N-ethyl carbazole derivative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of fluorescence sensing, and particularly discloses an N-ethyl carbazole derivative as well as a preparation method and application thereof. On the basis of a'double key-lock 'design strategy, the N-ethyl carbazole derivative provided by the invention is used as a fluorescent probe, on one hand, the N-ethyl carbazole derivative has good response to a viscosity environment under an acidic condition, and quantitative detection of viscosity can be realized; and on the other hand, the probe shows good selectivity and sensitivity on viscosity recognition under the acidic condition, is not easily interfered by other potential interferents such as anions, cations, biomolecules and other common active substances in the viscosity detection process, and has the advantages of high sensitivity and high selectivity on viscosity detection. The N-ethyl carbazole derivative can be used as a sensitive off-on viscosity fluorescent probe, and has an extremely important application value in the fields of biosensors, fluorescent probes, fluorescence imaging, food detection, organic light-emitting devices and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence sensing, and particularly relates to N-ethylcarbazole derivatives, their preparation methods and applications. Background Art

[0002] Viscosity, as an important physical and chemical parameter in organisms, has a significant impact on signal transmission, substance diffusion, and the speed of interactions between biomolecules. The execution of cell functions such as apoptosis, autophagy, ferroptosis, and oxidative stress is closely related to cell viscosity. Since there are differences in the viscosities of organelles in different regions within biological cells, changes in cell viscosity will directly affect the functions of these organelles. When cell viscosity changes, it may interfere with key biochemical reactions such as material transport, signal transduction, and metabolic processes within the cell, thereby leading to abnormal cell functions. Therefore, monitoring changes in organelle viscosity in the cellular microenvironment is of great significance. Developing viscosity detection probes helps to clarify the mechanisms of related diseases caused by abnormal organelle viscosity and further understand cell functions.

[0003] Traditional methods for detecting viscosity mainly include capillary viscometers, rotational viscometers, electrochemical analysis methods, etc., but these methods cannot be used for viscosity detection in organisms. Fluorescent probe technology has always been regarded as a powerful tool for sensing and imaging in organisms due to its advantages such as high sensitivity, strong specificity, non-destructive imaging, and high spatio-temporal resolution. The design method of viscosity fluorescent probes generally follows the principle of "fluorescent molecular rotors". Its design usually incorporates a fluorophore containing an electron donor and an acceptor, as well as a rotatable part. When the viscosity of the environment is low, the rapid rotation of the rotor causes energy loss through non-radiative transitions, resulting in weak fluorescence or even quenching. When the viscosity of the environment increases, the viscosity hinders the normal rotation of the rotor, and the energy is released in a radiative manner and shows an increase in fluorescence. Currently reported viscosity detection probes are basically single response sites, and are easily affected by complex biological environments during detection and undergo non-specific activation, resulting in false positive results. Summary of the Invention

[0004] The purpose of the present invention is to provide, in view of the above deficiencies of the prior art, N-ethylcarbazole derivatives, their preparation methods and applications. This compound has good selectivity and high sensitivity to viscosity under acidic conditions, and at the same time has significant imaging ability in the tumor microenvironment with a high viscosity expression, and is expected to achieve the diagnosis and treatment of related diseases by monitoring viscosity concentration.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions: The first aspect of the present invention is to provide an N-ethylcarbazole derivative, and the structural formula of the N-ethylcarbazole derivative is shown as Formula I or Formula II, Formula I; or Formula II.

[0006] The second aspect of the present invention is to provide a method for preparing the above-mentioned N-ethylcarbazole derivative, comprising the following steps: S1. Under the protection of an inert gas, piperidine is added to a mixed solution of 6-bromo-9-ethylcarbazole-3-carbaldehyde and benzothiazole-2-acetonitrile, and a first heating reaction is carried out. After the reaction is completed, vacuum drying is carried out, and the crude product is purified and separated to obtain compound NEI-1; S2. Compound NEI-1 and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester are dissolved in a mixed solution of an organic solvent and H2O, and then potassium carbonate and Pd(dppf)Cl2 are added. Under an inert atmosphere, a second heating reaction is carried out. After completion, filtration is carried out, the filtrate is extracted multiple times, the organic phases are combined, dried over anhydrous sodium sulfate, the organic solvent is removed under reduced pressure, and the crude product is purified and separated to obtain compound NEI-2.

