Tumor-targeted near-infrared copper ion fluorescent probe as well as preparation and application thereof

Abstract

The invention discloses a near-infrared copper ion fluorescent probe with tumor targeting property as well as a preparation method and application of the near-infrared copper ion fluorescent probe in biological imaging, and belongs to the technical field of analytical chemistry and biology. The near-infrared copper ion fluorescent probe disclosed by the invention can efficiently detect copper ions and specifically target CXCR4 high-expression tumor cells, the near-infrared emission characteristic of the probe avoids biological autofluorescence interference, the imaging definition and sensitivity are improved, and the high selectivity and sensitivity of the probe ensure that the copper ions are accurately detected in a complex biological environment. The kit is suitable for early tumor diagnosis and research; the preparation method is simple, low in cost and suitable for large-scale production. The probe disclosed by the invention not only can monitor the change of copper ions in tumors in real time and reveal a tumor mechanism, but also has wide clinical application prospects including tumor staging, treatment monitoring and the like. The 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-targeted near-infrared copper ion fluorescent probe and a preparation and application thereof. Background Art

[0002] Copper (II) ion (Cu 2+ ) As an essential trace element in organisms, copper plays a vital role in a variety of biological processes, including enzyme catalysis, regulation of protein function, DNA replication and repair, cell differentiation and proliferation, etc. Studies have shown that compared with normal tissues, tumors have a higher demand for copper. Clinical studies have shown that the copper ion level in the serum or tumor tissue of cancer patients is significantly increased, and higher copper ion levels often represent poor clinical staging and prognosis. Copper can increase genomic instability, promote angiogenesis, and upregulate tumor-promoting signaling pathways, thereby enhancing the metastasis and invasiveness of tumors. However, excessive copper ions have been shown to induce tumor cell death through pathways such as apoptosis or oxidative stress, and the newly discovered copper death also provides a new potential target for tumor treatment. Therefore, in tumor cells, the high demand and abnormal accumulation of copper ions not only promote the growth, metastasis and invasion of tumors, but also provide a new idea for tumor treatment-affecting the fate of tumor cells by regulating copper ion levels; however, due to the heterogeneity and complexity of tumor cells, how to accurately, quickly and in real time detect the copper ion content in tumor cells has become an urgent problem to be solved.

[0003] Traditional methods for detecting copper ions, such as atomic absorption spectroscopy and inductively coupled plasma mass spectrometry, have high accuracy, but they have limitations such as complex operation, long time consumption, and inability to detect in real time in situ, making it difficult to meet the needs of rapid detection in clinical and scientific research. In complex biological environments, especially inside tumor cells, it is still challenging to achieve specific detection of copper ions. 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 changes in concentration.

[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 fluorescent signals, thereby achieving quantitative detection of target molecules. In tumor cells, fluorescent probes can penetrate the cell membrane and enter the cell, reflecting the dynamic changes of specific molecules in the cell in real time, providing important information for the diagnosis and treatment of tumors.

[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 vigorous growth and metabolism of tumor cells, CXCL12 can attract endothelial stem cells and synergize with multiple growth factors such as vascular epidermal growth factor (VEGF) to increase the growth of endothelial cells to form a large number of new small blood vessels. At the same time, CXCR4 is highly expressed on the surface of many types of cancer cells, including ovarian cancer cells, and plays an important role in their growth and their 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 view of the difficulty of copper ion detection in tumor cells, how to achieve high sensitivity, high selectivity and real-time detection of copper ions in a complex tumor environment, and at the same time give fluorescent probes tumor specificity to accurately reflect the copper ion level in tumor cells, so as to 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, which was then added with boron tribromide at 0-5°C for reaction, 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 is dissolved at 0-5°C, and boron tribromide is added for reaction. The mixture is neutralized, extracted, dried, and purified by column chromatography to obtain compound D4. Under argon protection, compound D4 is mixed with 1-ethyl-2-methylquinolinium iodide, and acetic anhydride is added for reaction. The mixture is 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, 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 the compound D3 or compound D5 obtained in S2 with the compound D7 obtained in S3, extracting, purifying, vacuum concentrating, drying, adding a hydrochloric acid solution containing dioxane to react again, purifying to 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 in the extraction of S1 are 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.

[0032] S2: The molar ratio of compound D1 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 10-14h.

[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] S2: the molar ratio of compound D1 to boron tribromide is 1:20-22, the reaction is carried out at room temperature for 14-18 hours, the extraction is carried out using dichloromethane as a solvent for 2-4 times, and the column chromatography purification is carried out using dichloromethane and methanol in a volume ratio of 30:1 as a 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 in 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 the 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 to the detection and imaging of exogenous copper ions in tumor cells, wherein the tumor targeting is targeting CXCR4 receptor.

