Fluoroboron dipyrrole compounds, autophagy-related protein probes, their preparation methods and applications

Abstract

This invention belongs to the field of biomedical technology, and particularly relates to fluoroboron dipyrrole compounds, autophagy-related protein probes, their preparation methods, and applications. The fluoroboron dipyrrole compounds possess pH sensitivity, target autophagy-related proteins (such as LC3 or P62 proteins), and exhibit high fluorescence quantum yields. They can be used for efficient and rapid screening of potential autophagy-related protein ligands, autophagy inhibitors, autophagy agonists, and molecular gels at the cellular level. Furthermore, this invention provides compounds that can serve as ligands for autophagy-related proteins. These compounds exhibit good affinity for LC3 protein and can be used to construct ATTEC molecules for the degradation of target substances such as proteins, organelles, and nucleic acids.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to fluoroboron dipyrrole (BODIPY) compounds, autophagy-related protein probes, their preparation methods and applications. Background Technology

[0002] Small molecule drugs have rapidly dominated the modern pharmaceutical market due to their potential oral bioavailability, ability to cross biological barriers, and ease of chemical synthesis and modification. Small molecule drugs exert their effects as enzyme inhibitors, enzyme activators, and receptor antagonists by occupying the active binding sites of their targets. However, only 15% of the available drug targets encoded by the human genome can be targeted by small molecule drugs. The remaining proteins typically have smooth surfaces with almost no binding sites, and some protein targets function through protein-protein interactions (PPIs). These "undruggable" proteins significantly limit the development of small molecule drugs.

[0003] Targeted protein degradation (TPD) is a breakthrough technology in the fields of chemical biology and medicinal chemistry in recent years. TPD technology offers new therapeutic strategies for "undruggable" proteins. Among them, protein hydrolysis-targeting chimeras (PROTACs) are currently the most widely used and mature technology in TPD research. PROTACs are heterobifunctional molecules that recruit proteins of interest (POIs) by hijacking endogenous E3 ligases, inducing POI ubiquitination, and subsequently degrading them via the ubiquitin-proteasome system (UPS). Although PROTACs have strong degradation capabilities, they are overly dependent on a limited number of E3 ligases, and genomic alterations to E3 ligases can lead to acquired resistance to PROTACs. Furthermore, PROTAC molecules can only degrade intracellular, soluble, and short-lived proteins, limiting their application in many diseases caused by extracellular proteins and protein aggregation, such as Huntington's disease and Alzheimer's disease.

[0004] Besides proteases, lysosomes are also ubiquitous metabolic organelles within cells, capable of degrading various substances such as proteins, nucleic acids, and organelles through autophagy. Autophagy has long been associated with multiple aspects of regulating immune responses, including pathogen capture, metabolic regulation, and cellular homeostasis. Current research indicates that autophagy is linked to the development of metabolic diseases, malignant tumors, immune dysregulation, and neurodegenerative diseases. Therefore, lysosomal degradation of biomolecules can greatly enrich the toolbox of targeted protein degradation and expand its application in human diseases. This has spurred the development of novel degradation technologies based on the autophagy-lysosomal pathway (ALP). Current novel degradation technologies include lysosome-targeting chimeras (LYTAC), autophagy-targeting chimeras (AUTAC), autophagy-targeting chimeras (AUTOTAC), and autophagosome-tethering compounds (ATTEC). These new technologies have the potential to expand the range of degradable targets from extracellular proteins to organelles.

[0005] LC3 protein, as a marker protein in autophagy, not only mediates autophagy but is also involved in processes such as the formation and fusion of autophagosomes. The technique of using LC3 protein to degrade proteins is called ATTEC technology. Currently, compounds that can be used as ligands for LC3 protein include 8F20, 10O5, AN1, AN2, and their analogues. However, the synthesis of ATTEC molecules based on existing ligands has shown corresponding limitations. 8F20 molecules are chiral, making their synthesis relatively difficult. 10O5 and AN1 have poor solubility, resulting in poor solubility for some ATTEC molecules; furthermore, these compounds exhibit cis-trans isomerism, leading to difficulties in compound synthesis and structural identification. AN2 molecules and their analogues are coumarin compounds, with poor solubility and low synthetic yields. These problems seriously hinder the development of ATTEC technology.

[0006] Similar to the LC3 protein, the P62 protein is also a key protein in the autophagy process. P62 can target LC3 through self-aggregation and participate in autophagy. The degradation technology mediated by P62 is called AUTOTAC technology. It is important to note that P62 can degrade not only monomeric proteins but also mediate the degradation of protein aggregates. Considering that P62 targets the LC3 protein after self-aggregation during autophagy, multiple target proteins can be degraded simultaneously during P62-mediated degradation, and the degradation efficiency may be superior to that of ATTEC molecules.

[0007] Molecular glues also induce the adjacency of PPIs, subsequently forming ternary complexes that degrade target proteins. They can be effective even at low concentrations, achieving long-term degradation of POIs. Compared to PROTACs and ATTEC technologies, molecular glues have smaller molecular weights, resulting in better oral bioavailability and pharmacokinetic properties. However, the discovery of new molecular glues often occurs accidentally, and the rational design of molecular glues remains a challenge. Summary of the Invention

[0008] The technical problem to be solved by this invention:

[0009] Based on the current state of development of target protein degradation technology, this invention proposes a novel fluoroboron dipyrrole compound, an autophagy-related protein probe, its preparation method, and its applications. The fluoroboron dipyrrole compound exhibits pH sensitivity, targeting properties for autophagy-related proteins (such as LC3 or P62 proteins), and high fluorescence quantum yield. It can be used for efficient and rapid screening of potential autophagy-related protein ligands, autophagy inhibitors, autophagy agonists, and molecular gels at the cellular level. Furthermore, this invention provides compounds that can serve as ligands for autophagy-related proteins. These compounds exhibit good affinity for LC3 protein and can be used to construct ATTEC molecules for the degradation of target substances such as proteins, organelles, and nucleic acids.

[0010] The present invention achieves the above objectives through the following technical solutions:

[0011] In a first aspect, the present invention provides fluoroboron dipyrrole compounds of general formula I or pharmaceutically acceptable salts or solvates thereof: R1, R2, and R3 are independently selected from hydrogen, halogen, hydroxyl, and C. 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 6-12 Aryl, C 5-12 heteroaryl; optionally, the C 6-12 Aryl, C 5-12 The heteroaryl group is independently and optionally selected by one or more halogens, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH; Linking group A is C1-C 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 alkynyl chain; and R 10 -Selected from halogen anions.

