Amphiphilic organic near-infrared thermal activation delayed fluorescence molecule as well as preparation method and application thereof

Abstract

The invention provides an amphiphilic organic near-infrared thermal activation delayed fluorescence molecule as well as a preparation method and application thereof, and relates to the technical field of organic long-life thermal activation delayed fluorescence materials. The molecule takes a hydrophobic donor-acceptor structure as a skeleton, a hydrophobic donor is grafted with a hydrophilic high-molecular compound chain, and an acceptor is coupled with a targeting group; the preparation method comprises the following steps: firstly, synthesizing N-[(3-chloropropyl) / 3-propyl (triphenyl) phosphine / (2-ethyl) morpholine]-4-ethynyl benzamide and 4-bromo-7-acetamido (indoline / indole / carbazole / 10H-phenothiazine)-2, 1, 3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE) intermediates, then reacting the intermediates with [1, 1, 2] thiadiazole, and carrying out a reaction to obtain the compound. The preparation method comprises the following steps: reacting 1, 1-bis (diphenylphosphine) ferrocene] palladium dichloride, cuprous iodide and triethylamine. The nanoparticles of the amphiphilic organic near-infrared thermal activation delayed fluorescence molecule have the service life of 39 microseconds, are used for I / II type combined photodynamic therapy guided by service life imaging, and can realize high-contrast detection and accurate treatment on tumor focuses.

Description

Technical Field

[0001] The invention belongs to the technical field of organic luminescent materials, and in particular relates to an amphiphilic organic near-infrared thermally activated delayed fluorescent molecule and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy, as a promising cancer treatment method, has been approved for clinical use due to its minimally invasive nature, unique selectivity, resistance to chemotherapy drugs, few side effects, and simple operation. During photodynamic therapy, the photosensitizer is first excited from the ground state (S0) to the singlet state (S1) under light, and then transforms to the triplet state (T1) through the intersystem crossing (ISC) process (F. Fang, et al. Small 2022, 18, 2106215.). The triplet photosensitizer transfers electrons to the adjacent oxygen-containing substrate to form hydroxyl radicals (HO·), superoxide anions (O2 -· ) and other free radicals, known as type I photodynamic therapy, or transferring energy to surrounding molecular oxygen ( 3 O2) produces singlet oxygen ( 1 O2), known as type II photodynamic therapy. Despite significant advances in the design and development of photodynamic therapy over the past few decades, the vast majority of treatments kill cancer cells via only one type of cytotoxic reactive oxygen species. In this case, the oxygen concentration, substrate, temperature, or redox properties in the tumor microenvironment can easily interfere with the efficiency of photodynamic therapy.

[0003] At present, some different photodynamic therapy synergistic methods have been proposed, such as combining type I photodynamic therapy and type II photodynamic therapy with multiple reactive oxygen species, which can avoid the above problems to a certain extent. However, different types of reactive oxygen species will also offset each other and affect the effect of photodynamic therapy. For example, Qi et al. reported two AIE PSs, TPPM and MeOTPPM, to overcome the hypoxia defect of tumors in photodynamic therapy. MeOTPPM produces a large number of hydroxyl radicals, while singlet oxygen is significantly insufficient, which is the opposite of the case of TPPM (X. Zhao, et al. Chemical Engineering Journal 2021, 410, 128133). Yoon et al. replaced the two oxygen atoms in traditional naphthylimide (MANI-S) with sulfur atoms, producing about 27 times the superoxide anion, while only a small amount of singlet oxygen was produced (V.-N. Nguyen, et al. Am. Chem. Soc., 2019, 141, 16243-16248.). Therefore, it is reasonable to assume that photosensitizers with enhanced intersystem crossing (ISC) processes to generate multiple reactive oxygen species and each reactive oxygen species at high yields are essential for the combined Type-Ⅰ and Type-Ⅱ mechanisms. At present, the most commonly used strategy to promote the intersystem crossing (ISC) process is to introduce metal atoms into photosensitizers to enhance spin-orbit coupling (J. Zhang, et al. ACS Nano, 2023, 17, 23430-23441.). However, the inherent cytotoxicity and difficulty in degradation of metal-containing photosensitizers seriously hinder their clinical application. Therefore, exploring the precise design of photosensitizers through organic chemical design and synthesis to enhance the intersystem crossing (ISC) process, thereby achieving the joint development of type Ⅰ and type Ⅱ photodynamic therapy, will bring more benefits to the clinic.

[0004] Organic thermally activated delayed fluorescent materials have extremely small singlet-triplet energy gap (ΔE ST ), since the energy conversion between S1 and T1 states is easy to achieve, it provides the possibility to achieve efficient intersystem crossing (ISC) and achieve a breakthrough in reactive oxygen species from the perspective of multi-species and high productivity. At the same time, excitons in the triplet state (T1) can be upconverted to the singlet state (S1) through heat-assisted reverse intersystem crossing (RISC), resulting in unique long-lived emission, usually ranging from microseconds to seconds, which can be used for lifetime imaging to eliminate short-lived background interference. Therefore, it is of great practical significance to develop new thermally activated delayed fluorescence photosensitizers to meet the above requirements at the same time.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide an amphiphilic organic near-infrared thermally activated delayed fluorescent molecule and a preparation method and application thereof to solve the above problems.

