Fluorescent probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a fluorescent probe as well as a preparation method and application thereof. The SA-beta-gal fluorescent probe prepared by the invention is novel in structure, high in sensitivity, high in response speed and excellent in pH stability, and can be used for detecting the activity of beta-galactosidase in cells, tissues and living bodies in real time; the obtained fluorescent probe compound is used for evaluating the feasibility of the curative effect of the anti-aging drug, the drug screening process can be completed by a single person within several days, and the period and cost of an existing anti-aging drug screening method at present are greatly shortened; in addition, according to the preparation method of the SA-beta-gal fluorescent probe, raw materials are easy to obtain, reaction conditions are controllable, synthesis is simple, and the SA-beta-gal fluorescent probe has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry and more specifically relates to a fluorescent probe and a preparation method and application thereof. Background Art

[0002] Aging is a ubiquitous biological process in which all organ systems accumulate damage due to various stressors, gradually leading to irreversible functional decline. Cellular senescence is a major pathogenic factor in the aging process. Due to the lack of specific anti-aging targets, the screening and development of anti-aging drugs is currently based on the lifespan phenotypes of model organisms such as Caenorhabditis elegans, Drosophila, and mice.

[0003] Caenorhabditis elegans has a short life cycle and a clear genetic background, making it a classic model organism for studying aging. Anti-aging drugs are screened by observing the effects of drugs on nematode lifespan, locomotion, reproductive ability, and changes in gene expression related to aging. Fruit flies reproduce quickly and are easy to raise and genetically manipulate. In screening for anti-aging drugs in fruit flies, the effects of drugs on fruit fly lifespan, climbing ability, learning and memory ability, etc. can be examined. Mice are closer to humans in terms of physiological structure and genetics, making them an ideal mammalian model for studying anti-aging drugs. Researchers can intervene with drugs in mice and observe their lifespan, body composition (such as the ratio of fat to muscle), behavioral characteristics (such as cognitive function and motor coordination), physiological and biochemical indicators (such as blood sugar, blood lipids, and inflammatory factor levels), and pathological changes in tissues and organs. However, these methods are often time-consuming and laborious.

[0004] β-galactosidase (SA-β-gal or β-gal), a glycoside hydrolase, is highly expressed and abnormally accumulated in senescent cells. In particular, the activity of senescence-associated β-galactosidase in lysosomes is significantly elevated, making it a core biomarker of cellular aging. The accumulation of senescent cells is closely associated with a variety of age-related diseases, such as degenerative diseases, tumors, and chronic inflammation. Therefore, targeted regulation of β-gal activity has become a key area of anti-aging research.

[0005] In recent years, fluorescent probes based on β-gal activity have become a research hotspot due to their high sensitivity, real-time nature, and high-throughput potential. For example, prior art discloses a class of SA-β-gal fluorescent probes, which work by enzymatically cleaving the glycosidic bond between the fluorophore and the galactose moiety, causing the fluorescent signal to "light up" or shift in wavelength. These SA-β-gal fluorescent probes have a detection limit as low as 0.17 U / L for β-galactosidase. However, existing probes are significantly affected by matrix effects in complex biological samples (such as serum and tissue sections). The actual detection limit in clinical samples often exceeds the theoretical value, making them unable to meet the demand for ultra-low β-gal concentrations (<0.001 U / L) required for early disease diagnosis. Research on aging-related diseases requires dynamic monitoring of β-gal activity gradients, but low-sensitivity probes struggle to capture subtle fluctuations in enzyme activity during the early stages of aging, hindering understanding of the mechanisms of the aging process. Furthermore, these studies have not fully evaluated the impact of pH on probe performance. Ideally, probes should be pH-stable to adapt to diverse microenvironments.

[0006] Therefore, developing novel probes with ultra-high sensitivity (detection limit <0.0005 U / L), excellent membrane permeability, and anti-interference capabilities is crucial to overcoming current technological bottlenecks. Such probes will advance the study of aging mechanisms from the macroscopic phenotypic to the molecular level and provide powerful tools for the precise screening of anti-aging drugs. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing SA-β-gal fluorescent probe, such as poor sensitivity and need to improve pH stability, and to provide a fluorescent probe.

[0008] The purpose of the present invention is to provide a method for preparing the fluorescent probe compound.

[0009] Another object of the present invention is to provide the use of one or more of the fluorescent probe compounds in the preparation of β-galactosidase detection products.

[0010] Another object of the present invention is to provide the use of one or more of the fluorescent probe compounds in the preparation of β-galactosidase imaging products.

[0011] Another object of the present invention is to provide a reagent composition.

[0012] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0013] The present invention protects a fluorescent probe compound having a structure shown in formula (I):

[0014]

[0015] Wherein, said R1 is selected from hydrogen or R2 is selected from hydrogen or acetyl.

[0016] Preferably, the fluorescent probe compound is selected from any one of the following structural formulas:

[0017]

[0018] Furthermore, the difference between TZ0813 and TZ0813A is that the four hydroxyl groups of TZ0813 are converted into acetoxy groups. As TZ0813A enters the cell, its acetoxy groups will be hydrolyzed into hydroxyl groups. Therefore, TZ0813 and TZ0813A have the same ability to detect SA-β-gal. However, the acetoxy groups are more hydrophobic than the hydroxyl groups, so TZ0813A can better enter the cell.

[0019] Furthermore, the fluorescent probe compound also includes a pharmaceutically acceptable salt, a prodrug or a solvate thereof.

[0020] The preparation method of the fluorescent probe compound protected by the present invention comprises the following steps: mixing compound 1 and a basic catalyst in a first organic solvent, adding triphosgene, and fully reacting at 70-110° C., adding a second organic solvent containing compound 2 and an organic base to the fully reacted system, reacting at room temperature, and completing the reaction, followed by post-treatment to obtain the target compound I-1;

[0021] When R2 of the fluorescent probe compound is selected from hydrogen, a basic reagent-methanol mixture is added to the obtained target compound I-1, and a deacetylation reaction is carried out at room temperature. After post-treatment, the target compound I-2 is obtained:

[0022]

[0023] Furthermore, the above-mentioned compound 1 and compound 2 can be obtained by purchase or self-production.

[0024] Preferably, the alkaline catalyst is selected from one or more of 4-dimethylaminopyridine, 4-pyrrolidinylpyridine, 5,6,9,10-tetrahydro-4H,8H-pyrido[3,2,1-IJ][1,6]naphthyridine, and 4,4,10,10-tetramethyl-5,6,9,10-tetrahydro-4H,8H-pyridyl[3,2,1-IJ][1,6]naphthyridine.

[0025] Preferably, the alkaline agent is selected from one or more of sodium methoxide, ammonia, potassium hydroxide and potassium carbonate.

[0026] Preferably, the organic base is selected from one or more of triethylamine, diisopropylethylamine, N-methylmorpholine, and 2,6-lutidine.

[0027] Preferably, the first organic solvent includes one or more of toluene, xylene, and acetonitrile.

[0028] Preferably, the second organic solvent includes one or more of dichloromethane, acetone, acetonitrile, and N,N-dimethylformamide.

[0029] Preferably, the sufficient reaction time is 2 to 4 hours, preferably 3 hours.

[0030] Preferably, the reaction is stirred overnight.

[0031] Furthermore, in the preparation of the target compound I-1, the post-treatment is silica gel column chromatography, specifically, the reactant is chromatographed on a silica gel column (DCM / MeOH 15:1).

[0032] Furthermore, in the preparation of the target compound I-2, the post-treatment includes removing the solvent and chromatography. Specifically, after the reaction is completed, the solvent is removed to obtain a crude product, and the crude product is chromatographed on a silica gel column (DCM / MeOH=1:1) to obtain the target product.

[0033] The present invention protects the use of one or more fluorescent probe compounds in the preparation of a β-galactosidase detection product.

[0034] Furthermore, the β-galactosidase detection product is used to detect the activity of β-galactosidase in vivo or in vitro.

[0035] The present invention protects a method for detecting β-galactosidase for non-disease diagnosis purposes, wherein the method uses one or more of the fluorescent probe compounds to detect β-galactosidase.

[0036] Furthermore, the detection limit of the β-galactosidase is less than 1.5×10 4 U / mL.

