A kidney-cleared long-circulating near-infrared fluorescent probe and its preparation method and application

Abstract

A renal-cleared, long-circulating near-infrared fluorescent probe, its preparation method, and applications. This renal-cleared, long-circulating near-infrared fluorescent probe utilizes a sulfonated calixarene derivative as a renal-clearing moiety and a cyanine dye derivative as a fluorophore, coupled via a click reaction. This probe exhibits broad spectrum compatibility with a variety of fluorophores and exhibits excellent water solubility, biocompatibility, rapid renal clearance, extended blood circulation time, and tumor targeting. As a fluorescent probe, it enables more precise non-invasive imaging of diseases and intraoperative navigation.

Description

Technical Field

[0001] The present 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 crucial role in clinical diagnosis and treatment. Assisted by optical instruments, optical imaging uses optical contrast agents to visualize biomolecules, cells, tissues, and organisms in real time and in multiple dimensions. It is widely used in biomolecule detection and imaging, drug distribution and metabolism tracking, disease detection and diagnosis, and other fields. It offers advantages such as being noninvasive, non-ionizing, and possessing high temporal and spatial resolution and sensitivity. Contrast agents, also known as contrast agents, are substances that enhance the contrast of medical imaging images by altering parameters such as tissue density, signal intensity, or the speed of sound, making the tissue or organ in the image more clearly visible. Traditional optical contrast agents are primarily cleared through the reticuloendothelial system and often accumulate in the liver and spleen, leading to potential organ toxicity. In contrast, renal-clearable optical agents (RCOAs) are rapidly excreted through the kidneys, effectively reducing the risk of intracellular catabolism and potential toxicity to normal organs. Therefore, developing new RCOAs or endowing traditional contrast agents with metabolic characteristics of renal clearance is a key factor in expanding their biological applications and promoting their clinical translation.

[0003] Sulfonated calixarene has remarkable biological properties, such as high bioavailability and oral absorption, and has excellent potential in biomedical applications. It also has good biocompatibility and non-cytotoxicity. In vitro evaluations have shown that sulfonated calixarene at concentrations 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, the use of sulfonated calixarene as a renal clearance carrier for optical contrast agents has sufficient theoretical feasibility.

[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 their absorption and emission are located in the near-infrared range, the absorption and autofluorescence intensity of biological tissues are low, so they are used as developer with advantages such as low background interference, high sensitivity, and non-invasiveness. They have 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 the liver and spleen). 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 that 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 more serious skin accumulation. ICG (indocyanine green) is the only near-infrared contrast agent approved by the U.S. FDA for clinical use. It strongly binds 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 a pharmacokinetic behavior comparable to ICG, but it has a longer emission wavelength and, due to its extended conjugation and increased planarity, has stronger hydrophobicity, resulting in serious aggregation in water and weak fluorescence. However, because the stability of the aggregates formed in water is improved, the photothermal efficiency is improved, and therefore it is widely used in the photothermal treatment 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 describes a renal-cleared, long-circulating near-infrared fluorescent compound, its preparation method, and its applications. By describing the compound's structure, synthesis and preparation, and application scenarios, this patent will provide researchers and technical developers in related fields with a new tool for efficient, accurate, and reliable fluorescence detection. Summary of the Invention

[0007] The purpose of the present invention is to overcome the problems of traditional renal-cleared fluorescent probes such as short blood circulation time, insufficient tumor targeting, difficulty in preparing polymer / macromolecular compounds, and unstable shawl differences, 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 renal-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 renal-cleared long-circulating near-infrared fluorescent compound in surgical navigation resection and phototherapy of breast cancer.

[0011] To achieve the above objectives, the present invention adopts the following technical solutions.

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

[0013]

[0014]

[0015] Here, n is an integer from 1 to 5.

[0016] The present invention uses a sulfonated calixarene as a renal clearance and long-circulation unit, with an azide group modified at its lower edge, a cyanine dye as a fluorophore (chromophore), and an alkyne modified at one end. The final probe is obtained through a copper-catalyzed azide-alkyne cycloaddition reaction. The fluorophore has near-infrared fluorescence emission and a good fluorescence quantum yield, enabling 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 due to the introduction of the sulfonated calixarene.

