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

Abstract

To provide activity-based probe compounds, compositions, and methods of use.SOLUTION: Activity-based probe compounds for use in labeling a cysteine protease are provided. The compounds are targeted to the protease through a specific targeting element. The compounds additionally include a detectable element, such as a fluorescent label, a radiolabel, or a chelator. In some cases, the compounds additionally include a quenching element that is released upon reaction with the protease. Also provided are compositions comprising the compounds and methods for using the compounds, for example in labeling a protease in an animal and in visualizing a tumor in an animal.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 61 / 794,296, filed March 15, 2013, the disclosure of which is incorporated herein by reference in its entirety. Statement of government support This invention was made with government support under Project No. 5R01EB005011 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0002] Background of the Invention Various technologies are currently being developed for use in the areas of molecular imaging and disease monitoring. Optical fluorescence imaging, in particular, is an approach that is beginning to show promise as a clinical tool, given its sensitivity, specificity, and non-invasiveness. The specificity of fluorescent optical probes can sometimes be provided by their biological targets. For example, optical probes recognized by enzyme targets in biological samples often produce highly specific signals only when the probe's fluorescence becomes uninhibited upon enzymatic reaction. Ideally, the fluorescent moiety of the probe remains bound to its enzyme target even after the fluorescent signal is activated by the enzymatic reaction. Such fluorescent activity-based probes (ABPs) have been described for protease targets. Blum et al. (2009) PLoS One 4:e6374; doi:10.1371 / journal.pone.0006374. ABPs are distinguishable from simple fluorescent substrates by the permanent covalent bond resulting from the reaction of the ABP with the active site catalytic residue of the enzyme. Although fluorescent substrates may be seen as advantageous due to signal amplification resulting from catalytic turnover by their target enzymes, APBs have been shown to exhibit increased rates of tissue uptake and prolonged retention of the probe in target tissues due to their covalent modification of the target enzyme.

[0003] Target enzymes of interest for use with fluorescence-based optical probes are proteases, particularly cysteine ​​proteases. Cysteine ​​cathepsins are a family of proteases that play important roles in health and disease. Reiser et al. (2010) J. Clin. Invest. 120:3421-31. Although their function has been primarily described as contributing to the endosomal pathway, accumulating evidence indicates that they are key regulators of matrix degradation, suggesting that they also have functions in extracellular contexts. Broemme & Wilson (2011) Role of Cysteine ​​Cathepsins in Extracellular Proteolysis. Biology of Extracellular Matrix Volume 2 23-51. In addition, members of the cysteine ​​cathepsin family have been shown to be key players in the development and progression of various types of cancer. Mohamed & Sloane (2006) Nat. Rev. Cancer (2006) 6:764-75; Palermo & Joyce (2008) Trends Pharmacol. Sci. 29:22-8. Furthermore, changes in the expression of endogenous inhibitors of cathepsins, cysteines, have been observed in cancer. Cox (2009) Cystatins and cancer. Front. Biosci. 14:463-74. These observations highlight the importance of tools that allow direct assessment of the activity of these proteases in the context of the wild-type tumor microenvironment, combined with potential changes in the intracellular and extracellular milieu. Various ABPs targeting the cysteine ​​cathepsin family have been synthesized. Edgington et al. (2011) Curr. Opin. Chem. Biol. 15:798-805. In particular, fluorescently quenched ABPs (qABPs) ) has proven to be a powerful tool for noninvasive optical imaging of cancer and subsequent characterization of target cathepsins at the histological, cellular, and protein levels. Blum et al. (2007) Nat. Chem. Biol. 3:668-77, Verdoes et al. (2012) Chem. Biol. 19:619-28.

[0004] Activity-based inhibitors of dipeptidyl peptidase I based on 2,3,5,6-tetrafluorophenoxyarylmethylketone reactive groups have been reported (Deu et al., (2010) Chem Biol. 17:808-819), but these inhibitors were non-peptidic and did not contain detectable groups.

[0005] Quenched activity-based peptidic inhibitors for use in fluorescent imaging of cells, including active proteases such as cathepsins, have also been reported. See, for example, U.S. Patent Application Publication No. 2007 / 0036725. These probes use an ester-linked acyloxymethylketone reactive group to bind to the protease active site. In some cases, activity-based fluorescent probes are non-peptidic. See, for example, WO 2012 / 118715. In some cases, activity-based probes are used to radiolabel their target enzymes. See, for example, WO 2009 / 124265. However, there remains a need in the art for novel activity-based fluorescent probes of cysteine ​​proteases that have higher cellular uptake, target a broader spectrum of cysteine ​​protease activities, and offer increased sensitivity of detection. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US Patent Application Publication No. 2007 / 0036725 [Patent Document 2] International Publication No. 2012 / 118715 [Patent Document 3] International Publication No. 2009 / 124265 [Non-patent literature]

[0007] [Non-Patent Document 1] Blum et al. (2009) PLoS One 4:e6374;doi:10.1371 / journal.pone.0006374 [Non-patent document 2] Reiser et al. (2010) J.Clin.Invest.120:3421-31 [Non-patent document 3] Broemme&Wilson(2011) Role of Cysteine ​​Cathepsins in Extracellular Proteolysis.Biology of Extracellular Matrix Volume 2 23-51 [Non-patent document 4] Mohamed&Sloane (2006) Nat.Rev.Cancer(2006) 6:764-75 [Non-patent document 5] Palermo&Joyce (2008)Trends Pharmacol.Sci.29:22-8 [Non-patent document 6] Cox(2009)Cystatins and cancer. Front.Biosci.14:463-74 [Non-Patent Document 7] Edgington et al. (2011)Curr.Opin.Chem.Biol.15:798-805 [Non-patent document 8] Nat.Chem.Biol.3:668-77,Verdoes et al. (2012)Chem.Biol.19:619-28 [Non-Patent Document 9] Deu et al. (2010) Chem Biol.17:808-819 Summary of the Invention [Means for solving the problem]

[0008] The present invention addresses these and other problems by providing compounds, compositions, and methods of using the compounds and compositions to label cysteine ​​proteases.

[0009] In particular, in accordance with one aspect of the present invention, a compound of structural formula (I) [ka] (In the formula, L is an ether bond leaving element; T is a targeting element, D is a detectable element) The compound is represented by:

[0010] In some embodiments of the invention, the D group is a fluorescent label, a radiolabel, or a chelator.

[0011] In certain embodiments, the D group is a fluorescent label, especially fluorescein, Oregon Green, bora-diaza-indecene, rhodamine, or a cyanine label, and in certain embodiments, the fluorescent label is a cyanine label such as Cy5.

[0012] In some embodiments of the present invention, the T group targets the compound to a cysteine ​​protease. In certain embodiments, the T group is a non-peptide targeting element, such as an element containing a triazole structure, including various specific compounds containing a triazole structure. In certain other embodiments, the T group is a peptidic targeting element.

[0013] In some compound embodiments, the DT-group is [ka] and wherein L1 is a linker; AA1 is an amino acid side chain, U is O, N or S; R1 is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl or a protecting group, optionally substituted with 1 to 3 A groups; Each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkoxy, or alkanoyl. alkylamino, aryl, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylalkyl, heterocyclylalkoxy, heterocyclylalkanoyl, heterocyclylalkamino, hydroxyl, thio, amino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidinyl, urea, halo, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfonamido, or azido.

[0014] In certain compound embodiments, L1 is an optionally substituted alkyl linker, wherein each carbon atom is optionally replaced by a heteroatom.

[0015] In other particular compound embodiments, AA1 is an aralkyl amino acid side chain, optionally substituted with 1 to 3 A groups.

[0016] In yet other particular compound embodiments, U is O.

[0017] In certain embodiments, the D group is a fluorescent label, a radiolabel, or a chelator.

[0018] In particular embodiments, the D group is a fluorescent label, more particularly fluorescein, Oregon Green, bora-diaza-indecene, rhodamine, or a cyanine label. In more particular embodiments, the fluorescent label is a cyanine label such as Cy5.

[0019] In some compound embodiments, the L group comprises a quencher, and in more particular embodiments, L is L2-L3-Q, where L2 is a phenoxy group, L3 is a linker, and Q is a quencher.

[0020] In certain embodiments, L is [ka] where each Y is independently an electron-withdrawing group or hydrogen.

[0021] In more particular embodiments, each Y is independently halogen or hydrogen, and in more particular embodiments, L is [ka] and L3 is an optionally substituted alkyl linker, wherein each carbon atom is optionally replaced with a heteroatom.

[0022] In more particular embodiments, L is [ka] wherein R is a QSY quencher and n is an integer from 1 to 16.

[0023] In a preferred embodiment, the QSY-quencher is a hydrophobic QSY-quencher, and more particularly, the hydrophilic QSY-quencher is a sulfo-QSY-quencher.

[0024] In some embodiments of the present invention, a compound represented by structural formula (II) [ka] wherein D is a fluorescent label, L3 is a linker, and Q is a quencher.

[0025] In a more particular embodiment, structural formula (III) [ka] where R is a QSY quencher, D is a cyanine dye, and m and n are independently integers from 1 to 16. The R group can be QSY21 or sulfo-QSY21, and the D group can be Cy5, in some of these embodiments.

[0026] Specific compound embodiments of the present invention include the following: [ka] Contains, where R=QSY21 and n=6, R = sulfo-QSY21 and n = 6, R=QSY21 and n=2 and R=sulfo-QSY21 and n=2.

[0027] According to another aspect, the present invention provides a composition for use in labeling a protease in an animal, comprising a compound of the present disclosure and a pharmaceutically acceptable carrier.

[0028] According to yet another aspect, the present invention provides a method for labeling a protease in an animal comprising administering to the animal a composition of the present disclosure.

[0029] The present invention further provides a method for visualizing a tumor in an animal, comprising administering a composition of the present disclosure to the animal and measuring a detectable signal generated in the animal from the reaction of the composition with a cathepsin cysteine ​​protease, wherein the detectable signal is associated with a tumor in the animal.

