Fluorophore free fluorescent probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of analytical chemistry, and relates to a fluorophore free fluorescent probe as well as a preparation method and application thereof, in particular to a fluorescent probe for detecting tumor cells sensitive to ferroheme, ferrous ions and artemisinin compounds as well as a preparation method and application thereof. The fluorescent dye of the lightening type probe is located on one side of carbon free radicals generated after probe molecules are broken. The synthesized probe can be used for detecting ferroheme with high sensitivity, and also can be used for detecting ferrous ions. Free fluorescent dye is mainly generated after the fluorescent probe molecule reacts with ferroheme, and the fluorescent probe molecule shows obvious advantages when the low-concentration probe molecule detects tumor cells. When the fluorescent probe is used for detecting tumor cells sensitive to artemisinin compounds by using a flow cytometry, the fluorescent probe has the advantages of easiness in operation, high selectivity, high sensitivity and the like. Therefore, the method has potential practical application value in evaluating the killing ability of artemisinin compounds to different tumor cells.

Description

Technical Field

[0001] The invention relates to a fluorophore-free fluorescent probe and a preparation method thereof, and application thereof in detecting heme or ferrous ions and tumor cells sensitive to artemisinin compounds, belonging to the technical field of analytical chemistry. Background Art

[0002] Artemisinin (ART) is a sesquiterpene lactone compound isolated from the Asteraceae plant Artemisia annua by Chinese scientist Tu Youyou in 1972. It has significant antimalarial activity. By modifying the C10 of the artemisinin structure, compounds including dihydroartemisinin, artemether, and artesunate were obtained, which improved the poor stability and low water solubility of artemisinin, significantly improved the antimalarial activity of artemisinin, and played an important role in the treatment of malaria. With the continuous deepening of research, researchers have found that artemisinin compounds not only show obvious efficacy in antimalarial, but also have good effects in anti-tumor, and their anti-cancer activity has received more and more attention. Studies have shown that artemisinin compounds can inhibit the growth of myeloid leukemia cells and eventually lead to cell apoptosis.

[0003] There is evidence that the level of heme is the key to the antimalarial and antitumor effects of artemisinin compounds. Heme can interact with the peroxide bridge bond in the structure of artemisinin compounds, thereby breaking it, first generating oxygen free radicals, and then rearranging into carbon free radicals to exert its effects. By increasing or inhibiting the level of heme in tumor cells, the inhibitory effect of artemisinin compounds on tumor cells can be significantly increased or decreased. Therefore, the development of a tool that can detect the level of heme in different tumor cells will hopefully screen out tumor cells that are sensitive to artemisinin compounds, and will also help to further understand how these compounds exert their antitumor effects. Traditional methods for detecting heme levels generally detect total heme levels. Genetically encoded probes for detecting heme developed in recent years are also interfered by hemin and zinc porphyrin (ZnPP), and the operation is more complicated. In addition, the level of heme in cells is lower than that in malarial parasites, so a simple, sensitive and practical method is needed to detect the level of heme in different tumor cells.

[0004] Fluorescent probes can react or bind specifically with the object to be detected through recognition groups, causing changes in the photophysical properties of the probe itself, thereby causing changes in the fluorescence signal. According to the changes in the fluorescence signal, the specific object to be detected can be detected. The technology of using fluorescent probe analysis to detect has been successfully applied to the research of many diseases due to its advantages such as simple operation, good selectivity, high sensitivity and real-time online monitoring. The peroxide bridge bond in artemisinin compounds is the group that specifically reacts with heme, so the peroxide bridge bond can be used to develop a fluorescent probe for detecting heme. Similar to artemisinin compounds, the peroxide bridge bond in the structure of hawkclaw acetone is also the key to its antimalarial effect. After reacting with heme, it also involves a free radical-mediated process: first, oxygen free radicals are generated, and then rearranged into carbon free radicals. However, unlike the rearrangement process of artemisinin compounds, which occurs inside the molecule, hawkclaw acetone will break after rearrangement, turning the entire molecule into two parts. This feature can be used to develop free radical-mediated light-up fluorescent probes to detect heme and tumor cells sensitive to artemisinin compounds.

