Water-soluble near-infrared two-region small molecular fluorescent compound as well as preparation method and application thereof

Abstract

The invention relates to a water-soluble near-infrared two-region small molecular fluorescent compound as well as a preparation method and application thereof. The compound has a symmetrical conjugated skeleton structure as shown in a general formula (I), X and Y are independently O, S or Se, and R1-R7 are controllable hydrophilic or neutral substituents and can endow good water solubility and tissue penetrability. The compound has strong fluorescence emission in the wave band of 900-1500 nm, the quantum yield is higher than 0.5%, and the compound has excellent light stability and biocompatibility. The preparation method is simple, efficient and suitable for large-scale production. The compound can be widely applied to in-vivo imaging scenes such as kidney imaging, bladder metabolism monitoring, whole-body blood vessel and lymphatic system imaging, tumor tracing and intraoperative navigation, is discharged through urine after injection, is clear in metabolic pathway and low in biological accumulation risk, and has good in-vivo safety and clinical transformation potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic functional materials and biomedical imaging, and particularly relates to a water-soluble near-infrared second region (NIR-II) small molecule fluorescent compound, a preparation method thereof, and an application thereof in in-vivo imaging. This compound can be widely applied to biomedical research and diagnosis and treatment fields such as in-vivo fluorescence imaging, kidney metabolism tracking, blood vessel imaging, and lymphatic system imaging. Background Art

[0002] Near-infrared second region (Near-Infrared II, NIR-II, wavelength range 900–1700 nm) biomedical imaging technology has become a research hotspot in the fields of life science and medicine in recent years due to its advantages such as deep tissue penetration, small scattering, low background autofluorescence, and high imaging resolution. Compared with traditional visible light and near-infrared first region (NIR-I, 700–900 nm), NIR-II fluorescent probes have greater potential in deep tissue imaging, real-time imaging, and the combination of precise diagnosis and treatment, and have been widely applied in multiple directions such as blood vessel imaging, tumor navigation, brain function imaging, and kidney metabolism observation. The current mainstream NIR-II fluorescent materials mainly include: (1) Quantum dot-based fluorescent probes (such as PbS, Ag2S, etc.), which have excellent optical properties but have problems of high toxicity and poor biodegradability. (2) Rare earth nanomaterials, with strong luminescence stability, but complex synthesis, large particle size, and obvious accumulation in the liver and spleen, which are not conducive to clinical transformation. (3) Polymer / small molecule aggregation-induced emission materials (AIE), with partially controllable synthesis, but generally poor water solubility, often requiring polymer coating or micelle carriers. (4) Organic small molecule NIR-II fluorescent probes, which have become the research focus in recent years because of their clear structure, controllable metabolism, and low toxicity, and are particularly suitable for in-vivo imaging applications.

[0003] Although small molecule fluorescent probes have certain advantages, there are still the following technical bottlenecks: (1) Poor water solubility: Many small molecule NIR-II dyes are hydrophobic aromatic structures and need to rely on nano-carriers or surface modification to enter the aqueous phase, increasing the synthesis complexity and biological uncertainty; (2) Limited optical performance: The emission wavelengths of some small molecules are not sufficient to enter the NIR-II region, or the quantum yield is relatively low, and the imaging effect is not ideal; (3) Poor biological compatibility: It is not easily metabolized and cleared by the biological system and is easily accumulated in organs such as the liver and spleen; (4) Complicated preparation route and difficult industrial scale-up: Some NIR-II small molecules require multiple-step modification and high-cost raw materials, which are not conducive to large-scale application.

