A near-infrared fluorescent probe for detecting bone regeneration vitality and its preparation method

Abstract

This invention discloses a near-infrared fluorescent probe for detecting bone regeneration vitality and its preparation method, belonging to the field of biochemistry. Using rhodamine as the fluorescent core, the invention utilizes quinoline ring expansion to increase the conjugated area and extend emission. The brightness and stability of the probe are controlled by adjusting the steric hindrance of the aromatic ring at position 9, constructing a series of stable, high-brightness probe cores with low pKa. By introducing a phosphate ester group as a responder while simultaneously quenching the fluorescence of the core, a near-infrared fluorescent probe, SiRd-MOP, for detecting bone regeneration vitality is obtained. The preparation method of this invention has high yield and mild reaction conditions. The prepared probe can specifically and sensitively respond to alkaline phosphatase (ALP), and has successfully achieved ALP activity monitoring in osteogenic differentiation models and throughout the entire process of fracture regeneration and repair in mice. It can be used for real-time assessment of bone regeneration vitality and has very high biological application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemical technology, and particularly relates to a near-infrared fluorescent probe for detecting bone regeneration activity and a preparation method thereof. BACKGROUND

[0002] Bone tissue regeneration and repair is a complex and orderly process, which can be generally divided into four stages: initial inflammation, cartilage callus formation, hard callus formation and bone remodeling. Timely and accurate assessment of bone repair status is crucial for judging the healing process, predicting complication risks and optimizing treatment plans. During bone repair, active osteoblasts secrete a tissue non-specific alkaline phosphatase (ALP), which removes its inhibition of mineralization by hydrolyzing pyrophosphate, and the inorganic phosphate produced thereby provides raw materials for mineralization, thereby driving the matrix mineralization process. ALP is a key biomarker reflecting early osteogenic activity and bone matrix mineralization process. Therefore, real-time, dynamic and non-invasive monitoring of ALP activity changes at the injury site can provide direct and key biological information for bone repair progress and real-time assessment of bone regeneration activity.

[0003] Currently, common ALP detection methods include serum biochemical analysis and immunohistochemical staining. However, serum ALP only reflects the total amount of ALP in the whole body, cannot locate the fracture site and lacks dynamic information. Immunohistochemical staining completely relies on ex vivo samples and cannot perform in vivo in situ detection of enzyme activity.

[0004] Fluorescence imaging technology provides a powerful tool for in vivo biomarker monitoring due to its high sensitivity, high spatiotemporal resolution and non-invasiveness. Among them, near-infrared window fluorescent probes exhibit significant advantages in deep tissue in vivo imaging due to their deep tissue penetration ability and low background autofluorescence. In recent years, a variety of ALP fluorescent probes have been reported. However, due to the large pH change range during bone regeneration, traditional ALP probes usually only respond to ALP in the alkaline range, making it difficult to capture the osteogenic signal in the early acidic microenvironment. In addition, active oxygen produced during the inflammation stage also hinders the continuous monitoring of ALP activity during the whole bone regeneration process. Therefore, developing a new type of near-infrared ALP response probe with wide-range pH applicability and anti-active oxygen interference for bone regeneration activity detection is of great significance for clinical bone repair and regeneration research. SUMMARY

[0005] To solve the above technical problems, the purpose of the present application is to provide a near-infrared fluorescent probe for detecting bone regeneration activity and a preparation method thereof, which effectively solves the problems of low pKa, narrow pH response range, poor chemical stability and difficulty in achieving bone regeneration repair activity detection of existing ALP probes.

[0006] The technical scheme for solving the above technical problems of the present application is as follows: a near-infrared fluorescent probe for detecting bone regenerative activity is provided, and the structural formula of the probe is:

[0007] ;

[0008] wherein R1 and R2 are H, CH3, OCH3 or OPh respectively.

[0009] Further, the structural formula of the near-infrared fluorescent probe is .

[0010] The present application also provides a preparation method of the above-mentioned near-infrared fluorescent probe for detecting bone regenerative activity, comprising the following steps:

[0011] (1) dissolving m-bromoaniline in a first organic solvent, adding iodine and hydrochloric acid as catalyst, and heating and stirring to obtain a first intermediate;

[0012] (2) dissolving the first intermediate in a second organic solvent containing a weak base, adding iodomethane, and heating and stirring to obtain a second intermediate;

[0013] (3) dissolving 2-bromo-4-methoxybenzoic acid in the second organic solvent, adding dichlorosulfoxide, and stirring to react to obtain a third intermediate;

[0014] (4) adding the second intermediate and anhydrous aluminum chloride into the second organic solvent, adding the third intermediate, and heating and stirring to react to obtain a fourth intermediate;

[0015] (5) dissolving the fourth intermediate in the second organic solvent, adding sodium borohydride, and stirring to react to obtain a fifth intermediate;

[0016] (6) dissolving the fifth intermediate in the second organic solvent, adding trifluoroacetic acid and triethylsilane, and stirring to react to obtain a sixth intermediate;

[0017] (7) dissolving the sixth intermediate in the second organic solvent, adding a strong base under stirring, and then adding dimethyldichlorosilane under stirring to react to obtain a seventh intermediate;

[0018] (8) dissolving the seventh intermediate in the first organic solvent, adding potassium permanganate under stirring to react to obtain an eighth intermediate;

[0019] (9) dissolving the eighth intermediate in the second organic solvent, adding boron tribromide under stirring to react to obtain a ninth intermediate;

[0020] (10) dissolving the ninth intermediate, imidazole and tert-butyl dimethylchlorosilane in the second organic solvent, and stirring to react to obtain a tenth intermediate;

[0021] (11) Dissolve the bromobenzene ring compound in a second organic solvent, add a strong base under stirring, then add the tenth intermediate dissolved in the second organic solvent, stir the reaction to obtain the precursor compound SiRd;

[0022] Among them, the structural formula of the brominated cyclic compound is R1 and R2 are H, CH3, OCH3, or OPh, respectively;

[0023] (12) Dissolve the precursor compound in a second organic solvent, add a weak base and phosphorus oxychloride to react, add water to hydrolyze, and obtain a near-infrared fluorescent probe for detecting bone regeneration vitality.

[0024] Furthermore, in step (1), the mass-to-volume ratio of m-bromoaniline to the first organic solvent is 90-110 g: 250 mL.

[0025] Furthermore, in step (1), the mass-to-volume ratio of m-bromoaniline to the first organic solvent is 100 g: 250 mL.

[0026] Furthermore, in step (2), the molar volume ratio of the first intermediate, the weak base, the second organic solvent, and iodomethane is 0.1-0.2 mol: 0.2-0.3 mol: 240-260 mL: 17-18 mL.

[0027] Furthermore, in step (2), the molar volume ratio of the first intermediate, the weak base, the second organic solvent, and iodomethane is 0.14 mol: 0.21 mol: 250 mL: 17.44 mL.

[0028] Furthermore, in step (3), the mass-to-volume ratio of 2-bromo-4-methoxybenzoic acid, the second organic solvent, and thionyl chloride is 7-9 g: 30 mL: 10 mL.

