Alkenyl indole derivative as well as preparation method and application thereof in field of RNA (Ribonucleic Acid) fluorescent probes

Abstract

The invention belongs to the technical field of RNA (Ribonucleic Acid) detection, and particularly relates to an alkenyl indole derivative as well as a preparation method and application thereof in the field of RNA fluorescent probes. The alkenyl indole derivative takes alkenyl indole as a mother nucleus, is a novel functional fluorescent probe, can be used for dyeing RNA (Ribonucleic Acid) in cells, and widens the range of fluorescent probe types. The process of detecting RNA by using the compound as a fluorescent probe is not interfered by other components, and the compound shows excellent anti-interference performance and high fluorescence intensity. Besides, the preparation process is simple, raw materials are easy to obtain, reaction conditions are mild, and the product is stable in structure and convenient to store, has important application value in the fields of RNA detection and biological function research, and particularly shows wide application prospects in the fields of living cell imaging, molecular diagnosis and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of RNA detection, and in particular relates to an alkenyl indole derivative and a preparation method thereof and application thereof in the field of RNA fluorescent probes. Background Art

[0002] Nucleic acids are a class of large biological molecules usually located in the cell nucleus. They are mainly responsible for the storage and transmission of genetic information of organisms. They are the general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids are widely present in all animal and plant cells, microorganisms, viruses and bacteriophages. They are one of the basic substances of life and play a decisive role in life phenomena such as growth, development, inheritance and variation of organisms. As an important information carrier and functional molecule, RNA plays a key role in biological processes such as gene reverse transcription, transcriptional regulation and protein translation. Studies in recent years have shown that RNA not only plays an important role in life activities such as gene expression regulation, telomere stability maintenance, RNA splicing regulation and virus replication, but its abnormal folding is also closely related to the occurrence and development of various diseases such as cancer and neurodegenerative diseases. Therefore, the development of molecular probes that can specifically recognize RNA has important scientific significance and application value for revealing disease mechanisms, achieving early diagnosis and promoting the development of targeted drugs.

[0003] At present, the research on fluorescent probes for nucleic acids is mainly concentrated in the field of DNA, and some probes have successfully achieved in vitro and in vivo detection of DNA. However, the research on fluorescent probes that can specifically recognize RNA is relatively scarce, and the existing probes generally have insufficient photostability, which makes it difficult to meet the needs of long-term dynamic monitoring; high cytotoxicity limits their application in living bodies; poor membrane permeability affects their effective enrichment in cells and other technical bottlenecks. This limits the practical application of RNA probes in the biomedical field. Therefore, the development of a fluorescent probe that can specifically recognize RNA, has excellent photostability and biocompatibility, so as to achieve real-time tracking of the dynamic folding process of RNA in vivo and in vitro and in-depth research on its biological functions has become a technical problem that needs to be solved urgently.

[0004] Indole is a benzopyrrole aromatic heterocyclic compound. Indole and its derivatives have shown significant advantages in the field of fluorescent probe design due to their good biocompatibility and excellent fluorescence properties. Due to their rigid conjugated structure and strong modifiability, the molecular structure of alkenyl indole compounds is highly modifiable. By introducing specific functional groups, highly selective recognition of RNA and precise regulation of fluorescence signals can be achieved, providing new ideas for the development of high-performance RNA probes. Summary of the invention

[0005] In order to solve the above-mentioned deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide an alkenyl indole derivative, which can be used as a fluorescent probe for specific and selective detection of RNA in vivo and in vitro.

[0006] Another object of the present invention is to provide a method for preparing the above alkenyl indole derivatives.

[0007] Another object of the present invention is to provide the application of the alkenyl indole derivatives.

[0008] The fourth object of the present invention is to provide an RNA fluorescent probe.

[0009] The purpose of the present invention is achieved through the following technical solutions: An alkenyl indole derivative is a compound having a structure as shown in formula (A) or a pharmaceutically acceptable salt thereof:

[0010] (A) Wherein, R is selected from anisole, methylpyridin-1-ium, benzaldehyde or N-methyl-4-phenylvinylpyridin-1-ium.

[0011] The alkenyl indole derivative is preferably a compound having a structure as shown in formula (I), formula (II) or formula (III) or a pharmaceutically acceptable salt thereof.

