Design, preparation and application of targeted activated enriched near-infrared probe and photosensitizer

Abstract

The invention provides a design, a preparation method and application of a targeted activated enriched near-infrared probe and a photosensitizer. Specifically, the invention provides a novel molecular design, preparation and application method, so that the fluorescence signal or photosensitive performance can be started under the action of disease-related enzyme or chemical environment, and the fluorescence signal or photosensitive performance can be effectively enriched at the periphery of disease-related cells and tissues. The compound is shown as a formula I, wherein each variable is defined in the text. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the fields of biomarkers and biomedicine, and particularly relates to the design, preparation and application of targeted activation enrichment type near-infrared probes and photosensitizers. Background Art

[0002] The abnormal expression of enzymes or disease microenvironment factors (such as ROS) is closely related to the occurrence and development of many diseases. For example, the level of alkaline phosphatase (ALP) in the human body is closely related to various diseases such as cancer and liver injury. The activity of γ-glutamyl transpeptidase (GGT) is abnormal in diseases such as hepatotoxicity, asthma, diabetes and cancer. N-acetyl-β-D-glucosaminidase (NAG) has been proven to be a biomarker of kidney injury in clinical practice. The abnormal expression of carboxylesterase (CE) in physiology and pathology is closely related to the occurrence of liver cancer. The disease microenvironment factor H 2 O 2 is related to diseases of liver injury and kidney injury,

[0003] and is closely related to kidney injury. Living organisms are extremely complex systems that are constantly undergoing dynamic changes. Therefore, realizing the real-time in-vivo monitoring of enzyme activity and ROS generation in living organisms is of great significance for disease prevention, early diagnosis and treatment.

[0004] To achieve highly sensitive and real-time detection of enzyme activity and ROS, based on the ability of enzymes and ROS to specifically catalyze the conversion of their substrates into products, fluorescence-enhanced probes activated by enzymes and ROS have been developed. Such probes generally consist of a fluorophore and an enzyme or ROS recognition group. The fluorescence signal of the fluorophore in this type of probe is in the off state due to the quenching effect of the recognition group. During detection, when the target enzyme or ROS specifically hydrolyzes the recognition group, the fluorescence turns on, thereby achieving an increase in the intensity of the fluorescence signal or a change in the absorption and emission wavelengths of the fluorescence, and simultaneously realizing the catalytic amplification of the fluorescence signal. Enzyme- or ROS-activated fluorescence-enhanced probes generally detect the sensitivity of the probe to enzymes in solution, and the detection limit of the probe is detected through enzyme kinetics. However, different from the relatively homogeneous, closed, and static general detection solution, living organisms are a more complex, highly dynamic, and open heterogeneous environment. After being activated by enzymes or ROS, the fluorescent signal molecules are prone to be interfered by the dynamic physiological environment, quickly diffuse from the detection site, and be excreted from the living organism, resulting in a decrease in the signal-to-noise ratio and sensitivity of the enzyme or ROS activity detection by the fluorescent probe. General fluorescence-enhanced probes do not have the ability to retain signals. During the imaging of enzymes or ROS in living animals, especially enzymes located outside the cell membrane, such as glutamyl transferase, alkaline phosphatase, etc., the activated fluorescent signal molecules are directly in contact with the blood and are more likely to quickly diffuse from the enzyme detection site and be excreted from the body with the blood flow, resulting in a decrease in the sensitivity of the fluorescent probe when detecting enzymes or ROS in living animals and making it difficult to achieve the expected fluorescence imaging effect. Therefore, in the complex, dynamic, and open living organisms, the application of fluorescent probes is more challenging. Therefore, developing near-infrared fluorescent probes that can achieve great enrichment at the detection site is of great significance for improving the detection sensitivity of enzyme activity or ROS in vivo.

[0005] The special catalytic activity of enzymes not only plays an important role in the diagnosis of diseases but also has a huge impact on the treatment of diseases. Photodynamic therapy (PDT) has currently become an important method for clinical tumor treatment due to its minimally invasive nature, negligible drug resistance, and good treatment effect. Photodynamic therapy mainly induces the death of tumor cells by generating highly toxic reactive oxygen species (ROS) in tumor tissues under the excitation of light with a specific wavelength by photosensitizers. Therefore, the targeted delivery of photosensitizers in living tumor tissues is one of the keys to photodynamic therapy. To achieve this goal, a series of activatable photosensitizers based on tumor-related enzyme activities have been successively developed, achieving selective activation in tumor tissues. However, since living animals are a highly dynamic living system, the activated photosensitizers are prone to be quickly cleared from the tumor tissues, significantly reducing the effect of photodynamic therapy. Therefore, achieving effective enrichment of photosensitizers in tumor tissues is of great significance for improving the photodynamic therapy effect and specificity of photosensitizers.

[0006] In summary, there is an urgent need in the art to develop a class of probes and photosensitizers that can be enriched at the target site. SUMMARY OF THE INVENTION

[0007] The object of the present invention is to provide a design, preparation method and application of a class of targeted activation-enriched near-infrared probes and photosensitizers. Specifically, the present invention provides a class of novel molecular design, preparation and application methods, which can turn on the fluorescence signal or photosensitive performance under the action of disease-related enzymes or chemical environments, and can be effectively enriched around disease-related cells and tissues.

[0008] In the first aspect of the present invention, a compound is provided, and the compound is shown as Formula I;

[0009]

[0010] In Formula I,

[0011] R 1 is a group that can be specifically hydrolyzed by a biomacromolecular enzyme or a group that can be specifically activated by a disease microenvironment factor; and when specifically hydrolyzed or specifically activated, R 1 and X 1 the bond between them is broken and R 1 leaves;

[0012] X 1 is O or NH;

[0013] L is a linking group that can leave by itself after R 1 leaves;

[0014] R 2 is a water-soluble group;

[0015] FP is a fluorescent group or a photosensitive group.

[0016] In another preferred example, the compound can be used as a probe or a photosensitizer; preferably, a far-infrared probe or a photosensitizer.

[0017] In another preferred example, there is at least one aromatic ring in FP, and are respectively connected to adjacent ring atoms on the aromatic ring.

[0018] In another preferred example, the aromatic ring is a benzene ring or a 5- or 6-membered heteroaromatic ring.

[0019] In another preferred example, the aromatic ring is an aromatic ring fused with other rings.

[0020] In another preferred example, the aromatic ring is a benzene ring.

[0021] In another preferred example, the aromatic ring is the benzene ring in the fused ring shown by the following formula

[0022]

[0023] wherein, X 1 is O or S.

[0024] In another preferred example, the ability to be specifically hydrolyzed by a biomacromolecular enzyme means that: when R 1 is a group that can be specifically hydrolyzed by a biomacromolecular enzyme, the biomacromolecular enzyme can recognize this group, and under the action of the biomacromolecular enzyme, X 1 -R 1 is hydrolyzed so that the bond between X 1 and R 1 is broken.

[0025] In another preferred example, the biomacromolecular enzyme includes: alkaline phosphatase, γ-glutamyl transpeptidase, aminopeptidase, Fapα enzyme, Hepsin enzyme.

[0026] In another preferred example, the enzyme recognition groups that can be specifically hydrolyzed by alkaline phosphatase include:

[0027]

[0028] In another preferred example, the enzyme recognition groups that can be specifically hydrolyzed by γ-glutamyl transpeptidase include:

[0029]

[0030] In another preferred example, the enzyme recognition groups that can be specifically hydrolyzed by aminopeptidase include:

[0031]

[0032] In another preferred example, the enzyme recognition groups that can be specifically hydrolyzed by Fapα enzyme include:

[0033]

[0034] In another preferred example, the enzyme recognition groups of Hepsin enzyme include:

[0035]

[0036] In another preferred example, the enzyme recognition groups that can be specifically hydrolyzed by a biomacromolecular enzyme are selected from the following group:

[0037]

[0038] In another preferred embodiment, the ability to be specifically activated by disease microenvironment factors means that when R 1 is a group capable of being specifically activated by disease microenvironment factors, under the action of disease microenvironment factors, X 1 and R 1 The bond between them can be broken.

[0039] In another preferred embodiment, the disease microenvironment factors include: reactive oxygen species (ROS).

[0040] In another preferred embodiment, the reactive oxygen species (ROS) include: peroxides (such as H 2 O 2 , ONOO - ), superoxide anion

[0041] In another preferred embodiment, the groups that can be specifically activated by peroxides include:

[0042]

[0043] In another preferred embodiment, the groups that can be specifically activated by superoxide anion include:

[0044]

[0045] In another preferred embodiment, the group capable of being specifically activated by disease microenvironment factors is selected from the group consisting of:

[0046]

[0047] In another preferred embodiment, R 1 is selected from the group consisting of:

[0048]

[0049] In another preferred embodiment, L is selected from the group consisting of:

[0050] In another preferred embodiment, L is selected from the group consisting of: wherein, * represents the connection position with X 1 .

[0051] In another preferred embodiment, X 1 is O, and L is selected from the group consisting of:

[0052] In another preferred embodiment, X 1 is NH, and L is

[0053] In another preferred embodiment, R 2is a water-soluble group linked to the carbamide. When the compound is activated, the carbamide bond breaks and the water-soluble group leaves.

[0054] In another preferred example, the water-soluble group includes: an anion-cation pair (such as ), a group containing at least three carboxyl functional groups (such as ), a group containing at least two sulfonic acid functional groups (SO 3 - ), (such as ).

[0055] In another preferred example, R 2 is selected from the group consisting of:

[0056]

[0057] In another preferred example, the FP moiety has a structure shown in Formula II-1 or Formula II-2:

[0058]

[0059] Wherein,

[0060] a represents the position connected to ;

[0061] b represents the position connected to ;

[0062] R 3 is -C 1-6 alkyl, -C 1-6 alkyl-R Fu ; wherein, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu is selected from the group consisting of: -COOH, -N 3 , -NH 2 , -N + (C 1-6 alkyl) 3 );

[0063] R 4 、R 5 、R 6 and R 7 are each independently H or a heavy atom I, Br;

[0064] R 8 is H, F, Cl, or Br;

[0065] X 2 is O or S;

[0066] X 3are I, Br, and Cl.

[0067] In another preferred example, the FP moiety has a structure shown in Formula III-1 or III-2:

[0068]

[0069] wherein,

[0070] a represents the position connected to ;

[0071] b represents the position connected to ;

[0072] R 3 is -C 1-6 alkyl, -C 1-6 alkyl-R Fu ; wherein, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu is selected from the group consisting of: -COOH, -N 3 , -NH 2 , -N + (C 1-6 alkyl) 3 );

[0073] R 4 、R 5 、R 6 and R 7 are each independently H or a heavy atom (such as I, Br);

[0074] R 8 is H, F, Cl, or Br;

[0075] X 2 is O or S;

[0076] X 3 is I, Br, or Cl;

[0077] In another preferred example, the FP moiety has a structure shown in Formula IV-1 or IV-2:

[0078]

[0079] wherein,

[0080] a represents the position connected to ;

[0081] b represents the position connected to ;

[0082] X 2is O or S;

[0083] X 4 is O or NR 3 ;

[0084] R 3 is -C 1-6 alkyl, -C 1-6 alkyl-R Fu ; wherein, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu is selected from the group consisting of: -COOH, -N 3 -, -NH 2 -, -N + (C 1-6 alkyl) 3 );

[0085] R 4 、R 5 、R 6 and R 7 are each independently H or a heavy atom (such as I, Br);

[0086] R 8 is H, F, Cl, or Br.

[0087] In another preferred example, the FP moiety has a structure shown in Formula V-1 or V-2:

[0088]

[0089] wherein,

[0090] a represents the position connected to ;

[0091] b represents the position connected to ;

[0092] X 2 is O or S;

[0093] X 5 is O or S;

[0094] R 9 is NEt 2 、NPh 2 、or NCH 3 Ph;

[0095] R 8 is H, F, Cl, or Br.

