Activity-based probe compounds, compositions, and methods of use thereof

Abstract

The present invention provides activity-based probe compounds for labeling cysteine proteases. The compounds target proteases through specific target elements. The compounds also include a detectable element, such as a fluorescent label, a radiolabel or a chelating agent. In some cases, the compound also includes a quenching element that is released upon reaction with the protease. The invention also provides compositions comprising the compounds, and methods of using the compounds, such as labeling proteases in an animal body and visualizing a tumor in an animal body.

Description

[0001] This application is a divisional application of the patent application with the application number 2014800282877, the application date of March 15, 2014, and the invention title of "Activity-Based Probe Compounds, Compositions, and Methods of Use Thereof". Technical Field

[0002] Cross-Reference to Related Applications

[0003] This application claims the benefit of U.S. Provisional Application No. 61 / 794,296, filed on March 15, 2013, the disclosure of which is incorporated herein by reference in its entirety.

[0004] Statement Regarding Government Support

[0005] This invention was made under a government support project with the project number 5R01NS045684-07 (awarded by the National Institutes of Health, USA). The government has certain rights in this invention. Background Art

[0006] Currently, various technologies are being developed for the fields of molecular imaging and disease monitoring. In particular, optical fluorescence imaging is a method that is beginning to show potential as a clinical tool due to its sensitivity, specificity, and non-invasiveness. The specificity of a fluorescent optical probe can be provided by a biological target in some cases. For example, an optical probe that is recognized by an enzyme target in a biological sample often produces a very specific signal if the fluorescence of the probe is only released in the case of an enzyme reaction. Ideally, even when the fluorescent signal has been activated by an enzyme reaction, the fluorescent moiety of the probe remains attached to its enzyme target. The use of such activity-based probes (ABPs) for protease targets has been disclosed. Blum et al., (2009) PLoS One 4: e6374; doi:10.1371 / journal.pone.0006374. The permanent covalent bond formed through the reaction between the ABP and the catalytic residue of the active part of the enzyme enables the ABP to be distinguished from a simple fluorescent substrate. Although the signal amplification due to the catalytic turnover by the target enzyme may seem to make the fluorescent substrate advantageous, it has been found that the APB shows increased tissue uptake kinetics and an extended probe retention period in the target tissue due to the covalent modification of the target enzyme.

[0007] Among them, the target enzyme to be used with the fluorescence-based optical probe is a protease, particularly a cysteine protease. This cysteine protease is a protease family that plays an important role in health and disease. Reiser et al., (2010) J. Clin. Invest. 120:3421-31. Although their functions have been mainly described as restricted to the endosomal pathway, however, more and more evidence proves that they are the main regulators of matrix degradation, indicating that they also act extracellularly. & Wilson (2011) Role of Cysteine Cathepsins in Extracellular Proteolysis. Biology of Extracellular Matrix Volume 2 23-51. In addition, it has been shown that the cysteine cathepsin family is a major player in the development and progression of several types of cancer. Mohamed & Sloane (2006) Nat. Rev. Cancer (2006) 6:764-75; Palermo & Joyce (2008) Trends Pharmacol. Sci. 29:22-8. In addition, alterations in the expression of endogenous inhibitors of cysteine cathepsins (cystatins) have been observed in cancer. Cox (2009) Cystatins and cancer. Front. Biosci. 14:463-74. These observations, combined with the potential changes in the intracellular and extracellular environments, emphasize the importance of tools that allow direct assessment of the activity of these proteases in the context of the native tumor microenvironment. Several ABPs against the cysteine cathepsin family have been synthesized. Edgington et al., (2011) Curr. Opin. Chem. Biol. 15:798-805. In particular, fluorescence quenching ABPs (qABPs) have been proven to be powerful tools for non-invasive optical imaging of cancer and subsequent characterization of target tissue proteases at the tissue level, cell level, and protein level. Blum et al., (2007) Nat. Chem. Biol. 3:668-77; Verdoes et al. (2012) Chem. Biol. 19:619-28.

[0008] Inhibitors of activity-based dipeptidyl peptidase I based on the 2,3,5,6-tetrafluorophenoxy arylmethyl ketone reactive group have been reported (Deu et al., (2010) Chem Biol. 17:808-819), but these inhibitors are non-peptidic and do not include detectable groups.

[0009] Fluorescent quenched activity-based peptide inhibitors for imaging cells containing active proteases, such as cathepsins, have also been reported. See, for example, U.S. Patent Application Publication No. 2007 / 0036725. These probes employ an ester-linked acyloxymethyl ketone reactive group to bind to the protease active site. In some cases, the activity-based fluorescent probes are non-peptide probes. See, for example, PCT International Publication No. WO 2012 / 118715. In some cases, activity-based probes are used to radiolabel their target enzymes. See, for example, PCT International Publication No. WO 2009 / 124265.

[0010] However, there remains a need in the art for novel activity-based fluorescent probes for cysteine proteases that have higher cellular uptake rates, target a broader spectrum of cysteine protease activities, and provide improved detection sensitivity. SUMMARY OF THE INVENTION

[0011] The present invention addresses the above and other problems by providing compounds, compositions, and methods of using the compounds and compositions for labeling cysteine proteases.

[0012] In particular, according to one aspect of the present invention, there is provided a compound represented by Structural Formula (I):

[0013]

[0014] wherein

[0015] L is an ether-linked leaving element;

[0016] T is a target element;

[0017] D is a detectable element.

[0018] In some embodiments of the present invention, the D group is a fluorescent label, a radiolabel, or a chelator.

[0019] In certain embodiments, the D group is a fluorescent label, and even more specifically, fluorescein, Oregon green, BODIPY dye, rhodamine, or a cyanine label. In still more specific embodiments, the fluorescent label is a cyanine label, such as Cy5.

[0020] In some embodiments of the present invention, the T group targets the compound to cysteine proteases. In certain embodiments, the T group is a non-peptide target element (e.g., an element containing a triazole structure), including (e.g.) various specific elements containing a triazole structure. In certain other embodiments, the T group is a peptide target element.

[0021] In some embodiments of the compounds, the D-T-group is

[0022]

[0023] wherein L 1 is a linker;

[0024] AA 1 is an amino acid side chain;

[0025] U is O, N or S;

[0026] R 1 is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclic, heterocyclicalkyl or a protecting group, and is optionally substituted by 1 to 3 A groups; and

[0027] each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkanamino, aryl, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkanamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkanamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclic, heterocycloxy, heterocyclamino, heterocyclicalkyl, heterocyclicalkoxy, heterocyclicalkanoyl, heterocyclicalkanamino, hydroxy, thio, amino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidyl, ureido, halo, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfamino or azido.

[0028] In embodiments of certain compounds, L 1 is an optionally substituted alkyl linker, wherein each carbon atom is optionally replaced by a heteroatom.

[0029] In embodiments of other certain compounds, AA 1 is an aralkyl amino acid side chain and is optionally substituted by 1 to 3 A groups.

[0030] In embodiments of still other certain compounds, U is O.

[0031] In some embodiments, the D-group is a fluorescent label, a radioactive label or a chelator.

[0032] In certain embodiments, the D-group is a fluorescent label, and even more specifically is fluorescein, Oregon Green, BODIPY dye, rhodamine or a cyanine label. In still more specific embodiments, the fluorescent label is a cyanine label, such as Cy5.

[0033] In some embodiments of the compounds, the L group comprises a quencher. In more specific embodiments, L is L 2 -L 3 -Q, where L 2 is phenoxy, L 3 is a linker, and Q is a quencher.

[0034] In specific embodiments, L is

[0035]

[0036] where each Y is independently an electron-withdrawing group or hydrogen.

[0037] In more specific embodiments, each Y is independently halogen or hydrogen. In even more specific embodiments, L is

[0038] and

[0039] L 3 is an optionally substituted alkyl linker, where each carbon atom is optionally replaced by a heteroatom.

[0040] In even more specific embodiments, L is

[0041] where

[0042] R is a QSY quencher and n is an integer from 1 to 16.

[0043] In preferred embodiments, the QSY quencher is a hydrophilic QSY quencher. More specifically, the hydrophilic QSY quencher is a sulfo-QSY quencher.

[0044] In some embodiments of the present invention, the compound is a compound represented by structural formula (II):

[0045]

[0046] where D is a fluorescent label, L 3 is a linker, and Q is a quencher.

[0047] In more particular embodiments, the compound is a compound represented by structural formula (III):

[0048]

[0049] where R is a QSY quencher, D is a cyanine dye, and m and n are independently integers from 1 to 16. In some embodiments, the R group can be QSY21 or sulfo-QSY21, and the D group can be Cy5.

[0050] The specific compounds of the present invention include the following compounds:

[0051]

[0052] wherein R = QSY21 and n = 6;

[0053] R = sulfo-QSY21 and n = 6;

[0054] R = QSY21 and n = 2; and

[0055] R = sulfo-QSY21 and n = 2.

[0056] According to another aspect of the present invention, the present invention provides a composition comprising the compound of the present invention and a pharmaceutically acceptable carrier, which is used for labeling proteases in animals.

[0057] According to still another aspect of the present invention, the present invention provides a method for labeling proteases in animals, the method comprising the following steps:

[0058] Administering the composition of the present invention to an animal.

[0059] The present invention also provides a method for visualizing tumors in animals, the method comprising the following steps:

[0060] Administering the composition of the present invention to an animal, and measuring a detectable signal generated in the animal due to the reaction between the composition and cathepsin cysteine protease, wherein the detectable signal is related to tumors in the animal.

[0061] In a specific method embodiment, the detectable signal is a fluorescence signal. In other specific method embodiments, the fluorescence signal is generated at the tumor margin. Brief Description of the Drawings

[0062] Figure 1 .a) Structures of qABP GB137(1) and probes 2 to 8 synthesized in this work. b) Labeling profiles of probes 1 to 8 at 1 μM in live RAW cells. c) Concentration-dependent labeling of probes 1 and 8 in live RAW cells. d) Total cathepsin labeling intensity of probes 1-8 relative to 5 μM GB137(1) in live RAW cells.

[0063] Figure 2. a) Concentration-dependent labeling of probe 8 on RAW cell lysates at pH 5.5. b) Labeling time course of 0.5 μM probe 8 in live RAW cells. c) Labeling inhibition and serum stability of probes 1 and 8 by pretreatment with JPM-OEt (50 μM) in live RAW cells. d) Live cell fluorescence micrographs of RAW cells exposed to 1 μM probe 8 (upper row) or micrographs co-localized with lysotracker (second row, scale bar: 10 μm).

[0064] Figure 3 . a) Time-course plots of non-invasive optical imaging of tumor-bearing mice injected with probes 8 and 1 (right panel). The bottom panel represents the optimal fluorescence contrast at each time point. b) Time-dependent tumor-specific fluorescence (tumor-background) in mice treated with probe 1 or 8 (n = 3; data represent mean ± standard error). c) Ex vivo tumor fluorescence (upper panel) and in vivo fluorescently labeled proteins (lower panel) visualized by in-gel fluorescence scanning after SDS-PAGE. d) Fluorescence intensities at the end points of non-invasive optical imaging (shown in a), ex vivo tumor imaging, and in-gel fluorescence labeling (shown in c). Intensities relative to probe 1 are shown (n = 3; data represent mean ± standard error). e) Micrograph of fluorescence of tumor tissue sections treated with probe 8 (left panel), where CD68 immunostaining was performed (middle panel) and nuclear staining (DAPI - right panel, scale bar: 50 μm). f) Three-dimensional reconstruction by CLSM of tumor tissue sections treated with probe 8 (red), where CD68 immunostaining was performed (green) and nuclear staining (DAPI - blue).

[0065] Figure 4 . a) Immunoprecipitation of cathepsin labeled with BMV109. b, c) Concentration-dependent labeling of probes 1 - 8 in live RAW cells. The plots in b) and c) were electrophoresed on the same gel, respectively.