[0030] In certain method embodiments, the detectable signal is a fluorescent signal. In other certain method embodiments, the fluorescent signal is produced at the tumor margin. The present invention provides, for example, the following items. (Item 1) Formula (I) [ka] (In the formula, L is an ether bond leaving element, T is a targeting element, D is a detectable element) 1. A compound for use in labeling a protease having the formula: (Item 2) 2. The compound according to item 1, wherein D is a fluorescent label, a radiolabel or a chelator. (Item 3) 3. The compound according to item 2, wherein D is a fluorescent label. (Item 4) 4. The compound according to item 3, wherein the fluorescent label is a fluorescein, Oregon green, bora-diaza-indecene, rhodamine or cyanine label. (Item 5) 5. The compound according to item 4, wherein the fluorescent label is a cyanine label. (Item 6) 6. The compound according to item 5, wherein the cyanine label is Cy5. (Item 7) The compound according to item 1, wherein T targets the compound to a cysteine ​​protease. 2. The compound according to item 1, wherein T is a non-peptide targeting element. (Item 9) 9. The compound according to item 8, wherein the non-peptide targeting element comprises a triazole structure. (Item 10) The following compounds: [ka] Item 10. The compound according to item 9, selected from one of: (Item 11) 2. The compound according to item 1, wherein T is a peptidic targeting element. (Item 12) DT-, [ka] wherein L1 is a linker, AA1 is an amino acid side chain, U is O, N or S; R1 is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl or a protecting group, optionally substituted with 1 to 3 A groups; each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylalkyl, heterocyclylalkoxy, heterocyclylalkanoyl, heterocyclylalkamino, hydroxyl, thio, amino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidinyl, urea, halo, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfonamido, or azide. (Item 13) Item 13. The compound according to item 12, wherein L1 is an optionally substituted alkyl linker, wherein each carbon atom is optionally replaced with a heteroatom. (Item 14) 13. The compound according to item 12, wherein AA1 is an aralkyl amino acid side chain, optionally substituted with 1 to 3 A groups. (Item 15) Item 13. The compound according to item 12, wherein U is O. (Item 16) 13. The compound according to item 12, wherein D is a fluorescent label, a radiolabel or a chelator. (Item 17) 17. The compound according to item 16, wherein D is a fluorescent label. (Item 18) 18. The compound according to item 17, wherein the fluorescent label is a fluorescein, Oregon green, bora-diaza-indecene, rhodamine or cyanine label. (Item 19) 19. The compound according to item 18, wherein the fluorescent label is a cyanine label. (Item 20) 20. The compound according to item 19, wherein the cyanine label is Cy5. (Item 21) 21. The compound according to any one of items 1 to 20, wherein L comprises a quencher. (Item 22) L is L2-L3-Q, L2 is a phenoxy group, L3 is a linker, 22. The compound according to item 21, wherein Q is a quencher. (Item 23) L [ka] 23. The compound according to item 22, wherein each Y is independently an electron-withdrawing group or hydrogen. (Item 24) 24. The compound according to item 23, wherein each Y is independently halogen or hydrogen. (Item 25) L [ka] and L3 is an optionally substituted alkyl linker, wherein each carbon atom is optionally replaced with a heteroatom. (Item 26) L, [ka] and R is a QSY quencher, 26. The compound according to item 25, wherein n is an integer of 1 to 16. (Item 27) 27. The compound according to item 26, wherein the QSY quencher is a hydrophilic QSY quencher. (Item 28) 28. The compound according to item 27, wherein the hydrophilic QSY quencher is a sulfo-QSY quencher. (Item 29) Formula (II) [ka] wherein D is a fluorescent label; L3 is a linker, Q is a quencher. (Item 30) Formula (III) [ka] wherein R is a QSY quencher; D is a cyanine dye, m and n are independently integers from 1 to 16. 29. The compound according to item 29, having the formula: (Item 31) 31. The compound according to item 30, wherein R is QSY21 or sulfo-QSY21 and D is Cy5. (Item 32) The following compounds [ka] where R=QSY21 and n=6, R = sulfo-QSY21 and n = 6, R=QSY21 and n=2 and 32. The compound according to item 31, wherein R=sulfo-QSY21 and n=2). (Item 33) A compound according to any one of items 1 to 32 and a pharmaceutically acceptable carrier. , a composition for use in labeling proteases in animals. (Item 34) 34. A method for labeling a protease in an animal, comprising administering to the animal the composition of claim 33. (Item 35) Administering the composition of item 33 to the animal; measuring a detectable signal generated in said animal from the reaction of the composition with a cathepsin cysteine ​​protease; wherein said detectable signal is associated with a tumor in said animal. (Item 36) 36. The method of claim 35, wherein the detectable signal is a fluorescent signal. (Item 37) 37. The method of claim 36, wherein the fluorescent signal occurs at a tumor margin. [Brief explanation of the drawings]

[0031] [Figure 1] Figure 1 shows a) the structures of qABPs GB137(1) and probes 2-8 synthesized in this study. b) Labeling profiles of probes 1-8 in live RAW cells at 1 μM. c) Concentration-dependent labeling by probes 1 and 8 in live RAW cells. d) Total cathepsin labeling intensity of probes 1-8 in live RAW cells relative to 5 μM GB137(1).

[0032] [Figure 2]Figure 2: a) Concentration-dependent labeling of RAW cell lysates with probe 8 at pH 5.5. b) Labeling time course with 0.5 μM probe 8 in live RAW cells. c) Inhibition of labeling and serum stability of probes 1 and 8 in live RAW cells pretreated with JPM-OEt (50 μM). d) Live-cell fluorescence microscopy of RAW cells exposed to 1 μM probe 8 (top row of panels) and colocalization with Lysotracker (second row of panels, scale bar 10 μm).

[0033] [Figure 3-1] Figure 3. a) Noninvasive optical imaging time course of tumor-bearing mice injected with probes 8 and 1 (right panel). The bottom panel shows the optimal fluorescence contrast at each time point. b) Time-dependent tumor-specific fluorescence (tumor minus background) for mice treated with probes 1 or 8 (n = 3, data represent mean ± standard deviation). c) Ex vivo tumor fluorescence (upper panel) and in vivo fluorescently labeled proteins after SDS-PAGE visualized by in-gel fluorescent scanning (lower panel). d) Fluorescence intensity at the end points of noninvasive optical imaging (shown in a), ex vivo tumor imaging, and in-gel fluorescent labeling (shown in c). Intensity is depicted for probe 1 (n = 3, data represent mean ± standard deviation). e) Fluorescence microscopy of probe 8 (left panel)-treated tumor tissue sections with CD68 immunostaining (center panel) and nuclear staining (DAPI—right panel, scale bar 50 μm). f) 3D reconstruction of CLSM of probe 8 (red) treated tumor tissue section with CD68 immunostaining (green) and nuclear staining (DAPI-blue). [Figure 3-2]Figure 3. a) Noninvasive optical imaging time course (right panel) of tumor-bearing mice injected with probes 8 and 1. The bottom panel shows the optimal fluorescence contrast at each time point. b) Time-dependent tumor-specific fluorescence (tumor minus background) for mice treated with probes 1 or 8 (n = 3, data represent mean ± standard deviation). c) Ex vivo tumor fluorescence (upper panel) and in vivo fluorescently labeled proteins after SDS-PAGE visualized by in-gel fluorescent scanning (lower panel). d) Fluorescence intensity at the end points of noninvasive optical imaging (shown in a), ex vivo tumor imaging, and in-gel fluorescent labeling (shown in c). Intensity is depicted for probe 1 (n = 3, data represent mean ± standard deviation). e) Fluorescence microscopy of probe 8 (left panel)-treated tumor tissue sections with CD68 immunostaining (center panel) and nuclear staining (DAPI—right panel, scale bar 50 μm). f) 3D reconstruction of CLSM of probe 8 (red) treated tumor tissue section with CD68 immunostaining (green) and nuclear staining (DAPI-blue).

[0034] [Figure 4] Figure 4: a) Immunoprecipitation of BMV109-labeled cysteine ​​cathepsins. b, c) Concentration-dependent labeling with probes 1 to 8 in live RAW cells. Panels in b) and c) were run on the same gel.

[0035] [Figure 5-1]Figure 5. a) Noninvasive optical imaging of tumor-bearing mice 8 hours after injection of probes 1, 2, 6, or 8. The lower panel shows the optimal fluorescence contrast at each time point. b) Time-dependent tumor-specific fluorescence (tumor minus background) for mice treated with probes 1, 2, 6, or 8 (n = 3, data represent mean ± standard deviation). c) Ex vivo tumor fluorescence (upper panel) and in vivo fluorescently labeled proteins visualized by in-gel fluorescent scanning after SDS-PAGE (lower panel). d) Fluorescence intensity at the end of noninvasive optical imaging (shown in a), ex vivo tumor imaging, and in-gel fluorescent labeling (shown in c). Intensity is depicted relative to probe 1 (n = 3, data represent mean ± standard deviation). e) Fluorescence microscopy of tumor sections treated with probe 8 (first, third, and fourth columns) with CD68 immunostaining (second, third, and fourth columns) and nuclear staining (DAPI - third and fourth columns, scale bar 50 μm). No probe control (middle row panels) and isotype controls for immunostaining (bottom row panels) are depicted. f) Colocalization diagram for probe 8 (Cy5) and CD68 (FITC). [Figure 5-2]Figure 5. a) Noninvasive optical imaging of tumor-bearing mice 8 hours after injection of probes 1, 2, 6, or 8. The lower panel shows the optimal fluorescence contrast at each time point. b) Time-dependent tumor-specific fluorescence (tumor minus background) for mice treated with probes 1, 2, 6, or 8 (n = 3, data represent mean ± standard deviation). c) Ex vivo tumor fluorescence (upper panel) and in vivo fluorescently labeled proteins visualized by in-gel fluorescent scanning after SDS-PAGE (lower panel). d) Fluorescence intensity at the end of noninvasive optical imaging (shown in a), ex vivo tumor imaging, and in-gel fluorescent labeling (shown in c). Intensity is depicted relative to probe 1 (n = 3, data represent mean ± standard deviation). e) Fluorescence microscopy of tumor sections treated with probe 8 (first, third, and fourth columns) with CD68 immunostaining (second, third, and fourth columns) and nuclear staining (DAPI - third and fourth columns, scale bar 50 μm). No probe control (middle row panels) and isotype controls for immunostaining (bottom row panels) are depicted. f) Colocalization diagram for probe 8 (Cy5) and CD68 (FITC). [Figure 5-3]Figure 5. a) Noninvasive optical imaging of tumor-bearing mice 8 hours after injection of probes 1, 2, 6, or 8. The lower panel shows the optimal fluorescence contrast at each time point. b) Time-dependent tumor-specific fluorescence (tumor minus background) for mice treated with probes 1, 2, 6, or 8 (n = 3, data represent mean ± standard deviation). c) Ex vivo tumor fluorescence (upper panel) and in vivo fluorescently labeled proteins visualized by in-gel fluorescent scanning after SDS-PAGE (lower panel). d) Fluorescence intensity at the end of noninvasive optical imaging (shown in a), ex vivo tumor imaging, and in-gel fluorescent labeling (shown in c). Intensity is depicted relative to probe 1 (n = 3, data represent mean ± standard deviation). e) Fluorescence microscopy of tumor sections treated with probe 8 (first, third, and fourth columns) with CD68 immunostaining (second, third, and fourth columns) and nuclear staining (DAPI - third and fourth columns, scale bar 50 μm). No probe control (middle row panels) and isotype controls for immunostaining (bottom row panels) are depicted. f) Colocalization diagram for probe 8 (Cy5) and CD68 (FITC). DETAILED DESCRIPTION OF THE INVENTION

[0036] Cysteine ​​cathepsins are a family of proteases that play important roles in both normal cellular physiology and the pathology of many human diseases. Accordingly, numerous substrate- and activity-based probe (ABP) classes have been developed to study the function of these enzymes. Provided herein, in some embodiments, is a class of quenched fluorescence activity-based probes containing phenoxymethyl ketone (PMK) electrophiles. These reagents exhibit enhanced and broad reactivity toward cysteine ​​cathepsins, leading to dramatically improved in vitro and in vivo labeling properties compared to previously reported ABPs. The probes are further demonstrated herein to highlight tumors in mice with unprecedented signal intensity and contrast. These novel reagents enable the study of cysteine ​​cathepsins at the organism, tissue, cellular, and protein levels in various models of human disease.