[0029] Furthermore, in step (3), the mass-volume ratio of 2-bromo-4-methoxybenzoic acid, the second organic solvent, and thionyl chloride is 8 g: 30 mL: 10 mL.

[0030] Furthermore, in step (4), the mass-volume ratio of the second intermediate, anhydrous aluminum chloride, the third intermediate, and the second organic solvent is 7-9 g: 4-5 g: 8-9 g: 40 mL.

[0031] Furthermore, in step (4), the mass-volume ratio of the second intermediate, anhydrous aluminum chloride, the third intermediate, and the second organic solvent is 8 g: 4.03 g: 8.24 g: 40 mL.

[0032] Furthermore, in step (5), the mass-volume ratio of the fourth intermediate, sodium borohydride, and the second organic solvent is 1-2 g: 500 mg: 50 mL.

[0033] Furthermore, in step (5), the mass-volume ratio of the fourth intermediate, sodium borohydride, and the second organic solvent is 1 g: 500 mg: 50 mL.

[0034] Furthermore, in step (6), the mass-to-volume ratio of the fifth intermediate, the second organic solvent, trifluoroacetic acid, and triethylsilane is 1-2 g: 15 mL: 0.2 mL: 0.2 mL.

[0035] Furthermore, in step (6), the mass-to-volume ratio of the fifth intermediate, the second organic solvent, trifluoroacetic acid, and triethylsilane is 1 g: 15 mL: 0.2 mL: 0.2 mL.

[0036] Furthermore, in step (7), the mass-to-volume ratio of the sixth intermediate, the second organic solvent, the strong base, and dimethyldichlorosilane is 3.7-3.9 g: 40 mL: 6-7 mL: 1-2 mL.

[0037] Furthermore, in step (7), the mass-to-volume ratio of the sixth intermediate, the second organic solvent, the strong base, and dimethyldichlorosilane is 3.8 g: 40 mL: 6.88 mL: 1.25 mL.

[0038] Furthermore, in step (8), the mass-to-volume ratio of the seventh intermediate, potassium permanganate, and the first organic solvent is 6-8 g: 4-6 g: 100 mL.

[0039] Furthermore, in step (8), the mass-volume ratio of the seventh intermediate, potassium permanganate, and the first organic solvent is 7 g: 5 g: 100 mL.

[0040] Furthermore, in step (9), the mass-to-volume ratio of the eighth intermediate, the second organic solvent, and boron tribromide is 500 mg: 10 mL: 1.26 mL.

[0041] Furthermore, in step (10), the equivalent ratio of the ninth intermediate, imidazole and tert-butyldimethylchlorosilane is 1:1.1-1.3:1.1-1.3.

[0042] Furthermore, in step (10), the equivalent ratio of the ninth intermediate, imidazole and tert-butyldimethylchlorosilane is 1:1.2:1.2.

[0043] Furthermore, in step (11), the ratio of the bromobenzene ring compound, the tenth intermediate, the second organic solvent, and the strong base is 10 eq: 50 mg: 10 mL: 0.8 mL. Here, the second organic solvent refers to the amount used to dissolve the bromobenzene ring compound.

[0044] Further, in step (11), the tenth intermediate dissolved in the second organic solvent has a mass-volume ratio of 50 mg: 1 mL.

[0045] Furthermore, in step (11), the precursor compound has the following structural formula: ; where R1 and R2 are H, CH3, OCH3 or OPh, respectively.

[0046] Furthermore, in step (12), the mass-to-volume ratio of the precursor compound, the second organic solvent, the weak base, and trichloride is 10 mg: 5 mL: 0.2 mL: 0.2 mL.

[0047] Furthermore, in steps (1) and (8), the first organic solvent is acetone.

[0048] Furthermore, in steps (2)-(7) and (9)-(11), the second organic solvent is at least one of dichloromethane, acetonitrile, chloroform, methanol, THF and DMF.

[0049] Furthermore, in steps (7) and (11), the strong base is at least one of n-butyllithium and sec-butyllithium.

[0050] Furthermore, in steps (2) and (12), the weak base is at least one of pyridine, triethylamine, 4-dimethylaminopyridine, potassium carbonate, and cesium carbonate.

[0051] The present invention also provides the application of the above-mentioned near-infrared fluorescent probe for detecting bone regeneration activity in detecting alkaline phosphatase activity during osteogenic differentiation and bone regeneration.

[0052] Furthermore, the activity of endogenous alkaline phosphatase is detected using a near-infrared fluorescent probe for detecting bone regeneration vitality. This detection is not limited to a specific type of cell or animal, but is applicable to various bone regeneration and repair models.

[0053] The present invention has the following beneficial effects:

[0054] 1. This invention, based on silazane fluorescent derivatives, combines aromatic rings with different steric hindrance modifications to improve the brightness and stability of the fluorophore. By introducing phosphate ester bonds as ALP response sites, a sensitive and specific ALP-responsive probe with a wide pH response range and resistance to reactive oxygen species interference was designed and synthesized. This probe can be used to monitor ALP activity in osteogenic differentiation models and in vivo bone regeneration models. The fluorescent probe of this invention has a large Stokes shift (>50 nm), which can effectively avoid background light interference. Furthermore, it has a near-infrared emission wavelength (>650 nm), which can effectively reduce the interference of biological autofluorescence and improve the imaging signal-to-noise ratio. The fluorescent probe of this invention has high sensitivity and high selectivity for ALP, and can detect ALP activity in osteogenic differentiation models and throughout the entire process of mouse bone regeneration.

[0055] 2. The fluorescent probe of this invention uses rhodamine as the fluorescent core and utilizes quinoline ring expansion to increase the conjugated area and prolong emission. The brightness and stability of the probe are controlled by adjusting the steric hindrance of the aromatic ring at position 9, thus constructing a series of stable, high-brightness probe cores with low pKa. By introducing a phosphate ester group as a responding group while simultaneously quenching the fluorescence of the core, a near-infrared fluorescent probe, SiRd-MOP, for detecting bone regeneration activity is obtained. The preparation method of this invention has high yield and mild reaction conditions. The prepared probe can specifically and sensitively respond to alkaline phosphatase (ALP), and has successfully achieved ALP activity monitoring in osteogenic differentiation models and throughout the entire process of fracture regeneration and repair in mice. It can be used for real-time assessment of bone regeneration activity and has very high biological application prospects. Attached Figure Description

[0056] Figure 1 This is a synthetic route diagram of the preparation method of the present invention;

[0057] Figure 2 The 1H NMR spectrum of the precursor compound SiRd in Example 1;

[0058] Figure 3 The carbon spectrum of the precursor compound SiRd in Example 1;

[0059] Figure 4 The 1H NMR spectrum of the precursor compound SiRd-Me in Example 2;

[0060] Figure 5 The carbon spectrum of the precursor compound SiRd-Me in Example 2;

[0061] Figure 6 The 1H NMR spectrum of the precursor compound SiRd-Mo in Example 3;

[0062] Figure 7 The carbon spectrum of the precursor compound SiRd-Mo in Example 3;

[0063] Figure 8 The 1H NMR spectrum of the near-infrared fluorescent probe SiRd-MOP obtained in Example 3;

[0064] Figure 9 The carbon spectrum of the near-infrared fluorescent probe SiRd-MOP obtained in Example 3;

[0065] Figure 10 The phosphorus spectrum of the near-infrared fluorescent probe SiRd-MOP obtained in Example 3;

[0066] Figure 11 The precursor compound SiRd-MO was prepared in different concentrations of Cys, GSH, NaHS, NaClO, H2O2, and ONOO. - Normalized fluorescence intensity plots at the maximum emission wavelength; (a) Cys, (b) GSH, (c) NaHS, (d) NaClO, (e) H2O2, (f) ONOO - ;

[0067] Figure 12 The UV absorption and fluorescence spectra of SiRd-MO and SiRd-MOP are shown; (a) is the UV absorption spectrum, and (b) is the fluorescence spectrum.

