Naphthalimide fluorophore-containing enamine N-oxide, preparation thereof and application of enamine N-oxide as Fe < 2 + > fluorescent probe

Abstract

The invention discloses enamine N-oxide containing a naphthalimide fluorophore, preparation of the enamine N-oxide and application of the enamine N-oxide in a Fe < 2 + > fluorescent probe. The structural formula of the enamine N-oxide containing the naphthalimide fluorophore is shown as a formula (I). The invention also provides a general synthesis method of the compound, which comprises the following steps: reacting a structural fragment containing an amino functional group with 4-bromo-1, 8-naphthalic anhydride, and carrying out hydroxyl substitution, bromine substitution, alkynyl addition and the like to generate enamine N-oxide. When being used as a fluorescent probe, the compound has high sensitivity, excellent temporal-spatial resolution and light stability, is suitable for dynamically monitoring the change of the Fe < 2 + > content in the ferroptosis process and related protein marking research, provides a new-generation molecular tool for ferroptosis mechanism research, and has important application prospects in the fields of disease model construction and drug screening. # imgabs0 #

Description

Technical Field

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

[0002] As an essential trace element in the human body, iron 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. The imbalance of this ion homeostasis has been proven to be closely related to various pathological processes: the Fenton reaction mediated by Fe 2+ can cause oxidative stress damage by generating reactive oxygen species (ROS), and thus participate in the occurrence and development of hepatitis, neurodegenerative diseases, and tumors; while systemic Fe 2+ deficiency leads to metabolic disorders such as iron deficiency anemia.

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

[0004] Existing Fe 2+ detection techniques mainly include atomic absorption spectrometry, electrochemical analysis, 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, and insufficient spatio-temporal resolution, and it is difficult to meet the requirements of real-time dynamic monitoring in vivo. In contrast, fluorescent probe technology has become the preferred solution for in vivo metal ion analysis due to its high sensitivity (detection limit up to nM level), excellent spatio-temporal resolution (subcellular localization accuracy), and real-time visualization detection ability.

[0005] Currently reported Fe 2+Fluorescent probes can be classified into three categories according to the signal response mode: (1) Fluorescence-enhanced probes (such as chelation systems based on rhodamine B); (2) Fluorescence-quenched probes (such as phenanthroline derivatives); (3) Ratiometric probes (dual-emission wavelength correction type). However, the existing probes still have the following key bottlenecks in meeting the actual needs of ferroptosis research: (i) Insufficient mitochondrial targeting ability, making it difficult to accurately reflect the Fe 2+ dynamics in the core region of ferroptosis; (ii) Poor photostability, with significant signal attenuation during long-term tracking; (iii) Lack of design of synchronous control compounds, making it difficult to exclude the interference of factors such as microenvironmental ROS. Summary of the Invention

[0006] The object of the present invention is to address the above problems in the existing technologies, and provide a mitochondrial-targeted fluorescent probe based on the enamine N-oxide structure, its preparation method and application. By introducing a mitochondrial targeting group and optimizing the electron donor-acceptor structure, the probe can achieve efficient capture and stable tracing of mitochondrial Fe 2+ This technological breakthrough provides a new generation of molecular tools for the study of ferroptosis mechanisms and has important application prospects in the fields of disease model construction and drug screening.

[0007] To solve the above technical problems, the technical solution adopted in the present invention is as follows:

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

[0009]

[0010] In formula (I), R is selected from a structural fragment; wherein represents the site where R is connected to N.

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

[0012]

[0013] In the second aspect of the present invention, there is provided a preparation method of the above enamine N-oxide containing a naphthalimide fluorophore, including 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): Perform a hydroxyl substitution reaction on intermediate R1 to obtain intermediate R2;

[0016] Step (3): Mix intermediate R2 with propargyl bromide to perform a bromine substitution reaction to obtain intermediate R3;

[0017] Step (4): Mix intermediate R3 with diethylhydroxylamine to perform an addition reaction to obtain an enamine N-oxide containing a naphthalimide fluorophore represented by the general formula (I).

[0018] The synthetic route of the reaction is as follows:

[0019]

[0020] More specifically, the compound represented by the general formula (I) of the present invention can be prepared by the above method. However, the conditions of this method, such as reactants, solvents, the amounts of compounds used, reaction temperature, reaction time required, etc. are not limited to the above explanations. The compounds of the present invention can also be conveniently prepared by optionally combining various synthesis methods described in this specification or known in the art, and such combinations can be easily carried out by those skilled in the art to which the present invention pertains.

