An alkenyl indole derivative, a preparation method thereof, and an application in the field of RNA fluorescent probes

Abstract

The present invention belongs to the technical field of RNA detection, and particularly relates to an alkenyl indole derivative, a preparation method thereof, and an application in the field of RNA fluorescent probes. The alkenyl indole derivative uses alkenyl indole as the mother nucleus and is a novel functional fluorescent probe, which can stain RNA in cells and broadens the range of types of fluorescent probes. The process of using this compound as a fluorescent probe to detect RNA is not interfered by other components, showing excellent anti-interference performance and high fluorescence intensity. In addition, its preparation process is simple, the raw materials are easily available, the reaction conditions are mild, the product structure is stable, and it is convenient for storage. It has important application value in the fields of RNA detection and its biological function research, and particularly shows broad application prospects in the fields of live cell imaging, molecular diagnosis, etc.

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] To solve the deficiencies and drawbacks of the above-mentioned existing technologies, the primary object of the present invention is to provide a vinyl indole derivative, which can be used as a fluorescent probe for specifically and selectively detecting RNA in vitro and in vivo.

[0006] Another object of the present invention is to provide a preparation method of the above-mentioned vinyl indole derivative.

[0007] Still another object of the present invention is to provide an application of the above-mentioned vinyl indole derivative.

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

[0009] The objects of the present invention are achieved by the following technical solutions:

[0010] A vinyl indole derivative is a compound having a structure shown in formula (A) or a pharmaceutically acceptable salt thereof:

[0011]

[0012] (A)

[0013] Wherein, R is selected from anisole group, methylpyridin-1-ium group, benzaldehyde group or N-methyl-4-styrylpyridin-1-ium group.

[0014] The above-mentioned vinyl indole derivative is preferably a compound having a structure shown in formula (Ⅰ), formula (Ⅱ) or formula (Ⅲ) or a pharmaceutically acceptable salt thereof.

[0015]

[0016] Formula (Ⅰ) Formula (Ⅱ) Formula (Ⅲ)

[0017] The preparation method of the above-mentioned vinyl indole derivative comprises the following steps:

[0018] (1) Under nitrogen protection, indole, 2-chloropyridine, tripotassium phosphate, copper(I) iodide, trans-N,N'-dimethyl-1,2-cyclohexanediamine and solvent toluene are mixed and reacted at 90 - 110 °C for 24 - 36 h to obtain an intermediate having a structure shown in formula (a);

[0019] (2) Under nitrogen protection, the intermediate having a structure shown in formula (a) prepared in step (1), a phenylacetylene derivative, a catalyst, a first additive, and a second additive are dissolved in anhydrous ether as a solvent and reacted at 70 - 90 °C for 10 - 16 h to obtain a vinyl indole derivative having a structure shown in formula (Ⅰ) or formula (Ⅱ) or an intermediate having a structure shown in formula (b);

[0020] (3) Add the intermediate with formula (b) prepared in step (2) and N-methyl-4-methylpyridin-1-ium to anhydrous ethanol as the solvent, and then add piperidine; then react at 50 - 70 °C for 8 - 12 h to obtain an alkenylindole derivative with the structure shown in formula (Ⅲ).

[0021] The structure of the intermediate with formula (a) described in step (1) is as follows:

[0022]

[0023] Formula (a)

[0024] The phenylacetylene derivative described in step (2) is preferably 4-ethynylanisole, 4-ethynyl-1-methylpyridin-1-ium or 4-ethynylbenzaldehyde, and their structures are as follows:

[0025]

[0026] The described 4-ethynyl-1-methylpyridin-1-ium is preferably prepared by the following method:

[0027] Mix 4-ethynylpyridine, iodomethane and tetrahydrofuran; react at 66 °C for 3 h to obtain 4-ethynyl-1-methylpyridin-1-ium.

