A near-infrared fluorescent probe and a preparation method and application thereof

Abstract

The application provides a near-infrared fluorescent probe and a preparation method and application thereof, and belongs to the technical field of fluorescent probes.The application takes flavones and heterocyclic compounds as asymmetric molecular building blocks, and controls the electric property, steric hindrance and water solubility of the molecular building blocks through a molecular engineering strategy to prepare the near-infrared fluorescent probe.The near-infrared fluorescent probe has the advantages of long fluorescent emission wavelength, large stokes shift and the like, and effectively avoids the interference of biological background fluorescence.In the aspect of nucleic acid labeling, the near-infrared fluorescent probe has the significant advantages of high signal-to-noise ratio, good selectivity and good biocompatibility.The near-infrared fluorescent probe is imaged by using a laser confocal microscope and a structured illumination super-resolution fluorescence microscope, and the RNA in HeLa cells and mouse white blood cells is successfully traced and displayed, and the fine structure of nucleoli can be visualized at a high resolution.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescent probe technology, in particular to a near-infrared fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] Nucleic acid is one of the most basic substances of living organisms, which is mainly divided into two categories, namely deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Based on the central dogma, nucleic acid has a variety of key biological functions, such as participating in cell metabolism, regulating genetics and various diseases. Therefore, the accurate detection of nucleic acid is of great significance to life science research, disease evolution analysis and drug screening. It is worth noting that fluorescent detection has the advantages of good selectivity, high sensitivity and simple operation, and the fluorescent dye based on the design of molecular probe provides an important detection and analysis tool for the visualization of nucleic acid in living organisms.

[0003] A series of nucleic acid labeling dyes have been developed, such as Hoechst dye based on benzimidazole molecular building block and SYTO dye based on thiazole orange molecular building block. The above-mentioned commercial nucleic acid dyes have a single structure, and have the problems of short wavelength, small stokes shift and poor selectivity, for example, the emission wavelength of Hoechst 33342 is 450 nm, which is easily interfered by biological background fluorescence. Therefore, how to develop a new type of nucleic acid labeling molecular building block, especially considering the wavelength and specificity of the probe, will effectively break through the performance limitations of existing commercial dyes and the imaging application bottleneck. SUMMARY

[0004] The present application aims to provide a near-infrared fluorescent probe and a preparation method and application thereof, so as to solve the problem of single structure of nucleic acid probe in the prior art, and short wavelength, small stokes shift and poor selectivity.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides a near-infrared fluorescent probe, which has the following structure:

[0007] 、 、

[0008] (I) (II) (III)

[0009] 、 、

[0010] (IV) (V)

[0011]

[0012] (Ⅵ)

[0013] wherein R1 is independently selected from methyl, ethyl, , , , , , , , , or ; R2 is independently selected from hydrogen atom, methyl, ethyl, isopropyl, tert-butyl, , , , , or ; R3 is independently selected from hydrogen atom or N,N dimethylamino; n is independently selected from 1, 3, 5 or 7;

[0014] L in the structure of (III), (VI) is independently selected from sulfur atom, imino or isopropylidene, R4 is independently selected from hydrogen atom, N,N dimethylamino, methoxy, , or .

[0015] As a preferred, the near-infrared fluorescent probe has the structure as shown below:

[0016] , ,

[0017] (I-1) (II-1)

[0018] ,

[0019] (III-1) (IV-1)

[0020]

[0021] (V-1) (VI-1).

[0022] The present application provides a preparation method of the above-mentioned near-infrared fluorescent probe, comprising the following steps:

[0023] (1) mixing a heterocyclic compound and a halogen-containing compound in acetonitrile to carry out a reflux reaction to obtain compound 1;

[0024] (2) mixing compound 1, N,N diphenylformamidine and piperidine in methanol to carry out a reflux reaction to obtain compound 2;

[0025] (3) mixing compound 2 and mixed solvent, and then refluxing to obtain compound 3;

[0026] (4) mixing compound 3, compound 4 and piperidine in methanol, and then refluxing to obtain near-infrared fluorescent probe;

[0027] The compound 4 has the following structure:

[0028] or

[0029] wherein, R2 is independently selected from hydrogen atom, methyl, ethyl, isopropyl, tert-butyl, , , , , or ; R3 is selected from hydrogen atom or N,N- dimethylamino.

[0030] As preferred, in the step (1), the heterocyclic compound is or ; the halogen-containing compound is CH3-X, CH3CH2-X, , , , , , , , or ; the mass ratio of the heterocyclic compound and the halogen-containing compound is 900-1500 mg: 7.5-8.0 g; the mass-volume ratio of the heterocyclic compound and acetonitrile is 900-1500 mg: 8-25 mL; the temperature of the refluxing reaction is 70-90℃, and the refluxing reaction time is 8-16 h.

[0031] As preferred, in the step (2), the compound 1, N,N diphenylformamidinium and piperidine have a mass ratio of 150-500: 150-300: 130-260; the compound 1 and methanol have a mass-volume ratio of 150-500 mg: 5-10 mL; the temperature of the refluxing reaction is 50-70℃, and the refluxing reaction time is 4-6 h;

[0032] In the step (3), the mass-volume ratio of compound 2 and the mixed solvent is 30-90 mg:10-30 mL; the mixed solvent is a mixed solvent of acetone and acetyl chloride, wherein the volume ratio of acetone and acetyl chloride is 1:1-2; the temperature of the reflux reaction is 30-50℃, and the reflux reaction time is 1-24 h.

[0033] Preferably, in the step (4), the mass ratio of compound 3 and compound 4 is 30-600:30-60; the mass-volume ratio of compound 3 and methanol is 30-600 mg:1-8 mL; the volume ratio of methanol and piperidine is 1-8:0.5-2; the temperature of the reflux reaction is 40-70℃, and the reflux reaction time is 3-24 h.

[0034] The present application provides a preparation method of the near-infrared fluorescent probe, comprising the following steps:

[0035] (1) mixing a heterocyclic compound and a halogen-containing compound in acetonitrile to perform a reflux reaction to obtain compound 1;

[0036] (2) mixing compound 1, N,N diphenylformamidum and acetic anhydride to perform a reflux reaction to obtain compound 2;

[0037] (3) mixing compound 2, compound 4 and piperidine in methanol to perform a reflux reaction to obtain the near-infrared fluorescent probe;

[0038] The compound 4 has the following structure:

[0039] or

[0040] wherein R2 is independently selected from a hydrogen atom, a methyl group, an ethyl group, an isopropyl group, a tert-butyl group, , , , , or ; R3 is selected from a hydrogen atom or N,N a dimethylamino group.

