Indole-benzothiadiazole derivatives, processes for their preparation and use

Abstract

The application discloses an indole-benzothiadiazole derivative, a preparation method and application thereof. The structural general formula of the indole-benzothiadiazole derivative is shown in the figure, wherein R is selected from one of methyl piperazine, diethylamine, azetidine and pyrrolidine. The derivative can specifically bind and fluorescently label mitochondrial RNA in cells, and has good selectivity, high fluorescent signal-to-noise ratio and excellent anti-interference ability in a complex biological system. Meanwhile, the indole-benzothiadiazole derivative provided by the application has a simple synthesis route, is easy to obtain, has good stability, is convenient to store and use, can specifically bind RNA and produce strong fluorescent response, is suitable for real-time detection and imaging of mitochondrial RNA in vitro and in vivo, and is a high-efficiency mitochondrial RNA small-molecule fluorescent probe.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probes and RNA detection technology, and more specifically, to a class of indole-benzothiadiazole derivatives, their preparation methods, and applications. Background Technology

[0002] Mitochondria, as the "energy factories" of eukaryotic cells, play a crucial role in cell survival and physiological activity through their homeostasis. Mitochondrial RNA (mtRNA), as a core intermediate carrier of mitochondrial gene expression, not only participates in the synthesis of respiratory chain complex subunits, ribosomal RNA, and transfer RNA encoded by mitochondria themselves, but its expression level, processing modification status, and localization changes are also closely related to mitochondrial dysfunction. Studies have shown that mutations, abnormal degradation, or dysregulation of mtRNA can lead to mitochondrial respiratory chain dysfunction, subsequently triggering a series of major diseases, including neurodegenerative diseases (such as Alzheimer's and Parkinson's), hereditary mitochondrial diseases (such as Leber's disease), metabolic reprogramming of tumor cells, and aging-related diseases. Therefore, achieving highly specific recognition, real-time dynamic tracking, and functional regulation of mtRNA has become a core requirement in the biomedical field for elucidating the pathogenesis of mitochondrial-related diseases and developing early diagnostic technologies and targeted therapeutic strategies.

[0003] To meet the technical needs of mtRNA research, nucleic acid probe technology, with its advantages of strong designability and clear targeting, has become the mainstream tool for mtRNA detection and analysis. Traditional mtRNA detection probes mainly use DNA probes or conventional RNA probes as the core, binding to target mtRNA through the base complementary pairing principle, and achieving detection by combining fluorescent labeling, radioactive labeling, or enzyme-catalyzed signal amplification techniques. For example, DNA probes based on in situ hybridization (ISH) technology have been used for cellular localization analysis of mtRNA, while real-time quantitative polymerase chain reaction (qPCR) combined with a detection system of specific primers and probes has enabled the quantitative analysis of mtRNA expression levels.

[0004] However, the unique structural features and physiological environment of mitochondria present several insurmountable technical bottlenecks for traditional nucleic acid probes in mtRNA targeting applications: First, mitochondria have a double-membrane structure with a significant potential difference and ion gradient between the intermembrane space and the matrix. Traditional linear nucleic acid probes, with their large molecular weight and high hydrophilicity, cannot penetrate the mitochondrial membrane barrier autonomously and require delivery via vectors (such as liposomes or viral vectors). However, vector-mediated delivery not only increases cytotoxicity but may also lead to premature release of the probe into the cytoplasm, reducing mitochondrial targeting efficiency. Second, mtRNA is mainly found in the mitochondrial matrix, while the cytoplasm contains a large amount of nuclear gene-encoded RNA (nRNA). First, some nRNAs and mtRNAs share sequence homology. Traditional probes lack specificity in base pairing and are prone to cross-hybridization, resulting in excessively high background signals and failing to achieve high-specificity mtRNA recognition. Second, the mitochondrial matrix contains high concentrations of nucleases (such as RNase H) and reactive oxygen species (ROS). The phosphodiester bonds of traditional nucleic acid probes are easily degraded, resulting in poor stability and making it impossible to achieve long-term dynamic tracking of mtRNAs. Third, traditional probes mostly rely on single base pairing to achieve targeted binding. The binding affinity is significantly affected by environmental factors such as temperature and ionic strength, and is prone to unwinding under physiological conditions, further reducing the sensitivity and reliability of detection.

[0005] To address these issues, researchers in the field have attempted to optimize traditional probes through chemical modifications. For example, introducing lipid groups such as cholesterol and fatty acids to the probe tip can enhance membrane permeability, or thiomodifying the phosphodiester bond can improve nuclease resistance. However, these improvements still have significant limitations: while lipid modification can improve membrane permeability, it increases non-specific adsorption of the probe on the cell membrane surface; while thiomodification can enhance stability, it reduces the binding specificity of the probe to the target mtRNA, and it is difficult to simultaneously achieve a balance between membrane permeability, stability, and specificity. Furthermore, some studies have attempted to construct probes using artificial nucleic acid analogs such as peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). Although this can improve binding affinity and stability, the synthesis cost of such probes is high, and their rigid structure further reduces membrane permeability, making it difficult to promote their clinical translation and large-scale application.

[0006] Therefore, developing novel fluorescent probes capable of selectively recognizing RNA in vitro, effectively labeling mitochondria in cells, and whose fluorescence signal depends on RNA binding has become an important tool for deepening research on mtRNA function. The successful development of such probes is expected to provide new research methods for understanding the mechanisms of mitochondrial-related diseases, possessing significant scientific value and application potential. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art. The present invention provides a class of indole-benzothiadiazole derivatives, their preparation methods and applications.

