Responsive near-infrared first-zone fluorescent compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a responsive near-infrared first-zone fluorescent compound as well as a preparation method and application thereof. The compound mainly comprises a fluorescent probe of three functional modules including a fluorescent chromophore, a fibrosis specific enzyme response group and a quenching group, and on the basis, an active / passive targeting group of a targeted disease organ is introduced to form a more excellent fluorescent probe of four functional modules. The effect of the probe is verified in a time-varying progress experiment of a fibrosis disease model, and the probe not only has high specificity and sensitivity, but also has relatively good safety and biocompatibility; the near-infrared first-area fluorescent compound and pharmaceutically acceptable salts or solvates, enantiomers, diastereoisomers and tautomers thereof can be used for preparing fluorescent molecular probes, and diagnosis, intraoperative navigation treatment, tissue and organ function evaluation and the like can be more accurately carried out on diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a class of responsive near-infrared I fluorescent compounds and their preparation methods and applications. Background Technology

[0002] Fibrosis is a significant cause of morbidity and mortality worldwide. Fibrosis refers to the dysregulation of tissue repair responses following tissue damage, particularly during chronic inflammatory diseases. Common diseases associated with fibrosis include viral hepatitis, non-alcoholic fatty liver disease (NAFLD), chronic kidney disease, idiopathic pulmonary fibrosis (IPF), tuberculosis, myocardial fibrosis, and cystic fibrosis. The increasing prevalence of fibrosis poses a major health challenge, and untreated organ fibrosis can lead to organ failure.

[0003] Currently, commonly used clinical diagnostic methods include complete blood count, urinalysis, pathological tissue biopsy, and various imaging techniques applicable to the detection and monitoring of fibrosis, such as ultrasound (US), computed tomography (CT), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), positron emission tomography (PET), and optical imaging (OI). Tissue biopsy is the "gold standard" for diagnosing many diseases, but its invasiveness leads to poor patient compliance. Therefore, non-invasive diagnostic methods are particularly important for the clinical diagnosis of fibrotic diseases. Traditional imaging methods are also problematic due to their high cost, complex imaging processes, radiation exposure, low clarity and resolution, and susceptibility to interference from external factors such as infection and tissue degeneration, which can affect their accuracy.

[0004] In comparison, molecular optical imaging technology has advantages such as non-invasive imaging, superior spatial and temporal resolution, rapid diagnosis, simple operation, and high sensitivity and accuracy through the use of targets and biomarkers. However, the current problems and challenges of fluorescent molecular probes and fluorescence imaging technology in fibrosis detection are mainly: (1) lack of early diagnosis: due to the high incidence and long latency of fibrosis, it is still challenging to develop a molecular contrast agent that can monitor fibrosis in its early stages. (2) low signal-to-noise ratio: due to the low signal-to-noise ratio and high background signal of constant-brightness fluorescent probes, the sensitivity of probe detection is reduced. (3) poor specificity: currently, there are few bindings of fluorescent probes to key therapeutic targets that are highly related to fibrosis, making it difficult to develop effective anti-fibrotic therapies targeting current targets. Lack of in vitro detection: in vivo detection is expensive and time-consuming, while in vitro point-of-care diagnosis can greatly improve detection efficiency and save instrument and labor costs while ensuring accuracy. However, how to improve the specificity and sensitivity of fluorescent molecular probes and fluorescence imaging technology in fibrosis detection still needs further investigation. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a class of responsive near-infrared fluorescent compounds, their preparation methods, and applications. Targeting specific protein markers highly expressed during the development of fibrosis and precisely targeting fibrosis sites, an "activation" strategy is employed, using small-molecule fluorescent dyes with excellent near-infrared fluorescence (650-900 nm) characteristics as signal transducers to construct activation-type near-infrared probes for the diagnosis of fibrosis.

[0006] Construction of a novel activated near-infrared (NIIR) fluorescent probe system: Studies have shown that prolyl hydroxylase (PHD) and matrix metalloproteinases 2 / 9 (MMPs) are closely related to the development of liver and lung fibrosis. In addition to MMPs, transglutaminase 2 (TG2) also plays an important role in renal fibrosis. Currently, molecular probes, organically combined with multiple imaging modalities such as CT, MRI, and PET, are widely recognized as a core technology for multimodal imaging of fibrosis. Several research groups both domestically and internationally have achieved excellent results targeting fibrosis, with progress made in targeting lysine (Lys-Ald), elastin, and collagen. In the visible light region, certain components of biological tissues spontaneously fluoresce, but the intensity of scattered light from the sample is high, severely interfering with fluorescence detection and imaging tracer. Therefore, near-infrared (NIIR) fluorescent probes with low background interference and strong tissue penetration are gradually becoming a research hotspot. Of the two existing types of fluorescent probes—"always-on" and "activatable / responsive" probes—responsive probes have the advantage of not generating fluorescence signals until they are activated by a specific biological target, thus producing high-contrast fluorescence imaging. Activatable / responsive probes can be more targeted, modifying molecular structures and using disease biomarkers to track changes in real time, providing better navigation. Quenchers act like black holes, not producing fluorescence themselves, and can absorb all fluorescence signals emitted by reporter groups within a certain range, solving the problem of light pollution in experimental systems and improving the signal-to-noise ratio of fluorescence signals by reducing background signal. Therefore, due to their significant advantages of low background fluorescence values ​​and high signal-to-noise ratio, quencher groups can also be incorporated into molecular design to optimize activation / response.

[0007] To overcome the current lack of research on near-infrared fluorescent compounds in fluorescence imaging, the technical solution adopted in this invention is as follows: The first aspect of this invention provides a class of responsive near-infrared I fluorescent compounds, comprising four modules. ,in: R1 is a fluorescent chromophore, which is any one of the ZWCY series, ICG series, CY series, and Rhodamine series; R2 is a fibrosis-specific enzyme response group, which is any one of the mouse-derived / rat-derived / human-derived TG2, MMP9 / 2, and Cathepsin D response groups; R3 is a quenching group, and the quenching group is any one of the IRDye-QC series and the BQH series. R4 is an active / passive targeting group that targets diseased organs, which can be any one of the kidneys, liver, or lungs. The quenching group is selected based on the emission wavelength range of the fluorescent chromophore. The range of the IRDye-QC series quenching groups includes all listed fluorescent chromophores, and the range of the BQH series quenching groups includes the rhodamine series and some CY series fluorescent chromophores.

[0008] Furthermore, the responsive near-infrared fluorescent compound is a compound with any one of the structures of formulas (I) to (V): Where n is an integer from 0 to 20; n1 is an integer from 0 to 20; n2 is an integer from 3 to 100; and n3 is an integer from 1 to 50.