[0007] Furthermore, in step S1, the molar ratio of 6-bromo-9-ethylcarbazole-3-carbaldehyde to benzothiazole-2-acetonitrile is 10:1 to 1:10; the molar ratio of 6-bromo-9-ethylcarbazole-3-carbaldehyde to piperidine is 50:1 to 1:20.

[0008] Furthermore, both 6-bromo-9-ethylcarbazole-3-carbaldehyde and benzothiazole-2-acetonitrile are dissolved in an organic solvent, and the organic solvent is at least one of acetonitrile, methanol, ethanol, N,N-dimethylformamide, and N,N-dimethylacetamide; the temperature of the first heating reaction is 40~150 °C, and the time is 4~48 h.

[0009] Furthermore, in step S3, the molar ratio of compound NEI-1 to 4-(N,N-dimethylamino)phenylboronic acid pinacol ester is 1:1 to 1:25; the molar ratio of compound NEI-1 to potassium carbonate is 20:1 to 1:20; the molar ratio of compound NEI-1 to Pd(dppf)Cl2 is 40:1 to 1:10.

[0010] Furthermore, both compound NEI-1 and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester are dissolved in a mixed solution of an organic solvent and water, and the organic solvent is at least one of 1,4-dioxane, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide; the temperature of the second heating reaction is 20~200 °C, and the time is 2~48 h.

[0011] Furthermore, purification and separation are carried out by column chromatography.

[0012] The third aspect of the present invention is to provide the application of the above-mentioned N-ethylcarbazole derivative in detecting viscosity under acidic conditions, where the pH of the acidic condition is 2-6.

[0013] The fourth aspect of the present invention is to provide a viscosity fluorescent probe containing the N-ethylcarbazole derivative described in claim 1, and the viscosity fluorescent probe is used for cell imaging.

[0014] Furthermore, the detection solvent system of the viscosity fluorescent probe is selected from any one of alcohol-aqueous solution, acetonitrile-aqueous solution or dimethyl sulfoxide-aqueous solution.

[0015] Compared with the prior art, the beneficial effects brought by the technical solution provided by the present invention are as follows: (1) Based on the "double-key-lock" design strategy, the present invention provides an N-ethylcarbazole derivative. As a fluorescent probe, on the one hand, it has a good response to the viscosity environment under acidic conditions and can realize the quantitative detection of viscosity; on the other hand, the probe shows good selectivity and sensitivity in the recognition of viscosity under acidic conditions and is not easily interfered by other potential interfering substances such as anions, cations, biomolecules and other common active substances during the viscosity detection process, and has the advantages of high sensitivity and high selectivity for viscosity detection.

[0016] (2) The preparation method provided by the present invention is simple, and the prepared N-ethylcarbazole derivative can be used as a sensitive "turn-off-turn-on" type viscosity fluorescent probe, which has extremely important application values in the fields of biosensors, fluorescent probes, fluorescence imaging, food detection, organic light-emitting devices, etc.

[0017] (3) The N-ethylcarbazole derivative NEI-2 provided by the present invention has good cell permeability and biocompatibility as a "turn-off-turn-on" type fluorescent probe, can be applied to the field of cell imaging, has the ability to target lysosomes, and can efficiently and accurately realize the viscosity detection in biomedical and life systems.

