A tumor-targeted near-infrared copper ion fluorescent probe and its preparation and application

Abstract

The present invention discloses a tumor-targeting near-infrared copper ion fluorescent probe, a preparation method thereof, and its application in biological imaging, belonging to the fields of analytical chemistry and biotechnology. The near-infrared copper ion fluorescent probe disclosed in the present invention can efficiently detect copper ions and specifically target tumor cells with high expression of CXCR4. The near-infrared emission characteristics of the probe avoid interference from biological autofluorescence, improve imaging clarity and sensitivity, and its high selectivity and sensitivity ensure accurate detection of copper ions in complex biological environments. It is suitable for early diagnosis and research of tumors; the preparation method is simple, low-cost, and suitable for large-scale production. The probe of the present invention can not only monitor the changes of copper ions in tumors in real time and reveal the mechanism of tumors, but also has broad clinical application prospects, including tumor staging, treatment monitoring, etc. This invention provides important technical support for biomedical research and clinical diagnosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical applications, and in particular relates to a tumor-targeting near-infrared copper ion fluorescent probe and its preparation and application. Background Art

[0002] Copper (II) ions (Cu 2+ Copper, an essential trace element in living organisms, plays a crucial role in numerous biological processes, including enzyme catalysis, regulation of protein function, DNA replication and repair, and cell differentiation and proliferation. Studies have shown that tumors have a higher demand for copper than normal tissues. Clinical studies have shown that copper ion levels are significantly elevated in the serum or tumor tissues of cancer patients, and higher copper ion levels often correlate with poorer clinical stage and prognosis. Copper can increase genomic instability, promote angiogenesis, and upregulate pro-tumor signaling pathways, thereby enhancing tumor metastasis and invasiveness. However, excessive copper ion levels have been shown to induce tumor cell death through pathways such as apoptosis or oxidative stress. The newly discovered copper death mechanism also provides a new potential target for cancer therapy. Therefore, the high demand and abnormal accumulation of copper ions in tumor cells not only promotes tumor growth, metastasis, and invasion, but also provides a new approach for cancer treatment: regulating copper ion levels to influence tumor cell fate. However, due to the heterogeneity and complexity of tumor cells, accurate, rapid, and real-time detection of copper ion levels in tumor cells has become an urgent challenge.

[0003] Traditional methods for detecting copper ions, such as atomic absorption spectroscopy and inductively coupled plasma mass spectrometry, although highly accurate, have limitations such as complex operation, long time consumption, and inability to perform real-time in situ detection, making it difficult to meet the rapid detection needs in clinical and scientific research. In complex biological environments, especially inside tumor cells, achieving specific detection of copper ions remains challenging. The copper ion concentration in tumor cells may vary depending on disease progression, tumor type, and individual differences. Traditional detection methods may have limitations in sensitivity and accuracy, making it difficult to capture subtle concentration changes.

[0004] In recent years, fluorescent probe technology has been widely used in the biomedical field due to its advantages such as high sensitivity, high selectivity, real-time in situ detection, and good biocompatibility. Fluorescent probes can specifically bind to target molecules, causing changes in the fluorescent signal, thereby enabling quantitative detection of target molecules. In tumor cells, fluorescent probes can penetrate the cell membrane and enter the cell interior, reflecting the dynamic changes of specific molecules in real time, providing important information for tumor diagnosis and treatment.

[0005] CXCR4 is a specific receptor for the chemokine CXCL12. The CXCL12 / CXCR4 axis is associated with multiple mechanisms of tumor metastasis, proliferation, adhesion, and angiogenesis. Due to the high metabolic activity of tumor cells, CXCL12 attracts endothelial stem cells and, in synergy with various growth factors such as vascular epidermal growth factor (VEGF), increases endothelial cell growth and forms numerous new, tiny blood vessels. CXCR4 is also highly expressed on the surface of various cancer cells, including ovarian cancer cells, and plays a crucial role in their growth and ability to metastasize.

[0006] Therefore, given the core role of CXCR4 as a specific receptor for the chemokine CXCL12 in tumor biology, and its close connection with key processes such as tumor metastasis, proliferation, adhesion and angiogenesis, the development of a fluorescent probe that can simultaneously and specifically detect CXCR4 and copper ions has extremely high scientific value and clinical application prospects, and is of great significance for improving the accuracy of tumor diagnosis and guiding the formulation of tumor treatment plans. Summary of the Invention

[0007] In response to the difficulty of copper ion detection in tumor cells, how to achieve high sensitivity, high selectivity and real-time detection of copper ions in the complex tumor environment, while giving fluorescent probes tumor specificity to accurately reflect the copper ion level in tumor cells and provide a reliable basis for tumor diagnosis, staging and copper-based therapy, the present invention aims to provide a tumor-targeted near-infrared copper ion probe and its preparation and application.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides a tumor-targeting near-infrared copper ion fluorescent probe, wherein the fluorescent probe has any one of the following structures:

[0010]

[0011] The present invention provides a method for preparing the tumor-targeting near-infrared copper ion fluorescent probe, comprising:

[0012] S1, under argon protection, 4-diethylaminosalicylaldehyde and cesium carbonate were mixed, reacted with 2-bromocyclohex-1-ene-1-carbaldehyde, extracted, and purified by column chromatography to prepare compound D1;

[0013] The structural formula of the compound D1 is:

[0014]

[0015] S2, under argon protection, compound D1 in S1 was mixed with 1-ethyl-2-methylquinolinium iodide, and a solvent was added to react to obtain compound D2. Boron tribromide was added to react at 0-5°C, diluted with dichloromethane, quenched with sodium carbonate solution, extracted, filtered, and purified by column chromatography to obtain compound D3;

[0016]

[0017] The structural formula of the compound D2 is:

[0018] The structural formula of the compound D3 is:

[0019] Compound D1 obtained by S1 was dissolved at 0-5°C and boron tribromide was added for reaction. The mixture was neutralized, extracted, dried, and purified by column chromatography to obtain compound D4. Under argon protection, compound D4 was mixed with 1-ethyl-2-methylquinolinium iodide, acetic anhydride was added for reaction, filtered, dried, and purified by column chromatography to obtain compound D5.

