NTR-responsive kidney clearing type near-infrared fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses an NTR-responsive kidney clearing type near-infrared fluorescent probe as well as a preparation method and application thereof. The probe has a structure as shown in a formula (I) and is synthesized from a compound 1, 2, 4 and mono-(6-amino-6-deoxy)-beta-cyclodextrin through a three-step reaction, the process is simplified, and the yield is increased. The probe is sensitive in response to low-concentration NTR, fluorescence is remarkably enhanced at 713 nm under excitation of 680 nm, and in combination with the penetrability of near-infrared light, in-situ imaging of the living kidney can be achieved. The probe is rapidly metabolized through the kidney, the clearance rate of the probe in 6 hours is 87.8%, the residue is low, and the biological safety is high. The kit is used for preparing an AKI diagnostic reagent, the kidney injury is detected 66h earlier than that of traditional serum creatinine through urine fluorescence analysis, and the sensitivity is high; the probe can also be used as a multi-mode imaging agent and a diagnosis and treatment integrated probe to monitor the drug renal toxicity in real time. The invention provides an efficient noninvasive scheme for early diagnosis, dynamic evaluation and precise treatment of acute kidney injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology. More specifically, the present invention relates to an NTR-responsive kidney-clearing near-infrared fluorescent probe and its preparation method and application. Background Art

[0002] Acute kidney injury (AKI), as a very common clinical syndrome, has shown an obvious upward trend in the incidence rate globally in recent years. The increasing number of AKI patients has brought a heavy burden to the medical system. AKI is mainly manifested by a sharp decline in renal function within a short period of time, and this process develops rapidly and is extremely harmful. The decline in renal function will lead to the inability of metabolic wastes in the body to be excreted normally, thus accumulating in the body and causing a series of adverse consequences. At the same time, the decline in renal function will also disrupt the normal water, electrolyte and acid-base balance of the human body, making the internal environment of the patient in a disordered state, seriously affecting physical health. When metabolic wastes accumulate too much in the body, it will have toxic effects on multiple important systems such as the cardiovascular system and the nervous system, causing complications such as arrhythmia and disturbance of consciousness.

[0003] The causes of AKI are extremely diverse, covering various factors such as prerenal, renal and postrenal factors. Prerenal factors include severe dehydration, massive hemorrhage, heart failure, etc., which will lead to insufficient renal blood perfusion and then cause renal injury; renal factors are commonly seen in acute tubular necrosis, glomerulonephritis, drug poisoning, etc., and these diseases directly damage the organizational structure and function of the kidney; postrenal factors are mainly caused by urinary tract obstruction, such as stones, tumor compression of the ureter, etc., which hinder the normal excretion of urine, resulting in increased pressure in the kidney and affecting renal function. In addition, the pathogenesis of AKI is also very complex, involving multiple biological processes such as inflammatory response, oxidative stress, apoptosis, etc., and these processes are intertwined with each other, further increasing the difficulty of its research and treatment. It is precisely due to the complexity of the causes and mechanisms of AKI that it has the characteristics of high incidence and high mortality. This not only poses a great threat to the life and health of patients, but also becomes a serious public health problem globally, causing a heavy burden on social economy, including a large consumption of medical resources, the economic pressure of patients and their families, etc.

[0004] At present, in clinical practice, the diagnosis of AKI mainly relies on two relatively easy-to-obtain kidney function indicators: serum creatinine and urine volume. However, these two indicators have obvious shortcomings. Serum creatinine, as a traditional indicator for evaluating kidney function, is affected by many factors. For example, changes in blood volume will affect the concentration of serum creatinine. When the patient is in a state of dehydration or edema, the serum creatinine value may be biased; age is also an important factor. The muscle content of the elderly is relatively low, and the blood creatinine level itself may be low, which will also affect the accurate judgment of kidney function; people with high muscle content, such as athletes, may have a higher basal value of serum creatinine, making it more difficult to judge kidney damage; in addition, some drugs can also interfere with the metabolism of serum creatinine, resulting in inaccurate test results. Moreover, there is a significant lag in the response of serum creatinine to kidney damage. Usually, serum creatinine will gradually rise 24 to 36 hours after kidney damage occurs, which makes it difficult for doctors to detect the disease in the early stage of kidney damage and delay the best treatment time. Urine volume, as another commonly used diagnostic indicator, also has a low diagnostic sensitivity for AKI. In actual clinical practice, there are some AKI patients whose urine volume does not decrease significantly, which is the so-called non-oliguric AKI. For these patients, it is easy to miss the diagnosis by relying solely on urine volume indicators, which leads to the condition being ignored and further deteriorating.

[0005] Although in recent years, with the continuous deepening of medical research, some more sensitive biomarkers have been discovered, such as kidney injury molecules, neutrophil gelatinase-associated lipocalin, etc. These biomarkers have shown certain potential in the early diagnosis of AKI, and can change in the early stage of kidney injury, reflecting abnormal renal function more promptly than blood creatinine and urine volume. However, due to the limitations of these biomarkers in detection methods, detection costs, clinical standardization, etc., they have not yet been widely used in large-scale clinical practice. In addition, some detection methods require special instruments and professional technicians to operate, and the detection cost is high, which limits its promotion in primary medical institutions; and there are differences in detection standards between different laboratories, resulting in poor comparability of test results and difficulty in forming a unified diagnostic standard.

