Near-infrared fluorescent probes and uses thereof

Abstract

The application discloses a near-infrared fluorescent probe and application thereof, and belongs to the field of near-infrared imaging. The compound with the structure of formula I, especially the structure of formula I-a, can simultaneously produce a fluorescent response in the near-infrared region to beta-gal and / or viscosity, and can simultaneously produce a specific fluorescent response in the near-infrared region to the increase of beta-gal activity and the high-viscosity microenvironment in cancer cells through a double-lock mechanism, thereby being capable of being used for distinguishing tumor cells and normal cells and being capable of monitoring the tumor aging process.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of near-infrared imaging, more particularly, to a near-infrared fluorescent probe and its use in detecting β-gal and / or viscosity. BACKGROUND

[0002] Fluorescence visualization technology is widely used in the field of tumors. Fluorescent probes have been rapidly developed in recent years due to their high sensitivity, non-invasiveness, real-time visualization, and other characteristics. At present, there are a large number of fluorescent probes for cancer detection, which provides valuable information for the study of disease pathological processes and drug development, diagnosis and treatment.

[0003] Indocyanine green (ICG) is the most widely used near-infrared fluorescent dye approved by the US FDA, mainly used for liver function assessment, tumor surgery navigation, etc. However, as an "always-on" probe, ICG is easily disturbed by false positive signals in practical application, especially for patients with liver cirrhosis, the false positive rate is as high as 40%. On the other hand, common tumor treatment strategies such as radiotherapy, chemotherapy and immunotherapy often induce tumor cell senescence. Over-senescent tumor cells can promote changes in tumor malignant phenotypes such as acquired drug resistance, invasion and metastasis potential through senescence-associated secretory phenotype (SASP), and may lead to tumor recurrence after treatment. Therefore, it is particularly important to detect senescent cancer cells in tumor tissue. However, there is still a large gap in the research of fluorescent probes that can focus on tumors and their state changes during chemotherapy. Therefore, the development of fluorescent probes that can accurately image cancer cells and their state changes during chemotherapy will help to evaluate the effectiveness of anticancer therapy and guide individualized drug regimens.

[0004] Human β-galactosidase (β-gal) is a hydrolytic enzyme encoded by the GLB1 gene, and its main physiological function is to remove galactose residues in gangliosides, glycoproteins, sphingolipids and keratin sulfate, catalyze lactose to hydrolyze into galactose and glucose, and maintain the life activities of the body. In normal proliferating cells, β-gal is almost undetectable. However, β-gal expression is up-regulated in various cancers such as ovarian cancer, liver cancer and gastric cancer, and can be used as a biomarker for related cancer diagnosis in vivo and in vitro.

[0005] In addition, β-gal that can be specifically detected in senescent cells is defined as senescence-associated β-gal (SA-β-gal). SA-β-gal is known as the "gold standard" for senescent cell detection. At present, fluorescent probes responsive to β-gal have been used in tumor or senescence models, respectively.

[0006] Furthermore, because diagnostic strategies relying solely on a single biomarker are highly dependent on the sensitivity of the probe, test results are often affected by false positive signals generated by complex components in the human microenvironment. In the complex pathological context of tumors and aging, single-factor response probes may lack specificity, making it difficult to accurately identify and differentiate specific diseases.

[0007] Viscosity is an important microenvironment parameter that influences the interaction and transmission of intracellular biomolecules and chemical signals. Abnormal viscosity is closely related to pathological processes such as tumors, cardiovascular diseases, and liver ischemia-reperfusion injury. Viscosity-responsive fluorescent probes have been widely reported for disease-related imaging studies. These probes provide intuitive and convenient microscopic information by fluorescently imaging locally altered viscosity, aiding in the clinical diagnosis of complex pathological processes. However, current fluorescent probes for viscosity detection often suffer from insufficient specificity and small Stokes shifts.

[0008] In recent years, "dual-lock" probes have received increasing attention in the field of disease diagnosis. Dual-lock probes typically refer to activatable optical probes that possess two response mechanisms, releasing the full fluorescence signal only when interacting with two response signals. This strategy can effectively reduce false-positive interference from fluorescent probes based on single biomarkers, improve their spatial resolution and detection sensitivity, thereby enhancing the accuracy and reliability of diagnosis. Although there are some reports on dual-lock probes based on β-gal or viscosity, no dual-lock probe combining β-gal and viscosity has yet been used simultaneously to image tumors and monitor their dynamic changes during chemotherapy. Summary of the Invention

[0009] Based on this, the object of the present invention is to provide a new compound capable of near-infrared fluorescence imaging that is responsive to β-gal and / or viscosity, and to use the compound in near-infrared imaging of tumor cells to facilitate image guidance for patients with the disease during surgery.

[0010] The technical solution provided by this invention is as follows:

[0011] 1. Compound

[0012] The first aspect of this invention provides a class of compounds or their stereoisomers or isotopes having the structure shown in Formula I:

[0013]

[0014] Formula I;

[0015] Wherein, X - Selected from Cl - , Br - or I - .

[0016] The first aspect of the present application provides a compound or its stereoisomer, isotopologue, having the structure of formula I-a:

[0017]

[0018] Formula I-a (hereinafter referred to as ZW-gal).

[0019] Isotopologue refers to one or more atoms in a compound being replaced by its radioisotope. For example, any one or more hydrogen atoms in formula I or formula I-a being replaced by deuterium or tritium to form a new compound, which is within the scope of the present application. Stereoisomers include enantiomers, diastereomers, geometric isomers, conformational isomers, racemates, etc. Racemate refers to a mixture of a pair of enantiomers in equal amounts (1:1).

[0020] Unless otherwise specified, the compounds described in the present application include all possible stereoisomers thereof. They can exist in the form of racemates, enantiomers, diastereomers, tautomers, or in the form of enantiomerically or diastereomerically enriched forms. The present application includes all such isomeric forms and mixtures thereof.

[0021] The present application finds:

[0022] With the increase of the concentration of human β-galactosidase (β-gal), β-gal The compound of formula I-a exhibits enhanced and red-shifted fluorescence emission in the near-infrared range, which enables partial unlocking of fluorescence;

[0023] With the increase of the concentration of human β-galactosidase (β-gal),

[0024] With the increase of the concentration of human β-galactosidase (β-gal),

[0025] In the present application, a double-locking probe ZW-gal with the structure of formula I, particularly formula I-a, is designed and synthesized, which is based on the synergistic mechanism of specific response to β-gal and microenvironment viscosity.

[0026] The fluorescence emission of the probe ZW-gal is regulated by two mechanisms: (i) ZW-gal is hydrolyzed by β-gal to generate ZW-OH, exposing the hydroxyl group, which unlocks part of the fluorescence by enhancing the intramolecular charge transfer effect (ICT); and (ii) in a high viscosity microenvironment, the rotation of the intramolecular single bond is restricted, thereby inhibiting the twisted intramolecular charge transfer effect (TICT), significantly reducing the non-radiative transition, resulting in the enhancement of the fluorescence signal of the probe. Although β-gal or viscosity alone can cause the response of the probe ZW-gal, the synergistic response of β-gal and viscosity can unlock the full fluorescence of ZW-gal, and can achieve the optimal effect of significant enhancement of the fluorescence signal.

[0027] Cancer cells, in general, exhibit abnormally high levels of β-gal activity and simultaneous up-regulation of the viscosity of their microenvironment, and particularly senescent cancer cells, the up-regulation of β-gal activity and the viscosity of the microenvironment is more significant, therefore, the probe can distinguish normal cells from cancer cells, and further identify senescent cancer cells, particularly chemotherapy-induced senescent cancer cells, by near-infrared fluorescence imaging.

[0028] In addition, the compound of formula I-a structure can stain the nucleus of the cell, and based on the characteristic morphological changes (such as chromatin pyknosis, nuclear fragmentation, etc.) generated by the nucleus during the death process, it can achieve tracking and identification of the death process of chemotherapy-induced cancer cells.

