Fluorescent bifunctional probes for protease activity

Abstract

This document discloses aspects of compounds according to Formula I or Formula II. It also discloses aspects of compositions and kits comprising the compounds. The compounds can be used as probes for protease activity, such as cathepsin B activity. Additionally, the compounds can be used to identify and / or monitor cancers, such as colorectal cancer, prostate cancer, ovarian cancer, or breast cancer. It also discloses aspects of methods for preparing the compounds and for using the compounds to monitor protease activity in vitro or in vivo.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefits of U.S. Provisional Application No. 63 / 689,102, filed August 30, 2024, and U.S. Provisional Application No. 63 / 599,926, filed November 16, 2023, pursuant to 35 USC § 119(e). The entire contents of the foregoing applications are incorporated herein by reference.

[0003] Reference to the electronic sequence list

[0004] This application includes a sequence list in .XML format that has been submitted electronically, and that sequence list is hereby incorporated herein by reference in its entirety. The copy of the .XML file, created on October 25, 2024, is named “TP386526WO1.xml” and is 9,418 bytes in size. The sequence list contained in that .XML file is part of the specification and is hereby incorporated herein by reference in its entirety. Technical Field

[0005] This article discloses aspects of compounds suitable for use as fluorescent probes. Methods for preparing and using these compounds are also disclosed. Background Technology

[0006] Proteases, such as cathepsin B, are involved in the degradation of proteins and organelles, antigen presentation, and the execution of cell death pathways. However, tools for real-time monitoring of lysosomal protein degradation in living cells, such as imaging platforms, are lacking. Furthermore, aberrant protease activity may be associated with a variety of cancers, including colorectal cancer, prostate cancer, ovarian cancer, and breast cancer. Significant efforts have been devoted to the development of molecular and medical imaging techniques surrounding general protease activity, particularly cathepsin B. Summary of the Invention

[0007] This article discloses aspects of compounds of formula I.

[0008]

[0009] Or its salt. Regarding formula I, X is -dye- or -N(-dye)-, where the dye is a fluorescent dye or a phosphorescent dye, the quencher is the excited-state energy-quenching part, the peptide is a protease-cleavable peptide moiety and may be oriented in a forward (N-terminus to C-terminus) or reverse (C-terminus to N-terminus) direction, R is a reactive group suitable for conjugating the compound to the target molecule; and L 1 L 2 and L 3 Each of them is independently selected from the following connectors: covalent bonds, or a portion containing two or more covalent bonds and at least one atom selected from C, N, O, P or S.

[0010] In some respects, the compound has a structure according to formula IA or IB.

[0011]

[0012] .

[0013] This article also discloses aspects of compounds according to Formula II.

[0014] .

[0015] This document also discloses aspects of compositions comprising compounds according to any one of Formula I, Formula IA, Formula IB, or Formula II. In some aspects, the composition comprises a compound conjugated to a biomolecule, such as an antibody, enzyme, protein, oligonucleotide, or dextran. The composition may also comprise a solvent, buffer, surfactant, or a combination thereof. Aspects of kits comprising compounds or compositions are also disclosed.

[0016] This article also discloses an aspect of a method comprising contacting cells with the compounds disclosed herein, exposing the cells to light, and determining whether a signal, such as a fluorescence signal or a phosphorescence signal, is present.

[0017] The above and other objects, features and advantages of the present invention will become more apparent from the following detailed description with reference to the accompanying drawings. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating an exemplary mechanism of action of the disclosed compound.

[0019] Figure 2 This is a graph comparing the average intensity of the antibodies, showing the signal intensity after treating Her-2 positive SKBR3 cells with compound I-1 conjugated with trastuzumab (trade name Herceptin) (anti-Her2 antibody) or EGFR positive A431 cells with compound I-1 conjugated with cetuximab (anti-EGFR antibody).

[0020] Figure 3 This is a graph comparing the average intensity of the antibodies, showing the signal intensity after treating Her-2 and EGFR-negative MCF7 cells with compound I-1 conjugated to trastuzumab (trade name Herceptin) (anti-Her2 antibody) or cetuximab (anti-EGFR antibody).

[0021] Figure 4 This is a graph showing the signal ratio of SKBR3 / MCF7 (trastuzumab) and A431 / MCF7 (cetuximab) compared to the antibody, illustrating the signal from... Figure 2 and Figure 3The ratio of the average fluorescence intensity (MFI) of the positive signal to the negative signal.

[0022] Figure 5 These are digital images showing the signals observed in Her-2 positive SKBR3 cells, EGFR positive A431 cells, and Her-2 and EGFR negative MCF7 cells after treatment with cetuximab-compound I-1 or trastuzumab-compound I-1 conjugates.

[0023] Figure 6 This is a graph showing the flow cytometry analysis of the rituximab-compound I-1 conjugate, and provides the signal-to-background ratio of Ramos and Jurkat cells, both treated with the rituximab-compound I-1 conjugate, as determined by the ratio of the mean fluorescence intensity (MFI) of the Ramos population to the Jurkat population.

[0024] Figure 7 This is a graph showing the flow cytometry analysis of the rituximab-compound I-1 conjugate and provides the signal-to-background ratio of Ramos cells treated with the rituximab-compound I-1 conjugate, as determined by the ratio of the mean fluorescence intensity (MFI) of the stained population to the unstained population.

[0025] Figure 8 This is a graph comparing cell counts with DAPI staining, showing the populations of live and dead cells in cells treated with rituximab-compound I-1 and CD19-FITC.

[0026] Figure 9A This is a diagram comparing CD19-FITC staining with rituximab-compound I-1 staining, showing Ramos cells that are positive for both CD20 and CD19, while the control Jurkat cell population is negative for both CD20 and CD19.

[0027] Figure 9B It is CD19-Alexa Fluor ™ The image shows Ramos cells stained with 700 stain compared to rituximab (anti-CD20) - compound I-7 conjugate, revealing positivity for both CD19 and CD20 in both cells. The Hu IgG1 isotype control - compound I-7 conjugate was used as a negative control to show background staining caused by compound I-7.

[0028] Figure 9C It is shown Figure 9B A graph of mean fluorescence intensity (MFI) analyzed by flow cytometry.

[0029] Figure 10This is a digital image showing the signal of SKBR3 cells treated with trastuzumab compound I-1 at different concentrations of the cathepsin B inhibitor CA-074 (Me).

[0030] Figure 11A The graph shows the average fluorescence intensity versus concentration, illustrating that the intensity of the trastuzumab-compound I-1 conjugate decreases with increasing amounts of the cathepsin B inhibitor CA-074 (Me).

[0031] Figure 11B The graph shows the average fluorescence intensity versus concentration, indicating that the intensity of the trastuzumab-compound I-7 conjugate in SKBR3 cells decreases with increasing CA-074 (Me) concentration.

[0032] Figure 11C The graph shows the average fluorescence intensity versus concentration, illustrating that the intensity of the cetuximab-compound I-7 conjugate in A431 cells decreases with increasing CA-074 (Me) concentration.

[0033] Figure 12 This demonstrates the trastuzumab-compound I-1 conjugate with LysoTracker. ™ Red (top image) and trastuzumab – pHrodo ™ Colocalization of the Red conjugate (middle image), or the trastuzumab-compound I-1 conjugate with BacMam ™ Digital images of colocalization of Lamp1-GFP (base map) indicate that the conjugate is localized to lysosomes.

[0034] Figure 13A Digital images of SKBR3 and MCF7 cells stained with trastuzumab-compound I-7 conjugate are shown, illustrating that HER2-positive SKBR3 cells produce signaling, while HER2-negative MCF7 cells do not.

[0035] Figure 13B The figure shows Her-2 positive SKBR3 cells and EGFR positive A431 cells treated with trastuzumab-compound I-7 conjugate, cetuximab-compound I-7 conjugate, or human IgG1 (hIgG1)-compound I-7 conjugate (control), indicating the specific internalization of trastuzumab-compound I-7 conjugate in SKBR3 cells and the specific internalization of cetuximab-compound I-7 conjugate in A431 cells.

[0036] Figure 14Digital images of SKBR3 and MCF7 cells stained with trastuzumab-compound I-3 conjugate are shown, illustrating that HER2-positive SKBR3 cells produce a signal, while HER2-negative MCF7 cells do not.

[0037] Figure 15 This is a graph comparing the signal-to-noise ratio with the concentration of cathepsin B, showing the fluorescence response of compounds II-1 (Res) and I-1 (DQ) to cathepsin B from 30 to 120 minutes.

[0038] Figure 16 This is a graph comparing the signal-to-noise ratio with the concentration of cathepsin B, showing the fluorescence response of compound I-1 (DQ) to cathepsin B from 30 to 120 minutes.

[0039] Figure 17 This is a graph comparing absorbance to wavelength, showing the absorbance and antibody labeling level of each batch of compound I-1.

[0040] Figure 18A and Figure 18B This is a graph of signal and signal-to-noise ratio versus cathepsin B concentration, comparing the concentrations from compound II-1 ( Figure 18A ) and coumarin-based cathepsin B substrate (Z-LR-AMC) ( Figure 18B (The signal).

[0041] Figure 19A and Figure 19B It shows the fluorescence spectrum ( Figure 19A ) and the response of Z-LR-AMC substrates to cathepsin B ( Figure 19B (The image is shown.)

[0042] Figure 20A and Figure 20B It shows the fluorescence spectrum ( Figure 20A ) and the response of compound II-1 to cathepsin B ( Figure 20B (The image is shown.)

[0043] Figure 21 This is a graph comparing the signal concentrations of cathepsin B, specifically comparing the signals of compound II-1 and the chlororhodamine substrate (PEG-LR-chlororhodamine).

[0044] Figure 22A and Figure 22B This is a graph comparing the signal concentration of cathepsin B with that of compound II-1 ( Figure 22A ) and compound II-2 ( Figure 22B The fluorescence signal of ).

[0045] Figure 23This is a diagram showing the reactivity of the free acid of compound I-1 with various cathepsins (cathepsin B, cathepsin D, and cathepsin L).

[0046] Figure 24 This is a diagram showing the inhibition of cathepsin B and cathepsin L by compound I-1.

[0047] Figure 25 Digital images of SKBR3 cells treated with trastuzumab-compound I-7 conjugate or Hu IgG1 isotype control-compound I-7 conjugate and subsequently fixed with 4% PFA. Images were acquired immediately after fixation and 24 hours after fixation.

[0048] Figure 26 It is shown Figure 25 A graph showing the quantitative results of signal / background in the image (trastuzumab / Hu IgG1 isotype control). Detailed Implementation

[0049] I. Terms and Definitions

[0050] The following explanations of terms and methods are provided to better describe the invention and to guide those skilled in the art in the practice of the invention. Unless the context clearly indicates otherwise, the singular forms “an,” “a,” and “the / described” mean one / a kind or more than one / a kind. Unless the context clearly indicates otherwise, the term “or” means a single element or a combination of two or more of the stated alternative elements. As used herein, “comprising” means “including.” Thus, “comprising A or B” means “including A, B, or A and B” without excluding additional elements. All references, including patents and patent applications, are incorporated herein by reference.

[0051] Unless otherwise indicated, all figures expressing the amount, molecular weight, percentage, temperature, time, etc. of the components as used in this specification or claims should be understood to be modified by the term "about". Therefore, unless otherwise indicated, the numerical parameters stated are approximate values, implied or explicit, and may depend on the desired properties sought and / or the detection limits under standard test conditions / methods. When embodiments are directly and explicitly distinguished from the prior art discussed, embodiment numbers are not approximate values ​​unless the word "about" is used.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials to those described herein may be used in practice or testing of this disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0053] "alkyl" refers to an alkyl group having 1 to 25 carbon atoms. 1-25 Carbon atoms, typically 1 to 10 (C) 1-10 Carbon atoms, such as 1 to 6 (C 1-6 ) carbon atoms or 1 to 4 (C 1-4 The term refers to the saturated aliphatic hydrocarbon moiety of a carbon atom. It includes, for example, straight-chain, branched, and cyclic (cycloalkyl) hydrocarbon groups. A cycloalkyl group may contain three to twenty-five carbon atoms, for example, three to fifteen, three to ten, or three to six carbon atoms. Exemplary alkyl groups include, but are not limited to, methyl (CH3), ethyl (-CH2CH3), n-propyl (-CH2CH2CH3), isopropyl (-CH(CH3)2), n-butyl (-CH2CH2CH2CH3), isobutyl (-CH2CH2(CH3)2), sec-butyl (-CH(CH3)(CH2CH3), tert-butyl (-C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), neopentyl (-CH2C(CH3)3), cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl.

[0054] "Alkenyl" refers to a group having one or more double bonds and 2 to 25 carbon atoms (C10, C2 ... 2-25 Carbon atoms, typically 2 to 10 (C) 2-10 Carbon atoms, such as 2 to 6 (C) 2-6 ) carbon atoms or 2 to 4 (C 2-4 The unsaturated aliphatic hydrocarbon group of a carbon atom. This term includes, for example, straight-chain, branched, and cyclic (cycloalkenyl) hydrocarbon groups. Cycloalkenyl groups can contain three to twenty-five carbon atoms, for example, three to fifteen, three to ten, or three to six carbon atoms.

[0055] "Alkyne" refers to a group having one or more triple bonds and 2 to 25 (C) groups. 2-25 Carbon atoms, typically 2 to 10 (C) 2-10 Carbon atoms, such as 2 to 6 (C) 2-6 ) carbon atoms or 2 to 4 (C 2-4 The unsaturated aliphatic hydrocarbon group of a carbon atom. This term includes, for example, straight-chain, branched, and cyclic (cycloalkynyl) hydrocarbon groups. Cycloalkynyl groups can contain three to twenty-five carbon atoms, for example, three to fifteen, three to ten, or three to six carbon atoms.

[0056] "Amine" refers to a moiety -NR'2, where each R' is independently H or C. 1-10 Alkyl groups, or two R' groups together with the nitrogen to which they are attached, form 5- to 8-membered heterocyclic groups.

[0057] "Aminooxy" refers to the group -ONH2.

[0058] Unless otherwise specified, "aryl" refers to an aromatic carbocyclic group (e.g., 1,2,3,4-tetrahydroquinoline, benzo[a]dioxane, etc.) having 6 to 15 carbon atoms, either a monocyclic (e.g., phenyl) or multiple fused rings (at least one of which is aromatic). If any aromatic ring moiety contains a heteroatom, the group is heteroaryl rather than aryl. Aryl groups can be, for example, monocyclic, bicyclic, tricyclic, or tetracyclic.

[0059] "Azides" refers to a portion of -N3.

[0060] "Cetase B probe" refers to the compounds of formulas I, IA, IB and II disclosed herein.

