CN111943868B

CN111943868B - Diethylamine-containing azine hydrazine compound and preparation method and application thereof

Info

Publication number: CN111943868B
Application number: CN202010393997.7A
Authority: CN
Inventors: 唐本忠; 王志明; 胡蓉; 周凡; 崔娜; 邓棋云; 张可心; 印平安
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2020-05-11
Filing date: 2020-05-11
Publication date: 2023-04-21
Anticipated expiration: 2040-05-11
Also published as: CN111943868A; WO2021227206A1

Abstract

The invention discloses an azine hydrazine compound containing diethylamine, and a preparation method and application thereof. The method comprises the following steps: and (3) heating the diphenyl hydrazine derivative and the aryl containing the diethylamine in a solvent for reaction, and separating and purifying a reaction product to obtain the azine hydrazine compound containing the diethylamine. The invention also discloses application of the compound, and the identification and selective antibacterial effect on fungi are realized by regulating the interaction strength between the azine hydrazine compound and microorganisms so as to regulate the fluorescence intensity of the azine hydrazine compound. The diethylamine-containing azine hydrazine compound has remarkable aggregation-induced emission (AIE) property and active oxygen generation capacity, shows selective bacterial fluorescent staining capacity and antibacterial activity, and has good application prospect.

Description

Diethylamine-containing azine hydrazine compound and preparation method and application thereof

Technical Field

The invention belongs to the field of analysis and detection materials, and particularly relates to an azine hydrazine compound containing diethylamine, and a preparation method and application thereof.

Background

Humans have been fully saturated from ancient times with infectious diseases caused by a variety of pathogenic microorganisms (e.g., bacteria, fungi, viruses, and parasites). Antibiotics have been widely used for many years as the most effective drug for controlling epidemic diseases, but the evolved drug-resistant pathogenic bacteria have caused hundreds of thousands of deaths in developed countries each year due to the misuse and abuse of antibiotics in the fields of medical care, industrial and agricultural production, etc. (antibiotics used in agriculture and animal husbandry to help livestock growth). The data collected by the World Health Organization (WHO) from 129 member countries indicates that antibiotic resistance has emerged in every corner of the world, an increasing number of multi-drug resistant pathogenic infections have severely threatened human survival and health, and forced humans to enter the "post antibiotic age" where no drug is available, i.e., common infections and bruises are potentially fatal. The existing research results show that the traditional chemical synthesis and drug screening strategies all need to go through a long research and development process, and waste a great deal of resources while spending a great deal of money. More seriously, the development speed of new drugs often lags behind the appearance and diffusion speed of drug-resistant pathogenic bacteria, which makes the traditional drug development strategy have little effect in solving the problem of drug resistance. And for infection caused by microorganisms, a rapid differentiation of microorganisms and an effective antimicrobial strategy are of equal importance for the clinical treatment of microbial infections.

At present, a plurality of systems integrating diagnosis and treatment (diagnosis and treatment integration) have been developed, wherein fluorescent materials capable of effectively generating Reactive Oxygen Species (ROS) have good application prospects in microorganism diagnosis and treatment integration. Photodynamic therapy is a technique for diagnosing and treating diseases using photodynamic effects generated by light and photosensitizers. In recent years, the efficacy of the composition on local microbial infection of skin is remarkable, and the composition is called photodynamic antimicrobial chemotherapy (photodvnamic antimicrobial chemothempy, PACT), and pathogenic bacteria of the composition comprise staphylococcus aureus, pseudomonas aeruginosa, acinetobacter baumannii, escherichia coli, porphyromonas gingivalis, multiple drug resistant bacteria and the like. Furthermore, photodynamic antimicrobial chemotherapy is not prone to developing resistance. The reagents commonly used for PACT at present mainly comprise porphyrin photosensitizers, phthalocyanine photosensitizers and porphine photosensitizers. In addition, there are many photosensitizers based on organic small molecule fluorescent materials and polymer fluorescent materials, and the fluorescent materials not only can effectively sensitize and generate active oxygen, but also can obtain good effect in antibiosis, and the fluorescent property of the fluorescent materials can realize biological imaging and generate active oxygen in photosensitization. However, the substances have limited solubility, and aggregate can be formed under physiological conditions, so that the luminescence and the active oxygen generation of the substances are inhibited, and the further application of the substances in microorganism diagnosis and treatment integration is limited. The fluorescent material with aggregation-induced emission performance can utilize a process of forming an aggregation state, inhibit the dissipation of excited state energy by limiting intramolecular movement, improve the aggregation state luminous efficiency and active oxygen generation capacity, and is hopeful to develop into a novel material for microorganism diagnosis and treatment integration.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide an azine hydrazine compound containing diethylamine, and a preparation method and application thereof.