[0018] (4) The N-ethylcarbazole derivative provided by the present invention can be used as a sensitive "turn-off-turn-on" type viscosity fluorescent probe, which has extremely important application values in the fields of biosensors, fluorescent probes, fluorescence imaging, food detection, organic light-emitting devices, etc. Description of the Drawings

[0019] Figure 1 It is a graph showing the relationship between the fluorescence intensity and pH of the N-ethylcarbazole derivative NEI-2 provided by the present invention in a viscosity environment; Figure 2 It is a fluorescence spectrum diagram of 0% Gly and 95% Gly of the N-ethylcarbazole derivative NEI-2 provided by the present invention under different pH conditions; Figure 3Fluorescence spectra of the N-ethylcarbazole derivative NEI-2 provided by the present invention in a glycerol / water (pH = 4) system at different viscosities; Figure 4 Log(I 532 ) vs. Log(viscosity) linear relationship diagram of the N-ethylcarbazole derivative NEI-2 provided by the present invention in a glycerol / water (pH = 4) system with different volume ratios; Figure 5 Fluorescence response selectivity experiment of the N-ethylcarbazole derivative NEI-2 to viscosity and other related biological small molecules at 532 nm, where (1) 1 O2; (2) Cys; (3) GSH; (4) H2O2; (5) NO; (6) •OH; (7) ONOO; (8) NaClO; (9) Al 3+ ; (10) Ca 2+ ; (11) Cu 2+ ; (12) Fe 3+ ; (13) Mg 2+ ; (14) K + ; (15) Na + ; (16) Zn 2+ ; (17) NO3 - ; (18) SO3 2- ; (19) HSO3 - ; (20) SO4 2- ; (21) F - ; (22) Gly; Figure 6 Anti-interference experiment of the fluorescence response of the N-ethylcarbazole derivative NEI-2 to viscosity and other related biological small molecules at 532 nm, where (1) 1 O2; (2) Cys; (3) GSH; (4) H2O2; (5) NO; (6) •OH; (7) ONOO; (8) NaClO; (9) Al 3+ ; (10) Ca 2+ ; (11) Cu 2+ ; (12) Fe 3+ ; (13) Mg 2+ ; (14) K + ; (15) Na + ; (16) Zn 2+ ; (17) NO3 -； (18) SO3 2- ; (19) HSO3 - ; (20) SO4 2- ; (21) F - ; Figure 7 Cell imaging and cell localization map of N-ethylcarbazole derivative NEI-2. Detailed implementation manners To make the objectives, technical solutions and advantages of the present invention clearer, the following further describes in detail the specific implementation manners of the present invention with reference to specific embodiments and the accompanying drawings. For the embodiments where specific test methods, instrument devices or conditions are not specified, they are all carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0020] The synthesis route of N-ethylcarbazole derivative NEI-1 provided by the present invention is as follows: , Compound 1 is 6-bromo-9-ethylcarbazole-3-carbaldehyde, CAS: 24301-72-2; Compound 1 can be obtained commercially or synthesized by itself. The specific synthesis process is to slowly drop POCl3 into 6-bromo-9-ethylcarbazole. After stirring and reacting for a period of time, the reaction solution is poured into a large amount of ice-water bath, and the pH of the system is adjusted to neutral with an aqueous sodium carbonate solution, followed by suction filtration, washing with water, and the crude product is purified and separated by column chromatography to obtain Compound 1. Both 6-bromo-9-ethylcarbazole and POCl3 are dissolved in an organic solvent, and the organic solvent is at least one of N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, dimethyl sulfoxide, methanol, and ethanol. The molar ratio of 6-bromo-9-ethylcarbazole to POCl3 is 10:1 to 1:10, the stirring temperature is -20 to 50 °C, and the time is 0.5 to 48 h.

[0021] It should be noted that the molar ratio of 6-bromo-9-ethylcarbazole to POCl3 is 10:1 to 1:10, including but not limited to 9:2, 8:3, 7:4, 6:5, 5:5, 5:6, 4:7, 3:8, 2:9, 1:10. At the same time, it should include but not limited to a specific value included between any two of the above values, such as but not limited to 4:3 included between 7:4 and 6:5, etc., and will not be elaborated one by one.

