RU2815018C2 - Sulfoxonium ylide derivatives as probes for cysteine protease - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to a compound of formula I or a salt thereof, where R₁ is selected from the group consisting of (C₁-C₈) alkyl, (C₁-C₈) hydroxyalkyl, (C₁-C₈) haloalkyl, (C₃-C₈) cycloalkyl, (C₂-C₈) alkenyl and (C₂-C₈) alkynyl; R₂ is selected from the group consisting of (C₁-C₈) alkyl, (C₁-C₈) hydroxyalkyl, (C₁-C₈) haloalkyl, (C₃-C₈) cycloalkyl, (C₂-C₈) alkenyl and (C₂-C₈) alkynyl; R₃ represents a side chain of an alpha amino acid selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, methionine, ethionine, phenylalanine, tyrosine, levodopa, tryptophan, cysteine, homocysteine, selenocysteine, selenicomocysteine, selenomethyonin, selenesetionine, lysine, hyistidine, arginine, ornithin, asparagic acid, glutamic acid, serin, homeserin, o-methylgom squeezer, o-ejolgomoserin, treonin, asparagin and glutamine, where the indicated side chain is not necessary for amino protection group and is optionally further substituted with one or more identical or different R_3x substituents, where R_3x is selected from the group consisting of hydroxy, halogen, (C₁-C₄) alkyl, (C₁-C₄) hydroxyalkyl, (C₁-C₄) haloalkyl, (C₁-C₄) alkoxy and (C₁-C₄) haloalkoxy; R₄ is selected from the group consisting of a hydrogen atom and (C₁-C₄) alkyl; R₅ represents a detectable element that is a fluorescent label that is a cyanine label; X represents: (i) connection; or (ii) a biradical fragment of formula II which is linked to the substituent R₅ through an amino group, where R₆ represents a side chain of an alpha amino acid selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, methionine, ethionine, phenylalanine, tyrosine, levodopa, tryptophan, cysteine, homocysteine, selenocysteine, selenogomocysteine, selenomethionine, selenoethionine, lysine, histidine, arginine, ornithine, aspartic acid, glutamic acid, serine, homoserine, O- methylhomoserine, O-ethylhomoserine, threonine, asparagine and glutamine, wherein the said side chain is optionally substituted with an amino protecting group and optionally further substituted with one or more identical or different R_6x substituents, wherein R_6x is selected from the group consisting of hydroxy, halogen, (C₁-C₄) alkyl, (C₁-C₄) hydroxyalkyl, (C₁-C₄) haloalkyl, (C₁-C₄) alkoxy and (C₁-C₄) haloalkoxy; R₇ is selected from the group consisting of a hydrogen atom and (C₁-C₄) alkyl; and wherein the amino protecting group is selected from the group consisting of benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), p-toluenesulfonyl (Tos), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf), mesityl-2-sulfonyl (Mts), 4-methoxy-2,3,6-trimethylphenylsulfonyl (Mtr), acetamido, and phthalimido. The invention also relates to compositions acting as probes based on cathepsin X inhibitors containing a compound of formula I, as well as methods of detecting the activity of cathepsin X.

EFFECT: new compounds that can be used in medicine as activity-based probes for cysteine proteases such as cathepsin X, as well as methods of detecting cysteine protease activity and related diagnostic or therapeutic methods.

46 cl, 25 dwg, 1 tbl, 14 ex

Description

Field of technology

[0001] The present invention relates to compounds that can be used as activity-based probes and/or inhibitors of cysteine proteases such as cathepsin X, methods for detecting cysteine protease activity, and related diagnostic and therapeutic methods and applications.

State of the art

[0002] According to Lechtenberg et al., ACS Chem. Biol. 2015, 10, 945-951, “proteases are central mediators of numerous physiological processes.” Proteolytic cleavage events underlie protein degradation, enzyme activation and protein maturation and regulate a wide range of pathways from cell death, cell migration and proliferation, inflammation and immune response, to blood coagulation (Rawlings ND and Salvesen G. (2012) Handbook of Proteolytic Enzymes, Third edition, Academic Press, Waltham, MA). On the other hand, aberrant proteolysis is often associated with serious disorders. In addition, proteases are typically expressed in the cell or secreted as inactive zymogens that require activation through processes such as proteolytic cleavage or dimerization. Protease activation underlies tight temporal and spatial regulation, and thus protease location is generally not an ideal marker of protease function. In contrast, the spatiotemporal location of the active form of a given protease is essential to understanding its function. To this end, activity-based probes have been developed for various proteases (Deu E., Verdoes M. and Bogyo M. (2012), New approaches for dissecting protease functions to improve probe development and drug discovery. Nat. Struct. Mol. Biol. 19, 9−16). These probes are designed as active site protease inhibitors to specifically label the active protease and thus are effective research and diagnostic tools. In addition, these probes further pave the way for the development of potent inhibitors of selected proteases for potential therapeutic applications (Deu et al. 2012, Nat. Struct. Mol. Biol. 19, 9-16).”

[0003] Among the target enzymes of interest are cysteine proteases, including cysteine cathepsins and, in particular, cathepsin X.

[0004] Cysteine cathepsins are a family of lysosomal proteases that are frequently activated in various human malignancies and are involved in various oncogenic processes such as angiogenesis, proliferation, apoptosis and invasion. The cysteine cathepsin family constitutes the largest family of cathepsins with 11 proteases in humans belonging to clan CA, family CI a: cathepsins B, C (also known as cathepsin J and dipeptidyl peptidase 1), F, H, K (also known as cathepsin 02), L, O, S, W, V (also known as cathepsin L2) and Z (also known as cathepsin X and cathepsin P). Cathepsins are emerging as important players in tumor progression, making them potential targets for the treatment of a wide range of human cancers.

[0005] Cathepsin X (also referred to as cathepsin Z/P) is a cathepsin cysteine protease that is unique among its family members in that it exhibits strong carboxypeptidase activity. Cathepsin X is believed to underlie many human diseases. Its expression has been associated with several types of cancer and neurodegenerative diseases, although its role in normal physiology is still poorly understood. Progress in our understanding of its function has been hampered by the lack of available tools that can specifically measure the proteolytic activity of cathepsin X.

[0006] Cathepsin X promotes adhesion and maturation of macrophages and dendritic cells (Obermajer et al., 2008) and suppresses clathrin-dependent phagocytosis through cleavage profiles (Pečar Fonović and Kos, 2015). Cathepsin X regulates hormone signaling, where its cleavage into bradykinin, kallidin or angiotensin leads to changes in specificity towards their cognate receptors and divergent signaling downstream (Nägler et al., 2010). Cathepsin X is also expressed by neurons, where its cleavage by α-enolase regulates neurite survival and outgrowth (Obermajer et al., 2009). In addition, cathepsin X expression is high in amyloid plaques, where it may exert protective effects against neurodegenerative disorders such as Alzheimer's disease (Hafner et al., 2013; Wendt et al., 2007), and in the spinal cord for neuropathic pain ( Leichsenring et al., 2008). Cathepsin X mRNA has been reported to be upregulated in normal regions of the MS brain (Huynh et al., 2014) and has been implicated in the generation of IL-1β (Allan et al., 2017; Orlowski et al., 2015) and in mediating neuroinflammation (Allan et al., 2017). It is also upregulated in the tumor microenvironment of breast (Edgington-Mitchell et al., 2015), pancreatic (Akkari et al., 2014), prostate (Nägler et al., 2004) and gastric (Bernhardt et al., 2010; Krueger et al., 2005), where it likely promotes tumor invasion. Thus, cathepsin X is a promising clinical biomarker and therapeutic target for various diseases.

[0007] Like most cathepsins, cathepsin X is synthesized as a zymogen, which is activated in the acidic environment of endolysosomes. Once activated, it can also be negatively regulated by endogenous inhibitors, although probably not by cystatin C or stefin A (Duivenvoorden et al., 2017; Nägler et al., 1999). In addition to its proteolytic functions, cathepsin X can also promote integrin-mediated signaling through the Arg-Gly-Asp (RGD) motif in its prodomain (Akkari et al., 2014). As a result of these complex post-translational regulatory mechanisms, traditional biochemical methods that measure total protein levels rarely reflect the pool of proteolytically active enzyme. Therefore, the ability to specifically measure its activity in natural environments is essential to accurately determine its proteolytic functions in health and disease.

[0008] To this end, efforts have focused on developing fluorescent activity-based probes (ABPs) for cathepsin X. Fluorescent ABPs are small molecules that contain an electrophilic group (active group), a recognition sequence that confers selectivity, and a fluorophore for detection (Edgington and Bogyo, 2013; Edgington et al., 2011; Sanman and Bogyo, 2014). In the active state, the protease initiates a nucleophilic attack on the active group, resulting in the formation of a covalent irreversible bond. Assessment of probe labeling can then be used to quantify protease activity using SDS-PAGE (fluorescence in gel), fluorescence microscopy, flow cytometry, or optical imaging of whole tissues or organisms. Importantly, the covalent nature of probe binding allows target confirmation through immunoprecipitation with specific antibodies or affinity purification followed by proteomic analysis.

[0009] Probes with absolute specificity for cathepsin X have not previously been reported. BMV109, a fluorescently quenched ABP with a tetrafluorophenoxymethyl ketone group, is a pan-cathepsin probe targeting cathepsin X, B, S, and L (Verdoes et al., 2013; Edgington-Mitchell et al., 2015). Because the size of cathepsin X is similar to cathepsin B, one of the most abundant and ubiquitously expressed cathepsins, it may be difficult to clearly separate the two proteases by SDS-PAGE. This prevents accurate quantification by in-gel fluorescence. MGP140 is an activity-based epoxy probe that exhibits greater specificity for cathepsin X than BMV109, but also reacts strongly with cathepsin B (Paulick and Bogyo, 2011). If mice are pretreated with GB11-NH2, an inhibitor of cathepsin B, S and L, before injection with MGP140, specific labeling of cathepsin X can be achieved. However, such manipulation of the system leads to hyperactivation of cathepsin X, possibly compensatory for the loss of cathepsin B activity. Thus Thus, it is critical to develop activity-based probes with increased specificity for cathepsin X so that its physiological activity can be assessed.

[0010] Shaw discloses the synthesis of peptidyl sulfonium salts and their inhibitory effect on cysteinyl proteases, papain and cathepsin B (Shaw, 1988).

[0011] Gordon et al., US Patent No. 5,223,486, disclose peptidyl diazomethyl ketones and peptidyl sulfonium salts as inhibitors of the tumor procoagulant cysteine protease.

[0012] Paulick et al. describe the synthesis and characterization of several fluorescent activity-based probes bearing either an acyloxymethyl ketone (AOMK) or an epoxide active group designed to target cathepsin X. The epoxide-based probes were found to label cathepsin X, whereas the AOMK probes were unable to label this protease (Paulick and Bogyo, 2011).

[0013] Thus, there remains a need for compounds that function as probes (or inhibitors) based on the activity of cysteine proteases, such as cysteine cathepsins and, more specifically, cathepsin X. Such compounds are useful for studying the role of the corresponding cysteine protease in normal physiology and for diseases. In addition, such compounds may be useful in the diagnosis and/or treatment of diseases associated with corresponding cysteine protease activity.

[0014] The inventors of the present invention attempted to search for new potential active groups for cysteine proteases and unexpectedly discovered that the compounds described below, bearing a sulfoxonium ylide moiety as the active group, can function as activity-based probes for cysteine proteases such as cysteine cathepsins , and more specifically cathepsin X, with increased potency and selectivity compared to previously described probes (such as BMV109 and MGP140).

Objectives and essence of the invention

[0015] An object of some embodiments of the present invention is to provide activity-based probe compounds that enable the detection of cysteine protease activity in biological samples, such as cell lysates or tissue lysates.

[0016] An object of some embodiments of the present invention is to provide activity-based probe compounds that enable the detection of cysteine protease activity in living cells.

[0017] An object of some embodiments of the present invention is to provide activity-based probe compounds that allow detection of cysteine protease activity in vivo.

[0018] The purpose of some embodiments of the present invention is to provide activity-based probe compounds that allow the detection of cathepsin X activity in biological samples, such as cell lysates or tissue lysates.

[0019] The purpose of some embodiments of the present invention is to provide activity-based probe compounds that allow the detection of cathepsin X activity in living cells.

[0020] An object of some embodiments of the present invention is to provide activity-based probe compounds that allow detection of cathepsin X activity in vivo.

[0021] The object of some embodiments of the present invention is to provide activity-based probes that detect cysteine protease activity with increased selectivity compared to the activity-based probes BMV109 and MGP140.

[0022] The object of some embodiments of the present invention is to provide activity-based probe compounds that detect cathepsin X activity with increased selectivity compared to the activity-based probes BMV109 and MGP140.

[0023] It is an object of some embodiments of the present invention to provide activity-based probe compounds that detect cathepsin X activity with high efficiency for cathepsin X, such as increased efficiency for cathepsin X compared to the activity-based probes, BMV109 and MGP140.

[0024] An object of some embodiments of the present invention is to provide activity-based probe compounds that allow detection of cathepsin X activity (e.g., in cell lysates, tissue lysates, living cells, and in vivo) and that do not exhibit cross-reactivity with cathepsin B and /or cathepsin L.

[0025] The purpose of some embodiments of the present invention is to provide cysteine protease inhibitors.

[0026] It is an object of some embodiments of the present invention to provide cathepsin X inhibitors.

[0027] It is an object of some embodiments of the present invention to provide cathepsin X inhibitors that do not cross-react with cathepsin B and/or cathepsin L.

[0028] The object of some embodiments of the present invention is to provide compounds that can be used to diagnose a disease associated with cysteine protease activity.

[0029] An object of some embodiments of the present invention is to provide compounds that can be used to diagnose a disease associated with cathepsin X activity.

[0030] The purpose of some embodiments of the present invention is to provide compounds that can be used for the diagnosis of a disease selected from the group consisting of cancer, inflammatory diseases and neurodegenerative diseases.

[0031] An object of some embodiments of the present invention is to provide compounds that can be used to treat a disease associated with cysteine protease activity.

[0032] An object of some embodiments of the present invention is to provide compounds that can be used to treat a disease associated with cathepsin X activity.

[0033] The object of some embodiments of the present invention is to provide compounds that can be used to treat a disease selected from the group consisting of cancer, inflammatory diseases and neurodegenerative diseases.

[0034] It should be understood that the above objects also apply to the corresponding methods, as well as compounds/compositions for use in the corresponding method.

[0035] In some embodiments, the present invention provides a compound of Formula I:

[0036]

[0037] or salts thereof,

Where:

R ₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl;

R ₂ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl;

R ₃ represents an alpha amino acid side chain;

R ₄ is selected from the group consisting of a hydrogen atom and (C ₁ -C ₄ ) alkyl;

R ₅ is selected from the group consisting of a detectable element, an amino protecting group, (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl, (C ₂ -C ₈ ) alkynyl, (C ₁ -C ₈ ) alkylcarbonyl, (C ₁ -C ₈ ) hydroxyalkylcarbonyl, (C ₁ -C ₈ ) haloalkylcarbonyl, (C ₃ -C ₈ ) cycloalkylcarbonyl, (C ₁ -C ₈ ) alkyloxycarbonyl, benzyloxycarbonyl and a hydrogen atom;

X represents:

(i) communication; or

(ii) a biradical fragment of formula II or III, which is linked to the R ₅ substituent via an amino group,

[0038]

Where:

R ₆ represents an alpha amino acid side chain;

R ₇ is selected from the group consisting of a hydrogen atom and (C ₁ -C ₄ ) alkyl;

R ₈ represents an alpha amino acid side chain;

R ₉ is selected from the group consisting of a hydrogen atom and (C ₁ -C ₄ ) alkyl; And

n is 1, 2, 3 or 4.

[0039] In some embodiments, the present invention provides a compound (Formula I) having a formula selected from the following group of formulas:

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] or salts thereof.

[0048] In some embodiments, the present invention provides a compound (Formula I) having the following formula:

[0049]

[0050] or salts thereof.

[0051] In some embodiments, the present invention provides a composition comprising a compound of Formula I as described herein, or a salt thereof, and an excipient.

[0052] In some embodiments, the present invention provides a composition comprising a compound of Formula I as described herein, or a salt thereof, and an excipient, wherein the compound contains at least one detectable element.

[0053] In some embodiments, the present invention provides a method for detecting cysteine protease activity, comprising:

(1) contacting the cysteine protease with an activity-based probe compound containing a sulfoxonium ylide moiety as an active group, and

(2) subsequent cysteine protease analysis, including measurement of the detected signal.

[0054] In some embodiments, the present invention provides a method for detecting cysteine protease activity in vitro, comprising:

(1) contacting the biological sample with the compound with an activity-based probe containing a sulfoxonium ylide moiety as an active group, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

[0055] In some embodiments, the present invention provides a method for detecting cysteine protease activity in a biological sample obtained from a subject, comprising:

(1) contacting the biological sample in vitro with an activity-based probe compound containing a sulfoxonium ylide moiety as an active group, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

[0056] In some embodiments, the present invention provides a method for detecting cysteine protease activity, comprising:

(1) contacting the cysteine protease with a compound of formula I as described herein, or a salt thereof, or a composition as described herein, and

(2) subsequent cysteine protease analysis, including measurement of the detected signal.

[0057] In some embodiments, the present invention provides a method for detecting cysteine protease activity in vitro, comprising:

(1) contacting the biological sample with a compound of Formula I as described herein, or a salt thereof, or a composition as described herein, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

[0058] In some embodiments, the present invention provides a method for detecting cysteine protease activity in a biological sample obtained from a subject, comprising:

(1) contacting the biological sample in vitro with a compound of Formula I as described herein, or a salt thereof, or a composition as described herein, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

[0059] In some embodiments, the present invention provides a method for detecting cysteine protease activity, comprising:

(1) administering to the subject an activity-based probe compound containing a sulfoxonium ylide moiety as the active group,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

[0060] In some embodiments, the present invention provides a method for detecting cysteine protease activity, comprising:

(1) administering to the subject a compound of Formula I as described herein, or a salt or composition thereof, as described herein,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

[0061] In some embodiments, the present invention provides a method for in vivo detection of cysteine protease activity in a subject, comprising:

(1) administering to the subject an activity-based probe compound containing a sulfoxonium ylide moiety as an active group, and

(2) follow-up examination of the subject, including measurement of the detected signal.

[0062] In some embodiments, the present invention provides a method for in vivo detection of cysteine protease activity in a subject, comprising:

(1) administering to the subject a compound of formula I, as described herein, or a salt or composition thereof, as described herein, and

(2) subsequent examination of the subject, including measurement of the detected signal.

[0063] In some embodiments, the present invention provides a method of inhibiting a cysteine protease, comprising contacting the cysteine protease with a compound of Formula I as described herein, or a salt thereof, or a composition as described herein.

[0064] In some embodiments, the present invention provides a method of inhibiting a cysteine protease in vitro, comprising contacting a biological sample with a compound of Formula I as described herein, or a salt thereof, or a composition as described herein.

[0065] In some embodiments, the present invention provides a method of inhibiting a cysteine protease in a biological sample obtained from a subject, comprising contacting the biological sample in vitro with a compound of Formula I as described herein, or a salt thereof, or a composition as described herein.

[0066] In some embodiments, the present invention provides a method for in vivo inhibition of a cysteine protease in a subject, comprising administering to the subject a compound of Formula I as described herein, or a salt or composition thereof, as described herein.

[0067] In some embodiments, the present invention provides a method for diagnosing a disease associated with cysteine protease activity in a subject, comprising:

(1) contacting a biological sample obtained from a subject in vitro with a compound of Formula I as described herein, or a salt thereof, or a composition as described herein, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

[0068] In some embodiments, the present invention provides a method for diagnosing a disease associated with cysteine protease activity in a subject, comprising:

(1) administering to the subject a compound of Formula I as described herein, or a salt or composition thereof, as described herein,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

[0069] In some embodiments, the present invention provides a method for in vivo diagnosis of a disease associated with cysteine protease activity in a subject, comprising:

(1) administering to the subject a compound of formula I, as described herein, or a salt or composition thereof, as described herein, and

(2) follow-up examination of the subject, including measurement of the detected signal.

[0070] In some embodiments, the present invention provides a compound of Formula I as described herein, or a salt thereof, for use in a method for diagnosing a disease associated with cysteine protease activity in a subject, wherein the method includes:

(1) administering to the subject a compound of formula I as described herein, or a salt thereof,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

[0071] In some embodiments, the present invention provides a composition as described herein for use in a method for diagnosing a disease associated with cysteine protease activity in a subject, wherein the method includes:

(1) administering to the subject the composition as described herein,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

[0072] In some embodiments, the present invention provides a compound of Formula I as described herein, or a salt thereof, for use in a method for diagnosing a disease associated with cysteine protease activity in a subject in vivo, wherein the method includes:

(1) administering to the subject a compound of Formula I as described herein, or a salt thereof, and

(2) follow-up examination of the subject, including measurement of the detected signal.

[0073] In some embodiments, the present invention provides a composition as described herein for use in a method for diagnosing in vivo a disease associated with cysteine protease activity in a subject, wherein the method includes:

(1) administering to the subject the composition as described herein, and

(2) follow-up examination of the subject, including measurement of the detected signal.

[0074] In some embodiments, the present invention provides a method of treating a disease associated with cysteine protease activity, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I as described herein, or a salt thereof, or a therapeutically effective amount of a composition, as described here.

[0075] In some embodiments, the present invention provides a compound of Formula I as described herein, or a salt thereof, for use in the treatment of a disease associated with cysteine protease activity.

[0076] In some embodiments, the present invention provides a composition for use in treating a disease associated with cysteine protease activity, comprising a compound of Formula I as described herein, or a salt thereof, and a pharmaceutically acceptable excipient.

[0077] In some embodiments, the present invention relates to the use of a compound of Formula I as described herein, or a salt thereof, in the manufacture of a medicament for the treatment of a disease associated with cysteine protease activity.

[0078] In some embodiments, the present invention relates to the use of a composition described herein in the manufacture of a medicament for the treatment of a disease associated with cysteine protease activity.

[0079] In some embodiments, the present invention provides a method for preparing a compound bearing a chloromethyl ketone group, comprising reacting a compound bearing a sulfoxonium ylide moiety to produce a compound bearing a chloromethyl ketone group.

[0080] In some embodiments, the present invention provides a method for preparing an activity-based probe compound bearing an acyloxymethyl ketone group as the active group, comprising:

(i) preparing a chloromethyl ketone group-bearing intermediate by reacting a compound containing a sulfoxonium ylide moiety to produce a chloromethyl ketone group-bearing compound; And

(ii) further processing the compound containing the chloromethylketone moiety to obtain said probe compound based on activity.

Brief description of the figures

[0081] In FIG. Figure 1A shows the structure of the sCy5-Val-SY probe.

In fig. 1B shows the proposed binding mechanism of the sCy5-Val-SY probe to a cysteine protease.

In fig. Figure 2A shows the results of Example 3 (labeling of RAW264.7 lysates with sCy5-Val-SY (at 1 μM for 20 min) or BMV109 (at 1 μM for 20 min) alone or after pretreatment with 10 μM MDV-590 (cathepsin S inhibitor ) or JPM-OEt (cysteine cathepsin pan-inhibitor).

In fig. 2A-1 shows the results of Example 3 (labeling of RAW264.7 lysates with the indicated concentrations of sCy5-Val-SY for 20 min according to in-gel fluorescence analysis).

In fig. 2A-2 shows the results of Example 3 (labeling of RAW264.7 lysates with 1 μM sCy5-Val-SY for the indicated times, as determined by in-gel fluorescence analysis).

In fig. 2B shows the results of Example 3 (immunoprecipitation of samples labeled with sCy5-Val-SY (labeling at 1 μM for 20 min) with a cathepsin X-specific antibody).

In fig. 2C shows the results of Example 4 (labeling of spleen lysates from wild-type or cathepsin X-deficient mice with sCy5-Val-SY or BMV109).

In fig. 2D shows the results of Example 5 (labeling living RAW264.7 cells with increasing doses of sCy5-Val-SY or BMV109 for 2 hours).

In fig. 2D-1 shows the results of Example 5 (labeling of live (intact) RAW264.7 cells with 1 μM sCy5-Val-SY for the indicated time, analyzed using in-gel fluorescence).

In fig. 2E shows the results of Example 5 (immunoprecipitation of samples labeled with sCy5-Val-SY (labeling live cells at 1 μM for 2 hours) with a cathepsin X-specific antibody).

In fig. 2F shows the results of Example 5 (labeling of live RAW264.7 cells with and without overnight pretreatment with 10 μM MDV-590, sCy5-Val-SY, or BMV109 (1 μM, 2 h).

In fig. 3A shows the results of example 6 (labeling of RAW264.7 lysates with the indicated probe based on sulfoxonium ylide or BMV109 (the probe was added in increasing concentrations of 0.01, 0.05, 0.1, 0.5, 1.5 μM, as indicated by the arrow with increasing background)).

In fig. 3B shows the results of Example 7 (labeling of kidney lysates with the indicated probe based on sulfoxonium ylide or BMV109 (the probe was added in increasing concentrations of 0.1, 0.5, 1.5 μM, as indicated by the arrow with increasing background)).

In fig. 3C shows the results of Example 8 (labeling of living RAW264.7 cells with the indicated sulfoxonium ylide probe or BMV109 (probe added at increasing concentrations of 0.1, 0.5, 1, 5 μM, as indicated by the arrow with increasing background)).

In fig. 3D shows the results of Example 9 (labeling of living MDA-MB-231HM cells with the indicated probe based on sulfoxonium ylide or BMV109 (the probe was added in increasing concentrations of 0.1, 0.5, 1, 5 μM, as indicated by the arrow with increasing background)).

In fig. 4A shows the results of Example 10 (SDS-PAGE and gel fluorescence of tissues from mice that were not treated with probe (NP), sCy5-Nle-SY, or BMV109. BMV109-labeled samples were excised from the same gel and presented at the same gain setting. as other samples in the corresponding tissue. Gains for each tissue were set individually to reflect the optimal contrast for labeling cathepsin X. An autofluorescent band was observed in the no-probe control.

In fig. 4B shows the results of Example 10 (immunoprecipitation of liver and kidney samples with cathepsin X-specific antibody).

In fig. Figure 5 shows the results of Example 10 (confocal microscopy of cathepsin X labeling in kidneys with sCy5-Nle-SY. Kidney sections from sCy5-Nle-SY-injected mice or no-probe controls were analyzed for sCy5 fluorescence or cathepsin X immunoreactivity.

In fig. 6A shows the results of Example 11 (labeling of live RAW264.7 cells with the indicated AOMK probes and sulfoxonium ylide (in increasing concentrations of 0.1, 0.5, 1 and 5 μM, as indicated by the arrow with increasing background), as analyzed by fluorescence in gel. In the top panel, the gain settings are the same for all samples. In the bottom panel, the gain settings were individually set to display the optimal contrast for the AOMK probes.

In fig. 6B shows the results of Example 12 (in vivo colon labeling with the sCy5-Nle-AOMK probe. The top band is the autofluorescent protein that appears in controls without probe).

In fig. Figure 7 shows the results of experiments to identify proteases labeled with the probe. (A, C-G): Immunoprecipitation of probe-labeled lysates (RAW264.7 cells or kidney, with the indicated probe introduced into live cells or lysates as indicated) with cathepsin-specific antibodies. (B) Labeling of live RAW264.7 cells with and without overnight pretreatment with cathepsin S inhibitors MDV590 (10 μM) or Z-FL (20 μM) with sCy5-Nle-SY (1 μM, 2 h).

In fig. 8A shows the results of Example 13 (labeling of lysates prepared from human oral squamous cell carcinoma tissue or patient-matched normal oral mucosa, sCy5-Nle-SY (incubation at 5 µM for 20 min) and in-gel fluorescence analysis, and also immunoblotting of samples).

In fig. 8B shows the results of Example 13 (immunoprecipitation of human oral carcinoma tissue lysates labeled with Cy5-Nle-SY with cathepsin X-specific antibody).

Figure 9 shows the results of Example 14 (labeling RAW264.7 cells that were pretreated with Boc-Val-SY (0, 10 or 100 μM) overnight with sCy5-Val-SY, and fluorescence analysis of lysed cells in gel).

Detailed Description of the Invention

[0082] When describing the present invention, the following terms should be used as defined below.

[0083] As used herein, the singular forms include plural references unless the context clearly indicates otherwise.

[0084] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art.

[0085] The term “activity-based probe” is intended to have the same meaning as commonly understood by those skilled in the art. Activity-based probes (ABPs) are small molecules that covalently bind to the active site of an enzyme (e.g., protease) or group of enzymes based on activity (i.e., the labeling reaction requires enzyme activity). ABPs typically include three elements: (i) an electrophilic group called the “active group,” (ii) a linker or recognition sequence, and (iii) a detection element or “reporter moiety” for detection. The enzyme attacks the electrophilic active group, resulting in the formation of a covalent adduct, which can then be detected either directly (for example, if the element to be detected is a fluorescent tag) or by two-step labeling (for example, post-labeling a modification of the ligation marker).

[0086] The term "detectable element" or "reporter group/fragment" refers to a functional group in a compound (activity-based probe) that can be detected using methods including, but not limited to, optical methods (e.g., fluorescence measurement or UV absorbance -radiation), radiography, biochemical methods (for example, using an immunochemical reagent such as an antibody), etc. The term "detectable element" includes functional groups that can be detected "directly" (eg, by measuring fluorescence after SDS-PAGE analysis), as well as functional groups that can be detected after performing a secondary labeling step and subsequent detection of the secondary label. An example of such groups is a biotin tag, which can be detected, for example, after secondary labeling with fluorescently labeled streptavidin and subsequent fluorescence measurement. Another example of such groups is a click chemical tag (bioorthogonal ligation marker), which can be detected, for example, after secondary labeling with a fluorescent tag using a click chemical (bioorthogonal) reaction and subsequent fluorescence measurement.

