Cationic macrocyclic photosensitizer, its preparation method and application

Abstract

The present application relates to a kind of cation macrocycle photosensitizer and its preparation method and application;Through triphenylamine derivative and p-benzyl bromide synthesis four cation macrocycle 1 molecule, 1 can be used as photosensitizer, the molecule can be through I / II type light reaction path to produce active oxygen, and under the condition of oxygen deficiency, biological molecule (NADH, BH4) is damaged to a large extent.In addition, further through the acylhydrazone bond of pH response, the polyethylene glycol (PEG) chain is modified on the molecule, obtains polyethylene glycol modified four cation macrocycle 1-PEG, through macrocyclic cation cavity, load tumor drug, self-assembly forms supramolecular nanoparticle, realizes the combined treatment of photodynamic and drug, system has remote operation and non-invasive, with drug release and acylhydrazone bond break, fluorescence emission intensity also gradually enhances, can be effectively navigated by fluorescence imaging drug treatment to tumor, thus it has wide application prospect in cancer diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug technology, and in particular relates to a cationic macrocyclic photosensitizer, its preparation method and application. Background Technology

[0002] Photodynamic therapy (PDT) is a novel cancer treatment method that generates reactive oxygen species through photoexcitation of photosensitizers. Compared with treatments such as surgery, chemotherapy, and radiotherapy, it has advantages such as high treatment efficiency, no drug resistance, minimal invasiveness, and low systemic side effects, and has gradually become an alternative clinical treatment for some cancers. However, traditional small-molecule photosensitizers often suffer from poor water solubility, easy photobleaching, weak selective recognition, and poor therapeutic effects. Therefore, designing and constructing highly efficient photosensitizers that combine water solubility, photostability, fluorescence imaging capabilities, and tumor targeting has become a research hotspot in this field and has broad clinical application prospects.

[0003] Photosensitizers typically generate highly toxic reactive oxygen species (ROS) for photodynamic therapy through two different mechanisms (type I and type II PDT). The difference between these two PDT mechanisms lies in the fact that, in type I PDT, the photosensitizer transfers electrons to oxygen molecules to generate superoxide anion radicals (·O2). – In type II PDT, the photosensitizer transfers energy to oxygen molecules to form singlet oxygen. 1 O2). Currently, several photosensitizers have been approved and successfully used in clinical treatment, most of which are generated through type II PDT. 1 O2 is used for photodynamic therapy (PDT). However, in the hypoxic environment of tumors, type II photosensitizers consume oxygen molecules, and the therapeutic effect decreases with decreasing oxygen concentration, thus limiting the therapeutic efficacy of type II PDT. Meanwhile, in type I PDT, although oxygen mainly participates in electron transfer processes, it is both consumed and generated, and can be reused. However, the factors affecting type I PDT are very complex, including substrate concentration, photosensitizer redox potential, and the affinity between the photosensitizer and substrate. Therefore, developing photosensitizers with simple molecular structures and well-defined photodynamic mechanisms has significant scientific research value and practical application significance.

[0004] In recent years, supramolecular photodynamic therapy based on macrocyclic molecules has become a hot research topic. For example, strategies such as supramolecular assembly and macrocyclic confinement can be used to address the luminescence quenching problem caused by molecular aggregation, thereby enhancing its luminescence and photodynamic therapy capabilities. Furthermore, existing commercial photosensitizers can be modified and optimized through multiple non-covalent interactions to reduce phototoxicity and improve targeted therapeutic effects, endowing supramolecular assemblies with responsiveness to biological stimuli, ultimately enabling selective imaging of cancer cells and precise targeted tumor diagnosis and treatment. Despite significant progress, developing biocompatible and safe supramolecular photodynamic therapy systems remains a crucial challenge to be addressed in this field. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a cationic macrocyclic photosensitizer, its preparation method, and its application.

[0006] The technical solution adopted in this invention is: a cationic macrocyclic compound with the structure shown in Formula 1;

[0007]

[0008] Preferably, the compound shown in Formula 1 is modified with polyethylene glycol, and the structure is shown in Formula 2.

[0009]

[0010] A method for preparing cationic macrocyclic compounds involves reacting 4-bromo-N,N-di(4-(pyridin-4-yl)phenyl)amine, 4-formylphenylboronic acid, tetratriphenylphosphine palladium, and 1,4-dioxane to generate 4'-(di(4-(pyridin-4-yl)phenyl)amine)-[1,1'-biphenyl]-4-carboxaldehyde, which is then reacted with p-phenylbenzyl bromide to generate the tetracationic macrocyclic compound shown in Formula 1.

[0011] Preferably, the polyethylene glycol chain is further modified onto the cationic macrocycle via acylhydrazone bonds to obtain compound of formula 2.