[0041] Preferably, in the step (1), the heterocyclic compound is ; the halogen-containing compound is CH3-X, CH3CH2-X, , , , , , , , or The mass ratio of the heterocyclic compound and the halogen-containing compound is 1.3-1.6:1.4-1.8; the mass-volume ratio of the heterocyclic compound and acetonitrile is 1.3-1.6 g:10-20 mL; the temperature of the reflux reaction is 70-90 DEG C, and the reflux reaction time is 6-10 h.

[0042] As preferred, in step (2), the mass ratio of the compound 1 and N,N The mass ratio of the compound 2 and the compound 4 is 50-55:30-35, the mass-volume ratio of the compound 2 and methanol is 50-55 mg:3-8 mL, the volume ratio of the methanol and piperidine is 3-8:1-3, the temperature of the reflux reaction is 40-60 DEG C, and the reflux reaction time is 1-3 h.

[0043] The application provides application of the near-infrared fluorescent probe in preparation of DNA or RNA stock solution and in cell imaging.

[0044] The application has the following beneficial effects:

[0045] The application uses flavone and a heterocyclic compound as asymmetric molecular building blocks, and controls the electric property, steric hindrance and water solubility of the molecular building blocks through a molecular engineering strategy to prepare a near-infrared fluorescent probe. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 The absorption spectrum of the near-infrared fluorescent probe prepared in Example 1 in different kinds of solvents;

[0047] Figure 2 The fluorescence emission spectrum of the near-infrared fluorescent probe prepared in Example 1 in different kinds of solvents;

[0048] Figure 3 The absorption spectrum of the near-infrared fluorescent probe prepared in Example 2 in different kinds of solvents;

[0049] Figure 4 The fluorescence emission spectrum of the near-infrared fluorescent probe prepared in Example 2 in different kinds of solvents;

[0050] Figure 5 The absorption spectrum of the near-infrared fluorescent probe prepared in Example 3 in different kinds of solvents;

[0051] Figure 6 The fluorescence emission spectrum of the near-infrared fluorescent probe prepared in Example 3 in different kinds of solvents;

[0052] Figure 7 The absorption spectrum of the near-infrared fluorescent probe prepared in Example 4 in different kinds of solvents;

[0053] Figure 8 The fluorescence emission spectrum of the near-infrared fluorescent probe prepared in Example 4 in different kinds of solvents;

[0054] Figure 9 The fluorescence emission spectrum of the near-infrared fluorescent probe prepared in Example 1 in Tris-HCl solution with pH = 7.2 after being combined with DNA or RNA, respectively;

[0055] Figure 10 The fluorescence intensity enhancement multiple of the near-infrared fluorescent probe prepared in Example 1 in Tris-HCl solution with pH = 7.2 after being combined with DNA or RNA, respectively, compared with the free state;

[0056] Figure 11 The laser confocal microscope imaging diagram formed after the near-infrared fluorescent probe prepared in Example 1 is dyed to live HeLa cells;

[0057] Figure 12 The structured light illumination super-resolution microscope imaging diagram formed after the near-infrared fluorescent probe prepared in Example 1 is dyed to live HeLa cells;

[0058] Figure 13 The structured light illumination super-resolution microscope imaging diagram formed after the near-infrared fluorescent probe prepared in Example 1 is dyed to mouse white blood cells. DETAILED DESCRIPTION

[0059] The present application provides a kind of near-infrared fluorescent probe, the near-infrared fluorescent probe has the structure as shown in the following:

[0060] 、 、

[0061] (Ⅰ) (Ⅱ) (Ⅲ)

[0062] 、 、

[0063] (IV) (V)

[0064]

[0065] (VI)

[0066] wherein, R1 is independently selected from methyl, ethyl, 、 、 、 、 、 、 、 、 or ; R2 is independently selected from hydrogen atom, methyl, ethyl, isopropyl, tert-butyl, 、 、 、 、 or ; R3 is independently selected from hydrogen atom or N,N dimethylamino; n is independently selected from 1, 3, 5 or 7;

[0067] L in the structure of (III), (VI) is independently selected from sulfur atom, imino or isopropylidene, R4 is independently selected from hydrogen atom, N,N dimethylamino, methoxy, 、 or .

[0068] In the present application, the near-infrared fluorescent probe has the structure as shown below:

[0069] 、 、

[0070] (I-1) (II-1)

[0071] 、

[0072] (III-1) (IV-1)

[0073]

[0074] (V-1) (VI-1).

[0075] The present application provides a preparation method of the near-infrared fluorescent probe as described above, comprising the following steps:

[0076] (1) mixing a heterocyclic compound and a halogen-containing compound in acetonitrile to perform a reflux reaction to obtain compound 1;

[0077] (2) mixing compound 1, N,N - mixing diphenylformamidinium and piperidine in methanol to perform a reflux reaction to obtain compound 2;

[0078] (3) mixing compound 2 and a mixed solvent to perform a reflux reaction to obtain compound 3;

[0079] (4) mixing compound 3, compound 4, and piperidine in methanol to perform a reflux reaction to obtain a near-infrared fluorescent probe;

[0080] The compound 4 has the structure shown below:

[0081] or

[0082] wherein R2 is independently selected from a hydrogen atom, a methyl group, an ethyl group, an isopropyl group, a tert-butyl group, , , , , or ; R3 is selected from a hydrogen atom or N,N- a dimethylamino group.

[0083] In the present application, R2 in the compound 4 is preferably an isopropyl group, a tert-butyl group, , , , , or , and is further preferably , , , , or ; and R3 is preferably N,N- a dimethylamino group.

[0084] In the present application, in the step (1), the heterocyclic compound is or ; and the halogen-containing compound is CH3-X, CH3CH2-X, , , , , , , , or , preferably CH3-X, CH3CH2-X, , , , or , further preferably CH3-X or CH3CH2-X; the mass ratio of the heterocyclic compound and the halogen-containing compound is 900-1500 mg: 7.5-8.0 g, preferably 920-1400 mg: 7.6-7.9 g, further preferably 931-1400 mg: 7.75-7.8 g; the mass-volume ratio of the heterocyclic compound and acetonitrile is 900-1500 mg: 8-25 mL, preferably 920-1400 mg: 10-22 mL, further preferably 931-1400 mg: 10-20 mL; the temperature of the reflux reaction is 70-90°C, preferably 75-85°C, further preferably 80°C, and the reflux reaction time is 8-16 h, preferably 10-15 h, further preferably 10-12 h.

[0085] In the present application, in the step (2), the mass ratio of the compound 1, N,N - the mass ratio of diphenylformamidinium and piperidine is 150-500: 150-300: 130-260, preferably 200-449: 157-294: 136-255; the mass-volume ratio of the compound 1 and methanol is 150-500 mg: 5-10 mL, preferably 200-449 mg: 5-7 mL; the temperature of the reflux reaction is 50-70°C, preferably 55-65°C, further preferably 60°C, and the reflux reaction time is 4-6 h, preferably 4.5-5.5 h, further preferably 5 h.

