CN111635449A

CN111635449A - Lupeol pyridine quaternary ammonium salt derivative and preparation method and application thereof

Info

Publication number: CN111635449A
Application number: CN202010654852.8A
Authority: CN
Inventors: 韩泽平; 何金花; 黎毓光
Original assignee: Guangzhou Panyu Central Hospital
Current assignee: Guangzhou Panyu Central Hospital
Priority date: 2020-07-09
Filing date: 2020-07-09
Publication date: 2020-09-08
Anticipated expiration: 2040-07-09
Also published as: CN111635449B

Abstract

The invention discloses lupeol pyridine quaternary ammonium salt derivatives, and a preparation method and application thereof, and belongs to the field of medicine synthesis. The invention provides lupeol pyridine quaternary ammonium salt derivatives and application thereof in treating and/or preventing cancers, wherein the molecular formula of the derivatives is C₃₈H₅₉IN₂O₂Molecular weight 702.1. The invention takes lupeol as an initial raw material to synthesize a water-soluble lupeol pyridine quaternary ammonium salt derivative, and pharmacological experiments prove that the derivative has obvious inhibition effect on various tumor cell strains, has obvious anticancer effect compared with lupeol, has low toxicity on normal cell strains, has good hydrophilicity, is easy to dissolve in solvents such as water, ethanol and DMSO, has better solubility than lupeol, and has great research and development potential; is expected to be developed into antitumor drugs with oneDefinite medical value and market prospect.

Description

Lupeol pyridine quaternary ammonium salt derivative and preparation method and application thereof

Technical Field

The invention belongs to the field of drug synthesis, and relates to lupeol pyridine quaternary ammonium salt derivatives, and a preparation method and application thereof.

Background

Cancer, i.e., malignant tumor, is a serious disease that endangers human health caused by multigenic mutations, multifactorial causes, and multistage development. Recent data show 18078957 cases of new cancer cases and 9555027 cases of cancer death in 2018 worldwide. Therefore, various works for preventing and treating cancer are actively carried out, and how to effectively treat cancer has become a urgent necessity. The malignant tumor treatment method mainly comprises surgical treatment, radiotherapy, chemotherapy, biological treatment and the like, the chemotherapy is still one of the main means for treating the tumor at present and in a long term in the future, but the side effect caused by chemotherapy seriously reduces the life quality of patients. In the face of a plurality of tumor diseases at present, the existing antitumor drugs far from meeting the requirements are urgently needed to be researched and developed.

Lupeol (lupeol) is a pentacyclic triterpene compound derived from herbal plants such as Lupeol, fructus Mangifera Indicae and herba Taraxaci, and has a molecular formula of C₃₀H₅₀O, molecular weight 426.7174, has effects of anti-inflammatory, antioxidant, anti-infection, anti-hyperglycemia, anti-asthma, antirheumatic, protecting heart, protecting nerve, protecting liver and regulating immunity, etc., and suggests that lupeol has wide pharmacological activity and multiple action mechanisms and targets. In the anti-tumor research, lupeol has good inhibitory effect on various tumor cells, including colorectal cancer, prostate cancer, liver cancer, lung cancer, bladder cancer and the like, and the mechanism of lupeol relates to the change of various intracellular signal pathways. However, lupeol is insoluble in water and, although it is miscible with diethyl ether, benzene, petroleum ether, and hot ethanol, its solubility is not good. In research experiments, warm absolute ethanol and Dimethylsulfoxide (DMSO) were often used in a 1: 1 as a solvent, but the dissolution effect is still not ideal, drug crystals are easy to precipitate, the research on the anti-tumor aspect of the drug is limited, and the development of the drug to clinical application is further hindered.

Disclosure of Invention

In order to overcome the disadvantages and shortcomings of the prior art, the invention provides a lupeol pyridine quaternary ammonium salt derivative. The derivative is prepared by connecting lupeol and hydrophilic group, and has molecular formula of C₃₈H₅₉IN₂O₂Molecular weight 702.1; the compound has good hydrophilicity, and the compound is tested for anti-tumor activity, thereby providing the lupeol derivative with certain medicinal value.