[0066] Figure 5. a) Non-invasive optical images of tumor-bearing mice 8 hours after injection of probe 1, 2, 6, or 8. The bottom graph represents the optimal fluorescence contrast at each time point. b) Time-dependent tumor-specific fluorescence (tumor-background) of mice treated with probe 1, 2, 6, or 8 (n = 3; data represent mean ± standard error). c) Ex vivo tumor fluorescence (upper panel) and in vivo fluorescently labeled proteins (lower panel) visualized by in-gel fluorescence scanning after SDS-PAGE. d) Fluorescence intensities at the endpoints of non-invasive optical imaging (shown in a), ex vivo tumor imaging, and in-gel fluorescence labeling (shown in c). Intensities relative to probe 1 are shown (n = 3; data represent mean ± standard error). e) Fluorescence micrographs of tumor tissue sections treated with probe 8 (first, third, and fourth columns), where CD68 immunostaining was performed (second, third, and fourth columns) and nuclear staining (DAPI, third and fourth columns, scale bar: 50 μm). Un-probed control (graph in the middle row) and isotype control for immunostaining (lower graph) are shown. f) Co-localization map of probe 8 (Cy5) and CD68 (FITC). Detailed Description

[0067] Cysteine cathepsins are a family of proteases that play important roles in normal cell physiology and in the pathology of many human diseases. Accordingly, many classes of substrates and activity-based probes (ABPs) have been developed to study the functions of these enzymes. Provided herein is a class of probes based on quenched fluorescence activity that, in some embodiments, comprise a phenoxymethyl ketone (PMK) electrophile. Compared to previously reported ABPs, these reagents exhibit enhanced spectral reactivity towards cysteine cathepsins, resulting in substantially improved labeling performance both in vitro and in vivo. Further, it is demonstrated herein that the probes display tumors in mice with unprecedented signal intensity and contrast. These new reagents enable the study of cysteine cathepsins at the organismal, tissue, cellular, and protein levels in multiple models of human disease.

[0068] Compound

[0069] Accordingly, in some aspects, the present invention provides a novel compound for labeling proteases, particularly cathepsins. The compounds of the present invention can be compounds of formula (I):

[0070]

[0071] wherein

[0072] L is an ether-linked leaving element;

[0073] T is a targeting element; and

[0074] D is a detectable element.

[0075] The target element T of the compounds of the present invention can be a peptide or non-peptide structure, preferably targeting the compounds to cysteine proteases.

[0076] Non-limiting examples of non-peptide structural elements incorporated into the compounds of the present invention for these purposes are those described in PCT International Publication No. WO2012 / 118715, the entire content of which is incorporated herein by reference. In a preferred embodiment, the non-peptide target element comprises a triazole structure.

[0077] Specific examples of the compounds of the present invention having a non-peptide target element are:

[0078]

[0079] Non-limiting examples of peptide structural elements that can be incorporated into the compounds of the present invention for targeting the compounds to cysteine proteases (especially cathepsin cysteine) are those described in PCT International Publication No. WO2009 / 124265, the entire content of which is incorporated herein by reference.

[0080] In some embodiments of the compounds of the present invention, D-T- is

[0081]

[0082] wherein L 1 is a linker;

[0083] AA 1 is an amino acid side chain;

[0084] U is O, N or S;

[0085] R 1 is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclic, heterocyclicalkyl or a protecting group, and is optionally substituted by 1 to 3 A groups; and

[0086] Each A is independently an alkyl group, alkenyl group, alkynyl group, alkoxy group, alkanoyl group, alkanamino group, aryl group, aryloxy group, arylamino group, aralkyl group, aralkoxy group, aralkanoyl group, aralkanamino group, heteroaryl group, heteroaryloxy group, heteroarylamino group, heteroaralkyl group, heteroaralkoxy group, heteroaralkanoyl group, heteroaralkanamino group, cycloalkyl group, cycloalkenyl group, cycloalkylalkyl group, cycloalkoxy group, cycloalkanoyl group, cycloalkanamino group, heterocyclic group, heterocycloxy group, heterocyclicamino group, heterocyclicalkyl group, heterocyclicalkoxy group, heterocyclicalkanoyl group, heterocyclicalkanamino group, hydroxy group, thio group, amino group, alkanoylamino group, aroylamino group, aralkanoylamino group, alkylcarboxyl group, carbonate group, carbamate group, guanidyl group, ureido group, halo group, trihalomethyl group, cyano group, nitro group, phosphoryl group, sulfonyl group, sulfamino group or azido group.

[0087] As used herein, the term "alkyl" refers to saturated aliphatic groups, including straight-chain alkyl, branched-chain alkyl, cycloalkyl (alicyclic), alkyl-substituted cycloalkyl, and cycloalkyl-substituted alkyl. In some embodiments, the straight-chain or branched-chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., straight-chain C 1 -C 30 , branched-chain C 3 -C 30 ), more specifically 20 or fewer. Similarly, some cycloalkyls have 3 to 10 carbon atoms in their ring structure, more specifically 5, 6, or 7 carbons in the ring structure.

[0088] In addition, as used throughout the specification, examples, and claims, the term "alkyl" (or "lower alkyl") is intended to include both "unsubstituted alkyl" and "substituted alkyl", where substituted alkyl means that the alkyl moiety has a substituent that replaces hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can (e.g.) include halo groups, hydroxy groups, carbonyl groups (such as keto groups, carboxyl groups, alkoxycarbonyl groups, formyl groups, or acyl groups), thiocarbonyl groups (such as thioesters, thioacetates, or thiocarboxylates), alkoxy groups, phosphoryl groups, phosphates, phosphonates, hypophosphites, amino groups, amides, amidines, imines, cyano groups, nitro groups, azido groups, thio groups, alkylthio groups, sulfates, sulfonates, sulfamoyl groups, sulfamino groups, sulfonyl groups, heterocyclic groups, aralkyl groups, aromatic or heteroaromatic moieties. Those skilled in the art will understand that the moieties substituted on the hydrocarbon chain, if desired, can themselves be substituted. For example, the substituents of substituted alkyl can include substituted and unsubstituted forms of amino groups, azido groups, imino groups, amido groups, phosphoryl groups (including phosphonates and hypophosphites), and sulfonyl groups (including sulfates, sulfamino groups, sulfamoyl groups, and sulfonates), as well as ethers, alkylthio groups, carbonyl groups (including ketones, aldehydes, carboxylic acids, and esters), -CF 3 , -CN, etc. Exemplary substituted alkyls are described below. Cycloalkyl can be further substituted by alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl, -CF 3, substituted with -CN etc.

[0089] In some specific embodiments, as used herein, the term "alkoxy" refers to a lower alkyl group in which oxygen is connected. Exemplary alkoxy groups include methoxy, ethoxy, propoxy, n-butoxy, etc.

[0090] As used herein, the term "alkenyl" refers to an aliphatic group containing at least one double bond, and is intended to include "unsubstituted alkenyl" and "substituted alkenyl", the latter referring to an alkenyl moiety having substituents that replace hydrogen on one or more carbons of the alkenyl. Such substituents may occur on one or more carbons that include or do not include one or more of the double bonds. In addition, unless stability is inhibited, such substituents include all those substituents expected to be used for alkyl groups as discussed above. For example, substitution of alkenyl by one or more alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups is contemplated.

[0091] When used in conjunction with chemical groups such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, the term "C x-y " is intended to include groups containing x to y carbon atoms in the chain. For example, the term "C x-y " refers to a substituted or unsubstituted saturated hydrocarbon group, including straight-chain alkyl and branched-chain alkyl groups containing x to y carbon atoms in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc. "C 0 -alkyl" indicates that the terminal group is hydrogen or a bond if internal. The terms "C 2-y -alkenyl" and "C 2-y -alkynyl" refer to substituted or unsubstituted unsaturated aliphatic group analogs that are similar to the above alkyl groups in terms of length and possible degree of substitution, but contain at least one double bond or triple bond, respectively.

[0092] As used herein, the term "alkylamino" refers to an amino group substituted by at least one alkyl group.

[0093] As used herein, the term "alkylthio" refers to a thio group substituted by an alkyl group, and can be represented by the general formula alkyl-S-.

[0094] As used herein, the term "alkynyl" refers to an aliphatic group containing at least one triple bond and is intended to include "unsubstituted alkynyl" and "substituted alkynyl", the latter referring to an alkynyl moiety having substituents that replace hydrogen on one or more carbons of the alkynyl. Such substituents may occur on one or more carbons that include or do not include one or more triple bonds. Additionally, unless stability is inhibited, such substituents include all those substituents contemplated for alkyls as discussed above. For example, substitution of an alkynyl by one or more alkyls, cycloalkyls, heterocyclics, aryls, or heteroaryls is contemplated.

[0095] As used herein, the term "amide" refers to the following group:

[0096]

[0097] wherein R x and R y each independently represent hydrogen or a hydrocarbon group or R x and R y together with the N atom to which they are attached form a heterocyclic ring structure having 4 to 8 atoms.

[0098] The terms "amine" and "amino" are well known in the art and refer to unsubstituted and substituted amines and their salts, such as the group represented by the following formula:

[0099]

[0100] wherein R x , R y and R z each independently represent hydrogen or a hydrocarbon group or R x and R y together with the N atom to which they are attached form a heterocyclic ring structure having 4 to 8 atoms.

[0101] As used herein, the term "aminoalkyl" refers to an alkyl substituted by an amino group.

[0102] As used herein, the term "aralkyl" refers to an alkyl substituted by an aryl group.

[0103] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups, wherein each atom of the ring is carbon. In certain embodiments, the ring is a 5- to 7-membered ring, and in more specific embodiments is a 6-membered ring. The term "aryl" also includes polycyclic systems having two or more rings, wherein two or more carbons are common to two adjacent rings, wherein at least one of the adjacent rings is aromatic, for example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclic. Aryl includes benzene, naphthalene, phenanthrene, phenol, aniline, etc.

[0104] The term "carbamate group" is well known in the art and refers to the following group:

[0105]

[0106] wherein R x and R y independently represent hydrogen or a hydrocarbon group or R x and R y together with their connecting atoms form a heterocyclic ring structure having 4 to 8 atoms.

[0107] As used herein, the term "cycloalkyl" refers to a non-aromatic saturated or unsaturated ring in which each atom of the ring is carbon. In certain embodiments, the ring of the cycloalkyl contains 3 to 10 atoms, and in more specific embodiments, 5 to 7 atoms.

[0108] The term "carbonate group" is well known in the art and refers to the group -OCO 2 -R x wherein R x represents a hydrocarbon group.

[0109] As used herein, the term "carboxyl" refers to the group represented by the formula -CO 2 H.

[0110] As used herein, the term "ester" refers to the group -C(O)OR x wherein R x represents a hydrocarbon group.

[0111] As used herein, the term "ether" refers to a hydrocarbon group connected to another hydrocarbon group through oxygen. Thus, the ether substituent of a hydrocarbon group can be hydrocarbon group -O-. Ethers can be symmetric or asymmetric. Examples of ethers include, but are not limited to, heterocyclic -O- heterocyclic and aryl -O- heterocyclic. Ethers include "alkoxyalkyl" groups, which can be represented by the general formula alkyl -O- alkyl.

[0112] The term "guanidyl" is well known in the art and can be represented by the following general formula:

[0113]

[0114] wherein R x and R y independently represent hydrogen or a hydrocarbon group.

[0115] As used herein, the terms "halo" and "halogen" refer to halogen and include fluorine, chlorine, bromine, and iodine.

[0116] As used herein, the terms "hetaralkyl" and "heteroaralkyl" refer to an alkyl group substituted with a heteroaryl group.

[0117] The terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic monocyclic structures, which are 5- to 7-membered rings in certain specific embodiments, more specifically 5- to 6-membered rings, the ring structure of which includes at least one heteroatom, in some embodiments, 1 to 4 heteroatoms, and in more specific embodiments, one or two heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic systems having two or more rings, wherein two or more carbons are common to two adjacent rings, wherein at least one of the rings is heteroaromatic, for example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclic. Heteroaryl includes, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, etc.

[0118] As used herein, the term "heteroatom" refers to an atom of any element other than carbon or hydrogen. Typical heteroatoms are nitrogen, oxygen, and sulfur.

