WO2021227206A1 - 一种含二乙胺的吖嗪联肼类化合物及其制备方法与应用 - Google Patents

一种含二乙胺的吖嗪联肼类化合物及其制备方法与应用 Download PDF

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WO2021227206A1
WO2021227206A1 PCT/CN2020/097562 CN2020097562W WO2021227206A1 WO 2021227206 A1 WO2021227206 A1 WO 2021227206A1 CN 2020097562 W CN2020097562 W CN 2020097562W WO 2021227206 A1 WO2021227206 A1 WO 2021227206A1
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diethylamine
azine
hydrazine compound
hydrazine
dpnap
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French (fr)
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唐本忠
王志明
胡蓉
周凡
崔娜
邓棋云
张可心
印平安
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华南理工大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
    • C07C251/72—Hydrazones
    • C07C251/88—Hydrazones having also the other nitrogen atom doubly-bound to a carbon atom, e.g. azines
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1007—Non-condensed systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Definitions

  • the invention belongs to the field of analysis and detection materials, and specifically relates to an azine hydrazine compound containing diethylamine and a preparation method and application thereof.
  • the pathogenic bacteria targeted include Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli , Porphyromonas gingivalis and multi-drug resistant bacteria.
  • photodynamic antimicrobial chemotherapy is not prone to drug resistance.
  • reagents for PACT mainly include porphyrin photosensitizers, phthalocyanine photosensitizers, and porphin photosensitizers.
  • Fluorescent materials with aggregation-induced luminescence properties can use the process of forming an aggregate state to inhibit the energy dissipation of the excited state by limiting intramolecular motion, and improve the luminous efficiency of the aggregate state and the ability to generate active oxygen. It is expected to develop into a new type of Integrated materials for microbiological diagnosis and treatment.
  • the purpose of the present invention is to provide an azine hydrazine compound containing diethylamine and a preparation method and application thereof.
  • the primary objective of the present invention is to provide an aryl-diphenyl-azine hydrazine compound containing diethylamine.
  • Another object of the present invention is to provide a method for preparing the diethylamine-containing aryl-diphenyl-azine hydrazine compound.
  • Another object of the present invention is to provide the application of the above-mentioned diethylamine-containing aryl-diphenyl-azine hydrazine compounds as fluorescent probe materials in the fields of biological analysis and clinical medical detection.
  • Ar represents an aromatic group or its derivative structure
  • the substituents R1-R10 are selected from hydrogen, alkyl, hydroxyl, alkoxy, nitro, cyano, amino, mercapto, halogen, diethylamine, and phenyl.
  • Tolyl naphthyl, furanyl, thienyl, pyrrolyl, pyridyl, pyranyl, quinolinyl, indolyl, carboxyl or its derivatives, carbazolyl or anilino.
  • the azine hydrazine compound containing diethylamine has the structural formula:
  • Ar represents an aromatic group or its derivative structure
  • substituents R1 and R2 are selected from hydrogen, alkyl, hydroxyl, alkoxy, nitro, cyano, amino, mercapto, halogen, diethylamine, phenyl, One of tolyl, naphthyl, furyl, thienyl, pyrrolyl, pyridyl, pyranyl, quinolinyl, indolyl, carboxyl or derivatives thereof, carbazolyl or anilino.
  • R 1 and R 2 are each selected from hydrogen or one of the following structural formulas:
  • the substituents R1 and R2 are both hydrogen and hydroxyl
  • Ar is a benzene ring or a benzene ring derivative
  • the diethylamine-containing aryl salicylaldehyde-diphenyl-azine hydrazine preferably has a structural formula as described in any one of the following:
  • A1-A5 are hydrogen, alkyl, hydroxy, alkoxy, nitro, cyano, amino, mercapto, halogen substituent, diethylamine, phenyl, tolyl, naphthyl, furanyl, thienyl, One of pyrrolyl, pyridyl, pyranyl, quinolinyl, indolyl, carboxyl or derivatives thereof, carbazolyl or anilino.
  • azine hydrazine compound preferably has a structural formula as described in any one of the following:
  • B1-B9 are hydrogen, alkyl, hydroxyl, alkoxy, nitro, cyano, amino, mercapto, halogen substituent, phenyl, tolyl, naphthyl, furyl, thienyl, pyrrolyl, pyridine
  • the A1-A8 and B1-B8 are respectively selected from hydrogen or one of the following structural formulas:
  • the substituents R1 and R2 are both hydrogen and hydroxyl
  • Ar is furan, thiophene, pyrrole, pyridine, pyran, quinoline (containing isoquinoline), indole, carbazole, anilino group or derivatives thereof
  • the aryl-diphenyl-azine hydrazine compound preferably has a structural formula as described in any one of the following:
  • A1-A10 are selected from hydrogen, alkyl, hydroxyl, alkoxy, nitro, cyano, amino, mercapto, halogen, diethylamine, phenyl, tolyl, naphthyl, furyl, and thienyl. , Pyrrolyl, pyridyl, pyranyl, quinolinyl, indolyl, carboxyl or its derivatives, carbazolyl or anilino.
  • diethylamine-containing azine hydrazine compound has any one of the following structural formulas:
  • the present invention provides a preparation method of azine hydrazine compounds containing diethylamine, which comprises the following steps:
  • the diphenylhydrazine derivative and the diethylamine-containing aryl group are dissolved in a solvent, and then heated to react for separation and purification to obtain the diethylamine-containing azine hydrazine compound.
  • the method of separation and purification is chromatographic column separation.
  • the molar ratio of the diphenylhydrazine derivative and the diethylamine-containing aryl group is 1:1-1:10.
  • the solvent is methanol, ethanol, acetic acid, tetrahydrofuran, toluene, benzene, chloroform, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide and N-methylpyrrolidone More than one of them.
  • the temperature of the heating reaction is 20-150° C.
  • the time of the heating reaction is 1-24 h.
  • the invention provides applications of diethylamine-containing azine hydrazine compounds in the preparation of selective antibacterial drugs and selective imaging reagents for fungi.
  • the selective imaging of the fungus is specifically:
  • the fluorescence signal can only be observed on the fungus.
