Dyes containing 1,4-diazabicyclo[2.2.2]octane and preparation method and application thereof

Abstract

This paper describes a class of dyes containing 1,4-diazabicyclo[2.2.2]octane, their preparation methods, and applications, belonging to the fields of optical diagnostics and biomedicine. The dye comprises the dye molecule and 1,4-diazabicyclo[2.2.2]octane covalently coupled to the dye. 1,4-diazabicyclo[2.2.2]octane directly quenches singlet oxygen generated by the dye, affecting the triplet state of the dye, thereby improving its anti-bleaching ability and photostability, and reducing phototoxicity during fluorescence imaging. Furthermore, the introduction of 1,4-diazabicyclo[2.2.2]octane significantly improves the brightness of the dye, inhibits photoscintillation, and enhances its water solubility. Its protonation characteristic enhances the dye's mitochondrial targeting ability. These compounds, by reducing singlet oxygen and enhancing the local photothermal and reactive oxygen species production in mitochondria, induce potent cell necrosis, apoptosis, and immunogenic cell death, comprehensively improving anticancer efficacy. These compounds exhibit excellent photostability and low skin toxicity, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the fields of optical diagnosis and treatment and biomedicine, and relates to a class of dyes containing 1,4-diazabicyclo[2.2.2]octane, their preparation methods and applications. Background Technology

[0002] In recent years, organic small molecule dyes have been widely used in disease diagnosis and treatment research due to their excellent photophysical properties. Dye-based fluorescence imaging enables highly sensitive, high spatiotemporal resolution visualization, while their photodynamic therapy (PDT) and photothermal therapy (PTT) properties allow for precise photoresponsive treatment of lesions, offering significant advantages such as non-invasiveness, real-time monitoring, and integrated diagnosis and treatment. This has become an important development direction for the diagnosis and treatment of major diseases such as cancer. However, existing organic dyes still face a series of unresolved bottlenecks that greatly limit their diagnostic and therapeutic effects in the complex in vivo environment.

[0003] First, traditional organic dyes are prone to structural damage under light exposure, exhibiting insufficient photostability and easily undergoing photobleaching during continuous laser excitation. This significantly reduces the persistence of imaging signals and hinders long-term dynamic monitoring. Second, many dyes possess significant hydrophobic properties, easily accumulating and quenching under aqueous or physiological conditions. This not only reduces fluorescence quantum yield and imaging sensitivity but also affects their distribution in vivo, blood circulation, and targeting ability. In photodynamic and photothermal therapy applications, organic dyes, upon photoexcitation, can generate reactive oxygen species (such as singlet oxygen) or convert to localized high temperatures, thereby inducing tumor cell apoptosis or necrosis. However, most dyes absorb in the visible light region, easily accumulating in tissues such as skin after entering the body, producing phototoxic reactions even under natural light. This is one of the main side effects of current phototherapy agents in clinical applications. Furthermore, many small-molecule dyes have limited nonradiative transition efficiency and low photothermal conversion capacity, making it difficult to meet the demand for highly efficient photothermal agents in deep tumor treatment.

[0004] Numerous studies have shown that singlet oxygen is not only a core factor leading to dye photobleaching but also a key active species responsible for reducing photothermal conversion efficiency and causing skin phototoxicity. Uncontrolled singlet oxygen generated under light conditions attacks the structure of the dye itself, further exacerbating photodegradation and structural damage. Therefore, controlling singlet oxygen could potentially significantly improve the photostability of dyes, reduce skin toxicity, and enhance the therapeutic selectivity and overall efficacy in photodynamic and photothermal therapy. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention designs and synthesizes dye molecules containing 1,4-diazabicyclo[2.2.2]octane (DABCO). The introduction of DABCO gives the dye the following advantages:

[0006] First, DABCO is an excellent singlet oxygen quenching group and also has excellent water solubility. Combining DABCO with fluorescent dyes can not only improve the photostability of the dyes, inhibit photobleaching, and prolong imaging time, but also significantly improve the water solubility of the dyes and inhibit aggregation.

[0007] Second: DABCO is easily protonated and has excellent mitochondrial targeting ability. Its mitochondrial localization ability is consistent with that of the commonly used mitochondrial targeting group triphenylphosphine, which can significantly improve the mitochondrial targeting ability of the dye, and its water solubility is better than that of triphenylphosphine.

[0008] Third: In photothermal therapy, photodynamic therapy, or sonodynamic therapy, DABCO not only powerfully induces mitochondrial damage and reactive oxygen species caused by mitochondrial stress, but also achieves higher photothermal conversion. This photodynamic and photothermal process surrounding mitochondria can effectively induce cancer cell death and activate potent immunogenic cell death.

[0009] Fourth: During photothermal therapy, photodynamic therapy, or sonodynamic therapy, DABCO can quench singlet oxygen produced by dyes under visible light irradiation, reducing dye-induced skin phototoxicity and improving the safety of clinical treatment. In fluorescence imaging applications, DABCO can quench singlet oxygen produced by dyes under light source irradiation, reducing dye phototoxicity.

[0010] A class of dyes containing 1,4-diazabicyclo[2.2.2]octane, the molecular structure of which is shown below:

[0011] or

[0012] Wherein, X represents a different type of dye, selected from cyanine dyes, rhodamine, fluorescein, porphyrin, phthalocyanine, fluoroborpyrrole, coumarin, NBD (nitrobenzoxadiazole), etc. Alternatively, it can be a nano-form of the dye.

[0013] Wherein, Z is a linking group, and according to the different DABCO structures in formulas (I) and (II), it is selected from the following linking groups, such as... , , , , , , , , , , , , , , , n and p are independent integers from 0 to 10. The order of the DABCO and dye linkages on Z is interchangeable. The linking groups are not limited to the structures described above; any structure that covalently links DABCO and the dye is acceptable.

[0014] X is shown in the table below:

[0015]

[0016]

[0017] R1, R3, and R4 are each independently selected from H and sulfonic acid groups;

[0018] R5 is independently selected from H and Cl; R6 is independently selected from H and alkyl groups with 1-10 carbon atoms.

[0019] R2 is independently selected from alkyl groups with 1-10 carbon atoms, haloalkyl groups with 1-10 carbon atoms, alkenyl groups with 2-10 carbon atoms, alkynyl groups with 2-10 carbon atoms, alkoxycarbonyl alkyl groups with 1-10 carbon atoms, sulfonic acid groups with 1-10 carbon atoms, carboxylic acid groups with 1-10 carbon atoms, aralkyl groups with 6-12 carbon atoms, cycloalkyl groups with 3-10 carbon atoms, or... .

[0020] a is an integer from 1 to 7; x is an integer from 0 to 100.

[0021] Each Y atom is an anion, and the anions are selected from BF4. - Cl - ,Br - I - NO3 - SO4 2- PF6 - OAc - ClO4 - CH3COO - CF3COO - CH3SO3 - Or CF3SO3 - m is an integer from 1 to 3.

[0022] The structures containing -COOH and -SO3H mentioned above can be the acid prototypes of the structures, or the corresponding medically acceptable salts, such as sodium salts and potassium salts.

[0023] Its preferred structure is as follows:

[0024]

[0025] This invention provides a method for synthesizing a dye containing 1,4-dioxabicyclo[2.2.2]octane, characterized in that the preparation method is as follows:

[0026]

[0027] Using piperazine as a raw material, it undergoes a substitution reaction with a compound of formula (S1) or formula (S2) to obtain a compound of formula (A1) or formula (A2), and then undergoes an ammonolysis reaction or a condensation reaction with a compound of formula (S3) or formula (S4) to obtain a compound of formula (B1) or formula (B2). Finally, it undergoes a condensation reaction with a dye molecule with a carboxyl terminus to obtain a dye molecule modified with DABCO.

