Novel targeting near-infrared fluorescent probe NFA-NIR-Cys as well as preparation method and application of novel targeting near-infrared fluorescent probe NFA-NIR-Cys

Abstract

The invention discloses a novel targeting near infrared fluorescent probe NFA-NIR-Cys as well as a preparation method and application thereof. The preparation method of the near-infrared fluorescent probe comprises the following steps: (1) preparing a compound 2 by taking fluorenic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and a compound 1 as raw materials; (2) preparing a compound 4 by taking the compound 3, N, N-diisopropylethylamine and acryloyl chloride as raw materials; (3) preparing a compound 5 by taking 1, 1, 2-trimethyl-1H-benzo [e] indole, p-toluenesulfonic acid propargyl ester and the compound 4 as raw materials; and (4) preparing the near-infrared fluorescent probe NFA-NIR-Cys by taking the compound 5, the compound 2 and cuprous iodide as raw materials. The preparation method is simple and high in yield, and the prepared near-infrared fluorescent probe is rapid in response, wide in detection range and high in sensitivity and can be applied to detection of expression of Cys in cells.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthesis and analysis and detection, and in particular to a novel targeted near-infrared fluorescent probe NFA-NIR-Cys and a preparation method and application thereof. Background Art

[0002] Cancer, as one of the major public health issues, has seriously threatened human health. Early detection and timely treatment of cancer are urgently needed. Tumor markers are characteristic substances produced directly by tumor cells or induced by non-tumor cells through tumor cells. Biothiols are not only involved in the regulation of physiological processes, but also considered as tumor markers. It is of practical significance to use tumor markers for early cancer screening.

[0003] Cysteine ​​(Cys) is an active thiol in biological systems and a common amino acid in the growth process of organisms. Its molecular structure contains a sulfhydryl group and can participate in many physiological and pathological changes. Cysteine ​​can not only maintain the stability of redox in the body, but also regulate cell activity. Disorders in cysteine ​​levels in living cells can lead to a series of diseases, such as skin damage, liver damage, edema, lethargy, cardiovascular disease and growth retardation. Compared with traditional detection methods, fluorescent probes have excellent biocompatibility and high spatiotemporal resolution. They are mainly composed of fluorophores and recognition groups and have been considered as a new detection tool for biological thiols. Given the importance of Cys in physiological activities, the development of fluorescent probes for Cys detection has always been the focus of researchers.

[0004] Cyclooxygenase (COX) or prostaglandin endoperoxide synthase (PGH synthase) is an important enzyme in the synthesis of prostaglandins from arachidonic acid. COX is found on the luminal side of the endoplasmic reticulum and the nuclear membrane and exists in at least 2 isoforms, COX-1 and COX-2. COX-2 expression levels in normal cells are very low, but COX-2 can be induced by inflammatory stimuli and cytokines and tumor growth factors. COX-2 is expressed in a large number of human tumors (primary and metastatic) or cancer cells, and these cells may play a role in cancer progression, such as increasing cancer cell proliferation, cell survival and tumor invasion. COX-2 can be used as a target for several anticancer and nonsteroidal anti-inflammatory drug compounds, which are widely used for anti-inflammatory purposes. Indomethacin (IMC) is a nonsteroidal anti-inflammatory drug that is classified as a non-selective COX-2 inhibitor because it can also interfere with the homeostatic functions regulated by COX-1. The binding of IMC on COX-2 occurs through reversible competitive inhibition. However, when the molecular design is optimized to conjugate IMCs with fluorescent dyes, their selectivity for COX-2 can be significantly improved. Recently, several fluorophore-linked IMCs have been reported as COX-2 targets.

[0005] At present, some specific fluorescent probes for detecting Cys or COX-2 have been developed, such as a fluorescent probe based on chalcone fluorescent dye for detecting Cys in lysosomes. The probe combines morpholine with chalcone fluorophores to enable it to target lysosomes. When the probe is excited at 400nm, after adding Cys, the fluorescence at 568nm is significantly enhanced, and a large Stokes shift (168nm) occurs, and the color changes from light yellow to bright yellow visible to the naked eye. A fluorescent probe based on coumarin targeting COX-2, the probe contains a diethylamino substituent to produce an electronic effect. When the probe is excited at 375nm, intramolecular charge transfer (ICT) occurs, causing interference lines of the benzene ring, and a resonance state is formed through the "push-pull" effect on the electronic charge. By using the fluorescence polarization method to determine the affinity of its dissociation constant (Kd), it was found that the probe has a strong binding affinity with COX-2 (Kd = 0.007nmol / L). By studying the specificity of the probe for cancer cells that overexpress COX-2 and measuring time-dependent cell uptake, the blue fluorescence signal increased over time, and the fluorescence intensity of cancer cells was higher than that of normal cells, which could distinguish the two types of cells. In addition, after lipopolysaccharides (LPS) induced COX-2 expression in normal cells, the intracellular fluorescence was brighter, indicating that the compound may be a useful probe for monitoring cellular inflammation and is useful for drug development.