[0086] In the present application, in the step (3), the mass-volume ratio of the compound 2 and the mixed solvent is 30-90 mg: 10-30 mL, preferably 40-80 mg: 15-25 mL, further preferably 50-70 mg: 20 mL; the mixed solvent is a mixed solvent of acetone and acetyl chloride, wherein the volume ratio of acetone and acetyl chloride is 1: 1-2, preferably 1: 1; the temperature of the reflux reaction is 30-50°C, preferably 35-45°C, further preferably 40°C, and the reflux reaction time is 1-24 h, preferably 3 h, 5 h, 10 h, 15 h, 24 h, further preferably 3 h, 10 h, 24 h.

[0087] In the present application, in the step (4), the mass ratio of the compound 3 and the compound 4 is 30-600:30-60, preferably 40-500:35-55, further preferably 49-478:40-44; the mass-volume ratio of the compound 3 and the methanol is 30-600 mg:1-8 mL, preferably 40-500 mg:2-7 mL, further preferably 49-478:2-5 mL; the volume ratio of the methanol and the piperidine is 1-8:0.5-2, preferably 2-7:0.5-1.5, further preferably 2-5:0.5-1; the temperature of the reflux reaction is 40-70℃, preferably 45-65℃, further preferably 50-60℃, and the time of the reflux reaction is 3-24 h, preferably 3 h, 5 h, 10 h, 15 h, 24 h, further preferably 3 h, 10 h, 24 h.

[0088] The present application provides a preparation method of the near-infrared fluorescent probe, comprising the following steps:

[0089] (1) mixing the heterocyclic compound and the halogen-containing compound in acetonitrile to perform a reflux reaction to obtain the compound 1;

[0090] (2) mixing the compound 1, N,N - the diphenylformamidine and the acetic anhydride to perform a reflux reaction to obtain the compound 2;

[0091] (3) mixing the compound 2, the compound 4 and the piperidine in methanol to perform a reflux reaction to obtain the near-infrared fluorescent probe;

[0092] The compound 4 has the following structure:

[0093] or

[0094] wherein R2 is independently selected from a hydrogen atom, a methyl group, an ethyl group, an isopropyl group, a tert-butyl group, , , , , or ; R3 is selected from a hydrogen atom or N,N a dimethylamino group.

[0095] In the present application, R2 in the compound 4 is preferably an isopropyl group, a tert-butyl group, , , , , or , further preferably , , , , or R3 is preferably... N,N- Dimethylamino.

[0096] In this invention, in step (1), the heterocyclic compound is The halogen-containing compound is CH3-X, CH3CH2-X, , , , , , , , or Preferably CH3-X, CH3CH2-X, , , , or The heterocyclic compound and the halogenated compound are preferably CH3-X or CH3CH2-X; the mass ratio of the heterocyclic compound to the halogenated compound is 1.3~1.6:1.4~1.8, preferably 1.4~1.5:1.5~1.7, and more preferably 1.45~1.49:1.56~1.65; the mass-volume ratio of the heterocyclic compound to the acetonitrile is 1.3~1.6g:10~20mL, preferably 1.4~1.5g:12~18mL, and more preferably 1.45~1.49g:14~16mL; the reflux reaction temperature is 70~90℃, preferably 75~85℃, and more preferably 80℃, and the reflux reaction time is 6~10h, preferably 6h, 8h, or 10h, and more preferably 8h.

[0097] In this invention, in step (2), compound 1 and N,NThe mass ratio of diphenylmethanemid is 800~1200:700~800, preferably 900~1100:720~780, more preferably 1000:740~771; the mass-volume ratio of compound 1 to acetic anhydride is 800~1200 mg:8~12 mL, preferably 900~1100 mg:9~11 mL, more preferably 1000 mg:10 mL; the reflux reaction temperature is 100~120℃, preferably 105~115℃, more preferably 110℃; the reflux reaction time is 10~14 h, preferably 11 h, 12 h, 13 h, 14 h, more preferably 12 h, 13 h; in step (3), the... The mass ratio of compound 2 to compound 4 is 50~55:30~35, preferably 51~54:31~34, and more preferably 52~53:31~33; the mass-volume ratio of compound 2 to methanol is 50~55 mg:3~8 mL, preferably 51~54 mg:4~7 mL, and more preferably 52~53 mg:5~6 mL; the volume ratio of methanol to piperidine is 3~8:1~3, preferably 4~7:1~2, and more preferably 5~6:1; the reflux reaction temperature is 40~60℃, preferably 45~55℃, and more preferably 50℃; the reflux reaction time is 1~3 h, preferably 1 h, 2 h, or 3 h, and more preferably 2 h.

[0098] This invention provides an application of the aforementioned near-infrared fluorescent probe in the preparation of DNA or RNA storage solutions and in cell imaging.

[0099] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0100] Example 1

[0101] (1) 931 mg of 4-methylpyridine (Py) and 7.8 g of iodoethane were dissolved in 20 mL of anhydrous acetonitrile and refluxed at 80 °C for 10 h. After the reaction was completed, the solvent was evaporated and then separated by column chromatography (the mobile phase was dichloromethane and methanol, with a volume ratio of 10:1) to obtain 2.16 g of compound 1 (Et-Py). Compound 1 was a yellow oily liquid with a yield of 87%.

[0102]

[0103] 1 ¹H NMR (400 MHz, CDCl₃, ppm): δ 1.72 (t, J= 8.0 Hz, 3H, -CH3), 2.70(s, 3H, -CH3), 4.93 (q, J 1 = 4.0 Hz, J 2 = 8.0 Hz, 2H, -CH2-), 7.94 (d, J = 4.0Hz, 2H, pyridine-H), 9.29 (d, J = 4.0 Hz, 2H, pyridine-H).

[0104] 13 C NMR (100 MHz, CDCl3, ppm) δ : 17.22, 22.41, 56.65, 129.03, 143.86,159.07.

[0105] Mass spectrometry (ESI-MS, m / z): [M] + calcd. for [C8H 12 N] + 122.0970:found 122.0986.

[0106] (2) 200 mg of compound 1 (Et-Py), 157 mg of N,N -diphenylformamidinium, 136 mg of piperidine, dissolved in 5 mL of methanol, refluxed at a temperature of 60 °C for 5 h, after the reaction was completed, the solvent was spin-dried, and column chromatography was performed (the mobile phase was dichloromethane and methanol, the volume ratio of dichloromethane and methanol was 10:1) to obtain 61 mg of compound 2 (Et-Py-M), which was a brown solid, and the yield was 22%.