[0037] Furthermore, the response time of the β-galactosidase is less than 7 minutes.

[0038] The present invention protects the use of one or more fluorescent probe compounds in the preparation of β-galactosidase imaging products.

[0039] The present invention protects a reagent composition containing one or more of the fluorescent probe compounds.

[0040] Compared with the prior art, the present invention has the following advantages: the SA-β-gal fluorescent probe prepared by the present invention has a novel structure and high sensitivity (detection limit <1.5×10 4U / mL), fast response speed (response time <7min), excellent pH stability (strong fluorescence intensity at pH 4-9), and can detect the activity of β-galactosidase in cells, tissues and living bodies in real time; the feasibility of using the obtained fluorescent probe compound to evaluate the efficacy of anti-aging drugs, the drug screening process can be completed by a single person within a few days, greatly shortening the cycle and cost of the current anti-aging drug screening method. In addition, the preparation method of the above-mentioned SA-β-gal fluorescent probe has readily available raw materials, controllable reaction conditions, and simple synthesis, and has broad application prospects; in addition, the preparation method of the SA-β-gal fluorescent probe of the present invention has readily available raw materials, controllable reaction conditions, and simple synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the H NMR spectrum of TZ0812 in DMSO-d6.

[0042] Figure 2 This is the carbon NMR spectrum of TZ0812 in DMSO-d6.

[0043] Figure 3 This is the high-resolution mass spectrum of TZ0812.

[0044] Figure 4 This is the H NMR spectrum of TZ0813A in DMSO-d6.

[0045] Figure 5 This is the carbon NMR spectrum of TZ0813A in DMSO-d6.

[0046] Figure 6 This is the high-resolution mass spectrum of TZ0813A.

[0047] Figure 7 This is the H NMR spectrum of TZ0813 in DMSO-d6.

[0048] Figure 8 This is the carbon NMR spectrum of TZ0813 in DMSO-d6.

[0049] Figure 9 This is the high-resolution mass spectrum of TZ0813.

[0050] Figure 10 This is a statistical diagram of the effects of different pH on fluorescent probe compounds under AOβ-gal.

[0051] Figure 11 This is a statistical diagram of the detection limit determination results of TZ0805-TZ0813.

[0052] Figure 12Figure a shows the green channel fluorescence image (upper) and intensity image (lower) of 9L / LacZ cells after incubation with TZ0812 for different times, scale bar = 10 μm, green channel: λex / λem = 488 nm / 520-560 nm; Figure b shows the correlation intensity statistics of the fluorescence in the green channel of 9L / LacZ cells after incubation with TZ0812; Figure c shows the SA-β-gal positive area statistics of HUVEC cells, MRC-5 cells and 9L / LacZ cells in different cells; Figure d shows the X-gal staining detection of HUVEC cells, MRC-5 cells and 9L / LacZ cells before and after ROS and Dox stimulation, scale bar = 20 μm; Figure e shows the different groups of cells (including young HUVEC cells, Dox-induced senescent HUVEC cells (before and after D-galactose treatment) and H2O2-induced senescent HUVEC cells (before and after D-galactose treatment)) under TZ0812 ( Figure 2 shows the green channel fluorescence images of HUVEC cells stained with TZ0812 (P28, 20 μM), scale bar = 10 μm, green channel: λex / λem = 488 nm / 520-560 nm; Figure f shows the relative intensity statistics of green channel fluorescence of HUVEC cells in different groups; Figure g shows the fluorescence images of young MRC-5 cells (P28) and senescent MRC-5 cells (P40) after treatment with TZ0812, scale bar = 10 μm, green channel: λex / λem = 488 nm / 520-560 nm; Figure h shows the relative intensity statistics of green channel fluorescence of MRC-5 cells in different groups; in the figure, Yng, young; Sct, senescent; Dox-Sct, doxorubicin-induced senescent cells; Dox-Sct-D, doxorubicin-induced senescent cells treated with D-galactose; H2O2-Sct, H2O2-induced senescent cells; H2O2-Sct-D, H2O2-induced senescent cells treated with D-galactose.

[0053] Figure 13Figure a shows the fluorescence images of 9L / LacZ cells after incubation with TZ0813 (10 μM) and Hoechst 33258 for different time periods, scale bar = 10 μm, blue channel: λex / λem = 405 nm / 420-460 nm, green channel: λex / λem = 488 nm / 520-560 nm; Figure b shows the fluorescence images of TZ0813A and LysoTracker Fluorescence image (left) and fluorescence intensity data statistics (right) of co-localization detection of Red in Dox-induced HUVEC cells, scale bar = 10 μm; Figure c shows the green channel fluorescence image (upper) and intensity image (lower) of Dox-induced senescent HUVEC cells after incubation with TZ0813A (10 μM) for different times, as well as the data statistics of the relative intensity of the green channel (right), scale bar = 10 μm; green channel: λex / λem = 488 nm / 520-560 nm; Figure d shows the green channel fluorescence image of different groups of cells stained with TZ0813A (10 μM), including young HUVEC cells, Dxo-induced senescent HUVEC cells (before and after D-galactose treatment) and H2O2-induced senescent HUVEC cells (before and after D-galactose treatment), scale bar = 10 μm, green channel: λex / λem = 488 nm / 520-560 nm; e shows fluorescence images of young MRC-5 cells (P28) and senescent MRC-5 cells (P40) after treatment with TZ0813A (10 μM), scale bar = 10 μm, green channel: λex / λem = 488 nm / 520-560 nm; f-g show statistical data of the relative fluorescence intensity in the green channel of HUVEC cells (f) and MRC-5 cells (g) in different groups; h shows a schematic diagram of the TZ0813A molecular modification strategy; Yng, young; Sct, senescent; Dox-Sct, doxorubicin-induced senescent cells; Dox-Sct-D, doxorubicin-induced senescent cells treated with D-galactose; H2O2-Sct, H2O2-induced senescent cells. H2O2-Sct-D, H2O2-induced senescent cells treated with D-galactose.

[0054] Figure 14Figure a is a schematic diagram of the experimental design process for doxorubicin-induced mice and metformin-treated mice, naturally aged mice, and young mice; Figure b is the fluorescence image and X-gal staining detection image of kidney sections of different groups of mice after treatment with TZ0812 and TZ0813A, including young control mice, doxorubicin-induced aged mice, doxorubicin-induced metformin-treated mice, 3-month-old mice, and 25-month-old mice, scale bar = 50 μm; Figure c is a statistical data graph of the relative intensity of green fluorescence in different groups after staining with TZ0812 or TZ0813A; Yng, young; Met, metformin; Dox, doxorubicin; Dox-Met, aged mice induced by doxorubicin and treated with metformin.

[0055] Figure 15 Figure a is a flow chart of the efficacy evaluation method based on TZ0813A; Figures b to f are statistical graphs of the changes in SA-β-gal in senescent cells at different drug concentrations (10μM and 100μM) using TZ0813A.

[0056] Figure 16 Figure a is a schematic diagram of the experimental design of psoriasis mice and the treatment process of TZ0813A; Figure b is a statistical graph of the data of IF detection of psoriasis mouse skin based on p21, p16 and β-gal; Figure c is a representative image of in vivo fluorescence imaging of control mice and psoriasis mice after taking TZ0813A; Figure d is a statistical graph of the radiation efficiency of different groups of mice. DETAILED DESCRIPTION

[0057] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0058] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0059] Figure 12 a means Figure 12 Figure a in Figure 12 b means Figure 12 The naming of other figures is similar to Figure b in .

[0060] Example 1 Synthesis of TZ0805

[0061] The synthetic route of TZ0805 is as follows:

[0062]

[0063] The specific preparation steps of TZ0805 are as follows:

[0064] (1) Synthesis of intermediate M1, 6-bromo-2-propyl-1H-benzo[de]isoquinoline-1,3(2H)-dione:

[0065] Dissolve 6-bromo-1H,3H-benzo[de]isochromene-1,3-dione (5.4 g, 19.49 mmol, 1 eq.) and propan-1-amine (1.38 g, 23.35 mmol, 1.2 eq.) in ethanol (20 mL). Reflux the reaction for 8 hours, then cool to room temperature to precipitate. Filter the mixture, rinse the filtrate three times with petroleum ether, and yield the yellow product without further purification. 1 HNMR(400MHz,Chloroform-d)δ8.61(dd,J=7.3,1.1Hz,1H),8.51(dd,J=8.4,1.2Hz,1H),8.37(d,J=7.8Hz,1H), 7.99(d,J=7.8Hz,1H),7.81(dd,J=8.5,7.3Hz,1H),4.16–4.07(m,2H),1.81–1.70(m,2H),1.01(t,J=7.4Hz,3H).