[0028] The structure of the intermediate with formula (b) described in step (2) is as follows:

[0029]

[0030] Formula (b)

[0031] The molar ratio of indole, 2-chloropyridine, tripotassium phosphate, copper(I) 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; among them, if the molar amounts of 2-chloropyridine, tripotassium phosphate, copper(I) iodide and trans-N,N'-dimethyl-1,2-cyclohexanediamine solvent are too large, it will cause waste, and if they are too small, it will lead to a decrease in conversion rate and a reduction in atom utilization rate.

[0032] The molar ratio to volume ratio (mmol:mL) of indole and toluene described in step (1) is 1:5; among them, if the amount of toluene used is too large, it will cause waste, and if it is too small, it will lead to a decrease in yield and an increase in by-products.

[0033] The conditions of the reaction described in step (1) are preferably a reaction at 110 °C for 24 h; since trans-N,N'-dimethyl-1,2-cyclohexanediamine is used as a ligand, if the reaction temperature is too high, it will decompose or undergo structural changes, thus 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 of the copper catalyst and the reaction efficiency; when the reaction time at 110 °C is too short and the reaction is not fully carried out, the yield will decrease; when the reaction time at 110 °C is too long, the yield does not increase significantly; if the reaction temperature of 110 °C is reduced to 90 °C, the reaction time needs to be extended to 36 h.

[0034] 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 will decrease significantly, and even the reaction will not occur.

[0035] The molar ratio of the intermediate having the formula (a), the phenylacetylene derivative, the catalyst, the first additive, and the second additive described in step (2) is preferably 1:1.5:0.02:0.02:0.02; if the molar ratios of the intermediate, the phenylacetylene derivative, the catalyst, the first additive, and the second additive are too large, it will cause waste, and if they are too small, the conversion rate will decrease.

[0036] The molar ratio of the intermediate having the formula (a) described in step (2) to anhydrous ether in terms of amount of substance and volume (mmol: mL) is 1:5; if anhydrous ether is replaced with other solvents, the yield will decrease, and even the reaction will not occur; if the amount of anhydrous ether used is too much, it will cause waste, and if it is too little, the yield will decrease and the amount of by-products will increase.

[0037] The conditions of the reaction described in step (2) are preferably a reaction at 80 °C for 12 - 16 h.

[0038] The molar ratio of the intermediate having the formula (b) described in step (3) to N-methyl-4-methylpyridin-1-ium is preferably 1:1.1.

[0039] The molar ratio of the intermediate having the formula (b) described in step (3) to anhydrous ethanol (EtOH) in terms of amount of substance and volume (mmol: mL) is 1:(2 - 5); if the volume of anhydrous ethanol is too much, it will cause waste, and if it is too little, the dissolution will be insufficient and the reaction yield will decrease.

[0040] The molar ratio of the intermediate having the formula (b) described in step (3) to pyridine is preferably 10:1.5.

[0041] The conditions of the reaction described in step (3) are preferably a reaction at 60 °C for 10 h; 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. If the reaction time at 60 °C is too short, the reaction is not complete and the yield decreases; if the reaction time at 60 °C is too long, the yield does not increase significantly.

[0042] The N-methyl-4-methylpyridin-1-ium described in step (3) is preferably prepared by the following method:

[0043] Under nitrogen protection, 4-methylpyridine, methyl iodide and tetrahydrofuran are mixed and reacted at 66 °C for 6 h; N-methyl-4-methylpyridin-1-ium is obtained.

[0044] The application of the alkenylindole derivative in the field of RNA fluorescent probes.

[0045] An RNA fluorescent probe comprising the above alkenylindole derivative.

[0046] The synthetic route of the present invention is as follows:

[0047] When the alkenylindole derivative has the structures shown in formula (I) and formula (II), the synthetic route is as follows:

[0048]

[0049] When the alkenylindole derivative has the structure shown in formula (III), the synthetic route is as follows:

[0050]

[0051] The present invention has the following advantages and effects compared with the prior art:

[0052] (1) The present invention uses alkenylindole as the parent nucleus to prepare a series of alkenylindole derivatives. As fluorescent probes, this series of alkenylindole derivatives can cover the visible light region to the near-infrared region I, have good luminescence performance and large Stokes shift, and are a new type of full-spectrum coverage fluorescent probe. The present invention further broadens the range of existing fluorescent probe types.