[0012] Preferably, R1 and R2 are independently selected from hydrogen and C. 1-6 Alkyl groups (e.g., -CH3, -Et); R3 is selected from hydrogen, C 1-6 Alkyl groups (e.g., -CH3, -Et), C 6-12 Aryl (e.g., phenyl), C 5-12 Heteroaryl (e.g., pyridyl); optionally, the aryl or heteroaryl group is selected independently by one or more halogens, C 1-6 Alkyl groups (e.g., -CH3, -Et), C 1-6 The functional group is substituted with an alkoxy group (e.g., -OCH3); the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH; the linking group A is C1-C. 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 alkynyl chain; R 10 - Selected from halogen anions (e.g., Br) - I - ).

[0013] In a second aspect, the present invention provides fluoroboron dipyrrole compounds of general formula II or pharmaceutically acceptable salts or solvates thereof: R1, R2, R4, R5, and R6 are independently selected from hydrogen, halogen, hydroxyl, and C. 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 6-12 Aryl, C 5-12 heteroaryl; optionally, the C 6-12 Aryl, C 5-12 The heteroaryl group is independently and optionally selected by one or more halogens, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH; Linking group A is C1-C 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 alkynyl chain; and R 10 - Selected from halogen anions.

[0014] Preferably, R1 and R2 are independently selected from hydrogen and C. 1-6Alkyl groups (e.g., -CH3, -Et); R4 and R5 are independently selected from hydrogen, halogens (e.g., -Br), -OH, and C. 1-6 Alkyl group (e.g., -OCH3); R6 is selected from halogens (e.g., -Br, -I); the linking group A is C1-C. 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 alkynyl chain; R 10 - Selected from halogen anions (e.g., Br) - I - ).

[0015] Thirdly, the present invention provides fluoroboron dipyrrole compounds of general formula III or their pharmaceutically acceptable salts or solvates: R1 and R2 are independently selected from hydrogen, halogen, hydroxyl, and C. 1-6 Alkyl, C 1-6 alkoxy- or halogen-substituted C 1-6 Alkyl, C 6-12 Aryl, C 5-12 heteroaryl; optionally, the C 6-12 Aryl, C 5-12 The heteroaryl group is optionally surrounded by one or more elements, each independently selected from halogens, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH; Linking group A is C1-C 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 alkynyl chain; and R 10 - Selected from halogen anions.

[0016] Preferably, R1 and R2 are independently selected from hydrogen and C. 1-6 Alkyl groups (e.g., -CH3, -Et); linking group A is C1-C 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 alkynyl chain; R 10 - Selected from halogen anions (e.g., Br) - I - ).

[0017] Fourthly, the present invention provides fluoroboron dipyrrole compounds of general formula IV or pharmaceutically acceptable salts or solvates thereof: R1, R2, and R9 are independently selected from hydrogen, halogen, hydroxyl, and C. 1-6 Alkyl, C 1-6 alkoxy- or halogen-substituted C 1-6 Alkyl, C 6-12 Aryl, C 5-12 heteroaryl; optionally, the C 6-12 Aryl, C 5-12 The heteroaryl group is independently and optionally selected by one or more halogens, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH; R7 is selected from -O-, -NH-, -CH2NH- (benzene ring attached to the left and A group attached to the right), -CH2O- (benzene ring attached to the left and A group attached to the right), and -CONH- (benzene ring attached to the left and A group attached to the right); R8 is selected from hydrogen, -OBn, and -OCH2CH2Ph; Linking group A is C1-C 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 alkynyl chain; and R 10 - Selected from halogen anions.

[0018] Preferably, R1 and R2 are independently selected from hydrogen and C. 1-6 Alkyl group (e.g., -CH3, -Et); R7 is selected from -O-, -NH-, -CH2NH- (benzene ring attached to the left), -CH2O- (benzene ring attached to the left), -CONH- (benzene ring attached to the left); R8 is selected from hydrogen, -OBn, -OCH2CH2Ph; R9 is selected from hydrogen, halogen (e.g., -F); the connecting group A is C1-C. 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 alkynyl chain; R 10 - Selected from halogen anions (e.g., Br) - I - ).

[0019] Fifthly, the present invention provides compositions comprising a pharmaceutically acceptable carrier and the said fluoroboron dipyrrole compound or a pharmaceutically acceptable salt or solvate thereof.

[0020] Sixthly, the present invention provides the use of the aforementioned fluoroboron dipyrrole compounds or their pharmaceutically acceptable salts or solvates, or the compositions thereof, in the preparation of products for screening autophagy-related protein ligands, autophagy agonists, autophagy inhibitors, and / or products for screening molecular gels. The products include, but are not limited to, detection reagents or kits.

[0021] Preferably, the product is an LC3 protein probe or a P62 protein probe. For example, an LC3 protein probe or a P62 protein probe based on the autophagy pathway.

[0022] Preferably, the autophagy-associated protein ligand is a compound of general formula V or a pharmaceutically acceptable salt or solvate thereof: R 11 R 12 R 13 R 14 R 15 Independently selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkyl groups, CO2Me, halogen-substituted C 1-6 Alkyl, C 6-12 Aryl, C 5-12 Heteroaryl (5 to 12-membered heteroaryl); optionally, the C 6-12 Aryl, C 5-12 The heteroaryl group is independently and optionally selected by one or more halogens, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH.

[0023] Ideally, R 11 Selected from hydrogen, -OH; R 13 and R 15 Independently selected from hydrogen, C 1-6 Alkyl groups (e.g., -CH3, -Et), -CO2Me, C 6-12 Aryl (e.g., phenyl); R 12 Selected from halogens and halogen-substituted C 1-6 Alkyl groups (e.g., -CF3); R 14 Selected from hydrogen, C 1-6 Alkyl groups (e.g., -CH3).

[0024] In a seventh aspect, the present invention provides compounds of general formula V or pharmaceutically acceptable salts or solvates thereof: R 11R 12 R 13 R 14 R 15 Independently selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkyl groups, CO2Me, halogen-substituted C 1-6 Alkyl, C 6-12 Aryl, C 5-12 Heteroaryl (5 to 12-membered heteroaryl); optionally, the C 6-12 Aryl, C 5-12 The heteroaryl group is independently and optionally selected by one or more halogens, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH.

[0025] Ideally, R 11 Selected from hydrogen, -OH; R 13 and R 15 Independently selected from hydrogen, C 1-6 Alkyl groups (e.g., -CH3, -Et), -CO2Me, C 6-12 Aryl (e.g., phenyl); R 12 Selected from halogens and halogen-substituted C 1-6 Alkyl groups (e.g., -CF3); R 14 Selected from hydrogen, C 1-6 Alkyl groups (e.g., -CH3).