[0006] However, most of these probes have short emission wavelengths (<550nm), which is not conducive to fluorescence imaging of living systems, and have poor targeting. Compared with traditional fluorescent probes, near-infrared fluorescent probes have the advantages of higher signal-to-noise ratio, deeper tissue penetration, higher spatial resolution and real-time visibility. As asymmetric analogs of cyanine dyes, semi-cyanine dyes have long wavelengths and excellent imaging capabilities and have become a potential fluorophore. In addition, targeted probes for specific identification and detection of organelles remain a research hotspot. Therefore, there is a high demand for the development of targeted probes that can distinguish Cys, homocysteine ​​(homocysteine, Hcy) and glutathione (glutathione, GSH) and specifically detect them, which has practical application prospects. Summary of the invention

[0007] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a novel targeted near-infrared fluorescent probe NFA-NIR-Cys and a preparation method and application thereof, so as to solve the problems of the existing probes having short emission wavelength, insufficient sensitivity and poor targeting.

[0008] The technical solution of the present invention to solve the above technical problems is as follows:

[0009] Provided is an infrared fluorescent probe, the structural formula of which is as follows:

[0010]

[0011] The method for preparing the fluorescent probe comprises the following steps:

[0012] (1) Synthesis of compound 2: Dissolve niflumic acid (NFA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1-ethyl-(3-dimethylaminopropyl)carbonyldiimide, EDCI), 4-dimethylaminopyridine (4-dimethylaminopyridine, DMAP) and compound 1 in a solvent and stir to obtain compound 2;

[0013] (2) Synthesis of compound 4: Under the protection of a rare gas, compound 3 and N,N-diisopropylethylamine (DIPEA) were dissolved in a solvent, stirred, acryloyl chloride was added, and stirred again to obtain compound 4;

[0014] (3) Synthesis of Compound 5: 1,1,2-trimethyl-1H-benz[e]indole and propargyl p-toluenesulfonate were mixed for reaction, and after the reaction, acetic anhydride and Compound 4 were added, piperidine was added dropwise, and the reaction was continued to obtain Compound 5;

[0015] (4) Synthesis of near-infrared fluorescent molecular probe NFA-NIR-Cys: Compound 5, compound 2 and cuprous iodide are dissolved in a solvent and reacted to obtain a product;

[0016] The chemical reaction formula of the above preparation method is as follows:

[0017]

[0018] Furthermore, the above-mentioned solvent includes: N,N-dimethylformamide (DMF).

[0019] Furthermore, in step (1), the molar mass ratio of NFA, EDCI, DMAP and compound 1 is 1:2-4:0.5-1.5:0.5-1.5.

[0020] Furthermore, in step (2), the molar mass ratio of compound 3, DIPEA and acryloyl chloride is 1:0.5-1.5:1.5-2.5.

[0021] Furthermore, in step (3), the molar mass ratio of 1,1,2-trimethyl-1H-benz[e]indole, propargyl p-toluenesulfonate and compound 4 is 1:2-3:0.5-1.5.

[0022] Furthermore, in step (4), the molar mass ratio of compound 5, compound 2 and cuprous iodide is 1:2-4:1-3.

[0023] Furthermore, the stirring temperature in step (1) is 20 to 25° C. and the stirring time is 10 to 15 hours.

[0024] Furthermore, in step (2), the stirring temperature is -2 to 4°C and the stirring time is 20 to 40 minutes.

[0025] Furthermore, in step (2), the stirring temperature is 20 to 25° C. and the stirring time is 8 to 12 hours.

[0026] Furthermore, in step (3), the mixed reaction temperature is 40 to 60° C. and the time is 10 to 15 hours.

[0027] Furthermore, in step (3), the reaction temperature is continued at 50 to 70° C. and the reaction time is 1 to 3 hours.

[0028] Furthermore, in step (4), the reaction temperature is 20-25° C. and the reaction time is 8-12 h.

[0029] Application of the above-mentioned near-infrared fluorescent probe in cancer cell targeted imaging agents.

[0030] A cancer cell-targeted fluorescent probe kit, comprising the near-infrared fluorescent probe according to claim 1.

[0031] The present invention has the following beneficial effects:

[0032] (1) The near-infrared fluorescent probe prepared by the present invention has a fast response, a wide detection range, and a high sensitivity. It can be used to detect changes in endogenous and exogenous Cys in cells and can effectively distinguish cancer cells that overexpress COX-2 from normal cells.

[0033] (2) The present invention has near-infrared fluorescence emission, which can effectively avoid the interference of self-absorption and biological background fluorescence, and can avoid errors caused by uneven probe concentration and probe distribution.

