A naphthaleneimide compound and its preparation method and application

Abstract

A naphthaleneimide compound, its preparation method, and application. The present invention provides a naphthaleneimide compound, the structure of which is shown in Formula I: R is selected from one of the following: The present invention also provides a method for preparing the naphthaleneimide compound. The compound has the ability to accurately trace mitochondrial autophagy and has the ability to release peroxynitrite (ONOO) ‑ The responsiveness and mitochondrial targeting capabilities of the ELISA can be applied to prepare a kit for tracking oxidative stress-induced mitophagy and / or peroxynitrite activation.

Description

Technical Field

[0001] The invention relates to the field of fine chemical technology, and in particular to a naphthaleneimide compound and its preparation method and application. Background Art

[0002] Mitochondria are essential for maintaining human health, acting as energy generators and participating in many important metabolic processes. Mitochondrial autophagy is a lysosome-dependent process that is used to clear dysfunctional mitochondria and maintain the integrity of normally functioning mitochondria in response to mitochondrial damage. Reactive oxygen species (ROS) are key signaling molecules in oxidative stress that can trigger mitochondrial dysfunction and mitophagy. Dysregulation of mitophagy is associated with a variety of diseases, such as tumors, neurodegenerative diseases, inflammation, aging, etc. Monitoring and tracking the mitophagy process triggered by oxidative stress is of great significance for elucidating its role in maintaining cellular homeostasis, physiological and pathological processes.

[0003] Currently reported fluorescent probes for detecting mitophagy are primarily based on changes in mitochondrial pH, viscosity, polarity, and membrane potential. However, only a few studies have comprehensively monitored the entire mitophagy process, including induction and progression. Because ROS plays a key role as an indicator in oxidative stress-induced mitophagy models, combining ROS detection with mitophagy monitoring will provide new insights into the biological mechanisms of mitophagy. Summary of the Invention

[0004] The present invention develops a ratiometric fluorescent probe NA-DP, which is specifically used to detect peroxynitrite (ONOO - ) and subsequent oxidative stress-induced mitophagy. Diphenylphosphinyl and triphenylphosphine groups were incorporated into the naphthalimide backbone to give the probe ONOO - response and mitochondrial targeting ability. - The effect of NA-DP on ONOO is based on the addition-elimination reaction mechanism, so even under the interference of other reactive oxygen species, the probe NA-DP - Also showed high selectivity. - In the presence of triphenylphosphine, the diphenylphosphine group is converted to a hydroxyl group, enhancing the electron-donating ability of the donor and thus the intramolecular charge transfer (ICT) effect, generating a ratiometric fluorescence signal. Subsequently, based on the mitochondrial targeting ability of the triphenylphosphine group and the fluorescence stability of the probe in acidic pH environments, the overlap between the NA-DP probe channel and the lysosomal tracer (LysoTracker) channel was observed to successfully track oxidative stress-induced mitophagy in cells.

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. Therefore, in a first aspect of the present invention, the present invention provides a naphthalene imide compound, the structure of which is shown in Formula I:

[0006]

[0007] R is selected from One of them.

[0008] Preferably, R is

[0009] R is When, compound I corresponds to compound NA-DP; R is When , compound I corresponds to compound NAB-DP.

[0010] In a second aspect of the present invention, the present invention provides a method for preparing the naphthalimide compound described in the first aspect of the present invention, wherein the naphthalimide compound is prepared from compound II and compound III, and the reaction formula for preparing the naphthalimide compound from compound II and compound III is as follows:

[0011]

[0012] R is When, compound II corresponds to compound NA-OH; R is When , compound II corresponds to compound NAB-OH.

[0013] In one or more embodiments of the present invention, compound II and compound III are used to prepare the naphthalene imide compound, comprising the following steps: adding compound II and compound III to a reaction vessel, conducting a first reaction, adding DCM (dichloromethane) after the first reaction is complete, filtering, concentrating the filtrate, and purifying to obtain the naphthalene imide compound.

[0014] In one or more embodiments of the present invention, the first reaction is carried out in a solvent, and the solvent is CH3CN (acetonitrile).

