A class of fluorescent compounds of indole-dione and indole-quinoxaline derivatives, their preparation methods and uses

Abstract

This invention discloses a class of fluorescent compounds containing indole-diones and indole-quinoxaline derivatives, their preparation methods, and uses, belonging to the field of fluorescent compound technology. This invention provides a novel class of fluorescent compounds containing indole-diones and indole-quinoxaline derivatives with structures shown in general formulas (I) and (II). These compounds exhibit low toxicity and long emission wavelengths, showing significant application potential in the field of bioimaging.

Description

Technical Field

[0001] This invention relates to the field of fluorescent compound technology, specifically to a class of novel indole-dione and indole-quinoxaline derivative fluorescent compounds, their preparation methods, and uses. Background Technology

[0002] In chemical biology research and medical diagnostics, biofluorescence imaging has become one of the most commonly used methods for monitoring and tracking physiological changes and behavioral patterns in organisms. Due to its high sensitivity, ease of operation, real-time detection capabilities in living systems, and good biocompatibility, fluorescence tracing has become a novel and powerful technique. Various fluorescent molecules have been used as selective bioimaging tools, such as some metal ions, cations, anions, various enzymes, and biological organs and tissues. Despite these advantages, the feasibility of using fluorescence technology for specific applications is often limited by the availability of suitable fluorescent molecules.

[0003] Indolone compounds are fluorophores with high fluorescence quantum yield and high stability, and their skeletons are often used in the luminescent portion of probes. The unique chemical structure of the fluorophore endows it with unique photophysical properties and related chemical reactivity. Fluorescent molecular probes with metal ion recognition capabilities have attracted widespread attention, and the design and synthesis of novel fluorescent probe molecules has become a recent research hotspot. Due to the excellent optical properties of indoquinoxaline and its derivatives, they are often used as fluorophores or recognition groups in chemical sensors, playing an important role in the synthesis of fluorescent sensing molecules, magnetic complexes, ion recognition probes, and bioimaging. Indoquinoxaline is an important class of nitrogen-containing heterocyclic compounds, in which the electron-rich indole unit fuses with the electron-deficient quinoline moiety to form an embedded donor-acceptor (DA) system. Besides its significant biological applications in the pharmaceutical field, indoquinoxaline has also been studied for use in optoelectronic devices. It has good thermal stability and can be used as a photoinitiator. The electronic properties of indoquinoxaline are sensitive to the substitution position and strength of the electron-donating group and the solvent used; its photoelectronic properties and thermal stability can be tuned by modifying the donor substituents in the DA framework.

[0004] However, in addition to fluorescence properties, bioimaging requires compounds to successfully enter cells and exert their fluorescence properties. The structure-activity relationships of different compounds in cell imaging are unclear, and some compounds, although possessing certain fluorescence properties, cannot be successfully imaged, becoming one of the barriers to effective imaging with fluorescent compounds.

[0005] Therefore, developing effective bioimaging methods for rapidly tracking targets in complex biological systems is of great significance for advancing the integrated diagnosis and treatment of related diseases. Summary of the Invention

[0006] To address the aforementioned issues, this invention aims to tandem indole-dione, cyano, and o-phenylenediamine structures to obtain compounds with better fluorescence performance that can be applied in the field of bioimaging technology. This provides novel fluorescent compounds containing indole-dione and indolequinoxaline derivatives, which can then be used in bioimaging.

[0007] The present invention mainly solves the above-mentioned technical problems through the following technical solutions.

[0008] [Compound]

[0009] A class of structures represented by general formula (I) or general formula (II) containing indoledione and indolequinoxaline derivative fluorescent compounds or pharmaceutically acceptable salts thereof:

[0010]

[0011] in,

[0012] R1 and R3 are independently selected from substituted or unsubstituted aromatic ring groups; the substituents are selected from: C1-4 alkoxy, C1-4 alkyl, amino-NR a R b ;R a R b Each is independently selected from H and C1-4 alkyl groups;

[0013] R2 is independently selected from: O, -C(CN)2;

[0014] R4 and R5 are independently selected from: H, -CH2COOR c ;R c Selected from H and C1-4 alkyl groups.

[0015] In one embodiment of the present invention, the aromatic ring group includes phenyl and naphthyl groups.

[0016] In one embodiment of the present invention, the substituent on the aromatic ring group may specifically be methoxy, amino, methyl, ethyl, or propyl.

[0017] Specifically,

[0018] In one embodiment of the present invention, R1 and R3 can be specifically selected from:

[0019] .