[0020]

[0021] The structural formula of the compound D4 is:

[0022] The structural formula of the compound D5 is:

[0023] S3, di-tert-butyl dicarbonate reacts with 1,4,8,11-tetraazacyclotetradecane, and the mixture is purified by column chromatography to obtain compound D6, compound D6, anhydrous dichloromethane, p-chloromethylbenzoyl chloride, and triethylamine are mixed for reaction, extracted, purified, concentrated, and dissolved in acetone, and sodium iodide is added to evaporate the solvent to obtain compound D7;

[0024]

[0025]

[0026] The structural formula of the compound D6 is:

[0027] The structural formula of the compound D7 is

[0028] S4, reacting compound D3 or compound D5 obtained in S2 with compound D7 obtained in S3, extracting, purifying, concentrating in vacuo, drying, adding a hydrochloric acid solution containing dioxane, and reacting again to purify and obtain a fluorescent probe that targets the CXCR4 receptor and simultaneously detects copper ions;

[0029]

[0030] The molar ratio of 4-diethylamino salicylaldehyde, cesium carbonate and 2-bromocyclohex-1-ene-1-carboxaldehyde in S1 is 2-3:4-5:1, the reaction temperature is 24-26° C., and the reaction time is 16-20 h.

[0031] The solvents used for the extraction in S1 are water and dichloromethane, and the mobile phase used for the column chromatography purification is petroleum ether and ethyl acetate in a volume ratio of 6:1.

[0032] The molar ratio of compound D1 in S2 to 1-ethyl-2-methylquinolinium iodide is 1:1, the solvent is acetic anhydride, the reaction temperature is 100-120° C., and the reaction time is 10-14 h.

[0033] The molar ratio of compound D2 to boron tribromide in S2 is 1:20-25, and the reaction is carried out at room temperature for 20-26 hours. The column chromatography purification uses dichloromethane and methanol in a volume ratio of 30:1 as the mobile phase.

[0034] The molar ratio of compound D1 to boron tribromide in S2 is 1:20-22, and the reaction is carried out at room temperature for 14-18 hours. The extraction is performed using dichloromethane as a solvent for 2-4 times, and the column chromatography purification is performed using dichloromethane and methanol in a volume ratio of 30:1 as the mobile phase;

[0035] The molar ratio of the compound D4 to 1-ethyl-2-methylquinolinium iodide is 1:1-2, the reaction temperature is 100-120° C., and the reaction time is 8-12 hours.

[0036] The molar ratio of di-tert-butyl dicarbonate to 1,4,8,11-tetraazacyclotetradecane in S3 is 2-3:1, and the reaction is carried out at room temperature for 16-20 hours. The column chromatography purification adopts a mobile phase of petroleum ether and ethyl acetate with a volume ratio of 20:1; the molar ratio of compound 6, p-chloromethylbenzoyl chloride and triethylamine is 1:20-30:20-30, and the reaction is carried out at room temperature for 3-5 hours.

[0037] The molar ratio of the compound D3 or compound D5 to the compound D7 is 1:2-3, and the reaction is carried out at 50-70° C. for 3-5 hours.

[0038] The present invention provides the application of the tumor-targeting near-infrared copper ion fluorescent probe for detecting and imaging exogenous copper ions in tumor cells, wherein the tumor targeting is targeting the CXCR4 receptor.

[0039] Compared with the prior art, the present invention achieves the following technical effects:

[0040] The present invention provides a tumor-targeting near-infrared copper ion fluorescent probe, which has 1) efficient copper ion detection and specific tumor targeting: the probe achieves rapid and highly selective detection of copper ions through a specific coordination reaction with copper ions, which not only improves the accuracy of detection but also reduces interference from other metal ions in the biological environment. At the same time, the probe can be specifically enriched in tumor cells with high CXCR4 expression, providing a powerful tool for early diagnosis of tumors; 2) excellent fluorescence performance: the probe's emission wavelength is in the near-infrared region (685nm), which can effectively avoid interference from biological autoluminescence signals and improve the signal-to-noise ratio and clarity of fluorescence imaging; the probe has a large Stokes shift, which helps to reduce the interference of excitation light on emission light, further improving the sensitivity and accuracy of fluorescence detection; 3) strong tissue penetration and real-time imaging capability: the probe can respond to changes in copper ions in tumor cells in real time and quickly, achieving dynamic imaging. Therefore, the fluorescent probe of the present invention has significant advantages and broad application prospects in copper ion detection and tumor imaging, providing strong technical support for the diagnosis, treatment and basic research of tumors.

[0041] The preparation method of the tumor-targeting near-infrared copper ion fluorescent probe provided by the present invention is prepared through a multi-step organic synthesis reaction. The reaction conditions are mild and easy to control, and the purification steps are simple, making it suitable for large-scale production. The raw materials are readily available and the cost is low, which is conducive to practical application. Through the carefully designed molecular structure, the fluorescent probe can specifically recognize and bind to the CXCR4 receptor and exhibit a highly sensitive response to copper ions. This high selectivity and sensitivity ensure the accuracy and reliability of the probe in complex biological environments.

[0042] The fluorescent probe provided by the present invention can deeply understand the molecular mechanisms of tumor growth, metastasis and treatment by detecting changes in copper ions in tumor cells, and can be used to evaluate the efficacy of tumor targeted therapy, providing a new tool for the detection and imaging of exogenous copper ions in tumor cells. Due to the high expression of CXCR4 receptors in various tumor cells, this fluorescent probe has potential clinical application value in the early diagnosis, staging, treatment monitoring and prognosis evaluation of tumors. Through further research and optimization, this probe can also be used for the research and treatment of other diseases related to CXCR4 receptors and copper ion metabolism. The tumor-targeting near-infrared copper ion fluorescent probe provided by the present invention has important technical effects and broad application prospects in biomedical research and clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The UV absorption spectra of the probes DHX-Cu1 and DHX-Cu2 of the present invention before and after responding to copper ions (0-50 μM); (A) is the probe DHX-Cu1, and (B) is the probe DHX-Cu2;

[0044] Figure 2 The fluorescence titration spectra of the probes DHX-Cu1 and DHX-Cu2 of the present invention before and after responding to copper ions (0-50 μM); (A) is the probe DHX-Cu1, and (B) is the probe DHX-Cu2;

[0045] Figure 3 The selective and interfering fluorescence spectra of the fluorescent probes DHX-Cu1 and DHX-Cu2 for copper ions according to the present invention are shown in Figure 1; (A) is the probe DHX-Cu1, and (B) is the probe DHX-Cu2.