[0066] (2) Synthesis of intermediate M2, i.e., 6-methoxy-2-propyl-1H-benzo[de]isoquinoline-1,3(2H)-dione:

[0067] Intermediate M1 (2 g, 6.29 mmol, 1 eq.), CH3ONa (2.67 g, 49.42 mmol, 7.85 eq.) and CuSO4·5H2O (158 mg, 0.98 mmol, 0.15 eq.) were dissolved in 15 mL of methanol and refluxed for 12 hours. The reaction mixture was dried and washed with cold water to give a yellow solid product. 1 H NMR(400MHz,Chloroform-d)δ8.57(t,J=6.5Hz,1H),8.52(t,J=7.2Hz,2H),7.67(t,J=7.8Hz, 1H),7.05–6.98(m,1H),4.13(d,J=11.5Hz,5H),1.76(h,J=7.4Hz,2H),1.02(t,J=7.4Hz,3H).

[0068] (3) Synthesis of intermediate TZ08FL, 7-hydroxy-2-propyl-1H-benzo[de]isoquinoline-1,3(2H)-dione: Intermediate M2 (1 g, 3.71 mmol) and HI (10 mL) were added to a round-bottom flask and heated under reflux for 15 hours. The mixture was filtered to obtain a crude product. The crude product was separated by silica gel column chromatography (PE / EA 1:1) to obtain TZ08FL as an orange solid with a yield of 77.01%.

[0069] TZ08FL (77.01%). 1 H NMR (400MHz, DMSO-d6) δ11.85(s,1H),8.52(d,J=8.3Hz,1H),8.46(d,J=7.2Hz,1H),8.35(d,J=8.2Hz,1H),7 .75(t,J=7.8Hz,1H),7.15(d,J=8.2Hz,1H),4.01–3.94(m,2H),1.63(q,J=7.4Hz,2H),0.91(t,J=7.4Hz,3H).

[0070] (4) Synthesis of TZ0805, i.e., 2-propyl-6-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1H-benzo[de]isoquinoline-1,3(2H)-dione: TZ08FL (200 mg, 783.47 μmol, 1 eq) and Cs2CO3 (510.54 mg, 1.57 mmol, 2 eq) were placed in a reaction flask with 5 mL of DMF and reacted at room temperature for 30 min. To the stirred system was then added (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triacetate (483.25 mg, 1.18 mmol, 1.5 eq). The reaction was monitored by TLC (PE:EA = 1:1) until completion. The product was purified by column chromatography (PE:EA = 3:1) and dried to yield a white solid. This solid (150 mg, 256.16 μmol / eq.) was dissolved in MeOH at 0°C. MeONa (110.71 mg, 2.05 mmol, 8 eq.) dissolved in MeOH was then added dropwise to the reaction system, which was then warmed to room temperature. After completion of the reaction, the mixture was neutralized with hydrochloric acid, and the solvent was removed under reduced pressure. The crude product was then chromatographed on a silica gel column (DCM / MeOH = 3:1) to yield TZ0805 in a 30.31% yield. 1H NMR (400MHz, DMSO-d6): δ8.72(d,J=8.4Hz,1H),8.52(d,J=7.0Hz,1H),8.43(s,1H),7.86(s,1H),7.47(d,J=8.3Hz,1H),5.50(d,J=5.0Hz,1H), 5.23(d,J=7.6Hz,1H),5.02(d,J=5.3Hz,1H),4.81–4.58(m,2H),4.07–3.92(m,2H),3.78(s,2H),1.65(q,J=7.3Hz,2H),0.92(t,J=7.2Hz,3H). 13 CNMR (101MHz, DMSO-d6): δ163.64,162.97,158.32,132.87,131.19,128.95,128.62,126.42,123.07,1 21.88,115.25,109.74,101.12,75.94,72.98,70.28,68.06,60.28,41.07,20.91,11.40.HR-ESIMSm / z calcd.for C 21 H 24 NO8 + ,418.1496,found[M+H] + ,418.1498.

[0071] Example 2 Synthesis of TZ0806

[0072] The synthetic route of TZ0806 is as follows:

[0073]

[0074] The specific preparation steps of TZ0806 are as follows:

[0075] (1) Synthesis of intermediate M3, i.e., (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-formylphenoxy)tetrahydro-2H-pyran-3,4,5-triacetate: 4-Hydroxybenzaldehyde (200 mg, 1.64 mmol, 1 eq) dissolved in 1 M NaOH solution was added dropwise to (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triacetate (1.01 g, 2.46 mmol, 1.5 eq) dissolved in acetone at room temperature. After the reaction, the pH was adjusted with hydrochloric acid. The mixture was then purified on a silica gel column (PE:EA = 6:1) to obtain product M3 in a yield of 12.42%. 1H NMR (400MHz, Chloroform-d): δ9.93(s,1H),7.88–7.82(m,2H),7.11(d,J=8.7Hz,2H),5.52(dd,J=10.4,7.9Hz,1H) ,5.47(d,J=3.4Hz,1H),5.17(d,J=7.9Hz,1H),4.26–4.08(m,4H),2.19(s,3H),2.06(d,J=1.0Hz,6H),2.02(s,3H).

[0076] (2) Synthesis of intermediate M4, i.e., (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triacetate: NaBH4 was added dropwise to EtOH (10 mL) containing intermediate M3 (1 g, 2.21 mmol) at room temperature. When TLC (PE / EA 1:1) showed that intermediate M3 was completely consumed, saturated aqueous NH4Cl solution was added to quench the reaction mixture. The solvent was removed by evaporation under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography using (PE / EA = 4:1) as the eluent to obtain intermediate M4 as a white solid in a yield of 72.92%. 1 H NMR(400MHz,DMSO-d6)δ:7.27(d,J=8.6Hz,2H),6.95(d,J=8.6Hz,2H),5.42(d, J=7.8Hz,1H),5.36–5.33(m,1H),5.29(dd,J=10.3,3.5Hz,1H),5.21(dd,J=10.3 ,7.8Hz,1H),5.10(t,J=5.7Hz,1H),4.44(d,J=5.6Hz,2H),4.41(d,J=6.5Hz,1H) ,4.10(dt,J=7.4,4.2Hz,2H),2.16(s,3H),2.03(d,J=11.8Hz,6H),1.95(s,3H).

[0077] (3) Synthesis of TZ0806, i.e., 2-propyl-6-((4-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)-1H-benzo[de]isoquinoline-1,3(2H)-dione: I2 (837.79 mg, 3.3 mmol, 1.5 eq.), PPh3 (865.79 mg, 3.3 mmol, 1.5 eq.), imidazole (749.06 mg, 11 mmol, 5 eq.) and DCM (10 mL) were added to a reaction flask and placed in ice water under argon protection. Intermediate M4 (1 g, 2.2 mmol, 1 eq) was dissolved in 20 mL of DCM and added dropwise to the above mixture. After completion of the reaction, as determined by TLC (PE / EA 1:1), the system was washed with saturated aqueous Na2S2O3. The mixture was purified by silica gel column chromatography, eluting with (PE / EA 5:1). The resulting solid was dissolved in anhydrous DMF (15 mL), and compound TZ08FL (452.35 mg, 1.77 mmol, 1 eq) and Cs2CO3 (1.15 g, 3.54 mmol, 2 eq) were added. The mixture was stirred at room temperature overnight. After purification by silica gel column chromatography, eluting with (PE / EA 1:1), the product was dissolved in MeOH at 0°C. MeONa was dissolved in MeOH and added dropwise to the reaction system, which was then warmed to room temperature. After completion of the reaction, the mixture was neutralized with HCl, and the solvent was removed under reduced pressure. The crude product was then chromatographed on silica gel (DCM / MeOH = 5:1) to yield TZ0806 in a 40.01% yield. 1 HNMR (600MHz, DMSO-d6): δ8.51(d,J=8.4Hz,1H),8.47(d,J=7.3Hz,1H),8.43(d,J=8.3Hz,1H),7.82–7 .73(m,1H),7.53(d,J=8.7Hz,2H),7.42(d,J=8.4Hz,1H),7.11(d,J=8.7Hz,2H),5.40(s,2H),5.18(s, 1H),4.87(d,J=7.7Hz,2H),4.66(s,1H),4.52(s,1H),4.01–3.95(m,2H),3.72(s,1H),3.58(dd,J=17. 1,5.4Hz,3H),3.51(dd,J=9.9,3.8Hz,1H),3.44(s,1H),1.64(h,J=7.4Hz,2H),0.92(t,J=7.4Hz,3H). 13C NMR (101MHz, DMSO-d6): δ163.59,162.93,159.35,157.60,133.14,131.06,129.55,129.06,128.61,128.39,126.45,1 22.95,121.88,116.34,114.29,107.42,100.98,75.55,73.31,70.27,68.15,60.40,41.03,20.90,11.39.HR-ESIMSm / z calcd.for C 28 H 29 NO9Na + ,546.1735,found[M+Na] + ,546.1705.

