Lipid droplet targeted naphthalimide thermally activated delayed fluorescent compound and application thereof in tumor photodynamic radiosensitization therapy

Abstract

The invention provides a lipid droplet targeted naphthalimide thermally activated delayed fluorescent compound and application thereof in tumor photodynamic radiosensitization therapy, and an NIOH-Cz molecule has a plurality of excellent characteristics and shows a wide application prospect. The fluorescent compound adopts a D-A structure, the singlet-triplet energy level difference is small, efficient intersystem crossing is promoted, a large amount of active oxygen can be effectively generated under illumination, and a foundation is laid for photodynamic therapy. The material has a large Stokes shift of 165 nm, reduces self-absorption, and improves the light penetration depth and the signal-to-noise ratio. The fluorescence lifetime in a normal oxygen PBS solution reaches 1.78 [mu] s, and imaging and treatment are facilitated. The molecule has good photodynamic therapy and radiotherapy sensitization effects, and also has biological safety. The structure and the synthesis method are simple, the solution state keeps TADF characteristics, the lipid droplet targeting property is good, and the selective accumulation of tumor tissues can be improved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound and an application thereof in photodynamic radiosensitization therapy for tumors. Background Art

[0002] Radiotherapy is one of the important means of clinically treating malignant tumors. According to statistics, about 50% of cancer patients need to receive radiotherapy during treatment. However, the radiotherapy resistance of tumor cells and the damage of radiation to normal tissues are the key factors restricting the effect of radiotherapy. In order to improve the effect of radiotherapy, researchers have developed a variety of radiosensitizers.

[0003] The radiosensitizers commonly used in clinical practice currently include the following categories:

[0004] Small molecule chemotherapy drugs: such as 5-fluorouracil, cisplatin, paclitaxel, etc. These drugs can enhance the radiosensitivity of tumor cells through different mechanisms of action (such as interfering with DNA synthesis, promoting DNA double-strand breaks, regulating cell cycle, etc.). However, this type of drug has the following disadvantages:

[0005] a) Serious side effects: Radiotherapy combined with these chemotherapy drugs often leads to severe systemic toxicity, such as bone marrow suppression, nephrotoxicity, etc., which makes it difficult for patients to bear.

[0006] b) Poor targeting: These drugs have effects on both normal cells and tumor cells, making it difficult to achieve specific sensitization of tumors.

[0007] Nitroimidazole radiosensitizers: such as pimonidazole, nimozole, and sodium glycidazole independently developed in my country. The mechanism of action of this type of drug is that it is reduced to a cytotoxic substance in an oxygen-deficient environment, thereby selectively killing hypoxic tumor cells. However, this type of drug also has the following problems:

[0008] a) The selectivity is not ideal: Although it has a certain selectivity for hypoxic cells, the overall selectivity for tumors is still not ideal.

[0009] b) Dose limitation: High-dose clinical application often causes severe neurotoxicity, while low doses are difficult to achieve the ideal sensitization effect.

[0010] c) Limited effectiveness: The sensitization effect on tumor cells in non-hypoxic areas is limited.

[0011] Nanomaterial radiotherapy sensitizers: In recent years, researchers have tried to use nanomaterials to develop new radiotherapy sensitizers. For example, nanoparticles based on high atomic number elements such as gold, platinum, and hafnium can improve the effect of radiotherapy by enhancing the photoelectric effect and Compton scattering. However, this type of nanomaterial still faces the following challenges:

[0012] a) Biosafety: The long-term biosafety of some inorganic nanomaterials still needs further evaluation.

[0013] b) Metabolic clearance: Some nanomaterials are difficult to degrade and eliminate in the body, which may bring potential long-term toxicity risks.

[0014] c) Insufficient targeting: Although nanomaterials can utilize the EPR effect to be enriched in tumor tissues, their targeting to specific structures within tumor cells (such as lipid droplets) is still insufficient.

[0015] Recent studies have shown that abnormal lipid droplet metabolism in tumor cells is closely related to radioresistance. Pagliari et al. found that in order to cope with the increase in reactive oxygen species (ROS) produced after radiotherapy, tumor cells need to regulate their ROS scavenging system, and lipid droplets play an important role in regulating excessive oxidative stress. The more lipid droplets there are, the stronger the survival ability of tumor cells after radiotherapy, which suggests that the lipid droplet content may contribute to the generation of radioresistance. Cruz et al. further found that targeting lipid metabolism can improve the effect of radiotherapy. However, no radiosensitizer targeting lipid droplets has been developed yet.

[0016] In terms of photosensitizers, thermally activated delayed fluorescence (TADF) materials have attracted the attention of researchers in recent years due to their unique photophysical properties. TADF materials have a small excited singlet-excited triplet energy level difference (ΔE ST ), which is conducive to achieving efficient intersystem crossing, thereby generating a large amount of reactive oxygen species (ROS). For example, Zhang et al. reported a TADF nanoparticle for photodynamic therapy, which showed good anti-tumor effect. The TADF material developed by Kabe and Adachi exhibits the characteristics of long-lasting luminescence, providing new possibilities for bioimaging and photodynamic therapy. However, the existing TADF photosensitizers still have the following shortcomings:

[0017] a) Lack of specific targeting: Most TADF photosensitizers lack the ability to target specific organelles such as lipid droplets.