[0061] "Cyclooctyne" refers to a strained cyclic alkyne containing a ring of eight carbon atoms linked by seven single bonds and one triple bond. Exemplary cyclooctynes ​​include, but are not limited to, those containing... , , , , and .

[0062] "Ester" refers to a partial -CO2R', where R' is C 1-10 alkyl.

[0063] "Free acid" refers to a compound that contains an acidic moiety (such as a carboxylic acid or hydroxyl moiety) as a reactive group.

[0064] "Heteroaryl" refers to an aromatic ring system containing 5 to 15 ring atoms, wherein the ring atoms include at least one carbon atom and usually multiple carbon atoms, and at least one (such as one to five) heteroatoms. The heteroatoms can be nitrogen, phosphorus, oxygen, silicon, or sulfur atoms. The heteroaryl moiety can be a monocyclic moiety or can contain multiple rings, such as in a bicyclic or tricyclic ring system, provided that at least one of the rings contains a heteroatom.

[0065] A "heterocyclic group" refers to a non-aromatic ring system comprising 3 to 15 ring atoms, containing at least one carbon atom and usually multiple carbon atoms, and at least one (such as one to five) heteroatoms. The heteroatoms can be nitrogen, phosphorus, oxygen, silicon, or sulfur atoms. The heterocyclic group portion can be a monocyclic portion or can contain multiple rings, such as in bicyclic or tricyclic ring systems, provided that at least one of the rings contains a heteroatom. Such polycyclic portions can include fused or bridged ring systems and spirocyclic systems; and any nitrogen, phosphorus, carbon, silicon, or sulfur atom in the heterocyclic group portion can optionally be oxidized to various oxidation states.

[0066] "Acylhydrazide" refers to part of -C(O)NHNH2.

[0067] "Hydrazine" refers to a portion of -NHNH2.

[0068] "Hydroxy group" refers to a portion of -OH.

[0069] "Hydroxyamine" refers to a portion of -NH2OH.

[0070] "Glyoxal" refers to a portion of -C(O)C(O)H.

[0071] "Isocyanate" refers to a portion of -NCO.

[0072] "Isothiocyanate" refers to a portion of -NCS.

[0073] "Maleimide" refers to a portion of... .

[0074] "Oxadiazepine" refers to a portion of... .

[0075] A “peptide” is a compound that contains amino acid residues linked together by peptide bonds. As used herein, a peptide compound has 2 to 10 or more amino acid residues. In some respects, peptide compounds have 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues.

[0076] "Sulfonamide" refers to the part -SO2NR'2, where each R' is independently H or C. 1-10 Alkyl groups, or two R' groups together with the nitrogen to which they are attached, form 5- to 8-membered heterocyclic groups or 5- to 6-membered heteroaryl groups.

[0077] "Sulfonyl halide" refers to a partial -SO2X, where X is a halogen group, such as F, Br, Cl or I, preferably F, Br or Cl.

[0078] "Thioheterocyclic alkynes" refers to strained cyclic alkynes with sulfur atoms replacing carbon atoms, and includes 3,3,6,6-tetramethylthioheptyne (TMTH), TMTH-sulfinimide (TMTHSI), and compounds described by de Almeida et al., Angew. Chem. Int. Ed. Engl. 51:2443 (2012).

[0079] "Thiols" refers to some -SH groups.

[0080] "Vinyl" refers to the part -CH=CH2.

[0081] Unless otherwise defined herein, the term "biomolecule" refers to a molecule of any biological origin. In some respects, a biomolecule is selected from amino acids, peptides, proteins, antibodies, antibody fragments, enzymes, receptors, monosaccharides, polysaccharides, carbohydrates, lectins, ion complexes, nucleotides, oligonucleotides, nucleic acids, aptamers, haptens, drugs, toxins, lipids, phospholipids, lipoproteins, glycoproteins, hormones, lipopolysaccharides, liposomes, lipophilic polymers, non-biological organic polymers, polymeric microparticles, biological particles, animal cells, plant cells, bacteria, yeast, viruses, virus-like microparticles, and ligands. In some respects, a biomolecule is an antibody.

[0082] As used herein, the term "connector" refers to a single covalent bond or a portion containing a series of stable covalent bonds, typically incorporating 1 to 40 multivalent atoms selected from the group consisting of C, N, O, S, and P, which covalently attach the fluorescent compound to another portion, such as a chemically reactive group or a biological or non-biological component. The number of multivalent atoms in a connector can be, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, or greater, up to 40 or more. Connectors can be linear or non-linear; some connectors have overhanging side chains or overhanging functional groups, or both. Examples of such connectors include water-solubilizing groups, such as polyethylene glycol (PEG) groups. Examples of such overhanging portions are hydrophilic modifiers, such as solubilizing groups, such as, for example, sulfonyl groups. In some respects, connectors consist of any combination of single, double, triple, or aromatic carbon-carbon, carbon-nitrogen, nitrogen-nitrogen, carbon-oxygen, and carbon-sulfur bonds.

[0083] Unless otherwise defined herein, the term "PEG" or "polyethylene glycol" refers to any polyethylene glycol moiety - [CH2-CH2-O]. n - where n is an integer. In some respects, n is chosen from 3, 4, and 5.

[0084] Unless otherwise defined herein, the term "reactive group" means a chemically reactive portion as will be understood by one of ordinary skill in the art, and generally refers to an attachment site of another substance. A reactive group is a portion of the compounds disclosed herein capable of chemically reacting with functional groups on different compounds to form covalent bonds, such as carboxylic acids or succinimides. Reactive groups typically include nucleophiles, electrophiles, and photoactivated groups. Examples of reactive groups include, but are not limited to, alkenes, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, acylhydrazines, diazo groups, diazonium salts, nitro groups, nitriles, thiols, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, ketals, acid anhydrides, sulfate esters, hyposulfonic acid isonitriles, amidines, imides, imine esters, nitrones, hydroxylamines, aminooxy groups, oximes, isohydroxamic acids, and thioisohydroxamic acids. Propylene, orthoesters, sulfites, enamines, acetylenes, urea, pseudourea, aminourea, carbodiimide, carbonates, carbamates, carbamoyl halides, imides, azides, azo compounds, oxyazo compounds, nitroso compounds, acrylamides, activated esters of carboxylic acids, acyl azides, acyl halides, hydroxyl groups, alkyl halides, sulfonates, amines, aniline, aryl halides, aziridines, borate esters, diazonides, epoxides, diols, haloacetamides, halotriazins, imide esters, sulfonyl halides, and thiols. Reactive functional groups also include those groups used in the preparation of bioconjugates, such as succinimide esters (SE), N-hydroxysuccinimide esters, sulfonyl-succinimide esters, and other substituted succinimide esters; maleimides; reactive phenyl esters, such as dibromophenyl esters, nitrobenzene esters, thiophenyl esters, substituted thiophenyl esters, sulfonyl dichlorophenyl (SDP) esters, sulfonyl tetrafluorophenyl (STP) esters, tetrafluorophenyl (TFP) esters, pentafluorophenyl (PFP) esters; nitrotriacetic acid (NTA); aminoglucan; acetoxymethyl ester (AM); isocyanates, cyanates, isothiocyanates, thiocyanates; thioheptyne, and cyclooctyne, such as dibenzocyclooctyne (DIBO) or dibenzozacyclooctyne (DBCO). Methods for preparing each of these functional groups are well known in the art, and their application or modification for a particular purpose is within the capabilities of those skilled in the art (see, for example, Sandler and Karo, eds.). Organic Functional Group Preparations ,Academic Press, San Diego, 1989).

[0085] Selected examples of reactive groups and the parts they form and the connecting bonds are shown in Table 1, where the reactions of electrophilic and nucleophilic groups produce covalent bonds.

[0086] Table 1: Examples of certain reactive groups that form useful covalent bonds

[0087]

[0088]

[0089] II.Compounds

[0090] The disclosed compounds can be used as probes for protease activity, for example, to monitor lysosomal protein degradation pathways by conjugating the probe to a target protein. The probes can also act as activity-based lysosomal trackers in living cells and can be used to visualize lysosomal localization and antibody and protein degradation. The disclosed compounds contain a protease-cleavable peptide moiety that can be cleaved by enzymes such as cathepsin B, a lysosomal-specific enzyme. Outside the lysosome, the disclosed compounds exhibit very little or no background fluorescence, and the protease-cleavable peptide moiety is cleaved only when localized to the protease-rich environment of the lysosome, which separates the dye from the quencher, thereby generating a fluorescent signal from the unquenched dye.

[0091] In some respects, the compound has a structure according to general formula I.

[0092]

[0093] Or its salt.

[0094] With respect to formula I, X is either a -dye- or -N(-dye)-. In some respects, X is a -dye-, and the compound has a structure according to formula IA.

[0095]

[0096] In other respects, X is -N(-dye)-, and the compound has a structure according to formula IB.

[0097]

[0098] In terms of substitution, the compound has a structure according to general formula II.

[0099]

[0100] Or its salt.

[0101] Regarding Formulas I, IA, IB, and II: the peptide is a peptide moiety that can be cleaved by a protease and can be oriented in a forward (N-terminus to C-terminus) or reverse (C-terminus to N-terminus) direction; the dye is a fluorescent or phosphorescent dye; R is a reactive group suitable for conjugating the compound to a target molecule; if present, the quencher is the excited-state energy quenching moiety; and L 1 L 2 and L 3Each of them is independently selected from the following connectors: covalent bonds, or a portion containing two or more covalent bonds and at least one atom selected from C, N, O, P or S.

[0102] Especially regarding formula II, L 2 It is a covalent bond or a self-eliminating joint.

[0103] A. Quenching agent

[0104] The quencher may have an absorption range of 250 nm to 1,000 nm. In some aspects, the quencher comprises a quenching moiety selected from the following: azo dye-based quenchers, cyanine-based quenchers, rhodamine-based quenchers, azidophthalocyanine-based quenchers, heme chloride quenchers, malachite green, malachite green analogs, or combinations thereof. In some aspects, the quencher comprises one quenching moiety, but in other aspects, the quencher comprises two or more quenching moieties, such as two, three, four, or five quenching moieties.

[0105] In some respects, quenchers are derived from azobenzene quenchers, xanthonium dyes, nitrobenzoxadiazole compounds, or malachite green. In other respects, quenchers are or contain derivatives of:

[0106] , ,

[0107] , , , , , , , , , , , , or .

[0108] B. Dyes

[0109] The dye may be any fluorescent or phosphorescent dye suitable for use in conjunction with quenchers as disclosed herein. In some aspects, the dye is a cyanine dye, rhodamine or a rhodamine analog dye, fluorescein or a fluorescein analog dye, xanthine or a xanthine analog dye, BODIPY dye, coumarin dye, phthalocyanine dye, porphyrin dye, pyrene dye, fluorene or polyfluorene dye, excited-state intramolecular proton transfer (ESIPT) dye, aggregation-induced emission dye, metal complex dye, or combinations thereof. In some aspects, the dye is a fluorescein dye, rhodamine, group 14 rhodamine dye, phospharhodamine, cyanine dye having a 2-, 3-, 5-, 7-, or 9-methylene structure, or coumarin.

[0110] In some respects, dyes are derived from

[0111] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0112] In some aspects of Formula II, the dye is an acridinone dye, a fluorescein dye or an analogue thereof, a coumarin, a xanthine-hybrid cyanine dye, a phenolic dye, or an aniline dye. In other aspects, the dye is selected from...

[0113]

[0114] C. peptides

[0115] A peptide is any peptide that is cleaved by a target protease. In some respects, a peptide is a cathepsin B-cleavable peptide. In some respects, a peptide is selected from Gly-Gly-Phe-Gly (SEQ ID NO: 1), Val-Cit, Val-Cit-PAB, Gly-Gly, Gly-Gly-Gly, Phe-Lys, Val-Ala, Val-Gly, Val-Val, Ala-Ala-Asn, Gly-Phe-Leu-Gly (SEQ ID NO: 2), Arg-Arg, Phe-Arg-Arg-Gly (SEQ ID NO: 3), Gly-Phe-Leu-Gly-Lys (SEQ ID NO: 4), Lys-Lys, Lys-Lys-PAB, Gly-Arg-Arg-Gly-Lys-Gly-Gly (SEQ ID NO: 5), Gly-Glu-Leu-Gly (SEQ ID NO: 6). 6) Val-Arg, Glu-Arg or Gly-Ile-Val-Arg-Ala-Lys (SEQ ID NO: 7), wherein PAB is a para-aminobenzyl group.

[0116] In one respect, the peptide is Val-Cit.

[0117] In one respect, the peptide is Val-Cit-PAB.

[0118] Unexpectedly, it was found that the orientation of the protease-cleavable peptide moiety in the compounds presented herein affects the ability of the cleaved compounds to be immobilized with a fixative (such as formaldehyde). For conjugates having a protease-cleavable peptide moiety with a positive orientation (i.e., the dye is located on the N-terminal side of the peptide moiety, and the quencher is located on the C-terminal side of the peptide moiety), after protease cleavage, the remaining peptide moiety is retained on the dye, as shown in Scheme 1 below.

[0119]

[0120] However, for compounds containing a peptide moiety that is cleaved by a protease in a reverse orientation (i.e., the quencher is located on the N-terminal side of the peptide moiety and the dye is located on the C-terminal side of the peptide moiety), after cleavage, both the peptide moiety and the quencher are removed from the conjugate, leaving the dye and a free amine group, which can be used to react with a fixative (such as formaldehyde), as shown in Scheme 2 below.

[0121]

[0122] D. Reactive group R

[0123] In Formula I, R is a reactive group suitable for conjugating the compound to a target molecule. R can be acrylamide, activated ester of carboxylic acid, carboxylic acid ester, acyl azide, acyl nitrile, aldehyde, alkyl halide, acid anhydride, aniline, amine, aryl halide, azide, aziridine, borate ester, diazonium, haloacetamide, haloalkyl, halotriazine, hydrazine, imide ester, isocyanate, isothiocyanate, maleimide, phosphoramide, photoactivated group, reactive platinum complex, silyl halide, sulfonyl halide, and thiol. In some embodiments, the reactive group is selected from acrylamide, carboxylic acid, activated ester of carboxylic acid, acyl azide, acyl halide, hydroxyl group, aldehyde, alkyl halide, sulfonate, amine, acid anhydride, aniline, aryl halide, azide, aziridine, borate, carbodiimide, carbonate, carbamate, carbamoyl halide, diazonium, epoxide, glycol, haloacetamide, halomethyl, halotriazine, hydrazine, hydroxylamine, aminooxy, imide ester, iodoacetamide, isothiocyanate, ketone, maleimide. , sulfonyl halides, thiols, succinimides, substituted succinimides, sulfonyl-succinimides, reactive phenyl esters, dibromophenyl esters, nitrobenzene esters, sulfonodichlorophenyl esters, sulfonotetrafluorophenyl esters, tetrafluorophenyl esters, pentafluorophenyl esters, thiophenyl esters, substituted thiophenyl esters, hypozinotriacetic acid, isocyanates, cyanates, aminoglucan, acetoxymethyl esters, thioheptyne, and cyclooctyne, such as dibenzocyclooctyne (DIBO) or dibenzozacyclooctyne (DBCO). In some respects, R is an ester, isocyanate, isothiocyanate, maleimide, vinyl moiety, thiol, amine, hydroxyl, hydrazine, acylhydrazine, hydroxylamine, aminooxy, a clickable handle for click chemistry (e.g., azide, alkyne, tetrazine or transcyclooctene (TCO)), sulfonyl halide (e.g., sulfonyl bromide, sulfonyl chloride or sulfonyl fluoride), sulfonamide (e.g., sulfonyl triazole), glyoxal, oxaziridine or photoreactive moiety (e.g., benzophenone, aziridine or azide compound).