[0053] (2) The alkenylindole derivative provided by the present invention is 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 specifically detect nucleolar RNA in cells, and the process of detecting RNA is not interfered by other components, showing excellent anti-interference performance and high fluorescence intensity, providing ideas and directions for the in vivo and in vitro real-time tracking of RNA and the in-depth study of its biological functions.

[0054] (3)The preparation method of the alkenylindole derivatives provided by the present invention not only uses simple and readily available raw materials, but also has a simple operation process, mild reaction conditions, stable product structure, and is convenient for storage, solving the problems in the prior art that the indole conjugated skeleton cannot be effectively extended, resulting in poor application of indole in the field of fluorescent probes.

[0055] (4)The present invention has important application value in the fields of RNA detection and its biological function research, especially showing broad application prospects in the fields of live cell imaging, molecular diagnosis, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 is the absorption spectrum result diagram of the fluorescent probe PyIC of the present invention before and after interacting with different RNAs.

[0057] Figure 2 is the fluorescence spectrum result diagram of the fluorescent probe PyIC of the present invention before and after interacting with different RNAs.

[0058] Figure 3 is the result diagram of the change of the fluorescence spectrum of the fluorescent probe PyIC of the present invention with the increase of RNA.

[0059] Figure 4 is the result diagram of the cytotoxicity of the fluorescent probe PyIC of the present invention.

[0060] Figure 5 is the result diagram of endogenous RNA cell imaging of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0061] Example 1. Synthesis of (E)-2-(4-methoxystyryl)-1-(pyridin-2-yl)-1H-indole (PyIA)

[0062] The present invention will be further described in detail below in conjunction with the examples and the drawings, but the implementation manners of the present invention are not limited thereto.

[0063] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0064]

[0065] (1) In a dry Schlenk reaction tube, indole (1.0 mmol), 2-chloropyridine (1.2 mmol), tripotassium phosphate (2.1 mmol), copper(I) iodide (0.05 mmol), and trans-N,N'-dimethyl-1,2-cyclohexanediamine (0.20 mmol) were added successively. Using 5 mL of toluene as the solvent, the system was then purged with nitrogen three times to remove oxygen. Then the reaction system was heated to 110 °C and stirred magnetically for 24 h under nitrogen protection. After the reaction, it was cooled to room temperature, and the solvent was removed under reduced pressure to obtain the 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%).

[0066] (2) In a dry Schlenk reaction tube, 1-(pyridin-2-yl)-1H-indole (1 mmol) prepared in step (1), 4-ethynylanisole (1.5 mmol), manganese pentacarbonyl bromide (0.02 mmol), N,N-diisopropylethylamine (0.02 mmol), and benzoic acid (0.02 mmol) were added successively. Using 5 mL of anhydrous diethyl ether as the solvent, the system was then purged with nitrogen three times to remove oxygen. Then the reaction system was heated to 80 °C and stirred magnetically for 12 h under nitrogen protection. After the reaction, it was cooled to room temperature, extracted three times with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the 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-methoxystyryl)-1-(pyridin-2-yl)-1H-indole (PyIA) (white solid, yield 85%).

[0067] The NMR characterization results of the fluorescent probe PyIA with an alkenylindole as the core are as follows:

[0068] 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 N2ONa + [M+Na] + :349.1425, Found: 349.1397。

[0069] The spectral properties of the above PyIA in aqueous solvent were detected, and the test results are shown in Table 1.

