An organic dye based on phenothiazine structure, its synthesis method and application

Abstract

This invention belongs to the field of fluorescent probes and photodynamic therapy (PDT) applications, specifically relating to an organic dye based on a phenothiazine structure, its synthesis method, and its applications. Using phenothiazine and 4,4'-dimethoxytriphenylamine as electron donors and methylpyridinium salt as electron acceptors, and with phenothiazine as the reaction site, this invention synthesizes an organic dye based on a phenothiazine structure for detecting hypochlorite and for tumor photodynamic therapy. This dye is the first of its kind synthesized in this invention, using readily available raw materials, a simple process, and is industrially feasible. Furthermore, this dye exhibits high selectivity and high sensitivity for hypochlorite detection, enabling rapid detection of hypochlorite accompanied by fluorescence activation. It can specifically image tumor cells, such as HeLa cells. Simultaneously, this dye generates reactive oxygen species (ROS) under light conditions, which can be used to assess the efficacy of fluorescence imaging-guided tumor photodynamic therapy. It has broad value in the detection of hypochlorite signaling molecules and in tumor photodynamic therapy in the biomedical field.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probes and photodynamic therapy applications, specifically relating to an organic dye based on a phenothiazine structure, its synthesis method, and its application. Background Technology

[0002] Hypochlorous acid or hypochlorite (HClO / ClO) - Reactive oxygen species (ROS), produced by the catalytic action of hydrogen peroxide and chloride ions under the action of myeloperoxidase (MPO), are among the reactive oxygen species present in living organisms. Simultaneously, as an inflammatory substance, it exists in high concentrations within tumor cells. - Abnormal accumulation of ClO₂ is closely related to the pathogenesis of tumors. Therefore, achieving ClO₂ accumulation in organisms, especially tumor cells, is crucial. - The detection of ClO content is of great significance. Several traditional methods have been proposed. - Detection methods include iodometric titration, electrochemical analysis, potentiometric analysis, chromatography, and colorimetric chemical sensors. Among these, fluorescence analysis has attracted widespread attention due to its advantages over traditional detection methods, such as good selectivity, high temporal and spatial resolution, and high sensitivity.

[0003] Cancer, a disease with high mortality, high invasiveness, and high recurrence rates, threatens the lives and health of an increasing number of people. Photodynamic therapy (PDT) is a pioneering and effective anti-cancer modality that relies on photoactivated photosensitizers (PSs) to generate cytotoxic reactive oxygen species. PDT for cancer treatment has low side effects and high selectivity, and shows great promise in clinical applications. Therefore, developing effective methods for cancer diagnosis and treatment is of great significance.

[0004] Compared to normal cells, tumor cells, such as cervical cancer (HeLa) cells, contain a relatively higher concentration of ClO₂. - Therefore, this characteristic can be used to distinguish and image normal cells from tumor cells. To date, several small organic molecule fluorescent probes have been reported for use with ClO₂. - While these small organic molecule probes can be used for cell imaging, they are rarely used for imaging-guided photodiodes (PDTs). Furthermore, most PSs do not have the ability to specifically image tumor cells.

[0005] Phenothiazine is a heterocyclic compound with a non-planar "butterfly" structure. As a fluorophore nucleus, it possesses advantages such as ease of functionalization and a large Stokes shift. The sulfur atom in the phenothiazine nucleus is readily converted to ClO₂. - It is oxidized to sulfoxide (S=O). Therefore, this property can be used to design and synthesize a class of fluorescent probes based on the phenothiazine structure. The synthesized probes have the specific recognition of ClO. -It can image tumor cells and also has the ability to perform fluorescence imaging-guided photodynamic therapy on tumors. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide an organic dye based on the phenothiazine structure, its synthesis method and application.

[0007] The first aspect of this invention is to provide an organic dye based on a phenothiazine structure, the general structural formula of which is:

[0008]

[0009] Wherein, X is S or O, and R is H or a halogen or a C1-C20 straight-chain alkoxy or a C1-C20 branched-chain alkoxy.