[0009] Furthermore, the fibrosis-specific enzyme-responsive group is a mouse / rat / human TG2-responsive sequence. Mouse-derived MMP2 / 9 response sequences Mouse / rat / human cathepsin D response sequences Any one of them.

[0010] Furthermore, the quenching group is of the IRDye-QC series. , Or BHQ-1 -N3 BHQ-2-N3 BHQ-3-N3 Any one of them.

[0011] Furthermore, the targeting group is , Integrin αvβ3, αvβ5, αvβ6, collagen, or any one of the passively targeted alkyne / carboxyl-DSPE-PEG.

[0012] Furthermore, the collagen is any one of type I, III, or IV collagen.

[0013] Furthermore, the MW of the PEG is any one of 1000, 2000, 3400, and 5000.

[0014] Furthermore, the responsive near-infrared I fluorescent compound has any one of the following structures: .

[0015] A second aspect of this invention provides a method for preparing the above-mentioned type of responsive near-infrared I fluorescent compound, comprising the following steps: Step S1: Compound B undergoes a condensation reaction with compound C to obtain an intermediate compound; the intermediate compound undergoes a click reaction with compound A to obtain compound D; compound D undergoes a substitution reaction with NH2-NH-FMOC to obtain near-infrared region I fluorescent compound I as shown in formula (I); Step S2: Compound E undergoes a condensation reaction with compound DSPE-PEG to obtain compound F; compound F undergoes a condensation reaction with compound C to obtain compound G; compound G undergoes a click reaction with compound A to obtain near-infrared region I fluorescent compound II as shown in formula (II); Step S3: ICG-COOH undergoes a condensation reaction with compound B to obtain compound H; compound H undergoes a click reaction with compound A to obtain compound I; compound I undergoes a substitution reaction with NH2-NH-FMOC to obtain near-infrared region I fluorescent compound III as shown in formula (III); Step S4: CY7 undergoes a condensation reaction with compound B to obtain compound J; compound J undergoes a click reaction with compound A to obtain compound K; compound K undergoes a substitution reaction with NH2-NH-FMOC to obtain near-infrared region I fluorescent compound IV as shown in formula (IV); Step S5: RhB undergoes a condensation reaction with compound B to obtain compound L; compound L undergoes a click reaction with compound A to obtain compound M; compound M undergoes a substitution reaction with NH2-NH-FMOC to obtain near-infrared 1 region fluorescent compound V as shown in formula (V); The structural formulas of compounds A, B, C, D, E, F, G, H, I, J, K, L, and M are shown below: .

[0016] Furthermore, the condensation reaction is carried out at a temperature of 25-120°C for 6-24 hours; the substitution reaction is carried out at a temperature of 25-120°C for 6-24 hours; and the click reaction is carried out at a temperature of 0-60°C for 8-24 hours.

[0017] Furthermore, the substitution reaction is carried out in the presence of a base, wherein the base is one or more of sodium hydroxide, potassium hydroxide, sodium hydride, potassium carbonate, cesium carbonate, sodium acetate, sodium ethoxide, triethylamine, pyridine, HATU, HBTU, and DIPEA.

[0018] Furthermore, the preparation method is carried out in the presence of an organic solvent, which is selected from one or more of methanol, ethanol, acetonitrile, dichloromethane, tetrahydrofuran, acetic acid, acetic anhydride, N,N-dimethylformamide, diethyl ether, DMSO, and toluene.

[0019] The third aspect of this invention provides the application of the above-mentioned class of responsive near-infrared I fluorescent compounds and their pharmaceutically acceptable salts or solvates, enantiomers, diastereomers, and tautomers in the preparation of fluorescent molecular probes.

[0020] Further, the pharmaceutically acceptable salts are hydrochloride, hydrobromide, nitrate, methyl nitrate, sulfate, hydrogen sulfate, aminosulfate, phosphate, acetate, glycolate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, para-aminosalicylic acid, glycolate, lactate, heptanoate, phthalate, oxalate, succinate, benzoate, and o-acetoxybenzoic acid. The salt, chlorobenzoate, methylbenzoate, mononitrobenzene, hydroxybenzoate, methoxybenzoate, mandelate, tannic acid salt, formate, stearate, ascorbate, palmitate, oleate, pyruvate, dihydroxynaphthyl salt, propionate, laurate, glutamate, propionate, estolate, methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-aminobenzenesulfonate, p-toluenesulfonate (toluenesulfonate), and naphthalene-2-sulfonate.

[0021] Furthermore, the fluorescent molecular probe is used for specific responses in the progression of fibrosis and for in vitro urine analysis.

[0022] This invention utilizes near-infrared 1-region fluorescent chromophores with excellent optical properties. Easily modifiable near-infrared fluorescent chromophores are selected, and targeted modifications are applied to construct activating fluorescent probes. Near-infrared 1-region fluorescent compounds use a specific main chain structure as the chromophore / fluorophore, and then connect biomarker-responsive groups, targeting groups, and quenching groups to both ends to obtain biomarker-responsive fluorescent tracers. The rationally designed activating near-infrared 1-region fluorescent probes mainly include four functional modules: fluorescent chromophores, fibrinogen-specific enzyme-responsive groups, targeting groups, and quenching groups. First, fluorescent chromophores with good optical properties and modification sites are synthesized and screened. Second, quenching groups are designed and screened based on the principle of fluorescence quenching, and then synthesized. Furthermore, based on the pathological characteristics of fibrosis, biomarkers were identified and corresponding peptide responsive groups were designed. These peptide responsive groups were then modified onto fluorescent chromophores to synthesize molecular probes that can be activated by fibrosis-related specific enzymes. Finally, the above modules were connected into a whole through a click chemistry reaction using azide groups pre-reserved on quenching groups to obtain activated molecular probes. The water solubility, optical properties, stability, response sensitivity, and response specificity of the probes were tested to screen for molecular probes with excellent performance. The metabolic pathways and toxic side effects of the molecular probes in mice were studied, and the recovery rate, biodistribution, and organ and tissue toxicity of the activated naked probes in urine were evaluated to screen for probes with good biocompatibility. The design and synthesis of activated near-infrared 1 fluorescent molecular probes that respond to specific biomarkers with high selectivity and specificity, and the effective enrichment and activation of signals at fibrotic lesion sites, are prerequisites for achieving in situ imaging and in vitro urine detection. This study explores in vitro urine detection methods for fibrosis. Using various rat / mouse fibrosis models, we investigated the sensitivity and specificity of probe responses at fibrotic lesion sites, analyzed the quantitative relationship between kidney / liver / urine signal intensity and the degree of fibrosis, and established a non-invasive and sensitive monitoring system for imaging / urine detection.

