阅读说明：本技术 二苄基二硫代氨基甲酸铜药物治疗三阴性乳腺癌的应用 (Application of copper dibenzyl dithiocarbamate medicament to treatment of triple negative breast cancer ) 是由 吴超君 于 2021-04-16 设计创作，主要内容包括：本发明涉及二苄基二硫代氨基甲酸铜药物治疗三阴性乳腺癌的应用,二苄基二硫代氨基甲酸铜药物的具体制备方法如下：步骤一：二苄基二硫代氨基甲酸钠的合成,将0.06moL的质量为11.8g的二苄胺和150ml的乙腈在冰水浴中搅拌,加入0.09mol的质量为6.96g二硫化碳6.96g和0.09mol的质量分数为50%的氢氧化钠,搅拌反应16小时后出抽滤,将滤液旋蒸后得到产品,将产品用乙醚洗涤后干燥得产物13.04g,粗产品用乙腈重结晶得到二苄基二硫代氨基甲酸钠的质量为11.1g；在双硫仑抗癌结构基础上,重新合成新的药物二苄基二硫代氨基甲酸铜,不需要体内转化,且脂溶性提高,在实际实验过程中,二苄基二硫代氨基甲酸铜有很好的对耐药性三阴性乳腺癌具有抑制效果。(The invention relates to an application of a copper dibenzyl dithiocarbamate medicament in treating triple negative breast cancer, wherein the specific preparation method of the copper dibenzyl dithiocarbamate medicament comprises the following steps: the method comprises the following steps: synthesizing sodium dibenzyl dithiocarbamate, namely stirring 0.06moL of dibenzylamine with the mass of 11.8g and 150ml of acetonitrile in ice water bath, adding 0.09moL of carbon disulfide with the mass of 6.96g and 0.09moL of sodium hydroxide with the mass fraction of 50%, stirring for reacting for 16 hours, performing suction filtration, performing rotary evaporation on the filtrate to obtain a product, washing the product with diethyl ether, drying to obtain 13.04g of the product, and recrystallizing the crude product with acetonitrile to obtain 11.1g of sodium dibenzyl dithiocarbamate; on the basis of a disulfiram anticancer structure, a new medicament copper dibenzyl dithiocarbamate is synthesized again, in-vivo transformation is not needed, the fat solubility is improved, and in the actual experimental process, the copper dibenzyl dithiocarbamate has a good effect of inhibiting drug-resistant triple negative breast cancer.)

1. The application of the copper dibenzyl dithiocarbamate medicament in treating triple negative breast cancer is characterized in that the specific preparation method of the copper dibenzyl dithiocarbamate medicament is as follows:

the method comprises the following steps: preparation of sodium dibenzyl dithiocarbamate

Stirring 0.06moL of dibenzylamine with the mass of 11.8g and 150ml of acetonitrile in an ice water bath, adding 6.96g of carbon disulfide with the mass of 6.96g and 0.09moL of sodium hydroxide with the mass fraction of 50% into the mixture, stirring the mixture to react for 16 hours, performing suction filtration, performing rotary evaporation on the filtrate to obtain a product, washing the product with diethyl ether, drying the product to obtain 13.04g of product, and recrystallizing the crude product with acetonitrile to obtain 11.1g of sodium dibenzyl dithiocarbamate;

step two: preparation of copper dibenzyl dithiocarbamate

8ml of N, N-dimethylformamide, 1mmol of sodium dibenzyldithiocarbamate (0.295 g in mass), and 1mmol of CuCl (0.171 g in mass) were charged in a beaker₂·2H₂O and 2ml of H₂O stirring to dissolve completely, transferring the solution to 25ml of polytetrafluoroethyleneAnd (3) reacting the mixture in a stainless steel high-pressure reaction kettle with an inner lining at the temperature of 80 ℃ for 72 hours, cooling the mixture to room temperature at the speed of 10 ℃/min after the reaction is finished, and separating out dark green crystals on the inner wall of the reaction kettle, namely the copper dibenzyl dithiocarbamate.

