PRIORITY

This application claims priority to United States Provisional Application Number 62/222,586, filed 23 September 2015 and to United States Provisional Application Number 62/344,251 , filed 01 June 2016. The entire content of these United States Provisional

Applications is hereby incorporated herein by reference.

BACKGROUND

Apoptosis is a complex mechanism of programmed cell death that is controlled by multiple biochemical events leading to morphological cell changes and eventual cell death. The apoptosis process begins when apoptotic signals cause regulatory proteins to initiate an apoptosis pathway. The primary pathways targeted include mitochondrial functionality, transduced signals via adaptor proteins to the apoptotic mechanism, and drug induced increases in calcium within the cell. Apoptosis culminates in coordinated cell death that requires energy and, unlike cell death occurring by necrosis, does not induce an inflammatory response.

Apoptosis is a critical event in numerous processes within the body. For example, embryonic development relies on apoptosis, and tissues that turn over rapidly require tight regulation to avoid serious pathological consequences. Certain medical conditions, such as cancer, are characterized by insufficient apoptosis (insufficient cell death) and uncontrolled cell proliferation brought on in part by the failure to regulate apoptosis. In treating such conditions, apoptosis can be induced by various means. For example, chemical induction of apoptosis can be achieved by administering drugs such as chemotherapeutic agents that initiate apoptosis. In some medical conditions, including neuropathies such as Alzheimer's disease, excess apoptosis can damage organs. Apoptosis can also be indicative of tissue damage, such as damaged heart tissue following ischemia or reperfusion insults.

Inflammation is an organism's reaction to harmful stimuli, such as pathogens, damaged cells, or irritants, and an attempt by the organism (e.g. a human body) to initiate a healing process and remove the cause of the inflammation. An inflammatory reaction typically involves an organism's local vascular system, immune system, and cells within the injured tissue.

Chronic inflammation is characterized by a shift in cell types at the site of inflammation and simultaneous destruction and healing of tissue. Cellular pathways leading to both apoptosis and inflammation involve the activation of members of a family of proteases known as caspases. At least 14 members of the caspase family have been identified in vertebrates, and at least 8 are known to be involved in apoptotic cell death (see Saunders, et ah, Anal. Biochem., 284, 114-24 (2000)). Caspases are a group of highly specific cysteine proteases that cleave aspartic acid peptide bonds within proteins. Caspases collaborate in the proteolytic cascade by activating themselves and each other. Apoptosis-related caspases can be divided into two categories: "initiator" caspases (e.g., caspase-2, caspase-8, caspase-9 and caspase- 10), and downstream "effector" caspases (e.g., caspase-3, caspase-6, caspase-7 and caspase-14). Initiator caspases mediate their oligomerization and autoactivation in response to specific upstream signals, and can activate effector caspases by cleaving their inactive pro-forms. Activated effector caspases continue the apoptotic process by cleaving protein substrates within a cell. Inhibitors of caspases can thus regulate the initiation and/or effector enzymes within the apoptotic caspase chain reaction by inhibiting these processes.

Other caspases (e.g., caspase-1 , caspase-4, caspase-5, caspase-1 1 and caspase-13) are involved in inflammatory pathways. One such inflammation-related caspase (i.e., caspase-1) was identified as the IL-1 converting enzyme (ICE-1) required for activation of the IL-1 beta and IL-18 cytokines in inflammatory responses. The detection of active caspases involved in inflammatory pathways indicates an acute or chronic inflammatory response - e.g., inflammation associated with inflammatory diseases such as rheumatoid arthritis or atherosclerosis.

During apoptosis and/or inflammation, high levels of active caspases are expressed. One way to block apoptosis and/or inflammation involves the use of peptidic or synthetic caspase inhibitors, which bind to active caspases within cells. Historically, broad spectrum, peptidic pan- caspase inhibitors such as Z-VAD-FMK have routinely been used in vitro in scientific research and drug development screening to block caspase activity. A more potent caspase inhibitor may prove valuable for in vitro testing, in that it may allow for the use of less product and potentially shorten test duration.

In in vivo applications, caspase inhibitors have been shown in various animal models to inhibit post myocardial infarction apoptosis, to reduce lesion volume and neurological deficit resulting from stroke or ischemia, to reduce post-traumatic apoptosis and neurological deficit in traumatic brain injury (TBI), and to be effective in treating fulminant liver destruction, liver disease and sepsis (see e.g. Yaoita et al. (1998) Circulation, 97: 276-281 ; Endres et al. (1998) J. Cerebral Blood Flow and Metabolism, 18: 238-247; Cheng et al. (1998) J. Clin. Invest. 101 : 1992-1999; Yakovlev et al. (1997) J. Neurosci. 17: 7415-7424; Rodriquez et al. (1996) J. Exp. Med., 184: 2067-2072); Pockros et al. (2007) HEPATOLOGY, Vol. 46, No. 2). More recently, Caserta et al found that Q-VD-OPH, a next generation broad spectrum caspase inhibitor, was significantly more effective in preventing apoptosis in vivo than widely used caspase inhibitors such as Z-VAD-FMK (see Q-VD-OPH, a broad spectrum caspase inhibitor with anti-apoptotic properties. Apoptosis. 2003 Aug 8(4):345-52). Additionally, the pan caspase inhibitor emricasan (a potent inhibitor of both apoptotic and inflammatory caspases) has recently been investigated as a therapeutic in various models of liver disease. Recent research efforts also include the use of caspase inhibitors in combination with various therapeutics for the treatment of cancer (ref: Brumatti et al., The Caspase-8 Inhibitor Emricasan Combines With the SMAC Mimetic

Binnapant to Induce Necroptosis and Treat Acute Myeloid Leukemia, Sci Trans Med 8:339ra69, 2016).

It is hoped that caspase inhibitors may someday be used in vivo as potential therapeutics for certain conditions involving apoptosis and/or inflammation, including (but not limited to) neurodegenerative diseases such as Alzheimer's and Multiple Sclerosis, liver disease, spinal atrophy, stroke, traumatic brain injury, myocardial infarction, fibrotic diseases (kidney fibrosis, idiopathic pulmonary fibrosis, diabetic nephropathy, liver fibrosis, non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC), systemic sclerosis, corneal fibrosis), and inflammatory conditions related to metabolic disease. However, the use of existing caspase inhibitors in vivo has met with considerable challenges involving undesirable pharmacological effects and cytotoxicity, due in part to the dose of inhibitor required to achieve a sufficient anti-apoptotic and/or anti-inflammatory effect. Available information suggests the need for safe, stable, caspase-selective, cell-permeant, irreversible caspase inhibitors with increased potency (i.e. enhanced caspase binding affinity and kinetics), suitable for both in vitro and in vivo

applications. Inflammatory pathways also involve the expression of a family of proteases called cathepsins. The cysetine cathepsins, in particular (cathepsins B, C, F, H, K, L, O, S, W, X/Z) are often highly upregulated or overexpressed during conditions where inflammation is present, such as cancer (especially with tumor invasion, angiogenesis, metastasis, or tumor associated macrophages (TAMS) in the tumor microenvironment), auto-immune diseases (e.g. lupus, psoriasis, Crohn's disease, Sjogren's syndrome, celiac disease), neurodegenerative diseases (e.g. Alzheimers), traumatic brain injury, arthritis, hepatitis (including alcohol-related and NASH), pancreatitis, liver fibrosis and steatosis (including HCV-associated), pulmonary fibrosis, renal fibrosis and cardiac fibrosis (ref: Golde et al., Science 255: 728-730, 1992; Munger et al., Biochem. J. 31 1 : 299-305, 1995; Iwata et al., Arthritis and Rheumatism 40: 499-509, 1997; Yan et al, Biol. Chem. 379: 113-123, 1998; Joyce et al., Cancer Cell 5: 443-453, 2004; Turk et al., Biochimica et Biophysica Acta. 1824 (2012) 68-88; Hook et al., Frontiers in Neurology 6:article 178, 2015). Because cysteine cathepsins (like caspases) play a critical role in a number of diseases, they are increasingly becoming attractive targets for numerous therapeutic

applications.

One way to block cathepsin expresion involves the use of peptidic or synthetic cathepsin inhibitors which bind to (and as a result, inhibit the activity of) cysteine cathepsins. As with caspase inhibitors, cysteine cathepsin inhibitors are used in vitro in scientific research and drug development screening. For example, cathepsin inhibitors such as Z-FA-FMK (for cathepsin B) are widely available and have been used extensively for in vitro applications. As with caspase inhibitors, more potent cathepsin inhibitors may prove valuable for in vitro testing, in that they may allow for the use of less product and potentially shorten test duration.

Targeting cysteine cathepsins in vivo for therapeutic purposes is a continuing effort, with several commercial projects currently focusing on inhibition of cathepsin B, L, S or K for various clinical applications such as neuropathic pain and Alzheimers, liver fibrosis (associated with HCV, non-alcoholic steatohepatititis or "NASH", alcoholic steatohepatitis, non-alcoholic fatty liver disease), cirrhosis, and various conditions associated with metabolic disease.

In regard to cancer, in addition to the implication of cysteine cathepsin upregulation in tumor metastasis and invasion, mounting evidence suggests that cysteine cathepsins are also upregulated in cells within the tumor environment, such as tumor associated macrophages (TAMs). Cathepsin inhibitors which can also target upregulation of cathepsins in the tumor microenvironment (e.g. within TAMs) might someday represent a viable approach to cancer prevention or treatment, and cathepsin inhibitor compounds designed to target both the tumor and tumor microenvironment have already been proposed (ref: Mikhaylov, et al. Ferri- Liposomes as a Novel MRI- Visible Drug Delivery System for Targeting Tumours and their Microenvironment, Nat. Nanotechnol., 6 (2011), pp. 594-602; Salpeter, et al. A Novel Cysteine cathepsin inhibitor Yields Macrophage Cell Death and Mammary Tumor Regression, Oncogene, (2015), 1 -13).

Cathepsin inhibitors have also been used as anti-viral agents. Examples of cathepsin inhibitors used to block virus replication were illustrated in Van der Linden, et al., Cysteine Cathepsins as Anti-Ebola Agents. ACS Infect. Dis., 2016, 2 (3), pp 173-179 and in US2009/0203629A1 (Hepatitis C related). Recent studies suggest that blocking cathepsin activity may not only address the inflammation and tissue injury associated with some viruses such as HCV, but also the overall viral burden itself.

As with caspase inhibitors, there is a need for safe, stable, selective, cell-permeant, cathepsin inhibitors with increased potency (i.e. enhanced cathepsin binding affinity and kinetics), suitable for both in vitro and in vivo applications.

SUMMARY

The present invention provides compositions and methods for caspase and cysteine cathepsin inhibition. In particular, a new class of highly potent, cell membrane permeant, anti- apoptotic and/or anti-inflammatory peptide based caspase and cathepsin inhibitors is provided. The compounds of this invention are capable of forming irreversible covalent bonds to the active site of a caspase or cysteine cathepsin and inhibiting the activity of that enzyme.

One potential application of the invention is in cell based high throughput screening (HTS) testing where rapid inhibition of the apoptotic or inflammatory pathway is desired.

Another potential application is in cell based high throughput screening (HTS) testing where minimal concentration of inhibitor is desired.

Another potential application is the inhibition of caspase or cysteine cathepsin activity in a cell-free system by adding any one of the compounds described in the invention to the purified caspase(s) or cathepsin(s) that it targets.

Another potential application (for the pharmaceutically acceptable derivatives of these compounds) is in the treatment of a variety of mammalian disease states or conditions associated with an increase in cellular apoptosis and/or inflammation, including (but not limited to) myocardial infarction, stroke, traumatic brain injury, fulminant liver destruction, endotoxic shock, sepsis, septic shock, chronic hepatitis (including virus related hepatitis, alcoholic steatohepatitis and non-alcoholic steatohepatitis or "NASH"), pancreatitis, viral infection, fibrosis, implant or transplant rejection, auto-immune diseases, arthritis, neurological conditions (e.g. Alzheimer's Disease), cancer, and ototoxicity.

In one embodiment the invention provides a compound of formula (I):

wherein: Ui is absent or is a fluorinated group comprising 1-10 carbon atoms and one or more fluorine atoms; and U ₂ is absent or is a fluorinated group comprising 1 -10 carbon atoms and one or more fluorine atoms; wherein at least one of Ui and U ₂ is present;

X is a molecule that will recognize a cysteine protease; and

J is a reactive group that binds to a cysteine protease.

One aspect of the invention concerns a method for preventing and/or treating a caspase- mediated or cathepsin-associated disease or condition in a subject in need thereof, comprising administering to said subject an effective amount of a compound represented by any of the above molecules.

Another related aspect of the invention concerns the use of a compound represented by any of the above molecules for the manufacture of a medication for the prevention and/or treatment of caspase-mediated or cathepsin-associated diseases or conditions in a subject in need thereof.

One aspect of the invention concerns a method of treating excessive apoptosis or inflammation affected by caspase activity in a cell or a tissue, the method comprising: contacting the cell or tissue with an effective amount of one or more compounds represented by any of the above formulas.

A final aspect of the invention concerns the use of a compound represented by any of the above molecules to inhibit caspase or cathepsin activity in a cell-free system.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: TFA-VAD(OMe)-FMK vs Z-VAD(OMe)-FMK vs Q-VD-OPH, Percent Inhibition of Caspase 3 at Varying Inhibitor Concentration. Competition Assay using active Caspase-3 and Ac- V AD- AFC (Enzo Lifesciences). Fluorescence kinetic reads performed with a Microplate Reader (M2e, Molecular Devices).

