Caspase-2 inhibitor compounds

Abstract

The present invention relates to a compound of formula (I): or a pharma- ceutically acceptable salt, solvate or stereoisomer thereof, pharmaceutical compositions containing them, and their use in the treatment and / or prevention of diseases or disorders mediated by caspase-2.

Description

[Technical field]

[0001] The present invention relates to novel peptide derivatives, pharmaceutical compositions containing said derivatives, and their use in the prevention and / or treatment of caspase-2 mediated diseases or disorders. [Background technology]

[0002] Caspases are a family of evolutionarily conserved cysteine-dependent endoproteases that hydrolyze their substrates after specific aspartic acid residues (Lamkanfi, 2002). Caspases are associated with a wide range of biological activities, including apoptosis (Ramirez and Salvesen, 2018), inflammation (Vande Walle, 2016), cell differentiation (Fernando, 2002), and metabolism (Shalini, 2015).

[0003] Caspases are classified into two main groups: those involved in regulating inflammatory processes (-1, -4, -5, -11, -12) and those central to the induction and execution of apoptosis (2, -3, -7, -8, -9, -10) (Shalini, 2015). Apoptotic caspase zymogens are classified as either initiator or executioner caspases based on their role in the apoptotic program. Initiator caspases (e.g., caspase-2, -8, and -9), which are important in upstream signaling, become activated by the binding of adaptor proteins to their prodomains. This binding promotes oligomerization into high molecular weight protein complexes, which trigger dimerization and processing to the active enzymes. Once active, initiator caspases activate executioner caspases (e.g., caspase-3 and -7) via proteolytic cleavage. Executioner caspases then proceed to cleave protein substrates, leading to the systematic disorganization of the cell and ultimately cell death.

[0004] Initiator caspase activation during apoptosis is mediated by two major pathways: the mitochondrial or intrinsic pathway and the death receptor or extrinsic pathway. The intrinsic pathway is activated in response to cellular stress (e.g., ROS, cytotoxic drugs, DNA damage), leading to the activation of initiator caspase-2 (Shalini, 2015). Active caspase-2 cleaves Bid, and truncated Bid (tBid) activates mitochondrial permeabilization through regulation of Bax and Bak (Enoksson, 2004). Mitochondrial cytochrome c is then released into the cytosol and induces oligomerization of Apaf-1, forming a large heptameric complex known as the apoptosome (Bao, 2007). The apoptosome recruits and activates the initiator caspase, caspase-9, which can directly cleave and activate the executioner caspases, caspase-3 and -7 (Bao, 2007).

[0005] The extrinsic apoptotic pathway is induced at the plasma membrane by ligand binding to its extracellular death receptor (e.g., TNFR, Fas, TRAIL) (Gaur, 2003). This mediates the recruitment and activation of caspase-8 or -10 through the death-inducing signaling complex (DISC), which contains the FAS-associated death domain protein (FADD) and / or the TNFR-associated death domain protein (TRADD) and other components. Caspase-8 also cleaves BID into a truncated form (tBID), which participates in the mitochondrial pathway to amplify the apoptotic response (Li, 1998). Once the initiator caspases are activated via the extrinsic or intrinsic apoptotic pathway, they mediate the activation of the effector caspases-3, -6 and -7 (Bao, 2007).

[0006] Caspase-2, originally named Need-2 (in mice) or Ich-1 (in humans), shares 55% similarity with C. elegans (Yuan, 1993; Wang, 1994; Kumar, 1994) and is the most conserved caspase across species. Caspase-2 contains an N-terminal caspase recruitment domain (CARD) followed by a large subunit (p19) that contains the active site and a small subunit (p12). Thus, caspase-2 is most similar to caspase-9, the initiator of the intrinsic pathway of apoptosis (Li and Yuan, 2008). However, in contrast to conventional initiator caspases such as caspase-9 or the apical caspase of extrinsic apoptosis, caspase-8, caspase-2 does not process apoptotic effectors that need to be cleaved by initiators for their activation, such as caspases-3, -6 or -7 (Guo et al., 2002; Van de Craen et al., 1999). In contrast to other initiator caspases, caspase-2 undergoes autocatalytic cleavage after dimerization and does not require cleavage for its initial activation (BC Baliga 2004).

[0007] Caspase-2 has several unique features, including the presence of a nuclear localization signal that plays a central role in triggering the apoptotic pathway following DNA damage in several cellular models. In addition, caspase-2 has been reported to mediate non-apoptotic signaling pathways, including de novo lipogenesis (Kim, 2018), metabolic regulation (Nutt, 2005), tumor suppression (Kumar, 1995), mitotic abnormalities (Vitale, 2011), cell cycle regulation (Sidi, 2008) and DNA repair (Vigneswara, 2020).

[0008] Loss of viable cells in healthy tissues due to increased apoptosis contributes to the development and progression of many human disorders as well as environmental, medical toxicity and pathogens (Singh, 2019).

[0009] Increased caspase-2-induced apoptosis has been reported in various diseases or conditions. Loss of retinal ganglion cells (RGCs) after optic nerve injury is a hallmark of certain human eye diseases, such as ischemic optic neuropathy. In a rat optic nerve transection model, caspase-2 was found to be expressed and cleaved in RGCs (Ahmed, 2011). In neonatal ischemic brain injury, ischemic injury triggers multiple pathways of oxidative stress, inflammation, and excitotoxicity, leading to massive cell death in the ischemic area by apoptosis mediated by caspase-2 activation (Carlsson, 2011; Chauvier, 2011). In stroke, apoptosis of cerebral neurons is the main pathological change occurring in the peri-infarct zone after transient global ischemia, which leads to ischemia / reperfusion (I / R) injury. Apoptosis of cerebral neurons can lead to hemiparesis, death, or cognitive impairment after stroke (Turkmen, 2011). In animal models of stroke, caspase-2 expression and activation increased after I / R. In Alzheimer's disease (AD) and other tauopathies, tau protein forms fibrils, which are thought to be neurotoxic. Caspase-2 cleaves tau at Asp314, which leads to impaired cognitive and synaptic function in animal and cell models. The cleavage product, Δtau314, is resistant to fibrillization and is present at higher levels in the brains of cognitively impaired mice and humans with AD. Expression of a tau mutant that resisted caspase-2 cleavage prevented tau from infiltrating spines, repositioning glutamate receptors, and impairing synaptic function in cultured neurons, preventing memory impairment and neurodegeneration in mice (Zhao, 2016). · Caspase-2 promotes obesity, metabolic syndrome, and nonalcoholic fatty liver disease (Machado, 2016; Kim, 2018). Caspase-2 expression was strongly correlated with the severity of liver disease in patients with NAFLD (Machado, 2015; Kim, 2018).

[0010] Strong evidence of increased apoptosis having a role in many diseases has prompted efforts to drug this pathway for therapeutic benefit. The first generation of caspase inhibitors were reversible aldehyde peptides that showed limited therapeutic benefit due to the lack of selectivity and the different warheads used.

[0011] In the first generation of inhibitors, several peptide sequences were developed that were thought to selectively inhibit different caspases, e.g., Ac-DEVD-CHO (a preferential inhibitor of caspase-3 and caspase-7) and Ac-VDVAD-CHO (a preferential inhibitor of caspases-2, -3, and -7).

[0012] To effectively inhibit apoptosis, selectivity for caspase-3 is crucial due to the high concentration of this caspase compared to other caspases in almost all tissues and its promiscuous nature (McStay, 2008). Furthermore, inhibitors that are non-selective for caspase-2 have limited clinical benefit in direct apoptosis inhibition because they act downstream of the mitochondrial outer membrane pore and cannot reverse the major damage to mitochondrial function induced by caspase-2-mediated pore formation (Singh, 2019). Moreover, selectivity may also be important in situations where caspase-8 (extrinsic apoptosis) is also activated and selective caspase-2 (intrinsic apoptosis) inhibition is required.

[0013] The inhibitor Ac-VDVAD-CHO is more potent against caspase-3 than caspase-2. This raises some questions about the validity of the data generated with this reagent when used in a cellular context as a "selective" caspase-2 inhibitor. Several publications have already indicated specificity concerns regarding existing inhibitors and highlight the urgent need for more selective inhibitors in the caspase field (Pereira, 2008; Berger, 2006; McStay, 2008; Benkova, 2009; Yun, 2007; Krumschnabel, 2009; Kitevska, 2009; Schweizer, 2007; Poreba, 2019 ("Caspase selective reagents for diagnosing apoptotic mechanicisms", Poreba et al., Cell Death and Differentiation). In this paper (mainly in the supplementary section), we provided a detailed kinetic analysis of caspase inhibitors / probes created based on natural amino acid sequences.

[0014] There are several types of warheads used in caspase inhibitor design. However, they all function similarly. Their mechanism of action relies on the nucleophilic attack of an active site cysteine ​​on the electrophilic center, thus forming a transitional (reversible) or covalent (irreversible) caspase-inhibitor complex (Evans, 2006). The most important feature of a particular warhead is its ability to target only the active site cysteine ​​residues, omitting other free nucleophiles in the proteome. Therefore, the catalytic mechanism of the target proteolytic enzyme plays a key role in the selection of an appropriate warhead.

[0015] It is important that the thiol group of the catalytic cysteine ​​residue in cysteine ​​proteases is more polarizable than the hydroxyl group on the catalytic serine or threonine, and therefore the electrophiles used as warheads for cysteine ​​proteases may be more flexible than those for serine or threonine proteases (Powers, 2002). Thus, caspases have been most widely studied with inhibitors containing warheads such as diazomethylketones, epoxides, and halo- and acyloxy-methylketones. The main advantages of these warheads are their ease of synthesis, good bioavailability, and active site Cys selective reactivity. The most used commercially available caspase inhibitor warheads are fluoro- (FMK or -CH2F), chloro- (CMK or -CH2Cl), or acyloxy-methylketone (AOMK) (Poreba, 2015, ("Small molecule active site directed tools for studying human caspases" Poreba et al., Chemical Reviews, 2015)). The main advantages of these warheads are their ease of synthesis, good bioavailability, and Cys-selective reactivity at the active site (Sanman, 2014; Powers, 2002).

[0016] FMK-based inhibitors were the first and have dominated research in this field so far. One advantage of FMK is that the ketone reagent penetrates the plasma membrane and is relatively non-toxic to cells (Van Noorden, 2001 ("The history of Z-VAD-FMK, a tool for understanding the significance of caspase inhibition" Van Noorden, Acta Histochemica, 2001)). The use of FMK inhibitors also has some disadvantages: cross-reactivity of such probes with other cysteine ​​proteases such as legumain, cathepsin B and cathepsin H, as well as the generation of high labeling background from non-specific binding of the reactive FMK group: (Rozman-Pungercar, 2003; Schotte, 1999). Inhibitors containing the FMK warhead have been shown to be toxic in vivo, especially due to the release of fluoroacetate groups in the liver, which leads to inhibition of aconitase. Therefore, the development of inhibitors bearing the FMK group was halted at the preclinical stage due to their hepatotoxicity (Citarella, 2020).

[0017] AOMKs are the weakest electrophiles in this group and therefore are most suitable for the development of caspase inhibitors, as they have little cross-reactivity with other biological nucleophiles. Thus, the weaker electrophilicity allows them to react more specifically with caspases and show reduced cross-reactivity with other Cys-dependent proteases (Poreba et al., Chemical Reviews (2015) BI-BJ).

[0018] Down-modulation of caspase-2 expression effectively prevents apoptosis in several models. In a model of retinal ganglion cell (RGC) loss after optic nerve injury, inhibition of caspase-2 expression by siRNA had a neuroprotective effect by significantly enhancing RGC survival for at least 30 days (Ahmed, 2011). In neonatal ischemic brain injury, genetic (Carlsson, 2011) or pharmacological (Chauvier, 2011) inhibition of caspase-2 reduces cortical and white matter damage in the neonatal brain after excitotoxicity, arterial insult, and hypoxia. In stroke apoptosis, treatment with microRNA (miR-1247-3p) inhibited caspase-2 expression and attenuated neuronal apoptosis (Zhang, 2019). In animal models of Alzheimer’s disease (AD) and other tauopathies, reducing levels of caspase-2 restored long-term memory in mice with pre-existing deficits (Zhao, 2016). · Caspase-2 depletion protected methionine / choline deficient (MCD) diet-induced steatohepatitis mice from hepatocyte apoptosis and fibrosis progression (Machado, 2015).

[0019] Compounds capable of inhibiting caspase-2 activity have been reported, for example, in WO 2005 / 105829 and EP 2670774. However, these known caspase-2 inhibitors also have too high activity against caspase-3. They do not qualify as selective caspase-2 inhibitors. More recently, a series of reversible caspase-2 inhibitors have been reported. When evaluated in vitro on human recombinant caspases, these compounds were found to preferentially inhibit caspase-2 but have moderate efficacy in cellular assays and structural properties that are not compatible with in vivo use (Maillard, 2011). WO 2017 / 162674 and WO 2019 / 068538 disclose peptide compounds containing five amino acid units as caspase-2 inhibitors.

[0020] (2019) also disclosed caspase-2 inhibitors composed of five amino acid units in the L-stereochemistry. Compound NH-23-C2 (NH-Idc-hGlu-Thr(Bzl)-Ser-Asp) was found to have the highest caspase-2 inhibitory activity among the compounds assayed, yet still had no selectivity for caspase-3 and limited selectivity for caspase-8.

[0021] Thus, more specifically, there remains a need for potent and / or selective caspase-2 inhibitors with significantly reduced activity against caspase-3 / caspase-8.It would be highly advantageous to provide more selective and efficient caspase-2 inhibitors for use in the prevention and / or treatment of diseases and / or injuries involving caspase-2 activity, such as non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD), obesity, metabolic syndrome, liver cirrhosis, neonatal cerebral ischemia, cardiac ischemia and chronic degenerative diseases, such as Alzheimer's disease.

[0022] It would also be highly advantageous to provide more effective and selective caspase-2 inhibitors for use as activity-based probes to specifically detect caspase-2 activity.

