Preparation and application of fluorescent probe for detecting hypochlorous acid

Abstract

The invention discloses preparation and application of a fluorescent probe for detecting hypochlorous acid, and the probe comprises phenothiazine (PZ), quinoline malononitrile (QM), hemicyanine and other structures, and has high anti-interference capability and remarkable Stokes shift. Under various stimuli, the probe can monitor the HClO level in RAW 264.7 and 4T1 cells in a red channel, can record the correlation between the HClO concentration and the breast cancer recurrence progress, is used for diagnosing the recurrent breast cancer and tracking the treatment progress of the recurrent breast cancer, and monitors the recurrence state of breast tumors through intracellular HClO fluorescence imaging. The method has theoretical guiding significance and good application prospects in the aspects of promoting early diagnosis of the recurrent breast cancer, development of therapeutic drugs and the like.

Description

Technical Field

[0001] The invention relates to the technical field of medical detection, and in particular to the preparation and application of a fluorescent probe for detecting hypochlorous acid in a breast cancer recurrence model. Background Art

[0002] Breast cancer (BC) has become one of the most common malignant tumors in women, posing a serious threat to human health. Treatment options for breast cancer usually depend on pathological type, stage, hormone sensitivity, patient age and overall health status. The main treatment options for breast cancer include surgery, chemotherapy, biological therapy, targeted therapy and hormone therapy. Among them, surgical resection is one of the methods for treating primary breast cancer. However, some tiny lesions may be missed during surgery, which can easily lead to breast cancer recurrence, with a recurrence probability between 10% and 41%. Therefore, early diagnosis of postoperative recurrent breast cancer is of great clinical significance for improving the survival rate of related patients.

[0003] At present, traditional methods for detecting postoperative recurrence of breast cancer mainly rely on X-ray screening, computed tomography, magnetic resonance imaging, positron emission tomography, ultrasound imaging, and histopathological biopsy. However, these tests may bring radioactive risks or invasive problems to patients. At the same time, traditional imaging methods present bright signals and nonspecific aggregation in normal or other inflammatory tissues, resulting in poor signal specificity and low sensitivity of tumor recognition, leading to misdiagnosis and increasing the treatment cost of patients. Therefore, it is urgent to establish some new methods for diagnosing recurrent breast cancer.

[0004] Fluorescence imaging has been widely used to monitor the progression of life activities with excellent sensitivity, non-invasiveness, and high spatiotemporal resolution. Reactive oxygen species (ROS) are essential for maintaining the balance of redox homeostasis in normal biological processes. Among reactive oxygen species, hypochlorous acid (HClO) is one of the most important endogenous ROS species in organisms and has a strong oxidative capacity. Under the catalysis of myeloperoxidase, chloride in biological systems can react with hydrogen peroxide to generate HClO. Studies have found that HClO can not only regulate the redox balance in cells by participating in various cell signaling processes, but also eliminate harmful pathogens and microorganisms and the response of the immune system. More and more studies have shown that improper regulation of HClO may aggravate the progression of BC and promote the metastasis of BC cells. Therefore, HClO may be a valuable biomarker for the diagnosis and treatment tracking of recurrent BC.

[0005] Compared with traditional monitoring methods, phenothiazine is a recognition group that reacts well to hypochlorous acid and is also an excellent electron donor. As a strong acceptor, the indole unit has been widely used in the construction of cyanine and hemicyanine dyes. Quinolinemalononitrile is also an electron acceptor and has been shown to have typical AIE characteristics. By cleverly connecting the above three building blocks, a fluorescent probe for the detection of HClO was designed and synthesized, aiming to design an optimal probe with excellent anti-interference and anti-quenching capabilities for the detection of HClO content in vivo. The results showed that the fluorescent probe has near-infrared fluorescence, high selectivity and stable fluorescence signals, has a rapid and highly sensitive response to HClO, and can be successfully used to diagnose the recurrence of early breast tumors. Summary of the invention

