An enamine n-oxide containing a naphthalimide fluorophore, its preparation and use as a fe2+ fluorescent probe

Abstract

The application discloses an enamine N-oxide containing a naphthalimide fluorescent group, a preparation method of the enamine N-oxide and application of the enamine N-oxide in a fluorescent probe. 2+ The application also provides a general synthesis method of the compound, wherein an amino functional group containing structural fragment is reacted with 4-bromo-1,8-naphthalic anhydride, and then the enamine N-oxide is generated through steps of hydroxyl substitution, bromine substitution and alkyne addition. 2+ When the compound is applied as the fluorescent probe, the compound has high sensitivity, excellent space-time resolution and light stability, is suitable for dynamically monitoring changes of Fe 2+ content in the iron death process and related protein marker research, and provides a new generation of molecular tools for iron death mechanism research, and has important application prospects in the fields of disease model construction and drug screening.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of organic small molecule fluorescent probes, and particularly relates to an enamine N-oxide containing a naphthalimide fluorophore, preparation thereof and application thereof as a Fe 2+ fluorescent probe. BACKGROUND

[0002] Iron, as an essential trace element in human body, plays a core role in key physiological processes such as oxygen transport, DNA metabolism, heme synthesis and nerve conduction. Intracellular iron exists in the form of Fe 2+ / Fe 3+ dynamic equilibrium, in which the reducing microenvironment makes Fe 2+ the main active form. Imbalance of this ion homeostasis has been proven to be closely related to various pathological processes: Fe 2+ mediated Fenton reaction can cause oxidative stress damage by producing reactive oxygen species (ROS), and thus participate in the occurrence and development of hepatitis, neurodegenerative diseases and tumors; and systemic Fe 2+ deficiency causes metabolic disorders such as iron deficiency anemia.

[0003] In recent years, ferroptosis has attracted widespread attention as a new type of iron-dependent programmed cell death. Its characteristic performance is the lipid peroxidation cascade reaction caused by the abnormal increase of intracellular Fe 2+ concentration, which is closely related to tumor treatment resistance, neurodegenerative pathology, etc. Therefore, dynamic monitoring of the change of Fe 2+ concentration in living cells (especially mitochondrial microenvironment) has important scientific value for elucidating the molecular mechanism of ferroptosis and developing targeted regulation strategies.

[0004] Existing Fe 2+ detection techniques mainly include atomic absorption spectrometry, electrochemical analysis and spectrophotometry, etc. Although these methods have the advantage of low detection limit, they generally have defects such as complex sample pretreatment, inability to achieve in-situ detection, insufficient spatiotemporal resolution, etc., and are difficult to meet the needs of real-time dynamic monitoring in vivo. In comparison, fluorescent probe technology has become the preferred solution for in vivo metal ion analysis, with high sensitivity (detection limit of nM level), excellent spatiotemporal resolution (subcellular localization accuracy) and real-time visual detection capability.

[0005] Currently reported Fe 2+The fluorescent probes can be divided into three categories according to the signal response mode: (1) fluorescence enhancement type probes (such as a chelating system based on rhodamine B); (2) fluorescence quenching type probes (such as phenanthroline derivatives); (3) ratio type probes (double emission wavelength correction type). However, the existing probes still have the following key bottlenecks in the actual needs of the study of ferroptosis: (i) insufficient mitochondrial targeting ability, which is difficult to accurately reflect the Fe 2+ dynamics in the core area of ferroptosis; (ii) poor light stability, and significant signal attenuation during long-term tracking; (iii) lack of design of synchronous control compounds, which is difficult to exclude the interference of microenvironment ROS and other factors. SUMMARY

[0006] The purpose of the present application is to solve the problems in the prior art, and provide a mitochondrial-targeting fluorescent probe based on enamine N-oxide structure, and a preparation method and application thereof. By introducing a mitochondrial-targeting group and optimizing the electron donor-acceptor structure, efficient capture and stable tracing of the probe to mitochondrial Fe 2+ are achieved. This technology provides a new generation of molecular tools for the study of ferroptosis mechanism, and has important application prospects in the fields of disease model construction and drug screening.

[0007] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect, the present application provides an enamine N-oxide containing a naphthalimide fluorophore, or an optical isomer, a racemate, a single enantiomer, a possible diastereoisomer thereof, or a pharmaceutically acceptable salt, a prodrug, a deuterated derivative, a hydrate, a solvate thereof; the structural formula of the enamine N-oxide containing the naphthalimide fluorophore is shown as formula (I).

[0009]

[0010] In formula (I), R is selected from a structural fragment; wherein represents the position of the connection between R and N.

[0011] That is, the structural formula of the enamine N-oxide containing the naphthalimide fluorophore is selected from any one of the following:

[0012]

[0013] In a second aspect, the present application provides a preparation method of the above-mentioned enamine N-oxide containing a naphthalimide fluorophore, comprising the following steps:

[0014] Step (1), reacting an R structural fragment containing an amino functional group with 4-bromo-1,8-naphthalic anhydride to obtain an intermediate R1;

[0015] Step (2), hydroxyl substitution reaction on intermediate R1 to obtain intermediate R2;

[0016] Step (3), bromine substitution reaction on intermediate R2 mixed with bromine propargyl to obtain intermediate R3;

[0017] Step (4), addition reaction on intermediate R3 mixed with diethyl hydroxylamine to obtain the enamine N-oxide containing naphthalimide fluorescent group shown in general formula (I).

[0018] The reaction synthesis route is as follows:

[0019]

[0020] More specifically, the compound shown in general formula (I) of the present application can be prepared by the above method, however, the conditions of the method, such as the reactants, solvents, the amount of compounds used, reaction temperature, reaction time required, etc. are not limited to the above explanations. The compound of the present application can also be conveniently prepared by optionally combining various synthesis methods described in the present specification or known in the art, and such combination can be easily carried out by a person skilled in the art to which the present application belongs.