2. The use of copper dibenzyl dithiocarbamate in the drug for the treatment of triple negative breast cancer according to claim 1, wherein the temperature in the ice-water bath is between 0 ℃ and 4 ℃.

3. The use of copper dibenzyl dithiocarbamate for the treatment of triple negative breast cancer according to claim 1, wherein the specific steps are as follows:

s1, firstly establishing paclitaxel resistant cells, wherein the MDA-MB-231 cell strain belongs to triple negative breast cancer, inducing the paclitaxel resistance, establishing a drug resistant cell line, JL-1912 comparing with anthracycline drugs, and comparing IC₅₀；

Firstly, establishing an MDA-MB-231 taxol-resistant cell line, and screening the acting concentration of taxol;

secondly, screening drug-resistant cell strains;

thirdly, calculating the drug resistance times of the drug-resistant cell strains;

s2, adding epirubicin, copper dibenzyldithiocarbamate and complete culture solution into the paclitaxel drug-resistant cells respectively, and carrying out control culture and then carrying out color comparison.

4. The use of copper dibenzyl dithiocarbamate in the treatment of triple negative breast cancer according to claim 3, wherein the specific operation steps of the first step are as follows:

laying cells in a 96-well plate in advance, wherein the laying density is 1x105 cells/ml, the volume is 100 mu L per well, after the cells are attached to the wall, serum-free synchronous culture is carried out for 24 hours, culture media containing paclitaxel with different concentrations and 100 mu L per well are respectively added, after the cells are cultured for 72 hours, each well of culture solution is sucked, 100 mu L of fresh serum-free 1640 culture medium and 10 mu LMTT detection reagent are added, the mixture is placed for 4 hours at 37 ℃, cell supernatant is sucked and discarded, MTT reagent lysate of 10 percent SDS is added, and the mixture is fully mixed with the culture solutionDissolving precipitate, reading light absorption value at 490nm, selecting the concentration with cell survival rate higher than 90% as subsequent action concentration, and calculating IC of paclitaxel on MDA231 cell₅₀And (4) concentration.

5. The use of copper dibenzyl dithiocarbamate in the treatment of triple negative breast cancer according to claim 3, wherein the specific steps of the second step are as follows:

the method comprises the steps of pre-paving cells in a 6-well plate, wherein the paving density is 1x105 cells/ml, the volume is 1500 mu L per well, replacing a culture medium containing paclitaxel after the cells are attached to the wall, continuously acting the cells, replacing the culture medium once every 3-5 days, increasing the acting concentration of the paclitaxel by one time when the cells do not die obviously, repeatedly increasing the acting concentration of the paclitaxel for treating MDA231 cells for 4-5 months, and confirming MDA231 paclitaxel resistant cell strains MDA 231-TAXO.

6. The use of copper dibenzyl dithiocarbamate in the treatment of triple negative breast cancer according to claim 3, wherein the third step is performed by the following steps:

pre-spreading MDA231 and MDA231-TAXO cells in a 96-well plate, wherein the density of the spread plate is 1x 105/mL, the volume is 100 mu L/well, the cells are subjected to serum-free synchronous culture for 24h after being attached to the wall, culture media containing paclitaxel with different concentrations and 100 mu L/well are respectively added, after the cells are cultured for 72h, culture solution in each well is sucked, 100 mu L of fresh serum-free 1640 culture medium and 10 mu L of MTT detection reagent are added, the cells are placed for 4h at 37 ℃, cell supernatant is sucked and discarded, MTT reagent lysate of 10% SDS is added, precipitates are fully dissolved and precipitated, the light absorption values are read at 490nm, and the semi-inhibitory concentration IC of the MDA231 and MDA231-TAXO cells are calculated₅₀Obtaining the drug resistance multiple, wherein the drug resistance multiple = MDA231-TAXO cell IC50/MDA231 cell IC_50，5.4RI =8.13, available to the cells.

