Fluorophore free fluorescent probe, preparation method and application thereof

Abstract

The application belongs to the technical field of analytical chemistry, and relates to a class of free fluorophore type fluorescent probes and a preparation method and application thereof, in particular to a class of fluorescent probes for detecting tumor cells sensitive to ferriheme, divalent iron ions and artemisinin compounds and a preparation method and application thereof. The fluorescent dye of the point-on type probe is located on the side of the carbon free radical generated after the probe molecule is broken. The synthesized probe can detect ferriheme with high sensitivity and can also detect divalent iron ions. After the fluorescent probe molecule reacts with ferriheme, free fluorescent dyes are mainly generated, which has obvious advantages in detecting tumor cells at low concentrations of probe molecules. When the fluorescent probe is used to detect tumor cells sensitive to artemisinin compounds by using a flow cytometer, the fluorescent probe has the advantages of easy operation, high selectivity, high sensitivity and the like. Therefore, the fluorescent probe has potential practical application value in evaluating the killing ability of artemisinin compounds on different tumor cells.

Description

Technical Field

[0001] This invention relates to a class of fluorophore-free fluorescent probes, their preparation methods, and their applications in detecting heme or ferrous ions and in detecting tumor cells sensitive to artemisinin-like compounds, belonging to the field of analytical chemistry technology. Background Technology

[0002] Artemisinin (ART) is a sesquiterpene lactone compound isolated from Artemisia annua (a plant in the Asteraceae family) by Chinese scientist Tu Youyou in 1972. It possesses significant antimalarial activity. By modifying the C10 structure of artemisinin, compounds including dihydroartemisinin, artemether, and artesunate were obtained, improving the poor stability and low water solubility of artemisinin and significantly enhancing its antimalarial activity, playing a crucial role in the treatment of malaria. With ongoing research, scientists have discovered that artemisinin-like compounds not only exhibit significant efficacy in antimalarial treatment but also show promising antitumor effects, with their anticancer activity attracting increasing attention. Studies have indicated that artemisinin-like compounds can inhibit the growth of myeloid leukemia cells, ultimately leading to apoptosis.

[0003] Current evidence suggests that the level of heme is crucial for the antimalarial and antitumor effects of artemisinin-like compounds. Heme interacts with the peroxide bridges in the structure of artemisinin-like compounds, causing them to break and generate oxygen free radicals, which then rearrange into carbon free radicals to exert their effects. By increasing or inhibiting the level of heme in tumor cells, the inhibitory effect of artemisinin-like compounds on tumor cells can be significantly enhanced or diminished. Therefore, developing a tool to detect the level of heme in different tumor cells is expected to screen for tumor cells sensitive to artemisinin-like compounds and will also help to further understand how these compounds exert their antitumor effects. Traditional methods for detecting heme levels generally measure total heme levels. Gene-encoding probes for detecting heme developed in recent years are also susceptible to interference from hemin and zinc porphyrin (ZnPP) and are relatively complex to operate. In addition, the level of heme in cells is relatively low compared to that in malaria parasites, so a simple, sensitive, and practical method is needed to detect the level of heme in different tumor cells.

[0004] Fluorescent probes can specifically react or bind to analytes through recognition groups, altering the probe's photophysical properties and causing changes in fluorescence signals. These changes allow for the detection of specific analytes. Fluorescent probe analysis has been successfully applied in various disease studies due to its advantages such as ease of operation, high selectivity, high sensitivity, and real-time online monitoring. The peroxide bridge in artemisinin-like compounds is a group that specifically reacts with heme; therefore, it can be used to develop fluorescent probes for detecting heme. Similar to artemisinin-like compounds, the peroxide bridge in the structure of scutellarin is also key to its antimalarial effect. Its interaction with heme involves a free radical-mediated process: first, an oxygen free radical is generated, then rearranges into a carbon free radical. However, unlike artemisinin-like compounds where the rearrangement occurs internally, scutellarin undergoes a breakage after rearrangement, splitting the molecule into two parts. This characteristic can be used to develop free radical-mediated, glowing fluorescent probes to detect heme and tumor cells sensitive to artemisinin-like compounds.

[0005] In addition, the binding affinity between the fluorophore generated after probe activation and the protein is also a crucial factor to consider in probe design. Proteins can sometimes reduce the fluorescence signal of fluorophores bound to them; therefore, fluorophore-free probes exhibit better detection performance at low concentrations. Summary of the Invention

[0006] With the increasing discovery of the antitumor effects of artemisinin-like compounds, distinguishing which tumor cells are sensitive to these compounds has become a crucial issue. Addressing the challenges currently faced in detecting artemisinin-sensitive tumor cells, this invention, based on the characteristic that scutellarin reacts with heme to form oxygen free radicals, which then rearrange into carbon free radicals, causing the entire molecule to break into two parts, adjusts the positions of the fluorescent groups and fluorescence quenching groups on both sides of the scutellarin structural skeleton. A small-molecule fluorescent probe with a fluorescent dye located on the carbon free radical side generated after the probe molecule breaks down is designed and synthesized. The synthesized probe can detect heme and artemisinin-sensitive tumor cells with high sensitivity, and can also detect ferrous ions. The fluorophore containing free radicals generated after the fluorescent probe molecule reacts with heme preferentially binds to water in the reaction system, mainly generating a free fluorophore. Compared to existing fluorescent probes where the fluorophore is located on the other side of the probe molecule, the fluorescent probe provided by this invention can reduce the binding of the fluorescent dye portion generated after fragmentation to the detected tumor cell protein, thereby reducing the influence of the protein on the fluorescent dye and showing significant advantages in the detection of low-concentration probe molecules. Furthermore, this invention also provides a method for probe preparation and an operational procedure for its application.

[0007] The objective of this invention is to develop a class of fluorescent probes that are highly sensitive, practical, and easy to operate for detecting heme or ferrous ions, as well as tumor cells sensitive to artemisinin-like compounds.

[0008] The fluorescent probe involved in this invention reacts with heme in tumor cells, causing the probe molecule to break into two parts. The fluorescence quenching group and the fluorophore can no longer undergo fluorescence resonance energy transfer, resulting in a sharp increase in the fluorescence signal of the probe molecule. The synthesized fluorescent probe can effectively distinguish tumor cells sensitive to artemisinin-like compounds, and features simple operation, reliable results, low detection limit, and high selectivity.

[0009] Technical solution

[0010] This invention includes the following compounds or pharmaceutically acceptable salts thereof:

[0011]

[0012] The fluorescent probes used in this invention for detecting heme, ferrous ions, and tumor cells sensitive to artemisinin-like compounds have the following general structural formula XII:

[0013]

[0014] in:

[0015] Compound XII contains the following 8 isomers:

[0016]

[0017] The fluorescent probe involved in this invention for detecting heme, ferrous ions, and distinguishing artemisinin-based tumor cells is a free radical-mediated, turn-on fluorescent probe based on the scutellarin skeleton.

[0018] On the other hand, the present invention provides a method for preparing compound XII, the method comprising the following steps:

[0019] (1) Compound I and methylene blue were added to the photoreactor, dissolved in acetonitrile, oxygen was introduced, the xenon lamp was turned on, and the reaction was carried out at 0°C until completion. The oxygen supply was stopped, the mixture was transferred to room temperature, and then p-toluenesulfonic acid was added. The reaction was continued at room temperature for 10-12 hours. The reaction solvent was removed under reduced pressure, and the intermediate compound II was obtained by silica gel column chromatography purification.

