Near-infrared nile blue fluorescent dye as well as synthesis method and application thereof

Abstract

The invention discloses a near-infrared nile blue fluorescent dye and a synthesis method and application thereof, and the dye has excellent fluorescence intensity and is suitable for near-infrared region fluorescence imaging. A series of novel fluorescent dyes NRO-N are successfully prepared by replacing diethylamino of Nile blue with various tertiary amine structures, introducing alkyl groups or sulfonyl groups with different lengths at different positions of molecules, replacing anions with different anions and adjusting the luminous intensity and solubility of the Nile blue dye. Wherein the fluorescence intensity of NBO-2-ClO4 in a phosphoric acid buffer solution is higher than that of a contrast molecule diethylamino Nile blue. A new tool is provided for the fluorescent dye for near-infrared region imaging, the preparation process is simple and efficient, large-scale production is easy, and the fluorescent dye has wide clinical application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organic dyes, and relates to a novel nile blue compound, a synthesis method thereof and application thereof. BACKGROUND

[0002] In recent years, chronic non-communicable diseases have become the main cause of current resident deaths, and cancer is a difficult point that is difficult to solve. With the continuous development of microtechnologies, the cognition of human beings on the physiological process of living beings gradually transits from the macro level to the micro field. In recent years, with the in-depth understanding of the disease process, it is found that the morphology, distribution and content of various cell contents such as nucleic acids, mitochondria, polypeptides and enzymes in cells are closely related to diseases. Fluorescent probes are widely used in the fields of biological imaging, medical diagnosis and post-treatment evaluation due to their good stability, high sensitivity, strong anti-interference, good biocompatibility and adjustable functions, and provide important research tools for clinical medical workers in early diagnosis of tumors and detection of malignant tumors.

[0003] Nile blue dye, as an organic derivative with phenoxazine as the parent body, also inherits the excellent light stability, thermal stability and antioxidant property of phenoxazine compounds, and the maximum peak position of the absorption and emission spectrum is usually above 600 nm wavelength, which has very excellent photophysical properties and plays an important role in the field of fluorescent probes. However, the fluorescence of this kind of dye in water is often weakened due to aggregation, leading to a decrease in imaging accuracy, and is often accompanied by high dark toxicity, which may damage normal tissues during imaging. In summary, the traditional oxazine dye has obvious defects in the photoluminescence intensity in water and biological safety. Therefore, it is an urgent problem to be solved to improve the photoluminescence intensity of nile blue dye and reduce the dark toxicity by a new strategy. SUMMARY

[0004] The present application aims at the above-mentioned problems, and provides a novel near-infrared nile blue fluorescent dye, a preparation method and application thereof. The preparation method replaces the diethylamino group of the conventional nile blue with a tertiary amine group with different electron-donating ability and introduces C1-4 alkyl or C1-4 alkylsulfonyl in the molecule, and replaces the anion to inhibit aggregation quenching, so that a novel fluorescent dye with stronger fluorescence intensity in DCM or PBS is obtained. Some prepared fluorescent dyes have higher fluorescence intensity and better solubility, the preparation process is simple, and is easy to popularize.

[0005] The first aspect of the present application is to protect a kind of near-infrared nile blue fluorescent dye, which is absorbed in red light band 620nm-750nm, and its structure is as general formula I:

[0006]

[0007] The R1 is selected from tertiary amine groups; more preferably, the R1 is selected from one of diethylamino, octahydro-4H-quinolineazinyl, 1,1,7,7-tetramethyloctahydro-4H-quinolineazin-4-yl, aziridine, aziridine, N-methylpiperazinyl, N,N-dimethylpiperazinyl, 1,4-diethylpiperazinyl, and N,N-propanesulfonic acid ethylamino.

[0008] R2 is a C1-4 alkyl or C1-4 alkylsulfonyl group; wherein, the C1-4 alkylsulfonyl group includes methanesulfonic acid group, ethanesulfonic acid group, propanesulfonic acid group and butanesulfonic acid group;

[0009] The X - Selected from chloride ions (Cl) - ), bromide ions (Br) - ), iodide ions (I) - ), perchlorate ions (ClO4) - ), hexafluorophosphate ions (PF6) - ), antimony hexafluorophosphate ion (SbF6) - Boron tetrafluoride ions (BF4) - ), tetra(4-fluorophenyl)borate anion (FPhB) - ), tetrafluorophenylboronic acid ion (TBFB) - Tetra[3,5-bis(trifluoromethyl)phenyl]borate anion (TFM) - ) and Δ-tris(tetrachloro-1,2-phenylenediol) phosphate (V) anion (TRIS) - One of them.

