Kidney clearance type long-circulating near-infrared fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a kidney clearance type long-circulating near-infrared fluorescent probe as well as a preparation method and application thereof. The kidney clearance type long-circulation near-infrared fluorescent probe takes a sulfonated calixarene derivative as a kidney clearance fragment and a derivative with a cyanine dye structure as a fluorophore, and the sulfonated calixarene derivative and the derivative are coupled through a click reaction, so that the probe has certain broad-spectrum property and can adapt to various fluorophores; and the hydrogel has the advantages of favorable water solubility, favorable biocompatibility, high kidney removal efficiency, longer blood circulation time and certain tumor targeting property. And as a fluorescent probe, non-invasive imaging, intraoperative navigation and the like can be more accurately carried out on diseases.

Description

Technical Field

[0001] The invention relates to the field of fluorescent dye molecules, and in particular to a kidney-cleared long-circulating near-infrared fluorescent compound and a preparation method and application thereof. Background Art

[0002] Optical imaging technology has become an important research method in the biomedical field and plays a vital role in the clinical diagnosis and treatment process. With the assistance of optical instruments, optical imaging technology uses optical contrast agents to visualize and monitor biomolecules, cells, tissues and organisms in real time and in multiple dimensions. It is widely used in the fields of biomolecule detection imaging, drug distribution metabolism tracking, disease detection and diagnosis, etc., with the advantages of non-invasive, non-ionizing radiation, high temporal and spatial resolution and high sensitivity. Contrast agent, also known as contrast agent, is a substance that can improve the contrast of medical imaging images. It makes the tissue or organ in the image more visible by changing parameters such as the density, signal intensity or sound speed of tissues or organs. Traditional optical contrast agents are mainly cleared by the reticuloendothelial system and usually accumulate in the liver and spleen, leading to potential organ toxicity. In contrast, renal-clearable optical agents (RCOAs) can be quickly excreted from the body through the kidneys, which can effectively reduce the risk of contrast agent degradation and metabolism in cells and the potential toxicity to normal organs. Therefore, developing new RCOAs or endowing traditional contrast agents with metabolic characteristics of renal clearance are key factors in expanding their biological applications and promoting their clinical transformation.

[0003] Sulfonated calixarene has remarkable biological properties, such as high bioavailability, oral absorption, excellent potential in biomedical applications, and good biocompatibility and non-cytotoxicity. In vitro evaluation shows that sulfonated calixarene at a concentration of up to 5mM has no hemolytic toxicity, does not stimulate neutrophils, and does not cause nonspecific immune responses. In addition, sulfonated calix[4]arene can be rapidly cleared through urine without accumulating in the liver [Chemical Communications, 2006 (23): 2425-2438.]. Therefore, it is theoretically feasible to use sulfonated calixarene as a renal clearance carrier for optical contrast agents.

[0004] In the field of optical imaging, cyanines have been shown to be the most promising class of dyes for biomedical applications, with advantages such as large molar absorption coefficient, high fluorescence quantum yield, and good stability. In addition, since its absorption and emission are located in the near-infrared range, the absorption and autofluorescence intensity of biological tissues are low, and thus it is used as a developer, with advantages such as small background interference, high sensitivity, and non-invasiveness, and has been widely used in the fields of protein and nucleic acid labeling, gene sequencing, animal in vivo imaging, and clinical angiography. However, traditional cyanine dyes have a highly lipophilic conjugated polyene center, which strongly binds to plasma proteins in serum after intravenous injection, and is mainly absorbed and metabolized by the mononuclear phagocyte system (such as liver and spleen), and some can continue to accumulate on the skin lipid membrane [Angewandte Chemie International Edition, 2015, 127 (51): 15654-15658.]. For example, IR783, IR820, and ICG are water-soluble cyanine dyes, which are widely used in the optical diagnosis and treatment of various diseases. IR783 has better water solubility and higher singlet oxygen quantum yield among the three, and is widely used in near-infrared bioimaging and photodynamic therapy. It is mainly metabolized by the liver and has serious skin accumulation. ICG (indocyanine green) is the only near-infrared contrast agent approved by the U.S. FDA for clinical use. It is strongly bound to plasma proteins and is quickly cleared from the blood, mainly metabolized by the liver [Angewandte Chemie International Edition, 2015, 127 (51): 15654-15658.]. IR820 has pharmacokinetic behavior comparable to ICG, but it has a longer emission wavelength, and due to its extended conjugation and increased planarity, it has stronger hydrophobicity, resulting in severe aggregation in water and weak fluorescence. However, due to the improved stability of the aggregates formed in water and the improved photothermal efficiency, it is widely used in the photothermal therapy of diseases and is called "new indocyanine green".

