CN112552344A

CN112552344A - Labeled compound containing multiple anchor points as well as preparation method and application thereof

Info

Publication number: CN112552344A
Application number: CN201910854447.8A
Authority: CN
Inventors: 谢贺新; 宋恒; 陈叶锋
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2019-09-10
Filing date: 2019-09-10
Publication date: 2021-03-26

Abstract

The invention provides a high-efficiency biomacromolecule covalent labeling compound (a compound shown in a formula I) containing multiple anchor points, and a preparation method and application thereof. The marking reagent or the compound has an o, p-methylene benzoquinone (o, p-QM) precursor, and can generate methylene benzoquinone (QM) active intermediates for multiple times, so that the aim of marking proteins for multiple times is fulfilled, and the low-efficiency mark caused by quenching of the QM active intermediates by other nucleophilic reagents can be remarkably reduced, so that the aim of efficiently and stably marking biomacromolecules is fulfilled. Tumor cells expressing alkaline phosphatase (ALP) can be specifically labeled and imaged live.

Description

Labeled compound containing multiple anchor points as well as preparation method and application thereof

Technical Field

The invention belongs to the field of biological markers and biological medicines, and particularly relates to a high-efficiency biomacromolecule marker compound containing multiple anchor points, and a preparation method and application thereof.

Background

Methylene benzoquinone (QMs) is a very reactive intermediate, and the structures which are most widely applied in nature are ortho-methylene benzoquinone (o-QMs) and para-methylene benzoquinone (p-QMs). For example, many animals and plants utilize QMs as a defense; QMs is involved in the anticancer process of epirubicin and its derivatives. In addition, QMs is also highly electrophilic and susceptible to attack by nucleophiles from living systems, and thus has a very strong potential to capture biomacromolecules and establish covalent bonds therewith. With this property, QMs is also widely used in research in the life science field for developing highly efficient and stable bioconjugate reagents. For example, as early as 1990 scientists introduced the chemistry of o-QMs reactive intermediates to "capture" and "anchor" nucleophilic groups in living macromolecules into the working mechanisms of glucosidase activity inhibitors. Subsequently, inhibitors, capture agents and detection means based on such strategies are further applied in the development of enzymes, antibodies and specific cell sorting. This strategy is largely limited by the low specificity of the QMs reactive intermediate due to the complex and numerous material bases involved in life processes and constituting organisms. QMs as a self-anchored imaging reagent is widely applied to the activity detection of various important biological enzymes such as phosphatase, glycosidase, lipase, sulfatase, beta-lactamase and the like. However, current QM-based probe molecules are less efficient at labeling biological macromolecules. Moreover, the existing studies still fail to provide a satisfactory answer.

In view of the above, there is an urgent need in the art to develop a covalent biomacromolecule labeling reagent with high efficiency and high sensitivity.

Disclosure of Invention

The invention aims to provide a biomacromolecule covalent labeling reagent with high efficiency and high sensitivity.

In a first aspect of the invention, there is provided a compound of formula I,

in the formula I, the compound is shown in the specification,

a is selected from the following group: a target recognition group (preferably, phosphate, glucoside, deoxynucleoside, peptide compound), a leaving group (preferably, selected from a hydroxyl protecting group, an amino protecting group, a sulfhydryl protecting group), or a two-time or multi-time release functional structure;

in the group A, the target recognition group is a group which can be recognized by biological macromolecules and interacts with the target recognition group so as to generate phenolic hydroxyl, phenolic sulfhydryl or amino; the leaving group is a group which is sensitive to induction by light, heat or chemistry and which is capable of cleaving the covalent bond between A-X to produce a phenolic hydroxyl group, a phenolic thiol group or an amino group; the structure with the secondary or multiple release function refers to a group which can be activated by a trigger and then hydrolyzed to release an o, p-QM intermediate; and the structure of the secondary or multiple release function is shown as formula A

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

x is O, NH or S;

LG is capable of reacting with gem-difluoro (CHF)₂) The aromatic ring parent nucleus jointly forms a multi-anchor point functional group;

l is a linking group;

r is selected from the group consisting of: a reporter group, a therapeutic group; wherein, the reporter group refers to a group which can be detected by fluorescence, radiation and/or magnetic signals, and the therapeutic group refers to a drug molecule and a drug precursor with therapeutic functions;

in a further preferred embodiment of the method,

a is selected from the following group: phosphoric acid group (-H)₂PO₃) (ii) a A glycoside; deoxidationA nucleoside; a 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);

x is selected from the group consisting of: o, NH, S;

LG is selected from the group consisting of: F. cl, Br, OAc, OCONHR^b、R^bSO₂、-NR^b ₃ ⁺(ii) a And R is^bEach independently selected from the group consisting of: C1-C6 alkyl, substituted or unsubstituted phenyl (preferably, C₆H₅NO₂)；

L is a linking group;

r is selected from the group consisting of: a reporter group, a drug group; wherein, the reporter group refers to a group which can be detected by fluorescence, radiation and/or magnetic signals, and the drug group refers to drug molecules and drug precursors with therapeutic functions;

unless otherwise specified, the substitution refers to the substitution of one or more hydrogens in the group with a substituent selected from the group consisting of: halogen (preferably, F, Cl, Br, I), C1-C6 alkyl, C1-C6 haloalkyl.

In another preferred embodiment, L is-Wa-L¹-Wb-; and Wb is linked to R; in the group L, the alkyl group,

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 or 2}-five-or six-membered ring containing 1-3 nitrogen heteroatoms- (CH)₂) _{0.1 or 2}-preferably (c) the (c) is (c),

)；

L¹is one or more selected from the group consisting ofA linking group composed of a plurality of unit structures: a substituted or unsubstituted C1-C4 alkylene group, a five-or six-membered ring containing 1 to 3 nitrogen heteroatoms (preferably, it is

) (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；

R_aEach independently selected from the group consisting of: H. C1-C6 alkyl, C1-C6 haloalkyl, or substituted or unsubstituted C3-C6 cycloalkyl (preferably, R_aIs H).

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

In another preferred embodiment, the radioactive group capable of being detected is referred to as being detected¹⁸F-labeled group.

In another preferred embodiment, a is selected from the group consisting of: phosphoric acid group (-H)₂PO₃)、

And

and R' is selected from the group consisting of: C1-C6 alkyl, C1-C6 haloalkyl; preferably, a is a phosphate group.

In another preferred embodiment, X is O.

In another preferred embodiment, LG is selected from the group consisting of: F. OCONHR^b(ii) a And R is^bEach independently selected from the group consisting of: C1-C6 alkyl (preferably, Et, Me), substituted or unsubstituted phenyl (preferably, C₆H₅NO₂)。

In another preferred embodiment, Wa is- (CH)₂)_{0.1 or 2}Containing 1 to 3 ofFive-or six-membered ring of nitrogen hetero atoms- (CH)₂)_{0.1 or 2}-preferably (c) the (c) is (c),

)。

in another preferred embodiment, Wb is selected from the group consisting of: none, NR_a-CO。

In another preferred embodiment, L¹Selected from the group consisting of: - (CH)₂)_n1-NHCO-(CH₂-O-CH₂)_n2-(CH₂)_n1-、 -(CH₂)_n3-、-(CH₂)_n1-Ar¹-(CH₂)_n1-NHCO-(CH₂-O-CH₂)_n2-(CH₂)_n1-。

In another preferred embodiment, R is a reporter group; preferably, R is a group capable of fluorescing.