[0021] In a third aspect of the present application, the above-mentioned enamine N-oxide containing naphthalimide fluorescent group is provided for use as a Fe 2+ detection fluorescent probe.

[0022] Further, the application specifically refers to the dynamic detection of the change of Fe 2+ content in the mitochondria of living cells during the process of inducing cellular ferroptosis.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] The present application provides a compound based on the structure of enamine N-oxide and its pharmaceutical composition, hydrate, isotopic derivative, chiral isomer, alloform, salt, prodrug and preparation, etc., and applies it to a Fe 2+ detection fluorescent probe. The probe has high sensitivity, excellent spatial and temporal resolution and light stability, and is suitable for dynamic monitoring of the change of Fe 2+ content during the process of ferroptosis and related protein marker research, which provides a new generation of molecular tool for ferroptosis mechanism research, and has important application prospects in the fields of disease model construction and drug screening. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The response mechanism of the fluorescent probe.

[0026] Figure 2 The ultraviolet absorption spectrum of the probe FP, MiFP, wherein A is FP and B is MiFP.

[0027] Figure 3 The fluorescence emission spectra of the probes FP and MiFP at 369 nm, wherein A is FP and B is MiFP.

[0028] Figure 4 The fluorescence intensity changes of the probes FP and MiFP after responding to different metal ions, wherein A is FP and B is MiFP.

[0029] Figure 5 The fluorescence titration experiment results of the probes FP and MiFP after responding to different concentrations of Fe 2+ , wherein A is FP and B is MiFP.

[0030] Figure 6 The pH stability test results of the probes FP and MiFP after incubation under different pH conditions, wherein A is FP and B is MiFP.

[0031] Figure 7 The fluorescence emission spectra test results of the probes FP and MiFP at different time points, wherein A is FP and B is MiFP.

[0032] Figure 8 The Hep3B cell imaging diagram of the probe FP.

[0033] Figure 9 The mitochondrial localization imaging diagram of the probe MiFP.

[0034] Figure 10 The Hep3B cell imaging diagram of the probe MiFP.

[0035] Figure 11 The cell imaging diagram of the probe MiFP in the process of inducing cell ferroptosis. DETAILED DESCRIPTION

[0036] The present application is further described below in conjunction with the accompanying drawings and examples, but the present application is not limited in the scope of the described examples.

[0037] Example 1:

[0038] The preparation of the compound FP includes the following steps:

[0039]

[0040] Take 4-bromo-1,8-naphthalic anhydride (1135 mg, 5 mmol) in a 100 mL round bottom flask, add super dry 1,4-dioxane (50 mL) to completely dissolve it, add 70% aqueous ethylamine solution (386 mg, 6 mmol) dropwise, heat the reaction to reflux, stir overnight. TLC monitor the reaction is complete, the reaction is cooled to room temperature, pour into water (200 mL), resulting in a light yellow precipitate, reduced pressure filter, filter cake washed with water 3 times, vacuum drying, to get a light yellow solid R1 (1282 mg), yield 84%. 1 HNMR (500 MHz, DMSO) δ 11.84 (s, 1H), 8.50 (d, J = 8.3 Hz, 1H), 8.43 (d, J = 7.2 Hz, 1H), 8.32 (d, J = 8.2 Hz, 1H), 7.76-7.70 (m, 1H), 7.14 (d, J = 8.2 Hz, 1H), 4.06 (q, J = 7.0 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, CDC13) δ 163.4, 163.4, 133.2, 131.9, 131.1, 131.0, 130.6, 130.2, 128.9, 128.0, 123.2, 122.3, 35.6, 13.3. HRMS (ESI): m / z [M+H] + calcd. for C14H10BrNO2: 303.9895; found: 303.9961.

[0041] Take intermediate R1 (304 mg, 1 mmol), N-hydroxysuccinimide (127 mg, 1.1 mmol) and potassium carbonate (456 mg, 3.3 mmol) in a 25 mL round bottom flask, add super dry DMSO (3 mL), the reaction is heated to 80 °C, stir for 1.5 h. TLC monitor the reaction is complete, the reaction is added to 40 mL of water to get a mixture, with hydrochloric acid solution (1M) to adjust the pH to acidic (pH = 1), resulting in a yellow precipitate, reduced pressure filter, filter cake washed with water 3 times, vacuum drying, to get a yellow solid R2 (237 mg), yield 97%. 1 HNMR (500 MHz, DMSO) δ 11.84 (s, 1H), 8.50 (d, J = 8.3 Hz, 1H), 8.43 (d, J = 7.2 Hz, 1H), 8.32 (d, J = 8.2 Hz, 1H), 7.76-7.70 (m, 1H), 7.14 (d, J = 8.2 Hz, 1H), 4.06 (q, J = 7.0 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H). 13CNMR (126 MHz, DMSO) δ 163.9, 163.2, 160.6, 133.9, 131.4, 129.5, 129.2, 125.9, 122.8, 122.2, 113.1, 110.4, 34.9, 13.7. HRMS (ESI): m / z [M+H] 242.0808. + calcd. for C14H11NO3: 242.0739; found: 242.0808.

