2, 3-diphenyl quinoline compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of synthesis of organic compounds, and particularly relates to a 2, 3-diphenyl quinoline compound as well as a preparation method and application thereof. The invention relates to a 2, 3-diphenyl quinoline compound, and the structure of the 2, 3-diphenyl quinoline compound is as follows: in the formula, R1 and R2 are respectively and independently selected from H or C1-C20 alkyl. The preparation method of the 2, 3-diphenyl quinoline compound comprises the following steps: mixing o-iodoaniline, styrene, an organic catalyst and an organic solvent S1, reacting to prepare an intermediate M, and reacting the intermediate M with a compound N to obtain the 2, 3-diphenyl quinoline compound. The 2, 3-diphenyl quinoline compound disclosed by the invention has high biocompatibility, low cytotoxicity and light stability, and has a potential application prospect in cell imaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic compound synthesis, and particularly relates to a 2,3-diphenylquinoline compound, a preparation method thereof, and an application thereof. Background Art

[0002] Quinoline is an important class of nitrogen-containing aromatic heterocyclic compounds, and nitrogen heterocyclic compounds are essential for clinically important drugs and are widely used in organic synthesis. At the same time, they have extremely high application value in the fields of medicine and materials, etc.

[0003] In biochemistry and clinical diagnosis, the visualization of target molecules in vitro or in vivo has very important practical significance. At present, fluorescence imaging techniques, imaging experiments, and analysis methods related to the visualization of target molecules all need to be realized by means of fluorescent molecules, and these fluorescent molecules are required to have characteristics such as simple structure, high sensitivity, good photostability, good biocompatibility, easy synthesis, and low cost.

[0004] Lipid droplets (LDs) are membrane-bound organelles that are ubiquitous in mammals, mainly present in the cytoplasm and a small part in the nucleus. LDs play a crucial role in various cellular activities. It can be used to store lipids and prevent cellular lipid poisoning. Many diseases are also related to the dysregulation of the LD life cycle and physiological functions. Such as obesity, non-alcoholic fatty liver, atherosclerosis, lipodystrophy, etc. In addition, LDs interact with most organelles in the cell, such as the nucleus, mitochondria, and endoplasmic reticulum, etc. LDs also have important significance for maintaining the normal functions of organelles. It can protect the endoplasmic reticulum during endoplasmic reticulum stress and can also protect mitochondria from damage during autophagy. Therefore, the development of LD imaging probes in living cells and fixed cells is of great significance in cell biology and biomedical research. Summary of the Invention

[0005] The purpose of the present invention is to provide a 2,3-diphenylquinoline compound. The 2,3-diphenylquinoline compound has high biocompatibility, low cytotoxicity, and photostability, and has potential application prospects in cell imaging.

[0006] The second purpose of the present invention is to provide a preparation method of a 2,3-diphenylquinoline compound.

[0007] The third purpose of the present invention is to provide an application of a 2,3-diphenylquinoline compound.

[0008] The technical solution of the 2,3-diphenylquinoline compound of the present invention is as follows: A 2,3-diphenylquinoline compound, the structure of the 2,3-diphenylquinoline compound is as follows: Among them, R 1 and R 2 are each independently selected from H or C 1 -C 20 alkyl group.

[0009] The technical solution of the preparation method of the 2,3-diphenylquinoline compound of the present invention is as follows: A preparation method of a 2,3-diphenylquinoline compound as described in claim 1, comprising the following steps: mixing o-iodoaniline, styrene, an organic catalyst and an organic solvent S1, reacting to prepare an intermediate M, and then reacting the intermediate M with a compound N to obtain the product.

[0010] Preferably, o-iodoaniline, styrene and tetrakis(triphenylphosphine)palladium are dissolved in an organic solvent S1, heated to 60-80 °C and reacted at this temperature. After the reaction is completed, the intermediate M is obtained by separation. The organic solvent S1 is at least one of triethylamine, acetone and n-butanol.

[0011] More preferably, the molar ratio of o-iodoaniline, styrene and tetrakis(triphenylphosphine)palladium is 1:1-2:0.01-0.1.

[0012] Preferably, the intermediate M, the compound N and aluminum trichloride are dissolved in an organic solvent S2, and the reaction is carried out under an LED lamp. After the reaction is completed, the reaction is quenched and the 2,3-diphenylquinoline compound is obtained by separation.