[0046] Figure 4 Figure 1 is a diagram showing the binding patterns of the fluorescent probes DHX-Cu1 and DHX-Cu2 of the present invention with the CXCR4 receptor protein (3odu); (A) is the probe DHX-Cu1, and (B) is the probe DHX-Cu2;

[0047] Figure 5 The CXCR4 receptor antagonism and IC of the fluorescent probe of the present invention on CCRF-CEM cells with high expression of CXCR4 receptor 50 Value graph; (A) is the probe DHX-Cu1, (B) is the probe DHX-Cu2, (C) is the positive drug AMD3100, and (D) is the antagonistic effect of different compounds (5 μM) on CXCR4 receptor;

[0048] Figure 6 The laser confocal imaging images of the fluorescent probe of the present invention specifically recognizing exogenous copper ions in A2780 cells; wherein, (A1), (A2) and (A3) are control groups, in which only DMSO was added; (B1), (B2) and (B3) were incubated with only 10 μM probe DHX-Cu1 for 15 min; (C1), (C2) and (C3) were incubated with 2 μM Cu (GTSM) for 2 h and then incubated with 10 μM probe DHX-Cu1 for 15 min; (D1), (D2) and (D3) were first incubated with 200 μM BCS as a copper ion chelator for 4 h and then incubated with 10 μM probe DHX-Cu1 for 15 min; after treatment, each group was directly imaged with FV3000 laser confocal imaging, scale bar 20 μM;

[0049] Figure 7These are laser confocal imaging images of the fluorescent probe of the present invention specifically recognizing exogenous copper ions in A2780 cells; (A) Control group, only DMSO was added; (B) Incubation with only 10 μM probe DHX-Cu2 for 15 min; (C) Cu(GTSM) 2 μM was incubated for 2 h, followed by incubation with 10 μM DHX-Cu2 for 15 min; (D) 200 μM BCS was first incubated as a copper ion chelator for 4 h, followed by incubation with 10 μM probe DHX-Cu2 for 15 min. After treatment, each group was directly imaged using FV3000 laser confocal imaging. Scale bar 20 μM.

[0050] Figure 8 These are laser confocal images of the fluorescent probe of the present invention on SKOV3 ovarian cancer cells with high expression of CXCR4 and IOSE80 normal ovarian cells with low expression of CXCR4; (A1) is the bright field of the probe DHX-Cu1 on normal ovarian cells ISOE80; (B1) is the bright field of the probe DHX-Cu2 on normal ovarian cells ISOE80; (C1) is the bright field of the probe DHX-Cu1 on SKOV3 ovarian cancer cells with high expression of CXCR4; (D1) is the bright field of the probe DHX-C u2 Bright field of SKOV3 ovarian cancer cells with high expression of CXCR4, (A2) cells were incubated with 10μM probe DHX-Cu1 for 30 minutes; (C2) cells were incubated with 10μM probe DHX-Cu1 for 30 minutes; (B2) cells were incubated with 10μM probe DHX-Cu2 for 30 minutes; (D2) cells were incubated with 10μM probe DHX-Cu2 for 30 minutes; after treatment, each group was directly imaged with FV3000 laser confocal imaging, scale bar 20μM. DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0052] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0053] In the present invention, unless otherwise specified, all experimental materials used are commercially available products well known to those skilled in the art.

[0054] Example 1

[0055] This embodiment provides a tumor-targeting near-infrared copper ion fluorescent probe DHX-Cu1, and the specific preparation steps are as follows:

[0056] (1) Preparation of Compound D1

[0057]

[0058] 4-Diethylaminosalicylaldehyde (2.93 g, 19.1 mmol) and cesium carbonate (11.05 g, 33.9 mmol) were weighed and added to a 100 mL two-necked flask. The mixture was evacuated and filled with argon. Then, the compound 2-bromocyclohex-1-ene-1-carbaldehyde (CAS-38127-47-8) (4.32 g, 9 mmol) dissolved in DMF was added to the two-necked flask filled with argon, and the mixture was stirred at 25°C for 18 h. After the reaction, the reaction solution was extracted with a mixed solvent of water and dichloromethane, and the organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain a crude product, which was then purified by column chromatography (petroleum ether:ethyl acetate = 6:1) to obtain 2.31 g of compound D1 as an orange-yellow oil with a yield of 42%.

[0059] 1 H NMR (400MHz, Chloroform-d) δ10.24(s,1H),7.01(d,J=9.1Hz,1H),6.70–6.36(m,6H),2.56–2.43(m,4H),2.37(t,J=5.9Hz,3H),1.82-1.39(m,3H).

[0060] The structural formula of compound D1 is:

[0061] (2) Preparation of Compound D2

[0062]

[0063] Compound D1 (484 mg, 2 mmol) and 1-ethyl-2-methylquinolinium iodide (CAS-606-55-3) (600 mg, 2 mmol) were added to a 100 mL two-necked flask. The mixture was evacuated and supplemented with argon. Acetic anhydride (10 mL) was added as a solvent, and the mixture was refluxed at 110° C. for 12 h. After the reaction, water was added, the mixture was filtered under reduced pressure using a Buchner funnel, and dried to obtain 672.3 mg of a bluish-purple solid compound D2 with a yield of 85%.