[0124] In some respects, R is selected from: , , , , , , Where R' is an aromatic or aliphatic group, , , , , , , , , , , , , , , , , , , , , , , , , , Where R” is an aromatic group or a substituted aromatic group, such as a phenyl or a substituted phenyl group, , and .

[0125] E. Connector

[0126] Connector L 1 L 2 and L 3 Each of these is independently a covalent bond or a portion containing two or more covalent bonds and at least one atom selected from C, N, O, P, or S. In some respects, L 1 L 2 and L 3 Each of these is independently selected from the following connectors: polyethylene glycol (PEG), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted unsaturated alkyl, alkenyl, alkoxy, alkanoyl, alkylamino, aryloxy, arylamino, aralkyl, arylalkoxy, arylanoyl, arylamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylalkyl, heteroarylalkoxy, heteroarylanoyl, heteroarylamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalanoyl, heterocyclic, heterocyclicoxy, heterocyclicamino, heterocyclic alkyl, heterocyclic alkoxy, heterocyclic alkanoyl, heterocyclic alkanoyl, heterocyclic alkanoyl, alkanoylamino, arylanoylamino, alkylcarboxyl, carbonate, carbamate, guanidine, urea, thiourea, phosphoryl, sulfonyl, sulfonamide, or ketone.

[0127] In some respects, L 1 L 2 and L 3 Each of the following is independently selected from: polyethylene glycol (PEG), substituted or unsubstituted C6 aryl groups, substituted or unsubstituted 5- to 10-membered heteroaryl groups containing one, two, or three heteroatoms selected from N, O, or S, substituted or unsubstituted C6 aryl groups. 1-6 Alkyl, substituted or unsubstituted C 2-6 Alkenyl, substituted or unsubstituted C 2-6 alkynyl group, C 1-6 Alkoxy, C 2-6 Alkyl (C 2-6 Alkyl-C(=O)-), C 1-6Alkylamino, C6 aryloxy, C6 arylamino, C 7-9 Araneyl, C 7-9 Arylalkoxy, C 7-9 Araneyl, C 7-9 Arylamino, (substituted or unsubstituted, 5- to 10-membered heteroaryl groups comprising 1, 2, or 3 heteroatoms selected from N, O, or S) -O-, (substituted or unsubstituted, 5- to 10-membered heteroaryl groups comprising 1, 2, or 3 heteroatoms selected from N, O, or S) -NH-, (substituted or unsubstituted, 5- to 10-membered heteroaryl groups comprising 1, 2, or 3 heteroatoms selected from N, O, or S) -C 1-6 Alkyl-, (substituted or unsubstituted 5- to 10-membered heteroaryl groups comprising one, two, or three heteroatoms selected from N, O, or S)-C 1-6 Alkyl-O-, (substituted or unsubstituted 5- to 10-membered heteroaryl groups comprising one, two, or three heteroatoms selected from N, O, or S)-C 1-6 Alkyl-C(=O)-, (substituted or unsubstituted 5- to 10-membered heteroaryl groups containing one, two, or three heteroatoms selected from N, O, or S)-C 1-6 Alkyl-NH-, C 3-7 cycloalkyl, C 3-7 Cycloalkenyl, C 3-7 Cycloalkylalkyl, C 3-7 Cycloalkoxy, C 3-7 Cycloalkyl acyl, C 3-7 Cycloalkylamino, substituted or unsubstituted, 5- to 10-membered heterocyclic group comprising 1, 2, or 3 heteroatoms selected from N, O, or S; (substituted or unsubstituted, 5- to 10-membered heterocyclic group comprising 1, 2, or 3 heteroatoms selected from N, O, or S) -O-; (substituted or unsubstituted, 5- to 10-membered heterocyclic group comprising 1, 2, or 3 heteroatoms selected from N, O, or S) -NH-; (substituted or unsubstituted, 5- to 10-membered heterocyclic group comprising 1, 2, or 3 heteroatoms selected from N, O, or S) -C 1-6 Alkyl- (substituted or unsubstituted 5- to 10-membered heterocyclic group comprising one, two, or three heteroatoms selected from N, O, or S) -C 1-6 Alkyl-O-, (substituted or unsubstituted 5- to 10-membered heterocyclic group comprising one, two, or three heteroatoms selected from N, O, or S)-C 1-6 Alkyl-C(=O)-, (substituted or unsubstituted 5- to 10-membered heterocyclic group containing one, two, or three heteroatoms selected from N, O, or S)-C 1-6 Alkyl-NH-, C 1-6 Alkylamino (-C 1-6 Alkyl-C(=O)NH-), -C6 aryl-C(=O)NH-, aralkylamide (-C6 aryl-C1-6 alkyl-C(=O)NH-), alkyl carboxyl group (-C 1-6 Alkyl-C(=O)-), carbonate (-OC(=O)O-), carbamate (-OC(=O)NH-), guanidinyl (-NHC(=NH)NH-), urea (-NHC(=O)NH-), thiourea (-NHC(=S)NH-), phosphoryl (-P(=O)(OH)-), sulfonyl (-SO2-), sulfonamide (-SO2NH-), or ketone (-C(=O)-).

[0128] In some respects, L 1 L 2 and L 3 Each of them is independently selected from -KLJ-, where each of K and J is independently a bond, -C(=O)-, -NR. a -、-OC(=O)-、-C(=O)O-、-OC(=O)NH-、-NHC(=O)O-、-SO2-、-SO2NH-、-NHSO2-、-NHC(=S)NH-、-P(=O)(OH)-、-NHC(=O)NH-、-NHC(=NH)NH-、-OC(=O)O- or -O-; and

[0129] L is -C 1-20 Alkyl-,-[(CH2CH2)O] x (CH2CH2)-,-(CH2) y -(substituted or unsubstituted C6 aryl)-(CH2) y -,-(CH2) y -(substituted or unsubstituted 5- to 10-membered heterocyclic group containing one, two, or three heteroatoms selected from N, O, or S)-(CH2) y -,-(CH2) y -(substituted or unsubstituted 5- to 10-membered heteroaryl groups containing one, two, or three heteroatoms selected from N, O, or S)-(CH2) y -,-(CH2) y -(substituted or unsubstituted C) 3-8 Alicyclic group)-(CH2) y - For each independent L, x is 1 to 6, and each y is independently 0 to 6, and R a It is H, C 1-4 Alkyl, 2,4-disulfonic acid benzyl ( ), sulfonyl alanine (cystic acid) residues ( ) or monosulfonic acid benzyl ( ).

[0130] In some respects, L1 L 2 or L 3 At least one of them is or contains PEG.

[0131] In some respects, L 1 L 2 or L 3 At least one of them is -KLJ-, where L is -[(CH2CH2)O] x (CH2CH2)-. In some respects, L 1 L 2 or L 3 At least two of them are -KLJ-, where each L is independently -[(CH2CH2)O]. x (CH2CH2)-.

[0132] In some aspects of Equation II, L 2 It is a self-eliminating connector and can be selected in any orientation.

[0133] , , or .

[0134] Exemplary compounds according to Formula I include:

[0135] ;

[0136] ;

[0137] ;

[0138] ;

[0139] ;

[0140] ;

[0141] ;

[0142] ;

[0143] ;

[0144] ;

[0145] ;

[0146] ;

[0147] ;

[0148] ;

[0149] ;

[0150] ;

[0151] ;and

[0152] .

[0153] Exemplary compounds according to Formula II include, but are not limited to:

[0154] or

[0155] .

[0156] Salt

[0157] In some aspects of Formula I, IA, IB, or II, the disclosed compounds are free base compounds, i.e., non-salts, and therefore do not contain one or more charged portions and one or more counterions. However, in other aspects of Formula I, IA, IB, or II, the disclosed compounds are salts. In such aspects, the salt may contain one or more counterions, such as 1, 2, 3, 4, 5, or more counterions. The counterion may be any suitable counterion, and when the salt contains more than one counterion, the counterions may be the same or different. In some aspects, the counterion is or contains substantially charged organic compounds, such as: trialkylammonium ions, such as triethylammonium ions; alkali metal ions, such as sodium ions, lithium ions, and / or potassium ions; and / or alkaline earth metal ions, such as magnesium ions and / or calcium ions.

[0158] target molecules

[0159] In any aspect of Formula I, IA, IB, or II, the target molecule may be any molecule suitable for conjugation with the disclosed compound. In some aspects, the target molecule is a biomolecule and may be a biomolecule containing an amine functional group. In some aspects, the biomolecule is selected from amino acids, peptides, proteins, antibodies, antibody fragments, enzymes, receptors, monosaccharides, polysaccharides, carbohydrates, lectins, ion complexes, nucleotides, oligonucleotides, nucleic acids, aptamers, haptens, drugs, toxins, lipids, phospholipids, lipoproteins, glycoproteins, hormones, lipopolysaccharides, liposomes, lipophilic polymers, non-biological organic polymers, polymer microparticles, biological particles, animal cells, plant cells, bacteria, yeast, viruses, virus-like microparticles, and ligands. In some aspects, the biomolecule is an antibody, antibody fragment, antibody-drug conjugate, enzyme, protein, peptide, aptamer, lectin, oligonucleotide, carbohydrate, hapten, drug, toxin, or dextran. In some aspects, the biomolecule is an antibody fragment.

[0160] III. Compositions and Kits

[0161] This document also discloses aspects of compositions comprising compounds as disclosed herein conjugated to a biomolecule. The biomolecule may be any biomolecule suitable for conjugation to the disclosed compounds. In some aspects, the biomolecule has an amine moiety suitable for conjugation to the compound. The biomolecule may be selected for uptake by cells or tissues containing a target protease, such that the conjugated compound can be used to interact with the protease. In some aspects, the biomolecule is an antibody, enzyme, protein, oligonucleotide, or dextran. And in any aspect, the composition may also contain one or more of a solvent, buffer, surfactant, or combinations thereof.

[0162] This document also discloses kits comprising the disclosed compounds or combinations thereof. In some aspects, the kit comprises one or more of the disclosed compounds and / or compounds conjugated to biomolecules. The kit may also comprise one or more of a solvent, buffer, surfactant, base suitable for conjugation, purification column, collection tube, or combinations thereof. In some aspects, the kit may include compounds that perform two or more functions in the kit. For example, the kit may contain sodium bicarbonate, which can act as both a buffer and a base.

[0163] The surfactant may be any surfactant suitable for use in conjunction with the disclosed compounds and compositions thereof. In some aspects, the surfactant is selected from polyoxyethylene lauryl ether (Brijn). ™ -35), polyoxyethylene (20) sorbitan monolaurate (Tween ™ -20), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton) ™ X-100), octylphenoxypolyethoxyethanol (Nonidet)™ P-40) or combinations thereof.

[0164] The buffer may be any buffer suitable for use with the disclosed compounds and their compositions, such as aqueous buffers, bicarbonate buffers, phosphate buffers, carbonate buffers, borate buffers, or combinations thereof. In some aspects, the buffer is selected from phosphate-buffered saline (PBS), tris(hydroxymethyl)aminomethane-HCl (Tris-HCl), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES), or combinations thereof. For example, bicarbonate buffer (pH about 8.3) may be used for the disclosed aspects of compounds containing succinimide esters; phosphate buffer (pH about 7.2 to about 8) may be used for the disclosed aspects of compounds containing thiol-reactive functional groups; and / or carbonate or borate buffer (pH about 9) may be used for the disclosed aspects of compounds containing isothiocyanate or dichlorotriazine reactive groups.

[0165] The solvent may be any solvent suitable for use with the disclosed compounds and their compositions. In some aspects, the solvent is selected from water, DMSO, methanol, or combinations thereof.

[0166] In some respects, the kit contains one or more of the following: a surfactant selected from polyoxyethylene lauryl ether (Brijn) ™ -35), polyoxyethylene (20) sorbitan monolaurate (Tween ™ -20), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton) ™ X-100), octylphenoxypolyethoxyethanol (Nonidet) ™ P-40) or combinations thereof; a buffer selected from phosphate-buffered saline (PBS), tris(hydroxymethyl)aminomethane-HCl (Tris-HCl), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES) or combinations thereof; and / or a solvent selected from water, DMSO, methanol or combinations thereof.

[0167] IV. Synthesis

[0168] The disclosed compounds can be prepared as illustrated below and as will be understood by one of ordinary skill in the art of organic synthesis. An exemplary synthesis may include the following first reaction steps according to Scheme 3.

[0169]

[0170] Regarding option 3, PG is suitable for preventing L. 3The compound has protecting groups reacting at both ends, and RG is a reactive group suitable for conjugation with a quencher. Protecting and reactive groups are disclosed herein and are known to those skilled in the art. Exemplary groups are provided in the examples herein and include BOC groups, amine and carboxylic acid groups, and their activated analogs. Further information regarding protecting groups can be found in Wuts and Greene, “Greene's Protective Groups in Organic Synthesis,” 4th edition, Wiley.

[0171] In some respects, L 3 The compound and quencher are combined in a solvent and in the presence of a base (such as an organic base, e.g., a trialkylamine base). Suitable solvents include, but are not limited to, DMF, THF, acetonitrile, or combinations thereof.

[0172] An exemplary second reaction step is shown in Scheme 4.

[0173]

[0174] Regarding option 4, L 3 - Quenching agent compounds are deprotected and conjugated to peptide moieties. Deprotection methods are disclosed herein and are known to those skilled in the art. Further information on specific deprotection methods for different protecting groups can be found in Wuts and Greene, “Greene's Protective Groups in Organic Synthesis”.

