Caspase-2 inhibitor compounds

Abstract

The present invention relates to a compound of formula (II): TIFF2026501655000073.tif65170 or a pharmaceutically 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 involved in 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 major groups: those involved in regulating inflammatory processes (-1, -4, -5, -11, and -12) and those central to the induction and execution of apoptosis (-2, -3, -7, -8, -9, and -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 into active enzymes. Once activated, 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 organized disassembly of the cell and ultimately to 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), resulting in the activation of initiator caspase-2 (Shalini, 2015). Active caspase-2 cleaves Bid, and cleaved Bid (tBid) activates mitochondrial permeabilization through regulation of Bax and Bak (Enoksson, 2004). Mitochondrial cytochrome c is then released into the cytosol and induces the 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 apoptosis 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 via 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 to form a cleaved form (tBID), which participates in the mitochondrial pathway to amplify the apoptotic response (Li, 1998). Once initiator caspases are activated via the extrinsic or intrinsic apoptosis pathway, they mediate the activation of 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 the large subunit (p19) and small subunit (p12) that contain the active site. 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 must 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 possesses several unique features, including the presence of a nuclear localization signal that plays a central role in triggering apoptotic pathways after DNA damage in several cellular models. Furthermore, 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] The 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 a variety of diseases or conditions.

[0010] 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).

[0011] 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).

[0012] In stroke, apoptosis of cerebral neurons is the major pathological change occurring in the peri-infarct zone after transient global ischemia, which leads to ischemia / reperfusion (I / R) injury. Cerebral neuron apoptosis can lead to hemiplegia, death, or cognitive impairment after stroke (Turkmen, 2011). In animal models of stroke, caspase-2 expression and activation increased after I / R.

[0013] 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 cognitive and synaptic dysfunction in animal and cell models. The cleavage product, Δtau314, is resistant to fibrillation and is present at higher levels in the brains of cognitively impaired mice and humans with AD. Expression of a tau mutant that resists 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).

[0014] 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).

[0015] 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.

[0016] In the first generation of inhibitors, several peptide sequences were developed that were thought to selectively inhibit different caspases, such as 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).

[0017] To effectively inhibit apoptosis, selectivity for caspase-3 is crucial due to the high concentration of this caspase relative to other caspases in almost all tissues and its promiscuous nature (McStay, 2008). Furthermore, non-selective inhibitors of caspase-2 have limited clinical benefit in direct apoptosis inhibition because they act downstream of the mitochondrial outer membrane pore and cannot reverse the significant damage to mitochondrial function induced by caspase-2-mediated pore formation (Singh, 2019). Furthermore, selectivity may also be important in situations where caspase-8 (extrinsic apoptosis) is also activated and selective caspase-2 (intrinsic apoptosis) inhibition is required.

[0018] The inhibitor Ac-VDVAD-CHO is more potent against caspase-3 than caspase-2. This raises some questions about the validity of data generated with this reagent when used in a cellular environment as a "selective" caspase-2 inhibitor. Several publications have already raised specificity concerns with existing inhibitors, highlighting 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 mechanisms," Poreba et al., Cell Death and Differentiation). In this paper (mainly in the supplemental section), we provided a detailed kinetic analysis of caspase inhibitors / probes created based on natural amino acid sequences.

[0019] 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 transient (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 ​​residue, excluding other free nucleophilic species in the proteome. Therefore, the catalytic mechanism of the target protease plays a key role in selecting an appropriate warhead.

[0020] Importantly, the thiol group of the catalytic cysteine ​​residue in cysteine ​​proteases is more polarizable than the hydroxyl group on the catalytic serine or threonine; therefore, electrophiles used as warheads for cysteine ​​proteases may be more flexible than those for serine or threonine proteases (Powers, 2002). Therefore, caspases have been most widely studied with inhibitors containing warheads such as diazomethyl ketones, epoxides, and halo- and acyloxy-methyl ketones. The main advantages of these warheads are their ease of synthesis, good bioavailability, and Cys-selective reactivity at the active site. The most commonly used commercially available caspase inhibitor warheads are fluoro- (FMK or -CHF), chloro- (CMK or -CHCl), or acyloxy-methyl ketones (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 of the active site (Sanman, 2014; Powers, 2002).

[0021] FMK-based inhibitors were the first and have dominated research in this field to date. 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 several drawbacks, including 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 nonspecific binding of the reactive FMK group (Rozman-Pungercar, 2003; Schotte, 1999). Inhibitors containing FMK warheads have been shown to be toxic in vivo, particularly 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 in the preclinical stage due to their hepatotoxicity (Citarella, 2020).

[0022] AOMK is the weakest electrophile in this group and is therefore most suitable for the development of caspase inhibitors because it exhibits little cross-reactivity with other biological nucleophiles. Therefore, the weaker electrophilicity allows them to react more specifically with caspases and exhibit reduced cross-reactivity with other Cys-dependent proteases (Poreba et al., Chemical Reviews (2015) BI-BJ).

[0023] Down-modulation of caspase-2 expression effectively prevents apoptosis in several models.

[0024] 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).

[0025] 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.

[0026] In stroke apoptosis, treatment with microRNA (miR-1247-3p) inhibited caspase-2 expression and attenuated neuronal apoptosis (Zhang, 2019).

[0027] In animal models of Alzheimer's disease (AD) and other tauopathies, reducing caspase-2 levels restored long-term memory in mice with pre-existing deficits (Zhao, 2016).

[0028] Caspase-2 depletion protected mice with methionine / choline-deficient (MCD) diet-induced steatohepatitis from hepatocyte apoptosis and fibrosis progression (Machado, 2015).

[0029] Compounds capable of inhibiting caspase-2 activity have been reported, for example, in International Publication No. 2005 / 105829 and European Patent No. 2670774. However, these known caspase-2 inhibitors also have excessively high activity against caspase-3, making them unqualified 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 had moderate efficacy in cellular assays and possessed structural characteristics incompatible with in vivo use (Maillard, 2011). International Publication Nos. 2017 / 162674 and 2019 / 068538 disclose peptide compounds containing five amino acid units as caspase-2 inhibitors.

[0030] (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) had the highest caspase-2 inhibitory activity among the compounds assayed, but still showed no selectivity for caspase-3 and limited selectivity for caspase-8.

[0031] Thus, more specifically, there remains a need for potent and / or selective caspase-2 inhibitors that have significantly reduced activity against caspase-3 / caspase-8. In particular, 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 in which caspase-2 activity is implicated, such as nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD), obesity, metabolic syndrome, liver cirrhosis, neonatal cerebral ischemia, cardiac ischemia, and chronic degenerative diseases such as Alzheimer's disease.

[0032] 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. Summary of the Invention

[0033] The compounds of the present invention are intended to meet these needs. [Brief explanation of the drawings]

[0034] [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" caspase. The concentration of active caspase was determined by active site titration. [Figure 2] Raw data for calculation of the kb / I inhibition parameter of NH-23-C2 synthetic inhibitors against caspase-2. The kb / I parameter was measured under pseudo-first-order kinetic conditions ([I] >> [E]). NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-ACC was used as the substrate. ACC fluorescence was monitored using wavelengths of 355 nm (excitation) and 460 nm (emission). Second-order inhibition rates (kb / I) were determined in at least three independent experiments and presented as the mean value. GraphPad Prism 7 software was used for the calculation. [Figure 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 using the Morrison equation, and the IC50 parameter was calculated using the formula: IC50 = Ki × (1 + [S] / Km), where [S] is the substrate concentration used in the assay and Km is the Michaelis-Menten constant for the substrate. All measurements were performed at least three times, 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. [Figure 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 was 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 was 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 the 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 / sec, relative fluorescence units / sec) was set to 100%, and the cleavage rates of other substrates in the series were adjusted accordingly. The concentrations of individual substrates were 10 µM, and the concentration of combinatorial substrates was 100 µM. The caspase-2 concentration was 10 nM. DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description of the Invention In the context of the present invention, the following terms have the meanings detailed below.

[0036] "C 1-6 The term "alkyl" 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 ("alkyl") and attached to the rest of the molecule by a single bond, including, but not limited to, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, etc. Preferably, "alkyl" refers to methyl or ethyl.

[0037] "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, but not limited to, for example, cyclopropyl, cyclohexyl, or cyclopentyl.

[0038] "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, but not limited to, for example, phenyl, naphthyl, etc. Preferably, "aryl" refers to phenyl.