[0064] 1H NMR (400MHz, DMSO-d6) δ8.66(d,J=9.1Hz,1H),8.57(d,J=9.3Hz,1H),8.49(d,J=14.7Hz,1H),8.31( d,J=9.0Hz,1H),8.15(d,J=7.9Hz,1H),7.98(t,J=7.9Hz,1H),7.72(t,J=7.5Hz,1H),7.29(d,J=8.5H z,1H),7.20(d,J=2.5Hz,1H),7.02(s,1H),6.76(dd,J=8.5,2.5Hz,1H),6.63(d,J=14.8Hz,1H),4.87 (q,J=7.2Hz,2H),3.86(s,3H),2.71-2.53(m,4H),1.76(p,J=6.5,6.0Hz,2H),1.50(t,J=7.1Hz,3H).

[0065] The structural formula of compound D2 is:

[0066] (3) Preparation of Compound D3

[0067]

[0068] Compound D2 (396 mg, 1 mmol) was added to a 100 mL round-bottom flask, evacuated, and filled with argon. Once the system was filled with argon, anhydrous dichloromethane (20 mL) was added as the solvent. Boron tribromide (20 mmol) was slowly added at 0°C, and the mixture was allowed to react at room temperature for 24 h. After the reaction, saturated sodium carbonate solution was added to quench the reaction. The aqueous phase was extracted with dichloromethane three times. The organic phase was collected and dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the crude product. The product was purified by column chromatography (dichloromethane:methanol = 30:1) to obtain 270 mg of a bluish-purple solid, Compound D3, in a 70% yield.

[0069] 1 H NMR(600MHz,DMSO-d6)δ10.36(s, 1 H),8.66(d,J=6.0Hz, 1H),8.60–8.49(m,2H),8.33(d,J=12.0Hz,1H),8.21(d,J=6.0Hz,1H),8.01(t,J=6.0Hz,1H),7.76(t,J=6.0Hz,1H),7.24(d,J=6. 0Hz,1H),7.05(s,1H),6.93(s,1H),6.67(m,2H),4.89(q,J=6.0Hz,6.0Hz,2H),2.62(m,4H),1.78(s,2H),1.51(t,J=6.0Hz,3H).

[0070] The structural formula of compound D3 is:

[0071] (4) Preparation of Compound D4

[0072]

[0073] Compound D1 (242 mg, 1 mmol) was dissolved in anhydrous dichloromethane (10 mL). Boron tribromide (5 g, 20 mmol) was slowly added dropwise at 0°C. The mixture was stirred at room temperature for 16 hours, then poured into ice water (50 mL) and neutralized with saturated sodium bicarbonate. The filtrate was extracted three times with anhydrous dichloromethane. The organic phase was then dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (dichloromethane:methanol = 30:1) to obtain 140 mg of compound D4 as a yellow solid in a 61% yield.

[0074] 1 H NMR(400MHz,DMSO-d6)δ10.20(d,J=10.6Hz,2H),7.19(d,J=8.2Hz,1H),6.92(s,1H),6 .65-6.57(m,2H),2.54(d,J=6.2Hz,2H),2.28(t,J=6.0Hz,2H),1.61(p,J=6.0Hz,2H).

[0075] The structural formula of compound D4 is:

[0076] (5) Preparation of Compound D5

[0077]

[0078] Compound D4 (458 mg, 2 mmol) and 1-ethyl-2-methylquinolinium iodide (CAS-606-55-3) (657 mg, 2.2 mmol) were added to a reaction flask. The mixture was evacuated, supplemented with argon, and acetic anhydride (10 mL) was added as the solvent. The reaction was refluxed at 110°C for 10 h. Water was then added, the mixture was filtered under reduced pressure using a Buchner funnel, and dried. The crude product was purified by column chromatography (dichloromethane:methanol = 15:1) to afford 420 mg of compound D5 as a purple-red solid in a 53% yield.

[0079] 1 H NMR(400MHz,)δ7.63(d,J=15.5Hz,1H),7.55(d,J=7.8Hz,2H),7.51(d,J=9.6Hz,2 H),7.20(d,J=8.5Hz,1H),7.00(d,J=2.3Hz,1H),6.82–6.77(m,1H),6.70(d,J=10 .6Hz,2H),6.60(d,J=15.5Hz,1H),5.20(s,2H),3.21(s,6H),3.07(s,2H),2.50(d ,J=39.2Hz,12H),2.13(s,4H),1.71(s,2H),1.28(d,J=40.7Hz,4H),1.03(s,6H).

[0080] The structural formula of compound D5 is:

[0081] (6) Preparation of Compound D6

[0082]

[0083] Di-tert-butyl dicarbonate (7 mL, 30 mmol) was diluted with ethanol (5 mL) and slowly added dropwise to a solution of 1,4,8,11-tetraazacyclotetradecane (CAS-295-37-4) (2.4 g, 12 mmol) in anhydrous dichloromethane (10 mL) at 0°C. The mixture was allowed to react at room temperature for 18 h. After the reaction, the crude product was purified by column chromatography (petroleum ether:ethyl acetate = 20:1) to obtain a colorless transparent liquid. The product was concentrated in vacuo and dried to obtain 3.1 g of white solid compound D6 with a yield of 52%.

[0084] 1 H NMR (400MHz, Chloroform-d) δ 4.19-3.98 (m, 2H), 3.57-3.26 (m, 16H), 1.72 (d, J = 19.5Hz, 4H), 1.46 (s, 27H).

