Near-infrared photosensitizer targeting mitochondrial DNA G-quadruplex and preparation and application thereof

Abstract

The application discloses a near-infrared photosensitizer targeting mitochondrial DNA G-quadruplex and a preparation and application thereof, the photosensitizer takes fluorinated quaternary ammonium pyrroloquinoline as an electron-withdrawing mother nucleus, constructs a D-π-A push-pull electron skeleton with furan bridged benzene selenium diazole as an electron-donating unit, introduces a heavy atom effect of selenium atoms to improve singlet oxygen generation efficiency, and naturally targets and enriches mitochondria through a cation quaternary ammonium structure; intramolecular rotation in a free state causes fluorescence quenching, after specific binding with mtDNA G4, molecular rotation is limited, an ICT effect is turned on near-infrared fluorescence, target-specific signal activation and synchronous photodynamic activity are realized. The molecule has a mild synthesis process, raw materials are easily obtained in commercialization, has multiple functions of G4 quantitative detection, in-situ visualization of cell organelles, in-vivo tumor imaging and targeted photodestruction, has high specificity and high biological safety, and provides a new small-molecule tool platform for precise and visual photodynamic diagnosis and treatment of tumors.

Description

Technical Field

[0001] This invention belongs to the field of organic near-infrared photosensitizer technology, specifically, it relates to near-infrared photosensitizers that target mitochondrial DNA G-quadruplexes and their preparation and application. Background Technology

[0002] Precision oncology diagnosis and treatment is a core development direction in modern oncology medicine. Photodynamic therapy (PDT) has outstanding advantages such as being minimally invasive, spatially and temporally precise and controllable, and free from chemotherapy resistance. Photosensitizers, as core functional molecules, directly determine the imaging signal-to-noise ratio and tumor-killing effect. Mitochondrial DNA G-quadruplex (mtDNA G4) is a unique non-classical nucleic acid secondary structure in mitochondria. It is abnormally highly expressed in various tumor cells and participates in the regulation of mitochondrial energy metabolism, oxidative stress balance, and apoptosis pathways, making it a highly promising tumor-specific endogenous target.

[0003] Existing mtDNA-targeting G4 photosensitizers have many insurmountable drawbacks: most G4-recognizing photosensitizers have absorption and emission wavelengths in the visible light range, resulting in shallow penetration into biological tissues, severe interference from autofluorescence background, and poor contrast in in vivo imaging; iridium-based targeting probes have high production costs and potential biotoxicity due to long-term in vivo accumulation; traditional hemicyanine derivatives have low fluorescence activation rates and insufficient singlet oxygen generation efficiency, leading to weak photodynamic therapy; most probes only target the cell nucleus and cannot specifically enrich mitochondria, making it difficult to achieve in-situ visualization of mitochondria and targeted photodamage to organelles; existing probe imaging functions and photodynamic therapy functions are disconnected, making it impossible to achieve integrated diagnosis and treatment of "single molecule, targeted recognition - fluorescence activation - reactive oxygen species killing".

[0004] The natural isoindigo backbone exhibits good binding ability to the G-quadruplex, but conventional oxygen-containing isoindigo molecules have weak spin-orbit coupling, resulting in low singlet oxygen yield. Current research lacks near-infrared photosensitizers for mtDNA G4 that incorporate selenium heavy atoms and fluorinated quaternary ammonium cations to construct a D-π-A push-pull electron system. Selenium atoms can enhance the heavy atom effect, improve intersystem crossing efficiency, and significantly increase... 1 O2 production; the fluorinated quaternary ammonium pyrrolidine quinazoline cation is naturally enriched in mitochondria, and furan-bridged selenide diazole regulates photoelectric properties. Therefore, the accurate identification and quantitative detection of mitochondrial DNA G-quadruplex (mtDNA G4) in cells and organisms has crucial clinical and basic research value for elucidating the pathological molecular mechanisms of tumor mitochondrial metabolic disorders and apoptosis imbalance, as well as for achieving early and accurate tumor diagnosis and targeted photodynamic intervention. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome the shortcomings of existing photosensitizers targeting mitochondrial DNA G-quadruplexes, and to provide a near-infrared photosensitizer targeting mitochondrial DNA G-quadruplexes, its preparation, and its application. The photosensitizer uses fluoroquaternary ammonium pyrrolo[2,1-b]quinazoline as the electron-withdrawing core and benzo[selenodiazole]furan conjugated units as electron-donating photosensitizing recognition groups. Its signal activation mechanism relies on the π-π stacking, hydrogen bonding, and electrostatic multiple interactions between the photosensitizer molecule and the mtDNA G-quadruplex, restricting the molecule's free rotation, blocking intramolecular nonradiative transition channels, restoring intramolecular charge transfer (ICT) effects, and simultaneously activating near-infrared fluorescence signals and singlet oxygen generation pathways, achieving highly specific visualization of mitochondrial G-quadruplexes and targeted photodynamic killing. ISe-FMA can precisely locate tumor lesions through near-infrared fluorescence imaging, guide targeted photodynamic therapy, and efficiently eliminate tumor cells highly expressing mtDNA G-quadruplexes. This molecular design strategy significantly improves the recognition specificity of G-quadruplexes and the efficiency of photosensitive therapy, providing a novel functional molecular tool for basic tumor pathology research, early diagnosis, and photodynamic intervention.