[0039] The term "halogen" refers to bromo, chloro, iodo or fluoro.

[0040] The term "C1-C6 haloalkyl" refers to an alkyl group as defined above in which at least one of the hydrogen atoms has been replaced with a halogen atom, such as CF3, CCl3, CHF2, CH2F, CF2CF3.

[0041] "C 1-6 The term "alkoxyl" refers to C 1-6alkyl is an alkyl group 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.

[0042] "(C6-C 10 The term "aryl(C-C)alkyl" refers to an aryl group as defined above that is attached to the rest of the molecule via an alkyl group as defined above. Preferably, (C-C 10 )Aryl(C1-C6)alkyl is (C6)aryl(C1-C3)alkyl, for example, benzyl.

[0043] "5- to 10-membered heterocyclyl" refers to a stable 5- to 10-membered ring group, preferably a 5- or 6-membered ring, which consists of carbon atoms and 1 to 5, preferably 1 to 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, and octahydro-pyrrolopyrazine.

[0044] "5- to 10-membered heteroaryl" refers to a stable 5- to 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.

[0045] As understood in the art, there may be some degree of substitution for the residues defined above. Therefore, any group of the present invention may have substitution. In this document, when a substituent of a group of the present invention is mentioned, it means that the specific residue may be substituted at one or more available positions by one or more substituents. The aforementioned substituents include, but are not limited to, for example, 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- to 10-membered heterocyclyl, 5- to 10-membered heteroaryl, —CN, —NO2, —OR, —SR, —C(O)R, —C(O)OR, —OC(O)R, —C(O)NR2, —NR2, and —S2R, 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 ) independently selected from alkyl, 5- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl.

[0046] The compounds of the present invention may be in the form of a salt, solvate or stereoisomer, preferably a pharmaceutically acceptable salt, solvate or stereoisomer.

[0047] As previously mentioned, the present invention also provides "salts" of the compounds described herein. By way of example, the aforementioned salts may be acid addition salts, base addition salts, or metal salts, and can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical processes known to those skilled in the art. See generally G.S. 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; S.M. 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 the appropriate base or acid, for example, in water or an organic solvent, or 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 hydrochloride, hydrobromide, hydroiodide, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, and the like; and organic acid addition salts such as acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate, p-toluenesulfonate, camphorsulfonate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, and the like. Examples of base addition salts include salts of phenyl, hydroxybenzoate, methyl, hydroxybenzoate ...

[0048] The term " solvate " according to the present invention should be understood to mean any form of the active compound according to the present invention that is combined with another molecule (possibly polar solvent) through non-covalent bond.Examples of solvate include hydrate and alcoholate.Solvation method is generally known in the state of the art.

[0049] As used herein, the term "stereoisomer" 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.

[0050] The term "chiral center" refers to a carbon atom to which four different groups are attached.

[0051] 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; one enantiomer rotates the plane of polarized light in one direction and the other enantiomer rotates the plane of polarized light in the opposite direction, and when present in equal amounts, form a racemate.

[0052] The terms "racemate" or "racemate" refer to a mixture of equal amounts of enantiomers, wherein the mixture is optically inactive.

[0053] 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.

[0054] 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.

[0055] The compounds of the present invention have chiral centers, and therefore can 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 their mixtures (including racemic mixtures, enantiomerically enriched mixtures, and diastereomeric enriched mixtures) are considered to be within the scope of the present invention.

[0056] The term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and that, when administered to humans, do not normally cause allergic or similar adverse reactions, such as stomach upset, dizziness, etc. Preferably, as used herein, the term "pharmaceutically 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 particularly in humans.

[0057] As used herein, "fluorophore" refers to a molecule or moiety that is capable of re-emitting light upon optical excitation.

[0058] As used herein, the term "warhead" refers to a moiety present on the compounds of the present invention that can covalently bind to the active site of the target enzyme (caspase-2), thereby achieving 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.

[0059] 1) Brady KD. Bimodal inhibition of caspase-1 by aryloxymethyl and acyloxymethyl ketones.Biochemistry.1998;37:8508-15.

[0060] 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.

[0061] Poreba et al., Caspase selective reagents for diagnosing apoptotic mechanisms, CDD 2019.

[0062] As used herein, the term "prodrug" refers to a compound that is a drug precursor that releases the drug (or "activity") in vivo after administration through some chemical or physiological process (e.g., hydrolysis, enzymatic cleavage or hydrolysis, or metabolic conversion to the desired drug form). The present invention includes within its scope prodrugs such as compounds of the invention methylated at P1 (Asp), where in vivo demethylation of the P1 group releases the active ingredient.

[0063] Compounds of the Invention The present application exemplifies potent and selective caspase-2 ligands. In particular, the present application provides compounds of formula (I): TIFF2026501655000002.tif73170 or a salt, solvate or stereoisomer thereof, During the ceremony, R2 and R3 may each independently be H, or R2 and R3 may be absent, resulting in the deprotonated form CO(O -) is obtained, During the ceremony, n is 1, 2 or 3; A is an amine group, and R1 is a reactive group.

[0064] The Examples herein demonstrate that even minor modifications at P2 of compounds of formula (I) have a significant impact on the binding affinity of the resulting analogs (see Example 2). In this sense, it is clear that Ser->Lys, Orn, or Dab substitutions are tolerated by caspase-2, but this is not the case for caspase-3 and caspase-8 (see Tables 3-5). This unexpected result translates into increased selectivity of these compounds for caspase-2, with the Dab analogs in particular being 5-fold more selective than caspase-3 and over 20-fold more selective than caspase-8 (see Tables 6-7).

[0065] Thus, the present specification discloses certain ligands of formula (I) that have a free primary amine group at P2. Specifically, the present application demonstrates that the peptide corresponding to Idc-hGlu-Thr(Bzl)-[Lys / Orn / Dab]-Asp-OR1 is indeed a potent and selective ligand for caspase 2 (see Examples 2 and 3).

[0066] However, peptide-based therapeutic agents tend to exhibit rapid in vivo proteolysis, poor oral bioavailability, and poor pharmacokinetics. More precisely, peptide-based caspase inhibitors have limited clinical use due to their poor pharmacokinetic properties, such as poor oral bioavailability, cell permeability, and metabolic stability.

[0067] Over the past few years, many advances have been made in the incorporation of unnatural amino acids, methylation of carboxyl groups, fluorine substituents, engineered salt bridges (S. Xiong, J. Virol. 2017, 91), introduction of D-amino acids (B.D. Welch, J. Virol. 2010, 84), backbone bridges (O. Bolarinwa, Org. Biomol. Chem. 2018, 16, 7878-7882), hydrogen bond surrogates (D. Wang, Chem. Int. Ed. 2008, 47, 1879-1882), foldamers (W.S. Horne, Proc. Natl. Acad. Sci. USA 2009,106,14751-14756.), cyclization (M.K. Lee, Exp. Mol. Med. 2006,38,18-26.), multimerization (C.Whang, J.Med.Chem. 2022,65,4,2809-2819), conjugation to small molecules (M.Ferrer, Nat.Struct.Biol. 1999,6,953-960), or to different classes of lipids (Y.Zhu, H.Chong, J.Virol. 2019,93,e02312-02318), as well as PEGylation (C.Wang, Viruses Several concepts and strategies have been reported to improve the inhibitor properties of peptides, such as the use of hydroxybenzoates (D. Wensel, J. Virol. 2019, 11, 811) or dual-targeting inhibitors (D. Wensel, J. Virol. 2019, 93, e00907-00919). The resulting derivatives may exhibit increased activity but, more importantly, improved metabolic stability compared to wild-type peptides.

[0068] Methylation of the carboxyl group of the side chain of an amino acid confers beneficial properties to peptides, such as increased solubility in aqueous and / or organic solvents, resistance to proteolysis, diffusion across membranes, and increased oral bioavailability (Gordon DJ, J. Pept. Res. 2002; 60: 34-55, Adessi C, Biol. Chem. 2003; 278(16): 13905-13911, Kokkoni N. Biochemistry. 2006; 45: 9906-9918). Therefore, in the present invention, we methylated the side chains of aspartic acid (R3 in Formula I) and homoglutamic acid (R2 in Formula I) because this is expected to increase cell membrane permeability, as successfully demonstrated in Z-VAD-fmk, a common inhibitor used to treat caspases in cell-based experiments. In this sense, in the case of Z-VAD-fmk, methylation of the aspartic acid side chain significantly increased cell permeability without affecting the activity of the molecule.

