Oxophenothiazine salt compound as well as preparation method and application thereof

Abstract

The invention discloses an oxophenothiazine salt compound as well as a preparation method and application thereof. The novel oxophenothiazine salt (MBO) fluorescent dye is researched and developed by adopting'no heavy atom effect 'and utilizing bridging sulfur atoms of oxygen substituted phenothiazine salt to carry out same-group atom substitution. According to the oxophenothiazine salt, the spin-orbit coupling (SOC) constant of methylene blue (MB) and the intersystem crossing rate (kISC) from the singlet state to the triplet state are reduced, the fluorescence imaging capacity of the oxophenothiazine salt is enhanced, and compared with methylene blue, the oxophenothiazine salt has better safety and lower cytotoxicity. The novel fluorescent dye has the functions of blue staining and fluorescence imaging on lymph nodes, and is suitable for preparing tumor sentinel lymph node biopsy and intraoperative lymph node tracing and cleaning reagents.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to an oxo-phenothiazine salt compound and a preparation method and application thereof. Background Art

[0002] Gastric cancer is one of the most common malignant tumors. In 2022, the number of new cases of gastric cancer worldwide was about 968,000, and the number of deaths from gastric cancer was about 660,000. Gastric cancer is highly malignant and progresses rapidly, and it is very easy to metastasize to lymph nodes.

[0003] The overall strategy for gastric cancer treatment is comprehensive treatment with surgery as the main approach. Radical surgery includes complete resection of the primary lesion and thorough clearance of regional lymph nodes. The intraoperative tracing and clearance of lymph nodes are related to the success of radical surgery. The eighth edition of the International Union Against Cancer / American Joint Committee on Cancer recommends the clearance of at least 16 groups of lymph nodes for radical gastric cancer resection. The Japanese Gastric Cancer Management Protocol also recommends the clearance of at least 15 groups of lymph nodes during surgery. This shows that the detection and clearance of gastric cancer lymph nodes play an important role in surgical treatment.

[0004] The "Healthy China Action-Cancer Prevention and Control Action Implementation Plan (2023-2030)" requires strengthening cancer prevention and control, screening, early diagnosis and treatment, and scientific research to steadily improve the level of standardized diagnosis and treatment, and effectively reduce the harm of cancer. Lymph node biopsy and tracing have always been the focus and difficulty of gastric cancer surgery. For tracers, the appropriate particle size is an important basis for lymph node development. The pore size between capillary lymphatic cells is about 100-500nm, which is much larger than the pore size of the capillary endothelium. Injecting tracers of appropriate particle size around the tumor can selectively enter the capillary lymphatic vessels, thereby developing the lymphatic vessels and lymph nodes. At present, the main methods for gastric cancer lymph node tracing are dye method, fluorescence method and radionuclide method.

[0005] Common lymph node tracers in clinical practice include indocyanine green (ICG), methylene blue, nanocarbon, and radionuclides. As a near-infrared fluorescent dye, indocyanine green is radiation-free and low-cost, but its fluorescence penetration distance is less than 1 cm, and fluorescence imaging cannot clearly show deep lymphatic drainage and metastasis. Blue dyes have a strong affinity for nucleic acids, among which the color development time of methylene blue is longer than that of isosulfan blue and patent blue, but the allergic reaction of methylene blue is higher than that of the two. The advantage of nanocarbon is that it can clearly identify and remove lymph nodes during surgery, but its retention time in tissues is too long, resulting in persistent black staining after surgery. Radionuclide tracers can provide high-quality lymph imaging for lymph node tracing, but the regulations and operational requirements for radionuclide radiation safety limit the promotion and use of radionuclide tracers. In addition, existing lymph node tracers can only achieve single white light or fluorescence tracing. In clinical practice, in order to improve the detection rate of lymph nodes, two or more tracers are often used in combination to improve the detection rate of lymph nodes, but this also brings complexity and instability to the operation.

[0006] Therefore, it is of great significance to develop a new fluorescent dye that can be used for lymph node tracing. Summary of the invention

[0007] Purpose of the invention: The purpose of the present invention is to provide an oxo-phenothiazine salt compound and a preparation method and application thereof in view of the deficiencies in the prior art.

