CN110981897A - Zinc (II) complex, synthesis method and application thereof in antitumor drugs - Google Patents

Abstract

The invention discloses a zinc (II) complex, a synthesis method and application thereof in antitumor drugs, wherein the synthesis method comprises the steps of taking tert-butyl (2-formylpyridine-3-yl) carbamate and 4, 4-diethyl thiosemicarbazone, taking alcohols and hydrochloric acid as solvents, carrying out reaction, adjusting the pH value to be neutral, generating yellow precipitates, collecting the precipitates, washing to obtain a ligand; taking the obtained ligand and zinc nitrate, taking alcohols and acetonitrile as solvents, carrying out reaction, standing the reactant, separating out yellow crystals, and collecting the crystals to obtain the zinc (II) complex. The zinc (II) complex has obvious anti-tumor activity and can be used for treating tumors.

Description

Zinc (II) complex, synthesis method and application thereof in antitumor drugs

Technical Field

The invention relates to the technical field of medicines, in particular to a zinc (II) complex, a synthetic method and application thereof in antitumor medicines.

Background

Cancer (mainly malignant tumor) is one of the most serious diseases endangering human health, and is listed as one of five major difficult and complicated diseases in the world by the world health organization. Platinum drugs are FDA-approved metal-based drugs that are widely used in the treatment of various malignancies. However, resistance to tumors and toxicity to normal cells limit platinum drugs. Thiosemicarbazones have a number of biological activities such as antitumor, antiviral, antimalarial and antibacterial properties, and some thiosemicarbazones have been used in clinical stage I/II cancer therapy, for example, 3-amino-2-pyridinecarboxaldehyde thiosemicarbazone and bis-2-pyridylketal-4-cyclohexane-4-methylaminothiourea (Stacy, A. E., et al. Journal of Medicinal Chemistry, 2016, 59, 4965-4984). In general, some metal complexes of thiosemicarbazones exhibit greater antitumor activity in vitro and in vivo than the ligand alone. The zinc (II) complex of thiosemicarbazone acts synergistically with copper ions in lysosomes to produce reactive oxygen species through redox reactions and produce cytotoxicity. At present, no published report of the synthesis and application of a zinc (II) metal complex using 3-amino-2 pyridine formaldehyde-condensed 4, 4-diethyl thiosemicarbazone as a ligand exists.

How to design a zinc (II) complex which has reasonable structure, obvious in-vitro anti-tumor activity and better potential medicinal value and is expected to be used for preparing various anti-tumor medicaments and a synthetic method thereof are the problems to be solved at present.

Disclosure of Invention

The invention aims to solve the technical problem of providing a synthesis method of a zinc (II) metal complex with 3-amino-2 pyridine formaldehyde-condensed 4, 4-diethyl thiosemicarbazone hydrochloride as a ligand and an application of the complex in antitumor drugs.

The zinc (II) metal complex is a zinc (II) metal complex taking 3-amino-2 pyridine formaldehyde 4, 4-diethyl thiosemicarbazone hydrochloride as a ligand, and is a compound with a structure shown in the following formula (I) or a pharmaceutically acceptable structure thereof:

（I）。

the synthesis method of the compound shown in the formula (I) comprises the following steps:

1) taking (2-formylpyridine-3-yl) carbamic acid tert-butyl ester and 4, 4-diethyl thiosemicarbazone, taking alcohols and hydrochloric acid as solvents for reaction, adjusting the pH value to be neutral, generating a precipitate, collecting the precipitate, and washing to obtain ligand 3-amino-2 pyridine carboxaldehyde 4, 4-diethyl thiosemicarbazone hydrochloride;

2) taking ligand 3-amino-2 pyridine formaldehyde 4, 4-diethyl thiosemicarbazone and zinc nitrate, taking alcohols as a solvent, reacting, standing reactants, crystallizing, and collecting crystals to obtain the target compound.