[0087] Thus, a “bioorthogonal ligation marker” is a functional group present in the compounds of the present invention at the initial stage of probe labeling (in vivo or ex vivo contact of the protease/biological sample/subject with the compounds of the present invention), which provides subsequent attachment of a secondary label (corresponding to the actually detected label) in a secondary labeling step using, for example, a click-chemical (bioorthogonal) reaction, which is carried out in vitro.

[0088] Click chemistry labels and corresponding click chemistry reactions for secondary labeling, i.e., attaching a label that is actually to be detected, are described, for example, in Martell et al., Applications of Copper-Catalyzed Click Chemistry in Activity -Based Protein Profiling, Molecules 2014, 19, 1378-1393, and in Willems et al., Bioorthogonal chemistry: Applications in activity-based protein profiling, Acc. Chem. Res. 2011, 44, 718-729.

[0089] The term “cysteine protease activity” refers to the proteolytic activity of a cysteine protease. In the case of cathepsin X, the term "cysteine protease activity" or "cathepsin X activity" refers to proteolytic activity, which is carboxypeptidase activity.

[0090] Cathepsin X, S, B and L each belong to the cysteine protease family. The term "cathepsin X" refers to a protein derived from the CTSZ gene locus. "Cathepsin X" is also called "cathepsin Z" or "cathepsin P". The term "cathepsin S" refers to a protein derived from the CTSS gene locus. The term "cathepsin B" refers to a protein derived from the CTSB gene locus. The term "cathepsin L" refers to a protein derived from the CTSL gene locus.

[0091] The term “patient” means a subject, particularly a person, who has developed a clinical manifestation of a particular symptom or symptoms indicating a need for treatment, is being treated prophylactically or prophylactically for a condition, or has been diagnosed with a condition to be treated.

[0092] The term “subject” is intended to include mammalian subjects, in particular human subjects, and includes the definition of the term “patient” and does not exclude subjects who are completely normal in all respects or with respect to a particular condition.

[0093] As used herein, the term “therapeutically effective” refers to the amount of drug or level of drug administration required to obtain the desired therapeutic result.

[0094] As used herein, terms like “administration” or “administer” include various routes of administration, such as intravenous, subcutaneous, intramuscular, oral, intranasal, sublingual, or topical.

[0095] The term “tissue sample” or “biopsy tissue material” refers to a sample of biological tissue obtained from a subject, such as a sample obtained by excision, needle aspiration, biopsy forceps, or smear. Tissue samples also include mucosal biopsies and stool samples. The tissue sampled may be living, dead, healthy or diseased and contain a heterogeneous mixture of cell types and extracellular factors

[0096] “Mucosal biopsy material” is typically obtained by sampling mucus that has accumulated on the surface of other tissue, such as mucous membranes or the epithelium of the intestinal tract. Mucosal biopsy material contains cells isolated from tissue on which mucus has accumulated.

[0097] The term "sputum sample" refers to a sample that is a mixture of saliva and mucus coughed up from the respiratory tract. The "sputum sample" can be obtained either invasively or non-invasively. Invasive methods include oropharyngeal or endotracheal suction while the patient is intubated, and the resulting contents are collected in a sputum trap. Non-invasive methods collect contents produced when a subject coughs, sometimes after spraying saline to liquefy the secretions.

[0098] The term "fecal sample" or "stool sample" refers to a sample taken from the stool of a subject. The stool samples contain cells isolated from the gastrointestinal tract and cell isolates from the subject's gastrointestinal tract.

[0099] The term "tissue sample lysate" refers to a solution obtained by lysing the cells of a tissue sample. The term "lysis" or "lysis" refers to the disintegration or rupture of cell membranes, resulting in the release of cell contents and/or subsequent cell death. Lysis can be accomplished, for example, by mechanical, enzymatic or osmotic disruption of cell membranes.

[0100] As used herein, the term “cysteine protease activity-associated disease” means a disease in which cysteine protease activity is involved in the pathogenesis of the disease. In a "cysteine protease-associated disease", the level of cysteine protease activity in a disease state or affected area of the body (eg, body part, organ, pathological tissue, including tumor tissue) deviates from the corresponding level of cysteine protease activity detected in a state without pathology or in a corresponding area of the body without pathology. In some embodiments, the level of cysteine protease activity in a disease state or diseased body region is elevated compared to the corresponding level of cysteine protease activity detected in a non-pathological state or in a corresponding non-pathological body region. For example, in a disease state or in an area without pathology, the level of cysteine protease activity may be below the detectable limit, whereas in a disease state or in an affected area, the level of cysteine protease activity is above the detectable limit. Diseases associated with cysteine protease activity include celiac disease, gastrointestinal motility disorders, pain, pruritus, skin disease, diet-induced obesity, metabolic disorder, asthma, rheumatoid arthritis, periodontitis, inflammatory gastrointestinal disease, functional gastrointestinal disorder. -intestinal tract, cancer, fibrotic disease, metabolic dysfunction, neurological disease, chronic obstructive pulmonary disease (COPD) and infection. In some embodiments, the “cysteine protease activity-associated disease” is a “cathepsin X activity-associated disease.”

[0101] As used herein, the terms "inflammatory gastrointestinal disease", "inflammatory gastrointestinal disorder", "inflammatory gastrointestinal disorder" or "inflammatory gastrointestinal disease" refer to gastrointestinal diseases, i.e. e. diseases affecting the gastrointestinal tract, namely the mouth, esophagus, stomach, small intestine, large intestine (colon) and rectum, as well as additional digestive organs (for example, tongue, salivary glands, pancreas, liver and gallbladder), in which there is inflammation of one or more parts of the gastrointestinal tract. Inflammatory diseases of the gastrointestinal tract include, for example, inflammatory bowel disease, infectious diarrhea, mesenteric ischemia, diverticulitis and necrotizing enterocolitis (NEC).

[0102] The term "inflammatory bowel disease" or "IBD" refers to a collection of diseases characterized by chronic and recurrent inflammation in the gastrointestinal tract. IBD primarily includes ulcerative colitis (UC) and Crohn's disease (CD), both of which are associated with diarrhea, rectal bleeding, increased urge to defecate, and pain, but also includes less common diseases such as acute colitis, immuno-oncologic colitis , colitis due to chemotherapy/radiation therapy, colitis due to graft-versus-host syndrome (GvHD), collagenous colitis, lymphocytic colitis, microscopic colitis, diversion colitis, Behçet's disease and colitis of undetermined etiology and pouchitis.

[0103] As used herein, the term “functional gastrointestinal disorders,” “functional gastrointestinal disorders,” or “functional gastrointestinal diseases” refers to disorders of the gut-brain interface. It is a group of disorders classified by gastrointestinal symptoms associated with any combination of the following factors: dysmotility, visceral hypersensitivity, altered mucosal and immune function, altered gut microbiota, and altered central nervous system (CNS) processing. The term "functional" is usually applied to disorders in which the body's normal functioning is disrupted in terms of intestinal motility, the sensitivity of intestinal nerves, or the way the brain controls some of these functions. However, there are no structural abnormalities that can be seen with endoscopy, x-rays, or blood tests. Thus, these disorders are largely identified by symptom characteristics. Functional gastrointestinal disorders include irritable bowel syndrome, functional chest pain, functional dyspepsia, nausea and vomiting, functional constipation, functional diarrhea, fecal incontinence, functional anorectal pain and functional defecation disorders

[0104] The term “infection” refers to the process or condition in which an infectious agent (eg, such as pathogenic bacteria, fungi, protozoa, viruses, prions, viroids, nematodes and other helminths) enters and multiplies in the body tissues of an infected subject.

[0105] The term “cancer” refers to a collection of diseases characterized by uncontrolled abnormal cell growth with the potential to invade or spread to other parts of the body. Cancer can affect any tissue and is named after the tissue from which it develops. The term "oral cancer" refers to oral cancer, i.e., any growth of cancerous tissue located in the oral cavity of a subject. Typical histological types of oral cancer are teratoma, adenocarcinoma arising from the major or minor salivary gland, lymphoma from the tonsils or other lymphoid tissue, or melanoma from pigment-producing cells of the oral mucosa. The most common type of oral cancer is squamous cell carcinoma, which occurs in the tissues lining the mouth and lips, with less common types including Kaposi's sarcoma. Oral cancer most often affects the tongue, but can also develop on the floor of the mouth, the lining of the cheeks, gums, lips or roof of the mouth. The term "breast cancer" refers to cancer of the breast. Examples of breast cancer include ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), Paget's nipple cancer, phyllodes tumor, and angiosarcoma. The term "prostate cancer" refers to cancer of the prostate. Examples of prostate cancer include adenocarcinomas of the prostate. The term “colorectal cancer” refers to cancer of the colon and/or rectum. Examples of colorectal cancer are adenocarcinomas, carcinoid tumors, gastrointestinal stromal tumors (GISTs), lymphomas, and sarcomas originating from the colon or rectum.

[0106] The term "alpha amino acid" refers to natural and non-natural alpha amino acids.

[0107] The term "naturally occurring amino acid" includes proteinogenic and non-proteinogenic amino acids.

[0108] With respect to alpha amino acid side chains, the term “structural analogue” refers to a side chain in which the CH ₂ group is replaced by O, S or NH, and/or where the -CH ₂ -CH ₂ - group is replaced by - CH=CH- group.

[0109] With respect to alpha amino acid side chains, the term “homolog” refers to a side chain that extends by one CH ₂ group.

[0110] The term "(Cy-Cz)" when used in combination with a chemical group such as alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, aryl, etc., indicates the possible number of carbon atoms in the group (i.e. from y to z carbon atoms).

[0111] As used herein, the term “alkyl” refers to a straight or branched chain alkyl group. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, isobutyl, t-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl and 2,2-dimethylpropyl.

[0112] As used herein, the term “haloalkyl” refers to a straight or branched chain alkyl group in which the hydrogen atoms of that group are partially or completely replaced by halogen atoms. Examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl and the like. In one embodiment, 1 to 3 hydrogen atoms are replaced by halogen atoms.

[0113] As used herein, the term “hydroxyalkyl” refers to a straight or branched chain alkyl group in which at least one hydrogen atom of that group is replaced by a hydroxy group. In some embodiments, one or two hydrogen atoms are replaced with a hydroxy group. In some embodiments, one hydrogen atom is replaced by a hydroxy group.

[0114] As used herein, the term “cycloalkyl” means a saturated monocyclic hydrocarbon radical. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl and cyclodecyl.

[0115] As used herein, the term “alkenyl” means at least one straight or branched chain unsaturated hydrocarbon radical, i.e., a straight or branched chain hydrocarbon radical having at least one double carbon carbon bond. Examples of alkenyl groups include, for example, vinyl, allyl (2-propen-1-yl), 1-propen-1-yl, 2-propen-2-yl, metallyl (2-methylprop-2-en-1-yl) , 2-buten-1-yl, 3-buten-1-yl, 2-penten-1-yl, 3-penten-1-yl, 4-penten-1-yl, 1-methylbut-2-en-1 -yl, 2-ethylprop-2-en-1-yl, etc.

[0116] As used herein, the term “alkynyl” means a straight or branched chain hydrocarbon radical having at least one carbon-carbon triple bond. Examples of alkynyl groups include ethynyl, propargyl (2-propyn-1-yl, also called prop-2-yn-1-yl), 1-propyn-1-yl (also called prop-1-yn-1-yl), 1-methylprop-2-yn-1-yl, 2-butyn-1-yl, 3-butyn-1-yl, 1-pentyn-1-yl, 3-pentyn-1-yl, 4-pentyn-1- yl, 1-methylbut-2-in-1-yl, 1-ethylprop-2-in-1-yl, etc.

[0117] As used herein, the term “aryl” refers to groups derived from monocyclic or polycyclic aromatic hydrocarbons by removing a hydrogen atom from a ring carbon atom. Examples of aryl groups include phenyl and naphthyl.

[0118] As used herein, the term “heteroaryl” refers to groups derived from heteroarenes by removing a hydrogen atom from any ring atom. Examples of heteroaryl groups include those derived from pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, indole and the like. Typical heteroatoms of heteroarenes are nitrogen, oxygen and sulfur.

[0119] The term “halogen” or “halogen atom” as used herein means fluorine, bromine, chlorine or iodine, in particular fluorine or chlorine.

[0120] As used herein, the term “alkoxy” refers to a straight or branched chain alkyl group that is linked via an oxygen group to the rest of the molecule. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butyloxy, 2-butyloxy, isobutyloxy, tert-butyloxy and the like.

[0121] The term "alkylcarbonyl" refers to a straight or branched alkyl group that is bonded via a carbonyl group carbon atom (C=O) to the rest of the molecule.

[0122] The term "amino protecting group" refers to a chemical group that renders the amino group non-reactive, but which can also be removed to reduce the amino group. Examples of amino protecting groups include benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), p-toluenesulfonyl (Tos), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf), mesityl-2-sulfonyl (Mts), 4-methoxy-2,3,6-trimethylphenylsulfonyl ( Mtr), acetamido, phthalimido, etc. Other amino protecting groups are known to those skilled in the art, including, for example, those described by Green and Wuts (Protective Groups in Organic Synthesis, 4th ed. 2007, Wiley-Interscience, New York) and P. Kocienski (Thieme, 2005).

[0123] As used herein, the term “(C ₆ -C ₁₀ ) arylmethyl” means a methyl group substituted with a (C ₆ -C ₁₀ ) aryl group. An example of a (C ₆ -C ₁₀ ) arylmethyl group is benzyl.

[0124] As used herein, the term “(C ₃ -C ₉ ) heteroarylmethyl” refers to a methyl group substituted with a (C ₃ -C ₉ ) heteroarylmethyl group. An example of a (C ₃ -C ₉ ) heteroarylmethyl group is (1H-indol-3-yl)methyl.

[0125] The term "sulfo" as used herein is recognized in the art and refers to the group -SO ₃ H or a salt form thereof (such as a pharmaceutically acceptable salt).

[0126] Formulas indicating positively or negatively charged atoms or groups (such as N ⁺ or SO ^{3 -} ) mean salt forms of the corresponding formula (including "internal salts" in the case of zwitterions).

[0127] For purposes of the present invention, the term “salt” includes salts of inorganic acids such as hydrochloride, hydrobromide, sulfate, phosphate, and the like; and salts of organic acids such as myristate, formate, acetate, trifluoroacetate, maleate, tartrate, bitartrate and the like; sulfonates such as methanesulfonate, benzenesulfonate, p-toluenesulfonate and the like; and amino acid salts such as arginate, aspartate, glutamate and the like. The term "salt" includes solvates, such as hydrates, of the corresponding salt.

[0128] In some embodiments, the term “salt” as used herein means a diagnostically and/or pharmaceutically acceptable salt. In some embodiments of the present invention, where the compound is administered or intended to be administered to a subject, the term “salt” means a pharmaceutically acceptable salt or a diagnostically and pharmaceutically acceptable salt.

[0129] As used herein, the term “pharmaceutically acceptable salt” means a salt of a compound of the present invention that is safe and effective for topical or systemic use in mammals and that has the desired biological activity. The counterion is suitable for the intended use, is non-toxic, and does not interfere with the desired biological effect of the compound. Pharmaceutically acceptable salts in the context of the present invention include those listed in the IUPAC Handbook of Pharmaceutically Acceptable Salts (Wermuth, CG and Stahl, PH, Pharmaceutical Salts: Properties, Selection and Use - A Handbook, Verlag Helvetica Chimica Acta (2002)).

[0130] As used herein, the term "diagnostically acceptable salt" refers to a salt of a compound of the present invention that is suitable and effective for the desired diagnostic method. Its counterion does not interfere with the reaction required to detect the target protein or the detection method.

[0131] In some embodiments, the compounds of the present invention are in the form of a trifluoroacetate salt, for example, after HPLC purification in an elution solvent including trifluoroacetic acid (TFA).

[0132] In some embodiments, “excipient” means a diagnostically and/or pharmaceutically acceptable excipient. In some embodiments of the present invention, where the composition is administered or intended to be administered to a subject, the term “excipient” refers to a pharmaceutically acceptable excipient or a diagnostically and pharmaceutically acceptable excipient.

[0133] In wavy line formulas, the wavy line represents or indicates the point of connection with the rest of the molecule.

[0134] A compound of formula (I) (and optionally as further defined by formulas II and III) may contain one or more asymmetric centers and thus may give enantiomers, diastereomers and other stereoisomeric forms. Unless specifically stated otherwise, the description covers the compounds in all such possible forms, as well as their racemic and resolved forms, or any mixture thereof. When a Compound of formula (I) contains an olefinic double bond or other center of geometric asymmetry, and unless specifically stated otherwise, it is intended to include all "geometric isomers", for example, both the E and Z geometric isomers. Unless specifically stated otherwise , all "tautomers", such as the ketone-enol, amide-imide acid, lactam-lactim, enamine-imine, amine-imine and enamine-enimine tautomers, are also covered herein.

[0135] As used herein, the terms “stereoisomer”, “stereoisomeric form”, and the like. are general terms for all isomers of individual molecules, which differ only in the orientation of their atoms in space. It includes enantiomers and isomers of compounds with more than one chiral center that are not mirror images of each other ("diastereomers").

[0136] The term "chiral center" refers to a carbon atom to which four different groups are attached.

[0137] The term "enantiomer" or "enantiomer" refers to a molecule that is not superimposed on its mirror image and is therefore optically active when the enantiomer rotates the plane of polarized light in one direction and its mirror image rotates the plane of polarized light in the opposite direction .

[0138] The term "racemic" refers to a mixture of equal parts of enantiomers that is optically inactive.

[0139] The term “separation” refers to the separation, concentration, or depletion of one of the two enantiomeric forms of a molecule. Optical isomers of the compound of formula (I) can be prepared by known methods such as chiral chromatography or the formation of diastereomeric salts from an optically active acid or base.

[0140] Optical purity can be expressed in units of enantiomeric excess (% ee), which is determined by the formula:

[0141]

[0142] In one embodiment, the invention relates to compounds having absolute stereochemistry as defined by Formulas IA (and as optionally further defined by Formulas IIA and IIIA).

[0143] The compounds of the present invention can be synthesized using standard synthetic chemical techniques, for example, using the methods described in the Examples section below. Other useful synthetic methods are described, for example, in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, ^7th Ed., (Wiley, 2013); Carey and Sundberg, Advanced Organic Chemistry ^4th 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 incorporated herein by reference in their entirety). Compounds are typically synthesized using starting materials that are generally available from commercial sources or that can be readily prepared using methods well known to those skilled in the art. See, for example, Fiesers' Reagents for Organic Synthesis, Volumes 1-27 (Wiley, 2013), or Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, including applications.

[0144] Connections

[0145] In some embodiments, the present invention provides a compound of Formula I:

[0146]

[0147] or salts thereof.

Where:

R ₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl;

R ₂ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C8) alkynyl;

R ₃ represents an alpha amino acid side chain;

R ₄ is selected from the group consisting of a hydrogen atom and (C ₁ -C ₄ ) alkyl;

R ₅ is selected from the group consisting of a detectable element, an amino protecting group, (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl, (C ₂ -C ₈ ) alkynyl, (C ₁ -C ₈ ) alkylcarbonyl, (C ₁ -C ₈ ) hydroxyalkylcarbonyl, (C ₁ -C ₈ ) haloalkylcarbonyl, (C ₃ -C ₈ ) cycloalkylcarbonyl, (C ₁ -C ₈ ) alkyloxycarbonyl, benzyloxycarbonyl and a hydrogen atom;

[0148] X represents:

(i) communication; or

(ii) a diradical fragment of formula II or III, which is connected to the R ₅ substituent through an amino group:

[0149]

Where:

R ₆ represents an alpha amino acid side chain;

R ₇ is selected from the group consisting of a hydrogen atom and (C ₁ -C ₄ ) alkyl;

R ₈ represents an alpha amino acid side chain;

R ₉ is selected from the group consisting of a hydrogen atom and (C ₁ -C ₄ ) alkyl; And

n is 1, 2, 3 or 4.

[0150] In some embodiments, a compound of Formula I is characterized in that R ₃ is a naturally occurring alpha amino acid side chain or a structural isomer, homologue, and/or structural analogue of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different _R3x substituents, wherein:

R _3x is selected from the group consisting of hydroxy, halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and ( C ₁ -C ₄ ) haloalkoxy.

[0151] In some embodiments, R ₃ is a side chain of a proteinogenic alpha amino acid or a structural isomer, homologue, and/or structural analogue of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different _R3x substituents, wherein:

R _3x is selected from the group consisting of hydroxy, halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and ( C ₁ -C ₄ ) haloalkoxy.

[0152] In some embodiments, R ₃ is a side chain of a proteinogenic alpha amino acid other than lysine, or a structural isomer or homolog of said side chain,

wherein said side chain or structural isomer or homologue thereof is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different R _3x substituents, wherein:

R _3x is selected from the group consisting of hydroxy, halogen, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ - _C 4) haloalkoxy.

[0153] In some embodiments, R ₃ is a side chain of an alpha amino acid selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, methionine, ethionine, phenylalanine, Tyrosin, levodopa, tripophane, cysteine, homocysteine, selenocysteine, selenicomocysteine, selenometionine, selenetionine, lysine, histidine, arginine, ornitin, asparaginic acid, glutamic acid, serin, homoseerine, o-methylgomsserin, o-e-echomoserin, trionin, asparagin and glutern or a structural isomer, homologue and/or structural analogue of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different R _3x substituents, wherein

R _3x is selected from the group consisting of hydroxy, halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and ( C ₁ -C ₄ ) haloalkoxy.

[0154] In some embodiments, R ₃ is a side chain of an alpha amino acid selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, methionine, ethionine, phenylalanine, Tyrosin, levodopa, tripophane, cysteine, homocysteine, selenocysteine, selenicomocysteine, selenometionine, selenetionine, lysine, histidine, arginine, ornitin, asparaginic acid, glutamic acid, serin, homoseerine, o-methylgomsserin, o-e-echomoserin, trionin, asparagin and glutern or a structural isomer, homologue and/or structural analogue of said side chain,

wherein said side chain or structural isomer or homolog thereof is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different R3x substituents, wherein:

R _3x is selected from the group consisting of hydroxy, halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and ( C ₁ -C ₄ ) haloalkoxy. In some such embodiments, the alpha amino acid is selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, methionine, ethionine, phenylalanine, tyrosine, levodopa, tryptophan, cysteine, homocysteine , selenocysteine, selenohomocysteine, selenomethionine and selenethionine. In some such embodiments, the alpha amino acid is selected from the group consisting of alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, phenylalanine, and tryptophan. In some such embodiments, the alpha amino acid is selected from the group consisting of valine, norvaline, leucine, isoleucine, norleucine, and homonorleucine. In some such embodiments, the alpha amino acid is selected from the group consisting of valine, leucine, isoleucine, and norleucine. In some such embodiments, the alpha amino acid is leucine or norleucine. In some such embodiments, the alpha amino acid is norleucine.

[0155] In some embodiments, R ₃ is selected from the group consisting of hydrogen, (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl, (C ₂ -C ₈ ) alkynyl, (C ₆ -C ₁₀ ) arylmethyl, (C ₃ -C ₉ ) heteroarylmethyl and -CH ₂ CH ₂ CH ₂ CH ₂ N (R _3a )(R _3b ); Where:

R _3a is selected from the group consisting of a detectable element, an amino protecting group, a hydrogen atom and a (C ₁ -C ₈ ) alkyl; And

R _3b is selected from the group consisting of a hydrogen atom and a (C ₁ -C ₄ ) alkyl atom. In some embodiments, R ₃ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₂ -C ₈ ) alkenyl, (C ₂ -C ₈ ) alkynyl, (C ₆ -C ₁₀ ) arylmethyl, (C ₃ -C ₉ ) heteroarylmethyl and -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a ) (R _3b ). In some embodiments, R ₃ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₆ -C ₁₀ ) arylmethyl, (C ₃ -C ₉ ) heteroarylmethyl, and -CH ₂ CH ₂ CH ₂ CH ₂ N( _R3a )( _R3b ). In some embodiments, R ₃ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, benzyl, (1H-indol-3-yl)methyl, and -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ). In some embodiments, R ₃ is selected from the group consisting of (C ₁ -C ₆ ) alkyl, benzyl, (1H-indol-3-yl)methyl, and -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ). In some embodiments, R ₃ is selected from the group consisting of ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, n-pentyl, benzyl, (1H-indol-3-yl)methyl, and -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ). In some embodiments, R _3b is a hydrogen atom.

[0156] In some embodiments, R ₃ is not an amino protecting group, a hydrogen atom, or a (C ₁ -C ₈ ) alkyl when X is a bond. In some embodiments, R ₃ is -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ) when X is a bond.

[0157] In other such embodiments, R _3a is a detectable element (regardless of the nature of X). In yet other such embodiments, R _3a is a hydrogen atom. In still other such embodiments, R _3a is an amino protecting group. In yet other such embodiments, R _3a is (C ₁ -C ₈ ) alkyl.

[0158] In some embodiments, R _3b is a hydrogen atom.

[0159] In some embodiments, R ₃ is selected from the group consisting of hydrogen, (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl, (C ₂ -C ₈ ) alkynyl, (C ₆ -C ₁₀ ) arylmethyl and (C ₃ -C ₉ ) heteroarylmethyl. In some such embodiments, R ₃ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₂ -C 8 ) alkenyl, (C ₂ -C ₈ ) alkynyl, (C ₆ -C ₁₀ ) arylmethyl, and (C ₃ -C ₉ ) heteroarylmethyl. In some such embodiments, R ₃ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₆ -C ₁₀ ) arylmethyl, and (C _{3 -C9} ) heteroarylmethyl. In some such embodiments, R ₃ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, benzyl, and (1H-indol-3-yl)methyl. In some such embodiments, R ₃ is selected from the group consisting of (C ₁ -C ₆ ) alkyl, benzyl, and (1H-indol-3-yl)methyl. In some such embodiments, R ₃ is selected from the group consisting of ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, n-pentyl, benzyl, and (1H-indole)-3-yl)methyl. In some such embodiments, R ₃ is selected from the group consisting of n-butyl, sec-butyl, and isobutyl. In some such embodiments, R ₃ is n-butyl.

[0160] In some embodiments, the compound of Formula I is characterized in that R ₃ is -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ); Where:

R _3a is selected from the group consisting of a detectable element, an amino protecting group, a hydrogen atom and a (C ₁ -C ₈ ) alkyl; And

R _3b is selected from the group consisting of a hydrogen atom and a (C ₁ -C ₄ ) alkyl atom. In still other such embodiments, R _3a does not represent an amino protecting group, a hydrogen atom, or a (C ₁ -C ₈ ) alkyl when X is a bond. In still other such embodiments, R _3a is a hydrogen atom (regardless of the nature of X). In still other such embodiments, R _3a is an amino protecting group. In yet other such embodiments, R _3a is (C ₁ -C ₈ ) alkyl. In some embodiments, R _3b is a hydrogen atom.

[0161] In some embodiments, R ₁ is (C ₁ -C ₈ ) alkyl. In some such embodiments, R ₁ is methyl.

[0162] In some embodiments, R ₂ is (C ₁ -C ₈ ) alkyl. In some such embodiments, R ₂ is methyl.

[0163] In some embodiments, R ₄ represents a hydrogen atom.

[0164] In some embodiments, R ₅ is selected from the group consisting of a detectable element, an amino protecting group, and a hydrogen atom.

[0165] In some embodiments, R ₆ is:

(i) a side chain of a naturally occurring alpha amino acid, or

(ii) a side chain of a proteinogenic alpha amino acid, or

(iii) an alpha amino acid side chain selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, methionine, ethionine, phenylalanine, tyrosine, levodopa, tryptophan, cysteine, homocysteine, selenocysteine, selenohomocysteine, selenomethionine, selenoethionine, lysine, histidine, arginine, ornithine, aspartic acid, glutamic acid, serine, homoserine, O-methylhomoserine, O-ethylhomoserine, threonine, asparagine and glutamine, or

(iv) a phenylalanine side chain,

or a structural isomer, homologue and/or structural analog of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different R _6x substituents, wherein:

R _6x is selected from the group consisting of hydroxy, halogen, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and ( C ₁ -C ₄ ) haloalkoxy.

[0167] In some embodiments, R ₆ is a phenylalanine side chain or a structural isomer, homologue, and/or structural analogue of said side chain.

[0168] In some embodiments, R ₈ is:

(i) a side chain of a naturally occurring alpha amino acid, or

(ii) a side chain of a proteinogenic alpha amino acid, or

(iii) an alpha amino acid side chain selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, methionine, ethionine, phenylalanine, tyrosine, levodopa, tryptophan, cysteine, homocysteine, selenocysteine, selenohomocysteine, selenomethionine, selenoethionine, lysine, histidine, arginine, ornithine, aspartic acid, glutamic acid, serine, homoserine, O-methylhomoserine, O-ethylhomoserine, threonine, asparagine and glutamine, or

(iv) a phenylalanine side chain,

or a structural isomer, homologue and/or structural analog of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different _R8x substituents, wherein:

R _8x is selected from the group consisting of hydroxy, halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and ( C ₁ -C ₄ ) haloalkoxy.

[0170] In some embodiments, R ₇ is a hydrogen atom.

[0171] In some embodiments, R ₉ is a hydrogen atom.

[0172] In some embodiments, when the substituent is an amino protecting group, the amino protecting group may be selected from the group consisting of benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), p-toluenesulfonyl (Tos), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf) , mesityl-2-sulfonyl (Mts), 4-methoxy-2,3,6-trimethylphenylsulfonyl (Mtr), acetamido and phthalimido. In some such embodiments, the amino protecting group is benzyloxycarbonyl.

[0173] In some of the above embodiments, the compound of formula I exhibits absolute stereochemistry in accordance with formula IA:

[0174]

[0175] In certain such embodiments, in a compound of formula I or IA, X is (i) a bond or (ii) a diradical moiety of formula II or III exhibiting absolute stereochemistry according to formulas IIA and IIIA, respectively:

[0176]

[0177] In some embodiments, X represents a relationship.

