Tandem double-locking liver targeting fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a tandem double-locking liver targeting fluorescent probe as well as a preparation method and application thereof. The probe is a compound LDMTY. Through experiments, the applicant finds that the probe shows high sensitivity and selectivity to HClO and beta-gal, the lowest detection limits reach 0.046 mu M and 2.54 * 10 <-4 > U / mL respectively, overexpression of HClO and beta-gal in HCC can be recognized at the same time, the capacity of targeting and accumulating in the liver is achieved, hepatocellular carcinoma can be accurately detected, and the application prospect is wide. And promising potential and value are shown in the aspect of HCC detection.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a tandem dual-locked liver-targeted fluorescent probe, a preparation method thereof, and an application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is a primary liver cancer with a high mortality rate, and its incidence and mortality are continuously increasing globally. As is well known, early diagnosis is of great significance for improving the survival rate of cancer patients. Research shows that changes in the levels of specific biomarkers, such as enzymes, pH value, metal ions, oxygen, and glutathione, usually already exist in the early stages of cancer. Identifying and validating cancer biomarkers with high specificity and robustness is of great significance for the early screening, timely diagnosis, accurate prognosis, and prevention of tumor progression of cancer. Biomarker imaging is rapidly becoming an effective strategy for disease diagnosis. As a bioimaging technology, fluorescent probes have developed rapidly in recent years and have become a popular bioimaging technology with high sensitivity, high selectivity, non-invasive real-time observation, intuitive operation, and small molecular weight. So far, a large number of fluorescent probes have been reported for detecting liver disease biomarkers including hepatocellular carcinoma, providing valuable and rich information for the research of the pathological process of diseases, drug development, diagnosis, and treatment. However, the design and application of most fluorescent probes mainly focus on the detection of a single biomarker, largely ignoring the great potential contained in the combined detection of multiple biomarkers. The fundamental limitation of such probes for detecting a single biomarker is that a single biomarker is overexpressed in multiple diseases, resulting in the lack of specificity required for accurate disease diagnosis by such probes, thus increasing the risk of false positives.

[0003] Reactive oxygen species (ROS) have long been associated with tumors, mainly generated by the reaction of myeloperoxidase catalyzing hydrogen peroxide and chloride ions. Due to tumor metabolic disorders, the concentration of ROS in cancer cells is about ten times that of normal cells. Among the many ROS members, endogenous hypochlorous acid (HClO) is regarded as the "representative molecule" of the ROS family due to its unique biological stability and is widely considered the "spokesperson" of ROS. An increase in endogenous HClO levels may be associated with various diseases, such as neurodegenerative diseases, arthritis, atherosclerosis, drug-induced liver injury, and liver cancer. This extensive correlation also means that relying solely on an increase in endogenous HClO levels cannot accurately diagnose hepatocellular carcinoma. Currently, there have been many reports on fluorescent probes for detecting endogenous HClO, which have excellent selectivity, high sensitivity, and rapid response to HClO. However, many of these probes also have limitations, especially the lack of specificity for detecting specific diseases when imaging endogenous HClO in various diseases. In addition, some reported probes for liver cancer detection cannot specifically target the liver. Therefore, this application aims to introduce another biomarker overexpressed in hepatocellular carcinoma as the target molecule, and then form a switch for detecting hepatocellular carcinoma, thus constituting a tandem double-lock probe molecule. In addition, we hope to add a liver-targeting moiety to the probe molecule to promote the accumulation of the probe molecule in the liver and further enhance the detection ability. Thereby improving the specificity and selectivity of liver cancer detection.

[0004] β-galactosidase (β-gal) is considered an important biomarker for cellular senescence and cancer development and is associated with the progression of various cancers. As a typical glycoside hydrolase, β-gal can specifically catalyze the hydrolysis of β-galactoside substances, breaking the glycosidic bond to generate the corresponding monosaccharide products. This unique enzymatic property makes it not only of great value in the early diagnosis of cancer. It is worth noting that galactose can be recognized by the asialoglycoprotein receptor (ASGPR) overexpressed on hepatocytes, and the galactose-functionalized carrier can be selectively endocytosed into hepatocytes, with the "cluster glycoside effect". Therefore, the probe molecule is linked to the galactose structure through a β-galactoside bond, having the dual advantages of hepatocyte-targeted imaging and a β-gal-specific responsive recognition site. In recent years, many fluorescent probes with β-gal as the recognition moiety have been reported for the highly selective detection and imaging of β-gal in organisms. These probes are commonly used for imaging cancer cells or senescent cells, as well as other applications, fully confirming the association between elevated β-gal levels in organisms and diseases such as cancer and senescence. However, it should be particularly noted that since β-gal can show upregulated expression levels under various physiological and pathological conditions, it is difficult to achieve accurate differential diagnosis of HCC relying solely on the single indicator of increased β-gal activity.

[0005] Since HClO and β-gal are not specific biomarkers for a particular disease, there are limitations in detecting them alone for diagnosing a specific disease. However, they are both overexpressed in HCC, which prompted the inventor team of this application to consider integrating them as co-responsive biomarkers into the same probe system. By constructing a dual biomarker-activated logic gating mechanism, limitations such as the lack of diagnostic specificity caused by single biomarker detection can be effectively overcome. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a tandem dual-locked liver-targeted fluorescent probe, its preparation method and application. This probe has obvious liver targeting ability and can simultaneously recognize the overexpression of HClO and β-gal in hepatocellular carcinoma, so as to accurately detect hepatocellular carcinoma.

[0007] To solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A tandem dual-locked liver-targeted fluorescent probe, denoted as compound LDMTY, whose structure is shown in the following formula:

[0009]

[0010] The preparation method of the above tandem dual-locked liver-targeted fluorescent probe includes the following steps:

[0011] 1) Take compound 3 and compound 4 and place them in an organic solvent, with or without adding a base catalyst, and react under heating or non-heating conditions, then remove the solvent to obtain intermediate compound 5;

[0012] 2) Place compound 5 and phenyl isothiocyanate in an organic solvent and react under heating or non-heating conditions, then remove the solvent to obtain intermediate compound 6;

[0013] 3) Place compound 6 in an organic solvent, add a basic reagent, and react under heating or non-heating conditions, then remove the solvent to obtain the crude target product;

[0014] The structures of compound 3, compound 4, compound 5 and compound 6 are as follows:

[0015]

[0016] In step 1) of the above preparation method, compound 3 involved can be synthesized with reference to existing literature (Journal of Photochemistry & Photobiology, A: Chemistry 453 (2024) 115597); compound 4 involved can be directly purchased from the market or synthesized with reference to existing literature (Li Z, Ren M, Wang L, Dai L, Lin W, Development of a two-photon fluorescent probe for the selective detection of β-galactosidase in living cells and tissues, Journal of Materials Chemistry B. 2019, 7(21): 3431-3437. Li Z, Ren M, Wang L, Dai L, Lin W, Development of a red-emissive two-photon fluorescent probe for sensitive detection of beta-galactosidase in vitro and in vivo, Sensors and Actuators B: Chemical. 2020, 307: 127643.).