[0004] Therefore, in order to solve the problems in the prior art and address the multiple technical challenges of existing materials in terms of water solubility, photo-stability, biocompatibility, and application scenarios, it is urgently necessary to develop a new type of small molecule NIR-II fluorescent compound with a simple structure, adjustable wavelength, good water solubility, biocompatible metabolism, and controllable synthesis process to meet the dual needs of life science research and clinical translation. Summary of the Invention

[0005] The present invention aims to provide a NIR-II small molecule fluorescent probe, which is a NIR-II small molecule compound with a symmetrical conjugated skeleton, modified with natural water-soluble groups, good biocompatibility, and tissue penetration ability, novel structure, good water solubility, and excellent biocompatibility. It has a high quantum yield, good photo-stability, and tissue penetration ability, and is applicable to fields such as in vivo vascular imaging, lymphatic system imaging, tumor tracing, and treatment.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a water-soluble near-infrared region II small molecule fluorescent compound, and the compound has the following general formula (I):

[0008]

[0009] Wherein X and Y are each independently selected from one of O, S, and Se

[0010] R1 and R2 are each independently selected from H and one of the following 6 groups,

[0011]

[0012] Among them, in the structural formulas of the 6 groups, n is an integer taken from 0 to 20.

[0013] R3, R4, R5, R6, and R7 are each independently selected from H and one of the following 34 groups,

[0014]

[0015] Among them, in the structural formulas of the 34 groups, m is an integer taken from 0 to 20.

[0016] As a specific embodiment, the water-soluble near-infrared region II organic fluorescent small molecule compound is:

[0017]

[0018] That is, in the general formula (I), R1 is R2 is R3 is H, R4 is H, R5 is R6 is H and R7 is H.

[0019] The molecular weight of compound HS-1 is 856.23 Da, logP = -1.2, and the water solubility > 10 mg / mL. In its symmetric D-π-A-π-D framework, the bridging of Se atoms causes the emission to redshift to 1300 nm, and the terminal disulfonic acid groups ensure water solubility.

[0020] Furthermore, the fluorescence emission wavelength of the water-soluble organic fluorescent small molecule compound in the second near-infrared region is 900 - 1500 nm.

[0021] As a specific embodiment, the water-soluble organic fluorescent small molecule compound in the second near-infrared region is

[0022]

[0023] In the second aspect of the present invention, there is provided a preparation method of the above-mentioned regulable metabolism organic fluorescent small molecule compound, including:

[0024]

[0025] React compound 1 with n-butyllithium at a temperature of -70 °C to -80 °C, and then add tributyltin chloride, and react for 10 - 15 hours to obtain compound 2;

[0026] React compound 2 with compound 3 in toluene under palladium catalysis to obtain compound 4;

[0027] After reducing compound 4 with zinc powder, add N-sulfinylaniline, trimethylchlorosilane and pyridine to react to obtain compound 5;

[0028] React compound 5 with N-bromosuccinimide in N,N-dimethylformamide and acetonitrile to obtain compound 6;

[0029] React compound 6 with compound 7 under palladium catalysis in THF and an aqueous solution of NaHCO3 to obtain the water-soluble small molecule fluorescent compound in the second near-infrared region.

[0030] As a specific embodiment, the method specifically includes:

[0031] Synthesis of compound 2: Dissolve compound 1 in tetrahydrofuran, add it to a round-bottom reaction flask, the reaction temperature is -70 to -80 °C, preferably -78 °C, continuously stir for 10 - 60 minutes, add n-butyllithium dropwise, after the addition is complete, stir for 2 hours, and then add tributyltin chloride dropwise. After reacting for 10 - 15 hours, terminate the reaction. Preferably, the molar ratio of compound 1, n-butyllithium and tributyltin chloride is 1:(1 - 2):(1 - 2), for example, 1:1.3:1.3; the tetrahydrofuran is an ultra-dry solvent.

[0032] Synthesis of Compound 4: Compound 2, Compound 3 and tetrakis(triphenylphosphine)palladium were successively added into a round-bottom reaction flask. Toluene as the solvent was added, and the reaction was carried out for 15 - 20 h, and then the reaction ended. The molar ratio of Compound 2, Compound 3 and tetrakis(triphenylphosphine)palladium was 1:(0.2 - 0.6):(0.02 - 0.06), for example 1:0.5:0.05, to obtain Compound 4.