[0037] compound Thus, in some aspects, the present disclosure provides novel compounds for use in labeling protease enzymes, particularly cathepsins. The compounds of the present disclosure have the formula (I): [ka] (In the formula, L is an ether bond leaving element, T is a targeting element, D is a detectable element) The compound may be:

[0038] The targeting element T of the compound can be a peptidic or non-peptidic structure, and preferably targets the compound to cysteine ​​proteases.

[0039] For these purposes, non-limiting examples of non-peptidic structural elements usefully incorporated into the compounds are described in WO 2012 / 118715, which is incorporated herein in its entirety. In a preferred embodiment, the non-peptidic targeting element has a triazole structure.

[0040] Specific examples of compounds of the invention having non-peptide targeting moieties are: [ka] is.

[0041] Non-limiting examples of peptidic structural elements that can be usefully incorporated into the present compounds to target the compounds to cysteine ​​proteases, particularly cysteine ​​cathepsins, are described in WO 2009 / 124265, the entire contents of which are incorporated herein by reference.

[0042] In some embodiments of the compound, DT- is [ka] where: L1 is a linker, AA1 is an amino acid side chain, U is O, N or S; R1 is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl or a protecting group, optionally substituted with 1 to 3 A groups; Each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylalkyl, heterocyclylalkoxy, heterocyclyl It is tetracyclylalkanoyl, heterocyclylalkamino, hydroxyl, thio, amino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidinyl, urea, halo, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfonamido or azido.

[0043] As used herein, the term "alkyl" refers to the radical of a saturated aliphatic group, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, a straight-chain or branched-chain alkyl contains 30 or fewer carbon atoms in its backbone (e.g., C1-C6 for a straight chain). 30 , C3~C for branching difference 30 Likewise, some cycloalkyls have from 3-10 carbon atoms in their ring structure, and more particularly have 5, 6 or 7 carbons in the ring structure.

[0044] Furthermore, as used throughout the specification, examples, and claims, the term "alkyl" (or "lower alkyl") is intended to include both "unsubstituted alkyl" and "substituted alkyl," the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such structures can include, for example, halo, hydroxyl, carbonyl (such as keto, carboxy, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, thio, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For example, substituents of substituted alkyls can include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfates, sulfonamides, sulfamoyl, and sulfonates), and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN, and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CF3, -CN, and the like.

[0045] As used herein, the term "alkoxy" refers to an alkyl group, in certain embodiments a lower alkyl group, having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, t-butoxy, and the like.

[0046] As used herein, the term "alkenyl" refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl," the latter of which refers to an alkenyl moiety having substituents replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents may occur on one or more carbons that are either included or not included in one or more double bonds. Furthermore, such substituents include all of those contemplated for alkyl groups, as discussed above, except where stability would be impaired. For example, substitution of alkenyl groups with one or more alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl groups is contemplated.

[0047] When used with a chemical moiety, such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, the term "C x-y " is meant to include groups containing x to y carbons in the chain. For example, the term "C x-y -alkyl" includes trifluoromethyl and and haloalkyl groups such as 2,2,2-trifluoroethyl, and the like. "C0-alkyl" refers to a substituted or unsubstituted saturated hydrocarbon group containing x to y carbons in the chain, including straight-chain alkyl and branched-chain alkyl groups, including haloalkyl groups such as 2,2,2-trifluoroethyl, and the like. "C0-alkyl" refers to a hydrogen atom in the group at a terminal position, or if internal, a bond. 2-y -alkenyl" and "C 2-y "-Alkynyl" refers to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to alkyl as described above, but having at least one double or triple bond, respectively.

[0048] As used herein, the term "alkylamino" means an amino group substituted with at least one alkyl group.

[0049] As used herein, the term "alkylthio" means a thiol group substituted with an alkyl group and may be represented by the general formula alkyl-S-.

[0050] As used herein, the term "alkynyl" refers to an aliphatic group containing at least one triple bond and is intended to include both "unsubstituted alkynyl" and "substituted alkynyl," the latter of which refers to an alkynyl moiety having substituents replacing hydrogen on one or more carbons of the alkynyl group. Such substituents may occur on one or more carbons that are included or not included in one or more triple bonds. Furthermore, such substituents include all of those contemplated for alkyl groups, as discussed above, except where stability would be impaired. For example, substitution of alkynyl groups with one or more alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl groups is contemplated.

[0051] As used herein, the term "amide" refers to the group [ka] where R x and R y each independently represents hydrogen or a hydrocarbyl group, or R x and R y together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0052] The terms "amine" and "amino" are art-recognized and refer to unsubstituted and substituted amines and their salts, e.g., [ka] means a moiety that can be represented by the formula: x , R y and R z each independently represents hydrogen or a hydrocarbyl group, or R x and R y together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0053] As used herein, the term "aminoalkyl" refers to an alkyl substituted with an amino group. means a group.

[0054] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group.

[0055] As used herein, the term "aryl" includes substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. In certain embodiments, the ring is 5- to 7-membered, and in more specific embodiments, it is a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two connected rings, where at least one ring is aromatic and the other cyclic rings can be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0056] The term "carbamate" is art-recognized and refers to a group [ka] where R x and R y independently represent hydrogen or a hydrocarbyl group, or R x and R y together with the atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0057] As used herein, the term "cycloalkyl" means a non-aromatic saturated or unsaturated ring in which each atom of the ring is carbon. In certain embodiments, the cycloalkyl ring contains 3 to 10 atoms, and in more particular embodiments, 5 to 7 atoms.

[0058] The term "carbonate" is art-recognized and refers to a group -OCO-R x where Rx represents a hydrocarbyl group.

[0059] As used herein, the term "carboxy" means a group represented by the formula -CO2H.

[0060] As used herein, the term "ester" refers to a group -C(O)OR x where R x represents a hydrocarbyl group.

[0061] As used herein, the term "ether" refers to a hydrocarbyl group linked to another hydrocarbyl group through an oxygen atom. Thus, the ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. Ethers can be either symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl groups," which can be represented by the general formula alkyl-O-alkyl.

[0062] The term "guanidinyl" is art-recognized and has the general formula [ka] where R x and R y independently represent hydrogen or hydrocarbyl.

[0063] As used herein, the terms "halo" and "halogen" mean halogen and include chloro, fluoro, bromo, and iodo.

[0064] As used herein, the terms "hetaralkyl" and "heteroaralkyl" refer to an alkyl group substituted with a hetaryl group.

[0065] The terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic single ring structures, in certain embodiments, 5- to 7-membered rings, and more particularly 5- to 6-membered rings, whose ring structures contain at least one heteroatom, in some embodiments 1 to 4 heteroatoms, and more particularly 1 to 2 heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, where at least one ring is heteroaromatic and the other cyclic rings can be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0066] As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Typical heteroatoms are nitrogen, oxygen, and sulfur.

[0067] The terms "heterocyclyl," "heterocycle," and "heterocyclic" refer to substituted or unsubstituted non-aromatic ring structures, in certain embodiments, 3- to 10-membered rings, more particularly 3- to 7-membered rings, whose ring structures contain at least one heteroatom, in some embodiments 1 to 4 heteroatoms, and more particularly 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, where at least one ring is heterocyclic and the other cyclic rings, for example, can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0068] As used herein, the term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocycle group.

[0069] As used herein, the term "hydrocarbyl" means a group that has no =0 or =S substituents and that is typically bonded through a carbon atom, with at least one carbon-hydrogen bond and a primarily carbon backbone, but which may optionally contain heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered hydrocarbyl for purposes of this specification, although groups that have an =0 substituent on the bonded carbon are not. ) acetyl and ethoxy (attached through an oxygen rather than a carbon). Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

[0070] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0071] The term "lower," when used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups in which 10 or fewer, and in certain embodiments, 6 or fewer, non-hydrogen atoms are present in the substituent. For example, "lower alkyl" refers to an alkyl group containing 10 or fewer, and in certain embodiments, 6 or fewer, carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, and alkoxy substituents defined herein are, in the description, lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, and lower alkoxy, respectively, whether they appear alone or in combination with other substituents, such as hydroxyalkyl and aralkyl (where, for example, atoms in the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

[0072] The terms "polycyclyl," "polycycle," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings." Each of the rings of a polycycle can be substituted or unsubstituted. In certain embodiments, each ring of a polycycle contains 3 to 10 atoms, more particularly 5 to 7 atoms in the ring.

[0073] The term "substituted" refers to a moiety having substituents replacing a hydrogen on one or more backbone carbons. It is understood that "substituted" or "substituted with" includes the implicit proviso that such substitution is in accordance with the allowed valencies of the substituted atom and substituent, and that the substitution results in a stable compound that does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, etc., under the conditions in which the compound is to be used. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituent of organic compounds described herein that satisfies the valence of the heteroatom. Substituents can include any of the substituents described herein, such as halogen, hydroxyl, carbonyl (such as keto, carboxy, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

[0074] Unless specifically stated as "unsubstituted," references to chemical moieties herein include substituted or unsubstituted. For example, reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.

