Application of an organic photoacid generator in the preparation of antitumor drugs

Abstract

This invention relates to the field of biomedical technology, specifically to the application of an organic photosensitive acid generator in the preparation of antitumor drugs. In this invention, an organic photoresponsive molecular system is constructed. The organic photosensitive acid generator possesses the synergistic effect of photoacid therapy (PAT) and photodynamic therapy (PDT), enhancing the efficacy of photoactivated therapy and improving the reliability and effectiveness of tumor treatment. Under ultraviolet light irradiation of a specific wavelength, the organic photosensitive acid generator produces a large amount of H₂. + Reactive oxygen species (ROS) can damage the DNA, proteins, and lipids of tumor cells, thereby inducing cell death.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of an organic photoacid generator in the preparation of antitumor drugs. Background Technology

[0002] Organic photoresponsive molecular switches possess excellent spatiotemporal control capabilities and non-invasive activation properties, demonstrating enormous application potential in fields such as bioimaging and precision medicine. Through rational molecular design, these molecular systems can achieve controlled release of functional groups upon photoexcitation, thereby inducing a series of biological reactions. They have become key molecular tools driving the innovative development of photocontrolled therapy strategies, providing important support for precise regulation of the treatment process and enhancing treatment specificity.

[0003] Photodynamic therapy (PDT) is a classic light-controlled therapy. Its core mechanism involves the interaction of an excited-state photosensitizer with oxygen molecules to generate reactive oxygen species (ROS), which induce apoptosis to achieve a therapeutic effect. However, this therapy faces significant limitations in clinical application: the tumor microenvironment is generally hypoxic, which severely inhibits the ROS generation efficiency of PDT and significantly weakens the therapeutic effect. To address this issue, type I photodynamic therapy has emerged. It generates ROS through an electron transfer reaction between a photosensitizer and a substrate, maintaining therapeutic activity even under low oxygen concentration conditions, effectively compensating for the high oxygen dependence of traditional type II photodynamic therapy.

[0004] Besides oxidative stress, intracellular pH homeostasis is crucial for tumor metabolic regulation. Tumor cells possess unique metabolic characteristics: extracellular acidification and intracellular alkalization. This abnormal pH gradient provides favorable conditions for tumor cell proliferation and tolerance to hypoxic environments. Studies have confirmed that inducing intracellular acidification can effectively inhibit tumor cell glycolysis, interfere with their adaptation to hypoxic environments, disrupt their metabolic homeostasis, and thus inhibit tumor progression. Photoacid therapy (PAT) can release H+ through light-controlled release... + Precise regulation of intracellular pH has led to its being considered a potential adjuvant therapy in hypoxic tumor environments. In recent years, integrating the functions of photosensitive oxidative stress (PAT) and photosensitive catalytic decomposition (PDT) into a single molecular system to synergistically induce oxidative stress and metabolic imbalance under unified photoexcitation conditions has become an important development direction in the field of photocontrolled tumor therapy, aiming to improve the therapeutic effect of hypoxic tumors through multi-mechanism synergy. However, the design strategies and structural types of related materials are not yet diverse enough, necessitating the development of novel functionally integrated photoresponsive molecular systems. Summary of the Invention

[0005] Based on this, the present invention provides an application of an organic photoacid generator in the preparation of antitumor drugs, which at least solves one problem in the prior art.

[0006] In a first aspect, the present invention provides the use of an organic photosensitive acid generator in the preparation of an antitumor drug, wherein the organic photosensitive acid generator has a structure as shown in one of the following formulas:

[0007] , , , ;

[0008] Wherein, X is F, Cl, Br or I; Y is C, Si, O, S or Se; Z is O or S; and R1, R2, R3, R4 are independently selected from hydrogen and C1-C8 alkyl groups.

[0009] In a second aspect, the present invention provides an antitumor drug comprising an organic photosensitive acid generator, said organic photosensitive acid generator having a structure as shown in one of the following formulas:

[0010] , , , ;

[0011] Wherein, X is F, Cl, Br or I; Y is C, Si, O, S or Se; Z is O or S; and R1, R2, R3, R4 are independently selected from hydrogen and C1-C8 alkyl groups.