[0008] The first objective of this invention is to provide a class of indole-benzothiadiazole derivatives.

[0009] A second objective of this invention is to provide a method for preparing indole-benzothiadiazole derivatives.

[0010] A third objective of this invention is to provide applications of indole-benzothiadiazole derivatives.

[0011] The fourth objective of this invention is to provide a testing product.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution:

[0013] This invention provides a class of indole-benzothiadiazole derivatives, the structural formula of which is:

[0014] ;

[0015] R is selected from one of methylpiperazinyl, diethylamino, aziridine, and pyrrolidinyl.

[0016] This invention develops a novel indole-benzothiadiazole derivative, which is a "fluorescence-activated" probe: its fluorescence is at background levels in the absence of a target or when interacting with DNA. Upon binding to single- or double-stranded RNA (ssRNA / dsRNA) or G-quadruplex RNA (G4 RNA), the fluorescence signal is specifically "activated," resulting in significant fluorescence enhancement. This effectively distinguishes between DNA and RNA and exhibits excellent selective recognition of RNA structures. The indole-benzothiadiazole derivative provided by this invention can be used as a fluorescent probe. Its red fluorescence emission originates from the entire extended indole-benzothiadiazole conjugated system, reflecting the photophysical properties of their combined interaction. Simultaneously, this derivative specifically recognizes mitochondrial RNA, exhibits strong anti-interference capabilities, and demonstrates high binding selectivity and recognition specificity for mitochondrial RNA in both live and fixed cell systems. It features a high signal-to-noise ratio, stable fluorescence signal, and is suitable for real-time, dynamic fluorescence imaging. It can be used for real-time, dynamic fluorescence imaging and detection of mitochondrial RNA, enabling visualization and tracking of mitochondrial RNA distribution and changes. The indole-benzothiadiazole derivative uses the dimethylindole fragment as a mitochondrial targeting group, which guides the probe to accumulate in mitochondria; while the benzothiadiazole fragment, as the recognition and signaling core, is responsible for specifically binding RNA and participating in fluorescence emission. The covalent connection between the two achieves functional synergy, enabling the compound to target mitochondria and detect RNA in situ, and can be better used for the detection, labeling and dynamic tracing of mitochondrial RNA and functional studies.

[0017] Furthermore, the indole-benzothiadiazole derivative has one of the following structural formulas:

[0018] .

[0019] This invention provides a method for preparing indole-benzothiadiazole derivatives, wherein 1,2,3,3-tetramethyl-3H-indole-1-onium iodide and benzothiadiazole derivatives are dissolved in organic solvent E and reacted at 70-85°C for 10-15 h. After the reaction is completed, the indole-benzothiadiazole derivatives are obtained by concentration, washing, drying and purification.

[0020] The structural formula of the benzothiadiazole derivative is selected from one of the following structural formulas: .

[0021] This invention is based on the classic Knoevenagel condensation reaction, which involves aldol-alcohol condensation under a weakly alkaline environment. The reaction between 1,2,3,3-tetramethyl-3H-indole-1-onium iodide and benzothiadiazole derivatives is highly reactive, requiring only the addition of a weakly alkaline environment provided by organic solvents such as ethanol, without the need for additional weakly alkaline reagents.

[0022] Preferably, the organic solvent E is selected from one or more of methanol, isopropanol, n-butanol, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, 1,4-dioxane, acetic acid, or water.

[0023] Preferably, the molar ratio of the 1,2,3,3-tetramethyl-3H-indole-1-onium iodide and the benzothiadiazole derivative is 1:(1-2).

[0024] Specifically, the reaction raw material 1,2,3,3-tetramethyl-3H-indole-1-onium iodide (ID-I) used in this invention can be a commercially available compound or synthesized in-house. As a preferred method, this invention also provides a method for synthesizing ID-I: 2,3,3-trimethylindole and iodomethane are dissolved in organic solvent A and reacted at 60-130°C for 8-24 hours. After the reaction, the organic solvent A is removed by vacuum concentration to obtain a crude product. Diethyl ether is added to the crude product, and the mixture is stirred, washed, and the solid is collected by filtration to obtain 1,2,3,3-tetramethyl-3H-indole-1-onium iodide, whose structural formula is:

[0025] .

[0026] Preferably, the organic solvent A is selected from one or more of acetonitrile, dimethyl sulfoxide, sulfolane, or toluene.

[0027] Preferably, the molar ratio of 2,3,3-trimethylindole to iodomethane is 1:(1.2-10).

[0028] Preferably, the present invention also provides a method for synthesizing the benzothiadiazole derivative as a reactant, comprising the following steps:

[0029] (1) Synthesis of intermediate raw material S-1: 4-bromo-7-methylbenzo[C][1,2,5]thiadiazole, a bromide reagent, and azobisisobutyronitrile were dissolved in organic solvent B and reacted at 80-120℃ for 4-16 h. After the reaction was completed, the mixture was washed, dried, and purified to obtain 4-bromo-7-(dibromomethyl)benzo[C][1,2,5]thiadiazole, which is intermediate raw material S-1. Its structural formula is as follows:

[0030] .

[0031] (2) Synthesis of intermediate raw material S-2: Intermediate raw material S-1 and silver nitrate were reacted with each other in a mixed system of water and organic solvent C at reflux temperature for 4-8 h. After the reaction was completed, the mixture was extracted, washed, dried and purified to obtain 7-bromo-4-aldehyde benzo[c][1,2,5]thiadiazole, which is intermediate raw material S-2. Its structural formula is as follows:

[0032] .