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

In another preferred embodiment, R is selected from the group consisting of:

in another preferred embodiment, L is selected from the group consisting of:

-(CH₂)_n1-Ar¹-(CH₂)_n1-NHCO-(CH₂-O-CH₂)_n2-(CH₂)_n1-NHCO-、

-(CH₂)_n1-Ar¹-(CH₂)_n3-、

-CONH-(CH₂)_n1-Ar¹-(CH₂)_n1-NHCO-(CH₂-O-CH₂)_n2-(CH₂)_n1-NHCO-；

wherein n1 is 0, 1, 2 or 3; n2 is 1, 2, 3, 4 or 5; n3 is 2, 3, 4, 5, 6, 7, 8 or 9;

Ar¹is a compound containing 1-3 nitrogen heteroatomsA five or six membered ring (preferably,

)。

in another preferred embodiment, L is selected from the group consisting of:

in another preferred embodiment, the compound is represented by formula IV,

wherein LG, L and R are defined as above.

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

In another preferred embodiment, the compound of formula I is selected from the group consisting of:

in a second aspect the present invention provides a process for the preparation of a compound as described in the first aspect, comprising the steps of:

(i) reacting a compound of formula Ia with N₂-L²-r (ib) to obtain a compound of formula I;

L²is ≡ L¹-Wb (≡ means a carbon-carbon triple bond),

A、X、LG、L¹and Wb is as previously defined.

In another preferred embodiment, the compound of formula I is represented by formula IV, and the process for preparing the compound of formula IV comprises the steps of:

(i) reacting a compound of formula IVa with N₂-L²-R (Ib) to give a compound of formula IV.

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

In another preferred embodiment, there is provided a process for the preparation of compound 4 comprising the steps of

(1) Preparation of compound 1:

(1a) reacting compound 1a (2-bromo-1- [ 4-hydroxy-3- (hydroxymethyl) phenyl) in an inert solvent 1a (preferably DMF)]-ethan-1-one) with NaN₃Reacting to obtain an azide intermediate; and

(1b) reacting the azido intermediate obtained in step (1a) with diethyl phosphite (5) and carbon tetrachloride in the presence of N, N-Diisopropylethylamine (DIPEA) and 4-Dimethylaminopyridine (DMAP) in an inert solvent 1b, preferably, ultra dry (anhydrous) acetonitrile, to give compound 1;

(2) preparation of compound 2:

(2a) reacting compound 1 with dess-martin oxidant (DMP) in an inert solvent 2a, preferably dichloromethane, to give an intermediate of compound 2,

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

(3) preparation of compound 3:

(3a) reacting compound 2 with NaBH in an inert solvent 3a, preferably methanol₄To give an intermediate of compound 3;

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

(4) preparation of compound 4:

compound 4 is reacted with trimethylbromosilane (TMSBr) in an inert solvent 4, preferably anhydrous acetonitrile, to give compound 4.

In a third aspect of the invention, there is provided a test reagent or test composition comprising a compound according to the first aspect and a detectably acceptable carrier.

In a fourth aspect of the invention, there is provided a pharmaceutical composition comprising a compound according to the first aspect and a detectably acceptable carrier.

In a fifth aspect of the invention, there is provided an intermediate of formula Ia,

in formula II, A, X, LG is as previously defined.

In another preferred embodiment, the intermediate is represented by formula IVa

In a sixth aspect of the invention there is provided a method of non-therapeutically inhibiting ALP overexpression in vitro comprising the steps of: contacting a subject with a compound of formula I, thereby inhibiting ALP overexpression.

In another preferred embodiment, the subject is a cell.

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

In another preferred embodiment, the disease associated with overexpression of ALP is a tumor.

In an eighth aspect of the present invention, there is provided a method for labeling a cell overexpressing ALP, comprising the steps of: contacting a subject with a compound of formula I, thereby inhibiting ALP overexpression.

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

In a tenth aspect of the invention, a use of a compound as in the first aspect, is characterized by (i) for labeling, tracking and/or imaging cells overexpressing ALP; and/or (ii) for the preparation of a composition for labeling, tracking and/or imaging cells overexpressing ALP; and/or (iii) for the preparation of a medicament or composition for the treatment of a disease associated with overexpression of ALP.

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

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

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

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a graph showing the labeling of a fluorescent probe ALP-6 and its structural analogs to a protein under the activation of alkaline phosphatase (ALP);

FIG. 2 is a non-specific binding diagram of a fluorescent probe ALP-6 and its structural analogs to Bovine Serum Albumin (BSA);

FIG. 3 is a cellular image of the fluorescent probe ALP-0, 2, 5 and 6;

FIG. 4 is a graph showing the labeling of a protein by the fluorescent probe ALP NIR-2 and its analogues under activation of alkaline phosphatase (ALP) and its non-specific binding to a non-enzymatic protein;

FIG. 5 is a photograph of an image of a cell with the fluorescent probe ALP NIR-0, 1 and 2.

Figure 6 shows the general principle of action of a compound or agent according to the first aspect.

Detailed Description

The inventors have extensively and intensively studied and unexpectedly found a class of probe molecules or compounds based on QM chemical reaction processes with multiple "anchor" characteristics or labeling reagents containing the probe molecules or compounds). The marking reagent can generate QM active intermediates for many times, so that the protein can be marked for many times, the low-efficiency marking caused by quenching of the QM active intermediates by other nucleophilic reagents can be reduced, and the effect of marking biomacromolecules efficiently and stably is finally achieved. The present invention has been completed based on this inventor.

Term(s) for

Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent refers to a straight or branched chain hydrocarbon radical having the indicated number of carbon atoms (i.e., C)_1-8Representing 1-8 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.

Herein, unless otherwise specified, the term "substituted" means that one or more hydrogen atoms on a group are replaced with a substituent selected from the group consisting of: halogen, unsubstituted or halogenated C1-C6 alkyl.

Unless otherwise specified, all occurrences of a compound in the present invention are intended to include all possible optical isomers, such as a single chiral compound, or a mixture of various chiral compounds (i.e., a racemate). In all compounds of the present invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.

As used herein, "covalently labeled compound", "labeling compound", "covalently labeled molecule", "labeling molecule", "compound of formula I", "probe molecule of the invention" and "compound of the invention" may be used interchangeably to refer to a compound of formula I as described in the first aspect.

As used herein, a group "derived from" a compound or drug (e.g., a drug group as described herein) refers to a drug or compound that has lost some of the group or atoms (e.g., 1H atom) to form a group that can be attached to another moiety, such as an L group of the present application, and retain or substantially retain the original compound or drug activity.

As used herein, "structure with secondary or multiple release function" refers to a structure in which the trigger agent site is cleaved by the trigger agent (X-a cleavage) to generate a first QM intermediate that rapidly leaves through electronic rearrangement, thereby generating the core QM intermediate with the "multi-anchor" structure to which this patent refers. An exemplary structure is shown below (wherein A 'is as defined for the group A in formula I, and more preferably, A' is selected from the group consisting of a targeting recognition group, a leaving group).