[0029] The first aspect of the present application also provides a compound or its racemate, stereoisomer, isotopologue, having the structure shown in the following formula I-b:

[0030]

[0031] Formula I-b.

[0032] The first aspect of the present application also provides a compound or its stereoisomer, isotopologue, having the structure shown in the following formula I-c:

[0033]

[0034] Formula I-c.

[0035] As a preferred embodiment of any one of the compounds of the first aspect of the present application or its stereoisomer, isotopologue, wherein the compound has a maximum absorption wavelength and a maximum fluorescence emission wavelength in the range of 500 nm to 800 nm.

[0036] Specifically, the compound of formula I-a structure has a maximum absorption wavelength of 540±5 nm.

[0037] Specifically, the compound of formula I-a structure has a maximum fluorescence emission wavelength of 620±5 nm.

[0038] In particular, the compound of structure of Formula I-a exhibits a maximum fluorescence emission wavelength of 665 ± 5 nm in response to β-gal.

[0039] As a preferred embodiment of any one of the first aspect of the present application, the compound or its stereoisomer, isotopologue selectively images tumor cells and is used to evaluate the therapeutic response of tumor to chemotherapy, including but not limited to detecting the aging, necrosis and other processes of tumor. Preferably, the tumor cells are selected from any one or more of hepatocarcinoma tumor cells, ovarian cancer cells, gastric cancer tumor cells, colon cancer tumor cells, breast cancer tumor cells or head and neck cancer tumor cells.

[0040] 2. Near-infrared fluorescent probe

[0041] The second aspect of the present application provides a near-infrared fluorescent probe having the structure shown in the following Formula I:

[0042]

[0043] Formula I;

[0044] wherein, the X - is selected from Cl - , Br - or I - .

[0045] As a preferred embodiment of any one of the second aspect of the present application, the near-infrared fluorescent probe has the structure shown in the following Formula I-a:

[0046]

[0047] Formula I-a (hereinafter referred to as ZW-gal).

[0048] As a preferred embodiment of any one of the second aspect of the present application, the near-infrared fluorescent probe has the structure shown in the following Formula I-b:

[0049]

[0050] Formula I-b.

[0051] As a preferred embodiment of any one of the second aspect of the present application, the near-infrared fluorescent probe has the structure shown in the following Formula I-c:

[0052]

[0053] Formula I-c.

[0054] As described above, the compound based on the structure of Formula I-a responds to β-gal and viscosity, which can be used as a near-infrared fluorescent probe for detecting β-gal and viscosity.

[0055] As a preferred embodiment of any one of the second aspect of the present application, the near-infrared fluorescent probe has a maximum absorption wavelength and a maximum fluorescence emission wavelength in the range of 500 nm to 800 nm.

[0056] Specifically, the compound based on the structure of Formula I-a has a maximum absorption wavelength of 540 ± 5 nm.

[0057] Specifically, the compound based on the structure of Formula I-a has a maximum fluorescence emission wavelength of 620 ± 5 nm.

[0058] Specifically, the compound based on the structure of Formula I-a exhibits a maximum fluorescence emission wavelength of 665 ± 5 nm in response to β-gal.

[0059] Since abnormal high levels of β-gal activity and simultaneous up-regulation of microenvironment viscosity are usually exhibited in tumor cells, especially in senescent tumor cells, the probe can distinguish normal cells from cancer cells and identify chemotherapy-induced cancer cell senescence through near-infrared fluorescence imaging.

[0060] As a preferred embodiment of any one of the second aspect of the present application, the near-infrared fluorescent probe selectively images tumor cells. Preferably, the tumor cells are selected from any one or more of hepatocarcinoma tumor cells, ovarian cancer cells, gastric cancer tumor cells, colon cancer tumor cells, breast cancer tumor cells, or head and neck cancer tumor cells.

[0061] 3. Composition

[0062] The third aspect of the present application provides a composition comprising the compound of any one of the first aspect of the present application or a stereoisomer, isotopologue thereof, and a pharmaceutically acceptable carrier or excipient.

[0063] 4. Use of a near-infrared imaging agent

[0064] The fourth aspect of the present application provides the use of the compound of any one of the first aspect of the present application or a stereoisomer, isotopologue thereof, or the composition of any one of the third aspect of the present application for preparing a near-infrared imaging agent.

[0065] As described above, the compound based on the structure of Formula I-a has fluorescence emission in the near-infrared region range when responding to β-gal or viscosity, respectively, and the compound based on the structure of Formula I-a can be used as a near-infrared imaging agent for responding to β-gal or viscosity.

[0066] Further, based on the fact that the compound of structure I-a exhibits a significantly enhanced fluorescence emission in the near-infrared region range upon the synergistic response of β-gal and viscosity, the compound of structure I-a can be used as a near-infrared imaging reagent for the dual-lock response of β-gal and viscosity.

[0067] 5. Use of a compound for the preparation of a reagent for detecting human β-galactosidase

[0068] The fifth aspect of the present application provides the use of the compound of any one of the first aspect of the present application or a stereoisomer, an isotopologue thereof, or the composition of any one of the third aspect of the present application for the preparation of a reagent for detecting human β-galactosidase (β-gal).

[0069] As a preferred embodiment of the use of any one of the fifth aspect of the present application, the compound has the structure of general formula I.

[0070] As a preferred embodiment of the use of any one of the fifth aspect of the present application, the compound is selected from one or more of the compounds of formula I-a, formula I-b, and formula I-c, and most preferably is the compound of formula I-a.

[0071] As previously described, the compound of structure I-a exhibits enhanced and red-shifted fluorescence emission in the near-infrared region range upon the increase of the concentration of β-gal; the compound of structure I-a can be used as a near-infrared imaging reagent for detecting β-gal.

[0072] 6. Use of a compound for the preparation of a reagent for detecting viscosity

[0073] The sixth aspect of the present application provides the use of the compound of any one of the first aspect of the present application or a stereoisomer, an isotopologue thereof, or the composition of any one of the third aspect of the present application for the preparation of a reagent for detecting viscosity.

[0074] As a preferred embodiment of the use of any one of the sixth aspect of the present application, the compound has the structure of general formula I.

[0075] As a preferred embodiment of the use of any one of the sixth aspect of the present application, the compound is selected from one or more of the compounds of formula I-a, formula I-b, and formula I-c, and most preferably is the compound of formula I-a.

[0076] As previously described, the compound of structure I-a exhibits enhanced fluorescence emission in the near-infrared region range upon the increase of the viscosity; the compound of structure I-a can be used as a near-infrared imaging reagent for detecting viscosity.

[0077] 7. Use of a compound for the preparation of a reagent for detecting human β-galactosidase and viscosity

[0078] The seventh aspect of the present application provides use of the compound according to any one of the first aspect of the present application or a stereoisomer, an isotopologue thereof or the composition according to any one of the third aspect of the present application for the preparation of a reagent for detecting human β-galactosidase and viscosity.

[0079] As a preferred embodiment of the use according to any one of the seventh aspect of the present application, the compound has the structure shown in general formula I.

[0080] As a preferred embodiment of the use according to any one of the seventh aspect of the present application, the compound is selected from one or more of the compounds shown in formula I-a, formula I-b and formula I-c, and most preferably the compound is shown in formula I-a.

[0081] As previously described, with the increase of β-gal concentration and viscosity, the compound with the structure of formula I-a shows significantly enhanced and red-shifted fluorescence emission in the near-infrared range, and the compound with the structure of formula I-a can be used as a near-infrared imaging reagent for detecting β-gal and viscosity.

[0082] 8. Use for the preparation of a reagent for detecting tumor cells

[0083] The eighth aspect of the present application provides use of the compound according to any one of the first aspect of the present application or a stereoisomer, an isotopologue thereof or the composition according to any one of the third aspect of the present application for the preparation of a reagent for detecting tumor cells, wherein the reagent is capable of selectively imaging at least one tumor cell.