[0007] In order to achieve the above objectives, the present invention particularly adopts the following technical solutions:

[0008] The invention provides an amphiphilic organic near-infrared thermally activated delayed fluorescent molecule. The amphiphilic organic near-infrared thermally activated delayed fluorescent molecule uses a hydrophobic donor-acceptor (DA) structure as a skeleton, the hydrophobic donor is grafted with a hydrophilic polymer compound, and the receptor is coupled with a targeting group.

[0009] The hydrophobic donor includes one or more of 5-acetylaminoindoline, 5-acetylaminoindole, 3-acetylaminocarbazole or 3-acetylamino-10H-phenothiazine.

[0010] The acceptor includes 2,1,3-benzothiadiazole.

[0011] The chemical formula of the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule is: NX-4-ethynylbenzamide-acetylamino Y-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid, wherein X is any one of 3-chloropropyl, 3-propyl (triphenyl) phosphine, and (2-ethyl) morpholine, and Y is any one of indoline, indole, carbazole, and 10H-phenothiazine;

[0012] Preferably, the general structural formula of the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule includes any one of the general formula I, general formula II, general formula III or general formula IV:

[0013] The general formula I is:

[0014] The general formula II is:

[0015] The general formula III is:

[0016] The general formula IV is:

[0017] Wherein, R1 is any one of the targeting groups; and R2 is a hydrophilic polymer compound.

[0018] The targeting group is selected from any one of an endoplasmic reticulum targeting group, a mitochondrial targeting group, and a lysosome targeting group.

[0019] The endoplasmic reticulum targeting group includes N-(3-chloropropyl)-4-ethynylbenzamide, the structural formula of which is:

[0020] The mitochondrial targeting group includes N-3-propyl (triphenyl) phosphine-4-ethynyl benzamide, the structural formula of which is:

[0021] The lysosomal targeting group includes N-(2-ethyl)morpholine-4-ethynylbenzamide, which has the structural formula:

[0022] Preferably, the hydrophilic polymer compound is a water-soluble phospholipid-polyethylene glycol-carboxylic acid (DSPE-PEG 2000 -COOH).

[0023] The present invention also provides a method for preparing the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule, which comprises the following steps:

[0024] S1: Add 4-ethynylbenzoic acid and O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate into a reaction container, inject dry dimethylformamide under argon atmosphere, add N-ethyldiisopropylamine to obtain a mixture, and stir at 0-8°C for 10-20 minutes, then add any one of 3-chloropropylamine hydrochloride, 3-aminopropyl(triphenyl)phosphine bromide or N-(2-aminoethyl)morpholine, and stir at room temperature overnight to obtain compound A: N-[(3-chloropropyl) / 3-propyl(triphenyl)phosphine / (2-ethyl)morpholine]-4-ethynylbenzamide; wherein the compound A includes compound A-1, compound A-2, and compound A-3;

[0025] The compound A-1 is N-(3-chloropropyl)-4-ethynylbenzamide; the compound A-2 is N-[3-propyl(triphenyl)phosphine]-4-ethynylbenzamide; the compound A-3 is N-(2-ethyl)morpholine-4-ethynylbenzamide;

[0026] Preferably, in step S1, the synthetic routes of compound A-1, compound A-2 and compound A-3 can be:

[0027]

[0028] S2: Take any one of 5-nitroindole, 5-nitroindole, 3-nitrocarbazole or 3-nitro-10H-phenothiazine, add it into a dry dimethyl sulfoxide solution together with 4,7-dibromo-2,1,3-benzothiadiazole, copper, octadecanoyl-6 and potassium carbonate, stir at 160°C for 0.5-1 hour under nitrogen atmosphere, and the obtained intermediate compound is 4-bromo-7-nitroindole-2,1,3-benzothiadiazole, 4-bromo -7-nitroindole-2,1,3-benzothiadiazole, 4-bromo-7-nitrocarbazole-2,1,3-benzothiadiazole, 4-bromo-7-nitro-10H-phenothiazine-2,1,3-benzothiadiazole; then, the intermediate and stannous chloride dihydrate are added to anhydrous ethanol at a molar mass ratio of 1.3-1.4:13.2-13.4, heated to reflux under a nitrogen atmosphere, and stirred overnight to obtain compound B;

[0029] Wherein, the compound B includes compound B-1, compound B-2, compound B-3 and compound B-4;

[0030] The compound B-1 is 4-bromo-7-aminoindoline-2,1,3-benzothiadiazole; the compound B-2 is 4-bromo-7-aminoindole-2,1,3-benzothiadiazole; the compound B-3 is 4-bromo-7-aminocarbazole-2,1,3-benzothiadiazole; the compound B-4 is 4-bromo-7-amino-10H-phenothiazine-2,1,3-benzothiadiazole;

[0031] Preferably, in step S2, the synthetic routes of compound B-1, compound B-2, compound B-3 and compound B-4 can be:

[0032]