[0010]

[0011] For the technical solution described above, a further preferred embodiment is that the fluorescent dye is selected from one of NBO-1-X, NBO-2-X, NBO-3-X, NBO-4-X, NBO-5-X, NBO-6-X, NBO-7-X, NBO-8-X, and NBO-9-X;

[0012]

[0013] The second aspect of this application is to protect a method for preparing the aforementioned near-infrared Nile blue fluorescent dye, comprising the following steps:

[0014] When R2 is a C1-4 alkyl group

[0015] The synthesis of this kind of fluorescent dye is only five steps, first, the intermediate 1 is synthesized by the reaction of C1-4 alkyl iodide and 1-naphthylamine, then the intermediate 2 is synthesized by the introduction of diazonium salt in p-nitroaniline; then the intermediate 3 is synthesized by the reaction of intermediate 1; then the target compound with different anions is obtained by ion exchange with different anions X- by dissolving the aniline compound containing R1 group and intermediate 3 in anhydrous DMF, adding perchloric acid and heating to a certain temperature under inert gas protection and stirring.

[0016]

[0017] When R2 is C1-4 alkylsulfonyl;

[0018] The synthesis of this kind of fluorescent dye is only five steps, first, the intermediate 1 is synthesized by the reaction of C1-4 alkyl iodide and 1-naphthylamine, then the intermediate 2 is synthesized by the introduction of diazonium salt in p-nitroaniline; then the intermediate 3 is synthesized by the reaction of intermediate 1; then the target compound with different anions is obtained by ion exchange with different anions X- by dissolving the aniline compound containing R1 group and intermediate 3 in anhydrous DMF, adding perchloric acid and heating to a certain temperature under inert gas protection and stirring.

[0019]

[0020] The specific preparation steps are as follows:

[0021] (1) When R2 is C1-4 alkyl, the general preparation method of Nile blue fluorescent dye

[0022] 1) 1-naphthylamine, potassium carbonate and C1-4 alkyl iodide are mixed in ethanol, the mixture is heated to 80°C and stirred. After the reaction is completed, the solvent is distilled off under reduced pressure and column chromatography is carried out to obtain intermediate 1.

[0023] 2) 4-nitroaniline dissolved in propionic acid / acetic acid / DMF is mixed with concentrated hydrochloric acid. The mixture is heated to 70°C, then cooled to 0°C. Sodium nitrite aqueous solution is added dropwise to the mixture. After the dropwise addition is completed, the system is stirred at 0°C. After the reaction is completed, a solution of intermediate 2 is obtained;

[0024] 3) DMF solution containing intermediate 1 is added dropwise to the solution of intermediate 2, after the dropwise addition is completed, the system is stirred at 0°C for a certain time, then warmed to room temperature and stirred. After the reaction is completed, the solution is basified with ammonia water. Filtration to obtain solid intermediate 3;

[0025] 4) Intermediate 3 and aniline compound containing R1 group are dissolved in anhydrous DMF, perchloric acid is added, heated to a certain temperature and stirred, after the reaction is completed and cooled, suction filtration, distillation under reduced pressure to remove the solvent and column chromatography to obtain the target compound with perchlorate anion;

[0026] 5) the salt containing different X - The salt containing different X - of the target compound.

[0027] Further preferably, the salt containing different X - of the above technical solution is selected from one of sodium chloride, sodium bromide, sodium iodide, sodium perchlorate, sodium hexafluorophosphate, sodium hexafluoroantimonate, sodium tetrafluoroborate, lithium tetra(4-fluorophenyl)borate, lithium tetra(pentafluorophenyl)borate, lithium tetra[3,5-bis(trifluoromethyl)phenyl]borate, and Δ-tris(tetrachloro-1,2-benzene diolato)phosphate (V) sodium.

[0028] Further preferably, the molar ratio of 1-naphthylamine, alkyl iodide and potassium carbonate in step 1) of the above technical solution is (0.8-1.2):(0.8-1.2):(2-4); further preferably, it is 1:1:3.

[0029] Further preferably, the eluent for column chromatography separation in step 1) of the above technical solution is a petroleum ether-ethyl acetate system with a volume ratio of 100:1.

[0030] Further preferably, the volume ratio of propionic acid / acetic acid / DMF in step 2) of the above technical solution is (0.8-1.2):(4-6):(3-5); further preferably, it is 1:5.42:4.28.

[0031] Further preferably, the molar ratio of 4-nitroaniline, concentrated hydrochloric acid and nitrous acid in step 2) of the above technical solution is (0.8-1.2):(15-25):(0.8-1.5); further preferably, it is 1:20:1.

[0032] Further preferably, the molar ratio of N-ethyl naphthylamine and intermediate 1 in step 3) of the above technical solution is (0.8-1.2):(2-4); further preferably, it is 1:3.

[0033] Further preferably, the molar ratio of intermediate 2, aniline compound containing R1 group and perchloric acid in step 4) of the above technical solution is (0.8-1.2):(0.8-1.2):(15-30); further preferably, it is 1.01:1:22.2.