In the synthesis method, the raw materials of tert-butyl (2-formylpyridine-3-yl) carbamate and 4, 4-diethyl thiosemicarbazone; the alcohol substance is preferably methanol and/or ethanol, the methanol is preferably methanol with the volume concentration of 20-80%, and the ethanol is preferably ethanol with the volume concentration of 20-80%; when the alcohol is selected from the combination of methanol and ethanol, the ratio of methanol to ethanol can be any ratio.

In the above synthesis method, the reaction is preferably performed in a range from 20 ℃ to the reflux temperature of the solvent (i.e., alcohol), the reaction may be performed at normal temperature, a conventional heating reaction or reflux reaction, preferably a reflux reaction, is further preferably performed in a range from 50 ℃ to the reflux temperature of the solvent, more preferably in a range from 60 ℃ to the reflux temperature of the solvent (i.e., alcohol), whether the reaction is complete or not can be followed by Thin Layer Chromatography (TLC), and in step 1), when the reaction is performed in a range from 50 ℃ to the reflux temperature of the solvent, the reaction time is generally controlled to be 1-24 hours.

In step 1 of the above synthesis method, the collected precipitate is usually washed with one or more of methanol, ethanol, ether and water. The ratio of the amounts of material of tert-butyl (2-formylpyridin-3-yl) carbamate and 4, 4-diethylthiosemicarbazone in this step is a stoichiometric ratio, typically 1: 1. The amount of the solvent is preferably such that the raw material to be reacted can be dissolved, and usually, 1 mmol of tert-butyl (2-formylpyridin-3-yl) carbamate or 1 mmol of 4, 4-diethylthiosemicarbazone is dissolved in 2 to 20mL of the solvent. In a specific dissolving step, the tert-butyl (2-formylpyridin-3-yl) carbamate and the 4, 4-diethyl thiosemicarbazone are respectively dissolved by using solvents and then mixed together for reaction; or mixing (2-formylpyridin-3-yl) carbamic acid tert-butyl ester and 4, 4-diethyl thiosemicarbazone, and adding the solvent.

In step 2 of the above synthesis method, in order to improve the product purity, it is preferable to filter the reactant obtained by the reaction, collect the filtrate, and then allow the filtrate to stand and crystallize. In the step, the standing is preferably performed at a relatively low temperature, and the standing and crystallization is usually performed at 0-8 ℃. Further, when standing for crystallization, a layer of film is covered on the opening of the container for containing the filtrate, and then a plurality of small holes are formed on the film to slowly volatilize the solvent, so as to obtain better crystallization effect (namely, obtain crystals with larger grains and higher crystal yield). In this step, the mass ratio of ligand 3-amino-2-pyridinecarboxaldehyde 4, 4-diethylthiosemicarbazone to zinc nitrate is stoichiometric, typically 1: 1. The amount of the solvent is preferably that the raw materials for reaction can be dissolved, and usually, 2-10 mL of the solvent is used for dissolving 1 mmol of the ligand 3-amino-2-pyridine carboxaldehyde 4, 4-diethyl thiosemicarbazone or zinc nitrate. In the specific dissolving step, the ligand 3-amino-2 pyridine carboxaldehyde 4, 4-diethyl thiosemicarbazone and zinc nitrate are respectively dissolved by using solvents and then mixed together for reaction; or mixing the ligand 3-amino-2 pyridine formaldehyde-condensed 4, 4-diethyl thiosemicarbazone with zinc nitrate and then adding the solvent.

The invention also discloses application of the compound shown in the formula (I) in preparing antitumor drugs.

The invention further comprises an antitumor drug prepared by using the compound shown in the formula (I) as an active ingredient.

Compared with the prior art, the invention has the beneficial effects that:

compared with the prior art, the invention provides a novel zinc (II) complex taking 3-amino-2-pyridine formaldehyde 4, 4-diethyl thiosemicarbazone as a ligand and a synthesis method thereof, and the in vitro anti-tumor activity research of the applicant on the obtained zinc (II) complex shows that the zinc (II) complex has obvious in vitro anti-tumor activity on HepG-2, MCF-7, NCI-H460, T24, Hela and A549 tumor cell strains, has better potential medicinal value and is expected to be used for preparing various anti-tumor medicaments.

