Targeted lysosome aggregation-induced emission type I photosensitizer as well as preparation method and application thereof

Abstract

The invention provides a lysosome-targeted aggregation-induced emission I-type photosensitizer and a preparation method and application thereof.The photosensitizer takes indoline as an electron donor and pyridine cations as an electron acceptor, has the aggregation-induced emission characteristic, can rapidly mark lysosome and has the excellent photodynamic cancer cell killing effect, and the photosensitizer has the advantages that the photosensitizer is simple in preparation method and easy to popularize. Wide application prospects are realized in the fields of biological fluorescence imaging and photodynamic therapy.

Description

Technical Field

[0001] This invention relates to the field of fluorescence imaging and photodynamic therapy, specifically to a type I photosensitizer that targets lysosomes and induces aggregation-induced emission, its preparation method, and its application. Background Technology

[0002] Lysosomes are important acidic organelles within cells, containing various hydrolytic enzymes and organic substances. They play a crucial role in the digestion, breakdown, and recycling of intracellular metabolites and participate in numerous life processes. Directly targeting lysosomes with photosensitizers can enhance the efficacy of photodynamic therapy (PDT) because reactive oxygen species (ROS) generated directly within lysosomes overcome the challenges posed by their limited migration distance (less than 20 nanometers) and short lifespan (less than 40 nanoseconds). This highlights the necessity of photosensitizers that can both highly specifically target lysosomes and efficiently generate ROS, thereby achieving effective cancer treatment. Currently used photosensitizers, such as Photofrin and Temoporfin, primarily generate type II ROS, exhibiting high oxygen dependence, weak PDT efficacy, and are prone to fluorescence quenching and reduced ROS generation capacity after aggregation at high concentrations. In contrast, aggregation-induced emission (AIE) materials show significant fluorescence enhancement and increased ROS generation after aggregation. Therefore, developing aggregation-induced emission type I photosensitizers with lysosome targeting is of great significance for advancing fluorescence imaging-guided PDT.

[0003] Therefore, it is urgent to study how to solve the problems of poor photostability of lysosomal-targeted fluorescent dyes and unsatisfactory photodynamic therapy of photosensitizers in existing technologies. Summary of the Invention

[0004] The purpose of this invention is to provide a type I photosensitizer that targets lysosomes and induces aggregation-induced emission, along with its preparation method and application, in order to solve the problems of poor photostability of existing commercially available lysosome-targeting fluorescent dyes and unsatisfactory photodynamic therapeutic effects of commercially available photosensitizers.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A type I photosensitizer that targets lysosome aggregation-induced luminescence, wherein the photosensitizer has the structural formula shown in Formula I:

[0006] in, In Equation I, R1 can be any one of R11, R12, and R13; R11 is -(CH2)nCH3, where n = any value from 0, 1, ..., 12; R12 is -(CH2)nCH2Br, where n = any value from 0, 1, ..., 12; The structural formula of R13 is shown in Formula II, which is:

[0007] In Equation II, n = any value among 0, 1, ..., 12; R2 in Formula I is selected from at least one of substituted or unsubstituted C1-C2 alkyl, substituted or unsubstituted C1-C2 alkoxy, nitro, haloalkyl, and formyl.

[0008] To achieve the above objectives, the present invention also provides the following technical solutions: A method for preparing the above-mentioned photosensitizer, the method comprising the following steps: S1, 1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole, bromobenzene, NaOBu-t, Pd(OAc)2 and P(t-Bu)3, and anhydrous toluene were mixed and refluxed under nitrogen protection, filtered, concentrated, and the crude product was purified by silica gel column chromatography. S2, under nitrogen protection, darkness, and low temperature conditions, an acetone solution of N-bromosuccinimide and 4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole was added to a double-necked round-bottom flask to carry out the reaction. After the reaction was completed, water was added to terminate the reaction, the resulting reactants were evaporated, and then dichloromethane and water were added and washed with dichloromethane. The organic phase was collected, dried with anhydrous sodium sulfate, and then the solvent was evaporated to remove the solvent. The crude product was purified by silica gel column chromatography using petroleum ether as the eluent. S3. In a double-necked round-bottom flask equipped with a condenser, 7-bromo-4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole, 5-formyl-2-thiopheneboronic acid, [1,1'-bis(triphenylphosphine)ferrocene]palladium dichloride and potassium carbonate were added. Under nitrogen protection, a mixed solvent of toluene and methanol was added as the reaction solvent, and dichloromethane was added as the organic phase. The dichloromethane was washed successively with water and a saturated sodium chloride aqueous solution. Then, the solution was purified and separated using a silica gel column. S4, 5-[7-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indolyl)]thiophene-2-carboxaldehyde and 2-[4-(pyridin-4-yl)phenyl]acetonitrile were mixed and dissolved in anhydrous methanol, CH3ONa was added, the reaction was stirred and then the solvent was removed by rotary evaporation under reduced pressure. DCM / MeOH was used as the elution solvent, and the purification and separation were performed by silica gel column chromatography. S5, under nitrogen protection, 2-[4-(pyridin-4-yl)phenyl]-3-[5-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indol-7-yl)thiophen-2-yl]acrylonitrile and acetonitrile were added to the reaction vessel, and iodoethane was added to the reaction vessel using a syringe. After reflux, a saturated acetone solution containing KPF6 was added to displace the ions, and then water was added to terminate the reaction. The reaction product was extracted with dichloromethane, and the organic solvent was removed under vacuum. The product was purified and separated using a silica gel column.

[0009] To achieve the above objectives, the present invention also provides the following technical solutions: Applications of the aforementioned photosensitizers or photosensitizers obtained by the methods described above in the fields of fluorescence imaging and photodynamic therapy.

[0010] Other applicable fields will become apparent from the description provided in this invention.

[0011] The descriptions and specific examples in the invention summary are intended to be illustrative only and are not intended to limit the scope of the invention.

[0012] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects: 1. This invention provides a lysosome-targeted high-performance type I photosensitizer, which combines aggregation-induced emission properties, excellent photostability, and high efficiency in killing cancer cells, overcoming the problems of insufficient photostability of existing lysosome-targeting dyes and poor photodynamic efficacy of commercial type I photosensitizers.

[0013] 2. The specific structure of the photosensitizer provided by the present invention uses indoline as an electron donor and pyridine cation as an electron acceptor. The strong intramolecular charge transfer in this photosensitizer helps to reduce the singlet-triplet energy level difference, thereby improving intersystem crossing to facilitate the generation of reactive oxygen species.

[0014] 3. The photosensitizer provided by the present invention has the advantages of simple synthesis method, high synthesis yield, cheap and readily available raw materials, and is suitable for large-scale preparation.

[0015] 4. This photosensitizer exhibits excellent lysosomal targeting and localization capabilities. Co-localization experiments with commercial lysosomal probes show that its Pearson co-localization coefficient is as high as 0.87, indicating precise localization.

