A near-infrared fluorescent compound for early diagnosis and treatment of glioma and a preparation method and application thereof

Abstract

The application discloses a near-infrared fluorescent compound for early diagnosis and treatment of glioma and a preparation method and application thereof, and a structure formula of the compound is shown in the following. The near-infrared fluorescent compound for early diagnosis and treatment of glioma prepared by the application has a near-infrared fluorescent emission wavelength, can effectively reduce background interference and reduce the possibility of false positive results in the imaging of glioma; meanwhile, the compound can distinguish glioma cells from normal cells through fluorescence intensity, especially has a high degree of distinction for U87MG cells, has application potential for early diagnosis of glioma and auxiliary imaging in the process of surgical resection. The near-infrared fluorescent compound prepared by the application shows spectral anti-tumor activity on different glioma cell strains, realizes integration of diagnosis and treatment of glioma, and is a multifunctional small molecule capable of being used for diagnosis and treatment of glioma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a near-infrared fluorescence for early diagnosis and treatment of gliomas, its preparation method, and its application. Background Technology

[0002] Gliomas are considered among the most aggressive and difficult-to-cure brain tumors. Current treatments for gliomas include surgical resection, radiotherapy, and chemotherapy (such as temozolomide), but these methods have several limitations, such as incomplete tumor resection, drug resistance, and recurrence. Despite advancements in modern neurosurgery, radiosurgery, and chemotherapy, the survival rate for glioma patients remains low. Therefore, early detection and diagnosis are crucial.

[0003] Fluorescence imaging, with its real-time and high-resolution characteristics, has been widely used in the biomedical field. For gliomas, fluorescence imaging technology improves diagnostic accuracy, surgical navigation, and treatment monitoring. It also provides real-time monitoring during surgery, assisting surgeons in clearly observing fluorescently labeled tumor areas under a microscope for more precise tumor resection. 5-Aminolevulinic acid (5-ALA), as a fluorescent dye, has been proven effective in completely removing tumors and prolonging progression-free survival, and has therefore been incorporated into the surgical treatment of malignant gliomas. However, its use still has some limitations, including potential false-positive and false-negative results, and the inability to re-dose. Therefore, there is an urgent need to develop high-performance fluorescent probes for the diagnosis and surgical guidance of gliomas.

[0004] Near-infrared fluorescence typically refers to fluorescence emitting wavelengths between 650 nm and 900 nm. Fluorescent probes in this wavelength range can penetrate biological tissues better, which is beneficial for imaging deep structures. Furthermore, biological tissues in the near-infrared range usually have low autoluminescence; therefore, near-infrared probes can improve the signal-to-noise ratio of biological imaging, making target areas clearer and easier to observe. Near-infrared fluorescent probes have enormous potential and application prospects in biomedical imaging, diagnosis, treatment monitoring, and disease research.

[0005] Compared to near-infrared fluorescent probes, fluorescent drugs not only enable tumor imaging and early diagnosis but also possess excellent anti-tumor activity. They effectively eliminate residual tumor cells during adjuvant resection surgery and have great potential for subsequent treatment after tumor resection. Currently, there is a lack of multifunctional organic compounds that can achieve both diagnosis and treatment of gliomas using a single molecule; therefore, there is an urgent need to develop glioma diagnostic and therapeutic compounds with superior performance. Summary of the Invention

[0006] Objective of the Invention: Addressing the problems existing in the prior art, this invention provides a near-infrared fluorescent compound for the early diagnosis and treatment of gliomas. This diagnostic compound can distinguish glioma cells from brain microvascular endothelial cells under flow cytometry and confocal microscopy. Simultaneously, this compound exhibits anti-tumor activity against different types of glioma cells and U87MG3D tumor cell spheroids. This invention combines early diagnosis and treatment of gliomas into a single drug molecule. Through the specific fluorescent compound designed in this invention, early diagnosis and subsequent treatment of gliomas can be effectively achieved.

[0007] The present invention also provides a method for preparing and applying the near-infrared fluorescent compound for early diagnosis and treatment of glioma.