[0017] 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 alkyne group can be coupled with the sulfonated calixarene through a click reaction to obtain a renal-cleared long-circulating near-infrared fluorescent compound.

[0018] The renal-cleared, long-circulating near-infrared fluorescent compound provided by the present invention can be prepared into dosage forms for a variety of administration modes, including intravenous injection, spraying, and intraperitoneal injection. Taking intravenous administration as an example, the renal-cleared, long-circulating near-infrared fluorescent compound described in the present invention has a renal clearance rate of >40% within 24 hours, significantly increased blood circulation time, and can be rapidly enriched in tumors, enabling in vivo detection and visual identification of lesions. It also provides an important tool for fluorescence-guided surgery and is expected to become a new auxiliary means for precision medicine.

[0019] The preparation method of the above-mentioned renal-cleared long-circulation near-infrared fluorescent compound comprises the following steps:

[0020] The compound CnACN was 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.

[0021] 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, quenched by adding methanol, and then hydrochloric acid solution (1M) was added and stirred at 50°C for 0.5 hours. After removing the organic solvent under reduced pressure, a white precipitate was obtained, which was filtered and further purified by silica gel column chromatography to obtain compound CnANH 2。

[0022] Compound SCnANH was prepared as Figure 1 As shown, 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 diethyl ether, and stirred for 20 minutes. The mixture was allowed to stand, and the supernatant was decanted. Diethyl ether was then added and stirred for 20 minutes. The precipitate was filtered, and the filter cake was ground in diethyl ether, filtered again, and dried in vacuo to obtain SCnANH.

[0023] Compound SC4AN3 was prepared as follows Figure 1 As shown, compound SCnANH is dissolved in water 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.

[0024] 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), sodium L-ascorbate (1.3 equivalents), and CuSO4·5H2O (0.1 equivalent) are dissolved in water. This aqueous solution is then added to the probe solution and stirred at 60°C for 24 hours. The mixture is separated and purified by rapid preparative liquid chromatography (spherical C18 (AQ) column, 20μm to 45μm), eluted with water and acetonitrile, and freeze-dried to obtain compounds of Formulas (I) to (III).

[0025] The preparation method of 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 is subjected to a copper-catalyzed azide-alkyne cycloaddition reaction in a mixed solvent of tetrahydrofuran, ethanol and water at 60° C., and then separated by a water-resistant C18 column to obtain compounds of formula (I) to formula (III).

[0026] 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 scope of protection of the present invention.

[0027] Preferably, the fluorescent probe is a renal-cleared, long-circulating fluorescent probe.

[0028] 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, heptanoate, 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.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 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. Both 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., and 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

[0031] Figure 1 Synthesis route of a renal-cleared long-circulating near-infrared fluorescent probe described in the present invention

[0032] Figure 2 Absorption spectra of the renal-cleared long-circulating near-infrared fluorescent probe SC4-RCOAs (5 μM) and commercial control molecules IR783, IR820 and ICG (5 μM)

[0033] Figure 3 Fluorescence spectra of the renal-cleared long-circulating near-infrared fluorescent probe SC4-RCOAs (5 μM) and commercial control molecules IR783, IR820 and ICG (5 μM)

[0034] Figure 4 Pharmacokinetic evaluation of the renal-cleared, long-circulating near-infrared fluorescent probes described herein. (a) Blood pharmacokinetic curves and (b) cumulative urinary excretion curves of each probe (200 μM) following tail vein injection. n = 3, mean ± SD.

[0035] Figure 5 The renal-cleared, long-circulating near-infrared fluorescent probe SC4-CyP3 described in this invention was used for in vivo imaging: (a) Fluorescence imaging of SC4-CyP3 and ICG (200 μM) in BALB / c-nu mice bearing subcutaneous 4T1-LUC1 tumors; (b) Comparison of probe accumulation and fluorescence intensity at the tumor site; (c) Tumor-to-muscle signal-to-noise ratio of SC4-CyP3 and ICG (marked by red and yellow circles). Fluorescence conditions were the same as above. n = 3, mean ± SD.