[0018] b) Poor water solubility: Many TADF molecules are hydrophobic and require complex packaging or modification before they can be used in aqueous solutions.

[0019] c) The synergistic effects of photodynamic therapy and radiosensitization have not been fully explored.

[0020] In summary, the existing radiotherapy sensitizers and photosensitizers still have many deficiencies in terms of selectivity, toxicity, side effects, targeting, etc. Therefore, we propose a lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound and its application in tumor photodynamic radiosensitization therapy. Summary of the invention

[0021] The purpose of the present invention is to address the problems raised by the existing background technology. In order to achieve the above invention purpose, the present invention provides the following technical solutions: a lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound, the molecular structure of the TADF compound NIOH-Cz is:

[0022]

[0023] Among them, the NIOH-Cz molecule is composed of an electron donor (D) and an electron acceptor (A) connected by a conjugated skeleton, and adopts a DA-type molecular structure, in which the carbazole group serves as an electron donor and the naphthylimide group serves as an electron acceptor. The two groups are connected by a single bond to regulate the torsion angle between the donor and the acceptor and separate the HOMO and LUMO orbitals.

[0024] A method for synthesizing a lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound, wherein the synthesis route of NIOH-Cz is:

[0025]

[0026] As a preferred technical solution of the present invention, the following specific steps are included:

[0027] Step 1, synthesis of NIOMe-Br:

[0028] Compound 1 (13853.5 mg, 50 mmol), p-Anisidine (6773.25 mg, 55 mmol), and anhydrous ethanol (150 mL) were added to a dried 250 mL single-mouth bottle in sequence, heated to reflux, and stirred vigorously overnight. After overnight reaction, heating was stopped and cooled to room temperature. Filtered, washed with ethanol (50 mL × 3), and dried in an oven at 80 degrees to obtain the product. 17.500 g, yield: 91.6%.

[0029] Step 2, synthesis of NIOMe-Cz:

[0030] In a dried 250 mL single-mouth bottle, NIOMe-Br (229.3 mg, 0.6 mmol), Pd(dppf)Cl 2 (32.9 mg, 0.045 mmol), t-BuOK (151.5 mg, 2.25 mmol), Cz (150.5 mg, 0.9 mmol), add ultra-dry toluene (10 mL), and quickly add P(t-Bu) under argon atmosphere. 3 (18.2 mg, 0.09 mmol), heated to reflux overnight. After the reaction, cooled to room temperature, the solvent was removed by distillation under reduced pressure. Silica gel column chromatography (200-300 mesh silica gel, eluent: PE / EA=4 / 1). 215 mg, yield: 76.6%;

[0031] Step 3, Synthesis of NIOH-Cz:

[0032] In a dried 50 mL single-mouth bottle, NIOMe-Cz (163.0 mg, 0.35 mmol) was added, dissolved in ultra-dry dichloromethane (5 mL), and 1 M BBr was added. 3 The mixture was diluted with dichloromethane (1.75 mL, 1.75 mmol), replaced with argon three times, and stirred at room temperature. The reaction progress was monitored by TLC. After the reaction, a large amount of dichloromethane was added for dilution, washed with water three times, washed with saturated brine three times, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. Silica gel column chromatography (200-300 mesh silica gel, eluent: PE / EA=2 / 1) was used for separation. 108 mg, yield: 68.0%.

[0033] As a preferred technical solution of the present invention, the structure of the fluorescent compound is characterized as: NIOH-Cz

[0034] a) 1 H NMR (500MHz, Chloroform-D): δ8.86(d,J=7.7Hz,1H),8.73(dd,J 1 =

[0035] 7.2,J 2 =1.2Hz,1H),8.26-8.21(m,2H),7.96(d,J=7.7Hz,1H),7.88(dd,J 1 =8.5,J 2 =1.2Hz,1H),7.67(dd,J 1 =8.5,J 2 =7.2Hz,1H),7.43-7.34(m,4H),7.20-7.16(m,2H),7.08-7.05(m,2H),6.94-6.89(m,2H),6.73-6.65(m,1H).

[0036] b) 13 C NMR (126MHz, DMSO-d6): δ163.72,163.38,157.27,141.34,139.08,131.43,131.38,129.93,129.45,129.24,128.5 7,128.30,127.90,126.77,126.60,123.80,123.20,123.07,120.84,120.62,115.47,109.86,59.76,20.76,14.08.

[0037] c)HRMS(ESI)m / z:[M+H] + Calculate value for C 30 H 19 N 2 O 3 :455.13902; measured value:455.13818.

[0038] Application of a lipid droplet-targeted naphthylimide heat-activated delayed fluorescence compound in photodynamic radiosensitization therapy of tumors.

[0039] A lipid droplet-targeted naphthylimide-based heat-activated delayed fluorescence compound NIOH-Cz is used for photodynamic radiosensitization therapy of tumors.

[0040] A method for photodynamic radiosensitization of tumors using a lipid droplet-targeted naphthylimide thermally activated delayed fluorescent compound, characterized in that it comprises using an effective amount of NIOH-Cz, followed by light irradiation and radiotherapy.