[0033] S3: O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate and phospholipid-polyethylene glycol-carboxylic acid (DSPE-PEG 2000 -COOH) is added into a container, and in a nitrogen atmosphere, dry dimethylformamide is injected, followed by N-ethyldiisopropylamine, and stirred at 0-8°C for 0.5-1 hour, and then compound B obtained by S2 is added, and the reaction is carried out at room temperature for 23-25 ​​hours to obtain compound C; wherein the compound C includes compound C-1, compound C-2, compound C-3 and compound C-4;

[0034] Preferably, the compound C-1 is 4-bromo-7-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE), and its preparation process is: O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate and phospholipid-polyethylene glycol-carboxylic acid (DSPE-PEG 2000 -COOH) was added into a container, and dry dimethylformamide was injected under nitrogen atmosphere, followed by N-ethyldiisopropylamine, and stirred at 0-8°C for 0.5-1 hour, and then compound B-1 obtained by S2 was added, and reacted at room temperature for 23-25 ​​hours to obtain the compound C-1;

[0035] Preferably, the compound C-2 is 4-bromo-7-acetylaminoindole-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE), and its preparation process is: O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate and phospholipid-polyethylene glycol-carboxylic acid (DSPE-PEG 2000 -COOH) is added to a container, and dry dimethylformamide is injected under a nitrogen atmosphere, followed by N-ethyldiisopropylamine, and stirred at 0-8°C for 0.5-1 hour, and then compound B-2 obtained by S2 is added, and the reaction is carried out at room temperature for 23-25 ​​hours to obtain the compound C-2;

[0036] Preferably, the compound C-3 is 4-bromo-7-acetylaminocarbazole-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE), and its preparation process is: O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate and phospholipid-polyethylene glycol-carboxylic acid (DSPE-PEG 2000 -COOH) is added to a container, and under a nitrogen atmosphere, dry dimethylformamide is injected, followed by N-ethyldiisopropylamine, and stirred at 0-8°C for 0.5-1 hour, and then the compound B-3 obtained by S2 is added, and the reaction is carried out at room temperature for 23-25 ​​hours to obtain the compound C-3;

[0037] Preferably, the compound C-4 is 4-bromo-7-acetylaminophenothiazine-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE), and its preparation process is: O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate and phospholipid-polyethylene glycol-carboxylic acid (DSPE-PEG 2000 -COOH) is added to a container, and dry dimethylformamide is injected under a nitrogen atmosphere, followed by N-ethyldiisopropylamine, and stirred at 0-8°C for 0.5-1 hour, and then the compound B-4 obtained by S2 is added, and the reaction is carried out at room temperature for 23-25 ​​hours to obtain the compound C-4;

[0038] S4: Add compound C obtained in S3, compound A obtained in S1, [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride and cuprous iodide to a container containing dry tetrahydrofuran, add triethylamine under an argon atmosphere, and stir at 65° C. for 11-13 hours to obtain the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule;

[0039] Optionally, the amphiphilic organic near-infrared thermally activated delayed fluorescent molecular compound can be: N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindole-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE), N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindole-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE), N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindole-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid ( PEG-DSPE), N-(3-chloropropyl)-4-ethynylbenzamide-acetylamino (10H-phenothiazine)-2,1,3-benzothiadiazole-polyethylene glycol-phospholipids (PEG-DSPE), N-[3-propyl (triphenyl) phosphine]-4-ethynylbenzamide-acetylaminoindole-2,1,3-benzothiadiazole-polyethylene glycol-phospholipids (PEG-DSPE), N-[3-propyl (triphenyl) phosphine]-4-ethynylbenzamide-acetylaminoindole-2,1,3-benzothiadiazole-polyethylene glycol-phospholipids (PEG -DSPE), N-[3-propyl(triphenyl)phosphine]-4-ethynylbenzamide-acetylaminocarbazole-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE), N-[3-propyl(triphenyl)phosphine]-4-ethynylbenzamide-acetylamino(10H-phenothiazine)-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE), N-(2-ethyl)morpholine-4-ethynylbenzamide-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-D SPE), N-(2-ethyl)morpholine-4-ethynylbenzamide-acetylaminoindole-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE), N-(2-ethyl)morpholine)-4-ethynylbenzamide-acetylaminocarbazole-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE), N-(2-ethyl)morpholine-4-ethynylbenzamide-acetylamino(10H-phenothiazine)-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE);

[0040] Optionally, any one of the compounds C-1, C-2, C-3 or C-4 obtained in S3, any one of the compounds A-1, A-2 or A-3 obtained in S1, [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride and cuprous iodide are added to a container containing dry tetrahydrofuran, and triethylamine is added under an argon atmosphere, followed by stirring at 65°C for 11-13 hours to obtain the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule.