[0033] (3) Synthesis of benzothiadiazole derivatives of the reaction raw material: intermediate raw material S-2, amine compound and base are dissolved in organic solvent D and reacted at 80-100℃ for 12-18h. After the reaction is completed, the benzothiadiazole derivatives of the reaction raw material are obtained by washing, drying and purification.

[0034] Preferably, the reflux temperature is 100~120℃.

[0035] Preferably, the amine compound is selected from one of N-methylpiperazine, diethylamine, aziridine, and pyrrolidine.

[0036] Preferably, the brominating agent is selected from one or more of N-bromosuccinimide, N-bromoacetamide, N-chlorosuccinimide, N-iodosuccinimide, or 1,3-dibromo-5,5-dimethylhydantoin.

[0037] Preferably, the organic solvent B is selected from one or more of chlorobenzene, o-dichlorobenzene, mesitylene, 1,2-dichloroethane, diphenyl ether, anisole, 1,3-dimethyl-2-imidazolinone, or N-methylpyrrolidone.

[0038] Preferably, the organic solvent C is selected from one or more of benzene, toluene, xylene, dichloromethane, chloroform, 1,2-dichloroethane, tetrahydrofuran, or 1,4-dioxane.

[0039] More preferably, in the synthesis of intermediate raw material S-2, the volume ratio of water to organic solvent C is 1:(1.5-4).

[0040] Preferably, the alkali is selected from one or more of cesium carbonate, potassium phosphate, sodium carbonate, triethylamine, N,N-diisopropylethylamine, pyridine, 1,8-diazabicycloundec-7-ene, potassium tert-butoxide, sodium hydride, sodium methoxide, sodium bicarbonate, or potassium hydroxide.

[0041] Preferably, the organic solvent D is selected from one or more of benzene, toluene, xylene, dichloromethane, chloroform, 1,2-dichloroethane, tetrahydrofuran, or 1,4-dioxane.

[0042] Preferably, the organic solvent E is selected from one or more of methanol, isopropanol, n-butanol, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, 1,4-dioxane, acetic acid, or water.

[0043] Preferably, the molar ratio of the 4-bromo-7-methylbenzo[C][1,2,5]thiadiazole, the brominizing agent, and azobisisobutyronitrile is 1:(2-5):(0.05-0.2); the molar ratio of the 4-bromo-7-(dibromomethyl)benzo[C][1,2,5]thiadiazole and silver nitrate is 1:(2-3); and the molar ratio of the 7-bromo-4-aldehyde benzo[C][1,2,5]thiadiazole, the amine compound, and the base is 1:(1.2-2):(1.5-3).

[0044] The preparation method provided by this invention has clear steps, mild reaction conditions, and the raw materials and reagents used (such as amine compounds, brominating reagents, basic reagents and various alternative solvents) are all common or commercially available, making it easy to operate and scale up.

[0045] Therefore, the present invention provides the application of the above-mentioned indole-benzothiadiazole derivative as a fluorescent probe.

[0046] This invention provides the application of the above-mentioned indole-benzothiadiazole derivative in the detection or labeling of mitochondrial RNA.

[0047] This invention provides the use of the above-mentioned indole-benzothiadiazole derivative in the preparation of products for detecting or labeling mitochondrial RNA.

[0048] Preferably, the indole-benzothiadiazole derivative is used as a fluorescent probe for detecting or labeling mitochondrial RNA in cells.

[0049] Preferably, the indole-benzothiadiazole derivative is used as a fluorescent probe in mitochondrial RNA tracking and labeling.

[0050] More preferably, the detection includes fluorescence imaging of mitochondrial RNA in live or immobilized cells.

[0051] In addition, the present invention also provides a detection product containing the above-mentioned indole-benzothiadiazole derivative.

[0052] The present invention has the following beneficial effects:

[0053] The indole-benzothiadiazole derivative provided by this invention possesses excellent fluorescence properties, making it suitable as a fluorescent probe. It specifically binds to and fluorescently labels mitochondrial RNA within cells, exhibiting good selectivity, a high fluorescence signal-to-noise ratio, and excellent anti-interference capabilities in complex biological systems. Its stable biofluorescence signal is suitable for real-time, dynamic fluorescence imaging, enabling visualization and tracking of mitochondrial RNA distribution and changes. This provides new methods and ideas for developing more fluorescent probes for mitochondrial RNA detection. Furthermore, the preparation method of the derivative provided by this invention has clear steps, mild reaction conditions, and uses common or commercially available raw materials and reagents (such as amine compounds, brominating reagents, basic reagents, and various alternative solvents). This facilitates operation and scale-up, making it easier to prepare and better suited for real-time detection and imaging of mitochondrial RNA in vitro and in vivo, demonstrating good application potential in related biological research and disease diagnosis. Attached Figure Description

[0054] Figure 1 For compound 1-1 in Example 8 1 H NMR spectrum.

[0055] Figure 2 For compound 1-1 in Example 8 13 C NMR spectrum.

[0056] Figure 3 For compound 2-1 in Example 9 1 H NMR spectrum.

[0057] Figure 4 For compound 2-1 in Example 9 13 C NMR spectrum.

[0058] Figure 5 Compound 3-1 in Example 10 1 H NMR spectrum.

[0059] Figure 6 Compound 3-1 in Example 10 13 C NMR spectrum.

[0060] Figure 7 For compound 4-1 in Example 11 1 H NMR spectrum.

[0061] Figure 8 For compound 4-1 in Example 11 13 C NMR spectrum.