[0119] The terms "heterocyclic group", "heterocycle", and "heterocyclic" refer to substituted or unsubstituted non-aromatic ring structures, which are 3- to 10-membered rings in certain specific embodiments, more specifically 3- to 7-membered rings, the ring structure of which includes at least one heteroatom, in some embodiments 1 to 4 heteroatoms, and in more specific embodiments one or two heteroatoms. The terms "heterocyclic group" and "heterocyclic" also include polycyclic systems having two or more rings, wherein two or more carbons are two adjacent rings, wherein at least one of the rings is a heterocyclic group, for example, a polycyclic system, and the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclic. Heterocyclic group includes, for example, piperidine, piperazine, pyrrolidine, morpholine, lactone, and lactam, etc.

[0120] As used herein, the term "heterocyclic alkyl" refers to an alkyl group substituted with a heterocyclic group.

[0121] As used herein, the term "hydrocarbyl" refers to a group bonded through carbon atoms that has no =O or =S substituents, and generally has at least one carbon-hydrogen bond and a predominantly carbon backbone, but may optionally include heteroatoms. Thus, for this purpose, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered hydrocarbyls, but substituents such as acetyl (which has an =O substituent on the connecting carbon) and ethoxy (which is connected through oxygen rather than carbon) are not considered hydrocarbyls. The hydrocarbyls include, but are not limited to, aryl, heteroaryl, carbocyclic, heterocyclic, alkyl, alkenyl, alkynyl, and combinations thereof.

[0122] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0123] When the term "lower" is used in conjunction with a chemical group (such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy), it refers to a group that includes ten or fewer non-hydrogen atoms in the substituent. And in certain embodiments, six or fewer. "Lower alkyl", for example, refers to an alkyl group containing ten or fewer carbon atoms, and in specific embodiments, six or fewer carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl substituents as defined herein are lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, and lower alkoxy, whether they appear alone or in combination with other substituents, as defined in hydroxyalkyl and aralkyl (in this case, for example, when counting the carbon atoms in the alkyl substituent, the atoms in the aryl are not counted).

[0124] The terms "polycyclyl", "polycycle", and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclic), where two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings". Each ring of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle is a ring containing 3 to 10 atoms, more specifically 5 to 7 atoms.

[0125] The term "substituted" refers to a group having substituents replacing hydrogen on one or more carbon atoms of the backbone. It should be understood that "substitution" or "substituted" includes implicit conditions that such substitution is in accordance with the allowed valences of the substituted atoms and substituents, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation (such as by rearrangement, cyclization, elimination, etc.) under its conditions of use. As used herein, "substituted" is intended to include all permitted substituents of organic compounds. Broadly speaking, permitted substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. For suitable organic compounds, the permitted substituents can be one or more and the same or different. For the purposes of this invention, a heteroatom (such as nitrogen) can have a hydrogen substituent and / or any permitted substituent of an organic compound that satisfies the valence of the heteroatom. Substituents can include any of the substituents described herein, e.g., halogen, hydroxy, carbonyl (such as ketone, carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioester, thioacetate, or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidino, imino, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfamino, sulfonyl, heterocyclic, aralkyl, aromatic or heteroaromatic moieties. Those skilled in the art will understand that if appropriate, the substituted moieties on a hydrocarbon chain can be substituted themselves.

[0126] Unless otherwise specified, the chemical moiety "unsubstituted" as referred to herein should be understood to include substituted variants. For example, the mention of an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.

[0127] The term "sulfate" is well known in the art and refers to the group -OSO 3 H or a pharmaceutically acceptable salt thereof.

[0128] The term "sulfonamide" is well known in the art and refers to a group represented by the following general formula:

[0129]

[0130] wherein R x and R y independently represent hydrogen or a hydrocarbon group.

[0131] The term "sulfoxide" is well known in the art and refers to the group -S(O)-R x wherein R x represents a hydrocarbon group.

[0132] The term "sulfonyl" or "sulfonate group" is well known in the art and refers to the group -SO 3H or a pharmaceutically acceptable salt thereof.

[0133] The term "sulfone" is well known in the art and refers to the group -S(O) 2 -R x wherein R x represents a hydrocarbon group.

[0134] As used herein, the term "thioalkyl" refers to an alkyl group substituted by a mercapto group.

[0135] As used herein, the term "thioester" refers to the group -C(O)SR x or -SC(O)R x wherein R x represents a hydrocarbon group.

[0136] As used herein, the term "thioether" is equivalent to an ether in which oxygen is replaced by sulfur.

[0137] The term "ureido" is well known in the art and can be represented by the following general formula:

[0138]

[0139] wherein R x and R y independently represent hydrogen or a hydrocarbon group.

[0140] The compounds of the present invention are generally synthesized using standard synthetic chemical techniques, for example, using the methods described in the Examples section below. Other useful synthetic techniques (e.g.) are described in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th Ed., (Wiley, 2013); Carey and Sundberg, Advanced Organic Chemistry 4 thEds., Vols. A and B (Plenum 2000, 2001); Fieser's Reagents for Organic Synthesis, Volumes 1 - 27 (Wiley, 2013); Rodd's Chemistry of Carbon Compounds, Volumes 1 - 5 and Supplements (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1 - 81 (Wiley, 2013); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989) (the entire contents of which are incorporated herein by reference). The starting materials used for these compounds are generally commercially available or can be readily prepared using methods well known to those skilled in the art. See, for example, Fieser's Reagents for Organic Synthesis, Volumes 1 - 27 (Wiley, 2013) or Beilstein's Handbuch der organischen Chemie, 4, Aufl. ed. Springer - Verlag, Berlin, including supplements.

[0141] When referring to the components of the compounds of the present invention, the term "residue derived from..." can be used to describe a residue formed by the reaction of a first reactive functional group on a first component and a second reactive functional group on a second component. In an exemplary embodiment, an amine group on a first component can react with an activated carboxyl group on a second component, thereby forming a residue comprising one or more amide moieties. The present invention encompasses other arrangements of the first and second reactive functional groups. For example, as will be understood by those skilled in the art, a reaction between an azide - substituted first component and an alkyne - substituted second component, catalyzed by a ketone or non - ketone, produces a residue containing a triazole via a well - known "click" reaction. See Kolb et al., (2001) Angew. Chem. Int. Ed. Engl. 40:2004; Evans (2007) Aus. J. Chem. 60:384. Exemplary methods for generating non - peptide fluorescent imaging probes using "click" reactions are provided in PCT International Publication No. WO 2012 / 118715. The adaptation of these methods to produce or modify the compounds of the present invention falls within the skill of the art.

[0142] Those skilled in the art will understand that a protecting group is reversibly attached to the desired position of a molecule to control the reaction of other reagents at that position. Protecting groups useful in the practice of the present invention are well known in the art. See, for example, Greene's Protective Groups in Organic Synthesis, 4th th edition, edited by P.G.M. Wuts and T.W. Greene (Wiley-Interscience, 2006); and Protecting Groups by P. Kocienski (Thieme, 2005).

[0143] The L 1 group of the compounds of the present invention is a linker group that connects the detectable element D to the target element. As will be understood by those skilled in the art, this group can be any suitable linker. The L 1 group is preferably an alkyl linker group, where the alkyl is optionally substituted, and in addition, the carbon in the linker is optionally replaced by a heteroatom to an extent that the resulting structure is chemically stable. Such substitution and replacement should be understood to include the insertion of groups within the linker, such as ethers, thioethers, disulfides, esters, amides, carbonates, carbamates, and the like. The preferred linker length is in the range of 5 to 40 bond lengths, and it can be branched, straight-chain, or contain a ring. In some cases, the linker can include double bonds. Depending on specific needs, they can be hydrophobic or hydrophilic as required.

[0144] It should also be understood that the connection between the L 1 group and the detectable element D can be any suitable chemical connection, as will be understood by those skilled in the art. For example, in some cases, the compounds of the present invention may be conveniently prepared by including in the precursor of the detectable element a moiety that reacts with a specific chemical group (such as an amino group, a thiol group, etc.). The detectable element can be readily attached to the target element at such a position through the reaction of this moiety on the target element. It can thus be understood that even though the details of the connection structure are not explicitly shown, these types of connections are within the scope of the compounds disclosed herein.

[0145] As will be understood by those skilled in the art, the AA 1 group of the compounds of the present invention can be any natural or unnatural amino acid side chain. In a preferred embodiment, the AA 1 group is an aralkyl amino acid side chain optionally substituted by 1 to 3 A groups. In an even more preferred embodiment, the AA 1 group is a phenylalanine side chain.

[0146] In a preferred compound, the U group is O.

[0147] In certain embodiments, the detectable element of the compounds of the invention is a fluorescent label, a radioactive label, a chelating agent, etc. Examples of radioactive labels and chelating agents suitable for use in these compounds are described in PCT International Publication No. 2009 / 124265.

[0148] In a preferred embodiment of the compounds of the invention, the detectable element is a fluorescent label. As is known to those of ordinary skill in the art, when stimulated by absorption of incident electromagnetic radiation, a fluorescent label emits electromagnetic radiation, preferably visible light. A wide variety of fluorescent labels are commercially available, including those having a reactive moiety for coupling the label to a reactive group such as an amino group, a thiol group, etc. For example, see, The Molecular Handbook—A Guide to Fluorescent Probes and Labeling Technologies.

[0149] An example of a fluorescent label is fluorescein, which is widely used for immunofluorescent labeling. Fluorescein is a xanthene dye having a maximum absorbance at 495 nanometers. A related fluorophore is Oregon Green, a fluorinated derivative of fluorescein.

[0150] In some embodiments, the fluorescent label used in the detectable element of the compounds of the invention can be a pH-dependent fluorophore. As will be understood by those skilled in the art, for example, such fluorescent labels as used in the compounds labeled “LES12” and “LES13” shown below exhibit a fluorescence spectrum that depends on the pH of the labeled environment and can thus be used to report environmental information about the label after the reaction, such as information about the location or type of protease labeled by the reacting compound. Various pH-dependent fluorophores of labels useful in the detectable element of the compounds of the invention are known. For example, see The Molecular Handbook—A Guide to Fluorescent Probes and Labeling Technologies, the entire contents of which are incorporated herein by reference.

[0151] Other exemplary fluorescent labels suitable for use in the compounds of the invention are BODIPY dyes, rhodamines, and cyanine dyes. In particular, BODIPY dyes are represented by 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (boron-dipyrromethene) and are known as dyes. Various derivatives of these dyes are known and are considered suitable for use as the detectable element in the compounds of the invention. For example, see Chen et al., (2000) J. Org. Chem. 65:2900-2906.

[0152] Another class of useful fluorescent labels employed in the compounds of the present invention are the IRDye infrared dyes available from Li-Cor (www.licor.com). Non-limiting examples of these dyes are IRDye 800CW, IRDye 680RD, IRDye680LT, IRDye 750, IRDye700DX, IRDye 800RS, and IRDye 650.

[0153] Rhodamine dyes are a class of dyes based on the rhodamine ring structure. Rhodamines include, in particular, tetramethylrhodamine (TMR) and carboxytetramethylrhodamine (TAMRA). TMR is a very common fluorophore used to prepare protein conjugates (especially antibody and avidin conjugates), while TAMRA is a dye commonly used for oligonucleotide labeling and automated nucleic acid sequencing. Rhodamine exists as a natural complement to fluorescein-based fluorophores, providing a longer wavelength emission maximum and thus offering the opportunity for multicolor labeling or staining.

[0154] The sulfonated rhodamine series fluorophores known as Alexa Fluor dyes are also included in the group of rhodamine dyes. The Alexa Fluor dyes introduced by Molecular Probes exemplify a great advance in modern fluorescence technology. For more intense fluorescence emission, these sulfonated rhodamine derivatives exhibit higher quantum yields than spectrally similar probes and have several additional improved properties, including enhanced photostability, absorption spectra matched to common laser lines, pH insensitivity, and high water solubility.

