A phenothiazine / phenoxazine-based organic compound, its preparation method and applications

Abstract

The present invention discloses phenothiazine / phenoxazine-based organic compounds, their preparation methods, and applications. The phenothiazine / phenoxazine-based organic compounds of the present invention have good photophysical properties, high biocompatibility, small molecular weight, large Stokes shift, and excellent targeting performance. They can accurately stain subcellular structures in biological cells and effectively locate them in organelles in a wash-free manner, thereby achieving subcellular structure images with a high signal-to-noise ratio. The phenothiazine / phenoxazine-based organic compounds of the present invention have low cytotoxicity and large Stokes shift as small molecule fluorescent dyes. Compared with commercial organelle dyes, the fluorescent dyes of the present invention can specifically accumulate in cell mitochondria, cell membranes, lysosomes, endoplasmic reticulum, and lipid particles, and have excellent photostability, and can be used to observe the microstructure of cells and cell-to-cell interactions for a long time.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis, and in particular relates to an organic compound based on phenothiazine / phenoxazine, and a preparation method and application thereof. Background Art

[0002] Real-time monitoring of cell status is a crucial task in biological and medical research. This is particularly true for cellular substructures such as mitochondria, lysosomes, and the endoplasmic reticulum. Each organelle has a specific function, and many diseases are associated with organelle dysfunction. By monitoring organelles, scientists can gain a deeper understanding of their functions and their interactions within the cell. It can also assist physicians in diagnosing diseases and understanding their pathogenesis, thereby guiding treatment and research. Furthermore, many drugs target specific molecules within organelles or trigger specific pathways. By monitoring organelles, researchers can effectively evaluate drug efficacy, optimize drugs, and accelerate drug development.

[0003] Fluorescence imaging is a commonly used method for detecting organelles. It boasts high sensitivity, high selectivity, non-invasiveness, and real-time visualization, and has been widely used in scientific research, for example, to precisely locate tumor cells, study cellular interactions, and evaluate drug efficacy. By conjugating groups that target specific organelles to fluorescent dyes, the location and distribution of organelles in cells can be visualized. Organic small molecule fluorescent probes are easy to prepare and highly biocompatible, and their biological and medical applications are increasingly mature. However, after staining cells with commercial dyes, excess dye often needs to be washed away from the culture dish with phosphate-buffered saline (PBS) to reduce background noise during fluorescence imaging and obtain images with a better signal-to-noise ratio. Washing the culture dish with PBS is not only time-consuming and laborious, but can also damage the cell's microstructure, hindering the observation of cellular interactions. Therefore, there is an urgent need to develop a wash-free cell dye with excellent photophysical properties and good biocompatibility. Summary of the Invention

[0004] In order to overcome the problems existing in the above-mentioned prior art, one object of the present invention is to provide two organic compounds based on phenothiazine / phenoxazine. A second object of the present invention is to provide a method for preparing organic compounds based on phenothiazine / phenoxazine. A third object of the present invention is to provide applications of organic compounds based on phenothiazine / phenoxazine. A fourth object of the present invention is to provide an organic environmentally sensitive fluorescent dye. A fifth object of the present invention is to provide a wash-free cell dye.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides an organic compound based on phenothiazine / phenoxazine, the structural formula of which is shown in Formula (A), Formula (B) or Formula (C):

[0007]

[0008] In formula (A), formula (B) or formula (C), n is independently 1-12; X is independently oxygen or sulfur; Z is independently hydrogen or nitro;

[0009] R1 is an alkylamino group, an alkylheterocyclicamino group, an alkyl group, an alkoxy group or a phenylphosphino group;

[0010] R2 is an amide group, a sulfonyl group, an aromatic heterocyclic group, a fused heterocyclic group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylaminocarbonyl group, an arylaminocarbonyl group, or a substituted or unsubstituted alkoxy group; Y is O or NH;

[0011] The R3 is an alkyl group, an alkoxy group or an aralkyl group.

[0012] Preferably, R1 is a dialkylamino group, a trialkylamino group, an alkylheterocyclicamino group, a C3-C15 alkyl group, a C5-C27 alkoxy group or a phenylphosphino group.

[0013] More preferably, the R1 is as shown in Formula 1 to Formula 12:

[0014]

[0015] Wherein, the M is any one of iodide ion, chloride ion, bromide ion, alkyl sulfonate, tetrafluoroborate, and perchlorate.

[0016] Preferably, R2 is a sulfonamide group, a fused heterocyclic group, an aromatic aminocarbonyl group, a substituted or unsubstituted alkoxy group; the substituent of the substituted alkoxy group is a halogen.

[0017] More preferably, the R2 is as shown in Formula 13-Formula 18:

[0018]

[0019] Preferably, R3 is a C6-C14 alkyl group, a C7-C25 alkoxy group or an aralkyl group.

[0020] More preferably, the R3 is as shown in Formula 19-Formula 22:

[0021]

[0022] The second aspect of the present invention provides a method for preparing the phenothiazine / phenoxazine-based organic compound according to the first aspect of the present invention. When the phenothiazine / phenoxazine-based organic compound is as represented by formula (A), the preparation method comprises the following steps:

[0023] The halogenated alkyl phenothiazine / phenoxazine compound represented by formula (a) is reacted with R1H to obtain a phenothiazine / phenoxazine-based organic compound represented by formula (A); the reaction formula is as follows:

[0024]

[0025] Wherein, K is iodine, chlorine, bromine or fluorine;

[0026] When the phenothiazine / phenoxazine-based organic compound is as shown in formula (B), the preparation method thereof comprises the following steps:

[0027] The acetoxy phenothiazine / phenoxazine compound represented by formula (b) is reacted with R2-YH to obtain a phenothiazine / phenoxazine-based organic compound represented by formula (B); the reaction formula is as follows:

[0028]

[0029] When the phenothiazine / phenoxazine-based organic compound is as shown in formula (C), the preparation method thereof comprises the following steps:

[0030] The acetoxy phenothiazine / phenoxazine compound represented by formula (b) is reacted with the compound represented by formula (c) to obtain a phenothiazine / phenoxazine-based organic compound represented by formula (C); the reaction formula is as follows:

[0031]

[0032] Wherein, n, X, Z, R1, R2, YY, and R3 are as described in the first aspect.

[0033] Preferably, in the preparation of the phenothiazine / phenoxazine-based organic compound represented by formula (A), the reaction temperature is 90-110°C.

[0034] Preferably, in the preparation of the phenothiazine / phenoxazine-based organic compound represented by formula (A), the reaction time is 10-50 h.

[0035] Preferably, in the preparation of the phenothiazine / phenoxazine-based organic compound represented by formula (B), the reaction temperature is 15-120°C.

[0036] Preferably, in the preparation of the phenothiazine / phenoxazine-based organic compound represented by formula (B), the reaction time is 10-30 h.

[0037] Preferably, in the preparation of the phenothiazine / phenoxazine-based organic compound represented by formula (C), the reaction temperature is 15-40°C.

[0038] Preferably, in the preparation of the phenothiazine / phenoxazine-based organic compound represented by formula (C), the reaction time is 6-18 h.