[0034] (3) The preparation method of the present invention is simple, the raw materials are cheap and easily available, the synthesis process is simple, the yield is high, and it is suitable for large-scale production and promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the absorption spectrum of Example 2 in the PBS-CH3CN buffer system, where 0-200 μmol / L Cys reacts with 10 μmol / L probe NFA-NIR-Cys;

[0036] Figure 2 This is the fluorescence response diagram of 0-200 μmol / L Cys and 10 μmol / L probe NFA-NIR-Cys in PBS-CH3CN buffer system in Example 3;

[0037] Figure 3 This is the absorption spectra of Example 4 in which 10 μmol / L probe NFA-NIR-Cys reacts with 200 μmol / L different analytes in a PBS-CH3CN buffer system;

[0038] Figure 4 The fluorescence emission diagram of the probe NFA-NIR-Cys reacting with different analytes in the PBS-CH3CN buffer system in Example 5;

[0039] Figure 5 The fluorescence response diagram of the probe NFA-NIR-Cys and different analytes in the PBS-CH3CN buffer system in Example 5;

[0040] Figure 6 This is the fluorescence response diagram of the probe NFA-NIR-Cys in Example 6 at 723 nm when different analytes were added to the PBS-CH3CN buffer system and then Cys was added, wherein the left side of the bar graph is the experimental group without adding Cys, and the right side is the experimental group with adding Cys;

[0041] Figure 7 The graphs of the fluorescence intensity of the probe NFA-NIR-Cys in Example 7 and the Cys solutions of 0 μmol / L, 100 μmol / L, 150 μmol / L, and 200 μmol / L, respectively, versus time, wherein the curves from top to bottom are the Cys solutions of 0 μmol / L, 100 μmol / L, 150 μmol / L, and 200 μmol / L, respectively;

[0042] Figure 8 The fluorescence intensity diagram of the probe containing or not containing Cys at 723 nm in Example 8 under the PBS-CH3CN buffer system at different pH ranges;

[0043] Fig. 9 This is a linear correlation diagram of the fluorescence intensity of the probe LY-Cys at 723 nm and the Cys concentration in the PBS-CH3CN buffer system in Example 8;

[0044] Fig.10 This is a response mechanism diagram between the probe NFA-NIR-Cys and Cys in Example 9;

[0045] Fig.11 This is the high-resolution mass spectrum of the probe NFA-NIR-Cys after the reaction with Cys in Example 9;

[0046] Fig.12 This is a graph showing the activity of probe NFA-NIR-Cys in ID8 cells measured by CCK-8 method in Example 10;

[0047] Fig.13 This is the confocal fluorescence imaging image of Example 11 probe NFA-NIR-Cys after incubation with different cells for 30 minutes;

[0048] Fig.14 This is a comparison of the fluorescence intensity of the probe NFA-NIR-Cys in Example 11 after incubation with different cells for 30 minutes;

[0049] Fig.15 This is the confocal fluorescence imaging of Example 11 in which 250 μmol / L Cys, GSH, and Hcy were respectively added to ID8 cells and incubated for 30 min, and then the probe NFA-NIR-Cys was added and incubated for 30 min;

[0050] Fig.16 This is a fluorescence intensity comparison diagram of Example 11 in which 250 μmol / L Cys, GSH, and Hcy were respectively added to ID8 cells and incubated for 30 min, and then the probe NFA-NIR-Cys was added and incubated for 30 min;

[0051] Fig.17 This is the confocal fluorescence imaging of Example 11 in which N-ethylmaleimide (NEM) was first added to ID8 cells, NEM+Cys was incubated for 30 minutes, and then the probe NFA-NIR-Cys was added and incubated for 30 minutes;

[0052] Fig.18 This is a fluorescence intensity comparison diagram of Example 11 in which NEM and NEM+Cys were first added to ID8 cells and incubated for 30 minutes, and then the probe NFA-NIR-Cys was added and incubated for 30 minutes;

[0053] Fig.19 This is the confocal fluorescence imaging of Example 11 in which NFA and IMC were first added to ID8 cells and incubated for 30 minutes, and then the probe NFA-NIR-Cys was added and incubated for 30 minutes;

[0054] Fig. 20 This is a fluorescence intensity comparison chart of Example 11 in which NFA and IMC were first added to ID8 cells and incubated for 30 minutes, and then the probe NFA-NIR-Cys was added and incubated for 30 minutes. DETAILED DESCRIPTION

[0055] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0056] Embodiment 1:

[0057] A method for preparing a near-infrared fluorescent probe comprises the following steps:

[0058] (1) The synthesis method of compound 1 refers to Emilie M, Anne-Sophie B, Elodie Q, et al. Intracellular location matters: rationalization of the anti-inflammatory activity of a manganese (II) superoxide dismutase mimic complex [J]. ChemComm, 2020, 56: 7885-7888.