[0015] In one or more embodiments of the present invention, the compound II is prepared from compound IV and 2-hydroxyisoindole-1,3-dione. The reaction formula for preparing compound II from compound IV and 2-hydroxyisoindole-1,3-dione is as follows:

[0016]

[0017] R is When compound IV corresponds to compound NA-Br; R is When , compound II corresponds to compound NAB-Br.

[0018] In one or more embodiments of the present invention, compound IV and 2-hydroxyisoindole-1,3-dione are used to prepare compound II, which comprises the following steps: adding compound IV and 2-hydroxyisoindole-1,3-dione to a reaction vessel, conducting a second reaction, and after the second reaction is complete, adding water, adjusting the pH, filtering, and washing the filter residue to obtain compound II.

[0019] In one or more embodiments of the present invention, the second reaction is carried out in a solvent, and the solvent is DMSO (dimethyl sulfoxide).

[0020] In one or more embodiments of the present invention, the second reaction temperature is controlled to be 75-85° C.; the second reaction is carried out under the protection of an inert gas; and the pH is adjusted to be 2-3.

[0021] In a third aspect of the present invention, the present invention provides a probe comprising the naphthaleneimide compound for detecting peroxynitrite-induced mitochondrial autophagy according to the first aspect of the present invention.

[0022] In the fourth aspect of the present invention, the present invention provides use of the naphthalimide compound described in the first aspect of the present invention or the fluorescent probe described in the third aspect of the present invention in preparing a kit for tracing oxidative stress-induced mitophagy and / or peroxynitrite activation.

[0023] Preferably, the present invention provides use of the naphthalimide compound described in the first aspect of the present invention or the fluorescent probe described in the third aspect of the present invention in a kit for detecting peroxynitrite-induced mitophagy.

[0024] The beneficial effects of the present invention are:

[0025] 1. The present invention provides a fluorescent probe for tracing oxidative stress-induced mitophagy and peroxynitrite activation. By modifying the hydroxynaphthalimide fluorophore containing a triphenylphosphine group with a diphenylphosphinyl bromide, targeted dual detection is achieved, avoiding the inability of traditional probes to visualize the complete mitophagy process in living cells, and having the ability to accurately trace mitophagy.

[0026] 2. The probe exhibits a ratiometric fluorescence response to peroxynitrite, and its fluorescence pH stability ensures consistent and significant fluorescence signal intensity during mitophagy tracing. The addition-elimination reaction mechanism of the diphenylphosphinyl group interacting with peroxynitrite greatly improves the selectivity of the probe, while the mitochondrial targeting of triphenylphosphine significantly enhances the accuracy of mitophagy tracing. The probe successfully achieved in situ dynamic imaging of oxidative stress-induced mitophagy in cell experiments, providing new insights into the development of probes for monitoring the entire process of mitophagy, from induction to development.

[0027] 3. The fluorescent probe prepared by the present invention combines diphenylphosphinyl and triphenylphosphine groups into the naphthalimide skeleton, so that the probe has ONOO - responsiveness and mitochondrial targeting capabilities.

[0028] 4. The probe NA-DP prepared by the present invention depends on ONOO - Addition-elimination reaction with diphenylphosphinyl to ONOO - It also shows high selectivity. - In the presence of triphenylphosphine, the diphenylphosphine group is converted to a hydroxyl group, enhancing the electron-donating capacity of the donor and thus the intramolecular charge transfer (ICT) effect, generating a ratiometric fluorescence signal. Subsequently, due to the mitochondrial targeting ability of the triphenylphosphine group and the stability of the probe's fluorescence intensity in acidic pH environments, it was successfully used to track oxidative stress-induced mitophagy in cells by observing the overlap between the NA-DP probe channel and the LysoTracker channel.

