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WO2024239539A1 - 一种两性离子荧光化合物及其制备方法和应用 - Google Patents

一种两性离子荧光化合物及其制备方法和应用 Download PDF

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WO2024239539A1
WO2024239539A1 PCT/CN2023/127719 CN2023127719W WO2024239539A1 WO 2024239539 A1 WO2024239539 A1 WO 2024239539A1 CN 2023127719 W CN2023127719 W CN 2023127719W WO 2024239539 A1 WO2024239539 A1 WO 2024239539A1
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compound
formula
represented
reaction
zwitterionic
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黄佳国
周娅
刘彪翔
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Sun Yat Sen University
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    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • A—HUMAN NECESSITIES
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    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
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    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
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    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
    • 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
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    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to the field of biomedical technology, and more specifically, to a zwitterionic fluorescent compound and a preparation method and application thereof.
  • the urinary system is composed of kidneys, ureters, bladder, and urethra, and is an important excretion pathway for human metabolic products. During the excretion process, abnormal metabolic waste, foreign microorganisms, drugs, etc. in the body can easily cause infection and damage to surrounding tissues and cells, leading to a series of diseases.
  • the current clinical diagnosis of urinary system diseases often relies on traditional imaging methods. However, these methods have defects such as high ionizing radiation, low sensitivity, invasiveness, and high cost, making it difficult to diagnose early and implement intervention.
  • optical imaging/detection technology Compared with traditional imaging technology, optical imaging/detection technology has the advantages of high sensitivity, strong specificity, high safety, and convenience and popularity. Therefore, the use of non-invasive, non-ionizing radiation, highly specific and sensitive fluorescence imaging technology to detect urinary system diseases has excellent application prospects.
  • the probes used for kidney disease detection mainly include inorganic nanoprobes and organic molecular probes.
  • the metabolism of inorganic nanoprobes is limited by the pore size of the glomerular basement membrane, and has strict selectivity for the size of nanoprobes. Only inorganic nanoparticles with a hydrated diameter of less than 6nm and a low protein binding rate can be effectively excreted through the kidneys.
  • inorganic nanomaterials have a slow metabolism rate and can be captured and accumulated by the endothelial reticular system of the liver when circulating in the body, resulting in long-term, potential biological toxicity.
  • organic molecular probes have the advantages of fast metabolism, high biocompatibility, and modifiable structure. They have a wide range of applications in the biomedical field, including cell imaging, tumor diagnosis and treatment, and clinical intraoperative navigation.
  • the use of organic molecules to construct renal clearance fluorescent probes is mainly achieved by coupling hydrophilic macromolecules (such as polyethylene glycol, cyclodextrin, polysaccharide compounds, etc.) to achieve good water solubility and renal metabolism effects.
  • hydrophilic macromolecules such as polyethylene glycol, cyclodextrin, polysaccharide compounds, etc.
  • the patent with publication number CN115947946A provides a hydrophilic organic macromolecule renal clearance type fluorescent probe.
  • hydrophilic organic macromolecule labeled molecular probes are easily degraded in the body; in addition, fluorescent coupling labeled hydrophilic macromolecules are usually polymers, so there are often problems such as unstable synthesis process and large differences between different batches, which affect subsequent biological applications.
  • the primary purpose of the present invention is to overcome the problems of the existing organic macromolecular renal clearance fluorescent probe being easily degraded in vivo and the unstable product batches, and to provide a zwitterionic fluorescent compound.
  • a further object of the present invention is to provide a method for preparing the above zwitterionic fluorescent compound.
  • a further object of the present invention is to provide the use of the above zwitterionic fluorescent compound or its pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, tautomer in the preparation of fluorescent probes.
  • a zwitterionic fluorescent compound having a structure shown in any one of formulas (I) to (V):
  • R1 is R2 is -OH, -NH2 , A is a self-eliminating group, B is a biomarker response group; n is an integer of 0 to 50, n1 is an integer of 0 to 20, and n2 is an integer of 0 to 20.
  • the present invention uses a specific main structure as a fluorophore (chromophore), connects a specific R2 group (-OH, -NH2 or containing a biomarker response group) to a specific substitution position at one end of the main structure, and connects a sulfonic acid group and a fragment containing a quaternary ammonium to a specific substitution position at the other end of the main structure, respectively, to obtain a zwitterionic fluorescent compound.
  • chromophore chromophore
  • the fluorophore has a good quantum yield
  • the biomarker response group can be activated by the corresponding biomarker, thereby causing changes in the molecular structure and optical properties, generating a stronger fluorescent signal, and can be monitored in real time using fluorescent imaging technology
  • the zwitterionic fluorescent compound not only has high sensitivity, good biocompatibility and optical stability, but also has good water solubility and renal clearance efficiency through coordination with the substitution group or fragment.
  • the zwitterionic fluorescent compounds provided by the present invention can be used as fluorescent probes and prepared into preparations for various modes of administration (intravenous injection, intraperitoneal injection or spraying). Taking the intravenous injection mode as an example, after the zwitterionic fluorescent compounds of the present invention are intravenously injected, since the expression content of biomarkers in healthy tissues is very low, the biomarker response group of the fluorescent probe is not activated, and no change in the probe electron cloud density occurs, so no obvious fluorescent optical signal is detected in healthy tissues.
  • biomarkers When the urinary system is lesioned, biomarkers are expressed in large quantities, and the zwitterionic fluorescent compounds metabolized by the kidneys are activated, releasing strong fluorescent signals, which can be monitored in real time by fluorescent imaging, and dynamic monitoring of lesions in the urinary system and visual identification of lesion areas can be achieved, providing more accurate guidance for precision surgical treatment, so as to improve surgical efficacy and patient prognosis, and is expected to provide a new auxiliary method for human disease diagnosis and treatment.
  • the self-eliminating group A is N-(2-aminoeliminating group A
  • the self-eliminating group A is When , the left connection site is connected to the biomarker response group B; the self-eliminating group A is When the left connection site is connected to B; the self-eliminating group A is When , the right joining site is connected to B.
  • the biomarker response group B can be selected according to different target biomarkers.
  • the present invention provides a series of different biomarker response groups.
  • the biomarker response group B is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-amino
  • n is an integer of 1-20.
  • n1 is an integer of 1-10.
  • n2 is an integer from 1 to 10.
  • the preparation method of the above zwitterionic fluorescent compound comprises the following steps:
  • the condensation reaction in step S1 is carried out at a temperature of 45 to 100° C. and for a time of 5 to 12 hours.
  • the solvent used for the condensation reaction in step S1 is anhydrous ethanol, anhydrous methanol, N,N-dimethylformamide Or one or more of acetic anhydride
  • the selected activating agent is one or more of potassium carbonate, cesium carbonate, sodium acetate or potassium acetate.
  • the temperature of the demethylation protection reaction in step S2 is 0-30° C., and the time is 5-12 h; the temperature of the reduction reaction in step S2 is 45-100° C., and the time is 5-12 h.
  • the solvent used in the demethylation protection reaction in step S2 is one or more of ethanol, dichloromethane, methanol or N,N-dimethylformamide
  • the activating agent used is one or more of boron tribromide, aluminum chloride, sodium ethanethiolate or trimethylsilane iodide
  • the solvent used in the reduction reaction in step S2 is one or more of ethanol, methanol, N,N-dimethylformamide or water
  • the reducing agent used is one or more of palladium carbon, hydrogen, stannous chloride or iron powder.