[0070] Table 1 Spectral properties of PyIA in aqueous solvent

[0071]

[0072] Note: λ abs max is the maximum absorption wavelength, λ em max is the maximum emission wavelength, ε is the maximum molar absorptivity, Φ is the fluorescence quantum yield; Stokes shift is λ em max - λ abs max 。

[0073] Example 2 Synthesis of (E)-1-methyl-4-[2-(1-(pyridin-2-yl)-1H-indol-2-yl)vinyl]pyridin-1-ium (PyIB)

[0074]

[0075] (1) In a dry Schlenk reaction tube, 4-ethynylpyridine (1 mmol) and methyl iodide (1.1 mmol) were successively added, and 5 mL of tetrahydrofuran was used as the solvent. Subsequently, the system was purged with nitrogen three times to remove oxygen; then the reaction system was heated to 66 °C and stirred magnetically for 3 h under nitrogen protection; after the reaction was completed, it 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%).

[0076] (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), manganese pentacarbonyl bromide (0.02 mmol), N,N-diisopropylethylamine (0.02 mmol), and benzoic acid (0.02 mmol) were successively added. Using 5 mL of anhydrous ether as the solvent, the system was then purged with nitrogen three times to remove oxygen. Then the reaction system was heated to 80 °C, and under nitrogen protection, magnetic stirring was started and the reaction was carried out for 16 h. After the reaction was completed, it was cooled to room temperature, extracted 3 times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was 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%).

[0077] The NMR characterization results of the fluorescent probe PyIB with an alkenylindole as the core are as follows:

[0078] 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+) : Calculated for C 21 H 18 N3 + [M] +: 312.1563, Found: 312.1541。

[0079] The spectral properties of the above PyIB in aqueous solvent were detected, and the test results are shown in Table 2.

[0080] Table 2 Spectral properties of PyIB in aqueous solvent

[0081]

[0082] Note: λ abs max is the maximum absorption wavelength, λ em max is the maximum emission wavelength, ε is the maximum molar absorptivity, Φ is the fluorescence quantum yield, and Stokes shift is λ em max - λ abs max .

[0083] Example 3 Synthesis of 1-methyl-4-((E)-4-((E)-2-(1-(pyridin-2-yl)-1H-indol-2-yl)vinyl)styryl)pyridin-1-ium (PyIC)

[0084]

[0085] (1) In a dry Schlenk reaction tube, 1-(pyridin-2-yl)-1H-indole (1 mmol) prepared in Example 1, 4-ethynylbenzaldehyde (1.5 mmol), manganese pentacarbonyl bromide (0.02 mmol), N,N-diisopropylethylamine (0.02 mmol), and benzoic acid (0.02 mmol) were successively added. 5 mL of anhydrous ether was used as the solvent, and then the system was purged with nitrogen three times to remove oxygen. Then the reaction system was heated to 80 °C and stirred magnetically for 12 h under nitrogen protection. After the reaction was completed, it was cooled to room temperature, extracted with ethyl acetate three times, and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was 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%).

[0086] (2) In a dry Schlenk reaction tube, 4-methylpyridine (1 mmol) and methyl iodide (1.1 mmol) were successively added, with 5 mL of tetrahydrofuran as the solvent. Subsequently, nitrogen was introduced to displace the system three times to exclude oxygen. Then, the reaction system was heated to 66 °C, and magnetic stirring was started under nitrogen protection for 6 h. After the reaction was completed, it 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%).

[0087] (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 make it homogeneous. Then, the reaction system was heated to 60 °C and continuously stirred for 10 h. After the reaction was completed, it was cooled to room temperature, and the ethanol solvent was removed by vacuum distillation to obtain a crude product. The crude product was dissolved in ethyl acetate (50 mL), washed successively with saturated sodium bicarbonate solution (20 mL × 2) and deionized water (20 mL × 2), and the organic phases were combined after liquid separation. After drying over anhydrous sodium sulfate, the solvent was removed under reduced pressure to obtain a light orange oil. It was purified by gradient elution using silica gel column chromatography (eluent: dichloromethane and methanol), specifically: gradually transitioning from low polarity (pure dichloromethane) to the target ratio of dichloromethane:methanol = 10:1 (V:V); the target fraction was collected, and after rotary evaporation, compound 1-methyl-4-((E)-4-((E)-2-(1-(pyridin-2-yl)-1H-indol-2-yl)vinyl)styryl)pyridin-1-ium (PyIC) (orange solid, yield 85%) was obtained.

