Hydrazine compounds and their preparation methods and uses

Abstract

The present invention provides a hydrazine compound, its preparation method, and its use as a buffered fluorescent probe (BFP) in lysosomes and autophagosomes. The hydrazine compound Z11 of the present invention can be used to prepare a buffered fluorescent probe (BFP) for ultra-long-term stable imaging within the physiological pH range. The buffered fluorescent probe is bleached within the lysosome and / or autophagosome, and the intact external probe is exchanged into the lysosome and / or autophagosome to continue fluorescence imaging. This strategy not only provides fluorescence imaging of the newly generated target (lysosome and / or autophagosome), but also ensures that the photobleached fluorescent probe within the target (lysosome and / or autophagosome) is effectively replaced by the new, intact fluorescent probe surrounding the target (lysosome and / or autophagosome), thus ensuring the stability of fluorescence imaging.

Description

Technical Field

[0001] The present invention relates to the field of inspection and detection technology, and in particular to a hydrazine compound and a preparation method thereof, and use of the hydrazine compound as a buffered fluorescent probe (BFP) in lysosomes and autophagosomes. Background Art

[0002] Under normal circumstances, cells contain distinct subcellular organelle compartments, separated from the surrounding cytoplasm by membranes and performing specific functions. These functions require different pH values, resulting in varying pH levels in different regions of the same cell. For example, the pH of the nucleus ranges from 7.2-7.4, the pH of mitochondria is approximately 8.0, the pH of the Golgi apparatus ranges from 6.0-6.7, and the pH of lysosomes ranges from 4.0-5.5. Understanding and measuring pH variations in various organelles is crucial for exploring molecular mechanisms within cells and related diseases.

[0003] Lysosomes, as crucial acidic organelles in eukaryotic cells, are essential components of the cell. They contain over 60 acidic hydrolases, cathepsins, and various specific membrane proteins. They degrade macromolecules and related cellular components, participating in processes such as plasma membrane repair, protein degradation, pathogen clearance, endocytosis, and autophagy. Abnormal lysosomal pH or abnormal pH fluctuations can lead to lysosomal dysfunction and lysosomal defects, ultimately causing cellular dysfunction and potentially inducing lysosomal storage diseases (such as Tay-Sachs syndrome, glycogen storage disease type II, and inclusion disease), neurodegenerative diseases (such as Alzheimer's disease), shock, rheumatoid arthritis, and cancer. Therefore, dynamic monitoring of pH changes in lysosomes and lysosomal autophagy is of great significance for understanding the molecular mechanisms of intracellular life processes and for the diagnosis and treatment of lysosomal-related diseases. As the "digestive organ" of the cell, research on the lysosome remains a hot topic in life sciences.

[0004] Common methods for measuring pH in cells and organelles include electrochemical sensing, nuclear magnetic resonance (NMR), and surface-enhanced Raman spectroscopy (SERS). These methods typically require high-precision instrumentation and complex processing, limiting their application in real-time pH monitoring at the living cell level. Fluorescence detection, also known as fluorescent probes, is a powerful tool for studying subcellular structures. Its advantages include non-destructive testing, high spatiotemporal resolution, and real-time dynamic monitoring, making it a popular approach for bioimaging analysis.

[0005] However, existing fluorescent probes for lysosomal pH suffer from various drawbacks. For example, neutral red, acridine orange, and LysoTracker lack specificity for lysosomal localization. Once the pH within the lysosome increases, these probes will exit the lysosome, resulting in fluorescence quenching. Prolonged incubation with lysosomes can also lead to increased pH within the lysosome. Furthermore, large fluorescent probes designed based on the lysosome as a site of metabolic activity are highly toxic and unsuitable for long-term lysosomal tracking. Therefore, developing novel lysosomal pH probes that are low in toxicity, highly sensitive, highly selective, capable of real-time dynamic monitoring within the physiological pH range, and resistant to photobleaching is of great value. Summary of the Invention

[0006] In a first aspect, the present invention provides a hydrazine compound.