Drawings

FIG. 1 is a schematic diagram of a 3-amino-2-pyridinecarboxaldehyde 4, 4-diethyl thiosemicarbazone hydrochloride single crystal;

FIG. 2 is a view showing a single crystal structure of a zinc complex;

FIG. 3 is a schematic diagram of a cell cycle experiment in a control group;

FIG. 4 is a schematic diagram of an experiment in which a ligand inhibits the cell cycle;

FIG. 5 is a schematic diagram of an experiment for inhibiting cell cycle by a zinc (II) complex;

FIG. 6 is a schematic diagram of the apoptosis experiment in the control group;

FIG. 7 is a schematic diagram of an apoptosis assay for ligand cells;

FIG. 8 is a schematic diagram of the apoptosis experiment of zinc (II) complex.

Detailed Description

The present invention will be better understood from the following detailed description of specific examples, which should not be construed as limiting the scope of the present invention.

In the following examples and experimental examples, the abbreviations used have the following meanings:

zinc (II) complex: the compound is a zinc (II) complex which takes 3-amino-2 pyridine formaldehyde-condensed 4, 4-diethyl thiosemicarbazone as a ligand, namely the compound shown as the formula (I) in the invention.

Ligand: represents ligand 3-amino-2 pyridine carboxaldehyde 4, 4-diethyl thiosemicarbazone:

DMSO, DMSO: dimethyl sulfoxide.

Example 1: and (3) synthesizing a zinc (II) complex.

The synthetic route is as follows:

the specific synthesis method comprises the following steps:

1) dissolving 1 mmol of tert-butyl (2-formylpyridin-3-yl) carbamate (0.222 g) in 20ml of ethanol (the concentration of the solvent ethanol is 50 v/v%), stirring at 60 ℃ for 15min to prepare a solution, dropwise adding the solution into 20ml of ethanol (the concentration of the solvent ethanol is 50 v/v%) solution added with 1 mmol of 4, 4-diethyl thiosemicarbazone (0.147 g), dropwise adding 3 drops of concentrated hydrochloric acid, carrying out reflux stirring at 60 ℃ for reaction for 24h, adjusting the pH value of the solution to be neutral to obtain a light yellow precipitate, filtering the light yellow precipitate, washing the precipitate with absolute ethanol and diethyl ether for 3 times respectively, and drying to obtain ligand 3-amino-2 pyridine carboxaldehyde 4, 4-diethyl thiosemicarbazone hydrochloride. Recrystallizing with ethanol to obtain light yellow crystal;

the obtained yellowish crystal is subjected to element analysis, mass spectrometry and X-ray single crystal diffraction analysis, and the specific spectral characteristics are as follows:

(1) elemental analysis:

the chemical formula is as follows: c₁₁H₁₇N₅SCl. The elemental analysis results were: calculated values: c, 46.07, H, 5.97 and N, 24.42. Test values are: c, 46.32, H, 5.57 and N, 24.21.

(2) Mass spectrometry analysis:

calculated values: 250.12, respectively; test values are: 250.12.

(3) x-ray single crystal diffraction analysis:

the structure diagram of the single crystal is determined as shown in FIG. 1, and the crystal structure data and the bond angle of part of the bond length of the obtained complex are shown in the following tables 1 to 3, respectively:

table 1: ligand crystal structure data.

Table 2: part of the bond length of the ligand.

Table 3: partial bond angle of the ligand.

