A type of labeled compound containing multiple "anchor points" and its preparation method and application

Abstract

The present invention provides a highly efficient biomacromolecule covalent labeling compound (compound of formula I) containing multiple "anchor points" and its preparation method and application. The labeling reagent or compound of the present invention has an o, p-methylene benzoquinone (o, p-QM) precursor, which can generate methylene benzoquinone (QM) active intermediates multiple times, thereby achieving the purpose of multiple protein labeling, and can significantly reduce the inefficient labeling caused by quenching of QM active intermediates by other nucleophilic reagents, thereby achieving the purpose of "efficient and stable" labeling of biomacromolecules. It can specifically label tumor cells expressing alkaline phosphatase (ALP) and perform live cell imaging on them.

Description

Technical Field

[0001] The present invention belongs to the field of biomarkers and biomedicine, and specifically relates to a class of highly efficient biomacromolecule marker compounds containing multiple "anchor points" and a preparation method and application thereof. Background Art

[0002] Quinone methylidene (QMs) is a class of highly reactive active intermediates, and the most widely used structures in nature are o-quinone methylidene (o-QMs) and p-quinone methylidene (p-QMs). For example, many animals and plants use QMs as a means of defense; QMs participate in the anti-cancer process of doxorubicin and its derivative epirubicin. In addition, QMs are also very susceptible to attack by nucleophiles from life systems due to their extremely high electrophilicity, thus having a very strong potential to capture biological macromolecules and establish covalent bonds with them. Due to this property, QMs are also widely used in research in the field of life sciences to develop efficient and stable bioconjugation reagents. For example, as early as 1990, scientists introduced the chemical process of o-QMs active intermediates "capturing" and "anchoring" nucleophilic groups in life macromolecules into the working mechanism of glucosidase activity inhibitors. Subsequently, inhibitors, capture agents and their detection methods based on this strategy have also been further applied to the research and development of enzymes, antibodies and specific cell sorting. Since the material basis involved in life processes and the composition of organisms is both complex and numerous, this strategy is largely limited by the low specificity of QMs active intermediates. QMs, as "self-anchoring" imaging agents, have also been widely used in the activity detection of many important biological enzymes such as phosphatases, glycosidases, lipases, sulfatases, and β-lactamases. However, the current QM-based probe molecules have low labeling efficiency for biomacromolecules. And existing research has still not given a satisfactory answer.

[0003] In summary, there is an urgent need in the art to develop a biomacromolecule covalent labeling reagent with high efficiency and high sensitivity. Summary of the invention

[0004] The purpose of the present invention is to provide a biomacromolecule covalent labeling reagent with high efficiency and high sensitivity.

[0005] In the first aspect of the present invention, there is provided a compound of formula I,

[0006]

[0007] In Formula I,

[0008] A is selected from the following group: a targeting recognition group (preferably, phosphate, glycoside, deoxynucleoside, peptide compound), a leaving group (preferably, selected from: hydroxyl protecting group, amino protecting group, thiol protecting group), or a secondary or multiple release functional structure;

[0009] In the A group, the targeting recognition group is a group that can be recognized by the biomacromolecule and interact with the targeting recognition group to generate a phenolic hydroxyl group, a phenolic thiol group or an amino group; the leaving group refers to a group that is sensitive to light, heat, or chemical induction and can break the covalent bond between AX to generate a phenolic hydroxyl group, a phenolic thiol group or an amino group; the structure of the secondary or multiple release function refers to a group that can be hydrolyzed and release an o,p-QM intermediate after being activated by a trigger; and the structure of the secondary or multiple release function is shown in formula A

[0010]

[0011] In formula A, A' is selected from the following group: a target recognition group, a leaving group;

[0012] X is O, NH or S;

[0013] LG is a functional group that can form multiple "anchor points" with geminal difluoride (CHF2) and aromatic ring core;

[0014] L is a linking group;

[0015] R is selected from the following group: a reporter group, a therapeutic group; wherein the reporter group refers to a group that can be detected by fluorescence, radiation and / or magnetic signals, and the therapeutic group refers to a drug molecule and a drug precursor having a therapeutic function;

[0016] In another preferred embodiment,

[0017] A is selected from the following group: phosphate group (-H2PO3); glycoside; deoxynucleoside; hydroxyl, amino or thiol protecting group (preferably, a photosensitive protecting group; more preferably, and R" is selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl);

[0018] X is selected from the group consisting of O, NH, S;

[0019] LG is selected from the group consisting of F, Cl, Br, OAc, OCONHR b , R b SO2, -NR b 3 + ; and R b Each independently selected from the following group: C1-C6 alkyl, substituted or unsubstituted phenyl (preferably, C6H5NO2);

[0020] L is a linking group;

[0021] R is selected from the following group: a reporter group, a drug group; wherein the reporter group refers to a group that can be detected by fluorescence, radiation and / or magnetic signals, and the drug group refers to a drug molecule and a drug precursor having a therapeutic function;

[0022] Unless otherwise specified, the substitution refers to the replacement of one or more hydrogen atoms in the group by a substituent selected from the group consisting of halogen (preferably F, Cl, Br, I), C1-C6 alkyl, and C1-C6 haloalkyl.

[0023] In another preferred embodiment, L is -Wa-L 1 -Wb-; and Wb is connected to R; in the L group,

[0024] Wa and Wb are each independently selected from the group consisting of none, O, S, NR a ,CO,COO,SO,SO2,CO-NR a NR a -CO, SO-N(R a )、N(R a )-SO、NR a -COO, COO-NR a NR a -SO2, SO2-NR a , CS-NR a NR a -CS、N(R a )-CO-NR a 、-(CH2) 0、1或2 - a five- or six-membered ring containing 1 to 3 nitrogen heteroatoms -(CH2) 0、1或2 -(Preferably, );

[0025] L 1 is a linking group consisting of one or more unit structures selected from the group consisting of a substituted or unsubstituted C1-C4 alkylene group, a five- or six-membered ring containing 1-3 nitrogen heteroatoms (preferably, ), (substituted or unsubstituted C1-C2 alkylene)-O, (substituted or unsubstituted C1-C2 alkylene)-O-(substituted or unsubstituted C1-C2 alkylene), (substituted or unsubstituted C1-C2 alkylene)-S, (substituted or unsubstituted C1-C2 alkylene)-S-(substituted or unsubstituted C1-C2 alkylene), NR a 、CO、COO、CO-NR a NR a -CO;

[0026] R aEach is independently selected from the following group: H, C1-C6 alkyl, C1-C6 haloalkyl, or substituted or unsubstituted C3-C6 cycloalkyl (preferably, R a for H).

[0027] In another preferred embodiment, the drug group is derived from a drug selected from the following group: antibiotics (preferably, cephalosporins, vancomycin); avibactam; sunitinib, amantadine, acyclovir.

[0028] In another preferred embodiment, the radioactive group capable of being detected is 18 F-labeled group.

[0029] In another preferred embodiment, A is selected from the following group: phosphate group (-H2PO3), and R" is selected from the following group: C1-C6 alkyl, C1-C6 haloalkyl; preferably, A is a phosphate group.

[0030] In another preferred embodiment, X is O.

[0031] In another preferred embodiment, LG is selected from the following group: F, OCONHR b ; and R b Each is independently selected from the following group: C1-C6 alkyl (preferably Et, Me), substituted or unsubstituted phenyl (preferably C6H5NO2).

[0032] In another preferred embodiment, Wa is -(CH2) 0、1或2 - a five- or six-membered ring containing 1 to 3 nitrogen heteroatoms -(CH2) 0、1或2 -(Preferably, ).

[0033] In another preferred embodiment, Wb is selected from the following group: None, NR a -CO.

[0034] In another preferred embodiment, L 1 Selected from the group consisting of: -(CH2) n1 -NHCO-(CH2-O-CH2) n2 -(CH2) n1 -、-(CH2) n3 -、-(CH2) n1 -Ar 1 -(CH2) n1 -NHCO-(CH2-O-CH2) n2 -(CH2) n1 -.

[0035] In another preferred embodiment, R is a reporter group; preferably, R is a group capable of emitting fluorescence.

[0036] In another preferred embodiment, the group capable of emitting fluorescence is fluorescein or a fluorescence-enhancing dye.