[0107]

[0108] 1 H NMR (400 MHz, DMSO- d 6 , ppm): δ 1.44 (t, J = 8.0Hz, 3H, -CH3),4.27 (q, J 2 = 8.0Hz, 2H, -CH2-), 5.88 (d, J = 12.0 Hz, 1H, alkene-H), 7.03 (t, J= 8.0 Hz, 1H, phenyl-H), 7.32 (t, J = 8.0 Hz, 2H, phenyl-H), 7.35 (t, J =8.0 Hz, 2H,phenyl-H), 7.78 (t, J = 4.0 Hz, 2H, phenyl-H), 8.42 (d, J =8.0 Hz, 2H,pyridine-H), 8.52 (d, J = 12.0 Hz, 1H, alkene-H), 10.34 (s, 2H, amide-H).

[0109] 13 C NMR (100 MHz, DMSO- d 6 , ppm) δ :16.09, 53.27, 98.68, 115.73, 118.99,122.56, 129.53, 140.52, 141.39, 142.34, 154.88.

[0110] Mass spectrometry (ESI-MS, m / z): [M] + calcd. for [C 15 H 17 N2] + 225.1383:found 225.1386.

[0111] (3) 50 mg of compound 2 (Et-Py-M) was dissolved in 10 mL of acetone and 10 mL of acetyl chloride, and reacted at a temperature of 40°C for 24 h. After the reaction was completed, the solvent was spin-dried, and column chromatography was performed (the mobile phase was dichloromethane and methanol, and the volume ratio of dichloromethane to methanol was 10:1) to obtain 48 mg of compound 3 (Et-Py-Ac), which was a red solid, with a yield of 69%.

[0112]

[0113] 1 H NMR (400 MHz, DMSO- d 6 , ppm): δ 1.46 (t, J = 8.0 Hz, 3H, -CH3), 1.99(s, 3H, -CH3), 4.42 (q,J 2 = 8.0 Hz, 2H, -CH2-), 5.39 (d, J = 12.0 Hz, 1H,alkene-H), 7.43 (d, J = 8.0 Hz, 2H, phenyl-H), 7.58 (t, J = 8.0 Hz, 1H,phenyl-H), 7.64 (t, J = 8.0 Hz, 2H, phenyl-H), 8.72 (d, J = 4.0 Hz, 2H,pyridine-H), 8.77 (d, J = 12.0 Hz, 1H, alkene-H).

[0114] 13 C NMR (100 MHz, DMSO- d 6 , ppm) δ : 16.65, 55.14, 106.99, 122.96,129.13, 129.95, 130.88, 138.41, 143.74, 153.83, 170.07.

[0115] Mass spectrometry (ESI-MS, m / z): [M] + calcd. for [C 17 H 19 ON2] + 267.1487:found 267.1492.

[0116] (4) 478 mg of compound 3 (Et-Py-Ac), 44 mg of compound 4 (F-1) were dissolved in 5 mL of methanol, 1 mL of piperidine was added, and the reaction was carried out at 60°C for 24 h. After the reaction was completed, the solvent was rotary evaporated, and column chromatography was performed (the mobile phase was dichloromethane and methanol, and the volume ratio of dichloromethane and methanol was 10:1) to obtain near-infrared fluorescent probe 50 mg as a black solid, with a yield of 85%.

[0117]

[0118] 1 H NMR (400 MHz, DMSO- d 6 , ppm): δ 1.50 (t,J = 8.0 Hz, 3H, -CH3), 3.05(s, 6H, -CH3), 4.41 (q, J = 8.0 Hz, 2H, -CH2-), 6.61 (d, J = 12.0 Hz, 1H,alkene-H), 6.66 (s, 1H, phenyl-H), 6.71 (d, J = 12.0 Hz, 1H, alkene-H), 6.80(d, J = 8.0 Hz, 1H, alkene-H), 7.58 (t, J = 8.0 Hz, 3H, phenyl-H), 7.61 (d, J = 8.0 Hz, 1H, phenyl-H), 7.84 (d, J = 8.0 Hz, 1H, phenyl-H), 8.04 (d, J = 4.0Hz, 2H, pyridine-H), 8.11 (d, J = 8.0 Hz, 2H, phenyl-H), 8.45 (t, J = 12.0 Hz,1H, phenyl-H), 8.65 (d, J = 8.0 Hz, 2H, pyridine-H).

[0119] 13 C NMR (100 MHz, DMSO- d 6 , ppm) δ: 16.22, 98.24, 108.97, 110.50,120.35, 121.65, 124.35, 125.34, 128.78, 132.18, 136.74, 139.10, 142.43,151.76, 152.49, 153.29.

[0120] Mass spectrometry (ESI-MS, m / z): [M] + calcd. for [C 27 H 27 N2O] + 395.2123:found 395.2113.

[0121] Example 2

[0122] (1) 1.4 g of 7-methylquinoline, 7.75 g of iodoethane, dissolved in 10 mL of anhydrous acetonitrile, was refluxed at a temperature of 80°C for 12 h, after the reaction was completed, the solvent was spin-dried, and column chromatography was performed (the mobile phase was dichloromethane and methanol, the volume ratio of dichloromethane and methanol was 10:1) to obtain compound 1 (Et-Q) 2.9 g, compound 1 was a yellow solid, the yield was 97%.

[0123]

[0124] 1 H NMR (400 MHz, DMSO- d 6 , ppm): δ 1.60 (t, J = 8.0 Hz, 3H, -CH3), 3.02(s, 3H, -CH3), 5.06 (q, J = 8.0 Hz, 2H, -CH2-), 8.08 (m, 2H, phenyl-H), 8.27(t, J = 8.0 Hz, 1H, phenyl-H), 8.56 (d, J = 8.0 Hz, 1H, phenyl-H), 8.61 (d, J = 8.0 Hz, 1H, phenyl-H).

[0125] 13 C NMR (100 MHz, DMSO- d 6 , ppm) δ: 15.22, 19.65, 52.50, 119.18,122.72,127.18, 128.88, 129.50, 135.08, 136.51, 147.99, 158.33.

[0126] Mass spectrometry (ESI-MS, m / z): [M] + calcd. for [C 12 H 14 N] + 172.1121:found 172.1118.

[0127] (2) 449 mg of compound 1 (Et-Q), 294 mg of 2-bromo-4,6-dimethoxy-1,3,5-triazine, 0.5 mL of pyridine, 0.5 mL of acetonitrile, was refluxed at a temperature of 80°C for 12 h, after the reaction was completed, the solvent was spin-dried, and column chromatography was performed (the mobile phase was dichloromethane and methanol, the volume ratio of dichloromethane and methanol was 10:1) to obtain compound 2 (Et-Q) 0.5 g, compound 2 was a yellow solid, the yield was 97%. N,N- Diphenylformamidinium, 255 mg of piperidine, dissolved in 7 mL of methanol, refluxed at a temperature of 60 °C for 5 h, after the reaction was completed, the solvent was rotary evaporated, and column chromatography was performed (the mobile phase was dichloromethane and methanol, the volume ratio of dichloromethane and methanol was 10:1), to obtain compound 2 (Et-Q-M) 103 mg, compound 2 was a yellow solid, the yield was 17%.