Methylene benzoquinone (QMs)

Methylene benzoquinone (QMs) is a very reactive intermediate, and the structures which are most widely applied in nature are ortho-methylene benzoquinone (o-QMs) and para-methylene benzoquinone (p-QMs). For example, many animals and plants utilize QMs as a defense; QMs is involved in the anticancer process of epirubicin and its derivatives. In addition, QMs is also highly electrophilic and susceptible to attack by nucleophiles in living systems, and thus has a very strong potential to capture biomacromolecules and establish covalent bonds therewith. With this property, QMs is also widely used in research in the life science field for developing highly efficient and stable bioconjugate reagents. Subsequently, inhibitors, capture agents and detection means based on such strategies are further applied in the development of enzymes, antibodies and specific cell sorting. This strategy is largely limited by the low specificity of the QMs reactive intermediate due to the complex and numerous material bases involved in life processes and constituting organisms. Specifically, it is possible that QMs intermediate with high reactivity diffuses rapidly to the surroundings after the formation of target protein catalytic site due to molecular thermal movement, if it reacts rapidly and is linked with nucleophilic group of target protein catalytic reaction site; otherwise the activated QMs intermediate will covalently attach to a nucleophilic site in a nearby protein. Therefore, the former process may affect the structure and activity of the target enzyme, and then the target enzyme activity inhibition effect is generated, so that the method can be used for capturing protein and developing enzyme inhibitors; the latter can reduce the influence of chemical modification on the structure and activity of target protease, not only can more accurately reflect the activity of enzyme, but also can bring the 'waterfall amplification' effect of marking a plurality of proteins at the later stage through 'one-point triggering' of the enzyme, and further embodies the advantages of QMs intermediate in the biological application of chemical modification imaging reagents. QMs as a self-anchored imaging reagent is widely used in the activity detection and biomarker process of various important biological enzymes such as phosphatase, glycosidase, lipase, sulfatase, beta-lactamase, etc.

Generally, QM-based labeled probe molecules structurally comprise: an enzyme recognition moiety, a potential capture group, a linker arm, and a reporter group. Wherein the enzyme recognition site plays a role in searching for a target substance and initiating a 'self-anchoring' process; the potential capture group, the QM precursor, performs a potential biomolecule "anchor" function, and plays a crucial role in the efficient covalent labeling of proteins. Currently, there are two main types of conventional QMs precursors: (1) o-monofluoromethylphenol or o-difluoromethylphenol derivative (o-QM precursor); (2) 4-alkylphenols, with leaving groups (such as fluorine, carbamate and carboxylate) attached at the benzyl (4-alkyl) position (p-QM precursor). Both are the spontaneous leaving of the leaving group induced by electron transfer after hydrolytic activation of the target enzyme, releasing the highly reactive QMs reactive intermediate in situ, which in turn initiates covalent linkage 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, so that the proper modification of the molecular structure of the probe is predicted, the specific recognition of the target enzyme to the probe is not influenced, and even the functionality of the molecule can be increased by the probe. For example, the detaching group of a p-QM type fluorescent probe is engineered to be a fluorescence quenching group, resulting in a fluorescent "off-on" low background, covalently labeled fluorescent probe.

By the above explanation, the enzyme activity-based "self-anchored" biomacromolecule labeling process involving methylenequinone (QM) should have promising application and development prospects. However, the main reason affecting the practical application of this strategy is the efficiency of labeling of the molecule with the biomacromolecule. Its labeling efficiency is mainly limited by the type of QMs, the rate of formation, the magnitude of electrophilic reactivity, and the stability of the finally formed covalent conjugate. If the marking efficiency is relatively low, the practical application of this strategy is significantly hampered, and prior studies have still not given a satisfactory answer. Therefore, the relation between the structure and the performance of QMs is researched, a self-anchoring type biomacromolecule labeling reagent with high labeling efficiency is searched, the signal-to-noise ratio of the reagent molecules in practical application can be effectively improved, the detection sensitivity of the reagent is improved, and the method develops a way for researching the fields of the biomarker technology and the biomedicine.

Multiple anchor points "

In the present invention, the compound (formula I) or the reagent according to the first aspect is a fluorescent probe obtained by linking an enzyme recognition group to a reporter group having a fluorescent response ability via a linker. The agent (or compound) promotes the breakage of A-X bond covalent bond under the action of a targeting recognition group, and rapidly generates a Quinone Methylene (QM) active intermediate with high reaction activity, and the active intermediate is easily attacked by nucleophilic reagent in a biological macromolecule structure, so that a covalent bond is formed between the active intermediate and the biological macromolecule. In addition, the reagent structure always contains a plurality of QM precursors, so that the reagent structure has the capability of activating and labeling the biological macromolecules for multiple times, namely the characteristic of multiple 'anchor points', and the reagent has the properties of targeted activation and efficient covalent labeling of the biological macromolecules, and the action principle is approximately shown in figure 6.

ALP

ALP, an important hydrolase in the body, is not only involved in the dephosphorylation process of various biomolecules, including nucleic acids and proteins, but also other small molecules; and has close relationship with the occurrence and development of a plurality of diseases (such as osteoblastic bone cancer, prostatic cancer and hepatitis), therefore, ALP is also used as an important biomarker in molecular biology and even clinical diagnosis. There have also been many studies on the detection method of ALP activity and distribution, among which the development of QM-based "self-anchoring" fluorescent probes is not rare. In view of the above, the present invention adopts ALP as a model enzyme, designs a fluorescent probe molecule, and intensively studies the relationship between the structure of a QM active intermediate as a capture unit and the labeling efficiency thereof, thereby screening and determining that a QM precursor structure having multiple leaving groups as a capture unit is advantageous for the practical application of the covalent bond labeling reagent. And realizes the activity detection of ALP in vitro and in vivo and the living cell imaging of the ALP over-expression tumor cells.

High-efficiency biological macromolecule covalent labeling compound containing multiple anchor points

The invention provides a high-efficiency biological macromolecule covalent labeling compound (or a compound shown in a formula I) containing multiple anchor points, wherein the reagent generates a high-reactivity O-or P-QM intermediate under the action of alkaline phosphatase hydrolysis phosphate ester bonds, and the high-reactivity O-or P-QM intermediate is covalently linked with nucleophilic groups in a protein structure around an activation site, so that the reagent has the property of detecting or labeling ALP.