[0088] The NMR characterization results of the fluorescent probe PyIC with an alkenylindole as the core are as follows:

[0089] 1 H NMR (400 MHz, DMSO- d 6) δ 9.00 – 8.95 (m, 2H), 8.27 (dd, J = 3.2, 1.7Hz, 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。

[0090] The spectral properties of the above PyIC in aqueous solvent were detected, and the test results are shown in Table 3.

[0091] Table 3 Spectral properties of PyIC in aqueous solvent

[0092]

[0093] Note: λ abs max is the maximum absorption wavelength, λ em max is the maximum emission wavelength, ε is the maximum molar absorptivity, Φ is the fluorescence quantum yield, and Stokes shift is λ em max - λ abs max 。

[0094] Example 4 Spectra of the fluorescence probe PyIC interacting with different active substances

[0095] In this example, the absorption spectra and fluorescence spectra of the fluorescence probe PyIC before and after interacting with different active substances were detected. The specific experimental procedure was as follows:

[0096] The fluorescence probe PyIC prepared in Example 3 was dissolved in 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 fluorescence probe PyIC. It was evenly divided into 6 portions, with at least 3 replicates for each treatment; 2-fold equivalents of different types of RNA (all commercially available) were added to each portion of the stock solution; after mixing evenly, its absorption spectrum and fluorescence emission spectrum were measured. Among them, the excitation wavelength for measuring the fluorescence spectrum was 410 nm, and both the excitation and emission slit widths were 20 nm.

[0097] Figure 1Absorption spectra of the fluorescent probe PyIC of the present invention before and after interacting with different RNAs. Among them, different types of nucleic acids are shown in Table 4, which are miR-18a, miR-20a, miR-17, pre-miR-21, MALAT1, and SS, a total of 6 types. The abscissa is the wavelength (nm), and the ordinate is the absorbance. From Figure 1 it can be seen that when no RNA is 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. After adding MALAT1, the absorption peak intensity at 410 nm in its absorption spectrum increases significantly.

[0098] Figure 2 Fluorescence spectra of the fluorescent probe PyIC of the present invention before and after interacting with different RNAs. Among them, the abscissa is the wavelength (nm); the ordinate is the fluorescence value. From Figure 2 it can be seen that when RNA is added, the fluorescence value of the fluorescent probe PyIC increases. After adding MALAT1, the emission peak intensity at 640 nm increases significantly.

[0099] Table 4 Nucleic acid sequences

[0100]

[0101] Example 5 Spectral response of the fluorescent probe PyIC to RNA

[0102] In this example, the fluorescence spectra of the fluorescent probe PyIC before and after interacting with different concentrations of the active substance were detected. The specific experimental process is as follows:

[0103] Take the fluorescent probe PyIC prepared in Example 3 and dissolve it in a neutral (pH = 7.0) Tris-HCl buffer solution (20 mM NaCl, 20 mM Tris) to prepare a stock solution of 20 μmol / L fluorescent probe PyIC; add different equivalents of MALAT1 to each stock solution so that its concentration is 0 - 40 μM. After mixing evenly, measure its fluorescence emission spectrum. The excitation wavelength for measuring the fluorescence spectrum is 410 nm, and the excitation and emission slit widths are both 1.5 nm.

[0104] As Figure 3 shown, the change in the fluorescence spectrum of the fluorescent probe PyIC of the present invention with the addition of MALAT1. Among them, the abscissa is the wavelength (nm), and the ordinate is the relative fluorescence intensity. From Figure 3 it can be seen that as the added equivalent of MALAT1 increases, the emission peak intensity at 640 nm gradually increases.