[0155] Cyanine dyes correspond to the related dyes Cy2, Cy3, Cy5, Cy7, and their families of derivatives, which are based on a partially saturated indole azacycle nucleus with two aromatic units linked by polyolefin bridges of different carbon numbers. These probes exhibit fluorescence excitation and emission spectra similar to many traditional dyes (such as fluorescein and tetramethylrhodamine), but they have enhanced water solubility, photostability, and higher quantum yields. Most cyanine dyes are more environmentally stable than their traditional counterparts, making their fluorescence emission intensity less sensitive to pH and the organic mounting medium. In a manner similar to Alexa Fluors, the excitation wavelengths of the Cy series of synthetic dyes are specifically tuned to be used by common laser and arc discharge light sources, and fluorescence emission can be detected using combinations of traditional filters. Cyanine dyes are readily available as reactive dyes or fluorophores. Cyanine dyes generally have a broader absorption spectrum than the members of the Alexa Fluor family, making them somewhat more versatile in the selection of laser excitation sources for confocal microscopy.

[0156] In a preferred embodiment, the detectable element of the compounds of the present invention is the cyanine dye Cy5.

[0157] In some embodiments, within the detectable element of the compounds of the present invention, it is beneficial to include multiple fluorescent labels, radioactive labels, chelating agents, etc. For example, the following exemplary compounds labeled "LES12" and "LES13" include two different fluorescent labels within separate detection elements. As can be understood by those skilled in the art, such multiple labels can be achieved using conventional coupling chemistry. For example, the fluorescent labels in the "LES12" and "LES13" compounds are coupled using "click" chemistry. Examples of useful intermediate compounds for synthesizing compounds containing multiple labels within the detectable element by "click" chemistry are shown below ("WL938"). This compound contains an azide group and thus can readily react with a suitable alkyne-containing reagent in a "click" reaction. As can be understood by those of ordinary skill in the art, the positions of the alkyne and azide groups can also be reversed if desired.

[0158] In some embodiments, the compounds of the present invention are compounds of formula (I), wherein T is a peptide target element, and the compounds are further described in the statements numbered below:

[0159] 1. A compound of formula (I), wherein D-T- is a short detectable peptidyl; and

[0160] L is an ether-linked leaving element.

[0161] 2. The compound of statement 1, wherein the detectable peptidyl contains 1 to 4 amino acid residues.

[0162] 3. The compound of statement 2, wherein D-T is:

[0163] wherein

[0164] each AA 1 , AA 2 , AA 3 and AA 4 is independently an amino acid side chain or -L 1 -D;

[0165] each R A is independently hydrogen or R 1 ;

[0166] R B is hydrogen, R 1 , -C(O)R 1 , -C(O)OR 1 , -C(O)SR 1 or -C(O)N(R1 )(R A );

[0167] R 1 is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclic group, heterocyclic group alkyl, protecting group or -L 1 -D, and is optionally substituted by 1 to 3 A groups;

[0168] Each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkanamino, aryl, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkanamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkanamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclic group, heterocyclic oxy group, heterocyclic group amino, heterocyclic group alkyl, heterocyclic group alkoxy, heterocyclic group alkanoyl, heterocyclic group alkanamino, hydroxy, thio group, amino, alkanoyl amino, aroyl amino, aralkanoyl amino, alkyl carboxyl, carbonate, carbamate, guanidyl, ureido, halo group, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfamino or azide group; and

[0169] L 1 is a linker.

[0170] 4. The compound of claim 3, wherein

[0171] each R A is hydrogen; and

[0172] R B is -C(O)OR 1 .

[0173] 5. The compound of claim 3, wherein D-T- is

[0174]

[0175] each AA 1 and AA 2 are independently amino acid side chains or -L 1 -D; and

[0176] each R A is hydrogen.

[0177] 6. The compound of claim 3, wherein D-T- is

[0178]

[0179] each AA 1 , AA 2 and AA 3is independently an amino acid side chain or -L 1 -D; and

[0180] each R A is hydrogen.

[0181] 7. The compound of claim 3, wherein D-T- is

[0182]

[0183] each AA 1 、AA 2 、AA 3 and AA 4 is independently an amino acid side chain or -L 1 -D; and

[0184] each R A is hydrogen.

[0185] 8. The compound of claim 3, wherein L 1 is an optionally substituted alkyl linker, wherein each carbon atom is optionally replaced by a heteroatom.

[0186] 9. The compound of claim 3, wherein R B is -C(O)OR 1 .

[0187] 10. The compound of claim 3, wherein D is a fluorescent label, a radioactive label or a chelating agent.

[0188] 11. The compound of claim 10, wherein D is a fluorescent label.

[0189] 12. The compound of claim 11, wherein the fluorescent label is fluorescein, Oregon Green, BODIPY dye, rhodamine or a cyanine label.

[0190] 13. The compound of claim 12, wherein the fluorescent label is a cyanine label.

[0191] 14. The compound of claim 13, wherein the cyanine label is Cy5.

[0192] As can be understood by those skilled in the art, the AA 1 、AA 2 、AA 3 and AA 4 groups of the compounds of the present invention can independently be any natural or unnatural amino acid side chain or the group "-L 1"-D". In a preferred embodiment, the group is an aralkyl amino acid side chain optionally substituted with 1 to 3 A groups. In an even more preferred embodiment, the group is the side chain from phenylalanine. In other preferred embodiments, the group is the side chain from an acidic amino acid residue, such as from an aspartic acid or glutamic acid residue, or from an alkyl amino acid residue (such as alanine, leucine, isoleucine, valine) or from any combination of other such amino acid residues. Side chains from other amino acid residues (such as lysine, arginine, tyrosine, glutamine, asparagine, etc.) are also preferably included in the compounds of the present invention.

[0193] In AA 1 、AA 2 、AA 3 or AA 4 group is an "L 1 -D" group compound embodiment, in L 1 The linker element can be provided by an amino acid side chain. For example, a lysine residue conveniently provides an aminoalkyl for reaction with a suitably activated detectable element.

[0194] A "short" detectable peptide group is defined herein as a detectable peptide group having at most 10 amino acid residues.

[0195] The ether-linked leaving element L of the compounds of the present invention affects the reactivity of the compounds with the active site of their target enzymes and can also affect the specificity for targeting specific enzymes. The ether bond of the leaving element in these compounds is in contrast to the ester bond in other activity-based probes such as acyloxymethyl ketone (AOMK). Compared to ester linkages or other types of probes, an ether-linked leaving element, for example, a phenol ether-linked leaving element, can have improved in vivo stability.

[0196] In some embodiments, the ether-linked leaving element of the compounds of the invention includes a quencher. The term "quencher" refers to a chemical entity that modulates the luminescence of a fluorophore. In some cases, the quencher itself can be a fluorescent molecule that emits fluorescence at a characteristic wavelength that is different from the label for which fluorescence quenching occurs. Thus, when appropriately conjugated with other dyes, a fluorophore can act as a quencher and vice versa. In these cases, an increase in fluorescence from the acceptor molecule (the acceptor having a different wavelength from the donor label) can respectively report the interaction of the labeled compound with its environment (e.g., the active site of a target enzyme). In some cases, the quencher itself does not fluoresce (i.e., the quencher is a "dark acceptor"). Such quenchers include, for example, dabcyl, methyl red, QSY diaryl rhodamine dyes, etc. In particular, dabcyl (4-dimethylamino-benzenediazo) benzoic acid) is a common dark quencher that has been widely used in many assays such as "molecular beacons" for DNA detection (U.S. Patent No. 5,989,823). The BHQ series of diazo dyes, known as "black hole quenchers", have a broad range of absorption that overlaps well with the luminescence of many fluorophores (PCT International Publication No. WO01 / 86001). QSY from Molecular Probes is another example of a dark quencher dye that has been widely used as a quencher in many bioassays (U.S. Patent No. 6,399,392).

[0197] In particular, QSY7 is a non-fluorescent diaryl rhodamine derivative (U.S. Patent Application Publication No. 2005 / 0014160). QSY21 is a non-fluorescent diaryl rhodamine chromophore that has strong absorption within the visible spectrum and is an effective fluorescence quencher. Fluorophore / quencher pairs are further described in U.S. Patent Application Publication No. 2004 / 0241679.

[0198] IRDye QC-1 (available from LI-COR) is another example of a non-fluorescent dye that is suitable for use as a quencher for the compounds of the invention. It effectively quenches fluorescence generated by a broad range of fluorophores (including wavelength ranges from the visible light expected to the near-infrared region).

[0199] In some embodiments of the compounds of the invention, the leaving group element L is L 2 -L 3 -Q, where L 2 is phenoxy, L 3 is a linker, and Q is a quencher. The leaving group element can be, for example:

[0200]

[0201] Where each Y is independently an electron-withdrawing group or hydrogen. In such compounds, each Y can independently be a halogen or hydrogen. In certain compounds, the L group is, for example:

[0202]

[0203] As will be understood by those of ordinary skill in the art, the L of the above leaving group 3 The linker group can be any suitable linker. In particular, the L 3 The linker group can, for example, be the L as described above 1 group.

[0204] In certain compounds, the L group is, for example:

[0205]

[0206] Where R is a QSY quencher and n is an integer from 1 to 8. In certain embodiments, the QSY quencher is a hydrophilic quencher, such as, for example, a sulfo-QSY quencher.

[0207] In some specific embodiments, the compounds of the present disclosure have the structure of formula (II):

[0208]

[0209] In some more specific embodiments, the compounds of the present disclosure have the structure of formula (III):

[0210]

[0211] In these embodiments, m and n are independently integers from 1 to 16.

[0212] In some embodiments, R is QSY21 or sulfo-QSY21, and D is Cy5.

[0213] Specific non-limiting compound embodiments of the present invention include:

[0214]

[0215] Where R = QSY21 and n = 6;

[0216] R = sulfo-QSY21 and n = 6;

[0217] R = QSY21 and n = 2; and

[0218] R = sulfo-QSY21 and n = 2.

[0219] In some embodiments, the compounds of the present invention are compounds having the structure of formula (IV):

[0220]

[0221] wherein T is a peptide target element, and the compound is further described in the claims numbered as follows:

[0222] 1. A compound of formula (IV), wherein D-T- is a short detectable peptidyl;

[0223] L 3 is a linker; and

[0224] Q is a quencher.

[0225] 2. The compound of claim 1, wherein the detectable peptidyl comprises 1 to 4 amino acid residues.

[0226] 3. The compound of claim 2, wherein D-T is:

[0227] wherein

[0228] each AA 1 、AA 2 、AA 3 and AA 4 is independently an amino acid side chain or -L 1 -D;

[0229] each R A is independently hydrogen or R 1 ;

[0230] R B is hydrogen, R 1 、-C(O)R 1 、-C(O)OR 1 、-C(O)SR 1 or -C(O)N(R 1 )(R A );

[0231] R 1 is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclic group, heterocyclic group alkyl, a protecting group or -L 1 -D, and is optionally substituted by 1 to 3 A groups;

[0232] Each A is independently an alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkanamino, aryl, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkanamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkanamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclic, heterocycloxy, heterocyclamino, heterocyclalkyl, heterocyclalkoxy, heterocyclalkanoyl, heterocyclalkanamino, hydroxy, thio, amino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidyl, ureido, halo, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfamino or azido; and

[0233] L 1 is a linker.

[0234] 4. The compound of claim 3, wherein

[0235] each R A is hydrogen; and

[0236] R B is -C(O)OR 1 .

[0237] 5. The compound of claim 3, wherein D-T- is

[0238]

[0239] each AA 1 and AA 2 are independently an amino acid side chain or -L 1 -D; and

[0240] each R A is hydrogen.

[0241] 6. The compound of claim 3, wherein D-T- is

[0242]

[0243] each AA 1 , AA 2 and AA 3 are independently an amino acid side chain or -L 1 -D; and

[0244] each R A is hydrogen.

[0245] 7. The compound of claim 3, wherein D-T- is

[0246]

[0247] each AA1 , AA 2 , AA 3 and AA 4 is independently an amino acid side chain or -L 1 -D; and

[0248] each R A is hydrogen.