[0006] To solve the above-mentioned technical problems, the basic concept of the technical solution adopted by the present invention is as follows:

[0007] This invention provides a near-infrared photosensitizer targeting the G-quadruplex of mitochondrial DNA. The photosensitizer is named ISe-FMA, with the chemical name (E)-3-((5-(7-(dimethylamino)benzo[c][1,2,5]selenodiazol-4-yl)furan-2-yl)methylene)-6,7-difluoro-4-methyl-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-4-iodonium salt, and its chemical structural formula is shown in Formula I.

[0008]

[0009] Formula I.

[0010] Another aspect of this invention provides a method for preparing a near-infrared photosensitizer targeting the G-quadruplex of mitochondrial DNA. The preparation process is as follows: firstly, compound 1, quaternary ammonium salt intermediate 2, compound 3, and dimethylamine-substituted benzo[a]selenide diazole compound 4 are synthesized stepwise, followed by bromination and Suzuki coupling to obtain furan-bridged selenide diazole aldehyde intermediate 6; quaternary ammonium salt intermediate 2 and furan-bridged selenide diazole aldehyde intermediate 6 are dissolved in anhydrous acetic acid, and sodium acetate is added as a catalyst to carry out a Knoevenagel condensation reaction at 80°C for 2 h; after vacuum concentration of the reaction solution, it is purified by silica gel column chromatography using dichloromethane / methanol (20:1, v / v) as the eluent to obtain the blue-purple solid product ISe-FMA with a yield of 34%.

[0011] In another aspect, the present invention provides applications of the above-mentioned near-infrared photosensitizer, including but not limited to: (1) applications in in vitro qualitative / quantitative detection of DNA / RNA G-quadruplexes; (2) applications in confocal fluorescence imaging reagents for mitochondrial DNA G-quadruplexes in live cells; (3) applications in the preparation of in vitro tumor photodynamic killing agents targeting mitochondrial G4; (4) applications in tumor photodynamic therapy guided by near-infrared fluorescence imaging in tumor-bearing mice; and (5) applications in the preparation of in vivo tumor near-infrared fluorescence imaging-guided photodynamic therapy integrated drugs.

[0012] This invention develops a near-infrared photosensitizer, ISe-FMA, targeting the mitochondrial DNA G-quadruplex D-π-A type, for high-contrast tumor imaging and targeted photodynamic ablation. Benefiting from the advantages of selenium heavy atom effects, cationic mitochondrial targeting framework, and long-wavelength near-infrared absorption and emission, fluoroquinazoline-benzoselenide diazole photosensitizers are suitable for deep in vivo tumor diagnosis and treatment. This invention constructs ISe-FMA based on a design strategy of "target-bound activation of ICT and simultaneous generation of reactive oxygen species": in its free state, the molecule freely rotates, consuming excitation energy, resulting in weak fluorescence and extremely low singlet oxygen production; when the molecule specifically binds to the mtDNA G-quadruplex, multiple intermolecular forces restrict single bond rotation, reopening the ICT pathway, while selenium atoms enhance intersystem crossing efficiency, generating a large amount of singlet oxygen. Experiments have confirmed that ISe-FMA possesses excellent binding affinity for G-quadruplexes, high fluorescence activation rate, and excellent singlet oxygen generation capability, providing a general design approach for the development of mitochondrial G-quadruplex-targeted therapeutic molecules. It also provides a highly efficient reagent for exploring the pathological mechanisms of malignant tumors, early imaging diagnosis, and photodynamic targeted therapy.

[0013] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art.

[0014] (1) This invention introduces selenium atoms into the photosensitive framework to construct a heavy atom system, which significantly enhances the spin-orbit coupling effect, strengthens the triplet population, and achieves a singlet oxygen yield of 21.8%. At the same time, it redshifts the absorption and emission wavelengths of molecules, greatly improving the near-infrared live imaging performance.