[0006] Due to the lack of early diagnostic methods for AKI, many patients are already in the middle or late stages of the disease when they seek medical treatment, missing the best time for treatment, which is one of the main reasons for poor prognosis. Early diagnosis is crucial for the treatment and prognosis of AKI patients. If kidney damage can be detected in time and effective treatment measures can be taken in the early stages, the mortality rate of patients can be significantly reduced, the quality of life of patients can be improved, and the waste of medical resources can be reduced. Therefore, the development of a new method that can sensitively diagnose AKI has become an important issue that needs to be urgently addressed in the current field of nephrology.

[0007] As the end product of human metabolism, urine is not regulated by homeostatic mechanisms and is not stored in the body. It can reflect the body's metabolic status in real time, so urine markers have unique advantages in reflecting the true condition of the body at the moment, and have shown great potential in the field of early diagnosis of AKI. However, the types of urine biomarkers currently known are relatively limited. Some important substances closely related to kidney damage, such as reactive oxygen species and intracellular enzymes, cannot be evaluated by traditional urine analysis methods because they are not secreted into the urine. This limits the application of urine markers in the early diagnosis of AKI to a certain extent.

[0008] The emergence of kidney-cleared fluorescent probes provides a new idea for solving this problem. After entering the body, this type of fluorescent probe can preferentially enrich at the disease lesion site by virtue of its special structure and properties, and then react specifically with a specific target. During the reaction, the fluorescent probe undergoes structural changes and transforms into a fluorescent artificial urine biomarker. These artificial urine biomarkers are excreted from the body with urine, and the level of the disease target can be indirectly assessed by detecting the fluorescence intensity in urine. This urine fluorescence analysis strategy based on artificial urine biomarkers cleverly circumvents many challenges faced by in vivo fluorescence imaging, such as the attenuation of fluorescent signals in the body and poor tissue penetration. Moreover, urine samples are easy to collect, low-cost, and have the advantages of immediacy and non-invasiveness, and are highly accepted by patients. In this context, the relevant detection technology of nitroreductase (NTR) response has become a research hotspot. In the prior art, the nitroreductase-responsive kidney-cleared fluorescent probe disclosed in the invention with publication number CN118852493A has shown certain value in the diagnosis of AKI. The probe can show a specific fluorescence enhancement response to nitroreductase and can be used as an artificial urine biomarker for the diagnosis of early AKI, and to a certain extent, can detect AKI ahead of traditional clinical methods.

[0009] However, this technology still has some limitations. In terms of the sensitivity of nitroreductase (NTR) response, although it can detect the presence of nitroreductase and produce a fluorescent response, the change in its fluorescent signal is not significant when faced with low concentrations of nitroreductase, which makes it difficult to accurately capture the slight changes in nitroreductase levels in early renal damage, affecting the accuracy of early diagnosis. In terms of detection limit, the lower detection limit of this probe is relatively high, and it cannot effectively detect extremely small changes in nitroreductase in the body, which may miss some patients in the very early stages of the disease and delay diagnosis.

[0010] In addition, such probes in the prior art can generally only be used as fluorescence probes to detect the approximate level of nitroreductase and cannot achieve clear fluorescence imaging in vivo in situ. In a complex biological environment, simply detecting the presence or approximate content of nitroreductase makes it difficult to intuitively present its distribution in kidney tissues or cells, which is not conducive to doctors' comprehensive understanding of the occurrence and development process of diseases and limits the in-depth assessment of the condition of AKI.

[0011] Therefore, developing a technology that has more advantages in terms of the response sensitivity and detection limit of nitroreductase (NTR) and can achieve precise fluorescence imaging is of great significance for the early diagnosis of AKI. It is expected to break through the limitations of existing diagnostic technologies, provide more accurate and comprehensive diagnostic information for clinicians, and thus improve the prognosis of AKI patients. Summary of the Invention

[0012] One object of the present invention is to solve at least the above problems and provide at least the advantages described hereinafter.

[0013] Another object of the present invention is to provide a kidney-clearing near-infrared fluorescence probe responsive to NTR, which significantly improves the response sensitivity and detection limit for NTR, can accurately detect low-concentration NTR in early kidney injury, and achieve fluorescence imaging in vivo in situ to intuitively present the distribution of kidney lesions. Its preparation method is simple and low-cost, and it has important application value in the fields of early diagnosis of AKI, drug efficacy monitoring, and diagnosis and treatment integration, providing a highly efficient and non-invasive detection means for clinical practice.

[0014] To achieve these objects and other advantages according to the present invention, there is provided a kidney-clearing near-infrared fluorescence probe responsive to NTR, which has the structure of formula (I): (I); Wherein, n ranges from 1 to 10.

[0015] The present invention further claims the preparation method of the kidney-clearing near-infrared fluorescence probe responsive to NTR, including: Step 1: React compound 1 and compound 2 as raw materials in a first solvent for 0.5 to 10 h to obtain compound 3; Step 2: Heat and reflux the compound 3 obtained in step 1 and compound 4 in a second solvent for 2 to 24 h to obtain compound 5; Step 3: Dissolve the compound 5 obtained in step 2 in a third solvent, and react with mono-(6-amino-6-deoxy)-β-cyclodextrin at 10 to 50 °C for 2 to 36 h in the presence of 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate and a base, and separate and purify to obtain the compound of formula (I); Among them, the structures of Compound 1, Compound 2, Compound 3, Compound 4 and Compound 5 are shown in Formula (Ⅱ), Formula (Ⅲ), Formula (Ⅳ), Formula (Ⅴ) and Formula (Ⅵ) respectively: (Ⅱ); (Ⅲ); (Ⅳ); (Ⅴ); (Ⅵ).