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

[0022] Furthermore, the above use is specifically: the dynamic detection of the change in the content of Fe 2+ in the mitochondria of living cells during the induction of ferroptosis.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] The present invention provides a compound based on the enamine N-oxide structure and its pharmaceutical compositions, hydrates, isotope derivatives, chiral isomers, allosteric isomers, salts, prodrugs, and preparations, etc., and applies them as an Fe 2+ detection fluorescent probe. This probe has high sensitivity, excellent spatiotemporal resolution, and photostability, and is suitable for dynamically monitoring the change in the content of Fe 2+ during ferroptosis and related protein marker research, providing a new generation of molecular tools for the study of ferroptosis mechanisms and having important application prospects in the fields of disease model construction and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0026] Figure 2 are the ultraviolet absorption spectra of the probes FP and MiFP, where A is FP and B is MiFP.

[0027] Figure 3 Fluorescence emission spectra of probes FP and MiFP at 369 nm, where A is FP and B is MiFP.

[0028] Figure 4 Changes in fluorescence intensity of probes FP and MiFP after responding to different metal ions, where A is FP and B is MiFP.

[0029] Figure 5 For probes FP and MiFP with different concentrations of Fe 2+ Results of fluorescence titration experiments after response, where A is FP and B is MiFP.

[0030] Figure 6 Results of pH stability tests of probes FP and MiFP after incubation under different pH conditions, where A is FP and B is MiFP.

[0031] Figure 7 Results of fluorescence emission spectrum tests of probes FP and MiFP at different time points, where A is FP and B is MiFP.

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

[0033] Figure 9 Mitochondrial localization imaging diagram of probe MiFP.

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

[0035] Figure 11 Cell imaging diagram of probe MiFP during the induction of ferroptosis. Detailed implementation manners

[0036] The present invention will be further described below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the scope of the described embodiments.

[0037] Example 1:

[0038] Preparation of compound FP, including the following steps:

[0039]

[0040] Weigh 4-bromo-1,8-naphthalic anhydride (1135 mg, 5 mmol) into a 100 mL round-bottom flask, add ultradry 1,4-dioxane (50 mL) to completely dissolve it, and gradually add 70% aqueous ethylamine solution (386 mg, 6 mmol). Heat the reaction solution to reflux and stir the reaction overnight. After monitoring the reaction to completion by TLC, cool the reaction solution to room temperature, pour it into water (200 mL), and a pale yellow precipitate is produced. Filter it under reduced pressure, wash the filter cake with water 3 times, and dry it in vacuo to obtain a pale yellow solid R1 (1282 mg), with a yield of 84%. 1 HNMR(500MHz,CDCl 3 )δ8.63(d,J=7.3Hz,1H),8.56-8.49(m,1H),8.39(d,J=7.8Hz,1H),8.01(d,J=7.8Hz,1H),7.82(dd,J=8.3,7.5Hz,1H),4.23(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H). 13 C NMR(126MHz,CDCl 3 )δ163.4,163.4,133.2,131.9,131.1,131.0,130.6,130.2,128.9,128.0,123.2,122.3,35.6,13.3.HRMS(ESI):m / z[M+H] + calcd.forC14H10BrNO2:303.9895;found:303.9961.

[0041] Weigh the intermediate R1 (304 mg, 1 mmol), N-hydroxysuccinimide (127 mg, 1.1 mmol) and potassium carbonate (456 mg, 3.3 mmol) into a 25 mL round-bottom flask, add ultradry DMSO (3 mL), heat the reaction solution to 80 °C, and stir the reaction for 1.5 h. After monitoring the reaction to completion by TLC, add the reaction solution to 40 mL of water to obtain a mixture, adjust the pH to acidic (pH = 1) with hydrochloric acid solution (1 M), and a yellow precipitate is produced. Filter it under reduced pressure, wash the filter cake with water 3 times, and dry it in vacuo to obtain a yellow solid R2 (237 mg), with a yield of 97%. 1 H NMR(500MHz,DMSO)δ11.84(s,1H),8.50(d,J=8.3Hz,1H),8.43(d,J=7.2Hz,1H),8.32(d,J=8.2Hz,1H),7.76-7.70(m,1H),7.14(d,J=8.2Hz,1H),4.06(q,J=7.0Hz,2H),1.21(t,J=7.1Hz,3H). 13CNMR(126MHz, 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] + calcd. for C14H11NO3: 242.0739; found: 242.0808.