[0023] The compounds of the present invention are aimed at meeting these needs. [Brief description of the drawings]

[0024] [Figure 1] P2 substrate screening for caspase-2, caspase-3 and caspase-8. The x-axis represents the abbreviation of the L-amino acid at the P2 position in NH-Idc-hGlu-Thr(Bzl)-P2-Asp-ACC, while the y-axis represents the rate of substrate hydrolysis (RFU / s) per 10 nM of specific activity or "active site titrated" caspases. The concentration of active caspases was determined by active site titration. [Diagram 2]Raw data for calculation of kobs / I inhibition parameters of NH-23-C2 synthetic inhibitors against caspase-2. kobs / I parameters were measured under pseudo-first-order kinetic conditions ([I]>>[E]). NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-ACC was used as substrate. ACC fluorescence was monitored using wavelengths of 355 nm (excitation) and 460 nm (emission). Second-order inhibition rates (kobs / I) were determined in at least three independent experiments and presented as mean values. GraphPad Prism 7 software was used for calculations. [Diagram 3] Raw data for determining the Ki and IC50 parameters of the NH-23-C2 inhibitor against caspase-2. The Ki parameter was measured using the Morrison equation (Copeland, 2000). NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-ACC was used as the ACC fluorogenic substrate for caspase-2. ACC fluorescence was monitored using wavelengths of 355 nm (excitation) and 460 nm (emission). The Ki parameter was calculated from the Morrison equation and the IC50 parameter was calculated using the equation: IC50 = Ki × (1 + [S] / Km), where [S] is the substrate concentration used in the assay and Km is the Michaelis-Menten constant of the substrate. All measurements were performed at least in triplicate and data were analyzed using GraphPad Prism 7 software. [Figure 4] Lipid accumulation in HepG2 cells. Intracellular lipid accumulation was measured as relative fluorescence (fluorescence 493, BODIPY / fluorescence 503, DAPI). Results are expressed as mean ± SEM (n=10). *p<0.0001 vs control **p<0.05, *** p<0.001 vs NASH [Diagram 5] Lipid accumulation in HepG2 cells. NH-23-C2 and compound 1 were used at 10, 20 and 25 mM. Intracellular lipid accumulation was measured as relative fluorescence (fluorescence 493, BODIPY / fluorescence 503, DAPI). Results are expressed as mean ± SEM (n=10). *p<0.0001 vs control, **p<0.05, *** p<0.001 vs NASH. [Figure 6]Substrate selectivity of recombinant caspase-2 tested against five fluorogenic substrates with the general formula: NH-Idc-hGlu-P3-Dab-Asp-ACC, where P3 is Thr(Bzl), Glu(Chx), Glu, or Val. The cleavage rate of the best substrate (expressed as RFU / sec, relative fluorescence units / sec) was set to 100% and the cleavage rates of the other substrates were adjusted accordingly. The substrate concentration was 10 μM and the caspase-2 concentration was 10 nM. [Figure 7] Substrate selectivity of recombinant caspase-2 tested against 25 fluorescent substrates with the general formula NH-Idc-P4-P3-Dab-Asp-ACC, where P4 is Asp, hGlu, Ile, Leu or hLeu, and P3 is Thr(Bzl), Glu(Chx), Glu, Val or Abu. Results are presented as bar graphs (variant 1) or heap maps (variant 2). The cleavage rate of the best substrate (expressed as RFU / s, relative fluorescence units / s) was set to 100% and the cleavage rates of the other substrates were adjusted accordingly. The substrate concentration was 10 μM and the caspase-2 concentration was 10 nM. [Figure 8] Analysis of subsite cooperativity of caspase-2 at P4 and P3 positions using five combinatorial fluorogenic substrates with an equimolar mixture of natural amino acids at the P3 position (NH-Idc-P4-Mix-Dab-Asp-ACC, left) and ten individual fluorogenic substrates with defined amino acids at the P3 position: glutamic acid-Glu (NH-Idc-P4-Glu-Dab-Asp-ACC, center) or glutamic acid cyclohexyl ester-Glu(Chx) (NH-Idc-P4-Glu(Chx)-Dab-Asp-ACC, right). The cleavage rate of the best substrate from each series (expressed as RFU / s, relative fluorescence units / s) was set to 100% and the cleavage rates of the other substrates in the series were adjusted accordingly. The concentration of the individual substrates was 10 μM and that of the combinatorial substrates was 100 μM. The caspase-2 concentration was 10 nM. Detailed Description of the Invention

[0025] In the context of the present invention, the following terms have the meanings detailed below.

[0026] "C 1-6 The term "alkyl" refers to a group that does not contain unsaturation and has 1 to 6, preferably 1 to 3, alkyl groups ("C 1-3 alkyl"), more preferably 1 or 2 ("C 1-2 "Alkyl" refers to a straight or branched hydrocarbon chain residue consisting of carbon and hydrogen atoms having 10 carbon atoms (e.g., 100 to 200 carbon atoms ...

[0027] "C 3-7 The term "cycloalkyl" refers to a saturated or partially saturated monocyclic or bicyclic aliphatic group having 3 to 7, preferably 3 to 6, carbon atoms attached to the remainder of the molecule by a single bond, including, for example and without limitation, cyclopropyl, cyclohexyl, or cyclopentyl.

[0028] "C 6-10 The term "aryl" refers to an aromatic group having 6 to 10, preferably 6 or 10, carbon atoms containing one or two aromatic nuclei including, for example and without limitation, phenyl, naphthyl, etc. Preferably, "aryl" refers to phenyl.

[0029] The term "halogen" refers to bromo, chloro, iodo or fluoro.

[0030] "C 1 -C 6 The term "haloalkyl" refers to an alkyl group in which at least one of the hydrogen atoms is replaced with a halogen atom, e.g., CF 3 , CCl 3 , CHF 2 , C.H. 2 F, C.F. 2 CF 3 "R" refers to an alkyl group as defined above replaced by "R" or "R";

[0031] "C 1-6The term "alkoxy" refers to 1-6 The alkyl group is as defined above, preferably C 1-3 Alkyl group of the formula -OC 1-6 Refers to an alkyl group. 1-6 Examples of alkoxyl include methoxy, ethoxy, propoxy, iso-propoxy, butoxy, tert-butoxy, iso-butoxy and sec-butoxy, preferably methoxy.

[0032] "(C 6 -C 10 )Aryl(C 1 -C 6 The term "alkyl" refers to an aryl group as defined above that is attached to the remainder of the molecule via an alkyl group as defined above. 6 -C 10 )Aryl(C 1 -C 6 )Alkyl is (C 6 )Aryl(C 1 -C 3 ) alkyl, for example benzyl.

[0033] "5-10 membered heterocyclyl" refers to a stable 5-10 membered ring group, preferably a 5 or 6 membered ring, which consists of carbon atoms and 1-5, preferably 1-4, heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and can be partially or fully saturated. For purposes of this invention, a heterocycle can be a monocyclyl or bicyclyl ring system. Examples of such heterocycles include, but are not limited to, pyrrolidine, piperidine, tetrahydropyridine, piperazine, morpholine, thiomorpholine, diazepane, tetrahydrofuran, tetrahydropyran, octahydro-pyrrolopyrazine.

[0034] "5-10 membered heteroaryl" refers to a stable 5-10 membered aromatic ring group, preferably a 5 or 6 membered aromatic ring, consisting of carbon atoms and 1 to 5, preferably 1 to 4, heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. For purposes of this invention, heteroaryl can be a monocyclyl or bicyclyl ring system. Examples of such heteroaryls include, but are not limited to, thiophene, furan, pyrrole, thiazole, oxazole, isothiazole, isoxazole, imidazole, pyrazole, triazole, oxadiazole, thiadiazole, tetrazole, tetrazole oxide, oxadiazolone, pyridine, pyrimidine, dihydroindolone, benzimidazole, benzothiazole, benzofuran, indole, purine, and quinoline.

[0035] As understood in the art, there may be some substitutions for the residues defined above. Thus, any group of the present invention may have substitutions. Reference to the substituents of the groups of the present invention in this document indicates that the particular residue may be substituted at one or more available positions by one or more substituents. The aforementioned substituents include, for example, in a non-limiting sense, halogen, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5-10 membered heterocyclyl, 5-10 membered heteroaryl, -CN, -NO 2 , -OR, -SR, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NR 2 , -NR 2 and -SO 2 R, where each R is H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-7 Cycloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl.

[0036] The compounds of the invention may be in the form of a salt, solvate or stereoisomer, preferably a pharma- ceutically acceptable salt, solvate or stereoisomer.

[0037] As already indicated, the present invention also provides the "salts" of the compounds described herein. By way of example, the aforementioned salts can be acid addition salts, base addition salts or metal salts, and can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical processes known to those skilled in the art. See generally GS Paulekuhn et al., "Trends in Active Pharmaceutical Ingredient Salt Selection based on Analysis of the Orange Book Database", J.Med.Chem., 2007, 50:6665-72, SM Berge et al., "Pharmaceutical Salts", J Pharm Sci., 1977, 66:1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth, Eds.Wiley-VCH and VHCA Zurich, 2002. Such salts are generally prepared by reacting the free acid or base form of the aforementioned compound with a stoichiometric amount of an appropriate base or acid, for example, in water or in an organic solvent or in a mixture of the two. Nonaqueous media like ether, ethyl acetate, ethanol, acetone, isopropanol, or acetonitrile are generally preferred.Illustrative examples of acid addition salts include inorganic acid addition salts, such as hydrochlorides, hydrobromides, hydroiodides, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, and the like; and organic acid addition salts, such as acetates, maleates, fumarates, citrates, oxalates, succinates, tartrates, malates, mandelates, methanesulfonates, p-toluenesulfonates, camphorsulfonates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, and the like. Examples of base addition salts include salts of inorganic bases, such as ammonium salts, and organic bases, such as ethylenediamine, ethanolamine, N,N-dialkyleneethanolamine, triethanolamine, glutamine, amino acid base salts, and the like. Examples of metal salts include, for example, sodium, potassium, calcium, magnesium, aluminum, and lithium salts.

[0038] The term "solvate" according to the present invention should be understood to mean any form of the active compound according to the present invention in which another molecule (possibly polar solvent) is bound via non-covalent bond.Examples of solvate include hydrate and alcoholate.Solvation methods are generally known in the state of the art.

[0039] As used herein, the term "stereoisomers" is a general term for all isomers of individual molecules that differ only in the orientation of their atoms in space. Thus, stereoisomeric compounds are molecules that are non-superimposable mirror images of one another and include enantiomers and diastereomers.

[0040] The term "chiral center" refers to a carbon atom to which four different groups are attached.

[0041] The terms "enantiomer" and "mirror" refer to one of two stereoisomers of a compound that are non-superimposable mirror images of each other. Enantiomeric compounds are optically active, with one enantiomer rotating the plane of polarized light in one direction and the other enantiomer rotating the plane of polarized light in the opposite direction, and when present in equal amounts, form a racemate.

[0042] The terms "racemate" or "racemate" refer to a mixture having equal amounts of enantiomers, wherein the mixture is optically inactive.

[0043] The terms "diastereomer" and "diastereomeric form" refer to stereoisomers of a compound with two or more chiral centers that are not mirror images of one another.

[0044] As will be understood, the terms enantiomerically enriched or diastereomerically enriched describe a mixture of two enantiomers or diastereomers in which one enantiomer or diastereomer is present in greater amount than the other enantiomer or diastereomer.

[0045] The compounds of the present invention have chiral centers and can therefore exist in different stereoisomeric forms, such as enantiomeric or diastereomeric forms.Therefore, any given compound referred to herein is intended to represent any one of racemic compounds, one or more enantiomeric forms, and one or more diastereomeric forms.All stereoisomers, including the enantiomers and diastereoisomers of the compounds referred to herein, and mixtures thereof, including racemic mixtures, enantiomerically enriched mixtures, and diastereomeric enriched mixtures, are considered to be within the scope of the present invention.

[0046] The term "pharmacologically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not normally cause allergic or similar adverse reactions when administered to humans, such as stomach upset, dizziness, etc. Preferably, as used herein, the term "pharmacologically acceptable" means approved by a government regulatory agency or listed in the United States Pharmacopoeia or another generally recognized pharmacopoeia for use in animals, and more specifically, in humans.

[0047] As used herein, "fluorophore" refers to a molecule or moiety that is capable of re-emitting light upon optical excitation.

[0048] As used herein, the term "warhead" refers to a moiety present on the compound of the present invention that can covalently bind to the active site of the target enzyme (caspase-2) and thereby achieve an irreversible inhibitory effect. Those skilled in the art will recognize that certain reactive functional groups can act as warheads. Note that "covalent binding" does not equal "irreversible inhibition". For example, aldehydes bind covalently but reversibly. Furthermore, some AOMKs can act as reversible inhibitors. 1) Brady KD. Bimodal inhibition of caspase-1 by aryloxymethyl and acyloxymethyl ketones.Biochemistry.1998;37:8508-15. 2) Brady KD, Giegel DA, Grinnell C, Lunney E, Talanian RV, Wong W et al. A catalytic mechanism for caspase-1 and for bimodal inhibition of caspase-1 by activated aspartic ketones.Bioorg Med Chem.1999;7:621-31.

[0049] Poreba et al., Caspase selective reagents for diagnosing apoptotic mechanisms, CDD 2019.

[0050] Compounds of the Invention In a first aspect, the present invention provides a compound of formula (I): P 5 -P 4 -P 3 -AA-P 1 -R 1 (I) The present invention relates to a compound of the formula:

[0051] Each of the elements P5, P4, P3, AA, P1 and R1 are described in detail throughout the present invention. Note that the present invention encompasses any combination of any of these six elements (subunits) within formula I above, and each of these elements is defined throughout the present specification.

[0052] Note that each subunit P5-P1 is linked to its neighboring subunit via a covalent peptide bond between the a-amino group of one amino acid subunit and the a-carboxyl group of the neighboring amino acid. In the case of substrates, R1 is linked to P1 via a covalent peptide bond between the amino group of the R1 subunit and the a-carboxyl group of the P1 amino acid. In the case of inhibitors, P1 is linked to R1 via a covalent bond between the a-carboxyl group of the P1 amino acid and the R1 moiety, which can be a number of moieties such as acyloxymethylketones (AOMK) or simple hydrogen atoms (in aldehydes) as shown throughout this specification.

[0053] 1.AA In any of the formulae of the present invention, AA, in particular of formula I, is 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5-10 membered heterocyclyl, 5-10 membered heteroaryl, -NO 2 , -C(O)R, -C(O)OR and -SO 2 R, wherein each R is H, C, 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl.

[0054] In a preferred embodiment, the optional substituents on AA are halogen, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6) alkyl, -C(O)R and -C(O)OR, where each R is selected from H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6- 10 aryl and (C6-10)aryl(C1-6)alkyl, for example, halogen, methyl, cyclohexyl, benzyl, -C(O)OMe, -C(O)OPh, and -C(O)OBn or -C(O)OBzl.

[0055] As used herein, the term "basic amino acid" refers to a naturally occurring or synthetic amino acid having a side chain capable of accepting a proton and becoming positively charged under physiological conditions.

[0056] Examples of basic amino acids according to the present invention include histidine (His), arginine (Arg), arginine homologs, lysine (Lys), acetyl lysine (Lys(Ac)), ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), 4-aminocyclohexyl-alanine (Aca), 4-amino-phenylalanine (Apa), aminomethylcyclohexyl-alanine (Ama), 4-aminomethyl-phenylalanine (Amp), 4-guanine, 4-aminomethyl-phenylalanine (Amp), ... 4-Pyridinyl-alanine (Gpa), 4-guanidine-cyclohexylalanine (Gca), citrulline (Cit), 4-piperidinyl-alanine (Pia), 3-(2-pyridyl)alanine (2-Pal), 3-(3-Pal)alanine (3-Pal), 3-(4-pyridyl)alanine (4-Pal), or homologs and / or beta derivatives thereof, which, if an acidic group is present, can exist as a deprotonated, negatively charged entity. [ka] [ka]

[0057] Notwithstanding the above, AA may also be represented by an amino acid in the form of a proline derivative selected from any of the formulas consisting of: [ka]

[0058] In certain aspects of the invention, acidic or basic residues may be present in the compounds. Those skilled in the art will understand that basic residues may be present in either the unprotonated form or the positively charged protonated form, and acidic residues may be present in either the deprotonated form or the non-deprotonated form, with the deprotonated form carrying a negative charge.

[0059] As used herein, the term "homolog" preferably refers to a methylene group (-CH 2 -) is added to or deleted from the side chain.

[0060] A β derivative refers to an amino acid in which the amino group is attached to the carbon β to the carboxyl group, instead of to the carbon adjacent to the carboxyl group.

[0061] In a preferred embodiment, AA is a compound of formula (II) or a salt, solvate or stereoisomer thereof: [ka] and During the ceremony, n is selected from 0, 1, 2, 3, 4, 5 and 6; m is selected from 0, 1, 2 and 3; p is selected from 0 and 1; X is absent or C 3-7 Cycloalkyl and C 6-10 aryl; Y is -NR a R b , [ka] where R a , R b and R c , H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5-10 membered heterocyclyl, 5-10 membered heteroaryl, -NO 2 , -C(O)R, -C(O)OR and -SO 2 R, where each R is selected from H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl.

[0062] According to a preferred embodiment of the invention, p is 0 and AA is of the formula: [ka] Based on wherein n, m, X and Y are as defined above.

[0063] According to one embodiment of the present invention, n is selected from 1, 2 and 3.

[0064] In preferred embodiments, m+n is 1, 2, 3, 4, 5 or 6, more preferably 1, 2, 3, 4 and 5.

[0065] Preferably, X is absent or selected from cyclohexyl and phenyl.

[0066] According to another embodiment, AA is of the formula [ka] Based on n is selected from 0, 1, 2, 3, 4, 5 and 6; p is selected from 0 and 1; Y is -NR a R b , [ka] where R a , R b and R c , H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5-10 membered heterocyclyl, 5-10 membered heteroaryl, -NO 2 , -C(O)R, -C(O)OR and -SO 2 R, where each R is selected from H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl.

