Specific VASH compound (SVC) inhibitors for heart disease

Abstract

The present invention relates to a compound of formula (I) for use in the prevention and / or treatment of heart failure, cardiomyopathy, myocardial infarction-induced cardiac dysfunction in subjects requiring it, and / or for the improvement of cardiac function: [Formula 1] JPEG2026509764000079.jpg41166 or its pharmaceutically acceptable salts and / or solvates.

Description

[Technical Field]

[0001] The present invention relates to novel specific vash compounds (SVCs) in the prevention and / or treatment of heart disease, particularly heart failure and cardiomyopathy. [Background technology]

[0002] Heart failure (HF) is defined as a condition in which the heart is unable to supply sufficient blood flow to the body's organs and tissues. Heart failure is often characterized by diastolic dysfunction, i.e., insufficient ventricular filling due to the inability of the myocardium to relax sufficiently. Diastolic dysfunction is characteristic of hypertrophic cardiomyopathy (HCM) (Maron et al., 1995) and heart failure with preserved ejection fraction (HFpEF). These conditions are often characterized by prolonged and impaired left ventricular (LV) relaxation, delayed LV filling, and increased diastolic LV stiffness.

[0003] The prevalence of heart failure continues to rise, and its high mortality rate highlights the need for new therapies. In particular, heart failure with preserved ejection fraction (HFpEF), which is estimated to account for about half of all heart failure cases, currently has limited treatment options. In many cases of HFpEF, myocardial relaxation is slow, contributing to impaired pumping function. The delayed myocardial relaxation in HFpEF is also due to increased internal resistance resulting from microtubule (MT) densification and stabilizing post-translational modifications such as α-tubulin detyrosination (Chen et al., 2018). Therefore, targeting MT to reduce the overall stiffness of cardiomyocytes may improve relaxation rate without increasing energy expenditure.

[0004] Microtubules (MTs) are the primary cytoskeletal components found in all eukaryotic cells. MTs are formed by the polymerization of α-tubulin and β-tubulin heterodimers and function as mechanotransducers, converting altered contractile forces into intracellular signals. MTs also act as compressive resistance elements, mechanically inhibiting cardiomyocyte contraction. MTs act as viscoelastic resistance elements, inhibiting sarcomere shortening, particularly in pathological conditions involving MT proliferation, potentially impairing cardiac function. Post-translational modifications (PTMs) of MTs alter their mechanical properties and binding interactions (Roll-Mecak, 2020). Detyrosinization, a PTM of α-tubulin, has recently been shown to affect MT-dependent mechanotransduction in both cardiomyopathy and skeletal muscle disease (Kerr JP et al., 2015). This specific PTM is significantly increased in patient tissues and preclinical models of cardiac disease (Chen et al., 2018). In myocardial insufficiency, maladaptation of cardiomyocytes to chronic stress correlates with increased density of the MT network, VASH protein levels, and / or detyrosination activity. The increased density of the MT network and the exacerbation of disease-related detyrosination activity mechanically interfere with cardiomyocyte function through contractile dysfunction, leading to compensatory mechanisms and severe myocardial remodeling.

[0005] The development of safe and effective treatments for heart failure, particularly those that minimize the increased risk of ischemia or arrhythmias associated with current costly symptomatic efforts, remains a pressing need. Over the past five years, researchers have identified modified cardiomyocyte (CM) microtubules (MTs) as a novel therapeutic target for HF treatment. Significant microtubule densification and increased detyrosination activity are observed in human and mouse HF from various origins (Tsutsui H. et al, 1993; Cheng G. et al., 2008), leading to cardiomyocyte rigidity and impaired contractile movement. Reversing this post-translational modification by forcibly expressing a reverse enzyme called tubulin tyrosine ligase (TTL) is sufficient to reduce the rigidity of dysfunctional human and mouse cardiomyocytes and myocardium and improve contractility. However, the use of viral expression vectors for cardiovascular applications remains complex. Of particular note and significance to cardiovascular medical knowledge is that addressing the detyrosinized state caused by TTL expression leads to improvements in cardiac dynamics that occur independently of calcium circulation, and significantly reduces the risks associated with new therapeutic approaches that reduce detyrosinization.

[0006] Gene therapy that reduces detyrosine conversion has been shown to improve cardiac function in patients with heart failure. Cardiac gene therapy holds great potential, but it faces many hurdles, and achieving safe and efficient transduction to the heart is a major challenge, especially in large animals.