[0033] Synthesis of Compound 5: Compound 4, zinc powder and inorganic salt ammonium chloride were added into a reaction flask, and a mixed solvent of methanol and dichloromethane was added. After reacting for 1 - 3 h, the reaction was terminated and the reaction solution was extracted. The intermediate crude product, N-sulfinylaniline, trimethylchlorosilane and pyridine were added into the reaction flask. The reaction was carried out for 4 - 8 h, and then the reaction was terminated. Preferably, the molar ratio of Compound 4, zinc powder and inorganic salt was 1:(50 - 150):(30 - 50), for example 1:100:40.

[0034] Synthesis of Compound 6: Compound 4 and N-bromosuccinimide were added into a reaction flask, and hydrobromic acid was added. The reaction solvent was a mixed solution of N,N-dimethylformamide and acetonitrile. The reaction was carried out overnight. The reaction was terminated. Preferably, the molar ratio of Compound 5 and N-bromosuccinimide was 1:(1 - 5), for example 1:2.5; the volume ratio of N,N-dimethylformamide and acetonitrile was (1 - 3):(1 - 3), for example 2:1.

[0035] Synthesis of Compound 8: Compound 6, 7 and the catalyst tetrakis(triphenylphosphine)palladium were successively added into a reaction flask. The solvent was a mixed solution of tetrahydrofuran and NaHCO3, and the reaction system lasted for 15 - 20 h. The reaction was terminated. Preferably, in the above step, the molar ratio of Compound 5, 6 and tetrakis(triphenylphosphine)palladium was 1:(2 - 3):(0.01 - 0.5), for example 1:2.5:0.1.

[0036] The key points of the method include: controlling side reactions in the low-temperature lithiation step with a yield of 85%; selectively removing nitro groups in the zinc powder reduction step to avoid skeleton destruction; bromination site-directed modification to ensure precise introduction of substituents.

[0037] In the third aspect of the present invention, there is provided a use of the above-mentioned water-soluble near-infrared II-region organic fluorescent small molecule compound as a near-infrared II-region fluorescence imaging probe in kidneys, bladders and systemic blood vessels in vivo.

[0038] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0039] The water-soluble near-infrared II (NIR-II) small molecule fluorescent compound provided by the present invention has the following remarkable beneficial effects:

[0040] 1. Reasonable structural design and flexible construction

[0041] The present invention adopts a D–π–A–π–D type symmetric conjugated backbone structure. The introduction of heteroatom bridges (Y and Z are O, S, Se) in the core structure enhances the electron delocalization ability, extends the conjugation path, and effectively regulates the molecular energy level and emission wavelength. By introducing hydrophilic substituents with adjustable lengths (such as PEG, sulfonate, carboxylic acid or quaternary ammonium salt) at the end, the water solubility and biodistribution characteristics can be flexibly regulated, with extensive structural tunability and function expansion space.

[0042] 2. Good water solubility and biocompatibility

[0043] Compared with existing hydrophobic organic small molecules or quantum dot-based NIR-II fluorescent probes, the compounds of the present invention introduce hydrophilic groups through the molecule itself (non-surface modification), significantly improving the overall hydrophilicity of the molecule. They can be directly dissolved in the aqueous phase system without additional coating or micelle carriers, avoiding complex post-treatment processes. In addition, the molecular structure does not contain heavy metals or low-toxicity groups, showing excellent biocompatibility and safety, and is suitable for long-term in vivo imaging.

[0044] 3. Excellent fluorescence performance and adjustable emission wavelength

[0045] The compounds of the present invention have strong fluorescence emission signals in the second near-infrared region (900 - 1500 nm), with a wide excitation wavelength range, and are compatible with a variety of light source systems. By regulating the donor, bridging group, and acceptor units, the adjustable range of the emission wavelength can be achieved from 1000 nm to 1300 nm; the quantum yield is better than that of similar small molecules, up to more than 2% (in aqueous solution), with a high imaging signal-to-noise ratio and low background, meeting the requirements of various high-resolution in vivo imaging.

[0046] 4. Strong photo-stability and suitable for long-term in vivo imaging

[0047] The molecules of the present invention have good photo-stability under strong light conditions, are not prone to photo-bleaching or structural degradation, and can achieve long-term and high-repetition-rate in vivo imaging operations, suitable for continuous monitoring or multi-time point imaging experiments.