[0075] The term "sulfate" is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.

[0076] The term "sulfonamide" is art-recognized and can be represented by the general formula [ka] wherein R x and R y independently represent hydrogen or hydrocarbyl.

[0077] The term "sulfoxide" is art-recognized and refers to the group -S(O)-R x where R x represents a hydrocarbyl.

[0078] The terms "sulfo" or "sulfonate" are art-recognized and refer to the group -SO3H or a pharmaceutically acceptable salt thereof.

[0079] The term "sulfone" is art-recognized and refers to the group -S(O)-R x where R x represents a hydrocarbyl.

[0080] As used herein, the term "thioalkyl" refers to an alkyl group substituted with a thiol group.

[0081] As used herein, the term "thioester" refers to the group -C(O)SR x or -SC(O)R x where R x represents a hydrocarbyl.

[0082] As used herein, the term "thioether" is equivalent to an ether, where the oxygen is replaced by a sulfur.

[0083] The term "urea" is art-recognized and has the general formula [ka] where R x and R y independently represent hydrogen or hydrocarbyl.

[0084] The compounds of the present invention are generally synthesized using standard synthetic chemistry techniques, for example, using the methods described in the Examples section below. Other useful synthetic techniques are described, for example, in March's Advanced Organic Chemistry: Reactions,Mechanisms,and Structure,7th Ed.,(Wiley,2013);Carey and Sundberg,Advanced Organic Chemistry 4 th Ed., Vols. A and B (Plenum 2000, 2001); Fiesers' Reagents for Organic Synthesis, Volumes 1-27 (Wiley, 2013); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-81 (Wiley, 2013); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), all of which are incorporated by reference. The compounds are generally readily synthesized using starting materials that are commonly available from commercial sources or prepared using methods well known to those skilled in the art. See, for example, Fiesers' Reagents for Organic Synthesis, Volumes 1-27 (Wiley, 2013), including supplements, or Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin.

[0085] When referring to components of a compound 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 with a second reactive functional group on a second component to form a covalent bond. In an exemplary embodiment, an amine group on a first component can react with an activated carboxyl group on a second component to form a residue containing one or more amide moieties. Other arrangements of first and second reactive functional groups are encompassed by the present invention. For example, copper-catalyzed or copper-free reaction of an azide-substituted first component with an alkyne-substituted second component yields a thiazole-containing residue through the well-known "click" reaction, as will be understood by those skilled in the art. See Kolb et al. (2001) Angew. Chem. Int. Ed. Engl. 40:2004; Evans (2007) Aus. J. Chem. 60:384. An exemplary method for generating non-peptide fluorescent imaging probes using the "click" reaction is provided in WO 2012 / 118715. The adaptation of these methods to produce or modify the claimed compounds is within the skill of the art.

[0086] Those skilled in the art will understand that protecting groups are reversibly attached to a desired location on a molecule to control the reaction of other agents at that location. 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, 4 by P. G. Wuts and T. W. Greene. th edition, (Wiley-Interscience, 2006); and Protecting Groups by P. Kocienski (Thieme, 2005).

[0087] The L1 group of the present compounds is a linker group that connects the detectable element D to the targeting element. This group can be any suitable linker, as will be understood by those skilled in the art. The L1 group is preferably an alkyl linker group, where the alkyl linker is optionally substituted, and carbons within the linker are optionally replaced with heteroatoms to the extent that the resulting structure is chemically stable. Such substitutions and replacements should be understood to include intervening groups within the linker, such as ethers, thioethers, disulfides, esters, amides, carbonates, carbamates, and the like. Preferred linkers range in length from 5 to 40 bonds and can be branched, linear, or contain rings. Linkers can optionally contain double bonds. They can be hydrophobic or hydrophilic, as desired according to specific requirements.

[0088] The linkage between the L group and the detectable element D may be any suitable linkage, as will be understood by those skilled in the art. It should be further understood that the bond may be chemical. For example, the present compound can be conveniently prepared by including a moiety reactive with a specific chemical group, such as an amino group, a thiol group, etc., in the detectable element precursor in some cases. In such a state, the detectable element can be simply connected to the targeting element through the reaction of this group on the targeting element. These types of bonds are therefore understood to be within the scope of the disclosed compounds, even if the structural details of the connection are not explicitly shown.

[0089] The AA1 group of the present compounds can be any natural or unnatural amino acid side chain, as will be understood by those skilled in the art. In a preferred embodiment, the AA1 group is an aralkyl amino acid side chain optionally substituted with one to three A groups. In a further preferred embodiment, the AA1 group is a phenylalanine side chain.

[0090] In preferred compounds, the U group is O.

[0091] The detectable element of the present compounds is, in certain embodiments, a fluorescent label, a radiolabel, a chelator, etc. Examples of suitable radiolabels and chelators for use in these compounds are described in WO 2009 / 124265.

[0092] In a preferred embodiment of the present compound, the detectable element is a fluorescent label. As known by those skilled in the art, a fluorescent label emits electromagnetic radiation, preferably visible light, when stimulated by the absorption of incident electromagnetic radiation. A wide variety of fluorescent labels are commercially available, including labels having reactive moieties useful for binding the label to reactive groups such as amino groups, thiol groups, etc. See, for example, The Molecular Probes® Handbook - A Guide to Fluorescent Probes and Labeling Technologies.

[0093] An example of a fluorescent label is fluorescein, which is widely used in immunofluorescence labeling. Fluorescein is a xanthene dye with an absorption maximum at 495 nanometers. A related fluorophore is Oregon Green, a fluorinated derivative of fluorescein.

[0094] In some embodiments, the fluorescent label used in the detectable element of the compounds of the present invention may be a pH-dependent fluorophore. For example, such fluorescent labels, as shown below, as used in the compounds labeled "LES12" and "LES13," exhibit fluorescence spectra that depend on the pH of the label's environment, as will be understood by those skilled in the art, and are therefore useful in reporting information about the label's environment after a reaction, such as the type or location of the protease labeled by the reactive compound. The pH-dependent fluorescence of various labels usefully included in the detectable element of the compounds may be well known. See, for example, The Molecular Probes® Handbook—A Guide to Fluorescent Probes and Labeling Technologies, the entire contents of which are incorporated herein by reference.

[0095] Other exemplary fluorescent labels suitable for use in the present compounds are boradiazaindecene, rhodamine, and cyanine dyes. In particular, boradiazaindecene dyes are represented by 4,4-difluoro-4-bora-3a,4a-diaza-s-indecene, known as BODIPY® dyes. Various derivatives of these dyes are known and may be suitable for use as detectable elements in the compounds of the present disclosure. See, for example, Chen et al. (2000) J. Org. Chem. 65:2900-2906.

[0096] Another class of fluorescent labels that may be beneficially used in the compounds of the present invention is Li-Cor (www and IRDye infrared dyes available from iCor.com. Non-limiting examples of these dyes are IRDye 800CW, IRDye 680RD, IRDye 680LT, IRDye 750, IRDye 700DX, IRDye 800RS, and IRDye 650.

[0097] Rhodamine dyes are a class of dyes based on the rhodamine ring structure. Rhodamines include tetramethylrhodamine (TMR), a very common fluorophore for preparing protein conjugates, especially antibody and avidin conjugates, and carboxytetramethylrhodamine (TAMRA), a dye commonly used for oligonucleotide labeling and automated nucleic acid sequencing. Rhodamines are established as a natural complement to fluorescein-based fluorophores, offering longer wavelength emission maxima and thus opening up opportunities for multicolor labeling or staining.

[0098] Also included within the rhodamine dye family are the sulfonated rhodamine series of fluorophores known as Alexa Fluor dyes. A dramatic advance in modern fluorophore technology is exemplified by the Alexa Fluor dyes, introduced by Molecular Probes. These sulfonated rhodamine derivatives exhibit higher quantum yields for more intense fluorescence emission than spectrally similar probes, and possess a variety of additional improved properties, including enhanced photostability, absorption spectra matched to common laser lines, pH insensitivity, and a high degree of water solubility.

[0099] Cyanine dyes represent a family of related dyes, Cy2, Cy3, Cy5, Cy7, and their derivatives, based on a partially saturated indole nitrogen heterocyclic nucleus with two aromatic units linked via a polyalkane bridge of varying carbon numbers. These probes exhibit fluorescence excitation and emission profiles similar to many of the traditional dyes, such as fluorescein and tetramethylrhodamine, but with enhanced water solubility, photostability, and higher quantum yields. Most cyanine dyes are more environmentally stable than their traditional counterparts, rendering their fluorescence emission intensity less sensitive to pH and organic mounting media. In a manner similar to Alexa Fluors, the excitation wavelengths of the Cy series of synthetic dyes are specifically tailored for use with common laser and arc discharge sources, and fluorescence emission is detectable with traditional filter combinations. Cyanine dyes are readily available as reactive dyes or fluorophores. Cyanine dyes generally have broader absorption spectra than members of the Alexa Fluor family, making them somewhat more versatile in the selection of laser excitation sources for confocal microscopy.

[0100] In a preferred embodiment, the detectable element of the compound is the cyanine dye, Cy5.

[0101] In some embodiments, it may be beneficial to include multiple fluorescent labels, radiolabels, chelators, etc. within the detectable element of the compounds of the present invention. For example, the exemplary compounds labeled "LES12" and "LES13" below contain two different fluorescent labels within a single detectable element. Such multiple labeling can be achieved using routine conjugation chemistry, as will be understood by those skilled in the art. For example, the fluorescent labels within the "LES12" and "LES13" compounds were linked using "click" chemistry. An example of an intermediate compound useful in the synthesis of compounds containing multiple labels within the detectable element via "click" chemistry is shown below ("WL938"). This compound contains an azide group and can therefore be easily reacted with a suitable alkyne-containing reagent in a "click" reaction. The positions of the alkyne and azide groups can also be reversed, if desired, as will be understood by those skilled in the art.