[0008] Technical Solution: To achieve the above objectives, the present invention provides a near-infrared fluorescent compound for early diagnosis and treatment of gliomas, characterized in that the compound has the following structural formula:

[0009]

[0010] The present invention discloses a method for preparing a near-infrared fluorescent drug compound for early diagnosis and treatment of glioma, comprising the following steps:

[0011] (1) Under inert gas protection, [(S)-1-(4-bromophenyl)ethyl] tert-butyl carbamate, cesium carbonate, S-(-)-1,1'-binaphthyl-2,2'-bisdiphenylphosphine and palladium acetate were mixed, and then an organic solvent was added. After heating and stirring, N-methylpiperazine was added. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was extracted and purified to obtain compound W3.

[0012] (2) Add W3 to hydrochloric acid, stir, add organic solvent, heat and stir, and after the reaction is complete, remove the solvent by rotary evaporation to obtain compound W4. Under the protection of inert gas, mix cyanoacetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, compound W4, and 4-dimethylaminopyridine organic solvent, stir in an ice bath until the temperature returns to room temperature, remove the solvent by rotary evaporation after the reaction is complete, and purify to obtain compound W5.

[0013] (3) Under the protection of an inert gas, compound 6-(dimethylamino)quinoline-2-carboxaldehyde and compound W5 were mixed, an organic solvent was added, the mixture was heated and stirred, and after the reaction was complete, the solvent was removed by rotary evaporation and purified to obtain the compound, which was named QP-4.

[0014] In step (1), the molar ratio of [(S)-1-(4-bromophenyl)ethyl]carbamate tert-butyl ester, cesium carbonate, S-(-)-1,1'-binaphthyl-2,2'-bis(diphenylphosphine), palladium acetate, and N-methylpiperazine is 1:1.4:0.06:0.04:3-1:2:0.12:0.08, the reaction temperature is 100-120℃, and the reaction time is 10-14h.

[0015] Preferably, the molar ratio of [(S)-1-(4-bromophenyl)ethyl]carbamate tert-butyl ester, cesium carbonate, S-(-)-1,1'-binaphthyl-2,2'-bis(diphenylphosphine), palladium acetate, and N-methylpiperazine is 1:1.5:0.06:0.04:3, the reaction temperature is 100℃, and the reaction time is 12h.

[0016] In step (2), the molar ratio of cyanoacetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine compound, and compound W4 is 1:1.5:0.25:1-1:3:0.5:2, and the reaction temperature is 8-12 h after cooling to room temperature in an ice bath.

[0017] Preferably, in step (2), the molar ratio of cyanoacetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine compound, and compound W3 is 1:1.5:0.25:1, and the reaction temperature is 12h after cooling to room temperature in an ice bath.

[0018] In step (3), the molar ratio of compound 6-(dimethylamino)quinoline-2-carboxaldehyde and compound W5 is 3:1-5:1, the reaction temperature is 40-70℃, and the reaction time is 2-5h.

[0019] Preferably, in step (3), the molar ratio of compound 6-(dimethylamino)quinoline-2-carboxaldehyde and compound W5 is 3:1, the reaction temperature is 60℃, and the reaction time is 2h.

[0020] Preferably, the preparation includes the following steps:

[0021] (1) Dissolve 4-amino-N,N-dimethylaniline in hydrogen chloride solution, then add crotonaldehyde, stir the mixture at room temperature for 1 h, then add toluene and heat and stir. After the reaction is complete, remove the solvent by rotary evaporation, extract and purify to obtain compound W1;

[0022] (2) Add the compound selenium dioxide to 1,4-dioxane, heat for 30 min, then add compound W1, heat and stir, and after the reaction is complete, filter and purify with diatomaceous earth to obtain compound W2 (6-(dimethylamino)quinoline-2-carboxaldehyde).

[0023] (3) Under nitrogen protection, [(S)-1-(4-bromophenyl)ethyl] tert-butyl carbamate, cesium carbonate, S-(-)-1,1'-binaphthyl-2,2'-bisdiphenylphosphine, and palladium acetate were mixed, and then toluene was added. After heating and stirring for 20 min, N-methylpiperazine was added. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was extracted and purified to obtain compound W3.