[0096] In another preferred example, the FP moiety has a structure shown in Formula VI-1 or VI-2:

[0097]

[0098] Among them,

[0099] a represents the position connected to the connection position;

[0100] b represents the position connected to the connection position;

[0101] X 2 is O or S;

[0102] X 5 is O or S;

[0103] R 10 and R 11 each independently represents C 1-6 alkyl (such as Et) or Ph;

[0104] R 8 is H, F, Cl or Br.

[0105] In another preferred example, the water-soluble group is a water-soluble group that can be connected to the carbamide structure. When the compound is activated, the carbamide breaks and the water-soluble group leaves.

[0106] In another preferred example, the exemplary compound is selected from Table A

[0107] Table A

[0108]

[0109]

[0110] In the second aspect of the present invention, a probe is provided, wherein the probe is a compound represented by Formula I; wherein, X 1 , X 2 , R 1 and L are defined as in the first aspect; and the FP moiety is defined as follows.

[0111] In another preferred example, the FP moiety has a structure represented by Formula II-1 or II-2; wherein, R 4 , R 5 , R 6 and R 7 are all H, and a, b, R 3 , R 8 , X 2 and X 3 are defined as in the first aspect.

[0112] In another preferred example, the FP moiety has a structure represented by Formula III-1 or III-2; wherein, R4 , R 5 , R 6 and R 7 are all H, and a, b, R 3 , R 8 , X 2 and X 3 are as defined in the first aspect.

[0113] In another preferred example, the FP moiety has the structure shown in Formula IV-1 or IV-2; wherein, R 4 , R 5 , R 6 and R 7 are all H, and a, b, R 8 , X 2 and X 4 are as defined in the first aspect.

[0114] In another preferred example, the FP moiety has the structure shown in Formula V-1 or V-2; wherein, a, b, R 8 , R 9 , X 2 and X 5 are as defined in the first aspect.

[0115] In another preferred example, the FP moiety has the structure shown in Formula VI-1 or VI-2; wherein, a, b, R 8 , R 10 , R 11 , X 2 and X 5 are as defined in the first aspect. In the third aspect of the present invention, a photosensitizer is provided, wherein the photosensitizer is a compound shown in Formula I; wherein, X 1 , X 2 , R 1 and L are as defined in the first aspect; and the FP moiety is defined as follows.

[0116] In another preferred example, the FP moiety has the structure shown in Formula II-1 or II-2; wherein, R 4 , R 5 , R 6 and R 7 are each independently H or a heavy atom, and at least one of R 4 , R 5 , R 6 and R 7 is a heavy atom; a, b, R 3 , R 8 , X 2 and X 3 are as defined in the first aspect.

[0117] In another preferred example, the FP moiety has the structure shown in Formula III-1 or III-2; wherein, R 4 , R 5 , R 6 and R 7 are each independently H or a heavy atom, and at least one of R 4 , R 5 , R 6 and R 7 is a heavy atom; a, b, R 3 , R 8 , X 2 and X 3 are as defined in the first aspect.

[0118] In another preferred example, the FP moiety has the structure shown in Formula IV-1 or IV-2; wherein, R 4 , R 5 , R 6 and R 7 are each independently H or a heavy atom, and at least one of R 4 , R 5 , R 6 and R 7 is a heavy atom; a, b, R 8 , X 2 and X 4 are as defined in the first aspect.

[0119] In another preferred example, 1, 2 or 3 of R 4 , R 5 , R 6 and R 7 are heavy atoms.

[0120] In another preferred example, the heavy atoms are each independently I or Br.

[0121] In another preferred example, the photosensitizer is a near-infrared photosensitizer.

[0122] In the fourth aspect of the present invention, a composition is provided, wherein the composition comprises:

[0123] (1) The compound as described in the first aspect; and

[0124] (2) A pharmaceutically or detectably acceptable carrier.

[0125] In another preferred example, the composition is a probe composition.

[0126] In another preferred example, the composition is a photosensitizer composition.

[0127] In a fifth aspect of the present invention, there is provided a probe composition, wherein the probe composition comprises:

[0128] (1) a compound as described in the second aspect; and

[0129] (2) a pharmaceutically or detectably acceptable carrier.

[0130] In a sixth aspect of the present invention, there is provided a photosensitizer composition, wherein the photosensitizer composition comprises:

[0131] (1) a compound as described in the third aspect; and

[0132] (2) a pharmaceutically or detectably acceptable carrier.

[0133] In a seventh aspect of the present invention, there is provided a use of a compound as described in the first aspect or a pharmaceutically or detectably acceptable salt thereof, or a probe as described in the second aspect, or a composition as described in the fourth aspect, or a probe composition as described in the fifth aspect, wherein the use is one or more selected from the following groups:

[0134] (a) for the detection of an enzyme (preferably, the detection of the enzyme is non-therapeutic and non-diagnostic in vitro);

[0135] (b) for the detection of disease microenvironment factors (such as ROS) (preferably, the disease microenvironment factors (such as ROS) are non-therapeutic and non-diagnostic in vitro);

[0136] (c) for the preparation of a detection reagent for the detection of an enzyme or disease microenvironment factors (such as ROS);

[0137] (d) for the imaging of cells (preferably, the imaging of the cells is non-therapeutic and non-diagnostic in vitro);

[0138] (e) for the preparation of a detection reagent for cell imaging analysis;

[0139] (f) for tumor tissue or cell imaging (preferably, the imaging of the cells is non-therapeutic and non-diagnostic in vitro);

[0140] (g) for the preparation of a detection reagent for tumor tissue or cells;

[0141] In another preferred example, the enzyme includes: alkaline phosphatase, γ-glutamyl transpeptidase, aminopeptidase, Fapα enzyme, Hepsin enzyme.

[0142] In another preferred example, the detection of the enzyme includes the detection of the expression level of the enzyme and / or the detection of the activity of the enzyme.

[0143] In another preferred example, the detection reagent can be used to image tumor tissues or cells or detect the expression level or activity of cellular enzymes in a living subject.

[0144] In an eighth aspect of the present invention, there is provided a use of the compound as described in the first aspect or a pharmaceutically or detectably acceptable salt thereof, or the photosensitizer probe as described in the third aspect, or the composition as described in the fourth aspect, or the photosensitizer composition as described in the sixth aspect, wherein the use is one or more selected from the following groups:

[0145] (h) for photodynamic therapy of tumors;

[0146] (i) for preparing a photosensitizing therapeutic agent for photodynamic therapy of tumors.

[0147] In a ninth aspect of the present invention, there is provided a detection kit,

[0148] The kit includes: the compound as described in the first aspect or a pharmaceutically or detectably acceptable salt thereof, or the composition as described in the fourth aspect.

[0149] In a tenth aspect of the present invention, there is provided a method for imaging cells or tissues, the method comprising the steps of: incubating the cells or tissues to be imaged in the presence of the compound as described in the first aspect, and obtaining an image of the cells or tissues by an instrument (such as a fluorescence microscope).

[0150] In another preferred example, the method is non-therapeutic in vitro.

[0151] In an eleventh aspect of the present invention, there is provided a method for in vivo imaging, the method comprising the steps of:

[0152] (i) administering to a subject in need the compound as described in the first aspect or a pharmaceutically or detectably acceptable salt thereof, or the composition as described in the fourth aspect; and

[0153] (ii) imaging using an instrument.

[0154] In another preferred example, the detection subject is a human or non-human mammal (preferably including non-human primates or rodents such as rats or mice).

[0155] In another preferred example, the in vivo imaging is real-time imaging or static imaging; preferably, it is real-time imaging.

[0156] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0157] Figure 1.1 Shows the mechanism of action of the probe or photosensitizer of the present invention.

[0158] Figure 2.1 Shows the solution enzyme response verification of the near-infrared fluorescence probe ALPIN-6

[0159] Figure 2.2 Shows the imaging comparison of the near-red fluorescence probes ALPIN-6, ALPIN-5, ALPIN-4 on HeLa cells overexpressing alkaline phosphatase (ALP) and the imaging comparison of HeLa cells and HEK293 with weak ALP expression.

[0160] Figure 2.3 Shows the real-time imaging of HeLa cells by the near-red fluorescence probe ALPIN-6.

[0161] Figure 2.4 Shows the specific imaging of the near-red fluorescence probe ALPIN-6 on mixed HeLa and HEK293 cells.

[0162] Figure 2.5 Shows the real-time imaging of the near-infrared fluorescence probe ALPIN-6 on tumor-bearing mice loaded with HeLa cells.

[0163] Figure 2.6 Shows the calculation of the enrichment content of the near-red photosensitizer ALPIN-6I in HeLa cells.

[0164] Figure 2.7 Shows the ROS generation effect of the photosensitizer ALPIN-6I in HeLa cells.

[0165] Figure 2.8 Shows the effect of the photosensitizer ALPIN-6I on killing tumor cells.

[0166] Figure 2.9 Shows the effect of specifically killing tumor cells by the photosensitizer ALPIN-6I in mixed cells.

[0167] Figure 2.10 Shows the real-time imaging of the near-infrared photosensitizer ALPIN-6I on tumor-bearing mice loaded with HeLa cells.

[0168] Figure 2.11 Is the photodynamic therapy of the near-infrared photosensitizer ALPIN-6I on tumor-bearing mice loaded with HeLa cells.

[0169] Figure 3.1 Shows the imaging comparison of the near-red fluorescence and photoacoustic probes ALPIN-7, ALPIN-8 on HeLa cells overexpressing alkaline phosphatase (ALP) and the imaging of HEK293 with weak ALP expression.

[0170] Figure 3.2 It shows the real-time imaging of the near-infrared fluorescence photoacoustic probe ALPIN-7 on HeLa cells.

[0171] Figure 3.3 It shows the real-time imaging of the near-infrared fluorescence photoacoustic probe ALPIN-7 on tumor-bearing mice with HeLa cells. Specific embodiments

[0172] After long-term and in-depth research, the inventor of the present invention, in order to improve the diagnosis and treatment effects of probes and photosensitizers in diseases, for the first time developed a class of near-infrared probes and photosensitizers with the functions of activation and enrichment at the detection target based on the design strategy of methylenebenzoquinone, that is, under the primary trigger of a target enzyme or a factor in the target microenvironment, etc., the probes such as near-infrared probes or photosensitizers in the unactivated state can be realized. The probes or photosensitizers of the present invention can: 1) change from high hydrophilicity and low cell membrane permeability to high lipophilicity and cell membrane permeability, thereby reducing their clearance rate and enhancing the retention efficiency in the target; 2) anchor the fluorescent group or photosensitizer by forming a covalent bond through the reaction of the methylenebenzoquinone intermediate with macromolecules such as proteins or nucleophiles in cells; 3) activate the near-infrared fluorescence or photoacoustic signal or activate the photosensitizing activity under near-infrared light. This method greatly enhances the enrichment of signal molecules at the detection site and improves the detection sensitivity of near-infrared fluorescence probes and the photodynamic therapy efficiency of photosensitizers. Based on this, the inventor completed the present invention.

[0173] Terms

[0174] In this article, unless otherwise specified, the terms or abbreviations used have the general meanings well-known to those skilled in the art. For example, Ph represents phenyl.

[0175] Unless otherwise stated, the term "alkyl" itself or as part of another substituent A (such as the alkyl part in alkoxy, etc.) refers to a straight-chain or branched-chain hydrocarbon group with a specified number of carbon atoms (i.e., C 1-6 represents 1-6 carbons). Examples of alkyl include: methyl (Me), ethyl (Et), n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, etc. In the present invention, the preferred alkyls are those alkyls having 1-4 carbons, i.e., C 1-4 alkyl.

