Targeted activation enrichment near-infrared probes and photosensitizer design, preparation and application

Abstract

Provided are a class of targeted activation enrichment near-infrared probes and photosensitizers, and the design, preparation and application thereof. Specifically, provided are a class of novel molecular design, preparation and application methods, which enable the opening of fluorescence signals or photosensitive properties under the action of disease-related enzymes or chemical environments, and can be effectively enriched around disease-related cells and tissues. The compounds are shown as formula I, wherein each variable is defined in the specification.

Description

Design, preparation and application of targeted activation-enriched near-infrared probes and photosensitizers

[0001] This application claims priority to Chinese Patent Application No. 2023117032530 filed on December 12, 2023, and Chinese Patent Application No. 202411740178X filed on November 29, 2024. This application incorporates the entirety of the aforementioned Chinese patent applications. Technical Field

[0002] The present invention belongs to the field of biomarkers and biomedicine, and specifically relates to the design, preparation and application of targeted activation enrichment type near-infrared probes and photosensitizers. Background Art

[0003] Abnormal expression of enzymes or disease microenvironment factors (ROS, etc.) 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 a variety of diseases such as cancer and liver damage. γ-glutamyl transpeptidase (GGT) is abnormally active in diseases such as hepatotoxicity, asthma, diabetes and cancer. N-acetyl-β-D-glucosidase (NAG) has been proven to be a biomarker of kidney damage in clinical practice. Abnormal expression of carboxylesterase (CE) in physiology and pathology is closely related to the occurrence of liver cancer. The disease microenvironment factor H2O2 is related to diseases of liver damage and kidney damage.

[0004] Closely related to kidney damage. Living organisms are extremely complex systems that undergo dynamic changes at all times. Therefore, real-time in vivo monitoring of enzyme activity and ROS production in living organisms is of great significance for disease prevention, early diagnosis, and treatment.

[0005] To achieve highly sensitive and real-time detection of enzyme activity and ROS, researchers have developed enzyme- and ROS-activated fluorescence-enhanced probes based on the ability of enzymes and ROS to specifically catalyze the conversion of their substrates into products. These probes typically consist of a fluorophore and an enzyme or ROS recognition group. The fluorescence signal of the fluorophore in these probes is quenched by the recognition group, which turns on when the target enzyme or ROS-specific hydrolase recognizes the group. This results in enhanced fluorescence signal intensity or shifts in the absorption and emission wavelengths, while also achieving catalytic amplification of the fluorescence signal. Enzyme- or ROS-activated fluorescence-enhanced probes are typically tested in solution to measure the probe's sensitivity to the enzyme, with the detection limit determined by enzyme kinetics. However, unlike the relatively homogeneous, closed, and static nature of conventional detection solutions, living organisms present a more complex, highly dynamic, and open heterogeneous environment. The fluorescent signal molecules activated by enzymes or ROS are easily affected by the dynamic physiological environment and rapidly diffuse away from the detection site and are excreted from the organism, resulting in a decrease in the signal-to-noise ratio and sensitivity of fluorescent probes for enzyme or ROS activity detection. Generally, 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 and alkaline phosphatase, the activated fluorescent signal molecules come into direct contact with the blood, making it easier for them to diffuse rapidly from the enzyme detection site and be discharged from the body along with the blood flow. This results in a decrease in the sensitivity of the fluorescent probe when detecting enzymes or ROS in living animals, making it difficult to achieve the expected fluorescence imaging effect. Therefore, the application of fluorescent probes is more challenging in complex, dynamic, and open living organisms. Therefore, the development of near-infrared fluorescent probes that can achieve great enrichment at the detection site is of great significance for improving the sensitivity of enzyme activity or ROS detection in living organisms.

[0006] The special catalytic activity of enzymes plays an important role not only in the diagnosis of diseases, but also in the treatment of diseases.

[0007] Regarding tumors: Photodynamic therapy (PDT) has become an important method for clinical tumor treatment due to its minimally invasive nature, negligible drug resistance and good therapeutic effect. Photodynamic therapy mainly induces tumor cell death by producing highly toxic reactive oxygen species (ROS) in tumor tissue under the excitation of light of 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 activated photosensitizers based on the activity of tumor-related enzymes have been developed to achieve selective activation in tumor tissues. However, since living animals are a highly dynamic living system, activated photosensitizers are easily cleared from tumor tissues and significantly reduce the effect of photodynamic therapy. Therefore, achieving effective enrichment of photosensitizers in tumor tissues is of great significance to improving the photodynamic therapy effect and specificity of photosensitizers.

[0008] Regarding antibacterial treatments: Photodynamic therapy (PDT) is a treatment modality that uses light of a specific wavelength to activate photosensitizers, causing them to produce reactive oxygen molecules, inducing the death of diseased tissue. There are numerous reports on the application of photosensitizers in antibacterial applications, but these molecules, like traditional probes, are difficult to retain at the target site and easily diffuse in vivo.

[0009] In summary, there is an urgent need in this field to develop a class of probes and photosensitizers that can be enriched at target sites. Summary of the Invention

[0010] The purpose of the present invention is to provide a class of targeted activation-enrichment near-infrared probes and photosensitizers for design, preparation, and application. Specifically, the present invention provides a novel class of molecular design, preparation, and application methods that can activate fluorescent signals or photosensitivity in the presence of disease-related enzymes or chemical environments, and effectively enrich around disease-related cells and tissues. The compounds of the present invention can be linked to different specific activation groups according to different needs, thereby activating the molecules at the target location. For example, when targeting the tumor field, groups that are specifically activated in the tumor environment can be introduced into the molecule; when targeting β-lactamase-resistant bacteria, groups that are specifically activated in the β-lactamase environment can be introduced into the molecule.

[0011] In the first aspect of the present invention, a compound is provided, wherein the compound is as shown in Formula I;

[0012] In Formula I,

[0013] R1 is a group that can be specifically hydrolyzed by a biomacromolecule enzyme or a group that can be specifically activated by a disease microenvironmental factor; and after being specifically hydrolyzed or specifically activated, the bond between R1 and X1 is broken and R1 leaves;

[0014] X1 is a connecting bond, O or NH;

[0015] L is a linking group that can leave on its own when R1 leaves;

[0016] R2 is C 1-6 Alkyl or water-soluble groups;

[0017] The FP portion is a fluorescent group or a photosensitive group.

[0018] In another preferred embodiment, the ability to be specifically hydrolyzed by a biomacromolecular enzyme means that when R1 is a group that can be specifically hydrolyzed by a biomacromolecular enzyme, the biomacromolecular enzyme can recognize the group and, under the action of the biomacromolecular enzyme, hydrolyze X1-R1 to break the bond between X1 and R1.

[0019] In another preferred embodiment, the biomacromolecule enzyme is alkaline phosphatase, γ-glutamyl transpeptidase, aminopeptidase, Fapα enzyme, Hepsin enzyme or β-lactamase, preferably alkaline phosphatase or β-lactamase.

[0020] In another preferred embodiment, the group that can be specifically hydrolyzed by a biomacromolecule enzyme is: an enzyme recognition group that can be specifically hydrolyzed by alkaline phosphatase, an enzyme recognition group that can be specifically hydrolyzed by γ-glutamyl transpeptidase, an enzyme recognition group that can be specifically hydrolyzed by aminopeptidase, an enzyme recognition group that can be specifically hydrolyzed by Fapα enzyme, an enzyme recognition group that can be specifically hydrolyzed by Hepsin enzyme, or an enzyme recognition group that can be specifically hydrolyzed by β-lactamase.

[0021] In another preferred embodiment, the enzyme recognition group that can be specifically hydrolyzed by alkaline phosphatase is

[0022] In another preferred embodiment, the enzyme recognition group that can be specifically hydrolyzed by γ-glutamyl transpeptidase is

[0023] In another preferred embodiment, the enzyme recognition group capable of being specifically hydrolyzed by aminopeptidase is

[0024] In another preferred embodiment, the enzyme recognition group that can be specifically hydrolyzed by Fapα enzyme is

[0025] In another preferred embodiment, the enzyme recognition group capable of being recognized by Hepsin enzyme is

[0026] In another preferred embodiment, the enzyme recognition group that can be specifically hydrolyzed by β-lactamase is Among them, R 1a Select from the following group: C 1-6 Alkyl, phenyl, 5- or 6-membered heteroaryl, C 1-4 Alkylene-phenyl, C 1-4 Alkylene-(5 or 6 membered heteroaryl), water-soluble group, wherein the C 1-6 Alkyl, phenyl, 5- or 6-membered heteroaryl, C 1-4 Alkylene-phenyl, C 1-4 Alkylene-(5 or 6 membered heteroaryl) is optionally substituted with one or more halogen, C 1-6 Alkyl or halogenated C 1-6 Alkyl substituted; preferably, R 1a It is a water-soluble group;

[0027] R 2a Selected from the following group: H, -OC 1-4Alkyl; preferably, R 2a Selected from the group consisting of H, -OMe; More preferably, R 2a is H;

[0028] R 3a Selected from the following group: H, C 1-6 Alkyl, monovalent cations (such as Na + ), wherein the C 1-6 The alkyl group is optionally substituted with one or more halogen, C 1-6 Alkyl or halogenated C 1-6 Alkyl substituted; preferably R 3a Selected from the following group: H, C 1-6 Alkyl, monovalent cation; more preferably, R 3a is H;

[0029] R 4a and R 5a Each independently selected from the following group: H, C 1-4 Alkyl (such as methyl or ethyl); or R 4a and R 5a Together with the carbon atom to which it is connected, it forms C 3-4 Cycloalkyl; preferably, R 4a and R 5a Each independently selected from the following group: H, C 1-4 Alkyl (such as methyl or ethyl); more preferably, R 4a and R 5a is H;

[0030] n is 0, 1, or 2; preferably, n is 0.

[0031] In another preferred embodiment, the ability to be specifically activated by disease microenvironmental factors means that when R1 is a group that can be specifically activated by disease microenvironmental factors, the bond between X1 and R1 can be broken under the action of disease microenvironmental factors.

[0032] In another preferred embodiment, the disease microenvironmental factor is infection by drug-resistant bacteria expressing β-lactamase or reactive oxygen species (ROS).

[0033] In another preferred embodiment, the active oxygen is preferably peroxide or superoxide anion.

[0034] In another preferred embodiment, the group that can be specifically activated by peroxide is:

[0035] In another preferred embodiment, the group that can be specifically activated by superoxide anions is

[0036] In another preferred embodiment, the group that can be specifically activated by disease microenvironmental factors is a group that can be specifically activated by peroxides or a group that can be specifically activated by superoxide anions.

[0037] In another preferred embodiment, the water-soluble group is a -L1-W1-L2-water-soluble portion; wherein L1 and L2 are each independently absent or a divalent linking group, and W1 is absent or a linking group formed by a click chemistry reaction.

[0038] In another preferred embodiment, L1 and L2 are each independently absent or a divalent linking group that is stable and non-cleavable in a cell or in vivo environment, preferably absent or a C1-C4 alkylene group.

[0039] In another preferred embodiment, W1 is none or

[0040] In another preferred embodiment, the water-soluble portion is selected from the following group: a group containing an anion-cation pair, a group containing one or more carboxyl groups, and a group containing one or more sulfonic acid groups.

[0041] In another preferred embodiment, the group containing one or more carboxyl groups is a group containing 2 or 3 carboxyl groups, for example

[0042] In another preferred embodiment, the group containing one or more sulfonic acid groups is a group containing 2 or 3 sulfonic acid groups, for example

[0043] In another preferred embodiment, the group containing an anion-cation pair is wherein a is 1, 2, 3 or 4, preferably 2 or 3.

[0044] In another preferred embodiment, the water-soluble group is Preferably

[0045] In another preferred embodiment, the water-soluble group is a water-soluble group that can be connected to a carbonamide structure. When the compound is activated, the carbonamide is disconnected and the water-soluble group leaves.

[0046] In another preferred embodiment, R1 is selected from the following group:

[0047] where R 1a 、R 2a 、R 3a 、R 4a 、R 5a and n are as defined above; preferably, R1 is where R 1a is a water-soluble group; more preferably, R1 is

[0048] In another preferred embodiment, X1 is a connecting bond or O, preferably O.

[0049] In another preferred embodiment, L is selected from the following group: C1-C4 alkylene; preferably, L is selected from the following group: C1-C4 alkylene; wherein * represents the connection position with X1; more preferably, L is

[0050] In another preferred embodiment, R2 is C 1-6 Alkyl or water-soluble group; preferably, R2 is ethyl, Preferably ethyl,

[0051] In another preferred embodiment, the FP portion has a structure as shown in Formula II-1 or Formula II-2:

[0052] in,

[0053] a represents The location of the connection;

[0054] b stands for The location of the connection;

[0055] R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu Select from the following groups: -COOH, -N3, -NH2, -N + (C 1-6 alkyl)3);

[0056] R4, R5, R6 and R7 are each independently H, Br or I;

[0057] R8 is H, F, Cl, or Br;

[0058] X2 is O or S;

[0059] X3 is I, Br, or Cl;

[0060] Preferably, the FP portion is

[0061] In another preferred embodiment, the compound is as shown in Formula I;

[0062] In Formula I,

[0063] R1 is a group that can be specifically hydrolyzed by a biomacromolecule enzyme; and after being specifically hydrolyzed, the bond between R1 and X1 is disconnected and R1 leaves; the biomacromolecule enzyme is alkaline phosphatase or β-lactamase;

[0064] X1 is a connecting bond, O or NH;

[0065] L is a linking group that can leave on its own when R1 leaves;

[0066] R2 is C 1-6 Alkyl or water-soluble group; the water-soluble group is -L1-W1-L2-water-soluble part;

[0067] L1 and L2 are each independently C1-C4 alkylene;

[0068] W1 is

[0069] The water-soluble portion is selected from the group consisting of: A group containing 2 or 3 carboxyl groups, or a group containing 2 or 3 sulfonic acid groups, wherein a is 1, 2, 3 or 4;

[0070] The FP portion has a structure as shown in Formula II-1 or Formula II-2:

[0071] in,

[0072] a represents The location of the connection;

[0073] b stands for The location of the connection;

[0074] R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu Select from the following groups: -COOH, -N3, -NH2, -N + (C 1-6 alkyl)3);

[0075] R4, R5, R6 and R7 are each independently H, Br or I;

[0076] R8 is H, F, Cl, or Br;

[0077] X2 is O or S;

[0078] X3 is I, Br, or Cl.

[0079] In another preferred embodiment, the compound is as shown in Formula I;

[0080] In Formula I,

[0081] R1 is where R 1a 、R 2a 、R 3a 、R 4a 、R 5a and n is as defined in claim 3 or 4;

[0082] X1 is a connecting bond or O;

[0083] L is or C1-C4 alkylene;

[0084] R2 is C 1-6 Alkyl or water-soluble group; the water-soluble group is -L1-W1-L2-water-soluble part;

[0085] L1 and L2 are each independently C1-C4 alkylene;

[0086] W1 is

[0087] The water-soluble portion is selected from the group consisting of: A group containing 2 or 3 carboxyl groups, or a group containing 2 or 3 sulfonic acid groups, wherein a is 1, 2, 3 or 4;

[0088] The FP portion has a structure as shown in Formula II-1 or Formula II-2:

[0089] in,

[0090] a represents The location of the connection;

[0091] b stands for The location of the connection;

[0092] R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu Select from the following groups: -COOH, -N3, -NH2, -N + (C 1-6 alkyl)3);

[0093] R4, R5, R6 and R7 are each independently H, Br or I;

[0094] R8 is H, F, Cl, or Br;

[0095] X2 is O or S;

[0096] X3 is I, Br, or Cl.

[0097] In another preferred embodiment, the compound is as shown in Formula I;

[0098] In Formula I,

[0099] R1 is a group that can be specifically hydrolyzed by a biomacromolecule enzyme; and after being specifically hydrolyzed, the bond between R1 and X1 is broken and R1 leaves; the biomacromolecule enzyme is alkaline phosphatase;

[0100] X1 is O or NH;

[0101] L is a linking group that can leave on its own when R1 leaves;

[0102] R2 is a water-soluble group; the water-soluble group is a -L1-W1-L2-water-soluble part;

[0103] L1 and L2 are each independently C1-C4 alkylene;

[0104] W1 is

[0105] The water-soluble portion is selected from the group consisting of: A group containing 2 or 3 carboxyl groups, or a group containing 2 or 3 sulfonic acid groups, wherein a is 1, 2, 3 or 4;

[0106] The FP portion has a structure as shown in Formula II-1 or Formula II-2:

[0107] in,

[0108] a represents The location of the connection;

[0109] b stands for The location of the connection;

[0110] R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu Select from the following groups: -COOH, -N3, -NH2, -N + (C 1-6 alkyl)3);

[0111] R4, R5, R6 and R7 are each independently H, Br or I;

[0112] R8 is H, F, Cl, or Br;

[0113] X2 is O or S;

[0114] X3 is I, Br, or Cl.

[0115] In another preferred embodiment, the compound is as shown in Formula I;

[0116] In Formula I,

[0117] R1 is

[0118] X1 is 0;

[0119] L is a linking group that can leave on its own when R1 leaves;

[0120] R2 is a water-soluble group; the water-soluble group is a -L1-W1-L2-water-soluble part;

[0121] L1 and L2 are each independently C1-C4 alkylene;

[0122] W1 is

[0123] The water-soluble portion is a is 2 or 3;

[0124] The FP portion has a structure as shown in Formula II-1:

[0125] in,

[0126] a represents The location of the connection;

[0127] b stands for The location of the connection;

[0128] R3 is -C 1-6 alkyl;

[0129] R4, R5, R6 and R7 are each independently H, Br or I;

[0130] R8 is H, F, Cl, or Br;

[0131] X2 is O or S;

[0132] X3 is I, Br, or Cl.

[0133] In another preferred embodiment, the compound is as shown in Formula I;

[0134] In Formula I,

[0135] R1 is

[0136] X1 is 0;

[0137] L is

[0138] R2 is a water-soluble group; the water-soluble group is a -L1-W1-L2-water-soluble part;

[0139] L1 and L2 are each independently C1-C4 alkylene;

[0140] W1 is

[0141] The water-soluble portion is a is 2 or 3;

[0142] The FP portion is

[0143] In another preferred embodiment, the compound is as shown in Formula IA;

[0144] in

[0145] n = 0, 1, or 2;

[0146] R 1a Select from the following group: C 1-6 Alkyl, phenyl, 5- or 6-membered heteroaryl, C 1-4 Alkylene-phenyl, C 1-4 Alkylene-(5- or 6-membered heteroaryl), water-soluble group;

[0147] R 2a Selected from the following group: H, -OC 1-4 alkyl;

[0148] R 3a Selected from the following group: H, C 1-6 Alkyl, monovalent cation;

[0149] R 4a and R 5a Each independently selected from the following group: H, C 1-4 alkyl;

[0150] R 6a Select from the following group: C 1-6 Alkyl, water-soluble group;

[0151] R 7a C 1-6alkyl;

[0152] R 8a 、R 9a 、R 10a and R 11a Each independently selected from the group consisting of H, Br or I;

[0153] It is a monovalent acid ion;

[0154] The water-soluble group is a -L1-W1-L2-water-soluble part;

[0155] L1 and L2 are each independently C1-C4 alkylene;

[0156] W1 is

[0157] The water-soluble portion is Or a group containing 2 or 3 carboxyl groups, a is 2 or 3.

[0158] In another preferred embodiment, the compound is as shown in Formula IA;

[0159] in

[0160] n=0;

[0161] R 1a It is a water-soluble group;

[0162] R 2a Selected from the following group: H, -OC 1-4 alkyl;

[0163] R 3a Selected from the following group: H, C 1-6 Alkyl, monovalent cation;

[0164] R 4a and R 5a Each independently selected from the following group: H, C 1-4 alkyl;

[0165] R 6a Select from the following group: C 1-6 Alkyl, water-soluble group;

[0166] R 7a C 1-6 alkyl;

[0167] R 8a 、R 9a 、R 10a and R 11a Each independently selected from the group consisting of H, Br or I;

[0168] It is a monovalent acid ion;

[0169] The water-soluble group is a -L1-W1-L2-water-soluble part;

[0170] L1 and L2 are each independently C1-C4 alkylene;

[0171] W1 is

[0172] The water-soluble portion is a is 2 or 3.

[0173] In another preferred embodiment, the compound is as shown in Formula IA;

[0174] in

[0175] n=0;

[0176] R 1a It is a water-soluble group;

[0177] R 2a is H;

[0178] R 3a is H;

[0179] R 4a and R 5a is H;

[0180] R 6a Select from the following group: C 1-6 Alkyl, water-soluble group;

[0181] R 7a C 1-6 alkyl;

[0182] R 8a 、R 9a 、R 10a and R 11a Each independently selected from the group consisting of H, Br or I;

[0183] It is a monovalent acid ion;

[0184] The water-soluble group is a -L1-W1-L2-water-soluble part;

[0185] L1 and L2 are each independently C1-C4 alkylene;

[0186] W1 is

[0187] The water-soluble portion is a is 2 or 3.

[0188] In the first aspect of the present invention, a compound is provided, wherein the compound is as shown in Formula I;

[0189] In Formula I,

[0190] R1 is a group that can be specifically hydrolyzed by a biomacromolecule enzyme or a group that can be specifically activated by a disease microenvironmental factor; and after being specifically hydrolyzed or specifically activated, the bond between R1 and X1 is broken and R1 leaves;

[0191] X1 is O or NH;

[0192] L is a linking group that can leave on its own when R1 leaves;

[0193] R2 is a water-soluble group;

[0194] FP is a fluorescent group or a photosensitive group.

[0195] In another preferred embodiment, the compound can be used as a probe or photosensitizer; preferably, a far-infrared probe or photosensitizer.

[0196] In another preferred embodiment, there is at least one aromatic ring in FP, and They are respectively connected to adjacent ring atoms on the aromatic ring.

[0197] In another preferred embodiment, the aromatic ring is a benzene ring or a 5- or 6-membered heteroaromatic ring.

[0198] In another preferred embodiment, the aromatic ring is an aromatic ring fused with other rings.

[0199] In another preferred embodiment, the aromatic ring is a benzene ring.

[0200] In another preferred embodiment, the aromatic ring is a benzene ring in the ring as shown below:

[0201] Wherein, X1 is O or S.

[0202] In another preferred embodiment, the ability to be specifically hydrolyzed by a biomacromolecular enzyme means that when R1 is a group that can be specifically hydrolyzed by a biomacromolecular enzyme, the biomacromolecular enzyme can recognize the group and, under the action of the biomacromolecular enzyme, hydrolyze X1-R1 to break the bond between X1 and R1.

[0203] In another preferred embodiment, the biomacromolecule enzymes include: alkaline phosphatase, γ-glutamyl transpeptidase, aminopeptidase, Fapα enzyme, and Hepsin enzyme.