[0017] In step 1) of the above preparation method, the addition of a base catalyst can improve the yield of intermediate compound 5. The base catalyst can be one or a combination of two or more selected from potassium carbonate, cesium hydroxide, cesium carbonate, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium tert-butoxide, sodium tert-butoxide, 1,8-diazabicycloundec-7-ene, pyridine, 4-dimethylaminopyridine, triethylamine, and N,N-diisopropylethylamine. The dosage of the base catalyst is preferably 1 to 10 times the molar amount of compound 3.

[0018] In step 3) of the above preparation method, the addition of a basic reagent for reaction is to remove the acetyl (Ac) protecting group on the galactose structure of compound 6. The basic reagent can be one or a combination of two or more selected from sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium methoxide, and ammonia water, etc.

[0019] The organic solvents involved in each step of the above preparation method can be one or a combination of two or more selected from methanol, ethanol, propanol, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), acetonitrile, ether, benzene, and toluene, preferably ethanol, N,N-dimethylformamide, dimethyl sulfoxide, acetonitrile, or toluene. The amount of the organic solvent used can be determined as needed, and generally, it is appropriate to be able to fully dissolve the raw materials to participate in the reaction. Specifically, based on 0.1 mmol of compound 3, compound 5, or compound 6, the total amount of the organic solvent used for all raw materials in each step is usually 1-10 mL. The dosage ratio of the reaction raw materials in each step is usually their stoichiometric ratio.

[0020] For the reaction temperature, in steps 1) and 3), it is preferred that the reaction is carried out at room temperature; while in step 2), it is preferred that the reaction is carried out under heating conditions, and more preferably, the reaction is refluxed under the condition of ≥50 °C. The reaction is tracked and detected by TLC until it is complete.

[0021] The products obtained in steps 1) and 2) of the above preparation method are both crude products of intermediate products. Preferably, the obtained intermediate products are purified before being used in the subsequent steps. Specifically, the crude products can be purified by means such as silica gel column chromatography. The eluent used in chromatography can be a mixed solvent composed of ethyl acetate and petroleum ether in a volume ratio of 1:1 to 1:10, or a mixed solvent composed of dichloromethane and methanol in a volume ratio of 30:1 to 60:1.

[0022] To improve the purity of the target product, the method of the present invention further includes the step of purifying the obtained crude target product. Purification is preferably carried out by means of silica gel column chromatography. The eluent used in chromatography can be a mixed solvent composed of ethyl acetate and petroleum ether in a volume ratio of 1:1 to 1:10, or a mixed solvent composed of dichloromethane and methanol in a volume ratio of 20:1 to 40:1.

[0023] The applicant found through experiments that the probe of the present invention shows high sensitivity and selectivity to HClO and β-gal, and the lowest detection limits reach 0.046 μM and 2.54×10 -4 U / mL respectively, can simultaneously recognize the overexpression of HClO and β-gal in HCC, and has obvious liver targeting ability, and can accurately detect HCC.

[0024] Based on the above findings, the present invention includes the application of the above tandem double-locked liver-targeted fluorescent probe in the qualitative or quantitative detection of the contents of HClO and β-gal in vitro.

[0025] Further included is the application of the above-mentioned tandem dual-locked liver-targeted fluorescent probe in HClO and β-gal imaging in cells or in vivo. During cell imaging, the probe is excited at 488 nm, and its emission light is collected in the range of 540 - 580 nm. During mouse imaging, the probe is excited at 535 nm, and its emission light is collected at 600 nm.

[0026] Further also included is the application of the above-mentioned tandem dual-locked liver-targeted fluorescent probe in detecting hepatocellular carcinoma in cells or in vivo.

[0027] Compared with the prior art, the present invention uses the fluorescent dye rhodamine with low cytotoxicity and high fluorescence efficiency, and selects a thiocyanate that can rapidly undergo a cyclization reaction with HClO as the HClO-responsive switch. Under the stimulation of HClO, the hemithiocarbamoyl moiety in the fluorescent probe molecule is transformed into dithiazole, resulting in the transformation of the structure from the spirolactone form (non-fluorescent) to the open-ring form (fluorescent). On the other hand, this structure is coupled with a galactose fragment through an ether bond to form a β-galactoside bond connection, serving as another switch of the probe molecule. These two switches together constitute a tandem dual-locked fluorescent probe, namely compound LDMTY. LDMTY shows high sensitivity and selectivity towards HClO and β-gal, with the lowest detection limits being 0.046 μM and 2.54×10 -4 U / mL, respectively. In addition, the tandem dual-locked mechanism effectively reduces false positives and improves the specificity and accuracy of differentiating hepatocellular carcinoma cells from normal cells. In addition, the inventor team of the present application also utilizes the cluster glycoside effect to endow LDMTY with the ability to target and accumulate in the liver, which is of great significance for the detection of hepatocellular carcinoma. As far as the inventor team of the present application knows, the designed probe is the first tandem dual-locked fluorescent probe that can accurately detect hepatocellular carcinoma by simultaneously recognizing the overexpression of HClO and β-gal in hepatocellular carcinoma and the obvious liver-targeting ability. As an innovative tandem dual-locked visual analysis tool, LDMTY demonstrates promising potential and value in HCC detection. Description of the Drawings

[0028] Figure 1 Schematic diagram of the response mechanism of LDMTY to β-gal and HClO. (a) Conceptual schematic diagram of LDMTY when encountering β-gal and HClO and the subsequent fluorescence changes; (b) DFT calculation reveals the distribution characteristics of molecular orbitals (LUMO diagram) during the response process of LDMTY; (c) HRMS obtained after incubating LDMTY with β-gal and HClO.

[0029] Figure 2 DFT calculation reveals the distribution characteristics of molecular orbitals (HOMO diagram) during the response process of LDMTY.

[0030] Figure 3 Characteristic spectra of LDMTY towards β-gal and HClO. (a) Absorption and fluorescence spectra obtained after adding HClO (0 - 50 μM) to an LDMTY (10 μM) solution; (b) Absorption spectra obtained after incubating HClO (0 - 50 μM) and β-gal (2 U / mL) with LDMTY (10 μM) for 30 min; (c) Fluorescence spectra obtained after incubating HClO (0 - 50 μM) and β-gal (2 U / mL) with LDMTY (10 μM) for 30 min; (d) Fluorescence spectra obtained after incubating HClO (40 μM) and β-gal (0 - 3 U / mL) with LDMTY (10 μM) for 30 min; (e) Time-dependent fluorescence response of LDMTY (10 μM) under the treatment of HClO (40 μM) and β-gal (2 U / mL); (f) Changes in fluorescence intensity of LDMTY (10 μM) before and after reacting with HClO (40 μM) and β-gal (2 U / mL) under different pH conditions. λ ex / λ em = 535 / 561 nm, slit: 5 nm / 5 nm.

[0031] Figure 4 Fluorescence and absorption spectra of LDMTY over time during the reaction with HClO. (a) Fluorescence spectra; (b) Absorption spectra. After incubating with β-gal (2 U / mL) for 25 min, HClO (40 μM) was added and incubated for 5 min. λ ex / λ em = 535 / 561 nm, slit: 5 nm / 5 nm.