[0085] The structural formula of compound D6 is:

[0086] (7) Preparation of Compound D7

[0087]

[0088] Compound D4 (1 g, 2 mmol) was added to a 100 mL two-necked flask, and the mixture was evacuated and supplemented with argon. Anhydrous dichloromethane, p-chloromethylbenzoyl chloride (CAS-876-08-4) (4.725 g, 25 mmol), and triethylamine (CAS-121-44-8) (3.46 mL, 25 mmol) were added to the two-necked flask in sequence at 0°C. The mixture was reacted for 1 h at 0°C, and then the ice bath was removed and the mixture was reacted for 4 h at room temperature. After the reaction, anhydrous dichloromethane and water were added and extracted. The organic phase was taken and the process was repeated three times. The organic phase was collected and dried over anhydrous sodium sulfate, filtered, and the crude product was removed by rotary evaporation. The obtained organic phase was purified by column chromatography (petroleum ether: ethyl acetate = 10:1) and concentrated in vacuo to obtain 456 mg of a white solid. The white solid was then directly dissolved in acetone solution, and sodium iodide (4.2 g, 28 mmol) was added. The mixture was reacted for 4 h, and then the solvent was evaporated under low pressure to obtain 850 mg of light yellow solid compound D7 with a yield of 57%.

[0089] 1 H NMR (400MHz, Chloroform-d) δ7.40 (d, J = 7.8Hz, 2H), 7.32 (s, 2H), 4.44 (s, 2H), 3.50 (d, J = 95.9Hz, 16H), 1.82 (d, J = 30.3Hz, 4H), 1.52-1.22 (m, 27H).

[0090] The structural formula of compound D7 is:

[0091] (8) Preparation of fluorescent probe DHX-Cu1

[0092]

[0093] D3 (382 mg, 1 mmol), compound D7 (1.86 g, 2.5 mmol), potassium tert-butoxide (168 mg, 1.5 mmol) were added to the reaction flask, and the mixture was evacuated and filled with argon. Anhydrous N, N-dimethylformamide was then added and the reaction was carried out at 60°C for 4 h. After the reaction, the organic phase was extracted with dichloromethane and water, and the crude product was obtained by rotary evaporation. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 2:1), concentrated in vacuo, and dried to obtain a purple solid product. Hydrochloric acid (5 mL, 1 mol / L, dioxane solution) was then added and the reaction was carried out at 0°C for 4 h. After the reaction was completed, DHX-Cu1 was purified by column chromatography (dichloromethane: methanol = 1:1) to obtain 245 mg of a purple solid with a yield of 35%.

[0094] 1 H NMR(400MHz,DMSO-d6)δ9.78(s,3H),8.33(d,J=34.1Hz,1H),8.18(d,J=12.8Hz,1H),7 .98(d,J=6.1Hz,1H),7.84(s,1H),7.65(s,1H),7.38(s,1H),7.20(d,J=21.7Hz,4H),6 .69(s,1H),6.60-6.49(m,1H),6.31(d,J=13.9Hz,1H),4.93(s,2H),4.56(s,2H),2.97 (dd,J=114.6,54.9Hz,16H),2.28-2.19(m,4H),1.85(s,4H),1.44(s,2H),1.17(s,3H). 13C NMR(151MHz,DMSO-d6)δ160.82,156.29,154.72,154.04,141.90,138.65,134.76,130.49,128.71,128.44,128.20,128.01,127.31,1 21.02,118.55,112.65,111.28,102.44,79.33,79.27,79.19,69.95,55.37,45.81,29.10,28.57,28.52,28.36,24.51,20.62,13.80.H RMS[M+H] + :C 44 H 52 N5O3 + , Exact Mass, 698.4065, found, 698.4045.

[0095] The structural formula of the fluorescent probe DHX-Cu1 is:

[0096] Example 2

[0097] This example provides a tumor-targeted near-infrared copper ion fluorescent probe DHX-Cu2 based on Example 1. The specific preparation steps are as follows:

[0098]

[0099] D5 (382 mg, 1 mmol), compound D7 (1.86 g, 2.5 mmol), potassium tert-butoxide (168 mg, 1.5 mmol) were added to the reaction flask, and the mixture was evacuated and filled with argon. Anhydrous N, N-dimethylformamide was then added and the reaction was carried out at 60°C for 4 h. After the reaction, the organic phase was extracted with dichloromethane and water, and the crude product was obtained by rotary evaporation. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 2:1), concentrated in vacuo, and dried to obtain a purple solid product. Hydrochloric acid (5 mL, 1 mol / L, dioxane solution) was then added and the reaction was carried out at 0°C for 4 h. After the reaction was completed, DHX-Cu2 was purified by column chromatography (dichloromethane: methanol = 1:1) to obtain 213 mg of a purple solid with a yield of 30%.

[0100] 1 H NMR(400MHz,)δ7.63(d,J=15.5Hz,1H),7.55(d,J=7.8Hz,2H),7.51(d,J=9.6Hz,2 H),7.20(d,J=8.5Hz,1H),7.00(d,J=2.3Hz,1H),6.82-6.77(m,1H),6.70(d,J=10 .6Hz,2H),6.60(d,J=15.5Hz,1H),5.20(s,2H),3.21(s,6H),3.07(s,2H),2.50(d ,J=39.2Hz,12H),2.13(s,4H),1.71(s,2H),1.28(d,J=40.7Hz,4H),1.03(s,6H). 13C NMR(101MHz,DMSO)δ169.29,159.60,157.00,153.36,151.08,137.80,132.41, 127.73,127.45,127.32,126.69,125.89,123.72,120.79,115.39,114.67,113. 81,111.79,110.53,101.77,72.41,69.26,42.31,41.94,40.07,39.87,39.66, 39.45,39.24,39.03,38.82,38.09,31.63,27.42,24.11,22.05,20.36,13.92.H RMS[M+H]+ :C 44 H 52 N6O3, Exact Mass, 713.4134, found, 713.4150.

[0101] The structural formula of the fluorescent probe DHX-Cu2 is:

[0102] Example 3

[0103] In this example, based on Examples 1-2, the ultraviolet absorption spectra of the fluorescent probes DHX-Cu1 and DHX-Cu2 provided by the present invention before and after responding to copper ions were measured.

[0104] Weigh the probes DHX-Cu1 and DHX-Cu2 separately, dissolve them in dimethyl sulfoxide (DMSO) to prepare a probe stock solution with a molar concentration of 1.0mM, and store at 4-8°C for later use. Prepare a copper ion standard solution with a molar concentration of 10mM using distilled water. Take 50μL of the fluorescent probe stock solution and dissolve it in 9.5mL of phosphate buffer solution (10mM, pH=7.0) to prepare a 5μM fluorescent probe solution (5μM PBS, 10mM, pH=7.0). Add 50μM copper ion solution to the fluorescent probe solution, react for 15 minutes, and then detect the UV spectrum. For specific results, see the attached figure. Figure 1 shown.