[0037] In another preferred embodiment, R is selected from the following group:

[0038]

[0039] In another preferred embodiment, L is selected from the following group:

[0040] -(CH2) n1 -Ar 1 -(CH2) n1 -NHCO-(CH2-O-CH2) n2 -(CH2) n1 -NHCO-,

[0041] -(CH2) n1 -Ar 1 -(CH2) n3 -、

[0042] -CONH-(CH2) n1 -Ar 1 -(CH2) n1 -NHCO-(CH2-O-CH2) n2 -(CH2) n1 -NHCO-;

[0043] Wherein, n1=0, 1, 2 or 3; n2=1, 2, 3, 4 or 5; n3=2, 3, 4, 5, 6, 7, 8 or 9;

[0044] Ar 1 is a five- or six-membered ring containing 1 to 3 nitrogen heteroatoms (preferably, ).

[0045] In another preferred embodiment, L is selected from the following group:

[0046]

[0047] In another preferred embodiment, the compound is as shown in Formula IV,

[0048]

[0049] Wherein, LG, L, and R are as defined above.

[0050] In another preferred embodiment, A, X, LG, L and R are corresponding groups in the specific compounds in Table 1 (preferably, ALP-6 and ALP NIR-2).

[0051] In another preferred embodiment, the compound of formula I is selected from the following group:

[0052]

[0053] In a second aspect of the present invention, there is provided a method for preparing the compound as described in the first aspect, comprising the steps of:

[0054] (i) reacting a compound of formula Ia with N2-L 2 -R(Ib) to obtain a compound of formula I;

[0055]

[0056] L 2 ≡-L 1 -Wb (≡ refers to the carbon-carbon triple bond),

[0057] A, X, LG, L 1 and Wb are as defined above.

[0058] In another preferred embodiment, the compound of formula I is as shown in formula IV, and the method for preparing the compound of formula IV comprises the steps of:

[0059]

[0060] (i) reacting a compound of formula IVa with N2-L 2 -R(Ib) to obtain a compound of formula IV.

[0061] In another preferred embodiment, the compound of formula Ia or the compound of formula IVa is compound 4

[0062]

[0063] In another preferred embodiment, a method for preparing compound 4 is provided, comprising the steps of

[0064]

[0065] (1) Preparation of compound 1:

[0066] (1a) reacting compound 1a (2-bromo-1-[4-hydroxy-3-(hydroxymethyl)phenyl]-ethan-1-one) with NaN3 in an inert solvent 1a (preferably DMF) to obtain an azidation intermediate; and

[0067] (1b) reacting the azidation intermediate obtained in step (1a) with diethyl phosphite (5) and carbon tetrachloride in an inert solvent 1b (preferably, ultra-dry (anhydrous) acetonitrile) in the presence of N,N-diisopropylethylamine (DIPEA) and 4-dimethylaminopyridine (DMAP) to obtain compound 1;

[0068] (2) Preparation of Compound 2:

[0069] (2a) reacting compound 1 with Dess-Martin periodinane (DMP) in an inert solvent 2a (preferably dichloromethane) to obtain an intermediate of compound 2,

[0070] (2b) reacting the intermediate obtained in step (2a) with diethylaminosulfur trifluoride (DAST) in an inert solvent 2b (preferably ethanol) to obtain compound 2;

[0071] (3) Preparation of compound 3:

[0072] (3a) reacting compound 2 with NaBH4 in an inert solvent 3a (preferably methanol) to obtain an intermediate of compound 3;

[0073] (3b) reacting the intermediate of step (3a) with ethyl isocyanate in an inert solvent 3b (preferably triethylamine (TEA)) to obtain compound 4;

[0074] (4) Preparation of compound 4:

[0075] Compound 4 is reacted with trimethylsilyl bromide (TMSBr) in an inert solvent 4 (preferably, anhydrous acetonitrile) to obtain compound 4.

[0076] In the third aspect of the present invention, a detection reagent or a detection composition is provided, wherein the detection reagent or the detection composition comprises the compound as described in the first aspect and a carrier acceptable in detection.

[0077] In the fourth aspect of the present invention, a pharmaceutical composition is provided, comprising the compound as described in the first aspect and a carrier acceptable in detection.

[0078] In the fifth aspect of the present invention, an intermediate is provided, wherein the intermediate is as shown in Formula Ia,

[0079]

[0080] In Formula II, A, X, and LG are as defined above.

[0081] In another preferred embodiment, the intermediate is as shown in formula IVa

[0082]

[0083] In a sixth aspect of the present invention, there is provided an in vitro non-therapeutic method for inhibiting ALP overexpression, comprising the steps of contacting a subject with a compound of formula I, thereby inhibiting ALP overexpression.

[0084] In another preferred embodiment, the object is a cell.

[0085] In the seventh aspect of the present invention, a method for treating and / or preventing a disease associated with ALP overexpression is provided, comprising the steps of administering the compound of the first aspect or the pharmaceutical composition of the fourth aspect to a subject in need thereof.

[0086] In another preferred embodiment, the disease associated with ALP overexpression is a tumor.

[0087] In an eighth aspect of the present invention, a method for marking cells overexpressing ALP is provided, characterized in that it comprises the step of: contacting a subject with a compound of formula I, thereby inhibiting ALP overexpression.

[0088] In the ninth aspect of the present invention, a method for diagnosing a disease associated with ALP overexpression in a patient is provided, comprising the steps of administering the compound as described in the first aspect or the detection reagent or detection composition as described in the third aspect to a subject in need thereof.

[0089] In another preferred embodiment, the disease associated with ALP overexpression is a tumor.

[0090] In the tenth aspect of the present invention, a use of the compound of the first aspect is characterized in that: (i) it is used to label, track and / or image cells overexpressing ALP; and / or (ii) it is used to prepare a composition for labeling, tracking and / or imaging cells overexpressing ALP; and / or (iii) it is used to prepare a drug or composition for treating a disease associated with ALP overexpression.

[0091] In another preferred embodiment, when the use of the compound of formula I is (i) and / or (ii), R in formula I is a reporting group.

[0092] In another preferred embodiment, when the use of the compound of formula I is (iii), R in formula I is a therapeutic group.

[0093] In another preferred embodiment, the labeling, tracking and / or imaging of cells overexpressing ALP is non-therapeutic in vitro.

[0094] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 This is a diagram of protein labeling by the fluorescent probe ALP-6 and its structural analogs under the activation of alkaline phosphatase (ALP);

[0096] Figure 2This is the non-specific binding diagram of the fluorescent probe ALP-6 and its structural analogs with bovine serum albumin (BSA);

[0097] Figure 3 Cell imaging for fluorescent probes ALP-0, 2, 5, and 6;

[0098] Figure 4 The fluorescent probe ALP NIR-2 and its analogs label proteins under the activation of alkaline phosphatase (ALP) and their non-specific binding to non-enzyme proteins;

[0099] Figure 5 Cell imaging images of fluorescent probes ALP NIR-0, 1 and 2.

[0100] Figure 6 The general working principle of the compound or reagent as described in the first aspect is shown. DETAILED DESCRIPTION

[0101] After extensive and in-depth research, the inventor unexpectedly discovered a class of probe molecules or compounds based on the QM chemical reaction process with multiple "anchor point" characteristics or labeling reagents containing the probe molecules or compounds). The labeling reagent of the present invention can generate QM active intermediates multiple times, so it can label proteins multiple times and can reduce the inefficient labeling caused by quenching of QM active intermediates by other nucleophilic reagents, ultimately achieving the effect of "efficient and stable" labeling of biomacromolecules. Based on this, the inventor completed the present invention.

[0102] the term

[0103] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent refers to a straight or branched chain hydrocarbon radical (i.e., C 1-8 represents 1 to 8 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0104] As used herein, unless otherwise specified, the term "substituted" means 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.

[0105] Unless otherwise specified, in the present invention, all compounds appearing are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). Among all compounds of the present invention, each chiral carbon atom can be optionally in R configuration or S configuration, or a mixture of R configuration and S configuration.

[0106] As used herein, "covalently labeled compound", "labeled compound", "covalently labeled molecule", "labeled molecule", "compound of Formula I", "probe molecule of the present invention" and "compound of the present invention" can be used interchangeably to refer to the compound of Formula I as described in the first aspect.