[0013] More preferably, the structure of the compound N is as follows: Among them, R 3 and R 4 are each independently selected from H or C 1 -C 20 alkyl group.

[0014] Even more preferably, the molar ratio of the intermediate M to the compound N is 1-1.5:1, and the molar ratio of the compound N to aluminum trichloride is 1:0.1-1.5.

[0015] More preferably, the organic solvent S2 is at least one of acetonitrile, chloroform, toluene and trichloromethane.

[0016] The technical solution of the application of the 2,3-diphenylquinoline compound of the present invention is as follows: An application of a 2,3-diphenylquinoline compound in cell imaging, and the structure of the 2,3-diphenylquinoline compound is as follows: Among them, R 1 and R2 Each independently selected from H or C 1 -C 20 alkyl

[0017] Use of a 2,3-diphenylquinoline compound as described in the above structural formula and preparation method as a fluorescent probe for lipid droplets

[0018] Beneficial effects: The preparation steps of this compound are simple, and it has the advantages of high biocompatibility, low cytotoxicity, rapid staining and good photostability. It can specifically target lipid droplets and has good color development performance, and can be used as a fluorescent probe for lipid droplets in biomedical research Description of the drawings

[0019] Figure 1 Is the UV / visible absorption spectrum of the compound 2-(4-N,N'-dimethyl-phenyl)-3-phenyl-quinoline (Compound Q-NMe 2 ) prepared in Example 2, the solvent is ethyl acetate, the concentration is 10 uM, and the scanning wavelength is 250-500 nm Figure 2 Is the fluorescence spectrum of the compound Q-NMe prepared in Example 2 2 , the solvent is ethyl acetate, the concentration is 10 uM, λex = 360 nm (excitation light wavelength), silt width: 5 nm / 5nm Figure 3 In, a and b are the cell viabilities of LO2 cells and Hela cells after exposure to the compound Q-NMe at concentrations of 0, 1, 5, 10 and 20 μM 2 for 24 h Figure 4 Is the fluorescence image (a), bright field image (b), and merged image (c) of the compound Q-NMe of LO2 cells without oleic acid induction 2 ; Figure 5 Is the Oil Red O staining image of LO2 cells after incubation with 250 μM oleic acid for 24 h Figure 6 Is the lipid droplet co-localization analysis of the compound Q-NMe 2 and Nile Red in LO2 cells. Among them, a is the fluorescence staining image of the compound Q-NMe 2 ; b is the fluorescence staining image of Nile Red; c is the merged image of the fluorescence images of the compound Q-NMe 2 and Nile Red; d is the co-localization scatter plot of the compound Q-NMe 2 and Nile Red (Pearson correlation coefficient is 0.929); e is the co-localization line plot of the compound Q-NMe 2 and Nile Red distributed along the line segment Figure 7For compound Q-NMe 2 And co-localization analysis of lipid droplets between Nile Red and compound Q-NMe in Hela cells. Among them, a is the fluorescence staining image of compound Q-NMe 2 ; b is the fluorescence staining image of Nile Red; c is the merged image of compound Q-NMe 2 And the fluorescence image of Nile Red; d is the co-localization scatter plot of compound Q-NMe 2 And Nile Red (Pearson correlation coefficient is 0.950); e is the co-localization line plot of compound Q-NMe 2 And Nile Red along the line segment Figure 8 For LO2 cells incubated with 5 μM compound Q-NMe 2 At 37 °C for 5 min, 10 min and 20 min; Figure 9 Among them, a is the fluorescence image of LO2 cells stained with 5 μM compound Q-NMe 2 After laser irradiation for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 min; b is the curve of signal intensity changing with laser irradiation time, and the laser power is 100 W. Detailed implementation manners

[0020] The embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present invention, and should not be construed as a limitation to the present invention.