The invention also aims to provide application of the lupeol pyridine quaternary ammonium salt derivative.

The invention carries out the activity detection experiment of human tumor cells and normal epithelial cells on the synthesized lupeol pyridine quaternary ammonium salt derivative, and the result shows that the lupeol pyridine quaternary ammonium salt derivative can obviously inhibit the proliferation of various tumor cells, has higher inhibition rate than lupeol, has small toxicity to the normal epithelial cells of human, is easy to dissolve in solvents such as water, ethanol, DMSO and the like, has better solubility than lupeol, and has great research and development potential.

The invention also aims to provide a preparation method of the lupeol pyridine quaternary ammonium salt derivative.

The purpose of the invention is realized by the following technical scheme:

the invention provides a lupeol pyridine quaternary ammonium salt derivative, which has a structural formula shown as a formula I:

the invention also provides application of the lupeol pyridine quaternary ammonium salt derivative or the pharmaceutically acceptable salt thereof in preparing a medicament for treating and/or preventing cancer.

Preferably, the cancer is selected from bladder cancer, kidney cancer, neuroblastoma, liver cancer, nasopharyngeal cancer, prostate cancer, colon cancer, etc.;

the effective concentration of the lupeol pyridine quaternary ammonium salt derivative or the pharmaceutically acceptable salt thereof is 1-80 mu mmol/L of cell suspension, further 2-80 mu mmol/L of cell suspension, and further 2-64 mu mmol/L of cell suspension, wherein the concentration of the cell suspension is 2.5 × 10⁷cell/L suspension.

The dosage form of the medicine is oral dosage form or injection dosage form;

the medicament contains one or more pharmaceutically acceptable carriers or excipients.

A composition for treating and/or preventing cancer, wherein the active ingredient is the lupeol pyridine quaternary ammonium salt derivative or the pharmaceutically acceptable salt thereof.

The invention also provides a preparation method of the lupeol pyridine quaternary ammonium salt derivative, which comprises the following steps:

(1) dissolving the compound 1 in an organic solvent, adding the compound 2, reacting at room temperature, and monitoring by TLC until the compound 1 completely reacts; then adding an organic solvent to dilute the reaction solution, washing with saturated saline solution, drying the organic phase, filtering, and concentrating the filtrate to dryness to obtain a crude product of the compound 3, wherein the crude product is directly used in the next step without further purification;

(2) dissolving the crude product of the compound 3 by using an organic solvent, adding the compound 4 into the solution, and stirring the solution for reaction; cooling to room temperature after the reaction is finished, adding an organic solvent to dilute the reaction solution, washing with saturated saline solution, drying the organic phase, filtering, concentrating the filtrate to dryness to obtain a crude product, purifying the crude product by using a silica gel column chromatography, and obtaining a white-like solid compound 5 by using a dichloromethane-methanol (40: 1) eluent, wherein the total yield of the two steps is 36.86%;

(3) taking the compound 5 and dry acetonitrile, stirring and dissolving, adding methyl iodide, and reacting at room temperature after the addition is finished; at the moment, more solids are separated out, the filtration is carried out, and a filter cake is recrystallized by acetonitrile; preparing a tan solid compound A, namely a lupeol pyridine quaternary ammonium salt derivative; the yield of this step was 36.65%.