[0008] The oxo-phenothiazine salt of the present invention uses oxygen to replace the bridging sulfur atom of the phenothiazine salt, and uses the "heavy atom-free effect" to perform homologous atom substitution. The oxo-phenothiazine salt reduces the spin-orbit coupling (SOC) constant of methylene blue and the intersystem crossing rate (k ISC ), enhancing its fluorescence imaging capability, thus enabling dual tracing of lymph nodes by white light and fluorescence, and having better safety and lower cytotoxicity compared to methylene blue.

[0009] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0010] The present invention provides an oxo-phenothiazine salt compound having the following structural formula:

[0011]

[0012] The chemical name is: 3,7-bis(dimethylamino)dibenzo[1,2-b:1',2'-e][1,4]oxathiaphenylthiazine salt.

[0013] The present invention also provides a method for preparing the oxo-phenothiazine salt compound, comprising the following steps:

[0014] (1) reacting 3-dimethylaminophenol with sodium nitrite to prepare an intermediate product, 4-dimethylaminobenzene-1,2-diol;

[0015]

[0016] (2) reacting the intermediate product obtained in step (1) with 3-dimethylaminophenol to prepare the oxyphenothiazine salt compound MBO.

[0017]

[0018] Preferably, in step (1), the intermediate product is prepared by the following method:

[0019] 3-Dimethylaminophenol was dissolved in hydrochloric acid, sodium nitrite was slowly added in portions, and the mixture was stirred for reaction. After the reaction was completed, the mixture was filtered, washed and dried to obtain the intermediate product 4-dimethylaminobenzene-1,2-diol.

[0020] Furthermore, the reaction temperature is ≤5°C and the reaction time is 2h.

[0021] Preferably, in step (2), the oxo-phenothiazine salt compound MBO is prepared by the following method:

[0022] Dissolve 3-dimethylaminophenol in 90% isopropanol, heat and stir to obtain a mixed solution; mix the intermediate product obtained in step (1) with perchloric acid, add it to the mixed solution, distill to obtain a crude product, filter and collect the precipitate, wash and dry it to obtain the oxo-phenothiazine salt compound MBO.

[0023] Furthermore, the heating temperature is 80°C.

[0024] The 90% isopropanol is prepared by mixing isopropanol and water in a volume ratio of 9:1.

[0025] A further preferred embodiment of the present invention is that the method for preparing the oxo-phenothiazine salt compound comprises the following steps:

[0026] (1) 3-dimethylaminophenol is dissolved in hydrochloric acid, sodium nitrite is slowly added in portions, the solution temperature is kept below 5° C. and stirred for 2 h. After the reaction is completed, the precipitate is filtered using a Buchner funnel, and the obtained precipitate is washed with a pre-cooled 2M hydrochloric acid solution and dried to obtain a yellow intermediate product 4-dimethylaminobenzene-1,2-diol;

[0027] (2) dissolving 3-dimethylaminophenol in 90% isopropanol, stirring at 80° C. under argon protection to obtain a mixed solution; mixing the intermediate product obtained in step (1) and perchloric acid in 90% isopropanol, and adding the mixture into the mixed solution in four equal portions for 1 hour; rotary distilling off a small amount of solvent, adding 90% isopropanol to the residue, and repeating the process three times within 4 hours to obtain a crude product by rotary distillation, and purifying the product by silica gel column chromatography with a gradient of CHCl3 / MeOH of 10:1-10:3 (v / v), washing, and drying to obtain a dark blue solid, i.e., the oxophenothiazine salt compound.

[0028] The invention also provides application of the oxo-phenothiazine salt compound as a fluorescent dye.

[0029] The present invention also provides the use of the oxo-phenothiazine salt compound in preparing a tumor sentinel lymph node biopsy reagent.

[0030] The present invention also provides the use of the oxo-phenothiazine salt compound in preparing a lymph node tracer during tumor surgery.

[0031] The present invention also provides the use of the oxo-phenothiazine salt compound in preparing a lymph node dissection reagent during tumor surgery.

[0032] Methylene blue is a traditional tracer dye that can quickly penetrate tissues to stain lymph nodes. However, methylene blue releases ROS under white light and during imaging, causing a certain degree of phototoxicity and tissue inflammatory response. Therefore, using the methylene blue skeleton for modification to effectively avoid the above problems is a new idea for developing a new type of non-toxic lymph node tracer. By replacing the bridging sulfur atom of phenothiazine salts with oxygen and using the "heavy atom-free effect" to replace atoms of the same group, oxy-phenothiazine salts reduce the spin-orbit coupling (SOC) constant of methylene blue and the intersystem crossing rate (k) from singlet to triplet. ISC ), enhancing its fluorescence imaging capability, thus enabling dual tracing of lymph nodes using white light and fluorescence.