Typically, the invention provides a high-efficiency biological macromolecule covalent labeling compound (namely, a compound shown in formula I) containing multiple anchor points, the reagent is shown in formula I,

in the formula (I), the compound is shown in the specification,

a is a targeted recognition group (such as phosphate, glycoside, 3-oxo-n-butyl or deoxynucleoside) capable of being recognized by a biological macromolecule (such as enzyme, protein, nucleic acid, etc.) and interacting therewith to generate a phenolic hydroxyl group (mercapto or amino), or a group (such as hydroxyl (OH) or a hydroxyl, mercapto or amino protecting group (such as a photosensitive protecting group (photosensitive reagent)) which is sensitive to an external means (such as light, heat or chemical induction) and causes cleavage of the covalent bond between A and X to generate a phenolic hydroxyl group (mercapto or amino); or a structure having a "secondary release function", e.g., catalytic hydrolysis to first form one or more leaving functional groups (e.g., o, p-QM potential), which are then removed to release the o, p-QM intermediate described herein;

x is O, NH or S;

LG with gem-difluoro (CHF)₂) The aromatic ring mother nucleus together form a multi-anchor functional group, wherein LG can be F, Cl, Br, OAc, OCONHR^a(R^a＝Et、Me、C₆H₅NO₂Etc.), sulfones (R)^bSO₂) Or quaternary ammonium salts (e.g. -NR)^c ₃ ⁺) And the like common leaving groups;

l is a connecting group constructed in various ways, such as an amido bond, an ester bond, an ether bond or a connecting group established in a click chemistry mode;

r is a reporter group with fluorescent, radioactive or magnetic signals, or a drug molecule and a prodrug with therapeutic functions.

Preferably, the photosensitive protecting group is

(UV light triggered) or

(NIR light triggered).

Preferably, the therapeutically functional drug molecules and prodrugs are those derived from, for example, but not limited to, antibiotics (cephalosporins, vancomycin) for antibacterial, resistance to drug resistance; abamebactam, an inhibitor for inhibiting serine beta-lactamase; sunitinib selectively targets and acts on multiple receptor tyrosine kinases, blocks tumor growth and directly attacks tumor cells, and achieves the aim of resisting tumors; amantadine and acyclovir are used for resisting virus.

In another preferred embodiment, the multi-anchor-point high-efficiency biomacromolecule covalent labeling compound is represented by formula II, wherein Reporter is a Reporter group with fluorescent, radioactive or magnetic signals:

in another preferred embodiment, the biomacromolecule covalent labeling compound with multiple "anchor points" is represented by formula III, wherein Drug is a Drug molecule and a prodrug with therapeutic functions:

further, R in the reagent is a reporter group with fluorescence, and the structure of a fluorescent probe (ALP-6) of which the reporter group is fluorescein is as follows:

further, the preferential fluorescent probe can be selectively recognized and hydrolyzed by alkaline phosphatase, and generates QM intermediates having active chemical properties, thereby serving to covalently label ALP and its peripheral proteins.

Further, ALP-6 is specifically activated by alkaline phosphatase (ALP) hydrolysis and generates O-or P-QM intermediates which are then covalently linked to nucleophilic groups in the protein structure surrounding the activation site, thereby acting as a marker and detection of ALP activity or labeling of tumor cells overexpressing ALP.

Further, R in the reagent is a reporter group with fluorescence, and the structure of a fluorescent probe (ALP NIR-2) of which the reporter group is a fluorescence-enhanced dye is as follows:

preferably, ALP NIR-2 can be covalently labeled on a target protein and peripheral proteins thereof through the same process as ALP-6 after ALP hydrolysis activation, and plays roles in ALP activity detection and ALP over-expression tumor cell localization; and the fluorescent intensity of the probe can be increased compared with that before the reaction through the establishment of a dye group conjugated system before and after the reaction, thereby achieving the purposes of relatively improving the target signal intensity, reducing the noise and improving the detection result. In addition, the emission wavelength of the fluorescent probe is positioned in a near infrared region, so that the characteristics of strong tissue permeability and small tissue damage of near infrared light can be utilized to lay a foundation for realizing in-vivo imaging of ALP in tumor of a tumor-bearing mouse.

Preparation method

The following describes more specifically the processes for the preparation of the compounds of formula (I) according to the invention, but these particular processes do not constitute any limitation of the invention. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains. Generally, in the preparative schemes, each reaction is typically in an inert solvent. The reaction time is usually 0.1 to 60 hours, preferably 0.5 to 48 hours.

Typically, the method for preparing the multi-anchor high-efficiency biological macromolecule covalent labeling compound (compound of formula I) provided by the invention comprises the following steps:

(i) reacting a compound of formula Ia (preferably formula IVa) in an inert solvent iCompounds such as compounds 4) and N₂-L²-R (Ib) to obtain a compound of formula I (or a compound of formula IV).

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

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

Preferably, the reaction time of step (i) is 0.1-2 hours (e.g. 0.5 hours), and/or the reaction temperature is room temperature (e.g. 20-30 ℃).

In one embodiment, compound 4 of the present invention can be prepared by reference to the following scheme:

in one embodiment, the compounds of formula I of the present invention may be prepared by the following scheme:

the process mainly introduces an enzyme recognition site through the phosphoric acid reaction of phenolic hydroxyl; subsequently, a plurality of potential leaving groups are introduced into the QM parent core structure by modifying the potential QM parent core structure; as the final covalent bond binding "anchor"; the subsequent introduction of a late-modifiable azide group into the latent QM core is accomplished, followed by the linkage between the enzyme recognition group, the nucleophile capture group and the fluorescent reporter group by a "click chemistry reaction" between azide and alkyne groups.

In one embodiment, compound 1 is prepared by the following method:

2-bromo-1- [ 4-hydroxy-3- (hydroxymethyl) phenyl]-ethan-1-one with NaN₃Adding the mixture into a reaction bottle, and adding the mixture into N, N-Dimethylformamide (DMF);

reacting the reaction system with stirring at room temperature for 12 hours, and recrystallizing to obtain an azide intermediate of the raw material;

adding the intermediate, N-Diisopropylethylamine (DIPEA) and 4-Dimethylaminopyridine (DMAP) into a reaction bottle, adding ultra-dry acetonitrile, cooling to-20 ℃, and dropwise adding carbon tetrachloride and Diethyl phosphite (5, Diethyl phosphate); and after dropping, transferring the reaction solution to room temperature, stirring and reacting for 1 hour, spin-drying the solvent, adding dichloromethane for dissolving, washing with saturated saline solution, drying with anhydrous magnesium sulfate, concentrating, and quickly separating and purifying by silica gel column chromatography to obtain the compound 1.

In one embodiment, compound 2 is prepared by the following method:

adding the compound 1 and dess-martin oxidant (DMP) into a reaction bottle, and adding dichloromethane for dissolving; reacting the reaction system at room temperature for 2 hours, adding saturated salt water, separating an organic phase, extracting a water phase by using dichloromethane, combining the organic phase, drying by using anhydrous magnesium sulfate, concentrating, and directly using an obtained intermediate crude product for the next experiment without further purification;

adding the intermediate crude product into a reaction bottle, adding redistilled dichloromethane for dissolving, and dropwise adding Diethylaminosulfur trifluoride (DAST) under an ice bath condition;

after dripping, stirring the reaction system in an ice bath for 2 hours for reaction, then quenching the reaction system by using water, extracting a water layer by using dichloromethane, collecting an organic phase, drying and concentrating the organic phase, and quickly separating and purifying the organic phase by using silica gel column chromatography to obtain the compound 2;

in another embodiment, compound 3 is prepared by the following method:

adding the compound 2 into a reaction bottle, adding methanol for dissolving, cooling in ice bath, and then adding NaBH₄(ii) a Stirring the reaction system under an ice bath condition for reaction for 0.5 hour, adding saturated ammonium chloride aqueous solution after the reaction is finished to terminate the reaction, extracting an aqueous phase by using dichloromethane, and drying and concentrating a combined organic phase to obtain a reaction intermediate crude product;

adding the crude product, Ethyl isocyanate (6, Ethyl isocyanate) and Triethylamine (TEA) into a reaction bottle, and adding redistilled dichloromethane to dissolve;

heating and refluxing the reaction liquid for 12 hours, adding water to quench excessive ethyl isocyanate after the reaction is finished, extracting with dichloromethane, combining organic phases, drying with anhydrous magnesium sulfate, concentrating, and performing silica gel column chromatography to obtain the compound 3.