[0005] Therefore, utilizing the renal clearance and easy modification properties of sulfonated calixarene to develop single-molecule, long-circulating, near-infrared renal-cleared fluorescent probes is of great significance for biological imaging, disease diagnosis, and urinary system diagnosis and treatment.

[0006] This application aims to describe a renal-cleared long-circulating near-infrared fluorescent compound and its preparation method and application. By describing the compound structure, synthesis and preparation, and application scenarios in detail, this patent will provide researchers and technical developers in related fields with a new tool to achieve efficient, accurate and reliable fluorescence detection. Summary of the invention

[0007] The purpose of the present invention is to overcome the problems of short blood circulation time, insufficient tumor targeting, difficulty in preparing polymer / macromolecular compounds, and unstable shoulder differences of traditional renal-cleared fluorescent probes, and to provide a renal-cleared long-circulation near-infrared fluorescent compound.

[0008] A further object of the present invention is to provide a method for preparing the above-mentioned kidney-cleared long-circulating near-infrared fluorescent compound.

[0009] A further object of the present invention is to provide the use of the above-mentioned renal-cleared long-circulating near-infrared fluorescent compound or its pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, tautomer in the preparation of fluorescent probes.

[0010] A further object of the present invention is to provide the use of the above-mentioned kidney-cleared long-circulating near-infrared fluorescent compound in surgical navigation resection and phototherapy of breast cancer.

[0011] To achieve the above purpose, the present invention adopts the following technical solution.

[0012] A kidney-cleared long-circulation near-infrared fluorescent compound having a chemical structure shown in any one of formulas (I) to (III):

[0013] Here, n is an integer from 1 to 5.

[0014] The present invention uses sulfonated calixarene as a renal clearance and long circulation unit, its lower edge is modified with an azide group, a cyanine dye is used as a fluorophore (chromophore), one end is modified with an alkynyl group, and the final probe is obtained by a copper-catalyzed azide-alkynyl cycloaddition reaction. Among them, the fluorophore has near-infrared fluorescence emission and a good fluorescence quantum yield, and can realize in vivo optical imaging. This type of probe not only has a high signal-to-noise ratio and good biocompatibility, but also has good water solubility and rapid renal clearance efficiency due to the introduction of sulfonated calixarene.

[0015] It should be noted that in formula (I) to formula (III), the fluorophores are not limited to the three types mentioned above. In principle, any fluorophore that can modify the alkynyl group can be coupled with the sulfonated calixarene through a click reaction to obtain a renal-cleared long-circulation near-infrared fluorescent compound.

[0016] The kidney-cleared long-circulating near-infrared fluorescent compound provided by the present invention can be prepared into dosage forms of various administration forms, including intravenous injection, spraying, and intraperitoneal injection. Taking the intravenous administration form as an example, the kidney-cleared long-circulating near-infrared fluorescent compound described in the present invention has a renal clearance rate of >40% within 24 hours after intravenous administration, and a significantly increased blood circulation time compared to the corresponding commercial probe. It can be quickly enriched in the tumor, realizing the in vivo detection and visual identification of the lesion site. In addition, it also provides an important tool for fluorescent navigation surgery, which is expected to provide a new auxiliary means for human precision medicine.

[0017] The method for preparing the above-mentioned kidney-cleared long-circulation near-infrared fluorescent compound comprises the following steps:

[0018] The compound CnACN is prepared as Figure 1 As shown, compound CnA is heated to 80°C in acetonitrile to completely dissolve it, then cooled to room temperature, sodium methoxide is added, and stirred at room temperature for 1 hour, finally sodium methoxide is added and stirred at room temperature for 1 hour, finally bromoacetonitrile is added and stirred at 80°C for 24 hours, the reaction is monitored by thin layer chromatography, and finally silica gel column chromatography is used to separate to obtain compound CnACN.