[0028] In step a, piperazine undergoes a substitution reaction with compound of formula (S1) to obtain compound of formula (A1);

[0029] Step d involves a substitution reaction between piperazine and compound (S2) to obtain compound (A2);

[0030] Preferably, the substitution reaction conditions are as follows: piperazine and compound (S1) or compound (S2) are dissolved in a first organic solvent, an activator is added, the reaction is carried out at 80-120 °C for 8-12 hours, the solvent is removed, and the product is obtained by separation and purification.

[0031] Preferably, the first organic solvent is selected from one or more of acetonitrile, toluene, dioxane, and N,N-dimethylformamide;

[0032] Preferably, the activator is selected from one or more of triethylamine, N,N-diisopropylethylamine, potassium carbonate, and cesium carbonate;

[0033] Preferably, the reaction temperature is 80-120 °C, more preferably 110 °C;

[0034] Preferably, the reaction time is 8 h, 10 h or 12 h, more preferably 12 h;

[0035] Preferably, the molar ratio of piperazine to formula (S1) or formula (S2) in the substitution reaction is 1:1;

[0036] Step b involves the reaction of compound (A1) with compound (S3) via ammonolysis to obtain compound (B1).

[0037] Step e involves a condensation reaction between compound (A2) and compound (S4) to obtain compound (B2).

[0038] Preferably, the ammonolysis reaction conditions are as follows: the compound of formula (A1) and the compound of formula (S3) are mixed and heated, reacted at 80 °C for 10 hours, and the unreacted compound of formula (S3) is removed by rotary evaporation under reduced pressure to obtain the product.

[0039] Preferably, the condensation reaction conditions are as follows: compound (A2) is refluxed in thionyl chloride for 6 hours, excess thionyl chloride is removed by vacuum distillation, the residue is dissolved in a second organic solvent, compound S4 and a base solution in the second organic solvent are added dropwise, the reaction is carried out overnight at room temperature, and the product is obtained by separation and purification.

[0040] Preferably, the second organic solvent is selected from one or more of dichloromethane, acetonitrile, tetrahydrofuran, and N,N-dimethylformamide, and more preferably dichloromethane;

[0041] Preferably, the base is selected from triethylamine or N,N-diisopropylethylamine.

[0042] Step c involves a condensation reaction between compound (B1) and compound (S5) to obtain compound (C1).

[0043] Step f involves a condensation reaction between compound (B2) and compound (S5) to obtain compound (C2).

[0044] Preferably, the condensation reaction conditions are as follows: dissolve compound (B1) or compound (B2) and compound (S5) in a third organic solvent, add O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and a base, react overnight at room temperature, and after the reaction is completed, separate and purify to obtain the product;

[0045] Preferably, the third organic solvent is selected from one or more of N,N-dimethylformamide, tetrahydrofuran, and acetonitrile, and more preferably N,N-dimethylformamide;

[0046] Preferably, the base is selected from triethylamine or N,N-diisopropylethylamine.

[0047] Step g involves a nucleophilic reaction between DABCO and compound (S3) to obtain compound (C3);

[0048] Preferably, the nucleophilic reaction conditions are as follows: DABCO and the compound of formula (S3) are dissolved in a fourth organic solvent, reacted at 80-120 °C for 8-12 hours, and after removing the solvent, the product is obtained by separation and purification.

[0049] Preferably, the fourth organic solvent is selected from one or more of acetonitrile, toluene, dioxane, and N,N-dimethylformamide;

[0050] Preferably, the reaction temperature is 80-120 °C, more preferably 110 °C;

[0051] Preferably, the reaction time is 8 h, 10 h or 12 h, more preferably 12 h;

[0052] Preferably, the molar ratio of piperazine to formula (S1) or formula (S2) in the substitution reaction is 1:4.

[0053] Uses of dyes containing 1,4-diazabicyclo[2.2.2]octane, wherein the compounds are used for biomolecular labeling, fluorescent labeling, immunofluorescence analysis, flow cytometry analysis, single-molecule detection, or for fluorescence imaging using microscopy techniques selected from: confocal microscopy, wide-field fluorescence microscopy, two-photon microscopy, stimulated emission depletion microscopy, random optical reconstruction microscopy, or structured light illumination microscopy. The fluorescence imaging targets are cells, tissues, or living animals; the fluorescence imaging mode is long-term imaging.

[0054] Uses of dyes containing 1,4-diazabicyclo[2.2.2]octane for mitochondrial imaging. These dyes can not only interact with mitochondria via electrostatic interaction between the dye and DABCO, but can also be further modified to introduce other groups such as amino groups and haloalkyl groups for covalent cross-linking with mitochondria, thereby enabling longer-duration, higher-resolution imaging of mitochondria.

[0055] The use of dyes containing 1,4-diazabicyclo[2.2.2]octane is characterized in that the dyes are preferred for research related to biomacromolecules. This includes any one or more of the following: protein purification, screening of candidate small molecule drugs that bind to proteins, protein interactions, protein imaging, localization, and tracking. The protein imaging includes multicolor imaging with other imaging tags, including Halo tags and / or SNAP tags.

[0056] Application of dyes containing 1,4-diazabicyclo[2.2.2]octane in photodynamic therapy.

[0057] Application of dyes containing 1,4-diazabicyclo[2.2.2]octane in photothermal therapy.

[0058] Application of dyes containing 1,4-diazabicyclo[2.2.2]octane in sonodynamic therapy.

[0059] The use of dyes containing 1,4-diazabicyclo[2.2.2]octane or pharmaceutically acceptable salts thereof in the treatment of cancer. Characterized by the fact that the cancers are lung cancer, breast cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, ovarian cancer, prostate cancer, testicular cancer, nasopharyngeal cancer, esophageal cancer, malignant lymphoma, squamous cell carcinoma of the head and neck, thyroid cancer, and osteosarcoma, etc.

[0060] Dyes containing 1,4-diazabicyclo[2.2.2]octane can be used in the form of small dye molecules, organic or inorganic nanoparticles of any form.

[0061] The beneficial effects of this invention: This invention provides the synthesis and application of a class of dyes containing 1,4-diazabicyclo[2.2.2]octane. Using piperazine as a raw material, DABCO with different chain lengths and amino or hydroxyl links is synthesized sequentially through substitution, ammonolysis, or condensation reactions. Then, it undergoes an amide condensation reaction with a photosensitizer having a carboxyl terminus to obtain a photosensitizer modified with DABCO. Simultaneously, DABCO can also be covalently combined with dyes modified with terminal haloalkanes. DABCO is an excellent singlet oxygen quenching group, and also possesses excellent water solubility and mitochondrial targeting ability. Combining DABCO with fluorescent dyes not only improves the photostability of the dye, inhibits photobleaching, and prolongs imaging time, but also significantly improves the water solubility of the dye and inhibits aggregation. In photodynamic therapy, photothermal therapy, or sonodynamic therapy, DABCO can not only strongly induce mitochondrial damage and reactive oxygen species induced by mitochondrial stress, but also achieve higher photothermal conversion. This photodynamic and photothermal process around mitochondria can effectively induce cancer cell death and activate potent immunogenic cell death. DABCO can quench singlet oxygen produced by dyes under visible light irradiation, reducing dye-induced skin phototoxicity and improving the safety of clinical treatment. Therefore, the 1,4-diazabicyclo[2.2.2]octane-containing dyes provided by this invention have great application potential in bioimaging, mitochondrial imaging, photodynamic therapy, photothermal therapy, and sonodynamic therapy, and are also suitable for diseases caused by other viruses and bacteria. These dyes are simple to synthesize, the raw materials are readily available, and they have great commercial potential, making them excellent diagnostic and therapeutic reagents. Attached Figure Description

[0062] Figure 1 These are the ultraviolet absorption spectra of compounds Cy5, Cy5-D, Cy5.5, and Cy5.5-D.