[0034] Further preferably, the reaction temperature in step 4) of the above technical solution is in the range of 120-150°C; further preferably, it is 140°C.

[0035] For the technical solutions described above, further preferably, the eluent for column chromatography separation in step 4) is a dichloromethane-methanol system with a volume ratio of 20-100:1.

[0036] For the technical solutions described above, further preferably, the molar ratio of the target compound with perchlorate anions in step 5) to the salt containing different X - is (0.8-1.2):(5-15); further preferably, 1:10.

[0037] For the technical solutions described above, further preferably, the eluent for column chromatography separation in step 5) is a dichloromethane-methanol system with a volume ratio of 20-100:1.

[0038] (2) Preparation method of Nile blue fluorescent dye with sulfonamide group when R2=C1-4alkylsulfonyl

[0039] 1) Dissolve 1-naphthylamine and C1-4sulfonic acid lactone in n-butanol, reflux, after the reaction is completed and cooled, filter and wash with cold methanol to obtain intermediate 1;

[0040] 2) Mix 4-nitroaniline dissolved in propionic acid / acetic acid / DMF with concentrated hydrochloric acid. Heat the mixture to 70°C, then cool to 0°C. Add aqueous sodium nitrite solution dropwise to the mixture. After the dropwise addition is complete, the system is stirred at 0°C. After the reaction is completed, a solution of intermediate 1 is obtained;

[0041] 3) Add a DMF solution containing N-ethyl naphthylamine to the intermediate 2 solution dropwise, after the dropwise addition is complete, the system is stirred at 0°C for a certain period of time, then warmed to room temperature and stirred. After the reaction is completed, the solution is basified with ammonia water. Filter to obtain solid intermediate 3;

[0042] 4) Dissolve intermediate 3 and an aniline compound containing R1group in anhydrous DMF, add perchloric acid, heat to a certain temperature and stir, after the reaction is completed and cooled, filter, remove the solvent by vacuum distillation, and perform column chromatography to obtain the target compound with perchlorate anions;

[0043] 5) Dissolve the salt containing different X - and the target compound with perchlorate anions in methanol, stir at room temperature, after the end, filter, remove the solvent by vacuum distillation, and perform column chromatography to obtain the target compound with X - anions.

[0044] Among them, for the technical solutions described above, further preferably, the above-mentioned salt containing different X -The salt of the formula (I) is selected from one of sodium chloride, sodium bromide, sodium iodide, sodium perchlorate, sodium hexafluorophosphate, sodium hexafluoroantimonate, sodium tetrafluoroborate, sodium ortho-carborane, sodium tetracarbonylborate, lithium tetra(4-fluorophenyl)borate, lithium tetra(pentafluorophenyl)borate, lithium tetra[3,5-bis(trifluoromethyl)phenyl]borate, and sodium delta-tris(tetrachloro-1,2-benzenediolato)phosphate (V).

[0045] For the technical solution described above, further preferably, the molar ratio of 1-naphthylamine to the corresponding sulfolactone in the above step 1) is (0.8-1.2):(0.7-1.5); further preferably 1:1.1.

[0046] For the technical solution described above, further preferably, the volume ratio of propionic acid / acetic acid / DMF in the above step 2) is (0.8-1.2):(4-6):(3-5); further preferably 1:5.42:4.28.

[0047] For the technical solution described above, further preferably, the molar ratio of 4-nitroaniline, concentrated hydrochloric acid and nitrous acid in the above step 2) is (0.8-1.2):(15-25):(0.8-1.5); further preferably 1:20:1.

[0048] For the technical solution described above, further preferably, the molar ratio of N-ethylnaphthylamine to intermediate 1 in the above step 3) is (0.8-1.2):(2-4); further preferably 1:3.

[0049] For the technical solution described above, further preferably, the molar ratio of intermediate 2, aniline compound containing R1 group and perchloric acid in the above step 4) is (0.8-1.2):(0.8-1.2):(15-30); further preferably 1.01:1:22.2.

[0050] For the technical solution described above, further preferably, the reaction temperature in the above step 4) is in the range of 120-150°C; further preferably 140°C.

[0051] For the technical solution described above, further preferably, the eluent for column chromatography separation in the above step 4) is a dichloromethane-methanol system with a volume ratio of 20-100:1.

[0052] For the technical solution described above, further preferably, the molar ratio of the target compound with anion of perchlorate ion to the salt containing different X - in the above step 5) is (0.8-1.2):(5-15); further preferably 1:10.

[0053] For the technical solutions described above, further preferably, the eluent for column chromatography separation in step 5) is a dichloromethane-methanol system with a volume ratio of 20-100:1.