[0026] Eighthly, the present invention provides the use of the said compound or a pharmaceutically acceptable salt or solvate thereof in the preparation of ATTEC molecules. Attached Figure Description

[0027] Figure 1 The fluorescence intensity of the LC3 protein probe at different pH values ​​is shown.

[0028] Figure 2 Fluorescence images of the probe XXH-LC3-BOD (10 μM) in HeLa cells treated with different concentrations of compound 10O5 are shown.

[0029] Figure 3 The results show the screening of LC3 protein probes for LC3 protein ligands, autophagy inhibitors, autophagy agonists and molecular gels, and the competition curves are plotted based on fluorescence intensity.

[0030] Figure 4 The SPR results for LD5 are shown. The left graph shows the concentration-dependent curve. The right graph shows the response at different concentrations.

[0031] Figure 5The diagram shows the ability of LD5 to degrade the PCSK9 protein in the construction of PCSK9-ATTEC(PD1). The left panel shows the Western blot results, and the right panel shows the statistical results. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and the following embodiments. It should be understood that the drawings and the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0033] Terminology Explanation

[0034] As used in this article, "mn" refers to the range from m to n, as well as the subranges and individual point values ​​within it. For example, the expression "C1-C20" or "C1-20" covers the range of 1 to 20 carbon atoms and should be understood to also cover any subrange and each point value within it, such as C2-C5, C3-C4, C1-C2, C1-C3, C1-C4, C1-C5, C1-C6, and C1, C2, C3, C4, C5, C6, C7, C8, etc. For example, the expressions "C3-C10" or "C3-10" should also be understood in a similar way, such as encompassing any subrange and point value contained therein, such as C3-C9, C6-C9, C6-C8, C6-C7, C7-C10, C7-C9, C7-C8, C8-C9, etc., as well as C3, 4, 5, 6, 7, 8, 9, 10, etc. Other similar expressions in this document should also be understood in a similar way.

[0035] The terms “halogen” or “halogenated” as used herein should be understood to refer to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I) atoms.

[0036] As used herein, the term "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, connected to the rest of the molecule by single bonds. An alkyl group can have 1–20 carbon atoms, i.e., C1–C2. 20 Alkyl groups, such as C 1-4 Alkyl, C 1-3 Alkyl, C 1-2 Alkyl, C3 alkyl, C4 alkyl, C 1-6 Alkyl, C 3-6Alkyl. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or their isomers. A "subunit" is a group obtained by removing a hydrogen atom from a carbon atom containing a free valence electron, having two connection sites for attachment to the rest of the molecule. For example, "alkylene" or "alkyl subunit" refers to a saturated straight-chain or branched divalent hydrocarbon group.

[0037] As used herein, the term "alkylene" refers, alone or in combination with other groups, to a straight-chain or branched saturated divalent hydrocarbon group. For example, the term "C 1-20 "Alkylene" refers to alkylene groups having 1-20 carbon atoms, such as methylene, ethylene, propylene, butylene, pentylene, hexylene, 1-methylethylene, 2-methylethylene, methylpropylene, or ethylpropylene. The term "cycloalkylene" refers to a cyclic, saturated divalent hydrocarbon group. For example, the term "C..." 3-6 "Cycloalkylene" refers to cycloalkyl subunits having 3-6 carbon atoms, such as cyclopropyl subunits, cyclobutyl subunits, cyclopentyl subunits, cyclohexyl subunits, etc. The term "alkoxy subunit" refers to "-O-alkylene" or "alkylene-O-". "C 1-8 Examples of "alkoxy subunits" include, but are not limited to, -O-methylene, -O-ethylene, -O-propylene, -O-butylene, methylene-O-, ethylene-O-, propylene-O-, butylene-O-, etc.

[0038] As used herein, the term "alkenyl" refers to an unsaturated aliphatic hydrocarbon group consisting of a straight or branched chain of carbon and hydrogen atoms, possessing at least one double bond. Alkenyl groups can have 2–20 carbon atoms, i.e., "C6H ... 2-20 "Alkenyl", such as C2-4 alkenyl, C3-4 alkenyl. Non-limiting examples of alkenyl include, but are not limited to, vinyl, allyl, (E)-2-methyl vinyl, (Z)-2-methyl vinyl, (E)-but-2-alkenyl, (Z)-but-2-alkenyl, (E)-but-1-alkenyl, (Z)-but-1-alkenyl, etc.

[0039] As used herein, the term "alkynyl" refers to an unsaturated aliphatic hydrocarbon group consisting of a straight or branched chain of carbon and hydrogen atoms, possessing at least one triple bond. Alynyl groups can have 2–20 carbon atoms, i.e., "C6H2O". 2-8 "Alkyne group", such as C2-4 alkynyl group, C 3-4 Alkynyl. Non-limiting examples of alkynyl include, but are not limited to, ethynyl, prop-1-alkynyl, prop-2-alkynyl, but-1-alkynyl, but-2-alkynyl, but-3-alkynyl, etc.

[0040] As used herein, the term "cyclic hydrocarbon group" refers to a saturated or unsaturated non-aromatic cyclic hydrocarbon group composed of carbon and hydrogen atoms, preferably containing one or two rings. The cyclic hydrocarbon group can be a monocyclic, fused polycyclic, bridged, or spirocyclic structure. The cyclic hydrocarbon group can have 3-10 carbon atoms, i.e., "C 3-10 "Cyclic hydrocarbon group", such as C 3-8 Cyclic hydrocarbon group, C 3-6 Cyclic hydrocarbon group, C5 cyclic hydrocarbon group, C6 cyclic hydrocarbon group, C7 cyclic hydrocarbon group. Non-limiting examples of cyclic hydrocarbon groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclic [2.2.1]heptyl and spiro[3.3]heptyl. The term also covers cases where the C atom can be substituted with an oxygen (=O).

[0041] As used in this article, the term "cycloalkyl" refers to a saturated cycloalkyl group.

[0042] As used herein, the term "heterocyclic group" or "heterocyclic hydrocarbon group" refers to a monocyclic or bicyclic ring system (ternary to decacyclic, ternary to octagonal, ternary to heptary, quaternary to hexacyclic) having, for example, 3 to 10 (suitably 3 to 8, more preferably 3 to 7, especially 4 to 6) ring atoms, wherein at least one ring atom (e.g., 1 or 3) is a heteroatom selected from N, O, S, and P, and the remaining ring atoms are C. The ring system may be saturated (or understood as the corresponding "heterocyclic alkyl") or unsaturated (i.e., having one or more double and / or triple bonds within the ring). "Heterocyclic group" or "heterocyclic hydrocarbon group" is not aromatic. The term also covers cases where the C atom may be substituted with an oxo (=O) and / or the S atom on the ring may be substituted with one or two oxo (=O) atoms and / or the P atom on the ring may be substituted with one or two oxo (=O) atoms.