[0012] Preferably, the specific steps include:

[0013] Step 1: 4'-(di(4-(pyridin-4-yl)phenyl)amine, 4-formylphenylboronic acid, tetratriphenylphosphine palladium, 1,4-dioxane and potassium carbonate were reacted to synthesize 4'-(di(4-(pyridin-4-yl)phenyl)amine)-[1,1'-biphenyl]-4-carboxaldehyde;

[0014] Step 2: Dissolve 4'-(bis(4-(pyridin-4-yl)phenyl)amine)-[1,1'-biphenyl]-4-carboxaldehyde and p-phenylbenzyl bromide in an organic solvent, redissolve the obtained solid substance in a mixture of methanol and water, and add ammonium hexafluorophosphate to the solution to prepare the tetracationic macrocycle 1 shown in Formula 1;

[0015] Step 3: Dissolve polyethylene glycol monomethyl ether, triethylamine, and p-toluenesulfonyl chloride in dichloromethane and react to obtain PEG. 5000 4-Methylbenzenesulfonate; PEG 5000 4-Methylbenzenesulfonate reacts with ethyl p-hydroxybenzoate to give PEG. 5000 4-ethyl benzoate; PEG 5000 Ethyl 4-benzoate was dissolved in ethanol, and hydrazine hydrate was added to react and give PEG. 5000 -4-benzoylhydrazide;

[0016] Step 4: Combine the PEG obtained in Step 2 with the PEG obtained in Step 3. 5000- -4-benzoylhydrazine dissolved in anhydrous methanol, with the addition of trifluoroacetic acid and Molecular sieves were used to react and obtain polyethylene glycol-modified tetracationic macrocyclic 1-PEG as shown in Formula 2.

[0017] Application of cationic macrocyclic compounds in photosensitizers.

[0018] A drug-loaded nanoparticle is obtained by loading a drug into the macrocyclic cation cavity of a tetracationic macrocyclic compound and then self-assembling it into supramolecular nanoparticles.

[0019] Preferably, the drug is an antitumor drug;

[0020] Preferably, the drug is pemetrexed (PEM).

[0021] Preferably, the tetracationic macrocyclic compound shown in Formula 2 is dissolved in dichloromethane, an aqueous drug solution is added, the mixture is ultrasonically mixed, the organic phase is removed, and the mixture is lyophilized to obtain drug-loaded nanoparticles.

[0022] The application of cationic macrocyclic compounds or drug-loaded nanoparticles as shown in Formula 1 or Formula 2 in fluorescent tumor tracer reagents and / or tumor therapeutic drugs.

[0023] Preferably, in the microacidic environment of a tumor, the compound shown in Formula 2 or the drug-loaded nanoparticles release macrocyclic molecules after the acylhydrazone bond is broken, resulting in enhanced fluorescence, thereby achieving tumor tracing.

[0024] Alternatively, when the compound shown in Formula 2 or the drug-loaded nanoparticles are exposed to light, they generate one or more of ROS, singlet oxygen, and superoxide anion free radicals, which disrupt the redox balance in tumor cells, causing tumor cell damage and death.

[0025] The advantages and positive effects of this invention are: it provides a polyethylene glycol-modified tetracationic macrocyclic compound that can be used as a photosensitizer, exhibiting good luminescent properties and suitable for cell imaging and in vivo imaging; when irradiated with light greater than 420 nm, it can efficiently generate singlet oxygen and superoxide anion free radicals, oxidizing intracellular biomolecules, disrupting the intracellular redox balance, and causing cell damage and death; the polyethylene glycol-modified tetracationic macrocyclic compound has the potential for application in two-photon imaging and photodynamic therapy;

[0026] 1. It has a large cation cavity, which has the potential to complex with negatively charged drugs and can be used for drug delivery to achieve the purpose of photodynamic drug combination therapy; after being combined with anti-tumor drugs, the self-assembled nanoparticles have water solubility, photostability, good luminescence properties and imaging capabilities; the nanoparticles have high anti-tumor ability, are non-invasive and have low side effects, and can guide the treatment process through fluorescence in vivo imaging function. Attached Figure Description

[0027] Figure 1 Synthetic route of tetracationic macrocycle (1);

[0028] Figure 2 Characterization of 4'-(bis(4-(pyridin-4-yl)phenyl)amine)-[1,1'-biphenyl]-4-carboxaldehyde compounds; (a) 1 (a) HNMR spectrum (400MHz, DMSO-d6); (b) 13 C10 NMR spectrum (100MHz, DMSO-d6);

[0029] Figure 3 Characterization of compound 1; (a) 1 (a) 1H NMR spectrum (400MHz, CD3CN); (b) 13 (c) CNMR spectrum (100MHz, CD3CN); HR-mass spectrum;

[0030] Figure 4 Compound PEG 5000 4-Methylbenzenesulfonate 1 Characterized by 1H NMR (400MHz, DMSO-d6);

[0031] Figure 5 Compound PEG 5000 ethyl 4-benzoate 1 Characterized by 1H NMR (400MHz, DMSO-d6);

[0032] Figure 6 Compound PEG 5000 -4-benzoylhydrazide1 Characterized by 1H NMR (400MHz, DMSO-d6);

[0033] Figure 7 Compound 1-PEG 1 Characterized by 1H NMR (400MHz, DMSO-d6);

[0034] Figure 8 Characterization of particle size and surface potential of compound 1-PEG; (a) DLS and TEM characterization of 1-PEG; (b) Zeta potential characterization of 1-PEG;

[0035] Figure 9 The degree of acylhydrazone bond cleavage in 1-PEG molecules under different pH buffer solutions; (a) fluorescence emission spectra of 1-PEG in different pH buffer solutions; (b) release curves of 1-PEG at different pH over time;

[0036] Figure 10 Characterization of the inclusion interaction between compound 1 and PEM; (a) Job plot of 1 and PEM; (b) Fluorescence emission plot of PEM titrated with 1; (c) Nonlinear least squares analysis results calculated based on the difference in fluorescence emission intensity at 560 nm.