[0010] Preferably, R is OCH3 and X is S.

[0011] A second aspect of the present invention is to provide a method for synthesizing an organic dye based on a phenothiazine structure as described above, comprising the following steps:

[0012] (1) Compound 1 and compound 3 react to give compound 2;

[0013] (2) Compound 2 is reacted with 1,4-dimethylpyridine-1-onium iodide to obtain the phenothiazine-based organic dye as described in claim 1 or 2.

[0014] The structural formula of compound 1 is:

[0015]

[0016] The structural formula of compound 3 is:

[0017]

[0018] The structural formula of compound 2 is:

[0019]

[0020] Preferably, in step (1), under the protection of an inert gas, compound 1 and compound 3 are dissolved in 1,4-dioxane, Cs2CO3 and catalyst are added, the reaction solution is stirred and reacted under the protection of an inert gas, and after the reaction is completed, the mixture is extracted, dried and separated to obtain compound 2.

[0021] Preferably, the molar ratio of compound 1, compound 3, Cs2CO3 and catalyst is 1:2.1:3:0.05.

[0022] Preferably, the catalyst is Pd(PPh3)4.

[0023] Preferably, in step (2), under the protection of an inert gas, anhydrous ethanol is used as a solvent to add compound 2, 1,4-dimethylpyridine-1-onium iodide and piperidine, and the mixture is heated to reflux and stirred to react. After the reaction is completed and cooled, the mixture is concentrated and purified to obtain the organic dye based on the phenothiazine structure.

[0024] Preferably, the molar ratio of compound 2 to 1,4-dimethylpyridine-1-onium iodide is 1:3.

[0025] A third aspect of the present invention provides an application of an organic dye based on a phenothiazine structure, as described above, for the detection of hypochlorite, wherein the phenothiazine organic dye exhibits fluorescence activation after reacting with hypochlorite.

[0026] A fourth aspect of the present invention provides an application of the organic dye described above in the preparation of a tumor photodynamic therapy drug, wherein the phenothiazine-type organic dye is capable of killing tumor cells by light irradiation, the tumor cells including cervical cancer cells and liver cancer cells.

[0027] The beneficial effects of this invention are reflected in:

[0028] (1) This invention synthesizes a novel type of organic dye for hypochlorite detection using phenothiazine and 4,4'-dimethoxytriphenylamine as electron donors, methylpyridinium salt compounds as electron acceptors, and phenothiazine as the reaction site. This organic dye is the first of its kind synthesized in this invention. The raw materials are readily available, the process is simple, it can be industrialized, and it has strong commercial value.

[0029] (2) The organic dye provided by this invention reacts with ClO in an EtOH / H2O (v / v = 1 / 1) solution. - After the reaction, both the ultraviolet-visible absorption spectrum and the fluorescence emission spectrum changed significantly, thus enabling the visual detection of hypochlorite ions by this type of organic dye.

[0030] (3) The organic dyes provided by this invention exhibit good selectivity and anti-interference ability for the detection of hypochlorite ions, H2O2,·OH, t BUOO - , t BUOOH, 1 O2, NO, glutathione (GSH), cysteine ​​(Cys), homocysteine ​​(Hcy), glutamate (Glu), histidine (His), K + Na + ,Fe 2+ Mg 2+ SO4 2- S2O5 2- HSO3 - ,F- ,Cl - Plasma, biothiols, and amino acids do not affect the dye's effect on ClO. - The detection caused interference.

[0031] (4) The organic dye provided by the present invention has a high sensitivity for the detection of hypochlorite, reaching the nanomolar level, which provides support for the detection of endogenous hypochlorite in cells.

[0032] (5) The organic dyes provided by this invention can rapidly enter cells and achieve endogenous and exogenous ClO in HeLa cells. - The detection.

[0033] (6) The organic dye provided by the present invention has a strong ROS generation efficiency, showing a stronger ROS generation efficiency than the commercial dye RoseBengal. In addition, ROS generation can also be clearly detected in cells.