[0102] In some embodiments, the compounds of the invention are compounds of formula (I), where T is a peptidic targeting element, and the compounds are further described in the following numbered sentences: 1. A compound of formula (I) wherein DT- is a short detectable peptidic group and L is an ether bond leaving element. 2. The compound of sentence 1, wherein the detectable peptidic group comprises 1 to 4 amino acid residues. 3.DT [ka] wherein each AA1, AA2, AA3, and AA4 is independently an amino acid side chain or -L1-D; Each R A are independently hydrogen or R1; R B is hydrogen, R1, -C(O)R1, -C(O)OR1, -C(O)SR1 or -C(O)N(R1)(R A ) and R1 is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, a protecting group, or -L1-D-, optionally substituted with 1 to 3 A groups; each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylalkyl, heterocyclylalkoxy, heterocyclylalkanoyl, heterocyclylalkamino, hydroxyl, thio, amino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidinyl, urea, halo, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfonamido, or azido; L1 is the linker, compound of sentence 2. 4.Each R A is hydrogen and R B The compound of sentence 3, where is -C(O)OR1. 5.DT is, [ka] and each AA1 and AA2 is independently an amino acid side chain or -L1-D; Each R A is hydrogen, the compound in sentence 3. 6.DT, [ka] and each AA1, AA2, and AA3 is independently an amino acid side chain or L1-D; Each R A is hydrogen, the compound in sentence 3. 7.DT, [ka] and each AA1, AA2, AA3, and AA4 is independently an amino acid side chain or -L1-D; Each R A is hydrogen, the compound in sentence 3. 8. The compound of sentence 3, wherein L1 is an optionally substituted alkyl linker, each carbon atom being optionally replaced with a heteroatom. 9.R B The compound of sentence 3, where is -C(O)OR1. 10. The compound of sentence 3, where D is a fluorescent label, a radiolabel, or a chelator. 11.The compound in sentence 10 where D is a fluorescent label. 12. The compound of sentence 11, wherein the fluorescent label is a fluorescein, Oregon green, bora-diaza-indecene, rhodamine, or cyanine label. 13. The compound in sentence 12, in which the fluorescent dye is a cyanine label. 14. The compound of sentence 13, in which the cyanine label is Cy5.

[0103] In these embodiments of the present compounds, the AA1, AA2, AA3, and AA4 groups can independently be any natural or unnatural amino acid side chain, or the group "-L1-D," as understood by one of skill in the art. In a preferred embodiment, the group is an aralkylamino side chain, optionally substituted with one to three A groups. In a further 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 the side chain 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 other such amino acid residue, in any combination. Side chains from other amino acid residues, such as lysine, arginine, tyrosine, glutamine, asparagine, and the like, are also preferred in the present compounds.

[0104] In compound embodiments in which the AA1, AA2, AA3, or AA4 group is a "-L1-D" group, the L1 linker moiety can be provided by an amino acid side chain. For example, a lysine residue conveniently provides an amino-alkyl group for reaction with a suitably activated detectable element.

[0105] A "short" detectable peptidic group is defined herein as a detectable peptidic group having up to 10 amino acid residues.

[0106] The ether-linked leaving element L of the present compounds can affect the reactivity of the compounds with their target enzyme active sites and thus affect the targeting specificity for a particular enzyme. The ether linkage of the leaving element in these compounds contrasts with the ester linkage of other activity-based probes, such as acyloxymethyl ketones (AOMKs). For example, an ether-linked leaving element, such as a phenol ether-linked leaving element, can provide improved stability in vivo relative to ester-linked or other types of probes.

[0107] In some embodiments, the ether-linked leaving element of the present compounds includes a quencher. The term "quencher" refers to a chemical entity that modulates the emission of a fluorophore. In some cases, a quencher can itself be a fluorescent molecule that emits fluorescence at a characteristic wavelength different from that of the label whose fluorescence is quenched. Thus, a fluorophore can act as a quencher when appropriately attached to another dye, or vice versa. In these situations, an increase in fluorescence from the acceptor molecule, which is of a wavelength different from that of the donor label, can separately report the interaction of the labeled compound with its environment, such as the active site of a target enzyme. In some cases, the quencher does not itself fluoresce (i.e., the quencher is a "dark acceptor"). Such quenchers include, for example, dabcyl, methyl red, and QSY diarylrhodamine dyes. In particular, dabcyl (4-dimethylamino-phenylazo)benzoate) is a common dark quencher 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, called "Black Hole Quenchers," offer a broad range of absorption that overlaps well with the emission of many fluorophores. WO 01 / 86001. Molecular Probes' QSY series dyes are other examples of dark quencher dyes that have been used extensively as quenching reagents in many bioassays. U.S. Patent No. 6,399,392.

[0108] In particular, QSY7 is a non-fluorescent diarylrhodamine derivative. U.S. Patent Application Publication No. 2005 / 0014160. QSY21 is a non-fluorescent diarylrhodamine chromophore with strong absorption in the visible spectrum and is an effective fluorescence quencher. Further fluorophore / quencher pairs are exemplified in U.S. Patent Application Publication No. 2004 / 0241679.

[0109] IRDye QC-1 (available from Li-Cor) is another example of a non-fluorescent dye suitable for use as a quencher in the present compounds. It effectively quenches fluorescence from a wide range of fluorophores, including wavelengths ranging from the visible to the near-infrared.

[0110] In some embodiments of the compounds, the leaving group element L is L2-L3-Q, where L2 is a phenoxy group, L3 is a linker, and Q is a quencher. [ka] 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 can be, for example, [ka] is.

[0111] The L3 linker group of the leaving element can be any suitable linker, as will be appreciated by those skilled in the art. In particular, the L3 linker group can be an L1 group, for example, as described above.

[0112] In certain other compounds, the L group is, for example: [ka] 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.

[0113] In some particular embodiments, compounds of the present disclosure have the structure of formula (II): [ka]

[0114] In some more specific embodiments, the compounds of the present disclosure have the structure of formula (III): [ka]

[0115] In these embodiments, m and n are independently integers from 1 to 16.

[0116] In some embodiments, R is QSY21 or sulfo-QSY21 and D is Cy5.

[0117] Specific non-limiting compound embodiments of the present invention include: [ka] Including, where R=QSY21 and n=6, R = sulfo-QSY21 and n = 6, R=QSY21 and n=2 and R=sulfo-QSY21 and n=2.

[0118] In some embodiments, the compound of the present invention is a compound having the structure of formula (IV): [ka] where T is a peptidic targeting element, and the compounds are further described in the numbered sentences below. 1. where DT- is a short detectable peptidic group; L3 is a linker, The compound of formula (IV), wherein Q is a quencher. 2. The compound of sentence 1, wherein the detectable peptidic group comprises 1 to 4 amino acid residues. 3.DT [ka] wherein each AA1, AA2, AA3, and AA4 is independently an amino acid side chain or -L1-D; Each R A are independently hydrogen or R1; R B is hydrogen, R1, -C(O)R1, -C(O)OR1, -(C(O)SR1 or -C (O)N(R1)(R A ) and R1 is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, a protecting group, or -L1-D-, optionally substituted with 1 to 3 A groups; Each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, hetero cyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylalkyl, heterocyclylalkoxy, heterocyclylalkanoyl, heterocyclylalkamino, hydroxyl, thio, amino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidinyl, urea, halo, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfonamido, or azide, and L1 is a linker. 4.Each R A is hydrogen and R B The compound of sentence 3, where is -C(O)OR1. 5.DT- is [ka] and each AA1 and AA2 is independently an amino acid side chain or -L1-D; Each R A is hydrogen, the compound in sentence 3. 6.DT- is [ka] each AA1, AA2, and AA3 is independently an amino acid side chain or L1-D; Each R A is hydrogen, the compound in sentence 3. 7.DT, [ka] each AA1, AA2, AA3, and AA4 is independently an amino acid side chain or -L1-D; Each R A is hydrogen, the compound in sentence 3. 8. The compound of sentence 3, wherein L1 is an optionally substituted alkyl linker, each carbon atom being optionally replaced with a heteroatom. 9.R B The compound of sentence 3, where is -C(O)OR1. 10. The compound of sentence 3, where D is a fluorescent label, a radiolabel, or a chelator. 11.The compound in sentence 10 where D is a fluorescent label. 12. The compound of sentence 11, wherein the fluorescent label is a fluorescein, Oregon green, bora-diaza-indecene, rhodamine, or cyanine label. 13. The compound in sentence 12, in which the fluorescent dye is a cyanine label. 14. The compound of sentence 13, in which the cyanine label is Cy5.

[0119] In these embodiments of the present compounds, the AA1, AA2, AA3, and AA4 groups can independently be any natural or unnatural amino acid side chain, or the group "-L1-D," as understood by one of skill in the art. In a preferred embodiment, the group is an aralkylamino side chain, optionally substituted with one to three A groups. In a further 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 the side chain 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 other such amino acid residue, in any combination. Side chains from other amino acid residues, such as lysine, arginine, tyrosine, glutamine, asparagine, and the like, are also preferred in the present compounds.

[0120] In compound embodiments in which the AA1, AA2, AA3, or AA4 group is a "-L1-D" group, the L1 linker moiety can be provided by an amino acid side chain. For example, a lysine residue conveniently provides an amino-alkyl group for reaction with a suitably activated detectable element.

[0121] A "short" detectable peptidic group is defined herein as a detectable peptidic group having up to 10 amino acid residues.

[0122] Other specific non-limiting compound embodiments of the present invention include: [ka] [ka] Contains

[0123] Pharmaceutical Compositions In another aspect, the present invention provides pharmaceutical compositions comprising a compound of the present invention and a pharmaceutically acceptable carrier. Such compositions are useful, for example, for imaging tissues in animals, and for assessing the activity of enzymes, such as protease enzymes, in animals. In particular, for compounds of the present invention that label cathepsins, pharmaceutical compositions can be useful as tools for non-invasive optical imaging of cancer cells.

[0124] Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline, or other solvents or excipients such as glycols, glycerol, oils such as olive oil or injectable organic esters. In certain embodiments, when such pharmaceutical compositions are administered to humans, the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to affect delayed release of the drug or to selectively target one or more cells, tissues, or organs. Pharmaceutical compositions can be in unit dosage forms, such as tablets, capsules, sprinkle capsules, granules, powders, syrups, suppositories, injections, etc. The composition can also be present in a transdermal delivery system, for example, a skin patch.

[0125] Pharmaceutically acceptable carriers may contain physiologically acceptable agents that, for example, 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 a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. Pharmaceutical compositions may also contain liposomes or other polymer matrices into which, for example, the compounds of the present invention can be incorporated. For example, liposomes composed of phospholipids or other lipids are relatively simple to prepare and administer, non-toxic, physiologically acceptable, and metabolically stable. It is a possible carrier.

[0126] The phrase "pharmaceutically acceptable" is used herein to mean compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, and are commensurate with a reasonable benefit / risk ratio.