  • the temperature of the substitution reaction in step S3 is 45-100° C., and the time is 5-12 h; the temperature of the condensation reaction in step S3 is 0-45° C., and the time is 8-24 h.
  • the solvent selected for the substitution reaction in step S3 is one or more of anhydrous methanol, anhydrous N,N-dimethylformamide, anhydrous acetonitrile or anhydrous tetrahydrofuran;
  • the solvent selected for the condensation reaction in step S3 is one or more of anhydrous methanol, anhydrous N,N-dimethylformamide, anhydrous dichloromethane or anhydrous acetonitrile
  • the condensation reagent selected is one or more of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), dicyclohexylcarbodiimide (DCC) or benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBop
  • the compound 2 in step S1 is prepared by the following steps:
  • the substitution reaction in step S11 is carried out at 80-150° C. for 24-72 hours, and the solvent is one or more of N,N-dimethylformamide, toluene or o-dichlorobenzene.
  • the compound 3 in step S1 is prepared by the following steps:
  • the substitution reaction temperature in step S12 is 80-150° C.
  • the time is 24-72 h
  • the solvent is one or more of N,N-dimethylformamide, toluene or o-dichlorobenzene;
  • the compound 7 in step S1 is prepared by the following steps:
  • the bromination reaction temperature in step S13 is 0 to 30°C, the time is 4 to 12 hours, and the solvent is N,N-dimethyl One or more of formamide, dichloromethane, tetrahydrofuran or acetonitrile;
  • the cyclization reaction temperature in step S14 is 0-30° C.
  • the time is 4-12 h
  • the solvent is one or more of N,N-dimethylformamide, dichloromethane, tetrahydrofuran or acetonitrile
  • the activator is one or more of potassium carbonate, cesium carbonate, sodium acetate or potassium acetate.
  • the fluorescent probe is a renal-cleared fluorescent probe.
  • the fluorescent probe is a constantly-on fluorescent probe or an excitation fluorescent probe.
  • the pharmaceutically acceptable salt is hydrochloride, hydrobromide, nitrate, methylnitrate, sulfate, bisulfate, aminosulfate, phosphate, acetate, glycolate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, para-aminosalicylate, glycolate, lactate, enanthate, phthalate, oxalate, succinate, benzoate, o-acetyl At least one of oxybenzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, mandelate, tannate, formate, stearate, ascorbate, palmitate, oleate, pyruvate, bishydroxynaphthoate, malonate, laurate, glutarate
  • the present invention has the following beneficial effects:
  • the zwitterionic fluorescent compound provided by the present invention uses a derivative of a hemicyanine dye structure as a fluorophore, modifies a sulfonic acid group on the indole ring, and respectively connects an R2 group (-OH, -NH2 or a biomarker response group) and a quaternary ammonium-containing fragment at both ends. It not only has high sensitivity, good biocompatibility and optical stability, but also has good water solubility and renal clearance efficiency through coordination with the substituted group or fragment. As a fluorescent probe, it can more accurately diagnose diseases at an early stage, navigate intraoperatively, evaluate tissue and organ functions, etc., and can play an important role in future medical optical examinations, with excellent application prospects.
  • FIG1 is a schematic diagram of a method for preparing the zwitterionic fluorescent compound ZWCYOH1 of Example 1;
  • FIG2 is a schematic diagram of a method for preparing the zwitterionic fluorescent compound ZWCYOH2 of Example 2;
  • FIG3 is a schematic diagram of a method for preparing the zwitterionic fluorescent compound ZWCYOH3 of Example 3;
  • FIG4 is a schematic diagram of a method for preparing the zwitterionic fluorescent compound ZWCYNH 2 of Example 4;
  • FIG5 is a schematic diagram of a method for preparing the zwitterionic fluorescent compound ZWCYNH 2 P of Example 5;
  • FIG6 is a diagram of the ultraviolet absorption spectra of the zwitterionic fluorescent compounds ZWCYOH1, ZWCYOH2, ZWCYOH3, and ZWCYNH 2 of Examples 1 to 4;
  • FIG7 is a fluorescence spectra of zwitterionic fluorescent compounds ZWCYOH1, ZWCYOH2, ZWCYOH3, and ZWCYNH 2 of Examples 1 to 4;
  • FIG8 shows the urine recovery rate within 24 hours after injection of zwitterionic fluorescent compounds ZWCYOH1, ZWCYOH2, ZWCYOH3, and ZWCYNH2 of Examples 1 to 4 ;
  • FIG9 is a graph showing the fluorescence spectrum change of the zwitterionic fluorescent compound ZWCYNH 2 P in Example 5 in response to in vitro protease;
  • FIG. 10 is an in vivo imaging diagram of the zwitterionic fluorescent compound ZWCYNH 2 P of Example 5 in an acute kidney injury mouse model;
  • FIG. 11 is an in vitro organ imaging diagram of the zwitterionic fluorescent compound ZWCYNH 2 P of Example 5 in an acute renal injury mouse model.
  • This embodiment provides a zwitterionic fluorescent compound, and a schematic diagram of a preparation method thereof is shown in FIG1 , which specifically comprises the following steps:
  • This embodiment provides a zwitterionic fluorescent compound, and a schematic diagram of a preparation method thereof is shown in FIG2 , which specifically includes the following steps:
  • This embodiment provides a zwitterionic fluorescent compound, and a schematic diagram of a preparation method thereof is shown in FIG3 , which specifically includes the following steps:
  • This embodiment provides a zwitterionic fluorescent compound, and a schematic diagram of a preparation method thereof is shown in FIG4 , which specifically comprises the following steps:
  • This embodiment provides a zwitterionic fluorescent compound, which is obtained by attaching a biomarker response group to ZWCYNH2 in Example 4.
  • the schematic diagram of the preparation method is shown in FIG5 , and specifically comprises the following steps:
  • Spectral test Prepare PBS solution and methanol solution of 0.05 mg/mL constant-bright fluorescent probes ZWCYOH1, ZWCYOH2, ZWCYOH3, and ZWCYNH2 .
  • Use ultraviolet spectrometer to measure the absorption spectrum of each sample at 400-900 nm, and use fluorescence spectrometer to measure the fluorescence spectrum of each sample (excitation wavelength: 660 nm).
  • the test results are shown in Figures 6 and 7.
  • the fluorophores of the probes are all hemicyanine structures, so there is no obvious difference in the maximum absorption and maximum emission wavelengths of the spectrum.
  • Renal clearance efficiency test Prepare 0.05 mg/mL PBS solution of the ever-bright fluorescent probes ZWCYOH1, ZWCYOH2, ZWCYOH3, and ZWCYNH 2. Take 12 Balb/c mice and randomly divide them into 3 groups, 3 mice in each group, and inject 75 ⁇ L of the probe solution into the tail vein of the mice. After injection, place the mice individually in a clean metabolic cage, collect urine within 24 hours and record the volume. Use high performance liquid chromatography to prepare a standard curve of probe concentration-chromatographic peak area, and calculate the recovery rate of the probe in mouse urine.