  • the selective antibacterial is specifically:
  • the principle of the present invention is: forming intramolecular hydrogen bonds with the electrons through the ortho-position hydroxyl group and the N atom in the hydrazine, and realizing the effective red shift of the fluorescence spectrum through the excited state intramolecular proton transfer (ESIPT); Intramolecular hydrogen bonding and diphenyl internal rotation restriction make the structure have significant aggregation-induced luminescence properties; the introduction of multiple hydroxyl groups makes the intramolecular movement restricted to increase the fluorescence quantum yield; through the introduction of alkalescent diethylamine It can enhance the interaction between molecules and microorganisms; directly conjugated diethylamine with the luminescent center, can regulate the interaction with microorganisms and then regulate the ionization of diethylamine to form a strong charge transfer (CT) state, which has a strong influence on fluorescence Control, realize the specific recognition of fungi; use the good light absorption capacity of azines to realize energy transfer, sensitize the surrounding oxygen molecules to produce active oxygen, and
  • the present invention has the following advantages and beneficial effects:
  • the hydrazine structure is selected as the conjugation bridging motif.
  • the form of alternating single and double bonds is used to maintain the conjugation degree of the probe molecule, and on the other hand,
  • the lone pair of electrons of the N atom can further increase the degree of conjugation, reduce the energy gap between the singlet state and the triplet state, and improve the ability of sensitization to generate active oxygen;
  • the diethylamine-containing azine hydrazine compound provided by the present invention introduces a phenolic hydroxyl structure at both ends of the hydrazine, and forms an ESIPT state (intramolecular proton transfer in the excited state) with the lone pair of N electrons, which is effective Increase the Stokes shift to prevent molecular self-absorption;
  • the free rotation of diphenyl groups introduces the RIR (intramolecular restricted rotation) mechanism, which strengthens the AIE performance of such molecules and increases the fluorescence quantum yield of the molecules;
  • the diethylamine-containing azine hydrazine compound provided by the present invention introduces diethylamine into the aromatic group, and has a strong binding ability with gram-positive bacteria and fungi; the diethylamine is combined with the luminescent center Conjugation connection makes it responsive to acidic environment and acidic molecules on the surface of gram-positive bacteria. After ionization, it forms a strong CT state and quenches fluorescence, helping to achieve selective imaging of fungi.
  • Figure 1 is the hydrogen spectrum of DPNAP in deuterated chloroform
  • Figure 2 shows the normalized UV absorption spectrum and fluorescence emission spectrum of DPNAP in H2O/THF (99:1, v/v) solution
  • Figure 3 shows the fluorescence emission spectra of DPNAP in different ratios of water and THF
  • Figure 4 shows the fluorescence emission spectra of DPNAP in different solvents (n-hexane, triethylamine, ethyl acetate, tetrahydrofuran, ethanol, isopropanol, dimethyl sulfoxide);
  • FIG. 5 shows the emission spectra of DPNAP in hydrochloric acid (HCl), water and sodium hydroxide (NaOH) solutions respectively;
  • Figure 6 is a graph showing changes in the fluorescence intensity of the active oxygen probe DCFH under light conditions of DPNAP and photosensitizer Ce6;
  • Figure 7 shows the results of confocal imaging after DPNAP interacts with different microorganisms and their mixed samples
  • Figure 8 is a graph showing changes in fluorescence intensity after DPNAP is combined with teichoic acid (LTA) on the surface of Gram-positive bacteria;
  • Figure 9 shows the statistical results of the plate antibacterial experiment of DPNAP against Staphylococcus aureus under dark and light conditions
  • Figure 10 shows the statistical results of the plate antibacterial experiment of DPNAP against Candida albicans under dark and light conditions
  • Figure 11 shows the statistical results of the plate antibacterial experiment of DPNAP on E. coli under dark and light conditions
  • Figure 12 (A) is the scanning electron micrograph of Staphylococcus aureus and DPNAP under dark and light conditions; (B) is the scanning electron micrograph of the fungus and DPNAP under dark and light conditions; (C) is the E. coli and Scanning electron micrograph under dark and light conditions after combining DPNAP;
  • Figure 13 shows the results (A) and statistical results (B) of the plate antibacterial experiment of DPNAP on mixed bacterial samples.
  • Figure 14 shows the statistical results of the plate antibacterial experiment of DPNAP on the super bacteria MRSA
  • Figure 15 is an effect diagram of wound healing in a mouse infection model treated in different ways
  • Figure 16 shows the NMR data of O-DPAS in deuterated chloroform
  • Figure 17 is the normalized ultraviolet absorption spectrum and fluorescence emission spectrum of O-DPAS in H2O/THF (99:1, v/v) solution;
  • Figure 18 shows the fluorescence emission spectra of O-DPAS in different solvents (n-hexane, triethylamine, ethyl acetate, tetrahydrofuran, ethanol, isopropanol, dimethyl sulfoxide);
  • Figure 19 is the result of confocal imaging after DO-DPAS interacts with different microorganisms and their mixed samples
  • Figure 20 shows the NMR data of DO-DPAS in deuterated chloroform
  • Figure 21 is the normalized ultraviolet absorption spectrum and fluorescence emission spectrum of DO-DPAS in H2O/THF (99:1, v/v) solution;
  • Figure 22 shows the fluorescence emission spectra of DO-DPAS in different solvents (n-hexane, triethylamine, ethyl acetate, tetrahydrofuran, ethanol, isopropanol, dimethyl sulfoxide).
  • Figure 1 is the hydrogen spectrum of DPNAP, which proves the correctness of its structure.
  • FIG. 2 shows the normalized UV absorption spectrum and fluorescence emission spectrum of DPNAP in H 2 O/THF (99:1, v/v) solution;
  • Figure 3 shows the fluorescence emission spectrum of DPNAP in different ratios of water and THF .
  • [DPNAP] 10 ⁇ M;
  • ⁇ ex 412 nm.
  • the maximum emission peak position is around 548nm in both the solution state and the aggregate state, and the strong luminescence with a large Stokes shift (136nm) originates from the proton transfer in the excited state.
  • Process ESIPT
  • Figure 4 shows the fluorescence emission spectra of DPNAP in different solvents (n-hexane, triethylamine, ethyl acetate, tetrahydrofuran, ethanol, isopropanol, dimethyl sulfoxide).
  • FIG. 5 shows the fluorescence emission spectra of DPNAP under different pH environments.
  • Figure 6 is a characterization of the ability of DPNAP to generate reactive oxygen species, in which the commercial photosensitizer Ce6 is used as a reference.