[0063] Figure 2 These are the fluorescence emission spectra of compounds Cy5, Cy5-D, Cy5.5, and Cy5.5-D.

[0064] Figure 3 The compounds Cy5 and Cy5-D were subjected to 200 mW / cm 2 The change in absorption at 645 nm wavelength after 30 min of illumination; Cy5.5 and Cy5.5-D after 200 mW / cm 2 Changes in absorbance at 685 nm wavelength after 30 min of illumination.

[0065] Figure 4 The compounds Cy5 and Cy5-D were subjected to 1200 mW / cm 2 The change in absorption at 645 nm wavelength after 4 min of illumination; Cy5.5 and Cy5.5-D after 1200 mW / cm 2 The change in absorption at a wavelength of 685 nm after 4 min of illumination.

[0066] Figure 5 It refers to the cellular dark toxicity of compounds Cy5 and Cy5-D.

[0067] Figure 6 These are intracellular mitochondrial images of compounds Cy5 and Cy5-D at shooting times of 0 s, 2 s, and 4 s.

[0068] Figure 7 These are the results of colocalization experiments of compounds Cy5 and Cy5-D with mitochondrial imaging dyes.

[0069] Figure 8 The normalized trend of UV absorption at 415 nm after co-incubation of compounds Cypate (Cy) and Cypate-D (CyD) with DPBF indicates the generation of singlet oxygen.

[0070] Figure 9 These are the results of thermal cycling experiments on compounds Cypate (Cy) and Cypate-D (CyD).

[0071] Figure 10 The results show the intracellular stability of compounds Cypate (Cy) and Cypate-D (CyD) after light exposure.

[0072] Figure 11 These are the results of colocalization experiments of compounds Cypate (Cy) and Cypate-D (CyD) with mitochondrial imaging dyes.

[0073] Figure 12 These are the cellular ICD results (CRT, HMGB1, and ATP) for compounds Cypate (Cy) and Cypate-D (CyD).

[0074] Figure 13 The changes in tumor volume, tumor weight, and mouse body weight in mice treated with compounds Cypate (Cy) and Cypate-D (CyD) are shown.

[0075] Figure 14 The compounds IR780, IR780-D, and the IR780-DABCO mixture were subjected to a wavelength of 655 nm and a wavelength of 200 mW / cm². 2The trend of absorbance change after 30 minutes of light exposure;

[0076] Figure 15 The results show the intracellular stability of compounds IR780 and IR780-D after light exposure.

[0077] Figure 16 These are the results of colocalization experiments of compounds IR780 and IR780-D with mitochondrial imaging dyes.

[0078] Figure 17 These are the cellular ICD results (CRT and HMGB1) for compounds IR780 and IR780-D.

[0079] Figure 18 The results show the changes in tumor weight, tumor volume, and mouse body weight in mice treated with compounds IR780, IR780-D, IR780-N (nanomedicine), and IR780-DN (nanomedicine).

[0080] Figure 19 This describes the skin damage in mice under visible light irradiation after administration of compounds Cypate (Cy) and Cypate-D (CyD).

[0081] Figure 20 These are the UV absorption spectra of TPP-QD1 in different solvents.

[0082] Figure 21 The changes in UV absorption of TPP-QD1 and the control compound TPP under white light irradiation (4W) were used to assess their stability. Detailed Implementation

[0083] The technical solution of the present invention will be described in detail below through specific embodiments. It should be understood that the following specific embodiments are only exemplary, and any modifications or changes that do not depart from the design of the technical solution of the present invention should be within the scope of protection of the claims of the present invention.

[0084] The specific embodiments of the present invention are described in detail below with reference to the technical solutions:

[0085] Example 1:

[0086]

[0087] Step a: Piperazine (1.00 g, 11.61 mmol), ethyl 2,3-dibromopropionate (3.02 g, 11.61 mmol), and triethylamine (2.35 g, 23.22 mmol) were mixed in toluene (50 mL) and reacted at 80 °C for 10 hours, monitored by TLC. After the reaction was stopped, the mixture was cooled to room temperature, filtered, and the filtrate was concentrated by rotary evaporation under reduced pressure to give a brownish-yellow oil. Compound A was purified by silica gel column chromatography (DCM:MeOH = 20:1, v / v) to obtain compound A.

[0088] Step b: The mixture of compound A (1.00 g, 5.43 mmol) and ethylenediamine (5 mL) was stirred at 80 °C for 10 hours. After the reaction of the starting materials was completed by TLC monitoring, the mixture was cooled to room temperature and the ethylenediamine was removed by rotary evaporation under reduced pressure to obtain a brown oily substance, which is compound B1.

[0089] Step c: Compound Cy3 (100 mg, 0.22 mmol), compound B1 (65.00 mg, 0.33 mmol, 1.5 equivalents), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (248.61 mg, 0.66 mmol, 3 equivalents), and N,N-diisopropylethylamine (56.48 mg, 0.44 mmol, 2 equivalents) were mixed and dissolved in N,N-dimethylformamide (5 mL). The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure. The residue was purified by semi-preparative HPLC (C18 silica gel column, mobile phase 70% ACN + 30% H2O, 50 min, flow rate 4 mL / min) to obtain the target product Cy3-D in 45% yield. The product structure was identified by HRMS [M+H]. + :638.4225.

[0090] Example 2:

[0091]

[0092] The synthesis methods for steps a and b are the same as in Example 1. Compound Cy3-CC (100 mg, 0.18 mmol), compound B1 (106.64 mg, 0.54 mmol, 3 equivalents), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (204.52 mg, 0.54 mmol, 3 equivalents) and N,N-diisopropylethylamine (46.35 mg, 0.36 mmol, 2 equivalents) were mixed and dissolved in N,N-dimethylformamide (5 mL). The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure. The residue was purified by semi-preparative HPLC (C18 silica column, mobile phase 70% ACN + 30% H2O, 50 min, flow rate 4 mL / min) to obtain the target product Cy3-DD in 39% yield. The product was identified by HRMS [M+H]. + :918.6124.

[0093] Example 3:

[0094]

[0095] The synthesis method was the same as in Example 1, except that compound Cy5 was used instead of compound Cy3. The structure of the product Cy5-D was identified by HRMS and HNMR. HRMS [M+H] + :664.4381. 1H NMR (400 MHz, Methanol-d4) δ 8.22 (t, J = 13.1Hz, 2H), 7.52 – 7.46 (m, 2H), 7.40 (t, J = 7.7 Hz, 2H), 7.32 – 7.20 (m, 4H), 6.63 (t, J = 12.4 Hz, 1H), 6.27 (dd, J = 13.7, 4.8 Hz, 2H), 4.09 (t, J = 7.4Hz, 2H), 3.84 (dd, J = 15.7, 6.4 Hz, 1H), 3.61 (s, 3H), 3.51 – 3.39 (m, 2H), 3.34 (d, J = 1.6 Hz, 4H), 3.27 (d, J = 2.7 Hz, 1H), 3.22 (d, J = 9.4 Hz, 3H), 3.14 (s, 2H), 3.03 (q, J = 8.6, 8.1 Hz, 2H), 2.20 (t, J = 7.4 Hz, 2H), 1.81(q, J = 7.3 Hz, 2H), 1.75 – 1.58 (m, 14H), 1.51 – 1.42 (m, 2H).