[0078] Example 3 Synthesis of TZ0807

[0079] The synthetic route of TZ0807 is as follows:

[0080]

[0081] The specific preparation steps of TZ0807 are as follows:

[0082] (1) Synthesis of intermediate M5, i.e. (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-formyl-2-methoxyphenoxy)tetrahydro-2H-pyran-3,4,5-triacetate: The synthesis steps of M5 are similar to those of M3. 1 H NMR (400MHz, Chloroform-d): δ9.88 (s, 1H), 7.41 (d, J = 9.1Hz, 2H), 7.22 (d, J = 8. 0Hz,1H),5.54(dd,J=10.3,8.1Hz,1H),5.45(d,J=2.7Hz,1H),5.11(dd,J=10.5, 3.3Hz,1H),5.05(d,J=7.9Hz,1H),4.19(ddd,J=28.8,11.2,6.6Hz,2H),4.05(t, J=6.6Hz,1H),3.89(s,3H),2.17(s,3H),2.07(s,3H),2.05(s,3H),2.01(s,3H).

[0083] (2) Synthesis of intermediate M6, i.e. (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-(hydroxymethyl)-2-methoxyphenoxy)tetrahydro-2H-pyran-3,4,5-triacetate:

[0084] The synthesis steps of M6 are similar to those of M4. 1 H NMR (400MHz, Chloroform-d): δ7.10 (d, J=8.1Hz, 1H), 6.95 (s, 1H), 6.84 (d, J=8 .1Hz,1H),5.50(dd,J=10.2,8.2Hz,1H),5.43(d,J=2.9Hz,1H),5.09(dd,J=10. 5,3.3Hz,1H),4.88(d,J=8.0Hz,1H),4.64(s,2H),4.26–4.11(m,2H),3.96(t,J =6.7Hz,1H),3.83(s,3H),2.17(s,3H),2.08(s,3H),2.04(s,3H),2.01(s,3H).

[0085] (3) Synthesis of TZ0807, i.e., 6-((3-methoxy-4-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)-2-propyl-1H-benzo[de]isoquinoline-1,3(2H)-dione: The synthesis steps of TZ0807 are similar to those of TZ0806. 1 H NMR(400MHz,DMSO-d6)δ8.56(d,J=8.3Hz,1H),8.48(dd,J=14.9,7.8Hz,2H),7.81(t,J=7.9Hz,1H) ,7.45(d,J=8.4Hz,1H),7.23(s,1H),7.12(q,J=8.3Hz,2H),5.39(s,2H),5.08(d,J=5.4Hz,1H),4. 87(dd,J=24.4,6.7Hz,2H),4.61(t,J=5.1Hz,1H),4.50(d,J=4.5Hz,1H),4.03–3.95(m,2H),3.81( s,3H),3.74–3.68(m,1H),3.64–3.36(m,5H),1.66(dt,J=14.8,7.4Hz,2H),0.92(t,J=7.4Hz,3H). 13C NMR (101MHz, DMSO-d6): δ163.62,162.96,159.41,149.06,146.72,133.18,131.09,129.35,128.64,128.46,126.49,122.99,121.90,120.6 9,115.26,114.32,112.80,107.44,100.63,75.49,73.54,70.55,70.22,68.12,60.33,55.76,41.04,20.90,11.39.HR-ESIMSm / zcalcd.for C 29 H 31 NO 10 Na + ,576.1840,found[M+Na] + ,576.1881.

[0086] Example 4 Synthesis of TZ0808

[0087] The synthetic route of TZ0808 is as follows:

[0088]

[0089] The specific preparation steps of TZ0808 are as follows:

[0090] (1) Synthesis of intermediate M7, i.e., (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-formyl-2-methylphenoxy)tetrahydro-2H-pyran-3,4,5-triacetate: 6-4-Hydroxy-3-methylbenzaldehyde (200 mg, 1.47 mmol, 1 eq) and Ag2O (510.62 mg, 2.02 mmol, 1.5 eq.) were dissolved in 5 mL of DCM. The system was protected from light. (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triacetate (664.45 mg, 1.62 mmol, 1.1 eq.) dissolved in DCM (5 mL) was added, and the mixture was stirred overnight. After completion of the reaction, the mixture was filtered through celite to obtain a crude product. The crude product was separated by column chromatography on silica gel (PE / EA 3:1) to obtain white solid intermediate M7 with a yield of 51.66%. 1H NMR (400MHz, DMSO-d6): δ9.88(s,1H),7.86–7.70(m,2H),7.22(d,J=8.5Hz,1H),5.60(d,J=6.3Hz,1H),5.39(s ,1H),5.32(s,2H),4.60–4.48(m,1H),4.13(s,2H),2.15(d,J=7.3Hz,6H),2.04(d,J=9.0Hz,6H),1.97(s,3H). 13 C NMR (101MHz, DMSO-d6): δ192.14,170.47,170.35,170.04,169.78,159.69,132.15,131.40,130.34,127.89 ,114.49,97.72,71.10,70.35,68.57,67.66,61.79,20.96,20.90,20.87,20.81,17.91,15.92.HR-ESIMSm / z calcd.forC 22 H 26 O 11 Na + ,489.1367,found[M+Na] + ,489.1371.

[0091] (2) Synthesis of intermediate M8, i.e. (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-(hydroxymethyl)-2-methylphenoxy)tetrahydro-2H-pyran-3,4,5-triacetate: The synthesis steps of M8 are similar to those of M4. 1 H NMR (400MHz, DMSO-d6): δ7.10(d,J=7.4Hz,2H),6.97(d,J=8.3Hz,1H),5.35–5.33(m,1H),5.31(s,1H),5.30–5.22(m,2H),4. 43(d,J=6.4Hz,1H),4.39(s,2H),4.12(qd,J=11.3,6.3Hz,2H),2.16(s,3H),2.06(d,J=7.4Hz,6H),2.02(s,3H),1.95(s,3H). 13C NMR (101MHz, DMSO-d6): δ170.49,170.33,170.05,169.73,154.15,137.20,129.62,126.70,125.58, 115.03,98.88,70.76,70.52,68.83,67.74,62.90,61.83,20.96,20.89,20.83,16.05.HR-ESIMSm / z calcd.forC 22 H 28 O 11 Na + ,491.1524,found[M+Na] + ,491.1531.