In another embodiment, compound 1 is prepared by the following method:

under the protection of nitrogen, placing the compound 3 in a reaction bottle, adding ultra-dry acetonitrile for dissolving, and dropwise adding trimethyl bromosilane (TMSBr) under an ice bath condition;

after the dripping is finished, the reaction solution is placed at room temperature and stirred for reaction for 12 hours, after the reaction is finished, saturated ammonium chloride aqueous solution is used for quenching, and then the reaction solution is separated and purified by a reverse C18 column and is frozen and dried to obtain the compound 4.

In another embodiment, the compound of formula I is prepared by the following process:

the detection probe is obtained by reacting compound 4 with compound 7 (or 8) in an inert solvent.

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

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

Preferably, the reaction time is 0.1-2 hours (e.g., 0.5 hour), and/or the reaction temperature is room temperature (e.g., 20-30 deg.C)

Preferably, the method further comprises the step of isolating and/or purifying the detection probe; preferably, the step of isolating and/or purifying the detection probe is purification using a reverse phase C18 preparative column, freeze-drying.

The main advantages of the invention include:

(a) the compound of the present invention has the ability to produce QM active intermediates several times, and thus can label proteins several times;

(b) the compounds of the invention can reduce the ineffective target caused by quenching of QM active intermediate by other nucleophiles).

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

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise specified, in the embodiments of the present invention,¹H-NMR、¹³C-NMR was measured with a Bruker 400Mz type instrument using deuterated chloroform (CDCl)₃) Deuterated dimethyl sulfoxide (d)₆-DMSO), internal standard Tetramethylsilane (TMS); all solvents were chromatographically, analytically or chemically pure.

EXAMPLE 1.1 preparation of Compound 1

2-bromo-1- [ 4-hydroxy-3- (hydroxymethyl) phenyl]-Ethan-1-one (4g, 16.3218mmol) with NaN₃(1.53 g, 23.6mmol) was added to a reaction flask and N, N-Dimethylformamide (DMF) was added;

reacting the reaction system with stirring at room temperature for 12 hours, and recrystallizing to obtain 2.1168g of azidation intermediate of the raw material;

the above intermediate (500mg, 2.41mmol) was added to a reaction flask with N, N-diisopropylethylamine (DIPEA,1.0495 mL, 6.025mmol) and 4-dimethylaminopyridine (DMAP,29.9mg, 0.241mmol), ultra-dry acetonitrile was added, cooled to-20 ℃ and carbon tetrachloride (2.32mL, 24.1mmol) and Diethyl phosphite (5, Diethyl phoshite, 357. mu.L, 2.77mmol) were added dropwise;

after completion of the dropwise addition, the reaction mixture was stirred at room temperature for 1 hour, the solvent was dried by spinning, and then methylene chloride was added to dissolve the reaction mixture, and the mixture was washed with saturated brine, dried over anhydrous magnesium sulfate, concentrated and then subjected to silica gel column chromatography to purify the product quickly to obtain Compound 1(521mg, 63%).

The prepared compound 1 has the spectral characteristics that:¹H NMR(400MHz,CDCl₃)8.02(s,1H), 7.86(dd,J＝8.5,2.1Hz,1H),7.35(d,J＝8.5Hz,1H),4.72(s,2H),4.56(s,2H), 4.31–4.21(m,4H),1.38(t,J＝7.1Hz,6H).¹³C 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).

EXAMPLE 1.2 preparation of Compound 2

Compound 1(190.37mg, 0.55mmol) and dess-martin oxidizer (DMP,251.2mg, 0.592mmol) were added to a reaction flask and dissolved by adding dichloromethane (2 mL);

reacting the reaction system at room temperature for 2 hours, adding 5mL of saturated saline solution, separating an organic phase, extracting an aqueous phase with dichloromethane (5mL multiplied by 3), combining the organic phases, drying with anhydrous magnesium sulfate, concentrating, and directly using an obtained intermediate crude product for the next experiment without further purification;

adding the crude intermediate (180.5mg) into a reaction bottle, adding 5mL of redistilled dichloromethane for dissolving, and dropwise adding Diethylaminosulfur trifluoride (DAST, 136 mu L and 1.109mmol) under an ice bath condition;

after dropping, the reaction was stirred in an ice bath for 2 hours, followed by quenching with 2mL of water, extracting the aqueous layer with dichloromethane (5mL × 3) and collecting the organic phase, which was dried, concentrated and then rapidly separated and purified by silica gel column chromatography to obtain compound 2(144mg, 72%);

the prepared compound 2 has the spectral characteristics that:¹H 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).¹³C NMR(100MHz,CDCl₃)

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＝6.2 Hz),54.96,16.14(d,J＝6.6Hz).

EXAMPLE 1.3 preparation of Compound 3

Compound 2(201.6mg, 0.555mmol) was added to a reaction flask, dissolved in 5mL of methanol and cooled in an ice bath, followed by the addition of NaBH₄(31.5mg，0.833mmol)；

Stirring the reaction system under an ice bath condition for reaction for 0.5 hour, adding 2mL of saturated ammonium chloride aqueous solution after the reaction is finished to stop the reaction, extracting an aqueous phase by using dichloromethane (10mL multiplied by 3), and drying and concentrating a combined organic phase to obtain 161.3mg of a crude reaction intermediate product;

the crude product, Ethyl isocyanate (6, Ethyl isocyanate, 175. mu.L, 2.208mmol) and triethylamine (TEA, 307. mu.L, 2.208mmol) were added to a reaction flask, and 3mL of redistilled dichloromethane were added to dissolve;

the reaction mixture was refluxed for 12 hours, and after completion of the reaction, 2mL of water was added to quench excess ethyl isocyanate, and the mixture was extracted with methylene chloride (10 mL. times.3), and the organic phases were combined, dried over anhydrous magnesium sulfate, concentrated, and subjected to silica gel column chromatography to obtain Compound 3(115mg, 47%).

The prepared compound 3 has the spectral characteristics that: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, 1H),4.28–4.18(m,4H),3.61–3.43(m,2H),3.30–3.18(m,2H),1.36(t,J＝7.1 Hz,6H),1.15(t,J＝7.2Hz,3H).¹³C 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＝6.2 Hz),55.21,36.00,16.00(d,J＝6.6Hz),15.03.

EXAMPLE 1.4 preparation of Compound 4

Under the protection of nitrogen, placing the compound 3(115.2mg, 0.264mmol) in a reaction bottle, adding ultra-dry acetonitrile for dissolving, and dropwise adding trimethyl bromosilane (TMSBr,174 mu L, 1.320mmol) under the ice bath condition;

after dropping, the reaction mixture was left to stand at room temperature and stirred for reaction for 12 hours, and after completion of the reaction, the reaction mixture was quenched with a saturated aqueous solution of ammonium chloride, and then the reaction mixture was subjected to separation and purification by a reverse C18 column to obtain the compound 4(34 mg, 35%) after freeze-drying.