[0006] 1. Technical issues to be resolved In view of the defects of the prior art and the problem of early diagnosis of clinical recurrent breast cancer, the present invention provides a preparation and application of a fluorescent probe for detecting hypochlorous acid. The probe can not only extend its excitation wavelength, but also serve as a fluorophore of hypochlorous acid by introducing an indole group and a quinoline malononitrile group at both ends of phenothiazine. This fluorophore can oxidize the sulfur atom in phenothiazine to form a sulfoxide structure in response to hypochlorous acid, so as to achieve faster release and produce obvious fluorescence color changes, and can more sensitively detect the content of hypochlorous acid in biological samples, avoid interference from substances in serum samples, solve the problem of poor accuracy and sensitivity of existing detection methods, help understand the microscopic process of early diagnosis of recurrent breast cancer, and help further develop drugs for the treatment of breast cancer recurrence.

[0007] (II) Technical solution In order to solve the above technical problems, the present invention adopts the following technical solutions: Preparation and application of a fluorescent probe for detecting hypochlorous acid, the structure of the probe is as follows:

[0008] Where R 1 , R 2 H, F, Cl, Br, I, NO 2 , C 1-18 Alkyl, CN, CONHNH 2 、COOR、OH、OCH 3 One of them, R 3 , R 4 It is one of F, Cl, Br, and I.

[0009] The present invention discloses a method for preparing a fluorescent probe for detecting hypochlorous acid, comprising the following steps: (1) Preparation of 10-ethyl-10H-phenothiazine-3-carboxaldehyde 10-Ethyl-10H-phenothiazine was added into a flask containing a suitable solvent and stirred under argon protection. 3 Dilute in a suitable solvent and add dropwise to the above solution at a suitable temperature. Stir the mixture continuously, then move to a suitable temperature and stir again. After cooling to room temperature, pour the mixture into ice water and 2 CO 3 The solution was extracted with dichloromethane, washed three times with brine, and washed with anhydrous Na 2 SO 4 The organic layer was concentrated by rotary evaporation and the residue was purified by silica gel column using petroleum ether / ethyl acetate as eluent to give 10-ethyl-10H-phenothiazine-3-carbaldehyde; (2) Preparation of 10-ethyl-10H-phenothiazine-3,7-dicarboxaldehyde 10-Ethyl-10H-phenothiazine-3-carboxaldehyde was added to a flask containing a suitable solvent and stirred under argon protection. 3 Dilute in a suitable solvent and add dropwise to the above solution at a suitable temperature. Stir the mixture continuously, then move to a suitable temperature and stir again. After cooling to room temperature, pour the mixture into ice water and 2 CO 3 The solution was extracted with dichloromethane, washed three times with brine, and washed with anhydrous Na 2 SO 4 The organic layer was concentrated by rotary evaporation and the residue was purified by silica gel column using a suitable solvent as eluent to obtain 10-ethyl-10H-phenothiazine-3,7-dicarbaldehyde; (3) Preparation of (E)-2-(1-ethyl-2-(2-(10-ethyl-7-formyl-10H-phenothiazin-3-yl)vinyl)quinolin-4(1H)-ylidene)malononitrile 2-(1-ethyl-2-methylquinolin-4(1H)-ylidene)malononitrile and 10-ethyl-10H-phenothiazine-3,7-dicarbaldehyde are dissolved in a suitable mixed solvent. Piperidine is slowly injected through a syringe under argon protection. The reaction mixture is stirred at a suitable temperature. After cooling to room temperature, the mixture is concentrated by rotary evaporation, and the residue is purified by a silica gel column using a suitable solvent as an eluent to obtain (E)-2-(1-ethyl-2-(2-(10-ethyl-7-formyl-10H-phenothiazin-3-yl)vinyl)quinolin-4(1H)-ylidene)malononitrile; (4) Preparation of fluorescent probes (E)-2-(1-ethyl-2-(2-(10-ethyl-7-formyl-10H-phenothiazin-3-yl)vinyl)quinolin-4(1H)-ylidene)malononitrile and 5-methoxy-1,2,3,3-tetramethyl-3H-indole-1-iodide were dissolved in a suitable mixed solvent. Piperidine was injected through a syringe under argon protection. The reaction mixture was stirred at a suitable temperature. After cooling to room temperature, the mixture was concentrated by rotary evaporation, and the residue was purified by silica gel column using a suitable solvent as eluent to obtain a hypochlorous acid fluorescent probe.