[0054] The third aspect of the present application is to protect the use of the compound of general formula I in the preparation of optical detection reagents for non-diagnostic treatment, especially in the preparation of reagents for in vivo imaging. The present application emphasizes its application in the field of non-disease diagnosis, especially focusing on the diagnosis of non-disease that can effectively image abnormal cell proliferation, and the diagnosis of non-disease that requires deep tissue penetration and high selective cell uptake. Specifically, it includes: as a near-infrared imaging reagent for tumor diagnosis, covering the development of in vivo imaging reagents based on deep tissue penetration characteristics, the design of in vitro three-dimensional culture system tracing reagents for abnormal cell proliferation behavior, and the preparation of drug delivery evaluation reagents using high selective cell uptake performance

[0055] The compound of general formula I includes oxonitroblue compounds with various tertiary amines, C1-4 alkyl or C1-4 alkylsulfonyl groups, and different anions.

[0056] Compared with the prior art, the present application has the following beneficial effects:

[0057] 1) The high-brightness fluorescent dye based on nitroblue provided by the present application has strong fluorescence intensity in PBS, achieving higher precision in disease imaging.

[0058] 2) By making slight structural changes, such as introducing alkyl or sulfonyl groups at different positions, introducing different anions, and introducing non-planar tertiary amine structures, the aggregation of the fluorescent dye in PBS can be effectively adjusted, reducing the fluorescence weakening caused by dye aggregation in water. Different tertiary amine structures have different electron-donating abilities, which can effectively regulate the excited state properties of the fluorescent dye, achieving precise optimization of the fluorescence imaging effect.

[0059] 3) Compared with the reported nitroblue, some of the nitroblue fluorescent dyes reported in the present application have stronger fluorescence intensity in water, which is beneficial to achieve clearer and finer fluorescence imaging.

[0060] 4) Absorption and emission in the near-infrared region have deeper penetration depth and less attenuation of fluorescence signal, which is beneficial to in vivo imaging. In addition, the energy of such light is low, which greatly reduces the toxicity of cells and tissues.

[0061] 5) As can be seen from the examples, the synthesis route of the fluorescent dye of the present application is clear, the steps are short, the yield is high, it is convenient for large-scale preparation, and the purification process is simple, which is beneficial to subsequent clinical transformation and application. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1is the nuclear magnetic hydrogen spectrum of NBO-1-CIO4;

[0063] Figure 2 is the high resolution mass spectrum of NBO-1-CIO4;

[0064] Figure 3 is the nuclear magnetic hydrogen spectrum of NBO-2-CIO4;

[0065] Figure 4 is the high resolution mass spectrum of NBO-2-CIO4;

[0066] Figure 5 is the high resolution mass spectrum of NBO-1-TBFB (positive mode);

[0067] Figure 6 is the high resolution mass spectrum of NBO-1-TBFB (negative mode);

[0068] Figure 7 is the nuclear magnetic hydrogen spectrum of NBO-8-Cl;

[0069] Figure 8 is the high resolution mass spectrum of NBO-8-Cl;

[0070] Figure 9 are the absorption and emission spectra of NBO-1-CIO4, NBO-2-CIO4, NBO-1-TBFB, NBO-8-Cl and NBO in dichloromethane.

[0071] Figure 10 are the absorption and emission spectra of NBO-1-CIO4, NBO-2-CIO4, NBO-1-TBFB, NBO-8-Cl and NBO in PBS. DETAILED DESCRIPTION

[0072] The present application will be further illustrated by the following examples and figures, which are intended to be illustrative only and not limiting. The methods of operation described in the following examples, unless otherwise indicated, are conventional methods.

[0073]

[0074] Example 1

[0075] This example relates to the synthesis of an oxonidyl blue fluorescent dye, having the following structure:

[0076]

[0077] Example 1.1

[0078]

[0079] Iodoethane (0.544 g, 3.49 mmol), 1-naphthylamine (0.5 g, 3.49 mmol) and potassium carbonate (1.45 g, 10.48 mmol) were mixed in 20 mL of ethanol. The mixture was heated to 80 °C and stirred for 6 h. After completion of the reaction, the mixture was cooled to room temperature and the solvent was removed under reduced pressure. The crude product was purified by column chromatography using 100:1 petroleum ether-ethyl acetate as eluent to obtain the intermediate 1.

[0080] Example 1.2

[0081]

[0082] 4-Nitroaniline (0.414 g, 3.00 mmol) dissolved in propionic acid / acetic acid / DMF (0.7 mL:3.8 mL:3 mL) was mixed with concentrated hydrochloric acid (5 mL). The mixture was heated to 70 °C until the substrate dissolved and then cooled to 0 °C. A solution of sodium nitrite (0.207 g, 3.00 mmol) in water (1 mL) was added dropwise to the mixture and stirred at 0 °C for 2 h. After completion of the reaction, the intermediate compound 2 was obtained.