Wherein the structural formulas of the compound 1, the compound 3 and the compound 5 are respectively as follows:

the structural formulas of the compound 2 and the compound 4 are respectively as follows:

preferably, the organic solvent is dichloromethane;

preferably, the concentration of the compound 1 in the step (1) is 100 mmol/L;

preferably, the molar ratio of compound 1 to compound 2 in step (1) is 1: 1.1;

preferably, the reaction time at room temperature in the step (1) is 2 h;

preferably, the volume ratio of the amount of the organic solvent used for diluting the reaction solution in the step (1) to the amount of the organic solvent used for dissolving the compound 1 is 1: 1.

preferably, the compound 4 in the step (2) is used in an amount of 1: 1.2, adding;

preferably, the stirring reaction condition in the step (2) is that the stirring reaction is carried out for 17 hours at 40 ℃;

preferably, the volume ratio of the amount of the organic solvent used for diluting the reaction solution in the step (2) to the amount of the organic solvent used for dissolving the compound 3 is 1: 1.

preferably, the molar ratio of the compound 5 to the methyl iodide in the step (3) is 1: 7.3.

preferably, the reaction time at room temperature in the step (3) is 17 h.

Compared with the prior art, the invention has the following advantages and effects:

the invention provides lupeol pyridine quaternary ammonium salt derivatives shown as a formula I and application thereof in treating and/or preventing cancers. The invention takes lupeol as a starting material to synthesize a water-soluble lupeol pyridine quaternary ammonium salt derivative, and pharmacological experiments prove that the derivative has obvious inhibition effect on various tumor cell strains, has obvious anticancer effect compared with lupeol, has low toxicity on normal cell strains, is expected to be developed into an antitumor drug, and has certain medical value and market prospect.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

The experimental procedures for specific experimental conditions not specified in the following examples are generally performed according to conventional experimental conditions or according to experimental conditions recommended by the manufacturers. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.

Example 1: preparation of compound a (lupeol pyridiniumquat derivative):

compound 1(1.28g, 3.0mmol) was dissolved in 30mL of Dichloromethane (DCM), compound 2(0.53g, 3.3mmol) was added and reacted at room temperature for 2h, monitored by TLC until compound 1 was reacted completely. The reaction was diluted with 30mL of dichloromethane, washed twice with 30mL each time of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness to give 1.42g of crude compound 3, which was used in the next step without further purification.

The crude compound 3 was dissolved in 30mL of dichloromethane, to which was added compound 4(0.39g, 3.6mmol), and the reaction stirred at 40 ℃ for 17 h. After the reaction, the reaction mixture was cooled to room temperature, 30mL of dichloromethane was added to dilute the reaction mixture, the reaction mixture was washed twice with 20mL of saturated brine each time, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness to obtain a crude product, which was purified by silica gel column chromatography using dichloromethane-methanol (volume ratio 40: 1) as an eluent to obtain 0.62g of 5 as a white-like solid compound in a total yield of 36.86% in two steps. ESI-MS⁺m/z：561.3[M+H]⁺，583.4[M+Na]⁺。

A10 mL single vial was charged with Compound 5(0.61g, 1.1mmol) and 8mL of dry acetonitrile, and the mixture was dissolved with stirring, and 0.5mL (about 7.3eq) of iodomethane was added thereto, after the addition was completed, the reaction flask was sealed, and the reaction was allowed to proceed at room temperature for 17 hours. At this point more solid precipitated, filtered and the filter cake recrystallized from acetonitrile. 0.28g of a tan solid compound A was obtained, the yield of this step being 36.65%.

ESI-MS⁺m/z：703.4[M+H]⁺，575.5[M-I^-]。

¹H-NMR(500M，CD₃OD)8.846-8.830(d，2H，J＝8.0Hz)，7.972-7.956(d，2H，J＝8.0Hz)，4.704(s，1H)，4.627-4.585(m，3H)，4.389(s，3H)，4.356-4.309(m，1H)，2.465-2.395(m，1H)，1.988-1.933(m，1H)，1.757-1.705(m，8H)，1.532-1.209(m，16H),1.102-0.814(m，20H)。

EXAMPLE 2 inhibition of human tumor cell proliferation assay by Compound A of the invention

1. Dissolving the medicine: using warm absolute ethanol and DMSO in a 1: 1, dissolving lupeol to prepare 7811.57 mu M total solution; compound A of the present invention was dissolved in DMSO to prepare 14228.6. mu.M total solution. The experiment was performed with complete medium dilution to the corresponding working concentration.

