-cd ] carbazole-5-carboxylic acid:

step e: 2g of the compound intermediate 4 obtained in example 1 was dissolved in a mixed solvent of ethanol, chloroform and water in volumes of 75ml, 25ml and 10ml, respectively, and 2.2g of potassium hydroxide was added thereto, and after stirring and reacting at room temperature for 8 hours, the solvent was concentrated in vacuo after adjusting the pH to neutral, followed by addition of chloroform: extracting the reaction product with 15ml of water for three times, and combining and concentrating organic phases to obtain a crude product;

step f: and e, performing preparative separation on the crude product in the step e by using mobile phase reagents of methanol and chloroform in a volume ratio of 1:99, and combining concentrated collected liquids to obtain formula 1b (1.8g) with the yield of 93%.

Example 4

Synthetic route to ethyl (3aR, 3a1S, 5R, 5aR, 10bR) -9- (((3S, 5S, 7S, 9S) -9- (ethoxycarbonyl) -5-ethyl-5-hydroxy-1, 4,5,6,7,8,9, 10-octahydro-2H-3, 7-methylene [1] azacycloundec [5,4-b ] indol-9-yl) -3 a-ethyl-6-formyl-5-hydroxy-8-methoxy-3 a, 3a1,4,5,5a, 6,11, 12-octahydro-1H-indolo [8,1-cd ] carbazole-5-carboxylate:

step e: dissolving 2g of the compound 1b obtained in example 3 in a mixed solvent of DMF and chloroform, wherein the volumes of the solvents are 75ml and 20ml respectively, adding 1g of CDI at room temperature, stirring for 30 minutes, adding 0.35ml of ethanol, stirring at 50 ℃ for reaction for 12 hours, adding water, filtering and drying to obtain a crude product;

step f: and e, performing preparative separation on the crude product in the step e by using mobile phase reagents of methanol and chloroform in a volume ratio of 1:99, and combining concentrated collected liquid to obtain formula 1c (2g) with the yield of 92.9%.

Example 5

Respectively and precisely transferring a proper amount of vincristine stock solution, and diluting with a DMEM medium to prepare a series of solutions with the concentrations of 10, 20, 30, 50, 100, 200, 300, 400 and 500 mu g/mL. Secondly, respectively and precisely transferring a proper amount of 1a, 1b and 1c sample stock solution, and diluting the sample stock solution with a proper amount of DMEM culture medium to prepare a series of solutions with the concentrations of 10, 20, 30, 50, 100, 200, 300, 400 and 500 mu g/mL. ③ precisely transferring a proper amount of DMSO stock solution respectively, and diluting the DMSO stock solution by DMEM to prepare a DMEM series solution with DMSO contents of 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.5%, 2% and 3%.

Cytotoxicity studies were performed using the MTT method. According to the cell density of 1 × 105 cells/well, the normal human neuron cell suspension is inoculated into a 96-well plate and placed in a cell culture box for 24 h. The samples 10, 20, 30, 50, 100, 200, 300, 400, 500 mu g/mL and 1a containing vincristine with different concentrations and 100 mu L of DMEM medium are added, blank wells and 100 mu L of PBS buffer solution are additionally arranged as negative controls, and 6 duplicate wells are arranged in each group. After the cells are cultured in the cell culture box for 12 hours, 20 mu L of MTT solution with the concentration of 0.5mg/mL is added into each hole, the cells are cultured in the culture box for 4 hours again, supernatant is removed, 100 mu L of DMSO solution is added into each hole, the cells are placed on a shaking table for 30min until blue crystals are completely dissolved, and an absorbance OD value is measured at 490nm by using an enzyme labeling instrument. The survival of the cells at each concentration was calculated from the measured OD values and the maximum safe concentration of vincristine and 1a, 1b, 1c samples in normal neuronal cells was determined as shown in figure 1.

As shown in the results of cytotoxicity examination in FIG. 1, the samples 1a, 1b and 1c are less toxic to cells, wherein the toxicity to normal neuronal cells is significantly increased when the DMSO content exceeds 1.5%, so that DMSO can be used for corresponding cell experiments, but the content is preferably controlled below 1%. Toxicity studies of vincristine and 1a, 1b, 1c samples showed increasing concentrations, but from comparative results, the 1a, 1b, 1c samples showed more than 3 times higher cell viability than vincristine, which also indicates that the 1a, 1b, 1c samples would have lower neurotoxicity.

Example 6

The results of the study in example 5 show that the 1a, 1b, 1c samples are less neurotoxic than vincristine, and theoretically have less tumor cell killing effect than vincristine. In contrast, tumor cells have a relatively complex mechanism for drug absorption due to their complex intracellular and extracellular environments and over-expression of various protein factors. In order to fully prove the killing effect of vincristine and 1a, 1b, 1c samples on tumor cells, screening tests of the antitumor activity of the vincristine and the 1a, 1b, 1c samples are carried out.