[0084] As a preferred embodiment of the use according to any one of the eighth aspect of the present application, the compound has the structure shown in general formula I.

[0085] As a preferred embodiment of the use according to any one of the eighth aspect of the present application, the compound is selected from one or more of the compounds shown in formula I-a, formula I-b and formula I-c, and most preferably the compound is shown in formula I-a.

[0086] Tumor cells have higher β-gal concentration and viscosity increases synchronously compared with normal cells, and therefore, the compound with the structure of formula I-a can be used as a near-infrared imaging reagent for detecting tumor cells.

[0087] As a preferred embodiment of the use according to any one of the eighth aspect of the present application, the tumor cell is selected from any one or more of hepatocarcinoma tumor cells, ovarian cancer cells, gastric cancer tumor cells, colon cancer tumor cells, breast cancer tumor cells or head and neck cancer tumor cells.

[0088] As a preferred embodiment of the use according to any one of the eighth aspect of the present application, the tumor cell has a characteristic selected from:

[0089] a tumor cell characterized by increased viscosity; and / or

[0090] tumor cells characterized by increased human β-galactosidase activity.

[0091] As a preferred embodiment of any one of the uses according to the eighth aspect of the present application, the tumor cells are senescent tumor cells.

[0092] Tumor cells undergo senescence, and the β-gal activity in the cells increases significantly, accompanied by a synchronous increase in viscosity. Therefore, the compound of formula I-a can be used as a near-infrared imaging reagent for detecting senescent tumor cells.

[0093] The compound of formula I-a can image the characteristic morphological changes (such as chromatin pyknosis, nuclear fragmentation, etc.) of the nucleus during cell death by staining the nucleus. Therefore, the compound of formula I-a can be used as a near-infrared imaging reagent for detecting chemotherapy-induced tumor cell death.

[0094] 9. Use of a compound according to any one of the first aspect of the present application or a stereoisomer, isotopologue thereof or a composition according to any one of the third aspect of the present application in the preparation of an agent for image-guided surgery on a subject suffering from a disease, wherein the agent is visualized by irradiation with excitation light at the site of the surgery, and the disease is cancer.

[0095] The ninth aspect of the present application provides the use of a compound according to any one of the first aspect of the present application or a stereoisomer, isotopologue thereof or a composition according to any one of the third aspect of the present application in the preparation of an agent for image-guided surgery on a subject suffering from a disease, wherein the agent is visualized by irradiation with excitation light at the site of the surgery, and the disease is cancer.

[0096] As a preferred embodiment of any one of the uses according to the ninth aspect of the present application, the wavelength of the excitation light is in the range of 500 nm to 600 nm.

[0097] As a preferred embodiment of any one of the uses according to the ninth aspect of the present application, the compound has the structure shown in general formula I.

[0098] As a preferred embodiment of any one of the uses according to the ninth aspect of the present application, the compound is selected from one or more of the compounds shown in formula I-a, formula I-b and formula I-c, and most preferably is the compound shown in formula I-a.

[0099] Under the excitation of the excitation light described above, the compound of formula I-a has enhanced fluorescence emission intensity at the tumor cells of a subject suffering from cancer, based on which the tumor cells can be accurately located to facilitate the surgery.

[0100] 10. A method for preparing a compound

[0101] The tenth aspect of the present application provides a method for preparing a compound according to the first aspect of the present application of formula I-a or a stereoisomer, isotopologue thereof, comprising the following steps:

[0102] (1) reacting compound 5-bromo-2-(3,4-vinyl dioxy thiophene) formaldehyde and 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) naphthalene-2-ol to obtain compound 1;

[0103] The structural formula of the compound 1 is as shown below:

[0104]

[0105] (2) reacting compound 1 and bromo tetraacetyl galactoside to obtain compound 2;

[0106] The structural formula of the compound 2 is as shown below:

[0107]

[0108] (3) deprotecting compound 2 to obtain compound 3;

[0109] The structural formula of the compound 3 is as shown below:

[0110]

[0111] (4) reacting compound 3 and 1,2,3,3-tetramethyl-3H-indole iodide to obtain compound shown in formula I-a, which is a β-gal and viscosity double-locked near-infrared fluorescent compound (ZW-gal);

[0112]

[0113] Formula I-a.

[0114] Preferably, in step (1), the molar ratio of compound 5-bromo-2-(3,4-vinyl dioxy thiophene) formaldehyde and 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) naphthalene-2-ol is 1: (1-4).

[0115] Preferably, in step (1), a catalyst is used to catalyze the reaction, and the catalyst is tetrakis triphenyl phosphine palladium.

[0116] Preferably, in step (1), the molar ratio of compound 5-bromo-2-(3,4-vinyl dioxy thiophene) formaldehyde and the catalyst is 1:0.05.

[0117] Preferably, in step (1), the reaction is carried out in the presence of a base, and the base is sodium carbonate.

[0118] Preferably, in step (1), the molar ratio of compound 5-bromo-2-(3,4-vinyl dioxy thiophene) formaldehyde and the base is 1:4.

[0119] Preferably, in step (1), the reaction is carried out in a solvent, and the solvent is a mixture of dioxane and water.

[0120] Preferably, in step (1), the reaction is carried out under a protective gas atmosphere, and the protective gas is nitrogen.

[0121] Preferably, in step (1), the temperature of the reaction is 60-80°C, and the reaction time is 16-24 h.

[0122] Preferably, in step (1), after the reaction, a step of post-treatment is further included.

[0123] The reaction liquid obtained after the reaction in step (1) is filtered through diatomite, the liquid is rotary dried, and is separated and purified by silica gel column chromatography, and the developing agent is a mixture of petroleum ether and ethyl acetate at a volume ratio of 10:1, to prepare compound 1.

[0124] Preferably, in step (2), the molar ratio of compound 1 to bromotetraacetyl galactoside is 1:(1-2).

[0125] Preferably, in step (2), the reaction is carried out in the presence of a phase transfer catalyst, and the phase transfer catalyst is tetrabutylammonium bromide.

[0126] Preferably, in step (2), the molar ratio of compound 1 to the phase transfer catalyst is 1:0.125.

[0127] Preferably, in step (2), the reaction is carried out in the presence of a base, and the base is selected from sodium hydroxide.

[0128] Preferably, in step (2), the concentration of the base is 10M.

[0129] Preferably, in step (2), the reaction is carried out in a solvent, and the solvent is selected from dichloromethane.

[0130] Preferably, in step (2), the temperature of the reaction is room temperature, and the reaction time is 3-5 h.

[0131] Preferably, in step (2), after the reaction, a step of post-treatment is further included.

[0132] The reaction mixture is separated in dichloromethane and saturated brine, the organic phase is dried in anhydrous magnesium sulfate, and the solvent is extracted. The crude product is separated and purified by silica gel column chromatography, and the developing agent is a mixture of dichloromethane and methanol at a volume ratio of 200:1, to prepare compound 2.

[0133] Preferably, in step (3), the deprotection treatment is carried out in the presence of a base, and the base is sodium methoxide.

[0134] Preferably, in step (3), the molar ratio of compound 2 and base is 1:2.

[0135] Preferably, in step (3), the reaction is carried out in a solvent, and the solvent is methanol.

[0136] Preferably, in step (3), the temperature of the reaction is room temperature, and the time of the reaction is 30 min.

[0137] Preferably, in step (3), after the reaction, a step of post-treatment is further included.

[0138] The reaction liquid obtained after the reaction in step (3) is filtered and dried to obtain compound 3.

[0139] Preferably, in step (4), the molar ratio of compound 3 and 1,2,3,3-tetramethyl-3H-indolium iodide is 1:(1~1.5).

[0140] Preferably, in step (4), the reaction is carried out in the presence of a base, and the base is piperidine.

