CN105669712A - N-heterocyclic carbene silver complexes with antibiosis bioactivity, and preparation method thereof - Google Patents

Abstract

The invention discloses N-heterocyclic carbene silver complexes with antibiosis bioactivity, and a preparation method thereof, and relates to an antibiosis activity complex and a preparation method thereof. Three N-heterocyclic carbene silver complexes are prepared through reacting imidazolium salts with Ag2O, and the imidazolium salt ligand is characterized in that the nitrogen atom in di(1-imidazolyl)methane is structurally modified with an amide compound to obtain an amide functionalized bisimidazolium salt ligand. The method has the advantages of simple reaction operation, easy post-treatment and easy product separation. A result of the biological antibacterial activity researches of the complexes shows that the three carbene silver complexes have good antibacterial activity and have stronger Escherichia coli inhibition effect than Staphylococcus aureus, and the structure of silver carbine has certain influences on the antibacterial activity.

Description

Nitrocyclic carbene silver complex with antibacterial biological activity and preparation method thereof

technical field

The invention relates to an antibacterial active complex and a preparation method thereof, in particular to a nitrogen heterocyclic carbene silver complex with antibacterial biological activity and a preparation method thereof.

Background technique

N-heterocyclic carbene transition metal complexes are a hotspot in organometallic chemistry. This is mainly due to the excellent catalytic performance of N-heterocyclic carbene transition metal complexes in catalyzing the formation of chemical bonds such as C-C, C-N, C-O, and C-S. N-heterocyclic carbene can form stable complexes with almost all transition metal elements in the periodic table, among which there are many research reports on metal complexes such as Pd, Pt, Ni, Ir, Rh, Ru, Cu, etc., while silver carbene metal The transfer route is an important method for the synthesis of other N-heterocyclic carbene metal complexes. In addition, silver carbene complexes also have antibacterial and antiseptic uses, and some even show high activity in antitumor. In terms of anti-cancer, different silver complexes have a similar mode of action, mainly by releasing silver ions and then entering cells, thereby disrupting cell function. At present, the biggest problem existing in the existing silver complex drugs is that due to the instability of the silver complexes, the rapid release of silver ions causes the drugs to quickly lose their corresponding efficacy. In order to solve this problem, we designed and introduced a ligand N-heterocyclic carbene with strong coordination ability to form a silver nitrogen heterocyclic carbene complex with good stability. The synthesis method mainly uses imidazolium salt and Ag2O, Ag2CO3 or AgOAc reaction, the reaction is not sensitive to water and air, the reaction conditions are mild, the operation is simple, the formed Ag-carbene bond has large bond energy, good stability, and is not easy to dissociate rapidly.

Contents of the invention

The object of the present invention is to provide a nitrogen-heterocyclic carbene silver complex with antibacterial biological activity and a preparation method thereof. The present invention has selected representative Gram-negative bacteria Escherichia coli and Gram-positive bacteria gold Staphylococcus aureus introduces an amidated functional group on the nitrogen atom of the nitrogen heterocyclic carbene, and obtains a silver nitrogen heterocyclic carbene complex with good coordination stability. These complexes are binuclear silver carbene prepared by reacting imidazolium salt with Ag ₂ 0 under light-shielding conditions. The preparation method is simple, the reaction conditions are mild, and they are stable to water and air.

The purpose of the present invention is achieved through the following technical solutions:

Nitrocyclic carbene silver complexes with antibacterial biological activity. The complexes are all prepared by reacting imidazolium salts with Ag ₂ O in the dark. The formed silver carbene contains 4 Ag-carbene bonds, and the imidazole ring The structure of the group connected by N atoms is diverse, and its general structure is as follows:

.