[0005] In addition, the ability of the fluorophore produced after probe activation to bind to proteins is also an important consideration in probe design. Proteins sometimes reduce the fluorescence signal of fluorophores bound to proteins, so fluorophore-free probes have better detection effects at low concentrations. Summary of the invention

[0006] With the continuous proposal of the anti-tumor effect of artemisinin compounds, how to distinguish which tumor cells are sensitive to such compounds has become a key issue. In view of the challenges currently faced by the detection of sensitive tumor cells of artemisinin compounds, the present invention forms oxygen free radicals after the reaction of hawk claw methyl ester with heme, and then rearranges into carbon free radicals, so that the entire molecule is broken into two parts. On the basis of the existing invention, the positions of the fluorescent groups and fluorescence quenching groups on both sides of the hawk claw methyl ester structural skeleton are adjusted, and a light-up type small molecule fluorescent probe with a fluorescent dye located on the side of the carbon free radical generated after the probe molecule is broken is designed and synthesized. The synthesized probe can detect heme and tumor cells sensitive to artemisinin compounds with high sensitivity, and can also detect divalent iron ions. The fluorophore containing free radicals generated by the fluorescent probe molecule after reacting with heme preferentially combines with water in the reaction system, mainly generating free fluorophores. Compared with the fluorescent probes of the prior art in which the fluorophore is located on the other side of the probe molecule, the fluorescent probe provided by the present invention can reduce the binding of the fluorescent dye part generated after the breakage with the tumor cell protein to be detected, reduce the influence of the protein on the fluorescent dye, and show obvious advantages in the detection of low-concentration probe molecules. In addition, the present invention further provides a method for preparing the probe and an operating process for its application.

[0007] The object of the present invention is to develop a type of fluorescent probe which has high sensitivity, strong practicability and is easy to operate for detecting heme, or divalent iron ions, and tumor cells which are sensitive to artemisinin compounds.

[0008] The fluorescent probe involved in the present invention can react with heme in tumor cells, causing the probe molecule to break into two parts, and fluorescence resonance energy transfer cannot continue to occur between the fluorescence quenching group and the fluorophore, thereby causing the fluorescence signal of the probe molecule to increase sharply. The synthesized fluorescent probe can effectively distinguish tumor cells that are sensitive to artemisinin compounds, and has the characteristics of simple operation, reliable results, low detection limit, and strong selectivity.

[0009] Technical Solution

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

[0011]

[0012] The fluorescent probe for detecting heme, divalent iron ions and tumor cells sensitive to artemisinin compounds in the present invention has the following structural formula XII:

[0013]

[0014] in:

[0015] Compound XII contains the following 8 isomers:

[0016]

[0017] The present invention relates to a fluorescent probe for detecting heme, divalent iron ions and distinguishing tumor cells sensitive to artemisinin compounds, which is a free radical-mediated, turn-on fluorescent probe based on a galactoside skeleton.

[0018] In another aspect, the present invention provides a method for preparing compound XII, which method involves the following steps:

[0019] (1) Compound I and methylene blue were added to a photoreactor, dissolved in acetonitrile, oxygen was introduced, and the xenon lamp reactor was turned on. The reaction was carried out at 0°C until completion, and the oxygen was stopped. The reaction 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 purified by silica gel column chromatography.

[0020]

[0021] The reaction is preferably carried out under the irradiation of a xenon lamp in a photoreactor with the ice-water mixture refluxed and stirred peripherally to prevent the local temperature from being too high during the reaction at 0°C under the irradiation of the xenon lamp.

[0022] The reaction solvent is preferably acetonitrile.

[0023] The reaction time at 0°C is preferably 0.5-2 hours depending on the amount of feed, and the reaction time at room temperature is preferably 10-12 hours.

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

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

[0026]

[0027] The ozone is preferably introduced into the reaction flask by bubbling, so that the ozone is more evenly distributed in the reaction solvent.

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

[0029] The reaction solvent is preferably dichloromethane.

[0030] Adjust the ozone introduction time according to the feed ratio, stop introducing ozone until the reaction solution turns light blue, continue bubbling oxygen for a few minutes, introduce argon, and exhaust excess oxygen.

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

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

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

[0034]

[0035] The reaction solvent is preferably dichloromethane.

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

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

[0038] (4) The intermediate compound IV obtained in step (3) and sodium borohydride are placed in a reaction bottle; methanol is added at -15°C and the reaction is continued for 40 minutes. After the reaction is completed, water is added to quench the reaction, and the mixture is extracted with dichloromethane. The organic phase is collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the intermediate compound V.

[0039]

[0040] The reaction solvent is preferably added last in the reaction addition sequence.

[0041] The reaction solvent is preferably methanol.

[0042] The reaction temperature is preferably -15°C.

[0043] The reaction time is controlled between 40 minutes and 1 hour. Too long a time may result in the reduction of the carbonyl group on the cyclohexane.

[0044] In the post-treatment, water is preferably added first to quench the excess sodium borohydride to prevent the carbonyl group on the cyclohexane from being reduced due to the increase in temperature caused by direct concentration.

[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, and the mixture was transferred to room temperature and continued to react until completion. The reaction solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography to obtain the intermediate compound VI.

[0046]

[0047] The reaction solvent is preferably dichloromethane, which is convenient for removal during subsequent treatment.