[0016] 5. The photosensitizer of this invention exhibits highly efficient type I photodynamic activity. Experimental data show that its hydroxyl radical generation rate is 7 times that of the traditional photosensitizer Rose Bengal. In in vitro cell experiments, at 30 mW / cm², [the rate is significantly higher]. 2 After 10 minutes of white light irradiation, only 1 μM of the photosensitizer is needed to reduce the survival rate of cancer cells to an extremely low level (<1%).

[0017] 6. Compared with existing photosensitizers, the photosensitizer provided by this invention has significantly improved killing ability. Under the same test conditions, the cancer cell survival rate of existing photosensitizers Rose Bengal and dihydroporphyrin e6 is higher than 85%, while the cancer cell survival rate of the photosensitizer provided by this invention is less than 1%. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other solutions can be obtained based on these drawings without creative effort.

[0019] Figure 1 The hydrogen NMR spectrum of Lyso-PSs ICPy provided in Example 1 of this invention; Figure 2 The carbon NMR spectrum of Lyso-PSs ICPy provided in Example 1 of this invention; Figure 3 This refers to the high-resolution mass spectrometry of Lyso-PSs ICPy provided in Embodiment 1 of the present invention; Figure 4 The UV absorption spectra of Lyso-PSs ICPy in different solvents provided in Example 1 of the present invention are shown, where the horizontal axis is wavelength, the vertical axis is molar absorptivity, and the concentration of the fluorescent probe is 10 μM. Figure 5 The normalized fluorescence spectra of Lyso-PSs ICPy in different solvents provided in Example 1 of the present invention are shown, where the horizontal axis is wavelength, the vertical axis is fluorescence intensity, and the concentration of the fluorescent probe is 10 μM. Figure 6(a) shows the fluorescence emission of Lyso-PSs ICPy provided in Example 1 of the present invention in DMSO / toluene mixed solvents with different toluene volume fractions, where the horizontal axis is wavelength in nm and the vertical axis is fluorescence intensity, and the concentration of the fluorescent probe is 10 μM. Figure 6(b) shows the fluorescence emission of Lyso-PSs ICPy provided in Example 1 of the present invention in DMSO / toluene mixed solvents with different toluene volume fractions, where the horizontal axis is the toluene volume fraction in vol%, the vertical axis is the relative value of fluorescence intensity, and the concentration of the fluorescent probe is 10 μM. Figure 7 The diagram shows the singlet oxygen production of Lyso-PSs ICPy provided in Example 1 of this invention, where the horizontal axis represents the illumination time in seconds and the vertical axis represents the absorbance ratio of 9,10-anthratridiyl-bis(methylene)dicarboxylic acid (ABDA). Figure 8 The graph shows the generation of hydroxyl radicals in Lyso-PSs ICPy provided in Example 1 of this invention, where the horizontal axis represents the illumination time and the vertical axis represents the fluorescence intensity ratio of hydroxyphenyl fluorescein (HPF). Figure 9 This is a confocal fluorescence imaging image of lysosome colocalization of mouse breast cancer cells (4T1) labeled with Lyso-PSs ICPy, provided in Example 1 of this invention. Figure 10 The fluorescence signal retention values ​​of Lyso-PSs ICPy and commercial lysosomes provided in Example 1 of this invention under continuous fluorescence imaging; Figure 11 A schematic diagram illustrating cell viability in an in vitro photodynamic therapy test using Lyso-PSs ICPy provided in Embodiment 1 of the present invention and the prior art photosensitizers Rose Bengal and dihydroporphyrin e6. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. It should be understood that the specific embodiments described herein are only for illustration and explanation of the present invention and are not intended to limit the present invention.

[0021] Any specific numerical values ​​disclosed herein (including the endpoints of numerical ranges) are not limited to their exact values, but should be understood to also include values ​​close to the exact value, such as all possible values ​​within ±5% of the exact value. Furthermore, with respect to the disclosed numerical ranges, one or more new numerical ranges can be obtained by arbitrarily combining the endpoint values ​​of the range, the endpoint values ​​with specific point values ​​within the range, and the specific point values ​​themselves; these new numerical ranges should also be considered as specifically disclosed herein.

[0022] The terminology used in this invention is for the purpose of describing specific exemplary embodiments only and is not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” as used herein are intended to include the plural forms as well. The terms “comprising,” “including,” “containing,” and “having” are inclusive and thus describe the presence of said features, elements, compositions, steps, integers, operations, and / or components, but do not exclude the presence or inclusion of one or more other features, integers, steps, operations, elements, components, and / or sets thereof. Although the open-ended term “comprising” should be understood as a non-limiting term used to describe and claim the various embodiments described in this invention, in some aspects it may instead be understood as a more restrictive and limiting term, such as “consisting of” or “essentially composed of.” Thus, for any given embodiment describing a composition, material, component, element, feature, integer, operation, and / or process step, the invention also particularly includes embodiments consisting of or substantially consisting of such compositions, materials, components, elements, features, integers, operations, and / or process steps. In the case of “consisting of…”, the alternative embodiments exclude any additional compositions, materials, components, elements, features, integers, operations and / or process steps. In the case of “essentially composed of…”, any additional compositions, materials, components, elements, features, integers, operations and / or process steps that substantially affect the essential and novel characteristics are excluded from such embodiments. However, any compositions, materials, components, elements, features, integers, operations and / or process steps that do not substantially affect the essential and novel characteristics may be included in the embodiments.

[0023] Any method steps, processes, and operations described in this invention should not be construed as necessarily requiring them to be performed in the specific order discussed or shown, unless explicitly specified. It should also be understood that, unless otherwise stated, additional or alternative steps may be used.

[0024] In this invention, except where expressly stated, any matters or issues not mentioned are directly applicable to those known in the art without any modification. Furthermore, any embodiment described in this invention can be freely combined with one or more other embodiments described in this invention, and the resulting technical solutions or concepts are considered part of the original disclosure or original record of this invention, and should not be regarded as new content not disclosed or anticipated by this invention, unless those skilled in the art consider the combination to be clearly unreasonable.

[0025] Unless otherwise stated, the terms used herein have the same meaning as commonly understood by those skilled in the art, and if a term is defined herein and its definition differs from the common understanding in the art, the definition herein shall prevail.

[0026] Unless otherwise stated, when % is mentioned in this document, it refers to wt%.

[0027] First aspect This invention provides a type I photosensitizer for targeting lysosome aggregation-induced emission, the structure of which is shown in Formula I. The molecular core employs a strong push-pull electron system composed of an indoline-pyridine cation. Under photoexcitation, a significant intramolecular charge transfer occurs from the electron-rich indoline (electron donor) to the electron-deficient pyridine cation (electron acceptor). This effect significantly reduces the energy level difference between the singlet and triplet states, thereby greatly improving the efficiency of intersystem crossing. The triplet excited-state molecule, with its longer lifetime, has a higher probability of undergoing electron transfer reactions with surrounding substrates, preferentially following the type I reaction pathway, and efficiently converting oxygen or water molecules into reactive oxygen species such as hydroxyl radicals.