[0022] It should also be noted that the stirring temperature can be any temperature between -20 °C and 50 °C; the reaction time can be determined according to whether the materials react completely to obtain the optimal time, and the time is at least in the range of 0.5 to 48 h.

[0023] Compound 6-bromo-9-ethylcarbazole-3-carbaldehyde can be prepared by adopting the above synthesis method.

[0024] The specific process for synthesizing the compound NEI-1 is as follows: Under the protection of an inert gas, piperidine is added to a mixed solution of compound 1 and benzothiazole-2-acetonitrile (CAS: 56278-50-3), and the mixture is heated for reaction. After the reaction is completed, the organic solvent is removed under vacuum, and the crude product is purified and separated by column chromatography to obtain compound NEI-1. Both compound 1 and benzothiazole-2-acetonitrile are dissolved in an organic solvent, and the organic solvent is at least one of acetonitrile, methanol, ethanol, N,N-dimethylformamide, N,N-dimethylacetamide. The molar ratio of compound 1 to piperidine is 50:1 to 1:20, the reaction temperature is 40 to 150 °C, and the time is 4 to 48 h.

[0025] It should be noted that the molar ratio of compound 1 to piperidine is 50:1 to 1:20, including but not limited to 45:1, 30:1, 20:1, 10:1, 5:1, 1:5, 1:10, 1:15, 1:20. At the same time, it should include but not be limited to specific values included between any two of the above values, such as but not limited to 1:1 included between 5:1 and 1:5, etc., and will not be elaborated one by one.

[0026] It should also be noted that the reaction temperature can be any temperature between 40 and 150 °C; the reaction time can be determined according to whether the materials have reacted completely to determine the optimal time, and the time is at least in the range of 4 to 48 h.

[0027] Compound NEI-1 can be prepared by using the above synthesis method.

[0028] The synthesis route of the N-ethylcarbazole derivative NEI-2 provided by the present invention is as follows: , The specific process for synthesizing the compound NEI-2 is as follows: Compound NEI-1 and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester (CAS: 171364-78-6) are dissolved in a mixed solution of an organic solvent and H2O, and then potassium carbonate and Pd(dppf)Cl2 are added. Under an inert atmosphere, the mixture is heated for reaction. After completion, it is filtered, the filtrate is extracted multiple times, the organic phases are combined, dried over anhydrous sodium sulfate, the organic solvent is removed under reduced pressure, and the crude product is purified and separated by column chromatography to obtain compound NEI-2. Both compound NEI-1 and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester are dissolved in a mixed solution of an organic solvent and water, and the organic solvent is at least one of 1,4-dioxane, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide. The molar ratio of compound NEI-1 to potassium carbonate is 20:1 to 1:20, the molar ratio of compound NEI-1 to Pd(dppf)Cl2 is 40:1 to 1:10, the reaction temperature is 20 to 200 °C, and the time is 2 to 48 h.

[0029] It should be noted that the molar ratio of compound NEI-1 to potassium carbonate is 20:1 to 1:20, including but not limited to 20:1, 10:1, 5:1, 1:5, 1:10, 1:15, 1:20. At the same time, it should include but not be limited to specific values included between any two of the above values. For example, but not limited to 0.44:1.1 included between 2:1 and 1:2, etc., which will not be elaborated one by one.

[0030] It should also be noted that the molar ratio of compound NEI-1 to Pd(dppf)Cl2 is 40:1 to 1:10, including but not limited to 39:1, 38:1, 35:1, 30:1, 20:1, 10:1, 5:1, 1:5, 1:10, 1:15, 1:20. At the same time, it should include but not be limited to specific values included between any two of the above values. For example, but not limited to 3.5:0.14 included between 5:1 and 1:5, etc., which will not be elaborated one by one.