Based on the above prior art, a primary object of the present invention is to provide a diethylamine-containing aryl-diphenyl-azine hydrazines.

Another object of the present invention is to provide a process for producing the above diethylamine-containing aryl-diphenyl-azine hydrazines.

It is still another object of the present invention to provide the use of the above-mentioned diethylamine-containing aryl-diphenyl-azine hydrazines as fluorescent probe materials in the fields of bioanalytical and clinical medicine detection, etc.

The object of the invention is achieved by at least one of the following technical solutions.

The structural general formula of the azine hydrazine compounds provided by the invention is shown as follows:

wherein Ar represents an aromatic group or a derivative structure thereof, and the substituent groups R1-R10 are respectively selected from one of hydrogen, alkyl, hydroxyl, alkoxy, nitro, cyano, amino, sulfhydryl, halogen atom, diethylamine, phenyl, tolyl, naphthyl, furyl, thienyl, pyrrolyl, pyridyl, pyranyl, quinolinyl, indolyl, carboxyl or derivative groups thereof, carbazolyl or anilino.

Preferably, the diethylamine-containing azine hydrazines have the structural formula

Wherein Ar represents an aromatic group or a derivative thereof, and the substituent R1 and R2 are respectively selected from one of hydrogen, alkyl, hydroxyl, alkoxy, nitro, cyano, amino, sulfhydryl, halogen atom, diethylamine, phenyl, tolyl, naphthyl, furyl, thienyl, pyrrolyl, pyridyl, pyranyl, quinolinyl, indolyl, carboxyl or a derivative thereof, carbazolyl or anilino.

The R is ¹ 、R ² Selected from hydrogen or one of the following structural formulas:

further, when the substituents R1, R2 are hydrogen, hydroxy, and Ar is a benzene ring or a benzene ring derivative, the diethylamine-containing aryl salicylaldehyde-diphenyl-azine hydrazines preferably have a structural formula as set forth in any one of the following:

wherein A1-A5 are one of hydrogen, alkyl, hydroxyl, alkoxy, nitro, cyano, amino, sulfhydryl, halogen substituent, diethylamine, phenyl, tolyl, naphthyl, furyl, thienyl, pyrrolyl, pyridyl, pyranyl, quinolinyl, indolyl, carboxyl or derivative groups thereof, carbazolyl or anilino.

Further, when the substituents R1, R2 are each hydrogen, hydroxyl, ar is a naphthalene ring or a derivative thereof, an anthracene ring or a derivative thereof, a phenanthrene ring or a derivative thereof, the aryl-diphenyl-azine-dihydrazide compound preferably has a structural formula as described in any one of the following:

wherein B1-B9 are one of hydrogen, alkyl, hydroxyl, alkoxy, nitro, cyano, amino, sulfhydryl, halogen substituent, phenyl, tolyl, naphthyl, furyl, thienyl, pyrrolyl, pyridyl, pyranyl, quinolinyl, indolyl, carboxyl or derivative groups thereof, carbazolyl or anilino.