[0019] The preparation of compound CnANH2 is as follows Figure 1 As shown, the compound CnACN was first dissolved in anhydrous tetrahydrofuran, the reaction was placed in an ice bath, BH3 / THF solution was slowly added, and the mixture was stirred for 1 hour before returning to room temperature. The reaction solution was heated to 70°C and stirred for 24 hours. The reaction solution was cooled to room temperature, methanol was added to quench, and then hydrochloric acid solution (1M) was added and stirred at 50°C for 0.5 hours. After the organic solvent was removed under reduced pressure, a white precipitate was obtained, which was filtered and further purified by silica gel column chromatography to obtain the compound CnANH 2。

[0020] The compound SCnANH was prepared as Figure 1 As shown, the compound CnANH2 was mixed with concentrated sulfuric acid in an ice-water bath, heated to 55°C and stirred for 24 hours. The reaction solution was cooled to room temperature, added dropwise to ether and stirred for 20 minutes, allowed to stand, the supernatant was decanted, ether was added again and stirred for 20 minutes, the precipitate was filtered, the filter cake was ground in ether and filtered again, and vacuum dried to obtain SCnANH.

[0021] Compound SC4AN3 was prepared as Figure 1 As shown, compound SCnANH is dissolved in water together with potassium carbonate and copper sulfate pentahydrate, 1H-imidazole-1-sulfonyl azide hydrochloride is added, and the mixture is stirred at room temperature for 24 hours. The product is recrystallized from acetonitrile, filtered and dried in vacuo to obtain compound SC4AN3.

[0022] The preparation of compounds of formula (I) to formula (III) is as follows Figure 1 As shown, the dye molecule (CyP1, CyP2 or CyP3, 1 equivalent) is dissolved in a mixed solvent of tetrahydrofuran and ethanol; SC4AN3 (1 equivalent), L-sodium ascorbate (1.3 equivalents) and CuSO4·5H2O (0.1 equivalent) are dissolved in water, and then the aqueous solution is added to the probe solution and stirred at 60°C for 24 hours. The compounds of formula (I) to (III) are obtained by separation and purification by rapid preparative liquid chromatography (spherical C18 (AQ) column, 20 μm to 45 μm), eluted with water and acetonitrile, and freeze-dried.

[0023] A method for preparing a kidney-cleared long-circulation near-infrared fluorescent compound comprises the following steps: Figure 1 The compound shown (CyP1, CyP2 or CyP3) and Figure 1 The compound SC4AN3 shown is subjected to a copper-catalyzed azide-alkyne cycloaddition reaction at 60° C. in a mixed solvent of tetrahydrofuran, ethanol and water, and then separated by a water-resistant C18 column to obtain compounds shown in formula (I) to formula (III).

[0024] The use of the above-mentioned renal-cleared long-circulating near-infrared fluorescent compound or its pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, tautomer in the preparation of fluorescent probes is also within the protection scope of the present invention.

[0025] Preferably, the fluorescent probe is a renal-cleared, long-circulating fluorescent probe.

[0026] Preferably, the pharmaceutically acceptable salt is hydrochloride, hydrobromide, nitrate, methylnitrate, sulfate, bisulfate, aminosulfate, phosphate, acetate, glycolate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, para-aminosalicylate, glycolate, lactate, enanthate, phthalate, oxalate, succinate, benzoate, o-acetyl At least one of oxybenzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, mandelate, tannate, formate, stearate, ascorbate, palmitate, oleate, pyruvate, bishydroxynaphthoate, malonate, laurate, glutarate, glutamate, propionate lauryl sulfate, methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-aminobenzenesulfonate, p-toluenesulfonate (toluenesulfonate) or naphthalene-2-sulfonate.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The renal-cleared long-circulating near-infrared fluorescent probe provided by the present invention uses a cyanine dye derivative as a fluorophore and a sulfonated calixarene derivative as a renal clearance and long-circulation unit. The two are efficiently and simply obtained through a click reaction. It not only has good fluorescence properties, excellent water solubility, and good biocompatibility, but also has faster renal clearance efficiency, better signal-to-noise ratio and tumor targeting. It can more accurately locate the lesion site, perform intraoperative navigation and tissue and organ function evaluation, etc., can play an important role in future medical imaging examinations and surgeries, and has broad application prospects and commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Synthesis route of a renal-cleared long-circulating near-infrared fluorescent probe described in the present invention

[0030] Figure 2 Absorption spectra of the renal-cleared long-circulating near-infrared fluorescent probe SC4-RCOAs (5 μM) and commercial reference molecules IR783, IR820 and ICG (5 μM) of the present invention

[0031] Figure 3 Fluorescence spectra of the renal-cleared long-circulating near-infrared fluorescent probe SC4-RCOAs (5 μM) and commercial reference molecules IR783, IR820 and ICG (5 μM) of the present invention

[0032] Figure 4 Pharmacokinetic evaluation of the renal-cleared long-circulating near-infrared fluorescent probe of the present invention. (a) Blood pharmacokinetic curve of each probe (200 μM) injected into the tail vein and (b) cumulative urine excretion rate curve. n=3, mean ± standard deviation.