  • the fluorescence intensity of the reactive oxygen probe dichlorofluorescein (DCFH) mixed with DPNAP gradually increased. Although the increase was not as good as Ce6, the overall increase was obvious, indicating that DPNAP has a stronger The sensitization ability to produce reactive oxygen species.
  • Example 2 The compound in Example 1 is used to identify and kill bacteria
  • the strain was co-stained with 5 ⁇ M DPNAP in PBS buffer solution at 37°C for 20 minutes, and then centrifuged at 7100 rpm for 1 minute, the stained strain was placed in 10 ⁇ L PBS buffer solution and stored in the freezer Used for laser scanning confocal microscope inspection.
  • Figure 7 shows the results of confocal imaging after DPNAP interacts with different microorganisms and their mixed samples. It can be seen from the figure that DPNAP can selectively image fungi (Candida albicans and Saccharomyces cerevisiae), while in Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis) and negative bacteria (E. coli) , Pseudomonas aeruginosa), no fluorescent signal can be detected on the surface. Even in the mixed samples (Candida albicans, Escherichia coli and Staphylococcus aureus), it only binds to the fungus Candida albicans, realizing selective imaging of the fungus.
  • DPNAP can selectively image fungi (Candida albicans and Saccharomyces cerevisiae), while in Gram-positive bacteria (Staphylococcus aureus, Enterococcus f
  • Figure 8 shows the changes in fluorescence intensity after DPNAP is combined with teichoic acid (LTA), which is unique to the surface of Gram-positive bacteria. It can be seen from the figure that as the concentration of LTA increases, the fluorescence of DPNAP continues to decrease, indicating that the fluorescence of DPNAP will be quenched by the LTA on the surface of the bacteria and will not emit light after the combination of DPNAP and Gram-positive bacteria. The positive bacteria do not emit light when combined.
  • LTA teichoic acid
  • Figure 9 shows the statistical results of the plate antibacterial experiment of DPNAP against Staphylococcus aureus. It can be seen from Figure 9 that under dark conditions, the inhibitory rate of 5 ⁇ M DPNAP against Staphylococcus aureus is close to 50%, and as the concentration increases, the antibacterial effect continues to increase. Under light conditions, even at a concentration as low as 0.1 ⁇ M, its antibacterial effect is close to 100%, indicating the excellent antibacterial effect of DPNAP on Staphylococcus aureus.
  • Figure 10 shows the statistical results of the plate antibacterial experiment of DPNAP against Candida albicans. It can be seen from the figure that under dark conditions, DPNAP has almost no antibacterial effect on Candida albicans. But under light conditions, its antibacterial effect is obvious, and its IC50 value is lower than 0.5 ⁇ M. It shows that DPNAP has a good photodynamic therapy effect on Candida albicans.
  • Figure 11 shows the statistical results of the plate antibacterial experiment of DPNAP on E. coli. It can be seen from the figure that DPNAP has no obvious inhibitory effect on E. coli no matter under dark conditions or light conditions. It shows that DPNAP has basically no antibacterial activity against Escherichia coli.
  • Figure 12 is a scanning electron microscope image of the bacterial surface in the dark and under light conditions after DPNAP is combined with microorganisms. From Figure 12 (A), (B) and (C), it can be seen that the cell wall structure of the three microorganisms is intact in the dark and under the light conditions, and there is no obvious damage, indicating that the antibacterial activity is not caused by the obvious damage of the cell wall structure.
  • Table 1 shows the changes in the concentration of extracellular nucleic acid. It can be seen from Table 1 below that for Staphylococcus aureus, compared with the blank group, after Staphylococcus aureus interacts with DPNAP, the concentration of extracellular nucleic acid in the dark group and the light group increased to a certain extent. Combined with the scanning electron microscope results, It shows that the cell wall structure is damaged to a certain extent after the interaction with DPNAP, which causes the leakage of nucleic acid. The ROS generated after light will further damage the cell wall structure, and ultimately lead to effective killing of Staphylococcus aureus; and for Candida albicans, its light The extracellular nucleic acids in the dark group and the dark group are the same as those in the blank group.
  • Nucleic acid concentration changes combined with scanning electron microscopy results can prove that simple binding will not damage the structure of the cell wall, and the reactive oxygen species generated after light will cause the inactivation of biological macromolecules and ultimately achieve effective killing of fungi; for E. coli, binding Fluorescence imaging results show that DPNAP will not bind to Escherichia coli because it does not exhibit antibacterial activity. It can be seen that the difference in binding between DPNAP and the three microorganisms ultimately achieves the selective killing of the three microorganisms.
  • Figure 13 is a graph (A) and statistical results (B) of the plate antibacterial experiment effect of DPNAP on mixed bacterial samples. It can be seen from parts (A) and (B) of Figure 13 that after adding DPNAP, most of the Staphylococcus aureus is basically killed under dark conditions. After continuing to light, all Staphylococcus aureus and Candida albicans were inhibited, and only Escherichia coli remained. Achieve selective antibacterial in the mixed bacteria sample.
  • Figure 14 shows the statistical results of the plate antibacterial experiment of DPNAP on the super bacterium MRSA. It can be seen from Figure 14 that under dark conditions, its antibacterial effect at a concentration of 10 ⁇ M is less obvious than that of ordinary Staphylococcus aureus, but its antibacterial effect on super bacteria is very significant under light conditions.
  • Figure 15 is the application effect diagram of DPNAP in in vivo antibacterial. It can be seen from Figure 15 that compared with the infected mice treated with phosphate buffered saline (PBS) only, the healing speed of DPNAP is greatly improved after smearing and lighting, and the effect is consistent with that of cefalotin; while DPNAP smearing In the future, if light is not used, the healing speed will be slowed down, indicating that the active oxygen produced by DPNAP light can effectively inhibit the growth of bacteria and improve the healing speed of wounds, which is expected to be used in the treatment of wound infections.
  • PBS phosphate buffered saline
  • Figure 16 shows the hydrogen spectrum of DO-DPAS.
  • Figure 17 shows the normalized UV absorption spectrum and fluorescence emission spectrum of DO-DPAS in a H 2 O/THF (99:1, v/v) solution.
  • Figure 18 shows the fluorescence emission spectra of DO-DPAS in different solvents (n-hexane, triethylamine, ethyl acetate, tetrahydrofuran, ethanol, isopropanol, dimethyl sulfoxide).