[0204] In another preferred embodiment, the enzyme recognition group that can be specifically hydrolyzed by alkaline phosphatase includes:

[0205] In another preferred embodiment, the enzyme recognition group that can be specifically hydrolyzed by γ-glutamyl transpeptidase includes:

[0206] In another preferred embodiment, the enzyme recognition group that can be specifically hydrolyzed by aminopeptidase includes:

[0207] In another preferred embodiment, the enzyme recognition group that can be specifically hydrolyzed by Fapα enzyme includes:

[0208] In another preferred embodiment, the enzyme recognition group capable of being recognized by Hepsin enzyme includes:

[0209] In another preferred embodiment, the enzyme recognition group capable of being specifically hydrolyzed by a biomacromolecule enzyme is selected from the following group:

[0210] In another preferred embodiment, the ability to be specifically activated by disease microenvironmental factors means that when R1 is a group that can be specifically activated by disease microenvironmental factors, the bond between X1 and R1 can be broken under the action of disease microenvironmental factors.

[0211] In another preferred embodiment, the disease microenvironmental factors include reactive oxygen species (ROS).

[0212] In another preferred embodiment, the reactive oxygen species (ROS) include: peroxides (such as H2O2, ONOO - ), superoxide anion

[0213] In another preferred embodiment, the base that can be specifically activated by peroxide includes:

[0214] In another preferred embodiment, the group that can be specifically activated by superoxide anions includes:

[0215] In another preferred embodiment, the group that can be specifically activated by disease microenvironmental factors is selected from the following group:

[0216] In another preferred embodiment, R1 is selected from the following group:

[0217] In another preferred embodiment, L is selected from the following group:

[0218] In another preferred embodiment, L is selected from the following group: Where * represents the connection position with X1.

[0219] In another preferred embodiment, X1 is O, and L is selected from the following group:

[0220] In another preferred embodiment, X1 is NH, and L is

[0221] In another preferred embodiment, R2 is a water-soluble group connected to the carbonamide. When the compound is activated, the carbonamide is disconnected and the water-soluble group leaves.

[0222] In another preferred embodiment, the water-soluble group includes: an anion-cation pair (such as ), a group containing at least three carboxyl functional groups (such as ), containing at least two sulfonic acid functional groups (SO3 - ) groups (such as ).

[0223] In another preferred embodiment, R2 is selected from the following group:

[0224] In another preferred embodiment, the FP portion has a structure as shown in Formula II-1 or Formula II-2:

[0225] in,

[0226] a represents The location of the connection;

[0227] b stands for The location of the connection;

[0228] R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu Select from the following groups: -COOH, -N3, -NH2, -N + (C 1-6 alkyl)3);

[0229] R4, R5, R6 and R7 are each independently H or a heavy atom I, Br;

[0230] R8 is H, F, Cl, or Br;

[0231] X2 is O or S;

[0232] X3 is I, Br, or Cl.

[0233] In another preferred embodiment, the FP portion has a structure as shown in Formula III-1 or III-2:

[0234] in,

[0235] a represents The location of the connection;

[0236] b stands for The location of the connection;

[0237] R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu Select from the following groups: -COOH, -N3, -NH2, -N + (C 1-6 alkyl)3);

[0238] R4, R5, R6 and R7 are each independently H or a heavy atom (such as I, Br);

[0239] R8 is H, F, Cl, or Br;

[0240] X2 is O or S;

[0241] X3 is I, Br, or Cl;

[0242] In another preferred embodiment, the FP portion has a structure as shown in Formula IV-1 or IV-2:

[0243] in,

[0244] a represents The location of the connection;

[0245] b stands for The location of the connection;

[0246] X2 is O or S;

[0247] X4 is O or NR3;

[0248] R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu Select from the following groups: -COOH, -N3, -NH2, -N+ (C 1-6 alkyl)3);

[0249] R4, R5, R6 and R7 are each independently H or a heavy atom (such as I, Br);

[0250] R8 is H, F, Cl, or Br.

[0251] In another preferred embodiment, the FP portion has a structure as shown in Formula V-1 or V-2:

[0252] in,

[0253] a represents The location of the connection;

[0254] b stands for The location of the connection;

[0255] X2 is O or S;

[0256] X5 is O or S;

[0257] R9 is NEt2, NPh2, or NCH3Ph;

[0258] R8 is H, F, Cl, or Br.

[0259] In another preferred embodiment, the FP portion has a structure as shown in Formula VI-1 or VI-2:

[0260] in,

[0261] a represents The location of the connection;

[0262] b stands for The location of the connection;

[0263] X2 is O or S;

[0264] X5 is O or S;

[0265] R 10 and R 11 Each independently is C 1-6 Alkyl (such as Et) or Ph;

[0266] R8 is H, F, Cl or Br.

[0267] In another preferred embodiment, the water-soluble group is a water-soluble group that can be connected to a carbonamide structure. When the compound is activated, the carbonamide is disconnected and the water-soluble group leaves.

[0268] In another preferred embodiment, the exemplary compounds are selected from Table 1

[0269] Table 1

[0270] In a second aspect of the present invention, a probe is provided, wherein the probe is a compound as shown in Formula I; wherein X1, X2, R1 and L are as defined in the first aspect; and the FP portion is defined as follows.

[0271] In another preferred embodiment, the FP portion has a structure as shown in formula II-1 or II-2; wherein R4, R5, R6 and R7 are all H, and a, b, R3, R8, X2 and X3 are as defined in the first aspect.

[0272] In another preferred embodiment, the FP portion has a structure as shown in formula III-1 or III-2; wherein R4, R5, R6 and R7 are all H, and a, b, R3, R8, X2 and X3 are as defined in the first aspect.

[0273] In another preferred embodiment, the FP portion has a structure as shown in Formula IV-1 or IV-2; wherein R4, R5, R6 and R7 are all H, and a, b, R8, X2 and X4 are as defined in the first aspect.

[0274] In another preferred embodiment, the FP portion has a structure as shown in Formula V-1 or V-2; wherein a, b, R8, R9, X2 and X5 are as defined in the first aspect.

[0275] In another preferred embodiment, the FP portion has a structure shown in Formula VI-1 or VI-2; wherein a, b, R8, R 10 、R 11 , X2 and X5 are as defined in the first aspect. In a third aspect of the present invention, a photosensitizer is provided, wherein the photosensitizer is a compound as shown in Formula I; wherein X1, X2, R1 and L are as defined in the first aspect; and the FP portion is defined as follows.

[0276] In another preferred embodiment, the FP portion has a structure as shown in formula II-1 or II-2; wherein, R4, R5, R6 and R7 are each independently H or a heavy atom, and at least one of R4, R5, R6 and R7 is a heavy atom; a, b, R3, R8, X2 and X3 are as defined in the first aspect.

[0277] In another preferred embodiment, the FP portion has a structure as shown in formula III-1 or III-2; wherein, R4, R5, R6 and R7 are each independently H or a heavy atom, and at least one of R4, R5, R6 and R7 is a heavy atom; a, b, R3, R8, X2 and X3 are as defined in the first aspect.

[0278] In another preferred embodiment, the FP portion has a structure as shown in formula IV-1 or IV-2; wherein, R4, R5, R6 and R7 are each independently H or a heavy atom, and at least one of R4, R5, R6 and R7 is a heavy atom; a, b, R8, X2 and X4 are as defined in the first aspect.

[0279] In another preferred embodiment, one, two or three of R4, R5, R6 and R7 are heavy atoms.

[0280] In another preferred embodiment, the heavy atoms are each independently I or Br.

[0281] In another preferred embodiment, the photosensitizer is a near-infrared photosensitizer.

[0282] In a fourth aspect of the present invention, a composition is provided, wherein the composition comprises:

[0283] (1) the compound according to the first aspect; and

[0284] (2) A pharmaceutically or biologically acceptable carrier.

[0285] In another preferred embodiment, the composition is a probe composition.

[0286] In another preferred embodiment, the composition is a photosensitizer composition.

[0287] In a fifth aspect of the present invention, a probe composition is provided, wherein the probe composition comprises:

[0288] (1) the compound according to the second aspect; and

[0289] (2) A pharmaceutically or biologically acceptable carrier.

[0290] In a sixth aspect of the present invention, a photosensitizer composition is provided, wherein the photosensitizer composition comprises:

[0291] (1) the compound according to the third aspect; and

[0292] (2) A pharmaceutically or biologically acceptable carrier.

[0293] In the seventh aspect of the present invention, there is provided a use of the compound according to the first aspect or a pharmaceutically or diagnostically acceptable salt thereof, or the probe according to the second aspect, or the composition according to the fourth aspect, or the probe composition according to the fifth aspect, wherein the use is one or more selected from the following groups:

[0294] (a) for enzyme detection (preferably, the enzyme detection is in vitro non-therapeutic and non-diagnostic);

[0295] (b) for the detection of disease microenvironmental factors (such as ROS) (preferably, the disease microenvironmental factors (such as ROS) are non-therapeutic and non-diagnostic in vitro);

[0296] (c) for preparing a detection reagent for detecting an enzyme or a disease microenvironment factor (such as ROS);

[0297] (d) for imaging cells (preferably, the imaging of cells is in vitro non-therapeutic and non-diagnostic);

[0298] (e) for preparing a detection reagent for imaging analysis of cells;

[0299] (f) for imaging tumor tissue or cells (preferably, the imaging of the cells is in vitro non-therapeutic and non-diagnostic);

[0300] (g) for preparing a detection reagent for tumor tissue or cells;

[0301] In another preferred embodiment, the enzymes include: alkaline phosphatase, γ-glutamyl transpeptidase, aminopeptidase, Fapα enzyme, and Hepsin enzyme.

[0302] In another preferred embodiment, the detection of the enzyme includes detection of the expression level of the enzyme and / or detection of the activity of the enzyme.

[0303] In another preferred embodiment, 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.

[0304] In an eighth aspect of the present invention, there is provided a use of the compound according to the first aspect or a pharmaceutically or diagnostically acceptable salt thereof, or the photosensitizer probe according to the third aspect, or the composition according to the fourth aspect, or the photosensitizer composition according to the sixth aspect, wherein the use is one or more selected from the following group:

[0305] (h) Use in photodynamic therapy for tumors;

[0306] (i) Used for preparing photosensitizing therapeutic agents for photodynamic therapy of tumors.

[0307] In a ninth aspect of the present invention, a detection kit is provided.

[0308] The kit comprises: the compound described in the first aspect or a pharmaceutically or diagnostically acceptable salt thereof or the composition described in the fourth aspect.

[0309] In the tenth aspect of the present invention, a method for imaging cells or tissues is provided, which comprises the steps of: incubating 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).

[0310] In another preferred embodiment, the method is non-therapeutic in vitro.

[0311] In an eleventh aspect of the present invention, a method for in vivo imaging is provided, the method comprising the steps of:

[0312] (i) administering the compound of the first aspect or a pharmaceutically or biochemically acceptable salt thereof, or the composition of the fourth aspect to a subject in need thereof; and

[0313] (ii) Using the instrument for imaging.

[0314] In another preferred embodiment, the detection subject is a human or a non-human mammal (preferably, including a non-human primate or a rodent such as a rat or a mouse).

[0315] In another preferred embodiment, the in vivo imaging is real-time imaging or static imaging; preferably, it is real-time imaging.

[0316] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one.

[0317] The inventors have conducted long-term and in-depth research. In order to improve the diagnostic and therapeutic effects of probes and photosensitizers in diseases, the inventors have developed for the first time a class of near-infrared probes and photosensitizers with the function of opening and enriching at the detection target site based on the design strategy of methylene benzoquinone, that is, under the one-time triggering of the target enzyme or the factors in the target microenvironment, the probe in the unopened state, such as the near-infrared probe or photosensitizer, can be converted to high lipophilicity and cell permeability, thereby reducing its clearance rate and enhancing the retention efficiency in the target; 2) by forming a methylene benzoquinone intermediate to react with macromolecules such as proteins or nucleophiles in cells to form a covalent bond to achieve the anchoring of the fluorescent group or photosensitizer; 3) turn on the near-infrared fluorescence or photoacoustic signal or turn on the photosensitivity under near-infrared light. This method greatly enhances the enrichment of signal molecules at the detection site, improves the detection sensitivity of the near-infrared fluorescent probe and the photodynamic therapy efficiency of the photosensitizer. Based on this, the inventors completed the present invention.

[0318] Near-infrared probes and photosensitizers and their applications

[0319] The present invention provides a variety of near-infrared probes and photosensitizers (also referred to herein as compounds of the present invention) with the function of opening and enriching the target, as well as their preparation methods and their applications in in vivo imaging and treatment. The near-infrared probe or photosensitizer provided by the present invention itself has good water solubility, and its fluorescent signal or photosensitivity is in a closed state at a non-target position, but under a single trigger of the target enzyme or disease microenvironment (ROS, etc.), it will achieve: 1) from high hydrophilicity and low cell permeability to high lipophilicity and cell permeability, thereby reducing its clearance rate and enhancing the retention efficiency in the target; 2) by forming a methylene quinone intermediate and reacting with a macromolecule such as a protein or a nucleophilic reagent in the cell to form a covalent bond to achieve the anchoring of the fluorescent group or photosensitizer; 3) turning on the near-infrared fluorescence or photoacoustic signal or turning on the photosensitivity under near-infrared light. This type of near-infrared probe or photosensitizer is composed of a recognition group of an enzyme or disease microenvironment (ROS, etc.), a near-infrared fluorescent or photoacoustic group or a photosensitizer, a leaving group, and a hydrophilic group connected to the leaving group. Because the near-infrared fluorescent probe or photosensitizer provided by the present invention has good hydrophilicity, it can be freely transported to tumor tissue in vivo, and under the selective action of the target enzyme or disease microenvironment (ROS, etc.), the recognition group can be caused to leave, thereby promoting the leaving group connected to the highly hydrophilic functional group to leave and form a methylene benzoquinone intermediate (QM) with high lipophilicity and high reactivity. This not only reduces the clearance rate of the near-infrared fluorescent probe or photosensitizer of the present invention in tumor tissue, but also effectively improves cell permeability and facilitates cellular uptake. In addition, the nucleophilic residues on the protein in the tumor cell can react with the methylene benzoquinone intermediate to form a covalent bond, achieving its effective enrichment in the tumor and simultaneously opening the near-infrared fluorescent photoacoustic or photosensitizing activity.

[0320] In one embodiment, the present invention provides a class of target activation enrichment type near-infrared probes and photosensitizers (i.e., compounds of the present invention, or compounds having a hydrophilic group that can leave), the structure of which is shown in the general formula (I):

[0321] Formula I wherein,

[0322] R1 is a group that can be specifically hydrolyzed by a biomacromolecule enzyme or a group that can be specifically activated by a disease microenvironmental factor; and after being specifically hydrolyzed or specifically activated, the bond between R1 and X2 is broken and R1 leaves;

[0323] X1 is O or NH;

[0324] L is a linking group that can leave on its own when R1 leaves;

[0325] R2 is a water-soluble group connected to the carbonamide. When the compound is activated, the carbonamide is disconnected and the water-soluble group leaves.

[0326] The FP portion is a fluorescent group or a photosensitive group.

[0327] In another preferred embodiment, after the compound is specifically hydrolyzed or specifically activated, the bond between X1-R is broken, and L leaves by itself under the influence of the electron transfer caused by the breaking of the bond between X1-R1 and the departure of R1. It breaks down under the influence of the electron transfer from L, causing R2 to leave.

[0328] In further embodiments, R1 is selected from the group consisting of:

[0329] L is selected from the following group: Wherein, * represents the connection position with X2; and / or

[0330] R2 is selected from the group consisting of:

[0331] In other embodiments, the FP moiety has the structure shown in Formula II:

[0332] in,

[0333] a represents The location of the connection;

[0334] b stands for The location of the connection;

[0335] R3 is -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 groups: -COOH, -N3, -NH2, -N + (C 1-6 R4, R5, R6 and R7 are each independently H or a heavy atom I, Br. R8 is H, F, Cl, or Br.

[0336] X2 is O or S.

[0337] X3 is I, Br, or Cl.

[0338] In further embodiments, the FP moiety has the structure shown below:

[0339] in,

[0340] a represents The location of the connection;

[0341] b stands for The location of the connection;

[0342] R3 is -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 groups: -COOH, -N3, -NH2, -N + (C 1-6 R4, R5, R6 and R7 are each independently H or a heavy atom I, Br. R8 is H, F, Cl, or Br.

[0343] X2 is O or S.

[0344] X3 is I, Br, or Cl.

[0345] In further embodiments, the FP moiety has the structure shown below:

[0346] in,

[0347] a represents The location of the connection;

[0348] b stands for The location of the connection;

[0349] X2 is O or S.

[0350] X4 is O or NR3;

[0351] R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu is a modifiable functional group or a positively charged group; preferably, R Fu Select from the following groups: -COOH, -N3, -NH2, -N + (C 1-6 Alkyl)3.

[0352] R4, R5, R6 and R7 are each independently H or a heavy atom such as I, Br.

[0353] R8 is H, F, Cl, or Br.

[0354] In further embodiments, the FP moiety has the structure shown below:

[0355] in,

[0356] a represents The location of the connection;

[0357] b stands for The location of the connection;

[0358] X2 is O or S.

[0359] X5 is O or S.

[0360] R9 is NEt2, NPh2, NCH3Ph.

[0361] R8 is H, F, Cl, or Br.

[0362] In further embodiments, the FP moiety has the structure shown below:

[0363] in,

[0364] a represents The location of the connection;

[0365] b stands for The location of the connection;

[0366] X2 is O or S.

[0367] X5 is O or S.

[0368] R 10 、R 11 For Et, Ph.

[0369] R8 is H, F, Cl, or Br.

[0370] In further embodiments, the FP moiety, X1, X2, R1 and L are as defined in the first aspect.

[0371] In other embodiments, the FP moiety, X1, X2, R1 and L are as defined in the first aspect and are each independently a corresponding group of the specific compounds shown in Table A.

[0372] In other embodiments, the FP moiety, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 、R 11 , X1, X2, X3, X4, X5, and L are each independently the corresponding group of the specific compound shown in Table A.

[0373] In further embodiments, the near-infrared probe or photosensitizer is selected from Table A.

[0374] In another embodiment, a near-infrared fluorescent probe as shown below is provided, namely, compound ALPIN-6;

[0375] In another embodiment, there is provided a near-infrared basic photosensitizer as shown below, namely compound ALPIN-6I;

[0376] In another embodiment, there is provided a near-infrared fluorescent and photoacoustic probe, compound ALPIN-7, as shown below;

[0377] In another embodiment, the near-infrared probe and photosensitizer provided by the present invention with the function of opening and enriching the target point are targeted to the target. After the enzyme or disease microenvironment active factor hydrolyzes the recognition group (even if the bond in X2-R1 is broken, the connecting group L leaves on its own), thereby leaving the leaving group (carbonamide structure) and the water-soluble group (R2) of the near-infrared probe or photosensitizer in the unopened state, thereby triggering the following response: 1) from high hydrophilicity and low cell permeability to high lipophilicity and cell permeability, thereby reducing its clearance rate and enhancing the retention efficiency in the target; 2) by forming a methylene benzoquinone intermediate and reacting with a macromolecule such as a protein or a nucleophilic reagent in the cell to form a covalent bond to achieve the anchoring of the fluorescent group or photosensitizer; 3) turning on the near-infrared fluorescence or photoacoustic signal or turning on the photosensitivity under near-infrared light. Its working principle is shown in Figure 1, in which an alkaline phosphatase detection probe is taken as an example.

[0378] In another embodiment, the present invention provides near-infrared probes (such as compounds ALPIN-6 and ALPIN-7) for real-time imaging of alkaline phosphatase (ALP) activity in vitro and in vivo.

[0379] In another embodiment, the present invention also provides use of a near-infrared photosensitizer (such as ALPIN-6I) as an ALP-activated photosensitizer in photodynamic therapy for tumors.

[0380] Experiments have shown that the (especially preferred) near-infrared fluorescent probes or photosensitizers provided by the present invention, which have the function of activating and enriching the target site, can effectively realize the visualization imaging of related enzymes in vivo and in vitro, greatly enhancing the enrichment efficiency of the probe, and improving the imaging sensitivity and specificity. In addition, the photosensitizer can not only achieve the selective killing of tumor cells overexpressing the target enzyme under co-incubation conditions, but also leave other cells unaffected. More importantly, the intravenous injection of this photosensitizer into tumor-bearing mice can completely eliminate tumors overexpressing the target enzyme in all tested mice, and no tumor recurrence was observed during the recovery process. At the same time, no detectable toxicity was observed in other tissues.

[0381] In another aspect of the present invention, a compound or a salt thereof is provided, wherein the compound is as shown in Formula IA;

[0382] in

[0383] n = 0, 1, or 2;

[0384] R 1a Selected from the group consisting of: substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted 5- or 6-membered heteroaryl, substituted or unsubstituted C 1-4 Alkylene-phenyl, substituted or unsubstituted C 1-4 Alkylene-5 or 6-membered heteroaryl, group containing a water-soluble portion;

[0385] R 2a Selected from the group consisting of: H, -OC1-4 alkyl;

[0386] R 3a Selected from the group consisting of H, substituted or unsubstituted C 1-6 Alkyl, monovalent cations (such as Na + );

[0387] R 4a and R 5a Each independently selected from the following group: H, C 1-4 Alkyl (such as methyl, ethyl); or R 4a and R 5a Together with the carbon atom to which it is connected, it forms C 3-4 Cycloalkyl;

[0388] R 6a Selected from the group consisting of: substituted or unsubstituted C 1-6 Alkyl groups, groups containing water-soluble moieties;

[0389] R 7a is substituted or unsubstituted C 1-6 alkyl;

[0390] R 8a 、R 9a 、R 10a and R 11a are each independently selected from the group consisting of: H, heavy atoms;

[0391] It is a monovalent acid ion;

[0392] The substitution refers to that one or more hydrogen atoms on the group are replaced by a substituent selected from the group consisting of halogen, unsubstituted or halogenated C1-C6 alkyl.

[0393] In another preferred embodiment, the salt refers to a chemically or pharmaceutically acceptable salt.

[0394] In another preferred embodiment, the group containing a water-soluble portion is -L1-W1-L2-water-soluble portion; wherein L1 and L2 are each independently none or a divalent linking group, and W1 is none or a linking group formed by a click chemistry reaction.

[0395] In another preferred embodiment, L1 and L2 are each independently a divalent linking group that is absent or stable and non-cleavable in a cell or in vivo environment.

[0396] In another preferred embodiment, L1 and L2 are each independently zero or a C1-C4 alkylene group.

[0397] In another preferred embodiment, the linking group formed by the click chemistry reaction is

[0398] In another preferred embodiment, W1 is none or

[0399] In another preferred embodiment, the water-soluble portion is selected from the following group: a group containing an anion-cation pair, a group containing one or more carboxyl groups, and a group containing one or more sulfonic acid groups.

[0400] In another preferred embodiment, the group containing one or more carboxyl groups is a group containing 2 or 3 carboxyl groups.

[0401] In another preferred embodiment, the group containing one or more sulfonic acid groups is a group containing 2 or 3 sulfonic acid groups.

[0402] In another preferred embodiment, the portion containing an anion-cation pair comprises: Wherein, a is 1, 2, 3 or 4.

[0403] In another preferred embodiment, the group containing one or more carboxyl groups includes:

[0404] In another preferred embodiment, the group containing one or more sulfonic acid groups includes:

[0405] In another preferred embodiment, a is 2 or 3.