[0033] Beneficial effects:

[0034] (1) The present invention introduces oxygen atoms into methylene blue and uses the "heavy atom-free effect" to perform homologous atom substitution to develop a new type of fluorescent dye, oxy-phenothiazine salt (MBO). Oxy-phenothiazine salt reduces the spin-orbit coupling (SOC) constant of methylene blue and the intersystem crossing rate (k) from singlet to triplet. ISC ), which can realize blue staining of lymph nodes and fluorescent tracing of lymph nodes at the same time, thus obtaining the ability of a single tracer to have both white light and fluorescent dual tracing.

[0035] (2) The novel fluorescent dye MBO prepared by the present invention can be retained in the lymph nodes for up to 3 hours, and a single administration can be achieved to ensure the tracing of the lymph nodes throughout the operation.

[0036] (3) Compared with methylene blue MB, the novel fluorescent dye MBO prepared by the present invention has higher safety and lower biological toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The oxo-phenothiazine salt compound of the present invention is 1 H NMR spectrum;

[0038] Figure 2 The oxo-phenothiazine salt compound of the present invention is 13 C NMR spectrum;

[0039] Figure 3 is the HR-MS spectrum of the oxo-phenothiazine salt compound of the present invention;

[0040] Figure 4 The physical picture of the novel fluorescent dye MBO and methylene blue MB of the oxo-phenothiazine salt of the present invention ( Figure 4 a), Absorption spectra of MBO and methylene blue MB ( Figure 4 b), Fluorescence spectra of MBO and methylene blue MB ( Figure 4 c);

[0041] Figure 5 This is a photo of the blue staining of lymph nodes after footpad injection of the novel fluorescent dyes MBO and MB of the oxo-phenothiazine salt of the present invention ( Figure 5 a), Fluorescence images of lymph nodes in vivo after MBO and MB footpad injection ( Figure 5 b);

[0042] Figure 6 Fluorescent imaging of lymph nodes using the new fluorescent dyes MBO and MB, which are oxo-phenothiazine salts, in cases where the blue-stained lymph nodes cannot be identified with the naked eye due to damaged blood vessels;

[0043] Figure 7 The fluorescence spectrum of SOSG under MBO illumination ( Figure 7 a), Fluorescence spectrum of SOSG under MB illumination ( Figure 7 b), Statistical line graph of SOSG spectral intensity under illumination conditions for MBO and MB ( Figure 7 c), Fluorescence images of ROS produced by MBO and MB in MFC cells under dark and light conditions ( Figure 7 d);

[0044] Figure 8 H&E staining results under illumination for MB footpad injection ( Figure 8a), H&E staining results under illumination conditions after MBO footpad injection ( Figure 8 b), Fluorescence quantitative PCR results of TNF-α production under light and dark conditions after MB and MBO footpad injection ( Figure 8 c), Fluorescence quantitative PCR results of IL-6 production under light and dark conditions after MB and MBO footpad injection ( Figure 8 d), CCK-8 results of cell viability of MFC cells incubated with different concentrations of MB and MBO in the dark ( Figure 8 e), CCK-8 results of cell viability of MFC cells incubated with different concentrations of MB and MBO under light conditions ( Figure 8 f). DETAILED DESCRIPTION

[0045] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0046] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0047] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0048] Example 1 Preparation of Oxyphenothiazine Salt Compounds

[0049] (1) Preparation of 4-dimethylaminobenzene-1,2-diol

[0050]

[0051] Dissolve 3-dimethylaminophenol (2 g, 14.58 mmol) in 5 ml of cold 6M HCl solution, and add NaNO2 (1.06 g, 15.31 mmol) in batches over 1 h, while keeping the solution temperature below 5 ° C. Continue stirring the reaction mixture for 2 h. After the reaction is completed, filter the precipitate with a Buchner funnel and wash it with cold 2M HCl solution. Dry the product to obtain an intermediate yellow solid for the next step.