[0059] For the synthesis method of compound 3, please refer to the literature Yu Chen, Yumin Wang, Xianghua Wu, et al. Discriminative detection of cysteine / homocysteine ​​and glutathione in HeLacells by dual-channel fluorescent probe [J]. Dyes and Pigments, 2021, 186: 109015.

[0060] (2) Synthesis of Compound 2: NFA (2.56 g, 9.08 mmol), EDCI (5.22 g, 27.24 mmol), DMAP (1.10 g, 9.08 mmol) and Compound 1 (2.5 mL, 9.08 mmol) were dissolved in 50 mL DMF and stirred at room temperature for 12 h. The mixture was extracted with saturated sodium chloride solution and ethyl acetate three times, and the organic solvent was dried with anhydrous Na2SO4. The mixture was separated by column chromatography under the conditions of 1:1 volume ratio of petroleum ether and dichloromethane to obtain a colorless oil compound 2 with a yield of 40%. The NMR test results of Compound 2 are as follows: 1 H NMR (500MHz, MeOD) δ8.34-8.31(m,1H),8.24-8.17(m,2H),7.74(dd,J=8.2,1.7Hz,1H),7.41(t,J=8.0Hz,1H),7.24(d,J=7.7Hz,1H) ,6.78(dd,J=7.8,4.8Hz,1H),4.26(t,J=6.6Hz,2H),3.26(t,J=6.8Hz,2H),1.78-1.70(m,2H),1.61-1.54(m,2H),1.47-1.39(m,4H).

[0061] (3) Synthesis of compound 4: Under argon protection, compound 3 (0.114 g, 0.5 mmol) and DIPEA (0.1 mL, 0.5 mmol) were dissolved in 10 mL of dichloromethane solution, stirred at 0°C for 30 min, then acryloyl chloride (0.15 mL, 1 mmol) was added and stirred at room temperature for 10 h. After the reaction was completed, it was extracted with saturated sodium chloride solution and ethyl acetate three times, the organic solvent was dried with anhydrous Na2SO4, and separated by column chromatography. The column chromatography conditions were a volume ratio of petroleum ether to dichloromethane of 1:1, and a yellow solid compound 4 was obtained with a yield of 20%. The NMR test results of compound 4: 1H NMR (500MHz, CDCl3) δ10.34(s,1H),7.20(d,J=8.3Hz,1H),6.99(s,1H),6.91(d,J=8.3Hz,1H),6.72-6.61(m,2H),6 .34(dd,J=17.3,10.5Hz,1H),6.08(d,J=10.5Hz,1H),2.66-2.57(m,2H),2.47(t,J=6.0Hz,2H),1.79-1.72(m,2H).

[0062] (4) Synthesis of Compound 5: A mixture of 1,1,2-trimethyl-1H-benz[e]indole (210 mg, 1 mmol) and propargyl p-toluenesulfonate (0.4 mL, 2.3 mmol) was reacted at 50°C for 12 h. After cooling to room temperature, 10 mL of acetic anhydride and compound 4 (0.268 g, 0.9 mmol) were added to the reaction mixture, and 0.5 mL of piperidine was slowly added dropwise. The mixture was reacted at 60°C for 2 h. The organic layer was extracted with ether and saturated sodium chloride solution, dried with anhydrous Na2SO4, filtered, and separated by column chromatography. The column chromatography conditions were a volume ratio of petroleum ether to dichloromethane of 1:1, and a dark blue solid compound 5 was obtained with a yield of 75%. The NMR test results of compound 5 are as follows: 1 H-NMR (500MHz, CDCl3) δ8.80(d,J=13.6Hz,1H),8.19(d,J=7.7Hz,1H),8.05(dd,J=16.0,8.1Hz,2H),7. 79(d,J=8.5Hz,1H),7.70(d,J=7.3Hz,1H),7.60(t,J=7.3Hz,1H),7.42(d,J=8.3Hz,1H),7.21(d,J=9.5H z,1H),7.15-7.05(m,2H),6.97(d,J=7.7Hz,1H),6.72(d,J=17.3Hz,1H),6.40(dd,J=17.1,10.4Hz,1H) ,6.16(d,J=10.4Hz,1H),5.85(s,2H),2.93(s,2H),2.76(s,2H),2.49(s,1H),1.98(s,2H),1.78(s,6H). 13C-NMR (126MHz, DMSO-d6): δ=179.67,163.88,160.22,152.77,152.36,145.79 ,138.43,135.85,134.64,132.50,132.11,130.89,130.79,130.18,129.98,1 28.67,128.28,127.36,127.08,126.40,122.61,119.81,119.64,115.10,112.50,109.99,105.33,77.61,76.21,52.43,35.80,28.67,27.12,23.67,19.85.