[0016] Preferably, the first solvent in Step 1 is N,N-dimethylformamide; the second solvent in Step 2 is selected from one of N,N-dimethylformamide, ethanol, methanol, tetrahydrofuran, and dichloromethane; the third solvent in Step 3 is selected from one of dimethyl sulfoxide, N,N-dimethylformamide, ethanol, methanol, and acetonitrile.

[0017] Preferably, the base in Step 3 is selected from one of potassium carbonate, cesium carbonate, pyridine, piperidine, dimethylamine, triethylamine, and N,N-diisopropylethylamine.

[0018] Preferably, the molar ratio of Compound 5, mono-(6-amino-6-deoxy)-β-cyclodextrin, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate and the base in Step 3 is 1:0.5~3:1~6:2~8.

[0019] The present invention further claims the application of the NTR-responsive renal clearance near-infrared fluorescence probe in the preparation of in vivo in-situ fluorescence imaging agents, and the in vivo in-situ fluorescence imaging agents can be used to prepare AKI diagnostic products and multimodal molecular imaging products.

[0020] According to the above application, the multimodal molecular imaging product includes a near-infrared fluorescence module and other imaging modules. Among them, the probe main structure of the near-infrared fluorescence module shows the NTR-responsive near-infrared fluorescence signal as shown in Formula (Ⅰ) for dynamic functional imaging; the other imaging modules can be magnetic nanoparticles, such as magnetite, which are integrated onto the surface of the probe main body through covalent coupling or physical adsorption to endow magnetic resonance imaging ability for high-resolution localization of anatomical structures; or radioactive isotopes, which label the probe main body through chelating agents to achieve single-photon emission computed tomography or positron emission tomography; or gold nanoparticles, which are used as photoacoustic imaging enhancers to generate acoustic signals through near-infrared light excitation to provide high-contrast imaging of deep tissues.

[0021] Preferably, the AKI diagnostic product comprises a reagent stock solution A and a phosphate buffer solution. The reagent stock solution A comprises a solution of a kidney-clearing near-infrared fluorescent probe responsive to NTR shown in formula (I) with a concentration of 1-10 mM. The phosphate buffer solution has a concentration of 10-100 mM, a pH of 5-9, and is selected from at least one of Na2HPO4, KH2PO4, and NaH2PO4.

[0022] The present invention further claims the use of the kidney-clearing near-infrared fluorescent probe responsive to NTR in the preparation of an optical molecular probe.

[0023] The present invention further claims the use of the kidney-clearing near-infrared fluorescent probe responsive to NTR in the preparation of a theranostic probe.

[0024] According to the above application, one implementation is as follows: Integrate a treatment module, such as a drug molecule, into the probe molecular structure. When the probe locates the kidney lesion area (high NTR expression site) through near-infrared fluorescence imaging, the nitro group in its structure is reduced under the catalysis of NTR, triggering the release or activation of the treatment module. For example, the release of a targeted drug: Couple a kidney injury repair drug, such as an antioxidant or an anti-inflammatory molecule, to the probe structure, and release the drug to the lesion site under NTR response to directly and precisely repair the damaged tissue.

[0025] The present invention further claims the use of the kidney-clearing near-infrared fluorescent probe responsive to NTR in real-time monitoring of the degree of kidney injury caused by drugs.

[0026] According to the above application, one implementation is as follows: The probe is introduced into the body through intravenous injection. The hydrophilic groups in its structure endow it with kidney targeting and are limitedly enriched in kidney tissues. When drugs (such as cisplatin, aminoglycoside antibiotics, etc.) cause kidney injury, the expression of NTR in the damaged area is significantly up-regulated, and the nitro group of the probe is specifically reduced by NTR, triggering an enhancement of the near-infrared fluorescence signal (excitation 680 nm, emission 713 nm). The experimental animals are imaged in real time at different time points (such as 0, 6, 12, 24 h) after drug administration. The near-infrared light has strong penetrability, and the change in fluorescence intensity in the kidney area can be clearly shown. By quantitatively analyzing the fluorescence signal intensity, the NTR activity level can be directly reflected, and the degree of kidney injury can be indirectly characterized. Continuous imaging shows that the fluorescence signal increases with the prolongation of drug action time, revealing the progression rate of kidney injury. For example, the fluorescence intensity after 24 h of cisplatin induction is higher than that after 6 h, indicating that the injury continues to deteriorate. Traditional kidney injury markers such as serum creatinine (CRE), blood urea nitrogen (BUN), and urinary NAG, NGAL, etc. are synchronously detected to verify the consistency and higher sensitivity between the fluorescence signal and clinical indicators. Further, the present invention also verifies the alleviating effect of the drug combined with a protective agent. When the drug is combined with a protective agent, such as N-acetylcysteine (NAC), the fluorescence signal intensity of the probe is significantly reduced, reflecting the alleviating effect of the protective agent on kidney injury. For example, the fluorescence intensity of the cisplatin + NAC group shows an obvious decrease compared with the pure cisplatin group, and the kidney protection effect of NAC is verified in real time.