[0107] As used herein, a group "derived from" a compound or drug (such as the drug group described in the present application) means that a part of the group or atom (such as 1 H atom) is lost from the drug or compound to form a group that can be connected to other parts such as the L group of the present application and maintain or substantially maintain the activity of the original compound or drug.

[0108] As used herein, "a structure with secondary or multiple release functions" means that the triggering agent action site in the structure breaks the covalent bond (XA break) under the action of the triggering agent, generating the first QM intermediate, which leaves quickly through electronic rearrangement, thereby generating the core QM intermediate with a "multi-anchor" structure involved in this patent. An exemplary structure is shown in the following formula (wherein, the definition of A' is the same as that of the A group in Formula I, and more preferably, A' is selected from: a targeting recognition group, a leaving group).

[0109]

[0110] Quinone Methylene (QMs)

[0111] Quinone methylidene (QMs) is a class of highly reactive active intermediates. The most widely used structures in nature are o-quinone methylidene (o-QMs) and p-quinone methylidene (p-QMs). For example, many animals and plants use QMs as a means of defense; QMs participate in the anti-cancer process of doxorubicin and its derivative epirubicin. In addition, QMs are also very susceptible to attack from nucleophilic reagents in living systems due to their extremely high electrophilicity, thus having a very strong potential to capture biomacromolecules and establish covalent bonds with them. Due to this property, QMs are also widely used in research in the field of life sciences to develop efficient and stable bioconjugation reagents. Subsequently, inhibitors, capture agents and their detection methods based on this strategy have also been further applied to the research and development of enzymes, antibodies and specific cell sorting. Because the material basis involved in life processes and the composition of organisms is both complex and numerous, this strategy is largely limited by the low specificity of QMs active intermediates. Specifically, it may be because of molecular thermal motion that the highly reactive QMs intermediates are formed at the catalytic site of the target protein and then diffuse rapidly to the surroundings. If they react and connect rapidly with the nucleophilic group at the catalytic reaction site of the target protein, otherwise the activated QMs intermediates will covalently connect with the nucleophilic site in the nearby protein. It can be inferred that the former process may affect the structure and activity of the target enzyme, and then produce an inhibitory effect on the target enzyme activity, which can be used for protein capture and the development of enzyme inhibitors; while the latter can reduce the impact of chemical modification on the structure and activity of the target protease, which can not only more accurately reflect the activity of the enzyme, but also bring about the "waterfall amplification" effect of many proteins being labeled in the later stage through the "one-point triggering" of the enzyme, which then reflects the advantages of QMs intermediates in chemically modified imaging reagents in biological applications. QMs, as a "self-anchoring" imaging reagent, has also been widely used in the activity detection and biolabeling process of many important biological enzymes such as phosphatases, glycosidases, lipases, sulfatases, and β-lactamases.

[0112]

[0113] Generally, the structure of QM-based labeled probe molecules includes: enzyme recognition part, potential capture group, linker and reporter group. The enzyme recognition part plays the role of searching for target substances and initiating the "self-anchoring" process; the potential capture group, i.e., QM precursor, plays the role of "anchoring" potential biomolecules and plays a vital role in the process of effective covalent labeling of proteins. At present, there are two main types of traditional QMs precursors: (1) o-monofluoromethylphenol or o-difluoromethylphenol derivatives (o-QM precursors); (2) 4-alkylphenols, whose benzyl (4-alkyl) is connected to leaving groups (such as fluorine, carbamate and carboxylate) (p-QM precursors). Both of them induce the spontaneous departure of leaving groups through electron transfer after the target enzyme is hydrolyzed and activated, and release highly reactive QMs active intermediates in situ, which then trigger the covalent connection between the probe molecule and the biomacromolecule. The potential "anchor point" of the p-QM precursor is far away from the recognition group of the enzyme, which indicates that the proper modification of the probe molecular structure will not only not affect the specific recognition of the probe by the target enzyme, but can even increase the functionality of the probe molecule. For example, the detaching group of the p-QM type fluorescent probe is transformed into a fluorescence quenching group to obtain a low background, covalently labeled fluorescent probe with fluorescence "off-on".

[0114] Through the above explanation, the "self-anchoring" biomacromolecule labeling process based on enzyme activity involving quinone methylene (QM) should have applications and development prospects that are worth looking forward to. However, the main reason affecting the practical application of this strategy is the labeling efficiency of the molecule on the biomacromolecule. Its labeling efficiency is mainly limited by the type of QMs, the rate of formation, the level of electrophilic reaction activity, and the stability of the covalent conjugate finally formed. If the labeling efficiency is relatively low, it will obviously hinder the practical application of this strategy, and existing research has still not been able to give a satisfactory answer. Therefore, studying the relationship between the structure and performance of QMs and finding "self-anchoring" biomacromolecule labeling reagents with high labeling efficiency can effectively improve the signal-to-noise ratio of the reagent molecules in practical applications, improve the detection sensitivity of the reagents, and expand the research on biomarker technology and biomedicine.

[0115] Multiple "anchor points"

[0116] In the present invention, as described in the first aspect, the compound (Formula I) or reagent, the reagent (or compound) is a fluorescent probe obtained by connecting an enzyme recognition group to a reporter group having a fluorescent response ability through a connecting arm. Under the action of the targeted recognition group, the reagent (or compound) promotes the breaking of the covalent bond of the AX bond, and rapidly generates a quinone methylene (QM) active intermediate with high reactivity, which is highly susceptible to the attack of nucleophilic reagents in the biomacromolecule structure, and then forms a covalent bond with the biomacromolecule. In addition, since the reagent structure always contains multiple QM precursors, it has the ability to activate and label biomacromolecules multiple times, that is, multiple "anchor point" characteristics, so that the reagent has the properties of targeted activation and efficient covalent labeling of biomacromolecules, and its working principle is roughly as follows Figure 6 shown.

[0117] ALP

[0118] As an important hydrolase in the body, ALP is not only involved in the dephosphorylation process of various biomolecules, including nucleic acids and proteins, and other small molecules; but also closely related to the occurrence and development of many diseases (such as osteoblastic bone cancer, prostate cancer, hepatitis), so ALP is also used as an important biomarker in molecular biology and even clinical diagnosis. Therefore, there are many studies on the detection methods of ALP activity and distribution, among which there is no lack of development of "self-anchoring" fluorescent probes based on QM. In view of the above reasons, the present invention uses ALP as a model enzyme, designs fluorescent probe molecules, and deeply studies the relationship between the structure of the QM active intermediate as a capture unit and its labeling efficiency, from which the QM precursor structure with multiple leaving groups is screened and determined as a capture unit for the practical application of this type of covalent labeling reagent. And the activity detection of ALP in vitro and in vivo and the live cell imaging of ALP overexpressing tumor cells are realized.

[0119] Highly efficient covalent labeling of biomacromolecules with multiple "anchor points"

[0120] The present invention provides a highly efficient biomacromolecule covalent labeling compound (or compound of formula I) containing multiple "anchor points". The reagent generates a highly reactive O- or P-QM intermediate under the action of alkaline phosphatase hydrolyzing the phosphate bond, which is covalently linked to the nucleophilic group in the protein structure around the activation site, thereby enabling the reagent to detect or label ALP.

[0121] Typically, the present invention provides a class of highly efficient biomacromolecule covalent labeling compounds containing multiple "anchor points" (i.e., compounds of formula I), wherein the reagent is as shown in formula I,

[0122]

[0123] In the formula,

[0124] A is a targeted recognition group (such as phosphate, glycoside, 3-oxo-n-butyl or deoxynucleoside) that can be recognized by biological macromolecules (such as enzymes, proteins and nucleic acids) and interact with them to produce phenolic hydroxyl (thiol or amino) groups, or a group (such as hydroxyl (OH) or hydroxyl, thiol or amino protecting group (such as photosensitive protecting group (photosensitive reagent))) that is sensitive to external means (such as light, heat or chemical induction) and promotes the cleavage of the covalent bond between AX to produce phenolic hydroxyl (thiol or amino) groups; or a structure with a "secondary release function", for example, one or more removable functional groups (such as o,p-QM latent bodies) are first formed by catalytic hydrolysis, and then the o,p-QM intermediate described herein is released by the departure of the group;

[0125] X is O, NH or S;

[0126] LG, geminal difluoride (CHF2), and aromatic ring core together form multiple "anchor" functional groups, among which LG can be F, Cl, Br, OAc, OCONHR a (R a =Et, Me, C6H5NO2, etc.), sulfone (R b SO2) or quaternary ammonium salts (such as -NR c 3 + ) and other common leaving groups;

[0127] L is a linking group constructed in various ways, such as an amide bond, an ester bond, an ether bond, or a linking group established by a "click chemistry" method;

[0128] R is a reporter group with fluorescent, radioactive or magnetic signals, or a drug molecule or drug precursor with therapeutic function.