[0007] In parallel with this, inhibition of detyrosinases has also shown considerable therapeutic potential. Vasohibinases (VASH1 and 2), in coordination with or independently of their chaperone, the small vasohibin-binding protein (SVBP), remove C-terminal tyrosine residues from polymerized MT α-tubulin (Van der Laan S. et al., 2019). VASH1 was recently identified as the major detyrosinase in human and mouse myocardium, and VASH1 KD has been found to be sufficient to reduce CM stiffness and improve left ventricular diameter shortening and contraction and relaxation velocities in failing human CM. Of particular note is VASH1 KD in CM in patients with heart failure with preserved ejection fraction (HFpEF), Ca 2+ This resulted in remarkably rapid relaxation independent of transitional changes (Chen, CY et al., 2020). Since VASH1 does not bind to and block free tubulin, VASH1 KD does not exhibit clear MT depolymerization activity in CM (Chen, CY et al., 2020), but instead specifically reduces dTyr while maintaining network dynamics and density. Preventing VASH2-dependent detyrosinization substantially limits the reduction in left ventricular ejection fraction after acute myocardial infarction in mice without affecting infarct size or cardiac remodeling (Yu X. et al., 2021).

[0008] While currently available VASH inhibitors have shortcomings, improvements are being made. Parthenolide (PTL), a highly reactive sesquiterpene lactone and FDA-approved chemotherapeutic agent, does indeed directly inhibit VASH activity, albeit only at high concentrations (Aillaud, C. et al., 2017). In CM, 10 μM PTL (<IC50) results in a slight decrease in dTyr (30-40%) after 2 hours of treatment (Kerr, JP et al., 2015; Robison, P. et al., 2016; Schuldt, M. & Kuster, 2020), but it cannot be repurposed for product development due to numerous potential off-target effects that limit its long-term use, including modulation of NF-κB, STAT3, and JNK signaling pathways (Caporizzo, MA, Chen, CY & Prosser, B. L, 2019).

[0009] Therefore, there is a need to provide more specific VASH compound (SVC) inhibitors as a solution.

[0010] In 2017, epoxide-Y (Epo-Y) was reported and used in targeted chemical proteomics screening, identifying a detyrosinase complex and demonstrating its potency as a VASH inhibitor more than 10 times stronger than PTL (Aillaud, C. et al., 2017).

[0011] However, the inventors have recently developed SVC inhibitors that show a 10- to 1000-fold improvement in VASH inhibition compared to Epo-Y. These SVC inhibitors readily cross the cell membrane and effectively reduce intracellular dTyr, improving cardiomyocyte function. The inventors tested these compounds in ZSF1 obese rats, which are considered the best rodent model of heart failure with ejection fraction (HFpEF) to date, as they exhibit three important comorbidities associated with human HFpEF: hypertension, obesity, and diabetes. The inventors demonstrated that these compounds effectively improve diastolic function. In particular, the SVC inhibitors were shown to significantly reduce muscle cell stiffness in ZSF1 obese muscle cells compared to ZSF1 lean controls, improving contraction and relaxation dynamics. Furthermore, advantageously, these compounds are clearly tolerable when delivered intravenously or orally in vivo and are effective in reducing detyrosination. [Overview of the Initiative]

[0012] Therefore, the present invention is useful for treating patients with heart failure and / or cardiomyopathy, particularly patients with reduced ejection fraction or sustained ejection fraction heart failure (e.g., HFrEF or HFpEF, respectively), and / or Ca 2+ This invention relates to compositions and methods useful for reducing cardiac stiffness and improving contractility, independently of Ca 2+ It can be combined with other therapies, including those involving dependent pathways ("combination therapy").

[0013] A first aspect of the present invention is a method for improving cardiac function in a subject of interest, wherein the subject of interest is a compound of formula (I): [ka] [In the formula, X is [ka] Or -NH-CH2-, R 1is,

Chem.

Chem.

[0014] In other words, the present invention also relates to the use of the compound of formula (I) for the prevention and / or treatment of heart failure, cardiomyopathy, myocardial infarction-induced cardiac dysfunction and / or for the improvement of cardiac function in a subject in need thereof:

Chem.