[0127] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or excipient, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in carrying or transporting a compound of interest from one organ or body part to another. Each carrier must be "acceptable" in that it is compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve 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 carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; and (9) oils such as peanut oil, cotton oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil. Examples of suitable non-toxic and compatible substances include: (10) glycerols 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) buffers such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffer solution, and (21) other non-toxic and compatible substances used in pharmaceutical formulations. See The Science and Practice of Pharmacy, 20th ed. (Alfonso R. Gennaro ed.), 2000.

[0128] Pharmaceutical compositions containing the compounds of the present invention can be administered to a subject by any of a number of routes, including, for example, oral (e.g., in aqueous or non-aqueous solutions or suspensions, such as drenches, tablets, boluses, powders, granules, or pastes for application to the tongue), sublingual, anal, rectal, or vaginal (e.g., as pessaries, creams, or foams), parenteral (e.g., as a sterile solution or suspension, including intramuscular, intravenous, subcutaneous, or intrathecal), nasal, intraperitoneal, subcutaneous, transdermal (e.g., as a patch applied to the skin), or topical (e.g., as a cream, ointment, or spray applied to the skin). The compounds can also be formulated for inhalation. In certain embodiments, the compounds of the present invention can be simply dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable for those routes can be found, for example, in U.S. Pat. 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 patents cited therein.

[0129] Labeling and visualization methods In another aspect, the present invention provides a method for visualizing a tumor in an animal, comprising administering to the animal a composition of the present invention.

[0130] In yet another aspect, the present invention provides a method for visualizing tumors in an animal, comprising the steps of administering to the animal a composition of the present invention and measuring a detectable signal generated in the animal from the reaction of the composition with a cathepsin cysteine ​​protease, wherein the detectable signal The virus is associated with tumors in animals.

[0131] In some embodiments of the method, the detectable signal is a fluorescent signal. In some embodiments, the fluorescent signal occurs at the tumor margin.

[0132] Administration of peptide imaging agents to animals is well understood by those of skill in the art. In a preferred embodiment, the agent is administered by injection, although any other suitable means of administration is considered within the scope of the present invention.

[0133] The methods of the present invention are directed to the labeling and visualization of proteases, particularly cysteine ​​proteases, in animals. Suitable animals include animals expressing cysteine ​​proteases, particularly in tumor cells. In a preferred embodiment, the animal is a mammal. In a highly preferred embodiment, the animal is a human. In another preferred embodiment, the animal is a livestock or pet.

[0134] In some embodiments, the method of the present invention includes measuring a detectable signal generated in an animal. Methods for measuring a detectable signal include, but are not limited to, imaging methods, such as fluorescent imaging methods. In some embodiments, the fluorescent imaging system is, for example, the Xenogen IVIS100 system, although any suitable imaging system can be used.

[0135] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of the invention or any embodiment thereof. Having now described the invention in detail, this will be more clearly understood by reference to the following examples, which are included herein for purposes of illustration only and are not intended to limit the invention. [Example]

[0136] Synthesis and characterization of quenched fluorescent cysteine ​​cathepsin imaging probes containing novel phenoxymethyl ketone (PMK) electrophiles The goal of this study was to develop a qABP with improved overall in vivo properties compared to existing qABPs available for noninvasive optical imaging of cancer. Therefore, we decided to optimize the probe's three key elements: the quencher, the linker, and the electrophilic "warhead." One of the biggest obstacles of the cysteine ​​cathepsin qABPs reported to date is their relatively 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 biodispersion of the probe. The length of the spacer connecting the electrophile and quencher was also varied to reduce the lipophilicity of the qABP. Finally, novel electrophiles were explored to increase the range of potential cathepsin targets. Because various members of the cysteine ​​cathepsin family are upregulated in various cancers (Mohamed & Sloane (2006) Nat. Rev. Cancer (2006) 6:764-75), brighter fluorescent signals in tumors would be expected if the probe targeted a broad spectrum of cysteine ​​cathepsin activity. To obtain more pan-reactive probes, the size of the electrophile was reduced, increasing activity. It has previously been shown that 2,3,5,6-tetrafluoro-substituted phenoxymethyl ketone (PMK) electrophiles have greater reactivity toward cysteine ​​dipeptidyl aminopeptidases compared with 2,6-dimethylbenzoic acid-derived acyloxymethyl ketones (AOMKs). Deu et al. (2010) Chem Biol. 17:808-819. The smaller size of PMK also results in pan-reactivity because some binding grooves of cysteine ​​cathepsins are sterically restricted. (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. Furthermore, phenolic ethers are presumed to be more stable in vivo compared to AOMK electrophiles, which contain ester bonds that can be degraded by esterases.

[0137] As a starting point for this study, seven analogs (2–8) of qABP GB137 (1) were synthesized. (Blum et al., (2007) Nat. Chem. Biol. 3:668–77) (Figure 1a). These compounds represent all combinations of two electrophiles, two quenchers, and two linker lengths. All probes were synthesized using an optimized, solution-chemistry-based procedure as described in the accompanying Scheme 1 below. Probe specificity and potency were first tested by labeling intact RAW264.7 cells (a murine leukemia-monocyte-macrophage cell line) (Figure 1b). Various trends were observed in the probe properties. All sulfo-QSY21-functionalized qABPs (2, 4, 6, and 8) showed stronger total cathepsin labeling compared to the more hydrophobic QSY21-containing probes (1, 3, 5, and 7). Interestingly, changing the spacer length from a hexyl to an ethyl linker did not have a dramatic effect on the labeling profile. Perhaps the most striking observation was that qABPs with PMK electrophiles exhibited broader cysteine ​​cathepsin labeling profiles compared to their AOMK counterparts. Probes 5–8 exhibited robust cathepsin X labeling, while sulfo-QSY21-functionalized probes 6 and 8 were capable of labeling higher molecular weight preforms of cathepsin L. The identity of the fluorescently labeled cathepsins was determined by immunoprecipitation (Figure 4a). Various other interesting trends were observed when performing titration labeling experiments in live RAW cells (Figures 1c, d, 4b, c). The most hydrophobic qABPs (1 and 5) reached a reduced maximum in labeling intensity at 0.5 μM, suggesting that their reduced water solubility leads to probe precipitation at higher concentrations. The shorter spacer length appears to be beneficial, with all probes having an ethyl spacer, which provides brighter labeling compared to their hexyl-containing counterparts. A clear difference in selectivity is observed when comparing AOMKs with PMKs. AOMK qABPs preferentially label cathepsins S and L, and only at higher concentrations label cathepsin B.Surprisingly, AOMK qABPs 2–4 label cathepsin X, although previous studies have shown that various other related AOMKs fail to label this target (Paulick & Bogyo (2011) ACS Chem. Biol. 6:563–72). PMK qABPs also label all target cysteine ​​cathepsins with equal intensity, even at lower probe concentrations. Together, these experiments demonstrate that increasing hydrophilicity improves labeling intensity and that the new PMK qABPs have a broader, more pan-cysteine ​​cathepsin labeling profile.

[0138] Because PMK qABP8 was the most optimal in terms of total labeling intensity and broad cathepsin reactivity, we decided to proceed with this probe for further in vivo studies. To further define target selectivity, RAW cell lysates were labeled with increasing concentrations of qABP8 at pH 5.5. These results showed that the probe was most potent for cathepsins B and X, with labeling observed at concentrations as low as 5 nM. However, labeling of all cathepsins (B, S, L, and X) saturated at 500 nM of probe (Figure 2a). When the probe was used for time-course labeling of live RAW cells at a set concentration of 500 nM, rapid saturation of cathepsin X was observed, followed by slower labeling of cathepsins S, L, and B, with an increase in cathepsin B labeling signal even at 120 min (Figure 2b). These data suggest that the probe is most likely able to access the pool of cathepsin X due to its location within the cell or on the cell surface. Cathepsins B and X are accessible to different extents by the probe. These results also suggest that the PMK probe may have alternate locations in cells where it can be located. To test the stability of the novel PMK probe, we examined the effect of serum exposure on labeling in RAW cells (Fig. 1c). While 4 hours of serum pre-exposure to the original AOMK probe 1 resulted in a loss of approximately 70% of target labeling, more than 80% of labeling was maintained for PMK qABP8. Pretreatment of cells with the cysteine ​​cathepsin inhibitor JPM-OEt also blocked more than 90% of this labeling. Given the stability and improved labeling properties of the PMK probe, we next performed live-cell fluorescence microscopy studies. These results confirmed that the probe produced a bright, specific labeling signal and that the majority of the probe-labeled cathepsin remained in lysosomes (Fig. 2d).

[0139] Given the positive live-cell labeling qualities of the novel PMK electrophiles, the best-performing PMK qABPs 2, 6, and 8 were tested in an orthotopic mouse model of breast cancer (Tao et al., (2008) BMC Cancer 8:228). Furthermore, these PMK probes were compared with the original AOMK probe 1 (Figures 3 and 5). 4T1 cells were implanted into the mammary fat pads of Balb / c mice, digits 2 and 7, and tumor growth was monitored. Once tumors were established, mice were injected with equimolar amounts of qABPs (20 nmol) through the tail vein, and Cy5 fluorescence was noninvasively imaged over time (Figures 3a and 3b). Again, these results confirmed that qABP 8 proved superior. Robust tumor-specific activation of fluorescence was observable for probe 8, particularly in the tumor region, with high total contrast. This signal continued to increase over time until the end of the time course. Ultimately, probe 8 achieved a greater than 20-fold enhancement of tumor-specific fluorescent signal compared to probe 1. Good tumor-specific contrast was also observed for probe 6 and, to a lesser extent, for probe 2, although both were greater than 10-fold greater than probe 1 (Figures 5a, b). After completion of the time course, tumors were removed and tumor fluorescence was measured ex vivo, followed by homogenization and analysis of fluorescently labeled proteins by SDS-PAGE (Figures 3c and 5c). Quantification of ex vivo fluorescence and total cysteine ​​cathepsin labeling showed trends similar to those seen in noninvasive optical imaging studies (Figures 3d and 5d). To determine the cellular source of probe fluorescence, immunofluorescent staining of tumor tissue sections from probe-labeled mice was performed using the macrophage marker CD68 (Figures 3e and 5e). Cy5 fluorescence localized to CD68-positive cells; however, not all CD68-positive cells were also positive for probe 8, suggesting a different activation state of tumor-associated macrophages. Detailed analysis by confocal laser scanning microscopy (CLSM) confirmed that all cells positive for probe 8 were also positive for CD68, but the probe-labeled cathepsin and CD68 signals did not colocalize to the same vesicles (Fig. 3f and Fig. 5f).Taken together, these data confirm that increasing the hydrophilicity of the quencher, shortening the spacer, and introducing a more reactive and less sterically restricted electrophilic trap resulted in qABPs with broad cysteine ​​cathepsin reactivity and overall improved in vivo properties.