  • ZWCYOH1, ZWCYOH2, ZWCYOH3, and ZWCYNH2 can all be metabolized through the urinary system and exist in the excreted urine. Among them, the charge distribution of the constant-brightness probes ZWCYOH2, ZWCYOH3, and ZWCYNH2 is more balanced, and the urine recovery rates of the three are higher, which are 88%, 91%, and 93%, respectively. This proves that the zwitterionic fluorescent probe provided by the present invention has good renal clearance efficiency and can realize the detection of urinary system diseases.
  • test of the responsiveness of the activation probe A 10 ⁇ M activation probe ZWCYNH 2 P solution was incubated with the apoptotic protease caspase-8 in a buffer solution at 37°C. The fluorescence spectrum of the solution was measured. The test results are shown in FIG9 . The polypeptide sequence of the probe was recognized and cleaved by the caspase-8 protease, the fluorescence emission wavelength of the solution was red-shifted, and the fluorescence intensity increased.
  • mice were randomly divided into 6 groups, namely blank control group (control group), NAC control group, model group (12h group, 24h group, 48h group, 72h group), three mice in each group.
  • Control group intraperitoneal injection of 0.4mL normal saline
  • AKI (acute kidney injury) model group intraperitoneal injection of 20mg/kg cisplatin solution, and imaging examination was performed at 12h, 24h, 48h, and 72h after injection
  • NAC group tail vein injection of 400mg/kg NAC solution, 30min later, intraperitoneal injection of 20mg/kg cisplatin solution, and imaging examination was performed at 48h after cisplatin injection. Finally, the mice were euthanized and imaging of each organ was performed.
  • the test results are shown in Figure 11.
  • the signal of the liver is weaker than that of the kidney, proving that the probe is mainly metabolized by the kidney rather than the liver.
  • the kidney signal of each group increases with the extension of cisplatin injection time, that is, the expression of capase-8 protein gradually increases, which can achieve early diagnosis of the disease.

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Abstract

本发明涉及一种两性离子荧光化合物及其制备方法和应用。该两性离子荧光化合物以半花菁染料结构的衍生物作为荧光团，以及在两端分别连接R2基团(-OH、-NH2或含有生物标志物响应基团)和含有季铵的片段，不仅具有较高的灵敏度、较好的生物相容性和光学稳定性，而且通过和取代基团或片段的配合而具有良好的水溶性与肾清除效率，作为荧光探针，可更为精准地对疾病进行早期诊断，术中导航治疗，组织器官功能评价等。

Description

一种两性离子荧光化合物及其制备方法和应用 技术领域
本发明涉及生物医学技术领域，更具体地，涉及一种两性离子荧光化合物及其制备方法和应用。
背景技术
泌尿系统由肾脏、输尿管、膀胱、尿道组成，是人体代谢产物的重要排泄途径。在排泄过程中，异常的体内代谢废物、外来微生物、药物等易引起周围组织、细胞的感染和损伤，进而导致一系列疾病。当前泌尿系统疾病的临床诊断常依赖于传统影像学手段，然而这些方法存在高电离辐射、灵敏度低、有创及成本高等缺陷，难以早期诊断并实施干预。与传统影像学技术相比，光学成像/检测技术具有高灵敏度、特异性强、安全性高以及便捷易普及等优势。因此，利用非侵入性、无电离辐射、高特异性和灵敏度的荧光成像技术检测泌尿系统疾病具有极好的应用前景。
目前用于肾脏疾病检测的探针主要包括无机纳米探针与有机分子探针。无机纳米探针的代谢受肾小球基底膜孔径的限制，对纳米探针大小具有严格的选择性，仅水合直径小于6nm且具有低蛋白结合率的无机纳米颗粒才能够有效的通过肾脏排出。其次，无机纳米材料的代谢速度较慢，在体内循环时可被肝脏的内皮网状系统捕获蓄积，导致长期的、潜在的生物毒性。此外，无机纳米探针的制备规模受限、价格高昂、合成生产重现性与定量存在困难。相比之下，有机分子探针具有代谢速度快、生物相容性高、结构可修饰等优势，在生物医学领域具有广泛的应用包括细胞成像，肿瘤诊断治疗及临床术中导航等。利用有机分子构建肾清除型的荧光探针主要通过偶联亲水性大分子(如聚乙二醇、环糊精、多糖化合物等)来实现良好的水溶性和肾脏代谢效果。比如，公开号为CN115947946A的专利就提供了一种亲水性的有机大分子肾清除型的荧光探针。然而由于体内存在大量复杂的糖蛋白酶，导致此类亲水性有机大分子标记的分子探针容易在体内发生降解；此外，荧光偶联标记亲水性大分子通常属于聚合物，故常出现合成工艺不稳定，不同批次间存在较大差异等问题，影响后续生物应用。