[0020]

[0021] The reaction is preferably carried out under xenon lamp irradiation in a photoreactor, with the ice-water mixture being refluxed and stirred around the periphery to prevent excessively high local temperatures during the 0°C reaction process under xenon lamp irradiation.

[0022] Acetonitrile is preferred as the reaction solvent.

[0023] The optimal reaction time at 0℃ is 0.5-2 hours depending on the amount of material fed, and the optimal reaction time at room temperature is 10-12 hours.

[0024] For silica gel column chromatography purification, petroleum ether / ethyl acetate is preferred as the eluent, and gradient elution is preferred as the elution method.

[0025] (2) Ozone was bubbled into the dichloromethane-78°C cooled solution of intermediate compound II obtained in step (1) until the reaction solution changed from colorless to light blue. After the reaction was completed, the solution was transferred to room temperature, dimethyl sulfide was added, and the mixture was stirred for 30 minutes. The reaction solvent was removed under reduced pressure, and intermediate compound III was obtained by silica gel column chromatography.

[0026]

[0027] The preferred method for introducing ozone into the reaction flask is bubbling, which allows for a more uniform distribution of ozone in the reaction solvent.

[0028] The preferred reaction temperature is -78℃, and anhydrous ethanol is preferably cooled using a low-temperature reactor.

[0029] The preferred reaction solvent is dichloromethane.

[0030] Adjust the ozone introduction time according to the feed ratio until the reaction liquid turns light blue, then stop the ozone introduction, continue bubbling with oxygen for a few minutes, introduce argon gas, and remove excess oxygen.

[0031] Add dimethyl sulfide at 2.5 equivalents of intermediate II, and continue stirring at room temperature for 30 minutes.

[0032] For silica gel column chromatography purification, petroleum ether / ethyl acetate is preferred as the eluent, and gradient elution is preferred as the elution method.

[0033] (3) The intermediate compound III obtained in step (2) was dissolved in dichloromethane, and 1-triphenylphosphine-2-propanone was added at 0°C. The mixture was stirred for 15 minutes and then transferred to room temperature to continue the reaction for 33 hours. After the reaction was completed, the reaction solvent was removed under reduced pressure and purified by silica gel column chromatography to obtain intermediate compound IV.

[0034]

[0035] The preferred reaction solvent is dichloromethane.

[0036] The preferred reaction time at 0℃ is 15 minutes, and the preferred reaction time at room temperature is 30-35 hours.

[0037] For silica gel column chromatography purification, petroleum ether / ethyl acetate is preferred as the eluent, and gradient elution is preferred as the elution method.

[0038] (4) Place the intermediate compound IV obtained in step (3) and sodium borohydride in a reaction flask; add methanol at -15℃ and continue the reaction for 40 minutes. After the reaction is complete, add water to quench the reaction, extract with dichloromethane, collect the organic phase, dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain intermediate compound V.

[0039]

[0040] The preferred order for adding reactants is to add the reaction solvent last.

[0041] Methanol is preferred as the reaction solvent.

[0042] The preferred reaction temperature is -15℃.

[0043] The reaction time should be controlled between 40 minutes and 1 hour. If the time is too long, the carbonyl group on cyclohexane may be reduced.

[0044] Post-processing preferably involves quenching excess sodium borohydride with water to prevent the carbonyl group on cyclohexane from being reduced due to increased concentration temperature.

[0045] (5) The intermediate compound V obtained in step (4) was dissolved in dichloromethane at 0°C, followed by the addition of 4-nitrophenyl chloroformate and pyridine. The mixture was then transferred to room temperature and the reaction was continued until completion. The reaction solvent was removed under reduced pressure, and the intermediate compound VI was obtained by silica gel column chromatography purification.

[0046]

[0047] The preferred reaction solvent is dichloromethane, which is easy to remove during subsequent processing.

[0048] The intermediate compound V, 4-nitrophenyl chloroformate, is added, preferably at a temperature of 0°C.

[0049] The preferred reaction temperature is room temperature, and the preferred reaction time is 5 hours.

[0050] If a white precipitate forms after the reaction is complete, it is preferable to filter the mixture and then remove the reaction solvent under reduced pressure.

[0051] For silica gel column chromatography purification, petroleum ether / ethyl acetate is preferred as the eluent, and gradient elution is preferred as the elution method.

[0052] (6) Dissolve intermediate compound VI obtained in step (5) in dichloromethane, add Boc-ethylenediamine and 4-dimethylaminopyridine at room temperature, continue stirring until the reaction is complete, wash with saturated ammonium chloride solution and saturated sodium chloride solution in sequence, separate the organic layer, dry with anhydrous sodium sulfate, filter, remove solvent under reduced pressure, and purify by silica gel column chromatography to obtain intermediate compound VII.

[0053]

[0054] The preferred reaction temperature is room temperature.

[0055] The optimal reaction time is 5 hours.

[0056] The preferred reaction solvent is dichloromethane, which has good solubility in the raw materials and is easily removed under reduced pressure.

[0057] The standard for completion of washing is that the organic layer becomes colorless and transparent.

[0058] For silica gel column chromatography purification, petroleum ether / ethyl acetate is preferred as the eluent, and gradient elution is preferred as the elution method.

[0059] (7) The intermediate compound VII, lithium borohydride obtained in step (6), was dissolved in anhydrous diethyl ether at -6°C under an argon atmosphere. The mixture was stirred at -6°C until the reaction was complete. The mixture was then quenched with water, extracted with dichloromethane, and the organic phase was separated. The mixture was dried with anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure to obtain intermediate compound VIII.

[0060]

[0061] The preferred reaction solvent is anhydrous diethyl ether.

[0062] The reaction is preferably carried out under anhydrous and oxygen-free conditions. Before adding the reaction solvent, the air in the reaction flask must be replaced with argon gas to ensure an oxygen-free environment.

[0063] The preferred reaction temperature is -6℃.

[0064] The optimal reaction time is 6 hours.

[0065] After the reaction is complete, water quenching is the preferred post-treatment method.

[0066] (8) Dissolve intermediate compound VIII obtained in step (7) in dichloromethane, add 4-nitrophenyl chloroformate and pyridine at 0°C, react at room temperature for 4 days, quench the reaction with water, separate the organic phase, wash with saturated ammonium chloride solution, dry with anhydrous sodium sulfate, filter, remove solvent under reduced pressure, and purify by silica gel column chromatography to obtain intermediate compound IX.

[0067]

[0068] The reaction is preferably carried out at 0°C, and the reaction continues at room temperature.

[0069] The optimal reaction time at room temperature is 4 days.

[0070] The reaction is preferably carried out in dichloromethane.

[0071] For silica gel column chromatography purification, petroleum ether / ethyl acetate is preferred as the eluent, and gradient elution is preferred as the elution method.

[0072] (9) The intermediate compound IX obtained in step (8) was dissolved in dichloromethane, N,N-diisopropylethylamine was added until the solution was clear and transparent, 4-dimethylaminopyridine was added, and the reaction was continued at room temperature. After the reaction was completed, the solution was washed with saturated ammonium chloride solution and saturated sodium chloride solution in sequence, dried with anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the intermediate compound X was purified by thin-layer chromatography.

[0073]

[0074] The reaction is preferably carried out in dichloromethane for ease of subsequent processing.