[0048] 5. Simple synthesis method and scalable production

[0049] The synthesis route provided by the present invention has simple steps, mild reaction conditions, high reaction yields, and the key intermediates can be stably stored and used for subsequent modification, with good industrialization prospects. Especially in the synthesis process, the use of highly toxic metal elements or high-pressure and high-temperature reactions is avoided, which is beneficial to environmental protection and process safety.

[0050] 6. Good in vivo distribution and metabolic characteristics

[0051] The compounds of the present invention have a moderate molecular weight and high water solubility. After intravenous injection, they can be rapidly distributed to tissues such as the kidneys, bladder, and blood vessels. Their metabolic pathways in the body are clear and they can be excreted through urine, possessing rapid metabolism and reducing the risk of bioaccumulation. They are particularly suitable for renal function imaging and pharmacokinetic studies.

[0052] 7. Wide range of applications and good application prospects

[0053] As a NIR-II imaging probe, the said compound is not only applicable to the imaging of blood vessels, kidneys, and lymphatic systems in basic medical research, but also can be extended to application scenarios such as preclinical tumor tracing, intraoperative navigation, and drug delivery evaluation. It is especially suitable for active targeting imaging and treatment after ligand modification.

[0054] In summary, a class of novel-structured and superior-property NIR-II small molecule compounds proposed by the present invention are superior to the prior art in terms of structural design, synthesis process, imaging performance, and biocompatibility, and have outstanding scientific research and industrialization value. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 It is the hydrogen spectrum of HS-1.

[0056] Figure 2 Among them, (a) is the absorption spectrum of HS-1; (b) is the emission spectrum of HS-1.

[0057] Figure 3 It is the comparison of the stability of HS-1 and ICG nanoparticles.

[0058] Figure 4 It is the bladder imaging of mice with HS-1 at different time points.

[0059] Figure 5 It is the kidney imaging of mice with HS-1.

[0060] Figure 6 It is the whole-body blood vessel imaging of mice with HS-1.

[0061] Figure 7 It is the hemolysis experiment of cells with different concentrations of HS-1.

[0062] Figure 8 It is the HE staining of various organs of mice after continuous injection of HS-1 for 3 days.

[0063] Figure 9 It is the blood routine and blood biochemical indexes of mice after continuous injection of HS-1 for 3 days. DETAILED DESCRIPTION OF THE INVENTION

[0064] The present invention will be specifically described below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention, rather than limiting the present invention.

[0065] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of any contradiction, this specification shall prevail.

[0066] Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment, etc. used in the present invention can be obtained through market purchases or by existing methods.

[0067] Example 1: Synthesis of Compound HS-1

[0068]

[0069] Synthesis of Compound 10: Compound 9 (660 mg, 2.57 mmol) and the solvent tetrahydrofuran (20 mL) were successively added to a round-bottom reaction flask, and the reaction flask was placed in a low-temperature reaction kettle with the temperature set at -78°C. After 30 minutes, n-butyllithium (1.24 mL, 3.09 mmol) was added. After 2 hours, tributyltin chloride (0.84 mL, 3.09 mmol) was added. After 12 hours, ethyl acetate was added to the reaction solution to quench the reaction, and it was extracted three times with distilled water to obtain Compound 2. Without purification, it was directly fed for subsequent reactions.

[0070] Synthesis of Compound 12: Compound 10 (529.5 mg, 0.84 mmol), 2,5-dibromo-3,4-dinitrothiophene (Compound 11, 0.34 mmol, 111.65 mg), the catalyst tetrakis(triphenylphosphine)palladium (0.04 mmol, 38.87 mg), and toluene (80 mL) were successively added to a round-bottom reaction flask. The inside of the reaction flask was under an argon atmosphere, and the temperature was 110°C. After 18 h, the reaction ended. The reaction temperature was cooled to room temperature, ethyl acetate was added to terminate the reaction, it was extracted three times with distilled water, once with saturated brine, dried over anhydrous magnesium sulfate, and separated by silica gel column chromatography to obtain Compound 4 (205.93 mg, yield 72%).