[0012] Due to the adoption of the above technical solutions, the embodiments of the present invention have at least the following beneficial effects: an organic photoresponsive molecular system is constructed, and the organic photoacid generator has the synergistic effect of photoacid therapy (PAT) and photodynamic therapy (PDT), which can enhance the effect of photoactivated therapy and improve the reliability and effectiveness of tumor treatment; under ultraviolet light irradiation of a specific wavelength, the organic photoacid generator produces a large amount of H + Reactive oxygen species (ROS) can damage the DNA, proteins, and lipids of tumor cells, thereby inducing cell death. Attached Figure Description

[0013] Figure 1 This is the absorption spectrum of CzC after light irradiation in an embodiment of the present invention.

[0014] Figure 2 This is a graph showing the relationship between the absorption intensity of CzC at 370nm and the irradiation time after light irradiation in an embodiment of the present invention.

[0015] Figure 3 These are thin-layer chromatographic images of CzC before and after light irradiation in an embodiment of the present invention.

[0016] Figure 4 This is a photograph of pH test paper labeled with CzC solution exposed to light, as shown in an embodiment of the present invention.

[0017] Figure 5 The example of this invention is CzC after being irradiated with light in deuterated chloroform. 1 H nuclear magnetic resonance spectrum.

[0018] Figure 6 This is the thermogravimetric analysis spectrum of CzC in an embodiment of the present invention.

[0019] Figure 7 This is a differential scanning calorimetry (DSC) spectrum of CzC in an embodiment of the present invention.

[0020] Figure 8 This is a schematic diagram of the crystal structure and intermolecular interactions of CzC in an embodiment of the present invention.

[0021] Figure 9 This is a schematic diagram of the stacking method of CzC crystals in an embodiment of the present invention.

[0022] Figure 10 This is a laser confocal scanning microscope image of 4T-1 cells incubated with CzC in an embodiment of the present invention.

[0023] Figure 11 This is a quantitative analysis diagram of 4T-1 cells at different time points after incubation with CzC in an embodiment of the present invention.

[0024] Figure 12 This is a graph showing the detection results of pH value and reactive oxygen species level in 4T-1 cells treated with CzC in an embodiment of the present invention.

[0025] Figure 13 This is a quantitative analysis diagram of glutathione in 4T-1 cells after different treatments in an embodiment of the present invention.

[0026] Figure 14 This is a quantitative analysis diagram of superoxide dismutase in 4T-1 cells after different treatments in this embodiment of the invention.

[0027] Figure 15 The figure shows the cytotoxicity test results of different concentrations of CzC on 4T-1 cells in an embodiment of the present invention.

[0028] Figure 16 This is a diagram showing the fluorescence staining results of CzC-induced 4T-1 cell death in an embodiment of the present invention.

[0029] Figure 17 This figure shows the results of flow cytometry analysis of apoptosis in 4T-1 cells after different treatments in this embodiment of the invention.

[0030] Figure 18 This is a quantitative analysis diagram of the apoptosis level of 4T-1 cells detected by flow cytometry after different treatments in this embodiment of the invention.

[0031] Figure 19 This is a Western blot image showing the results of protein immunoblotting detection of apoptosis-related proteins in cancer cells treated with CzC in an embodiment of the present invention.

[0032] Figure 20 This is a quantitative analysis diagram of the expression levels of Bax, Bcl-2, and Caspase-3 in an embodiment of the present invention.

[0033] Figure 21 This is a graph showing the relative tumor volume growth curves of mice after different treatments within 14 days in an embodiment of the present invention.

[0034] Figure 22 This is a graph showing the weight changes of mice over 14 days after different treatments in this embodiment of the invention.

[0035] Figure 23 This is a graph showing the analysis of liver function indicators in mice after different treatments in this embodiment of the invention.

[0036] Figure 24 This is a graph showing the analysis of renal function indicators in mice after different treatments in this embodiment of the invention.

[0037] Figure 25 This is a graph showing the analysis of blood routine indicators of mice after different treatments in this embodiment of the invention.