[0043] The heterocyclic groups used in this article can be, for example, four-membered rings, such as azirmonobutylene or oxobutylene; or five-membered rings, such as tetrahydrofuranyl, dioxalyl, pyrrolyl, imidazoalkyl, pyrazolealkyl, pyrrolinyl, oxopyrrolyl, 2-oxoimidazolidin-1-yl; or six-membered rings, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazine, 1,1-dioxo-1,2-thiazin-2-yl or trithiaalkyl; or seven-membered rings, such as diazirmonobutylene. The heterocyclic group may optionally be benzofused.

[0044] Heterocyclic groups can be bicyclic, without restriction, such as five-membered fused five-membered rings, such as hexahydrocyclopentane[C]pyrrole-2(1H)-yl ring; or five-membered fused six-membered bicyclic rings, such as hexahydropyrrolo[1,2-a]pyrazine-2(1H)-yl ring.

[0045] Heterocycles can be unsaturated, meaning they can contain one or more double bonds, without limitation. For example, unsaturated heterocycles containing nitrogen atoms can be 1,6-dihydropyrimidine, 1,2-dihydropyrimidine, 1,4-dihydropyrimidine, 1,6-dihydropyridine, 1,2-dihydropyridine, 1,4-dihydropyridine, 2,3-dihydro-IH-pyrrole, 3,4-dihydro-IH-pyrrole, 2,5-dihydro-IH-pyrroleyl, 4H-[1,3,4]thiadiazinyl, 4,5-dihydrooxazolyl, or 4H-[1,4]thiazinyl rings. Unsaturated heterocycles containing oxygen atoms can be 2H-pyran, 4H-pyran, or 2,3-dihydrofuran. Unsaturated heterocycles containing sulfur atoms can be 2H-thiaran or 4H-thiaran. Heterocycles can be benzofused, without limitation, for example, dihydroisoquinoline rings.

[0046] As used herein, the term "hydrocarbon chain" refers to a chain-like group composed of carbon and hydrogen atoms, which may be straight or branched. The hydrocarbon chain may be saturated (i.e., alkylene) or unsaturated, meaning it may contain one or more carbon-carbon double or triple bonds. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2-), 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,7-heptene (-CH2CH2CH2CH2CH2CH2-).

[0047] The term "optional" as used in this article means that the position may or may not have a substituent.

[0048] The "compound" described in this invention includes all stereoisomers, geometric isomers, tautomers, and isotopes.

[0049] The "compound" described in this invention may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all isomers include, such as enantiomers and diastereomers. Compounds containing asymmetric carbon atoms in this invention may be isolated in optically active pure form or in racemic form; the optically active pure form may be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0050] The "compound" described in this invention also includes geometric isomers; the geometric isomers may exist as mixtures or as separate E or Z structural forms.

[0051] The "compound" described in this invention also includes tautomer forms; the tautomer forms originate from the exchange of a single bond with an adjacent double bond and are accompanied by the migration of a proton.

[0052] The "compound" described in this invention also includes atoms of all isotopes, whether intermediates or the final compound; isotopic atoms include those having the same number of protons but different mass numbers, for example, hydrogen isotopes include deuterium and tritium. Furthermore, if desired, for example for specific therapeutic or diagnostic purposes, the compounds of this invention may incorporate isotopes or radioactive isotopes known in the art, such as... 3 H, 15 O、 13 C or 15 Nitrogen isotopes.

[0053] The "pharmaceutically acceptable salt" as described in this invention refers to a pharmaceutically acceptable salt that maintains the pharmacological activity of its parent compound while improving its physicochemical or metabolic properties. Such salts include acid addition salts or base addition salts prepared from pharmaceutically acceptable acids or bases (including organic acids, inorganic acids, organic bases, and inorganic bases), or mixtures thereof. In this invention, suitable inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or similar acids; suitable organic acids include fumaric acid, tartaric acid, lactic acid, acetic acid, citric acid, trifluoromethanesulfonic acid, mandelic acid, salicylic acid, or their analogues.

[0054] The compounds according to the invention can also exist in the form of solvates, such as hydrates (hemihydrates, monohydrates, dihydrates, trihydrates, etc.).

[0055] This invention provides a novel fluoroboron dipyrrole (BODIPY) compound of general structural formula I or a pharmaceutically acceptable salt or solvate thereof: R1, R2, and R3 are independently selected from hydrogen, halogen, hydroxyl, and C. 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 6-12 Aryl, C 5-12 Heteroaryl, wherein the C 6-12 Aryl, C 5-12 The heteroaryl group is optionally surrounded by one or more elements, each independently selected from halogens, C 1-6 Alkyl, C 1-6The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH; Linking group A is C1-C 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 alkynyl chain; and R 10 - Selected from halogen anions (e.g., I) - ).

[0056] In some technical solutions, R1 and R2 are independently selected from hydrogen, -CH3, and -Et; R3 is independently selected from hydrogen, -CH3, -Et, and C. 6-12 Aryl (e.g., phenyl), C 5-12 The aryl or heteroaryl group (e.g., pyridyl) is optionally substituted by one or more functional groups, each independently selected from -CH3, -Et, halogen, and -OCH3; the linking group A is C1-C. 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 Alkyne chain.

[0057] In a preferred embodiment of the present invention, R1 and R2 are C 1-6 alkyl.

[0058] In a preferred embodiment of the present invention, R3 is a methyl group.

[0059] In a preferred embodiment of the present invention, the linking group A is a C5 alkyl chain.

[0060] As an example, the fluoroboron dipyrrole (BODIPY) compound is:

[0061] As an example, the present invention also provides a method for preparing fluoroboron dipyrrole compounds with the above-described structure:

[0062] The present invention also provides compositions comprising a pharmaceutically acceptable carrier and a fluoroboron dipyrrole compound of general formula I or a pharmaceutically acceptable salt or solvate thereof.

[0063] The present invention also provides the use of fluoroboron dipyrrole compounds of general formula I or pharmaceutically acceptable salts or solvates thereof, or the compositions thereof, in the preparation of products for screening ligands for autophagy-related proteins (e.g., LC3 protein), autophagy agonists, autophagy inhibitors, and products for screening molecular gels.

[0064] In some embodiments, the product is a fluorescent probe with a fluoroboron dipyrrole structure. The probe is a small molecule fluorescent probe, such as an LC3 protein probe based on the autophagy pathway. This probe exhibits good pH responsiveness and LC3 protein targeting.

[0065] Experimental verification has shown that the LC3 protein probe can be used for efficient detection and screening of LC3 protein ligands, autophagy agonists and inhibitors, as well as efficient detection and screening of molecular gels, providing new methods and ideas for screening protein ligands at the cellular level.