[0129] Preferably, the photosensitive protecting group is (UV light trigger) or (NIR light triggered).

[0130] Preferably, the drug molecules and drug precursors with therapeutic functions are derived from, for example (but not limited to) antibiotics (cephalosporin, vancomycin), which are used for antibacterial and anti-drug resistance; avibactam, which inhibits serine β-lactamase inhibitors; sunitinib, which selectively targets multiple receptor tyrosine kinases, blocks tumor growth and directly attacks tumor cells to achieve anti-tumor purposes; amantadine and acyclovir, which are used for antiviral.

[0131] In another preferred embodiment, the highly efficient biomacromolecule covalent labeling compound with multiple "anchor points" is as shown in Formula II, wherein Reporter is a reporter group with fluorescent, radioactive or magnetic signals:

[0132]

[0133] In another preferred embodiment, the biomacromolecule covalently labeled compound with multiple "anchor points" is as shown in Formula III, wherein Drug is a drug molecule and a drug precursor with therapeutic function:

[0134]

[0135] Furthermore, R in the reagent is a fluorescent reporter group, and the structure of the fluorescent probe (ALP-6) whose reporter group is fluorescein is:

[0136]

[0137] Furthermore, the preferential fluorescent probe can be selectively recognized and hydrolyzed by alkaline phosphatase, and generate a QM intermediate with active chemical properties, thereby playing a role in covalently labeling ALP and its surrounding proteins.

[0138] Furthermore, ALP-6 can be specifically activated by alkaline phosphatase (ALP) hydrolysis to generate O- or P-QM intermediates, which are then covalently linked to nucleophilic groups in the protein structure around the activation site, thereby marking and detecting ALP activity or marking tumor cells that overexpress ALP.

[0139] Furthermore, R in the reagent is a fluorescent reporter group, and the structure of the fluorescent probe (ALP NIR-2) whose reporter group is a fluorescence-enhanced dye is:

[0140]

[0141] Preferably, after ALP hydrolysis activation, ALP NIR-2 will go through the same process as ALP-6, and can not only covalently label the target protein and its surrounding proteins, play the role of ALP activity detection and ALP overexpression tumor cells localization; but also can establish a dye group conjugation system before and after the reaction, so that the fluorescence intensity of the probe is increased compared with that before the reaction, thereby achieving the purpose of relatively improving the target signal intensity, reducing noise, and improving the detection results. In addition, since the emission wavelength of the fluorescent probe is in the near-infrared region, the characteristics of near-infrared light with strong tissue penetration and little tissue damage can be used to lay the foundation for the in vivo imaging of ALP in tumor-bearing mice.

[0142] Preparation method

[0143] The preparation method of the compound of formula (I) of the present invention is described in more detail below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such a combination can be easily carried out by a technician in the field to which the present invention belongs. Usually, in the preparation process, each reaction is usually in an inert solvent. The reaction time is usually 0.1 hour-60 hours, preferably 0.5-48 hours.

[0144] Typically, the method for preparing a highly efficient biomacromolecule covalently labeled compound (compound of formula I) with multiple "anchor points" provided by the present invention comprises the steps of:

[0145]

[0146] (i) in an inert solvent i, reacting a compound of formula Ia (preferably, a compound of formula IVa such as compound 4) with N2-L 2 -R(Ib) to obtain a compound of formula I (or a compound of formula IV).

[0147] Preferably, the inert solvent i is selected from the group consisting of dimethyl sulfoxide (DMSO), water, or a combination thereof.

[0148] Preferably, step (i) reacts the compound of formula Ib in the presence of vitamin C, copper sulfate and tris(3-hydroxypropyltriazolylmethyl)amine.

[0149] Preferably, the reaction time of step (i) is 0.1 to 2 hours (eg, 0.5 hours), and / or the reaction temperature is room temperature (eg, 20-30° C.).

[0150] In a specific embodiment, compound 4 of the present invention can be prepared according to the following process:

[0151]

[0152]

[0153] In a specific embodiment, the compound of formula I of the present invention can be prepared by the following process:

[0154]

[0155] This process mainly introduces enzyme recognition sites through the phosphorylation reaction of phenolic hydroxyl groups; then, multiple potential leaving groups are introduced into the QM core structure by modifying the potential QM core structure; as the final covalent bond "anchor point"; - then the purpose of introducing azide groups that can be later modified into the potential QM core is achieved, and then the connection between the enzyme recognition group, the nucleophile capture group and the fluorescent reporter group is achieved through the "click chemistry reaction" between the azide group and the alkyne group.

[0156] In one specific embodiment, compound 1 is prepared by the following method:

[0157] Add 2-bromo-1-[4-hydroxy-3-(hydroxymethyl)phenyl]-ethan-1-one and NaN3 to a reaction flask and add N,N-dimethylformamide (DMF);

[0158] After the reaction system was stirred at room temperature for 12 hours, the azidation intermediate of the raw material was obtained by recrystallization;

[0159] The above intermediate, N, N-diisopropylethylamine (DIPEA) and 4-dimethylaminopyridine (DMAP) are added to a reaction bottle, ultra-dry acetonitrile is added, cooled to -20°C and carbon tetrachloride and diethyl phosphite (5, Diethyl phosphite) are added dropwise; after the addition is completed, the reaction solution is transferred to room temperature and stirred for reaction for 1 hour, the solvent is dried and dichloromethane is added to dissolve, washed with saturated brine, dried over anhydrous magnesium sulfate, concentrated, and then quickly separated and purified by silica gel column chromatography to obtain the compound 1.

[0160] In one specific embodiment, compound 2 is prepared by the following method:

[0161] Compound 1 and Dess-Martin periodinane (DMP) were added to a reaction flask, and dichloromethane was added to dissolve; the reaction system was reacted at room temperature for 2 hours, saturated brine was added, the organic phase was separated, the aqueous phase was extracted with dichloromethane, the organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated, and the obtained crude intermediate product was directly used in the next experiment without further purification;

[0162] The above intermediate crude product was added into a reaction flask, redistilled dichloromethane was added to dissolve it, and diethylaminosulfur trifluoride (DAST) was added dropwise under ice bath condition;

[0163] After the addition was completed, the reaction system was stirred in an ice bath for 2 hours, then quenched with water, the aqueous layer was extracted with dichloromethane and the organic phase was collected. The organic phase was dried and concentrated, and then quickly separated and purified by silica gel column chromatography to obtain the compound 2;

[0164] In another specific embodiment, compound 3 is prepared by the following method:

[0165] Compound 2 was added to a reaction flask, methanol was added to dissolve it and cooled in an ice bath, and then NaBH4 was added; the reaction system was stirred in an ice bath for 0.5 hours, and after the reaction was completed, a saturated aqueous solution of ammonium chloride was added to terminate the reaction, and the aqueous phase was extracted with dichloromethane. The combined organic phases were dried and concentrated to obtain a crude reaction intermediate;

[0166] The crude product, ethyl isocyanate (6, Ethyl isocyanate) and triethylamine (TEA) were added into a reaction flask, and redistilled dichloromethane was added to dissolve;

[0167] The reaction solution was heated to reflux for 12 hours. After the reaction was completed, water was added to quench the excess ethyl isocyanate, and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous magnesium sulfate, concentrated, and subjected to silica gel column chromatography to obtain the compound 3.

[0168] In another specific embodiment, compound 1 is prepared by the following method:

[0169] Under nitrogen protection, compound 3 was placed in a reaction bottle, ultra-dry acetonitrile was added to dissolve it, and trimethylsilyl bromide (TMSBr) was added dropwise under ice bath conditions;

[0170] After the addition was completed, the reaction solution was stirred at room temperature for 12 hours. After the reaction was completed, it was quenched with a saturated aqueous ammonium chloride solution. The reaction solution was then separated and purified by a reverse C18 column and freeze-dried to obtain the compound 4.