[0028] The lone pair of electrons in the nitrogen atom of the indoline unit makes it basic. In the cellular environment, this molecule selectively enters and remains in acidic lysosomes (pH 4.5-5.0). In the acidic environment, the nitrogen atom is protonated, giving the entire molecule a positive charge. This creates an electrostatic attraction with the negatively charged components in the lysosomal membrane and also greatly enhances the molecule's hydrophilicity, making it "captured" in the lysosomal lumen and difficult to diffuse out, ultimately achieving the precise targeting demonstrated by a colocalization coefficient as high as 0.87.

[0029]

[0030] in, In Equation I, R1 can be any one of R11, R12, and R13; R11 is -(CH2)nCH3, where n = any value from 0, 1, ..., 12; R12 is -(CH2)nCH2Br, where n = any value from 0, 1, ..., 12; The structural formula of R13 is shown in Formula II, which is:

[0031] In Equation II, n = any value among 0, 1, ..., 12; R2 in Formula I is selected from at least one of substituted or unsubstituted C1-C2 alkyl, substituted or unsubstituted C1-C2 alkoxy, nitro, haloalkyl, and formyl.

[0032] In some embodiments of the present invention, R2 is any one of -CH3, -CH2CH3, -OCH3, -NO2, -CF3, and -CHO. By introducing the above functional groups at the R2 position, the electron cloud density and spatial packing mode of the molecular core can be precisely controlled, thereby optimizing performance. Specifically: Introducing electron-withdrawing groups (such as -NO2, -CF3, -CHO): These groups further enhance the intensity of intramolecular charge transfer effects, significantly reducing the singlet-triplet energy level difference and thus greatly promoting intersystem crossing processes. This leads to an increased triplet excited-state yield, making the molecule more prone to generating type I reactive oxygen species via electron transfer pathways. Therefore, these substituents are primarily designed to achieve higher photodynamic therapy efficiency.

[0033] Introducing electron-donating groups (such as -CH3, -OCH3): These groups moderately weaken the overall charge transfer intensity of the molecule. Their primary function is to optimize the spatial arrangement of the molecule in the aggregated state. By introducing appropriate steric hindrance or altering intermolecular interactions, they effectively suppress fluorescence quenching caused by aggregation, thereby enhancing its aggregation-induced emission properties. This results in photosensitizers having a higher signal-to-noise ratio during imaging, making them more suitable for image-guided photodynamic therapy.

[0034] Second aspect This invention provides a method for preparing the photosensitizer described in the first aspect above, the method comprising the following steps: S1, mixing 1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole, bromobenzene, NaOBu-t, Pd(OAc)2 and P(t-Bu)3, and anhydrous toluene, and refluxing under nitrogen protection, filtering, concentrating, and purifying the crude product by silica gel column chromatography; S2, under nitrogen protection, darkness, and low temperature conditions, adding N-bromosuccinimide and 4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole to a two-necked round-bottom flask. The reaction was carried out with an acetone solution of indole. After the reaction was completed, water was added to terminate the reaction. The resulting reactants were evaporated, and then dichloromethane and water were added. The mixture was washed with dichloromethane, and the organic phase was collected. The organic phase was dried with anhydrous sodium sulfate, and then the solvent was evaporated. The crude product was purified by silica gel column chromatography using petroleum ether as the eluent. S3, in a double-necked round-bottom flask equipped with a condenser, 7-bromo-4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole, 5-formyl-2-thiopheneboronic acid, [1,1'-bis(triphenylphosphine)ferrocene]palladium dichloride and carbonate were added. Potassium was added under nitrogen protection as a mixed solvent of toluene and methanol as the reaction solvent, and dichloromethane was added as the organic phase. The dichloromethane was washed successively with water and a saturated sodium chloride aqueous solution, and then purified and separated using a silica gel column. For S4, 5-[7-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indolyl)]thiophene-2-carboxaldehyde and 2-[4-(pyridin-4-yl)phenyl]acetonitrile were mixed and dissolved in anhydrous methanol, CH3ONa was added, and after stirring, the reaction solvent was removed by rotary evaporation under reduced pressure. DCM / MeOH was used as the elution solvent. The product was purified and separated using a silica gel column chromatography method. In step S5, under nitrogen protection, 2-[4-(pyridin-4-yl)phenyl]-3-[5-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indol-7-yl)thiophen-2-yl]acrylonitrile and acetonitrile were added to a reaction vessel, and iodoethane was added using a syringe. After reflux, a saturated acetone solution containing KPF6 was added to displace the ions, and water was added to terminate the reaction. The reaction product was extracted with dichloromethane, and the organic solvent was removed under vacuum. Purification and separation were then performed using a silica gel column chromatography method. This method has the advantages of inexpensive raw materials, simple synthesis, and high yield.

[0035] In some embodiments of the present invention, in step S1, the amount of 1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole is 1 part by weight, the amount of bromobenzene is 1.3 parts by weight, the amount of NaOBu-t is 0.85 parts by weight, the amount of Pd(OAc)2 is 0.03 parts by weight, the amount of P(t-Bu)3 is 0.04 parts by weight, and the amount of anhydrous toluene is 40 parts by weight.

[0036] It is worth noting that excessive bromobenzene will lead to waste, while insufficient bromobenzene will result in incomplete reaction of 1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole, and the absence of bromobenzene will prevent the reaction from occurring. Excessive NaOBu-t will lead to waste, while insufficient NaOBu-t will result in incomplete reaction, and the absence of NaOBu-t will prevent the reaction from occurring. Insufficient Pd(OAc)2 will result in a slow reaction rate and long reaction time, while excessive Pd(OAc)2 will lead to waste and increased synthesis costs, and the absence of Pd(OAc)2 will prevent the reaction from occurring. Excessive Pd(t-Bu)3 will lead to waste and increased synthesis costs, while insufficient Pd(t-Bu)3 will lead to a slow reaction rate, and the absence of Pd(t-Bu)3 will prevent the reaction from occurring.

[0037] In some embodiments of the present invention, in step S1, the temperature during the reflux process is 120°C, the reflux duration is 12 h, and the reflux flow rate is 2-3 drops / second. It is worth noting that the volume of "each drop" can specifically refer to any one of 0.1 mL, 0.2 mL, 0.5 mL, 1 mL, or 1.5 mL, or a range of any two of these values, or any value within that range.

[0038] It is worth noting that if the reflux temperature is too high, the toluene will not condense sufficiently, the pressure in the closed system will increase, causing the nitrogen balloon to rupture, the system to be exposed to oxygen, and the catalyst to be oxidized by the air and become ineffective; if the reflux temperature is too low, the reaction rate will be reduced, the reaction time will be prolonged, or even no reaction will occur.

[0039] If the reflux time is too long, it will lead to the formation of byproducts and reduce the yield; if the reflux time is too short, it will lead to incomplete reaction and reduce the yield.

[0040] In some embodiments of the present invention, in step S1, the filtration operation uses rapid qualitative filter paper and vacuum filtration. The purpose is to filter and remove inorganic salts, facilitating subsequent processing of the reaction stock solution.

[0041] In some embodiments of the present invention, in step S1, the concentration process is vacuum rotary evaporation at a temperature of 60°C, the purpose of which is to quickly remove organic solvents.