[0141] Preferably, in step (4), the molar ratio of compound 3 and base is 1:0.25.

[0142] Preferably, in step (4), the reaction is carried out in a solvent, and the solvent is acetonitrile.

[0143] Preferably, in step (4), the temperature of the reaction is 70~100℃, and the time of the reaction is 1~3 h.

[0144] Preferably, in step (4), after the reaction, a step of post-treatment is further included.

[0145] The reaction liquid obtained after the reaction in step (4) is filtered by diatomite, the liquid is spin-dried and separated and purified by silica gel column chromatography, the developing agent is a mixture of dichloromethane and methanol at a volume ratio of 20:1, and high-purity ZW-gal is prepared.

[0146] In the present application, considering the changes of beta-gal and microenvironment viscosity in tumor and its aging process, a kind of double-lock probe about beta-gal and viscosity is designed, especially the compound ZW-gal, through the response accumulation of two elevated biomarkers, ZW-gal can accurately distinguish a variety of tumor cells and normal cells with high specificity, and realize the intuitive and non-destructive fluorescence tracking of tumor cell state in the process of chemotherapy, and real-time imaging of cancer cell aging and death process induced by chemotherapy. The probe is successfully applied to a mouse liver cancer model, realizes the monitoring of tumor and its aging process, and completes the image-guided radical resection. ZW-gal provides a powerful tool for related drug research and development, efficacy diagnosis, disease detection and prognosis evaluation.

[0147] Beneficial effects

[0148] Compared with the prior art, the beneficial effects of the technical scheme of the present application are:

[0149] (1) The present application designs a new compound, the compound within the structural range of general formula I, especially the compound of formula I-a, produces a fluorescent response in the near-infrared region to beta-gal and / or viscosity, and can be used for the detection or near-infrared imaging of beta-gal and / or viscosity;

[0150] Further, since the beta-gal activity and viscosity increase in tumor cells, the compound within the structural range of general formula I, especially the compound of formula I-a, can realize the differentiation of tumor cells and normal cells, and be effectively used for near-infrared imaging of tumor cells;

[0151] Still further, since the beta-gal activity and viscosity further increase in senescent tumor cells, the compound within the structural range of general formula I, especially the compound of formula I-a, can be effectively used for near-infrared imaging of senescent tumor cells, especially chemotherapy-induced tumor senescence. In addition, the compound of formula I-a can also stain the cell nucleus, and based on the characteristic morphological changes (such as chromatin pyknosis, nuclear fragmentation, etc.) produced in the death process of the cell nucleus, realize the tracking and identification of the death process of chemotherapy-induced cancer cells.

[0152] (2) Experiments show that the compound of formula I-a (ZW-gal) has a significant fluorescent response to beta-gal and / or viscosity, and can be used for the detection of beta-gal and / or viscosity.

[0153] (3) Cell experiments show that the compound of formula I-a (ZW-gal) has a significant fluorescent response to a variety of tumor cells (HepG2, SKOV-3, SGC-7901, etc.), and can be used for near-infrared fluorescence imaging of tumor cells;

[0154] Animal experiments show that the compound of formula I-a (ZW-gal) has a faster response time to tumor cells than the traditional positive control ICG.

[0155] (4) The compound of formula I-a (ZW-gal) can simultaneously image the mouse liver cancer and its aging in vivo, and can be sprayed for surgical navigation to completely remove the tumor, and has good application and development potential.

[0156] (5) The compound of formula I-a (ZW-gal) has good imaging characteristics, such as near-infrared I region emission, rapid fluorescence response, large enough stokes shift, excellent stability, good selectivity, and no obvious toxicity in cells and mice, and provides a new practical tool for imaging tumors and chemotherapy to induce their aging or death. BRIEF DESCRIPTION OF DRAWINGS

[0157] Figure 1 is the compound of formula I-a (ZW-gal double-lock probe) 1 HNMR spectrum.

[0158] Figure 2 is the compound of formula I-a (ZW-gal) 13 CNMR spectrum.

[0159] Figure 3 is the mass spectrum of the compound of formula I-a (ZW-gal).

[0160] Figure 4 is the liquid chromatogram of the compound of formula I-a (ZW-gal).

[0161] Figure 5 is the optical property characterization of the compound of formula I-a (ZW-gal);

[0162] Among them, unless otherwise specified, the solvent is PBS / DMSO (7:3, v / v):

[0163] (a): ZW-gal (10 μM); ZW-OH (10 μM);

[0164] (b): ZW-gal (10 μM); ZW-OH (10 μM);

[0165] (c) ZW-gal (10 μΜ); ZW-gal + Glycerol (ZW-gal 10 μΜ + Glycerol 50%); ZW-gal + β-gal (ZW-gal 10 μΜ + β-gal 10 U / mL); ZW-gal + β-gal + Glycerol (ZW-gal 10 μΜ + β-gal 10 U / mL + Glycerol 50%); β-gal (10 U / mL);

[0166] (d) ZW-gal (10 μΜ); ZW-gal + Glycerol (ZW-gal 10 μΜ + Glycerol 50%); ZW-gal + β-gal (ZW-gal 10 μΜ + β-gal 10 U / mL); ZW-gal + β-gal + Glycerol (ZW-gal 10 μΜ + β-gal 10 U / mL + Glycerol 50%);

[0167] (e) ZW-gal + β-gal (ZW-gal 10 μΜ + β-gal 0 to 0.013 U / mL);

[0168] (f) ZW-gal + β-gal + Glycerol (ZW-gal 10 μΜ, β-gal 10 U / mL, solution viscosity 1.005 cP to 1412 cP);

[0169] (g) ZW-gal (10 μΜ); β-gal (10 U / mL);

[0170] (h) ZW-gal (10 μΜ); ZW-gal + β-gal (ZW-gal 10 μΜ + β-gal 10 U / mL); ZW-gal + 50% Glycerol (ZW-gal 10 μΜ + Glycerol 50%); ZW-gal + β-gal + 50% Glycerol (ZW-gal 10 μΜ + β-gal 10 U / mL + Glycerol 50%); Light conditions (λex= 540 nm, excitation light continuous illumination for 1 h, collect the fluorescence intensity at 665 nm, data interval is recorded every 5 min);

[0171] (i) ZW-gal aqueous solution in dioxane (20% to 80%), ZW-gal concentration is 10 μΜ;

[0172] (j) Represented from left to right in order: 0, ZW-gal (10 μΜ), 1, ZW-gal (10 μΜ) + L-Glu (100 μΜ), 2, ZW-gal (10 μΜ) + lysozyme (100 μΜ), 3, ZW-gal (10 μΜ) + Gln (100 μΜ), 4, ZW-gal (10 μΜ) + L-Arg (100 μΜ), 5, ZW-gal (10 μΜ) + Phospholipase (100 μΜ), 6, ZW-gal (10 μΜ) + Tyr (100 μΜ), 7, ZW-gal (10 μΜ) + BSA (100 μΜ), 8, ZW-gal (10 μΜ) + H2O2 (100 μΜ), 9, ZW-gal (10 μΜ) + Carboxylesterase (100 μΜ), 10, ZW-gal (10 μΜ) + Trypsin (100 μΜ), 11, ZW-gal (10 μΜ) + Elastase (100 μΜ), 12, ZW-gal (10 μΜ) + Leu (100 μΜ), 13, ZW-gal (10 μΜ) + Ala (100 μΜ), 14, ZW-gal (10 μΜ) + TBHP (100 μΜ), 15, ZW-gal (10 μΜ) + Cys (100 μΜ), 15, ZW-gal (10 μΜ) + GOD (100 μΜ), 17, ZW-gal (10 μΜ) + CuBr2 (100 μΜ), 18, ZW-gal (10 μΜ) + GSH (100 μΜ), 19, ZW-gal (10 μΜ) + NaSH (100 μΜ), 20, ZW-gal (10 μΜ) + a-Chymotrypsin (100 μΜ), 21, ZW-gal (10 μΜ) + Ureas (100 μΜ), 22, ZW-gal (10 μΜ) + Asp (100 μΜ), 23, ZW-gal (10 μΜ) + Vc (100 μΜ), 24, ZW-gal (10 μΜ) + Gly (100 μΜ), 25, ZW-gal (10 μΜ) + FeSO4 (100 μΜ), 26, ZW-gal (10 μΜ) + β-gal (10 U / mL), 27, ZW-gal (10 μΜ) + β-gal (10 U / mL) + glycerol (50%).