[0039] Preferably, the molar ratio of the haloalkyl phenothiazine / phenoxazine compound represented by formula (a) to R1H is 1:(1-6).

[0040] Preferably, the molar ratio of the acetoxy phenothiazine / phenoxazine compound represented by formula (b) to R2-YH is 1:(1-2).

[0041] Preferably, the molar ratio of the acetoxy phenothiazine / phenoxazine compound represented by formula (b) to the compound represented by formula (c) is 1:(1-2).

[0042] Preferably, the haloalkyl phenothiazine / phenoxazine compound represented by formula (a) is prepared by a preparation method comprising the following steps:

[0043] The phenothiazine / phenoxazine compound represented by formula (a1) is reacted with a dihalogenated alkane to obtain a haloalkyl phenothiazine / phenoxazine compound represented by formula (a); the reaction formula is as follows:

[0044]

[0045] Wherein, n, X, Z, and K are as described above.

[0046] More preferably, the preparation process of the haloalkyl phenothiazine / phenoxazine compound represented by formula (a) includes the use of a reducing agent. Further preferably, the reducing agent is sodium hydride.

[0047] Preferably, the preparation of the phenothiazine / phenoxazine-based organic compound further includes the use of an organic solvent, and the organic solvent is selected from one of dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, tetrahydrofuran, dioxane, acetone, acetonitrile, ethylene glycol dimethyl ether, toluene, xylene, N,N-dimethylformamide or N,N-dimethylacetamide.

[0048] Preferably, the method for preparing the phenothiazine / phenoxazine-based organic compound as shown in formula (B) or (C) further comprises using a catalyst 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU).

[0049] Preferably, the preparation method of the phenothiazine / phenoxazine-based organic compound represented by formula (B) may further use 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as catalysts.

[0050] The third aspect of the present invention further provides use of the phenothiazine / phenoxazine-based organic compound according to the first aspect of the present invention in a fluorescent dye.

[0051] The fourth aspect of the present invention further provides an organic environmentally sensitive fluorescent dye, comprising the phenothiazine / phenoxazine-based organic compound.

[0052] Preferably, the organic environment-sensitive fluorescent dye is used in detecting polar solvents / non-polar solvents.

[0053] The fifth aspect of the present invention further provides a wash-free cell dye comprising the phenothiazine / phenoxazine-based organic compound.

[0054] Preferably, the wash-free cell dye is used to locate mitochondria, lysosomes, cell membranes, endoplasmic reticulum or lipid droplets.

[0055] The beneficial effects of the present invention are:

[0056] (1) The phenothiazine / phenoxazine-based organic compounds described in the present invention have good photophysical properties, high biocompatibility, small molecular weight, large Stokes shift, and excellent targeting performance. They can accurately stain subcellular structures in biological cells and effectively locate mitochondria, lysosomes, cell membranes, endoplasmic reticulum, and lipid droplets in a wash-free manner, thereby achieving subcellular structure images with a high signal-to-noise ratio.

[0057] (2) The phenothiazine / phenoxazine-based organic compounds described in the present invention have low cytotoxicity and large Stokes shift as small molecule fluorescent dyes, with a fluorescence emission peak located at 550-650 nm. Compared with commercial organelle dyes, the fluorescent dyes of the present invention can specifically accumulate in cell mitochondria, cell membranes, lysosomes, endoplasmic reticulum, and lipid particles. The Stokes shift of the dyes of the present invention can reach a maximum of 236 nm in dichloromethane solution. They also have excellent photostability and can be used for long-term observation of cellular microstructures and intercellular interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Absorption and fluorescence spectra of compounds PTZ-Lyso (a) and PTZ-2NO2-Lyso (b) in dichloromethane;

[0059] Figure 2 Absorption and fluorescence spectra of compounds PXZ-ER (a) and PXZ-2NO2-ER (b) in dichloromethane;

[0060] Figure 3 is the absorption and fluorescence spectra of compound 10a in dichloromethane;

[0061] Figure 4 Fluorescence spectra of compounds PTZ-Lyso (a) and PTZ-2NO2-Lyso (b) in tetrahydrofuran and methanol;

[0062] Figure 5 Fluorescence spectra of compounds PXZ-ER (a) and PXZ-2NO2-ER (b) in tetrahydrofuran and methanol;

[0063] Figure 6 is the fluorescence spectrum of compound 10a in tetrahydrofuran and methanol;

[0064] Figure 7 For the compound PTZ-Lyso and the commercial lysosomal dye LysoTracker TM Colocalization of Green DND-26 in Hela cells;

[0065] Figure 8 The compound PTZ-Mito and the commercial mitochondrial dye MitoTracker TM Colocalization of Green FM in Hela cells;

[0066] Figure 9 Co-localization of the compound PTZ-Memb and the commercial cell membrane dye CellMask Green Plasma MembraneStain in Hela cells;

[0067] Figure 10 The compound PTZ-S-ER and the commercial endoplasmic reticulum dye ER-Tracker TM Green Co-localization of FLGlibenclamide in Hela cells;

[0068] Figure 11 Co-localization of the compound PTZ-Lipid and lipid droplet dye BODIPY 493 / 503 in Hela cells;

[0069] Figure 12 The compound PTZ-2NO2-Lyso and the commercial lysosomal dye LysoTracker TM Colocalization of Green DND-26 in Hela cells

[0070] Figure 13 The compound PTZ-2NO2-Mito and the commercial mitochondrial dye MitoTracker TM Colocalization of Green FM in Hela cells;

[0071] Figure 14Co-localization of compound PTZ-2NO2-Memb and commercial cell membrane dye CellMask Green PlasmaMembrane Stain in Hela cells;

[0072] Figure 15 The compound PTZ-2NO2-ER and the commercial endoplasmic reticulum dye ER-Tracker TM Green Co-localization of FLGlibenclamide in Hela cells;

[0073] Figure 16 Co-localization of the compound PTZ-2NO2-Lipid and lipid droplet dye BODIPY 493 / 503 in Hela cells;

[0074] Figure 17 The compound PXZ-Mito and the commercial mitochondrial dye MitoTracker TM Colocalization of Red FM in Hela cells;

[0075] Figure 18 The compound PXZ-ER and the commercial endoplasmic reticulum dye ER-Tracker TM Red(BODIPY TM Co-localization of TR and Glibenclamide in Hela cells;

[0076] Figure 19 The compound PXZ-2NO2-ER and the commercial endoplasmic reticulum dye ER-Tracker TM Red(BODIPY TM Co-localization of TR and Glibenclamide in Hela cells;

[0077] Figure 20 Compound 10a and the commercial lysosomal dye LysoTracker TM Colocalization of Green DND-26 in HeLa cells. DETAILED DESCRIPTION

[0078] The present invention is further described in detail below through specific examples. Unless otherwise specified, the raw materials used in the following examples can be obtained from conventional commercial sources or prepared and isolated by simple synthesis; unless otherwise specified, the processes used are conventional processes in the art.