[0069] Modification of organic molecules with fluorine substituents is a widely used strategy for improving small molecule drugs due to fluorine's ability to productively influence molecular properties such as conformation, the pKa values ​​of adjacent functional groups, and hydrophobicity. This often leads to improved interactions with proteins, enzymes, receptors, or membranes, thereby improving pharmacological properties, cell permeability, bioavailability, and activity (S. Purser, Chem. Soc. Rev. 2008, 37, 320-330). Furthermore, due to the significant stability of the fluorine covalent bond, these molecules often exhibit improved metabolic stability.

[0070] The ready accessibility of fluorine-containing amino acids (J. Moschner, Chem. Rev. 2019, 119, 10718-10801; J. Leppkes, J. Fluorine Chem. 2020, 232, 109453) has sparked renewed interest in these building blocks for drug design (H. Mei, Eur. J. Med. Chem. 2020, 186, 111826) and peptide and protein engineering. Selective incorporation of fluorine-containing amino acids into peptides allows for the modulation of distinct pharmacokinetic and physicochemical functionalities (e.g., structure, folding, or stability against thermal and chemical denaturation) of the resulting biopolymers. Furthermore, this can improve the metabolic stability, biological activity, cell permeability, and bioavailability of peptide-based drugs (G. Akcay, J. Fluorine Chem. 2009, 130, 1178-1182; M. Salwiczek, Chem. Soc. Rev. 2012, 41, 2135-2171; ENG Marsh, Acc. Chem. Res. 2014, 47, 2878-2886; AABerger, Acc. Chem. Res. 2017, 50, 2093-2103; S. Huhmann, Eur. J. Org. Chem. 2018, 3667-3679; R. Smits, Curr. Top. Med. Chem. 2006, 6, 1483-1498).

[0071] In this application, we attempted to introduce fluorinated amino acid analogs and methylation in the carboxy group of the amino acid side chain into compounds targeting caspase-2 to improve their drug performance.

[0072] Therefore, in the present invention, the inventors provide a compound of general formula (II): TIFF2026501655000003.tif80170 or a salt, solvate or stereoisomer thereof (In the formula, In the heterocycle at the P5 position TIFF2026501655000004.tif11170 (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; R2 and R3 are hydrogen or C, including branched alkyl and cycloalkyl. 1-4 independently selected from alkyl, R4, R5, R6 and R7 are independently hydrogen, halogen, or C, including branched alkyl and cycloalkyl. 1-4 alkyl, wherein each C 1-4 The alkyl is optionally substituted with 1 to 3 halogen atoms, x and z are each independently selected from 0, 1, or 2, and n is selected from 0, 1, 2, or 3; R8 and R9 are independently selected from hydrogen, halogen, or C1-4 alkyl, including branched alkyl and cycloalkyl, wherein each C1-4 alkyl is optionally substituted with 1 to 3 halogen atoms.

[0073] R1 is 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, particularly 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).

[0074] Note that for 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: The fluorophore-like portion is ACC in TIFF2026501655000005.tif27170.

[0075] More specifically, R1 has the formula: and a moiety such as a fluorophore (used in connection with a substrate) selected from the group of During the ceremony, W is selected from -NH- and -O-; R10 to R13 are H, C 1-6 Alkyl and C 3-7 independently selected from cycloalkyl, aryl, heteroaryl, CF, —CHCOOH, —CHCONHR, and —CHOR; and R14 is selected from H, C1-6 alkyl and C3-7 cycloalkyl.

[0076] Examples of R1 moieties as fluorophores can be selected from any of the following lists: TIFF2026501655000007.tif28170

[0077] Other examples of moieties potentially useful as fluorophores can be selected from the group consisting of: TIFF2026501655000008.tif104170

[0078] In the context of this invention, a fluorophore (or fluorescent dye, similar to a chromophore) is a fluorescent chemical compound that can re-emit light upon photoexcitation. Fluorophores or substrates typically contain several linked 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 represents -NH-, such as ACC.Additional fluorophores in R1 include 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-1, PO-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 Ca2+ 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 yellow fluorescent protein) Oregon Green 488 Oregon Green 488 antibody conjugate pH 8.0 Fluo-3 BCECF pH 9.0 SBFI-Na+ Indo-1, Ca2+ saturation 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, LysoSensor Yellow pH 9.0 Indo-1, Ca2+ saturation 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, and Cy2 LysoSensor Green.

[0079] On the other hand, for 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 acyloxymethylketone (AOMK) or a simple hydrogen atom (in an aldehyde).

[0080] Thus, in another embodiment, R1 may be a warhead, and in particular, R1 may be TIFF2026501655000009.tif100170 or a salt, solvate or stereoisomer thereof; During the ceremony, q is 0, 1, 2, 3, 4 or 5; Each Z1 is C 1-6 Alkyl, halogen, -CN, -NO2 and C 1-6 are independently selected from alkoxyl, Z2 is a halogen; The Z3 is a C 1-6 Alkyl, C 6-10 Aryl and (C 6-10 ) Aryl (C 1-6 ) alkyl.

[0081] Specific examples of warheads useful in the present invention can be selected from any of the following (all of the following compounds are directly bound to the a-COOH group of the P1 subunit): TIFF2026501655000010.tif147170TIFF2026501655000011.tif218170

[0082] Preferably, R1 has the formula: TIFF2026501655000012.tif21170 Warhead, During the ceremony, q is 0, 1, 2, 3 or 4; Each Z1 is C 1-6 Alkyl, halogen, -CN and C 1-6 are independently selected from alkoxyl.

[0083] In one embodiment, Z1 is independently selected from halogen, methyl and -OMe, preferably F, Cl and Me (methyl).

[0084] In a further embodiment, R1 is of the formula Selected from the group TIFF2026501655000013.tif20170, where each Z1 is C1-6 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).

[0085] In a preferred embodiment, R1 is of the formula I have TIFF2026501655000014.tif19170.

[0086] In another preferred embodiment, R1 is represented by the following formula: TIFF2026501655000015.tif36170 (in the formula, j is 0 or 1, R 10 , R 11 , R 12 , R 13 and R 14 are each independently hydrogen, halogen, or C, including branched alkyl and cycloalkyl. 1-4 selected from alkyl, phenyl, phenoxy, nitro, nitrile, PhCO, NHAc, where each alkyl is optionally substituted with 1 to 3 halogen atoms).

[0087] It should be noted that in certain embodiments, any of R10 and R11, R14 and R13, R13 and R12, or R11 and R12 together form a fused phenyl ring that forms an additional aromatic ring, preferably a naphthyl group.

[0088] In another preferred embodiment, R1 incorporates the P1 group to form a group having the formula: TIFF2026501655000016.tif28170

[0089] In another embodiment, we provide herein a compound of general formula (II) or a salt, solvate or stereoisomer thereof: (In the formula, In the heterocycle at the P5 position TIFF2026501655000017.tif10170 (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; R4, R5, R6 and R7 are each independently selected from hydrogen, halogen, e.g., fluorine or trifluoromethyl; x and z are each 1; n is 0, 1, 2 or 3; R8 and R9 are each independently selected from hydrogen, halogen, such as fluorine or trifluoromethyl; R2 and R3 are independently selected from hydrogen or C 1-4 alkyl, preferably selected from methyl or ethyl, and R1 is a chemically reactive group as defined above).

[0090] In another embodiment, we provide herein a compound of general formula (II) or a salt, solvate or stereoisomer thereof: (In the formula, In the heterocycle at the P5 position TIFF2026501655000018.tif11170 (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; R4, R5, R6 and R7 are each independently selected from hydrogen or halogen, such as fluorine; x and z are each 1, n is 0, 1, 2 or 3; R8 and R9 are each independently selected from hydrogen, halogen, such as fluorine or trifluoromethyl; R2 and R3 are independently selected from hydrogen or methyl; and R1 is a chemically reactive group as defined above).

[0091] In another embodiment, we provide herein a compound of general formula (II) or a salt, solvate or stereoisomer thereof: (In the formula, In the heterocycle at the P5 position TIFF2026501655000019.tif11170 (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; R4 and R6 are each independently selected from halogen, e.g., fluorine; R5 and R7 are both hydrogen; x and z are each 1, n is 0, 1, 2 or 3; R8 and R9 are each independently selected from hydrogen, halogen, such as fluorine or trifluoromethyl; R2 and R3 are independently selected from hydrogen or methyl; and R1 is a chemically reactive group as defined above).