因此，开发单分子、无标记的肾清除型的荧光探针对于泌尿系统疾病的诊疗具有重要意义。
发明内容
本发明的首要目的是克服上述现有现有的有机大分子肾清除型的荧光探针在体内易降解，且产品批次不稳定的问题，提供一种两性离子荧光化合物。
本发明的进一步目的是提供上述两性离子荧光化合物的制备方法。
本发明的进一步目的是提供上述两性离子荧光化合物或其药学上可接受的盐、溶剂化物、对映异构体、非对映异构体、互变异构体在制备荧光探针中的应用。
本发明的上述目的通过以下技术方案实现：
一种两性离子荧光化合物，具有式(Ⅰ)～式(Ⅴ)任一所示的结构：

其中，R1为R2为-OH、-NH2、 A为自消除基团，B为生物标志物响应基团；n为0～50的整数，n1为0～20的整数，n2为0～20的整数。
本发明以特定的主体结构作为荧光团(发色团)，在主体结构的一端的特定取代位上连接特定的R2基团(-OH、-NH2或含有生物标志物响应基团)，在主体结构的另一端的特定取代位上分别连接磺酸基和含有季铵的片段，获得两性离子荧光化合物。其中，荧光团具有较好的量子产率，生物标志物响应基团可被对应的生物标志物激活，进而引起分子结构与光学性能的改变，产生更强的荧光信号，并可利用荧光成像技术实时监测；该两性离子荧光化合物不仅具有较高的灵敏度、较好的生物相容性和光学稳定性，而且通过和取代基团或片段的配合而具有良好的水溶性与肾清除效率。
需要说明的是，式(Ⅰ)～式(Ⅴ)中都属于吲哚类化合物结构，在本发明中可以等同替换；比多一个苯环，共轭效应增强。两者都具有共轭效应，随着苯基个数增加，紫外吸收波长逐渐增长，也都可以发射出荧光，并且荧光波长也逐渐增加。
本发明提供的两性离子荧光化合物可作为荧光探针，制备成多种给药方式(静脉注射、腹腔注射或者喷洒)的制剂。以静脉注射方式为例，本发明的两性离子荧光化合物经静脉注射后，由于健康组织生物标志物表达含量非常低，荧光探针的生物标志物响应基团未被激活，不发生探针电子云密度的改变，因此在健康组织中不会检测到明显的荧光光学信号。当泌尿系统发生病变，生物标志物大量表达，经肾脏代谢的两性离子荧光化合物被激活，释放较强的荧光信号，可通过荧光成像实时监测，实现泌尿系统病变部位的动态监测以及病变区域的可视化识别，为手术的精准化治疗提供更精确的指导，以提高手术疗效与患者预后，有望为人类的疾病诊断和治疗提供一种新的辅助方法。
优选地，所述自消除基团A为
应当理解的是，自消除基团A为时，左侧连接位点与生物标志物响应基团B连接；自消除基团A为时，左侧连接位点与B连接；自消除基团A为时，右侧连接位点与B连接。
生物标志物响应基团B可根据目标生物标志物的不同进行选择，本发明在此提供一系列不同的生物标志物响应基团。
优选地，所述生物标志物响应基团B为
经研究，上述生物标志物响应基团与生物标志物之间特异性激发的关系如下：


优选地，所述n为1～20的整数。
优选地，n1为1～10的整数。
优选地，n2为1～10的整数。
上述两性离子荧光化合物的制备方法，包括如下步骤：
S1.式(1)所示的化合物1和式(6)所示的化合物6发生缩合反应，得到式(8)所示的化合物8；
或式(1)所示的化合物1和式(7)所示的化合物7发生缩合反应，得到式(9)所示的化合物9；
或式(2)所示的化合物2和式(6)所示的化合物6发生缩合反应，得到式(12)所示的化合物12；
或式(2)所示的化合物2和式(7)所示的化合物7发生缩合反应，得到式(13)所示的化合物13；
或式(3)所示的化合物3和式(6)所示的化合物6发生缩合反应，得到式(16)所示的化合物16；
或式(3)所示的化合物3和式(7)所示的化合物7发生缩合反应，得到式(17)所示的化合物17；
或式(4)所示的化合物4和式(6)所示的化合物6发生缩合反应，得到式(20)所示的化合物20；
或式(4)所示的化合物4和式(7)所示的化合物7发生缩合反应，得到式(21)所示的化合物21；
或式(5)所示的化合物5和式(6)所示的化合物6发生缩合反应，得到式(24)所示的化合物24；
或式(5)所示的化合物5和式(7)所示的化合物7发生缩合反应，得到式(25)所示的化合物25；
S2.化合物8发生脱甲基保护反应，得式(10)所示的化合物10；
或化合物12发生脱甲基保护反应，得式(14)所示的化合物14；
或化合物16发生脱甲基保护反应，得式(18)所示的化合物18；
或化合物20发生脱甲基保护反应，得式(22)所示的化合物22；
或化合物24发生脱甲基保护反应，得式(26)所示的化合物26；
或化合物9经还原反应，得式(11)所示的化合物11；
或化合物13经还原反应，得式(15)所示的化合物15；
或化合物17经还原反应，得式(19)所示的化合物19；
或化合物21经还原反应，得式(23)所示的化合物23；
或化合物25经还原反应，得式(27)所示的化合物27；
S3.化合物10与R2‐H发生取代反应，即得如式(Ⅰ)所示两性离子荧光化合物；
或化合物14与R2‐H发生取代反应，即得如式(Ⅱ)所示两性离子荧光化合物；
或化合物18与R2‐H发生取代反应，即得如式(Ⅲ)所示两性离子荧光化合物；
或化合物22与R2‐H发生取代反应，即得如式(Ⅳ)所示两性离子荧光化合物；
或化合物26与R2‐H发生取代反应，即得如式(Ⅴ)所示两性离子荧光化合物；
或化合物11与R2‐H发生缩合反应，即得如式(Ⅰ)所示两性离子荧光化合物；
或化合物15与R2‐H发生缩合反应，即得如式(Ⅱ)所示两性离子荧光化合物；
或化合物19与R2‐H发生缩合反应，即得如式(Ⅲ)所示两性离子荧光化合物
或化合物23与R2‐H发生缩合反应，即得如式(Ⅳ)所示两性离子荧光化合物；
或化合物27与R2‐H发生缩合反应，即得如式(Ⅴ)所示两性离子荧光化合物；
优选地，步骤S1中所述缩合反应的温度为45～100℃，时间为5～12h。
优选地，步骤S1中所述缩合反应选用的溶剂为无水乙醇、无水甲醇、N,N-二甲基甲酰胺 或乙酸酐中的一种或几种，选用的活化剂为碳酸钾、碳酸铯、醋酸钠或醋酸钾中的一种或几种。
优选地，步骤S2中所述脱甲基保护反应的温度为0～30℃，时间为5～12h；步骤S2中所述还原反应的温度为45～100℃，时间为5～12h。
优选地，步骤S2中所述脱甲基保护反应选用的溶剂为乙醇、二氯甲烷、甲醇或N,N-二甲基甲酰胺中的一种或几种，选用的活化剂为三溴化硼、氯化铝、乙硫醇钠或三甲基碘硅烷中的一种或几种；步骤S2中所述还原反应选用的溶剂为乙醇、甲醇、N,N-二甲基甲酰胺或水中的一种或几种，选用的还原剂为钯碳、氢气、氯化亚锡或铁粉中的一种或几种。
优选地，步骤S3中所述取代反应的温度为45～100℃，时间为5～12h；步骤S3中所述缩合反应的温度为0～45℃，时间为8～24h。