[0007] To achieve the above object, the technical solution of the present invention is:

[0008] A hydrazine compound Z11 has the following chemical structure:

[0009] .

[0010] In a second aspect, the present invention provides a method for preparing the compound Z11.

[0011] Compound Z11 is prepared by reacting salicylic acid hydrazide with 4-bromo-1,8-naphthalene dicarboxylic anhydride, and then replacing the bromine with 4-hydroxyethylpiperazine. The synthetic route of Z11 is as follows:

[0012]

[0013] Compound Z11 is prepared by reacting salicylic acid hydrazide with 4-bromo-1,8-naphthalene dicarboxylic anhydride, and then replacing the bromine with 4-hydroxyethylpiperazine. The steps are as follows:

[0014] (1) Synthesis of compound 3: Compound 1 was dissolved in ethanol, compound 2 was added, stirred and refluxed for 4 h, cooled to room temperature, filtered, the filter cake was washed with ethanol (5 mL × 2), and dried under reduced pressure at 50°C to obtain compound 3;

[0015] (2) Synthesis of Z11: Compound 3 was dissolved in DMSO, and N-(2-hydroxyethyl)piperazine and sodium carbonate were added. The mixture was stirred and heated to 90°C for reaction. TLC was monitored. When the spots of compound 3 disappeared, the reaction was stopped. The mixture was cooled to room temperature, diluted with purified water, and 1 mol / L hydrochloric acid was added to adjust the pH to 6. The mixture was filtered, and the filter cake was washed with purified water and dried under reduced pressure at 50°C to obtain compound Z11.

[0016] In a third aspect, the present invention provides use of compound Z11 in preparing a buffering fluorogenic probe (BFP).

[0017] Furthermore, the present invention provides use of compound Z11 in preparing a buffered fluorescent probe (BFP) for stable imaging of lysosomes and / or autophagosomes and / or autophagy processes.

[0018] The present invention provides use of compound Z11 in preparing a kit for tracking pH changes in lysosomes and / or autophagosomes and / or autophagy processes.

[0019] The compound Z11 of the present invention can be prepared into a buffered fluorescent probe (BFP) with ultra-long-term stable imaging in a physiological pH range.

[0020] The invention relates to use of the compound Z11 in the preparation of a buffered fluorescent probe (BFP) for ultra-long-term stable imaging of lysosomes and / or autophagosomes and / or autophagy processes in a physiological pH range.

[0021] Specifically, the compound Z11 described in the present invention is used in the preparation of a buffered fluorescent probe (BFP) for ultra-long-term stable imaging in the physiological pH range. The specific physiological pH range fluorescent probe refers to a fluorescent probe whose fluorescence change (intensity) has a good linear correlation with the physiological pH (pH5.4-pH7.4), providing a good detection method for pH changes in lysosomes and / or autophagosomes or the autophagy process.

[0022] Specifically, in the novel buffered fluorogenic probe (BFP) for ultra-long-term stable imaging in the physiological pH range described in the present invention, the specific "buffering fluorogenic probe" (BFP) refers to a buffer pool formed at a relatively high concentration outside the analyte (lysosome or / and autophagosome), the fluorescent probe exchange rate is greater than the photobleaching rate, and the probe that is not bound to protons outside the analyte (lysosome or / and autophagosome) will not produce a significant fluorescent signal.

[0023] Beneficial effects

[0024] The present invention provides a compound Z11 that can be used to prepare a buffered fluorescent probe (BFP) for ultra-long-term stable imaging in the physiological pH range. Specifically, ultra-long-term stable imaging is achieved through a buffering strategy, namely, a pH-buffered fluorogenic probe (BFP) strategy is used to address the issue of photostability in dynamic imaging of lysosomes and / or autophagy processes. The buffered fluorescent probe is bleached within the lysosome and / or autophagosome, and the intact external probe is exchanged into the lysosome and / or autophagosome to continue fluorescence color development. This strategy not only provides fluorescence color development of the newly generated target (lysosome and / or autophagosome), but also ensures that the photobleached fluorescent probe inside the target (lysosome and / or autophagosome) is effectively replaced by the new and intact fluorescent probe surrounding the external environment, thereby ensuring the stability of fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the 1H NMR spectrum of compound 3;