[0010] Furthermore, the solvent is any one or more of DMF, DMSO, acetonitrile, acetone, ethanol, tetrahydrofuran, dichloromethane, methanol, petroleum ether, and ethyl acetate.

[0011] Furthermore, the reaction temperature is 25-150°C.

[0012] Furthermore, the reaction time is 2 h-48 h.

[0013] Furthermore, the reduced pressure concentration is carried out at a pressure of 0.1 Mpa-10 Mpa, and the eluent is petroleum ether / ethyl acetate or dichloromethane / methanol, and the volume ratio thereof is 100:1-1:1.

[0014] Furthermore, the silica gel column chromatography separation uses a 100-800 mesh silica gel column; the structure of the 10-ethyl-10H-phenothiazine is as follows: ; The structure of 2-(1-ethyl-2-methylquinolin-4(1H)-ylidene)malononitrile is as follows: ;

[0015] The structure of 5-methoxy-1,2,3,3-tetramethyl-3H-indole-1-iodide is as follows: ;

[0016] Where R 1 , R 2 H, F, Cl, Br, I, NO 2 , C 1-18 Alkyl, CN, CONHNH 2 、COOR、OH、OCH 3 One of them, R 3 , R 4 It is one of F, Cl, Br, and I.

[0017] The present invention also provides application of the fluorescent probe in detecting breast cancer recurrence.

[0018] Furthermore, the detection of breast cancer recurrence by the fluorescent probe is carried out in breast cancer model mice.

[0019] The fluorescent probe provided by the present invention has a fluorescence property whose emission wavelength is greater than 600 nm by introducing an indole group and a quinoline malononitrile group at both ends of phenothiazine. The fluorescent probe can monitor the changes of HClO in living cells and cancer cells under physiological conditions and convert them into changes in its fluorescence signal, which can be used for monitoring breast cancer recurrence and evaluating the efficacy of drug treatment for recurrent breast cancer, providing a preliminary basis for the subsequent clinical efficacy of recurrent breast cancer.

[0020] (III) Beneficial effects The beneficial effects produced by the present invention are: 1. The present invention successfully develops a fluorescent probe with a new structure by introducing indole groups and quinoline malononitrile groups at both ends of phenothiazine to provide it with a built-in recognition group for hypochlorous acid, thereby improving the sensitivity of the probe.

[0021] Compared with existing detection methods, this fluorescent probe has the following advantages: (1) Lower detection limit and higher sensitivity; (2) It is less affected by factors such as instruments and microenvironment, and can obtain effective high-resolution detection in vivo and in vitro, especially in deep tissues. It can also track and monitor hypochlorous acid in the body with high sensitivity and specificity, and can achieve real-time detection of recurrent breast cancer. (3) The substance being tested can be quantitatively analyzed through changes in fluorescence intensity without being interfered by the background of biological samples.

[0022] 2. By using this fluorescent probe to detect recurrent breast cancer in mice, it was found that this probe can determine the progression of recurrent breast cancer through real-time efficacy testing, reducing the risk of invasive testing.

[0023] 3. Compared with traditional detection methods, the detection of hypochlorous acid using the fluorescent probe proposed in the present invention is simple to operate, has low reagent consumption cost, high accuracy, and has good clinical transformation application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below.

[0025] Figure 1 is the molecular structure formula of the fluorescent probe.

[0026] Figure 2 The preparation route equation for the fluorescent probe.

[0027] Figure 3H NMR spectrum of PZ-CHO, an intermediate compound used to prepare fluorescent probes.

[0028] Figure 4 H NMR spectrum of intermediate compound 5 prepared for fluorescent probe.

[0029] Figure 5 H NMR spectrum of QP-CHO, an intermediate compound for the preparation of fluorescent probes.

[0030] Figure 6 This is the H NMR spectrum of the fluorescent probe QPIO.

[0031] Figure 7 This is the NMR carbon spectrum of the fluorescent probe QPIO.