[0033] Figure 5 The renal-cleared long-circulating near-infrared fluorescent probe SC4-CyP3 of the present invention is used for in vivo imaging: (a) fluorescence imaging of SC4-CyP3 and ICG (200 μM) in subcutaneous 4T1-LUC1 tumor-bearing BALB / c-nu mice; (b) probe aggregation fluorescence intensity comparison at the tumor site; (c) tumor-muscle signal-to-noise ratio of SC4-CyP3 and ICG (marked by red and yellow circles). Fluorescence conditions are the same as before, n=3, mean±SD.

[0034] Figure 6 The renal-cleared long-circulating near-infrared fluorescent probe SC4-CyP3 of the present invention is used for in vivo imaging: (a) Fluorescence imaging of SC4-CyP3 (200 μM) in BALB / c-nu mice bearing mammary 4T1-LUC1 tumors; (b) quantification of probe aggregation fluorescence intensity at the tumor site (tumor marked by red circle). Fluorescence conditions are the same as before, n=3, mean±SD. Figure 7The renal-cleared long-circulating near-infrared fluorescent probe SC4-CyP3 of the present invention is used to guide the surgical resection of in situ 4T1-LUC1 breast tumors. Abbreviations: Fl., fluorescence; Lu., bioluminescence; BF, bright field; T1, tumor 1; T2, tumor 2. DETAILED DESCRIPTION

[0035] The content of the present invention will be described in detail below in conjunction with specific embodiments. It should be noted that the following description is an explanation of the present invention rather than a limitation, and various changes can be made within the scope of the present invention.

[0036] Example 1

[0037] This embodiment provides a renal-cleared long-circulation near-infrared fluorescent compound, and a schematic diagram of its preparation method is shown in Figure 1 , taking n=1 as an example, the preparation method when n=2 to 5 is similar to that when n=1, specifically comprising the following steps:

[0038] 1. Add C4A (n = 1) (1.0 g, 2.4 mmol) and acetonitrile (150 mL) to a 500 mL round-bottom flask, heat to 80 ° C to completely dissolve C4A, then cool to room temperature, add sodium methoxide (0.19 g, 3.5 mmol) and stir at room temperature for 1 h, finally add bromoacetonitrile (0.56 g, 4.7 mmol) and stir at 80 ° C for 24 h. Separate the target product by silica gel column chromatography, and elute with ethyl acetate / petroleum ether (v:v, 1:3) to obtain compound C4ACN (n = 1). C4ACN, white solid, yield 32%. 1 H NMR(400MHz,Chloroform-d)δ9.27(s,1H),8.50(s,2H),7.09(q,J=7.5Hz,6H),7.00(d,J=7.6Hz,2H),6.95(t,J=7.6Hz,1H),6.71(d t,J=11.1,7.5Hz,3H),5.03(s,2H),4.34(d,J=13.3Hz,2H),4.24(d,J=13.9Hz,2H),3.59(d,J=13.4Hz,2H),3.50(d,J=13.9Hz,2H). 13 C NMR(100MHz,Chloroform-d)δ150.7,150.1,148.7,133.4,130.1,129.1,128. 8,128.4,128.3,128.1,127.56,127.5,122.1,121.1,114.5,60.5,31.7,31.5.

[0039] 2. In a 100mL round-bottom flask, C4ACN (0.6g, 1.3mmol) was dissolved in anhydrous tetrahydrofuran (10mL), the reaction solution was placed in an ice-water bath, BH3 / THF solution (15mL, 1M) was added and stirred for 1h, and after returning to room temperature, the reaction solution was heated to 70°C and stirred for 24h. After the reaction solution was cooled to room temperature, methanol (5mL) was added to quench the reaction, and then hydrochloric acid solution (15mL, 1M) was added and stirred at 50°C for 0.5h. After the organic solvent was removed under reduced pressure, a white precipitate was obtained, which was filtered and further purified by silica gel column chromatography. Compound C4ANH (n=1) was obtained by eluting with dichloromethane / methanol (v:v, 1:50). C4ANH, white solid, yield 77%. 1 H NMR(400MHz,Chloroform-d)δ7.13–6.98(m,8H),6.90(t,J=7.5Hz,1H),6.69( t,J=7.5Hz,3H),4.37(d,J=13.1Hz,2H),4.30–4.19(m,4H),3.60–3.31(m,6H). 13 C NMR (100MHz, DMSO-d6) δ159.7,155.2,153.0,135.4,130.9,130.8,129.8,128.6,128.1,124.4,118.1,114.7,69.6,40.7,34.2,31.5.