[0092] In another embodiment, we provide herein a compound of general formula (II) or a salt, solvate or stereoisomer thereof: (In the formula, In the heterocycle at the P5 position TIFF2026501655000020.tif11170 (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; R4 and R6 are each independently selected from halogen, e.g., fluorine; R5 and R7 are both hydrogen; x and z are each 1, n is 0, 1, 2 or 3; R8 and R9 are both hydrogen; R2 and R3 are independently selected from hydrogen or methyl; and R1 is a chemically reactive group as defined above).

[0093] In yet another embodiment, we provide herein a compound of general formula (III): TIFF2026501655000021.tif79170 or a salt, solvate or stereoisomer thereof, wherein: In the heterocycle at the P5 position TIFF2026501655000022.tif10170 (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; R2 and R3 are hydrogen or C, including branched alkyl and cycloalkyl. 1-4 independently selected from alkyl, R4, R5, R6 and R7 are independently hydrogen, halogen, or C, including branched alkyl and cycloalkyl. 1-4 alkyl, wherein each C 1-4 The alkyl is optionally substituted with 1 to 3 halogen atoms, x and z are each independently selected from 0, 1, or 2, and n is 0, 1, 2, or 3; R8 and R9 are independently selected from hydrogen, halogen, or C1-4 alkyl, including branched alkyl and cycloalkyl, wherein each C1-4 alkyl is optionally substituted with 1 to 3 halogen atoms; j is 0 or 1, R 10 , R 11 , R 12 , R 13 and R 14 are each independently selected from hydrogen, halogen, or C1-4 alkyl, including branched alkyl and cycloalkyl, phenyl, phenoxy, nitro, nitrile, PhCO, NHAc, where each alkyl is optionally substituted with 1 to 3 halogen atoms.

[0094] In certain embodiments, R 10 and R 11 , R 14 and R 13 , R 13 and R 12 , or R 11 and R 12It should be noted that any of the following may be taken together to form a fused phenyl ring forming an additional aromatic ring, preferably a naphthyl group.

[0095] In yet another embodiment, we provide herein a compound of general formula (III), or a salt, solvate or stereoisomer thereof: (In the formula, In the heterocycle at the P5 position TIFF2026501655000023.tif10170 (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; R4, R5, R6 and R7 are each independently selected from hydrogen, halogen, e.g., fluorine or trifluoromethyl; x and z are each 1; n is 0, 1, 2 or 3; R8 and R9 are each independently selected from hydrogen, halogen, such as fluorine or trifluoromethyl; R2 and R3 are independently selected from hydrogen or C 1-4 alkyl, preferably selected from methyl or ethyl, j is 0 or 1, and R 10 , R 11 , R 12 , R 13 and R 14 are each independently selected from hydrogen, halogen, or C1-4 alkyl, including branched alkyl and cycloalkyl, phenyl, phenoxy, nitro, nitrile, PhCO, NHAc, where each alkyl is optionally substituted with 1 to 3 halogen atoms.

[0096] In certain embodiments, R 10 and R 11 , R 14 and R 13 , R 13 and R 12 , or R 11 and R 12It should be noted that any of the following may be taken together to form a fused phenyl ring forming an additional aromatic ring, preferably a naphthyl group.

[0097] In yet another embodiment, we provide herein a compound of general formula (III), or a salt, solvate or stereoisomer thereof, wherein: In the heterocycle at the P5 position TIFF2026501655000024.tif11170 (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; R4, R5, R6 and R7 are each independently selected from hydrogen, halogen, e.g., fluorine or trifluoromethyl; x and z are each 1; n is 0, 1, 2 or 3; R8 and R9 are each independently selected from hydrogen, halogen, e.g., fluorine or trifluoromethyl; R2 and R3 are independently selected from hydrogen or methyl; j is 0 or 1, and R 10 , R 11 , R 12 , R 13 and R 14 are each independently selected from hydrogen, halogen, or C1-4 alkyl, including branched alkyl and cycloalkyl, phenyl, phenoxy, nitro, nitrile, PhCO, NHAc, where each alkyl is optionally substituted with 1 to 3 halogen atoms.

[0098] In certain embodiments, R 10 and R 11 , R 14 and R 13 , R 13 and R 12 , or R 11 and R 12 It should be noted that any of the following may be taken together to form a fused phenyl ring forming an additional aromatic ring, preferably a naphthyl group.

[0099] In yet another embodiment, we provide herein a compound of general formula (IV), or a salt, solvate or stereoisomer thereof: TIFF2026501655000025.tif79170 (in the formula, In the heterocycle at the P5 position TIFF2026501655000026.tif10170 (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; R2 and R3 are independently hydrogen or C, including branched alkyl and cycloalkyl. 1-4 Preferably, R2 and R3 are independently selected from hydrogen, methyl or ethyl; R4 and R6 are each independently hydrogen, halogen, or C, including branched alkyl and cycloalkyl. 1-4 alkyl, wherein each C 1-4 The alkyl is optionally substituted with 1 to 3 halogen atoms. Preferably, R4 and R6 are each independently selected from hydrogen, halogen, such as fluorine or trifluoromethyl; x and z are each independently selected from 0, 1 or 2, and n is 0, 1, 2 or 3. Preferably, x and z are 1 and n is 0, 1, 2 or 3; R8 and R9 are independently hydrogen, halogen, or C, including branched alkyl and cycloalkyl. 1-4 alkyl, wherein each C 1-4 The alkyl is optionally substituted with 1 to 3 halogen atoms. Preferably, R8 and R9 are independently selected from hydrogen or halogen, such as fluorine; j is 0 or 1, and R 10 , R 11 , R 12 , R 13 and R 14are each independently selected from hydrogen, halogen, or C alkyl, including branched alkyl and cycloalkyl, phenyl, phenoxy, nitro, nitrile, PhCO, NHAc, where each alkyl is optionally substituted with 1 to 3 halogen atoms. 14 or R 10 is selected from halogen, for example fluorine, methyl or trifluoromethyl, R 11 , R 12 and R 13 are each hydrogen or a halogen, such as fluorine.

[0100] In yet another embodiment, we provide herein a compound of general formula (V), or a salt, solvate or stereoisomer thereof: TIFF2026501655000027.tif75170 (in the formula, In the heterocycle at the P5 position TIFF2026501655000028.tif10170 (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; R2 and R3 are independently selected from hydrogen or methyl; R4 and R6 are each independently selected from hydrogen or halogen, such as fluorine; n is 0, 1, 2 or 3; j is 0 or 1, and R 14 or R 10 is selected from halogen, e.g., fluorine, or methyl; R 11 and R 13 are each hydrogen or a halogen, such as fluorine, If j is 0, R 14 , R 10 is a halogen, e.g., fluorine, and R 11 and R 13 is a halogen, e.g., fluorine).

[0101] Note that all structures above encompass all possible forms (e.g., one deprotonated acid, two deprotonated acids with the amine in both states), and that the P5 position may be present as shown, or may be an oxidized derivative in which an additional double bond is present.

[0102] In certain embodiments, the compound of formula (II) is selected from the group consisting of the following compounds (protonated or deprotonated) or salts, solvates, or stereoisomers thereof: TIFF2026501655000029.tif69170

[0103] In particular, the following chemical structures (protonated or deprotonated) or salts, solvates or stereoisomers thereof selected from any one of the following lists are specifically encompassed by the present invention: TIFF2026501655000030.tif229170

[0104] More specifically, the following chemical structures (protonated or deprotonated) or a salt, solvate, or stereoisomer thereof selected from any one of the following lists are specifically encompassed by the present invention: TIFF2026501655000031.tif184170

[0105] Note that in Example 6, the inhibitory activity of some of the above compounds was tested.

[0106] Pharmaceutical Composition 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.

[0107] Thus, in a further aspect, the present invention relates to a pharmaceutical composition comprising a compound of any of the above formulae defined herein, wherein R1 is warhead such as AOMK or a salt, solvate or stereoisomer thereof, and at least one pharmaceutically acceptable excipient.

[0108] 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 Excipients" by Rowe CR; Paul JS; Marian EQ, sixth Edition, 2009.