[0062] Figure 9This is a bar chart comparing the relative fluorescence intensities measured after titrating different types of nucleic acids with the compounds in Example 12 (A in the figure represents compound 1-1; B represents compound 2-1; C represents compound 3-1; and D represents compound 4-1).

[0063] Figure 10 This is a co-localization imaging image of compound 1-1 in Example 13 and a commercial mitochondrial / lysosomal fluorescent probe.

[0064] Figure 11 This is a co-localization imaging image of compound 2-1 in Example 13 and a commercial mitochondrial / lysosomal fluorescent probe.

[0065] Figure 12 This is a co-localization imaging image of compound 3-1 in Example 13 and a commercial mitochondrial / lysosomal fluorescent probe.

[0066] Figure 13 This is a co-localization imaging image of compound 4-1 in Example 13 and a commercial mitochondrial / lysosomal fluorescent probe.

[0067] Figure 14 The images show compounds 1-1 and 2-1 from Example 14 in HeLa cells after RNase and DNase digestion.

[0068] Figure 15 The images show compounds 3-1 and 4-1 from Example 14 in HeLa cells after RNase and DNase digestion. Detailed Implementation

[0069] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0070] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0071] Example 1: Synthesis of 1,2,3,3-Tetramethyl-3H-indole-1-onium iodide (ID-I)

[0072] Synthesis of the reactant ID-I: 1.6 g (10.0 mmol) of 2,3,3-trimethylindole was weighed and placed in a dry, pressure-resistant reaction tube. A magnetic stir bar was added, followed by the addition of iodomethane (1.70 g, 12 mmol) and 20 mL of acetonitrile. The mixture was stirred until the solid was completely dissolved, and then the reaction tube was sealed. The temperature was raised to 110 °C and the reaction was stirred for 18 h. After the reaction was completed, the system was cooled to room temperature, and the acetonitrile was removed by concentration under reduced pressure to obtain the crude product. Diethyl ether (5 mL × 3) was added to the crude product, and the mixture was stirred and washed to remove excess iodomethane and low-polarity impurities. The yellow solid (2.4 g, 80% yield) was collected by filtration.

[0073] The synthetic route is as follows: .

[0074] Example 2: Synthesis of 4-bromo-7-(dibromomethyl)benzo[c][1,2,5]thiadiazole (S-1)

[0075] Synthesis of intermediate S-1: 4-Bromo-7-methylbenzo[C][1,2,5]thiadiazole (2.4 g, 10 mmol) was weighed and placed in a dry three-necked flask with a magnetic stir bar and a reflux condenser (with a nitrogen balloon attached to the top for inert gas protection). 30 mL of chlorobenzene was added under nitrogen atmosphere and stirred until the substrate was fully dissolved. Then, N-bromosuccinimide (NBS, 3.9 g, 22 mmol) and azobisisobutyronitrile (AIBN, 250 mg, 1.5 mmol) were added sequentially. The mixture was heated to 110 °C in an oil bath and refluxed with stirring for 8 h. After the reaction, the system was cooled to room temperature, and the succinimide byproduct was removed by filtration. The filtrate was transferred to a separatory funnel and washed with saturated brine (50 mL × 3). The separated organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at ≤60 °C to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v) Using a ratio of 5:1 as the eluent, a pale yellow solid product (2.9 g, yield 72%) was obtained.

[0076] The synthetic route is as follows: .

[0077] Example 3: Synthesis of 7-bromo-4-aldehyde benzo[c][1,2,5]thiadiazole (S-2)

[0078] Synthesis of intermediate S-2: S-1 (398.9 mg, 1.0 mmol) was weighed and placed in a dry two-necked flask. A magnetic stir bar and a reflux condenser were added. 10 mL of toluene was added to the flask and stirred to dissolve S-2. Then, silver nitrate (424.7 mg, 2.5 mmol) and 5 mL of distilled water were added. The mixture was heated to the reflux temperature of toluene (about 110 °C) and stirred for 6 h. After the reaction was completed, the system was cooled to room temperature. The silver bromide precipitate formed in the reaction was removed by filtration. The filtrate was transferred to a separatory funnel to separate the toluene organic phase. The aqueous phase was extracted with toluene (5 mL × 3). All organic phases were combined, washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography with petroleum ether / ethyl acetate (v / v = 3:1) as the eluent to obtain a pale yellow solid product (178.6 mg, yield 70%).

[0079] The synthetic route is as follows: .

[0080] Example 4: Synthesis of 7-(4-methylpiperazin-1-yl)benzo[c][1,2,5]thiadiazole-4-carboxaldehyde (S-1-1)

[0081] Synthesis of reactant S-1-1: S-2 (255.1 mg, 1.0 mmol), N-methylpiperazine (120.2 mg, 1.2 mmol), and potassium carbonate (276.4 mg, 2.0 mmol) were weighed and placed in a dry two-necked flask. 10 mL of benzene was added, and the flask was equipped with a magnetic stir bar and a reflux condenser. The mixture was heated to 90 °C and stirred for 15 h. After the reaction was completed, the system was cooled to room temperature, and the insoluble inorganic salts were removed by filtration. The filtrate was transferred to a separatory funnel and washed with saturated brine (10 mL × 3). The separated organic phase was dried over anhydrous sodium sulfate for 30 min, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 2:1) as the eluent to obtain a yellow solid product (230.9 mg, yield 65%).

[0082] The synthetic route is as follows: .