[0176] Near-infrared probes and photosensitizers and their applications

[0177] The present invention provides a variety of near-infrared probes and photosensitizers (which may also be referred to as the compounds of the present invention herein) having the functions of turning on and enriching at the detection target, their preparation methods, and their applications in in vivo imaging and treatment, etc. The near-infrared probes or photosensitizers provided by the present invention have good water solubility themselves. At non-target positions, their fluorescence signals or photosensitive properties are in the off state, but under the primary triggering of target enzymes or disease microenvironments (such as ROS), the following will be achieved: 1) changing from high hydrophilicity and low cell membrane permeability to high lipophilicity and cell membrane permeability, thereby reducing their clearance rate and enhancing the retention efficiency in the target; 2) anchoring the fluorescent group or photosensitizer by reacting with macromolecules such as proteins or nucleophilic reagents in cells through the formation of a methylene benzoquinone intermediate to form a covalent bond; 3) turning on the near-infrared fluorescence or photoacoustic signal or turning on the photosensitive activity under near-infrared light. This type of near-infrared probe or photosensitizer is composed of a recognition group for enzymes or disease microenvironments (such as ROS), a near-infrared fluorescence or photoacoustic group or photosensitive group, a leaving group, and a hydrophilic group connected to the leaving group. Because the near-infrared fluorescence probes or photosensitizers provided by the present invention have good hydrophilicity themselves, they can be freely transported to tumor tissues in vivo, and under the selective action of target enzymes or disease microenvironments (such as ROS), the recognition group can leave, thereby promoting the leaving of the leaving group connected to the highly hydrophilic functional group to form a methylene benzoquinone intermediate (QM) with high lipophilicity and high reactivity. This can not only reduce the clearance rate of the near-infrared fluorescence probes or photosensitizers of the present invention in tumor tissues, but also effectively improve cell membrane permeability and facilitate cell uptake. Moreover, the nucleophilic residues on proteins in tumor cells can react with the methylene benzoquinone intermediate to form a covalent bond, realizing their effective enrichment in tumors, and at the same time turning on the near-infrared fluorescence photoacoustic or photosensitive activity.

[0178] In a specific embodiment, the present invention provides a class of target-activated enrichment-type near-infrared probes and photosensitizers (i.e., the compounds of the present invention, or compounds with a leaving hydrophilic group), and their structures are shown in general formula (I):

[0179]

[0180] Formula I wherein,

[0181] R 1 is a group that can be specifically hydrolyzed by a biomacromolecule enzyme or a group that can be specifically activated by a disease microenvironment factor; and when specifically hydrolyzed or specifically activated, R 1 and X 2 the bond between them is broken and R 1 leaves;

[0182] X 1 is O or NH;

[0183] L is when R1 A linking group that can leave on its own after leaving;

[0184] R 2 is a water-soluble group linked to the carbamide. When the compound is activated, the carbamide breaks, and the water-soluble group leaves.

[0185] The FP moiety is a fluorescent group or a photosensitive group.

[0186] In another preferred example, after the compound is specifically hydrolyzed or specifically activated, the bond between X 1 and -R breaks, and L leaves on its own under the influence of the electron transfer caused by the breakage of the bond between X 1 and -R 1 breaks, and R 1 leaves under the influence of the electron transfer caused by the leaving of L, breaks under the influence of the electron transfer caused by the leaving of L, so that R 2 leaves.

[0187] In another embodiment, R 1 is selected from the group consisting of:

[0188]

[0189] and / or,

[0190] L is selected from the group consisting of: wherein, * represents the connection position with X 2 ; and / or

[0191] R 2 is selected from the group consisting of:

[0192]

[0193] In another embodiment, the FP moiety has the structure shown in Formula II:

[0194]

[0195] wherein,

[0196] a represents the connection position with ;

[0197] b represents the connection position with ;

[0198] R 3 is -C 1-6 alkyl, -C 1-6 alkyl-R Fu ; wherein, R Fu is a modifiable functional group or a positively charged group; R Fu is selected from the group consisting of: -COOH, -N 3 、-NH 2 、-N + (C 1-6 alkyl) 3 . R 4 , R 5 , R 6 and R 7 Each is independently H or a heavy atom I, Br. 8 They are H, F, Cl, and Br.

[0199] X 2 It is O or S.

[0200] X 3 It is I, Br, and Cl.

[0201] In other embodiments, the FP moiety has the structure shown below:

[0202]

[0203] in,

[0204] a represents The location of the connection;

[0205] b represents The location of the connection;

[0206] R 3 For -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu is a modifiable functional group or a positively charged group; R Fu Select from the following group: -COOH, -N 3 、-NH 2 、-N + (C 1-6 alkyl) 3 . R 4 , R 5 , R 6 and R 7 Each is independently H or a heavy atom I, Br. 8 They are H, F, Cl, and Br.

[0207] X 2 It is O or S.

[0208] X 3 It is I, Br, and Cl.

[0209] In other embodiments, the FP moiety has the structure shown below:

[0210]

[0211] Wherein,

[0212] a represents the position connected to ;

[0213] b represents the position connected to ;

[0214] X 2 is O or S.

[0215] X 4 is O or NR 3 ;

[0216] R 3 is -C 1-6 alkyl, -C 1-6 alkyl-R Fu ; wherein, R Fu is a modifiable functional group or a positively charged group; preferably, R Fu is selected from the group consisting of: -COOH, -N 3 -, -NH 2 -, -N + (C 1-6 alkyl) 3 .

[0217] R 4 、R 5 、R 6 and R 7 are each independently H or a heavy atom such as I, Br.

[0218] R 8 is H, F, Cl, Br.

[0219] In another embodiment, the FP moiety has the structure shown below:

[0220]

[0221] Wherein,

[0222] a represents the position connected to ;

[0223] b represents the position connected to ;

[0224] X 2 is O or S.

[0225] X 5 is O or S.

[0226] R 9 is NEt 2 、NPh 2 、NCH 3 Ph.

[0227] R 8 is H, F, Cl, Br.

[0228] In a further embodiment, the FP moiety has the structure shown below:

[0229]

[0230] wherein,

[0231] a represents the position connected to ;

[0232] b represents the position connected to ;

[0233] X 2 is O or S.

[0234] X 5 is O or S.

[0235] R 10 、R 11 are Et, Ph.

[0236] R 8 is H, F, Cl, Br.

[0237] In a further embodiment, the FP moiety, X 1 、X 2 、R 1 and L are as defined in the first aspect.

[0238] In a further embodiment, the FP moiety, X 1 、X 2 、R 1 and L are each independently the corresponding groups of the specific compounds shown in Table A as defined in the first aspect.

[0239] In a further embodiment, the FP moiety, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、X 1 、X 2 、X3 , X 4 , X 5 , and L are each independently the corresponding group of the specific compound shown in Table A.

[0240] In another embodiment, the near-infrared probe or photosensitizer is selected from Table A.

[0241] In another embodiment, a near-infrared fluorescent probe as shown below, namely compound ALPIN-6, is provided;

[0242]

[0243] In another embodiment, a near-infrared basic photosensitizer as shown below, namely compound ALPIN-6I, is provided;

[0244]

[0245] In another embodiment, a near-infrared fluorescence and photoacoustic probe as shown below, namely compound ALPIN-7, is provided;

[0246]

[0247] In another embodiment, after the near-infrared probe and photosensitizer provided by the present invention having the function of turning on and enriching at the detection target are targeted to the target, an enzyme or a disease microenvironment active factor hydrolyzes the recognition group (even if the bond in X 2 -R 1 is broken, and at the same time the linking group L leaves by itself), realizing the leaving of the leaving group (carbamide structure) and the water-soluble group (R 2 ) of the near-infrared probe or photosensitizer in the unturned-on state, thereby triggering the following responses: 1) changing from high hydrophilicity and low cell membrane permeability to high lipophilicity and cell membrane permeability, thereby reducing its clearance rate and enhancing the retention efficiency in the target; 2) realizing the anchoring of the fluorescent group or photosensitizer by reacting with macromolecules such as proteins or nucleophiles in cells to form a covalent bond through the formation of a methylene benzoquinone intermediate; 3) turning on the near-infrared fluorescence or photoacoustic signal or turning on the photosensitizing activity under near-infrared light. Its principle of action is as shown in Figure 1.1 , Figure 1.1 taking the alkaline phosphatase detection probe as an example.

[0248] In another embodiment, the present invention provides near-infrared probes (such as compounds ALPIN-6 and ALPIN-7) for real-time imaging of the activity of alkaline phosphatase (ALP) in vitro and in vivo.

[0249] In another embodiment, the present invention also provides the use of near-infrared photosensitizers (such as ALPIN-6I) as photosensitizers activated by ALP in the photodynamic therapy of tumors.

[0250] Experiments show that the (especially preferred) near-infrared fluorescent probes or photosensitizers provided by the present invention, which are near-infrared fluorescent probes with the function of turning on and enriching at the detection target, can effectively realize the visualization imaging of related enzymes in vitro and in vivo, greatly enhance the enrichment efficiency of the probes, improve the imaging sensitivity and specificity, and the photosensitizer can not only selectively kill the target enzyme overexpressing tumor cells under co-incubation conditions, while other cells are not affected. More importantly, treating tumor-bearing mice with this photosensitizer by intravenous injection can completely eliminate the tumors with overexpression of the target enzyme in all tested mice, and no tumor recurrence is seen during their recovery process, and no detectable toxicity is seen in other tissues.

[0251] The main advantages of the present invention include

[0252] (a) The probes (such as ALPIN-6 and ALPIN-7) and photosensitizers (such as ALPIN-6I) of the present invention have the ability of "one trigger and three responses", that is, after being triggered once by an enzyme or an active factor in the disease microenvironment, the following three responses are triggered: 1) changing from high hydrophilicity and low cell membrane permeability to high lipophilicity and cell membrane permeability, thereby reducing its clearance rate and enhancing the retention efficiency in the target; 2) anchoring the fluorescent group or photosensitizer by reacting with macromolecules such as proteins or nucleophiles in cells to form covalent bonds through the formation of methylene benzoquinone intermediates; 3) turning on the near-infrared fluorescence or photoacoustic signal or turning on the photosensitive activity under near-infrared light.

[0253] (b) The probes (such as ALPIN-6 and ALPIN-7) and photosensitizers (such as ALPIN-6I) of the present invention have the function of turning on and enriching at the detection target.

[0254] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0255] The following will introduce the specific implementation manners of the preparation methods of the near-infrared fluorescent probes and photosensitizers of the present invention with the function of turning on and enriching at the detection target. Note: In the implementation of the present invention, 1 H-NMR, 13 C-NMR was measured with a Bruker 400Mz instrument, and the measurement solvent was deuterated chloroform (CDCl 3 ), deuterated dimethyl sulfoxide (d 6 -DMSO), and the internal standard was tetramethylsilane (TMS); all solvents were chromatographically pure, analytically pure or chemically pure.

[0256] Preparation Example 1: Synthesis of Near-Infrared Probe

[0257] Taking the preparation method of a near-infrared fluorescent probe (ALPIN-6) as an example, this example describes the preparation process of a near-infrared probe with a water-soluble group that can leave.