[0071] Synthesis of Compound 13: Zinc powder (8.3 g, 69.23 mmol), Compound 12 (970.7 mg, 0.58 mmol), methanol (40 mL), dichloromethane (40 mL), and inorganic salt ammonium chloride (1.11 g, 20.77 mmol) were added to a round-bottom reaction flask. The reaction was carried out at 25 °C for 2 h. After diluting the reaction solution with dichloromethane, it was extracted three times with saturated sodium chloride. It was dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain an intermediate product. The intermediate product, N-sulfinylaniline (494 mg, 3.56 mmol), trimethylchlorosilane (4.74 mmol, 514 mg), and ultradry pyridine (4 mL) were successively added to the reaction flask. The reaction was carried out at 25 °C for 6 h. After completion, the reaction solution was diluted with dichloromethane and extracted three times with 1 M hydrochloric acid solution, dried over anhydrous magnesium sulfate, concentrated to obtain a crude product, and purified Compound 13 (505.45 mg, yield 52.8%) was obtained by column chromatography separation.

[0072] Synthesis of Reactant 14: Compound 13 (1.86 g, 2.19 mmol), N,N-dimethylformamide (10 mL), acetonitrile (5 mL), and N-bromosuccinimide (0.81 g, 4.83 mmol) were successively added to the flask. The reaction was carried out at 65 °C for 12 h. After the reaction solution temperature was restored to room temperature, the reaction solution was diluted with dichloromethane and extracted three times with saturated sodium bicarbonate solution, dried over anhydrous magnesium sulfate to obtain Compound 14, and this compound was directly fed into the next step of the reaction.

[0073] Synthesis of Compound 16: Compound 14 (1.14 g, 2.1 mmol), catalyst tetrakis(triphenylphosphine)palladium (97.15 mg, 0.08 mmol), tetrahydrofuran (50 mL), 15% sodium bicarbonate solution (10 mL), and Compound 15 (848.3 mg, 0.84 mmol) were successively added to the reaction flask. The reaction flask was equipped with a reflux condenser, and a nitrogen environment was maintained in the reaction flask. The reaction temperature was 80 °C. The reaction was ended after 18 h. The reaction solution temperature was cooled to room temperature, the reaction solution was diluted with dichloromethane, and extracted three times with saturated sodium chloride to obtain a crude product. Compound 7 (777.29 mg, yield 55%) was obtained by column chromatography separation.

[0074] Figure 1 of 1 The 1H NMR spectrum is completely consistent with the molecular structure of HS-1, verifying the precise modification of its symmetric conjugate skeleton and hydrophilic substituents.

[0075] Example 2. Synthesis of Compound HS-2

[0076]

[0077] Synthesis of Compound 18: Compound 17 (660 mg, 2.57 mmol) and the solvent tetrahydrofuran (20 mL) were successively added to a round-bottom reaction flask, which was placed in a low-temperature reaction kettle with the temperature set at -78 °C. After 30 minutes, n-butyllithium (1.24 mL, 3.09 mmol) was added. After 2 hours, tributyltin chloride (0.84 mL, 3.09 mmol) was added. After 12 hours, ethyl acetate was added to the reaction solution to quench the reaction, and it was extracted three times with distilled water to obtain Compound 2. Without purification, it was directly used for the subsequent reaction without further purification.

[0078] Synthesis of Compound 20: Compound 18 (529.5 mg, 0.84 mmol), 2,5-dibromo-3,4-dinitrothiophene (Compound 11, 0.34 mmol, 111.65 mg), the catalyst tetrakis(triphenylphosphine)palladium (0.04 mmol, 38.87 mg), and toluene (80 mL) were successively added to a round-bottom reaction flask. The inside of the reaction flask was under an argon atmosphere, and the temperature was 110 °C. After 18 h, the reaction ended. The reaction temperature was cooled to room temperature, and ethyl acetate was added to terminate the reaction. It was extracted three times with distilled water and once with saturated brine, dried over anhydrous magnesium sulfate, and separated by silica gel column chromatography to obtain Compound 4 (205.93 mg, yield 72%).