[0105] By the attached Figure 1 The data shows that Figure 1 In the figure, (A) shows the probe DHX-Cu1, and (B) shows the probe DHX-Cu2. Before the addition of copper ions, both probes (DHX-Cu1 and DHX-Cu2) exhibited a maximum absorption peak at 560 nm. This indicates that the probe molecules have a characteristic absorption peak at this wavelength. After the addition of copper ions and a 15-minute reaction, the absorption peaks of both probes at 560 nm decreased significantly. This change indicates that the probe molecules interacted with the copper ions, forming a new substance or complex. The decrease in the absorption peak is due to the change in the distribution of electrons in the fluorescent probe molecules after the probe molecules bind to the copper ions, resulting in quenching of the fluorescence signal.

[0106] The above data support that the DHX-Cu1 and DHX-Cu2 probes can specifically bind to copper ions to form new substances or complexes. This binding can be monitored by changes in the absorption peak in the UV spectrum, providing an experimental basis for the application of the probes in copper ion detection.

[0107] Example 4

[0108] In this example, based on Examples 1-2, the fluorescence spectra of the fluorescent probes DHX-Cu1 and DHX-Cu2 of the present invention before and after responding to copper ions were measured.

[0109] Different concentrations (0-50 μM) of copper ion solution were gradually added to the DHX-Cu1 and DHX-Cu2 (5 μM PBS, 10 mM, pH = 7.0) probe solutions. After 15 minutes of reaction, the fluorescence intensity changes of the mixed solutions at emission wavelengths of 685 nm and 760 nm were measured with an excitation wavelength of 565 nm. After the addition of copper ions to the fluorescent probe solution, the fluorescent probe showed a quenched fluorescence signal change. For specific results, see the attached Figure 2 shown.

[0110] By the attached Figure 2 The data shows that Figure 2 In the figure, (A) is the probe DHX-Cu1, and (B) is the probe DHX-Cu2. 2+ ) increases, the fluorescence intensity of the two fluorescent probes decreases rapidly. When the copper ion concentration is greater than 40 μM, the rate of fluorescence intensity decreases gradually slows down until a certain equilibrium is reached, at which point the fluorescence intensity is only one-tenth of the fluorescence intensity when no copper ions are added. Therefore, this fluorescent probe can be used to detect copper ions in aqueous solution. Among them, the fluorescence emission wavelengths of DHX-Cu1 and DHX-Cu2 probes are 685nm and 760nm, respectively, which are located in the near-infrared region. This wavelength is conducive to penetrating biological tissues, reducing biological autofluorescence, and is suitable for biological imaging.

[0111] Example 5

[0112] Based on Examples 1-2, this example determines the selective anti-interference properties of the fluorescent probes DHX-Cu1 and DHX-Cu2 of the present invention with respect to copper ions. To test the selectivity of the fluorescent probes for copper ions, the responses of the fluorescent probes to different metal ion analytes are explored.

[0113] To test the selectivity of the fluorescent probe for copper ions, the response of the fluorescent probe to various metal ion analytes was investigated. Accurately weigh a certain mass of the following metal salts: LiNO₃, MgCl₂, CaCl₂, FeCl₃·6H₂O, HgCl₂, CrCl₃·6H₂O, AgNO₃, CdCl₂·H₂O, NiCl₂·6H₂O, Cu(NO₃)₂·3H₂O, ZnCl₂, [Cu(CH₃CN)₄][PF₆], dissolve them in distilled water and dilute to 10 mL. Ultrasonic mixing was performed to prepare 10 mM analyte standard solutions, which were then stored at 4-8°C until ready for use. 50 μM solutions of different metal analytes were gradually added to the probe solutions of DHX-Cu1 and DHX-Cu2 (5 μM PBS, 10 mM, pH = 7.0), respectively. After the mixture reacted for 15 minutes, the fluorescence emission spectra were measured at an excitation wavelength of 565 nm and an emission wavelength of 685 nm or 760 nm. The specific results are shown in the attached Figure 3 shown.

[0114] By the attached Figure 3 The data shows that Figure 3 In the figure, (A) is the probe DHX-Cu1, and (B) is the probe DHX-Cu2. Selectivity: For both DHX-Cu1 and DHX-Cu2, after the addition of copper ions (50 μM), the fluorescence intensity of the fluorescent probe is significantly reduced, while the fluorescence response of the fluorescent probe to other metals (50 μM) can be weak. Interference: After adding other analytes to the fluorescent probe solution and then adding copper ions, the fluorescence intensity is significantly reduced, indicating that the change in fluorescence intensity caused by copper ions will not be interfered with by other metal ions. The above results show that the fluorescent probes DHX-Cu1 and DHX-Cu2 have excellent selectivity and low interference for copper ions, and can be used for the detection of copper ions.

[0115] Example 6

[0116] This example, based on Examples 1-2, studies the binding mechanism between the fluorescent probe provided by the present invention and the CXCR4 receptor protein (3odu).

[0117] The fluorescent probe of the present invention produces a strong near-infrared fluorescence signal at 685nm or 760nm in a PBS buffer solution under an excitation wavelength of 565nm. When reacting with copper ions in the solution, the copper ions coordinate with the 1,4,8,11-tetraazacyclic ring in the probe, quenching the fluorescence signal at the emission wavelength of 685nm or 760nm.

[0118] Data source and processing: The SDF format files of the fluorescent probe were obtained using the Pubchem database, and the structural files of the CXCR4 receptor protein (3odu) were collected from the PDB database. The receptor protein was preprocessed using Pymol software, including the removal of water molecules and small molecule ligands, and hydrogenation and charge processing were performed using AutoDock Tools. Finally, the data were saved in pdbqt format for molecular docking.