[0015] (2) The cationic quaternary ammonium nucleus of the present invention naturally targets and enriches mitochondria, and after binding only to mtDNA G4, it simultaneously activates fluorescence signal and photodynamic activity, reducing off-target damage to normal tissues. It can achieve imaging-guided precision phototherapy by non-invasively tracing tumor lesions through in vivo near-infrared fluorescence.

[0016] (3) Verification of technical effectiveness:

[0017] ① High-contrast fluorescence response: After binding with c-Myc DNA G4 in vitro, the fluorescence activation factor is up to 58 times, the Stokes shift is 98nm, and the background interference is extremely low.

[0018] ② Cell targeting specificity: The fluorescence signal intensity of tumor cells is much higher than that of normal cells, and the Pearson correlation coefficient for mitochondrial colocalization is 0.95;

[0019] ③ In vivo anti-tumor effect: The tumor growth inhibition rate of the ISe-FMA combined with 680nm light treatment group was 72%, and there was no pathological damage to the major organs of mice, demonstrating excellent biosafety.

[0020] The specific embodiments of the present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0021] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention, but do not constitute an undue limitation of the invention. Obviously, the drawings described below are merely some embodiments, and those skilled in the art can obtain other drawings based on these drawings without creative effort. In the drawings:

[0022] Figure 1 The synthetic route of the photosensitizer ISe-FMA described in the embodiments of the present invention;

[0023] Figure 2 This is a schematic diagram illustrating the design principle of the photosensitizer ISe-FMA described in an embodiment of the present invention;

[0024] Figure 3 intermediate 2 in dimethyl sulfoxide-d6 (DMSO-d6) 1 H NMR spectrum;

[0025] Figure 4 intermediate 2 in dimethyl sulfoxide-d6 (DMSO-d6) 13 C NMR spectrum;

[0026] Figure 5 The photosensitizer ISe-FMA in dimethyl sulfoxide-d6 (DMSO-d6) 1 H NMR spectrum;

[0027] Figure 6 The photosensitizer ISe-FMA in dimethyl sulfoxide-d6 (DMSO-d6) 13 C NMR spectrum;

[0028] Figure 7 The above is the ESI-MS spectrum of the photosensitizer ISe-FMA described in the embodiments of the present invention;

[0029] Figure 8Figures show the in vitro spectral and molecular characterization of ISe-FMA: (a) Schematic diagram of the overall principle of using a near-infrared photosensitizer targeting mitochondrial G-quadruplexes for tumor imaging and photodynamic therapy. (b) Normalized UV absorption and near-infrared fluorescence spectra of ISe-FMA before and after interaction with c-Myc DNA G4. (c) SOSG singlet oxygen fluorescence intensity of the ISe-FMA and c-Myc DNA G4 system under different illumination durations. (d) Fluorescence spectrum of 2.5 μM ISe-FMA mixed with gradient concentrations of c-Myc DNA G4. (e) Linear fitting relationship between 768 nm fluorescence intensity and 0.1-3.0 μM c-Myc DNA G4. (f) 768 nm fluorescence intensity of 2.5 μM ISe-FMA coexisting with various nucleic acids (5.0 μM). (g) Job curve of ISe-FMA binding to c-Myc DNA G4 (total concentration fixed at 10 μM). (h) Circular dichroism of c-Myc DNA G4 with and without ISe-FMA. Data are expressed as mean ± standard deviation (n=3).

[0030] Figure 9 The following are images from the ISe-FMA live-cell confocal imaging, singlet oxygen, and cytotoxicity experiments of this invention: (a) Confocal imaging of MCF-7 cells after ISe-FMA co-staining with fluorescent probes of mitochondria, lysosomes, and nuclei, respectively. (b) ISe-FMA fluorescence imaging of MCF-7 cells after treatment with DNase I, RNase A, and RNase A combined with DNase I. (c) Fluorescence imaging of MCF-7 cells after treatment with valamicin, ionomycin, and potassium ions. (d) Quantification of the average fluorescence intensity of each group corresponding to (c). (e) Fluorescence imaging of MCF-7 cells after pretreatment with hypoxia, 2-MeOE2, and KC7F2. (f) Quantification of the average fluorescence intensity of each group corresponding to (e). (g) Flow cytometry results of A549, MCF-7, HeLa, and HEK293T cells with and without ISe-FMA incubation. (h) SOSG singlet oxygen confocal images of MCF-7 cells in different treatment groups. (i) Quantitative analysis of singlet oxygen fluorescence corresponding to plot (h). (j) Viability of MCF-7 cells treated with different concentrations of ISe-FMA in the dark / under light. (k) Cell viability of each cell line after treatment with ISe-FMA in the dark and under light. Scale bar 10 μm. All data are expressed as mean ± standard deviation (n=3).