[0406] In another preferred embodiment, the group containing a water-soluble portion is selected from the following group:

[0407] In another preferred embodiment, R 1a and R 6a At least one of the groups is a group containing a water-soluble portion.

[0408] In another preferred embodiment, R 1a It is a group containing a water-soluble portion.

[0409] In another preferred embodiment, R 6a It is a group containing a water-soluble portion.

[0410] In another preferred embodiment, R 1a and R 6a All of them are groups containing a water-soluble portion.

[0411] In another preferred embodiment, the heavy atoms are each independently I or Br.

[0412] In another preferred embodiment, n=0.

[0413] In another preferred embodiment, R 2a Selected from the group consisting of H, -OMe.

[0414] In another preferred embodiment, R 2a For H.

[0415] In another preferred embodiment, R 3a Selected from the following group: H, C 1-6 Alkyl, monovalent cations (such as Na + ).

[0416] In another preferred embodiment, R 3a For H.

[0417] In another preferred embodiment, R 4a and R 5a Each independently selected from the following group: H, C 1-4 alkyl.

[0418] In another preferred embodiment, R 4a and R 5a Each is independently selected from the group consisting of H, methyl, and ethyl.

[0419] In another preferred embodiment, R 7a C 1-6 alkyl.

[0420] In another preferred embodiment, R 7a For ethyl.

[0421] In another preferred embodiment, Selected from the following group: I - Br - , and Cl - .

[0422] In another preferred embodiment, For I - .

[0423] In another preferred embodiment, the compound or a salt thereof is used as a probe (near-infrared probe or near-infrared fluorescent probe).

[0424] In another preferred embodiment, when the compound or its salt is used as a probe, R 8a 、R 9a 、R 10a and R 11a Both are H.

[0425] In another preferred embodiment, the compound or its salt is used as a photosensitizer.

[0426] In another preferred embodiment, when the compound or its salt is used as a photosensitizer, R8, R9, R 10 and R 11 At least one of them is a heavy atom, and the rest are H.

[0427] In another preferred embodiment, R 8a 、R 9a 、R 10a and R 11a 1, 2 or 3 of them are heavy atoms.

[0428] In another preferred embodiment, the heavy atoms are each independently I or Br.

[0429] In another preferred embodiment, n, R 1a 、R 2a 、R 3a 、R 4a 、R 5a 、R 6a 、R 7a 、R 8a 、R 9a 、R 10a and R 11a Each independently represents a corresponding group in the specific compounds described in the examples of this application.

[0430] In another preferred embodiment, the compound is selected from the following group:

[0431] In another preferred embodiment, when the compound or its salt is used as a probe, the compound is BIN-3.

[0432] In another preferred embodiment, when the compound or its salt is used as a photosensitizer, the compound is BIN-3I.

[0433] In the second aspect of the present invention, a probe is provided, wherein the probe is a compound or a salt thereof as shown in Formula I; wherein n, R 1a 、R 2a 、R 3a 、R 4a 、R5a 、R 6a 、R 7a and as defined in the first aspect; and R 8a 、R 9a 、R 10a and R 11a Both are H.

[0434] In another preferred embodiment, the salt is a pharmaceutically or biologically acceptable salt.

[0435] In another preferred embodiment, the probe is BIN-3.

[0436] In the third aspect of the present invention, a photosensitizer is provided, wherein the photosensitizer is a compound or a salt thereof as shown in Formula I; wherein n, R 1a 、R 2a 、R 3a 、R 4a 、R 5a 、R 6a 、R 7a and as defined in the first aspect; and R 8a 、R 9a 、R 10a and R 11a At least one of them is a heavy atom, and the others are independently selected from H or heavy atoms.

[0437] In another preferred embodiment, the salt is a pharmaceutically or biologically acceptable salt.

[0438] In another preferred embodiment, R 8a 、R 9a 、R 10a and R 11a 1, 2 or 3 of them are heavy atoms.

[0439] In another preferred embodiment, the heavy atoms are each independently I or Br.

[0440] In another preferred embodiment, the photosensitizer is a near-infrared photosensitizer.

[0441] In another preferred embodiment, the photosensitizer is BIN-3I.

[0442] In a fourth aspect of the present invention, a composition is provided, wherein the composition comprises:

[0443] (1) the compound according to the first aspect or a salt thereof; and

[0444] (2) A pharmaceutically or biologically acceptable carrier.

[0445] In another preferred embodiment, the salt is a pharmaceutically or biologically acceptable salt.

[0446] In another preferred embodiment, the composition is a probe composition.

[0447] In another preferred embodiment, the composition is a photosensitizer composition.

[0448] In a fifth aspect of the present invention, a probe composition is provided, wherein the probe composition comprises:

[0449] (1) The probe according to the second aspect; and

[0450] (2) A pharmaceutically or biologically acceptable carrier.

[0451] In a sixth aspect of the present invention, a photosensitizer composition is provided, wherein the photosensitizer composition comprises:

[0452] (1) the photosensitizer according to the third aspect; and

[0453] (2) A pharmaceutically or biologically acceptable carrier.

[0454] In the seventh aspect of the present invention, there is provided a use of the compound or salt thereof according to the first aspect, the probe according to the second aspect, the composition according to the fourth aspect, or the probe composition according to the fifth aspect, wherein the use is one or more selected from the following group:

[0455] (a) for the detection of β-lactamase (preferably, the detection of β-lactamase is in vitro non-therapeutic and non-diagnostic);

[0456] (b) for the detection or imaging of drug-resistant bacteria (preferably, the detection or imaging of the bacteria is in vitro non-therapeutic and non-diagnostic);

[0457] (c) Used for preparing detection reagents for detecting or imaging drug-resistant bacteria.

[0458] In another preferred embodiment, the drug-resistant bacteria are drug-resistant bacteria expressing β-lactamase.

[0459] In another preferred embodiment, the detection or imaging of the bacteria includes: determining whether the detected bacteria express β-lactamase and the amount of β-lactamase expressed.

[0460] In another preferred embodiment, the detection reagent can be used to detect drug-resistant bacteria in living subjects.

[0461] In an eighth aspect of the present invention, there is provided a use of the compound or salt thereof according to the first aspect, the photosensitizer according to the third aspect, the composition according to the fourth aspect, or the photosensitizer composition according to the sixth aspect, wherein the use is one or more selected from the following group:

[0462] (a) for killing drug-resistant bacteria (preferably, the killing of drug-resistant bacteria is non-therapeutic in vitro);

[0463] (b) for preparing a medicament for killing drug-resistant bacteria or for treating or preventing a disease caused by infection with drug-resistant bacteria.

[0464] In a ninth aspect of the present invention, a detection kit is provided.

[0465] The kit comprises: the compound or salt thereof as described in the first aspect or the composition as described in the fourth aspect.

[0466] In the tenth aspect of the present invention, a method for imaging drug-resistant bacteria is provided, the method comprising the steps of incubating the compound or salt thereof described in the first aspect with an object, and obtaining an image of the drug-resistant bacteria using an instrument (such as a fluorescence microscope).

[0467] In another preferred embodiment, the method is non-therapeutic in vitro.

[0468] In another preferred embodiment, the objects include: bacteria, cells, tissues, culture medium (such as a culture dish in which bacteria are cultured).

[0469] β-lactamase-activatable probes or photosensitizers

[0470] β-lactamases are highly efficient at hydrolyzing antibiotics during the catalytic process. While bacterial expression of β-lactamases can contribute to antibiotic resistance, it also provides a unique opportunity for detecting and even selectively killing resistant bacteria in vivo. Since the development of the first fluorescent β-lactamase probe by Qian Yongjian's group in 1998, a large number of fluorescent β-lactamase substrates have been reported. In in vitro solution-based tests, most of these probes have shown high sensitivity and selectivity in detecting β-lactamase activity. However, the use of fluorescent probes to monitor bacterial infections in living animals remains largely elusive. This challenge is primarily due to the fact that β-lactamases are primarily located in the bacterial periplasm or are excreted, and the internal environment of living animals is complex and variable.

[0471] For in vivo imaging with fluorescent probes, not only is it necessary to enhance the fluorescence intensity as in in vitro experiments, but it is also crucial to retain or even enrich the activated fluorescent groups at the target site. In addition, the excitation / emission of near-infrared (NIR) fluorescence is also crucial for in vivo imaging. The near-infrared band can minimize the interference of autofluorescence in the body and achieve deeper tissue penetration. We use the same strategy in photosensitizers that can achieve photodynamic therapy, introducing iodine atoms into the near-infrared dye to enhance the heavy atom effect. 1The yield of O2 can produce more active oxygen after near-infrared light irradiation, thereby achieving the purpose of retaining imaging and inhibiting or even killing drug-resistant bacteria, realizing the application of integrated diagnosis and treatment.

[0472] In this study, we combined the design of traditional β-lactamase-activatable fluorescent probes with a quinone methylene (QM)-based self-anchoring strategy and a design that alters hydrophilicity and lipophilicity to develop novel β-lactamase near-infrared fluorescent probes and photosensitizers for fluorescent imaging and treatment of bacterial infections in mice. This novel fluorescent probe and photosensitizer not only enhances fluorescence emission upon selective activation by β-lactamase, like traditional fluorescent probes and photosensitizers, but also effectively accumulates on β-lactamase-expressing bacteria by altering their hydrophilicity and forming covalent bonds with protein macromolecules on or near the bacteria. These characteristics give them unique advantages in in vivo imaging and photodynamic therapy, enabling the selective monitoring and killing of drug-resistant bacteria.

[0473] Labeled compounds and photosensitizers of the present invention

[0474] One objective of the present invention is to provide a strategy for the enrichment, detection, and treatment of drug-resistant bacteria by β-lactamase activation. Specifically, the present invention utilizes the β-lactam structure as the recognition group of the fluorescent probe and the photosensitizer, and combines it with methylene benzoquinone with variable hydrophilicity and variable charge, and adopts a multiple response design strategy to design a near-infrared fluorescent probe and photosensitizer for β-lactamase to detect and kill drug-resistant bacteria.

[0475] Another object of the present invention is to provide a synthesis method

[0476] Another object of the present invention is to provide a specific application of the probe and photosensitizer to selectively label drug-resistant bacteria after enzyme activation and to enrich and detect and kill drug-resistant bacteria in vivo.

[0477] In one aspect of the present invention, there is provided a compound represented by formula IA, wherein formula IA represents a β-lactamase near-infrared probe and a β-lactamase near-infrared photosensitizer.

[0478] Wherein, each group is as defined in the first aspect.

[0479] In some embodiments, n=0, 1, 2; and / or,

[0480] R 1a Selected from the group consisting of: substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted 5- or 6-membered heteroaryl, substituted or unsubstituted C 1-4 Alkylene-phenyl, substituted or unsubstituted C 1-4Alkylene-five-membered or six-membered heteroaryl and water-soluble groups such as anions and cations Two carboxylic acids Three carboxylic acids Disulfonic acid etc.; and / or,

[0481] R 2a Selected from the group consisting of H, OMe; and / or

[0482] R 3a Selected from the group consisting of H, substituted or unsubstituted C 1-6 Alkyl groups, monovalent cations such as Na + and / or,

[0483] R 4a , R 5a Selected from the group consisting of H, methyl or ethyl, or R 4a , R 5a At the same time and / or,

[0484] R 6a Selected from the group consisting of water-soluble groups such as anionic and cationic pairs connected to the carbonamide structure Two carboxylic acids Three carboxylic acids Disulfonic acid When the enzyme is activated, the carbonamide structure will break off the water-soluble group and leave; and / or,

[0485] R 7a Selected from substituted or unsubstituted C 1-6 Alkyl; and / or,

[0486] R 8a 、R 9a 、R 10a 、R 11a is H (near-infrared fluorescent probe) or I, Br (photosensitizer), and the introduction of I or Br as a heavy atom will enhance the efficiency of the photosensitizer in generating ROS; and / or,

[0487] X is I, Br, Cl, etc.

[0488] The second aspect of the present invention is to provide the following compounds and their synthesis methods

[0489] Probe structural formula (II)

[0490] Photosensitizer structural formula (III)

[0491] The third aspect of the present invention provides a test method for reagents (II) and (III) of the second aspect, and demonstrates their use for highly sensitive and selective detection and killing of drug-resistant bacteria expressing β-lactamase in vitro and in vivo.

[0492] The present invention designs a near-infrared enrichment probe and photosensitizer that can be activated by β-lactamase hydrolysis. The ultraviolet absorption, fluorescence spectrum, HPLC analysis, SDS-PAGE and near-infrared fluorescence scanning experiments before and after in vitro enzymatic hydrolysis show that the

[0493] The β-lactamase probe and photosensitizer are selectively activated by β-lactamase, releasing a fluorescent signal and covalently binding to an affinity reagent. Subsequently, fluorescence imaging and probe enrichment on β-lactamase-expressing resistant bacteria were performed, demonstrating that the probe and photosensitizer selectively label and accumulate on resistant bacteria. Finally, a series of in vivo imaging experiments demonstrated that the probe can be retained and even accumulated at sites of resistant bacterial infection in highly dynamic living organisms. The fluorescence intensity can be used to rapidly monitor the effectiveness of inhibitors or antibiotics in treating bacteria in real time.

[0494] the term

[0495] In this document, unless otherwise specified, the terms or abbreviations used have the general meanings familiar to those skilled in the art. For example, Ph represents phenyl.

[0496] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent A (e.g., the alkyl portion of alkoxy, etc.) refers to a straight-chain or branched hydrocarbon radical (i.e., C 1-6 = represents 1-6 carbons). Examples of alkyl groups include: methyl (Me), ethyl (Et), n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, etc. In the present invention, preferred alkyl groups are those having 1-4 carbons, i.e., C 1-4 alkyl.

[0497] The main advantages of the present invention include one or more of the following:

[0498] (a) The compounds of the present invention can be linked to different specific activating groups according to different needs, thereby activating the molecule at the target site. For example, when targeting tumors, a group specifically activated in the tumor environment can be introduced into the molecule; when targeting β-lactamase-resistant bacteria, a group specifically activated in the β-lactamase environment can be introduced into the molecule.

[0499] (b) The probes (such as ALPIN-6 and ALPIN-7) and photosensitizers (such as ALPIN-6I) of the present invention have a "one-touch three-response" capability, that is, after being triggered once by an enzyme or disease microenvironment active factor, they trigger the following three responses: 1) changing from high hydrophilicity and low cell permeability to high lipophilicity and cell permeability, thereby reducing its clearance rate and enhancing its retention efficiency in the target; 2) anchoring of the fluorescent group or photosensitizer by forming a methylene quinone intermediate to react with macromolecules such as proteins or nucleophilic reagents in cells to form a covalent bond; 3) turning on near-infrared fluorescence or photoacoustic signals or turning on photosensitivity under near-infrared light.

[0500] (c) The probes (such as ALPIN-6 and ALPIN-7) and photosensitizers (such as ALPIN-6I) of the present invention have the functions of opening and enriching the target site.

[0501] (d) After being selectively hydrolyzed by β-lactamase, the probe or photosensitizer of the present invention can not only enhance the fluorescence intensity like most traditional β-lactamase probes and photosensitizers, but also significantly enrich bacteria expressing β-lactamase.

[0502] (e) The probe and photosensitizer of the present invention exhibit excellent selectivity. Experiments have demonstrated that they can selectively fluorescently label β-lactamase-positive bacteria, preventing cross-labeling even in the presence of other susceptible bacteria. This allows for easy, wash-free, and real-time observation of drug-resistant bacteria.

[0503] (f) The probe or photosensitizer of the present invention can selectively accumulate at the infection site, thereby enabling the in vivo observation of β-lactamase-expressing bacterial infection with a low signal-to-noise ratio, sensitivity, and non-invasiveness through the probe of the present invention. The photosensitizer of the present invention can accurately kill drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0504] FIG1 shows the mechanism of action of the probe or photosensitizer of the present invention.

[0505] Figure 2 shows the solution enzyme response verification of the near-infrared fluorescent probe ALPIN-6.

[0506] Figure 3 shows the solution enzyme response verification of the near-infrared fluorescent probe ALPIN-6.

[0507] Figure 4 shows the solution enzyme response verification of the near-infrared fluorescent probe ALPIN-6.

[0508] Figure 5 shows the solution enzyme response verification of the near-infrared fluorescent probe ALPIN-6.

[0509] Figure 6 shows the solution enzyme response verification of the near-infrared fluorescent probe ALPIN-6.

[0510] Figure 7 shows the imaging comparison of alkaline phosphatase (ALP)-overexpressing HeLa cells using near-infrared fluorescent probes ALPIN-6, ALPIN-5, and ALPIN-4, as well as the imaging comparison of HeLa cells and ALP-weakly expressing HEK293 cells.

[0511] Figure 8 shows real-time imaging of HeLa cells using the near-infrared fluorescent probe ALPIN-6.

[0512] FIG9 shows the specific imaging of HeLa and HEK293 mixed cells by the near-infrared fluorescent probe ALPIN-6.

[0513] FIG10 shows the real-time imaging of HeLa cell-bearing tumor mice using the near-infrared fluorescent probe ALPIN-6.

[0514] FIG11 shows the imaging of HeLa cell-bearing tumor mice using the near-infrared fluorescent probe ALPIN-6.

[0515] FIG12 shows the real imaging of HeLa cell-bearing tumor mice using the near-infrared fluorescent probe ALPIN-6.

[0516] FIG13 shows the distribution of the near-infrared fluorescent probe ALPIN-6 in HeLa cell-bearing tumor mice.

[0517] FIG14 shows the calculation of the enrichment content of the near-infrared photosensitizer ALPIN-6I in HeLa cells.

[0518] FIG15 shows the effect of photosensitizer ALPIN-6I on ROS production in HeLa cells.

[0519] FIG16 shows the effect of photosensitizer ALPIN-6I in killing tumor cells.

[0520] FIG17 shows the effect of photosensitizer ALPIN-6I in specifically killing tumor cells when mixed with cells.

[0521] FIG18 shows the real-time imaging of HeLa cell-bearing tumor mice using the near-infrared photosensitizer ALPIN-6I.

[0522] FIG19 shows the photodynamic therapy of HeLa cell-bearing tumor mice by the near-infrared photosensitizer ALPIN-6I.

[0523] Figure 20 shows the photodynamic therapy of HeLa cell-bearing tumor mice by the near-infrared photosensitizer ALPIN-6I.

[0524] Figure 21 shows the photodynamic therapy of HeLa cell-bearing tumor mice using the near-infrared photosensitizer ALPIN-6I.

[0525] FIG22 shows a comparison of the imaging of alkaline phosphatase (ALP)-overexpressing HeLa cells and the imaging of ALP-weakly expressing HEK293 cells using near-infrared fluorescence and photoacoustic probes ALPIN-7 and ALPIN-8.

[0526] FIG23 shows the real-time imaging of HeLa cells using the near-infrared fluorescent photoacoustic probe ALPIN-7.

[0527] FIG24 shows the real-time imaging of HeLa cell-bearing tumor mice using the near-infrared fluorescent photoacoustic probe ALPIN-7.

[0528] FIG25 shows the fluorescence intensity of the near-infrared fluorescent photoacoustic probe ALPIN-7 in HeLa cell-bearing tumor mice.

[0529] FIG26 shows the fluorescence distribution of the near-infrared fluorescent photoacoustic probe ALPIN-7 in HeLa cell-bearing tumor mice.

[0530] FIG27 is a graph showing the UV absorption and fluorescence intensity of BIN-3 before and after incubation with β-lactamase TEM-1 according to an embodiment of the present invention.

[0531] FIG28 is a graph showing the UV absorption and fluorescence intensity spectra of BIN-2 before and after incubation with β-lactamase TEM-1 according to an embodiment of the present invention.

[0532] FIG29 is a graph showing the UV absorption and fluorescence intensity spectra of BIN-3 before and after incubation with β-lactamase TEM-1 according to an embodiment of the present invention.

[0533] FIG30 is a spectrum of ultraviolet absorption and fluorescence intensity after incubation of BIN-2 and β-lactamase TEM-1 according to an embodiment of the present invention.

[0534] FIG31 is a spectrum of ultraviolet absorption and fluorescence intensity after incubation of BIN-3I in Example of the present invention with β-lactamase TEM-1.

[0535] FIG32 is a graph showing the UV absorption and fluorescence intensity of BIN-3I before and after incubation with β-lactamase TEM-1 according to an embodiment of the present invention.

[0536] FIG33 is a graph showing the UV absorption and fluorescence intensity of BIN-3I before and after incubation with β-lactamase TEM-1 according to an embodiment of the present invention.

[0537] FIG34 is a graph showing the UV absorption and fluorescence intensity of BIN-2I before and after incubation with β-lactamase TEM-1 according to an embodiment of the present invention.

[0538] Figure 35 is a histogram of the fluorescence intensity of BIN-3 and BIN-2 (5 μM) in the examples of the present invention against different enzymes and proteins. 1: TEM-1 (10 nM); 2: TEM-1 (10 nM) + inhibitor (100 μM); 3: lysozyme (2 U / mL); 4: β-glucosidase (5 μM); 5: lipase (2 U / mL); 6: bovine serum albumin (10 μM); 7: glutathione (2 μM); 8: lysine (1 mM); 9: cysteine ​​(1 mM); 10: tryptophan (1 mM); λ ex / λ em =687 / 714nm.

[0539] Figure 36 is the SDS-PAGE and near-infrared fluorescence two-color imaging of the probes BIN-3, BIN-2, photosensitizers BIN-3I and BIN-2I (10 μM) after incubation with TEM-1 and nucleophilic reagent BSA according to the embodiment of the present invention.

[0540] Figure 37 is the SDS-PAGE and near-infrared fluorescence two-color imaging of the probes BIN-3, BIN-2, photosensitizers BIN-3I and BIN-2I (10 μM) after incubation with TEM-1 and nucleophilic reagent BSA according to the embodiment of the present invention.

[0541] FIG38 is a fluorescence imaging diagram of the main probe BIN-3 and the control probe BIN-2 and bla-positive bacteria and bla-negative bacteria;

[0542] FIG39 is fluorescence imaging of the main probe BIN-3 and the control probe BIN-2 with bla-negative bacteria before and after washing;

[0543] FIG40 shows the selective imaging of mixed bacteria by the main probe BIN-3 and the control probe BIN-2.

[0544] FIG41 is an enrichment experiment of BIN-3 and bla-positive bacteria according to an embodiment of the present invention.

[0545] FIG42 is an enrichment experiment of BIN-2 and bla-positive bacteria according to an embodiment of the present invention.

[0546] FIG43 is a diagram showing blood routine, blood biochemistry, and HE tissue staining sections of BIN-3 and BIN-2 in mice according to examples of the present invention.

[0547] FIG44 shows representative fluorescence images of the whole body of mice after intravenous injection of the probe or after simultaneous intramuscular injection of the inhibitor (100 mM, 50 μL) before and after the probe injection.

[0548] FIG45 shows the temporal trend of the fluorescence intensity of the right thigh.

[0549] FIG46 shows the temporal trends of the fluorescence intensity of the right thigh and the signal-to-noise ratio of the whole-body fluorescence signal.

[0550] Figure 47 shows photographs of the left and right thighs of infected mice observed with the naked eye 10 hours after the probe was injected into the muscles.

[0551] Figure 48 shows fluorescent images of infected thighs and major organs 10 hours after probe injection.

