An oxo-phenothiazine salt compound and its preparation method and application

Abstract

The present invention discloses an oxy-phenothiazine salt compound, its preparation method, and application. The present invention adopts the "heavy atom-free effect" and utilizes the bridging sulfur atom of the oxygen-substituted phenothiazine salt to perform homologous atom substitution to develop a novel fluorescent dye, an oxy-phenothiazine salt (MBO). The oxy-phenothiazine salt of the present invention reduces the spin-orbit coupling (SOC) constant of methylene blue (MB) and the intersystem crossing rate (k) from the singlet state to the triplet state. ISC ), enhancing its fluorescence imaging capabilities and offering improved safety and lower cytotoxicity compared to methylene blue itself. The novel fluorescent dye of the present invention combines blue staining of lymph nodes with fluorescence imaging, making it suitable for use in the preparation of reagents for sentinel lymph node biopsy, intraoperative lymph node tracing, and clearance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to an oxophenothiazine salt compound and a preparation method and application thereof. Background Art

[0002] Gastric cancer is one of the most common malignant tumors. In 2022, there were approximately 968,000 new cases of gastric cancer worldwide, and approximately 660,000 deaths from the disease. Gastric cancer is highly malignant, progresses rapidly, and is prone to lymph node metastasis.

[0003] The overall strategy for gastric cancer treatment is a comprehensive approach, primarily based on surgery. Radical surgery involves complete resection of the primary lesion and thorough clearance of regional lymph nodes. Intraoperative lymph node mapping and the extent of clearance are crucial to the success of radical resection. The eighth edition of the International Union Against Cancer / American Joint Committee on Cancer guidelines recommends clearance of at least 16 lymph nodes for radical gastric cancer resection. The Japanese Guidelines for the Management of Gastric Cancer also recommend clearance of at least 15 lymph nodes. This demonstrates the crucial role that lymph node detection and clearance play 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, 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. Tracers with appropriate particle size are injected around the tumor, which 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. Indocyanine green is a near-infrared fluorescent dye that 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 them, 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 it stays in the tissue for too long, resulting in persistent black staining after surgery. Radionuclide tracers can provide high-quality lymphatic 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 its preparation method and application in view of the shortcomings of 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 utilizes 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) from the singlet state to the triplet state. 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]oxathiazole salt.

[0013] The present invention also provides a method for preparing the oxophenothiazine 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] 3-Dimethylaminophenol is dissolved in 90% isopropanol, heated and stirred to obtain a mixed solution; the intermediate product obtained in step (1) is evenly mixed with perchloric acid, added to the above mixed solution, and distilled to obtain a crude product, and the precipitate is collected by filtration, washed, and dried to obtain the oxophenothiazine salt compound MBO.

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

[0024] The 90% isopropyl alcohol is prepared by mixing isopropyl alcohol 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 oxophenothiazine salt compound comprises the following steps:

[0026] (1) 3-Dimethylaminophenol was dissolved in hydrochloric acid, and sodium nitrite was slowly added in portions, and the solution temperature was kept below 5°C and stirred for 2 hours. After the reaction was completed, the precipitate was filtered with a Buchner funnel, and the obtained precipitate was 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 and stirring at 80° C. under argon protection to obtain a mixed solution; uniformly mixing the intermediate product obtained in step (1) with 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, which is purified by silica gel column chromatography with a gradient of CHCl3 / MeOH in a ratio of 10:1 to 10:3 (v / v), and washed and dried to obtain a dark blue solid, i.e., the oxophenothiazine salt compound.

[0028] The present 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 to modify and 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 the phenothiazine salt with oxygen and using the "heavy atom-free effect" to replace the same group of atoms, the oxy-phenothiazine salt reduces the spin-orbit coupling (SOC) constant of methylene blue and the intersystem crossing rate (k) from the singlet state to the triplet state. 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, oxyphenothiazine salt (MBO). Oxyphenothiazine salt reduces the spin-orbit coupling (SOC) constant and the intersystem crossing rate (k) from singlet to triplet of methylene blue. ISC ), which can achieve blue staining of lymph nodes while also performing fluorescent tracing of lymph nodes, thereby obtaining the ability of a single tracer to have both white light and fluorescent dual tracing capabilities.