[0012]

[0013] Formula (I) Formula (II) Formula (III) The preparation method of the alkenyl indole derivative comprises the following steps: (1) Under nitrogen protection, indole, 2-chloropyridine, tripotassium phosphate, cuprous iodide, trans-N,N'-dimethyl-1,2-cyclohexanediamine and solvent toluene are mixed and reacted at 90-110° C. for 24-36 hours to obtain an intermediate represented by formula (a); (2) Under nitrogen protection, the intermediate of formula (a) obtained in step (1), the phenylacetylene derivative, the catalyst, the first additive and the second additive are dissolved in anhydrous ether as a solvent, and reacted at 70-90° C. for 10-16 hours to obtain an alkenyl indole derivative having a structure as shown in formula (I) or formula (II) or an intermediate of formula (b); (3) Adding the intermediate of formula (b) obtained in step (2) and N-methyl-4-methylpyridin-1-ium to anhydrous ethanol as a solvent, and then adding piperidine; then reacting at 50-70° C. for 8-12 hours to obtain an olefinic indole derivative having a structure as shown in formula (III).

[0014] The structure of the intermediate represented by formula (a) in step (1) is as follows:

[0015] Formula (a) The phenylacetylene derivative described in step (2) is preferably 4-ethynylanisole, 4-ethynyl-1-methylpyridin-1-ium or 4-ethynylbenzaldehyde, and its structure is shown below:

[0016] The 4-ethynyl-1-methylpyridin-1-ium is preferably prepared by the following method: Mix 4-ethynylpyridine, iodomethane and tetrahydrofuran; react at 66° C. for 3 h to obtain 4-ethynyl-1-methylpyridin-1-ium.

[0017] The structure of the intermediate represented by formula (b) in step (2) is as follows:

[0018] Formula (b) The molar ratio of indole, 2-chloropyridine, tripotassium phosphate, cuprous iodide and trans-N,N'-dimethyl-1,2-cyclohexanediamine solvent described in step (1) is preferably 1:1.2:2.1:0.05:0.2; wherein, too much amount of 2-chloropyridine, tripotassium phosphate, cuprous iodide and trans-N,N'-dimethyl-1,2-cyclohexanediamine solvent will cause waste, while too little amount will lead to decreased conversion rate and reduced atomic utilization.

[0019] The amount-to-volume ratio (mmol:mL) of indole to toluene in step (1) is 1:5. If the amount of toluene is too much, it will cause waste, while if the amount of toluene is too little, it will lead to a decrease in yield and an increase in by-products.

[0020] The reaction conditions described in step (1) are preferably 110°C for 24h. Since trans-N,N'-dimethyl-1,2-cyclohexanediamine is used as a ligand, if the reaction temperature is too high, it will decompose or change its structure, thereby losing its promoting effect on the reaction and affecting the catalytic efficiency of the reaction. If the reaction temperature is too low, its coordination effect cannot be fully exerted, affecting the activity and reaction efficiency of the copper catalyst. When the reaction time at 110°C is too short and not completed, the yield is reduced. When the reaction time at 110°C is too long, the yield is not significantly improved. If the reaction temperature is reduced from 110°C to 90°C, the reaction time needs to be extended to 36h.

[0021] The catalyst described in step (2) is manganese pentacarbonyl bromide (MnBr(CO)5), the first additive is N,N-diisopropylethylamine (DIPEA), and the second additive is benzoic acid (PhCO2H); if other catalysts and additives are used in the present invention, the yield is significantly reduced, or even the reaction does not occur.

[0022] The molar ratio of the intermediate represented by formula (a), the phenylacetylene derivative, the catalyst, the first additive and the second additive in step (2) is preferably 1:1.5:0.02:0.02:0.02; too large a molar ratio of the intermediate, the phenylacetylene derivative, the catalyst, the first additive and the second additive will cause waste, while too small a molar ratio will lead to a decrease in conversion rate.

[0023] The amount-to-volume ratio (mmol:mL) of the intermediate represented by formula (a) to anhydrous ether in step (2) is 1:5. If the anhydrous ether is replaced with other solvents, the yield will decrease or even the reaction will not occur. Excessive use of anhydrous ether will cause waste, while too little will result in a decrease in yield and an increase in by-products.

[0024] The reaction conditions in step (2) are preferably 80° C. for 12-16 h.

[0025] The molar ratio of the intermediate of formula (b) to N-methyl-4-methylpyridin-1-ium in step (3) is preferably 1:1.1.