The nitrogen heterocyclic carbene silver complex with antibacterial biological activity, the silver nitrogen heterocyclic carbene complex with antibacterial biological activity, the filter paper method is used to measure the inhibition of the silver carbene complex to Escherichia coli and Staphylococcus aureus Ability to measure different concentrations of sample solutions to obtain the size of the diameter of the inhibition zone to determine the sensitivity of the strain to the compound; greater than Escherichia coli and Staphylococcus aureus, as the sample concentration increases, the diameter of the inhibition zone gradually increases , the inhibitory effect is enhanced with the increase of the concentration, and the diameter of the inhibition zone for Escherichia coli is larger than that of Staphylococcus aureus.

A method for preparing a nitrogen-heterocyclic carbene-silver complex with antibacterial biological activity, the method comprising the following processes:

a) Preparation of bis(1-imidazolyl)methane. Grind NaOH solid into powder and dissolve in 15ml of acetonitrile, add imidazole under stirring, stir at 60°C until the solution turns into a white suspension, slowly add dibromomethane in 5ml of acetonitrile solution dropwise, the dropwise addition is completed within 1.5 hours, then heat up to reflux React for 3.5 hours; filter after the reaction is completed, wash with acetonitrile, and remove the filtrate by rotary evaporation to obtain a white flake solid, which is dissolved in 10ml acetone and filtered to remove insoluble matter, the filtrate is cooled and crystallized, filtered, and vacuum-dried to obtain white needle-shaped crystals;

b) Preparation of N-chloroacetyl chloride aniline compound, weigh the aniline compound and place it in a 100ml two-necked reaction bottle, add 20ml of dichloromethane as a solvent, add 3~5 drops of triethylamine, and drop it with constant pressure at room temperature Slowly add 10ml of chloroacetyl chloride dichloromethane solution to the above solution through a liquid funnel, and stir at room temperature for 2 hours; after the reaction, remove the solvent by rotary evaporation, add 50ml of water for washing, and filter to obtain a white solid;

c) Preparation of the imidazolium salt ligand, the compound bis(1-imidazolyl)methane was dissolved in 10ml of acetonitrile, and then N-chloroacetyl chloride aniline compound was added after it was dissolved, reacted at 100°C for 12h, filtered after the reaction was completed, and Wash with acetonitrile and ether, and dry in vacuo to obtain a white solid;

d) Preparation of silver carbene complexes, respectively dissolve the imidazolium salt ligand in 5ml of anhydrous methanol, then add Ag ₂ O, stir for 24 hours under dark conditions, the Ag ₂ O solution turns into a white suspension, and the reaction ends After filtration, it was dried in the dark to obtain a white solid.

Advantage and effect of the present invention are:

1. An amidated functional group is introduced on the nitrogen atom of the nitrogen heterocyclic carbene to obtain a silver nitrogen heterocyclic carbene complex with good coordination stability. These complexes are binuclear silver carbene prepared by reacting imidazolium salt with Ag ₂ 0 under light-shielding conditions. The preparation method is simple, the reaction conditions are mild, and they are stable to water and air.

2. Silver carbene complexes have good inhibitory activity against both Escherichia coli and Staphylococcus aureus. As the concentration of the compound increases, the antibacterial effect tends to increase significantly. When the nitrogen atom of the imidazole ring is connected with different groups, the inhibitory effect Bacteria are also different.

detailed description

The present invention will be described in detail below in conjunction with examples.

1. Preparation method and reaction conditions of silver nitrogen heterocyclic carbene complexes with antibacterial activity

a) Preparation of bis(1-imidazolyl)methane. Grind NaOH solid into powder and dissolve in 15ml of acetonitrile, add imidazole under stirring, stir at 60°C until the solution becomes a white suspension, slowly add dibromomethane in 5ml of acetonitrile solution dropwise, and heat up to reflux for 3.5 hours. After the reaction is completed, filter, wash with acetonitrile, and remove the filtrate by rotary evaporation to obtain a white flake solid, which is dissolved in 10 ml of acetone and filtered to remove insoluble matter. The filtrate is crystallized by cooling, filtered, and dried in vacuo to obtain white needle-shaped crystals.