Figure 2: TFA-VAD(OMe)-FMK vs Z-VAD(OMe)-FMK vs Q-VD-OPH, Inhibition

Rate Constant Comparison. Competition Assay using active Caspase-3 and Ac- V AD- AFC (Enzo Lifesciences). Fluorescence kinetic reads performed with a Microplate Reader (M2e, Molecular Devices).

Figure 3: Structure of TFA-VAD(OMe)-FMK (aka Trifluoroacetyl-L-valyl-L-alanyl-L- aspartic acid methyl ester fluoromethyl ketone).

Figure 4: TFA-VAD(OMe)-FMK is more potent at inhibiting staurosporine-induced caspase activity compared to Z-VAD(OMe)-FMK and Q-VD(OMe)-FMK. Inhibitors (10 uM) were added to Jurkat cells (human T lymphocyte cell line) for 15 minutes. 1 uM Staurosporine (protein kinase inhibitor) was then added for 3.5 hours to induce apoptosis. After 3.5 hours, CAS-MAP active caspase labeling reagent (FAM-VAD(OMe)-FMK) was added for 20 minutes. Cells were then analyzed by flow cytometry. An increase in FAM-VAD-FMK fluorescence intensity correlates with caspase activity.

Figure 5: TFA-VAD(OMe)-FMK is more potent than Z-VAD(OMe)-FMK at inhibiting apoptosis. Jurkat cells were incubated with the indicated concentrations of TFA-VAD(OMe)- FMK or Z-VAD(OMe)-FMK for 15 minutes prior to stimulation with 1 μΜ Staurosporine for 4 hours, 5 μΜ Camptothecin (topoisomerase I inhibitor) for 4 hours or 100 ng/ml anti-TRAIL R2 agonist antibody for 24 hours. Cells were labeled with Annexin V - Alexa Fluor 488 and a cell impermeable DNA dye (SYTOX AADvanced) and analyzed by flow cytometry. % Apoptosis was calculated by adding the percentage of cells in the Annexin V positive/SYTOX negative and Annexin V positive/SYTOX positive populations. Data were normalized to no inhibitor samples (unstimulated = 0%, stimulated = 100%) and percent inhibition was calculated.

Figure 6: TFA-VAD(OMe)-FMK inhibits staurosporine-induced apoptosis after 24 hours in cell culture. Jurkat cells were incubated with 10 μΜ TFA-VAD(OMe)-FMK or 10 μΜ TFA-VAD(OMe)-FMK for 24 hours in RPMI 1640 media with 10% FBS. After 24 hours, cells were stimulated for 4 hours with 1 μΜ staurosporine to induce apoptosis followed by labeling with Annexin V - Alexa Fluor 488 and a cell impermeable DNA dye (SYTOX AADvanced). Stained cells were analyzed by flow cytometry. % Apoptosis was calculated by adding the percentage of cells in the Annexin V positive/SYTOX negative and Annexin V positive/SYTOX positive populations.

Figure 7: TFA-VAD(OMe)-FMK, TFA-VAD(OMe)-TPH, TFA-VD(OMe)-TPH, TFA- EVD-TPH and TFA-6E8D-TPH inhibit caspase-3/7 activity in Hep G2 cells (human hepatocyte carcinoma cell line). Hep G2 cells were incubated with the indicated concetrations of caspase inhibitor for 15 minutes prior to a 24 hour treatment with 2 μg/ml anti-TRAIL R2 agonist antibody to induce caspase activity. After 24 hours, a caspase-3/7 specific fluorescent substrate (MP39) was added to each sample in a duel function cell lysis/caspase activity buffer. After 2 hours of substrate incubation, fluorescence was determined using a fluorescence microplate reader. Relative Fluorescent Units were normalized to no inhibitor samples (no anti-TRAIL R2 antibody = 0%, + anti-TRAIL R2 antibody = 100%) and percent inhibition was calculated. Figure 8: TFA-VAD(OMe)-FMK, TFA-VAD(OMe)-TPH, TFA-VD(OMe)-TPH, TFA- EVD-TPH and TFA-6E8D-TPH inhibit staurosporine-induced apoptosis in Jurkat cells. Jurkat cells were incubated with 5 μΜ of the indicated caspase inhibitor for 15 minutes prior to stimulation with 1 μΜ staurosporine for 4 hours. Cells were labeled with Annexin V - Alexa Fluor 488 and a cell impermeable DNA dye (SYTOX AADvanced) and analyzed by flow cytometry. % Apoptosis was calculated by adding the percentage of cells in the Annexin V positive/SYTOX negative and Annexin V positive/SYTOX positive populations. Data were normalized to no inhibitor samples (no staurosporine = 0%, + staurosporine = 100%) and percent inhibition was calculated.

Figure 9: TFA-VAD(OMe)-FMK, TFA-VAD(OMe)-TPH, TFA-VD(OMe)-TPH, TFA-

EVD-TPH and TFA-6E8D-TPH inhibit anti-TRAIL R2 antibody-induced apoptosis in Jurkat cells. Jurkat cells were incubated with the indicated concentrations of caspase inhibitor for 15 minutes prior to stimulation with 100 ng/ml anti-TRAIL R2 agonist antibody for 24 hours. Cells were labeled with Annexin V - Alexa Fluor 488 and a cell impermeable DNA dye (SYTOX AADvanced) and analyzed by flow cytometry. % Apoptosis was calculated by adding the percentage of cells in the Annexin V positive/SYTOX negative and Annexin V positive/SYTOX positive populations. Data were normalized to no inhibitor samples (no anti-TRAIL R2 antibody = 0%, + anti-TRAIL R2 antibody = 100%) and percent inhibition was calculated.

Figure 10. RAW 264.7 cells were incubated with TFA-FK-TPH, TFA-FR-TPH or E64d at the indicated concentrations for 1 hour prior to a 2 hour incubation with 1 μΜ BMV109. Cells were then collected, lysed in hypotonic lysis buffer and protein concentrations were determined. 50 μg of cell lysate was separated by SDS-PAGE on 15% Mini-PROTEAN TGX precast gels (Bio Rad). Gels were scanned using a Typhoon FLA 9500 (Cy5).

DETAILED DESCRIPTION

The present disclosure relates to cysteine protease inhibitors that covalently bind to cysteine proteases (e.g. active caspases or cathepsins), and methods of using such inhibitors to block caspase-mediated apoptosis, caspase-mediated inflammation, and cathepsin expression associated with any of the cathepsin related conditions mentioned herein. The present disclosure further relates to kits comprising the present cysteine protease inhibitors and instructions for their use.

Without being bound to any particular theory, it is believed that the presence of F in the compounds of the invention enhances cell permeability and or improves inhibitor-enzyme binding. This is because fluorine is a small and dense atom, only about 10% bigger in diameter than hydrogen and fluorine-containing molecules are hydrophobic.

Definitions

As used herein, the term "biological sample" refers to any type of material of biological origin, including but not limited to a blood sample, tissue sample, cell suspension, cellular extract, or tissue homogenate.

As used herein, the term "cysteine protease" includes enzymes that degrade proteins using a nucleophilic cysteine thiol. In one embodiment, the cysteine protease is a caspase or cysteine cathepsin. In one embodiment, the cysteine protease is a caspase. In one embodiment, the cysteine protease is a cysteine cathepsin.

As used herein, the term "zn vitro " refers to processes or procedures performed on a biological sample outside a living organism. For example, in vitro administration includes administering (i.e., delivering, applying, etc.) a cysteine protease inhibitor to a biological sample that is outside a living organism.

As used herein, the term "m vivo ^' " refers to processes or procedures performed inside a living, multicellular organism. For example, in vivo administration includes administering a cysteine protease inhibitor to a living subject (e.g., a mammal, such as a human).

As used herein, the term "incidence" refers to the occurrence rate, frequency of an event, or the quantifiable degree to which an event occurs.

As used herein, the term "incubating", when used with respect to incubating a sample with a cysteine protease inhibitor, refers to exposure conditions (e.g., time, temperature, pH, etc.) sufficient for the formation of caspase-inhibitor or cathepsin-inhibitor complexes.

As used herein, the term "N-terminal group" refers to a moiety attached to the N- terminal position of the recognition sequence of the cysteine protease inhibitor. The N-terminal group in the caspase inhibitors (and certain cathepsin inhibitors) of this invention is a

trifluoroacetyl (TFA) group.

As used herein, the term "C-terminal group" refers to a moiety attached to the C-terminal position of the recognition sequence of the cysteine protease inhibitor. The C-terminal group end of the molecule, in certain cathepsin inhibitors described in this invention, contains a

trifluoroacetyl (TFA) group.

As used herein, the term "reactive group that binds to a cysteine protease" includes groups that are capable of interacting with or reacting with the catalytic site of a cysteine protease.

In one embodiment, a reactive group is capable of interacting with or reacting with the catalytic site of a caspase or cysteine cathepsin. In one embodiment, a reactive group is capable of forming a covalent bond with a cysteine protease. In one embodiment, a reactive group is capable of forming a covalent bond with a caspase or cysteine cathepsin. In one embodiment, a reactive group is a leaving group that can be displaced to form a covalent bond. In one embodiment, for example, the reactive group can be FMK, PMK, OPH, or TPH. In one embodiment, for example, the reactive group can be a halogen atom, a substituted or unsubstituted phenol group, a substituted or unsubstituted benzoylate group, a substituted or unsubstituted BMK group, a substituted or unsubstituted FMK group, and a substituted or unsubstituted PMK group. In one embodiment, for example, the reactive group can be a halogen atom (e.g. F or CI) or a (C ₁ -C4) alkyl group that is substituted with one or more halogen atoms. In one embodiment, for example, the reactive group can be a portion of a cysteine protease inhibitor that covalently binds to the active catalytic site of a cysteine protease.

As used herein, the term "recognition sequence" refers to a portion of the cysteine protease inhibitors comprising a sequence of 1 to 8 natural or unnatural amino acids or synthetic analogs thereof, which is selective for one or more cysteine proteases.

As used herein, the term "synthetic analog" in regard to an amino acid refers to a (chemically) substituted natural amino acid, an unsubstituted unnatural amino acid, or a substituted unnatural amino acid.

As used herein, the term "unnatural amino acid" refers to an amino acid (or conformation thereof) not normally found in nature (i.e. not found in the genetic code of any organisms, not encoded into proteins). Synthetic analogs are a type of unnatural amino acid.

As used herein, the term "substituted," with respect to a group, means the group is substituted with one or more (e.g., 1 , 2, 3, 4 or 5) substituents independently selected from halo, cyano, nitro, carboxy, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) haloalkyl, (C ₁ -C ₄ ) alkoxy, (C] -C ₄ ) haloalkoxy, (Ci-C ₄ ) alkanoyl, (CrC ₄ ) alkoxycarbonyl, (CrC ₄ ) alkanoyloxy, amino, (CrC ₄ ) alkylamino, and ((C _! -C ₄ )alkyl) ₂ amino.

As used herein, the following abbreviations have the meanings given.

AOMK = acyloxymethyl ketone

BMK = benzoyloxymethyl ketone DE = L-aspartyl-L-glutamic acid

FK = phenylalyl lysine

FMK = fluoromethyl ketone

=

6E(OMe)8D(OMe) =

HD = L-histidyl-L-aspartic acid

IE = L-isoleucyl-L-glutamic acid

LAD = L-leucyl-L-alanyl-L-aspartic acid

LE = L-leucyl-L-glutamic acid

NMe = N-methyl

OMe = methyl ester

OPH = 2,6-difluoro substituted PMK

PMK = phenoxymethyl ketone

TD = L-threonyl-L-aspartic acid

TFA = trifluoroacetyl

TPH = 2,3,5,6-tetrafluoro substituted PMK

VAD = L-valyl-L-alanyl-L-aspartic acid VD = L-valyl-L-aspartic acid

WE = L-tryptophanyl-L-glutamic acid

YE = L-tyrosinyl-L-glutamic acid

Z = carbobenzyloxy

N-Terminal Group (Protecting Group)

The N-terminal group may serve to protect the recognition sequence during synthesis of a cysteine protease inhibitor. In at least one embodiment, an N-terminal protecting group may be present during synthesis of an intermediate precursor of a cysteine protease inhibitor, but may be removed and replaced with a different N-terminal moiety - i.e., the N-terminal group of a cysteine protease inhibitor intermediate may be different from the N-terminal group of a final cysteine protease inhibitor.