[0552] Figure 49 is a graph showing the quantification of fluorescence intensity in infected thighs and major organs 10 hours after probe injection.

[0553] Figure 50 shows fluorescence images of different numbers of bla-positive E. coli in the thigh of a mouse infected with the probe after intravenous injection. The black circle in the figure represents the area where the whole body signal was obtained, and the inset is λ ex / em = Fluorescence image at 660 / 710 nm.

[0554] Figure 51 is the quantification of fluorescence intensity in the infected right thigh.

[0555] FIG52 shows the signal-to-noise ratio of the fluorescence signal of the thigh infected with different numbers of bacteria and the whole body signal.

[0556] Figure 53 shows the mCherry channel (λ ex / em =580 / 620nm) and near infrared channel (λ ex / em =660 / 710 nm) whole-body fluorescence image taken 10 hours after injection of the main probe.

[0557] Figure 54 is a photograph of a thigh infected with bacteria with or without skin peeled off 10 hours after probe injection.

[0558] Figure 55 is a fluorescent image of the posterior thigh taken using mCherry and near-infrared channels 10 hours after injection of the main probe. DETAILED DESCRIPTION

[0559] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which no specific conditions are specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0560] The following will introduce the specific implementation of the method for preparing the near-infrared fluorescent probe and photosensitizer with the function of opening and enriching the target site. 1 H-NMR, 13C-NMR was measured using a Bruker 400Mz instrument. The solvents were deuterated chloroform (CDCl3) and deuterated dimethyl sulfoxide (d6-DMSO), and the internal standard was tetramethylsilane (TMS). All solvents were of chromatographic, analytical, or chemical grade.

[0561] Preparation Example 1: Near-infrared probe synthesis

[0562] This example takes the preparation method of a near-infrared fluorescent probe (ALPIN-6) as an example to describe the preparation process of a near-infrared probe with a removable water-soluble group.

[0563] The structure of ALPIN-6 is as follows:

[0564] The preparation process of ALPIN-6 is as follows:

[0565] a. Preparation of Compound 3

[0566] To a 25 mL Schlenk reaction tube, 1 (524.2 mg, 0.78 mmol), 2 (948.0 mg, 6.77 mmol), and KHCO (255.1 mg, 2.55 mmol) were added and the N was replaced by vacuum three times. Under N protection, 4 mL of ultra-dry DMF was added and the reaction was heated to 75 ° C with stirring for 4 h. The reaction was stopped and cooled to room temperature. 10 mL of ethyl acetate was added to dilute the reaction and washed three times with water (10 mL). The organic phase was separated by a separatory funnel, dried over anhydrous sodium sulfate, filtered, mixed with silica gel, and separated by column chromatography. The product was washed off with DCM:CH OH = 5:1 and dried on a rotary evaporator to obtain 293.3 mg of a blue solid with a yield of 66%. The spectral characteristics are: 1 H NMR (400MHz, CD3OD) δ8.74(d,J=14.8Hz,1H),7.63(d,J=7.6Hz,1H),7.56–7.45(m,4H),7.46–7.38(m,1H),6.88(s,1H),6.44(d,J=15.2Hz,1 H),4.69(s,2H),4.29(t,J=7.4Hz,2H),2.78(t,J=5.8Hz,2H),2.72(t,J=5.8Hz,2H),2.00–1.88(m,4H),1.82(s,6H),1.08(t,J=7.4Hz,3H). 13C NMR (101MHz, CD3OD) δ176.92,162.43,159.58,153.96,144.99,141.80,141.70,135.36,128.79,128.09,126.76,126.58,126.32 ,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 NO3(MI) + 442.2382, found 442.2377.

[0567] b. Preparation of Compound 5

[0568] Dye 3 (50.5 mg, 0.089 mmol), KI (142.8 mg, 0.860 mmol), KHCO (49.4 mg, 0.493 mmol), and 18-crown-6 (20.3 mg, 0.077 mmol) were added to a 10 mL reaction tube and the atmosphere was replaced with N2 by vacuum three times. Under N2 protection, 1.0 mL of ultra-dry DMF was added, followed by 4 (48.4 mg, 0.159 mmol). The reaction was stirred at room temperature for 4 h, and the reaction was stopped. DCM (10.0 mL) was added for dilution, and the mixture was washed with water (5.0 mL). The organic phase was separated using a separatory funnel, dried over anhydrous sodium sulfate, filtered, mixed with silica gel, and separated by column chromatography. The product was washed off with DCM:CH3OH = 10:1 and dried on a rotary evaporator 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, CDCl3) 11H NMR (600 MHz, CD3OD) 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 13C NMR (151 MHz, CD3OD) 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 NO7P(M-I) + 732.3090, found 732.3089.

[0569] c. Preparation of compound ALPIN-6

[0570] Compound 5 (33.5 mg, 0.040 mmol) was added to a 10 mL reaction vial, 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 was stirred at room temperature for 6 h. The mixture was diluted with DCM, washed with water, dried over anhydrous sodium sulfate, and filtered. The filtrate was dried on a rotary evaporator to obtain compound 7, which was used in the next step without further purification.

[0571] To a 10 mL Schlenk reaction tube, 7 (22.8 mg), 1,3-dimethylbarbituric acid (7.8 mg, 0.05 mmol), and Pd(PPh3)4 (5.8 mg, 0.005 mmol) were added. The mixture was evacuated and replaced with nitrogen three times. 0.5 mL each of MeCN and DCM were added. The reaction was stirred at room temperature for 1 h. The reaction solution was then spin-dried to dryness. 9 (10.2 mg, 0.043 mmol), CuSO4 (3.5 mg, 0.022 mmol), ascorbic acid (17.5 mg, 0.099 mmol), DMSO (0.5 mL), and H2O (0.5 mL) were added. The reaction was stirred at room temperature for 1 h. High-performance preparative liquid chromatography (HPLC) yielded 2.1 mg of the title compound in a three-step yield of 13%.

[0572] The spectral characteristics are: 1 H NMR(600MHz,d6-DMSO)δ8.57(d,J=15.0Hz,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=15.0Hz,1H),5.34(s,2H),5.10(s,2H),4.92(t,J=6. 9Hz,2H),4.40(t,J=6.9Hz,2H),4.27(d,J=6.0Hz,2H),3.85(t,J=6.9Hz,2H),3.07(s,6H),2.73–2.68(m,4H),2.4 4(t,J=6.9Hz,2H),1.99–1.94(m,2H),1.86–1.82(m,6H),1.79(s,6H),0.99(t,J=7.2Hz,3H).HRMS(ESI)m / zcalcd for C 47 H 58 N6O 11 PS(MI) + 945.3616, found 945.3624.

[0573] Preparation Example 2: Synthesis of Near-Infrared Photosensitizer

[0574] This example takes the preparation method of the near-infrared photosensitizer (ALPIN-6I) as an example to describe the preparation process of the near-infrared photosensitizer.

[0575] The structure of ALPIN-6I is as follows:

[0576] The preparation process of ALPIN-6I is as follows:

[0577] a. Preparation of Compound 11

[0578] Compound 5-iodo-2,3,3-trimethyl-3H-indole 10 (5.0 g, 17.537 mmol) was added to a 100 mL reaction flask, followed by 30 mL of ultra-dry ACN and 1-iodopropane (8.5 mL, 87.683 mmol). The reaction was heated under reflux for 18 hours. Acetonitrile was dried on a rotary evaporator, acetone was added, and the mixture was heated to 60°C. The solid precipitated after cooling and filtration to obtain 7.489 g of a pink solid with a yield of 94%. The spectral characteristics are as follows: 1 H NMR(600MHz,d6-DMSO)δ8.32(d,J=1.2Hz,1H),8.00(dd,J=8.4,0.6Hz,1H),7.82(d,J=8.4Hz,1H),4.4 1(t,J=7.8Hz,2H),2.83(s,3H),1.89–1.80(m,2H),1.54(s,6H),0.98(t,J=7.2Hz,3H).HRMS(ESI)m / z calcd for C 14 H 19 IN(MI) + 328.0557, found 328.0563.

[0579] b. Preparation of Compound 12

[0580] Anhydrous sodium acetate (81.3 mg, 0.991 mmol), 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde (805.0 mg, 4.664 mmol), and compound 11 (4.443 g, 9.762 mmol) were added to a reaction flask. The atmosphere was replaced with nitrogen three times, and 8 mL of ethanol was added. The reaction was stirred at 60°C for 16 h. After mixing and column chromatography, 2.806 g of a green solid was obtained in a 66% yield. The chromatographic characteristics are as follows: 1H NMR (400MHz, CDCl3) δ8.32(d,J=13.6Hz,2H),7.70(d,J=8.0Hz,2H),7.65(s,2H),7.00(d,J=8.0Hz,2H),6.26(d,J=13. 6Hz,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=6.8Hz,6H).HRMS(ESI)m / z calcd for C 36 H 42 ClI2N2(MI) + 791.1120, found 791.1134.

[0581] c. Preparation of Compound 13

[0582] Compound 12 (1.973 g, 2.147 mmol), 2 (1.572 g, 11.217 mmol), and KHCO₃ (676.1 mg, 6.753 mmol) were added to a reaction flask. The atmosphere was replaced with N₂ three times, and 5 mL of DMF was added. The reaction was stirred at 75°C for 3 h. The mixture was diluted with ethyl acetate, washed three times with water, and dried over anhydrous sodium sulfate. The sample was mixed and purified by column chromatography to obtain 730.0 mg of a blue compound in a 49% yield. The chromatographic characteristics are as follows: 1 HNMR(600MHz,d6-DMSO)δ7.85(d,J=13.2Hz,1H),7.74(d,J=1.2Hz,1H),7.6 9(s,1H),7.57(dd,J=8.4,1.2Hz,1H),7.37(s,1H),6.92(d,J=8.4Hz,1H),6. 19(s,1H),5.76(s,1H),4.40(s,2H),3.84(t,J=7.2Hz,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=7.2Hz,3H). 13C NMR (151MHz, d6-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 / z calcd for C 29 H 31 INO3(MI) + 568.1343, found 568.1350.

[0583] d. Preparation of Compound 14

[0584] Compound 13 (82.3 mg, 0.118 mmol), 4 (79.0 mg, 0.240 mmol), KI (205.3 mg, 1.237 mmol), KHCO (63.6 mg, 0.635 mmol), and 18-crown-6 (31.7 mg, 0.120 mmol) were added to a reaction flask. The atmosphere was replaced with nitrogen three times, and 1 mL of DMF was added. The reaction was stirred at room temperature for 6 h. The product was diluted with DCM, washed three times with water, dried over anhydrous sodium sulfate, mixed, and purified by column chromatography to obtain 57.1 mg of a blue compound in a 50% yield. The spectral characteristics are as follows: 1 H NMR (600MHz, CDCl3) 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). 13C NMR (151MHz, CDCl3) δ175.11, 163.56, 159.97, 154.33, 150.46 (d, J = 6.8Hz), 144.88, 143.55, 141.59,137.92,137.12,132.69,131.95(d,J=7.6Hz),131.82,130.92,128.69,127.19,126. 57,120.28(d,J=4.8Hz),118.86,115.89,115.45,113.32,101.65,99.15,90.34,70.16,68.9 8(d,J=5.6Hz),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 INO7P(MI) + 834.2051, found 834.2056.

[0585] e. Preparation of Compound 15

[0586] Compound 14 (57.1 mg, 0.059 mmol) and 6 (31.6 mg, 0.144 mmol) were added to a reaction flask. DCM and Et3N (29 μL, 0.205 mmol) were then added and stirred at room temperature for 12 h. The product was diluted with DCM, washed three times with water, dried over anhydrous sodium sulfate, mixed, and purified by column chromatography to obtain 19.6 mg of a blue compound in a 48% yield. The chromatographic characteristics are: 1H NMR (600MHz, d6-DMSO) δ8.54(d,J=12.0Hz,1H),8.24(s,1H),7.89(d,J=12.0Hz,1H),7.79(t,J=6.0Hz,1H),7.60(d,J=6.0 Hz,2H),7.58–7.55(m,2H),7.52(d,J=6.0Hz,1H),7.32(d,J=6.0Hz,2H),7.19(s,1H),6.54(d,J=12.0Hz,1H),6.01–5.87(m ,2H),5.41(s,2H),5.34(d,J=18.0Hz,2H),5.22(d,J=12.0Hz,2H),5.12(s,2H),4.70–4.58(m,4H),4.35(t,J=6.0Hz,2H),3 .81(d,J=6.0Hz,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=6.0Hz,3H).HRMS(ESI)m / z calcd for C 46 H 49 IN2O8P(MI) + 915.2266, found 915.2270.

[0587] f. Preparation of compound ALPIN-6I

[0588] To a 10 mL Schlenk reaction tube, 15 (26.6 mg, 0.029 mmol), 1,3-dimethylbarbituric acid (7.8 mg, 0.05 mmol), and Pd(PPh3)4 (4.5 mg, 0.0039 mmol) were added. The mixture was evacuated and replaced with nitrogen three times. 0.5 mL each of MeCN and DCM was added. After stirring at room temperature for 30 min, the reaction solution was spin-dried and 9 (10.2 mg, 0.043 mmol), CuSO4 (2.1 mg, 0.013 mmol), ascorbic acid (22.1 mg, 0.125 mmol), DMSO (0.5 mL), and H2O (0.5 mL) were added. The reaction was stirred at room temperature for 1 h. High-performance preparative liquid chromatography (HPLC) yielded 4.7 mg of the title compound in a three-step yield of 15%. The chromatographic characteristics are as follows: 1H NMR(600MHz,d6-DMSO)δ8.55(d,J=14.7Hz,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=14.8Hz,1H),5.36(s,2H),5.10(s,2H),4.93(t,J =6.9Hz,2H),4.34(t,J=6.8Hz,2H),4.28(d,J=5.7Hz,2H),3.85(t,J=6.9Hz,2H),3.07(s,6H),2.73–2.68(m,4H) ,2.45(t,J=6.8Hz,2H),2.01–1.93(m,2H),1.86–1.79(m,6H),1.78(s,6H),0.96(t,J=7.4Hz,3H).HRMS(ESI)m / z calcd for C 47 H 57 N6O 11 PSI(MI) + 1071.2583,found 1071.2585.

[0589] Preparation Example 3: Preparation method of alkaline phosphatase near-infrared fluorescence and photoacoustic probe (ALPIN-7) with the function of opening and enriching at the detection target site.

[0590] The structure of ALPIN-7 is as follows:

[0591] The preparation process of ALPIN-7 is as follows:

[0592] a. Preparation of Compound 23

[0593] Preparation of Compound 21: To a 25 mL Schlenk reaction tube, add 2-hydroxy-4-iodobenzoic acid (1.24 g, 4.7 mmol), K2CO3 (1.98 g, 14.1 mmol), 18-C-6 (127.6 mg, 0.47 mmol), and 5 mL of ultra-dry DMF. Then add allyl bromide (1.2 mL, 14.1-14.1 mmol) and stir overnight at room temperature. Stop the reaction, dilute with 20 mL of ethyl acetate, and wash three times with 20 mL of water. Separate the organic phase with a separatory funnel, dry over anhydrous sodium sulfate, filter, and mix with silica gel. Separate by column chromatography and dry on a rotary evaporator to obtain 1.256 g of a white solid in 88% yield. Spectral characteristics: 1H NMR (400MHz, CDCl3) δ7.53(d,J=8.2Hz,1H),7.34(dd,J=8.2,1.6Hz,1H),7.30(d,J=1.2Hz,H),6.09–5.96(m,2H),5.54(dd,J=17.2,1.6H z, 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).

[0594] Preparation of compound 22: To a 25 mL Schlenk reaction tube, 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), and KOH (1.5 g, 26.6 mmol). The atmosphere was replaced with nitrogen three times, and then 4 mL of DMSO and 1 mL of H2O were added. The mixture was stirred and reacted at 110°C overnight. The reaction was stopped, cooled to room temperature, and then acidified with 1 M aqueous HCl. The mixture was diluted with 20 mL of ethyl acetate and washed three times with 20 mL of water. The organic phase was separated using a separatory funnel, dried over anhydrous sodium sulfate, filtered, mixed with silica gel, and separated by column chromatography. The mixture was dried on a rotary evaporator to obtain 752.6 mg of a white solid in a 67% yield. Spectral characteristics: 1 H NMR (400MHz, CDCl3) δ8.03 (d, J=8.4Hz, 1H), 6.98 (dd, J=8.4, 1.6Hz, 1H), 6.89 (d, J=1 .6Hz,1H),6.14–6.04(m,1H),5.45–5.52(m,2H),4.77(d,J=5.6Hz,2H),3.69(s,1H).

[0595] Preparation of compound 23: To a 10 mL Schlenk reaction tube, add 22 (435.2 mg, 2.1 mmol). N2 was replaced three times, followed by the addition of 4.2 mL of dry THF. Then, under nitrogen, 6.3 mL of a solution of lithium aluminum tetrahydride in tetrahydrofuran was added dropwise. Stir at room temperature overnight. Stop the reaction, slowly add water dropwise in an ice bath, adjust to acidity with 1 M aqueous HCl, dilute with 20 mL of ethyl acetate, and wash three times with 20 mL of water. Separate the organic phase with a separatory funnel, dry over anhydrous sodium sulfate, filter, and rotary evaporator to obtain the title compound, which was directly used in the next step.

[0596] b. Preparation of Compound 24

[0597] To a 10 mL Schlenk reaction tube, IR780 (108.2 mg, 0.16 mmol), 23 (117.1 mg, 0.75 mmol), and KHCO (48.3 mg, 0.48 mmol) were added. The atmosphere was replaced with nitrogen three times, and 0.5 mL of dry DMF was added. The reaction was stirred at 75°C for 2 h. The reaction was stopped, diluted with 20 mL of ethyl acetate, and washed three times with 20 mL of water. The organic phase was separated using a separatory funnel, dried over anhydrous sodium sulfate, filtered, mixed with silica gel, and separated by column chromatography. 54.2 mg of the blue compound was obtained by rotary evaporation to dryness in a 62% yield. Spectral characteristics: 1 H NMR (400MHz, CDCl3) δ8.11(d,J=13.6Hz,1H),7.58(s,1H),7.52(d,J=7.6Hz,1H),7.43–7.39(m,2H),7.30–7.25(m,2H),6.86(s,1H),6.19(d,J=13 .6Hz,1H),4.65(s,2H),4.09(t,J=7.2Hz,2H),2.75(t,J=5.2Hz,2H),2.6 3(t,J=5.6Hz,2H),1.92–1.61(m,4H),1.73(s,6H),1.03(t,J=7.6Hz,3H). 13 C NMR (101MHz, CDCl3) δ175.83,158.76,146.42,144.86,144.47,143.50 (d,J=3.2Hz),142.27,138.62,134.31,133.85,132.4 0,128.70,128.44,126.03,124.54,115.07,114.57,103.69,63.24,52.94,49.10,35.81,31.57,29.91,24.62,24.28,14.23.

[0598] c. Preparation of Compound 26

[0599] Dye 24 (54.2 mg, 0.076 mmol), KI (31.2 mg, 0.186 mmol), KHCO3 (19.7 mg, 0.197 mmol), and 18-crown-6 (3.4 mg, 0.013 mmol) were added to a 10 mL reaction tube and the N2 was replaced by vacuum three times. Under N2 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, the reaction was stopped, and 10.0 mL of DCM was added to dilute the mixture. The mixture was washed with water (5.0 mL). The organic phase was separated with a separatory funnel, dried over anhydrous sodium sulfate, filtered, mixed with silica gel, quickly passed through a column, and spin-dried to obtain a blue solid compound 25. Compound 25 was added to a 10 mL reaction tube, followed by 1 mL of DCM, 6 (42.3 mg, 0.19 mmol), and TEA (37 μL, 0.27 mmol). The reaction was stirred at room temperature overnight. The reaction was stopped and diluted with 10.0 mL of DCM. The mixture was washed with water (5.0 mL). The organic phase was separated using a separatory funnel, dried over anhydrous sodium sulfate, filtered, mixed with silica gel, and separated by column chromatography. The mixture was then dried to obtain 40.7 mg of a blue solid compound with a two-step yield of 57%. Spectral characteristics: 1 H NMR (600MHz, CDCl3) δ9.34(d,J=14.4Hz,1H),8.55–8.53(m,2H),8.49(t,J=7.2Hz,1H),8.44–8.41(m,3H),8.34(d,J= 7.8Hz,1H),8.28–8.26(m,2H),8.22(s,2H),7.55(d,J=14.4Hz,1H),7.00–6.93(m,2H),6.40(dd,J=17.4,1.2Hz,2H), 6.29(dd,J=10.8,1.2Hz,2H),6.21(d,J=17.4Hz,4H),5.72–5.62(m,4H),5.31(t,J=7.2Hz,2H),5.01(s,3H),3.77(t, J=5.6Hz,2H),3.67(t,J=5.6Hz,2H),3.27(s,1H),2.98–2.95(m,2H),2.81(s,6H),2.28(s,2H),2.07(t,J=7.2Hz,3H). 13C NMR (151MHz, CDCl3) δ177.29,160.47,160.23,157.51,145.23,142.07,141.41,136.40,135.76,1 33.06,132.42,132.00(d,J=6.9Hz),131.13,129.20,128.76,127.53,127.07,126.87,123.08,12 2.70,120.36(d,J=4.4Hz),117.16,115.25,112.53,107.35,105.08,79.64,71.64,70.07,68.96( d, J=6.0Hz), 61.49, 50.77, 46.94, 32.47, 30.89, 29.70, 28.43, 26.66, 21.31, 20.24, 14.13, 11.44.

[0600] d. Preparation of compound ALPIN-7

[0601] To a 10 mL Schlenk reaction tube, 26 (25.7 mg, 0.028 mmol), 1,3-dimethylbarbituric acid (14.3 mg, 0.825 mmol), and Pd(PPh3)4 (6.5 mg, 0.006 mmol) were added. The nitrogen atmosphere was replaced by vacuum three times, and 0.5 mL each of MeCN and DCM was added. After stirring at room temperature for 1 hour, the reaction solution was spin-dried to dryness, and 9 (6.5 mg, 0.027 mmol), CuSO4 (4.4 mg, 0.027 mmol), ascorbic acid (19.4 mg, 0.11 mmol), DMSO (0.5 mL), and H2O (0.5 mL) were added. The reaction was stirred at room temperature for 1 hour. High-performance preparative liquid chromatography (HPLC) yielded 4.5 mg of the title compound in a three-step yield of 15%. Spectral characteristics: 1H NMR(600MHz,d6-DMSO)δ8.28(d,J=14.4Hz,1H),8.11(s,1H),7.94–7.86(m,2H),7.83(d,J=7.2Hz,1H),7.80–7.75(m,4H),7.62(d,J=1 9.8Hz,1H),7.57(t,J=7.8Hz,1H),7.50(t,J=7.8Hz,1H),7.48(d,J=8.4Hz,1H),7.40(s,1H),7.21(d,J=8.4Hz,2H),6.79(d,J=15Hz,1H ),5.26(s,2H),5.09(s,2H),4.92(t,J=7.2Hz,2H),4.44(t,J=7.2Hz,2H),4.27(d,J=5.4Hz,2H),3.85(t,J=6.9Hz,2H),3.07(s,6H),2 .77(s,2H),2.70(s,2H),2.45(t,J=6.9Hz,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=7.4Hz,3H).