[0178] In certain other embodiments, X is a diradical moiety of formula II. In some such embodiments, X is a diradical moiety of Formula IIA.

[0179] In some other embodiments, X is a diradical moiety of Formula III. In some such embodiments, X is a diradical moiety of Formula IIIA. In some such embodiments, n is 1.

[0180] In some embodiments, the detectable element is selected from the group consisting of a fluorescent tag, a biotin tag, a radiolabel, a chelator (eg, for a radiolabel), and a bioorthogonal ligation tag.

[0181] A detectable element, such as a fluorescent tag, a biotin tag, a radiolabel, a chelator, or a bioorthogonal ligation marker, may include a linker for inclusion in the compounds of the present invention (i.e., to attach the detectable element or tag to the rest of the molecule) . Suitable linkers are known to those skilled in the art. Examples of linkers that can be used in the compounds of the present invention are described in WO 2012/118715 A2 (see page 18, lines 9-18), the contents of which are incorporated herein by reference. The linker may also include a polyethylene glycol (PEG) moiety, such as PEG-4, PEG-6 or PEG-8, for attachment to the rest of the molecule.

[0182] The definition of the term "radioactive label" and examples of radioactive labels that can be used in the compounds of the present invention are described in WO 2009/124265 A1 (see page 11, line 25 - page 13, line 3), the contents of which incorporated herein by reference.

[0183] The definition of the term "chelator" and examples of chelators that can be used in the compounds of the present invention are described in WO 2009/124265 A1 (see page 10, line 26 - page 11, line 14), the contents of which are incorporated herein via link.

[0184] The definition of the term “biorthogonal ligation marker” and examples of bioorthogonal ligation marker that can be used in the compounds of the present invention and corresponding “click” reactions are described, for example, in Martell et al., Applications of Copper-Catalyzed Click . Chemistry in Activity-Based Protein Profiling, Molecules 2014, 19, 1378-1393, which is incorporated herein by reference. Adaptation of these methods for the preparation or modification of the compounds of the present invention is within the competence of those skilled in the art.

[0185] Bioorthogonal reactions or click reactions for attaching a secondary label include:

A. traceless combination of azides by Staudinger ligation with triarylphosphines to form an amide bond,

B. cycloaddition of tetrazine using 1,2,4,5-tetrazine and a strained diene (trans-cyclooctene),

C. a copper(I) catalyzed azide-alkyne cycloaddition reaction (CuAAC) between an azide and a terminal alkyne to form a 1,4-disubstituted 1,2,3-triazole, and

D. a variant of the azide-alkyne cycloaddition without the presence of copper using a strained alkyne to speed up the reaction.

[0186] In this regard, reference is made in particular to FIG. 1B and FIG. 2 in Martell et al., Molecules 2014, 19, 1378-1393, the contents of which are incorporated herein by reference.

[0187] Thus, in some embodiments, the bioorthogonal ligation marker contains a functional group selected from the group consisting of an azide, 1,2,4,5-tetrazine, and an alkyne (such as a terminal alkyne). These functional groups allow the addition of a secondary label using one of the above bioorthogonal reactions (A)-(D).

[0188] In some embodiments, the detectable element is a fluorescent label. As is known to those skilled in the art, fluorescent tags emit electromagnetic radiation, preferably visible light, when stimulated by absorption of incident electromagnetic radiation. A wide variety of fluorescent labels are commercially available, including labels containing reactive groups, which can be used to link the label to reactive groups, such as, for example, amino groups, thiol groups, and the like. See, for example, The Molecular Probes® Handbook - A Guide to Fluorescent Probes and Labeling Technologies, which is incorporated herein by reference in its entirety.

[0189] Examples of fluorescent labels that can be used in the compounds of the present invention are described in WO 2018/119476 A1 (see paragraphs [0084]-[0095]) and in WO 2012/118715 A2 (see page 15, page 18 to page 17, line 12, and page 18, line 19 to page 21, line 1), the contents of which are incorporated herein by reference. Such fluorescent tags may include a linker for inclusion in the compounds of the present invention, for example, as described in WO 2012/118715 A2 (see page 18, lines 9-18), the contents of which are incorporated herein by reference.

[0190] In some embodiments, the detectable element is a fluorescent label. In some such embodiments, the fluorescent label is selected from the group consisting of fluorescein, Oregon green (a fluorinated derivative of fluorescein), boradiazaidecene dye, rhodamine dye (such as tetramethylrhodamine and carboxytetramethylrhodamine), benzopyryllium dye, coumarin dye, cyanine label, or benzoindole label ( e.g. indocyanine green).

[0191] Commercially available examples of such dyes include BODIPY® dyes (boradiase-indecene dyes), Alexa Fluor® series dyes (sulfonated rhodamines), DyLight® series dyes (containing, for example, sulfonated or unsulfonated coumarin, rhodamine, benzopyryl, or cyanine in as the main structure), IRDye® series dyes and cyanine (Cy) dyes (e.g. Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, sCy3, sCy5 and sCy7). Such cyanine tags can be purchased, for example, from Abcam, Tocris, GoldBio, ThermoFisher, Kerafast, Lumiprobe, AAT Bioquest or W&J Pharmachem.

[0192] In some embodiments, the fluorescent label is a cyanine label. In some such embodiments, the fluorescent label is a cyanine label selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, sCy3, sCy5 and sCy7. In some such embodiments, the fluorescent label is Cy5 or sCy5. In some embodiments, the fluorescent label is sCy5.

[0193] In some embodiments, the fluorescent label is a cyanine label having a formula selected from the following group of formulas:

[0194]

[0195] where in each of the above formulas,

A is selected from the group consisting of CH ₂ , C(CH ₃ ) ₂ , C(C ₂ H ₅ ) ₂ , NH, N(CH ₃ ), N(C ₂ H ₅ ), O, S and Se;

R ₁₀ is selected from the group consisting of $-(CH ₂ )pC(=O)-& and $-(CH ₂ ) _q -C(=O)-NH-[CH ₂ CH ₂ O] _r -CH ₂ CH ₂ -C(=O)-&;

Where:

p is equal to 2, 3, 4, 5, 6, 7 or 8;

q is equal to 2, 3, 4, 5, 6, 7 or 8;

r is equal to 2, 3, 4, 5, 6, 7 or 8;

$ represents the point of connection with the nitrogen atom of the cyanine group; and & represents the point of connection with the rest of the molecule;

R ₁₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl and (C ₆ -C ₁₀ ) aryl; And

R ₁₂ represents H or sulfo group. In some embodiments, p is 5, q is 5, and r is 4.

[0196] In some embodiments, where the fluorescent label is a cyanine label having one of the above formulas,

A is selected from the group consisting of CH ₂ , C(CH ₃ ) ₂ and C(C ₂ H ₅ ) ₂ ;

R ₁₀ is selected from the group consisting of $-(CH ₂ )pC(=O)-& and $-(CH ₂ ) _q -C(=O)-NH-[CH ₂ CH ₂ O] _r -CH ₂ CH ₂ -C(=O)-&;

Where:

p equals 2, 3, 4, 5 or 6;

q is equal to 2, 3, 4, 5 or 6;

r is 2, 3, 4, 5 or 6;

$ represents the point of attachment to the nitrogen atom of the cyanine group; and & represents the point of connection with the rest of the molecule;

R ₁₁ represents (C ₁ -C ₈ ) alkyl; And

R ₁₂ represents H or sulfo group. In some embodiments, p is 5, q is 5, and r is 4.

[0197] In some embodiments, where the fluorescent label is a cyanine label having one of the above formulas,

A is C(CH ₃ ) ₂ or C(C ₂ H ₅ ) ₂ ;

R ₁₀ is selected from the group consisting of $-(CH ₂ ) _p -C(=O)-& and $- (CH ₂ ) _q -C(=O)-NH-[CH ₂ CH ₂ O] _r -CH ₂ CH ₂ -C(=O)-&;

Where:

p equals 2, 3, 4, 5 or 6;

q is equal to 2, 3, 4, 5 or 6;

r is 2, 3, 4, 5 or 6;

$ represents the point of attachment to the nitrogen atom of the cyanine group; and & represents the point of connection with the rest of the molecule;

R ₁₁ is methyl, ethyl or propyl; And

R ₁₂ represents H or sulfo group. In some embodiments, p is 5, q is 5, and r is 4.

[0198] In some embodiments, where the fluorescent label is a cyanine label having one of the above formulas,

A represents C(CH ₃ ) ₂ ;

R ₁₀ is selected from the group consisting of $-(CH ₂ ) _p -C(=O)-& and $- (CH ₂ ) _q -C(=O)-NH-[CH ₂ CH ₂ O] _r -CH ₂ CH ₂ -C(=O)-&;

Where:

p is 4, 5 or 6;

q is 4, 5 or 6;

r is 3, 4, 5 or 6;

$ represents the point of connection with the nitrogen atom of the cyanine group; and & represents the point of connection with the rest of the molecule;

R ₁₁ represents methyl or ethyl; And

R ₁₂ represents H or sulfo group. In some embodiments, p is 5, q is 5, and r is 4.

[0199] In some embodiments, where the fluorescent label is a cyanine label having one of the above formulas,

A represents C(CH ₃ ) ₂ ;

R ₁₀ represents $-(CH ₂ ) _p -C(=O)-&; Where:

p is 4, 5 or 6; And

$ represents the point of attachment to the nitrogen atom of the cyanine group; and & represents the point of connection with the rest of the molecule;

R ₁₁ represents methyl or ethyl; And

R ₁₂ represents a sulfo group. In some embodiments, p is equal to 5.

[0200] In some embodiments, where the fluorescent label is a cyanine label having one of the above formulas,

A represents C(CH ₃ ) ₂ ;

R ₁₀ is $-(CH ₂ ) _q -C(=O)-NH-[CH ₂ CH ₂ O] _r -CH ₂ CH ₂ -C(=O)-&;

Where:

q is 4, 5 or 6;

r is 3, 4, 5 or 6;

$ represents the point of attachment to the nitrogen atom of the cyanine group; and & represents the point of connection with the rest of the molecule;

R ₁₁ represents methyl or ethyl; And

R ₁₂ is H. In some embodiments, p is 5 and r is 4.

[0201] In some embodiments, where the fluorescent label is a cyanine label having one of the above formulas, R ₅ is a detectable element, and & is a junction point with X.

[0202] In some embodiments, the fluorescent label is a cyanine label having a formula selected from the following group of formulas:

[0203]

[0204]

[0205]

[0206] where in each of the above formulas,

the wavy line represents the connection point with the rest of the molecule;

and R ₁₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl and (C ₆ -C ₁₀ ) aryl. In some such embodiments, R11 is (C ₁ -C ₈ ) alkyl. In some such embodiments, R ₁₁ is methyl or ethyl.

[0207] In some embodiments, the fluorescent label is a cyanine label having the formula,

[0208]

where the wavy line represents the connection point with the rest of the molecule; and R ₁₁ is methyl or ethyl.

[0209] In some embodiments, where the fluorescent label is a cyanine label having one of the above formulas, R ₅ represents the detectable element, and the wavy line represents the connection point with X.

[0210] In some embodiments, a compound of formula I (or IA) contains at least one detectable element, such as one, two or three detectable elements. In some such embodiments, a compound of formula I (or IA) as defined above contains a single detectable element.

[0211] In some embodiments, R ₅ is a detectable element.

[0212] In some embodiments, R ₃ carries a detectable element. In some such embodiments, R ₃ is a lysine side chain or a structural isomer, homologue, and/or structural analogue of said side chain, wherein said side chain or structural isomer, homologue, and/or structural analogue is substituted with a detectable element and optionally further substituted with one or more identical or different substituents R _3x , where R _3x is selected from the group consisting of hydroxy, halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy. In some such embodiments, R ₃ is -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ); Where:

R _3a represents a detectable element; And

R _3b is selected from the group consisting of a hydrogen atom and a (C ₁ -C ₄ ) alkyl atom.

[0213] In some embodiments, where R ₃ carries a detectable element, R ₅ is selected from the group consisting of an amino protecting group, (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl, (C ₂ -C ₈ ) alkynyl, (C ₁ -C ₈ ) alkylcarbonyl, (C ₁ -C ₈ ) hydroxyalkylcarbonyl, (C ₁ -C ₈ ) haloalkylcarbonyl, (C ₃ -C ₈ ) cycloalkylcarbonyl, (C ₁ -C ₈ ) alkyloxycarbonyl, benzyloxycarbonyl and a hydrogen atom. In some such embodiments, R ₅ is an amino protecting group.

[0214] In some embodiments, X is a diradical moiety of formula II or IIA, and R ₆ carries a detectable element. In some such embodiments, R ₆ is a lysine side chain or a structural isomer, homologue, and/or structural analogue of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is substituted with a detectable element and optionally further substituted with one or more identical or different R _6x substituents, wherein R _6x is selected from the group consisting of hydroxy, a halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy.

[0215] In some embodiments, X is a diradical moiety of formula III or IIIA, and R ₆ carries a detectable element. In some such embodiments, R ₆ is a lysine side chain or a structural isomer, homologue, and/or structural analogue of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is substituted with a detectable element and optionally further substituted with one or more identical or different R _6x substituents, wherein R _6x is selected from the group consisting of hydroxy, a halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy.

[0216] In some embodiments, X is a diradical moiety of formula III or IIIA, and R ₈ carries a detectable element. In some such embodiments, _R8 is a lysine side chain or a structural isomer, homologue, and/or structural analogue of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is substituted with a detectable element and optionally further substituted with one or more identical or different R _8x substituents, wherein R _8x is selected from the group consisting of hydroxy, a halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy.

[0217] In some embodiments, a compound of Formula I is a compound having one of the following formulas (with absolute stereochemistry as indicated):

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226] or its salt.

[0227] In some preferred embodiments, the compound of formula I is a compound having the following formula (with absolute stereochemistry as indicated):

[0228]

[0229] or its salt.

[0230] Songs

[0231] In some embodiments, the present invention provides a composition comprising a compound of Formula I (or IA) as described above, or a salt thereof, and an excipient (eg, a pharmaceutically and/or diagnostically acceptable excipient).

[0232] Pharmaceutically acceptable excipients that can be used in the compositions of the present invention are known to those skilled in the art. Examples of such pharmaceutically acceptable excipients include, for example, those described in paragraphs [0114] - [0118] in WO 2018/119476, the contents of which are hereby incorporated into the present disclosure.

[0233] In some embodiments, the composition is an aqueous solution containing, for example, water, phosphate buffered saline, or a buffer solution as a pharmaceutically acceptable excipient. Such compositions can be used, for example, for intravenous injection.

[0234] Methods for detecting cysteine protease activity

[0235] In the methods for detecting cysteine protease activity of the present invention, only proteolytically active forms of the corresponding cysteine protease(s) are detected.

[0236] The detected signal is measured after the interaction of the activity-based probe compound and the cysteine protease has resulted in the formation of a covalent bond. The measured detection signal is emitted by the labeled enzyme, i.e., the detectable element of the activity-based probe compound covalently attached to the cysteine protease. In some embodiments, the detectable signal is measured after the labeled enzyme has been subjected to a secondary labeling step.

[0237] The concept of detecting enzyme activity using activity-based probes and associated detection methods and underlying experimental protocols are known to those skilled in the art (see, e.g., Edgington and Bogyo, 2013; Edgington-Mitchell, L.E., and Bogyo, M. (2016). Detection of Active Caspases During Apoptosis Using Fluorescent Activity-Based Probes. Methods Mol Biol. 1419, 27-39; and Edgington-Mitchell, L. E., Bogyo, M., and Verdoes, M. (2017). Live Cell Imaging and Profiling of Cysteine Cathepsin Activity Using a Quenched Activity-Based Probe Methods Mol Biol 1491, 145-159 (the contents of which are incorporated herein by reference in their entirety). Those skilled in the art will know how to appropriately adapt these methods/protocols for use in the methods of the present invention.

[0238] In vitro detection methods

[0239] In some embodiments, the present invention provides a method for detecting cysteine protease activity, comprising:

(1) contacting the cysteine protease with an activity-based probe compound containing a sulfoxonium ylide moiety as an active group, and

(2) subsequent cysteine protease analysis, including measurement of the detected signal.

In some such embodiments, the method is an in vitro method.

[0240] In some embodiments, the present invention provides a method for detecting cysteine protease activity in vitro, comprising:

(1) contacting the biological sample with an activity-based probe compound containing a sulfoxonium ylide moiety as an active group, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

[0241] In some embodiments, the present invention provides a method for detecting cysteine protease activity in a biological sample obtained from a subject, comprising:

(1) contacting the biological sample in vitro with an activity-based probe compound containing a sulfoxonium ylide moiety as an active group, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

[0242] In some embodiments of the above methods, the activity probe compound contains a sulfoxonium ylide moiety having the formula:

[0243]

[0244] where:

R ₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl; And

R ₂ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl. In some embodiments, R ₁ is methyl and R ₂ is methyl.

[0245] In some embodiments, the present invention provides a method for detecting cysteine protease activity, comprising:

(1) contacting the cysteine protease with a compound of formula I as described herein, or a salt thereof, or a composition as described herein, and

(2) subsequent cysteine protease analysis, including measurement of the detected signal.

In some such embodiments, the method is an in vitro method.

[0246] In some embodiments, the present invention provides a method for detecting cysteine protease activity in vitro, comprising:

(1) contacting the biological sample with a compound of Formula I as described herein, or a salt thereof, or a composition as described herein, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

[0247] In some embodiments, the present invention provides a method for detecting cysteine protease activity in a biological sample obtained from a subject, comprising:

(1) contacting the biological sample in vitro with a compound of Formula I as described herein, or a salt thereof, or a composition as described herein, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

[0248] In some embodiments of each of the above methods, step (2) includes performing at least one analytical method selected from the group consisting of gel electrophoresis followed by in-gel fluorescence, gel electrophoresis followed by radiography, gel electrophoresis and subsequent immunoblotting, fluorescence microscopy, flow cytometry, ex vivo optical imaging, radiography, affinity purification and subsequent mass spectrometry, affinity purification and subsequent proteomics.

[0249] In some embodiments of each of the above methods, the detected signal is measured by measuring fluorescence. In some such embodiments, at least one analytical method is selected from the group consisting of gel electrophoresis followed by in-gel fluorescence, fluorescence microscopy, and flow cytometry. In some such embodiments, at least one analytical method is selected from gel electrophoresis followed by in-gel fluorescence and fluorescence microscopy. In some such embodiments, said compound comprises a detectable element in the form of a fluorescent label (eg, as described herein). In some other embodiments, the compound comprises a detectable element in the form of a bioorthogonal ligation marker, and step (2) includes secondary labeling using click chemistry to incorporate a fluorescent label before performing the at least one analytical method. In some other embodiments, the compound contains a detectable element in the form of biotin, and step (2) includes secondary labeling with fluorescently labeled streptavidin or secondary labeling with a fluorescently labeled biotin-specific antibody before performing at least one analytical method.

[0250] In some embodiments of each of the above methods, at least one analytical method is selected from the group consisting of radiography, gel electrophoresis, and post-radiography. In some such embodiments, said compound comprises a detectable element in the form of a radioactive tracer. In some other embodiments, the compound comprises a detectable element as a chelator for a radioactive label. In some other embodiments, the compound comprises a detectable element in the form of a bioorthogonal ligation marker, and step (2) includes secondary labeling by click chemistry to incorporate a radiolabel or a radiolabel chelator before performing the at least one analytical method.

[0251] In some embodiments of each of the above methods, at least one analytical method is selected from the group consisting of affinity purification followed by mass spectrometry, affinity purification followed by proteomics. In some such embodiments, said compound comprises a detectable element in the form of a biotin tag. In some other embodiments, the compound comprises a detectable element as a bioorthogonal ligation marker, and step (2) includes secondary labeling using click chemistry to incorporate a biotin tag before performing at least one analytical procedure. In the case of biotin labeling, affinity purification can be carried out using, for example, streptavidin-coated beads or beads coated with a biotin-specific antibody.

[0252] In some embodiments, affinity purification can be performed using beads coated with an antibody specific for a particular label. In some such embodiments, said compound includes said label as a detectable element. In some other embodiments, the compound contains a detectable element in the form of a bioorthogonal ligation marker, and step (2) includes secondary labeling using click chemistry to incorporate the tag before affinity purification is performed.

[0253] In some embodiments of each of the above methods, at least one analytical method is selected from the group consisting of gel electrophoresis and subsequent immunoblotting. In some such embodiments, the compound contains a detectable element in the form of a biotin tag, and step (2) includes secondary labeling, for example with HRP-labeled streptavidin, before performing the at least one analytical method. In some other embodiments, the compound comprises a detectable element in the form of a bioorthogonal ligation marker, and step (2) includes secondary labeling using click chemistry to apply a biotin label and subsequent labeling, for example, with HRP-labeled streptavidin, until at least at least one analytical method.

[0254] In some embodiments, the gel electrophoresis is one-dimensional or two-dimensional gel electrophoresis (such as SDS-PAGE or native PAGE). In some embodiments, the gel electrophoresis is SDS-PAGE.

[0255] In vitro detection methods with pre-administration to the subject

[0256] In some embodiments, the present invention provides a method for detecting cysteine protease activity, comprising:

(1) administering to the subject an activity-based probe compound containing a sulfoxonium ylide moiety as the active group,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

[0257] In some such embodiments, the activity-based probe compound is administered intravenously. In some embodiments of this method, the activity probe compound contains a sulfoxonium ylide moiety having formula (IV):

[0258]

[0259] where:

R ₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl; And

R ₂ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ - _C8 ) alkenyl and (C ₂ -C ₈ ) alkynyl. In some embodiments, R ₁ is methyl and R ₂ is methyl.

[0260] In some embodiments, the present invention provides a method for detecting cysteine protease activity, comprising:

(1) administering to the subject a compound of Formula I as described herein, or a salt or composition thereof, as described herein,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

In some such embodiments, the compound or a salt or composition thereof is administered intravenously.

[0261] In some embodiments of each of the above methods, step (3) includes performing at least one analytical method selected from the group consisting of gel electrophoresis followed by in-gel fluorescence, gel electrophoresis followed by radiography, gel electrophoresis and subsequent immunoblotting, fluorescence microscopy, flow cytometry, ex vivo optical imaging, radiography, affinity purification and subsequent mass spectrometry, affinity purification and subsequent proteomics.

[0262] In some embodiments of each of the above methods, the detected signal is measured by measuring fluorescence. In some such embodiments, at least one analytical method is selected from the group consisting of gel electrophoresis followed by in-gel fluorescence, fluorescence microscopy, and flow cytometry. In some such embodiments, at least one analytical method is selected from gel electrophoresis followed by in-gel fluorescence and fluorescence microscopy. In some such embodiments, said compound comprises a detectable element in the form of a fluorescent label (eg, as described herein). In some other embodiments, the compound comprises a detectable element as a bioorthogonal ligation marker, and step (3) includes secondary labeling using click chemistry to incorporate a fluorescent label before performing the at least one analytical method. In some other embodiments, the compound contains a detectable element in the form of biotin, and step (3) includes secondary labeling with fluorescently labeled streptavidin or secondary labeling with a fluorescently labeled biotin-specific antibody before performing the at least one analytical method.

[0263] In some embodiments of each of the above methods, at least one analytical method is selected from the group consisting of radiography, gel electrophoresis, and post-radiography. In some such embodiments, said compound comprises a detectable element in the form of a radioactive tracer. In some other embodiments, the compound comprises a detectable element as a chelator for a radioactive label. In some other embodiments, the compound comprises a detectable element in the form of a bioorthogonal ligation marker, and step (3) includes secondary labeling using click chemistry to apply a radiolabel or a radiolabel chelator before performing the at least one analytical method.

[0264] In some embodiments of each of the above methods, at least one analytical method is selected from the group consisting of affinity purification followed by mass spectrometry, affinity purification followed by proteomics. In some such embodiments, said compound comprises a detectable element in the form of a biotin tag. In some other embodiments, the compound comprises a detectable element as a bioorthogonal ligation marker, and step (3) includes secondary labeling with click chemistry to incorporate a biotin tag prior to performing the at least one analytical method. In the case of biotin labeling, affinity purification can be carried out using, for example, streptavidin-coated beads or beads coated with a biotin-specific antibody. In some embodiments, affinity purification can be performed using beads coated with an antibody specific for a particular label. In some such embodiments, said compound contains said label as a detectable element. In some other embodiments, the compound contains a detectable element in the form of a bioorthogonal ligation marker, and step (3) includes secondary labeling using click chemistry to incorporate the tag before affinity purification is performed.

[0265] In some embodiments of each of the above methods, at least one analytical method is selected from the group consisting of gel electrophoresis and subsequent immunoblotting. In some such embodiments, the compound contains a detectable element in the form of a biotin tag, and step (3) includes secondary labeling, for example with HRP-labeled streptavidin, before performing the at least one analytical method. In some other embodiments, said compound comprises a detectable element in the form of a bioorthogonal ligation marker, and step (3) includes secondary labeling with cline chemistry to apply a biotin label and subsequent labeling, for example, with HRP-labeled streptavidin, to at least at least one analytical method.

[0266] In some embodiments, the gel electrophoresis is one-dimensional or two-dimensional gel electrophoresis (such as SDS-Page or native PAGE). In some embodiments, the gel electrophoresis is SDS-PAGE. In some embodiments, the subject is a human.

[0267] In vivo detection methods

[0268] In some embodiments, the present invention provides a method for in vivo detection of cysteine protease activity in a subject, comprising:

(1) administering to the subject an activity-based probe compound containing a sulfoxonium ylide moiety as an active group, and

(2) subsequent examination of the subject, including measurement of the detected signal.

In some such embodiments, the activity-based probe compound is administered intravenously. In some embodiments of this method, the activity probe compound contains a sulfoxonium ylide moiety having formula (IV):

[0269]

[0270] where:

R ₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl; And

R ₂ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl. In some embodiments, R ₁ is (C ₁ -C ₈ ) alkyl, and R ₂ is (C ₁ -C ₈ ) alkyl. In some embodiments, R ₁ is methyl and R ₂ is methyl.

[0271] In some embodiments, the present invention provides a method for detecting in vivo cysteine protease activity in a subject, comprising:

(1) administering to the subject a compound of formula I, as described herein, or a salt or composition thereof, as described herein, and

(2) subsequent examination of the subject, including measurement of the detected signal.

In some such embodiments, the compound or a salt or composition thereof is administered intravenously.

[0272] In some embodiments of each of the above methods (in vivo), the detected signal is measured using in vivo optical imaging, radiography, or positron emission tomography. In some such embodiments, the detected signal is measured using radiography or positron emission tomography. In some such embodiments, said compound comprises a detectable element in the form of a radioactive tracer. In some other embodiments, the compound comprises a detectable element as a chelator for a radioactive label. In some embodiments, the subject is a human.

[0273] Methods of inhibiting cysteine proteases

[0274] In some embodiments, the present invention provides a method of inhibiting a cysteine protease, comprising contacting the cysteine protease with a compound of Formula I as described herein, or a salt thereof, or a composition as described herein. In some such embodiments, the method is an in vitro method.

[0275] In some embodiments, the present invention provides a method of inhibiting a cysteine protease in vitro, comprising contacting a biological sample with a compound of Formula I as described herein, or a salt thereof, or a composition as described herein.

[0276] In some embodiments, the present invention provides a method of inhibiting a cysteine protease in a biological sample obtained from a subject, comprising contacting the biological sample in vitro with a compound of Formula I as described herein, or a salt thereof, or a composition as described herein.

[0277] In some embodiments, the present invention provides a method of in vivo inhibition of a cysteine protease in a subject, comprising administering to the subject a compound of Formula I as described herein, or a salt or composition thereof, as described herein. In some such embodiments, the compound or a salt or composition thereof is administered intravenously.

[0278] In some embodiments of cysteine protease inhibition methods as described herein, the compound of Formula I as described herein does not contain a detectable element such as a fluorescent label, a biotin label, a radiolabel, a chelator, and a bioorthogonal ligation marker.

[0279] Cysteine proteases

[0280] In methods for detecting cysteine protease activity as described herein, and in methods for inhibiting cysteine protease as described herein, the cysteine protease may, for example, be a mammalian cysteine protease. In some embodiments, the cysteine protease is a human cysteine protease. In some embodiments, the cysteine protease is a cysteine cathepsin. In some embodiments, the cysteine protease is a mammalian cysteine cathepsin. In some embodiments, the cysteine protease is a human cysteine cathepsin. In some embodiments, the cysteine protease is cathepsin S and/or cathepsin X. In some embodiments, the cysteine protease is cathepsin X. In certain embodiments, the cysteine protease is mammalian cathepsin X. In some embodiments, the cysteine protease is human cathepsin X.

[0281] In some embodiments of methods for detecting cysteine protease activity as described herein, cathepsin X activity is detected, and cathepsin B activity and/or cathepsin L activity is not detected. In some embodiments, cathepsin X activity and cathepsin S activity are detected, and cathepsin B activity and/or cathepsin L activity are not detected.

[0282] In some embodiments of cysteine protease inhibition methods as described herein, cathepsin X is inhibited and cathepsin B and/or cathepsin L are not inhibited. In some embodiments, cathepsin X and cathepsin S are inhibited, and cathepsin B and/or cathepsin L are not inhibited.

[0283] In methods for detecting cysteine protease activity in vitro as described herein, the identity of the labeled protein can be analyzed, for example, by subjecting an aliquot of the probe-labeled sample to an immunoprecipitation assay (e.g., precipitation with an antibody specific for the corresponding cysteine protease (e.g., cathepsin X).

[0284] Diagnostic methods and corresponding compounds/compositions for diagnostic use

[0285] In some embodiments, the present invention provides a method for diagnosing a disease associated with cysteine protease activity in a subject, comprising:

(1) contacting a biological sample obtained from a subject in vitro with a compound of Formula I as described herein, or a salt thereof, or a composition as described herein, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

In some such embodiments, the method comprises detecting cysteine protease activity in accordance with any of the methods described herein in the "Detection Methods (In Vitro)" section.