[0027] The present invention has at least the following beneficial effects: First, the NTR-responsive kidney-clearing near-infrared fluorescence probe structure provided by the present invention can still produce a significant fluorescence response under a low concentration of nitroreductase, accurately capturing the weak changes in NTR levels in early kidney injury; Second, the present invention simplifies the synthesis process of the NTR-responsive kidney-clearing near-infrared fluorescence probe through multi-step reactions and specific solvent selection, which is suitable for large-scale preparation; Third, the NTR-responsive kidney-clearing near-infrared fluorescence probe provided by the present invention can be applied to the preparation of AKI diagnostic products, multimodal molecular imaging products, and theranostic probes, providing a complete solution from detection to treatment; Fourth, the NTR-responsive kidney-clearing near-infrared fluorescence probe provided by the present invention can also be used to realize the real-time monitoring of the degree of kidney injury caused by drugs, providing a dynamic evaluation tool for drug research and development and personalized treatment; Fifth, the AKI diagnostic product provided by the present invention can be used for the naked-eye visualization judgment of AKI based on the urine color, providing available means for the bedside detection, rapid self-examination, large-scale screening, etc. of AKI; Sixthly, the AKI diagnostic product provided by the present invention ensures the stability of the probe in a complex biological environment through the design of a specific stock solution and buffer system, enhancing the repeatability and clinical practicability of the detection results.

[0028] Other advantages, objectives, and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. Description of the Drawings

[0029] Figure 1 1H NMR comparison charts of the NTR-responsive kidney-clearing near-infrared fluorescent probe, compound 3, and mono-(6-amino-6-deoxy)-β-cyclodextrin synthesized in Example 1 of the present invention; 1 1H NMR comparison charts; Figure 2 Absorption spectra of the NTR-responsive kidney-clearing near-infrared fluorescent probe before and after reacting with nitroreductase in another embodiment of the present invention. Among them, curve a is the absorption spectrum of the 10 μM NTR-responsive kidney-clearing near-infrared fluorescent probe itself, and curve b is the absorption spectrum of the 10 μM NTR-responsive kidney-clearing near-infrared fluorescent probe after reacting with 2.0 μg / mL NTR; Figure 3 Fluorescence response charts of the NTR-responsive kidney-clearing near-infrared fluorescent probe to different concentrations of nitroreductase in another embodiment of the present invention; Figure 4 Survival rates of HK-2 cells after co-incubation with different concentrations of the AKI diagnostic product for 24 h in another embodiment of the present invention; Figure 5 Confocal imaging charts of HK-2 cells treated with cisplatin for different times after incubation with the AKI diagnostic product in another embodiment of the present invention; Figure 6 Confocal imaging charts of HK-2 cells pretreated with coumarin and N-acetylcysteine and then co-cultured with the AKI diagnostic product after cisplatin induction in another embodiment of the present invention; Figure 7 Kidney clearance rate curves of the AKI diagnostic product in mice in another embodiment of the present invention; Figure 8 In vivo fluorescence imaging charts of AKI mice induced by cisplatin for different times in another embodiment of the present invention; Figure 9 Changes in urine color and fluorescence of AKI mice induced by cisplatin for different times in another embodiment of the present invention; Figure 10 Changes in serum CRE, BUN, and urine NAG and NGAL contents of AKI mice induced by cisplatin for different times in another embodiment of the present invention. Detailed implementation manners

[0030] The present invention will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement it according to the description in the specification.

[0031] It should be understood that terms such as "having", "comprising", and "including" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0032] Example 1 (taking n = 2 as an example) An NTR-responsive renal clearance near-infrared fluorescent probe has the following structure: ; Its specific synthesis route is as follows: Its specific synthesis steps are as follows: 1. Dissolve compound 1 (0.23 g, 1 mmol) and K2CO3 (0.41 g, 3 mmol) in DMF. After stirring for 30 min, add 4-nitrobenzyl bromide (0.54 g, 2.5 mmol). React at room temperature for 3 h, then extract with ethyl acetate and saturated brine. The organic phase is dried with anhydrous Na2SO4. After removing the solvent under reduced pressure, the residue obtained is purified by silica gel column chromatography to obtain intermediate 3, which is a pale yellow solid.

[0033] As Figure 1 , the NMR and high-resolution mass spectrometry results of intermediate 3 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ10.20 (s, 1H), 8.20 (dd, 2H, J = 2.8, 6.0 Hz), 8.01 (d, 1H, J = 2.8 Hz), 7.65(d, 2H, J = 8.8 Hz),7.19-7.16 (m, 1H), 6.83-6.76 (m, 2H), 5.25 (s, 2H), 2.54-2.51 (m, 2H), 2.29 (t, 2H, J = 5.6 Hz), 1.34-1.62 (m, 2H). 1313C NMR (400 MHz, DMSO-d6) δ 186.3, 162.3, 161.0, 160.2, 152.7, 150.8, 146.3, 128.0, 127.0, 125.8, 123.3, 114.2, 111.7, 111.0, 100.5, 62.0, 30.8, 29.0, 20.0. High-resolution mass spectrometry: C 21 H 18 NO 5+ , [M + H + ; Calculated value: 364.1179; Measured value: 364.1184.

[0034] 2. Dissolve intermediate 3 (0.36 g, 1 mmol) and compound 4 (0.27 g, 1 mmol) in ethanol and reflux for 7 h. After cooling, remove the solvent under reduced pressure. The residue obtained is purified by silica gel column chromatography to obtain the small molecule compound HN. Compound HN is a blue solid.

