CN111825623B

CN111825623B - [2- (5' -fluorouracil) acetic acid- (N-ethyl) piperazinyl dithiocarbamate ] anhydride and application thereof

Info

Publication number: CN111825623B
Application number: CN202010612432.3A
Authority: CN
Inventors: 刘明星; 王溥照; 李洁南; 王友运
Original assignee: Hubei University of Technology
Current assignee: Hubei University of Technology
Priority date: 2020-06-30
Filing date: 2020-06-30
Publication date: 2021-04-23
Anticipated expiration: 2040-06-30
Also published as: CN111825623A

Abstract

The invention belongs to the technical field of preparation of antitumor drugs, and particularly relates to design and synthesis of a 5-fluorouracil and dithiocarbamate compound P1 and application thereof in preparation of antitumor drugs, in particular to [2- (5' -fluorouracil) acetic acid- (N-ethyl) piperazinyl dithiocarbamate ] anhydride and application thereof in preparation of antitumor drugs. After certain derivatization of 5-fluorouracil, the derivative is combined with (N-ethyl) piperazinyl dithiocarbamate through a chemical bond, and a potential multi-target antitumor new drug is designed and synthesized. The cytotoxicity test verifies the anti-tumor activity of the compound, and the compound is used in combination with copper ions, so that the inhibition capacity of the growth of corresponding tumor cells is obviously enhanced. The tumor cell clone formation experiment further shows the capability of inhibiting the cell proliferation activity, and the cell metastasis experiment also shows the inhibition effect of the tumor cell metastasis capability.

Description

[2- (5' -fluorouracil) acetic acid- (N-ethyl) piperazinyl dithiocarbamate ] anhydride and application thereof

Technical Field

The invention belongs to the technical field of preparation of antitumor drugs, and particularly relates to design and synthesis of a 5-fluorouracil and dithiocarbamate compound P1 and application thereof in preparation of antitumor drugs, in particular to [2- (5' -fluorouracil) acetic acid- (N-ethyl) piperazinyl dithiocarbamate ] anhydride and application thereof in preparation of antitumor drugs.

Background

5-fluorouracil (5-FU) is a common pyrimidine antimetabolite and has been successfully developed to date as the first choice therapeutic drug for gastric cancer, rectal cancer, breast cancer and the like. However, 5-fluorouracil has low lipid solubility, obvious first-pass elimination and more adverse reactions in clinical application, further application of the 5-fluorouracil is limited, and a plurality of excellent drugs are obtained after proper derivatization of the 5-fluorouracil, compared with 5-fluorouracil, the drugs have the advantages of enhanced clinical curative effect, reduced toxicity and convenient administration mode, which indicates that structural modification of the 5-fluorouracil is an important way for solving the defects of the 5-fluorouracil.

The dithiocarbamate derivative is generally used as a vulcanization accelerator for a rubber article. Meanwhile, the derivatives also have biological activities of killing insects, resisting bacteria, resisting fungi, detoxifying agents for heavy metals, resisting tumors and the like. The sulfur atom in the dithioamino group can form a stable complex with transition metal ions, so that the complex becomes a multi-effect pharmacophore, and a batch of compounds with good anti-tumor activity are obtained by screening. The anticancer mechanism of disulfiram is reported, and the disulfiram metabolizes in vivo to generate two molecules of diethyl dithiocarbamate ions, the ions and copper ions form an active anticancer compound, and the compound can be firmly combined with NPL4 protein in a p97-NPL4 pathway, inhibit the quality control function of the compound, enable a large amount of defective protein to be accumulated in cancer cells and finally induce the apoptosis of the cancer cells.

The principle of drug combination is a common drug structure modification means in medicinal chemistry. The disadvantages are that: due to the large molecular structure of the combined medicament, the combined medicament is often in large contact with the envisaged target. Such as: sometimes the stereoselectivity of the split drug molecule is altered; sometimes, the pharmacodynamic group is masked by a group with a larger ortho position to form larger steric hindrance; and the difficulty of bond breaking is also related to the structure of the connected group. After the structure of the drug is changed, the absorption, transportation, metabolism and the like of the drug in the body are changed, which leads to the complex situation of pharmacodynamics. Such as: the spliced molecules generally have larger relative molecular mass, so the permeability of the cells is poorer; the molecular structure is large, the property is relatively unstable, and the decomposition is easy, so that the practical medicinal value is not available; in addition, some molecules are too stable in vivo, are not sensitive to hydrolytic enzymes, and cannot rapidly and quantitatively release target drugs, so that the molecules also have no clinical significance.