[0079] Example 1

[0080] This embodiment provides a phenothiazine-based organic compound PTZ-Mito, the preparation method of which is as follows:

[0081] S1. Preparation of nitro-phenothiazine 1a. The preparation method and reaction formula thereof are as follows:

[0082]

[0083] Phenothiazine (2.00 g, 10.04 mmol) was dissolved in a mixture of tetrahydrofuran and acetic acid (5:2) in an ice-water bath and stirred for 10 minutes. Sodium nitrite (2.08 g, 30.11 mmol) was dissolved in 4 mL of water and slowly added to the reaction solution, which was stirred at room temperature for 12 hours. After the reaction was complete, 100 mL of aqueous solution was added, filtered, dried, and recrystallized from toluene to obtain the black intermediate product 1a (1.9 g, 77% yield).

[0084] S2. Preparation of haloalkyl phenothiazine 2a. The preparation method and reaction formula thereof are as follows:

[0085]

[0086] Under a nitrogen atmosphere, 1,6-dibromohexane (399.51 mg, 1.64 mmol) and sodium hydride (98 mg, 4.09 mmol) were added to N,N-dimethylformamide and stirred at room temperature for 10 minutes. Then, 1a (200.00 mg, 0.82 mmol) was added and the reaction was continued for 12 hours. After the reaction was completed, ice water was added, extracted with ethyl acetate, and the organic phase was dried and column chromatography was performed to obtain an oily product 2a (250 mg, 75% yield). The NMR characterization of the product was as follows: 1 H NMR (400MHz, DMSO-d6) δ8.05(dd,J=9.1,2.7Hz,1H),7.95(d,J=2.7Hz,1H),7.26(ddd,J=8.2,7.2,1.6Hz,1H),7.22–7.09(m,3H),7. 04(td,J=7.4,1.1Hz,1H), 3.97(t,J=7.0Hz,2H), 3.49(t,J=6.7Hz,2H), 1.72(ddt,J=32.0,13.5,6.6Hz,4H), 1.40(q,J=3.2Hz,4H).

[0087] S3. Preparation of the organic compound PTZ-Mito. The preparation method and reaction formula thereof are as follows:

[0088]

[0089] 2a (100 mg, 0.25 mmol) was dissolved in N,N-dimethylformamide, and triphenylphosphine (77.27 mg, 0.29 mmol) was added. The mixture was allowed to react at 100°C for 48 hours. After the reaction, the mixture was extracted with ethyl acetate, the organic phase was dried, and column chromatography was performed to obtain the product PTZ-Mito (140 mg, 82% yield). The product was characterized by NMR: 1 H NMR (400MHz, Methanol-d4) δ8.04(dd,J=9.0,2.6Hz,1H),7.88(dtd,J=8.8,4.1,2.0Hz,4H),7.80–7.68(m,12H),7.22(ddd,J=8 .6,7.3,1.6Hz,1H),7.12–6.98(m,4H),4.01(t,J=6.5Hz,2H),1.74(p,J=6.7Hz,2H),1.65–1.49(m,6H),1.33(d,J=6.5Hz,2H).

[0090] Example 2

[0091] This embodiment provides a phenothiazine-based organic compound PTZ-Lyso, the preparation method and reaction formula of which are as follows:

[0092]

[0093] 2a (100 mg, 0.25 mmol) from Example 1 was dissolved in N,N-dimethylformamide, and morpholine (106.94 mg, 1.23 mmol) was added. The mixture was allowed to react at 100°C for 12 hours. After the reaction, the mixture was extracted with ethyl acetate, the organic phase was dried, and column chromatography was performed to obtain the product PTZ-Lyso (74 mg, 73% yield). The product was characterized by NMR: 1 H NMR(400MHz, DMSO-d6)δ8.05(dd,J=9.1,2.7Hz,1H),7.96(d,J=2.7Hz,1H),7.75–7 .64(m,1H),7.26(ddd,J=8.7,7.2,1.6Hz,1H),7.22–7.09(m,3H),7.05(td,J=7.5, 1.1Hz,1H),4.23(t,J=6.6Hz,1H),3.97(t,J=6.9Hz,2H),3.51(t,J=4.6Hz,4H),2. 26(s,3H),2.22–2.07(m,2H),1.76–1.59(m,3H),1.37(qd,J=10.0,8.8,6.4Hz,4H).

[0094] Example 3

[0095] This embodiment provides a phenothiazine-based organic compound PTZ-2NO2-Lyso, the preparation method of which is as follows:

[0096] S1. Preparation of nitro-phenothiazine 1b. The preparation method and reaction formula thereof are as follows:

[0097]

[0098] Phenothiazine (2.00 g, 10.04 mmol) was dissolved in a mixture of tetrahydrofuran and acetic acid (ratio 5:2) in an ice-water bath and stirred for 10 minutes. Sodium nitrite (4.16 g, 60.22 mmol) was dissolved in 4 mL of water and slowly added to the reaction solution. Stir at 40°C for 12 hours. After the reaction was complete, 100 mL of aqueous solution was added, filtered, dried, and recrystallized from toluene to obtain the black intermediate product 1b (2.3 g, 79% yield).

[0099] S2. Preparation of haloalkyl phenothiazine 2b. The preparation method and reaction formula thereof are as follows:

[0100]

[0101] Under a nitrogen atmosphere, 1,6-dibromohexane (506 mg, 2.07 mmol) and sodium hydride (99 mg, 4.15 mmol) were added to N,N-dimethylformamide and stirred at room temperature for 10 minutes. Then, 1b (300.00 mg, 1.04 mmol) was added and the reaction was continued for 12 hours. After the reaction was completed, ice water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried and column chromatography was performed to obtain the oily product 2b (340 mg, 72% yield). The NMR characterization of the product was as follows: 1 H NMR(400MHz,Chloroform-d)δ8.09(dd,J=9.0,2.6Hz,2H),7.99(d,J=2.6Hz,2H),6.94(d,J=9.0Hz,2H),3. 99 (dd, J = 7.9, 6.4 Hz, 2H), 3.41 (t, J = 6.6 Hz, 2H), 1.87 (ddt, J = 8.7, 5.3, 2.8 Hz, 4H), 1.51 (p, J = 3.5 Hz, 4H).

[0102] S3. Prepare the organic compound PTZ-2NO2-Lyso. The preparation method and reaction formula thereof are as follows:

[0103]

[0104] Using the method for synthesizing the molecular PTZ-Lyso, PTZ-2NO2-Lyso was obtained (yield: 65%). The NMR characterization of the product was: 1H NMR (400MHz, DMSO-d6) δ8.09(dd,J=9.1,2.7Hz,2H),8.03(d,J=2.6Hz,2H),7.28(d,J=9.1Hz,2H),4.05(t,J=7.0Hz,2 H), 3.51 (t, J = 4.6Hz, 4H), 2.18 (t, J = 7.2Hz, 2H), 1.70 (p, J = 7.1Hz, 2H), 1.45–1.32 (m, 4H), 1.28 (q, J = 8.3, 7.8Hz, 2H).