[0035] The NMR and high-resolution mass spectrometry results of compound HN are as follows: 11H NMR (400 MHz, DMSO-d6) δ 8.57 (d, 1H, J = 14.8 Hz), 8.31 (d, 1H, J = 8.8 Hz), 7.81 - 7.76 (m, 3H), 7.70 (d, 1H, J = 8.0 Hz), 7.58 - 7.47 (m, 4H), 7.20 (d, 1H, J = 2.4 Hz), 7.11 (d, 2H, J = 8.8 Hz), 6.57 (d, 1H, J = 14.8 Hz), 5.47 (s, 2H), 5.42 (t, 2H, J = 7.2 Hz), 2.72 (t, 2H, J = 6.0 Hz), 2.67 (t, 2H, J = 6.0 Hz), 2.19 (t, 2H, J = 7.2 Hz), 1.83 - 1.79 (m, 4H), 1.76 (s, 6H), 1.60 - 1.52 (m, 2H), 1.45 - 1.39 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 177.6, 174.3, 160.9, 160.3, 153.7, 147.2, 145.0, 144.2, 142.2, 141.4, 132.8, 129.0, 128.9, 128.6, 127.3, 127.2, 123.8, 122.8, 115.9, 114.1, 113.4, 104.7, 102.2, 68.9, 54.9, 50.5, 48.6, 44.7, 33.4, 28.4, 27.5, 27.2, 25.6, 24.2, 23.6. High resolution mass spectrometry: C 38 H 39 N2O6 + , [M + ; Calculated value: 619.2803; Measured value: 619.2811.

[0036] 3. Dissolve compound HN (0.62 g, 1 mmol), N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 0.27 g, 1.5 mmol), and N,N-diisopropylethylamine (0.09 g, 1.5 mmol) in DMF and stir at room temperature for half an hour. Then add mono-(6-amino-6-deoxy)-β-cyclodextrin (β-CD, 1.13 g, 1 mmol) and continue the reaction for 24 h. Subsequently, extract with ethyl acetate and saturated brine three times, centrifuge to collect the blue flocculent precipitate in the aqueous phase, wash three times with deionized water, and obtain the NTR-responsive kidney-clearing near-infrared fluorescent probe (HN-CD) as a blue powder after freeze-drying. High-resolution mass spectrometry: C 80 H 108 N3O 39 + , [M + ; Calculated value: 1734.6554; Measured value: 1734.6554.

[0037] Figure 1 For the obtained NTR-responsive kidney-clearing near-infrared fluorescent probe, intermediate 3, and β-CD 1 1H NMR comparison chart. It can be seen that the characteristic peaks of small molecule compound 5 and mono-(6-amino-6-deoxy)-β-cyclodextrin appear in the 1H NMR spectrum of the NTR-responsive kidney-clearing near-infrared fluorescent probe, proving the successful synthesis of the NTR-responsive kidney-clearing near-infrared fluorescent probe.

[0038] Example 2 Optical response of the NTR-responsive kidney-clearing near-infrared fluorescent probe (HN-CD) to nitroreductase (NTR) Weigh 17.3 mg of the NTR-responsive kidney-clearing near-infrared fluorescent probe obtained in Example 1 and dissolve it in 10 mL of dimethyl sulfoxide to prepare a probe mother liquor with a concentration of 1 mM. Add 50 µL of the probe mother liquor to a certain amount of phosphate buffer solution (0.01 M), then add nitroreductase solutions with different concentrations and make up to 5 mL. After reacting at 37°C for 60 min, measure the absorption spectrum and fluorescence emission spectrum. When measuring the fluorescence emission spectrum, excite at 680 nm.

[0039] The measurement results are as Figure 2 and Figure 3 shown. It can be seen from Figure 2 that after the compound HN-CD reacts with nitroreductase, its maximum absorption wavelength redshifts from 600 nm to 660 nm. Figure 3Fluorescence response diagram of a kidney-clearing near-infrared fluorescent probe responsive to NTR for nitroreductase, where the concentration range of nitroreductase is 0 - 2.0 μg / mL, and the concentrations of nitroreductase from bottom to top are 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 μg / mL. It can be seen from the above experimental results that the kidney-clearing near-infrared fluorescent probe responsive to NTR has almost no fluorescence itself. After adding nitroreductase, the fluorescence at 713 nm is significantly enhanced and shows concentration dependence, indicating that the kidney-clearing near-infrared fluorescent probe responsive to NTR has a sensitive fluorescence enhancement response to nitroreductase, and the detection limit is as low as 5.0 ng / mL. This means that the probe provided by the present invention can detect extremely low concentrations of nitroreductase, and it can detect changes in nitroreductase in the early stage of the disease or in trace pathological tissues, which is helpful for the early detection and diagnosis of diseases.

[0040] When measuring the fluorescence emission spectrum of the kidney-clearing fluorescent probe responsive to nitroreductase disclosed in the publication number CN118852493A, it is excited at 425 nm. After adding nitroreductase, the fluorescence of the kidney-clearing fluorescent probe responsive to nitroreductase is significantly enhanced at 500 - 550 nm (visible light region). The kidney-clearing near-infrared fluorescent probe responsive to NTR obtained in the present invention uses an excitation wavelength of 680 nm and an emission wavelength of 713 nm (near-infrared region), which significantly improves the tissue penetration depth of the fluorescent probe and is suitable for in vivo in situ imaging of deep organs such as the kidney, broadening the application range of the kidney-clearing near-infrared fluorescent probe responsive to NTR and having the function of preparing AKI diagnostic products such as in vivo in situ imaging reagents. At the same time, near-infrared imaging technology has strong compatibility with existing medical equipment, facilitating its promotion to clinical multimodal molecular diagnosis, enabling the kidney-clearing near-infrared fluorescent probe responsive to NTR to have the functions of preparing multimodal molecular imaging products and preparing integrated diagnosis and treatment probes. Near-infrared light can greatly reduce the interference of autofluorescence of biological tissues, reduce biological background interference, and improve the signal-to-noise ratio and sensitivity of detection. The specific fluorescence enhancement response at 713 nm in this application also avoids cross-interference with biological background fluorescence (such as 400 - 650 nm), improving the specificity of detection. Near-infrared light causes little damage to tissues and supports long-term, non-invasive dynamic evaluation of drug nephrotoxicity or the progression of AKI, which also enables the kidney-clearing near-infrared fluorescent probe responsive to NTR to have the function of real-time monitoring of the degree of kidney damage caused by drugs.