[0128]

[0129] 1 H NMR (400 MHz, DMSO- d 6 , ppm) δ 1.48 (t, J = 8.0 Hz, 3H, -CH3), 4.70(q, J = 8.0 Hz, 2H, -CH2-), 6.73 (d, J = 12.0 Hz, 1H, alkene-H), 7.14 (t, J =8.0 Hz, 1H, phenyl-H), 7.43 (m, 4H, phenyl-H), 7.82 (t, J = 8.0 Hz, 1H,phenyl-H), 8.06 (t, J = 8.0 Hz, 1H, phenyl-H), 8.16 (d, J = 8.0 Hz, 1H,phenyl-H), 8.23 (d, J = 8.0 Hz, 1H, phenyl-H), 8.16 (d, J = 8.0 Hz, 1H,phenyl-H), 8.34 (d, J = 8.0 Hz, 1H, phenyl-H), 8.69 (d, J = 8.0 Hz, 1H,phenyl-H), 8.90 (d, J = 12.0 Hz, 1H, alkene-H), 11.01 (s, 1H, amino-H).

[0130] 13 C NMR (100 MHz, DMSO- d 6 , ppm) δ14.82, 49.78, 95.88, 109.85, 116.51, 118.31, 123.75, 125.07, 129.65, 134.06, 137.42, 139.89, 143.34, 145.87, 153.86.

[0131] Mass spectrometry (ESI-MS, m / z): [M] + calcd. for [C 19 H 19 N2] + 275.1543: found 275.1549.

[0132] (3) 70 mg of compound 2 (Et-Q-M) was dissolved in 10 mL of acetone and 10 mL of acetyl chloride, and reacted at a temperature of 40°C for 3 h. After the reaction was completed, the solvent was evaporated and column chromatography was performed (mobile phase: dichloromethane and methanol, volume ratio of dichloromethane and methanol: 10:1) to obtain 49 mg of compound 3 (Et-Q-Ac) as a yellow solid, with a yield of 64%.

[0133]

[0134] 1 H NMR (400 MHz, DMSO- d 6 , ppm): δ 1.53 (t, J = 8.0 Hz, 3H, -CH3), 2.08(s, 3H, -CH3), 4.95 (q, J = 8.0 Hz, 2H, -CH2-), 6.02 (d, J = 12.0 Hz, 1H,alkene-H), 7.53 (d, J = 8.0 Hz, 2H, phenyl-H), 7.66 (d, J = 8.0 Hz, 1H,phenyl-H), 7.70 (m, 2H, phenyl-H), 7.86 (t, J = 8.0 Hz, 1H, phenyl-H), 7.92(d, J = 8.0 Hz, 1H, phenyl-H), 8.17 (t, J = 8.0 Hz, 1H, phenyl-H), 8.38 (d,J = 8.0 Hz, 1H, phenyl-H), 8.46 (d, J = 8.0 Hz, 1H, phenyl-H), 8.88 (d, J =12.0 Hz, 1H, alkene-H), 9.22 (d, J = 8.0 Hz, 1H, phenyl-H).

[0135] 13 C NMR (100 MHz, DMSO- d 6 , ppm) δ : 15.58, 52.22, 103.23, 115.62,119.63, 125.70, 126.21, 129.01, 129.55, 131.04, 135.39, 137.75, 138.40,147.14, 153.34, 170.27.

[0136] Mass spectrometry (ESI-MS, m / z): [M] + calcd. for [C 21 H 21 ON2] + 317.1648:found 317.1643.

[0137] (4) 49 mg of compound 3 (Et-Q-Ac), 40 mg of compound 4 (F-1) were dissolved in 2 mL of methanol, 0.5 mL of piperidine was added, and the reaction was carried out at a temperature of 50°C for 3 h. After the reaction was completed, the solvent was rotary evaporated, and column chromatography was carried out (the mobile phase was dichloromethane and methanol, the volume ratio of dichloromethane and methanol was 10:1) to obtain near-infrared fluorescent probe 23 mg as a black solid, with a yield of 38%.

[0138]

[0139] 1 H NMR (400 MHz, DMSO- d 6 , ppm): δ 1.53 (t, J = 8.0 Hz, 3H, -CH3), 3.09(s, 6H, -CH3), 4.80 (q, J= 8.0 Hz, 2H, -CH2-), 6.73 (d, J = 4.0 Hz, 1H,phenyl-H), 6.92 (dd, J 1 = 8.0 Hz, J 2 = 2.4 Hz, 1H, phenyl-H), 7.03 (d, J =12.0 Hz, 1H, alkene-H), 7.59 (m, 4H, phenyl-H), 7.81 (s, 1H, phenyl-H), 7.88(d, J = 8.0 Hz, 2H, phenyl-H), 8.10 (t, J = 8.0 Hz, 1H, phenyl-H), 8.17 (d, J = 4.0 Hz, 2H, phenyl-H), 8.31 (d, J = 8.0 Hz, 1H, phenyl-H), 8.41 (d, J = 8.0Hz, 1H, phenyl-H), 8.67 (d, J = 8.0 Hz, 1H, phenyl-H), 8.79 (t, J = 12.0 Hz,1H, alkene-H), 8.87 (d, J = 4.0 Hz, 1H, phenyl-H).

[0140] 13 C NMR (100 MHz, DMSO- d 6 , ppm) δ : 14.94, 50.34, 54.88, 97.92, 101.03,109.26, 111.29, 112.73, 116.13, 118.22, 124.30, 125.14, 125.51, 126.37,127.49, 128.68, 131.90, 134.02, 137.61, 139.62, 141.92, 143.52, 151.55,152.81, 153.78.

[0141] Mass spectrometry (ESI-MS, m / z): [M]+ calcd. for [C 31 H 29 N2O] + 445.2274:found 445.2249.

[0142] Example 3

[0143] (1) 1.49 g of benzothiazole, 1.56 g of iodoethane were dissolved in 15 mL of anhydrous acetonitrile, and the reaction was carried out at a temperature of 80°C for 8 h. After the reaction was completed, the solvent was evaporated, and column chromatography was performed (the mobile phase was dichloromethane and methanol, and the volume ratio of dichloromethane and methanol was 10:1) to obtain 246 mg of compound 1 (Et-BT), which was a brown solid, and the yield was 8.1%.

[0144]

[0145] 1 H NMR (400 MHz, DMSO- d 6 , ppm): δ 1.46 (t, J = 8.0 Hz, 3H, -CH3), 3.21(s, 3H, -CH3), 4.77 (q, J 2 = 8.0 Hz, 2H, -CH2-), 7.81 (t, J = 8.0 Hz, 1Hphenyl-H), 7.90 (t, J = 8.0 Hz, 1H phenyl-H), 8.34 (d, J = 8.0 Hz, 1H,phenyl-H), 8.45 (d, J = 8.0 Hz, 1H, phenyl-H).