Disclosure of Invention

In view of the deficiencies of the prior art and to fill the technical gap in the field, the first object of the present invention is to provide a compound P1: [2- (5' -Fluorouracil) acetic acid- (N-ethyl) piperazinyldithiocarbamic acid]Anhydrides of the formula:

the molecular formula is as follows: c₁₃H₁₇FN₄O₃S₂

Molecular weight: 360.07261

The compound is prepared by properly deriving 5-fluorouracil and connecting the derivative with (N-ethyl) piperazine dithiocarbamate through acid anhydride bond, thereby preparing a novel derivative with better solubility as a prodrug. The product is decomposed under the action of human lactonase, so that the curative effect is exerted, and the two medicines can mutually promote on the pharmacological activity, thereby achieving the purposes of enhancing the curative effect, reducing the toxic and side effects and improving the bioavailability of the medicines. And the medicament after the formation of the ester can be released only by hydrolysis of esterase, so that the possibility of prolonging the action time is provided, and the research and development are worth.

The second purpose of the invention is to provide an application of the compound P1 in preparing anti-tumor drugs.

The compound is subjected to preliminary biological activity evaluation, and the result shows that the compound has obvious effects of inhibiting the growth of tumor cells MCF-7, U87MG, HepG2 and Hela.

The experiment of the invention proves that: the introduction of (N-ethyl) piperazinyl dithiocarbamate group increases the activity of 5-fluorouracil derivative, which indicates that (N-ethyl) piperazinyl dithiocarbamate group has synergistic effect on the anticancer activity of 5-fluorouracil. When the compound P1 is used in combination with copper ions, the antitumor activity of the compound P1 is obviously enhanced, which indicates that the compound P1 is metabolized to (N-ethyl) piperazinyl dithiocarbamate under the action of enzymes in tumor cells, and forms a chelate with the copper ions, so that the activity is enhanced. When the compound P1 is used in combination with copper ions (preferably, the compound P1 is used in combination with copper ions according to the molar ratio of 1: 1), the proliferation activity and the metastatic capacity of tumor cells can be inhibited, and the tumor cells can be blocked in the G1 stage.

The compound P1 can be prepared by the following synthetic process route:

compared with the prior art, the invention has the advantages and beneficial effects that:

1. provides a new medicinal compound with outstanding advantages;

2. the preparation method of the novel compound has the advantages of simple operation steps, less side reactions, easily obtained raw materials and green and environment-friendly solvent.

Drawings

FIG. 1 is a drawing showing the preparation of sodium (N-ethyl) piperazinyldithiocarbamate prepared in example III¹H NMR spectrum;

FIG. 2 is the [2- (5' -fluorouracil) acetic acid- (N-ethyl) piperazinyldithiocarbamic acid prepared in example IV]Of anhydrides¹H NMR spectrum;

FIG. 3 is the [2- (5' -fluorouracil) acetic acid- (N-ethyl) piperazinyldithiocarbamic acid prepared in example IV]Of anhydrides¹³A C NMR spectrum;

FIG. 4 is a graph showing the results of the cytotoxicity test of U87MG in example V;

FIG. 5 is a graph showing the results of the cytotoxicity test in HepG2 in example five;

FIG. 6 is a graph showing the results of the cytotoxicity test of MCF-7 in example five;

FIG. 7 is a graph showing the results of the Hela cytotoxicity test in example five;

FIG. 8 is a graph showing the results of the experiments for detecting apoptosis of MCF-7 cells in the sixth example, wherein p is <0.05 and p is < 0.01;

fig. 9 is a graph showing the results of the apoptosis test of U87MG in example six, wherein p is <0.05 and p is < 0.01;

FIG. 10 is a graph showing the results of the experiments for cloning MCF-7 cells in example seven;