[0026] Figure 2 It is the high-resolution mass spectrometry (HRMS) of Z11;

[0027] Figure 3 is the infrared spectrum (FT-IR) of Z11;

[0028] Figure 4 It's Z11 1 H NMR spectrum;

[0029] Figure 5 It's Z11 13 C NMR spectrum;

[0030] Figure 6 Figure 3 is the UV absorption spectrum of Z11 (4.0 μM) in DMSO / Tris-HCl (1:20, v / v) solutions at pH values ranging from 2.0 to 11.0. The insert shows visible light images of probe Z11 in DMSO / Tris-HCl (1:20, v / v) solutions at pH 5.4 and pH 7.8.

[0031] Figure 7 Z11 (4.0 μM) was dissolved in DMSO / Tris-HCl (1:20, v / v) at pH values ranging from 3.5 to 10 (λ ex =405 nm, Ex / Em slit = 3 / 3 nm). Inserts are images of probe Z11 in DMSO / Tris-HCl (1:20, v / v) at pH 5.4 and pH 7.8 under UV light (365 nm).

[0032] Figure 8 The nonlinear fitting plot of the fluorescence intensity of Z11 (4.0 μM) at 530 nm and pH value (3.5-10.0) is shown in the figure. The linear relationship between the fluorescence intensity of Z11 at 530 nm and pH value (5.4-7.8) is shown in the figure. 2 =0.9966, equation: y=-42.937x+391.65;

[0033] Figure 9 The fluorescence changes of Z11 (4.0 μM) in DMSO / Tris-HCl (1:20, v / v) from pH 5.4 to pH 7.8 were observed under UV light (365 nm) and the color changes under visible light were observed;

[0034] Figure 10 The fluorescence intensity response (530 nm) of Z11 (4.0 μM) in DMSO / Tris-HCl (1:20, v / v) at pH 5.4, 6.8, and 7.8 when different ions were added: 1: blank, 2: HPO4 2- , 3:HCO 3- , 4:CH3COO - , 5:Cu 2+ , 6:Hg 2+ ,7:Br - ,8:Ca 2+ ,9:Co 2+ , 10:Cd 2+ , 11:Na + , 12:Cl - ,13:Ni 2+ , 14:SO4 2- , 15:Mg 2+ , 16:F - ,17:Fe 3+ , 18:Zn 2+ ,19:Ag + ,20:Mn 2+ , 21:Pb 2+ , 22:K + , 23:I - ,24:Fe 2+ ,25:NO3 - , that is, the interference experimental spectrum of other ions under complex physiological conditions of Z11;

[0035] Figure 11 The time course of the fluorescence intensity of Z11 (4.0 μM) in DMSO / Tris-HCl (1:20, v / v) at different pH values (5.4, 6.8, and 7.8), i.e., the experimental spectra of the photostability of Z11 under different pH conditions;

[0036] Figure 12 The fluorescence intensity of Z11 (4.0 μM) changes when the pH value changes between 5.4 and 7.4 in DMSO / Tris-HCl (1:20, v / v), i.e., the reversibility experimental spectrum of Z11 at different pH values.