[0171] In another specific embodiment, the compound of formula I is prepared by the following method:

[0172] Compound 4 is reacted with compound 7 (or 8) in an inert solvent to obtain the detection probe.

[0173] Preferably, compound 4 is reacted with compound 7 (or 8) in the presence of vitamin C, copper sulfate and tris(3-hydroxypropyltriazolemethyl)amine.

[0174] Preferably, the inert solvent is dimethyl sulfoxide (DMSO) and / or water.

[0175] Preferably, the reaction time of the reaction is 0.1 to 2 hours (such as 0.5 hours), and / or the reaction temperature is room temperature (such as 20-30° C.)

[0176] Preferably, the method further comprises the step of separating and / or purifying the detection probe; more preferably, the step of separating and / or purifying the detection probe comprises purification using a reverse phase C18 preparative column and freeze drying.

[0177] The main advantages of the present invention include:

[0178] (a) The compounds of the present invention have the ability to generate QM-active intermediates multiple times, and thus can label proteins multiple times;

[0179] (b) The compounds of the present invention can reduce the ineffectiveness caused by quenching of QM active intermediates by other nucleophiles).

[0180] (c) The compound or labeling reagent of the present invention has high labeling efficiency and stable labeling.

[0181] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0182] Unless otherwise specified, in the embodiments of the present invention, 1 H-NMR, 13 C-NMR was measured using a Bruker 400Mz instrument. The measuring solvents were deuterated chloroform (CDCl3) and deuterated dimethyl sulfoxide (d6-DMSO), and the internal standard was tetramethylsilane (TMS). All solvents were chromatographically pure, analytically pure, or chemically pure.

[0183] Example 1.1 Preparation of Compound 1

[0184]

[0185] 2-Bromo-1-[4-hydroxy-3-(hydroxymethyl)phenyl]-ethan-1-one (4 g, 16.3218 mmol) and NaN3 (1.53 g, 23.6 mmol) were added to a reaction flask and added to N,N-dimethylformamide (DMF);

[0186] The reaction system was stirred at room temperature for 12 hours, and then recrystallized to obtain 2.1168 g of the azidation intermediate of the raw material;

[0187] The above intermediate (500 mg, 2.41 mmol) was added to a reaction flask with N,N-diisopropylethylamine (DIPEA, 1.0495 mL, 6.025 mmol) and 4-dimethylaminopyridine (DMAP, 29.9 mg, 0.241 mmol), and ultra-dry acetonitrile was added, cooled to -20 °C, and carbon tetrachloride (2.32 mL, 24.1 mmol) and diethyl phosphite (5, Diethyl phosphite, 357 μL, 2.77 mmol) were added dropwise;

[0188] After the addition was completed, the reaction solution was transferred to room temperature and stirred for 1 hour. The solvent was dried and dichloromethane was added to dissolve the solution. The solution was washed with saturated brine, dried over anhydrous magnesium sulfate, concentrated, and quickly separated and purified by silica gel column chromatography to obtain compound 1 (521 mg, 63%).

[0189] The spectrum characteristics of the prepared compound 1 are: 1 H NMR(400MHz, CDCl3)8.02(s,1H),7.86(dd,J=8.5,2.1Hz,1H),7.35(d,J=8.5 Hz,1H),4.72(s,2H),4.56(s,2H),4.31–4.21(m,4H),1.38(t,J=7.1Hz,6H). 13 C NMR (100MHz, CDCl3) 192.27, 152.26 (d, J = 7.0Hz), 133.80 (d, J = 6.2Hz), 131.60, 129.55,128.78,120.62,65.44(d,J=6.2Hz),59.28,54.88,16.07(d,J=6.6Hz).

[0190] Example 1.2 Preparation of Compound 2

[0191]

[0192] Compound 1 (190.37 mg, 0.55 mmol) and Dess-Martin periodinane (DMP, 251.2 mg, 0.592 mmol) were added to a reaction flask, and dichloromethane (2 mL) was added to dissolve;

[0193] The reaction system was reacted at room temperature for 2 hours, 5 mL of saturated brine was added, the organic phase was separated, the aqueous phase was extracted with dichloromethane (5 mL × 3), the organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated. The obtained crude intermediate product was used directly in the next experiment without further purification;

[0194] The above intermediate crude product (180.5 mg) was added to a reaction flask, 5 mL of redistilled dichloromethane was added to dissolve it, and diethylaminosulfur trifluoride (DAST, 136 μL, 1.109 mmol) was added dropwise under ice bath conditions;

[0195] After the addition, the reaction system was stirred in an ice bath for 2 hours, then quenched with 2 mL of water, the aqueous layer was extracted with dichloromethane (5 mL × 3) and the organic phase was collected. The organic phase was dried and concentrated, and then quickly separated and purified by silica gel column chromatography to obtain compound 2 (144 mg, 72%).

[0196] The spectrum characteristics of the prepared compound 2 are: 1 H NMR(400MHz, CDCl3)8.17(s,1H),8.05(dd,J=8.7,2.1Hz,1H),7.64(d,J=8.8Hz, 1H), 6.95 (t, J = 54.8Hz, 1H), 4.57 (s, 2H), 4.32–4.19 (m, 4H), 1.40–1.36 (m, 6H). 13 C NMR (100MHz,CDCl3)

[0197] 191.48,152.96(q,J=6.0Hz),132.19,131.21,127.11(t,J=6.0Hz),126.16(td,J=23.4,7.8Hz ), 120.73 (d, J = 2.2Hz), 110.73 (t, J = 238.6Hz), 65.56 (d, J = 6.2Hz), 54.96, 16.14 (d, J = 6.6Hz).

[0198] Example 1.3 Preparation of Compound 3

[0199]

[0200] Compound 2 (201.6 mg, 0.555 mmol) was added to a reaction flask, 5 mL of methanol was added to dissolve and cooled in an ice bath, and then NaBH4 (31.5 mg, 0.833 mmol) was added;

[0201] The reaction system was stirred in an ice bath for 0.5 hours. After the reaction was completed, 2 mL of saturated ammonium chloride aqueous solution was added to terminate the reaction, and the aqueous phase was extracted with dichloromethane (10 mL×3). The combined organic phases were dried and concentrated to obtain 161.3 mg of a crude reaction intermediate product.

[0202] The above crude product, ethyl isocyanate (6, Ethyl isocyanate, 175 μL, 2.208 mmol) and triethylamine (TEA, 307 μL, 2.208 mmol) were added to a reaction flask, and 3 mL of redistilled dichloromethane was added to dissolve;

[0203] The reaction solution was heated under reflux for 12 hours. After the reaction was completed, 2 mL of water was added to quench the excess ethyl isocyanate, and the mixture was extracted with dichloromethane (10 mL×3). The organic phases were combined, dried over anhydrous magnesium sulfate, concentrated, and subjected to silica gel column chromatography to obtain compound 3 (115 mg, 47%).

[0204] The spectrum characteristics of the prepared compound 3 are: 1H NMR(400MHz, CDCl3)7.60(s,1H),7.50–7.42(m,2H),6.93(t,J=55.1Hz,1H),5.88–5.83(m,1H),4.87(s,1 H),4.28–4.18(m,4H),3.61–3.43(m,2H),3.30–3.18(m,2H),1.36(t,J=7.1Hz,6H),1.15(t,J=7.2Hz,3H). 13 C NMR (100MHz, CDCl3) 154.82, 148.54 (q, J = 5.9Hz), 135.07, 130.22, 125.64 (td, J = 22.9, 7.2Hz), 124.66 (t, J = 5.8Hz) ,120.41(d,J=1.8Hz),111.06(t,J=237.7Hz),73.86,65.11(d,J=6.2Hz),55.21,36.00,16.00(d,J=6.6Hz),15.03.

[0205] Example 1.4 Preparation of Compound 4

[0206]

[0207] Under nitrogen protection, compound 3 (115.2 mg, 0.264 mmol) was placed in a reaction bottle, ultra-dry acetonitrile was added to dissolve it, and trimethylsilyl bromide (TMSBr, 174 μL, 1.320 mmol) was added dropwise under ice bath conditions;

[0208] After the addition was completed, the reaction solution was stirred at room temperature for 12 hours. After the reaction was completed, it was quenched with a saturated aqueous ammonium chloride solution. The reaction solution was then separated and purified by a reverse C18 column and freeze-dried to obtain the compound 4 (34 mg, 35%).