[0024] (4) Add W3 to hydrochloric acid, stir, then add 1,4-dioxane and methanol, heat and stir, and after the reaction is complete, remove the solvent by rotary evaporation. No purification is required to obtain compound W4.

[0025] (5) Under argon protection, cyanoacetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and compound W4 were mixed, dichloromethane was added, and the mixture was stirred in an ice bath until the temperature returned to room temperature. After the reaction was complete, the solvent was removed by rotary evaporation and purified to obtain compound W5.

[0026] (6) Under argon protection, compound W2 and compound W5 were mixed, ethanol and piperidine were added, the mixture was heated and stirred, and after the reaction was complete, the solvent was removed by rotary evaporation and purified to obtain compound QP-4.

[0027] The reaction formula is shown below:

[0028]

[0029] Furthermore, the specific steps for preparing the compound are as follows:

[0030] 4-Amino-N,N-dimethylaniline (36.7 mmol, 1 eq) was dissolved in 4 mol / L (66 mL) HCl solution, followed by the addition of crotonaldehyde (73.5 mmol, 2 eq). The mixture was stirred at room temperature for 1 h. Toluene (35 mL) was then added, and the mixture was refluxed overnight at 115 °C. After cooling to room temperature, the toluene layer was removed, and the aqueous layer was neutralized with saturated sodium hydroxide solution. The solution was extracted with dichloromethane, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated by filtration under reduced pressure. The crude product was purified by column chromatography to give a sandy brown compound W1.

[0031] Selenium dioxide (25.1 mmol, 1.3 eq) was mixed with dioxane (140 mL) and heated at 80 °C for 30 min. Then, compound W1 (9.2 mmol, 1 eq) was added, and the mixture was stirred at 80 °C for 14 h. After cooling to room temperature, the mixture was filtered through diatomaceous earth, and the filter residue was washed several times with a small amount of dichloromethane. The filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to give a yellow solid compound W2 (6-(dimethylamino)quinoline-2-carboxaldehyde).

[0032] Under argon protection, a two-necked flask was used to add [(S)-1-(4-bromophenyl)ethyl]carbamate tert-butyl ester (0.5 mmol, 1 eq) and cesium carbonate (0.7 mmol, 1.5 eq), followed by S-(-)-1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (0.03 mmol, 0.06 eq) and palladium acetate (0.02 mmol, 0.04 eq). Toluene (10 mL) was added, and the mixture was heated to reflux at 100 °C for 20 min. Then, N-methylpiperazine (1.5 mmol, 3 eq) was added, and the reaction was allowed to proceed overnight for 12 h. The reaction solution was cooled to room temperature, toluene was removed, and the mixture was extracted with dichloromethane and saturated brine. After drying with anhydrous sodium sulfate, the crude product was purified by column chromatography to obtain a yellowish-brown solid compound W3.

[0033] Compound W3 (0.1 mmol) was added to 4 mol / L (2 ml) hydrochloric acid, stirred, and then 1,4-dioxane (1 ml) and methanol (1 ml) were added. The mixture was stirred at 50 °C for 1 h. After cooling to room temperature, the solvent was removed by rotary evaporation. No purification was required to obtain the crude product compound W4.

[0034] Under argon protection, cyanoacetic acid (1 mmol, 1 eq) was added to the reaction flask, followed by 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.5 mmol, 1.5 eq), 4-dimethylaminopyridine (0.25 mmol, 0.25 eq), compound W4 (1 mmol, 1 eq), dichloromethane (4 mL), and triethylamine (2 mL). The mixture was stirred in an ice bath until it returned to room temperature, and the reaction was allowed to proceed overnight for 12 h. After the reaction was complete, the product was extracted directly with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain a pale yellow solid compound W5.

[0035] Under argon protection, compounds W2 (0.3 mmol, 3 eq) and W5 (0.1 mmol, 1 eq) were added to a reaction flask, dissolved in anhydrous ethanol (5 mL), and then piperidine (0.1 mL) was added. The mixture was stirred at 60 °C for 2 h. After cooling to room temperature, the solvent was removed by rotary evaporation, and the crude product was purified by column chromatography to obtain the red solid compound QP-4.