[0031] Figure 10The following are experimental images of near-infrared fluorescence imaging and in vivo photodynamic therapy in nude mice bearing tumors according to the present invention: (a) Representative near-infrared fluorescence images of tumor-bearing mice injected intratumorally with PBS, ISe-FMA alone, Pt-ttpy pretreatment + ISe-FMA, and TMPYP4 pretreatment + ISe-FMA, respectively. (b) Quantitative fluorescence intensity of tumors in each group corresponding to (a) (n=3). (c) Near-infrared fluorescence images of tumor-bearing nude mice at different time points after intratumoral injection of ISe-FMA. (d) Fluorescence change curve of tumor area over time (n=3). (e) Schematic diagram of the complete experimental process of tumor photodynamic therapy. (f) Tumor growth curves of each experimental group (n=4). (g) Photograph of the actual tumor removed at the end of the experiment. (h) Box plot of tumor weight in each group (n=4). (i) Weight change curve of mice in each group during the experimental period. (j) H&E pathological sections of tumor tissue in each group. All data are expressed as mean ± standard deviation. Statistical analysis was performed using a two-tailed t-test (***p < 0.001, **p < 0.01).

[0032] It should be noted that these accompanying drawings and textual descriptions are not intended to limit the scope of the invention in any way, but rather to illustrate the concept of the invention to those skilled in the art by referring to specific embodiments. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0034] Example 1

[0035] Preparation process of fluoroquaternary ammonium pyrrolo[2,1-b]quinazoline compound 1 and quaternary ammonium salt intermediate 2, refer to route 1.

[0036]

[0037] Route 1

[0038] The specific steps are as follows:

[0039] Synthesis of Compound 1:

[0040] 2-Amino-4,5-difluorobenzoic acid (0.85 g, 5 mmol) was mixed with 2-pyrrolidone (0.75 mL, 10 mmol), and 20 mL of phosphorus oxychloride was added as a solvent and dehydrating agent. The mixture was heated in an oil bath at 110°C for 12 h. After the reaction, excess phosphorus oxychloride was removed under vacuum, the reaction solution was diluted with ice water, and sodium hydroxide aqueous solution was slowly added dropwise to neutralize the solution. The organic phase was extracted with ethyl acetate, the organic layer was dried with anhydrous sodium sulfate, and the crude product was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with petroleum ether / ethyl acetate (10 / 1, v / v) as the eluent to give a white solid with a yield of 57%, which was named compound 1.

[0041] Synthesis of intermediate 2:

[0042] Compound 1 (0.56 g, 2.5 mmol) was dissolved in 10 mL of anhydrous acetonitrile. 0.75 mL of iodomethane was added to the system. The system was sealed in a high-pressure reactor and stirred at 80°C for 72 h. After the reaction system was cooled to room temperature, the solid product was collected by filtration. The solid was washed several times with anhydrous acetonitrile and dried under vacuum at low temperature to obtain a yellow solid with a yield of 68%, which was named intermediate 2.

[0043] The intermediate 2 prepared in this embodiment was tested, and the results are as follows: Figures 3 to 4 As shown: Figure 3 intermediate 2 in dimethyl sulfoxide-d6 (DMSO-d6) 1 H NMR spectrum; Figure 4 intermediate 2 in dimethyl sulfoxide-d6 (DMSO-d6) 13 C NMR spectrum;

[0044] in, Figure 3 The hydrogen spectrum was analyzed as follows 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 8.42 (1H, d, J= 8.0 Hz, Ar-H), 8.40 (1H, d, J = 4.0 Hz, Ar-H), 4.32 (2H, t, J = 8.0 Hz, CH2), 4.00 (3H, s, CH3), 3.74 (2H, t, J = 8.0 Hz, CH2), 2.36 (2H, t, J = 8.0 Hz, CH2).

[0045] Figure 4 The carbon spectrum analysis is as follows: 13C NMR (100 MHz, DMSO-d6) δ (ppm): 168.34, 156.50,153.81, 150.76, 148.59, 137.13, 117.60, 115.70, 109.49, 51.05, 34.07, 19.39.

[0046] The above results prove that intermediate 2 has been successfully synthesized in this embodiment, and its structural formula is as follows:

[0047] .

[0048] Example 2

[0049] The preparation process of the photosensitizer ISe-FMA is shown in reference route 2.