[0042] Weigh the intermediate R2 (122 mg, 0.5 mmol) and potassium carbonate (138 mg, 1 mmol) into a 25 mL round-bottom flask, add ultra-dry DMF (2 mL) to dissolve, and add propargyl bromide (89 mg, 0.75 mmol) dropwise. Heat the reaction solution to 80 °C and stir the reaction overnight. After monitoring the reaction to completion by TLC, add the reaction solution to 30 mL of water to obtain a mixture, and add ethyl acetate (20 mL × 3) to extract the mixture through a separatory funnel. After separating and combining the organic layers, wash with saturated NaCl and dry with anhydrous NaSO 4 Dry. The desiccant is removed by filtration, and the filtrate is concentrated under reduced pressure to obtain the crude product. The crude product is separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 7:1, v:v) to obtain a pale yellow solid R3 (103 mg), with a yield of 73%. 1 H NMR(500MHz, CDCl 3 ) 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(126MHz, CDCl 3 ) 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] + calcd. for C17H13NO3: 280.0895; found: 280.0966.

[0043] Weigh the intermediate R3 (70 mg, 0.25 mmol) into a 25 mL round-bottom flask, add 20% trifluoroethanol / chloroform (2 mL) to completely dissolve it, and dropwise add diethylhydroxylamine (111 mg, 1.24 mmol). Heat the reaction solution to 60 °C and stir for 24 h. Remove the solvent under reduced pressure, 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] The preparation of compound MiFP includes the following steps:

[0046]

[0047] Weigh 3-bromopropylamine hydrobromide (10 g, 45.68 mmol) and triphenylphosphine (11.98 g, 45.68 mmol) into a 200 mL round-bottom flask, add ultra-dry acetonitrile (60 mL) to completely dissolve them, heat the reaction solution to reflux, and stir for 18 h. After monitoring the reaction to completion by TLC, cool the reaction solution to room temperature. White precipitate forms in the flask. Filter under reduced pressure, wash the filter cake with ethyl acetate three times, and dry in vacuo to obtain the white solid R4 (15.23 g) with a yield of 69%. 11H 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 13C 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] Weigh 4 - bromo - 1,8 - naphthalic anhydride (499 mg, 1.8 mmol) and intermediate R4 (953 mg, 1.98 mmol) into a 100 mL round - bottom flask, add anhydrous ethanol (30 mL) and a small amount of triethylamine (3 mL) to dissolve them completely. Heat the reaction solution to reflux and stir the reaction overnight. After monitoring the completion of the reaction by TLC, remove the solvent under reduced pressure. The crude product is separated and purified by silica gel column chromatography (dichloromethane:methanol = 30:1, v:v) to obtain an orange solid R5 (778 mg) with a yield of 75%. 1 1H NMR (500 MHz, CDCl 3 ) 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 13C NMR (126 MHz, CDCl 3 ) 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] Weigh the intermediate R5 (660 mg, 1 mmol), N-hydroxysuccinimide (127 mg, 1.1 mmol) and potassium carbonate (456 mg, 3.3 mmol) into a 25 mL round-bottom flask, add ultradry DMSO (3 mL), and heat to 80 °C and stir for 3 h. After monitoring the reaction to completion by TLC, add the reaction solution to 40 mL of water to obtain a mixture, adjust the pH to acidic (pH = 1) with hydrochloric acid solution (1 M), an orange precipitate is produced, filter it under reduced pressure, wash the filter cake with water 3 times, and dry it in vacuo to obtain an orange solid R6 (503 mg) with a yield of 84%. 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] Weigh the intermediate R6 (503 mg, 0.84 mmol) and potassium carbonate (233 mg, 1.69 mmol) into a 25 mL round-bottom flask, add ultradry DMF (3 mL), add propargyl bromide (150 mg, 1.26 mmol) dropwise, heat the reaction solution to 80 °C, and stir overnight. After monitoring the reaction to completion by TLC, add the reaction solution to 50 mL of water to obtain a mixture, add ethyl acetate (30 mL × 3) to extract the mixture through a separatory funnel, separate and combine the organic layers, wash with saturated NaCl, and dry over anhydrous Na2SO4. The desiccant is removed by filtration, and the filtrate is concentrated under reduced pressure to obtain the crude product. The crude product is separated and purified by silica gel column chromatography (dichloromethane:methanol = 30:1, v:v) to obtain an orange solid R7 (471 mg) with a yield of 88%. 1 H NMR (500 MHz, CDCl 3)δ8.50(dd, J = 8.4, 1.1Hz, 1H), 8.41(dd, J = 7.3, 1.1Hz, 1H), 8.38(d, J = 8.3Hz, 1H), 7.82 - 7.65(m, 15H), 7.62(d, J = 7.4Hz, 1H), 7.14(d, J = 8.4Hz, 1H), 5.04(d, J = 2.4Hz, 2H), 4.37(t, J = 7.1Hz, 2H), 3.98 - 3.87(m, 2H), 2.68(t, J = 2.4Hz, 1H), 2.15(dd, J = 15.9, 9.5Hz, 2H). 13 C NMR(126MHz, CDCl 3 )δ164.2, 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] Weigh the intermediate R7(400mg, 0.63mmol) into a 25mL round-bottom flask, add 20% trifluoroethanol / chloroform(4mL) to dissolve it completely, add diethylhydroxylamine(281mg, 3.15mmol) dropwise, heat the reaction solution to 60°C, and stir the reaction for 24h. Cool the reaction solution to room temperature, remove the solvent under reduced pressure to obtain the crude product. The crude product is separated and purified by silica gel column chromatography(dichloromethane: methanol: ammonia water = 95:5:0.5, v:v:v) to obtain the probe MiFP(219mg) with a yield of 48%. 1 H NMR(500MHz, DMSO)δ8.52(d, J = 7.0Hz, 1H), 8.37(d, J = 6.2Hz, 1H), 8.21(d, J = 8.5Hz, 1H), 7.90 - 7.76(m, 15H), 7.60(t, J = 7.7Hz, 1H), 6.88(d, J = 8.5Hz, 1H), 4.22(t, J = 7.2Hz, 2H), 3.77(t, J = 15.1Hz, 2H), 2.93(t, J = 7.3Hz, 4H), 1.96(dd, J = 15.2, 7.1Hz, 2H), 1.21(t, J = 7.3Hz, 10H). 1313C NMR(126MHz, DMSO) δ 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 Tests of Probes FP and MiFP