[0038] Figure 26 This is a histological analysis diagram of hematoxylin-eosin stained sections of major organs of mice after different treatments in this embodiment of the invention. Detailed Implementation

[0039] The following will provide a clear and complete description of the concept and technical effects of the present invention, so as to fully explain the purpose, solution and effects of the present invention.

[0040] In a first aspect, the present invention provides the use of an organic photosensitive acid generator in the preparation of an antitumor drug, wherein the organic photosensitive acid generator has a structure as shown in one of the following formulas:

[0041] , , , ;

[0042] Wherein, X is F, Cl, Br or I; Y is C, Si, O, S or Se; Z is O or S; and R1, R2, R3, R4 are independently selected from hydrogen and C1-C8 alkyl groups.

[0043] The organic photoacid generator can simultaneously generate reactive oxygen species (ROS) and protons (H+) under single-wavelength light excitation. +The release of ) allows for the synergistic therapeutic effects of photodynamic therapy and photoacid therapy.

[0044] In some optional embodiments, the antitumor drug is an antitumor photodynamic therapy drug, and the excitation wavelength of the antitumor photodynamic therapy drug is 365 nm. Under 365 nm laser irradiation, the organic photoacid generator can produce a large amount of ROS and H₂. + The two work synergistically to influence the intracellular environment of tumor cells. On the one hand, H... + Changes in secretion levels can alter intracellular acid-base balance, affecting normal cellular physiological functions. On the other hand, ROS production disrupts intracellular redox balance, leading to inhibition of antioxidant systems (glutathione and superoxide dismutase), preventing the timely removal of excess ROS, thus exacerbating cellular oxidative damage and promoting apoptosis. The aforementioned organic photoacid generator exerts its anti-tumor effect by activating Caspase family proteases (Caspase-3) to initiate the tumor cell apoptosis program.

[0045] In some optional embodiments, X is Cl; Y is C; Z is O; and R1, R2, R3, and R4 are independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0046] In some optional embodiments, the organic photoacid generator has the structure shown in the following formula:

[0047] .

[0048] In a second aspect, the present invention provides an antitumor drug comprising an organic photosensitive acid generator, said organic photosensitive acid generator having a structure as shown in one of the following formulas:

[0049] , , , ;

[0050] Wherein, X is F, Cl, Br or I; Y is C, Si, O, S or Se; Z is O or S; and R1, R2, R3, R4 are independently selected from hydrogen and C1-C8 alkyl groups.

[0051] In some optional embodiments, the antitumor drug is an anti-breast cancer drug.

[0052] In some optional embodiments, the antitumor drug further includes pharmaceutically acceptable excipients.

[0053] In some alternative embodiments, the pharmaceutically acceptable excipient is a solvent or diluent.

[0054] In some alternative embodiments, the pharmaceutically acceptable excipient is water.

[0055] In a typical embodiment, the organic photoacid generator CzC is prepared according to the following steps:

[0056] 3-Chloro-2-thiophenecarboxylic acid (0.61 g, 3.75 mmol), 3,6-di-tert-butylcarbazole (0.70 g, 2.5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 1.44 g, 7.50 mmol), and 4-dimethylaminopyridine (DMAP, 0.76 g, 6.25 mmol) were dissolved in 10 mL of dichloromethane (CH2Cl2). The reaction mixture was stirred at 50 °C in the dark for 5 hours. After the reaction was completed, the reaction mixture was extracted with a water / dichloromethane mixture, and the resulting organic phase was dried over anhydrous magnesium sulfate (MgSO4). The crude product was purified by rapid column chromatography (eluent: petroleum ether:dichloromethane = 3:1) to give a yellow solid powder product CzC. The chemical equation for this reaction is as follows:

[0057]

[0058] Characterization data of product CzC: 1 H NMR (400 MHz, CDCl3, ), δ (ppm): 7.98(2H, s),7.60 (1H, d, J = 4.1 Hz), 7.49 (2H, d, J = 8.8 Hz), 7.40 (2H, t, J = 5.3 Hz), 7.05(1H, d, J = 3.5 Hz), 1.43 (18H, s); 13 C NMR (101 MHz, CDCl3, ), δ (ppm): 160.58,147.32, 136.89, 131.11, 129.32, 129.02, 127.79, 126.58, 124.85, 116.12,114.93, 34.93, 31.82.