[0052] (2) Preparation of 3,7-bis(dimethylamino)dibenzo[1,2-b:1',2'-e][1,4]oxathiazine salt (MBO oxathiazine salt compound)

[0053]

[0054] 3-Dimethylaminophenol (1.5 g, 9.03 mmol) was dissolved in 90% isopropanol (i-PrOH / H2O 9 / 1, volume ratio, 10 mL) and stirred at 80°C in a 100 ml three-necked round-bottom flask under argon protection. 4-Dimethylaminobenzene-1,2-diol (1.49 g, 10.83 mmol) and HClO4 (70%, 35 μl) were mixed evenly in 90% isopropanol (5 mL) and added to the above heated and stirred solution in four equal portions for 1 h. 10 ml of the solvent was rotary distilled and 10 ml of 90% isopropanol was added to the reaction mixture. This was repeated three times within 4 hours. The crude product was obtained by rotary distillation and purified by silica gel column chromatography with a gradient of CHCl3 / MeOH from 10:1 to 10:3 (v / v). The powder was washed with AcOEt and Et2O, and the product was dried under vacuum to obtain 600 mg of a dark blue solid, namely, an oxophenothiazine salt compound, with a yield of 25%.

[0055] The hydrogen spectrum of the oxo-phenothiazine salt compound MBO is shown Figure 1 , carbon spectrum Figure 2 And mass spectrometry Figure 3 .

[0056] 1 H NMR (400MHz, MeOD) δ (ppm) 7.82 (d, J = 9.6 Hz, 2H), 7.43 (dd, J = 9.6, 2.7 Hz, 2H), 6.98 (d, J = 2.7 Hz, 2H), 3.44 (s, 12H).

[0057] 13 C NMR (100MHz, MeOD): δ (ppm) 155.84, 139.56, 136.99, 135.96, 119.97, 107.36, 41.50ppm.

[0058] HR-MS (ESI, positive charge mode, m / z): [M-ClO4] + , calculated value 268.21523, measured value 268.14444.

[0059] Example 2: The new fluorescent dye MBO of oxo-phenothiazine salt can realize double tracking of lymph nodes by white light and fluorescence

[0060] In order to verify the dual white light and fluorescence tracing of lymph nodes by the new fluorescent dye MBO of oxo-phenothiazine salt, the absorption spectrum and fluorescence spectrum of the synthesized MBO were firstly detected.

[0061] Five-week-old, male, BALB / c mice weighing approximately 20 g were purchased from Nanjing Jicui Pharmaceutical Co., Ltd., and the MFC cell line was purchased from Wuhan Pronocell Life Science Co., Ltd.

[0062] The gastric cancer cells MFC with good growth status were injected into the right foot pad of mice. The number of gastric cancer cells injected into each mouse was 5×10 5 / mouse, to establish a footpad lymph node metastasis model. Two weeks later, the footpad was injected with MBO (3 mg / kg) dissolved in PBS. The footpad was dissected upward to observe whether the popliteal lymph nodes were stained blue and how long the blue staining lasted. In addition, the fluorescent imaging of the popliteal lymph nodes was observed by in vivo imaging of mice after MBO injection. The footpad of mice in the control group was injected with MB (3 mg / kg) dissolved in PBS. The experimental results are shown in Figure 4 .

[0063] Figure 4 a is a physical picture of MBO and methylene blue MB. Figure 4 b is the absorption spectrum of MBO and methylene blue MB detected by UV-vis spectrometer. Figure 4 c is the fluorescence spectra of MBO and methylene blue MB detected by Horiba FluoroMax-4 fluorescence spectrometer.

[0064] It can be seen from the figure that: compared with methylene blue itself, MBO has fluorescence imaging; and the fluorescence signal generated by MB is very weak, while the fluorescence signal of MBO is very strong, indicating that the bridging sulfur atom of the oxygen-substituted phenothiazine salt enhances the fluorescence imaging ability of MBO.

[0065] Blue-stained popliteal lymph nodes can be seen within minutes of MBO and MB footpad injection, and the blue staining time of lymph nodes can reach 3 hours ( Figure 5 a); using in vivo imaging (CRI maestro system (λ ex / λ em =650 / 665nm, CRI, USA) to observe the fluorescence imaging of lymph nodes. After MBO injection, the fluorescence imaging of popliteal lymph nodes was visible. The fluorescence of MB was weak and the fluorescence imaging of lymph nodes could not be performed ( Figure 5 b) It was demonstrated that compared with MB itself, the new fluorescent dye MBO based on oxo-phenothiazine salt can achieve dual white light and fluorescence tracing of lymph nodes.