[0053] 1 Experimental Reagents

[0054] 1.1 Probe Stock Solution

[0055] Weigh a certain amount of probes FP and MiFP, completely dissolve them with DMSO to obtain a stock mother solution with a concentration of 10 mM. When conducting tests, pipette the probe stock solution into HEPES (50 mM, pH 7.4) buffer solution for dilution to obtain test solutions with corresponding concentrations.

[0056] 1.2 Ionic Compound Solution

[0057] Weigh a certain amount of Na 2 SO 4 , K 2 SO 4 , CuSO 4 , MgSO 4 , Fe 2 (SO4) 3 , FeSO 4 , ZnSO 4 , Li 2 SO 4 , NiSO 4 , CoSO 4 Dissolve them in HEPES (50 mM, pH 7.4) buffer solution to obtain a metal ion stock mother solution with a concentration of 20 mM. When conducting tests, pipette the mother solution into the prepared probe solution for reaction.

[0058] 2 Experimental Methods

[0059] 2.1 Spectral Detection Method

[0060] Use HEPES buffer solution containing 0.1% DMSO as the blank background for zero adjustment. Pipette 2 mL of the probe FP test solution prepared at an appropriate concentration into a quartz cuvette, and detect the ultraviolet-visible absorption spectrum and fluorescence emission spectrum.

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

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

[0063] 2.2 Design idea of fluorescence probe spectral detection

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

[0065] 3 Experimental results

[0066] According to the above experimental method, the ultraviolet absorption spectrum and fluorescence emission spectrum of the probes FP and MiFP were tested. The mother solutions of the probes FP and MiFP were diluted into 50 μM test solutions with HEPES (50 mM, pH 7.4) buffer. 200 μM of Fe 2+ solution was added to the FP mother solution, and 100 μM of Fe 2+ solution was added to the MiFP mother solution and mixed well. After incubation for 1 h, ultraviolet absorption spectrum detection was carried out. The results are shown in Figure 2 .