[0079] Synthesis of Compound 21: Zinc powder (8.3 g, 69.23 mmol), Compound 20 (970.7 mg, 0.58 mmol), methanol (40 mL), dichloromethane (40 mL), and the inorganic salt ammonium chloride (1.11 g, 20.77 mmol) were added to a round-bottom reaction flask. The reaction was carried out at 25 °C for 2 h. After diluting the reaction solution with dichloromethane, it was extracted three times with saturated sodium chloride. It was dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain an intermediate product. The intermediate product, N-phenylsulfinylbenzenamine (494 mg, 3.56 mmol), trimethylchlorosilane (4.74 mmol, 514 mg), and ultra-dry pyridine (4 mL) were successively added to the reaction flask. The reaction was carried out at 25 °C for 6 h. After completion, the reaction solution was diluted with dichloromethane, extracted three times with 1 M hydrochloric acid solution, dried over anhydrous magnesium sulfate, concentrated to obtain a crude product, and separated by column chromatography to obtain pure Compound 13 (505.45 mg, yield 52.8%).

[0080] Synthesis of Reactant Compound 22: Compound 21 (1.86 g, 2.19 mmol), N,N-dimethylformamide (10 mL), acetonitrile (5 mL), and N-bromosuccinimide (0.81 g, 4.83 mmol) were successively added to a flask. The reaction was carried out at 65 °C for 12 h. After the reaction solution temperature returned to room temperature, the reaction solution was diluted with dichloromethane, extracted three times with saturated sodium bicarbonate solution, and dried over anhydrous magnesium sulfate to obtain Compound 14, which was directly used for the next reaction.

[0081] Synthesis of Compound 24: Compound 22 (1.14 g, 2.1 mmol), the catalyst tetrakis(triphenylphosphine)palladium (97.15 mg, 0.08 mmol), tetrahydrofuran (50 mL), 15% sodium bicarbonate solution (10 mL), and Compound 15 (848.3 mg, 0.84 mmol) were successively added to a reaction flask. The reaction flask was equipped with a condenser reflux device, and a nitrogen environment was maintained in the reaction flask. The reaction temperature was 80 °C. The reaction was terminated after 18 h. The temperature of the reaction solution was cooled to room temperature, the reaction solution was diluted with dichloromethane, and extracted three times with saturated sodium chloride to obtain a crude product. Compound 7 (777.29 mg, yield 55%) was obtained by column chromatography separation.

[0082] Figure 1 of 1 The 1H NMR spectrum is completely consistent with the molecular structure of HS-2, verifying the precise modification of its symmetric conjugated skeleton and hydrophilic substituents. The vascular imaging effect of HS-2 is the same as that of HS-1 in Example 1.

[0083] Example 3. Determination of Absorption Spectrum and Emission Spectrum

[0084] HS-1 prepared in Example 1 was dissolved in water, and its absorption and emission spectra were measured using a UV-Vis-NIR spectrophotometer and a fluorescence spectrometer, respectively. The results are as Figure 2 shown.

[0085] Figure 2 The absorption spectrum of a shows that the maximum absorption wavelength is about ~750 nm, and the emission wavelength is between 900 - 1500 nm, indicating that the compound has a wide excitation window and is compatible with a variety of NIR-II excitation light sources.

[0086] Figure 2 The fluorescence spectrum of b shows: a strong fluorescence emission peak appears at 1000 nm, and the emission range extends to 1500 nm, meeting the requirements of NIR-II imaging.

[0087] Quantum yield (QY): The QY in aqueous solution was measured to be 1.2% (>0.5%) by the reference method, which is significantly better than traditional NIR-II dyes (such as ICG QY < 0.1%).

[0088] In summary, HS-1 has excellent NIR-II optical properties and meets the requirements of high-resolution in vivo imaging.