[0075] The amount of N,N-diisopropylethylamine used is based on the standard that the reaction solution becomes clear and transparent, so that all the hydrochloride fluorescent dye is converted into free ammonia and dissolved in the reaction solvent.

[0076] The preferred reaction temperature is room temperature, and the reaction time is maintained at 48 hours.

[0077] After the reaction is complete, washing with a saturated ammonium chloride solution and a saturated sodium chloride solution can remove N,N-diisopropylethylamine and 4-dimethylaminopyridine from the reaction system, facilitating subsequent separation.

[0078] Thin-layer chromatography is preferred as the separation method, petroleum ether / ethyl acetate is preferred as the developing solvent, and dichloromethane / methanol is preferred as the subsequent eluent.

[0079] (10) Dissolve the intermediate compound X obtained in step (9) in methanol, add trifluoroacetic acid, react at room temperature until complete, remove the reaction solvent under reduced pressure to obtain intermediate compound XI, which can be directly used in the next step of the reaction.

[0080]

[0081] The reaction is preferably carried out in a methanol solution.

[0082] Trifluoroacetic acid used for Boc removal is added dropwise to ensure a complete reaction.

[0083] The preferred reaction time is 18 to 20 hours.

[0084] After the reaction is complete, it is preferable to proceed directly to the next step without any post-processing.

[0085] (11) Disperse red activated by 4-nitrophenyl chloroformate of the intermediate compound XI obtained in step (10) was dissolved in dichloromethane, and N,N-diisopropylethylamine was added to adjust the pH of the reaction solution to >9. Then 4-dimethylaminopyridine was added, and the reaction was continued at room temperature until completion. The solvent was removed under reduced pressure, and the compound XII was purified by thin-layer chromatography.

[0086]

[0087] The reaction is preferably carried out in dichloromethane, which is beneficial for the occurrence of the reaction and post-processing.

[0088] The pH value of the reaction solution is preferably detected using pH test paper.

[0089] The preferred separation method is to use dichloromethane / methanol as the developing solvent in thin-layer chromatography to separate and purify compound XII, followed by elution with dichloromethane / methanol (V / V 10 / 1).

[0090] All raw materials used in this invention are commercially available, or can be simply prepared from raw materials known in the art by methods known to those skilled in the art or methods disclosed in the prior art.

[0091] On the other hand, the present invention also provides a method for distinguishing tumor cells sensitive to artemisinin-like compounds using the above-mentioned compound XII, including but not limited to the following method: different tumor cells are seeded into cell culture dishes and grown for 24 hours; cell culture medium containing compound XII is added to the tumor cells to be tested and incubated for 1 hour in the form of medium change; cells are collected; the average fluorescence intensity of different tumor cells is detected by flow cytometry; and tumor cells sensitive to artemisinin-like compounds can be detected by comparing the average fluorescence intensity of different tumor cells.

[0092] The beneficial effects of this invention are: this type of fluorescent probe can detect related products of heme or ferrous ions, identify tumor cells sensitive to artemisinin-like compounds, including U937 cells, MV-4-11 cells, etc., with low detection sensitivity, fast response speed, strong practicality and simple operation.

[0093] From the above description and common knowledge known to those skilled in the art, the various advantages of such fluorescent probes can be understood:

[0094] (1) This type of fluorescent probe is used to detect tumor cells that are sensitive to artemisinin compounds. It has the advantages of being easy to operate, highly selective, and highly sensitive.

[0095] (2) After reacting with heme in tumor cells, this type of fluorescent probe molecule breaks into two parts. The fluorescent dye part containing free radicals preferentially reacts with water, and most of it remains in a free state, which can reduce the impact of binding proteins on the fluorescent dye itself. Compared with existing fluorescent probes where the fluorescent dye is located on the other side of the probe molecule, it shows a significant advantage in detecting tumor cells at low concentrations. Attached Figure Description

[0096] Figure 1 Mass spectrum of free fluorescent dye after the probe XIId reacts with heme.

[0097] Figure 2 Mass spectrum of α,β unsaturated ketone containing dispersed red fraction after probe XIId reacts with heme.

[0098] Figure 3 Changes in probe fluorescence intensity caused by the reaction of probe XIId with heme under ultraviolet light (365nm).

[0099] Figure 4 The change in probe fluorescence intensity caused by the reaction of probe XIId with heme, where hemin is the base for heme and L-SA is sodium ascorbate.

[0100] Figure 5 The change in probe fluorescence intensity caused by the reaction of probe XIId with ferrous ions, L-SA: sodium ascorbate, Fe 2+ : Ferrous ions (Fe2+).

[0101] Figure 6 Comparison of proteins in U937 cell lysates after the reaction of probe XIId and probe XIII with heme.

[0102] Figure 7 Comparison of probe XIId and probe XIII after reaction with heme to label bovine serum albumin (BSA): bovine serum albumin.

[0103] Figure 8 Comparison of labeled proteins after incubation of probe XIId and probe XIII with U937 cells.

[0104] Figure 9 Using flow cytometry, we compared the changes in cell fluorescence intensity after incubating U937 cells with different concentrations of probe XIId and probe XIII.

[0105] Figure 10 The proliferation of different tumor cells under the action of 10 μM dihydroartemisinin was compared using chemiluminescence cell viability assay. (DHA: dihydroartemisinin)

[0106] Figure 11 The changes in cell fluorescence intensity after incubation of probe XIId with different tumor cells were compared using flow cytometry. Detailed Implementation

[0107] Example 1: Synthesis of fluorescent probe XIId:

[0108]

[0109] (1) Synthesis of intermediate II

[0110] Compound I (1.5 g, 9.15 mmol) and methylene blue (374 mg, 1 mmol) were added to a photoreactor, followed by 20 mL of acetonitrile. Oxygen was bubbled through the reactor, and the xenon lamp was turned on. The reaction was carried out at 0 °C for 1 hour. The oxygen bubbling was stopped, and the mixture was transferred to room temperature. P-toluenesulfonic acid (500 mg, 2.9 mmol) was added, and the reaction was continued at room temperature for 12 hours. The reaction solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate (100 / 1-15 / 1, v / v)). This reaction was repeated 7 times to give a colorless oily intermediate II (5.67 g, 45%). HRESI-MS m / z: 197.05 [M+H] + (calcd 197.12 for C) 11 H 17 O3 + ).

[0111] (2) Synthesis of intermediate IIId

[0112] Ozone was bubbled into a 50 mL solution of intermediate compound II (5.0 g, 25.5 mmol) in dichloromethane cooled to -78 °C until the reaction solution changed from colorless to pale blue, approximately 1 hour. After the reaction was complete, the ozone generator was turned off, oxygen was continued to be introduced for 5 minutes, the oxygen cylinder was turned off, argon was introduced for 5 minutes, the mixture was transferred to room temperature, dimethyl sulfide (4.8 mL, 63.8 mmol) was added, and the mixture was stirred for 30 minutes. The reaction solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate (50 / 1-6 / 1, v / v)) to give a colorless oily intermediate IIId (808 mg, 16%). 1 H NMR (500MHz, CDCl3) δ9.33 (s, 1H), 4.42 (s, 1H), 2.61 (d, J = 13.0Hz, 1H), 2.46-2. 39(m,4H),1.90(d,J=12.4Hz,1H),1.53(s,3H),1.28(d,J=6.6Hz,3H).HRESI-MS m / z:199.0966[M+H] +(calcd 199.0965 for C) 10 H 15 O4 + ).