[0020] In one embodiment of the present invention, R4 may be selected from: H, COOH, or COOEt.

[0021] In one embodiment of the present invention, most preferably, the specific structure of the fluorescent compound containing indole-dione and indole-quinoxaline derivatives is as follows:

[0022]

[0023] In one embodiment of the present invention, pharmaceutically acceptable salts include inorganic salts or organic salts; wherein, inorganic salts include hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, hydrogen sulfate, nitrate, phosphate, and acid phosphate; and the organic salts are selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, and ascorbate.

[0024] [Synthesis Method]

[0025] The present invention also provides a method for preparing the above-mentioned fluorescent compounds containing indole-dione and indole-quinoxaline derivatives.

[0026] .

[0027] Specifically, the following steps are included:

[0028] The 1-substituted alkyne was dissolved in THF, and TEA solution was added. Then Pd(PPh3)2Cl2 and CuI were added to the mixed solution. The mixture was purged three times with N2 and microwaved for 15 min. The resulting reaction solution was filtered using a vacuum funnel. The solid was washed with methanol and dried to obtain compound 2.

[0029] Compound 2 and malondicyanate were added to H2O:EtOH (volume ratio = 7:3) and reacted in an oil bath at 75°C for 20 min. The resulting reaction solution was filtered using a vacuum filter funnel, the solid was washed with methanol, and dried to obtain compound 3.

[0030] Compound 2 and o-phenylenediamine were added to 3 ml of glacial acetic acid and reacted in an oil bath at 80 °C for 10 min. After the reaction was complete, the reaction solution was filtered using a vacuum filter funnel, the solid was washed with methanol, and dried to obtain compound 4.

[0031] Compound 4 was added to 1 ml of tetrahydrofuran (THF). Under ice bath conditions, an aqueous solution of LiOH was slowly added dropwise to the reaction solution. After reacting at room temperature for 10 min, the mixture was refluxed in an oil bath for 2 h. After the reaction was complete, the solvent was evaporated, and 6 M hydrochloric acid was added to adjust the pH to 2-3. A yellow solid precipitated out. The solid was filtered using a vacuum funnel, washed with water, and dried to obtain compound 5.

[0032] [use]

[0033] This invention also provides the application of the above-mentioned fluorescent compounds containing indoledione and indolequinoxaline derivatives or their pharmaceutically acceptable salts in the field of fluorescent probe preparation.

[0034] The present invention also provides the application of the above-mentioned fluorescent compounds containing indoledione and indolequinoxaline derivatives or their pharmaceutically acceptable salts in the field of bioimaging.

[0035] The present invention also provides a bioimaging reagent comprising the above-mentioned fluorescent compound or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients.

[0036] Beneficial effects:

[0037] The fluorescent compounds of this invention, containing indole-dione and indole-quinoxaline derivatives, exhibit excellent fluorescence properties, with a maximum excitation wavelength range of 350-410 nm and a maximum emission wavelength range of 400-550 nm. The fluorescent compounds synthesized in this invention contain a quinoxaline structure, which has certain applications in the biological field. Furthermore, cytotoxicity experiments show high cell viability and virtually no cytotoxicity, providing a new technical means in the field of cell imaging and having significant implications for the diagnosis and treatment of malignant diseases such as tumors. Attached Figure Description

[0038] Figure 1 The absorption spectra of compounds I and II in DMSO are shown (c = 100 μM).

[0039] Figure 2 The emission spectra of compounds I and II in DMSO are shown (c = 100 μM).

[0040] Figure 3 The graph shows the results of the toxicity test of the compound in Example 1 on normal human cells.

[0041] Figure 4 This is a graph showing the fluorescence imaging results of the compound in Example 1 in HeLa cells. Detailed Implementation

[0042] Example 1

[0043]

[0044] Add compound 1 (400 mg, 1.62 mmol) to 5 mL of DMF, add K2CO3 (670 mg, 4.86 mmol), stir for 10 minutes in an ice bath, and slowly add ethyl bromoacetate (325 mg, 1.94 mmol) to the mixture. Incubate at room temperature for 1 hour. After the reaction is complete as detected by thin-layer chromatography (TLC), add the reaction solution dropwise to ice water. A solid precipitates out. Filter the solid using a vacuum funnel, wash it three times with water, and dry it to obtain compound 2 (compound I-7). No further purification is required.