Preferably, in the above structural formula, the A1-A8 and B1-B8 are respectively selected from hydrogen or one of the following structural formulas:

further, when the substituents R1, R2 are each hydrogen, hydroxy, ar is furan, thiophene, pyrrole, pyridine, pyran, quinoline (including isoquinoline), indole, carbazole, anilino or derivative thereof, the aryl-diphenyl-azine-based compound preferably has a structural formula as described in any one of the following:

further, the diethylamine-containing azine hydrazine compound has a structural formula as follows

Wherein A1-A10 are respectively selected from one of hydrogen, alkyl, hydroxyl, alkoxy, nitryl, cyano, amino, sulfydryl, halogen atom, diethylamine, phenyl, tolyl, naphthyl, furyl, thienyl, pyrrolyl, pyridyl, pyranyl, quinolinyl, indolyl, carboxyl or derivative groups thereof, carbazolyl or anilino.

Further, the diethylamine-containing azine hydrazine compound has any one of the following structural formulas:

the invention provides a preparation method of an azine hydrazine compound containing diethylamine, which comprises the following steps:

dissolving diphenyl hydrazine derivative and aryl containing diethylamine in a solvent, then heating for reaction, separating and purifying to obtain the azine hydrazine compound containing diethylamine.

Preferably, the separation and purification mode is chromatographic column separation.

Further, the structural formula of the diphenyl hydrazine derivative is one of the following structural formulas:

further, the structural formula of the diethylamine-containing aryl group is shown as follows:

the method comprises the steps of carrying out a first treatment on the surface of the The molar ratio of the diphenyl hydrazine derivative to the aryl containing diethylamine is 1:1-1:10.

further, the solvent is more than one of methanol, ethanol, acetic acid, tetrahydrofuran, toluene, benzene, chloroform, methylene dichloride, N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone.

Further, the temperature of the heating reaction is 20-150 ℃, and the time of the heating reaction is 1-24h.

The invention provides application of an azine hydrazine compound containing diethylamine in preparing a selective antibacterial drug and a selective imaging reagent of fungi.

The selective imaging of the fungi is specifically as follows:

the azine hydrazines are added into a bacterial culture medium, and only fluorescent signals can be observed on fungi under a fluorescent microscope or a laser scanning confocal microscope.

The selective antibacterial agent is specifically as follows:

after the azine hydrazine compounds are co-cultured with different microorganisms, the antibacterial activity of the azine hydrazine compounds is researched by a classical flat sterilization experiment, most gram-positive bacteria can be killed after the azine hydrazine compounds are added with medicines, all the gram-positive bacteria and fungi can be killed after continuous illumination, and the growth of the gram-negative bacteria is not influenced.

The principle of the invention is as follows: an intramolecular hydrogen bond is formed to electrons through the ortho-hydroxyl and N atoms in hydrazine, and the effective red shift of the fluorescence spectrum is realized through intramolecular proton transfer (ESIPT) in an excited state; the structure has obvious aggregation-induced luminescence property by utilizing the intramolecular hydrogen bond and the limited rotation in diphenyl under the aggregation state; the fluorescence quantum yield is improved by limiting the intramolecular movement of the hydroxyl groups; the interaction between the molecule and the microorganism can be enhanced by introducing alkalescent diethylamine; the diethylamine is directly conjugated with the luminous center, and the ionization of the diethylamine can be regulated and controlled to form a strong Charge Transfer (CT) state through the interaction with microorganisms, so that the fluorescence intensity is regulated and controlled, and the specific identification of fungi is realized; the azine structure is utilized to realize energy transfer by utilizing good light absorption capacity, surrounding oxygen molecules are sensitized to generate active oxygen, and photodynamic treatment of microbial infection is realized.

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

(1) According to the azine hydrazines compound containing diethylamine, a hydrazine structure is selected as a conjugated bridging element, on one hand, the conjugated degree of a probe molecule is kept by using a single double bond alternating mode, on the other hand, the conjugated degree of an N atom lone pair electron can be further increased, the energy difference between a singlet state and a triplet state is reduced, and the capability of generating active oxygen by sensitization is improved;

(2) According to the diethylamine-containing azine hydrazine compound, phenolic hydroxyl structures are introduced at two ends of hydrazine, ESIPT states (proton transfer in molecules under excited states) are formed by lone pair electrons of N, so that Stokes displacement is effectively increased, and self-absorption of molecules is prevented; free rotation of diphenyl introduces a RIR (intramolecular limited rotation) mechanism, enhancing AIE performance of such molecules and improving fluorescence quantum yield of the molecules;

(3) The azine hydrazine compound containing diethylamine provided by the invention has stronger binding capacity with gram-positive bacteria and fungi by introducing diethylamine into an aromatic group; diethylamine is conjugated with a luminescence center, so that the diethylamine has responsiveness to acidic environment and acidic molecules on the surface of gram-positive bacteria, and forms a strong CT state to quench fluorescence after ionization, thereby helping to realize selective imaging of fungi.