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

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

[0050] If a white precipitate is generated after the reaction is completed, it is preferably filtered and then the reaction solvent is removed under reduced pressure.

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

[0052] (6) The intermediate compound VI obtained in step (5) is dissolved in dichloromethane, and Boc-ethylenediamine and 4-dimethylaminopyridine are added at room temperature. The mixture is stirred until the reaction is completed, and washed with saturated ammonium chloride solution and saturated sodium chloride solution in sequence. The organic layer is separated, dried over anhydrous sodium sulfate, filtered, and the solvent is removed under reduced pressure. The intermediate compound VII is purified by silica gel column chromatography to obtain the intermediate compound VII.

[0053]

[0054] The reaction temperature is preferably room temperature.

[0055] The reaction time is preferably 5 hours.

[0056] The reaction solvent is preferably dichloromethane, which has good solubility for the raw materials and can be easily removed under reduced pressure.

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

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

[0059] (7) The intermediate compound VII obtained in step (6) and lithium borohydride are dissolved in anhydrous ether at -6°C under an argon atmosphere, and the reaction is continued to be stirred at -6°C until the reaction is completed. Water is added to quench the reaction, and the reaction is extracted with dichloromethane. The organic phase is separated, dried over anhydrous sodium sulfate, filtered, and the solvent is removed under reduced pressure to obtain the intermediate compound VIII.

[0060]

[0061] The reaction solvent is preferably anhydrous ether.

[0062] The reaction is preferably carried out in the absence of water and oxygen. Before adding the reaction solvent, the air in the reaction bottle needs to be replaced with argon to ensure an oxygen-free environment.

[0063] The reaction temperature is preferably -6°C.

[0064] The reaction time is preferably 6 hours.

[0065] After the reaction is completed, it is preferably post-treated by quenching with water.

[0066] (8) The intermediate compound VIII obtained in step (7) was dissolved in dichloromethane, 4-nitrophenyl chloroformate and pyridine were added at 0°C, and the reaction was allowed to react at room temperature for 4 days. Water was added to quench the reaction, and the organic phase was separated, washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The intermediate compound IX was purified by silica gel column chromatography.

[0067]

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

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

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

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

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

[0073]

[0074] The reaction is preferably carried out in dichloromethane to facilitate subsequent treatment.

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

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

[0077] After the reaction is completed, washing with saturated ammonium chloride solution and saturated sodium chloride solution can remove N, N-diisopropylethylamine and 4-dimethylaminopyridine in the reaction system, which is convenient for subsequent separation.

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

[0079] (10) The intermediate compound X obtained in step (9) is dissolved in methanol, trifluoroacetic acid is added, and the reaction is completed at room temperature. The reaction solvent is removed under reduced pressure to obtain the intermediate compound XI, which is directly used in the next step reaction.

[0080]

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

[0082] The trifluoroacetic acid used for Boc removal is added dropwise to facilitate a complete reaction.

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

[0084] After the reaction is completed, it is preferred to directly proceed to the next step without post-treatment.

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

[0086]

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

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

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

[0090] Various raw materials used in the present 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 compounds using the above-mentioned compound XII, including but not limited to the following method: inoculating different tumor cells into a cell culture dish and growing them for 24 hours, adding a cell culture medium containing compound XII to the tumor cells to be detected in the form of liquid replacement and incubating for 1 hour, collecting the cells, and using a flow cytometer to detect the average fluorescence intensity of different tumor cells. The average fluorescence intensity of different tumor cells can be compared to detect tumor cells sensitive to artemisinin compounds.

[0092] The beneficial effects of the present invention are: this type of fluorescent probe can detect related products of heme or divalent iron ions, identify tumor cells sensitive to artemisinin compounds, including U937 cells, MV-4-11 cells, etc., have 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, 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 and has the advantages of easy operation, strong selectivity, and high sensitivity.

[0095] (2) After reacting with the heme in tumor cells, this type of fluorescent probe molecule breaks into two parts, among which the fluorescent dye part containing free radicals reacts with water first, and most of it is in a free state, which can reduce the impact of binding to proteins on the fluorescent dye itself. Compared with the fluorescent probes with fluorescent dyes located on the other side of the probe molecule, it shows obvious advantages in detecting tumor cells at low concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0097] Figure 2 , Mass spectrum of α, β-unsaturated ketone containing disperse red part after probe XIId reacted with heme.

[0098] Figure 3 , the change 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, hemin: hemoglobin, L-SA: sodium ascorbate.

[0100] Figure 5 , the change of probe fluorescence intensity caused by the reaction of probe XIId with divalent iron ions, L-SA: sodium ascorbate, Fe 2+ : Divalent iron ion.

[0101] Figure 6 , probe XIId, and invented probe XIII respectively reacted with heme to label proteins in U937 cell lysate.