[0105] Example 6 Cytotoxicity of the fluorescent probe PyIC

[0106] In this example, the cytotoxicity of the fluorescent probe PyIC was detected. The specific experimental procedure was as follows:

[0107] HeLa cells (source: Thermo Fisher Scientific) were passaged with complete medium and placed in a cell incubator with 5% CO2, 95% air, and 37°C for proliferation culture. Then, they were transferred to a 96-well plate at a ratio of 8000 cells / well and cultured for 24 h to allow the cells to adhere. Subsequently, different concentrations of the fluorescent probe PyIC were added to these cells, and the final concentrations were 0, 5, 10, 15, 20, 25, 30, 35, 40, and 50 μM, respectively. After culturing for another 24 h, 10 μL of CCK-8 solution was added to each well, and the culture plate was gently shaken to mix evenly to avoid air bubbles. After continuing to culture for 1 h, the absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. The control group was carried out under the same conditions without adding the fluorescent probe PyIC.

[0108] Figure 4 shows the cytotoxicity results of the fluorescent probe PyIC. Among them, the abscissa is the concentration of the added fluorescent probe PyIC, and the ordinate is the cell survival rate. As Figure 4 can be seen, the fluorescent probe PyIC has relatively low cytotoxicity.

[0109] Example 7 Endogenous RNA Cellular Imaging of Fluorescent Probe PyIC

[0110] In this example, the endogenous RNA cellular imaging of the fluorescent probe PyIC was detected. The specific experimental procedure was as follows:

[0111] (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 terminating the digestion, the cells were collected by centrifugation at 1000 rpm for 5 min. Subsequently, the cells were resuspended in complete medium, and the cell density was adjusted to 5×10 5 cells / mL. They were inoculated into a confocal special culture dish and placed in a constant temperature incubator at 37°C and 5% CO2 for adherent culture for 24 h.

[0112] (2) The experiment was divided into a blank control group and four PyIC treatment groups, a total of five groups. Among them, the blank control group was directly replaced with fresh complete medium and continued to be cultured. The PyIC treatment groups were added with preheated complete medium containing 5 μM PyIC probe (dissolved in DMSO, final concentration <0.1%) and continued to be incubated for 40 min under dark conditions.

[0113] (3)After the culture is completed, remove the culture medium and gently wash the cells 3 times with pre-warmed phosphate buffer. The blank control group and one group treated with PyIC are immediately subjected to live cell imaging; the remaining three groups are: untreated (blank) group, DNase-treated group, and RNase-treated group, which are respectively subjected to: no treatment, addition of DNase solution (ThermoFisher DNase I, 1 mL containing 10 μg / mL DNase I and 1 mM Ca 2+ / Mg 2+ PBS solution), addition of RNase solution (Sigma RNase A, 1 mL containing 50 μg / mL RNase A PBS solution), and then incubated at 37 °C for 1 h for live cell imaging.

[0114] The specific imaging method is as follows: Imaging is performed using a laser scanning confocal microscope. Set the 488 nm argon ion laser as the excitation light source, and the emission detection window is 570 - 640 nm. Bright field channels, fluorescence channels, and superimposed images are collected respectively.

[0115] Figure 5 It is the result diagram of endogenous RNA cell imaging. Figure 5 In (A) of Figure 5 , (a)-(c) are the blank group without adding the fluorescent probe PyIC; (d)-(f) are the probe PyIC group with a final concentration of 5 μM of the fluorescent probe PyIC added; the bright field channels are (a) and (d), the fluorescence channels are (b) and (e), and the superimposed images are (c) and (f); ex = 488 nm, em = 570 - 640 nm. From Figure 5 the blank control group of (a)-(c), it can be seen that there is no fluorescent signal in the cells without adding the fluorescent probe PyIC; from Figure 5 the PyIC fluorescent probe group of (d)-(f), it can be seen that when the fluorescent probe PyIC is added, bright fluorescence appears in the cells, indicating that the probe successfully binds to the endogenous and emits specific signals. The superimposed image can clearly show the co-localization relationship between the probe localization and the cell structure. Figure 5 In (B) of

[0116] The results of the present invention show that alkenyl indole derivatives have high specificity for RNA. Therefore, they can be developed into RNA fluorescent probes to track the dynamic folding process of RNA in vivo and in vitro and study its impact on the life activities of organisms.