[0037] Figure 13 Z11 is dissolved in DMSO-D6 solution and 1 equivalent of TFA-D1 is added. 1 H NMR diagram, i.e., Z11 nuclear magnetic titration diagram;

[0038] Figure 14 Gaussian 09 software was used to calculate the density functional theory of Z11 and Z11-H using the DFT / B3LYP / 6-31G (d, p) theoretical level. + The energy calculation spectrum of HOMO and LUMO orbitals of Z11 is shown in Figure 2. The high occupied molecular orbital (HOMO) electron density cloud of Z11 is distributed in 1,8-naphthalimide and piperazine groups, while Z11-H + The HOMO of Z11 and Z11-H is mainly located on the 1,8-naphthalene imide group. + The outermost occupied molecular orbitals (LOMOs) of Z11 and Z11-H are all located on the 1,8-naphthalene imide group. + The energy gaps are 2.37 ev and 3.53 ev, respectively, and become higher;

[0039] Figure 15 The results of the CCK-8 assay for the cytotoxicity of probe Z11 were obtained. Z11 was co-incubated with A549 cells for 24 h. The horizontal axis represents the concentration of Z11, and the vertical axis represents the absorbance measured by a microplate reader after the corresponding CCK-8 reagent produced a color reaction in a living cell environment. The absorbance reflects the number of living cells.

[0040] Figure 16 are fluorescence images of A549 cells stained with different concentrations of Z11;

[0041] Figure 17 Bright field, fluorescence field, and superimposed field images of A549 cells stained with 20 μM Z11 for different time periods;

[0042] Figure 18 This is the result of lysosome fluorescence colocalization imaging. DAPI fluorescent dye labels the cell nucleus, which is displayed in the blue channel, and Lyso-Tracker Red labels the lysosomes, which is displayed in the red channel. Z11 fluorescence imaging is displayed in the green channel. By superimposing the fluorescence images of the three channels, the overlap between Z11 and Lyso-Tracker Red can be evaluated.

[0043] Figure 19 This is the imaging of A549 cells by Z11 under the condition of changing the intracellular pH;

[0044] Figure 20 A549 cells were stained with Z11 and observed directly under a fluorescence microscope without washing with PBS buffer. Group E was continuously irradiated under the excitation light source of the fluorescence microscope, while group UE turned off the light source after the imaging was completed. The two groups were imaged at regular intervals of 0 min, 5 min, 10 min, and 15 min. DETAILED DESCRIPTION

[0045] The present invention is described in detail below by specific examples. It is pointed out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above invention. The raw materials and reagents used in the present invention are all commercially available products. Unless otherwise specified, the parts and percentages described in the present invention are all parts by weight, and the percentages are all mass percentages.

[0046] The present invention provides a buffered fluorescent probe capable of ultra-long-term stable imaging within a physiological pH range. The probe's hydroxyethylpiperazine and salicyl moieties are lysosomal-targeting. N-hydroxyethylpiperazine, a pH-responsive group, undergoes a significant change in its intramolecular charge before and after protonation, enabling Z11 to measure the pH of lysosomes and / or autophagosomes and / or the autophagic process. The probe can also be applied to imaging pH changes during the autophagic process of lysosomes and / or autophagosomes.

[0047] According to the embodiments of the method for synthesizing a buffered fluorescent probe for ultralong-term stable imaging within the physiological pH range of this application, Z11 is a lysosomal fluorescent probe within the physiological pH range, exhibiting ultralong-term stable imaging, a buffering effect, good photostability, resistance to photobleaching, excellent fluorescence response, and low fluorescence toxicity. Furthermore, it can monitor pH changes in lysosomes and / or autophagosomes and / or the autophagic process over a long period of time.

[0048] Synthesis of compound Z11

[0049] It is obtained by reacting salicylic acid hydrazide with 4-bromo-1,8-naphthalene dicarboxylic anhydride, and then replacing the bromine with 4-hydroxyethylpiperazine. The synthetic route of Z11 is shown below.