[0026] The amount-to-volume ratio (mmol:mL) of the intermediate of formula (b) and anhydrous ethanol (EtOH) in step (3) is 1:(2-5); too much volume of anhydrous ethanol will cause waste, while too little volume of anhydrous ethanol will lead to insufficient dissolution and reduce the reaction yield.

[0027] The molar ratio of the intermediate of formula (b) to pyridine in step (3) is preferably 10:1.5.

[0028] The reaction conditions in step (3) are preferably 60°C for 10 hours. When the reaction temperature (60°C) is too low, the solubility of the raw materials decreases, reducing the reaction yield. When the temperature is too high, more by-products are generated. When the reaction time at 60°C is too short, the reaction is not completed, and the yield is reduced. When the reaction time at 60°C is too long, the yield is not significantly improved.

[0029] The N-methyl-4-methylpyridin-1-ium described in step (3) is preferably prepared by the following method: Under nitrogen protection, 4-methylpyridine, iodomethane and tetrahydrofuran were mixed and reacted at 66°C for 6h to obtain N-methyl-4-methylpyridin-1-ium.

[0030] The application of the alkenyl indole derivatives in the field of RNA fluorescent probes.

[0031] An RNA fluorescent probe comprises the alkenyl indole derivative.

[0032] The synthetic route of the present invention is as follows: When the alkenyl indole derivative has a structure as shown in formula (I) or formula (II), the synthesis route is as follows:

[0034] When the alkenyl indole derivative has a structure as shown in formula (III), the synthesis route is as follows:

[0036] Compared with the prior art, the present invention has the following advantages and effects: (1) The present invention uses alkenyl indole as the parent core to prepare a series of alkenyl indole derivatives. The series of alkenyl indole derivatives can be used as fluorescent probes, covering the visible light region to the near-infrared region, and have good luminescence properties and a large Stokes shift. They are a new type of full-spectrum fluorescent probe. The present invention further broadens the range of existing fluorescent probes.

[0037] (2) The alkenyl indole derivatives provided by the present invention are a new type of functional nucleic acid fluorescent probe, which has the characteristics of low cytotoxicity, good membrane permeability, excellent photostability and biocompatibility. It can realize the specific detection of nucleolar RNA in cells. The RNA detection process is not interfered by other components, and exhibits excellent anti-interference performance and high fluorescence intensity, which provides ideas and directions for the real-time tracking of RNA in vivo and in vitro and the in-depth study of its biological functions.

[0038] (3) The preparation method of the alkenyl indole derivatives provided by the present invention not only has simple and readily available raw materials, but also has a simple operation process, mild reaction conditions, stable product structure, and is easy to store. It solves the problem that the prior art cannot effectively extend the indole conjugated skeleton, resulting in poor application of indole in the field of fluorescent probes.

[0039] (4) The present invention has important application value in the field of RNA detection and its biological function research, especially in the fields of living cell imaging and molecular diagnosis, showing broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the absorption spectrum result diagram of the fluorescent probe PyIC of the present invention before and after the action with different RNAs.

[0041] Figure 2 It is a fluorescence spectrum result diagram of the fluorescent probe PyIC of the present invention before and after the action with different RNAs.

[0042] Figure 3 This is a result diagram of the fluorescence spectrum change of the fluorescent probe PyIC of the present invention as RNA is added.

[0043] Figure 4 This is a graph showing the cytotoxicity of the fluorescent probe PyIC of the present invention.

[0044] Figure 5 This is a result diagram of endogenous RNA cell imaging of the present invention. DETAILED DESCRIPTION

[0045] Example 1 Synthesis of (E)-2-(4-methoxyphenylvinyl)-1-(pyridin-2-yl)-1H-indole (PyIA)

[0046] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0047] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0048]

[0049] (1) In a dry Schlenk reaction tube, indole (1.0 mmol), 2-chloropyridine (1.2 mmol), tripotassium phosphate (2.1 mmol), cuprous iodide (0.05 mmol), trans-N,N'-dimethyl-1,2-cyclohexanediamine (0.20 mmol) and 5 mL of toluene were added in sequence, and then nitrogen was introduced into the system three times to exclude oxygen; the reaction system was then heated to 110°C and magnetic stirring was turned on under nitrogen protection for 24 h; after the reaction was completed, the reaction was cooled to room temperature and the solvent was removed under reduced pressure to obtain a crude product; the crude product was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 20:1 (V:V)) to obtain the target product 1-(pyridin-2-yl)-1H-indole (transparent oil, yield 85%).