[0034] (7) The organic dyes provided by this invention have low cell dark toxicity, but show strong phototoxicity under white light irradiation, indicating that such photosensitizers have high biological safety and excellent photodynamic therapy effect.

[0035] In summary, this invention provides an organic dye based on a phenothiazine structure. This organic dye is simple to prepare, exhibits high selectivity and sensitivity for hypochlorite detection, and enables rapid detection of hypochlorite accompanied by fluorescence activation, allowing for specific imaging of tumor cells. Furthermore, this dye generates ROS under illumination both before and after hypochlorite response, demonstrating photodynamic ability. This allows for fluorescence-guided photodynamic therapy to kill tumor cells, demonstrating its broad application value in the biomedical field for the detection of hypochlorite signaling molecules and in tumor and photodynamic therapy. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0037] Figure 1 This is the synthesis route diagram for Example 1;

[0038] Figure 2 A is the organic dye synthesized in Example 1, reacted with ClO in EtOH / H2O (v / v = 1 / 1) solvent (dye concentration: 10 μM). -(100μM) UV-Vis absorption spectra before and after the response Figure 2 B represents the reaction of the dye in EtOH / H2O (v / v = 1 / 1) solvent (dye concentration: 10 μM) with ClO. - Fluorescence emission spectra before and after the response (100 μM);

[0039] Figure 3 A is the organic dye synthesized in Example 1, reacted with ClO in EtOH / H2O (v / v = 1 / 1) solvent (dye concentration: 10 μM). - A bar chart showing the fluorescence intensity of (100 μM) or other test samples (100 μM). Figure 3 B represents the dye in the reaction of EtOH / H2O (v / v = 1 / 1) (concentration: 10 μM) and ClO. - Fluorescence intensity histogram after the reaction of H2O2 (1), H2O2 (1), and OH (2) with other test samples (100 μM); Test samples: H2O2 (1), ·OH (2), t BUOO - (3), t BUOOH(4), 1 O2(5),NO(6),GSH(7),Cys(8),Hcy(9),L-Glu(10),L-His(11),K + (12),Na + (13),Fe 2+ (14),Mg 2+ (15), SO4 2- (16),S2O5 2- (17), HSO3 - (18),F - (19),Cl - (20),ClO - (21), Blank(22);

[0040] Figure 4 A represents the organic dye (10 μM) synthesized in Example 1 and different concentrations of ClO₂. - Fluorescence spectrum changes after reaction (0, 10, 30, 50, 70, 80, 90, 95, 100 μM) Figure 4 B represents the fluorescence intensity of the organic dye (10 μM) synthesized in Example 1 and its correlation with ClO. - Linear relationship graph of concentration (2-10 μM);

[0041] Figure 5 A shows the fluorescence intensity change of the ROS indicator DCFH-DA under white light irradiation caused by the dye TPTPy synthesized in Example 1. Figure 5B is the dye TPTPy synthesized in Example 1, which is reacted with ClO - Images showing the fluorescence changes of DCFH-DA under the same conditions after the response. Figure 5 C is an image showing the fluorescence change of DCFH-DA under the same conditions caused by the commercial dye Rose Bengal. Figure 5 D represents adding only ClO - Images showing fluorescence changes in DCFH-DA under the same conditions;

[0042] Figure 6 The dye TPTPy synthesized in Example 1 reacts with endogenous and exogenous ClO in normal human umbilical vein endothelial cells (HUVEC) and cervical cancer cells (HeLa). - The fluorescence confocal images after the reaction: (A1-A3) HeLa cells were cultured with TPTPy (10 μM) for 2 h; (B1-B3) HeLa cells were first cultured with NAC (100 μM) for 0.5 h, then with TPTPy (10 μM) for 2 h; (C1-C3) HeLa cells were first cultured with NaClO (100 μM) for 0.5 h, then with TPTPy (10 μM) for 2 h; (D1-D3) HeLa cells were first cultured with NAC (100 μM) for 0.5 h, then with NaClO (100 μM) for 0.5 h, and finally with TPTPy (10 μM) for 2 h; (E1-E3) HUVEC cells were cultured with TPTPy (10 μM) for 2 h.