[0105] Example 4

[0106] This embodiment provides a phenothiazine-based organic compound PTZ-2NO2-Mito, the preparation method and reaction formula of which are as follows:

[0107]

[0108] The method for synthesizing the PTZ-Mito molecule was used, and the substrate was 2b from Example 3 to obtain the PTZ-2NO2-Mito molecule (yield: 82%). The NMR characterization of the product was as follows: 1 H NMR (400MHz, Methanol-d4) δ8.10 (dd, J=9.0, 2.6Hz, 2H), 7.99 (d, J=2.6Hz, 2H), 7.90 (td, J=5.8, 2.9Hz, 3H), 7.79–7. 72(m,12H),7.18(d,J=9.1Hz,2H),4.09(t,J=6.7Hz,2H),1.77(q,J=7.0Hz,2H),1.68–1.53(m,6H),1.34-1.33(m,2H).

[0109] Example 5

[0110] This embodiment provides a phenothiazine-based organic compound 10a, the preparation method and reaction formula of which are as follows:

[0111]

[0112] 1a (200.00 mg, 0.82 mmol) from Example 1 and 2-bromo-N,N-diethylethylamine hydrobromide (176.94 mg, 0.98 mmol) were dissolved in a mixed solution of dichloromethane / acetone (3 mL / 3 mL), and 3 mL of sodium hydroxide aqueous solution (3 g sodium hydroxide / 3 mL water) was added. The mixture was reacted at room temperature for 24 hours. After the reaction was completed, the mixture was extracted with dichloromethane, the organic phase was dried, and the product 10a (210.00 mg, 74% yield) was obtained by column chromatography. The NMR characterization of the product was as follows: 1H NMR(400MHz,Chloroform-d)δ8.04(dd,J=9.0,2.6Hz,1H),7.96(d,J=2.6Hz,1H),7.21(ddd,J=8.2,7.4,1.6Hz,1H),7.12(dd, J=7.6,1.6Hz,1H),7.05–6.96(m,3H),4.05(t,J=6.7Hz,2H),2.89–2.80(m,2H),2.64(q,J=7.1Hz,4H),1.08(t,J=7.1Hz,6H).

[0113] Example 6

[0114] This embodiment provides a phenothiazine-based organic compound PTZ-Memb, the preparation method of which is as follows:

[0115] S1. Preparation of acetoxyphenothiazine 4a. The preparation method and reaction formula thereof are as follows:

[0116]

[0117] 1a (1.00 g, 4.09 mmol) of Example 1 and tert-butyl bromoacetate (958.24 mg, 4.91 mmol) were dissolved in a mixed solution of dichloromethane / acetone (10 mL / 10 mL), and 10 mL of sodium hydroxide aqueous solution (10 g sodium hydroxide / 10 mL water) was added and reacted at room temperature for 24 hours. After the reaction was completed, dichloromethane was used for extraction, the organic phase was dried, and column chromatography was performed to obtain product 3a (1.10 g, 75% yield). The nuclear magnetic resonance characterization of the product was as follows: 1 H NMR (400MHz, Acetone-d6) δ8.05(dd,J=9.1,2.7Hz,1H),7.93(d,J=2.6Hz,1H),7.24(ddd,J=8.2,7.3,1.6Hz,1H),7.17(dd,J =7.7,1.6Hz,1H),7.07(td,J=7.5,1.1Hz,1H),6.92(d,J=9.1Hz,1H),6.85(dd,J=8.2,1.1Hz,1H),4.70(s,2H),1.53(s,9H).

[0118] 3a (1.00 g, 2.79 mmol) was dissolved in dichloromethane, and 10 mL of trifluoroacetic acid was added. The reaction was allowed to proceed at 60°C for 6 hours. After the reaction was complete, the organic phase was removed and column chromatography was performed to obtain product 4a (0.60 g, 71% yield). The product was characterized by NMR: 1HNMR(400MHz,DMSO-d6)δ13.33(s,1H),8.03(dd,J=9.1,2.7Hz,1H),7.94(d,J=2.7Hz,1H),7.25– 7.13(m,2H),7.07–7.01(m,1H),6.84(d,J=9.1Hz,1H),6.77(dd,J=8.2,1.1Hz,1H),4.70(s,2H).

[0119] S2. Prepare compound PTZ-Memb. Its preparation method and reaction formula are as follows:

[0120]

[0121] 4a (100 mg, 0.33 mmol) was dissolved in 5 mL of N,N-dimethylformamide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 151 mg, 0.39 mmol) was added. The mixture was stirred at room temperature for 15 minutes. 3-(Dodecylamino)-1-propanesulfonic acid (111 mg, 0.36 mmol) and N,N-diisopropylethylamine (85 mg, 0.66 mmol) were added and reacted at room temperature for 12 hours. After the reaction was completed, the organic phase was removed by extraction and column chromatography to obtain the product PTZ-Memb (120 mg, 61% yield). The product was characterized by NMR: 1 H NMR(400MHz, Methanol-d4)δ8.00(ddd,J=13.2,9.1,2.7Hz,1H),7.91(t,J=2.3Hz,1H),7.22–7.12(m,1H),7.08 (ddd,J=7.6,2.8,1.6Hz,1H),7.00(tdd,J=7.5,3.4,1.1Hz,1H),6.72–6.62(m,2H),4.92(s,2H),3.83–3.66(m,3 H),3.61(t,J=7.3Hz,1H),3.52(dt,J=17.4,7.7Hz,2H),3.24(q,J=7.4Hz,2H),2.98–2.84(m,2H),2.30–2.06(m, 2H), 1.80 (s, 1H), 1.68 (d, J = 8.8Hz, 1H), 1.43 (d, J = 4.6Hz, 2H), 1.38 (dd, J = 6.7, 3.4Hz, 12H), 0.98–0.85 (m, 3H).

[0122] Example 7

[0123] This embodiment provides a phenothiazine-based organic compound PTZ-2NO2-Memb, the preparation method and reaction formula of which are as follows:

[0124] Using the synthetic method for preparing molecule 4a, the substrate 1bb of Example 3 was used to obtain molecule 4b (yield 72%). The NMR characterization of the product was: 1 H NMR (400 MHz, DMSO-d6) δ 8.07 (dd, J = 9.1, 2.7 Hz, 2H), 8.01 (d, J = 2.7 Hz, 2H), 6.93 (d, J = 9.2 Hz, 2H), 4.81 (s, 2H).

[0125]

[0126] Using the synthetic method for preparing the PTZ-Memb molecule, the PTZ-2NO2-Memb molecule was obtained (yield 56%). The NMR characterization of the product is: 1 H NMR (400MHz, Methanol-d4) δ8.04 (ddd, J=11.8, 9.1, 2.6Hz, 2H), 7.96 (dd, J=2.7, 1.4 Hz,2H),6.75(dd,J=9.1,2.5Hz,2H),5.02(s,2H),3.77–3.68(m,1H),3.62(t,J=7.2Hz ,1H),3.52(dt,J=13.3,7.8Hz,2H),3.20(t,J=7.2Hz,1H),3.05–2.86(m,4H),2.31–2. 08(m,3H),1.81(s,1H),1.68(d,J=8.2Hz,2H),1.50–1.32(m,14H),0.93–0.89(m,3H).