[0042] In some embodiments of the present invention, the silica gel column used in step S1 has a mesh size of 200-300. It is understood that the higher the mesh size of the silica gel, the better the compound separation effect.

[0043] In some embodiments of the present invention, the low-temperature condition in step S2 is 0~4°C. It is worth noting that if the temperature is too high, it will lead to the formation of byproducts; if the temperature is too low, it will result in a slow reaction rate and prolonged reaction time.

[0044] In some embodiments of the present invention, in step S2, the amount of N-bromosuccinimide is 0.84 parts by weight, and the amount of 4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole is 1 part by weight. It is worth noting that excessive NBS will result in multiple bromine residues, which are byproducts, while insufficient N-bromosuccinimide will lead to incomplete reaction of the reactants. Therefore, it is necessary to strictly adhere to this ratio.

[0045] In some embodiments of the present invention, in step S2, the mass ratio of 4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole to acetone in the acetone solution is 1:40. It should be noted that using too little acetone solvent will result in insoluble reactants and incomplete reaction, while using too much acetone solvent will lead to acetone waste and increased costs.

[0046] In some embodiments of the present invention, in step S2, when adding water to terminate the reaction, the amount of water added needs to satisfy the ratio of indoline to water of 1:3. It should be noted that if too little water is added, the reaction cannot be terminated; if too much water is added, it has no effect on the reaction.

[0047] In some embodiments of the present invention, in step S2, the evaporation temperature is 40°C and the evaporation time is 10 minutes when evaporating the obtained reactants. It is understood that excessively high temperatures during rotary evaporation can cause the solvent to boil violently, while excessively low temperatures can result in slow solvent evaporation. Insufficient rotary evaporation time can lead to solvent residue, while prolonged rotary evaporation time has no effect.

[0048] In some embodiments of the present invention, in step S2, when adding dichloromethane and water, the amount of dichloromethane added is 30 parts by weight, and the amount of water added is 30 parts by weight. It should be noted that if too little dichloromethane is added, the crude product after rotary evaporation will not dissolve, and the purpose of liquid-liquid extraction cannot be achieved. Adding more has no significant effect. Similarly, if too little water is added, residual inorganic salts in the crude product will not be removed; adding more water has no significant effect.

[0049] In some embodiments of the present invention, in step S2, when drying the organic phase with anhydrous sodium sulfate, the amount of anhydrous sodium sulfate used is 1 part by weight. It should be noted that adding too little will result in a small amount of water in the organic solvent, causing blockage during column chromatography. Adding a larger amount has no significant effect.

[0050] In some embodiments of the present invention, in step S2, during the evaporation to remove the solvent, the evaporation temperature is 30°C and the evaporation time is 5 minutes. It should be noted that excessively high temperatures during rotary evaporation can cause the solvent to boil violently, while excessively low temperatures can result in slow solvent evaporation. Insufficient evaporation time can lead to solvent residue, while prolonged evaporation time has no effect.

[0051] In some embodiments of the present invention, in step S3, the volume of the double-necked round-bottom flask equipped with a condenser is 100 mL. It is understood that organic synthesis reactions require the solvent volume to be 1 / 3 to 2 / 3 of the solvent volume in the reaction flask.

[0052] In some embodiments of the present invention, in step S3, the amount of 7-bromo-4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole is 1 part by weight, the amount of 5-formyl-2-thiopheneboronic acid is 1 part by weight, the amount of [1,1'-bis(triphenylphosphine)ferrocene]palladium dichloride is 0.3 parts by weight, and the amount of potassium carbonate is 2.2 parts by weight.

[0053] It should be noted that adding too little 5-formyl-2-thiopheneboronic acid will lead to incomplete reaction of 7-bromo-4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole; while adding too much will result in waste and increase the synthesis cost. Similarly, adding too little 1,1'-bis(triphenylphosphine)ferrocene]palladium dichloride will result in a slow reaction rate and prolonged reaction time, while adding too much will result in waste and increase the synthesis cost. Likewise, adding too little potassium carbonate will result in incomplete reaction, while adding too much will result in waste and increase the synthesis cost.

[0054] In some embodiments of the present invention, in step S3, the mass ratio of toluene to methanol in the mixed solvent is 1:1. This ratio allows for better dissolution of the reactants.

[0055] In some embodiments of the present invention, in step S3, the amount of dichloromethane added is 30 parts by weight. It is worth noting that adding too little will result in incomplete dissolution of the crude product.

[0056] In some embodiments of the present invention, in step S3, the silica gel column has a mesh size of 200-300. Silica gel columns within this range provide better separation performance.

[0057] In some embodiments of the present invention, in step S4, the amount of 5-[7-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indolyl)]thiophene-2-carboxaldehyde is 1 part by weight, the amount of 2-[4-(pyridin-4-yl)phenyl]acetonitrile is 1.2 parts by weight, the amount of anhydrous methanol is 30 parts by weight, and the amount of CH3ONa is 1 part by weight.

[0058] It should be noted that adding too little 2-[4-(pyridin-4-yl)phenyl]acetonitrile will lead to incomplete reaction of 5-[7-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indolyl)]thiophene-2-carboxaldehyde, while adding too much will result in waste of raw materials and increase synthesis costs. Adding too little anhydrous methanol will result in insufficient dissolution of reactants and incomplete reaction, while adding too much will result in solvent waste and increase synthesis costs. Adding more CH3ONa has no significant effect on the reaction, while adding too little will slow down the reaction rate and prolong the reaction time.

[0059] In some embodiments of the present invention, during step S4, the vacuum rotary evaporation process is carried out at a pressure of -90 kPa, a temperature of 40°C, a rotation speed of 55 rpm, and a processing time of 10 min. It is worth noting that if the vacuum pressure is too low, the rotary evaporation time will be prolonged; a high vacuum pressure has no effect on the experiment; a high temperature will cause the solvent to boil violently; a low temperature will prolong the rotary evaporation time; the rotation speed has no effect on the experiment; a processing time that is too short will result in incomplete solvent evaporation; and a processing time that is too long has no effect on the experiment.

[0060] In some embodiments of the present invention, in step S4, when DCM / MeOH is used as the eluent, the mass ratio of DCM to MeOH is 168:1, the amount of DCM is 1000 parts by weight, and the amount of MeOH is 5.95 parts by weight. Only within this range can the compound and impurities be rapidly separated. If the mass ratio of DCM / MeOH is greater than 168:1, the separation time will be long; if the mass ratio is less than 168:1, the compound and impurities will not be separated.

[0061] In some embodiments of the present invention, in step S4, the silica gel column has a mesh size of 200-300. Silica gel columns of this mesh size provide good chromatographic separation.

[0062] In some embodiments of the present invention, in step S5, the amount of 2-[4-(pyridin-4-yl)phenyl]-3-[5-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indol-7-yl)thiophen-2-yl]acrylonitrile is 1 part by weight, the amount of acetonitrile is 100 parts by weight, and the amount of iodoethane is 20 parts by weight. Acetonitrile, as a solvent, is wasted if used in large quantities, increasing synthesis costs; if used in small quantities, the reactants become insoluble, reducing the synthesis yield. Iodoethane, as a reactant, has no effect on the reaction if added in large quantities, but leads to incomplete reaction of the reactants if added in small quantities.