2. Cell culture: 16 tumor cell lines (5637 cells of bladder cancer, 786-O cells of kidney cancer, SK-N-SH cells of neuroblastoma, Huh7 cells of liver cancer, Cne2 cells of nasopharyngeal carcinoma, PC3 cells of prostate cancer, Colo205 cells of colon cancer, DLD-1 cells of colon cancer, HCT-116 cells of colon cancer, HT-29 cells of colon cancer, Lovo cells of colon cancer, O cells of RK colon cancer, SW480 cells of colon cancer, SW620 cells of colon cancer, Caco2 cells of colon cancer, THC-8307 cells of colon cancer, 16 cell lines are all conventional commercial cell lines) used for experiments are placed in corresponding culture media containing 10% Fetal Bovine Serum (FBS) or 20% FBS, and the cells are subjected to CO treatment at 37 ℃, saturation humidity and 5% of volume fraction₂The culture medium corresponding to each cell line is shown in Table 1.

TABLE 1 culture Medium for individual tumor cells

MTT experiment, namely taking tumor cells in logarithmic growth phase for experiment, respectively suspending the tumor cells by using corresponding 10% FBS or 20% FBS culture medium, and adjusting the cell density to be 2.5 × 10⁴cell/mL suspension, 200. mu.L per well inoculated 96-well plate, 37 ℃, 5% CO₂Incubate overnight to allow the cells to adhere well. The culture medium in each well was aspirated, and 200. mu.L of the compound A of the present invention or lupeol diluted in the corresponding complete medium was added to each well to give final concentrations of 2. mu.M, 4. mu.M, 8. mu.M, 16. mu.M, 32. mu.M, and 64. mu.M, respectively. Each concentration was set to 8 replicates. Simultaneously, wells inoculated with cells without drug were set as controlsIn the group, the control group was not added with drugs, and 200. mu.L/well of complete medium was added, and the wells to which only medium was added without inoculated cells were set as blank wells, and 200. mu.L/well of complete medium was added. 5% CO₂Incubate at 37 ℃ for 48 h. mu.L of MTT20 per well at 5% CO₂Incubating for 4h in an incubator at 37 ℃, terminating the culture, absorbing and removing supernatant, adding 150 mu L DMSO into each hole, shaking the mixture in a shaking table for 10min to fully dissolve crystals, and measuring the absorbance A value at 490 nm. Inhibition rate of cell proliferation ═ a_{Control group}-A_{Blank group})/(A_{Drug action group}-A_{Blank group}) × 100%, half maximal Inhibitory Concentration (IC) was calculated using GraphPad Prism statistics software₅₀)。

4. The experimental results are as follows: according to the above test method, the IC of compound A of the present invention and lupeol in 48 hours on 16 human tumor cells such as bladder cancer 5637 cell, kidney cancer 786-O cell, neuroblastoma SK-N-SH cell, liver cancer Huh7 cell, nasopharyngeal carcinoma Cne2 cell, prostate cancer PC3 cell, colon cancer Colo205 cell, colon cancer DLD-1 cell, colon cancer HCT-116 cell, colon cancer HT-29 cell, colon cancer Lovo cell, colon cancer RKO cell, colon cancer SW480 cell, colon cancer SW620 cell, colon cancer Caco2 cell, colon cancer THC-8307 cell and the like₅₀Values, see table 2.

MTT results show that the compound A and lupeol have different degrees of proliferation inhibition effects on 16 tumor cells and are concentration-dependent. Except 4 strains of cells such as colon cancer Colo205 cells, HT-29 cells, RKO cells, Caco2 cells and the like, the inhibiting effect of the compound A on the other 12 strains of tumor cells is higher than that of lupeol (P)<0.05). Wherein, the IC of the compound A of the invention on HCT-116 cells of colon cancer and SK-N-SH cells of neuroblastoma₅₀The content of the compound is as low as 5.549 +/-0.33 mu M and 8.362 +/-0.81 mu M, and the inhibition effect is obvious.