[0036] The present invention relates to the application of the near-infrared fluorescent compound for early diagnosis and treatment of glioma in the preparation of drugs that inhibit tumor growth and kill tumor cells.

[0037] The application of the near-infrared fluorescent compound for early diagnosis and treatment of glioma described in this invention in the preparation of a diagnostic and therapeutic glioma cell drug.

[0038] The compound effectively distinguishes between glioma cells and microvascular endothelial cells in the preparation of a therapeutic glioma cell drug.

[0039] The compound targets lysosomes of glioma cells.

[0040] Furthermore, the compound exhibits good inhibitory and killing activity against different types of glioma cells and U87MG3D tumor cell spheres. The different types of glioma cells refer to 2D cultured U87MG, U343MG, U251MG, SHSY5Y, GL261, and other cells; while U87MG3D tumor cell spheres refer to 3D cultured 3D cell spheres.

[0041] The present invention relates to the application of near-infrared fluorescent compounds for early diagnosis and treatment of gliomas in the preparation of drugs or reagents for near-infrared fluorescence, flow cytometry fluorescence detection, and confocal fluorescence imaging.

[0042] The compound exhibits near-infrared fluorescence and emits near-infrared fluorescence in PBS solution; it can also be used in flow cytometry fluorescence detection and confocal fluorescence imaging.

[0043] Furthermore, the fluorescent compound of the present invention can emit near-infrared fluorescence with a maximum wavelength of 674 nm under excitation light of 350 nm-600 nm in PBS.

[0044] Preferably, the fluorescent compound QP-4 exhibits maximum intensity near-infrared fluorescence when excited by 476 nm excitation light in PBS.

[0045] The fluorescent compound QP-4 described in this invention can target the lysosomes of glioma cells. Furthermore, after co-incubation with U87MG cells, the fluorescent compound QP-4 can selectively accumulate in the lysosomes of U87MG cells under confocal imaging.

[0046] The application of the fluorescent compound QP-4 described in this invention in the identification of glioma cells and normal cells, further demonstrates that the fluorescent compound QP-4 can identify glioma cells and brain microvascular endothelial cells by fluorescence intensity under flow cytometry and confocal fiber microscopy.

[0047] The application of the fluorescent compound QP-4 described in this invention in the treatment of glioma further demonstrates that the fluorescent compound QP-4 can significantly inhibit the proliferation of glioma cells such as U87MG, U343MG, U251MG, SHSY5Y, and GL261, and can significantly kill U87MG3D tumor cell spheres, that is, it has a significant killing ability against solid tumors.

[0048] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0049] 1. The near-infrared fluorescent compound QP-4 prepared by this invention for the early diagnosis and treatment of glioma has a near-infrared fluorescence emission wavelength, which can effectively reduce background interference and reduce the possibility of false positive results in glioma imaging.

[0050] 2. The near-infrared fluorescent compound QP-4 prepared in this invention can selectively aggregate in the lysosomes of glioma cells, and can be used as an important analytical tool for detecting lysosomal changes during the pathological process of glioma cells.

[0051] 3. The near-infrared fluorescent compound QP-4 prepared in this invention can distinguish glioma cells from normal cells by fluorescence intensity, especially showing high discrimination against U87MG cells. It has potential applications in the diagnosis of early gliomas and as an auxiliary imaging agent during surgical resection.

[0052] 4. The near-infrared fluorescent compound QP-4 prepared in this invention exhibits spectral antitumor activity against different glioma cell lines. QP-4 integrates the diagnosis and treatment of glioma and is a multifunctional small molecule that can be used for the diagnosis and treatment of glioma. Attached Figure Description

[0053] Figure 1 The hydrogen spectrum of W3 prepared in Example 1 of this invention;

[0054] Figure 2 The carbon spectrum of W3 prepared in Example 1 of this invention;

[0055] Figure 3 The hydrogen spectrum of W5 prepared in Example 1 of this invention;

[0056] Figure 4 The carbon spectrum of W5 prepared in Example 1 of this invention;

[0057] Figure 5 The hydrogen spectrum of QP-4 prepared in Example 1 of this invention;

[0058] Figure 6 The carbon spectrum of QP-4 prepared in Example 1 of this invention;

[0059] Figure 7 This is the mass spectrum of QP-4 prepared in Example 1 of the present invention;

[0060] Figure 8 The excitation and fluorescence emission spectra of QP-4 in PBS (1% DMSO) are shown.