[0063] In some embodiments of the present invention, in step S5, the concentration of KPF6 in the saturated acetone solution containing KPF6 is 10 wt%. KPF6 is potassium hexafluorophosphate.

[0064] In some embodiments of the present invention, in step S5, the reaction temperature in the reaction vessel is 95°C, and the reaction time is 6 hours. It should be noted that if the reaction temperature is higher than 95°C, it has no significant effect on the reaction; if the reaction temperature is lower than 95°C, the reaction time should be extended. If the reaction time is too short, the reactants will not react completely, resulting in a reduced yield; if the reaction time is long, it has no effect on the reaction yield.

[0065] In some embodiments of the present invention, in step S5, the reflux treatment is carried out at a temperature of 66°C for 1 hour, with a flow rate of 2-3 drops / second. It is worth noting that the volume of "each drop" can specifically refer to any one of 0.1mL, 0.2mL, 0.5mL, 1mL, or 1.5mL, or a range of any two values, or any value within that range. It should be noted that if the reaction temperature is too low, anion exchange will be incomplete; if the reaction temperature is too high, it will have no effect on the reaction. Similarly, if the time is too short, anion exchange will be incomplete; if the time is too long, it will have no effect on the reaction. The flow rate reflects the reaction temperature; a too-fast flow rate indicates a too-high reaction temperature, and a too-slow flow rate indicates a too-low reaction temperature.

[0066] Third aspect The present invention provides the application of the photosensitizer of the first aspect above, or the photosensitizer obtained by the method of the second aspect above, in the field of fluorescence imaging and photodynamic therapy.

[0067] Example 1 This embodiment synthesizes the lysosomal targeted photosensitizer Lyso-PSs ICPy through the following steps: 1. Synthesis of intermediate 4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole (compound M2):

[0068] Compound M1, namely 1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole, bromobenzene, NaOBu-t, Pd(OAc)2, P(t-Bu)3, and anhydrous toluene were mixed and refluxed under nitrogen protection for 12 h at a reaction temperature of 120 °C. After cooling to room temperature, the reaction mixture was filtered and the filtrate was concentrated. The crude product was purified by silica gel column chromatography using petroleum ether as solvent, and a white liquid was finally obtained. The amount of compound M1, namely 1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole, was 1.50 g, equivalent to 9.42 mmol; the amount of bromobenzene was 1.48 g, equivalent to 9.42 mmol; the amount of NaOBu-t was 1.36 g, equivalent to 14.13 mmol; the amount of Pd(OAc)2 was 0.063 g, which was 3 mol% of M1; the amount of P(t-Bu)3 was 0.137 g, which was 5 mol% of M1; and the amount of anhydrous toluene was 75 mL. The yield of the above compound M2 was 75%.

[0069] 2. Synthesis of intermediate 7-bromo-4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole (compound M3):

[0070] In a 250 mL double-necked round-bottom flask, N-bromosuccinimide was added and reacted under nitrogen protection, in the dark, and at 0 °C. During the reaction, an acetone solution of 4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole (i.e., compound M2 mentioned above) was added. The reaction was carried out for 4 h, and the reaction progress was monitored by thin-layer chromatography. After the reaction was complete, 15 mL of water was added to terminate the reaction. The resulting reactants were evaporated. Then, dichloromethane and water were added, and the mixture was washed three times with DCM. The organic phase was collected and dried with anhydrous sodium sulfate. After evaporating the solvent, the crude product was purified by silica gel column chromatography using petroleum ether as the eluent to obtain a white liquid. The amount of N-bromosuccinimide used was 1.309 g; the amount of acetone solution of 4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole was 41.573 g, containing 1.573 g of 4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole and 40 g of acetone; in the above-mentioned evaporation treatment of the obtained reactants, the evaporation temperature was 40℃ and the evaporation time was 10 min; the amount of dichloromethane added was 30 g and the amount of water added was 30 g; the mass of the collected organic phase was 2.2 g, and 2 g of anhydrous sodium sulfate was used for the above-mentioned drying; the solvent was evaporated at 30℃ for 10 min to facilitate subsequent silica gel column chromatography; the mass of the white liquid obtained in this step was 1.99 g, and the yield was 95%.

[0071] 3. Synthesis of intermediate 5-[(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indol-7-yl)]thiophene-2-carboxaldehyde (compound M4):

[0072] In a 100 mL double-necked round-bottom flask equipped with a condenser, 7-bromo-4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole (i.e., the above-mentioned compound M3), 5-formyl-2-thiopheneboronic acid, [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride, and potassium carbonate were added. Under nitrogen protection, a mixed solvent of toluene and methanol was added as the reaction solvent. The reaction was refluxed at 75 °C for 16 h. After the reaction was completed, the above reaction solvent was removed by rotary evaporation under reduced pressure. Dichloromethane was then added as the organic phase, and the organic phase was washed successively with water and saturated sodium chloride aqueous solution. PE / DCM was used as the elution solvent, and the mixture was purified and separated by passing it through a 200-300 mesh silica gel column to obtain a deep yellow solid. The amount of 7-bromo-4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole (i.e., the above-mentioned compound M3) used was 1.000 g, equivalent to 3.18 mmol; the amount of 5-formyl-2-thiopheneboronic acid used was 0.645 g, equivalent to 4.13 mmol; the amount of [1,1'-bis(triphenylphosphine)ferrocene]palladium dichloride used was 0.3 g, equivalent to 0.41 mmol; the amount of potassium carbonate used was 2.20 g, equivalent to 15.9 mmol; in the above-mentioned mixed solvent of toluene and methanol, the volume ratio of toluene to methanol was 1:1, and the amount of the mixed solvent used was 40 mL; in the above-mentioned rotary evaporation under reduced pressure, the pressure was -90 °C. The pressure was 100 mL; the temperature was 40 °C; the rotation speed was 55 rpm; the amount of dichloromethane used was 100 mL; the Chinese name of PE is petroleum ether; the Chinese name of DCM is dichloromethane; the volume ratio of PE to DCM was 2:1; the mass of the resulting dark yellow solid was 0.824 g, and the yield was 75%.

[0073] The resolved data of the proton NMR spectrum of the above compound M4 are as follows: 1 H NMR (600 MHz, DMSO-d6) δ9.83 (s, 1H), 7.96 (d, J = 4.0 Hz, 1H), 7.59 (s, 1H), 7.55 (d, J = 4.0 Hz,1H), 7.48 (d, J = 8.4 Hz, 1H), 7.39 (t, J = 7.8 Hz, 2H), 7.33 (d, J = 7.4 Hz,2H), 7.04 (t, J = 7.2 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 4.94 (t, J = 6.6 Hz,1H), 3.87 (t, J = 7.5 Hz, 1H), 2.10-1.98 (m, 1H), 1.83 (ddd, J = 17.0, 13.0,6.4 Hz, 2H), 1.73 (dt, J = 13.1, 3.5 Hz, 1H), 1.63 (dtt, J = 12.5, 6.1, 3.2Hz, 1H), 1.41-1.34 (m, 1H).