[0102] Figure 7 , probe XIId, and the invented probe XIII respectively reacted with heme to label bovine serum albumin. BSA: bovine serum albumin.

[0103] Figure 8 , probe XIId, and the invented probe XIII are compared with the labeled proteins after incubation with U937 cells.

[0104] Fig. 9 , The changes in cell fluorescence intensity were compared using flow cytometry after different concentrations of probe XIId and the invented probe XIII were incubated with U937 cells.

[0105] Fig.10 , The proliferation of different tumor cells under the action of 10μM dihydroartemisinin was compared using chemiluminescent cell viability assay. DHA: dihydroartemisinin.

[0106] Fig.11 , Use flow cytometry to compare the changes in cell fluorescence intensity after probe XIId was incubated with different tumor cells. DETAILED DESCRIPTION

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

[0111] (2) Synthesis of intermediate IIId

[0112] Ozone was bubbled into a dichloromethane-78°C cooled solution (50 mL) of the intermediate compound II (5.0 g, 25.5 mmol) until the reaction solution changed from colorless to light blue, about 1 hour. After the reaction was completed, the ozone generator was turned off and oxygen was continued to flow for 5 minutes. The oxygen bottle was turned off and argon was passed for 5 minutes. The mixture was transferred to room temperature and dimethyl sulfide (4.8 mL, 63.8 mmol) was added. The mixture was stirred for 30 minutes, and the reaction solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate (50 / 1-6 / 1, v / v)) to give a colorless oily compound 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 199.0965for C 10 H 15 O4 + ).

[0113] (3) Synthesis of intermediate IVd

[0114] The intermediate compound IIId (800 mg, 4.04 mmol) was dissolved in 20 mL of dichloromethane, and a dichloromethane solution (10 mL) of 1-triphenylphosphine-2-propanone (1.542 g, 4.84 mmol) was added, and stirred at 0°C for 15 minutes. The reaction was continued at room temperature for 30 hours. After the reaction was completed, the reaction was concentrated by rotary evaporation, and the reaction solvent was removed under reduced pressure. 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 239.1278for C 13 H 19 O4 + )

[0115] (4) Synthesis of intermediate Vd

[0116] The intermediate compound IVd (460 mg, 1.93 mmol) and sodium borohydride (220 mg, 5.8 mmol) were placed in a reaction bottle, 20 mL of methanol was added at -15 °C, and the reaction was continued for 40 minutes. After the reaction was completed, 20 mL of water was added to quench the reaction, and the mixture was extracted with dichloromethane (40 mL × 2). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a colorless oily intermediate compound Vd. The next step of the reaction was carried out directly without post-treatment.

[0117] (5) Synthesis of intermediate VId

[0118] The intermediate compound Vd (460 mg, 1.92 mmol) was dissolved in 150 mL of dichloromethane, and 4-nitrophenyl chloroformate (1.156 g, 5.75 mmol) and pyridine (938 μL, 9.58 mmol) were added at 0°C, and 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 compound 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 428.1316for C 20 H 23 NNaO8 + ).

[0119] (6) Synthesis of Intermediate VIId

[0120] The 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), and the reaction was carried out at room temperature for 5 hours. The mixture was washed with saturated ammonium chloride solution and saturated sodium chloride solution in sequence 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 a colorless oily compound 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 327.1914for C 16 H 27 N2O5 + ).

[0121] (7) Synthesis of Intermediate VIIId

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

[0123] (8) Synthesis of intermediate IXd

[0124] The intermediate compound VIIId (310 mg, 0.724 mmol) was dissolved in 30 mL of dichloromethane, and 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, and 50 mL of water was added to quench the reaction. 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 product was purified by silica gel column chromatography (petroleum ether / ethyl acetate (100 / 1-1 / 1, v / v)) to give a colorless oily compound intermediate IXd (150 mg, 35.0%), which was directly used in the next step reaction.

[0125] (9) Synthesis of intermediate Xd

[0126] The intermediate compound IXd (60 mg, 0.1 mmol), the hydrochloride fluorescent dye (BODIPY-NH2 . 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 reaction was carried out at room temperature for 48 hours. After the reaction was completed, it was washed with saturated ammonium chloride solution (10 mL × 3) and saturated sodium chloride solution (10 mL × 3) in sequence, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. Thin layer chromatography (petroleum ether / ethyl acetate (1 / 2, v / v)) was used for purification, and dichloromethane / methanol (10 / 1) was used for elution to obtain an orange-red solid compound 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 735.4059for C 35 H 54 BF2N6O8 + )