TABLE 2 comparison of Lupeol pyridine quaternary ammonium salt derivatives and IC50 of Lupeol on various tumor cells

EXAMPLE 3 toxicity test on Normal human epithelial cells with Compound A of the present invention

1. Dissolving the medicine: the compound A of the invention is dissolved by DMSO to prepare 14228.6 mu M total solution, and the total solution is diluted to corresponding working concentration by complete culture medium for experiment.

2. Cell culture: 6 normal epithelial cells (conventional commercial cell lines) such as human prostate epithelial RWPE-1 cell, liver HL-7702 cell, colon epithelial HCoEpiC cell, nasopharyngeal epithelial NP69 cell, mammary epithelial MCF-10A cell and alveolar epithelial HPAEpic cell were placed in corresponding culture media containing 10% Fetal Bovine Serum (FBS) or no FBS, and at 37 deg.C, saturation humidity, and volume fraction of 5% CO₂The cells were cultured in an incubator and the corresponding media for each cell are shown in Table 3.

TABLE 3 culture Medium for each of the Normal cells

MTT experiment, namely taking cells in logarithmic growth phase for experiment, respectively suspending the cells by using corresponding 10% fetal bovine serum or FBS-free culture medium, and adjusting the cell density to 2.5 × 10⁴cell/mL suspension, 200. mu.L per well inoculated 96-well plate, 37 ℃, 5% CO₂Incubate overnight to allow the cells to adhere well. The culture medium in each well was aspirated, and 200. mu.L/well of Compound A of the present invention diluted with the corresponding complete medium was added to the wells to give final concentrations of 1. mu.M, 2. mu.M, 4. mu.M, 8. mu.M, 16. mu.M, and 32. mu.M, respectively. Each concentration was set to 8 replicates. Meanwhile, a hole which is not added with medicine and only inoculates cells is set as a control group, the control group is not added with medicine and only 200 mu L/hole of complete culture medium is added, a hole which is not added with the inoculated cells and only added with the culture medium is set as a blank hole, and 200 mu L/hole of complete culture medium is added. 5% CO₂Incubate at 37 ℃ for 48 h. MTT 20. mu.L/well at 5% CO₂Incubating for 4h in an incubator at 37 ℃, terminating the culture, absorbing and removing supernatant, adding 150 mu L DMSO into each hole, shaking the mixture in a shaking table for 10min to fully dissolve crystals, measuring the absorbance A value at 490nm, and calculating the cell survival rate. Survival rate ═ 1- (a)_{Control group}-A_{Blank group})/(A_{Drug action group}-A_{Blank group})]×100％。

4. The experimental results are as follows: according to the above test methods, the effect of the compound A of the present invention on the survival rate of 6 normal epithelial cells such as human prostate epithelial RWPE-1 cells, liver HL-7702 cells, colon epithelial HCoEpiC cells, nasopharyngeal epithelial NP69 cells, mammary epithelial MCF-10A cells and alveolar epithelial HPAEpiC cells within 48 hours was determined, and is shown in Table 4.

MTT results show that compared with a control group, the compound A has no obvious influence on the survival rate of human normal liver HL-7702 cells, colon epithelial HCoEpiC cells, nasopharyngeal epithelial NP69 cells and alveolar epithelial HPAEpic cells (P is more than 0.05). The compound A can reduce the survival rate (P <0.05) of the prostate epithelial RWPE-1 cells and the breast epithelial MCF-10A cells only at a high concentration of 32 mu M, and has small influence degree, which indicates that the compound A has small toxicity to normal cells and low toxic and side effects, and is suitable for the development of antitumor drugs.