Drawings

FIG. 1 is a hydrogen diagram of DPNAP in deuterated chloroform;

FIG. 2 shows DPNAP at H ₂ Normalized ultraviolet absorbance spectra and fluorescence emission spectra in O/THF (99:1, v/v) solution;

FIG. 3 is a graph showing fluorescence emission spectra of DPNAP at various ratios of water and THF;

FIG. 4 is a graph showing fluorescence emission spectra of DPNAP in various solvents (n-hexane, triethylamine, ethyl acetate, tetrahydrofuran, ethanol, isopropanol, dimethylsulfoxide);

FIG. 5 is a graph showing the emission spectra of DPNAP in hydrochloric acid (HCl), water and sodium hydroxide (NaOH) solutions, respectively;

FIG. 6 is a graph showing the change in fluorescence intensity of the active oxygen probe DCFH under illumination conditions for DPNAP and photosensitizer Ce 6;

FIG. 7 is a graph showing confocal imaging results after DPNAP has interacted with different microorganisms and mixed bacterial samples thereof;

FIG. 8 is a graph showing changes in fluorescence intensity after DPNAP binds to teichoic acid (LTA) on the surface of gram-positive bacteria;

FIG. 9 is a graph showing the statistical results of the plate antibacterial experiments of DPNAP against Staphylococcus aureus under dark and light conditions;

FIG. 10 is a graph showing the statistical result of the plate antibacterial experiment of DPNAP on Candida albicans under dark and light conditions;

FIG. 11 is a graph showing the statistical result of the antibacterial experiment of DPNAP against E.coli under dark and light conditions;

FIG. 12 (A) is a scanning electron microscope image of Staphylococcus aureus in dark and light conditions after combination with DPNAP; (B) Scanning electron microscope images of fungi combined with DPNAP under dark and light conditions; (C) The scanning electron microscope image is a scanning electron microscope image under dark and illumination conditions after the combination of the escherichia coli and the DPNAP;

FIG. 13 is a graph showing the antibacterial activity of DPNAP on a mixed bacterial sample.

FIG. 14 is a graph showing the statistical result of the plate antibacterial experiment of DPNAP on super bacteria MRSA;

FIG. 15 is a graph showing the effect of wound healing after treatment in a different manner in a mouse infection model;

FIG. 16 is a graph of nuclear magnetic data of O-DPAS in deuterated chloroform;

FIG. 17 is a diagram of O-DPAS at H ₂ Normalized ultraviolet absorbance spectra and fluorescence emission spectra in O/THF (99:1, v/v) solution;

FIG. 18 is a graph showing fluorescence emission spectra of O-DPAS in different solvents (n-hexane, triethylamine, ethyl acetate, tetrahydrofuran, ethanol, isopropanol, dimethylsulfoxide);

FIG. 19 is a graph showing confocal imaging results of DO-DPAS interaction with different microorganisms and mixed bacterial samples thereof;

FIG. 20 is a graph of nuclear magnetic data of DO-DPAS in deuterated chloroform;

FIG. 21 is a diagram of DO-DPAS at H ₂ Normalized ultraviolet absorbance spectra and fluorescence emission spectra in O/THF (99:1, v/v) solution;

FIG. 22 is a graph showing fluorescence emission spectra of DO-DPAS in different solvents (n-hexane, triethylamine, ethyl acetate, tetrahydrofuran, ethanol, isopropanol, dimethylsulfoxide).

Detailed Description

The following examples are presented to further illustrate the practice of the invention, but are not intended to limit the practice and protection of the invention. It should be noted that the following processes, if not specifically described in detail, can be realized or understood by those skilled in the art with reference to the prior art. The reagents or apparatus used were not manufacturer-specific and were considered conventional products commercially available.