[0023] The specific excitation correspondence between the above-mentioned biomarker response groups and the biomarkers is shown in Table 1 below: Table 1 Compared with the prior art, the beneficial effects of the present invention are: This invention addresses the challenges of fibrosis diagnosis by leveraging the high signal-to-noise ratio and strong tissue penetration of the near-infrared 1 region to develop an activated near-infrared 1 region fluorescent probe for non-invasive diagnosis of renal and hepatic fibrosis. For renal fibrosis, after intravenous injection, the probe targets the fibrotic tissue, is specifically recognized and cleaved by the TG2 enzyme, and the quenching group is deactivated, exposing the fluorescent group and emitting a fluorescent signal. This signal is then metabolized by the kidneys and excreted in the urine, enabling non-invasive in vitro diagnosis of renal fibrosis via urine. For hepatic and pulmonary fibrosis, the same fluorescent group is combined with the liver fibrosis-specific recognition enzyme MMP2 / 9. After intravenous injection, the probe targets the fibrotic lesion, and after recognition and cleavage, the fluorescent cluster is excreted in the urine, allowing for in vitro urine diagnosis.

[0024] Specifically, the fluorescent probe designed in this invention has the following characteristics and advantages: (1) Non-invasive diagnosis of renal fibrosis and liver fibrosis can be achieved by activation-type near-infrared fluorescence imaging. This targeted and "activation-type" strategy can effectively overcome the interference of non-specific fluorescence signals. Combined with the high tissue penetration depth and high spatiotemporal resolution characteristics of near-infrared fluorescence, the sensitivity and signal-to-noise ratio of detection can be significantly improved, enabling rapid diagnosis and providing a new approach for disease diagnosis and treatment assessment.

[0025] (2) The use of in vitro urine testing greatly improves the convenience of testing. The advantages of in vitro diagnostic testing, such as speed, accuracy and efficiency, make it have great potential for further transformation and application. Attached Figure Description

[0026] Figure 1 The diagram shows the synthesis of each compound in Examples 1-16.

[0027] Figure 2 The results show the biocompatibility and stability of near-infrared fluorescent compound formula I.

[0028] Figure 3 The fluorescence spectrum changes of near-infrared region I fluorescent compound I in the presence or absence of TG2 enzyme.

[0029] Figure 4 Metabolic experiments of near-infrared fluorescent compound formula I in healthy mice and sections of different organs.

[0030] Figure 5 The results show in vivo fluorescence imaging, in vitro kidney imaging, and in vitro urine detection of near-infrared fluorescent compound I in different groups of a UUO-modeled mouse model of renal fibrosis.

[0031] Figure 6In vitro fluorescence imaging and quantitative mapping of different organs in a near-infrared I fluorescent compound, Formula II, in a carbon tetrachloride-modeled mouse model of liver fibrosis.

[0032] Figure 7 In vitro fluorescence imaging and quantitative mapping of different organs in a bleomycin-induced pulmonary fibrosis mouse model, using near-infrared region I fluorescent compound formula II.

[0033] Figure 8 In vivo fluorescence imaging and quantification of near-infrared region I fluorescent compound III in a FA-modeled mouse model of renal fibrosis.

[0034] Figure 9 In vivo fluorescence imaging and quantification of near-infrared region I fluorescent compound IV in a FA-modeled mouse model of renal fibrosis.

[0035] Figure 10 In vivo fluorescence imaging and quantification of near-infrared region I fluorescent compound V in a mouse model of renal fibrosis modeled by adenine. Detailed Implementation

[0036] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0037] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0038] The structures of the relevant compounds in the following examples are as follows: Example 1: Synthesis of compound M2 A solvent-free solution of compound M1 (477.0 mg, 3.0 mmol) and 1,4-dibromobutane (0.7 mL, 6.0 mmol) was stirred overnight at room temperature. The reaction mixture was washed three times with ethyl acetate (EA), and the resulting precipitate was dried under vacuum to give compound M2 (750.0 mg, 89% yield) as a white solid. 1H NMR (500 MHz, CDCl3): δ 7.83 (d, J =6.5 Hz, 1H), 7.67-7.48 (m, 3H), 4.87 (t, Hz, 2H), 3.54 (d, Hz, 2H), 3.18 (s,2H), 2.19-2.11 (m, 2H), 2.01 (s, 3H), 1.63 (s, 6H). LRMS (ESI) m / z: [M+H] + Calcd for C 15 H 22 BrN + 296.09; Found 296.06.

[0039] Example 2: Synthesis of compound M3 Compound M2 (590.0 mg, 2.0 mmol) and sodium azide (260.0 mg, 4.0 mmol) were dissolved in dry N,N-dimethylformamide (DMF) (10 mL) and stirred overnight at 50 °C. After cooling to room temperature, the reaction mixture was quenched with ice water and extracted three times with dichloromethane (DCM). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to give compound M3 (370.1 mg, 72% yield), which was used directly in the next reaction without further purification. 1 H NMR (400MHz, CDCl3): δ 7.14–7.08 (m, 2H), 6.78–6.74 (m, 1H), 6.52 (d, J = 7.6 Hz, 1H), 3.87–3.84 (m, 2H), 3.54 (t, J = 6.8 Hz, 2H), 3.30 (t, J = 6.8 Hz, 2H), 1.78–1.71 (m, 2H), 1.66–1.63 (m, 2H), 1.33 (s, 6H); 13 C NMR (101 MHz, CDCl3)δ 161.6, 145.8, 137.6, 127.6, 122.0, 118.5, 105.1, 73.4, 51.4, 44.3, 41.8,30.1, 26.7, 23.7; LRMS (ESI) m / z: [M+H] + Calcd for C 15 H 22 N4 + 258.36; Found 258.41.

[0040] Example 3: Synthesis of compound M5 Compound M4 (244.8 mg, 1.2 mmol) and tin chloride (907.2 mg, 4.8 mmol) were dissolved in methanol (MeOH) (20 mL) and stirred overnight at 90 °C. After cooling to room temperature, the mixture was quenched with water and extracted three times with dichloromethane. The organic layers were combined, dried over sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography (eluting with a gradient of petroleum ether / ethyl acetate = 20:1–5:1) to give compound M5 (179.6 mg, 86% yield) as a pink solid. 1 H NMR(400 MHz, Methanol-d4) δ 7.16 (d, J = 8.0 Hz, 1H), 6.74 (d, J = 2.0 Hz, 1H), 6.63 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 2.21 (s, 3H), 1.27 (s, 6H); 13 C NMR (126MHz, DMSO-d6) δ 181.6, 147.2, 146.5, 143.9, 119.4, 112.2, 107.8, 52.6, 23.0,14.7; LRMS (ESI) m / z: [M+H] + Calcd for C 11 H 15 N2175.25; Found 175.23.