The N-terminal group on final caspase inhibitors and some final cysteine cathepsin inhibitors described in this invention is a trifluoroacetyl (TFA) group. In certain final cysteine cathepsin inhibitors described in this invention, the trifluoroacetyl (TFA) group is located on the C-terminal end of the Recognition Sequence. It is believed that a TFA group on the N-Terminal or C-Terminal position of a peptide based cysteine protease inhibitor enhances binding to the enzyme and/or enhances cell membrane permeability of the cysteine protease inhibitor. Recognition Sequence

Each recognition sequence comprises one or more natural or unnatural, synthetically modified amino acids and is able to bind to one or more caspases or cysteine cathepsins. In general, amino acids of the recognition sequence may be L, D, or D/L racemates. A recognition sequence may allow the cysteine protease inhibitor to target structurally similar caspases or cysteine cathepsins with the same or different affinities and kinetics. For instance, the recognition sequence VAD (valine-alanine-aspartic acid) is a poly-caspase inhibitor that binds to all or most known caspases (Garcia-Calvo et al., J. Biol. Chem. 273(49):32608-13 (1998)), and thus all of the disclosed reactive groups present in caspase inhibitors containing the VAD recognition sequence will covalently bind to all or most active caspases. Accordingly, in at least one embodiment, the caspase inhibitor is designed to react with multiple caspases to allow inhibition of all or most caspases. Similarly, the FK recognition sequence is designed to react with multiple cysteine cathepsins (B/L/S/X/Z). Accordingly, the FK containing cysteine cathepsin inhibitors described herein are considered "poly" cysteine cathepsin inhibitors.

In at least one other embodiment, the caspase inhibitor is designed to react selectively with caspase- 1, thus allowing for detection of inflammation in particular. For instance, caspase inhibitors containing the recognition sequence WEHD (SEQ ID NO:l) or YVAD (SEQ ID NO:2) will bind covalently to caspase-1 over other apoptosis-associated caspases.

In at least one other embodiment, the caspase inhibitor is designed to react with two caspases that have similar substrate selectivity. For instance, caspase inhibitors containing the recognition sequence DEVD (SEQ ID NO:3) will recognize and allow detection of both caspase-3 and caspase-7.

In at least one embodiment, the caspase inhibitor recognition sequence is selected from inhibitor recognition sequences listed in Table 1. Table 1 identifies well known recognition sequences of caspase inhibitors disclosed herein and their corresponding ability to selectively bind to all or most active caspases (i.e., poly-caspase), inflammation-related active caspases (e.g., caspase-1), or apoptosis-related active caspases (e.g., caspase-3/7 or caspase-8).

Table 1

In one embodiment, the molecule that will recognize a cysteine protease (i.e. the cysteine protease inhibitor recognition sequence) is selected from:

Examples of synthetic or "unnatural" amino acids include (but are not limited to):

In at least one embodiment, the cysteine cathepsin inhibitor recognition sequence is FK, FR, GGR (including synthetic analogs thereof). Reactive Group - Binding Mechanism to Active Caspase (Illustrative Example)

Following the administration of a caspase inhibitor, a multi-step binding mechanism results in the formation of a covalent bond between the reactive group and the -SH moiety of the cysteine residue in the active catalytic site of the caspase. According to at least one embodiment, the caspase inhibitor binds irreversibly and covalently to the caspase.

In at least one embodiment, the caspase inhibitors comprise a reactive group that enables the inhibitor to covalently bind to an active caspase. Typically, the reactive group includes a moiety that leaves as the reactive group reacts with the active caspase. The reactive group can be tailored to recognize and bind to specific types of caspases, such as caspases associated with apoptosis, caspases associated with inflammation, or caspases associated with both.

A representative example of the multi-step binding mechanism is illustrated below for caspase inhibitors that comprise a ketone that reacts with the cysteine residue in the catalytic site of an active caspase. This same general mechanism (nucleophilic attack on the electrophilic leaving group) applies to cysteine cathepsin inhibitors described in this invention. For the epoxide containing cysteine cathepsin inhibitors described herein, the epoxysuccinyl group irreversibly binds to an active thiol group on target cysteine cathepsins to form a thioether linkage.

Binding Mechanism of DEVD-FMK to Cysteine Residue in Catalytic Site of Active

Caspase

Formation of Thiohemiketal Intermediate I:

Formation of Three-Membered Sulfonium Intermediate II:

Formation of Stable Covalent Bond:

As shown above, the three-membered sulfonium Intermediate II rearranges to yield the final thioether adduct.

Reactive Group - Binding Mechanism of Epoxide Containing Inhibitor to Cysteine Cathepsin

General Structure of Cysteine Protease Inhibitors

In one embodiment the caspase inhibitors (and select cysteine cathepsin inhibitors) of the present disclosure have the following general structure: (U)-X-J-(U), where U must be present on at least one end of the molecule and may be attached directly or indirectly (i.e. via linker) to the C-terminal and/or N-terminal end of the molecule, and where U is a fluorinated group or perfluoroalkyl group comprising 1-10 carbon atoms and one or more fluorine atoms (e.g. a trifluoroacetyl (TFA) group); X is any molecule that will recognize a cysteine protease (e.g. a peptide-based caspase or cysteine cathepsin recognition sequence containing 1-8 amino acids or synthetic analogs thereof), and J is any reactive group that binds to a caspase or cysteine cathepsm (e.g. a halo-methyl ketone such as fluoro-methyl ketone (FMK), a phenoxy-methyl ketone (PMK), or a substituted phenoxy-methyl ketone (such as a 2,3,5,6 tetrafluoro phenoxy- methyl ketone a.k.a. "TPH" or a 2,6 difluoro phenoxy-methyl ketone a.k.a. "OPH"), an acyloxy- methyl ketone a.k.a. "AOMK" or an epoxide).

In one embodiment the caspase inhibitors of the present disclosure are further described by the following formula:U-X-NH-CH(R)-CO-CH ₂ -J,where U is any fluorinated group or perfluoroalkly group (e.g. a trifluoroacetyl (TFA) group), X is any molecule that will recognize an active caspase (e.g. a peptide having 1-8 amino acids or synthetic analogs thereof), R is CH2- C02-CH3 for live cells and in vivo applications or CH2-C02-H for cell-free systems, and J is any molecule that will react with active caspases; for example, a halo-methyl ketone such as fluoro-methyl ketone (FMK), a phenoxy-methyl ketone (PMK), or a substituted phenoxy-methyl ketone (such as a 2,3,5,6 tetrafluoro phenoxy-methyl ketone a.k.a. "TPH" or a 2,6 difluoro phenoxy-methyl ketone a.k.a. "OPH"), an acyloxy-methyl ketone a.k.a. "AOMK" or an epoxide).

In this general structure, U comprises the N-terminal group, X-NH-CH(R)-CO-CH ₂ comprises the recognition sequence, and J comprises the reactive group.

In one embodiment, X is selected from one or more naturally occurring amino acids, one or more synthetic amino acids and combinations thereof. X may be one or more amino acids selected from alanine, aspartic acid, glutamic acid, histidine, glycine, arginine, lysine, methionine, proline, isoleucine, phenylalanine, leucine, threonine, tryptophan, valine, tyrosine, glutamine, serine, pyrrolysine, selenocysteine, norvaline, norleucine and synthetic analogs thereof (where applicable).

In one embodiment, J is capable of binding at least one caspase selected from caspase- 1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase- 10, caspase- 1 1, caspase- 12, caspase- 13 and caspase- 14. J may be selected from BMK, FMK, PMK, OPH, TPH and AOMK.

In one embodiment, J is capable of binding at least one caspase selected from caspase- 1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase- 10, caspase- 1 1, caspase- 12, caspase- 13 and caspase- 14. J may be selected from FMK, PMK, OPH, TPH and AOMK. In one embodiment, J is capable of binding to at least one cysteine cathepsin selected from Cathepsins B, C, F, H, K, L, O, S, W, X/Z. J may be selected from FMK, PMK, OPH, TPH and AOMK.

In one embodiment, J is selected from a halogen, a phenol group, a benzoylate group, a BMK group, an FMK group, and a PMK group, in which the groups mentioned herein may be substituted or unsubstituted.

In one embodiment, J is selected from BMK, FMK, and PMK. In at least one embodiment, the J halogen is F, CI, or Br. In at least one embodiment, the J phenol is a compound of Formula A:

Formula A

wherein X _1? X ₂ , X ₃ , X ₄ , and X ₅ is each individually selected from H, F, CI, alkyl, aryl, aralkyl, amino, nitro, and carboxy. In one embodiment, at least one of Xj, X ₂ , X ₃ , X , and X5 is H. In at least one embodiment, alkyl is C _1-10 alkyl, for example, Ci _-6 alkyl. In at least one embodiment, the J benzoylate is a compound of Formula B:

Formula B

wherein X _1? X ₂ , X ₃ , X ₄ , and X ₅ is each individually selected from H, F, CI, alkyl, aryl, aralkyl, amino, nitro, or carboxy. In one embodiment, at least one of X], X ₂ , X ₃ , X ₄ , and X ₅ is H. In at least one embodiment, alkyl is C _1-10 alkyl, for example, Ci _-6 alkyl.

In one embodiment, the cysteine protease inhibitor has a molecular weight of about 300- 1500 Daltons.

The cysteine protease inhibitors are typically cell permeant (i.e., exhibit good cell membrane permeability) and can typically selectively target cysteine proteases (i.e. caspases and cysteine cathepsins) of interest inside the cells of a living organism or a biological sample.

In at least one embodiment, the cysteine protease inhibitors do not undergo facile metabolism, and possess a long half-life (e.g., more than 6 hours) throughout the life of the permeated cell.

Additionally, since the cysteine protease inhibitors bind to caspases as illustrated in the representative binding mechanisms shown above, each cysteine protease inhibitor forms a metabolite having the formula U-X-NH-CH(R)-CO-CH ₂ -S-Cys-Enzyme, wherein U, X, and R are as defined above.

Representative Examples of Caspase Inhibitors

An example of caspase inhibitors of the present disclosure includes, but is not limited to TFA-VAD(OMe)-FMK:

-VD(OMe)-FMK

TFA-VAD-FMK

TFA-VADOMe)-2,3,5,6-Tetrafluorophenoxymethyl Ketone

TFA-VAD-2,3,5,6-Tetrafluorophenoxymethyl Ketone

TFA-(2-Oxoacetamido)(Propanamido)-4-Oxo-5-(2,3,5,6-Tetraf luorophenoxy) Pentanoic Acid

TFA-Asp-3,4 Difluorophenylalanine-Val-Asp-2,3,5,6 Tetrafluorophenoxymethyl Ketone

TFA-Asp-Pentafluorophenylalanine-Val-Asp-2,3,5,6 Tetrafluorophenoxymethyl Ketone

TFA-EVD-2,3 ₅ 5,6-Tetrafluorophenoxymethyl Ketone

TFA-E(OMe)VD(OMe)-2,3,5,6-Tetrafluorophenoxymethyl Ketone

TFA-6E8D-2,3,5,6 Tetrafluorophenoxymethyl Ketone

TFA-6E(OMe)8D(OMe)-2,3,5,6 Tetrafluorophenoxymethly Ketone

Representative Examples of Cysteine Cathepsin Inhibitors

Examples of cysteine cathepsin inhibitors of the present disclosure include, but are not limited to:

(R,R at the epoxide) Administration or Delivery of Cysteine Protease Inhibitors

Examples of organs or targets applicable to the use of cysteine protease inhibitors include, but are not limited to: eye, breast, heart, brain and central nervous system (CNS), kidneys, lungs, liver, skin, pancreas, skeletal system, connective tissue (e.g., joints), stomach, upper gastrointestinal tract, lower gastrointestinal tract, circulatory system (e.g., blood), lymphatic system, sexual organs (male and female), prostate, embryologic tissue, muscular system, and gallbladder.

In at least one embodiment, the cysteine protease inhibitor is delivered in vitro to a biological sample (e.g., a blood sample, tissue sample, cell suspension, cellular extract, or tissue homogenate) by direct application of the inhibitor to the sample. Given that the cysteine protease inhibitors exhibit good cell permeability, there is no need to use additional reagents to facilitate the entry of the inhibitors into cells. In one instance, the cysteine protease inhibitor is reconstituted in DMSO, further diluted in phosphate buffered saline (PBS) or cell culture media and applied directly to cells in a cell culture dish. Upon application, the cysteine protease inhibitor is allowed to incubate with the cells under the conditions sufficient for the formation of caspase-inhibitor complexes.

In at least one other embodiment, the cysteine protease inhibitor is administered in vivo to a subject (e.g., animal or human) intravenously, intraperitoneally, intramuscularly, subcutaneously, topically, and/or by direct application to a target organ. The cysteine protease inhibitor is diluted with a suitable excipient (in some cases a pharmaceutically acceptable excipient) and administered in an effective amount, which is an amount that is sufficient to provide meaningful results with respect to the intended purpose - e.g., therapeutic, drug development, etc. Suitable excipients include, but are not limited to, adhesives, binders, bulking agents, carriers, colors, diluents, disintegrating agents, fillers, glidants, granulating agents, lubricating agents, polymers, preservatives, wetting agents, and combinations thereof. One or more excipients may be selected from sucrose, lactose, cellulose, methyl cellulose, gelatin, polyvinylpyrrolidone, polyethylene glycol and water. Kits

In general, the kits of the present disclosure are used for blocking apoptotic activity and/or conditions associated with inflammatory activity in a biological sample. The kit may comprise one or more cysteine protease inhibitors. The kit may further include packaging materials with instructions for using the components of the kit, such as how to use the cysteine protease inhibitors provided in the kit, storage conditions, etc. Components of the kit may be provided in separate containers (e.g., vials) or combined.