优选地，步骤S3中所述取代反应选用的溶剂为无水甲醇、无水N,N-二甲基甲酰胺、无水乙腈或无水四氢呋喃中的一种或几种；步骤S3中所述缩合反应选用的溶剂为无水甲醇、无水N,N-二甲基甲酰胺、无水二氯甲烷或无水乙腈中的一种或几种，选用的缩合试剂为2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(HATU)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI)、二环己基碳二亚胺(DCC)或六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBop)中的一种或几种，选用的活化剂为碳酸钾、N,N-二异丙基乙胺、三乙胺、吡啶或甲氧苄啶中的一种或几种。
优选地，步骤S1中所述化合物2通过如下步骤制备得到：
S11.式(28)所示的化合物28与式(29)所示的化合物29发生取代反应得到化合物2；
更为优选地，步骤S11所述的取代反应为80～150℃，时间为24～72h，溶剂为N,N-二甲基甲酰胺、甲苯或邻二氯苯中的一种或几种。
优选地，步骤S1中所述化合物3通过如下步骤制备得到：
S12.式(30)所示的化合物30与式(31)所示的化合物31发生取代反应得到化合物3；
更为优选地，步骤S12所述的取代反应温度为80～150℃，时间为24～72h，溶剂为N,N-二甲基甲酰胺、甲苯或邻二氯苯中的一种或几种；
优选地，步骤S1中所述化合物7通过如下步骤制备得到：
S13.式(32)所示的化合物32发生溴代反应得式(33)所示的化合物33；
S14.化合物33与式(34)所示的化合物34发生成环反应得到化合物7；
更为优选地，步骤S13所述的溴代反应温度为0～30℃，时间为4～12h，溶剂为N,N-二甲基 甲酰胺、二氯甲烷、四氢呋喃或乙腈中的一种或几种；
更为优选地，步骤S14所述的成环反应温度0～30℃，时间为4～12h，溶剂为N,N-二甲基甲酰胺、二氯甲烷、四氢呋喃或乙腈中的一种或几种，活化剂为碳酸钾、碳酸铯、醋酸钠或醋酸钾中的一种或几种。
上述两性离子荧光化合物或其药学上可接受的盐、溶剂化物、对映异构体、非对映异构体、互变异构体在制备荧光探针中的应用也在本发明的保护范围内。
优选地，所述荧光探针为肾清除型的荧光探针。
优选地，所述荧光探针为常亮型荧光探针或激发型荧光探针。
优选地，所述药学上可接受的盐为盐酸盐、氢溴酸盐、硝酸盐、甲基硝酸盐、硫酸盐、硫酸氢盐、氨基硫酸盐、磷酸盐、乙酸盐、羟基乙酸盐、苯基乙酸盐、丙酸盐、丁酸盐、异丁酸盐、戊酸盐、马来酸盐、羟基马来酸盐、丙烯酸盐、延胡索酸盐、苹果酸盐、酒石酸盐、柠檬酸盐、水杨酸盐、对氨基水杨酸盐、乙醇酸盐、乳酸盐、庚酸盐、邻苯二甲酸盐、草酸盐、琥珀酸盐、苯甲酸盐、邻乙酰氧基苯甲酸盐、氯苯甲酸盐、甲基苯甲酸盐、二硝基苯甲酸盐、羟苯酸盐、甲氧基苯甲酸盐、扁桃酸盐、丹宁酸盐、甲酸盐、硬脂酸盐、抗坏血酸盐、棕榈酸盐、油酸盐、丙酮酸盐、双羟奈酸盐、丙二酸盐、月桂酸盐、戊二酸盐、谷氨酸盐、丙酸酯月桂硫酸盐、甲磺酸盐、乙磺酸盐、2-羟基乙磺酸盐、苯磺酸盐、对氨基苯磺酸盐、对甲苯磺酸盐(甲苯磺酸盐)或萘-2-磺酸盐中的至少一种。
与现有技术相比，本发明的有益效果是：
本发明提供的两性离子荧光化合物以半花菁染料结构的衍生物作为荧光团，在吲哚环上修饰磺酸基，以及在两端分别连接R2基团(-OH、-NH2或含有生物标志物响应基团)和含有季铵的片段，不仅具有较高的灵敏度、较好的生物相容性和光学稳定性，而且通过和取代基团或片段的配合而具有良好的水溶性与肾清除效率，作为荧光探针，可更为精准地对疾病进行早期诊断，术中导航治疗，组织器官功能评价等，可在未来的医学光学检查中发挥重要的作用，具有极好的应用前景。
附图说明
图1为实施例1的两性离子荧光化合物ZWCYOH1的制备方法的示意图；
图2为实施例2的两性离子荧光化合物ZWCYOH2的制备方法的示意图；
图3为实施例3的两性离子荧光化合物ZWCYOH3的制备方法的示意图；
图4为实施例4的两性离子荧光化合物ZWCYNH2的制备方法的示意图；
图5为实施例5的两性离子荧光化合物ZWCYNH2P的制备方法的示意图；
图6为实施例1～4的两性离子荧光化合物ZWCYOH1、ZWCYOH2、ZWCYOH3、ZWCYNH2的紫外吸收光谱图；
图7为实施例1～4的两性离子荧光化合物ZWCYOH1、ZWCYOH2、ZWCYOH3、ZWCYNH2的荧光光谱图；
图8为实施例1～4的两性离子荧光化合物ZWCYOH1、ZWCYOH2、ZWCYOH3、ZWCYNH2注射后24h内的尿液回收率；
图9为实施例5的两性离子荧光化合物ZWCYNH2P体外蛋白酶响应的荧光光谱变化；
图10为实施例5的两性离子荧光化合物ZWCYNH2P在急性肾损伤小鼠模型中的活体成像图；
图11为实施例5的两性离子荧光化合物ZWCYNH2P在急性肾损伤小鼠模型中的离体器官成像图。
具体实施方式
为了更清楚、完整的描述本发明的技术方案，以下通过具体实施例进一步详细说明本发明，应当理解，此处所描述的具体实施例仅用于解释本发明，并不用于限定本发明，可以在本发明权 利限定的范围内进行各种改变。
实施例1
本实施例提供一种两性离子荧光化合物，其制备方法的示意图见图1，具体包括如下步骤：
1.取100mL的双口圆底烧瓶，氩气氛围下，依次加入无水二氯甲烷(30mL)、N,N-二甲基甲酰胺(6mL，77.5mmol)、三溴化磷(2mL，21.4mmol)，常温搅拌30min后，加入化合物A1(2mL,19.3mmol)，常温搅拌过夜。反应结束，将反应液缓慢滴加到冰冷的饱和碳酸氢钠溶液中，旋干。乙酸乙酯萃取三次，无水硫酸钠干燥，旋干，即得淡黄色至棕黄色油状液体化合物B1。无需进一步纯化处理，直接投入下一步反应。
2.取100mL的双口圆底烧瓶，氩气氛围下，依次加入化合物C1(741mg,4.9mmol)、碳酸铯(3.9g,12.1mmol)、化合物B1(764mg,4.1mmol)、N,N-二甲基甲酰胺(20mL)，常温搅拌过夜。反应结束后，旋干，乙酸乙酯萃取三次，无水硫酸钠干燥后，旋干。粗产物通过柱层析分离纯化，洗脱体系为石油醚：乙酸乙酯＝15:1。收集洗脱纯化后的产物，旋干即得淡黄色至棕黄色固体化合物D1(560mg,产率58％)。1H NMR(400MHz,CDCl3):δ10.32(s,1H),7.08(d,J＝9.1Hz,1H),6.69–6.63(m,3H),3.84(s,2H),2.60–2.54(m,1H),2.47–2.41(m,1H),1.74–1.68(m,1H).LRMS(ESI)m/z:[M+H]+Calcd for C15H15O3243.10；Found 243.10.。
3.取100mL圆底烧瓶，依次加入化合物E1(3.76g,20mmol)、醋酸(30mL)、化合物F1(3.2mL,30mmol)、无水醋酸钠(3.2g,38mmol)，油浴加热至80℃，反应过夜。反应结束后无需处理，直接上样柱层析，用二氯甲烷：甲醇＝15:1洗脱目标产物。旋干，即得粉色粉末状化合物G1(4.0g,产率84％)。1H NMR(400MHz,DMSO-d6):δ7.63(s,1H),7.55(d,J＝7.9Hz,1H),7.34(d,J＝7.9Hz,1H),2.21(s,3H),1.25(s,6H).LRMS(ESI)m/z:[M+H]+Calcd for C11H14NO3S 240.07；Found 240.19.。