[0041] Preferably, in S1, the molar mass ratio of 4-ethynylbenzoic acid, O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate, N-ethyldiisopropylamine and 3-chloropropylamine hydrochloride or 3-aminopropyl (triphenyl)phosphine bromide or N-(2-aminoethyl)morpholine is 1.8-2.2: 2.3-2.7: 5.8-6.2: 2.2-2.8;

[0042] Optionally, S1 further comprises dissolving the product obtained by stirring overnight at room temperature in S1 in ethyl acetate, washing with water, hydrochloric acid, sodium bicarbonate aqueous solution and brine in sequence, drying the organic layer over anhydrous sodium sulfate, filtering and vacuum drying, and then separating and purifying by column chromatography using petroleum ether / ethyl acetate as eluent to obtain the compound A;

[0043] Preferably, in S2, the molar mass ratio of the 5-nitroindoline or 5-nitroindole or 3-nitrocarbazole or 3-nitro-10H-phenothiazine to 4,7-dibromo-2,1,3-benzothiadiazole, copper, octadecanoyl-6, and potassium carbonate is 15-25:8-10:65-70:12-15:60-65, respectively;

[0044] Optionally, S2 further comprises: after stirring at 160° C. for 0.5-1 hour as described in S2, pouring the system into ether after cooling, leaving overnight, filtering out the solvent, vacuum drying, and separating and purifying the crude product by column chromatography, wherein the eluent is petroleum ether / dichloromethane, and then vacuum drying to obtain an intermediate compound; the intermediate compound is any one of 4-bromo-7-nitroindole-2,1,3-benzothiadiazole, 4-bromo-7-nitroindole-2,1,3-benzothiadiazole, 4-bromo-7-nitrocarbazole-2,1,3-benzothiadiazole, and 4-bromo-7-nitro-10H-phenothiazine-2,1,3-benzothiadiazole;

[0045] Further preferably, S2 further comprises: after the system stirred overnight is cooled, a saturated sodium bicarbonate solution is added thereto until the system is neutral, extracted with dichloromethane, rotary evaporated, the obtained solid is vacuum dried, the crude product is separated and purified by column chromatography, the eluent is petroleum ether / dichloromethane, and then vacuum dried to obtain compound B;

[0046] Preferably, in S3, the O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate, phospholipid-polyethylene glycol-carboxylic acid (DSPE-PEG 2000 -COOH), N-ethyldiisopropylamine, and compound B have a molar mass ratio of 0.14-1.16: 0.14-0.16: 0.9-1.1: 0.05-0.1;

[0047] Optionally, S3 further comprises: after the reaction at room temperature for 23-25 ​​hours, dialyzing the product for 48-72 hours until the dialysate is substantially colorless, and freeze-drying to obtain the compound C;

[0048] Preferably, in S4: the molar mass ratio of compound C obtained in S3, compound A obtained in S1, [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, cuprous iodide, and triethylamine is 0.4-0.8: 0.2-0.8: 0.08-0.12: 0.14-0.16: 0.9-1.1;

[0049] Optionally, the S4 further comprises: after stirring at 65° C. for 11-13 hours, rotary evaporation, dialyzing the product for 48-72 hours until the dialysate is substantially colorless, and freeze-drying to obtain the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule.

[0050] The present invention also provides an application of the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule, which is used for lifetime imaging-guided type I / II combined photodynamic therapy. High-contrast detection and accurate treatment of tumor lesions are performed through lifetime imaging-guided type I / II combined photodynamic therapy.

[0051] Beneficial effects of the present invention:

[0052] The amphiphilic organic near-infrared thermally activated delayed fluorescent molecule provided by the present invention has a hydrophobic donor-acceptor (DA) structure as a skeleton, is grafted with a targeting group and a hydrophilic polymer compound chain, and can be further self-assembled into nanoparticles through π-π stacking and hydrogen bonding. ST )~0.084eV promotes the intersystem crossing (ISC) process, thereby generating a variety of reactive oxygen species, including high-yield hydroxyl radicals, superoxide anions and singlet oxygen, which can also quickly target and stably accumulate in tumor tissues; it can be used for lifetime imaging-guided type I / II combined photodynamic therapy, and the yield of each reactive oxygen species (hydroxyl radicals, superoxide anions and singlet oxygen) is very high, and the generation efficiency of total reactive oxygen species and various types of reactive oxygen species are higher than existing photosensitizers, thereby enhancing the type I / II combined photodynamic therapy capability.

[0053] The preparation method provided by the invention has the advantages of simple operation, simple and controllable conditions, cheap and readily available raw materials, low preparation cost, clear reaction route, high yield, strong repeatability, and is suitable for industrial promotion.

[0054] The amphiphilic organic near-infrared thermally activated delayed fluorescent nanoparticles provided by the present invention have a lifetime of up to 39 μs, can be used as lifetime imaging signals, eliminate short-lifetime background interference of tissues, accurately guide the in vivo photodynamic therapy process, and are suitable for high-contrast detection and accurate treatment of tumor lesions. They provide accurate and reliable lifetime bioimaging and advanced photodynamic therapy in clinical biomedical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0056] Figure 1 This is a hydrogen nuclear magnetic resonance spectrum of compound A-1 provided in Example 1 in deuterated chloroform;

[0057] Figure 2 This is the nuclear magnetic resonance spectrum of compound B-1 provided in Example 1 in deuterated chloroform;

[0058] Figure 3 The nuclear magnetic resonance spectrum of compound C-1 provided in Example 1 in deuterated chloroform;

[0059] Figure 4 The H NMR spectrum of the compound N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE) provided in Example 1 in deuterated chloroform;

[0060] Figure 5 The absorption and emission spectra of the nanoparticles of the amphiphilic organic near-infrared thermally activated delayed fluorescent molecules provided in Example 2 in phosphate buffer;

[0061] Figure 6 A schematic diagram of the process of using the nanoparticles of the amphiphilic organic near-infrared thermally activated delayed fluorescent molecules provided in Example 2 to generate reactive oxygen species by lifetime imaging;

[0062] Figure 7 A transient phosphorescence decay curve of the nanoparticles of the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule provided in Example 2 in a phosphate buffer solution;

[0063] Figure 8 This is an electron paramagnetic resonance (EPR) spectrum of the active oxygen test of the nanoparticles of the amphiphilic organic near-infrared thermally activated delayed fluorescent molecules provided in Example 2.