[0113] (3) Synthesis of intermediate IVd

[0114] Intermediate compound IIId (800 mg, 4.04 mmol) was dissolved in 20 mL of dichloromethane, and a dichloromethane solution (10 mL) of 1-triphenylphosphinyl-2-propanone (1.542 g, 4.84 mmol) was added. The mixture was stirred at 0 °C for 15 minutes and reacted at room temperature for 30 hours. After the reaction was completed, the mixture was concentrated by rotary evaporation, the reaction solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate (100 / 1-1 / 1, v / v)) to give a colorless oily compound IVd (481 mg, 50%). 1 H NMR (400MHz, CDCl3) δ6.50(d,J=16.4Hz,1H),6.03(d,J=16.4Hz,1H),4.40(q,J=4.0,2.8Hz,1H),2.69(dq,J=13.7,3.5Hz,1H),2.54(dt,J=15.9,2.8H z,1H),2.42(qd,J=6.9,3.5Hz,1H),2.38-2.31(m,1H),2.23(s,4H),1.91( ddd,J=13.7,3.1,1.6Hz,1H),1.62(s,3H),1.27(d,J=6.8Hz,3H).HRESI-MS m / z:239.1283[M+H] + (calcd 239.1278 for C) 13 H 19 O4 + )

[0115] (4) Synthesis of intermediate Vd

[0116] Intermediate compound IVd (460 mg, 1.93 mmol) and sodium borohydride (220 mg, 5.8 mmol) were placed in a reaction flask. 20 mL of methanol was added at -15 °C, and the reaction was continued for 40 minutes. After the reaction was complete, 20 mL of water was added to quench the reaction. The mixture was extracted with dichloromethane (40 mL × 2), and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a colorless oily intermediate, Vd. No further processing was required before proceeding to the next reaction step.

[0117] (5) Synthesis of intermediate VId

[0118] Intermediate compound Vd (460 mg, 1.92 mmol) was dissolved in 150 mL of dichloromethane. 4-Nitrophenyl chloroformate (1.156 g, 5.75 mmol) and pyridine (938 μL, 9.58 mmol) were added at 0 °C. The mixture was transferred to room temperature and the reaction was continued for 5 hours. After the reaction was completed, the reaction solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate (100 / 1-4 / 1, v / v)) to give a white solid intermediate VId (343 mg, 44.3%). 1 H NMR (500MHz, CDCl3) δ8.30-8.23(m,2H),7.53-7.46(m,2H),5.63(dd,J=16.2,0.8Hz,1 H),5.42(dd,J=16.2,7.6Hz,1H),5.24-5.10(m,1H),4.41-4.39(m,1H),2.72-2.64(m, 2H),2.48-2.36(m,1H),2.28(ddd,J=15.6,4.5,1.1Hz,1H),2.14-2.11(m,1H),1.89(d dd,J=13.5,3.2,1.8Hz,1H),1.59(s,3H),1.43(d,J=6.5Hz,3H),1.29(d,J=6.8Hz,3H). 13 C NMR (125MHz, CDCl3) δ207.53,155.89,152.15,145.52,135.70,127.43,125.41,1 22.48,82.14,82.08,48.89,43.73,36.94,28.53,22.37,20.22,11.20.HRESI-MS m / z:428.1325[M+Na] + (calcd 428.1316 for C) 20 H 23 NNaO8 + ).

[0119] (6) Synthesis of intermediate VIId

[0120] Intermediate compound VId (337 mg, 0.83 mmol) was dissolved in 20 mL of dichloromethane, followed by the addition of Boc-ethylenediamine (399 mg, 2.5 mmol) and 4-dimethylaminopyridine (305 mg, 2.5 mmol). The mixture was reacted at room temperature for 5 hours. The mixture was washed successively with saturated ammonium chloride solution and saturated sodium chloride solution until the dichloromethane layer was nearly colorless and transparent. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate (50 / 1-1 / 2, v / v)) to give colorless oily intermediate VIId (316 mg, 89.5%). 1 H NMR (500MHz, CDCl3) δ5.50 (d, J = 16.1Hz, 1H), 5.39-5.27 (m, 1H), 5.15-5.10 (m ,1H),5.04(s,1H),4.39-4.37(m,1H),3.50-3.03(m,4H),2.73-2.63(m,2H),2. 41-2.36(m,1H),2.27(dd,J=15.8,4.5Hz,1H),2.12-2.09(m,1H),1.86(ddd,J =13.8,2.9,1.6Hz,1H),1.56(s,3H),1.43(s,9H),1.29-1.22(m,6H).HRESI-MS m / z: 327.1915 [M-Boc+2H] + (calcd 327.1914 for C) 16 H 27 N2O5 + ).

[0121] (7) Synthesis of intermediate VIIId

[0122] Intermediate compound VIId (310 mg, 0.73 mmol) and lithium borohydride (160 mg, 7.3 mmol) were dissolved in 20 mL of anhydrous diethyl ether at -6 °C under an argon atmosphere. The reaction was continued at -6 °C for 12 hours. After the reaction was completed, 40 mL of water was added to quench the reaction. The mixture was extracted with dichloromethane (30 mL × 3) to separate the organic phase. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure to obtain a colorless oily intermediate VIIId (310 mg, 99.4%), which was directly used in the next reaction.

[0123] (8) Synthesis of intermediate IXd

[0124] Intermediate compound VIIId (310 mg, 0.724 mmol) was dissolved in 30 mL of dichloromethane. 4-Nitrophenyl chloroformate (437 mg, 2.17 mmol) and pyridine (353 μL, 3.62 mmol) were added at 0 °C. The reaction was continued at room temperature for 4 days. The reaction was quenched with 50 mL of water. The organic phase was separated, washed with saturated ammonium chloride solution (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The solution was purified by silica gel column chromatography (petroleum ether / ethyl acetate (100 / 1-1 / 1, v / v)) to give colorless oily intermediate IXd (150 mg, 35.0%), which was directly used in the next reaction.

[0125] (9) Synthesis of intermediate Xd

[0126] The intermediate compound IXd (60 mg, 0.1 mmol) and the hydrochloride fluorescent dye (BODIPY-NH2) were added. . HCl (33 mg, 0.11 mmol) was dissolved in 8 mL of dichloromethane. N,N-diisopropylethylamine was added until the hydrochloride fluorescent dye was completely dissolved. 4-Dimethylaminopyridine (37 mg, 0.3 mmol) was added, and the mixture was reacted at room temperature for 48 hours. After the reaction was completed, the mixture was washed successively with saturated ammonium chloride solution (10 mL × 3) and saturated sodium chloride solution (10 mL × 3). The mixture was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The mixture was purified by thin-layer chromatography (petroleum ether / ethyl acetate (1 / 2, v / v)). The solution was eluted with dichloromethane / methanol (10 / 1) to give an orange-red solid intermediate Xd (71 mg, 98.6%). 1 H NMR (400MHz, CDCl3) δ7.58 (s, 1H), 7.25 (s, 1H), 6.41 (dd, J = 4.0, 2.1Hz, 1H), 6.16 (s, 1H), 5 .99-5.96(m,1H),5.72-5.56(m,2H),5.22-4.95(m,4H),3.91(t,J=3.8Hz,1H),3.61-3.33(m ,2H),3.25-3.12(m,6H),2.56(s,3H),2.51(s,3H),2.14-1.99(m,2H),1.91-1.79(m,1H),1. 66(q,J=2.6Hz,1H),1.53(s,3H),1.42(s,9H),1.25(d,J=6.5Hz,3H),1.09(d,J=7.1Hz,3H). 13C NMR (100MHz, CDCl3) δ160.94,157.44,156.57,145.54,143.16,137.93,134.39,134.17,130.11,124.37,123.64,115.91,80.77,7 9.70,77.99,70.66,69.25,43.16,41.61,40.42,38.13,32.72,30.70,28.82,28.50,22.61,19.05,16.29,15.12,13.04.HRESI-MS m / z:718.3815[M+H] + (calcd 718.3793forC 35 H 51 BF2N5O8 + );735.4072[M+NH4 + ] + (calcd 735.4059 for C) 35 H 54 BF2N6O8 + )