[0045] NMR mass spectrometry data: 1 H NMR (600 MHz, DMSO- d 6) δ 7.65 (d, J = 7.8 Hz, 1H), 7.62 (dd, J = 7.2, 2.4 Hz, 2H), 7.50 - 7.48 (m, 2H), 7.48 (s, 2H), 7.34 (dd, J =7.8, 1.2 Hz, 1H), 4.65 (s, 2H), 4.18 (q, J = 7.2 Hz, 2H), 1.23 (t, J = 7.2 Hz, 3H).MS (ESI): m / z calcd. For C 22 H 18 N₂NaO₄ 397.10 [M+Na+CH₃CN] + Found 397.05 [M+Na+CH3CN] + .

[0046] Add 2 (50 mg, 0.2 mmol) and malondicyandiamide (16 mg, 0.24 mmol) to 2 ml of H2O:EtOH (volume ratio = 7:3), and heat in an oil bath at 75°C. 0 After reacting at C for 20 min, the reaction was detected to be complete by thin-layer chromatography (TLC). The resulting reaction solution was filtered using a vacuum funnel, the solid was washed with methanol, and dried to obtain compound 3 (compound I-19), which did not require further purification.

[0047] NMR mass spectrometry data: 1 H NMR (600 MHz, DMSO-d6) δ 11.37 (s, 1H), 7.91 (d, J =8.1 Hz, 1H), 7.63 (dd,J = 7.8, 1.8 Hz, 2H), 7.54 - 7.41 (m, 3H), 7.31 (dd, J =8.1, 1.2 Hz, 1H), 7.04 (s, 1H). 13 C NMR (151 MHz, DMSO-d6) δ 163.79, 149.54, 146.45, 131.83, 130.69, 129.81, 128.92, 126.01, 125.98, 121.30, 118.70, 113.43, 113.09, 111.57, 95.02, 89.27, 80.71. MS (ESI): m / z calcd. ForC 19 H9N3NaO 318.07.15 [M+Na] + Found 317.70 [M+Na] + .

[0048] Add 2 (50 mg, 0.15 mmol) and o-phenylenediamine (16 mg, 0.15 mmol) to 3 ml of glacial acetic acid, and heat in an oil bath at 80°C. 0 After reacting at C for 10 min, the reaction was detected by thin-layer chromatography (TLC) to indicate complete reaction. The resulting reaction solution was then filtered using a vacuum funnel. The solid was washed with methanol and dried to obtain compound 4 (compound II-7), which required no further purification.

[0049] NMR mass spectrometry data: 1 H NMR (600 MHz, DMSO-d6) δ 8.44 (d, J = 7.8 Hz, 1H), 8.31 (d, J = 7.8 Hz, 1H), 8.13 (d, J = 8.1 Hz, 1H), 8.10 (s, 1H), 7.87 (t, J =7.2 Hz, 1H), 7.82 - 7.77 (m, 1H), 7.64 (s, 2H), 7.61 (d, J = 8.1 Hz, 1H), 7.48(s, 3H), 5.75 (s, 1H), 5.45 (s, 1H), 4.19 (q, J = 6.6 Hz, 2H), 1.23 (t, J= 6.6Hz, 3H).MS (ESI): m / z calcd. For C 19 H9N3NaO 406.15 [M+H] + Found 406.05 [M+H] + .

[0050] Compound 4 (30 mg, 0.07 mmol) was added to 1 ml of tetrahydrofuran (THF). Under ice bath conditions, an aqueous solution of LiOH (8 mg, 0.33 mmol) was slowly added dropwise to the reaction solution. After reacting at room temperature for 10 min, the mixture was refluxed in an oil bath for 2 h. After the reaction was detected by thin-layer chromatography (TLC) to be complete, the solvent was evaporated, and 6 M hydrochloric acid was added to adjust the pH to 2-3. A yellow solid precipitated out. The solid was filtered using a vacuum funnel, washed with water, and dried to obtain compound 5 (compound II-13).

[0051] NMR mass spectrometry data: 1 H NMR (600 MHz, DMSO-d6) δ 8.42 (d, J = 7.8 Hz, 1H), 8.29 (d, J = 9.3 Hz, 1H), 8.13 (d, J = 9.3 Hz, 1H), 8.08 (s, 1H), 7.87 - 7.84 (m, 1H), 7.78 (td, J = 7.8, 7.0, 1.3 Hz, 1H), 7.65 (d, J = 4.2 Hz, 1H), 7.64 (d, J = 1.8Hz, 1H), 7.59 (dd, J = 7.8, 1.2 Hz, 1H), 7.49 (d, J = 1.8 Hz, 1H), 7.48 (d, J =1.8 Hz, 2H), 5.34 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 169.56, 145.55, 144.35, 139.87, 139.20, 138.95, 131.52, 129.53, 129.19, 128.88, 127.54, 126.68, 124.76, 124.54, 122.40, 121.95, 118.69, 113.54, 91.45, 89.87, 42.59. MS (ESI): m / z calcd. For C 24 H 14 N3O2376.12 [MH] + Found 376.05 [MH] + .