[0037] Figure 11 Characterization of the particle size and surface potential of the PEM@1-PEG composite; (a) DLS and TEM characterization of PEM@1-PEG; (b) Zeta potential characterization of PEM@1-PEG.

[0038] Figure 12 The ultraviolet absorption and fluorescence emission spectra of compound 1;

[0039] Figure 13 Fluorescence emission spectra of compounds 1, 1-PEG, and PEM@1-PEG;

[0040] Figure 14 Photoluminescence spectra of 1 in different solvents; (a) 1 in a mixed solvent of DMF and H2O; (b) 1 in a mixed solvent of DMSO and H2O;

[0041] Figure 15 The photostability of 1 molecule was characterized and compared with commercial photosensitizers; (a) UV-Vis absorption spectra of 1 solution under light irradiation at different times; (b) UV-Vis absorption spectra of RB solution under light irradiation at different times.

[0042] Figure 16Characterization of ROS generation of 1,1-PEG or PEM@1-PEG molecules using DCFH fluorescent probes; (a), (b), and (c) are fluorescence emission diagrams of DCFH fluorescent probes after different illumination times in the presence of 1,1-PEG or PEM@1-PEG; (d), (e), and (f) are fluorescence emission intensity differences of DCFH fluorescent probes after different illumination times in the presence of 1,1-PEG or PEM@1-PEG.

[0043] Figure 17 Characterization of ROS generation by DHR123 fluorescent probe on 1,1-PEG or PEM@1-PEG molecules; (a), (b), and (c) are fluorescence emission diagrams of DHR123 fluorescent probe after different illumination times in the presence of 1,1-PEG or PEM@1-PEG; (d), (e), and (f) are fluorescence emission intensity differences of DHR123 fluorescent probe after different illumination times in the presence of 1,1-PEG or PEM@1-PEG.

[0044] Figure 18 Characterization of superoxide anion radical generation by DHE fluorescent probes in 1,1-PEG or PEM@1-PEG molecules; (a), (b), and (c) are fluorescence emission diagrams of DHE fluorescent probes after different illumination times in the presence of 1,1-PEG or PEM@1-PEG; (d), (e), and (f) are fluorescence emission intensity differences of DHE fluorescent probes after different illumination times in the presence of 1,1-PEG or PEM@1-PEG.

[0045] Figure 19 Characterization of singlet oxygen generation in 1,1-PEG or PEM@1-PEG molecules using ABDA UV probes; (a) UV absorption spectra of ABDA UV probe mixed with 1 after different irradiation times, (b) UV absorption spectra of ABDA UV probe mixed with 1-PEG after different irradiation times, (c) UV absorption spectra of ABDA UV probe mixed with PEM@1-PEG after different irradiation times.

[0046] Figure 20Characterization of NADH oxidation by 1,1-PEG or PEM@1-PEG molecules under illumination; (a) UV absorption spectra of NADH in the presence of 1-PEG under normoxic conditions after different illumination times; (b) UV absorption spectra of NADH in the presence of 1-PEG under hypoxic conditions after different illumination times; (c) UV absorption intensity differences of NADH in the presence of 1-PEG under different illumination times; (d) UV absorption spectra of NADH in the presence of 1-PEG under normoxic conditions after different illumination times; (e) UV absorption spectra of NADH in the presence of 1-PEG under hypoxic conditions after different illumination times. (f) Ultraviolet absorption spectra of NADH in the presence of 1-PEG after different light exposure times; (g) Ultraviolet absorption spectra of NADH in the presence of PEM@1-PEG under normal oxygen conditions after different light exposure times; (h) Ultraviolet absorption spectra of NADH in the presence of PEM@1-PEG under hypoxia conditions after different light exposure times; (i) Ultraviolet absorption spectra of NADH in the presence of PEM@1-PEG after different light exposure times.

[0047] Figure 21 Characterization of BH4 oxidation by 1,1-PEG or PEM@1-PEG molecules under illumination; (a) UV absorption spectra of BH4 in the presence of 1-PEG under normal oxygen conditions after different illumination times; (b) UV absorption spectra of BH4 in the presence of 1-PEG under hypoxia conditions after different illumination times; (c) UV absorption intensity differences of BH4 in the presence of 1-PEG under different illumination times; (d) UV absorption spectra of BH4 in the presence of 1-PEG under normal oxygen conditions after different illumination times; (e) UV absorption spectra of BH4 in the presence of 1-PEG under hypoxia conditions after different illumination times. (f) shows the UV absorption spectra of BH4 under different illumination times in the presence of PEG; (g) shows the UV absorption intensity difference of BH4 under different illumination times in the presence of 1-PEG under normal oxygen conditions; (h) shows the UV absorption spectra of BH4 under different illumination times in the presence of PEM@1-PEG under hypoxic conditions; (i) shows the UV absorption intensity difference of BH4 under different illumination times in the presence of PEM@1-PEG.