[0249] 8. The compound of claim 3, wherein L 1 is an optionally substituted alkyl linker, wherein each carbon atom is optionally replaced by a heteroatom.

[0250] 9. The compound of claim 3, wherein R B is -C(O)OR 1 .

[0251] 10. The compound of claim 3, wherein D is a fluorescent label, a radioactive label or a chelating agent.

[0252] 11. The compound of claim 10, wherein D is a fluorescent label.

[0253] 12. The compound of claim 11, wherein the fluorescent label is fluorescein, Oregon Green, BODIPY dye, rhodamine or a cyanine label.

[0254] 13. The compound of claim 12, wherein the fluorescent label is a cyanine label.

[0255] 14. The compound of claim 13, wherein the cyanine label is Cy5.

[0256] As can be understood by those skilled in the art, the AA 1 , AA 2 , AA 3 and AA 4 groups can independently be any natural or non-natural amino acid side chain or the group "-L 1 -D". In a preferred embodiment, the group is an aralkyl amino acid side chain optionally substituted with 1 to 3 A groups. In an even more preferred embodiment, the group is the side chain from phenylalanine. In other preferred embodiments, the group is the side chain from an acidic amino acid residue, such as from an aspartic acid or glutamic acid residue, or the side chain from an alkyl amino acid residue, such as alanine, leucine, isoleucine, valine, or any combination of other such amino acid residues. Side chains from other amino acid residues (such as lysine, arginine, tyrosine, glutamine, asparagine, etc.) are also preferably included in the compounds of the present invention.

[0257] In AA 1 , AA 2 , AA 3 or AA4 The group is "L 1 -D" group, in the compound embodiment, in L 1 The linker element can be provided by an amino acid side chain. For example, a lysine residue conveniently provides an aminoalkyl for reacting with a suitably activated detectable element.

[0258] A "short" detectable peptide group is defined herein as a detectable peptide group having at most 10 amino acid residues.

[0259] Other specific non-limiting compound embodiments of the present invention include:

[0260]

[0261]

[0262] Pharmaceutical composition

[0263] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable carrier. Such compositions are (for example) used for imaging within tissues of an animal body and are also used to evaluate the activity of enzymes (such as proteases) in an animal body. In particular, for the compounds of the present invention that label cathepsin, the pharmaceutical composition can be used as a non-invasive optical imaging tool for cancer cells.

[0264] Pharmaceutically acceptable carriers are known in the art and (for example) include aqueous solutions such as water or physiological buffer saline or other solvents or excipients (such as ethylene glycol, glycerol, oils (such as olive oil) or injectable organic esters). In a specific embodiment, when such a pharmaceutical composition is administered to humans, the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients can be selected (for example) to achieve slow release of the medicament or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in unit dosage form (such as tablets, capsules, sprinkle capsules, granules, powders, syrups, suppositories, or injections, etc.). The composition can also be present in a transdermal delivery system (for example, a skin patch).

[0265] A pharmaceutically acceptable carrier may comprise a physiologically acceptable agent which is used, for example, to stabilize or increase the absorption of the compounds of the present invention. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins or other stabilizers or excipients. The selection of the pharmaceutically acceptable carrier, including the physiologically acceptable agent, depends, for example, on the route of administration of the composition. The pharmaceutical composition may also include liposomes or other polymeric matrices in which, for example, the compound pharmaceutical composition of the present invention has been incorporated. Liposomes composed of, for example, phospholipids or other lipids are non-toxic, physiologically acceptable and metabolizable carriers which can be manufactured and administered relatively simply.

[0266] As used herein, the phrase "pharmaceutically acceptable" is used to refer to those compounds, materials, compositions, and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0267] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or excipient, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. See Remington: The Science and Practice of Pharmacy, 20th ed. (Alfonso R. Gennaro, ed.), 2000.

[0268] The pharmaceutical compositions containing the compounds of the present invention can be administered to a subject by any one of a variety of administration routes, which include, for example, oral (e.g., as a drench in an aqueous or non-aqueous solution or suspension, tablets, pills, powders, granules, pastes applied to the tongue); sublingual; perianal, rectal or vaginal (e.g., as a vaginal suppository, cream or foam); parenteral (including intramuscular, intravenous, subcutaneous or intrathecal, e.g., in the form of a sterile solution or suspension); nasal; intraperitoneal; subcutaneous; transdermal (e.g., as a patch applied to the skin); or topical (e.g., applied to the skin as a cream, ointment or spray). The compounds can also be formulated for inhalation. In certain embodiments, the compounds of the present invention can simply be dissolved or suspended in sterile water. Details of suitable administration routes and the routes of administration suitable for administering the compositions can be found, for example, in U.S. Patent Nos. 6,110,973; 5,763,493; 5,731,000; 5,541,231; 5,427,798; 5,358,970; and 4,172,896, as well as the references cited in these patent documents.

[0269] Method for labeling and visualization

[0270] In another aspect, the present invention provides a method for visualizing a tumor in an animal, the method comprising the step of administering to the animal a composition of the present invention.

[0271] In another aspect, the present invention provides a method for visualizing a tumor in an animal, the method comprising administering to the animal a composition of the present invention and measuring a detectable signal in the animal resulting from the reaction between the composition and a cysteine cathepsin, wherein the detectable signal is related to the tumor in the animal.

[0272] In some embodiments of the method, the detectable signal is a fluorescence signal. In some embodiments, the fluorescence signal is generated at the tumor margin.

[0273] The administration of peptide imaging agents to animals is well known to those of ordinary skill in the art. In a preferred embodiment, the agent is administered by injection, but other suitable modes of administration are also considered to be within the scope of the present invention.

[0274] The methods of the present invention are directed to the labeling and visualization of proteases (especially cysteine proteases) in animals. Suitable animals include those that express cysteine proteases, especially those that express cysteine proteases in tumor cells. In a preferred embodiment, the animal is a mammal. In a highly preferred embodiment, the animal is a human. In other preferred embodiments, the animal is a domestic animal or a pet.

[0275] In some embodiments, the method of the present invention includes the step of measuring a detectable signal generated in an animal. Methods of measuring a detectable signal include, but are not limited to, imaging methods such as fluorescence imaging. In some embodiments, for example, the fluorescence imaging system (e.g.) is the Xenogen IVIS100 system, but any suitable system can be used.

[0276] It will be apparent to those skilled in the art that other suitable modifications and adaptations can be made to the methods and applications described herein without departing from the scope of the present invention or any of its embodiments. Although the present invention has been described in detail, it will be understood more clearly by reference to the following examples, which are included herein for illustrative purposes only and are not intended to limit the present invention.

[0277] Examples

[0278] Synthesis and characterization of a quenched fluorescence cysteine cathepsin imaging probe containing a novel phenoxymethyl ketone (PMK) electrophile Synthesis and characterization

[0279] The aim of this work was to develop a qABP with overall improved in vivo performance compared to existing qABPs for non-invasive optical imaging of cancer. Therefore, three main components of the probe were decided to be optimized: the quencher, the linker, and the electrophilic "warhead". One of the biggest drawbacks of cysteine cathepsin qABPs reported to date is poor water solubility. Therefore, a sulfonate group was introduced into the QSY21 quencher (Xing et al., (2005) J. Am. Chem. Soc. 127:4158-9) to improve water solubility and thereby improve the biodistribution of the probe. The length of the spacer connecting the electrophile and the quencher was also varied to reduce the lipophilicity of the qABP. Finally, new electrophiles were explored to increase the range of possible cathepsin targets. Since the expression of some members of the cysteine protease family is upregulated in a variety of cancers (Mohamed & Sloane (2006) Nat. Rev. Cancer (2006) 6:764-75), brighter fluorescence signals would be expected if broad-spectrum cysteine protease activity is detected. To obtain a more pan-reactive probe, the size of the electrophile was reduced and the reactivity was increased. It has previously been shown that the 2,3,5,6-tetrafluorosubstituted phenoxymethyl ketone (PMK) electrophile has higher reactivity towards cysteine dipeptidyl aminopeptidase compared to the 2,6-dimethylbenzoic acid-derived acyloxymethyl ketone (AOMK). Deu et al., (2010) Chem Biol. 17:808-819. The small size of PMK can also enhance pan-reactivity because the binding pockets of some cysteine cathepsins are sterically restricted. Blum et al., (2005) Nat. Chem. Biol. 1:203-9; Blum et al., (2007) Nat. Chem. Biol. 3:668-77; Paulick & Bogyo (2011) ACS Chem. Biol. 6:563-72. In addition, the phenol ether is expected to be more stable in vivo compared to the AOMK electrophile (which contains an ester bond that can be degraded by esterases).

[0280] As a starting point for this study, seven analogs (2-8) of qABP GB137 were synthesized ( Figure 1 a). Blum et al., (2007) Nat. Chem. Biol. 3:668-77. These compounds represent all combinations of two electrophiles, two quenchers, and two linker lengths. All probes were synthesized using a preferred protocol based on solution chemistry as described in the description related to Scheme 1 below. Initially, the specificity and potency of the probes were tested by labeling intact RAW 264.7 cells (a murine monocytic leukemia macrophage cell line) ( Figure 1b). Observe several trends in the probe properties. Compared to the probes (1, 3, 5, and 7) containing the more hydrophobic QSY21, all of the sulfo-QSY21-functionalized qABPs (2, 4, 6, and 8) exhibited stronger overall cathepsin labeling performance. Interestingly, the change in the spacer length, which occurred by changing the hexyl linker to an ethyl linker, did not significantly affect the labeling behavior. Perhaps the most striking finding was that the qABPs with the PMK electrophile exhibited a broader cysteine protease labeling profile compared to their AOMK counterparts. Probes 5 to 8 showed strong cathepsin X labeling, and the sulfo-QSY21-functionalized probes 6 and 8 were able to label higher molecular weight cathepsin L precursors. The properties of the fluorescently labeled cathepsins were determined using immunoprecipitation ( Figure 4 a). After performing titration labeling experiments in live RAW cells, observe other trends of interest ( Figure 1 c, d. Figure 4 b, c). The most hydrophobic qABPs (1 and 5) reached a maximum in the reduced labeling intensity at 0.5 μM, which suggests that their reduced water solubility caused the probe to precipitate at higher concentrations. The shorter spacer length seems to be beneficial, as the probes carrying the ethyl spacer had brighter labeling compared to the probes containing hexyl. When compared to the AOMK with PMK, significant differences in selectivity were observed. The AOMK qABPs preferentially labeled cathepsin S and L and only labeled cathepsin B at higher concentrations. Unexpectedly, the AOMK qABP 2 - 4 labeled cathepsin X, although previous studies had shown that some other related AOMKs could not label this target (Paulick & Bogyo (2011) ACS Chemical Biology 6:563 - 72). Even at lower probe concentration levels, the PMK qABPs labeled all of the target cysteine cathepsins with equal intensity. In summary, these experiments demonstrated that increased hydrophilicity improved the labeling intensity and that this novel PMK qABP had a broader and more comprehensive cysteine cathepsin labeling profile.

[0281] Since PMK qABP 8 was the most optimized in terms of overall labeling intensity and spectral cathepsin reactivity, it was decided to further conduct in vivo studies with this probe. To further determine the selectivity of the target, RAW cell lysates were labeled with qABP 8 at gradually increasing concentrations at pH 5.5. These results showed that the probe was most effective for cathepsin B and X, as labeling was observed at concentrations as low as 5 nM. However, the labeling of all cathepsins (B, S, L, X) was saturated with a 500 nM probe. Figure 2a). When the probe was used for live RAW cell labeling in real-time at a set concentration of 500 nm, cathepsin X was observed to saturate rapidly, followed by slower labeling of cathepsins S, L, and B, where the cathepsin B labeling signal increased progressively, even at 120 minutes ( Figure 2 b). These data suggest that the probe may enter the cathepsin X pool most rapidly, which may be due to its localization within or on the cell surface. It also suggests that cathepsins B and X may be other locations where the probe enters the cell to different extents. To test the stability of the new PMK probe, the effect of serum exposure on labeling in RAW cells was examined ( Figure 1 c). While a 4-hour serum pre-exposure of the initial AOMK probe 1 resulted in nearly 70% loss of target labeling, more than 80% of the labeling was retained for PMK qABP 8. Cells pretreated with the cysteine cathepsin inhibitor JPM-OET also blocked more than 90% of the labeling. Given the stability and improved labeling properties of the PMK probe, fluorescence microscopy studies of live cells were then carried out. These results confirmed that the probe produced bright and specific labeling signals, and most of the probe-labeled cathepsins resided in lysosomes ( Figure 2 d).