[0286] In some embodiments, the present invention provides a method for diagnosing a disease associated with cysteine protease activity in a subject, comprising:

(1) administering to the subject a compound of Formula I as described herein, or a salt or composition thereof, as described herein,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

In some such embodiments, the method includes determining cysteine protease activity in accordance with any of the methods described herein in the section "Methods of Detection (In Vitro) with Pre-Administration to a Subject."

[0287] In some embodiments, the present invention provides a method for in vivo diagnosis of a disease associated with cysteine protease activity in a subject, comprising:

(1) administering to the subject a compound of formula I, as described herein, or a salt or composition thereof, as described herein, and

(2) subsequent examination of the subject, including measurement of the detected signal.

In some such embodiments, the method includes determining cysteine protease activity in accordance with any of the methods described herein in the "In Vivo Detection Methods" section.

[0288] In some embodiments, the present invention provides a compound of Formula I as described herein, or a salt thereof, for use in a method for diagnosing a disease associated with cysteine protease activity in a subject, wherein the method includes:

(1) administering to the subject a compound of formula I as described herein, or a salt thereof,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

In some such embodiments, the method comprises determining cysteine protease activity in accordance with any of the methods described herein in the section "Methods of Detection (In Vitro) with Pre-Administration to a Subject."

[0289] In some embodiments, the present invention provides a composition as described herein for use in a method for diagnosing a disease associated with cysteine protease activity in a subject, wherein the method includes:

(1) administering to the subject the composition as described herein,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

In some such embodiments, the method includes determining cysteine protease activity in accordance with any of the methods described herein in the section "Methods of Detection (In Vitro) with Pre-Administration to a Subject."

[0290] In some embodiments, the present invention provides a compound of Formula I as described herein, or a salt thereof, for use in a method for diagnosing in vivo a disease associated with cysteine protease activity in a subject, wherein the method includes:

(1) administering to the subject a compound of Formula I as described herein, or a salt thereof, and

(2) subsequent examination of the subject, including measurement of the detected signal.

In some such embodiments, the method includes determining cysteine protease activity in accordance with any of the methods described herein in the "In Vivo Detection Methods" section.

[0291] In some embodiments, the present invention provides a composition described herein for use in a method for diagnosing a disease associated with cysteine protease activity in a subject in vivo, wherein the method includes:

(1) administering to the subject the composition as described herein, and

(2) subsequent examination of the subject, including measurement of the detected signal.

In some such embodiments, the method includes determining cysteine protease activity in accordance with any of the methods described herein in the "In Vivo Detection Methods" section.

[0292] Biological samples

[0293] In some embodiments, the biological sample is selected from the group consisting of cells, cell lysates, tissue samples, tissue lysates, and body fluids. In some embodiments, the biological sample is a cell lysate or tissue lysate, such as a purified cell lysate or a purified tissue lysate. In some embodiments, the biological sample is living cells. In some embodiments of the detection method as described above, live cells are lysed and purified between step (1) and step (2) and between steps (2) and (3), respectively. In some embodiments, the biological sample is obtained from a human.

[0294] In some embodiments, the biological sample is obtained from the oral cavity (such as the oral mucosa), lungs, brain, spinal cord, pancreas, stomach, prostate, liver, bone marrow, colon (such as distal colon or proximal colon), rectum, mammary gland, skin, mucous membrane or mucus of a subject, or from the feces or sputum of a subject (such as a mammal, more specifically a human subject). The biological sample may be tumor tissue obtained from the oral cavity, lung, brain, spinal cord, pancreas, stomach, prostate, liver, bone marrow, colon (eg, distal colon or proximal colon), rectum, breast, skin, mucous membrane or mucus of the subject.

[0295] In some such embodiments, the biological sample is obtained from the oral cavity (such as the oral mucosa) of the subject. In some such embodiments, the biological sample is a cell lysate or a tissue lysate. In some embodiments, the biological sample is an oral biopsy material. In some embodiments, the biological sample is a biopsy material from the oral mucosa.

[0296] In some embodiments, the biological sample is obtained from the gastrointestinal tract of a subject. In some such embodiments, the biological sample is a cell lysate or a tissue lysate. In some such embodiments, the biological sample is an oral biopsy specimen, an esophageal specimen, a stomach specimen, a small intestinal specimen, a colon specimen, a proximal colon specimen, a distal colon specimen, a rectal specimen, a stool specimen, or a mucosal biopsy specimen. shells. In some embodiments, in which the biological sample is obtained from the gastrointestinal tract of a subject, the biological sample is a mucosal biopsy material selected from the group consisting of an oral mucosal biopsy material, an esophageal mucosal biopsy material, a thin mucosal biopsy material intestine, colon mucosal biopsy material or rectal mucosal biopsy material.

[0297] In some embodiments, the biological sample is obtained from the colon (such as the distal colon or proximal colon) of the subject. In some such embodiments, the biological sample is a cell lysate or a tissue lysate. In some embodiments, the biological sample is a colon biopsy. In some embodiments, the biological sample is a biopsy material from the colon mucosa. In some embodiments, in which the biological sample is obtained from the colon, the biological sample is a stool sample.

[0298] In some embodiments, the biological sample is obtained from the prostate gland of a subject. In some such embodiments, the biological sample is a cell lysate or a tissue lysate. In some embodiments, the biological sample is a prostate biopsy material.

[0299] In some embodiments, the biological sample may be cells, cell lysates, tissue samples, and tissue lysates obtained from the mammary gland of a female subject (particularly a female subject).

[0300] In some embodiments, the biological sample is a tissue lysate selected from the group consisting of an oral biopsy sample, a lung sample, a brain sample, a spinal cord sample, a pancreas sample, a stomach sample, a prostate sample, a liver sample, a bone marrow sample, a colon sample (eg, a distal colon sample and a proximal colon sample), a rectal sample, a breast sample, a skin sample, a mucosal biopsy material, a stool sample, a sputum sample, and a tumor sample.

[0301] Methods of treatment and corresponding compounds/compositions for use in treatment

[0302] In some embodiments, the present invention provides a method of treating a disease associated with cysteine protease activity, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula I, as described herein, or a therapeutically effective amount of a compound of a composition, as described herein .

[0303] In some embodiments, the present invention provides a compound of formula I, as described herein, for use in the treatment of a disease associated with cysteine protease activity.

[0304] In some embodiments, the present invention provides a composition for use in treating a disease associated with cysteine protease activity, comprising a compound of Formula I as described herein and an excipient.

[0305] In some embodiments, the present invention relates to the use of a compound of formula I, as described herein, in the manufacture of a medicament for the treatment of a disease associated with cysteine protease activity.

[0306] In some embodiments, the present invention relates to the use of a composition described herein in the manufacture of a medicament for the treatment of a disease associated with cysteine protease activity.

[0307] In some of the above embodiments, the compound of formula I as described herein, when administered or used (or for use) in the treatment of a disease, does not contain a detectable element, such as a fluorescent label, a biotin label, a radiolabel, a chelator, and a bioorthogonal marker for ligation.

[0308] Diseases

[0309] In some embodiments, the disease (to be diagnosed or treated) associated with cysteine protease activity is selected from the group consisting of celiac disease, a gastrointestinal motility disorder, pain, itching, a skin disease (such as atopic dermatitis), diet-induced obesity, metabolic disorder (including but not limited to non-alcoholic steatohepatitis (NASH), liver and pancreatic disease), asthma, rheumatoid arthritis, periodontitis, inflammatory disease (including inflammatory disease of the gastrointestinal tract such as inflammatory bowel disease), functional gastrointestinal disorders (eg, irritable bowel syndrome, functional chest pain, functional dyspepsia, nausea and vomiting, functional constipation, functional diarrhea, fecal incontinence, functional anorectal pain and functional defecation disorder), cancer, fibrotic disease, metabolic dysfunction, neurological disease and neurodegenerative disease.

[0310] In some embodiments, the disease (to be diagnosed or treated) associated with cysteine protease activity is selected from the group consisting of cancer, inflammatory disease, and neurodegenerative disease.

[0311] In some embodiments, the disease (to be diagnosed or treated) is a cancer selected from the group consisting of breast cancer, brain cancer (glioblastoma), bone marrow cancer, pancreatic cancer, lung cancer, prostate cancer, liver cancer (hepatocellular carcinoma), oral cancer, colorectal cancer and stomach cancer.

[0312] In some embodiments, the disease (to be diagnosed or treated) is an inflammatory disease selected from the group consisting of inflammatory disease of the gastrointestinal tract, pancreatitis, and infection. In some such embodiments, the inflammatory gastrointestinal disease is selected from the group consisting of inflammatory bowel disease, infectious diarrhea, mesenteric ischemia, diverticulitis, and necrotizing enterocolitis (NEC). In some embodiments, the inflammatory disease of the gastrointestinal tract is an inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is selected from the group consisting of ulcerative colitis, Crohn's disease, diversion colitis, colitis of undetermined etiology and pauchitis, microscopic colitis, immuno-oncological colitis, colitis due to chemotherapy/radiation therapy, colitis due to graft-versus-host syndrome "(GvHD), acute colitis, Behçet's disease, collagenous colitis, lymphocytic colitis. In some embodiments, the inflammatory gastrointestinal disease is selected from ulcerative colitis and Crohn's disease.

[0313] In some embodiments, the disease (to be diagnosed or treated) is an inflammatory disease selected from the group consisting of ulcerative colitis, Crohn's disease, diversion colitis, colitis of undetermined etiology and pauchitis, microscopic colitis, immuno-oncological colitis, chemotherapy-induced colitis /radiation therapy, graft-versus-host syndrome (GvHD) colitis, acute colitis, Behcet's disease, collagenous colitis, lymphocytic colitis, infectious diarrhea, mesenteric ischemia, diverticulitis and necrotizing enterocolitis (NEC), pancreatitis and infection.

[0314] In some embodiments, the disease (to be diagnosed or treated) is an inflammatory disease selected from inflammatory bowel diseases. In some such embodiments, the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

[0315] In some embodiments, the disease (to be diagnosed or treated) is a neurodegenerative disease selected from the group consisting of Alzheimer's disease, multiple sclerosis, and neuropathic pain.

[0316] In some embodiments related to the diagnosis of a disease, wherein the disease is breast cancer, the biological sample is a sample, as described above, that is obtained from the mammary gland of a subject, for example, from tumor tissue located in the mammary gland.

[0317] In some embodiments related to the diagnosis of a disease wherein the disease is brain cancer, the biological sample is a sample as described above that is obtained from the brain of a subject, such as from tumor tissue located in the brain.

[0318] In some embodiments related to the diagnosis of a disease wherein the disease is bone marrow cancer, the biological sample is a sample as described above that is obtained from the bone marrow of a subject, such as from tumor tissue located in the bone marrow.

[0319] In some embodiments related to the diagnosis of a disease, wherein the disease is pancreatic cancer, the biological sample is a sample as described above that is obtained from the pancreas of a subject, for example, from tumor tissue located in the pancreas.

[0320] In some embodiments related to the diagnosis of a disease, where the disease is lung cancer, the biological sample is a sample as described above that is obtained from the lungs of a subject, for example, from tumor tissue located in the lung.

[0321] In some embodiments related to the diagnosis of a disease, wherein the disease is prostate cancer, the biological sample is a sample as described above that is obtained from the prostate gland of a subject, for example, from tumor tissue located in the prostate gland.

[0322] In some embodiments related to the diagnosis of a disease, where the disease is liver cancer, the biological sample is a sample as described above that is obtained from the liver of a subject, for example from tumor tissue located in the liver.

[0323] In some embodiments related to the diagnosis of a disease, where the disease is oral cancer, the biological sample is a sample as described above that is obtained from the oral cavity of a subject, such as from tumor tissue located in the oral cavity.

[0324] In some embodiments related to the diagnosis of a disease wherein the disease is colorectal cancer, the biological sample is a sample as described above that is obtained from the colon or rectum of a subject, such as from tumor tissue located in the colon or rectum .

[0325] In some embodiments related to the diagnosis of a disease, where the disease is gastric cancer, the biological sample is a sample as described above that is obtained from the stomach of a subject, for example from tumor tissue located in the stomach.

[0326] In some embodiments related to the diagnosis of a disease, where the disease is an inflammatory gastrointestinal disease or a functional gastrointestinal disorder, the biological sample is a sample as described above that is obtained from the gastrointestinal tract (such like the colon) of the subject.

[0327] In some embodiments related to the diagnosis of a disease, where the disease is inflammatory bowel disease, the biological sample is a sample as described above that is obtained from the gastrointestinal tract (such as the colon) of a subject.

[0328] In some embodiments, related to the diagnosis of a disease, where the disease is ulcerative colitis, Crohn's disease, diversion colitis, colitis of undetermined etiology and pouchitis, microscopic colitis, immuno-oncological colitis, colitis due to chemotherapy/radiation therapy, colitis associated with a syndrome graft versus host disease (GvHD), acute colitis, Behçet's disease, collagenous colitis, lymphocytic colitis, infectious diarrhea, mesenteric ischemia, diverticulitis or necrotizing enterocolitis (NEC), a biological specimen is a specimen as described above that is obtained from a gastrointestinal tract intestinal tract (eg, large intestine) of the subject.

[0329] In some embodiments related to the diagnosis of a disease, where the disease is pancreatitis, the biological sample is a sample as described above obtained from the pancreas of the subject.

[0330] In some embodiments related to the diagnosis of a disease, where the disease is an infection, the biological sample is a sample, as described above, obtained from an infected area or part of the body of a subject.

[0331] In some embodiments related to diagnosing a disease, wherein the disease is Alzheimer's disease, the biological sample is a sample as described above that is obtained from the brain of a subject.

[0332] In some embodiments related to diagnosing a disease, wherein the disease is multiple sclerosis, the biological sample is a sample as described above that is obtained from the brain of a subject.

[0333] In some embodiments related to the diagnosis of a disease, where the disease is neuropathic pain, the biological sample is a sample as described above obtained from the spinal cord of the subject.

[0334] In some embodiments, the biological sample includes a sample obtained from pathological tissue of a subject (eg, tumor tissue, inflamed tissue, and/or infected tissue). In some embodiments, the biological sample includes a sample obtained from pathological tissue of a subject and a sample obtained from normal (non-pathological) tissue from the same subject as a control.

[0335] In some embodiments, the disease (to be diagnosed or treated) is oral cancer. In some such embodiments, the oral cancer is oral squamous cell carcinoma. For diagnosing oral cancer, such as oral squamous cell carcinoma, the biological sample may be a tissue sample (tissue lysate) obtained from the oral cavity of a subject, such as tumor tissue located in the oral cavity. In some such embodiments, the biological sample is a biopsy material from the oral mucosa obtained from a subject (eg, including biopsy material from tumor tissue and from normal (non-pathological) tissue from the same subject).

[0336] In some embodiments, the disease associated with cysteine protease activity is a disease associated with cysteine protease activity, wherein the cysteine protease is a mammalian cysteine protease. In some embodiments, the cysteine protease is a human cysteine protease. In some embodiments, the cysteine protease is a cysteine cathepsin. In some embodiments, the cysteine protease is a mammalian cysteine cathepsin. In some embodiments, the cysteine protease is a human cysteine cathepsin. In some embodiments, the cysteine protease is cathepsin S and/or cathepsin X. In some embodiments, the cysteine protease is cathepsin X. In certain embodiments, the cysteine protease is mammalian cathepsin X. In some embodiments, the cysteine protease is human cathepsin X.

[0337] In some embodiments, the disease associated with cysteine protease activity is a disease associated with cathepsin X activity.

[0338] Synthesis of chloromethyl ketones via a sulfoxonium ylide intermediate

[0339] Many of the reported probes based on cysteine protease activity include active groups from acyloxymethyl ketone (AOMK) or phenoxymethyl ketone (PMK) (Edgington et al., 2009; 2012; 2013; Oresic Bender et al., 2015; Verdoes et al., 2013; 2012). The synthesis of these electrophiles requires the formation of chloromethyl ketone intermediates, a process that has historically been achieved through the formation of diazomethane, an extremely explosive yellow gas. The present inventors have now discovered a new synthetic route to access chloromethyl ketones via a sulfoxonium ylide intermediate (Scheme 2) that does not require the formation of diazomethanes, thereby avoiding this potentially dangerous reaction and thus providing a safer alternative to previously used methods. This method also eliminates the need for N-methyl-N-nitroso-p-toluenesulfonamide (Diazald®), a product that was recently discontinued by Sigma Aldrich.

[0340] Thus, in some embodiments, the present invention provides a method for preparing a compound containing a chloromethyl ketone group, comprising reacting a compound bearing a sulfoxonium ylide moiety to produce a compound bearing a chloromethyl ketone group.

[0341] In some embodiments, the present invention provides a method for preparing an activity-based probe compound bearing an acyloxymethyl ketone moiety or a phenoxymethyl ketone moiety as an active group, comprising:

(i) preparing an intermediate containing a chloromethyl ketone group by reacting a compound containing a sulfoxonium ylide moiety to produce a compound bearing a chloromethyl ketone group; And

(ii) subsequent processing of the compound containing the chloromethyl ketone group to obtain the specified probe compound based on activity.

[0342] In some of the above embodiments, the method includes reacting a compound bearing a sulfoxonium ylide moiety with hydrochloric acid to produce a compound containing a chloromethyl ketone moiety. In some such embodiments, the compound bearing the sulfoxonium ylide moiety is reacted with hydrochloric acid at an elevated temperature. In some such embodiments, the compound bearing the sulfoxonium ylide moiety is reacted with hydrochloric acid at an elevated temperature and in an organic solvent. In some such embodiments, the organic solvent includes an ether, such as tetrahydrofuran.

[0343] In some embodiments, the sulfoxonium ylide moiety has formula (IV):

[0344]

[0345] where:

R ₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl; And

R ₂ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl. In some embodiments, R ₁ is (C ₁ -C ₈ ) alkyl, and R ₂ is (C ₁ -C ₈ ) alkyl. In some embodiments, wherein R ₁ is methyl and R ₂ is methyl.

[0346] Using the improved method described above, three AOMK probes carrying Lys, Phe and Nle were successfully generated, suggesting that this method can be widely applied to the synthesis of various ABPs. Comparison of the synthesized AOMK probes with sulfoxonium ylide probes carrying identical recognition sequences showed that the corresponding sulfoxonium ylide probes were more potent and specific than the acyloxymethyl ketone analogues, further confirming the utility of the new sulfoxonium ylide active moiety. Thus, sulfoxonium ylide probes represent a clear advance in the tools available to study the function of cysteine proteases such as cathepsin X.

[0347] In a further aspect, the invention provides a compound having a formula selected from the group of formulas consisting of:

[0348]

[0349]

[0350]

[0351] or salts thereof.

Detailed Description of the Invention

[0352] The present invention is now more fully described with reference to accompanying examples. However, it should be understood that the following description is illustrative only and should in no way be construed as limiting the invention.

[0353] Examples

[0354] Synthesis and characterization of compounds

[0355] General information

[0356] All reagents were purchased from commercial suppliers and used without further purification.

[0357] Sulfo-Cy5 (free carboxylic acid form) was purchased from W&J Pharmachem.

[0358] All solvents used were HPLC grade. All water-sensitive reactions were carried out in anhydrous solvents under an argon atmosphere.

[0358] RP-HPLC was performed on a Phenomenex Luna C-8 column (100 Å, 10 μm, 250 x 21.5 mm) using a Waters 600 semipreparative HPLC incorporating a Waters 486 UV detector. The elution profile was a linear gradient from 0 -60% buffer A to buffer B (buffer A: 0.1% TFA in water; buffer B: 0.1% TFA in acetonitrile) for 60 min at a flow rate of 15 ml/min. The identity of the compound was confirmed by ESI-MS using a Shimadzu LCMS2020 instrument incorporating a Phenomenex Luna C-8 column (100 Å, 3 μm, 100 × 2.00 mm). The elution profile was 0.1% TFA in water for 4 min followed by a linear gradient from 0–60% buffer A to buffer B over 10 min at a flow rate of 0.2 ml/min.

[0359] Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker Avance III Nanobay spectrometer coupled to a BACS 60 automatic sample changer. The spectrometer was equipped with a PABBO BB-1H/DZ-GRD 5 mm probe. 1H NMR spectra were obtained at 400 MHz. Each resonance was assigned according to the following condition: chemical shift (δ) measured in parts per million (ppm) relative to the residual non-deuterated solvent peak of chloroform (unless otherwise stated) as an internal reference relative to trimethylsilane (= 0), multiplicity, coupling constants (J Hz), number of protons and assignment. Multiplicities are designated as (c) singlet, (e) doublet, (dd) doublet of doublets, (t) triplet, (q) quartet, or (m) multiple.

[0360] Carbon nuclear magnetic resonance ( ¹³ C NMR) spectra were obtained at 100 MHz. Each resonance was assigned according to the following condition: chemical shift (δ) measured in parts per million (ppm) relative to the residual non-deuterated solvent peak of chloroform (unless otherwise stated) as an internal reference relative to trimethylsilane (= 0), number of carbon atoms, and assignment.

[0361] Liquid chromatography mass spectrometry (LCMS) was performed on an Agilent UHPLC/MS 1260 instrument (G1311A 1200 series quaternary pump, autosampler: G1329A 1200 series thermostated autosampler, detector: G1314B 1200 series variable wavelength detector). The elution profile was a linear gradient of 5–100% B over 2.5 min at a flow rate of 0.5 mL/min. Solvent A: water 0.1% formic acid, solvent B: acetonitrile 0.1% formic acid. Liquid chromatography conditions; Analysis by reverse phase HPLC, Column: Poroshell 120 EC-C18 3.0 x 50 mm 2.7 µm, column temperature: 35°C, injection volume: 1 µl, detection: monitored at 254 nm and 214 nm. Mass spectrometer conditions: ion source: quadrupole, ion mode: API-ES, drying gas temperature: 350°C. Major ion peaks (m/z) are indicated with major peak intensities in parentheses.

[0362] High resolution mass spectra (HRMS) were acquired on an Agilent 6224 TOF LC/MS mass spectrometer coupled to an Agilent 1290 Infinity (Agilent, Palo Alto, CA). All data were recorded and the reference mass was corrected using a dual-spray electrospray ionization (ESI) source. Mass spectrometer conditions: ionization mode: electrospray ionization; drying gas flow: 11 l/min, solvent A=aqueous solution of 0.1% formic acid, solvent B=acetonitrile/0.1% formic acid. The found and calculated ion peaks (m/z) are shown.

[0363] All HPLC analytical analyzes were performed on an Agilent 1260 Infinity Analytical HPLC coupled to a 1260 Degasser: G1322A, 1260 Binary Pump: G1312B, 1260 HiP ALS Autosampler: G1367E, 1260 TCC: G1316A and 1260 DAD Detector: G42 12B. The column used was a Zorbax Eclipse Plus C18 Rapid Resolution 4.6 mm × 100 mm 3.5 μm. The volume of the injected sample was 2 μL, which was treated in 0.1% TFA in acetonitrile with a gradient from 5 to 100% over 10 min at a flow rate of 1 ml/min. The analysis was performed at 214 nm and 254 nm.

[0364] Example 1: Synthesis of Sulfoxonium Ylide Compounds

[0365] Compounds containing sulfoxonium ylide (probes sCy5-AA-SY) were synthesized according to reaction scheme 1 and in accordance with the general methods A, B and E described below.

[0366]

Scheme 1. Synthesis of sCy5-AA-SY probes. i) 4-nitrophenylchloroformate, Et ₃ N, DMAP, CH ₂ Cl ₂ , 0°C, 6 hours. ii) SOMe ₃ ⁺ I ^- KOtBu, THF, reflux → 0°C. iii) TPA/CH ₂ Cl ₂ mixture (1:1), iv) sulfo-Cy5, PyClock, DIPEA, DMF, room temperature, 18 h.

General Method A. Preparation of Nitrophenyl Esters

Triethylamine (1.2 eq) was added to a solution of Boc-protected amino acid (1 eq) in CH ₂ Cl ₂ (5 ml). The stirred mixture was cooled to 0°C and 4-nitrophenyl chloroformate (1.2 eq) was added. After 10 min, DMAP (0.1 eq) was added and the mixture was stirred at 0°C for 6 h. The reaction mixture was further diluted with CH ₂ Cl ₂ (15 ml) and washed with saturated NaHCO ₃ (10 ml), 0.1 M HCl solution (10 ml), brine (10 ml), and then dried (MgSO ₄ ), filtered and the solvent was evaporated in vacuo to obtain the crude product.

[0368] General Method B. Preparation of Sulfoxonium Ylides

Trimethylsulfoxonium iodide (4 equiv) was suspended in dry THF (5 ml) and KOtBu (4 equiv) was added. The mixture was stirred at reflux for 3 hours in the dark. The reaction mixture was cooled to 0°C, a solution of nitrophenyl ether (1 eq) in THF was added dropwise and stirred for 18 hours. The reaction was quenched with H ₂ O (30 ml) and the solution was concentrated in vacuo to remove THF. The remaining aqueous solution was extracted with EtOAc (3 x 30 ml). The combined organic extract was washed with brine (15 ml), dried (MgSO ₄ ), filtered and the solvent was removed in vacuo to obtain the crude product.

[0369] General Method E: Sulfocyanine 5 Labeling of Sulfoxonium Ylides

Boc-protected sulfoxonium ylide (1 equiv) was treated with a 1:1 mixture of TFA and CH ₂ Cl ₂ (2 ml) and stirred at ambient temperature for 1 hour. Volatile components were removed under a stream of nitrogen. DMF (300 μl) and DIPEA (4 equiv) were added to the resulting residue. Separately, Sulfo-Cy5 (1 equiv) and PyClock (2 equiv) were dissolved in DMF (300 μl) and DIPEA (4 equiv) and stirred for 2 min before adding to the above solution. The reaction mixture was stirred at ambient temperature for 18 h in the dark.

[0370] Example 1.1: Synthesis and Characterization of Nitrophenyl Esters Intermediates

[0371] 4-Nitrophenyl (tert-butoxycarbonyl)-L-valinate (SJM-724-20)

[0372]

[0373] Ice-cold dry DMF (20 mL) was slowly added to 4-nitrophenyl chloroformate (2.27 g, 11.2 mmol) and stirred for 10 minutes under nitrogen. The mixture was then gradually heated to ambient temperature. A solution of boc-L-valine (2.02 g, 9.30 mmol) in DMF (7 mL) was added dropwise, followed by triethylamine (1.50 mL, 10.8 mmol). Stirring was continued for 1 hour. The reaction mixture was diluted with EtOAc (50 ml) and washed with H ₂ O (3 x 30 ml) and brine (30 ml). The organic layer was dried (MgSO ₄ ), filtered and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography (SiO ₂ , 10-13% EtOAc:petroleum alcohol) gave the product as a viscous, colorless oil (2.01 g, 64%). HRMS (ESI ⁺ ): found: m/z 361.1386 (M+Na) ⁺ , C ₁₆ H ₂₂ N ₂ NaO ₆ ⁺ calculated m/z 361.1370. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.29-8.23 (m, 2H), 7.31-7.25 (m, 2H), 5.05 (d, J=8.4 Hz, 1H), 4.44 (dd, J=8.6, 5.1 Hz, 1H), 2.37-2.24 (m, 1H), 1.45 (s, 9H), 1.09 (d , J=6.8 Hz, 3H), 1.03 (d, J=6.9 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 170.6 (C), 155.9 (C), 155.3 (C), 145.7 (C), 125.4 (CH), 122.5 ( CH), 80.6 (C), 59.1 (CH), 31.2 (CH), 28.4 (CH3), 19.3 ( _CH3 ), 17.9 ( _CH3 ). LC-MS (ESI +) m/z: 339.3 (M+H) ⁺ (40%), 360.8 (M+Na) ⁺ (80%).

[0374] 4-Nitrophenyl (tert-butoxycarbonyl)-L-isoleucinate (SJM-724-64)

[0375]

[0376] The title compound was prepared by Method A from boc-L-isoleucine (200 mg, 0.86 mmol), 4-nitrophenyl chloroformate (209 mg, 1.04 mmol), DMAP (11 mg, 0.09 mmol) and Et ₃ N (145 µl, 1.04 mmol) in CH ₂ Cl ₂ to give the crude product as a colorless oil. Purification by column chromatography (SiO ₂ , petroleum alcohol:EtOAc 9:1) gave the product as a white solid (259 mg, 85%). ¹ H NMR (401 MHz, CDCl ₃ ) δ 8.31-8.25 (m, 2H), 7.33-7.27 (m, 2H), 5.03 (d, J=7.8 Hz, 1H), 4.49 (dd, J=8.3, 5.2 Hz, 1H), 2.11-1.97 (m, 1H), 1.65-1.52 (m, 1H), 1 .47 (s, 9H), 1.38-1.23 (m, 1H), 1.07 (d, J=6.8 Hz, 3H), 1.00 (t, J=7.4 Hz, 3H). LC-MS (ESI ⁺ ) m/z: 252.9 (M - Boc+ H) ⁺ (100%), 374.9 (M+Na) ⁺ (60%).

[0377] 4-nitrophenyl (tert-butoxycarbonyl)-L-leucinate (SJM-724-76)

[0378]

[0379] The title compound was prepared by Method A from boc-L-leucine (200 mg, 0.86 mmol), 4-nitrophenyl chloroformate (209 mg, 1.04 mmol), DMAP (11 mg, 0.09 mmol) and Et ₃ N (145 µl, 1.04 mmol) in CH ₂ Cl ₂ to give the crude product as a yellow oil. Purification by column chromatography (SiO ₂ , petroleum alcohol:EtOAc 9:1) gave the product as a white solid (192 mg, 63%). ¹ H NMR (401 MHz, CDCl ₃ ) δ 8.32-8.24 (m, 2H), 7.35-7.28 (m, 2H), 4.91 (d, J=7.8 Hz, 1H), 4.59-4.44 (m, 1H), 1.89-1.73 (m, 2H), 1.73-1.62 (m, 1H), 1.46 (s, 9H) , 1.03 (d, J=2.1 Hz, 3H), 1.02 (d, J=1.9 Hz, 3H). LC-MS (ESI ⁺ ) m/z: 252.9 (M - Boc+H) ⁺ (100%), 374.9 (M+Na) ⁺ (55%).