[0041] As a preferred technical solution of the present invention, the TADF compound NIOH-Cz specifically targets lipid droplets in tumor cells.

[0042] As a preferred technical solution of the present invention, the TADF compound NIOH-Cz enhances the sensitivity of tumor cells to radiation during radiotherapy.

[0043] As a preferred technical solution of the present invention, the TADF compound NIOH-Cz is used to enhance the tumor treatment effect.

[0044] Compared with the prior art, the present invention has the following beneficial effects: 1. Efficient ROS generation capability:

[0045] The NIOH-Cz molecule achieves a small singlet-triplet energy level difference (ΔE) through a carefully designed DA structure. ST =0.05eV), promoting an efficient intersystem crossing (ISC) process. This enables the molecule to effectively generate triplet excitons under light conditions, thereby generating a large amount of reactive oxygen species (ROS). As shown above, NIOH-Cz produced significantly more ROS under light compared to the control group, which laid the foundation for its application in photodynamic therapy.

[0046] 2. Large Stokes shift:

[0047] NIOH-Cz molecules exhibit a large Stokes shift (λ) of 165 nm. abs =415nm,λ em=580nm). This property effectively reduces the self-absorption effect and increases the light penetration depth in biological tissues. The large Stokes shift also helps to improve the signal-to-noise ratio, giving NIOH-Cz an advantage in biological imaging and photodynamic therapy.

[0048] 3. Long fluorescence lifetime:

[0049] NIOH-Cz exhibits a long luminescence lifetime of microseconds (1.78 μs) in normoxic PBS solution. This long lifetime property enables NIOH-Cz to be distinguished from the short-lived autofluorescence in vivo, which is beneficial for improving the signal-to-noise ratio of imaging and the time window of photodynamic therapy.

[0050] 4. Excellent photodynamic therapy effect:

[0051] As mentioned above, under light conditions, NIOH-Cz not only significantly reduced the survival rate of A549 cells, but also significantly inhibited the growth of xenograft tumors derived from radiotherapy-resistant cell lines. This efficient photodynamic therapy effect is due to the high ROS production ability and good cellular uptake properties of NIOH-Cz.

[0052] 5. Good radiotherapy sensitization effect:

[0053] In vitro and in vivo experiments showed that compared with RT treatment alone or PDT treatment alone, NIOH-Cz-mediated PDT combined with radiotherapy showed a significant tumor inhibitory effect, revealing that targeting lipid droplets is a new and effective radiosensitization strategy.

[0054] 6. Good biosafety:

[0055] In vitro and in vivo studies have shown that NIOH-Cz-mediated PDT combined with radiotherapy has good biosafety. This good biocompatibility provides a guarantee for its safe use in biomedical applications.

[0056] 7. Simple molecular structure and synthesis method:

[0057] NIOH-Cz adopts a simple DA structure design, and the synthesis method is direct and efficient, which not only reduces the preparation cost, but also increases the possibility of large-scale production and clinical transformation.

[0058] 8. Maintain TADF properties in solution state:

[0059] Unlike many materials that need to be in solid or aggregated state to exert TADF effect, NIOH-Cz still maintains good TADF properties in solution state, which makes it more suitable for application in biological environment.

[0060] 9. Good lipid droplet targeting:

[0061] Based on the molecular structure characteristics of NIOH-Cz, it has good lipid droplet targeting. This targeting can improve the selective accumulation of photosensitizers in tumor tissues and further enhance the therapeutic effect.

[0062] 10. Multifunctional application potential:

[0063] As mentioned above, NIOH-Cz can be used not only for photodynamic therapy, but also for radiosensitization, biological imaging, etc. This versatility provides the possibility for the development of comprehensive diagnosis and treatment strategies.

[0064] 11. Provide a platform for subsequent improvements:

[0065] The structure of NIOH-Cz provides a basis for further functionalization and performance optimization. For example, its targeting and bioavailability can be enhanced by introducing specific targeting groups or constructing nanocarrier systems.

[0066] The NIOH-Cz molecule provided by the present invention achieves excellent properties such as efficient ROS generation, long fluorescence lifetime, and large Stokes shift through its unique molecular design, showing application prospects in tumor photodynamic therapy and radiotherapy sensitization. Its simple structure and synthesis method, as well as good biocompatibility, lay the foundation for subsequent clinical translation research. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The molecular structure diagram of NIOH-Cz provided by the present invention;

[0068] Figure 2 The synthetic route of NIOH-Cz provided by the present invention;

[0069] Figure 3 The present invention provides (a) absorption spectra of NIOH-Cz in DMSO and PBS; (b) emission spectra of NIOH-Cz in DMSO and PBS, λ ex =415nm;

[0070] Figure 4 The present invention provides (a) NIOH-Cz molecular structure, the electron density distribution of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), and the energy difference ΔE between the singlet state and the triplet state ST Figure. (b) Photoexcited transient fluorescence decay lifetime curve of NIOH-Cz in PBS; (c) Low-temperature fluorescence phosphorescence spectrum of NIOH-Cz in 2-methyltetrahydrofuran;

[0071] Figure 5The flow cytometry analysis of NIOH-Cz uptake and mean fluorescence intensity histogram at different time points in A549 cells provided by the present invention;