[0209] The spectral characteristics of the prepared compound 4 are: 1 H NMR(400MHz,d6-DMSO)7.60(s,1H),7.54(d,J=8.4Hz,1H),7.48–7.39(m,2H),7.11(t,J=55.1Hz,1H ),5.80(t,J=5.6Hz,1H),3.64(d,J=5.6Hz,2H),3.00(qd,J=10.8,7.0Hz,2H),1.00(t,J=7.2Hz,3H). 13C NMR(100MHz,d6-DMSO)154.93,149.29(q,J=6.0Hz),134.74,130.24,125.10(td,J=22.7, 6.2Hz), 123.96 (t, J = 4.7Hz), 120.97, 111.65 (t, J = 235.1Hz), 72.79, 54.49, 35.25, 15.02.

[0210] Example 1.5 Synthesis of Compound 7

[0211]

[0212] 5-Carboxyfluorescein (200.0 mg, 0.532 mmol), N-hydroxysuccinimide (91.8 mg, 0.798 mmol) and EDCI (153.0 mg, 0.798 mmol) were placed in a reaction bottle, evacuated and replaced with nitrogen three times, 2 mL of dry DMF was added under nitrogen protection until it was completely dissolved, and the reaction was stirred in an ice bath for 4 h. The reaction was completed after HPLC monitoring, and a large amount of ether was added to the reaction solution for precipitation and centrifugation to finally obtain 144.5 mg of crude product 7-1

[0213]

[0214] The raw material 7-2 (2.1631 g, 4.865 mmol) and EDCI (1.3989 g, 7.297 mmol) were dissolved in 8 mL DCM, and propargylamine (347 μL, 5.838 mmol) was added under ice bath conditions. The reaction solution was slowly heated to room temperature and continued to stir. After TLC monitoring until the raw material 2 completely disappeared, 10 mL of saturated brine was added, and extracted with DCM (5 mL×3). The organic phases were combined, dried over anhydrous magnesium sulfate, concentrated, and quickly separated by silica gel column chromatography to obtain 1.1144 g of crude product 3. Under ice bath conditions, 100.0 mg of intermediate 7-3 (0.333 mmol) was dissolved in 2 mL of DCM / TFA / Tips (10:9:1) mixed solution, stirred for 30 min under ice bath, and the reaction was complete after HPLC monitoring. After the solvent was spin-dried, TFA was removed by vacuum to obtain the crude product of intermediate 4. 23.67 mg of intermediate 1 (0.05 mmol) was weighed and dissolved in 40 μL DMF, and then 30 μL of intermediate 7-4 (C DMF =2M, 0.06mmol) and 42μL TEA (0.3mmol), shaken at room temperature for 1h, and then the reaction was complete after HPLC monitoring. A large amount of ether was added for precipitation and centrifuged to finally obtain 21.4mg of compound 7

[0215] Low-resolution mass spectrometry data of compound 7: MS (ESI) m / z calculated value C30 H 26 N2O9(M+H) + 559.2, measured value 559.2; (M+Na) + 581.2, measured value 581.2.

[0216] Example 1.6 Preparation of fluorescent probe (ALP-6)

[0217]

[0218] Compound 4 (1.54 mg, 2.76 μmol) and compound 7 (C DMF =636.8mM, 5μL, 1.79μmol) was placed in a reaction bottle, and dimethyl sulfoxide (DMSO, 15L), water, vitamin C (1.3mg, 7.16mol), copper sulfate (30g, 0.18mol) and tris(3-hydroxypropyltriazolemethyl)amine (THPTA, 80g, 0.18mol) were added;

[0219] The reaction system was reacted at room temperature for 0.5 hours. After the reaction was completed, the mixture was purified using a reverse phase C18 preparative column and freeze-dried to obtain a light yellow compound, namely the fluorescent probe (ALP-6, 1.0 mg, 40%).

[0220] The spectral characteristics of ALP-6 are: HRMS (ESI) m / z calculated value C 42 H 41 F2N6O 15 P(MH) - 937.2263, measured value 937.2256.

[0221] Compound 8 can be prepared by referring to the method of the prior art.

[0222] Example 1.7 Preparation of fluorescent probe (ALP NIR-2)

[0223]

[0224] Compound 4 (2.90 mg, 7.63 μmol) and compound 8 (2.22 mg, 4.59 μmol) were placed in a reaction bottle, and dimethyl sulfoxide (DMSO), water, vitamin C, copper sulfate and tris(3-hydroxypropyltriazolylmethyl)amine were added;

[0225] The reaction system was reacted at room temperature for 0.5 hours. After the reaction was completed, the product was purified using a reverse phase C18 preparative column and freeze-dried to obtain a blue compound, namely the fluorescent probe (ALP NIR-2, 1.0 mg, 31%).

[0226] Spectral characteristics: HRMS (ESI) m / z calculated value C 46 H 44 F2N5O8P(MH) - 862.2823, measured value 862.2819.

[0227] Example 1.8 Fluorescent probe

[0228] The other compounds in the list can be prepared by referring to the method of Example 1.5 and / or 1.6.

[0229] Table 1. Chemical structures of ALP detection probes

[0230]

[0231]

[0232] Wherein, A represents general formula A, and B represents general formula B.

[0233] ALP-1, 3, 4, 5 and ALP NIR-1 are single "anchor" ALP detection probes, ALP-2, 6 and ALP NIR-2 are multi-"anchor" detection probes (B) of the present invention, and ALP-0 and ALP NIR-1 are "anchor-free" ALP detection probes.

[0234] Example 2.1 Labeling of proteins by ALP-6 and its analogs under alkaline phosphatase (ALP) activation

[0235] Experimental conditions: 7.5 μM fluorescent probe (ALP-6 and its structural derivatives ALP-0-5) and 12.5 U / mL target enzyme ALP (12.5 μg) were incubated in a 37°C water bath in the dark for 1 hour; or to further simulate the physiological environment, 1.5 μg BSA was added to the above reaction solution as the surrounding protein of the target enzyme, and incubated in the dark for 1 hour under the same experimental conditions. Then, the above protein samples were subjected to SDS-PAGE gel electrophoresis under a constant voltage of 150 V, and the protein gel was subjected to protein in-gel fluorescence scanning under the Cy2 channel (below) and Coomassie blue staining (above) (results see Figure 1 ).

[0236] Experimental results: Under simulated physiological conditions, the fluorescent probe ALP-6 and its analogs can not only be activated and label the target protein ALP and its surrounding proteins (such as BSA) after specific recognition and hydrolysis by alkaline phosphatase; Figure 1It can be seen that although different fluorescent probes have only slight differences in their structures, their labeling effects on proteins in the system are significantly different; and among them, ALP-3, 4, 5 and 6 have relatively outstanding labeling effects on proteins. Through structural comparison and analysis, the above-mentioned fluorescent molecules all have potential p-QM structures.

[0237] Experimental conclusion: By modifying the molecular structure of the fluorescent probe, we can obtain "self-anchoring" fluorescent probe molecules ALP-3, 4, 5 and 6 with relatively ideal protein covalent labeling efficiency and all containing potential p-QM structures. This shows that this type of potential p-QM structure is more conducive to the establishment of covalent bonds between the capture group and the protein nucleophilic group; and its existence can more effectively improve the labeling efficiency of "self-anchoring" fluorescent probes based on QM intermediates for biomacromolecules.

[0238] Test Example 2.2 Nonspecific binding of ALP-6 and its analogs to bovine serum albumin (BSA)

[0239] Experimental conditions: 7.5 μM fluorescent probes (ALP-6 and its structural derivatives ALP-0 to 5) were incubated with 3 μg BSA in a 37°C water bath in the dark for 1 hour without alkaline phosphatase (ALP) catalysis. Then, the above protein samples were subjected to SDS-PAGE gel electrophoresis at a constant voltage of 150 V, and the protein gel was scanned for protein in-gel fluorescence under the Cy 2 channel (lower) and stained with Coomassie blue (upper) (results see Figure 2 ).