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

[0040] The present invention provides a tumor-targeted near-infrared copper ion fluorescent probe, which has 1) efficient copper ion detection and specific tumor targeting: the probe realizes 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 the interference of other metal ions in the biological environment; at the same time, the probe can be specifically enriched in tumor cells with high expression of CXCR4, providing a powerful tool for early diagnosis of tumors; 2) excellent fluorescence performance: the emission wavelength of the probe is in the near-infrared region (685nm), which can effectively avoid the interference of 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, and further improves the sensitivity and accuracy of fluorescence detection; 3) strong tissue penetration and real-time imaging capability: the probe can respond to the changes of copper ions in tumor cells in real time and quickly to achieve dynamic imaging; therefore, the fluorescent probe of the present invention has significant advantages and broad application prospects in copper ion detection and tumor imaging, and provides 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 by a multi-step organic synthesis reaction, the reaction conditions are mild and easy to control, and the purification step is simple, which is suitable for large-scale production; the raw materials are easily available, the cost is low, and it is conducive to practical application; through the carefully designed molecular structure, the fluorescent probe can specifically recognize and bind to the CXCR4 receptor, and at the same time exhibits a high-sensitivity response to copper ions, and 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 is 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, the fluorescent probe has potential clinical application value in early diagnosis, staging, treatment monitoring and prognosis evaluation of tumors, and through further research and optimization, the 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); wherein (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); wherein (A) is the probe DHX-Cu1, and (B) is the probe DHX-Cu2;

[0045] Figure 3 The fluorescence spectra of the fluorescent probes DHX-Cu1 and DHX-Cu2 of the present invention for copper ion selection and interference; wherein (A) is the probe DHX-Cu1, and (B) is the probe DHX-Cu2;

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

[0047] Figure 5 The CXCR4 receptor antagonism and IC 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 antagonism of different compounds (5 μM) to the CXCR4 receptor;

[0048] Figure 6 The laser confocal imaging diagram of the fluorescent probe of the present invention specifically recognizing exogenous copper ions in A2780 cells; wherein, (A1), (A2) and (A3) are control groups, 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; each group was directly imaged with FV3000 laser confocal imaging after treatment, scale bar 20 μM;

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

[0050] Figure 8 The 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 min; (C2) cells were incubated with 10μM probe DHX-Cu1 for 30 min; (B2) cells were incubated with 10μM probe DHX-Cu2 for 30 min; (D2) cells were incubated with 10μM probe DHX-Cu2 for 30 min; each group was directly imaged with FV3000 laser confocal imaging after treatment, scale bar 20μM. DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme 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 described embodiments 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 creative work 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 the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

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

[0054] Example 1

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

[0056] (1) Preparation of Compound D1

[0057]

[0058] Weigh 4-diethylamino salicylaldehyde (2.93 g, 19.1 mmol) and cesium carbonate (11.05 g, 33.9 mmol), add to a 100 mL two-necked flask, evacuate, replenish argon, and then add the compound 2-bromocyclohex-1-ene-1-carboxaldehyde (CAS-38127-47-8) (4.32 g, 9 mmol) dissolved in DMF to a two-necked flask filled with argon, and stir at 25 ° C for 18 h; after the reaction, extract the reaction solution with a mixed solvent of water and dichloromethane, dry the organic layer with anhydrous sodium sulfate, filter, and remove the solvent by rotary evaporation to obtain a crude product, which is 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 and the mixture was filtered under reduced pressure using a Buchner funnel and dried to obtain 672.3 mg of a blue-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, vacuumed, supplemented with argon, and anhydrous dichloromethane (20 mL) was added as solvent after the system was filled with argon. 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, a saturated sodium carbonate solution was added to quench the reaction; the aqueous phase was extracted with dichloromethane, and the organic phase was collected and dried with anhydrous sodium sulfate after three times of extraction, filtered, and rotary evaporated to obtain a crude product, which was purified by column chromatography (dichloromethane: methanol = 30: 1) to obtain 270 mg of a blue-purple solid compound D3 with a yield of 70%.

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

[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 the reaction flask, vacuumed, supplemented with argon, acetic anhydride (10 mL) was added as solvent, and refluxed at 110°C for 10 h; then water was added and 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 obtain 420 mg of purple-red solid compound D5, with a yield of 53%.