[0050] (2) In a dry Schlenk reaction tube, 1-(pyridin-2-yl)-1H-indole (1 mmol), 4-ethynylanisole (1.5 mmol), pentacarbonylmanganese bromide (0.02 mmol), N,N-diisopropylethylamine (0.02 mmol), benzoic acid (0.02 mmol) and 5 mL of anhydrous ether were added in sequence, and nitrogen was then introduced into the system three times to remove oxygen; the reaction system was then heated to 80°C. The reaction was allowed to stir magnetically under nitrogen protection for 12 h. After the reaction was completed, the mixture was cooled to room temperature and extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (eluent: dichloromethane: petroleum ether = 1:1 (V:V)) to obtain the target product (E)-2-(4-methoxyphenylvinyl)-1-(pyridin-2-yl)-1H-indole (PyIA) (white solid, yield 85%).

[0051] The NMR characterization results of the fluorescent probe PyIA with alkenyl indole as the parent core are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.62 (dd, J = 4.9, 1.1 Hz, 1H), 7.96 (td, J = 7.7, 2.0 Hz, 1H), 7.52 – 7.47 (m, 1H), 7.43 – 7.37 (m, 2H), 7.27 (dd, J =9.1, 4.1 Hz, 3H), 7.06 (d, J = 16.3 Hz, 1H), 7.04 – 6.99 (m, 2H), 6.93 (s,1H), 6.84 (d, J = 16.3 Hz, 1H), 6.78 (d, J = 8.7 Hz, 2H), 3.63 (s, 3H). HRMS(ESI+):Calculated for C 22 H 18 N2O + [M+Na] + :349.1425, Found: 349.1397.

[0052] The spectral properties of the above PyIA in water solvent were tested, and the test results are shown in Table 1.

[0053] Table 1 Spectral properties of PyIA in water solvent

[0054] Note: abs max is the maximum absorption wavelength, λ em max The maximum emission wavelength, ε is the maximum molar absorption coefficient, Φ is the fluorescence quantum yield; Stokes shift is λ em max - λ abs max .

[0055] Example 2 Synthesis of (E)-1-methyl-4-[2-(1-(pyridin-2-yl)-1H-indol-2-yl)vinyl]pyridin-1-ium (PyIB)

[0056]

[0057] (1) In a dry Schlenk reaction tube, 4-ethynylpyridine (1 mmol), iodomethane (1.1 mmol), and 5 mL of tetrahydrofuran were added in sequence as solvent, and then nitrogen was introduced into the system three times to exclude oxygen; the reaction system was then heated to 66°C and magnetic stirring was turned on under nitrogen protection for 3 h; after the reaction was completed, the reaction was cooled to room temperature and the solvent was removed under reduced pressure to obtain the target product 4-ethynyl-1-methylpyridin-1-ium (black solid, yield 98%).

[0058] (2) In a dry Schlenk reaction tube, 1-(pyridin-2-yl)-1H-indole (1 mmol) prepared in Example 1, 4-ethynyl-1-methylpyridin-1-ium (1.5 mmol) prepared in step (1), pentacarbonyl manganese bromide (0.02 mmol), N,N-diisopropylethylamine (0.02 mmol), benzoic acid (0.02 mmol) and 5 mL of anhydrous ether were added in sequence, and then nitrogen was introduced into the system three times to remove oxygen; the reaction system was then heated to 80°C. , and open the magnetic stirring to react for 16 hours under nitrogen protection; after the reaction, cool to room temperature, extract with ethyl acetate three times, combine the organic phases, dry over anhydrous sodium sulfate, and remove the solvent under reduced pressure to obtain a crude product; the crude product is purified by silica gel column chromatography (eluent: dichloromethane: methanol = 30:1 (V:V)) to obtain the target product (E)-1-methyl-4-[2-(1-(pyridin-2-yl)-1H-indol-2-yl)vinyl]pyridin-1-ium (PyIB) (light yellow solid, yield 74%).