[0041] Example 4: Synthesis of compound M6 A solution of compound M5 (340.0 mg, 2.0 mmol) and 1,2-oxothiacyclopentane 2,2-dioxide (10.0 mL) was stirred at room temperature for 12 hours. After removing the solvent under vacuum, the residue was purified by recrystallization (methanol / dichloromethane) to give compound M6 (721.0 mg, 67% yield) as a purple solid. 1H NMR (400 MHz, Methanol-d4) δ 7.65(d, J = 8.8 Hz, 1H), 7.25 (d, J = 2.4 Hz, 1H), 6.94 (dd, J = 8.8 Hz, 2.4 Hz,1H), 4.62 (t, J = 8.0 Hz, 2H), 3.63 (t, J = 8.0 Hz, 4H), 2.99 (t, J = 6.8 Hz, 2H), 2.90–2.86 (m, 7H), 2.38–2.31 (m, 2H), 2.18–2.05 (m, 4H), 1.57 (s, 6H); 13 C NMR (125 MHz, D2O) δ 183.4, 144.5, 117.2, 116.6, 111.1, 68.7, 54.5, 54.3,52.7, 47.9, 47.4, 46.3, 42.5, 24.2, 22.8, 21.8, 21.1, 17.7, 16.2, 12.1; LRMS(ESI) m / z: [M+H] + Calcd for 539.65; Found 539.24.

[0042] Example 5: Synthesis of compound IRDye-QC Compound M3 (257.0 mg, 1.0 mmol), compound M6 (538.0 mg, 1.0 mmol), N-[(3-(Anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline (hydrochloride) (359.0 mg, 1.0 mmol), Ac2O (1.0 mL, 10.6 mmol), and NaOAc (492.0 mg, 6.0 mmol) were dissolved in ethanol (EtOH) (8 mL) and stirred overnight at 70 °C. After cooling to room temperature, the mixture was quenched with water and extracted three times with ethyl acetate to remove excess starting material. The aqueous phase was evaporated under vacuum. Purification was performed by reversed-phase high-performance liquid chromatography to give the blue solid compound IRDye-QC (223.4 mg, 24% yield). 1H NMR (500 MHz, DMSO-d6) δ 8.27 (d, J = 15.0 Hz,1H), 7.85 (d, J = 13.0 Hz, 1H), 7.54 (d, J = 9.0 Hz, 1H), 7.43–7.41 (m, 2H),7.27 (t, J = 8.0 Hz, 1H), 7.11 (d, J = 8.0 Hz, 1H), 7.03 (t, J = 8.0 Hz, 1H), 6.90–6.84 (m, 2H), 5.87 (d, J = 13.0f Hz, 1H), 4.56 (t, J = 8.0 Hz, 2H), 3.96(t, J = 7.5 Hz, 2H), 3.55–3.52 (m, 4H), 2.73 (t, J = 6.0 Hz, 2H), 2.66 (t, J= 6.0 Hz, 2H), 2.59 (t, J = 6.5 Hz, 2H), 2.10–2.05 (m, 2H), 1.88–1.84 (m,14H), 1,70 (s, 12H), 1.61 (s, 12H); 13 C NMR (126 MHz, DMSO-d6) δ 172.2, 172.1,164.6, 148.5, 145.5, 144.2, 143.3, 143.2, 139.5, 135.0, 130.2, 128.3, 128.0,124.7, 122.2, 114.8, 111.3, 108.8, 108.3, 106.4, 95.9, 69.1, 53.3, 50.4,50.4, 50.3, 49.6, 48.5, 47.6, 47.0, 41.7, 40.0, 39.9, 39.7, 39.5, 39.4, 39.2,39.0, 29.1, 27.9, 27.3, 25.7, 25.7, 24.5, 23.6, 22.9, 22.2, 21.2, 20.8, 18.7,14.0, 12.6; LRMS (ESI) m / z: [M] - Calcd for C 43 H 54 ClN6O9S3 - 930.58; Found 930.16.

[0043] Example 6: Synthesis of compound M8 A mixed solution of 4-hydrazinobenzenesulfonic acid (1.88 g, 10.0 mmol), 3-methyl-2-butanone (1.6 mL, 15.0 mmol), sodium acetate (1.6 g, 19.0 mmol), and acetic acid (AcOH) (20 mL) was stirred overnight at 110 °C. After cooling to room temperature, the reaction mixture was quenched with water and extracted with ethyl acetate to remove excess starting material. The aqueous phase was evaporated under vacuum, and the residue was dissolved in methanol, filtered, and concentrated to give compound M8 (2.0 g, 84% yield) as a pink solid. 1 H NMR (400MHz, Methanol-d4) δ 7.84–7.80 (m, 2H), 7.47-7.45 (m, 1H), 1.97 (s, 3H), 1.32 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 188.9, 172.0, 153.7, 145.2, 145.1,125.1, 119.2, 118.22, 53.3, 22.5, 15.2; LRMS (ESI) m / z: [M+H] + Calcd forC 11 H 13 NO3S + 239.29; Found 239.54.

[0044] Example 7: Synthesis of compound M9 Compound M8 (478.0 mg, 2.0 mmol) and (3-bromopropyl)trimethylammonium bromide (991.8 mg, 3.8 mmol) were dissolved in benzene (10.0 mL) and stirred at 110 °C for 3 days. After removing the solvent under reduced pressure, the residue was purified by recrystallization (methanol / dichloromethane) to give compound M9 (476.0 mg, 70% yield) as a pink solid. 1 H NMR (400 MHz, D2O)δ 8.15 (s, 1H), 8.04–7.95 (m, 2H), 4.66 (t, J = 8.4 Hz, 2H), 3.70 (t, J = 8.0Hz, 2H), 3.38 (s, 9H), 3.24 (s, 3H), 2.55 (m, 2H), 1.66 (s, 3H). 13C NMR (126MHz, DMSO-d6) δ 198.9, 149.1, 141.7, 141.1, 126.3, 120.7, 115.2, 61.9, 54.6,52.6, 44.8, 21.9, 21.3, 14.8; LRMS (ESI) m / z: [M] + Calcd for C 17 H 27 N2O3S + 339.17; Found 339.38.