[0032] Figure 5 Ion selectivity test of LDMTY towards β-gal and HClO: Interferents (50 μM) were added to LDMTY (10 μM) respectively to obtain (a) absorption spectra and (b) fluorescence spectra. λ ex / λ em = 535 / 561 nm, slit: 5 nm / 5 nm.

[0033] Figure 6Ion and enzyme selectivity tests of LDMTY for β-gal and HClO. (a) Absorption spectra and (b) fluorescence spectra were obtained by adding various interferents (40 μM) after incubating β-gal (2 U / mL) with LDMTY solution (10 μM) at 37 °C for 30 min. (c) Absorption spectra and (d) fluorescence intensities were obtained by adding various interferents (enzymes and proteins) to LDMTY solution (10 μM) respectively. (e) Absorption spectra and (f) fluorescence intensities were obtained by adding 40 μM HClO to LDMTY (10 μM) and incubating at 37 °C for 10 min, and then adding various enzymes (2 U / mL) respectively. λ ex / λ em = 535 / 561 nm, slit: 5 nm / 5 nm.

[0034] Figure 7 Cytotoxicity assay of LDMTY in LO2, HepG2, and HONE1 cells. LDMTY was incubated with cells for 24 h, and cell viability was observed by the CCK8 method. Error bars represent mean ± standard deviation (n = 10).

[0035] Figure 8 Visual fluorescence images of LDMTY in LO2 cells in the presence of exogenous HClO and β-gal. (a) Fluorescence images of the tandem double-lock switch state of LDMTY (10 μM) in LO2 cells after 60 min of incubation in the presence of exogenous HClO and β-gal. Scale bar: 10 μm. (b) Average fluorescence intensity of the LO2 cells in (a). Error bars represent mean ± SD (n = 3), ns P>0.05, ****p<0.0001.

[0036] Figure 9 Evaluation of the imaging ability of LDMTY for intracellular endogenous HClO using LO2 cells as a model. (a) Fluorescence imaging of endogenous HClO and β-gal in live LO2 cells after incubation with LDMTY (10 μM) for 60 min. Scale bar: 10 μm. (b) Average fluorescence intensity of the LO2 cells in the relevant experimental groups. Error bars represent mean ± SD (n = 3), ns P>0.05, ****p<0.0001.

[0037] Figure 10Fluorescence images and fluorescence intensities of LDMTY in LO2, HepG2, and HONE1 cells. (a) Fluorescence images of LDMTY (10 μM) in LO2 cells, HONE1 cells, and HepG2 cells. Images were taken at 30 min and 60 min after incubation, respectively. In another group, galactose (100 μM) was used as an inhibitor and pretreated for 3 h, then LDMTY was added and incubated for 60 min. Scale bar: 10 μm. (b) Average fluorescence intensity of HepG2 cells in the relevant experimental groups. Error bars represent mean ± SD (n = 3), ***p < 0.001, ****p < 0.0001.

[0038] Figure 11 Fluorescence imaging and fluorescence intensity of LDMTY in zebrafish in the presence of exogenous HClO and β-gal. (a) Fluorescence images of the tandem dual-lock switch state of LDMTY in live zebrafish in the presence of exogenous HClO and β-gal. Scale bar: 200 μm. (b) Average fluorescence intensity of zebrafish in the relevant experimental groups. Error bars represent mean ± standard deviation (n = 3), ns P > 0.05, ***p < 0.001, ****p < 0.0001.

[0039] Figure 12 Fluorescence images of LDMY (10 μM) in live zebrafish in the presence of exogenous HClO and β-gal. β-gal (0.5 U / mL) and HClO (10 μM) were added to the water for incubating zebrafish, then incubated for 30 min, then LDMY was added and cultured for 60 min. Scale bar: 200 μm.

[0040] Figure 13 Fluorescence imaging and fluorescence intensity of LDMTY in zebrafish with endogenous HClO. (a) Fluorescence imaging of LDMTY (10 μM) in zebrafish with endogenous HClO. Scale bar: 200 μm. (b) Average fluorescence intensity of zebrafish in the relevant experimental groups. Error bars represent mean ± SD (n = 3), ****p < 0.0001.

[0041] Figure 14 Fluorescence imaging of LDMTY in healthy mice and tumor-bearing mice. (a) LDMTY (0.5 mM, 100 μL) was subcutaneously injected into healthy mice. (b) Half an hour after injecting β-gal (0.1 U) and HClO (5 μM) into the tumor site of tumor-bearing mice, LDMTY (0.5 mM, 100 μL) was injected. Images were collected at 0, 5, 30, and 60 min after injection. Error bars represent mean ± standard deviation (n = 3), ns P > 0.05, ****p < 0.0001.

[0042] Figure 15 HRMS for LDMTY: C 37 H 38 The calculated molecular weight of N4O8S is [M+H] + : 699.2483, found in the HRMS(ESI+) spectrum: 699.2506.

[0043] Figure 16 HRMS(LDMS) obtained after incubating LDMTY with HClO. C 37 H 38 N4O8 + The calculated molecular weight is [M] + : 665.2606, found in the HRMS(ESI+) spectrum: 665.2606.

[0044] Figure 17 HRMS(LDMY) obtained after incubating LDMTY with β-gal. C 31 H 28 N4O 3S The calculated molecular weight is [M+H] + : 537.1882, found in the HRMS(ESI+) spectrum: 537.1882.

[0045] Figure 18 1H NMR (600 MHz) spectrum of compound 3 in DMSO-d6 1

[0046] Figure 19 1H NMR (600 MHz) spectrum of compound 5 in DMSO-d6 1

[0047] Figure 20 13C NMR (150 MHz) spectrum of compound 5 in DMSO-d6 13

[0048] Figure 21 1H NMR (600 MHz) spectrum of compound 6 in DMSO-d6 1

[0049] Figure 22 13C NMR (150 MHz) spectrum of compound 6 in DMSO-d6 13

[0050] Figure 23 1H NMR spectrum (600 MHz, DMSO-d6) of LDMTY 1

[0051] Figure 24 1H NMR spectrum of LDMTY 13 ​​​​​​13C NMR spectrum (150 MHz, DMSO-d6). Detailed implementation mode

[0052] In order to better explain the technical solution of the present invention, the present invention will be further described in detail below in conjunction with embodiments, but the implementation modes of the present invention are not limited thereto.

[0053] Compound 3 involved in the following examples was prepared according to the following synthetic route:

[0054]

[0055] (a) Trifluoroacetic acid, reflux at 90 °C for 24 h, yield 93%. (b) Hydrazine hydrate, ethanol, reflux at 90 °C for 4 h, yield 91%.