[0059] The NMR characterization results of the fluorescent probe PyIB with alkenyl indole as the parent core are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.81 (d, J = 6.5 Hz, 2H), 8.67 (dd, J =5.0, 1.9 Hz, 1H), 8.61 (s, 1H), 8.37 (dd, J = 7.1, 2.2 Hz, 1H), 8.32 – 8.24(m, 2H), 8.21 (d, J = 6.5 Hz, 2H), 8.13 (td, J = 7.6, 2.1 Hz, 1H), 7.67 (d, J = 8.2Hz, 1H), 7.63 (d, J = 16.2 Hz, 1H), 7.51 – 7.43 (m, 1H), 7.42 (dd, J =4.5, 2.7 Hz, 1H), 7.41 – 7.38 (m, 1H), 4.18 (s, 3H). HRMS (ES+) : Calculatedfor C 21 H 18 N3 + [M] + : 312.1563,Found:312.1541.

[0060] The spectral properties of the above-mentioned PyIB in water solvent were detected, and the test results are shown in Table 2.

[0061] Table 2 Spectral properties of PyIB in water solvent

[0062] Note: abs max is the maximum absorption wavelength, λ em max The maximum emission wavelength, ε is the maximum molar absorption coefficient, Φ is the fluorescence quantum yield, and Stokes shift is λ em max - λ abs max .

[0063] Example 3 Synthesis of 1-methyl-4-((E)-4-((E)-2-(1-(pyridin-2-yl)-1H-indol-2-yl)vinyl)phenylvinyl)pyridin-1-ium (PyIC)

[0064]

[0065] (1) In a dry Schlenk reaction tube, 1-(pyridin-2-yl)-1H-indole (1 mmol), 4-ethynylbenzaldehyde (1.5 mmol), pentacarbonylmanganese bromide (0.02 mmol), N,N-diisopropylethylamine (0.02 mmol), benzoic acid (0.02 mmol) and 5 mL of anhydrous ether were added in sequence, and then nitrogen was introduced into the system three times to remove oxygen; the reaction system was then heated to 80° C, and open the magnetic stirring to react for 12 hours under nitrogen protection; after the reaction, cool to room temperature, extract with ethyl acetate three times, combine the organic phases, dry over anhydrous sodium sulfate, and remove the solvent under reduced pressure to obtain a crude product; the crude product is purified by silica gel column chromatography (eluent: dichloromethane: methanol = 30:1 (V:V)) to obtain (E)-4-(2-(1-(pyridin-2-yl)-1H-indol-2-yl)vinyl)benzaldehyde (light yellow solid, yield 82%).

[0066] (2) In a dry Schlenk reaction tube, 4-methylpyridine (1 mmol), iodomethane (1.1 mmol), and 5 mL of tetrahydrofuran were added in sequence as solvent, and then nitrogen was introduced into the system three times to exclude oxygen; the reaction system was then heated to 66°C, and magnetic stirring was turned on under nitrogen protection for 6 h; after the reaction was completed, the reaction was cooled to room temperature, and the solvent was removed under reduced pressure to obtain the target product N-methyl-4-methylpyridin-1-ium (black solid, yield 98%).

[0067] (3) In a dry Schlenk reaction tube, (E)-4-(2-(1-(pyridin-2-yl)-1H-indol-2-yl)vinyl)benzaldehyde (10 mmol, 3.24 g) prepared in step (1) and N-methyl-4-methylpyridin-1-ium (11 mmol, 1.19 g) prepared in step (2) were dissolved in anhydrous ethanol (EtOH, 30 mL), piperidine (1.5 mmol, 0.129 g) was added as a reaction catalyst, and the mixture was stirred at room temperature for 10 min to mix well; then the reaction system was heated to 60°C and stirred for 10 h; after the reaction was completed, the mixture was cooled to room temperature and the ethanol solvent was removed by vacuum distillation to obtain a crude product; the crude product was treated with ethyl acetate ( The product was dissolved in 20% ethanol (50 mL) and washed with saturated sodium bicarbonate solution (20 mL × 2) and deionized water (20 mL × 2) in sequence. The organic phases were combined after separation and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain a light orange oil. The product was purified by gradient elution using silica gel column chromatography (eluent: dichloromethane and methanol), specifically: gradually transitioning from low-grade (pure dichloromethane) to the target ratio of dichloromethane: methanol = 10:1 (V:V). The target components were collected and rotary evaporated to obtain the compound 1-methyl-4-((E)-4-((E)-2-(1-(pyridin-2-yl)-1H-indol-2-yl)vinyl)phenylvinyl)pyridin-1-ium (PyIC) (orange solid, yield 85%).