[0045] Example 8: Synthesis of compound M10 Compound M9 (340.0 mg, 1.0 mmol), (E)-2-chloro-3-(hydroxymethylene)cyclohex-1-encarbaldehyde (86.0 mg, 0.5 mmol), and sodium acetate (123.0 mg, 1.5 mmol) were dissolved in ethanol (10 mL) and stirred at 70 °C for 12 hours under an argon atmosphere. After cooling to room temperature, the mixture was filtered, and the filtrate was concentrated under vacuum. The crude product was washed with ethyl acetate (3 × 20 mL) and dichloromethane (3 × 20 mL) to give compound M10 (293.0 mg, 72% yield) as a green solid. 1 H NMR(400 MHz, DMSO-d6) δ 8.31 (d, J = 14.0 Hz, 2H), 7.83 (s, 2H), 7.71 (d, J =8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 6.34 (d, J = 14.0 Hz, 2H), 4.25 (m,4H), 3.07 (s, 18H), 2.74 (m, 4H), 2.17 (m, 6H), 1.89 (m, 4H), 1.71 (s, 12H).LRMS (ESI) m / z: [M+H] + Calcd for C 42 H 59 ClN4O6S2 + 815.52; Found 815.43.

[0046] Example 9: Synthesis of compound M11 Compound M10 (1.62 g, 2.0 mmol) and 3-(4-aminophenyl)propionic acid (990.0 mg, 6.0 mmol) were dissolved in DMF / water (90 ml / 10 ml) and stirred overnight at 90 °C. After cooling to room temperature, the mixture was concentrated under vacuum. Purification was performed by reversed-phase high-performance liquid chromatography to give compound M11 (1.31 g, 70% yield) as a blue solid. 1 H NMR (400MHz, DMSO-d6) δ 8.25 (s, 2H), 7.69 (m, 1H), 7.53-7.50 (m, 4H), 7.14-7.12 (m,2H), 7.07 (m, 2H), 6.90-6.88 (m, 2H), 3.96 (m, 4H), 3.44-3.42 (m, 4H), 3.05(s, 18H), 2.72-2.68 (m, 2H), 261-2.58 (m, 4H), 2.43-2.39 (m, 2H), 2.06 (m,4H), 1.83-1.80 (m, 2H), 1.29 (s, 12H). LRMS (ESI) m / z: [MH] - Calcd forC 51 H 67 N5O8S2 + 941.25; Found 940.64.

[0047] Example 10: Synthesis of compound M12 Compound M11 (941.0 mg, 1.0 mmol), peptide (82.2 mg, 0.1 mmol), HATU (152.0 mg, 0.4 mmol), HBTU (151.6 mg, 0.4 mmol), and DIPEA (36.5 μL, 0.2 mmol) were dissolved in DMF (10 mL) and stirred overnight at room temperature. The mixture was concentrated under vacuum. Purification was performed by reversed-phase high-performance liquid chromatography to give a blue solid compound M12 (69.8 mg, 40% yield). 1H NMR (500 MHz, D2O) δ 10.54 (s, 5H), 10.20(s, 1H), 9.90–9.86 (m, 2H), 9.48 (d, J = 8.0 Hz, 1H), 9.39 (s, 1H), 9.24 (s,1H), 8.21 (s, 1H), 7.60 (s, 1H), 7.31 (s, 1H), 6.89 (s, 1H), 6.80 (s, 1H),6.63 (s, 1H), 6.53 (s, 2H), 6.32 (s, 6H), 5.84 (s, 2H), 5.75 (d, J = 24.5 Hz,1H), 5.69 (s, 2H), 5.44 (s, 16H), 5.32 (s, 10H), 5.26 (s, 1H), 5.21 (s, 1H),4.97 (s, 3H), 4.87 (s, 2H), 4.58 (d, J = 67.0 Hz, 4H), 4.43–4.28 (m, 6H),4.22 (s, 4H), 4.08 (s, 4H), 3.74 (s, 2H), 3.54 (s, 12H), 3.49 (s, 16H), 3.46(s, 6H), 3.12 (d, 2H); 13 C NMR (126 MHz, DMSO-d6) δ 174.4, 172.4, 172.2,171.7, 171.6, 171.5, 171.3, 170.8, 170.5, 143.6, 143.2, 139.8, 134.5, 130.1,126.4, 124.9, 120.0, 118.9, 108.9, 97.4, 84.2, 71.8, 63.0, 60.34, 52.8, 49.9,48.6, 47.8, 47.3, 46.8, 40.8, 39.0, 38.9, 37.5, 34.2, 32.3, 31.9, 31.0, 29.5,28.2, 26.6, 25.0, 23.6, 21.9, 20.7, 18.7, 17.3, 15.6, 15.2, 14.9, 14.6, 14.5;LRMS (ESI) m / z: [M] - Calcd for C 88 H 122 N 14 O 19 S2 -1744.17; Found 1743.80.

[0048] Example 11: Synthesis of compound M13 A solution of compound M12 (177.4 mg, 1.0 mmol), NH2-NH-FMOC (31.3 mg, 0.1 mmol), HATU (190.0 mg, 0.5 mmol), HBTU (189.5 mg, 0.5 mmol), DIPEA (73.0 μL, 0.4 mmol), and DMF (5 mL) was stirred overnight at room temperature. Piperidine / DMF solvent was then added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The mixture was concentrated and purified by reversed-phase high-performance liquid chromatography to give compound M13 (53.0 mg, 30% yield) as a blue solid. 1 H NMR (500 MHz, D2O) δ 10.54 (s, 5H), 10.20 (s, 1H), 9.90–9.78 (m, 2H), 9.47 (d, J = 7.5 Hz, 1H), 9.41 (s, 1H), 9.29 (s, 1H), 8.21 (s, 1H), 7.60 (s,1H), 7.31 (s, 1H), 6.94 (s, 1H), 6.80 (s, 1H), 6.63 (s, 1H), 6.53 (s, 2H),6.45–6.32 (m, 6H), 5.90–5.78 (m, 2H), 5.73 (d, J = 24.0 Hz, 1H), 5.65 (s,2H), 5.44 (s, 16H), 5.32 (s, 10H), 5.26 (s, 1H), 5.21 (s, 1H), 4.97 (s, 3H),4.87 (s, 2H), 4.65 (s, 4H), 4.52–4.30 (m, 10H), 4.22 (s, 4H), 4.08 (s, 4H), 3.74 (s, 2H), 3.62 (s, 12H), 3.54 (s, 16H), 3.46 (s, 6H), 3.11 (d, J = 6.0Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 174.5, 172.5, 172.3, 171.7, 171.6,171.3, 170.8, 170.5, 143.9, 143.0, 140.0, 134.6, 130.1, 126.5, 124.8, 120.0,118.8, 109.3, 98.1, 84.2, 71.7, 63.0, 60.4, 52.8, 50.0, 49.6, 48.1, 47.3,46.8, 40.7, 39.0, 38.9, 37.5, 35.6, 34.2, 32.3,31.9,31.0,29.5,29.1,29.0,28.2,27.0,26.6,25.0,23.6,22.6,21.9,20.9,17.3,15.5,15.1,14.9,14.5;LRMS (ESI) m / z: [M+H] + Calcd for C 88 H 130 N 18 O 17 S2 + 1776.23; Found 1776.92.