[0258] The structure of ALPIN-6 is as follows:

[0259]

[0260] The preparation process of ALPIN-6 is as follows:

[0261]

[0262] a. Preparation of Compound 3

[0263] Add 1 (524.2 mg, 0.78 mmol), 2 (948.0 mg, 6.77 mmol), and KHCO 3 (255.1 mg, 2.55 mmol) to a 25 mL Schlenk reaction tube, evacuate and replace with N 2 three times. Under N 2 protection, add 4 mL of ultra-dry DMF, heat to 75 °C and stir for 4 h. Stop the reaction, cool to room temperature, add 10 mL of ethyl acetate for dilution, wash with water three times (10 mL each), separate the organic phase with a separatory funnel, dry over anhydrous sodium sulfate, filter by suction, mix with silica gel, separate by column chromatography, and elute the product with DCM:CH 3 OH = 5:1. Rotate and evaporate to dryness to obtain 293.3 mg of blue solid, with a yield of 66%. The spectral characteristics are as follows: 1 H NMR (400 MHz, CD 3 OD) δ 8.74 (d, J = 14.8 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.56 - 7.45 (m, 4H), 7.46 - 7.38 (m, 1H), 6.88 (s, 1H), 6.44 (d, J = 15.2 Hz, 1H), 4.69 (s, 2H), 4.29 (t, J = 7.4 Hz, 2H), 2.78 (t, J = 5.8 Hz, 2H), 2.72 (t, J = 5.8 Hz, 2H), 2.00 - 1.88 (m, 4H), 1.82 (s, 6H), 1.08 (t, J = 7.4 Hz, 3H). 13 C NMR (101 MHz, CD 3OD) δ 176.92, 162.43, 159.58, 153.96, 144.99, 141.80, 141.70, 135.36, 128.79, 128.09, 126.76, 126.58, 126.32, 122.33, 114.83, 114.19, 112.17, 102.28, 100.79, 58.57, 50.26, 45.93, 28.57, 27.08, 23.67, 20.73, 20.31, 10.23. HRMS(ESI) m / z calcd for C 31 H 32 NO 3 (M - I) + 442.2382, found 442.2377.

[0264] b. Preparation of Compound 5

[0265] Dye 3 (50.5 mg, 0.089 mmol), KI (142.8 mg, 0.860 mmol), KHCO 3 (49.4 mg, 0.493 mmol), 18 - crown - 6 (20.3 mg, 0.077 mmol) were added to a 10 mL reaction tube and evacuated and replaced with N 2 three times. Under N 2 protection, 1.0 mL of ultradry DMF was added, and then 4 (48.4 mg, 0.159 mmol) was added. The reaction was stirred at room temperature for 4 h. The reaction was stopped, 10.0 mL of DCM was added for dilution, washed with water (5.0 mL), and the organic phase was separated with a separatory funnel, dried over anhydrous sodium sulfate, filtered by suction, mixed with silica gel, and separated by column chromatography. DCM:CH 3 OH = 10:1 to elute the product. The product was dried by rotary evaporation to obtain 50.1 mg of a blue solid compound with a yield of 67%. The spectral characteristics are as follows: 1 H NMR (400 MHz, CDCl 3 ) 1 H NMR (600 MHz, CD 3OD) δ 8.74 (d, J = 15.0 Hz, 1H), 7.68 (d, J = 7.2 Hz, 1H), 7.58 (s, 1H), 7.53 - 7.51 (m, 2H), 7.48 - 7.44 (m, 4H), 7.27 (d, J = 7.8 Hz, 2H), 7.12 (s, 1H), 6.48 (d, J = 14.4 Hz, 1H), 6.00 - 5.78 (m, 2H), 5.30 (s, 2H), 5.28 (d, J = 18.0 Hz, 2H), 5.08 (d, J = 10.2 Hz, 2H), 4.70 (s, 2H), 4.44 - 4.47 (m, 2H), 4.31 (t, J = 7.2 Hz, 4H), 2.81 - 2.78 (m 2H), 2.72 - 2.70 (m, 2H), 1.96 - 1.92 (m, 4H), 1.84 (s, 6H), 1.08 (t, J = 7.2 Hz, 3H). 13 C NMR (151 MHz, CD 3 OD) δ 177.32, 162.31, 159.46, 153.89, 153.05 (d, J = 6.7 Hz), 145.40, 141.93, 141.56, 134.90, 134.33 (d, J = 7.8 Hz), 130.95, 129.65 (d, J = 2.5 Hz), 128.76, 128.43, 127.29, 126.74, 126.00, 124.85, 122.50, 120.19 (d, J = 5.1 Hz), 115.44, 115.06, 114.53, 112.06, 102.64, 99.28, 82.38, 70.35, 70.28, 69.59, 66.38 (d, J = 5.5 Hz), 58.27, 50.47, 48.46, 43.36, 28.62, 27.20, 26.06, 23.92, 20.18, 15.15. HRMS (ESI) m / z calcd for C 44 H 47 NO 7 P(M - I) + 732.3090, found 732.3089.

[0266] c. Preparation of Compound ALPIN - 6

[0267] Compound 5 (33.5 mg, 0.040 mmol) was added to a 10 mL reaction flask, followed by 0.5 mL of ultra-dry DCM, 6 (16.7 mg, 0.076 mmol), and triethylamine (20 μL, 0.144 mmol). The reaction mixture was stirred at room temperature for 6 h. Then it was diluted with DCM, washed with water, dried over anhydrous sodium sulfate, filtered by suction, and the filtrate was evaporated to dryness by a rotary evaporator to obtain Compound 7, which was directly used in the next step without further purification.

[0268] To a 10 mL Schlenk reaction tube was added 7 (22.8 mg), 1,3-dimethylbarbituric acid (7.8 mg, 0.05 mmol), Pd(PPh 3 ) 4 (5.8 mg, 0.005 mmol), and the tube was evacuated and backfilled with N 2 three times. Then 0.5 mL of MeCN and 0.5 mL of DCM were added. After stirring at room temperature for 1 h, the reaction solution was evaporated to dryness. Then 9 (10.2 mg, 0.043 mmol), CuSO 4 (3.5 mg, 0.022 mmol), ascorbic acid (17.5 mg, 0.099 mmol), DMSO (0.5 mL), and H 2 O (0.5 mL) were added. The reaction mixture was stirred at room temperature for 1 h, and the target compound (2.1 mg) was obtained by high-performance preparative liquid chromatography with a three-step yield of 13%.

[0269] The spectral characteristics are as follows: 1 H NMR (600 MHz, d 6 -DMSO) δ 8.57 (d, J = 15.0 Hz, 1H), 8.22 (s, 1H), 8.11 (s, 1H), 7.91 - 7.87 (m, 1H), 7.64 - 7.62 (m, 2H), 7.60 (s, 1H), 7.57 - 7.50 (m, 4H), 7.24 - 7.22 (m, 2H), 6.59 (d, J = 15.0 Hz, 1H), 5.34 (s, 2H), 5.10 (s, 2H), 4.92 (t, J = 6.9 Hz, 2H), 4.40 (t, J = 6.9 Hz, 2H), 4.27 (d, J = 6.0 Hz, 2H), 3.85 (t, J = 6.9 Hz, 2H), 3.07 (s, 6H), 2.73 - 2.68 (m, 4H), 2.44 (t, J = 6.9 Hz, 2H), 1.99 - 1.94 (m, 2H), 1.86 - 1.82 (m, 6H), 1.79 (s, 6H), 0.99 (t, J = 7.2 Hz, 3H). HRMS (ESI) m / z calcd for C 47 H 58 N 6 O11 PS(M-I) + 945.3616, found 945.3624.

[0270] Preparation Example 2: Synthesis of Near-Infrared Photosensitizer

[0271] In this example, the preparation process of the near-infrared photosensitizer was described by taking the preparation method of the near-infrared photosensitizer (ALPIN-6I) as an example.

[0272] The structure of ALPIN-6I is as follows:

[0273]

[0274] The preparation process of ALPIN-6I is as follows:

[0275]

[0276] a. Preparation of Compound 11

[0277] Compound 5-iodo-2,3,3-trimethyl-3H-indole 10 (5.0 g, 17.537 mmol) was added to a 100 mL reaction flask, and then 30 mL of ultradry ACN and 1-iodopropane (8.5 mL, 87.683 mmol) were added. The mixture was heated under reflux for 18 h. The acetonitrile was evaporated to dryness using a rotary evaporator, and acetone was added and heated to 60 °C. The solid was precipitated by cooling, and the pink solid (7.489 g) was obtained by filtration with a yield of 94%. The spectral characteristics were as follows: 1 H NMR(600MHz, d 6 -DMSO) δ 8.32 (d, J = 1.2 Hz, 1H), 8.00 (dd, J = 8.4, 0.6 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 4.41 (t, J = 7.8 Hz, 2H), 2.83 (s, 3H), 1.89 - 1.80 (m, 2H), 1.54 (s, 6H), 0.98 (t, J = 7.2 Hz, 3H). HRMS(ESI) m / z calcd for C 14 H 19 IN(M-I) + 328.0557, found 328.0563.

[0278] b. Preparation of Compound 12

[0279] Anhydrous sodium acetate (81.3 mg, 0.991 mmol), 2-chloro-3-(hydroxy-methylene)-1-cyclohexene-1-carbaldehyde (805.0 mg, 4.664 mmol), and Compound 11 (4.443 g, 9.762 mmol) were added to the reaction flask.

[0280] N 2 Replace three times, then add 8 mL of ethanol, and stir and react at 60 °C for 16 h. Mix the sample and perform column chromatography to obtain 2.806 g of green solid, with a yield of 66%. The spectral characteristics are as follows: 1 H NMR (400 MHz, CDCl 3 ) δ 8.32 (d, J = 13.6 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H), 7.65 (s, 2H), 7.00 (d, J = 8.0 Hz, 2H), 6.26 (d, J = 13.6 Hz, 2H), 4.44 - 3.93 (m, 4H), 2.94 - 2.60 (m, 4H), 2.07 - 1.83 (m, 6H), 1.71 (s, 12H), 1.06 (t, J = 6.8 Hz, 6H). HRMS (ESI) m / z calcd for C 36 H 42 ClI 2 N 2 (M - I) + 791.1120, found 791.1134.

[0281] c. Preparation of Compound 13

[0282] Add compound 12 (1.973 g, 2.147 mmol), 2 (1.572 g, 11.217 mmol), and KHCO 3 (676.1 mg, 6.753 mmol) into the reaction flask, replace with N 2 three times, then add 5 mL of DMF, and stir and react at 75 °C for 3 h. Dilute with ethyl acetate, wash three times with water, dry over anhydrous sodium sulfate, mix the sample and perform column chromatography to obtain 730.0 mg of blue compound, with a yield of 49%. The spectral characteristics are as follows: 1 H NMR (600 MHz, d 6 -DMSO) δ 7.85 (d, J = 13.2 Hz, 1H), 7.74 (d, J = 1.2 Hz, 1H), 7.69 (s, 1H), 7.57 (dd, J = 8.4, 1.2 Hz, 1H), 7.37 (s, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.19 (s, 1H), 5.76 (s, 1H), 4.40 (s, 2H), 3.84 (t, J = 7.2 Hz, 2H), 2.72 - 2.56 (m, 4H), 1.84 - 1.76 (m, 2H), 1.72 - 1.64 (m, 2H), 1.61 (s, 6H), 0.93 (t, J = 7.2 Hz, 3H). 13 C NMR (151 MHz, d 6-DMSO)δ162.73,157.81,157.32,143.42,141.79,139.32,138.75,136.41,130.43,129.54,124.76,116.76,116.43 ,110.76,101.43,94.65,84.15,58.89,54.82,46.65,28.00,27.06,23.91,20.83,19.43,11.08.HRMS(ESI)m / zcalcd for C 29 H 31 INO 3 (MI) + 568.1343, found 568.1350.