[0092] (3) Synthesis of TZ0808, i.e., 6-((3-methyl-4-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)-2-propyl-1H-benzo[de]isoquinoline-1,3(2H)-dione: The synthesis of TZ0808 is similar to that of TZ0806. 1 H NMR (400MHz, DMSO-d6): δ8.52(dd,J=13.8,6.9Hz,2H),8.48–8.41(m,1H),7.80(q,J=7.8Hz,1H),7.48–7 .40(m,1H),7.36(d,J=8.1Hz,2H),7.12(d,J=8.0Hz,1H),5.36(s,2H),5.17(d,J=5.1Hz,1H),4.89(d,J=5 .5Hz,1H),4.80(d,J=7.7Hz,1H),4.66(t,J=5.0Hz,1H),4.54(d,J=4.2Hz,1H),3.99(t,J=7.0Hz,2H),3.7 1(s,1H),3.57(t,J=23.8Hz,4H),3.42(s,1H),2.25(s,3H),1.64(q,J=7.3Hz,2H),0.92(t,J=7.4Hz,3H). 13C NMR (101MHz, DMSO-d6): δ163.61,162.95,159.41,155.83,133.17,131.07,130.52,128.72,128.62,128.43,127.05,127.00,126. 45,122.96,121.89,114.62,114.26,107.39,101.54,75.51,73.32,70.39,68.15,60.42,41.03,20.90,16.13,11.39.HR-ESIMSm / z calcd.for C 29 H 31 NO9Na + ,560.1891,found[M+Na] + ,560.1857.

[0093] Example 5 Synthesis of TZ0809 and TZ0810

[0094] The synthetic routes of TZ0809 and TZ0810 are as follows:

[0095]

[0096] The specific preparation steps of TZ0809 and TZ0810 are as follows:

[0097] (1) Synthesis of intermediate M9, i.e. (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-formyl-2-nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triacetate: The synthesis of M9 is similar to that of M7. 1 H NMR (400MHz, Chloroform-d): δ9.98 (s, 1H), 8.30 (d, J = 2.0Hz, 1H), 8.07 (dd, J = 8. 7,2.1Hz,1H),7.48(d,J=8.6Hz,1H),5.59(dd,J=10.4,7.8Hz,1H),5.49(d,J=3.5 Hz,1H),5.21(d,J=7.9Hz,1H),5.13(dd,J=10.4,3.4Hz,1H),4.26(dd,J=10.8,6. 5Hz,1H),4.21–4.12(m,2H),2.19(s,3H),2.13(s,3H),2.08(s,3H),2.02(s,3H).

[0098] (2) Synthesis of intermediate M10, i.e. (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-(hydroxymethyl)-2-nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triacetate: The synthesis of M10 is similar to that of M4. 1 H NMR(400MHz,Chloroform-d):10.56(s,1H),8.05(s,1H),7.52(d,J=8.6Hz,1H) ,7.14(s,1H),5.40(d,J=3.5Hz,1H),5.29(s,6H),5.01(dd,J=10.5,3.4Hz,1H), 4.85(d,J=12.4Hz,1H),4.61(d,J=12.4Hz,1H),4.55(d,J=8.0Hz,1H),4.17(dd ,J=11.4,6.7Hz,2H),3.94–3.89(m,1H),2.16(s,3H),2.06(s,6H),1.98(s,3H).

[0099] (3) Synthesis of TZ0809, i.e., 6-((3-nitro-4-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)-2-propyl-1H-benzo[de]isoquinoline-1,3(2H)-dione: The synthesis of TZ0809 is similar to that of TZ0806. 1 H NMR (400MHz, DMSO-d6): δ8.58(d,J=8.4Hz,1H),8.53–8.40(m,2H),8.13(s,1H),7.86(s,1H),7.82(t,J =7.8Hz,1H),7.51(d,J=8.6Hz,1H),7.45(d,J=8.3Hz,1H),5.49(s,2H),5.19(d,J=4.9Hz,1H),5.08(d,J =7.6Hz,1H),4.91(s,1H),4.68(t,J=5.2Hz,1H),4.62(d,J=4.0Hz,1H),4.03–3.91(m,2H),3.72(s,1H), 3.66(s,1H),3.63–3.47(m,3H),3.42(s,1H),1.99(s,1H),1.65(q,J=7.3Hz,2H),0.92(t,J=7.4Hz,3H). 13C NMR(101MHz,DMSO-d6):δ163.58,162.93,159.00,149.58,140.01,133.71,133.10,131.12,129.62,128.61,128.43,126.55,124.36,122.88,121.90,117.29,114.61,107.45,101.10,75.84,73.34,70.00,68.94,68.00,60.29,41.05,20.90,11.38.HR-ESIMSm / z calcd.for C 28 H 28 N2O 11 Na + ,591.1585,found[M+Na] + ,591.1553.

[0100] (4) Synthesis of TZ0810, i.e., 6-((3-amino-4-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)-2-propyl-1H-benzo[de]isoquinoline-1,3(2H)-dione: I2 (762.3 mg, 3 mmol, 1.5 eq), PPh3 (787.78 mg, 3 mmol, 1.5 eq), and imidazole (681.57 mg, 10.01 mmol, 5 eq) were dissolved in 5 mL of DCM and cooled to 0°C. M10 (1 g, 2 mmol, 1 eq) dissolved in DCM (10 mL) was added. The reaction was complete as determined by TLC (PE / EA 1:1). The mixture was washed with saturated Na2S2O3 water and purified by silica gel column chromatography, eluting with (PE / EA 5:1). The resulting solid was dissolved in anhydrous DMF (15 mL). A stirred solution of compound TZ08FL (389.62 mg, 1.53 mmol, 1 eq) and Cs2CO3 (994.59 mg, 3.54 mmol, 2 eq) in dry DMF (10 mL) was added to the above solution. After completion of the reaction, separation was performed using a silica gel column (PE / EA 1:1) to obtain a white solid. This solid (0.3 g, 407.23 μmol, 1 eq) and Pd / C (241.14 mg, 2.04 mmol, 5 eq) were stirred in DCM (5 mL) under hydrogen overnight. The solvent was then removed to obtain the crude product, which was dissolved in MeOH at 0°C. MeONa was dissolved in MeOH and added dropwise to the reaction system, and the reaction temperature was raised to room temperature. After the reaction, the mixture was neutralized with hydrochloric acid, and the solvent was removed under reduced pressure. The crude product was then subjected to column chromatography on silica gel (DCM / MeOH=4:1) to obtain TZ0810 with a yield of 10.34%. 1H NMR (400MHz, DMSO-d6): δ8.56(d,J=8.3Hz,1H),8.50(d,J=7.2Hz,1H),8.45(d,J=8.0Hz,1H),7.82(t,J=7.8Hz, 1H),7.41(d,J=8.4Hz,1H),7.00(d,J=8.1Hz,1H),6.84(d,J=2.1Hz,1H),6.65(dd,J=8.2,2.1Hz,1H),5.52–5.44 (m,1H),5.29(s,2H),5.12(s,2H),4.93(s,1H),4.75(s,1H),4.63(s,1H),4.49(d,J=7.7Hz,1H),4.04–3.94(m, 2H),3.71(s,1H),3.57(dd,J=10.9,7.7Hz,2H),3.52–3.45(m,3H),1.64(q,J=7.4Hz,2H),0.91(t,J=7.4Hz,3H). 13 C NMR (101MHz, DMSO-d6): δ144.49,139.46,133.25,131.12,126.49,123.05,113.68,107. 52,103.43,75.51,72.91,72.26,70.78,70.52,67.94,60.25,20.90,11.38.HR-ESIMSm / z calcd.forC 28 H 30 N2O9Na + ,561.1844,found[M+Na] + ,561.1850.

[0101] Example 6 Synthesis of TZ0811 and TZ0812

[0102] The synthetic routes of TZ0811 and TZ0812 are as follows:

[0103]

[0104] The specific preparation steps of TZ0811 and TZ0812 are as follows:

[0105] (1) Synthesis of intermediate M11, i.e., (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triacetate: M4 (200 mg, 440.11 μmol, 1 eq) was mixed with 4-nitrophenyl chloroformate (106.45 mg, 528.14 μmol, 1.2 eq), dissolved in DCM (5 mL), and pyridine (10 μL) was added and stirred at room temperature. The reaction was monitored by TLC until completion. The mixture was spin-dried and purified on a silica gel column (PE / EA=10:1) to obtain M11 as a white solid in a yield of 45.2%. 1 H NMR (400MHz, Chloroform-d): δ8.27 (d, J=9.1Hz, 2H), 7.38 (t, J=9.5Hz, 4H), 7. 03(d,J=8.5Hz,2H),5.48(dd,J=12.7,3.0Hz,2H),5.24(s,2H),5.12(dd,J=10. 5,3.4Hz,1H),5.07(d,J=7.9Hz,1H),4.23(dd,J=11.6,7.2Hz,1H),4.16(dd,J= 11.2, 6.2Hz, 1H), 4.07 (t, J = 6.6Hz, 1H), 2.19 (s, 3H), 2.06 (s, 6H), 2.02 (s, 3H).