[0040] 3. Add C4ANH (0.5 g, 1.1 mmol) to a 50 mL round-bottom flask and mix with concentrated sulfuric acid (10 mL) in an ice-water bath, then stir at 55 °C for 24 h. Cool the reaction solution to room temperature, drop into ether (180 mL) and stir for 20 min, let stand, pour off the supernatant, add ether (150 mL) and stir for 20 min, filter the precipitate, grind the filter cake in ether (10 mL), filter again, and vacuum dry to obtain SC4ANH (n=1). SC4ANH, off-white solid, yield 89%. 1 H NMR(400MHz,D2O)δ7.66(s,2H),7.58(s,2H),7.48(d,J=4.8Hz,4H),3.98(q ,J=16.3Hz,6H),3.79(t,J=4.9Hz,2H),3.67(d,J=14.3Hz,2H),2.58(s,2H). 13C NMR (100MHz, D2O) δ156.1,152.6,138.4,135.5,135.0,134.1,129.8,129.5,128.4,127.7,126.3,126.1,125.9,125.6,67.4,38.6,35.0,30.7.

[0041] 4. SC4ANH (0.3 g, 0.38 mmol), K2CO3 (0.14 g, 1.02 mmol) and CuSO4·5H2O (0.95 mg, 0.0038 mmol) were dissolved in H2O (4 mL), and then 1H-imidazole-1-sulfonyl azide hydrochloride (78 mg, 0.46 mmol) was added and stirred at room temperature for 24 h. The product was recrystallized from acetonitrile (50 mL), filtered and dried in vacuo to obtain SC4A-N3 (n=1). Pale yellow solid, yield 92%. 1 H NMR(400MHz,D2O)δ7.66(s,2H),7.61(d,J=10.5Hz,4H),7.49(s,2H),4.22(d,J=14.9Hz,2 H), 4.13–4.06 (m, 2H), 4.03 (d, J = 14.3Hz, 2H), 3.87 (dd, J = 19.3, 14.7Hz, 4H), 3.58 (s, 2H). 13 C NMR (100MHz, D2O) δ155.5,154.2,138.3,134.0,133.9,132.90,129.6,129.0,128.9,126.4,126.1,125.8,125.6,125.2,72.6,50.2,31.9,31.5.