[0127] Example 8

[0128] This embodiment provides a phenothiazine-based organic compound PTZ-S-ER, the preparation method and reaction formula of which are as follows:

[0129]

[0130] 4a (150 mg, 0.50 mmol) from Example 6 was dissolved in 5 mL of N,N-dimethylformamide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 283 mg, 0.74 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. N-(2-aminoethyl)-4-methylbenzenesulfonamide (127 mg, 0.60 mmol) and N,N-diisopropylethylamine (128 mg, 0.99 mmol) were added, and the mixture was reacted at room temperature for 12 hours. After the reaction was completed, the mixture was extracted, the organic phase was removed, and the product PTZ-S-ER (190 mg, 76% yield) was obtained by column chromatography. The NMR characterization of the product was as follows: 1 H NMR(400MHz,Chloroform-d)δ8.03–7.97(m,2H),7.74–7.69(m,2H),7.30(d,J=8.0Hz,2H),7.22–7.14(m,2H),7.02(td,J=7.5,1.1Hz,1H) ,6.88–6.78(m,2H),6.34(t,J=6.0Hz,1H),4.60(t,J=6.3Hz,1H),4.52(s,2H),3.20(q,J=6.6Hz,2H),2.60(q,J=6.7Hz,2H),1.35(s,3H).

[0131] Example 9

[0132] This embodiment provides a phenothiazine-based organic compound PTZ-2NO2-ER, the preparation method and reaction formula of which are as follows:

[0133]

[0134] Using the synthetic method for preparing the molecule PTZ-S-ER, the molecule PTZ-2NO2-ER was obtained (yield 42%). The NMR characterization of the product was: 1 H NMR (400MHz, DMSO-d6) δ8.55(t,J=5.8Hz,1H),8.03(dd,J=9.0,2.7Hz,2H),7.99(d,J=2.7Hz,2H),7.76–7.68(m,3H ),7.47–7.37(m,2H),6.87(d,J=9.2Hz,2H),4.61(s,2H),3.22(q,J=6.1Hz,2H),2.85(q,J=6.2Hz,2H),2.39(s,3H).

[0135] Example 10

[0136] This embodiment provides a phenothiazine-based organic compound PTZ-L-ER, the preparation method of which is as follows:

[0137] S1. Prepare compound 5a. The preparation method and reaction formula thereof are as follows:

[0138]

[0139] 4a (500.00 mg, 1.65 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (385.15 mg, 2.48 mmol) were dissolved in dichloromethane solution, and N-hydroxysuccinimide (285.53 mg, 2.48 mmol) was added and reacted at room temperature for 4 hours. After the reaction was completed, the organic phase was removed and column chromatography was performed to obtain product 5a (310.00 mg, 49% yield). The product was characterized by nuclear magnetic resonance (NMR) as follows: 1 H NMR (400MHz, DMSO-d6) δ8.08–7.99(m,2H),7.24(tt,J=7.8,1.5Hz,2H),7.10(td,J=7.3,1.1Hz,1H),7.00–6.87(m,2H),5.45(s,2H),2.86(s,4H).

[0140] S2. Prepare the organic compound PTZ-L-ER. The preparation method and reaction formula thereof are as follows:

[0141]

[0142] 5a (200.00 mg, 0.52 mmol) was dissolved in 2 mL of dichloromethane and slowly added dropwise to a dichloromethane solution containing hexamethylenediamine (121.25 mg, 1.04 mmol) and allowed to react at room temperature for 1 hour. After the reaction was completed, the organic phase was removed and column chromatography was performed to obtain 6a. 6a (50.00 mg, 0.12 mmol) and p-toluenesulfonic acid chloride (47.60 mg, 0.25 mmol) were dissolved in dichloromethane and allowed to react at room temperature for 12 hours. After the reaction was completed, the mixture was extracted three times, the organic phase was dried, and column chromatography was performed to obtain the product PTZ-L-ER (53.00 mg, 76% yield). The NMR characterization of the product was as follows: 1H NMR(400MHz,Chloroform-d)δ8.05–7.99(m,2H),7.76–7.71(m,2H),7.32(d,J =8.0Hz,2H),7.25–7.17(m,2H),7.08(td,J=7.5,1.1Hz,1H),6.90–6.80(m,2H ),6.37(t,J=6.0Hz,1H),4.64(t,J=6.3Hz,1H),4.54(s,2H),3.24(q,J=6.6Hz ,2H),2.88(q,J=6.7Hz,2H),1.35(dq,J=18.7,7.1Hz,6H),1.12–1.01(m,2H).

[0143] Example 11

[0144] This embodiment provides a phenothiazine-based organic compound PTZ-Lipid, the preparation method and reaction formula of which are as follows:

[0145]

[0146] 4a (200.00 mg, 0.66 mmol) from Example 6 was dissolved in toluene (10 mL) and triethylene glycol monomethyl ether (2 ml), heated to 80°C, stirred for 15 minutes, and then 2 drops of concentrated sulfuric acid were added. The temperature was raised to 110°C and the reaction was allowed to proceed for 24 hours. After the reaction was completed, the mixture was extracted with ethyl acetate, the organic phase was dried, and column chromatography was performed to obtain the product PTZ-Lipid (115.00 mg, 39% yield). The product was characterized by NMR: 1 H NMR(400MHz,Chloroform-d)δ8.00(dd,J=8.9,2.6Hz,1H),7.96(d,J=2.6Hz,1H),7.20–7.08(m,2H),7.03(td,J=7.5,1.1Hz,1H),6.7 0–6.63(m,2H),4.62(s,2H),4.51–4.45(m,2H),3.83–3.75(m,2H),3.68(ddd,J=9.2,4.0,2.1Hz,6H),3.63–3.54(m,2H),3.39(s,3H).

[0147] Example 12

[0148] This embodiment provides a phenothiazine-based organic compound PTZ-2NO2-Lipid, the preparation method and reaction formula of which are as follows:

[0149]

[0150] Using the synthetic method for preparing the PTZ-Lipid molecule, the PTZ-2NO2-Lipid molecule was obtained (yield 32%). The NMR characterization of the product was: 1 H NMR(400MHz,Chloroform-d)δ8.05(dd,J=9.0,2.6Hz,2H),7.99–7.96(m,2H),6.75(d,J=9.0Hz,2H),4 .68(s,2H),4.54–4.49(m,2H),3.85–3.76(m,2H),3.75–3.64(m,6H),3.62–3.55(m,2H),3.38(s,3H).

[0151] Example 13

[0152] This embodiment provides a phenoxazine-based organic compound PXZ-Lyso, the preparation method of which is as follows:

[0153] S1. Preparation of nitrophenoxazines 1c and 1d. The preparation methods and structural formulas thereof are as follows:

[0154]

[0155] The base material is phenoxazine, and the molecules 1c and 1d were prepared using the method for synthesizing 1a and 1b.