[0096] Example 4:

[0097]

[0098] The synthesis method was the same as in Example 1, except that compound Cy5.5 was used to replace compound Cy3. The structure of the product Cy5.5-D was identified by HRMS and HNMR. HRMS [M+H] + :764.4694. 1H NMR (400 MHz, DMSO-d6) δ 8.46 (t, J = 13.1Hz, 2H), 8.26 (dd, J = 9.0, 2.3 Hz, 2H), 8.17 – 7.99 (m, 5H), 7.83 (d, J =5.7 Hz, 1H), 7.80 – 3.74 (s, 4H), 3.22 – 2.99 (m, 10H), 2.86 (t, J = 18.7 Hz, 2H), 2.06 (t, J =7.3 Hz, 2H), 1.74 (d, J = 7.5 Hz, 2H), 1.56 (p, J = 7.6 Hz, 2H), 1.39 (t, J =8.0 Hz, 2H).

[0099] Example 5:

[0100]

[0101] The synthesis method was the same as in Example 1, but compound Cy3 was substituted with compound Cy3.5. The structure of the product Cy3.5-D was identified by HRMS [M+H]. + :738.4538.

[0102] Example 6:

[0103]

[0104] The synthesis method was the same as in Example 1, but compound Cy3 was replaced with compound Cypate. The structure of the product Cypate-D was identified by HRMS and HNMR. HRMS [M+H] + :806.4436. 1H NMR (400 MHz, Methanol-d4) δ 8.23 ​​(d, J= 8.5 Hz, 2H), 8.12 – 8.04 (m, 2H), 8.00 (td, J = 7.6, 6.5, 4.0 Hz, 5H), 7.67– 7.60 (m, 4H), 7.49 (tt, J = 7.2, 4.2 Hz, 2H), 6.62 (td, J = 12.6, 5.1 Hz,2H), 6.39 (dd, J = 20.3, 13.6 Hz, 2H), 4.50 (dt, J = 15.2, 7.0 Hz, 4H), 3.76(t, J = 8.8 Hz, 1H), 3.52 – 3.46 (m, 1H), 3.41 – 3.34 (m, 2H), 3.24 (dd, J =14.1, 4.6 Hz, 7H), 3.14 (d, J = 7.6 Hz, 2H), 2.99 (q, J = 8.5, 7.2 Hz, 2H), 2.91 – 2.85 (m, 2H), 2.72 (d, J = 6.8 Hz, 2H), 2.00 (s, 12H).

[0105] Example 7:

[0106]

[0107] The synthesis method was the same as in Example 1, except that compound IR780 was used to replace compound Cy3. The structure of product IR780-D was identified by HRMS and HNMR. HRMS [M+H] + :837.4884. 1H NMR (500 MHz, Chloroform-d) δ 8.60 (d, J =14.1 Hz, 2H), 7.74 (d, J = 8.1 Hz, 2H), 7.39 (s, 1H), 7.27 (d, J = 8.1 Hz,4H), 7.15 (d, J = 7.8 Hz, 3H), 6.99 (d, J = 8.0 Hz, 2H), 6.03 (d, J = 13.8Hz, 2H), 3.90 (s, 4H), 3.76 (s, 1H), 3.57 (s, 2H), 3.39 (s, 2H), 3.22 (s,2H), 3.07 (s, 2H), 2.93 (s, 5H), 2.65 (s, 4H), 1.97 (s, 2H), 1.81 (q, J = 7.4Hz, 6H), 1.37 (d, J = 10.2 Hz, 12H), 0.99 (t, J = 7.4 Hz, 6H).

[0108] The structure of the product IR813-DD (-CC) was identified by HRMS, and the HRMS [M+H] structure was [0.05]. + :1060.5734.

[0109] Example 8:

[0110]

[0111] The synthesis method was the same as in Example 1, but compound Cy3 was replaced with compound Cy3B. The structure of the product Cy3B-D was identified by HRMS, HRMS [M+H] + :742.3429.

[0112] Example 9:

[0113]

[0114] The synthesis method was the same as in Example 1, except that compound DY549 was used to replace compound Cy3. The structure of product DY549-D was identified by HRMS, and the HRMS result was [M+H]. + :1122.3698.

[0115] Example 10:

[0116]

[0117] The synthesis method was the same as in Example 1, but compound ATTO655 was used to replace compound Cy3. The structure of the product ATTO655-D was identified by HRMS, and the HRMS result was [M+H].+ :709.3538.

[0118] Example 11:

[0119]

[0120] The synthesis method was the same as in Example 1, but compound ATTO647N was used to replace compound Cy3. The structure of the product ATTO647N-D was identified by HRMS, HRMS [M+H] + :827.5378.

[0121] Example 12:

[0122]

[0123] The synthesis method was the same as in Example 1, but compound ATTO565 was used to replace compound Cy3. The structure of the product ATTO565-D was identified by HRMS [M+H]. + :692.3602.

[0124] Example 13:

[0125]

[0126] The synthesis method was the same as in Example 1, except that compound Alexa633 was used to replace compound Cy3. The structure of the product Alexa633-D was identified by HRMS, and the HRMS result was [M+H]. + :1088.1975.

[0127] Example 14:

[0128]

[0129] The synthesis method was the same as in Example 1, but compound TMR was used instead of compound Cy3. The structure of the product TMR-D was identified by HRMS, HRMS[M+H]. + :612.2976.

[0130] Example 15:

[0131]

[0132] The synthesis method was the same as in Example 1, except that compound Alexa568 was used instead of compound Cy3. The structure of the product Alexa568-D was identified by HRMS, and the HRMS result was [M+H]. + :932.3365.

[0133] Example 16:

[0134]

[0135] The synthesis method was the same as in Example 1, except that compound Alexa532 was used instead of compound Cy3. The structure of the product Alexa532-D was identified by HRMS, and the HRMS result was [M+H]. + :808.2840.

[0136] Example 17:

[0137]

[0138] The synthesis method was the same as in Example 1, but compound JF646 was used to replace compound Cy3. The structure of product JF646-D was identified by HRMS, and HRMS [M+H]... + :786.2113.

[0139] Example 18:

[0140]

[0141] The synthesis method was the same as in Example 1, but compound ATTO532 was used to replace compound Cy3. The product structure ATTO532-D was identified by HRMS, HRMS [M+H]. + :728.2214.

[0142] Example 19:

[0143]

[0144] The synthesis method was the same as in Example 1, but compound C1 was substituted for compound Cy3. The structure of product D1 was identified by HRMS, HRMS [M+H] + :557.1958.

[0145] Example 20:

[0146]

[0147] Steps a, b, and c were performed as in Example 1, with compound Ra-110-1 replacing compound Cy3. The resulting product was dissolved in 5 mL of a 1 mol / L LiOH solution and 5 mL of a dioxane-based mixed solvent, refluxed for 2 h, and the reaction mixture was cooled to room temperature. Extraction was performed with ethyl acetate, and the aqueous phase was adjusted to neutral with dilute hydrochloric acid, followed by a second extraction. The organic phase was evaporated to dryness, and the crude product was purified by semi-preparative HPLC (CY38 silica gel column, mobile phase 70% ACN + 30% H2O, 50 min, flow rate 4 mL / min), with a yield of 62%. The structure of the obtained product Ra-110-1-D was identified by HRMS [M+H]. + :584.2663.