[0109] 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 dosage form. The aforementioned pharmaceutical dosage forms of the pharmaceutical composition are prepared according to conventional methods known to those skilled in the art.

[0110] 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.

[0111] 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 pharmaceutically acceptable wetting agents, such as sodium lauryl sulfate.Solid oral compositions 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 optionally be coated, particularly with enteric coating, according to methods well known in conventional pharmaceutical practice.

[0112] 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.

[0113] The formulations referred to may be prepared using standard methods such as those described or referred to in the European Pharmacopoeia and the United States Pharmacopoeia and similar reference texts.

[0114] The compounds or compositions of the present invention may be administered by any suitable method, such as oral, sublingual, intranasal, intraocular, parenteral, subcutaneous, intramuscular, intravenous, or transdermal administration.

[0115] Generally, the effective amount of a compound of the present invention to be administered will depend on the relative efficacy of the compound selected, the severity of the disorder being treated and / or prevented, and the patient's body weight. The active compound can be administered one or more times daily, for example, 1, 2, 3, or 4 times daily, 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, an effective dosage of a compound of the present invention is about 500 mg / kg body weight / day or less. In another embodiment, an effective dosage of a compound of the present invention is about 100 mg / kg body weight / day or less. In another embodiment, an effective dosage ranges from about 0.01 mg / kg body weight / day to about 100 mg / kg body weight / day of a compound of the present invention. In another embodiment, an effective dosage ranges from about 0.02 mg / kg body weight / day to about 50 mg / kg body weight / day, and in another embodiment, from about 0.025 mg / kg body weight / day to about 20 mg / kg body weight / day.

[0116] Uses of the Compounds of the Invention Compounds of the invention in which R1 is a group that includes a fluorophore moiety can be used as activity-based probes for determining caspase-2 activity.

[0117] Therefore, in another aspect, the present invention refers to the in vitro use of a compound of the invention as defined herein, or a salt, solvate or stereoisomer thereof, wherein R1 is a group comprising a fluorophore moiety, for determining caspase-2 activity.

[0118] In certain embodiments, caspase-2 activity in a cell or tissue can be determined by contacting a compound of the invention as defined herein, or a salt, solvate or stereoisomer thereof, wherein R1 is a group comprising a fluorophore moiety, with a sample comprising said cell or tissue and measuring the fluorescent signal emitted upon light excitation.

[0119] Compounds of any of the above formulas of the present 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. Thus, compounds of the present invention in which R1 is a group can be used to prevent or treat diseases or disorders involving caspase-2 activity.

[0120] Thus, in another aspect, the present invention relates to a compound of the invention wherein R1 is a warhead such as AOMK, or a salt, solvate or stereoisomer thereof, for use as a medicament.

[0121] In another aspect, the present invention provides 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; and prevention of cytotoxicity. prevention of cytotoxicity due to physical factors such as radiation and acoustic trauma, especially chemically mediated cytotoxicity; skin injuries; sterile inflammatory diseases, such as 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, such as bacterial infections, especially infections by bacteria producing pore-forming toxins, influenza virus infections and single-stranded RNA rhabdoviridae infections, such as Maraba virus or vesicular stomatitis virus; diseases caused by pathogenic bacteria, such as Brucella, Staphylococcus aureus aureus and Salmonella; dyslipidemia; obesity; metabolic syndrome; and non-alcoholic fatty liver disease, e.g., non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver disease (NAFLD), or a salt, solvate or stereoisomer thereof.

[0122] In a preferred embodiment, the present invention relates to a compound of the present invention, wherein R1 is 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).

[0123] 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.

[0124] The term "ameliorate" in the context of the present invention is understood to mean any improvement to the condition of the treated patient.

[0125] 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.

[0126] In one embodiment, any of the methods or uses described herein may further comprise administering to the patient at least one other therapeutic agent.

[0127] 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. [Example]

[0128] 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 (Louiseville, KY, USA), PE Biosciences Limited (Hong Kong, China), and Combi-Blocks (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 purchased from Iris Biotech. GmbH. Anhydrous HOBt was purchased from Creosalus. 2,4,6-Trimethylpyridine (2,4,6-collidine), acetonitrile (ACN, HPLC gradient grade), and triisopropylsilane (TIPS) were purchased from Sigma-Aldrich. N,N'-Dimethylformamide (DMF, analytically pure), methanol (MeOH), dichloromethane (DCM), AcOH, diethyl ether (EtO), and phosphorus pentoxide (PO) 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 HPLC-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). The LC-MS assay was run from 95% phase A to 5% phase A over 20 min. All compounds were at least 95% pure.

[0129] 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 previously. Unless otherwise specified, all amino acids have the L configuration.

[0130] 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. Next, the Fmoc-protecting group was 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 minimal 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 three times with DMF. The Fmoc-ACC-OH coupling was repeated using 1.5 equivalents of the above reagent to improve the coupling yield. 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 h and repeated using 1.5 equivalents of Fmoc-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. The resin was washed six times with DMF, three times with DCM, and three times with MeOH, and dried overnight over PO in a desiccator. The resulting resin (approximately 12 g) was divided into 100 mg portions and used for the synthesis of individual caspase-2 substrates.

[0131] P2 substrate synthesis ACC-labeled fluorescent substrates of the general formula NH-Idc-hGlu-Thr(Bzl)-P2-Asp-ACC were synthesized using a MultiChem 48-well synthesizer (FlexChem, SciGene, CA, USA). For each substrate, 100 mg and 0.05 mmol of NH2-Asp(tBu)-ACC resin were placed in 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 thirty 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 preincubated with 3 equivalents (x30) of HATU (4.5 mmol, 1.72 g) and 2,4,6-collidine (4.5 mmol, 600 μL) in a minimal amount of DMF for 1 minute and poured onto the resin. The P3 coupling reaction was carried out for 3 hours, 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 preincubated with 3 equivalents (x30) of HATU (4.5 mmol, 1.72 g) and 2,4,6-collidine (4.5 mmol, 600 mL) in a minimal 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 preincubated 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 hours, followed by a ninhydrin test. The Fmoc group was then removed from each substrate using 20% ​​piperidine in DMF. The resin in each well was washed six times with DMF, three times with DCM, and three times with MeOH, and then dried overnight over PO in a desiccator. 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 hours (shaking once every 15 minutes). The solution from each well was collected separately, and the remaining resin was washed with TFA. The substrate was then precipitated with ice-cold EtO for 30 minutes and centrifuged. The supernatant was then discarded, and the pellet was resuspended in ice-cold EtO 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.

[0132] Preparation of recombinant caspases Detailed protocols for the expression and purification of human apoptotic caspases can be found elsewhere (Stennicke, 1999).

[0133] Enzyme kinetics research Screening of P2 substrates for caspases-2, -3, and -8 was performed using an fMax spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA) operating in fluorescence kinetic mode in 96-well Corning (Corning, NY, USA) plates. ACC 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 no. 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. All enzymes were preincubated for 15 min before any activity assay. Substrates were screened at a concentration of 10 mM, and the caspase concentrations were as follows: caspase-2 5 nM, caspase-3 15 nM, and caspase-8 40 nM. The total volume in a single well was 100 mL. The 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 the rate of fluorescence release / production (RFU / sec relative fluorescence units / sec) per 10 nM of caspase.

[0134] result We aimed to develop highly specific and selective caspase-2 substrates by reexamining its catalytic selectivity at the P2 (also referred to as AA throughout this invention) position. According to the generally accepted notion, "specific" refers to high kinetic parameters (e.g., high kcat / KM for substrates and high kob / I for inhibitors), while "selective" means that some reagents (substrates / inhibitors) target only one enzyme. The primary goal of this portion of the present 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 as the most selective substrate for caspase-2 (Poreba et al., Cell Death & Differentiation 2019, 26, 2695-2709). From the WRMP23 formula described above, the following Scaffold 1 (see Scaffold 1 below) was designed, which forms the basis for the de novo development of highly specific and selective caspase-2 substrates and inhibitors of the present invention: TIFF2026501655000032.tif51170

[0135] Scaffold 1. NH-Idc-hGlu-Thr(Bzl)-AA-Asp-R1. R1: -ACC (substrate) or -AOMK (inhibitor). R3: H. R2: H. AA: P2.

[0136] 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 Figure 1 and Table 1).