[0156] S2. Preparation of haloalkylphenoxazine 2c. The preparation method and structural formula thereof are as follows:

[0157]

[0158] Molecule 2c was prepared using the method for preparing 2a (yield 32%). The NMR characterization of the product was: 1 H NMR(400MHz,Chloroform-d)δ7.75(dd,J=9.0,2.6Hz,1H),7.44(d,J=2.6Hz,1H),6.8 7(td,J=7.7,1.6Hz,1H),6.79(td,J=7.7,1.5Hz,1H),6.71–6.66(m,1H),6.57(dd,J=8 .0,1.5Hz,1H),6.43(d,J=8.9Hz,1H),3.62–3.52(m,2H),3.46(t,J=6.6Hz,2H),1.97 –1.88(m,2H),1.72(p,J=7.7Hz,2H),1.60–1.54(m,2H),1.48(dd,J=10.6,5.3Hz,2H).

[0159] S3. Prepare the organic compound PXZ-Lyso, the preparation method and structural formula of which are as follows:

[0160]

[0161] The molecule PXZ-Lyso was prepared using the method for preparing PTZ-Lyso (yield 62%). The NMR characterization of the product is: 1 HNMR(400MHz,Chloroform-d)δ7.74(dd,J=8.9,2.6Hz,1H),7.43(d,J=2.6Hz,1H),6.86(t d,J=7.7,1.6Hz,1H),6.78(td,J=7.7,1.4Hz,1H),6.67(dd,J=7.8,1.6Hz,1H),6.55(dd,J= 8.0,1.5Hz,1H),6.41(d,J=9.0Hz,1H),3.75(t,J=4.7Hz,4H),3.58–3.50(m,2H),2.47(t, J=4.7Hz,4H),2.42–2.34(m,2H),1.78–1.64(m,2H),1.63–1.51(m,2H),1.54–1.36(m,4H).

[0162] Example 14

[0163] This embodiment provides a phenoxazine-based organic compound PXZ-Mito, the preparation method and reaction formula of which are as follows:

[0164]

[0165] The substrate was molecule 2c of Example 14, and the molecule PXZ-Mito was prepared using the method for preparing PTZ-Mito (yield 71%). The NMR characterization of the product was: 1 H NMR (400MHz, Methanol-d4) δ7.94–7.91(m,1H),7.91–7.88(m,2H),7.84–7.73(m,14H),7.40(d,J=2.6Hz,1H),6.87(dd,J=7.7,1.6Hz,1H),6.78( dd,J=7.7,1.5Hz,1H),6.74(dd,J=8.0,1.4Hz,1H),6.71–6.67(m,2H),3. 70–3.61(m,2H),1.68(ddd,J=23.0,15.0,7.8Hz,8H),1.57–1.49(m,2H).

[0166] Example 15

[0167] This embodiment provides a phenoxazine-based organic compound PXZ-Lipid, the preparation method of which is as follows:

[0168] S1. Preparation of acetoxyphenoxazines 3c and 3d. The preparation methods and structural formulas thereof are as follows:

[0169]

[0170] Molecules 3c and 3d were prepared using the methods for synthesizing 3a and 3b.

[0171] The NMR characterization of product 3c is as follows: 1 H NMR(400MHz,Chloroform-d)δ13.33(s,1H),δ7.79(dd,J=8.9,2.6Hz,1H),7.55(d,J=2.5Hz,1H),6.87(dtd,J=19.4,7.5 ,1.6Hz,2H),6.76(dd,J=7.7,1.7Hz,1H),6.47(dd,J=7.8,1.6Hz,1H),6.39(d,J=8.9Hz,1H),4.22(s,2H),1.51(s,9H).

[0172] The NMR characterization of product 3d is: 1 H NMR (400 MHz, Chloroform-d) δ7.85 (dd, J = 8.9, 2.5 Hz, 2H), 7.62 (d, J = 2.5 Hz, 2H), 6.51 (d, J = 8.9 Hz, 2H), 4.29 (s, 2H), 1.53 (s, 9H).

[0173]

[0174] Molecules 4c and 4d were prepared using the methods for synthesizing 4a and 4b.

[0175] The NMR characterization of product 4c is as follows: 1 H NMR (400MHz, DMSO-d6) δ13.36 (s, 1H), δ7.79 (dd, J=8.8, 2.7Hz, 1H), 7.56 (d, J=2.7Hz, 1H), 6.84 (dtd, J=19.4, 7.5, 1.6Hz, 2H), 6.76 (dd, J = 7.8, 1.5Hz, 1H), 6.47 (dd, J = 7.9, 1.6Hz, 1H), 6.37 (d, J = 8.9Hz, 1H), 4.22 (s, 2H).

[0176] The NMR characterization of product 4d is: 1H NMR (400MHz, DMSO-d6) δ7.85 (dd, J = 8.8, 2.4Hz, 2H), 7.62 (d, J = 2.5Hz, 2H), 6.51 (d, J = 8.9Hz, 2H), 4.29 (s, 2H).

[0177] S2. Prepare compound PXZ-ER. Its preparation method and reaction formula are as follows:

[0178]

[0179] The molecule PXZ-ER was prepared using the method for preparing PTZ-ER (yield 76%). The NMR characterization of the product was as follows: 1 HNMR(400MHz,DMSO-d6)δ8.39(t,J=5.8Hz,1H),7.74(dd,J=9.0,2.6Hz,1H),7.71–7.65(m,3H),7.44–7.38(m,3H),6.85(dtd,J=20.2,7 .5,1.6Hz,2H),6.77(dd,J=7.7,1.7Hz,1H),6.66–6.58(m,2H),4.31(s,2H),3.15(q,J=6.4Hz,2H),2.77(q,J=6.5Hz,2H),2.39(s,3H).

[0180] Example 16

[0181] This embodiment provides a phenoxazine-based organic compound PXZ-Lipid, the preparation method and reaction formula of which are as follows:

[0182]

[0183] The molecule PXZ-Lipid was prepared using the method for preparing PTZ-Lipid (yield 46%). The NMR characterization of the product was: 1 H NMR (400MHz, Chloroform-d) δ7.79(dd,J=8.9,2.6Hz,1H),7.56(d,J=2.5Hz,1H),6.87(dtd,J=16.7,7.5,1.6Hz,2H),6.77(dd,J=7.6,1.8Hz,1H),6.49(dd ,J=7.6,1.8Hz,1H),6.44(d,J=8.9Hz,1H),4.44–4.40(m,2H),4.39(s,2H),3 .78–3.73(m,2H),3.66(q,J=2.6,2.1Hz,6H),3.60–3.54(m,2H),3.39(s,3H).

[0184] Example 17

[0185] This embodiment provides a phenoxazine-based organic compound PXZ-Memb, the preparation method and reaction formula of which are as follows:

[0186]

[0187] The molecule PXZ-Memb was prepared using the method for preparing PTZ-Memb (yield 35%). The NMR characterization of the product was: 1 H NMR (400MHz, DMSO-d6) δ7.71(dd,J=9.0,2.6Hz,1H),7.43(d,J=2.6Hz,1H),6.87(dd,J=7.6,2.0H z,1H),6.83(t,J=2.1Hz,1H),6.82–6.77(m,2H),6.57–6.54(m,1H),4.75(s,2H),3.55(t,J=7.5Hz ,2H),3.26(t,J=7.3Hz,2H),2.54(d,J=4.1Hz,1H),2.40–2.34(m,1H),1.96(t,J=7.4Hz,2H),1.8 3–1.74(m,1H),1.66(s,1H),1.46(t,J=7.1Hz,2H),1.34(s,2H),1.29–1.22(m,14H),0.85(s,3H).