[0066] This invention provides a novel fluoroboron dipyrrole compound of general structural formula IV or a pharmaceutically acceptable salt or solvate thereof: R1, R2, and R9 are independently selected from hydrogen, halogen, hydroxyl, and C. 1-6 Alkyl, C 1-6 alkoxy- or halogen-substituted C 1-6 Alkyl, C 6-12 Aryl, C 5-12 Heteroaryl, wherein the C 6-12 Aryl, C 5-12 The heteroaryl group is optionally surrounded by one or more elements, each independently selected from halogens, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH; R7 is selected from -O-, -NH-, -CH2NH-, -CH2O-, -CONH-; the left side of R7 is attached to the benzene ring, and the right side is attached to the A group; R8 is selected from hydrogen, -OBn, and -OCH2CH2Ph; Linking group A is C1-C 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 alkynyl chain; and R 10 - Selected from halogen anions.

[0067] In some technical solutions, R1 and R2 are independently selected from hydrogen and C. 1-6 Alkyl groups (e.g., -CH3, -Et); R7 is selected from -O-, -NH-, -CH2NH-, -CH2O-, -CONH-; R8 is selected from hydrogen, -OBn, -OCH2CH2Ph; R9 is selected from hydrogen, halogens (e.g., -F); R 10 - Selected from halogen anions (e.g., Br) - I- ); Linking group A is C1-C 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 Alkyne chain.

[0068] In a preferred embodiment of the present invention, R1 and R2 are C 1-6 Alkyl group.

[0069] In a preferred embodiment of the present invention, R7 is -O-.

[0070] In a preferred embodiment of the present invention, R8 is -OBn.

[0071] In a preferred embodiment of the present invention, R9 is hydrogen.

[0072] In a preferred embodiment of the present invention, the R 10 - For I - .

[0073] As an example, the fluoroboron dipyrrole compound is:

[0074] This invention also provides a method for preparing the aforementioned fluoroboron dipyrrole compounds:

[0075] The present invention also provides compositions comprising a pharmaceutically acceptable carrier and a fluoroboron dipyrrole compound of formula IV or a pharmaceutically acceptable salt or solvate thereof.

[0076] The present invention also provides the use of fluoroboron dipyrrole compounds of general formula IV or pharmaceutically acceptable salts or solvates thereof in the preparation of products for the detection of autophagy-related protein ligands, autophagy agonists, autophagy inhibitors and molecular gels.

[0077] In some embodiments, the product is a fluorescent probe with a fluoroboron dipyrrole structure. The probe is a small molecule fluorescent probe, such as a P62 protein probe based on the autophagy pathway. The probe exhibits good pH responsiveness and P62 protein targeting.

[0078] Experimental verification has shown that the probe can be used for efficient detection and screening of P62 protein ligands, autophagy agonists and inhibitors, as well as efficient detection and screening of molecular gels, providing new methods and ideas for screening protein ligands at the cellular level.

[0079] This invention also provides compounds represented by structural formula V or pharmaceutically acceptable salts or solvates thereof: R 11 R 12 R 13 R 14 R 15 Independently selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkyl groups, CO2Me, halogen-substituted C 1-6 Alkyl, C 6-12 Aryl, C 5-12 Heteroaryl, wherein the C 6-12 Aryl, C 5-12 The heteroaryl group is optionally surrounded by one or more elements, each independently selected from halogens, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH.

[0080] In some technical solutions, R 11 Selected from hydrogen, -OH; R 13 and R 15 Independently selected from hydrogen, -CO2Me, C 1-6 Alkyl groups (e.g., -CH3, -Et), C 6-12 Aryl (e.g., phenyl); R 12 C independently selected from halogens and halogen-substituted C 1-6 Alkyl groups (e.g., -CF3); R 14 Selected from hydrogen, C 1-6 Alkyl groups (e.g., -CH3).

[0081] In a preferred embodiment of the present invention, the R 11 For -OH, R 12 For -Cl, R 13 R 14 For -CH3, R 15 It is hydrogen.

[0082] As an example, the compound is:

[0083] As an example, the preparation method of the compound is as follows:

[0084] The compound represented by general formula V of the present invention has a good affinity for LC3 protein and can be used as an LC3 protein ligand to construct ATTEC molecules for the degradation of proteins, organelles and nucleic acids.

[0085] In summary, this invention provides fluoroboron dipyrrole compounds, autophagy-related protein probes, their preparation methods, and applications. The fluoroboron dipyrrole compounds possess pH sensitivity, target autophagy-related proteins (such as LC3 or P62 proteins), and exhibit high fluorescence quantum yields. They can be used for efficient and rapid screening of potential autophagy-related protein ligands, autophagy inhibitors, autophagy agonists, and molecular gels at the cellular level, providing new cellular-level insights for the discovery of ligands, autophagy agonists, autophagy inhibitors, and molecular gels in targeted protein degradation technology, and enriching the field of targeted protein degradation. Furthermore, this invention provides compounds that can serve as ligands for autophagy-related proteins. These compounds exhibit good affinity for LC3 protein and can be used to construct ATTEC molecules for the degradation of target substances such as proteins, organelles, and nucleic acids.

[0086] The present invention will be further described in detail below with reference to specific embodiments. It should also be understood that the following embodiments are only for further illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below. The following synthesis schemes only list the preparation methods of some compounds in the present invention. Referring to commonly used techniques and existing technologies in the art, those skilled in the art can use similar methods to synthesize the compounds of the present invention based on the above synthesis schemes. Unless otherwise specified, the eluent ratio refers to the volume ratio.

[0087] Example 1: Preparation of LC3 protein probe based on autophagy pathway

[0088] 1) Preparation of compound X-1

[0089] Under ice bath conditions, 4-diethylaminobenzoic acid (2 g, 10.36 mmol) was dissolved in ultradry dichloromethane (20 mL), and then oxalyl chloride (3.9 g, 30.95 mmol) was added dropwise. The mixture was reacted at room temperature for 30 minutes, and then evaporated to dryness to obtain the acyl chloride, which was used directly in the next step. Under ice bath conditions, benzotriazole (1.35 g, 11.34 mmol) and triethylamine (2 g, 19.8 mmol) were dissolved in ultradry dichloromethane (30 mL), and the prepared acyl chloride was added dropwise to the above solution. The mixture was reacted at room temperature for 2 hours. Silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) gave a pale yellow solid (1.89 g, yield 62%). m / z: 295.2 [M+]. 1HNMR (400MHz, DMSO-d6) δ8.25(m,2H),8.08(d,2H),7.77(t,1H),7.61(t,1H),6.86(d,2H),3.5(q,4H),1.18(t,6H).