[0602] Preparation Example 4: Preparation of probe

[0603] Example 4.1: Synthesis of probe BIN-3

[0604] The synthetic route of the main probe BIN-3 is as follows:

[0605] Step 1: Compound 2 (238.7 mg, 0.532 mmol), KI (151.9 mg, 0.915 mmol), and ultra-dry DMF (1.2 mL) were added to a reaction flask and stirred at room temperature for 0.5 h. KHCO3 (38.6 mg, 0.386 mmol), 18-crown-6 (24.7 mg, 0.093 mmol), and compound 1 (103.1 mg, 0.181 mmol) were then added. The reaction was stopped after 1.5 h. DCM (20 mL) was added to the reaction solution for dilution, washed with saturated NaCl solution, washed with water, and extracted with DCM three times (30 mL x 3). The combined DCM was dried over anhydrous Na2SO4, concentrated in vacuo on a rotary evaporator, and purified by column chromatography to obtain 99.4 mg of blue solid compound 3 in a yield of 56%. 1H NMR (600MHz, CD3OD) δ8.75(d,J=15.0Hz,1H),7.56(s,1H),7.55–7.48(m,3H),7.45(s,1H),7.40(t,J=7.2Hz,1H),7.30( d,J=8.4Hz,2H),7.23(s,1H),6.79(d,J=8.4Hz,2H),6.52(d,J=14.4Hz,1H),5.79(d,J=4.8Hz,1H),5.36(d,J=12.0Hz,1H ),5.28–5.21(m,2H),5.15(d,J=4.8Hz,1H),4.98(d,J=12.6Hz,1H),4.68–4.60(m,2H),4.33(t,J=7.2Hz,2H),3.76–3.63 (m,5H),2.86–2.69(m,4H),2.51–2.41(m,4H),2.24(s,1H),2.01–1.91(m,4H),1.89–1.80(m,6H),1.08(t,J=7.2Hz,3H). 13 C NMR (151MHz, CD3OD) δ177.80,173.06,165.13,162.19,161.79,160.03,158.67,153.88,145.41,142.09 ,141.67,134.02,130.00,129.14,128.78,127.60,126.90,126.80,126.45,126.05,125.39,122.33,115 .76,114.23,113.54,112.47,103.18,99.16,81.84,69.02,67.62,66.95,59.42,58.30,57.66,54.32,50.58,46.06,34.03,28.72,27.46,27.07,25.10,23.73,20.85,20.24,14.02,10.19. HRMS (ESI) m / z calcd: C 50 H 52 N3O8S(MI) + 854.3470, measured value: 854.3472.

[0606] Step 2: Compound 3 (70.9 mg, 0.072 mmol) was added to a 10 mL schlenk tube and replaced with nitrogen three times. Et-NCO (58 μL, 0.723 mmol) and DBTDL (18 μL, 0.029 mmol) were dissolved in DMF (700 μL) and then added to the reaction tube. The reaction was stirred at room temperature for 4 h. After the reaction was completed, the reaction solution was diluted with DCM (10 mL), washed with saturated NaCl solution, washed with water, and extracted with DCM three times (20 mL x 3). The combined DCM was dried over anhydrous Na2SO4, dried under vacuum, and purified by column chromatography to obtain 63.5 mg of blue solid compound 4 with a yield of 83%. 1 H NMR (600MHz, CD3OD) δ8.74(d,J=14.4Hz,1H),7.56(d,J=8.4Hz,1H),7.54–7.47(m,3H),7.42(t,J=7.2Hz,1H),7.38(s,1H),7.30 (d,J=8.4Hz,2H),7.20(s,1H),6.78(d,J=8.4Hz,2H),6.55(d,J=15.0Hz,1H),5.80(d,J=4.8Hz,1H),5.36(d,J=12.0Hz,1H),5.28 –5.20(m,2H),5.17–5.06(m,3H),4.96(d,J=12.6Hz,1H),4.34(t,J=7.2Hz,2H),3.77–3.62(m,5H),3.15(q,J=7.2Hz,2H),2.85– 2.71(m,4H),2.50–2.41(m,4H),2.24(s,1H),2.01–1.92(m,4H),1.90–1.79(m,6H),1.12(t,J=7.2Hz,3H),1.08(t,J=7.8Hz,3H). 13C NMR (151MHz, CD3OD) δ178.13,173.07,165.14,162.14,161.37,160.02,158.95,157.10,154.23,145.58,142. 19,141.62,133.25,130.02,128.83,127.88,127.84,127.10,126.80,125.86,125.51,124.42,122.36,115.6 5,114.28,113.53,112.65,103.69,99.42,81.85,69.03,67.63,67.08,60.71,59.41,57.65,54.33,50.71,46.17,35.30,34.04,28.75,27.38,27.01,25.21,23.71,20.91,20.20,14.04,13.98,10.19. HRMS (ESI) m / z calcd: C 53 H 57 N4O9S(MI) + 925.3841, measured value: 925.3841.

[0607] Step 3: Compound 4 (15.2 mg, 0.014 mmol) and DCM (1.0 mL) were added to a 25 mL reaction flask in sequence, and the mixture was cooled to 0°C in an ice-water bath. 20 μL of water, 20 μL of triisopropylsilane and 400 μL of trifluoroacetic acid (TFA) were dissolved in 1.0 mL of DCM, and the mixed solution was slowly added dropwise to the reaction flask. After 1.5 h of reaction, the reaction of the raw material was completely completed by HPLC analysis. The reaction was stopped, and 7 mL of 1 M NaHCO3 solution was added. The mixture was washed with water and extracted with DCM 3 times (10 mL x 3). The combined organic phase was dried over anhydrous Na2SO4, dried under vacuum, dissolved in 4.0 mL of acetonitrile, and purified by a reverse phase C18 preparative column. The prepared solution was dried with acetonitrile and then freeze-dried to obtain 10.0 mg of a blue solid product, compound 5, with a yield of 74%. 1H NMR (600MHz, CD3OD) δ8.78(d,J=15.0Hz,1H),7.69(d,J=7.2Hz,1H),7.59(s,1H),7.58–7.49(m,3H),7.46(t,J=7.8Hz,1H), 7.36(s,1H),6.54(d,J=15.0Hz,1H),5.75(d,J=4.8Hz,1H),5.58(d,J=13.2Hz,1H),5.22–5.04(m,4H),4.34(t,J=7.2Hz,2H ), 3.67 (d, J = 18.6 Hz, 1H), 3.61 (d, J = 18.0 Hz, 1H), 3.15 (q, J = 7.2 Hz, 2H), 2.82–2.76 (m, 2H), 2.74–2.69 (m, 2H), 2.46–2.39 (m, 4H), 2.22 (s, 1H), 1.98–1.92 (m, 4H), 1.88–1.82 (m, 6H), 1.12 (t, J = 7.2 Hz, 3H), 1.07 (t, J = 7.2 Hz, 3H). HRMS (ESI) m / z calculated value: C 45 H 49 N4O8S(MI) + 805.3266, measured value: 805.3267.

[0608] Step 5: Compound 5 (7.1 mg, 0.008 mmol), compound 6 (13.2 mg, 0.056 mmol), anhydrous copper sulfate (15.1 mg, 0.095 mmol), and ascorbic acid (14.9 mg, 0.085 mmol) were dissolved in DMSO (0.5 mL) and H2O (0.1 mL). The mixture was reacted at room temperature for 1 h. After the reaction was terminated, the product was purified using a reverse phase C18 preparative column to obtain 6.2 mg of a blue solid BIN-3 in a 70% yield. HRMS (ESI) m / z calculated value: C 52 H 65 N8O 11 S2(MI) + 1041.4209, measured value: 1041.4219.

[0609] Example 4.2: Synthesis of probe BIN-2

[0610] The synthetic route of the control probe BIN-2 is as follows:

[0611] Step 1: Compound 7 (197.2 mg, 0.439 mmol), KI (134.1 mg, 0.878 mmol), and ultra-dry DMF (1.0 mL) were added to the reaction flask in sequence and stirred at room temperature for 0.5 h. KHCO3 (31.0 mg, 0.310 mmol), 18-crown-6 (20.0 mg, 0.074 mmol), and compound 2 (62.4 mg, 0.116 mmol) were then added. The reaction was stopped after 3 h. The reaction solution was diluted with DCM (20 mL), washed with saturated NaCl solution, washed with water, and extracted with DCM three times (30 mL x 3). The combined DCM was dried over anhydrous Na2SO4, concentrated in vacuo on a rotary evaporator, and purified by column chromatography to obtain 62.2 mg of blue solid compound 8 with a yield of 57%. 1 H NMR(600MHz,CD3OD)δ8.75(d,J=15.0Hz,1H),7.60–7.49(m,3H),7.47–7.40(m,2H),7.38(s,1H),7.29(d,J=9.0Hz,2H),7 .10(d,J=1.8Hz,1H),6.90(dd,J=9.0,2.4Hz,1H),6.79(d,J=8.4Hz,2H),6.54(d,J=15.0Hz,1H),5.78(d,J=4.8Hz,1H),5. 30(d,J=12.0Hz,1H),5.23(d,J=12.0Hz,1H),5.15–5.09(m,2H),4.92(d,J=12.6Hz,1H),4.34(t,J=7.8Hz,2H),3.76–3.6 0(m,5H),2.85–2.68(m,4H),2.52–2.42(m,4H),2.25(s,1H),2.01–1.91(m,4H),1.89–1.78(m,6H),1.08(t,J=7.2Hz,3H). 13C NMR(151MHz,CD3OD)δ178.12,173.07,165.07,162.03,161.53,161.47,160.02,154.34,145.71,1 42.17,141.61,133.41,130.13,128.83,128.68,127.52,127.09,126.88,126.14,125.52,122.38 ,116.23,114.26,114.05,113.53,112.64,103.61,100.96,81.88,69.06,67.61,66.89,59.41,57.67,54.34,50.69,46.17,34.04,28.71,27.27,27.01,25.39,23.67,20.91,20.24,14.05,10.19.

[0612] Step 2: Compound 8 (25.2 mg, 0.027 mmol) and DCM (1.0 mL) were added to a 25 mL reaction flask in sequence, and the mixture was cooled to 0°C in an ice-water bath. 20 μL of water, 20 μL of triisopropylsilane and 400 μL of trifluoroacetic acid (TFA) were dissolved in 1.0 mL of DCM, and the mixed solution was slowly added dropwise to the reaction flask. After 1.5 h of reaction, the reaction of the raw material was completed by HPLC analysis. The reaction was stopped, and 7 mL of 1 M NaHCO3 solution was added, washed with water, and extracted with DCM 3 times (10 mL x 3). The combined organic phase was dried over anhydrous Na2SO4, dried under vacuum, dissolved in 4.0 mL of acetonitrile, and purified by a reversed-phase C18 preparative column. The prepared solution was dried with acetonitrile and then freeze-dried to obtain 18.6 mg of a blue solid product BIN-1 with a yield of 85%. 1H NMR(600MHz,d6-DMSO)δ8.91(d,J=8.4Hz,1H),8.61(d,J=15.0Hz,1H),7.75(d,J=7.2Hz,1H),7.72(d,J=7.8Hz,1H),7.59–7.5 2(m,2H),7.52–7.45(m,2H),7.29(d,J=1.8Hz,1H),7.03(dd,J=8.4,1.8Hz,1H),6.60(d,J=15.0Hz,1H),5.74(dd,J=8.4,4.8H z,1H),5.20–5.11(m,2H),4.97(d,J=12.0Hz,1H),4.41(t,J=7.2Hz,2H),3.71(d,J=18.6Hz,1H),3.60(d,J=18.6Hz,1H),2.77 (s,1H),2.75–2.71(m,2H),2.71–2.65(m,2H),2.45–2.32(m,4H),1.88–1.81(m,4H),1.80–1.73(m,6H),0.99(t,J=7.2Hz,3H). 13 C NMR(151MHz,d6-DMSO)δ178.34,171.66,165.37,163.52,161.46,160.74,154.23,145.41, 142.74,141.95,133.39,129.44,129.36,127.67,127.63,126.88,124.56,123.18,116.24, 114.40,114.11,113.86,105.14,102.00,83.86,71.95,67.52,59.55,57.94,50.98,46.55,34.06,28.91,28.10,28.01,25.74,24.03,21.43,20.37,14.50,11.48. HRMS (ESI) m / z calculated: C 41 H 42 N3O6S(MI) + 704.2789, measured value: 704.2798.

[0613] Step 3: Compound BIN-1 (9.1 mg, 0.011 mmol), compound 6 (11.3 mg, 0.048 mmol), anhydrous copper sulfate (11.6 mg, 0.073 mmol), and ascorbic acid (13.2 mg, 0.075 mmol) were dissolved in DMSO (0.5 mL) and H2O (0.1 mL). The mixture was reacted at room temperature for 1 h. After the reaction was stopped, the mixture was purified using a reverse phase C18 preparative column to obtain 8.0 mg of blue solid BIN-2 with a yield of 69%. 1 H NMR (600MHz, d6-DMSO) δ8.92(d,J=8.4Hz,1H),8.61(d,J=15.0Hz,1H),7.96(d,J=6. 6Hz,1H),7.75(d,J=7.2Hz,1H),7.72(d,J=8.4Hz,1H),7.58–7.53(m,2H),7.51–7.4 5(m,2H),7.29(d,J=2.4Hz,1H),7.03(dd,J=8.4,1.8Hz,1H),6.60(d,J=15.0Hz,1H) ,5.73(dd,J=7.8,4.8Hz,1H),5.19–5.11(m,2H),4.96(d,J=12.0Hz,1H),4.90(t,J=6 .6Hz,2H),4.41(t,J=7.2Hz,2H),3.85(t,J=7.2Hz,2H),3.72(d,J=18.6Hz,1H),3.6 1(d,J=18.0Hz,1H),3.54–3.51(m,2H),3.06(s,6H),2.90–2.87(m,2H),2.75–2.72( m, 2H), 2.71–2.66 (m, 2H), 2.58–2.54 (m, 2H), 2.47 (t, J = 6.6 Hz, 2H), 2.04–1.96 (m, 2H), 1.88–1.80 (m, 4H), 1.80–1.74 (m, 6H), 0.99 (t, J = 7.2 Hz, 3H). HRMS (ESI) m / z calculated value: C 48 H 58 N7O9S2(MI) + 940.3732, measured value: 940.3738.

[0614] Example 5: Preparation of photosensitizer

[0615] Example 5.1: Synthesis of photosensitizer BIN-3I

[0616] The synthesis method of photosensitizer BIN-3I is as follows:

[0617] Step 1: Compound 2 (40 mg, 0.060 mmol), KI (108 mg, 0.651 mmol), and ultra-dry DMF (0.4 mL) were added to a reaction flask and stirred at room temperature for 0.5 h. KHCO3 (32 mg, 0.032 mmol), 18-crown-6 (16 mg, 0.060 mmol), and compound 9 (84 mg, 0.187 mmol) were then added. The reaction was stopped after 1.5 h. DCM (20 mL) was added to the reaction solution for dilution, washed with saturated NaCl solution, washed with water, and extracted with DCM three times (30 mL x 3). The combined DCM was dried over anhydrous Na2SO4, concentrated in vacuo on a rotary evaporator, and purified by column chromatography to give 16.4 mg of blue solid compound 10 in a yield of 25%. 1 H NMR (400MHz, DMSO-d6) δ = 8.93 (d, J = 8.5, 1H), 8.54 ( d, J = 14.8, 1H), 8.02 ( d, J = 1.7, 1H), 7.87 ( dd, J = 8.3, 1.7, 1H), 7.69 ( s, 1H) ,7.62(s,1H),7.44(d,J=8.3,1H),7.30(d,J=8.6,2H),7.16(s,1H),6.81(d,J=8.6,2H),6.46(d,J=14.8,1H),5.79(dd,J=8.4 ,4.8,1H),5.29–5.20(m,2H),5.19(d,J=4.9,1H),5.03(d,J=12.1,1H),4.94(d,J=12.2,1H),4.51(s,2H),3.81(s,3H),3.74– 3.63(m,5H),2.80–2.73(m,2H),2.69(d,J=12.2,2H),2.40–2.37(m,4H),2.34–2.33(m,1H),1.85(s,2H),1.75(d,J=5.6,6H).

[0618] Step 2: Compound 10 (2 mg, 0.002 mmol) was added to a reaction flask, 3-isocyanatoprop-1-yne (1.5 mg, 0.018 mmol) and DBTDL (1.3 mg, 0.002 mmol) were dissolved in DMF (100 μL) and then added to a reaction tube. The reaction was stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was diluted with DCM (2 mL), washed with saturated NaCl solution, washed with water, and extracted with DCM three times (2 mL x 3). The combined DCM was dried over anhydrous Na2SO4, dried under vacuum, and purified by column chromatography to obtain 2 mg of blue solid compound 11 with a yield of 93%. 1H NMR (400MHz, DMSO-d6) δ=8.94(d,J=8.5,1H),8.54(d,J=14.9,1H),8.04(d,J=1.6,1H),7.89(dd,J=8.4,1.6,1H),7.77(t,J=5.7,1H),7.54(d, J=5.7,2H),7.47(d,J=8.5,1H),7.30(d,J=8.6,2H),7.18(s,1H),6.80 (d,J=8.6,2H),6.52(d,J=15.0,1H),5.80(dd,J=8.5,4.9,1H),5.26(q, J=12.0,2H),5.18(d,J=4.9,1H),5.05(d,J=7.0,1H),4.93(d,J=12.1, 1H),3.84(s,3H),3.82–3.77(m,2H),3.73–3.63(m,5H),3.12(d,J=2.7, 1H),2.77–2.73(m,2H),2.72–2.66(m,2H),2.40–2.37(m,4H),2.18(t, J=7.3,1H),1.83(d,J=6.3,2H),1.76(d,J=4.8,6H),1.58–1.45(m,2H).

[0619] Step 3: Compound 11 (2 mg, 0.002 mmol) was added to a 25 mL reaction flask in sequence, cooled to 0°C in an ice-water bath, 10 μL of water, 10 μL of triisopropylsilane and 200 μL of trifluoroacetic acid (TFA) were dissolved in 1.0 mL of DCM, and the mixed solution was slowly added dropwise to the reaction flask. After 2 h of reaction, HPLC analysis showed that the raw material reaction was complete. The reaction was stopped, 5 mL of 1 M NaHCO3 solution was added, washed with water, and extracted with DCM 3 times (10 mL x 3). The combined organic phases were dried over anhydrous Na2SO4 and dried in vacuo to obtain the crude product compound 12 without purification.

[0620] Step 4: Compound 12 (2 mg, 0.002 mmol), compound 13 (1 mg, 0.006 mmol), anhydrous copper sulfate (0.3 mg, 0.002 mmol), and ascorbic acid (1.4 mg, 0.008 mmol) were dissolved in DMSO (0.1 mL) and H2O (0.1 mL). The reaction was carried out at room temperature for 1 h. After the reaction was stopped, the product was purified using a reverse phase C18 preparative column to obtain 1 mg of a blue solid BIN-3I with a yield of 59%. 1H NMR (400MHz, DMSO-d6) δ=8.92(d,J=8.4,1H),8.55(d,J=14.8,1H),8.11(d,J=1.7,1H),8.05(s,1H),7.92(s,1H),7.91–7.85(m,2H),7.55(s ,1H),7.53(s,1H),7.49(d,J=2.6,1H),6.50(d,J=14.9,1H),5.73(dd, J=8.4,4.8,1H),5.68(t,J=6.9,1H),5.59(dd,J=7.7,6.1,1H),5.36(d ,J=12.9,1H),5.15(d,J=4.9,1H),5.10(d,J=3.9,1H),5.02(d,J=13.0 ,1H),4.28(d,J=5.9,2H),3.83(s,3H),3.63(d,J=12.3,2H),3.20(s,1 H),3.16(s,1H),2.86–2.80(m,2H),2.72(d,J=3.9,2H),2.68–2.66(m, 2H),2.54(s,4H),2.33(p,J=1.8,2H),1.82(s,2H),1.78–1.71(m,6H).

[0621] Example 5.2: Synthesis of photosensitizer BIN-2I

[0622] The synthetic route of the control photosensitizer BIN-2I is as follows:

[0623] Step 1: Compound 2 (10 mg, 0.016 mmol), KI (35 mg, 0.210 mmol), and ultra-dry DMF (0.4 mL) were added to a reaction flask and stirred at room temperature for 0.5 h. KHCO3 (7.7 mg, 0.077 mmol), 18-crown-6 (4 mg, 0.016 mmol), and compound 14 (24.6 mg, 0.055 mmol) were then added. The reaction was stopped after 1.5 h. DCM (20 mL) was added to the reaction solution for dilution, washed with saturated NaCl solution, washed with water, and extracted with DCM three times (30 mL x 3). The combined DCM was dried over anhydrous Na2SO4, concentrated in vacuo on a rotary evaporator, and purified by column chromatography to obtain 5.8 mg of blue solid compound 15 in a yield of 35%.

[0624] Step 2: Compound 15 (2 mg, 0.002 mmol) was added sequentially to a 25 mL reaction flask, cooled to 0°C in an ice-water bath, 10 μL of water, 10 μL of triisopropylsilane and 200 μL of trifluoroacetic acid (TFA) were dissolved in 1.0 mL of DCM, and the mixed solution was slowly added dropwise to the reaction flask. After 2 h of reaction, HPLC analysis showed that the reaction of the raw materials was complete. The reaction was stopped, 5 mL of 1 M NaHCO3 solution was added, washed with water, and extracted with DCM 3 times (10 mL x 3). The combined organic phases were dried over anhydrous Na2SO4 and dried in vacuo to obtain the crude product compound 16 without purification.

[0625] Step 3: Compound 16 (2 mg, 0.002 mmol), compound 13 (1 mg, 0.006 mmol), anhydrous copper sulfate (0.3 mg, 0.002 mmol), and ascorbic acid (1.4 mg, 0.008 mmol) were dissolved in DMSO (0.1 mL) and H2O (0.1 mL). The mixture was reacted at room temperature for 1 h. After the reaction was stopped, a reverse phase C18 preparative column was used to prepare and purify the blue solid BIN-2I. Mass spectrometric data of BIN-2I: UPLC-MS (EI), C 43 H 42 IN6O 10 S + (MI) + Calculated value: 961.17, measured value: 961.17.

[0626] Test Example 1: Verification of Solution Enzyme Response of Near-Infrared Fluorescent Probe ALPIN-6

[0627] a. Near-infrared fluorescent probe absorption and fluorescence test conditions

[0628] The probe's absorption spectrum was measured using a UV-Vis spectrophotometer with a wavelength scan range of 400 to 800 nm. Fluorescence spectra were measured using a fluorimeter with an excitation wavelength of 687 nm, excitation and emission slit widths of 3 nm, and a wavelength scan range of 697 to 850 nm. Fluorescence intensity changes over time were measured using a microplate reader (SpectraMax i3) using a 96-well plate with a default total volume of 100 μL, an excitation wavelength of 687 nm, an emission wavelength of 714 nm, and excitation and emission slit widths of 15 nm. The PMT setting was the default for the microplate reader, with 10 flashes / read. Unless otherwise specified, the PBS buffer (1X, pH 7.4) used in fluorescence and absorption assays contained 0.1% surfactant CHAPS (3-[3-(cholamidopropyl)dimethylammonium]-1-propanesulfonic acid inner salt), the probe ALPIN-6 concentration was 10 μM, the final ALP concentration was 2 U / mL, and the final β-mercaptoethanol concentration was 140 mM. The samples were incubated in a 37°C water bath for 30 min.