[0083] Example 5: Synthesis of 7-(diethylamino)benzo[c][1,2,5]thiadiazole-4-carboxaldehyde (S-2-1)

[0084] Synthesis of reactant S-2-1: S-2 (255.1 mg, 1.0 mmol), diethylamine (87.8 mg, 1.2 mmol), and potassium carbonate (276.4 mg, 2.0 mmol) were weighed and placed in a dry two-necked flask. 10 mL of benzene was added, and the flask was equipped with a magnetic stir bar and a reflux condenser. The mixture was heated to 90 °C and stirred for 15 h. After the reaction was completed, the system was cooled to room temperature, and the insoluble inorganic salts were removed by filtration. The filtrate was transferred to a separatory funnel and washed with saturated brine (10 mL × 3). The separated organic phase was dried over anhydrous sodium sulfate for 30 min, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 2:1) as the eluent to obtain a yellow solid product (242.2 mg, yield 71%).

[0085] The synthetic route is as follows: .

[0086] Example 6: Synthesis of 7-(azacyclobutane-1-yl)benzo[c][1,2,5]thiadiazole-4-carboxaldehyde (S-3-1)

[0087] Synthesis of reactant S-3-1: S-2 (255.1 mg, 1.0 mmol), aziridine (68.5 mg, 1.2 mmol), and potassium carbonate (276.4 mg, 2.0 mmol) were weighed and placed in a dry two-necked flask. 10 mL of benzene was added, and the flask was equipped with a magnetic stir bar and a reflux condenser. The mixture was heated to 90 °C and stirred for 15 h. After the reaction was completed, the system was cooled to room temperature, and the insoluble inorganic salts were removed by filtration. The filtrate was transferred to a separatory funnel and washed with saturated brine (10 mL × 3). The separated organic phase was dried over anhydrous sodium sulfate for 30 min, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 2:1) as the eluent to obtain a yellow solid product (206.1 mg, yield 63%).

[0088] The synthetic route is as follows: .

[0089] Example 7: Synthesis of 7-(pyrrolidone-1-yl)benzo[c][1,2,5]thiadiazole-4-carboxaldehyde (S-4-1)

[0090] Synthesis of reactant S-4-1: S-2 (255.1 mg, 1.0 mmol), pyrrolidine (85.3 mg, 1.2 mmol), and potassium carbonate (276.4 mg, 2.0 mmol) were weighed and placed in a dry two-necked flask. 10 mL of benzene was added, and the flask was equipped with a magnetic stir bar and a reflux condenser. The mixture was heated to 90 °C and stirred for 15 h. After the reaction was completed, the system was cooled to room temperature, and the insoluble inorganic salts were removed by filtration. The filtrate was transferred to a separatory funnel and washed with saturated brine (10 mL × 3). The separated organic phase was dried over anhydrous sodium sulfate for 30 min, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 2:1) as the eluent to obtain a yellow solid product (238.0 mg, yield 70%).

[0091] The synthetic route is as follows: .

[0092] Example 8: Synthesis of (E)-1,3,3-trimethyl-2-(2-(7-(4-methylpiperazin-1-yl)benzo[c][1,2,5]thiadiazol-4-yl)vinyl)-3H-indole-1-onium iodide (1-1)

[0093] Synthesis of indole-benzothiadiazole derivative 1-1: S-1-1 (355.3 mg, 1.0 mmol) and ID-I (300.9 mg, 1.0 mmol) were weighed and placed in a dry two-necked flask equipped with a magnetic stir bar and a reflux condenser. 15 mL of ethanol was added and stirred to dissolve the solid completely. The mixture was then heated to the reflux temperature of ethanol (approximately 78 °C) and stirred for 12 h. After the reaction was completed, the system was cooled to room temperature and the ethanol was removed by vacuum concentration using a rotary evaporator to obtain the crude product. 10 mL of dichloromethane was added to the crude product to dissolve it. The solution was transferred to a separatory funnel and washed with saturated brine (10 mL × 2). The separated organic phase was dried over anhydrous sodium sulfate for 30 min, filtered, and the filtrate was concentrated under vacuum. The crude product was purified by silica gel column chromatography using dichloromethane / methanol (v / v = 10:1) as the eluent to obtain a reddish-brown solid product (409.4 mg, yield 60%).

[0094] The synthetic route is as follows: .

[0095] Indole-benzothiadiazole derivative 1-1 1 H NMR spectrum as follows Figure 1 As shown, its 13 C NMR spectra as follows Figure 2 As shown, the product's 1 H NMR and 13 The characterization results of C NMR are as follows:

[0096] 1 H NMR (500 MHz, DMSO-D6) δ 8.54 (d, J = 15.7 Hz, 1H), 8.43 (d, J =8.6 Hz, 1H), 8.23 ​​(d, J = 15.6 Hz, 1H), 7.78 (dd, J = 12.4, 7.7 Hz, 2H), 7.55(t, J = 7.7 Hz, 1H), 7.49 (t, J = 7.4 Hz, 1H), 7.09 (d, J = 8.7 Hz, 1H), 4.17(s, 4H), 3.97 (s, 3H), 2.29 (s, 3H), 1.78 (s, 6H), 1.19 (s, 1H).

[0097] 13C NMR (126 MHz, DMSO-D6) δ 186.99, 180.39, 155.29, 154.85, 149.23,147.38, 142.90, 142.19, 136.87, 128.93, 128.07, 122.84, 115.91, 114.08,109.44, 108.97, 54.41, 51.09, 48.87, 33.39, 27.39, 26.30.