[0064] Fig. 9 Representative photos of tumors 6 days after treatment with the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule nanoparticles provided in Example 2. DETAILED DESCRIPTION

[0065] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0066] Example 1

[0067] This embodiment provides an amphiphilic organic near-infrared thermally activated delayed fluorescent molecule and a preparation method thereof, which specifically comprises the following steps:

[0068] S1: Synthesis of Compound A-1: ​​N-(3-chloropropyl)-4-ethynylbenzamide:

[0069] 4-Ethynylbenzoic acid (0.3 g, 2 mmol) and O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate (1 g, 2.5 mmol) were added to the reaction vessel. Dry dimethylformamide was injected under argon atmosphere, and N-ethyldiisopropylamine was added. The mixture was stirred at 4°C for 15 minutes. Then, 3-chloropropylamine hydrochloride (0.3 g, 2.5 mmol) was added and stirred at room temperature overnight. The product was dissolved in ethyl acetate, washed with water, hydrochloric acid, sodium bicarbonate solution and brine in sequence. The organic layer was dried over anhydrous sodium sulfate, filtered and dried in vacuo, and then separated and purified by column chromatography using petroleum ether / ethyl acetate = 2:1 (v / v) as the eluent to obtain a white solid, namely compound A-1: ​​N-(3-chloropropyl)-4-ethynylbenzamide. The yield of the product was calculated to be 92%, and it was measured: 1H NMR (400MHz, Chloroform-d) δ7.71 (d, J = 8.3 Hz, 2H), 7.51 (d, J = 8.3 Hz, 2H), 6.61 (s, 1H), 3.60 (dt, J = 19.6, 6.4 Hz, 4H), 3.19 (s, 1H), 2.09 (p, J = 6.5 Hz, 2H). The spectrum is shown in Figure 1 As shown;

[0070] S2: Synthesis of compound B-1: 4-bromo-7-aminoindoline-2,1,3-benzothiadiazole:

[0071] 5-Nitroindole (5 g, 17 mmol), 4,7-dibromo-2,1,3-benzothiadiazole (1.4 g, 8.5 mmol), copper (4.35 g, 68 mmol), octadecacrown-6 (3.59 g, 13.5 mmol) and potassium carbonate (9.4 g, 63 mmol) were added to a dry dimethyl sulfoxide solution, stirred at 160°C for 0.5 hour under a nitrogen atmosphere, poured into ether after cooling, left overnight, filtered to remove the solvent, and dried in vacuo. The crude product was separated and purified by column chromatography with petroleum ether / dichloromethane = 7:3 (v / v) as the eluent. After vacuum drying, an orange-red solid compound 4-bromo-7-nitroindole-2,1,3-benzothiadiazole was obtained with a yield of 52%. It was measured that: 1H NMR (400MHz, Chloroform-d) δ8.09 (s, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 7.9 Hz, 1H), 7.41 (d, J = 7.9 Hz, 1H), 6.76 (d, J = 8.8 Hz, 1H), 4.52 (t, J = 8.3 Hz, 2H), 3.35 (t, J = 8.5 Hz, 2H). Afterwards, the compound 4-bromo-7-nitroindole-2,1,3-benzothiadiazole (0.5 g, 1 .33mmol) and stannous chloride dihydrate (3g, 13.3mmol) were added to anhydrous ethanol, heated to reflux under nitrogen atmosphere, stirred overnight, and after cooling, saturated sodium bicarbonate solution was added to the reaction solution until the solution was neutral, extracted with dichloromethane, and rotary evaporated. The obtained solid was vacuum dried, and the crude product was separated and purified by column chromatography. The eluent was petroleum ether / dichloromethane = 1:8 (v / v). After vacuum drying, compound B-1: 4-bromo-7-aminoindoline-2,1,3-benzothiadiazole was obtained as a dark purple solid with a yield of 80%. It was measured: 1H NMR (400MHz, Chloroform-d, δ, ppm) δ7.67 (d, J = 7.7 Hz, 1H), 7.23 (s, 1H), 6.95 (d, J = 7.5 Hz, 1H), 6.68 (s, 1H), 6.51 (s, 1H), 4.42 (s, 2H), 3.53 (s, 2H), 3.15 (s, 2H). The spectrum is shown in Figure 2 As shown;

[0072] S3: Synthesis of compound C-1: 4-bromo-7-acetylaminoindoline-2,1,3-benzothiadiazole-phospholipid-polyethylene glycol-phospholipid (PEG-DSPE):