[0629] b. HPLC analysis of changes before and after near-infrared probe and enzyme hydrolysis

[0630] The probe ALPIN-6 (10 μM) and ALP (2 U / mL) were incubated in a PBS buffer solution in a 37°C water bath for 30 min. The reaction solution was centrifuged at 12,000 rpm for 10 min using a 10 kDa ultrafiltration membrane to remove the protein. 100 μL of the filtrate was taken for HPLC analysis at a wavelength of 600 nm.

[0631] c. Gel imaging of proteins covalently labeled with near-infrared probes

[0632] According to experimental requirements, a total volume of 25 μL of gel electrophoresis sample was prepared: fluorescent probe ALPIN-6 or ALPIN-5 (10 μM) was incubated with different proteomic samples (ALP 8 μM, BSA protein 5 μM) at 37°C for 2 hours, and then 2 μL of gel electrophoresis loading buffer (5x) was added and mixed thoroughly. An 8% gel was prepared according to the Beyotime kit, and 20 μL of the above sample was loaded into each well. The gel was run on an SDS-PAGE gel electrophoresis apparatus at 120 V for 2-3 hours to obtain an image of the gel. The gel was then scanned with an Odyssey CLx Image Studio near-infrared dual-color scanner system (λ ex / em =685nm / 720nm), and the scanned images were analyzed for protein fluorescence bands. The protein gel was then stained with Coomassie Brilliant Blue solution on a shaker for 30 minutes to mark the protein band positions. The Coomassie Brilliant Blue solution was then washed off the gel with a Coomassie Brilliant Blue eluent (ethanol:acetic acid:water = 1:2:7), and a mobile phone photo was taken.

[0633] The results are shown in Figures 2-6. As can be seen from Figures 2-6, the absorption of the fluorescent probe ALPIN-6 with "one-touch, three-response" of the present invention red-shifted after reacting with ALP, and the fluorescence intensity increased. In the presence of sufficient nucleophile β-ME, the absorption and fluorescence changes were more pronounced (Figures 2, 3). HPLC verification also confirmed that the probe ALPIN-6 reacted with the ALP enzyme (Figure 5). In the specificity verification, only the probe and ALP enzyme group achieved a significant fluorescence enhancement (Figure 5). In the protein labeling effect experiment, the labeling effect of the probe ALPIN-6 after reacting with the enzyme was the best (Figure 6). (In Figure 6, M: protein marker; 1: ALP; 2: ALP + ALPIN-6; 3: ALP + ALPIN-5; 4: BSA; 5: BSA + ALPIN-6; 6: ALP + BSA + ALPIN-6).

[0634] 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.

[0635] Test Example 2: Biological Experimental Verification of Near-Infrared Probe ALPIN-6

[0636] Test Example 2.1: Comparison of Imaging of Alkaline Phosphatase (ALP)-Overexpressing HeLa Cells and ALP-Weakly Expressing HEK293 Cells Using Near-Infrared Fluorescent Probes ALPIN-6, ALPIN-5, and ALPIN-4 ( FIG. 7 )

[0637] Experimental conditions: 1 μM of the fluorescent probes ALPIN-6, ALPIN-5, and ALPIN-4 were incubated with HeLa cells (overexpressing ALP on their cell membranes) and normal HEK293 cells (underexpressing ALP) in culture dishes for 30 minutes at 37°C. Alternatively, HeLa cells were pretreated with the alkaline phosphatase inhibitor Na₃VO₄ for 1 hour and then incubated with the probes for 30 minutes. The probe-containing culture medium was then discarded, and the cells were washed once with PBS buffer (pH 7.4). 1 mL of phenol red-free DMEM was added, and the cells were imaged using a confocal fluorescence microscope (Cy 5.5 channel).

[0638] The experimental results are shown in Figure 7. As shown in Figure 7, the "one-touch, three-response" fluorescent probe ALPIN-6 of the present invention not only achieves more pronounced fluorescence imaging of ALP-overexpressing cells, compared to the conventional self-anchoring probe ALPIN-5 (structure shown in Figure 7) and the conventional fluorescent probe ALPIN-4 (structure shown in Figure 7), but also exhibits a more pronounced fluorescence signal than that of an ALP-low-expressing cell line (HEK293). Furthermore, in the ALP inhibitor group experiment, the fluorescence signal of HeLa cells labeled by the fluorescent probe ALPIN-6 was weakened, indicating that the initiation of the fluorescent probe's labeling depends on the specific hydrolysis of phosphate bonds by alkaline phosphatase.

[0639] Experimental conclusion: After the fluorescent probe ALPIN-6 reacts with the ALP enzyme in the cells, it can have a better cell enrichment effect, reduce the diffusion of the fluorescent probe in the cell imaging experiment, reduce the background signal, and greatly improve the detection sensitivity.

[0640] Test Example 2.2: Real-time Imaging of HeLa Cells Using the Near-Infrared Fluorescent Probe ALPIN-6 ( FIG. 8 )

[0641] Experimental conditions: Cultured HeLa cells were plated in a confocal dish, 0.1 μM fluorescent probe ALPIN-6 was added to the HeLa cells, and cell imaging was performed at different time periods using a confocal fluorescence microscope (Cy 5.5 channel).

[0642] The experimental results are shown in Figure 8. As shown in Figure 8, when real-time imaging of HeLa cells using the "one-touch, three-response" fluorescent probe ALPIN-6 occurs, a fluorescent signal rapidly appears on the cell membrane, consistent with the expression of the ALP enzyme on the HeLa cell membrane. Over time, the fluorescent signal gradually appears inside the cell and becomes increasingly stronger. At 20 minutes, the fluorescent signal intensity on the cell surface is more than 2000-fold higher than the background fluorescence.

[0643] Experimental Conclusion: The fluorescent probe ALPIN-6 can visualize ALP enzyme activity in HeLa cells in real time. This is because the probe switches from hydrophilic to lipophilic properties upon enzyme activation, enhancing cellular uptake. Furthermore, the self-anchoring of the methylene benzoquinone limits the penetration and diffusion of the activated fluorescent signal, significantly increasing the accumulation of the fluorescent signal in tumor cells and improving the probe's ability to visualize enzyme activity in real time.

[0644] Test Example 2.3: Specific Imaging of HeLa and HEK293 Mixed Cells by Near-Infrared Fluorescent Probe ALPIN-6 ( FIG. 9 )

[0645] Experimental conditions: Cultured HeLa and HEK293 cells (stable expression of green fluorescent protein) were plated in a confocal dish. 1 μM of the fluorescent probe ALPIN-6 was added to the mixed cells. After incubation in a 37°C incubator for 20 minutes, the cells were imaged using a fluorescence microscope.

[0646] The experimental results are shown in FIG9 . As can be seen from FIG9 , HEK293 cells stably transfected with yellow fluorescent protein only have yellow fluorescence signals but no red fluorescence. Red fluorescence only appears in HeLa cells without green fluorescence.

[0647] Experimental conclusion: After mixing ALP-overexpressing HeLa cells and ALP-weakly expressing HEK293 cells, the fluorescent probe ALPIN-6 can specifically image ALP-overexpressing HeLa cells. This is due to the "one-touch, three-response" design strategy that can achieve great enrichment of the probe at the detection site.

[0648] Test Example 2.4: Real-time Imaging of HeLa Cell-Bearing Tumor Mice Using the Near-Infrared Fluorescent Probe ALPIN-6 (Figures 10-13)

[0649] Experimental conditions:

[0650] Construction of HeLa tumor model in nude mice:

[0651] (1) Preparation of HeLa cells: The culture and passage of cells were performed in the same manner as before. After digestion and centrifugation of the cultured cells, they were washed twice with 1 mL of PBS each time. After washing, the cells were centrifuged and the supernatant was discarded. DMEM without FBS was added and mixed. The cells were counted and then matrigel was added. After mixing, the cells were placed in an ice bath for later use.

[0652] (2) Implantation of HeLa cells in nude mice: BALB / c female nude mice aged 4 to 6 weeks were implanted subcutaneously on the right hind thigh using a 1 mL sterile medical syringe containing 4×10 HeLa cells. 6 200 μL of a mixture of DMEM (without FBS) and Matrigel (1:4) was added. Grow the cells at 25°C for 2 to 3 weeks with self-sufficiency of water and food until the tumors grew to approximately 0.1 cm. 3 Animal imaging experiments were performed.

[0653] (3) In vivo imaging of HeLa tumor model nude mice by tail vein injection of probe: First, the experimental mice were divided into 3 groups, 3 mice in each group, and the whole body background fluorescence imaging (λ) of 6 nude mice was collected using IVIS Lumina XRMS Series III live animal imaging system. ex =660nm,λ em=710 nm). The fluorescent probe ALPIN-6 or ALPIN-4 was then prepared into a 40 μM PBS solution. 200 μL of the prepared probe solution was injected into the tail vein of tumor model mice using an insulin syringe. Whole-body fluorescence data of the experimental model mice were then collected using a live animal imaging device at six 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 Na3VO4 solution was first injected orally into the tumor. Half an hour later, the same concentration of the probe ALPIN-6 was injected, and imaging was performed over the same time period.

[0654] (4) Anatomical tissue imaging of nude mice with HeLa tumor model: 36 hours after the probe was injected into the tail vein of the nude mice, the mice were killed by cervical dislocation, and the tumors, heart, lungs, liver, spleen, and kidneys were removed by dissection, and fluorescence imaging of the organs and tissues was performed.

[0655] The experimental results are shown in Figures 10-13 and Table 2. As shown in Figures 10-13, a clear fluorescent signal appeared in the tumors of the ALPIN-6 group after intravenous injection, and this signal persisted until 36 hours later. In contrast, the fluorescent signal in the tumors of the group pre-injected with the inhibitor was significantly reduced. The control group, the ALPIN-4 group, also showed a fluorescent signal at 1 hour, but it was significantly weaker than that of the ALPIN-6 group and disappeared after 2 hours. This result is consistent with the fluorescent signal observed in the tumors of tumor-bearing mice after autopsy.

[0656] Table 2

[0657] Experimental conclusion: In a highly dynamic living animal system, ALPIN-6 has better tumor signal enrichment and retention effects, improves the signal intensity of living tumor detection, is beneficial to the detection of living tumor tissue, and plays a very important role in surgical navigation and resection of tumor tissue.

[0658] Test Example 3: Biological Experimental Verification of Near-Infrared Photosensitizer ALPIN-6I

[0659] Test Example 3.1: Calculation of the enrichment content of the near-infrared photosensitizer ALPIN-6I in HeLa cells

[0660] Experimental conditions: 4 μM, 1.5 mL photosensitizer ALPIN-6I was incubated with HeLa cells (100,000) that overexpressed ALP on the cell membrane surface in a 37°C incubator for 1 hour, then the cells were collected and incubated, washed twice with phenol red-free DMEM, the washing solution was collected, and 200 μL RIPA cell lysis buffer was added. After the cells were fully lysed, the lysate was collected. ALP enzyme and BSA were added to the collected incubation and washing solutions to ensure that the probes retained in the solution could be fully activated. The fluorescence intensity was then measured using a fluorimeter. Three groups were tested in parallel, and the probe concentration was calculated based on the standard curve. The photosensitizer concentration was calculated in the same way for the cell lysate, and then based on the volume of the cells (the volume of a single cell is 2.6×10 3 μm 3 =2.6×10 -12 L) Calculate the concentration in cells.

[0661] The experimental results are shown in Figure 14. As shown in Figure 14, the photosensitizer concentration in HeLa cells was as high as 2.4 mM, while the total probe concentration in the cell incubation and wash solutions was 0.00087 mM. The fluorescence signal intensity in the cells was over 2000 times greater than the photosensitizer concentration in the incubation solution. Furthermore, after incubation with the photosensitizer and centrifugation, the cell pellet collected showed a distinct blue coloration for ALPIN-6I, while no blue fluorescence was observed for the control photosensitizer, ALPIN-4I.

[0662] Experimental conclusion: The photosensitizer ALPIN-6I has a very high enrichment effect in ALP-overexpressing HeLa cells, which is very beneficial to improving the photodynamic therapy effect of the photosensitizer.

[0663] Test Example 3.2: Effect of photosensitizer ALPIN-6I on ROS production in HeLa cells

[0664] Experimental conditions: HeLa cells or HEK293 cells were incubated with the photosensitizer ALPIN-6I (0, 1, 2, 4 μM) or ALPIN-4I (4 μM) and the ROS detection probe H2DCFDA (10 μM) in a 37°C incubator for 30 minutes, washed twice with PBS, and then irradiated with 660 nm, 36 mW light for 2 minutes, and fluorescence imaging was performed using a fluorescence microscope.

[0665] The experimental results are shown in Figure 15. As shown in Figure 15, as the concentration of the photosensitizer ALPIN-6I increases, the green fluorescence of DCF increases, indicating greater ROS production. In the ALPIN-6I group, no green fluorescence was produced without illumination; nor was green fluorescence produced in the negative HEK293 cell group. Furthermore, under the same conditions, the green fluorescence in the control photosensitizer group, ALPIN-4I, was much weaker than that in ALPIN-6I.

[0666] Experimental Conclusions: The photosensitizer ALPIN-6I is specifically activated in ALP-overexpressing HeLa cells, but not in negative cells. It produces ROS only under light conditions. Furthermore, the efficiency of ROS production is significantly higher than that of the control photosensitizer, ALPIN-4I. This is attributed to the accumulation of the photosensitizer ALPIN-6I in tumor cells.

[0667] Test Example 3.3: Effect of photosensitizer ALPIN-6I in killing tumor cells

[0668] Experimental conditions: After incubating HeLa cells or HEK293 cells with the photosensitizer ALPIN-6I or ALPIN-4I (4 μM) in a 37°C incubator for 1 hour, the incubation medium was aspirated and new FBS-containing culture medium was added. The cells were then irradiated with 660 nm, 36 mW light for 10 minutes and cultured in a 37°C cell culture incubator for 24 hours. The cells were stained with the Bio-Time Live-Dead Cell Staining Kit and then imaged using a fluorescence microscope.

[0669] The experimental results are shown in Figure 16. As shown in Figure 16, only the HeLa cells in the ALPIN-6I plus illumination group were able to stain with PI (red indicates cell death). In contrast, the control probe ALPIN-4I plus illumination group and the PBS plus illumination group only stained with AM (green indicates live cells). Similar results were observed in the negative HEK293 cells in the illumination group and the ALPIN-6I no illumination group.

[0670] Experimental conclusion: Only in the presence of both ALP and light can the photosensitizer ALPIN-6I be activated and kill tumors. Moreover, under the same conditions, the photodynamic therapy effect of ALPIN-6I is better than that of the control photosensitizer ALPIN-4I.

[0671] Test Example 3.4: Effect of photosensitizer ALPIN-6I in specifically killing tumor cells in mixed cells ( FIG. 17 )

[0672] Experimental conditions: The photosensitizer ALPIN-6I (4 μM) was mixed with HeLa cells and HEK293 cells (stable expression of yellow fluorescent protein) and incubated in a 37°C incubator for 30 minutes. The incubation medium was then aspirated and new FBS-containing culture medium was added. The cells were then irradiated with 660 nm, 36 mW light for 10 minutes and cultured in a 37°C cell culture incubator for 24 hours. The cells were stained with PI (dead cells) using the Beyotime Live-Dead Cell Staining Kit, and fluorescence imaging was performed using a fluorescence microscope.

[0673] The experimental results are shown in FIG17 . As can be seen from FIG17 , only HeLa cells can be stained red by PI, while HEK293 cells stably expressing yellow fluorescent protein cannot be stained by PI, which indicates that HeLa cells are killed, while HEK293 cells are alive.

[0674] Experimental Conclusion: In a mixed cell population, the photosensitizer ALPIN-6I specifically kills HeLa tumor cells overexpressing ALP, while having no effect on normal cells. This is because the photosensitizer ALPIN-6I, after being activated by the enzyme, remains within the tumor cells and does not diffuse into the solution and be taken up by normal cells.

[0675] Test Example 3.5: Real-time imaging of HeLa cell-bearing tumor mice using near-infrared photosensitizer ALPIN-6I

[0676] Experimental conditions: First, the experimental tumor-bearing mice were divided into 3 groups, 3 in each group, and the whole-body background fluorescence images (λ ex =660nm,λ em =710 nm). The photosensitizer ALPIN-6I or ALPIN-4I was then prepared into a 40 μM PBS solution. 200 μL of the prepared probe solution was injected into the tail vein of tumor model mice using an insulin syringe. Whole-body fluorescence data of the experimental model mice were then collected using a live animal imaging device at six different time points (1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, and 24 hours). For the inhibitor group, 50 μL of a 10 mM Na3VO4 solution was first injected into the tumor in situ. Half an hour later, the probe ALPIN-6I at the same concentration was injected, and imaging was performed over the same time period.

[0677] The experimental results are shown in Figure 18. As shown in Figure 18, after intravenous injection of the probe, a clear fluorescent signal appeared on the tumor in the ALPIN-6I group 1 hour after injection, and the signal remained significant up to 24 hours after injection. In contrast, the fluorescent signal in the tumor in the group pre-injected with the inhibitor was significantly weakened. The control probe ALPIN-4I group also showed a fluorescent signal at 1 hour, but it was significantly weaker than that in the ALPIN-6I group, and the fluorescent signal disappeared after 2 hours.

[0678] Experimental conclusion: In a highly dynamic living animal system, ALPIN-6I has better tumor signal enrichment and retention effects.

[0679] Test Example 3.6: Photodynamic therapy of HeLa cell tumor-bearing mice using near-infrared photosensitizer ALPIN-6I

[0680] Experimental conditions: First, the experimental tumor-bearing mice were divided into 3 groups, 4 mice in each group, and the photosensitizer 1.1 mg kg was injected into the tail vein. -1 One hour later, the mice were irradiated with 660 nm, 0.5 W light for 10 minutes, and again after 24 hours. Then, the photosensitizer was injected again 48 hours later, and the mice were irradiated with light again after 1 hour and again after 72 hours. The tumor-bearing mice were then allowed to recover on their own for 15 days.

[0681] The experimental results are shown in Figures 19-21 and Table 3. As shown in Figures 19-21, the tumors of the tumor-bearing mice in the ALPIN-6I group treated with photodynamic therapy completely disappeared after 4 days of treatment, and the tumor-bearing mice recovered and no tumor recurrence was observed after 18 days of treatment, while the tumors of the tumor-bearing mice in the control group did not disappear and gradually increased in size.

[0682] Table 3

[0683] Experimental conclusion: In a highly dynamic living animal system, ALPIN-6I has the ability to target tumor tissue and can achieve enrichment in tumor tissue, enhancing the effect of photodynamic therapy, and can completely eliminate tumor tissue without recurrence.

[0684] Test Example 4: Biological Experimental Verification of Near-Infrared Fluorescence and Photoacoustic Probe ALPIN-7

[0685] Test Example 4.1: Comparison of imaging of alkaline phosphatase (ALP)-overexpressing HeLa cells and ALP-weakly expressing HEK293 cells using near-infrared fluorescence and photoacoustic probes ALPIN-7 and ALPIN-8

[0686] Experimental conditions: 5 μM of the fluorescent probes ALPIN-7 and ALPIN-8 (structures shown in Figure 22) were incubated in culture dishes with HeLa cells overexpressing ALP on their cell membranes and HEK293 cells, normal tissue cells with low ALP expression, for 30 minutes at 37°C. Alternatively, HeLa cells were pretreated with the alkaline phosphatase inhibitor Na₃VO₄ for 1 hour and then incubated with the probes for 30 minutes. The probe-containing culture medium was then discarded, and the cells were washed once with PBS buffer (pH 7.4). 1 mL of phenol red-free DMEM was added, and the cells were imaged using a confocal fluorescence microscope (Cy 5.5 channel).

[0687] The experimental results are shown in Figure 22. As shown in Figure 22, the ALPIN-7 probe not only achieved more pronounced fluorescence imaging of ALP-overexpressing cells compared to the control probe ALPIN-8, but also exhibited a more pronounced fluorescence signal than the ALP-low-expressing cell line (HEK293). Furthermore, in the ALP inhibitor group, the fluorescence signal of HeLa cells labeled with the ALPIN-7 fluorescent probe was weakened, indicating that the initiation of fluorescent probe labeling depends on the specific hydrolysis of phosphate bonds by alkaline phosphatase.

[0688] Experimental conclusion: After the fluorescent probe ALPIN-7 reacts with the ALP enzyme in the cells, it can have a better cell enrichment effect, reduce the diffusion of the fluorescent probe in the cell imaging experiment, reduce the background signal, and greatly improve the detection sensitivity.

[0689] Test Example 4.2: Real-time Imaging of HeLa Cells Using the Near-Infrared Fluorescent Photoacoustic Probe ALPIN-7

[0690] Experimental conditions: Cultured HeLa cells were plated in a confocal dish, 1 μM fluorescent probe ALPIN-7 was added to the HeLa cells, and cell imaging was performed at different time periods using a confocal fluorescence microscope (Cy 5.5 channel).

[0691] The experimental results are shown in Figure 23. As shown in Figure 23, when the ALPIN-7 probe was used to image HeLa cells in real time, a fluorescent signal first appeared rapidly on the cell membrane, consistent with the expression of the ALP enzyme on the HeLa cell membrane. Over time, the fluorescent signal gradually appeared inside the cell, and the intensity of the fluorescent signal inside the cell increased with time.

[0692] Experimental Conclusion: The fluorescent probe ALPIN-7 can visualize ALP enzyme activity in HeLa cells in real time. This is because the probe switches from hydrophilic to lipophilic properties upon enzyme activation, enhancing cellular uptake. Furthermore, the self-anchoring of the methylene benzoquinone limits the penetration and diffusion of the activated fluorescent signal, significantly increasing the accumulation of the fluorescent signal in tumor cells and improving the probe's ability to visualize enzyme activity in real time.

[0693] Test Example 4.3: Real-time imaging of HeLa cell-bearing tumor mice using near-infrared fluorescent photoacoustic probe ALPIN-7

[0694] Experimental conditions:

[0695] Construction of HeLa tumor model in nude mice:

[0696] (1) Preparation of HeLa cells: The culture and passage of cells were performed in the same manner as before. After digestion and centrifugation of the cultured cells, they were washed twice with 1 mL of PBS each time. After washing, the cells were centrifuged and the supernatant was discarded. DMEM without FBS was added and mixed. The cells were counted and then matrigel was added. After mixing, the cells were placed in an ice bath for later use.

[0697] (2) Implantation of HeLa cells in nude mice: BALB / c female nude mice aged 4 to 6 weeks were implanted subcutaneously on the right hind thigh using a 1 mL sterile medical syringe containing 4×10 HeLa cells. 6 200 μL of a mixture of DMEM (without FBS) and Matrigel (1:4) was added. Grow the cells at 25°C for 2 to 3 weeks with self-sufficiency of water and food until the tumors grew to approximately 0.1 cm. 3 Animal imaging experiments were performed.