[0283] d. Preparation of Compound 14

[0284] Compound 13 (82.3 mg, 0.118 mmol), 4 (79.0 mg, 0.240 mmol), KI (205.3 mg, 1.237 mmol), KHCO 3 (63.6 mg, 0.635 mmol), 18-crown-6 (31.7 mg, 0.120 mmol) were added to the reaction bottle, N 2 Replace three times, add 1 mL DMF, stir and react at room temperature for 6 hours. Dilute with DCM, wash three times with water, dry with anhydrous sodium sulfate, mix and column chromatograph to obtain 57.1 mg of blue compound with a yield of 50%. The spectral characteristics are: 1 H NMR (600 MHz, CDCl 3 )δ8.59(d,J=14.4Hz,1H),7.80(d,J=1.2Hz,1H),7.74(dd,J=8.4,1.2Hz,1H),7.65(s,1H),7.41(d,J=7.8Hz ,2H),7.38(s,1H),7.24(d,J=8.4Hz,2H),7.05-6.96(m,2H),6.20(d,J=14.4Hz,1H),5.99-5.89(m,2H),5.37 (dd,J=17.4,1.2Hz,2H),5.27(dd,J=10.2,0.6Hz,2H),5.17(s,2H),4.76(s,2H),4.69-4.60(m,4H),4.11(t ,J=7.2Hz,2H),2.77-2.69(m,2H),2.67-2.59(m,2H),1.95-1.86(m,4H),1.78(s,6H),1.04(t,J=7.2Hz,3H). 1313C NMR (151 MHz, CDCl 3 ) δ 175.11, 163.56, 159.97, 154.33, 150.46 (d, J = 6.8 Hz), 144.88, 143.55, 141.59, 137.92, 137.12, 132.69, 131.95 (d, J = 7.6 Hz), 131.82, 130.92, 128.69, 127.19, 126.57, 120.28 (d, J = 4.8 Hz), 118.86, 115.89, 115.45, 113.32, 101.65, 99.15, 90.34, 70.16, 68.98 (d, J = 5.6 Hz), 59.51, 50.05, 46.37, 28.96, 28.22, 24.20, 20.87, 20.21, 11.45. HRMS (ESI) m / z calcd for C 42 H 46 INO 7 P(M-I) + 834.2051, found 834.2056.

[0285] e. Preparation of Compound 15

[0286] Compound 14 (57.1 mg, 0.059 mmol) and 6 (31.6 mg, 0.144 mmol) were added to a reaction flask, and then DCM and Et 3 N (29 μL, 0.205 mmol) were added, and the mixture was stirred at room temperature for 12 h. It was diluted with DCM, washed with water three times, dried over anhydrous sodium sulfate, mixed with silica gel, and purified by column chromatography to obtain 19.6 mg of a blue compound with a yield of 48%. The spectral characteristics are as follows: 1 1H NMR (600 MHz, d 6 -DMSO) δ 8.54 (d, J = 12.0 Hz, 1H), 8.24 (s, 1H), 7.89 (d, J = 12.0 Hz, 1H), 7.79 (t, J = 6.0 Hz, 1H), 7.60 (d, J = 6.0 Hz, 2H), 7.58 - 7.55 (m, 2H), 7.52 (d, J = 6.0 Hz, 1H), 7.32 (d, J = 6.0 Hz, 2H), 7.19 (s, 1H), 6.54 (d, J =

[0287] 12.0 Hz, 1H), 6.01 - 5.87 (m, 2H), 5.41 (s, 2H), 5.34 (d, J = 18.0 Hz, 2H), 5.22 (d, J = 12.0 Hz, 2H), 5.12 (s, 2H), 4.70 - 4.58 (m, 4H), 4.35 (t, J = 6.0 Hz, 2H), 3.81 (d, J = 6.0 Hz, 2H), 3.13 (s, 1H), 2.78 - 2.65 (m, 4H), 1.88 - 1.79 (m, 4H), 1.78 (s, 6H), 0.97 (t, J = 6.0 Hz, 3H). HRMS(ESI) m / z calcd for C 46 H 49 IN 2 O 8 P(M - I) + 915.2266, found 915.2270.

[0288] f. Preparation of compound ALPIN - 6I

[0289] Add 15 (26.6 mg, 0.029 mmol), 1,3 - dimethylbarbituric acid (7.8 mg, 0.05 mmol), Pd(PPh 3 ) 4 (4.5 mg, 0.0039 mmol) into a 10 mL Schlenk reaction tube, evacuate and replace with N2 three times, and add 0.5 mL of MeCN and 0.5 mL of DCM each. After stirring the reaction at room temperature for 30 min, rotary evaporate the reaction solution, add 9 (10.2 mg, 0.043 mmol), CuSO 4 (2.1 mg, 0.013 mmol), ascorbic acid (22.1 mg, 0.125 mmol), DMSO (0.5 mL), and H 2 O (0.5 mL), stir the reaction at room temperature for 1 h, and prepare the target compound (4.7 mg) by high - performance preparative liquid chromatography with a three - step yield of 15%. The spectral characteristics are as follows: 11H NMR(600MHz, d6-DMSO) δ 8.55 (d, J = 14.7 Hz, 1H), 8.22 (s, 1H), 8.12 (s, 1H), 7.91 - 7.86 (m, 1H), 7.65 - 7.61 (m, 2H), 7.60 (s, 1H), 7.57 - 7.49 (m, 4H), 7.24 - 7.22 (m, 2H), 6.53 (d, J = 14.8 Hz, 1H), 5.36 (s, 2H), 5.10 (s, 2H), 4.93 (t, J = 6.9 Hz, 2H), 4.34 (t, J = 6.8 Hz, 2H), 4.28 (d, J = 5.7 Hz, 2H), 3.85 (t, J = 6.9 Hz, 2H), 3.07 (s, 6H), 2.73 - 2.68 (m, 4H), 2.45 (t, J = 6.8 Hz, 2H), 2.01 - 1.93 (m, 2H), 1.86 - 1.79 (m, 6H), 1.78 (s, 6H), 0.96 (t, J = 7.4 Hz, 3H). HRMS(ESI) m / z calcd for C 47 H 57 N 6 O 11 PSI(M - I) + 1071.2583, found 1071.2585.

[0290] Test Example 1: Verification of Solution Enzyme Response of Near-Infrared Fluorescent Probe ALPIN-6

[0291] a. Absorption and Fluorescence Test Conditions of Near-Infrared Fluorescent Probe

[0292] The full absorption spectrum of the probe was measured by a UV-Vis spectrophotometer, with a wavelength scanning range of 400 nm to 800 nm. The full fluorescence spectrum was measured by a fluorescence spectrometer, with an excitation wavelength of 687 nm, excitation and emission slit widths of 3 nm, and a wavelength scanning range of 697 nm to 850 nm. The change in fluorescence intensity over time was measured by a microplate reader (SpectraMax i3), using a 96-well plate. The default total volume in the plate was 100 μL, the excitation wavelength was 687 nm, the emission wavelength was 714 nm, the excitation and emission slit widths were 15 nm, the PMT was set to the default value of the microplate reader, and 10 flashes / read. Unless otherwise specified, the PBS buffer (1X, pH 7.4) solutions used for fluorescence and absorption tests both contained 0.1% surfactant CHAPS (3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate inner salt), the concentration of probe ALPIN-6 was 10 μM, the final concentration of ALP was 2 U / mL, the final concentration of β-mercaptoethanol was 140 mM, and the incubation was carried out in a 37°C water bath for 30 min.

[0293] b. HPLC analysis of the changes in the near-infrared probe before and after enzymatic hydrolysis

[0294] After incubating the probe ALPIN-6 (10 μM) with ALP (2 U / mL) in PBS buffer solution in a water bath at 37 °C for 30 min, the reaction solution was centrifuged at 12000 r / min for 10 min using a 10 kDa ultrafiltration membrane to remove proteins. 100 μL of the filtrate was taken for HPLC analysis at a wavelength of 600 nm.

[0295] c. Covalent labeling of the near-infrared probe on protein gel imaging

[0296] According to the experimental requirements, the preparation of the gel electrophoresis loading sample with a total volume of 25 μL: After incubating the fluorescent probe ALPIN-6 or ALPIN-5 (10 μM) with different protein group samples (ALP 8 μM, BSA protein 5 μM) at 37 °C for 2 h, 2 μL of the gel electrophoresis loading buffer (5x) was added and mixed well for later use. An 8% gel was prepared according to the Beyotime kit. 20 μL of the above sample was loaded into each well. After running on a SDS-PAGE gel electrophoresis instrument at 120 V for 2 - 3 h to obtain the imaging gel, it was scanned with an Odyssey CLx Image Studio near-infrared two-color scanner system (λ ex / em = 685 nm / 720 nm), and the protein fluorescence bands in the scanned imaging map were analyzed. Then the protein gel was added to the Coomassie Brilliant Blue solution and stained on a shaker for 30 min to mark the positions of the protein bands. Then the Coomassie Brilliant Blue on the gel was washed off with the Coomassie Brilliant Blue solution eluent (ethanol:acetic acid:water = 1:2:7), and a mobile phone was used to take pictures.

[0297] The results are as Figure 2.1 shown. As Figure 2.1 can be seen, after the reaction of the fluorescent probe ALPIN-6 with "one-touch triple response" of the present invention with ALP, the absorption undergoes a red shift and the fluorescence intensity increases. And when there is enough nucleophile β-ME, the absorption and fluorescence changes are more obvious ( Figure 2.1 a, b), and HPLC verification also proves that the probe ALPIN-6 reacts with ALP enzyme ( Figure 2.1 c). In the specificity verification, only the probe and the ALP enzyme group achieved obvious fluorescence enhancement ( Figure 2.1 d). In the protein labeling effect experiment, the labeling effect of the probe ALPIN-6 after reacting with the enzyme is the best ( Figure 2.1 e). ( Figure 2.1 In e, M: protein marker; 1: ALP; 2: ALP + ALPIN-6; 3: ALP + ALPIN-5; 4: BSA; 5: BSA + ALPIN-6; 6: ALP + BSA + ALPIN-6).

[0298] Experimental conclusion: The fluorescent probe ALPIN-6 can specifically respond to ALP enzyme and has a good protein labeling effect after reacting with the enzyme.

[0299] Test Example 2: Biological experiment verification of near-infrared probe ALPIN-6

[0300] Test Example 2.1: Imaging comparison of near-infrared fluorescent probes ALPIN-6, ALPIN-5, and ALPIN-4 on HeLa cells with overexpressed alkaline phosphatase (ALP) and imaging of HEK293 with weak ALP expression ( Figure 2.2 )

[0301] Experimental conditions: 1 μM of fluorescent probes ALPIN-6, ALPIN-5, and ALPIN-4 were respectively incubated with HeLa cells with overexpressed ALP on the cell membrane surface and normal tissue cells HEK293 with low ALP expression in a culture dish in an incubator at 37 °C for 30 min; or HeLa cells were pretreated with alkaline phosphatase inhibitor Na 3 VO 4 for 1 hour and then co-incubated with the probe for 30 min. Subsequently, the medium containing the probe was discarded and washed once with PBS buffer (pH = 7.4); 1 mL of phenol red-free DMEM was added, and cell imaging (Cy 5.5 channel) was performed using a confocal fluorescence microscope.

[0302] The experimental results are as Figure 2.2 shown. It can be Figure 2.2 seen that the fluorescent probe ALPIN-6 with "one-touch triple response" of the present invention, compared with the general self-anchoring probe ALPIN-5 (structure as Figure 2.2 shown) and the ordinary fluorescent probe ALPIN-4 (structure as Figure 2.2 shown), can not only achieve more obvious fluorescence imaging of ALP overexpressing cells, but also has more obvious fluorescence signals than the ALP low-expressing cell line (HEK293). In addition, in the ALP inhibitor group experiment, the fluorescence signal of the fluorescent probe ALPIN-6 for labeling HeLa cells weakened, indicating that the initiation of the labeling effect of the fluorescent probe depends on the property of alkaline phosphatase specifically hydrolyzing the phosphate ester bond.

[0303] Experimental conclusion: After reacting with ALP enzyme in cells, the fluorescent probe ALPIN-6 can have a better cell enrichment effect, reduce the diffusion of the fluorescent probe in cell imaging experiments, reduce the background signal, and greatly improve the detection sensitivity.

[0304] Test Example 2.2: Real-time imaging of HeLa cells with near-infrared fluorescent probe ALPIN-6 ( Figure 2.3 )

[0305] Experimental conditions: The cultured HeLa cells were spread evenly in a confocal dish, and 0.1 μM of the fluorescent probe ALPIN-6 was added to the HeLa cells. Confocal fluorescence microscopy was used to image the cells at different time intervals (Cy 5.5 channel).