[0127] (10) Synthesis of intermediate XId

[0128] Intermediate compound Xd (35 mg, 0.049 mmol) was dissolved in 3 mL of methanol, and trifluoroacetic acid (400 μL) was added in batches over 5 hours. The reaction was continued at room temperature for 20 hours. The reaction solvent was removed under reduced pressure to obtain intermediate compound XId, which could be used directly in the next reaction without post-treatment.

[0129] (11) Synthesis of fluorescent probe XIId

[0130] The intermediate compound XId obtained in step 10, 4-nitrophenyl chloroformate activated disperse red (Activated Qucher, 28 mg, 0.059 mmol), was dissolved in 3 mL of dichloromethane. N,N-diisopropylethylamine was added to adjust the pH of the reaction solution to >9, followed by the addition of 4-dimethylaminopyridine (18 mg, 0.147 mmol). The reaction was continued at room temperature for 20 hours. The solvent was removed under reduced pressure, and the solution was initially purified by thin-layer chromatography (dichloromethane / methanol (20 / 1, v / v)). Preparative liquid chromatography was used for further purification (conditions: isocratic separation, acetonitrile / water (72 / 28, v / v), 3 mL / min) to obtain a dark red solid fluorescent probe XIId (7.7 mg, 16.4%). 1H NMR(500MHz,CDCl3)δ8.32(d,J=8.6Hz,2H),8.09(d,J=8.8Hz,2H),7.97(d,J=8.7Hz,2H),7.55(s,1H),7.20(d,J=3.7Hz,1H),7.06(d,J=8.0Hz,2H),6.38(s,1H),6.16(s,1H),5.66-5.62(m,2H),5.20-5.14(m,9.8Hz,1H),5.04(s,1H),4.43-4.13(m,2H),3.92(s,1H),3.81(s,2H),,3.73-3.31(m,4H),3.29-2.95(m,6H),2.55(s,3H),2.49(s,3H),2.15-1.97(m,2H),1.89-1.83(m,1H),1.68-1.63(m,1H),1.52(s,3H),1.47-1.39(m,1H),1.36-1.30(m,4H),1.25(t,J=7.3Hz,3H),1.07(d,J=7.1Hz,3H). 13 C NMR(125MHz,CDCl3)δ161.09,160.71,160.40,160.08,159.76,157.61,156.81,156.61,147.07,145.67,143.29,137.73,134.42,134.23,130.14,129.79,125.29,124.38,123.74,120.80,116.50,115.85,115.57,114.22,80.88,78.06,70.91,69.53,61.38,50.94,47.68,42.94,41.18,40.67,38.01,32.64,32.57,30.49,28.73,22.52,19.04,16.31,15.13,13.19,12.40.HRESI-MS m / z:958.4451[M+H] + (calcd 958.4441for C 47 H 59 BF2N9O 10 + );980.4239[M+Na + ] + (calcd980.4260for C 47 H 58 BF2N9NaO 10 + )。

[0131]

[0132] (12) Hydrochloride fluorescent dye (BODIPY-NH2) . Synthesis of HCl

[0133]

[0134] 1) Synthesis of compound BN-1

[0135] Fmoc-beta-alanine (6.22 g, 20 mmol) was dissolved in 70 mL of anhydrous tetrahydrofuran, followed by the addition of 2,2'-dithiodipyridine (6.6 g, 30 mmol) and triphenylphosphine (10.48 g, 40 mmol). The reaction mixture was stirred under an argon atmosphere for 24 hours. In a separate clean reaction flask, pyrrole (8.04 g, 120 mmol) was dissolved in 90 mL of anhydrous tetrahydrofuran. Under an argon atmosphere, 1 M methyl magnesium bromide (90 mL, 90 mmol) was added to the pyrrole tetrahydrofuran solution using a constant pressure dropping funnel at -78 °C. The reaction was continued at -78 °C for 30 minutes, then the temperature was raised to -20 °C and reacted for 30 minutes. Finally, the temperature was lowered to -78 °C and stirred for 30 minutes. The thioester mixture that had reacted for 24 hours was added to the reaction solution of pyrrole and methyl magnesium bromide using a constant pressure dropping funnel at -78°C and stirred for another 30 minutes. The mixture was then transferred to room temperature and reacted for another 30 minutes. After the reaction was complete, the reaction was quenched with saturated ammonium chloride solution (300 mL), diluted with anhydrous diethyl ether (300 mL), and the organic layer was separated. The organic layer was washed with water (200 × 3 mL) and brine (500 mL), respectively, dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by silica gel column chromatography (petroleum ether / dichloromethane / ethyl acetate (50 / 50 / 1-1 / 1 / 1, v / v / v)) to give a white solid compound BN-1 (5.15 g, 71.5%). 1 H NMR (400MHz, DMSO-d6) δ11.81(s,1H),7.88(d,J=7.5Hz,2H),7.68(d,J=7.5Hz,2H),7.41(t,J=7.4Hz,2H),7.37-7.28(m,3H),7.09-7 .07(m,1H),7.00-6.93(m,1H),6.20-6.18(m,1H),4.29(d,J=7.0Hz,2H),4.24-4.18(m,1H),3.35-3.29(m,2H),2.92(t,J=7.0Hz,2H). 13C NMR(100MHz,DMSO-d6)δ188.22,156.59,144.45,141.28,132.22,128.13,127.59,126.01,125.71,120.64 ,117.18,110.31,65.85,47.26,40.68,40.47,40.26,40.05,39.84,39.63,39.43,38.20,37.13.HRESI-MS m / z:361.1542[M+H] + (calcd 361.1547 for C) 22 H 21 N2O3 + ).