[0068] Figure 13 The results are shown in Figure 1. (a) is the UV absorption spectrum of SiRd-MO as a function of pH, and (b) is the fluorescence spectrum of SiRd-MO as a function of pH.

[0069] Figure 14 Normalized plot of fluorescence intensity at the maximum emission wavelength of SiRd-MO as a function of pH;

[0070] Figure 15 The UV absorption and fluorescence emission spectra of SiRd-MOP and ALP responses are shown.

[0071] Figure 16 The maximum fluorescence intensity of SiRd-MOP and ALP before and after response varies with pH in buffer solutions of different pH values;

[0072] Figure 17 The results of SiRd-MOP's determination of the limit of detection of ALP are shown in the figure; (a) is the relationship between fluorescence intensity at the maximum emission wavelength and ALP concentration, and (b) is the linear fitting of fluorescence intensity in the range of 0-3 U / L ALP.

[0073] Figure 18The graphs show the response spectra and fluorescence intensity of SiRd-MOP and ALP at different concentrations as a function of SiRd-MOP concentration; (a) shows the response spectra of SiRd-MOP and ALP at different concentrations; (b) shows the fluorescence intensity at the maximum emission wavelength as a function of SiRd-MOP concentration.

[0074] Figure 19 The graphs show the response kinetics and catalytic rate relationship between SiRd-MOP and ALP at different concentrations; (a) shows the response kinetics between SiRd-MOP and ALP at different concentrations, and (b) shows the relationship between SiRd-MOP concentration and catalytic rate.

[0075] Figure 20 Figure 1 shows the experimental results of the selectivity and anti-interference of the SiRd-MOP and ALP responses.

[0076] Figure 21 Figure showing the CCK-8 cytotoxicity assay results for SiRd-MO and SiRd-MOP;

[0077] Figure 22 The results of the assay for the detection ability of SiRd-MOP on ALP in osteogenic differentiated cells are shown in the figure; (a) is the confocal imaging results of MC3T3-E1 and SiRd-MOP after co-incubation at different osteogenic induction stages, and (b) is the statistical graph of intracellular SiRd-MOP fluorescence intensity at different induction times.

[0078] Figure 23 In vivo and in vitro imaging of mice injected with SiRd-MOP via tail vein at different time points after fracture, and bright field images of the femur.

[0079] Figure 24 The results of ALP in situ imaging experiments in the fracture model are shown in Figure 1; (a) is a statistical graph of femoral fluorescence intensity in in vivo imaging of mice, and (b) is a statistical graph of femoral fluorescence intensity in ex vivo imaging of mice.

[0080] Figure 25 Figure 1 shows the results of the frozen section imaging experiment of the femur; (a) shows the frozen section imaging of the mouse femur, (b) shows the statistical results of SiRd-MOP fluorescence intensity in the frozen section of the mouse femur, and (c) shows the immunofluorescence staining results of the frozen section of the mouse femur. Detailed Implementation

[0081] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0082] Example 1

[0083] A near-infrared fluorescent probe for detecting bone regeneration vitality is prepared by means of the following steps (synthetic route diagram shown in Figure 1):

[0084] (1) Dissolve 100 g of m-bromoaniline in 250 mL of acetone solution, add iodine and hydrochloric acid for catalysis, reflux at 80 °C overnight, remove solvent by evaporation after the reaction is complete, purify the black viscous substance by silica gel column chromatography, and precipitate after cooling to obtain the first intermediate (yield 87%). 1 H NMR (400 MHz, DMSO- d 6) δ 6.81 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 2.1Hz, 1H), 6.53 (dd, J = 8.0, 2.0 Hz, 1H), 6.12 – 6.08 (m, 1H), 5.27 (t, J = 1.7Hz, 1H), 1.84 (d, J = 1.4 Hz, 3H), 1.17 (s, 6H). 13 C NMR (101 MHz, DMSO- d 6) δ146.2, 129.1, 127.3, 125.3, 121.4, 119.6, 117.9, 114.4, 51.8, 31.5, 18.5.

[0085] Its synthetic route is as follows:

[0086] ;

[0087] (2) Dissolve 0.14 mol of the first intermediate and 0.21 mol of potassium carbonate in 250 mL of acetonitrile solution, add 17.44 mL of iodomethane, reflux overnight, and after the reaction is complete, filter with diatomaceous earth to remove potassium carbonate, collect the filtrate and remove the solvent by vortexing, purify the crude product by silica gel column chromatography, and precipitate after cooling to obtain the second intermediate (yield 98%). 1 H NMR (400 MHz, Chloroform- d ) δ 6.87 (d, J = 8.0 Hz, 1H), 6.75 (dd, J = 8.1, 1.9 Hz, 1H), 6.61(d, J = 1.9 Hz, 1H), 5.29 (q, J= 1.5 Hz, 1H), 2.77 (s, 3H), 1.95 (d, J = 1.4Hz, 3H), 1.30 (s, 6H). 13 C NMR (101 MHz, Chloroform- d ) δ 146.4, 130.1, 127.5,124.4, 122.5, 122.0, 118.7, 113.3, 77.4, 77.0, 76.7, 56.5, 30.7, 27.4, 18.5,18.5.

[0088] Its synthetic route is as follows:

[0089] ;

[0090] (3) Dissolve 8 g of 2-bromo-4-methoxybenzoic acid in 30 mL of chloroform solution, add 10 mL of thionyl chloride and reflux. After the solution is clear, remove the solvent to obtain the third intermediate.

[0091] Its synthetic route is as follows:

[0092] ;

[0093] (4) Add 8 g of the second intermediate and 4.03 g of anhydrous aluminum chloride to 40 mL of anhydrous DCM, mix and stir for 10 min, add 8.24 g of the third intermediate, heat to reflux overnight, after the reaction is complete, quench with water at 0 °C, then extract with dichloromethane, collect the organic phase and remove the solvent by vortexing, and purify the crude product by silica gel column chromatography to obtain the fourth intermediate (yield 68%). 1 H NMR (400 MHz, Chloroform- d ) δ 7.35 (d, J = 8.6 Hz, 1H), 7.21 (s, 1H), 7.14 (d, J = 2.5 Hz, 1H), 6.85 (dd, J = 8.6, 2.4 Hz, 1H), 6.64 (s, 1H), 5.28(t, J = 1.5 Hz, 1H), 3.84 (s, 3H), 2.85 (s, 3H), 1.88 (d, J = 1.4 Hz, 3H), 1.35 (s, 6H). 13 C NMR (101 MHz, Chloroform- d) δ 193.7, 161.4, 148.4, 133.7, 132.0,129.8, 127.6, 126.8, 125.3, 123.9, 122.1, 120.8, 118.80, 114.9, 112.9, 77.4,77.0, 76.7, 57.5, 55.6, 31.1, 28.5, 18.4.