[0072] Figure 6 The co-localization imaging provided by the present invention detects the lipid droplet targeting of NIOH-Cz (10 μM). NIOH-Cz (green signal), excitation wavelength 488 nm, collection wavelength 500-700 nm; Nile Red (red signal), excitation wavelength 561 nm, collection wavelength 580-700 nm;

[0073] Figure 7 The present invention provides (a) NIOH-Cz (20 μM) under white light irradiation (20 mW / cm 2 ) ROS generation capacity of NIOH-Cz (10 μM) in A549 cells after laser irradiation for 10 min. DCFH-DA (10 μM) was used as a ROS detection probe, with an excitation wavelength of 480 nm and a maximum emission wavelength of 525 nm. (b) ROS generation capacity of NIOH-Cz (10 μM) in A549 cells after laser irradiation for 10 min. DCFH-DA (10 μM) was used as a ROS indicator probe, with an excitation wavelength of 480 nm and a maximum emission wavelength of 525 nm. ex =488nm,λ em =500-600nm;

[0074] Figure 8 The present invention provides (a) CCK8 method to detect the light and dark toxicity of different concentrations of NIOH-Cz in A549 cells (20mW / cm 2 , 10min). (b) Live / dead staining evaluation under different laser intensities (20mW / cm 2 or 50mW / cm 2 , 10min), killing ability of NIOH-Cz (10μM) on A549 cells. Calcein AM (green signal, living cells), λ ex =488nm,λ em =500-550nm; PI (red signal, dead cells), λ ex =561nm,λ em =590-640nm;

[0075] Fig. 9 The present invention provides (a) confocal imaging of NIOH-Cz in CMT167, CMT167-R, A549, and A549-R cells. NIOH-Cz:λ ex =488nm,λ em=500-700nm. (b) Fluorescence quantitative analysis of fluorescence images using ImageJ software. Mean±SD, n=4. (c) Two-photon confocal imaging of NIOH-Cz in A549 and A549-R cells. NIOH-Cz:λ ex =880nm,λ em =525±20nm. Flow cytometry analysis of the uptake and mean fluorescence intensity histogram of NIOH-Cz (d) and commercial lipid droplet dye Nile Red (e) in A549 and A549-R cells;

[0076] Fig.10 The present invention provides (a) live / dead staining to evaluate the killing ability of PDT and X-ray combined treatment on A549-R cells. (b) Annexin V-FITC / PI double staining flow cytometry to detect cell apoptosis. (c) Immunofluorescence staining to detect the expression of γ-H2AX in different treatment groups. (d) WB detection of the expression of γ-H2AX in different treatment groups;

[0077] Fig.11 (a) Schematic diagram of the treatment scheme provided by the present invention. (b) Graph of tumor volume changes during treatment. (c) Graph of nude mouse weight changes during treatment. (d) Tumor images of different treatment groups after treatment. (e) Tumor weights of different treatment groups after treatment. (f) H&E staining of tumor tissues after different treatments;

[0078] Fig.12 The H&E tissue staining images of the five main organs of the A549-R xenograft tumor-bearing nude mice after different treatments provided by the present invention. DETAILED DESCRIPTION

[0079] 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 described clearly and completely in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0080] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents some embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. It should be noted that the embodiments of the present invention and the features and technical solutions in the embodiments can be combined with each other without conflict. It should be noted that similar numbers and letters represent similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0081] Example 1: A lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound, the molecular structure of the TADF compound NIOH-Cz is:

[0082]

[0083] Among them, the NIOH-Cz molecule is composed of an electron donor (D) and an electron acceptor (A) connected by a conjugated skeleton, and adopts a DA-type molecular structure, in which the carbazole group serves as an electron donor and the naphthylimide group serves as an electron acceptor. The two groups are connected by a single bond to regulate the torsion angle between the donor and the acceptor and separate the HOMO and LUMO orbitals.

[0084] A method for synthesizing a lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound. The synthetic route and molecular structure of NIOH-Cz are as follows:

[0085]

[0086] The specific steps include:

[0087] Step 1, synthesis of NIOMe-Br:

[0088] Compound 1 (13853.5 mg, 50 mmol), p-Anisidine (6773.25 mg, 55 mmol), and anhydrous ethanol (150 mL) were added to a dried 250 mL single-mouth bottle in sequence, heated to reflux, and stirred vigorously overnight. After overnight reaction, heating was stopped and cooled to room temperature. Filtered, washed with ethanol (50 mL × 3), and dried in an oven at 80 degrees to obtain the product. 17.500 g, yield: 91.6%.