[0282] Given the new PMK electrophile-positive live cell labeling properties, the best-performing PMK qABP 2, 6, and 8 were tested in an in situ mouse model of breast cancer. Tao et al., (2008) BMC Cancer 8:228. Additionally, these PMK probes were compared to the initial AOMK probe 1 ( Figure 3 and Figure 5 ). 4T1 cells were transplanted into the mammary fat pads numbered 2 and 7 of Balb / c mice, and tumor growth was monitored. When tumors formed, the mice were injected via the tail vein with an equimolar amount of qABP (20 nmol), and non-invasive imaging of Cy5 fluorescence was carried out over time ( Figure 3 a, b). These results once again confirmed that qABP 8 proved superior. For probe 8, strong tumor-specific fluorescence activation could be clearly observed within the tumor region with high overall contrast. The signal continued to increase over time until the end of the time course.

[0283] Ultimately, probe 8 achieved a tumor-specific fluorescence signal enhancement 20-fold higher than that of probe 1. Good tumor-specific contrast was also observed for probe 6, and to a lesser extent for probe 2, yet both exceeded probe 1 by a factor of 10 ( Figure 5a, b). After completion of the time course, the tumors were excised and the ex vivo tumor fluorescence was measured, followed by homogenization and analysis of the fluorescently labeled proteins by SDS-PAGE( Figure 3 c and Figure 5 c). Quantification of the ex vivo fluorescence and of the total cathepsin labeling showed similar trends as in the non-invasive optical imaging studies( Figure 3 d and 5d). To determine the cellular origin of the probe fluorescence, immunofluorescence staining of tumor tissue sections from probe-labeled mice with the macrophage marker CD68 was performed( Figure 3 e and Figure 5 e). The fluorescence of Cy5 was localized to CD68-positive cells; however, not all CD68-positive cells were probe 8-positive, indicating different activation states of tumor-associated macrophages. More detailed analysis using laser confocal scanning microscopy (CLSM) confirmed that all cells positive for probe 8 were CD68-positive, but the cathepsin and CD68 signals labeled by the probe did not co-localize to the same vesicles( Figure 3 f and Figure 5 f). In summary, these data confirm that increasing the hydrophilicity of the quencher, shortening the spacer and introducing a more reactive and less sterically hindered nucleophilic trap result in qABP with broad cathepsin reactivity and overall improved in vivo properties.

[0284] Although very different functions have been described for some family members of cysteine proteases (Conus & Simon (2010) Swiss Med. Wkly. 140: w13042), other effects are redundant, and alterations in the activity of one cathepsin can affect the activity of other cathepsins. For example, loss of cathepsin B is compensated by increased activity of cathepsin X (Sevenich et al., (2010) Proc. Natl Acad. Sci. USA 107: 2497 - 502) and upregulation of cathepsin B leads to downregulation of cathepsin L (Gopinathan et al., (2012) Gut 61: 877 - 84). Thus, broad-spectrum probes are highly valuable as they facilitate the readout of multiple cysteine cathepsins in one assay and enable comparison of the activities of individual cathepsins with each other. The effectiveness of such broadly reactive ABPs has been demonstrated by the broad serine hydrolase fluorophosphonate probe (Liu et al., (1999) Proc. Natl Acad. Sci. USA 96: 14694 - 9) and the broadly reactive protease probe MV151 (Verdoes et al., (2006) Chem. Biol. 13: 1217 - 26). In addition, since the qABP of PMK is highly reactive towards cathepsin X, these scaffolds can be used to prepare qABPs selective for cysteine cathepsins that are still poorly understood (Paulick & Bogyo (2011) ACS Chem. Biol. 6: 563 - 72).

[0285] In summary, a new class of quenched-fluorescence-based activity probes has been synthesized that bear PMK electrophiles with higher reactivity and broader selectivity compared to previously reported AOMK-based probes. By introducing a sulfonated quencher and shortening the spacer between the electrophile and the quencher, the hydrophilicity of the qABP was further increased, resulting in higher water solubility and improved in vivo properties, leading to enhanced contrast in non-invasive optical imaging of cancer.

[0286] Methods

[0287] General

[0288] All resins and reagents were purchased from commercial suppliers and used without further purification. All solvents used were HPLC grade. All water-sensitive reactions were carried out in anhydrous solvents under a positive pressure of argon. Reactions were analyzed by LC-MS using an API 150EX single quadrupole mass spectrometer (Applied Biosystems). Using a C18 column, using The detector 100 (Amersham Pharmacia Biotech) was used for reverse-phase HPLC. NMR spectra were recorded on a Varian 400 MHz (400 / 100), Varian 500 MHz (500 / 125), or Varian Inova 600 MHz (600 / 150 MHz) equipped with a pulsed field gradient accessory. Chemical shifts were shown in ppm (δ) relative to tetramethylsilane as an internal standard. Coupling constants were shown in Hz. Fluorescent gels were scanned using a Typhoon 9400 flatbed laser scanner (GE Healthcare). Gel-internal labeling intensities were quantified using Image J software. Statistical analysis was performed using Microsoft Excel, and s.e.m. was calculated by dividing s.d. by the square root of n. Fluorescent microscopy images were acquired in a Zeiss confocal LSM 710 and Zeiss Axiovert inverted microscope (Carl Zeiss) equipped with 10×, 40×, and 63× objectives. Slidebook software was used to control the microscope and camera and for data analysis (Intelligent Imaging Innovations).

[0289] qABP synthesis

[0290] The synthetic schemes for the synthesis of the following compounds are described in Scheme 1 shown below.

[0291] 2,6-Dimethyl-4-((6-(tritylamino)hexyl)carbamoyl)benzoic acid (11a). Mono-trityl 1,6-hexanediamine acetate (9a) (117.2 mg, 0.28 mmol) was dissolved in DCM and washed with saturated aqueous NaHCO 3 and dried over Na 2 SO 4 and concentrated in vacuo. The amine was dissolved in DMF and HOBt monohydrate (43 mg, 0.28 mmol, 1 equiv), and EDC (54 mg, 0.28 mmol, 1 equiv) and 2,6-dimethyl-p-benzoic acid (10) (54.4 mg, 0.28 mmol, 1 equiv) were added. The reaction mixture was stirred overnight and then concentrated in vacuo. The crude product was purified by flash column chromatography (DCM → DCM solution of 5% MeOH), then dissolved in DCM and washed with water, dried over MgSO 4 to give 70 mg of the product (0.13 mmol, isolated yield: 47%).

[0292] 2,6-Dimethyl-4-((2-(triphenylamino)ethyl)carbamoyl)benzoic acid (11b). Mono-triphenylethylenediamine acetate (9b) (97.9 mg, 0.27 mmol) was dissolved in DCM and washed with saturated aqueous NaHCO 3 and dried over Na 2 SO 4 and concentrated in vacuo. The amine was dissolved in DMF and HOBt monohydrate (43 mg, 0.28 mmol, 1.04 equiv), and EDC (61 mg, 0.32 mmol, 1.2 equiv) and 2,6-dimethyl-p-benzoic acid (10) (52 mg, 0.27 mmol, 1 equiv) were added. The reaction mixture was stirred overnight and then concentrated in vacuo. The crude product was purified by flash column chromatography (DCM → DCM solution of 5% MeOH), then dissolved in DCM and washed with water, dried over MgSO 4 to give 28 mg of product (0.06 mmol, isolated yield: 22%).

[0293] 2,3,5,6-Tetrafluoro-4-hydroxy-N-(6-(triphenylamino)hexyl)benzamide (13a). Mono-triphenyl-1,6-hexanediamine acetate (9a) (117.2 mg, 0.28 mmol) was dissolved in DCM and washed with saturated aqueous NaHCO 3 and dried over Na 2 SO 4 and concentrated in vacuo. The amine was dissolved in DMF and HOBt monohydrate (43 mg, 0.28 mmol, 1 equiv), and EDC (54 mg, 0.28 mmol, 1 equiv) and 2,3,5,6-tetrafluoro-4-hydroxybenzoic acid (12) (59 mg, 0.28 mmol, 1 equiv) were added. The reaction mixture was stirred overnight and then concentrated in vacuo. The crude product was purified by flash column chromatography (15% -> 30% ethyl acetate in hexane) to give 90 mg of product (0.16 mmol, isolated yield: 58%).

[0294] 2,3,5,6-Tetrafluoro-4-hydroxy-N-(2-(triphenylamino)ethyl)benzamide (13b). Mono-triphenylethylenediamine acetate (9b) (100 mg, 0.28 mmol) was dissolved in DCM and washed with saturated aqueous NaHCO 3 and dried over Na 2 SO 4Dry and concentrate in vacuo. The amine was dissolved in DMF and HOBt monohydrate (43 mg, 0.28 mmol, 1 equiv), and EDC (54 mg, 0.28 mmol, 1 equiv) and 2,3,5,6-tetrafluoro-4-hydroxybenzoic acid (12) (59 mg, 0.28 mmol, 1 equiv) were added. The reaction mixture was stirred overnight and then concentrated in vacuo. The crude product was purified by flash column chromatography (20% -> 35% ethyl acetate in hexanes) to afford 90 mg of the product (0.18 mmol, isolated yield: 65%). 1 H NMR (400 MHz, DMSO) δ = 8.77 (t, J = 6.0, 1H), 7.39 (d, J = 7.8, 6H), 7.27 (t, J = 7.7, 6H), 7.17 (t, J = 7.2, 3H), 3.40 - 3.35 (m, 2H), 2.86 - 2.77 (m, 1H), 2.14 - 2.04 (m, 2H).

[0295]

[0296] (Sulso-QSY21: Sulfo-QSY21)

[0297] Scheme 1. Reagents and conditions: i. EDC, HOBt, DMF. ii. a) KF, DMF. b) 1% TFA, DCM. iii. a) QSY21-NHS or Sulfo-QSY21-NHS, DiPEA, DMSO; b) TFA / DCM = 1 / 1; c) Cy5-NHS, DiPEA, DMSO. iv. a) KF, DMF, 80 °C. b) 1% TFA, DCM.

[0298] Intermediate 15. Potassium fluoride (3 mg, 52 μmol, 3 equiv) was suspended in DMF by sonication for 5 min, and then carboxylic acid 11a (10 mg, 19 μmol, 1.1 equiv) was added. The reaction mixture was stirred for 10 min, and then chloromethyl ketone 14 (9.7 mg, 17.3 μmol, 1 equiv) was added. After 2 h, the reaction mixture was concentrated in vacuo. The crude product was dissolved in a DCM solution of 1% TFA and stirred for 30 min, and then quenched by adding triisopropylsilane until the solution became colorless. After co-evaporation with toluene (3x), the title compound was purified by HPLC (preparative reverse-phase C 18 column, CH 3 CN / H 2 O 0.1% TFA, 15:85 to 55:45 over 20 min; 5 mL / min) and then lyophilized to afford white powder 15 (3.12 mg, 3.46 μmol, yield over two steps: 20%).