[0073] O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate (0.05 g, 0.15 mmol) and phospholipid-polyethylene glycol-carboxylic acid (DSPE-PEG 2000 -COOH) (0.3 g, 0.15 mmol) was added to a 20 mL round-bottom flask. In a nitrogen atmosphere, dry dimethylformamide was injected, followed by 13 mL of N-ethyldiisopropylamine. The mixture was stirred at room temperature for 1 hour, and then 4-bromo-7-aminoindoline-2,1,3-benzothiadiazole (0.02 g, 0.075 mmol) was added. The mixture was reacted at room temperature for 24 hours. The reaction solution was poured into ice water, filtered, and the product was washed with water until the filtrate was basically colorless. The product was dried in vacuo to obtain compound C-1: 4-bromo-7-acetylaminoindoline-2,1,3-benzothiadiazole-poly(ethylene glycol) Diol-phospholipid (PEG-DSPE) is a purple solid with a yield of 89%. The measured results are: 1HNMR (400MHz, Chloroform-d, δ, ppm) 7.54, 7.53 (d, 1H), 7.43 (s, 1H), 7.36 (s, 1H), 7.13, 7.12 (d, J = 3.5Hz, 1H), 7.09 (s, 1H), 4.18 (s, 2H), 3.76 (t, J = 5.3Hz, 2H), 3.52 (t, J = 6.0Hz, 2H), 3.49 (s, 3H). The spectrum is shown in Figure 3 As shown;

[0074] S4: Synthesis of compound N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE):

[0075] Compound 4-bromo-7-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE) (1 g, 0.5 mmol), N-(3-chloropropyl)-4-ethynylbenzamide (0.13 mg, 0.6 mmol), [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium (0.073 g, 0.1 mmol), and cuprous iodide (0.029 mg, 0.15 mmol) were added to a mixture containing 2 In a round-bottom flask containing 0 mL of dry tetrahydrofuran, triethylamine (140 μL, 1 mmol) was added under argon protection, and then stirred at 65° C. for 12 hours, rotary evaporated, and the product was washed with a large amount of water for multiple times until the filtrate was basically colorless, and vacuum dried to obtain the compound N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE) as a purple solid with a yield of 93%. 1H NMR(400MHz,Chloroform-d,δ,ppm)δ7.76,7.75(d,J=7.4Hz,3H),7.61,7.59(d,J=6.6Hz,3H),7.49(d,J=7.3Hz,1H),7.40(d,J=7.3Hz,2H ), 4.70 (s, 2H), 4.26 (s, 2H), 3.76 (t, J = 5.0Hz, 2H), 3.72 (t, J = 4.8Hz, 2H), 3.55 (t, J = 5.0Hz, 2H), 3.52 (t, J = 3.8Hz, 2H), 3.49 (s, 3H). The spectrum is as follows. Figure 4 shown.

[0076] The synthesis method of this embodiment is as follows:

[0077]

[0078] Example 2

[0079] The compound N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE) prepared in Example 1 was self-assembled in phosphate buffer (PBS) to prepare N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE) nanoparticles.

[0080] The absorption and emission spectra of the nanoparticles prepared in phosphate buffered saline (PBS) in Example 2 were tested. Figure 5 As shown, Figure 5The absorption and emission spectra of N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE) nanoparticles in phosphate buffer provided in Example 2 are shown in FIG. Figure 5 It can be seen that compared with the compound N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE) in organic solvent, the nanoparticles have a similar absorbance peak at around 525nm, while the emission wavelength is about 690nm and is significantly red-shifted, which is due to the aggregation state caused by π-π stacking and hydrogen bonding.

[0081] The self-assembled N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE) nanoparticles have a low singlet-triplet energy gap (ΔE ST ) value, thus providing enhanced intersystem crossing (ISC) process and direct reverse intersystem crossing (RISC), thus promoting TADF emission, such as Figure 6 As shown, Figure 6 Schematic diagram of the process of using the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule nanoparticles provided in Example 2 to generate reactive oxygen species for lifetime imaging, Figure 6 It can be seen that after illumination, some of the near-infrared thermally activated delayed fluorescent nanoparticles in the triplet state (3PS*) interact with singlet oxygen or substrates and are converted into three reactive oxygen species, hydroxyl radicals, superoxide anions, and singlet oxygen, in aqueous solution. Therefore, the intersystem crossing (ISC) process is enhanced to produce a variety of reactive oxygen species with high productivity of each reactive oxygen species, which can enhance type I / type II combined photodynamic therapy. At the same time, the excitons in the triplet state (T1) can be upconverted to the singlet state (S1) through heat-assisted reverse intersystem crossing (RISC), resulting in unique long-lived emission.

[0082] N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE) nanoparticles were dispersed in phosphate buffer (PBS) and their fluorescence lifetime was tested. The transient photoluminescence decay curve is shown in Figure 7 As shown, Figure 7 The transient phosphorescence decay curve of the nanoparticles of the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule provided in Example 2 in phosphate buffer is shown in FIG. Figure 7 It can be seen that the nanoparticles show a long life of 39 μs, indicating that the amphiphilic organic near-infrared thermally activated delayed fluorescence molecules provided by the present invention can be used as lifetime imaging signals to eliminate the short-lifetime background interference of tissues and accurately guide the in vivo photodynamic therapy process.