[0119] Molecular docking: Using CXCR4 receptor protein as receptor and fluorescent probe as ligand, molecular docking was performed using the VINA algorithm built into the Pyrx software. The binding strength between the fluorescent probe and the receptor protein was evaluated by calculating the binding energy. The binding pattern diagram of the fluorescent probe and CXCR4 receptor protein (3odu) is shown in the attached figure. Figure 4 shown.

[0120] By the attached Figure 4 It can be seen that the binding mode of the fluorescent probe of the present invention and the CXCR4 receptor protein (3odu) was calculated. The binding energy was calculated. DHX-Cu1 formed conventional hydrogen bond interactions with amino acid residues such as Leu208, Ile215, Leu216, Val242, Leu246, and Phe249 of the CXCR4 receptor protein, and the calculated specific binding energy was -11.1 kcal / mol, indicating that DHX-Cu1 has strong binding activity with the CXCR4 receptor. DHX-Cu2 formed conventional hydrogen bond interactions with amino acid residues such as Leu208, Ile215, Leu216, and Val242 of the CXCR4 receptor protein. Its specific binding energy was -11.9 kcal / mol, slightly higher than that of DHX-Cu1, further demonstrating the strong binding ability of the fluorescent probe to the CXCR4 receptor.

[0121] Example 7

[0122] This example, based on Examples 1-2, measures the antagonistic effects of the fluorescent probes DHX-Cu1 and DHX-Cu2 of the present invention on the CXCR4 receptor.

[0123] The CXCR4 receptor antagonist effect of the fluorescent probe was investigated by a 12G5 antibody competitive binding experiment. The CCRF-CEM cells were grouped and then different concentrations of probes (0.5-10000nM) were added, incubated at 4°C for 4h, and then the cells were washed with flow cytometry buffer, and the antibody 12G-5 (PE anti-human CD184 (CXCR4) Antibody, Biolegend) 100W / mL was added to incubate the cells for 60min. At the same time, a negative control was prepared according to the same method. A FACScan (BD) flow cytometer was used to test the fluorescence intensity (PE / PI), and the signal channel was Ex / Em: 488nm / 575nm. The mean fluorescence intensity (MFI) was used as a quantitative method to measure and calculate the hAB inhibition percentage to calculate the inhibitory efficiency of the compound. For specific data, see the attached. Figure 5 shown.

[0124] By the attached Figure 5 The data shows that Figure 5 In the figure, (A) is the probe DHX-Cu1, (B) is the probe DHX-Cu2, (C) is the positive drug AMD3100, and (D) is the antagonistic effect of different compounds (5 μM) on CXCR4 receptor. 50 The values ​​were 274.1nM and 386.5nM, respectively, which were close to those of the positive drug AMD3100 (IC 50 =238.6nM), and the CXCR4 antagonism of the two probes was at the submolar level. Therefore, the fluorescent probe was proven to have excellent CXCR4 receptor antagonism in vitro. At the same time, since the probe solution concentration for copper ions is 5μM, the receptor antagonism of AMD3100, DHX-Cu1, DHX-Cu2, D5 and D7 compounds at a concentration of 5μM was measured respectively; the inhibition efficiency of DHX-Cu1 and DHX-Cu2 on 12G5 antibody was 85% and 78%, while the inhibition rate of D5 and D7 compounds containing only fluorophores was only 15% and 12%. Figure 2 It can be seen that DHX-Cu1 and DHX-Cu2 have CXCR4 receptor targeting performance and excellent imaging performance, and can monitor copper ions at the micromolar level in tumor cells that highly express CXCR4 receptors.

[0125] Example 8

[0126] In this example, based on Examples 1-2, a laser confocal microscopy experiment was conducted to investigate the specific recognition of exogenous copper ions by the fluorescent probes DHX-Cu1 and DHX-Cu2 of the present invention in A2780 cells.

[0127] A2780 cells were seeded in confocal microplates and divided into the control group, which was incubated with DMSO only for 30 minutes; the DHX-Cu1 group was incubated with the probe 10 μM for 30 minutes; the Cu supplemented group was first incubated with Cu (GTSM) 2 μM as exogenous copper ions for 4 hours, washed with PBS, and then incubated with the probe (DHX-1 or DHX-Cu2) 10 μM (HBSS) for 15 minutes; the Cu deficient group was first incubated with BCS 400 μM as a copper ion chelator for 6 hours, washed with PBS, and then incubated with the probe (DHX-1 or DHX-Cu2) 10 μM (HBSS) for 30 minutes. After each treatment, the cells were imaged directly, as shown in the attached figure. Figure 6 and attached Figure 7 shown.

[0128] By the attached Figure 6-7 The data show that specific recognition: the fluorescence intensity of the Cu supplemented group decreased significantly, which directly proves that the DHX-Cu1 and DHX-Cu2 probes can specifically recognize and bind to exogenous copper ions in cells, resulting in changes in fluorescence signals; rapid response: the probes can respond to changes in copper ion concentration within a relatively short period of time (15 to 30 minutes), indicating that they have high sensitivity and rapid response capabilities; indicating that the probes can achieve rapid and real-time detection of exogenous copper ions in cells; the probes of the present invention have potential application value in the biomedical field, and can be used to monitor changes in intracellular copper ion levels, evaluate the status of copper metabolism-related diseases, and serve as a tool for drug screening and development.

[0129] Example 9

[0130] In this example, based on Examples 1-2, laser confocal microscopy experiments were conducted on SKOV3 ovarian cancer cells with high CXCR4 expression and IOSE-80 normal ovarian cells with low CXCR4 expression using the fluorescent probes DHX-Cu1 and DHX-Cu2 of the present invention.

[0131] SKOV3 cells and IOSE-80 normal ovarian cells were inoculated into confocal microplates, and the cells were incubated with 10 μM fluorescent probe (DHX-1 or DHX-Cu2) for 30 min. After washing with PBS three times, laser confocal imaging of the cells was performed directly to observe and record the fluorescence signals emitted by the cells. For detailed results, see the attached Figure 8 shown.