[0015] Another aspect of the present invention is a method for treating a cardiovascular disease selected from heart failure, cardiomyopathy, and myocardial infarction-induced cardiac dysfunction in a subject of interest, wherein an effective dose of a compound of formula (I) is administered to the subject of interest: [ka] [In the formula, X is [ka] Or -NH-CH2-, R 1 teeth, [ka] or NR 1a R 1b And here, R 1a H is R 1b This is a C1-C6 alkyl group that is either unsubstituted or substituted with C(O)-O-C1-C6 alkyl or C(O)-NH-C1-C6 alkyl. R is OR 2 And here, R 2 This is a C1-C6 fatty acid chain (the above fatty acid chain may be substituted in some cases), or a C1-C6 alkyl-aryl compound. R 3 These are OH or O-C1-C6 fatty acid chains. Y is -(CH2) m -(m=2) or [ka] And, R 5 It is C(O)OH or O-C1-C6 alkyl, R 6 NH-CH(R 7 )-(CH2) n -R 8 (Here, R 7 H is R 8 [where n is an aryl compound, n is 1, 2, or 3, preferably 1), or an O-C1-C6 fatty acid chain. The method involves administering either a pharmaceutically acceptable salt and / or solvate thereof.

[0016] Another aspect of the present invention is combination therapy in which compound (I), as defined in the present invention, is combined with current standard treatments for heart failure, cardiomyopathy, and / or myocardial infarction-induced cardiac dysfunction.

[0017] Another aspect of the present invention is the use of a conjugate comprising a compound (I) as defined above, or optionally a labeled biomolecule, or an unlinked fragment of the compound (I), as a research tool for research and development activities of cardiac dysfunction or heart failure. Specific Description of the Invention

[0018] Detailed explanation definition Unless otherwise defined herein, technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art.

[0019] The terms “one (a)” or “one (an)” refer to one or more. Therefore, the terms “one (a)” (or “one (an)”), “one or more” and “at least one” are used interchangeably herein.

[0020] The words "comprise, comprises, comprising" are to be interpreted comprehensively, not exclusively. The words "consist, consisting" and their variations are to be interpreted exclusively, not comprehensively. Various embodiments herein are shown using the words "comprise," but in other circumstances, the relevant embodiments are also intended to be interpreted and described using the words "consist" or "essentially consisting of."

[0021] The term "approximately" includes fluctuations of up to +10% unless otherwise specified.

[0022] In this specification, the term “therapy” refers to any protocol, method, and / or agent that can be used to prevent, manage, treat, and / or improve a disease, disorder, or condition in a patient. The patient is at risk of developing a disease, disorder, or condition, or is suspected of having developed a disease, disorder, or condition, or has been diagnosed with a disease, disorder, or condition. Non-limiting examples of therapy include the administration of compositions (e.g., pharmaceutical compositions), physical therapy, physiotherapy, psychotherapy, etc. Therapy may also include the possibility of combining multiple therapies, in which case the various therapies may be sequential, simultaneous, or mixed. If therapy involves the administration of a composition, the composition is administered in an amount, method, and / or form that is effective in treating or preventing the patient’s disease, disorder, or condition. Therapy may require two or more administrations of the composition.

[0023] As used herein, the term “treating” or “treatment” means improvement of a patient’s disease, disorder, or condition at a clinical, histological, and / or biochemical level. The term “treating” or “treatment” includes, in particular, improving, to a statistically significant extent or to a degree detectable by those skilled in the art, any clinical, histological, and / or biochemical symptom or parameter associated with a patient’s disease, disorder, or condition, or inhibiting, mitigating, or delaying the progression or exacerbation of a patient’s disease, disorder, or condition (including secondary disorders caused by the disease, disorder, or condition). In some embodiments, a treatment is evaluated on a population basis, and the therapy is considered to “treat” a particular disease, disorder, or condition if a statistically significant improvement in the patient’s disease, disorder, or condition is observed in the population affected by the disease, disorder, or condition.