Embodiments

In one aspect, the invention relates to a kit containing instructions for use and at least one cysteine protease inhibitor compound of the following formula: (U)-X-J-(U), where at least one U must be present and where U may be attached either directly or indirectly (i.e. via linker) to the C-terminal or N-terminal end of the molecule, and where U is a fluorinated group or perfluoroalkyl group comprising 1-10 carbon atoms and one or more fluorine atoms (e.g. a trifluoroacetyl (TFA) group); X is any molecule that will recognize a cysteine protease (e.g. a peptide-based caspase or cysteine cathepsin recognition sequence containing 1-8 amino acids or synthetic analogs thereof), and J is any reactive group that binds to a caspase or cysteine cathepsin (e.g. a halo-methyl ketone such as fluoro-methyl ketone (FMK), a phenoxy-methyl ketone (PMK), or a substituted phenoxy-methyl ketone (such as a 2,3,5,6 tetrafluoro phenoxy- methyl ketone a.k.a. "TPH" or a 2,6 difluoro phenoxy-methyl ketone a.k.a. "OPH"), an acyloxy- methyl ketone a.k.a. "AOMK" or an epoxide).

In another aspect, the invention relates to a kit containing instructions for use and at least one caspase inhibitor compound of the following formula: U-X-NH-CH(R)-CO-CH ₂ -J, where U is any fluorinated group or perfluoroalkly group (e.g. a trifluoroacetyl (TFA) group), X is any molecule that will recognize an active caspase (e.g. a peptide having 1-8 amino acids or synthetic analogs thereof), R is CH2-C02-CH3 for live cells and in vivo applications or CH2- C02-H for cell-free systems, and J is any molecule that will react with active caspases, e.g. a halo-methyl ketone such as fluoro-methyl ketone (FMK), a phenoxy-methyl ketone (PMK), or a substituted phenoxy-methyl ketone (such as a 2,3,5,6 tetrafluoro phenoxy-methyl ketone ("TPH") or a 2,6 difluoro phenoxy-methyl ketone a.k.a. "OPH"), an acyloxy-methyl ketone a.k.a. "AOMK" or an epoxide). In another aspect, the "X" for the caspase or cysteine cathepsin inhibitor comprises at least one amino acid selected from alanine, aspartic acid, glutamic acid, histidine, glycine, arginine, lysine, methionine, proline, isoleucine, phenylalanine, leucine, threonine, tryptophan, valine, tyrosine, glutamine, serine, pyrrolysine, selenocysteine, norvaline, norleucine and synthetic analogs thereof (where applicable).

In another aspect, the compound is selected from the group consisting of:

TFA-WEHD-FMK TFA-IETD-FMK TFA-DEVD-FMK

TFA-WEHD-PMK TFA-IETD-PMK TFA-DEVD-PMK

TFA-WEHD-OPH TFA-IETD-OPH TFA-DEVD-OPH

TFA-WEHD-TPH TFA-IETD-TPH TFA-DEVD-TPH

TFA-WEHD-AOMK TFA-IETD-AOMK TFA-DEVD-AOMK

TFA-LEHD-FMK TFA-LETD-FMK TFA-VDVAD-FMK

TFA-LEHD-PMK TFA-LETD-PMK TFA-VDVAD-PMK

TFA-LEHD-OPH TFA-LETD-OPH TFA-VDVAD-OPH

TFA-LEHD-TPH TFA-LETD-TPH TFA-VDVAD-TPH TFA-LEHD-AOMK TFA-LETD-AOMK TFA-VDVAD-AOMK

TFA-6E8D-FMK TFA-EVD-FMK

TFA-6E8D-PMK TFA-EVD-PMK

TFA-6E8D-OPH TFA-EVD-OPH

TFA-6E8D-TPH TFA-EVD-TPH

TFA-6E8D-AOMK TFA-EVD-AOMK

In another aspect, the compound is selected from the group consisting of:

TFA-VAD(OMe)-FMK TFA-YVAD(OMe)-FMK TFA-WE(OMe)HD(OMe)-FMK TFA-VAD(OMe)-PMK TFA-YVAD(OMe)-PMK TFA-WE(OMe)HD(OMe)-PMK TFA-VAD(OMe)-OPH TFA-YVAD(OMe)-OPH TFA-WE(OMe)HD(OMe)-OPH TFA-VAD(OMe)-AOMK TFA-YVAD(OMe)-AOMK TFA-WE(OMe)HD(OMe)-AOMK TFA-IE(OMe)TD(OMe)-FMK TFA-D(OMe)E(OMe)VD(OMe)-FMK

TFA-IE(OMe)TD(OMe)-PMK TFA-D(OMe)E(OMe)VD(OMe)-PMK

TFA-IE(OMe)TD(OMe)-OPH TFA-D(OMe)E(OMe)VD(OMe)-OPH

TFA-IE(OMe)TD(OMe)-AOMK TFA-D(OMe)E(OMe)VD(OMe)-AOMK

TFA-LE(OMe)HD(OMe)-FMK TFA-LE(OMe)TD(OMe)-FMK

TFA-LE(OMe)HD(OMe)-PMK TFA-LE(OMe)TD(OMe)-PMK

TFA-LE(OMe)HD(OMe)-OPH TFA-LE(OMe)TD(OMe)-OPH

TFA-LE(OMe)HD(OMe)-AOMK TFA-LE(OMe)TD(OMe)-AOMK

TFA-VD(OMe)VAD(OMe)-FMK TFA-VD(OMe)-FMK

TFA-VD(OMe)VAD(OMe)-PMK TFA-VD(OMe)-PMK

TFA-VD(OMe)VAD(OMe)-OPH TFA-VD(OMe)-OPH

TFA-VD(OMe)VAD(OMe)-AOMK TFA-VD(OMe)-AOMK

TFA-VAD-FMK TFA-YVAD-FMK TFA-WEHD-FMK

TFA-VAD-PMK TFA-YVAD-PMK TFA-WEHD-PMK

TFA-VAD-OPH TFA-YVAD-OPH TFA-WEHD-OPH

TFA-VAD-AOM TFA-YVAD-AOMK TFA-WEHD-AOMK

TFA-IETD-FMK TFA-DEVD-FMK

TFA-IETD-PMK TFA-DEVD-PMK

TFA-IETD-OPH TFA-DEVD-OPH TFA-IETD-AOMK TFA-DEVD-AOMK

TFA-LEHD-FMK TFA-LETD-FMK

TFA-LEHD-PMK TFA-LETD-PMK

TFA-LEHD-OPH TFA-LETD-OPH

TFA-LEHD-AOMK TFA-LETD-AOM

TFA-VDVAD-FMK TFA-VD-FMK

TFA-VDVAD-PMK TFA-VD-PMK

TFA-VDVAD-OPH TFA-VD-OPH

TFA-VDVAD-AOMK TFA-VD-AOMK

and pharmaceutically acceptable derivatives thereof.

In another aspect, the compound is selected from the group consisting of:

TFA-VAD(OMe)-FMK

TFA-VD(OMe)-FMK

TFA-VAD-FMK

TFA-VD-FMK

TF A- VD(OMe)-2,3,5,6-Tetrafluorophenoxymethyl Ketone

TFA-VAD OMe)-2,3,5,6-Tetrafluorophenoxymethyl Ketone

TF A- VD-2,3,5,6-Tetrafluorophenoxymethyl Ketone

TFA-VAD-2,3,5,6-Tetrafluorophenoxymethyl Ketone

TFA-EVD-2,3,5,6-Tetrafluorophenoxymethyl Ketone

TFA-E(OMe)VD(OMe)-2,3,5,6-Tetrafluorophenoxymethyl Ketone

TFA-6E8D-2,3,5,6 Tetrafluorophenoxymethyl Ketone

TFA-6E(OMe)8D(OMe)-2,3,5,6 Tetrafluorophenoxymethly Ketone

TFA-(2-Oxoacetamido)(Tropanamido)-4-Oxo-5-(2,3,5,6-Tetrafluo rophenoxy) Pentanoic Acid

TFA-Asp-3,4 Difluorophenylalanine-Val-Asp-2,3,5,6 Tetrafluorophenoxymethyl Ketone

TFA-Asp-PentafluorophenyIalanine-VaI-Asp-2,3,5,6 Tetrafluorophenoxymethyl Ketone

and pharmaceutically acceptable derivatives thereof.

In another embodiment, the compound is selected from the group consisting of:

TFA-FK-FMK TFA-FR-FMK TFA-GGR-FMK

TFA-FK-PMK TFA-FR-PMK TFA-GGR-PMK

TFA-FK-OPH TFA-FR-OPH TFA-GGR-OPH

TFA-FK-TPH TFA-FR-TPH TFA-GGR-TPH

TFA-FK-AOMK TFA-FR-AOMK TFA-GGR-AOMK

and pharmaceutically acceptable derivatives thereof.

In another embodiment, the compound contains an epoxide based "J" reactive group and is selected from the group consisting of:

and pharmaceutically acceptable derivatives thereof.

In another embodiment, the compound contains at least one cathepsin inhibitor and is selected from the group consisting of:

harmaceutically acceptable derivatives thereof.

In another embodiment, the present invention relates to a composition comprising a caspase inhibitor or cysteine cathepsin inhibitor and an excipient (which may be selected from sucrose, lactose, cellulose, gelatin, polyvinylpyrrolidone, trehalose, cyclodextrin, hyaluronic acid, polyethylene glycol, DMSO and water.

In another embodiment, the present invention relates to a method of inhibiting apoptosis and/or inflammation in a living organism by administering in vivo a caspase inhibitor or cathepsin inhibitor to the living organism.

In another embodiment, the present invention relates to a method of inhibiting apoptosis and/or inflammation in a cell population by administering in vitro a caspase inhibitor or cathepsin inhibitor to a biological sample and incubating the sample with the inhibitor under conditions sufficient to form caspase-inhibitor complexes. The biological sample may be selected from a blood sample, tissue sample, cell suspension, cellular extract, or tissue homogenate.

In another embodiment, the compound is a compound of formula (la): UrX-J (la).

In another embodiment, the compound is a compound of formula (lb):

X-J-U ₂ (lb). In another embodiment, the compound is a compound of formula (Ic): CF ₃ -C(=0)-X-J (Ic).

In another embodiment, X is a molecule that will recognize a caspase or cysteine cathepsin.

In another embodiment, X is a peptide that contains 1-8 amino acids.

In another embodiment, X is a peptide that contains 2-5 amino acids.

In another embodiment, X is a peptide selected from the group consisting of: VD, VAD, EVD, D-3-V-D, L-E-H-D (SEQ ID NO:9), L-E-T-D (SEQ ID NO: 10), D-E-V-D (SEQ ID NO:3), D-E-P-D (SEQ ID NO:7), D-29-V-D, D-34-V-D, 26-34-V-D, 26-3-V-D, 26-E-V-D, 31- E-T-D, 31-E-23-D, 29-E-T-D, 6-E-8-D, D-E-1 1-D, D-30-11-D, D-30-V-D, P-L-A-D (SEQ ID NO:8), I-L-A-D (SEQ ID NO:l 1), I-L-38-D, I-F-P-D (SEQ ID NO:12), D-3-V-D, D-34-V-D, 31-E-23-D, and P-L-A-D (SEQ ID NO:8), wherein the numbers 3, 6, 8, 1 1, 23, 26, 29, 30, 31, 34, and 38 represent the following structures:

see Berger, A. B., et al., 2006, Mol. Cell 23, 509-21.

In another embodiment, J is selected from the group consisting of AMOK, BMK, FMK, OPH, PMK, and TPH.

In another embodiment, J is selected from the group consisting of AMOK, OPH, PMK, and TPH.

In another embodiment, J is TPH.

In another embodiment, the compound is selected from the group consisting of: TFA- EVD-TPH; TFA-6E8D-TPH; TFA-VAD(OMe)-TPH; TF A- VD(OMe)-TPH ; TFA-FK-TPH; and TFA-F -TPH.

In another embodiment, the compound is a compound of formula (Id): (U)-X-J-(U) (Id)

wherein:

U is present on at least one end of the molecule and consists of a fluorinated group comprising 1-10 carbon atoms and one or more fluorine atoms;

X is any molecule that will recognize a cysteine protease; and

J is any reactive group that binds to a caspase or cysteine cathepsin.

In another embodiment, U is a trifluoroacetyl (TFA) group that may be connected directly or indirectly (i.e. via linker) to either the C-terminal end or to the N-terminal end J group.

In another embodiment, the compound is a compound of formula (Ie): (U)-X-NH-CH(R)-CO-CH ₂ -J(U) (Ie)

wherein:

U is a fluorinated group comprising 1-10 carbon atoms and one or more fluorine atoms X is a peptide-based caspase recognition sequence comprising 1-8 amino acids; R is -CH ₂ C0 ₂ CH ₃ , -CH ₂ C0 ₂ H; and

J is any reactive group that binds to a caspase.

In another embodiment, U is a trifluoroacetyl (TFA) group that may be connected directly or indirectly (i.e. via linker) to either the C-terminal end or to the N-terminal end J group.

In another embodiment, X comprises at least one amino acid selected from alanine, aspartic acid, glutamic acid, histidine, glycine, arginine, lysine, methionine, proline, isoleucine, phenylalanine, leucine, threonine, tryptophan, valine, tyrosine, glutamine, serine, pyrrolysine, selenocysteine, norvaline, norleucine and synthetic analogs thereof (where applicable).