[0698] (3) In vivo imaging of HeLa tumor model nude mice by tail vein injection of probe: First, the experimental mice were divided into 3 groups, 3 mice in each group, and the whole body background fluorescence imaging (λ) of 6 nude mice was collected using IVIS Lumina XRMS Series III live animal imaging system. ex =660nm,λ em =710 nm). The fluorescent probe ALPIN-7 or ALPIN-8 was then prepared into a 40 μM PBS solution. 200 μL of the prepared probe solution was injected into the tail vein of tumor model mice using an insulin syringe. Whole-body fluorescence data of the experimental model mice were then collected using a live animal imaging device at six different time points (1 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 48 h). In the inhibitor group, 50 μL of a 10 mM Na3VO4 solution was first injected into the tumor in situ. Half an hour later, the probe ALPIN-7 at the same concentration was injected, and imaging was performed over the same time period.

[0699] (4) Anatomical tissue imaging of nude mice with HeLa tumor model: 36 hours after the probe was injected into the tail vein of the nude mice, the mice were killed by cervical dislocation, and the tumors, heart, lungs, liver, spleen, and kidneys were removed by dissection, and fluorescence imaging of the organs and tissues was performed.

[0700] The experimental results are shown in Figures 24-26. As shown in Figures 24-26, a clear fluorescent signal appeared in the tumors of the ALPIN-7 group after intravenous injection of the probe, and this signal persisted until 36 hours later. In contrast, the fluorescent signal in the tumors of the group pre-injected with the inhibitor was significantly reduced. The control probe ALPIN-8 group also showed a fluorescent signal at 1 hour, but it was significantly weaker than that of the ALPIN-7 group and disappeared after 2 hours. This result is consistent with the fluorescent signal observed in the tumors of tumor-bearing mice after dissection.

[0701] Experimental conclusion: In a highly dynamic living animal system, ALPIN-7 has better tumor signal enrichment and retention effects, improves the signal intensity of living tumor detection, is beneficial to the detection of living tumor tissue, and plays a very important role in surgical navigation and resection of tumor tissue.

[0702] Test Example 5: UV absorption and fluorescence spectra of enzymatic hydrolysis

[0703] Experimental conditions: Probes BIN-3 and BIN-2 were prepared into a 5 μM working solution in PBS (pH = 7.4), the final concentration of β-lactamase TEM-1 was 100 nM, the final concentration of nucleophile β-mercaptoethanol was 10 mM, the reaction system was 100 μL, and incubated at 37°C for 15 minutes. Photosensitizers BIN-3I and BIN-2I were prepared into a 10 μM working solution in PBS (pH = 7.4), the final concentration of β-lactamase TEM-1 was 100 nM, the final concentration of nucleophile β-mercaptoethanol was 100 μM, the reaction system was 100 μL, and incubated at 37°C for 15 minutes. Subsequently, all UV absorption spectra were measured at room temperature using a Shimadzu UV spectrophotometer with a spectral range of 400-800 nm. All fluorescence spectra were measured using a fluorimeter with an excitation setting of 680 nm and an emission spectrum within the range of 690-850 nm.

[0704] Experimental results: The UV absorption spectra of the near-infrared probe are shown in Figures 27 and 28. The absorption of the main probe BIN-3 and the control probe BIN-2 red-shifts after enzymatic hydrolysis. The absorption spectra of the main probes also red-shift after enzymatic hydrolysis and attack by affinity reagents. At the same time, the absorption spectra of the main probes do not change after incubation with nucleophiles. The fluorescence spectra of Figures 29 and 30 show that the fluorescence of the main probes BIN-3 and the control probes BIN-2 turns on after enzymatic hydrolysis. The fluorescence of the main probe BIN-3 is further enhanced after being attacked by nucleophiles after enzymatic hydrolysis. The UV absorption spectra of the photosensitizers are shown in Figures 31 and 32. The absorption spectra of the main photosensitizer BIN-3I and the control photosensitizer BIN-2I change after enzymatic hydrolysis. At the same time, the absorption spectra of the main photosensitizers do not change after incubation with nucleophiles. The fluorescence spectra results, shown in Figures 33 and 34, show that the primary photosensitizer BIN-3I and the control photosensitizer BIN-2I exhibit fluorescence upon enzymatic hydrolysis. Furthermore, the fluorescence of the primary photosensitizer BIN-3I further intensifies upon attack by a nucleophile following enzymatic hydrolysis. This indicates that, by mimicking the in vivo dynamics through the addition of a large amount of the affinity reagent β-mercaptoethanol, the enzymatic hydrolysis of the fluorescent probe BIN-3 / photosensitizer BIN-3I forms a quinone methylene intermediate, which is then attacked by a large amount of nucleophiles, leading to a secondary fluorescence onset.

[0705] Test Example 6: HPLC analysis of enzymatic hydrolysis

[0706] Experimental conditions: The probe was prepared into a 5 μM working solution in PBS (pH = 7.4), the photosensitizer was prepared into a 10 μM working solution in PBS (pH = 7.4), the final concentration of β-lactamase TEM-1 was 100 nM, the final concentration of nucleophilic reagent β-mercaptoethanol was 10 mM, the reaction system was 100 μL, incubated at 37°C for 15 minutes, and the hydrolysis was detected by HPLC at a wavelength of 660 nm.

[0707] The experimental results showed that the main probe BIN-3 could be hydrolyzed by β-lactamase TEM-1. The HPLC peak shape of the main probe BIN-3 after enzymatic hydrolysis was a series of peaks, indicating that an unstable methylene benzoquinone intermediate was formed at this time. After enzymatic hydrolysis, in the presence of sufficient nucleophilic reagent β-mercaptoethanol, a single stable peak was formed. This peak was verified by mass spectrometry to be a stable structure covalently formed by the thiol group and the methylene benzoquinone.

[0708] The experimental results showed that the control probe BIN-2 and the control probe BIN-2 could be hydrolyzed by β-lactamase TEM-1, and the hydrolysis product was compound 7 The photosensitizer results showed that, similar to the probe, the main photosensitizer BIN-3I formed a series of irregular peaks after enzymatic hydrolysis, forming an unstable methylene benzoquinone intermediate. However, in the presence of sufficient β-mercaptoethanol after enzymatic hydrolysis, a single peak was formed. The control photosensitizer BIN-2I was hydrolyzed by TEM-1 to form compound 14. It can be seen that, unlike the hydrolysis of the comparison / control probe / photosensitizer which directly obtains a hydrolysis product, the hydrolysis of the probe / photosensitizer of the present application is a double-opening process, which is first hydrolyzed by enzyme and then by the action of nucleophilic reagent, and finally converted into a single stable product.

[0709] Test Example 7: Enzyme Selective Recognition Experiment

[0710] Experimental conditions: The probe was prepared into a 5 μM working solution in PBS (pH = 7.4), and a series of enzymes and proteins were added. The reaction system was 100 μL and incubated at 37°C for 30 minutes. The fluorescence intensity of all groups was then tested using a microplate reader, with the excitation and emission wavelengths set to 680 nm and 720 nm, respectively.

[0711] The results are shown in Figure 35, which shows that the main probe BIN-3 and the control probe BIN-2 only respond to β-lactamase TEM-1, but have no response to other enzymes and proteins, indicating that the probe has good selectivity for β-lactamase and can avoid interference from other enzymes and proteins in the body.

[0712] Test Example 8: SDS-PAGE and near-infrared fluorescence dual-color scanning experiment

[0713] Experimental conditions: 12% SDS-PAGE separation gel and 5% stacking gel were prepared for standby use. The probe and photosensitizer were prepared into a 10 μM working solution in PBS (pH = 7.4) and incubated with different proteins at 37°C for 2 hours. The concentrations of β-lactamase TEM-1 and BSA were 5 μM and 10 μM, respectively. For the enzyme inhibitor group, the inhibitor avibactam (1 mM) was incubated with TEM-1 for 30 minutes before incubating with the probe. SDS-PAGE loading buffer was then added to the sample and mixed. 15 μL of sample was loaded per group and electrophoresed using a gel electrophoresis instrument. After completion, near-infrared fluorescence dual-color scanning was used to obtain fluorescent bands. Finally, the gel was stained with Coomassie Brilliant Blue and photographed using a gel imaging system.

[0714] The results are shown in Figures 36 and 37. The main probe BIN-3 / main photosensitizer BIN-3I showed red fluorescence after incubation with TEM-1, while the control probe BIN-2 / control photosensitizer BIN-2I showed no fluorescence after incubation with TEM-1 and the inhibitor group. In the group with the additional protein BSA, the main probe BIN-3 / main photosensitizer BIN-3I was also able to be labeled on the affinity protein BSA band after being activated by TEM-1, while the control probe BIN-2 / control photosensitizer BIN-2I did not have the function of labeling proteins. This shows that after the main probe BIN-3 / main photosensitizer BIN-3I is activated by TEM-1, the methylene benzoquinone intermediate after the recognition group leaves can be covalently linked to and labeled with proteins or surrounding affinity proteins.

[0715] Test Example 9: Cell / bacteria biocompatibility

[0716] Experimental conditions: Bacterial growth curve assay, CCK-8 assay and red blood cell hemolysis assay were used to detect the in vitro biocompatibility of the probe.

[0717] In the growth curve experiment of the probe on bacteria, the experiment was carried out in a clean bench. First, the Escherichia coli producing / non-producing β-lactamase was revived on the LB agar plate, and then a single clone colony was picked and placed in 1 mL of culture medium and cultured in a shaker at 37 ° C overnight. The next day, the bacterial solution was diluted 10 6 times, the time at this time is 0h, and then 50μM probe is added and placed on a shaker for culture. At regular intervals, a certain amount of bacterial solution is taken out for gradient dilution and then dropped on the agar plate. After culture overnight, the number of bacteria is counted to examine the survival of bacteria and judge the effect of the probe on bacterial growth.

[0718] The results showed that the growth trends of Escherichia coli producing / non-producing β-lactamase were roughly the same with and without the probe, indicating that the probe had good biocompatibility with bacteria.

[0719] In the CCK-8 experiment, the experiment was carried out in a clean bench. Human embryonic kidney cells (HEK293 cells) with good adhesion were digested with trypsin, collected in a centrifuge tube and centrifuged at 1000 rpm for 3 minutes. After centrifugation, the supernatant was removed and the cells were diluted to 1x10 5 Cells / mL was used as the working suspension, and 100 μL was added to each well of a transparent 96-well plate. The plate was cultured in a cell culture incubator for 24 hours until the cells adhered. After the culture medium was gently discarded with a pipette, different concentrations (10, 20, 40, and 100 μM) of culture medium containing the probe were added. At the same time, DMEM and cells without drug were used as blank and positive controls, respectively, and the plates were cultured in a cell culture incubator for another 24 hours. Then, according to the instructions of the CCK-8 kit, 10 μL of CCK-8 solution was added to each well, cultured at 37°C for 3 hours, and the absorbance at 450 nm was read using a microplate reader.

[0720] The results showed that the cell survival rate of the group with added probes was not lower than that of the group without added probes, indicating that the probes are highly safe for cells.

[0721] In the hemolysis assay, fresh Balb / C mouse red blood cells (RBCs) were washed with PBS buffer and then diluted to a 5% (v / v) RBC working solution. The compound was then serially diluted in PBS buffer, starting at a 200 μM concentration and followed by a dichotomous dilution from 200 μM to 3.125 μM. 50 μL of compound was then pipetted into a clear 96-well plate. 50 μL of RBC working solution was then added to the plate, bringing the final volume to 100 μL / well. PBS and 0.1% Triton X-100 treatment groups served as blank and positive controls, respectively. After incubation at 37°C for 1 hour, the plate was centrifuged at 3500 rpm for 5 minutes, and 80 μL of the supernatant from each well was transferred to a new clear well. The absorbance at 405 nm was measured using a microplate reader to calculate the percentage of hemolysis.

[0722] The experimental results showed that at an added concentration of 100 μM, the hemolysis rate of the probe was comparable to that of the blank group, indicating that the probe had no hemolytic toxicity to mouse red blood cells and had high biocompatibility.

[0723] Test Example 10: Probe Imaging Experiment on Bacteria

[0724] Experimental conditions: Bacterial experiments were performed in a clean bench. First, the bacteria producing / non-producing β-lactamases were revived on LB agar plates. Then, single colonies were picked and placed in 1 mL of culture medium. The culture was shaken at 37°C for 3 to 5 hours until the bacteria reached the logarithmic growth phase. The bacteria were collected in sterile EP tubes, centrifuged at 5000 rpm, washed twice with PBS, and resuspended in PBS at a concentration of 1 x 10 7CFU of bacteria were incubated with the main probe BIN-3 and the control probe BIN-2 at 37°C for 2 hours. Another group of cleaning probes was set up. After the incubation, the bacterial suspension was washed twice with PBS. The bacteria were then dropped onto a glass slide and imaged under a fluorescence microscope. The channel was Cy5.5, and the fluorescence of Cy5.5 was colored red.

[0725] Experimental Results: As shown in Figure 38, both the primary probe BIN-3 and the control probe BIN-2 produced red fluorescence on the bacteria after normal incubation. However, after pre-treatment with inhibitors, no fluorescence was observed. After subsequent washing with PBS, as shown in Figure 39, the primary probe BIN-3 group still exhibited fluorescence, while the control probe BIN-2 group showed almost no fluorescence. This demonstrates that the primary probe BIN-3 can label drug-resistant bacteria without washing, preventing their spread.

[0726] For the selective imaging experiment of the probe and mixed bacteria expressing β-lactamase and mCherry fluorescent protein, the steps of culturing bacteria are the same as above. The difference is that the two bacteria are mixed evenly before imaging, and then the probe is added and incubated with it at 37°C for 2 hours. After that, the bacteria are dropped on a glass slide and imaged under a fluorescence microscope. The channels are mCherry and Cy5.5, respectively. The fluorescence of mCherry is colored green and the fluorescence of Cy5.5 is colored red.

[0727] Experimental Results: As shown in Figure 40, in a mixed bacterial imaging experiment, the main probe BIN-3 clearly shows accurate labeling of resistant bacteria expressing β-lactamases, while no fluorescence signal is observed for bacteria expressing mCherry. However, the control probe BIN-2 group not only fluoresces on resistant bacteria but also on mCherry-expressing bacteria. This demonstrates that the main probe BIN-3 can eliminate interference from bacteria that do not express β-lactamases and covalently label them. In contrast, the control probe, lacking an anchoring group, is affected by the diffused fluorophore and exhibits fluorescence signals even on bacteria that do not express β-lactamases.

[0728] Test Example 11: Probe enrichment investigation

[0729] Experimental conditions: Bacterial experiments were performed in a clean bench. β-lactamase-producing Enterobacter cloacae was first revived on LB agar plates. Single colonies were then picked and placed in 1 mL of culture medium and cultured in a shaker at 37°C. Bacteria were collected in sterile EP tubes, centrifuged at 5000 rpm, washed twice with PBS, and resuspended in PBS at a concentration of 1 x 10 9After incubating CFU of bacteria with the main probe BIN-3 and the control probe BIN-2 at 37°C for 2 hours, the culture medium was collected and the fluorescence intensity of the culture medium was measured. The bacteria were resuspended in lysis buffer and the fluorescence intensity of the bacterial lysate was measured to infer the content of the probe in the bacteria.

[0730] The experimental results are shown in Figures 41-42. After incubation of the main probe BIN-3 with β-lactamase-expressing Enterobacter cloacae and rinsing with PBS, the bacteria appeared green after incubation with the main probe BIN-3, while the bacteria incubated with the control probe BIN-2 showed no other color. Fluorescence measurements of the bacterial supernatant and lysate were performed to estimate the probe concentrations in the bacteria and culture medium. Figures 41-42 show that the probe concentration in the bacteria expressing the main probe BIN-3 was over 1500 times that in the culture medium, significantly higher than the concentration in the culture medium. Despite the structural similarity between the control probe and the main probe, the concentration of the control probe BIN-2 was 150 times lower than that in the bacteria expressing the main probe BIN-3. Furthermore, analysis of the probe concentration in the culture medium revealed that the majority of the BIN-2 probe remained in the culture medium, rather than being enriched in drug-resistant bacteria like BIN-3. This demonstrates that the main probe can enrich bacteria, a property particularly useful for detecting expressing bacteria in highly dynamic environments, such as living animals.

[0731] Test Example 12: In vivo safety

[0732] Experimental Conditions: In vivo safety evaluation of the probes was conducted by measuring blood routine tests, blood biochemical parameters, and tissue staining. Female BALB / c mice, 4-6 weeks old, were obtained from the Shanghai Laboratory Animal Research Center. Mice were housed at 25°C with ample food and water. For tissue staining, mice were divided into three groups, each receiving saline, the primary probe BIN-3, and the control probe BIN-2 (1 μmol / kg) via tail vein injection. Twenty-four hours after saline or probe injection, mice were euthanized and autopsied. Organs (heart, lungs, liver, spleen, and kidneys) were sectioned and stained with hematoxylin and eosin (H&E). For blood routine tests, mice were divided into three groups, each receiving saline, BIN-3, and BIN-2 (1 μmol / kg) via tail vein injection. Twenty-four hours after injection, blood samples were drawn from each group and sent for blood routine testing.

[0733] Experimental Results: As shown in Figure 43, 24 hours after intravenous injection of BIN-3 or BIN-2, we examined a series of blood biochemical parameters in these mice, including white blood cell (WBC), red blood cell (RBC), hemoglobin, hematocrit, mean hematocrit, mean hemoglobin, mean hemoglobin concentration, platelet count, procalcitonin, total protein, albumin, and urea, as well as H&E staining of major organs. The results showed no significant differences between mice injected with BIN-2, BIN-3, and saline, demonstrating the high biocompatibility of these probes in mice.

[0734] Test Example 13: Real-time imaging of bacteria in vivo

[0735] Experimental conditions: The in vivo imaging experiment of the probe was carried out by injecting the probe into the infected model mice, and then performing IVIS real-time fluorescence imaging on the mice. The mice were selected from 4-6 week old BALB / c female mice from Shanghai Experimental Animal Research Center. The mice were kept at a temperature of 25°C and provided with sufficient food and water during the feeding process. The real-time in vivo imaging groups of mice were set up with 5 mice per group, including the main probe BIN-3, the main probe BIN-3 and the inhibitor, and the control probe BIN-2. The probe concentration was 1 μmol / kg, and the inhibitor avibactam concentration was 100 mM. Escherichia coli encoding bla (5x 10 8 CFU) were injected into the right posterior thigh muscle of mice to establish a mouse infection model. One hour after the right posterior thigh muscle of mice was infected with bacteria, BIN-3 or BIN-2 (1 μmol / kg) was injected into the mice through the tail vein. In the inhibitor group, avibactam (100 mM) was incubated with bacteria at 37°C for 2 hours and then injected into the right posterior thigh muscle of mice, followed by tail vein injection of the probe BIN-3. Whole-body fluorescence images were acquired 1, 2, 3, 4, 6, 8, and 10 hours after probe injection using the IVIS Lumina XRMS Series III imaging system, and the fluorescence intensity was quantified by ROI measurement using Living Image software.

[0736] Experimental results: As shown in Figures 44-49 and Table 4, a clear fluorescence signal was observed at the site of bacterial infection in mice 1 hour after injection of the main probe BIN-3. Over the next few hours, the fluorescence intensity continued to increase, and 3 hours after injection of the main probe BIN-3, the signal-to-noise ratio (SBR) exceeded 10. Ten hours after probe injection, the fluorescence signal was still detectable at the infection site, with an SBR of approximately 9. The effective retention of the active fluorophore at the detection site expanded the observation window and improved the signal-to-noise ratio and detection sensitivity in imaging. In addition, pretreatment of the bacteria with the inhibitor avibactam effectively reduced the fluorescence intensity, indicating that the observed fluorescence was generated by BIN-3 hydrolysis mediated by bla. As a control, we also evaluated the in vivo imaging performance of the control probe BIN-2. This probe produced fluorescence at the bacterial infection site 1 hour after tail vein injection, but the fluorescence intensity and SBR were lower than those of BIN-3. Over time, BIN-2 fluorescence at the bacterial infection site rapidly decreased, and the SBR dropped to nearly 1 5 hours after probe injection, comparable to the background signal. In contrast, the self-anchored probe BIN-3 produced significantly stronger fluorescence and SBR at the infection site. In vitro fluorescence imaging demonstrated that the self-anchored main probe BIN-3 produced a significantly stronger fluorescence signal at the bacterially infected right thigh compared to the control probe BIN-2. Fluorescence signals in other major organs of the mouse, such as the heart, liver, spleen, lungs, and kidneys, remained very weak, demonstrating the high in vivo specificity of the main probe BIN-3 (Figure 46). When the thigh skin of mice injected with BIN-3 was peeled, a distinct blue-green coloration at the infection site was clearly visible to the naked eye. This coloration is characteristic of hemicyanine dyes, but not seen in mice treated with BIN-2 or the inhibitor (Figure 47). Quantification of fluorescence intensity in isolated organs also showed that the main probe group had the highest fluorescence intensity in the right thigh infected with bacteria, while the fluorescence intensity of the inhibitor group and the control probe group was significantly weaker than that of the main probe group. These results indicate that BIN-3 has remarkable retention efficiency at the activation site in highly dynamic animals, which may be due to the novel hydrophilicity-switchable anchor design based on methylene benzoquinone.

[0737] Table 4

[0738] Test Example 14: In vivo bacterial sensitivity imaging

[0739] Experimental conditions: The in vivo sensitivity imaging experiment of the probe was carried out by injecting the probe into mice infected with different numbers of myositis models, and then performing IVIS fluorescence imaging on the mice. The mice were 4-6 weeks old BALB / c female mice from the Shanghai Experimental Animal Research Center. The mice were kept at a temperature of 25°C, and were provided with sufficient food and water during the feeding process. The in vivo sensitivity imaging group of mice was set to 3 per group, and the concentration of the main probe BIN-3 was 1μmol / kg. Different concentrations were set for the four thighs of each mouse. The left upper limb was the normal saline control group, and the right upper limb, left lower limb and right lower limb were set to 5x 10 6 CFU, 5x 10 7 CFU and 5x 10 8 CFU were detected by tail vein probe 1 hour after bacterial injection. Mice were imaged with IVIS Lumina XRMS Series III live fluorescence imaging 5 hours later, and fluorescence intensity was quantified by ROI measurement using Living Image software.

[0740] Experimental Results: As shown in Figures 50-52 and Tables 5-6, one hour after injection of the main probe BIN-3, fluorescent signals of varying intensities were observed at sites infected with different bacteria in mice. The intensity of the fluorescent signal was proportional to the number of bacteria. Quantification of the fluorescence intensity at different sites revealed that the greater the number of bacteria, the higher the fluorescence intensity. This result was also confirmed by the signal-to-noise ratio of the infection site to the systemic signal.