[0056] Preparation of Compound 2: 2-(4-Diethylamino)-2-hydroxybenzoyl)benzoic acid (2.5 g, 8 mmol) and resorcinol (881 mg, 8 mmol) were dissolved in trifluoroacetic acid (21 mL, 42 mmol). The mixture was refluxed for 24 hours. After removing the solvent, the resulting red solid mixture was purified by silica gel chromatography using dichloromethane (DCM) / methanol (MeOH) (30 / 1, v / v) as the eluent to obtain Compound 2 as a red solid (2.88 g, yield: 93%). 1 1H NMR (600 MHz, DMSO-d6) δ 10.03 (s, 1H), 7.89 (d, J = 7.7 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.16 (d, J = 7.7 Hz, 1H), 6.60 (s, 1H), 6.45 (s, 2H), 6.41–6.29 (m, 3H), 3.25 (d, J = 7.1 Hz, 4H), 0.99 (d, J = 1.8 Hz, 6H). 13 13C NMR (150 MHz, DMSO-d6) δ 168.83, 159.48, 152.41, 152.29, 149.25, 135.41, 129.95, 129.66, 129.03, 128.65, 126.61, 124.56, 124.12, 112.37, 110.02, 108.54, 104.86, 102.26, 96.98, 84.07, 43.82, 12.34.

[0057] Preparation of Compound 3: Compound 2 (550 mg, 1.42 mmol) was dissolved in anhydrous ethanol (20 mL). Then, an excess of hydrazine hydrate (2.5 mL, 48 mmol) was added. The mixture was refluxed for 4 hours. After removal of the solvent, the solid mixture was purified by silica gel chromatography using DCM / MeOH (30 / 1, v / v) as the eluent to obtain Compound 3 as a white solid (520 mg, yield 91%). The 1 1H NMR spectrum of Compound 3 is as follows Figure 18 shown, and the spectral data are as follows: 1 1H NMR (600 MHz, DMSO-d6) δ 9.93 (s, 1H), 7.77 (dd, J = 6.7, 1.9 Hz, 1H), 7.56–7.42 (m, 2H), 6.96 (dd, J = 6.6, 1.7 Hz, 1H), 6.59 (d, J = 2.3 Hz, 1H), 6.42 (dd, J = 8.6, 2.4 Hz, 1H), 6.40–6.32 (m, 4H), 4.31 (s, 2H), 3.29 (d, J = 7.1 Hz, 4H), 1.05 (t, J = 7.0 Hz, 6H).

[0058] Example 1: Preparation of Compound LDMTY

[0059]

[0060] (c) N,N-dimethylformamide, cesium carbonate, at room temperature for 12 h, with a yield of 65%. (d) Phenyl isothiocyanate, acetonitrile, refluxed at 80 °C for 12 h, with a yield of 57%. (f) Potassium carbonate, methanol, at room temperature for 3 h, with a yield of 62%.

[0061] 1) Preparation of Compound 5: Compound 3 (518 mg, 1.29 mmol) and Compound 4 (530 mg, 1.29 mmol) were dissolved in N,N-dimethylformamide (DMF, 20 mL). Cesium carbonate (2.1 g, 6.495 mmol) was added as a catalyst. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, DMF was removed by extraction with saturated sodium chloride solution and dichloromethane. Finally, the solid mixture was purified by silica gel chromatography using dichloromethane / methanol (30 / 1, v / v) as the eluent to obtain Compound 5 as a white solid (610 mg, yield 65%). The 1 1H NMR spectrum and 13 13C NMR spectrum of Compound 5 are as follows Figure 19 and Figure 20 shown, and the spectral data are as follows:

[0062] 11H NMR (600 MHz, DMSO-d6) δ 7.47–7.40 (m, 1H), 7.16–7.10 (m, 2H), 6.61 (dd, J = 12.6, 6.5 Hz, 1H), 6.57 (s, 1H), 6.26 (d, J = 8.9 Hz, 1H), 6.19 (t, J = 9.5 Hz, 1H), 6.06–5.96 (m, 3H), 5.15 (d, J = 7.8 Hz, 1H), 5.01 (s, 1H), 4.93 (d, J = 10.6 Hz, 1H), 4.87 (t, J = 9.1 Hz, 1H), 4.10 (d, J = 6.7 Hz, 1H), 4.05 (d, J = 12.8 Hz, 2H), 3.76 (t, J = 5.4 Hz, 2H), 2.95–2.92 (m, 4H), 1.78 (s, 3H), 1.74–1.63 (m, 6H), 1.59 (s, 3H), 0.70 (t, J = 7.2 Hz, 6H).

[0063] For compound 5 13 13C NMR (150 MHz, DMSO-d6) representative signals were δ 170.49, 170.41, 170.35, 170.05, 169.78, 166.23, 166.10, 157.08, 157.04, 152.93, 152.90, 152.86, 152.83, 151.81, 151.70, 148.68, 133.11, 129.54, 129.33, 128.88, 128.65, 128.53, 128.03, 127.94, 123.76, 123.66, 122.85, 114.40, 112.84, 108.72, 104.97, 103.92, 97.78, 97.52, 70.88, 70.51, 68.54, 67.54, 65.04, 64.98, 61.73, 44.09, 20.70, 20.68, 20.66, 20.62, 12.63.

[0064] 2) Preparation of compound 6: Dissolve compound 5 (480 mg, 0.656 mmol) and phenyl isothiocyanate (0.5 ml, 3.28 mmol) in acetonitrile (20 mL). Reflux the mixture for 12 hours. After the reaction is completed, dry the solution by distillation under reduced pressure. Then, purify it by silica gel column chromatography using dichloromethane / methanol (40 / 1, v / v) as the eluent to obtain compound 6 as a white solid (320 mg, yield: 57%). The 1 1H NMR spectrum and 13 13C NMR spectrum are shown respectively as Figure 21 and Figure 22 follows, and the spectral data are as follows:

[0065] 1 1H NMR (600 MHz, DMSO-d6) δ 9.53 (d, J = 67.6 Hz, 1H), 8.86 (s, 1H), 7.95–7.91 (m, 1H), 7.65 (dt, J = 15.9, 7.7 Hz, 2H), 7.17 (d, J = 8.1 Hz, 4H), 7.08 (d, J = 8.1 Hz, 4H), 6.92 (s, 4H), 6.59 (s, 1H), 6.33 (s, 3H), 5.55 (d, J = 7.9 Hz, 1H), 5.38 (d, J = 4.4 Hz, 1H), 5.30 (d, J = 11.0 Hz, 1H), 5.24 (d, J = 8.5 Hz, 1H), 4.51 (s, 1H), 4.17–4.07 (m, 2H), 3.35–3.32 (m, 4H), 2.15 (d, J = 3.3 Hz, 3H), 2.08 (d, J = 3.4 Hz, 2H), 2.02 (t, J = 4.2 Hz, 4H), 1.97–1.94 (m, 3H), 1.06 (s, 6H).

[0066] Of compound 6 13 13C NMR (150 MHz, DMSO-d6) representative signals are δ 181.29, 170.07, 169.96, 169.65, 169.32, 169.28, 153.24, 153.14, 148.66, 138.76, 133.88, 129.71, 129.06, 128.84, 127.67, 125.78, 125.19, 124.33, 123.17, 121.87, 118.22, 113.10, 103.92, 103.00, 97.39, 97.31, 96.76, 70.65, 70.53, 70.26, 68.27, 68.22, 67.34, 67.26, 65.70, 61.64, 61.43, 43.76, 20.52, 20.49, 20.46, 20.40, 12.40.