4.取100mL圆底烧瓶，依次加入化合物G1(1.5g,6.0mmol)、化合物H1(3.1g,12.0mmol)、甲苯(35mL)后，油浴加热至100℃，反应24h。反应结束后，旋干，用二氯甲烷洗涤，即得粉色粉末状化合物I1。无需进一步纯化处理，直接投入下一步反应。
5.取50mL圆底烧瓶，氩气氛围下，依次加入化合物I1(170mg,0.5mmol)、化合物D1(137mg,0.6mmol)、无水醋酸钠(246mg,3.0mmol)、无水乙醇(15mL)后，油浴加热至55℃，反应过夜。反应完成后，旋干，冰浴下加入三溴化硼(1.5mL,15.6mmol)，继续搅拌过夜。反应结束后将反应液缓慢滴加到冰冷的饱和碳酸氢钠溶液中，旋干，高效液相色谱法分离，即得绿色粉末化合物J1(90mg,产率33％)，即为两性离子荧光化合物，记为ZWCYOH1。1H NMR(400MHz,DMSO-d6):δ11.02(s,1H),8.62(d,J＝14.6Hz,1H),7.91(s,1H),7.73(d,J＝8.2Hz,1H),7.68(s,1H),7.56(dd,J＝16.6,8.5Hz,2H),7.02(s,1H),6.90(dd,J＝8.5,2.2Hz,1H),6.45(d,J＝14.6Hz,1H),4.37(t,J＝7.3Hz,2H),3.08(s,9H),2.80–2.67(m,4H),2.21(s,2H),1.78(s,6H),1.23(s,4H).LRMS(ESI)m/z:[M+H]+Calcd for C31H38N2O5S 550.25；Found 549.92.。
本实施例提供两性离子荧光化合物的结构式如下：
其可以作为常亮型荧光探针。
实施例2
本实施例提供一种两性离子荧光化合物，其制备方法的示意图见图2，具体包括如下步骤：
1、取50mL圆底烧瓶，依次加入化合物A2(576mg,4.0mmol)、叔丁醇(15mL)、叔丁 醇钾(538mg,4.8mmol)、化合物B2(0.4mL,4.0mmol)。70℃搅拌过夜。反应结束后，旋干，正己烷萃取三次，取水相，调节溶液pH至1，旋干，干燥。加入甲醇(5mL)复溶，过滤，取滤液，直接投入下一步。
2、取50mL圆底烧瓶，加入化合物C2的甲醇溶液(即第1步得到的滤液)后，加入1mol/L氢氧化钠溶液(14mL,14.0mmol)。油浴升温至70℃，反应过夜。反应结束，旋干调节溶液pH至1后，旋干，干燥。加入20mL丙酮复溶，过滤，取滤液旋干，即得化合物D2(300mg,产率36％)。LRMS(ESI)m/z:[M+H]+Calcd for C8H17O4S 209.08；Found 209.12.。
3、取50mL的圆底烧瓶，依次加入化合物D2(300mg,1.4mmol)、化合物E2(188mg,1.0mol)、醋酸(10mL)。油浴升温至95℃回流过夜。反应结束后，用异丙醇溶液洗涤，过滤，取滤渣即为化合物F2(176mg,产率48％)。LRMS(ESI)m/z:[M+H]+Calcd for C14H19NO6S2362.07；Found 362.23.。
4、取100mL圆底烧瓶，依次加入化合物F2(1.3g,3.0mmol)、邻二氯苯(30mL)、化合物H1(3.2g,12.0mmol)、碘化钠(900mg,6mmol)后，油浴加热至100℃，反应24h。反应结束后，旋干，加入乙酸乙酯洗涤，即得化合物H2。无需进一步纯化处理，直接投入下一步反应。
5、取100mL双口圆底烧瓶，依次加入化合物H2(567mg,1.2mmol)、化合物D1(410mg,1.8mmol，实施例1已制得)、无水醋酸钠(590mg,2.4mmol)、无水乙醇(30mL)后，油浴加热至55℃，反应过夜。反应完成后，旋干，冰浴下加入三溴化硼(1.5mL,15.6mmol)，继续搅拌过夜。反应结束后将反应液缓慢滴加到冰冷的饱和碳酸氢钠溶液中，旋干，高效液相色谱法分离，即得绿色粉末化合物I2(249mg,产率31％)，即为两性离子荧光化合物，记为ZWCYOH2。1H NMR(500MHz,Methanol-d4):δ8.55(s,1H),7.91(d,J＝10.4Hz,1H),7.80(s,2H),7.53(d,J＝9.0Hz,1H),7.41(d,J＝8.6Hz,1H),6.86(d,J＝9.0Hz,2H),6.77(s,2H),6.61(d,J＝8.1Hz,2H),6.29(d,J＝13.5Hz,3H),4.57(s,1H),4.27–4.20(m,1H),3.85(s,1H),3.67(s,1H),3.19(dd,J＝26.2,16.2Hz,1H),2.82(s,1H),2.66(d,J＝23.7Hz,1H),2.33(s,1H),1.95(s,1H),1.81(s,1H),1.60(s,1H),1.31(d,J＝14.9Hz,1H),1.14(s,1H).LRMS(ESI)m/z:[M+H]+Calcd for C34H43N2O8S2671.25；Found 671.77.。
本实施例提供两性离子荧光化合物的结构式如下：
其可以作为常亮型荧光探针。
实施例3
本实施例提供一种两性离子荧光化合物，其制备方法的示意图见图3，具体包括如下步骤：
1、取100mL圆底烧瓶，依次加入化合物A3(10.3g,65.0mmol)、水(16mL)、氢氧化钠(2.6g,65.0mmol)后，常温搅拌5h。反应结束，静置分层。收集有机相于一新的100mL圆底烧瓶，加入1,2-二氯乙烷(20mL)和化合物B3(8.5mL,97.5mmol)，油浴升温至50℃，反应过夜。反应完成，加入乙酸乙酯洗涤，即得化合物C3(13.7g,产率87％)。1H NMR(400MHz,D2O):δ3.64(s,2H),3.44(dd,J＝8.4,5.6Hz,4H),3.07(s,6H),2.91(s,2H),2.19(dd,J＝11.9,7.1Hz,4H).
2、取100mL的圆底烧瓶，依次加入化合物G1(4.0g,16.0mmol，实施例1已制得)、邻二 氯苯(30mL)、化合物C3(4.0g,12.0mmol)、碘化钠(1.8g,12mmol)，油浴升温至100℃，反应24h。反应结束后，旋干，高效液相色谱法分离，即得粉色固体化合物D3(3.0g,产率42％)。1H NMR(500MHz,Methanol-d4):δ8.23–8.09(m,2H),8.06(s,1H),4.84–4.61(m,1H),3.88–3.76(m,2H),3.72–3.55(m,1H),3.46–3.37(m,1H),3.27–3.21(m,1H),2.92(s,1H),2.60(d,J＝47.6Hz,1H),2.32(d,J＝6.4Hz,1H),1.74(s,4H).LRMS(ESI)m/z:[M-H]-Calcd for C19H29N2O6S2445.15；Found 445.22.