[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 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 oxophenothiazine salt compound of the present invention;

[0040] Figure 4 The physical picture of the novel fluorescent dye MBO and methylene blue MB 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 novel fluorescent dyes MBO and MB, both oxo-phenothiazine salts, in cases where 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 of MBO and MB under illumination conditions ( 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 conditions 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 experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. 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] 3-Dimethylaminophenol (2 g, 14.58 mmol) was dissolved in 5 ml of cold 6 M HCl solution. NaNO2 (1.06 g, 15.31 mmol) was added portionwise over 1 hour while maintaining the solution temperature below 5°C. The reaction mixture was stirred for 2 hours. After the reaction was complete, the precipitate was filtered using a Buchner funnel and washed with cold 2 M HCl solution. The product was dried to obtain a yellow solid intermediate for use in 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, v / v, 10 mL) and stirred at 80°C in a 100 mL three-necked round-bottom flask under argon. 4-Dimethylaminobenzene-1,2-diol (1.49 g, 10.83 mmol) and HClO₄ (70%, 35 μl) were mixed in 90% isopropanol (5 mL) and added to the heated and stirred solution in four equal portions over 1 hour. A 10 mL volume of solvent was rotary distilled, and 10 mL of 90% isopropanol was added to the reaction mixture. This process was repeated three times over 4 hours. Rotary distillation afforded the crude product, which was purified by silica gel column chromatography using a CHCl₃ / MeOH gradient of 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, ie, 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, a phenothiazine salt, can achieve dual white light and fluorescence tracing of lymph nodes

[0060] In order to verify the dual white light and fluorescence tracing of lymph nodes by the novel 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] MFC gastric cancer cells 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, a footpad lymph node metastasis model was established. Two weeks later, MBO dissolved in PBS (3 mg / kg) was injected into the footpad. The footpad was dissected upwards to observe whether the popliteal lymph nodes were stained blue and how long the blue staining lasted. In addition, after the injection of MBO, the mice were imaged in vivo to observe the fluorescence imaging of the popliteal lymph nodes. The control group mice were injected with MB dissolved in PBS (3 mg / kg) in the footpad. 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] As can be seen from the figure, MBO has fluorescence imaging compared to methylene blue itself; 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 the 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, a novel oxo-phenothiazine salt, can achieve dual white light and fluorescence tracing of lymph nodes.

[0066] Example 3: The novel fluorescent dye MBO, a phenothiazine salt, can achieve intraoperative fluorescent tracing of lymph nodes

[0067] 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 footpad. The experimental results are shown in Figure 6 .

[0068] from Figure 6 It can be seen that even after the damaged blood vessels make it impossible to identify the blue-stained lymph nodes with the naked eye, MBO can still accurately locate and clearly image them under fluorescence imaging, and has the ability to trace the lymph nodes during surgery.

[0069] Example 4: The novel fluorescent dye MBO, a phenothiazine salt, generates 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 production of ROS was observed using a SOSG probe (10 μM) purchased from Biyuntian Biotechnology Co., Ltd. The signal strength of SOSG on the Horiba FluoroMax-4 fluorescence spectrometer was positively correlated with the amount of ROS produced. 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 Biyuntian 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 amount of green fluorescence expression 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 containing oxo-phenothiazine salt, reduces tissue inflammatory response compared to MB.

[0074] ROS can promote the recruitment of immune cells, exacerbate local cell damage, and play a key role in inflammatory responses. Five-week-old, male, BALB / c mice weighing approximately 20 g were purchased from Nanjing Jicui Pharmaceutical. Each mouse was injected with MBO and MB (3 mg / kg in PBS) into the right footpad and illuminated with LED light (500 mW, 10 min) twice, 24 hours apart. Footpad tissue was harvested 48 hours later, fixed, embedded, and cryopreserved for detection of tissue immune cell infiltration and inflammatory cytokine production. CCK8 was also used to investigate the effects of different concentrations of MBO and MB on cell viability under light and dark conditions.

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

[0076] Fluorescence quantitative PCR was used to detect the production of tissue inflammatory factors in 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 Kangwei Century 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 used for 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 injection of 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 8,000 MFC cells was added to each well of a 96-well plate. Different concentrations of MBO and MB were added to each well (experimental wells As). Control wells Ac (containing cells, culture medium, CCK-8 solution, but no drug) and blank wells Ab (containing culture medium, CCK-8 solution, but no cells or drug) were set up. After treatment with darkness and light (LED light, 500 mW, 1 min), 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 another 2 hours. The absorbance at 450 nm was measured using a microplate reader. The cell viability 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 MB has an IC of 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 oxophenothiazine 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 that has not been reported in other studies. This fluorescent dye has the ability to dually trace lymph nodes with white light and fluorescence, enabling biopsy of tumor sentinel lymph nodes, intraoperative tracing and clearance of lymph nodes, and at the same time, 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 may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. Use of an oxophenothiazine salt compound in the preparation of a tumor sentinel lymph node biopsy reagent, characterized in that: The compound has the following structural formula: 。 2. Use of an oxophenothiazine salt compound in the preparation of a lymph node tracer during tumor surgery, characterized in that: The compound has the following structural formula: 。 3. Use of an oxophenothiazine salt compound in the preparation of a lymph node dissection reagent during tumor surgery, characterized in that: The compound has the following structural formula: 。

Images