[0090] 2) Preparation of compound X-2

[0091] Compound X-1 (1.89 g, 6.43 mmol) and pyrrole (0.52 g, 7.71 mmol) were dissolved in ultradry dichloromethane (20 mL), and then titanium tetrachloride (13 mL) was added dropwise. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, methanol (8 mL) was added to quench the reaction mixture. The solution was purified by silica gel column chromatography (dichloromethane:methanol = 100:1) to give a yellow solid (1.03 g, yield 66%). m / z: 243.1 [M+]. 1 H NMR (400MHz, DMSO-d6) δ11.77(s,1H),7.78(d,2H),7.10(s,1H),6.78(m,1H),6.74(d,2H),6.24(dd,1H),3.5(q,4H),1.18(t,6H).

[0092] 3) Preparation of compound X-3

[0093] Compound X-2 (120 mg, 0.5 mmol) was dissolved in ultradry dichloromethane (10 mL) under ice bath conditions, followed by the dropwise addition of POCl3 (0.105 mL), and the reaction was stirred at room temperature for 1 hour. Subsequently, a solution of 2,4-dimethyl-3-ethylpyrrole (185 mg, 1.5 mmol) in ultradry dichloromethane (5 mL) was added, and the reaction was carried out overnight at room temperature. Purification by silica gel column chromatography (dichloromethane:methanol = 20:1) yielded a red solid (100 mg, yield 68.59%). m / z: 348.2 [M+]. 1 H NMR(400MHz,DMSO-d6)δ12.10(s,1H),7.69(s,1H),7.30(d,2H),6.94(d,2H),6.78(s,1H), 6.62(d,1H),3.54(d,4H),2.46(d,2H),2.41(s,3H),1.69(s,3H),1.19(t,6H),1.08(t,3H).

[0094] 4) Preparation of compound X-4

[0095] Compound X-3 (170 mg, 0.58 mmol) was dissolved in toluene (10 mL) under ice bath conditions, followed by the addition of triethylamine (0.24 mL), and the reaction was stirred at room temperature for 1 hour. Then, under ice bath conditions, a boron trifluoride ether solution (0.3 mL) was slowly added dropwise to the reaction mixture. After the addition was complete, the reaction was also heated to 120 °C and reacted at 120 °C for 2 hours. After the reaction was complete, a saturated ammonium chloride solution was added to the reaction mixture, and the mixture was extracted with DCM. Silica gel column chromatography (petroleum ether: dichloromethane = 6:1) yielded a dark red solid (200 mg, yield 87.7%). m / z: 396.2 [M+]. 1 H NMR(400MHz,DMSO-d6)δ7.59(s,1H),7.23(d,2H),6.79(d,2H),6.49(s,1H),6.40(d ,1H),3.42(d,4H),2.38(d,2H),1.63(s,3H),1.23(s,3H),1.15(t,6H),1.00(t,3H).

[0096] 5) Preparation of compound X-5

[0097] Phosphorus oxychloride (1.82 mL, 19.9 mmol) was dissolved in ultradry DMF (1.82 mL) under ice bath conditions, stirred for 5 minutes, and then stirred at room temperature for 1 hour. Subsequently, compound X-4 (200 mg, 0.5 mmol) was dissolved in ultradry dichloromethane (10 mL) and added dropwise to the above phosphorus oxychloride DMF solution, and the reaction was stirred overnight. After the reaction was complete, the reaction was quenched by adding saturated sodium bicarbonate solution, and extracted with dichloromethane. Silica gel column chromatography (petroleum ether:dichloromethane = 6:1) gave a dark red solid (48 mg, yield 22.4%). m / z: 424.2 [M+]. 1 H NMR(400MHz,DMSO-d6)δ9.77(s,1H),8.16(s,1H),7.28(m,2H),6.82(d,2H),6.75(d ,1H),3.44(q,4H),2.61(s,3H),2.42(q,2H),1.70(s,3H),1.16(t,6H),1.02(t,3H).

[0098] 6) Preparation of compound X-6

[0099] Compound 5,7-dihydroxy-4-methyl-2H-benzopyran-2-one (1 g, 5.2 mmol), 1,5-diiodopentane (1.68 g, 5.2 mmol), and potassium carbonate (0.72 g, 5.5 mmol) were dissolved in DMF (30 mL), and the mixture was stirred at room temperature for 6 hours. The reaction was carried out by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1) to give a white solid (400 mg, yield 19.8%). m / z: 389.0 [M+].

[0100] 7) Preparation of compound X-7

[0101] Compound X-6 (400 mg, 1 mmol) and 2,3,3-trimethylindole (164 mg, 1 mmol) were dissolved in acetonitrile (10 mL) and refluxed at 85 °C for 48 hours under nitrogen protection. After the reaction was complete, the mixture was recrystallized from acetone to give a pale purple solid (293 mg, yield 52%). m / z: 419.0 [M+]. 1 H NMR(400MHz,DMSO-d6)δ10.52(s,1H),7.98(dd,2H),7.84(dd,1H),7.62(dd,1H),6.33(d,1H),6.30(d,1H) ,5.91(d,1H),4.50(t,2H),4.02(t,2H),2.85(s,3H),2.42(d,3H),1.89(m,4H),1.59(m,2H),1.53(s,6H).

[0102] 8) Preparation of compound X-8

[0103] Compound X-5 (100 mg, 0.18 mmol) and compound X-7 (77 mg, 0.18 mmol) were dissolved in ethanol (10 mL) and refluxed at 85 °C for 12 h under nitrogen protection. The solution was then purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to give a black solid (114 mg, yield 67%). m / z: 825.4 [M+]. 1H NMR(400MHz,DMSO-d6)δ10.48(s,1H),8.74(s,1H),8.49(d,1H),7.83(m,2H), 7.54(m,2H),7.47(d,1H),7.30(m,2H),7.23(s,1H),6.82(d,2H),6.28(m,2H), 5.82(d,1H),4.63(t,2H),3.97(t,2H),3.43(q,4H),2.62(s,3H),2.43(d,2H), 2.34(d,3H),1.71(s,6H),1.67(s,3H),1.23(s,6H),1.15(t,6H),1.03(t,3H).

[0104] Example 2: Preparation of P62 protein probe based on autophagy pathway

[0105] 1) Preparation of compound M-9

[0106] 3,4-Dibenzyloxybenzaldehyde (500 mg, 1.58 mmol) and sodium borohydride (356 mg, 9.4 mmol) were dissolved in anhydrous methanol (10 mL) and reacted at room temperature for 1 hour under nitrogen protection. After the reaction was complete, the solution was evaporated to dryness, quenched with dilute hydrochloric acid, extracted with ethyl acetate, and evaporated to dryness to give a white solid (420 mg, yield 83%). m / z: 321.4 [M+].