[0306] The experimental results are as Figure 2.3 shown. It can be seen from Figure 2.3 that when the fluorescent probe ALPIN-6 with "one-touch triple response" was used to image HeLa cells in real time, the fluorescent signal first appeared rapidly on the cell membrane, which was consistent with the expression of ALP enzyme on the HeLa cell membrane. As time passed, the fluorescent signal gradually appeared inside the cells, and the fluorescent signal inside the cells became stronger with the extension of time. At 20 min, the intensity of the fluorescent signal on the cells was more than 2000 times stronger than the background fluorescence.

[0307] Experimental conclusion: The fluorescent probe ALPIN-6 can visualize the activity of ALP enzyme in HeLa cells in real time. After the enzyme activates the probe, it is converted from hydrophilic to lipophilic, which enhances the cellular uptake and the self-anchoring of methylene benzoquinone, restricts the penetration and diffusion of the turned-on fluorescent signal, greatly enhances the accumulation of the fluorescent signal in tumor cells, and improves the ability of the probe to visualize enzyme activity in real time.

[0308] Test Example 2.3: Specific imaging of HeLa and HEK293 mixed cells by the near-infrared fluorescent probe ALPIN-6( Figure 2.4 )

[0309] Experimental conditions: The cultured HeLa and HEK293 cells (stably transfected with green fluorescent protein) were spread evenly in a confocal dish, and 1 μM of the fluorescent probe ALPIN-6 was added to the mixed cells. After incubating in an incubator at 37 °C for 20 min, fluorescence microscopy was used to image the cells.

[0310] The experimental results are as Figure 2.4 shown. It can be seen from Figure 2.4 that there was only yellow fluorescent signal in the HEK293 cells stably transfected with yellow fluorescent protein, and no red fluorescence. The red fluorescence only appeared in the HeLa cells without green fluorescence.

[0311] Experimental conclusion: After mixing HeLa cells with overexpressed ALP and HEK293 cells with weak ALP expression, the fluorescent probe ALPIN-6 can specifically image HeLa cells with overexpressed ALP, which benefits from the fact that this "one-touch triple response" design strategy can achieve a great enrichment of the probe at the detection site.

[0312] Test Example 2.4: Real-time imaging of HeLa cell-bearing tumor mice by the near-infrared fluorescent probe ALPIN-6Figure 2.5 )

[0313] Experimental conditions:

[0314] Construction of HeLa tumor model nude mice:

[0315] (1) Preparation of HeLa cells: The cell culture and passage were the same as before. After the cultured cells were digested and centrifuged, they were washed twice with PBS, 1 mL each time. After washing, they were centrifuged, the supernatant was discarded, and DMEM without FBS was added and mixed evenly. After counting, Matrigel was added, and after mixing evenly, it was placed in an ice bath for standby.

[0316] (2) Implantation of HeLa cells into tumor model nude mice: Female BALB / c nude mice at 4 - 6 weeks old were implanted subcutaneously on the right hind thigh with 200 μL of a mixed solution of DMEM (without FBS) containing 4×10 6 HeLa cells and Matrigel (1:4) using a 1 mL sterile medical syringe. They were grown at 25°C with water and food provided ad libitum for 2 - 3 weeks. When the tumor grew to about 0.1 cm 3 , animal imaging experiments were performed.

[0317] (3) In vivo imaging of tail vein injection of probes in HeLa tumor model nude mice: First, the experimental animals were divided into 3 groups, 3 mice in each group. The whole body background fluorescence imaging (λ ex = 660 nm, λ em = 710 nm) of 6 nude mice was collected using an IVIS Lumina XRMS Series III in vivo animal imaging system. Then, the fluorescent probes ALPIN - 6 or ALPIN - 4 were prepared into a PBS solution with a concentration of 40 μM. A insulin syringe was used to inject 200 μL of the prepared probe solution into the tail vein of the tumor model mice. Subsequently, the whole body fluorescence data of the experimental model mice were collected using an in vivo animal imager at 6 different time points (1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 24 h, and 36 h). In the inhibitor group, 50 μL of a 10 mM Na 3 VO 4 solution was injected in situ into the tumor, and half an hour later, the probe ALPIN - 6 with the same concentration was injected, and then imaging was performed at the same time period.

[0318] (4) Anatomical tissue imaging of HeLa tumor model nude mice: After 36 h of injecting the probe via the tail vein, the nude mice were killed by cervical dislocation. After dissecting to obtain the tumor, heart, lungs, liver, spleen, and kidneys, fluorescence imaging of the organs and tissues was performed.

[0319] The experimental results are as Figure 2.5 shown, and by Figure 2.5It can be seen that after intravenous injection of the probe, obvious fluorescence signals appeared on the tumors in the ALPIN-6 injection probe group 1 hour later, and obvious fluorescence signals were still present on the tumors until 36 hours. However, the fluorescence signals on the tumors in the group pre-injected with the inhibitor were significantly weakened; the control probe ALPIN-4 group also showed fluorescence signals at 1 hour, but they were significantly weaker than those in the ALPIN-6 group, and the fluorescence signals on the tumors disappeared 2 hours later. This result was also consistent with the fluorescence signals on the tumors after dissecting the tumor-bearing mice.

[0320] Experimental conclusion: In a highly dynamic in vivo animal system, ALPIN-6 has better tumor signal enrichment and retention effects, improves the signal intensity of in vivo tumor detection, is beneficial to the detection of in vivo tumor tissues, and plays a very important role in surgical navigation for removing tumor tissues.

[0321] Test Example 3: Biological experiment verification of near-infrared photosensitizer ALPIN-6I

[0322] Test Example 3.1: Calculation of the enrichment content of near-infrared photosensitizer ALPIN-6I in HeLa cells

[0323] Experimental conditions: Incubate 4 μM, 1.5 mL of photosensitizer ALPIN-6I with 100,000 HeLa cells with overexpressed ALP on the cell membrane surface in a 37 °C incubator for 1 hour, then collect the incubation, wash it 2 times with phenol red-free DMEM, collect the washing solution, add 200 μL of RIPA cell lysate, fully lyse the cells, and collect the lysate. Add ALP enzyme and BSA to the collected incubation solution and washing solution to ensure that the remaining probes in the solution can be fully activated, then measure the fluorescence intensity with a fluorescence spectrometer, perform parallel tests in three groups, and calculate the probe concentration according to the standard curve. Calculate the photosensitizer concentration in the cell lysate in the same way, and then calculate the concentration in the cells according to the volume of the cells (the volume of a single cell is 2.6×10 3 μm 3 =2.6×10 -12 L).

[0324] The experimental results are as Figure 2.6 shown. It can be Figure 2.6 seen that the concentration of the photosensitizer in HeLa cells is as high as 2.4 mM, while the total concentration of the probe in the cell incubation solution and washing solution is 0.00087 mM. The fluorescence signal intensity in the cells is more than 2000 times that of the photosensitizer concentration in the incubation solution. And after the cells were incubated with the photosensitizer and centrifuged, in the collected cell precipitate, ALPIN-6I had obvious blue color, and no blue fluorescence was seen in the control photosensitizer ALPIN-4I.

[0325] Experimental conclusion: The photosensitizer ALPIN-6I has a very high enrichment effect in HeLa cells with overexpressed ALP, which is very beneficial for improving the photodynamic therapy effect of the photosensitizer.

[0326] Test Example 3.2: ROS generation effect of the photosensitizer ALPIN-6I in HeLa cells

[0327] Experimental conditions: The photosensitizer ALPIN-6I (0, 1, 2, 4 μM) or ALPIN-4I (4 μM) and the ROS detection probe H 2 DCFDA (10 μM) were incubated with HeLa cells or HEK293 cells in an incubator at 37 °C for 30 min, washed twice with PBS, and then irradiated with light at 660 nm and 36 mW for 2 min, followed by fluorescence imaging using a fluorescence microscope.

[0328] The experimental results are as Figure 2.7 shown. It can be seen from Figure 2.7 that as the concentration of the photosensitizer ALPIN-6I increases, the green fluorescence of DCF becomes stronger, indicating that more ROS are generated. Without light irradiation, no green fluorescence is produced in the ALPIN-6I group; and no green fluorescence is also produced in the negative cell HEK293 group. Moreover, under the same conditions, the green fluorescence in the control photosensitizer group ALPIN-4I is much weaker than that in ALPIN-6I.

[0329] Experimental conclusion: The photosensitizer ALPIN-6I can be specifically activated in HeLa cells with overexpressed ALP, cannot be activated in negative cells, and can only generate ROS under light irradiation. Moreover, the efficiency of generating ROS is significantly higher than that of the control photosensitizer group ALPIN-4I. This benefits from the enrichment of the photosensitizer ALPIN-6I in tumor cells.

[0330] Test Example 3.3: Effect of the photosensitizer ALPIN-6I on killing tumor cells

[0331] Experimental conditions: The photosensitizer ALPIN-6I or ALPIN-4I (4 μM) was incubated with HeLa cells or HEK293 cells in an incubator at 37 °C for 1 h, the incubation solution was aspirated, and then fresh medium containing FBS was added. Then, it was irradiated with light at 660 nm and 36 mW for 10 min, placed in an incubator at 37 °C for 24 h, stained using the Beyotime live / dead cell staining kit, and then fluorescence imaging was performed using a fluorescence microscope.

[0332] The experimental results are as Figure 2.8 shown. It can be seen from Figure 2.8It can be seen that only the HeLa cells in the ALPIN-6I plus light irradiation group can be stained with PI (red represents cell death). In the control probe ALPIN-4I plus light irradiation group and the PBS plus light irradiation group, only AM staining is observed (green represents living cells), and the same results are obtained in the negative cell HEK293 plus light irradiation group and the ALPIN-6I without light irradiation group.

[0333] Experimental conclusion: Only when both ALP and light are present can the photosensitizer ALPIN-6I be activated to kill tumors, and under the same conditions, the photodynamic therapy effect of ALPIN-6I is better than that of the control photosensitizer ALPIN-4I.

[0334] Test Example 3.4: Effect of the photosensitizer ALPIN-6I on specifically killing tumor cells in mixed cells ( Figure 2.9 )

[0335] Experimental conditions: The photosensitizer ALPIN-6I (4 μM) was mixed with a mixed cell of HeLa cells and HEK293 cells (stably transfected with yellow fluorescent protein) and incubated in a 37 °C incubator for 30 min. Then, the incubation solution was aspirated, and a new medium containing FBS was added. After that, it was irradiated with light at 660 nm and 36 mW for 10 min, and then cultured in a 37 °C cell incubator for 24 h. PI staining (for dead cells) was performed using the Beyotime Live / Dead Cell Staining Kit, and then fluorescence imaging was carried out using a fluorescence microscope.

[0336] The experimental results are as Figure 2.9 shown. It can be Figure 2.9 seen that only HeLa cells can be stained red with PI, while HEK293 cells stably transfected with yellow fluorescent protein cannot be stained with PI, indicating that HeLa cells are killed while HEK293 cells survive.

[0337] Experimental conclusion: In mixed cells, the photosensitizer ALPIN-6I can specifically kill HeLa tumor cells with overexpression of ALP without affecting normal cells. This is because after being activated by the enzyme, the photosensitizer ALPIN-6I can be retained in tumor cells and will not diffuse into the solution and then be taken up by normal cells.

[0338] Test Example 3.5: Real-time imaging of near-infrared photosensitizer ALPIN-6I on HeLa cell-bearing tumor mice

[0339] Experimental conditions: First, the experimental animals, tumor-bearing mice, were divided into 3 groups with 3 mice in each group. The whole-body background fluorescence imaging of 6 nude mice was collected using the IVIS Lumina XRMS Series III in vivo animal imaging system (λ ex = 660 nm, λ em= 710 nm). Then, the photosensitizer ALPIN-6I or ALPIN-4I was formulated into a PBS solution with a concentration of 40 μM. An insulin syringe was used to inject 200 μL of the prepared probe solution into the tail vein of tumor model mice. Subsequently, whole-body fluorescence data of the experimental model mice were collected using a live animal imager at 6 different time points (1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 24 h). In the inhibitor group, 50 μL of a 10 mM Na 3 VO 4 solution was injected in situ into the tumor, and after half an hour, the probe ALPIN-6I with the same concentration was injected. Then, imaging was performed during the same time period.