[0136] 2) Synthesis of compound BN-2

[0137] Compound BN-1 (1.08 g, 3 mmol) and 2,4-dimethylpyrrole (501 μL, 4.8 mmol) were dissolved in 50 mL of dichloromethane at 0 °C and stirred for 10 min. Then, phosphorus oxychloride (600 μL, 600 mmol) was added dropwise to the reaction solution at 0 °C, and the reaction was continued for 1 h. The reaction solution was heated to 35 °C and stirred for 20 h. Then, N,N-diisopropylethylamine (2.62 mL, 12 mmol) and boron trifluoride ether (1.7 mL, 12 mmol) were added, and the reaction was carried out at room temperature for 4 h. After the reaction was completed, the solution was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / dichloromethane / ethyl acetate (10 / 10 / 1-2 / 2 / 1, v / v / v)). The reaction was repeated 5 times to give a red solid compound BN-2 (2.58 g, 35.3%). 1 H NMR (400MHz, DMSO-d6) δ7.90(d,J=7.5Hz,2H),7.67(d,J=7.4Hz,2H),7.62(d,J=6.1Hz ,2H),7.42(t,J=7.4Hz,2H),7.35(dd,J=7.4,1.2Hz,1H),7.31(dd,J=7.7,2.4Hz,1H),6 .48(dd,J=4.0,2.1Hz,1H),6.42(s,1H),4.37(d,J=6.7Hz,2H),4.21(t,J=6.5Hz,1H), 3.26(dd,J=8.8,5.5Hz,2H),3.17(d,J=5.2Hz,1H),3.12(t,J=7.6Hz,2H),2.48(s,6H). 13C NMR(100MHz,DMSO-d6)δ161.16,156.71,146.54,144.41,143.75,141.33,138.15,134.19,133.98,128.1 5,127.61,125.62,124.71,124.29,120.67,116.51,65.92,47.28,43.05,30.51,16.16,15.13.HRESI-MS m / z:466.2100[MF] + (calcd 466.2097 for C) 28 H 26 BFN3O2 + ).

[0138] 3) Hydrochloride fluorescent dye (BODIPY-NH2) . Synthesis of HCl

[0139] BN-2 (2.58 g, 5.32 mmol) was dissolved in 100 mL of dichloromethane. 211 μL LBU was added in four portions, every 15 minutes, for a total reaction time of 1 hour. Then, 10 mL of 0.5 N hydrochloric acid aqueous solution was slowly added dropwise, and the reaction was continued at room temperature for another 15 minutes. The mixture was filtered, and the solid was washed with ethyl acetate and dichloromethane to obtain an orange-red solid hydrochloride fluorescent dye (BODIPY-NH2). . HCl, 1.17 g, 73.1%. 1 H NMR(400MHz, DMSO-d6)δ8.42(s,3H),7.66-7.65(m,1H),7.60(dd,J=4.1,1.2Hz,1H),6.53(dd,J =4.0, 2.1Hz, 1H), 6.46 (d, J = 1.0Hz, 1H), 3.41-3.36 (m, 2H), 3.04 (t, J = 8.5Hz, 2H), 2.50 (s, 6H). 13 C NMR (100MHz, DMSO-d6) δ161.63,146.42,141.02,138.07,133.56,133.40,124.77,124.03,116.23,40.21,26.62,15.98,14.69.HRESI-MS m / z:264.1742[M-Cl] + (calcd 264.1478 for C) 13 H 17 BF2N3 + ).

[0140] (12) Synthesis of Disperse Red Activated by 4-Nitrophenyl Chloroformate

[0141]

[0142] Disperse Red (62.8 mg, 0.2 mmol) was dissolved in 5 mL of dichloromethane and cooled to 0 °C. 4-Nitrophenylchloroformate (121 mg, 0.6 mmol) and pyridine (106 μL, 1.0 mmol) were added, and the mixture was reacted at room temperature for 2 hours. After the reaction was complete, the solution was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate (50 / 1-5 / 1, v / v)) to obtain a crude dark red solid of 4-nitrophenylchloroformate activated disperse Red (Activated Qucher, 67 mg). This crude product can be used directly for the synthesis of compound XII without further purification. HRESI-MS m / z: 480.1514 [M+H] + (calcd 480.1514 for C) 23 H 22 N5O7 + ).

[0143] Experimental Example 1: Interaction of Probe XIId with Heme

[0144]

[0145] Fluorescent probe XIId (0.957 mg) and hemin (0.651 mg) were each dissolved in 1 mL of dimethyl sulfoxide (DMSO) to prepare a 1 mM working solution. Sodium ascorbate L-SA (1.98 mg) was dissolved in 1 mL of water to prepare a 10 mM working solution. 10 μL of each of these three solutions was added to 170 μL of 1×PBS and reacted at 37 °C for 2 h to obtain a free fluorescent dye product resulting from the reaction of free radicals with water and a disperse red product containing α,β-unsaturated ketones. Both products were identified by high-resolution mass spectrometry. The free fluorescent dye product resulting from the reaction of free radicals with water: HRESI-MS m / z: 434.2156 [MH] + (calcd 434.2068forC 21 H 27 BF2N3O4 - ()( Figure 1 Disperse Red product containing α,β-unsaturated ketones: HRESI-MS m / z: 541.2412 [M+H] + (calcd 541.2405 for C) 26 H 33 N6O7 + ()( Figure 2 This indicates that the fluorescent probe molecule can react with Heme and break into two parts, thus enabling its detection. When the reaction solution is placed under a UV lamp (365nm), obvious fluorescence can be observed. Figure 3 ).

[0146] Experiment Example 2: Changes in fluorescence intensity of probe XIId interacting with Heme using an ELISA reader.

[0147] The 1 mM fluorescent probe XIId solution and heme solution from Experiment 1 were diluted to prepare a 0.1 mM working solution. Sodium ascorbate L-SA (0.198 mg) was dissolved in 1 mL of water to prepare a 1 mM working solution. 1 μL of each of these three solutions was added to 97 μL of 1×PBS and incubated at 37°C for 1 h. Detection was performed using a microplate reader. A significant increase in fluorescence signal was observed at 530 nm under an excitation wavelength of 480 nm. Figure 4 ).

[0148] Experiment Example 3: Changes in fluorescence intensity of probe XIId interacting with ferrous ions using an ELISA reader.

[0149] The 1 mM fluorescent probe XIId solution from Experiment 1 was diluted to a 0.1 mM working solution. Ferrous sulfate heptahydrate (0.278 mg) was dissolved in 1 mL of water to prepare a 1 mM solution, which was then diluted to a 0.1 mM working solution. Sodium ascorbate L-SA (0.198 mg) was dissolved in 1 mL of water to prepare a 1 mM working solution. 1 μL of each of these three solutions was added to 97 μL of 1×PBS and incubated at 37°C for 1 h. The results were then analyzed using a microplate reader. An increase in fluorescence signal was observed at 530 nm under an excitation wavelength of 480 nm. Figure 5 ).

[0150] Experimental Example 4: 10% SDS-PAGE fluorescent gel detection probe XIId, and the invented probe XIII, after reacting with heme, labeled the proteins in U937 cell lysates and bovine serum albumin.

[0151] The 1 mM fluorescent probe XIId solution and heme solution from Experimental Example 1 were diluted to prepare 0.1 mM and 0.5 mM working solutions. The invented probe XIII (0.941 mg dissolved in 1 mL DMSO) was prepared to prepare a 1 mM DMSO solution and diluted to prepare 0.1 mM and 0.5 mM working solutions. Sodium ascorbate L-SA (1.98 mg) was dissolved in 1 mL water to prepare a 10 mM solution and diluted to prepare 1 mM and 5 mM working solutions.

