Naphthalimide compound as well as preparation method and application thereof

Abstract

The invention discloses a naphthalimide compound as well as a preparation method and application thereof. The invention provides a naphthalimide compound. The structure of the naphthalimide compound is shown as a formula I, wherein # imgabs 0 # R is selected from one of # imgabs 1 #. The invention also provides a preparation method of the naphthalimide compound. The fluorescent probe has the capability of accurately tracing mitochondrial autophagy, also has peroxynitrite ONOO-response and mitochondrial targeting capabilities, and can be applied to preparation of a kit for tracing oxidative stress induced mitochondrial autophagy and / or peroxynitrite activation.

Description

Technical Field

[0001] The invention patent 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. Mitochondria are a lysosome-dependent process that is used to clear dysfunctional mitochondria and maintain the integrity of properly 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 mainly based on changes in mitochondrial pH, viscosity, polarity, and membrane potential. However, only a few studies involve comprehensive monitoring of the entire mitophagy process, including induction and development. Since ROS plays a key role as an indicator in the oxidative stress-induced mitophagy model, combining ROS detection with mitophagy monitoring will provide a new perspective for further elucidation of the biological mechanism of mitophagy. Summary of the invention

[0004] The present invention has developed 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. Due to the diphenylphosphinyl and ONOO - The action of the probe NA-DP on ONOO is based on the addition-elimination reaction mechanism, so even under the interference of other reactive oxygen species, the - It also shows high selectivity. - In the presence of triphenylphosphine, the diphenylphosphine group is converted to a hydroxyl group, which enhances 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 an acidic pH environment, the mitochondrial autophagy induced by oxidative stress in cells was successfully traced by observing the overlap between the NA-DP probe channel and the lysosomal tracer (LysoTracker) channel.

[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 the 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 container, performing 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, and 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, 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 vessel, a second reaction, and after the second reaction is complete, adding water, adjusting the pH, filtering, and washing the filter residue to obtain the 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 the third aspect of the present invention, the present invention provides a probe comprising the naphthalimide 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 the 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 the preparation of a kit for tracing mitochondrial autophagy and / or peroxynitrite activation induced by oxidative stress.

[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 mitochondrial autophagy.

[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 that the fluorescence signal has consistent and significant intensity during the tracing of mitophagy. The addition-elimination reaction mechanism of the interaction between diphenylphosphinoyl and peroxynitrite greatly improves the selectivity of the probe, while the mitochondrial targeting of triphenylphosphine significantly improves the accuracy of tracing mitophagy. 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.

[0027] 3. The fluorescent probe prepared by the present invention combines diphenylphosphinyl and triphenylphosphine groups into the naphthalimide backbone, so that the probe has ONOO - Response 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 NA-DP, the diphenylphosphinyl group is converted to a hydroxyl group, which enhances the electron-donating ability 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 fluorescence intensity of the probe in an acidic pH environment, 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 For NA-DP to ONOO - Ratio response plot of ;

[0031] in, Figure 1 A. Figure 1 B respectively shows the 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 450nm and 550nm changing with time in the presence of (20μM);

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

[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 For 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 The fluorescence imaging and fluorescence ratio (G / B) quantitative analysis diagram 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 The fluorescence imaging and fluorescence ratio (G / B) quantitative analysis diagram 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 diagram of the fluorescent probe (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 diagrams of the co-localization of the fluorescent probes (NA-DP, NAB-DP) provided by the present invention and the lysosomal tracer (LysoTracker) (100 nM) respectively after carbonyl cyanide-3-chlorophenylhydrazone (CCCP) (50 μM) was used to induce mitochondrial autophagy in HeLa cells;

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

[0050] Figure 6 B is a fluorescence imaging image of the co-localization of NAB-DP (10 μM) and lysosomal tracer (LysoTracker) (100 nM) after mitochondrial autophagy in HeLa cells was induced by 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 to limit the scope of the present invention. In the following examples, if specific conditions are not specified, they are carried out according to normal conditions or conditions recommended by the manufacturer. The methods used are conventional methods known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise specified, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the recorded content may also be applied to the present invention.

[0052] Example 1-1

[0053]

[0054] NA-Br (500 mg, 0.76 mmol), 2-hydroxyisoindole-1,3-dione (248 mg, 1.52 mmol) and K2CO3 (315 mg, 2.28 mmol) were added to a 50 mL round-bottom flask. After dehydration and deoxygenation of the reaction system, DMSO (10 mL) was injected. The reaction mixture was heated to 80 ° C with stirring and stirred for 8 h under N2 protection. After cooling to room temperature, 80 mL of H2O was poured into the above solution, and the pH was adjusted to 2-3 by dropping 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, yield 80.2%).

[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, found 516.1697.

[0056] Example 1-2

[0057]

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

[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] NAB-Br (500 mg, 1.51 mmol), 2-hydroxyisoindole-1,3-dione (491 mg, 3.00 mmol) and K2CO3 (624 mg, 4.52 mmol) were added to a 50 mL round-bottom flask. After dehydration and deoxygenation of the reaction system, DMSO (10 mL) was injected. The reaction mixture was heated to 80 ° C with stirring and stirred for 10 h under N2 protection. After cooling to room temperature, 80 mL of H2O was poured into the above solution, and the pH was adjusted to 2-3 by dropping dilute hydrochloric acid. The orange-red precipitate was filtered and washed three times with H2O to obtain compound NAB-OH as a light yellow powder (346 mg, yield 85.3%).