[0042] Intermediate R2(122 mg, 0.5 mmol) and potassium carbonate (138 mg, 1 mmol) were weighed into a 25 mL round bottom flask, dissolved in super dry DMF (2 mL), propargyl bromide (89 mg, 0.75 mmol) was added dropwise, the reaction was warmed to 80 °C and stirred overnight. After TLC monitoring the reaction was complete, the reaction was added to 30 mL water to give a mixture, ethyl acetate (20 mL x 3) was added to extract the mixture through a separatory funnel, the organic layer was separated and combined, washed with saturated NaCl, dried over anhydrous NaS04. The drying agent was removed by filtration, the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 7: 1, v:v) to give yellow solid R3(103 mg) in 73% yield. 1 H NMR (500 MHz, CDC13) δ 8.62-8.54 (m, 3H), 7.75-7.66 (m, 1H), 7.17 (d, J = 8.3 Hz, 1H), 5.02 (d, J = 2.3 Hz, 2H), 4.23 (q, J = 7.1 Hz, 2H), 2.63 (t, J = 2.3 Hz, 1H), 1.33 (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, CDC13) δ 164.2, 163.6, 158.4, 132.9, 131.6, 129.4, 128.6, 126.16, 123.6, 122.5, 116.0, 106.6, 56.6, 35.4, 13.4. HRMS (ESI): m / z [M+H] 280.0966. + calcd. for C17H13NO3: 280.0895; found: 280.0966.

[0043] Take the intermediate R3 (70 mg, 0.25 mmol) in a 25 mL round-bottom flask, add 20% trifluoroethanol / chloroform (2 mL) to completely dissolve it, dropwise add diethyl hydroxylamine (111 mg, 1.24 mmol), and warm the reaction to 60°C and stir for 24 h. Remove the solvent under reduced pressure, and separate and purify the crude product by silica gel column chromatography (dichloromethane:methanol:ammonia water = 97:3:0.3, v:v:v) to obtain the probe FP (55 mg) with a yield of 60%. 1 H NMR (500 MHz, MeOD) δ 8.22 (dd, J = 8.3, 1.1 Hz, 1H), 8.09 (dd, J = 7.3, 1.1 Hz, 1H), 8.08-8.04 (m, 1H), 7.44-7.38 (m, 1H), 6.92 (d, J = 8.3 Hz, 1H), 6.70 (dt, J = 13.4, 5.1 Hz, 1H), 6.50 (d, J = 13.2 Hz, 1H), 4.91 (dd, J = 5.0, 1.3 Hz, 2H), 3.94 (q, J = 7.1 Hz, 2H), 3.39 (ddd, J = 32.3, 12.4, 7.1 Hz, 4H), 1.24 (t, J = 7.2 Hz, 6H), 1.15 (d, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, MeOD) δ 163.7, 163.2, 158.9, 139.4, 132.6, 130.7, 128.5, 128.1, 125.6, 124.0, 122.8, 121.6, 114.6, 106.3, 65.4, 63.9, 34.8, 12.2, 7.2. HRMS (ESI): m / z [M+H] + calcd. for C21H24N2O4: 369.1736; found: 369.1808.

[0044] Example 2:

[0045] Preparation of compound MiFP, comprising the following steps:

[0046]

[0047] Take 3-bromopropylamine hydrobromide (10 g, 45.68 mmol) and triphenylphosphine (11.98 g, 45.68 mmol) in a 200 mL round-bottom flask, add super-dry acetonitrile (60 mL) to completely dissolve it, warm the reaction to reflux, and stir for 18 h. After TLC monitoring shows that the reaction is complete, cool the reaction to room temperature, and a white precipitate is produced in the flask. Filter under reduced pressure, wash the filter cake with ethyl acetate 3 times, and dry under vacuum to obtain white solid R4 (15.23 g) with a yield of 69%. 1H NMR (500 MHz, DMSO) δ 7.93 - 7.79 (m, 15H), 3.84 - 3.75 (m, 2H), 3.01 (s, 2H), 1.92 - 1.81 (m, 2H). 13 C NMR (126 MHz, DMSO) δ 135.6, 134.1, 134.1, 130.9, 130.8, 118.9, 118.2. HRMS (ESI): m / z [M-HBr] - calcd. for C21H24BrNP + : 320.1557; found: 320.1568.

[0048] To a 100 mL round bottom flask, 4-bromo-1,8-naphthalic anhydride (499 mg, 1.8 mmol) and intermediate R4 (953 mg, 1.98 mmol) were weighed in, and added with anhydrous ethanol (30 mL) and a small amount of triethylamine (3 mL) to completely dissolve, the reaction was heated to reflux and stirred overnight. After TLC monitoring the reaction was complete, the solvent was removed under reduced pressure, and the crude product was separated and purified by silica gel column chromatography (dichloromethane:methanol = 30:1, v:v) to obtain orange solid R5 (778 mg) with a yield of 75%. 1 H NMR (500 MHz, CDCl3) δ 8.50 (d, J = 7.9 Hz, 2H), 8.25 (d, J = 9 Hz, 1H), 7.96 (d, J = 7.9 Hz, 1H), 7.88 - 7.66 (m, 16H), 4.40 (t, J = 7.2 Hz, 2H), 4.10 - 4.01 (m, 2H), 2.15 (dt, J = 9.5, 7.4 Hz, 2H). 13 C NMR (126 MHz, CDCl3) δ 163.4, 163.3, 135.2, 135.2, 133.7, 133.6, 133.5, 132.1, 131.2, 131.0, 130.7, 130.6, 130.4, 128.7, 128.1, 122.5, 121.6, 118.3, 117.6, 40.6, 29.7, 21.3. HRMS (ESI): m / z [M+H] + calcd. for [C33H26Br NO2P] + : 578.0879; found: 578.0878