[0068] The NMR characterization results of the fluorescent probe PyIC with alkenyl indole as the parent core are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 9.00 – 8.95 (m, 2H), 8.27 (dd, J = 3.2,1.7 Hz, 1H), 7.92 (d, J = 7.7 Hz, 2H), 7.86 – 7.78 (m, 2H), 7.53 (ddd, J =8.5, 6.2, 1.5 Hz, 2H), 7.52 (s, 4H), 7.51 – 7.43 (m, 1H), 7.41 (td, J = 6.5,1.2 Hz, 1H), 7.27 – 7.15 (m, 4H), 7.11 – 7.03 (m, 2H), 4.26 (d, J = 1.0 Hz,3H). HRMS (ES+): Calculated for C 29 H 24 N3 +[M] + :414.2017 ,Found:414.2026.

[0069] The spectral properties of the above PyIC in water solvent were detected, and the test results are shown in Table 3.

[0070] Table 3 Spectral properties of PyIC in water solvent

[0071] Note: abs max is the maximum absorption wavelength, λ em max The maximum emission wavelength, ε is the maximum molar absorption coefficient, Φ is the fluorescence quantum yield, and Stokes shift is λ em max - λ abs max .

[0072] Example 4 Spectra of the fluorescent probe PyIC interacting with different active substances

[0073] This example detects the absorption spectrum and fluorescence spectrum of the fluorescent probe PyIC before and after the action of different active substances. The specific experimental process is as follows: The fluorescent probe PyIC prepared in Example 3 was dissolved in a neutral (pH=7.0) Tris-HCl buffer solution (20 mM NaCl, 20 mM Tris) to prepare a stock solution with a concentration of 20 μmol / L of the fluorescent probe PyIC, which was evenly divided into 6 portions, with at least 3 replicates for each treatment; 2 times the equivalent of different types of RNA (all commercially available) were added to each stock solution; after mixing evenly, the absorption spectrum and fluorescence emission spectrum were measured, wherein the excitation wavelength for measuring the fluorescence spectrum was 410 nm, and the excitation and emission slit widths were both 20 nm.

[0074] Figure 1 The absorption spectra of the fluorescent probe PyIC of the present invention before and after the action with different RNAs, wherein the different types of nucleic acids are shown in Table 4, namely miR-18a, miR-20a, miR-17, pre-miR-21, MALAT1, SS, a total of 6 types. The horizontal axis is wavelength (nm), and the vertical axis is absorbance. Figure 1 It can be seen that when RNA is not added, the maximum absorption wavelength of the fluorescent probe PyIC is around 410 nm. When RNA is added, the absorption peak intensity at 410 nm in the absorption spectrum of the fluorescent probe PyIC increases, and after adding MALAT1, the absorption peak intensity at 410 nm in its absorption spectrum increases significantly.

[0075] Figure 2The fluorescence spectra of the fluorescent probe PyIC of the present invention before and after the interaction with different RNAs are shown in Figure 1, where the horizontal axis is the wavelength (nm) and the vertical axis is the fluorescence value. Figure 2 It can be seen that when RNA is added, the fluorescence value of the fluorescent probe PyIC increases, and after the addition of MALAT1, its emission peak intensity at 640nm increases significantly.

[0076] Table 4 Nucleotide sequences

[0077] Example 5 Spectral response of fluorescent probe PyIC to RNA

[0078] This example detects the fluorescence spectra of the fluorescent probe PyIC before and after the action of active substances with different concentrations. The specific experimental process is as follows: The fluorescent probe PyIC prepared in Example 3 was dissolved in a neutral (pH=7.0) Tris-HCl buffer solution (20mMNaCl, 20mM Tris) to prepare a 20μmol / L stock solution of the fluorescent probe PyIC; different equivalents of MALAT1 were added to each stock solution to make the concentration 0-40μM, and the fluorescence emission spectrum was measured after mixing evenly. The excitation wavelength of the fluorescence spectrum was measured to be 410nm, and the excitation and emission slit widths were both 1.5nm.

[0079] like Figure 3 As shown in the figure, the fluorescence spectrum of the fluorescent probe PyIC of the present invention changes with the addition of MALAT1, wherein the horizontal axis is the wavelength (nm) and the vertical axis is the relative fluorescence intensity. Figure 3 It can be seen that with the increase of the added equivalent of MALAT1, its emission peak intensity at 640nm gradually increases.