[0067] The mother solution of the probe FP was diluted into a 0.25 μM test solution with HEPES (50 mM, pH 7.4) buffer. 20 μM of ferrous sulfate solution was added to it and mixed well. After incubation for 1 h, fluorescence emission spectrum detection was carried out. The mother solution of the probe MiFP was diluted into a 1 μM test solution with HEPES (50 mM, pH 7.4) buffer. 40 μM of ferrous sulfate solution was added to it and mixed well. After incubation for 1 h, fluorescence emission spectrum detection was carried out. 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 adding Fe 2+After that, the absorption peak at 369 nm was significantly weakened, and the absorption peak at 445 nm was significantly enhanced. It can be seen that the absorption peak at 369 nm is the absorption peak of probe FP, and the absorption peak at 445 nm is the absorption peak of intermediate R2. In the presence of Fe 2+ , probe FP was reduced to intermediate R2. Figure 3 The fluorescence emission spectrum results of probe FP in 2+ showed that the excitation wavelength of probe FP was 369 nm, the emission wavelength range was 390 - 600 nm, and the maximum emission wavelength was located at 453 nm. It was found that the fluorescence intensity of probe FP at 453 nm was significantly weakened. The above results indicate that probe FP can be used as a quenching fluorescence probe for detecting Fe

[0069] Figure 2 The ultraviolet absorption spectrum results of probe MiFP in 2+ showed that there was a strong absorption peak at 373 nm and a weak absorption peak at 450 nm without adding Fe 2+ . After adding Fe 2+ , the absorption peak at 373 nm was significantly weakened, and the absorption peak at 450 nm was significantly enhanced. It can be seen that the absorption peak at 373 nm is the absorption peak of probe MiFP, and the absorption peak at 450 nm is the absorption peak of intermediate R6. In the presence of Fe Figure 3 , probe MiFP was reduced to intermediate R6. The fluorescence emission spectrum results of probe MiFP in 2+ showed that the excitation wavelength of probe MiFP was 373 nm, the emission wavelength range was 390 - 600 nm, and the maximum emission wavelength was located at 454 nm. The fluorescence intensity of probe MiFP at 454 nm was significantly weakened. The above results indicate that probe MiFP is a quenching fluorescence probe for detecting Fe

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

[0071] The mother solutions of probes FP and MiFP were respectively diluted into test solutions of 0.25 and 1 μM using HEPES (50 mM, pH 7.4) buffer. Different metal ions (Fe 2+ , Fe 3+ , Na+, Cu 2+ , Mg 2+ , Co 2+ , Li+, Ni+, K+, Zn 2+ ) were added to them respectively, and after thorough mixing and incubation for 1 h, fluorescence emission spectrum tests were carried out. The results are shown in Figure 4 . The results showed that under the excitation of an excitation wavelength of 369 nm, only when Fe 2+After that, the fluorescence emissions of probe FP at 453 nm and probe MiFP at 454 nm decreased significantly, while the introduction of other metal ions had a weak or almost no effect on the fluorescence intensities of probes FP and MiFP, demonstrating that probes FP and MiFP have good selective responses to Fe 2+ ions.

[0072] Using HEPES (50 mM, pH 7.4) buffer, the stock solutions of probes FP and MiFP were diluted into test solutions of 0.25 and 1 μM respectively. Different concentrations of Fe 2+ solutions were added, and after incubation for 1 h, a fluorescence titration experiment was carried out. The results are shown in Figure 5 . The results showed that in the concentration range of 0 - 40 μM of Fe 2+ , there was a good linear relationship between the fluorescence intensity at the maximum emission wavelength of 453 nm of probe FP and the concentration of Fe 2+ , and the linear coefficient R 2 = 0.99143. In the concentration range of 0 - 60 μM of Fe 2+ , there was a good linear relationship between the fluorescence intensity at the maximum emission wavelength of 454 nm of probe MiFP and the concentration of Fe 2+ , and the linear coefficient R2 = 0.98927. According to the formula LOD = 3σ / k, the detection limits of probes FP and MiFP for Fe 2+ were 4.41 μM and 9.97 μM respectively, showing high response sensitivities.

[0073] Using HEPES buffer, the stock solutions of probes FP and MiFP were diluted into test solutions of 0.25 and 0.5 μM respectively. A solution of Fe 2+ with a concentration of 10 μM was added, and after incubation for 1 h under different pH (2 - 11) conditions, a pH stability test was carried out. The results are shown in Figure 6 . The results showed that the fluorescence intensities of probes FP and MiFP themselves gradually decreased with the increase of pH value, and the change trend was relatively gentle under physiological conditions (pH = 6 - 8). In the acidic to neutral environment (pH = 2 - 7), after adding Fe 2+ , the fluorescence response changes of probes FP and MiFP gradually increased with the increase of pH value, and there was the strongest fluorescence response at pH = 7; under alkaline conditions (pH = 9 - 11), Fe 2+ would form Fe(OH) 2 precipitate, and at the same time it was easily oxidized to Fe(OH) 3 precipitate, losing the response ability, so the fluorescence responses of probes FP and MiFP gradually weakened with the increase of pH value. The above results showed that probes FP and MiFP had good Fe 2+ response abilities under physiological conditions (pH = 7.4).