[0063] (5) Synthesis of near-infrared fluorescent probe NFA-NIR-Cys: Compound 5 (0.09 g, 0.175 mmol), compound 2 (0.215 g, 0.525 mmol) and cuprous iodide (0.0669 g, 0.35 mmol) were dissolved in 10 mL DMF, reacted at room temperature for 10 h, extracted with saturated sodium chloride solution and ethyl acetate three times, dried the organic solvent with anhydrous Na2SO4, and separated by column chromatography. The column chromatography conditions were a volume ratio of dichloromethane to methanol of 20:1, and a dark blue solid near-infrared fluorescent probe NFA-NIR-Cys was obtained with a yield of 25%. NMR detection results of near-infrared fluorescent probe NFA-NIR-Cys: 1H-NMR(500MHz,CDCl3)δ10.40(s,1H),9.11(s,1H),8.79(d,J=15.2Hz,1H),8.41(dd,J=4.7,1.8Hz,1H),8.34(d,J=8.5Hz,1H),8.26(dd,J=7.8,1.9Hz,1H),8.17(d,J=8.4Hz,1H),8.11(d,J=6.8Hz,2H),8.02(d,J=8.2Hz,1H),7.89(d,J=7.3Hz,1H),7.66(t,J=7.3Hz,1H),7.57(t,J=7.4Hz,1H),7.50-7.30(m,3H),7.23-7.19(m,1H),7.15(d,J=1.9Hz,1H),7.05(dd,J=8.4,2.1Hz,1H),7.00(s,1H),6.82(dd,J=7.8,4.7Hz,1H),6.71(d,J=17.8Hz,1H),6.39(dd,J=17.3,10.5Hz,1H),6.28(s,2H),6.15(d,J=10.5Hz,1H),4.41(t,J=7.2Hz,2H),4.31(t,J=6.5Hz,2H),2.90(s,2H),2.72(d,J=5.6Hz,2H),1.98(d,J=7.8Hz,6H),1.77(dd,J=14.5,6.8Hz,6H),1.52-1.46(m,2H),1.42(s,2H). 13 C NMR(126MHz,CDCl3)δ180.34,167.39,163.85,159.10,155.63,152.98,152.83,152.34,145.57,140.35,140.15,139.80,139.18,135.89,133.72,133.14,131.59,131.10,130.84,130.32,130.27,129.98,129.09,127.88,127.81,127.35,127.25,126.27,125.62,123.34,122.11,119.67,118.73,116.91,115.60,114.11,113.34,109.31,107.56,107.05,65.19,52.66,50.28,41.98,29.99,29.53,28.34,27.67,26.09,25.42,24.39,20.19[M + ]:calcd for[C 54 H50 FN6O5 + ]:919.3789;found919.3800.

[0064] Example 2: Fluorescence titration experiment of near-infrared fluorescent probe NFA-NIR-Cys

[0065] In order to study the response of the near-infrared fluorescent probe NFA-NIR-Cys to Cys, the NFA-NIR-Cys prepared in Example 1 was used as the test object, and the ultraviolet absorption of 10 μmol / L probe NFA-NIR-Cys in the buffer system of CH3CN / PBS=4 / 6 (0.01 mol / L, pH=7.4) in the presence or absence of Cys was evaluated.

[0066] The experimental results are as follows Figure 1 The probe NFA-NIR-Cys shown in the figure has an ultraviolet absorption peak at 620nm and a shoulder peak at around 660nm. When the Cys concentration increases, the original absorption peak at 620nm decreases, and the absorption peak at 660nm red-shifts, which shows that the probe responds to Cys. When 200μmol / L Cys is added, the maximum absorption peak is reached. As the concentration of Cys increases, an isosbestic point appears at 640nm, indicating that a nucleophilic attack occurs between Cys and the probe, and Cys releases the intrinsic near-infrared fluorescent dye to restore the ICT structure. Cys shows a good linear relationship with the absorption spectrum.

[0067] Example 3: UV titration experiment of near-infrared fluorescent probe NFA-NIR-Cys

[0068] In order to study the sensitivity of NFA-NIR-Cys in detecting Cys under 650nm excitation, a series of fluorescence titration experiments were conducted using the NFA-NIR-Cys prepared in Example 1 as the test object. The probe NFA-NIR-Cys was added to a CH3CN / PBS=4 / 6 (0.01mol / L, pH=7.4) system, and different concentrations of Cys were added to react for 15 minutes and then the fluorescence data was recorded.

[0069] The experimental results are as follows Figure 2 As shown, when no Cys was added, almost no fluorescence signal was observed for the probe NFA-NIR-Cys. As the amount of Cys added increased, the emission peak at 723 nm increased. When the added Cys was 200 μmol / L, the fluorescence emission intensity reached the maximum value. During the Cys change process, the fluorescence intensity at 723 nm increased by nearly 30 times.