[0080] Example 6 Cytotoxicity of fluorescent probe PyIC

[0081] This example detects the cytotoxicity of the fluorescent probe PyIC, and the specific experimental process is as follows: HeLa cells (source: Thermo Fisher Scientific) were passaged with complete culture medium and placed in a cell culture incubator at 5% CO2, 95% air, and 37°C for proliferation culture; then transferred to a 96-well plate at a ratio of 8,000 cells / well and cultured for 24 hours to allow the cells to adhere to the wall; then different concentrations of the fluorescent probe PyIC were added to these cells, making the final concentrations 0, 5, 10, 15, 20, 25, 30, 35, 40, and 50 μM, and cultured again for 24 hours; then 10 μL of CCK-8 solution was added to each well, and the culture plate was gently shaken to mix and avoid bubbles; after continuing to culture for 1 hour, the absorbance was measured at a wavelength of 450 nm using an enzyme reader. The control group was carried out under the same conditions without the addition of the fluorescent probe PyIC.

[0082] Figure 4 is the cytotoxicity result of the fluorescent probe PyIC, where the horizontal axis is the concentration of the added fluorescent probe PyIC and the vertical axis is the cell survival rate. Figure 4 It can be seen that the fluorescent probe PyIC has less cytotoxicity.

[0083] Example 7 Endogenous RNA Cell Imaging with Fluorescent Probe PyIC

[0084] This example detects endogenous RNA cell imaging using the fluorescent probe PyIC, and the specific experimental process is as follows: (1) Hela cells in the logarithmic growth phase with intact morphology were digested with 0.25% trypsin-EDTA solution at 37°C for 2 min. After digestion, the cells were collected by centrifugation at 1000 rpm for 5 min and then resuspended in complete culture medium. The cell density was adjusted to 5×10 5 cells / mL, inoculated into a confocal culture dish, and placed in a 37°C, 5% CO2 constant temperature incubator for 24 h; (2) The experiment was divided into five groups, including one blank control group and four PyIC treatment groups. The blank control group was directly replaced with fresh complete culture medium for continued culture; the PyIC treatment group was added with preheated complete culture medium containing 5 μM PyIC probe (dissolved in DMSO, final concentration <0.1%) and incubated for 40 min in the dark. (3) After the culture was completed, the culture medium was removed and the cells were gently washed three times with preheated phosphate buffer. The blank control group and one PyIC-treated group were then immediately subjected to live cell imaging. The remaining three groups were: no treatment (blank group), DNase enzyme treatment group, and RNase enzyme treatment group, which were: no treatment, addition of DNase solution (ThermoFisher DNase I, 1 mL containing 10 µg / mL DNase I and 1 mM Ca2+ / Mg 2+ PBS solution), added RNase solution (Sigma RNase A, 1 mL of PBS solution containing 50 µg / mL RNase A), and then placed at 37°C for 1 h for live cell imaging.

[0085] The specific imaging method is: using a laser scanning confocal microscope for imaging, setting a 488nm argon ion laser as the excitation light source, the emission detection window is 570-640nm, and collecting bright field channel, fluorescence channel and superimposed image respectively.

[0086] Figure 5 This is the result of endogenous RNA cell imaging. Figure 5 In (A), (a)-(c) are blank groups without the addition of fluorescent probe PyIC; (d)-(f) are probe PyIC groups with the addition of fluorescent probe PyIC at a final concentration of 5 μM; the bright field channels are (a) and (d), the fluorescent channels are (b) and (e), and the overlays are (c) and (f); ex=488nm, em=570-640nm. Figure 5 (a)-(c) The blank control group shows that when the fluorescent probe PyIC is not added, there is no fluorescent signal in the cells; Figure 5 (d)-(f) PyIC fluorescent probe group shows that when the fluorescent probe PyIC is added, the cells show bright fluorescence, indicating that the probe successfully binds to the endogenous and emits a specific signal. The superimposed image can clearly show the co-localization relationship between the probe localization and the cell structure. Figure 5 (B) is the imaging image of the fluorescent probe PyIC in Hela cells and the RNase and DNase enzymatic degradation of the compound in Hela cells. Figure 5 As can be seen in the figure, the fluorescent probe PyIC mainly stains nucleolar RNA in Hela cells. When DNAse is added for digestion, the staining does not disappear. On the contrary, after RNAse is added for digestion, the cell staining disappears, which indicates that the fluorescent probe PyIC stains nucleolar RNA.