[0127] (10) Synthesis of intermediate XId

[0128] The 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, and the reaction solvent was removed under reduced pressure to obtain the intermediate compound XId, which was directly used in the next step 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, and N,N-diisopropylethylamine was added to adjust the reaction solution pH to > 9, followed by the addition of 4-dimethylaminopyridine (18 mg, 0.147 mmol), and the reaction was continued at room temperature for 20 hours. The solvent was removed under reduced pressure, and the compound was initially purified by thin layer chromatography (dichloromethane / methanol (20 / 1, v / v)), and further purified by preparative liquid chromatography (conditions for 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, and then 2,2'-disulfide dipyridine (6.6 g, 30 mmol) and triphenylphosphine (10.48 g, 40 mmol) were added, and the reaction mixture was stirred under an argon atmosphere for 24 hours; in another clean reaction bottle, pyrrole (8.04 g, 120 mmol) was dissolved in 90 mL of anhydrous tetrahydrofuran, and under an argon atmosphere, 1 M methylmagnesium bromide (90 mL, 90 mmol) was added to the tetrahydrofuran solution of pyrrole using a constant pressure dropping funnel at -78°C, and the reaction was continued at -78°C for 30 minutes, the temperature was raised to -20°C for reaction for 30 minutes, and then the temperature was lowered to -78°C and stirred for 30 minutes. The thioester mixture that had been reacted for 24 hours was added to the reaction solution of pyrrole and methylmagnesium bromide using a constant pressure dropping funnel at -78°C and continued to stir for 30 minutes. The mixture was transferred to room temperature and reacted for 30 minutes. After the reaction was completed, the reaction was quenched with saturated ammonium chloride solution (300 mL), diluted with anhydrous ether (300 mL), and the organic layer was separated and washed with water (200×3 mL) and brine (500 mL), respectively, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography (petroleum ether / dichloromethane / ethyl acetate (50 / 50 / 1-1 / 1 / 1, v / v / v)) to obtain 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 361.1547for 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 minutes. Then, phosphorus oxychloride (600 μL, 600 mmol) was added dropwise to the reaction solution at 0°C and the reaction was continued for 1 hour. The reaction solution was heated to 35°C and stirred for 20 hours. Then, N,N-diisopropylethylamine (2.62 mL, 12 mmol) and boron trifluoride ether (1.7 mL, 12 mmol) were added and reacted at room temperature for 4 hours. After the reaction was completed, it 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 obtain 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 466.2097for 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, and 211 μL of DBU was added every 15 minutes for four times, and the reaction lasted for 1 hour. Then 10 mL of 0.5 N aqueous hydrochloric acid solution was slowly added dropwise, and the reaction continued for 15 minutes at room temperature. The solid was filtered and 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 264.1478for C 13 H 17 BF2N3 + ).

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

[0141]

[0142] Disperse red (62.8 mg, 0.2 mmol) was dissolved in 5 mL of dichloromethane, cooled to 0°C, 4-nitrophenyl chloroformate (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 completed, the mixture 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 product of dark red solid 4-nitrophenyl chloroformate activated disperse red (67 mg), which can be directly used in the synthesis of compound XII without further purification. HRESI-MS m / z: 480.1514 [M+H] + (calcd 480.1514for 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, and 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 the 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 of the reaction of free radicals with water and a dispersed red product containing α, β unsaturated ketones. Both products were confirmed by high-resolution mass spectrometry. The free fluorescent dye product of the reaction of free radicals with water: HRESI-MS m / z: 434.2156 [MH] + (calcd 434.2068forC 21 H 27 BF2N3O4 - )( Figure 1 ). Dispersed red product containing α, β unsaturated ketone: HRESI-MS m / z: 541.2412 [M+H] + (calcd 541.2405for C 26 H 33 N6O7 + )( Figure 2 ). This indicates that the fluorescent probe molecule can react with heme and then break into two parts, which can be used for the detection of heme. The reaction solution was moved to an ultraviolet lamp (365nm) for observation, and obvious fluorescence ( Figure 3 ).

[0146] Experimental Example 2: Changes in the fluorescence intensity of the probe XIId and Heme detected by an ELISA instrument

[0147] The 1 mM fluorescent probe XIId solution and hemin solution in Experimental Example 1 were diluted to 0.1 mM working solution, and 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 the three solutions was added to 97 μL of 1× PBS, incubated at 37°C for 1 h, and detected using an ELISA reader. Under an excitation wavelength of 480 nm, a significant increase in the fluorescence signal was observed at 530 nm ( Figure 4 ).

[0148] Experimental Example 3: Changes in the fluorescence intensity of the probe XIId interacting with divalent iron ions detected by an ELISA instrument

[0149] The 1 mM fluorescent probe XIId solution in Experimental Example 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, 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 the three solutions was added to 97 μL of 1×PBS, incubated at 37°C for 1 h, and detected using an enzyme reader. Under an excitation wavelength of 480 nm, an increase in the fluorescence signal was observed at 530 nm ( Figure 5 ).