[0067] 3) Preparation of compound LDMTY: Dissolve compound 6 (300 mg, 0.346 mmol) and potassium carbonate (150 mg, 1.086 mmol) in methanol (20 mL). Stir the mixture at room temperature for 3 hours. After the reaction is completed, distill and dry the solution under reduced pressure. Then, use dichloromethane / methanol (20 / 1, v / v) as the eluent and purify the solid mixture by silica gel chromatography to obtain compound LDMTY as a white solid (150 mg, yield: 62%). The melting point (m.p.) is 167–169 °C. The HRMS spectrum of compound LDMTY, 11H NMR spectrum and 13 13C NMR spectrum are shown in Figure 15 , Figure 23 and Figure 24 respectively. The spectral data are as follows:

[0068] HRMS(ESI+) Calcd for C 37 H 38 N4O8S [M + H] + : 699.2483. found: 699.2506.

[0069] 1 1H NMR (600 MHz, DMSO-d6) δ 9.53 (s, 1H), 8.85 (s, 1H), 7.92 (d, J = 7.4 Hz, 1H), 7.68 - 7.61 (m, 2H), 7.35 - 6.96 (m, 7H), 6.88 (d, J = 8.7 Hz, 1H), 6.66 (s, 1H), 6.33 (d, J = 68.9 Hz, 3H), 5.22 (t, J = 4.2 Hz, 1H), 4.89 (dt, J = 13.5, 3.6 Hz, 2H), 4.75–4.69 (m, 1H), 4.55 (d, J = 4.5 Hz, 1H), 3.72 (dd, J = 9.1, 4.5 Hz, 1H), 3.67–3.48 (m, 5H), 3.34–3.31 (m, 5H), 1.06 (s, 6H).

[0070] The 13 13C NMR (150 MHz, DMSO-d6) representative signals of LDMTY are δ 181.34, 166.75, 158.50, 153.29, 153.10, 150.54, 148.69, 138.75, 133.87, 129.63, 129.00, 127.80, 125.84, 125.29, 124.28, 123.14, 112.78, 111.97, 108.35, 103.92, 102.95, 101.10, 96.98, 75.71, 75.54, 73.25, 70.20, 69.85, 68.25, 68.13, 65.83, 60.55, 60.41, 43.73, 12.49.

[0071] Example 2: Preparation of Compound LDMTY

[0072] Repeat Example 1, except that cesium carbonate is not added in step 1).

[0073] Finally, 131.4 mg of white solid was obtained with a yield of 14%. It was characterized by 1H-NMR, 13C-NMR and high-resolution mass spectrometry and determined to be compound LDMTY.

[0074] Example 3: Preparation of Compound LDMTY

[0075] Repeat Example 1, with the differences as follows:

[0076] In step 1), DMSO was used instead of DMF. After the reaction, DMSO was removed by extraction with saturated sodium chloride solution and dichloromethane, and potassium carbonate was used instead of cesium carbonate.

[0077] In step 2), toluene was used instead of acetonitrile, and the reaction was refluxed at 100 °C until the reaction was complete.

[0078] In step 3), ethanol was used instead of methanol, and sodium hydroxide was used instead of potassium carbonate. When the crude product was purified by silica gel column, the eluent was changed to ethyl acetate / petroleum ether with a volume ratio of 1:3.

[0079] Finally, 135.5 mg of white solid was obtained with a yield of 56%. It was characterized by 1H-NMR, 13C-NMR and high-resolution mass spectrometry and determined to be compound LDMTY.

[0080] Example 4: Preparation of Compound LDMTY

[0081] Repeat Example 1, with the differences as follows:

[0082] In step 1), ethanol was used instead of DMF. After the reaction, ethanol was removed by rotary evaporation under reduced pressure, and potassium hydroxide was used instead of cesium carbonate.

[0083] In step 2), DMSO was used instead of acetonitrile, and the reaction was refluxed at 180 °C until the reaction was complete. When the intermediate product was purified by silica gel column, the eluent was changed to ethyl acetate / petroleum ether with a volume ratio of 1:3.

[0084] In step 3), benzene was used instead of methanol, and sodium methoxide was used instead of potassium carbonate.

[0085] Finally, 123 mg of white solid was obtained with a yield of 51%. It was characterized by 1H-NMR, 13C-NMR and high-resolution mass spectrometry and determined to be compound LDMTY.

[0086] Example 5: Preparation of Compound LDMTY

[0087] Repeat Example 1, with the differences as follows:

[0088] In step 2), DMF was used instead of acetonitrile, and the reaction was refluxed at 80 °C until the reaction was complete.

[0089] In step 3), DMF was used instead of methanol, and potassium hydroxide was used instead of potassium carbonate.

[0090] Finally, 258 mg of white solid was obtained with a yield of 46%. It was characterized by 1H NMR, 13C NMR and high-resolution mass spectrometry and determined to be compound LDMTY.

[0091] Experimental example: Detection limit and imaging ability of the tandem dual-locked liver-targeted fluorescent probe LDMTY of the present invention

[0092] 1. Experimental part

[0093] 1.1 Instruments and materials

[0094] All reactants were purchased commercially and did not require further purification. β-galactosidase (β-gal, produced from Escherichia coli) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Using Bruker 600 MHz (Switzerland) of deuterated dimethyl sulfoxide (DMSO-d6) as the internal standard and TMS as the internal standard, record 1 1H NMR and 13 13C NMR spectra. Mass spectrometry analysis was carried out using Thermo Fisher Scientific LTQ FTUltra (USA). Ultraviolet-visible absorption and fluorescence spectra were obtained using Agilent Technologies Cary 3500 ultraviolet-visible spectrophotometer (USA) and Agilent Technologies Cary Eclipse (USA) at room temperature, respectively. Cytotoxicity assays were performed using a microplate spectrophotometer (Tecan Infinite infirite200pro, Switzerland). Cell and zebrafish image analysis was performed using a multiphoton laser confocal scanning microscope (Fv3000, Japan). Zebrafish fertilized eggs were cultured in an illumination incubator (GZX-250E). Imaging of mice was performed using an AniView600 multimode in vivo animal imaging system (China).

[0095] 1.2 Detection of LDMTY in solution

[0096] First, dissolve LDMTY in DMSO to prepare a 1 mM stock solution and store it at 4 °C. Take 0.02 mL of the 1 mM stock solution and adjust the volume to 2 mL with 0.1 M PBS buffer (pH 7.4, containing 10% DMSO). Transfer 1 mL into a 1 cm 3 quartz cell for absorbance or fluorescence measurement. The reaction system was incubated at 37 °C for 30 min (unless otherwise specified) in PBS buffer before detection.