[0032] Figure 8 This is the high-resolution mass spectrum of the fluorescent probe QPIO.

[0033] Fig. 9 This is the standard curve of the fluorescent probe QPIO for detecting hypochlorous acid.

[0034] Fig.10 This is the UV spectrum of the fluorescent probe QPIO before and after the reaction with hypochlorous acid.

[0035] Fig.11 The fluorescence spectra of the fluorescent probe QPIO before and after the reaction with hypochlorous acid.

[0036] Fig.12 The fluorescent probe QPIO was used for early diagnosis and prognostic evaluation of recurrent breast cancer model mice. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0039] Example 1: Preparation of fluorescent probe for detecting hypochlorous acid Preparation route as attached Figure 2 As shown, R 1 For H, R 2 For H, R 3 For H, R 4 Taking H as an example, the fluorescent probe is named QPIO.

[0040] Preparation of compound PZ-CHO: Compound 4 (3.00 g, 13.20 mmol) was added to a flask containing 10 mL of anhydrous DMF and stirred for 10 minutes under argon protection. 3 (10 mL, 125.00 mmol) was diluted in 20 mL of anhydrous DMF and added dropwise to the above solution at 0°C. The mixture was stirred continuously for 60 minutes and then moved to 90°C and stirred for another 20 hours. After cooling to room temperature, the mixture was poured into ice water and quenched with K 2 CO 3 The solution was adjusted to pH 7-8. The solution was extracted with DCM, washed 3 times with brine, and washed with anhydrous Na 2 SO 4 The organic layer was concentrated by rotary evaporation and the residue was purified by silica gel column using petroleum ether / ethyl acetate (v / v, 10 / 1, R f = 0.5) as the eluent to obtain compound PZ-CHO (2.90 g, 86%). 1 H NMR (400 MHz, DMSO- d 6 ): δ [ppm]: 9.79 (s, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.58 (s,1H), 7.23 (t, J = 6.8 Hz, 1H), 7.16 (d, J = 6.8 Hz, 2H), 7.08 (d, J = 7.6 Hz, 1H),7.00 (t, J = 7.2 Hz, 1H), 3.99 (q, J = 6.8 Hz, 2H), 1.32 (t, J = 6.8 Hz, 3H).

[0041] Preparation of compound 5: Compound PZ-CHO (2.00 g, 7.83 mmol) was added into a flask containing 10 mL of anhydrous DMF and stirred for 10 minutes under argon protection. 3 (6 mL, 80.00 mmol) was diluted in 20 mL of anhydrous DMF and added dropwise to the above solution at 0°C. The mixture was stirred continuously for 60 minutes and then moved to 90°C and stirred for another 20 hours. After cooling to room temperature, the mixture was poured into ice water and quenched with K 2 CO 3 The pH of the solution was adjusted to about 7-8. The solution was extracted with DCM and washed with anhydrous Na2 SO 4 The organic layer was concentrated by rotary evaporation, and the residue was passed through a silica gel column using petroleum ether / ethyl acetate (v / v, 2 / 1, R f = 0.5) to obtain pure compound 5 (1.80 g, 81%). 1 H NMR (400 MHz, DMSO- d 6 ): δ [ppm]:9.81 (s, 2H), 7.74 (d, J = 8.6 Hz, 2H), 7.61 (s, 2H), 7.24 (d, J = 8.6 Hz, 2H),4.06 (q, J = 6.8 Hz, 2H), 1.34 (t, J = 7.0 Hz, 3H).