[0050]

[0051] Route 2

[0052] The specific steps are as follows:

[0053] Synthesis of compound 3:

[0054] 2,3-Difluoro-o-phenylenediamine (1.6 g, 12.6 mmol) and selenium dioxide (1.53 g, 13.9 mmol) were added to 15 mL of anhydrous ethanol and refluxed at 80 °C for 1 h under an argon atmosphere. The mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography with petroleum ether / ethyl acetate (20 / 1, v / v) as the eluent to give a yellow solid with a yield of 90%, which was named compound 3.

[0055] Synthesis of compound 4:

[0056] Compound 3 (4-fluorobenzo[c][1,2,5]selenodiazole, 0.6 g, 3 mmol), 1.5 mL of triethylamine, and 2 mol / L tetrahydrofuran dimethylamine solution (10 equivalents) were added to an argon-protected high-pressure reactor. Anhydrous ethanol was used as the solvent, and the mixture was stirred at 150 °C for 24 h. After the reaction was completed, the mixture was cooled to room temperature, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography with petroleum ether / ethyl acetate (10 / 1, v / v) to give a red solid with a yield of 85%, which was named compound 4.

[0057] Synthesis of compound 5:

[0058] Compound 4 (0.26 g, 1 mmol) was dissolved in dichloromethane, and NBS (0.2 g, 1.1 mmol) was added in portions at 0 °C. The mixture was stirred at room temperature for 1 h, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography with petroleum ether / ethyl acetate (20 / 1, v / v) as the eluent to give an orange-red solid in a yield of 66%, which was named compound 5.

[0059] Synthesis of intermediate 6:

[0060] Compound 5 (0.61 g, 2 mmol), 5-formylfuran-2-boronic acid (0.31 g, 2.6 mmol), Pd(dppf)Cl2 (0.14 g, 0.2 mmol), and potassium carbonate (0.14 g, 1 mmol) were added to 1,4-dioxane, and stirred at 95 °C for 24 h under nitrogen protection. After extraction and drying, the mixture was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with petroleum ether / ethyl acetate (10 / 1, v / v) as the eluent to give a yellow solid in 50% yield, which was named intermediate 6.

[0061] Synthesis of ISe-FMA:

[0062] Intermediate 2 (0.036 g, 0.1 mmol), intermediate 6 (0.032 g, 0.1 mmol), and sodium acetate (0.027 g, 0.2 mmol) were added to anhydrous acetic anhydride (3 mL), stirred at 80 °C for 2 h, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography with DCM / MeOH (20 / 1, v / v) as the eluent to obtain a blue-purple solid with a yield of 34%, named ISe-FMA.

[0063] The photosensitizer ISe-FMA prepared in this embodiment was tested, and the results are as follows: Figures 5 to 7 As shown, they are respectively: Figure 5 The photosensitizer ISe-FMA in dimethyl sulfoxide-d6 (DMSO-d6) 1 H NMR spectrum; Figure 6 The photosensitizer ISe-FMA in dimethyl sulfoxide-d6 (DMSO-d6) 13 C NMR spectrum; Figure 7 The above is the ESI-MS spectrum of the photosensitizer ISe-FMA described in the embodiments of the present invention;

[0064] in, Figure 5 The hydrogen spectrum analysis is as follows: 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.09 (1H, s,Ar-H), 7.70 (1H, s, Ar-H), 7.69 (1H, d, J = 2.0 Hz, Ar-H), 7.38 (1H, d, J =12.0 Hz, Ar-H), 7.26 (1H, d, J = 4.0 Hz, Ar-H), 7.17 (1H, d, J = 8.0 Hz, Ar-H), 5.75 (1H, s, Ar-H), 5.32 (1H, d, J = 4.0 Hz, CH=), 4.23 (6H, s, 2 ×CH3), 4.15 (2H, d, J = 4.0 Hz, CH2), 4.13 (2H, d, J = 4.0 Hz, CH2), 1.41 (3H, s, CH3).

[0065] Figure 6 The carbon spectrum analysis is as follows: 13 C NMR (100 MHz, DMSO-d6) δ (ppm): 170.07, 167.44,165.93, 148.51, 143.80, 134.21, 132.77, 132.04, 131.02, 129.93, 129.12,118.79, 79.44, 78.96, 67.57, 56.06, 38.71, 30.38, 28.84, 24.38, 22.87, 19.03,14.75, 11.36.

[0066] Figure 7 The ESI-MS mass spectrometry resolution was: ISe-FMA [M+H] + The theoretical molecular weight is 540.44; the actual measured molecular weight is 540.41.

[0067] The above results prove that the photosensitizer ISe-FMA has been successfully synthesized in this embodiment, and its structural formula is as follows:

[0068] .