[0049] Example 12: Synthesis of near-infrared I fluorescent compound M14 (ZWCYF) Compound M13 (17.8 mg, 0.01 mmol), IRDye-QC (9.3 mg, 0.01 mmol), copper sulfate·5H2O (2.4 mg, 0.01 mmol), and sodium ascorbate (VcNa) (1.9 mg, 0.01 mmol) were dissolved in DMSO / water (v / v = 1:1.6 mL) and stirred overnight at room temperature. After the reaction was complete, the mixture was placed in a dialysis membrane with a molecular weight cutoff of 1.0 kDa and dialyzed with water for 24 hours. The solution was then lyophilized to obtain the product compound M14 as a blue solid (18.8 mg, 70% yield), which was characterized by 1H NMR spectroscopy using CD3OD. 1H NMR (500 MHz, Methanol-d4) δ 9.40 (d,J = 13.0 Hz, 1H) δ 8.50 (dd, J = 13.5, 6.0 Hz, 2H), 8.07 (d, J = 13.0 Hz,1H), 7.56 (d, J = 9.5 Hz, 2H), 7.49 (d, J = 9.0 Hz, 2H), 7.39–7.24 (m, 11H), 7.10–6.93 (m, 13H), 6.82 –6.74 (m, 3H), 5.92 (d, J = 12.5 Hz, 2H), 5.36 (t, J= 4.5 Hz, 4H), 4.69–4.64 (m, 8H), 4.0–3.89 (m, 14H), 3.67 (d, J = 7.0 Hz, 16H), 3.60–3.54 (m, 12H), 3.43 (td, J = 6.5, 2.5 Hz, 24H), 3.00 (t, J = 6.0Hz, 8H), 2.90 (d, MALDI-TOF MS found: 2692.989.

[0050] Example 13: Synthesis of ZWCYL, a near-infrared I fluorescent compound Similar to the synthesis of the fluorescent compound ZWCYF. In the first step, compound E1 (93.1 mg, 0.1 mmol), DSPE-PEG2000 (26.3 mg, 0.1 mmol), HATU (18.9 mg, 0.05 mmol), HBTU (18.8 mg, 0.05 mmol), and DIPEA (8.0 μL, 0.04 mmol) were dissolved in DMF. After stirring overnight at room temperature, product F1 was separated by liquid chromatography. In the second step, compound F1 (17.7 mg, 0.01 mmol), compound C (9.4 mg, 0.02 mmol), HATU (3.4 mg, 0.01 mmol), HBTU (2.9 mg, 0.01 mmol), and DIPEA were dissolved in DMF. After stirring overnight at room temperature, product G1 was separated by liquid chromatography. In step three, G1 (27.0 mg, 0.01 mmol) and compound A1 (0.9 mg, 0.01 mmol), copper sulfate·5H2O (2.4 mg, 0.01 mmol), and sodium ascorbate (VcNa) (1.9 mg, 0.01 mmol) were dissolved in DMSO / water (v / v = 1:1.6 mL). After stirring overnight at room temperature, the mixture was placed in a dialysis membrane with a molecular weight cutoff of 1.0 kDa and dialyzed with water for 24 hours. The solution was then lyophilized to obtain the product compound ZWCYL, a blue solid, with MALDI-TOF MS results of 5500-5700.

[0051] Example 14: Synthesis of ICGF, a near-infrared I fluorescent compound Similar to the synthesis of the fluorescent compound ZWCYF, the following steps were taken: ICG-COOH (730.6 mg, 1 mmol), E1 (822 mg, 1 mmol), HATU (190.0 mg, 0.5 mmol), HBTU (189.5 mg, 0.5 mmol), DIPEA (73.0 μL, 0.4 mmol), and DMF (5 mL) were stirred overnight at room temperature, followed by liquid chromatography separation of product H1. In the second step, H1 (14.7 mg, 0.01 mmol) was dissolved with compound A1 (0.9 mg, 0.01 mmol), copper sulfate·5H2O (2.4 mg, 0.01 mmol), and sodium ascorbate (VcNa) (1.9 mg, 0.01 mmol) in DMSO / water (v / v = 1:1.6 mL). After stirring overnight at room temperature, the mixture was placed in a dialysis membrane with a molecular weight cutoff of 1.0 kDa and dialyzed with water for 24 hours. The solution was then lyophilized to obtain product compound I1. In the third step, compound I1 (2.4 mg, 0.01 mmol) was mixed with NH2-NH-FMOC (1.9 mg, 0.01 mmol), HATU (190.0 mg, 0.5 mmol), HBTU (189.5 mg, 0.5 mmol), DIPEA (73.0 μL, 0.4 mmol), and DMF (5 mL) and stirred overnight at room temperature. Piperidine / DMF solvent was then added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The mixture was concentrated and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain compound ICGF. MALDI-TOF MS found: 2482.361.

[0052] Example 15: Synthesis of the near-infrared I fluorescent compound CY7F Similar to the synthesis of the fluorescent compound ZWCYF, compounds CY7 (682.8 mg, 1 mmol), E1 (822 mg, 1 mmol), HATU (190.0 mg, 0.5 mmol), HBTU (189.5 mg, 0.5 mmol), DIPEA (73.0 μL, 0.4 mmol), and DMF (5 mL) were stirred overnight at room temperature, followed by liquid chromatography separation of product J1. In the second step, J1 (14.2 mg, 0.01 mmol) was dissolved in DMSO / water (v / v = 1:1.6 mL) with compound A1 (0.9 mg, 0.01 mmol), copper sulfate·5H2O (2.4 mg, 0.01 mmol), and sodium ascorbate (VcNa) (1.9 mg, 0.01 mmol). After stirring overnight at room temperature, the mixture was placed in a dialysis membrane with a molecular weight cutoff of 1.0 kDa and dialyzed with water for 24 hours. The solution was then lyophilized to obtain product compound K1. In the third step, compound K1 (2.3 mg, 0.01 mmol) was mixed with NH2-NH-FMOC (1.9 mg, 0.01 mmol), HATU (190.0 mg, 0.5 mmol), HBTU (189.5 mg, 0.5 mmol), DIPEA (73.0 μL, 0.4 mmol), and DMF (5 mL) and stirred overnight at room temperature. Piperidine / DMF solvent was then added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The mixture was concentrated and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain compound CY7F. MALDI-TOF MS found: 2432.916.