[0031] It should also be noted that the reaction temperature can be any temperature between 20 and 200 °C; the reaction time can be determined according to whether the materials react completely to determine the optimal time, and the time is at least in the range of 2 to 48 h.

[0032] Compound NEI-2 can be prepared by adopting the above synthesis method.

[0033] The following takes specific examples as representatives to elaborate on the synthesis methods of compounds NEI-1 and NEI-2 provided by the present invention.

[0034] Example 1 The present example provides a preparation method of compounds NEI-1 and NEI-2, which is specifically as follows: (1) Under ice bath conditions, dissolve 6-bromo-9-ethylcarbazole (1.1 g, 4 mmol) in 10 mL of DMF. Under stirring, slowly dropwise add POCl3 (3.0 eq.). During the reaction process, control the temperature at 0 °C, and then gradually raise the temperature to room temperature for reaction. After the reaction is completed, pour the reaction solution into a large amount of ice water bath. A pale yellow solid precipitates. Under stirring, slowly add saturated sodium carbonate aqueous solution to adjust the pH of the system to neutral. Filter by suction, wash with water, and dry under vacuum. The crude product is separated and purified by a medium-pressure chromatography column with ethyl acetate / petroleum ether with a volume ratio of 3:1 as the eluent to obtain product compound 1, and the yield is 80%.

[0035] The results of 1H NMR for structure confirmation are as follows: 11H NMR (400 MHz, CDCl3, δ ppm): 9.88 (s, 1H), 8.85(s, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.34 (m, 2H),7.60 (d, J = 8.0 Hz, 1H), 4.53 (m, 2H), 1.37 (t, J = 8.0 Hz, 3H).

[0036] (2) Dissolve compound 1 (600 mg, 2 mmol) and benzothiazole-2-acetonitrile (248 mg, 2 mmol) in 10 mL of acetonitrile. Under nitrogen protection, add a catalytic amount of piperidine (30 μL), and react overnight under reflux at 90 °C. After the reaction is completed, cool to room temperature, remove the organic solvent under vacuum. The crude product is separated and purified by medium-pressure chromatography column with dichloromethane / petroleum ether with a volume ratio of 5:1 as the eluent to obtain a yellow solid powder, which is compound NEI-1, and the yield is 65%.

[0037] The results of 1H NMR for structure confirmation are as follows: 1 1H NMR (400 MHz, CDCl3, δ ppm): 8.34 (s, 1H),8.18(d, J = 8.0 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.58(d, J = 8.0 Hz, 1H), 7.38 (m, 3H),7.53 (m, 2H), 7.62 (m, 1H), 4.51 (m, 2H),1.34 (t, J = 8.0 Hz, 3H).

[0038] (3) Dissolve compound NEI-1 (200 mg, 0.44 mmol) and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester (271 mg, 1.1 mmol) in a mixed solution of 1,4-dioxane and H2O (4:1, 5 mL). Add potassium carbonate (151 mg, 1.1 mmol) and Pd(dppf)Cl2 (16 mg, 0.022 mmol) respectively. Under nitrogen protection, reflux at 100 °C for 8 hours. After the reaction is completed, filter the reaction solution, extract it with dichloromethane (50 mL) three times, combine the organic phases, dry over anhydrous sodium sulfate, remove the organic solvent under reduced pressure. The crude product is separated and purified by medium-pressure chromatography column with dichloromethane / methanol with a volume ratio of 10:1 as the eluent to obtain an orange solid powder, which is compound NEI-2, and the yield is 80%.