[0152] 1×10 6U937 cells were seeded into 6-well plates and grown for 12 hours. Cells were then collected, and 530 μL of 0.1% NP40 / PBS solution was added. The cells were lysed at 4°C for 15 minutes and centrifuged at 15,000 rpm for 20 minutes at 4°C. The supernatant was collected, and 97 μL of each supernatant was added to five EP tubes, labeled as groups A, B, C, D, and E. Group A: Add 2 μL DMSO and 1 μL water. Group B: Add 1 μL of 0.1 mM probe XIId solution, 1 μL of 0.1 mM hemin solution, and 1 μL of 1 mM sodium ascorbate L-SA solution. Group C: Add 1 μL of 0.5 mM probe XIId solution, 1 μL of 0.5 mM hemin solution, and 1 μL of 5 mM sodium ascorbate L-SA solution. Group D: Add 1 μL of 0.1 mM probe XIII solution, 1 μL of 0.1 mM hemin solution, and 1 μL of 1 mM sodium ascorbate L-SA solution. Group E: Add 1 μL of 0.5 mM probe XIII solution, 1 μL of 0.5 mM hemin solution, and 1 μL of 5 mM sodium ascorbate L-SA solution. The reaction was carried out at 37℃ with shaking for 2 hours. 40 μL of each group was taken and 10 μL of 5× loading buffer was added. The mixture was boiled at 95℃ for 5 minutes and then added to 10% SDS-PAGE fluorescent gel at a rate of 20 μL per well. The gel was run and fluorescence was developed (Cy2, 473 nm excitation).

[0153] Prepare a 10 μM bovine serum albumin (BSA) solution, and take 97 μL of each solution into 5 EP tubes, labeling them as group FJ. Add 2 μL of BSA to group F. DMSO, 1 μL water, 1 μL of 0.1 mM probe XIId solution, 1 μL of 0.1 mM hemin solution, 1 μL of 1 mM sodium ascorbate L-SA solution added to Group G, 1 μL of 0.5 mM probe XIId solution, 1 μL of 0.5 mM hemin solution, 1 μL of 5 mM sodium ascorbate L-SA solution added to Group H, 1 μL of 0.1 mM probe XIII solution, 1 μL of 0.1 mM hemin solution, 1 μL of 1 mM sodium ascorbate L-SA solution added to Group I, 1 μL of 0.5 mM probe XIII solution, 1 μL of 0.5 mM hemin solution, 1 μL of 5 mM sodium ascorbate L-SA solution added to Group J. The reaction was carried out at 37℃ with shaking for 2 hours. 40 μL of each group was taken and 10 μL of 5× loading buffer was added. The mixture was boiled at 95℃ for 5 minutes and then added to 10% SDS-PAGE fluorescent gel at a rate of 20 μL per well. The gel was run and fluorescence was developed (Cy2, 473 nm excitation).

[0154] From the results ( Figure 6 As can be seen from 7), the number of fluorescently labeled proteins in cell lysate and bovine serum albumin is significantly reduced by probe XIId compared to the invented probe XIII, indicating that the fluorescent dye of probe XIId mainly exists in free form and is less affected by fluorescence changes caused by interaction with proteins.

[0155] Experimental Example 5: 10% SDS-PAGE fluorescent gel detection probe XIId and the invented probe XIII were used to label protein status in U937 cells.

[0156] 1×10 6 Seven U937 cells were seeded into 35 mm culture dishes, numbered A, G, and G. After 12 hours of growth, the following media were added to the culture dish: Group A: 1 mL of medium containing 1 μL DMSO; Group B: 1 mL of medium containing probe XIId at a final concentration of 0.1 μM; Group C: 1 mL of medium containing probe XIId at a final concentration of 1 μM; Group D: 1 mL of medium containing probe XIId at a final concentration of 5 μM; Group E: 1 mL of medium containing probe XIII at a final concentration of 0.1 μM; Group F: 1 mL of medium containing probe XIII at a final concentration of 1 μM; and Group G: 1 mL of medium containing probe XIII at a final concentration of 5 μM. The cells were then incubated for 3 hours. Cells were collected into 7 EP tubes, and 60 μL of 0.1% NP40 / PBS solution was added to each tube. The cells were lysed at 4°C for 15 minutes, centrifuged at 15,000 rpm at 4°C for 20 minutes, and the supernatant was collected. The protein concentration was adjusted to be consistent using the BCA method. 40 μL of the supernatant was taken from each group, and 10 μL of 5× loading buffer was added. The cells were boiled at 95°C for 5 minutes. 20 μL of the supernatant was added to 10% SDS-PAGE fluorescent gel, and the gel was run and fluorescently developed (Cy2, 473 nm excitation).

[0157] From the results ( Figure 8 As can be seen from the data, the number of fluorescently labeled proteins by probe XIId in U937 cells is significantly less than that of proteins labeled by the previously invented probe XIII. This indicates that probe XIId can be cleaved into two parts by heme in cells. Combining the mass spectrometry results after the reaction of probe XIId with heme and the labeling of cell lysate and bovine serum albumin, it can be concluded that the free radical-containing fluorescent dye portion generated after probe XIId reacts with heme in cells mainly exists in a free form.

[0158] Experiment Example 6: Flow cytometry was used to detect changes in cell fluorescence intensity after incubation of U937 cells with different concentrations of probe XIId and the invented probe XIII.

[0159] 5×10 5 Eleven U937 cells were seeded into 35 mm culture dishes, designated as group AK. After 24 hours of growth, group A was treated with a medium change containing 1 μL of [a specific ingredient]. 1 mL of DMSO medium was added to each of the following groups: Group B: 1 mL of medium containing probe XIId with a final concentration of 0.1 μM; Group C: 1 mL of medium containing probe XIId with a final concentration of 0.5 μM; Group D: 1 mL of medium containing probe XIId with a final concentration of 1 μM; Group E: 1 mL of medium containing probe XIId with a final concentration of 5 μM; Group F: 1 mL of medium containing probe XIId with a final concentration of 10 μM; Group G: 1 mL of medium containing probe XIII with a final concentration of 0.1 μM; Group H: 1 mL of medium containing probe XIII with a final concentration of 0.5 μM; Group I: 1 mL of medium containing probe XIII with a final concentration of 1 μM; Group G: 1 mL of medium containing probe XIII with a final concentration of 5 μM; Group K: 1 mL of medium containing probe XIII with a final concentration of 10 μM. All groups were incubated for 1 hour. Cells were collected into 11 EP tubes, each group was resuspended in 1 mL of PBS, and 10,000 cells were collected using flow cytometry and the average fluorescence intensity was measured in the FITC channel for comparison.

[0160] From the results ( Figure 9 As can be seen from the data, the fluorescence intensity of U937 cells was significantly stronger when incubated with low concentrations (0.1-1 μM) of probe XIId than that of probe XIII at the same concentration. Since the fluorescent dye of probe XIId is mainly in a free state after interacting with heme in the cells, it is less affected by protein on fluorescence intensity, thus exhibiting better sensitivity. Free heme in U937 cells can be detected even with low concentrations of probe XIId. As the probe concentration increases, the free fluorescent dye reaches saturation, and excess fluorescent dye gradually diffuses outside the cell. Protein-bound fluorescent dye cannot diffuse outside the cell, therefore, the fluorescence intensity of probe XIII increases when incubated at high concentrations (10 μM). Considering that high-concentration probe incubation may affect cell viability in practical applications, and that the concentration of free heme in tumor cells is in the nM range, probe XIId, which provides higher fluorescence intensity in U937 cells when incubated at low concentrations, is more effective in distinguishing tumor cells sensitive to artemisinin-based compounds and also helps save on detection costs.

[0161] Experiment Example 7: Chemiluminescence assay for the growth of different tumor cells in the presence of 10 μM dihydroartemisinin (DHA).