[0029] 5. The fluorescent probe prepared by the present invention for accurately tracing oxidative stress-induced mitophagy and peroxynitrite activation has successfully achieved in situ dynamic imaging of oxidative stress-induced mitophagy, providing new insights for the development of probes for monitoring the entire process of mitophagy from induction to occurrence and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 NA-DP to ONOO - Ratio response plot of ;

[0031] in, Figure 1 A. Figure 1 B respectively NA-DP (20 μM) probe in ONOO - Spectral graph of absorption and fluorescence changes in the presence of (20 μM) for 40 min;

[0032] Figure 1 C is NA-DP (20 μM) in ONOO -Spectral graph of the fluorescence intensity at 450 nm and 550 nm as a function of time in the presence of (20 μM);

[0033] Figure 1 D. Figure 1 E were NA-DP (20 μM) and different concentrations of ONOO - Spectral diagram of absorption and fluorescence changes after 15 minutes of action (0-30μM);

[0034] Figure 1 F is the fluorescence intensity of NA-DP (20 μM) after 15 min of reaction (I 550 nm / I 450 nm )With ONOO - Concentration (0-30 μM) change curve.

[0035] Figure 2 NA-DP to ONOO - selectivity and pH stability;

[0036] in, Figure 2 A represents the presence of ONOO after 40 min of incubation. - The fluorescence changes of NA-DP (20 μM) at 550 nm (1: blank control; 2: ONOO - ;3: H2O2; 4: NaClO; 5: ·OH; 6: 1 O2;7:O2 ·- ;8: Cys; 9: GSH; 10: HCy; 11: Na + ;12:K + ;13:Ca 2 + );

[0037] Figure 2 B is with ONOO - Normalized absorption spectra of NA-OH and NA-DP before and after incubation;

[0038] Figure 2 C. Figure 2 D are the fluorescence spectra of NA-DP and NA-OH in PBS buffer (10 mM, V / V, EtOH / PBS = 1 / 1) as the pH changes.

[0039] Figure 3 Fluorescence imaging and fluorescence ratio (G / B) quantitative analysis of the Control group, SIN-1 group, and SIN-1 / UA group in Example 5;

[0040] in, Figure 3 A is the fluorescence imaging of the Control group, SIN-1 group, and SIN-1 / UA group in Example 5;

[0041] Figure 3 B is a quantitative analysis graph of the fluorescence ratio (G / B) of the Control group, SIN-1 group, and SIN-1 / UA group in Example 5.

[0042] Figure 4 Fluorescence imaging and fluorescence ratio (G / B) quantitative analysis of the Control group, APAP group, and APAP / NAC group in Example 6;

[0043] in, Figure 4 A is the fluorescence imaging of the Control group, APAP group, and APAP / NAC group in Example 6;

[0044] Figure 4 B is a quantitative analysis graph of the fluorescence ratio (G / B) of the Control group, APAP group, and APAP / NAC group in Example 6.

[0045] Figure 5 Co-localization fluorescence imaging of the fluorescent probes (NA-DP, NAB-DP) provided by the present invention with a mitochondrial tracer (MitoTracker) (100 nM) and a lysosomal tracer (LysoTracker) (100 nM) in HeLa cells;

[0046] in, Figure 5 A is the co-localization fluorescence imaging of NA-DP (10 μM) with mitochondrial tracer (MitoTracker) (100 nM) and lysosomal tracer (LysoTracker) (100 nM) in HeLa cells;

[0047] Figure 5 B is the co-localization fluorescence imaging of NAB-DP (10 μM) with mitochondrial tracer (MitoTracker) (100 nM) and lysosomal tracer (LysoTracker) (100 nM) in HeLa cells.

[0048] Figure 6 The fluorescence imaging images of the fluorescent probes (NA-DP and NAB-DP) provided by the present invention co-localizing with the lysosomal tracer (LysoTracker) (100 nM) after HeLa cells were induced to undergo mitophagy using carbonyl cyanide-3-chlorophenylhydrazone (CCCP) (50 μM) are shown;

[0049] in, Figure 6A is a fluorescence imaging image of co-localization of NA-DP (10 μM) and lysosomal tracer (LysoTracker) (100 nM) after HeLa cells were induced to undergo mitophagy using carbonyl cyanide-3-chlorophenylhydrazone (CCCP) (50 μM);

[0050] Figure 6 B is a fluorescence imaging image of the colocalization of NAB-DP (10 μM) and lysosomal tracer (LysoTracker) (100 nM) after HeLa cells were induced to undergo mitophagy using carbonyl cyanide-3-chlorophenylhydrazone (CCCP) (50 μM). DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with specific examples and accompanying drawings, but the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. In the following examples, if no specific conditions are specified, the conditions according to normal conditions or manufacturer's recommendations are carried out, and the method used, if not otherwise specified, is a conventional method well known in the art, and the consumables and reagents used, if not otherwise specified, are commercially available. Unless otherwise indicated, the professional and scientific terms used herein are identical in meaning to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied to the present invention.