[0340] The experimental results are as Figure 2.10 shown. As Figure 2.10 can be seen, after intravenous injection of the probe, obvious fluorescence signals appeared on the tumors in the group injected with the probe ALPIN-6I after 1 h, and obvious fluorescence signals were still present on the tumors until 24 h. However, the fluorescence signals on the tumors in the group pre-injected with the inhibitor were significantly weakened; obvious fluorescence signals also appeared in the control probe ALPIN-4I group at 1 h, but were significantly weaker than those in the ALPIN-6I group, and the fluorescence signals on the tumors disappeared after 2 h.

[0341] Experimental conclusion: In a highly dynamic live animal system, ALPIN-6I has better tumor signal enrichment and retention effects.

[0342] Test Example 3.6: Photodynamic therapy of HeLa cell-bearing tumor mice with near-infrared photosensitizer ALPIN-6I

[0343] Experimental conditions: First, the tumor-bearing mice were divided into 3 groups, with 4 mice in each group. The photosensitizer was injected into the tail vein at a dose of 1.1 mg / kg. -1 . After 1 h, light irradiation was performed at 660 nm with a power of 0.5 W for 10 min, and then again after 24 h; then, the photosensitizer was injected again after 48 h, light irradiation was performed after 1 h, and again after 72 h. Subsequently, the tumor-bearing mice were allowed to recover on their own for 15 days.

[0344] The experimental results are as Figure 2.11 shown. As Figure 2.11 can be seen, the tumors of the tumor-bearing mice in the ALPIN-6I group of photodynamic therapy completely disappeared 4 days after treatment, and the tumor-bearing mice recovered and no tumor recurrence was observed 18 days after treatment. However, the tumors of the tumor-bearing mice in the control group did not disappear and gradually increased.

[0345] Experimental conclusion: In a highly dynamic live animal system, ALPIN-6I has the ability to target tumor tissues, can achieve enrichment in tumor tissues, enhance the effect of photodynamic therapy, can completely eliminate tumor tissues and will not recur.

[0346] Preparation Example 3: Preparation method of alkaline phosphatase near-infrared fluorescence and photoacoustic probe (ALPIN-7) with the function of turning on and enriching at the detection target.

[0347] The structure of ALPIN-7 is as follows:

[0348]

[0349] The preparation process of ALPIN-7 is as follows:

[0350]

[0351] a. Preparation of Compound 23

[0352]

[0353] Prepare Compound 21: Add 2-hydroxy-4-iodobenzoic acid (1.24 g, 4.7 mmol), K 2 CO 3 (1.98 g, 14.1 mmol), 18-C-6 (127.6 mg, 0.47 mmol), 5 mL of ultra-dry DMF, and then add allyl bromide (1.2 mL, 14.1 mmol). Stir and react overnight at room temperature. Stop the reaction, add 20 mL of ethyl acetate to dilute, wash with water three times (20 mL each), separate the organic phase with a separatory funnel, dry over anhydrous sodium sulfate, filter by suction, mix with silica gel, separate by column chromatography, and rotary evaporate to dryness to obtain 1.256 g of white solid, with a yield of 88%. Spectral characteristics: 1 H NMR (400 MHz, CDCl 3 ) δ 7.53 (d, J = 8.2 Hz, 1H), 7.34 (dd, J = 8.2, 1.6 Hz, 1H), 7.30 (d, J = 1.2 Hz, H), 6.09 - 5.96 (m, 2H), 5.54 (dd, J = 17.2, 1.6 Hz, 1H), 5.41 (dd, J = 15.6, 1.6 Hz, 1H), 5.32 (dd, J = 10.8, 1.6 Hz, 1H), 5.27 (dd, J = 10.8, 1.6 Hz, 1H), 4.79 (d, J = 5.6, 2H), 4.60 (d, J = 4.8, 2H).

[0354] Prepare Compound 22: Add 21 (1.83 g, 5.32 mmol), 1,2-ethanedithiol (890 μL, 10.64 mmol), copper sulfate pentahydrate (66.4 mg, 0.266 mmol), KOH (1.5 g, 26.6 mmol), N 2Replace three times, then add 4 mL of DMSO and 1 mL of H 2 O, stir and react overnight at 110 °C. Stop the reaction, cool to room temperature, then add 1 M HCl aqueous solution to adjust to acidity, dilute with 20 mL of ethyl acetate, wash three times with water (20 mL each), separate the organic phase with a separatory funnel, dry over anhydrous sodium sulfate, filter by suction, mix with silica gel, separate by column chromatography, and rotary evaporate to dryness to obtain 752.6 mg of a white solid with a yield of 67%. Spectral characteristics: 1 1H NMR (400 MHz, CDCl 3 ) δ 8.03 (d, J = 8.4 Hz, 1H), 6.98 (dd, J = 8.4, 1.6 Hz, 1H), 6.89 (d, J = 1.6 Hz, 1H), 6.14 - 6.04 (m, 1H), 5.45 - 5.52 (m, 2H), 4.77 (d, J = 5.6 Hz, 2H), 3.69 (s, 1H).

[0355] Preparation of compound 23: Add 22 (435.2 mg, 2.1 mmol) to a 10 mL Schlenk reaction tube, N 2 Replace three times, then add 4.2 mL of dry THF, and then dropwise add 6.3 mL of a tetrahydrofuran solution of lithium aluminum hydride under nitrogen protection, and stir overnight at room temperature. Stop the reaction, slowly add water dropwise in an ice bath, adjust to acidity with 1 M HCl aqueous solution, dilute with 20 mL of ethyl acetate, wash three times with water (20 mL each), separate the organic phase with a separatory funnel, dry over anhydrous sodium sulfate, filter by suction, and directly use the obtained target compound in the next step after rotary evaporation to dryness.

[0356] b. Preparation of compound 24

[0357] Add IR780 (108.2 mg, 0.16 mmol), 23 (117.1 mg, 0.75 mmol), and KHCO 3 (48.3 mg, 0.48 mmol) to a 10 mL Schlenk reaction tube, N 2 Replace three times, then add 0.5 mL of dry DMF, and stir and react at 75 °C for 2 h. Stop the reaction, add 20 mL of ethyl acetate to dilute, wash three times with water (20 mL each), separate the organic phase with a separatory funnel, dry over anhydrous sodium sulfate, filter by suction, mix with silica gel, separate by column chromatography, and rotary evaporate to dryness to obtain 54.2 mg of a blue compound with a yield of 62%. Spectral characteristics: 1 1H NMR (400 MHz, CDCl 3)δ8.11(d, J = 13.6 Hz, 1H), 7.58(s, 1H), 7.52(d, J = 7.6 Hz, 1H), 7.43 - 7.39(m, 2H), 7.30 - 7.25(m, 2H), 6.86(s, 1H), 6.19(d, J = 13.6 Hz, 1H), 4.65(s, 2H), 4.09(t, J = 7.2 Hz, 2H), 2.75(t, J = 5.2 Hz, 2H), 2.63(t, J = 5.6 Hz, 2H), 1.92 - 1.61(m, 4H), 1.73(s, 6H), 1.03(t, J = 7.6 Hz, 3H). 13 C NMR(101MHz, CDCl 3 )δ175.83, 158.76, 146.42, 144.86, 144.47, 143.50(d, J = 3.2 Hz), 142.27, 138.62, 134.31, 133.85, 132.40, 128.70, 128.44, 126.03, 124.54, 115.07, 114.57, 103.69, 63.24, 52.94, 49.10, 35.81, 31.57, 29.91, 24.62, 24.28, 14.23.

[0358] c. Preparation of Compound 26

[0359] Dye 24 (54.2 mg, 0.076 mmol), KI (31.2 mg, 0.186 mmol), KHCO 3 (19.7 mg, 0.197 mmol), 18 - crown - 6 (3.4 mg, 0.013 mmol) were added to a 10 mL reaction tube and evacuated and replaced with N 2 three times. Under N 2 protection, 1.0 mL of ultra - dry DMF was added, and then 4 (87.4 mg, 0.279 mmol) was added. The reaction was stirred at room temperature for 4 h, and then the reaction was stopped. 10.0 mL of DCM was added for dilution, and it was washed with water (5.0 mL). The organic phase was separated with a separatory funnel, dried over anhydrous sodium sulfate, filtered by suction, mixed with silica gel, and quickly passed through a column. After rotary evaporation, a blue solid compound 25 was obtained. Compound 25 was added to a 10 mL reaction tube, 1 mL of DCM was added, and then 6 (42.3 mg, 0.19 mmol) and TEA (37 μL, 0.27 mmol) were added. The reaction was stirred at room temperature overnight, and then the reaction was stopped. 10.0 mL of DCM was added for dilution, and it was washed with water (5.0 mL). The organic phase was separated with a separatory funnel, dried over anhydrous sodium sulfate, filtered by suction, mixed with silica gel, separated by column chromatography, and rotary evaporation gave 40.7 mg of a blue solid compound with a two - step yield of 57%. Spectral characteristics: 11H NMR (600 MHz, CDCl 3 ) δ 9.34 (d, J = 14.4 Hz, 1H), 8.55 - 8.53 (m, 2H), 8.49 (t, J = 7.2 Hz, 1H), 8.44 - 8.41 (m, 3H), 8.34 (d, J = 7.8 Hz, 1H), 8.28 - 8.26 (m, 2H), 8.22 (s, 2H), 7.55 (d, J = 14.4 Hz, 1H), 7.00 - 6.93 (m, 2H), 6.40 (dd, J = 17.4, 1.2 Hz, 2H), 6.29 (dd, J = 10.8, 1.2 Hz, 2H), 6.21 (d, J = 17.4 Hz, 4H), 5.72 - 5.62 (m, 4H), 5.31 (t, J = 7.2 Hz, 2H), 5.01 (s, 3H), 3.77 (t, J = 5.6 Hz, 2H), 3.67 (t, J = 5.6 Hz, 2H), 3.27 (s, 1H), 2.98 - 2.95 (m, 2H), 2.81 (s, 6H), 2.28 (s, 2H), 2.07 (t, J = 7.2 Hz, 3H). 13 13C NMR (151 MHz, CDCl 3 ) δ 177.29, 160.47, 160.23, 157.51, 145.23, 142.07, 141.41, 136.40, 135.76, 133.06, 132.42, 132.00 (d, J = 6.9 Hz), 131.13, 129.20, 128.76, 127.53, 127.07, 126.87, 123.08, 122.70, 120.36 (d, J = 4.4 Hz), 117.16, 115.25, 112.53, 107.35, 105.08, 79.64, 71.64, 70.07, 68.96 (d, J = 6.0 Hz), 61.49, 50.77, 46.94, 32.47, 30.89, 29.70, 28.43, 26.66, 21.31, 20.24, 14.13, 11.44.