[0039] The results of the hydrogen spectrum for structural confirmation are as follows: 1 H NMR (400 MHz, d6-DMSO, δ ppm): 8.97-8.96 (d, J = 4.0, 1 H), 8.51 (s, 1 H), 8.39-8.36 (m, 2 H), 8.19-8.17 (d, J = 8.0, 1 H), 8.09-8.07 (d, J = 8.0, 1 H), 7.86-7.84 (d, J = 8.0, 1 H,), 7.79-7.77 (m, 1 H), 7.74-7.71 (m, 1 H), 7.64-7.51 (m, 3 H), 7.53-7.48 (m, 1 H), 6.88-8.85 (d, J = 12.0, 2 H), 4.60-4.56 (dd, J1 = J2 = 8.0, 2 H), 2.96 (s, 6H), 1.38 (t, J = 8.0, 3H).

[0040] The results of the carbon spectrum are as follows: 13 C NMR (400 MHz, d6-DMSO, δ ppm): 164.62, 153.55, 149.95, 149.64, 142.64, 139.49, 134.54, 127.69, 127.47, 126.34, 125.92, 123.55, 123.34, 123.26, 122.83, 117.76, 117.72, 113.39, 100.89, 40.66, 14.35.

[0041] The organic compound of the N-ethylcarbazole derivative prepared in Example 1 was subjected to the tests of Examples 2-6, and the specific data and analysis are as follows.

[0042] Example 2 This example provides an application of an N-ethylcarbazole derivative, specifically the fluorescence responsiveness of NEI-2 to viscosity under different pH conditions.

[0043] A series of solutions with pH values ranging from 2 to 12 were accurately prepared using a pH meter. Subsequently, different pH mixed solutions containing 95% glycerol were prepared. Finally, a stock solution of the organic compound NEI-2 of the N-ethylcarbazole derivative prepared in Example 1 was added to make its final concentration 10 μM. In addition, different pH mixed solutions with a final concentration of 10 μM of NEI-2 were also prepared separately. After mixing evenly, the fluorescence emission spectra were measured in a fluorescence emission spectrometer with a fluorescence excitation wavelength of 440 nm and a slit of 10 / 10 nm, and the test results are as Figure 1 and Figure 2 shown.

[0044] It can be seen from Figure 1 that under the viscosity environment, the fluorescence intensity of the probe is closely related to pH. The fluorescence increases significantly in the acidic range, and the fluorescence is the strongest when pH = 4. When pH is in the neutral or alkaline condition, there is almost no fluorescence. The change of light can also be clearly seen under a 365 nm ultraviolet lamp. In addition, further research found that in the absence of glycerol, as Figure 2 shown, the fluorescence intensity of 0% glycerol is weak under different pH conditions. The above results further confirm that the N-ethylcarbazole derivative NEI-2 is a fluorescence probe with dual responses to viscosity and pH. Similar effects can also be achieved using the N-ethylcarbazole derivative NEI-1.

[0045] Example 3 This example provides an application of the N-ethylcarbazole derivative, specifically the fluorescence response detection of NEI-2 to viscosity under acidic conditions.

[0046] An aqueous solution with a pH of 4 was mixed with glycerol. In this mixed system, the volume ratio of glycerol was gradually increased from 0% to 95%. Subsequently, the organic compound NEI-2 of the N-ethylcarbazole derivative prepared in Example 1 was added to these mixed systems with different viscosities at a final concentration of 10 μM and mixed thoroughly. Fluorescence spectra were scanned in a fluorescence emission spectrometer with a fluorescence excitation wavelength of 440 nm and a slit of 10 / 10 nm, and the fluorescence spectra of different viscosity systems are as Figure 3 shown.

[0047] It can be seen from Figure 3 that when the volume ratio of glycerol in the system gradually increases, the fluorescence intensity of NEI-2 shows a significant increasing trend, which provides important information about the viscosity sensitivity of the probe. When the glycerol volume ratio is 95%, the fluorescence intensity at 532 nm increases by about 163 times. Under a 365 nm ultraviolet lamp, it can be seen that when the viscosity is large, the fluorescence of the probe changes from colorless to bright yellow light. As Figure 4 known, LogI532 There is a good linear relationship with Log(viscosity), and the linear equation is Y = -0.3801 + 1.5081X (R 2 = 0.9941). Among them, Y represents LogI 532 , and X represents Log(viscosity). The above data indicate that the N-ethylcarbazole derivative NEI-2 is a fluorescent probe capable of quantitatively detecting viscosity.