  • Example 4 The compound of Example 3 is used for bacterial imaging
  • the strain was co-stained with 5 ⁇ M DO-DPAS in PBS buffer solution at 37°C for 20 minutes, and then centrifuged at 7100 rpm for 1 minute. The stained strain was placed in 10 ⁇ L PBS buffer solution and stored in Used in the freezer for laser scanning confocal microscope inspection.
  • Figure 19 shows the results of confocal imaging after DO-DPAS interacts with different microorganisms. It can be seen from Figure 19 that the imaging effect of DO-DPAS is not ideal because the fluorescence quantum yield is too low, and the fluorescence signal of DO-DPAS is almost invisible on the surface of microorganisms. It is proved that the introduction of multiple hydroxyl groups can increase the fluorescence quantum yield of the molecule, which is conducive to microbial imaging.
  • Figure 20 is a hydrogen spectrum of O-DPAS.
  • Figure 21 shows the normalized ultraviolet absorption spectrum and fluorescence emission spectrum of O-DPAS in a H 2 O/THF (99:1, v/v) solution.
  • Figure 22 shows the fluorescence emission spectra of O-DPAS in different solvents (n-hexane, triethylamine, ethyl acetate, tetrahydrofuran, ethanol, isopropanol, dimethyl sulfoxide).

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Abstract

本发明公开了一种含二乙胺的吖嗪联肼类化合物及其制备方法与应用。该方法包括：将二苯基肼衍生物与含二乙胺的芳基在溶剂中，进行加热反应，反应产物经分离提纯后即得到所述含二乙胺的吖嗪联肼类化合物。本发明同时公开了上述化合物的应用，通过调控吖嗪联肼类化合物与微生物之间的相互作用强弱进而调节吖嗪联肼类化合物的荧光强度，实现对真菌的识别及选择性抗菌。本发明的含二乙胺的吖嗪联肼类化合物，具有显著的聚集诱导发光(AIE)性质产生活性氧的能力，表现出选择性的细菌荧光染色能力及抗菌活性，具有很好的应用前景。

Description

一种含二乙胺的吖嗪联肼类化合物及其制备方法与应用 技术领域
本发明属于分析检测材料领域，具体涉及一种含二乙胺的吖嗪联肼类化合物及其制备方法与应用。
背景技术
人类自古就饱受由各种致病微生物(例如:细菌，真菌，病毒和寄生虫)引起的感染性疾病的侵害。多年来，抗生素作为最有效的控制流行性疾病的药物被广泛使用，但因抗生素在医疗卫生、工农业生产等领域的乱用和滥用(农牧业中使用抗生素来帮助牲畜生长)，进化而来的耐药性致病菌每年已造成发达国家数十万人的死亡。世界卫生组织(WHO)收集的来自129个成员国的相关数据表明，抗生素的耐药性已经出现在了世界上的每一个角落，日益增加的多药耐药性致病菌感染，已经严重威胁了人类的生存和健康，已迫使人类进入无药可医的“后抗生素时代”，即普通的感染和轻伤也有可能致命。已有的研究成果表明，传统的化学合成和药物筛选策略都需要经历漫长研发过程，花费大量金钱的同时，浪费大量的资源。更为严重的是，新药的研发速度常常落后于耐药性致病菌的出现和扩散速度，这使得传统药物开发策略在解决耐药性的难题上收效甚微。而针对微生物导致的感染，微生物的快速区分及有效的抗菌策略对于临床治疗微生物感染有着同样重要的意义。
目前，已经发展很多集诊断和治疗为一体化(诊疗一体化)的体系，其中，具有能够有效产生活性氧(ROS)的一类荧光材料在微生物诊疗一体化中展现了良好的应用前景。而光动力疗法是利用光和光敏剂产生的光动力效应进行疾病诊断和治疗的一种技术。近年发现，对皮肤局部微生物感染疗效显著，称为光动力抗微生物化学疗法(photodvnamic antimicrobial chemothempy，PACT)，针对的病原菌包括金黄色葡萄球菌、铜绿假单胞菌、鲍曼不动杆菌、大肠杆菌、牙龈卟啉单胞菌及多重耐药菌等。此外，光动力抗微生物化学疗法不易产生耐药性。目前常用的用于PACT的试剂主要包括卟啉光敏剂、酞菁类光敏剂、卟吩类光敏剂。除此之外，还有很多基于有机小分子荧光材料和聚合物荧光材料的光敏剂，此类荧光材料不仅能有效敏化产生活性氧在抗菌中也取得了良好的效果，其荧光性质还能实现生物成像，在光敏产生活性氧。但是此类物质溶解性有限，在生理条件下会形成聚集体，导致其发光及活性氧的产生都受到抑制，限制了其在微生物诊疗一体化中的进一步应用。具有聚集诱导发光性能的荧光材料，能够利用形成聚集态这样一个过程，通过限制分子内运动抑制激发态能量的耗散，提高聚集态发光效率及活性氧产生能力，有望发展成一类新型的用于微生物诊疗一体化的材料。