[0052] Example 1-1

[0053]

[0054] A 50 mL round-bottom flask was charged with NA-Br (500 mg, 0.76 mmol), 2-hydroxyisoindole-1,3-dione (248 mg, 1.52 mmol), and K2CO3 (315 mg, 2.28 mmol). After dehydration and deoxygenation, DMSO (10 mL) was injected. The reaction mixture was heated to 80°C with stirring and stirred under N2 for 8 h. After cooling to room temperature, 80 mL of H2O was added to the solution, and the pH was adjusted to 2-3 by dropwise addition of dilute hydrochloric acid. The orange-red precipitate was filtered and washed three times with H2O to obtain the product, NA-OH, as a yellow powder (363 mg, 80.2% yield).

[0055] 1H NMR (400MHz, DMSO-d6) δ8.49(dd,J=8.2,1.3Hz,1H),8.36(dd,J=7.3,1.4Hz,1H),8.18(d,J=8.5Hz,1H),7.90-7.85(m,3H),7.8 4-7.69(m,12H),7.58(t,J=7.8Hz,1H),6.80(d,J=8.5Hz,1H),4.19(t,J=6.7Hz,2H),3.79-3.70(m,2H),1.92(d,J=14.5Hz,2H). 13 C NMR (101MHz, DMSO-d6) δ166.98,164.21,162.96,134.99,134.43,133.77,133.67,130.80,130.59,130.34,130. 22,129.95,124.51,123.79,121.47,118.80,117.94,111.46,107.29,20.72,18.53,18.02.HRMS-ESI(m / z):[M] + Calcd.for C 33 H 27 NO3P + 516.1724,found516.1697.

[0056] Example 1-2

[0057]

[0058] To a 25 mL Schlenk tube, NA-OH (100 mg, 0.17 mmol), compound III (78 mg, 0.20 mmol), and K2CO3 (47 mg, 0.34 mmol) were added. After dehydration and deoxygenation, ultra-dry CH3CN (2 mL) was added via syringe under a N2 atmosphere. The reaction was stirred at room temperature for 8 h and then diluted with DCM (4 mL). The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. Purification on a silica gel column using DCM / MeOH (25:1, v / v) as the eluent afforded compound NA-DP as an orange solid (93 mg, 61.3% yield).

[0059] 1H NMR (400MHz, DMSO-d6) δ8.55(d,J=8.4Hz,1H),8.48(d,J=6.8Hz,1H),8.42(d,J=8.3Hz,1H),7.95-7.86(m,7H),7.82-7.72(m,13H), 7.66-7.60(m,2H),7.59-7.53(m,6H),7.43-7.32(m,3H),5.40(s,2H),4.21(t,J=7.2Hz,2H),3.83-3.66(m,2H),2.10-1.94(m,2H). 13 C NMR(101MHz,DMSO-d6)δ163.83,163.14,159.16,150.59,150.52,134.97,133.73,13 3.63,133.08,132.86,132.42,131.59,131.49,131.41,131.07,130.31,130.19,130 .05,129.51,129.07,128.94,128.78,128.46,126.50,122.90,122.08,120.81,120. 76,118.75,117.89,114.62,107.37,69.73,20.53,18.51,17.99.HRMS-ESI(m / z):[M] + Calcd.for C 52 H 42 BNO5P2 + 822.2533, found 822.2502.

[0060] Example 2-1

[0061]

[0062] To a 50 mL round-bottom flask, add NAB-Br (500 mg, 1.51 mmol), 2-hydroxyisoindole-1,3-dione (491 mg, 3.00 mmol), and KCO (624 mg, 4.52 mmol). After dehydration and deoxygenation, DMSO (10 mL) was injected. The reaction mixture was heated to 80°C with stirring and stirred under N for 10 h. After cooling to room temperature, 80 mL of H2O was added to the solution, and the pH was adjusted to 2-3 by dropwise addition of dilute hydrochloric acid. Filtration afforded an orange-red precipitate, which was washed three times with H2O to afford compound NAB-OH as a light yellow powder (346 mg, 85.3% yield).