b) Preparation of N-chloroacetyl chloride aniline compounds. Weigh the aniline compound and put it in a 100ml two-necked reaction bottle, add 20ml of dichloromethane as solvent, add 3~5 drops of triethylamine dropwise, and slowly distill 10ml of chloroacetyl chloride in dichloromethane solution with a constant pressure dropping funnel at room temperature Add dropwise to the above solution, and stir at room temperature for 2 hours. After the reaction was completed, the solvent was removed by rotary evaporation, 50 ml of water was added for washing, and a white solid was obtained by filtration. Aniline compounds include: aniline, o-chloroaniline, and p-methylaniline.

c) Preparation of imidazolate ligands. Dissolve the compound bis(1-imidazolyl)methane in 10ml of acetonitrile, and then add N-chloroacetyl chloride aniline compound after it dissolves, react at 100°C for 12h, filter after the reaction is completed, wash with acetonitrile and ether, and dry in vacuo to obtain white solid. N-chloroacetyl chloride aniline compounds include N-phenyl-chloroacetamide, N-(2-chlorophenyl)-chloroacetamide, N-phenyl-chloroacetamide.

d) Preparation of silver carbene complexes. Dissolve the imidazolium salt ligand in 5ml of anhydrous methanol, then add Ag ₂ O, and stir for 24 hours in the dark. The color of Ag ₂ O gradually becomes lighter, and the solution becomes a white suspension. After the reaction, filter to avoid Light drying gave a white solid.

2. Determination of antibacterial activity of silver carbene complexes in vitro

a) Selection of strains. Select representative Escherichia coli and Staphylococcus aureus, Escherichia coli is a Gram-negative short bacillus with a size of about 0.5×1-3μm, has flagella, can move, and has no spores; Staphylococcus aureus is a spherical Gram Positive bacteria are arranged in beads under the microscope, with a size of about 0.8 μm, no flagella, no spores, and most of them have no capsule. The optimum growth temperature is 37°C, and the optimum growth pH is 7.4.

b) Activation and preparation of strains. The strains selected before the experiment were stored at -80°C and must be activated before use. The activation adopts the two-generation culture method, which is beneficial to improve the activity of the strain. The specific operation is as follows: use a sterilized inoculation loop to pick the strains refrigerated in the refrigerator, inoculate them into a sterilized 50ml petri dish, and then culture them at 37°C for 24 hours to observe the culture status. If the culture medium is turbid, it means that the growth condition of the strain is good, otherwise it needs to be inoculated and cultured again. Similar to this operation, the cultured first-generation strains are then cultured for the second generation. Due to its high concentration, the activated strains will be too dense for direct coating, which is not conducive to observation. Therefore, the strains must be diluted to carry out the coating experiment. The dilution method is to use a pipette gun to draw 100 μl of the activated strain, put it in 100ml of sterilized distilled water, mix it evenly, and set it aside.

c) Preparation of medium. The raw materials for the liquid medium include 1.0g of peptone, 0.5g of yeast extract, 1.0g of sodium chloride, and 100ml of distilled water. The solid medium is based on the liquid medium and then 2.0g of agar is added to make it solidify. Prepare in a shaped bottle, mix well and store in a sealed seal. The medium must be sterilized before use. The sterilization equipment is an autoclave, and the method is to maintain the pressure at 0.1MPa~0.125Mpa for 15 minutes.

d) Preparation of samples. Silver carbene complexes with 4 different concentrations (0.01mg/ml, 0.1mg/ml, 1mg/ml, 10mg/ml) were prepared in DMF respectively.

e) Preparation of plates. Quickly import the solid medium sterilized by the autoclave into the plate while it is hot, spread it flat to make it about 0.15cm thick, then let it stand, slowly solidify, and after solidification, put it into a 37°C incubator for culture and make a laboratory test. Bacteria detection.

f) Preparation of tablets. Punch the filter paper into small discs with a pore size of 5 mm, and then put the filter paper into a petri dish for sterilization treatment. After sterilization, soak it in sample solutions of different concentrations for use.

g) Coating of strains. Add 10 μl of diluted culture solution to the surface of the solid medium, and spread it evenly with a spreader, then spread the filter paper soaked in the sample solution on the surface of the medium, spread 4 pieces on each plate, and carry out 3 times for each concentration Parallel experiments.

h) Place the above-mentioned plate loaded with the tablets in an incubator at 37° C. for 24 hours, and observe the phenomenon. As a result, it was found that transparent circles-inhibition circles of different sizes appeared on the agar medium, and the antibacterial activity of the sample could be seen by measuring the diameter of the inhibition circles.