[0089] Example 4. Determination of Stability

[0090] Stability comparison between HS-1 and ICG: The fluorescence intensity retention rates of the two compounds were compared under continuous light irradiation in PBS. The results are as Figure 3 shown.

[0091] ICG group: Under the irradiation of 808 nm laser (200 mW / cm 2 ), the fluorescence retention rate was higher than 95% within 30 min, the ICG retention rate was less than 20%, and the fluorescence intensity decayed by more than 80% within 30 min, indicating that ICG was prone to photobleaching.

[0092] HS-1 group: The fluorescence intensity remained > 95% under the same conditions, proving that its molecular structure had strong resistance to photo-oxidative degradation.

[0093] In summary, it can be seen that the photostability of HS-1 is significantly better than that of the clinically commonly used dye ICG and is suitable for long-term dynamic imaging.

[0094] Example 4. HS-1 in vivo metabolism and bladder imaging

[0095] HS-1 was injected into nude mice via the tail vein at a dose of 200 μL and a concentration of 1 mg / mL. Imaging acquisitions were performed at the following time points after administration: 0 min, 3 min, 30 min, 1 h, 3 h, 6 h, and 12 h after injection. The mice were placed under anesthesia and placed on the imaging platform with their abdomens facing up. Fluorescence signal images were acquired using 808 nm laser excitation, and the results are as Figure 4 shown.

[0096] It can be seen from Figure 4 that:

[0097] t = 3 min: The signal first appeared in the bladder area, indicating rapid renal filtration.

[0098] t = 30 min: The fluorescence intensity reached the peak, and the half-life (t1 / 2) was 25 min.

[0099] t = 12 h: The signal completely disappeared, and the metabolic clearance rate was > 95%.

[0100] Metabolic pathway: Excreted through urine, without enterohepatic circulation accumulation.

[0101] In summary, it can be seen that HS-1 has the characteristics of rapid metabolism, high biosafety, and is suitable for dynamic monitoring of renal function.

[0102] Example 6. Method applied to kidney imaging

[0103] HS-1 was injected into nude mice via the tail vein at a dose of 200 μL and a concentration of 1 mg / mL. 30 minutes after injection, the mice were placed under anesthesia and placed on the imaging platform with their abdomens facing up. Fluorescence signal images were acquired using 808 nm laser excitation.

[0104] Kidney-specific imaging is as Figure 5 shown:

[0105] Imaging effect: 30 minutes after injection, a high-intensity signal (signal-to-noise ratio > 20) appears in the bilateral kidney regions with clear boundaries. The molecular weight (1312) and hydrophilic group (sulfonic acid group) of HS-1 promote glomerular filtration and avoid binding to plasma proteins.

[0106] In summary, HS-1 is an efficient kidney-specific probe suitable for the evaluation of acute kidney injury and the study of drug nephrotoxicity.

[0107] Example 7: Method for vascular imaging

[0108] Inject HS-1 into nude mice via the tail vein at a dose of 200 μL and a concentration of 1 mg / mL. Immediately after injection, place the mice under anesthesia, place them abdomen-up on the imaging platform, and use 808 nm laser excitation to collect fluorescence signal images. The results are as Figure 6 shown.

[0109] Vascular imaging: Visualization occurs immediately after injection, and microvessels with a diameter < 0.5 mm (such as mesenteric blood vessels) can be distinguished, with a penetration depth > 5 mm.

[0110] Tumor targeting: The signal intensity in the tumor region increases by 15 times within 2 hours (vs. surrounding tissues), indicating significant passive targeting (EPR effect).

[0111] Intraoperative navigation: Real-time display of the tumor boundary to guide precise resection.

[0112] Conclusion: HS-1 has important application potential in the integration of vascular imaging and tumor diagnosis and treatment.

[0113] Example 8: Biodistribution and safety evaluation

[0114] 1. Hemolysis experiment: Incubate HS-1 solutions at different concentrations (25, 50, 75, 100 μg / mL) with mouse red blood cells, measure the absorbance at 540 nm of the supernatant, and calculate the hemolysis rate.