[0053] Example 16: Synthesis of the near-infrared I fluorescent compound RhF Similar to the synthesis of the fluorescent compound ZWCYF, compounds RhB (479 mg, 1 mmol), E1 (822 mg, 1 mmol), HATU (190.0 mg, 0.5 mmol), HBTU (189.5 mg, 0.5 mmol), DIPEA (73.0 μL, 0.4 mmol), and DMF (5 mL) were stirred overnight at room temperature, followed by liquid chromatography separation of product L1. In the second step, L1 (11.9 mg, 0.01 mmol) was dissolved in DMSO / water (v / v = 1:1.6 mL) with compounds A1 (0.9 mg, 0.01 mmol), copper sulfate·5H2O (2.4 mg, 0.01 mmol), and sodium ascorbate (VcNa) (1.9 mg, 0.01 mmol). After stirring overnight at room temperature, the mixture was placed in a dialysis membrane with a molecular weight cutoff of 1.0 kDa and dialyzed with water for 24 hours. The solution was then lyophilized to obtain product compound M1. In the third step, a solution of compound M1 (2.1 mg, 0.01 mmol) with NH2-NH-FMOC (1.9 mg, 0.01 mmol), HATU (190.0 mg, 0.5 mmol), HBTU (189.5 mg, 0.5 mmol), DIPEA (73.0 μL, 0.4 mmol), and DMF (5 mL) was stirred overnight at room temperature. Piperidine / DMF solvent was then added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The mixture was concentrated and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain compound RhF. MALDI-TOF MS found: 2230.874.

[0054] Test Example 1: Biocompatibility and Stability Experiment Calculations showed that ZWCYF has a logD value of -0.71, indicating good water solubility and high bioavailability. Simultaneously, the photostability of the near-infrared I fluorescent compound ZWCYF dissolved in FBS was tested as described in Example 12. The ZWCYF solution and FBS solution were incubated at 37 °C for 24 hours. The fluorescence intensity of the mixture was measured using a fluorescence spectrophotometer, and the experiment was repeated three times. Next, pH stability was tested. The ZWCYF solution (10 µM) was incubated in different buffer solutions with pH values ​​ranging from 4.0 to 8.5. The fluorescence intensity of the fluorescent compound was measured using a fluorescence spectrophotometer. The test results are as follows: Figure 2 As shown, ZWCYF exhibits good stability and certain photostability within a pH range of 4.0-8.5.

[0055] Test Example 2: Spectral Testing A 10 µM solution of the near-infrared I fluorescent compound from Example 12 was reacted with TG2 in ultrapure water at 37 °C. The UV-Vis and fluorescence spectra of the solution were then measured. The test results are as follows: Figure 3 As shown, from Figure 3 Calculations show that the near-infrared fluorescent compound of the present invention has a very high sensitivity to TG2 enzyme response, and the maximum emission wavelength of the TG2 enzyme-responsive near-infrared fluorescent probe of the present invention is 800 nm.

[0056] Test Example 3: In vivo stability and biocompatibility study Male Balb / c mice (6-8 weeks old) were intravenously injected with ZWCYF solution. Twenty-four hours post-injection, mice were anesthetized for dorsal and ventral imaging, followed by euthanasia for peritoneal and excised organ imaging. Fluorescence intensity was assessed using imaging analysis. Major organs (heart, liver, spleen, lungs, and kidneys) were removed from male Balb / c mice and placed in 4% paraformaldehyde (PFA) for histological examination. Control mice were injected with the same volume of PBS. Metabolic experiments of the obtained near-infrared fluorescent compound (Formula I) in healthy mice and sections of different organs are shown below. Figure 4 As shown in the figure, the metabolic experiment shows that it is mainly distributed in the kidneys, indicating that ZWCYF has good renal clearance. At the same time, compared with the control group, the sections of different organs of mice after the metabolic experiment show that ZWCYF has good biocompatibility.

[0057] Test Example 4: UUO Fibrosis Mouse Experiment Establishment of a UUO fibrosis mouse model: Male Balb / c mice aged 6-8 weeks were randomly selected and divided into two groups. Unilateral ureteral ligation (using 4-0 silk suture) was performed via laparotomy. The control group received 0.2 mL of physiological saline. Imaging examinations were performed using an IVIS spectroscopy system on days 0, 7, and 21 after UUO treatment. Mouse weight and symptoms were monitored daily during the experiment, and blood and urine samples were collected at different time points after UUO treatment.

[0058] Real-time in vivo NIRF imaging in the UUO mouse model: For the UUO mouse model, real-time NIRF imaging was performed within 2 hours after intravenous injection of ZWCYF (10 μmol / kg) 7 and 21 days after modeling. Fluorescence images were acquired using an IVIS spectrumCT system with excitation wavelength of 700±10 nm, emission wavelength of 800±10 nm, and acquisition time of 1 s. Mice were euthanized 2 hours after injection of the near-infrared I fluorescent compound of Example 12, and ex vivo organs were collected.

[0059] In vitro urine analysis in the UUO mouse model: Urine samples were collected from live mice via metabolic cages 24 hours prior. Approximately 300 μL of collected urine samples were centrifuged at 3000 rpm for 10 minutes, and the supernatant was filtered through a 0.22 μm syringe filter. 100 μL of urine samples were incubated with ZWCYF (10 μM) and calcium chloride in PBS solution (10 mM, pH 7.4) at 37°C for 3 hours. Fluorescence was then measured and quantitatively analyzed using an IVIS optical imaging system at a time of 2 s (excitation wavelength 700 ± 10 nm, emission wavelength 790 ± 10 nm). The in vivo fluorescence imaging, in vitro kidney imaging, and in vitro urine analysis results of the near-infrared 1-region fluorescent compound formula I in different groups of the UUO-modeled renal fibrosis mouse model are shown below. Figure 5 As shown in the figure, after UUO surgery, due to the formation of hydronephrosis and hydroureter, the fluorescence signal intensity of the obstructed kidney was significantly greater than that of the contralateral kidney, suggesting that TG2 gradually increases during the progression of renal fibrosis. These in vivo observations were further validated by in vitro urine fluorescence imaging studies, demonstrating that this near-infrared I fluorescent compound has the advantages of high selectivity, high sensitivity, and high spatial resolution in in vivo imaging.

[0060] Test Example 5: Mouse Model Experiment of Liver Fibrosis To induce liver fibrosis, mice were intraperitoneally injected with carbon tetrachloride (carbon tetrachloride / olive oil = 1 / 4 (v / v), 2.5 mL / kg body weight, twice a week). The fluorescent probe ZWCYL was detected at days 0, 14, and 28 post-treatment. Figure 6 The isolated liver organs, after 14 and 28 days of liver fibrosis, showed increasing fluorescence signals, indicating that the fibrosis process was well detected.