[0299] Intermediate 16. Potassium fluoride (3 mg, 52 μmol, 3 equiv) was suspended in DMF by sonication for 5 min, followed by the addition of carboxylic acid 11b (9.5 mg, 20 μmol, 1.1 equiv). The reaction mixture was stirred for 10 min, followed by the addition of chloromethyl ketone 14 (10 mg, 17.9 μmol, 1 equiv). After 1.5 h, the reaction mixture was concentrated in vacuo, the crude product was dissolved in a DCM solution of 1% TFA, and stirred for 30 min, followed by quenching by the addition of triisopropylsilane until the solution became colorless. After co-evaporation with toluene (3x), Intermediate 16 was purified by HPLC (preparative reversed-phase C 18 column, CH 3 CN / H 2 O 0.1% TFA, from 15:85 to 55:45 over 20 min; 5 mL / min), followed by lyophilization to give a white powder (3.99 mg, 4.57 μmol, 26% yield over two steps). 1 1H NMR (500 MHz, CD 3 OD) δ 7.80 (s, 1H), 7.42 (s, 1H), 7.35 - 7.18 (m, 10H), 5.06 (s, 2H), 4.85 - 4.78 (m, 2H), 4.42 (dd, J = 13.1, 6.2 Hz, 1H), 4.37 (dd, J = 10.1, 4.0 Hz, 1H), 3.64 (t, J = 5.7 Hz, 2H), 3.18 (t, J = 4.8 Hz, 2H), 3.12 (dd, J = 13.7, 7.0 Hz, 1H), 3.01 (t, J = 7.3 Hz, 2H), 2.94 (dd, J = 13.6, 8.9 Hz, 1H), 2.41 (s, 3H), 2.34 (s, 3H), 1.92 - 1.82 (m, 1H), 1.67 - 1.57 (m, 1H), 1.49 - 1.26 (m, 4H), 1.42 (s, 9H).

[0300] Intermediate 17. Potassium fluoride (6.3 mg, 108 μmol, 3 equiv) was suspended in DMF by sonication for 5 min, followed by the addition of phenol 13a (21.5 mg, 39 μmol, 1.1 equiv). The reaction mixture was stirred for 10 min, followed by the addition of chloromethyl ketone 14 (20 mg, 36 μmol, 1 equiv). The reaction mixture was stirred at 80 °C for 5 h, followed by concentration in vacuo. The crude product was dissolved in a DCM solution of 1% TFA, and stirred for 30 min, followed by quenching by the addition of triisopropylsilane until the solution became colorless. After co-evaporation with toluene (3x), it was purified by HPLC (preparative reversed-phase C 18 column, CH 3 CN / H 2Purified with 0.1% TFA in a ratio of 25:75 to 70:30 for 20 minutes at a flow rate of 5 mL / min, followed by lyophilization to obtain a white powder (16.6 mg, 17.5 μmol, with a two-step yield of 49%). 1 H NMR (500 MHz, CD 3 OD) δ 7.29 (m, 10H), 5.07 (s, 2H), 4.86 (m, 2H), 4.44 (m, 2H), 3.41 (t, J = 6.8, 2H), 3.10 (dd, J = 13.5, 7.0, 1H), 3.02 (t, J = 6.8, 2H), 2.97 - 2.91 (m, 3H), 1.93 - 1.81 (m, 1H), 1.73 - 1.62 (m, 4H), 1.62 - 1.53 (m, 1H), 1.51 - 1.46 (m, 4H), 1.43 (s, 9H), 1.45 - 1.25 (m, 4H).

[0301] Intermediate 18. Potassium fluoride (6.3 mg, 108 μmol, 3 equiv) was suspended in DMF by sonication for 5 minutes, and then phenol 13b (19.4 mg, 39 μmol, 1.1 equiv) was added. The reaction mixture was stirred for 10 minutes, and then chloromethyl ketone 14 (20 mg, 36 μmol, 1 equiv) was added. The reaction mixture was stirred at 80 °C for 3 hours and then concentrated in vacuo. The crude product was dissolved in a DCM solution of 1% TFA and stirred for 30 minutes, and then quenched by adding triisopropylsilane until the solution became colorless. After co-evaporation with toluene (3x), it was purified by HPLC (preparative reversed-phase C 18 column, CH 3 CN / H 2 O 0.1% TFA, in a ratio of 20:80 to 60:40 for 20 minutes at a flow rate of 5 mL / min, followed by lyophilization to obtain the title compound as a white powder ((15.4 mg, 17.3 μmol, with a two-step yield of 48%). 1 H NMR (400 MHz, CD 3 OD) δ = 7.36 - 7.12 (m, 10H), 5.05 (s, 2H), 4.86 - 4.81 (m, 2H), 4.42 - 4.37 (m, 2H), 3.64 (t, J = 6.5, 2H), 3.14 (t, J = 6.5, 2H), 3.08 (dd, J = 13.9, 7.2, 1H), 2.99 (t, J = 6.5, 2H), 2.91 (dd, J = 13.9, 8.4, 1H), 1.90 - 1.78 (m, 1H), 1.62 - 1.48 (m, 1H), 1.41 (s, 9H), 1.46 - 1.20 (m, 4H).

[0302] Probe 1 (GB137). Intermediate 15 (1.5 mg, 1.7 μmol) was dissolved in DMSO (50 μl), and QSY21-NHS (1.39 mg, 1.7 μmol, 1 equiv) and DiPEA (1.5 μl, 8.5 μmol, 5 equiv) were added. After 1 h, the QSY21 amide was purified by HPLC (preparative reverse-phase C 18 column, CH 3 CN / H 2 O 0.1% TFA, 40:60 to 80:20 over 20 min; 5 mL / min), and then lyophilized. To remove the Boc protecting group, the resulting dark blue powder was dissolved in TFA / DCM (1 / 1) and reacted for 30 min, followed by co-evaporation with toluene (3x) to give 2.42 mg of the corresponding TFA salt (1.6 μmol, 95% yield over two steps). The amine was dissolved in DMSO (50 μl), and Cy5-NHS (1.3 mg, 1.76 μmol, 1.1 equiv) and DiPEA (1.4 μl, 8 μmol, 5 equiv) were added. After 1 h, it was purified by HPLC (preparative reverse-phase C 18 column, CH 3 CN / H 2 O 0.1% TFA, 40:60 to 75:25 over 20 min; 5 mL / min), followed by lyophilization to give Probe 1 as a dark blue powder (2.0 mg, 0.99 μmol, 62%).

[0303] Probe 2 (BMV122). Intermediate 15 (1.5 mg, 1.7 μmol) was dissolved in DMSO (50 μl), and sulfo-QSY21-NHS (1.66 mg, 1.7 μmol, 1 equiv) and DiPEA (1.5 μl, 8.5 μmol, 5 equiv) were added. After 1 h, the sulfo-QSY21 amide was purified by HPLC (preparative reverse-phase C 18 column, CH 3 CN / H 2 O 0.1% TFA, 30:70 to 70:30 over 20 min; 5 mL / min), and then lyophilized. To remove the Boc protecting group, the resulting dark blue powder was dissolved in TFA / DCM (1 / 1), and reacted for 30 min, followed by co-evaporation with toluene (3x) to give 2.29 mg of the corresponding TFA salt (1.39 μmol, 81% yield over two steps). The amine was dissolved in DMSO (50 μl), and Cy5-NHS (1.1 mg, 1.5 μmol, 1.1 equiv) and DiPEA (1.2 μl, 7 μmol, 5 equiv) were added. After 1 h, it was purified by HPLC (preparative reverse-phase C 18Column, CH 3 CN / H 2 O 0.1% TFA, from 15:85 to 50:50 over 20 min; 5 mL / min), then lyophilized to obtain probe 2 as a dark blue powder (1.83 mg, 0.84 μmol, 61%).

[0304] Probe 3 (BMV145). Intermediate 16 (1.5 mg, 1.7 μmol) was dissolved in DMSO (50 μl), and QSY21-NHS (1.39 mg, 1.7 μmol, 1 equiv) and DiPEA (1.5 μl, 8.5 μmol, 5 equiv) were added. After 1 h, by HPLC (preparative reverse-phase C 18 Column, CH 3 CN / H 2 O 0.1% TFA, from 40:60 to 80:20 over 20 min; 5 mL / min) QSY21 amide, then lyophilized. To remove the Boc protecting group, the resulting dark blue powder was dissolved in TFA / DCM (1 / 1) and reacted for 30 min, then co-evaporated with toluene (3x) to obtain 0.86 mg of the corresponding TFA salt (0.6 μmol, isolated yield of 35% over two steps). The amine was dissolved in DMSO (50 μl), and Cy5-NHS (0.5 mg, 0.66 μmol, 1.1 equiv) and DiPEA (0.57 μl, 3.3 μmol, 5 equiv) were added. After 1 h, purified by HPLC (preparative reverse-phase C 18 Column, CH 3 CN / H 2 O 0.1% TFA, from 40:60 to 75:25 over 20 min; 5 mL / min), then lyophilized to obtain probe 3 as a dark blue powder (0.67 mg, 0.34 μmol, 57%).

[0305] Probe 4 (BMV146). Intermediate 16 (1.0 mg, 1.2 μmol) was dissolved in DMSO (50 μl), and sulfo-QSY21-NHS (1.25 mg, 1.2 μmol, 1 equiv) and DiPEA (1.05 μl, 6 μmol, 5 equiv) were added. After 1 h, by HPLC (preparative reverse-phase C 18 Column, CH 3 CN / H 20.1% TFA, from 20:80 to 80:20 for 20 min; 5 mL / min) sulfo-QSY21 amide, then lyophilized. To remove the Boc protecting group, the resulting dark blue powder was dissolved in TFA / DCM (1 / 1) and reacted for 30 min, then co-evaporated with toluene (3x) to give 1.06 mg of the corresponding TFA salt (0.66 μmol, 55% yield over two steps). The amine was dissolved in DMSO (50 μl), and Cy5-NHS (0.55 mg, 0.73 μmol, 1.1 equiv) and DiPEA (0.64 μl, 3.65 μmol, 5 equiv) were added. After 1 h, purification by HPLC (preparative reverse-phase C 18 column, CH 3 CN / H 2 O 0.1% TFA, from 15:85 to 50:50 for 20 min; 5 mL / min), followed by lyophilization to give the probe 4 as a dark blue powder (0.63 mg, 0.3 μmol, 45%).

[0306] Probe 5 (BMV118). Intermediate 17 (1.2 mg, 1.3 μmol) was dissolved in DMSO (50 μl), and QSY21-NHS (1.0 mg, 1.3 μmol, 1 equiv) and DiPEA (1.13 μl, 6.5 μmol, 5 equiv) were added. After 2 h, purification by HPLC (preparative reverse-phase C 18 column, CH 3 CN / H 2 O 0.1% TFA, from 40:60 to 80:20 for 20 min; 5 mL / min) to purify the QSY21 amide, then lyophilized. To remove the Boc protecting group, the resulting dark blue powder was dissolved in TFA / DCM (1 / 1) and reacted for 30 min, then co-evaporated with toluene (3x) to give 2.0 mg of the corresponding TFA salt (1.3 μmol, quantitative after two steps). The amine was dissolved in DMSO (50 μl), and Cy5-NHS (1.0 mg, 1.3 μmol, 1 equiv) and DiPEA (1.1 μl, 6.5 μmol, 5 equiv) were added. After 1 h, purification by HPLC (preparative reverse-phase C 18 column, CH 3 CN / H 2 O 0.1% TFA, from 40:60 to 85:15 for 20 min; 5 mL / min), followed by lyophilization to give the probe 5 as a dark blue powder (1.91 mg, 0.94 μmol, 72%).

[0307] Probe 6 (BMV119). Intermediate 17 (1.2 mg, 1.3 μmol) was dissolved in DMSO (50 μl), and sulfo - QSY21 - NHS (1.35 mg, 1.3 μmol, 1 equiv) and DiPEA (1.13 μl, 6.5 μmol, 5 equiv) were added. After 1 h, the sulfo - QSY21 amide was purified by HPLC (preparative reversed - phase C 18 column, CH 3 CN / H 2 O 0.1% TFA, from 30:70 to 90:10 over 20 min; 5 mL / min), and then lyophilized. To remove the Boc protecting group, the resulting dark blue powder was dissolved in TFA / DCM (1 / 1), and the reaction was carried out for 30 min, followed by co - evaporation with toluene (3x) to give 1.98 mg of the corresponding TFA salt (0.9 μmol, 70% yield over two steps). The amine was dissolved in DMSO (50 μl), and Cy5 - NHS (0.7 mg, 0.9 μmol, 1.1 equiv) and DiPEA (0.8 μl, 4.5 μmol, 5 equiv) were added. After 1 h, it was purified by HPLC (preparative reversed - phase C 18 column, CH 3 CN / H 2 O 0.1% TFA, from 15:85 to 50:50 over 20 min; 5 mL / min), followed by lyophilization to give probe 6 as a dark blue powder (1.63 mg, 0.74 μmol, 82%).