[0106] (2) Synthesis of Intermediate M12, 7-nitro-2-propyl-1H-benzo[de]isoquinoline-1,3(2H)-dione: 6-Nitro-1H,3H-benzo[de]isochromene-1,3-dione (1 g, 4.11 mmol, 1 eq) and propan-1-amine (291.7 mg, 4.93 mmol, 1.2 eq) were dissolved in EtOH and heated under reflux overnight. The resulting solid was used in the next reaction without further purification.

[0107] (3) Synthesis of TZ08F1, i.e., 6-amino-2-propyl-1H-benzo[de]isoquinoline-1,3(2H)-dione: M13 (500 mg, 1.76 mmol, 1 eq), NH4Cl (470.41 mg, 8.79 mmol, 5 eq), MeOH (7 mL), and H2O (3 mL) were mixed and heated to 50°C. After 15 minutes, Fe (491.12 mg, 8.79 mmol, 5 eq) was quickly added to the solution, and the mixture was heated to 80°C. After 3 hours, TLC analysis (DCM / MeOH 20:1) showed that all M13 was consumed. The mixture was filtered through celite while hot and purified by silica gel column chromatography (DCM / MeOH = 10:1) to obtain the product TZ08F1 in a yield of 35%. 1 H NMR (400MHz, DMSO-d6) δ11.86(s,1H),8.53(d,J=8.3Hz,1H),8.47(d,J=7.2Hz,1H),8.36(d,J=8.2Hz,1H),7 .76(t,J=7.8Hz,1H),7.16(d,J=8.2Hz,1H),4.03–3.90(m,2H),1.64(q,J=7.4Hz,2H),0.91(t,J=7.4Hz,3H).

[0108] (4) Synthesis of TZ0811, i.e., 2-propyl-6-((4-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)amino)-1H-benzo[de]isoquinoline-1,3(2H)-dione: TZ08F1 (200 mg, 786.51 μmol, 1 eq), M11 (584.72 mg, 2.36 mmol, 1.2 eq), and DMAP (288.26 mg, 2.36 mmol, 3 eq) were added to a clean reactor, followed by the addition of 5 mL of DMF, and the mixture was stirred at room temperature overnight. After the reaction, the system was extracted with water and purified on a silica gel column (DCM / MeOH 20:1) to obtain a yellow solid. The solid (200 mg, 289.56 μmol, 1 eq) was dissolved in MeOH at 0°C. MeONa (125.15 mg, 2.32 mmol, 8 eq.) was dissolved in MeOH and added dropwise to the reaction system. The reaction temperature was then raised to room temperature. After completion of the reaction, the mixture was neutralized with hydrochloric acid, and the solvent was removed under reduced pressure. The crude product was then subjected to column chromatography on silica gel (DCM / MeOH = 5:1) to obtain TZ0811 in a 7.39% yield. 1H NMR (400MHz, DMSO-d6): δ8.70(d,J=8.5Hz,1H),8.55–8.43(m,2H),8.34–8.12(m,1H),7 .82(t,J=7.9Hz,1H),7.44(d,J=8.4Hz,2H),7.07(d,J=8.4Hz,2H),5.21(s,2H),5.18(d ,J=5.0Hz,1H),4.85(d,J=7.5Hz,2H),4.66(d,J=5.3Hz,1H),4.51(s,1H),4.00(t,J=7. 3Hz,2H),3.71(s,1H),3.62–3.46(m,6H),1.65(q,J=7.4Hz,2H),0.92(t,J=7.4Hz,3H). 13 C NMR (101MHz, DMSO-d6): δ169.59,163.51,162.94,140.33,131.63,131.54,131.44,130.85,129.22,128.71,128.31,126.36, 123.98,122.27,119.32,117.43,41.09,39.52,31.13,30.37,29.81,29.00,28.69,24.08,22.08,20.86,11.37.HR-ESIMSm / z calcd.for C 28 H 32 N2O8 + ,523.2075,found[M+H] + ,523.2085.

[0109] (5) Synthesis of TZ0812, i.e., 2-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl(1,3-dioxo-2-propyl-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)carbamate:

[0110] A solution of TZ08F1 (111.92 mg, 440.11 μmol, 2 eq) and DMAP (161.31 mg, 1.32 mmol, 6 eq) in toluene was heated to 70°C and stirred for 20 minutes. BTC (261.19 mg, 880.23 μmol, 4 eq) was added and the mixture was heated to 110°C and refluxed for 3 hours. A solution of compound M4 (500 mg, 1.10 mmol, 5 eq.) and TEA (305.88 μL, 2.20 mmol, 10 eq.) in DCM (5 mL) was then added dropwise via syringe to the stirred mixture, maintaining the internal temperature around 0°C. The reaction vessel was then warmed from ice water to room temperature and stirred overnight. The mixture was chromatographed on a silica gel column (DCM / MeOH = 15:1) to yield a yellow solid. This solid (100 mg, 136.11 μmol, 1 eq.) was dissolved in MeOH at 0°C. MeONa (58.82 mg, 1.09 mmol, 8 eq) was dissolved in MeOH and added dropwise to the reaction system. The reaction temperature was then raised to room temperature. The mixture was then neutralized with hydrochloric acid, and the solvent was removed under reduced pressure. The crude product was then subjected to column chromatography on silica gel (DCM / MeOH = 1:1) to obtain TZ0812. 1 HNMR(400MHz,Chloroform-d)δ8.77(s,1H),8.44(s,2H),8.18(d,J=8.0Hz,1H),7.71(d,J=7.5Hz,1H),7.32(s,2H),6.99(s,2H),6.68( s,1H),5.11(s,1H),4.82(s,2H),4.60(s,3H),4.47(s,1H),3.96(s,2H),3.68(s,1H),3.50(d,J=25.6Hz,4H),1.60(s,2H),0.89(s,3H).

[0111] 13C NMR (101MHz, CDCl3) δ169.98,169.85,169.57,169.24,163.51,162.96,156.45,155.41,153.96,140 .70,137.09,131.70,131.44,130.93,130.68,130.16,129.27,128.82,128.35,127.86,126.39,123. 84,122.24,118.14,117.07,116.42,116.15,98.00,97.72,70.37,70.29,70.14,68.42,68.35,67.22 ,66.17,62.37,61.29,41.09,31.14,29.82,28.99,20.86,20.49,20.44,20.40,20.34,11.36.HR-ESI MSm / z calcd.forC 29 H 30 N2O 10 Na + ,589.1793,found[M+Na] + ,589.1790..TZ0812 specific hydrogen spectrum, carbon spectrum, mass spectrum data such as Figures 1 to 3 shown.

[0112] Example 7 Synthesis of TZ0813A and TZ0813

[0113] The synthetic routes of TZ0813A and TZ0813 are as follows:

[0114]

[0115] The specific preparation steps of TZ0813A and TZ0813 are as follows:

[0116] (1) Synthesis of intermediate M13, i.e., 2-(3-morpholinopropyl)-6-nitro-1H-benzo[de]isoquinoline-1,3(2H)-dione: The synthesis process of M13 is similar to that of M12. 1 H NMR(400MHz,Chloroform-d)δ8.86(d,J=7.8Hz,1H),8.75(d,J=6.4Hz,1H),8.70(d,J=8.0Hz,1H),8.43(d,J=8.0Hz ,1H),8.08–7.93(m,1H),4.35–4.20(m,2H),3.61(s,4H),2.55(t,J=6.8Hz,2H),2.47(s,4H),1.98(p,J=7.0Hz,2H).

[0117] (2) Synthesis of TZ08F3, i.e. 6-amino-2-(3-morpholinopropyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione: The synthesis process of TZ08F3 is similar to that of TZ08F1. 1 H NMR (400MHz, DMSO-d6) δ8.64(d,J=8.3Hz,1H),8.43(d,J=7.2Hz,1H),8.19(d,J=8.4Hz,1H),7.65(t,J=7.8 Hz,1H),7.49(s,2H),6.85(d,J=8.4Hz,1H),4.07(t,J=6.8Hz,2H),3.64(s,4H),2.78(s,6H),1.93(s,2H).