[0173] Figure 6 Cytotoxicity test of the compound of formula I-a (ZW-gal, control (0 μΜ), 10 μΜ, 20 μΜ, 30 μΜ, 40 μΜ, 50 μΜ).

[0174] Figure 7 Figure 8 is the near-infrared fluorescence imaging application of the compound of formula I-a (ZW-gal) to tumor cells and their state changes during chemotherapy, wherein the Control group (ZW-gal, 10 μM), the D-gal group (ZW-gal (10 μM) + D-gal (β-gal inhibitor, concentration 1 mM)), the β-gal group (ZW-gal (10 μM) + β-gal (10 U / mL)), the Dex group (ZW-gal (10 μM) + Dex (concentration 5 μM)), and the β-gal + Dex group (ZW-gal (10 μM) + β-gal (10 U / mL) + Dex (concentration 5 μM)).

[0175] Figure 8 Figure 9 is the application of the compound of formula I-a (ZW-gal) to specifically recognize liver cancer and its aging in a mouse model; wherein:

[0176] (a) Schematic diagram of DOX-treated cancer cells;

[0177] (b) Imaging diagram of the compound ZW-gal (10 μM) to cancer cells (HepG2, SKOV-3, SGC-7901) and non-cancer cells (LO2, 293T, GES-1);

[0178] (c) Average fluorescence intensity output of cancer cells or non-cancer cells in the near-infrared (NIR) channel;

[0179] (d) Imaging diagram of the compound ZW-gal (10 μM) to cancer cells (HepG2) and aged cancer cells (DOX (200 nM) + HepG2);

[0180] (e) Relative fluorescence intensity of ZW-gal in HepG2 and aged HepG2 cells;

[0181] (f) Imaging diagram of the compound ZW-gal (10 μM) to monitor the state and death process of cancer cells (HepG2) induced by high-dose DOX (2 μM).

[0182] Figure 9 Figure 10 is the application of the compound of formula I-a (ZW-gal) to in vivo imaging of tumors and their aging and surgical navigation; a subcutaneous tumor model was established by subcutaneously injecting H22 cells (3 × 10 7 cells / 100 μL) into the armpit of Kunming mice (6 weeks old); when the tumor volume reached about 100 mm 3At the same time, DOX (10 mg / kg bw, 100 μL, intravenous injection, once) was injected to induce tumor senescence, while saline was injected as a control. Mice were divided into the following three treatment groups: (1) Senescent tumor + ZW-gal; (2) Tumor + ZW-gal; (3) Tumor + ICG; each group had 3 mice; one week later, the mice with tumor growth were subjected to imaging experiments; among them, senescent tumor + ZW-gal group: intratumoral injection of ZW-gal (100 μM, 100 μL); tumor + ZW-gal group: intratumoral injection of ZW-gal (100 μM, 100 μL); tumor + ICG group: intratumoral injection of ICG (100 μM, 100 μL);

[0183] (a) Schematic diagram of in vivo imaging protocol for tumors and tumor aging; sc, subcutaneous injection; iv, tail vein injection; it, intratumoral injection;

[0184] (b) In vivo imaging results of H22 tumor-bearing mice after intratumoral injection of ZW-gal or ICG (100 μM, 100 μL);

[0185] (c) Fluorescence intensity ratio at different time points (tumor site / normal site); I tumor : Total fluorescence intensity in the tumor region at this moment; I normal : Total fluorescence intensity of the symmetrical normal region at this moment; data are expressed as mean ± standard deviation (n=3);

[0186] (d) X-gal (1 mg / mL, 20 hours) staining results of major mouse organs;

[0187] (e) Ex vivo fluorescence imaging of tumors and normal organs 15 minutes after injection;

[0188] (f) ZW-gal-guided tumor detection and resection surgery.

[0189] Figure 10 Pathological evaluation of normal tissues and tumors in mice using H&E staining. Detailed Implementation

[0190] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0191] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0192] As used herein, the term "about" is used to provide flexibility to a given term, measurement, or value. The degree of flexibility of a particular variable will be readily determined by one of skill in the art.

[0193] Concentrations, amounts, and other numerical data can be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be construed as having been followed to following the principle of including and disclosing every single numerical value falling within the range even though the exact values are not written out. For instance, a range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited values of about 1 to about 4.5, but also include an implicit range of values from about 1 to about 4.5, such as, for example, values like 1, 1.1, 1.2, 1.3, 1.4, etc. and also

[0194] It should be noted that MTT method is used for cytotoxicity test, which is a colorimetric cytotoxicity / viability detection technique, and the core principle is that succinate dehydrogenase in mitochondria of living cells can reduce yellow water-soluble tetrazolium salt MTT (3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide) to water-insoluble blue-violet formazan crystal.

[0195] Raw materials: dimethyl sulfoxide (DMSO), phosphate buffer (PBS), dexamethasone (Dex) and other chemicals and reagents were purchased from commercial suppliers.

[0196] β-gal was purchased from Merck (CAS: 9031-11-2; product number: G5160).

[0197] ICG was provided by Nuoyuan Medical Instrument Co., Ltd.

[0198] Kunming mice were purchased from Guangxi Medical University.

[0199] The application will be further described below in conjunction with specific examples.

[0200] Example 1: Preparation of ZW-gal

[0201] This example provides a near-infrared fluorescent compound ZW-gal, which has the structure shown in the following formula I-a:

[0202]

[0203] Formula I-a.

[0204] The synthetic route of compound ZW-gal is shown as follows:

[0205]

[0206] The preparation method specifically comprises the following steps:

[0207] (a) Preparation of compound 1:

[0208] 5-bromo-2-(3,4-vinyl bis o-thiophene) formaldehyde (249.08 mg, 1 mmol) was dissolved in a mixed solvent of dioxane and water (6 mL, 1 / 1, v / v), and stirred until completely dissolved. 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-ol (337.67 mg, 1.25 mmol), tetrakis triphenyl phosphine palladium (57.778 mg, 0.05 mmol), and sodium carbonate (423.96 mg, 4 mmol) were sequentially added to a round-bottom flask. Nitrogen protection, 80°C reaction for 18 h. After the reaction was completed, the obtained reaction solution was filtered through diatomite, the liquid was rotary evaporated and separated and purified by silica gel column chromatography, and the developing agent was petroleum ether: ethyl acetate at 10:1 (by volume ratio). Compound 1 (yellow solid, 260 mg) was prepared at a yield of 83.33%. Compound 1 was accumulated by repeating the experiment.

[0209] (b) Preparation of compound 2:

[0210] Compound 1 (595.07 mg, 1 mmol) was placed in a round-bottom flask and dissolved in dichloromethane (20 mL). Bromotetraacetyl galactoside salt (616.8 mg, 1.5 mmol) and tetrabutylammonium hydrogen sulfate (40.30 mg, 0.125 mmol) were sequentially added. 10M sodium hydroxide was added to the reaction system under stirring at room temperature, and stirring was performed at room temperature for 3 h. After the reaction was completed, the reaction mixture was separated in dichloromethane and saturated brine. The organic phase was dried over anhydrous magnesium sulfate, and the solvent was extracted. The crude product was separated and purified by silica gel column chromatography, and the developing agent was dichloromethane:methanol at 200:1 (by volume ratio). Compound 2 (yellow solid, 403 mg) was prepared at a yield of 62.76%.