[0043] Figure 7 The image shows the fluorescence imaging of the organic dye synthesized in Example 1 in HeLa cells, which enhanced the ROS indicator DCFH-DA after irradiation with white light for different durations.

[0044] Figure 8 This is a graph showing the phototoxicity and dark toxicity data of the organic dye TPTPy synthesized in Example 1 in HeLa cells. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0046] Example 1

[0047] Synthesis of TPTPy, an organic dye based on phenothiazine structure for the detection of hypochlorite and photodynamic therapy of tumors.

[0048] (1) Synthesis of compound 2

[0049]

[0050] A 50 mL two-necked round-bottom flask was used as a solvent. 15 mL of 1,4-dioxane and 5 mL of ultrapure water were added, along with 658 mg (1.0 mmol) of compound 1 and 499 mg (2.1 mmol) of 5-aldehyde-2-thiophene borate pinacol ester. Then, 975 mg (3.0 mmol) of Cs₂CO₃ was added, followed by 60 mg of Pd(PPh₃)₄ as a catalyst. Under argon protection, the reaction mixture was thoroughly mixed and heated to 100 °C for 12 h. After the reaction was complete, the mixture was cooled to room temperature, and 30 mL of ultrapure water was added. The mixture was then extracted multiple times with dichloromethane. The extracted organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was separated and purified by silica gel column chromatography using (petroleum ether / ethyl acetate / dichloromethane, v / v = 4 / 1 / 1) as the mobile phase, yielding 0.41 g of orange-yellow solid compound 2, with a yield of 57%. 1 H NMR(400MHz,DMSO-d6)δ9.86(s,2H),7.99(s,2H),7.63(s,2H),7.51(s,2H),7.38(d,J=7.8Hz,2H ), 7.21 (d, J = 7.0Hz, 6H), 6.99 (d, J = 7.9Hz, 3H), 6.92 (s, 3H), 6.24 (d, J = 8.4Hz, 2H), 3.76 (s, 6H).

[0051] (2) Synthesis of dye TPTPy

[0052]

[0053] A 50 mL two-necked round-bottom flask was filled with 20 mL of anhydrous ethanol as solvent. 722 mg (1.0 mmol) of compound 2, 1.17 g (5.0 mmol) of 1,4-dimethylpyridine-1-onium iodide, and 0.2 mL of piperidine were added. After thoroughly mixing the reaction mixture, the mixture was heated to 80 °C and reacted for 24 h under argon protection. After the reaction was complete, the mixture was allowed to cool to room temperature to obtain the crude product. The crude product was purified by neutral alumina column chromatography using dichloromethane / methanol (v / v = 10 / 1) as the mobile phase to obtain 532 mg of the deep purple compound TPTPy. The yield was 46%. 1H NMR (400MHz, DMSO-d6) δ8.82(d,J=6.6Hz,4H),8.18(t,J=12.1Hz,6H),7.51(dd,J=17.5,3.7Hz,4H),7.44(d,J=1.8Hz,2H),7.29(m,2H),7. 22(d,J=8.7Hz,6H), 7.12(d,J=15.9Hz,2H), 7.00(d,J=8.9Hz,4H), 6.94(d,J=8.8Hz,2H), 6.26(d,J=8.6Hz,2H), 4.22(s,6H), 3.77(s,6H).

[0054] Example 2

[0055] The organic dye TPTPy from Example 1 was subjected to UV and fluorescence emission spectra after responding to hypochlorous acid, as shown in the following figures. Figure 2 As shown in A and 2B. The maximum absorption wavelength of TPTPy is at 490 nm, which is similar to that of ClO. - After the reaction, the dye's maximum absorption peak shifted to 445 nm. Meanwhile, TPTPy and ClO... - The fluorescence intensity changed significantly before and after the reaction. TPTPy without the addition of ClO - The previous fluorescence emission signal was very weak, but after adding ClO - Subsequently, the dye solution exhibited a significant fluorescence "turn-on" phenomenon, showing a strong fluorescence signal peak at 585 nm. Simultaneously, upon the addition of ClO... - Afterwards, we can observe with the naked eye that the solution changes from its original light pink to yellow, and under 365nm ultraviolet light, the solution emits a clearly visible orange-red fluorescence.