[0061] Test Example 6: Mouse Model Experiment of Pulmonary Fibrosis To induce pulmonary fibrosis, bleomycin (5 mg / mL, 100 μL) was injected intraperitoneally on days 1, 5, 8, 11, and 15. The fluorescent probe ZWCYL was injected 30 minutes after treatment on days 0, 14, and 28. Different organs were then dissected for fluorescence imaging and reading. The results are as follows: Figure 7 As shown, the fluorescence imaging trends of isolated lungs are consistent with the progression of pulmonary fibrosis.

[0062] Test Example 7: FA-induced renal fibrosis mouse model experiment Male Balb / c mice aged 6-8 weeks were randomly assigned to groups and treated with freshly prepared folic acid solution (250 mg / kg, dissolved in sodium bicarbonate, intraperitoneal injection). The control group received saline (0.2 mL). Mice were imaged using an IVIS spectroscopy system at days 0, 7, 14, and 21 after FA treatment. Mouse weight and symptoms were monitored daily throughout the experiment. In vivo fluorescence imaging of the ICGF probe was performed at different time points after saline or FA treatment, and the results are shown below. Figure 8 As the degree of fibrosis increases, the fluorescence signal of the ICGF probe also increases, indicating that the near-infrared I region fluorescent compound has excellent performance.

[0063] Using a FA-induced renal fibrosis mouse model, the probe compound CY7F was injected on days 0 and 14 for NIRF imaging. The excitation wavelength was 700±10 nm, the emission wavelength was 800±10 nm, and the acquisition time was 1 s. The in vivo fluorescence imaging and quantitative results of the obtained near-infrared I fluorescent compound IV in the FA-modeled renal fibrosis mouse model are shown below. Figure 9 As shown, a significant fluorescence intensity signal was observed at the kidney site 30 minutes after injection.

[0064] Test Example 8: Adenine-induced renal fibrosis mouse model experiment Adenine nephropathy was induced in 6-8 week old C57BL / 6 mice by feeding them a diet containing 0.2% adenine. Healthy mice served as the control group. In vivo fluorescence imaging was performed on days 0 and 14 by injection of the probe RhF compound. The excitation wavelength was 700±10 nm, the emission wavelength was 800±10 nm, and the acquisition time was 1 s. Figure 10 Significant NIRF signals were observed in the kidneys of live mice 30 minutes after injection.

[0065] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A class of responsive near-infrared I fluorescent compounds, characterized in that, It has four modules. ,in: R1 is a fluorescent chromophore, which is any one of the ZWCY series, ICG series, CY series, and Rhodamine series; R2 is a fibrosis-specific enzyme response group, which is any one of the mouse-derived / rat-derived / human-derived TG2, MMP9 / 2, and Cathepsin D response groups; R3 is a quenching group, and the quenching group is any one of the IRDye-QC series and the BQH series. R4 is an active / passive targeting group that targets diseased organs, which can be any one of the kidneys, liver, or lungs. The quenching group is selected based on the emission wavelength range of the fluorescent chromophore. The range of the IRDye-QC series quenching groups includes all listed fluorescent chromophores, and the range of the BQH series quenching groups includes the rhodamine series and some CY series fluorescent chromophores.

2. The type of responsive near-infrared I fluorescent compound according to claim 1, characterized in that, The responsive near-infrared fluorescent compound is a compound with any one of the structures of formulas (I) to (V): Where n is an integer from 0 to 20; n1 is an integer from 0 to 20; n2 is an integer from 3 to 100; and n3 is an integer from 1 to 50.

3. The type of responsive near-infrared I fluorescent compound according to claim 1, characterized in that, The fibrosis-specific enzyme-responsive group is a mouse / rat / human TG2-responsive sequence. Mouse-derived MMP2 / 9 response sequences Mouse / rat / human cathepsin D response sequences Any one of them.

4. The type of responsive near-infrared I fluorescent compound according to claim 1, characterized in that, The quenching group is an IRDye-QC series. , Or BHQ-1 -N3 BHQ-2-N3 BHQ-3-N3 Any one of them.

5. The type of responsive near-infrared I fluorescent compound according to claim 1, characterized in that, The targeting group is , Integrin αvβ3, αvβ5, αvβ6, collagen, or any one of the passively targeted alkyne / carboxyl-DSPE-PEG.

6. A class of responsive near-infrared I fluorescent compounds according to claim 1 or 2, characterized in that, The responsive near-infrared I fluorescent compound has any one of the following structures: 。 7. A method for preparing a type of responsive near-infrared fluorescent compound as described in claim 2, characterized in that, Includes the following steps: Step S1: Compound B and compound C undergo a condensation reaction to obtain an intermediate compound; The intermediate compound reacts with compound A via a click reaction to give compound D. Compound D then reacts with NH2-NH-FMOC via a substitution reaction to give near-infrared region I fluorescent compound I as shown in formula (I). Step S2: Compound E undergoes a condensation reaction with compound DSPE-PEG to obtain compound F; compound F undergoes a condensation reaction with compound C to obtain compound G; compound G undergoes a click reaction with compound A to obtain near-infrared region I fluorescent compound II as shown in formula (II); Step S3: ICG-COOH undergoes a condensation reaction with compound B to obtain compound H; compound H undergoes a click reaction with compound A to obtain compound I; compound I undergoes a substitution reaction with NH2-NH-FMOC to obtain near-infrared region I fluorescent compound III as shown in formula (III); Step S4: CY7 undergoes a condensation reaction with compound B to give compound J; Compound J reacts with compound A via a click reaction to give compound K. Compound K then reacts with NH2-NH-FMOC via a substitution reaction to give near-infrared region I fluorescent compound IV as shown in formula (IV). Step S5: RhB undergoes a condensation reaction with compound B to obtain compound L; compound L undergoes a click reaction with compound A to obtain compound M; compound M undergoes a substitution reaction with NH2-NH-FMOC to obtain near-infrared 1 region fluorescent compound V as shown in formula (V); The structural formulas of compounds A, B, C, D, E, F, G, H, I, J, K, L, and M are shown below: 。 8. The method for preparing a class of responsive near-infrared I fluorescent compounds according to claim 7, characterized in that, The condensation reaction is carried out at a temperature of 25-120°C for 6-24 hours; the substitution reaction is carried out at a temperature of 25-120°C for 6-24 hours; and the click reaction is carried out at a temperature of 0-60°C for 6-24 hours.

9. The use of a class of responsive near-infrared fluorescent compounds as described in any one of claims 1-6, and their pharmaceutically acceptable salts or solvates, enantiomers, diastereomers, and tautomers in the preparation of fluorescent molecular probes.

10. The application according to claim 9, characterized in that, The fluorescent molecular probes are used for specific responses in the progression of fibrosis and for in vitro urine analysis.

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