3、取100mL双口圆底烧瓶，依次加入化合物D3(760mg,1.7mmol)、化合物D1(470mg,2.1mmol，实施例1已制得)、无水醋酸钠(845mg,10mmol)、N,N-二甲基甲酰胺(5mL)后，油浴加热至55℃反应过夜。反应完成后，旋干，冰浴下加入三溴化硼(1.5mL,15.6mmol)，继续搅拌过夜。反应结束后将反应液缓慢滴加到冰冷的饱和碳酸氢钠溶液中，旋干，高效液相色谱法分离，即得绿色粉末化合物E3(379mg,产率34％)，即为两性离子荧光化合物，记为ZWCYOH3。1H NMR(500MHz,Methanol-d4):δ8.29(d,J＝13.7Hz,1H),7.86(s,1H),7.82(d,J＝7.8Hz,1H),7.78(d,J＝6.7Hz,1H),7.45(d,J＝9.0Hz,1H),7.22(d,J＝8.3Hz,1H),6.76(dd,J＝9.1,1.6Hz,1H),6.56(s,1H),6.04(d,J＝13.5Hz,1H),3.63–3.54(m,6H),3.16(s,6H),2.83(dd,J＝23.2,16.7Hz,12H),2.26(dd,J＝20.9,7.4Hz,6H).LRMS(ESI)m/z:[M+H]+Calcd for C33H41N2O8S2657.23；Found 657.68.
本实施例提供的两性离子荧光化合物的结构式如下：
其可以作为常亮型荧光探针。
实施例4
本实施例提供一种两性离子荧光化合物，其制备方法的示意图见图4，具体包括如下步骤：
1、取100mL双口圆底烧瓶，氩气氛围下，依次加入化合物A4(500mg,3.0mmol)、碳酸铯(3.6g,10.8mmol)、N,N-二甲基甲酰胺(20mL)、化合物B1(1.4g,5.0mmol，实施例1已制得)。反应置于常温搅拌过夜。乙酸乙酯萃取三次，无水硫酸钠干燥后，旋干。粗产物通过柱层析分离纯化，洗脱体系为石油醚：乙酸乙酯＝10:1。收集洗脱纯化后的产物，旋干即得淡黄色至棕黄色固体化合物B4(629mg,产率82％)。1H NMR(400MHz,CDCl3):δ10.38(s,1H),7.93(d,J＝10.2Hz,2H),7.28(d,J＝8.2Hz,1H),6.70(s,1H),2.68–2.62(m,2H),2.47(t,J＝5.7Hz,2H),1.80–1.73(m,2H).LRMS(ESI)m/z:[M+H]+Calcd for C14H11NO4258.08；Found 258.26.。
2、取100mL双口圆底烧瓶，氩气氛围下，依次加入化合物D3(1.0g,2.2mmol，实施例3已制得)、化合物B4(310mg,1.2mmol)、无水醋酸钠(590mg,7.2mmol)、N,N-二甲基甲酰胺(15mL)、酸酐(15mL)后，油浴加热至55℃反应过夜。反应完成后，旋干。加入甲醇(30mL)、氯化亚锡(4.7g,25.0mmol)的浓盐酸溶液，油浴加热至55℃反应过夜。反应完成后，滴加饱和碳酸氢钠溶液，直至pH至8。旋干，高效液相色谱法分离，即得绿色粉末化合物C4(366mg,产率56％)，即为两性离子荧光化合物，记为ZWCYNH2。1H NMR(500MHz,Methanol-d4):δ8.50(d,J＝13.5Hz,1H),7.78(dd,J＝30.2,13.5Hz,2H),7.43(d,J＝8.7Hz,1H),7.28(d,J＝8.1Hz,1H),7.16(s,1H),7.01(s,1H),6.83(d,J＝8.5Hz,1H),6.14(d,J＝13.7Hz,1H),3.60–3.46(m,4H),3.07(s,3H),2.79(d,J＝12.3Hz,2H),2.72(d,J＝22.0Hz,2H),2.11(t,J＝7.2Hz,3H),1.86(s,2H),1.74(d,J＝8.9Hz,2H),1.51(dd,J＝14.1,7.1Hz,3H),1.47–1.37(m,3H),1.21(d,J＝34.8Hz,6H).LRMS(ESI)m/z:[M-H]-Calcd for C33H40N3O7S2654.24；Found 654.20.。
本实施例提供的两性离子荧光化合物的结构式如下：
其可以作为常亮型荧光探针。
实施例5
本实施例提供一种两性离子荧光化合物，其在实施例4的ZWCYNH2上接上生物标志物响应基团后得到，其制备方法的示意图见图5，具体包括如下步骤：
1、取50mL圆底烧瓶，依次加入化合物A5(130mg,0.2mmol)、O-(7-氮杂苯并三唑-1-基)-N,N,N’,N’-四甲基脲六氟磷酸盐(140mg,0.4mmol)、无水N,N-二甲基甲酰胺(15mL)和N,N-二异丙基乙胺(30μL,0.2mmol)。常温搅拌20分钟后，加入实施例4得到的化合物C4(50mg,0.08mmol)，继续常温搅拌，反应过夜。反应结束，旋干后，无需其它处理，即投入下一步。
5.取25mL圆底烧瓶，加入化合物B5、三氟乙酸(1mL,13mmol)。搅拌8h。反应结束，旋干，用高效液相色谱分离，即得蓝色粉末化合物C5(26mg,产率29％)，即为两性离子荧光化合物，记为ZWCYNH2P。1H NMR(500MHz,D2O):δ8.58(d,J＝14.3Hz,1H),8.44(s,1H),7.95(s,1H),7.89(d,J＝8.3Hz,1H),7.81(s,1H),7.38(d,J＝8.3Hz,1H),7.33–7.25(m,2H),6.32(d,J＝14.5Hz,1H),4.71(dd,J＝9.7,6.1Hz,363H),4.64–4.56(m,5H),4.42–4.32(m,3H),4.22(dd,J＝13.7,7.5Hz,4H),4.02(d,J＝7.5Hz,1H),3.69(d,J＝7.2Hz,2H),3.66–3.59(m,2H),3.56–3.43(m,4H),3.10(s,2H),2.92(dd,J＝18.2,11.0Hz,2H),2.71–2.58(m,4H),2.36–2.13(m,6H),1.99(s,1H),1.72(s,2H),1.57(d,J＝4.4Hz,2H),1.40–1.20(m,6H),0.85–0.71(m,4H).LRMS(ESI)m/z:[M-H]-Calcd for C54H72N7O17S21154.45；Found 1154.45.。
本实施例提供的两性离子荧光化合物结构式如下：
其可以作为激活型荧光探针。
性能测试
1.光谱测试：配制0.05mg/mL常亮型荧光探针ZWCYOH1、ZWCYOH2、ZWCYOH3、ZWCYNH2的PBS溶液和甲醇溶液。用紫外光谱仪测定各样品400～900nm的吸收光谱图，用荧光光谱仪测定各样品的荧光光谱图(激发波长：660nm)。测试结果如图6与图7所示，由于各 探针的荧光团均为半花菁结构，故光谱的最大吸收与最大发射波长无明显差异。
2.肾清除效率测试：配制0.05mg/mL常亮型荧光探针ZWCYOH1、ZWCYOH2、ZWCYOH3、ZWCYNH2的PBS溶液。取12只Balb/c小鼠，随机分成3组，每组3只，小鼠尾静脉注射探针溶液75μL。注射后将小鼠单独放置在干净的代谢笼中，收集24h内的尿液并记录体积。用高效液相色谱制备探针浓度-色谱峰面积的标准曲线，计算小鼠尿液中探针的回收率。