[0098] Example 9: Synthesis of (E)-2-(2-(7-(diethylamino)benzo[c][1,2,5]thiadiazol-4-yl)vinyl)-1,3,3-trimethyl-3H-indole-1-onium iodide (2-1)

[0099] Synthesis of indole-benzothiadiazole derivative 2-1: S-2-1 (340.9 mg, 1.0 mmol) and ID-I (300.9 mg, 1.0 mmol) were weighed and placed in a dry two-necked flask equipped with a magnetic stir bar and a reflux condenser. 15 mL of ethanol was added and stirred to dissolve the solid completely. The mixture was then heated to the reflux temperature of ethanol (approximately 78 °C) and stirred for 12 h. After the reaction was completed, the system was cooled to room temperature and the ethanol was removed by vacuum concentration using a rotary evaporator to obtain the crude product. 10 mL of dichloromethane was added to the crude product to dissolve it. The solution was transferred to a separatory funnel and washed with saturated brine (10 mL × 2). The separated organic phase was dried over anhydrous sodium sulfate for 30 min, filtered, and the filtrate was concentrated under vacuum. The crude product was purified by silica gel column chromatography using dichloromethane / methanol (v / v = 10:1) as the eluent to obtain a reddish-brown solid product (359.6 mg, yield 55%).

[0100] The synthetic route is as follows: .

[0101] Indole-benzothiadiazole derivative 2-1 1 H NMR spectrum as follows Figure 3 As shown, its 13 C NMR spectra as follows Figure 4 As shown, the product's 1 H NMR and 13 The characterization results of C NMR are as follows:

[0102] 1H NMR (500 MHz, DMSO-D6) δ 8.41 (d, J = 15.3 Hz, 1H), 8.28 (d, J =9.0 Hz, 1H), 8.01 (d, J = 15.2 Hz, 1H), 7.66 (d, J = 7.5 Hz, 1H), 7.59 (d, J= 8.0 Hz, 1H), 7.42 (t, J = 7.7 Hz, 1H), 7.34 (t, J = 7.4 Hz, 1H), 6.78 (d, J= 9.1 Hz, 1H), 3.99 – 3.95 (m, 4H), 3.81 (s, 3H), 1.67 (s, 6H), 1.23 (t, J =7.0 Hz, 6H).

[0103] 13 C NMR (126 MHz, DMSO-D6) δ 179.16, 155.57, 149.09, 146.59, 146.13,142.17, 141.97, 129.43, 128.51, 127.01, 122.42, 113.56, 113.10, 106.86,106.00, 50.28, 47.28, 32.54, 26.31, 12.65.

[0104] Example 10: Synthesis of (E)-2-(2-(7-(azacyclobutane-1-yl)benzo[c][1,2,5]thiadiazol-4-yl)vinyl)-1,3,3-trimethyl-3H-indole-1-onium iodide (3-1)

[0105] Synthesis of indole-benzothiadiazole derivative 3-1: S-3-1 (339.9 mg, 1.0 mmol) and ID-I (300.9 mg, 1.0 mmol) were weighed and placed in a dry two-necked flask equipped with a magnetic stir bar and a reflux condenser. 15 mL of ethanol was added and stirred to dissolve the solid completely. The mixture was then heated to the reflux temperature of ethanol (approximately 78 °C) and stirred for 12 h. After the reaction was completed, the system was cooled to room temperature and the ethanol was removed by vacuum concentration using a rotary evaporator to obtain the crude product. 10 mL of dichloromethane was added to the crude product to dissolve it. The solution was transferred to a separatory funnel and washed with saturated brine (10 mL × 2). The separated organic phase was dried over anhydrous sodium sulfate for 30 min, filtered, and the filtrate was concentrated under vacuum. The crude product was purified by silica gel column chromatography using dichloromethane / methanol (v / v = 10:1) as the eluent to obtain a reddish-brown solid product (372.1 mg, yield 57%).

[0106] The synthetic route is as follows: .

[0107] Indole-benzothiadiazole derivative 3-1 1 H NMR spectrum as follows Figure 5 As shown, its 13 C NMR spectra as follows Figure 6 As shown, the product's 1 H NMR and 13 The characterization results of C NMR are as follows:

[0108] 1 H NMR (500 MHz, DMSO-D6) δ 8.48 (d, J = 15.1 Hz, 1H), 8.36 (d, J =8.9 Hz, 1H), 8.06 (s, 1H), 7.74 (d, J = 7.4 Hz, 1H), 7.66 (d, J = 7.9 Hz,1H), 7.50 (t, J = 7.7 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 6.68 (d, J = 8.9 Hz,1H), 3.88 (d, J = 5.4 Hz, 3H), 2.05 (t, J = 6.8 Hz, 4H), 1.76 (s, 4H), 1.20(d, J = 14.7 Hz, 1H).

[0109] 13 C NMR (126 MHz, DMSO-D6) δ 179.63, 155.97, 149.89, 147.62, 146.70,142.82, 142.75, 132.03, 129.21, 127.59, 123.16, 114.06, 113.71, 107.92,106.23, 53.36, 50.92, 33.18, 29.50, 27.12.