[0150] Experimental Example 4: 10% SDS-PAGE fluorescent gel detection of probe XIId and the invented probe XIII respectively reacted with heme to label proteins in U937 cell lysate and bovine serum albumin

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

[0152] 1×10 6U937 cells were inoculated into 6-well plates and grown for 12 hours. The cells were collected and lysed by adding 530 μL 0.1% NP40 / PBS solution. The cells were lysed in a 4°C refrigerator for 15 minutes. The cells were centrifuged at 15,000 rpm at 4°C for 20 minutes. The supernatant was collected and 97 μL was added to five EP tubes respectively, marked as groups A, B, C, D and E. 2 μL DMSO and 1 μL water were added to group A, 1 μL 0.1 mM probe XIId solution, 1 μL 0.1 mM hemin solution, and 1 μL 1 mM sodium ascorbate L-SA solution were added to group B, 1 μL 0.5 mM probe XIId solution, 1 μL 0.5 mM hemin solution, and 1 μL 5 mM sodium ascorbate L-SA solution were added to group C, 1 μL 0.1 mM probe XIII solution, 1 μL 0.1 mM hemin solution, and 1 μL 1 mM sodium ascorbate L-SA solution were added to group D, and 1 μL 0.5 mM probe XIII solution, 1 μL 0.5 mM hemin solution, and 1 μL 5 mM sodium ascorbate L-SA solution were added to group E. Incubate at 37°C with shaking for 2 hours. Take 40 μL from each group, add 10 μL 5× loading buffer, boil at 95°C for 5 minutes, add 20 μL per well to 10% SDS-PAGE fluorescent gel, run the gel and perform fluorescence development (Cy2, 473 nm excitation).

[0153] Prepare a 10 μM solution of bovine serum albumin (BSA), take 97 μL of each into 5 EP tubes, mark them as FJ groups, add 2 μL of DMSO, 1 μL water, 1 μL 0.1mM probe XIId solution, 1 μL 0.1mM hemin solution, and 1 μL 1mM sodium ascorbate L-SA solution were added to group G, 1 μL 0.5mM probe XIId solution, 1 μL 0.5mM hemin solution, and 1 μL 5mM sodium ascorbate L-SA solution were added to group H, 1 μL 0.1mM probe XIII solution, 1 μL 0.1mM hemin solution, and 1 μL 1mM sodium ascorbate L-SA solution were added to group I, and 1 μL 0.5mM probe XIII solution, 1 μL 0.5mM hemin solution, and 1 μL 5mM sodium ascorbate L-SA solution were added to group J. Incubate the mixture at 37°C with shaking for 2 hours. Take 40 μL of each sample and add 10 μL of 5× loading buffer. Boil at 95°C for 5 minutes. Add 20 μL of the sample to each well of 10% SDS-PAGE fluorescent gel. Run the gel and perform fluorescence development (Cy2, 473 nm excitation).

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

[0155] Experimental Example 5: 10% SDS-PAGE fluorescent gel detection probe XIId and invented probe XIII respectively labeled proteins in U937 cells

[0156] 1×10 6 U937 cells were inoculated into 35 mm culture dishes, a total of 7 dishes, numbered as groups AG, and grown for 12 hours. 1 mL of culture medium containing 1 μL DMSO was added to group A in the form of medium replacement, 1 mL of culture medium containing probe XIId with a final concentration of 0.1 μM was added to group B, 1 mL of culture medium containing probe XIId with a final concentration of 1 μM was added to group C, 1 mL of culture medium containing probe XIId with a final concentration of 5 μM was added to group D, 1 mL of culture medium containing probe XIII with a final concentration of 0.1 μM was added to group E, 1 mL of culture medium containing probe XIII with a final concentration of 1 μM was added to group F, and 1 mL of culture medium containing probe XIII with a final concentration of 5 μM was added to group G. The cells were incubated for 3 hours. Collect cells into 7 EP tubes, add 60 μL 0.1% NP40 / PBS solution to each tube, lyse in a 4°C refrigerator for 15 minutes, centrifuge at 4°C, 15000 rpm for 20 minutes, collect the supernatant, adjust the protein concentration to the same by BCA method, take 40 μL from each group, add 10 μL 5× loading buffer, boil at 95°C for 5 minutes, add 20 μL per well to 10% SDS-PAGE fluorescent gel, run the gel and perform fluorescence development (Cy2, 473 nm excitation).

[0157] From the results ( Figure 8 ), it can be seen from the data that the proteins fluorescently labeled by probe XIId in U937 cells are also significantly less than those labeled by the invented probe XIII. This firstly indicates that probe XIId can be broken 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 part generated by probe XIId after the reaction with heme in cells mainly exists in a free form.