[0175] Peptides and L can be combined using any suitable technique. 3 Partial conjugation. Typically, peptides contain a reactive (such as an unprotected) amine or carboxylic acid moiety, and this moiety is conjugated with L... 3The carboxylic acid moiety is partially conjugated with a suitable reactive group, such as a carboxylic acid or an amine. Conjugation methods are disclosed herein and are known to those skilled in the art. In some aspects, the carboxylic acid moiety is activated and treated with the amine moiety in a suitable solvent and in the presence of a base (such as an organic base (e.g., a trialkylamine or pyridine) or an inorganic base (e.g., a carbonate base, such as potassium carbonate or sodium carbonate)). Suitable activation methods include, but are not limited to: treatment with O-(N-succinimide)-N,N,N',N'-tetramethylurea tetrafluoroborate (TSTU); formation of acyl chlorides, such as by treatment with thionyl chloride; treatment with 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and a base (such as diisopropylethylamine (DIPEA)); treatment with carbonyl diimidazole (CDI); or treatment with carbodiimide (such as dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)).

[0176] The third exemplary reaction step is shown in Scheme 5.

[0177]

[0178] Regarding scheme 5, each PG is independently suitable for preventing L. 1 and L 2 Protecting groups that react with dyes. Typically, dyes contain reactive moieties suitable for conjugation with amines, such as carboxylic acid moieties. The dye is reacted with L... using any suitable technique. 1 -NH-L 2 Partial conjugation. In some respects, the dye comprises a carboxylic acid moiety that forms an amide bond with the NH moiety. The carboxylic acid may be activated prior to conjugation, such as by using the activators disclosed herein. In other respects, the carboxylic acid reacts with an amine in the presence of ((7-azabenzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate) (PyAOB) and a suitable base (such as an organic base, e.g., a trialkylamine base).

[0179] An exemplary fourth reaction step is shown in Scheme 6.

[0180]

[0181] Regarding scheme 6, the product of scheme 3 is first deprotected using a suitable technique. The deprotection method is disclosed herein and is known to those skilled in the art. Then, the quencher from scheme 4—L... 3 - Peptide compounds treat deprotected compounds. In some respects, L 2The peptide contains an acid moiety, which is first activated and then reacts with an amine on the peptide moiety. In some respects, the acid moiety is activated by treatment with 3,5-dichloro-4-((dimethylamino)(dimethylimino)methoxy)benzenesulfonate and N,N-dimethylaminopyridine (DMAP). Alternative activation techniques are disclosed herein.

[0182] Then, in the presence of a suitable base (such as an organic base, such as a trialkylamine base, such as triethylamine), a quencher -L 3 - Peptide compounds are used to treat activated compounds.

[0183] In some cases, the disclosed compound can be prepared as illustrated in Scheme 7 below and as will be understood by one of ordinary skill in the art of organic synthesis. An exemplary synthesis may include the following three reaction steps according to Scheme 7: reacting a reactive dye with a protected L... 1 and L 2 The linker of the group is coupled, followed by a deprotection reaction to generate a bifunctional dye-linker (step (1)). In step (2), the bifunctional dye-linker reacts with a reactive protecting peptide to form an intermediate, which is further deprotected to form a bifunctional dye-peptide conjugate. In step (3), the bifunctional dye-peptide conjugate can be coupled with a reactive quencher to generate a dye-peptide-quencher intermediate having a reactive group R1. The R1 of the intermediate is converted into a reactive group R to generate the desired compound, which can be conjugated to biomolecules and other targets.

[0184]

[0185] V. Application

[0186] The disclosed compounds can be used as fluorescent probes and for real-time monitoring of endosome sorting and lysosomal degradation of proteins in living cells, and can also be used as indicators of protease activity. Real-time monitoring of protein endosome sorting and lysosomal degradation can be used, for example, in drug development. Typically, a target protein (such as an antibody-drug conjugate or ADC) binds to a degrading agent, which binds to a cell surface receptor, and the complex is internalized into the cell. The receptor then cycles back to the cell surface, while the protein is transported to a lysosome containing enzymes that degrade the protein. However, questions often remain regarding the destination of the degrading agent and whether the target protein is actually degraded in the lysosome, enters another compartment, or is recycled back to the cell surface.

[0187] The compounds disclosed herein can be used to provide a reliable indicator of lysosomal entry, emitting a bright fluorescent signal upon cleavage by proteases, such as cathepsin B cleavage occurring in lysosomes. The compounds are typically selected or synthesized to have a peptide moiety suitable for use as a substrate for proteases such as cathepsin B. The disclosed compounds are readily conjugated to biomolecules such as antibodies and other proteins, enabling live-cell dynamic assessment of endosome sorting and lysosomal degradation, and providing sensitive, specific, and bright indicators without genetic engineering. These compounds fill an unmet need for real-time monitoring of endosome sorting and lysosomal degradation of proteins in live cells. The disclosed compounds, or compositions comprising these compounds optionally conjugated to biomolecules, are suitable for in vitro, ex vivo, and in vivo assays. The data provided herein demonstrate that the compounds work with high sensitivity in imaging and flow cytometry, and that they are specific for cellular uptake and lysosomal degradation.

[0188] Typically, the presence or absence of fluorescence is monitored after a sample (such as cells or tissue) has been contacted with the disclosed compound or a combination thereof. Generally, the absence of fluorescence indicates the absence of protease activity, while the presence of fluorescence indicates protease activity. In some examples, different samples were tested using the same compound, and the sample with higher protease activity produced a fluorescence signal faster and with a higher overall intensity than the sample with lower activity. In other examples testing two different substrates, relative fluorescence intensity was used to investigate protease activity. The data is also used to track the delivery of the disclosed compound or a combination thereof, or a disclosed compound conjugated to a biomolecule.

[0189] The disclosed compounds or combinations thereof may be used to detect diseases and / or conditions indicating abnormal protease activity. Such diseases and / or conditions include, but are not limited to, cancer. In some aspects, the disclosed compounds may be used as cathepsin B probes and thus may be used to identify and / or monitor cathepsin B activity in cells and / or tissues. In some aspects, the disclosed compounds, which may be used as cathepsin B probes, may be used to identify and / or monitor cancers such as colorectal cancer, prostate cancer, ovarian cancer, or breast cancer.

[0190] In some aspects, methods of using the disclosed compounds may include contacting cells with the disclosed compounds or compositions, exposing the cells to light (such as visible or UV light), and determining the presence or absence of a fluorescence or phosphorescence signal. Exposing the cells to light and determining the presence or absence of a fluorescence or phosphorescence signal may include exposing the cells to light at a first wavelength and detecting the presence or absence of light having a second wavelength different from the first wavelength. The cells may be in vitro cells or isolated cells. In some aspects, the cells are in vivo.

[0191] Cell contact may include incubating cells for a time interval sufficient to promote the entry of the compound or composition into the cells. The time interval may be 1 hour or less to 24 hours or longer, such as 2 hours to 16 hours.

[0192] In some aspects, the method includes conjugating the compounds disclosed herein with a biomolecule to form a biomolecule-compound conjugate, contacting a cell with the biomolecule-compound conjugate, incubating the cell for a time interval sufficient to allow the biomolecule-compound conjugate to enter the cell, irradiating the cell with light of an appropriate wavelength, and detecting the fluorescence emission of the biomolecule-compound conjugate.

[0193] In some aspects, the method involves contacting a sample with a compound disclosed herein or a biomolecule-compound conjugate disclosed herein. Samples may include live cells, intracellular fluid, extracellular fluid, biological fluids, serum, bio-fermentation media, environmental samples, industrial samples, proteins, peptides, buffer solutions, biological fluids or chemical reactors, blood, saliva, urine, water, soil, wastewater, seawater, pharmaceuticals, food, or beverages.

[0194] In some respects, a sample or medium in which the compounds, conjugates, or compositions disclosed herein are present is irradiated with a selected wavelength of light to produce a detectable optical response, which is then observed using a device for detecting the optical response. Equipment that can be used to irradiate the compounds, conjugates, and compositions disclosed herein includes, but is not limited to, handheld ultraviolet lamps, mercury arc lamps, xenon lamps, lasers, and laser diodes. These irradiation sources are optically integrated into laser scanners, fluorescence microplate readers, standard or microfluorometers, or chromatographic detectors.

[0195] In some aspects, methods for analysis or detection using the compounds, conjugates, or compositions provided herein are provided. More specifically, detection can be performed using optical devices. In some embodiments, fluorescence emission is optionally detected by visual inspection or using any of the following devices: CCD camera, video camera, photographic film, laser scanning equipment, fluorometer, photodiode, quantum counter, epifluorescence microscope, scanning microscope, flow cytometer, fluorescence microplate reader, or by means of amplification of the signal, such as a photomultiplier tube. When examining samples using a flow cytometer, fluorescence microscope, or fluorometer, the instrument is optionally used to distinguish and differentiate the fluorescent compounds, conjugates, or compositions provided herein from those of a second fluorophore. In the case of examining samples using a flow cytometer, the examination of the sample optionally includes separating particles within the sample based on fluorescence response using a sorting device.

[0196] The following embodiments are provided to illustrate certain features of certain aspects of this disclosure, but the scope of the claims should not be limited to those features illustrated.

[0197] Example

[0198] Example 1: Synthesis of Compound I-6

[0199] 1. Synthesis of Compound 4

[0200]

[0201] Compound 1 (1.400 g) and O-(N-succinimide)-N,N,N',N'-tetramethylurea tetrafluoroborate (TSTU) (741 mg) were dissolved in DMF (30 mL). TEA (1.400 mL) was added to the solution, and the solution was stirred at room temperature until TLC showed the disappearance of compound 1 (approximately 1 hour) and the formation of a new compound (2). Then, compound 3 (493 mg) was added to the solution with stirring, and the mixture was stirred until TLC showed the disappearance of compound 2 (30-60 minutes). The solvent was evaporated by rotary evaporation, and the residue was dissolved in dichloromethane (DCM) (150 mL). The DCM solution was washed twice with water (2 × 150 mL). The organic layer was dried over Na2SO4, filtered, and evaporated by rotary evaporation. The residue was purified by silica gel column chromatography, eluting with DCM solution in methanol. The desired fractions were combined and evaporated by rotary evaporation to give compound 4 (1.35 g) as a blue solid. The proton NMR of the product is consistent with its structure.

[0202] 2. Synthesis of Compound 7

[0203]

[0204] Compound 4 (50 mg) was dissolved in a mixture of DCM (1 mL) and TFA (trifluoroacetic acid) (1 mL). The solution was stirred at room temperature until TLC showed that compound 4 had disappeared (approximately 30 minutes). The solution was evaporated by rotary evaporation, and the residue was co-evaporated twice with DCM-toluene (1:1). The residue was further dried under high vacuum for 1 hour and then used unpurified for the next step. The residue was dissolved in DMF (1 mL), and compound 6 (28 mg) and compound HATU (27 mg) were added to the solution. DIEA (0.1 mL) was added, and the solution was stirred at room temperature under argon for 1 hour. The solvent was evaporated under high vacuum. The residue was purified by silica gel column chromatography using DCM-methanol as the eluent. The solvent was evaporated to give compound 7 as a blue solid. Yield: 60 mg (80% after two steps).

[0205] 3. Synthesis of Compound 8

[0206]

[0207] Compound 7 (10 mg) was dissolved in a mixture of DCM (1 mL) and TFA (trifluoroacetic acid) (1 mL). The solution was stirred at room temperature until TLC showed that compound 7 had disappeared (approximately 30 minutes). The solution was evaporated by rotary evaporation, and the residue was co-evaporated twice with DCM-toluene (1:1). The residue was further dried under high vacuum for 1 hour and then used unpurified for the next step.

[0208] 4. Synthesis of Compound 11

[0209]

[0210] Compound 9 (174 mg, 1.5 equivalents), compound 10 (300 mg, 1 equivalent), and PyAOP (7-azabenzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate) (203 mg, 1.5 equivalents) were dissolved in 12 mL of DMF. Triethylamine (TEA) (0.18 mL, 5 equivalents) was added to the solution with stirring under an inert atmosphere. The mixture was stirred at room temperature until TLC showed the disappearance of compound 10 (approximately 45 minutes). The solvent was evaporated by rotary evaporation to give a blue solid. The solid was purified by reversed-phase column chromatography, eluting with triethylacetate ammonium buffer followed by water / methanol. The solvent was evaporated, followed by lyophilization, to give compound 11 (330 mg) as a light blue solid.

[0211] 5. Synthesis of Compound 12

[0212]

[0213] Compound 11 (330 mg) was dissolved in TFA (trifluoroacetic acid) (5 mL). The solution was stirred at room temperature until TLC showed that compound 11 had disappeared (approximately 2 hours). The TFA was evaporated by rotary evaporation, and the residue was co-evaporated twice with CH3CN-toluene (1:1). The residue was purified by reversed-phase column chromatography, eluting with triethylacetate ammonium buffer followed by water / acetonitrile. The solvent was evaporated, followed by lyophilization, to give compound 12 as a blue solid. Yield: 280 mg (68% after two steps).

[0214] Synthesis of 6.14

[0215]

[0216] Compound 12 (23 mg), compound 13 (13 mg), and N,N-dimethylaminopyridine (DMAP) (4.5 mg) were dissolved in anhydrous DMF (1 mL). The solution was stirred at room temperature until TLC showed the disappearance of compound 12 (approximately 1 hour). The solvent was evaporated by rotary evaporation. The residue was purified by reversed-phase column chromatography, eluting with water / acetonitrile. The solvent was evaporated, followed by lyophilization, to give compound 14 as a blue solid. Yield: 27 mg (79%).

[0217] 7. Synthesis of Compound I-6

[0218]

[0219] Compound 14 (25 mg, 1.7 equivalents), compound 8 (9 mg, 1 equivalent), and triethylamine (16 µL) were dissolved in anhydrous DMF (1 mL). The solution was stirred at room temperature until TLC showed the disappearance of compound 8 (approximately 1 hour). The solvent was evaporated by rotary evaporation. The residue was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / acetonitrile. The solvent was evaporated, followed by lyophilization, to give compound I-6 as a blue solid. Yield: 9 mg (46%). The LCMS of the product was consistent with its structure. MS: 808 [M+3] 3+ 1212 [M+3] 2+ .

[0220] Example 2: Synthesis of Compound I-1

[0221] 1. Synthesis of Compound 16

[0222]

[0223] Compound 5 was synthesized starting with 1.2 g of compound 4, using the method described in Example 1. Compound 15 (845 mg), anhydrous DMF (60 mL), and triethylamine (550 µL) were added to a flask containing compound 5. The solution was stirred at room temperature until TLC showed that compound 5 had disappeared (approximately 1 hour). The solvent was evaporated using a rotary evaporator. The solid was washed twice with ethyl acetate (2 × 250 mL) by sonication and stirring. The solid was collected by filtration and dried under high vacuum to give compound 16 as a blue solid. Compound 16 was ready for use in the next step without further purification.