[0211] (c) Preparation of compound 3:

[0212] Compound 2 (642.14 g, 1 mmol) was placed in a round-bottom flask and dissolved in methanol (20 mL). Sodium methoxide (108.05 mg, 2 mmol) was added and stirred at 37°C for 30 min. After the reaction was completed, the precipitate was filtered and vacuum dried to obtain compound 3 (orange solid, 240 mg) at a yield of 50.58%.

[0213] (d) Preparation of compound ZW-gal:

[0214] Compound 3 (474.48 mg, 1 mmol) was dissolved in acetonitrile (20 mL) in a round-bottom flask, and then 1,2,3,3-tetramethyl-3H-indole iodide (361.4 mg, 1.2 mmol) and piperidine (21.29 mg, 0.25 mmol) were added sequentially. The mixture was stirred at 90 °C for 2 h. After the reaction was complete, the resulting reaction solution was filtered through diatomaceous earth, the liquid was evaporated to dryness, and purified by silica gel column chromatography with dichloromethane:methanol at a volume ratio of 20:1. High-purity ZW-gal (black solid, 220 mg) was obtained with a yield of 29.04%.

[0215] by nuclear magnetic resonance hydrogen spectrum ( 1 H NMR, carbon NMR ( 13 The structure of compound ZW-gal was characterized by C1NMR, high-resolution mass spectrometry (HRMS), and high-performance liquid chromatography. The structural determination results are as follows:

[0216] like Figure 1 As shown, 11H NMR (400 MHz, DMSO-d6) δ 8.38 (s, 1H), 8.29 (d, J = 15.6 Hz, 1H), 7.98 (d, J = 9.0 Hz, 1H), 7.91 (q, J = 8.8 Hz, 2H), 7.83 (dd, J = 7.7, 3.4 Hz, 2H), 7.60 (t, J = 7.6 Hz, 1H), 7.58 – 7.47 (m, 2H), 7.33 (dd, J = 8.9, 2.4 Hz, 1H), 7.16 (d, J = 15.6 Hz, 1H), 5.27 (d, J = 5.2 Hz, 1H), 5.04 (d, J = 7.7 Hz, 1H), 4.97 (d, J = 5.7 Hz, 1H), 4.77 (t, J = 5.6 Hz, 1H), 4.74– 4.38 (m, 5H), 4.00 (s, 3H), 3.76 (t, J = 4.0 Hz, 1H), 3.72 – 3.62 (m, 2H), 3.59 (q, J = 5.5 Hz, 1H), 3.53 (q, J = 5.5, 5.1 Hz, 1H), 3.47 (ddd, J = 9.1, 5.5, 2.8 Hz, 1H), 1.74 (s, 6H).

[0217] As Figure 2 shown 13 13C NMR (126 MHz, DMSO-d6) δ 180.02, 156.96, 149.57, 143.28, 142.43, 139.74, 134.59, 130.58, 129.42, 129.38, 129.14, 128.85, 128.51, 127.24, 126.33, 124.75, 123.19, 120.43, 114.82, 113.98, 110.80, 101.33, 76.06, 73.86, 70.81, 68.43, 66.49, 65.29, 60.68, 51.67, 34.04, 29.47, 26.55.

[0218] As Figure 3 shown, HRMS (ESI): m / z: Chemical Formula: C35H36INO8S, [M-I] + found 630.2346.

[0219] The purity of the compound ZW-gal as shown in Figure 4 is: 98.66%.

[0220] The preparation steps of compounds I-b and I-c can refer to compound I-a.

[0221] Example 2: Study on the spectral properties of compound ZW-gal

[0222] Experimental method:

[0223] (I) UV-Vis absorption spectrum and fluorescence emission spectrum of ZW-gal

[0224] ZW-gal or its β-gal hydrolysis product ZW-OH was prepared into a 10 mM stock solution with DMSO as the solvent; β-gal was prepared into a 1 U / μL stock solution with ultrapure water as the solvent. In order to carry out spectral measurement, the stock solution of ZW-gal or ZW-OH was diluted to 10 µM using PBS / DMSO mixed solvent (7:3, v / v). In the wavelength range of 200 ~ 900 nm, the ultraviolet-visible (UV-Vis) absorption spectrum of each diluted sample was recorded, and the maximum absorption wavelength of ZW-gal and ZW-OH was about 540 nm. Excitation at 540 nm to obtain the fluorescence spectrum of the two compounds.

[0225] As shown in Figure 5 (a) and (b), the compound ZW-gal has a maximum excitation wavelength E x max = 540 nm and a maximum fluorescence emission wavelength E m max = 665 nm, indicating that the molecule is a near-infrared fluorescent probe.

[0226] (II) Fluorescence emission spectrum of ZW-gal in response to enzyme

[0227] ZW-gal was mixed with PBS / DMSO (7:3, v / v) mixed solvent, then the corresponding enzyme (such as β-gal, etc.) was added, and incubation was carried out within 30 minutes. The final concentration of compound ZW-gal was 10 μM.

[0228] (III) Fluorescence emission spectrum of ZW-gal in response to viscosity

[0229] Under high viscosity conditions, the reaction mixture (ZW-gal or ZW-gal + β-gal) was mixed with glycerol. The final concentration of compound ZW-gal was 10 μM, and the volume ratio of glycerol in the high viscosity group was 50%.

[0230] As shown in Figure 5As shown in (c) and (d), the response to viscosity or β-gal alone can only unlock a portion of the fluorescence reporter capability of ZW-gal, exhibiting a clear dual-lock response characteristic. When β-gal combines with increased viscosity, the probe achieves a superimposed response, further enhancing near-infrared fluorescence emission, which holds promise for accurate reporting of pathological conditions.

[0231] like Figure 5 As shown in (e), with increasing β-gal concentration, the probe fluorescence intensity gradually increases or the maximum emission wavelength gradually red-shifts, reaching its maximum fluorescence intensity at a concentration of 0.013 U / mL. A linear regression analysis was performed on the fluorescence at the maximum emission wavelength against the enzyme concentration to obtain the probe's working curve, and its limit of detection (LOD) was calculated to be 2.72 × 10⁻⁶. -3 U / mL.

[0232] (iv) Fluorescence emission spectra of ZW-gal in response to β-gal and viscosity

[0233] The excitation wavelength was 540 nm, and the fluorescence intensity was measured to evaluate the dual-lock response properties of the probe compound ZW-gal. A 10 μM ZW-gal solution was added to glycerol-water mixed solutions (0%–90%) containing different concentrations of β-gal (10 U / mL), and the reaction was carried out at 37 °C for 15 min. The fluorescence intensity at 540 nm was measured to perform quantitative titration of ZW-gal against glycerol.

[0234] like Figure 5 As shown in (f), the ZW-OH generated by the reaction of ZW-gal and β-gal exhibits a 24.6-fold increase in fluorescence intensity at the maximum emission wavelength in response to viscosities (glycerol) ranging from 1.005 cP to 1412 cP. The increased ambient viscosity improves the rigidity of the probe structure, restricts the rotation of intramolecular covalent bonds in ZW-OH, suppresses the TICT effect to reduce nonradiative transitions, and thus significantly increases luminescence.

[0235] (v) Time-kinetic experiments of the probe compound ZW-gal

[0236] ZW-gal was mixed with PBS / DMSO (7:3, v / v) assay buffer (pH 4.5), then β-gal was added and incubated for 6 minutes. The excitation wavelength was 540 nm to complete the time-kinetic experiment of the probe compound ZW-gal.

[0237] like Figure 5 As shown in (g), the response of ZW-gal to β-gal is completed rapidly within 2 minutes, surpassing most similar probes, which is beneficial for the practical application of the probe compound ZW-gal in real-time cancer imaging.