[0087] The results of the present invention indicate that alkenyl indole derivatives have high specificity for RNA, and therefore can be developed into RNA fluorescent probes to track the dynamic folding process of RNA in vivo and in vitro, and study its influence on the life activities of organisms.

[0088] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An alkenyl indole derivative, characterized in that A compound having a structure as shown in formula (A) or a pharmaceutically acceptable salt thereof: (A); wherein R is selected from anisole, methylpyridin-1-ium, benzaldehyde or N-methyl-4-phenylvinylpyridin-1-ium.

2. The method for preparing an alkenyl indole derivative according to claim 1, characterized in that The following steps are included: (1) Under nitrogen protection, indole, 2-chloropyridine, tripotassium phosphate, cuprous iodide, trans-N,N'-dimethyl-1,2-cyclohexanediamine and solvent toluene are mixed and reacted at 90-110° C. for 24-36 hours to obtain an intermediate represented by formula (a); (2) Under nitrogen protection, the intermediate of formula (a) obtained in step (1), the phenylacetylene derivative, the catalyst, the first additive and the second additive are dissolved in anhydrous ether as a solvent, and reacted at 70-90° C. for 10-16 hours to obtain an alkenyl indole derivative having a structure as shown in formula (I) or formula (II) or an intermediate of formula (b); (3) adding the intermediate of formula (b) obtained in step (2) and N-methyl-4-methylpyridin-1-ium to anhydrous ethanol as a solvent, and then adding piperidine; then reacting at 50-70° C. for 8-12 hours to obtain an alkenyl indole derivative having a structure as shown in formula (III); The structure of the intermediate represented by formula (a) in step (1) is as follows: Formula (a); The structure of the intermediate represented by formula (b) in step (2) is as follows: Formula (b); The structure of the alkenyl indole derivative having the structure shown in formula (I) or (II) described in step (2) is as follows: Formula (I); Formula (II); The structure of the alkenyl indole derivative having the structure shown in formula (III) described in step (3) is as follows: Formula (III).

3. The method for preparing an alkenyl indole derivative according to claim 2, characterized in that: The phenylacetylene derivative described in step (2) is 4-ethynylanisole, 4-ethynyl-1-methylpyridin-1-ium or 4-ethynylbenzaldehyde, and its structure is shown below: 。 4. The method for preparing an alkenyl indole derivative according to claim 3, characterized in that: The 4-ethynyl-1-methylpyridin-1-ium is prepared by the following method: 4-Ethynylpyridine, iodomethane and tetrahydrofuran were mixed and reacted at 66° C. for 3 h to obtain 4-ethynyl-1-methylpyridin-1-ium.

5. The method for preparing alkenyl indole derivatives according to claim 2, characterized in that: The molar ratio of indole, 2-chloropyridine, tripotassium phosphate, cuprous iodide and trans-N,N'-dimethyl-1,2-cyclohexanediamine solvent described in step (1) is 1:1.2:2.1:0.05:0.

2.

6. The method for preparing alkenyl indole derivatives according to claim 2, characterized in that: The catalyst in step (2) is manganese pentacarbonyl bromide, the first additive is N,N-diisopropylethylamine, and the second additive is benzoic acid; The molar ratio of the intermediate represented by formula (a), the phenylacetylene derivative, the catalyst, the first additive and the second additive in step (2) is 1:1.5:0.02:0.02:0.

02.

7. The method for preparing alkenyl indole derivatives according to claim 2, characterized in that: The molar ratio of the intermediate of formula (b) to N-methyl-4-methylpyridin-1-ium in step (3) is 1:1.1; The molar ratio of the intermediate of formula (b) to pyridine in step (3) is 10:1.

5.

8. The method for preparing alkenyl indole derivatives according to claim 2, characterized in that: The N-methyl-4-methylpyridin-1-ium described in step (3) is prepared by the following method: Under nitrogen protection, 4-methylpyridine, iodomethane and tetrahydrofuran were mixed and reacted at 66°C for 6h to obtain N-methyl-4-methylpyridin-1-ium.

9. Use of the alkenyl indole derivative according to claim 1 in the field of RNA fluorescent probes.

10. An RNA fluorescent probe, characterized in that The invention comprises the alkenyl indole derivative according to claim 1.

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