[0067] According to one embodiment of the present invention, n is selected from 1, 2, 3, 4 and 5.

[0068] In a preferred embodiment, p is 0.

[0069] According to one embodiment of the present invention, R a , R b and R c , H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6) alkyl, -C(O)R and -C(O)OR, where each R is H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl and (C 6-10 )Aryl(C 1-6 ) alkyl. In certain embodiments, R a , R b and R c is independently selected from H, methyl, cyclohexyl, benzyl, -C(O)OMe, -C(O)OPh, or -C(O)OBn. a , R b and R c is H.

[0070] In one embodiment, AA is an optionally substituted amino acid selected from histidine (His), arginine (Arg), lysine (Lys), ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), 4-aminocyclohexyl-alanine (Aca), 4-amino-phenylalanine (Apa), aminomethylcyclohexyl-alanine (Ama), 4-aminomethyl-phenylalanine (Amp), 4-guanidine-phenylalanine (Gpa), 4-guanidine-cyclohexylalanine (Gca), citrulline (Cit), 4-piperidinyl-alanine (Pia), 3-(2-pyridyl)alanine (2-Pal), 3-(3-pyridyl)alanine (3-Pal), 3-(4-pyridyl)alanine (4-Pal), or homologs and / or beta derivatives thereof.

[0071] Note further that AA may also be Ser, as described below in section "P5."

[0072] In a preferred embodiment, AA is an amino acid selected from histidine (His), arginine (Arg), lysine (Lys), ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), 4-aminocyclohexyl-alanine (Aca), 4-amino-phenylalanine (Apa), aminomethylcyclohexyl-alanine (Ama), 4-aminomethyl-phenylalanine (Amp), 4-guanidine-phenylalanine (Gpa), 4-guanidine-cyclohexylalanine (Gca), citrulline (Cit), 4-piperidinyl-alanine (Pia), 3-(2-pyridyl)alanine (2-Pal), 3-(3-pyridyl)alanine (3-Pal), 3-(4-pyridyl)alanine (4-Pal), or homologs and / or beta derivatives thereof, which may be selected from the group consisting of halogen, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) Alkyl, 5-10 membered heterocyclyl, 5-10 membered heteroaryl, -CN, -NO 2 , -OR, -SR, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NR 2 , -NR 2 and -SO 2 R, wherein each R is H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-7 Cycloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl. Preferably, halogen, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, -C(O)R and -C(O)OR, where each R is H, C1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl. More preferably, halogen, methyl, cyclohexyl, benzyl, -C(O)OMe, -C(O)OPh and -C(O)OBn.

[0073] AA may be substituted with one or more groups as defined above, preferably with one, two or three groups, more preferably with one or two groups, even more preferably with one group.

[0074] Preferably, when AA is substituted, it is N-substituted, i.e., substituted at one or more of the nitrogen atoms present in the side chain of the amino acid.

[0075] In one embodiment, AA is C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5-10 membered heterocyclyl, 5-10 membered heteroaryl, -NO 2 , -C(O)R, -C(O)OR and -SO 2 R, wherein each R is selected from H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl. 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6) alkyl, -C(O)R and -C(O)OR, where each R is H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl. More preferably, halogen, methyl, cyclohexyl, benzyl, -C(O)OMe, -C(O)OPh and -C(O)OBn.

[0076] 2.P5 P5 is preferably a compound within formula I of the following formula (III): [ka] and In the formula, A, B, C and D are H, halogen, OR', NHR', C 1-6 Alkyl, C 3-7 Cycloalkyl, -OR', -SR', -OC(O)R', -C(O)R', -C(O)OR' and -C(O)NR' 2 where each R' is independently selected from H, C 1-6 Alkyl and C 3-7 cycloalkyl, one of A, B, C or D represents a carboxamide group in the form of a peptide bond linking P5 to the remainder of the final molecule (position P4) as shown in formula (I); wherein H, I, J and K are independently carbon or nitrogen, preferably carbon atoms.

[0077] TIFF2024524493000011.tif10170 (also referred to herein as the dotted line) represents a bond that may or may not be present. If present, it combines with an already existing single bond to form a double bond. Note that when a double bond is formed in combination with an already existing single bond, AA can be Ser (serine), among other optional further options described in section "AA" above. It should be noted that further examples of P5 are derived from any of the following Idc derivatives selected from the group consisting of: [ka]

[0078] Such derivatives are linked to P4 by formation of a peptide bond from a carboxylic acid present in each derivative of P5 to the a-amine group of P4.

[0079] 3.P4 P4 is represented by the following formula (IV): [ka] is a compound within formula I: wherein a is selected from 0, 1, 2, 3, 4, 5 and 6; Y and X are H, fluorine, C 1-6 Alkyl, C 3-7 cycloalkyl, -OR', -SR', -OC(O)R', -C(O)R', -C(O)OR' and -C(O)NR'2, where each R' is H, C 1-6 Alkyl and C 3-7 cycloalkyl; R2 and R3 are H, C 1-3 Alkyl and C 3-7 independently selected from cycloalkyl or -CO(OR9); W is S or absent, and if W is absent, -CH 2 (R2R3) group is -CH 2 is directly linked to the (XY) group, Z is NH or CH 2 and b is 0 or 1; C is 0 or 1; R9 is H, C 1-6 Alkyl or C 3-7 R9 is also R 2 and R3 may be absent such that it is in the deprotonated form CO(O-).

[0080] 4.P1 P1 is a compound represented by the formula (V): [ka] is a compound within formula I: wherein a is selected from 0, 1, 2, 3, 4, 5 and 6; X and Y are H, fluorine, C 1-6 Alkyl, C 3-7 cycloalkyl, -OR', -SR, -OC(O)R', -C(O)R', -C(O)OR' and -C(O)NR'2, where each R' is H, C 1-6 Alkyl and C 3-7 cycloalkyl; b is 0 or 1; R 2 , H, C 1-6 Alkyl and C 3-7 R2 may also be absent such that -CO(OR2) is in the deprotonated form CO(O-).

[0081] On the other hand, and in an alternative embodiment, we further present herein a scheme for the synthesis of Asp (aspartic acid) masked inhibitors. [ka]

[0082] Thus, P1 may be a compound within formula I of the alternative formula (V) as follows: [ka] may be X and Y are H, fluorine, C 1-6 Alkyl, C 3-7 cycloalkyl, -OR', -SR, -OC(O)R', -C(O)R', -C(O)OR' and -C(O)NR'2, where each R' is H, C 1-6Alkyl and C 3-7 cycloalkyl; b is 0 or 1; R* is a chemical group selected from hydrogen, methyl, or ethyl that is the target of esterases, hydrolytic enzymes that break down esters into acids and alcohols in a chemical reaction with water called hydrolysis. Note that in this particular alternative, the R1 subunit as described in formula (I) is not present since the structure has been replaced with R*.

[0083] 5.P3 P3 is a glutamic acid cyclohexyl ester, Glu (especially L-Glu), Thr(Bzl) (threonine benzyl ether) or a Glu analogue such as L-Glu(o-Me), L-Glu(o-CHX) or L-Glu(o-Bzl) or a compound of formula (VI): [ka] and a derivative selected from the compounds In the formula, R 4 ~R 6 is H, fluorine, hydroxyl (-OH), -SH or C 1-6 alkyl, preferably methyl; R 7 and R 8 are independently selected from the group consisting of H, fluorine and a methyl group.

[0084] In particular, P3 is selected from glutamic acid cyclohexyl ester (Glu(Chx)), L-glutamic acid methyl ester (Glu(me)), aminobutyric acid (Abu), Thr(Bzl) (threonine benzyl ether), or any naturally occurring amino acid selected from the group consisting of Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Glu (glutamic acid), Gln (glutamine), Gly (glycine), His (histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Nle (norleucine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine) and Val (valine).

[0085] 6.R1 R1 is preferably a chemically reactive group. As described in Gehringer et al. (J.Med.Chem.2019,62,(12)5673-5724), incorporating a chemically reactive group into a drug molecule can confer certain advantages, especially in the field of enzyme inhibition, where the group can form a covalent bond between the drug and the enzyme, thereby enhancing the inhibition profile. R1 can be a fluorophore (used in conjunction with a substrate) or a warhead (used in conjunction with an inhibitor).

[0086] Note that in the case of the substrates exemplified below (such as those containing a fluorophore), R1 is linked to the P1 subunit via a covalent peptide bond between the amino group (or any other group shown as W) of the R1 subunit and the a-carboxyl group of the P1 amino acid. Preferably, R1 has the formula: [ka] The moiety is a fluorophore such as ACC.

[0087] More specifically, R 1 is the expression: [ka] and a moiety such as a fluorophore (used in connection with a substrate) selected from the group During the ceremony, W is selected from -NH- and -O-; R10 to R13 are H and C 1-6 Alkyl and C 3-7 Cycloalkyl, aryl, heteroaryl, CF 3 , -CH 2 COOH, -CH 2 CONHR14, and CH 2 Independently selected from OR14, R14 is selected from H, C1-6 alkyl, and C3-7 cycloalkyl.

[0088] Examples of R1 moieties as fluorophores can be selected from any of the following lists: [ka]

[0089] Other examples of moieties potentially useful as fluorophores can be selected from the group consisting of: [ka]

[0090] In the context of the present invention, a fluorophore (or fluorescent dye, similar to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores or substrates typically contain several bound aromatic groups, or planar or cyclic molecules with several π bonds. Fluorophores are particularly used to stain tissues, cells or materials in various analytical methods, namely fluorescence imaging and spectroscopy, where W in the above formula will represent -NH-, such as ACC. R 1Additional fluorophores in the range are: Indo-1, Ca-saturated Indo-1 Ca2+ Cascade Blue BSA pH 7.0 Cascade Blue LysoTracker Blue Alexa 405 LysoSensor Blue pH 5.0 LysoSensor Blue DyLight 405 DyLight 350 BFP (blue fluorescent protein) Alexa 350 7-Amino-4-methylcoumarin pH 7.0, Aminocoumarin AMCA conjugate coumarin 7-Hydroxy-4-methylcoumarin 7-Hydroxy-4-methylcoumarin pH 9.0 6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0 Hoechst 33342 Pacific Blue Hoechst 33258 Hoechst 33258-DNA Pacific Blue antibody conjugate pH 8.0 PO-PRO-1PO-PRO-1-DNA POPO-1 433nm POPO-1-DNA DAPI-DNA DAPI Marina Blue SYTOX Blue-DNA CFP (cyan fluorescent protein) eCFP (enhanced cyan fluorescent protein) 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) Indo-1, Ca-free 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid) BO-PRO-1-DNA BOPRO-1 BOBO-1-DNA SYTO 45-DNA evoglow-Pp1 evoglow-Bs1 evoglow-Bs2 Auramine O DiO LysoSensor Green pH 5.0 Cy2 LysoSensor Green Fura-2, high Ca Fura-2 Ca2 SYTO 13-DNA YO-PRO-1-DNA YOYO-1-DNA eGFP (enhanced green fluorescent protein) LysoTracker Green GFP (S65T) BODIPY FL, MeOH Sapphire BODIPY FL conjugate MitoTracker Green MitoTracker Green FM Fluorescein 0.1M NaOH Calcein pH 9.0 Fluorescein pH 9.0 Calcein Fura-2, Ca-free Fluo-4 FDA DTAF Fluorescein Fluorescein antibody conjugate pH 8.0 CFDA FITC Alexa Fluor 488 hydrazide-water DyLight 488 5-FAM pH 9.0 FITC antibody conjugate pH 8.0 Alexa 488 Rhodamine 110 Rhodamine 110 pH 7.0 Acridine orange Alexa Fluor 488 antibody conjugate pH 8.0 BCECF pH 5.5 PicoGreen dsDNA quantification reagent SYBR Green I Rhodamine Green pH 7.0 CyQUANT GR-DNA NeuroTrace 500 / 525, green fluorescent Nissl stain RNA DansylCadaverine Rhodol green antibody conjugate pH 8.0 Fluoro-Emeral Nissl Fluorescein dextran pH 8.0 Rhodamine green 5-(and-6)-carboxy-2',7'-dichlorofluorescein pH 9.0 DansylCadaverine, MeOH eYFP (enhanced green fluorescent protein) Oregon Green 488 Oregon Green 488 antibody conjugate pH 8.0 Fluo-3 BCECF pH 9.0 SBFI-Na+ Indo-1, Ca2+ saturated Indo-1 Ca2+ Cascade Blue BSA pH 7.0 Cascade Blue LysoTracker Blue Alexa 405 LysoSensor Blue pH 5.0 LysoSensor Blue DyLight 405 DyLight 350 BFP (Blue Fluorescent Protein) Alexa 350 7-Amino-4-methylcoumarin pH 7.0, Aminocoumarin AMCA conjugate coumarin 7-Hydroxy-4-methylcoumarin 7-Hydroxy-4-methylcoumarin pH 9.0 6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0 Hoechst 33342 Pacific Blue Hoechst 33258 Hoechst 33258-DNA Pacific Blue antibody conjugate pH 8.0 PO-PRO-1PO-PRO-1-DNA POPO-1 433nm POPO-1-DNA DAPI-DNA DAPI Marina Blue SYTOX Blue-DNA CFP (cyan fluorescent protein) eCFP (enhanced cyan fluorescent protein) 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) Indo-1, Ca-free 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid) BO-PRO-1-DNA BOPRO-1 BOBO-1-DNA SYTO 45-DNA evoglow-Pp1 evoglow-Bs1 evoglow-Bs2 Auramine O DiO LysoSensor Green pH5.0 Cy2 LysoSensor Green LysoSensor Yellow pH9.0 Indo-1, Ca2+ saturated Indo-1 Ca2+ Cascade Blue BSA pH 7.0 Cascade Blue LysoTracker Blue Alexa 405 LysoSensor Blue pH 5.0 LysoSensor Blue DyLight 405 DyLight 350 BFP (Blue Fluorescent Protein) Alexa 350 7-Amino-4-methylcoumarin pH 7.0, Aminocoumarin AMCA conjugate coumarin 7-Hydroxy-4-methylcoumarin 7-Hydroxy-4-methylcoumarin pH 9.0 6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0 Hoechst 33342 Pacific Blue Hoechst 33258 Hoechst 33258-DNA Pacific Blue antibody conjugate pH 8.0 PO-PRO-1PO-PRO-1-DNA POPO-1 433nm POPO-1-DNA DAPI-DNA DAPI Marina Blue SYTOX Blue-DNA Choose from common fluorophores including CFP (cyan fluorescent protein) eCFP (enhanced cyan fluorescent protein) 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) Indo-1, Ca-free 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid) BO-PRO-1-DNA BOPRO-1 BOBO-1-DNA SYTO 45-DNA evoglow-Pp1 evoglow-Bs1 evoglow-Bs2 Auramine O DiO LysoSensor Green pH 5.0 Cy2 LysoSensor Green.

[0091] On the other hand, in the case of inhibitors, P1 is linked to R1 via a covalent bond between the a-carboxyl group of the P1 amino acid and the R1 moiety, which can be a number of moieties such as acyloxymethylketones (AOMK) or a simple hydrogen atom (in an aldehyde).

[0092] Thus, in another embodiment, R1 may be a warhead, and in particular, R1 may be [ka] or a salt, solvate or stereoisomer thereof; During the ceremony, q is 0, 1, 2, 3, 4 or 5; each Z 1 is C 1-6 Alkyl, halogen, -CN, -NO 2 and C 1-6 independently selected from alkoxyl, Z 2 is a halogen, Z 3 is C 1-6 Alkyl, C 6-10 Aryl and (C 6-10 )Aryl(C 1-6 ) alkyl.

[0093] Specific examples of warheads useful in the present invention can be selected from any of the following (all of the following compounds are directly attached to the a-COOH group of the P1 subunit): [ka] TIFF2024524493000024.tif229170

[0094] Preferably, R1 has the formula: [ka] The warhead of During the ceremony, q is 0, 1, 2, 3 or 4; each Z 1 is C 1-6 Alkyl, halogen, -CN and C 1-6 alkoxyl.

[0095] In one embodiment, Z 1 are independently selected from halogen, methyl and -OMe, preferably F, Cl and Me (methyl).