The prepared compound 4 has the spectral characteristics that:¹H 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＝6.0 Hz),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.

EXAMPLE 1.5 Synthesis of Compound 7

Placing 5-carboxyfluorescein (200.0mg, 0.532mmol), N-hydroxysuccinimide (91.8mg, 0.798mmol) and EDCI (153.0mg, 0.798mmol) in a reaction bottle, vacuumizing, replacing with nitrogen for 3 times, adding 2mL dry DMF under nitrogen protection until completely dissolving, stirring and reacting for 4h under ice bath condition, monitoring reaction by HPLC, adding a large amount of diethyl ether into reaction liquid for precipitation and centrifugation to obtain 144.5mg crude product 7-1

Dissolving 7-2(2.1631g, 4.865mmol) and EDCI (1.3989g, 7.297mmol) in 8mL DCM, adding propargylamine (347 μ L, 5.838mmol) under ice bath condition, slowly heating the reaction solution to room temperature, stirring, detecting by TLC until 2 of the raw material disappears completely, adding 10mL saturated saline, extracting with DCM (5mL × 3), combining the organic phases, drying with anhydrous magnesium sulfate, concentrating, and separating by silica gel column chromatography1.1144g of crude product 3 are obtained. 100.0mg of intermediate 7-3(0.333mmol) was dissolved in 2mL of a mixture of DCM/TFA/Tips (10:9:1) under ice-bath conditions, the reaction was stirred for 30min under ice-bath and monitored by HPLC for completion, after solvent spin-drying, TFA was removed by vacuum-pumping to give crude intermediate 4. 23.67mg of intermediate 1(0.05mmol) were weighed out and dissolved in 40. mu.L of DMF, followed by the addition of 30. mu.L each of intermediates 7-4 (C)_DMFAfter shaking the reaction for 1h at rt and then monitoring the reaction completion by HPLC, 2M, 0.06mmol) and 42 μ L TEA (0.3mmol), 21.4mg of compound 7 was obtained by precipitation with copious amounts of diethyl ether and centrifugation

Low resolution mass spectral data for compound 7: MS (ESI) calculation of m/z C₃₀H₂₆N₂O₉(M+H)⁺559.2, found 559.2; (M + Na)⁺581.2, found 581.2.

EXAMPLE 1.6 preparation of fluorescent Probe (ALP-6)

Compound 4(1.54mg, 2.76. mu. mol), compound 7 (C)_DMF636.8mM, 5 μ L, 1.79 μmol) was placed in a reaction flask, and dimethyl sulfoxide (DMSO,15L), water, vitamin C (1.3mg, 7.16mol), copper sulfate (30g,0.18mol), and tris (3-hydroxypropyltriazolomethyl) amine (THPTA,80g,0.18mol) were added;

the reaction system was reacted at room temperature for 0.5 hour, and the reaction was terminated, purified by reverse phase C18 preparative column, and freeze-dried to obtain a pale yellow compound, i.e., the fluorescent probe (ALP-6,1.0mg, 40%).

The spectrum of ALP-6 is characterized by: HRMS (ESI) m/z calculated value C₄₂H₄₁F₂N₆O₁₅P(M-H)^-937.2263, found 937.2256.

Compound 8 can be prepared according to prior art methods.

EXAMPLE 1.7 preparation of fluorescent Probe (ALP NIR-2)

Placing compound 4(2.90mg, 7.63. mu. mol) and compound 8(2.22mg, 4.59. mu. mol) in a reaction flask, adding dimethyl sulfoxide (DMSO), water, vitamin C, copper sulfate and tris (3-hydroxypropyl triazolylmethyl) amine;

the reaction system was reacted at room temperature for 0.5 hour, and the reaction was completed, purified by reverse phase C18 preparative column, and freeze-dried to obtain the blue compound, i.e., the fluorescent probe (ALP NIR-2,1.0mg, 31%).

The spectrogram is characterized in that: HRMS (ESI) m/z calculated value C₄₆H₄₄F₂N₅O₈P(M-H)^-862.2823, found 862.2819.

EXAMPLE 1.8 fluorescent probes

The other compounds in the list may be prepared by reference to the methods of example 1.5 and/or 1.6.

TABLE 1 chemical Structure of ALP detection Probe

Wherein A represents a general formula A, and B represents a general formula B.

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 no "anchor" ALP detection probes.

Example 2.1 labeling of proteins with alkaline phosphatase (ALP) activation by ALP-6 and analogs thereof

The experimental conditions are as follows: respectively incubating 7.5 mu M fluorescent probe (ALP-6 and its structural derivative ALP-0-5) and 12.5U/mL target enzyme ALP (12.5 mu g) in a water bath at 37 ℃ in the dark for 1 hour; alternatively, 1.5. mu.g of BSA as a peripheral protein of the target enzyme was added to the reaction mixture in a manner similar to the physiological environment, and the mixture was incubated under the same experimental conditions for 1 hour under dark conditions. The above protein samples were then subjected to SDS-PAGE gel electrophoresis under constant pressure conditions of 150V, and the protein gels were subjected to in-gel fluorescence scanning (bottom) and Coomassie blue staining (top) under Cy 2 channel (results see FIG. 1).

The experimental results are as follows: under simulated physiological conditions, the fluorescent probe ALP-6 and the analogue thereof can be activated and realize the labeling of a target protein ALP and surrounding proteins (such as BSA) after the specific recognition and hydrolysis of alkaline phosphatase; moreover, as can be seen from fig. 1, although the structures of different fluorescent probes are slightly different, the labeling effects on proteins in the system are obviously different; and the labeling effect of ALP-3, 4, 5 and 6 on proteins is relatively outstanding, and the fluorescent molecules have potential p-QM structures through structural comparison and analysis.

And (4) experimental conclusion: by modifying the structure of the fluorescent probe molecule, the 'self-anchoring' fluorescent probe molecules ALP-3, 4, 5 and 6 which have relatively ideal covalent labeling efficiency on proteins and all contain potential p-QM structures can be obtained. The potential p-QM structure is more favorable for establishing covalent bonds between capture groups and protein nucleophilic groups; and the existence of the QM intermediate can more effectively improve the labeling efficiency of the QM intermediate-based self-anchored fluorescent probe on the biomacromolecule.

Test example 2.2 non-specific binding of ALP-6 and analogs thereof to Bovine Serum Albumin (BSA)

The experimental conditions are as follows: respectively, 7.5 mu M fluorescent probes (ALP-6 and the structural derivative ALP-0-5) were incubated with 3 mu g BSA in a water bath at 37 ℃ for 1 hour in the absence of alkaline phosphatase (ALP) catalysis and in the absence of light. The above protein samples were then subjected to SDS-PAGE gel electrophoresis under constant pressure conditions of 150V, and the protein gels were subjected to in-gel fluorescence scanning (bottom) and Coomassie blue staining (top) under Cy 2 channel (results see FIG. 2).