[0188] Example 18

[0189] This embodiment provides a phenoxazine-based organic compound PXZ-2NO2-ER, the preparation method and structural formula of which are as follows:

[0190]

[0191] The molecule PXZ-2NO2-ER was prepared using the method for preparing PTZ-2NO2-ER (yield 78%). The NMR characterization of the product is: 1 H NMR(400MHz, DMSO-d6)δ8.46(t,J=5.8Hz,1H),7.80(dd,J=9.0,2.6Hz,2H),7.73–7.62(m,3H),7.51(d,J=2.6Hz,2H), 7.40(d,J=8.0Hz,2H), 6.79(d,J=9.0Hz,2H), 4.43(s,2H), 3.16(q,J=6.4Hz,2H), 2.78(q,J=6.5Hz,2H), 2.38(s,3H).

[0192] Example 19

[0193] This embodiment provides a phenoxazine-based organic compound PXZ-2NO2-Lipid, the preparation method and structural formula of which are as follows:

[0194]

[0195] The molecule PXZ-2NO2-Lipid was prepared using the method for preparing PTZ-2NO2-Lipid (yield 40%). The NMR characterization of the product is: 1 H NMR(400MHz,Chloroform-d)δ7.87–7.81(m,2H),7.62(d,J=2.6Hz,2H),6.59(dd,J=8.9,2.2Hz,2H) ,4.50–4.42(m,4H),3.76(dt,J=6.6,2.5Hz,2H),3.70–3.66(m,6H),3.61–3.56(m,2H),3.39(s,3H).

[0196] Example 20

[0197] This embodiment provides a phenoxazine-based organic compound PXZ-2NO2-Memb, the preparation method and structural formula of which are as follows:

[0198]

[0199] The molecule PXZ-2NO2-Memb was prepared using the method for preparing PTZ-2NO2-Memb (yield 38%). The NMR characterization of the product is: 1 H NMR(400MHz, DMSO-d6)7.76(dd,J=9.0,2.6Hz,2H),7.51(d,J=2.6Hz,2H),6.80(d,J= 9.1Hz,2H),4.93(s,2H),3.56(t,J=7.4Hz,2H),3.26(t,J=7.4Hz,2H),2.55(dd,J=8. 9,6.4Hz,1H),2.38(dd,J=8.9,6.4Hz,1H),2.03–1.93(m,2H),1.79(q,J=7.6Hz,1H), 1.67(s,1H),1.50–1.42(m,2H),1.35(s,2H),1.29–1.22(m,14H),0.86–0.83(m,3H).

[0200] Experimental testing

[0201] 1. Spectral property test of representative compounds

[0202] The compounds PTZ-Lyso and PTZ-2NO2-Lyso were dissolved in dichloromethane solution and the absorption and emission spectra of the molecules were tested. The absorption / emission spectra of the molecule PTZ-Lyso are shown in FIG. Figure 1 As shown in Figure a, the maximum absorption wavelength is 432 nanometers, the maximum emission wavelength is 668 nanometers, and the Stokes shift is 236 nanometers. The absorption / emission spectrum of the molecule PTZ-2NO2-Lyso is shown in Figure a. Figure 1 As shown in Figure b, its maximum absorption peak is similar to that of PTZ-Lyso at 436 nm, but its maximum emission peak is significantly blue-shifted to 615 nm. The Stokes shift is 179 nm. The absorption and emission spectra of PXZ-ER and PXZ-2NO2-ER were measured in the same manner. The absorption / emission spectra of PXZ-ER are shown in Figure 2. Figure 2 As shown in Figure a, the maximum absorption wavelength is 442 nanometers, the maximum emission wavelength is 610 nanometers, and the Stokes shift is 168 nanometers. The absorption / emission spectrum of the molecule PTZ-2NO2-Lyso is shown in Figure a. Figure 2 As shown in Figure b, its maximum absorption peak is 452 nm, the maximum emission wavelength is 563 nm, and the Stokes shift is 111 nm. The absorption and emission spectra of molecule 10a were measured using the same method. The absorption / emission spectra of molecule 10a are shown in Figure b. Figure 3 As shown, the maximum absorption peak is 425 nm, the maximum emission peak is 602 nm, and the Stokes shift is 177 nm.

[0203] 2. Fluorescence spectra of representative compounds in different solvents

[0204] The fluorescence spectra of PTZ-Lyso and PTZ-2NO2-Lyso in polar solvent methanol and non-polar solvent tetrahydrofuran were tested respectively. The results are as follows Figure 4 As shown in the figure, both compounds exhibit strong fluorescence intensity in tetrahydrofuran, while under the same conditions, there is almost no fluorescence in methanol solution, indicating that the fluorescence intensity of the two compounds is extremely sensitive to the polarity of the solvent. In non-polar solvents, the fluorescence is greatly enhanced (PTZ-Lyso on the left and PTZ-2NO2-Lyso on the right). Under the same conditions, the molecule PXZ-ER ( Figure 5 Figure a), PXZ-2NO2-ER( Figure 5 Figure b), 10a( Figure 6 ) all exhibited the same properties, indicating that organic small molecule fluorescent dyes based on phenoxazine / phenothiazine have obvious polar environmental sensitivity.

[0205] 3. Organelle localization experiment

[0206] LysoTracker TMGreen DND-26 was added to HeLa cells and incubated at 37°C, 5% CO2 for 30 minutes. Then, the PTZ-Lyso dye of the present invention was added and cultured for another 30 minutes. No need to use PBS to wash out the excess dye, and laser confocal imaging was performed directly. Figure 7 As shown, the present invention can overlap well with commercial lysosomal dyes, with a colocalization coefficient of 0.91, indicating that PTZ-Lyso is a lysosomal targeting dye. The same method was used to test PTZ-Mito and the commercial mitochondrial dye MitoTracker TM Green FM's co-localization capability. Figure 8 As shown, PTZ-mito overlaps well with commercial mitochondrial dyes, with a colocalization coefficient of 0.96, indicating that PTZ-mito is a mitochondrial-targeted dye.

[0207] The commercial cell membrane dye CellMask Green Plasma Membrane Stain was added to HeLa cells, incubated for 5 minutes, and then the excess dye was removed and washed once with PBS. The PTZ-Memb of the present invention was added and incubated for another 5 minutes, and then laser confocal imaging was performed. The results are shown in FIG. Figure 9 As shown in the figure, PTZ-Memb can be effectively localized to the cell membrane, and the colocalization coefficient with commercial cell membrane dyes is 0.86, indicating that PTZ-Memb is a cell membrane-targeted dye.