[0148] Example 21:

[0149]

[0150] The synthesis method was the same as in Example 1, except that compound Ra-110-2 was used to replace compound Ra-110-1. The structure of the product Ra-110-2-D was identified by HRMS, and the HRMS result was [M+H]. + :584.2663.

[0151] Example 22:

[0152]

[0153] The synthesis method was the same as in Example 1, except that compound Temra-1 was used to replace compound Ra-110-1. The structure of the product Temra-1-D was identified by HRMS, and the HRMS result was [M+H]. + :640.3289.

[0154] Example 23:

[0155]

[0156] The synthesis method was the same as in Example 1, except that compound Temra-2 was used to replace compound Ra-110-1. The structure of the product Temra-2-D was identified by HRMS, and the HRMS result was [M+H]. + :640.3289.

[0157] Example 24:

[0158]

[0159] The synthesis method was the same as in Example 1, but compound Hp was used to replace compound Cy3. The structure of the product Hp-D was identified by HRMS, HRMS [M+H] + :779.4166.

[0160] Example 25:

[0161]

[0162] The synthesis method followed Example 1, replacing compound Cy3-CC with compound HpD. The structure of the product HpD-D was identified by HRMS, and HRMS [M+H] was obtained. + :743.3955.

[0163] Example 26:

[0164]

[0165] The synthesis method for steps a and b is described in Example 1;

[0166] Step c: Compound B1 (150 mg, 0.75 mmol) and triethylamine (76.13 mg, 0.75 mmol, 1 equivalent) were dissolved in dichloromethane (5 mL). The mixture was placed in an ice bath, and a solution of triphosgene (85.26 mg, 0.30 mmol, 0.4 equivalent) in dichloromethane (2 mL) was added to the mixture. The reaction was then heated to room temperature and allowed to proceed overnight. The solvent was removed by rotary evaporation, and the residue was dissolved in dichloromethane (3 mL). Compound C2 (100 mg, 0.30 mmol) in dichloromethane (5 mL) was added dropwise under ice bath conditions. The reaction was then heated to room temperature and allowed to proceed for 10 hours, with the reaction monitored by TLC. After the reaction, the solvent was removed by rotary evaporation under reduced pressure. The residue was then purified by preparative HPLC (CY38 silica gel column, mobile phase 70% ACN + 30% H2O, 50 min, flow rate 4 mL / min) to obtain the target product D2 in 51% yield. The product was identified by HRMS [M+H]. + :781.3231;

[0167] Example 27:

[0168]

[0169] The synthesis method follows the same procedure as in Example 26, but with compound ZnPC replacing compound C2. The structure of the product ZnPC-D was identified by HRMS, and the HRMS result was [M+H]. + :874.2590.

[0170] Example 28:

[0171]

[0172] The synthesis method follows the same procedure as in Example 26, but with compound TPP replacing compound C2. The structure of the product TPP-D was identified by HRMS, [M+H]. + :903.3540.

[0173] Example 29:

[0174]

[0175] The synthesis method follows the same procedure as in Example 26, with compound ZnPC-Q replacing compound C2. The structure of the product ZnPC-QD was identified by HRMS. HRMS [M+H] + :1765.7993.

[0176] Example 30:

[0177]

[0178] The synthesis method follows the same procedure as in Example 26, but with compound TPP replacing compound C2. The structure of the product TPP-QD was identified by HRMS, and the HRMS result was [M+H]. + :874.2590.

[0179] Example 31:

[0180]

[0181] The synthesis method was the same as in Example 1, but compound ICG was used instead of compound Cy3. The structure of the product ICG-D was identified by HRMS and HNMR. HRMS [M+H] + :912.4888. 1 H NMR (400 MHz, DMSO-d6) δ 8.25 (dd, J = 8.8,4.6 Hz, 2H), 8.05 (td, J = 8.2, 4.4 Hz, 5H), 7.98 (d, J = 12.6 Hz, 2H), 7.83(s, 1H), 7.78 (d, J = 9.2 Hz, 2H), 7.69 (d, J = 8.9 Hz, 1H), 7.65 (d, J = 7.8Hz, 2H), 7.50 (q, J = 7.3 Hz, 2H), 6.57 (dt, J = 21.2, 12.7 Hz, 3H), 6.37 (d,J = 13.7 Hz, 1H), 4.27 – 4.14 (m, 4H), 3.73 (d, J = 9.3 Hz, 1H), 2.90 (d, J =4.3 Hz, 2H), 2.57 (d, J = 7.2 Hz, 2H), 2.06 (t, J = 7.1 Hz, 3H), 2.01 – 1.96(m, 1H), 1.91 (s, 10H), 1.89 (s, 4H), 1.56 (d, J = 7.8 Hz, 3H), 1.40 (d, J =7.3 Hz, 2H).

[0182] Example 32:

[0183]

[0184] The synthesis method was the same as in Example 1, but compound Cum was used instead of compound Cy3. The structure of the product Cum-D was identified by HRMS, HRMS [M+H] +:442.2376.

[0185] Example 33:

[0186]

[0187] The synthesis method was the same as in Example 1, but compound BODIPY was used instead of compound Cy3. The structure of the product BODIPY-D was identified by HRMS, and the HRMS result was [M+H]. + :473.2570.

[0188] Example 34:

[0189]

[0190] The synthesis method was the same as in Example 1, but compound ATTO495 was used to replace compound Cy3. The structure of the product ATTO495-D was identified by HRMS [M+H]. + :533.3395.

[0191] Example 35:

[0192]

[0193] The synthesis method was the same as in Example 1, but compound OG was used to replace compound Cy3. The structure of the product OG-D was identified by HRMS, HRMS [M+H] + :593.1770.

[0194] Example 36:

[0195]

[0196] Compound Cy3-Cl1 (200 mg, 0.37 mmol) and compound DABCO (165 mg, 1.48 mmol, 4.0 equivalents) were mixed in acetonitrile (10 mL), and the mixture was then refluxed at 80 °C for 10 h. After the reaction was complete, the mixture was cooled to room temperature, and the solvent was removed by rotary evaporation. The residue was purified by semi-preparative HPLC (C18 silica gel column, mobile phase 70% ACN + 30% H2O, 50 min, flow rate 4 mL / min) to obtain the target product Cy3-D1 (60 mg, yield 30.18%). The product was identified using HRMS [M+H]. + : 539.4025.

[0197] Example 37:

[0198]

[0199] The synthesis method was the same as in Example 36, but compound Ra-110-1-Br was used to replace compound Cy3-Cl1. The structure of the product Ra-110-1-D1 was identified by HRMS [M+H]. + :514.2365.

[0200] Example 38:

[0201]

[0202] The synthetic method followed Example 36, replacing compound Cy3-Cl1 with compound Ra-110-2-Br. The structure of the product Ra-110-2-D1 was identified by HRMS [M+H]. + :514.2365.

[0203] Example 39:

[0204]

[0205] The synthesis method was the same as in Example 36, but with compound Temra-1-Br replacing compound Cy3-Cl1. The structure of the product Temra-1-D1 was identified by HRMS, HRMS [M+H] + :570.2991.

[0206] Example 40:

[0207]

[0208] The synthesis method was the same as in Example 36, but with compound Temra-2-Br replacing compound Cy3-Cl1. The structure of the product Temra-2-D1 was identified by HRMS, HRMS [M+H] + :570.2991.

[0209] Example 41:

[0210]

[0211] The synthesis method follows the same procedure as in Example 36, but with compound Cy5-Cl1 substituted for compound Cy3-Cl1. The structure of the product Cy5-D1 was identified by HRMS [M+H]. + :565.4181.