[0070] Example 4: UV-selective experiment of near-infrared fluorescent probe NFA-NIR-Cys

[0071] In order to study the selectivity of the probe NFA-NIR-Cys for Cys, the NFA-NIR-Cys prepared in Example 1 was used as the test object, and the probe was reacted with different amino acids (Blank, Val, Arg, Lys, His, Pro, Leu, Glu, Thr, Asp, Gly, Tyr, Iso, Met, Phe, Ser, Ala, GSH, Hcy, Cys) in a CH3CN / PBS=4 / 6 (0.01mol / L, pH=7.4) system, and the absorption was recorded after 15 min of reaction.

[0072] The experimental results are as follows Figure 3 As shown, after adding amino acids such as Val, Arg, Lys, His, Pro, Leu, Glu, Thr, Asp, Gly, Tyr, Iso, Met, Phe, Ser, and Ala, no red-shifted absorption peak appeared. After adding two amino acids, GSH and Hcy, a tiny absorption peak appeared. Only after adding Cys did there be an obvious change, producing a large red-shifted absorption peak, indicating that the probe has good selectivity for Cys and can specifically detect Cys.

[0073] Example 5: Fluorescence selection experiment of near-infrared fluorescent probe NFA-NIR-Cys

[0074] In order to study the selectivity of the probe NFA-NIR-Cys for Cys, the NFA-NIR-Cys prepared in Example 1 was used as the test object. The fluorescence changes of the probe and different amino acids (Blank, Val, Arg, Lys, His, Pro, Leu, Glu, Thr, Asp, Gly, Tyr, Iso, Met, Phe, Ser, Ala, GSH, Hcy, Cys) in CH3CN / PBS=4 / 6 (0.01mol / L, pH=7.4) system were tested. Different amino acids were added at 200μmol / L and reacted for 15min.

[0075] The experimental results are as follows Figure 4 As shown in the figure, the fluorescence signal of amino acids such as Val, Arg, Lys, His, Pro, Leu, Glu, Thr, Asp, Gly, Tyr, Iso, Met, Phe, Ser, and Ala did not change significantly. The fluorescence intensity of GSH and Hcy increased slightly. Only when 200 μmol / L Cys was added, the fluorescence intensity increased significantly, indicating that the probe is sensitive to Cys and can distinguish Cys, Hcy, GSH and other amino acids. In order to take into account the error of the experiment, the selectivity experiment was repeated three times, and the error results were calculated and analyzed, as shown in the figure. Figure 5As shown, reproducible experiments also demonstrated that the probe was sensitive to Cys and could distinguish Cys, Hcy, GSH and other amino acids.

[0076] Example 6: Anti-interference experiment of near-infrared fluorescent probe NFA-NIR-Cys

[0077] In order to further study the high selectivity of the probe NFA-NIR-Cys for Cys, a competition experiment was conducted using the NFA-NIR-Cys prepared in Example 1 as the test object. The probe NFA-NIR-Cys 10 μmol / L was reacted with different amino acids (Blank, Val, Arg, Lys, His, Pro, Leu, Glu, Thr, Asp, Gly, Tyr, Iso, Met, Phe, Ser, Ala, GSH, Hcy, Cys) in a CH3CN / PBS=4 / 6 (0.01 mol / L, pH=7.4) system, and its fluorescence was tested after 15 min of reaction.

[0078] The experimental results are as follows Figure 6 As shown in the figure, when Cys was not added to the test object, there was no significant change in the fluorescence intensity at 723nm. When Cys was further added to the probe solution containing various other amino acids, each sample produced a significant fluorescence signal at 723nm. These results show that the probe has a strong ability to resist interference from other amino acids in Cys recognition.

[0079] Example 7: Study on the temporal response of near-infrared fluorescent probe NFA-NIR-Cys to Cys

[0080] In order to further study the reaction rate of the probe NFA-NIR-Cys to Cys, the NFA-NIR-Cys prepared in Example 1 was used as the test object, and the changes in the fluorescence intensity of the probe in the presence of Cys of 0 μmol / L, 100 μmol / L, 150 μmol / L, and 200 μmol / L within 30 min were monitored.

[0081] The experimental results are as follows Figure 7 As shown in the figure, without adding Cys, the fluorescence intensity does not change with time. When Cys is added, the fluorescence intensity of NFA-NIR-Cys increases rapidly within 15 minutes and stabilizes after 30 minutes, indicating that NFA-NIR-Cys can react quickly to Cys.

[0082] Example 8: Study on the detection limit of Cys by near-infrared fluorescent probe NFA-NIR-Cys

[0083] In order to detect the sensitivity of NFA-NIR-Cys to Cys under 650nm excitation, the NFA-NIR-Cys prepared in Example 1 was used as the test object, and the probe 5 μmol / L was placed in the CH3CN / PBS=4 / 6 (0.01 mol / L, pH=7.4) system, and several groups of parallel experiments were performed to calculate the standard deviation. The fluorescence intensity of Cys between 0-40 μmol / L was then tested, and the fluorescence data were collected for graphical analysis.