[0110] Example 11: Synthesis of (E)-1,3,3-trimethyl-2-(2-(7-(pyrrolidone-1-yl)benzo[c][1,2,5]thiadiazol-4-yl)vinyl)-3H-indole-1-onium iodide (4-1)

[0111] Synthesis of indole-benzothiadiazole derivative 4-1: S-4-1 (338.9 mg, 1.0 mmol) and ID-I (300.9 mg, 1.0 mmol) were weighed and placed in a dry two-necked flask equipped with a magnetic stir bar and a reflux condenser. 15 mL of ethanol was added and stirred to dissolve the solid completely. The mixture was then heated to the reflux temperature of ethanol (approximately 78 °C) and stirred for 12 h. After the reaction was completed, the system was cooled to room temperature and the ethanol was removed by vacuum concentration using a rotary evaporator to obtain the crude product. 10 mL of dichloromethane was added to the crude product to dissolve it. The solution was transferred to a separatory funnel and washed with saturated brine (10 mL × 2). The separated organic phase was dried over anhydrous sodium sulfate for 30 min, filtered, and the filtrate was concentrated under vacuum. The crude product was purified by silica gel column chromatography using dichloromethane / methanol (v / v = 10:1) as the eluent to obtain a reddish-brown solid product (391.1 mg, yield 60%).

[0112] The synthetic route is as follows: .

[0113] Indole-benzothiadiazole derivative 4-1 1 H NMR spectrum as follows Figure 7 As shown, its 13 C NMR spectra as follows Figure 8 As shown, the product's 1 H NMR and 13 The characterization results of C NMR are as follows:

[0114] 1 H NMR (500 MHz, DMSO-D6) δ 8.46 (d, J = 15.3 Hz, 1H), 8.34 (d, J =8.7 Hz, 1H), 8.03 (d, J = 15.1 Hz, 1H), 7.73 (d, J = 7.4 Hz, 1H), 7.65 (d, J= 8.0 Hz, 1H), 7.50 (td, J = 7.7, 1.2 Hz, 1H), 7.41 (td, J = 7.4, 0.9 Hz,1H), 6.44 (d, J = 8.7 Hz, 1H), 3.86 (s, 3H), 2.58 – 2.49 (m, 2H), 2.04 (s,4H), 1.75 (s, 6H).

[0115] 13C NMR (126 MHz, DMSO-D6) δ 179.13, 155.26, 146.93, 146.23, 142.37,142.33, 128.79, 127.13, 122.73, 118.23, 113.56, 113.24, 105.61, 104.40,50.45, 32.69, 26.67, 16.78, 1.29.

[0116] Example 12: Fluorescence spectroscopy testing of indole-benzothiadiazole derivatives for different nucleic acid selectivity

[0117] The indole-benzothiadiazole derivatives prepared in Examples 8-11 were diluted to a working concentration (1 μM) using Tris buffer (10 mM Tris, 60 mM KCl, pH 7.4). Different types of nucleic acid solutions were added, with the nucleic acid sequences and types shown in Table 1. The mixture was gently mixed and incubated at room temperature in the dark for 15 min. A Tris solution containing only an equal amount of the compound and no nucleic acid was used as a blank control. Detection was performed using a fluorescence spectrometer: the optimal excitation wavelength of the compound was set, and the scanning range was 500-750 nm. The fluorescence emission spectra of each sample under these conditions were recorded.

[0118] Table 1. Nucleic acid sequences and types used in this invention

[0119]

[0120] Note: In the sequence listing, "r" refers to the base "U"; the 4a4t sequence is set to be skipped because it has less than 10 bases.

[0121] The measurement results are as follows Figure 9 As shown, in vitro selectivity experiments demonstrated that the indole-benzothiadiazole derivatives prepared in Examples 8-11 are "fluorescence-activated" probes: their fluorescence is at background levels (F / F0≈1) in the absence of a target or when interacting with DNA; however, upon binding to single- or double-stranded RNA (ssRNA / dsRNA) or G-quadruplex RNA (G4 RNA), the fluorescence signal is specifically "activated," resulting in significant fluorescence enhancement. These results demonstrate that such compounds can effectively distinguish between DNA and RNA, exhibiting excellent selective recognition ability for RNA structures.

[0122] Example 13: Co-localization imaging of compounds and commercial organelle dyes

[0123] HeLa cells were seeded in confocal culture dishes and cultured to an appropriate density. Then, they were co-stained with the target small molecule probes (indole-benzothiadiazole derivatives 1-1, 2-1, 3-1, and 4-1) prepared in Examples 8-11, along with their corresponding deep red mitochondrial or lysosomal dyes. The staining concentration of small molecule 1-1 was 10 μM, while the staining concentrations of small molecules 2-1, 3-1, and 4-1 were all 0.5 μM. After staining, imaging was performed using a confocal microscope: small molecule 1-1 was excited using a 488 nm laser channel, while small molecules 2-1, 3-1, and 4-1 were excited using a 561 nm laser channel; both deep red organelle dyes were excited using a 640 nm laser channel. By comparing the superposition of signals from different channels, the degree of co-localization between each small molecule probe and specific organelles was analyzed.

[0124] The results are as follows Figures 10 to 13 As shown, confocal imaging results indicate that the fluorescence signal of the indole-benzothiadiazole derivative provided by this invention highly overlaps with mitochondrial dyes, while exhibiting low co-localization with lysosomal dyes, thus confirming its excellent mitochondrial targeting.

[0125] Example 14: Staining of fixed cells based on enzymatic digestion

[0126] Suitable HeLa cells were seeded in confocal culture dishes, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, and then divided into three groups for treatment: (1) control group: incubated with PBS buffer only; (2) DNase treatment group: incubated with buffer containing DNase I (1U / μL) at 37℃ for 3h; (3) RNase treatment group: incubated with buffer containing RNase A (100μg / mL) at 37℃ for 3h. After enzymatic digestion, the cells in each group were thoroughly washed with PBS, and co-stained with DAPI using the target small molecule probes prepared in Examples 8-11 (at the staining concentration in Example 13 above), and the retention of fluorescence signals was observed by confocal microscopy.