[0224] 2. Synthesis of Compound 17

[0225]

[0226] Compound 16 (900 mg) was dissolved in DCM (15 mL), and the solution was placed in an ice bath. Cold trifluoroacetic acid (15 mL) was added to the solution with stirring under argon atmosphere. The solution was stirred in an ice bath for approximately 1 hour. The reaction mixture was added dropwise over 7–10 minutes to diethyl ether (approximately 800 mL) with vigorous stirring. A blue precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by filtration and washed with diethyl ether. The solid was purified by silica gel column chromatography, eluting with DCM-methanol. The desired fractions were combined and evaporated using rotary evaporation. The solid was dissolved in 15 mL of a 10% methanol / DCM mixture. The solution was filtered to remove silica gel. The solution was evaporated and dried under high vacuum to give compound 17 (450 mg, 50%) as a blue solid.

[0227] 3. Synthesis of Compound I-1

[0228]

[0229] Compound 12 (240 mg), compound 13 (160 mg), and N,N-dimethylaminopyridine (DMAP) (37 mg) were dissolved in anhydrous DMF (10 mL). The solution was stirred at room temperature until TLC showed the disappearance of compound 12 (approximately 1.5 h). The reaction mixture was added dropwise to ethyl acetate (approximately 400 mL) under vigorous stirring. A blue precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by filtration and washed with ethyl acetate. The solid was transferred to a 250 mL round-bottom flask and dried under high vacuum for 1 h to give crude compound 14. Compound 14 was used unpurified for the next step.

[0230] Compound 17 (143 mg), anhydrous DMF (10 mL), and triethylamine (103 µL) were added to a round-bottom flask containing crude compound 14. The solution was stirred at room temperature until TLC showed the disappearance of compound 17 (approximately 1 hour). The reaction mixture was added dropwise over 2–3 minutes to vigorously stirred diethyl ether (approximately 300 mL). A blue precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by filtration and washed with diethyl ether. After drying, the solid was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / acetonitrile. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound I-1 as a blue solid. Yield: 140 mg (45% after two steps). The LCMS of the product was consistent with the structure. MS: 837 [M+3] 3+ 1256 [M+3] 2+ .

[0231] Example 3: Synthesis of Compound I-2

[0232] 1. Synthesis of Compound 19

[0233]

[0234] Compound 18 (150 mg), compound 3 (46 mg), and triethylamine (132 µL) were dissolved in 3 mL of DMF. The solution was stirred at room temperature under an inert atmosphere until compound 18 disappeared on TLC (approximately 1 hour). The solvent was evaporated by rotary evaporation. The residue was dissolved in dichloromethane (DCM) (50 mL). The DCM solution was washed twice with water (2 × 50 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated by rotary evaporation. The residue was purified by silica gel column chromatography, eluting with a methanol-DCM solution. The desired fractions were combined and evaporated by rotary evaporation to give compound 19 (110 mg, 69%) as a red solid. The proton NMR of the product was consistent with its structure.

[0235] 2. Synthesis of Compound 22

[0236]

[0237] Compound 19 (75 mg) was dissolved in a mixture of DCM (2 mL) and TFA (trifluoroacetic acid) (2 mL). The solution was stirred at room temperature until TLC showed that compound 19 had disappeared (approximately 30 minutes). The solution was evaporated by rotary evaporation, and the residue was co-evaporated twice with DCM-toluene (1:1). The residue was further dried under high vacuum for 1 hour and then used unpurified for the next step.

[0238] Compound 15 (as shown in Example 2) (58 mg), anhydrous DMF (2 mL), and triethylamine (63 µL) were added to the flask containing compound 19. The solution was stirred at room temperature until TLC showed that compound 19 had disappeared (approximately 1 hour). The solvent was evaporated using a rotary evaporator. The solid was washed twice with ethyl acetate (2 × 250 mL) by sonication and stirring. The solid was collected by filtration and dried under high vacuum to give compound 21 as a red solid. Compound 21 can be used in the next step without further purification.

[0239] Crude compound 21 was dissolved in DCM (2 mL), and the solution was placed in an ice bath. Cold trifluoroacetic acid (2 mL) was added to the solution with stirring under argon atmosphere. The solution was stirred in an ice bath for approximately 1 hour. The reaction mixture was added dropwise over 7–10 minutes to diethyl ether (approximately 100 mL) under vigorous stirring. A red precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by filtration and washed with diethyl ether. The solid was purified by silica gel column chromatography, eluting with DCM-methanol. The desired fractions were combined and evaporated using rotary evaporation. The solid was dissolved in 10 mL of a 10% methanol / DCM mixture. The solution was filtered to remove silica gel. The solution was evaporated and dried under high vacuum to give compound 22 (50 mg, 42% after 3 steps) as a red solid.

[0240] 3. Synthesis of Compound 24

[0241]

[0242] Compound 9 (18 mg), compound 23 (30 mg), and PyAOP (7-azabenzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate) (27 mg) were dissolved in 1.5 mL of DMF. Triethylamine (TEA) (18 µL) was added to the solution with stirring under an inert atmosphere. The mixture was stirred at room temperature until TLC showed the disappearance of compound 23 (approximately 45 min). The solvent was evaporated by rotary evaporation to give a deep purple solid. TFA (trifluoroacetic acid) (1 mL) was added to the residue. The solution was stirred at room temperature for 2 h. The TFA was evaporated by rotary evaporation, and the residue was co-evaporated twice with CH3CN-toluene (1:1). The residue was purified by reversed-phase column chromatography. The solvent was evaporated, followed by lyophilization, to give compound 24 as a deep purple solid. Yield: 35 mg (79% after two steps).

[0243] 4. Synthesis of Compound 25

[0244]

[0245] Compound 24 (20 mg), compound 13 (13 mg), and N,N-dimethylaminopyridine (DMAP) (4.0 mg) were dissolved in anhydrous DMF (1.5 mL). The solution was stirred at room temperature until TLC showed that compound 24 had disappeared (approximately 1 hour). The reaction mixture was added dropwise to ethyl acetate (approximately 40 mL) under vigorous stirring. A deep purple precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by filtration and washed with ethyl acetate. The solid was transferred to a 50 mL round-bottom flask and dried under high vacuum for 1 hour to give crude compound 25. Compound 25 was used unpurified for the next step.

[0246] 5. Synthesis of compound I-2

[0247]

[0248] Compound 22 (9.3 mg), anhydrous DMF (1 mL), and triethylamine (18 µL) were added to a round-bottom flask containing crude compound 25. The solution was stirred at room temperature until TLC showed the disappearance of compound 22 (approximately 1 hour). The reaction mixture was added dropwise over 2–3 minutes to a vigorously stirred diethyl ether (approximately 12 mL). A deep purple precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by centrifugation and washed with diethyl ether. After drying, the solid was purified by reversed-phase column chromatography, eluting with triethylacetate ammonium buffer followed by water / acetonitrile. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound I-2 as a deep purple solid. Yield: 9 mg (41% after two steps). The product was consistent with its structure by LCMS. MS: 820 M 3+ 1230 M 2+ .

[0249] Example 4: Synthesis of Compound I-3

[0250] 1. Synthesis of Compound 27

[0251]

[0252] Compound 26 (12 mg), compound 23 (from Example 3) (20 mg), and PyAOP (7-azabenzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate) (13 mg) were dissolved in 1 mL of DMF. Triethylamine (TEA) (15 µL) was added to the solution with stirring under an inert atmosphere. The mixture was stirred at room temperature until TLC showed the disappearance of compound 23 (approximately 45 min). The solvent was evaporated by rotary evaporation to give a deep purple solid. TFA (trifluoroacetic acid) (1 mL) was added to the residue. The solution was stirred at room temperature for 2 hours. The TFA was evaporated by rotary evaporation, and the residue was co-evaporated twice with CH3CN-toluene (1:1). The residue was purified by reversed-phase column chromatography, eluting with triethylacetate ammonium buffer followed by water / methanol. The solvent was evaporated, followed by lyophilization, to give compound 27 as a deep purple solid. Yield: 25 mg (79% after two steps).

[0253] 2. Synthesis of Compound 29

[0254]

[0255] Compound 27 (24.4 mg), compound 28 (12 mg), anhydrous DMF (0.5 mL), and N,N-diisopropylethylamine (DIEA) (5 µL) were added to a vial. The solution was stirred at room temperature until TLC showed the disappearance of compound 27. Piperidine (0.1 mL) was added to the reaction vial, and the solution was stirred for 30 minutes. The reaction mixture was added dropwise over 2-3 minutes to approximately 12 mL of vigorously stirred ethyl acetate. A deep purple precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by centrifugation and washed with ethyl acetate. After drying, the solid was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / methanol. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound 29 as a deep purple solid. Yield: 24 mg (80% after two steps).

[0256] 3. Synthesis of Compound 30

[0257]

[0258] Compound 29 (20.2 mg), compound 18 (13 mg), anhydrous DMF (0.5 mL), and triethylamine (TEA) (10 µL) were mixed in a vial. The solution was stirred at room temperature until TLC showed the disappearance of compound 29 (approximately 1 hour). The reaction mixture was added dropwise over 2–3 minutes to ethyl acetate (approximately 12 mL) under vigorous stirring. A deep purple precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by centrifugation and washed with ethyl acetate. After drying, the solid was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / methanol. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound 30 as a deep purple solid. Yield: 20 mg (80%).

[0259] 4. Synthesis of compound I-3

[0260]

[0261] Compound 30 (10.5 mg), compound 13 (3 mg), and N,N-dimethylaminopyridine (DMAP) (1.1 mg) were dissolved in anhydrous DMF (0.5 mL). The solution was stirred at room temperature until TLC showed the disappearance of compound 30 (approximately 1 hour). The solvent was evaporated by rotary evaporation. The residue was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / acetonitrile. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound I-3 as a deep purple solid. Yield: 11 mg (93%). The LCMS of the product was consistent with the structure. MS: 855 [M+2] 3+1284 [M+4] 2+ .

[0262] Example 5: Synthesis of Compound I-4

[0263] 1. Synthesis of Compound 33

[0264]

[0265] Compound 31 (54 mg), compound 32 (20 mg), and triethylamine (40 µL) were dissolved in anhydrous DMF (2 mL). The solution was stirred at room temperature until compound 31 disappeared on TLC (approximately 1 hour). The solvent was evaporated by rotary evaporation. The residue was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / methanol. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound 33 as a brown solid. Yield: 44 mg (80%).

[0266] 2. Synthesis of Compound 34

[0267]

[0268] Compound 33 (40 mg), compound 13 (18 mg), and N,N-dimethylaminopyridine (DMAP) (6.5 mg) were dissolved in anhydrous DMF (1.2 mL). The solution was stirred at room temperature until TLC showed that compound 33 had disappeared (approximately 1 hour). The solvent was evaporated by rotary evaporation. The residue was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / acetonitrile. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound 34 as a brown solid. Yield: 40 mg (73%).

[0269] 3. Synthesis of Compound 37

[0270]

[0271] Compound 34 (12 mg), compound 22 (from Example 3) (12 mg), and triethylamine (10 µL) were dissolved in anhydrous DMF (0.5 mL). The solution was stirred at room temperature until TLC showed that compound 34 had disappeared (approximately 1 hour). PyAOP (7-azabenzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate (8 mg) and compound 36 (8 mg) were added, and the solution was stirred at room temperature for another hour. The solvent was evaporated by rotary evaporation to give a deep purple solid. TFA (trifluoroacetic acid) (1 mL) was added to the residue. The solution was stirred at room temperature for 2 hours. The TFA was evaporated by rotary evaporation, and the residue was co-evaporated twice with CH3CN-toluene (1:1). The residue was purified by reversed-phase column chromatography, eluting with triethylacetate ammonium buffer followed by water / methanol. The solvent was evaporated, followed by lyophilization, to give compound 37 as a deep purple solid. Yield: 7.5 mg (30% after three steps).

[0272] 4. Synthesis of compound I-4

[0273]

[0274] Compound 37 (6 mg), compound 13 (3 mg), and N,N-dimethylaminopyridine (DMAP) (2 mg) were dissolved in anhydrous DMF (0.5 mL). The solution was stirred at room temperature until TLC showed the disappearance of compound 37 (approximately 1 hour). The solvent was evaporated by rotary evaporation. The residue was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / acetonitrile. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound I-4 as a deep purple solid. Yield: 6 mg (88%). The LCMS of the product was consistent with the structure. MS: 819 [M+3] 3+ 1229 [M+4] 2+ .

[0275] Example 6: Synthesis of Compound I-5

[0276] 1. Synthesis of Compound 38

[0277]

[0278] Compound 31 (30 mg), compound 26 (26 mg), and triethylamine (25 µL) were dissolved in anhydrous DMF (1.5 mL). The solution was stirred at room temperature until TLC showed that compound 31 had disappeared (as shown in Example 5) (approximately 2 hours). The solvent was evaporated by rotary evaporation to give a brown solid. TFA (trifluoroacetic acid) (1 mL) was added to the residue. The solution was stirred at room temperature for 2 hours. The TFA was evaporated by rotary evaporation, and the residue was co-evaporated twice with CH3CN-toluene (1:1). The residue was purified by reversed-phase column chromatography, eluting with triethylacetate ammonium buffer followed by water / methanol. The solvent was evaporated, followed by lyophilization, to give compound 38 as a brown solid. Yield: 29 mg (72% after two steps).

[0279] 2. Synthesis of Compound 39

[0280]

[0281] Compound 38 (13.6 mg), compound 28 (13 mg), anhydrous DMF (0.5 mL), and N,N-diisopropylethylamine (DIEA) (5 µL) were added to a vial. The solution was stirred at room temperature until TLC showed the disappearance of compound 38. Piperidine (0.1 mL) was added to the reaction vial, and the solution was stirred for 30 minutes. The reaction mixture was added dropwise over 2-3 minutes to approximately 12 mL of vigorously stirred ethyl acetate. A brown precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by centrifugation and washed with ethyl acetate. After drying, the solid was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / methanol. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound 39 as a brown solid. Yield: 10.8 mg (60% after two steps).

[0282] 3. Synthesis of Compound 40

[0283]

[0284] Compound 39 (10 mg), compound A (8 mg), anhydrous DMF (0.5 mL), and triethylamine (TEA) (10 µL) were mixed in a vial. The solution was stirred at room temperature until TLC showed the disappearance of compound 39 (approximately 1 hour). The reaction mixture was added dropwise over 2–3 minutes to ethyl acetate (approximately 12 mL) under vigorous stirring. A deep purple precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by centrifugation and washed with ethyl acetate. After drying, the solid was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / methanol. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound 40 as a deep purple solid. Yield: 9.5 mg (71%).