[0024] As used herein, the terms “prevention,” “prevention,” and “preventing” refer to the reduction of the risk to an individual of acquiring or developing a given disease, disorder, or condition. The term “prevention” may also include delaying the onset of, and / or reducing the frequency and / or intensity of, clinical, histological, and / or biochemical symptoms or parameters associated with the given disease, disorder, or condition. In some embodiments, prevention is assessed on a population basis and is considered “preventing” a disease, disorder, or condition if, in a population susceptible to that particular disease, disorder, or condition, a statistically significant reduction in the risk of acquiring or developing that disease, disorder, or condition, and / or a statistically significant delay in onset, and / or a statistically significant reduction in the frequency and / or intensity of clinical, histological, and / or biochemical symptoms or parameters associated with that disease, disorder, or condition is observed.

[0025] As used herein, the term “administration” means oral administration, suppository administration, topical contact, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intrafocal administration, subarachnoid administration, intracranial administration, intranasal administration, or subcutaneous administration, or implantation of a sustained-release device to a subject, such as a mini osmotic pump. Administration may be by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatine, gingival, nasal cavity, vagina, rectum, or percutaneous). Parenteral administration includes, for example, intravenous, intramuscular, intra-arterial, intradermal, subcutaneous, intraperitoneal, intracardiac, and intracranial administration. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, and transdermal patches.

[0026] The term "co-administration" means that the compositions described herein are administered simultaneously with, immediately before, or immediately after, the administration of one or more additional therapies. In certain embodiments, the compositions are specifically targeted to the heart (e.g., via direct injection).

[0027] As used herein, the term “combination” refers to the possibility of any arrangement of various components (e.g., the compound of formula (I) according to the present invention and another treatment). Such arrangements include mixtures of the above components, as well as separate combinations for combined or sequential administration. The present invention encompasses combinations comprising each component at equimolar concentrations, as well as combinations of significantly different concentrations. It is understood that the optimal concentration of each component in a combination can be determined by those skilled in the art.

[0028] "Current standard of care for heart failure, cardiomyopathy, and myocardial infarction-induced cardiac dysfunction" refers to recognized treatment plans for cardiovascular diseases selected from heart failure, cardiomyopathy, and myocardial infarction-induced cardiac dysfunction, such as those listed in the 2021 ESC guidelines for the diagnosis and treatment of acute and chronic heart failure (McDonagh TA et al., 2021).

[0029] "(The subject) that needs it" means animals, preferably mammals, including humans. In a particular preferred embodiment, the subject that needs it is a human having or being prone to developing a cardiac disorder, in particular heart failure, cardiomyopathy, or myocardial infarction-induced cardiac dysfunction.

[0030] Heart failure is a condition that develops when the heart is unable to pump enough blood to meet the body's needs. This can occur when the heart is not filled with enough blood, or when the heart weakens and cannot pump properly.

[0031] Heart failure can have a sudden onset (acute) or develop gradually over time as the heart weakens (chronic). It can affect one or both sides of the heart. Left and right heart failure may have different causes. Often, heart failure is caused by other medical conditions damaging the heart, including coronary artery disease and subsequent myocardial infarction, cardiac inflammation, hypertension, cardiomyopathy, or arrhythmias. Heart failure can also damage the liver or kidneys. Other potential complications include pulmonary hypertension or other cardiac conditions such as arrhythmias, valvular heart disease, and sudden cardiac arrest.

[0032] Cardiomyopathy refers to a problem with the heart muscle that makes it difficult for the heart to pump blood effectively. There are many types and causes of cardiomyopathy, and people of all ages can be affected. Depending on the type of cardiomyopathy, the heart muscle may become thicker, harder, or larger than normal. This weakens the heart and can lead to arrhythmias, heart failure, or even cardiac arrest. Cardiomyopathy includes dilated, hypertrophic, ischemic, hereditary, and idiopathic types.

[0033] In certain embodiments, heart failure or cardiomyopathy in the present invention encompasses chronic heart failure or cardiomyopathy.

[0034] In another specific embodiment, heart failure or cardiomyopathy in the present invention encompasses acute heart failure (AHF) syndromes, including post-myocardial infarction syndrome, post-cardiac surgery syndrome, post-cardiac arrest syndrome, hypertensive crisis conditions, acute symptoms of non-ischemic cardiomyopathy, and acute exacerbations of chronic cardiomyopathy of various etiologies.

[0035] As used in this invention, the term "stereoisomer" refers to conformational stereoisomers, and more specifically, optical isomers.

[0036] In this invention, optical isomers arise particularly from differences in the spatial positions of substituents bonded to X. Therefore, the carbon or nitrogen atoms of the X group to which the substituents are bonded represent chiral or chiral centers. Thus, optical isomers that are not mirror images of each other are called "diastereoisomers," and optical isomers that are mirror images that cannot be superimposed are called "enantiomers."