[0096] In a further embodiment, R 1 is the formula [ka] is selected from the group In the formula, each Z 1 is C 1-6 Alkyl, halogen, -CN and C 1-6 It is independently selected from alkoxyl, preferably from halogen, methyl and -OMe, more preferably from F, Cl and Me (methyl).

[0097] In a preferred embodiment, R 1 is the formula [ka] has.

[0098] In the following, the formula (I): P 5 -P 4 -P 3 -AA-P 1 -R 1 Compound or a salt, solvate or stereoisomer thereof Reference will now be made to specific embodiments of the invention, including but not limited to the following:

[0099] In a preferred embodiment, the compound of formula (I) is characterized in that AA is as defined above, preferably AA is of formula (II): [ka] This refers to the compound During the ceremony, n is selected from 0, 1, 2, 3, 4, 5 and 6; m is selected from 0, 1, 2 and 3; p is selected from 0 and 1; X is absent or C 3-7 Cycloalkyl and C 6-10 aryl; Y is -NRa R b , [ka] where R a , R b and R c , H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5-10 membered heterocyclyl, 5-10 membered heteroaryl, -NO 2 , -C(O)R, -C(O)OR and -SO 2 R, where each R is selected from H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl.

[0100] According to a preferred embodiment of the invention, p is 0 and AA is of the formula: [ka] Based on wherein n, m, X and Y are as defined above.

[0101] According to one embodiment of the present invention, n is selected from 1, 2 and 3.

[0102] In preferred embodiments, m+n is 1, 2, 3, 4, 5 or 6, more preferably 1, 2, 3, 4 and 5.

[0103] Preferably, X is absent or selected from cyclohexyl and phenyl.

[0104] According to another embodiment, AA is of the formula [ka] Based on n is selected from 0, 1, 2, 3, 4, 5 and 6; p is selected from 0 and 1; Y is -NR a R b , [ka] where R a , R b and R c , H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5-10 membered heterocyclyl, 5-10 membered heteroaryl, -NO 2 , -C(O)R, -C(O)OR and -SO 2 R, where each R is selected from H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl.

[0105] According to one embodiment of the present invention, n is selected from 1, 2, 3, 4 and 5.

[0106] In a preferred embodiment, p is 0.

[0107] According to one embodiment of the present invention, R a , R b and R c , H, C 1-6 Alkyl, C 3-7Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, -C(O)R and -C(O)OR, where each R is H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl and (C 6-10 )Aryl(C 1-6 ) alkyl. In certain embodiments, R a , R b and R c is independently selected from H, methyl, cyclohexyl, benzyl, -C(O)OMe, -C(O)OPh, or -C(O)OBn. a , R b and R c is H.

[0108] In one embodiment, AA is an optionally substituted amino acid selected from histidine (His), arginine (Arg), an arginine homolog, lysine (Lys), acetyl lysine, ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), 4-aminocyclohexyl-alanine (Aca), 4-amino-phenylalanine (Apa), aminomethylcyclohexyl-alanine (Ama), 4-aminomethyl-phenylalanine (Amp), 4-guanidine-phenylalanine (Gpa), 4-guanidine-cyclohexylalanine (Gca), citrulline (Cit), 4-piperidinyl-alanine (Pia), 3-(2-pyridyl)alanine (2-Pal), 3-(3-pyridyl)alanine (3-Pal), 3-(4-pyridyl)alanine (4-Pal), or homologs and / or beta derivatives thereof.

[0109] In a more preferred embodiment, AA is histidine (His), arginine (Arg), lysine (Lys), ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), 4-aminocyclohexyl-alanine (Aca), 4-amino-phenylalanine (Apa), aminomethylcyclohexyl-alanine (Ama), 4-aminomethyl-phenylalanine (Amp), 4-guanidine-phenylalanine (Gpa), 4-guanidine-cyclohexylalanine (Gca), citrulline (Cit), 4-piperidinyl-alanine (Pia), 3-(2-pyridyl)alanine (2-Pal), 3-(3-pyridyl)alanine (3-Pal), 3-(4-pyridyl)alanine (4-Pal), or homologs thereof and and / or beta derivatives, which are optionally substituted with at least one group selected from halogen, C1-6 alkyl, C3-7 cycloalkyl, C1-6 haloalkyl, C6-10 aryl, (C6-10)aryl(C1-6)alkyl, 5-10 membered heterocyclyl, 5-10 membered heteroaryl, -CN, -NO2, -OR, -SR, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NR2, -NR2 and -S02R, where each R is independently selected from H, C1-6 alkyl, C1-6 haloalkyl, C3-7 cycloalkyl, C6-10 aryl, (C6-10)aryl(C1-6)alkyl, 5-10 membered heterocyclyl, and 5-10 membered heteroaryl. Preferred are halogen, C1-6 alkyl, C3-7 cycloalkyl, C1-6 haloalkyl, C6-10 aryl, (C6-10)aryl(C1-6)alkyl, -C(O)R and -C(O)OR, where each R is independently selected from H, C1-6 alkyl, C3-7 cycloalkyl, C1-6 haloalkyl, C6-10 aryl, (C6-10)aryl(C1-6)alkyl. More preferred are halogen, methyl, cyclohexyl, benzyl, -C(O)OMe, -C(O)OPh and -C(O)OBn.

[0110] More preferably, AA is an amino acid selected from lysine (Lys), ornithine (Orn), diaminobutyric acid (Dab) or diaminopropionic acid (Dap); P5 is represented by the formula (III): [ka] is a compound of In the formula, A and B are hydrogen atoms, and H, I, J and K are carbon atoms; TIFF2024524493000034.tif11170 (also referred to herein as the dotted line) represents a bond that may or may not be present. If present, it combines with an already present single bond to form a double bond. P4 is selected from the group consisting of isoleucine, leucine, homoleucine, Glu, hGlu (homoglutamic acid) and Asp, in some cases, the amino acid has an acidic group in the side chain, in which case any of these amino acid residues may be in deprotonated or protonated form; P1 is Asp or aspartic acid, which amino acid residue may be in deprotonated or protonated form; P3 is Thr(Bzl) (threonine benzyl ether) or a compound of formula (VI): [ka] and derivatives thereof selected from In the formula, R 4 ~R 6 can be independently selected from H, F or methyl; R 7 and R 8is independently selected from the group consisting of H, F and a methyl group. Alternatively, P3 can be selected from any naturally occurring amino acid selected from the group consisting of glutamic acid cyclohexyl ester (Glu(Chx)), L-glutamic acid methyl ester (Glu(me)), aminobutyric acid (Abu), or Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Glu (glutamic acid), Gln (glutamine), Gly (glycine), His (histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Nle (norleucine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine) and Val (valine); R1 is: [ka] or a salt, solvate or stereoisomer thereof, During the ceremony, q is 0, 1, 2, 3, 4 or 5; each Z 1 is C 1-6 Alkyl, halogen, -CN, -NO 2 and C 1-6 independently selected from alkoxyl, Z 2 is a halogen, Z 3 is C 1-6 Alkyl, C 6-10 Aryl and (C 6-10 )Aryl(C 1-6 ) alkyl. Preferably, R1 has the formula: [ka] The warhead of During the ceremony, q is 0, 1, 2, 3 or 4; each Z 1 is C 1-6 Alkyl, halogen, -CN and C1-6 alkoxyl.

[0111] In one embodiment, Z 1 are independently selected from halogen, methyl and -OMe, preferably F, Cl and Me.

[0112] In a further embodiment, R 1 is the formula [ka] is selected from the group In the formula, each Z 1 is C 1-6 Alkyl, halogen, -CN and C 1-6 It is independently selected from alkoxyl, preferably halogen, methyl and -OMe, more preferably F, Cl and Me.

[0113] In a preferred embodiment, R 1 is the formula [ka] (Me is a methyl group).

[0114] More specifically, R 1 is the expression: [ka] and a moiety such as a fluorophore (used in connection with a substrate) selected from the group During the ceremony, W is selected from -NH- and -O-; R10 to R13 are H and C 1-6 Alkyl and C 3-7 Cycloalkyl, aryl, heteroaryl, CF 3 , -CH 2 COOH, -CH 2 CONHR14, and CH 2 Independently selected from OR14, R14 is H, C 1-6Alkyl and C 3-7 cycloalkyl.

[0115] In another preferred embodiment, R1 is ACC.

[0116] In a preferred embodiment, the compound of formula (I) has formula VII: [ka] Compound or a salt, solvate or stereoisomer thereof, During the ceremony, - n is 1, 2, 3 or 4, -A is -NR a R b , [ka] where R a , R b and R c , H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5-10 membered heterocyclyl, 5-10 membered heteroaryl, -NO 2 , -C(O)R, -C(O)OR and -SO 2 R, where each R is selected from H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) independently selected from alkyl, 5- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl, preferably A is selected from the group consisting of -COOH or -NH2; - P1, P3, P4, P5 and R1 are as defined in any of the above embodiments, and preferably each of these compounds is characterized as follows: P5 is represented by the formula (III): [ka] is a compound of In the formula, A and B are hydrogen atoms, and H, I, J and K are independently carbon or nitrogen atoms; TIFF2024524493000044.tif10170 (also referred to herein as a dotted line) represents a bond that may or may not be present, and if present, it combines with an already present single bond to form a double bond; P4 is selected from the group consisting of isoleucine, leucine, homoleucine, hGlu (homoglutamic acid), Glu, norleucine (Nle), threonine (Thr), tyrosine (Tyr), valine (Val), norvaline (Nva), tert-leucine (Tle), and aspartic acid or Asp, in some cases, the amino acid has an acidic group in the side chain, in which case any of these amino acid residues may be in deprotonated or protonated form; P1 is Asp or aspartic acid, which amino acid residue may be in the deprotonated or protonated form; P3 is Thr(Bzl) (threonine benzyl ether) or a group of formula (VI): [ka] and derivatives thereof selected from the compounds In the formula, R 4 ~R 6 can be independently selected from H, F or methyl; R 7 and R 8is independently selected from the group consisting of H, F and a methyl group. Alternatively, P3 is selected from glutamic acid cyclohexyl ester (Glu(Chx)), L-glutamic acid methyl ester (Glu(me)), aminobutyric acid (Abu), or any naturally occurring amino acid selected from the group consisting of Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Glu (glutamic acid), Gln (glutamine), Gly (glycine), His (histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Nle (norleucine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine) and Val (valine); R1 is: [ka] or a salt, solvate or stereoisomer thereof, During the ceremony, q is 0, 1, 2, 3, 4 or 5; each Z 1 is C 1-6 Alkyl, halogen, -CN, -NO 2 and C 1-6 independently selected from alkoxyl, Z 2 is a halogen, Z 3 is C 1-6 Alkyl, C 6-10 Aryl and (C 6-10 )Aryl(C 1-6 ) alkyl. Preferably, R1 has the formula: [ka] The warhead of During the ceremony, q is 0, 1, 2, 3 or 4; each Z 1 is C 1-6 Alkyl, halogen, -CN and C1-6 alkoxyl.

[0117] In one embodiment, Z 1 are independently selected from halogen, methyl and -OMe, preferably F, Cl and Me.

[0118] In a further embodiment, R 1 is the formula [ka] is selected from the group In the formula, each Z 1 is C 1-6 Alkyl, halogen, -CN and C 1-6 It is independently selected from alkoxyl, preferably halogen, methyl and -OMe, more preferably F, Cl and Me.

[0119] In a preferred embodiment, R 1 is the formula [ka] The present invention has or consists solely of:

[0120] More specifically, R 1 is the expression: [ka] and a moiety such as a fluorophore (used in connection with a substrate) selected from the group During the ceremony, W is selected from -NH- and -O-; R10 to R13 are H and C 1-6 Alkyl and C 3-7 Cycloalkyl, aryl, heteroaryl, CF 3 , -CH 2 COOH, -CH 2 CONHR14, and CH 2 Independently selected from OR14, R14 is H, C 1-6Alkyl and C 3-7 cycloalkyl.

[0121] In another preferred embodiment, R1 is ACC.

[0122] In a preferred embodiment, the compound of formula (I) has formula VII: [ka] Compound or a salt, solvate or stereoisomer thereof, During the ceremony, - n is 2, 3 or 4, -A is -NR a R b group, preferably an amine group, P5 is represented by the formula (III): [ka] is a compound of wherein A and B are hydrogen atoms, and H, I, J and K are independently carbon atoms; TIFF2024524493000053.tif9170 represents a bond that may or may not be present, and if present, it combines with an already present single bond to form a double bond, P4 is selected from the group consisting of isoleucine, leucine, homoleucine, hGlu (homoglutamic acid), Glu, and Asp or aspartic acid, in some cases, the amino acid has an acidic group in the side chain, in which case any of these amino acid residues may be in deprotonated or protonated form; P1 is Asp or aspartic acid, which amino acid residue can be in the protonated or deprotonated form; P3 is Thr(Bzl) (threonine benzyl ether) or a group of formula (VI): [ka] and derivatives thereof selected from the compounds In the formula, R 4 ~R 6 can be independently selected from H, F or methyl; R 7 and R 8 is independently selected from the group consisting of H, F and a methyl group. Alternatively, P3 is selected from glutamic acid cyclohexyl ester (Glu(Chx)), L-glutamic acid methyl ester (Glu(me)), aminobutyric acid (Abu), or any naturally occurring amino acid selected from the group consisting of Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Glu (glutamic acid), Gln (glutamine), Gly (glycine), His (histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Nle (norleucine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine) and Val (valine); R1 is a formula [ka] is selected from the group In the formula, each Z 1 is C 1-6 Alkyl, halogen, -CN and C 1-6 It is independently selected from alkoxyl, preferably halogen, methyl and -OMe, more preferably F, Cl and Me.

[0123] In one embodiment, R 1 is the formula [ka] Or have ACC.

[0124] In a preferred embodiment, the compound of formula (I) has formula VIII: [ka] Compound or a salt, solvate or stereoisomer thereof, During the ceremony, R2 and R3 are independently H, C1-6 alkyl, or C3-7 cycloalkyl, where R2 and / or R 3 may be absent to result in the deprotonated form CO(O-), TIFF2024524493000058.tif9170 (dotted line) represents a bond that may or may not be present; if present, it combines with an already present single bond to form a double bond; In the formula, AA is an optionally N-substituted basic amino acid as defined above, in particular AA is of formula (II): [ka] or a salt, solvate or stereoisomer thereof, During the ceremony, n is selected from 0, 1, 2, 3, 4, 5 and 6; m is selected from 0, 1, 2 and 3; p is selected from 0 and 1; X is not present or C 3-7 Cycloalkyl and C 6-10 aryl; Y is -NR a R b , [ka] where R a , R b and R c , H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5-10 membered heterocyclyl, 5-10 membered heteroaryl, -NO 2 , -C(O)R, -C(O)OR and -SO 2 R, where each R is selected from H, C 1-6 Alkyl, C3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl.

[0125] According to a preferred embodiment of the invention, p is 0 and AA is of the formula: [ka] Based on wherein n, m, X and Y are as defined above.

[0126] According to one embodiment of the present invention, n is selected from 1, 2 and 3.

[0127] In preferred embodiments, m+n is 1, 2, 3, 4, 5 or 6, more preferably 1, 2, 3, 4 and 5.

[0128] Preferably, X is absent or selected from cyclohexyl and phenyl.

[0129] According to another embodiment, AA is of the formula [ka] Based on n is selected from 0, 1, 2, 3, 4, 5 and 6; p is selected from 0 and 1; Y is -NR a R b , [ka] where R a , R b and R c , H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5-10 membered heterocyclyl, 5-10 membered heteroaryl, -NO 2 , -C(O)R, -C(O)OR and -SO 2 R, where each R is selected from H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl.

[0130] According to one embodiment of the present invention, n is selected from 1, 2, 3, 4 and 5.

[0131] In a preferred embodiment, p is 0.

[0132] According to one embodiment of the present invention, R a , R b and R c , H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, -C(O)R and -C(O)OR, where each R is H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl and (C 6-10 )Aryl(C 1-6 ) alkyl. In certain embodiments, R a , R b and R c is independently selected from H, methyl, cyclohexyl, benzyl, -C(O)OMe, -C(O)OPh, or -C(O)OBn. a , Rb and R c is H.