[0146] 13 C NMR (100 MHz, DMSO- d 6 , ppm) δ: 13.26, 17.02, 44.88, 116.67,124.58, 127.95, 128.14, 129.01, 129.31, 129.47, 140.39, 176.60.

[0147] Mass spectrometry (ESI-MS, m / z): [M] +calcd. for [C 10 H 12 NS] + 178.0685:found 178.0678.

[0148] (2) 1000 mg of compound 1 (Et-BT), 771 mg of N,N dimethylformamide was dissolved in 10 mL of acetic anhydride and reacted at a temperature of 110°C for 12 h. After the reaction was completed, the solvent was evaporated and column chromatography was performed (mobile phase: dichloromethane and methanol, volume ratio of dichloromethane and methanol: 10:1) to obtain 504 mg of compound 2 (Et-BT-Ac) as a yellow solid, and the yield of compound 2 was 47.6%.

[0149]

[0150] 1 H NMR (400 MHz, CDCl3, ppm): δ 1.37 (t, J = 8.0 Hz, 3H, -CH3), 2.11(s, 3H, -CH3), 4.67 (q, J 2 = 8.0 Hz, 2H, -CH2-), 5.72 (d, J = 16.0 Hz, 1H,alkene-H), 7.47 (d, J = 8.0 Hz, 2H, phenyl-H), 8.45 (d, J = 8.0 Hz, 1H,phenyl-H), 7.63-7.77 (m, 5H, phenyl-H), 7.97 (d, J = 16.0 Hz, 1H, phenyl-H),7.99 (d, J = 16.0 Hz, 1H, phenyl-H), 8.99 (d, J = 12.0 Hz, 1H, alkene-H).

[0151] 13 C NMR (100 MHz, DMSO- d 6 , ppm) δ: 12.94, 23.04, 43.68, 95.85, 115.95,124.22, 126.96, 127.75, 128.06, 128.38, 129.06, 130.01, 130.28, 130.73,136.93, 140.61, 145.38, 170.01, 171.37.

[0152] Mass spectrometry (ESI-MS, m / z): [M] + calcd. for [C 19 H 19 ON2S] + 323.1213: found 323.1201.

[0153] (3) 53 mg of compound 2 (Et-BT-Ac), 31 mg of compound 4 (F-1), 1 mL of piperidine and 5 mL of methanol were mixed and refluxed at a temperature of 50 °C for 2 h. After the reaction was completed, the solvent was spin-dried, and column chromatography was performed (the mobile phase was dichloromethane and methanol, and the volume ratio of dichloromethane to methanol was 10:1) to obtain 20 mg of near-infrared fluorescent probe as a black solid, with a yield of 30.7%.

[0154]

[0155] 1 H NMR (400 MHz, CDCl3, ppm): δ 1.42 (t, J = 8.0 Hz, 3H, -CH3), 3.14(s, 6H, -CH3), 4.55 (q, J 2 = 8.0 Hz, 2H, -CH2-), 6.83 (d, J = 4.0 Hz, 1H,phenyl-H), 7.01 (dd, J 1 = 8.0 Hz, J 2 = 4.0 Hz, 1H, phenyl-H), 7.06 (d, J =16.0 Hz, 2H, alkene-H), 7.56 (t, J = 8.0 Hz, 1H, phenyl-H), 7.61-7.64 (m, 3H,phenyl-H), 7.69 (t, J = 8.0 Hz, 1H, phenyl-H), 7.92 (s, 1H, phenyl-H), 7.96(dd, J 1 = 8.0 Hz, J 2= 8.0 Hz, 1H, phenyl-H), 8.23 (t, J = 8.0 Hz, 1H, phenyl-H), 8.23 (t, J = 4.0 Hz, 2H, phenyl-H), 8.59 (t, J = 12.0 Hz, 1H, alkene-H).

[0156] Mass spectrometry (ESI-MS, m / z): [M] + calcd. for [C 29 H 27 ON2S] + 451.1839: found 451.1841.

[0157] Example 4

[0158] Compound 3 (Et-Py-Ac) in Example 1, 53 mg, compound 4 (F-2), 43 mg, were dissolved in 5 mL of methanol, 1 mL of piperidine was added, and the reaction was carried out at 60°C for 24 h. After the reaction was completed, the solvent was rotary evaporated, and column chromatography was performed (the mobile phase was dichloromethane and methanol, the volume ratio of dichloromethane and methanol was 10:1) to obtain near-infrared fluorescent probe 25 mg, compound IV-1 was black solid, and the yield was 33%.

[0159]

[0160] 1 H NMR (400 MHz, DMSO- d 6 , ppm): δ 1.48 (t, J = 8.0 Hz, 3H, -CH3), 1.92(m, 4H, -CH2-), 2.78 (t, J = 8.0 Hz, 2H, -CH2-), 2.89 (t, J = 4.0 Hz, 2H, -CH2-), 3.27 (s, 4H, -CH2-), 4.36 (q, J = 8.0 Hz, 2H, -CH2-), 6.52 (d, J = 16.0Hz, 1H, alkene-H), 6.63 (d, J= 12.0 Hz, 1H, alkene-H), 7.45 (s, 1H, phenyl-H), 7.56 (m, 4H, phenyl-H), 7.97 (d, J = 4.0 Hz, 2H, pyridine-H), 8.08 (d, J = 8.0 Hz, 2H, phenyl-H), 8.43 (t, J = 16.0 Hz, 1H, alkene-H), 8.57 (d, J =4.0 Hz, 2H, pyridine-H).

[0161] 13 C NMR (100 MHz, DMSO- d 6 , ppm) δ: 16.23, 20.19, 21.02, 27.09, 48.55,49.12, 53.82, 54.92, 100.14, 106.31, 108.32, 109.82, 119.03, 119.76, 120.31,121.09, 125.14, 128.64, 129.89, 132.45, 137.76, 139.44, 142.07, 145.34,148.67, 151.44, 153.16.

[0162] Mass spectrometry (ESI-MS, m / z): [M] + calcd. for [C 31 H 31 N2O] + 447.2431:found 447.2404.

[0163] Example 5

[0164] 65 mg of compound 3 (Et-Q-Ac) in Example 2, 59 mg of compound 4 (F-2), were dissolved in 5 mL of methanol, 1 mL of piperidine was added, and the reaction was carried out at a temperature of 60 °C for 24 h. After the reaction was completed, the solvent was rotary evaporated, and column chromatography was performed (the mobile phase was dichloromethane and methanol, and the volume ratio of dichloromethane and methanol was 10:1) to obtain near-infrared fluorescent probe 32 mg, compound V-1 was a black solid, and the yield was 38%.