[0117] 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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An alkenyl indole derivative, characterized in that It is a compound having the structure shown in formula (Ⅲ) or a pharmaceutically acceptable salt thereof:

2. The method for preparing the alkenylindole derivative according to claim 1, wherein It comprises the following steps: (1) Under nitrogen protection, indole, 2-chloropyridine, tripotassium phosphate, copper(I) iodide, trans-N,N'-dimethyl-1,2-cyclohexanediamine and the solvent toluene are mixed and reacted at 90 - 110 °C for 24 - 36 h to obtain an intermediate having the structure shown in formula (a); (2) Under nitrogen protection, the intermediate having the structure shown in formula (a) prepared in step (1), a phenylacetylene derivative, a catalyst, a first additive, and a second additive are dissolved in the solvent anhydrous ether and reacted at 70 - 90 °C for 10 - 16 h to obtain an intermediate having the structure shown in formula (b); (3) The intermediate having the structure shown in formula (b) prepared in step (2) and the compound having the structure shown in formula (c) are added to the solvent anhydrous ethanol, and then piperidine is added; then it is reacted at 50 - 70 °C for 8 - 12 h to obtain an alkenylindole derivative having the structure shown in formula (Ⅲ); The structure of the intermediate having the structure shown in formula (a) described in step (1) is as follows: The structure of the intermediate having the structure shown in formula (b) described in step (2) is as follows: The phenylacetylene derivative described in step (2) is p-ethynylbenzaldehyde, and its structure is as follows: The structure of the compound having the structure shown in formula (c) described in step (3) is as follows: The catalyst described in step (2) is manganese pentacarbonyl bromide, the first additive is N,N-diisopropylethylamine, and the second additive is benzoic acid.

3. The method for preparing an alkenylindole derivative according to claim 2, wherein: The reaction conditions in step (2) are reaction at 80 °C for 12 - 16 h.

4. The method for preparing an alkenylindole derivative according to claim 2, wherein: The molar ratio of indole, 2-chloropyridine, tripotassium phosphate, copper(I) iodide to trans-N,N'-dimethyl-1,2-cyclohexanediamine solvent described in step (1) is 1:1.2:2.1:0.05:0.

2.

5. The method for preparing an alkenylindole derivative according to claim 2, wherein: The molar ratio of the intermediate having the structure shown in formula (a), the phenylacetylene derivative, the catalyst, the first additive to the second additive described in step (2) is 1:1.5:0.02:0.02:0.

02.

6. The method for preparing an alkenylindole derivative according to claim 2, wherein: The molar ratio of the intermediate having the structure shown in formula (b) to the compound having the structure shown in formula (c) described in step (3) is 1:1.1; The molar ratio of the intermediate having the structure shown in formula (b) to piperidine described in step (3) is 10:1.

5.

7. The method for preparing an alkenylindole derivative according to claim 2, wherein: The compound having the structure shown in formula (c) described in step (3) is prepared by the following method: Under nitrogen protection, 4-methylpyridine, iodomethane and tetrahydrofuran are mixed and reacted at 66 °C for 6 h; to obtain the compound having the structure shown in formula (c).

8. Use of the alkenylindole derivative according to claim 1 in the preparation of an RNA fluorescent probe.

9. An RNA fluorescent probe, characterized in that Comprising the alkenylindole derivative according to claim 1.

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