[0097] 1.3 Calculate the in vitro detection limit (LOD) of LDMTY and density functional theory

[0098] The calculation method of the detection limit (LOD) is as follows: where σ is the standard deviation of the blank solution, is the average value of the blank measurements, xi is each blank measurement value, n is the number of blank measurements tested (n = 5), and k is the slope of the linear calibration curve between the fluorescence intensity (at a wavelength of 561 nm) and the concentration of HClO or β-gal.

[0099] The molecular structure was optimized using the density functional theory (DFT) method, with the B3LYP density functional and the 6-31G(d) basis set. The calculations were performed in the Gaussian 16 software package. The structure was fully optimized by this software to ensure the accuracy of the molecular geometry, and the visualization of the frontier molecular orbitals was achieved using Gaussian View.

[0100] 1.4 Cell culture and activity assay

[0101] LO2 cells (human normal liver cells), HepG2 cells (human hepatoma cells), and HONE1 cells (human nasopharyngeal carcinoma cells) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and penicillin-streptomycin (0.5 U / mL penicillin and 0.5 g / mL streptomycin) in cell culture flasks at 37 °C, 5% carbon dioxide, and constant humidity. Each cell line was seeded in cell culture dishes for 24 hours. All cells were washed 3 times with PBS buffer before imaging. Cell image analysis was performed using a multiphoton laser confocal scanning microscope (Fv3000, Japan). The probe LDNTY was excited at 488 nm, and its emission light was collected in the range of 540 - 580 nm.

[0102] First, LO2 cells, HepG2 cells, and HONE1 cells were seeded into 96-well plates containing growth medium and cultured at 37 °C for 24 hours. Subsequently, different concentrations (12.5, 25, 50, 100, and 200 μM) of LDMTY were introduced into the wells. The control group was given an equal volume of PBS solution containing 10% DMSO. Each experiment was repeated 10 times to ensure robustness. After adding LDMTY, the cells were further incubated at 37 °C and 5% carbon dioxide for 24 hours. At the end of the incubation period, 10 μL of the Cell Counting Kit-8 (CCK-8) reagent was introduced into the wells. Then, after incubation at 37 °C for 24 hours, the absorbance was measured at 450 nm using a microplate spectrophotometer (Tecan Infite 200pro). The cell viability was calculated according to the following formula: Cell viability (%) = (average absorbance of the treatment group / average absorbance of the control group) × 100%.

[0103] 1.5 Zebrafish rearing and imaging

[0104] Using zebrafish larvae from the new zebrafish biomedical R & D model center of Guangxi Medical University (Nanning, China), zebrafish fertilized eggs were cultured at 28 °C in a medium (GZX-250E) supplemented with 1-phenyl-phenyl-2-thiourea (PTU) to reduce pigment production in zebrafish. Zebrafish were subjected to image analysis using a multiphoton laser confocal scanning microscope (Fv3000, Japan), λ ex / λ em = 488 / 540 - 580 nm.

[0105] 1.6 Mouse fluorescence imaging

[0106] The nude mice used in this study were purchased from the Animal Experiment Center of Guangxi Medical University and were housed in a specific pathogen-free (SPF) environment to ensure their health status. A hepatocellular carcinoma (HCC) model was established by subcutaneous inoculation of HepG2 cells in the axillary region of 5-week-old nude mice. All experimental procedures were carried out strictly in accordance with the protocol approved by the Animal Care and Use Committee of Guangxi Medical University (approval number: HYLL-2021-133) and complied with the ethical guidelines for animal research. Mouse in vivo fluorescence imaging was performed using an AniView600 multimode in vivo animal imaging system (China), with the excitation wavelength set at 535 nm and the emission wavelength at 600 nm.

[0107] 2. Results and discussion

[0108] 2.1. Response mechanism of the probe

[0109] To confirm the induction mechanism of LDMTY towards β-gal and HClO, we first conducted reaction experiments of LDMTY with β-gal and HClO, and characterized the products by high-resolution mass spectrometry. The molecular weight of LDMTY is 698.2410, which is consistent with [M + H] in HRMS + = 699.2410 ([[]] Figure 15 ). After incubation with HClO, a peak at m / z 665.2623 appeared ([[]] Figure 16 ). In the sample incubated with β-gal, a peak at m / z 537.1963 appeared ([[]] Figure 17 ). While in the sample incubated with both β-gal and HClO, a peak at m / z 503.2092 appeared ([[]] Figure 1c). The results of the three HRMS were all in line with expectations, so the expected response mechanism was confirmed. Based on the HRMS data and the theoretical framework mastered by the inventor team of this application, we believe that the activation process of LDMTY is as follows: Under the oxidative stimulation of HClO, the thiosemicarbazide part of LDMTY undergoes a structural transformation to form a dithiazole heterocyclic structure. This conformational change triggers the opening of the spiro ring to generate an iminium salt cation, and the solution changes from a colorless state to a red state. However, the phenolic hydroxyl group protected by the galactose fragment in the rhodamine molecule inhibits the intramolecular charge transfer (ICT) effect. When the β-gal enzyme specifically hydrolyzes the β-galactosidic bond fragment, the free phenolic hydroxyl group is released, relieving its inhibitory effect on ICT ( Figure 1 a). At this point, the probe molecule LDMTY emits a strong fluorescence signal, indicating successful activation. The fluorescence activation of this probe strictly depends on the synergistic effect of β-gal and HClO, and finally forms a complete ICT conjugated system, thereby achieving a highly specific fluorescence response.

[0110] To further verify the response mechanism of LDMTY, we performed density functional theory (DFT) calculations to analyze the electronic structure changes of LDMTY. Figure 1 b and Figure 2 As shown in the figure, in the unactivated state, the π electron cloud in the HOMO orbital of LDMTY is mainly concentrated on the benzene ring of the rhodamine core connected to the phenylthiourea part. When LDMTY is activated and converted to LDMMS by the combined action of HClO and β-gal, the π electron cloud significantly extends to the entire rhodamine core, indicating the effective extension of the conjugated system. In contrast, when LDMTY reacts with HClO or β-gal alone, the degree of delocalization of the π electron cloud is low, indicating that the extension of the conjugated system is limited. In addition, DFT calculations show that the LUMO-HOMO energy gap of the activated product LDMMS (3.298014 eV) is significantly smaller than that of the unactivated LDMTY (4.224183 eV). Although the energy gap also decreases when LDMTY reacts with HClO or β-gal alone (3.778043 eV and 4.000993 eV, respectively), the reduction is relatively small. These calculation results further support the proposed response mechanism: HClO induces the opening of the spiro ring, while β-gal cleaves the β-galactosidic bond, ultimately leading to the formation of an extended conjugated system. This finding theoretically confirms the fluorescence activation mechanism of LDMTY under the combined action of HClO and β-gal.