[0048] Figure 22 Cell viability characterization after incubation with 1, 1-PEG or PEM@1-PEG; (a) Cell viability of A549 cells incubated with different concentrations of 1; (b) Cell viability of A549 cells after light irradiation following incubation with different concentrations of 1; (c) Cell viability of A549 cells under different treatments of 1-PEG+hv, PEM, PEM@1-PEG, and PEM@1-PEG+hv.

[0049] Figure 23After co-incubating cells with 1,1-PEG or PEM@1-PEG and commercial organelle localizers, confocal imaging was used to characterize the cells at a scale bar of 20 μm.

[0050] Figure 24 1. The generation of reactive oxygen species and superoxide anion free radicals in cells, scale bar 20 μm;

[0051] Figure 25 The generation of reactive oxygen species and superoxide anion free radicals by 1-PEG in cells, scale bar at 20 μm;

[0052] Figure 26 Results of changes in body weight and tumor volume in mice after different sample treatments; (a) In vivo fluorescence imaging of mice; (b) Changes in body weight of mice in different treatment groups over time; (c) Changes in tumor volume of mice in different treatment groups over time; (d) Representative tumor images of each treatment group at the end of the experiment.

[0053] Figure 27 The results of H&E staining of heart, liver, spleen, lung, kidney and tumor tissues of mice after different sample treatments.

[0054] Figure 28 It has a 1 crystal structure. Detailed Implementation

[0055] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0056] This invention relates to a cationic macrocyclic photosensitizer, its preparation method, and its applications. First, a tetracationic macrocyclic (1) molecule is synthesized from a triphenylamine derivative and p-phenylbenzyl bromide. 1 exhibits good water solubility, photostability, good luminescence properties, and imaging capabilities. Further, a polyethylene glycol (PEG) chain can be modified onto this molecule via acylhydrazone bonds to impart pH responsiveness to the system, yielding a polyethylene glycol-modified tetracationic macrocyclic (1-PEG). Both 1 and 1-PEG can be used as photosensitizers.

[0057] The preparation method of tetracationic macrocyclic (1) is as follows:

[0058] Step 1: 4'-(di(4-(pyridin-4-yl)phenyl)amine, 4-formylphenylboronic acid, tetratriphenylphosphine palladium, 1,4-dioxane and potassium carbonate were reacted to synthesize 4'-(di(4-(pyridin-4-yl)phenyl)amine)-[1,1'-biphenyl]-4-carboxaldehyde;

[0059] Step 2: Dissolve 4'-(di(4-(pyridin-4-yl)phenyl)amine)-[1,1'-biphenyl]-4-carboxaldehyde and p-benzyl bromide in an organic solvent, redissolve the obtained solid substance in a mixture of methanol and water, and add ammonium hexafluorophosphate to the solution to prepare a tetracationic macrocycle (1).

[0060] The preparation of polyethylene glycol-modified tetracationic macrocyclic 1-PEG based on step 1 includes the following steps:

[0061] Step 3: Dissolve polyethylene glycol monomethyl ether, triethylamine, and p-toluenesulfonyl chloride in dichloromethane and react to obtain PEG. 5000 4-Methylbenzenesulfonate; PEG 5000 4-Methylbenzenesulfonate reacts with ethyl p-hydroxybenzoate to give PEG. 5000 4-ethyl benzoate; PEG 5000 Ethyl 4-benzoate was dissolved in ethanol, and hydrazine hydrate was added to react and give PEG. 5000 -4-benzoylhydrazide;

[0062] Step 4: Combine the PEG obtained in Step 2 with the PEG obtained in Step 3. 5000- -4-benzoylhydrazine dissolved in anhydrous methanol, with the addition of trifluoroacetic acid and Molecular sieves were used to react and obtain polyethylene glycol-modified tetracationic macrocyclic 1-PEG.

[0063] The preparation process of 1- and 1-PEG and their modes of action in cells are as follows: Figure 1 As shown, both the prepared 1-PEG and 1-PEG exhibit luminescent properties and can be used for fluorescence imaging. Due to the influence of the modified PEG chain, 1-PEG and 1-PEG show differences in fluorescence intensity. After 1-PEG enters the cell, the low pH environment of tumor cells promotes the cleavage of acylhydrazone bonds, leading to the release of the 1-cyclic molecule and inducing fluorescence enhancement. Therefore, based on the EPR effect, 1-PEG can be passively targeted and spontaneously transported to the tumor site; then, the release of 1-PEG through acylhydrazone bond cleavage achieves a change in fluorescence intensity. Thus, 1-PEG can be used to trace the therapeutic process of tumors.

[0064] Upon exposure to light greater than 420 nm, xenon lamps efficiently generate singlet oxygen and superoxide anion free radicals, which can oxidize intracellular biomolecules, disrupt the intracellular redox balance, and cause cell damage and death. Macrocyclic molecules initially target the mitochondria within the cell; as photodynamic therapy progresses, the macrocyclic molecules translocate from the mitochondria to the cell nucleus, and the cell gradually begins apoptosis. Therefore, irradiation with xenon lamps greater than 420 nm can achieve highly efficient photodynamic therapy.