[0074] 4. Synthesis of 2-[4-(pyridin-4-yl)phenyl]-3-[5-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indol-7-yl)thiophen-2-yl]acrylonitrile (compound M5):

[0075] 5-[7-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indolyl)]thiophene-2-carboxaldehyde, i.e., the above compound M4, and 2-[4-(pyridin-4-yl)phenyl]acetonitrile were mixed and dissolved in anhydrous methanol. CH3ONa was added, and the reaction was stirred at room temperature. After the reaction was completed, the reaction solvent was removed by rotary evaporation under reduced pressure. DCM / MeOH was used as the elution solvent, and the solution was purified and separated by silica gel column chromatography with a mesh size of 200-300 to obtain a red solid. The amount of 5-[7-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indolyl)]thiophene-2-carboxaldehyde, i.e., the above compound M4, was 0.200 g, equivalent to 0.579 mmol; the amount of 2-[4-(pyridin-4-yl)phenyl]acetonitrile was 0.146 g, equivalent to 0.753 mmol; the amount of anhydrous methanol was 30 mL; the amount of CH3ONa was 60 mg; in the above-mentioned room temperature stirring reaction, the stirring speed was 300 rpm, and the reaction time was 24 h; in the above-mentioned reduced pressure rotary evaporation, the pressure was -90 kPa, the temperature was 40 °C, and the time was 10 min; the Chinese name of DCM is dichloromethane, and the Chinese name of MeOH is methanol; the mass ratio of DCM to MeOH was 168:1; the mass of the red fixative obtained above was 0.271 g, and the yield was 90%.

[0076] The resolved data of the proton NMR spectrum of the above compound M5 are as follows: 1H NMR (600 MHz, Chloroform-d)δ 8.71 (dd, J = 15.5, 4.8 Hz, 2H), 7.77 (d, J = 6.7 Hz, 2H), 7.72 (d, J = 8.5Hz, 1H), 7.65 (d, J = 8.3 Hz, 1H), 7.62 (d, J = 5.9 Hz, 1H), 7.59 (d, J = 4.8Hz, 1H), 7.56 (d, J = 4.0 Hz, 1H), 7.46 (s, 1H), 7.41 (dd, J = 8.3, 2.0 Hz,1H), 7.40-7.35 (m, 1H), 7.35-7.27 (m, 2H), 7.25-7.21 (m, 1H), 7.19-7.15 (m,1H), 7.11 (dd, J = 8.4, 2.0 Hz, 1H), 7.03 (q, J = 4.0, 3.5 Hz, 1H), 4.91-4.77(m, 1H), 3.91-3.75 (m, 1H), 1.98-1.86 (m, 2H), 1.85-1.76 (m, 2H), 1.61-1.39 (m, 2H).

[0077] 5. Synthesis of compound Lyso-PSs ICPy:

[0078] Under argon protection, the above-mentioned 2-[4-(pyridin-4-yl)phenyl]-3-[5-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indol-7-yl)thiophen-2-yl]acrylonitrile (compound M5) and acetonitrile were added to the reaction vessel, and iodoethane was added to the reaction vessel via a syringe; the reaction was refluxed; after the reaction was completed, a saturated acetone solution of KPF6 was added to displace the ions; then water was added to terminate the reaction, and the product was extracted with CH2Cl2; the organic solvent was removed under vacuum, and the crude product was purified on a silica gel column using a mixture of dichloromethane and methanol; The amount of 2-[4-(pyridin-4-yl)phenyl]-3-[5-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indol-7-yl)thiophen-2-yl]acrylonitrile, i.e., compound M5, was 0.15 g, equivalent to 0.283 mmol; the amount of acetonitrile was 15 mL; the amount of iodoethane was 3 mL; the reflux reaction was carried out at 95 °C for 6 h; and the saturated acetone of KPF6 was used. In the solution, the content of KPF6 is 10wt% and the content of acetone is 50mL; in the above-mentioned water addition to terminate the reaction, the amount of water added is 20mL; in the above-mentioned extraction of the product with CH2Cl2, the amount of CH2Cl2 used is 30mL; in the above-mentioned vacuum conditions, the vacuum degree is -90kPa, the temperature is 40℃, and the treatment time is 10min; in the above-mentioned mixture of dichloromethane and methanol, the volume ratio of dichloromethane to methanol is 100:1.

[0079] The resolved data of the proton NMR spectrum of the above compound Lyso-PSs ICPy are as follows: 1H NMR (600 MHz, DMSO-d6) δ 9.19-9.10 (m, 2H), 8.65-8.56 (m, 2H), 8.48 (s, 1H), 8.29-8.20 (m,2H), 7.96 (d, J = 8.6 Hz, 2H), 7.82-7.75 (m, 1H), 7.63-7.52 (m, 2H), 7.47(dt, J = 8.4, 2.8 Hz, 1H), 7.42-7.37 (m, 2H), 7.36-7.31 (m, 2H), 7.10-7.02(m, 1H), 6.98 (d, J = 8.4 Hz, 1H), 4.96 (ddd, J = 8.8, 6.4, 2.2 Hz, 1H), 4.62(q, J = 7.2 Hz, 2H), 3.90 (t, J = 8.8 Hz, 1H), 2.09 (dddd, J = 12.5, 10.4,9.0, 6.5 Hz, 1H), 1.85 (ddt, J = 13.2, 10.6, 6.6 Hz, 2H), 1.65 (dh, J = 9.4, 3.0 Hz, 1H), 1.57 (t, J = 7.3 Hz, 3H), 1.44-1.32 (m, 1H), 1.27-1.21 (m, 1H), 1.17 (t, J = 7.1 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 153.33, 151.08,147.86, 144.45, 141.58, 138.13, 137.27, 137.18, 136.09, 134.18, 132.76,129.32, 128.83, 125.98, 125.91, 124.16, 123.08, 122.33, 122.13, 122.09,119.60, 118.02, 107.48, 102.34, 68.35, 55.46, 44.36, 34.77, 33.04, 23.95, 16.20; High-resolution mass spectrometry C 37 H 32 N3S + The calculated value is C 37 H 32 N3S + : 550.2312, experimental value is 550.2318.

[0080] Example 2 The compound Lyso-PSs ICPy obtained in Example 1 was dissolved in DMSO solution to prepare a 1 mM fluorescent dye stock solution, and test solutions of different concentrations were prepared as needed to detect changes in its fluorescence spectrum. Specifically, the singlet oxygen generation capacity, hydroxyl radical generation capacity, intracellular lysosomal fluorescence imaging, and in vitro photodynamic therapy were examined.

[0081] Example 3 The UV absorption and fluorescence emission spectra of the Lyso-PSs ICPy obtained in Example 1 were tested in acetonitrile, chloroform, ethanol, dimethyl sulfoxide, and water, respectively. Each time, 30 μL of the fluorescent dye stock solution obtained in Example 2 was taken and added to 2970 μL of chloroform, tetrahydrofuran, methanol, 1,4-dioxane, and toluene to prepare a 10 μM fluorescent dye test solution, and the ultraviolet absorption and fluorescence emission spectra were tested.