[0066] Example 3: The novel fluorescent dye MBO of oxo-phenothiazine salt can realize the fluorescent tracing of lymph nodes during surgery

[0067] In order to simulate the tracing of lymph nodes by MBO during surgery, the footpad lymph node metastasis model constructed in Example 2 was used. Two weeks later, MBO (3 mg / kg) dissolved in PBS was injected into the footpad. The footpad was dissected upwards. After the blue-stained popliteal lymph nodes were observed, the surrounding blood vessels were damaged with a sharp instrument to simulate intraoperative bleeding. When the blue-stained popliteal lymph nodes could no longer be observed with the naked eye, in vivo imaging (CRI maestro system (λ ex / λ em=650 / 665nm, CRI, USA) to observe the fluorescence imaging of lymph nodes. The control group of mice was injected with MB dissolved in PBS (3mg / kg) in the foot pad. The experimental results are shown in Figure 6 .

[0068] from Figure 6 It can be seen that after the damaged blood vessels have made it impossible to identify the blue-stained lymph nodes with the naked eye, MBO can still be accurately located and clearly imaged under fluorescent imaging, and has the ability to trace the lymph nodes during surgery.

[0069] Example 4: The novel fluorescent dye MBO, a phenothiazine salt, produces less ROS in white light and imaging compared to methylene blue

[0070] In order to explore the ROS generated by MBO and MB during white light and imaging, reactive oxygen species probes were used for observation.

[0071] MB and MBO were placed in a PBS solution (1 μM), and illuminated with LED light (500 mW) at different time points. The SOSG probe (10 μM) purchased from Bio-Tech Biotechnology Co., Ltd. was used to observe the generation of ROS. The signal strength of SOSG on the Horiba FluoroMax-4 fluorescence spectrometer is positively correlated with the amount of ROS generated, such as Figure 7 As shown in ac, under light conditions, MBO produces less reactive oxygen species than MB.

[0072] At the same time, 32 μM of MBO and MB were added to the MFC cell culture plate cultured in 1640+10% FBS. The DCF probe purchased from Biotech Biotechnology Co., Ltd. was used to evaluate the production of ROS in the cells under LED light (500 mW). A fluorescence microscope (Leica, Germany) was used to observe the expression of green fluorescence in the cells. The more green fluorescence, the more ROS was produced. Figure 7 d shows that MBO produces less ROS than MB under light conditions.

[0073] Example 5: MBO, a novel fluorescent dye of oxo-phenothiazine salt, reduces tissue inflammatory response compared to MB

[0074] ROS can promote the recruitment of immune cells, aggravate local cell damage, and play a key role in inflammatory response. 5-week-old, male, BALB / c mice weighing about 20 g were purchased from Nanjing Jicui Pharmaceutical. Each mouse was injected with MBO and MB (3 mg / kg, PBS system) in the right footpad and illuminated with LED (500 mW, 10 min) twice, with an interval of 24 hours. After 48 hours, the footpad tissue was obtained, fixed, embedded, and frozen for detection of tissue immune cell infiltration and inflammatory factor production, respectively. CCK8 was used to observe the effects of different concentrations of MBO and MB on cell activity under light and dark conditions.

[0075] The foot pad tissue was fixed with formalin and embedded in paraffin, and 5 μm sections were cut and stained with hematoxylin and eosin. After staining, the sections were sealed with neutral resin. Figure 8 a and 8b are the results of H&E staining of the footpad after MB and MBO injection, respectively. The results showed that the footpad injected with MBO had less immune cell infiltration under light conditions compared with MB.

[0076] Fluorescence quantitative PCR was used to detect the production of tissue inflammatory factors such as MBO and methylene blue MB under light conditions. The PCR reaction system, primers and reaction program settings are as follows.