[0069] Example 3

[0070] In vitro spectral and molecular interaction performance analysis of photosensitizer ISe-FMA:

[0071] (1) Investigate the basic spectral response characteristics of ISe-FMA and c-Myc DNA G4;

[0072] Specific experimental procedures: Prepare 2.5 μM ISe-FMA buffer solution, add different concentration gradients of c-MycDNA G4, and incubate at 37℃ for 30 min. The UV spectral scanning range is 300-800 nm, the fluorescence excitation wavelength is 670 nm, and the emission acquisition range is 700-850 nm. The excitation and emission slit widths are both set to 8 nm. Singlet oxygen detection uses SOSG fluorescent indicator, with an excitation wavelength of 504 nm and an emission wavelength of 530 nm. Normalized absorbance and fluorescence curves with and without G4 are collected simultaneously. Fluorescence linearity curves are obtained by setting a 0.1-3.0 μM gradient of G4. Selectivity tests are performed using various nucleic acid substrates. Job binding curves and circular dichroism spectroscopy are also measured. Each experiment is repeated in triplicate.

[0073] Experimental results are as follows Figure 8 As shown. Figure 8 'a' is a schematic diagram illustrating the overall principle of using mitochondrial G-quadruplex-targeting photosensitizers for tumor imaging and photodynamic therapy. (See diagram for reference.) Figure 8 As shown in b, the maximum absorption peak of free ISe-FMA is located at 625 nm. After the addition of c-Myc DNAG4, the absorption significantly red-shifts to 670 nm, and the near-infrared fluorescence increases simultaneously and significantly. Figure 8 c. With prolonged illumination, the SOSG fluorescence intensity of the ISe-FMA+G4 system continuously increases, demonstrating that singlet oxygen can be efficiently generated under illumination. Figure 8 d. As the concentration of c-Myc DNAG4 increases, the fluorescence signal at 768 nm gradually increases. As shown in Figure 8e, the fluorescence intensity at 768 nm shows a good linear relationship with the G4 concentration in the range of 0.1-3.0 μM. Figure 8 f, ISe-FMA only shows a significant fluorescence enhancement for various G-quadruplexes, with no significant response to ssDNA, dsDNA, or ordinary RNA, demonstrating excellent target specificity. For example... Figure 8 g. The Job curve showed that the binding ratio of the probe to c-Myc DNAG4 was 1:2 (total nucleic acid + probe concentration was fixed at 10 μM). Figure 8 h, no significant change was observed in the circular dichroism of c-MycDNA G4 before and after the addition of ISe-FMA, proving that probe binding does not disrupt the parallel secondary structure of G4. All data are expressed as mean ± standard deviation (n=3).

[0074] (2) Investigate the molecular recognition binding mechanism between ISe-FMA and G4.

[0075] Specific experimental steps: DFT theoretical calculations were performed using Gaussian 16, and molecular docking simulations were conducted using AutoDock Vina. The interaction between ISe-FMA and c-Myc DNA G4 was then comprehensively analyzed by combining the CD spectrum, titration experiments, and Job curve data mentioned above.

[0076] Experimental results: ISe-FMA relies on π-π stacking, electrostatic attraction and hydrogen bonding to bind the G4 quadrant; intramolecular rotation is restricted to block nonradiative transitions and restore the ICT fluorescence pathway. At the same time, selenium heavy atoms enhance spin-orbit coupling efficiency and promote intersystem crossing. Under illumination, singlet oxygen is generated efficiently, fully elucidating the dual response mechanism of target activation imaging and photodynamics.

[0077] Example 4

[0078] Study on ISe-FMA live-cell mitochondrial-targeted imaging and in vitro photodynamic effects:

[0079] (1) ISe-FMA subcellular organelle colocalization and intracellular G4-specific imaging

[0080] Specific experimental procedures: MCF-7, HeLa, A549, and HEK293T cells were seeded into culture dishes and cultured in DMEM medium containing 10% fetal bovine serum and double antibiotics at 37°C and 5% CO2 for 24 h. Cells were incubated with 2 μM ISe-FMA for 0.5 h and co-stained with Mito-Tracker Green, Lyso-Tracker Green, and Hoechst 33342, respectively. Nucleic acid was digested using DNase I, RNase, and a mixture of RNase and DNase in different groups. Valamidcin and ionomycin were used to regulate mitochondrial potassium ion concentration. Hypoxic culture, 2-MeOE2, and KC7F2 inhibition of glucose metabolism pretreatment groups were established. Competitive blocking experiments were conducted using two types of G4 inhibitors, Pt-ttpy and TMPYP4. A confocal imaging scale bar of 10 μm was used, with 5 parallel observations per group.