[0165]

[0166] 1 H NMR (400 MHz, DMSO- d 6 , ppm): δ 1.45 (t, J = 8.0 Hz, 3H, -CH3), 1.90(m, 4H, -CH2-), 2.30 (t, J = 8.0 Hz, 2H, -CH2-), 2.90 (t, J = 4.0 Hz, 2H, -CH2-), 3.26 (s, 4H, -CH2-), 4.50 (q, J = 8.0 Hz, 2H, -CH2-), 6.70 (d, J = 4.0Hz, 1H, phenyl-H), 6.90 (dd, J 1 = 8.0 Hz, J 2 = 2.4 Hz, 1H, phenyl-H), 7.05 (d, J = 12.0 Hz, 1H, alkene-H), 7.57 (m, 2H, phenyl-H), 7.79 (s, 1H, phenyl-H),7.83 (d, J = 8.0 Hz, 2H, phenyl-H), 8.07 (t, J = 8.0 Hz, 1H, phenyl-H), 8.15(d, J = 4.0 Hz, 2H, phenyl-H), 8.26 (d, J = 8.0 Hz, 1H, phenyl-H), 8.40 (d, J = 8.0 Hz, 1H, phenyl-H), 8.56 (d, J = 8.0 Hz, 1H, phenyl-H), 8.79 (t, J =12.0 Hz, 1H, alkene-H), 8.92 (d, J = 4.0 Hz, 1H, phenyl-H).

[0167] 13 C NMR (100 MHz, DMSO- d 6, ppm) δ: 14.32, 22.15, 28.02, 29.76, 49.64,51.77, 103.42, 107.55, 116.23, 117.04, 118.76, 112.25. 123.22, 128.78,129.91, 133.08, 140.42, 145.45, 152.46, 172.32.

[0168] Mass spectrometry (ESI-MS, m / z): [M] + calcd. for [C 35 H 33 N2O] + 497.2588:found 497.2590.

[0169] Example 6

[0170] 60 mg of compound 3 (Et-BT-Ac) in Example 3, 54 mg of compound 4 (F-2), were dissolved in 5 mL of methanol, 1 mL of piperidine was added, and the reaction was carried out at a temperature of 60°C for 24 h. After the reaction was completed, the solvent was spin-dried, and column chromatography was performed (the mobile phase was dichloromethane and methanol, and the volume ratio of dichloromethane to methanol was 10:1) to obtain near-infrared fluorescent probe 36 mg, compound VI-1 as a black solid, with a yield of 47%.

[0171]

[0172] 1 H NMR (400 MHz, DMSO- d 6 , ppm): δ 1.43 (t, J = 8.0 Hz, 3H, -CH3), 1.87(m, 4H, -CH2-), 2.29 (t, J = 8.0 Hz, 2H, -CH2-), 2.93 (t, J = 4.0 Hz, 2H, -CH2-), 3.25 (s, 4H, -CH2-), 4.55 (q, J = 8.0 Hz, 2H, -CH2-), 7.03(dd, J 1 = 8.0Hz, J 2= 4.0 Hz, 1H, phenyl-H), 7.11 (d, J = 16.0 Hz, 2H, alkene-H), 7.55 (t, J = 8.0 Hz, 1H, phenyl-H), 7.58-7.66 (m, 2H, phenyl-H), 7.72 (t, J = 8.0 Hz,1H, phenyl-H), 7.93 (s, 1H, phenyl-H), 7.80 (dd, J 1 = 8.0 Hz, J 2 = 4.0 Hz, 2H,phenyl-H), 8.15 (d, J = 8.0 Hz, 1H, phenyl-H), 8.26 (t, J = 4.0 Hz, 2H,phenyl-H), 8.55 (t, J = 12.0 Hz, 1H, alkene-H).

[0173] 13 C NMR (100 MHz, DMSO- d 6 , ppm) δ: 13.92, 22.13, 29.73, 48.62, 51.73,89.27, 103.29, 117.26, 122.06, 122.25, 122.93, 123.76, 128.69, 129.38,130.46, 139.27, 148.16, 152.38, 152.76, 172.25.

[0174] Mass spectrometry (ESI-MS, m / z): [M] + calcd. for [C 33 H 31 N2OS] + 503.2152: found 503.2155.

[0175] Performance determination:

[0176] (1) Absorption and fluorescence spectrum test: the near-infrared fluorescent probes prepared in examples 1-4 were respectively dissolved in dimethyl sulfoxide to prepare a solution with a concentration of 1.0 x 10 -3mol / L stock solution, then 2 mL of dimethyl sulfoxide (DMSO), PBS buffer (PBS), dichloromethane (DCM), acetonitrile (CH3CN), tetrahydrofuran (THF), methanol (MeOH) were taken respectively, 20 μL of the prepared stock solution was added to each of them, and then they were mixed uniformly and transferred to optical quartz cuvettes (10 x 10 mm) for testing their absorption and fluorescence spectra, and the test results are shown in Figures 1-8 The maximum emission wavelength of compound I-1 is in the near-infrared region at about 680 nm, and the Stokes shift reaches 115 nm Figure 1 , Figure 2 ); the maximum emission wavelength of compound II-1 is in the near-infrared region at about 752 nm Figure 3 , Figure 4 ); the maximum emission wavelength of compound III-1 is in the near-infrared region at about 695 nm Figure 5 , Figure 6 ); the maximum emission wavelength of compound IV-1 is in the near-infrared region at about 714 nm, and the Stokes shift reaches 108 nm Figure 7 , Figure 8 ).

[0177] (2) Spectral response test of DNA and RNA: the near-infrared fluorescent probe prepared in Example 1 was dissolved in dimethyl sulfoxide to prepare a stock solution with a concentration of 1.0 x 10 -3 mol / L. 5 mg of DNA and RNA were taken respectively, and 1 mL of Tris-HCl solution with pH = 7.2 was added to each of them to prepare a nucleic acid stock solution with a concentration of 5.0 g / L.

[0178] The stock solution of DNA or RNA was diluted to 1.0 g / L respectively, and then the near-infrared fluorescent probe stock solution was added to make the final concentration of the near-infrared fluorescent probe 1.0 x 10 -5 mol / L, and then they were mixed uniformly and transferred to optical quartz cuvettes (10 x 10 mm) for testing their fluorescence spectra, and the test results are shown in Figure 9 , Figure 10 Without adding nucleic acid, the near-infrared fluorescent probe stock solution has almost no fluorescence signal; after adding nucleic acid, the fluorescence signal of the near-infrared fluorescent probe is significantly enhanced with 560 nm as the excitation wavelength, and the maximum emission peak appears in the near-infrared region at 680 nm, and the fluorescence intensity of the near-infrared fluorescent probe increases by 127 times or 410 times for DNA and RNA respectively, and the fluorescence intensity of the near-infrared fluorescent probe after binding with RNA is 3.2 times that after binding with DNA.