[0059] Irradiation was performed using light of different wavelengths at a concentration of 2.0 × 10⁻⁶. -5 We studied the photophysical properties of CzC in M-based CzC solutions before and after illumination. The results are as follows: Figure 1As shown, CzC exhibits significant photochromic behavior visible to the naked eye under 365 nm illumination. With prolonged irradiation time, the UV absorption spectrum of CzC changes significantly. In the absence of UV irradiation, its absorption spectrum shows a main peak near 305 nm and a shoulder peak at 318 nm. After prolonged UV irradiation, new absorption peaks gradually appear at 345 nm and 365 nm. Notably, this photoresponse process occurs very rapidly, with chemical equilibrium reached within 40 seconds (e.g., ...). Figure 2 (As shown).

[0060] Thin-layer chromatography (TLC) analysis was performed on the CzC solution before and after illumination, and the results are as follows: Figure 3 As shown, after a certain period of ultraviolet light irradiation, a new compound with higher polarity appeared in the CzC solution. Using a dip pen dipped in a dichloromethane solution of CzC as ink, writing was done on pH test paper. Then, the paper was irradiated with 365 nm ultraviolet light; the color of the pH test paper changed from yellow to deep red (as shown in the image). Figure 4 (As shown). These results demonstrate that CzC is an excellent photoacid generator (PAG) material.

[0061] To further elucidate the photochemical reaction mechanism of CzC, the reaction of CzC in deuterated chloroform (CDCl3) after irradiation with ultraviolet light (365 nm) was detected. 1 The reaction kinetics were characterized by changes in the H nuclear magnetic resonance (NMR) spectrum. The results are as follows: Figure 5 As shown, under continuous ultraviolet light irradiation, a series of new peaks appeared near 7.9 ppm, 8.1 ppm and 8.8 ppm.

[0062] The thermal stability of CzC was investigated using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Figure 6 and Figure 7 As shown, CzC exhibits excellent thermal decomposition temperatures (above 200℃). To investigate the factors affecting the thermal stability of CzC, the intermolecular interactions of CzC were analyzed, such as... Figure 8 and Figure 9 As shown in the crystal structure, CzC exhibits C–H···O (2.709 Å) and π-π interactions, indicating that its intermolecular interactions are relatively strong, thus contributing to its high level of thermal stability.

[0063] The photoactivation performance of CzC in the mouse breast cancer cell line (4T-1, purchased from the American Type Culture Collection (ATCC)) was evaluated in vitro. Specifically, CzC (10 μM) was incubated with 4T-1 cells in the dark for different times (0 h, 0.5 h, 1 h, 2 h, 4 h), and cell uptake was monitored using confocal laser scanning microscopy (CLSM). The results are as follows: Figure 10 and Figure 11 As shown, after 1 hour of incubation, 4T-1 cells showed obvious blue-green fluorescence derived from CzC in the cytoplasm, and the fluorescence intensity continued to increase with the extension of incubation time, reaching a maximum value at 4 hours of incubation. This indicates that CzC can be effectively taken up by cells.

[0064] The study investigated the H produced by CzC within cells. + And reactive oxygen species (ROS), specifically, 4T-1 cells were introduced at a concentration of 3 × 10⁻⁶ cells per well. 6 Cells were seeded at a density of [number] cells per well in six-well plates and cultured in DMEM medium for 24 hours; subsequently, they were divided into the following groups:

[0065] Control group: No lighting is applied;

[0066] Laser Illumination Unit: A 365nm laser (power density 10W / cm²) is used in a light-protected environment. 2 Irradiate for 15 minutes.

[0067] CzC group (CzC): CzC (10 μM) was added, and no light was applied;

[0068] CzC+ Illumination Assembly (CzC+Laser): 10 μM CzC is added, and a 365 nm laser (power density 10 W / cm²) is used in a light-protected environment. 2 Irradiate for 15 minutes.