FIG. 11 is a graph showing the result of the colony formation experiment of U87MG cells in example VII;

FIG. 12 is a graph showing the results of clone formation experiments of HepG2 cell in example seven;

in FIGS. 10-12, p <0.05, p < 0.01;

FIG. 13 is a graph showing the results of the Transwell experiment on MCF-7 cells in example eight;

FIG. 14 is a graph showing the results of the Transwell experiment on U87MG cells in example eight;

FIG. 15 is a graph showing the results of the Transwell experiment on HepG2 cells in example eight;

in FIGS. 13-15, p <0.05, p < 0.01;

FIG. 16 is a graph showing the results of the cell cycle assay of MCF-7 cells in example nine;

FIG. 17 is a graph showing the results of the cell cycle test of U87MG cells in example nine.

Detailed Description

The following detailed description of the preparation and application of compound P1 of the present invention will be provided by the applicant in conjunction with specific examples to facilitate the clear understanding of the present invention by those skilled in the art. It should be understood that the following examples should not be construed as limiting the scope of the claims of the present application in any way.

Examples one-four are examples of the preparation of compound P1, using reagents which are all commercially available, all grades being analytical pure, and a concentrated hydrochloric acid concentration of 37 wt%.

EXAMPLE one, Synthesis of 5-Fluorouracil-1-acetic acid

Adding 0.2mol of potassium hydroxide and 40ml of water into a three-neck flask, stirring and dissolving, adding 0.04mol of 5-fluorouracil, heating to 60 ℃, stirring for 30min, slowly dropwise adding a bromoacetic acid aqueous solution containing 0.072mol of bromoacetic acid, continuing to react to the end point after the dropwise adding is finished (TLC tracking), adjusting the pH to 5.4 by using concentrated hydrochloric acid, putting the mixture into a 4 ℃ refrigerator for cooling for 2h, filtering if precipitates are separated out, adjusting the pH to 2 by using concentrated hydrochloric acid, putting the mixture into an ice box for cooling for 12h, filtering, washing the precipitates by using pure water for 3 times, recrystallizing by using needle-shaped pure water, and drying to obtain 4.9g (yield: 64%) of 5-fluorouracil-1-acetic acid.¹H NMR(400MHz,dmso)δ13.23(s,1H),11.92(d,J＝5.0Hz,1H),8.06(d,J＝6.8Hz,1H),4.35(s,2H).m.p.280.3～280.9℃.

EXAMPLE Synthesis of 5-Fluorouracil-1-acetyl chloride

0.02mol of 5-fluorouracil-1-acetic acid prepared in example one was dissolved in 20mL of dichloromethane, cooled to 0 ℃, 15mL of thionyl chloride was slowly added dropwise, the reaction was continued to the end point by increasing the temperature to room temperature after completion of the dropwise addition (TLC tracing), and 3.17g of 5-fluorouracil-1-acetyl chloride was obtained as a pale yellow solid after spin-drying (yield 76%).¹H NMR(400MHz,dmso)δ11.88(d,J＝5.0Hz,1H),8.08(dd,J＝6.8,1.8Hz,1H),4.35(s,2H).m.p.270.2～272.0℃.

EXAMPLE III Synthesis of sodium (N-Ethyl) piperazinyldithiocarbamate

1g (25mmol) of sodium hydroxide and 8ml of methanol are added to the flask, 2.85g (25mmol) of N-ethylpiperazine is added to the flask, the temperature is reduced to below 10 ℃, 3ml of carbon disulfide is slowly added dropwise to the flask, the reaction is carried out till the end point (TLC tracking) at room temperature, and the solvent is evaporated to dryness under reduced pressure to obtain 4.6g (87%) of yellow solid.¹H NMR(400MHz,dmso)δ8.08(d,J＝6.6Hz,1H),4.30(s,2H),3.04(s,4H),2.67(s,4H),2.47(d,J＝5.8Hz,2H),0.99(t,J＝7.0Hz,3H).