[0036] Figure 6 The renal-cleared, long-circulating near-infrared fluorescent probe SC4-CyP3 described in this invention was used for in vivo imaging: (a) Fluorescence imaging of SC4-CyP3 (200 μM) in BALB / c-nu mice bearing 4T1-LUC1 mammary tumors; (b) Quantification of probe accumulation and fluorescence intensity at the tumor site (tumor marked by a red circle). Fluorescence conditions were the same as above. n = 3, mean ± SD.

[0037] Figure 7 The renal-cleared, long-circulating near-infrared fluorescent probe SC4-CyP3 described in this invention is used to guide surgical resection of orthotopic 4T1-LUC1 breast tumors. Abbreviations: Fl., fluorescence; Lu., bioluminescence; BF, bright field; T1, tumor 1; T2, tumor 2. DETAILED DESCRIPTION

[0038] The present invention will be described in detail below with reference to specific embodiments. It should be noted that the following description is intended to explain the present invention rather than to limit it, and various changes can be made within the scope of the present invention.

[0039] Example 1

[0040] This embodiment provides a renal-cleared long-circulating near-infrared fluorescent compound, the schematic diagram of its preparation method is shown in Figure 1 , below takes n=1 as an example, when n=2 to 5, the preparation method is similar to that of n=1, specifically comprising the following steps:

[0041] 1. A 500 mL round-bottom flask was charged with C4A (n=1) (1.0 g, 2.4 mmol) and acetonitrile (150 mL). The temperature was raised to 80°C to completely dissolve C4A. The mixture was then cooled to room temperature, and sodium methoxide (0.19 g, 3.5 mmol) was added. The mixture was stirred at room temperature for 1 h. Finally, bromoacetonitrile (0.56 g, 4.7 mmol) was added and the reaction was stirred at 80°C for 24 h. The desired product was isolated by silica gel column chromatography using ethyl acetate / petroleum ether (v:v, 1:3) as the eluent to obtain compound C4ACN (n=1). C4ACN was obtained as a white solid in a 32% yield. 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.

[0042] 2. In a 100 mL round-bottom flask, C4ACN (0.6 g, 1.3 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL). The reaction mixture was placed in an ice-water bath, and BH3 / THF solution (15 mL, 1 M) was added and stirred for 1 h. After returning to room temperature, the reaction mixture was heated to 70°C and stirred for 24 h. After the reaction mixture cooled to room temperature, methanol (5 mL) was added to quench the reaction. Hydrochloric acid solution (15 mL, 1 M) was then added and stirred at 50°C for 0.5 h. The organic solvent was removed under reduced pressure to obtain a white precipitate, which was filtered and further purified by silica gel column chromatography. Elution with dichloromethane / methanol (v:v, 1:50) afforded compound C4ANH (n=1) as a white solid in a 77% yield. 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.

[0043] 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. Stir at 55°C for 24 h. Cool the reaction solution to room temperature and add it dropwise to diethyl ether (180 mL) with stirring for 20 min. Allow to stand, decant the supernatant, add diethyl ether (150 mL) and stir for 20 min. Filter the precipitate, triturate the filter cake in diethyl ether (10 mL), filter again, and dry in vacuo to obtain SC4ANH (n=1). SC4ANH is obtained as an off-white solid in an 89% yield. 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.

[0044] 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). 1H-imidazole-1-sulfonyl azide hydrochloride (78 mg, 0.46 mmol) was then added and stirred at room temperature for 24 h. The product was recrystallized from acetonitrile (50 mL), filtered, and dried in vacuo to afford SC4A-N3 (n=1) as a pale yellow solid in a 92% yield. 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.

[0045] 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). TMThe product was purified by chromatography on a spherical C18 (AQ) column (20 μm to 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), and freeze-dried to afford compounds SC4-CyP1 (n=1), SC4-CyP2 (n=1), and SC4-CyP3 (n=1). SC4-CyP1 was obtained as a dark green solid in a 70% yield. 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.

[0046] Example 2

[0047] This embodiment provides a renal-cleared, long-circulating near-infrared fluorescent compound. The performance test is based on the case of n=1. The performance test of n=2 to 5 is similar to that of n=1, and specifically includes the following:

[0048] 1. Spectral test: The absorption and fluorescence spectra of SC4-CyP1 (5 μM), SC4-CyP1 (5 μM), and SC4-CyP3 (5 μM) were measured by UV-visible 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.