[0042] 5. Dissolve the dye molecule (CyP1, CyP2 or CyP3, 1 eq) in a mixed solvent of tetrahydrofuran (1 mL) and ethanol (2 mL); dissolve SC4AN3-Na (1 eq), sodium L-ascorbate (1.3 eq) and CuSO4·5H2O (0.1 eq) in water (3 mL), then add the aqueous solution to the probe solution and stir at 60°C for 24 h. Separate and purify by rapid preparative liquid chromatography (SepaBean TMmeachine T, spherical C18 (AQ) column, 20 μm-45 μm), eluted with water and acetonitrile (0-5 min: 100% water, 5-10 min: 80% water and 20% acetonitrile, 10-15 min: 80% water and 20% acetonitrile), freeze-dried to obtain compounds SC4-CyP1 (n=1), SC4-CyP2 (n=1) and SC4-CyP3 (n=1). SC4-CyP1, dark green solid, yield 70%. 1 HNMR(400MHz, DMSO-d6:D2O=7:3)δ8.23(d,J=11.4Hz,2H),7.59–7.52(m, 2H),7.49–7.35(m,10H),7.35–7.21(m,5H),4.40(s,2H),4.15(s,6H),3.9 1(d,J=13.1Hz,4H),3.49(d,J=12.8Hz,4H),2.63(dt,J=14.9,9.6Hz,8H), 1.88–1.77(m,4H),1.72(dd,J=14.6,8.4Hz,4H),1.61(d,J=18.9Hz,14H). 13 C NMR (101MHz, DMSO-d6:D2O=7:3) δ172.7,1726,163.5,152.3,150.1,148.6,143.9,143.6,1 43.4,142.4,142.3,141.5,141.4,140.0,139.0,133.5,129.2,127.6,127.30,127.2,126.9 ,126.8,126.5,125.6,123.8,122.9,111.9,111.7,101.8,74.6,51.0,49.4,49.4,44.1,43 .8,39.8,36.6,31.4,31.3,31.1,27.9,26.6,26.4,26.2,24.7,22.5,20.8.HRMS(MALDI-TOF MS):C 70 H 71 ClN5O 19 S5 3- , m / z calculated value [M] 3- 493.4351, found 493.4350. SC4-CyP2, dark green solid, yield 67%. 1H NMR(400MHz, DMSO-d6:D2O=7:3)δ8.30(d,J=40.7Hz,4H),8.04(d,J=16.1Hz,5H),7.73–7.24(m,16H),4.43–4.18(m,8H) ,3.90(d,J=13.2Hz,4H),3.51(d,J=11.2Hz,4H),2.72–2.60(m,8H),1.90(d,J=15.6Hz,16H),1.77(s,4H),1.68(s,2H). 13 C NMR (100MHz, DMSO-d6:D2O=7:3)δ173.8,163.5,154.7,152.4,152.3,150.2,148.1,1 46.9,143.9,142.5,140.0,139.0,134.0,133.5,131.8,131.0,130.4,128.4,127.9,1 27.6,127.4,127.3,126.8,126.5,125.6,124.0,122.7,112.0,101.3,74.2,51.1,51 .0,50.1,44.3,31.4,27.4,26.9,26.7,26.3,26.1,24.7,22.5,20.9.HRMS(MALDI-TOF MS):C 78 H 75 ClN5O 19 S5 3- , m / z calculated value [M] 3- 526.7788, found 526.7784. SC4-CyP3, dark green solid, yield 58%. 1 H NMR(400MHz, DMSO-d6:D2O=7:3)δ8.21(t,J=9.0Hz,2H),8.08–7.88(m,6H),7. 77–7.55(m,4H),7.53(d,J=9.1Hz,1H),7.48(t,J=7.0Hz,2H),7.45–7.19(m,9H ),4.35(s,2H),4.21–4.01(m,6H),3.86(d,J=13.0Hz,4H),3.49(t,J=12.3Hz,4 H),2.70–2.59(m,4H),1.97–1.82(m,12H),1.74(td,J=16.3,15.6,9.5Hz,8H). 13C NMR (100MHz, DMSO-d6:D2O=7:3)δ173.3,172.6,163.4,152.5,152.4,150.0,147.1,144.1,1 40.2,139.2,133.8,133.4,131.6,130.8,130.4,128.2,128.0,127.6,127.1,126.8,126.6, 125.2,124.0,122.6,112.0,111.8,105.6,74.8,54.0,51.1,50.9,50.8,50.1,44.0,43.6,4 2.4,36.5,31.4,31.1,27.2,26.9,26.7,26.1,24.9,22.5,17.4,16.1,12.6.HRMS(MALDI-TOF MS):C 75 H 72 N5O 19 S5 3- , m / z calculated value [M] 3- 502.1147, measured value 502.1150.

[0043] Example 2

[0044] This embodiment provides a renal-cleared long-circulation near-infrared fluorescent compound. The performance test is performed using n=1 as an example. When n=2 to 5, the performance is similar to that of n=1. Specifically, the performance test includes the following:

[0045] 1. Spectral test: The absorption and fluorescence spectra of SC4-CyP1 (5 μM), SC4-CyP1 (5 μM) and SC4-CyP3 (5 μM) were tested by UV-Vis spectrophotometer and fluorescence spectrophotometer in HEPES (10 mM, pH = 7.40) buffer solution, methanol and 30% FBS (diluted with HEPES). The test results are as follows: Figure 2 and Figure 3 shown.