[0049] Intermediate R5 (660 mg, 1 mmol), N-hydroxysuccinimide (127 mg, 1.1 mmol) and potassium carbonate (456 mg, 3.3 mmol) were weighed into a 25 mL round bottom flask, and super dry DMSO (3 mL) was added. The reaction was stirred at 80 °C for 3 h. After the reaction was completed as monitored by TLC, the reaction was added to 40 mL of water to give a mixture, which was adjusted to pH 1 with hydrochloric acid solution (1 M) to produce an orange precipitate. The precipitate was filtered under reduced pressure and washed with water three times. The filter cake was dried under vacuum to give orange solid R6 (503 mg) in 84% yield. 1 H NMR (500 MHz, DMSO) δ 8.52 (dd, J = 8.2, 1.1 Hz, 1H), 8.40 (dd, J = 7.3, 1.1 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 7.90 - 7.74 (m, 15H), 7.67 - 7.62 (m, 1H), 6.94 (d, J = 8.4 Hz, 1H), 4.21 (t, J = 7.1 Hz, 2H), 3.76 (dd, J = 14.6, 7.0 Hz, 2H), 1.96 (dd, J = 15.0, 7.3 Hz, 2H). 13 C NMR (126 MHz, DMSO) δ 164.6, 163.4, 135.4, 134.8, 134.2, 134.1, 131.2, 131.0, 130.7, 130.6, 130.3, 124.9, 124.2, 121.9, 119.1, 118.4, 111.9, 21.2, 19.0, 18.6. HRMS (ESI): m / z [M+H] + calcd. for [C33H27NO3P] + : 516.1723; found: 516.1726.

[0050] Intermediate R6 (503 mg, 0.84 mmol) and potassium carbonate (233 mg, 1.69 mmol) were weighed into a 25 mL round bottom flask, and super dry DMF (3 mL) was added. Bromopropyne (150 mg, 1.26 mmol) was added dropwise, and the reaction was stirred at 80 °C overnight. After the reaction was completed as monitored by TLC, the reaction was added to 50 mL of water to give a mixture, which was extracted by a separatory funnel with ethyl acetate (30 mL x 3). The combined organic layers were separated and washed with saturated NaCl and dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 30:1, v:v) to give orange solid R7 (471 mg) in 88% yield. 1H NMR (500 MHz, CDC13) δ 8.50 (dd, J = 8.4, 1.1 Hz, 1H), 8.41 (dd, J = 7.3, 1.1 Hz, 1H), 8.38 (d, J = 8.3 Hz, 1H), 7.82 - 7.65 (m, 15H), 7.62 (d, J = 7.4 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 5.04 (d, J = 2.4 Hz, 2H), 4.37 (t, J = 7.1 Hz, 2H), 3.98 - 3.87 (m, 2H), 2.68 (t, J = 2.4 Hz, 1H), 2.15 (dd, J = 15.9, 9.5 Hz, 2H). 13 C NMR (126 MHz, CDC13) δ 164.2, 163.6, 158.7, 135.2, 133.6, 133.6, 133.4, 131.7, 130.7, 130.6, 129.2, 129.0, 126.1, 123.4, 121.8, 118.3, 117.6, 115.1, 106.8, 56.8, 40.3, 29.7, 21.4, 21.0, 20.6. HRMS (ESI): m / z [M + H] + calcd for [C36H29NO3P] + : 554.1880; found: 554.1880.

[0051] Intermediate R7 (400 mg, 0.63 mmol) was weighed into a 25 mL round bottom flask, 20% trifluoroethanol / chloroform (4 mL) was added to dissolve it completely, diethylhydroxylamine (281 mg, 3.15 mmol) was added dropwise, the reaction was warmed to 60 °C and stirred for 24 h. The reaction was cooled to room temperature and the solvent was removed under reduced pressure to obtain the crude product. The crude product was separated and purified by silica gel column chromatography (dichloromethane:methanol:ammonia water = 95:5:0.5, v:v:v) to obtain probe MiFP (219 mg) with a yield of 48%. 1 H NMR (500 MHz, DMSO) δ 8.52 (d, J = 7.0 Hz, 1H), 8.37 (d, J = 6.2 Hz, 1H), 8.21 (d, J = 8.5 Hz, 1H), 7.90 - 7.76 (m, 15H), 7.60 (t, J = 7.7 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 4.22 (t, J = 7.2 Hz, 2H), 3.77 (t, J = 15.1 Hz, 2H), 2.93 (t, J = 7.3 Hz, 4H), 1.96 (dd, J = 15.2, 7.1 Hz, 2H), 1.21 (t, J = 7.3 Hz, 10H). 13C NMR (126 MHz, DMSO) d 164.6, 163.4, 135.4, 134.8, 134.2, 134.1, 131.2, 130.9, 130.7, 130.6, 130.3, 121.9, 119.1, 118.4, 111.8, 79.8, 53.2, 45.1, 41.8, 21.2, 11.5. HRMS (ESI): m / z [M+H] + calcd for [C40H40N2O4P] + : 643.2720; found: 643.2717.

[0052] Example 3: Spectral test of probes FP, MiFP

[0053] 1 Experimental reagents

[0054] 1.1 Probe stock solution

[0055] A certain amount of probe FP, MiFP was weighed and completely dissolved in DMSO to obtain a stock solution with a concentration of 10 mM. When testing, the probe stock solution was added to the HEPES (50 mM, pH 7.4) buffer for dilution to obtain the corresponding concentration of test solution.

[0056] 1.2 Ionic compound solution

[0057] A certain amount of Na2SO4, K2SO4, CuSO4, MgSO4, Fe2(SO4)3, FeSO4, ZnSO4, Li2SO4, NiSO4, CoSO4 was weighed and dissolved in HEPES (50 mM, pH 7.4) buffer to obtain a metal ion stock solution with a concentration of 20 mM. When testing, the stock solution was added to the prepared probe solution for reaction.