[0021] I. Specific embodiments of the 2,3-diphenylquinoline compounds of the present invention are as follows: Example 1 2-(4-N,N'-dimethyl-phenyl)-3-phenyl-quinoline (compound Q-NMe 2 ), and its structural formula is as follows: II. Specific embodiments of the preparation method of the 2,3-diphenylquinoline compounds (compound Q-NMe 2 ) of the present invention are as follows: Example 2 The preparation method of compound Q-NMe in Example 1 2 Comprises the following steps: (1) Preparation of intermediate M: Take a 50 mL dry two-necked round-bottom flask and displace it with nitrogen three times. Add o-iodoaniline (2.2 g, 10 mmol), styrene (2.1 g, 20 mmol) and tetrakis(triphenylphosphine)palladium (578 mg, 0.5 mmol) into the round-bottom flask, then add 10 mL of triethylamine to the round-bottom flask, heat to 80 °C and react for 24 h. After the reaction is completed, remove the excess solvent and separate by column chromatography to obtain intermediate M (1.93 g) with a yield of 99%. The synthetic route is as follows: 。

[0022] (2) Dissolve intermediate M (586 mg, 3 mmol), 4-(dimethylamino)benzaldehyde (300 mg, 2 mmol) and aluminum trichloride (400 mg, 3 mmol) in 40 mL of acetonitrile and 10 mL of chloroform, place it under a 20 W blue LED lamp at room temperature and react for 48 h. After the reaction is completed, quench the reaction with triethylamine, remove the excess solvent and separate by column chromatography to obtain 2-(4-N,N'-dimethyl-phenyl)-3-phenyl-quinoline (520 mg, compound Q-NMe 2 ) with a yield of 80%. The synthetic route is as follows: Characterization data of 2-(4-N,N'-dimethyl-phenyl)-3-phenyl-quinoline (compound Q-NMe 2 ): Yellow solid; melting point, 58 °C; 1 H NMR (500 MHz, CDCl 3 ) δ 8.16 (d, J J = 8.4 Hz, 1H), 8.05 (s, 1H), 7.78(d, J J = 7.6 Hz, 1H), 7.70 – 7.64 (m, 1H), 7.47 (t, J J = 7.5 Hz, 1H), 7.37 (d, J J =8.7 Hz, 2H), 7.34 – 7.25 (m, 5H),6.59 (d, J J = 8.9 Hz, 2H), 2.92 (s, 6H). 13 C NMR(125 MHz, CDCl 3) δ 158.3, 150.3, 147.5, 140.9, 137.6, 134.4, 131.2, 129.7, 129.3, 129.2, 128.3, 128.2, 127.4, 127.0, 126.9, 126.0, 111.7, 40.4. HRMS (ESI) for: C 23 H 20 N 2 [M + H]+: calcd 325.1699, found 325.1700。

[0023] Figure 1 The UV-visible absorption spectrum of 2-(4-N,N'-dimethyl-phenyl)-3-phenyl-quinoline (Compound Q-NMe 2 ) prepared in this example indicates that Compound Q-NMe 2 has absorption at wavelengths less than 425 nm under light illumination and can be excited by light with wavelengths lower than this band; Figure 2 The fluorescence spectrum of Compound Q-NMe 2 at 360 nm shows that its maximum fluorescence wavelength is approximately 470 nm, which is blue light.

[0024] Example 3 Differing from Example 2, in step (2), when the molar ratio of intermediate M, compound N (4-(dimethylamino)benzaldehyde), and aluminum trichloride is 1:1:0.1, the yield of Compound Q-NMe 2 is 30%.

[0025] Example 4 Differing from Example 2, in step (2), when the molar ratio of intermediate M, compound N (4-(dimethylamino)benzaldehyde), and aluminum trichloride is 1:1:1, the yield of Compound Q-NMe 2 is 70%.

[0026] Example 5 Differing from Example 2, in step (2), when the solvent is 50 mL of acetonitrile, the yield of Compound Q-NMe 2 is 70%.

[0027] Example 6 Differing from Example 2, in step (2), when the solvent is 50 mL of chloroform, the yield of Compound Q-NMe 2 is 64%.

[0028] Example 7 Different from Example 2, in step (2), when the solvent is 50 mL of toluene, the yield of compound Q-NMe 2 is 43%.

[0029] III. Specific examples of the biomedical applications of the compound Q-NMe of the present invention are as follows: 2 The compound (for the sake of convenient writing, hereinafter uniformly referred to as compound Q-NMe prepared in Example 2 is used for the following tests. 2 Designation).

[0030] 1. Cytotoxicity Before its application to cell imaging, the cytotoxicity of compound Q-NMe 2 is determined by the CCK-8 experiment.