[0063] 1H NMR (400MHz, DMSO-d6) δ11.87(s,1H),8.52(d,J=8.3Hz,1H),8.46(d,J=7.2Hz,1H),8.35(d,J=8.1Hz,1H),7.75(t,J=7.8 Hz,1H),7.15(d,J=8.2Hz,1H),4.01(t,J=7.4Hz,2H),1.59(p,J=7.4Hz,2H),1.33(h,J=7.3Hz,2H),0.91(t,J=7.3Hz,3H). 13 CNMR(101MHz,DMSO-d6)δ163.64,162.97,160.23,133.51,131.08,129.14,128.84, 125.55,122.34,121.77,112.59,109.93,29.76,19.86,13.76.HRMS-ESI(m / z):[MH] - Calcd.forC 16 H 14 NO3 - 268.0979,found 268.0976.

[0064] Example 2-2

[0065]

[0066] To a 25 mL Schlenk tube, add NAB-OH (100 mg, 0.37 mmol), compound III (173 mg, 0.45 mmol), and KCO (103 mg, 0.75 mmol). After dehydration and deoxygenation, ultra-dry CHCN (5 mL) was added via syringe under a nitrogen atmosphere. Stir at room temperature for 12 h and then diluted with DCM (10 mL). The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified on a silica gel column using DCM / MeOH (100:1, v / v) as the eluent to afford compound NAB-DP as a yellow solid (155 mg, 72.5% yield).

[0067] 1H NMR (400MHz, CDCl3) δ8.63-8.49(m,3H),7.91(dd,J=12.6,6.8Hz,4H),7.69(t,J=7.6Hz,1H),7.60-7.53(m,2H),7.52-7.46(m,4H),7.40(d,J=8.6Hz ,2H),7.28(d,J=8.9Hz,2H),7.06(d,J=8.3Hz,1H),5.26(s,2H),4.16(t,J =7.6Hz,2H),1.71-1.67(m,2H),1.49-1.40(m,2H),0.97(t,J=7.4Hz,3H). 13 C NMR (101MHz, CDCl3) δ164.61,164.02,159.70,151.15,151.07,133.37,132.76,132.73,131.96,131.85,131.69,131.56,130.19,129.51,129.2 1,128.88,128.77,128.75,126.15,123.68,122.58,121.30,121.25,11 5.52,106.45,70.31,40.22,30.37,20.52,13.99.HRMS-ESI(m / z):[M+H] + Calcd.for C 35 H 31 NO5P + 576.1935,found576.1918,[M+Na] + Calcd.for C 35 H 30 NNaO5P + 598.1754, found 598.1744.

[0068] Example 3 Fluorescent probe for ONOO - Ratio response

[0069] The probe NA-DP prepared in Example 1-2 and ONOO - Added to PBS buffer (10mM, pH=7.4, V / V, EtOH / PBS=1 / 1), the concentration of NA-DP was 20μM, ONOO - The concentration is 20 μM, Figure 1 A. Figure 1 B respectively NA-DP (20 μM) probe in ONOO - Spectral graph of absorption and fluorescence changes in the presence of (20 μM) for 40 min;

[0070] Figure 1 C is NA-DP (20 μM) in ONOO - Fluorescence intensity variation spectra at 450nm and 550nm over time at 20μM (40μM) are plotted. The figure shows a significant decrease in the UV-vis absorption of NA-DP at 370nm within 40 minutes, while an increase at 450nm causes the solution color to change from colorless to yellow. Simultaneously, the fluorescence emission peak at 550nm significantly increases, while that at 450nm decreases, causing the solution's fluorescence color to change from blue to yellow. Figure 1 D. Figure 1 E were NA-DP (20 μM) and different concentrations of ONOO - Spectral diagram of absorption and fluorescence changes after 15 minutes of action (0-30μM); Figure 1 F is the fluorescence intensity of NA-DP (20 μM) after 15 min of reaction (I 550 nm / I 450nm )With ONOO - The concentration (0-30μM) curve. As shown in the figure, with the increase of ONOO - The concentration increased from 0μM to 30μM, and after 15min of response, the absorption and emission spectra showed a good linear change. Therefore, the fluorescence intensity lg(I 550 nm / I 450 nm ) and ONOO - There is a good linear correlation between the concentrations (R 2 =0.9810), and the detection limit (LOD) was 0.31 μM, indicating that the probe has the ability to sensitively detect low concentrations of ONOO - The self-correction capability of the ratiometric fluorescence signal further improves the detection accuracy.