[0158] Experimental Example 6: Changes in cell fluorescence intensity after different concentrations of probe XIId and invented probe XIII were incubated with U937 cells by flow cytometry

[0159] 5×10 5 U937 cells were inoculated into 35 mm culture dishes, a total of 11 dishes, numbered as group AK, and grown for 24 hours. 1 μL of 1 mL of DMSO culture medium was added, 1 mL of culture medium containing probe XIId with a final concentration of 0.1 μM was added to group B, 1 mL of culture medium containing probe XIId with a final concentration of 0.5 μM was added to group C, 1 mL of culture medium containing probe XIId with a final concentration of 1 μM was added to group D, 1 mL of culture medium containing probe XIId with a final concentration of 5 μM was added to group E, 1 mL of culture medium containing probe XIId with a final concentration of 10 μM was added to group F, 1 mL of culture medium containing probe XIII with a final concentration of 0.1 μM was added to group G, 1 mL of culture medium containing probe XIII with a final concentration of 0.5 μM was added to group H, 1 mL of culture medium containing probe XIII with a final concentration of 1 μM was added to group I, 1 mL of culture medium containing probe XIII with a final concentration of 5 μM was added to group G, and 1 mL of culture medium containing probe XIII with a final concentration of 10 μM was added to group K, and incubated for 1 hour. The cells were collected into 11 EP tubes and resuspended in 1 mL PBS for each group. 10,000 cells were collected using a flow cytometer and the average fluorescence intensity was detected under the FITC channel for comparison.

[0160] From the results ( Fig. 9 ) It can be seen that when the probe XIId is incubated at a low concentration (0.1-1 μM), the fluorescence intensity of U937 cells is significantly stronger than that of the probe XIII group at the same concentration. Since the fluorescent dye of probe XIId is mainly in a free state after reacting with heme in the cell, the fluorescence intensity is less affected by the protein, so it has better sensitivity, and the free heme in U937 cells can be detected when the probe XIId is used at a low concentration. With the increase of the probe concentration, the free fluorescent dye will reach saturation as the concentration increases, and the excess fluorescent dye will gradually diffuse to the outside of the cell. The protein-bound fluorescent dye cannot diffuse to the outside of the cell, so when the probe is incubated at a high concentration (10 μM), the fluorescence intensity of probe XIII becomes higher. Considering that high-concentration probe incubation may affect cell viability in actual application, and the concentration of free heme in tumor cells is at the nM level, probe XIId that makes U937 cells have a higher fluorescence intensity when incubated at a low concentration is more conducive to distinguishing tumor cells sensitive to artemisinin compounds, and is also conducive to saving detection costs.

[0161] Experimental Example 7: Chemiluminescence detection of the growth of different tumor cells in the presence of 10 μM dihydroartemisinin (DHA)

[0162] Well-growing MV-4-11 cells, U937 cells, Hela cells, HCT-116 cells, U87MG cells, and HepG2 cells were inoculated into 96-well plates, 50 μL per well, 5000 cells, and then 50 μL of culture medium containing dihydroartemisinin (DHA) at a final concentration of 10 μM was added. 200 μL of PBS solution was added to the outermost circle of wells in the 96-well plate to prevent liquid evaporation from affecting the experimental results. Three replicate wells were set up, and the cells were placed in a 37°C CO2 incubator for incubation and growth for 72 hours. After that, 100 μL of detection reagent was added to each well, and incubated at 37°C for 10 minutes. The chemiluminescence value of each well was detected by an enzyme reader, and the percentage of cell proliferation was calculated compared with the blank group. From the results ( Fig.10 ) It can be seen that dihydroartemisinin has different killing abilities on different tumor cells, among which MV-4-11 cells and U937 cells showed obvious cell killing ability.

[0163]

[0164] Experimental Example 8: Comparison of mean fluorescence intensity of probe XIId after incubation with different tumor cells by flow cytometry

[0165] 5×10 5 MV-4-11 cells, U937 cells, Hela cells, HCT-116 cells, U87MG cells, and HepG2 cells were inoculated into 35 mm culture dishes, two dishes for each cell type, a total of 12 dishes, and grown for 24 hours. In the form of medium replacement, 1 mL of culture medium containing 1 μL DMSO was added to one dish of each cell, and 1 mL of culture medium containing probe XIId with a final concentration of 5 μM was added to the other dish, and incubated for 1 hour. The cells were collected into 12 EP tubes, each tube was resuspended with 1 mL PBS, and 10,000 cells were collected using a flow cytometer to detect the average fluorescence intensity under the FITC channel for comparison.