[0058] 2 Experimental method

[0059] 2.1 Spectral detection method

[0060] The HEPES buffer containing 0.1% DMSO was used as blank background zero, and 2 mL of the probe FP test solution prepared at the appropriate concentration was taken in a quartz cuvette using a pipette, and the ultraviolet-visible absorption spectrum and fluorescence emission spectrum were detected.

[0061] The collection range of the ultraviolet-visible absorption spectrum was 300-600 nm.

[0062] The excitation wavelength of the fluorescence emission spectrum was 369 nm, the emission wavelength range was 390-600 nm, the maximum emission wavelength was 453 nm, and the slit width was Wex=5 nm, Wem=5 nm.

[0063] 2.2 Fluorescent probe spectral detection design ideas

[0064] As shown in Figure 1 , the probe takes naphthalimide as the fluorescent group, and the enamine N-oxide as the Fe 2+ recognition group. The probe solution itself shows strong blue fluorescence. After reaction with Fe 2+ , elimination reaction occurs, releasing 4-hydroxynaphthalimide fluorescent group, and the fluorescence intensity is significantly weakened, thereby achieving the effect of recognizing Fe 2+ .

[0065] 3 Experimental results

[0066] The above experimental method was used to test the ultraviolet absorption spectrum and fluorescence emission spectrum of the probe FP and MiFP. The probe FP and MiFP mother liquor were diluted with HEPES (50 mM, pH 7.4) buffer to a test solution of 50 μM, 200 μM Fe 2+ solution was added to the FP mother liquor, and 100 μM Fe 2+ solution was added to the MiFP mother liquor, and mixed thoroughly. After incubation for 1 h, ultraviolet absorption spectrum detection was performed. The results are shown in Figure 2 .

[0067] The probe FP mother liquor was diluted with HEPES (50 mM, pH 7.4) buffer to a test solution of 0.25 μM, and 20 μM ferrous sulfate solution was added, mixed thoroughly, and incubated for 1 h. Fluorescence emission spectrum detection was performed. The probe MiFP mother liquor was diluted with HEPES (50 mM, pH 7.4) buffer to a test solution of 1 μM, and 40 μM ferrous sulfate solution was added, mixed thoroughly, and incubated for 1 h. Fluorescence emission spectrum detection was performed. The results are shown in Figure 3 .

[0068] Figure 2 The ultraviolet absorption spectrum results of the probe FP in 2+ show that there is a strong absorption peak at 369 nm and a weak absorption peak at 445 nm without the addition of Fe 2+ . After the addition of Fe 2+ , the absorption peak at 369 nm is significantly weakened, and the absorption peak at 445 nm is significantly enhanced. It can be seen that 369 nm is the absorption peak of the probe FP, and 445 nm is the absorption peak of the intermediate R2. Under the action of Fe 2+ , the probe FP is reduced to the intermediate R2. Figure 3The fluorescence emission spectrum results of the probe FP in the above solution show that the excitation wavelength of the probe FP is 369 nm, the emission wavelength range is 390-600 nm, and the maximum emission wavelength is at 453 nm. It can be found that the fluorescence intensity of the probe FP at 453 nm is significantly weakened. The above results show that the probe FP can be used as a fluorescence probe for detecting Fe 2+ by quenching.

[0069] Figure 2 The ultraviolet absorption spectrum results of the probe MiFP in the above solution show that there is a strong absorption peak at 373 nm and a weak absorption peak at 450 nm when no Fe 2+ is added. After Fe 2+ is added, the absorption peak at 373 nm is significantly weakened, and the absorption peak at 450 nm is significantly enhanced. It can be seen that the absorption peak of the probe MiFP is at 373 nm, and the absorption peak of the intermediate R6 is at 450 nm. Under the action of Fe 2+ , the probe MiFP is reduced to the intermediate R6. Figure 3 The fluorescence emission spectrum results of the probe MiFP in the above solution show that the excitation wavelength of the probe MiFP is 373 nm, the emission wavelength range is 390-600 nm, and the maximum emission wavelength is at 454 nm. The fluorescence intensity of the probe MiFP at 454 nm is significantly weakened. The above results show that the probe MiFP is a fluorescence probe for detecting Fe 2+ by quenching.

[0070] Example 4: Performance research of the probes FP and MiFP

[0071] The probe FP and MiFP mother liquor were respectively diluted into 0.25 μM and 1 μM test solutions using HEPES (50 mM, pH 7.4) buffer solution. Different metal ions (Fe 2+ , Fe 3+ , Na+, Cu 2+ , Mg 2+ , Co 2+ , Li+, Ni+, K+, Zn 2+ ) were added into the test solutions, and the mixtures were fully mixed and incubated for 1 h, and then fluorescence emission spectrum test was performed. The results are shown in Figure 4 . The results show that under the excitation of the excitation wavelength 369 nm, only after Fe 2+ is introduced, the fluorescence emission of the probe FP at 453 nm and the probe MiFP at 454 nm is significantly reduced, while the introduction of other metal ions has little or almost no effect on the fluorescence intensity of the probes FP and MiFP, proving that the probes FP and MiFP have good selective response to Fe 2+ .

[0072] The probe FP, MiFP mother liquor was diluted into 0.25, 1 μM test solution using HEPES (50 mM, pH 7.4) buffer, different concentrations of Fe 2+ solution were added, and after 1 h of incubation, fluorescence titration experiment was performed. The results are shown in Figure 5 . The results show that in the range of 0-40 μM Fe 2+ concentration, the fluorescence intensity of the probe FP at the maximum emission wavelength of 453 nm shows a good linear relationship with the Fe 2+ concentration, and the linear coefficient R 2 = 0.99143. In the range of 0-60 μM Fe 2+ concentration, the fluorescence intensity of the probe MiFP at the maximum emission wavelength of 454 nm shows a good linear relationship with the Fe 2+ concentration, and the linear coefficient R2= 0.98927. According to the formula LOD=3σ / k, the detection limit of the probe FP, MiFP for Fe 2+ is 4.41 μM, 9.97 μM, respectively, with high response sensitivity.