[0065] 1. Possessing a large cation cavity, it has the potential to complex with negatively charged drugs, thus serving as a drug carrier for drug delivery. Anticancer drugs are loaded into the macrocyclic cation cavity for drug delivery to tumor cells (tissues). In some embodiments of this invention, the anticancer drug may be pemetrexed (PEM). 1 or 1-PEG is mixed with pemetrexed at a molar ratio of 1:1 to form a complex, enabling photodynamic therapy combined with drug delivery. 1-PEG and PEM are mixed and incubated, self-assembling to form nanoparticles with a diameter of approximately 250 nm, giving them permeability and retention effects in vivo, making them more easily accumulated in tumor tissue sites. The cells contain polyglutamate synthase, which competes with the macrocyclic molecules for pemetrexed, ensuring sufficient drug release. This allows for simultaneous photodynamic therapy and chemotherapy. The assembly is irradiated externally using a light source greater than 420 nm, making it non-invasive and reducing harm to the organism.

[0066] These supramolecular nanoparticles exhibit highly efficient tumor treatment effects. Through combined therapy, they achieve highly effective cancer treatment. The system also features remote operability and non-invasiveness. As the drug is released and the acylhydrazone bond breaks, the fluorescence emission intensity gradually increases. Fluorescence imaging can effectively guide the drug to treat the tumor, thus showing broad application prospects in cancer diagnosis and treatment.

[0067] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0068] Example 1: Preparation of a tetracationic macrocycle (1)

[0069]

[0070] 1.1 Synthesis of 4'-(bis(4-(pyridin-4-yl)phenyl)amine)-[1,1'-biphenyl]-4-carboxaldehyde

[0071] The following solutions were synthesized according to literature: 4-bromo-N,N-bis(4-(pyridin-4-yl)phenyl)amine, 4-formylphenylboronic acid (1.2 g, 2.51 mmol), tetrakis(triphenylphosphine)palladium (0.5 g, 0.43 mmol), 1,4-dioxane (60 mL), and potassium carbonate solution (0.5 M, 25 mL). The mixture was stirred and refluxed for 24 h under an inert atmosphere. After the reaction was complete, the reactants were cooled to room temperature, extracted with dichloromethane, and dried over anhydrous sodium sulfate. The target compound b (90% yield) was purified by silica gel column chromatography (dichloromethane / methanol). Characterization results are as follows: Figure 2 As shown.

[0072] 1.2 Synthesis of Tetracationic Macrocycle (1)

[0073] 4'-(bis(4-(pyridin-4-yl)phenyl)amine)-[1,1'-biphenyl]-4-carboxaldehyde (100 mg, 0.2 mmol) and p-phenylbenzyl bromide (54.2 mg, 0.4 mmol) were dissolved in acetonitrile (100 mL) and heated under reflux for 4 days under inert gas protection. After the reaction was completed and cooled to room temperature, the mixture was filtered, and the residue was washed with acetonitrile. The collected solid was dissolved in a mixture of methanol and water, and ammonium hexafluorophosphate was added to the solution. The precipitate was collected by filtration and purified by silica gel column chromatography (acetonitrile / dichloro) to give target compound 1 (yield: 10%). Characterization results are as follows: Figure 3 As shown.

[0074] The crystal structure of compound 1 was studied by evaporating diethyl ether into a 1% acetonitrile solution to obtain the crystal structure of one molecule, as shown below. Figure 28 As shown.

[0075] Example 2: Preparation of polyethylene glycol-modified tetracationic macrocyclic (1-PEG)

[0076] 2.1 PEG 5000 Preparation of 4-methylbenzenesulfonate

[0077]

[0078] Polyethylene glycol monomethyl ether (5g, 1mmol, M) n =5000), triethylamine (1 mL), and p-toluenesulfonyl chloride (0.95 g, 5 mmol) were dissolved in dichloromethane (40 mL) and reacted at room temperature for two hours. After the reaction was complete, the solution was concentrated and washed with saturated sodium carbonate solution. The organic layer was dried with anhydrous sodium sulfate, and diethyl ether was added to produce a large amount of precipitate. The precipitate was filtered and dried under vacuum to obtain the target compound c; the characterization results are as follows. Figure 4 As shown.

[0079] 2.2 PEG 5000 Preparation of ethyl 4-benzoate

[0080] PEG 5000 4-Methylbenzenesulfonate (1.0 g, 2 mol) and ethyl p-hydroxybenzoate (731.1 mg, 4.4 mmol) were dissolved in N,N-dimethylformamide, and potassium carbonate (8.8 mmol, 1.2 g) was added. The mixture was heated and stirred for 12 h under an inert atmosphere. After the reaction was complete and cooled to room temperature, the solution was added dropwise to diethyl ether to obtain a precipitate, which was then filtered. The residue was dissolved in water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated, and added dropwise to diethyl ether. The mixture was then filtered and dried under vacuum to obtain the target compound d. The characterization results are as follows: Figure 5 As shown.

[0081] 2.3 PEG 5000 Preparation of 4-benzoylhydrazide

[0082] PEG 5000 Ethyl 4-benzoate (1.0 g, 0.2 mmol) was dissolved in ethanol, and hydrazine hydrate (1.0 g, 20 mmol) was added. The mixture was heated and stirred under reflux for 12 h in an inert atmosphere. After the reaction was completed, the mixture was cooled to room temperature, the solvent was removed under low pressure, the residue was dissolved in dichloromethane, and washed several times with water. The organic phase was dried over anhydrous sodium sulfate, concentrated, and added dropwise to diethyl ether. The mixture was filtered and dried under vacuum to obtain the target compound e. The characterization results are as follows: Figure 6 As shown.