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

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

[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 detector 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 fixative, Triton X-100, 5% BSA blocking solution, DAPI staining solution, ammonium ferrous sulfate hexahydrate, FP probe solution, bipyridine.

[0082] 2 Experimental purpose

[0083] To determine whether the molecular probe can enter the cells and explore the binding sites of the biomacromolecules it targets.

[0084] 3 Experimental method

[0085] Hep3B cells were seeded in 35 mm glass-bottom culture dishes, and the number of cells to be tested was controlled to be 10 5 cells / dish, divided into four groups, and placed at 37 °C, 5% CO​2 Cultivate for 12 h in an incubator. After the cells are cultured to an appropriate density, discard the original culture medium, wash 3 times with PBS buffer. The four groups of culture dishes are divided into a control group and three experimental groups. The subsequent experimental steps are as follows:

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

[0087] Experimental groups:

[0088] (1) Fe 2+ Group: Add 1 mL of serum-free medium solution containing 100 μM ammonium ferrous sulfate hexahydrate to the culture dish. After incubating for 30 min, discard the original culture medium, wash 3 times with PBS buffer, add serum-free medium solution containing 10 μM FP, and incubate for 1 h;

[0089] (2) Fe 2+ +Bpy group: Add 1 mL of serum-free medium solution containing 100 μM ammonium ferrous sulfate hexahydrate (stock solution is 10 mM PBS solution) to the culture dish. After incubating for 30 min, discard the original culture medium, wash 3 times with PBS buffer, add 1 mL of serum-free medium solution containing 10 μM FP and 1 mM bipyridine (stock solution is 100 mM DMSO solution), and incubate for 1 h;

[0090] (3) Bpy group: Add 1 mL of serum-free medium solution containing 10 μM FP and 1 mM bipyridine to the culture dish and incubate for 1 h.

[0091] After staining is completed, remove the drug-containing culture medium, wash 3 times with PBS buffer, and perform fluorescence imaging with a laser scanning confocal microscope at an excitation wavelength of 405 nm. The collected fluorescence emission range is 430 - 530 nm.

[0092] 4 Experimental results

[0093] The intracellular fluorescence imaging of probe FP on human hepatocellular carcinoma cells Hep3B was measured by the above experimental method. The results are as Figure 8 shown. The results show that after incubating human hepatocellular carcinoma cells (Hep3B) with probe FP (10 μM) for 1 h and observing through a confocal microscope, FP shows strong fluorescence emission in the blue fluorescence channel. After introducing exogenous Fe 2+ , the fluorescence intensity decreases significantly. After introducing the iron chelator bipyridine, the fluorescence intensity recovers. The above results indicate that probe FP has good responsiveness to Fe 2+ in living cells and shows corresponding changes in fluorescence intensity.

[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 detector 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 fixative, Triton X-100, 5% BSA blocking solution, DAPI staining solution, mitochondrial red fluorescent probe MTDR, ammonium ferrous sulfate hexahydrate, MiFP probe solution, bipyridine, ferroptosis inducer Erastin, ferroptosis inhibitor Ferrostatin-1;

[0100] Experimental Purpose

[0101] Determine whether the molecular probe can enter the cells and explore the binding sites of the biomacromolecules it targets

[0102] 3 Experimental Methods

[0103] 3.1 Co-localization Imaging Experimental Method

[0104] Inoculate well-growing Hep3B cells into a 35 mm glass-bottom culture dish, control the number of cells to be tested at 10 5 cells / dish, and place them in an incubator at 37 °C and 5% CO 2 for 12 h.

[0105] Discard the original medium and wash 3 times with PBS buffer. Add 1 mL of serum-free medium solution containing 10 μM MiFP (the mother liquor is a 10 mM DMSO solution) to the culture dish and incubate in the incubator for 1 h.

[0106] Discard the original medium, wash 3 times with PBS buffer, add 1 mL of serum-free medium solution containing 200 nM mitochondrial red fluorescent probe MTDR (the mother liquor is a 1 mM DMSO solution), and incubate for 30 min.