[0094] Its synthetic route is as follows:

[0095] ;

[0096] (5) Dissolve 1 g of the fourth intermediate in 50 mL of methanol, stir in an ice bath, slowly add 500 mg of sodium borohydride in portions, stir overnight at room temperature, and when the reaction is complete, quench the system with water at 0 °C, extract with dichloromethane, collect the organic phase, dry the organic phase with anhydrous sodium sulfate and remove the solvent by rotary evaporation, and purify the crude product by silica gel column chromatography to obtain the fifth intermediate (yield 96%). 1 H NMR (400 MHz, DMSO- d 6) δ 7.18 (d, J = 8.6 Hz, 1H), 7.12 (d, J = 2.6 Hz, 1H), 6.92 (d, J = 7.9 Hz, 2H), 6.56 (s, 1H), 5.91 (d, J = 5.1 Hz, 1H), 5.69 (d, J =5.3 Hz, 1H), 5.37 (d, J = 1.5 Hz, 1H), 3.73 (s, 3H), 2.69 (s, 3H), 1.77 (d, J =1.4 Hz, 3H), 1.23 (d, J = 11.9 Hz, 6H). 13 C NMR (101 MHz, Chloroform- d ) δ159.3, 145.7, 133.9, 130.4, 129.2, 128.0, 127.6, 124.0, 123.6, 122.9, 122.4,118.0, 114.2, 113.3, 77.4, 77.0, 76.7, 73.6, 56.6, 55.5, 30.8, 27.8, 27.1,18.4.

[0097] Its synthetic route is as follows:

[0098] ;

[0099] (6) Dissolve 1 g of the fifth intermediate in 15 mL of anhydrous DCM, stir for 10 min in an ice bath, add 0.2 mL of trifluoroacetic acid and 0.2 mL of triethylsilane, stir overnight at room temperature, and after the reaction is complete, quench the system with NaHCO3 aqueous solution, extract with dichloromethane, collect the organic phase and remove the solvent by rotary evaporation, and purify the crude product by silica gel column chromatography to obtain the sixth intermediate (yield 76%). 1 HNMR (400 MHz, Chloroform- d ) δ 7.14 (d, J = 2.7 Hz, 1H), 6.87 (d, J = 8.5 Hz,1H), 6.77 – 6.73 (m, 2H), 6.71 (s, 1H), 5.29 (d, J = 1.5 Hz, 1H), 4.01 (s,2H), 3.77 (s, 3H), 2.78 (s, 3H), 1.86 (d, J = 1.5 Hz, 3H), 1.30 (s, 6H). 13 C NMR (101 MHz, Chloroform- d ) δ 158.4, 144.9, 131.9, 130.6, 130.6, 127.5, 125.9,125.5, 124.8, 124.8, 122.9, 117.8, 114.4, 113.4, 77.4, 77.1, 76.7, 56.5,55.5, 40.2, 30.5, 27.3, 18.4.

[0100] Its synthetic route is as follows:

[0101] ;

[0102] (7) Dissolve 3.8 g of the sixth intermediate in 40 mL of anhydrous THF, cool the system to -78 °C, slowly inject 6.88 mL of n-butyllithium, and after the mixture reacts at -78 °C for 1 h, add 1.25 mL of dimethyldichlorosilane, continue to react at -78 °C for 1-2 min, then raise to room temperature and stir overnight. After the reaction is complete, add water to quench the system, extract with dichloromethane, collect the organic phase and remove the solvent by spin rotation to obtain the seventh intermediate;

[0103] Its synthetic route is as follows:

[0104] ;

[0105] (8) Dissolve 7 g of the seventh intermediate in 100 mL of acetone, cool to 10 °C, slowly add 5 g of potassium permanganate in batches for oxidation, stir overnight at room temperature, filter after the reaction is complete, collect the filtrate, remove the solvent by vortex, and purify the crude product by silica gel column chromatography to obtain the eighth intermediate (yield 38%). 1 H NMR (400 MHz, Chloroform- d ) δ 8.46 (d, J = 8.8 Hz,1H), 8.16 (s, 1H), 7.08 (d, J = 2.7 Hz, 1H), 7.05 (dd, J = 8.8, 2.7 Hz, 1H), 6.57 (s, 1H), 5.33 (d, J = 1.5 Hz, 1H), 3.90 (s, 3H), 2.95 (s, 3H), 2.08 (d, J = 1.4 Hz, 3H), 1.38 (s, 6H), 0.46 (s, 6H). 13 C NMR (101 MHz, cdcl3) δ 185.3,161.5, 147.2, 141.0, 140.9, 134.6, 132.0, 130.0, 129.1, 127.8, 125.1, 123.4,117.7, 114.9, 112.4, 77.4, 77.0, 76.7, 57.3, 55.3, 31.1, 28.7, 18.7, -1.2.

[0106] Its synthetic route is as follows:

[0107] ;

[0108] (9) Dissolve 500 mg of the eighth intermediate in 10 mL of anhydrous DCM, add boron tribromide (1.26 mL) of anhydrous DCM solution dropwise under ice bath, monitor the end of the reaction by TLC, quench the mixture in water, extract with dichloromethane, collect the organic phase and remove the solvent by vortexing, and purify the crude product by silica gel column chromatography to obtain the ninth intermediate (yield 88%). 1 HNMR (400 MHz, DMSO- d 6) δ 10.20 (s, 1H), 8.13 (d, J= 8.7 Hz, 1H), 7.88 (s,1H), 7.03 (d, J = 2.6 Hz, 1H), 6.92 (dd, J = 8.7, 2.6 Hz, 1H), 6.67 (s, 1H), 5.46 (d, J = 1.5 Hz, 1H), 2.90 (s, 3H), 1.95 (d, J = 1.4 Hz, 3H), 1.33 (s, 6H), 0.40 (s, 6H). 13 C NMR (101 MHz, Chloroform- d ) δ 186.4, 159.7, 147.5, 141.9,141.8, 133.5, 132.4, 130.0, 128.6, 127.7, 125.3, 123.4, 119.4, 117.6, 112.6,77.4, 77.1, 76.8, 57.4, 31.1, 28.8, 18.6, -1.4.