[0133] In one embodiment, AA is an optionally substituted amino acid selected from histidine (His), an arginine homolog, lysine (Lys), acetyl lysine, ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), 4-aminocyclohexyl-alanine (Aca), 4-amino-phenylalanine (Apa), aminomethylcyclohexyl-alanine (Ama), 4-aminomethyl-phenylalanine (Amp), 4-guanidine-phenylalanine (Gpa), 4-guanidine-cyclohexylalanine (Gca), citrulline (Cit), 4-piperidinyl-alanine (Pia), 3-(2-pyridyl)alanine (2-Pal), 3-(3-pyridyl)alanine (3-Pal), 3-(4-pyridyl)alanine (4-Pal), or homologs and / or beta derivatives thereof.

[0134] In a more preferred embodiment, AA is histidine (His), arginine (Arg), lysine (Lys), ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), 4-aminocyclohexyl-alanine (Aca), 4-amino-phenylalanine (Apa), aminomethylcyclohexyl-alanine (Ama), 4-aminomethyl-phenylalanine (Amp), 4-guanidine-phenylalanine (Gpa), 4-guanidine-cyclohexylalanine (Gca), citrulline (Cit), 4-piperidinyl-alanine (Pia), 3-(2-pyridyl)alanine (2-Pal), 3-(3-pyridyl)alanine (3-Pal), 3-(4-pyridyl)alanine (4-Pal), or homologs thereof and and / or beta derivatives, which are optionally substituted with at least one group selected from halogen, C1-6 alkyl, C3-7 cycloalkyl, C1-6 haloalkyl, C6-10 aryl, (C6-10)aryl(C1-6)alkyl, 5-10 membered heterocyclyl, 5-10 membered heteroaryl, -CN, -NO2, -OR, -SR, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NR2, -NR2 and -S02R, where each R is independently selected from H, C1-6 alkyl, C1-6 haloalkyl, C3-7 cycloalkyl, C6-10 aryl, (C6-10)aryl(C1-6)alkyl, 5-10 membered heterocyclyl, and 5-10 membered heteroaryl. Preferred are halogen, C1-6 alkyl, C3-7 cycloalkyl, C1-6 haloalkyl, C6-10 aryl, (C6-10)aryl(C1-6)alkyl, -C(O)R and -C(O)OR, where each R is independently selected from H, C1-6 alkyl, C3-7 cycloalkyl, C1-6 haloalkyl, C6-10 aryl, (C6-10)aryl(C1-6)alkyl. More preferred are halogen, methyl, cyclohexyl, benzyl, -C(O)OMe, -C(O)OPh and -C(O)OBn.

[0135] More preferably, AA is an amino acid selected from lysine (Lys), ornithine (Orn), diaminobutyric acid (Dab) or diaminopropionic acid (Dap); R1 is, [ka] or a salt, solvate or stereoisomer thereof, During the ceremony, q is 0, 1, 2, 3, 4 or 5; each Z 1 is C 1-6 Alkyl, halogen, -CN, -NO 2 and C 1-6 independently selected from alkoxyl, Z 2 is a halogen, Z 3 is C 1-6 Alkyl, C 6-10 Aryl and (C 6-10 )Aryl(C 1-6 ) alkyl.

[0136] Preferably, R1 has the formula: [ka] The warhead of During the ceremony, q is 0, 1, 2, 3 or 4; each Z 1 is C 1-6 Alkyl, halogen, -CN and C 1-6 alkoxyl.

[0137] In one embodiment, Z 1 are independently selected from halogen, methyl and -OMe, preferably F, Cl and Me.

[0138] In a further embodiment, R 1 is the formula [ka] is selected from the group In the formula, each Z 1 is C 1-6 Alkyl, halogen, -CN and C 1-6 It is independently selected from alkoxyl, preferably halogen, methyl and -OMe, more preferably F, Cl and Me. In a preferred embodiment, R 1 is the formula [ka] has. In another embodiment, R 1 is the expression: [ka] may be a portion of the substrate selected from the group During the ceremony, W is selected from -NH- and -O-; R10 to R13 are H, C1-6 alkyl and C3-7 cycloalkyl, aryl, heteroaryl, CF 3 , -CH 2 COOH, -CH 2 CONHR14, and CH 2 Independently selected from OR14, R14 is selected from H, C1-6 alkyl, and C3-7 cycloalkyl.

[0139] In another preferred embodiment, R1 is ACC.

[0140] In a preferred embodiment, the compound of formula (I) has formula IX: [ka] Compound or a salt, solvate or stereoisomer thereof, During the ceremony, R2 and R3 are independently H, C1-6 alkyl, or C3-7 cycloalkyl, where R2 and R 3is the deprotonated form CO(O - ) does not have to be present to achieve TIFF2024524493000070.tif9170 represents a bond that may or may not be present, and if present, it combines with an already present single bond to form a double bond, During the ceremony, R1 is as defined in any of the above embodiments, preferably R1 is of the formula: [ka] is selected from the group In the formula, each Z 1 is C 1-6 Alkyl, halogen, -CN and C 1-6 It is independently selected from alkoxyl, preferably halogen, methyl and -OMe, more preferably F, Cl and Me.

[0141] In a preferred embodiment, R 1 is the formula [ka] or has ACC, - n is 1, 2, 3 or 4, -A is -NR a R b , [ka] where R a , R b and R c , H, C 1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5-10 membered heterocyclyl, 5-10 membered heteroaryl, -NO 2 , -C(O)R, -C(O)OR and -SO 2 R, where each R is selected from H, C1-6 Alkyl, C 3-7 Cycloalkyl, C 1-6 Haloalkyl, C 6-10 Aryl, (C 6-10 )Aryl(C 1-6 ) alkyl, 5- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl, and preferably A is selected from the group consisting of a -COOH group or an NH2 group.

[0142] In a preferred embodiment, the compound of formula (I) has formula IX: [ka] Compound or a salt, solvate or stereoisomer thereof and R2 and R3 are H, C1-6 alkyl or C3-7 cycloalkyl, where R2 and R 3 may be absent, so that the deprotonated form CO(O - ) occurs, TIFF2024524493000075.tif9170 (dotted line) represents a bond that may or may not be present; if present, it combines with an already present single bond to form a double bond; During the ceremony, - n is 2, 3 or 4, -A is a -COOH group or -NR a R b groups, preferably amine groups, R1 being as defined above, preferably R 1 is the formula [ka] Or have ACC.

[0143] It should be noted that the above formula IX encompasses all possible forms (e.g., one acid deprotonated, two acids deprotonated together, the amine in both states). That is, formula IX encompasses all salts and solvates. A non-limiting example of one of these forms is: [ka]

[0144] In certain embodiments, the compound of formula (I) is [ka] TIFF2024524493000079.tif203170 (protonated or deprotonated), or a salt, solvate or stereoisomer thereof; In the formula, R 1 is as defined throughout this specification, Preferably, R 1 teeth, [ka] is selected from.

[0145] It should be noted that the six structures above cover all possible forms (e.g., one acid deprotonated, two acids deprotonated together, the amine in both states) and that position P5 can remain elongated or can be an oxidase derivative with an additional double bond present, as shown in formula III below: [ka] wherein A and B are hydrogen atoms, and H, I, J and K are independently carbon atoms; TIFF2024524493000082.tif8170 (dotted line) represents a bond that may or may not be present (as in the case of oxidase derivatives).

[0146] In certain embodiments, the compound of formula (I) is [ka] or a salt, solvate or stereoisomer thereof, selected from the group consisting of: Preferably, R1 in the above three structures is [ka] (also known as AOMK, acyloxymethyl ketone).

[0147] All three structures above cover all possible forms (e.g. one acid deprotonated, two acids deprotonated together, amine in both states) and position P5 may or may not be an oxidase oxidized derivative according to the following structure: [ka] wherein A and B are hydrogen atoms, and H, I, J and K are independently carbon atoms; TIFF2024524493000086.tif10170 (dotted line) represents a bond that may or may not be present (oxidase derivative).

[0148] In yet another preferred embodiment, the compound of formula (I) is a.NH-Idc-hGlu-Glu(Chx)-P2-Asp-R1; b.NH-Idc-hGlu-Glu-P2-Asp-R1; c. NH-Idc-hGlu-Glu(me)-P2-Asp-R1; and / or d.NH-Idc-hGlu-Val-P2-Asp-R1 or a salt, solvate or stereoisomer thereof, selected from the group consisting of: wherein P2 is selected from lysine (Lys), ornithine (Orn), diaminobutyric acid (Dab) or diaminopropionic acid (Dap), and preferably R1 is [ka] (also known as AOMK, acyloxymethyl ketone).

[0149] In yet another preferred embodiment, the compound of formula (I) is a compound of formula NH-Idc-hGlu-Mix-P2-Asp-R1 (protonated or deprotonated) or a salt, solvate or stereoisomer thereof, wherein P2 is selected from lysine (Lys), ornithine (Orn), diaminobutyric acid (Dab), or diaminopropionic acid (Dap), and wherein Mix is ​​selected from Ala (alanine), Arg (arginine), Asn (asparagine), R1 is any naturally occurring amino acid selected from the group consisting of Asp (aspartic acid), Gln (glutamine), Gly (glycine), His (histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Nle (leucine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine) and Val (valine), preferably R1 is [ka] (also known as AOMK, acyloxymethyl ketone).

[0150] Pharmaceutical Compositions Compounds of any of the above formulas of the present invention, in which R1 is a warhead such as AOMK, are irreversible inhibitors of caspase-2 and can therefore be used in the prevention or treatment of disorders or diseases mediated by this enzyme.

[0151] Thus, in a further aspect, the present invention provides a compound comprising R 1is a warhead such as AOMK or a salt, solvate or stereoisomer thereof, and at least one pharma- ceutical composition comprising a compound of any of the above formulae as defined herein, and at least one pharma- ceutically acceptable excipient.

[0152] The term "excipient" refers to a vehicle, diluent, or adjuvant administered with an active ingredient. Such pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water or saline and aqueous dextrose and glycerol solutions, especially injectable solutions, are preferably used as vehicles. Suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences" by EW Martin, 21st Edition, 2005, or "Handbook of Pharmaceutical" Rowe CR; Paul JS; Marian EQ, sixth Edition, 2009.

[0153] The excipients and auxiliary substances required to prepare the desired pharmaceutical dosage form of the pharmaceutical composition of the present invention depend, among other factors, on the selected pharmaceutical mode of administration. The aforementioned pharmaceutical dosage forms of the pharmaceutical composition are prepared according to conventional methods known to those skilled in the art.

[0154] Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules etc.) or liquid (solutions, suspensions or emulsions) composition for oral, topical or parenteral administration.

[0155] In one embodiment, the pharmaceutical composition is an oral delivery form.The dosage form suitable for oral administration can be tablets and capsules, and can contain conventional excipients known in the art, such as binders, for example, syrup, gum arabic, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example, lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine; lubricants for preparing tablets, for example, magnesium stearate; disintegrants, for example, starch, polyvinylpyrrolidone, sodium starch glycolate, or microcrystalline cellulose; or pharma-ceutically acceptable wetting agents, such as sodium lauryl sulfate.The solid oral composition can be prepared by conventional methods of blending, filling, or tableting. Such operations are conventional in the art.Tablets can be prepared, for example, by dry or wet granulation, and can be optionally coated, in particular with enteric coating, according to methods well known in normal pharmaceutical practice.

[0156] The pharmaceutical compositions may also be adapted for parenteral administration, such as sterile solutions, suspensions or lyophilized products in appropriate unit dosage forms. Suitable excipients, such as bulking agents, buffers or surfactants, may be used.

[0157] The formulations referred to may be prepared using standard methods, such as those described or referenced in the European Pharmacopoeia and the United States Pharmacopoeia and similar reference texts.

[0158] The compounds or compositions of the invention may be administered by any suitable method, such as oral, sublingual, intranasal, intraocular, parenteral, subcutaneous, intramuscular, intravenous, or transdermal administration.

[0159] In general, the effective amount of the compound of the invention to be administered will depend on the relative efficacy of the compound selected, the severity of the disorder to be treated and / or prevented, and the weight of the patient. The active compound can be administered one or more times per day, for example, 1, 2, 3 or 4 times per day, with a typical total daily dose ranging from about 0.01 mg / kg body weight / day to about 1000 mg / kg body weight / day. In another embodiment, the effective dosage of the compound of the invention is about 500 mg / kg body weight / day or less. In another embodiment, the effective dosage of the compound of the invention is about 100 mg / kg body weight / day or less. In another embodiment, the effective dosage is in the range of about 0.01 mg / kg body weight / day to about 100 mg / kg body weight / day of the compound of the invention. In another embodiment, it is in the range of about 0.02 mg / kg body weight / day to about 50 mg / kg body weight / day, and in another embodiment, it is in the range of about 0.025 mg / kg body weight / day to about 20 mg / kg body weight / day.

[0160] Uses of the Compounds of the Invention R 1 The compounds of the invention, in which A is a group that includes a fluorophore moiety, can be used as activity-based probes for determining caspase-2 activity.

[0161] Thus, in another aspect, the present invention provides a method for determining caspase-2 activity using ... 1 is a group comprising a fluorophore moiety, or a salt, solvate or stereoisomer thereof.

[0162] In certain embodiments, caspase-2 activity in cells or tissues is determined by R 1 is a group comprising a fluorophore moiety, or a salt, solvate or stereoisomer thereof, can be determined by contacting a sample comprising said cells or tissues and measuring the fluorescent signal emitted upon light excitation.

[0163] Compounds of any of the above formulas of the invention in which R1 is a warhead such as AOMK contain a group that reacts with the active site of the target enzyme (caspase-2) thereby providing an irreversible inhibitory effect. 1 The compounds of the invention based on the formula: can be used in the prevention or treatment of diseases or disorders in which caspase-2 activity is implicated.

[0164] Thus, in another aspect, the present invention provides a compound comprising R 1 is a warhead such as AOMK, or a salt, solvate or stereoisomer thereof.

[0165] In another aspect, the present invention relates to a method for the treatment of degenerative diseases, such as Alzheimer's disease, Huntington's disease, Parkinson's disease, mild cognitive impairment, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease, adrenoleukodystrophy; neonatal brain injury, particularly neonatal cerebral ischemia; traumatic brain injury; renal ischemia; hypoxic-ischemic injury; stroke-like situations brain injuries; cardiac ischemia; myocardial infarction; amyotrophic lateral sclerosis; retinal injury; ophthalmic diseases, such as age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, blunt eye injury, ischemic optic neuropathy, glaucoma; prevention of cytotoxicity prevention of cytotoxicity due to physical factors such as radiation and acoustic trauma, especially cytotoxicity mediated by chemicals; skin injuries; sterile inflammatory diseases, e.g. diabetes, atherosclerosis, gout, pseudogout, joint laxity, atherosclerosis, syndromes triggered by aluminum salts, non-arteritic ischemic optic neuropathy, glaucoma and metabolic diseases; non-sterile inflammatory diseases, e.g. bacterial infections, especially infections by bacteria producing pore-forming toxins, influenza virus infections and single-stranded RNA Rhabdoviridae infections, e.g. Maraba virus or vesicular stomatitis virus; diseases caused by pathogenic bacteria, e.g. Brucella, Staphylococcus aureus The present invention relates to a compound of the present invention, or a salt, solvate or stereoisomer thereof, wherein R1 is a warhead such as AOMK, for use in the prophylaxis and / or treatment of a disease or disorder associated with increased caspase-2 activation selected from: Propionibacterium aureus and Salmonella; dyslipidemia; obesity; metabolic syndrome; and non-alcoholic fatty liver disease, such as non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver disease (NAFLD).

[0166] In a preferred embodiment, the present invention relates to a compound of the present invention, wherein R1 is a warhead, e.g., AOMK, or a salt, solvate or stereoisomer thereof, for use in the prevention and / or treatment of non-alcoholic fatty liver disease (NAFLD).