[0050]

[0051] Synthesis of Compound 3: 2.77 g (0.01 mol) of Compound 1 was suspended in 30 mL of ethanol, and 1.67 g (0.011 mol) of Compound 2 was added. The mixture was stirred and refluxed for 4 h. The mixture was allowed to cool to room temperature and filtered. The filter cake was washed with ethanol (5 mL x 2) and dried under reduced pressure at 50°C to yield 3.62 g of Compound 3. Yield: 88%. Melting point: over 250°C 1 H NMR (attached Figure 1 )

[0052] Synthesis of Z11: Dissolve 0.822 g (0.002 mol) of compound 3 in 4 mL of DMSO. Add 0.32 g (0.0024 mol) of N-(2-hydroxyethyl)piperazine and 1.272 g (0.006 mol) of sodium carbonate. Stir and heat to 90°C. Monitor by TLC (ethyl acetate / petroleum ether: 1 / 2). Stop the reaction when the spots of compound 3 disappear. Cool the mixture, add 30 mL of purified water, and adjust the pH to 6 with 1 mol / L hydrochloric acid. Filter the mixture, wash the filter cake with purified water (10 mL x 3), and dry it under reduced pressure at 50°C to obtain 0.71 g of compound Z11 and 0.82 g of compound Z11. Yield: 90%. Melting point: 223.4–226.4°C. HRMS data: see attached. Figure 2 ,HRMS(positive-ESIMS)calcd for C 24 H 25 O5N4 (M+H) + :46.1825, Found:461.1813. FT-IR see attached Figure 3 . 1 H NMR Figure 4 , 1 H NMR (600 MHz, DMSO- d 6) δ 11.33 (s, 1H), 8.54 (d, J = 7.2 Hz,1H), 8.51 (d, J = 8.4 Hz, 1H), 8.46 (d, J = 8.1 Hz, 1H), 8.00 (dd, J = 7.9,1.5 Hz, 1H), 7.88 – 7.84 (m, 1H), 7.54 – 7.49 (m, 1H), 7.37 (d, J = 8.2 Hz,1H), 7.05 (d, J = 8.3 Hz, 1H), 7.01 (t, J= 7.5 Hz, 1H), 4.54 (s, 1H), 3.60(t, J = 6.2 Hz, 2H), 3.29 (s, 4H), 2.78 (s, 4H), 2.56 (t, J = 6.2 Hz, 2H). 13 CNMR see attached Figure 5 , 13 C NMR (151 MHz, DMSO- d 6) δ 167.02, 161.72, 159.45, 156.85,133.47, 131.92, 131.88, 129.73, 126.62, 125.90, 122.73, 119.58, 117.90,115.64, 115.43, 115.25, 60.63, 59.02, 53.56, 53.11.

[0053] Evaluation of fluorescence performance

[0054] The UV and fluorescence spectra of the probe under different pH conditions were studied. Z11 was dissolved in DMSO / Tris-HCl (1:20, v / v) solutions of different pH values and diluted to a concentration of 4 μmol / L. The UV absorption spectrum was scanned in the range of 350 nm-550 nm and the fluorescence spectrum was measured (λ ex =405 nm, gap 3 nm), record the fluorescence spectrum in the range of 450 nm-650 nm. See the attached UV scan Figure 6 . Fluorescence spectrum is attached Figure 7 In the UV spectrum, as the pH decreases, the absorption spectrum will red-shift and the absorbance will also decrease. In the fluorescence spectrum, 530 nm is the maximum fluorescence emission wavelength of Z11. As the pH decreases, its fluorescence intensity increases. Based on the fluorescence spectrum data, nonlinear fitting and linear curve fitting were performed on the fluorescence intensity at 530 nm and pH. The results are shown in the attached figure. Figure 8 (Inset is a linear fit graph). As pH increases, the fluorescence intensity of Z11 at 530 nm increases 23-fold. Within the pH range of 5.4 to 7.8, the fluorescence intensity of Z11 at 530 nm shows a good linear correlation with pH. The linear equation is: y = -42.937x + 391.65, with a correlation coefficient of r = 0.9966. Z11 solutions with pH values between 5.4 and 7.8 were observed under 365 nm UV light and visible light, respectively. The results are shown in the attached figure. Figure 9 As the pH decreases, its fluorescence gradually increases, gradually changing from a colorless and transparent solution to a yellow transparent solution.