[0083] The reactive oxygen species were tested on N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE) nanoparticles, such as Figure 8 As shown, Figure 8 The electron paramagnetic resonance (EPR) spectrum of the active oxygen test of the nanoparticles of the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule provided in Example 2 is shown in FIG. Figure 8 It can be seen that the generation of reactive oxygen species is evaluated by electron paramagnetic resonance (EPR) spectroscopy, 2,2,6,6-tetramethyl-4-piperidone (TEMP) is used as a spin trap for singlet oxygen, and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) is used as a spin trap to identify hydroxyl radicals and superoxide anions. When the nanoparticles and the aerated solution of 2,2,6,6-tetramethyl-4-piperidone (TEMP) are irradiated with light, a characteristic paramagnetic addition is observed, which matches the singlet oxygen signal, confirming that the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule provided by the present invention produces singlet oxygen.

[0084] The anticancer effect of the N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE) nanoparticles obtained in Example 2 was studied by intravenous injection into mice with RENCA orthotopic renal tumors, and photodynamic therapy was performed 7 days after surgery. 612nm laser irradiation was performed 1 hour after NP injection, with a cycle of 2 days, and continuous treatment for 6 days, each time for 10 minutes. A significant reduction in tumor area was observed from day 0 to day 6. The orthotopic renal tumors of mice with different treatment cycles were removed, and it was found that with the extension of the treatment cycle, the tumor wrapped around the outside of the kidney was significantly reduced, and the kidney shape gradually revealed, such as Fig. 9 shown.

[0085] Comparative Example 1

[0086] The compound in this comparative example 1 is an organic infrared photosensitizer material MeOTPPM (X.Zhao, et al.ChemicalEngineering Journal 2021,410,128133), and its chemical formula is:

[0087]

[0088] Comparative Example 2

[0089] The compound in this comparative example 2 is a photosensitizer ANONO2-Cy5 (X. Peng, et al. Adv. Mater. 2023, 35, 2305163), and its chemical formula is:

[0090]

[0091] The data comparison of active oxygen generation of the compounds provided in Example 2, Comparative Example 1 and Comparative Example 2 is shown in Table 1 below:

[0092] Table 1 Parameters of active oxygen types generated by compounds provided in Example 2, Comparative Example 1 and Comparative Example 2

[0093]

[0094] As shown in Table 1, the N-(3-chloropropyl)-4-ethynylbenzamide-acetylaminoindoline-2,1,3-benzothiadiazole-polyethylene glycol-phospholipid (PEG-DSPE) nanoparticles prepared in Example 2 of the present invention can generate three types of ROS ( 1 O2, HO· and O2 -· ), and the total ROS production is better than that of comparative examples 1 and 2. Therefore, the high production of multiple ROS improves the effect of type I / type II combined photodynamic therapy for diseases.

[0095] Please note that the technical features of the above embodiments can be combined arbitrarily. In order to make the description concise, all possible combinations of the technical features in the above embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. The above embodiments only express several implementation methods of the present application, and their descriptions are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all belong to the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be based on the attached claims.

Claims

1. An amphiphilic organic near-infrared thermally activated delayed fluorescent molecule, characterized in that: The amphiphilic organic near-infrared thermally activated delayed fluorescent molecule has a hydrophobic donor-acceptor (DA) structure as a skeleton, the hydrophobic donor is grafted with a hydrophilic polymer compound, and the receptor is coupled with a targeting group.

2. The amphiphilic organic near-infrared thermally activated delayed fluorescent molecule according to claim 1, characterized in that: The hydrophobic donor includes one or more of 5-acetylaminoindoline, 5-acetylaminoindole, 3-acetylaminocarbazole or 3-acetylamino-10H-phenothiazine.

3. The amphiphilic organic near-infrared thermally activated delayed fluorescent molecule according to claim 1, characterized in that: The acceptor includes 2,1,3-benzothiadiazole.

4. The amphiphilic organic near-infrared thermally activated delayed fluorescent molecule according to claim 1, characterized in that: The general structural formula includes any one of general formula I, general formula II, general formula III or general formula IV: The general formula I is: The general formula II is: The general formula III is: The general formula IV is: Wherein, R1 is any one of the targeting groups; and R2 is a hydrophilic polymer compound.

5. The amphiphilic organic near-infrared thermally activated delayed fluorescent molecule according to claim 1 or 4, characterized in that: The targeting group is selected from any one of an endoplasmic reticulum targeting group, a mitochondrial targeting group, and a lysosome targeting group.

6. The amphiphilic organic near-infrared thermally activated delayed fluorescent molecule according to claim 5, characterized in that: The endoplasmic reticulum targeting group includes N-(3-chloropropyl)-4-ethynylbenzamide, the structural formula of which is: The mitochondrial targeting group includes N-3-propyl (triphenyl) phosphine-4-ethynyl benzamide, the structural formula of which is: The lysosomal targeting group includes N-(2-ethyl)morpholine-4-ethynylbenzamide, which has the structural formula:

7. The amphiphilic organic near-infrared thermally activated delayed fluorescent molecule according to claim 1 or 4, characterized in that: The hydrophilic polymer compound is a water-soluble phospholipid-polyethylene glycol-carboxylic acid (DSPE-PEG 2000 -COOH).