[0069] After irradiation, cells from all groups were cultured for another 4 hours; the culture medium was discarded, and the wells were washed three times with phosphate-buffered saline (PBS); fluorescein diacetate (BCECF-AM, purchased from Beyotime, Shanghai, China) and fluorescein dichlorodihydroacetate (DCFH-DA, purchased from Beyotime, Shanghai, China) were used as indicators to detect the intracellular pH (i.e., H+) under light conditions. + Changes in (levels) and intracellular reactive oxygen species (ROS) levels. Results are as follows: Figure 12 As shown, strong BCECF-AM fluorescence signals were only observed in 4T-1 cells of the CzC+ light-illuminated group, indicating that CzC combined with laser treatment can generate a large amount of H+.+ Similarly, only the 4T-1 cells in the CzC+ light-illuminated group could easily show the strong green fluorescence of DCFH-DA, indicating that CzC can generate ROS within the cells under laser irradiation.

[0070] To further verify the oxidative stress effect of ROS generated by CzC on tumor cells, the levels of glutathione (GSH) and superoxide dismutase (SOD) were measured using a reduced glutathione assay kit (purchased from Nanjing Jiancheng Bioengineering Institute, catalog number A006-2-1) and a superoxide dismutase assay kit (purchased from Nanjing Jiancheng Bioengineering Institute, catalog number A001-3), respectively. The results are as follows: Figure 13 and Figure 14 As shown, the CzC+ light irradiation group resulted in a significant decrease in intracellular GSH and SOD levels in 4T-1 cells. This suggests that CzC and ROS may work synergistically to weaken the antioxidant capacity of tumor cells, prevent them from effectively clearing intracellular ROS, and lead to the accumulation of ROS in cells, further exacerbating oxidative damage to cells.

[0071] The in vitro killing effect of CzC on 4T-1 cells was evaluated using the Cell Counting Kit-8 (CCK-8) assay. Results are as follows: Figure 15 As shown, even with a high concentration (10 μM) of CzC, the survival rate of 4T-1 cells was above 90% without laser irradiation, indicating good biocompatibility. However, after irradiation with a 365 nm laser (10 W / cm², 15 min), the survival rate of 4T-1 cells decreased to 12.39%, indicating that CzC has excellent phototoxicity to 4T-1 cells.

[0072] These experimental results fully demonstrate that CzC, which has dual functions of photodynamic therapy and photoacid therapy, can generate a large amount of H. + And ROS. These substances disrupt acid-base balance, affect the intracellular environment, induce oxidative stress, and ultimately lead to apoptosis.

[0073] To further demonstrate the photoactivation effect of CzC, a live / dead cell staining method using calcein-AM (green fluorescence in live cells) and propidium iodide (red fluorescence in dead cells) was employed for visualization. The results are as follows: Figure 16 As shown, PI-positive red signals only appeared when 4T-1 cells were treated with both CzC and laser (365 nm, 10 W / cm², 15 min) simultaneously; while 4T-1 cells treated with CzC alone or with only 365 nm laser showed only green fluorescence of calcein-AM. These results indicate that CzC can effectively induce cancer cell death through a synergistic treatment strategy.

[0074] The apoptosis level of 4T-1 cells treated with different methods was detected by flow cytometry, and the results are as follows: Figure 17 and Figure 18 As shown, the proportion of apoptotic cells in 4T-1 cells increased from 0.18% in the blank group, 6.89% in the light-illuminated group, and 6.97% in the CzC group to 18.8% in the CzC+light-illuminated group. This indicates that the combined effect of CzC and laser caused CzC to produce more reactive oxygen species (ROS), which in turn aggravated oxidative stress and promoted cell apoptosis.