[0240] Experimental results: By properly adjusting the contrast, the fluorescent probe ALP-6 and its analogs have different degrees of non-specific binding to the non-enzyme protein BSA in the system without alkaline phosphatase hydrolysis activation. Among them, the non-specific binding signals of probes ALP-1, 2, 3 and 4 are relatively obvious, while the non-specific binding between fluorescent probes ALP-5 and 6 and BSA is very weak.

[0241] Experimental conclusion: This nonspecific binding between fluorescent probes and protein molecules is relatively common. This type of binding is not only the main reason for false positives when fluorescent probes detect target substances, but also greatly reduces the authenticity and practicality of fluorescent probes. Figure 2 The results show that the fluorescent probes ALP-5 and 6 show more accurate and real fluorescent signals when detecting the target enzyme ALP, and can more realistically reflect the covalent labeling efficiency of this type of fluorescent probe molecules on proteins after enzyme activation. Figure 1 The fluorescent probes ALP-5 and ALP-6 have excellent performance in protein covalent labeling after enzyme activation. The present invention discloses a class of highly efficient "self-anchoring" biomacromolecule covalent labeling compounds with good signal-to-noise ratio and capable of being specifically activated by enzymes.

[0242] Example 2.3 Cell Imaging Using ALP-0, 2, 5 and 6

[0243] Experimental conditions: In a 37°C incubator, 10 μM fluorescent probes ALP-0, 2, 5 and 6 were incubated with HeLa cells that overexpressed ALP on the cell membrane surface and normal tissue cells HEK293 that underexpressed ALP in a confocal culture dish for 1.5 hours; or HeLa cells were pretreated with alkaline phosphatase inhibitor p-BTO for 1 hour in advance and then incubated with different probes for 1.5 hours. The probe-containing culture medium was then discarded and washed three times with PBS buffer (pH = 7.4) to remove the probe molecules that were not covalently linked to the membrane surface; 1 mL of sterile PBS buffer (pH = 7.4) was added and cell imaging was performed using a fluorescence microscope (λ ex =470±20nm,λ em =515nm).

[0244] Experimental results:

[0245] The experimental results are as follows Figure 3 As shown, Figure 3 In the figure, the 7 groups on the left are the labeling experiments of fluorescent probes ALP-0, 2, 5 and 6 on the cell membrane under ALP activation on the surface of HeLa cell membrane; the 4 groups on the right are the labeling experiments of fluorescent probes ALP-0, 2, 5 and 6 on the HEK293 cell membrane with low expression of ALP. In the figure, FL is the green fluorescence signal; Hoechst is the cell nucleus signal stained by Hoechst 3342; BF is the cell morphology under white light irradiation; Merge is the composite image of the above fields of view; p-BTO ((-)-p-Bromotetramisole Oxalate) is a commercial ALP inhibitor. Scale bar = 50μm.

[0246] Depend on Figure 3It can be seen that the fluorescent probes ALP-2, 5, and 6 with covalent "anchor points" can not only recognize ALP overexpressing cells and clearly distinguish ALP low-expressing cell lines (HEK293) from high-expressing cell lines (HeLa) compared with the ordinary fluorescent probe ALP-0 without labeling ability; they can also use the QM active intermediates formed after ALP hydrolyzes the probe to "anchor" the cell membrane of positive cells. In addition, in the ALP inhibitor group experiment, the fluorescent signals of HeLa cell labeling by fluorescent probes ALP-2, 5, and 6 were weakened, which shows that the initiation of fluorescent probe labeling depends on the nature of alkaline phosphatase to specifically hydrolyze phosphate bonds. Again, by comparing the fluorescent semi-quantitative data of HeLa cell labeling by fluorescent probes ALP-5 and 6, it can be seen that fluorescent probe ALP-6 can more effectively detect target enzymes and label positive cells at the in vitro living cell level that is closer to the physiological environment.

[0247] Experimental conclusion: The fluorescent probe ALP-6 can achieve the identification and covalent labeling of tumor cells overexpressing ALP at the in vitro living cell level, thereby reducing the diffusion of the fluorescent probe in the cell imaging experiment, reducing the background signal, and improving the detection sensitivity. In addition, combined with the previous experimental results and conclusions, it can be seen that both fluorescent probes ALP-5 and 6 have potential p-QM structures and low nonspecific protein binding ability, and both show efficient protein labeling in gel running experiments; however, the fluorescent probe ALP-6 with multiple potential covalent bond "anchor points" can more effectively achieve covalent labeling of biomacromolecules at the cellular level, and has more practical application value and prospects in the fields of biomarkers and biomedicine.

[0248] Example 2.4 Protein labeling experiment of ALP NIR-2 and its analogs under alkaline phosphatase (ALP) activation and non-specific binding experiment with non-enzyme proteins

[0249] Experimental conditions: 5 μM enhanced fluorescent probe (ALP NIR-2 and its structural derivatives ALP-0, 1) and 1.5 μg BSA were incubated in a 37°C water bath in the dark for 2 hours with or without the target enzyme ALP catalysis; or 5 μM enhanced fluorescent probe was incubated with 5 U / mL ALP only in the dark for 2 hours under the same conditions. Subsequently, the above protein samples were subjected to SDS-PAGE gel electrophoresis at a constant voltage of 150 V, and the protein gel was subjected to protein in-gel fluorescence scanning under the Cy 5 channel (lower) and Coomassie blue staining (upper) (see Figure 4 ).

[0250] Experimental results: Under the specific recognition and hydrolysis of alkaline phosphatase, the enhanced "self-anchoring" fluorescent probes ALP NIR-1 and 2 can label the target enzyme and its surrounding proteins, while the protein bands in the lane where the enhanced fluorescent probe ALPNIR-0 without covalent labeling is located have no fluorescent signals, that is, ordinary probes cannot achieve the purpose of labeling the target enzyme and surrounding proteins. In addition, after pretreatment of ALP with the inhibitor p-BTO, the labeling effect of the covalently labeled fluorescent probes ALP NIR-1 and 2 on alkaline phosphatase was weakened. It was further confirmed that the covalent labeling effect of this type of "self-anchoring" probe on protein depends on the activation of the potential QM structure by the target enzyme. In addition, the results of protein in-gel fluorescence scanning showed that the enhanced fluorescent probe ALP NIR-2 containing multiple potential covalent bond "anchor points" had a slightly stronger covalent labeling effect on alkaline phosphatase and non-enzyme protein BSA than the single "anchor point" fluorescent probe ALP NIR-1.

[0251] Experimental conclusion: The experimental results show that the enhanced "self-anchoring" fluorescent probe molecules ALP NIR-1 and 2 with potential p-QM structure can effectively label the target enzyme and its surrounding proteins through covalent bonds after specific hydrolysis of the target enzyme. And the enhanced fluorescent probe ALP NIR-2 with multiple potential covalent bond "anchor points" can label proteins more efficiently. It further proves that the potential p-QM structure with multiple covalent bond "anchor points" is more conducive to the establishment of covalent bonds between the capture group and the protein nucleophilic group.

[0252] Example 2.5 Cell imaging using ALP NIR-0, 1 and 2

[0253] Experimental conditions: 5 μM enhanced fluorescent probes ALP NIR-0, 1 and 2 were incubated with HeLa cells overexpressing ALP on the cell membrane surface and normal tissue cells HEK293 with low ALP expression in a confocal culture dish for 1.5 hours; or HeLa cells were pretreated with alkaline phosphatase inhibitor p-BTO for 1 hour in advance and then incubated with different probes for 1.5 hours. The medium containing the probes was then discarded and washed three times with PBS buffer (pH = 7.4) to remove the probe molecules that were not covalently linked to the membrane surface; 1 mL of sterile PBS buffer (pH = 7.4) was added and cell imaging was performed using a fluorescence microscope (λ ex =682±12nm,λ em =721±41nm).

[0254] Experimental results:

[0255] The results are as follows Figure 5 As shown, Figure 5In the figure, the 5 columns on the left are the labeling experiments of ALP-positive cells (HeLa) and positive cells pretreated with inhibitors using fluorescent probes ALP NIR-0, 1 and 2; the 3 columns on the right are the labeling experiments of HEK293 cells with low ALP expression using fluorescent probes ALP-0, 1 and 2. Figure 5 FL is the green fluorescence signal; Hoechst is the cell nucleus signal stained by Hoechst 3342; BF is the cell morphology under white light irradiation; Merge is a composite image of the above fields of view; p-BTO ((-)-p-Bromotetramisole Oxalate) is a commercial ALP inhibitor. Scale bar = 50μm.