[0380] 4-nitrophenyl (S)-2-((tert-butoxycarbonyl)amino)hexanoate (SJM-724-116)

[0381]

[0382] The title compound was prepared by Method A from boc-L-norleucine (200 mg, 0.86 mmol), 4-nitrophenyl chloroformate (209 mg, 1.04 mmol), DMAP (11 mg, 0.09 mmol) and Et ₃ N (145 µl, 1.04 mmol) in CH ₂ Cl ₂ to give the crude product as a light yellow solid. Purification by column chromatography (SiO ₂ , petroleum alcohol:EtOAc 9:1) gave the product as a white solid (198 mg, 65%). HRMS (ESI ⁺ ): found: m/z 375.1534 (M+Na) ⁺ , for C ₁₇ H ₂₄ N ₂ NaO ₆ ⁺ calculated m/z 375.1527. 1H NMR (401 MHz, CDCl ₃ ) δ 8.32-8.25 (m, 2H), 7.33-7.27 (m, 2H), 4.99 (d, J=7.3 Hz, 1H ), 4.50 (dd, J=13.0, 7.4 Hz, 1H), 2.02-1.91 (m, 1H), 1.86-1.74 (m, 1H), 1, 51-1.35 (m, 13H), 0.95 (t, J=7.2 Hz, 3H). LC-MS (ESI ⁺ ) m/z: 252.9 (M - Boc+H) + (100%), 374.9 (M+Na) ⁺ (95%).

[0383] 4-nitrophenyl (tert-butoxycarbonyl)-L-phenylalaninate (SJM-724-68)

[0384]

[0385] The title compound was prepared by Method A from boc-L-phenylalanine (200 mg, 0.75 mmol), 4-nitrophenylchloroformate (182 mg, 0.90 mmol), DMAP (9 mg, 0.08 mmol) and Et ₃ N (126 µl, 0.90 mmol) in CH ₂ Cl ₂ to give the crude product as a white solid. Purification by column chromatography (SiO ₂ , CH ₂ Cl ₂ ) gave the product as a white solid (251 mg, 86%). ^1H NMR (401 MHz, CDCl ₃ ) δ 8.29-8.22 (m, 2H), 7.40-7.29 (m, 3H), 7.25-7.21 (m, 2H), 7.17-7.11 (m, 2H), 5.03 (d, J=7.0 Hz, 1H), 4.80 (dd, J=13.6, 6.4 Hz, 1H), 3 .31-3.15 (m, 2H), 1.45 (s, 9H). LC-MS (ESI ⁺ ) m/z: 286.9 (M - Boc+H) ⁺ (60%), 408.8 (M+Na) ⁺ (40%).

[0386] 4-Nitrophenyl (tert-butoxycarbonyl)-L-tryptonate (SJM-724-112)

[0387]

[0388] The title compound was prepared by Method A from boc-L-tryptophan (200 mg, 0.66 mmol), 4-nitrophenylchloroformate (159 mg, 0.79 mmol), DMAP (8 mg, 0.07 mmol) and Et ₃ N (110 µl, 0.79 mmol) in CH ₂ Cl ₂ to give the crude product as a yellow solid. Purification by column chromatography (SiO ₂ , petroleum alcohol:EtOAc2:1) gave the product as a white solid (200 mg, 72%). ¹ H NMR (401 MHz, CDCl ₃ ) δ 8.22-8.13 (m, 3H), 7.60 (d, J=7.9 Hz, 1H), 7.41 (d, J=8, 1 Hz, 1H), 7.25-7.22 (m, 1H), 7.16-7.11 (m, 2H), 7.03-6.98 (m, 2H), 5.15 (d , J=7.6 Hz, 1H), 4.91-4.80 (m, 1H), 3.50-3.35 (m, 2H), 1.45 (s, 9H). LC-MS (ESI ⁺ ) m/z: 325.9 (M - Boc+H) ⁺ (100%).

[0389] 4-Nitrophenyl N ^a -((benzyloxy)carbonyl)-Ne-(tert-butoxycarbonyl)-L-lysinate (SJM-724-40)

[0390]

[0391] Ice-cold dry DMF (20 mL) was slowly added to 4-nitrophenyl chloroformate (1.27 g, 6.31 mmol) and stirred for 10 minutes under nitrogen. The mixture was then gradually heated to ambient temperature. A solution of N ^a -benzyloxycarbonyl-Ne-(tert-butoxycarbonyl)-L-lysine (2.00 g, 5.26 mmol) in DMF (5 mL) was added dropwise, followed by triethylamine (879 μL, 6.31 mmol) . Stirring was continued for 20 hours. The reaction mixture was diluted with EtOAc (50 ml) and washed with H ₂ O (4 x 20 ml) and brine (20 ml). The organic layer was dried (MgSO ₄ ), filtered and concentrated under reduced pressure to give the crude product as a yellow oil. Purification by column chromatography (SiO ₂ , CH ₂ Cl ₂ :EtOAc 95:5) gave the product as a white solid (672 mg, 25%). ¹ H NMR (401 MHz, CDCl ₃ ) δ 8.27 (d, J=8.9 Hz, 2H), 7.40-7.27 (m, 7H), 5.63-5.49 (m, 1H), 5.14 (s, 2H), 4.64-4.49 (m, 2H), 3.15 (s, 2H), 2.10-1.82 (m, 2H), 1.56 -1.47 (m, 4H), 1.42 (s, 9H). LC-MS (ESI ⁺ ) m/z: 401.9 (M - Boc+H) ⁺ (100%), 524.8 (M+Na) ⁺ (20%).

[0392] (tert-Butoxycarbonyl)-L-phenylalanyl-L-valine (MS-4-178)

[0393]

[0394] Fmoc-Val-OH (679 mg, 2.0 mmol) was dissolved in a solution of DCM and triethylamine (842 μl, 6.0 mmol) and added to 2-chlorotrityl resin (1.0 g, 1 mmol) (1 mEq/g). The mixture was shaken at ambient temperature for 1 hour. The resin was washed with DCM (3-5 ml). A solution of MeOH (1 ml) in DCM (5 ml) and triethylamine (0.5 ml) was added to the resin and shaken for 30 min at ambient temperature. Fmoc was deprotected by treatment with 20% piperidine in DMF for 10 min and then washed with DMF (3-5 ml). Boc-Phe-OH (531 mg, 2.0 mmol) and PyBOP (1.04 g, 2.0 mmol) were dissolved in a solution of DCM and triethylamine (842 μL, 6.0 mmol) and added to the resin. The mixture was shaken at ambient temperature for 1 hour, and then drained, washed with DCM (3-5 ml) and dried in vacuum. The dipeptide was cleaved from the resin by treatment with 20% HFIP in DCM containing 1% TIPS for 2 hours. The resin was filtered and the filtrate was evaporated in vacuo to give crude product (180 mg).

[0395] 4-nitrophenyl (tert-butoxycarbonyl)-L-phenylalanyl-L-valinate (MS-4-182)

[0396]

[0397] To a solution of 4-nitrophenyl chloroformate (116 mg, 0.58 mmol) in dry THF (1 mL) was added dry DMF (150 μL) at 0°C. The reaction mixture was stirred for 10 min under a nitrogen atmosphere before gradually warming to ambient temperature. A solution of (tert-butoxycarbonyl)-L-phenylalanyl-L-valine (175 mg, 0.48 mmol) in THF (1 mL) and triethylamine (80 μL) was added dropwise to the above suspension. Stirring was continued for 20 minutes. The reaction mixture was diluted with EtOAc and washed with H ₂ O. The organic layer was dried (MgSO ₄ ), filtered and concentrated under reduced pressure to give the crude product (220 mg) as a pale yellow solid.

[0398] Example 1.2. Synthesis and characterization of sulfoxonium ylide compounds

[0399] tert-Butyl (S)-(1-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-4-methyl-2-oxopentan-3-yl)carbamate (SJM-724-24)

[0400]

[0401] The title compound was prepared by Method B from 4-nitrophenyl(tert-butoxycarbonyl)-L-valinate (SJM-724-20) (100 mg, 0.30 mmol). The resulting crude product was purified by column chromatography (SiO2, EtOAc) and recrystallized from diethyl ether to give the product as a white solid (58 mg, 67%). HRMS (ESI ⁺ ): found: m/z 292.1573 (M+H) ⁺ , for C ₁₃ H ₂₆ NO ₄ S ⁺ calculated m/z 292.1577. ¹ H NMR (401 MHz, CDCl ₃ ) δ 5.23 (d, J=8.4 Hz, 1H), 4.50 (s, 1H), 3.94 (dd, J=8.6, 5, 4 Hz, 1H), 3.42 (s, 3H), 3.39 (s, 3H), 2.10-1.99 (m, 1H), 1.43 (s, 9H), 0.94 ( d, J=6.7 Hz, 3H), 0.87 (d, J=6.8 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 187.89 (C), 155.94 (C), 79.11 (C), 70.07 (CH), 61.89 (CH), 42.25 ( CH ₃ ), 42.00 (CH ₃ ), 31.90 (CH), 28.43 (CH ₃ ), 19.60 (CH ₃ ), 17.66 (CH ₃ ). LC-MS (ESI ⁺ ) m/z: 291.9 (M+H) ⁺ (85%).

[0402] tert-Butyl ((3S,4S)-1-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-4-methyl-2-oxohexan-3-yl)carbamate (SJM-724-92)

[0403]

[0404] The title compound was prepared by Method B from 4-nitrophenyl (tert-butoxycarbonyl)-L-isoleucinate (SJM-724-64) (200 mg, 0.57 mmol). The resulting crude product was purified by column chromatography (SiO2, EtOAc:MeOH, 95:5) to give the product as a cream solid (126 mg, 73%). HRMS (ESI ⁺ ): found: m/z 306.1735 (M+H) ⁺ , C ₁₄ H ₂₈ NO ₄ S ⁺ calculated m/z 306.1734. ¹ H NMR (401 MHz, CDCl ₃ ) δ 5.21 (d, J=8.9 Hz, 1H), 4.49 (s, 1H), 3.97 (dd, J=8.7, 5, 7 Hz, 1H), 3.41 (s, 3H), 3.38 (s, 3H), 1.83-1.73 (m, 1H), 1.53-1.46 (m, 1H), 1.43 (s, 9H), 1.18-1.04 (m, 1H), 0.91 (d, J=6.7 Hz, 3H), 0.90 (t, J=7.5 Hz , 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 187.8 (C), 155.8 (C), 79.1 (C), 70.0 (CH), 61.3 (CH), 42.4 ( CH ₃ ), 42.1 (CH ₃ ), 38.5 (CH), 28.5 (CH ₃ ), 24.9 (CH ₂ ), 15.9 (CH ₃ ), 11.9 (CH ₃ ) . LC-MS (ESI ⁺ ) m/z: 305.9 (M+H) ⁺ (100%).

[0405] tert-Butyl (S)-(1-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-5-methyl-2-oxohexan-3-yl)carbamate (SJM-724-96)

[0406]

[0407] The title compound was prepared by Method B from 4-nitrophenyl (tert-butoxycarbonyl)-L-leucinate (SJM-724-76) (170 mg, 0.48 mmol). The resulting crude product was purified by column chromatography (SiO ₂ , EtOAc:MeOH, 95:5) to give the product as a white solid (118 mg, 80%). HRMS (ESI ⁺ ): found: m/z 306.1735 (M+H) ⁺ , C ₁₄ H ₂₈ NO ₄ S ⁺ calculated m/z 306.1734. ¹ H NMR (401 MHz, CDCl ₃ ) δ 5.04 (d, J=8.3 Hz, 1H), 4.51 (s, 1H), 4.16-4.06 (m, 1H), 3 .41 (s, 3H), 3.38 (s, 3H), 1.75-1.62 (m, 2H), 1.62-1.50 (m, 1H), 1.43 (s, 9H ), 0.94 (d, J=4.9 Hz, 3H), 0.93 (d, J=4.9 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 189.2 (C), 155.6 (C), 79.2 (C), 68.7 (CH), 55.6 (CH), 43.4 ( CH2), 42.4 (CH3), 42.1 ( _CH3 ), 28.5 ( _CH3 ), 25.0 (CH), 23.2 ( _CH3 ), 22.3 ( _CH3 ). LC-MS (ESI ⁺ ) m/z: 305.9 (M+H) ⁺ (80%).

[0408] tert-Butyl (S)-(1-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-2-oxoheptan-3-yl)carbamate (SJM-724-124)

[0409]

[0410] The title compound was prepared by Method B from 4-nitrophenyl (S)-2-((tert-butoxycarbonyl)amino)hexanoate (SJM-724-116) (170 mg, 0.48 mmol). The resulting crude product was purified by column chromatography (SiO2, EtOAc:MeOH, 95:5) to give the product as a white solid (128 mg, 87%). HRMS (ESI ⁺ ): found: m/z 306.1738 (M+H) ⁺ , C ₁₄ H ₂₈ NO ₄ S ⁺ calculated m/z 306.1734. ¹ H NMR (401 MHz, CDCl ₃ ) δ 5.19 (d, J=8.1 Hz, 1H), 4.49 (s, 1H), 4.00 (dd, J=13.4, 7, 5 Hz, 1H), 3.37 (s, 3H), 3.35 (s, 3H), 1.78-1.62 (m, 1H), 1.53-1.43 (m, 1H), 1.39 (s, 9H), 1.33-1.18 (m, 4H), 0.84 (t, J=7.0 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 188.6 (C), 155.6 (C), 79.1 (C), 68.9 (CH), 57.0 (CH), 42.3 ( CH ₃ ), 42.0 (CH ₃ ), 33.8 (CH2), 28.4 (CH ₃ ), 27.7 (CH ₂ ), 22.6 (CH ₂ ), 14.0 (CH ₃ ) . LC-MS (ESI ⁺ ) m/z: 306.0 (M+H) ⁺ (100%).

[0411] tert-Butyl (S)-(4-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-3-oxo-1-phenylbutan-2-yl)carbamate (SJM-724-72)

[0412]

[0413] The title compound was prepared by Method B from 4-nitrophenyl (tert-butoxycarbonyl)-L-phenylalaninate (SJM-724-68) (200 mg, 0.75 mmol). The resulting crude product was purified by column chromatography (SiO2, CH ₂ Cl ₂ ) to give the product as a beige solid (141 mg, 80%). HRMS (ESI ⁺ ): found: m/z 340.1579 (M+H) ⁺ , for C ₁₇ H ₂₆ NO ₄ S ⁺ calculated m/z 340.1577. ¹ H NMR (401 MHz, CDCl ₃ ) δ 7.29-7.17 (m, 5H), 5.22 (d, J=8.1 Hz, 1H), 4.32 (dd, J=14, 5, 7.4 Hz, 1H), 4.26 (s, 1H), 3.35 (s, 3H), 3.25 (s, 1H), 3.05-2.93 (m, 2H), 1.41 (s, 9H). LC-MS (ESI ⁺ ) m/z: 339.9 (M+H) ⁺ (100%).

[0414] tert-Butyl (S)-(4-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-1-(1H-indol-3-yl)-3-oxobutan-2-yl)carbamate (SJM -724-120)

[0415]

[0416] The title compound was prepared by Method B from 4-nitrophenyl (tert-butoxycarbonyl)-L-tryptophanate (SJM-724-112) (170 mg, 0.40 mmol). The resulting crude product was purified by column chromatography (SiO ₂ , EtOAc:MeOH 95:5) to give the product as a white solid (118 mg, 78%). HRMS (ESI ⁺ ): found: m/z 379.1697 (M+H) ⁺ , C ₁₉ H ₂₇ N ₂ O ₄ S + calculated m/z 379.1686. ¹ H NMR (401 MHz, CDCl ₃ ) δ 8.44 (s, 1H), 7.63 (d, J=7.9 Hz, 1H), 7.32 (d, J=8.1 Hz, 1H ), 7.13 (t, J=7.3 Hz, 1H), 7.08-6.98 (m, 2H), 5.40 (d, J=7.8 Hz, 1H), 4.37 (dd, J=13.3, 7.1 Hz, 1H), 4.25 (s, 1H), 3.19 (s, 3H), 3.26-3.09 (m, 2H), 2, 99 (s, 3H), 1.43 (s, 9H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 187.5 (C), 155.5 (C), 136.1 (C), 128.2 (C), 123.0 (CH), 121.9 ( CH), 119.3 (CH), 119.2 (CH), 111.7 (C), 111.3 (CH), 79.4 (C), 70.0 (CH), 57.8 (CH ), 41.8 (CH3), 41.6 ( _CH3 ), 29.3 (CH2), 28.54 ( _CH3 ). LC-MS (ESI ⁺ ) m/z: 378.9 (M+H) ⁺ (100%).

[0417] Benzyl tert-butyl (7-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-6-oxoheptane-1,5-diyl)(S)-dicarbamate (SJM-724-48)

[0418]

[0419] The title compound was prepared by Method B from 4-nitrophenyl-N ^a -((benzyloxy)carbonyl)-Ne-(tert-butoxycarbonyl)-L-lysinate (SJM-724-40) (200 mg, 0.40 mmol). The resulting crude product was purified by column chromatography (SiO2, EtOAc:MeOH, 9:1) to give the product as a white solid (138 mg, 76%). HRMS (ESI ⁺ ): found: m/ z 455.2216 (M+H) ⁺ , C ₂₂ H ₃₅ N ₂ O ₆ S ⁺ required m/z 455.2210. ¹ H NMR (401 MHz, CDCl ₃ ) δ 7.33-7.23 (m, 5H), 5.79 (d, J=7.9 Hz, 1H), 5.04 (s, 2H), 4 .72 (b.s, 1H), 4.52 (s, 1H), 4.07 (dd, J=13.1, 7.6 Hz, 1H), 3.32 (s, 3H), 3. 30 (s, 3H), 3.09-2.97 (m, 2H), 1.79-1.67 (m, 1H), 1.59-1.50 (m, 1H), 1.50- 1.26 (m, 4H), 1.38 (s, 9H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 187.6 (C), 156.1 (C), 136.7 (C), 128.5 (CH), 128.1 (CH), 128.0 ( CH), 78.9 (C), 69.5 (CH), 66.6 (CH2), 57.1 (CH), 42.0 ( _CH3 ), 41.8 ( _CH3 ), 40.3 (CH ₂ ), 33.4 (CH ₂ ), 29.7 (CH ₂ ), 28.4 (CH ₃ ), 22.5 (CH ₂ ). LC-MS (ESI ⁺ ) m/z: 454.9 (M+H) ⁺ (100%).

[0420] tert-Butyl ((S)-1-(((S)-1-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-4-methyl-2-oxopentan-3-yl)amino)- 1-oxo-3-phenylpropan-2-yl)carbamate (MS-4-186)

[0421]

[0422] The title compound was prepared by Method B from 4-nitrophenyl (tert-butoxycarbonyl)-L-phenylalanyl-L-valinate (MS-4-182) (200 mg, 0.41 mmol). The resulting crude product was purified by column chromatography (SiO ₂ , CHCl ₂ :MeOH 98:2) to obtain the title product (68 mg, 38%).

[0423] Example 1.3: Synthesis and Characterization of Sulfoxonium Ylide Probes

[0424] sCy5-Val-SY (SJM-724-28)

[0425]

[0426] The title compound was prepared by Method E from tert-butyl (S)-(1-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-4-methyl-2-oxopentan-3-yl)carbamate (SJM -724-24) (5 mg, 0.017 mmol). The resulting crude product was purified by RP-HPLC and lyophilized to give the product as a blue solid (7.9 mg, 56%). HRMS (ESI ⁺ ): found: m/z 830.3159 (M+H) ⁺ , C ₄₁ H ₅₆ N ₃ O ₉ S ₃ ⁺ calculated m/z 830.3173.

[0427] sCy5-Ile-SY (SJM-724-104)

[0428]

[0429] The title compound was prepared by Method E from tert-butyl ((3S,4S)-1-(dimethyl (oxo)-λ ⁶ -6-sulfanylidene)-4-methyl-2-oxohexan-3-yl)carbamate (SJM-724-92) (10 mg, 0.033 mmol). The resulting crude product was purified by RP-HPLC and lyophilized to give the product as a blue solid (3 mg, 11%). HRMS (ESI ⁺ ): found: m/z 844.3340 (M+H) ⁺ , C ₄₂ H ₅₈ N ₃ O ₉ S ₃ ⁺ calculated m/z 844.3330.

[0430] sCy5-Leu-SY (SJM-724-128)

[0431]

[0432] The title compound was prepared by Method E from tert-butyl (S)-(1-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-5-methyl-2-oxohexan-3-yl)carbamate (SJM -724-96) (10 mg, 0.033 mmol). The resulting crude product was purified by RP-HPLC and lyophilized to give the product as a blue solid (14.7 mg, 53%). HRMS (ESI ⁺ ): found: m/z 844.3328 (M+H) ⁺ , C ₄₂ H ₅₈ N ₃ O ₉ S ₃ ⁺ calculated m/z 844.3330.

[0433] sCy5-Nle-SY (SJM-724-132)

[0434]

[0435] The title compound was prepared by Method E from tert-butyl (S)-(1-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-2-oxoheptan-3-yl)carbamate (SJM-724-124 ) (10 mg, 0.033 mmol). The resulting crude product was purified by RP-HPLC and lyophilized to give the product as a blue solid (17.4 mg, 63%). HRMS (ESI ⁺ ): found: m/z 844.3335 (M+H) ⁺ , C ₄₂ H ₅₈ N ₃ O ₉ S ₃ ⁺ calculated m/z 844.3330.

[0436] sCy5-Phe-SY (SJM-724-80)

[0437]

[0438] The title compound was prepared by Method E from tert-butyl (S)-(4-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-3-oxo-1-phenylbutan-2-yl)carbamate (SJM -724-72) (10 mg, 0.029 mmol). The resulting crude product was purified by RP-HPLC and lyophilized to give the product as a blue solid (13.1 mg, 51%). HRMS (ESI ⁺ ): found: m/z 878.3157 (M+H) ⁺ , C ₄₅ H ₅₆ N ₃ O ₉ S ₃ ⁺ calculated m/z 878.3173.

[0439] sCy5-Trp-SY (SJM-724-80)

[0440]

[0441] The title compound was prepared by Method E from tert-butyl (S)-(4-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-1-(1H-indol-3-yl)-3-oxobutane -2-yl))carbamate (SJM-724-120) (10 mg, 0.026 mmol). The resulting crude product was purified by RP-HPLC and lyophilized to give the product as _a blue solid (6 mg, 25 HRMS ₍ ESI ⁺ ) _: found: 917.3277 (M+H)+, _C47H57N4O9 S ₃ ⁺ calculated m/z 917.3282.

[0442] Cbz-Lys(sCy5)-SY (SJM-724-100)

[0443]

[0444] The title compound was prepared by Method E from benzyl tert-butyl (7-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-6-oxoheptan-1,5-diyl)(S)-dicarbamate (SJM- 724-48) (10 mg, 0.022 mmol). The resulting crude product was purified by RP-HPLC and lyophilized to give the product as a blue solid (10 mg, 46%). HRMS (ESI ⁺ ): found: 993.3785 (M+H) ⁺ , C ₅₀ H ₆₅ N ₄ O ₁₁ S ₃ ⁺ calculated m/z 993.3806.

[0445] sCy5-Phe-Val-SY (MS-4-191)

[0446]

[0447] The title compound was prepared by Method E from tert-butyl ((S)-1-(((S)-1-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-4-methyl-2-oxopentane -3-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (MS-4-186) (10 mg, 0.022 mmol). The resulting crude product was purified by RP-HPLC and lyophilized to give the product as a blue solid (4.6 mg, 21%).

[0448] Example 2: Synthesis of acyloxymethyl ketones (AOMK)

Compounds having an active acyloxymethyl ketone group (AOMK) (sCy5-AA-AOMK probes) were synthesized according to reaction scheme 2 and in accordance with the general methods C, D and E described below.

[0450]

Scheme 2. Synthesis of sCy5-AA-AOMK probes via a sulfoxonium ylide intermediate. i) 4M HCl in dioxane, THF, reflux, 4 hours. ii) 2,6-dimethylbenzoic acid, KF, DMF, room temperature, 18 hours. iii) TFA/CH ₂ Cl ₂ (1:1), iv) Sulfo-Cy5, PyClock, DIPEA, DMF, room temperature, 18 h.

[0451] General Method C. Preparation of chloromethyl ketones

To a solution of sulfoxonium ylide (1 equiv) in dry THF (5 ml) was added 4 M HCl in dioxane (1.15 equiv). The solution was stirred at reflux for 4 hours. Solvents were removed in vacuo and the residue was treated with EtOAc (20 ml) and washed with H ₂ O (15 ml) and saturated NaHCO ₃ solution (15 ml). The organic layer was dried (MgSO ₄ ), filtered and concentrated in vacuo to give the crude product.

[0452] General Method D. Preparation of acyloxymethyl ketones (AOMK)

Potassium fluoride (3 equiv) was suspended in DMF (1 ml) and sonicated for 1 min. 2,6-dimethylbenzoic acid (1.1 eq) was added to the suspension and stirred at ambient temperature for 5 min. Chloromethyl ketone (1 eq) was added and the mixture was stirred at ambient temperature for 18 hours. DMF was removed in vacuo and the resulting residue was treated with EtOAc (20 ml) and washed with saturated NaHCO ₃ solution (15 ml). The organic layer was dried (MgSO ₄ ), filtered and the solvent was removed in vacuo to obtain the crude product.

[0453] General Method E: Sulfocyanine 5 (AOMK) labeling of acyloxymethyl ketones

Boc-protected acyloxymethyl ketone (AOMK) (1 eq) was treated with a 1:1 mixture of TFA and CH ₂ Cl ₂ (2 ml) and stirred at ambient temperature for 1 hour. Volatiles were removed under a stream of nitrogen. DMF (300 μl) and DIPEA (4 equiv) were added to the resulting residue. Separately, Sulfo-Cy5 (1 equiv) and PyClock (2 equiv) were dissolved in DMF (300 μL) and DIPEA (4 equiv) and stirred for 2 min before adding to the above solution. The reaction mixture was stirred at ambient temperature for 18 hours in the dark.

[0454] Example 2.1: Synthesis and Characterization of Chloromethyl Ketone Intermediates

[0455] tert-Butyl (S)-(1-chloro-2-oxoheptan-3-yl)carbamate (SJM-724-148)

[0456]

[0457] The title compound was prepared by Method C from tert-butyl (S)-(1-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-2-oxoheptan-3-yl)carbamate (SJM-724-124 ) (40 mg, 0.13 mmol). The resulting crude product was purified by column chromatography (SiO ₂ , petroleum alcohol:EtOAc, 9:1) to give the product as a white solid (20 mg, 58%). ¹ H NMR (401 MHz, CDCl ₃ ) δ 5.02 (d, J=6.4 Hz, 1H), 4.47 (dd, J=12.6, 7.7 Hz, 1H), 4.26 (d, J=4.8 Hz, 2H), 1.92-1.75 (m, 1H), 1.60-1.47 (m, 1H), 1.43 (s, 9H), 1, 39-1.25 (m, 4H), 0.90 (t, J=7.1 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 202.0 (C), 155.6 (C), 80.4 (C), 57.5 (CH), 46.8 (CH ₂ ), 31.3 (CH ₂ ), 28.4 (CH ₃ ), 27.6 (CH ₂ ), 22.5 (CH ₂ ), 13.9 (CH ₃ ). LC-MS (ESI ⁺ ) m/z: 164.1.0 (M - Boc+H) ⁺ (100%).

[0458] tert-Butyl (S)-(4-chloro-3-oxo-1-phenylbutan-2-yl) carbamate (SJM-724-152)

[0459]

[0460] The title compound was prepared by Method C from tert-butyl (S)-(4-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-3-oxo-1-phenylbutan-2-yl)carbamate (SJM -724-72) (50 mg, 0.15 mmol). The resulting crude product was purified by column chromatography (SiO2, petroleum alcohol:EtOAc, 9:1) to give the product as a white solid (30 mg, 68%). HRMS (ESI ⁺ ): found: m/z 320.1018 (M+Na) ⁺ , C ₁₅ H ₂₀ ClNNaO ₃ ⁺ calculated m/z 320.1024. ¹ H NMR (401 MHz, CDCl ₃ ) δ 7.35-7.23 (m, 3H), 7.19-7.13 (m, 2H), 5.06 (d, J=5.7 Hz, 1H), 4.66 (dd, J=13.0, 6.0 Hz, 1H), 4.17 (d, J=16.2 Hz, 1H), 3.98 (d, J=16.2 Hz, 1H), 3.08 (dd, J=13.8, 6.7 Hz, 1H), 2.99 (dd, J=13.4, 7.1 Hz, 1H), 1.40 (s , 9H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 201.5 (C), 155.3 (C), 135.7 (C), 129.3 (CH), 129.1 (CH), 127.5 ( CH), 80.6 (C), 58.6 (CH), 47.6 (CH ₂ ), 37.8 (CH ₂ ), 28.4 (CH ₃ ). LC-MS (ESI ⁺ ) m/z: 198.0 (M - Boc+H) ⁺ (100%).