[0071] Example 4 Fluorescent probe for ONOO - Selectivity, sensing mechanism and pH stability

[0072] The probe NA-DP prepared in Example 1-2 was respectively mixed with other competitive substances (1: blank control; 2: ONOO - ;3: H2O2; 4: NaClO; 5: ·OH; 6: 1 O2;7:O2 ·- ;8: Cys; 9: GSH; 10: HCy; 11: Na + ;12:K + ;13:Ca 2+) was added to PBS buffer (10 mM, pH = 7.4, V / V, EtOH / PBS = 1 / 1). After 40 min of incubation, the concentration of NA-DP was 20 μM and the concentration of other competing substances was 100 μM. The fluorescence intensity of NA-DP at 550 nm was detected. The results are as follows. Figure 2 As shown in A, after 40 min of incubation, ONOO - The fluorescence changes of NA-DP (20 μM) at 550 nm (1: blank control; 2: ONOO - ;3: H2O2; 4: NaClO; 5: ·OH; 6: 1 O2;7:O2 ·- ;8: Cys; 9: GSH; 10: HCy; 11: Na + ;12:K + ;13:Ca 2+ ). As can be seen from the figure, the fluorescence intensity of NA-DP at 550nm is only - In the presence of β-catenin, the fluorescence enhancement was observed within 40 min, while the fluorescence enhancement effect of other analytes was minimal. Figure 2 B is with ONOO - Normalized absorption spectra of NA-OH and NA-DP before and after incubation. - After the interaction, the absorption spectrum of NA-DP overlaps well with that of NA-OH, which indicates that the luminescent substance NA-OH is produced by hydrolysis of the NA-DP structure. Figure 2 C and D are the fluorescence spectra of NA-DP (20 μM) and NA-OH (20 μM) in PBS buffer (10 mM, V / V, EtOH / PBS = 1 / 1) as the pH changes. The emission spectra were obtained under 420 nm excitation. As can be seen from the figure, the absorption and emission of NA-DP are stable in the pH range of 4.0-10.0. - The absorbance of NA-OH changed to some extent in the pH range of 4.0 to 10.0, but the emission intensity of NA-OH remained significant. The above results indicate that NA-DP and NA-OH are pH stable, highlighting the potential of the probe to detect ONOO - The potential for mitophagy (pH 4.5-9.0) was then tracked.

[0073] Example 5 Exogenous ONOO in living cells - Fluorescence imaging.

[0074] Use ONOO -Donor 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazole hydrochloride (SIN-1) or ONOO - HeLa cells were pretreated with the scavenger uric acid (UA). The control group was treated with NA-DP (10 μM) for 30 minutes. The SIN-1 group was treated with SIN-1 (2 mM) for 3 hours, followed by NA-DP (10 μM) for 30 minutes.

[0075] The specific operation of the SIN-1 / UA group was as follows: HeLa cells were pre-incubated with UA (0.5 mM) for 1.5 h, then SIN-1 (2 mM) was added for 3 h, and finally NA-DP (10 μM) was added for 30 min. Figure 3 A is the fluorescence imaging of the Control group, SIN-1 group, and SIN-1 / UA group. Figure 3 B is a quantitative analysis of the fluorescence ratio (G / B) of the Control group, SIN-1 group, and SIN-1 / UA group. As shown in the figure, compared with the control group, SIN-1 pretreatment can lead to a significant increase in the fluorescence intensity ratio (G / B) of the green channel and blue channel of NA-DP in HeLa cells, while UA pretreatment cells significantly reduced the enhanced fluorescence intensity ratio (G / B) induced by SIN-1, thus confirming the effect of NA-DP on ONOO - Sensitivity to changes in levels. Blue channel: Ex / Em = 405 / 420-500 nm. Green channel: Ex / Em = 405 / 550-650 nm. Data are presented as mean ± SD of three replicate experiments. Statistical analysis: **P < 0.01, ***P < 0.001.