[0360] d. Preparation of Compound ALPIN - 7

[0361] Add 26 (25.7 mg, 0.028 mmol), 1,3 - dimethylbarbituric acid (14.3 mg, 0.825 mmol), and Pd(PPh 3 ) 4 (6.5 mg, 0.006 mmol) into a 10 mL Schlenk reaction tube and evacuate and replace with N 2Three times, add 0.5 mL of MeCN and DCM each. After stirring the reaction at room temperature for 1 h, rotary evaporate the reaction solution, add 9 (6.5 mg, 0.027 mmol), CuSO 4 (4.4 mg, 0.027 mmol), ascorbic acid (19.4 mg, 0.11 mmol), DMSO (0.5 mL), and H 2 O (0.5 mL), stir the reaction at room temperature for 1 h, and prepare the target compound 4.5 mg by high-performance preparative liquid chromatography, with a three-step yield of 15%. Spectral characteristics: 1 H NMR (600 MHz, d 6 -DMSO) δ 8.28 (d, J = 14.4 Hz, 1H), 8.11 (s, 1H), 7.94 - 7.86 (m, 2H), 7.83 (d, J = 7.2 Hz, 1H), 7.80 - 7.75 (m, 4H), 7.62 (d, J = 19.8 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.50 (t, J = 7.8 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.40 (s, 1H), 7.21 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 15 Hz, 1H), 5.26 (s, 2H), 5.09 (s, 2H), 4.92 (t, J = 7.2 Hz, 2H), 4.44 (t, J = 7.2 Hz, 2H), 4.27 (d, J = 5.4 Hz, 2H), 3.85 (t, J = 6.9 Hz, 2H), 3.07 (s, 6H), 2.77 (s, 2H), 2.70 (s, 2H), 2.45 (t, J = 6.9 Hz, 2H), 1.99 - 1.94 (m, 2H), 1.87 - 1.81 (m, 4H), 1.77 (s, 6H), 1.23 (s, 2H), 0.98 (t, J = 7.4 Hz, 3H).

[0362] Test Example 4: Biological Experiment Verification of Near-Infrared Fluorescence and Photoacoustic Probe ALPIN-7

[0363] Test Example 4.1: Imaging Comparison of Near-Infrared Fluorescence and Photoacoustic Probes ALPIN-7 and ALPIN-8 on HeLa Cells with Overexpressed Alkaline Phosphatase (ALP) and Imaging of HEK293 with Weak ALP Expression

[0364] Experimental Conditions: Incubate 5 μM fluorescent probes ALPIN-7 and ALPIN-8 (structures shown in Figure 3.1 ) with HeLa cells with overexpressed ALP on the cell membrane surface and normal tissue cells HEK293 with low ALP expression in a culture dish in a 37 °C incubator for 30 min; or pre-treat HeLa cells with the alkaline phosphatase inhibitor Na3 VO 4 Pretreat for 1 hour, and then co-incubate with the probe for 30 min. Subsequently, discard the medium containing the probe, and wash once with PBS buffer (pH = 7.4); add 1 mL of phenol red-free DMEM, and perform cell imaging (Cy 5.5 channel) using a confocal fluorescence microscope.

[0365] The experimental results are as Figure 3.1 shown, and it can be seen from Figure 3.1 that compared with the control probe ALPIN-8, the probe ALPIN-7 can not only achieve more obvious fluorescence imaging of ALP overexpressing cells, but also has more obvious fluorescence signals than ALP low-expressing cell lines (HEK293). In addition, in the experiment of the ALP inhibitor group, the fluorescence signal of the fluorescent probe ALPIN-7 for labeling HeLa cells weakened, indicating that the initiation of the labeling effect of the fluorescent probe depends on the property of alkaline phosphatase specifically hydrolyzing the phosphate ester bond.

[0366] Experimental conclusion: After reacting with the ALP enzyme in cells, the fluorescent probe ALPIN-7 can have a better cell enrichment effect, reduce the diffusion of the fluorescent probe in cell imaging experiments, reduce the background signal, and greatly improve the detection sensitivity.

[0367] Test Example 4.2: Real-time imaging of HeLa cells with near-infrared fluorescence photoacoustic probe ALPIN-7

[0368] Experimental conditions: The cultured HeLa cells were spread on a confocal dish, and 1 μM fluorescent probe ALPIN-7 was added to the HeLa cells, and cell imaging (Cy 5.5 channel) was performed at different time periods using a confocal fluorescence microscope.

[0369] The experimental results are as Figure 3.2 shown, and it can be seen from Figure 3.2 that when the probe ALPIN-7 was used for real-time imaging of HeLa cells, first, the fluorescence signal quickly appeared on the cell membrane, which was consistent with the expression of ALP enzyme on the HeLa cell membrane. As time passed, the fluorescence signal gradually appeared inside the cell, and the fluorescence signal inside the cell became stronger as time extended.

[0370] Experimental conclusion: The fluorescent probe ALPIN-7 can visualize the activity of ALP enzyme in HeLa cells in real time. Because after the enzyme activates the probe, it is converted from hydrophilic to lipophilic, which enhances the cell uptake and the self-anchoring of methylene benzoquinone, restricts the penetration and diffusion of the turned-on fluorescence signal, greatly enhances the accumulation of the fluorescence signal in tumor cells, and improves the ability of the probe to visualize the enzyme activity in real time.

[0371] Test Example 4.3: Real-time Imaging of HeLa Cell-bearing Tumor Mice with Near-infrared Fluorescent Photoacoustic Probe ALPIN-7

[0372] Experimental conditions:

[0373] Construction of HeLa tumor model nude mice:

[0374] (1) Preparation of HeLa cells: The cell culture and passage were the same as before. After culturing and digesting the cells and centrifuging, add PBS and wash twice, 1 mL each time. After washing and centrifuging, discard the supernatant, add DMEM without FBS and mix well, count, then add Matrigel, mix well and place in an ice bath for later use.

[0375] (2) Implantation of HeLa cells into tumor model nude mice: Female BALB / c nude mice aged 4 - 6 weeks were subcutaneously implanted with 200 μL of a mixed solution of DMEM (without FBS) containing 4×10 6 HeLa cells and Matrigel (1:4) in the right posterior thigh. At 25 °C, grow for 2 - 3 weeks with water and food provided ad libitum. When the tumor grows to about 0.1 cm 3 , perform animal imaging experiments.

[0376] (3) In vivo imaging of tail vein injection of the probe in HeLa tumor model nude mice: First, divide the experimental animals into 3 groups, 3 mice in each group. Use the IVIS Lumina XRMS Series III in vivo animal imaging system to collect the whole-body background fluorescence images (λ ex = 660 nm, λ em = 710 nm) of 6 nude mice. Then prepare the fluorescent probe ALPIN-7 or ALPIN-8 into a PBS solution with a concentration of 40 μM. Use an insulin syringe to inject 200 μL of the prepared probe solution into the tail vein of the tumor model mice. Subsequently, collect the whole-body fluorescence data of the experimental model mice at 6 different time points (1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 48 h). For the inhibitor group, first inject 50 μL of 10 mM Na 3 VO 4 solution into the tumor in situ, and then inject the same concentration of probe ALPIN-7 half an hour later, and then perform imaging at the same time period.

[0377] (4) Anatomical tissue imaging of HeLa tumor model nude mice: After 36 h of injecting the probe via the tail vein, kill the nude mice by cervical dislocation. After dissecting to obtain the tumor, heart, lung, liver, spleen, and kidney, perform fluorescence imaging of the organs and tissues.

[0378] The experimental results are as Figure 3.3 shown, as shown by Figure 3.3It can be seen that after intravenous injection of the probe, obvious fluorescence signals appeared on the tumors in the ALPIN-7 injection probe group 1 h later, and obvious fluorescence signals were still present on the tumors until 36 h. However, the fluorescence signals on the tumors in the group pre-injected with the inhibitor were significantly weakened; the control probe ALPIN-8 group also showed fluorescence signals at 1 h, but they were significantly weaker than those in the ALPIN-7 group, and the fluorescence signals on the tumors disappeared 2 h later. This result was also consistent with the fluorescence signals on the tumors after dissection of the tumor-bearing mice.

[0379] Experimental conclusion: In a highly dynamic in vivo animal system, ALPIN-7 has better tumor signal enrichment and retention effects, improves the signal intensity of in vivo tumor detection, is beneficial to the detection of in vivo tumor tissues, and plays a very important role in surgical navigation for tumor tissue resection.

[0380] All the documents mentioned in the present invention are cited in this application as references, as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A compound, characterized in that, the compound is shown as Formula I; In Formula I, R 1 is a group that can be specifically hydrolyzed by a biopolymer enzyme or a group that can be specifically activated by a disease microenvironment factor; and after being specifically hydrolyzed or specifically activated, R 1 and X 1 the bond between them is broken and R 1 leaves; X 1 is O or NH; L is a linking group that can leave on its own after R 1 has left; R 2 is a water-soluble group; the FP moiety is a fluorescent group or a photosensitive group.

2. The compound according to claim 1, characterized in that, R 1 selected from the group consisting of:

3. The compound according to claim 1, characterized in that, L is selected from the following group:

4. The compound according to claim 1, characterized in that, R 2 selected from the group consisting of:

5. The compound according to claim 1, characterized in that, the FP moiety has a structure shown as Formula II-1 or Formula II-2: wherein, a represents the position connected to ; b represents the position connected to ; R 3 is -C 1-6 alkyl, -C 1-6 alkyl-R Fu ; wherein, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu is selected from the group consisting of: -COOH, -N 3 , -NH 2 , -N + (C 1-6 alkyl) 3 ); R 4 、R 5 、R 6 and R 7 are each independently H or a heavy atom I, Br; R 8 is H, F, Cl, or Br; X 2 is O or S; X 3 is I, Br, Cl; or, the FP moiety has a structure shown as Formula III-1 or III-2: wherein, a represents the position connected to ; b represents the position connected to ; R 3 is -C 1-6 alkyl, -C 1-6 alkyl-R Fu ; wherein, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu is selected from the group consisting of: -COOH, -N 3 , -NH 2 , -N + (C 1-6 alkyl) 3 ); R 4 、R 5 、R 6 and R 7 are each independently H or a heavy atom (such as I, Br); R 8 is H, F, Cl, or Br; X 2 is O or S; X 3 is I, Br, or Cl; or, the FP moiety has a structure shown as Formula IV-1 or IV-2: wherein, a represents the position connected to ; b represents the position connected to ; X 2 is O or S; X 4 is O or NR 3 ; R 3 is -C 1-6 alkyl, -C 1-6 alkyl-R Fu ; wherein, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu is selected from the group consisting of: -COOH, -N 3 , -NH 2 , -N + (C 1-6 alkyl) 3 ); R 4 、R 5 、R 6 and R 7 are each independently H or a heavy atom (such as I, Br); R 8 is H, F, Cl, or Br; or, the FP moiety has a structure shown as Formula V-1 or V-2: wherein, a represents the position connected to ; b represents the position connected to ; X 2 is O or S; X 5 is O or S; R 9 is NEt 2 , NPh 2 , or NCH 3 Ph; R 8 is H, F, Cl, or Br; or, the FP moiety has a structure shown as Formula VI-1 or VI-2: wherein, a represents the position connected to ; b represents the position connected to ; X 2 is O or S; X 5 is O or S; R 10 and R 11 each independently is C 1-6 alkyl (such as Et) or Ph; R 8 is H, F, Cl or Br.

6. The compound according to claim 1, characterized in that, the compound is selected from Table A Table A 7. The compound according to any one of claims 1-6, characterized in that, the compound can be used as a probe or a photosensitizer.

8. A composition, characterized in that, the composition comprises: (1) The compound according to any one of claims 1-7; and (2) A pharmaceutically or detectably acceptable carrier.

9. Use of a compound according to any one of claims 1-7 or a composition according to claim 8, characterized in that, the use is one or more selected from the following group: (a) For the detection of an enzyme; (b) For the detection of disease microenvironment factors (such as ROS); (c) For the preparation of a detection reagent for the detection of an enzyme or disease microenvironment factors (such as ROS); (d) For the imaging of cells; (e) For the preparation of a detection reagent for cell imaging analysis; (f) For the imaging of tumor tissues or cells; (g) For the preparation of a detection reagent for tumor tissues or cells.

10. Use of a compound according to any one of claims 1-7 or a composition according to claim 8, characterized in that, the use is one or more selected from the following group: (h) For the photodynamic therapy of tumors; (i) For the preparation of a photosensitive therapeutic agent for the photodynamic therapy of tumors.

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