[0238] (VI) Light stability data of the probe compound ZW-gal

[0239] ZW-gal was added to different solvent systems (including PBS / DMSO mixed solvent at a volume ratio of 7:3, PBS / DMSO mixed solution added with 10 U / ml of β-gal, PBS / DMSO mixed solution added with 50% of glycerol, and PBS / DMSO mixed solvent added with 10 U / ml of β-gal and 50% of glycerol) and the reaction was carried out at 37°C for 30 minutes. Subsequently, the fluorescence intensity was monitored every 1 minute for 1 hour under continuous laser irradiation (λex= 540 nm). Thus, the light stability data of the probe compound ZW-gal were obtained.

[0240] As shown in (h) of FIG. 6, Figure 5 ZW-gal showed excellent light stability under different conditions.

[0241] (VII) Polarity stability of the probe compound ZW-gal

[0242] ZW-gal was added to mixed solvents of different polarity (1,4-dioxane / water, 20% ~ 80%, v / v) and the fluorescence spectrum was measured using an excitation wavelength of 540 nm. Thus, the polarity stability data of the probe compound ZW-gal were obtained.

[0243] As shown in (i) of FIG. 6, Figure 5 the fluorescence of the probe compound ZW-gal was not affected by the environmental polarity in the range of 20% to 80% of dioxane, showing good polarity stability.

[0244] (VIII) Species selectivity of the probe compound ZW-gal

[0245] Finally, some biologically relevant species were added to a 10 μM ZW-gal solution and reacted at 37°C for 30 minutes. The fluorescence intensity at 665 nm was then measured using an excitation wavelength of 540 nm. Related species of β-gal include: L-glutamate (L-Glu), lysozyme, glutamine (Gln), L-arginine (L-Arg), phospholipase, tyrosine (Tyr), bovine serum albumin (BSA), H2O2, carboxylesterase, trypsin, elastase, leucine (Leu), alanine (Ala), tert-butyl hydroperoxide (TBHP), L-cysteine ​​(Cys), glucose oxidase (GOD), CuBr2, glutathione (GSH), NaSH, α-chymotrypsin, urea derivatives (Ureas), L-aspartic acid (Asp), vitamin C, glycine (Gly), and FeSO4. In a PBS / DMSO mixed solvent at a volume ratio of 7:3, the concentration of β-gal was 10 U / ml, and the concentration of other species was 100 μM. This was used to evaluate the species selectivity of the probe compound ZW-gal.

[0246] like Figure 6 As shown in (j), ZW-gal does not react with other substances and does not cause interference. ZW-gal only responds in the presence of β-galactosidase (β-gal), exhibiting a redshift of the maximum emission wavelength to the near-infrared I region (665 nm) and enhanced fluorescence intensity. When glycerol is added to the test system to increase viscosity, the maximum emission wavelength of ZW-gal remains in the near-infrared I region (665 nm), but the fluorescence intensity is further amplified. This indicates that ZW-gal has good specificity for β-galactosidase, and the fluorescence signal increases with increasing system viscosity. This suggests that ZW-gal has good species selectivity and can be used for subsequent tests.

[0247] The above experiments comprehensively verify that ZW-gal has excellent optical properties and a dual-lock response to viscosity and β-gal, and has good potential for complex pathological imaging.

[0248] Example 3: Cytotoxicity test of compound ZW-gal

[0249] MTT method was used to determine the cytotoxicity of ZW-gal. HepG2 cells were inoculated in 96-well plates, about 5000 cells per well, 150 μL of culture solution, and incubated at 37°C for 24 h. Then different concentrations (0, 10, 20, 30, 40, 50 μM) of probe (ZW-gal) solution were added to each well, and incubated for 24 h. 5 μg / mL of MTT solution was added to each well and incubated for 4 h. After discarding the culture medium, 150 μL of DMSO was added to dissolve the formaldehyde crystals. Finally, the absorbance (OD) at 490 nm was measured.

[0250] Cell viability (%) = [(OD test - OD blank) / (OD control - OD blank )] × 100%.

[0251] The results are shown in Figure 7

[0252] When the concentration of ZW-gal was below 20 μM, the survival rate of HepG2 cells was above 80%. ZW-gal at 10 μM had no cytotoxicity, and the cell survival rate remained at 100%, which could be used for subsequent experiments.

[0253] Example 4: Verification of the dual-lock responsiveness of compound ZW-gal at the cell level

[0254] All groups of HepG2 cells were incubated with ZW-gal; exogenous addition of β-gal was selected to increase the content of intracellular β-gal, D-gal was added as a β-gal inhibitor, and Dex was used to induce an increase in intracellular viscosity. HepG2 cells were divided into four groups: exogenous addition of β-gal (β-gal group), D-gal inhibition of β-gal (D-gal group), Dex-induced increase in viscosity (Dex group), and combination of β-gal and Dex (β-gal + Dex group).

[0255] The results are shown in Figure 8

[0256] ​​D-gal pretreatment reduced the fluorescence intensity of ZW-gal in HepG2 to 16% of the control group. While β-gal pretreatment increased the fluorescence intensity to 1.68 times of the control group, which indicated that the probe was responsive to β-gal at the cellular level. Compared with the control group, the intracellular fluorescence intensity increased to 1.63 times after dexamethasone treatment, indicating that the intracellular probe still had viscosity response capability. When β-gal and dexamethasone were used to stimulate HepG2 cells at the same time to increase the intracellular β-gal content and viscosity, the fluorescence emission of ZW-gal could reach 4.53 times of the control group. The two-factor synergistic unlocking of the probe fluorescence significantly increased the fluorescence intensity (P < 0.0001). These results showed that ZW-gal was a double-locked probe at the cellular level, and the simultaneous increase of β-gal activity and viscosity was beneficial to complete activation.

[0257] Example 5: Application of compound ZW-gal for near-infrared fluorescence imaging of tumor cells and their state changes during chemotherapy

[0258] Human hepatoblastoma cell line (HepG2), human ovarian adenocarcinoma cell line (SKOV-3), human gastric cancer cell line (SGC-7901) were used as cancer cell model group, and human normal liver cell line (LO2), human embryonic kidney cell line (293T), human gastric mucosa epithelial cell line (GES-1) were used as normal control group. Fluorescence imaging of cancer cells and normal cells was performed by incubating the probe compound ZW-gal (10 μM, 30 min). During chemotherapy, high-concentration chemotherapeutic drugs directly kill cancer cells, and at the same time, part of the cancer cells enter the aging program, and the senescent cancer cells bring more malignant phenotype changes. Therefore, high-dose doxorubicin (DOX) was selected to induce cancer cell death, and low-dose DOX was selected to induce cancer cell senescence. Low-concentration DOX (200 nM) was used to construct a senescent cancer cell model, and the senescent model was verified to be successfully constructed by cell morphology and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) staining, as shown in (a) of Figure 8 .

[0259] Subsequently, the probe was incubated to perform fluorescence imaging of cancer cells and their senescence. High-concentration DOX (2 μM) was selected to stimulate HepG2 cancer cells, and ZW-gal was used to image the nuclear morphology to monitor the death of cancer cells during chemotherapy.

[0260] The results are shown in Figure 8 .

[0261] As shown in (b) and (c) of Figure 8 , the fluorescence intensity of the probe in cancer cells was significantly higher than that in non-cancer cells, and the probe could be widely used in various cancer cell types.

[0262] As shown in (d) and (e) of Figure 8 , low-dose chemotherapy drug doxorubicin (DOX) mimics chemotherapy drug to induce cancer cell senescence, and the imaging of the research probe in this application scenario is studied. A low concentration of DOX (200 nM) is used to construct a senescent cancer cell model, and the successful construction of the senescent model is verified by cell morphological flattening, vacuolization, and X-gal staining. Then, ZW-gal probe incubation is performed. Confocal imaging results show that when cancer cells undergo senescence, the emission of the probe is further significantly enhanced, which may be due to the increased expression of β-gal in senescent cancer cells and the increased cell viscosity.