[0046] 2. Pharmacokinetics and renal clearance test: BALB / c mice (n=3 for each probe) were injected with fluorescent molecules (200μM, 100μL) through the tail vein, and about 30μL of blood was collected by cutting the tail of heparinized capillary tubes at 1min, 3min, 5min, 10min, 15min, 20min, 30min, 1h, 1.5h, 2h, 3h, 4h, 5h, 6h, 8h, 12h, and 24h after injection. The blood was centrifuged at 3500r.pm for 15min, and the plasma was diluted with FBS. The fluorescence intensity was collected by NightOWL LB983 NC100 small animal imager, and the concentration of the probe in plasma was quantified according to the standard curve. After that, the pharmacokinetic parameters were calculated by the intelligent pharmacokinetic analysis of the software DAS (Data Analysis System). At the same time, in order to quantify the urinary excretion rate of the probe, urine was collected at 4h, 8h, 12h, and 24h after the injection of the probe. The fluorescence intensity was also collected by the NightOWL LB983 NC100 small animal imager, and the concentration of the probe in urine was quantified according to the standard curve.

[0047] The test results are as follows Figure 4 As shown in Figure 2, compared with their corresponding commercial probes (IR783, IR820 and ICG), SC4-CyP1, SC4-CyP2 and SC4-CyP3, respectively, the sulfonated calix[4]arene hardly changed the distribution speed (t 1 / 2α ), and significantly prolonged its elimination rate. The elimination half-life of IR820 and ICG (t 1 / 2β ) was only 0.08h (4.8min) and 0.058h (3.48min), while the elimination half-life of SC4-CyP2 and SC4-CyP3 was extended to 1.38h and 2.40h, and that of SC4-CyP1 was significantly extended to 7.45h. el ), and clearance rate (Cl) all showed the same results. This means that the blood circulation time of SC4-RCOAs is prolonged. Compared with the rapid clearance of IR820 and ICG, the bioavailability of SC4-RCOAs is improved. The drug-time curve and the area under the curve (AUC) also verified this result. The distribution rate from the central compartment to the peripheral compartment (K 12 ), the distribution rate from the peripheral chamber to the central chamber (K 21 ), and the apparent volume of distribution (V d ) show complex patterns, for example, K 12(IR783) >K 12(SC4-CyP1) , K 21(IR783) <K 21(SC4-CyP1)Compared with SC4-CyP1, IR783 can be distributed from the central chamber to the peripheral chamber faster, but will stay in the peripheral chamber tissue longer; while SC4-CyP2 and IR820 showed the opposite results, K 12(IR820) <K 12(SC4-CyP2) , K 21(IR820) >K 21(SC4-CyP2) ; For SC4-CyP3 and ICG, K 12(ICG) >K 12(SC4-CyP3) , K 21(ICG) >K 21(SC4-CyP3) , which reflects the rapid clearance rate of ICG (t 1 / 2β =3.48 min), and low bioavailability (AUC = 2.74% ID / g·h), while the bioavailability of SC4-CyP3 was significantly improved (AUC = 74.72% ID / g·h).

[0048] Sulfonated calix[4]arene changed the metabolic pathways of IR783, IR820 and ICG, causing them to be eliminated mainly through the kidneys and metabolized out of the body through urine. The cumulative urinary excretion rates of SC4-RCOAs within 24 hours were 53.08%, 40.44% and 79.34%, respectively.

[0049] The above results demonstrate that the renal-cleared long-circulation near-infrared fluorescent compound provided by the present invention has a longer blood circulation time and good renal clearance efficiency.

[0050] 3. In vivo tumor imaging. 4T1-LUC1 (4T1-Luciferase, luciferase-labeled 4T1) cells were used at 2×10 7 100 μL of cells were injected into the subcutaneous tissue of the back of BALB / c-nu mice and into the mammary fat pad of mice to establish subcutaneous and orthotopic breast tumor models. 3 To investigate the enrichment of SC4-CyP3 and ICG in tumors, SC4-CyP3 and ICG (200 μM, 100 μL) were injected into the tail vein of the two tumor models, and in vivo fluorescence imaging was performed at 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, and 72 h after injection (n = 3 for each probe).

[0051] like Figure 5As shown in the imaging of subcutaneous tumors, ICG has no obvious fluorescence at the tumor site due to its rapid clearance in the body. Through the fluorescence intensity ratio processing of the tumor and normal tissue sites, it can be seen that ICG shows the highest tumor signal-to-noise ratio at 8 hours; while SC4-CyP3 has obvious and continuous accumulation at the tumor site, with the strongest fluorescence at the tumor site at 24 hours, and still has a high level of accumulation at 72 hours. In addition, since the probes in the normal tissue site are gradually metabolized out of the body, SC4-CyP3 shows the highest tumor signal-to-noise ratio (TBR>2, Figure 5 ). In addition, in the imaging of breast tumors in situ ( Figure 6 ), the rate of SC4-CyP3 enrichment in the tumor was accelerated, the fluorescence in the tumor site reached the strongest at 4 h, and the clearance rate of SC4-CyP3 in breast in situ tumors was also accelerated compared with subcutaneous tumors.