[0137] TIFF2026501655000033.tif172170TIFF2026501655000034.tif27170

[0138] As shown in the table above, the inventors of the present invention have identified compounds of the formula We have found that compounds of the above formula, which have Lys at the AA position instead of serine, exhibit high caspase-2 activity and remarkable selectivity for caspase-3 or caspase-8, thus resulting in improved caspase-2 substrate inhibitors. We further found that compounds of the above formula, which have His at the AA position instead of serine, exhibit strong caspase-2 activity.

[0139] 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 (Louiseville, KY, USA), PE Biosciences Limited (Hong Kong, China), and Combi-Blocks (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 (EtO), and phosphorus pentoxide (PO) were obtained from POCh (Gliwice, Poland). Diazomethane for the synthesis of acyloxymethyl ketone (AOMK) inhibitors was generated according to the Aldrich Technical Bulletin (AL-180) protocol. All AOMK inhibitors were purified by reverse-phase HPLC on a Waters system (Waters M600 solvent delivery module and Waters M489 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) over 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). The LC-MS assay was run from 95% phase A to 5% phase A over 20 min. All compounds were at least 95% pure.

[0140] 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 NH-Idc-hGlu-Thr(Bzl)-AA-Asp-AOMK were synthesized according to a previously described general method (Poreba 2019, Poreba 2016). Ac-VDVAD-AOMK was synthesized as a control inhibitor. A detailed procedure for the synthesis of AOMK-based inhibitors is exemplified by 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 noted, all amino acids have the L configuration.

[0141] 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 in an ice / acetone bath at -10 °C for 10 min. 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. Next, a solution of the mixed anhydride was 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. To obtain Boc-Asp(tBu)-CHBr, 15 mL of a 1:2 solution of HBr (30% by weight in CHCOOH) 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 extracted with water (once), saturated aqueous NaHCO (twice), and brine (twice). The organic fraction was dried over MgSO 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, as determined by HPLC, was >95%, and the overall yield was >90%. In the next reaction, 1 equivalent of Boc-Asp(tBu)-CHBr was dissolved in a small amount of DMF, followed by the addition of KF (3 equivalents) and 2,6-dimethylbenzimic acid (2,6-DMBA) (1.2 equivalents). 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 extracted with 5% citric acid (twice), 5% aqueous NaHCO3 (twice), and brine (twice). The organic fraction was then dried over MgSO4 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 (NH2-Asp-AOMK) as a yellow oil, which was used without further purification (98% yield by HPLC) (Block B, Synthesis Scheme 1). In a separate synthesis, the 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 equivalents, 1 mmol, 383 mg) was dissolved in a minimum volume of anhydrous DCM, followed by the addition of 4.5 equivalents of DIPEA (1.5 mmol, 260 μL). The mixture was activated for 1 minute and poured onto the resin. The reaction mixture was stirred for 3 hours. The Fmoc group was then removed with 20% piperidine in DMF, and the resin was washed six times with DMF. Next, Fmoc-Thr(Bzl)-OH (3 equiv., 1 mmol, 431 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 μL) 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 six times with DMF. Next, Fmoc-hGlu(tBu)-OH (3 equiv., 1 mmol, 440 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 μL) 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 six 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 μL) was added. The mixture was poured onto the resin, and the reaction was allowed to proceed for 3 h. After this, the Fmoc group was removed with 20% piperidine in DMF, and the resin was washed six times with DMF, three times with DCM, and three times with MeOH. The resin was then dried overnight over PO in a desiccator. The peptide was then cleaved from the resin during a 45-minute 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:HO (v / v, 7:3) and lyophilized to obtain NH-Idc-hGlu(tBu)-Thr(Bzl)-Ser(tBu)-OH as a white powder (Block A, Synthesis Scheme 1). The peptide purity was >95% and was used for the synthesis of the NH-23-C2 inhibitor without further purification. Next, the peptide fragment (1 equivalent) was coupled with NH-Asp-AOMK (1 equivalent) in DMF using HATU (1 equivalent) and 2,4,6-collidine (5 equivalents) as coupling reagents. The reaction was carried out at room temperature for 2 hours, after which the mixture was injected onto an HPLC column, and NH-Idc-hGlu(tBu)-Thr(Bzl)-Ser(tBu)-Asp-AOMK was purified and lyophilized. The product was then dissolved in a 1:2 mixture of DCM:TFA 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 give the final compound: NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-AOMK (Block C, Synthesis Scheme 1). Molecular weight [m / z + H]. + The purity and cleavage were confirmed by LC-MS analysis. Other inhibitors were synthesized in a similar manner. The reference inhibitor Ac-VDVAD-AOMK (Ac-Val-Asp-Val-Ala-Asp-AOMK) was also synthesized using this procedure, except that the peptide fragment was additionally N-capped with an acetyl group. Acetic acid (5 equivalents) and HBTU (5 equivalents) were dissolved in a minimal amount of DMF and poured onto NH2-Val-Asp(tBu)-Val-Ala-resin (1 equivalent), and the acetylation reaction was carried out for 30 minutes. The N-acetylated peptide was then carried forward as described above. TIFF2026501655000036.tif173170

[0142] Synthetic Scheme 1. AOMK-based inhibitor synthesis procedure exemplified by the synthesis of NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-AOMK TIFF2026501655000037.tif21170

[0143] TIFF2026501655000038.tif128170

[0144] 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.

[0145] 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 parameter was measured under pseudo-first-order kinetic conditions ([I] >> [E]). Inhibitors (20 μL) were diluted in a 96-well plate and mixed with the appropriate substrate (20 μL): NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-ACC (50 μM) for caspase-2, Ac-DEVD-ACC (100 μM) for caspase-3, and Ac-LEHD-ACC (100 μM) for caspase-8. ACC fluorescence was monitored using wavelengths of 355 nm (excitation) and 460 nm (emission). The substrate-inhibitor mixture (40 μL total) was preincubated at 37°C for 15 min. In separate tubes, caspases were preincubated in assay buffer at 37°C. After 15 min, enzyme was added to the wells (60 μL per well, 100 μL total reaction volume), and fluorescence was immediately initiated and monitored for 30 min. Second-order inhibition rate (k obs / I) was determined in at least three independent experiments and presented as the mean value. GraphPad Prism7 software was used for calculations (see Figure 2).

[0146] Preparation of recombinant caspases Detailed protocols for the expression and purification of human apoptotic caspases can be found elsewhere (Stennicke, 1999).

[0147] Enzyme Dynamics Research-K i and IC 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 IC 50Inhibition parameters were measured. Prior to kinetic analysis, all caspases were active-site titrated using zVAD-fmk inhibitor (Cayman Chemical Company, Cat. No. 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. i Parameters were measured using the Morrison equation (Copeland, 2000). For each caspase, substrate hydrolysis was monitored using the appropriate ACC fluorogenic substrate: NH-Idc-hGlu-Thr(Bzl)-Ser-Asp-ACC (50 μM) for caspase-2, Ac-DEVD-ACC (100 μM) for caspase-3, and Ac-LEHD-ACC (100 μM) 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 fourfold higher than the caspase concentration in the assay. Caspase (60 μL) was first preheated in assay buffer in a 96-well plate at 37°C for 15 min and then preincubated with inhibitor (20 μL) for an additional 15 min at 37°C. Substrate (20 μL) 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 [S] was calculated using the formula [S], 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 three times, and data were analyzed using GraphPad Prism7 software.

[0148] The Ki and IC50 (for caspase-2) data are shown in FIG.

[0149] The results are expressed as kObs / I and the Ki and IC50 values ​​are shown in Tables 3-5 below. TIFF2026501655000039.tif55170

[0150] TIFF2026501655000040.tif55170

[0151] TIFF2026501655000041.tif55170

[0152] Selectivity results are also presented as split values ​​of k o b s / l , K i and IC 50 values ​​(see Tables 6 and 7 below). TIFF2026501655000042.tif66170

[0153] TIFF2026501655000043.tif65170

[0154] As previously 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 table above, the inventors of the present invention have found that compounds of the formula NH-Idc-hGlu-Thr(Bzl)-AA-Asp-AOMK, which have a Lys, Orn, Dab, or Dap group at the AA position instead of serine, for example, exhibit high caspase-2 inhibitory activity and remarkable selectivity toward caspase-3 or caspase-8, thus resulting in improved caspase-2 inhibitors.