[0056] Example 3

[0057] Selectivity tests were conducted on the organic dye TPTPy from Example 1. Figure 3 As shown in Figure A, the addition of various oxides, amino acids, and representative ions did not significantly alter the fluorescence emission of TPTPy. However, ClO - The addition of [a specific ingredient] can cause a significant "turn-on" phenomenon in the fluorescence of TPTPy. Furthermore, such as... Figure 3 As shown in B, in the presence of other competing samples, ClO - It will still cause TPTPy to appear and Figure 3 A similar fluorescence response phenomenon. Selectivity and anti-interference experiments demonstrate that the probe TPTPy exhibits a similar response to ClO. - It has excellent selectivity.

[0058] Example 4

[0059] A fluorescence titration experiment was performed on the organic dye TPTPy from Example 1, as follows: Figure 4 As shown in Figure A. The probe TPTPy itself has very weak fluorescence, which increases with ClO₂. - With the addition of [a specific component], the fluorescence emission intensity at 585 nm gradually increases. Figure 4 B is the probe TPTPy in ClO - Fluorescent titration experiments with concentrations ranging from 2 to 10 μM were conducted. The effect of dye TPTPy on ClO₂ was calculated by fitting a linear relationship to a straight line. - The detection limit of TPTPy is 185.38 nM. The results indicate that TPTPy has a detection limit of 185.38 nM for ClO₂. - It has very high sensitivity.

[0060] Example 5

[0061] The dye TPTPy and ClO in Example 1 - Experiments were conducted to test the ROS generation capability before and after the response, such as... Figure 5 As shown. DCFH-DA is an indicator of ROS. Figure 5 In A and 5B, the dye can be found to be reacted with ClO. - Before and after the response, the fluorescence of the solution at 525 nm was rapidly enhanced under illumination. Figure 5 Figure C shows the fluorescence enhancement of DCFH-DA under illumination using the commercial dye Rose Bengal. Figure D shows the fluorescence enhancement of DCFH-DA with only DCFH-DA and ClO. - The fluorescence spectrum after illumination shows that ClO was added. - It hardly enhances the fluorescence at 525 nm. Comparing images A, B, and C shows that TPTPy, when reacted with ClO... - It exhibits excellent ROS generation performance both before and after the response, while TPTPy also shows good performance when subjected to ClO. - The ROS generation efficiency before and after the response is higher than that of the commercial dye RoseBengal.

[0062] Example 6

[0063] The organic dye TPTPy in Example 1 inhibits the production of endogenous and exogenous ClO in human umbilical vein endothelial cells (HUVEC) and cervical cancer cells (HeLa). - Imaging detection was performed. For example... Figure 6 As shown in (A1-A3), HeLa cells themselves contain a large amount of ClO - Therefore, cells incubated with TPTPy produce obvious red fluorescence. Figure 6 (B1-B3) Clearing endogenous ClO in HeLa cells by using NAC -No obvious red fluorescence was observed when the cells were incubated with TPTPy again, indicating that TPTPy can be detected by endogenous ClO2 highly expressed in tumor cells. - Respond and light up. For example... Figure 6 As shown in (C1-C3), HeLa cells were incubated with TPTPy and exogenous ClO was added. - It exhibits a more pronounced red fluorescence. For example... Figure 6 As shown in (D1-D3), HeLa cells cleared endogenous ClO2 by incubation with NAC. - Then, exogenous ClO was added to the cells. - Finally, after incubating the cells with TPTPy, a distinct red fluorescence was observed. Figure 6 (E1-E3), for normal cells, such as human umbilical vein endothelial cells, because they do not have a high concentration of ClO₂... - Therefore, no obvious red fluorescence was produced after cells were incubated with TPTPy. This experiment demonstrates that the organic dye TPTPy can inhibit endogenous and exogenous ClO2 in cells. - It can perform monitoring and specifically illuminate its own ClO. - Highly expressed tumor cells.