Example 1

The following compounds were specifically synthesized according to the following synthetic route:

(1) Synthesis of Compound 2

The mixture of compound 1 (2 mmol) and excess hydrazine hydrate was stirred under reflux for 4 hours. After the completion of the reaction, the solvent and the remaining hydrazine hydrate were removed by rotary evaporation to give compound 2 as a transparent oil in 100% yield.

(2) Synthesis of Compound DPNAP

Compound 2 (1 mmol) and compound 3 (1.5 mmol) were stirred under reflux for 4 hours. After completion of the reaction, separation was performed with a chromatography silica gel column to obtain a yellow solid compound DPNAP (diethylamine-containing azine hydrazines) in a yield of 81.2%.

FIG. 1 is a hydrogen spectrum of DPNAP, which demonstrates the structural correctness.

FIG. 2 shows DPNAP at H ₂ Normalized ultraviolet absorbance spectra and fluorescence emission spectra in O/THF (99:1, v/v) solution; FIG. 3 is a fluorescence emission spectrum of DPNAP at different ratios of water and THF. [ DPNAP]＝10μM；λ _ex =412 nm. As can be seen from FIG. 2, the maximum emission peak position is around 548nm, the strong luminescence with large Stokes shift (136 nm) is derived from the excited state intramolecular proton transfer process (ESIPT) process, and the intramolecular hydrogen bond is protected and its free movement is suppressed, both in the solution state and in the aggregation stateEmitting intense ketonic luminescence. DPNAP increases with increasing water (poor solvent) content, and its fluorescence intensity increases, clearly confirming its AIE properties.

FIG. 4 is a fluorescence emission spectrum of DPNAP in different solvents (n-hexane, triethylamine, ethyl acetate, tetrahydrofuran, ethanol, isopropanol, dimethylsulfoxide). [ DPNAP]＝10μM；λ _ex =412 nm. As can be seen from fig. 4, the emission wavelength of DPNAP does not vary much in solvents of different polarities, maintaining excellent stability.

FIG. 5 is a fluorescence emission spectrum of DPNAP in different pH environments. [ DPNAP]＝10μM；λ _ex =412 nm. As can be seen from FIG. 5, DPNAP emits strong fluorescence in a neutral environment, and the fluorescence signal disappears in an acidic and basic condition. Both acidity and basicity are shown to disrupt the intramolecular hydrogen bonds, leading to quenching of fluorescence.

FIG. 6 is a representation of the ability of DPNAP to generate reactive oxygen species, with the commercial photosensitizer Ce6 as a reference. As the illumination time is prolonged, compared with a blank group, the fluorescent intensity of the active oxygen probe Dichlorofluorescein (DCFH) mixed with DPNAP is gradually increased, and the increase range is not as large as Ce6, but the overall increase is obvious, so that the DPNAP has stronger capability of sensitization to generate active oxygen.

Example 2: the compounds of example 1 are useful for identifying and killing bacteria

(1) Bacterial imaging experiments

a. The strains (E.coli, staphylococcus aureus, bacillus subtilis, enterococcus faecalis, pseudomonas aeruginosa, candida albicans and Saccharomyces cerevisiae) were inoculated in 5mL of the culture medium, and cultured at 37℃for 12 hours. The strain was then centrifuged at 7100 rpm for 1 min while washing three times with phosphate buffer (PBS, 10mm, ph=7.4), the supernatant was discarded, and the remaining strain was suspended in PBS and then diluted to 1.0 Optical Density (OD) at 600nm ₆₀₀ =1.0). For the mixed bacterial sample, candida albicans, escherichia coli and staphylococcus aureus are mixed together.

b. The strain was co-stained with DPNAP at a concentration of 5 μm in PBS buffer for 20 minutes at 37 ℃, then centrifuged at 7100 rpm for 1 minute, and the resulting stained strain was placed in 10 μl of PBS buffer and stored in a freezer for detection by a laser scanning confocal microscope.