[0075] Western blotting (WB) was used to further monitor apoptosis-related markers. Standardization was performed using β-actin as an internal control, and the expression levels of Caspase-3, Bcl-2 (anti-apoptotic protein), and Bax (pro-apoptotic protein) were detected. Results are as follows: Figures 19-20 As shown, compared with cancer cells treated with CzC alone, cancer cells in the CzC+ light-irradiated group exhibited higher Caspase-3 expression levels and lower Bcl-2 expression levels, indicating that CzC material activates Caspase family proteases, thereby initiating the apoptosis program.

[0076] Given the synergistic therapeutic effect of CzC at the cellular level, its in vivo anti-tumor efficacy was further evaluated. A 4T-1 orthotopic tumor model was constructed, and primary tumors were inoculated. When the primary tumor volume reached approximately 80 mm², the in vivo anti-tumor effect was assessed. 3 Balb / c mice aged 6-8 weeks were randomly divided into four groups (n=5 per group): blank control group, light-exposed group, CzC group, and CzC+ light-exposed group. On days 0, 3, and 6, phosphate-buffered saline (PBS) was injected orally into the primary tumor sites of mice in the blank control group and light-exposed group, while CzC solution (1 mg / mL, 80 μL) was injected orally into the primary tumor sites of mice in the CzC group and CzC+ light-exposed group. Two hours after injection, the primary tumors of mice in the light-exposed group and CzC+ light-exposed group were irradiated with 365 nm laser (10 W / cm²). 2 The mice were irradiated with laser light for 8 minutes. Subsequently, the average tumor volume of each group of mice was monitored over 14 days. To assess the efficacy of tumor treatment, tumor volume and body weight of the 4T-1 tumor model mice were recorded every two days after the first administration. Results are as follows: Figure 21 and Figure 22As shown, tumor growth in mice treated with 365nm laser alone or CzC alone was barely inhibited, indicating that neither treatment had any anti-tumor effect. In contrast, tumor growth in mice treated with CzC combined with 365nm laser was inhibited within 14 days, thanks to the synergistic effect of photoacoustic therapy and photodynamic therapy. Notably, the tumor volume in the CzC+light irradiation group was significantly smaller than in the other groups, suggesting that endogenous H+ in the tumor... + The synergistic consumption and PAT effect amplified the therapeutic effect, consistent with in vitro experimental results.

[0077] The biocompatibility of CzC in vivo was further evaluated through hematological tests, blood biochemical analysis, and hematoxylin-eosin (H&E) staining of major organs in healthy mice. First, blood samples were collected from healthy Balb / c mice 24 hours after intravenous injection of PBS (blank group) or CzC solution (CzC group). The collected blood samples were then used to further analyze key liver function indicators, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB), as well as key kidney function indicators, such as creatinine (CREA) and uric acid (UA). Results are as follows: Figure 23 and Figure 24 As shown, there were no significant differences in liver and kidney function indicators between the CzC group (right red bar) and the control group (left blue bar), indicating that CzC has good blood compatibility. Furthermore, as... Figure 25 As shown, routine blood analysis also confirmed that all parameters in the CzC group (right red bar) were within the normal range, indicating that CzC injection did not cause serious functional damage in the mice. Meanwhile, as... Figure 26 As shown, H&E staining results revealed no systemic toxicity symptoms in major organs, including the heart, liver, spleen, lungs, and kidneys. Overall, these findings indicate that CzC possesses excellent biosafety and biocompatibility.

[0078] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any embodiment that achieves the technical effects of the present invention by the same or equivalent means should fall within the protection scope of the present invention. Within the protection scope of the present invention, various modifications and variations can be made to the technical solutions and / or implementation methods.

Claims

1. The application of an organic photoacid generator in the preparation of drugs for tumor photodynamic therapy or photoacid therapy, characterized in that, The organic photoacid generator has the structure shown in the following formula: ； Wherein, X is F, Cl, Br or I; and R1 and R2 are independently selected from hydrogen and C1-C8 alkyl groups.

2. The application according to claim 1, characterized in that, The drug is a photodynamic therapy drug for tumors, and the excitation wavelength of the drug is 365nm.

3. The application according to claim 1 or 2, characterized in that, X is Cl, and R1 and R2 are independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

4. The application according to claim 3, characterized in that, The organic photoacid generator has the structure shown in the following formula: 。

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