[0111] 2.2. Characteristic spectra of LDMTY probe for HClO and β-gal

[0112] After obtaining the desired probe molecule, the optical properties of LDMTY for detecting β-gal and HClO in PBS buffer solution were investigated. The absorption spectra showed that when 0 - 50 μM HClO was added, two obvious absorption peaks appeared at 500 nm and 535 nm, but no significant fluorescence emission was observed at this time( Figure 3 a). However, when LDMTY was incubated with HClO and β-gal, the absorption peak at 500 nm gradually weakened, while the absorption peak at 535 nm gradually increased( Figure 3 b). According to the absorption spectra, 535 nm was selected as the excitation wavelength to obtain the fluorescence spectrum of LDMTY. The fluorescence intensity at 535 nm showed a linear relationship with the HClO concentration in the range of 0 - 40 μM( Figure 3 c). To further explore the performance of LDMTY, the response kinetics of LDMTY to HClO was studied. Next, we tested the concentration-dependent experiment of LDMTY. As Figure 3 shown in c, under the condition of the same β-gal concentration, with the gradual addition of HClO, the fluorescence emission gradually increased (220-fold). More importantly, the fluorescence intensity showed a linear relationship with the HClO concentration (0 - 40 μM), indicating that the HClO concentration in this range could be quantified by fluorescence, and the detection limit was 0.046 μM( Figure 3 c).

[0113] LDMTY and β-gal (0 - 3 U / mL) were incubated at 37 °C for 30 minutes, and then incubated with HClO (40 μM). The observed fluorescence change of the probe( Figure 3 d). As shown in the figure, the sample group containing β-gal (3 U / mL) showed a significant fluorescence enhancement when exposed to HClO (40 μM)( Figure 3 d). This finding indicates that when the β-gal concentration exceeds 3 U / mL, it can effectively cleave the galactose moiety. The fluorescence intensity at 561 nm showed a linear relationship with the β-gal concentration (0 - 3 U / mL), indicating that the β-gal concentration in this range could be quantified by fluorescence. Figure 3 The detection limit shown in d was 2.54×10 -4 U / mL, further verifying that the glycosidic bond connecting the galactose fragment in LDMTY is highly sensitive to β-gal.

[0114] The response kinetics of LDMTY to β-gal and the fluorescence changes at different pH values were further studied. As Figure 3As shown in e, after adding HClO and β-gal to the LDMTY solution, the fluorescence intensity increased rapidly within the initial 0 - 20 minutes. Subsequently, the growth rate gradually slowed down, and the fluorescence remained stable after 30 minutes. It is worth noting that the addition of HClO or β-gal alone did not cause a significant fluorescence response. These results indicate that while maintaining a double-locked state in vitro, LDMTY shows a rapid responsiveness to β-gal detection. Therefore, 30 minutes was selected as the optimal time point for subsequent detection experiments. As Figure 3 shown in f, significant pH changes did not cause large fluctuations in the fluorescence intensity of LDMTY in the groups containing only HClO or β-gal. This observation indicates that LDMTY has strong stability. In contrast, in the experimental group containing both HClO and β-gal, LDMTY showed the highest fluorescence intensity under neutral conditions (pH = 7.4). In addition, it maintained strong fluorescence in a slightly acidic environment, while the fluorescence intensity decreased significantly under alkaline and strongly acidic conditions. After incubating with β-gal and LDMTY for 30 minutes, an obvious fluorescence signal was observed 10 seconds after adding HClO. The fluorescence intensity reached its peak after 5 minutes( Figure 4 ). These findings indicate that LDMTY maintains strong fluorescence in a slightly acidic tumor microenvironment, suggesting its potential in tumor detection applications.

[0115] 2.3. Selectivity of LDMTY to HClO and β-gal

[0116] Subsequently, we investigated the selectivity of LDMTY to HClO and β-gal to verify its specific recognition ability and anti-interference performance in a complex biological environment. First, under the same test conditions, various interfering substances that might excite fluorescence were added to the LDMTY solution respectively. The results showed that the addition of these interfering substances did not significantly increase the fluorescence of the probe, and there was no increase in the absorption peak at 535 nm( Figure 5 ). Then, an ion selectivity test was carried out after incubating LDMTY with β-gal at 37 °C for 30 minutes. As Figure 6 shown in a and Figure 6 b, after adding HClO, the absorption and fluorescence of the LDMTY solution increased significantly. However, when other interfering substances were added, no obvious absorption and fluorescence changes were observed.

[0117] After the ion selectivity test, an enzyme selectivity test was carried out( Figure 6 from c to Figure 6f). Various enzymes or proteins were added to the LDMTY solution and then incubated at 37 °C for 30 minutes. The results showed that the addition of other enzymes or ions failed to induce significant fluorescence of the probe. Subsequently, HClO was added to the above mixture, and the color of each solution turned red. However, a significant fluorescence enhancement was only observed in the β-gal group ( Figure 6 f). The above results indicate that LDMTY can specifically recognize HClO and β-gal, has good selectivity and competitiveness, and these two substances are indispensable for the probe to excite significant fluorescence. It is expected that this probe can be applied to complex biological environments.

[0118] 2.4 Cell imaging of LDMTY

[0119] 2.4.1 Cytotoxicity of LDMTY

[0120] Inspired by the above satisfactory experimental results, we investigated the ability of LDMTY to perform bioimaging on cells. First, before the cell experiments, we used CCK8 to detect the cytotoxicity of LDMTY in LO2 cells, HepG2 cells, and HONE1 cells. As Figure 7 shown. As the dose of LDMTY ranged from 0 - 200 μM, LDMTY had good biocompatibility and low cytotoxicity to cells, indicating that LDMTY is a fluorescent probe suitable for imaging HClO and β-gal in organisms.

[0121] 2.4.2 Visualization of detecting HClO in living cells by LDMTY

[0122] After obtaining satisfactory low cytotoxicity results, we performed a series of experiments using LO2 cells to evaluate its tandem double-lock structure and the ability to image HClO and β-gal in the cellular environment. Initially, LO2 cells incubated with LDMTY alone showed minimal fluorescence. Notably, when HClO and β-gal were added separately, the fluorescence change of normal hepatocytes was very small and not statistically different from that when incubated with LDMTY alone (P > 0.05), indicating that the robust tandem double-lock structure was maintained at the cellular level. In addition, when LDMTY was added, strong fluorescence signals were observed in LO2 cells shortly after incubation with exogenous HClO (10 μM) and β-gal (1 U) in LO2 cells, indicating the high sensitivity of the probe to exogenous HClO and β-gal in hepatocytes ( Figure 8)。Subsequently, we used LO2 cells as a model system to evaluate the imaging ability of LDMTY for intracellular HClO. After adding lipopolysaccharide (LPS) and phorbol myristate acetate (PMA, a ROS inducer), the fluorescence signal was significantly enhanced (P<0.0001). However, when these cells were incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA, which consumes HClO), almost no fluorescence was observed ( Figure 9 ). This indicates that LDMTY has good imaging ability for intracellular endogenous HClO.

[0123] We further explored the potential of LDMTY to distinguish HCC cells. After LDMTY was applied to LO2, HepG2, and HONE1 cells respectively, there was almost no fluorescence in LO2 cells and almost no fluorescence in HONE1 cells. In contrast, the fluorescence intensity of HepG2 cells was significantly enhanced, about 3 times that of LO2 cells and 2 times that of HONE1 cells. These remarkable results suggest that LDMTY can distinguish cancer cells based on the differences in intracellular basal ROS and β-gal levels, especially in HCC cells, where its fluorescence signal characteristics are more prominent ( Figure 10 ). In summary, our research results indicate that LDMTY can effectively distinguish HCC cells from other cells based on the difference in fluorescence intensity, and thus has important potential in HCC diagnosis.