[0212] Example 42:

[0213]

[0214] The synthesis method was the same as in Example 36, but compound Cy5.5-Cl1 was used instead of compound Cy3-Cl1. The structure of the product Cy5.5-D1 was identified by HRMS [M+H]. + :665.4494.

[0215] Example 43:

[0216]

[0217] The synthesis method follows the same procedure as in Example 36, but with compound Cy5-Cl2 replacing compound Cy3-Cl1. The structure of the product Cy5-D2 was identified by HRMS. HRMS [M+H] + :746.5880.

[0218] Example 44:

[0219]

[0220] The synthesis method was the same as in Example 36, but compound Cy5.5-Cl2 was used instead of compound Cy3-Cl1. The structure of the product Cy5.5-D2 was identified by HRMS [M+H]. + :846.6193.

[0221] Example 45:

[0222]

[0223] The synthesis method was the same as in Example 36, but compound TPP-Br was used to replace compound Cy3-Cl1. The structure of the product TPP-QD1 was identified by HRMS. HRMS [M] 4+ :322.6940.

[0224] Example 46:

[0225] The ultraviolet absorption and fluorescence emission spectra of the dyes were determined. Using DMF as the solvent, a 10 mmol stock solution was prepared for Cy5, Cy5-D, Cy5.5, and Cy5.5-D. 10 μL of the stock solution was taken, diluted to 5 mL, and a 20 μM solution was prepared. The ultraviolet absorption spectra were then measured sequentially. Similarly, the fluorescence emission spectra of the four molecules were measured using the 20 μM solution. The excitation wavelengths for Cy5 and Cy5-D were 645 nm, and for Cy5.5 and Cy5.5-D, they were 685 nm. Figure 1 These are the ultraviolet absorption spectra of four dyes. Figure 2 These are the fluorescence emission spectra of four dyes.

[0226] Example 47:

[0227] The photostability of dyes was studied. The effect of light exposure on dye stability was investigated using UV-Vis. A wavelength of 660 nm and a power of 200 mW / cm² were used. 2 The compounds Cy5 (20 μM), Cy5-D (20 μM), Cy5.5 (20 μM), and Cy5.5-D (20 μM) were irradiated with a laser for a total of 30 minutes. UV-Vis measurements were performed every five minutes during the laser irradiation. The absorbance variation of the maximum absorption peak was normalized, and the results are as follows: Figure 3 As shown. A wavelength of 660nm was used, with a power of 1200 mW / cm². 2 The compounds Cy5 (5 μM), Cy5-D (25 μM), Cy5.5 (5 μM), and Cy5.5-D (5 μM) were irradiated with a laser for a total of 4 minutes. UV-Vis measurements were performed every 20 seconds during the laser irradiation. The absorbance variation of the maximum absorption peak was normalized, and the results are as follows: Figure 4 As shown. Figure 21 This is a photostability test of TPP-QD1, where TPP is the porphyrin control molecule. Figure 3 , Figure 4 and Figure 21 The results show that, compared with compounds Cy5 and Cy5.5 without DABCO groups and TPP, compounds Cy5-D, Cy5.5-D and TPP-QD1 with DABCO groups showed the smallest changes in ultraviolet absorption after laser irradiation and were more stable.

[0228] Example 48:

[0229] Cell safety study. The cell safety of compounds Cy5 and Cy5-D was assessed using the MTT assay. MCF-7 cells (5000 cells per well) were seeded into 96-well plates and incubated at 37 °C with 5% CO2 for 24 hours. After cell attachment, cells were treated with culture media containing different concentrations (0, 6.25, 12.5, 25, 50 μM) of the probe molecules. After 4 hours, the culture medium was replaced, and 10 μL of MTT solution was added to each well. After another 4 hours of incubation, the culture medium was removed, and 150 μL of DMSO was added to each well. The plates were then placed in a microplate reader, shaken at 37 °C for 1 min, and the absorbance at 490 nm was measured. Cell viability was calculated. The cell safety test results are shown below. Figure 5 As shown, the DABCO-modified dyes have good safety for cells.

[0230] Example 49:

[0231] Imaging using structured illumination microscopy (SIM microscopy). This study investigated mitochondrial imaging and photostability of compounds Cy5 and Cy5-D. MCF-7 cells were incubated for 24 hours. Culture media containing 50 nM of compounds Cy5 and Cy5-D were prepared, with 2 mL of the drug-containing medium added to each dish and incubated for 4 hours. The medium was then removed, and the cells were washed three times with PBS to remove any unadsorbed dye. Cells were then placed in 2 mL of serum-free cell culture medium per dish to maintain a mild environment, and imaging was performed using a microscope with an exposure time of 20 ms and a light intensity of 1%. Figure 6 This image shows intracellular mitochondrial images of compounds Cy5 and Cy5-D at imaging times of 0 s, 2 s, and 4 s. It can be seen that compound Cy5-D produces better imaging results than compound Cy5, with clearer mitochondrial filamentous structures. Furthermore, compound Cy5-D maintains luminescence for a longer period of irradiation and exhibits a longer quenching time, without significant damage to the mitochondrial structure. Therefore, compound Cy5-D demonstrates better photostability than compound Cy5.

[0232] Example 50:

[0233] DPBF singlet oxygen release assay. 1,4-Diphenylisobenzofuran (DPBF) was used as the singlet oxygen scavenger. The compounds Cypate (Cy, 1 μM) and Cypate-D (CyD) in the DPBF solution were detected at a wavelength of 655 nm with a power of 100 mW / cm². 2 The changes in UV absorption intensity after co-incubation with DPBF for a specified time under laser irradiation. Singlet oxygen release test results are as follows: Figure 8 As shown, compound Cypate-D showed a smaller decrease in absorbance at 417 nm compared to compound Cypate, indicating that the presence of the DABCO group can significantly reduce the release of singlet oxygen, thereby improving the photostability of the probe molecule.

[0234] Example 51:

[0235] Photothermal experiments: Thermal cycling experiments of compounds Cypate and Cypate-D. A wavelength of 808 nm and a power of 600 mW / cm² were used. 2 Using a laser as the light source, compounds Cypate (Cy) and Cypate-D (CyD) were irradiated for 3 minutes, respectively, followed by cooling to room temperature. This irradiation cycle was repeated three times, with temperature monitored every 20 seconds. The results are as follows: Figure 9 As shown, Cypate-D exhibits better stability compared to the compound Cypate.

[0236] Example 52:

[0237] Intracellular stability assay. MCF-7 cells (2 × 10⁶ cells per well) were used. 5 Cells were seeded in 6-well plates. After incubation for 4 h, cells in each group were exposed to an 808 nm laser for 0, 1, 3, and 5 min. Fluorescence signals of the two groups of cells at selected time points (0, 1, 3, and 5 min) after treatment were recorded by fluorescence imaging. Figure 10 The results show the intracellular stability of compounds Cypate and Cypate-D, indicating that compound Cypate-D has better stability in cells. Figure 15 The results show the intracellular stability of compounds IR780 and IR780-D, indicating that compound IR780-D has better stability in cells.