[0132] By the attached Figure 8The data show that SKOV3 cells show obvious red fluorescence signals, while IOSE-80 cells show weaker fluorescence signals, indicating that both fluorescent probes DHX-Cu1 and DHX-Cu2 can specifically recognize and bind to SKOV3 cells with high CXCR4 expression, while their binding ability to IOSE-80 cells with low CXCR4 expression is weaker, indicating that the fluorescent probes of the present invention have good selectivity for tumor cells and normal cells.

[0133] It can be seen that the present invention, by developing a fluorescent probe targeting the specific molecular marker CXCR4, can achieve accurate identification and positioning of cancer cells, provide new strategies and methods for cancer treatment, and is of great significance for early diagnosis, disease monitoring and targeted treatment of cancer.

[0134] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A tumor-targeting near-infrared copper ion fluorescent probe, characterized in that: The fluorescent probe structure is any one of the following: and .

2. The method for preparing the tumor-targeting near-infrared copper ion fluorescent probe according to claim 1, characterized in that: include: S1, under argon protection, 4-diethylaminosalicylaldehyde and cesium carbonate were mixed, reacted with 2-bromocyclohex-1-ene-1-carbaldehyde, extracted, and purified by column chromatography to prepare compound D1; The structural formula of the compound D1 is: S2, under argon protection, compound D1 in S1 was mixed with 1-ethyl-2-methylquinolinium iodide, and a solvent was added to react to obtain compound D2. Boron tribromide was added to react at 0-5°C, diluted with dichloromethane, quenched with sodium carbonate solution, extracted, filtered, and purified by column chromatography to obtain compound D3; The structural formula of the compound D2 is: ; The structural formula of the compound D3 is: ; Compound D1 obtained by S1 was dissolved at 0-5°C and boron tribromide was added for reaction. The mixture was neutralized, extracted, dried, and purified by column chromatography to obtain compound D4. Under argon protection, compound D4 was mixed with 1-ethyl-2-methylquinolinium iodide, acetic anhydride was added for reaction, filtered, dried, and purified by column chromatography to obtain compound D5. The structural formula of the compound D4 is: ; The structural formula of the compound D5 is: ; S3, di-tert-butyl dicarbonate reacts with 1,4,8,11-tetraazacyclotetradecane, and the mixture is purified by column chromatography to obtain compound D6, compound D6, anhydrous dichloromethane, p-chloromethylbenzoyl chloride, and triethylamine are mixed for reaction, extracted, purified, concentrated, and dissolved in acetone, and sodium iodide is added to evaporate the solvent to obtain compound D7; The structural formula of the compound D6 is: The structural formula of the compound D7 is ; S4, the compound D3 or compound D5 obtained in S2 is reacted with the compound D7 obtained in S3, extracted, purified, concentrated in vacuo, dried, added with a hydrochloric acid solution containing dioxane, and reacted again, purified, to obtain the near-infrared copper ion fluorescent probe compound according to claim 1.

3. The method for preparing the tumor-targeting near-infrared copper ion fluorescent probe according to claim 2, characterized in that: The molar ratio of 4-diethylaminosalicylaldehyde, cesium carbonate and 2-bromocyclohex-1-ene-1-carbaldehyde in S1 is 2~3:4~5:1, the reaction temperature is 24~26°C, and the reaction time is 16~20h; the solvent used in the extraction is water and dichloromethane, and the mobile phase used in the column chromatography purification is petroleum ether and ethyl acetate in a volume ratio of 6:

1.

4. The method for preparing the tumor-targeting near-infrared copper ion fluorescent probe according to claim 2, characterized in that: The molar ratio of compound D1 in S2 to 1-ethyl-2-methylquinolinium iodide is 1:1, the solvent is acetic anhydride, the reaction temperature is 100-120° C., and the reaction time is 10-14 h.

5. The method for preparing the tumor-targeting near-infrared copper ion fluorescent probe according to claim 2, characterized in that: The molar ratio of compound D2 to boron tribromide in S2 is 1:20-25, and the reaction is carried out at room temperature for 20-26 hours. The column chromatography purification uses dichloromethane and methanol in a volume ratio of 30:1 as the mobile phase.

6. The method for preparing the tumor-targeting near-infrared copper ion fluorescent probe according to claim 2, characterized in that: The molar ratio of compound D1 to boron tribromide in S2 is 1:20~22, and the reaction is carried out at room temperature for 14~18 hours. The extraction is performed using dichloromethane as a solvent for 2~4 times, and the column chromatography purification uses dichloromethane and methanol with a volume ratio of 30:1 as the mobile phase.

7. The method for preparing the tumor-targeting near-infrared copper ion fluorescent probe according to claim 2, characterized in that: The molar ratio of compound D4 described in S2 to 1-ethyl-2-methylquinolinium iodide is 1:1~2, the reaction temperature is 100~120℃, and the reaction time is 8~12h.

8. The method for preparing the tumor-targeting near-infrared copper ion fluorescent probe according to claim 2, characterized in that: The molar ratio of di-tert-butyl dicarbonate to 1,4,8,11-tetraazacyclotetradecane in S3 is 2~3:1, and the reaction is carried out at room temperature for 16h~20h. The column chromatography purification adopts a mobile phase of petroleum ether and ethyl acetate with a volume ratio of 20:1; the molar ratio of the compound D6, p-chloromethylbenzoyl chloride and triethylamine is 1:20~30:20~30, and the reaction is carried out at room temperature for 3h~5h.

9. The method for preparing the tumor-targeting near-infrared copper ion fluorescent probe according to claim 2, characterized in that: The molar ratio of the compound D3 or compound D5 to the compound D7 is 1:2-3, and the reaction is carried out at 50° C.-70° C. for 3 h-5 h.

10. Use of the tumor-targeting near-infrared copper ion fluorescent probe according to claim 1 in detecting exogenous copper ions in tumor cells and imaging for purposes other than disease diagnosis and treatment, characterized in that: The tumor targeting is targeting CXCR4 receptor.

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