[0042] Preparation of compound QP-CHO: Compound 3 (500 mg, 2.13 mmol) and compound 5 (730 mg, 2.55 mmol) were dissolved in 10 mL CH 3 CN / EtOH (v / v, 1 / 1) mixed solvent. Piperidine (0.20 mL, 1.88 mmol) was slowly injected through a syringe under argon protection. The reaction mixture was stirred at 75 ° C for 20 hours. After cooling to room temperature, the mixture was concentrated by rotary evaporation, and the residue was purified by silica gel column using dichloromethane / ethyl acetate (v / v, 10 / 1, R f = 0.5) as the eluent to obtain compound QP-CHO (750 mg, 70%). 1 H NMR (400 MHz, CDCl 3) δ [ppm]: 9.81 (s,1H), 9.13 (d, J = 8.0 Hz, 1H), 7.76 (t, J = 7.8 Hz, 1H), 7.70 – 7.61 (m, 2H),7.58 (s, 1H), 7.46 (t, J = 6.8 Hz, 1H), 7.31 – 7.29 (m, 2H), 7.20 (d, J =15.6 Hz, 1H), 7.12 (s, 1H), 6.94 (t, J = 7.6 Hz, 2H), 6.90 (s, 1H), 4.38 (t,J = 6.4 Hz, 2H), 4.01 (t, J = 6.4 Hz, 2H), 1.58 (s, 3H), 1.49 (t, J = 6.8 Hz,3H).

[0043] Preparation of fluorescent probe QPIO: QP-CHO (150 mg, 0.30 mmol) and compound 2 (150 mg, 0.45 mmol) were dissolved in 5 mL of ethanol mixed solvent. Piperidine (0.20 mL, 1.88 mmol) was injected by syringe under argon protection. The reaction mixture was stirred at 80 ° C for 10 hours. After cooling to room temperature, the mixture was concentrated by rotary evaporation, and the residue was purified by silica gel column using dichloromethane / methanol (v / v, 10 / 1, R f = 0.4) as the eluent to obtain the fluorescent probe QPIO (80 mg, 40%). 1 H NMR (400 MHz, DMSO-d 6) δ [ppm]: 8.93 (d, J = 8.0 Hz, 1H), 8.21 (d, J = 16.4 Hz, 1H), 8.10 (d, J = 8.8 Hz, 1H), 8.03 (s, 1H), 7.99 (d, J =8.4 Hz, 1H), 7.94 (t, J = 7.6 Hz, 1H), 7.77 (d, J = 9.2 Hz, 1H), 7.74 – 7.58(m, 6H), 7.51 (d, J = 6.8 Hz, 2H), 7.46 (s, 1H), 7.36 (d, J = 15.6 Hz, 1H),7.17 (m, 3H), 7.01 (s, 1H), 4.59 (q, J = 6.8 Hz, 2H), 4.08 (s, 3H), 4.06 (q,J = 7.0 Hz, 5H), 3.89 (s, 3H), 1.76 (s, 6H), 1.40 (t, J = 6.4 Hz, 3H), 1.36(t, J = 6.8 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6) δ [ppm]: 173.5, 158.3, 152.6,149.7, 136.6, 135.6, 134.2, 125.6, 121.1, 116.4, 115.8, 115.1, 113.9, 111.1,109.6, 109.1, 107.3, 56.6, 52.3, 49.4, 44.3, 27.8, 26.1, 24.6, 14.2, 12.8.HRMS for C 44 H 40 N 5 OS + [M] + , calculated: 686.2948; found: 686.2949.

[0044] Example 2: Calculation of standard curve for detection of hypochlorous acid using fluorescent probe QPIO

[0045] Sodium hypochlorite was dissolved in PBS solution (pH=7.4) to prepare 0, 10, 20, 30, 50, and 60 µM hypochlorous acid solutions. Then, 10 µg / mL of the fluorescent probe QPIO was added to the sodium hypochlorite solutions of different concentrations, and the solutions were placed in a water bath and incubated at 37 °C for 20 min. Finally, the fluorescence intensity was tested, with the excitation wavelength of 450 nm, the emission wavelength of 600 nm, and the slit width of 10 nm. The standard curve was calculated to be Y=0.42X+0.97, R 2 =0.999 (see attached Fig. 9 ).

[0046] Example 3: Spectral properties of the fluorescent probe itself

[0047] The new fluorescent probe (10 mg) was dissolved in dimethyl sulfoxide solution (10 mL) to prepare a 1 mg / mL probe test solution. The probe test solution was placed on an ultraviolet absorption spectrometer to scan the ultraviolet absorption spectrum in the range of 800-200 nm. Fig.10 As shown, the UV absorption spectrum ranges from 800 to 350 nm, and the central absorption wavelength is 600 nm. After adding 30 μM HClO to the above probe test solution, the UV absorption spectrum ranges from 380 to 600 nm, and the central absorption wavelength is 450 nm, indicating that the new probe can recognize and respond to HClO.