[0084] The experimental results are as follows Figure 8 and Fig. 9 As shown in Figure 2, when the Cys concentration was 0-40 μmol, the fluorescence intensity was well linearly correlated with the Cys concentration (R 2 =0.9907). According to the formula LOD=3S / K (S represents the standard deviation of the blank solution, K represents the slope of the linear regression), the detection result of the probe for Cys is 0.218 μmol / L. According to the analysis of relevant data, the results show that the probe has a high sensitivity to Cys, which is conducive to its practical application.

[0085] Example 9: Experimental verification of the reaction mechanism of near-infrared fluorescent probe NFA-NIR-Cys to Cys

[0086] According to the reported literature, the recognition mechanism of acrylate as the recognition group, the recognition mechanism of the probe NFA-NIR-Cys after adding Cys is as follows: Fig.10 As shown. The propylene ester part will undergo an addition reaction with cysteine, and then the amino group will attack the ester carbon group to form a seven-membered ring lactam, the propylene ester part will leave, the ICT effect will be restored, the hydroxyl part will be released, and a strong fluorescence signal will be generated. In order to prove the recognition mechanism of the probe, the NFA-NIR-Cys prepared in Example 1 was used as the test object, and the mixture after the reaction of the NFA-NIR-Cys probe and Cys was analyzed by high-resolution mass spectrometry.

[0087] The experimental results are as follows Fig.11 As shown, after adding 10 equivalents of Cys to the probe, two peaks at m / z = 865.3702 and 1040.4007 were observed in the mass spectrum. After analyzing the data, it was found that these two mass spectrum peaks were the fluorophore product and the intermediate product after the reaction, respectively, which preliminarily proved that the product after the reaction of the probe and Cys was consistent with the expected result.

[0088] Example 10: Cytotoxicity experiment of near-infrared fluorescent probe NFA-NIR-Cys on Cys

[0089] In order to explore whether the probe NFA-NIR-Cys can be used for cell or living body imaging, the fluorescent probe in the biological system has good biocompatibility and does not produce great toxicity to cells. In this regard, the NFA-NIR-Cys prepared in Example 1 was used as the test object, and the CCK-8 test method was used to evaluate the cytotoxicity of the probe. ID8 cells were selected for experimental research, and ID8 cells were cultured in different contents of NFA-NIR-Cys for 24 hours.

[0090] The experimental results are as follows Fig.12 As shown, with the increase of the concentration of the probe NFA-NIR-Cys, the relative cell viability of ID8 cells decreased slightly, but the cell survival rate was still as high as over 90%, indicating that NFA-NIR-Cys has low cytotoxicity and can be used for imaging of intracellular Cys.

[0091] Example 11: Cellular Imaging Experiment of Cys Using Near-Infrared Fluorescent Probe NFA-NIR-Cys

[0092] (1) In order to evaluate the selectivity of the probe for normal cells and cancer cells, three normal cells GES-1 (human gastric epithelial cells); RAW264.7 (leukemia cells); HIBEC (human bile duct epithelial cells) and three cancer cells ID8 (ovarian cancer); T98 (human glioma cells); 9L (glioma cells) were selected. The NFA-NIR-Cys prepared in Example 1 was used as the test object to conduct a cell fluorescence imaging experiment. The cells were incubated with 1 μmol / L of the probe solution for 30 minutes and then irradiated using the red light channel.

[0093] The experimental results are as follows Fig.13 , 14 As shown in the figure, a weak fluorescence signal was observed in normal cells by confocal laser scanning microscopy after 30 minutes. In contrast, the analyzed cancer cells all emitted bright red fluorescence (AB). Analyzing these data, it was concluded that the probe NFA-NIR-Cys can distinguish normal cells from cancer cells and has good selectivity for cancer cells. Fig.14 This is a comparison chart of the fluorescence intensity of the probe NFA-NIR-Cys after incubation with different cells for 30 minutes, which facilitates a more intuitive understanding of the difference in fluorescence intensity.

[0094] (2) In order to evaluate whether the probe NFA-NIR-Cys can selectively recognize Cys expression in cells, ID8 cells were selected and incubated with 250 μmol / L Cys, GSH and Hcy for 30 min, respectively. Then, 1 μmol / L probe NFA-NIR-Cys was incubated with the above ID8 cells for 30 min. The cells were observed by confocal laser scanning microscopy to analyze the induction of biothiols on cells.

[0095] The experimental results are as follows Fig.15 , 16 As shown, the probe NFA-NIR-Cys emits red fluorescence after treatment. Comparing with the cell imaging after adding Cys, GSH and Hcy, it is found that adding Cys can induce cells to emit brighter red fluorescence, while adding GSH and Hcy does not enhance the fluorescence signal of cells. This shows that the probe can be used to specifically detect exogenously added Cys.