[0115] The results are as Figure 7 shown. The hemolysis rate is less than 5% at all concentrations, showing no significant difference from the negative control (PBS), indicating that HS-1 has no destructive effect on red blood cells and has good blood compatibility.

[0116] 2. HE staining for tissue toxicity analysis: Collect the main organs of mice on the 7th day after drug administration, and observe tissue sections after HE staining.

[0117] The results are as Figure 8 shown, showing that the organizational structures of organs such as the heart, liver, spleen, lungs, and kidneys are intact, without pathological changes such as inflammation, bleeding, or necrosis, proving that HS-1 does not cause tissue toxicity in vivo and has good biosafety.

[0118] 3. Blood biochemistry and blood routine tests: Mouse blood samples were collected on the 1st and 7th days after administration to detect liver and kidney function (ALT, AST, BUN, CRE) and blood routine (WBC, RBC, HGB, etc.) indicators.

[0119] The results are as Figure 9 shown, indicating that all values are within the normal physiological range, and there is no significant difference between the experimental group and the control group, suggesting that HS-1 has no obvious blood toxicity.

[0120] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Obviously, those skilled in the art can make various changes and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A water-soluble small molecule fluorescent compound in the second near-infrared region, characterized in that, The compound has the following general structural formula (I): wherein X and Y are each independently selected from one of O, S, and Se R1 and R2 are each independently selected from H and one of the following 6 groups, wherein in the structural formulas of the 6 groups, n is an integer taken from 0 - 20; R3, R4, R5, R6, and R7 are each independently selected from H and one of the following 34 groups, wherein in the structural formulas of the 34 groups, m is an integer taken from 0 - 20.

2. The compound according to claim 1, wherein The fluorescence emission wavelength of the water-soluble small molecule fluorescent compound in the second near-infrared region is 900 - 1500 nm.

3. The compound according to claim 1, characterized in that, The quantum yield of the water-soluble small molecule fluorescent compound in the second near-infrared region in aqueous solution is greater than 0.5%.

4. The compound according to claim 1, wherein The water-soluble small molecule fluorescent compound in the second near-infrared region includes one of the following compounds:

5. A method for preparing the compound according to any one of claims 1-4, characterized in that, The method includes: Reacting compound 1 with n-butyllithium at a temperature of -70°C to -80°C, and then adding tributyltin chloride and reacting for 10 - 15 hours to obtain compound 2; Reacting compound 2 with compound 3 in toluene under palladium catalysis to obtain compound 4; Reducing compound 4 with zinc powder and then adding N-sulfinylaniline, trimethylchlorosilane, and pyridine to react to obtain compound 5; Reacting compound 5 with N-bromosuccinimide in N,N-dimethylformamide and acetonitrile to obtain compound 6; Reacting compound 6 with compound 7 under palladium catalysis in THF and an aqueous solution of NaHCO3 to obtain the water-soluble small molecule fluorescent compound in the second near-infrared region.

6. The preparation method according to claim 5, wherein The molar ratio of compound 1, n-butyllithium, and tributyltin chloride is 1:(1 - 2):(1 - 2).

7. The preparation method according to claim 5, characterized in that, The molar ratio of compound 2, compound 3, and tetrakis(triphenylphosphine)palladium is 1:(0.2 - 0.6):(0.02 - 0.06).

8. The preparation method according to claim 5, characterized in that, The molar ratio of compound 4, zinc powder, and inorganic salt is 1:(50 - 150):(30 - 50); the molar ratio of compound 5 and N-bromosuccinimide is 1:(1 - 5); the molar ratio of compound 5, 6, and tetrakis(triphenylphosphine)palladium is 1:(2 - 3):(0.01 - 0.5).

9. Use of the water-soluble small molecule fluorescent compound in the second near-infrared region according to any one of claims 1 - 3 in the preparation of an in-vivo imaging reagent.

10. The application according to claim 9, characterized in that, The in-vivo imaging includes one of kidney imaging, bladder and urinary system metabolism imaging, whole body vascular fluorescence imaging, lymphatic system imaging, tumor tracing, and intraoperative navigation.

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