[0055] Interference experiment

[0056] Different concentrations of interfering substances were added to Z11 solution with a concentration of 4.0 μM and pH values of 5.4, 6.8, and 7.8, respectively, in DMSO / Tris-HCl (1:20, v / v), and their fluorescence spectra were measured. Figure 10 , marked in the figure are 1: blank, 2: HPO4 2- (2.0 mM), 3:HCO3 - (2.0 mM), 4: CH3COO - (2.0 mM), 5: Cu 2+ (2.0 mM), 6:Hg 2 + (2.0 mM), 7:Br - (2.0 mM), 8:Ca 2+ (2.0 mM), 9:Co 2+ (2.0 mM), 10:Cd 2+ (0.2 mM), 11:Na + (1.5 M), 12:Cl - (1.5 M), 13:Ni 2+ (0.2 mM), 14: SO4 2- (0.2 mM), 15:Mg 2+ (2.0 mM), 16:F - (2.0 mM), 17:Fe 3+ (2.0 mM), 18:Zn 2+ (2.0 mM), 19: Ag + (2.0 mM), 20: Mn 2+ (2.0 mM), 21:Pb 2+ (2.0 mM), 22:K + (150 mM), 23:I - (2.0 mM), 24:Fe 2+ (2.0 mM), 25: NO3 - The results showed that the fluorescence signal of probe Z11 could be maintained with negligible changes in solutions with pH 5.4, pH 6.8 and pH 7.8, indicating that probe Z11 has good selectivity for H+.

[0057] Photostability and reversibility experiments

[0058] A Z11 solution with a concentration of 4.0 μM, pH 5.4, pH 6.8, and pH 7.8, and a solvent of DMSO / Tris-HCl (1:20, v / v) was irradiated continuously for 120 min using a fluorescence spectrometer. The fluorescence spectra were measured at 0 min, 5 min, 10 min, 15 min, 30 min, 45 min, 60 min, 80 min, 100 min, and 120 min, respectively. See the attached figure. Figure 11 The results showed that the probe Z11 was stable in solutions with pH 5.4, pH 6.8, and pH 7.8 after 120 min of irradiation. A Z11 solution with a concentration of 4.0 μM and a solvent of DMSO / Tris-HCl (1:20, v / v) was prepared. Hydrochloric acid and sodium hydroxide were added alternately to adjust the pH to 5.4 and 7.8, and its fluorescence spectrum was measured. Figure 12 The results showed that Z11 could stably generate and quench fluorescence alternately at 5.4 and 7.8, and its reversibility was good.

[0059] Nuclear magnetic titration and fluorescence generation mechanism research

[0060] In order to study the responsiveness of Z11 to pH, the nuclear magnetic titration of Z11 was studied. An equivalent amount of deuterated trifluoroacetic acid (TFA-D) was added to the DMSO-D6 solution of Z11, and its 1 H NMR spectrum and that of Z11 1 H NMR comparison, see attached Figure 13 As can be seen in the figure, the proton peaks at positions 2 and 3 exhibit significant chemical shifts, while the proton peaks at positions 1 and 4 exhibit some shifts due to the influence of adjacent carbon and hydrogen atoms. This indicates that the added acidic proton (by adjusting the pH) first binds to the nitrogen atom linking the hydroxyethyl group in Z11, generating fluorescence. Its pKa is 6.1.

[0061] In order to understand the photophysical properties of Z11 before and after acidification, Z11 and acidified Z11-H + Density functional theory (DFT) calculations were performed using Gaussian 09 software and the DFT / B3LYP / 6-31G (d, p) level of theory to calculate the Z11 and Z11-H + The energy of the HOMO and LUMO orbitals. Figure 14 Before acidification, the high occupied molecular orbital (HOMO) electron density cloud of Z11 is distributed in the 1,8-naphthaleneimide and piperazine groups, while the low occupied molecular orbital (LOMO) is located in the 1,8-naphthaleneimide group. The transfer of electrons during excitation causes the PET phenomenon, which suppresses the fluorescence of Z11. +, its high occupied molecular orbital (HOMO) and low occupied molecular orbital (LOMO) electron density clouds are distributed in 1,8-naphthalimide and piperazine groups, which inhibits electron transfer during excitation and the PET process, resulting in enhanced fluorescence. In addition, Z11 and Z11-H + The energy gaps of are 2.37 ev and 3.53 ev, respectively, which become higher, indicating that the absorption spectrum will red-shift with the decrease of pH value, which is consistent with the experimental results of ultraviolet absorption spectrum.