[0073] The probe FP, MiFP mother liquor was diluted into 0.25, 0.5 μM test solution using HEPES buffer, and 10 μM Fe 2+ solution was added, and after 1 h of incubation under different pH (2-11) conditions, pH stability test was performed. The results are shown in Figure 6 . The results show that the fluorescence intensity of the probe FP, MiFP itself gradually decreases with the increase of pH value, and the change trend is relatively flat under physiological conditions (pH=6-8). Under acidic to neutral environment (pH=2-7), after adding Fe 2+ , the fluorescence response change of the probe FP, MiFP gradually increases with the increase of pH value, and has the strongest fluorescence response at pH=7; under alkaline conditions (pH=9-11), Fe 2+ will generate Fe(OH)2precipitate, which is easily oxidized to Fe(OH)3precipitate, losing the response ability, so the fluorescence response of the probe FP, MiFP gradually weakens with the increase of pH value. The above results show that the probe FP, MiFP has good Fe 2+ response ability under physiological conditions (pH=7.4).

[0074] The probe FP mother liquor was diluted into 0.25 μM test solution using HEPES (50 mM, pH 7.4) buffer, and 20 μM Fe 2+solution, fluorescence emission spectrum test was carried out at different time points. The probe MiFP mother liquor was diluted into a test solution of 1 μM using a HEPES (50 mM, pH 7.4) buffer, 40 μM ferrous sulfate solution was added thereto, and fluorescence emission spectrum test was carried out at different time points. The results are as follows Figure 7 . The results show that the probes FP, MiFP and Fe 2+ respond rapidly within 5 min and reach a plateau within 60 min, and the fluorescence intensity no longer changes. It can be seen that the probes FP and MiFP can complete the response to Fe 2+ in a short time.

[0075] The above results show that the probes FP and MiFP have good Fe 2+ response ability under physiological conditions (pH = 7.4), better selective response, higher response sensitivity and can complete the response to Fe 2+ in a short time.

[0076] Example 5: Imaging study using molecular probe FP in living cells

[0077] 1. Experimental equipment and reagents

[0078] 1.1 Instruments

[0079] Portable LED ultraviolet flaw detection lamp (LUYOR), laser confocal microscope (Olympus, FV3000RS), constant temperature carbon dioxide incubator (THERMO).

[0080] 1.2 Reagents

[0081] DMEM medium, PBS, 4% paraformaldehyde tissue cell fixing solution, Triton X-100, 5% BSA blocking solution, DAPI staining solution, ferrous sulfate hexahydrate, FP probe solution, bipyridine.

[0082] 2. Experimental purpose

[0083] To determine whether the molecular probe can enter the cell and explore the binding site of the targeted biological macromolecule.

[0084] 3. Experimental method

[0085] Hep3B cells were inoculated in 35 mm glass bottom culture dishes, and the number of cells to be tested was controlled to be 10 5 cells / dish, and placed in a 37℃, 5% CO2 incubator for culture for 12 h. After the cells were cultured to the appropriate density, the original culture medium was discarded, and the cells were washed with PBS buffer for 3 times. Four groups of culture dishes were divided into one control group and three experimental groups, and the subsequent experimental steps were as follows:

[0086] Control group (Control): 1 mL of serum-free medium solution containing 10 μM FP was added to the culture dish, and incubated for 1 h.

[0087] Experimental group:

[0088] (1) Fe 2+ group: 1 mL of serum-free medium solution containing 100 μM ferrous ammonium sulfate hexahydrate was added to the culture dish, and after incubation for 30 min, the original medium was discarded, and the cells were washed with PBS buffer for 3 times, 1 mL of serum-free medium solution containing 10 μM FP was added, and incubated for 1 h;

[0089] (2) Fe 2+ +Bpy group: 1 mL of serum-free medium solution containing 100 μM ferrous ammonium sulfate hexahydrate (10 mM stock solution in PBS) was added to the culture dish, and after incubation for 30 min, the original medium was discarded, and the cells were washed with PBS buffer for 3 times, 1 mL of serum-free medium solution containing 10 μM FP and 1 mM bipyridyl (100 mM stock solution in DMSO) was added, and incubated for 1 h;

[0090] (3) Bpy group: 1 mL of serum-free medium solution containing 10 μM FP and 1 mM bipyridyl was added to the culture dish, and incubated for 1 h.

[0091] After the staining was completed, the drug-containing medium was removed, and the cells were washed with PBS buffer for 3 times, and fluorescence imaging was performed under excitation wavelength 405 nm by using a laser scanning confocal microscope, and the fluorescence emission range was 430-530 nm.

[0092] 4 Experimental results

[0093] The intracellular fluorescence imaging of probe FP on human liver cancer cells Hep3B was determined according to the above experimental method. The results are shown in Figure 8 The results show that after human liver cancer cells (Hep3B) were incubated with probe FP (10 μM) for 1 h, FP showed strong fluorescence emission in the blue fluorescence channel by confocal microscopy, and after the introduction of exogenous Fe 2+ , the fluorescence intensity was obviously reduced, and after the introduction of iron chelator bipyridyl, the fluorescence intensity was restored again. The above results show that the probe FP has good responsiveness to Fe 2+ in living cells, and presents corresponding fluorescence intensity changes.