[0089] Step 2, synthesis of NIOMe-Cz:

[0090] In a dried 250 mL single-mouth bottle, NIOMe-Br (229.3 mg, 0.6 mmol), Pd(dppf)Cl 2 (32.9 mg, 0.045 mmol), t-BuOK (151.5 mg, 2.25 mmol), Cz (150.5 mg, 0.9 mmol), add ultra-dry toluene (10 mL), and quickly add P(t-Bu) under argon atmosphere. 3 (18.2 mg, 0.09 mmol), heated to reflux overnight. After the reaction, cooled to room temperature, the solvent was removed by distillation under reduced pressure. Silica gel column chromatography (200-300 mesh silica gel, eluent: PE / EA=4 / 1). 215 mg, yield: 76.6%;

[0091] Step 3, Synthesis of NIOH-Cz:

[0092] In a dried 50 mL single-mouth bottle, NIOMe-Cz (163.0 mg, 0.35 mmol) was added, dissolved in ultra-dry dichloromethane (5 mL), and 1 M BBr was added. 3 The mixture was added with dichloromethane solution (1.75 mL, 1.75 mmol), replaced with argon three times, and stirred at room temperature. The reaction progress was monitored by TLC thin layer chromatography. After the reaction, a large amount of dichloromethane was added to dilute, washed with water three times, washed with saturated brine three times, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. Silica gel column chromatography (200-300 mesh silica gel, eluent: PE / EA=2 / 1) was used for separation. 108 mg, yield: 68.0%.

[0093] The structure of the fluorescent compound is characterized as: NIOH-Cz

[0094] a) 1 H NMR (500MHz, Chloroform-D): δ8.86(d,J=7.7Hz,1H),8.73(dd,J 1 =

[0095] 7.2,J 2 =1.2Hz,1H),8.26-8.21(m,2H),7.96(d,J=7.7Hz,1H),7.88(dd,J 1 =8.5,J 2 =1.2Hz,1H),7.67(dd,J 1 =8.5,J 2 =7.2Hz,1H),7.43-7.34(m,4H),7.20-7.16(m,2H),7.08-7.05(m,2H),6.94-6.89(m,2H),6.73-6.65(m,1H).

[0096] b) 13 C NMR (126MHz, DMSO-d6): δ163.72,163.38,157.27,141.34,139.08,131.43,131.38,129.93,129.45,129.24,128.5 7,128.30,127.90,126.77,126.60,123.80,123.20,123.07,120.84,120.62,115.47,109.86,59.76,20.76,14.08.

[0097] c)HRMS(ESI)m / z:[M+H] + Calculate value for C 30 H 19 N2 O 3 :455.13902; measured value: 455.13818.

[0098] Application of a lipid droplet-targeted naphthylimide heat-activated delayed fluorescence compound in photodynamic radiosensitization therapy of tumors.

[0099] A lipid droplet-targeted naphthylimide-based heat-activated delayed fluorescence compound NIOH-Cz is used for photodynamic radiosensitization therapy of tumors.

[0100] A method for photodynamic radiosensitization of tumors using a lipid droplet-targeted naphthylimide thermally activated delayed fluorescent compound, characterized in that it comprises using an effective amount of NIOH-Cz, followed by light irradiation and radiotherapy.

[0101] The TADF compound NIOH-Cz specifically targets lipid droplets in tumor cells.

[0102] The TADF compound NIOH-Cz enhances the sensitivity of tumor cells to radiation during radiotherapy.

[0103] The TADF compound NIOH-Cz is used in combination with other anti-tumor treatments to enhance the efficacy of tumor treatment.

[0104] Example 2: A lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound, wherein the TADF compound is NIOH-Cz.

[0105] The structure of TADF molecule NIOH-Cz:

[0106] The NIOH-Cz molecule is composed of an electron donor (D) and an electron acceptor (A) connected by a conjugated skeleton, using a DA-type molecular design strategy, in which the carbazole group acts as an electron donor and the naphthylimide group acts as an electron acceptor.

[0107] Molecular structure of NIOH-Cz:

[0108]

[0109] The meaning of the NIOH-Cz molecule is as follows:

[0110] a) The carbazole group has a strong electron-donating ability, which is conducive to the formation of intramolecular charge transfer (ICT) state.

[0111] b) The naphthalene imide group has a strong electron-withdrawing ability and can stabilize the ICT state.

[0112] c) The two groups are connected by a single bond, which can regulate the torsion angle between the donor and the acceptor, which is beneficial to separate the HOMO and LUMO orbitals and reduce the singlet-triplet energy level difference (ΔEST ).

[0113] A method for synthesizing a lipid droplet-targeted naphthylimide thermally activated delayed TADF compound NIOH-Cz, wherein the synthetic route of NIOH-Cz is:

[0114]

[0115] The specific steps of the synthetic route of NIOH-Cz are as follows:

[0116] Synthesis of NIOMe-Br:

[0117] Compound 1 (13853.5 mg, 50 mmol), p-Anisidine (6773.25 mg, 55 mmol), and anhydrous ethanol (150 mL) were added to a dried 250 mL single-mouth bottle in sequence, heated to reflux, and stirred vigorously overnight. After overnight reaction, heating was stopped and cooled to room temperature. Filtered, washed with ethanol (50 mL × 3), and dried in an oven at 80 degrees to obtain the product. 17.500 g, yield: 91.6%.