[0167] The term "treatment" or "treat" in the context of this specification means the administration of a compound or composition according to the invention to ameliorate or eliminate one or more symptoms associated with a disease or the aforementioned disease. "Treatment" also encompasses ameliorating or eliminating the physiological sequelae of a disease.

[0168] The term "ameliorate" in the context of the present invention is understood to mean any improvement to the condition of the patient being treated.

[0169] The term "prevention" or "preventing" in the context of this specification means the administration of a compound or composition according to the invention to reduce the risk of acquiring or developing a disease or one or more symptoms associated with the aforementioned disease.

[0170] In one embodiment, any of the methods or uses described herein may further comprise administering to the patient at least one other therapeutic agent.

[0171] The present invention is further illustrated by the following examples, which should not be construed in any way as limiting the scope of the invention as defined in the claims. EXAMPLES

[0172] Biological assays Example 1. Enzyme kinetic studies with substrates material and method reagent Fmoc-protected amino acids were purchased from Iris Biotech GmbH (Marktredwitz, Germany), Sigma-Aldrich (Poznan, Poland), Bachem (Torrance, CA, USA), Creosalus (Lowesville, KY, USA), PE Biosciences Limited (Hong Kong, China) and Combi-Block (San Diego, USA). Fmoc-ACC fluorescent dye was synthesized according to the procedure previously published by Maly et al. Rink Amide AM resin (200–300 mesh, loading 0.48 mmol / g), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), piperidine (PIP), diisopropylcarbodiimide (DICI) and trifluoroacetic acid (TFA) were obtained from Iris Biotech. GmbH. Anhydrous HOBt was purchased from Creosalus. 2,4,6-Trimethylpyridine (2,4,6-collidine), acetonitrile (ACN, HPLC gradient grade), triisopropylsilane (TIPS) were purchased from Sigma-Aldrich. N,N'-Dimethylformamide (DMF, analytically pure), methanol (MeOH), dichloromethane (DCM), AcOH, diethyl ether (Et2O), and phosphorus pentoxide (P2O5) were obtained from POCh (Gliwice, Poland). All ACC-labeled fluorescent substrates were purified using a semi-preparative Discovery® C8 column (10 μm particle size) with a Waters system (Waters M600 solvent delivery module and Waters Purification was performed by reversed-phase HPLC on a 100-millisecond LC-MS system (M2489 detector system). The solvent composition for substrate purification and LC-MS analysis was as follows: phase A (water / 0.1% TFA), phase B (ACN / 0.1% TFA). For purification, the assay was run with a linear gradient (95% phase A to 5% phase A) for 30 min. Purity and molecular weight [m / z+H] +was determined using an LC-MS Waters instrument using an analytical Discovery® C8 column (10 μm particle size). LC-MS assays were run from 95% phase A to 5% phase A in 20 min. All compounds were at least 95% pure.

[0173] Substrate synthesis Substrates with the general formula NH-Idc-hGlu-Thr(Bzl)-P2-Asp-ACC were synthesized on solid support according to the general method described above. Unless otherwise stated, all amino acids have the L configuration.

[0174] For all substrates synthesized, approximately 10 g (1 equiv., 4.8 mmol) of Fmoc-protected Rink Amide resin (0.48 mol / g) was placed in a 250 mL glass cartridge for solid-phase synthesis and swollen in DCM for 30 min. The DCM was then drained and the resin was washed three times with DMF. The Fmoc-protecting group was then removed in three cycles (5 min, 5 min, and 25 min) using 20% ​​piperidine in DMF, and the resin was washed six times with DMF. Approximately 2.5 equiv. of Fmoc-ACC-OH (12 mmol, 5.3 g) was preactivated with 2.5 equiv. of HOBt (12 mmol, 1.8 g) and 2.5 equiv. of DICI (12 mmol, 1.6 mL) in a minimum amount of DMF for 3 min and poured onto the resin. The cartridge was gently agitated for 30 min, and more DMF was added if the mixture became too dense. The reaction was carried out for 24 hours, followed by filtration and washing the resin with DMF three times. The Fmoc-ACC-OH coupling was repeated to improve the coupling yield using 1.5 equivalents of the above reagent. After 24 hours, the resin was washed three times with DMF and a ninhydrin test was performed to confirm complete Fmoc-ACC-OH coupling. The Fmoc group was then removed from ACC using 20% ​​piperidine in DMF, followed by washing the resin six times with DMF. Next, 2.5 equivalents of Fmoc-Asp(tBu)-OH (12 mmol, 5.3 g) were pre-incubated with 2.5 equivalents of HATU (12 mmol, 4.6 g) and 2.5 equivalents of 2,4,6-collidine (12 mmol, 1.6 mL) in DMF for 3 minutes and poured onto the H2N-ACC resin. The reaction was carried out for 24 hours and repeated using 1.5 equivalents of Fmoc-Asp(tBu)-OH / HATU / 2,4,6-collidine reagent for another 24 hours. The Fmoc-Asp(tBu)-ACC resin was then washed 3 times with DMF, followed by Fmoc deprotection with 20% piperidine in DMF, and the resin was washed 6 times with DMF, 3 times with DCM, 3 times with MeOH, and incubated in a desiccator for 30 min. 2 O 5 The resulting resin (approximately 12 g) was divided into 100 mg portions and used for the synthesis of individual caspase-2 substrates.

[0175] P2 substrate synthesis General formula NH-Idc-hGlu-Thr(Bzl)- P2-Asp-ACC ACC-labeled fluorescent substrate were synthesized using a MultiChem 48-well synthesizer (FlexChem, SciGene, CA, USA). For each substrate, 100 mg, 0.05 mmol of NH 2The -Asp(tBu)-ACC resin was placed into separate wells of a multi-well cartridge and DCM was added to swell the resin. The DCM was then filtered off and the resin was washed three times with DMF. In 30 1.5 mL tubes, 3 equivalents of various amino acids (0.15 mmol) were mixed with 1 mL of DMF containing 3 equivalents of HATU (0.15 mmol, 60 mg) and 3 equivalents of 2,4,6-collidine (0.15 mmol, 20 mL) and poured onto the resin. The P2 coupling reaction was carried out for 3 h, followed by a ninhydrin test. The Fmoc group was then removed from each substrate using 20% ​​piperidine in DMF, and the resin in each well was washed six times with DMF. Next, 3 equivalents (x30) of Fmoc-Thr(Bzl)-OH (4.5 mmol, 1.95 g) were pre-incubated with 3 equivalents (x30) of HATU (4.5 mmol, 1.72 g) and 2,4,6-collidine (4.5 mmol, 600 uL) in a minimum amount of DMF for 1 min and poured onto the resin. The P3 coupling reaction was carried out for 3 h, followed by a ninhydrin test. The Fmoc group was then removed from each substrate using 20% ​​piperidine in DMF, and the resin in each well was washed six times with DMF. Next, 3 equivalents (x30) of Fmoc-hGlu(tBu)-OH (4.5 mmol, 2.0 g) were pre-incubated with 3 equivalents (x30) of HATU (4.5 mmol, 1.72 g) and 2,4,6-collidine (4.5 mmol, 600 mL) in a minimum amount of DMF for 1 min and poured onto the resin. The P4 coupling reaction was carried out for 3 h, followed by a ninhydrin test. The Fmoc group was then removed from each substrate using 20% ​​piperidine in DMF, and the resin in each well was washed six times with DMF. Next, 3 equivalents (x30) of Fmoc-Idc-OH (4.5 mmol, 1.75 g) were pre-incubated with 3 equivalents (x30) of HATU (4.5 mmol, 1.72 g) and 2,4,6-collidine (4.5 mmol, 600 mL) in a minimum amount of DMF for 1 min and poured onto the resin. The P5 coupling reaction was carried out for 3 h, followed by a ninhydrin test. The Fmoc group was then removed from each substrate using 20% ​​piperidine in DMF, and the resin in each well was washed 6 times with DMF, 3 times with DCM, 3 times with MeOH, and incubated in a desiccator for 1 h at 4°C. 2 O 5The resin was dried overnight on ice. All substrates were cleaved from the resin using an ice-cold TFA / TPS / water (% v / v / v, 95 / 2.5 / 2.5) mixture for 2 h (shaking once every 15 min). The solution from each well was collected separately and the remaining resin was washed with TFA. The substrate was then washed with ice-cold Et 2 The supernatant was then discarded and the pellet was resuspended in ice-cold Et 2 The mixture was resuspended in 0 and centrifuged again. The supernatant was then discarded, the pellet was dried, and the crude product was dissolved in 1 mL of DMSO and purified by HPLC. The pure substrate was collected, frozen at -80°C, and lyophilized. The final product (white powder) was then dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 20 mM and stored at -80°C until use.

[0176] Preparation of recombinant caspases Detailed protocols for the expression and purification of human apoptotic caspases can be found elsewhere (Stennicke, 1999).

[0177] Enzyme kinetics research Operated in fluorescence kinetic mode in 96-well Corning (Corning, NY, USA) plates fMax Fluorescence Spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) We screened for P2 substrates for caspases-2, -3 and -8 usingACC fluorescence was monitored using wavelengths of 355 nm (excitation) and 460 nm (emission). Prior to kinetic analysis, all caspases were active site titrated using zVAD-fmk inhibitor (Cayman Chemical Company, catalog number 14467). Caspase assay buffer was 10% w / v sucrose, 1 M sodium citrate, 20 mM Pipes, 10 mM NaCl, 1 mM EDTA, and 10 mM DTT (pH=7.3). Buffers were prepared at room temperature and all kinetic assays were performed at 37°C. All enzymes were pre-incubated for 15 min before any activity assay. Substrates were screened at a concentration of 10 mM and caspase concentrations were as follows: caspase-25 nM, caspase-3 15 nM, and caspase-8 40 nM. The total volume in a single well was 100 mL. Total assay time was 30 min, but only the linear portion of the fluorescence progress curve was taken for analysis. Kinetic data were analyzed using Graph Pad Prism software. Data are presented as fluorescence release / production rate per 10 nM of caspase (RFU / sec relative fluorescence units / sec).

[0178] result By re-examining its catalytic selectivity at the P2 (also referred to as AA throughout the present invention) position, the inventors have determined that Highly specific and selective The aim of this study was to develop caspase-2 substrates. According to the generally accepted notion, "specific" means high kinetic parameters (e.g., high kcat / KM for substrates and high kobs / I for inhibitors), and "selective" means that some reagents (substrates / inhibitors) target only one enzyme. The main objective of this part of the invention was to synthesize and biochemically evaluate a series of pentapeptide fluorescent substrates designed based on the NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-ACC (WRMP23) formula, previously published to be the most selective substrate for caspase-2 (Poreba et al., Cell Death & Differentiation 2019, 26, 2695-2709). From the above-mentioned WRMP23 formula, the present invention Highly specific and selectiveThe following scaffold 1 (see Scaffold 1 below) was designed to form a basis for the de novo development of caspase-2 substrates and inhibitors: [ka]

[0179] Scaffold 1. NH-Idc-hGlu-Thr(Bzl)-AA-Asp-R 1 R1: -ACC (substrate) or -AOMK (inhibitor). R3: H. R2: H. AA: P2.

[0180] Results are presented herein as rates of substrate hydrolysis expressed as RFU / s, where RFU is relative fluorescence units per 10 nM of caspase (see FIG. 1 and Table 1).

[0181] [Table 1] [ka]

[0182] As shown in the table above, the inventors of the present invention have discovered compounds of the following formula: [ka]

[0183] In the formula, Lys at the AA position instead of serine exhibits higher caspase-2 activity and remarkable selectivity over caspase-3 or caspase-8, thus resulting in improved caspase-2 substrate inhibitors. The inventors further found that compounds of the above formula having His at the AA position instead of serine exhibit strong caspase-2 activity.

[0184] Example 2. Enzyme kinetic studies with inhibitors material and method reagent Fmoc-protected amino acids were purchased from Iris Biotech GmbH (Marktredwitz, Germany), Sigma-Aldrich (Poznan, Poland), Bachem (Torrance, CA, USA), Creosalus (Louisville, KY, USA), PE Biosciences Limited (Hong Kong, China) and Combi-Block (San Diego, USA). 2-Chlorotrityl chloride resin (100-200 mesh, loading 1.59 mmol / g), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), piperidine (PIP), diisopropylcarbodiimide (DICI) and trifluoroacetic acid (TFA) were purchased from Iris Biotech GmbH. 2,4,6-Trimethylpyridine (2,4,6-collidine), acetonitrile (ACN, HPLC gradient grade), triisopropylsilane (TIPS), hydrobromic acid solution (30 wt% HBr in acetic acid (AcOH)), N-methylmorpholine (NMM), tetrahydrofuran (THF anhydrous), isobutyl chloroformate (IBCF), and 2,6-dimethylbenzoic acid (2,6-DMBA) were purchased from Sigma-Aldrich. N,N'-Dimethylformamide (DMF, analytically pure), methanol (MeOH), dichloromethane (DCM), AcOH, diethyl ether (Et2O), and phosphorus pentoxide (P2O5) were obtained from POCh (Gliwice, Poland). Diazomethane for acyloxymethylketone (AOMK) inhibitor synthesis was generated according to the Aldrich Technical Bulletin (AL-180) protocol. All AOMK inhibitors were purified by reversed-phase HPLC on a Waters system (Waters M600 solvent delivery module and Waters M2489 detector system) using a semi-preparative Discovery® C8 column (10 μm particle size). The solvent composition for inhibitor purification and LC-MS analysis was as follows: Phase A (water / 0.1% TFA), Phase B (ACN / 0.1% TFA). For purification, the assay was run with a linear gradient (95% Phase A to 5% Phase A) for 30 min.Purity and molecular weight [m / z+H]. + was determined using an LC-MS Waters instrument using an analytical Discovery® C8 column (10 μm particle size). LC-MS assays were run from 95% phase A to 5% phase A in 20 min. All compounds were at least 95% pure.

[0185] Synthesis of P2 inhibitors Detailed kinetic analysis of fluorogenic substrates of apoptotic caspases at the P2 position allowed us to select caspase-2 selective tetrapeptide motifs and use them to design irreversible AOMK-tagged inhibitors. Inhibitors with the general formula of NH-Idc-hGlu-Thr(Bzl)-AA-Asp-AOMK were synthesized according to the general method previously described (Poreba 2019, Poreba 2016). As a control inhibitor, Ac-VDVAD-AOMK was synthesized. The detailed procedure of AOMK-based inhibitor synthesis is exemplified in the synthesis of NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-AOMK (NH-23-C2, Poreba et al., Cell Death & Differentiation 2019, 26, 2695-2709). All other inhibitors were synthesized and purified similarly. Unless otherwise stated, all amino acids have the L configuration.