The experimental results are as follows: by appropriate adjustment of the contrast, the fluorescent probe ALP-6 and its analogs have varying degrees of non-specific binding to the non-enzymatic protein BSA present in the system in the absence of alkaline phosphatase hydrolytic activation. The non-specific binding signals of probes ALP-1, 2, 3 and 4 were relatively significant, while the non-specific binding between the fluorescent probes ALP-5, 6 and BSA was very weak.

And (4) experimental conclusion: such non-specific binding between the fluorescent probe and the protein molecule is common, and the binding is not only a main reason for false positive of the fluorescent probe in the detection of the target substance, but also can greatly reduce the authenticity and the practicability of the fluorescent probe. The results of FIG. 2 show that the fluorescent signals displayed by the fluorescent probes ALP-5 and 6 during the detection of the target enzyme ALP are more accurate and real, and the fluorescent probes can truly reflect the covalent labeling efficiency of the fluorescent probe molecules on proteins after enzyme activation. With the combination of figure 1, the fluorescent probes ALP-5 and ALP 6 have excellent performance in covalent labeling of protein after enzyme activation, and the invention relates to a high-efficiency self-anchoring biomacromolecule covalent labeling compound which has good signal-to-noise ratio and can be specifically activated by enzyme.

Example 2.3 cellular imaging Using ALP-0, 2, 5 and 6

The experimental conditions are as follows: incubating 10 μ M fluorescent probes ALP-0, 2, 5 and 6 with HeLa cells overexpressing ALP on the cell membrane surface and HEK293 cells of normal tissues underexpressing ALP in a confocal culture dish for 1.5 hours in an incubator at 37 ℃; or the HeLa cells are pretreated for 1 hour by the alkaline phosphatase inhibitor p-BTO in advance and then incubated with different probes for 1.5 hours. The probe-containing medium was then discarded and washed three times with PBS buffer (pH 7.4) to remove probe molecules that did not establish covalent attachment to the membrane surface; 1mL of sterile PBS buffer (pH 7.4) was added, and cell imaging was performed using a fluorescence microscope (. lamda.)_ex＝470±20nm,λ_em＝515nm)。

The experimental results are as follows:

the experimental results are shown in FIG. 3, the left 7 groups are the labeling experiments of the fluorescent probes ALP-0, 2, 5 and 6 on the cell membrane under the activation of ALP on the HeLa cell membrane surface; the right 4 panels are labeled experiments with fluorescent probes ALP-0, 2, 5 and 6 on HEK293 cell membranes that express ALP poorly. FL in the figure is a green fluorescence signal; hoechst is a nucleus signal of Hoechst 3342 staining; BF is cell morphology under white light irradiation; merge is a composite map of the above several views; p-BTO ((-) -p-Bromotetramisole Oxalate) is a commercial inhibitor of ALP, Scale bar 50 μm.

As can be seen from FIG. 3, the fluorescent probes ALP-2, 5 and 6 with covalent bond "anchor point" can not only realize the identification of ALP over-expression cells, but also obviously distinguish ALP low-expression cell lines (HEK293) from high-expression cell lines (HeLa) compared with the ordinary fluorescent probe ALP-0 without labeling capability; and can realize 'anchoring' to the cell membrane of positive cells by utilizing QM active intermediate formed after ALP hydrolyzes the probe. In addition, in the ALP inhibitor group experiments, the fluorescent signals of the fluorescent probes ALP-2, 5 and 6 on the HeLa cell markers were reduced, which indicates that the initiation of the fluorescent probe labeling effect depends on the property of alkaline phosphatase specifically hydrolyzing phosphate bonds. Thirdly, by comparing the fluorescent semi-quantitative data of the fluorescent probes ALP-5 and 6 on the HeLa cell markers, it can be known that the fluorescent probe ALP-6 can more effectively detect the target enzyme and mark positive cells at the in vitro living cell level closer to the physiological environment.

And (4) experimental conclusion: the fluorescent probe ALP-6 can realize the identification and covalent labeling effect on tumor cells overexpressed by ALP at the level of in vitro living cells, thereby reducing the dispersion of the fluorescent probe in a cell imaging experiment, reducing background signals and improving the detection sensitivity. In addition, combining the previous experimental results and conclusions, it can be known that both the fluorescent probes ALP-5 and 6 have potential p-QM structure and low nonspecific protein binding force, and both exhibit high protein labeling effect in the running gel experiment; however, the fluorescent probe ALP-6 with a plurality of potential covalent bond anchor points can more effectively realize the covalent labeling effect of biological macromolecules at the cellular level, and has practical application value and prospect in the fields of biological labeling and biomedicine.

Example 2.4ALP NIR-2 and analogs thereof labeling of proteins with alkaline phosphatase (ALP) activation and non-specific binding to non-enzymatic proteins

The experimental conditions are as follows: incubating 5 μ M enhanced fluorescent probe (ALP NIR-2 and its structural derivatives ALP-0, 1) with 1.5 μ g BSA in water bath at 37 deg.C under the catalysis of target enzyme ALP for 2 hr in the absence or presence of light; alternatively, 5 μ M enhanced fluorescent probes were incubated with only 5U/mL ALP under the same conditions, protected from light, for 2 hours. The above protein samples were then subjected to SDS-PAGE gel electrophoresis under constant pressure conditions of 150V, and the protein gels were subjected to in-gel fluorescence scanning (bottom) and Coomassie blue staining (top) under Cy 5 channel (see FIG. 4).

The experimental results are as follows: under the specific recognition and hydrolysis action of alkaline phosphatase, the enhanced self-anchoring fluorescent probes ALP NIR-1 and 2 can both mark target enzyme and peripheral protein, and protein bands of lanes where the enhanced fluorescent probe ALP NIR-0 without covalent labeling action are not provided with fluorescent signals, namely, the common probe cannot achieve the purpose of marking the target enzyme and the peripheral protein. Furthermore, the covalently labelled fluorescent probe ALP NIR-1, 2 had a reduced labelling effect on alkaline phosphatase after pretreatment of ALP with the inhibitor p-BTO. It was further confirmed that the covalent labeling of proteins by this molecule of this type of "self-anchoring" probe depends on the activation of the potential QM structure by the target enzyme. In addition, the results of fluorescence scanning in protein gels show that the enhanced fluorescent probe ALP NIR-2 containing a plurality of potential covalent bond "anchor points" has slightly stronger covalent labeling effect on alkaline phosphatase and non-enzymatic protein BSA than the single "anchor point" fluorescent probe ALP NIR-1.

And (4) experimental conclusion: the experimental result shows that the enhanced self-anchored fluorescent probe molecules ALP NIR-1 and 2 with potential p-QM structures can effectively label target enzymes and surrounding proteins thereof through covalent bonds after the target enzymes are specifically hydrolyzed. And the enhanced fluorescent probe ALP NIR-2 with multiple potential covalent bond "anchors" enables more efficient labeling of proteins. It has further been shown that a potential p-QM structure with multiple covalent "anchor" linkages is more favorable for the establishment of covalent bonds between the capture group and the nucleophilic group of the protein.