[0285] 4. Synthesis of compound I-5

[0286]

[0287] Compound 40 (8.7 mg), compound 13 (1.7 mg), and N,N-dimethylaminopyridine (DMAP) (0.61 mg) were dissolved in anhydrous DMF (0.5 mL). The solution was stirred at room temperature until TLC showed the disappearance of compound 40 (approximately 1 hour). The solvent was evaporated by rotary evaporation. The residue was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / acetonitrile. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound I-5 as a deep purple solid. Yield: 7 mg (70%). The LCMS of the product was consistent with the structure. MS: 756 [M] 3+ 1134 [M+2] 2+ .

[0288] Example 7: Synthesis of Compound I-7

[0289] 1. Synthesis of Compound 41

[0290]

[0291] Compound 39 (19 mg), compound B (17 mg), anhydrous DMF (0.5 mL), and triethylamine (TEA) (10 µL) were mixed in a vial. The solution was stirred at room temperature until TLC showed the disappearance of compound 39 (approximately 1 hour). The reaction mixture was added dropwise over 2–3 minutes to ethyl acetate (approximately 12 mL) under vigorous stirring. A deep purple precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by centrifugation and washed with ethyl acetate. After drying, the solid was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / methanol. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound 40 as a deep purple solid. Yield: 24 mg (71%).

[0292] 4. Synthesis of compound I-7

[0293]

[0294] Compound 41 (23.9 mg), compound 13 (3.9 mg), and N,N-dimethylaminopyridine (DMAP) (1.4 mg) were dissolved in anhydrous DMF (0.5 mL). The solution was stirred at room temperature until TLC showed the disappearance of compound 41 (approximately 1 hour). The solvent was evaporated by rotary evaporation. The residue was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / acetonitrile. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound I-7 as a deep purple solid. Yield: 19 mg (70%). The LCMS of the product was consistent with the structure. MS: 809 [M+3] 3+ 1215 [M+6] 2+ .

[0295] Example 8: Synthesis of Compound I-8

[0296] 1. Synthesis of Compound 42

[0297]

[0298] Compound 26 (83 mg), compound 10 (100 mg), and PyAOP (7-azabenzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate) (68 mg) were dissolved in 3 mL of DMF. Triethylamine (TEA) (60 µL) was added to the solution with stirring under an inert atmosphere. The mixture was stirred at room temperature until TLC showed the disappearance of compound 10 (approximately 45 min). The solvent was evaporated by rotary evaporation to give a blue solid. TFA (trifluoroacetic acid) (2 mL) was added to the residue. The solution was stirred at room temperature for 2 h. The TFA was evaporated by rotary evaporation, and the residue was co-evaporated twice with CH3CN-toluene (1:1). The residue was purified by reversed-phase column chromatography, eluting with triethylacetate ammonium buffer followed by water / methanol. The solvent was evaporated, followed by lyophilization, to give compound 42 as a blue solid. Yield: 100 mg (70% after two steps).

[0299] 2. Synthesis of Compound 43

[0300]

[0301] Compound 42 (88 mg), compound 28 (50 mg), anhydrous DMF (4.5 mL), and N,N-diisopropylethylamine (DIEA) (28 µL) were added to a vial. The solution was stirred at room temperature until TLC showed the disappearance of compound 42. Piperidine (0.75 mL) was added to the reaction vial, and the solution was stirred for 30 minutes. The reaction mixture was added dropwise over 2-3 minutes to approximately 150 mL of vigorously stirred ethyl acetate. A blue precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by filtration and washed with ethyl acetate. After drying, the solid was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / methanol. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound 43 as a blue solid. Yield: 80 mg (74% after two steps).

[0302] 3. Synthesis of Compound 44

[0303]

[0304] Compound 43 (80 mg), compound 2 (48 mg), anhydrous DMF (2 mL), and triethylamine (TEA) (28 µL) were mixed in a vial. The solution was stirred at room temperature until compound 43 disappeared on TLC (approximately 1 hour). The solvent was evaporated to dryness. The solid was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / methanol. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound 44 as a blue solid. Yield: 95 mg (92%).

[0305] 4. Synthesis of compound I-8

[0306]

[0307] Compound 44 (42 mg), compound 13 (11 mg), and N,N-dimethylaminopyridine (DMAP) (2.9 mg) were dissolved in anhydrous DMF (1.5 mL). The solution was stirred at room temperature until TLC showed the disappearance of compound 44 (approximately 1.5 h). The reaction mixture was added dropwise over 2-3 minutes to vigorously stirred ethyl acetate (approximately 50 mL). A blue precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by filtration and washed with ethyl acetate. After drying, the residue was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / acetonitrile. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound I-3 as a blue solid. Yield: 36 mg (77%). The LCMS of the product was consistent with the structure. MS: 872 [M] 3+ 1308 [M] 2+ .

[0308] Example 9: Synthesis of Compound I-9

[0309]

[0310] Compound I-1 (20 mg), compound 45 (6 mg), and triethylamine (10 µL) were dissolved in anhydrous DMF (1 mL). The solution was stirred at room temperature until TLC showed the disappearance of compound 45 (approximately 4 hours). The solvent was evaporated to dryness. The residue was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / acetonitrile. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound I-9 as a blue solid. Yield: 12 mg (57%). The LCMS of the product was consistent with the structure. MS: 898 [M] 3+ 1348 [M] 2+ .

[0311] Example 10: Synthesis of Compound I-10

[0312]

[0313] Compound I-7 (20 mg), compound 45 (6 mg), and triethylamine (10 µL) were dissolved in anhydrous DMF (1 mL). The solution was stirred at room temperature until TLC showed the disappearance of compound I-7 (approximately 4.5 h). The solvent was evaporated to dryness. The residue was purified by reversed-phase column chromatography, eluting with triethylammonium acetate buffer followed by water / acetonitrile. The desired fractions were combined. The solvent was evaporated, followed by lyophilization, to give compound I-10 as a purple solid. Yield: 14.8 mg (70%). The LCMS of the product was consistent with the structure. MS: 653 [M] 4+ .

[0314] Example 11

[0315] Combining with biomolecules

[0316] Prepare a 2 mg / mL solution by dissolving 20 µg of lyophilized compound I-1 in 10 µL of DMSO. Add 10 µL of 1M sodium bicarbonate to 100 µg of trastuzumab (Herceptin), cetuximab, or rituximab, and adjust the volume to 90 µL in a 1.5 mL Eppendorf tube with 1x PBS. Add 10 µL of the 2 mg / mL compound I-1 solution, briefly vortex the mixture, and then incubate at 20 °C for 2 hours with gentle shaking on a oscillator. After conjugation, transfer the material to 0.5 mL of zebasite pre-equilibrated with 1x PBS. ™ Purification was performed using a dye and biotin removal column (Thermo Fisher Scientific). The purified material was collected in a clean 1.5 mL Eppendorf tube and centrifuged at 18 kJ x g for 10 minutes to remove any precipitate.

[0317] Quantitative

[0318] The conjugated labeled antibody was diluted to 1:4 in 100% DMSO, and Nanodrop was used. ™ In OD 280 and OD 662 The average of the next three measurements. The extinction coefficient of the dye in 80% DMSO is 344,500 M. -1 cm -1 And dyes in OD 280 The correction factor is 0.187.

[0319] Internalization assay – Imaging

[0320] In 96-well plates, cells were seeded at a density of 5,000 cells / well in 100 μL of cell culture medium and allowed to recover overnight. SKBR3 cells were used as a Her-2 positive cell line, A431 cells were EGFR positive, and MCF7 cells were both Her-2 and MCF7 negative.

[0321] After overnight recovery, 10x stock solutions of trastuzumab-compound I-1 (SKBR3+) or cetuximab-compound I-1 (A431+) were prepared in PBS at a concentration of 20 µg / mL and added in triplicate at 10 µL / well. The solutions were incubated at 37°C and 5% CO2 for 2–16 hours. Hoechst nuclear staining agent was added at a final concentration of 0.1 µg / mL 15 minutes before imaging.

[0322] For pHrodo ™ Red's co-localization assay involved cells with trastuzumab-pHrodo. ™ Co-incubated with Red (ThermoFisher Scientific), followed by washing and Hoechst staining. For use with LysoTracker... ™ In the co-localization assay conducted by Thermo Fisher Scientific, after 16 hours of incubation, cells were treated with 500 nM LysoTracker solution diluted in cell culture medium. ™ After 2 hours of red treatment, the sample was washed and stained with Hoechst.

[0323] 16 hours later, at EVOS ™ On the M7000 imaging system (Thermo Fisher Scientific), transmitted light, a DAPI filter for nuclear staining, and a pHrodo filter were used. ™ Red or LysoTracker ™ Images were acquired using an RFP filter for Red staining and a Cy5 filter set for staining compound I-1.

[0324] Quantification of signal (SKBR3 cells or A431 cells) and background (MCF7 cells) in CellInsight ™ The results were determined using high-content analysis on the CX7 LEDPro high-content screening platform (Thermo Fisher Scientific), using CircSpot thresholding and analyzing the average CircSpot intensity to determine the signal / background.

[0325] The conjugation and internalization of other compounds disclosed herein follow the methods described in Example 11 with respect to compound I-1.

[0326] Internalization assay – Flow cytometry

[0327] Ramos cells (CD20 and CD19 positive) or Jurkat cells (CD20 and CD19 negative) were centrifuged at 500 x g for 5 minutes, then centrifuged at 1 × 10⁻⁶. 6 Cells were resuspended in RPMI at a density of 100,000 cells / well to a final volume of 100 µL / well. A 10x stock solution of rituximab-compound I-1 was prepared in 1x PBS (pH 7.4) at 20 µg / mL and added at 10 µL / well to obtain a final concentration of 2 µg / mL. Cells were incubated overnight at 37°C and 5% CO2. After incubation, cells were washed in flow cytometry staining buffer. For double staining, 5 µL of anti-CD19-FITC was added and maintained for 30 min. Cells were washed 3× in 1x PBS and resuspended in 200 µL PBS + 3 µM DAPI. The solution was then used in Attune. ™ Cells were analyzed using an NxT flow cytometer (Thermo Fisher Scientific).

[0328] cathepsins B inhibition

[0329] As described above, SKBR3 cells were seeded at a density of 5,000 cells / well in 96-well plates. After overnight recovery, cells were treated for 2 hours with either the cathepsin B inhibitor CA-074 (Me) or the DMSO control, followed by serial dilutions from 100 µM to 98 nM, and then treated with trastuzumab compound I-1. After incubation at 37°C and 5% CO2, Hoechst nuclear staining agent was added at a concentration of 0.1 µg / mL and maintained for 15 minutes before imaging. (EVOS) ™ Images were acquired using transmitted light, a DAPI filter for nuclear staining, and a Cy5 filter for cathepsin B probe staining on the M7000.

[0330] In CellInsight ™ On the CX7 LED, quantitative results of the signal are determined through high intrinsic analysis, and the average CircSpot intensity is analyzed by using CircSpot thresholding.

[0331] result

[0332] Trastuzumab and cetuximab are therapeutic antibodies targeting the Her-2 receptor and EGFR, respectively. Upon binding, these antibodies are internalized and ultimately degraded by lysosomes. The cathepsin B probe compound I-1, possessing an amine-reactive functional group, is conjugated to both antibodies to monitor lysosomal degradation. SKBR3 cells were positive for Her-2, while A431 cells were positive for EGFR. MCF7 cells were used as a negative control because they were negative for both membrane receptors. No fluorescence signal was observed after initial treatment, but over time (data not shown), fluorescence in the Cy5 channel began to accumulate throughout the cells, forming bright punctate patterns. A bright signal was observed in the positive cell lines after 16 hours. Figure 2 and Figure 5 ), while no fluorescence was observed in the MCF7 negative cell line ( Figure 3 and Figure 5 (In CellInsight) ™ Quantitative analysis was performed on the CX7 LED Pro high-content screening platform (Thermo Fisher Scientific), and threshold segmentation of dotted staining was used to produce a high signal-to-background ratio. Figure 4 Importantly, these experiments were performed in cell culture medium and were not washed before imaging, allowing for real-time monitoring of cathepsin B activity and target degradation.

[0333] Numerous cathepsins are present in endosomes and lysosomes; therefore, to confirm the probe's specificity for cathepsin B, the cathepsin B-specific inhibitor CA-074 (Me) was used to inhibit its activity. SKBR3 cells were treated with different concentrations of CA-074 (Me) for 2 hours, followed by treatment with trastuzumab compound I-1. After incubation at 37°C / 5% CO2 for 16 hours, the cells were visualized in CellInsight under matched exposure conditions. ™ On the CX7 LED, fluorescence intensity was quantified by thresholding of dotted staining. Quantification of average fluorescence intensity indicates the dose response to CA-074 (Me) inhibition. Figure 10 ). In EVOS ™ Representative bright-field images acquired on the M7000 imaging system show punctate staining in untreated cells, while at higher concentrations, cathepsin B inhibition resulted in negative cathepsin B probe staining. Figures 11A-11C ).

[0334] SKBR3 cells using LysoTracker ™ Red staining (green) Figure 12 (Top image) or trastuzumab – pHrodo ™ Red co-staining (green) Figure 12 (middle image), and co-localization with trastuzumab-compound I-1 (red) was observed. The superimposed image shows overlapping yellow fluorescence.

[0335] Using trastuzumab-compound I-7 and human IgG1 isotype control-compound I-7 ( Figure 13A and Figure 13B ) or trastuzumab-compound I-3 conjugate ( Figure 14 ) stain SKBR3 cells, A431 cells, or MCF7 cells. In the FITC channel (green) ( Figure 13A (left image) and RFP channel ( Figure 14 In the left image, fluorescent dot staining was observed in SKBR3 cells; however, no signal was observed in any channel in the MCF7 negative cell line. Figure 13A and Figure 14 (See right figure). Quantitative analysis of the mean CircSpot intensity of SKBR3 and A431 cells treated with the compound I-7 antibody conjugate demonstrated the specific internalization of the trastuzumab-compound I-7 conjugate in SKBR3 cells and the cetuximab-compound I-7 conjugate in A431 cells. Figure 13B ).

[0336] Example 12

[0337] In the plate-based assay, cathepsin probes were tested against the cathepsin B enzyme to compare activity. One probe was compound II-2, which contains an LR cleavage site. The other probe was compound I-1.

[0338] Materials and methods

[0339] reaction buffer :

[0340] Basic buffer = phosphate-buffered saline, pH 7.4

[0341] Adjust the pH to pH 6 using concentrated phosphoric acid.