[0077] After RNA was extracted from the tissue, the extracted RNA was reverse transcribed into DNA using the Cowin Reverse Transcription Kit. The reaction system is shown in Table 1:

[0078] Table 1 Reaction system

[0079]

[0080] The DNA template obtained by the reaction was subjected to real-time quantitative qPCR reaction using the dye method (SYSB Green I). The primers, qPCR reaction system, and reaction program settings required for the reaction are shown in Table 2, Table 3, and Table 4, respectively:

[0081] Table 2 Reaction primers

[0082]

[0083] Table 3 qPCR reaction system

[0084]

[0085] Table 4 qPCR reaction procedure

[0086]

[0087]

[0088] The results are as follows Figure 8c and 8d showed that footpad-injected MBO produced less inflammatory factors TNF-α and IL-6 than MB under light conditions.

[0089] 100 μL of 1640+10% FBS culture system containing 8000 MFC cells was added to each well of a 96-well plate, and different concentrations of MBO and MB (experimental well As) were added to each well. At the same time, control wells Ac (containing cells, culture medium, CCK-8 solution, and no drugs) and blank wells Ab (containing culture medium, CCK-8 solution, and no cells and drugs) were set up. After dark and light (LED light, 500 mW, 1 min) treatment, the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. 10 μL of CCK-8 solution was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 2 hours. The absorbance at 450 nm was measured by an enzyme reader. The cell survival rate corresponding to different concentrations of MBO and MB was calculated as follows:

[0090] [(As-Ab) / (Ac-Ab)]×100%

[0091] The half-inhibitory concentrations (IC50) of MBO and MB under dark and light conditions were calculated by GraphPad. 50 ).

[0092] Figure 8 e shows that the IC of MBO in the dark 50 The IC of MB is 133 μM. 50 94 μM; Figure 8 f shows that MBO has a higher IC than MB under light conditions. 50 , which shows that MBO is less toxic to cells and has better biosafety.

[0093] Therefore, the new fluorescent dye MBO of oxo-phenothiazine salt can achieve dual tracing of lymph nodes by white light and fluorescence, and has smaller tissue inflammatory response and better safety compared with MB.

[0094] The tracer involved in the present invention is a new type of fluorescent dye, which has not been reported in other studies. The fluorescent dye has the ability to dually trace lymph nodes with white light and fluorescence, and can realize biopsy of tumor sentinel lymph nodes, intraoperative tracing and clearance of lymph nodes. Compared with MB, it has a smaller tissue inflammatory response and is safer.

[0095] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. An oxo-phenothiazine salt compound, characterized in that: It has the following structural formula:

2. A method for preparing an oxo-phenothiazine salt compound according to claim 1, characterized in that: The following steps are involved: (1) reacting 3-dimethylaminophenol with sodium nitrite to prepare an intermediate product, 4-dimethylaminobenzene-1,2-diol; (2) reacting the intermediate product obtained in step (1) with 3-dimethylaminophenol to prepare the oxyphenothiazine salt compound MBO.

3. The preparation method according to claim 2, characterized in that: In step (1), the intermediate product is prepared by the following method: 3-Dimethylaminophenol was dissolved in hydrochloric acid, sodium nitrite was slowly added in portions, and the mixture was stirred for reaction. After the reaction was completed, the mixture was filtered, washed and dried to obtain the intermediate product 4-dimethylaminobenzene-1,2-diol.

4. The preparation method according to claim 3, characterized in that: The reaction temperature is ≤5°C and the reaction time is 2h.

5. The preparation method according to claim 2, characterized in that: In step (2), the oxo-phenothiazine salt compound MBO is prepared by the following method: Dissolve 3-dimethylaminophenol in 90% isopropanol, heat and stir to obtain a mixed solution; mix the intermediate product obtained in step (1) with perchloric acid, add it to the mixed solution, distill to obtain a crude product, filter and collect the precipitate, wash and dry it to obtain the oxo-phenothiazine salt compound MBO.

6. The preparation method according to claim 5, characterized in that: The heating temperature is 80°C.

7. Use of the oxo-phenothiazine salt compound according to claim 1 as a fluorescent dye.

8. Use of the oxo-phenothiazine salt compound according to claim 1 in the preparation of a tumor sentinel lymph node biopsy reagent.

9. Use of the oxo-phenothiazine salt compound according to claim 1 in the preparation of a lymph node tracer during tumor surgery.

10. Use of the oxo-phenothiazine salt compound according to claim 1 in preparing a lymph node dissection reagent during tumor surgery.

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