7. The use of copper dibenzyl dithiocarbamate for the treatment of triple negative breast cancer according to claim 3, wherein the specific operation step of S2 is as follows:

the MDA231-TAXO cells are paved in a 96-well plate in advance, the paving density is 1x 105/mL, the volume is 100 mu L/well, the conventional culture is carried out for 24h, the concentration is set as an EPI group and a JL-1912 group, only complete culture solution is added into a control group, a culture medium without cells and substances to be detected is used for removing experiment background interference in blank holes, the culture is carried out for 20h after medicine addition, 20ul of 5mg/mL MTT solution is added into each hole, and the incubation is carried out for 4h continuously at 37 ℃; carefully remove the culture supernatant from the wells, add 150uL DMSO per well and shake for 10min to dissolve the crystals sufficiently, color: the 490nm wavelength is selected, the absorbance of each well is set on an enzyme linked immunosorbent assay instrument, and the result is recorded.

8. The use of copper dibenzyldithiocarbamate in the treatment of triple negative breast cancer according to claim 7, wherein the concentrations in the group of EPI are 0.5. mu.M, 1. mu.M, 2. mu.M and 5. mu.M, respectively, and the concentration in the group of JL-1912 is 0.5. mu.M, 1. mu.M, 2. mu.M and 5. mu.M.

Technical Field

The invention relates to the technical field of breast cancer treatment, in particular to application of copper dibenzyl dithiocarbamate medicaments to treatment of triple negative breast cancer.

Background

Breast cancer is the most common female cancer, accounting for about 23% of the total number of cancers, triple negative breast cancer is a heterogeneous group of breast cancers; chemotherapy is the only systemic treatment mode for improving prognosis at present, the main medicines are paclitaxel and anthracycline medicines, but no specific chemotherapeutic medicine has superiority in a specific breast cancer phenotype, even if the chemotherapy medicine has good reaction, the tumor can still be in a rapid progress state, and for a TNBC patient in late stage with chemotherapy failure, a new and more targeted treatment scheme is urgently required to be developed to achieve the treatment purpose; disulfiram is a drug discovered in the 19 th century and used for abstinence of alcohol, and related anticancer effects of disulfiram have renewed interest for people in recent 40 years, but at present, related researches at home and abroad are all carried out by using disulfiram or metal chelates thereof for anticancer effect research.

Disclosure of Invention

The invention aims to provide the application of the copper dibenzyl dithiocarbamate medicament in treating triple negative breast cancer, and the new medicament copper dibenzyl dithiocarbamate is synthesized again on the basis of a disulfiram anticancer structure, so that the in-vivo transformation is not needed, and the fat solubility is improved.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of the copper dibenzyl dithiocarbamate medicament in treating triple negative breast cancer comprises the following specific preparation method of the copper dibenzyl dithiocarbamate medicament:

the method comprises the following steps: preparation of sodium dibenzyl dithiocarbamate

Stirring 0.06moL of dibenzylamine with the mass of 11.8g and 150ml of acetonitrile in an ice water bath, adding 0.09moL of carbon disulfide with the mass of 6.96g and 0.09moL of sodium hydroxide with the mass fraction of 50%, stirring for reacting for 16 hours, performing suction filtration, performing rotary evaporation on the filtrate to obtain a product, washing the product with diethyl ether, drying to obtain a product 13.04g, and recrystallizing the crude product with acetonitrile to obtain sodium dibenzyl dithiocarbamate with the mass of 11.1 g;

step two: preparation of copper dibenzyl dithiocarbamate

8ml of N, N-dimethylformamide, 1mmol of sodium dibenzyldithiocarbamate (0.295 g in mass), and 1mmol of CuCl (0.171 g in mass) were charged in a beaker₂·2H₂O and 2ml of H₂Stirring to dissolve completely, transferring the solution into 25ml stainless steel high-pressure reaction kettle with polytetrafluoroethylene lining, reacting at 80 deg.C for 72 hr, and reacting at 10 deg.C/minCooling to room temperature, and precipitating dark green crystals on the inner wall of the reaction kettle, namely the copper dibenzyl dithiocarbamate.