[0107] After staining, discard the original medium, wash 3 times with PBS buffer, and perform fluorescence imaging using a laser scanning confocal microscope.

[0108] 3.2 Exogenous Fe 2+ Cell Imaging Experimental Method

[0109] Hep3B cells were seeded in 35-mm glass-bottom culture dishes, and the number of cells to be tested was controlled at 10 5 cells / dish. They were divided into four groups and placed in an incubator at 37 °C with 5% CO 2 for 12 h. The original culture medium was discarded, and the dishes were washed 3 times with PBS buffer. The four groups of culture dishes were divided into a control group and three experimental groups. The subsequent experimental steps were as follows:

[0110] Control group: Add 1 mL of serum-free medium solution containing 10 μM MiFP to the culture dish and incubate for 1 h.

[0111] Experimental groups:

[0112] (1) Fe 2+ group: Add 1 mL of serum-free medium solution containing 100 μM ammonium ferrous sulfate hexahydrate to the culture dish. After incubating for 30 min, discard the original culture medium, wash 3 times with PBS buffer, and then add 1 mL of serum-free medium solution containing 10 μM MiFP and incubate for 1 h;

[0113] (2) Fe 2+ +Bpy group: Add 1 mL of serum-free medium solution containing 100 μM ammonium ferrous sulfate hexahydrate (stock solution is 10 mM PBS solution) to the culture dish. After incubating for 30 min, discard the original culture medium, wash 3 times with PBS buffer, and then add 1 mL of serum-free medium solution containing 10 μM MiFP and 1 mM bipyridine (stock solution is 100 mM DMSO solution) and incubate for 1 h;

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

[0115] After staining, the drug-containing culture medium was removed, and the dishes were washed 3 times with PBS buffer. Fluorescence imaging was performed using a laser scanning confocal microscope at an excitation wavelength of 405 nm, and the collected fluorescence emission range was 430 - 530 nm.

[0116] 3.3 Experimental method for ferroptosis-induced cell imaging

[0117] Hep3B cells were seeded in 35-mm glass-bottom culture dishes, and the number of cells to be tested was controlled at 10 5 cells / dish. They were divided into four groups and placed in an incubator at 37 °C with 5% CO 2 for culture. The four groups of culture dishes were divided into a control group and three experimental groups. The subsequent experimental steps were as follows:

[0118] Experimental groups:

[0119] (1) Erastin group: Cells were cultured until they adhered to the wall. The original culture medium was discarded, and the cells were washed 3 times with PBS buffer. 2 mL of complete culture medium solution containing 10 μM ferroptosis inducer Erastin (the stock solution was a 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 until they adhered to the wall. The original culture medium was discarded, and the cells were washed 3 times with PBS buffer. 2 mL of complete culture medium solution containing 10 μM Erastin and 2 μM ferroptosis inhibitor Ferrostatin-1 (the stock solution was a 1 mM DMSO solution) was added to the culture dish and placed in the incubator for continued culture for 12 h;

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

[0122] Control group: Cultured synchronously with the experimental group without additional operations.

[0123] Remove the original culture medium, wash 3 times with PBS buffer, add 1 mL of serum-free culture medium solution containing 10 μM MiFP to the culture dish, and incubate for 1 h. After staining, remove the drug-containing culture medium, wash 3 times with PBS buffer, and perform fluorescence imaging with a laser scanning confocal microscope at an excitation wavelength of 405 nm. The collected fluorescence emission range is 430 - 530 nm.

[0124] 4 Experimental results

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

[0126] The response ability of the probe MiFP to Fe 2+ in cells was measured according to the above experimental method. The results are as Figure 10As shown. The results indicate that through confocal microscopy observation, MiFP exhibits strong fluorescence emission in the blue fluorescence channel. After introducing exogenous Fe 2+ , the fluorescence intensity significantly decreases. After introducing the iron chelator bipyridine, the fluorescence intensity recovers. These results suggest that the probe MiFP has good responsiveness to Fe 2+ in living cells and has potential application prospects in clinical practice.