[0166] From the results ( Fig.11) It can be seen that the average fluorescence intensity of U937 cells and MV-4-11 cells is the highest, and the average fluorescence intensity of the remaining Hela cells, HCT-116 cells, U87MG cells, and HepG2 cells decreases in turn. Since the anti-tumor effect of artemisinin compounds is closely related to the concentration of heme in tumor cells, it has been reported in the literature that when the concentration of heme in tumor cells increases or decreases, the anti-tumor effect of artemisinin compounds increases or decreases accordingly. Therefore, the concentration of heme in tumor cells sensitive to dihydroartemisinin is relatively high, and the fluorescence intensity after incubation with probe XIId should be significantly higher than that of tumor cells insensitive to dihydroartemisinin, which is consistent with the changes in the average fluorescence intensity in different tumor cells detected by flow cytometry, indicating that probe XIId can be used to detect tumor cells sensitive to artemisinin compounds.

Claims

1. The following compound 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. The method for preparing the compound according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Compound I and methylene blue are added to a photoreactor, dissolved in acetonitrile, oxygen is introduced, and the xenon light reactor is turned on. The reaction is carried out at 0°C until completion, oxygen is stopped, and the reaction is transferred to room temperature. Then p-toluenesulfonic acid is added, and the reaction is continued at room temperature until completion. The reaction solvent is removed under reduced pressure, and the intermediate compound II is purified; (2) bubbling ozone 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 mixture is transferred to room temperature, dimethyl sulfide is added, and stirring is continued until the reaction is completed. The reaction solvent is removed under reduced pressure, and purification is performed to obtain the intermediate compound III; (3) dissolving the intermediate compound III obtained in step (2) in dichloromethane, adding 1-triphenylphosphine-2-propanone, stirring at 0° C. for 15 minutes, and continuing to stir at room temperature. After the reaction is completed, removing the reaction solvent under reduced pressure, and purifying to obtain the intermediate compound IV; (4) placing the intermediate compound IV obtained in step (3) and sodium borohydride in a reaction flask; Methanol was added at -15°C, and the reaction was continued until completion, and water was added to quench, and the mixture was extracted with dichloromethane. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain intermediate compound V; (5) The intermediate compound V obtained in step (4) is dissolved in dichloromethane at 0° C., followed by the addition of 4-nitrophenyl chloroformate and pyridine, and the reaction is continued at room temperature until completion, and the reaction solvent is removed under reduced pressure, and the intermediate compound VI is purified; (6) dissolving the intermediate compound VI obtained in step (5) in dichloromethane, adding Boc-ethylenediamine and 4-dimethylaminopyridine at room temperature, continuing to stir until the reaction is completed, washing with saturated ammonium chloride solution and saturated sodium chloride solution in sequence, separating the organic layer, drying over anhydrous sodium sulfate, filtering, removing the solvent under reduced pressure, and purifying to obtain the intermediate compound VII; (7) The intermediate compound VII obtained in step (6) and lithium borohydride are dissolved in anhydrous ether at -6°C under an argon atmosphere, and stirring is continued until the reaction is completed. Water is added to quench the reaction, and the reaction is extracted with dichloromethane. The organic phase is separated, dried over anhydrous sodium sulfate, filtered, and the solvent is removed under reduced pressure to obtain the intermediate compound VIII; (8) The intermediate compound VIII obtained in step (7) is dissolved in dichloromethane, 4-nitrophenyl chloroformate and pyridine are added at 0° C., and the reaction is carried out at room temperature until completion. Water is added to quench the reaction, and the organic phase is separated, washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent is removed under reduced pressure to purify the intermediate compound IX; (9) The intermediate compound IX obtained in step (8) and the hydrochloride fluorescent dye are dissolved in dichloromethane, and N,N-diisopropylethylamine and 4-dimethylaminopyridine are added in sequence, and stirring is continued at room temperature. After the reaction is completed, the mixture is washed with a saturated ammonium chloride solution and a saturated sodium chloride solution in sequence, dried over anhydrous sodium sulfate, filtered, and the solvent is removed under reduced pressure to purify the intermediate compound X; (10) dissolving the intermediate compound X obtained in step (9) in methanol, adding trifluoroacetic acid, reacting at room temperature until completion, and removing the reaction solvent under reduced pressure to obtain the intermediate compound XI, which is directly used in the next step reaction; (11) The intermediate compound XI obtained in step (10) and the disperse red activated with 4-nitrophenyl chloroformate are dissolved in dichloromethane, and N,N-diisopropylethylamine is added to adjust the pH value of the reaction solution to alkaline, followed by the addition of 4-dimethylaminopyridine, and the reaction is continued at room temperature until completion, and the solvent is removed under reduced pressure and purified to obtain compound XII.

4. Use of the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in the preparation of products for detecting heme or ferrous ions.

5. Use of the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in the preparation of products related to detecting tumor cells sensitive to artemisinin compounds.

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