EXAMPLE Synthesis of tetrakis, [2- (5' -fluorouracil) acetic acid- (N-ethyl) piperazinyldithiocarbamic acid ] anhydride (Compound P1)

Adding 1.2g (5mmol) of sodium (N-ethyl) piperazinyldithiocarbamate and 10ml of THF into a reaction vessel, stirring, slowly dropwise adding 20ml of THF solution containing 1g (5mmol) of 5-fluorouracil-1-acetyl chloride, heating to 60 ℃ for reaction to the end point (TLC tracking), filtering, washing a filter cake with water,an off-white solid powder (1.2 g, 67%)¹H NMR(400MHz,dmso)δ7.94(d,J＝6.9Hz,1H),4.42–4.31(m,2H),4.14–3.88(m,4H),2.61–2.20(m,4H),1.00(dt,J＝12.6,7.1Hz,3H).¹³C NMR(101MHz,dmso)δ169.81(s),158.02(s),157.77(s),150.09(s),140.78(s),138.51(s),131.31(s),130.97(s),51.65(s),49.34(s),49.03(s),42.73(s),11.59(s).m.p.258.2～259.0℃.

In the following examples, human breast cancer cell MCF-7, glioma cell U87MG, liver cancer cell HepG2 and cervical cancer cell Hela were all cryopreserved materials in the laboratory of the present inventors (present in the group of the banian project by the special letter of university of north and lake).

EXAMPLE V cytotoxicity test

According to the counting result, a proper amount of each tumor cell suspension is taken and added into a blank high-sugar culture medium (DMEM high-sugar culture medium containing 10% fetal calf serum and 1% double-antibody solution (the double-antibody solution is prepared by the steps of weighing 1.5g of penicillin and 3.75g of streptomycin by using an analytical balance, adding the penicillin and the streptomycin into 250ml of PBS (the PBS concentration is 10mM, the pH is 7.4, the following differences are not repeated), carrying out ultrasonic treatment until the penicillin and the streptomycin are completely dissolved, filtering the penicillin and the streptomycin in a super-clean workbench, subpackaging the penicillin and the streptomycin in into 10ml of EP tubes, storing the dissolved penicillin and the dissolved and the filtered solutions in a refrigerator at-20 ℃ for later use, and evenly distributing the dissolved and the. The plate is labeled with the cell name and the plating date, and the 96-well plate is placed in an incubator (37 ℃, 5% CO)₂). After 24 hours, the cells enter the logarithmic growth phase after the adherent recovery of morphology. Diluting compound P1 (with storage concentration of 10mM and solvent of DMSO) with complete culture medium (DMEM high-sugar medium containing 10% fetal calf serum and 1% double antibody solution) to different concentrations, discarding the upper layer culture medium in 96-well plate, changing to culture medium (DMEM high-sugar medium containing 10% fetal calf serum and 1% double antibody solution, 100 μ l/well) containing 5-FU, compound P1, P1/Cu (equimolar P1 and copper gluconate), mixing, dissolving, and preparing into solution with different concentrations (the concentration refers to molar concentration of P1 or copper gluconate)₂) And (5) 24 h. MTT stock solution (0.5g MTT, completely dissolved in 100ml1 XPBS, dispensed at-20 deg.CFrozen in a refrigerator) and added to a 96-well plate MTT stock (20 μ l/well) and incubated at 37 ℃ for 4 hours. The 96-well plate was removed from the incubator, the supernatant was discarded, and 150. mu.l of DMSO (analytical grade) was added and dissolved on a shaker for 10 minutes. The absorbance at 490nm was measured on a microplate reader. The survival of the cells after treatment with different compounds was calculated. The survival rate of the control group is 100%, the survival rate of the cells treated by the compound P1 is (absorbance of the experimental group-absorbance of the blank group)/(absorbance of the control group-absorbance of the blank group) × 100%, the culture solution, the thiazole blue and the dimethyl sulfoxide are only added to the blank group, and the concentration of the drug in the control group is zero. The thiazole blue assay was repeated six times and the results are shown in FIGS. 4-7, corresponding data in tables 1-4.