[0140] Although very distinct functions have been described for several cysteine ​​cathepsin family members (Conus & Simon (2010) Swiss Med. Wkly. 140:w13042), other roles overlap, and altering the activity of one cathepsin can affect the activity of others. For example, loss of cathepsin B is compensated for 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). Therefore, broad-spectrum probes are highly valuable because they facilitate the readout of multiple cysteine ​​cathepsins in a single experiment and allow comparison of the activities of individual cathepsins relative to each other. The utility of such pan-reactive ABPs has been demonstrated using a pan-serine hydrolase fluorophosphonate probe (Liu et al., (1999) Pro It has been shown by the pan-reactive proteasome probe MV151 (Verdoes et al. (2006) Chem. Biol. 13:1217-26) and the PMK-based qABPs are highly reactive toward cathepsin X, and these scaffolds can be used to generate selective qABPs for this poorly understood cysteine ​​cathepsin (Paulick & Bogyo (2011) ACS Chem. Biol. 6:563-72).

[0141] In conclusion, a novel class of quenched fluorescence activity-based probes was synthesized with PMK electrophiles that possess greater reactivity and broader selectivity compared to previously reported AOMK-based probes. The hydrophilicity of qABPs was further increased by introducing a sulfonated quencher and shortening the spacer connecting the electrophile and quencher, resulting in greater water solubility, improved in vivo properties, and enhanced contrast in noninvasive optical imaging of cancer.

[0142] method General 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 performed 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). Reverse-phase HPLC was performed on an ÅKTA Explorer (Amersham Pharmacia Biotech) using a C18 column. 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 are given in ppm (δ) relative to tetramethylsilane as the internal standard. Coupling constants are given in Hz. Fluorescent gels were scanned using a Typhoon 9400 flatbed laser scanner (GE Healthcare). In-gel labeling intensity was quantified using Image J software. Statistical analysis was performed using Microsoft Excel, and sem was calculated by dividing sd by the square root of n. Fluorescence microscopy images were acquired on a Zeiss confocal LSM710 and Zeiss Axiovert 200M inverted microscope (Carl Zeiss) equipped with 10x, 40x, and 63x objectives. Slidebook software was used to control the microscope and camera and for data analysis (Intelligent Imaging Innovations).

[0143] qABP synthesis The synthetic scheme for the synthesis of the following compounds is depicted below in Scheme 1.

[0144] 2,6-Dimethyl-4-((6-(tritylamino)hexyl)carbamoyl)benzoate (11a). Mono-trityl 1,6-diaminohexaneacetate (9a) (117.2 mg, 0.28 mmol) was taken up in DCM, washed with saturated aqueous NaHCO, dried over NaSO, and concentrated in vacuo. The amine was dissolved in DMF, and HOBt monohydrate (43 mg, 0.28 mmol, 1 equiv.), EDC (54 mg, 0.28 mmol, 1 equiv.), and 2,6-dimethylterephthalic 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 was purified by flash column chromatography (DCM -> 5% MeOH in DCM) and then taken up in DCM, washed with water and dried over MgSO4 to give 70 mg (0.13 mmol, 47% isolated yield).

[0145] 2,6-Dimethyl-4-((2-(triamino)ethyl)carbamoyl)benzoate (11b). Mono-tritylethylenediamine acetate (9b) (97.9 mg, 0.27 mmol) was taken up in DCM, washed with saturated aqueous NaHCO, dried over NaSO, and concentrated in vacuo. The amine was dissolved in DMF, HOBt monohydrate (43 mg, 0.28 mmol, 1.04 equiv.), EDC (61 mg, 0.32 mmol, 1.2 equiv.), and 2,6-dimethylterephthalic acid (10) (52 mg, 0.27 mmol, 1 equiv.) were added, and the reaction mixture was stirred overnight and then concentrated in vacuo. The crude material was purified by flash column chromatography (DCM → 5% MeOH in DCM), subsequently taken up in DCM, washed with water, and dried over MgSO to give 28 mg (0.06 mmol, 22% isolated yield).

[0146] 2,3,5,6-Tetrafluoro-4-hydroxy-N-(6-(tritylamino)hexyl)benzamide (13a). Mono-trityl 1,6-diaminohexane acetate (9a) (117.2 mg, 0.28 mmol) was taken up in DCM, washed with saturated aqueous NaHCO, dried over NaSO, and concentrated in vacuo. The amine was dissolved in DMF, and HOBt monohydrate (43 mg, 0.28 mmol, 1 equiv.), 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 material was purified by flash column chromatography (15% → 30% ethyl acetate in hexanes) to give 90 mg (0.16 mmol, 58% isolated yield).

[0147] 2,3,5,6-Tetrafluoro-4-hydroxy-N-(2-(tritylamino)ethyl)benzamide (13b). Mono-tritylethylenediamine acetate (9b) (100 mg, 0.28 mmol) was taken up in DCM, washed with saturated aqueous NaHCO, dried over NaSO, and concentrated in vacuo. The amine was dissolved in DMF, and HOBt monohydrate (43 mg, 0.28 mmol, 1 equiv.), 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 material was purified by flash column chromatography (20% → 35% ethyl acetate in hexanes) to give 90 mg (0.18 mmol, 65% isolated yield). 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). [ka] 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.

[0148] Intermediate 15. Potassium fluoride (3 mg, 52 μmol, 3 equiv.) was sonicated for 5 min. The crude was suspended in DMF by filtration, followed by the addition of carboxylic acid 11a (10 mg, 19 μmol, 1.1 equiv). The reaction mixture was stirred for 10 min before the addition of chloromethyl ketone 14 (9.7 mg, 17.3 μmol, 1 equiv). After 2 h, the reaction mixture was concentrated in vacuo, and the crude material was taken up in 1% TFA in DCM and stirred for 30 min before being quenched by the addition of triisopropylsilane until the solution was colorless. After azeotroping with toluene (3×), the title compound was purified by HPLC (preparative reverse-phase C). 18 Purification by column, CHCN / H0 0.1% TFA, 15:85 to 55:45 over 20 min, 5 mL / min), followed by lyophilization gave 15 as a white powder (3.12 mg, 3.46 μmol, 20% over two steps).

[0149] Intermediate 16. Potassium fluoride (3 mg, 52 μmol, 3 equiv.) was suspended in DMF with sonication for 5 min, after which carboxylic acid 11b (9.5 mg, 20 μmol, 1.1 equiv.) was added. The reaction mixture was stirred for 10 min before the addition of chloromethyl ketone 14 (10 mg, 17.9 μmol, 1 equiv.). After 1.5 h, the reaction mixture was concentrated in vacuo, and the crude material was taken up in 1% TFA in DCM, stirred for 30 min, and then quenched by the addition of triisopropylsilane until the solution turned colorless. After azeotroping with toluene (3×), intermediate 16 was purified by HPLC (preparative reverse-phase C 18Purification by column, CH3CN / H2O 0.1% TFA, 15:85 to 55:45 over 20 min, 5 mL / min), followed by lyophilization gave a white powder (3.99 mg, 4.57 μmol, 26% over two steps). 1 H NMR (500 MHz, CD3OD) 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).

[0150] Intermediate 17. Potassium fluoride (6.3 mg, 108 μmol, 3 equiv.) was suspended in DMF with sonication for 5 min, after which phenol 13a (21.5 mg, 39 μmol, 1.1 equiv.) was added. The reaction mixture was stirred for 10 min before the addition of chloromethyl ketone 14 (20 mg, 36 μmol, 1 equiv.). The reaction mixture was stirred at 80° C. for 5 h and concentrated in vacuo. The crude material was taken up in 1% TFA in DCM, stirred for 30 min, and then quenched by the addition of triisopropylsilane until the solution turned colorless. After azeotroping with toluene (3×), the crude was purified by HPLC (preparative reverse-phase C). 18Purification by column, CH3CN / H2O 0.1% TFA, 25:75 to 70:30 over 20 min, 5 mL / min), followed by lyophilization gave the title compound as a white powder (16.6 mg, 17.5 μmol, 49% over two steps). 1 H NMR (500 MHz, CD3OD) 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).

[0151] Intermediate 18. Potassium fluoride (6.3 mg, 108 μmol, 3 equiv.) was added to the The crude was suspended in DMF by sonication, after which phenol 13b (19.4 mg, 39 μmol, 1.1 equiv.) was added. The reaction mixture was stirred for 10 min before the addition of chloromethyl ketone 14 (20 mg, 36 μmol, 1 equiv.). The reaction mixture was stirred at 80° C. for 3 h and concentrated in vacuo. The crude was taken up in 1% TFA in DCM, stirred for 30 min, and then quenched by the addition of triisopropylsilane until the solution turned colorless. After azeotroping with toluene (3×), the crude was purified by HPLC (preparative reverse-phase C). 18 Purification by column, CH3CN / H2O 0.1% TFA, 20:80 to 60:40 over 20 min, 5 mL / min), followed by lyophilization gave the title compound as a white powder (15.4 mg, 17.3 μmol, 48% over two steps). 1H NMR (400 MHz, CD3OD) 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).

[0152] Probe 1 (GB137). Intermediate 15 (1.5 mg, 1.7 μmol) was taken up 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 Purification was performed by column chromatography using CH3CN / HO 0.1% TFA (40:60 to 80:20, 5 mL / min, over 20 min), followed by lyophilization. To remove the Boc protecting group, the resulting dark blue powder was taken up in TFA / DCM (1 / 1) and allowed to react for 30 min before azeotroping with toluene (3x), yielding 2.42 mg of the corresponding TFA salt (1.6 μmol, 95% 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, the product was purified by HPLC (preparative reverse-phase C). 18 Column purification (CHCN / H2O 0.1% TFA, 40:60 to 75:25, 5 mL / min over 20 min) followed by lyophilization afforded probe 1 as a dark blue powder (2.0 mg, 0.99 μmol, 62%).