[0062] Cytotoxicity assay

[0063] The cytotoxicity of probe Z11 to A549 cells was measured by CCK-8 assay. A549 cells were plated onto 96-well plates with 1×10 cells per well. 3 The culture medium volume was maintained at 100 μL and incubated at 37°C in a 5% CO2 atmosphere for 12 h. A certain concentration of probe (0 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM) was added to every 5 wells and then incubated for 24 h. 100 μL of CCK-8 reagent was then added to each well and incubated for another 3 hours. Finally, the absorbance of each well at a wavelength of 450 nm was measured using a microplate reader, and a bar graph of the absorbance corresponding to each concentration was drawn. The results are shown in the attached figure. Figure 15 The results showed that the absorbance decreased slightly when the concentration of Z11 increased from 5.0 μM to 80.0 μM, indicating that the cytotoxicity to A549 cells was very low, and it has potential application prospects in imaging in living cells.

[0064] Optimal cell staining concentration

[0065] A549 cells were selected for cell fluorescence imaging detection using probe Z11. A549 cells were cultured in Ham's F-12K (Kaighn's) medium supplemented with 10% fetal bovine serum (FBS), 100 units / mL penicillin, and 100 units / mL streptomycin in an incubator with a CO2 concentration of 5%, a temperature of 37°C, and a humidity of 95%. Before each experiment, cells were counted and treated with trypsin when they reached 80-90% of the growth surface. The same procedure was followed for the following cell imaging projects. Fluorescence images after the experiment were acquired using a Nikon Ti2 inverted fluorescence microscope.

[0066] A549 cells (1×10 4 / cm 2) for 12 h, the culture medium was removed, the cells were washed three times with PBS buffer, 20 μM, 50 μM, and 100 μM Z11 were added for staining for 30 min and 1 h, respectively, and the cells were washed three times with PBS buffer and observed under a fluorescence microscope. Figure 16 The results showed that the fluorescence image signal-to-noise ratio at a concentration of 50 μM was higher, and the subcellular imaging effect was more obvious.

[0067] Optimal cell staining time

[0068] A549 cells (1×10 4 / cm 2 ) for 12 h, the culture medium was removed, the cells were washed three times with PBS buffer, 20 μM Z11 was added for staining, and the cells were washed three times with PBS buffer after 10 min, 30 min, 60 min, and 90 min, respectively, and then observed under a fluorescence microscope. Figure 17 The bright field, fluorescence field, and superimposed field of A549 cells stained with 20 μM Z11 for different time periods showed that the fluorescence of the cells was the strongest and the signal-to-noise ratio was the highest 30 minutes after staining.

[0069] Lysosome fluorescence colocalization experiment

[0070] A549 cells (1 × 10 5 / cm 2 ) were plated on a 35 mm glass-bottomed cell culture dish and incubated for 12 h. The culture medium was removed and the cells were washed three times with PBS buffer. 20 μM Z11 was added for staining for 1 h. Subsequently, 1 mL Lyso-Tracker Red was added for staining for 1 h and 1 μg / mL DAPI was added for staining for 30 min. The cells were then washed five times with PBS buffer. Finally, subcellular fluorescence imaging was observed in each channel under a Leica laser confocal fluorescence microscope. See Appendix. Figure 18 The results of lysosome fluorescence co-localization imaging showed that DAPI fluorescent dye can mark the cell nucleus, which is displayed by the blue channel, Lyso-Tracker Red can mark the lysosome, which is displayed by the red channel, and the fluorescence imaging of Z11 is displayed by the green channel. The image obtained by superimposing the fluorescence images of the three channels can be seen that the images of the red channel and the green channel overlap well, indicating that the probe can specifically stain lysosomes.