[0094] Example 6: Imaging study using molecular probe MiFP in living cells

[0095] 1 Experimental equipment and reagents

[0096] 1.1 Instruments

[0097] Portable LED ultraviolet flaw detection lamp (LUYOR), laser confocal microscope (Olympus, FV3000RS), constant temperature carbon dioxide incubator (THERMO)

[0098] 1.2 Reagents

[0099] DMEM medium, PBS, 4% paraformaldehyde tissue cell fixation solution, Triton X-100, 5% BSA blocking solution, DAPI staining solution, mitochondrial red fluorescent probe MTDR, ferrous sulfate hexahydrate, MiFP probe solution, bipyridine, ferroptosis inducer Erastin, ferroptosis inhibitor Ferrostatin-1;

[0100] Purpose of the experiment

[0101] To determine whether the molecular probe can enter the cell and explore the binding site of the targeted biological macromolecule

[0102] 3 Experimental method

[0103] 3.1 Co-localization imaging experimental method

[0104] Hep3B cells in good growth condition were inoculated in 35mm glass bottom culture dishes, and the number of cells to be tested was controlled at 10 5 cells / dish, and placed in a 37℃, 5% CO2 incubator for 12h of culture.

[0105] The original culture medium was discarded and washed with PBS buffer for 3 times. 1mL of serum-free medium solution containing 10μM MiFP (10mM DMSO solution) was added to the culture dish, and incubated in the incubator for 1h.

[0106] The original culture medium was discarded and washed with PBS buffer for 3 times, 1mL of serum-free medium solution containing 200nM mitochondrial red fluorescent probe MTDR (1mM DMSO solution) was added, and incubated for 30min.

[0107] After staining, the original culture medium was discarded and washed with PBS buffer for 3 times, and fluorescence imaging was performed using a laser scanning confocal microscope.

[0108] 3.2 Exogenous Fe 2+ Cell imaging experimental method

[0109] Hep3B cells were inoculated in 35mm glass bottom culture dishes, and the number of cells to be tested was controlled at 10 5 cells / dish, and placed in a 37℃, 5% CO2 incubator for 12h of culture. The original culture medium was discarded and washed with PBS buffer for 3 times, and the four culture dishes were divided into one control group and three experimental groups, and the subsequent experimental steps were as follows:

[0110] Control group (Control group): 1 mL of serum-free medium solution containing 10 μM MiFP was added to the culture dish and incubated for 1 h.

[0111] Experimental group:

[0112] (1) Fe 2+ Group: 1 mL of serum-free medium solution containing 100 μM ferrous ammonium sulfate hexahydrate was added to the culture dish, incubated for 30 min, the original culture medium was discarded, washed with PBS buffer for 3 times, 1 mL of serum-free medium solution containing 10 μM MiFP was added, and incubated for 1 h;

[0113] (2) Fe 2+ +Bpy group: 1 mL of serum-free medium solution containing 100 μM ferrous ammonium sulfate hexahydrate (10 mM PBS solution) was added to the culture dish, incubated for 30 min, the original culture medium was discarded, washed with PBS buffer for 3 times, 1 mL of serum-free medium solution containing 10 μM MiFP and 1 mM bipyridine (100 mM DMSO solution) was added, and incubated for 1 h;

[0114] (3) Bpy group: 1 mL of serum-free medium solution containing 10 μM MiFP and 1 mM bipyridine was added to the culture dish, and incubated for 1 h.

[0115] After staining, the drug-containing medium was removed, washed with PBS buffer for 3 times, and fluorescence imaging was performed under excitation wavelength 405 nm by laser scanning confocal microscope, and the collected fluorescence emission range was 430-530 nm.

[0116] 3.3 Iron death induction cell imaging experiment method

[0117] Hep3B cells were inoculated in 35 mm glass bottom culture dishes, and the number of cells to be tested was controlled to be 10 5 cells / dish, and placed in a 37°C, 5% CO2 incubator for culture. The four culture dishes were divided into one control group and three experimental groups, and the subsequent experimental steps were as follows:

[0118] Experimental group:

[0119] (1) Erastin group: the cells were cultured to adhere, the original culture medium was discarded, washed with PBS buffer for 3 times, 2 mL of complete culture medium solution containing 10 μM iron death inducer Erastin (10 mM DMSO solution) was added to the culture dish, and placed in the incubator for continued culture for 12 h;

[0120] (2) Erastin + Fer-1 group: cells were cultured to adhere, the original culture medium was discarded, washed with PBS buffer for 3 times, 2 mL of complete culture medium solution containing 10 μM Erastin and 2 μM Ferrostatin-1 (stock solution was 1 mM DMSO solution) was added in the culture dish, and placed in the incubator for continuous culture for 12 h;

[0121] (3) Fer-1 group: cells were cultured to adhere, the original culture medium was discarded, washed with PBS buffer for 3 times, 2 mL of complete culture medium solution containing 2 μM Ferrostatin-1 was added in the culture dish, and placed in the incubator for continuous culture for 12 h.

[0122] Control group (Control group): synchronous culture with the experimental group, no additional operation.

[0123] The original culture medium was removed, washed with PBS buffer for 3 times, 1 mL of serum-free medium containing 10 μM MiFP was added in the culture dish, and incubated for 1 h. After staining was completed, the drug-containing culture medium was removed, washed with PBS buffer for 3 times, and fluorescence imaging was performed under excitation wavelength 405 nm by laser scanning confocal microscope, and the collected fluorescence emission range was 430-530 nm.

[0124] 4 Experimental results

[0125] The targeting ability of probe MiFP to mitochondria in cells was determined according to the above experimental method. Co-localization experiments of probe MiFP and commercial mitochondrial deep red fluorescent dye (Mito-Tracker Deep Red FM, MTDR) were carried out in Hep3B cells. The results are shown in Figure 9 The results show that MiFP and MTDR both produce fluorescence emission at the mitochondrial site. The blue fluorescence signal emitted by MiFP has good overlap with the red fluorescence signal emitted by MTDR. The above results show that probe MiFP not only has good cell membrane permeability, but also can specifically target mitochondria.