[0118] (3) Synthesis of TZ0813A, i.e., (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-((((2-(3-morpholinopropyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)carbamoyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triacetate: A solution of TZ08F3 (210 mg, 618.75 μmol, 2 eq) and DMAP (226.78 mg, 1.86 mmol, 6 eq) in toluene was heated to 70°C, stirred for 20 minutes, and then BTC (367.20 mg, 1.24 mmol, 4 eq) was added and the temperature was raised to 110°C and refluxed for 3 hours. A solution of compound M4 (702.94 mg, 1.10 mmol, 5 eq.) and TEA (430.03 μL, 3.09 mmol, 10 eq.) in DCM (5 mL) was then added dropwise via syringe to the stirring mixture, maintaining the internal temperature around 0°C. The reaction vessel was then warmed from ice water to room temperature and stirred overnight. The mixture was chromatographed on a silica gel column (DCM / MeOH = 15:1) to yield TZ0813A (100 mg, 39.43%) as a yellow solid. 1H NMR (400MHz, DMSO-d6) δ10.35(s,1H),8.70(d,J=8.4Hz,1H),8.56–8.43(m,2H),8.20(d,J=8.1Hz,1H) ,7.83(t,J=7.8Hz,1H),7.48(d,J=8.0Hz,2H),7.04(d,J=8.0Hz,2H),5.48(d,J=7.6Hz,1H),5.35(s,1 H),5.29(d,J=10.5Hz,1H),5.23(s,3H),4.44(t,J=5.3Hz,1H),4.10(s,5H),3.38(s,5H),2.38(s,3H) ,2.29(s,5H),2.15(s,3H),2.02(d,J=14.7Hz,6H),1.95(s,3H),1.87–1.72(m,3H),0.89–0.76(m,1H). 13 C NMR(101MHz, CDCl3)170.38,170.23,170.15,169.38,164.21,163.72,163.18,162.7 6,157.24,152.98,138.93,132.54,131.29,130.46,130.28,128.97,126.69,125.87 ,123.48,122.88,117.12,116.72,99.51,71.11,70.79,68.62,67.49,66.84,66.49, 61.31,56.43,53.34,38.71,29.71,24.31,20.73,20.68,20.66,20.58.HR-ESIMSm / z calcd.for C 41 H 46 N3O 15 + ,820.2923,found[M+H] + ,820.2937.TZ0813A specific hydrogen spectrum, carbon spectrum, mass spectrum data such as Figures 4-6 shown.

[0119] (4) Synthesis of TZ0813, i.e., benzyl 4-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)2-(3-morpholinopropyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)carbamate: The synthesis process of TZ0813 is similar to that of TZ0812. 1H NMR (400MHz, DMSO-d6): δ10.36(s,1H),8.71(d,J=8.6Hz,1H),8.50(dd,J=9.7,7.7Hz,2H),8 .22(d,J=8.3Hz,1H),7.84(t,J=7.9Hz,1H),7.43(d,J=8.3Hz,2H),7.07(d,J=8.4Hz,2H),5. 21(s,2H),5.16(d,J=5.1Hz,1H),4.86(t,J=6.5Hz,2H),4.65(t,J=5.2Hz,1H),4.52(d,J=4. 6Hz,1H),4.11(t,J=7.0Hz,2H),3.70(t,J=4.0Hz,1H),3.61–3.40(m,9H),1.37–1.20(m,2H). 13 C NMR (101MHz, DMSO-d6): δ163.63,157.99,154.51,132.19,131.39,130.49,129.77,128.93,126. 87,124.37,122.83,118.67,116.75,101.36,76.00,73.82,70.76,68.59,66.89,60.85.HR-ESIMS m / z calcd.for C 33 H 38 N3O 11 + ,652.2501,found[M+H] + , the specific hydrogen spectrum, carbon spectrum, and mass spectrum data of 652.2507.TZ0813 are as follows Figures 7-9 shown.

[0120] Experimental performance test

[0121] (1) In vitro property test

[0122] 1. Effect of pH on the probe under AOβ-gal

[0123] 890 μL of PBS solution was added to each sample, with pH values of 3.13, 4.04, 5.02, 6.11, 7.04, 7.43, 7.95, 8.96, and 9.93, respectively. 10 μL of a 1 mM fluorescent probe solution was added to each sample, followed by 20 U / mL of AO β-gal enzyme in a volume of 100 μL to fully hydrolyze the probe and release the fluorophore, resulting in a total sample volume of 1 mL.

[0124] The results are as follows Figure 10As shown in the figure, compared with other probes TZ0806-TZ0811, the ability of probes TZ0812 and TZ0813 to detect β-gal showed stable and significant fluorescence signals in the pH range of 4-9, indicating that they have good potential for detecting intracellular β-gal.

[0125] 3. Determination of limit of detection (LOD) of TZ0805-TZ0813

[0126] Fluorescence emission spectra of β-gal solutions of varying concentrations at a wavelength of 550 nm were recorded using a fluorescence spectrometer, and the corresponding fluorescence intensities were measured. A standard curve was plotted with fluorescence intensity (F) as the ordinate and analyte concentration (C) as the abscissa. A linear fit was performed on the standard curve, yielding the linear regression equation: F = kC + b (where k is the slope and b is the intercept). Subsequently, the fluorescence intensity of blank samples (without β-gal) was measured multiple times (n = 15), and the standard deviation (SD) of the blank signals was calculated. Finally, the limit of detection (LOD) of the fluorescent probe at a wavelength of 550 nm for β-gal solutions of varying concentrations was calculated using the formula LOD = 3 × SD / ka, and the corresponding fluorescence intensities were measured. A standard curve was plotted with fluorescence intensity (F) as the ordinate and analyte concentration (C) as the abscissa. A linear fit was performed on the standard curve, yielding the linear regression equation: F = kC + b (where k is the slope and b is the intercept). Subsequently, the fluorescence intensity of blank samples (without β-gal) was measured multiple times (n = 15), and the standard deviation (SD) of the blank signals was calculated. Finally, the limit of detection (LOD) of the fluorescent probe was calculated according to the following formula: LOD = 3 × SD / k.

[0127] The results are as follows Figure 11 As shown in the figure, compared with probes TZ0805-TZ0811, TZ0812 and TZ0813 have the advantages of low detection limit, fast response speed and high pH stability.

[0128] (2) Cell experiments

[0129] 1. Construction of senescent cell model

[0130] HUVEC cells were uniformly seeded in six-well plates at densities of 30%, 60%, and 90%. These cells were incubated with medium containing 50 nM Dox or 200 μM H2O2 for varying periods of time to generate senescent HUVEC cells. Senescent MRC-5 cells (P40) were derived by passage young MRC-5 cells (P28) at a 1:2 ratio.

[0131] 2. X-gal staining of cells and tissues

[0132] The senescent β-galactosidase staining kit produced by Beyotime (Shanghai, China) was used to evaluate the senescent cells and kidney X-gal. The staining results were photographed under a microscope. All senescent cells were verified by X-gal staining. Figure 12 c, 12d, H2O2 and doxorubicin induced a significant increase in the number of X-gal positive cells in HUVEC cells and replicative senescent MRC-5 cells.

[0133] 3. TZ0812 imaging of 9L / LacZ cells at different times

[0134] 9L / lacZ cells were evenly plated in confocal imaging dishes. After all cells adhered, complete culture medium containing 20 μM TZ0812 was added to each dish. After incubation for 1, 3, 5, and 7 hours, the culture medium was replaced with normal culture medium and images were taken under a confocal microscope. λex / λem = 488 nm / 520-560 nm. Imaging results were quantified using ImageJ. Figure 12 As shown in a~b, it is proved that TZ0812 is hydrolyzed by β-gal in cells to produce fluorescence.