[0031] Experimental method: LO2 cells are stored in the laboratory of the applicant, and Hela cells are purchased from Beina Biotechnology (BNCC342189). The cytotoxicity experiment is completed according to the instructions of the Beijing Solarbio CCK-8 kit (CA1210, Solarbio). The specific steps are as follows. 100 μL of cell suspension is inoculated in a 96-well plate (1×10 4 cells / well), and the required concentration of compound Q-NMe 2 is added (0 μM, 1 μM, 5 μM, 10 μM, and 20 μM). After incubation in an incubator at 37°C and 5% CO 2 for 24 h, a working solution is prepared according to fresh medium:CCK-8 solution = 9:1, the old medium is replaced, and after incubation in the incubator for 2 h, the absorbance is measured at 450 nm using a microplate reader (BioTek, Vermont, USA).

[0032] As can be seen from Figure 3 a and 3b, when LO2 cells and Hela cells are exposed to compound Q-NMe 2 at a concentration of 0 - 20 μM for 24 h, the cell viability of both LO2 cells and Hela cells exceeds 90%, indicating that compound Q-NMe 2 has high biocompatibility and low cytotoxicity and has potential application prospects in cell imaging.

[0033] 2. Cell staining In an incubator at 37°C and 5% CO 2 , LO2 cells are incubated in a medium containing 5 μM of compound Q-NMe 2 for 20 min, and highly selective bright blue fluorescence in the cytoplasm is observed. The results are as shown in Figure 4 a-c. Not all cells are stained. Therefore, it is speculated that compound Q-NMe2 The targeted organelles may not be organelles such as mitochondria, endoplasmic reticulum or lysosomes.

[0034] LO2 is an immortalized human hepatocyte cell line. As is well known, hepatocytes are important storage sites for lipid droplets (LDs). Recent studies have shown that LDs are not simply energy storage devices within cells, but rather complex, active, and dynamically changing multifunctional organelles. LDs play important roles in physiological processes such as lipid metabolism, membrane trafficking, protein degradation, signal transduction, and embryonic development. In addition, LDs are also associated with a variety of human diseases, such as liver injury, diabetes, chronic kidney disease, etc. Therefore, the research on LDs has received increasing attention, and tracing LDs will be beneficial to the study of their functions.

[0035] According to Figure 4 , it is assumed that compound Q-NMe 2 is localized to LDs. To verify this hypothesis, oleic acid was used to induce the formation of LDs in LO2 cells and Hela cells to obtain better imaging effects.

[0036] Oil Red O staining: In an incubator at 37°C and 5% CO 2 , the cells were cultured in a medium containing 250 μM oleic acid for 24 h. After gently rinsing the cells twice with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 20 min. After washing three times with distilled water, the cells were immersed in 60% isopropanol for 20 - 30 s. The Oil Red O working solution (IO1720, Solarbio, Beijing) was dropped onto the cells and soaked for 10 - 20 min. The working solution was removed, and the cells were rinsed with 60% isopropanol for 20 - 30 s and then washed 2 - 3 times with PBS. The lipid droplets in the cells were observed under an ordinary optical microscope, and the results are as Figure 5 shown.

[0037] For further research, compound Q-NMe 2 and the commercial LDs dye Nile Red were co-stained in LO2 cells and Hela cells for co-localization imaging. Co-localization staining: 2×10 4 HeLa cells or LO2 cells on cell slides were incubated with a 5 μM compound Q-NMe 2 working solution for 20 min, and then incubated with Nile Red (1 mg / L) for 20 min. (Incubation conditions: in an incubator at 37°C and 5% CO 2 , and the working solution is the corresponding cell culture medium containing the corresponding concentration of the dye). After incubation, the cells were washed 2 - 3 times with PBS, and the staining was observed under a fluorescence microscope. Excitation wavelength: compound Q-NMe 2The wavelength is 330 - 385 nm; the wavelength of Nile Red is 532 - 554 nm. The gray values of the images were analyzed by ImageJ software. The co-localization analysis was completed by the Colocalization Finder plugin of ImageJ software.

[0038] As Figure 6 and Figure 7 shown, the staining of compound Q-NMe 2 was consistent with that of Nile Red in cells. Figure 6 d and 7d are the scatter plots of the two channels for co-localization in LO2 cells and Hela cells respectively (compound Q-NMe 2 / Nile Red). As can be seen from Fig. 6d and Figure 7 d, compound Q-NMe 2 showed a good co-localization coefficient with Nile Red (Pearson correlation coefficient > 0.90). In addition, as can be seen from Figure 6 e and Figure 7 e, compound Q-NMe 2 had stronger fluorescence intensity compared with Nile Red. The measurement results of the signal-to-noise ratio (SNR) showed that compound Q-NMe 2 showed higher SNR than Nile Red in LO2 cells and Hela cells ( Figure 6 a and 7a).