[0049] 2. Pharmacokinetics and Renal Clearance: BALB / c mice (n = 3 per probe) were injected with the fluorescent molecules (200 μM, 100 μL) via the tail vein. Blood was collected (approximately 30 μL) via tail-clipped heparinized capillary tubes at 1, 3, 5, 10, 15, 20, 30, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, and 24 hours after injection. The blood was centrifuged at 3500 rpm for 15 minutes. Plasma was diluted with FBS and fluorescence intensity was measured using a NightOWL LB983 NC100 small animal imager. Probe concentrations in plasma were quantified using a standard curve. Pharmacokinetic parameters were then calculated using the intelligent pharmacokinetic analysis software DAS (Data Analysis System). At the same time, in order to quantify the urinary excretion rate of the probe, urine was collected 4h, 8h, 12h, and 24h after the injection of the probe. The fluorescence intensity was also collected using the NightOWL LB983 NC100 small animal imager, and the concentration of the probe in urine was quantified according to the standard curve.

[0050] The test results are as follows Figure 4 As shown in the figure, compared with their corresponding commercial probes (IR783, IR820 and ICG), SC4-CyP1, SC4-CyP1 and SC4-CyP3, respectively, the sulfonated calix[4]arene hardly changed the distribution speed (t 1 / 2α ) and significantly prolonged their 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 prolonged to 1.38h and 2.40h, and that of SC4-CyP1 was significantly prolonged to 7.45h. el ), and clearance (Cl) 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 confirmed 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 apparent volume of distribution (V d ) showed 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 compartment to the peripheral compartment faster, but will stay in the peripheral compartment 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 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).

[0051] 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.

[0052] The above results demonstrate that the renal-cleared long-circulation near-infrared fluorescent compound provided by the present invention has a long blood circulation time and good renal clearance efficiency.

[0053] 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 accumulation of SC4-CyP3 and ICG in tumors, SC4-CyP3 and ICG (200 μM, 100 μL) were injected into the tail vein of 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).

[0054] 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 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 24 hours, and still has a high level of accumulation at 72 hours. Moreover, since the probe in the normal tissue site is gradually metabolized out of the body, SC4-CyP3 shows the highest tumor signal-to-noise ratio (TBR>2) at 72 hours. 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, and the fluorescence in the tumor site reached the strongest at 4 h. Moreover, the clearance rate of SC4-CyP3 in breast in situ tumors was also accelerated compared with that in subcutaneous tumors.

[0055] 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 four steps: 1) Fluorescence imaging of the tumor site at 72 hours and intraperitoneal injection of 100 μL of LUC1 substrate. Bioluminescence imaging of the tumor site was performed 10 minutes later. The location of bioluminescence indicated the location of the tumor cells. 2) Tumor resection was performed according to the location indicated by SC4-CyP3 fluorescence. 3) SC4-CyP3 fluorescence imaging and bioluminescence imaging were performed on the section surface after tumor resection to capture the remaining tumor tissue. The bioluminescence intensity was greatly reduced, and additional LUC1 substrate needed to be added to the section surface for imaging. 4) SC4-CyP3-mediated photodynamic therapy (808 nm, 1 W / cm 2 ,15min) and performed imaging again.

[0056] 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, guiding the resection of tumor; and it is not limited by the length of surgery during tumor resection; it also has high tumor selectivity and can detect smaller tumors (T2) in breast tissue. In contrast, bioluminescence has a short maintenance time and requires substrate supplementation during the resection 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 visualize 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, its distribution is highest in tumors and kidneys, indicating that it accumulates in large quantities in tumor tissues, and the residual probes in the body are still mainly metabolized by the kidneys.

[0057] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A renal-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 , where 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 method for preparing the renal-cleared long-circulating near-infrared fluorescent compound according to claim 1, wherein: The synthetic route is as follows: 。 5. Use of the renal-cleared, long-circulating near-infrared fluorescent compound or a pharmaceutically acceptable salt or tautomer thereof according to claim 1 in the preparation of a fluorescent probe.

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