[0186] Synthesis of NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-AOMK(NH-23-C2) A 0.2 M solution of Boc-Asp(tBu)-OH amino acid (5 mmol, 1.45 g) in anhydrous THF was stirred for 10 min at -10 °C in an ice / acetone bath. 4-Methylmorpholine (6.25 mmol, 1.25 equiv.) and isobutyl chloroformate (5.75 mmol, 1.15 equiv.) were then added. The reaction was carried out at -10 °C for 45 min. In a parallel experiment, diazomethane was generated according to the Aldrich Technical Bulletin (AL-180) protocol. A solution of the mixed anhydride was then added dropwise to ethereal diazomethane (16.6-21.4 mmol) at 0 °C. The mixture was stirred for 10 min, after this time the ice bath was removed and the reaction was carried out at room temperature for 2 h. Boc-Asp(tBu)-CH 2 To obtain Br, HBr (30% by weight CH 3 15 mL of a 1:2 solution of ethyl acetate (in COOH) and water was added dropwise to the mixture over 10 min. Immediately after, the mixture was diluted with ethyl acetate, transferred to a separatory funnel, and washed with water (1x), saturated aqueous NaHCO 3 (twice) and brine (twice). The organic fraction was extracted with MgSO 4 The mixture was dried over 100 ml of ethyl acetate and evaporated under reduced pressure. The product was obtained as a pale yellow oil and used in the synthesis without further purification. The product purity was >95% as determined by HPLC, and the overall yield was >90%. In the next reaction, 1 equivalent of Boc-Asp(tBu)-CH 2 Br was dissolved in a small amount of DMF, followed by the addition of KF (3 eq.) and 2,6-dimethylbenidine acid (2,6-DMBA) (1.2 eq.). The mixture was stirred for 25 min under an inert atmosphere of argon. After the reaction was complete (HPLC analysis), the solution was diluted with ethyl acetate, transferred to a separatory funnel and washed with 5% citric acid (twice), 5% NaHCO 3 The organic fraction was then extracted with aqueous solution (2 times) and brine (2 times). 4The mixture was dried over ice and evaporated under reduced pressure. The product was obtained as a yellow oil (yield >95%) and used in probe synthesis without further purification. Boc-Asp(tBu)-AOMK (100 mg / 0.213 mmol) was added to a solution of 25% TFA in DCM. Deprotection of the Boc and tBu groups was carried out for 30 min. The TFA and DCM were then evaporated under reduced pressure to give the final product (NH 2-Asp-AOMK) was obtained as a yellow oil and used without further purification (98% yield by HPLC) (Block B, Synthesis Scheme 1). In a separate synthesis, NH-Idc-hGlu(tBu)-Thr(Bzl)-Ser(tBu)-OH peptide fragment was synthesized using 2-chlorotrityl chloride resin (200 mg, 1.6 mmol / g, 0.33 mmol). Fmoc-L-Ser(tBu)-OH (3 eq, 1 mmol, 383 mg) was dissolved in a minimum volume of anhydrous DCM, followed by the addition of 4.5 eq of DIPEA (1.5 mmol, 260 uL), the mixture was activated for 1 min, and poured onto the resin. The reaction mixture was stirred for 3 h. The Fmoc group was then removed with 20% piperidine in DMF, and the resin was washed six times with DMF. Then, Fmoc-Thr(Bzl)-OH (3 eq. 1 mmol, 431 mg) and HATU (3 eq. 1 mmol, 380 mg) were dissolved in a minimum volume of DMF and 3 eq. of 2,4,6-collidine (1 mmol, 130 uL) was added. The mixture was poured onto the resin and the reaction was carried out for 3 hours. After this, the Fmoc group was removed with 20% piperidine in DMF and the resin was washed 6 times with DMF. Next, Fmoc-hGlu(tBu)-OH (3 eq. 1 mmol, 440 mg) and HATU (3 eq. 1 mmol, 380 mg) were dissolved in a minimum volume of DMF and 3 eq. of 2,4,6-collidine (1 mmol, 130 uL) was added. The mixture was poured onto the resin and the reaction was carried out for 3 hours. After this, the Fmoc group was removed with 20% piperidine in DMF and the resin was washed 6 times with DMF. Next, Fmoc-Idc-OH (3 equiv. 1 mmol, 386 mg) and HATU (3 equiv. 1 mmol, 380 mg) were dissolved in a minimum volume of DMF and 3 equiv. of 2,4,6-collidine (1 mmol, 130 uL) was added. The mixture was poured onto the resin and the reaction was carried out for 3 h. After this, the Fmoc group was removed with 20% piperidine in DMF and the resin was washed 6 times with DMF, 3 times with DCM and 3 times with MeOH. The resin was then dried overnight in a desiccator over P2O5. The peptide was then cleaved from the resin during a 45 min incubation in a mixture of DCM / TFE / AcOH (v / v / v, 8:1:1). The solution was then filtered and the solvent was removed under reduced pressure. The crude peptide was dissolved in acetonitrile:H 2NH-Idc-hGlu(tBu)-Thr(Bzl)-Ser(tBu)-OH was obtained as a white powder by dissolving in 1:2O (v / v, 7:3) and lyophilizing. The peptide purity was >95% and was used for the synthesis of NH-23-C2 inhibitor without further purification. The peptide fragment (1 eq.) was then coupled with NH2-Asp-AOMK (1 eq.) in DMF using HATU (1 eq.) and 2,4,6-collidine (5 eq.) as coupling reagents. The reaction was carried out at room temperature for 2 hours, after which the mixture was injected into HPLC and NH-Idc-hGlu(tBu)-Thr(Bzl)-Ser(tBu)-Asp-AOMK was purified and lyophilized. The product was then dissolved in a mixture of DCM:TFA 1:2 to remove the protecting groups. The reaction was carried out at room temperature for 1 hour and the DCM:TFA mixture was purged with argon. The crude product was purified by HPLC and lyophilized to obtain the final compound: NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-AOMK (Block C, Synthesis Scheme 1). Molecular weight [m / z+H] + The purity was confirmed by LC-MS analysis. In a similar manner, other inhibitors were synthesized. The reference inhibitor Ac-VDVAD-AOMK (Ac-Val-Asp-Val-Ala-Asp-AOMK) was also synthesized by this procedure, but in this case the peptide fragment was additionally N-capped with an acetyl group. Acetic acid (5 eq.) and HBTU (5 eq.) were dissolved in a minimum amount of DMF and poured onto NH2-Val-Asp(tBu)-Val-Ala-resin (1 eq.) and the acetylation reaction was carried out for 30 min. The N-acetylated peptide was then carried out as described above.

[0187] [ka] [ka]

[0188] [Table 2]

[0189] For the synthesis of NH-Idc-hGlu-Thr(Bzl)-Lys-Asp-AOMK, Fmoc-L-Lys(Boc)-OH was used instead of Fmoc-L-Ser(tBu)-OH. For the synthesis of NH-Idc-hGlu-Thr(Bzl)-Orn-Asp-AOMK, Fmoc-L-Orn(Boc)-OH was used instead of Fmoc-L-Ser(tBu)-OH. For the synthesis of NH-Idc-hGlu-Thr(Bzl)-Dab-Asp-AOMK, Fmoc-L-Dab(Boc)-OH was used instead of Fmoc-L-Ser(tBu)-OH. For the synthesis of NH-Idc-hGlu-Thr(Bzl)-Dap-Asp-AOMK, Fmoc-L-Dab(Boc)-OH was used instead of Fmoc-L-Ser(tBu)-OH. For the synthesis of NH-Idc-hGlu-Thr(Bzl)-Arg-Asp-AOMK, Fmoc-L-Arg(Pbf)-OH was used instead of Fmoc-L-Ser(tBu)-OH.

[0190] Enzyme kinetic studies - determination of kobs / I inhibition parameters The k of synthetic inhibitors against caspases-2, -3, and -8 was measured using an fMax spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA) operated in fluorescence kinetic mode in 96-well Corning (Corning, NY, USA) plates. obs Measurements of the / I inhibition parameter (second-order rate of enzyme inhibition) were performed. Prior to kinetic analysis, all caspases were active-site titrated using zVAD-fmk inhibitor (Cayman Chemical Company, catalog number 14467). The caspase assay buffer was 10% w / v sucrose, 1 M sodium citrate, 20 mM Pipes, 10 mM NaCl, 1 mM EDTA, and 10 mM DTT (pH = 7.3). Buffers were prepared at room temperature and all kinetic assays were performed at 37 °C. obsThe / I parameters were measured under pseudo-first-order kinetic conditions ([I]>>[E]). Inhibitors (20 uL) were diluted in a 96-well plate and mixed with the appropriate substrate (20 uL): NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-ACC (50 uM) for caspase-2, Ac-DEVD-ACC (100 uM) for caspase-3 and Ac-LEHD-ACC (100 uM) for caspase-8. ACC fluorescence was monitored using wavelengths of 355 nm (excitation) and 460 nm (emission). The substrate-inhibitor mixture (total 40 uL) was pre-incubated at 37°C for 15 min. Simultaneously, in separate tubes, caspases were pre-incubated in assay buffer at 37°C and after 15 min, enzymes were added to the wells (60 uL per well, total reaction volume 100 uL) and fluorescence was monitored immediately for 30 min. Second-order inhibition (k obs / I) was determined in at least three independent experiments and presented as the mean value. GraphPad Prism 7 software was used for calculations (see Figure 2).

[0191] Preparation of recombinant caspases Detailed protocols for the expression and purification of human apoptotic caspases can be found elsewhere (Stennicke, 1999).

[0192] Enzyme Dynamics Research-K i and I.C. 50 Determination of inhibition parameters The K of synthetic inhibitors against caspases-2, -3, and -8 was measured using an fMax spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA) operated in fluorescence kinetic mode in 96-well Corning (Corning, NY, USA) plates. i and I.C. 50Inhibition parameters were measured. Prior to kinetic analysis, all caspases were active site titrated using zVAD-fmk inhibitor (Cayman Chemical Company, Cat. No. 14467). Caspase assay buffer was 10% w / v sucrose, 1 M sodium citrate, 20 mM Pipes, 10 mM NaCl, 1 mM EDTA, and 10 mM DTT (pH=7.3). Buffers were prepared at room temperature and all kinetic assays were performed at 37°C. i Parameters were measured using the Morrison formula (Copeland, 2000). For each caspase, substrate hydrolysis was monitored using the appropriate ACC fluorogenic substrate: NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-ACC (50 uM) for caspase-2, Ac-DEVD-ACC (100 uM) for caspase-3, and Ac-LEHD-ACC (100 uM) for caspase-8. ACC fluorescence was monitored using wavelengths of 355 nm (excitation) and 460 nm (emission). To meet the criterion of reversible inhibitor kinetics, the minimum inhibitor concentration was at least 4-fold higher than the caspase concentration in the assay. Caspase (60 uL) was first pre-warmed in assay buffer in a 96-well plate for 15 min at 37°C and then pre-incubated with inhibitor (20 uL) for an additional 15 min at 37°C. Substrate (20 uL) was then added to the enzyme-inhibitor mixture and the reaction progress (ACC release) was monitored over time. i The parameters were calculated using the Morrison formula and IC 50 The parameters are given by the formula: IC 50 =K i ×(1+[S] / K m [Km] was calculated using the formula: where [S] is the substrate concentration used in the assay and Km is the Michaelis-Menten constant of the substrate. All measurements were performed at least in triplicate and data were analyzed using GraphPad Prism 7 software.

[0193] The Ki and IC50 (for caspase-2) data are shown in FIG.

[0194] The results are expressed as kobs / I and the Ki and IC50 values ​​are shown in Tables 3-5 below. [Table 3] [Table 4] [Table 5]

[0195] Selectivity results are also presented as split values ​​of kobs / I, Ki and IC50 values ​​(see Tables 6 and 7 below). [Table 6] [Table 7]

[0196] As already shown, the compounds NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-ACC and NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-AOMK have been disclosed as caspase-2 substrates and inhibitors in Poreba et al., Cell Death & Differentiation 2019, 26, 2695-2709. As shown in the above table, the inventors of the present invention have found that compounds of the formula NH-Idc-hGlu-Thr(Bzl)-AA-Asp-AOMK, for example with a Lys, Orn, Dab or Dap group at the AA position instead of serine, exhibit high caspase-2 inhibitory activity and remarkable selectivity towards caspase-3 or caspase-8, thus resulting in improved caspase-2 inhibitors.

[0197] Example 3. Intracellular lipid accumulation -material and method HepG2 cells (50.000 cells / well) were seeded in 12 multi-well culture dishes on cover glasses pre-coated with collagen (2 mg / mL collagen solution in PBS (BD Biosciences) incubated for 30 min at 37°C).

[0198] Cells were treated with the "NASH cocktail": DMEM containing 10% (v / v) FBS, 1% (v / v) PenStrep and 4.5 mg / mL glucose supplemented with 2 mM L-glutamine + fatty acids (100 μM sodium oleate and 100 μM palmitic acid), 100 nM insulin (all Sigma-Aldrich) and inflammatory cytokines (50 ng / mL tumor necrosis factor, TNF-α (Prospec), 25 ng / mL interleukin IL-1β (Petroteck) and 8 ng / mL transforming growth factor, TGF-β (R&D Systems)). Cells were exposed to this "NASH cocktail" for 24 h and KIN compounds were added simultaneously at different concentrations.

[0199] At the end of treatment, cells were washed with PBS and incubated with staining solution (BODIPY 493 / 503 (Thermo Fisher Scientific) for 15 min at 37°C. Cells were then washed three times with PBS and fixed in 4% PFA for 30 min at room temperature. After three further washes with PBS, coverslips were mounted on glass slides overnight at room temperature using one drop of Prolong® Gold antifade reagent (Invitrogen) with DAPI. Fluorescence micrographs were taken using an Axoimagen M1 microscope (Zeiss, Oberkochen, Germany) and the fluorescent signals (DAPI and BODIPY) were quantified using ImageJ (n=10). Intracellular lipid loading was calculated as a function of BODIPY™ 493 / 503 lipid dye area.

[0200] - result One metabolic process thought to initiate NASH is de novo lipogenesis (DNL), the rate of which is up to three times higher in patients (Lambert et al., 2014). DNL was speculated to contribute to the progression of NASH by increasing intracellular lipotoxin free fatty acids (FFA) in hepatocytes. Caspase-2 has been described to regulate the transcription of pro-lipogenic enzymes such as HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase) and HMGCS (hydroxymethylglutaryl-CoA synthase) (Kim, 2018). Thus, caspase-2 inhibition could prevent progression to NASH by lowering intracellular lipid concentrations in hepatocytes.

[0201] An in vitro cell model of NASH was used to test the efficacy of caspase-2 inhibitors in preventing intracellular accumulation of lipids. In this model, HepG2 cells are exposed to "NASH conditions" including lipogenic (glucose, insulin, fatty acids) as well as inflammatory and pro-apoptotic (TNF-α, IL-1β and TGF-β) triggers for 24 h. Cells show a significant increase in intracellular lipid load of at least 2-fold compared to non-triggered controls. This model has been shown to correlate with human NASH pathology (Boeckmans et al., 2019).

[0202] NASH-induced HepG2 cultures were treated with 10 μM KIN inhibitors for 24 h, and the inhibition of intracellular lipid accumulation was quantified as described in Materials and Methods. As shown in Figure 4, all caspase-2 inhibitors tested were able to inhibit lipid accumulation under NASH conditions. Compounds 1, 2, and 3 showed significantly higher inhibition rates than the NH-23-C2 compound.

[0203] The efficacy of caspase-2 inhibitors in preventing intracellular lipid accumulation was used to compare the efficacy of NH-23-C2 and compound 1. Cells were incubated with NASH conditions and 10, 20 and 25 μM of caspase-2 inhibitors to analyze their protection against lipid accumulation. As shown in Figure 5, compound 1 showed better efficacy than NH-23-C2 in inhibiting intracellular lipid accumulation under NASH conditions.

[0204] These experimental results provide experimental evidence that the compounds of the present invention capable of modulating the activity of caspase-2 can effectively inhibit lipid accumulation in hepatocytes.

[0205] Example 4. Kinetic assay -material and method Caspase-2 substrates were synthesized according to standard SPPS procedures, purified by HPLC, lyophilized, and dissolved in DMSO to a final concentration of 20 mM (Poreba et al., 2014, CDD). In particular, the following caspase-2 substrates were synthesized to perform this example: [ka] TIFF2024524493000102.tif167170TIFF2024524493000103.tif167170

[0206] Prior to the kinetic assay, the substrate (a few μL) was diluted to 1 μM (working concentration) in DMSO. Then, 1 μL of such substrate was spotted into a well of a 96-well plate, followed by the addition of 99 mL of caspase-2 in buffer. Immediately after adding the enzyme, the plate was placed in a fluorescence plate reader and the fluorescence was measured over time in kinetic mode (excitation 355 nm, emission 460 nm). The release of fluorescence was performed for 30 min and only the linear part of the plot was taken for analysis to calculate the reaction rate. The percentage of the best cleaved substrate was set to 100% and all other substrates were adjusted accordingly (% cleavage). The final concentration of the substrate was 10 μM and the final concentration of caspase-2 was 10 nM. The caspase-2 assay buffer was 20 mM Pipes, 100 mM NaCl, sucrose 10% (w / v), 10 mM DTT, pH 7.2-7.4. Caspase-2 was preincubated in buffer for 15 min at 37° C. before being added to the substrate.