[0127] The selective response ability of the probe MiFP to Fe 2+ in cells was measured according to the above experimental method, so as to detect the change of Fe 2+ content during the ferroptosis process of Hep3B cells. By incubating Hep3B cells with the ferroptosis inducer Erastin for 12 h to induce ferroptosis, and then staining with the probe MiFP to detect the change of Fe 2+ content. The results are as Figure 11 shown. The results show that after incubating Hep3B cells with the ferroptosis inducer Erastin for 12 h to induce ferroptosis, and then staining with the probe MiFP, a significant decrease in the fluorescence intensity in the blue fluorescence channel can be observed under confocal microscopy, while the fluorescence intensity recovers after introducing the ferroptosis inhibitor Ferrostatin-1. Meanwhile, in order to verify the labeling effect of the α,β-unsaturated imine ions generated by the reaction of the probe MiFP with Fe 2+ on proteins, we selected rhodamine B hydrazide as the fluorescence response probe for aldehyde groups. In the absence of ferroptosis, almost no fluorescence was observed in the red fluorescence channel of the cells. After ferroptosis occurred, with the increase of Fe 2+ content in the cells, the probe MiFP was reduced to form a large amount of protein aldehyde labeling and underwent a Schiff base reaction with rhodamine B hydrazide. Therefore, an obvious fluorescence image can be observed in the red fluorescence channel. The above results indicate that the probe MiFP can effectively detect the change of Fe 2+ content in cells during ferroptosis and simultaneously generate stable protein labeling, enabling the detection of related proteins during the ferroptosis process of cells.

[0128] In summary, in the present invention, a series of Fe 2+ selective fluorescence probes were synthesized by using naphthalimide as the fluorophore and enamine-N-oxide as the recognition group, and adding a targeting group to the side chain. They can achieve selective detection of Fe 2+ based on the enamine N-oxide structure and have no obvious response to other common metal ions. After being irradiated at wavelengths of 369 and 373 nm, the probe emits strong blue fluorescence. With the increase of Fe 2+ , and with the increase of Fe 2+With the addition of 2+ , the fluorescence intensity gradually decreases, and the recognition of Fe 2+ can be completed in a short time. At the same time, the probe has good biocompatibility and can be used to detect the change of intracellular Fe 2+ content during ferroptosis. The generated unsaturated imine fragment binds to the amino acid side chain to form a stable protein label, which can be recognized by other fluorescent probes. And it is expected to be used in iron ion-related biological research.

[0129] The above description of the present invention is illustrative rather than restrictive. Those skilled in the art understand that many modifications, variations or equivalents can be made within the spirit and scope defined by the claims, but they all fall within the protection scope of the present invention.

Claims

1. An enamine N-oxide containing a naphthalimide fluorophore, or an optical isomer, racemate, single enantiomer, possible diastereomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate thereof; characterized in that: The structural formula of the enamine N-oxide containing the naphthalimide fluorophore is shown in formula (I): In formula (I), R is selected from Structural fragment; Indicates the site where R and N are connected.

2. A method for preparing an enamine N-oxide containing a naphthalimide fluorophore as claimed in claim 1, characterized in that: The preparation method comprises the following steps: Step (1), reacting the R structural fragment containing an amino functional group with 4-bromo-1,8-naphthalene dicarboxylic anhydride to obtain an intermediate R1; Step (2), subjecting the intermediate R1 to a hydroxyl substitution reaction to obtain the intermediate R2; Step (3), mixing the intermediate R2 with propyne bromide to carry out a bromine substitution reaction to obtain the intermediate R3; Step (4), mixing the intermediate R3 with diethylhydroxylamine for addition reaction to obtain an enamine N-oxide containing a naphthalimide fluorophore as shown in the general formula (I); The synthetic route of the reaction is as follows:

3. The preparation method according to claim 2, characterized in that: In the hydroxyl substitution reaction, the intermediate R1, N-hydroxysuccinimide NHS, and potassium carbonate K2CO3 are dissolved in dimethyl sulfoxide DMSO and reacted at 80° C. for 1.5 h.

4. The preparation method according to claim 2, characterized in that: In the bromine substitution reaction, the intermediate R2 and potassium carbonate K2CO3 are dissolved in N,N-dimethylformamide DMF and reacted at 80° C. overnight.

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

6. The preparation method according to claim 2 or 5, characterized in that: In the addition reaction, the intermediate R3 is dissolved in a mixed solvent; the mixed solvent consists of trifluoroethanol TFE and chloroform CHCl3, wherein the volume fraction of trifluoroethanol TFE is 20%.

7. The enamine N-oxide containing a naphthalimide fluorophore as claimed in claim 1 is used as Fe 2+ Detection of fluorescent probes.

8. The use according to claim 7, characterized in that: The application is specifically: in the process of inducing cell ferroptosis, Fe 2+ Dynamic detection of content changes.

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