[0081] Experimental results are as follows Figure 9 As shown in Figure 9a, the ISe-FMA fluorescence signal highly overlaps with the mitochondrial probe, with a Pearson correlation coefficient of 0.95. It shows almost no co-localization with lysosomes or the cell nucleus, demonstrating precise mitochondrial targeting capability. Figure 9 b. After DNase treatment, cell fluorescence almost completely disappeared, while RNase treatment showed no significant change, proving that intracellular fluorescence originates from mitochondrial DNAG4. Figure 9 As shown in c, potassium ion regulating reagents can significantly alter the intensity of cell fluorescence. The corresponding quantitative fluorescence statistics for each group are as follows: Figure 9 As shown in d. Figure 9 e. Hypoxia induces an increase in G4, resulting in a significant rise in fluorescence; treatment with glucose metabolism inhibitors leads to a decrease in signal intensity. For example... Figure 9 f represents the quantitative results for the hypoxia and inhibitor groups. For example... Figure 9g, the flow cytometry fluorescence of tumor cells (MCF-7, HeLa, A549) was much higher than that of normal HEK293T cells, which could distinguish the differences in mtDNA G4 expression in cells.

[0082] (2) Detection of singlet oxygen in live cells and evaluation of cell phototoxicity

[0083] Specific experimental procedures: Cells were divided into a blank group, a light-only group, a probe-only light-avoidance group, and a probe + light-treatment group; incubated with 5μM ISe-FMA for 1 h, and treated with 680nm laser at 100mW / cm². 2 Irradiation for 10 min; SOSG indicator to label intracellular singlet oxygen; CCK-8 kit to determine cell viability under different probe concentrations and with or without light.

[0084] Experimental results are as follows Figure 9 h, only the ISe-FMA combined illumination group produced a large amount of singlet oxygen fluorescence. For example... Figure 9 i represents the quantitative fluorescence measurement of singlet oxygen. For example... Figure 9 Under light-protected conditions, the cell viability of 0-50 μM probes was above 88%, indicating good biocompatibility; however, MCF-7 cell viability significantly decreased after light exposure. Figure 9 k, HeLa and A549 tumor cells showed significant phototoxicity, while normal 293T cells showed minimal damage, demonstrating selective tumor killing effect. Scale bar 10 μm. All data are expressed as mean ± standard deviation (n=3).

[0085] Example 5

[0086] ISe-FMA tumor-bearing nude mice in vivo near-infrared imaging, in vivo photodynamic therapy and biosafety evaluation:

[0087] (1) Construction of tumor-bearing models and tumor-specific near-infrared imaging detection

[0088] Specific experimental procedures: Four-week-old female BALB / c nude mice were selected, and 2×10⁻⁶ mice were subcutaneously injected into the buttocks. 6 MCF-7 cells, when the tumor volume is approximately 80 mm. 3 Experiments were conducted. Experimental groups included: PBS blank control group, ISe-FMA group, G4 competitive ligand Pt-ttpy pretreatment group, and G4 unwinding agent TMPYP4 pretreatment group. Each group received an intratumoral injection of 50 μL or 50 μM probe solution. Whole-body near-infrared fluorescence was collected using an IVIS small animal imaging system at 0, 0.5, 1, 2, 5, and 12 h. The tumor ROI was delineated and the fluorescence intensity was statistically analyzed. There were 3 mice in each group.

[0089] Experimental results are as follows Figure 10As shown in a and 10b, only the ISe-FMA-injected group showed strong fluorescence signals in tumors. Pretreatment with a G4 inhibitor significantly reduced fluorescence, demonstrating that in vivo fluorescence is generated by the specific binding of mtDNA G4. Figure 10 c. On day 10, the tumor fluorescence reached its peak 60 minutes after injection and decreased significantly 12 hours later. The probe was mainly eliminated by liver metabolism.

[0090] (2) Evaluation of in vivo photodynamic therapy for tumor suppression

[0091] Specific experimental procedures: Tumor-bearing mice were randomly divided into 4 groups (n=4 per group): ① PBS control group; ② PBS + 680nm light irradiation group; ③ ISe-FMA light-avoidance group only; ④ ISe-FMA + light irradiation treatment group. The drug was administered intratumorally every 2 days, followed by 680nm laser irradiation of the tumor site for 10 minutes 1 hour after administration. The complete treatment cycle was 24 days. Tumor length and width and mouse weight were measured every other day. At the experimental endpoint, the tumor was dissected and weighed. Heart, liver, spleen, lung, kidney, and tumor tissues were fixed and prepared for H&E pathological sections.