Preferably, the temperature in the ice-water bath is 0-4 ℃.

Preferably, the specific operation steps of the application are as follows:

s1, firstly establishing paclitaxel resistant cells, wherein the MDA-MB-231 cell strain belongs to triple negative breast cancer, inducing the paclitaxel resistance, establishing a drug resistant cell line, JL-1912 comparing with anthracycline drugs, and comparing IC₅₀；

Firstly, establishing an MDA-MB-231 taxol-resistant cell line, and screening the acting concentration of taxol;

secondly, screening drug-resistant cell strains;

thirdly, calculating the drug resistance times of the drug-resistant cell strains;

s2, adding epirubicin, copper dibenzyldithiocarbamate and complete culture solution into the paclitaxel drug-resistant cells respectively, and carrying out control culture and then carrying out color comparison.

Preferably, the specific operation steps of the first step are as follows:

laying cells in a 96-well plate in advance, wherein the laying density is 1x105 cells/ml, the volume is 100 mu L per well, after the cells are attached to the wall, serum-free synchronous culture is carried out for 24 hours, culture media containing paclitaxel with different concentrations and 100 mu L per well are respectively added, after the cells are cultured for 72 hours, culture solution in each well is sucked, 100 mu L of fresh serum-free 1640 culture medium and 10 mu LMTT detection reagent are added, the cells are placed for 4 hours at 37 ℃, cell supernatant is sucked and discarded, MTT reagent lysate of 10 percent SDS is added, full dissolution and precipitation are carried out, the light absorption value is read at 490nm, the concentration of the cell survival rate higher than 90 percent is selected as the subsequent action concentration, and IC of the paclitaxel on MDA231 cells is calculated at the same time₅₀And (4) concentration.

Preferably, the specific operation steps of the second step are as follows:

the method comprises the steps of pre-paving cells in a 6-well plate, wherein the paving density is 1x105 cells/ml, the volume is 1500 mu L per well, replacing a culture medium containing paclitaxel after the cells are attached to the wall, continuously acting the cells, replacing the culture medium once every 3-5 days, increasing the acting concentration of the paclitaxel by one time when the cells do not die obviously, repeatedly increasing the acting concentration of the paclitaxel for treating MDA231 cells for 4-5 months, and confirming MDA231 paclitaxel resistant cell strains MDA 231-TAXO.

Preferably, the third step comprises the following specific operation steps:

pre-spreading MDA231 and MDA231-TAXO cells in a 96-well plate, wherein the density of the spread plate is 1x 105/mL, the volume is 100 mu L/well, the cells are subjected to serum-free synchronous culture for 24h after being attached to the wall, culture media containing paclitaxel with different concentrations and 100 mu L/well are respectively added, after the cells are cultured for 72h, culture solution in each well is sucked, 100 mu L of fresh serum-free 1640 culture medium and 10 mu L of MTT detection reagent are added, the cells are placed for 4h at 37 ℃, cell supernatant is sucked and discarded, MTT reagent lysate of 10% SDS is added, precipitates are fully dissolved and precipitated, the light absorption values are read at 490nm, and the semi-inhibitory concentration IC of the MDA231 and MDA231-TAXO cells are calculated₅₀Obtaining the drug resistance multiple, wherein the drug resistance multiple is MDA231-TAXO cell IC50/MDA231 cell IC₅₀5.4RI ═ 8.13, cells were available.