[0342] 1 mg / mL cysteine ​​= 8.25 mM

[0343] 0.1% Brij-35 surfactant

[0344] enzymes :

[0345] Recombinant human cathepsin B; BioLegend catalog number 557704

[0346] Activity = 2.5 U / µg; dilute before assay.

[0347] Enzyme preparation by 2X serial dilution

[0348] probe :

[0349] The 2X solution was prepared at 12 µM, therefore the final concentration was 6 µM.

[0350] Plate Reader :

[0351] Tecan SPARK

[0352] result

[0353] Figure 15 and Figure 16 Results were provided. The halogen-based probe (compound II-1) had a much higher signal than the dye-quencher probe (compound I-1), but both reagents showed an increasing signal over time and with increasing enzyme concentration.

[0354] Example 13

[0355] The extinction coefficients of fluorescent dyes containing compound I-1 or compounds containing the quenched portion of compound I-1 were measured in various solvent systems to identify solvent systems that would not cause spectral distortion in either component. The experiment was designed to determine the extinction of each component, which should be additive for the cathepsin B probe.

[0356] Materials and methods

[0357] The phalloid peptide conjugate of the dye moiety of compound I-1 and the free acid of the quencher moiety of compound I-1 were dissolved in DMSO at unknown high concentrations, and then diluted in various solvents. For both dyes, methanol was used as the reference solvent. For non-reference solvents, extinction was determined by comparing the values ​​with those in the reference solvent. All measurements were performed using quartz cuvettes (1 cm path length) on a Tecan SPARK plate reader.

[0358] in conclusion

[0359] The estimated extinction of the dye moiety of compound I-1 in DMSO is 220,700, and the estimated extinction in 90:10 DMSO:water is 228,000.

[0360] The estimated extinction of the quencher portion of compound I-1 in DMSO is 64,300, and the estimated extinction in 90:10 DMSO:water is 69,000.

[0361] If the dye spectrum is not distorted, the extinction of the compound I-1 cathepsin B probe, which contains both the dye and quencher moieties, is expected to be 285,000 in DMSO and 297,000 in 90:10 DMSO:water.

[0362] Example 14

[0363] Three batches of fluorescent cathepsin B probe compound I-1 were conjugated to trastuzumab (trade name = Herceptin) by reacting the substrate's SDP ester with the antibody. Free dye was removed by centrifugation column, and the degree of labeling was quantified.

[0364] Materials and methods

[0365] The SDP ester of compound I-1 was dissolved in DMSO and then reacted with the antibody in PBS buffer (pH approximately 9-10) containing 100 mM sodium bicarbonate. After incubation for >1 hour, the antibody was reacted with Zeba. ™ The crude conjugate was purified using a biotin and dye removal centrifuge column (ThermoFisher Scientific). The purified conjugate was diluted 10X in DMSO, resulting in a final solution containing 10% aqueous solution. Correction factors, as well as extinction coefficients of the substrate and antibody, were determined in the same buffer system.

[0366] Table 2

[0367]

[0368] in conclusion

[0369] Figure 17 The results are presented in Table 2 above. For all three batches of reactive cathepsin B probes, the labeling level was between 1 and 2 compounds: I-1 cathepsin B probe / antibody.

[0370] Example 15

[0371] The extinction coefficients of compound I-1 in various solvent mixtures were determined. The concentration of compound I-1 was determined by weighing (mass). The purpose of this experiment was to determine the degree of labeling on the antibody conjugate, which could not be diluted in methanol as it would precipitate.

[0372] Materials and methods

[0373] Approximately 5 mg of compound I-1 was weighed into a glass vial. Compound I-1 was dissolved in DMSO and then diluted in various solvent mixtures, including methanol, PBS-Tween-20, and mixtures of DMSO with water or PBS. Extinction was determined using Beer's Law from the solution concentration and the maximum absorbance of the dye.

[0374] in conclusion

[0375] In PBS-Tween, the spectrum of compound I-1 was highly distorted, resulting in a maximum absorbance at 608 nm instead of the expected approximately 660 nm. In methanol, DMSO, and high-DMSO aqueous mixtures, the spectrum was slightly distorted, with minor changes in the maximum absorption wavelength. For DMSO:water mixtures containing less than 70% DMSO, the spectrum was distorted (data not shown). The conclusion is that any aqueous solution containing ≥70% DMSO is suitable for determining labeling levels, but the extinction coefficient will vary between approximately 300 and 380,000 depending on the water content.

[0376] Example 16

[0377] A coumarin-based AMC-LR (7-amino-4-methylcoumarin-Leu-Arg) cathepsin B probe was selected as a positive control and its activity was compared in a plate-based assay against the cathepsin B enzyme. The test probe was compound II-1.

[0378] Materials and methods

[0379] reaction buffer :

[0380] Basic buffer = phosphate-buffered saline, pH 7.4

[0381] Adjust the pH to pH 6 using concentrated phosphoric acid.

[0382] 1 mg / mL cysteine ​​= 8.25 mM

[0383] 0.1% Brij-35 surfactant

[0384] enzymes :

[0385] Recombinant human cathepsin B; BioLegend catalog number 557704

[0386] Activity = 2.5 U / µg; dilute before assay.

[0387] The enzyme was prepared by serial dilution with 2X, and then mixed with the substrate at a 1:1 ratio.

[0388] probe:

[0389] The 2X solution was prepared at 12 µM, therefore the final concentration was 6 µM.

[0390] Plate Reader :

[0391] Tecan SPARK

[0392] in conclusion

[0393] Figures 18A-20B The results provided demonstrate that compound II-1 exhibits more than twice the signal of a coumarin-based AMC-LR probe, and at a slightly faster rate.

[0394] Example 17

[0395] In plate-based assays, the activity of cathepsin B enzyme-based probe candidates based on chlororhodamine (Cl-Rhod) was compared. The probes were compared to compound II-1, which also contains an LR cleavage site. However, in Cl-Rhod, the peptide is directly attached to the xanthine ring, while in compounds according to formula II (such as compound II-1), the peptide is attached to the dye via a self-eliminating linker. Additionally, the peptide is attached to a larger polycyclic moiety, for example, compared to coumarin.

[0396]

[0397] Materials and methods

[0398] reaction buffer :

[0399] Basic buffer = phosphate-buffered saline, pH 7.4

[0400] Adjust the pH to pH 6 using concentrated phosphoric acid.

[0401] 1 mg / mL cysteine ​​= 8.25 mM

[0402] 0.1% Brij-35 surfactant

[0403] enzymes :

[0404] Recombinant human cathepsin B; BioLegend catalog number 557704

[0405] Activity = 2.5 U / µg; dilute before assay.

[0406] The enzyme was prepared by serial dilution with 2X, and then mixed with the substrate at a 1:1 ratio.

[0407] probe :

[0408] The 2X solution was prepared at 12 µM, therefore the final concentration was 6 µM.

[0409] Plate Reader :

[0410] Tecan SPARK

[0411] in conclusion

[0412] Compared to the halogenated probe, Cl-Rhod showed no measurable activity against cathepsin B. Figure 21 ).

[0413] Example 18

[0414] In plate-based assays, compound II-2 was tested against cathepsin B to compare activity. The probe was compared with compound II-1, which also contains an LR cleavage site.

[0415] Materials and methods

[0416] reaction buffer :

[0417] Basic buffer = phosphate-buffered saline, pH 7.4

[0418] Adjust the pH to pH 6 using concentrated phosphoric acid.

[0419] 1 mg / mL cysteine ​​= 8.25 mM

[0420] 0.1% Brij-35 surfactant

[0421] enzymes :

[0422] Recombinant human cathepsin B; BioLegend catalog number 557704

[0423] Activity = 2.5 U / µg; dilute before assay.

[0424] The enzyme was prepared by serial dilution with 2X, and then mixed with the substrate at a 1:1 ratio.

[0425] probe :

[0426] The 2X solution was prepared at 12 µM, therefore the final concentration was 6 µM.

[0427] Plate Reader :

[0428] Tecan SPARK

[0429] in conclusion

[0430] Compound II-2 is active against cathepsin B, but its activity against cathepsin B is lower than that of compound I-1, and its signal-to-noise ratio is much lower. Figures 22A-22B ).

[0431] Example 19

[0432] The Val-Cit linker was initially thought to be specific for cathepsin B; however, recent studies have shown that it can also be cleaved by other cathepsins (Caculitan et al., Cancer Res. 77:7027 2017). To assess the confounding nature of the cathepsin B probes disclosed herein, the activities of compound I-1 against cathepsin B, cathepsin L, and cathepsin D were compared. Compound I-1 in its free acid form was titrated with recombinant cathepsin B, cathepsin D, and cathepsin L for 2 hours, and fluorescence was measured on a Tecan Spark plate reader. Surprisingly, cathepsin L exhibited approximately four times the activity of cathepsin B against compound I-1, while cathepsin D showed almost no activity under the same assay conditions. Figure 23 Therefore, the cathepsin B probe presented in this paper may not be specific to cathepsin B alone, but the probe does show specificity to certain lysosomal cathepsins and can prevent proteolytic release outside the lysosome.

[0433] Compound I-1 was used as a measure of enzyme activity to investigate the effect of inhibition on the activities of cathepsin B and cathepsin L. Recombinant cathepsin B and cathepsin L were titrated with the cathepsin B inhibitor CA-074 for 1 hour, followed by co-incubation with the free acid form of compound I-1 for 1 hour, and fluorescence was measured on a Tecan Spark. Although CA-074 effectively inhibited cathepsin B (IC50 = 20 μM), its inhibitory effect on cathepsin L was extremely low (IC50 = 1.875 nM), indicating that it is a good inhibitor for live cell inhibition. Figure 24 ).

[0434] Example 20

[0435] SKBR3 cells were treated with either trastuzumab-compound I-7 conjugate or Hu IgG1 isotype control-compound I-7 conjugate, followed by fixation with 4% PFA. EVOS were used. ™ The M7000 imaging system acquires images immediately after fixation and 24 hours after fixation. Figure 25 (In CellInsight) ™On the CX7 LED, quantitative results of the signal were determined through high intrinsic analysis. CircSpot thresholding was used to segment and analyze the average CircSpot intensity, and the results are shown below. Figure 26 These results indicate that conjugates of compounds containing peptides with “reverse” orientations, such as compound I-7, can be immobilized with 4% PFA.

[0436] Given that the principles of this disclosure can be applied to many possible embodiments, it should be understood that the aspects shown are merely preferred examples of this disclosure and should not be considered as limiting the scope of this disclosure. Rather, the scope of this disclosure is defined by the following claims. Therefore, we claim protection for all technologies falling within the scope and spirit of these claims.

[0437] Incorporated by reference

[0438] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference.

Claims

1. A compound according to formula I Or its salt, wherein: X is either -dye- or -N(-dye)-; Peptides are peptide portions that can be cleaved by proteases; R is a reactive group suitable for conjugating the compound to the target molecule; and L 1 L 2 and L 3 Each of them is independently selected from the following connectors: covalent bonds, or a portion containing two or more covalent bonds and at least one atom selected from C, N, O, P or S.

2. The compound according to claim 1, wherein the compound has a structure according to formula IA. 。 3. The compound according to claim 1, wherein the compound has a structure according to formula IB. 。 4. The compound according to any one of claims 1-3, wherein L 2 Attached to the N-terminus of the peptide.

5. The compound according to any one of claims 1-3, wherein L 2 Attached to the C-terminus of the peptide.

6. The compound according to any one of claims 1-5, wherein L 1 L 2 and L 3 Each of these is independently selected from the following connectors: polyethylene glycol (PEG), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted unsaturated alkyl, alkenyl, alkoxy, alkanoyl, alkylamino, aryloxy, arylamino, aralkyl, arylalkoxy, arylanoyl, arylamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylalkyl, heteroarylalkoxy, heteroarylanoyl, heteroarylamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalanoyl, heterocyclic, heterocyclicoxy, heterocyclicamino, heterocyclic alkyl, heterocyclic alkoxy, heterocyclic alkanoyl, heterocyclic alkanoyl, heterocyclic alkanoyl, alkanoylamino, arylanoylamino, alkylcarboxyl, carbonate, carbamate, guanidine, urea, thiourea, phosphoryl, sulfonyl, sulfonamide, or ketone.

7. The compound according to any one of claims 1-5, wherein L 1 L 2 and L 3 Each of the following is independently selected from: polyethylene glycol (PEG), substituted or unsubstituted C6 aryl groups, substituted or unsubstituted 5- to 10-membered heteroaryl groups containing one, two, or three heteroatoms selected from N, O, or S, substituted or unsubstituted C6 aryl groups. 1-6 Alkyl, substituted or unsubstituted C 2-6 Alkenyl, substituted or unsubstituted C 2-6 alkynyl group, C 1-6 Alkoxy, C 2-6 Alkyl (C 2-6 Alkyl-C(=O)-), C 1-6 Alkylamino, C6 aryloxy, C6 arylamino, C 7-9 Araneyl, C 7-9 Arylalkoxy, C 7-9 Araneyl, C 7-9 Arylamino, (substituted or unsubstituted, 5- to 10-membered heteroaryl groups comprising 1, 2, or 3 heteroatoms selected from N, O, or S) -O-, (substituted or unsubstituted, 5- to 10-membered heteroaryl groups comprising 1, 2, or 3 heteroatoms selected from N, O, or S) -NH-, (substituted or unsubstituted, 5- to 10-membered heteroaryl groups comprising 1, 2, or 3 heteroatoms selected from N, O, or S) -C 1-6 Alkyl-, (substituted or unsubstituted 5- to 10-membered heteroaryl groups comprising one, two, or three heteroatoms selected from N, O, or S)-C 1-6 Alkyl-O-, (substituted or unsubstituted 5- to 10-membered heteroaryl groups comprising one, two, or three heteroatoms selected from N, O, or S)-C 1-6 Alkyl-C(=O)-, (substituted or unsubstituted 5- to 10-membered heteroaryl groups containing one, two, or three heteroatoms selected from N, O, or S)-C 1-6 Alkyl-NH-, C 3-7 cycloalkyl, C 3-7 Cycloalkenyl, C 3-7 Cycloalkylalkyl, C 3-7 Cycloalkoxy, C 3-7 Cycloalkyl acyl, C 3-7 Cycloalkylamino, substituted or unsubstituted, 5- to 10-membered heterocyclic group comprising 1, 2, or 3 heteroatoms selected from N, O, or S; (substituted or unsubstituted, 5- to 10-membered heterocyclic group comprising 1, 2, or 3 heteroatoms selected from N, O, or S) -O-; (substituted or unsubstituted, 5- to 10-membered heterocyclic group comprising 1, 2, or 3 heteroatoms selected from N, O, or S) -NH-; (substituted or unsubstituted, 5- to 10-membered heterocyclic group comprising 1, 2, or 3 heteroatoms selected from N, O, or S) -C 1-6 Alkyl- (substituted or unsubstituted 5- to 10-membered heterocyclic group comprising one, two, or three heteroatoms selected from N, O, or S) -C 1-6 Alkyl-O-, (substituted or unsubstituted 5- to 10-membered heterocyclic group comprising one, two, or three heteroatoms selected from N, O, or S)-C 1-6 Alkyl-C(=O)-, (substituted or unsubstituted 5- to 10-membered heterocyclic group containing one, two, or three heteroatoms selected from N, O, or S)-C 1-6 Alkyl-NH-, C 1-6 Alkylamino (-C 1-6 alkyl-C(=O)NH-), -C6 aryl-C(=O)NH-, aralkylamide (-C6 aryl-C 1-6 alkyl-C(=O)NH-), alkyl carboxyl group (-C 1-6 Alkyl-C(=O)-), carbonate (-OC(=O)O-), carbamate (-OC(=O)NH-), guanidinyl (-NHC(=NH)NH-), urea (-NHC(=O)NH-), thiourea (-NHC(=S)NH-), phosphoryl (-P(=O)(OH)-), sulfonyl (-SO2-), sulfonamide (-SO2NH-), or ketone (-C(=O)-).