[0061] Figure 9 The mean fluorescence intensity of QP-4 cells after incubation with bEnd.3 and U87MG cells for different times;

[0062] Figure 10 The mean fluorescence intensity of QP-4 cells after incubation with bEnd.3 and different glioma cells for 30 min;

[0063] Figure 11 Confocal imaging of QP-4 cells after incubation with bEnd.3 and U87MG cells;

[0064] Figure 12 Colocalization fluorescence imaging of U87MG cells after incubation with QP-4 and the commercial mitochondrial dye Mito-Green;

[0065] Figure 13 Colocalization fluorescence imaging of U87MG cells after incubation with QP-4 and the commercial lysosomal dye Lyso-Green;

[0066] Figure 14 Cell survival rates after incubation of different concentrations of QP-4 with different cell types for 48 hours;

[0067] Figure 15 Morphological changes of 3D cell spheres of U87MG incubated with 5μm QP-4 for 5 days. Detailed Implementation

[0068] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0069] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0070] Example 1

[0071] The present invention relates to the preparation of near-infrared fluorescent drug compounds for early diagnosis and treatment of gliomas, comprising the following steps:

[0072]

[0073] 4-Amino-N,N-dimethylaniline (36.7 mmol) was dissolved in 4 mol / L (66 mL) hydrogen chloride solution, followed by the addition of crotonaldehyde (73.5 mmol). The mixture was stirred at room temperature for 1 h. Toluene (35 mL) was then added, and the mixture was refluxed overnight at 115 °C. After cooling to room temperature, the toluene layer was removed, and the aqueous layer was neutralized with saturated sodium hydroxide solution. The solution was extracted with dichloromethane, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated by filtration under reduced pressure. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 5:1 v / v) to give a sandy brown compound W1.

[0074] Selenium dioxide (25.1 mmol) was mixed with 1,4-dioxane (140 ml) and heated at 80 °C for 30 min. Then, compound W1 (9.2 mmol) was added, and the mixture was stirred at 80 °C for 14 h. After cooling to room temperature, the mixture was filtered through diatomaceous earth, and the filter residue was washed several times with a small amount of dichloromethane. The filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (dichloromethane:methanol = 250:1 v / v) to give a yellow solid compound W2 (6-(dimethylamino)quinoline-2-carboxaldehyde).

[0075] Under argon protection, a two-necked flask was used to add 0.5 mmol of [(S)-1-(4-bromophenyl)ethyl]carbamate tert-butyl ester and 0.7 mmol of cesium carbonate, followed by 0.03 mmol of S-(-)-1,1'-binaphthyl-2,2'-bis(diphenylphosphine) and 0.02 mmol of palladium acetate. Toluene (10 mL) was added, and the mixture was heated to reflux at 100 °C for 20 min. Then, N-methylpiperazine (1.5 mmol) was added, and the mixture was refluxed overnight for 12 h. The reaction solution was cooled to room temperature, toluene was removed, and the mixture was extracted with dichloromethane and saturated brine. After drying with anhydrous sodium sulfate, the crude product was purified by column chromatography (dichloromethane:methanol = 30:1 v / v) to obtain a yellowish-brown solid compound W3.

[0076] Compound W3 (0.1 mmol) was added to 4 mol / L (2 ml) hydrochloric acid, stirred, and then 1,4-dioxane (1 ml) and methanol (1 ml) were added. The mixture was stirred at 50 °C for 1 h. After cooling to room temperature, the solvent was removed by rotary evaporation. No purification was required to obtain the crude product compound W4.