[0308] Probe 7 (BMV108). Intermediate 18 (1.2 mg, 1.3 μmol) was dissolved in DMSO (50 μl), and QSY21 - NHS (1.2 mg, 1.4 μmol, 1.1 equiv) and DiPEA (1.13 μl, 6.5 μmol, 5 equiv) were added. After 1 h, the QSY21 amide was purified by HPLC (preparative reversed - phase C 18 column, CH 3 CN / H 2 O 0.1% TFA, from 30:70 to 70:30 over 20 min; 5 mL / min) to give a dark blue powder (1.43 mg, 0.99 μmol, 76%). To remove the Boc protecting group, the resulting dark blue powder was dissolved in TFA / DCM (1 / 1), and the reaction was carried out for 30 min, followed by co - evaporation with toluene. The TFA salt was dissolved in DMSO (50 μl), and Cy5 - NHS (0.83 mg, 1.1 μmol, 1.1 equiv) and DiPEA (0.88 μl, 5 μmol, 5 equiv) were added. After 1 h, it was purified by HPLC (preparative reversed - phase C 18 column, CH3 CN / H 2 O 0.1% TFA, from 30:70 to 70:30 over 20 minutes; 5 mL / min), followed by lyophilization to obtain probe 7 as a dark blue powder (0.95 mg, 0.48 μmol, 49%).

[0309] Probe 8 (BMV109). Intermediate 18 (5.8 mg, 6.5 μmol) was dissolved in DMSO (100 μL), and sulfo-QSY21-NHS (9.75 mg, 10.39 μmol, 1.6 equiv) and DiPEA (8.4 μL, 50.5 μmol, 7.8 equiv) were added. The mixture was stirred overnight. Purification of the sulfo-QSY21 amide was carried out by HPLC (preparative reversed-phase C 18 column, CH 3 CN / H 2 O 0.1% TFA, from 25:75 to 55:45 over 20 minutes; 5 mL / min), then lyophilized to obtain a dark blue powder. Subsequently, the Boc protecting group was removed by reaction in TFA / DCM (1 / 1) for 30 minutes, followed by co-evaporation with toluene (3x). The residue was dissolved in DMSO (250 μL), and Cy5-NHS (10.5 mg, 13.9 μmol, 2.1 equiv) and DiPEA (12 μL, 72 μmol, 11 equiv) were added. After 4 hours, purification was carried out by HPLC (preparative reversed-phase C 18 column, CH 3 CN / H 2 O 0.1% TFA, from 25:75 to 45:55 over 20 minutes; 5 mL / min), followed by lyophilization to obtain probe 8 as a dark blue powder (7.74 mg, 4.61 μmol, yield over three steps: 49%). 1 H NMR (600 MHz, CD 3CN) δ8.12 - 8.08 (m, 1H), 8.01 - 7.93 (m, 2H), 7.89 - 7.85 (m, 2H), 7.75 (dd, J = 12.0, 1.5 Hz, 2H), 7.72 (dd, J = 8.4, 1.7 Hz, 1H), 7.69 (dd, J = 8.3, 1.2 Hz, 1H), 7.66 (s, 2H), 7.62 - 7.57 (m, 2H), 7.51 (dd, J = 8.4, 5.1 Hz, 2H), 7.46 (d, J = 9.4 Hz, 2H), 7.41 - 7.35 (m, 3H), 7.24 (s, 1H), 7.22 (s, 1H), 7.21 - 7.14 (m, 6H), 7.13 - 7.09 (m, 6H), 7.05 (dd, J = 8.8, 4.6 Hz, 1H), 6.39 (t, J = 12.8 Hz, 1H), 6.11 (t, J = 12.6 Hz, 1H), 4.87 (q, J = 12.7 Hz, 2H), 4.83 (dd, J = 39.7, 14.1 Hz, 2H), 4.23 - 4.12 (m, 4H), 3.93 (q, J = 7.2 Hz, 2H), 3.86 (t, J = 7.4 Hz, 2H), 3.34 (dd, J = 6.7, 4.1 Hz, 2H), 3.28 - 3.15 (m, 9H), 3.04 - 2.92 (m, 3H), 2.80 - 2.74 (m, 1H), 2.45 (t, J = 11.9 Hz, 2H), 2.15 - 2.09 (m, 1H), 2.09 - 2.03 (m, 2H), 1.74 - 1.58 (m, 7H), 1.57 (s, 6H), 1.55 (s, 6H), 1.49 (dd, J = 15.1, 7.4 Hz, 4H), 1.35 - 1.22 (m, 7H), 1.20 (t, J = 7.3 Hz, 3H), 1.16 - 1.12 (m, 4H).

[0310] Cell culture and labeling of live cells and cell lysates

[0311] RAW cells were cultured in DMEM (GIBCO) medium supplemented with 10% fetal bovine serum (FBS; GIBCO), 100 units / mL penicillin, and 100 μg / mL streptomycin (GIBCO). 4T1 cells (ATCC) were cultured in RPMI (GIBCO) medium supplemented with 10% fetal bovine serum (GIBCO FBS), 100 units / mL penicillin, and 100 μg / mL streptomycin (GIBCO). All cells were incubated at 37 °C in 5% CO 2Cultured in a humidified incubator. Unless otherwise specified, for complete cell labeling, cells were exposed to the probe (in DMSO, 500x) in the medium and incubated at 37 °C for 2 hours. Where specified, cells were pre-incubated with inhibitor JPM-OET (in DMSO, 500x) for 1 hour or exposed to mouse serum (1 μl of the DMSO stock solution of the probe added to 9 μl of serum) 4 hours before adding the cells. After labeling, the cells were washed with PBS and resuspended in hypotonic lysis buffer (50 mM PIPES (pH 7.4), 10 mM potassium chloride, 5 mM magnesium chloride, 2 mM EDTA, 4 mM DTT, and 1% NP-40), placed on ice for 15 minutes, centrifuged at 4 °C for 30 minutes, after which the supernatant was collected and the protein concentration was determined using a BCA kit (Pierce). 40 μg of total protein was added to 4× SDS sample buffer and denatured by heating at 100 °C for 3 minutes, resolved by SDS-PAGE (15%), and the gel was scanned with a Typhoon imager (GE Healthcare) to visualize the labeled protease. The labeling intensity was quantified using Image J software. For cathepsin labeling in cell lysates, cells were collected, washed with PBS and resuspended in citrate buffer (50 mM citrate buffer (pH 5.5), 5 mM DDT, 0.5% CHAPS, 0.1% Triton X). After placing on ice for 15 minutes and centrifuging at 4 °C for 30 minutes, the supernatant was collected and the protein concentration was determined using a BCA kit (Pierce). At 37 °C, 40 μg of total protein was exposed to the specified probe (200x in DMSO) for 1 hour. 4× SDS sample buffer was added and the protein was denatured at 100 °C for 3 minutes and analyzed as described above. For live cell microscopy, RAW cells were seeded at a density of 1·10 5 cells in phenol red-free complete medium (in vitro system) in 35 mm glass-bottom dishes and cultured overnight. Cells were either exposed to DMSO or 1 μM of the probe (500x in DMSO) for 2 hours. For the last 1 hour, a green lysosome tracer (final concentration 200 nM, 1000x in DMSO) was added to the cells. Where specified, cells were pre-incubated with inhibitor JPM-OET (500x in DMSO) for 1 hour. Cells were imaged at 40x magnification in both the Cy5 and FITC channels using a Zeiss Axiovert 200 μM confocal microscope.

[0312] Animal model

[0313] All animal care and experiments were conducted in accordance with the current guidelines of the National Institutes of Health and the Stanford University Institutional Animal Care and Use Committee. Female BALB / c mice (6 - 8 weeks, Jackson Laboratories) were injected with 1·10 5 4T1 cells (ATCC) in PBS into fat pads numbered 2 and 7 under isoflurane anesthesia and tumor growth was monitored. Twenty-four hours before imaging, hair was removed from the area of interest using "Nair Lotion". On day 10, a specified probe (20 nmol; 0.8 nmol g -1 ) in a volume of 100 μL (PBS solution with 20% DMSO) was administered via the tail vein. After injection, the mice were non-invasively imaged at specified time points using an IVIS100 system (Xenogen). Images were analyzed using Living Image software (PerkinElmer). After the final time point, the mice were anesthetized with isoflurane and sacrificed by cervical dislocation. For ex vivo fluorescence measurements and assessment of in vivo probe labeling patterns, tumors were removed, imaged using an FMT 2500 (PerkinElmer), and the tissues were sonicated (1 minute on ice) in citrate buffer (50 mM citrate buffer (pH 5.5), 5 mM DDT, 0.5% CHAPS, 0.1% Triton X). After centrifugation at 4°C for 30 minutes, the supernatant was collected and the protein concentration was determined using a BCA kit (Pierce). At 100°C, 40 μg of total protein was denatured in SDS sample buffer for 3 minutes and analyzed as described above. For immunofluorescence, excised tumors were incubated in PBS solution with 4% PFA at 4°C for 6 hours, then incubated overnight in 30% sucrose solution and the tissues were frozen in OCT medium. 6-μm sections were fixed in acetone, blocked with PNB blocking buffer, and co-incubated overnight with rat anti-mouse CD68 (1:1000; Serotec). At room temperature, goat anti-rat conjugated with AlexaFluor-488 (1:500; Invitrogen) was incubated for 1 hour. Then the sections were stained with DAPI (2 μg / mL; Invitrogen) for five minutes and subsequently mounted in ProLong Gold mounting medium (Invitrogen). Subsequently, the tissues were visualized using a Zeiss Axiovert 200M microscope.

[0314] The full texts of all patents, patent publications, and other published literature mentioned herein are incorporated herein by reference as if each reference had been independently and specifically introduced herein.

[0315] Although specific embodiments have been provided, the above description is illustrative rather than restrictive. Any one or more features of the foregoing embodiments may be combined with one or more features of any other embodiment of the present invention in any manner. In addition, many variations of the present invention will become apparent to those skilled in the art after reading this specification. Therefore, the scope of the present invention should be determined by reference to the appended claims and the full scope of their equivalents.

Claims

1. A compound for labeling a protease, having the following formula: wherein D is a fluorescent label, and the fluorescent label is fluorescein, Oregon Green, BODIPY dye, rhodamine or cyanine label; L 3 is a joint; and Q is a quencher, and the quencher is a chemical entity that modulates the emission of a fluorophore.

2. The compound according to claim 1, wherein the fluorescent label is a cyanine label.

3. The compound according to claim 2, wherein the cyanine label is Cy5.

4. The compound according to claim 1, having the following formula: wherein R is a QSY quencher; D is a cyanine dye; and n is an integer from 1 to 16.

5. The compound according to claim 4, wherein R is QSY21 or sulfo-QSY21, and D is Cy5.

6. The compound according to claim 5, which is one of the following compounds: wherein R = QSY21 and n = 6; R = sulfo-QSY21 and n = 6; R = QSY21 and n = 2; and R = sulfo-QSY21 and n = 2.

7. The compound according to claim 1, wherein Q is QC-1.

8. A composition for labeling a protease in an animal, comprising the compound according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

9. The composition according to claim 8, for visualizing a tumor in an animal, the use comprising the following steps: administering the composition to the animal; and detecting a detectable signal generated in the animal due to the reaction between the composition and cathepsin cysteine protease, wherein the detectable signal is related to a tumor in the animal.

10. For the composition according to claim 9, wherein the detectable signal is a fluorescent signal.

11. For the composition according to claim 10, wherein the fluorescent signal is generated at the tumor margin.

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