[0263] As shown in (f) of Figure 9 , under the fluorescence imaging of ZW-gal, 2 μM of DOX induces the nuclei of HepG2 cells to undergo karyopyknosis, karyorrhexis, and karyolysis within 24 h, and the cell membrane structure disappears after 12 h, the cell contents leak, and the cell death mode presents obvious "necrosis" characteristics. This indicates that ZW-gal has good imaging stability and can outline the nuclear fluorescence image to intuitively and conveniently monitor the state and death process of cancer cells during chemotherapy.

[0264] In summary, ZW-gal can be used to distinguish various cancer cells from normal cells, image chemotherapy-induced tumor senescence, and also be used to track the state of cancer cells and monitor their death during chemotherapy. As a dual-lock probe of β-gal and viscosity, ZW-gal exhibits the significant advantage of "one needle with multiple uses".

[0265] Example 6: Application of dual-lock probe ZW-gal in specific recognition of liver cancer and its senescence in mouse models

[0266] A liver cancer model was constructed using mouse liver cancer (H22) cell lines: mouse liver cancer H22 (3 × 10 7 / 100 μL) was inoculated in the axillary fossa of Kunming mice, and when the tumor volume reached 100 mm 3 , DOX (10 mg / kg, 100 μL) was injected into the tail vein to induce senescence in one group, and the remaining mice were injected with the same dose of saline (Saline). Organ X - gal staining verified the successful construction of the tumor senescence model. Then, 100 μM, 100 μL of ZW-gal molecules were injected into each tumor (non-senescent), and fluorescence imaging was performed using a small animal live imaging device, and 100 μM, 100 μL of ICG molecules were injected into each tumor (non-senescent) as a control study, and fluorescence imaging was performed using a small animal live imaging device as Figure 9(a) and (b) of FIG. 1. In addition, the tumor margin was outlined by ZW-gal fluorescence imaging, and the intraoperative hepatocellular carcinoma was removed. Hematoxylin-eosin (H&E) staining was used to determine whether ZW-gal could effectively identify the tumor and assist in complete tumor resection.

[0267] The results are shown in FIG. 2. Figure 10 As shown in FIG. 2. Figure 9

[0268] As shown in FIG. 2. Figure 9 As shown in FIG. 2. tumor As shown in FIG. 2. normal The I / I ratio slowly increased and reached a maximum value of 1.85 at 1 h. The relatively slow response speed and the lower I / I ratio made ICG difficult to meet the application requirements of real-time and accurate imaging of fluorescence probes. tumor As shown in FIG. 2. normal The I / I ratio slowly increased and reached a maximum value of 1.85 at 1 h. The relatively slow response speed and the lower I / I ratio made ICG difficult to meet the application requirements of real-time and accurate imaging of fluorescence probes. tumor As shown in FIG. 2. normal The I / I ratio slowly increased and reached a maximum value of 1.85 at 1 h. The relatively slow response speed and the lower I / I ratio made ICG difficult to meet the application requirements of real-time and accurate imaging of fluorescence probes. tumor As shown in FIG. 2. normal The I / I ratio slowly increased and reached a maximum value of 1.85 at 1 h. The relatively slow response speed and the lower I / I ratio made ICG difficult to meet the application requirements of real-time and accurate imaging of fluorescence probes.

[0269] The success of the construction of the chemotherapy-induced tumor senescence model was verified by ex vivo organ X-gal staining (FIG. 3d). Figure 9 As shown in FIG. 3. tumor As shown in FIG. 3. normal The I / I ratio slowly increased and reached a maximum value of 1.85 at 1 h. The relatively slow response speed and the lower I / I ratio made ICG difficult to meet the application requirements of real-time and accurate imaging of fluorescence probes. tumor As shown in FIG. 3. normal The I / I ratio slowly increased and reached a maximum value of 1.85 at 1 h. The relatively slow response speed and the lower I / I ratio made ICG difficult to meet the application requirements of real-time and accurate imaging of fluorescence probes. Figure 9 As shown in FIG. 3. Figure 9 ​ZW-gal as a dual-lock probe of β-gal and viscosity can achieve in vivo imaging of tumor aging in 5 min and distinguish imaging of tumor in 15 min, and has good targeting in vivo, and can be used for rapid, convenient, non-invasive and real-time monitoring of tumor and its aging, evaluation of the treatment effect of chemotherapeutic drugs and guidance of drug treatment regimen.

[0270] As shown in (e) of the figure, Figure 10 As shown in (f) of the figure, ZW-gal can clearly illuminate the tumor site by spraying, and under the guidance of the fluorescence signal, the tumor can be completely removed. The fluorescence intensity of the tumor and its surrounding normal tissue has obvious difference.

[0271] As shown in (e) of the figure, ​ As shown in (f) of the figure, ZW-gal can clearly illuminate the tumor site by spraying, and under the guidance of the fluorescence signal, the tumor can be completely removed. The fluorescence intensity of the tumor and its surrounding normal tissue has obvious difference.

[0272] In summary, ZW-gal can not only be used for real-time monitoring of tumor and its aging in vivo imaging in a mouse model, but also can be used for fluorescence surgery navigation to better assist complete removal of the tumor by outlining the tumor boundary, and can be used as a practical, convenient and economical fluorescence tool for multiple clinical scenarios in the future.

[0273] The above is a schematic description of the present application and its embodiments, which is not restrictive, and the embodiments shown in the examples are only one of the embodiments of the present application, and the actual embodiments are not limited thereto. Therefore, if a person skilled in the art is inspired thereby, without departing from the spirit of the present application, similar embodiments and examples can be designed without creative design, which should belong to the protection scope of the present application.

Claims

1. A compound having the general formula shown in Formula I: Formula I; The X - Selected from Cl - , Br - Or I - .

2. A compound having the structure shown in formula I-a: Formula I-a.

3. The compound of claim 2, wherein the compound has a maximum absorption wavelength and a maximum fluorescence emission wavelength in the range of 500 nm to 800 nm.

4. A composition comprising the compound of any one of claims 1 to 3, and a pharmaceutically acceptable carrier or excipient.

5. Use of the compound of claim 1 in the preparation of near-infrared imaging reagents.

6. Use of the compound of claim 1 for preparing a reagent for detecting human β-galactosidase.

7. Use of the compound of claim 1 in the preparation of a reagent for detecting viscosity.

8. Use of the compound of claim 1 in the preparation of a reagent for detecting human β-galactosidase and viscosity.

9. The use of the compound of claim 1 in the preparation of a reagent for detecting tumor cells, wherein, The reagent is capable of imaging at least one type of tumor cell; The tumor cells have characteristics selected from the following: Tumor cells characterized by increased viscosity; and / or Tumor cells characterized by increased human β-galactosidase activity.

10. The use according to claim 9, wherein the tumor cells are selected from any one or more of liver cancer cells, ovarian cancer cells, gastric cancer cells, colon cancer cells, breast cancer cells, or head and neck cancer cells.

11. Use of the compound of claim 1 or the composition of claim 4 in the preparation of an image-guided agent for administering surgery to a subject with a disease, wherein, The reagent is exposed to excitation light at a designated surgical site to induce visualization; the disease is cancer.

12. The use according to claim 11, wherein the wavelength of the excitation light is in the range of 500 nm to 600 nm.

13. The method for preparing the compound according to claim 2, characterized in that, Includes the following steps: (1) Compound 5-bromo-2-(3,4-vinyldioxythiophene) formaldehyde and 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)naphthalene-2-ol were reacted to give compound 1; The structural formula of compound 1 is shown below: (2) Compound 1 reacts with bromotetraacetylgalactoside to give compound 2; The structural formula of compound 2 is shown below: (3) Compound 2 was deprotected to obtain compound 3; The structural formula of compound 3 is shown below: (4) Compound 3 reacts with 1,2,3,3-tetramethyl-3H-indole iodide to give the compound shown in formula Ia: Formula Ia.

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