[0082] See Figure 4 The maximum absorption wavelength of Lyso-PSs ICPy in different solvents is around 500-550 nm, while the maximum emission wavelength is between 650-750 nm. Figure 5 As shown, it has a large Stokes shift and near-infrared emission, which can avoid interference from self-absorption and biological autofluorescence, giving it a significant advantage in bioimaging.

[0083] Example 4 Referring to Figures 6(a) and 6(b), the fluorescence intensity of Lyso-PSs ICPy obtained in Example 1 above in DMSO / toluene mixed solutions with different toluene volume fractions is shown as the curves in the figures, where the concentration of Lyso-PSs ICPy is 10 μM.

[0084] Referring to Figures 6(a) and 6(b), DMSO is a good solvent for Lyso-PSs ICPy, while toluene is a poor solvent for Lyso-PSs ICPy; as the volume fraction of the poor solvent in the mixed solution increases, the fluorescence intensity gradually increases, indicating that Lyso-PSs ICPy has obvious aggregation-induced emission characteristics.

[0085] Example 5 A test solution was prepared by mixing 2970 μL of PBS buffer, 15 μL of 9,10-anthratridiyl-bis(methylene)dicarboxylic acid (ABDA, 10 mM, in dimethyl sulfoxide), and 15 μL of the photosensitizer obtained in Example 1 above (1 mM, in dimethyl sulfoxide) or the commercially available photosensitizers Rose Bengal (RB) or Chlorin e6 (Ce6) (1 mM, dissolved in dimethyl sulfoxide). The final concentration of ABDA was 50 μM, and the final concentration of the photosensitizer was 5 μM. The solution was prepared at 15 mw / cm². 2 Under white light irradiation, the absorption signal of the indicator was monitored. The decrease in the absorption signal at 378 nm indicated that... 1 The rate of O2 generation.

[0086] Example 6 2940 μL of PBS buffer, 30 μL of hydroxyphenylfluorescein (HPF) (1 mM, dimethyl sulfoxide) and 30 μL of the photosensitizer obtained in Example 1 above (1 mM, dimethyl sulfoxide) or commercial photosensitizer RB (1 mM, dimethyl sulfoxide) were mixed to make a final solution with a concentration of 10 μL of HPF and 10 μL of photosensitizer.

[0087] The mixed solution was subjected to 15 mW / cm 2 After white light irradiation, the generation rate of ·OH (hydroxyl radical) was characterized by monitoring the fluorescence signal of HPF (excitation wavelength 488 nm, emission wavelength 514 nm).

[0088] With increasing illumination time, the fluorescence signals of each group gradually increased, indicating that all groups possessed the ability to generate hydroxyl radicals. The fluorescence signal enhancement rate of Lyso-PSs ICPy was significantly stronger than that of commercial photosensitizers Rose Bengal and Chlorin e6. This indicates that Lyso-PSs ICPy is a photosensitizer primarily generating type I reactive oxygen species, enabling highly efficient photodynamic therapy under hypoxic conditions.

[0089] Example 7 Subcellular organelle colocalization experiment: 4T1 cells were seeded on confocal culture dishes and cultured overnight in a CO2 incubator (37°C, 5wt% CO2). Lyso-PSs ICPy (2 μM) obtained in Example 1 was added to the 4T1 cells and incubated for 2 hours. The cells were then washed three times with PBS and incubated for 30 minutes with different commercially available dyes, namely Lyso-Tracker Green (1 μM) or Mito-Tracker Green (200 nM) and Hoechst 33342 (10 μg / ml).

[0090] Cells were then imaged using a confocal laser scanning microscope (CLSM): the excitation wavelength of Lyso-PSs ICPy obtained in Example 1 was 490 nm, and the emission wavelength was 650-850 nm; the excitation wavelength of the mitochondrial green fluorescent probe was 488 nm, and the emission wavelength was 493-515 nm; the excitation wavelength of the lysosomal green fluorescent probe was 488 nm, and the emission wavelength was 493-515 nm; and the excitation wavelength of Hoechst 33342 was 405 nm, and the emission wavelength was 415-460 nm.

[0091] Colocalization analysis showed that the Lyso-PSs ICPy obtained in Example 1 had a high Pearson correlation coefficient with the lysosomal green fluorescent probe, indicating that the Lyso-PSs ICPy obtained in Example 1 has a significant lysosomal targeting effect.

[0092] Example 8 In vitro photodynamic therapy test: The viability of 4T1 cells was assessed using the MTT assay with thiazolyl blue (MTBD); cells were injected at a rate of 5 × 10⁶ cells / mL. 4 Cells were seeded at a density of [number] cells per well and cultured in 96-well plates for 24 h. Then, the cells were co-incubated with different concentrations of Lyso-PSs ICPy obtained in Example 1 above for 22 h, followed by replacement of the IDL-CPy solution with fresh DMEM (Dalberg modified Eagle medium). The cells were then cultured under white light (30 mw / cm²). 2After irradiation for 10 minutes, cells were cultured for another 12 hours. 10 μL of MTT solution (5 mg / mL, PBS) was added to each well, and the cells were cultured for another 4 hours. After removing the supernatant, 100 μL of DMSO (dimethyl sulfoxide) was added to each well to dissolve the crystals. Cell viability was assessed by recording the MTT absorbance at 550 nm using a microplate reader. Cells incubated only with culture medium in each group were designated as having 100% viability. The MTT solution mentioned above refers to a mixture of 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide and PBS, where 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide was the solute at a concentration of 5 mg / mL, and PBS was a phosphate buffer solution. Another Chinese name for MTT is "thiazolium blue".

[0093] The survival rate of 4T1 cancer cells showed a clear dose-dependent relationship with the concentration of Lyso-PSs ICPy photosensitizer obtained in Example 1: as the concentration of photosensitizer increased, the survival rate of cancer cells decreased significantly.

[0094] Compared with commercial photosensitizers Rose Bengal (RB) and Chlorin e6 (Ce6), the Lyso-PSs ICPy provided in Example 1 of this invention exhibits superior photodynamic killing effect on cancer cells. Under the same test conditions, at a photosensitizer concentration of 1 μM, the cancer cell survival rate of the RB and Ce6 groups was higher than 85%, while the cancer cell survival rate of Lyso-PSs ICPy was less than 1%. Therefore, the compound Lyso-PSs ICPy demonstrates excellent photodynamic properties.

[0095] Figure 10 The fluorescence signal retention values ​​of Lyso-PSs ICPy and commercial lysosomes provided in Example 1 of this invention under continuous fluorescence imaging; the commercial lysosomes were purchased through commercial channels and are branded as "Adamas Life", packaged in 50 μL, with an original number of C8328-50uL, and its full trade name is "Lyso-Detector Lysosome Green Fluorescent Probe".