[0107] 2) Preparation of compound M-10

[0108] M-9 (420 mg, 1.31 mmol) and 1,5-diiodopentane (0.40 mL, 2.69 mmol) were dissolved in DMF (10 mL), and potassium hydroxide (150 mg, 2.68 mmol) was added. The mixture was reacted at room temperature for 2 hours. After the reaction was complete, the mixture was extracted with ethyl acetate and purified by column chromatography to give a colorless oil (420 mg, yield 62%). m / z: 517.4 [M+].

[0109] 3) Preparation of compound M-11

[0110] M-10 (420 mg, 0.81 mmol) and 2,3,3-trimethylindole (130 mg, 0.81 mmol) were dissolved in acetonitrile (10 mL) and refluxed at 85 °C for 48 hours under nitrogen protection. After the reaction was complete, the mixture was recrystallized from acetone to give a pale purple solid (110 mg, yield 25%). m / z: 548.3 [M+].

[0111] 4) Preparation of compound M-12

[0112] M-11 (110 mg, 0.20 mmol) and X-5 (85 mg, 0.20 mmol) were dissolved in ethanol (10 mL) and refluxed at 90 °C for 48 hours under nitrogen protection. After the reaction was complete, the solution was evaporated to dryness and purified by column chromatography to give a dark purple solid (92 mg, 50% yield). m / z: 953.5 [M+].

[0113] Example 3: Preparation of LC3 protein ligand LD5

[0114] 1) Preparation of compound Z1

[0115] 3,6-Dichloro-4-methylpyridazine (1.0 g, 6.13 mmol), (S)-1-phenylethane-1-amine (743.45 mg, 6.13 mmol), N,N-diisopropylethylamine (2.14 mL, 12.27 mmol), and N-methylpyrrolidone (20 mL) were added to a microwave reaction tube. The reaction system was microwave-treated at 150 °C for 2 hours. After the reaction was complete, the reaction mixture was extracted with ethyl acetate (3 × 30 mL), the organic phase was collected, washed with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate to remove water, and then concentrated. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) to give compound Z1 (1.03 g, yield 67.77%). m / z: 246.1 [M+].

[0116] 2) Preparation of compound LD5

[0117] Compound Z1 (1.0 g, 4.04 mmol), (4-chloro-2-hydroxyphenyl)boronic acid (695 mg, 4.04 mmol), potassium fluoride (703 mg, 12.11 mmol), and tetrakis(triphenylphosphine)palladium (253.58 mg, 0.201 mmol) were dissolved in a solvent and added to a microwave reaction tube. The solvent was a mixture of ethylene glycol dimethyl ether (DME), ethanol, and water in a volume ratio of 7:3:2. The reaction system was microwave-treated at 120 °C for 1 hour. The reactants were extracted with ethyl acetate. The reaction mixture was purified by silica gel column chromatography to give compound LD5 (1.26 g, 76.54% yield). m / z: 339.1 [M+]. 1 H NMR(600MHz,DMSO-d6)δ10.27(s,1H),7.41(d,2H),7.31(t,2H),7.28(d,1H),7.20(m ,1H),7.15(d,1H),6.92(m,2H),6.68(s,1H),5.16(t,1H),2.00(s,3H),1.47(d,3H).

[0118] Example 4: Fluorescence spectral changes of LC3 protein probe based on autophagy pathway at different pH values

[0119] In a quartz cuvette, MeCN / PBS = 3 / 7 (3 mL) solutions with different pH values ​​were added (the pH was adjusted using 1 M dilute hydrochloric acid and 1 M sodium hydroxide solution), followed by 3 μL of DMSO solution for the probe until the final probe concentration was 10 μM. The changes in the fluorescence spectrum of the probe at different pH values ​​were then measured. Figure 1 The fluorescence intensity of the LC3 protein probe is shown at different pH values. Figure 1 It can be seen that the fluorescence intensity of the LC3 protein probe at 600 nm gradually increases as the pH decreases. This indicates that the fluoroboron dipyrrole compound is pH sensitive.

[0120] Example 5: Verification of the ability of LC3 protein probes based on the autophagy pathway to screen LC3 protein ligands, autophagy agonists, inhibitors, and molecular gels.

[0121] HeLa cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (BI) and 1% penicillin and streptomycin (Sigma). The incubator environment was 37°C, 95% air, and 5% carbon dioxide. Cells were seeded in confocal dishes, and LC3 protein probe (final concentration 10 μM) was co-incubated with 10O5 (concentrations of 0, 100 nM, 500 nM, and 10 μM) for 4 hours. Cell fluorescence images were then captured using a fluorescence confocal microscope.

[0122] Figure 2Fluorescence images of the probe XXH-LC3-BOD (10 μM) in HeLa cells treated with different concentrations of compound 10O5 are shown. Near-infrared signals were collected at 753–703 nm (λex = 543 nm). Figure 2 As shown, the confocal fluorescence intensity gradually decreases with increasing 10O5 concentration. Therefore, the LC3 protein probe designed in this invention can be used for screening LC3 protein ligands, autophagy agonists, inhibitors, and molecular gels.

[0123] Example 6: Detection of the ability of LC3 protein probes based on the autophagy pathway to screen LC3 protein ligands, autophagy agonists, inhibitors, and molecular gels.

[0124] In a 96-well plate, the probe (final concentration 10 μM) was co-incubated with compounds (10O5, 10O5-Br, 10O5-OCH3, LD5, LD11, LD13). The fluorescence intensity in different wells was detected by a microplate reader, and curves were plotted to screen potential LC3 protein ligands, autophagy agonists and inhibitors.

[0125] Figure 3 The results show the screening of LC3 protein probes for LC3 protein ligands, autophagy inhibitors, autophagy agonists, and molecular gels, with competition curves plotted based on fluorescence intensity. Figure 3 It was observed that the fluorescence intensity gradually decreased with increasing concentrations of known LC3 ligands (10O5, 10O5-Br, 10O5-OCH3) and compound LD5. This indicates that LD5 interacts with the LC3 protein and is a potential ligand for the LC3 protein.

[0126] Example 7: Binding affinity of compound LD5 to LC3 protein

[0127] The binding kinetics between the LC3 protein and the compound LD5 were characterized using surface plasmon resonance (SPR) Biacore technology. The LC3 protein was immobilized on the surface of a CM5 sensor chip via amine coupling, with a blank channel used as a negative control for each assay. Following immobilization, a series of analytes were systematically injected at concentrations in HBS-EP buffer, allowing for real-time capture of sensor map data corresponding to binding events occurring on the sensor surface. To ensure the integrity of the binding data, the electrode surface was rigorously regenerated with a high-pH solution after each injection cycle to remove bound analytes. The dissociation constant (Kd value) was determined by fitting the sensor map data to a 1:1 Langmuir binding model using the BIAevaluation software package, providing a comprehensive analysis of the molecular interactions under investigation.