Thus, it was confirmed that the obtained pale yellow solid product was 3-amino-2-pyridinecarboxaldehyde 4, 4-diethylthiosemicarbazone hydrochloride having the structural formula shown in the following formula (II):

(II)

2) 20ml of a methanol (solvent methanol concentration of 60 v/v%) solution containing 1 mmol of zinc nitrate (0.189 g) was added dropwise to 20ml of an ethanol (solvent ethanol concentration of 50 v/v%) solution containing 1 mmol of 3-amino-2-pyridinecarboxaldehyde 4, 4-diethyl-thiosemicarbazone ligand, the mixture was stirred under reflux at 60 ℃ for 2 hours, the reacted solution was filtered into a 50 ml beaker, and sealed with a wrap film, and 20 holes were punched with a needle to volatilize for several days at 4 ℃ to obtain yellow crystals.

The obtained yellow crystal is subjected to element analysis, mass spectrometry and X-ray single crystal diffraction analysis, and the specific spectral characteristics are as follows:

(1) elemental analysis:

the chemical formula is as follows: c₁₁H₁₇N₇O₆SZn. The elemental analysis results were: calculated values: c, 29.98, H, 3.89, N, 22.25 and O, 21.78. Test values are: c, 29.56, H, 4.03, N, 22.01 and O, 21.97.

(2) Mass spectrometry analysis:

calculated values: 438.03, respectively; test values are: 438.03.

(4) x-ray single crystal diffraction analysis:

the structure diagram of the single crystal is determined as shown in FIG. 2, and the crystal structure data and the bond angle of part of the bond length of the obtained complex are shown in the following tables 4-6, respectively:

table 2: crystal structure data of zinc complexes.

Table 2: the bond length of the complex.

Table 3: bond angle of the complex.

Therefore, it can be determined that the obtained yellow solid product is a zinc (II) metal complex using 3-amino-2-pyridinecarboxaldehyde 4, 4-diethylthiosemicarbazone as a ligand, and the structural formula of the complex is shown as the following formula (I):

(I)

experimental example 2: experiments on the antitumor activity of the zinc (II) complex of the present invention (prepared as described in example 1) and the ligand.

1) Cell lines and cell cultures

Tumor cell lines HepG-2, MCF-7, NCI-H460, T24, Hela and A549 tumor cell lines were obtained from the American Type Culture Collection ((Rockville MD, USA) by adding 10% fetal bovine serum and 100. mu.g mL into A5% volume incubator at 37 ℃^-1Streptomycin was cultured in RPM-1640 medium with 100 units per ml penicillin.

2) Cell growth inhibition assay (MTT method)

Note: MTT is named as 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2-H-tetrazolium bromide, and the Chinese chemical name is 3- (4, 5-dimethylthiazole-2) -2,5-diphenyl tetrazolium bromide, the trade name is: thiazole blue, a yellow dye.

After dissolving the zinc (II) complex with DMSO, diluting the zinc (II) complex into five gradients in sequence by using a complete culture medium, wherein the five gradients are 20 times of the working solution with the final concentration, and the contents of the cosolvent DMSO and the like are uniform except that the drug concentration is different among the gradients

Thus, the method can be used for the treatment of the tumor. Filtering with 0.22um microporous membrane for sterilization, and storing at 4 deg.C. Tumor cell line in log phase

HepG-2, MCF-7, NCI-H460, T24, Hela and A549, 0.18ml per well were inoculated into 96-well plates, respectively, at a cell concentration of about 0.4 to 0.5X 10⁵After 12H of culture and cell adherence, adding test compounds with different concentrations respectively, wherein 20ul of each well ensures that the final concentration is 0 mu M, 0.005 mu M, 0.01 mu M, 0.05 mu M, 0.1 mu M, 0.5 mu M and 1 mu M, and each gradient is provided with 4 multiple wells, wherein the final concentration of DMSO is less than 0.5%, simultaneously setting a corresponding negative control group (only cells and equal amount of DMSO without drugs in the culture solution), setting 4 multiple wells in each group, pouring out the culture solution after 72H of drug action, adding 100ul of DMSO, oscillating a flat plate oscillator for 10min to fully dissolve crystals, adjusting the blank control group to zero, measuring the absorbance (A) value after removing the bottom cell strain with 550nm/655nm double-wavelength by using an enzyme labeling instrument, and respectively calculating the light absorption (A) value of the compounds to Bel-7402, MCF-7, NCI-H460, T24 a and IC 549 a₅₀The results of the experiments are shown in Table 1, and it can be seen that the cellular activity of the zinc (II) complex on tumor cells Bel-7402, MCF-7, NCI-H460, T24 and Hela is significantly higher than that of the corresponding ligands.