[0048] Example 4 This example provides an application of an N-ethylcarbazole derivative, specifically the specific detection of viscosity by NEI-2 under acidic conditions.

[0049] Add the organic compound NEI-2 of the N-ethylcarbazole derivative prepared in Example 1 to an aqueous solution with a pH of 4 so that the final concentration of NEI-2 is 10 μM. Subsequently, add other potential interferents to this solution, such as reactive species ( 1 O 2、 Cys, GSH, H2O 2、 NO, •OH, ONOO, NaClO), cations (Al 3+ , Ca 2+ , Cu 2+ , Fe 3+ , Mg 2+ , K + , Na + , Zn 2+ ), anions (NO3 - , SO3 2- , HSO3 - , SO4 2- , F - ). After thoroughly shaking and mixing at room temperature, perform fluorescence spectral scanning in a fluorescence emission spectrometer with a fluorescence excitation wavelength of 440 nm and a slit of 10 / 10 nm. Subsequently, mix the aqueous solution with pH = 4 and glycerol. In this mixed system, the volume of glycerol is 95%. Add the organic compound NEI-2 of the N-ethylcarbazole derivative prepared in Example 1 to this mixed system at a final concentration of 10 μM. Then add the above interferents respectively. After thoroughly shaking and mixing at room temperature, perform fluorescence spectral scanning in a fluorescence emission spectrometer with a fluorescence excitation wavelength of 440 nm and a slit of 10 / 10 nm.

[0050] It can be seen from Figure 5 that in (1) 1 O2; (2) Cys; (3) GSH; (4) H2O2; (5) NO; (6) -OH; (7) ONOO; (8) NaClO; (9) Al 3+ ; (10) Ca2+ ; (11) Cu 2+ ; (12) Fe 3+ ; (13) Mg 2+ ; (14) K + ; (15) Na + ; (16) Zn 2+ ; (17) NO3 - ; (18) SO3 2- ; (19) HSO3 - ; (20) SO4 2- ; (21) F - In the presence of the relevant small molecules, the organic compound NEI-2 of N-ethylcarbazole derivative does not show fluorescence. However, when it comes into contact with glycerol, NEI-2 shows a strong fluorescence intensity. This result indicates that the organic compound NEI-2 of N-ethylcarbazole derivative has excellent selectivity for viscosity response in an acidic environment. Subsequently, the anti-interference performance of the N-ethylcarbazole derivative NEI-2 for viscosity response was evaluated. As Figure 6 shown, it was found that in the presence of other biological small molecules, the response of NEI-2 to viscosity was hardly affected. The above results indicate that the organic compound NEI-2 of N-ethylcarbazole derivative also has excellent anti-interference performance for viscosity response under acidic conditions.

[0051] Example 5 This example provides an application of N-ethylcarbazole derivative, specifically for fluorescence imaging of Hela cells.

[0052] The organic compound NEI-2 of N-ethylcarbazole derivative prepared in Example 1 was dissolved in cell culture medium and added to the pre-cultured Hela cells. The cells were incubated in a cell incubator at 37 °C and 5% CO2 for 2 h. The cells were washed three times with cold PBS to remove the NEI-2 solution that did not enter the cells. Then, the lysosome probe LTR was added and the incubation was continued for 30 min. After washing twice with cold PBS, confocal fluorescence microscopy was immediately used to observe the fluorescence imaging inside the cells. The excitation wavelength of the probe was 450 nm, and the receiving wavelength was the green channel. The excitation wavelength of the lysosome probe was 575 nm, and the receiving wavelength was the red channel.