[0135] 4. TZ0813 imaging of 9L / LacZ cells at different times

[0136] 9L / lacZ cells were evenly plated in confocal imaging dishes. After all cells adhered, complete medium containing 20 μM TZ0813 was added to each dish. After incubation for 30 minutes and 90 minutes, 20 μM Hoechst was added and incubated for 5 minutes. The culture medium was then replaced with normal medium and imaged under a confocal microscope. Blue channel: λex / λem = 405nm / 420-460nm; green channel: λex / λem = 488nm / 520-560nm. The results are shown in Figure 2. Figure 13 As shown in a, TZ0813 is hydrolyzed by β-gal in cells to produce fluorescence.

[0137] 5. TZ0813A imaging in 9L / LacZ cells at different times

[0138] 9L / lacZ cells were evenly plated in a confocal imaging dish. After all cells adhered, complete culture medium containing 20 μM TZ0813A was added to each dish. After incubation for 2 minutes, 10 minutes, 20 minutes, and 25 minutes, the culture medium was replaced with normal culture medium and images were taken under a confocal microscope. Green channel: λex / λem = 488nm / 520-560nm; imaging results were quantified using ImageJ. Figure 13 As shown in c, TZ0813A enters cells and is hydrolyzed by β-gal to produce fluorescence faster than TZ0813.

[0139] 6. TZ0813A lysosome co-localization experiment

[0140] Culture medium containing 20 μM TZ0813A was added to Dox-induced senescent HUVEC cells and incubated in a 37°C, 5% CO2 incubator for 7 hours. Lyso-Tracker Red (20 nM) was added to the culture medium, followed by incubation for 15 minutes and imaging. Green channel: λex / λem = 488 nm / 510-550 nm; red channel: λex / λem = 561 nm / 570-670 nm (Scale bar: 10 μm). Imaging results were quantified using ImageJ. Figure 13 As shown in b, most of TZ0813A is distributed in lysosomes after entering the cell.

[0141] 8. TZ0812 / TZ0813A cell imaging

[0142] Different groups of cells (including young HUVEC cells, Dox-induced senescent HUVEC cells (before and after D-galactose treatment), H2O2-induced senescent HUVEC cells (before and after D-galactose treatment), and young and senescent MRC-5 (P28) / (P40) cells) were evenly plated in confocal imaging dishes. Once all cells adhered, complete culture medium containing 20 μM TZ0812 or 10 μM TZ0813 was added to each dish. After incubation for two hours, the dish was replaced with normal complete culture medium and images were captured under a confocal microscope at λex / λem = 488 nm / 520-560 nm. Imaging results were quantified using ImageJ.

[0143] The results are as follows Figure 12 e~h, Figure 13 As shown in d-f, the fluorescence value of senescent cells treated with TZ0812 / TZ0813 was significantly enhanced compared with that of young cells. The fluorescence value of senescent cells treated with D-gal in advance decreased significantly. The results show that TZ0812 and TZ0813A have excellent ability to detect intracellular β-gal, which also indicates that TZ0812 and TZ0813A have rapid membrane permeability and lysosomal targeting (see Figure 13 h).

[0144] 4. TZ0812 / TZ0813A tissue imaging

[0145] like Figure 14The doxorubicin-induced premature aging mouse model shown in a was established by intraperitoneal injection of 10 mg / kg Dox twice every 10 days. Five days after the model was established, the aged mice were gavaged with metformin at a dose of 20 mg / kg once a day for 12 days. The naturally aged mouse model was a 25-month-old old mouse and a 3-month-old young mouse. We took their kidneys, embedded them, and cut them into 10 μM frozen sections. After incubation with PBS containing 20 μM TZ0812 or 10 μM TZ0813A for two hours, the images were taken under a confocal microscope at λex / λem=488nm / 520-560nm. The results are shown in Figure 14 As shown in b-c, the probe staining results are the same as those of the X-gal experiment, demonstrating that TZ0812 and TZ0813A are excellent probes for visually assessing the level of aging in mouse tissues.

[0146] 5. TZ0813A drug screening experiment

[0147] like Figure 15 As shown in a, we evenly plated Dox-induced senescent cells onto a 96-well plate. We then randomly selected 28 candidate drugs and cultured them with the senescent cells for three days (each drug was divided into two concentrations of 10μM and 100μM, with three replicate wells for each concentration). We then evaluated the SA-β-gal content in these cells to assess the anti-aging efficacy of the drugs. Three days later, the cells were incubated with 10μM TZ0813A for two hours. The fluorescence value of each well was read using a microplate reader (λex / λem = 460nm / 550nm). The results are shown in Figure 2. Figure 15 As shown in Figures b to f. In cells treated with high or low doses of hydrocortisone, metformin, phytol, crisaborole, fenalol, huperzine A-A, oxymatrine, and milrinone, fluorescence was significantly reduced, indicating that these drugs have the best anti-aging effects. Among them, metformin, phytol, huperzine A, and oxymatrine have been identified as potential anti-aging drugs. In contrast, the fluorescence intensity of the negative control group (cells treated with PBS for three days) remained essentially unchanged compared to the blank control group. This result validates the feasibility of using TZ0813A to evaluate drug efficacy. This drug screening process was completed by a single person within four days, greatly shortening the cycle and cost of existing anti-aging drug screening methods.

[0148] 6. In vivo imaging experiment of TZ0813A on psoriasis mice

[0149] like Figure 16 As shown in a, we used imiquimod (125 mg / kg) to apply on the back of mice for three consecutive days to establish a psoriasis model in mice, and then used immunofluorescence to verify the accumulation of senescent cells in the lesion part. Figure 16b. After TZ0813 (3.33 mg / kg) was applied topically to the affected skin, in vivo imaging was performed. Figure 16 As shown in Figures c and d, before TZ0813 administration, mice exhibited some autofluorescence. After administration of TZ0813, the fluorescence intensity between the control mice and the psoriasis phenotype mice differed significantly within 0.5 hours. A clear fluorescence signal was observed in the dorsal skin of the model mice, which gradually increased and peaked around 3 to 4 hours later. These results demonstrate that TZ0813A can visualize senescent cells in psoriasis lesions. This study demonstrates the use of TZ0813A to visualize senescent cells in a SA-β-gal-based psoriasis mouse model for the first time.

[0150] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A fluorescent probe compound, characterized in that The fluorescent probe compound has a structure shown in formula (I): Wherein, said R1 is selected from hydrogen or R2 is selected from hydrogen or acetyl.

2. The fluorescent probe compound according to claim 1, characterized in that The fluorescent probe compound is selected from any one of the following structural formulas:

3. The fluorescent probe compound according to claim 1 or 2, characterized in that The fluorescent probe compound also includes pharmaceutically acceptable salts, prodrugs or solvates thereof.

4. The method for preparing the fluorescent probe compound according to claim 1 or 2, characterized in that: The method comprises the following steps: mixing compound 1 and a basic catalyst in a first organic solvent, adding triphosgene, and fully reacting at 70-110° C., adding a second organic solvent containing compound 2 and an organic base to the fully reacted system, reacting at room temperature, and completing the reaction, followed by post-treatment to obtain the target compound I-1; When R2 of the fluorescent probe compound is selected from hydrogen, a basic reagent-methanol mixture is added to the obtained target compound I-1, and a deacetylation reaction is carried out at room temperature. After post-treatment, the target compound I-2 is obtained:

5. The preparation method according to claim 4, characterized in that: The alkaline catalyst is selected from one or more of 4-dimethylaminopyridine, 4-pyrrolidinylpyridine, 5,6,9,10-tetrahydro-4H,8H-pyrido[3,2,1-IJ][1,6]naphthyridine, and 4,4,10,10-tetramethyl-5,6,9,10-tetrahydro-4H,8H-pyridyl[3,2,1-IJ][1,6]naphthyridine.

6. The preparation method according to claim 4, characterized in that: The alkaline reagent is selected from one or more of sodium methoxide, ammonia, potassium hydroxide and potassium carbonate.

7. Use of one or more fluorescent probe compounds according to any one of claims 1 to 3 in the preparation of a β-galactosidase detection product.

8. A method for detecting β-galactosidase for non-disease diagnosis purposes, characterized in that: The method uses one or more fluorescent probe compounds according to any one of claims 1 to 3 to detect β-galactosidase.

9. Use of one or more fluorescent probe compounds according to any one of claims 1 to 3 in the preparation of a β-galactosidase imaging product.

10. A reagent composition, characterized in that Contains one or more fluorescent probe compounds according to any one of claims 1 to 3.

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