[0109] Its synthetic route is as follows:

[0110] ;

[0111] (10) The ninth intermediate (1 eq), imidazole (1.2 eq) and tert-butyldimethylchlorosilane (1.2 eq) were dissolved in anhydrous DMF and stirred overnight at room temperature. After the reaction was completed, the system was diluted with EA, the organic phase was washed three times with water, dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The residue was purified by silica gel column chromatography to obtain the tenth intermediate (yield 89%). 1 H NMR (400MHz, Chloroform- d ) δ 8.38 (d, J = 8.6 Hz, 1H), 8.15 (s, 1H), 7.02 (d, J = 2.6Hz, 1H), 6.97 (dd, J = 8.7, 2.6 Hz, 1H), 6.56 (s, 1H), 5.33 (d, J = 1.5 Hz,1H), 2.95 (s, 3H), 2.07 (d, J = 1.4 Hz, 3H), 1.38 (s, 6H), 1.01 (s, 9H), 0.45 (s, 6H), 0.26 (s, 6H).13 C NMR (101 MHz, Chloroform- d ) δ 185.4, 158.2, 147.2,141.0, 135.0, 131.9, 130.0, 129.2, 127.9, 125.1, 123.6, 123.4, 121.5, 112.4,77.3, 77.0, 76.7, 57.3, 31.1, 28.7, 25.7, 18.7, 18.3, -1.3, -4.3.

[0112] Its synthetic route is as follows:

[0113] ;

[0114] (11) Bromobenzene (10 eq) was dissolved in 10 mL of anhydrous THF, cooled to -78 °C, and 0.8 mL of sec-butyllithium was added. After reacting at -78 °C for 1 h, the tenth intermediate (50 mg) dissolved in 1 mL of anhydrous THF was added. The mixture was heated to room temperature and stirred for 30 min. The system was quenched with 2 N HCl aqueous solution, extracted with dichloromethane, and the organic phase was collected and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography to obtain the precursor compound SiRd (yield 52%). The 1H NMR and 1C NMR spectra of the precursor compound SiRd are shown below. Figure 2 and Figure 3 As shown; 1 H NMR (400 MHz, Chloroform- d ) δ 7.46 (d, J = 7.0 Hz, 3H), 7.22 (d, J = 7.8Hz, 2H), 7.01 (d, J = 10.0 Hz, 1H), 6.86 (d, J = 2.2 Hz, 1H), 6.72 (s, 1H), 6.55 (s, 1H), 6.24 (dd, J = 9.9, 2.3 Hz, 1H), 5.20 (s, 1H), 2.98 (s, 3H), 1.50 (s, 3H), 1.36 (s, 6H), 0.47 (s, 6H). 13 C NMR (101 MHz, Chloroform- d) δ 186.2,183.7, 158.7, 146.8, 146.0, 142.2, 142.1, 140.4, 135.6, 129.9, 129.5, 129.3,128.8, 127.9, 127.7, 126.9, 126.7, 125.9, 122.0, 115.8, 77.3, 77.0, 76.7,57.9, 31.2, 28.9, 17.7, -0.9. HRMS (ESI) C 28 H 30 NOSi [M+H] + , calculated for 424.2097, found 424.2099.

[0115] Its synthetic route is as follows:

[0116] ;

[0117] (12) Dissolve 10 mg of the precursor compound in 5 mL of anhydrous DCM. Using a three-way valve with a nitrogen-filled balloon, connect a vacuum system to remove water vapor and oxygen from the bottle three times. In an ice bath, inject 0.2 mL of pyridine and 0.2 mL of phosphorus oxychloride into the system in sequence. After reacting for 4 h, add ice water to the system and hydrolyze overnight. Adjust the pH of the aqueous phase to above 10 with triethylamine. Then wash the aqueous phase with dichloromethane until the organic phase is almost colorless. Add hydrochloric acid to adjust the pH back to acidic. Extract with dichloromethane again and collect the organic phase. Wash the organic phase with 0.1 N HCl aqueous solution to remove the triethylamine salt. Dry the organic phase with anhydrous sodium sulfate and remove the solvent by vortexing to obtain a near-infrared fluorescent probe for detecting bone regeneration vitality.

[0118] Example 2

[0119] The difference between Example 2 and Example 1 is that in step (11), bromobenzene is replaced with 2,6-dimethylbromobenzene. The specific steps are as follows:

[0120] 2,6-Dimethylbromobenzene (10 eq) was dissolved in 10 mL of anhydrous THF, cooled to -78 °C, and 0.8 mL of sec-butyllithium was added. After reacting at -78 °C for 1 h, the tenth intermediate (50 mg) dissolved in 1 mL of anhydrous THF was added. The mixture was heated to room temperature and stirred for 30 min. The system was quenched with 2 N HCl aqueous solution, extracted with dichloromethane, and the organic phase was collected and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography to give the precursor compound SiRd-Me (yield 43%). The 1H and 1C NMR spectra of the precursor compound SiRd-Me are shown below. Figure 4 and Figure 5 As shown; 1H NMR (400 MHz, DMSO- d 6) δ 7.31 – 7.24 (m, 1H), 7.19 (d, J = 7.5Hz, 2H), 6.88 (s, 1H), 6.77 – 6.68 (m, 2H), 6.33 (s, 1H), 6.06 (dd, J = 9.9,2.3 Hz, 1H), 5.33 (s, 1H), 2.95 (s, 3H), 1.93 (s, 6H), 1.36 (s, 3H), 1.30 (s,6H), 0.44 (s, 6H). 13 C NMR (101 MHz, DMSO- d 6) δ 182.9, 157.1, 146.7, 146.5,142.0, 140.0, 139.4, 135.5, 135.4, 130.6, 128.3, 127.7, 126.7, 126.4, 125.7,125.3, 122.1, 116.7, 58.2, 40.6, 40.4, 40.2, 40.0, 39.7, 39.5, 39.3, 31.6,29.2, 19.7, 17.6, -0.9. HRMS (ESI) C 30 H 34 NOSi [M+H] + , calculated for 452.2410, found 452.2409.

[0121] Its synthetic route is as follows:

[0122] .

[0123] Example 3

[0124] The difference between Example 3 and Example 1 is that in step (11), bromobenzene is replaced with 2,6-dimethoxybromobenzene. The specific steps are as follows:

[0125] 2,6-Dimethoxybromobenzene (10 eq) was dissolved in 10 mL of anhydrous THF, cooled to -78 °C, and 0.8 mL of sec-butyllithium was added. After reacting at -78 °C for 1 h, the tenth intermediate (50 mg) dissolved in 1 mL of anhydrous THF was added. The mixture was heated to room temperature and stirred for 30 min. The system was quenched with 2 N HCl aqueous solution, extracted with dichloromethane, and the organic phase was collected and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography to give the precursor compound SiRd-Mo (yield 43%). The 1H and 1C NMR spectra of the precursor compound SiRd-Mo are shown below. Figure 6 and Figure 7 As shown; 1 H NMR (400 MHz, Chloroform- d ) δ 7.39 (t, J = 8.4 Hz, 1H), 7.07(d, J = 9.9 Hz, 1H), 6.83 (d, J = 2.2 Hz, 1H), 6.70 (s, 1H), 6.68 (s, 1H), 6.66(d, J = 1.6 Hz, 2H), 6.25 (dd, J = 9.8, 2.2 Hz, 1H), 5.19 (d, J = 1.5 Hz, 1H), 3.67 (s, 6H), 2.95 (s, 3H), 1.55 (d, J = 1.4 Hz, 3H), 1.35 (s, 6H), 0.46 (s, 6H). 13 C NMR (101 MHz, cdcl3) δ 184.3, 157.6, 153.6, 146.8, 145.9, 141.8,141.4, 135.1, 129.9, 129.3, 128.4, 127.7, 127.6, 127.0, 126.2, 122.2, 117.3,115.4, 103.8, 77.3, 77.0, 76.7, 57.7, 56.1, 31.1, 29.0, 17.9, -1.1. HRMS(ESI) C 30 H 34 NO3Si [M+H] + , calculated for 484.2308, found 484.2300.