Preferably, the specific operation steps of S2 are as follows:

the MDA231-TAXO cells are paved in a 96-well plate in advance, the paving density is 1x 105/mL, the volume is 100 mu L/well, the conventional culture is carried out for 24h, the concentration is set as an EPI group and a JL-1912 group, only complete culture solution is added into a control group, a culture medium without cells and substances to be detected is used for removing experiment background interference in blank holes, the culture is carried out for 20h after medicine addition, 20ul of 5mg/mL MTT solution is added into each hole, and the incubation is carried out for 4h continuously at 37 ℃; carefully remove the culture supernatant from the wells, add 150uL DMSO per well and shake for 10min to dissolve the crystals sufficiently, color: the 490nm wavelength is selected, the absorbance of each well is set on an enzyme linked immunosorbent assay instrument, and the result is recorded.

Preferably, the concentration of the EPI group is 0.5. mu.M, 1. mu.M, 2. mu.M and 5. mu.M, respectively, and the concentration of the JL-1912 group is 0.5. mu.M, 1. mu.M, 2. mu.M and 5. mu.M.

The invention has the beneficial effects that: on the basis of a disulfiram anticancer structure, a new medicament copper dibenzyl dithiocarbamate is synthesized again, in-vivo transformation is not needed, the fat solubility is improved, and in the actual experimental process, the copper dibenzyl dithiocarbamate has a good effect of inhibiting drug-resistant triple negative breast cancer.

Drawings

FIG. 1 is a schematic representation of the three-dimensional structure of copper dibenzyl dithiocarbamate according to the present invention;

FIG. 2 is a graph showing the MDA231 and MDA231-TAXO cell half inhibitory concentration IC of the present invention₅₀Obtaining a drug resistance times table;

FIG. 3 is a graph plotting the concentration on the horizontal axis and the inhibition rate on the vertical axis according to the experimental data of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Specific examples are given below.

Referring to fig. 1, the application of copper dibenzyl dithiocarbamate in treating triple negative breast cancer is specifically disclosed in the following:

the method comprises the following steps: preparation of sodium dibenzyl dithiocarbamate

Stirring 0.06moL of dibenzylamine with the mass of 11.8g and 150ml of acetonitrile in an ice water bath, adding 0.09moL of carbon disulfide with the mass of 6.96g and 0.09moL of sodium hydroxide with the mass fraction of 50%, stirring for reacting for 16 hours, performing suction filtration, performing rotary evaporation on the filtrate to obtain a product, washing the product with diethyl ether, drying to obtain a product 13.04g, and recrystallizing the crude product with acetonitrile to obtain sodium dibenzyl dithiocarbamate with the mass of 11.1 g;

step two: preparation of copper dibenzyl dithiocarbamate

8ml of N, N-dimethylformamide, 1mmol of sodium dibenzyldithiocarbamate (0.295 g in mass), and 1mmol of CuCl (0.171 g in mass) were charged in a beaker₂·2H₂O and 2ml of H₂O stirring to dissolve completely, transferring the solution to 25ml teflon linerReacting in a high-pressure stainless steel reaction kettle at 80 ℃ for 72 hours, cooling to room temperature at the speed of 10 ℃/min after the reaction is finished, and precipitating dark green crystals on the inner wall of the reaction kettle, namely copper dibenzyl dithiocarbamate, wherein the structural formula of the copper dibenzyl dithiocarbamate is shown as follows;

as an embodiment of the invention, the temperature in the ice-water bath is 0-4 ℃.

As an embodiment of the invention, the specific operation steps of the application are as follows:

s1, firstly establishing paclitaxel resistant cells, wherein the MDA-MB-231 cell strain belongs to triple negative breast cancer, inducing the paclitaxel resistance, establishing a drug resistant cell line, JL-1912 comparing with anthracycline drugs, and comparing IC₅₀；

Firstly, establishing an MDA-MB-231 taxol-resistant cell line, and screening the acting concentration of taxol;

secondly, screening drug-resistant cell strains;

thirdly, calculating the drug resistance times of the drug-resistant cell strains;

s2, adding epirubicin, copper dibenzyldithiocarbamate and complete culture solution into the paclitaxel drug-resistant cells respectively, and carrying out control culture and then carrying out color comparison.