[0083] Example 1.3

[0084]

[0085] A solution of intermediate 1 (0.223 g, 1.004 mmol) in DMF (6 mL) was added dropwise to the mixture and stirred at 0 °C for 1 h, then warmed to room temperature and stirred for 24 h. After completion of the reaction, the solution was basified with aqueous ammonia (30%). The crude intermediate compound 3 was filtered. The crude product was used for the synthesis of the target product without further purification.

[0086] Compound NBO-1-CIO4

[0087]

[0088] Intermediate compound 3 (0.198 g, 0.534 mol) and 8-hydroxyquercetin (0.1 g, 0.527 mmol) were dissolved in dry DMF (5 mL) and perchloric acid (70%, 1 mL) was added dropwise to the reaction. The reaction was stirred at 140 °C under nitrogen atmosphere for 5 h. After completion of the reaction, dichloromethane (50 mL) was added to the mixture, which was washed with water (3 x 150 mL) and dried over anhydrous magnesium sulfate, filtered and concentrated. The obtained slurry of the crude product was purified by column chromatography using 100-20:1 dichloromethane-methanol as eluent to obtain the target compound NBO-1-CIO4: 1H NMR (400 MHz, DMSO) δ 9.39 (s, 1H), 8.71 (d, J = 7.7 Hz, 1H), 8.44 (d, J = 8.1 Hz, 1H), 7.89 (dd, J = 19.8, 12.2 Hz, 1H), 7.80 (t, J = 7.1 Hz, 1H), 7.44 (d, J = 16.2 Hz, 1H), 6.95 (s, 1H), 5.76 (s, 1H), 3.67 (d, J = 6.9 Hz, 2H), 3.58 (s, 4H), 2.87 (s, 4H), 1.99 (s, 4H), 1.36 (t, J = 6.8 Hz, 3H), [M+] of C24H24N3O+calcd 370.1914; found 370.1913. See Table 1 for NMR data. Figure 1 See Table 2 for mass spectrometry (HRMS) data. Figure 2 The results demonstrate the correct molecular structure.

[0089] Example 2

[0090] This example relates to the synthesis of a Nile Blue fluorescent dye, having the structure:

[0091]

[0092] Example 2.1

[0093]

[0094] Synthesis method same as Example 1.1

[0095] Example 2.2

[0096]

[0097] Synthesis method same as Example 1.2.

[0098] Example 1.3

[0099]

[0100] Synthesis method same as Example 1.3.

[0101] Compound NBO-2-CIO4

[0102]

[0103] Intermediate compound 2 (0.198 g, 0.534 mol) and tetramethylgueronidine (0.1 g, 0.408 mmol) were dissolved in dry DMF (5 mL), and perchloric acid (70%, 1 mL) was added dropwise to the reaction, which was stirred at 140 °C under nitrogen atmosphere for 5 h. After the reaction was completed, dichloromethane (50 mL) was added to the mixture, which was washed with water (3 x 150 mL) and dried over anhydrous magnesium sulfate, filtered and concentrated. The obtained crude product in slurry form was purified by column chromatography with an eluent ratio of 100-20:1 dichloromethane-methanol to obtain the target compound NBO-2-CIO4: 1 H NMR (400 MHz, DMSO) δ 9.42 (s, 1H), 8.81 (d, J = 8.0 Hz, 1H), 8.52 (d, J = 8.3 Hz, 1H), 7.93 (t, J = 7.6 Hz, 1H), 7.83 (t, J = 7.6 Hz, 1H), 7.70 (s, 1H), 7.13 (s, 1H), 3.79 - 3.72 (m, 2H), 3.68 (t, J = 5.8 Hz, 2H), 3.64 - 3.58 (m, 2H), 1.92 - 1.88 (m, 2H), 1.81 (t, J = 6.0 Hz, 2H), 1.59 (s, 6H), 1.40 - 1.35 (m, 9H), [M+] of calculated molecular formula C28H32N3O+ is 426.2540; the actual measurement is 426.2539. The detailed NMR data are shown in the following table: Figure 3 The detailed mass spectrometry (HRMS) detection results are shown in the following table: Figure 4 The molecular structure is correct.

[0104] Example 3

[0105] This example relates to the synthesis of a Nile blue fluorescent dye, which has the following structure:

[0106]

[0107] Example 3.1

[0108]

[0109] Synthesis method same as Example 1.1 Example 3.2

[0110]

[0111] Synthesis method same as Example 1.2.

[0112] Example 3.3

[0113]

[0114] Synthesis method same as Example 1.3.

[0115] Compound NBO-1-CIO4

[0116]

[0117] Synthetic method is the same as Example 1.4.