[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] NAB-OH (100 mg, 0.37 mmol), compound III (173 mg, 0.45 mmol) and K2CO3 (103 mg, 0.75 mmol) were added to a 25 mL Schlenk tube. After dehydration and deoxygenation of the reaction system, ultra-dry CH3CN (5 mL) was added with a syringe under N2 atmosphere. Stir at room temperature for 12 h and dilute with DCM (10 mL). The above reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column using DCM / MeOH (100:1, v / v) as eluent to obtain compound NAB-DP as a yellow solid (155 mg, yield 72.5%).

[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 shows the 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 - The fluorescence intensity change spectrum at 450nm and 550nm with time under (20μM); it can be seen from the figure that within 40min, the UV-vis absorption of NA-DP at 370nm decreased significantly, while the UV-vis absorption at 450nm increased, and the color of the solution changed from colorless to yellow. At the same time, the fluorescence emission peak at 550nm was significantly enhanced, the fluorescence emission peak at 450nm was weakened, and the fluorescence color of the solution changed from blue to yellow. Figure 1 D. Figure 1 E are NA-DP (20 μM) and different concentrations of ONOO - (0-30μM) Spectrum of absorption and fluorescence changes after 15min of action; 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 0 μM to 30 μM, and after 15 min of response, the absorption and emission spectra showed a good linear change. 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 competitive 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 5-nitro-2-nitro-2-yl-4 ... 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 after hydrolysis of the NA-DP structure. Figure 2 C and D are the fluorescence spectra of NA-DP (20μM) and NA-OH (20μM) in PBS buffer (10mM, V / V, EtOH / PBS=1 / 1) with pH changes, and the emission spectra were obtained under 420nm 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 show the pH stability of NA-DP and NA-OH, 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 scavenger uric acid (UA). The specific operation of the control group was: HeLa cells were added with NA-DP (10μM) for 30min. The specific operation of the SIN-1 group was: HeLa cells were added with SIN-1 (2mM) for 3h, and finally NA-DP (10μM) was added for 30min.

[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 the blue channel of NA-DP in HeLa cells, while the cells pretreated with UA significantly reduced the enhanced fluorescence intensity ratio (G / B) induced by SIN-1, thus confirming the effect of NA-DP on ONOO - Sensitivity of level changes. Blue channel: Ex / Em = 405 / 420-500nm. Green channel: Ex / Em = 405 / 550-650nm. Data are expressed as mean ± SD of three parallel 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, NA-DP (10 μM) was added to HepG2 cells for 30 min; in the APAP group, NA-DP (10 μM) was added to HepG2 cells after they were pre-incubated with 4 mM APAP for 48 h, and then NA-DP (10 μM) was added to the cells for 30 min; in the APAP / NAC group, NA-DP (10 μM) was added to the cells after they were co-incubated with 4 mM APAP and 1 mM N-acetylcysteine ​​(NAC) for 48 h, and then NA-DP (10 μM) was added to the cells 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 and the blue channel of NA-DP (10 μM) in HepG2 cells is low. However, after 48 h of pretreatment with 4 mM APAP, a significant increase in the G / B value was observed after the addition of NA-DP (10 μM), indicating that ONOO - Preincubation of HepG2 cells with N-acetylcysteine ​​(NAC) resulted in a significant decrease in the G / B value, which was consistent with ONOO - The results showed that NA-DP was effective in monitoring the progression of liver damage and evaluating liver protection interventions. Blue channel: Ex / Em = 405 / 420-500nm. Green channel: Ex / Em = 405 / 550-650nm. Data are presented as mean ± SD of three parallel experiments. Statistical analysis: **P < 0.01, ***P < 0.001.

[0081] Example 7 Mitochondrial targeting capability

[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 NA-DP and LysoTracker are combined shows a lower 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 a 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 incubated with NA-DP (10 μM) / NAB-DP (10 μM) and LysoTracker (100 nM) in sequence, 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 mitochondrial autophagy 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 mitochondrial autophagy was induced in HeLa cells using carbonyl cyanide-3-chlorophenylhydrazone (CCCP) (50 μM). During the mitochondrial autophagy 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 of the position pointed by the middle arrow is close to the merged color, indicating that the degree of autophagy continues to increase. As the CCCP treatment time increases, the merged fluorescence channel of the control compound NAB-DP and LysoTracker does not show enhanced PC under the same conditions, and the colors of the two channels at the position pointed by the arrow do not overlap at all. The above results confirm the ability of the probe NA-DP to accurately track mitochondrial autophagy. Among them, the green channel: Ex / Em = 405 / 550-650nm. 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, and 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 and the 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 mitophagy induced by oxidative stress 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 is to 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, which shall all be included in the scope of protection of the present invention.

Claims

1. A naphthaleneimide compound, characterized in that The structure of the naphthalene imide 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 naphthaleneimide compound according to claim 2, characterized in that: 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, performing 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, characterized in that: The first reaction is carried out in a solvent, and the solvent is acetonitrile.

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

6. The method for preparing a naphthaleneimide compound according to claim 5, characterized in that: 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 into a reaction container, performing 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 naphthaleneimide compound according to claim 6, characterized in that: The second reaction is carried out in a solvent, and the solvent is dimethyl sulfoxide.

8. The method for preparing a naphthaleneimide compound according to claim 6, characterized in that: 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 as claimed in claim 1.

10. Use of the naphthylimide compound according to claim 1 or the fluorescent probe according to claim 9 in preparing a kit for tracing mitochondrial autophagy and / or peroxynitrite activation induced by oxidative stress.

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