[0077] Under argon protection, cyanoacetic acid (1 mmol) was added to the reaction flask, followed by 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.5 mmol), 4-dimethylaminopyridine (0.25 mmol), compound W4 (1 mmol), dichloromethane (4 ml), and triethylamine (2 ml). The mixture was stirred in an ice bath until it returned to room temperature, and then stirred overnight. After the reaction was complete, the mixture was extracted directly with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (dichloromethane:methanol = 20:1 v / v) to give a pale yellow solid compound W5.

[0078] Under argon protection, compounds W2 (0.3 mmol) and W5 (0.1 mmol) were added to a reaction flask, dissolved in anhydrous ethanol (5 ml), and then piperidine (0.1 ml) was added. The mixture was stirred at 60 °C for 2 h. After cooling to room temperature, the solvent was removed by rotary evaporation, and the crude product was purified by column chromatography (dichloromethane:methanol = 10:1 v / v) to give the red solid compound QP-4. 1H NMR(400MHz,Chloroform-d)δ8.39(s,1H),8.03(d,J=9.4Hz,1H),7.94(d,J=8.5Hz,1H),7.62(d,J=8.5Hz,1H),7.39(dd,J=9.5,2.8Hz,1H),7.31(s,2H),6. 93(d,J=8.4Hz,2H),6.79-6.68(m,2H),5.23(p,J=7.0Hz,1H),3.24(t,J=5.0H z, 4H), 3.14 (s, 6H), 2.64 (t, J = 5.0Hz, 4H), 2.40 (s, 3H), 1.60 (d, J = 6.9Hz, 3H). 13 C NMR(101MHz,Chloroform-d)δ159.60,151.31,150.55,149.88,145.15,142.22,133.75,133.33,131.18,130.78,1 27.12,123.62,119.98,117.42,116.21,105.38,103.62,54.98,49.43,48.78,45.96,40.43,31.52,29.71,21.52.

[0079] The proton and carbon spectra of compounds W3, W5, and QP-4 prepared in this embodiment are shown below. Figures 1-6 As shown, the mass spectrum of QP-4 is as follows: Figure 7 As shown.

[0080] Example 2

[0081] Fluorescence performance detection of QP-4

[0082] A 1 mM stock solution of QP-4 was prepared using DMSO. This stock solution was then diluted in PBS (pH 7.4, 1% DMSO) for excitation and emission spectroscopy analysis. The results showed that ( Figure 8 The maximum excitation wavelength of QP-4 in PBS was 476 nm, and the maximum emission wavelength was 674 nm. QP-4 exhibited strong near-infrared fluorescence emission. These results indicate that QP-4 possesses near-infrared fluorescence emission properties, and that normal cells and tumor cells take up different amounts of the compound, suggesting its potential for early diagnosis of gliomas.

[0083] Example 3

[0084] Flow cytometry detection after different co-incubation times of QP-4, bEnd.3, and U87MG cells

[0085] U87MG and bEnd.3 cells were seeded in 6-well plates and cultured in a cell culture incubator (37℃, 5% CO2) until logarithmic growth. The cells were then co-incubated with 5 μM QP-4 medium at 30 min, 1 h, 2 h, and 3 h, respectively. Afterward, the cells were washed three times with PBS (pH 7.4) to remove excess drug. The cells were then digested with trypsin containing 0.25% EDTA for 30 seconds, followed by addition of serum-containing medium to stop the digestion. The cells were then centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in PBS (pH 7.4). After filtration through a 500-mesh filter, the same cell volume was maintained, and the mean fluorescence intensity of the FL3 (PerCP) channel was detected using flow cytometry. Significant differences in fluorescence intensity were observed between U87MG and bEnd.3 cells after different incubation times. Figure 9 The above results indicate that QP-4 can be used to distinguish glioma cells U87MG from mouse brain microvascular endothelial cells bEnd.3.

[0086] Example 4

[0087] After co-incubation of QP-4 with bEnd.3 and different glioma cells for 30 minutes, flow cytometry was used for detection.