[0083] 2.4 Preparation of polyethylene glycol-modified tetracationic macrocyclic (1-PEG)

[0084] The 1 (21.74 mg) prepared in Example 1 and PEG 5000- 4-Benzoylhydrazide (177.02 mg) was dissolved in anhydrous methanol, and trifluoroacetic acid (10 μL) was added. Molecular sieves were heated under reflux in an inert gas atmosphere for 24 h. After the reaction was complete, the solution was cooled to room temperature, filtered, the solvent was removed under low pressure, dissolved in water, and freeze-dried to obtain the target compound 1-PEG (yield: 85%). Characterization results are as follows: Figure 7 As shown.

[0085] A 1-PEG solution was dropped onto a copper grid and allowed to dry. The morphology of the 1-PEG was then imaged using a transmission electron microscope. The particle size and surface potential of the 1-PEG solution were measured using a Zeta potential analyzer and a wide-angle laser scattering instrument. The characterization results are as follows: Figure 8 As shown.

[0086] Example 3: Experiment on the degree of acylhydrazone bond cleavage in 1-PEG molecules under different pH buffer solutions

[0087] The 1-PEG prepared in Example 2 was placed in phosphate buffer solutions of different pH values, and its fluorescence emission intensity was measured using a fluorescence spectrometer. The 1-PEG solution was placed in a dialysis bag and dialyzed in buffer solutions of different pH values. Its ultraviolet absorption was measured at different time points, and the degree of acylhydrazone bond cleavage was calculated. The results are shown in Figure 9. It can be seen that the lower the pH value, the faster the acylhydrazone bond cleavage.

[0088] The interaction between molecules 1 and PEM was analyzed. Job curves of 1 and PEM were plotted using UV-Vis absorption spectroscopy to determine their bonding ratio. A titration experiment using PEM to 1 was conducted to detect the intermolecular interaction force between 1 and PEM. The characterization results are as follows: Figure 10 As shown.

[0089] Example 4: Preparation of a polyethylene glycol-modified tetracationic macrocyclic compound with pemetrexed (PEM@1-PEG)

[0090] Taking pemetrexed (PEM) as an example, the drug loading effect of the carrier 1-PEG was verified. 1-PEM (30 mg) prepared in Example 2 was dissolved in dichloromethane (1.5 mL), and an aqueous PEM solution (2 mg / mL, 2 mL) was added. The mixture was ultrasonically mixed, and the organic phase was evaporated. The resulting mixture was dialyzed for 12 h. After dialyzing, it was freeze-dried to obtain the polyethylene glycol-modified tetracationic macrocyclic compound PEM@1-PEG with pemetrexed.

[0091] PEM@1-PEG solution was dropped onto a copper grid and allowed to dry. The morphology of PEM@1-PEG was then imaged using a transmission electron microscope. The particle size and surface potential of the PEM@1-PEG solution were measured using a Zeta potential analyzer and a wide-angle laser scattering instrument. The results are as follows: Figure 11 As shown.

[0092] The prepared 1-PEG, 1-PEG, and PEM@1-PEG solutions were analyzed separately. The UV absorption spectrum of solution 1 was detected using a UV-Vis spectrometer, and the photoluminescence spectra of solutions 1-PEG and PEM@1-PEG were detected using a fluorescence emission spectrometer. The results are as follows: Figure 12 Figure 13 As shown. From Figure 13 It is known that unmodified 1-PEG produces higher fluorescence intensity than PEG modified with polyethylene glycol. When the PEG acylhydrazone bond breaks, 1-PEG is released, which can produce a change in fluorescence intensity.

[0093] Example 5: Study on the luminescence properties of 1

[0094] 5.1 Investigation 1: Photoluminescence Behavior Experiment in Different Solvents

[0095] One molecule was placed in solutions of different proportions of DMF / H2O or DMSO / H2O, and its luminescence behavior was tested using a fluorescence spectrometer; the results are as follows. Figure 14 As shown.

[0096] 5.2 Photostability Characterization Experiment of One Molecule

[0097] The changes in the UV absorption intensity of aqueous solution 1 after being placed under a xenon lamp for different durations were measured using a UV absorption spectrometer. Furthermore, a comparative experiment was conducted using the commercially available photosensitizer, red cinnabar (RB). Figure 15 As shown in (a), after 0-30 min of xenon lamp irradiation, one molecule exhibits the same UV-Vis absorption spectrum; as Figure 15As shown in (b), after being irradiated by a xenon lamp, the UV-Vis absorption spectrum of RB changed rapidly over time; the comparison shows that 1 molecule has better photostability.