[0096] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims. Furthermore, specific examples have been used in the specification to illustrate the principles and implementation methods of the present invention. The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention, and the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A type I photosensitizer that targets lysosome aggregation-induced luminescence, characterized in that, The structural formula of the photosensitizer is shown in Formula I: in, In Equation I, R1 can be any one of R11, R12, and R13; R11 is -(CH2)nCH3, where n = any value from 0, 1, ..., 12; R12 is -(CH2)nCH2Br, where n = any value from 0, 1, ..., 12; The structural formula of R13 is shown in Formula II, which is: In Equation II, n = any value among 0, 1, ..., 12; R2 in Formula I is selected from at least one of substituted or unsubstituted C1-C2 alkyl, substituted or unsubstituted C1-C2 alkoxy, nitro, haloalkyl, and formyl.

2. The photosensitizer according to claim 1, characterized in that, The R2 is any one of -CH3, -CH2CH3, -OCH3, -NO2, -CF3, and -CHO.

3. A method for preparing a photosensitizer as described in any one of claims 1 or 2, characterized in that, The method includes the following steps: S1, 1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole, bromobenzene, NaOBu-t, Pd(OAc)2, P(t-Bu)3, and anhydrous toluene were mixed and refluxed under nitrogen protection, filtered, concentrated, and the crude product was purified by silica gel column chromatography. S2, under nitrogen protection, darkness, and low temperature conditions, an acetone solution of N-bromosuccinimide and 4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole was added to a double-necked round-bottom flask to carry out the reaction. After the reaction was completed, water was added to terminate the reaction, the resulting reactants were evaporated, and then dichloromethane and water were added and washed with dichloromethane. The organic phase was collected, dried with anhydrous sodium sulfate, and then the solvent was evaporated to remove the solvent. The crude product was purified by silica gel column chromatography using petroleum ether as the eluent. S3. In a double-necked round-bottom flask equipped with a condenser, 7-bromo-4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole, 5-formyl-2-thiopheneboronic acid, [1,1'-bis(triphenylphosphine)ferrocene]palladium dichloride and potassium carbonate were added. Under nitrogen protection, a mixed solvent of toluene and methanol was added as the reaction solvent, and dichloromethane was added as the organic phase. The dichloromethane was washed successively with water and a saturated sodium chloride aqueous solution. Then, the solution was purified and separated using a silica gel column. S4, 5-[7-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indolyl)]thiophene-2-carboxaldehyde and 2-[4-(pyridin-4-yl)phenyl]acetonitrile were mixed and dissolved in anhydrous methanol, CH3ONa was added, the reaction was stirred and then the solvent was removed by rotary evaporation under reduced pressure. DCM / MeOH was used as the elution solvent, and the purification and separation were performed by silica gel column chromatography. S5, under nitrogen protection, 2-[4-(pyridin-4-yl)phenyl]-3-[5-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indol-7-yl)thiophen-2-yl]acrylonitrile and acetonitrile were added to the reaction vessel, and iodoethane was added to the reaction vessel using a syringe. After reflux, a saturated acetone solution containing KPF6 was added to displace the ions, and then water was added to terminate the reaction. The reaction product was extracted with dichloromethane, and the organic solvent was removed under vacuum. The product was purified and separated using a silica gel column.

4. The method according to claim 3, characterized in that, Step S1 has at least one of the following characteristics: The amount of 1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole is 1 part by weight, the amount of bromobenzene is 1.3 parts by weight, the amount of NaOBu-t is 0.85 parts by weight, the amount of Pd(OAc)2 is 0.03 parts by weight, the amount of P(t-Bu)3 is 0.04 parts by weight, and the amount of anhydrous toluene is 20 parts by weight. The temperature during the reflux process was 120℃, the reflux duration was 12h, and the reflux flow rate was 2~3 drops / second; The filtration process uses rapid qualitative filter paper and vacuum filtration. Concentration was achieved using vacuum rotary evaporation at a temperature of 60°C; The silica gel column used has a mesh size of 200-300.

5. The method according to claim 3, characterized in that, Step S2 has at least one of the following characteristics: The low-temperature condition is 0~4℃; The amount of N-bromosuccinimide used is 0.84 parts by weight, and the amount of acetone solution of 4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole used is 1 part by weight. In the acetone solution of 4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole, the mass ratio of 4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole to acetone is 1:

25. When water is added to terminate the reaction, the amount of water added satisfies the ratio of indoline to water = 1:3; The evaporation temperature of the reactants obtained by evaporation is 40°C, and the evaporation time is 10 min. When adding dichloromethane and water, the amount of dichloromethane added is 30 parts by weight, and the amount of water added is 30 parts by weight. When drying the organic phase with anhydrous sodium sulfate, the amount of anhydrous sodium sulfate used is 1 part by weight; During the solvent removal process, the evaporation temperature is 30°C and the evaporation time is 5 minutes.

6. The method according to claim 3, characterized in that, Step S3 has at least one of the following characteristics: The double-necked round-bottom flask equipped with a condenser has a volume of 100 mL; The amount of 7-bromo-4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indole is 1 part by weight, the amount of 5-formyl-2-thiopheneboronic acid is 1 part by weight, the amount of [1,1'-bis(triphenylphosphine)ferrocene]palladium dichloride is 0.3 parts by weight, and the amount of potassium carbonate is 2.2 parts by weight; In the mixed solvent of toluene and methanol, the mass ratio of toluene to methanol is 1:1; The amount of dichloromethane added is 30 parts by weight; The silica gel column in step S3 has a mesh size of 200-300.

7. The method according to claim 3, characterized in that, Step S4 has at least one of the following characteristics: The amount of 5-[7-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indolyl)]thiophene-2-carboxaldehyde is 1 part by weight, the amount of 2-[4-(pyridin-4-yl)phenyl]acetonitrile is 1.2 parts by weight, the amount of anhydrous methanol is 30 parts by weight, and the amount of CH3ONa is 1 part by weight; During the vacuum rotary evaporation process, the gas pressure is -90 kPa, the temperature is 40℃, the rotation speed is 55 rpm, and the processing time is 10 min. When DCM / MeOH is used as the eluent, the mass ratio of DCM to MeOH is 168:1, the amount of DCM is 1000 parts by weight, and the amount of MeOH is 5.95 parts by weight. The silica gel column in step S4 has a mesh size of 200-300.

8. The method according to claim 3, characterized in that, Step S5 has at least one of the following features: The amount of 2-[4-(pyridin-4-yl)phenyl]-3-[5-(4-phenyl-1,2,3,3a,4,8b-hexahydrocyclopentano[b]indol-7-yl)thiophen-2-yl]acrylonitrile is 1 part by weight, the amount of acetonitrile is 100 parts by weight, and the amount of iodoethane is 20 parts by weight; The concentration of KPF6 in the saturated acetone solution containing KPF6 is 10 wt%.

9. The method according to claim 3, characterized in that, Step S5 has at least one of the following features: In the reaction vessel, the reaction temperature is 95°C and the reaction time is 6 hours. In the reflux treatment, the temperature is 66℃, the duration is 1 hour, and the flow rate is 2~3 drops / second.

10. The use of the photosensitizer as described in claim 1 or 2, or the photosensitizer obtained by the method of any one of claims 3 to 9, in the field of fluorescence imaging and photodynamic therapy.

Images