[0088] U87MG, U343MG, SH-SY5Y, U251MG, GL261, and bEnd.3 cells were seeded in 12-well plates and cultured in a cell culture incubator (37℃, 5% CO2) until logarithmic growth. The cells were then co-incubated with 5 μM QP-4 medium at a final concentration for 30 minutes. After washing away excess drug, the cells were digested with trypsin containing 0.25% EDTA for 30 seconds–1 minute, followed by stopping the digestion with serum-containing medium. The cells were then centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS. After filtering through a 500-mesh filter, maintaining consistent cell volume, the mean fluorescence intensity of the FL3 (PerCP) channel was detected using flow cytometry. Significant differences in fluorescence intensity were observed between glioma cells and bEnd.3 cells after 30 minutes of incubation with different cell types. Figure 10 The above results indicate that QP-4 can be used to distinguish between different types of glioma cells and mouse brain microvascular endothelial cells (bEnd.3).

[0089] Example 5

[0090] Confocal imaging after incubation of QP-4 with bEnd.3 and U87MG

[0091] U87MG and bEnd.3 cells were seeded in confocal dishes and cultured in a cell culture incubator (37℃, 5% CO2) until confluence reached 70%. The cells were then co-incubated with complete medium at a final concentration of 5 μM QP-4 for 15 min. After incubation, the cells were washed three times with PBS, followed by the addition of incomplete medium without phenol red. The red channel (λ) was observed under a confocal microscope at 100x magnification. ex =488nm, λ em =662-737nm) imaging. The mean fluorescence intensity of QP-4 in U87MG cells was significantly higher than that in bEnd.3 cells ( =662-737nm). Figure 11 The above results indicate that QP-4 has the potential for early diagnosis of gliomas.

[0092] Example 6

[0093] Mitochondrial colocalization imaging after incubation with QP-4 and U87MG

[0094] U87MG cells were seeded in confocal dishes and cultured in a cell culture incubator (37℃, 5% CO2) until confluence reached 70%. Cells were co-incubated for 30 min with complete medium containing 200 nM commercial mitochondrial fluorescent dye (Mito-Green, Beyotime (C1048)). After incubation, the cells were washed three times with PBS. Subsequently, the cells were co-incubated for 15 min with complete medium containing 5 μM QP-4. After washing three times with PBS, incomplete medium without phenol red was added. The green and red channels were imaged separately under a confocal microscope at 100x magnification. (Green channel λ) ex =488nm, λ em =500-550nm; Red channel λ ex =488nm, λ em =662-737nm), the results show that ( Figure 12 The QP-4 compound is not located in mitochondria and has no significant targeting ability in mitochondria (Pearson coefficient: 0.73).

[0095] Example 7

[0096] Lysosomal colocalization imaging after incubation of QP-4 and U87MG

[0097] U87MG cells were seeded in confocal dishes and cultured in a cell culture incubator (37℃, 5% CO2) until confluence reached 70%. Cells were co-incubated for 30 min with complete medium containing 2 μM commercial lysosomal fluorescent dye (Lyso-Green), followed by three washes with PBS. Cells were then co-incubated for 15 min with complete medium containing 5 μM QP-4, followed by three washes with PBS. Finally, incomplete medium without phenol red was added. The green and red channels were imaged separately under a confocal microscope at 100x magnification. (Green channel λ) ex =488nm, λ em =500-550nm; Red channel λ ex =488nm, λ em =662-737nm), the results show that ( Figure 13 QP-4 has a significant targeting ability for lysosomes (Pearson coefficient: 0.94).

[0098] Example 7

[0099] QP-4 and cell viability and IC50 after incubation with different glioma cell lines 50

[0100] Different cell types were seeded into 96-well plates at 3000-5000 cells per well and cultured until adherent in a cell culture incubator (37℃, 5% CO2). Cells were co-incubated with QP-4 complete medium at different final concentrations for 48 hours, followed by incubation with 5 mg / mL MTT for another 4 hours. The medium was then discarded, and 150 μL of DMSO was added to each well. The plates were then shaken on a shaker for 10 minutes in the dark. The absorbance at 490 nm was measured using a microplate reader, and cell viability was calculated based on the absorbance. QP-4 showed good inhibitory activity against different glioma cell lines. Under the same system, the positive compound wp1066 (CAS: 857064-38-1) showed an IC50 inhibitory effect on U87MG cells. 50 =10.87 μM, and has low toxicity to normal cells bEnd.3 (see [link]). Figure 14 (and Table 1).