[0126] The response ability of probe MiFP to Fe 2+ in cells was determined according to the above experimental method. The results are shown in Figure 10 The results show that by confocal microscope observation, MiFP shows strong fluorescence emission in the blue fluorescence channel, and after the introduction of exogenous Fe 2+ , the fluorescence intensity is obviously reduced, and after the introduction of iron chelator bipyridine, the fluorescence intensity is restored. These results show that probe MiFP has good response to Fe 2+ in living cells, and has potential application prospect in clinic.

[0127] The effects of the probe MiFP on Fe in cells were determined using the experimental method described above. 2+ Selective responsiveness, thereby controlling Fe during ferroptosis in Hep3B cells. 2+ Changes in Fe content were detected. Ferrapoptosis was induced in Hep3B cells by incubating them with the ferroptosis inducer Erastin for 12 hours, followed by staining with the MiFP probe to detect Fe content. 2+ Changes in content. Results are as follows: Figure 11 As shown. The results indicated that incubating Hep3B cells with the ferroptosis inducer Erastin for 12 h induced ferroptosis. Subsequent staining with the MiFP probe revealed a significant decrease in fluorescence intensity in the blue fluorescence channel under confocal microscopy, which recovered upon introduction of the ferroptosis inhibitor Ferrostatin-1. Furthermore, to verify the interaction between the MiFP probe and Fe... 2+ To investigate the labeling effect of the α,β-unsaturated imine ions generated during the reaction on proteins, we used rhodamine B hydrazide as a fluorescent probe with an aldehyde group. In the absence of ferroptosis, almost no fluorescence was observed in the red fluorescence channel. However, after ferroptosis, fluorescence increased with the intracellular Fe... 2+ As the concentration increases, the probe MiFP is reduced, forming a large number of protein aldehyde labels, which then undergo a Schiff base reaction with rhodamine B hydrazide, resulting in a clear fluorescent image observed in the red fluorescence channel. These results indicate that the probe MiFP can effectively detect intracellular Fe during ferroptosis. 2+ Changes in protein content, along with the generation of stable protein markers, enable the detection of proteins involved in ferroptosis.

[0128] In summary, this invention utilizes a Fe group with naphthalimide as the fluorophore and enamine-N-oxide as the recognition group. 2+ Selective fluorescent probes and the addition of targeting groups to the side chains led to the synthesis of a series of Fe... 2+ Fluorescent probes. They are based on the enamine N-oxide structure, which can realize Fe 2+ The probe exhibits selective detection and shows no significant response to other common metal ions. After irradiation at wavelengths of 369 and 373 nm, it emits strong blue fluorescence, which, along with Fe... 2+ With the addition of [a specific ingredient], the fluorescence intensity gradually decreases, enabling the [process] of Fe [a specific substance] to be completed in a short time. 2+ The probe is capable of identifying iron. Furthermore, it possesses good biocompatibility and can be used to detect intracellular iron during ferroptosis. 2+ The change in concentration results in the unsaturated imine fragments produced by the reaction binding to amino acid side chains to form stable protein markers that can be recognized by other fluorescent probes. This technology holds promise for applications in iron-related biological research.

[0129] The above description of the present application is illustrative and not restrictive; many modifications and variations of the described embodiments will be apparent to those skilled in the art, in the spirit and scope of the claims.

Claims

1. An enamine N-oxide containing a naphthalimide fluorophore, or a pharmaceutically acceptable salt thereof; characterized in that, The structure of the naphthalimide fluorescent group-containing enamine N-oxide is shown as formula (I): R in formula (I) is selected from structural fragment; wherein represents the point of attachment of R to N.

2. A process for the preparation of an enamine N-oxide containing a naphthalimide fluorophore according to claim 1, characterized in that, The preparation method comprises the following steps: Step (1), reacting the R structure segment containing an amino functional group with 4-bromo-1,8-naphthalic anhydride to obtain an intermediate R1; Step (2), performing a hydroxyl substitution reaction on the intermediate R1 to obtain an intermediate R2; Step (3), mixing the intermediate R2 with bromopropargyl to perform a bromine substitution reaction to obtain an intermediate R3; Step (4), mixing the intermediate R3 with diethylhydroxylamine to perform an addition reaction to obtain the naphthalimide fluorescent group-containing enamine N-oxide shown in the general formula (I); The reaction synthesis route is as follows:

3. The preparation method according to claim 2, characterized in that, The hydroxyl substitution reaction dissolves the intermediate R1, N-hydroxysuccinimide (NHS) and potassium carbonate (K2CO3) in dimethyl sulfoxide (DMSO) together, and reacts at 80 DEG C for 1.5 h.

4. The preparation method according to claim 2, characterized in that, The bromine substitution reaction dissolves the intermediate R2 and potassium carbonate (K2CO3) in N,N-dimethylformamide (DMF) together, and reacts at 80 DEG C overnight.

5. The preparation method according to claim 2, characterized in that, The reaction temperature of the addition reaction is 60 DEG C, and the reaction time is 24 h.

6. The production method according to claim 2 or 5, characterized by, In the addition reaction, the intermediate R3 is dissolved in a mixed solvent; the mixed solvent is composed of trifluoroethanol (TFE) and chloroform (CHCl3), and the volume fraction of the trifluoroethanol (TFE) is 20%.

7. The naphthalimide fluorophore containing enamine N-oxide of claim 1 for use as a Fe 2+ application in detecting fluorescent probes.

8. Use according to claim 7, characterized in that, The application is specifically: dynamic detection of the change of Fe2+ content in the mitochondria of living cells in the process of inducing cellular ferroptosis.

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