测试结果如图8所示。ZWCYOH1、ZWCYOH2、ZWCYOH3、ZWCYNH2都可通过泌尿系统进行代谢而存在于排出的尿液中，其中，常亮型探针ZWCYOH2、ZWCYOH3、ZWCYNH2的电荷分布更加平衡，三者的尿液回收率更高，分别为88％、91％、93％，证明本发明提供的两性离子型荧光探针具有良好的肾清除效率，可实现泌尿系统疾病的检测。
ZWCYNH2P的肾清除效率测试结果相似，回收率达91％。
3.激活型探针的响应能力测试：将10μM的激活型探针ZWCYNH2P溶液与细胞凋亡蛋白酶caspase-8在37℃的缓冲溶液中孵育。测定溶液的荧光光谱变化。测试结果如图9所示，该探针的多肽序列被caspase-8蛋白酶识别并切割，溶液的荧光发射波长发生红移，荧光强度增加。
4.急性肾损伤小鼠模型的建立：取18只Balb/c小鼠，随机分成6组，分别为空白对照组(control组)、NAC对照组、模型组(12h组、24h组、48h组、72h组)，每组三只。(1)control组：腹腔注射0.4mL的生理盐水；(2)AKI(急性肾损伤)模型组：腹腔注射20mg/kg的顺铂溶液，并于注射后12h、24h、48h、72h进行影像学检查；(3)NAC组：尾静脉注射400mg/kg剂量的NAC溶液，30min后腹腔注射20mg/kg的顺铂溶液，于注射顺铂后的第48h进行影像学检查。最后，小鼠被安乐死，并对各器官进行成像。
测试结果如图10所示，探针ZWCYNH2P注射进入体内后，因control组和12h组中肾脏正常无病变，caspase-8表达量相对较低，探针荧光强度亦较低。而随着注射顺铂后时间的延长，顺铂逐步损伤肾脏组织，诱发急性肾损伤，导致肾脏中caspase-8蛋白的表达逐渐上升。探针注射进入体内，其多肽序列即被caspase-8蛋白酶识别并切割，暴露游离的氨基，恢复探针的“D-π-A”结构，表现出极强的荧光。
测试结果如图11所示，肝脏的信号比肾脏信号弱，证明探针主要经肾脏代谢，而不是肝脏代谢。同时，各组的肾脏信号随顺铂注射时间的延长而增强，即capase-8蛋白的表达逐渐增多，可实现疾病的早期诊断。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。

Claims (10)

  1. 一种两性离子荧光化合物，其特征在于，具有式(Ⅰ)～式(Ⅴ)任一所示的结构：
    其中，R1为-COO-、-SCN、-N3或R2为-OH、-NH2、A为自消除基团，B为生物标志物响应基团；n为0～50的整数，n1为0～20的整数，n2为0～20的整数。
  2. 根据权利要求1所述两性离子荧光化合物，其特征在于，所述自消除基团A为
  3. 根据权利要求1所述两性离子荧光化合物，其特征在于，所述生物标志物响应基团B为
  4. 根据权利要求1所述两性离子荧光化合物，其特征在于，所述n为1～20的整数。
  5. 根据权利要求1所述两性离子荧光化合物，其特征在于，具有如下所示结构：

  6. 权利要求1～5任一所述两性离子荧光化合物的制备方法，其特征在于，包括如下步骤：
    S1.式(1)所示的化合物1和式(6)所示的化合物6发生缩合反应，得到式(8)所示的化合物8；
    或式(1)所示的化合物1和式(7)所示的化合物7发生缩合反应，得到式(9)所示的化合物9；
    或式(2)所示的化合物2和式(6)所示的化合物6发生缩合反应，得到式(12)所示的化合物12；
    或式(2)所示的化合物2和式(7)所示的化合物7发生缩合反应，得到式(13)所示的化合物13；
    或式(3)所示的化合物3和式(6)所示的化合物6发生缩合反应，得到式(16)所示的化合物16；
    或式(3)所示的化合物3和式(7)所示的化合物7发生缩合反应，得到式(17)所示的化合物17；
    或式(4)所示的化合物4和式(6)所示的化合物6发生缩合反应，得到式(20)所示的化合物20；
    或式(4)所示的化合物4和式(7)所示的化合物7发生缩合反应，得到式(21)所示的化合物21；
    或式(5)所示的化合物5和式(6)所示的化合物6发生缩合反应，得到式(24)所示的化合物24；
    或式(5)所示的化合物5和式(7)所示的化合物7发生缩合反应，得到式(25)所示的化合物25；
    S2.化合物8发生脱甲基保护反应，得式(10)所示的化合物10；
    或化合物12发生脱甲基保护反应，得式(14)所示的化合物14；
    或化合物16发生脱甲基保护反应，得式(18)所示的化合物18；
    或化合物20发生脱甲基保护反应，得式(22)所示的化合物22；
    或化合物24发生脱甲基保护反应，得式(26)所示的化合物26；
    或化合物9经还原反应，得式(11)所示的化合物11；
    或化合物13经还原反应，得式(15)所示的化合物15；
    或化合物17经还原反应，得式(19)所示的化合物19；
    或化合物21经还原反应，得式(23)所示的化合物23；
    或化合物25经还原反应，得式(27)所示的化合物27；
    S3.化合物10与R2‐H发生取代反应，即得如式(Ⅰ)所示两性离子荧光化合物；
    或化合物14与R2‐H发生取代反应，即得如式(Ⅱ)所示两性离子荧光化合物；
    或化合物18与R2‐H发生取代反应，即得如式(Ⅲ)所示两性离子荧光化合物；
    或化合物22与R2‐H发生取代反应，即得如式(Ⅳ)所示两性离子荧光化合物；
    或化合物26与R2‐H发生取代反应，即得如式(Ⅴ)所示两性离子荧光化合物；
    或化合物11与R2‐H发生缩合反应，即得如式(Ⅰ)所示两性离子荧光化合物；
    或化合物15与R2‐H发生缩合反应，即得如式(Ⅱ)所示两性离子荧光化合物；
    或化合物19与R2‐H发生缩合反应，即得如式(Ⅲ)所示两性离子荧光化合物
    或化合物23与R2‐H发生缩合反应，即得如式(Ⅳ)所示两性离子荧光化合物；
    或化合物27与R2‐H发生缩合反应，即得如式(Ⅴ)所示两性离子荧光化合 物；
  7. 根据权利要求6所述两性离子荧光化合物的制备方法，其特征在于，步骤S1中所述缩合反应的温度为45～100℃，时间为5～12h。
  8. 根据权利要求6所述两性离子荧光化合物的制备方法，其特征在于，步骤S2中所述脱甲基保护反应的温度为0～30℃，时间为5～12h；步骤S2中所 述还原反应的温度为45～100℃，时间为5～12h。
  9. 根据权利要求6所述两性离子荧光化合物的制备方法，其特征在于，步骤S3中所述取代反应的温度为45～100℃，时间为5～12h；步骤S3中所述缩合反应的温度为0～45℃，时间为8～24h。
  10. 权利要求1～5任一所述两性离子荧光化合物或其药学上可接受的盐、溶剂化物、对映异构体、非对映异构体、互变异构体在制备荧光探针中的应用。
PCT/CN2023/127719 2023-05-23 2023-10-30 一种两性离子荧光化合物及其制备方法和应用 Pending WO2024239539A1 (zh)

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