[0101] Table 1. QP-4 and its IC50 values ​​for different cell types. 50 value

[0102]

[0103] Example 8

[0104] U87MG cells were seeded in 96-well plates with ultra-low adsorption, with 2000-3000 cells per well. After 4 days of culture, U87MG 3D cell spheroids were obtained. These were then cultured in medium with a final concentration of 5 μM QP-4 and 10 μM cisplatin, respectively, at 37°C in a 5% CO2 incubator. Cell changes were recorded daily, and the drug was administered again every other day for 5 consecutive days. On the fifth day, the cell spheroids were co-incubated with Calcein / PI for 30 min and then imaged under a confocal microscope (Beyotime, C2015S). The results are as follows: Figure 15 The results showed that the proportion of dead cells in cell spheres incubated with 5 μM MQP-4 for 5 days was significantly higher than that in cell spheres treated with 10 μM cisplatin, indicating that QP-4 has a good killing ability against U87MG3D cell spheres.

Claims

1. A near-infrared fluorescent compound for early diagnosis and treatment of gliomas, characterized in that, The structural formula of the compound is: 。 2. A method for preparing a near-infrared fluorescent drug compound for early diagnosis and treatment of glioma as described in claim 1, characterized in that, Includes the following steps: (1) Under inert gas protection, [(S)-1-(4-bromophenyl)ethyl] tert-butyl carbamate, cesium carbonate, S-(-)-1,1'-binaphthyl-2,2'-bis(diphenylphosphine) and palladium acetate were mixed, and then an organic solvent was added. After heating, N-methylpiperazine was added. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was extracted and purified to obtain compound W3, whose structure is as follows: ; (2) Add W3 to hydrochloric acid, stir, then add organic solvent, heat and stir. After the reaction is complete, remove the solvent by rotary evaporation to obtain compound W4, whose structure is: Under an inert gas atmosphere, cyanoacetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, compound W4, and 4-dimethylaminopyridine were added to an organic solvent and mixed. The mixture was stirred in an ice bath until it returned to room temperature. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was purified to obtain compound W5, whose structure is as follows: ; (3) Under the protection of an inert gas, compound 6-(dimethylamino)quinoline-2-carboxaldehyde and compound W5 were mixed, an organic solvent was added, the mixture was heated and stirred, and after the reaction was complete, the solvent was removed by rotary evaporation and purified to obtain the compound.

3. The preparation method according to claim 2, characterized in that, The reaction temperature in step (1) is 100-120℃ and the reaction time is 10-14h.

4. The preparation method according to claim 2, characterized in that, In step (2), the molar ratio of cyanoacetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine compound, and compound W4 is 1:1.5:0.25:1-1:3:0.5:2, and the reaction temperature is 8-12 h after cooling to room temperature in an ice bath.

5. The preparation method according to claim 2, characterized in that, In step (3), the molar ratio of compound 6-(dimethylamino)quinoline-2-carboxaldehyde to compound W5 is 3:1-5:1, the reaction temperature is 40-70℃, and the reaction time is 2-5h.

6. The use of the near-infrared fluorescent compound of claim 1 for early diagnosis and treatment of glioma in the preparation of a medicament for inhibiting tumor growth and killing tumor cells, wherein the tumor is a glioma.

7. The use of the near-infrared fluorescent compound of claim 1 for early diagnosis and treatment of glioma in the preparation of a medicament for the diagnosis and treatment of glioma cells.

8. The application according to claim 7, characterized in that, The compound effectively distinguishes between glioma cells and microvascular endothelial cells, and its application in the preparation of drugs for the diagnosis and treatment of glioma cells.

9. The application according to claim 7, characterized in that, The compound targets lysosomes of glioma cells.

10. The use of the near-infrared fluorescent compound of claim 1 for early diagnosis and treatment of glioma in the preparation of near-infrared fluorescent drugs or reagents.

11. The use of the near-infrared fluorescent compound of claim 1 for early diagnosis and treatment of glioma in the preparation of drugs or reagents for flow cytometry fluorescence detection and confocal fluorescence imaging.

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