[0118] Synthesis of NIOMe-Cz:

[0119] In a dried 250 mL single-mouth bottle, NIOMe-Br (229.3 mg, 0.6 mmol), Pd(dppf)Cl 2 (32.9 mg, 0.045 mmol), t-BuOK (151.5 mg, 2.25 mmol), Cz (150.5 mg, 0.9 mmol), add ultra-dry toluene (10 mL), and quickly add P(t-Bu) under argon atmosphere. 3 (18.2 mg, 0.09 mmol), heated to reflux overnight. After the reaction, cooled to room temperature, the solvent was removed by distillation under reduced pressure. Silica gel column chromatography (200-300 mesh silica gel, eluent: PE / EA = 4 / 1). 215 mg, yield: 76.6%

[0120] Synthesis of NIOH-Cz:

[0121] In a dried 50 mL single-mouth bottle, NIOMe-Cz (163.0 mg, 0.35 mmol) was added, dissolved in ultra-dry dichloromethane (5 mL), and 1 M BBr was added. 3The mixture was added with dichloromethane solution (1.75 mL, 1.75 mmol), replaced with argon three times, and stirred at room temperature. The reaction progress was monitored by TLC thin layer chromatography. After the reaction, a large amount of dichloromethane was added to dilute, washed with water three times, washed with saturated brine three times, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. Silica gel column chromatography (200-300 mesh silica gel, eluent: PE / EA=2 / 1) was used for separation. 108 mg, yield: 68.0%.

[0122] Characterization of NIOH-Cz:

[0123] Structural characterization:

[0124] NIOH-Cz:

[0125] a) 1 H NMR (500MHz, Chloroform-D): δ8.86(d,J=7.7Hz,1H),8.73(dd,J 1 =

[0126] 7.2,J 2 =1.2Hz,1H),8.26-8.21(m,2H),7.96(d,J=7.7Hz,1H),7.88(dd,J 1 =8.5,J 2 =1.2Hz,1H),7.67(dd,J 1 =8.5,J 2 =7.2Hz,1H),7.43-7.34(m,4H),7.20-7.16(m,2H),7.08-7.05(m,2H),6.94-6.89(m,2H),6.73-6.65(m,1H).

[0127] b) 13 C NMR (126MHz, DMSO-d6): δ163.72,163.38,157.27,141.34,139.08,131.43,131.38,129.93,129.45,129.24,128.5 7,128.30,127.90,126.77,126.60,123.80,123.20,123.07,120.84,120.62,115.47,109.86,59.76,20.76,14.08.

[0128] c)HRMS(ESI)m / z:[M+H] + Calculate value for C 30 H 19 N 2 O 3:455.13902; measured value: 455.13818.

[0129] Photophysical properties characterization:

[0130] The absorption spectra and fluorescence emission spectra of NIOH-Cz in different solvents were measured. The results showed that in DMSO solution, the maximum absorption peak (λ abs )=410nm, maximum emission peak (λ em )=590nm, Stokes shift=180nm; In PBS solution, the maximum absorption peak of NIOH-Cz (λ abs )=415nm, maximum emission peak (λ em )=580nm, Stokes shift=165nm. The absorption spectrum of NIOH-Cz has no obvious difference in DMSO and PBS, while the fluorescence spectrum shows obvious solvatochromic effect in solvents of different polarities, which indicates that NIOH-Cz has intramolecular charge transfer (ICT) effect. The lowest energy absorption band of NIOH-Cz at 415nm is the charge transfer absorption related to ICT from the carbazole part to the naphthylimide part. NIOH-Cz has a large Stokes shift (165nm) and strong ICT properties, which are in line with the typical characteristics of TADF compounds.

[0131] Through time-dependent density functional theory (TD-DFT) calculations and spectral tests, it was found that this type of fluorescent molecule has good TADF properties, with a fluorescence lifetime (τ) of 1.78 μs and ΔE ST =0.05eV.

[0132] Application of NIOH-Cz in extracorporeal photodynamic therapy:

[0133] To evaluate the potential application of NIOH-Cz in photodynamic therapy, we conducted in vitro cell experiments. Human non-small cell lung cancer cell line A549 and its radiotherapy-resistant cell line A549-R were used as model cell lines to investigate the photodynamic therapy effect of NIOH-Cz in vitro and in vivo.

[0134] Cellular Uptake and Lipid Droplet Targeting Analysis:

[0135] A549 cells were incubated with 10 μM NIOH-Cz for different time periods, and the uptake efficiency of NIOH-Cz in cells was analyzed by flow cytometry. The results showed that NIOH-Cz could enter cells after 10 minutes of incubation, and as the NIOH-Cz action time increased, the average fluorescence of the cells gradually increased, and the cell uptake of NIOH-Cz reached saturation at about 2 hours. These results indicate the effective cellular uptake of the photosensitizer NIOH-Cz.

[0136] NIOH-Cz and the commercial lipid droplet stain Nile Red were used to perform cell co-localization experiments in tumor cells. Its distribution in lipid droplets was analyzed by laser confocal scanning microscopy. Then, ImageJ was used to analyze the Pearson correlation coefficient in single cells, which was as high as 0.934, proving that NIOH-Cz has good specific labeling ability for intracellular lipid droplets.

[0137] Colocalization imaging was used to detect the lipid droplet targeting of NIOH-Cz (10 μM). NIOH-Cz (green signal), excitation wavelength 488 nm, collection wavelength 500-700 nm; Nile Red (red signal), excitation wavelength 561 nm, collection wavelength 580-700 nm.