[0064] Example 7

[0065] The ROS production capacity of the organic dye TPTPy from Example 1 in HeLa cells was tested, as shown in the following experiment. Figure 7 As shown, DCFH-DA is a ROS indicator that binds to ROS to produce green fluorescence. After incubation with TPTPy, HeLa cells showed almost no green fluorescence under no light conditions. However, after 2 min and 5 min of light treatment, the green fluorescence continuously increased. This indicates that TPTPy exhibits excellent ROS generation capacity within the cells.

[0066] Example 8

[0067] The phototoxicity and dark toxicity of the organic dye TPTPy from Example 1 were studied in HeLa cells. Figure 8 As shown in the figure, HeLa cells were incubated in 96-well plates with TPTPy at the concentration shown in the figure for 2 hours. After incubation, the cells were divided into two groups: one group was irradiated with white light for 10 minutes, and the other group was kept in darkness. After treatment, the cells were placed in an incubator and cultured for another 24 hours before cell viability testing was performed. TPTPy exhibited very low cytotoxicity in the dark, but showed high phototoxicity under light conditions. A TPTPy concentration of 30 μM showed good photodynamic therapy effects.

[0068] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. An organic dye based on a phenothiazine structure, characterized in that, The general structural formula of this organic dye is: ； in, R is OCH3, and X is S.

2. The method for synthesizing organic dyes based on phenothiazine structures as described in claim 1, characterized in that, Includes the following steps: (1) Compound 1 and compound 3 react to give compound 2; (2) Compound 2 was reacted with 1,4-dimethylpyridine-1-onium iodide to obtain the organic dye based on the phenothiazine structure as described in claim 1; The structural formula of compound 1 is: ； The structural formula of compound 3 is: ； The structural formula of compound 2 is: 。 3. The method for synthesizing organic dyes based on phenothiazine structures according to claim 2, characterized in that: In step (1), under the protection of an inert gas, compounds 1 and 3 are dissolved in 1,4-dioxane, Cs2CO3 and a catalyst are added, and the reaction solution is stirred under the protection of an inert gas. After the reaction is completed, the mixture is extracted, dried, separated and purified to obtain compound 2.

4. The method for synthesizing organic dyes based on phenothiazine structures according to claim 3, characterized in that: The molar ratio of compound 1, compound 3, Cs2CO3 and catalyst is 1:2.1:3:0.

05.

5. The method for synthesizing organic dyes based on phenothiazine structures according to claim 3, characterized in that: The catalyst is Pd(PPh3)4.

6. The method for synthesizing organic dyes based on phenothiazine structures according to claim 2, characterized in that: In step (2), under inert gas protection, anhydrous ethanol is used as solvent to add compound 2, 1,4-dimethylpyridine-1-onium iodide and piperidine, and the mixture is heated to reflux and stirred to react. After the reaction is completed and cooled, the mixture is concentrated and purified to obtain the organic dye based on the phenothiazine structure.

7. The method for synthesizing organic dyes based on phenothiazine structures according to claim 2, characterized in that: The molar ratio of compound 2 to 1,4-dimethylpyridine-1-onium iodide is 1:

3.

8. The application of an organic dye based on a phenothiazine structure as described in claim 1 for the detection of hypochlorite, wherein the application is for non-disease diagnosis and treatment purposes, characterized in that: The phenothiazine-structured organic dye exhibits fluorescence activation after reacting with hypochlorite.

9. The application of the phenothiazine-based organic dye as described in claim 1 in the preparation of tumor photodynamic therapy drugs, characterized in that: The phenothiazine-type organic dye can kill tumor cells, including cervical cancer cells and liver cancer cells, by means of light.

Images