[0128] Figure 4The SPR results for LD5 are shown. The left graph is the concentration-dependent curve, and the right graph shows the response at different concentrations (from top to bottom: 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM). Figure 4 The Kd value of compound LD5 with LC3 protein is 2.54 μM, indicating that the compound has a good affinity for LC3 protein.

[0129] Example 8: Compound LD5 was used to construct PCSK9-ATTEC(PD1).

[0130] After co-incubating Huh7 cells with different concentrations of PLD5 (0-5 μM) for 24 hours, the cells were lysed using Ripa lysis buffer to obtain protein samples, which were then analyzed by Western blot. First, the protein samples were separated by electrophoresis at a constant voltage of 80 V, and then the separated proteins were transferred to a PVDF membrane at a constant current of 300 mA. Next, the membrane was blocked with skim milk to reduce non-specific binding, and then incubated with specific PCSK9 and GAPDH primary antibodies to allow the primary antibodies to bind to the target proteins. After washing the membrane, a labeled secondary antibody (HRP-labeled goat anti-mouse lgG(H+L)) was added for incubation, followed by washing again to remove unbound secondary antibody. Finally, the relative amounts of PCSK9 and GAPDH proteins were detected by reacting the labeled secondary antibody with ECL reagent to generate a visible light signal.

[0131] Figure 5 The diagram shows the degradation ability of LD5 against PCSK9 protein in the construction of PCSK9-ATTEC(PD1). The left panel shows the Western blot results, and the right panel shows the statistical results. Figure 5 It can be seen that PCSK9-ATTEC (PD1) can significantly reduce the content of PCSK9 protein, indicating that compound LD5 can serve as a ligand for LC3 protein and be used for the construction of ATTEC molecules.

[0132] The above detailed embodiments further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above are merely one specific embodiment of the present invention and are not limited to the scope of protection of the present invention. The present invention can be embodied in various forms without departing from its essential characteristics. Therefore, the embodiments described herein are for illustrative purposes only and not for limitation. Since the scope of the present invention is defined by the claims rather than the specification, all changes falling within the scope defined by the claims, or their equivalents, should be understood to be included in the claims. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fluoroboron dipyrrole compound of general formula I, or a pharmaceutically acceptable salt or solvate thereof: R1, R2, and R3 are independently selected from hydrogen, halogen, hydroxyl, and C. 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 6-12 Aryl, C 5-12 heteroaryl; optionally, the C 6-12 Aryl, C 5-12 The heteroaryl group is independently and optionally selected by one or more halogens, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH; Linking group A is C1-C 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 alkynyl chain; and R 10 - Selected from halogen anions.

2. A fluoroboron dipyrrole compound of general formula II or a pharmaceutically acceptable salt or solvate thereof: R1, R2, R4, R5, and R6 are independently selected from hydrogen, halogen, hydroxyl, and C. 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 6-12 Aryl, C 5-12 heteroaryl; optionally, the C 6-12 Aryl, C 5-12 The heteroaryl group is independently and optionally selected by one or more halogens, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH; Linking group A is C1-C 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 alkynyl chain; and R 10 - Selected from halogen anions.

3. A fluoroboron dipyrrole compound of general formula III or a pharmaceutically acceptable salt or solvate thereof: R1 and R2 are independently selected from hydrogen, halogen, hydroxyl, and C. 1-6 Alkyl, C 1-6 alkoxy- or halogen-substituted C 1-6 Alkyl, C 6-12 Aryl, C 5-12 heteroaryl; optionally, the C 6-12 Aryl, C 5-12 The heteroaryl group is independently and optionally selected by one or more halogens, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH; Linking group A is C1-C 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 alkynyl chain; and R 10 - Selected from halogen anions.

4. A fluoroboron dipyrrole compound of formula IV or a pharmaceutically acceptable salt or solvate thereof: R1, R2, and R9 are independently selected from hydrogen, halogen, hydroxyl, and C. 1-6 Alkyl, C 1-6 alkoxy- or halogen-substituted C 1-6 Alkyl, C 6-12 Aryl, C 5-12 heteroaryl; optionally, the C 6-12 Aryl, C 5-12 The heteroaryl group is independently and optionally selected by one or more halogens, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH; R7 is selected from -O-, -NH-, -CH2NH-, -CH2O-, and -CONH-. R8 is selected from hydrogen, -OBn, and -OCH2CH2Ph; Linking group A is C1-C 20 Alkyl chains, ether chains, sulfur heterochains, nitrogen heterochains, C2-C 20 Alkenyl chain, C2-C 20 alkynyl chain; and R 10 - Selected from halogen anions.

5. A composition comprising a pharmaceutically acceptable carrier and a fluoroboron dipyrrole compound or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 4.

6. The use of any one of the fluoroboron dipyrrole compounds or pharmaceutically acceptable salts or solvates thereof according to any one of claims 1 to 4, or the composition according to claim 5, in the preparation of products for screening autophagy-related protein ligands, autophagy agonists, autophagy inhibitors, and / or products for screening molecular gels.

7. The application according to claim 6, characterized in that, The product is an LC3 protein probe or a P62 protein probe.

8. The application according to claim 6 or 7, characterized in that, The autophagy-associated protein ligand is a compound of general formula V or a pharmaceutically acceptable salt or solvate thereof: R 11 R 12 R 13 R 14 R 15 Independently selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkyl groups, CO2Me, halogen-substituted C 1-6 Alkyl, C 6-12 Aryl, C 5-12 heteroaryl; optionally, the C 6-12 Aryl, C 5-12 The heteroaryl group is optionally surrounded by one or more elements, each independently selected from halogens, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH.

9. A compound of formula V or a pharmaceutically acceptable salt or solvate thereof: R 11 R 12 R 13 R 14 R 15 Independently selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkyl groups, CO2Me, halogen-substituted C 1-6 Alkyl, C 6-12 Aryl, C 5-12 heteroaryl; optionally, the C 6-12 Aryl, C 5-12 The heteroaryl group is optionally surrounded by one or more elements, each independently selected from halogens, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted; the heteroaryl group has 1 to 3 atoms or atomic groups independently selected from O, N, S, NH.

10. The use of the compound of claim 9 or a pharmaceutically acceptable salt or solvate thereof in the preparation of ATTEC molecules.

Images