[0126] Its synthetic route is as follows:

[0127] .

[0128] The precursor compound SiRd-MO was further synthesized into a near-infrared fluorescent probe, SiRd-MOP, for detecting bone regeneration activity (yield 74%). The 1H, 1C, and 1N NMR spectra of the near-infrared fluorescent probe SiRd-MOP are shown below. Figure 8 , Figure 9 and Figure 10 As shown; 1 H NMR (400 MHz, Methanol- d 4) δ 7.71 (d, J = 2.4 Hz, 1H), 7.57 (t, J = 8.4Hz, 1H), 7.49 (s, 1H), 7.24 – 7.15 (m, 2H), 7.03 (s, 1H), 6.87 (d, J = 8.5 Hz,2H), 5.76 (s, 1H), 3.67 (s, 6H), 3.58 (s, 3H), 1.63 (d, J = 1.3 Hz, 3H), 1.58(s, 6H), 0.60 (s, 6H). 13 C NMR (101 MHz, Methanol- d 4) δ 163.5, 157.4, 157.1,157.0, 155.9, 153.8, 142.2, 136.4, 134.7, 133.5, 132.1, 131.3, 130.9, 126.2,126.2, 125.1, 124.4, 123.2, 121.6, 121.5, 115.4, 103.7, 62.7, 55.1, 48.2,48.0, 47.8, 47.6, 47.4, 47.1, 46.9, 33.7, 27.0, 16.0, -3.2. 31 P NMR (162 MHz, Methanol-) d 4) δ -4.80. HRMS (ESI) C 30 H 35 NO6PSi + [M + ], calculated for 564.1966, found 564.1967.

[0129] Its synthetic route is as follows:

[0130] .

[0131] Chemical stability of SiRd-MO (Example 1)

[0132] The precursor compound SiRd-Mo from Example 3 was prepared into a 5 mM DMSO stock solution, diluted to 10 μM with PBS, and then different concentrations of active sulfur (Cys, GSH, NaHS) or active oxygen (NaClO, H2O2, ONOO) were added. - After mixing, its fluorescence emission spectrum was scanned, and the fluorescence intensity at the maximum emission wavelength was normalized and plotted; the chemical stability of SiRd-MO is as follows: Figure 11 As shown.

[0133] Depend on Figure 11 It can be seen that the fluorescence intensity of SiRd-MO is relatively stable at physiological levels of reactive oxygen species and reactive sulfur, remaining above 80%, indicating that the precursor compound SiRd-MO is not affected by reactive oxygen species and reactive sulfur in vivo, and is suitable for in vivo experiments.

[0134] Experimental Example 2: Optical Performance Testing of SiRd-MO and SiRd-MOP

[0135] The near-infrared fluorescent probe SiRd-MOP obtained in Example 3 for detecting bone regeneration vitality was prepared as a 5 mM DMSO stock solution. SiRd-MO and SiRd-MOP were diluted to 10 μM solutions with Tris-HCl (pH=8). The UV absorption and fluorescence spectra were scanned and plotted. The UV absorption and fluorescence spectra of SiRd-MO and SiRd-MOP are shown below. Figure 12 As shown.

[0136] Depend on Figure 12 It can be seen that SiRd-MO has two absorption peaks, one at λ=360 nm and the other near λ=652 nm; while SiRd-MOP has only one main absorption peak at 558 nm.

[0137] Experimental Example 3: pH Response of SiRd-MO

[0138] The pH of the Tris solution was adjusted with HCl to prepare Tris-HCl buffer solutions with different pH values ​​(pH range 2-10). The SiRd-MO fluorescent stock solution was diluted with Tris-HCl buffer solutions of different pH values, and its UV absorption and fluorescence spectra in these buffer solutions were scanned. The results are as follows: Figure 13 As shown, the normalized plot of fluorescence intensity at its maximum emission wavelength as a function of pH is shown below. Figure 14 As shown.

[0139] Depend on Figure 13 It can be seen that as pH increases, the maximum absorption wavelength of SiRd-MO gradually redshifts, and the fluorescence intensity increases significantly. Figure 14 It can be seen that the pH response range of SiRd-MO is 4-6, and its pKa is 4.78 obtained by fitting.

[0140] Experimental Example 4: In vitro response test of SiRd-MOP and ALP

[0141] The SiRd-MOP fluorescent stock solution was diluted to 10 μM with Tris-HCl buffer, 100 U / L ALP was added, and the mixture was incubated at 37℃ for 30 min. Its UV absorption and fluorescence spectra were then scanned and plotted. Figure 15 As shown.

[0142] Depend on Figure 15 It can be seen that after the addition of ALP, its maximum absorption peak red-shifted to 652 nm, which is consistent with SiRd-MO, indicating that SiRd-MOP was converted into SiRd-MO after enzymatic digestion, and the fluorescence intensity increased by 25 times after the response.

[0143] Experimental Example 5: Optimal pH Screening for SiRd-MOP and ALP Responses

[0144] The pH of the Tris solution was adjusted with HCl to prepare Tris-HCl buffer solutions with different pH values ​​(pH range 3-10). The SiRd-MOP fluorescent stock solution was diluted with Tris-HCl buffer solutions of different pH values, and the emission spectra before and after the addition of ALP were scanned. The results are as follows: Figure 16 As shown.

[0145] Depend on Figure 16 It can be seen that the probe can respond to ALP when the pH is greater than 5, and the enzyme activity is the highest at pH 8, with the highest fluorescence enhancement after the response.

[0146] Experimental Example 6: Determination of the Limit of Detection for ALP by SiRd-MOP

[0147] The SiRd-MOP fluorescent stock solution was diluted to 10 μM with Tris-HCl buffer, and different concentrations of ALP were added. After incubation at 37 ℃ for 30 minutes, the fluorescence spectra were scanned and plotted. The results are shown below. Figure 17 As shown.

[0148] Depend on Figure 17It was observed that the fluorescence intensity measured after the reaction gradually increased with increasing ALP concentration. When the enzyme concentration increased to 8 U / L and above, the fluorescence intensity tended to stabilize, indicating that 10 U / L alkaline phosphatase was sufficient to catalyze all substrates simultaneously within 30 minutes. When the enzyme concentration was below 3 U / L, the fluorescence intensity showed a linear change with increasing enzyme concentration, indicating that within the 3 U / L enzyme concentration range, the amount of substrate greatly exceeded the enzyme's catalytic capacity. The slope of the linear equation obtained by fitting the equation was 722.17141. Thirteen consecutive scans were performed on the blank sample without ALP, and the standard deviation σ of its fluorescence intensity was calculated to be 3.883968. According to the formula for calculating the limit of detection (LOD=3σ / k), the detection limit of SiRd-MOP for ALP was 0.016135 U / L, indicating that the probe has high sensitivity to the enzyme.