TABLE 4 Effect of lupeol pyridinium Quaternary ammonium salt derivatives on survival of various human Normal cells

Note:^*p in comparison with the control group<0.05。

According to the content, the lupeol pyridine quaternary ammonium salt derivative with the anti-tumor activity and the preparation method thereof are provided, and the lupeol pyridine quaternary ammonium salt derivative is found for the first time to be capable of effectively inhibiting the survival and the growth of various tumor cell strains, has stronger inhibiting activity than lupeol, good solubility and small toxicity to normal cells, and has research and development potential and application prospect in clinical tumor treatment.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A lupeol pyridine quaternary ammonium salt derivative is characterized in that: the structural formula is shown as formula I:

2. use of lupeol pyridiniumnium salt derivative of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment and/or prevention of cancer.

3. Use according to claim 2, characterized in that:

the cancer is selected from bladder cancer, renal cancer, neuroblastoma, liver cancer, nasopharyngeal cancer, prostate cancer, and colon cancer.

4. Use according to claim 2, characterized in that:

the effective concentration of the lupeol pyridine quaternary ammonium salt derivative or the pharmaceutically acceptable salt thereof is 1-80 mu mmol/L of cell suspension, wherein the concentration of the cell suspension is 2.5 × 10⁷cell/L suspension.

5. Use according to claim 4, characterized in that:

the effective concentration of the lupeol pyridine quaternary ammonium salt derivative or the pharmaceutically acceptable salt thereof is 2-80 mu mmol/L of cell suspension.

6. Use according to claim 5, characterized in that:

the effective concentration of the lupeol pyridine quaternary ammonium salt derivative or the pharmaceutically acceptable salt thereof is 2-64 mu mmol/L of cell suspension.

7. Use according to claim 2, characterized in that:

the dosage form of the medicine is oral dosage form or injection dosage form;

8. A composition for use in the treatment and/or prevention of cancer, characterized in that: the lupeol pyridine quaternary ammonium salt derivative or the pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient.

9. The process for preparing lupeol pyridinium quaternary derivatives as claimed in claim 1, wherein: the method comprises the following steps:

(2) dissolving the crude product of the compound 3 by using an organic solvent, adding the compound 4 into the solution, and stirring the solution for reaction; and after the reaction is finished, cooling to room temperature, adding an organic solvent to dilute the reaction solution, washing with saturated saline solution, drying the organic phase, filtering, concentrating the filtrate to dryness to obtain a crude product, and purifying the crude product by using a silica gel column chromatography, wherein the eluent is a solvent with a volume ratio of 40: 1 to obtain a white solid compound 5;

(3) taking the compound 5 and dry acetonitrile, stirring and dissolving, adding methyl iodide, and reacting at room temperature after the addition is finished; at the moment, more solids are separated out, the filtration is carried out, and a filter cake is recrystallized by acetonitrile; preparing a tan solid compound A, namely a lupeol pyridine quaternary ammonium salt derivative;

10. the method of claim 9, wherein:

the organic solvent is dichloromethane;

the concentration of the compound 1 in the step (1) is 100 mmol/L;

the molar ratio of the compound 1 to the compound 2 in the step (1) is 1: 1.1;

the volume ratio of the amount of the organic solvent used for diluting the reaction solution in the step (1) to the amount of the organic solvent used for dissolving the compound 1 is 1: 1;

the dosage of the compound 4 in the step (2) is determined according to the molar ratio of the compound 1 to the compound 4, namely 1: 1.2, adding;

the volume ratio of the amount of the organic solvent used for diluting the reaction solution in the step (2) to the amount of the organic solvent used for dissolving the compound 3 is 1: 1;

the molar ratio of the compound 5 to the methyl iodide in the step (3) is 1: 7.3;

the room-temperature reaction time in the step (1) is 2 hours;

the stirring reaction condition in the step (2) is that stirring reaction is carried out for 17 hours at 40 ℃;

the reaction time at room temperature in the step (3) is 17 h.