发明内容
为了克服现有技术存在的上述不足，本发明的目的是提供一种含二乙胺的吖嗪联肼类化合物及其制备方法与应用。
基于以上现有技术，本发明的首要目的在于提供一种含二乙胺的芳基-二苯基-吖嗪联肼类化合物。
本发明的另一目的在于提供一种上述含二乙胺的芳基-二苯基-吖嗪联肼类化合物的制备 方法。
本发明的再一目的在于提供上述含二乙胺的芳基-二苯基-吖嗪联肼类化合物作为荧光探针材料在生物分析和临床医学检测等领域中的应用。
本发明的目的至少通过如下技术方案之一实现。
本发明提供的吖嗪联肼类化合物，其结构通式如下所示：
Figure PCTCN2020097562-appb-000001
其中，Ar表示芳香基团或其衍生结构，取代基R1-R10分别选自氢、烷基、羟基、烷氧基、硝基、氰基、氨基、巯基、卤素原子、二乙胺、苯基、甲苯基、萘基、呋喃基、噻吩基、吡咯基、吡啶基、吡喃基、喹啉基、吲哚基、羧基或其衍生基团、咔唑基或苯胺基中的一种。
优选地，所述含二乙胺的吖嗪联肼类化合物，其结构式为
Figure PCTCN2020097562-appb-000002
其中Ar表示芳香基团或其衍生结构，取代基R1、R2分别选自氢、烷基、羟基、烷氧基、硝基、氰基、氨基、巯基、卤素原子、二乙胺、苯基、甲苯基、萘基、呋喃基、噻吩基、吡咯基、吡啶基、吡喃基、喹啉基、吲哚基、羧基或其衍生基团、咔唑基或苯胺基中的一种。
所述R 1、R 2分别选自氢或以下结构式中的一种：
Figure PCTCN2020097562-appb-000003
进一步地，当所述取代基R1、R2均为氢、羟基，Ar为苯环或苯环衍生物时，所述含二 乙胺的芳基水杨醛-二苯基-吖嗪联肼类化合物优选具有如下任一项所述的结构式：
Figure PCTCN2020097562-appb-000004
其中，A1-A5为氢、烷基、羟基、烷氧基、硝基、氰基、氨基、巯基、卤素取代基、二乙胺、苯基、甲苯基、萘基、呋喃基、噻吩基、吡咯基、吡啶基、吡喃基、喹啉基、吲哚基、羧基或其衍生基团、咔唑基或苯胺基中的一种。
进一步地，当所述取代基R1、R2均为氢、羟基，Ar为萘环或其衍生物、蒽环或其衍生物、菲环或其衍生物时，所述芳基-二苯基-吖嗪联肼类化合物优选具有如下任一项所述的结构式：
Figure PCTCN2020097562-appb-000005
其中，B1-B9为氢、烷基、羟基、烷氧基、硝基、氰基、氨基、巯基、卤素取代基、苯基、甲苯基、萘基、呋喃基、噻吩基、吡咯基、吡啶基、吡喃基、喹啉基、吲哚基、羧基或其衍生基团、咔唑基或苯胺基中的一种。
优选地，上述结构式中，所述A1-A8、B1-B8分别选自氢或以下结构式中的一种：
Figure PCTCN2020097562-appb-000006
进一步地，当所述取代基R1、R2均为氢、羟基，Ar为呋喃、噻吩、吡咯、吡啶、吡喃、喹啉(含异喹啉)、吲哚、咔唑、苯胺基或其衍生基团时，所述芳基-二苯基-吖嗪联肼类化合物优选具有如下任一项所述的结构式：
Figure PCTCN2020097562-appb-000007
进一步地，所述的含二乙胺的吖嗪联肼类化合物，其结构式为
Figure PCTCN2020097562-appb-000008
其中，A1-A10分别选自氢、烷基、羟基、烷氧基、硝基、氰基、氨基、巯基、卤素原子、二乙胺、苯基、甲苯基、萘基、呋喃基、噻吩基、吡咯基、吡啶基、吡喃基、喹啉基、吲哚基、羧基或其衍生基团、咔唑基或苯胺基中的一种。
进一步地，所述含二乙胺的吖嗪联肼类化合物，具有如下任意一项结构式：
Figure PCTCN2020097562-appb-000009
本发明提供一种含二乙胺的吖嗪联肼类化合物的制备方法，包括以下步骤：
将二苯基肼衍生物与含二乙胺的芳基溶解在溶剂中，然后加热反应，分离提纯，得到所述含二乙胺的吖嗪联肼类化合物。
优选地，所述分离提纯的方式为层析柱分离。
进一步地，所述二苯基肼衍生物的结构式为如下所示结构式中的一种：
Figure PCTCN2020097562-appb-000010
进一步地，所述含二乙胺的芳基的结构式如下所示：
Figure PCTCN2020097562-appb-000011
；所述二苯基肼衍生物与含二乙胺的芳基的摩尔比为1：1-1：10。
进一步地，所述溶剂为甲醇、乙醇、乙酸、四氢呋喃、甲苯、苯、氯仿、二氯甲烷、N,N-二甲基甲酰胺，N,N-二甲基乙酰胺和N-甲基吡咯烷酮中的一种以上。
进一步地，所述加热反应的温度为20-150℃，加热反应的时间为1-24h。
本发明提供的含二乙胺的吖嗪联肼类化合物在制备选择性抗菌的药物和真菌的选择性成像试剂的应用。
所述真菌的选择性成像，具体为：
在细菌培养基中加入所述的吖嗪联肼类化合物，在荧光显微镜或激光扫描共聚焦显微镜下观察，仅能在真菌上观测到荧光信号。
所述选择性抗菌，具体为：
将所述的吖嗪联肼类化合物与不同微生物共培养后，用经典的平板杀菌实验，研究其抗菌活性，加入药物后能杀死绝大部分革兰氏阳性菌，继续光照后能够杀死所有革兰氏阳性菌及真菌，而对革兰氏阴性菌的生长没有影响。
本发明的原理为：通过邻位羟基与肼中的N原子故对电子形成分子内氢键，通过激发态 下的分子内质子转移(ESIPT)，实现荧光光谱的有效红移；利用聚集态下分子内氢键和二苯基内旋转受限使该结构具有显著聚集诱导发光性质；通过多个羟基的引入使其分子内运动受限提高荧光量子产率；通过引入偏碱性的二乙胺能够增强分子与微生物的相互作用；将二乙胺与发光中心直接共轭相连，能够通过调节与微生物相互作用进而调控二乙胺的离子化形成强的电荷转移(CT)态，对荧光强弱调控，实现真菌的特异性识别；利用吖嗪类结构良好的吸光能力实现能量转移，敏化周围的氧气分子产生活性氧，实现微生物感染的光动力治疗。