[0461] Benzyl tert-butyl(7-chloro-6-oxoheptane-1,5-diyl)(S)-dicarbamate (SJM-724-52)

[0462]

[0463] The title compound was prepared by Method C from benzyl tert-butyl (7-(dimethyl(oxo)-λ ⁶ -6-sulfanylidene)-6-oxoheptan-1,5-diyl) (S)-dicarbamate (SJM- 724-48) (130 mg, 0.29 mmol). The resulting crude product was purified by column chromatography (SiO ₂ , petroleum alcohol:EtOAc, 3:1) to give the product as a white solid (60 mg, 51%). HRMS (ESI ⁺ ): found: 435.1664 (M+Na) ⁺ , C ₂₀ H ₂₉ ClN ₂ NaO ₅ ⁺ calculated m/z 435.1657. ¹ H NMR (401 MHz, CDCl ₃ ) δ 7.38-7.27 (m, 5H), 5.68 (d, J=5.7 Hz, 1H), 5.09 (s, 2H), 4 .62 (s, 1H), 4.59-4.47 (m, 1H), 4.26 (d, J=2.1 Hz, 2H), 3.16-3.00 (m, 2H), 1.92-1.81 (m, 1H), 1.70-1.57 (m, 1H), 1.54-1.27 (m, 4H), 1.40 (s, 9H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 201.6 (C), 156.4 (C), 136.1 (C), 128.7 (CH), 128.4 (CH), 128.3 ( CH), 79.4 (C), 67.3 ( _CH2 ), 57.9 (CH), 46.6 ( _CH2 ), 39.6 ( _CH2 ), 30.8 ( _CH2 ), 29 .8 (CH ₂ ), 28.5 (CH ₃ ), 22.2 (CH ₂ ). LC-MS (ESI ⁺ ) m/z: 434.9.0 (M+Na) ⁺ (100%).

[0464] Example 2.2: Synthesis and Characterization of Acyloxymethyl Ketones (AOMK)

[0465] (S)-3-((tert-butoxycarbonyl)amino)-2-oxoheptyl 2,6-dimethylbenzoate (SJM-724-156)

[0466]

[0467] The title compound was prepared by Method D from tert-butyl (S)-(1-chloro-2-oxoheptan-3-yl)carbamate (SJM-724-148) (20 mg, 76 μmol). The resulting crude product was purified by column chromatography (SiO ₂ , petroleum alcohol:EtOAc, 95:5) to give the product as a white solid (18 mg, 63%). HRMS (ESI ⁺ ): found: m/z 400.2097 (M+Na) ⁺ , for C ₂₁ H ₃₁ NNaO ₅ ⁺ calculated m/z 400.2094. ¹ H NMR (401 MHz, CDCl ₃ ) δ 7.23-7.17 (m, 1H), 7.04 (d, J=7.8 Hz, 2H), 5.15-4.96 (m, 3H), 4.40 (dd, J=12.5, 7.7 Hz, 1H), 2.39 (s, 6H), 1.97-1.86 (m, 1H), 1.67-1 .54 (m, 1H), 1.45 (s, 9H), 1.41-1.30 (m, 4H), 0.94-0.88 (m, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 202.8 (C), 169.1 (C), 155.7 (C), 135.8 (C), 132.7 (C), 129.8 ( CH), 127.8 (CH), 80.4 (C), 66.9 (CH ₂ ), 57.0 (CH), 31.4 (CH ₂ ), 28.4 (CH ₃ ), 27, 4 (CH ₂ ), 22.5 (CH ₂ ), 20.0 (CH ₃ ), 14.0 (CH ₃ ). LC-MS (ESI ⁺ ) m/z: 399.9 (M+Na) + (100%).

[0468] (S)-3-((tert-butoxycarbonyl)amino)-2-oxo-4-phenylbutyl 2,6-dimethylbenzoate (SJM-724-172)

[0469]

[0470] The title compound was prepared by Method D from tert-butyl (S)-(4-chloro-3-oxo-1-phenylbutan-2-yl)carbamate (SJM-724-152) (25 mg, 84 µmol) . The resulting crude product was purified by column chromatography (SiO ₂ , petroleum alcohol:EtOAc, 95:5) to give the product as a white solid (27 mg, 78%). HRMS (ESI ⁺ ): found: 434.1944 (M+Na) ⁺ , C ₂₄ H ₂₉ NNaO ₅ ⁺ calculated m/z 434.1938. ¹ H NMR (401 MHz, CDCl ₃ ) δ 7.35-7.18 (m, 6H), 7.05 (d, J=7.6 Hz, 2H), 5.06 (d, J=7, 3 Hz, 1H), 4.94 (dd, J=43.1, 17.0 Hz, 2H), 4.62 (dd, J=14.0, 7.0 Hz, 1H), 3.18 ( dd, J=14.0, 6.4 Hz, 1H), 3.04 (dd, J=14.0, 7.2 Hz, 1H), 2.39 (s, 6H), 1.42 (s , 9H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 202.2 (C), 169.1 (C), 155.4 (C), 135.9 (C), 135.8 (C), 132.7 ( C), 129.8 (CH), 129.5 (CH), 128.9 (CH), 127.8 (CH), 127.3 (CH), 80.5 (C), 67.3 (CH2 ), 58.1 (CH), 37.5 (CH ₂ ), 28.4 (CH ₃ ), 20.0 (CH ₃ ). LC-MS (ESI ⁺ ) m/z: 433.8 (M+Na) ⁺ (100%).

[0471] (S)-3-(((benzyloxy)carbonyl)amino)-7-((tert-butoxycarbonyl)amino)-2-oxoheptyl 2,6-dimethylbenzoate (SJM-724-176)

[0472]

[0473] The title compound was prepared by Method D from benzyl tert-butyl (7-chloro-6-oxoheptan-1,5-diyl)(S)-dicarbamate (SJM-724-52) (50 mg, 121 μmol). The resulting crude product was purified by column chromatography (SiO ₂ , petroleum alcohol:EtOAc, 3:1) to give the product as a white solid (57 mg, 89%). HRMS (ESI ⁺ ): found: 549.2571 (M+Na) ⁺ , C ₂₉ H ₃₈ N ₂ NaO ₇ ⁺ calculated m/z 549.2571. ¹ H NMR (401 MHz, CDCl ₃ ) δ 7.38-7.29 (m, 5H), 7.23-7.17 (m, 1H), 7.04 (d, J=7.5 Hz, 2H), 5.66 (d, J=5.9 Hz, 1H), 5.11 (s, 2H), 5.12-4.95 (m, 2H), 4.65 (s, 1H), 4.52-4.41 (m, 1H), 3.18-3.00 (m, 2H), 2.39 (s, 6H), 2.04-1.89 (m, 1H), 1. 78-1.64 (m, 1H), 1.58-1.35 (m, 4H), 1.41 (s, 9H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 202.3 (C), 169.1 (C), 156.4 (C), 136.2 (C), 135.8 (C), 132.5 ( C), 129.9 (CH), 128.7 (CH), 128.4 (CH), 128.3 (CH), 127.8 (CH), 79.3 (C), 67.3 (CH ₂ ), 66.8 ( _CH2 ), 57.5 (CH), 39.7 ( _CH2 ), 30.9 ( _CH2 ), 29.8 ( _CH2 ), 28.5 ( _CH3 ), 22.0 (CH ₂ ), 20.0 (CH ₃ ). LC-MS (ESI ⁺ ) m/z: 426.9 (M-Boc+H) ⁺ (100%).

[0474] Example 2.3: Synthesis and Characterization of AcyloxymethylKetone (AOMK) Probes

[0475] sCy5-Nle-AOMK (SJM-724-160)

[0476]

[0477] The title compound was prepared by Method E from (S)-3-((tert-butoxycarbonyl)amino)-2-oxoheptyl-2,6-dimethylbenzoate (SJM-724-156) (8 mg, 21 μmol). The resulting crude product was purified by RP-HPLC and lyophilized to give the product as a blue solid (13.9 mg, 72%). HRMS (ESI ⁺ ): found: 916.3869 (M+H) ⁺ , C ₄ 9H ₆₂ N ₃ O ₁₀ S ₂ ⁺ calculated m/z 916.3871.

[0478] sCy5-Phe-AOMK (SJM-724-180)

[0479]

[0480] The title compound was prepared by Method E from (S)-3-((tert-butoxycarbonyl)amino)-2-oxo-4-phenylbutyl 2,6-dimethylbenzoate (SJM-724-172) (5 mg, 12 µmol). The resulting crude product was purified by RP-HPLC and lyophilized to give the product as a blue solid (6.5 mg, 56%). HRMS (ESI ⁺ ): found: 950.3718 (M+H) ⁺ , for C ₅₂ H ₆₀ N ₃ O ₁₀ S ₂ ⁺ calculated m/z 950.3715.

[0481] Cbz-Lys(sCy5)-AOMK (SJM-724-184)

[0482]

[0483] The title compound was prepared by Method E from (S)-3-((((benzyloxy)carbonyl)amino)-7-((tert-butoxycarbonyl)amino)-2-oxoheptyl 2,6-dimethylbenzoate (SJM- 724-176) (5 mg, 9 µmol) The resulting crude product was purified by RP-HPLC and lyophilized to give the product as a blue solid (6.5 mg, 64%). HRMS (ESI ⁺ ): found: 1065 .4351 (M+H) ⁺ , C ₅₇ H ₆₉ N ₄ O ₁₂ S ₂ ⁺ calculated m/z 1065.4348.

[0484] II. Probe testing

[0485] General information

[0486] Methods

[0487] Table of basic materials

Reagent or material Source Identifier Antibodies Mouse antibodies to cathepsin X/Z/P (polyclonal goat IgG) R&D AF1033 Alexa Fluor® 594 AffiniPure donkey anti-goat IgG (H+L) Jackson ImmunoResearch 705-585-003 Chemicals, peptides and recombinant proteins Sulfoxonium ylide probes Synthesized according to Section I above N/A BMV109 Synthesized in the laboratory (Verdoes et al., 2013) JPM-OEt Drug Synthesis & Chemistry Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute (Meara and Rich, 1996) MDV-590 Medivir Pharmaceuticals, Sweden N/A Basic commercial tests BCA Analysis Pierce PIE23223/PIE23224 Protein A/G PLUS-agarose Santa Cruz SC-2003 Experimental Models: Cell Lines RAW264.7 ATCC ATCC TIB-71 MDA-MB-231HM Dr Zhou Ou, Fudan University, Shanghai Cancer Center (Chang et al., 2007) Experimental models: microorganisms/strains Cathepsin X/Z Deficient Mice Yates Lab, University of Calgary (Sevenich et al., 2010) Wild type C57BL/6J mice Bred at Monash Animal Resesarch Platform, originally from The Jackson Laboratory 000664

[0488] Experimental model and details of research objects

[0489] Cell culture

RAW264.7 or MDA-MB-231HM cells were cultured in DMEM containing 10% fetal bovine serum and 1% antibiotic/antimycotic. RAW264.7 cells were passaged by rubber scraping, while MDA-MB-231HM cells were cultured with 0.02% EDTA in phosphate buffered saline (PBS).

[0490] Animals

All experiments involving animals were approved by the Monash University Animal Ethics Committee. Male C57BL/6J mice were obtained from Monash Animal Research Platform and used at 8–10 weeks of age. Flash-frozen spleens from wild-type and cathepsin X knockout mice as described in Sevenich et al., 2010 were obtained from the University of Calgary in accordance with the University of Calgary Institutional Animal Care and Use Committee requirements.

[0491] Method Details

[0492] Cell Lysate Labeling and SDS-PAGE Analysis

Cells were collected by scraping, washed once with PBS, and resuspended in lysis buffer containing 50 mM citrate [pH 5.5], 0.5% CHAPS, 0.1% Triton X-100, and 4 mM DTT. Cells were incubated on ice for at least 10 min with occasional shaking, followed by centrifugation (21× g at 4°C for 5 min). The cleared supernatants were then transferred to another tube and the protein concentration was determined using the BCA method. Total protein (50 μg) was aliquoted into tubes in a final volume of 20 μl of lysis buffer. Where indicated, JPM-OEt or MDV-590 was added from a 100× stock solution in DMSO and incubated at 37°C for 20 min before probe addition. The probe at the indicated concentration was added from a 100× stock solution in DMSO. Labeling was performed at 37°C for 20 min (unless otherwise stated), and reactions were quenched by adding 5× sample buffer (200 mM Tris-Cl [pH 6.8], 8% SDS, 0.04% bromophenol blue, 5% b-mercaptoethanol and 40% glycerol). The samples were then boiled for five minutes and the proteins were separated on a 15% SDS-PAGE gel. The gels were scanned on a Typhoon 5 flatbed laser scanner at excitation/emission wavelengths of 633/670 nm to detect sCy5 fluorescence.

[0493] Labeling living cells

RAW cells or MDA-MB-231HM cells were seeded in 12-well plates. Where indicated, MDV-590 or vehicle was added at a concentration of 10 μM from 10 mM DMSO for overnight incubation. When cell density reached 80%, the indicated probes were added at the indicated concentrations from a 1000× DMSO stock solution and incubated for the indicated period of time. The medium was then removed and replaced with PBS. Cells were then scraped and transferred to tubes, and lysis and SDS-PAGE analysis were performed as above except for skipping the probe addition step.

[0494] Tissue Analysis

Tissues were collected from healthy mice and flash frozen. During analysis, lysis buffer was added at a 10× volume:weight ratio and tissues were sonicated on ice. Cleared lysates were labeled with the indicated probe and analyzed as described above.

[0495] For in vivo labeled tissues, mice were first injected intravenously via the tail vein with sCy5-Nle-SY, BMV109, or sCy5-Nle-AOMK (50 nmol in 100 μl of 10% DMSO/PBS or vehicle control). Tissues were collected and analyzed as above, except that no additional probe was added.

[0496] Immunoprecipitation assay

The probe-labeled lysate above (in sample buffer) was separated into input or precipitated (50 μg of total protein in each case). The input sample was stored at -20°C. The precipitated sample was diluted with 500 μl IP buffer (PBS [pH 7.4], 0.5% NP-40, 1 mM EDTA). Goat anti-cathepsin X antibody (10 μl) was added along with 40 μl of a suspension of prewashed protein A/G agarose beads. Samples were rotated overnight at 4°C. The beads were then washed four times with IP buffer followed by a final wash with 0.9% NaCl. The beads were then resuspended in 2× sample buffer and boiled. Precipitated supernatants along with input samples were analyzed by fluorescent SDS-PAGE as above.

[0497] Confocal microscopy

Kidney tissues from mice treated with sCy5-Nle-SY (or vehicle control) as described above were fixed overnight in 4% paraformaldehyde in PBS followed by cryoprotection overnight in 30% sucrose. Tissues were placed in OCT, frozen on dry ice, and sectioned at 10 μm thickness. Immunostaining for cathepsin X was performed according to standard protocols. Briefly, sections were air dried, fixed in cold acetone for 10 min, air dried again, and then rehydrated in PBS. Sections were blocked in PBS containing 3% normal horse serum with 0.1% Triton X-100. Goat anti-cathepsin X was added in blocking buffer at a ratio of 1:100 overnight at 4°C. Sections were then washed and donkey anti-goat secondary antibody AlexaFluor594 was added at 1:500 for 1 h at room temperature. Sections were stained with DAPI for 5 min, washed, and treated with ProLong Diamond mounting medium. Staining was analyzed using a Leica SP8 inverted confocal microscope.

[0498] Example

3 - Labeling of sCy5-Val-SY in cell lysates

[0494] In Example 3, the sulfoxonium ylide probe sCy5-Val-SY was incubated with protein lysates obtained from RAW264.7 cells, an immortalized mouse macrophage line that contains high levels of active cysteine cathepsins (Verdoes et al., 2013). Cells were lysed in citrate buffer (pH 5.5) to provide optimal conditions for cathepsin activation, and probe was added.

- at 1 µM for 20 min,

- in various concentrations (0 µM, 0.01 µM, 0.05 µM, 0.1 µM, 0.5 µM or 1 µM) for 20 minutes; or

- at 1 µM for different periods of time (0, 1, 2, 5, 10, 20 and 30 min).

[0500] The lysates were then separated by SDS-PAGE and the gel was scanned for sCy5 fluorescence using a flatbed laser scanner. Exclusive concentration- and time-dependent labeling of a protease with a molecular mass of approximately 35 kDa was observed (Fig. 2A, Fig. 2A-1 and Fig. 2A-2). This labeling was prevented by pretreatment of the lysates with JPM-OEt, a pan-cysteine cathepsin inhibitor, confirming that this protease was a member of the cysteine cathepsin family (Fig. 2A). In contrast, MDV-590, a specific inhibitor of cathepsin S, did not compete for sCy5-Val-SY binding.

[0501] The labeling profile of sCy5-Val-SY was compared with that of BMV109, a pan-probe for cathepsins, repeating the above experiment using BMV109 as the probe added at a concentration of 1 μM for 20 min. The sCy5-Val-SY-tagged protease was found to have the same molecular weight as BMV109-tagged cathepsin X (Verdoes et al., 2013). This protease was indeed confirmed to be cathepsin X using immunoprecipitation of sCy5-Val-SY-labeled lysates (sCy5-Val-SY added at a concentration of 1 μM for 20 min) with a cathepsin X-specific antibody (Fig. 2B).

[0502] Example

4 - Labeling of sCy5-Val-SY in tissue lysates

Example 4 tested the ability of sCy5-Val-SY to label cathepsin X in mouse spleen lysates (from wild-type or cathepsin X-deficient mice) (probe addition at 1 μM for 20 min). As observed in macrophage lysates, the probe exhibited exceptional reactivity with cathepsin X in spleen lysates from wild-type mice, and this labeling was absent in lysates prepared from spleens from cathepsin X-deficient mice (Fig. 2C). In comparison, BMV109 (added at 1 μM for 20 min) strongly labeled cathepsin B and, to a lesser extent, cathepsin S and L (Fig. 2C).

[0504] In summary, the results of Examples 3 and 4 demonstrate the exceptional specificity of sCy5-Val-SY in cell and tissue lysates.

[0505] Example

5 - Labeling of sCy5-Val-SY in living cells

[0506] In Example 5, the permeability of the sCy5-Val-SY probe and its specificity profile in living RAW264.7 cells were assessed and compared with the BMV109 probe. After incubating the probe (at 1 µM) with live cells for increasing periods of time (0, 1, 5, 10, 15, 25, 35, 45, 55, 65, 90 and 120 min) or increasing probe concentration (0 µM , 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, and 1 μM each for 2 h), lysates were analyzed by measuring in-gel fluorescence as above. Here, time- and concentration-dependent labeling of two proteases was observed (Fig. 2D, Fig. 2D-1). The latter were identified as cathepsin X and S by immunoprecipitation (Fig. 2E) and competition with MDV-590 (Fig. 2F), respectively.

[0507] It was surprising to find cathepsin S labeling in living cells given its lack of binding to sCy5-Val-SY in cell lysates where high levels of cathepsin S activity were confirmed using the BMV109 probe. The findings suggest that The reactivity of cathepsin S with the sulfoxonium ylide probe depends on the labeling conditions. An attempt was made to investigate this by lysing cells in various buffers that could mimic the endosomal environment of cathepsin S, but cathepsin S labeling was not increased in the lysates (results not shown).

[0508] However, the sulfoxonium ylide probe demonstrated clear labeling of cathepsin X in lysates and live cells with significantly improved selectivity compared to BMV109 (Figure 2D,F). To our knowledge, the sulfoxonium ylide probe is the first covalent ABP for cathepsin X that also does not bind cathepsin B or L. As seen in FIG. 2A, 2C, 2D, and 2F, it is difficult to distinguish the labeling of cathepsin X from cathepsin B by BMV109 due to the similarity in size of the two proteases. However, sCy5-Val-SY allows clear detection of cathepsin X activity.

[0509] Example

6 - Labeling with sulfoxonium ylide probes in cell lysates

[0510] In Example 6, a small library of sulfoxonium ylide probes with different amino acids at the P1 position, prepared according to Example 1, was tested in RAW264.7 lysates and compared with BMV109. Increasing concentrations of probe were added to each probe (0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, and 5 μM for 20 min).

Tested sulfoxonium ylide probes: Cbz-Lys(sCy5)-SY sCy5-Val-SY sCy5-Ile-SY sCy5-Leu-SY sCy5-Nle-SY sCy5-Phe-SY sCy5-Phe-Val-SY

[0511] In RAW264.7 cell lysates, the probes carrying Ile, Leu, Nle and Phe all showed similar specificity for cathepsin X with sCy5-Val-SY, where sCy5-Nle-SY was the most active (Figure 3A). A probe carrying Cbz-Lys, in which sCy5 was attached via a lysine side chain, showed a loss of specificity, favoring cathepsin S over cathepsin X and B. sCy5-Phe-Val-SY, which included a P2 Phe residue, also showed loss specificity (Fig. 3A, Fig. 7). The labeling profile of this probe was similar to BMV109, although it showed improved activity for cathepsin X and S compared to BMV109.

[0512] Example

[0513] 7 - Labeling of sulfoxonium ylide probes in kidney lysates

[0513] In Example 7, sulfoxonium ylide probes prepared according to Example 1 were tested in kidney lysates and the results were compared to BMV109. Increasing concentrations of 0.1 μM, 0.5 μM, 1 μM, and 5 μM probe were added to each sulfoxonium ylide (and BMV109) probe over 20 min.

Tested sulfoxonium ylide probes: Cbz-Lys(sCy5)-SY sCy5-Val-SY sCy5-Ile-SY sCy5-Leu-SY sCy5-Nle-SY sCy5-Phe-SY sCy5-Phe-Val-SY

[0514] In kidney lysates, Leu and Nle had the greatest potency and specificity for cathepsin X, where Cbz-Lys, Phe and Phe-Val gave broader reactivity, and Val and Ile showed lower labeling (Figure 3B, Figure 7 ).

[0515] Example

8 - Labeling with sulfoxonium ylide probes in living RAW264.7 cells

[0516] In Example 8, sulfoxonium ylide probes prepared in accordance with Example 1 were applied to living RAW264.7 cells for two hours to examine the effectiveness and permeability of a series of sulfoxonium ylide probes in live cells, and the results were compared with BMV109 .

[0517] Increasing concentrations of 0.1 μM, 0.5 μM, 1 μM, and 5 μM probe were added to each sulfoxonium ylide probe (and BMV109) for 2 hours.

Tested sulfoxonium ylide probes: sCy5-Trp-SY Cbz-Lys(sCy5)-SY sCy5-Val-SY sCy5-Ile-SY sCy5-Leu-SY sCy5-Nle-SY sCy5-Phe-SY sCy5-Phe-Val-SY

[0518] Probes containing Trp, Val, Ile, Leu, Nle and Phe labeled with cathepsin X and S to a similar extent and with similar efficiency, while Cbz-Lys showed a preference for cathepsin S and Phe-Val labeled cathepsin B and L in addition to cathepsin X and S (Fig. 3C, Fig. 7).

[0519] Example

9 - Labeling with sulfoxonium ylide probes in living cells MDA-MB-231HM

[0520] Example 9 tested the specificity of sulfoxonium ylide probes for cathepsin X in a human breast cancer line known to express very low levels of cathepsin S, MDA-MB-231HM (Chang et al., 2007), and results were compared with BMV109. MDA-MB-231HM cells also allowed us to test whether sulfoxonium ylide probes could bind to human cathepsin X (in addition to the mouse cathepsin X shown previously).

[0521] Each sulfoxonium ylide probe (and BMV109) was added with increasing concentrations of probe (0.1 μM, 0.5 μM, 1 μM, and 5 μM).

Tested sulfoxonium ylide probes: sCy5-Trp-SY Cbz-Lys(sCy5)-SY sCy5-Val-SY sCy5-Ile-SY sCy5-Leu-SY sCy5-Nle-SY sCy5-Phe-SY sCy5-Phe-Val-SY

[0522] When the probes were incubated with MDA-MB-231 cells for shorter periods of time, very weak labeling of cathepsin X was observed (results not shown). However, clear labeling was observed after overnight incubation (Figure 3D). This likely reflects differences in the rate of endocytosis between macrophages and tumor cells and suggests that probes may enter the endolysosmal pathway directly rather than by diffusion across membranes. The sulfoxonium ylide probe series generally showed specific labeling of cathepsin X in these cells, with minimal cross-reactivity occurring at only 5 μM. sCy5-Lys-SY and especially sCy5-Phe-Val-SY showed the highest cross-reactivity with cathepsin B and L.

[0523] Example

10 - Characterization of sCy5-Nle-SY in vivo

[0524] Taking into account all the data obtained from cell and tissue lysates, live mouse macrophages and human cancer cells, the sulfoxonium ylide probe sCy5-Nle-SY was found to be the probe showing the highest potency and selectivity for cathepsin X. Thus , this probe was selected for in vivo studies.

[0525] In Example 10, the sCy5-Nle-SY probe, vehicle/no probe (NP) control, or BMV109 was administered intravenously to mice. After two hours of circulation, tissues were collected, lysed, and analyzed for probe labeling by fluorescent SDS-PAGE. Cathepsin X labeling was observed in the liver, kidney, colon, stomach, and spleen (Fig. 4A), and this was confirmed by immunoprecipitation with cathepsin X antibody (Fig. 4B). Although some cathepsin S labeling was also observed, the overall specificity profile was clearly improved compared to BMV109, which also strongly labels cathepsin B and L.

[0526] Additionally, following in vivo probe administration, cryosections of kidneys from mice injected with sCy5-Nle-SY or no-probe controls were prepared and analyzed by confocal microscopy for sCy5 fluorescence (red) or cathepsin X immunoreactivity (green) along with DAPI ( blue) to visualize the nucleus. The results are presented in Fig. 5. Strong punctate sCy5 fluorescence was observed, reminiscent of endolysosomal staining, and this signal overlapped significantly with immunoreactive cathepsin X (Fig. 5). Thus, sCy5-Nle-SY can be used to discriminate active cathepsin X versus total cathepsin X in tissues after in vivo administration.

[0527] Example

11 - Characteristics of AOMK and sulfoxonium ylide probes

[0528] In Example 11, the reactivity of the new AOMK probes prepared in Example 2 was compared with the corresponding sulfoxonium ylide probes prepared in Example 1 using a live RAW264.7 cell labeling experiment.

Tested AOMK probes: Tested sulfoxonium ylide probes: Cbz-Lys(sCy5)-AOMK Cbz-Lys(sCy5)-SY sCy5-Nle-AOMK sCy5-Nle-SY sCy5-Phe-AOMK sCy5-Phe-SY

[0529] Probes were added in increasing concentrations (0.1, 0.5, 1, 5 μM) for 2 hours and labeling was analyzed by in-gel fluorescence. The results are shown in Fig. 6A.

[0530] The AOMK probes were much less efficient than the ylide probes, indicating reduced reactivity. These probes labeled cathepsin B and S but not X (Figure 6A), consistent with previous data demonstrating limited reactivity of cathepsin X with the AOMK active moiety (Paulick and Bogyo, 2011).

[0531] Example

12 - Characterization of sCy5-Nle-AOMK in vivo

[0532] In Example 12, the AOMK probe sCy5-Nle-AOMK was tested in vivo and its tissue labeling was analyzed. The probe was administered intravenously to mice. After two hours of circulation, tissues were collected, lysed, and analyzed for probe labeling using fluorescent SDS-PAGE. Only weak labeling of cathepsin B and S was observed in the colon, but not in the other tissues examined (Fig. 6B).

[0533] Example

13 - Labeling of sCy5-Nle-SY in human tissues

[0534] To investigate the ability of sCy5-Nle-SY to label cathepsin X in human tissues, biopsies obtained from patients with oral squamous cell carcinoma were used. Normal oral mucosal tissue biopsied from the contralateral tongue of the same patients was used as a control.

[0535] Patients with human oral squamous cell carcinoma or matched subjects with normal oral mucosa were biopsied in accordance with protocols approved by the Institute's Review Board of the New York University Oral Cancer Center. The biopsy material was immediately frozen and stored at -80°C. Tissues were lysed by sonication in citrate buffer, and supernatants were cleared by centrifugation. Total protein concentration was assessed by BCA assay, and 80 μg was labeled with Cy5-Nle-SY. Lysates obtained from oral squamous cell carcinoma tissue or matched subjects with normal oral mucosa were incubated with sCy5-Nle-SY (5 μM for 20 min), and labeling was analyzed by in-gel fluorescence (fluorescent SDS-PAGE). Samples were also immunoblotted to determine total cathepsin X expression (n=10). In addition, sCy5-Nle-SY-labeled cancer lysates were immunoprecipitated with a cathepsin X-specific antibody to verify the identity of the labeled protein.

[0536] The results are shown in FIG. 8A and 8B. In all carcinoma samples examined, clear and selective labeling of cathepsin X was observed, which was confirmed by immunoprecipitation with a cathepsin X-specific antibody. Cathepsin X activity was significantly increased in carcinoma biopsies compared with normal tongue tissue, and this was confirmed by the increase in total cathepsin X measured using immunoblotting. The findings show that sCy5-Nle-SY can successfully detect cathepsin X activation in human tissues and reveal for the first time that cathepsin X is significantly activated in oral squamous cell carcinoma.

[0537] In summary, the results of these experiments demonstrate that the novel dimethylsulfoxonium ylide active moiety tested exhibits unique selectivity for the cysteine protease, cathepsin, in cell lysates, living cells, in vivo (e.g., mice) and in lysates tissues (mouse and human). The top probe of the sulfoxonium ylide probes tested, sCy5-Nle-SY, is the most potent and selective probe for cathepsin X to date, demonstrating exceptional specificity in cell lysates and cells that express low levels of cathepsin S. Although this probe does cross-react with cathepsin S in living macrophages and in vivo it does not result in cathepsin B or L labeling, a clear improvement over other cathepsin X-targeting probes (BMV109, MGP140). The use of sCy5-Nle-SY allows cathepsin X activity to be clearly measured by SDS-PAGE, whereas this was difficult with previous probes as a result of "distorting" levels of cathepsin B labeling. In addition, the active moiety from the sulfoxonium ylide was identified is stable enough to detect cathepsin X in vivo and that the sCy5-Nle-SY signal was bright enough to be detected by confocal microscopy.