[0153] Probe 2 (BMV122). Intermediate 15 (1.5 mg, 1.7 μmol) was taken up 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 Purification by column chromatography (CHCN / HO 0.1% TFA, 30:70 to 70:30, 5 mL / min, over 20 min) was followed by lyophilization. To remove the Boc protecting group, the resulting dark blue powder was taken up in TFA / DCM (1 / 1) and allowed to react for 30 min before azeotroping with toluene (3x), yielding 2.29 mg of the corresponding TFA salt (1.39 μmol, 81% 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, the product was purified by HPLC (preparative reverse-phase C 18 Purification by column (CHCN / H0 0.1% TFA, 15:85 to 50:50, 5 mL / min over 20 min) followed by lyophilization gave probe 2 as a dark blue powder (1.83 mg, 0.84 μmol, 61%).

[0154] Probe 3 (BMV145). Intermediate 16 (1.5 mg, 1.7 μmol) was taken up 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, CH3CN / H2O 0.1% TFA, 40:60 to 80:20, 5 mL / min over 20 min, followed by purification Lyophilization was performed. To remove the Boc protecting group, the resulting dark blue powder was taken up in TFA / DCM (1 / 1) and allowed to react for 30 min before azeotroping with toluene (3x) to give 0.86 mg of the corresponding TFA salt (0.6 μmol, 35% isolated yield 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, the product was analyzed by HPLC (preparative reverse-phase C 18 Purification by column (CHCN / H0 0.1% TFA, 40:60 to 75:25, 5 mL / min over 20 min) followed by lyophilization afforded probe 3 as a dark blue powder (0.67 mg, 0.34 μmol, 57%).

[0155] Probe 4 (BMV146). Intermediate 16 (1.0 mg, 1.2 μmol) was taken up 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, the sulfo-QSY21 amide was purified by HPLC (preparative reverse-phase C 18 Purification by column chromatography (CHCN / HO 0.1% TFA, 20:80 to 80:20, 5 mL / min) followed by lyophilization. To remove the Boc protecting group, the resulting dark blue powder was taken up in TFA / DCM (1 / 1) and allowed to react for 30 min before azeotroping with toluene (3x), yielding 1.06 mg of the corresponding TFA salt (0.66 μmol, 55% 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, the product was purified by HPLC (preparative reverse-phase C 18 Column purification (CHCN / H2O 0.1% TFA, 15:85 to 50:50, 5 mL / min over 20 min) followed by lyophilization afforded probe 4 as a dark blue powder (0.63 mg, 0.3 μmol, 45%).

[0156] Probe 5 (BMV118). Intermediate 17 (1.2 mg, 1.3 μmol) was taken up 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, the QSY21 amide was purified by HPLC (preparative reverse-phase C 18 Purification by column chromatography (CHCN / HO 0.1% TFA, 40:60 to 80:20, 5 mL / min, over 20 min) was followed by lyophilization. To remove the Boc protecting group, the resulting dark blue powder was taken up in TFA / DCM (1 / 1) and allowed to react for 30 min before azeotroping with toluene (3x) to give 2.0 mg of the corresponding TFA salt (1.3 μmol, quantitative over 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, the product was purified by HPLC (preparative reverse-phase C 18 Column purification (CHCN / H2O 0.1% TFA, 40:60 to 85:15 over 20 min, 5 mL / min) followed by lyophilization afforded probe 5 as a dark blue powder (1.91 mg, 0.94 μmol, 72%).

[0157] Probe 6 (BMV119). Intermediate 17 (1.2 mg, 1.3 μmol) was taken up 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 reverse-phase C 18 Purification by column chromatography (CHCN / HO 0.1% TFA, 30:70 to 90:10, 5 mL / min, over 20 min) was followed by lyophilization. To remove the Boc protecting group, the resulting dark blue powder was taken up in TFA / DCM (1 / 1) and allowed to react for 30 min before azeotroping with toluene (3x), yielding 1.98 mg of the corresponding TFA salt (0.9 μmol, 70% 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, the product was purified by HPLC (preparative reverse-phase C18 Column purification by CH3CN / H2O 0.1% TFA, 15:85 to 50:50, 5 mL / min over 20 min, followed by freezing Drying gave probe 6 as a dark blue powder (1.63 mg, 0.74 μmol, 82%).

[0158] Probe 7 (BMV108). Intermediate 18 (1.2 mg, 1.3 μmol) was taken up 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 reverse-phase C 18 Purification by column chromatography (CHCN / HO 0.1% TFA, 30:70 to 70:30, 5 mL / min) followed by lyophilization gave a dark blue powder (1.43 mg, 0.99 μmol, 76%). The Boc protecting group was then removed in TFA / DCM (1 / 1) for 30 min before azeotroping with toluene (3x). 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, the product was purified by HPLC (preparative reverse-phase C 18 Column purification (CHCN / H2O 0.1% TFA, 30:70 to 70:30 over 20 min, 5 mL / min) followed by lyophilization afforded probe 7 as a dark blue powder (0.95 mg, 0.48 μmol, 49% over two steps).

[0159] Probe 8 (BMV109). Intermediate 18 (5.8 mg, 6.5 μmol) was dissolved in DMSO (100 μL). 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 and the mixture was stirred overnight. Sulfo-QSY21 amide was purified by HPLC (preparative reverse-phase C). 18Purification by column chromatography (CHCN / HO 0.1% TFA, 25:75 to 55:45, 5 mL / min, over 20 min) followed by lyophilization gave a dark blue powder. The Boc protecting group was subsequently removed in TFA / DCM (1 / 1) for 30 min before azeotroping 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 h, the product was purified by HPLC (preparative reverse-phase C). 18 Column purification (CHCN / H2O 0.1% TFA, 25:75 to 45:55 over 20 min, 5 mL / min) followed by lyophilization afforded probe 8 as a dark blue powder (7.74 mg, 4.61 μmol, 71% over three steps). 1 H NMR (600 MHz, CD3CN) δ 8.12 - 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).

[0160] Cell culture and labeling of cell lysates RAW cells were cultured in DMEM (GIBCO) 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) supplemented with 10% fetal bovine serum (FBS; GIBCO), 100 units / mL penicillin, and 100 μg / mL streptomycin (GIBCO). All cells were cultured at 37°C in a 5% CO2 humidified incubator. For intact cell labeling, cells were exposed to probes (500x in DMSO) in culture medium and incubated at 37°C for 2 hours unless otherwise noted. Where indicated, cells were preincubated with the inhibitor JPM-OEt (500x in DMSO) for 1 hour or exposed to mouse serum (1 µl probe stock solution in DMSO added to 9 µl serum) for 4 hours before addition to cells. After labeling, cells were washed with PBS, resuspended in hypotonic lysis buffer (50 mM PIPES pH 7.4, 10 mM KCl, 5 mM MgCl2, 2 mM EDTA, 4 mM DTT, and 1% NP-40), placed on ice for 15 minutes, and centrifuged at 4°C for 30 minutes. The supernatant was collected, and the protein concentration was determined using a BCA kit (Pierce). 40 µg of total protein was denatured by adding 4x SDS-sample buffer and heating at 100°C for 3 minutes, resolved by SDS-PAGE (15%), and the labeled protease was visualized by scanning the gel with a Typhoon imager (GE Healthcare). Labeling intensity was quantified using Image J software. For cathepsin labeling in cell lysates, cells were harvested, 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 centrifugation for 15 minutes on ice and 30 minutes at 4°C, the supernatant was collected and the protein concentration was determined using a BCA kit (Pierce). 40 μg of total protein was exposed to the indicated probe (200× in DMSO) for 1 hour at 37°C. 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 cultured at 1:10 in complete medium without phenol red. 5 Cells were seeded at a density of 1000x in 35 mm glass-bottom dishes (In Vitro Scientific) and cultured overnight. Cells were exposed to either DMSO or 1 μM probe (500x in DMSO) for 2 hours. For the last hour, Lysotracker-Green (200 nM final concentration, 1000x in DMSO) was added to the cells. Where indicated, cells were preincubated with the inhibitor JPM-OEt (500x in DMSO) for 1 hour. Cells were imaged at 40x in the Cy5 and FITC channels using a Zeiss Axiovert 200M confocal microscope.

[0161] Animal models All animal handling and experiments were performed in accordance with current National Institutes of Health and Stanford University Institutional Animal Care and Use Committee guidelines. Female BALB / c mice (6–8 weeks, Jackson Laboratory) were anesthetized with isoflurane and given 1:10 PBS. 5 Fat pad numbers 2 and 7 were injected with 4T1 cells (ATCC) and tumor growth was monitored. 24 hours before imaging, hair in the target area was removed using "Nair lotion." On day 10, the indicated probes (20 nmol, 0.8 nmol g -1 ) was administered via the tail vein in a volume of 100 μL (20% DMSO in PBS). After injection, mice were non-invasively imaged at the indicated time points using the IVIS100 system (Xenogen). The results were analyzed using Living Image software (PerkinElmer). After the end point, mice were anesthetized with isofluorane and killed by cervical dislocation. For ex vivo fluorescence measurements and assessment of in vivo probe labeling profiles, tumors were removed and imaged using an FMT2500 (PerkinElmer). The tissues were sonicated (on ice for 1 minute) in citrate buffer (50 mM citrate buffer pH 5.5, 5 mM DDT, 0.5% CHAPS, 0.1% TritonX). After centrifugation at 4°C for 30 minutes, the supernatant was collected, and the protein concentration was determined using a BCA kit (Pierce). 40 μg of total protein was denatured in SDS-sample buffer at 100°C for 3 minutes and analyzed as described above. For immunofluorescence, excised tumors were incubated in 4% PFA solution in PBS for 6 hours at 4°C, followed by overnight incubation in 30% sucrose solution, and the tissues were frozen in OCT medium. 6-μm sections were fixed with acetone, blocked with PNB blocking buffer, and incubated overnight with rat anti-mouse CD68 (1:1000, Serotec). AlexaFluor-488-conjugated goat anti-rat (1:500; Invitrogen) was incubated for 1 hour at room temperature. Sections were then stained with DAPI (2 μg / mL; Invitrogen) for 5 minutes and then mounted in ProLong Gold Mounting Medium (Invitrogen). Tissues were then visualized using a Zeiss Axiovert 200M microscope.

[0162] All patents, patent publications, and other published references mentioned in this specification are hereby incorporated by reference in their entirety, just as if each was individually and specifically indicated to be incorporated by reference herein.

[0163] While specific examples have been provided, the foregoing description is illustrative and not limiting. Any one or more features of the above-described embodiments may be combined in any manner with one or more features of any other embodiment of the present invention. Moreover, many variations of the present invention will become apparent to those skilled in the art upon review of this specification. The scope of the present invention should therefore be determined by reference to the appended claims, along with their full scope of equivalents.

Claims

[Claim 1] Fluorescent activity.

Images