与现有技术相比，本发明具有如下优点和有益效果：
(1)本发明提供的含二乙胺的吖嗪联肼类化合物，选择联肼结构作为共轭桥联基元，一方面利用单双键交替的形式保持探针分子的共轭程度，另一方面N原子的孤对电子能够进一步增加共轭程度，降低单线态跟三线态之间的能极差，提高敏化产生活性氧的能力；
(2)本发明提供的含二乙胺的吖嗪联肼类化合物，在联肼两端引入酚羟基结构，与N的孤对电子形成ESIPT态(激发态下分子内的质子转移)，有效的增加斯托克斯位移，防止分子的自吸收现象；二苯基的自由转动引入RIR(分子内受限旋转)机制，强化了此类分子的AIE性能并提高了分子的荧光量子产率；
(3)本发明提供的含二乙胺的吖嗪联肼类化合物，在芳香基团引入二乙胺，与革兰氏阳性菌和真菌有较强的结合能力；将二乙胺与发光中心共轭连接，使其对酸性环境及革兰氏阳性菌表面的酸性分子具有响应性，离子化后形成强CT态而猝灭荧光，帮助实现对真菌的选择性成像。
附图说明
图1为DPNAP在氘代氯仿中的氢谱图；
图2为DPNAP在H2O/THF(99:1,v/v)的溶液中的归一化的紫外吸收光谱和荧光发射光谱图；
图3为DPNAP在不同水和THF比例下的荧光发射光谱图；
图4为DPNAP在不同溶剂(正己烷、三乙胺、乙酸乙酯、四氢呋喃、乙醇、异丙醇、二甲亚砜)中的荧光发射光谱图；
图5为DPNAP分别在盐酸(HCl)、水及氢氧化钠(NaOH)溶液中的发射光谱图；
图6为DPNAP及光敏剂Ce6在光照条件下，活性氧探针DCFH的荧光强度变化图；
图7为DPNAP与不同微生物及其混合菌样相互作用后的共聚焦成像结果图；
图8为DPNAP与革兰氏阳性菌表面的磷壁酸(LTA)结合以后荧光强度变化图；
图9为DPNAP在黑暗及光照条件下对金黄色葡萄球菌的平板抗菌实验统计结果图；
图10为DPNAP在黑暗及光照条件下对白色念珠菌的平板抗菌实验统计结果图；
图11为DPNAP在黑暗及光照条件下对大肠杆菌的平板抗菌实验统计结果图；
图12(A)为金黄色葡萄球菌与DPNAP结合后黑暗及光照条件下的扫描电镜图；(B)为真菌与DPNAP结合后黑暗及光照条件下的扫描电镜图；(C)为大肠杆菌与DPNAP结合后黑暗及光照条件下的扫描电镜图；
图13为DPNAP对混合细菌样品的平板抗菌实验结果图(A)及统计结果图(B)。
图14为DPNAP对超级菌MRSA的平板抗菌实验统计结果图；
图15为小鼠感染模型，用不同方式处理以后的伤口愈合情况效果图；
图16为O-DPAS在氘代氯仿中的核磁数据图；
图17为O-DPAS在H2O/THF(99:1,v/v)的溶液中的归一化的紫外吸收光谱和荧光发射光谱图；
图18为O-DPAS在不同溶剂(正己烷、三乙胺、乙酸乙酯、四氢呋喃、乙醇、异丙醇、二甲亚砜)中的荧光发射光谱图；
图19为DO-DPAS与不同微生物及其混合菌样相互作用后的共聚焦成像结果图；
图20为DO-DPAS在氘代氯仿中的核磁数据图；
图21为DO-DPAS在H2O/THF(99:1,v/v)的溶液中的归一化的紫外吸收光谱和荧光发射光谱图；
图22为DO-DPAS在不同溶剂(正己烷、三乙胺、乙酸乙酯、四氢呋喃、乙醇、异丙醇、二甲亚砜)中的荧光发射光谱图。
具体实施方式
以下结合实例对本发明的具体实施作进一步说明，但本发明的实施和保护不限于此。需指出的是，以下若有未特别详细说明之过程，均是本领域技术人员可参照现有技术实现或理解的。所用试剂或仪器未注明生产厂商者，视为可以通过市售购买得到的常规产品。
实施例1
按照如下合成路线，具体合成一下化合物：
Figure PCTCN2020097562-appb-000012
(1)化合物2的合成
在回流条件下将化合物1(2mmol)和过量水合肼的混合物搅拌4小时。在反应完成后，旋蒸除去溶剂和剩下的水合肼，得到透明油状化合物2，产率为100％。
(2)化合物DPNAP的合成
化合物2(1mmol)和化合物3(1.5mmol)在回流条件下搅拌4小时。在反应完成后，用层析硅胶柱进行分离，得到黄色固态化合物DPNAP(含二乙胺的吖嗪联肼类化合物)，产率为81.2％。
图1为DPNAP的氢谱，证明了其结构的正确性。
图2为DPNAP在H 2O/THF(99:1,v/v)的溶液中的归一化的紫外吸收光谱和荧光发射光谱；图3为DPNAP在不同水和THF比例下的荧光发射光谱。[DPNAP]＝10μM；λ ex＝412nm。由图2中可以看出，无论是在溶液态下还是在聚集态下，最大发射峰位置都是在548nm左右，具有大的Stokes位移(136nm)的强发光，源于激发态分子内质子转移过程(ESIPT)过程，而且由于分子内氢键被保护且其自由运动受到抑制，因此它可以发射强的酮式发光。DPNAP随着水(不良溶剂)含量的不断增加，其荧光强度也不断增加，清楚地证实了其AIE性质。
图4为DPNAP在不同溶剂(正己烷、三乙胺、乙酸乙酯、四氢呋喃、乙醇、异丙醇、二甲亚砜)中的荧光发射光谱。[DPNAP]＝10μM；λ ex＝412nm。由图4中可以看出，DPNAP 的发射波长在不同极性的溶剂中变化不大，保持了优异的稳定性。
图5为DPNAP在不同pH环境下的荧光发射光谱。[DPNAP]＝10μM；λ ex＝412nm。由图5中可以看出，DPNAP在中性环境下发射强烈的荧光，在酸性及碱性条件下，荧光信号消失。说明酸性及碱性均会破坏其分子内氢键，导致荧光的猝灭。
图6为DPNAP产生活性氧的能力表征，其中商业化光敏剂Ce6作为参比。随着光照时间的延长，相比于空白组，混有DPNAP的活性氧探针二氯荧光素(DCFH)荧光强度逐渐增加，其增加幅度虽不及Ce6，但是整体增加明显，说明DPNAP具有较强的敏化产生活性氧的能力。
实施例2：实施例1中化合物用于识别和杀灭细菌
(1)细菌成像实验