[0149] Experimental Example 7: Response Kinetics Test of SiRd-MOP to ALP

[0150] Different concentrations of SiRd-MOP were added to a Tris-HCl buffer solution containing ALP. After incubation at 37 °C for 30 minutes, their emission spectra were scanned, plotted, and linearly fitted. The results are shown in Figure 18. With increasing probe concentration, the fluorescence intensity gradually increased and showed a linear change. The linear equation obtained after linear fitting was y = 364.53887x + 201.96122. Subsequently, different concentrations of SiRd-MOP were added to a Tris-HCl buffer solution containing ALP, and the fluorescence intensity at 695 nm was recorded every 5 seconds. Figure 19 As shown. Substitute the data from the first 40 seconds of the reaction into the equation to calculate the enzyme catalytic rate. According to the Michaelis-Menten equation (V = (V...),... max ×[S]) / (K m +[S])) calculate the Michaelis constant (K m ) and the maximum reaction rate (V) of the enzyme-catalyzed reaction max The graph was then plotted. As the probe concentration increased, the reaction rate gradually increased, and the maximum catalytic rate (Vt) of ALP on the probe SiRd-MOP was calculated. ma x) is 8.27 μM / min, and the Michaelis constant (K) m The value was 6.97 μM, indicating that the enzyme had a high catalytic rate on the probe and a good affinity with it.

[0151] Experimental Example 8: Selectivity Test of SiRd-MOP Response to ALP

[0152] The SiRd-MOP fluorescent stock solution was diluted with Tris-HCl buffer solution, and 100 μM metal ions (Na+) were added separately. + K+ Mg 2+ Ca 2+ Cu 2+ Fe 2+ Fe 3+ 100 μM biothiols or amino acids (GSH, Lys, Cys, Tyr, Glu, Leu), 100 μM active species (HClO, H2O2, OH, 1O2), and 1 μg / mL enzymes (LAP, NQO1, NTR, MAO-A, MAO-B) were added and incubated at 37 ℃ for 30 minutes. After incubation, their fluorescence spectra were scanned. ALP was added, and the mixture was incubated again at 37 ℃ for 30 minutes, and the fluorescence spectra were scanned again. The results are as follows: Figure 20 As shown.

[0153] Depend on Figure 20 It can be seen that before the addition of ALP, SiRd-MOP did not respond to any of the analytes; after the addition of ALP, many analytes showed almost no interference with the response of SiRd-MOP and ALP, indicating that the probe has good response specificity and anti-interference ability.

[0154] Test Example 9: CCK-8 Cytotoxicity Assay

[0155] Taking MC3T3-E1 cells as an example, a toxicity test was performed on the probe. MC3T3-E1 cells in logarithmic growth phase were seeded in 96-well plates at 10,000 cells per well and cultured overnight at 37 °C with 5% CO2 in MEM Alpha medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (1000 kU / L). After complete cell adhesion, different concentration gradients of SiRd-MOP were added, with three replicates for each concentration, and a blank control was also included. After 24 hours of culturing following drug addition, the medium concentration on the SiRd-MOP side changed from blue to purple, indicating the in situ generation of SiRd-MO. Cell viability was monitored using the CCK-8 assay. Figure 21 As shown, SiRd-MO generated in situ from 10 μM SiRd-MOP exhibits less cytotoxicity to cells than direct incubation with 10 μM SiRd-MO. Furthermore, cell viability remained at approximately 90% after treatment with 10 μM SiRd-MOP, while cell viability remained at approximately 60% after treatment with 10 μM SiRd-MO. Overall, both molecules demonstrate good biocompatibility and are suitable for cell experiments.

[0156] Experimental Example 10: Detection ability of SiRd-MOP on ALP in osteoblasts

[0157] Taking MC3T3-E1 cells as an example, osteogenic differentiation was induced, and the detection ability of SiRd-MOP on ALP in osteogenic differentiated cells was tested. MC3T3-E1 cells that had grown to 80-90% confluence were digested from the culture dish to prepare a cell suspension. An appropriate amount of cell suspension was transferred to a confocal microscopy dish with a glass bottom, 1 mL of MEM Alpha complete medium was added, and the mixture was shaken well. The dish was then incubated in a humidified incubator at 37℃ and 5% CO2 for 24 h. When the cells were fully adhered and had grown to 60-70% confluence, the confocal microscopy dish was divided into an induction group and a control group. The old medium in the induction group was replaced with osteogenic differentiation induction medium, and the medium in the control group was replaced with MEM Alpha complete medium containing 5% FBS. On days 0, 3, 5, 7, 10, 14, and 21 of induced differentiation, the old culture medium was removed. Cells were gently rinsed with 1 mL of PBS buffer and then incubated at 37°C for 20 minutes in serum-free medium containing 2 μM SiRd-MOP, 1 μg / mL calcein, and 1 μg / mL nuclear dye (Heochst 33342). After washing away any dye that had not entered the cells, 1 mL of serum-free medium was added to maintain cell condition. Three-channel imaging was performed using a laser confocal microscope. Imaging conditions were: SiRd-MOP red channel: λ ex = 633 nm, λ em = 650 - 750 nm; Blue channel in the cell nucleus: λ ex = 405 nm, λ em = 430 -465 nm. The results are as follows: Figure 22 As shown, ALP secretion was fastest and its activity increased significantly around 5-7 days after induction of differentiation. After 7 days, the secretion rate gradually decreased, and the increase in fluorescence intensity was smaller, especially after 14 days, when the fluorescence intensity remained essentially unchanged. In contrast, the fluorescence intensity of the uninduced control group only increased slightly over time, but remained significantly lower than that of the induced group. On day 14, the fluorescence intensity of the control group increased somewhat, presumably because the prolonged culture time and high cell density caused partial differentiation, leading to the increased fluorescence intensity.

[0158] Experimental Example 11: In situ imaging of ALP in a fracture model

[0159] Using C57BL / 6 mice as an example, a fracture model was established to test the ability of SiRd-MOP to detect ALP activity during fracture repair and regeneration. Male C57BL / 6 mice aged 6-8 weeks underwent femoral fracture surgery. Hair was removed from the mice on days 3, 7, 14, 21, and 28 post-surgery to avoid imaging interference. Mice were anesthetized with 100 μL of SiRd-MOP (2 mg / mL) via tail vein injection. Small animal in vivo imaging (λ) was immediately initiated after isoflurane anesthesia using a small animal in vivo imaging system.ex = 640 nm, λ em = 700 nm). After the probe solution circulated in the mouse for about 15 minutes, the fluorescence signal at the fracture site reached its peak. The fluorescence signal at this point was recorded. Subsequently, the mouse was dissected, and the femur was removed for further in vitro imaging. The imaging results are as follows: Figure 23 As shown, the statistical results of in vivo and ex vivo imaging are as follows: Figure 24 As shown, the fluorescence intensity at the fracture site in mice with fracture modeling was significantly higher than that in the control group. Furthermore, as bone tissue repaired, the fluorescence intensity initially increased and then decreased, reaching its peak approximately 7-14 days post-surgery. The results of isolated femoral imaging were consistent with those of in vivo imaging. Bright-field images showed that after the fracture, the fracture ends were initially connected by connective tissue to form a callus. Subsequently, the callus gradually mineralized and hardened. After 28 days, femoral remodeling occurred, with the enlarged callus gradually shrinking and its morphology gradually returning to normal.