[0118] Compound NBO-1-TBFB

[0119]

[0120] Lithium tetrafluoroborate (1.462 g, 2.132 mmol) and NBO-1 (100 mg, 0.213 mmol) were dissolved in 20 ml of methanol, stirred at room temperature for 12 h, after the end of the extraction, the solvent was removed by distillation under reduced pressure and column chromatography, eluent ratio of 100-20: 1 dichloromethane-methanol system, to obtain NBO-1-TBFB target compound, mass spectrum (HRMS) detection results are shown in the following table: Figure 5 , 6. Only the peak of the tetrafluoroboric acid ion in the negative mode (C24BF20- [M-] is 678.9779, actually measured 678.9780), no peak of perchlorate ion (CIO4- [M-] is 98.9491), indicating complete ion exchange.

[0121] Example 4

[0122] This example relates to the synthesis of a Nile Blue fluorescent dye, the structure is as follows:

[0123]

[0124] Example 4.1

[0125]

[0126] 1-naphthylamine (0.5 g, 3.49 mmol) and propanesulfonic acid lactone (0.469 g, 3.84 mmol) were dissolved in n-butanol, refluxed overnight, after the reaction was completed and cooled to room temperature, the filter was washed with cold methanol to obtain the intermediate 1

[0127] Example 4.2

[0128]

[0129] Synthetic method is the same as Example 1.2.

[0130] Example 4.3

[0131]

[0132] Synthetic method is similar to Example 4.3.

[0133] Compound NBO-8-Cl

[0134]

[0135] Synthetic procedure was similar to Example 1.3. NBO-8-Cl: 1 H NMR (400 MHz, DMSO) δ 10.64 (s, 1H), 8.75 (d, J = 8.0 Hz, 1H), 8.60 (d, J = 8.2 Hz, 1H), 7.93 (t, J = 7.6 Hz, 1H), 7.82 (t, J = 8.8 Hz, 2H), 7.23 (d, J = 9.4 Hz, 1H), 7.06 (s, 1H), 6.90 (s, 1H), 3.85 (d, J = 6.7 Hz, 2H), 3.66 (q, J = 6.7 Hz, 4H), 2.70 (t, J = 6.6 Hz, 2H), 2.15 - 2.06 (m, 2H), 1.24 (t, 6H), [M+Na] of calculated molecular formula C23H25N3O4SNa+ was 462.1463; found 462.1451. See Figure 1 for detailed NMR data. Figure 7 HRMS results were shown in Figure 2, which indicated the correct molecular structure. Figure 8

[0136] Example 5

[0137] In this example, the absorption and emission spectra of the reported fluorescent dye NBO and the fluorescent dyes of the present application (NBO-1-C104, NBO-2-C104, NBO-1-TBFB and NBO-8-Cl) in solution were tested.

[0138] (1) The photophysical properties of NBO-1-C104, NBO-2-C104, NBO-1-TBFB, NBO-8-Cl and NBO were investigated by UV-Vis absorption and fluorescence emission spectra, with the fluorescent dyes diluted in dichloromethane (5 μΜ). As shown in Figure 3(a), the absorption peaks of NBO-1-C104, NBO-2-C104, NBO-1-TBFB, NBO-8-Cl and NBO in dichloromethane solution were obviously at 667, 663, 667, 632 and 628 nm, respectively. Figure 9 The fluorescence properties of NBO-1-C104, NBO-2-C104, NBO-1-TBFB, NBO-8-Cl and NBO in dichloromethane solution were also studied, as shown in Figure 3(b). The fluorescence peaks of NBO-1-C104, NBO-2-C104, NBO-1-TBFB, NBO-8-Cl and NBO in dichloromethane solution were at 680, 676, 680, 650 and 646 nm, respectively. Figure 9 ​As shown in (b), NBO exhibits the strongest red fluorescence in organic solvents, while the photoluminescence intensity (PL) of NBO-1-ClO4, NBO-2-ClO4, and NBO-1-TBFB in organic solvents is lower than that of NBO, although the wavelength is longer. However, the PL intensity of NBO-8-Cl in DCM is 1.28 times that of the previously reported fluorescent dye NBO, indicating that the introduction of appropriate sulfonic acid groups can enhance the PL intensity of the molecule in lipids, which is beneficial for lipid imaging (the excitation wavelengths in the fluorescence tests are all the maximum absorption wavelength of each dye minus 30 nm).

[0139] (2) The fluorescent dyes were diluted in PBS (5 μM), and the photophysical properties of NBO-1-ClO4, NBO-2-ClO4, NBO-1-TBFB, NBO-8-Cl, and NBO were investigated using UV-Vis absorption and fluorescence emission spectroscopy. Figure 10 As shown in (a), the absorption peaks of NBO-1-ClO4, NBO-2-ClO4, NBO-1-TBFB, NBO-8-Cl, and NBO in PBS solution are clearly observed at 664, 660, 618, 644, and 640 nm, respectively. This invention also investigates their fluorescence properties in PBS solution, such as... Figure 10 As shown in (b) above. Surprisingly, NBO-2-ClO4 exhibited the strongest red fluorescence in PBS, with a photoluminescence intensity 1.31 times that of the previously reported fluorescent dye NBO. The fluorescence of NBO-1-ClO4 was significantly weaker than that of NBO, indicating that the introduction of the four methyl groups inhibited molecular aggregation in PBS and increased the fluorescence intensity. Furthermore, the fluorescence of NBP-1-TBFB almost disappeared, presumably due to energy transfer between the tetrafluorophenylboronic acid ion and the dye, leading to fluorescence quenching. This suggests that not all anions are beneficial for increasing the photoluminescence intensity of the molecule. The fluorescence of NBO-8-Cl was slightly weaker than that of NBO, indicating that adjusting the position of the sulfonic acid group can regulate the molecular fluorescence intensity (the excitation wavelengths in the fluorescence tests were all the maximum absorption wavelengths of each dye minus 30 nm).