[0741] Table 5

[0742] Table 6

[0743] Test Example 15: In vivo selective imaging of drug-resistant bacteria

[0744] Experimental conditions: The in vivo selective imaging experiment of the probe was carried out by injecting the probe into the bodies of mice infected with different bacteria, and then performing IVIS real-time fluorescence imaging on the mice. The mice were 4-6 weeks old BALB / c female mice from the Shanghai Experimental Animal Research Center. The mice were kept at a temperature of 25°C, and sufficient food and water were provided during the feeding process. Three groups of mice were set up for in vivo sensitivity imaging, and the concentration of the main probe BIN-3 was 1 μmol / kg. As shown in Figure 53, sensitive Escherichia coli (which expresses mCherry fluorescent protein) was injected into the left thigh of the mouse, and bla-positive Escherichia coli was injected into the right thigh. One hour after the bacterial injection, the main probe BIN-3 was injected, and the mice were subjected to IVIS Lumina XRMS Series III in vivo fluorescence imaging, using the near-infrared channel (λ ex / em =660 / 710nm) and mCherry (λ ex / em=580 / 620nm) channel to image it.

[0745] Experimental Results: As shown in Figures 54-55, the primary probe BIN-3 selectively labeled bla-expressing bacteria on the right thigh, while fluorescence was detected only in the mCherry channel on the left thigh. After excising the mouse thigh skin, the characteristic blue-green hue of the hemicyanine dye was clearly visible on the right thigh (infected with bla-expressing E. coli), but not on the left thigh (infected with mCherry-expressing E. coli). These results demonstrate the primary probe's excellent selectivity for bla-expressing bacteria in mice.

[0746] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A compound or a salt thereof, characterized in that The compound is as shown in formula I; In Formula I, R1 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 after being specifically hydrolyzed or specifically activated, the bond between R1 and X1 is disconnected and R1 leaves; X1 is a connecting bond, O or NH; L is a linking group that can leave on its own when R1 leaves; R2 is C 1-6 Alkyl or water-soluble groups; The FP portion is a fluorescent group or a photosensitive group.

2. The compound or salt thereof according to claim 1, characterized in that The compound satisfies one or more of the following conditions: (1) The term "capable of being specifically hydrolyzed by a biomacromolecular enzyme" means that when R1 is a group that can be specifically hydrolyzed by a biomacromolecular enzyme, the biomacromolecular enzyme can recognize the group and, under the action of the biomacromolecular enzyme, hydrolyze X1-R1 so that the bond between X1 and R1 is broken; (2) The biomacromolecule enzyme is alkaline phosphatase, γ-glutamyl transpeptidase, aminopeptidase, Fapα enzyme, Hepsin enzyme or β-lactamase; (3) The group that can be specifically hydrolyzed by a biomacromolecule enzyme is: an enzyme recognition group that can be specifically hydrolyzed by alkaline phosphatase, an enzyme recognition group that can be specifically hydrolyzed by γ-glutamyl transpeptidase, an enzyme recognition group that can be specifically hydrolyzed by aminopeptidase, an enzyme recognition group that can be specifically hydrolyzed by Fapα enzyme, an enzyme recognition group that can be specifically hydrolyzed by Hepsin enzyme, or an enzyme recognition group that can be specifically hydrolyzed by β-lactamase; (4) The ability to be specifically activated by disease microenvironment factors means that when R1 is a group that can be specifically activated by disease microenvironment factors, the bond between X1 and R1 can be broken under the action of disease microenvironment factors; (5) The disease microenvironmental factors are infection by resistant bacteria expressing β-lactamase or reactive oxygen species; (6) The group that can be specifically activated by disease microenvironment factors is a group that can be specifically activated by peroxide or a group that can be specifically activated by superoxide anion; (7) The water-soluble group is a water-soluble part of -L1-W1-L2-; wherein L1 and L2 are each independently no or a divalent linking group, and W1 is no or a linking group formed by a click chemistry reaction; Preferably, the water-soluble group is Preferably (8) The water-soluble group is a water-soluble group that can be connected to the carbonamide structure. When the compound is activated, the carbonamide is disconnected and the water-soluble group leaves.

3. The compound or salt thereof according to claim 2, characterized in that The compound satisfies one or more of the following conditions: (1) The biomacromolecule enzyme is alkaline phosphatase or β-lactamase; (2) The enzyme recognition group that can be specifically hydrolyzed by alkaline phosphatase is (3) The enzyme recognition group that can be specifically hydrolyzed by γ-glutamyl transpeptidase is (4) The enzyme recognition group that can be specifically hydrolyzed by aminopeptidase is (5) The enzyme recognition group that can be specifically hydrolyzed by Fapα enzyme is (6) The enzyme recognition group capable of being recognized by Hepsin is (7) The enzyme recognition group that can be specifically hydrolyzed by β-lactamase is Among them, R 1a Select from the following group: C 1-6 Alkyl, phenyl, 5- or 6-membered heteroaryl, C 1-4 Alkylene-phenyl, C 1-4 Alkylene-(5 or 6 membered heteroaryl), water soluble group, wherein the C 1-6 Alkyl, phenyl, 5- or 6-membered heteroaryl, C 1-4 Alkylene-phenyl, C 1-4 Alkylene-(5 or 6 membered heteroaryl) is optionally substituted with one or more halogen, C 1-6 Alkyl or halogenated C 1-6 Alkyl substitution; R 2a Selected from the following group: H, -OC 1-4 alkyl; R 3a Selected from the following group: H, C 1-6 Alkyl groups, monovalent cations (such as Na + ), wherein the C 1-6 The alkyl group is optionally substituted with one or more halogen, C 1-6 Alkyl or halogenated C 1-6 Alkyl substitution; R 4a and R 5a Each independently selected from the following group: H, C 1-4 Alkyl (such as methyl or ethyl); or R 4a and R 5a Together with the carbon atom to which it is attached, it forms C 3-4 Cycloalkyl; n is 0, 1, or 2; (8) The active oxygen is preferably peroxide or superoxide anion; (9) The group that can be specifically activated by peroxide is: (10) The group that can be specifically activated by superoxide anions is (11) L1 and L2 are each independently a divalent linking group that is absent or stable and non-cleavable in a cell or in vivo environment; (12)W1 is None or (13) The water-soluble part is selected from the following group: a group containing an anion-cation pair, a group containing one or more carboxyl groups, and a group containing one or more sulfonic acid groups.

4. The compound or salt thereof according to claim 3, characterized in that The compound satisfies one or more of the following conditions: (1)R 1a It is a water-soluble group; (2)R 2a Selected from the group consisting of: H, -OMe; Preferably, R 2a is H; (3)R 3a Selected from the following group: H, C 1-6 Alkyl, monovalent cation; Preferably, R 3a is H; (4)R 4a and R 5a Each independently selected from the following group: H, C 1-4 Alkyl (such as methyl or ethyl); Preferably, R 4a and R 5a is H; (5)n is 0; (6) L1 and L2 are each independently zero or C1-C4 alkylene; (7) The group containing one or more carboxyl groups is a group containing 2 or 3 carboxyl groups, for example (8) The group containing one or more sulfonic acid groups is a group containing 2 or 3 sulfonic acid groups, for example (9) The group containing an anion-cation pair is wherein a is 1, 2, 3 or 4, preferably 2 or 3.

5. The compound or salt thereof according to claim 1, characterized in that The compound satisfies one or more of the following conditions: (1) R1 is selected from the following group: Where R 1a , R 2a , R 3a , R 4a , R 5a and n are as defined in claim 3 or 4; (2) X1 is a connecting bond or O; (3) L is selected from the group consisting of: C1-C4 alkylene; (4) R2 is C 1-6 Alkyl or water-soluble groups; (4) The FP portion has a structure as shown in Formula II-1 or Formula II-2: in, a represents The location of the connection; b represents The location of the connection; R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu Select from the following group: -COOH, -N3, -NH2, -N + (C 1-6 Alkyl)3); R4, R5, R6 and R7 are each independently H or a heavy atom (such as I, Br); R8 is H, F, Cl, or Br; X2 is O or S; X3 is I, Br, Cl; or, The FP part has a structure as shown in Formula III-1 or III-2: in, a represents The location of the connection; b represents The location of the connection; R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu Select from the following group: -COOH, -N3, -NH2, -N + (C 1-6 Alkyl)3); R4, R5, R6 and R7 are each independently H or a heavy atom (such as I, Br); R8 is H, F, Cl, or Br; X2 is O or S; X3 is I, Br, or Cl; Alternatively, the FP portion has a structure as shown in Formula IV-1 or IV-2: in, a represents The location of the connection; b represents The location of the connection; X2 is O or S; X4 is O or NR3; R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu Select from the following group: -COOH, -N3, -NH2, -N + (C 1-6 Alkyl)3); R4, R5, R6 and R7 are each independently H or a heavy atom (such as I, Br); R8 is H, F, Cl, or Br; or, The FP portion has a structure as shown in Formula V-1 or V-2: in, a represents The location of the connection; b represents The location of the connection; X2 is O or S; X5 is O or S; R9 is NEt2, NPh2, or NCH3Ph; R8 is H, F, Cl, or Br; or, The FP portion has a structure as shown in Formula VI-1 or VI-2: in, a represents The location of the connection; b represents The location of the connection; X2 is O or S; X5 is O or S; R 10 and R 11 Each independently is C 1-6 Alkyl (such as Et) or Ph; R8 is H, F, Cl or Br.

6. The compound or salt thereof according to claim 5, characterized in that The compound satisfies one or more of the following conditions: (1) R1 is Where R 1a It is a water-soluble group; Preferably, R1 is (2) X1 is 0; (3) L is selected from the group consisting of: C1-C4 alkylene; wherein * represents the connection position with X1; (4) R2 is ethyl, Preferably, ethyl, (5) The FP portion has a structure as shown in Formula II-1 or Formula II-2: in, a represents The location of the connection; b represents The location of the connection; R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu Select from the following group: -COOH, -N3, -NH2, -N + (C 1-6 Alkyl)3); R4, R5, R6 and R7 are each independently H, Br or I; R8 is H, F, Cl, or Br; X2 is O or S; X3 is I, Br, Cl; Preferably, the FP portion is 7. The compound or salt thereof according to claim 1, characterized in that X1 is O or NH; R2 is a water-soluble group.

8. The compound or salt thereof according to claim 7, characterized in that The compound satisfies one or more of the following conditions: (1) R1 is selected from the following group: (2) L is selected from the group consisting of: (3) R2 is selected from the group consisting of: (4) The FP portion has a structure as shown in Formula II-1 or Formula II-2: in, a represents The location of the connection; b represents The location of the connection; R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu Select from the following group: -COOH, -N3, -NH2, -N + (C 1-6 Alkyl)3); R4, R5, R6 and R7 are each independently H or a heavy atom I, Br; R8 is H, F, Cl, or Br; X2 is O or S; X3 is I, Br, Cl; or, The FP part has a structure as shown in Formula III-1 or III-2: in, a represents The location of the connection; b represents The location of the connection; R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu Select from the following group: -COOH, -N3, -NH2, -N + (C 1-6 Alkyl)3); R4, R5, R6 and R7 are each independently H or a heavy atom (such as I, Br); R8 is H, F, Cl, or Br; X2 is O or S; X3 is I, Br, or Cl; Alternatively, the FP portion has a structure as shown in Formula IV-1 or IV-2: in, a represents The location of the connection; b represents The location of the connection; X2 is O or S; X4 is O or NR3; R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu is a modifiable functional group or a positively charged group (preferably, R Fu Select from the following group: -COOH, -N3, -NH2, -N + (C 1-6 Alkyl)3); R4, R5, R6 and R7 are each independently H or a heavy atom (such as I, Br); R8 is H, F, Cl, or Br; or, The FP portion has a structure as shown in Formula V-1 or V-2: in, a represents The location of the connection; b represents The location of the connection; X2 is O or S; X5 is O or S; R9 is NEt2, NPh2, or NCH3Ph; R8 is H, F, Cl, or Br; or, The FP portion has a structure as shown in Formula VI-1 or VI-2: in, a represents The location of the connection; b represents The location of the connection; X2 is O or S; X5 is O or S; R 10 and R 11 Each independently is C 1-6 Alkyl (such as Et) or Ph; R8 is H, F, Cl or Br.

9. The compound or salt thereof according to claim 7, characterized in that The compound is selected from Table 1 Table 1 10. The compound or salt thereof according to any one of claims 1 to 7, characterized in that The compound is shown in Formula IA; in n = 0, 1, or 2; R 1a Select from the following group: C 1-6 Alkyl, phenyl, 5- or 6-membered heteroaryl, C 1-4 Alkylene-phenyl, C 1-4 Alkylene-(5 or 6 membered heteroaryl), water soluble group, wherein the C 1-6 Alkyl, phenyl, 5- or 6-membered heteroaryl, C 1-4 Alkylene-phenyl, C 1-4 Alkylene-(5 or 6 membered heteroaryl) is optionally substituted with one or more halogen, C 1-6 Alkyl or halogenated C 1-6 Alkyl substitution; R 2a Selected from the following group: H, -OC 1-4 alkyl; R 3a Selected from the following group: H, C 1-6 Alkyl groups, monovalent cations (such as Na + ), wherein the C 1-6 The alkyl group is optionally substituted with one or more halogen, C 1-6 Alkyl or halogenated C 1-6 Alkyl substitution; R 4a and R 5a Each independently selected from the following group: H, C 1-4 Alkyl (such as methyl or ethyl); or R 4a and R 5a Together with the carbon atom to which it is attached, it forms C 3-4 Cycloalkyl; R 6a Select from the following group: C 1-6 Alkyl, a group containing a water-soluble portion, wherein the C 1-6 The alkyl group is optionally substituted with one or more halogen, C 1-6 Alkyl or halogenated C 1-6 Alkyl substitution; R 7a C 1-6 Alkyl, wherein the C 1-6 The alkyl group is optionally substituted with one or more halogen, C 1-6 Alkyl or halogenated C 1-6 Alkyl substitution; R 8a , R 9a , R 10a and R 11a Each is independently selected from the group consisting of: H, heavy atoms; It is a monovalent acid ion.

11. The compound or salt thereof according to claim 10, characterized in that The compound meets one or more of the following conditions: (1) The group containing the water-soluble portion is -L1-W1-L2-water-soluble portion; wherein L1 and L2 are each independently no or a divalent linking group, and W1 is no or a linking group formed by a click chemistry reaction; Preferably, the water-soluble part is selected from the group consisting of: a group containing an anion-cation pair, a group containing one or more carboxyl groups, and a group containing one or more sulfonic acid groups; More preferably, the part containing anion and cation pairs comprises: Wherein, a is 1, 2, 3 or 4; The group containing one or more carboxyl groups includes: The group containing one or more sulfonic acid groups includes: (2)R 1a and R 6a At least one of the groups is a group containing a water-soluble portion.

12. The compound or salt thereof according to claim 10, characterized in that The compound is selected from the group consisting of:

13. The compound or salt thereof according to any one of claims 1 to 12, characterized in that The compound satisfies one of the following schemes: Solution 1: The compound is as shown in formula I; In Formula I, R1 is a group that can be specifically hydrolyzed by a biomacromolecule enzyme; and after being specifically hydrolyzed, the bond between R1 and X1 is disconnected and R1 leaves; the biomacromolecule enzyme is alkaline phosphatase or β-lactamase; X1 is a connecting bond, O or NH; L is a linking group that can leave on its own when R1 leaves; R2 is C 1-6 Alkyl or water-soluble group; the water-soluble group is -L1-W1-L2-water-soluble part; L1 and L2 are each independently C1-C4 alkylene; W1 is The water-soluble part is selected from the group consisting of: A group containing 2 or 3 carboxyl groups, or a group containing 2 or 3 sulfonic acid groups, wherein a is 1, 2, 3 or 4; The FP part has a structure as shown in Formula II-1 or Formula II-2: in, a represents The location of the connection; b represents The location of the connection; R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu Select from the following group: -COOH, -N3, -NH2, -N + (C 1-6 Alkyl)3); R4, R5, R6 and R7 are each independently H, Br or I; R8 is H, F, Cl, or Br; X2 is O or S; X3 is I, Br, Cl; Option 2: The compound is as shown in formula I; In Formula I, R1 is Where R 1a , R 2a , R 3a , R 4a , R 5a and n is as defined in claim 3 or 4; X1 is a connecting bond or O; L is or C1-C4 alkylene; R2 is C 1-6 Alkyl or water-soluble group; the water-soluble group is -L1-W1-L2-water-soluble part; L1 and L2 are each independently C1-C4 alkylene; W1 is The water-soluble part is selected from the group consisting of: A group containing 2 or 3 carboxyl groups, or a group containing 2 or 3 sulfonic acid groups, wherein a is 1, 2, 3 or 4; The FP part has a structure as shown in Formula II-1 or Formula II-2: in, a represents The location of the connection; b represents The location of the connection; R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu Select from the following group: -COOH, -N3, -NH2, -N + (C 1-6 Alkyl)3); R4, R5, R6 and R7 are each independently H, Br or I; R8 is H, F, Cl, or Br; X2 is O or S; X3 is I, Br, Cl; Option 3: The compound is as shown in formula I; In Formula I, R1 is a group that can be specifically hydrolyzed by a biomacromolecule enzyme; and after being specifically hydrolyzed, the bond between R1 and X1 is disconnected and R1 leaves; the biomacromolecule enzyme is alkaline phosphatase; X1 is O or NH; L is a linking group that can leave on its own when R1 leaves; R2 is a water-soluble group; the water-soluble group is a -L1-W1-L2-water-soluble part; L1 and L2 are each independently C1-C4 alkylene; W1 is The water-soluble part is selected from the group consisting of: A group containing 2 or 3 carboxyl groups, or a group containing 2 or 3 sulfonic acid groups, wherein a is 1, 2, 3 or 4; The FP part has a structure as shown in Formula II-1 or Formula II-2: in, a represents The location of the connection; b represents The location of the connection; R3 is -C 1-6 Alkyl, -C 1-6 Alkyl-R Fu ; Among them, R Fu Select from the following group: -COOH, -N3, -NH2, -N + (C 1-6 Alkyl)3); R4, R5, R6 and R7 are each independently H, Br or I; R8 is H, F, Cl, or Br; X2 is O or S; X3 is I, Br, Cl; Option 4: The compound is as shown in formula I; In Formula I, R1 is X1 is 0; L is a linking group that can leave on its own when R1 leaves; R2 is a water-soluble group; the water-soluble group is a -L1-W1-L2-water-soluble part; L1 and L2 are each independently a C1-C4 alkylene group; W1 is The water-soluble part is a is 2 or 3; The FP portion has a structure as shown in Formula II-1: in, a represents The location of the connection; b represents The location of the connection; R3 is -C 1-6 alkyl; R4, R5, R6 and R7 are each independently H, Br or I; R8 is H, F, Cl, or Br; X2 is O or S; X3 is I, Br, Cl; Option 5: The compound is as shown in formula I; In Formula I, R1 is X1 is 0; L is R2 is a water-soluble group; the water-soluble group is a -L1-W1-L2-water-soluble part; L1 and L2 are each independently C1-C4 alkylene; W1 is The water-soluble part is a is 2 or 3; The FP portion is Option 6: The compound is shown in Formula IA; in n = 0, 1, or 2; R 1a Select from the following group: C 1-6 Alkyl, phenyl, 5- or 6-membered heteroaryl, C 1-4 Alkylene-phenyl, C 1-4 Alkylene-(5- or 6-membered heteroaryl), water-soluble group; R 2a Selected from the following group: H, -OC 1-4 alkyl; R 3a Selected from the following group: H, C 1-6 Alkyl, monovalent cation; R 4a and R 5a Each independently selected from the following group: H, C 1-4 alkyl; R 6a Select from the following group: C 1-6 Alkyl, water-soluble group; R 7a C 1-6 alkyl; R 8a , R 9a , R 10a and R 11a Each independently selected from the group consisting of H, Br or I; It is a monovalent acid ion; The water-soluble group is -L1-W1-L2-water-soluble part; L1 and L2 are each independently C1-C4 alkylene; W1 is The water-soluble part is or a group containing 2 or 3 carboxyl groups, a is 2 or 3; Option 7: The compound is shown in Formula IA; in n＝0； R 1a It is a water-soluble group; R 2a Selected from the following group: H, -OC 1-4 alkyl; R 3a Selected from the following group: H, C 1-6 Alkyl, monovalent cation; R 4a and R 5a Each independently selected from the following group: H, C 1-4 alkyl; R 6a Select from the following group: C 1-6 Alkyl, water-soluble group; R 7a C 1-6 alkyl; R 8a , R 9a , R 10a and R 11a Each independently selected from the group consisting of H, Br or I; It is a monovalent acid ion; The water-soluble group is -L1-W1-L2-water-soluble part; L1 and L2 are each independently C1-C4 alkylene; W1 is The water-soluble part is a is 2 or 3; Option 8: The compound is shown in Formula IA; in n＝0； R 1a It is a water-soluble group; R 2a is H; R 3a is H; R 4a and R 5a is H; R 6a Select from the following group: C 1-6 Alkyl, water-soluble group; R 7a C 1-6 alkyl; R 8a , R 9a , R 10a and R 11a Each independently selected from the group consisting of H, Br or I; It is a monovalent acid ion; The water-soluble group is -L1-W1-L2-water-soluble part; L1 and L2 are each independently C1-C4 alkylene; W1 is The water-soluble part is a is 2 or 3.

14. A composition, wherein The composition comprises: (1) the compound or a salt thereof according to any one of claims 1 to 13; and (2) A pharmaceutically or biologically acceptable carrier.

15. A detection kit, characterized in that: The kit comprises: the compound or salt thereof according to any one of claims 1 to 13 or the composition according to claim 14.

16. Use of a compound or a salt thereof according to any one of claims 1 to 13 or a composition according to claim 14, characterized in that: The use is one or more selected from the following groups: (a) For enzyme detection; (b) Used for the detection of disease microenvironmental factors (such as ROS); (c) for preparing a detection reagent for detecting an enzyme or a disease microenvironment factor (such as ROS); (d) used for cell imaging; (e) Used to prepare detection reagents for imaging analysis of cells.

17. A use of a compound or a salt thereof according to any one of claims 7 to 9, characterized in that: The use is one or more selected from the following groups: (f) for imaging of tumor tissue or cells; (g) for preparing a detection reagent for tumor tissue or cells; (h) Used in photodynamic therapy for tumors; (i) Used for preparing photosensitizing therapeutic agents for photodynamic therapy of tumors.

18. Use of a compound or a salt thereof according to any one of claims 10 to 12, characterized in that: The use is one or more selected from the following groups: (a) Used for the detection of β-lactamase; (b) used for the detection or imaging of drug-resistant bacteria; (c) for preparing a detection reagent for detecting or imaging drug-resistant bacteria; or, The use is one or more selected from the following groups: (a) Used to kill drug-resistant bacteria; (b) for use in the preparation of a medicament for killing drug-resistant bacteria or for treating or preventing a disease caused by infection with drug-resistant bacteria.

19. A method for imaging drug-resistant bacteria, the method comprising the steps of: incubating the compound or salt thereof as described in any one of claims 10 to 12 with an object, and obtaining an imaging image of the drug-resistant bacteria by an instrument.

20. A detection kit, characterized in that: The kit comprises: the compound or salt thereof according to any one of claims 1 to 13 or the composition according to claim 14.

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