As an embodiment of the invention, the specific operation steps of the first step are as follows:

laying cells in a 96-well plate in advance, wherein the laying density is 1x105 cells/ml, the volume is 100 mu L per well, after the cells are attached to the wall, serum-free synchronous culture is carried out for 24 hours, culture media containing paclitaxel with different concentrations and 100 mu L per well are respectively added, after the cells are cultured for 72 hours, culture solution in each well is sucked, 100 mu L of fresh serum-free 1640 culture medium and 10 mu LMTT detection reagent are added, the cells are placed for 4 hours at 37 ℃, cell supernatant is sucked and discarded, MTT reagent lysate of 10 percent SDS is added, full dissolution and precipitation are carried out, the light absorption value is read at 490nm, the concentration of the cell survival rate higher than 90 percent is selected as the subsequent action concentration, and IC of the paclitaxel on MDA231 cells is calculated at the same time₅₀Concentration;

the second step comprises the following specific operation steps:

laying cells in a 6-hole plate in advance, wherein the laying density is 1x105 cells/ml, the volume is 1500 muL per hole, a culture medium containing paclitaxel is replaced after the cells are attached to the wall, the cells are continuously acted, the culture medium is replaced once every 3-5 days, when the cells do not obviously die, the action concentration of the paclitaxel is doubled, the time for treating MDA231 cells by repeatedly increasing the action concentration of the paclitaxel for 4-5 months is repeated, and MDA231 paclitaxel resistant cell strains MDA231-TAXO are confirmed;

the third step comprises the following specific operation steps:

pre-spreading MDA231 and MDA231-TAXO cells in a 96-well plate, wherein the density of the spread plate is 1x 105/mL, the volume is 100 mu L/well, the cells are subjected to serum-free synchronous culture for 24h after being attached to the wall, culture media containing paclitaxel with different concentrations and 100 mu L/well are respectively added, after the cells are cultured for 72h, culture solution in each well is sucked, 100 mu L of fresh serum-free 1640 culture medium and 10 mu L of MTT detection reagent are added, the cells are placed for 4h at 37 ℃, cell supernatant is sucked and discarded, MTT reagent lysate of 10% SDS is added, precipitates are fully dissolved and precipitated, the light absorption values are read at 490nm, and the semi-inhibitory concentration IC of the MDA231 and MDA231-TAXO cells are calculated₅₀Obtaining the drug resistance multiple, wherein the drug resistance multiple is MDA231-TAXO cell IC50/MDA231 cell IC₅₀5.4RI ═ 8.13, cells were available.

MDA231-TAXO resistance to TAXO

As an embodiment of the present invention, the specific operation steps of S2 are as follows:

the MDA231-TAXO cells are paved in a 96-well plate in advance, the paving density is 1x 105/mL, the volume is 100 mu L/well, the conventional culture is carried out for 24h, the concentration is set as an EPI group and a JL-1912 group, only complete culture solution is added into a control group, a culture medium without cells and substances to be detected is used for removing experiment background interference in blank holes, the culture is carried out for 20h after medicine addition, 20ul of 5mg/mL MTT solution is added into each hole, and the incubation is carried out for 4h continuously at 37 ℃; carefully remove the culture supernatant from the wells, add 150uL DMSO per well and shake for 10min to dissolve the crystals sufficiently, color: the 490nm wavelength is selected, the absorbance of each well is set on an enzyme linked immunosorbent assay instrument, and the result is recorded.

As an embodiment of the present invention, the concentrations of the EPI group are 0.5. mu.M, 1. mu.M, 2. mu.M and 5. mu.M, respectively, and the concentration of the JL-1912 group is 0.5. mu.M, 1. mu.M, 2. mu.M and 5. mu.M.

The concentration is plotted on the horizontal axis and the inhibition is plotted on the vertical axis, wherein the upper curve is JL-1912, the upper curve is copper dibenzyl dithiocarbamate, the lower curve is EPI, and the EPI is epirubicin.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.