TABLE 1U 87MG cytotoxicity test results (cell viability/%)

TABLE 2 results of HepG2 cytotoxicity test (cell viability/%)

TABLE 3 results of MCF-7 cytotoxicity assay (cell viability/%)

TABLE 4 Hela cytotoxicity test results (cell viability/%)

As can be seen from the figure, the cytotoxicity of P1 is similar to that of the positive control group 5-FU in the absence of copper ions, while the cytotoxicity of P1/Cu is obviously enhanced and is obviously stronger than that of the control group 5-FU in the presence of copper ions, which indicates that P1 is metabolized in cells to generate 5-FU and (N-ethyl) piperazinyl dithiocarbamate which jointly exert the antitumor effect, and the (N-ethyl) piperazinyl dithiocarbamate forms a chelate with the copper ions to obviously enhance the antitumor activity.

EXAMPLE six apoptosis detection

Each cell was cultured until after passage and tumor cells (15X 10) were plated in six-well plates⁴Per well), after 24 hours the cells recovered their morphology after adherence, replaced with an equal amount of fresh blank medium or fresh medium containing different concentrations of P1/Cu (2 mL/well), and incubated in an incubator for 48 hours (37 ℃, 5% CO)₂)。

A number of 5mL EP tubes were prepared and assigned serial numbers on crushed ice. The plates were removed and the supernatants collected in corresponding EP tubes. The remaining medium was washed out with 1 × PBS. Pancreatin was added for digestion, and all cells were collected after the cells became spotted and centrifuged at 1200rpm for 5 minutes. The supernatant was discarded and washed 2 times with 1 XPBS, at which time the cells had been transferred to 1.5ml EP tubes. The cells were flicked off by adding Annexin V binding solution and vortexed by adding FITC staining solution (2.5. mu.l/tube) and incubated on a shaker for 15 min. PI staining solution (5. mu.l/tube) was added, pipetted evenly and incubated on a shaker for 5 minutes. The cell density in each EP tube was adjusted (to give a cell count of 300 cells per second-500), and the results are shown in FIGS. 8-9, after sieving, in a flow cytometer.

The medium used in this example was the same as that used in the fifth example.

In this example, an Annexin V-FITC apoptosis detection kit was used 100 times, Shanghai Bibo biology.

As can be seen from FIGS. 8-9, the proportion of apoptotic cells was gradually increased with increasing concentration of either MCF-7 cells or U87MG cells, indicating that P1/Cu can exert an anti-tumor effect by promoting apoptosis of tumor cells.

EXAMPLE seventhly, clone formation experiment

Each cell was cultured until after passage and tumor cells (5000/well) were plated in six-well plates. After 24 hours, the cells were incubated for 7 days with the same amount of blank medium or medium containing different concentrations of P1/Cu to restore their adherent morphology. The supernatant was discarded, and the excess medium was washed out 2 times with 1 XPBS. The gel was fixed with methanol at 37 ℃ for 30 minutes, and excess methanol was washed off with 1 XPBS. The results of the photographs taken after staining with 1 Xcrystal violet stain (1mM) for 30 minutes and washing with 1 XPBS of excess stain are shown in FIGS. 10-12, and the corresponding data are shown in Table 5.

The medium used in this example was the same as that used in the fifth example.

As can be seen from the figure, the cell clone number gradually decreased with the increase of the concentration, and the cell clone number was already close to zero at the concentration of 62.5. mu.M, indicating that P1/Cu can exert an antitumor effect by inhibiting cell proliferation.

TABLE 5 clonogenic ratios (%) -for different cells at different concentrations

EXAMPLE eight, Transwell experiment

Each cell was cultured until after passage, suspended with serum-free medium and counted. Taking serum-free culture medium from EP tube to prepare solution containing 0, 15.625, 31.25, 62.5, 125, 250 μ M P1/Cu, and adding appropriate amount of cell suspension to ensure cell density of 1 × 10 per tube⁴Mu.l/100. mu.l of cell suspension prepared in serum-free medium was added to the upper chamber of the Transwell plate, and 600. mu.l of medium containing 2% fetal bovine serum was added to the lower chamber. Culturing in incubator (37 deg.C, 5% CO)₂) After 48 hours, the upper chamber was stained in Calcein-AM dye for 30 minutes after aspirating the liquid from the upper chamber. After the dyeing is finished, the excess dye liquor is washed away by ultrapure water, and cells on the upper surface of the upper chamber are wiped off by a cotton swab. The Transwell chamber was placed on a glass slide and photographed with an inverted fluorescence microscope, and the results are shown in FIGS. 13-15.