[0076] Example 6 Endogenous ONOO in living cells of drug-induced liver injury (DILI) - Fluorescence imaging

[0077] In the control group, HepG2 cells were treated with NA-DP (10 μM) for 30 min. In the APAP group, HepG2 cells were pre-incubated with 4 mM APAP for 48 h, followed by the addition of NA-DP (10 μM) for 30 min. In the APAP / NAC group, HepG2 cells were co-incubated with 4 mM APAP and 1 mM N-acetylcysteine ​​(NAC) for 48 h, followed by the addition of NA-DP (10 μM) for 30 min.

[0078] Figure 4 A is the fluorescence imaging of the Control group, APAP group, and APAP / NAC group.

[0079] Figure 4B is the quantitative analysis graph of the fluorescence ratio (G / B) of the Control group, APAP group, and APAP / NAC group.

[0080] As shown in the figure, HepG2 cells are used as a standard in vitro model for drug-induced liver injury (DILI) research. In the absence of acetaminophen (APAP), the fluorescence intensity ratio (G / B) of the green channel to the blue channel of NA-DP (10μM) in HepG2 cells is low. However, after 48h of pretreatment with 4mM APAP, a significant increase in the G / B value was observed after the addition of NA-DP (10μM), indicating that ONOO - Pre-incubation of HepG2 cells with N-acetylcysteine ​​(NAC) resulted in a significant decrease in the G / B value, which was similar to that of ONOO - These results demonstrate the effectiveness of NA-DP in monitoring the progression of liver injury and evaluating liver-protective interventions. Blue channel: Ex / Em = 405 / 420-500 nm. Green channel: Ex / Em = 405 / 550-650 nm. Data are presented as mean ± SD of three replicate experiments. Statistical analysis: **P < 0.01, ***P < 0.001.

[0081] Example 7 Mitochondrial targeting ability

[0082] HeLa cells were co-stained with NA-DP and the tracer Tracker and then observed using a confocal microscope. Figure 5 A. Figure 5 As shown in B, Figure 5 A is the co-localization fluorescence imaging of NA-DP (10 μM) with mitochondrial tracer (MitoTracker) (100 nM) and lysosomal tracer (LysoTracker) (100 nM) in HeLa cells. Figure 5 B is the co-localization fluorescence imaging of NAB-DP (10 μM) with mitochondrial tracer (MitoTracker) (100 nM) and lysosomal tracer (LysoTracker) (100 nM) in HeLa cells.

[0083] As can be seen from the figure, the fluorescence channel after merging NA-DP and LysoTracker showed a low Pearson correlation coefficient (PC) of 0.69, indicating that there is a certain degree of obvious localization between the NA-DP probe and the lysosomal compartment. When HeLa cells were co-stained with NA-DP and MitoTracker, a significantly higher PC value (0.88) was observed, demonstrating significant mitochondrial targeting ability. Fluorescence co-localization imaging experiments of NAB-DP and Tracker showed that the PC values ​​of MitoTracker (PC = 0.67) and LysoTracker (PC = 0.55) were very low, indicating that NAB-DP has poor mitochondrial targeting ability in the absence of the triphenylphosphine group. Among them, the blue channel: Ex / Em = 405 / 420-500nm. The red channel: Ex / Em = 561 / 570-650nm.