[0160] Experimental Example 12: Frozen Section Imaging of the Femur

[0161] The samples taken in Example 11, without decalcification, were directly embedded using OCT to prepare frozen sections. Laser confocal imaging was then performed directly on the sections under the following imaging conditions: λ ex = 639 nm, λ em = 650-757 nm. For example... Figure 25 As shown, the control group consisted of a normal-shaped femur with almost no ALP expression. On the third day after the fracture, the femur remained discontinuous, primarily due to inflammation, with low ALP activity and no initiation of bone repair. Between 7 and 14 days post-fracture, significant fluorescence signals appeared in the callus, indicating active bone repair with high ALP expression and activity. However, due to endochondral ossification, ALP expression was low in the chondrogenic portion of the callus. Between 21 and 28 days post-fracture, the fluorescence signal gradually weakened, the callus swelling gradually decreased, and the morphology gradually normalized, indicating the bone repair entering the remodeling stage. ALP activity gradually decreased, and osteogenic and osteoclast-remodeling processes reached equilibrium. Sp7 is a key marker for assessing early osteoblast lineage-specific differentiation and the initiation of bone formation. Immunofluorescence staining of the sp7 gene was performed on serial frozen sections. The expression location and intensity of Sp7 were consistent with ALP, further confirming that SiRd-MOP can be used for bone regeneration activity detection.

[0162] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A near-infrared fluorescent probe for detecting bone regeneration vitality, characterized in that, The structural formula is: ； Wherein, R1 and R2 are H, CH3, OCH3 or OPh respectively.

2. The method for preparing a near-infrared fluorescent probe for detecting bone remodeling activity according to claim 1, characterized in that, The method comprises the following steps: (1) m-bromoaniline is dissolved in a first organic solvent, and iodine and hydrochloric acid are added for catalysis, and heated and stirred to obtain a first intermediate; (2) the first intermediate is dissolved in a second organic solvent containing a weak base, and iodomethane is added, and heated and stirred to obtain a second intermediate; (3) 2-bromo-4-methoxybenzoic acid is dissolved in a second organic solvent, and thionyl chloride is added, and stirred to react to obtain a third intermediate; (4) the second intermediate and anhydrous aluminum chloride are added to the second organic solvent, and the third intermediate is added, and heated and stirred to react to obtain a fourth intermediate; (5) the fourth intermediate is dissolved in a second organic solvent, and sodium borohydride is added, and stirred to react to obtain a fifth intermediate; (6) the fifth intermediate is dissolved in a second organic solvent, and trifluoroacetic acid and triethylsilane are added, and stirred to react to obtain a sixth intermediate; (7) the sixth intermediate is dissolved in a second organic solvent, and a strong base is added under stirring, and then dimethyldichlorosilane is added and stirred to react to obtain a seventh intermediate; (8) the seventh intermediate is dissolved in a first organic solvent, and potassium permanganate is added under stirring to react to obtain an eighth intermediate; (9) the eighth intermediate is dissolved in a second organic solvent, and boron tribromide is added under stirring to react to obtain a ninth intermediate; (10) the ninth intermediate, imidazole and tert-butyl dimethyl chlorosilane are dissolved in a second organic solvent, and stirred to react to obtain a tenth intermediate; (11) a bromobenzene ring compound is dissolved in a second organic solvent, and a strong base is added under stirring, and then the tenth intermediate dissolved in a second organic solvent is added, and stirred to react to obtain a precursor compound; The bromo-substituted compound has the structural formula R1 and R2 are H, CH3, OCH3 or OPh, respectively. (12) the precursor compound is dissolved in a second organic solvent, and a weak base and phosphorus oxychloride are added to react, and hydrolyzed with water to obtain a near-infrared fluorescent probe for detecting bone regeneration activity.

3. The method for preparing a near-infrared fluorescent probe for detecting bone regenerative activity according to claim 2, wherein In step (1), the mass-volume ratio of m-bromoaniline and the first organic solvent is 90-110 g: 250 mL; In step (2), the amount ratio of the first intermediate, the weak base, the second organic solvent and iodomethane is 0.1-0.2 mol: 0.2-0.3 mol: 240-260 mL: 17-18 mL.

4. The method for preparing a near-infrared fluorescent probe for detecting bone regenerative activity according to claim 2, wherein In step (3), the mass-volume ratio of 2-bromo-4-methoxybenzoic acid, the second organic solvent and thionyl chloride is 7-9 g: 30 mL: 10 mL; In step (4), the mass-volume ratio of the second intermediate, anhydrous aluminum chloride, the third intermediate and the second organic solvent is 7-9 g: 4-5 g: 8-9 g: 40 mL.

5. The method for preparing a near-infrared fluorescent probe for detecting bone regenerative activity according to claim 2, wherein In step (5), the mass-volume ratio of the fourth intermediate, sodium borohydride and the second organic solvent is 1-2 g: 500 mg: 50 mL; In step (6), the mass-volume ratio of the fifth intermediate, the second organic solvent, trifluoroacetic acid and triethylsilane is 1-2 g: 15 mL: 0.2 mL: 0.2 mL.

6. The method for preparing a near-infrared fluorescent probe for detecting bone regenerative activity according to claim 2, wherein In step (7), the mass-volume ratio of the sixth intermediate, the second organic solvent, the strong base and dimethyldichlorosilane is 3.7-3.9 g: 40 mL: 6-7 mL: 1-2 mL; In step (8), the mass-volume ratio of the seventh intermediate, potassium permanganate and the first organic solvent is 6-8 g:4-6 g:100 mL.

7. The method for preparing the near-infrared fluorescent probe for detecting bone regeneration vitality as described in claim 2, characterized in that, In step (9), the mass-volume ratio of the eighth intermediate, the second organic solvent and boron tribromide is 500 mg:10 mL:1.26 mL; In step (10), the equivalent ratio of the ninth intermediate, imidazole and tert-butyldimethylsilyl chloride is 1:1.1-1.3:1.1-1.

3.

8. The method for preparing the near-infrared fluorescent probe for detecting bone regeneration vitality as described in claim 2, characterized in that, In step (11), the ratio of the use amount of the bromobenzene ring compound, the tenth intermediate, the second organic solvent and the strong base is 10 eq:50 mg:10 mL:0.8 mL; In step (12), the mass-volume ratio of the precursor compound, the second organic solvent, the weak base and phosphorus oxychloride is 10 mg:5 mL:0.2 mL:0.2 mL.

9. The method for preparing the near-infrared fluorescent probe for detecting bone regeneration vitality as described in claim 2, characterized in that, In step (1) and step (8), the first organic solvent is acetone; In step (2)-step (7) and step (9)-step (11), the second organic solvent is at least one of dichloromethane, acetonitrile, chloroform, methanol, THF and DMF; In step (7) and step (11), the strong base is at least one of n-butyllithium and sec-butyllithium; In step (2) and step (12), the weak base is at least one of pyridine, triethylamine, 4-dimethylaminopyridine, potassium carbonate and cesium carbonate.

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