[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 (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, and reacted 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, which 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 double-necked flask, and 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 double-necked flask in sequence at 0°C. The mixture was reacted at 0°C for 1 h, and then the ice bath was removed and the mixture was reacted at room temperature for 4 h. 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 mixed solution 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 argon gas was added under vacuum, and then anhydrous N, N-dimethylformamide was added, and the reaction was carried out at 60 ° C for 4 hours. After the reaction, the organic phase was extracted with dichloromethane and water, and the crude product was obtained by rotary evaporation, and then purified by column chromatography (petroleum ether: ethyl acetate = 2: 1), concentrated in vacuo, and dried to obtain a purple solid product. Then hydrochloric acid (5 mL, 1 mol / L, dioxane solution) was added, and the reaction was carried out at 0 ° C for 4 hours. After the reaction was completed, DHX-Cu1 was purified by column chromatography (dichloromethane: methanol = 1: 1) as a purple solid 245 mg, 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] Based on Example 1, this example provides a tumor-targeted near-infrared copper ion fluorescent probe DHX-Cu2, and 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 argon gas was added to the vacuum, and then anhydrous N, N-dimethylformamide was added, and the reaction was carried out at 60 ° C for 4 hours. After the reaction, the organic phase was extracted with dichloromethane and water, and the crude product was obtained by rotary evaporation, and then purified by column chromatography (petroleum ether: ethyl acetate = 2: 1), concentrated in vacuo, and dried to obtain a purple solid product. Then hydrochloric acid (5 mL, 1 moL / L, dioxane solution) was added, and the reaction was carried out at 0 ° C for 4 hours. After the reaction was completed, column chromatography purification (dichloromethane: methanol = 1: 1) was performed to obtain DHX-Cu2 as a purple solid 213 mg, 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 Example 1-2, the ultraviolet absorption spectra of the fluorescent probes DHX-Cu1 and DHX-Cu2 provided by the present invention before and after the response to copper ions are measured.

[0104] Weigh the probes DHX-Cu1 and DHX-Cu2 respectively, dissolve them in dimethyl sulfoxide solution (DMSO) to prepare a probe mother solution with a molar concentration of 1.0mM, and store it at 4-8°C for later use; prepare a copper ion with a molar concentration of 10mM as a standard solution with distilled water; take 50μL of the fluorescent probe mother 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 detect the UV spectrum. For specific results, see the attached Figure 1 shown.

[0105] By the attached Figure 1 The data shows that Figure 1 In the figure, (A) is the probe DHX-Cu1, and (B) is the probe DHX-Cu2. Before the addition of copper ions, both probes (DHX-Cu1 and DHX-Cu2) showed maximum absorption peaks at 560nm. This indicates that the probe molecule has a characteristic absorption peak at this wavelength; after adding copper ions and reacting for 15 minutes, the absorption peaks of both probes at 560nm showed a significant decrease. This change indicates that the probe molecule interacted with the copper ions to form 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 molecule after the probe molecule binds 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 peaks 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 Example 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 are measured.

[0109] Different concentrations (0-50 μM) of copper ion solution were gradually added to the probe solution of DHX-Cu1 and DHX-Cu2 (5 μM PBS, 10 mM, pH = 7.0). After 15 minutes of reaction, the fluorescence intensity changes of the mixed solution at the 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 40μM, the rate of fluorescence intensity decreases gradually slows down until a certain balance is reached, at which time the fluorescence intensity is only one-tenth of the fluorescence intensity when copper ions are not 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 of the fluorescent probes DHX-Cu1 and DHX-Cu2 of the present invention with copper ions. In order 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 different 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 make up to 10 mL, mix them evenly by ultrasonication, prepare a 10 mM analyte standard solution, and store it at 4-8 °C for later use. 50 μM of different metal analyte solutions were gradually added to the probe solutions of DHX-Cu1 and DHX-Cu2 (5 μM PBS, 10 mM, pH = 7.0), respectively. After the mixed solution 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 adding 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 copper ions, the fluorescence intensity is significantly reduced, indicating that the change in fluorescence intensity caused by copper ions will not be interfered by other metal ions. The above results show that the fluorescent probes DHX-Cu1 and DHX-Cu2 have excellent selectivity and low interference to copper ions, and can be used for the detection of copper ions.