[0127] The measurement results are as follows Figure 14 and Figure 15 As shown, after treatment with different nucleases, the fluorescence signal of the indole-benzothiadiazole derivative of the present invention in HeLa cells was significantly quenched after treatment with RNase A, but was basically retained after treatment with DNase I, which directly proves that its fluorescence generation depends on the binding of intracellular RNA.

[0128] The above experimental results show that the indole-benzothiadiazole derivative provided by this invention can specifically bind to RNA and generate a strong fluorescence response in vitro. In living cells, its fluorescence signal is highly co-localized with mitochondria, indicating that the indole-benzothiadiazole derivative can effectively enter cells, specifically bind to RNA in mitochondria, and generate a significant fluorescence-on signal, achieving specific labeling and imaging of mitochondrial RNA. It is a highly efficient small-molecule fluorescent probe for mitochondrial RNA. Furthermore, this invention uses a dimethylindole fragment as the mitochondrial targeting group, responsible for guiding the probe to accumulate in mitochondria; while the benzothiadiazole fragment acts as the recognition and signaling core, responsible for specifically binding RNA and participating in fluorescence emission. The covalent connection between the two achieves functional synergy, enabling the compound to target mitochondria and detect RNA in situ. Its red fluorescence emission originates from the entire extended indole-benzothiadiazole conjugated system, reflecting the photophysical properties of their combined action. It can specifically bind to and fluorescently label mitochondrial RNA in cells, and can be applied to the dynamic tracking and functional study of mitochondrial RNA in living cells.

[0129] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A class of indole-benzothiadiazole derivatives, characterized in that, The structural formula of the derivative is: ； R is selected from one of methylpiperazinyl, diethylamino, aziridine, and pyrrolidinyl.

2. The method for preparing the indole-benzothiadiazole derivative according to claim 1, characterized in that, 1,2,3,3-Tetramethyl-3H-indole-1-onium iodide and benzothiadiazole derivative were dissolved in organic solvent E and reacted at 70-85℃ for 10-15 h. After the reaction was completed, the indole-benzothiadiazole derivative was obtained by concentration, washing, drying and purification. The structural formula of the benzothiadiazole derivative is selected from one of the following structural formulas: 。 3. The preparation method according to claim 2, characterized in that, The organic solvent E is selected from one or more of methanol, isopropanol, n-butanol, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, 1,4-dioxane, acetic acid, and water.

4. The preparation method according to claim 2, characterized in that, The molar ratio of the 1,2,3,3-tetramethyl-3H-indole-1-onium iodide and the benzothiadiazole derivative is 1:(1-2).

5. The preparation method according to claim 2, characterized in that, The method for synthesizing the benzothiadiazole derivative is as follows: (1) Synthesis of intermediate raw material S-1: 4-bromo-7-methylbenzo[C][1,2,5]thiadiazole, bromide reagent and azobisisobutyronitrile were dissolved in organic solvent B and reacted at 80-120℃ for 4-16 h. After the reaction was completed, the intermediate raw material S-1 was obtained by washing, drying and purification. Its structural formula is as follows: ； (2) Synthesis of intermediate raw material S-2: Intermediate raw material S-1 and silver nitrate were reacted with each other in a mixed system of water and organic solvent C at reflux temperature for 4-8 hours. After the reaction was completed, the intermediate raw material S-2 was obtained by extraction, washing, drying and purification. Its structural formula is as follows: ； (3) Synthesis of benzothiadiazole derivatives of the reaction raw material: intermediate raw material S-2 is dissolved in organic solvent D with amine compound and base and reacted at 80-100℃ for 12-18h. After the reaction is completed, the benzothiadiazole derivatives of the reaction raw material are obtained by washing, drying and purification. The amine compound is selected from one of N-methylpiperazine, diethylamine, aziridine, and pyrrolidine.

6. The preparation method according to claim 5, characterized in that, The brominating reagent is selected from one or more of N-bromosuccinimide, N-bromoacetamide, and 1,3-dibromo-5,5-dimethylhydantoin; the organic solvent B is selected from one or more of chlorobenzene, o-dichlorobenzene, mesitylene, 1,2-dichloroethane, diphenyl ether, anisole, 1,3-dimethyl-2-imidazolinone, and N-methylpyrrolidone; the organic solvent C is selected from benzene, toluene, xylene, dichloromethane, chloroform, 1,2-dichloroethane, and tetrahydrobenzene. The base is selected from one or more of furan and 1,4-dioxane; the base is selected from one or more of cesium carbonate, potassium phosphate, sodium carbonate, triethylamine, N,N-diisopropylethylamine, pyridine, 1,8-diazabicycloundec-7-ene, potassium tert-butoxide, sodium hydride, sodium methoxide, sodium bicarbonate, and potassium hydroxide; the organic solvent D is selected from one or more of benzene, toluene, xylene, dichloromethane, chloroform, 1,2-dichloroethane, tetrahydrofuran, and 1,4-dioxane.

7. The preparation method according to claim 5, characterized in that, The molar ratio of 4-bromo-7-methylbenzo[C][1,2,5]thiadiazole, bromide reagent and azobisisobutyronitrile is 1:(2-5):(0.05-0.2); the molar ratio of intermediate raw material S-1 and silver nitrate is 1:(2-3); the molar ratio of intermediate raw material S-2, amine compound and base is 1:(1.2-2):(1.5-3).

8. The use of the indole-benzothiadiazole derivative of claim 1 in the preparation of products for detecting or labeling mitochondrial RNA.

9. A product for detecting mitochondrial RNA, characterized in that, Contains the indole-benzothiadiazole derivative of claim 1.

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