[0538] Example

14 - Boc-Val-SY as a cathepsin X inhibitor

[0539] To test the ability of sulfoxonium ylide compounds to function as cathepsin inhibitors, an unlabeled, boc-protected variant of Val-SY, i.e., tert-butyl (S)-(1-(dimethyl(oxo)-6-sulfanylidene)- 4-methyl-2-oxopentan-3-yl) carbamate (SJM-724-24). Live RAW264.7 cells were pretreated with Boc-Val-SY (0, 10, or 100 μM) overnight. Residual cathepsin X activity was measured by incubating live cells with sCy5-Val-SY for 2 hours. Cells were lysed, and tagged proteins were detected by in-gel fluorescence.

[0540] The results are shown in FIG. 9. Compared to controls, cells treated with 100 µM Boc-Val-SY showed a 71% and 51% reduction in cathepsin X and cathepsin S, respectively. The findings demonstrate the ability of Boc-Val-SY to compete for sCy5-Val-SY labeling and thus function as a cathepsin inhibitor.

[0541] The present examples, methods, procedures, specific compounds and molecules are provided to exemplify and illustrate the invention and are not to be construed in any way as limiting the scope of the invention, which is defined by the literal and equivalent scope of the appended claims. Any patents or publications mentioned herein indicate levels of skill in the field to which the patent relates and are intended to provide details of the invention that cannot be set forth in detail but will be understood by those skilled in the art. Such patents or publications are incorporated herein by reference to the same extent as if each were specifically and individually incorporated by reference and for the purpose of describing and enabling the use of said method or material.

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[0543] Additional embodiments of the invention include:

1. Compound of formula I

[0544]

or its salt,

Where:

R ₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl;

R ₂ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C8) alkynyl;

R ₃ represents an alpha amino acid side chain;

R ₄ is selected from the group consisting of a hydrogen atom and (C ₁ -C ₄ ) alkyl;

R ₅ is selected from the group consisting of a detectable element, an amino protecting group of amine, (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl , (C ₂ -C ₈ ) alkenyl, (C ₂ -C ₈ ) alkynyl, (C ₁ -C ₈ ) alkylcarbonyl, (C ₁ -C ₈ ) hydroxyalkylcarbonyl, (C ₁ -C ₈ ) haloalkylcarbonyl, (C ₃ - C ₈ ) cycloalkylcarbonyl, (C ₁ -C ₈ ) alkyloxycarbonyl, benzyloxycarbonyl and a hydrogen atom;

X represents:

(i) communication; or

(ii) a diradical fragment of formula II or III, which is connected to the R ₅ substituent through an amino group:

[0545]

Where:

R ₆ represents an alpha amino acid side chain;

R ₇ is selected from the group consisting of a hydrogen atom and (C ₁ -C ₄ ) alkyl;

R ₈ represents an alpha amino acid side chain;

R ₉ is selected from the group consisting of a hydrogen atom and (C ₁ -C ₄ ) alkyl; And

n is 1, 2, 3 or 4.

[0546] 2. A compound according to claim 1, wherein R ₃ represents a side chain of a naturally occurring alpha amino acid or a structural isomer, homologue and/or structural analogue of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different _R3x substituents, wherein:

R _3x is selected from the group consisting of hydroxy, halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and ( C ₁ -C ₄ ) haloalkoxy.

[0547] 3. A compound according to claim 1, wherein R ₃ represents a side chain of a proteinogenic alpha amino acid or a structural isomer, homologue and/or structural analogue of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different _R3x substituents, wherein:

R _3x is selected from the group consisting of hydroxy, halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and ( C ₁ -C ₄ ) haloalkoxy.

[0548] 4. A compound according to claim 1, wherein R ₃ represents a side chain of a proteinogenic alpha amino acid other than lysine, or a structural isomer or homolog of said side chain,

wherein said side chain or structural isomer or homologue thereof is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different R _3x substituents, wherein:

R _3x is selected from the group consisting of hydroxy, halogen, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ - _C 4) haloalkoxy.

[0549] 5. A compound according to claim 1, wherein R ₃ represents a side chain of an alpha amino acid selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, methionine , ethionine, phenylalanine, tyrosine, levodopa, tryptophan, cysteine, homocysteine, selenocysteine, selenagomocysteine, selenomethionine, selenoethionine, lysine, histidine, arginine, ornithine, aspartic acid, glutamic acid, serine, homoserine, O-methylhomoserine, O-ethylhomoserine, threon ina , asparagine and glutamine, or a structural isomer, homologue and/or structural analogue of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different _R3x substituents, wherein:

R _3x is selected from the group consisting of hydroxy, halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and ( C ₁ -C ₄ ) haloalkoxy.

[0549] 6. A compound according to claim 1, wherein R ₃ represents a side chain of an alpha amino acid selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, methionine , ethionine, phenylalanine, tyrosine, levodopa, tryptophan, cysteine, homocysteine, selenocysteine, selenagomocysteine, selenomethionine, selenoethionine, lysine, histidine, arginine, ornithine, aspartic acid, glutamic acid, serine, homoserine, O-methylhomoserine, O-ethylhomoserine, threon ina , asparagine and glutamine, or a structural isomer, homologue and/or structural analogue of said side chain,

wherein said side chain or structural isomer or homologue thereof is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different R _3x substituents, wherein:

R _3x is selected from the group consisting of hydroxy, halogen, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy.

[0551] 7. The compound of claim 6, wherein the alpha amino acid is selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, methionine, ethionine, phenylalanine, tyrosine, levodopa, tryptophan, cysteine, homocysteine, selenocysteine, selenohomocysteine, selenomethionine and selenethionine.

[0552] 8. The compound of claim 6, wherein the alpha amino acid is selected from the group consisting of alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, phenylalanine and tryptophan.

[0553] 9. The compound of claim 6, wherein the alpha amino acid is selected from the group consisting of valine, norvaline, leucine, isoleucine, norleucine and homonorleucine.

[0554] 10. The compound of claim 6, wherein the alpha amino acid is selected from the group consisting of valine, leucine, isoleucine and norleucine.

[0555] 11. The compound of claim 6, wherein the alpha amino acid is leucine or norleucine.

[0556] 12. The compound of claim 6, wherein the alpha amino acid is norleucine.

[0544]

[0557] 13. A compound according to claim 1, wherein R ₃ is selected from the group consisting of a hydrogen atom, (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ - C ₈ ) alkenyl, (C ₂ -C ₈ ) alkynyl, (C ₆ -C ₁₀ ) arylmethyl, (C ₃ -C ₉ ) heteroarylmethyl and -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ); Where:

R _3a is selected from the group consisting of a detectable element, an amino protecting group, a hydrogen atom and a (C ₁ -C ₈ ) alkyl; And

R _3b is selected from the group consisting of a hydrogen atom and a (C ₁ -C ₄ ) alkyl atom.

[0558] 14. The compound of claim 13, wherein R ₃ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₂ -C ₈ ) alkenyl, (C ₂ -C ₈ ) alkynyl, (C ₆ -C ₁₀ ) arylmethyl, (C ₃ -C ₉ ) heteroaryl methyl and -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a ) (R _3b ).

[0559] 15. The compound of claim 13, wherein R ₃ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₆ -C ₁₀ ) arylmethyl, (C ₃ -C ₉ ) heteroarylmethyl and -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ).

[0560] 16. The compound of claim 13, wherein R ₃ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, benzyl, (1H-indol-3-yl)methyl and -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ).

[0561] 17. The compound of claim 13, wherein R ₃ is selected from the group consisting of (C ₁ -C ₆ ) alkyl, benzyl, (1H-indol-3-yl)methyl and -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ).

[0562] 18. The compound of claim 13, wherein R ₃ is selected from the group consisting of ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, n-pentyl, benzyl, (1H-indole-3 -yl)methyl and -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ).

[0563] 19. A compound according to any one of claims 13 to 18, wherein if X is a bond, then R _3a is not an amino protecting group, a hydrogen atom or a (C ₁ -C ₈ ) alkyl.

[0564] 20. A compound according to any one of claims 13 to 18, wherein R _3a represents a detectable element.

[0565] 21. A compound according to any one of claims 13 to 18, wherein if X is a bond, then R ₃ is not -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ).

[0566] 22. A compound according to claim 1, wherein R ₃ is selected from the group consisting of a hydrogen atom, (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ - C ₈ ) alkenyl, (C ₂ -C ₈ ) alkynyl, (C ₆ -C ₁₀ ) arylmethyl and (C ₃ -C ₉ ) heteroarylmethyl.

[0567] 23. The compound of claim 22, wherein R ₃ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₂ -C 8 ) alkenyl, (C ₂ -C ₈ ) alkynyl, (C ₆ - C ₁₀ ) arylmethyl and (C ₃ -C ₉ ) heteroaryl methyl.

[0568] 24. The compound of claim 22, wherein R ₃ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₆ -C ₁₀ ) arylmethyl and (C _{3 -C9} ) heteroarylmethyl.

[0569] 25. The compound of claim 22, wherein R ₃ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, benzyl and (1H-indol-3-yl)methyl.

[0570] 26. The compound of claim 22, wherein R ₃ is selected from the group consisting of (C ₁ -C ₆ ) alkyl, benzyl and (1H-indol-3-yl)methyl.

[0571] 27. The compound of claim 22, wherein R ₃ is selected from the group consisting of ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, n-pentyl, benzyl and (1H-indole)- 3-yl)methyl.

[0572] 28. The compound of claim 22, wherein R ₃ is selected from the group consisting of n-butyl, sec-butyl and isobutyl.

[0573] 29. The compound of claim 22, wherein R ₃ is n-butyl.

[0574] 30. A compound according to claim 1, wherein R ₃ is -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ); Where:

R _3a is selected from the group consisting of a detectable element, an amino protecting group, a hydrogen atom and a (C ₁ -C ₈ ) alkyl; And

R _3b is selected from the group consisting of a hydrogen atom and a (C ₁ -C ₄ ) alkyl atom.

[0575] 31. The compound of claim 30, wherein if X is a bond, then R _3a is not an amino protecting group, a hydrogen atom or a (C ₁ -C ₈ ) alkyl.

[0576] 32. A compound according to any one of claims 13-18 and 30, wherein R _3a represents a hydrogen atom.

[0577] 33. The connection according to any one of claims. 13-18 and 30, where R _3a represents an amino protecting group.

[0578] 34. A compound according to any one of claims 13-18 and 30, wherein R _3a represents (C ₁ -C ₈ ) alkyl.

[0579] 35. A compound according to any one of claims 13-20 and 30-34, wherein R _3b represents a hydrogen atom.

[0580] 36. A compound according to any one of claims 1 to 35, wherein R ₁ represents (C ₁ -C ₈ ) alkyl.

[0581] 37. A compound according to any one of claims 1 to 36, wherein R ₁ represents methyl.

[0582] 38. A compound according to any one of claims 1 to 37, wherein R ₂ represents (C ₁ -C ₈ ) alkyl.

[0583] 39. A compound according to any one of claims 1 to 38, wherein R ₂ represents methyl.

[0584] 40. A compound according to any one of claims 1 to 39, wherein R ₄ represents a hydrogen atom.

[0585] 41. A compound according to any one of claims 1 to 40, wherein R ₅ is selected from the group consisting of a detectable element, an amino protecting group and a hydrogen atom.

[0586] 42. A compound according to any one of claims 1 to 41, wherein R ₆ represents a side chain of a naturally occurring alpha amino acid or a structural isomer, homologue and/or structural analogue of said side chain.

wherein said side chain or structural isomer, homolog and/or structural analogue is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different _R6x substituents, wherein:

R _6x is selected from the group consisting of hydroxy, halogen, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and ( C ₁ -C ₄ ) haloalkoxy.

[0587] 43. A compound according to any one of claims 1 to 41, wherein R ₆ represents a side chain of a proteinogenic alpha amino acid or a structural isomer, homologue and/or structural analogue of said side chain.

wherein said side chain or structural isomer, homolog and/or structural analogue is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different R _6x substituents, wherein

R _6x is selected from the group consisting of hydroxy, halogen, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and ( C ₁ -C ₄ ) haloalkoxy.

[0588] 44. A compound according to any one of claims 1 to 41, wherein R ₆ represents a side chain of an alpha amino acid selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine , homonorleucine, methionine, ethionine, phenylalanine, tyrosine, levodopa, tryptophan, cysteine, homocysteine, selenocysteine, selenogomocysteine, selenomethionine, selenoethionine, lysine, histidine, arginine, ornithine, aspartic acid, glutamic acid, serine, homoserine, O-methylhomoserine, Oh - ethyl homoserine, threonine, asparagine and glutamine, or a structural isomer, homologue and/or structural analogue of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different _R6x substituents, wherein:

R _6x is selected from the group consisting of hydroxy, halogen, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and ( C ₁ -C ₄ ) haloalkoxy.

[0589] 45. The compound of claim 44, wherein the alpha amino acid is phenylalanine.

[0590] 46. A compound according to any one of claims 1 to 41, wherein R ₆ represents a side chain of phenylalanine or a structural isomer, homolog and/or structural analogue of the specified side chain.

[0591] 47. A compound according to any one of claims 1 to 41, wherein R ₆ represents a phenylalanine side chain.

[0592] 48. A compound according to any one of claims 1 to 47, wherein R ₇ represents a hydrogen atom.

[0593] 49. A compound according to any one of claims 1 to 48, wherein R ₈ represents a side chain of a naturally occurring alpha amino acid or a structural isomer, homologue and/or structural analogue of said side chain.

wherein said side chain or structural isomer, homolog and/or structural analogue is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different _R8x substituents, wherein:

R _8x is selected from the group consisting of hydroxy, halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and ( C ₁ -C ₄ ) haloalkoxy.

[0594] 50. A compound according to any one of claims 1 to 48, wherein R ₈ represents a side chain of a proteinogenic alpha amino acid or a structural isomer, homologue and/or structural analogue of said side chain.

wherein said side chain or structural isomer, homologue and/or structural analogue is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different R _8x substituents, wherein

R _8x is selected from the group consisting of hydroxy, halogen, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, ( _{C 1} -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy.

[0595] 51. A compound according to any one of claims 1 to 48, wherein R ₈ represents a side chain of an alpha amino acid selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine , homonorleucine, methionine, ethionine, phenylalanine, tyrosine, levodopa, tryptophan, cysteine, homocysteine, selenocysteine, selenogomocysteine, selenomethionine, selenoethionine, lysine, histidine, arginine, ornithine, aspartic acid, glutamic acid, serine, homoserine, O-methylhomoserine, Oh - ethyl homoserine, threonine, asparagine and glutamine, or a structural isomer, homologue and/or structural analogue of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is optionally substituted with an amino protecting group or detectable element and optionally further substituted with one or more identical or different _R8x substituents, wherein _R8x is selected from the group consisting of hydroxy, a halogen atom, ( C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy.

[0596] 52. A compound according to any one of claims 1 to 51, wherein R ₉ represents a hydrogen atom.

[0597] 53. A compound according to any one of claims 1 to 52, wherein the amino protecting group is selected from the group consisting of benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), p- toluenesulfonyl (Tos), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf), mesityl -2-sulfonyl (Mts), 4-methoxy-2,3,6-trimethylphenylsulfonyl (Mtr), acetamido and phthalimido.

[0598] 54. A compound according to any one of claims 1 to 53, wherein the amino protecting group is benzyloxycarbonyl.

[0599] 55. A compound according to any one of claims 1 to 54, having formula IA:

[0600]

[0601] 56. A compound according to any one of claims 1 to 55, where the diradical fragment has formula IIA or IIIA:

[0602]

[0603] 57. A connection according to any one of claims 1 to 55, wherein X represents a bond.

[0604] 58. A compound according to any one of claims 1 to 55, wherein X is a diradical moiety of formula II.

[0605] 59. A compound according to any one of claims 1 to 55, wherein X is a diradical moiety of formula III.

[0606] 60. The compound of claim 56, wherein X is a diradical moiety of formula IIA.

[0607] 61. A compound according to claim 56, wherein X is a diradical moiety of formula IIIA.

[0608] 62. The connection according to any one of claims 1 to 61, where n is 1.

[0609] 63. The compound of any one of claims 1 to 62, wherein the detectable element is selected from the group consisting of a fluorescent label, a biotin label, a radiolabel, a chelator, and a bioorthogonal ligation marker.

[0610] 64. The compound according to any one of claims 1 to 63, wherein the detectable element is a fluorescent label.

[0611] 65. The compound of claim 64, wherein the fluorescent label is selected from the group consisting of fluorescein, Oregon green, boradiazaidecene dye, rhodamine dye, benzopyryllium dye, coumarin dye, cyanine label, or benzoindole label

[0612] 66. The compound of claim 64, wherein the fluorescent label is a cyanine label.

[0613] 67. The compound of claim 64, wherein the fluorescent label is a cyanine label having a formula selected from the following group of formulas:

[0614]

[0615] where in each of the above formulas,

A is selected from the group consisting of CH ₂ , C(CH ₃ ) ₂ , C(C ₂ H ₅ ) ₂ , NH, N(CH ₃ ), N(C ₂ H ₅ ), O, S and Se;

R ₁₀ is selected from the group consisting of $-(CH ₂ )pC(=O)-& and $-(CH ₂ ) _q -C(=O)-NH-[CH ₂ CH ₂ O] _r -CH ₂ CH ₂ -C(=O)-&;

Where:

p is equal to 2, 3, 4, 5, 6, 7 or 8;

q is equal to 2, 3, 4, 5, 6, 7 or 8;

r is equal to 2, 3, 4, 5, 6, 7 or 8;

$ represents the point of connection with the nitrogen atom of the cyanine group; and & represents the point of connection with the rest of the molecule;

R ₁₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl and (C ₆ -C ₁₀ ) aryl; And

R ₁₂ represents H or sulfo group.

[0616] 68. Connection according to paragraph 67, where:

A is selected from the group consisting of CH ₂ , C(CH ₃ ) ₂ and C(C ₂ H ₅ ) ₂ ;

R ₁₀ is selected from the group consisting of $-(CH ₂ )pC(=O)-& and $-(CH ₂ ) _q -C(=O)-NH-[CH ₂ CH ₂ O] _r -CH ₂ CH ₂ -C(=O)-&;

Where:

p equals 2, 3, 4, 5 or 6;

q is equal to 2, 3, 4, 5 or 6;

r is 2, 3, 4, 5 or 6;

$ represents the point of attachment to the nitrogen atom of the cyanine group; and & represents the point of connection with the rest of the molecule;

R ₁₁ represents (C ₁ -C ₈ ) alkyl; And

R ₁₂ represents H or sulfo group.

[0617] 69. Connection according to paragraph 67, where:

A is C(CH ₃ ) ₂ or C(C ₂ H ₅ ) ₂ ;

R ₁₀ is selected from the group consisting of $-(CH ₂ ) _p -C(=O)-& and $- (CH ₂ ) _q -C(=O)-NH-[CH ₂ CH ₂ O] _r -CH ₂ CH ₂ -C(=O)-&;

Where:

p equals 2, 3, 4, 5 or 6;

q is equal to 2, 3, 4, 5 or 6;

r is 2, 3, 4, 5 or 6;

$ represents the point of attachment to the nitrogen atom of the cyanine group; and & represents the point of connection with the rest of the molecule;

R ₁₁ is methyl, ethyl or propyl; And

R ₁₂ represents H or sulfo group.

[0618] 70. Connection according to paragraph 67, where:

A represents C(CH ₃ ) ₂ ;

R ₁₀ is selected from the group consisting of $-(CH ₂ ) _p -C(=O)-& and $- (CH ₂ ) _q -C(=O)-NH-[CH ₂ CH ₂ O] _r -CH ₂ CH ₂ -C(=O)-&;

Where:

p is 4, 5 or 6;

q is 4, 5 or 6;

r is 3, 4, 5 or 6;

$ represents the point of connection with the nitrogen atom of the cyanine group; and & represents the point of connection with the rest of the molecule;

R ₁₁ represents methyl or ethyl; And

R ₁₂ represents H or sulfo group.

[0619] 71. Connection according to paragraph 67, where:

A represents C(CH ₃ ) ₂ ;

R ₁₀ represents $-(CH ₂ ) _p -C(=O)-&; Where:

p is 4, 5 or 6; And

$ represents the point of attachment to the nitrogen atom of the cyanine moiety; and & represents the point of connection with the rest of the molecule;

R ₁₁ represents methyl or ethyl; And

R ₁₂ represents a sulfo group.

[0620] 72. Connection according to paragraph 67, where:

A represents C(CH ₃ ) ₂ ;

R ₁₀ is $-(CH ₂ ) _q -C(=O)-NH-[CH ₂ CH ₂ O] _r -CH ₂ CH ₂ -C(=O)-&;

Where:

q is 4, 5 or 6;

r is 3, 4, 5 or 6;

$ represents the point of attachment to the nitrogen atom of the cyanine group; and & represents the point of connection with the rest of the molecule;

R ₁₁ represents methyl or ethyl; And

R ₁₂ represents H.

[0621] 73. The connection according to any one of claims 67-72, where p is 5, q is 5 and r is 4.

[0622] 74. The compound of claim 64, wherein the fluorescent label is a cyanine label having a formula selected from the following group of formulas:

[0623]

[0624]

[0625]

[0626] where in each of the above formulas,

the wavy line represents the connection point with the rest of the molecule;

and R ₁₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl and (C ₆ -C ₁₀ ) aryl.

[0600]

[0627] 75. The compound of claim 74, wherein R ₁₁ is (C ₁ -C ₈ ) alkyl.

[0628] 76. The compound of claim 74, wherein R ₁₁ is methyl or ethyl.

[0629] 77. The compound of claim 64, wherein the fluorescent label is a cyanine label having the formula:

[0630]

[0631] where the wavy line represents the connection point with the rest of the molecule; and R ₁₁ is methyl or ethyl.

[0632] 78. A compound according to any one of claims 1 to 77, wherein the compound contains at least one detectable element.

[0633] 79. A compound according to any one of claims 1 to 77, wherein the compound contains one, two or three detectable elements.

[0634] 80. A compound according to any one of claims 1 to 77, wherein the compound contains a single detectable element.

[0635] 81. A compound according to any one of claims 1 to 80, wherein R ₅ represents a detectable element.

[0636] 82. A compound according to any one of claims 1 to 81, wherein R ₃ contains a detectable element.

[0637] 83. A compound according to any one of claims 1 to 82, wherein R ₃ represents a lysine side chain or a structural isomer, homologue and/or structural analogue of said side chain.

wherein said side chain or structural isomer, homolog and/or structural analogue is substituted with a detectable element and optionally further substituted with one or more identical or different R _3x substituents, wherein R _3x is selected from the group consisting of hydroxy, a halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy.

[0638] 84. The connection according to any one of claims 1 to 82,

where R ₃ represents -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ); Where:

R _3a represents a detectable element; And

R _3b is selected from the group consisting of a hydrogen atom and a (C ₁ -C ₄ ) alkyl atom.

[0639] 85. A compound according to any one of claims 82 to 84, wherein R ₅ is selected from the group consisting of an amino protecting group, (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl, (C ₂ -C ₈ ) alkynyl, (C ₁ -C ₈ ) alkylcarbonyl, (C ₁ -C ₈ ) hydroxyalkylcarbonyl, ( C ₁ -C ₈ ) haloalkylcarbonyl, (C ₃ -C ₈ ) cycloalkylcarbonyl, (C ₁ -C ₈ ) alkyloxycarbonyl, benzyloxycarbonyl and a hydrogen atom.

[0640] 86. A compound according to any one of claims 82 to 84, wherein R ₅ represents an amino protecting group.

[0641] 87. A compound according to any one of claims 1 to 86, wherein X is a diradical moiety of formula II or IIA and R ₆ carries a detectable element.

[0642] 88. A compound according to any one of claims 1 to 87, wherein X is a biradical fragment of formula II or IIA, and R ₆ is a lysine side chain or a structural isomer, homologue and/or structural analogue of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is substituted with a detectable element and optionally further substituted with one or more identical or different R _6x substituents, wherein R _6x is selected from the group consisting of hydroxy, a halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy.

[0643] 89. The compound of any one of items 1 to 86, wherein X is a diradical moiety of formula III or IIIA and R ₆ carries a detectable element.

[0644] 90. A compound according to any one of claims 1 to 86 and 89, where X is a diradical moiety of formula III or IIIA, and R ₆ is a lysine side chain or a structural isomer, homolog and/or structural analogue of said side chain,

wherein said side chain or structural isomer, homolog and/or structural analogue is substituted with a detectable element and optionally further substituted with one or more identical or different R _6x substituents, wherein R _6x is selected from the group consisting of hydroxy, a halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy.

[0645] 91. A compound according to any one of claims 1-86 and 89-90, wherein X is a diradical moiety of formula III or IIIA, and R ₈ carries a detectable element.

[0646] 92. A compound according to any one of claims 1-86 and 89-91, where X is a diradical moiety of formula III or IIIA, and R ₈ is a lysine side chain or a structural isomer, homologue and/or structural analogue of said side chain chains,

wherein said side chain or structural isomer, homolog and/or structural analogue is substituted with a detectable element and optionally further substituted with one or more identical or different R _8x substituents, wherein R _8x is selected from the group consisting of hydroxy, a halogen atom, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy.

[0647] 93. A compound selected from the group of formulas consisting of:

[0648]

[0649]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656] or its salt.

[0657] 94. Compound of formula:

[0658]

or its salt.

[0659] 95. A composition containing a compound according to any one of claims 1 to 94 or a salt and an excipient thereof.

[0660] 96. The composition of claim 95, wherein the composition contains a compound of any one of claims 78 to 94 or a salt and an excipient thereof.

[0661] 97. A method for detecting cysteine protease activity, comprising:

(1) contacting the cysteine protease with an activity-based probe compound containing a sulfoxonium ylide moiety as an active group, and

(2) subsequent cysteine protease analysis, including measurement of the detected signal.

[0662] 98. The method of claim 97, wherein the method is an in vitro method.

[0663] 99. A method for detecting cysteine protease activity in vitro, comprising:

(1) contacting the biological sample with an activity-based probe compound containing a sulfoxonium ylide moiety as an active group, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

[0664] 100. A method for detecting cysteine protease activity in a biological sample obtained from a subject, comprising:

(1) contacting the biological sample in vitro with an activity-based probe compound containing a sulfoxonium ylide moiety as an active group, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

[0665] 101. The method according to any one of claims 97 to 100, wherein the sulfoxonium ylide moiety has formula (IV):

[0666]

[0667] where:

R ₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ - _C8 ) alkynyl; And

R ₂ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl.

[0668] 102. The method of claim 101, wherein R ₁ is (C ₁ -C ₈ ) alkyl and R ₂ is (C ₁ -C ₈ ) alkyl.

[0669] 103. The method of claim 101, wherein R ₁ is methyl and R ₂ is methyl.

[0600] 104. A method for detecting cysteine protease activity, comprising:

(1) contacting the cysteine protease with a compound according to any one of claims 78 to 94 or a salt thereof, or with a composition according to claim 96, and

(2) subsequent cysteine protease analysis, including measurement of the detected signal.

[0671] 105. The method of claim 104, wherein the method is an in vitro method.

[0672] 106. A method for detecting cysteine protease activity in vitro, comprising:

(1) contacting the biological sample with a compound according to any one of claims 78 to 94 or a salt thereof, or with a composition according to claim 96, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

[0673] 107. A method for detecting cysteine protease activity in a biological sample obtained from a subject, comprising:

(1) contacting the biological sample in vitro with a compound according to any one of claims 78 to 94 or a salt thereof, or with a composition according to claim 96, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

[0674] 108. The method according to any one of claims 97 to 107, wherein step (2) includes performing at least one analytical method selected from the group consisting of gel electrophoresis and subsequent in-gel fluorescence, gel electrophoresis and subsequent radiography, gel electrophoresis and subsequent immunoblotting, fluorescence microscopy, flow cytometry, ex vivo optical imaging, radiography, affinity purification and subsequent mass spectrometry, affinity purification and subsequent proteomics.

[0675] 109. The method of any one of claims 97 to 108, wherein the detected signal is measured by measuring fluorescence.

[0676] 110. The method of claim 108 or 109, wherein at least one analytical method is selected from the group consisting of gel electrophoresis followed by in-gel fluorescence, fluorescence microscopy, and flow cytometry.

[0677] 111. The method of claim 108 or 109, wherein at least one analytical method is selected from gel electrophoresis followed by in-gel fluorescence and fluorescence microscopy.

[0678] 112. The method according to any one of claims 97 to 111, wherein said compound contains a detectable element in the form of a fluorescent label.

[0679] 113. The method according to any one of claims 109 to 111, wherein said compound comprises a detectable element in the form of a bioorthogonal marker for ligation, and wherein step (2) includes secondary labeling using click chemistry to incorporate a fluorescent label before performing, according to at least one analytical method.

[0680] 114. The method according to any one of claims 109 to 111, wherein said compound contains a detectable element in the form of biotin, and wherein step (2) comprises secondary labeling with fluorescently labeled streptavidin or secondary labeling with a fluorescently labeled biotin-specific antibody, before performing at least one analytical method.

[0681] 115. The method of any one of claims 108 to 114, wherein the gel electrophoresis is one-dimensional or two-dimensional gel electrophoresis.

[0682] 116. The method of any one of claims 108 to 115, wherein the gel electrophoresis is SDS-PAGE.

[0683] 117. The method of any one of claims 99-103 and 106-116, wherein the biological sample is selected from the group consisting of cells, cell lysates, tissue samples, tissue lysates, and body fluids.

[0684] 118. The method of claim 117, wherein the biological sample is a cell lysate or a tissue lysate.

[0685] 119. The method of claim 117, wherein the biological sample is a purified cell lysate or a purified tissue lysate.

[0686] 120. The method of claim 117, wherein the biological sample is living cells.

[0687] 121. The method of claim 120, wherein the living cells are lysed and purified between step (1) and step (2).