[0138] ROS-producing ability of NIOH-Cz:

[0139] DCFH-DA fluorescent probe was used to detect ROS generated by NIOH-Cz. 2 ), NIOH-Cz showed significant ROS production ability. Similarly, this molecule also showed good ROS production ability in A549 cells.

[0140] (a) NIOH-Cz (20 μM) under white light irradiation (20 mW / cm 2 ) ROS generation capacity of NIOH-Cz (10 μM) in A549 cells after laser irradiation for 10 min. DCFH-DA (10 μM) was used as a ROS detection probe, with an excitation wavelength of 480 nm and a maximum emission wavelength of 525 nm. (b) ROS generation capacity of NIOH-Cz (10 μM) in A549 cells after laser irradiation for 10 min. DCFH-DA (10 μM) was used as a ROS indicator probe, with an excitation wavelength of 480 nm and a maximum emission wavelength of 525 nm. ex =488nm,λ em =500-600nm.

[0141] Effects of extracorporeal photodynamic therapy:

[0142] NIOH-Cz significantly reduced the survival rate of A549 cells under light conditions, showing its good PDT effect.

[0143] 1. Cell survival rate: CCK-8 method was used to evaluate the survival rate of A549 cells after different concentrations of NIOH-Cz and light treatment. The results showed that NIOH-Cz could significantly reduce the cell survival rate under light conditions, and its effect increased with the increase of NIOH-Cz concentration.

[0144] 2. Live / dead cell staining: Calcein-AM and propidium iodide (PI) staining were used to observe cell activity. The results showed that NIOH-Cz could effectively kill A549 cells under light conditions, further proving its PDT effect.

[0145] External radiotherapy sensitization effect:

[0146] Laser confocal scanning microscopy, two-photon imaging and flow cytometry analysis showed that A549 radiotherapy-resistant cells (A549-R) had significantly more lipid droplets than A549 cells ( Fig. 9 ), indicating that lipid droplet content may contribute to radioresistance. Therefore, targeting lipid droplets is likely to become a new and effective radiosensitization strategy.

[0147] (a) Confocal imaging of NIOH-Cz in CMT167, CMT167-R, A549, and A549-R cells. NIOH-Cz:λ ex =488nm,λ em =500-700nm. (b) Fluorescence quantitative analysis of fluorescence images using ImageJ software. Mean±SD, n=4. (c) Two-photon confocal imaging of NIOH-Cz in A549 and A549-R cells. NIOH-Cz:λ ex =880nm,λ em =525±20 nm. Flow cytometry analysis of the uptake and mean fluorescence intensity histogram of NIOH-Cz (d) and commercial lipid droplet dye Nile Red (e) in A549 and A549-R cells.

[0148] The radiotherapy-resistant cells were treated with different treatments: ①PBS (Control group); ②NIOH-Cz; ③PBS+X-ray (6Gy) (RT treatment group); ④NIOH-Cz+white light irradiation (Light: 50mW / cm 2 , 10min) (PDT treatment group); ⑤NIOH-Cz+Light+X-ray (PDT+RT combined treatment group), through cell live / dead staining, cell apoptosis detection and γ-H2AX immunofluorescence staining, WB detection found that A549-R cells effectively induced cell death and apoptosis after receiving PDT combined with RT treatment ( Fig.10 a and 10b), and PDT treatment can aggravate DNA double-strand break damage and hinder damage repair, resulting in persistent damage ( Fig.10 c and 10d), indicating that NIOH-Cz-mediated PDT treatment produced a radiosensitizing effect.

[0149] In vivo radiosensitization effect:

[0150] The A549-R xenograft tumor-bearing nude mouse model was used to evaluate the therapeutic effect of radiosensitizers in living tumor treatment. Fig.11 The results showed that compared with RT alone or PDT alone, PDT combined with radiotherapy showed a significant tumor inhibition effect and had good biosafety.

[0151] 1. Tumor growth curve: Observe the tumor growth of mice in different treatment groups. The results show ( Fig.11 b), Tumor growth was significantly inhibited in the PDT combined with radiotherapy group.

[0152] 2. Body weight changes: Record the changes in body weight of mice in different treatment groups to evaluate the toxic and side effects of treatment. The results showed that ( Fig.11 c), there was no significant difference in the body weight changes of mice in each group, indicating the good safety of NIOH-Cz-mediated PDT treatment.

[0153] 3. Tumor weight: At the end of the experiment, the tumor weight of each group of mice was measured. The results showed ( Fig.11 d and 11e), the tumor weight was significantly reduced in the PDT combined with radiotherapy group.

[0154] 4. Hematoxylin-eosin (H&E) staining of tumor tissue: At the end of the experiment, tumor tissue was obtained and histologically analyzed by H&E staining. The results showed that compared with tumor cells in other groups, tumor cells were significantly necrotic after PDT combined with radiotherapy.

[0155] Through the above technical scheme, the present invention successfully designed and synthesized a new type of TADF small molecule NIOH-Cz targeting lipid droplets. This molecule has excellent photophysical properties, efficient ROS generation ability and good photodynamic therapy effect, and provides a potential new photosensitizer for photodynamic therapy and radiotherapy sensitization of tumors. This molecule effectively improves the effect of tumor radiotherapy, overcomes radiotherapy resistance, and reduces damage to normal tissues. It is a safe and efficient small molecule radiosensitizer.