The inhibitory effects of the three silver carbene complexes on Escherichia coli and Staphylococcus aureus are shown in the table below (the following results are the average values of the measurements):

It can be seen that the three silver carbene compounds have good inhibitory activity against both Escherichia coli and Staphylococcus aureus, but the inhibition of Escherichia coli is stronger than that of Staphylococcus aureus.

Claims (3)

1. there is the nitrogen heterocyclic carbene silver complex of antibiotic bioactive, it is characterised in that, described title complex is imidazole salts and Ag₂When O lucifuge, reaction is prepared from, and the silver card guest formed is containing 4 Ag-Cabbeen keys, and the unit structure being connected with imidazole ring atom N is various, and its general structure is as follows:

。

2. the nitrogen heterocyclic carbene silver complex with antibiotic bioactive according to claim 1, it is characterized in that, the described silver-colored aza ring carbene complex with antibacterial biological activity, filter paper method is adopted to measure silver carbene complex to the rejection ability of intestinal bacteria and streptococcus aureus, the sample solution of different concns is measured, be inhibited the size of loop diameter, judges that bacterial classification is to the sensitivity of compound; Being greater than intestinal bacteria and streptococcus aureus, along with the increase of sample concentration, suppress the diameter of circle to increase gradually, inhibition strengthens along with the increase of concentration, and the suppression loop diameter of intestinal bacteria is greater than streptococcus aureus.

3. there is the nitrogen heterocyclic carbene silver complex preparation method of antibiotic bioactive, it is characterised in that, described method comprises following process:

A) two (1-imidazolyl) methane is prepared, it is dissolved in 15ml acetonitrile by powdered for NaOH solid abrasive, imidazoles is added under stirring, 60 DEG C of stirrings, treat that solution turns into white suspension liquid, slowly dripping the 5ml acetonitrile solution adding methylene bromide, dropwise in 1.5 hours, then temperature rising reflux reacts 3.5 hours; Reaction is filtered after terminating, and with acetonitrile wash, revolves and steams removing filtrate, obtain white plates solid, be dissolved in 10ml acetone and be filtered to remove insolubles, filtrate crystallisation by cooling, filter, and vacuum-drying obtains white needle-like crystals;

B) preparation of N-chloroacetyl chloride amino benzenes compounds, take amino benzenes compounds and it is placed in 100ml two mouthfuls of reaction flasks, add 20ml methylene dichloride and make solvent, drip and add 3 ~ 5 triethylamines, with constant pressure funnel, the dichloromethane solution of 10ml chloroacetyl chloride is slowly added drop-wise to above-mentioned solution under room temperature, stirred at ambient temperature 2 hours; Reaction is revolved steaming and is removed solvent, add 50ml water washing, filter to obtain white solid after terminating;

C) preparation of corresponding imidazolium salts, is dissolved in 10ml acetonitrile by compound two (1-imidazolyl) methane, adds N-chloroacetyl chloride amino benzenes compounds after its dissolving again, 100 DEG C of reaction 12h, filter after having reacted, with acetonitrile, washed with diethylether, vacuum-drying, obtains white solid;

D) preparation of silver carbene complex, just corresponding imidazolium salts is dissolved in 5ml anhydrous methanol respectively, then adds Ag₂O, stirs 24h, Ag when lucifuge₂O solution turns into white suspension liquid, and reaction is filtered after terminating, and lucifuge is dry obtains white solid.