[0098] Example 6: Photodynamic therapy study of 1,1-PEG and PEM@1-PEG

[0099] 6.1 ROS generation of 1,1-PEG or PEM@1-PEG

[0100] ROS fluorescent probes DCFH and DHR123 were used to test 1,1-PEG or PEM@1-PEG, respectively. The fluorescence emission changes of DCFH solutions or DHR123 solutions containing 1,1-PEG or PEM@1-PEG molecules were measured by fluorescence emission spectroscopy under different illumination times. The results are as follows: Figure 16 Figure 17 As shown in the results of each test sample, it can be seen that the reaction system can produce obvious fluorescence changes with the increase of light exposure time, proving that ROS is generated in each reaction system; from the fluorescence emission intensity difference diagram, it can be seen that with the increase of time, the light conditions can promote the stable generation of ROS by 1,1-PEG or PEM@1-PEG.

[0101] 6.2 Generation of superoxide anion radicals from 1,1-PEG or PEM@1-PEG

[0102] 1,1-PEG or PEM@1-PEG was tested using the superoxide anion fluorescent probe DHE. Solutions containing 1,1-PEG or PEM@1-PEG molecules were placed under illumination for different durations, and the changes in fluorescence emission were measured using a fluorescence emission spectrometer. The results are shown in Figure 18. It can be seen that both 1,1-PEG and PEM@1-PEG molecules possess the ability to generate superoxide anion radicals under illumination.

[0103] 6.3 Singlet oxygen generation in 1,1-PEG or PEM@1-PEG

[0104] The singlet oxygen production of 1,1-PEG or PEM@1-PEG molecules was investigated using the fluorescent probe ABDA. ABDA solutions containing 1,1-PEG or PEM@1-PEG molecules were exposed to light for different durations, and the changes in UV absorption were measured using a UV-Vis absorption spectrometer. The results are as follows: Figure 19 As shown, 1,1-PEG or PEM@1-PEG molecules both have the ability to generate singlet oxygen under light irradiation.

[0105] 6.4 Study on the NADH oxidation ability of 1,1-PEG or PEM@1-PEG molecules under light irradiation

[0106] NADH solutions containing 1,1-PEG or PEM@1-PEG molecules were placed under light exposure for different time periods, and the changes in UV-Vis absorption were measured using a UV-Vis absorption spectrometer. The results are as follows: Figure 20 As shown, it can be seen that 1,1-PEG or PEM@1-PEG molecules have the ability to oxidize NADH molecules under light, which can disrupt the intracellular redox balance.

[0107] Study on the oxidation ability of 1,1-PEG or PEM@1-PEG molecules to BH4 under 6.5 light irradiation

[0108] Studies have found that BH4 is associated with inflammation and tumors. BH4 can promote tumor growth and regulate ubiquitination and proteasome activity through NOS-mediated S-nitrosation of target proteins. BH4 deficiency leads to increased protein ubiquitination and degradation in cells. Solutions of BH4 containing 1,1-PEG or PEM@1-PEG molecules were exposed to light for different durations, and changes in UV-Vis absorption were measured using a UV-Vis absorption spectrometer. Results are as follows... Figure 21 As shown, 1,1-PEG or PEM@1-PEG molecules both have the ability to oxidize BH4 molecules under light irradiation.

[0109] Example 7: Cellular experiments with 1,1-PEG or PEM@1-PEG

[0110] 7.1 Cell viability assay after incubation with 1,1-PEG or PEM@1-PEG

[0111] A549 cells were co-incubated with different concentrations of 1,1-PEG or PEM@1-PEG, and their dark toxicity and phototoxicity were measured. Figure 22 (a) and Figure 22 As shown in (b), in the dark toxicity test, cytotoxicity was observed when the concentration of 1 was 60 μM; in the phototoxicity test, cytotoxicity was observed when the concentration of 1 was 2 μM. Figure 22 As shown in (c), A549 cells were treated with 1-PEG+hv, PEM, PEM@1-PEG, and PEM@1-PEG+hv, respectively. The cells treated with PEM@1-PEG+light showed the lowest cell viability and the strongest ability to inhibit cancer cells.

[0112] 7.2 Cell localization studies of 1- and 1-PEG

[0113] A549 cells were co-incubated with 1,1-PEG and commercially available mitochondrial and nuclear localization agents, followed by confocal imaging. Results are as follows: Figure 23As shown, before light exposure, the distribution of 1 and 1-PEG molecules in cells showed good overlap with that of commercially available mitochondria; after light exposure, 1 and 1-PEG molecules were mainly concentrated in the cell nucleus, showing good overlap with commercially available nuclear localizers. This indicates that 1 and 1-PEG target mitochondria in cells without light exposure, and after light treatment, 1 and 1-PEG translocate from mitochondria to the cell nucleus. 7.3 Intracellular ROS and superoxide anion radical production of 1,1-PEG or PEM@1-PEG

[0114] A549 cells were co-incubated with the ROS fluorescent probe DCFH and 1 or 1-PEG, and after illumination, confocal imaging was performed to detect intracellular ROS production. The results are as follows: Figure 24 Figure 25 As shown, A549 cells can produce a large amount of ROS. A549 cells, the superoxide anion fluorescent probe DHE, and 1 or 1-PEG were then co-incubated. After illumination, confocal imaging was performed to detect the production of intracellular superoxide anions. The results are shown below. Figure 24 Figure 25 As shown, superoxide anions can also be produced in large quantities in A549 cells.