[0156] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the above specific implementation methods. Therefore, any modification or equivalent replacement of the present invention; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the invention are included in the scope of the claims of the present invention.

Claims

1. A lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound, characterized in that: The molecular structure of the TADF compound NIOH-Cz is: Among them, the NIOH-Cz molecule is composed of an electron donor (D) and an electron acceptor (A) connected by a conjugated skeleton, and adopts a DA-type molecular structure, in which the carbazole group serves as an electron donor and the naphthylimide group serves as an electron acceptor. The two groups are connected by a single bond to regulate the torsion angle between the donor and the acceptor and separate the HOMO and LUMO orbitals.

2. A method for synthesizing a lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound, characterized in that: The synthetic route molecular structure of NIOH-Cz is:

3. The method for synthesizing the lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound according to claim 2, characterized in that: The specific steps include: Step 1, Synthesis of NIOMe-Br: Compound 1 (13853.5 mg, 50 mmol), p-Anisidine (6773.25 mg, 55 mmol), and anhydrous ethanol (150 mL) were added to a dried 250 mL single-mouth bottle in sequence, heated to reflux, and stirred vigorously overnight; after overnight reaction, heating was stopped, and the mixture was cooled to room temperature; filtered, washed with ethanol (50 mL × 3), and dried in an oven at 80 degrees to obtain the product; 17.500 g, yield: 91.6%; Step 2, synthesis of NIOMe-Cz: NIOMe-Br (229.3 mg, 0.6 mmol), Pd(dppf)Cl2 (32.9 mg, 0.045 mmol), t-BuOK (151.5 mg, 2.25 mmol), Cz (150.5 mg, 0.9 mmol) were added to a dried 250 mL single-mouth bottle in sequence, and ultra-dry toluene (10 mL) was added. P(t-Bu)3 (18.2 mg, 0.09 mmol) was quickly added under an argon atmosphere, and heated to reflux overnight; after the reaction was completed, the mixture was cooled to room temperature and the solvent was removed by distillation under reduced pressure; silica gel column chromatography (200-300 mesh silica gel, eluent: PE / EA=4 / 1); 215 mg, yield: 76.6%; Step 3, Synthesis of NIOH-Cz: NIOMe-Cz (163.0 mg, 0.35 mmol) was added to a dried 50 mL single-mouth bottle in sequence, and ultra-dry dichloromethane (5 mL) was used to dissolve it. A 1 M BBr3 dichloromethane solution (1.75 mL, 1.75 mmol) was added, and the gas was replaced with argon three times and stirred at room temperature; TLC thin-layer chromatography was used to monitor the progress of the reaction; after the reaction, a large amount of dichloromethane was added to dilute, the product was washed with water three times, with saturated brine three times, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure; silica gel column chromatography was used for separation (200-300 mesh silica gel, eluent: PE / EA=2 / 1); 108 mg, yield: 68.0%.

4. The lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound according to claim 1, characterized in that: The structure of the fluorescent compound is characterized as: NIOH-Cz a) 1 H NMR(500MHz,Chloroform-D):δ8.86(d,J=7.7Hz,1H),8.73(dd,J1= 7.2, J2=1.2Hz, 1H), 8.26-8.21 (m, 2H), 7.96 (d, J=7.7Hz, 1H), 7.88 (dd, J1=8.5, J2=1.2Hz, 1H), 7.67 (dd, J1=8.5 ,J2=7.2Hz,1H),7.43-7.34(m,4H),7.20-7.16(m,2H),7.08-7.05(m,2H),6.94-6.89(m,2H),6.73-6.65(m,1H). b) 13 C NMR(126MHz,DMSO-d6):δ163.72,163.38,157.27,141.34,139.08,131.43,131.38,129.93,129.45,129.24,128.57,128.30,127.90,126.77,126.60,123.80,123.20,123.07,120.84,120.62,115.47,109.86,59.76,20.76,14.

08. c)HRMS(ESI)m / z:[M+H] + Calculate value for C 30 H 19 N2O3:455.13902; measured value:455.13818.

5. Use of a lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound according to claim 1 in photodynamic radiosensitization therapy for tumors.

6. A pharmaceutical composition prepared from the lipid droplet-targeted naphthylimide thermally activated delayed fluorescent compound NIOH-Cz according to claim 1, for use in photodynamic radiosensitization therapy of tumors.

7. A method for photodynamic radiosensitization of tumors using a lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound according to claim 1, characterized in that: It includes taking an effective amount of NIOH-Cz, followed by light irradiation and radiation therapy.

8. The lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound according to claim 1, characterized in that: The TADF compound NIOH-Cz specifically targets lipid droplets in tumor cells.

9. The lipid droplet-targeted naphthylimide thermally activated delayed fluorescence compound according to claim 1, characterized in that: The TADF compound NIOH-Cz enhances the sensitivity of tumor cells to radiation during radiotherapy.

10. The lipid droplet-targeting naphthylimide thermally activated delayed fluorescence compound according to claim 1, characterized in that: The TADF compound NIOH-Cz is used to enhance the tumor treatment effect.

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