a.菌种(大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、粪肠球菌、铜绿假单胞杆菌、白色念珠菌、酿酒酵母)接种在5mL的培养基中，在37℃培养12h。之后菌种以7100转离心1分钟，同时用磷酸盐缓冲液(PBS,10mM,pH＝7.4)冲洗三次，上清液弃去，留下的菌种悬浮在PBS中，然后在600nm下稀释到1.0个光密度(OD 600＝1.0)。针对混合菌样，则是将白色念珠菌、大肠杆菌及金黄色葡萄球菌混合在一起。
b.菌种在PBS缓冲溶液中与5μM浓度的DPNAP在37℃下共染20分钟，之后以7100转离心1分钟，得到的染色后菌种置于10μL PBS缓冲溶液中，并保存在冰柜中用于激光扫描共聚焦显微镜检测。
(2)抗菌实验
针对于单独菌液：
将金黄色葡萄球菌(OD 600＝0.4)稀释5倍后，分别与0.1、0.5、2、5、20μM的DPNAP避光相互作用10分钟，总体积为500μL，空白组为不含光敏剂分子的空白菌液，于白光(35mW·cm -2)下光照10分钟，对照组于黑暗条件下作用10分钟，之后稀释10 4倍至1mL，取100μL涂布于NB固体平板培养基上。将平板放置于37℃恒温培养箱培养16小时后，测定平板上菌落的数量。
用相同的条件处理白色念珠菌及大肠杆菌。
针对于混合菌样：
将金黄色葡萄球菌(OD 600＝1)、白色念珠菌(OD 600＝2)、大肠杆菌(OD 600＝1)分别取100μL、300μL、100μL，混匀后，与5μM的DPNAP避光相互作用10分钟，总体积为500μL，空白组为不含光敏剂分子的空白菌液，于白光(35mW·cm -2)下光照10分钟，对照组于黑暗条件下作用10分钟，之后稀释10 4倍至1mL，取100μL涂布于NB固体平板培养基上。将平板放置于37℃恒温培养箱培养16小时后，测定平板上菌落的数量。图7为DPNAP与不同微生物及其混合菌样相互作用后的共聚焦成像结果。由图中可以看出，DPNAP能选择性的成像真菌(白色念珠菌和酿酒酵母)，而在革兰氏阳性菌(金黄色葡萄球菌、粪肠球菌、枯草芽孢杆菌)及阴性菌(大肠杆菌、铜绿假单胞杆菌)表面检测不到荧光信号。即使在混合样品(白色念珠菌、大肠杆菌及金黄色葡萄球菌)中，也只与真菌白色念珠菌结合，实现了对真菌的选择性成像。
图8为DPNAP与革兰氏阳性菌表面特有的磷壁酸(LTA)结合后荧光强度变化。从图中可以看出，随着LTA浓度的增加，DPNAP的荧光不断降低，说明DPNAP与革兰氏阳性菌结合后其荧光会被细菌表面的LTA猝灭而不发光，最终实现与革兰氏阳性菌结合不发光。
图9为DPNAP对金黄色葡萄球菌的平板抗菌实验统计结果。从图9中可以看出，在黑暗条件下，5μM DPNAP对金黄色葡萄球菌的抑菌率就接近50％，并且随着浓度增加，抑菌效果不断增强。而在光照条件下，即使在0.1μM这样低的浓度下，其抑菌效果就接近100％，说明了DPNAP对于金黄色葡萄球菌优异的抗菌效果。
图10为DPNAP对白色念珠菌的平板抗菌实验统计结果。从图中可以看出，在黑暗条件下，DPNAP对白色念珠菌几乎没有抗菌效果。但是光照条件下，其抗菌效果明显，其IC50值低于0.5μM。说明DPNAP对于白色念珠菌具有良好的光动力治疗效果。
图11为DPNAP对大肠杆菌的平板抗菌实验统计结果。从图中可以看出，无论在黑暗条件或是光照条件下，DPNAP对于大肠杆菌均没有明显的抑制效果。说明DPNAP对于大肠杆菌基本没有抑菌活性。
图12为DPNAP与微生物结合后暗处及光照条件下细菌表面的扫描电镜成像。从图12的(A)(B)和(C)可以看出暗处及光照条件下三种微生物细胞壁结构完整，没有明显破坏，说明抗菌活性不是细胞壁结构的明显破坏导致。
表1为细胞外核酸浓度变化。从下表1可以看出，对于金黄色葡萄球菌来说，相比于空白组，金黄色葡萄球菌与DPNAP作用后，黑暗组及光照组的胞外核酸浓度有一定增加，结合扫描电镜结果，说明与DPNAP作用后其细胞壁结构有一定破坏致使核酸外泄，光照后产生的ROS会导致其细胞壁结构进一步遭到破坏，最终导致对金黄色葡萄球菌的有效杀伤；而对于白色念珠菌，其光照组及黑暗组的胞外核酸与空白组均一致。
表1
Figure PCTCN2020097562-appb-000013
核酸浓度变化结合扫描电镜结果，可以证明简单的结合不会破坏细胞壁的结构，光照以后产生的活性氧会导致生物大分子失活，最终实现对真菌的有效杀伤；而对于大肠杆菌来说，结合荧光成像结果，可以看出DPNAP不会与大肠杆菌结合，因为不表现出抗菌活性；由此可见，DPNAP与三种微生物结合的差异，最终实现了对三种微生物的选择性杀伤。
图13为DPNAP对混合细菌样品的平板抗菌实验效果图(A)及统计结果(B)。从图13的(A)和(B)部分中可以看出，加入DPNAP后，黑暗条件下，大部分的金黄色葡萄球菌基本被杀死。继续光照以后，所有的金黄色葡萄球菌及白色念珠菌均被抑制，只剩下大肠杆菌。实现了混合菌样中的选择性抗菌。
图14为DPNAP对超级菌MRSA的平板抗菌实验统计结果。从图14中可以看出，黑暗条件下，10μM浓度下其抑菌效果较普通金黄色葡萄球菌不明显，但是在光照条件下其对于超级菌的抗菌效果非常显著。
图15为DPNAP在活体抗菌中的应用效果图。从图15中可以看出，与仅用磷酸缓冲液(PBS)处理的感染小鼠相比，用DPNAP涂抹并光照以后，其愈合速度大幅度提高，且效果与头孢噻吩一致；而用DPNAP涂抹以后不采用光照，其愈合速度会减慢，说明DPNAP光照产生的活性氧能够有效抑制细菌的生长，提高伤口的愈合速度，有望用于伤口感染治疗。
实施例3
按照如下合成路线，具体合成一下化合物：
Figure PCTCN2020097562-appb-000014
(1)化合物5的合成
在回流条件下将化合物4(2mmol)和过量水合肼的混合物搅拌4小时。在反应完成后，旋蒸除去溶剂和剩下的水合肼，得到化合物5，产率为100％。
(2)化合物DO-DPAS的合成
化合物5(1mmol)和化合物3(1.5mmol)在回流条件下搅拌4小时。在反应完成后，用层析硅胶柱进行分离，得到黄色固态化合物DO-DPAS，产率为75％。