[0155] 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 at 37°C for 30 min).

[0156] Cells were treated with the "NASH cocktail": DMEM containing 4.5 mg / mL glucose supplemented with 10% (v / v) FBS, 1% (v / v) PenStrep, and 2 mM L-glutamine plus fatty acids (100 μM sodium oleate and 100 μM palmitic acid), 100 nM insulin (all from 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 at different concentrations simultaneously.

[0157] At the end of treatment, cells were washed with PBS and incubated with staining solution (BODIPY 493 / 503 (Thermo Fisher Scientific)) for 15 minutes at 37°C. Cells were then washed three times with PBS and fixed in 4% PFA for 30 minutes at room temperature. After three additional washes with PBS, coverslips were mounted on glass slides overnight at room temperature using one drop of Prolong® Gold antifade reagent (Invitrogen) containing 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.

[0158] 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 has been speculated to contribute to the progression of NASH by increasing intracellular lipotoxic 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). Therefore, caspase-2 inhibition could prevent progression to NASH by reducing intracellular lipid concentrations in hepatocytes.

[0159] An in vitro cell model of NASH was used to test the efficacy of caspase-2 inhibitors in preventing intracellular lipid accumulation. In this model, HepG2 cells are exposed to "NASH conditions" including lipogenic (glucose, insulin, fatty acids) and inflammatory and proapoptotic (TNF-α, IL-1β, and TGF-β) triggers for 24 hours. Cells exhibit a significant increase in intracellular lipid load of at least two-fold compared to non-triggered controls. This model has been shown to correlate with human NASH pathology (Boeckmans et al., 2019).

[0160] NASH-induced HepG2 cultures were treated with 10 μM of KIN inhibitors for 24 hours, and the inhibition of intracellular lipid accumulation was quantified as described in Materials and Methods. As shown in Figure 4, all tested caspase-2 inhibitors 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.

[0161] The efficacy of caspase-2 inhibitors in preventing intracellular lipid accumulation was compared between NH-23-C2 and Compound 1. Cells were incubated with NASH conditions and 10, 20, and 25 μM of the caspase-2 inhibitor to analyze its 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.

[0162] These experimental results provide experimental evidence that the compounds of the present invention that can regulate the activity of caspase-2 can effectively inhibit lipid accumulation in hepatocytes.

[0163] 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: TIFF2026501655000044.tif187170TIFF2026501655000045.tif176170TIFF2026501655000046.tif172170

[0164] Prior to the kinetic assay, substrate (a few μL) was diluted to 1 μM (working concentration) in DMSO. Next, 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 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 analyzed 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 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, 10% (w / v) sucrose, 10 mM DTT, pH 7.2–7.4. Caspase-2 was preincubated in buffer at 37°C for 15 min before being added to the substrate.

[0165] result The results are summarized in Figure 6 and detailed in the table below: TIFF2026501655000047.tif125170

[0166] To ensure the reliability of the data, all caspase 2 substrates were independently generated (synthesized, purified, and diluted in DMSO). The reference comparison value is the hydrolysis rate of the best substrate from this particular series of substrates: 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 highly 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 Figure 7.

[0167] 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. Next, the Fmoc-protecting group was 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 minimal amount of DMF for 3 minutes and then poured onto the resin. The reaction was allowed to proceed 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 preincubated 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 DMF for 3 min and poured onto the H2N-ACC resin. The reaction was carried out for 24 h and then repeated using 1.5 equivalents of the Fmoc-L-Asp(tBu)-OH / HATU / 2,4,6-collidine reagent for another 24 h. The Fmoc-L-Asp(tBu)-ACC resin was then washed three times with DMF, followed by Fmoc deprotection with 20% piperidine in DMF, and the resin was washed 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 allowed to proceed 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 six times with DMF. An isokinetic mixture of 19 natural amino acids (excluding cysteine ​​and including norleucine, which mimics methionine) was coupled to the P3 position. To do this, 5 equivalents of the isocratic 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 with DMF and preactivated for 3 minutes. The mixture was then poured onto the resin, and the cartridge was agitated for 3 hours. The slurry was filtered and washed three times with DMF. Complete coupling of P3 was confirmed by a ninhydrin test. Next, the Fmoc group was removed with 20% piperidine in DMF, and the H2N-Mix-Dab(Boc)-Asp(tBu)-ACC resin was divided into five portions (0.05 mmol each). P4 coupling was performed using a MultiChem 48-well synthesizer (FlexChem, SciGene, CA, USA). The five portions of the resin were placed in a 48-well cartridge and swelled with DCM for 30 minutes. The DCM was then filtered, and the resin was washed three times with DMF. In five 1.5 mL tubes, 2.5 equivalents of various 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. Next, 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 allowed to proceed 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 overnight over PO5 in a desiccator. All substrate was cleaved from the resin using an ice-cold TFA / TPS / water (% v / v / v, 95 / 2.5 / 2.5) mixture for 2 h (shaking 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 minutes 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.

[0168] One microliter of substrate (10 mM) was spotted into a well 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 minutes, and only the linear portion of the plot was analyzed to calculate reaction rates. The percentage of the best cleaved substrate was set to 100%, and all other substrates were adjusted accordingly (% cleavage).

[0169] Composition of the isokinetic mixture used in this particular synthesis (total amount - 1.2 mmol) TIFF2026501655000048.tif105170

[0170] The final concentration of 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, 10% (w / v) sucrose, 10 mM DTT, pH 7.2–7.4. Caspase-2 was preincubated in the buffer at 37 °C for 15 min before adding the substrate.

[0171] The results are shown in Figure 8 (left panel) and in the table below: TIFF2026501655000049.tif37170

[0172] 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).

[0173] Finally, to better represent the hydrolysis rate of the best substrates in each series shown in Examples 4 and 5, they were set to 100% and the other substrates were adjusted accordingly, as shown in Figure 8.

[0174] Example 6 Combining Warhead Building Blocks Scheme 1: TIFF2026501655000050.tif60170 Scheme 2: TIFF2026501655000051.tif46170 Scheme 3: TIFF2026501655000052.tif71170 Scheme 4: TIFF2026501655000053.tif48170Synthesis of Amino Acid Building Blocks: Scheme 4: TIFF2026501655000054.tif126170 General synthesis scheme for caspase-2 candidates: Example 1: Cas2_1:NH-Idc-hGlu-Thr(Bzl)-Dab-Asp-AOMK Example 2: Cas2_6:NH-Idc(7-F)-hGlu-Thr(Bzl)-Dab-Asp-AOMK Example 3: Cas2_9:NH-Idc(5-F)-hGlu-Thr(Bzl)-Dab-Asp-O(2,3,5,6-F4-Ph) Example 4: Cas2_9:NH-Idc(7-F)-hGlu-Thr(Bzl)-Dab-Asp-O(2,3,5,6-F4-Ph) TIFF2026501655000055.tif204170

[0175] result We tested the inhibitory activity of the following compounds: TIFF2026501655000056.tif92170

[0176] The results are shown in Table 8 below. TIFF2026501655000057.tif64170

Claims

1. Compounds of general formula (II): or a salt, solvate or stereoisomer thereof (In the formula, In the heterocycle at the P5 position (dotted line) represents a bond that may or may not be present, and if present, it forms a double bond together with an already present single bond; R ２ and R ３ are independently hydrogen or C, including branched alkyl and cycloalkyl １－４ alkyl, R ４ , R ５ , R ６ and R ７ are each independently hydrogen, halogen, or C, including branched alkyl and cycloalkyl. １－４ alkyl, wherein each C １－４ the alkyl is optionally substituted with 1 to 3 halogen atoms; x and z are each independently selected from 0, 1, or 2; n is 0, 1, 2, or 3; R ８ and R ９ are independently hydrogen, halogen, or C, including branched alkyl and cycloalkyl. １－４ alkyl, wherein each C １－４ The alkyl is optionally substituted with 1 to 3 halogen atoms, and R １ is a chemically reactive group such as a fluorophore or warhead).

2. A compound of general formula (II) according to claim 1, wherein In the heterocycle at the P5 position (dotted line) represents a bond that may or may not be present, and if present, it forms a double bond together with an already present single bond; R ４ , R ５ , R ６ and R ７ are each independently selected from hydrogen, trifluoromethyl, or halogen, such as fluorine; x and z are each 1; n is 0, 1, 2 or 3; R ８ and R ９ are each independently selected from hydrogen, halogen, e.g., fluorine, or trifluoromethyl; R ２ and R ３ are independently hydrogen or C １－４ alkyl, preferably selected from methyl or ethyl, and R １ is a chemically reactive group according to claim 1).