[0256] Compared with the ordinary enhanced fluorescent probe ALP NIR-0 without labeling ability, the enhanced fluorescent probes ALP-1 and 2 with covalent "anchor points" can not only realize the recognition of ALP overexpressing cells and clearly distinguish between ALP low-expressing cell lines (HEK293) and high-expressing cell lines (HeLa); but also can realize the "anchoring" of positive cells by using the QM active intermediates formed after ALP hydrolyzes the probe. In addition, in the ALP inhibitor group experiment, the fluorescence signal of the fluorescent probes ALP NIR-1 and 2 for HeLa cells with ALP inhibition was weakened, which shows that the initiation of the covalent labeling of the fluorescent probe depends on the process of ALP specifically hydrolyzing the phosphate bond to induce the production of QM intermediates. Again, by comparing the effects of fluorescent probes ALP NIR-1 and 2 on HeLa cell labeling, it can be seen that the enhanced fluorescent probe ALP NIR-2 with multiple potential covalent "target points" can more effectively and accurately detect the target enzyme and label positive cells at the in vitro living cell level that is closer to the physiological environment.

[0257] Experimental conclusion: The multi-"anchor point" enhanced fluorescent probe ALP NIR-2 can achieve the identification and precise positioning of tumor cells overexpressing ALP at the in vitro living cell level, thereby reducing the diffusion of fluorescent probes in cell imaging experiments, reducing background signals, and improving detection sensitivity. In addition, combined with the previous experimental results and conclusions, it can be seen that both enhanced fluorescent probes ALP NIR-1 and 2 have potential p-QM structures and efficient protein labeling effects; in addition, ALP NIR-2 has outstanding specific detection capabilities for ALP. Based on the above results, the enhanced fluorescent probe ALP NIR-2 with multiple potential covalent bond "anchor points" of the present invention can not only more effectively achieve covalent labeling of cell membrane surface proteins at the cellular level, but also has more promising prospects and practical application value in the fields of biomarkers and biomedicine.

[0258] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A compound of formula I, characterized in that In Formula I, A is a targeting recognition group; In the A group, the targeting recognition group is a group that can be recognized by the enzyme and interact with the enzyme to produce a phenolic hydroxyl group; X is O; LG is -O(C=O)NHR b ; Among them, R b Selected from the group consisting of C1-C6 alkyl, substituted or unsubstituted phenyl, C6H5NO2; L is a linking group and L is -Wa-L 1 -Wb-; and Wb is connected to R; In the L group, Wa is -(CH2) 1或2 - a five- or six-membered ring containing 1 to 3 nitrogen heteroatoms -(CH2) 0-2 -; Wb is selected from the group consisting of None, O, S, NR a 、CO、SO、SO2、CO-NR a NR a -CO, SO-N(R a )、N(R a )-SO、NR a -SO2, SO2-NR a , CS-NR a NR a -CS、N(R a )-CO-NR a 、-(CH2) 0、1或2 - a five- or six-membered ring containing 1 to 3 nitrogen heteroatoms -(CH2) 0-2 -; L 1 is a linking group consisting of one or more unit structures selected from the group consisting of substituted or unsubstituted C1-C4 alkylene, a five- or six-membered ring containing 1-3 nitrogen heteroatoms, (substituted or unsubstituted C1-C2 alkylene)-O, (substituted or unsubstituted C1-C2 alkylene)-S, NR a , CO; R a Each is independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 haloalkyl, or substituted or unsubstituted C3-C6 cycloalkyl; R is a reporter group; wherein the reporter group refers to a group that can emit fluorescence, radiation and / or magnetic signals; The substitution refers to that one or more hydrogen atoms in the group are replaced by a substituent selected from the group consisting of halogen, C1-C6 alkyl, and C1-C6 haloalkyl.

2. The compound of formula (I) according to claim 1, characterized in that A is selected from the following group: phosphate group, i.e. -H2PO3; glycoside group; deoxynucleoside group; X is selected from the following group: O; LG is -O(C=O)NHR b ; Among them, R b Selected from the group consisting of C1-C6 alkyl, substituted or unsubstituted phenyl, C6H5NO2; L is -Wa-L 1 -Wb-; and Wb is connected to R; In the L group, Wa is -(CH2) 1或2 - a five- or six-membered ring containing 1 to 3 nitrogen heteroatoms -(CH2) 0-2 -; Wb is selected from the group consisting of None, O, S, NR a 、CO、CO-NR a NR a -CO; L 1 is a linking group consisting of one or more unit structures selected from the group consisting of substituted or unsubstituted C1-C4 alkylene, a five- or six-membered ring containing 1-3 nitrogen heteroatoms, (substituted or unsubstituted C1-C2 alkylene)-O, (substituted or unsubstituted C1-C2 alkylene)-S, NR a , CO; R a Each is independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 haloalkyl, or substituted or unsubstituted C3-C6 cycloalkyl; R is selected from the following group: a reporter group; wherein the reporter group refers to a group capable of emitting fluorescence, radiation and / or magnetic signals; The substitution refers to that one or more hydrogen atoms in the group are replaced by a substituent selected from the group consisting of halogen, C1-C6 alkyl, and C1-C6 haloalkyl.

3. The compound according to claim 1 or 2, characterized in that L 1 is a linking group consisting of one or more unit structures selected from the group consisting of substituted or unsubstituted C1-C4 alkylene, a five- or six-membered ring containing 1 to 3 nitrogen heteroatoms, (substituted or unsubstituted C1-C2 alkylene)-O-(substituted or unsubstituted C1-C2 alkylene), (substituted or unsubstituted C1-C2 alkylene)-S-(substituted or unsubstituted C1-C2 alkylene), CO-NR a NR a -CO.

4. The compound according to claim 1 or 2, characterized in that The five- or six-membered ring containing 1 to 3 nitrogen heteroatoms -(CH2) 0-2 -for 5. The compound according to claim 1 or 2, characterized in that The five- or six-membered ring containing 1 to 3 nitrogen heteroatoms is 6. The compound according to claim 1 or 2, characterized in that R a For H.

7. The compound according to claim 1 or 2, characterized in that Halogen is F, Cl, Br or I.

8. The compound according to claim 1, characterized in that The compound has one or more of the following characteristics: (a) A is selected from the group consisting of a phosphate group, i.e., -H2PO3; (b) L is selected from the group consisting of: -(CH2) n1 -Ar 1 -(CH2) n1 -NHCO-(CH2-O-CH2) n2 -(CH2) n1 -NHCO-、-(CH2) n1 -Ar 1 -(CH2) n3 -; Wherein, n1=1 or 2; n2=1, 2, 3, 4 or 5; n3=2, 3, 4, 5, 6, 7, 8 or 9; Ar 1 is a five- or six-membered ring containing 1 to 3 nitrogen heteroatoms; and / or (c) R is a reporter group.

9. The compound according to claim 8, characterized in that Ar 1 for 10. The compound according to claim 8, characterized in that L is selected from the following group:

11. The compound according to claim 8, characterized in that Wa 12. The compound according to claim 8, characterized in that R is a group capable of emitting fluorescence.

13. The compound according to claim 1 or 2, characterized in that A is a phosphate group, i.e. -H2PO3.

14. The compound according to claim 13, characterized in that R b It is a C1-C6 alkyl group.

15. The compound according to claim 1, characterized in that The compound of formula I is selected from the group consisting of:

16. A detection reagent or a detection composition, characterized in that: The detection reagent or detection composition comprises the compound as claimed in claim 1 or 2 and a carrier acceptable in detection.

17. An intermediate, characterized in that The intermediate is shown in Formula Ia, In formula Ia, A, X, and LG are as defined in claim 1 or 2.

18. An in vitro non-therapeutic and non-diagnostic method for labeling cells overexpressing ALP, characterized in that: The method comprises the steps of contacting a subject with the compound of formula I as claimed in claim 1, thereby marking cells overexpressing ALP.

19. A use of the compound according to claim 1, characterized in that: (i) for in vitro non-therapeutic and non-diagnostic labeling, tracking and / or imaging of cells overexpressing ALP; and / or (ii) for preparing a composition for labeling, tracking and / or imaging of cells overexpressing ALP.

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