[0084] Example 8 Mitochondrial Autophagy Imaging

[0085] HeLa cells were sequentially incubated with NA-DP (10 μM) / NAB-DP (10 μM) and LysoTracker (100 nM), and then treated with carbonyl cyanide-3-chlorophenylhydrazone (CCCP) (50 μM). Figure 6 A is a fluorescence imaging image of the co-localization of NA-DP (10 μM) and lysosomal tracer (LysoTracker) (100 nM) after HeLa cells were induced to undergo mitophagy using carbonyl cyanide-3-chlorophenylhydrazone (CCCP) (50 μM). Figure 6 B is a fluorescence imaging image of co-localization of NAB-DP (10 μM) and lysosomal tracer (LysoTracker) (100 nM) after HeLa cells were induced to undergo mitophagy using carbonyl cyanide-3-chlorophenylhydrazone (CCCP) (50 μM). During the mitophagy process, the fluorescence of the green channel is clearly visible ( Figure 6 ), which is consistent with the pH response spectrum of NA-OH in solution. As the CCCP incubation time increases, the PC value of the combined fluorescence channel of NA-DP and LysoTracker gradually increases from 0.67 to 0.75. Figure 6 The fluorescence color at the location indicated by the middle arrow approaches the merged color, indicating a continuous increase in autophagy. With increasing CCCP treatment time, the merged fluorescence channel of the control compound NAB-DP and LysoTracker under the same conditions showed no enhanced PC, and the colors of the two channels at the location indicated by the arrow showed no overlap. These results confirm the ability of the probe NA-DP to accurately track mitophagy. For the green channel, Ex / Em = 405 / 550-650nm. For the red channel, Ex / Em = 561 / 570-650nm.

[0086] In summary, the novel probe for detecting peroxynitrite and subsequent oxidative-induced mitophagy of the present invention achieves a ratiometric fluorescence response to peroxynitrite by introducing a brominated diphenylphosphinyl group into a hydroxynaphthalimide fluorophore containing a triphenylphosphine group. The pH stability of the fluorescence ensures that the fluorescence signal has a consistent and significant intensity during the mitophagy tracing process. The addition-elimination reaction mechanism of the diphenylphosphinyl group with peroxynitrite greatly improves the selectivity of the probe, while the mitochondrial targeting of triphenylphosphine significantly improves the accuracy of mitophagy tracing. The probe successfully achieved in situ dynamic imaging of oxidative stress-induced mitophagy in cell experiments, providing new insights into the development of probes for monitoring the entire process of mitophagy from induction to occurrence and development.

[0087] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention, and all of these changes shall be included within the scope of protection of the present invention.

Claims

1. A naphthalimide compound, characterized in that The structure of the naphthaleneimide compound is shown in Formula I: R is selected from One of them.

2. A method for preparing the naphthalimide compound according to claim 1, characterized in that: The naphthalene imide compound is prepared from compound II and compound III, and the reaction formula for preparing the naphthalene imide compound from compound II and compound III is as follows:

3. The method for preparing a naphthalimide compound according to claim 2, wherein: The preparation of the naphthalene imide compound from compound II and compound III comprises the following steps: adding compound II and compound III into a reaction container, conducting a first reaction, adding dichloromethane after the first reaction is complete, filtering, concentrating the filtrate, and purifying to obtain the naphthalene imide compound.

4. The method for preparing a naphthaleneimide compound according to claim 3, wherein: The first reaction is carried out in a solvent, and the solvent is acetonitrile.

5. The method for preparing a naphthalimide compound according to claim 2, wherein: The compound II is prepared from compound IV and 2-hydroxyisoindole-1,3-dione. The reaction formula for preparing compound II from compound IV and 2-hydroxyisoindole-1,3-dione is as follows:

6. The method for preparing a naphthalimide compound according to claim 5, wherein: The preparation of compound II from compound IV and 2-hydroxyisoindole-1,3-dione includes the following steps: adding compound IV and 2-hydroxyisoindole-1,3-dione to a reaction container, conducting a second reaction, adding water after the second reaction is complete, adjusting the pH, filtering, and washing the filter residue to obtain the compound II.

7. The method for preparing a naphthalimide compound according to claim 6, wherein: The second reaction is carried out in a solvent, and the solvent is dimethyl sulfoxide.

8. The method for preparing a naphthalimide compound according to claim 6, wherein: The second reaction temperature is controlled at 75-85° C.; the second reaction is carried out under the protection of an inert gas; and the pH is adjusted to 2-3.

9. A probe, characterized in that: The invention comprises the naphthaleneimide compound according to claim 1.

10. Use of the naphthalimide compound according to claim 1 or the fluorescent probe according to claim 9 in preparing a kit for tracing oxidative stress-induced mitophagy and / or peroxynitrite activation.

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