[0207] result The results are summarized in Figure 6 and detailed in the table below: [Table 8]

[0208] To ensure the reliability of the data, all caspase 2 substrates were generated independently (synthesized, purified, diluted in DMSO). The reference comparison value is the hydrolysis rate of the best substrate from this particular series of substrates, namely NH-Idc-hGlu-Glu(Chx)-Dab-Asp-ACC. From this example, it is clear that when using a pentapeptide with the following scaffold NH-Idc-P4-P3-Dab-Asp-ACC, position P4 is very preferably hGlu, while P3 can be left open for at least one of the compounds Thr(Bzl), Glu(Chx), Glu(me), Glu or Val (as well as Abu). This is further illustrated in FIG. 7.

[0209] Example 5. -material and method A series of caspase-2 substrates with the general formula NH-Idc-P4-Mix-Dab-Asp-ACC [where P4 was either Asp, hGlu, Ile, Leu or hLeu]; Mix is ​​an equimolar mixture of natural amino acids and ACC is a fluorescent tag, were synthesized on solid support according to the general method described in Poreba et al., CDD, 2014. 500 mg (1 equiv., 0.24 mmol) of Fmoc-protected Rink Amide resin (0.48 mol / g) was placed in a glass cartridge for solid-phase synthesis and swollen in DCM for 30 min. The DCM was then drained and the resin was washed three times with DMF. The Fmoc-protecting group was then removed in three cycles (5 min, 5 min, and 25 min) using 20% ​​piperidine in DMF, and the resin was washed six times with DMF. Approximately 2.5 equivalents of Fmoc-ACC-OH (0.6 mmol, 265 mg) were preactivated with 2.5 equivalents of HATU (0.6 mmol, 228 mg) and 2.5 equivalents of 2,4,6-collidine (0.6 mmol, 80 μL) in a minimum amount of DMF for 3 minutes and poured onto the resin. The reaction was run for 4 hours, followed by filtration and washing the resin three times with DMF. After this, the resin was washed three times with DMF and a ninhydrin test was performed to confirm complete Fmoc-ACC-OH coupling. The Fmoc group was then removed from ACC using 20% ​​piperidine in DMF, followed by washing the resin six times with DMF. Next, 2.5 equivalents of Fmoc-L-Asp(tBu)-OH (0.6 mmol, 247 mg) were pre-incubated with 2.5 equivalents of HATU (0.6 mmol, 228 mg) and 2.5 equivalents of 2,4,6-collidine (0.6 mmol, 80 mL) in DMF for 3 min and poured onto the H2N-ACC resin. The reaction was carried out for 24 h and repeated using 1.5 equivalents of Fmoc-L-Asp(tBu)-OH / HATU / 2,4,6-collidine reagent for another 24 h. The Fmoc-Asp(tBu)-ACC resin was then washed three times with DMF, followed by Fmoc deprotection with 20% piperidine in DMF and washing the resin six times with DMF. Next, Fmoc-L-Dab(Boc)-OH was coupled to the P2 position.2.5 equivalents of this amino acid (0.6 mmol, 264 mg), 2.5 equivalents of HATU (0.6 mmol, 229 mg), and 2.5 equivalents of 2,4,6-collidine (0.6 mmol, 80 mL) were diluted in DMF and poured onto the H2N-mix-Asp(tBu)-ACC resin. The reaction was run for 3 h, followed by washing the slurry with DMF (6 times) and a ninhydrin test to confirm complete coupling. The Fmoc group was removed with 20% piperidine in DMF and the resin was washed 6 times with DMF. An isokinetic mixture of 19 natural amino acids (excluding cysteine ​​and including norleucine to mimic methionine) was coupled to the P3 position. To do this, 5 equivalents of the isokinetic mixture (1.2 mmol), 5 equivalents of HOBt (1.2 mmol, 180 mg), and 5 equivalents of DICI (1.2 mmol, 160 μL) were diluted in DMF and preactivated for 3 min. The mixture was then poured onto the resin and the cartridge was agitated for 3 h. The slurry was filtered and washed three times with DMF. Complete coupling of P3 was confirmed by ninhydrin test. Next, the Fmoc group was removed with 20% piperidine in DMF and the H2N-Mix-Dab(Boc)-Asp(tBu)-ACC resin was split into five portions (0.05 mmol each). P4 coupling was performed using a MultiChem 48-well synthesizer (FlexChem by SciGene, CA, USA). The five portions of the resin were placed in a 48-well cartridge and swelled with DCM for 30 min. The DCM was then filtered and the resin was washed three times with DMF. In five 1.5 mL tubes, 2.5 equivalents of different amino acids (0.12 mmol) were mixed with 1 mL of DMF containing 2.5 equivalents of HATU (0.12 mmol, 46 mg) and 2.5 equivalents of 2,4,6-collidine (0.12 mmol, 16 μL) and poured onto the resin. The P4 coupling reaction was carried out for 4 h, followed by a ninhydrin test. Finally, the Fmoc group was removed with 20% piperidine in DMF, and the resin was washed six times with DMF. Then, Fmoc-L-Idc-OH was coupled to the P5 position.2.5 equivalents of this amino acid (0.6 mmol, 231 mg), 2.5 equivalents of HATU (0.6 mmol, 229 mg), and 2.5 equivalents of 2,4,6-collidine (0.6 mmol, 80 mL) were diluted in DMF and poured into each portion of H2N-P4-Mix-Dab(Boc)-Asp(tBu)-ACC resin. The reaction was run for 3 h, followed by washing the slurry with DMF (6 times) and a ninhydrin test to confirm complete coupling. The Fmoc group was removed with 20% piperidine in DMF and the resin was washed 6 times with DMF, 3 times with DCM, and 3 times with MeOH. The resin was dried over P2O5 in a desiccator overnight. All substrates were cleaved from the resin using an ice-cold TFA / TPS / water (%v / v / v, 95 / 2.5 / 2.5) mixture for 2 h (shaking once every 15 min). The solution from each well was collected separately and the remaining resin was washed with TFA. The substrate was then precipitated with ice-cold Et2O for 30 min and centrifuged. The supernatant was then discarded and the pellet was resuspended in ice-cold Et2O and centrifuged again. The supernatant was then discarded and the pellet was dried, dissolved in 5 mL of a 1:1 water:ACN mixture, frozen at -80°C, and lyophilized. The final product (white powder) was then dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mM and stored at -80°C until use.

[0210] 1 μL of substrate (10 mM) was spotted into wells of a 96-well plate, followed by the addition of 99 mL of caspase-2 in buffer. Immediately after enzyme addition, the plate was placed in a fluorescence plate reader and fluorescence was measured over time in kinetic mode (excitation 355 nm, emission 460 nm). Fluorescence release was followed for 30 min and only the linear portion of the plot was taken for analysis to calculate reaction kinetics. The percentage of the best cleaved substrate was set to 100% and all other substrates were adjusted accordingly (% cleavage). Composition of the isokinetic mixture used in this particular synthesis (total amount - 1.2 mmol) [Table 9]

[0211] The final concentration of substrate was 10 μM and the final concentration of caspase-2 was 10 nM. Caspase-2 assay buffer was 20 mM Pipes, 100 mM NaCl, sucrose 10% (w / v), 10 mM DTT, pH 7.2-7.4. Caspase-2 was preincubated in buffer for 15 min at 37 °C before adding to the substrate. The results are shown in Figure 8 (left panel) and in the table below: [Table 10]

[0212] From the above table it is clear that P4 must be hGlu regardless of the natural amino acid used in P3 which can be any natural amino acid selected from the group consisting of Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Gln (glutamine), Gly (glycine), His (histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Nle (norleucine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine) and Val (valine).

[0213] Finally, to better represent the hydrolysis rate of the best substrates of each series shown in Examples 4 and 5, they were set to 100% and the other substrates were adjusted accordingly, as shown in FIG.

Claims

1. Formula (I): 【Chemical 1】 compound or a salt, solvate or stereoisomer thereof, wherein in the formula,[[]] -n is 1, 2, 3 or 4; -A is an amine group, wherein in the formula,[[]] P5 is a compound of formula (III): 【Chemical 2】 wherein A and B are hydrogen atoms, H, J and K are carbon atoms, and the dotted line represents a bond that may or may not be present, and when present, it forms a double bond in combination with the single bond that is already present; P4 is hGlu (homo-glutamic acid), where this amino acid residue may be in the deprotonated or protonated form; P1 is Asp or aspartic acid, and this amino acid residue may be in the protonated or deprotonated form; P3 is selected from cyclohexyl glutamate (Glu(Chx)), L-methyl glutamate (Glu(me)), Thr(Bzl) (threonine benzyl ether), aminobutyric acid (Abu), or any natural amino acid selected from the group consisting of Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Glu (glutamic acid), Gln (glutamine), Gly (glycine), His (histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Nle (norleucine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine) and Val (valine), wherein R1 is selected from the group consisting of [[]] 【Chemical 3】 wherein [[]] q is 0, 1, 2, 3, 4 or 5; Each Z １ is selected independently from １－６ C alkyl, halogen, -CN, -NO ２ and C １－６ alkoxyl, Z ２ is a halogen, Z ３ is C １－６ alkyl, C ６－１０ aryl and (C ６－１０ )aryl or (C １－６ )alkyl selected from, or or from a compound of the formula: 【Chemical Formula 4】 [[]] wherein W is selected from -NH- and -O-; R10 to R13 are independently selected from H, C1-6 alkyl and C3-7 cycloalkyl, aryl, heteroaryl, CF ３ , -CH ２ COOH, -CH ２ CONHR14, and CH ２ OR14, R14 is selected from H, C1-6 alkyl and C3-7 cycloalkyl, a compound.

2. R1 has a compound of the formula: 【Chemical Formula 5】 [[]] wherein q is 0, 1, 2, 3 or 4; Each Z １ is selected from C １－６ alkyl, halogen, -CN and C １－６ alkoxyl, or alternatively, R1 is a compound of the formula: 【Chemical Formula 6】 [[]] The compound according to claim 1.

3. P3 is selected from cyclohexyl glutamate (Glu(Chx)), L-methyl glutamate (Glu(me)), Glu or Thr(Bzl) (threonine benzyl ether), the compound according to claim 1 or 2.

4. The compound according to claim 1 or 2, wherein P3 is selected from any natural amino acid selected from the group consisting of Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Gln (glutamine), Gly (glycine), His (histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Nle (norleucine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine) and Val (valine).

5. The compound according to claim 1 or 2, wherein n is 2, 3 or 4.

6. The compound is of formula (IX): [Chemical Formula 7] or a salt, solvate or stereoisomer thereof, wherein R2 and R3 are independently H, and R2 and R ３ may not be present, resulting in the deprotonated form CO(O-), wherein n is 1, 2, 3 or 4, A is an amine group, R1 is selected from the group of formula 【Chemical Formula 8】

7. In the formula, each Z １ is selected independently from C １－６ alkyl, halogen, -CN and C １－６ alkoxyl, and is the compound according to claim 1 or 2. The compound is one of the following compounds a. NH-Idc-hGlu-Glu(Chx)-P2-Asp-R1 b. NH-Idc-hGlu-Glu-P2-Asp-R1 c. NH-Idc-hGlu-Glu(me)-P2-Asp-R1 d. NH-Idc-hGlu-Val-P2-Asp-R1 or a salt, solvate or stereoisomer thereof, and The compound according to claim 1 or 2, wherein P2 is selected from lysine (Lys), ornithine (Orn), diaminobutyric acid (Dab), or diaminopropionic acid (Dap).

8. The compound is one of the following compounds a. NH-Idc-hGlu-Glu(Chx)-P2-Asp-R1 b. NH-Idc-hGlu-Glu-P2-Asp-R1 c. NH-Idc-hGlu-Glu(me)-P2-Asp-R1 or a salt, solvate or stereoisomer thereof, and The compound according to claim 1 or 2, wherein P2 is selected from lysine (Lys), ornithine (Orn), diaminobutyric acid (Dab), or diaminopropionic acid (Dap).

9. The compound is one of the following compounds a. NH-Idc-hGlu-Glu(Chx)-Dab-Asp-R1; b. NH-Idc-hGlu-Glu-Dab-Asp-R1; and / or c. NH-Idc-hGlu-Glu(me)-Dab-Asp-R1 ​ The compound according to claim 1 or 2, which is selected from any one of them or a salt, solvate or stereoisomer thereof.

10. The compound is NH-Idc-hGlu-Mix-P2-Asp-R1 or a salt, solvate or stereoisomer thereof, wherein P2 is selected from lysine (Lys), ornithine (Orn), diaminobutyric acid (Dab), or diaminopropionic acid (Dap), and Mix is any natural amino acid selected from the group consisting of Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Gln (glutamine), Gly (glycine), His (histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Nle (norleucine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine) and Val (valine). The compound according to claim 1 or 2.

11. The compound according to claim 6, wherein P2 is lysine (Lys), ornithine (Orn) or diaminobutyric acid (Dab).

12. R1 is of the formula 【Chemical Formula 9】 having, wherein Me is a methyl (CH ３ ) group, the compound according to claim 7.

13. The compound is a compound of the formula 【Chemical 10】 or a salt, solvate or stereoisomer thereof, wherein the dotted line represents a bond that may or may not be present, and if present, it forms a double bond in combination with the single bond already present. The compound according to claim 6.

14. The compound is a compound of the formula 【Chemical 11】 or a salt, solvate or stereoisomer thereof, wherein the dotted line represents a bond that may or may not be present, and if present, it forms a double bond in combination with the single bond already present. The compound according to claim 6.

15. The compound is a compound of the formula 【Chemical 12】 or a salt, solvate or stereoisomer thereof, wherein the dotted line represents a bond that may or may not be present, and if present, it forms a double bond in combination with the single bond already present. The compound according to claim 6.

16. R1 is of the formula 【Chemical Formula 13】 consisting only of. The compound according to claim 13.

17. The compound according to claim 1 or 2, wherein all amino acids in the compound are in the L configuration.

18. A pharmaceutical composition comprising the compound according to claim 1 or 2 and optionally a pharmaceutically acceptable excipient.

19. A pharmaceutical composition comprising the compound according to claim 16 and a pharmaceutically acceptable excipient.

20. A medicament comprising the compound according to claim 1 or 2.

21. A medicament comprising the compound according to claim 16.

22. Degenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, Lewy body dementia, mild cognitive impairment, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease; neonatal brain injury, particularly neonatal cerebral ischemia; traumatic brain injury; renal ischemia; hypoxic-ischemic injury; stroke-like condition brain injury; cardiac ischemia; myocardial infarction; amyotrophic lateral sclerosis; retinal injury; eye diseases such as blunt eye injury, ischemic optic neuropathy and glaucoma; skin injury; aseptic inflammatory diseases such as diabetes, atherosclerosis, myocardial ischemia, gout, pseudogout, joint laxity, atherosclerosis, syndrome triggered by aluminum salts, non-arteritic ischemic optic neuropathy, glaucoma and metabolic diseases; non-aseptic inflammatory diseases such as bacterial infections, particularly infections by bacteria producing pore-forming toxins, influenza virus infection and single-stranded RNA rhabdovirus family infections such as Maraba virus or vesicular stomatitis virus; diseases caused by pathogenic bacteria such as Brucella, Staphylococcus aureus and Salmonella; dyslipidemia; obesity; metabolic syndrome; and non-alcoholic fatty liver diseases such as non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver disease (NAFLD), cirrhosis, primary sclerosing cholangitis and hepatocellular carcinoma, a medicament comprising the compound according to claim 1 or 2 for use in the prevention and / or treatment thereof.

23. A medicament comprising the compound according to claim 1 or 2 for use in the prevention and / or treatment of dyslipidemia, obesity, metabolic syndrome, cirrhosis, non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD).

24. A medicament comprising the compound according to claim 16 for use in the prevention and / or treatment of dyslipidemia, obesity, metabolic syndrome, cirrhosis, non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD).

25. A medicament comprising the compound according to claim 1 or 2 for use in the prevention and / or treatment of non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD).

26. A medicament comprising the compound according to claim 16 for use in the prevention and / or treatment of non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD).

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