[0208] ER-Tracker TM Green FL Glibenclamide) was added to HeLa cells, incubated for 40 minutes, excess dye was removed, and the cells were washed once with PBS. PTZ-S-ER of the present invention was added and incubated for another 40 minutes, and laser confocal imaging was performed. The results are shown in FIG. Figure 10 As shown, PTZ-S-ER can be effectively localized to the endoplasmic reticulum, and the colocalization coefficient with commercial endoplasmic reticulum dye is 0.92, indicating that PTZ-S-ER is an endoplasmic reticulum-targeted dye.

[0209] The lipid droplet dye BODIPY 493 / 503 was added to Hela cells, incubated for 30 minutes, and then the excess dye was removed and washed once with PBS. After adding PTZ-Lipid of the present invention and incubating for another 40 minutes, laser confocal imaging was performed. The results are shown in FIG. Figure 11 As shown in the figure, PTZ-Lipid can be effectively localized on lipid droplets, and the colocalization coefficient with lipid droplet dye is 0.90, indicating that PTZ-Lipid is a lipid droplet-targeted dye.

[0210] The colocalization ability of PTZ-2NO2-Lyso was tested using the method for testing the colocalization of PTZ-Lyso with commercial lysosomal dyes. The results are shown in Figure 2. Figure 12 As shown, PTZ-2NO2-Lyso can overlap well with commercial lysosomal dyes, with a colocalization coefficient of 0.92, indicating that PTZ-2NO2-Lyso is a lysosomal targeting dye.

[0211] The colocalization ability of PTZ-2NO2-Mito was tested using the method for testing the colocalization of PTZ-mito with commercial mitochondrial dyes. Figure 13 As shown, PTZ-2NO2-Mito overlaps well with commercial mitochondrial dyes, with a colocalization coefficient of 0.96, indicating that PTZ-2NO2-Mito is a mitochondrial-targeted dye.

[0212] The colocalization ability of PTZ-2NO2-Memb was tested using the method for testing the colocalization of PTZ-Memb with commercial cell membrane dyes. The results are as follows Figure 14 As shown, PTZ-2NO2-Memb can overlap well with commercial cell membrane dyes, with a colocalization coefficient of 0.83, indicating that PTZ-2NO2-Memb is a cell membrane-targeted dye.

[0213] The colocalization ability of PTZ-2NO2-ER was tested using the method for testing the colocalization of PTZ-S-ER with commercial endoplasmic reticulum dyes. Figure 15 As shown, PTZ-2NO2-ER and commercial endoplasmic reticulum dyes can overlap well with a colocalization coefficient of 0.93, indicating that PTZ-2NO2-ER is an endoplasmic reticulum-targeted dye.

[0214] The co-localization ability of PTZ-2NO2-Lipid was tested using the method for testing the co-localization of PTZ-Lipid and lipid droplet dyes. The results are as follows Figure 16 As shown in the figure, PTZ-2NO2-Lipid and lipid droplet dye can overlap well, with a colocalization coefficient of 0.93, indicating that PTZ-2NO2-ER is a lipid droplet targeting dye.

[0215] MitoTracker TM Red FM was added to HeLa cells and incubated at 37°C, 5% CO2 for 30 minutes. Then, the PXZ-Mito dye of the present invention was added and cultured for another 30 minutes. No need to wash the excess dye with PBS, and laser confocal imaging was performed directly. Figure 17 As shown, the present invention can overlap well with commercial lysosomal dyes, with a colocalization coefficient of 0.90, indicating that PXZ-Mito is a mitochondrial-targeted dye. The same method was used to test PXZ-ER and the commercial endoplasmic reticulum dye ER-Tracker.TM Red(BODIPY TM TR Glibenclamide) co-localization ability. Figure 18 As shown in Figure 2, PXZ-ER and commercial endoplasmic reticulum dyes can overlap well, with a colocalization coefficient of 0.91, indicating that PXZ-ER is an endoplasmic reticulum-targeted dye. The colocalization ability of PXZ-2NO2-ER was tested using the method for testing the colocalization of PXZ-ER and commercial endoplasmic reticulum dyes. The results are shown in Figure 2. Figure 19 As shown, PXZ-2NO2-ER can overlap well with the commercial endoplasmic reticulum dye, with a colocalization coefficient of 0.93, indicating that PXZ-2NO2-ER is an endoplasmic reticulum-targeted dye.

[0216] The colocalization ability of molecule 10a was tested using the method for testing the colocalization of PTZ-Lyso with commercial lysosomal dyes. Figure 20 As shown, 10a overlaps well with a commercial lysosomal dye, with a colocalization coefficient of 0.81, indicating that 10a is a lysosomal-targeted dye.

[0217] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An organic compound based on phenothiazine / phenoxazine, characterized in that The structural formula of the phenothiazine / phenoxazine-based organic compound is shown in Formula (B) or Formula (C): 、 ； In formula (B) or formula (C), n is independently 1-12; X is independently oxygen or sulfur; Z is independently hydrogen or nitro; The R2 is shown in Formula 13: ； Said Y is O or NH; The R3 is shown in Formula 19-20: 、 。 2. The method for preparing a phenothiazine / phenoxazine-based organic compound according to claim 1, characterized in that: When the phenothiazine / phenoxazine-based organic compound is as shown in formula (B), the preparation method thereof comprises the following steps: The acetoxy phenothiazine / phenoxazine compound represented by formula (b) is reacted with R2-YH to obtain a phenothiazine / phenoxazine-based organic compound represented by formula (B); the reaction formula is as follows: ； When the phenothiazine / phenoxazine-based organic compound is as shown in formula (C), the preparation method thereof comprises the following steps: The acetoxy phenothiazine / phenoxazine compound represented by formula (b) is reacted with the compound represented by formula (c) to obtain a phenothiazine / phenoxazine-based organic compound represented by formula (C); the reaction formula is as follows: ； Wherein, n, X, Z, R2, Y, and R3 are as described in claim 1.

3. The method for preparing phenothiazine / phenoxazine-based organic compounds according to claim 2, characterized in that: The reaction conditions of the preparation method are selected from the following: A) in the preparation of the phenothiazine / phenoxazine-based organic compound represented by formula (B), the reaction temperature is 15-120°C; B) in the preparation of the phenothiazine / phenoxazine-based organic compound represented by formula (B), the reaction time is 10-30 h; C) In the preparation of the phenothiazine / phenoxazine-based organic compound represented by formula (C), the reaction temperature is 15-40°C; D) In the preparation of the phenothiazine / phenoxazine-based organic compound represented by formula (C), the reaction time is 6-18 h.

4. The method for preparing phenothiazine / phenoxazine-based organic compounds according to claim 2, characterized in that: The molar ratio of the acetoxy phenothiazine / phenoxazine compound represented by formula (b) to R2-YH is 1:(1-2).

5. The method for preparing phenothiazine / phenoxazine-based organic compounds according to claim 2, characterized in that: The molar ratio of the acetoxy phenothiazine / phenoxazine compound represented by formula (b) to the compound represented by formula (c) is 1:(1-2).

6. Use of the phenothiazine / phenoxazine-based organic compound according to claim 1 in the preparation of fluorescent dyes.

7. A wash-free cell dye, characterized in that The invention comprises the phenothiazine / phenoxazine-based organic compound according to claim 1.

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