[0037] An equimolar mixture of two enantiomers of a chiral compound is called a racemic mixture or racemic compound.

[0038] In the context of the present invention, depending on the position of the substituent bonded to the X group, the compounds of the present invention may be diastereoisomers of structure (S,S), structure (R,R), structure (S,R), or structure (R,S), as exemplified below: [ka] If the positions of substituents are not indicated in the compound, the compound corresponds to any one of the diastereoisomers or a mixture of the diastereoisomers.

[0039] In certain embodiments, the compounds used in the present invention are diastereoisomers of the structure (S,S).

[0040] In the framework of this invention, the term "pharmaceutical composition" means a composition having preventive and curative properties.

[0041] For the purposes of this invention, the term "pharmaceutically acceptable" is intended to mean a composition that is useful in the preparation of pharmaceutical compositions and is generally safe and non-toxic with respect to pharmaceutical use.

[0042] The term "pharmaceutically acceptable salt and / or solvate" is intended, within the framework of the present invention, to mean a salt and / or solvate of a compound that is pharmaceutically acceptable as defined above and has the pharmacological activity of the corresponding compound.

[0043] Pharmaceutically acceptable salts include the following: (1) Acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid; or acid addition salts formed with organic acids such as acetic acid, benzenesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, succinic acid, dibenzoyl-L25 tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid and trifluoroacetic acid, and (2) Base addition salts formed when an acid proton present in the compound is substituted with a metal ion such as an alkali metal ion, alkaline earth metal ion, or aluminum ion; or when it is coordinately bonded with an organic or inorganic base. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, and tromethamine. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide.

[0044] Solvates of the compounds of the present invention that are acceptable for therapeutic use include conventional solvates, such as those formed in the presence of a solvent during the final step of the preparation of the compounds of the present invention. Examples include solvates formed in the presence of water (these solvates are also called hydrates) or ethanol.

[0045] "C x -C y The term "aliphatic chain" refers to a linear or branched hydrocarbon chain that is not aromatic, containing x to y carbon atoms, particularly 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms, and containing one or more fully saturated or unsaturated atoms. According to the present invention, the term "aliphatic chain" includes substituted or unsubstituted linear or branched alkyl groups, alkenyl groups, or alkynyl groups.

[0046] When used in this invention, the term "C1-C6 alkyl" refers to a linear or branched monovalent saturated hydrocarbon chain containing 1 to 6 carbon atoms, including, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl (also called ter-butyl), n-pentyl, n-hexyl, and the like.

[0047] When used in the present invention, the term "aryl" preferably refers to an aromatic hydrocarbon group comprising 6 to 12 carbon atoms and one or more fused rings, and is, for example, a phenyl or naphthyl group, but is not limited to these. Advantageously, the aryl is a phenyl group.

[0048] When used in this invention, the term "C1-C6-alkylaryl" refers to an alkyl group as defined above, each substituted with an aryl group as defined above. Advantageously, the "C1-C6-alkylaryl" is a benzyl group.

[0049] In the context of this invention, "optionally substituted" means that the group in question is, in particular, C1-C6 alkyl, NR a R b COR c CO2R d CONR e R f , OR g , N + R h R i R j (Here, R a ~R j This means that each atom is optionally substituted with one or more substituents that can be independently selected from H, C1-C6 alkyl, or aryl (preferably H or C1-C6 alkyl).

[0050] The term "peptide coupling" refers to a chemical reaction between an amine functional group and a carboxylic acid functional group. Peptide coupling is advantageous for diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), carbonyldiimidazole (CDI), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 0-(7-azobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), and (benzotriazole-1-yloxy)tripyrrolodinophosphonium hexafluorophosphate (PyBOP). The process is carried out in the presence of a coupling agent such as 7-azabenzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) or propylphosphonic anhydride, and optionally with an additive or base such as N-hydroxysuccinimide (NHS), N-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazole (HOOBt), l-hydroxy-7-azabenzotriazole (HAt), N-hydroxysylfosuccinimide (SulfoNHS), dimethylaminopyridine (DMAP), diisopropylethylamine (DIEA), or N-methylmorpholine (NMM).