TABLE 4 antitumor activity of ligands and zinc (II) complexes.

Note: bel-7402 is human liver cancer cell, MCF-7 is human breast cancer cell, NCI-H460 is large cell lung cancer cell, T24 human colon cancer cell, and Hela is human uterine cancer cell.

Experimental example 3: the zinc (II) complexes of the invention (prepared as described in example 1) and ligands inhibit cell cycle experiments.

Testing the cells stained by PI after 2 mu M of zinc (II) complex acts on MCF-724 h by a cell flow meter; the results are shown in fig. 3-5, the percentage of the cells in the G1 phase is increased by 9.8% and the percentage of the cells in the G2 phase is decreased by 4.39% compared with the blank control, which indicates that the ligand may pass through the G2 phase of the cell cycle, thereby affecting the cell proliferation; the percentage of cells in the G1 phase was increased by 19.62% and the percentage of cells in the G2 phase was decreased by 13.85% for the zinc (II) complex relative to the ligand, indicating that the zinc (II) complex might pass through the G2 phase of the cell cycle.

Experimental example 4: experiments on the promotion of apoptosis by the zinc (II) complex of the present invention (prepared as described in example 1) and ligand.

After 2 mu M of zinc (II) complex acts on MCF-724 h, cells stained by PI and Annexin V-FITC in a double-staining mode are tested by a cell flow meter; the results are shown in fig. 6-8, compared with the blank control, the number of apoptosis (sum of early apoptosis and late apoptosis) of the ligand is increased from 4.56% to 12.24%, while the cell survival rate is obviously reduced, which indicates that the ligand can promote apoptosis; the increase in the amount of apoptosis (sum of early apoptosis and late apoptosis) of the zinc (II) complex relative to the blank control from 4.56% to 34.02% indicates that the zinc (II) complex has a higher ability to promote apoptosis than the ligand.

The preferred embodiments and examples of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the embodiments and examples described above, and various changes can be made within the knowledge of those skilled in the art without departing from the concept of the present invention.

Claims (6)

1. A zinc (II) complex characterized by: the complex has antitumor activity and is a compound shown as the following formula (I) or a pharmaceutically acceptable salt thereof:

(I)。

2. the method of synthesizing a zinc (II) complex of claim 1, wherein: comprises the following steps of (a) carrying out,

step 1, taking tert-butyl (2-formyl pyridine-3-yl) carbamate and 4, 4-diethyl thiosemicarbazone, taking alcohols and hydrochloric acid as solvents, reacting, adjusting the pH value to be neutral, generating precipitates, collecting the precipitates, washing to obtain ligand 3-amino-2 pyridine carboxaldehyde 4, 4-diethyl thiosemicarbazone hydrochloride;

and 2, taking ligand 3-amino-2 pyridine formaldehyde 4, 4-diethyl-condensed-amino thiourea hydrochloride and zinc nitrate, taking alcohols and acetonitrile as solvents for reaction, standing reactants, crystallizing, and collecting crystals to obtain the target complex.

3. The method of claim 2, wherein the zinc (II) complex is synthesized by: the alcohol substance is methanol and/or ethanol.

4. The method of claim 2, wherein the zinc (II) complex is synthesized by: the reaction is carried out at a temperature ranging from 20 ℃ to the reflux temperature of the solvent.

5. Use of a zinc (II) complex according to claim 1 for the preparation of an antitumor medicament.

6. An antitumor agent prepared with the zinc (II) complex of claim 1 as an active ingredient.

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