[0115] Example 8: Mouse Tumor Treatment and Fluorescence Imaging Experiment

[0116] Tumor-bearing mice injected with A549 cells were treated in the following ways: I. saline, II. saline + hv, III. 1-PEG, IV. 1-PEG + hv, V. PEM@1-PEG, VI. PEM@1-PEG + hv. Tumor size and body weight changes were recorded for 14 days. The results are as follows: Figure 26 As shown in (a) and (b), the tumor volume of mice in group IV did not change significantly, while the tumor volume of mice in group VI showed no change, indicating that 1-PEG and PEM@1-PEG significantly inhibited tumor growth after light exposure. H&E staining was performed on tissue sections from the heart, liver, spleen, lung, kidney, and tumors of mice, and the results are shown below. Figure 27 As shown. Fluorescence in vivo imaging experiments were performed on mice 18 hours after tail vein injection of 1 or 1-PEG drug. The results are as follows. Figure 26 As shown in (c), unmodified 1-PEG can be distributed throughout the mouse body, while modified 1-PEG can target the tumor site. 1-PEG is more easily enriched in the tumor region than 1-PEG. The experimental results show that in group VI (PEM@1-PEG+hv), the combination of photodynamic therapy and drug treatment has the best effect.

[0117] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A cationic macrocyclic compound, characterized in that: The structure is shown in Equation 1; 2. The cationic macrocyclic compound according to claim 1, characterized in that: The compound shown in Formula 1 was modified with polyethylene glycol, and the structure is shown in Formula 2.

3. A method for preparing the cationic macrocyclic compound according to claim 1 or 2, characterized in that: 4-Bromo-N,N-Di(4-(pyridin-4-yl)phenyl)amine, 4-formylphenylboronic acid, tetra-triphenylphosphine palladium, and 1,4-dioxane are reacted to generate 4'-(di(4-(pyridin-4-yl)phenyl)amine)-[1,1'-biphenyl]-4-carboxaldehyde, which is then reacted with p-phenylbenzyl bromide to generate the cationic macrocyclic compound shown in Formula 1. Preferably, the polyethylene glycol chain is further modified onto the cationic macrocycle via acylhydrazone bonds to obtain compound of formula 2.

4. The method for preparing the cationic macrocyclic compound according to claim 3, characterized in that: Specifically, the steps include the following: Step 1: 4'-(di(4-(pyridin-4-yl)phenyl)amine, 4-formylphenylboronic acid, tetratriphenylphosphine palladium, 1,4-dioxane and potassium carbonate were reacted to synthesize 4'-(di(4-(pyridin-4-yl)phenyl)amine)-[1,1'-biphenyl]-4-carboxaldehyde; Step 2: Dissolve 4'-(bis(4-(pyridin-4-yl)phenyl)amine)-[1,1'-biphenyl]-4-carboxaldehyde and p-phenylbenzyl bromide in an organic solvent, redissolve the obtained solid substance in a mixture of methanol and water, and add ammonium hexafluorophosphate to the solution to prepare the tetracationic macrocycle 1 shown in Formula 1; Step 3: Dissolve polyethylene glycol monomethyl ether, triethylamine, and p-toluenesulfonyl chloride in dichloromethane and react to obtain PEG. 5000 4-Methylbenzenesulfonate; PEG 5000 4-Methylbenzenesulfonate reacts with ethyl p-hydroxybenzoate to give PEG. 5000 4-ethyl benzoate; PEG 5000 Ethyl 4-benzoate was dissolved in ethanol, and hydrazine hydrate was added to react and give PEG. 5000 -4-benzoylhydrazide; Step 4: Combine the PEG obtained in Step 2 with the PEG obtained in Step 3. 5000- -4-benzoylhydrazine dissolved in anhydrous methanol, with the addition of trifluoroacetic acid and Molecular sieves were used to react and obtain polyethylene glycol-modified tetracationic macrocyclic 1-PEG as shown in Formula 2.

5. The use of the cationic macrocyclic compound according to claim 1 or 2 in photosensitizers.

6. A drug-loaded nanoparticle, characterized in that: The drug is loaded into the macrocyclic cation cavity of the cationic macrocyclic compound of claim 1 or 2, and self-assembled to obtain supramolecular nanoparticles.

7. The drug-loaded nanoparticles according to claim 6, characterized in that: The drug is an anti-tumor drug; Preferably, the drug is pemetrexed.

8. The drug-loaded nanoparticles according to claim 6, characterized in that: The cationic macrocyclic compound shown in Formula 2 was dissolved in dichloromethane, and an aqueous drug solution was added. The mixture was ultrasonically mixed, the organic phase was removed, and the mixture was lyophilized to obtain drug-loaded nanoparticles.

9. The use of the cationic macrocyclic compound of claim 1 or 2 or the drug-loaded nanoparticles of any one of claims 6-8 in fluorescent tumor tracer reagents and / or tumor therapeutic drugs.

10. The application according to claim 9, characterized in that: In the microacidic environment of a tumor, the compound shown in Formula 2 or the drug-loaded nanoparticles release macrocyclic molecules after the acylhydrazone bond is broken, resulting in enhanced fluorescence, thereby achieving tumor tracing. Alternatively, when the compound shown in Formula 2 or the drug-loaded nanoparticles are exposed to light, they generate one or more of ROS, singlet oxygen, and superoxide anion free radicals, which disrupt the redox balance in tumor cells, causing tumor cell damage and death.

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