The medium used in this example was the same as that used in example five except that it contained no fetal calf serum or 2% fetal calf serum. That is, the culture medium + 1% double antibody solution was added to the upper chamber of the Transwell plate in this example, without fetal bovine serum; added to the lower chamber was culture medium + 1% double antibody solution + 2% fetal bovine serum.

As can be seen from the figure, the cells transferred to the lower surface of the upper chamber of the transwell plate gradually decreased with the increase of the concentration, which indicates that P1/Cu can exert an antitumor effect by decreasing the metastatic ability of tumor cells.

EXAMPLE nine cell cycle assays

Tumor cells (15X 10) in logarithmic growth phase were plated in six-well plates after each cell culture to passage⁴Hole/bore). After 24 hours, when the cells begin to enter the logarithmic growth phase after recovering from adherent morphology, the cells were incubated in an incubator (37 ℃ C., 5% CO) with the same amount of blank medium or different concentrations of P1/Cu (2 ml/well)₂)48h。

A number of 5ml EP tubes were prepared and assigned serial numbers on crushed ice. The plates were removed and the supernatants collected in corresponding EP tubes. The remaining medium was washed out with 1 × PBS. Pancreatin was added for digestion, and all cells were collected as blotches and centrifuged at 1200rpm for 5 minutes. The supernatant was discarded and washed 2 times with 1 XPBS, at which time the cells had been transferred to 1.5ml EP tubes. Fixation with pre-cooled 70% ethanol overnight at 4 ℃. The ethanol solution was carefully discarded by centrifugation at 300g and the cell pellet was flicked off with 1ml of 1 XPBS to remove the ethanol. Cells were flicked and incubated at 37 ℃ for 30 minutes by adding DNase-contaminated RNase (50. mu.l/tube), and after addition of PI (final concentration 50. mu.g/ml)/10% Triton, the cells were allowed to act on ice for 15 minutes, the cell density in each EP tube was adjusted (so that the number of cells detected per second was 300-500), and the cells were detected by flow cytometry after sieving. The results are shown in FIGS. 16-17.

The medium used in this example was the same as that used in the fifth example.

In this example, a cell cycle assay kit (100 times) was used, and Shanghai Bibo biology was used.

As can be seen from the graph, the cell proportion of MCF-7 cells in the G0/G1 phase increased from 36.6% to 45.8% and the cell proportion of U87MG cells in the G0/G1 phase increased from 44.9% to 52.8% with the increase of the concentration, suggesting that P1/Cu can exert an antitumor effect by blocking the cell division cycle.

Claims

1. Compound [2- (5' -fluorouracil) acetic acid- (N-ethyl) piperazinyl dithiocarbamic acid]An anhydride having the formula:

2. the use of the compound [2- (5' -fluorouracil) acetic acid- (N-ethyl) piperazinyldithiocarbamic acid ] anhydride according to claim 1 in the preparation of an anti-tumor medicament.

3. The use of the compound [2- (5' -fluorouracil) acetic acid- (N-ethyl) piperazinyldithiocarbamic acid ] anhydride as claimed in claim 1 for the manufacture of a medicament for inhibiting the growth of tumour cells MCF-7, U87MG, HepG2 or Hela.

4. The use of the compound [2- (5' -fluorouracil) acetic acid- (N-ethyl) piperazinyldithiocarbamic acid ] anhydride as claimed in claim 1 in combination with copper ions for the manufacture of a medicament for inhibiting the growth of tumour cells MCF-7, U87MG, HepG2 or Hela.

5. The use of the compound [2- (5' -fluorouracil) acetic acid- (N-ethyl) piperazinyldithiocarbamic acid ] anhydride as claimed in claim 1 in combination with copper ions for the manufacture of a medicament for inhibiting the proliferative and metastatic ability of tumor cells MCF-7, U87MG, HepG2 or Hela and causing the arrest of the G1 phase of tumor cells.