[0179] (3) Staining and fluorescence imaging test on living HeLa cells: HeLa cells were selected as the cell model, and the above-mentioned stock solution for the spectral response test of DNA and RNA was used. 10 μL of the stock solution was added to 1 mL of cell culture medium to prepare a near-infrared fluorescent probe solution with a concentration of 10 μmol / L. The HeLa cells were incubated in the solution for 60 min, and then washed with PBS solution for 3 times. Leica SP8 confocal microscope was used to perform microscopic fluorescence imaging on the HeLa cells stained by the near-infrared fluorescent probe. The results are shown in Figure 11 The near-infrared fluorescent probe has bright red fluorescence in the nucleolus region and cytoplasm of HeLa cells, and almost no fluorescence in the chromatin region of the nucleus. This is consistent with the response results of nucleic acids, proving that the near-infrared fluorescent probe can accurately bind to RNA and has a high signal-to-noise ratio.

[0180] (4) Staining and super-resolution fluorescence imaging of HeLa cells and mouse white blood cells: HeLa cells and mouse white blood cells were selected as the cell model for imaging. C57BL / 6J mice were used to collect whole blood by eyeball enucleation and blood collection method, and white blood cells were extracted from the whole blood by red blood cell lysis and centrifugation method. Then, the extracted white blood cells and HeLa cells were stained according to the method in (3) above, and were imaged using a structured light illumination super-resolution microscope. For HeLa cells, the results are shown in Figure 12 The near-infrared fluorescent probe can clearly show the nucleolus structure of HeLa cells. The bright part in the nucleolus is the dense fiber component (DFC) and granular component (GC) structure, and the dark part in the nucleolus is the fiber center (FC) structure. For mouse white blood cells, the results are shown in Figure 13 The near-infrared fluorescent probe can clearly show the super-resolution fluorescence image of the multi-leaf nucleus (containing RNA) of mouse white blood cells. The results of structured light illumination super-resolution fluorescence imaging show that the near-infrared fluorescent probe prepared in Example 1 has the ability to perform super-resolution fluorescence imaging on RNA.

[0181] The above-mentioned only is the preferred embodiment of the present application, it should be pointed out that, for the ordinary skilled in the art, without departing from the principles of the present application, can make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A near-infrared fluorescent probe, characterized by, The near-infrared fluorescent probe has a structure as shown in the following formula: 、 、 (Ⅰ-1) (Ⅱ-1) 、 、 (Ⅲ-1) (Ⅳ-1) (V-1) (VI-1).

2. The method for preparing a near-infrared fluorescent probe according to claim 1, characterized by, The method comprises the following steps: (1) mixing a heterocyclic compound and a halogen-containing compound in acetonitrile to perform a reflux reaction to obtain compound 1; (2) mixing compound 1, N,N-diphenylformamidine and piperidine in methanol to perform a reflux reaction to obtain compound 2; (3) mixing compound 2 and a mixed solvent to perform a reflux reaction to obtain compound 3; (4) mixing compound 3, compound 4 and piperidine in methanol to perform a reflux reaction to obtain the near-infrared fluorescent probe; The halogen-containing compound is CH3-CH2-X, wherein X is halogen; The heterocyclic compound is or ; The compound 4 has a structure as shown in the following formula: ； In step (3), the mixed solvent is a mixed solvent of acetone and acetyl chloride.

3. The production method according to claim 2, characterized by, In step (1), the mass ratio of the heterocyclic compound and the halogen-containing compound is 900-1500 mg: 7.5-8.0 g; The mass-volume ratio of the heterocyclic compound and acetonitrile is 900-1500 mg: 8-25 mL; the temperature of the reflux reaction is 70-90 DEG C, and the reflux reaction time is 8-16 h.

4. The method for preparing a near-infrared fluorescent probe according to claim 2 or 3, characterized by, In step (2), the mass ratio of compound 1, N,N-diphenylformamidine and piperidine is 150-500: 150-300: 130-260; the mass-volume ratio of compound 1 and methanol is 150-500 mg: 5-10 mL; the temperature of the reflux reaction is 50-70 DEG C, and the reflux reaction time is 4-6 h; In step (3), the mass-volume ratio of compound 2 and the mixed solvent is 30-90 mg: 10-30 mL; the volume ratio of acetone and acetyl chloride is 1: 1-2; the temperature of the reflux reaction is 30-50 DEG C, and the reflux reaction time is 1-24 h.

5. The method for preparing the near-infrared fluorescent probe according to claim 4, characterized in that, In step (4), the mass ratio of compound 3 and compound 4 is 30-600: 30-60; the mass-volume ratio of compound 3 and methanol is 30-600 mg: 1-8 mL; the volume ratio of methanol and piperidine is 1-8: 0.5-2; the temperature of the reflux reaction is 40-70 DEG C, and the reflux reaction time is 3-24 h.

6. The method of preparing a near-infrared fluorescent probe according to claim 1, characterized by, The method comprises the following steps: (1) mixing a heterocyclic compound and a halogen-containing compound in acetonitrile to perform a reflux reaction to obtain compound 1; (2) mixing compound 1, N,N - mixing diphenylformamidine and acetic anhydride and refluxing to obtain compound 2; (3) mixing compound 2, compound 4 and piperidine in methanol to perform a reflux reaction to obtain the near-infrared fluorescent probe; The halogen-containing compound is CH3-CH2-X, wherein X is halogen; The heterocyclic compound is ; The compound 4 has a structure as shown in the following formula: 。 7. The method for preparing the near-infrared fluorescent probe according to claim 6, characterized in that, In step (1), the mass ratio of the heterocyclic compound and the halogen-containing compound is 1.3-1.6: 1.4-1.8; the mass-volume ratio of the heterocyclic compound and acetonitrile is 1.3-1.6 g: 10-20 mL; the temperature of the reflux reaction is 70-90 DEG C, and the reflux reaction time is 6-10 h.

8. The method for preparing the near-infrared fluorescent probe according to claim 6 or 7, wherein, N,N in step (2), the compound 1 and ​ - the mass ratio of the compound 1 and the diphenylformamidine is 800~1200:700~800, the mass-volume ratio of the compound 1 and acetic anhydride is 800~1200 mg:8~12 mL, the temperature of the reflux reaction is 100~120℃, and the reflux reaction time is 10~14 h; in step (3), the mass ratio of the compound 2 and the compound 4 is 50~55:30~35, the mass-volume ratio of the compound 2 and methanol is 50~55 mg:3~8 mL, the volume ratio of the methanol and piperidine is 3~8:1~3, the temperature of the reflux reaction is 40~60℃, and the reflux reaction time is 1~3 h.

9. Use of the near-infrared fluorescent probe of claim 1 in the preparation of detecting DNA or RNA stock solution and in the cell imaging which is not for disease diagnosis or treatment.

Images