[0071] Cellular pH fluorescence imaging

[0072] A549 (1×10 4 / cm 2) cells were plated on a 24-well plate and incubated for 12 h. The culture medium was removed and the cells were washed three times with PBS buffer and stained with 50 μM Z11 for 1 h. The treated group was washed three times with PBS buffer and then incubated in high K medium at different pH values (pH 4, 5, 6, 7, 8, and 9). + The cells were further incubated in the presence of 10 μM nigericin in a PBS buffer (30 mM NaCl, 120 mM KCl, 1 mM CaCl2, 0.5 mM MgSO4, 1 mM NaH2PO4, 5 mM glucose, 20 mM HEPES, and 20 mM NaOAC) for 10 min. The control group was incubated in PBS buffer for 10 min. The cells were observed under a Nikon Ti2 inverted fluorescence microscope for fluorescence imaging. Figure 19 The results showed that when the intracellular pH was 4-5, the Z11-stained cell imaging fluorescence was strong, while when the pH was 6-9, the cell fluorescence intensity decreased significantly, indicating that the cells can intuitively monitor changes in intracellular pH.

[0073] No-wash cell fluorescence imaging

[0074] A549 (1×10 4 / cm 2 ) cells were plated on a 24-well plate and incubated for 12 h. The culture medium was removed, the cells were washed three times with PBS buffer, stained with 50 μM Z11 for 1 h, and observed directly under a fluorescence microscope. Group E was continuously irradiated under the fluorescent light source of the fluorescence microscope, while group UE was turned off after the imaging was completed. Both groups were imaged at 0 min, 5 min, 10 min, and 15 min, see Appendix. Figure 20 The results showed that after staining A549 cells, the cells were observed directly under a fluorescence microscope without washing with PBS buffer. The fluorescence of group E began to weaken over time. This was because Z11 was photobleached under the stimulation of an extremely high-intensity excitation light source. The fluorescence intensity of group UE did not change significantly over time. This was because each time the Z11 in the cells was photobleached, the Z11 in the cell environment that was not irradiated by the excitation light source could be replenished into the cells, achieving stable imaging.

Claims

1. A hydrazine compound having the chemical structure shown in Formula Z11 below:

2. The method for preparing compound Z11 according to claim 1, wherein: Salicylic acid hydrazide reacts with 4-bromo-1,8-naphthalene dicarboxylic anhydride to prepare compound 3, which is then replaced by 4-hydroxyethylpiperazine to obtain Z11. The synthetic route of Z11 is as follows:

3. The preparation method according to claim 2, wherein The steps include: (1) Synthesis of Compound 3: Compound 1 was dissolved in ethanol, compound 2 was added, stirred and refluxed for 4 h, cooled to room temperature, filtered, the filter cake was washed with ethanol, and dried under reduced pressure at 50°C to obtain compound 3; (2) Synthesis of Z11: Compound 3 was dissolved in DMSO, and N-(2-hydroxyethyl)piperazine and sodium carbonate were added. The mixture was stirred and heated to 90°C for reaction. The reaction was monitored by TLC. When the spots of compound 3 disappeared, the reaction was stopped. The mixture was cooled to room temperature, diluted with purified water, and 1 mol / L hydrochloric acid was added to adjust the pH to 6. The mixture was filtered, and the filter cake was washed with purified water and dried under reduced pressure at 50°C to obtain compound Z11.

4. Use of the compound Z11 as claimed in claim 1 in the preparation of a fluorescent probe.

5. Use of the compound Z11 as claimed in claim 1 in the preparation of a lysosomal buffered fluorescent probe (BFP).

6. Use of the compound Z11 according to claim 1 in preparing a buffered fluorescent probe (BFP) for ultra-long-term stable imaging in a physiological pH range of pH 5.4-pH 7.4.

Images