[0238] Example 53:

[0239] Mitochondrial targeting ability assay: MCF-7 cells were incubated for 24 hours. 10 μM of compound Cy5 and compound Cy5-D were added and incubated for 4 hours, respectively. The culture medium was then removed, and the cells were washed three times with PBS to remove any unentered compounds. Next, 2 mL of Mito-Tracker Green (100 nM, final concentration) diluted in serum-free medium was added to the dish and incubated for 30 minutes. The cell culture medium was removed, and the cells were washed three times with PBS to thoroughly remove any unentered Mito-Tracker Green. 2 mL of serum-free cell culture medium was added to the dish to provide a mild environment for imaging. Co-localization coefficients were calculated using ImageJ software. Mitochondrial co-localization experiments of compounds Cypate and Cypate-D, as well as compounds IR780 and IR780-D, were performed using the same method. Co-localization coefficients were calculated using ImageJ software. Figure 7 These are the results of co-localization experiments of compounds Cy5 and Cy5-D. The co-localization coefficient of Cy5-D is 0.86, which is higher than the co-localization coefficient of Cy5 (0.74), indicating that Cy5-D has a better mitochondrial targeting effect. Figure 11 These are the co-localization results of compounds Cypate (Cy) and Cypate-D (CyD). The co-localization coefficient of Cypate-D is 0.84, which is higher than the co-localization coefficient of Cypate (0.56), indicating that Cypate-D has a better mitochondrial targeting effect. Figure 16 The colocalization experiment results of compounds IR780 and IR780-D show that the colocalization coefficient of IR780-D is 0.90, which is higher than the colocalization coefficient of IR780 (0.69). Therefore, IR780-D has a better mitochondrial targeting effect.

[0240] Example 54:

[0241] Anticancer efficacy assay. The MTT assay was used to determine the killing effect of the dye on cancer cells under light or ultrasound conditions. Cancer cells (7000 cells per well) were seeded in 96-well plates and cultured for 24 hours. Subsequently, different concentrations of dye (Cypate, Cypate-D, IR780, or IR780-D) (concentrations of 0, 6.25, 12.5, 25, 50, and 100 µM) were added, and the cells were cultured for another 4 hours. Cells were washed with PBS and the culture medium was replaced with fresh medium. An 808 nm laser (1 W / cm²) was used. 2 Cells were irradiated and then cultured for another 12 hours. Cytotoxicity (IC50) 50 The results are shown in Table 1. The therapeutic light source for TPP-QD1 was 655 nm, 25 mW / cm². 2 Its cytotoxicity results were: IC50 50 (MCF-7) = 2.1 µM; IC 50 (A549) = 1.5 µM; IC 50 (4T1) = 2.1 µM; IC 50 (B16F10) = 2.5 µM; IC 50 (Hela) = 3.2 µM; IC 50 (HepG2) = 6.5 µM; IC 50 (AGS) = 5.2 µM; IC 50 (PANC-1) = 3.4 µM; IC 50 (SW620) = 0.1 µM; IC 50 (SK-OV-3) = 5.5 µM; IC 50 (PC-3) = 7.1 µM; IC 50 (KYSE-180) = 3.2 µM; IC 50 (SCC-75) = 1.9 µM; IC 50 (BC-PAP) = 1.6 µM; IC 50 (143B) = 7.5 µM.

[0242] In the sonodynamic therapy experiment, the representative molecule used was TPP-QD1. The ultrasound conditions were 1 W / cm². 2 At 1 MHz, its cytotoxicity results were: IC50 50 (MCF-7) = 5.2 µM; IC 50(A549) = 0.5 µM; IC 50 (4T1) = 1.2 µM; IC 50 (B16F10) = 7.5 µM; IC 50 (Hela) = 4.1 µM; IC 50 (HepG2) = 3.2 µM; IC 50 (AGS) = 2.2 µM; IC 50 (PANC-1) = 1.3 µM; IC 50 (SW620) = 1.9 µM; IC 50 (SK-OV-3) = 2.0 µM; IC 50 (PC-3) = 3.3 µM; IC 50 (KYSE-180) = 1.2 µM; IC 50 (SCC-75) = 2.7 µM; IC 50 (BC-PAP) = 1.8 µM; IC 50 (143B) = 1.9 µM.

[0243] Table 1

[0244]

[0245] Example 55:

[0246] Cellular ICD assay. MCF-7 cells were cultured for 24 h, then the medium was replaced with fresh medium containing 20 μM Cypate or Cypate-D, and incubated for another 4 h. After washing with PBS, fresh medium was added, and the cells were irradiated with an 808 nm near-infrared laser (0.6 W / cm²) for 5 min. The expression levels of CRT and HMGB1 were detected by Western blotting and confocal laser scanning microscopy (CLSM), and the release of 5′-adenosine triphosphate (ATP) was detected by an ATP assay kit. Figure 12These are the cellular ICD results for compounds Cypate and Cypate-D. The experimental results show that, compared to other control groups, Cypate-D, after treatment of tumor cells under light-activated conditions, significantly induced changes in immunogenic cell death-related markers, including promoting the exposure of calreticulin CRT to the cell membrane surface, inducing the release of high-mobility group box 1 (HMGB1) from the cell, and enhancing extracellular ATP secretion. These results indicate that Cypate-D has the ability to induce immunogenic cell death (ICD). The cellular ICD results for compounds IR780 and IR780-D are as follows: Figure 17 Experimental results showed that, after light exposure, IR780-D significantly induced changes in immunogenic cell death-related markers compared to other control groups, promoted CRT expression, and induced the release of HMGB1 from cells. IR780-D also demonstrated the ability to induce ICD.

[0247] Example 56:

[0248] Nanoparticle preparation. Human serum albumin (HSA, 6 mg) was dissolved in phosphate-buffered saline (PBS, 2 mL), and IR780-D or other small dye molecules (2 mg) were dissolved in N,N-dimethylformamide (DMF, 30 µL). The IR780-D solution was slowly added to the HSA solution under ultrasonic conditions, followed by vigorous stirring at room temperature for 6 h. Ultrafiltration with ultrapure water (3,500 rpm, 30 min per cycle) was used to remove unbound dye and excess HSA. The resulting nanoparticles were dispersed in ultrapure water, freeze-dried at low temperature, and stored at -20 °C.

[0249] Example 48:

[0250] In vivo tumor suppression experiment. Female BALB / c mice were selected, and 4T1 cells (5 × 10⁻⁶) were injected. 5 A tumor model was established by injecting 100 μL of PBS (100 cells) into the right dorsal region of BALB / c mice. The tumor volume was measured to be ~150 mm². 3 Mice were divided into groups and intravenously injected with saline, Cypate, or Cypate-D (10 mg / kg, 100 μL), respectively. Near-infrared light at 808 nm (0.6 W / cm²) was used for observation. 2 Phototherapy was administered to mice (5 min). Tumor volume and body weight were recorded every 2 days. After treatment, mice were sacrificed, and tumors and major organs (heart, liver, spleen, lung, and kidney) were fixed with paraformaldehyde (10% vol / vol). Sections were prepared and analyzed by hematoxylin-eosin (H&E) and immunofluorescence (Ki-67, TUNEL, CD4, and CD8) staining. The experimental procedures for compounds IR780 and IR780-D were the same as described above. Figure 13 The changes in tumor volume, tumor weight, and body weight in mice after administration of compounds Cypate and Cypate-D are shown. Figure 18 The changes in tumor volume, tumor weight, and body weight in mice after administration of compounds IR780 and IR780-D are shown.

[0251] Example 57:

[0252] Skin toxicity test. Healthy female BALB / c mice were randomly divided into saline group, Cypate group, and Cypate-D group. After intravenous injection of the drug, the mouse skin was exposed to 655 nm light (300 mW / cm²). 2 Apply the irradiation solution for 15 minutes. Monitor the irradiated area of ​​the mice regularly over the next 12 days. Sacrifice the mice 12 days after treatment. Figure 19 The images show the skin damage in mice after administration of compounds Cypate and Cypate-D. It can be seen that the skin of mice exposed to light after administration of Cypate-D showed almost no damage, and Cypate-D did not have significant skin toxicity after light treatment.