[0039] The above results indicate that compound Q-NMe 2 can precisely target LD and is expected to be used for fluorescence imaging of LD.

[0040] The imaging ability in living cells provides sufficient evidence for compound Q-NMe 2 to be used as a fluorescent dye for real-time dynamic monitoring of LD.

[0041] In addition, this application also explored the ability of compound Q-NMe 2 to stain fixed cells (fixed cell incubation conditions: temperature 15 - 25 °C, working solution is PBS buffer containing the corresponding concentration of dye), and optimized the incubation time of compound Q-NMe 2 for identifying LD. As Figure 8 shown, after fixing cells with 4% paraformaldehyde (PFA), compound Q-NMe 2 could still highly selectively target LD, thus realizing the staining of LD in fixed samples. In addition, after adding compound Q-NMe 2 to living cells or fixed LO2 cells, clear LD images could be obtained even after only incubating for 5 min.

[0042] The compound Q-NMe 2The photostability, and the results are as Figure 9 shown. It can be seen from Figure 9 that for the LO2 cells stained with 5 μM compound Q-NMe 2 , the fluorescence intensity still remained above 75% after 10 min of continuous laser irradiation. After high-intensity and long-time continuous irradiation, the blue fluorescence in the cells was still very bright, indicating that Q-NMe 2 has good photostability, indicating that compound Q-NMe 2 has good photostability.

[0043] In summary, compound Q-NMe 2 dye has low cytotoxicity to cells, rapid staining, simple steps, specific targeting of lipid droplets, good color development performance, and can be used as a fluorescent probe for lipid droplets in biomedical research.

[0044] In the present invention, the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0045] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A 2,3-diphenylquinoline compound, characterized in that, the structure of the 2,3-diphenylquinoline compound is as follows: Wherein, R 1 and R 2 are each independently selected from H or C 1 -C 20 alkyl.

2. A method for preparing the 2,3-diphenylquinoline compound according to claim 1, characterized in that, it includes the following steps: Mix o-iodoaniline, styrene, an organic catalyst and an organic solvent S1 and react to prepare an intermediate M, and then react the intermediate M with a compound N to obtain the product.

3. The method for preparing the 2,3-diphenylquinoline compound according to claim 2, characterized in that, Dissolve o-iodoaniline, styrene and tetrakis(triphenylphosphine)palladium in an organic solvent S1, heat to 60-80 °C and react at this temperature. After the reaction is completed, separate to obtain the intermediate M. The organic solvent S1 is at least one of triethylamine, acetone, and n-butanol.

4. The method for preparing the 2,3-diphenylquinoline compound according to claim 3, characterized in that, the molar ratio of o-iodoaniline, styrene and tetrakis(triphenylphosphine)palladium is 1:1-2:0.01-0.

1.

5. The method for preparing the 2,3-diphenylquinoline compound according to claim 2, characterized in that, Dissolve the intermediate M, the compound N and aluminum trichloride in an organic solvent S2, react under an LED lamp. After the reaction is completed, quench the reaction and separate to obtain the 2,3-diphenylquinoline compound.

6. The method for preparing the 2,3-diphenylquinoline compound according to claim 5, characterized in that, the structure of the compound N is as follows: Wherein, R 3 and R 4 are each independently selected from H or C 1 -C 20 alkyl.

7. The method for preparing the 2,3-diphenylquinoline compound according to claim 6, characterized in that, the molar ratio of the intermediate M to the compound N is 1-1.5:1, and the molar ratio of the compound N to aluminum trichloride is 1:0.1-1.

5.

8. The method for preparing the 2,3-diphenylquinoline compound according to claim 5, characterized in that, the organic solvent S2 is at least one of acetonitrile, chloroform, toluene, and trichloromethane.

9. Application of the 2,3-diphenylquinoline compound according to claim 1 or 2 in cell imaging.

10. Application of the 2,3-diphenylquinoline compound according to claim 1 or 2 as a fluorescent probe for lipid droplets.

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