[0041] Example 3 Preparation and cytotoxicity of AKI diagnostic products based on HN-CD Preparation of AKI diagnostic product: The AKI diagnostic product includes reagent stock solution A and phosphate buffer B. Reagent stock solution A is prepared by dissolving HN-CD in a certain volume of dimethyl sulfoxide, and phosphate buffer B is a NaH2PO4-K2HPO4 buffer solution with a concentration of 10 mM and a pH of 7.4.

[0042] Cytotoxicity analysis: The cytotoxicity of the diagnostic product was evaluated by the classical MTT method. Specifically, human renal cortical proximal tubular epithelial (HK-2) cells in the logarithmic growth phase were seeded in 96-well plates. After adherent culture for 24 h, DMEM containing different concentrations of reagent stock solution A was added and incubated for 24 h. The culture medium was discarded, and after washing 3 times with phosphate buffer B, 100 μL of MTT solution (0.5 mg / mL) was added to each well. After continued incubation for 4 h, the MTT was discarded, and after washing with phosphate buffer B, 150 μL of DMSO was added. Finally, the absorbance at 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated.

[0043] The measurement results are as Figure 4 shown. It can be seen from the figure that even when the concentration of HN-CD is as high as 20 μM, the cell survival rate can still remain above 80%, indicating that the AKI diagnostic product containing HN-CD has low cytotoxicity and high biocompatibility.

[0044] Example 4 The AKI diagnostic product was used to detect cisplatin-induced HK-2 cell damage: (1) HK-2 cells were cultured in DMEM medium containing 10% (v / v) fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin under the conditions of 37°C and 5% CO2.

[0045] (2) HK-2 cells in the logarithmic growth phase were seeded in a confocal dish. After culturing for 24 h, the culture medium was discarded and washed three times with phosphate buffer B. Then, the cells were co-incubated with a medium containing 10 μM cisplatin. After 12, 24, 36, and 48 h, the medium was removed, and the cells were washed three times with phosphate buffer B and then co-incubated with DMEM containing 10 μM reagent stock solution A for 2 h and then imaged under a confocal microscope. The imaging excitation wavelength was 635 nm, and the emission wavelength collection range was 650 - 750 nm.

[0046] The experimental results are as Figure 5 and 6 shown. From Figure 5It can be seen that after co-incubation with reagent stock solution A, normal cultured HK-2 cells showed almost no fluorescence. However, with the prolongation of cisplatin treatment time, the cell fluorescence gradually increased, suggesting that cisplatin can cause hypoxia in HK-2 cells and may up-regulate the expression of nitroreductase. From Figure 6 It can be seen that the fluorescence of cells treated with the nitroreductase inhibitor dicoumarin was significantly inhibited, indicating that the fluorescence in cells was indeed due to the up-regulation of nitroreductase by cisplatin, which in turn activated the fluorescence of the probe HN-CD. The intracellular fluorescence of cells pretreated with N-acetylcysteine was significantly reduced, indicating that N-acetylcysteine can alleviate cisplatin-induced cell damage and down-regulate the expression of nitroreductase. The above results show that the AKI diagnostic product prepared with the fluorescence probe HN-CD of the present invention can evaluate cisplatin-induced cell damage and intervention by fluorescence imaging.

[0047] Example 5 In vivo metabolic behavior of the AKI diagnostic product: This experiment was approved by the Animal Ethics and Use Committee of Shanxi Medical University. The experimental animals were 5-6-week-old male ICR mice, weighing 25 g ± 1 g per mouse, and all were raised in a sterile environment. Reagent stock solution A was diluted in phosphate buffer B at a dose of 5 μmol / kg and injected into the mice via the tail vein. The mice were placed in a metabolic cage, and urine was collected at different times (1, 3, 6, 9, 12, 24 h). After appropriate treatment, the content of HN-CD in the urine samples was analyzed using an enzyme-linked immunosorbent assay (ELISA) reader.

[0048] The measurement results are as Figure 7 shown. It can be seen that 6 h after injection, the renal clearance rate of HN-CD can reach 87.8%, and the clearance rate at 24 h can reach 92.1%. This indicates that the AKI diagnostic product based on HN-CD is mainly metabolized by the kidneys in vivo, rapidly excreted with urine, and has very little residue in the body. Therefore, this product has potential application prospects in the diagnosis of AKI based on urine fluorescence analysis.