[0688] 122. The method of any one of claims 99-103 and 106-121, wherein the biological sample is obtained from a human.

[0689] 123. A method for detecting cysteine protease activity, comprising:

(1) administering to the subject an activity-based probe compound containing a sulfoxonium ylide moiety as the active group,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

[0690] 124. The method of claim 123, wherein the activity-based probe compound is administered intravenously.

[0691] 125. The method according to claim 123 or 124, wherein the sulfoxonium ylide moiety has formula (IV):

[0692]

Where:

R ₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl; And

R ₂ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ - _C8 ) alkenyl and (C ₂ -C ₈ ) alkynyl.

[0693] 126. The method of claim 125, wherein R ₁ is (C ₁ -C ₈ ) alkyl and R ₂ is (C ₁ -C ₈ ) alkyl.

[0694] 127. The method of claim 125, wherein R ₁ is methyl and R ₂ is methyl.

[0695] 128. A method for determining cysteine protease activity, comprising:

(1) administering to the subject a compound according to any one of claims 78 to 94 or a salt thereof, or a composition according to claim 96,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

[0696] 129. The method of claim 128, wherein the compound or a salt or composition thereof is administered intravenously.

[0697] 130. The method according to any one of claims 123-129, wherein step (3) includes performing at least one analytical method selected from the group consisting of gel electrophoresis and subsequent in-gel fluorescence, gel electrophoresis and subsequent radiography, gel electrophoresis and subsequent immunoblotting, fluorescence microscopy, flow cytometry, ex vivo optical imaging, radiography, affinity purification and subsequent mass spectrometry, affinity purification and subsequent proteomics.

[0698] 131. The method according to any one of claims 123-130, wherein the detected signal is measured by fluorescence measurement.

[0699] 132. The method of claim 130 or 131, wherein at least one analytical method is selected from the group consisting of gel electrophoresis followed by in-gel fluorescence, fluorescence microscopy, and flow cytometry.

[0700] 133. The method of claim 130 or 131, wherein at least one analytical method is selected from gel electrophoresis followed by in-gel fluorescence and fluorescence microscopy.

[701] 134. The method according to any one of claims 123-133, wherein said compound contains a detectable element in the form of a fluorescent label.

[0702] 135. The method according to any one of claims 131-133, wherein said compound contains a detectable element in the form of a bioorthogonal marker for ligation, and step (3) includes secondary labeling using click chemistry to incorporate a fluorescent label before performing at least at least one analytical method.

[0703] 136. The method according to any one of claims 131-133, wherein said compound contains a detectable element in the form of biotin, and wherein step (3) comprises secondary labeling with a fluorescently labeled streptavidin or secondary labeling with a fluorescently labeled biotin-specific antibody, before performing at least one analytical method.

[0704] 137. The method of any one of claims 130-136, wherein the gel electrophoresis is one-dimensional or two-dimensional gel electrophoresis.

[0705] 138. The method of any one of claims 130-137, wherein the gel electrophoresis is SDS-PAGE.

[0706] 139. The method of any one of claims 123 to 138, wherein the biological sample is selected from the group consisting of cells, cell lysates, tissue samples, tissue lysates, and body fluids.

[0707] 140. The method of claim 139, wherein the biological sample is a cell lysate or a tissue lysate.

[0708] 141. The method of claim 139, wherein the biological sample is a purified cell lysate or a purified tissue lysate.

[0709]

[0710] 142. The method of any one of claims 123 to 141, wherein the subject is a human.

[0711] 143. A method for detecting cysteine protease activity in a subject in vivo, comprising:

(1) administering to the subject an activity-based probe compound containing a sulfoxonium ylide moiety as an active group, and

(2) subsequent examination of the object, including measurement of the detected signal.

[0712] 144. The method of claim 143, wherein the activity-based probe compound is administered intravenously.

[0713] 145. The method according to claim 143 or 144, wherein the sulfoxonium ylide moiety has formula (IV):

[0714]

Where:

R ₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl; And

R ₂ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl.

[0715] 146. The method of claim 145, wherein R ₁ is (C ₁ -C ₈ ) alkyl and R ₂ is (C ₁ -C ₈ ) alkyl.

[0716] 147. The method of claim 145, wherein R ₁ is methyl and R ₂ is methyl.

[0717] 148. A method for determining cysteine protease activity in a subject in vivo, comprising:

(1) administering to the subject a compound according to any one of claims 78 to 94 or a salt thereof, or a composition according to claim 96, and

(2) subsequent examination of the subject, including measurement of the detected signal.

[0718] 149. The method of claim 148, wherein the compound or a salt or composition thereof is administered intravenously.

[0719] 150. The method of any one of claims 143 to 149, wherein the detected signal is measured using in vivo optical imaging, radiography, or positron emission tomography.

[0720] 151. The method according to any one of claims 143-150, wherein the subject is a human.

[0721] 152. The method of any one of claims 97 to 151, wherein the cysteine protease is a mammalian cysteine protease.

[0722] 153. The method of any one of claims 97 to 151, wherein the cysteine protease is a human cysteine protease.

[0723] 154. The method of any one of claims 97 to 151, wherein the cysteine protease is a cysteine cathepsin.

[0724] 155. The method of any one of claims 97 to 151, wherein the cysteine protease is a mammalian cysteine cathepsin.

[0725] 156. The method of any one of claims 97 to 151, wherein the cysteine protease is a human cysteine cathepsin.

[0726] 157. The method of any one of claims 97 to 151, wherein the cysteine protease is cathepsin X.

[0727] 158. The method of any one of claims 97 to 151, wherein the cysteine protease is mammalian cathepsin X.

[0728] 159. The method of any one of claims 97 to 151, wherein the cysteine protease is human cathepsin X.

[0729] 160. The method of any one of claims 97 to 159, wherein cathepsin X activity is detected and cathepsin B activity and/or cathepsin L activity is not detected.

[0730] 161. The method according to any one of claims 97 to 159, wherein cathepsin X activity and cathepsin S activity are detected, and cathepsin B activity and/or cathepsin L activity are not detected.

[0731] 162. A method of inhibiting a cysteine protease, comprising contacting the cysteine protease with a compound of any one of claims 1 to 94 or a salt thereof, or a composition of claim 95.

[0732] 163. The method of claim 162, wherein the method is an in vitro method.

[0733] 164. A method of inhibiting cysteine protease in vitro, comprising contacting a biological sample with a compound according to any one of claims 1 to 94 or a salt thereof, or with a composition according to claim 95.

[0734] 165. A method of inhibiting a cysteine protease in a biological sample obtained from a subject, comprising contacting the biological sample in vitro with a compound of any one of claims 1 to 94 or a salt thereof, or a composition of claim 95.

[0735] 166. The method of claim 164 or 165, wherein the biological sample is selected from the group consisting of cells, cell lysates, tissue samples, tissue lysates, and body fluids.

[0736] 167. A method of in vivo inhibition of a cysteine protease in a subject, comprising administering to the subject a compound of any one of claims 1 to 94 or a salt thereof, or a composition of claim 95.

[0737] 168. The method of claim 167, wherein the compound or a salt or composition thereof is administered intravenously.

[0738] 169. The method of any one of claims 162 to 168, wherein the cysteine protease is a mammalian cysteine protease.

[0739] 170. The method of any one of claims 162 to 168, wherein the cysteine protease is a human cysteine protease.

[0740] 171. The method of any one of claims 162 to 168, wherein the cysteine protease is a cysteine cathepsin.

[0741] 172. The method of any one of claims 162 to 168, wherein the cysteine protease is a mammalian cysteine cathepsin.

[0742] 173. The method of any one of claims 162 to 168, wherein the cysteine protease is a human cysteine cathepsin.

[0743] 174. The method of any one of claims 162 to 168, wherein the cysteine protease is cathepsin X.

[0744] 175. The method of any one of claims 162 to 168, wherein the cysteine protease is mammalian cathepsin X.

[0745] 176. The method of any one of claims 162 to 168, wherein the cysteine protease is human cathepsin X.

[0746] 177. The method according to any one of claims 162-176, wherein cathepsin X is inhibited and cathepsin B and/or cathepsin L are not inhibited.

[0747] 178. The method according to any one of claims 162-176, wherein cathepsin X and cathepsin S are inhibited, and cathepsin B and/or cathepsin L are not inhibited.

[0748] 179. A method for diagnosing a disease associated with cysteine protease activity in a subject, comprising:

(1) contacting a biological sample obtained from a subject in vitro with a compound according to any one of claims 78 to 94 or a salt thereof, or with a composition according to claim 96, and

(2) subsequent analysis of the biological sample, including measurement of the detected signal.

[0749] 180. The method of claim 179, wherein the method comprises determining cysteine protease activity in accordance with the method of any one of claims 107-122 and 152-161.

[0750] 181. A method for diagnosing a disease associated with cysteine protease activity in a subject, comprising:

(1) administering to the subject a compound according to any one of claims 78 to 94 or a salt thereof, or a composition according to claim 96,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

[0751] 182. The method of claim 181, wherein the method comprises determining cysteine protease activity in accordance with the method of any one of claims 128-142 and 152-161.

[0752] 183. A method for diagnosing in vivo a disease associated with cysteine protease activity in a subject, comprising:

(1) administering to the subject a compound according to any one of claims 78 to 94 or a salt thereof, or a composition according to claim 96, and

(2) subsequent examination of the subject, including measurement of the detected signal.

[0753] 184. The method of claim 183, wherein the method comprises determining cysteine protease activity in accordance with the method of any one of claims 148 to 161.

[0754] 185. A compound according to any one of claims 78 to 94, or a salt thereof, for use in a method for diagnosing a disease associated with cysteine protease activity in a subject, wherein the method includes:

(1) administering to the subject a compound according to any one of claims 78 to 94 or a salt thereof,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

[0755] 186. A compound for use according to claim 185, wherein the method includes determining cysteine protease activity in accordance with the method of any one of claims 128-142 and 152-161.

[0756] 187. The composition of claim 96 for use in a method for diagnosing a disease associated with cysteine protease activity in a subject, wherein the method includes:

(1) administering to the subject the composition according to claim 96,

(2) subsequent collection of a biological sample from the subject; And

(3) subsequent analysis of the biological sample, including measurement of the detected signal.

[0757] 188. A composition for use according to claim 187, wherein the method comprises determining cysteine protease activity in accordance with the method of any one of claims 128-142 and 152-161.

[0758] 189. A compound according to any one of claims 78 to 94, or a salt thereof, for use in a method for in vivo diagnosis of a disease associated with cysteine protease activity in a subject, wherein the method includes:

(1) administering to the subject a compound according to any one of claims 78 to 94 or a salt thereof, and

(2) subsequent examination of the subject, including measurement of the detected signal.

[0759] 190. A compound for use according to claim 189, wherein the method comprises detecting cysteine protease activity in accordance with the method of any one of claims 148-161.

[0760] 191. The composition of claim 96 for use in a method for in vivo diagnosis of a disease associated with cysteine protease activity in a subject, wherein the method includes:

(1) administering to the subject the composition of claim 96, and

(2) subsequent examination of the subject, including measurement of the detected signal.

[0761] 192. A composition for use according to claim 191, wherein the method comprises detecting cysteine protease activity in accordance with the method of any one of claims 148-161.

[0762] 193. A method of treating a disease associated with cysteine protease activity, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any one of claims 1 to 94 or a salt thereof, or a therapeutically effective amount of a composition of claim 95.

[0763] 194. A compound according to any one of claims 1 to 94, or a salt thereof, for use in the treatment of a disease associated with cysteine protease activity.

[0764] 195. A composition for the treatment of a disease associated with cysteine protease activity, comprising a compound according to any one of claims 1 to 94 or a salt thereof and a pharmaceutically acceptable excipient.

[0765] 196. Use of a compound according to any one of claims 1 to 94 or a salt thereof in the manufacture of a medicament for the treatment of a disease associated with cysteine protease activity.

[0766] 197. Use of the composition according to claim 95 in the manufacture of a medicament for the treatment of a disease associated with cysteine protease activity.

[0767] 198. The method, use, compound for use, or composition for use according to any one of claims 179 to 197, wherein the disease is selected from the group consisting of celiac disease, gastrointestinal motility disorder, pain, itching, skin disease, obesity diet-induced, metabolic disorders, asthma, rheumatoid arthritis, periodontitis, inflammatory disease, functional gastrointestinal disorder, cancer, fibrotic disease, metabolic dysfunction, neurological disease and neurodegenerative disease.

[0768] 199. The method, use, compound for use, or composition for use according to claim 198, wherein the functional gastrointestinal disorder is selected from the group consisting of irritable bowel syndrome, functional chest pain, functional dyspepsia, nausea and vomiting, functional constipation, etc., functional diarrhea, fecal incontinence, functional anorectal pain and functional defecation disorders.

[0769] 200. The method, use, compound for use, or composition for use according to claim 199, wherein the functional gastrointestinal disorder is irritable bowel syndrome.

[0770] 201. The method, use, compound for use, or composition for use according to any one of claims 179 to 198, wherein the disease is selected from the group consisting of cancer, an inflammatory disease, and a neurodegenerative disease.

[0771] 202. The method, use, compound for use, or composition for use according to any one of claims 179 to 198, wherein the disease is cancer selected from the group consisting of breast cancer, brain cancer, bone marrow cancer, cancer pancreas, lung cancer, prostate cancer, liver cancer, oral cancer, colorectal cancer and stomach cancer.

[0772] 203. The method, use, compound for use, or composition for use according to any one of claims 179 to 198, wherein the disease is an inflammatory disease selected from the group consisting of inflammatory gastrointestinal disorder, pancreatitis, and infection.

[0773] 204. The method, use, compound for use, or composition for use according to claim 203, wherein the inflammatory gastrointestinal disorder is selected from the group consisting of inflammatory bowel disease, infectious diarrhea, mesenteric ischemia, diverticulitis, and necrotizing enterocolitis (NEC ).

[0774] 205. The method, use, compound for use or composition for use according to claim 204, wherein the inflammatory bowel disease is selected from the group consisting of ulcerative colitis, Crohn's disease, diversion colitis, colitis of undetermined etiology and pauchitis, microscopic colitis, immuno-oncological colitis, colitis due to chemotherapy/radiation therapy, colitis due to graft-versus-host syndrome (GvHD), acute colitis, Behçet's disease, collagenous colitis and lymphocytic colitis.

[0775] 206. The method, use, compound for use, or composition for use according to claim 204, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

[0776] 207. The method, use, compound for use or composition for use according to any one of claims 179 to 198, wherein the disease is a neurodegenerative disease selected from the group consisting of Alzheimer's disease, multiple sclerosis and neuropathic pain.

[0777] 208. The method, use, compound for use, or composition for use according to any one of claims 179 to 207, wherein the disease is a disease associated with cathepsin X activity.

[0778] 209. A method of producing a compound bearing a chloromethyl ketone group, comprising reacting a compound bearing a sulfoxonium ylide group to produce a compound bearing a chloromethyl ketone group.

[0779] 210. A method for preparing an activity-based probe compound bearing an acyloxymethyl ketone group or a phenoxymethyl ketone group as an active group, comprising:

(i) preparing an intermediate containing a chloromethyl ketone group by reacting a compound containing a sulfoxonium ylide moiety to produce a compound bearing a chloromethyl ketone group; And

(ii) further processing the compound containing the chloromethyl ketone group to obtain said probe compound based on activity.

[0780] 211. The method of claim 209 or 210, wherein the compound bearing the sulfoxonium ylide moiety is reacted with hydrochloric acid to produce a compound containing a chloromethyl ketone group.

[0781] 212. The method according to any one of claims 209-211, wherein the sulfoxonium ylide moiety has formula (IV):

[0782]

Where:

R ₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl; And

R ₂ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl.

[0783] 213. The method of claim 212, wherein R ₁ is (C ₁ -C ₈ ) alkyl and R ₂ is (C ₁ -C ₈ ) alkyl.

[0784] 214. The method of claim 212, wherein R ₁ is methyl and R ₂ is methyl.

[0785] 215. A compound selected from the group of formulas consisting of:

[0786]

[0787]

[0788]

or its salt.

Claims (152)

1. Compound of formula I or its salt, Where: R ₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl; R ₂ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl; R ₃ represents a side chain of an alpha amino acid selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, methionine, ethionine, phenylalanine, tyrosine, levodopa, tryptophan, cysteine , homocysteine, selenocysteine, selenohomocysteine, selenomethionine, selenoethionine, lysine, histidine, arginine, ornithine, aspartic acid, glutamic acid, serine, homoserine, O-methylhomoserine, O-ethylhomoserine, threonine, asparagine and glutamine, wherein said side chain is optionally substituted with an amino protecting group and optionally further substituted with one or more identical or different R _3x substituents, wherein R _3x is selected from the group consisting of hydroxy, halogen, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy; R ₄ is selected from the group consisting of a hydrogen atom and (C ₁ -C ₄ ) alkyl; R ₅ represents a detectable element that is a fluorescent label that is a cyanine label; X represents: (i) communication; or (ii) a diradical fragment of formula II, which is connected to the R ₅ substituent through an amino group: Where: R ₆ represents a side chain of an alpha amino acid selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, methionine, ethionine, phenylalanine, tyrosine, levodopa, tryptophan, cysteine , homocysteine, selenocysteine, selenohomocysteine, selenomethionine, selenoethionine, lysine, histidine, arginine, ornithine, aspartic acid, glutamic acid, serine, homoserine, O-methylhomoserine, O-ethylhomoserine, threonine, asparagine and glutamine, wherein said side chain is optionally substituted with an amino protecting group and optionally further substituted with one or more identical or different R _6x substituents, wherein R _6x is selected from the group consisting of hydroxy, halogen, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy; R ₇ is selected from the group consisting of a hydrogen atom and (C ₁ -C ₄ ) alkyl; And wherein the amino protecting group is selected from the group consisting of benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), p-toluenesulfonyl (Tos), 2,2,5,7,8- pentamethylchroman-6-sulfonyl (Pmc), 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf), mesityl-2-sulfonyl (Mts), 4-methoxy-2,3 ,6-trimethylphenylsulfonyl (Mtr), acetamido, and phthalimido. 2. Connection according to claim 1, where R ₃ represents an alpha amino acid side chain selected from the group consisting of alanine, alpha aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, phenylalanine and tryptophan. 3. A compound according to claim 1 or 2, wherein R ₃ represents an alpha amino acid side chain selected from the group consisting of valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, phenylalanine and tryptophan. 4. Compound of formula I or its salt, Where: R ₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl; R ₂ is selected from the group consisting of (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl and (C ₂ -C ₈ ) alkynyl; R ₃ is a lysine side chain, wherein said side chain is substituted with a detectable element and optionally further substituted with one or more identical or different R _3x substituents, wherein R _3x is selected from the group consisting of hydroxy, halogen, (C ₁ -C ₄ ) alkyl , (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy; R ₄ is selected from the group consisting of a hydrogen atom and (C ₁ -C ₄ ) alkyl; R ₅ is selected from the group consisting of amino protecting group, (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) hydroxyalkyl, (C ₁ -C ₈ ) haloalkyl, (C ₃ -C ₈ ) cycloalkyl, (C ₂ -C ₈ ) alkenyl, (C ₂ -C ₈ ) alkynyl, (C ₁ -C ₈ ) alkylcarbonyl, (C ₁ -C ₈ ) hydroxyalkylcarbonyl, (C ₁ -C ₈ ) haloalkylcarbonyl, (C ₃ -C ₈ ) cycloalkylcarbonyl , (C ₁ -C ₈ ) alkyloxycarbonyl, benzyloxycarbonyl and a hydrogen atom; X represents: (i) communication; or (ii) a diradical fragment of formula II, which is connected to the R ₅ substituent through an amino group: Where: R ₆ represents a side chain of an alpha amino acid selected from the group consisting of glycine, alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine, methionine, ethionine, phenylalanine, tyrosine, levodopa, tryptophan, cysteine , homocysteine, selenocysteine, selenohomocysteine, selenomethionine, selenoethionine, lysine, histidine, arginine, ornithine, aspartic acid, glutamic acid, serine, homoserine, O-methylhomoserine, O-ethylhomoserine, threonine, asparagine and glutamine, wherein said side chain is optionally substituted with an amino protecting group and optionally further substituted with one or more identical or different R _3x substituents, wherein R _3x is selected from the group consisting of hydroxy, halogen, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) hydroxyalkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy and (C ₁ -C ₄ ) haloalkoxy; R ₇ is selected from the group consisting of a hydrogen atom and (C ₁ -C ₄ ) alkyl; wherein the detectable element is a fluorescent label, wherein the fluorescent label is a cyanine label, and wherein the amino protecting group is selected from the group consisting of benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), p-toluenesulfonyl (Tos), 2,2,5,7,8- pentamethylchroman-6-sulfonyl (Pmc), 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf), mesityl-2-sulfonyl (Mts), 4-methoxy-2,3 ,6-trimethylphenylsulfonyl (Mtr), acetamido, and phthalimido. 5. The compound according to claim 4, where R ₅ represents an amino protecting group. 6. A compound according to any one of claims 1 to 5, where R ₁ represents (C ₁ -C ₈ ) alkyl and R ₂ represents (C ₁ -C ₈ ) alkyl. 7. The compound according to claim 6, where R ₁ and R ₂ represent methyl. 8. A compound according to any one of claims 1 to 7, where R ₄ represents a hydrogen atom. 9. Connection according to any one of claims 1-8, where R ₆ represents an alpha amino acid side chain selected from the group consisting of alanine, alpha-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, homonorleucine and phenylalanine. 10. The compound according to claim 9, where R ₆ represents a side chain of phenylalanine. 11. A compound according to any one of claims 1 to 10, where R ₇ represents a hydrogen atom. 12. A compound according to any one of claims 1 to 11, wherein the amino protecting group is selected from the group consisting of benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), and tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc). 13. A compound according to claim 12, wherein the amino protecting group is benzyloxycarbonyl. 14. A compound according to any one of claims 1 to 13, where the formula I has the formula IA and wherein the biradical moiety of formula II has formula IIA: 15. A compound according to any one of claims 1 to 14, where X represents a bond. 16. A compound according to any one of claims 1 to 15, wherein the cyanine label has a formula selected from the following group of formulas: where in each of the above formulas, A is selected from the group consisting of CH ₂ , C(CH ₃ ) ₂ , C(C ₂ H ₅ ) ₂ , NH, N(CH ₃ ), N(C ₂ H ₅ ), O, S and Se; R ₁₀ is selected from the group consisting of $-(CH ₂ ) _p -C(=O)-& and $-(CH ₂ ) _q -C(=O)-NH-[CH ₂ CH ₂ O] _r -CH ₂ CH ₂ -C(=O)-&; Where: p is equal to 2, 3, 4, 5, 6, 7 or 8; q is equal to 2, 3, 4, 5, 6, 7 or 8; r is equal to 2, 3, 4, 5, 6, 7 or 8; $ represents the point of connection with the nitrogen atom of the cyanine group; and & represents the point of connection with the rest of the molecule; R ₁₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl and (C ₆ -C ₁₀ ) aryl; And R ₁₂ represents H or sulfo group. 17. The compound according to claim 16, where the cyanine label has a formula selected from the following group of formulas: where in each of the above formulas, the wavy line represents the connection point with the rest of the molecule; and R ₁₁ is selected from the group consisting of (C ₁ -C ₈ ) alkyl and (C ₆ -C ₁₀ ) aryl. 18. The compound according to claim 17, where the cyanine label, has the formula: where the wavy line represents the connection point with the rest of the molecule; and R ₁₁ is methyl or ethyl. 19. A compound according to any one of claims 4 to 18, where R ₃ represents -CH ₂ CH ₂ CH ₂ CH ₂ N(R _3a )(R _3b ); Where: R _3a represents a detectable element; And R _3b is selected from the group consisting of a hydrogen atom and a (C ₁ -C ₄ ) alkyl atom. 20. The compound according to claim 1, which is a compound selected from the group of formulas consisting of: or its salt; wherein optionally the compound is a compound of the formula: or its salt. 21. The connection according to claim 4, which is or its salt. 22. A composition acting as activity probes or cathepsin X inhibitors, comprising an effective amount of a compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, and an excipient. 23. A composition acting as activity-based probes for cathepsin X in vitro, comprising an effective amount of a compound according to any one of claims 1 to 21 or a salt thereof, and an excipient. 24. A method for detecting cathepsin X activity in a biological sample obtained from a subject, comprising: (1) contacting the biological sample in vitro with a compound according to any one of claims 1 to 21 or a salt thereof, or with a composition according to claim 23, and (2) subsequent analysis of the biological sample, including measurement of the detected signal. 25. The method according to claim 24, where step (2) includes performing at least one analytical method selected from the group consisting of gel electrophoresis and subsequent gel fluorescence, gel electrophoresis and subsequent radiography, gel electrophoresis and subsequent immunoblotting, fluorescence microscopy, flow cytometry, ex vivo optical imaging, radiography, affinity purification and subsequent mass spectrometry, affinity purification and subsequent proteomics. 26. The method according to any one of claims 24, 25, where the detected signal is measured by fluorescence measurement; where optionally, at least one analytical method is selected from the group consisting of gel electrophoresis followed by in-gel fluorescence, fluorescence microscopy and flow cytometry. 27. Method according to any one of paragraphs 24-26, wherein the gel electrophoresis is one-dimensional or two-dimensional gel electrophoresis; and/or where the gel electrophoresis is SDS-PAGE. 28. Method according to any one of paragraphs 24-27, wherein the biological sample is selected from the group consisting of cells, cell lysates, tissue samples, tissue lysates, and body fluids. 29. Method according to claim 28, wherein the biological sample is a cell lysate or a tissue lysate; or wherein the biological sample is a purified cell lysate or a purified tissue lysate; or where the biological sample is living cells; or where live cells are lysed and purified between step (1) and step (2). 30. A method for detecting catapsin X activity, including: (1) administering to the subject a compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, or a composition according to claim 22, subsequently obtaining a biological sample from the subject; And subsequent analysis of the biological sample, including measurement of the detected signal. 31. The method according to claim 30, where step (3) includes performing at least one analytical method selected from the group consisting of gel electrophoresis and subsequent gel fluorescence, gel electrophoresis and subsequent radiography, gel electrophoresis and subsequent immunoblotting, fluorescence microscopy, flow cytometry, ex vivo optical imaging, radiography, affinity purification and subsequent mass spectrometry, affinity purification and subsequent proteomics. 32. The method according to any one of claims 30, 31, where the detected signal is measured by fluorescence measurement; and/or wherein at least one analytical method is selected from the group consisting of gel electrophoresis followed by in-gel fluorescence, fluorescence microscopy and flow cytometry. 33. The method according to any one of claims 30-32, where the gel electrophoresis is one-dimensional or two-dimensional gel electrophoresis; and/or where the gel electrophoresis is SDS-PAGE. 34. Method according to any one of paragraphs 30-34, wherein the biological sample is selected from the group consisting of cells, cell lysates, tissue samples, tissue lysates, and body fluids; wherein optionally the biological sample is a cell lysate or a tissue lysate; or wherein the biological sample is a purified cell lysate or a purified tissue lysate. 35. The method according to any one of claims 30-34, where the subject is a person. 36. A method for detecting catapsin X activity in a subject in vivo, comprising: (1) administering to the subject a compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, or a composition according to claim 22, and (2) subsequent examination of the subject, including measurement of the detected signal. 37. Method according to claim 36, where the detected signal is measured using in vivo optical imaging, radiography or positron emission tomography. 38. The method according to claim 36 or 37, where the subject is a person. 39. The method according to any one of claims 24 to 38, wherein cathepsin X is mammalian cathepsin X, and optionally human cathepsin X. 40. Method according to any one of paragraphs 24-39, wherein the activity of cathepsin X is detected, and the activity of cathepsin B and/or the activity of cathepsin L is not detected. 41. The method according to any one of claims 24 to 40, wherein the activity of cathepsin X and the activity of cathepsin S are detected, and the activity of cathepsin B and/or the activity of cathepsin L are not detected. 42. A compound according to any one of claims 1 to 21, or a pharmaceutically acceptable salt thereof, for use in a method for diagnosing a disease associated with cathepsin X activity in a subject, where the method includes: (1) administering to the subject a compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, (2) subsequent collection of a biological sample from the subject; And (3) subsequent analysis of the biological sample, including measurement of the detected signal; where optionally the method includes detecting cathepsin X activity in accordance with the method according to any one of claims 30-35 and 39-41. 43. The composition according to claim 22, for use in a method for diagnosing a disease associated with cathepsin X activity in a subject, where the method includes: (1) administering to the subject the composition according to claim 22, (2) subsequent collection of a biological sample from the subject; And (3) subsequent analysis of the biological sample, including measurement of the detected signal; where optionally the method includes detecting cathepsin X activity in accordance with the method according to any one of claims 30-35 and 39-41. 44. A compound according to any one of claims 1 to 21, or a pharmaceutically acceptable salt thereof, for use in a method for in vivo diagnosis of a disease associated with cathepsin X activity in a subject, where the method includes: (1) administering to the subject a compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, and (2) subsequent examination of the subject, including measurement of the detected signal; where optionally the method includes determining the activity of cathepsin X in accordance with the method according to any one of claims 36-38. 45. The composition according to claim 22 for use in a method for diagnosing in vivo a disease associated with cathepsin X activity in a subject, where the method includes: (1) administering to the subject the composition of claim 22, and (2) subsequent examination of the subject, including measurement of the detected signal; where optionally the method includes detecting cathepsin X activity in accordance with the method according to any one of claims 36-38. 46. The compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof in an effective amount for use in the treatment of a disease associated with cathepsin X activity, wherein the disease is selected from the group consisting of celiac disease, gastrointestinal motility disorder, pain, itching, skin disease, diet-induced obesity, metabolic disorder, asthma, rheumatoid arthritis, periodontitis, inflammatory disease, functional gastrointestinal disorder, cancer, fibrotic disease, metabolic dysfunction, neurological disease and neurodegenerative disease.