(2) Antibacterial experiments

For single bacterial liquid:

staphylococcus aureus (OD) ₆₀₀ =0.4) was diluted 5-fold, and interacted with DPNAP at 0.1, 0.5, 2, 5, 20 μΜ for 10 min in the dark, total volume 500 μl, blank group was blank bacterial liquid without photosensitizer molecule, and the mixture was diluted in white light (35 mw·cm ^-2 ) Illumination is carried out for 10 minutes, the control group is acted for 10 minutes under the dark condition, and then dilution is carried out for 10 minutes ⁴ Fold to 1mL, 100. Mu.L was plated on NB solid plate medium. After the plates were placed in a constant temperature incubator at 37℃for 16 hours, the number of colonies on the plates was measured.

Candida albicans and escherichia coli were treated with the same conditions.

For mixed bacterial samples:

staphylococcus aureus (OD) ₆₀₀ =1), candida albicans (OD ₆₀₀ =2), escherichia coli (OD ₆₀₀ =1) 100 μl, 300 μl, 100 μl, respectively, were mixed and interacted with 5 μΜ DPNAP for 10 min in the dark, the total volume was 500 μl, the blank group was a blank bacterial liquid without photosensitizer molecule, and the mixture was subjected to white light (35 mw·cm) ^-2 ) Illumination is carried out for 10 minutes, the control group is acted for 10 minutes under the dark condition, and then dilution is carried out for 10 minutes ⁴ Fold to 1mL, 100. Mu.L was plated on NB solid plate medium. After the plates were placed in a constant temperature incubator at 37℃for 16 hours, the number of colonies on the plates was measured. FIG. 7 shows confocal imaging results after DPNAP has interacted with different microorganisms and mixed bacterial samples thereof. As can be seen from the figure, DPNAP can selectively image fungi (candida albicans and saccharomyces cerevisiae), and no fluorescence signal can be detected on the surfaces of gram-positive bacteria (staphylococcus aureus, enterococcus faecalis and bacillus subtilis) and negative bacteria (escherichia coli and pseudomonas aeruginosa). Even in mixed samples (candida albicans, escherichia coli and staphylococcus aureus), the combination of the fluorescent dye and the candida albicans only realizes the selective imaging of fungi.

FIG. 8 shows the change in fluorescence intensity after DPNAP binds to teichoic acid (LTA) specific to the surface of gram-positive bacteria. As can be seen from the graph, with the increase of the concentration of LTA, the fluorescence of DPNAP is continuously reduced, which indicates that after DPNAP is combined with gram-positive bacteria, the fluorescence of DPNAP is quenched by LTA on the surface of bacteria and does not emit light, and finally, the combination with the gram-positive bacteria is realized and does not emit light.

FIG. 9 shows the statistical results of the antibacterial experiments of DPNAP against Staphylococcus aureus. As can be seen from fig. 9, under dark conditions, the antibacterial rate of 5 μm DPNAP against staphylococcus aureus was close to 50%, and the antibacterial effect was continuously enhanced as the concentration increased. In contrast, under the light condition, even at a concentration as low as 0.1. Mu.M, the antibacterial effect was close to 100%, indicating the excellent antibacterial effect of DPNAP against Staphylococcus aureus.

FIG. 10 shows the statistical results of the plate antibacterial experiments of DPNAP on Candida albicans. As can be seen from the figure, DPNAP has little antibacterial effect against candida albicans under dark conditions. However, under the illumination condition, the antibacterial effect is obvious, and the IC50 value is lower than 0.5 mu M. The DPNAP has good photodynamic treatment effect on candida albicans.

FIG. 11 shows the statistical results of the antibacterial experiments of DPNAP against E.coli. As can be seen from the figure, DPNAP has no obvious inhibitory effect on E.coli either under dark or light conditions. It was revealed that DPNAP had substantially no antibacterial activity against E.coli.

FIG. 12 is a scanning electron microscope image of bacterial surface in the dark and under light conditions after DPNAP has been combined with microorganisms. From FIGS. 12 (A), (B) and (C), it can be seen that the cell walls of the three microorganisms are intact in the dark and under light conditions, and that no significant damage is caused, indicating that the antibacterial activity is not caused by a significant damage of the cell wall structure.