[0049] After 9 hours of injection of the nitroreductase-responsive renal clearance fluorescent probe disclosed in Publication No. CN118852493A, the renal clearance rate of the fluorescent probe can reach 88.1%, and the clearance rate at 24 hours can reach 90.5%. The NTR-responsive renal clearance near-infrared fluorescent probe provided in this application achieved a clearance rate of 87.8% after 6 hours of injection. Compared with the 88.1% achieved in 9 hours in the above-mentioned published document, the detection window period was significantly shortened, supporting the rapid diagnosis of early AKI, and further verifying that the NTR-responsive renal clearance near-infrared fluorescent probe provided by the present invention has the function of real-time monitoring of the degree of kidney damage caused by drugs. The clearance rate of the NTR-responsive renal clearance near-infrared fluorescent probe provided in the application can reach 92.1% after 24 hours of injection, significantly higher than 90.5% in the above-mentioned published document, reducing the risk of probe residue in the body, improving biological safety, and further verifying that the NTR-responsive renal clearance near-infrared fluorescent probe provided by the present invention is suitable for in vivo in-situ imaging of deep organs such as the kidney. The faster clearance speed can shorten the waiting time of patients, support multiple detections and dynamic monitoring requirements, and further verify that the NTR-responsive renal clearance near-infrared fluorescent probe provided by the present invention has the function of real-time monitoring of the degree of kidney damage caused by drugs. While maintaining a high clearance rate and combining near-infrared fluorescence characteristics (excitation at 680 nm and emission at 713 nm), the NTR-responsive renal clearance near-infrared fluorescent probe provided by the present invention has both deep tissue imaging ability and rapid metabolism advantages, further verifying that the NTR-responsive renal clearance near-infrared fluorescent probe provided by the present invention has the function of preparing a diagnosis and treatment integrated probe.

[0050] Example 6 Diagnostic product for diagnosing cisplatin-induced AKI in mice based on in vivo fluorescence imaging (1) Establish a cisplatin-induced AKI model: The experimental animals were 5-6-week-old male ICR mice, weighing 25 g ± 1 g per mouse, and all were raised in a sterile environment. The mice were intraperitoneally injected with cisplatin (20 μmol / kg) for different times (6, 12, 24, 48, 72 h) to establish AKI model mice with different degrees. The N-acetylcysteine (NAC) intervention group was pre-injected with NAC into the mice before cisplatin injection, and then cisplatin was injected and induced for 48 h.

[0051] (2) Mouse fluorescence imaging: The reagent stock solution A was diluted in phosphate slow-release solution B at a dose of 5 μmol / kg and injected into the mice through the tail vein. Subsequently, fluorescence imaging was performed using a small animal in vivo imager at different time points.

[0052] The experimental results are as Figure 8As shown, 15 minutes after the AKI mice were injected with the diagnostic product, fluorescent signals appeared in their kidney regions. Moreover, the longer the cisplatin induction time, the stronger the fluorescent signals in the kidney regions of the mice. In contrast, almost no fluorescence was observed in the kidney regions of normal mice. That is, the fluorescence intensity in the kidneys was positively correlated with the severity of AKI in the mice, indicating that the diagnostic product could perform in-situ fluorescence imaging of AKI in mice and achieve rapid imaging diagnosis.

[0053] In CN118852493A, abnormal elevation of urine fluorescence in mice injected with a nitroreductase-responsive renal clearance fluorescent probe occurred 6 hours after cisplatin induction. In contrast, in this application, fluorescent signals could be detected in the kidney regions of AKI mice 15 minutes after injection of the diagnostic product (the above-mentioned published document required collecting urine for 3 hours for fluorescence analysis), achieving extremely early diagnosis of AKI, significantly shortening the clinical decision-making time. Moreover, in this application, the fluorescence intensity was positively correlated with the course of AKI (the longer the cisplatin induction time, the stronger the signal), providing a quantitative diagnosis basis, which was superior to the qualitative detection in the above-mentioned published document. The 15-minute rapid response combined with the quantitative detection ability could be integrated into emergency or point-of-care testing scenarios to improve the diagnostic efficiency of AKI. At the same time, the fluorescent signals continued to increase with the prolongation of the disease course, supporting continuous tracking of the development stage of AKI, realizing the function of preparing a diagnostic and therapeutic integrated probe, and also providing real-time basis for personalized treatment. In this application, almost no fluorescent signals were observed in the kidneys of normal mice, indicating that the NTR-responsive renal clearance near-infrared fluorescent probe provided by the present invention had high targeting to pathological regions, effectively avoiding false-positive interference.

[0054] Example 7 The diagnostic product detects cisplatin-induced AKI in mice based on urine fluorescence analysis (1) The diagnostic product was used for urine fluorescence analysis of AKI model mice: The reagent stock solution A was injected into AKI mice via the tail vein at a dose of 5 μmol / kg. The mice were placed in a metabolic cage, and urine was collected for 3 hours. The urine was centrifuged at 4500 rpm for 10 minutes and then subjected to fluorescence testing. The fluorescence excitation and emission wavelengths were 680 nm and 713 nm, respectively.

[0055] (2) Determination of serum creatinine (CRE) and blood urea nitrogen (BUN) levels in AKI model mice: Venous blood was taken from AKI model mice, centrifuged at 4500 rpm for 10 minutes, and the upper-layer serum was taken for clinical routine methods testing. CRE was measured by the sarcosine oxidase method, and BUN was measured by the urease method.

[0056] (3)Determination of the contents of N-acetyl-beta-D-glucosaminidase (NAG) and neutrophil gelatinase-associated lipocalin (NGAL) in the urine of AKI model mice: The AKI mice were placed in a metabolic cage to collect urine. After centrifuging the urine samples (4500 rpm, 10 min), the supernatant was taken to detect the contents of NAG and NGAL in the samples using the corresponding ELISA kits.