[0053] As Figure 7As shown, the cells treated with the N-ethylcarbazole derivative NEI-2 have obvious fluorescence. Through cell localization, it is found that the compound NEI-2 can be well localized in lysosomes. Research shows that the pH value range of lysosomes in healthy cells is 4.0 - 5.0. Compared with normal cells, the pH value of lysosomes in cancer cells is lower than that in normal cells. More importantly, cancer cells have a much higher viscosity than normal cells. It can be seen that the organic compound NEI-2 of the N-ethylcarbazole derivative can diagnose and treat diseases by monitoring the viscosity change in lysosomes, and this research plays an important role in the occurrence, development and pathological analysis of related diseases.

[0054] Without conflict, the above embodiments and the features in the embodiments in this article can be combined with each other.

[0055] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An N-ethylcarbazole derivative, characterized in that The structural formula of the N-ethylcarbazole derivative is shown in Formula I or Formula II, Formula I; or Formula II.

2. A method for preparing an N-ethylcarbazole derivative as claimed in claim 1, characterized in that: The following steps are involved: S1. Under the protection of inert gas, piperidine is added to a mixed solution of 6-bromo-9-ethylcarbazole-3-carboxaldehyde and benzothiazole-2-acetonitrile to carry out a first heating reaction. After the reaction is completed, vacuum drying is performed, and the crude product is purified and separated to obtain compound NEI-1; S2. Compound NEI-1 and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester are dissolved in a mixed solution of an organic solvent and H2O, and then potassium carbonate and Pd(dppf)Cl2 are added. A second heating reaction is carried out under an inert atmosphere. After completion, the reaction is filtered, and the filtrate is extracted multiple times. The organic phases are combined, dried over anhydrous sodium sulfate, and the organic solvent is removed under reduced pressure. The crude product is purified and separated to obtain compound NEI-2.

3. The preparation method according to claim 2, characterized in that In step S1, the molar ratio of 6-bromo-9-ethylcarbazole-3-carboxaldehyde to benzothiazole-2-acetonitrile is 10:1-1:10; the molar ratio of 6-bromo-9-ethylcarbazole-3-carboxaldehyde to piperidine is 50:1-1:

20.

4. The preparation method according to claim 3, characterized in that The 6-bromo-9-ethylcarbazole-3-carboxaldehyde and benzothiazole-2-acetonitrile are both dissolved in an organic solvent, and the organic solvent is at least one of acetonitrile, methanol, ethanol, N,N-dimethylformamide and N,N-dimethylacetamide; the temperature of the first heating reaction is 40-150° C., and the time is 4-48 h.

5. The preparation method according to claim 2, characterized in that: In step S3, the molar ratio of compound NEI-1 to 4-(N,N-dimethylamino)phenylboronic acid pinacol ester is 1:1~1:25; the molar ratio of compound NEI-1 to potassium carbonate is 20:1~1:20; and the molar ratio of compound NEI-1 to Pd(dppf)Cl2 is 40:1~1:

10.

6. The preparation method according to claim 5, characterized in that The compounds NEI-1 and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester are both dissolved in a mixed solution of an organic solvent and water, wherein the organic solvent is at least one of 1,4-dioxane, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide; the temperature of the second heating reaction is 20-200° C., and the time is 2-48 h.

7. The preparation method according to claim 4 or 6, characterized in that: Purification and separation were performed by column chromatography.

8. The use of the N-ethylcarbazole derivative for viscosity detection under acidic conditions as claimed in claim 1, characterized in that: The pH of the acidic condition is 2-6.

9. A viscosity fluorescent probe comprising the N-ethylcarbazole derivative according to claim 1, characterized in that: The viscosity fluorescent probe is used for cell imaging.

10. The viscosity fluorescent probe according to claim 9, characterized in that: The detection solvent system of the viscosity fluorescent probe is selected from any one of alcohol-water solution, acetonitrile-water solution or dimethyl sulfoxide-water solution.

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