3. The compound of general formula (II) according to claim 1 (In the formula, In the heterocycle at the P5 position (dotted line) represents a bond that may or may not be present, and if present, it forms a double bond together with an already present single bond; R ４ , R ５ , R ６ and R ７ are each independently selected from hydrogen or halogen, such as fluorine; x and z are each 1; n is 0, 1, 2 or 3; R ８ and R ９ are each independently selected from hydrogen, trifluoromethyl, or halogen, such as fluorine; R ２ and R ３ is independently selected from hydrogen or methyl, and R １ is a chemically reactive group according to claim 1).

4. The compound of general formula (II) according to claim 1 (In the formula, In the heterocycle at the P5 position (dotted line) represents a bond that may or may not be present, and if present, it forms a double bond together with an already present single bond; R ４ and R ６ are each independently selected from hydrogen or halogen, such as fluorine; R ５ and R ７ are both hydrogen, x and z are each 1; n is 0, 1, 2 or 3; R ８ and R ９ are each independently selected from hydrogen, trifluoromethyl, or halogen, such as fluorine; R ２ and R ３ is independently selected from hydrogen or methyl, and R １ is a chemically reactive group according to claim 1).

5. The compound of general formula (II) according to claim 1 (In the formula, In the heterocycle at the P5 position (dotted line) represents a bond that may or may not be present, and if present, it forms a double bond together with an already present single bond; R ４ and R ６ are each independently selected from hydrogen or halogen, such as fluorine; R ５ and R ７ are both hydrogen, x and z are each 1; n is 0, 1, 2 or 3; R ８ and R ９ are both hydrogen, R ２ and R ３ is independently selected from hydrogen or methyl, and R １ is a chemically reactive group according to claim 1).

6. 10. The compound of claim 1, or a salt, solvate or stereoisomer thereof, having the general formula (III): (In the formula, In the heterocycle at the P5 position (dotted line) represents a bond that may or may not be present, and if present, it forms a double bond together with an already present single bond; R ２ and R ３ are independently hydrogen or C, including branched alkyl and cycloalkyl １－４ alkyl, R ４ , R ５ , R ６ and R ７ are each independently hydrogen, halogen, or C, including branched alkyl and cycloalkyl. １－４ alkyl, wherein each C １－４ the alkyl is optionally substituted with 1 to 3 halogen atoms; x and z are each independently selected from 0, 1, or 2; n is 0, 1, 2, or 3; R ８ and R ９ is independently selected from hydrogen, halogen, or C1-4 alkyl, including branched alkyl and cycloalkyl, wherein each C1-4 alkyl is optionally substituted with 1 to 3 halogen atoms; j is 0 or 1, and R １０ , R １１ , R １２ , R １３ and R １４ are each independently selected from hydrogen, halogen, or C1-4 alkyl, including branched alkyl and cycloalkyl, phenyl, phenoxy, nitro, nitrile, PhCO, NHAc, where each alkyl is optionally substituted with 1 to 3 halogen atoms.

7. The compound according to claim 6 (In the formula, In the heterocycle at the P5 position (dotted line) represents a bond that may or may not be present, and if present, it forms a double bond together with an already present single bond; R ４ , R ５ , R ６ and R ７ are each independently selected from hydrogen, trifluoromethyl, or halogen, such as fluorine; x and z are each 1; n is 0, 1, 2 or 3; R ８ and R ９ are each independently selected from hydrogen, trifluoromethyl, or halogen, such as fluorine; R ２ and R ３ are independently hydrogen or C １－４ alkyl, preferably selected from methyl or ethyl, j is 0 or 1, and R １０ , R １１ , R １２ , R １３ and R １４ are each independently selected from hydrogen, halogen, or C1-4 alkyl, including branched alkyl and cycloalkyl, phenyl, phenoxy, nitro, nitrile, PhCO, NHAc, where each alkyl is optionally substituted with 1 to 3 halogen atoms.

8. 10. The compound of claim 1 having the general formula (IV), or a salt, solvate or stereoisomer thereof (In the formula, In the heterocycle at the P5 position (dotted line) represents a bond that may or may not be present, and if present, it forms a double bond together with an already present single bond; R ２ and R ３ are independently hydrogen or C, including branched alkyl and cycloalkyl. １－４ alkyl, preferably R ２ and R ３ is independently selected from hydrogen, methyl or ethyl; R ４ and R ６ are each independently hydrogen, halogen, or C, including branched alkyl and cycloalkyl. １－４ alkyl, wherein each C １－４ The alkyl is optionally substituted with 1 to 3 halogen atoms, preferably R ４ and R ６ are each independently selected from hydrogen, trifluoromethyl, or halogen, such as fluorine; x and z are each independently selected from 0, 1 or 2, and n is 0, 1, 2 or 3, preferably x and z are 1 and n is 0, 1, 2 or 3; R ８ and R ９ are independently hydrogen, halogen, or C, including branched alkyl and cycloalkyl. １－４ alkyl, wherein each C １－４ The alkyl is optionally substituted with 1 to 3 halogen atoms, preferably R ８ and R ９ are independently selected from hydrogen or halogen, such as fluorine; j is 0 or 1, and R １０ , R １１ , R １２ , R １３ and R １４ are each independently selected from hydrogen, halogen, or C1-4 alkyl including branched alkyl and cycloalkyl, phenyl, phenoxy, nitro, nitrile, PhCO, NHAc, where each alkyl is optionally substituted with 1 to 3 halogen atoms, preferably R １４ or R １０ is selected from halogen, such as fluorine, methyl or trifluoromethyl, R １１ , R １２ and R １３ are each hydrogen, or a halogen, such as fluorine).

9. 10. The compound of claim 1 having the general formula (V), or a salt, solvate or stereoisomer thereof (In the formula, In the heterocycle at the P5 position (dotted line) represents a bond that may or may not be present, and if present, it forms a double bond together with an already present single bond; R ２ and R ３ are independently selected from hydrogen or methyl; R ４ and R ６ are each independently selected from hydrogen or halogen, such as fluorine; n is 0, 1, 2 or 3; j is 0 or 1, and R １４ or R １０ is selected from halogen, for example fluorine, or methyl, and R １１ and R １３ are each hydrogen or a halogen, such as fluorine, When j is 0, R １４ , R １０ is a halogen, for example fluorine, and R １１ and R １３ is a halogen, for example fluorine).

10. 2. The compound of claim 1, wherein the compound is selected from any one of the following chemical structures (protonated or deprotonated) or a salt, solvate, or stereoisomer thereof: 。

11. 2. The compound of claim 1, wherein the compound is selected from any one of the following chemical compounds (protonated or deprotonated) or a salt, solvate, or stereoisomer thereof:

12. The compound of any one of claims 1 to 11, wherein all amino acids in the compound are in the L configuration.

13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12, and optionally a pharmaceutically acceptable excipient.

14. A compound according to any one of claims 1 to 12 for use as a pharmaceutical.

15. Degenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, dementia with Lewy bodies, mild cognitive impairment, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease; neonatal brain injury, in particular neonatal cerebral ischemia; traumatic brain injury; renal ischemia; hypoxic-ischemic injury; stroke-like brain injury; cardiac ischemia; myocardial infarction; amyotrophic lateral sclerosis; retinal damage; eye diseases such as blunt eye injury, ischemic optic neuropathy and glaucoma; skin injuries; sterile inflammatory diseases such as diabetes, atherosclerosis, myocardial ischemia, gout, pseudogout, joint laxity, atherosclerosis, syndromes triggered by aluminum salts, non-arteritic ischemic optic neuropathy, glaucoma and metabolic diseases; 13. A compound according to any one of claims 1 to 12 for the prevention and / or treatment of non-sterile inflammatory diseases, such as bacterial infections, in particular infections by bacteria that produce pore-forming toxins, influenza virus infections and single-stranded RNA Rhabdoviridae 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), liver cirrhosis, primary sclerosing cholangitis and hepatocellular carcinoma.

16. A compound according to any one of claims 1 to 12 for the prevention and / or treatment of dyslipidemia, obesity, metabolic syndrome, cirrhosis, non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD).

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