8. A method for preparing an amphiphilic organic near-infrared thermally activated delayed fluorescent molecule according to any one of claims 1 to 7, characterized in that: The preparation method comprises the following steps: S1: Add 4-ethynylbenzoic acid and O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate into a reaction container, inject dry dimethylformamide under argon atmosphere, add N-ethyldiisopropylamine to obtain a mixture, and stir at 0-8° C. for 10-20 minutes, then add any one of 3-chloropropylamine hydrochloride, 3-aminopropyl (triphenyl)phosphine bromide or N-(2-aminoethyl)morpholine, and stir at room temperature overnight to obtain compound A; S2: Take any one of 5-nitroindoleline, 5-nitroindole, 3-nitrocarbazole or 3-nitro-10H-phenothiazine, add it together with 4,7-dibromo-2,1,3-benzothiadiazole, copper, octadecanoyl-6 and potassium carbonate to a dry dimethyl sulfoxide solution, stir at 160° C. for 0.5-1 hour under a nitrogen atmosphere to obtain an intermediate compound, then add the intermediate compound and stannous chloride dihydrate in a molar mass ratio of 1.3-1.4:13.2-13.4 to anhydrous ethanol, heat under reflux under a nitrogen atmosphere, and stir overnight to obtain compound B; S3: O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate and phospholipid-polyethylene glycol-carboxylic acid (DSPE-PEG 2000 -COOH) was added into a container, and dry dimethylformamide was injected under nitrogen atmosphere, followed by N-ethyldiisopropylamine, and stirred at 0-8°C for 0.5-1 hour, and then compound B obtained by S2 was added, and reacted at room temperature for 23-25 ​​hours to obtain compound C; S4: Add compound C obtained in S3, compound A obtained in S1, [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride and cuprous iodide to a container containing dry tetrahydrofuran, add triethylamine under argon atmosphere, and stir at 65° C. for 11-13 hours to obtain the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule.

9. The preparation method according to claim 8, characterized in that: The preparation method also satisfies one or more of the following conditions: a. In S1, the molar mass ratio of 4-ethynylbenzoic acid, O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate, N-ethyldiisopropylamine and 3-chloropropylamine hydrochloride or 3-aminopropyl (triphenyl) phosphine bromide or N-(2-aminoethyl) morpholine is 1.8-2.2: 2.3-2.7: 5.8-6.2: 2.2-2.8; b. S1 further comprises dissolving the product obtained by stirring overnight at room temperature in S1 in ethyl acetate, washing with water, hydrochloric acid, sodium bicarbonate aqueous solution and brine in sequence, drying the organic layer over anhydrous sodium sulfate, filtering and drying in vacuo, and then separating and purifying by column chromatography using petroleum ether / ethyl acetate as eluent to obtain compound A; c. In S2, the molar mass ratio of 5-nitroindoline or 5-nitroindole or 3-nitrocarbazole or 3-nitro-10H-phenothiazine to 4,7-dibromo-2,1,3-benzothiadiazole, copper, octadecanoyl-6, and potassium carbonate is 15-25:8-10:65-70:12-15:60-65; d. The S2 further comprises: after stirring at 160° C. for 0.5-1 hour as described in S2, the system is cooled and poured into ether, left overnight, the solvent is filtered out, and vacuum dried. The crude product is separated and purified by column chromatography, the eluent is petroleum ether / dichloromethane, and then vacuum dried to obtain an intermediate compound of any one of 4-bromo-7-nitroindole-2,1,3-benzothiadiazole, 4-bromo-7-nitroindole-2,1,3-benzothiadiazole, 4-bromo-7-nitrocarbazole-2,1,3-benzothiadiazole, and 4-bromo-7-nitro-10H-phenothiazine-2,1,3-benzothiadiazole; Preferably, S2 further comprises: after the system stirred overnight is cooled, a saturated sodium bicarbonate solution is added thereto until the system is neutral, extracted with dichloromethane, rotary evaporated, the obtained solid is vacuum dried, the crude product is separated and purified by column chromatography, the eluent is petroleum ether / dichloromethane, and then vacuum dried to obtain compound B; e. In the S3, the O-(7-nitrogen benzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate, phospholipid-polyethylene glycol-carboxylic acid (DSPE-PEG 2000 -COOH), N-ethyldiisopropylamine, and compound B have a molar mass ratio of 0.14-1.16: 0.14-0.16: 0.9-1.1: 0.05-0.1; f. The S3 further comprises: after the reaction at room temperature for 23-25 ​​hours, dialyzing the product for 48-72 hours until the dialysate is substantially colorless, and freeze-drying to obtain the compound C; g. In S4: the molar mass ratio of compound C obtained in S3, compound A obtained in S1, [1,1-bis(diphenylphosphino)ferrocene] palladium dichloride, cuprous iodide, and triethylamine is 0.4 to 0.8: 0.2 to 0.8: 0.08 to 0.12: 0.14 to 0.16: 0.9 to 1.1; h. The S4 further comprises: after stirring at 65° C. for 11-13 hours, rotary evaporation, dialyzing the product for 48-72 hours until the dialysate is substantially colorless, and freeze-drying to obtain the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule.

10. A use of the amphiphilic organic near-infrared thermally activated delayed fluorescent molecule according to any one of claims 1 to 7, characterized in that: For lifespan imaging-guided type I / II combined photodynamic therapy.

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