[0115] Example 6

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

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

[0118] Data source and processing: The SDF format file of the fluorescent probe was obtained using the Pubchem database, and the structure file of the CXCR4 receptor protein (3odu) was collected from the PDB database; the receptor protein was preprocessed using Pymol software, including removing water molecules, small molecule ligands, etc., and AutoDockTools was used for hydrogenation and charge processing, and finally 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) is calculated. DHX-Cu1 forms 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 is -11.1kcal / mol, indicating that DHX-Cu1 has a strong binding activity with the CXCR4 receptor; DHX-Cu2 forms conventional hydrogen bond interactions with amino acid residues such as Leu208, Ile215, Leu216, and Val242 of the CXCR4 receptor protein. Its specific binding energy is -11.9kcal / mol, slightly higher than DHX-Cu1, which further proves the strong binding ability of the fluorescent probe with the CXCR4 receptor.

[0121] Example 7

[0122] This example is based on Example 1-2, and measures the antagonism of the fluorescent probes DHX-Cu1 and DHX-Cu2 of the present invention to the CXCR4 receptor.

[0123] The CXCR4 receptor antagonism of the fluorescent probe was investigated by the 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, 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 in the same way. A FACScan (BD) flow cytometer was used to test the fluorescence intensity (PE / PI), the signal channel was Ex / Em: 488nm / 575nm, and the mean fluorescence intensity (MFI) was used as a quantitative method to measure and calculate the hAB inhibition percentage to calculate the inhibition 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 antagonism of different compounds (5 μM) on the CXCR4 receptor. IC values ​​of receptor antagonism of DHX-Cu1 and DHX-Cu2 50 The values ​​were 274.1nM and 386.5nM, respectively, which were close to the positive drug AMD3100 (IC 50 =238.6nM), the CXCR4 antagonism of the two probes is at the sub-molar level. Therefore, it is proved in vitro that the fluorescent probe has excellent CXCR4 receptor antagonism. 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 rates of D5 and D7 compounds containing only fluorophores were only 15% and 12%, and the inhibition rate of the fluorophore-containing compounds was 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 Example 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 control group, which was incubated with DMSO only for 30 min, DHX-Cu1 group, which was incubated with probe 10 μM for 30 min, Cu supplemented group, which was first incubated with Cu (GTSM) 2 μM as exogenous copper ion for 4 h, then washed with PBS and then incubated with probe (DHX-1 or DHX-Cu2) 10 μM (HBSS) for 15 min, Cu deficient group, which was first incubated with BCS 400 μM as copper ion chelator for 6 h, then washed with PBS and then incubated with probe (DHX-1 or DHX-Cu2) 10 μM (HBSS) for 30 min. Cell imaging was performed directly after each treatment, 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 proved 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 concentrations within a 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 Example 1-2, laser confocal microscopy experiments were performed on SKOV3 ovarian cancer cells with high expression of CXCR4 and IOSE-80 normal ovarian cells with low expression of CXCR4 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 specific 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, which indicates that both fluorescent probes DHX-Cu1 and DHX-Cu2 can specifically recognize and bind to SKOV3 cells with high expression of CXCR4, while their binding ability to IOSE-80 cells with low expression of CXCR4 is weaker, which indicates that the fluorescent probe of the present invention has good selectivity for tumor cells and normal cells.

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

[0134] The above contents are 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:

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, which was then added with boron tribromide at 0-5°C for reaction, 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 is dissolved at 0-5°C, and boron tribromide is added for reaction. The mixture is neutralized, extracted, dried, and purified by column chromatography to obtain compound D4. Under argon protection, compound D4 is mixed with 1-ethyl-2-methylquinolinium iodide, and acetic anhydride is added for reaction. The mixture is 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, 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 reacts with the compound D7 obtained in S3, extracts, purifies, concentrates in vacuo, and after drying, adds a hydrochloric acid solution containing dioxane to react again, and purifies to obtain a fluorescent probe that targets the CXCR4 receptor and simultaneously detects copper ions.

3. The method for preparing the tumor-targeting near-infrared copper ion fluorescent probe according to claim 2, characterized in that: S1 The molar ratio of 4-diethylaminosalicylaldehyde, cesium carbonate and 2-bromocyclohex-1-ene-1-carboxaldehyde is 2-3:4-5:1, the reaction temperature is 24-26°C, and the reaction time is 16-20h; the solvent used for the extraction is 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.

4. The method for preparing the tumor-targeting near-infrared copper ion fluorescent probe according to claim 2, characterized in that: S2: The molar ratio of compound D1 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 10-14h.

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 in a volume ratio of 30:1 as a 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 in S2 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.

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 in 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. The tumor-targeting near-infrared copper ion fluorescent probe of claim 1 for detecting and imaging exogenous copper ions in tumor cells, characterized in that: The tumor targeting is targeting CXCR4 receptor.

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