[0124] 2.5 Fluorescent imaging of zebrafish

[0125] Based on the above satisfactory experimental results, to study whether LDMTY can be an effective tool for in vivo imaging of HClO and β-gal, we tested the tandem dual-lock ability of LDMTY for HClO and β-gal in zebrafish. Six-day-old healthy zebrafish were selected and incubated with LDMTY (10 μM) for 1 hour. As expected, no fluorescence was observed in zebrafish. Similarly, when zebrafish were incubated with HClO or β-gal alone for 30 minutes and then with LDMTY (10 μM) for 1 hour, the same phenomenon was observed, and no fluorescence signal was observed in zebrafish in vivo ( Figure 11 ). The results show that in organisms, LDMTY maintains a dual-locked fluorescence-off state.

[0126] Subsequently, we tested the ability of LDMTY to track HClO in zebrafish. Excitingly, in the experimental group containing both HClO and β-gal, the liver of zebrafish showed obvious fluorescence, and the fluorescence was concentrated in the liver of zebrafish, while in the LDMY experimental group without galactose structure, the fluorescence of zebrafish was not concentrated in the liver ( Figure 11 and Figure 12)。The results showed that LDMTY had high sensitivity to HClO and β-gal in vivo, causing the probe to emit fluorescence under the excitation of HClO and β-gal present in vivo. It was further demonstrated that fluorescent LDMTY had strong liver targeting ability. The liver targeting ability was attributed to the modification of its galactose structure, which could be recognized by overexpressed ASGPRs on hepatocytes. This recognition triggered the cluster glycoside effect, promoting the internalization of the probe into hepatocytes and enrichment in the liver. Then, we evaluated the effectiveness of LDMTY in imaging endogenous HClO in zebrafish. Fluorescent signals appeared in the livers of zebrafish after LPS and PMA treatment. However, when TPA was added to the zebrafish, the fluorescence intensity in the zebrafish decreased because TPA reduced the level of endogenous HClO produced by LPS and PMA stimulation( Figure 13 )。This observation demonstrated the sensitivity of LDMTY to changes in endogenous HClO concentration. In summary, this probe could effectively monitor endogenous HClO produced under drug stimulation.

[0127] 2.6 Fluorescent imaging of mice

[0128] We further evaluated the imaging performance of LDMTY in a mouse HCC model. First, the LDMTY solution was directly injected into the subcutaneous tissue of nude mice. Notably, no significant fluorescent signals were observed at the injection site at different time points (0, 5, 30, and 60 minutes), indicating that LDMTY showed high stability in vivo and did not spontaneously undergo fluorescence changes( Figure 14 a). Subsequently, we microinjected β-gal and HClO into the tumor sites of tumor-bearing mice to simulate the overexpression of β-gal and HClO in the HCC microenvironment. When the LDMTY solution was injected into the treated tumor sites, the fluorescent signals in the tumor regions gradually increased over time( Figure 14 b). These results indicated that LDMTY could specifically respond to HCC overexpressing β-gal and HClO, thus achieving selective fluorescence activation in tumor tissues. In summary, LDMTY demonstrated the potential to detect a mouse HCC model through fluorescent imaging, providing a new tool for future liver cancer diagnosis research.

[0129] 3. Conclusion

[0130] In summary, this study successfully developed a tandem dual-lock fluorescent probe, LDMTY, which is expected to achieve precise detection of liver cancer by detecting overexpressed β-gal and HClO in HCC cells. LDMTY has low cytotoxicity, which guarantees its application potential in vivo. Under normal physiological conditions, LDMTY is in a fluorescence-off state; while in the tumor microenvironment, it can specifically react with overexpressed β-gal and HClO, thus activating the fluorescence signal. Compared with the single-lock switch molecular probe, the tandem dual-lock LDMTY has a narrower disease detection range, reduces false positives, and improves the specificity and accuracy of detecting diseases in organisms. The experimental results show that LDMTY can effectively distinguish cancer cells from normal cells. It is worth noting that compared with other types of cancer cells, HCC cells show stronger fluorescence signals after treatment with LDMTY. In addition, after galactose modification, LDMTY exhibits significant liver-targeting ability, and this liver-specific localization property is of great significance for HCC detection. More importantly, LDMTY has been successfully applied to in vivo imaging studies of a mouse HCC model. Overall, as an innovative tandem dual-lock visualization analysis tool, LDMTY shows potential in the field of HCC diagnosis.

Claims

1. A tandem double-locked liver-targeted fluorescent probe, denoted as compound LDMTY, whose structure is shown as follows:

2. The preparation method of the tandem double-locked liver-targeted fluorescent probe according to claim 1, comprising the following steps: 1) Take compound 3 and compound 4 and place them in an organic solvent, with or without adding a base catalyst, and carry out a reaction under heating or non-heating conditions, remove the solvent, and obtain intermediate compound 5; 2) Place compound 5 and phenyl isothiocyanate in an organic solvent, and carry out a reaction under heating or non-heating conditions, remove the solvent, and obtain intermediate compound 6; 3) Place compound 6 in an organic solvent, add a basic reagent, and carry out a reaction under heating or non-heating conditions, remove the solvent, and obtain a crude target product; The structures of compound 3, compound 4, compound 5, and compound 6 are as follows:

3. The preparation method according to claim 2, characterized in that, In step 1), the base catalyst is one or a combination of two or more selected from potassium carbonate, cesium hydroxide, cesium carbonate, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium tert-butoxide, sodium tert-butoxide, 1,8-diazabicyclo[5.4.0]undec-7-ene, pyridine, 4-dimethylaminopyridine, triethylamine, and N,N-diisopropylethylamine.

4. The preparation method according to claim 2, characterized in that, In step 3), the basic reagent is one or a combination of two or more selected from sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium methoxide, and ammonia water.

5. The preparation method according to claim 2, characterized in that, The organic solvents involved in each step are one or a combination of two or more selected from methanol, ethanol, propanol, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, ether, benzene, and toluene.

6. The preparation method according to any one of claims 2 to 5, characterized in that, The prepared intermediate is purified before being used in the subsequent steps.

7. The preparation method according to any one of claims 2 to 5, characterized in that, It also includes the step of purifying the obtained crude target product.

8. The application of the tandem double-locked liver-targeted fluorescent probe according to claim 1 in the qualitative or quantitative detection of the contents of hypochlorous acid and β-galactosidase in vitro.

9. The application of the tandem double-locked liver-targeted fluorescent probe according to claim 1 in imaging hypochlorous acid and β-galactosidase in cells or in vivo.

10. The application of the tandem double-locked liver-targeted fluorescent probe according to claim 1 in detecting hepatocellular carcinoma in cells or in vivo.

Images