[0048] Example 4: Fluorescence detection behavior of fluorescent probe on HClO Accurately weigh 1.5 mg of the fluorescent probe and dissolve it in acetonitrile solution (15 mL) to prepare a 100 µg / mL probe test solution. Place the probe test solution in a colorimetric cell and place it in a fluorescence spectrophotometer to scan the fluorescence emission spectrum at a fixed excitation wavelength of 450 nm. Fig.11 As shown in FIG. 1 , the fluorescence emission spectrum ranges from 400 to 600 nm, and the central emission wavelength is 450 nm. The above probe test solution is mixed with 40 μM HClO and the fluorescence intensity at 600 nm is tested under 450 nm excitation on a fluorescence spectrophotometer. Fig. 9 The fluorescence enhancement is nearly 10,000 times, indicating that the new probe can respond to the fluorescence enhancement of HClO emitting red light. Different potential interfering substances (H 2 S. SO 2 , NO, H 2 O 2 、NO 2 - and NO 3), after reacting at room temperature for 10 min, the fluorescence intensity was then examined on a fluorescence spectrophotometer. It was found that other interfering substances except VPA did not cause changes in the probe fluorescence, indicating that the detection effect was good, the sensitivity was high, and interference could be effectively avoided.

[0049] Example 5: Early diagnosis of recurrent breast cancer model mice using fluorescent probe QPIO Weigh 1.5 mg of fluorescent probe accurately and dissolve it in DMSO solution (150 mL) to prepare 10 µg / mL probe test solution. Perform recurrent breast cancer detection on different groups of model mice. The Control group is a non-drug intervention group, and the DOX group and SDO group are different drug treatment groups. The disease progression of recurrent breast cancer mice is recorded by time point monitoring. Fig.12 As shown, after tumor resection, the fluorescence signal in the red channel was greatly weakened, indicating that the HClO level at the previous tumor site was reduced. During the tumor recurrence in mice, new tumor patches were observed with the naked eye on the 9th day, and their volume was the same as that of the previously removed tumor. The tumor recurrence rate reached 60% within 9 days after surgery. Under the intervention of SDO and DOX, near-infrared fluorescence imaging found that the fluorescence intensity of the mouse tumor site was significantly reduced compared with the control group, especially on the 7th and 9th days after surgery when the tumor recurrence rate was higher, indicating that the fluorescent probe QPIO can be used as an effective fluorescence imaging tool to provide valuable information for the early diagnosis and drug intervention of breast cancer tumor recurrence.

[0050] In summary, the present invention provides a preparation and application of a fluorescent probe for detecting hypochlorous acid, which can more sensitively perform quantitative detection of HClO in biological samples, avoid interference from substances in the sample, solve the problem of poor accuracy and sensitivity of existing detection methods, and has good clinical application prospects.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the protection scope of the present invention. In addition, after reading the technical content of the present invention, those skilled in the art may make various changes, modifications or variations to the present invention, and all of these equivalent forms also fall within the protection scope defined by the present application.

Claims

1. A fluorescent probe for detecting hypochlorous acid, characterized in that: The structure of the probe is as follows: ； Where R1, R2 are H, F, Cl, Br, I, NO2, C 1-18 One of alkyl, CN, CONHNH2, COOR, OH, OCH3; R3, R4 are one of F, Cl, Br, I.

2. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 1, characterized in that: The following steps are involved: (1) Preparation of 10-ethyl-10H-phenothiazine-3-carboxaldehyde 10-ethyl-10H-phenothiazine is added to a flask containing a suitable solvent and stirred under argon protection; POCl3 is diluted in a suitable solvent and added dropwise to the above solution at a suitable temperature; the mixture is stirred continuously, then moved to a suitable temperature and stirred again; after cooling to room temperature, the mixture is poured into ice water and the pH value is adjusted with a K2CO3 solution; the solution is extracted with dichloromethane, washed with brine 3 times, and dried over anhydrous Na2SO4; the organic layer is concentrated by rotary evaporation, and the residue is purified by a silica gel column using petroleum ether / ethyl acetate as an eluent to obtain 10-ethyl-10H-phenothiazine-3-carboxaldehyde; (2) Preparation of 10-ethyl-10H-phenothiazine-3,7-dicarboxaldehyde Add 10-ethyl-10H-phenothiazine-3-carboxaldehyde to a flask containing a suitable solvent and stir under argon protection; dilute POCl3 in a suitable solvent and add it dropwise to the above solution at a suitable temperature; stir the mixture continuously, then move to a suitable temperature and stir again; after cooling to room temperature, pour the mixture into ice water and adjust the pH value with K2CO3 solution; extract the solution with dichloromethane, wash with brine 3 times, and dry with anhydrous Na2SO4; concentrate the organic layer by rotary evaporation, and purify the residue by silica gel column using a suitable solvent as eluent to obtain 10-ethyl-10H-phenothiazine-3,7-dicarboxaldehyde; (3) Preparation of (E)-2-(1-ethyl-2-(2-(10-ethyl-7-formyl-10H-phenothiazin-3-yl)vinyl)quinolin-4(1H)-ylidene)malononitrile Dissolve 2-(1-ethyl-2-methylquinolin-4(1H)-ylidene)malononitrile and 10-ethyl-10H-phenothiazine-3,7-dicarbaldehyde in a suitable mixed solvent; slowly inject piperidine through a syringe under argon protection; keep the reaction mixture at a suitable temperature and stir again; after cooling to room temperature, concentrate the mixture by rotary evaporation, and purify the residue with a silica gel column using a suitable solvent as an eluent to obtain (E)-2-(1-ethyl-2-(2-(10-ethyl-7-formyl-10H-phenothiazin-3-yl)vinyl)quinolin-4(1H)-ylidene)malononitrile; (4) Preparation of fluorescent probes (E)-2-(1-ethyl-2-(2-(10-ethyl-7-formyl-10H-phenothiazin-3-yl)vinyl)quinolin-4(1H)-ylidene)malononitrile and 5-methoxy-1,2,3,3-tetramethyl-3H-indole-1-iodide are dissolved in a suitable mixed solvent; piperidine is injected through a syringe under the protection of argon gas, the reaction mixture is stirred at a suitable temperature, cooled to room temperature, and then the mixture is concentrated by rotary evaporation. The residue is purified by a silica gel column using a suitable solvent as an eluent to obtain a hypochlorous acid fluorescent probe.

3. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 2, characterized in that: The solvent is any one or more of DMF, DMSO, acetonitrile, acetone, ethanol, tetrahydrofuran, dichloromethane, methanol, petroleum ether, and ethyl acetate.

4. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 2, characterized in that: The reaction temperature is 25-150°C.

5. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 2, characterized in that: The reaction time is 2h-48h.

6. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 2, characterized in that: The reduced pressure concentration is carried out at a pressure of 0.1 Mpa-10 Mpa, and the eluent is petroleum ether / ethyl acetate or dichloromethane / methanol, and the volume ratio thereof is 100:1-1:

1.

7. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 2, characterized in that: The silica gel column chromatography separation adopts a 100-800 mesh silica gel column; the structure of the 10-ethyl-10H-phenothiazine is as follows: ； The structure of 2-(1-ethyl-2-methylquinolin-4(1H)-ylidene)malononitrile is as follows: ； The structure of 5-methoxy-1,2,3,3-tetramethyl-3H-indole-1-iodide is as follows: ； Where R1, R2 are H, F, Cl, Br, I, NO2, C 1-18 One of alkyl, CN, CONHNH2, COOR, OH, OCH3; R3, R4 are one of F, Cl, Br, I.

8. Use of the fluorescent probe according to claim 1 or 2 in detecting breast cancer recurrence.

9. The use according to claim 8, characterized in that: The detection of breast cancer recurrence by the fluorescent probe is carried out in breast cancer model mice.

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