Adding 7 cell lines including cervical cancer cell (HeLa), hepatocarcinoma cell (HepG2), lung cancer (A549), breast cancer (MCF-7), nasopharyngeal carcinoma (CNE-2Z), leukemia (K562) and neuroma cell (PC-12) into 10% fetal calf serum, culturing, placing in 5% CO²And culturing in a carbon dioxide incubator at 37.0 ℃. Taking cells in logarithmic growth phase to be planted in a 96-well plate, taking vincristine, 1a, 1b and 1c samples and DMEM negative control, taking DMSO as a solvent to prepare the administration concentration with gradient concentration of 10, 20, 30, 50, 100, 200, 300, 400 and 500 mu g/mL, adding thiazole blue (MTT) after 24 hours of action, incubating for 4 hours, detecting the absorbance under the wavelength of 570nm by using a microplate reader, taking effective data with the absorbance of 0.2-0.8 and the variance of less than 5 percent, analyzing and calculating the IC of the drugs on the tumor cells₅₀Values, as shown in fig. 2.

The results of the study in FIG. 2 show that the IC50 value for the 1a sample was lower than that of vincristine for each tumor cell (except for hepatoma cells), the IC50 value for the 1b sample was lower than that of vincristine for each tumor cell, and the IC50 value for the 1c sample was lower than that of vincristine for each tumor cell (except for hematopoietic stem cell cancerous cells). This suggests that, from a pharmacodynamic point of view, the cancer cells are targeted to several of the above-mentioned types: the samples 1a, 1b and 1c all have higher antitumor activity than vincristine, but 1a has poorer antitumor activity on liver cancer cells than vincristine, and 1c has poorer antitumor activity on hematopoietic stem cell cancerous cells (leukemia) than vincristine.

It is obvious from cytotoxicity test and anticancer activity data that the 1a, 1b, 1c sample shows low toxicity to normal cells, which is different from conventional knowledge that it also shows high activity to tumor cells, therefore, compared with the adverse reaction of neurotoxicity in clinical application of vincristine, the 1a, 1b, 1c compound is expected to reduce the clinical adverse reaction on the premise of maintaining the original or higher antitumor activity.

Since the above disclosure is only illustrative of the preferred embodiments of the present invention, it should be understood that the scope of the present invention is not limited thereto, and therefore, the present invention is intended to cover all equivalent variations within the scope of the present invention as defined in the appended claims.

Claims (3)

1. A preparation method of a bisindole alkaloid compound or a pharmaceutically acceptable salt thereof is characterized by comprising the following steps:

a. reacting the hydroxyl group of vincristine to obtain an intermediate 1;

b. selecting a reagent of potassium hydroxide, reacting the intermediate 1 and the potassium hydroxide at a mass ratio of 1:1.1 at room temperature for 5-6 hours, and extracting, concentrating and drying a reaction product to obtain an intermediate 2;

c. selecting a reagent as p-toluenesulfonyl chloride, reacting the intermediate 2 and the p-toluenesulfonyl chloride at a molar ratio of 1: 0.9-1.1 at 50-60 ℃ for 8-9 hours, and concentrating a reaction product to obtain an intermediate 3;

d. selecting a liquid ammonia solution with a reagent of calcium, wherein the intermediate 3: calcium: reacting liquid ammonia for 1-2 hours at room temperature according to the mass-volume ratio of 1g to 1.5-3 g to 60-80 ml, then adding bromobenzene until the solution turns blue, evaporating the liquid ammonia, and concentrating to obtain a crude product;

f. selecting chromatography to prepare and separate the crude product, mixing methanol and chloroform according to the volume ratio of 1:99 to obtain a mobile phase reagent, and combining concentrated collecting solution to obtain a formula 1;

the structure of formula 1 is as follows:

wherein R1 and R3 are each independently H, substituted or unsubstituted acyl, and R2 and R4 are each independently hydroxy, substituted or unsubstituted alkoxy;

if necessary, the method also comprises the following steps between the step d and the step f:

e. r of the crude product₁、R₂、R₃、R₄Any one of which is further derivatized.

2. A process for the preparation of a bisindole alkaloid compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein in step b, intermediate 1 is reacted in a solvent system comprising: mixing methanol, chloroform and water according to a volume ratio of 20-30: 1: 1; in step c, the intermediate 2 is reacted in the following solvent system: mixing pyridine and chloroform according to the volume ratio of 2.5-3: 1; in step d, the intermediate 3 is reacted in the following solvent system: and mixing toluene and chloroform according to the volume ratio of 2-5: 1.

3. The process for preparing a bisindole alkaloid compound according to claim 1, wherein the room temperature is 20 to 30 ℃.

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