Table 1 shows the extracellular nucleic acid concentration changes. As can be seen from table 1 below, for staphylococcus aureus, compared with a blank group, after action of staphylococcus aureus and DPNAP, the extracellular nucleic acid concentration of a dark group and an illumination group is increased to a certain extent, and in combination with the scanning electron microscope result, it is shown that the cell wall structure of the staphylococcus aureus is damaged to a certain extent after action of the staphylococcus aureus and DPNAP, so that the nucleic acid leaks, and ROS generated after illumination can cause the cell wall structure of the staphylococcus aureus to be further damaged, and finally effective killing of the staphylococcus aureus is caused; in the case of Candida albicans, the extracellular nucleic acids of the light group and the dark group are identical to those of the blank group.

TABLE 1

The concentration change of the nucleic acid is combined with the scanning electron microscope result, so that the simple combination can not damage the structure of the cell wall, active oxygen generated after illumination can cause the inactivation of biological macromolecules, and the effective killing of fungi is finally realized; in the case of E.coli, however, it can be seen that DPNAP does not bind to E.coli because it does not exhibit antibacterial activity in combination with the fluorescence imaging results; from this, it can be seen that the difference in the binding of DPNAP to three microorganisms eventually realizes selective killing of three microorganisms.

FIG. 13 is a graph showing the antibacterial activity of DPNAP on a mixed bacterial sample. As can be seen from fig. 13, most of staphylococcus aureus was substantially killed in dark condition after DPNAP addition. After continued illumination, all staphylococcus aureus and candida albicans were inhibited, leaving only escherichia coli. Realizes the selective antibiosis in the mixed bacterial sample.

FIG. 14 shows the statistical results of the plate antibacterial experiments of DPNAP on super bacteria MRSA. As can be seen from FIG. 14, the antibacterial effect is less obvious than that of the common Staphylococcus aureus at the concentration of 10 mu M under the dark condition, but the antibacterial effect on the superbacteria is very obvious under the illumination condition.

Fig. 15 is a graph showing the effect of DPNAP in living body antibacterial applications. As can be seen from fig. 15, the healing rate was greatly improved after the application of DPNAP and the irradiation with light, compared with the infected mice treated with Phosphate Buffer (PBS) alone, and the effect was consistent with that of cefalotin; and after the DPNAP is smeared, the light is not adopted, the healing speed is slowed down, so that the active oxygen generated by the DPNAP light can effectively inhibit the growth of bacteria, improve the healing speed of wounds and is hopeful to be used for wound infection treatment.

Example 3

(1) Synthesis of Compound 5

The mixture of compound 4 (2 mmol) and excess hydrazine hydrate was stirred under reflux for 4 hours. After the reaction was completed, the solvent and the remaining hydrazine hydrate were removed by rotary evaporation to give compound 5 in 100% yield.

(2) Synthesis of the Compound DO-DPAS

Compound 5 (1 mmol) and compound 3 (1.5 mmol) were stirred under reflux for 4 hours. After the completion of the reaction, separation was performed by a silica gel column chromatography to obtain a yellow solid compound DO-DPAS in 75% yield.

FIG. 16 is a hydrogen spectrum of DO-DPAS.

FIG. 17 is a diagram of DO-DPAS at H ₂ Normalized ultraviolet absorbance spectra and fluorescence emission spectra in O/THF (99:1, v/v) solution. [ DO-DPAS]＝10μM；λ _ex =401 nm. As can be seen from FIG. 17, the DO-DPAS maximum emission peak positions were all around 475 nm.

FIG. 18 is a fluorescence emission spectrum of DO-DPAS in different solvents (n-hexane, triethylamine, ethyl acetate, tetrahydrofuran, ethanol, isopropanol, dimethylsulfoxide). [ DO-DPAS]＝10μM；λ _ex =412 nm. As can be seen from fig. 18, the emission wavelength of DO-DPAS does not vary much in solvents of different polarities, maintaining excellent stability.