[0096] (3) In order to evaluate the probe NFA-NIR-Cys for endogenous detection of Cys in cells, NEM was used as a thiol blocker and the samples were divided into two groups. One group was incubated with NEM for 30 min, and the other group was incubated with NEM for 30 min and then 250 μmol / L Cys was added. Then, 1 μmol / L of the probe was incubated with the cells for 30 min and observed using confocal imaging.

[0097] The experimental results are as follows Fig.17 , 18 As shown, when NEM was not added, a strong fluorescence signal intensity was obtained in the red channel, representing the response of Cys originally present in the cells. After adding NEM, the original red fluorescence was observed to be greatly reduced in the red channel. This was due to the scavenging effect of NEM on biothiols, which led to a decrease in the Cys concentration in the cells. In another group, NEM and Cys were added at the same time, the scavenging effect of NEM on biothiols was inhibited, and a certain fluorescence response could be observed. The above results showed that the probe has the ability to specifically detect endogenous Cys expression in cells.

[0098] (4) In order to verify that the targeting ability of the probe NFA-NIR-Cys to cancer cells originated from the interaction between the targeting group and COX-2, IMC and NFA were co-incubated with ID8 cells for 30 min, and then the probe NFA-NIR-Cys was added and incubated for 30 min, and fluorescence measurement was performed.

[0099] The experimental results are as follows Fig.19 , 20 As shown, the experimental results show that compared with cells without COX-2 inhibitors, the cell imaging of the experimental group using COX-2 inhibitors shows a weaker fluorescence response. This is because the COX-2 inhibitor binds to COX-2 in the cell, inhibiting the targeting effect of the probe on COX-2 in the cancer cell. The experimental results prove that the ability of the probe NFA-NIR-Cys to target cancer cells comes from the interaction between the targeting group and COX-2.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A novel targeted near-infrared fluorescent probe NFA-NIR-Cys, characterized in that: The structural formula of the fluorescent probe is as follows:

2. The method for preparing the novel targeted near-infrared fluorescent probe according to claim 1, characterized in that: The following steps are involved: (1) Synthesis of Compound 2: Flunic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and Compound 1 are dissolved in a solvent and stirred to obtain Compound 2; (2) Synthesis of compound 4: Under the protection of a rare gas, compound 3 and N,N-diisopropylethylamine were dissolved in a solvent, stirred, acryloyl chloride was added, and stirred again to obtain compound 4; (3) Synthesis of Compound 5: 1,1,2-trimethyl-1H-benz[e]indole and propargyl p-toluenesulfonate were mixed for reaction, and after the reaction, acetic anhydride and Compound 4 were added, piperidine was added dropwise, and the reaction was continued to obtain Compound 5; (4) Synthesis of near-infrared fluorescent molecular probe NFA-NIR-Cys: Compound 5, compound 2 and cuprous iodide are dissolved in a solvent and reacted to obtain a product; The chemical reaction formula of the above preparation method is as follows:

3. The method for preparing the novel targeted near-infrared fluorescent probe according to claim 2, characterized in that: The solvent includes: N,N-dimethylformamide.

4. The method for preparing the novel targeted near-infrared fluorescent probe according to claim 2, characterized in that: In step (1), the molar mass ratio of the fluonic acid, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the 4-dimethylaminopyridine and the compound 1 is 1:2-4:0.5-1.5:0.5-1.

5.

5. The method for preparing the novel targeted near-infrared fluorescent probe according to claim 2, characterized in that: In step (2), the molar mass ratio of the compound 3, the N,N-diisopropylethylamine and the acryloyl chloride is 1:0.5-1.5:1.5-2.

5.

6. The method for preparing the novel targeted near-infrared fluorescent probe according to claim 2, characterized in that: In step (3), the molar mass ratio of the 1,1,2-trimethyl-1H-benz[e]indole, the propargyl p-toluenesulfonate and the compound 4 is 1:2-3:0.5-1.

5.

7. The method for preparing the novel targeted near-infrared fluorescent probe according to claim 2, characterized in that: In step (4), the molar mass ratio of the compound 5, the compound 2 and the cuprous iodide is 1:2-4:1-3.

8. The method for preparing the novel targeted near-infrared fluorescent probe according to claim 2, characterized in that: The stirring temperature of step (1) is 20-25°C and the time is 10-15h; the stirring temperature of step (2) is -2-4°C and the time is 20-40min, the further stirring temperature is 20-25°C and the time is 8-12h; the mixed reaction temperature of step (3) is 40-60°C and the time is 10-15h, the continued reaction temperature is 50-70°C and the time is 1-3h; the reaction temperature of step (4) is 20-25°C and the time is 8-12h.

9. Use of the novel targeted near-infrared fluorescent probe according to claim 1 in a targeted cancer cell imaging agent.

10. A fluorescent probe kit targeting cancer cells, characterized in that: It includes the novel targeted near-infrared fluorescent probe described in claim 1.

Images