8. The compound according to any one of claims 1-5, wherein L 1 L 2 and L 3 Each of them is independently selected from -KLJ-, where: Each of K and J is independently a bond, -C(=O)-, or -NR. a -, -OC(=O)-, -C(=O)O-, -OC(=O)NH-, -NHC(=O)O-, -SO2-, -SO2NH-, -NHSO2-, -NHC(=S)NH-, -P(=O)(OH)-, -NHC(=O)NH-, -NHC(=NH)NH-, -OC(=O)O- or -O-; L is -C 1-20 Alkyl-,-[(CH2CH2)O] x (CH2CH2)-,-(CH2) y -(substituted or unsubstituted C6 aryl)-(CH2) y -,-(CH2) y -(substituted or unsubstituted 5- to 10-membered heterocyclic group containing one, two, or three heteroatoms selected from N, O, or S)-(CH2) y -,-(CH2) y -(substituted or unsubstituted 5- to 10-membered heteroaryl groups containing one, two, or three heteroatoms selected from N, O, or S)-(CH2) y -,-(CH2) y -(substituted or unsubstituted C) 3-8 Alicyclic group)-(CH2) y -; x is 1 to 6; Each y is independently 0 to 6; and R a It is H, C 1-4 Alkyl, 2,4-disulfonic acid benzyl, sulfopropylamine residue or monosulfonic acid benzyl.

9. The compound of claim 8, wherein for at least one of L 1 , L 2 , and L 3 , L is -[(CH2CH2)0] x (CH2CH2)-.

10. The compound of claim 8, wherein for at least two of L 1 , L 2 , and L 3 , L is -[(CH2CH2)0] x (CH2CH2)-.

11. The compound according to any one of claims 1-10, wherein the dye is a cyanine dye, rhodamine or a rhodamine analog dye, fluorescein or a fluorescein analog dye, xanthine or a xanthine analog dye, BODIPY dye, coumarin dye, phthalocyanine dye, porphyrin dye, pyrene dye, fluorene or polyfluorene dye, excited-state intramolecular proton transfer (ESIPT) dye, aggregation-induced emission dye, metal complex dye, or a combination thereof.

12. The compound according to any one of claims 1-11, wherein the dye is a fluorescein dye, rhodamine, group 14 rhodamine dye, phospharhodamine, cyanine dye having a 2-, 3-, 5-, 7- or 9-methylene structure, or coumarin.

13. The compound according to any one of claims 1-11, wherein the quencher has an absorption of 250 nm to 1,000 nm.

14. The compound according to any one of claims 1-12, wherein the quencher comprises an azo dye-based quencher, a cyanine-based quencher, a rhodamine-based quencher, an azaphthalocyanine-based quencher, a heme chloride quencher, malachite green, malachite green analogs, or combinations thereof.

15. The compound of claim 14, wherein the quencher comprises two, three, four, or five quenching portions.

16. The compound according to any one of claims 1-15, wherein the quencher comprises azobenzene, , , , , , , , , , , , , , , or .

17. The compound according to any one of claims 1-16, wherein R is an ester, isocyanate, isothiocyanate, maleimide, vinyl moiety, thiol, amine, hydroxyl, hydrazine, acylhydrazine, hydroxylamine, aminooxy, clickable handle for click chemistry, sulfonyl halide, sulfonamide, glyoxal, oxazolidinium, carbonate, carbamoyl halide, or photoreactive moiety.

18. The compound of claim 17, wherein the clickable handle for click chemistry is selected from azides, alkynes, strained alkynes, cyclooctyne, thioheptyne, tetrazine, and trans-cyclooctene (TCO).

19. The compound according to any one of claims 1-18, wherein the peptide is a cathepsin B-cleavable peptide.

20. The compound according to any one of claims 1-19, wherein: The peptide is selected from Gly-Gly-Phe-Gly (SEQ ID NO: 1), Val-Cit, Val-Cit-PAB, Gly-Gly, Gly-Gly-Gly, Phe-Lys, Val-Ala, Val-Gly, Val-Val, Ala-Ala-Asn, Gly-Phe-Leu-Gly (SEQ ID NO: 2), Arg-Arg, Phe-Arg-Arg-Gly (SEQ ID NO: 3), Gly-Phe-Leu-Gly-Lys (SEQ ID NO: 4), Lys-Lys, Lys-Lys-PAB, Gly-Arg-Arg-Gly-Lys-Gly-Gly (SEQ ID NO: 5), Gly-Glu-Leu-Gly, (SEQ ID NO: 6) Val-Arg, Glu-Arg, or Gly-Ile-Val-Arg-Ala-Lys (SEQ ID NO: 7); and PAB is a para-aminobenzyl group.

21. The compound according to any one of claims 1-20, wherein the peptide is Val-Cit or Val-Cit-PAB.

22. The compound according to any one of claims 1-21, wherein the compound is a salt comprising one, two, three, four or five counterions.

23. The compound of claim 22, wherein the 1, 2, 3, 4 or 5 counterions comprise trialkylammonium ions.

24. The compound according to claim 23, wherein the trialkylammonium ion is a triethylammonium ion.

25. The compound according to claim 1, wherein the compound is selected from... ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ; or 。 26. A compound having general formula II or its salt, wherein: Peptides are peptide portions that can be cleaved by proteases; R is a reactive group suitable for conjugating the compound to the target molecule; and L 1 is a linker selected from a covalent bond, or a moiety comprising two or more covalent bonds and at least one atom selected from C, N, O, P, or S; and L 2 is a covalent bond or a self-elimination linker.

27. The compound according to claim 26, wherein L 1 It is selected from the following connectors: polyethylene glycol (PEG), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted unsaturated alkyl, alkenyl, alkoxy, alkanoyl, alkylamino, aryloxy, arylamino, aralkyl, arylalkoxy, arylanoyl, arylamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylalkyl, heteroarylalkoxy, heteroarylanoyl, heteroarylamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalanoyl, heterocyclic, heterocyclicoxy, heterocyclicamino, heterocyclic alkyl, heterocyclic alkoxy, heterocyclic alkanoyl, heterocyclic alkanoyl, heterocyclic alkanoyl, alkanoylamino, arylanoylamino, alkylcarboxyl, carbonate, carbamate, guanidine, urea, thiourea, phosphoryl, sulfonyl, sulfonamide or ketone.

28. The compound according to claim 26, wherein L 1 It is a connector selected from -KLJ-, wherein: Each of K and J is independently a bond, -C(=O)-, or -NR. a -, -OC(=O)-, -C(=O)O-, -OC(=O)NH-, -NHC(=O)O-, -SO2-, -SO2NH-, -NHSO2-, -NHC(=S)NH-, -P(=O)(OH)-, -NHC(=O)NH-, -NHC(=NH)NH-, -OC(=O)O- or -O-; L is -C 1-20 Alkyl-,-[(CH2CH2)O] x (CH2CH2)-,-(CH2) y -(substituted or unsubstituted C6 aryl)-(CH2) y -,-(CH2) y -(substituted or unsubstituted 5- to 10-membered heterocyclic group containing one, two, or three heteroatoms selected from N, O, or S)-(CH2) y -,-(CH2) y -(substituted or unsubstituted 5- to 10-membered heteroaryl groups containing one, two, or three heteroatoms selected from N, O, or S)-(CH2) y -,-(CH2) y -(substituted or unsubstituted C) 3-8 Alicyclic group)-(CH2) y -; x is 1 to 6; Each y is independently 0 to 6; and R a is H, C 1-4 alkyl, 2,4-disulfobenzyl, sulfopropylamine acid residue or monosulfobenzyl.

29. The compound according to any one of claims 26-28, wherein the dye is an acridinone dye, a fluorescein dye or an analogue thereof, a coumarin, a xanthine-hybrid cyanine dye, a phenolic dye or an aniline dye.

30. The compound according to any one of claims 26-29, wherein the dye is halogenated, DDAQ, fluorescein, or coumarin.

31. The compound according to any one of claims 26-30, wherein R is an ester, isocyanate, isothiocyanate, maleimide, vinyl moiety, thiol, amine, hydroxyl, hydrazine, acylhydrazine, hydroxylamine, aminooxy, clickable handle for click chemistry, sulfonyl halide, sulfonamide, glyoxal, oxazolidinium, carbonate, carbamoyl halide, or photoreactive moiety.

32. The compound of claim 31, wherein the clickable handle for click chemistry is selected from azides, alkynes, strained alkynes, cyclooctyne, thioheptyne, tetrazine, and trans-cyclooctene (TCO).

33. The compound according to any one of claims 26-32, wherein the peptide is a cathepsin B-cleavable peptide.

34. The compound according to any one of claims 26-33, wherein: The peptide is selected from Gly-Gly-Phe-Gly (SEQ ID NO: 1), Val-Cit, Val-Cit-PAB, Gly-Gly, Gly-Gly-Gly, Phe-Lys, Val-Ala, Val-Gly, Val-Val, Ala-Ala-Asn, Gly-Phe-Leu-Gly (SEQ ID NO: 2), Arg-Arg, Phe-Arg-Arg-Gly (SEQ ID NO: 3), Gly-Phe-Leu-Gly-Lys (SEQ ID NO: 4), Lys-Lys, Lys-Lys-PAB, Gly-Arg-Arg-Gly-Lys-Gly-Gly (SEQ ID NO: 5), Gly-Glu-Leu-Gly (SEQ ID NO: 6) Val-Arg, Glu-Arg, or Gly-Ile-Val-Arg-Ala-Lys (SEQ ID NO: 7); and PAB is a para-aminobenzyl group.

35. The compound according to any one of claims 26-34, wherein the peptide is Val-Cit or Val-Cit-PAB.

36. The compound of any one of claims 26-35, wherein L 1 is -[(CH2CH2)O] x (CH2CH2)-.

37. The compound according to any one of claims 26-36, wherein L 2 is selected from 、 、 or .

38. The compound according to any one of claims 26-37, wherein the compound is selected from: or .

39. A composition comprising a compound conjugated to a biomolecule according to any one of claims 1-38.

40. The composition of claim 39, wherein the biomolecule is an antibody, enzyme, protein, oligonucleotide, or dextran.

41. The composition according to any one of claims 38-40, further comprising one or more of a solvent, a buffer, a surfactant, or a combination thereof.

42. A reagent kit comprising: The compound according to any one of claims 1-38 or the composition according to any one of claims 39-41; as well as Solvent, buffer, surfactant, purification column, collection tube, sodium bicarbonate, or any combination thereof, or more thereof.

43. The kit according to claim 42, wherein the surfactant is polyoxyethylene lauryl ether (Brij). ™ -35), polyoxyethylene (20) sorbitan monolaurate (Tween ™ -20), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton) ™ X-100), octylphenoxypolyethoxyethanol (Nonidet) ™ P-40) or combinations thereof.

44. The kit according to claim 42 or 43, wherein the buffer is phosphate-buffered saline (PBS), tris(hydroxymethyl)aminomethane-HCl (Tris-HCl), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES), or a combination thereof.

45. The kit according to any one of claims 42-44, wherein the solvent comprises water, DMSO, methanol, or a combination thereof.

46. ​​A method comprising: Contact the cells with the compound according to any one of claims 1-38; Expose the cells to light; as well as Determine whether the signal exists.

47. A method comprising: Contact the sample with the compound according to any one of claims 1-38; Expose the sample to light; as well as Determine whether the signal exists.

48. The method of claim 46 or claim 47, wherein exposing the cell or sample to light and determining the presence or absence of the signal comprises exposing the cell or sample to light of a first wavelength and detecting the presence or absence of light of a second wavelength different from the first wavelength.

49. The method of claim 46 or claim 48, wherein the cell is an in vitro cell or an ex vivo cell.

50. The method of claim 46 or claim 48, wherein the cell is an in vivo cell.

51. A method comprising: The compound according to any one of claims 1-38 is conjugated with a biomolecule to form a biomolecule-compound conjugate.

52. The method of claim 51, further comprising: The time interval during which the cells are incubated is sufficient to allow the biomolecule-compound conjugate to enter the cells; The cells were irradiated with light of an appropriate wavelength to excite dye generation signals; as well as The signal from the biomolecule-compound conjugate is detected.

53. A method comprising: Contact the cells with the biomolecule-compound conjugate according to claim 51; The time interval during which the cells are incubated is sufficient to allow the biomolecule-compound conjugate to enter the cells; The cells were irradiated with light of an appropriate wavelength to excite dye generation signals; as well as The signal from the biomolecule-compound conjugate is detected.

54. A method comprising: Contact the cells with the composition according to any one of claims 39-41; The time interval during which the cells are incubated is sufficient to allow the composition to enter the cells; The cells were irradiated with light of an appropriate wavelength to excite dye generation signals; as well as The signal from the composition is detected.

55. A method comprising: Contact the sample with the compound according to any one of claims 1-38, or the composition according to any one of claims 39-41, or the biomolecule-compound conjugate according to claim 51; The time interval during which the sample is incubated is sufficient to allow the peptide to be enzymatically cleaved; Irradiate the sample with light of an appropriate wavelength that will excite the dye to generate a signal; as well as The signal from the sample is detected.

56. The method according to any one of claims 46-55, wherein the time interval is from 1 hour to 24 hours.

57. The method according to any one of claims 46-56, wherein the wavelength of the light is the wavelength of visible light.

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