[0162] Well-grown MV-4-11, U937, HeLa, HCT-116, U87MG, and HepG2 cells were seeded into 96-well plates, 50 μL per well (5000 cells per well). Then, 50 μL of culture medium containing 10 μM dihydroartemisinin (DHA) was added. 200 μL of PBS solution was added to the outermost ring of wells to prevent evaporation from affecting the experimental results. Three replicates were set up. After incubating the cells in a 37°C CO2 incubator for 72 hours, 100 μL of assay reagent was added to each well, and the cells were incubated at 37°C for 10 minutes. The chemiluminescence values ​​of each well were measured using a microplate reader, and the percentage of cell proliferation was calculated compared to the control group. Results (…) Figure 10 As can be seen from the data, dihydroartemisinin has different killing abilities against different tumor cells, among which MV-4-11 cells and U937 cells showed significant cell killing ability.

[0163]

[0164] Experiment Example 8: Flow cytometry comparison of the average fluorescence intensity of probe XIId after incubation with different tumor cells

[0165] 5×10 5 MV-4-11 cells, U937 cells, HeLa cells, HCT-116 cells, U87MG cells, and HepG2 cells were seeded into 35 mm culture dishes, two dishes per cell type, for a total of 12 dishes, and incubated for 24 hours. The medium was changed by adding 1 mL of medium containing 1 μL DMSO to one dish of each cell type, and 1 mL of medium containing probe XIId at a final concentration of 5 μM to the other dish, and incubating for 1 hour. Cells were then collected into 12 EP tubes, each resuspended in 1 mL PBS, and 10,000 cells were collected using flow cytometry. The average fluorescence intensity was measured in the FITC channel for comparison.

[0166] From the results ( Figure 11As can be seen, U937 cells and MV-4-11 cells had the highest average fluorescence intensity, while the average fluorescence intensity of the remaining HeLa cells, HCT-116 cells, U87MG cells, and HepG2 cells decreased in that order. Since the antitumor effect of artemisinin-based compounds is closely related to the concentration of heme in tumor cells, literature reports that the antitumor effect of artemisinin-based compounds is enhanced or weakened when the concentration of heme in tumor cells increases or decreases. Therefore, tumor cells sensitive to dihydroartemisinin have a correspondingly higher concentration of heme, and their fluorescence intensity after incubation with probe XIId should be significantly higher than that of tumor cells insensitive to dihydroartemisinin. This is consistent with the changes in average fluorescence intensity detected by flow cytometry in different tumor cells, indicating that probe XIId can be used to detect tumor cells sensitive to artemisinin-based compounds.

Claims

1. A compound of the following formula: ###0001### or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound XII includes the following isomers:

3. A process for the preparation of a compound according to claim 1, characterized in that, The preparation method comprises the following steps: (1) Compound I and methylene blue are added into a photo-reactor, dissolved in acetonitrile, oxygen is introduced, a xenon lamp photo-reactor is turned on, and the reaction is carried out at 0°C until completion; oxygen introduction is stopped, the temperature is transferred to room temperature, then p-toluenesulfonic acid is added, and the reaction is continuously carried out at room temperature until completion; the reaction solvent is removed under reduced pressure, and purification is carried out to obtain intermediate compound II; (2) Ozone is bubbled into a dichloromethane-78°C cooled solution of the intermediate compound II obtained in step (1) until the reaction solution changes from colorless to light blue; after the reaction is completed, the temperature is transferred to room temperature, dimethyl sulfide is added, and the stirring is continuously carried out until the reaction is completed; the reaction solvent is removed under reduced pressure, and purification is carried out to obtain intermediate compound III; (3) The intermediate compound III obtained in step (2) is dissolved in dichloromethane, 1-triphenylphosphine ethenyl-2-propanone is added, stirring is carried out at 0°C for 15 minutes, and the stirring is continuously carried out at room temperature; after the reaction is completed, the reaction solvent is removed under reduced pressure, and purification is carried out to obtain intermediate compound IV; (4) The intermediate compound IV obtained in step (3) and sodium borohydride are placed in a reaction bottle; methanol is added at -15°C, the reaction is continuously carried out until completion, water is added for quenching, dichloromethane is used for extraction, the organic phase is collected, anhydrous sodium sulfate is used for drying, filtration is carried out, and concentration is carried out under reduced pressure to obtain intermediate compound V; (5) The intermediate compound V obtained in step (4) is dissolved in dichloromethane at 0°C, then 4-nitrophenyl chloroformate and pyridine are added, the temperature is transferred to room temperature, the reaction is continuously carried out until completion, the reaction solvent is removed under reduced pressure, and purification is carried out to obtain intermediate compound VI; (6) The intermediate compound VI obtained in step (5) is dissolved in dichloromethane, Boc-ethylenediamine and 4-dimethylaminopyridine are added at room temperature, the stirring is continuously carried out until the reaction is completed, saturated ammonium chloride solution and saturated sodium chloride solution are used for washing in sequence, the organic phase is separated, anhydrous sodium sulfate is used for drying, filtration is carried out, the solvent is removed under reduced pressure, and purification is carried out to obtain intermediate compound VII; (7) The intermediate compound VII obtained in step (6) and lithium borohydride are dissolved in anhydrous ether under the protection of argon at -6°C, the stirring is continuously carried out until the reaction is completed, water is added for quenching, dichloromethane is used for extraction, the organic phase is separated, anhydrous sodium sulfate is used for drying, filtration is carried out, the solvent is removed under reduced pressure, and intermediate compound VIII is obtained; (8) The intermediate compound VIII obtained in step (7) is dissolved in dichloromethane, 4-nitrophenyl chloroformate and pyridine are added at 0°C, the reaction is carried out at room temperature until completion, water is added for quenching, the organic phase is separated, saturated ammonium chloride solution is used for washing, anhydrous sodium sulfate is used for drying, filtration is carried out, the solvent is removed under reduced pressure, and purification is carried out to obtain intermediate compound IX; (9) The intermediate compound IX obtained in step (8) and hydrochloride fluorescent dye are dissolved in dichloromethane, N,N-diisopropylethylamine and 4-dimethylaminopyridine are added in sequence, the stirring is continuously carried out at room temperature, after the reaction is completed, saturated ammonium chloride solution and saturated sodium chloride solution are used for washing in sequence, anhydrous sodium sulfate is used for drying, filtration is carried out, the solvent is removed under reduced pressure, and purification is carried out to obtain intermediate compound X; (10) The intermediate compound X obtained in step (9) is dissolved in methanol, trifluoroacetic acid is added, and the reaction is allowed to proceed to completion at room temperature. The reaction solvent is removed under reduced pressure to obtain intermediate compound XI, which is directly used in the next step; (11) The intermediate compound XI obtained in step (10) is dissolved in dichloromethane, and 4-nitrophenyl chloroformate-activated disperse red is added. N,N-diisopropylethylamine is added to adjust the pH of the reaction solution to be alkaline. Then 4-dimethylaminopyridine is added, and the reaction is allowed to proceed to completion at room temperature. The solvent is removed under reduced pressure, and purification is performed to obtain compound XII 4. Use of a compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, for the manufacture of a product for detecting a haem or ferrous ion related product.

5. Use of a compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, for the manufacture of a product for detecting tumour cells susceptible to artemisinin-like compounds.

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