In another embodiment, X comprises VAD(OMe), VD(OMe), VAD, VD,

E(OMe)VD(OMe), EVD, 6E8D, 6E(OMe)8D(OMe), FK, FR, GGR, WE(OMe)HD(OMe), IE(OMe)TD(OMe), D(OMe)E(OMe)VD(OMe), LE(OMe)HD(OMe), LE(OMe)TD(OMe), YVAD(OMe), VD(OMe)VAD(OMe) or any of the following: Asp-3,4 Difluorophenylalanine- Val-Asp, Asp-Pentafluorophenylalanine-Val-Asp, (2-Oxoacetamido)(Propanarnido)-4-Oxo- Pentanoic Acid, (Oxiran-2-yl) Carbonyl-L-Leucyl-3-(p-Hydroxyphenyl) Ethylamide.

In another embodiment, X is VAD(OMe), VD(OMe), VAD, VD, E(OMe)VD(OMe), EVD, 6E8D, 6E(OMe)8D(OMe), FK, FR, GGR, WE(OMe)HD(OMe), IE(OMe)TD(OMe), D(OMe)E(OMe)VD(OMe), LE(OMe)HD(OMe), LE(OMe)TD(OMe), YVAD(OMe),

VD(OMe)VAD(OMe) or any of the following: Asp-3,4 Difluorophenylalanine- Val-Asp, Asp- Pentafluorophenylalanine- Val-Asp, (2-Oxoacetamido)(Propanamido)-4-Oxo-Pentanoic Acid, (Oxiran-2-yl) Carbonyl-L-Leucyl-3-(p-Hydroxyphenyl) Ethylamide.

In another embodiment, J is any molecule that will react with active cysteine proteases, e.g.a halomethyl ketone (such as CMK, FMK), a substituted or unsubstituted phenoxymethyl ketone (PMK), an acyloxymethyl ketone (AOMK), or an epoxide.

In another embodiment, the compound is selected from:

TFA-VAD(OMe)-FMK TFA-YVAD(OMe)-FMK TFA-WE(OMe)HD(OMe)-FMK TFA-VAD(OMe)-PMK TFA-YVAD(OMe)-PMK TFA-WE(OMe)HD(OMe)-PMK TFA-VAD(OMe)-OPH TFA-YVAD(OMe)-OPH TFA-WE(OMe)HD(OMe)-OPH TFA-VAD(OMe)-TPH TFA-YVAD(OMe)-TPH TFA-WE(OMe)HD(OMe)-TPH TFA-VAD(OMe)-AOMK TFA-YVAD(OMe)-AOMK TFA-WE(OMe)HD(OMe)-AOMK TFA-IE(OMe)TD(OMe)-FMK TFA-D(OMe)E(OMe)VD(OMe)-FMK

TFA-IE(OMe)TD(OMe)-PMK TFA-D(OMe)E(OMe)VD(OMe)-PMK TFA-IE(OMe)TD(OMe)-OPH TFA-D(OMe)E(OMe)VD(OMe)-OPH

TFA-IE(OMe)TD(OMe)-TPH TFA-D(OMe)E(OMe)VD(OMe)-TPH

TFA-IE(OMe)TD(OMe)-AOMK TFA-D(OMe)E(OMe)VD(OMe)-AOMK

TFA-LE(OMe)HD(OMe)-FMK TFA-LE(OMe)TD(OMe)-FMK TFA-

TFA-LE(OMe)HD(OMe)-PMK TFA-LE(OMe)TD(OMe)-PMK TFA-EVD-PMK

TFA-LE(OMe)HD(OMe)-OPH TFA-LE(OMe)TD(OMe)-OPH TFA-EVD-OPH

TFA-LE(OMe)HD(OMe)-TPH TFA-LE(OMe)TD(OMe)-TPH TFA-EVD-TPH

TFA-LE(OMe)HD(OMe)-AOMK TFA-LE(OMe)TD(OMe)-AOMK TFA-EVD-AOMK

TFA-VD(OMe)VAD(OMe)-FMK TFA-VD(OMe)-FMK TFA-E(OMe)VD(OME)- FMK

TFA-VD(OMe)VAD(OMe)-PMK TFA-VD(OMe)-PMK TFA-E(OMe)VD(OMe)- PMK

TFA-VD(OMe)VAD(OMe)-OPH TFA-VD(OMe)-OPH TFA-E(OMe)VD(OMe)- OPH

TFA-VD(OMe)VAD(OMe)-TPH TFA-VD(OMe)-TPH TFA-E(OMe)VD(OMe)- TPH

TFA-VD(OMe)VAD(OMe)-AOMK TFA-VD(OMe)-AOMK TFA-E(OMe)VD(OMe)- AOMK

TFA-VAD-FMK TFA-VD-FMK TFA-6E8D-FMK TFA-6E(OMe)8D(OMe)- FMK

TFA-VAD-PMK TFA-VD-PMK TFA-6E8D-PMK TFA-6E(OMe)8D(OMe)- PMK

TFA-VAD-OPH TFA-VD-OPH TFA-6E8D-OPH TFA-6E(OMe)8D(OMe)- OPH

TFA-VAD-TPH TFA-VD-TPH TFA-6E8D-TPH TFA-6E(OMe)8D(OMe)- TPH

TFA-VAD-AOMK TFA-VD-AOMK TF A-6E8 D- AOMK TFA-6E(OME)8D(OMe)-

AOMK

TFA-GGR-FMK TFA-FK-FMK TFA-FR-FMK

TFA-GGR-PMK TFA-FK-PMK TFA-FR-PMK

TFA-GGR-OPH TFA-FK-OPH TFA-FR-OPH

TFA-GGR-TPH TFA-FK-TPH TFA-FR-TPH TFA-GGR-AOMK TFA-FK-AOMK TFA-FR-AOMK and the following compounds:

In another embodiment, the compound is a compound of formula (If):

CF ₃ -C(=0)-X-THP (If)

wherein X is a peptide that will recognize a caspase or cysteine cathepsin.

In another embodiment, the compound is a compound of formula (If):

CF ₃ -C(=0)-X-THP (If)

wherein X is a peptide that contains 1-8 amino acids.

In another embodiment, the compound is a compound of formula (If):

CF ₃ -C(=0)-X-THP (If)

wherein X is a peptide that contains 2-5 amino acids.

In another embodiment, the compound is a compound of formula (If):

CF ₃ -C(=0)-X-THP (If)

wherein X is a peptide selected from the group consisting of: E(OMe)-V-D, E(OMe)-V- D(OMe), E-V-D(OMe), 6-E(OMe)-8-D, 6-E(OMe)-8-D(OMe), 6-E-8D(OMe), V-A-D(OMe). V-D, V-D(OMe), F-K, F-R,VAD, EVD, D-3-V-D, L-E-H-D (SEQ ID NO:9), L-E-T-D (SEQ ID NO: 10), D-E-V-D (SEQ ID NO:3), D-E-P-D (SEQ ID NO:7), D-29-V-D, D-34-V-D, 26-34-V- D, 26-3-V-D, 26-E-V-D, 31-E-T-D, 31 -E-23-D, 29-E-T-D, 6-E-8-D, D-E-l 1-D, D-30-1 1 -D, D- 30-V-D, P-L-A-D (SEQ ID NO:8), I-L-A-D (SEQ ID NO:l 1), I-L-38-D, I-F-P-D (SEQ ID NO: 12), D-3-V-D, D-34-V-D, 31-E-23-D, and P-L-A-D (SEQ ID NO:8), wherein the numbers 3, 6, 8, 1 1, 23, 26, 29, 30, 31, 34, and 38 represent the following structures:

43

In another embodiment, X is a peptide selected from the group consisting of:

E(OMe)-V-D, E(OMe)-V-D(OMe), E-V-D(OMe), 6-E(OMe)-8-D, 6-E(OMe)-8-D(OMe), 6-E- 8D(OMe), V-A-D(OMe). V-D, V-D(OMe), F-K, F-R,VAD, EVD, and 6-E-8-D.

In another embodiment, X is a peptide selected from the group consisting of:

E(OMe)-V-D, E(OMe)-V-D(OMe), E-V-D(OMe), 6-E(OMe)-8-D, 6-E(OMe)-8-D(OMe), 6-E- 8D(OMe), V-A-D(OMe). V-D, V-D(OMe), VAD, EVD, and 6-E-8-D.

In another embodiment, X is a peptide selected from the group consisting of:

F-K and F-R. In another embodiment, J is acyloxymethyl, benzoyloxymethyl, fluoromethyl, 2,6- difluorophenoxymethyl, phenoxymethyl, or 2,3,5,6-tetrafluorophenoxymethyl, which form AMOK, BMK, FMK, OPH, PMK, or THP, respectively, when attached to the carboxy carbonyl group of X in a compound of the invention.

In another embodiment, a pharmaceutical composition is provided that comprises a compound of the invention and a pharmaceutically acceptable excipient, and optionally a therapeutic agent. In another embodiment, the excipient comprises sucrose, lactose, cellulose, gelatin, polyvinylpyrrolidone, trehalose, cyclodextrin, hyaluronic acid, polyethylene glycol or water. In another embodiment, the therapeutic agent is a cancer-targeted chemotherapeutic agent.

In another embodiment, a method for inhibiting a cysteine protease in vitro or in vivo is provided that comprises contacting the cysteine protease with a compound of the invention.

In another embodiment, a method for inhibiting a cysteine protease in vitro or in vivo is provided that comprises contacting the caspase with a compound of the invention. In another embodiment, the compound inhibits the cysteine protease by forming an irreversible covalent bond to the active site of a cysteine protease.

In another embodiment, a method for treating a disease or condition associated with an increase in cellular apoptosis and/or inflammation in a subject (e.g. animal or human) is provided that comprises administering to the subject a compound of the invention. In another embodiment, the disease or condition is myocardial infarction, stroke, traumatic brain injury, spinal atrophy, auto-immune diseases (e.g. Alzheimer's), fulminant liver destruction, endotoxic shock, sepsis, septic shock, liver disease such as chronic hepatitis (including virus-related hepatitis, alcoholic steatohepatitis and non-alcoholic steatohepatitis or "NASH"), pancreatitis, arthritis, viral infection, metabolic disease, cancer, implant or transplant rejection,

neurodegenerative diseases (e.g. multiple sclerosis), pulmonary fibrosis, cardiac fibrosis, renal fibrosis, liver fibrosis and cirrhosis or ototoxicity.

In another embodiment, a method for inhibiting apoptosis and/or inflammation

(including diseases or conditions associated with apoptosis and/or inflammation) in a subject (e.g. animal or human) is provided that comprises administering to the subject a compound of the invention.

In another embodiment, a method for inhibiting apoptosis and/or inflammation in a cell population is provided that comprises administering in vitro a compound of the invention to a biological sample to provide a resulting sample, and incubating the resulting sample under conditions sufficient to form caspase-inhibitor or cysteine cathepsin-inhibitor complexes. In another embodiment, the biological sample comprises blood sample, tissue, a cell suspension, a cellular extract, or a tissue homogenate.

In another embodiment, a method for preventing and/or treating a caspase-mediated or cysteine cathepsin-associated disease or condition in an subject (e.g. animal of human), is provided that comprises administering to the subject a compound of the invention.

In another embodiment, a kit comprising:

1 ) a compound of the invention; and

2) instructions for using the compound to inhibit a cysteine protease or to treat a cysteine protease-mediated disease or condition is provided.

In another embodiment, a kit comprising:

1) a compound of the invention; and

2) instructions for using the compound to inhibit a caspase or to treat a caspase- mediated disease or condition or a cysteine cathepsin-associated disease or condition.

In another embodiment, a method of inhibiting caspase or cysteine cathepsin activity in a cell-free system, is provided that comprises adding a compound of the invention to the purified caspases.