[0253] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A class of dyes containing 1,4-diazabicyclo[2.2.2]octane, characterized in that, The general structural formula of the dye is shown below: or ; Where X represents different types of dye molecules; Wherein, Z is a linking group, selected from the following structures: , , , , , , , , , , , , , , , n and p are independent integers between 0 and 10.

2. The dye containing 1,4-diazabicyclo[2.2.2]octane according to claim 1, characterized in that: In the structural formula, -ZX is selected from the following structures: ； ； Among them, the structures containing -COOH and -SO3H are the acid prototypes or their corresponding medically acceptable salts; R1, R3, and R4 are each independently selected from H and sulfonic acid groups; R5 is independently selected from H and Cl; R6 is independently selected from H and alkyl groups with 1-10 carbon atoms; R2 is selected from alkyl groups with 1-10 carbon atoms, haloalkyl groups with 1-10 carbon atoms, alkenyl groups with 2-10 carbon atoms, alkynyl groups with 2-10 carbon atoms, alkoxycarbonyl alkyl groups with 1-10 carbon atoms, sulfonic acid groups with 1-10 carbon atoms, carboxylic acid groups with 1-10 carbon atoms, aralkyl groups with 6-12 carbon atoms, cycloalkyl groups with 3-10 carbon atoms, or... ; a is an integer from 1 to 7; x is an integer from 0 to 100; Y is an anion, and the anion is selected from BF4. - Cl - ,Br - I - NO3 - SO4 2- PF6 - OAc - ClO4 - CH3COO - CF3COO - CH3SO3 - Or CF3SO3 - m is the number of anions, making the dye structure electrically neutral.

3. The dye containing 1,4-diazabicyclo[2.2.2]octane according to claim 1, characterized in that, The dye structure containing 1,4-diazabicyclo[2.2.2]octane is selected from: ； 。 4. A method for preparing a type of dye containing 1,4-diazabicyclo[2.2.2]octane according to any one of claims 1-3, characterized in that, Includes the following steps: ； Where n is an integer from 2 to 6; E1 and E2 are each independently selected from N or O; Preparation of compound C1: Step a: Piperazine and compound S1 undergo a substitution reaction to obtain compound A1; The substitution reaction conditions are as follows: piperazine and S1 are dissolved in a first organic solvent, an activator is added, the reaction is carried out at 80-120°C for 8-12 hours, the solvent is removed, and the product is obtained by separation and purification. Step b: Compound A1 undergoes an ammonolysis reaction with compound S3 to obtain compound B1; The ammonolysis reaction conditions are as follows: compound A1 and compound S3 are mixed and heated, reacted at 80 °C for 10 hours, and unreacted compound S3 is removed by rotary evaporation under reduced pressure to obtain the product. Step c: Compound B1 undergoes a condensation reaction with compound S5 to obtain compound C1; The condensation reaction conditions were as follows: compound B1 and compound S5 were dissolved in a third organic solvent, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate and a base were added, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was separated and purified. Preparation of compound C2: Step d: Piperazine and compound S2 undergo a substitution reaction to obtain compound A2; The substitution reaction conditions are as follows: piperazine and S2 are dissolved in a first organic solvent, and an activator is added. The reaction is carried out at 80-120°C for 8-12 hours. After removing the solvent, the product is obtained by separation and purification. The first organic solvent is selected from one or more of acetonitrile, toluene, dioxane, and N,N-dimethylformamide. The activator is selected from one or more of triethylamine, N,N-diisopropylethylamine, potassium carbonate, and cesium carbonate. Step e: Compound A2 undergoes a condensation reaction with compound S4 to obtain compound B2; The condensation reaction conditions are as follows: compound A2 is refluxed in thionyl chloride for 4-8 hours, excess thionyl chloride is removed by vacuum distillation, the residue is dissolved in a second organic solvent, compound S4 and a base solution in the second organic solvent are added dropwise, the reaction is carried out overnight at room temperature, and product B2 is obtained by separation and purification. Step f: Compound B2 undergoes a condensation reaction with compound S5 to obtain compound C2; The condensation reaction conditions were as follows: compound B2 and compound S5 were dissolved in a third organic solvent, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate and a base were added, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product C2 was obtained by separation and purification. Preparation of compound C3: Step g: Dissolve DABCO and compound S3 in a fourth organic solvent and react at 80-120℃ for 8-12 hours. After removing the solvent, separate and purify to obtain the product. The first organic solvent is selected from one or more of acetonitrile, toluene, dioxane, and N,N-dimethylformamide; the activator is selected from one or more of triethylamine, N,N-diisopropylethylamine, potassium carbonate, and cesium carbonate. The second organic solvent is selected from one or more of dichloromethane, acetonitrile, tetrahydrofuran, and N,N-dimethylformamide; The third organic solvent is selected from one or more of N,N-dimethylformamide, tetrahydrofuran, and acetonitrile; the base is selected from triethylamine or N,N-diisopropylethylamine. The fourth organic solvent is selected from one or more of acetonitrile, toluene, dioxane, and N,N-dimethylformamide.

5. The use of the dye containing 1,4-diazabicyclo[2.2.2]octane according to any one of claims 1 to 3, characterized in that, The compound is used for biomacromolecule labeling, fluorescent labeling, immunofluorescence analysis, flow cytometry analysis, single-molecule detection, or for fluorescence imaging using microscopy techniques; the microscopy techniques are selected from confocal microscopy, wide-field fluorescence microscopy, two-photon microscopy, stimulated emission depletion microscopy, random optical reconstruction microscopy, or structured light illumination microscopy.

6. The use according to claim 5, characterized in that, The object of the fluorescence imaging is a cell, tissue, or living animal; the mode of the fluorescence imaging is long-term imaging.

7. The use of a dye containing 1,4-diazabicyclo[2.2.2]octane according to any one of claims 1 to 3, characterized in that, The compound is used in mitochondrial imaging.

8. Use of the dye containing 1,4-diazabicyclo[2.2.2]octane according to any one of claims 1 to 3, characterized in that, When the compound is used for biomacromolecule labeling, it is used in protein purification, screening of candidate small molecule drugs that bind to proteins, protein interactions, protein imaging, localization, and tracking; the protein imaging includes multicolor imaging with Halo tags and / or SNAP tags.

9. The application of the dye containing 1,4-diazabicyclo[2.2.2]octane as described in any one of claims 1 to 3 in photothermal therapy, photodynamic therapy, and sonodynamic therapy.

10. The use of the dye containing 1,4-diazabicyclo[2.2.2]octane or a pharmaceutically acceptable salt thereof, as described in any one of claims 1 to 3, characterized in that: The use of the dyes containing 1,4-diazabicyclo[2.2.2]octane or pharmaceutically acceptable salts thereof in the treatment of cancer; The cancers mentioned are lung cancer, breast cancer, stomach cancer, liver cancer, pancreatic cancer, colorectal cancer, ovarian cancer, prostate cancer, testicular cancer, nasopharyngeal cancer, esophageal cancer, malignant lymphoma, squamous cell carcinoma of the head and neck, thyroid cancer, and osteosarcoma.

11. The application according to claim 9 or 10, wherein the dye containing 1,4-diazabicyclo[2.2.2]octane is used in the form of dye small molecules or any form of nanoparticles, wherein the carrier of the nanoparticles is an organic or inorganic nanocarrier, and the organic carrier is selected from liposomes and micelles.

Images