[0052] Following the above preparation process, by replacing the substrates accordingly, other fluorescent compounds containing indoledione and indolequinoxaline derivatives, as shown in Table 1, were obtained.

[0053] Table 1. Fluorescent compounds containing indole-dione and indolequinoxaline derivatives and their characterization data.

[0054]

[0055] [Example 2]

[0056] Spectroscopic experimental testing

[0057] Ultraviolet absorption: Weigh 1 mg of the compound, prepare a 1 mM concentration with DMSO, transfer 20 μL of the stock solution into a cuvette, add 180 μL of DMSO, place the cuvette in an ultraviolet spectrophotometer, and measure the excitation wavelength.

[0058] Fluorescence emission: Weigh 1 mg of the compound, prepare a 1 mM concentration with DMSO, transfer 20 μL of the stock solution into a cuvette, add 180 μL of DMSO, place the cuvette in a fluorescence spectrophotometer, and select the excitation wavelength obtained from the ultraviolet absorption experiment for measurement.

[0059] The test results obtained in Example 2 are shown below. Figure 1 , Figure 2 .

[0060] [Example 3]

[0061] Compound cytotoxicity test

[0062] 1) Materials:

[0063] Cell line: PBMC

[0064] Reagents: DMEM medium containing 20% ​​fetal bovine serum and 1% penicillin / streptomycin, 96-well white plate;

[0065] 2) Procedure: PBMC cells were collected, centrifuged, resuspended, and counted. Cells were seeded at a density of 10,000 cells / well in clear 96-well cell culture plates, with each well containing 80 μL of cell suspension (1640 containing 20% ​​FBS, Gibico). The culture plates were incubated at 37°C for 1 hour to equilibrate, under the conditions of 37°C + 5% CO2. Then, graded concentrations of compound II-13 were added. After three days, 30 μL of CTG solution (Promega) was added. After incubation at 37°C and 5% CO2 for 10 minutes, absorbance was recorded using a 96-well plate reader (Wallac EnVision Manager, Molecular Devices).

[0066] The test results obtained in Example 3 are shown below. Figure 3 As can be seen from the figure, compound II-13 did not exhibit cytotoxicity, indicating that the representative molecule II-13 can provide new technical means in the field of cell imaging.

[0067] [Example 4]

[0068] Cell imaging assay of the compound in HeLa cells

[0069] Confocal microscopy. HeLa cells were cultured on glass culture dishes for 12 hours before treatment. They were then incubated with 20 μM compounds II-13 and II-14 for 16 hours each, washed three times with PBS, fixed in 4% paraformaldehyde solution for 15 minutes, and washed again with PBS. Cell images were obtained using a Nikon Ti2-E+A1 confocal microscope.

[0070] The test results obtained in Example 4 are shown below. Figure 4 As shown in the figure, compound II-13 exhibits blue fluorescence in HeLa cells, indicating its superior performance in HeLa cell imaging. In contrast, compound II-14 showed no fluorescence signal in HeLa cells. This test demonstrates that although indoquinoxaline is a known fluorophore with high fluorescence quantum yield and high stability, the derivatization of its molecular structure has a complex impact on its successful intracellular luminescent imaging. This further illustrates that the successful development of an effective cell imaging structure is unpredictable, and underscores the necessity and importance of in-depth investigation into the fluorescence properties and structure-activity relationship of this fluorophore.

Claims

1. A fluorescent compound or a pharmaceutically acceptable salt thereof: characterized in that, Specifically, the structure is as follows: Ⅱ-13 。 2. The fluorescent compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized by, The pharmaceutically acceptable salt is selected from inorganic salts or organic salts; wherein the inorganic salt is selected from hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, nitrate, phosphate; the organic salt is selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartarate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, ascorbate.

3. The fluorescent compound or a pharmaceutically acceptable salt thereof according to claim 2, characterized by, The pharmaceutically acceptable salt is selected from bisulfate, acid phosphate.

4. Use of the fluorescent compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a biological imaging agent.

5. A bioimaging agent, characterized by, The fluorescent compound or a pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutical adjuvant.

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