In another embodiment, the compounds of the invention (e.g. the compounds of formula

I, la, lb, Ic, Id, and Ie) exclude the following compounds:

4- ¹⁸ F-B-GVAD(OMe)-BM ,

4- ¹⁸ F-B-GVAD(OMe)-FMK,

4- ¹⁸ F-B-GVAD(OMe)-PMK,

780-G-F ₆ V-AD(OMe)-BMK,

780-G-F ₆ V-AD(OMe)-FMK,

780-G-F ₆ V-AD(OMe)-PMK,

780-G-F ₆ V-AD-BMK,

780-G-F ₆ V-AD-FMK,

780-G-F ₆ V-AD-PMK,

ABzC-G-F ₆ V-AD(OMe)-BMK,

ABzC-G-F ₆ V-AD(OMe)-FMK, ABzC-G-F ₆ V-AD(OMe)-PMK,

ABzC-G-F ₆ V-AD-BMK,

ABzC-G-F ₆ V-AD-FMK,

ABzC-G-F ₆ V-AD-PMK,

BODIPY-FL-G-F ₆ V-AD(OMe)-BMK,

BODIPY-FL-G-F ₆ V-AD(OMe)-FMK,

BODIPY-FL-G-F ₆ V-AD(OMe)-PMK,

BODIPY-FL-G-F ₆ V-AD-BMK,

BODIPY-FL-G-F ₆ V-AD-FMK,

BODIPY-FL-G-F ₆ V-AD-PMK,

BODIPY-FL-GVAD(OMe)-BMK,

BODIPY-FL-GVAD(OMe)-FMK,

BODIPY-FL-GVAD(OMe)-PMK,

BODIPY-FL-GVAD-BMK,

BODIPY-FL-GVAD-FMK,

BODIPY-FL-GVAD-PMK,

FAM-G-F ₆ V-AD(OMe)-BMK,

FAM-G-F ₆ V-AD(OMe)-FMK

FAM-G-F ₆ V-AD(OMe)-PMK,

FAM-G-F ₆ V-AD-BMK,

FAM-G-F ₆ V-AD-FMK,

FAM-G-F ₆ V-AD-PMK,

N-AE-G-F ₆ V-AD(OMe)-BMK,

N-AE-G-F ₆ V-AD(OMe)-FMK,

N-AE-G-F ₆ V-AD(OMe)-PMK,

N-AE-G-F ₆ V-AD-BMK,

N-AE-G-F ₆ V-AD-FMK,

N-AE-G-F ₆ V-AD-PMK,

N-AM-G-F ₆ V-AD(OMe)-BMK,

N-AM-G-F ₆ V-AD(OMe)-FMK,

N-AM-G-F ₆ V-AD(OMe)-PMK,

N-AM-G-F ₆ V-AD-BMK, N-AM-G-F ₆ V-AD-FMK,

N-AM-G-F ₆ V-AD-PMK,

NOTA-G-F ₆ V-AD(OMe)-BMK,

NOTA-G-F ₆ V-AD(OMe)-FM ,

NOTA-G-F ₆ V-AD(OMe)-PMK,

NOTA-G-F ₆ V-AD-BMK,

NOTA-G-F ₆ V-AD-FMK,

NOTA-G-F ₆ V-AD-PMK,

ORGn-G-F ₆ V-AD(OMe)-BMK,

ORGn-G-F ₆ V-AD(OMe)-FMK,

ORGn-G-F ₆ V-AD(OMe)-PMK,

ORGn-G-F ₆ V-AD-BMK,

ORGn-G-F ₆ V-AD-FMK,

ORGn-G-F ₆ V-AD-PMK,

TFA-G-F ₆ V-AD(OMe)-BMK,

TFA-G-F ₆ V-AD(OMe)-FMK,

TFA-G-F ₆ V-AD(OMe)-PMK,

TFA-G-F ₆ V-AD-BM ,

TFA-G-F ₆ V-AD-FMK,

TFA-G-F ₆ V-AD-PMK,

TFA-GVAD(OMe)-BMK,

TFA-GVAD(OMe)-FMK,

TFA-GVAD(OMe)-PMK,

TFA-GVAD-BMK,

TFA-GVAD-FMK,

TFA-GVAD-PMK,

TFMCBz-G-F ₆ V-AD(OMe)-BMK,

TFMCBz-G-F ₆ V-AD(OMe)-FMK,

TFMCBz-G-F ₆ V-AD(OMe)-PMK,

TFMCBz-G-F ₆ V-AD-BMK,

TFMCBz-G-F ₆ V-AD-FMK,

TFMCBz-G-F ₆ V-AD-PMK, TFMCBz-GVAD(OMe)-BMK, TFMCBz-GVAD(OMe)-FMK,

TFMCBz-GVAD(OMe)-PMK,

TFMCBz-GVAD-BMK,

TFMCBz-GVAD-FMK,

TFMCBz-GVAD-PMK,

Z-G-F ₆ V-AD(OMe)-BMK,

Z-G-F ₆ V-AD(OMe)-FMK,

Z-G-F ₆ V-AD(OMe)-PMK,

Z-G-F ₆ V-AD-BMK,

Z-G-F ₆ V-AD-FMK,

Z-G-F ₆ V-AD-PMK,

BODIPY-FL-D(OMe)E(OMe)VD(OMe)-FMK, BODIPY-FL-IE(OMe)TD(OMe)-FMK, BODIPY-FL-LE(OMe)HD(O e)-FMK, BODIPY-FL-LE(OMe)TD(OMe)-FMK, BODIPY-FL-VAD(OMe)-FMK,

BODIPY-FL-W(NMe)E(OMe)HD(OMe)-FMK, BODIPY-FL-WE(OMe)HD(OMe)-FMK, BODIPY-FL-YVAD(OMe)-FMK,

BODIPY-FL-VD(OMe)VAD(OMe)-FMK, BODIPY-FL-F ₆ V-AD(OMe)-FMK

BODIPY-FL-D(OMe)E(OMe)VD(OMe)-PMK, BODIPY-FL-IE(OMe)TD(OMe)-PMK, BODIPY-FL-LE(OMe)HD(OMe)-PMK, BODIPY-FL-LE(OMe)TD(OMe)-PMK, BODIPY-FL-VAD(OMe)-PMK,

BODIPY-FL-W(NMe)E(OMe)HD(OMe)-PM , BODIPY-FL-WE(OMe)HD(OMe)-PMK, BODIPY-FL-YVAD(OMe)-PMK,

BODIPY-FL-VD(OMe)VAD(OMe)-PMK, BODIPY-FL-F ₆ V-AD(OMe)-PMK BODIPY-FL-D(OMe)E(OMe)VD(OMe)-BMK, BODIPY-FL-IE(OMe)TD(OMe)-BMK,

BODIPY-FL-LE(OMe)HD(OMe)-BM ,

BODIPY-FL-LE(OMe)TD(OMe)-BMK,

BODIPY-FL-VAD(OMe)-BMK,

BODIPY-FL-W(NMe)E(OMe)HD(OMe)-BMK, BODIPY-FL-WE(OMe)HD(OMe)-BMK,

BODIPY-FL-YVAD(OMe)-BMK,

BODIPY-FL-VD(OMe)VAD(OMe)-BMK, BODIPY-FL-F ₆ V-AD(OMe)-BMK

BODIPY-FL-D(OMe)E(OMe)VD(OMe)-OPH, BODIPY-FL-IE(OMe)TD(OMe)-OPH,

BODIPY-FL-LE(OMe)HD(OMe)-OPH,

BODIPY-FL-LE(OMe)TD(OMe)-OPH,

BODIPY-FL-VAD(OMe)-OPH,

BODIPY-FL-W(NMe)E(OMe)HD(OMe)-OPH, BODIPY-FL-WE(OMe)HD(OMe)-OPH,

BODIPY-FL-YVAD(OMe)-OPH,

BODIPY-FL-VD(OMe)VAD(OMe)-OPH, BODIPY-FL-F ₆ V-AD(OMe)-OPH

BODIPY-TMR-D(OMe)E(OMe)VD(OMe)-FMK, BODIPY-TMR-IE(OMe)TD(OMe)-FMK, BODIPY-TMR-LE(OMe)HD(OMe)-FMK, BODIPY-TMR-LE(OMe)TD(OMe)-FM , BODIPY-TMR-VAD(OMe)-FMK,

BODIPY-TMR-W(NMe)E(OMe)HD(OMe)-FMK, BODIPY-TMR-WE(OMe)HD(OMe)-FMK, BODIPY-TR-D(OMe)E(OMe)VD(OMe)-FMK, BODIPY-TR-IE(OMe)TD(OMe)-FMK,

BODIPY-TR-LE(OMe)HD(OMe)-FMK,

BODIPY-TR-LE(OMe)TD(OMe)-FMK,

BODIPY-TR-VAD(OMe)-FMK, BODIPY-TR-W(NMe)E(OMe)HD(OMe)-FMK,

BODIPY-TR-WE(OMe)HD(OMe)-FM ,

TFA-VAD(OMe)-FMK,

TFA-VAD(OMe)-PM ,

TFA-VAD(OMe)-BMK,

TFA-VAD(OMe)-OPH,

TFA-F ₆ V-AD(OMe)-FMK,

TFA-F ₆ V-AD(OMe)-PMK,

TFA-F ₆ V-AD(OMe)-OPH,

TFA-F ₆ V-AD(OMe)-BMK,

TFA-WE(OMe)HD(OMe)-FMK,

TFA-WE(OMe)HD(OMe)-BMK,

TFA-WE(OMe)HD(OMe)-PMK,

TFA-WE(OMe)HD(OMe)-OPH,

TFA-YVAD(OMe)-FMK,

TFA-YVAD(OMe)-BMK,

TFA-YVAD(OMe)-PMK,

TFA-YVAD(OMe)-OPH,

TFA-GWE(OMe)HD(OMe)-FMK,

TFA-GWE(OMe)HD(OMe)-BMK,

TFA-GWE(OMe)HD(OMe)-PMK,

TFA-GWE(OMe)HD(OMe)-OPH,

TFA-GYVAD(OMe)-FMK,

TFA-GYVAD(OMe)-BM ,

TFA-GYVAD(OMe)-PMK,

TFA-GYVAD(OMe)-OPH

TFMCBZ-VAD(OMe)-FMK,

TFMCBZ-VAD(OMe)-PMK,

TFMCBZ-VAD(OMe)-BM ,

TFMCBZ-VAD(OMe)-OPH,

TFMCBZ-F ₆ V-AD(OMe)-FMK,

TFMCBZ-F ₆ V-AD(OMe)-PMK, TFMCBZ-F ₆ V-AD(OMe)-OPH, TFMCBZ-F ₆ V-AD(OMe)-BMK, TFMCBZ-WE(OMe)HD(OMe)-FMK, TFMCBZ-WE(OMe)HD(OMe)-BMK, TFMCBZ-WE(OMe)HD(OMe)-PMK, TFMCBZ-WE(OMe)HD(OMe)-OPH, TFMCBZ-YVAD(OMe)-FMK,

TFMCBZ-YVAD(OMe)-BM ,

TFMCBZ-YVAD(OMe)-PMK,

TFMCBZ-YVAD(OMe)-OPH,

TFMCBZ-GWE(OMe)HD(OMe)-FMK, TFMCBZ-GWE(OMe)HD(OMe)-BMK, TFMCBZ-GWE(OMe)HD(OMe)-PMK, TFMCBZ-GWE(OMe)HD(OMe)-OPH, TFMCBZ-GYVAD(OMe)-FMK, TFMCBZ-GYVAD(OMe)-BMK, TFMCBZ-GYVAD(OMe)-PM , TFMCBZ-GYVAD(OMe)-OPH

1 ⁸ F-VAD(OMe)-FMK,

1 ⁸ F-VAD(OMe)-PMK,

^{I 8} F-VAD(OMe)-BMK,

¹⁸ F-VAD(OMe)-OPH,

¹⁸ F-F ₆ V-AD(OMe)-FMK,

¹⁸ F-F ₆ V-AD(OMe)-PMK,

1 ⁸ F-F ₆ V-AD(OMe)-OPH,

1 ⁸ F-F ₆ V- AD(OMe)-BMK,

¹⁸ F-WE(OMe)HD(OMe)-FMK,

¹⁸ F-WE(OMe)HD(OMe)-BMK, ¹⁸ F-WE(OMe)HD(OMe)-PMK, 1 ⁸ F-WE(OMe)HD(OMe)-OPH,

¹⁸ F-YVAD(OMe)-FMK,

¹⁸ F- YV AD(OMe)-BM , F- YV AD(OMe)-PMK,

1 ⁸ F-YVAD(OMe)-OPH,

1 ⁸ F-GWE(OMe)HD(OMe)-FMK,

1 ⁸ F-GWE(OMe)HD(OMe)-BMK,

1 ⁸ F-GWE(OMe)HD(OMe)-PMK,

1 ⁸ F-GWE(OMe)HD(OMe)-OPH,

1 ⁸ F-G YVAD(OMe)-FMK,

1 ⁸ F-GYVAD(OMe)-BMK,

1 ⁸ F-G YY AD(OMe)-PMK,

1 ⁸ F-GYVAD(OMe)-OPH,

BODIPY-W(NMe)E(OMe)HD(OMe)-BMK, BODIPY-W(NMe)E(OMe)HD(OMe)-FMK, BODIPY-W(NMe)E(OMe)HD(OMe)-PMK, ORGn-W(NMe)E(OMe)HD(OMe)-BMK, ORGn-W(NMe)E(OMe)HD(OMe)-FMK, ORGn-W(NMe)E(OMe)HD(OMe)-PMK, 780-W(N-TFM)E(OMe)HD(OMe)-BMK, 780-W(N-TFM)E(OMe)HD(OMe)-FMK, 780-W(N-TFM)E(OMe)HD(OMe)-PMK, ABzC-W(N-TFM)E(OMe)HD(OMe)-BMK, ABzC-W(N-TFM)E(OMe)HD(OMe)-FMK, ABzC-W(N-TFM)E(OMe)HD(OMe)-PMK, BODIPY-W(N-TFM)E(OMe)HD(OMe)-BMK, BODIPY-W(N-TFM)E(OMe)HD(OMe)-FMK, BODIPY-W(N-TFM)E(OMe)HD(OMe)-PMK, FAM-W(N-TFM)E(OMe)HD(OMe)-FMK, FAM-W(N-TFM)E(OMe)HD(OMe)-PMK, N-AE-W(N-TFM)E(OMe)HD(OMe)-BMK, N-AE-W(N-TFM)E(OMe)HD(OMe)-FMK, N-AE-W(N-TFM)E(OMe)HD(OMe)-PMK, N-AM-W(N-TFM)E(OMe)HD(OMe)-BMK, N-AM-W(N-TFM)E(OMe)HD(OMe)-FMK, N- AM- W(N -TFM)E(OMe)HD(OMe)-PMK, ORGn-W(N-TFM)E(OMe)HD(OMe)-BM , ORGn-W(N-TFM)E(OMe)HD(OMe)-FMK, ORGn-W(N-TFM)E(OMe)HD(OMe)-PM , BODIPY-W(N-Ac)E(OMe)HD(OMe)-BMK, BODIPY-W(N-Ac)E(OMe)HD(OMe)-FMK, BODlPY-W(N-Ac)E(OMe)HD(OMe)-PMK, ORGn-W(N-Ac)E(OMe)HD(OMe)-BMK, ORGn-W(N-Ac)E(OMe)HD(OMe)-FMK, ORGn-W(N-Ac)E(OMe)HD(OMe)-PMK, 780-W(N-TFA)E(OMe)HD(OMe)-BMK, 780-W(N-TFA)E(OMe)HD(OMe)-FMK, 780-W(N-TFA)E(OMe)HD(OMe)-PMK, BODIPY-W(N-TFA)E(OMe)HD(OMe)-BMK, BODIPY-W(N-TFA)E(OMe)HD(OMe)-FMK, BODIPY-W(N-TFA)E(OMe)HD(OMe)-PMK, F AM- W(N -TFA)E(OMe)HD(OMe)-BMK, FAM-W(N-TFA)E(OMe)HD(OMe)-FMK, FAM-W(N-TFA)E(OMe)HD(OMe)-PMK, ORGn-W(N-TFA)E(OMe)HD(OMe)-BMK, ORGn-W(N-TFA)E(OMe)HD(OMe)-FMK, ORGn-W(N-TFA)E(OMe)HD(OMe)-PMK, 780-5-F-W(NMe)E(OMe)HD(OMe)-BMK, 780-5-F-W(NMe)E(OMe)HD(OMe)-FMK, 780-5-F-W(NMe)E(OMe)HD(OMe)-PMK,