[0092] See the experimental procedure. Figure 10 e. For example Figure 10 f, In the ISe-FMA combined with light irradiation group, tumor growth was significantly inhibited, with a tumor inhibition rate of 72%. Figure 10 At the treatment endpoints of g and 10h, the tumor volume and weight in the treatment group were significantly smaller than those in the control group. For example... Figure 10 i. Throughout the entire cycle, there was no significant decrease in mouse body weight in any group, and the probe showed no significant systemic toxicity. For example... Figure 10 j. Tumor sections in the treatment group showed large areas of apoptotic vacuoles, while tumor cells in the blank group showed vigorous proliferation.

[0093] (3) Organ biosafety testing

[0094] Specific experimental steps: After treatment, the experimental animals were euthanized, major organs were removed to prepare H&E sections, and serum ALT, AST, CREA, BUN, UA, and liver and kidney biochemical indicators were detected simultaneously.

[0095] Experimental results: The cells in the heart, liver, spleen, lung, and kidney tissues were morphologically intact, without inflammation or necrosis; there were no statistically significant differences in liver and kidney function indicators among the groups, confirming the good biocompatibility of ISe-FMA in vivo. All data are expressed as mean ± standard deviation, and statistical analysis was performed using a two-tailed t-test (***p < 0.001, **p < 0.01).

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A near-infrared photosensitizer targeting mitochondrial DNA G-quadruplex, characterized in that, The photosensitizer is named ISe-FMA, with the chemical name (E)-3-((5-(7-(dimethylamino)benzo[c][1,2,5]selenodiazol-4-yl)furan-2-yl)methylene)-6,7-difluoro-4-methyl-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-4-iodonium salt, and its chemical structural formula is shown in Formula I: ； Formula I.

2. A method for preparing the near-infrared photosensitizer ISe-FMA targeting mitochondrial DNA G-quadruplex as described in claim 1, characterized in that, The preparation process is as follows: Sodium acetate was added to an anhydrous acetic anhydride system containing quaternary ammonium salt intermediates 2 and 6 as a catalyst, and a Knoevenagel condensation reaction was carried out at 80°C for 2 h. The reaction solution was concentrated under vacuum to remove the solvent, and the residue was purified by silica gel column chromatography with a volume ratio of dichloromethane:methanol = 20:1 to obtain a blue-purple solid ISe-FMA. Intermediate 2 of the quaternary ammonium salt is 6,7-difluoro-4-methyl-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-4-iodine quaternary ammonium salt, and intermediate 6 is 5-(7-(dimethylamino)benzo[c][1,2,5]selenodiazol-4-yl)furan-2-carboxaldehyde.

3. The preparation method according to claim 2, characterized in that, The synthesis steps of intermediate 6 are as follows: (1) Compound 4 was brominated by NBS to give compound 5, which is 7-bromo-N,N-dimethylbenzo[c][1,2,5]selenodiazole; (2) Compound 5 and 5-formylfuran-2-boronic acid were coupled via Suzuki reaction with Pd(dppf)Cl2 as catalyst and potassium carbonate as base. The reaction was carried out at 95°C for 24 h with 1,4-dioxane. The intermediate 6 was obtained by extraction and purification.

4. The preparation method according to claim 3, characterized in that, The synthesis steps of compound 4 are as follows: (1) Compound 3 was prepared by refluxing 2,3-difluoro-o-phenylenediamine with selenium dioxide and ethanol. Compound 3 is 4-fluorobenzo[c][1,2,5]selenodiazole. (2) Compound 3 was reacted with dimethylamine in a high-pressure autoclave at 150°C to give N,N-dimethylbenzo[c][1,2,5]selenodiazole, which is compound 4.

5. The application of the near-infrared photosensitizer targeting mitochondrial DNA G-quadruplex as described in claim 1, or the photosensitizer prepared by any of the methods described in claims 2-4, in the in vitro qualitative and quantitative detection of DNA / RNA G-quadruplexes.

6. The application of the near-infrared photosensitizer targeting mitochondrial DNA G-quadruplex as described in claim 1 or the photosensitizer prepared by any of the methods described in claims 2-4 in the preparation of confocal fluorescence imaging reagents for live cell mitochondrial DNA G-quadruplexes.

7. The use of the near-infrared photosensitizer targeting mitochondrial DNA G-quadruplex as described in claim 1 or the photosensitizer prepared by any of the methods described in claims 2-4 in the preparation of in vitro tumor photodynamic killing agents targeting mitochondrial G4.

8. The application of the near-infrared photosensitizer targeting mitochondrial DNA G-quadruplex as described in claim 1 or the photosensitizer prepared by any of the methods described in claims 2-4 in the preparation of in vivo tumor near-infrared fluorescence imaging-guided photodynamic therapy integrated drugs.

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