[0052] 4. Fluorescence-guided surgery and phototherapy. Surgical resection of the tumor (n=3) was performed 72 hours after tail vein injection of SC4-CyP3 (200 μM, 100 μL). The entire resection process was divided into 4 steps: 1) Fluorescence imaging of the tumor site at 72 hours, and intraperitoneal injection of 100 μL of LUC1 substrate. After 10 minutes, bioluminescence imaging of the tumor site was performed. The location of bioluminescence indicated the location of tumor cells; 2) Tumors were removed according to the location indicated by SC4-CyP3 fluorescence; 3) SC4-CyP3 fluorescence imaging and bioluminescence imaging were performed on the cut surface after tumor resection to capture the remaining tumor tissue. The bioluminescence intensity was greatly reduced, and additional LUC1 substrate needed to be dripped onto the cut surface for imaging; 4) SC4-CyP3-mediated photodynamic therapy (808 nm, 1 W / cm 2 ,15min) and performed imaging again.

[0053] like Figure 7 As shown in the figure, compared with bioluminescence, the fluorescence signal of SC4-CyP3 can clearly define the edge between tumor and normal tissue, guide the removal of tumor; and is not limited by the duration of surgery during tumor removal; it also has high tumor selectivity and can detect smaller tumors in breast tissue (T2). In contrast, bioluminescence has a short maintenance time and requires substrate supplementation during the removal process (Step 3). However, the LUC1 substrate has a certain amount of self-luminescence, which will interfere with the bioluminescence interacting with the tumor at high concentrations; in addition, bioluminescence fails to detect Figure 7Small tumors in the tumor (T2). Under the fluorescent guidance of SC4-CyP3, as much tumor tissue as possible was removed, but there was still residual tumor tissue that could not be removed manually. Since SC4-CyP3 has photodynamic properties, photodynamic therapy can be performed through the probes in the residual tumor tissue to kill the residual tumor cells (Step 4). Furthermore, H&E staining of the removed tumor tissue and the wound surface of photodynamic therapy confirmed that SC4-CyP3 can accurately develop the tumor edge and has a photodynamic killing effect on the residual tumor tissue ( Figure 7 Finally, the tissue biodistribution of SC4-CyP3 after 72 h of metabolism in two tumor models was investigated. Figure 7 As shown, it is most distributed in tumors and kidneys, indicating that it accumulates in large quantities in tumor tissues and that the residual probes in the body are still mainly metabolized by the kidneys.

[0054] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A kidney-cleared long-circulating near-infrared fluorescent compound, characterized in that: It has a chemical structure shown in any one of formula (I) to formula (III): Here, n is an integer from 1 to 5.

2. The renal-cleared long-circulating near-infrared fluorescent compound according to claim 1, characterized in that: The kidney-cleared long-circulating group is Wherein n is an integer from 1 to 5.

3. The renal-cleared long-circulating near-infrared fluorescent compound according to claim 1, characterized in that: The fluorophore structure is 4. The renal-cleared long-circulation near-infrared fluorescent compound according to claim 1, wherein the key intermediate is SCnA-N3, and the structure is:

5. The method for preparing the key intermediate SCnA-N3 as claimed in claim 4, characterized in that: The method comprises the following steps: the compound CnA shown in FIG1 is monosubstituted with a cyanoethyl group, borane is reduced to an amine, and 1H-imidazole-1-sulfonyl azide hydrochloride is added to obtain SC4AN3.

6. The method for preparing the renal-cleared long-circulating near-infrared fluorescent compound according to claim 1, characterized in that: The method comprises the following steps: reacting a compound (CyP1, CyP2 or CyP3) with a compound SC4AN3 in a mixed solvent of tetrahydrofuran, ethanol and water at 60°C by copper-catalyzed azide-alkyne cycloaddition reaction, and then separating the compounds represented by formula (I) to formula (III) through a water-resistant C18 column.

7. Use of any one of the renal-cleared long-circulating near-infrared fluorescent compounds according to claim 1 or its pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, and tautomers in the preparation of fluorescent probes.

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