ABzC-5-F-W(NMe)E(OMe)HD(OMe)-BMK, ABzC-5-F-W(NMe)E(OMe)HD(OMe)-FMK, ABzC-5-F-W(NMe)E(OMe)HD(OMe)-PMK, ABzC-5-F-W(N-TFM)E(OMe)HD(OMe)-BM , ABzC-5-F-W(N-TFM)E(OMe)HD(OMe)-FMK, ABzC-5-F-W(N-TFM)E(OMe)HD(OMe)-PMK, BODIPY-5-F-W(NMe)E(OMe)HD(OMe)-BMK, BODIPY-5-F-W(NMe)E(OMe)HD(OMe)-FMK, BODIPY-5-F-W(NMe)E(OMe)HD(OMe)-PMK, FAM-5-F-W(NMe)E(OMe)HD(OMe)-BMK, FAM-5-F-W(NMe)E(OMe)HD(OMe)-FMK, FAM-5-F-W(NMe)E(OMe)HD(OMe)-PMK, N-AE-5-F-W(NMe)E(OMe)HD(OMe)-BMK, N-AE-5-F-W(NMe)E(OMe)HD(OMe)-FMK, N-AE-5-F-W(NMe)E(OMe)HD(OMe)-PMK, N-AE-5-F-W(N-TFM)E(OMe)HD(OMe)-BMK, N-AE-5-F-W(N-TFM)E(OMe)HD(OMe)-FMK, N-AE-5-F-W(N-TFM)E(OMe)HD(OMe)-PMK, N-AM-5-F-W(NMe)E(OMe)HD(OMe)-BMK, N-AM-5-F-W(NMe)E(OMe)HD(OMe)-FMK, N-AM-5-F-W(NMe)E(OMe)HD(OMe)-PMK, N-AM-5-F-W(N-TFM)E(OMe)HD(OMe)-BMK, N-AM-5-F-W(N-TFM)E(OMe)HD(OMe)-FMK, N-AM-5-F-W(N-TFM)E(OMe)HD(OMe)-PMK, ORGn-5-F-W(NMe)E(OMe)HD(OMe)-BMK, ORGn-5-F-W(NMe)E(OMe)HD(OMe)-FMK, ORGn-5-F-W(NMe)E(OMe)HD(OMe)-PMK, 780-2-CF ₃ W(NMe)E(OMe)HD(OMe)-BMK, 780-2-CF ₃ W(NMe)E(OMe)HD(OMe)-FMK, 780-2-CF ₃ W(NMe)E(OMe)HD(OMe)-PMK, ABzC-2-CF ₃ W(NMe)E(OMe)HD(OMe)-BMK, ABzC-2-CF ₃ W(NMe)E(OMe)HD(OMe)-FMK, ABzC-2-CF ₃ W(NMe)E(OMe)HD(OMe)-PMK, ABzC-2-CF ₃ W(N-TFM)E(OMe)HD(OMe)-BMK, ABzC-2-CF ₃ W(N-TFM)E(OMe)HD(OMe)-FMK, ABzC-2-CF ₃ W(N-TFM)E(OMe)HD(OMe)-PMK, BODIPY-2-CF ₃ W(NMe)E(OMe)HD(OMe)-BMK, BODIPY-2-CF ₃ W(NMe)E(OMe)HD(OMe)-FMK, BODIPY-2-CF ₃ W(NMe)E(OMe)HD(OMe)-PMK, FAM-2-CF ₃ W(NMe)E(OMe)HD(OMe)-BMK, FAM-2-CF ₃ W(NMe)E(OMe)HD(O e)-FMK, FAM-2-CF ₃ W(NMe)E(OMe)HD(OMe)-PMK, N-AE-2-CF ₃ W(N-TFM)E(OMe)HD(OMe)-BMK, N-AE-2-CF ₃ W(N-TFM)E(OMe)HD(OMe)-FMK, N-AE-2-CF ₃ W(N-TFM)E(OMe)HD(OMe)-PMK, N-AM-2-CF ₃ W(N-TFM)E(OMe)HD(OMe)-BMK, N-AM-2-CF ₃ W(N-TFM)E(OMe)HD(OMe)-FMK, N-AM-2-CF ₃ W(N-TFM)E(OMe)HD(OMe)-PMK, ORGn-2-CF ₃ W(NMe)E(OMe)HD(OMe)-BMK, ORGn-2-CF ₃ W(NMe)E(OMe)HD(OMe)-FMK, ORGn-2-CF ₃ W(NMe)E(OMe)HD(OMe)-PMK, 780-6-CF ₃ W(NMe)E(OMe)HD(OMe)-BMK, 780-6-CF ₃ W(NMe)E(OMe)HD(OMe)-FMK, 780-6-CF ₃ W(NMe)E(OMe)HD(OMe)-PM ,

ABzC-6-CF ₃ W(NMe)E(OMe)HD(OMe)-BMK, ABzC-6-CF ₃ W(NMe)E(OMe)HD(OMe)-FMK, ABzC-6-CF ₃ W(NMe)E(OMe)HD(OMe)-PM , ABzC-6-CF ₃ W(N-TFM)E(OMe)HD(OMe)-BMK, ABzC-6-CF ₃ W(N-TFM)E(OMe)HD(OMe)-FMK, ABzC-6-CF ₃ W(N-TFM)E(OMe)HD(OMe)-PMK, BODIPY-6-CF ₃ W(NMe)E(OMe)HD(OMe)-BMK, BODIPY-6-CF ₃ W(NMe)E(OMe)HD(OMe)-FMK, BODIPY-6-CF ₃ W(NMe)E(OMe)HD(OMe)-PMK, FAM-6-CF ₃ W(NMe)E(OMe)HD(OMe)-BMK, FAM-6-CF ₃ W(NMe)E(OMe)HD(OMe)-FMK, FAM-6-CF ₃ W(NMe)E(OMe)HD(OMe)-PM , N-AE-6-CF ₃ W(N-TFM)E(OMe)HD(OMe)-BMK, N-AE-6-CF ₃ W(N-TFM)E(OMe)HD(OMe)-FMK, N-AE-6-CF ₃ W(N-TFM)E(OMe)HD(OMe)-PMK, N-AM-6-CF ₃ W(N-TFM)E(OMe)HD(OMe)-BMK, N-AM-6-CF ₃ W(N-TFM)E(OMe)HD(OMe)-FMK, N-AM-6-CF ₃ W(N-TFM)E(OMe)HD(OMe)-PMK,

ORGn-6-CF ₃ W(NMe)E(OMe)HD(OMe)-BMK,

ORGn-6-CF ₃ W(NMe)E(OMe)HD(OMe)-FMK, and

ORGn-6-CF ₃ W(NMe)E(OMe)HD(OMe)-PMK.

The invention is illustrated by the following non-limiting Examples. The examples below are specific to caspase inhibitors; however, they generally apply to the Cysteine cathepsin inhibitors described herein as well.

EXAMPLES

Example 1.

Synthesis of Caspase Inhibitors (General Structure)

According to at least one embodiment, the caspase inhibitors can be synthesized using liquid phase peptide synthesis (Bodanszky, PRINCIPLES OF PEPTIDE SYNTHESIS (1993) or solid phase peptide synthesis (Merrifield, J Amer Chem Soc 85(14):2149-54 (1963); Amblard et al., Mol. Biotechnol., 33(3):239-54 (2006)). An example of a suitable liquid phase peptide synthetic pathway is shown below, and generally involves building oligo peptides from the N-terminus of an amino acid by providing a first amino acid (1); protecting or blocking the N terminus of the amino acid using a protecting or blocking group (e.g., BOC or FMOC); coupling the first amino acid with a C-terminal ester of a second amino acid (2) by reacting the amino acids in the presence of a coupling agent (e.g., dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), usually in the presence of N-hydroxysuccinimide or 1 -hydroxybenzotriazole) to form a dipeptide (3); and deprotecting the dipeptide without removing any other protecting groups to yield the free dipeptide acid (4). Dipeptide (4) can be coupled to the suitably derivatized L- aspartic acid β-methyl ester to yield a desired caspase inhibitor or it can be coupled with a suitably protected amino acid to yield a fully protected tripeptide (5). If desired, by sequential deprotection of the C-terminus of 5 and analogous coupling to another suitably protected amino acid, a fully protected tetrapeptide may be constructed. Synthesized caspase inhibitors disclosed herein may be in the form of a waxy material or lyophilized powder.

The caspase inhibitor may be prepared by finishing the peptide chain with an aspartic acid portion and adding a fluoromethyl ketone (FMK), phenoxymethyl ketone (PMK), an OPH (2,6-difluorophenoxy ketone or 2,6-difluorophenoxymethyl ketone) group, or an acyloxymethyl ketone (AOMK) to the end of the peptide chain as a leaving group that is part of the reactive group and is positioned at the C-terminus of the recognition sequence of the caspase inhibitor. Alternatively, using a different protection strategy, FMOC-L-aspartic acid β-methyl ester may be made. From these molecules, FMK/PMK/OPH/AOMK can be attached to the a-carboxylic acid via a methylene group, introduced using diazomethane chemistry.

Example 2.

Characterization of Caspase Inhibitors: TFA-VAD(OMe)-FMK

Sequence: TFA-Val-Ala-Asp(OMe)-FMK

Molecular Weight: 429.4 amu

Solubility: 1 mg/mL in 10% Acetonitrile in H ₂ 0

Appearance: White Lyophilized Powder

Mass Spec: M ⁺ =430.1 1

Example 3 TFA-VAD(OMe)-FMK is more potent than Z-VAD(OMe)-FMK and Q- VD(OMe)-OPH at inhibiting purified caspase-3.

Inhibitors and substrate (Ac-VAD-AMC final assay concentration = 50 μΜ) were mixed in assay buffer (50 niM HEPES pH 7.2, 50 mM NaCl, 0.1% CHAPS, 10 mM EDTA, 5% glycerol, 10 mM DTT) in 96-well plates. Recombinant human active caspase-3 (final assay concentration = 0.5 U/reaction) was added to each sample for a total reaction volume of 100 μΐ. Immediately after addition of caspase-3, plates were read at 37 °C with an excitation of 405 nm, an emission of 505 nm and an emission cutoff of 495 nm every 30 seconds for 1 hour using a Molecular Devices SpectraMax M2 ^e plate reader.

All experiments were performed in duplicate, in two different days and three independent experiments. Prior to the inhibition experiment, the substrate was titrated with a constant concentration of enzyme, giving us a Km of 0.49782 μΜ, this number is used later to obtain the k _app . The amount of product was converted into percent of inhibited enzyme using the control or no inhibitor as a 0% inhibited and substrate only as our 100% inhibited.

The raw data were analyzed using a one-step exponential association curve to get the rates at different concentrations. Each curve was analyzed independently and only the curves which plateaued were used for further analysis by the rates obtained from said curves. These rates were then transformed into the apparent second order rate constant by using the

concentration of substrate, the concentration of enzyme, the concentration of inhibitor, the Km and the k value from the exponential association curve. The results showed Z-VAD-FMK having an apparent rate constant of 0.98 xl 0 ⁶ M ^"1 , sec ^"1 with Q-VD-OPH being a better inhibitor with a second order rate constant 1.6 xlO ⁶ M ^"1 , sec ^"1 and lastly TFA-VAD-FMK being the best inhibitor with a second order rate constant 7.5 xlO ⁶ M ^"1 , sec ^"1 . (Figure 1 and 2).

Example 4 TFA-VAD(OMe)-FMK is more potent at inhibiting staurosporine-induced caspase activity compared to Z-VAD(OMe)-FMK and Q-VD(OMe)-FMK.