图16为DO-DPAS的氢谱图。
图17为DO-DPAS在H 2O/THF(99:1,v/v)的溶液中的归一化的紫外吸收光谱和荧光发射光谱。[DO-DPAS]＝10μM；λ ex＝401nm。由图17中可以看出，DO-DPAS最大发射峰位置都是在475nm左右。
图18为DO-DPAS在不同溶剂(正己烷、三乙胺、乙酸乙酯、四氢呋喃、乙醇、异丙醇、二甲亚砜)中的荧光发射光谱。[DO-DPAS]＝10μM；λ ex＝412nm。由图18中可以看出，DO-DPAS的发射波长在不同极性的溶剂中变化不大，保持了优异的稳定性。
实施例4：实施例3中化合物用于细菌成像
a.菌种(大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、粪肠球菌、铜绿假单胞杆菌、白色念珠菌、酿酒酵母)接种在5mL的培养基中，在37℃培养12h。之后菌种以7100转离心1分钟，同时用磷酸盐缓冲液(PBS,10mM,pH＝7.4)冲洗三次，上清液弃去，留下的菌种悬浮在PBS中，然后在600nm下稀释到1.0个光密度(OD 600＝1.0)。
b.菌种在PBS缓冲溶液中与5μM浓度的DO-DPAS在37℃下共染20分钟，之后以7100转离心1分钟，得到的染色后菌种置于10μL PBS缓冲溶液中，并保存在冰柜中用于激光扫描共聚焦显微镜检测。
图19为DO-DPAS与不同微生物相互作用后的共聚焦成像结果。由图19中可以看出，DO-DPAS因为荧光量子产率太低，成像效果很不理想，再微生物表面几乎看不到DO-DPAS的荧光信号。证明多个羟基引入会提高分子的荧光量子产率，有利于微生物成像。
实施例5
Figure PCTCN2020097562-appb-000015
(1)化合物O-DPAS的合成
化合物5(1mmol)和化合物6(1.5mmol)在回流条件下搅拌4小时。在反应完成后，用层析硅胶柱进行分离，得到化合物NBADB，产率为87％。
图20为O-DPAS氢谱图。
图21为O-DPAS在H 2O/THF(99:1,v/v)的溶液中的归一化的紫外吸收光谱和荧光发射光谱。[O-DPAS]＝10μM；λ ex＝384nm。由图21中可以看出，O-DPAS最大发射峰位置都是在442nm左右。
图22为O-DPAS在不同溶剂(正己烷、三乙胺、乙酸乙酯、四氢呋喃、乙醇、异丙醇、二甲亚砜)中的荧光发射光谱。[O-DPAS]＝10μM；λ ex＝412nm。由图22中可以看出，O-DPAS的发射波长在不同极性的溶剂中变化不大，保持了优异的稳定性。
以上实施例仅为本发明较优的实施方式，仅用于解释本发明，而非限制本发明，本领域技术人员在未脱离本发明精神实质下所作的改变、替换、修饰等均应属于本发明的保护范围。

Claims (10)

  1. 一种含二乙胺的吖嗪联肼类化合物，其特征在于，结构通式如下所示：
    Figure PCTCN2020097562-appb-100001
    其中，Ar表示芳香基团或其衍生结构，取代基R1-R10分别选自氢、烷基、羟基、烷氧基、硝基、氰基、氨基、巯基、卤素原子、二乙胺、苯基、甲苯基、萘基、呋喃基、噻吩基、吡咯基、吡啶基、吡喃基、喹啉基、吲哚基、羧基或其衍生基团、咔唑基及苯胺基中的一种。
  2. 根据权利要求1所述的含二乙胺的吖嗪联肼类化合物，其特征在于，结构式为
    Figure PCTCN2020097562-appb-100002
    其中Ar表示芳香基团或其衍生结构，取代基R1、R2分别选自氢、烷基、羟基、烷氧基、硝基、氰基、氨基、巯基、卤素原子、二乙胺、苯基、甲苯基、萘基、呋喃基、噻吩基、吡咯基、吡啶基、吡喃基、喹啉基、吲哚基、羧基或其衍生基团、咔唑基或苯胺基中的一种。
  3. 根据权利要求1所述的含二乙胺的吖嗪联肼类化合物，其特征在于，结构式为
    Figure PCTCN2020097562-appb-100003
    其中，A1-A10分别选自氢、烷基、羟基、烷氧基、硝基、氰基、氨基、巯基、卤素原子、 二乙胺、苯基、甲苯基、萘基、呋喃基、噻吩基、吡咯基、吡啶基、吡喃基、喹啉基、吲哚基、羧基或其衍生基团、咔唑基或苯胺基中的一种。
  4. 根据权利要求3所述的含二乙胺的吖嗪联肼类化合物，其特征在于，具有如下任意一项结构式：
    Figure PCTCN2020097562-appb-100004
  5. 一种权利要求1-4任一项所述含二乙胺的吖嗪联肼类化合物的制备方法，其特征在于，包括以下步骤：
    将二苯基肼衍生物与含二乙胺的芳基溶解在溶剂中，然后加热反应，分离提纯，得到所述含二乙胺的吖嗪联肼类化合物。
  6. 根据权利要求5所述的含二乙胺的吖嗪联肼类化合物的制备方法，其特征在于，所述二苯基肼衍生物的结构式为如下所示结构式的一种：
    Figure PCTCN2020097562-appb-100005
  7. 根据权利要求5所述的含二乙胺的吖嗪联肼类化合物的制备方法，其特征在于，所述含二乙胺的芳基的结构式如下所示：
    Figure PCTCN2020097562-appb-100006
    所述二苯基肼衍生物与含二乙胺的芳基的摩尔比为1：1-1：10。
  8. 根据权利要求5所述的含二乙胺的吖嗪联肼类化合物的制备方法，其特征在于，所述溶剂为甲醇、乙醇、乙酸、四氢呋喃、甲苯、苯、氯仿、二氯甲烷、N,N-二甲基甲酰胺，N,N-二甲基乙酰胺和N-甲基吡咯烷酮中的一种以上。
  9. 根据权利要求5所述的含二乙胺的吖嗪联肼类化合物的制备方法，其特征在于，所述加热反应的温度为20-150℃，加热反应的时间为1-24h。
  10. 权利要求1-4任一项所述的含二乙胺的吖嗪联肼类化合物在制备选择性抗菌的药物和真菌的选择性成像试剂的应用。
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