Example 2.5 cell imaging Using ALP NIR-0, 1 and 2

The experimental conditions are as follows: incubating 5 μ M enhanced fluorescent probes ALP NIR-0, 1 and 2 with HeLa cells overexpressing ALP on the cell membrane surface and HEK293 cells of normal tissues underexpressing ALP on the cell membrane surface for 1.5 hours in a confocal culture dish respectively; or the HeLa cells are pretreated for 1 hour by the alkaline phosphatase inhibitor p-BTO in advance and then incubated with different probes for 1.5 hours. Followed byThe probe-containing medium was then discarded and washed three times with PBS buffer (pH 7.4) to remove probe molecules that did not establish covalent attachment to the membrane surface; 1mL of sterile PBS buffer (pH 7.4) was added, and cell imaging was performed using a fluorescence microscope (. lamda.)_ex＝682±12nm,λ_em＝ 721±41nm)。

The experimental results are as follows:

results are shown in FIG. 5, the left 5 columns are fluorescence probes ALP NIR-0, 1 and 2 for ALP positive cells (HeLa) and inhibitor pre-treatment positive cell labeling experiments; the right 3 columns show the labeling experiments of fluorescent probes ALP-0, 1 and 2 on HEK293 cells with low expression of ALP. FL in FIG. 5 is the green fluorescence signal; hoechst is a nucleus signal of Hoechst 3342 staining; BF is cell morphology under white light irradiation; merge is a composite map of the above several views; p-BTO ((-) -p-Bromotetramisole Oxalate) is a commercial inhibitor of ALP, Scale bar 50 μm.

Compared with the ordinary enhanced fluorescent probe ALP NIR-0 without the labeling capability, the enhanced fluorescent probes ALP-1 and 2 with covalent bond 'anchor points' can not only realize the identification of ALP over-expression cells, but also obviously distinguish ALP low-expression cell strains (HEK293) from high-expression cell strains (HeLa); moreover, the QM active intermediate formed after the ALP hydrolysis probe can be used to realize the 'anchoring' to the positive cells. In addition, in the ALP inhibitor group experiments, the fluorescent signals of the fluorescent probes ALP NIR-1 and 2 on ALP inhibited HeLa cells are weakened, which shows that the initiation of the covalent labeling effect of the fluorescent probes depends on the ALP to specifically hydrolyze the phosphoester bond to induce the process of producing QM intermediates. Thirdly, by comparing the effect of the fluorescent probes ALP NIR-1 and 2 on the labeling of HeLa cells, the enhanced fluorescent probe ALP NIR-2 with a plurality of potential covalent bond 'target points' can detect target enzymes and label positive cells more effectively and accurately at the in vitro living cell level closer to the physiological environment.

And (4) experimental conclusion: the multi-anchor-point enhanced fluorescent probe ALP NIR-2 can realize the identification and accurate positioning effects on tumor cells overexpressed by ALP at the level of in vitro living cells, thereby reducing the dispersion of the fluorescent probe in a cell imaging experiment, reducing background signals and improving the detection sensitivity. Furthermore, combining the results and conclusions of the previous experiments, it is clear that both enhanced fluorescent probes ALP NIR-1 and 2 have potential p-QM structure and highly efficient protein labeling; in addition, the specific detection capability of ALP NIR-2 on ALP is also more prominent. By combining the results, the enhanced fluorescent probe ALP NIR-2 with a plurality of potential covalent bond anchor points can more effectively realize the covalent labeling effect on the cell membrane surface protein at the cellular level, and has more promising prospect and practical application value in the fields of biological labeling and biomedicine.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1.A compound of formula I, characterized in that,

in the formula I, the compound is shown in the specification,

a is selected from the following group: a target recognition group, a leaving group, a secondary or multiple release functional structure;

x is selected from the group consisting of: o, NH, S;

l is a linking group;

r is selected from the group consisting of: a reporter group, a therapeutic group; wherein, the reporter group refers to a group which can be detected by fluorescence, radiation and/or magnetic signals, and the therapeutic group refers to a drug molecule and a drug precursor with therapeutic functions.

2. A compound of formula I according to claim 1,

a is selected from the following group: phosphoric acid group (-H)₂PO₃) (ii) a A glycoside; a deoxynucleoside; a 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);

x is selected from the group consisting of: o, NH or S;

L is L-Wa-L¹-Wb-; and Wb is linked to R;

in the group L, the alkyl group,

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 or 2}-five-or six-membered ring containing 1-3 nitrogen heteroatoms- (CH)₂)_0-2-preferably (c) the (c) is (c),

)；

L¹is 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 to 3 nitrogen heteroatoms (preferably, it is

(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；

R_aEach independently selected from the group consisting of: H. C1-C6 alkyl, C1-C6 haloalkyl, or substituted or unsubstituted C3-C6 cycloalkyl (preferably, R_aIs H);

r is selected from the group consisting of: a reporter group, a drug group; wherein, the reporter group refers to a group which can be detected or can emit fluorescence, radiation and/or magnetic signals, and the drug group refers to drug molecules and drug precursors with therapeutic functions;

3. The compound of claim 2, wherein the compound has one or more of the following characteristics:

(a) a is selected from the following group: phosphoric acid group (-H)₂PO₃)、

And R' is selected from the group consisting of: C1-C6 alkyl, C1-C6 haloalkyl; preferably, a is a phosphate group;

(b) x is O;

(c) LG is selected from the group consisting of: F. OCONHR^b(ii) a And R is^bEach independently selected from the group consisting of: C1-C6 alkyl (preferably, Et, Me), substituted or unsubstituted phenyl (preferably, C₆H₅NO₂)；

(d) Wa is- (CH)₂)_{0.1 or 2}-five-or six-membered ring containing 1-3 nitrogen heteroatoms- (CH)₂)_{0.1 or 2}- (preferably, Wa is

)；

(e) Wb is selected from the group consisting of: none, NR_a-CO；

(f)L¹Selected from the group consisting of: - (CH)₂)_n1-NHCO-(CH₂-O-CH₂)_n2-(CH₂)_n1-、-(CH₂)_n3-、-(CH₂)_n1-Ar¹-(CH₂)_n1-NHCO-(CH₂-O-CH₂)_n2-(CH₂)_n1-; and/or

(g) R is a reporter group; preferably, R is a group capable of fluorescing.

4. The compound of claim 1, wherein the compound of formula I is selected from the group consisting of:

5. a process for the preparation of a compound according to claim 2, comprising the steps of:

L²is ≡ L¹-Wb，

A、X、LG、L¹And Wb is as defined in claim 2.

6. A test reagent or test composition comprising a compound of claim 1 or 2 and a detectably acceptable carrier.

7. An intermediate, which is characterized in that the intermediate is shown as a formula Ia,

in the formula II, A, X, LG is as defined in claim 1 or 2.

8. A method for non-therapeutically inhibiting ALP overexpression in vitro comprising the steps of: contacting a subject with a compound of formula I, thereby inhibiting ALP overexpression.

9. A method of labeling cells overexpressing ALP comprising the steps of: contacting a subject with a compound of formula I, thereby inhibiting ALP overexpression.

10. Use of a compound according to claim 1, (i) for labeling, tracking and/or imaging of cells overexpressing ALP; and/or (ii) for the preparation of a composition for labeling, tracking and/or imaging cells overexpressing ALP; and/or (iii) for the preparation of a medicament or composition for the treatment of a disease associated with overexpression of ALP.