[0140] The above embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A class of Nile blue fluorescent dyes, characterized in that: Its absorption is located in the red light band of 620nm-750nm, and its structural formula is as shown in general formula I: R1 is selected from one of diethylamino, octahydro-4H-quinolinazinyl, 1,1,7,7-tetramethyloctahydro-4H-quinolinazin-4-yl, aziridine, aziridine, N-methylpiperazinyl, N,N-dimethylpiperazinyl, 1,4-diethylpiperazinyl, and N,N-propanesulfonic acid ethylamino. R2 is a C1-4 alkyl or C1-4 alkylsulfonyl group; wherein, the C1-4 alkylsulfonyl group includes methanesulfonic acid group, ethanesulfonic acid group, propanesulfonic acid group and butanesulfonic acid group; The X - It is selected from one of chloride ion, bromide ion, iodide ion, perchlorate ion, hexafluorophosphate ion, hexafluoroantimonate ion, boron tetrafluoride ion, tetra(4-fluorophenyl)boronic acid anion, tetrapentafluorophenylboronic acid ion, tetra[3,5-bis(trifluoromethyl)phenyl]boronic acid anion and Δ-tris(tetrachloro-1,2-phenylenediol)phosphate (V) anion.

2. The method for preparing near-infrared Nile blue fluorescent dyes according to claim 1, characterized in that: Includes the following steps: When R2 is a C1-4 alkyl group First, intermediate 1 is synthesized by reacting C1-4 iodoalkanes with 1-naphthylamine. Then, a diazonium salt is introduced into p-nitroaniline to synthesize intermediate 2. Intermediate 3 is then synthesized by reacting intermediate 1 with intermediate 2. Next, an aniline compound containing an R1 group and intermediate 3 are dissolved in anhydrous DMF, perchloric acid is added, and the mixture is heated to a certain temperature and stirred under inert gas protection. Finally, the intermediate is obtained by ion exchange with different anions X-. When R2 is a C1-4 alkylsulfonyl group; The synthetic route for this type of fluorescent dye consists of only five steps: introducing a C1-4 alkylsulfonyl group onto 1-naphthylamine to synthesize intermediate 1; introducing a diazonium salt into p-nitroaniline to synthesize intermediate 2; reacting intermediate 1 with intermediate 2 to synthesize intermediate 3; then dissolving an aniline compound containing an R1 group and intermediate 3 in anhydrous DMF, adding perchloric acid and heating to a certain temperature, and finally obtaining the product through ion exchange with different anions X-.

3. The preparation method according to claim 2, characterized in that: (1) When R2 is a C1-4 alkyl group, the steps include: 1) Mix 1-naphthylamine, potassium carbonate and C1-4 iodoalkane in ethanol, heat the mixture to 80°C and stir; after the reaction is complete, remove the solvent by vacuum distillation and perform column chromatography to obtain intermediate 1. 2) Mix 4-nitroaniline dissolved in propionic acid / acetic acid / DMF with concentrated hydrochloric acid; heat the mixture to 70°C, then cool it to 0°C; add sodium nitrite aqueous solution dropwise to the mixture; after the addition is complete, stir the system at 0°C; after the reaction is complete, a solution of intermediate 2 is obtained; 3) Add the DMF solution containing intermediate 1 dropwise to the solution of intermediate 2. After the addition is complete, mix the system at 0°C and then heat it to room temperature. After the reaction is complete, alkalize the solution with ammonia. Filter to obtain solid intermediate 3. 4) Dissolve intermediate 3 and aniline compounds containing R1 groups in anhydrous DMF, add perchloric acid, heat and stir, and after the reaction is completed and cooled, filter, remove the solvent by vacuum distillation, and perform column chromatography to obtain the target compound with perchlorate anion. 5) Containing different X - The target compound, whose salt and anion are perchlorate or chloride ions, was dissolved in methanol. The mixture was stirred at room temperature, and after stirring, filtered under vacuum, the solvent was removed by distillation, and column chromatography was performed to obtain the anion X. - The target compound.