Inhibitors (10 μΜ) were added to 1 x 10 ⁶ Jurkat cells (human T lymphocyte cell line) for 15 minutes in a total volume of 1 ml cell culture medium (RPMI + 10% FBS) at 37°C. 1 μΜ Staurosporine (protein kinase inhibitor) was added and cells were incubated for 3.5 hours at 37°C to induce apoptosis. After 3.5 hours, CAS-MAP active caspase labeling reagent (FAM- VAD(OMe)-FMK, 0.75 μΜ) was added and cells were incubated for 20 minutes at 37°C to label active caspases. Cells were collected, washed with PBS, and resuspended in 1 ml PBS for flow cytometry analysis using a Life Technologies Attune NxT acoustic focusing cytometer (488 nm excitation laser, 530 nm/30 nm band pass emission filter). An increase in FAM-VAD- FMK fluorescence intensity correlates with caspase activity. (Figure 4) Example 5 TFA-VAD(OMe)-FM is a more potent than Z-VAD(OMe)-FMK at inhibiting apoptosis.

Jurkat cells (1 x 10 ⁶ cells, 1 ml total volume in RPMI + 10% FBS) were incubated at 37°C with the indicated concentrations of TFA-VAD(OMe)-FMK or Z-VAD(OMe)-FMK for 15 minutes prior to apoptosis induction with 1 μΜ Staurosponne (protein kinase inhibitor) for 4 hours at 37°C, 5 μΜ Camptothecin (topoisomerase I inhibitor) for 4 hours at 37°C or 100 ng/ml anti-TRAIL R2 agonist antibody for 24 hours at 37°C. Cells were washed with annexin V binding buffer (140 mM NaCl, 2.5 mM CaC12, 10 mM HEPES pH 7.4) and resuspended in 100 μΐ annexin V binding buffer. 5 μΐ Annexin V - Alexa Fluor 488 (Thermo Scientific) was added to each sample and cells were incubated in the dark, at room temperature for 15 minutes. 1 ml of annexin V binding buffer was added to each sample followed by 1 μΐ SYTOX AADvanced (Thermo Scientific). Cells were incubated in the dark, at room temperature for 5 minutes prior to flow cytometry analysis using a Life Technologies Attune NxT acoustic focusing cytometer (488 nm excitation laser, 530 nm/30 nm band pass emission filter for annexin V- Alexa Fluor 488 and 488 nm excitation laser, 695 nm/40 nm band pass emission filter for SYTOX

AADvanced). % Apoptosis was calculated by adding the percentage of cells in the Annexin V positive/SYTOX negative and Annexin V positive/SYTOX positive populations. Data were normalized to no inhibitor samples (unstimulated = 0%, stimulated = 100%) and percent inhibition was calculated. (Figure 5)

Example 6 TFA-VAD(OMe)-FMK inhibits staurosporine-induced apoptosis after 24 hours in cell culture.

Jurkat cells (1 x 10 ⁶ cells, 1 ml total volume in RPMI + 10% FBS) were incubated with 10 μΜ TFA-VAD(OMe)-FMK or 10 μΜ Z-VAD(OMe)-FMK for 24 hours at 37°C. After 24 hours, apoptosis was induced with 1 μΜ staurosporine for 4 hours at 37°C. Cells were washed with annexin V binding buffer (140 mM NaCl, 2.5 mM CaC12, 10 mM HEPES pH 7.4) and resuspended in 100 μΐ annexin V binding buffer. 5 μΐ Annexin V - Alexa Fluor 488 (Thermo Scientific) was added to each sample and cells were incubated in the dark, at room temperature for 15 minutes. 1 ml of annexin V binding buffer was added to each sample followed by 1 μΐ SYTOX AADvanced (Thermo Scientific). Cells were incubated in the dark, at room

temperature for 5 minutes prior to flow cytometry analysis using a Life Technologies Attune

NxT acoustic focusing cytometer (488 nm excitation laser, 530 nm 30 nm band pass emission filter for annexin V- Alexa Fluor 488 and 488 nm excitation laser, 695 nm/40 nm band pass emission filter for SYTOX AADvanced). % Apoptosis was calculated by adding the percentage of cells in the Annexin V positive/SYTOX negative and Annexin V positive/SYTOX positive populations. (Figure 6)

Example 7 TFA-VAD(OMe)-FMK, TFA-VAD(OMe)-TPH, TFA-VD(OMe)-TPH, TFA- EVD-TPH and TFA-6E8D-TPH inhibit caspase-3/7 activity in Hep G2 cells.

Hep G2 cells (human hepatocyte carcinoma cell line) were seeded in 96 well plates at 1 x 10 ⁴ cells/well in DMEM + 10% FBS and incubated for 24 hours at 37°C. Inhibitors were added to the indicated concentrations and cells were incubated for 15 minutes at 37°C. Caspase activity was induced by treating cells with 2 μ§ ηύ anti-TRAIL R2 agonist antibody for 24 hours at 37°C in a total volume of 60 μΐ in DMEM + 10% FBS. After 24 hours, 60 μΐ of a 2X solution containing a caspase-3/7 specific fluorescent substrate (Ac-Asp-Phe(F ₅ )-Val-Asp-ACC, 2X = 200 μΜ) was added to each sample in a duel function cell lysis/caspase activity buffer (2X = 20% sucrose, 100 mM HEPES pH 7.4, 20 mM NaCl, 10 mM MgC12, 0.8 mM EGTA, 0.2% CHAPS, 1% TWEEN 20). After 2 hours of substrate incubation, fluorescence was determined with an excitation wavelength of 355 nm and an emission wavelength of 460 nm with a cutoff of 455 nm using a Molecular Devices SpectraMax M2 ^e plate reader. Relative Fluorescent Units were normalized to no inhibitor samples (no anti-TRAIL R2 antibody = 0%, + anti-TRAIL R2 antibody = 100%) and percent inhibition was calculated. (Figure 7)

Example 8 TFA-VAD(OMe)-FMK, TFA-VAD(OMe)-TPH and TFA-VD(OMe)-TPH inhibit staurosporine-induced apoptosis in Jurkat cells.

Jurkat cells (1 x 10 ⁶ cells, 1 ml total volume in RPMI + 10% FBS) were incubated with 5 μΜ of the indicated caspase inhibitor for 15 minutes prior to stimulation with 1 μΜ

staurosporine for 4 hours at 37°C. Cells were washed with annexin V binding buffer (140 mM NaCl, 2.5 mM CaC12, 10 mM HEPES pH 7.4) and resuspended in 100 μΐ annexin V binding buffer. 5 μΐ Annexin V - Alexa Fluor 488 (Thermo Scientific) was added to each sample and cells were incubated in the dark, at room temperature for 15 minutes. 1 ml of annexin V binding buffer was added to each sample followed by 1 μΐ SYTOX AADvanced (Thermo Scientific). Cells were incubated in the dark, at room temperature for 5 minutes prior to flow cytometry analysis using a Life Technologies Attune NxT acoustic focusing cytometer (488 nm excitation laser, 530 nm/30 nm band pass emission filter for annexin V- Alexa Fluor 488 and 488 nm excitation laser, 695 nm/40 nm band pass emission filter for SYTOX AADvanced). %

Apoptosis was calculated by adding the percentage of cells in the Annexin V positive/SYTOX negative and Annexin V positive/SYTOX positive populations. Data were normalized to no inhibitor samples (unstimulated = 0%, stimulated = 100%) and percent inhibition was calculated. TFA-EVD-TPH and TFA-6E8D-TPH inhibited apoptosis to a lesser extent than TFA-VAD-FMK, TFA-VAD-TPH and TFA-VD-TPH in staurosporine-induced Jurkat cells. (Figure 8)

Example 9 TFA-VAD(OMe)-FMK, TFA-VAD(OMe)-TPH, TFA-VD(OMe)-TPH, TFA- EVD-TPH and TFA-6E8D-TPH inhibit anti-TRAIL R2 antibody-induced apoptosis in Jurkat cells.

Jurkat Cells (1 x 10 ⁶ cells, 1 ml total volume in RPMI + 10% FBS) were incubated with the indicated concentrations of caspase inhibitor for 15 minutes prior to stimulation with 100 ng/ml anti-TRAIL R2 agonist antibody for 24 hours at 37°C. Cells were washed with annexin V binding buffer (140 mM NaCl, 2.5 mM CaC12, 10 mM HEPES pH 7.4) and resuspended in 100 μΐ annexin V binding buffer. 5 μΐ Annexin V - Alexa Fluor 488 (Thermo Scientific) was added to each sample and cells were incubated in the dark, at room temperature for 15 minutes. 1 ml of annexin V binding buffer was added to each sample followed by 1 μΐ SYTOX AADvanced (Thermo Scientific). Cells were incubated in the dark, at room temperature for 5 minutes prior to flow cytometry analysis using a Life Technologies Attune NxT acoustic focusing cytometer (488 nm excitation laser, 530 nm/30 nm band pass emission filter for annexin V- Alexa Fluor 488 and 488 nm excitation laser, 695 nm/40 nm band pass emission filter for SYTOX

AADvanced). % Apoptosis was calculated by adding the percentage of cells in the Annexin V positive/SYTOX negative and Annexin V positive/SYTOX positive populations. Data were normalized to no inhibitor samples (unstimulated = 0%, stimulated = 100%) and percent inhibition was calculated. (Figure 9) Example 10 TFA-FK-TPH and TFA-FR-TPH inhibit cathepsins in intact RAW 264.7 cells.

RAW 264.7 cells (mouse macrophage cell line) were plated in 6 well plates at 8 x 10 cells/well in DMEM + 10% FBS and allowed to grow overnight at 37°C. The next day, cells were treated with the indicated concentrations of TFA-FK-TPH, TFA-FR-TPH or E64d (a general cysteine cathepsin and calpain inhibitor) for 1 hour at 37°C followed by the addition of 1 μΜ BMV109 (a Cy5 labeled activity based probe that binds cathepsin B, L, S and X) for 2 hours at 37°C to label active cathepsins. Cells were washed with cold PBS, resuspended in 50 μΐ hypotonic lysis buffer (50 mM PIPES pH 7.4, 10 mM KC1, 5 mM MgC12, 2 mM EDTA, 1% Nonidet P-40, 4 mM DTT) and incubated on ice for 10 minutes. Lysates were spun at 17,000 x g for 10 minutes at 4°C and supernatants were collected and assayed for protein concentration using the Pierce 660 nm Protein Assay. 50 μg of cell lysate was separated by SDS-PAGE on 15% Mini-PROTEAN TGX precast gels (Bio-Rad). Gels were scanned using a GE Typhoon FLA 9500 (Cy5). TFA-FK-TPH was found to be a much more potent cathepsin inhibitor in intact RAW 264.7 cells than TFA-FR-TPH.

Example 11 Preparation of Representative Compounds of the Invention

As illustrated below, the following representative compounds of the invention were prepared by standard SPPS; the acid moieties were transformed to the 1 -(2,3,5,6- tetrafluorophenoxy)acetyl group by solution chemistry. Final products were confirmed by mass spectrometry.

TFA-EVD-TPH; Mass Spec [M+H] ⁺ =621.1

TFA-6E8D-TPH; Mass Sp ⁺ =724.8

TFA-VAD(OMe)-TPH; Mass Spec [M+H] ⁺ =575.9

TF A- VD(OMe)-TPH ; Mass Spec [M+H] ⁺ =505.1

TFA-FK-TPH; Mass Spec [M+H] ⁺ =552.1

TFA-FR-TPH; Mass Spec [M+H] ⁺ =581.2

Example 12 Preparation of Representative Compounds of the Invention

Representative compounds of the invention can also be prepared as illustrated below.

XC0 ₂ Bn 1 . CIC0 ₂ CH ₂ CHMe ₂₎ NM , THF

2. CH ₂ N ₂ , Et _z O

Joc N C0 ₂ H 3. HBr (aqueous) ^ _{Bqc HN} ^

101 (Tetrahedron, 1997, 53, 5325)

[Bi∞rg. Med. Chem. Lett , 1997, 19, 199) Intermediate compound 110 can be used to prepare compounds having D-TPH at the carboxy terminus. Similar intermediate compounds can be prepared to provide compounds having the functionality X-J at the carboxy terminus, wherein X is an amino acid or an appropriately protected amino acid and J has any of the values defined herein, by selection of the starting amino acid 101.

Solid-phase peptide synthesis (SPPS) can be used to prepare peptide sequences that can be incorporated in to compounds of the invention. Coupling of the carboxy terminus of a peptide sequences 111 with an intermediated of formula 110 followed by optional deprotection and optional functionalization of the N-terminus can provide compounds of the invention as illustrated below. 1. Solid-phase peptide synthesis (SPPS)

2. Cleave peptide from resin to C-

terminal C0 ₂ H (leave amino acid side

chains protected)

AA ₁ -AA _n -C0 ₂ H

111

1. PyBOP, DIEA, D F

2. Deprotection

N-terminal functionality

All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference (including United States Provisional Application Number 62/222586, International Patent Application Publication Number WO2015/175381 Al, United States Patent Number 8,673,904, United States Patent Application Publication Number 2009/0203629, Berger, A. B., et al., 2006, Mol. Cell 23, 509-21 ; Van der Linden, W., et al., 2016, ACS Infectious Diseases 2, 173-17; Albeck, A. and Estreicher, G., Tetrahedron, 1997, 53, 5325; and Ueno, H., et al., Bioorg. Med. Chem. Lett., 1997, 19, 199). The invention has been described with reference to various specific and preferred embodiments and

techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.