Example 4: the compound of example 3 was used for bacterial imaging

a. The strains (E.coli, staphylococcus aureus, bacillus subtilis, enterococcus faecalis, pseudomonas aeruginosa, candida albicans and Saccharomyces cerevisiae) were inoculated in 5mL of the culture medium, and cultured at 37℃for 12 hours. The strain was then centrifuged at 7100 rpm for 1 min while being washed three times with phosphate buffer (PBS, 10mm, ph=7.4), the supernatant was discarded, and the remaining strain was suspended in PBS and then diluted to 1 at 600 nm.0 Optical Density (OD) ₆₀₀ ＝1.0)。

b. The strain was co-stained with DO-DPAS at a concentration of 5. Mu.M in PBS buffer for 20 minutes at 37℃and then centrifuged at 7100 rpm for 1 minute, and the resulting stained strain was placed in 10. Mu.L of PBS buffer and stored in a freezer for detection by a laser scanning confocal microscope.

FIG. 19 shows confocal imaging results after DO-DPAS interaction with different microorganisms. As can be seen from FIG. 19, DO-DPAS has a very poor imaging effect due to a too low fluorescence quantum yield, and the fluorescent signal of DO-DPAS is hardly seen on the surface of the microorganism. The introduction of multiple hydroxyl groups has been shown to increase the fluorescence quantum yield of the molecules, which is beneficial for microbial imaging.

Example 5

(1) Synthesis of Compound O-DPAS

Compound 5 (1 mmol) and compound 6 (1.5 mmol) were stirred under reflux for 4 hours. After completion of the reaction, separation was performed by a chromatography silica gel column to obtain the compound NBADB in a yield of 87%.

FIG. 20 is a graph of O-DPAS hydrogen.

FIG. 21 is a diagram of O-DPAS at H ₂ Normalized ultraviolet absorbance spectra and fluorescence emission spectra in O/THF (99:1, v/v) solution. [ O-DPAS]＝10μM；λ _ex =384 nm. As can be seen from FIG. 21, the maximum emission peak positions of the O-DPAS were all around 442 nm.

FIG. 22 is a fluorescence emission spectrum of O-DPAS in different solvents (n-hexane, triethylamine, ethyl acetate, tetrahydrofuran, ethanol, isopropanol, dimethylsulfoxide). [ O-DPAS]＝10μM；λ _ex =412 nm. As can be seen from fig. 22, the emission wavelength of O-DPAS does not vary much in solvents of different polarities, maintaining excellent stability.

The above examples are only preferred embodiments of the present invention, and are merely for illustrating the present invention, not for limiting the present invention, and those skilled in the art should not be able to make any changes, substitutions, modifications and the like without departing from the spirit of the present invention.

Claims

1. An azine hydrazine compound containing diethylamine, which is characterized by the following structural formula:

2. a process for the preparation of diethylamine-containing azine hydrazines of claim 1 comprising the steps of:

dissolving a diphenyl hydrazine derivative and an aryl containing diethylamine in a solvent, then heating for reaction, and separating and purifying to obtain the azine hydrazine compound containing diethylamine; the structural formula of the diphenyl hydrazine derivative is one of the following structural formulas:

the structural formula of the aryl containing diethylamine is shown as follows:

3. the process for the preparation of diethylamine-containing azine-based compounds according to claim 2, wherein the molar ratio of said diphenylhydrazine derivatives to diethylamine-containing aryl groups is 1:1-1:10.

4. the process for producing a diethylamine-containing azine-based compound according to claim 2, wherein the solvent is one or more of methanol, ethanol, acetic acid, tetrahydrofuran, toluene, benzene, chloroform, methylene chloride, N-dimethylformamide, N-dimethylacetamide and N-methylpyrrolidone.

5. The process for the preparation of diethylamine-containing azine hydrazines according to any of the claims 2 to 4, characterized in that the heating reaction is carried out at a temperature of 20 to 150 ℃ for a time of 1 to 24 hours.

6. The use of a diethylamine-containing azine hydrazines of claim 1 for the preparation of a medicament for selective resistance against staphylococcus aureus or candida albicans and for the preparation of a selective imaging agent for the fungus candida albicans or saccharomyces cerevisiae.