4. The preparation method according to claim 3, characterized in that: The aforementioned containing different X - The salt is selected from one of sodium chloride, sodium bromide, sodium iodide, sodium perchlorate, sodium hexafluorophosphate, sodium hexafluoroantimonate, sodium boron tetrafluoride, lithium tetra(4-fluorophenyl)borate, lithium tetrapentafluorophenylborate, lithium tetra[3,5-bis(trifluoromethyl)phenyl]borate, and sodium Δ-tris(tetrachloro-1,2-phenylenediol)phosphate (V).

5. The preparation method according to claim 3, characterized in that: In step 1), the molar ratio of 1-naphthylamine, iodoalkane, and potassium carbonate is (0.8-1.2):(0.8-1.2):(2-4). In step 2), the volume ratio of propionic acid / acetic acid / DMF is (0.8-1.2):(4-6):(3-5); the molar ratio of 4-nitroaniline, concentrated hydrochloric acid, and nitrous acid is (0.8-1.2):(15-25):(0.8-1.5). In step 3), the molar ratio of N-ethylnaphthylamine to intermediate 1 is (0.8-1.2):(2-4); In step 4), the molar ratio of intermediate 2, the aniline compound containing the R1 group, and perchloric acid is (0.8-1.2):(0.8-1.2):(15-30). Step 5) The target compound with perchlorate ion as the anion is different from those containing different X. - The molar ratio of the salts is (0.8-1.2):(5-15); more preferably 1:

10. For the technical solution described above, a further preferred embodiment is that the eluent for column chromatography separation in step 5) is a dichloromethane-methanol system with a volume ratio of 20-100:

1.

6. The preparation method according to claim 2, characterized in that: (2) When R2 = C1-4 alkylsulfonyl group, the steps include: 1) Dissolve 1-naphthylamine and C1-4 sulfonyl lactone in n-butanol, reflux, and after the reaction is completed and cooled, filter and wash with cold methanol to obtain intermediate 1; 2) Mix 4-nitroaniline dissolved in propionic acid / acetic acid / DMF with concentrated hydrochloric acid; heat the mixture to 70°C, then cool it to 0°C; add sodium nitrite aqueous solution dropwise to the mixture; after the addition is complete, stir the system at 0°C; after the reaction is complete, a solution of intermediate 1 is obtained; 3) The DMF solution containing N-ethylnaphthylamine was added dropwise to the intermediate 2 solution. After the addition was complete, the system was stirred at 0°C for a certain period of time, then heated to room temperature and stirred. After the reaction was completed, the solution was alkalized with ammonia. The solid intermediate 3 was obtained by filtration. 4) Dissolve intermediate 3 and aniline compounds containing R1 groups in anhydrous DMF, add perchloric acid, heat to a certain temperature and stir, after the reaction is completed and cooled, filter, remove solvent by vacuum distillation, and then perform column chromatography to obtain the target compound with perchlorate ions as anions. 5) Containing different X - The target compound, whose salt and anion is perchlorate ion, was dissolved in methanol. The mixture was stirred at room temperature, and after stirring, the solvent was removed by filtration and vacuum distillation. Column chromatography was then performed to obtain the anion X. - The target compound.

7. The preparation method according to claim 6, characterized in that: The aforementioned containing different X - The salt is selected from one of sodium chloride, sodium bromide, sodium iodide, sodium perchlorate, sodium hexafluorophosphate, sodium hexafluoroantimonate, sodium borofluoride, sodium o-carborane, sodium ticarborane cobalt anion, lithium tetra(4-fluorophenyl)borate, lithium tetrapentafluorophenylborate, lithium tetra[3,5-bis(trifluoromethyl)phenyl]borate, and sodium Δ-tris(tetrachloro-1,2-phenylenediol)phosphate (V).

8. The preparation method according to claim 6, characterized in that: In step 1), the molar ratio of 1-naphthylamine to the corresponding sulfonyl lactone is (0.8-1.2):(0.7-1.5); In step 2), the volume ratio of propionic acid / acetic acid / DMF is (0.8-1.2):(4-6):(3-5); the molar ratio of 4-nitroaniline, concentrated hydrochloric acid, and nitrous acid is (0.8-1.2):(15-25):(0.8-1.5). In step 3), the molar ratio of N-ethylnaphthylamine to intermediate 1 is (0.8-1.2):(2-4); In step 4), the molar ratio of intermediate 2, the aniline compound containing the R1 group, and perchloric acid is (0.8-1.2):(0.8-1.2):(15-30). The reaction temperature range in step 4) is 120-150℃; Step 5) The target compound with perchlorate ion as the anion is different from those containing different X. - The molar ratio of the salts is (0.8-1.2):(5-15).

9. The use of the dye as described in claim 1 in the preparation of optical detection reagents for non-diagnostic and therapeutic purposes.

10. The application according to claim 9, characterized in that: This includes applications in the preparation of reagents for in vivo imaging.

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