[0057] The measurement results are as Figure 9 and Figure 10 shown. It can be seen from Figure 9 that after injecting the AKI diagnostic product, there was almost no fluorescence in the urine of normal mice, while the urine of mice treated with cisplatin for 6 h showed abnormalities, and the urine color changed visibly to the naked eye (gradually changed from yellow to blue-green), and the fluorescence intensity increased significantly. The longer the cisplatin treatment time, the stronger the urine fluorescence; while the fluorescence in the NAC intervention group was significantly inhibited, indicating that the diagnostic product can not only sensitively detect AKI based on urine fluorescence analysis, but also evaluate the degree of AKI in mice. It can be seen from Figure 10 that the NAG and NGAL in the urine of mice increased abnormally 48 h after cisplatin induction; while the serum CRE (clinical index) and BUN showed abnormalities only after 72 h, and there were no obvious changes in the BUN and CRE levels in the NAC intervention group. The above results show that the AKI diagnostic product of the present invention can not only diagnose AKI 66 h earlier than the current clinical method (CRE), but also has a higher diagnostic sensitivity than other reported diagnostic methods. At the same time, it can be judged by observing the color of the urine with the naked eye, and can quickly evaluate the progress of AKI with the help of a colorimetric test strip, and is expected to be used for bedside rapid detection, home self-testing, large-scale screening, etc.

[0058] In the published document CN118852493A, the urine of mice injected with a kidney-clearing fluorescent probe responsive to nitroreductase only showed an abnormal increase in fluorescence 6 h after cisplatin induction. This abnormality needed to be detected by excitation with a 365 nm fluorescent lamp or a fluorescence spectrophotometer, and there was no significant change in the urine color during this process, and it could not be judged with the naked eye.

[0059] The equipment quantity and processing scale described here are used to simplify the description of the present invention. The application, modification and variation of the NTR-responsive kidney-clearing near-infrared fluorescent probe agent of the present invention and its preparation method are obvious to those skilled in the art.

[0060] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those skilled in the art, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and the illustrated examples described herein.

Claims

1. A kidney-clearing near-infrared fluorescence probe for NTR response, characterized in that, It has the structure of formula (Ⅰ): （Ⅰ）； Among them, the value range of n is 1 to 10.

2. The preparation method of the NTR-responsive kidney-clearing near-infrared fluorescence probe according to claim 1, wherein, Including: Step 1: React compound 1 and compound 2 as raw materials in the first solvent for 0.5 to 10 h to obtain compound 3; Step 2: Heat and reflux the compound 3 obtained in Step 1 and compound 4 in the second solvent for 2 to 24 h to obtain compound 5; Step 3: Dissolve the compound 5 obtained in Step 2 in the third solvent, and in the presence of 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate and a base, react with mono-(6-amino-6-deoxy)-β-cyclodextrin at 10 to 50 °C for 2 to 36 h, and separate and purify to obtain the compound of formula (Ⅰ); Among them, the structures of compound 1, compound 2, compound 3, compound 4 and compound 5 are shown in formula (Ⅱ), formula (Ⅲ), formula (Ⅳ), formula (Ⅴ) and formula (Ⅵ) respectively: （Ⅱ）； （Ⅲ）； （Ⅳ）； （Ⅴ）； （Ⅵ）。 3. The preparation method according to claim 2, characterized in that, The first solvent described in Step 1 is N,N-dimethylformamide; the second solvent described in Step 2 is selected from one of N,N-dimethylformamide, ethanol, methanol, tetrahydrofuran, and dichloromethane; the third solvent described in Step 3 is selected from one of dimethyl sulfoxide, N,N-dimethylformamide, ethanol, methanol, and acetonitrile.

4. The preparation method according to claim 2, characterized in that, The base described in Step 3 is selected from one of potassium carbonate, cesium carbonate, pyridine, piperidine, dimethylamine, triethylamine, and N,N-diisopropylethylamine.

5. The preparation method of the NTR response according to claim 2, wherein, In Step 3, the molar ratio of compound 5, mono-(6-amino-6-deoxy)-β-cyclodextrin, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate and the base is 1:0.5 to 3:1 to 6:2 to 8.

6. Use of the NTR-responsive near-infrared fluorescence probe for renal clearance as described in claim 1 in the preparation of an in vivo in-situ fluorescence imaging agent, characterized in that, The in vivo in-situ fluorescence imaging agent can be used to prepare AKI diagnostic products and multimodal molecular imaging products.

7. The application according to claim 6, characterized in that, The AKI diagnostic product includes reagent stock solution A and phosphate buffer solution. The reagent stock solution A includes a solution of a kidney-clearing near-infrared fluorescence probe responsive to NTR shown in formula (Ⅰ) with a concentration of 1 to 10 mM. The concentration of the phosphate buffer solution is 10 to 100 mM, the pH is 5 to 9, and it is selected from at least one of Na2HPO4, KH2PO4, and NaH2PO4.

8. Use of the NTR-responsive kidney-clearing near-infrared fluorescence probe according to claim 1 in the preparation of an optical molecular probe.

9. Use of the NTR-responsive kidney-clearing near-infrared fluorescence probe according to claim 1 in the preparation of a diagnosis and treatment integrated probe.

10. Use of the NTR-responsive kidney-clearing near-infrared fluorescence probe according to claim 1 for real-time monitoring of the degree of kidney damage caused by drugs.

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