[0051] When used in this invention, the term "VASH or vasohibin (enzyme)" refers to tubulin carboxypeptidase enzymes (TCPases) involved in the microtubule detyrosination mechanism associated with muscular dystrophy, particularly heart failure, cardiomyopathy, and myocardial infarction-induced cardiac dysfunction.

[0052] The term "biomolecule" refers to molecules that possess biological properties. In the context of this invention, biomolecules include proteins, peptides, biomarkers, and, for example, but are not limited to these. Photolabeling agents such as rhodamine, coumarin derivatives, cyanine derivatives, or fluorescein; affinity probes such as biotin; or E3 ubiquitin ligase recruiters such as thalidomide, VH032, VH101, dBET1, dFKBP12, QCA570, ZNL-02-096, or d9A-2. It refers to.

[0053] The term "prodrug" refers to a generally pharmacologically inactive or less active derivative of an active drug that undergoes biotransformation in cellulo or in vitro to release the active drug through chemical or enzymatic cleavage. In the context of this invention, "pharmacologically inactive or less active derivative" is understood to mean that the prodrug does not exhibit adequate activity for inhibiting the VASH active site in vitro. However, in in cellulo and in vivo assays, prodrugs exhibit activity because such tests enable the necessary transformations to deliver the active drug. Prodrugs can offer many advantages over the parent drug, including increased cell permeability, solubility, improved stability, improved bioavailability, reduced side effects, and improved selectivity. The activation of prodrugs may involve many enzymes, including, but are not limited to, oxidoreductases such as CYP450 and DT-diaphorase, as well as hydrolases such as carboxylesterase and β-glucuronidase.

[0054] In the context of the present invention, prodrug compounds as defined herein may be inactive in vitro. However, prodrug compounds exhibit increased cell permeability. After cell permeability, prodrug compounds are hydrolyzed to provide the corresponding active drug, namely a potent VASH inhibitor.

[0055] Therefore, the first aspect of the present invention is a method for improving cardiac function in a subject of interest, wherein a compound of formula (I) is administered to the subject of interest: [ka] [In the formula, X is [ka] Or -NH-CH2-, R 1 teeth, [ka] or NR 1a R 1b And here, R 1a H is R 1b These are C1-C6 alkyl groups that are either unsubstituted or substituted with C(O)-O-C1-C6 alkyl or C(O)-NH-C1-C6 alkyl. R is OR 2 And here, R 2 This is a C1-C6 fatty acid chain (the above fatty acid chain may be substituted in some cases), or a C1-C6 alkyl-aryl compound. R 3 These are OH or O-C1-C6 fatty acid chains. Y is -(CH2) m -(m=2) or [ka] And, R 5 It is C(O)OH or O-C1-C6 alkyl, R 6 NH-CH(R 7 )-(CH2) n -R 8 (Here, R 7 H is R 8 [where n is an aryl compound, n is 1, 2, or 3, preferably 1), or an O-C1-C6 fatty acid chain. A method comprising administering either a pharmaceutically acceptable salt and / or solvate thereof.

[0056] In other words, the present invention relates to a compound of formula (I) for use in the prevention and / or treatment of heart failure, cardiomyopathy, myocardial infarction-induced cardiac dysfunction in subjects requiring it, and / or for the improvement of cardiac function: [ka] [wherein, X is [Chem.] or -NH-CH2-, R 1 is [Chem.] or NR 1a R 1b wherein R 1a is H, R 1b is unsubstituted or C(O)-O-C1-C6 alkyl-substituted C1-C6 alkyl, or C(O)-NH-C1-C6 alkyl, R is O-R 2 wherein R 2 is a C1-C6 aliphatic chain (the above aliphatic chain may be substituted), or C1-C6 alkyl-aryl, R 3 is OH or O-C1-C6 aliphatic chain, Y is -(CH2) m -(m = 2) or [Chem.] wherein, R 5 is C(O)OH or O-C1-C6 alkyl, R 6 is NH-CH(R 7 )-(CH2) n -R 8 (wherein R 7 is H, R 8 is aryl, n is 1, 2 or 3, preferably 1), or O-C1-C6 aliphatic chain) or a pharmaceutically acceptable salt and / or solvate thereof. Compound of formula (I) used in the present invention The compounds according to the present invention may be in the form of stereoisomers or mixtures of stereoisomers, for example, mixtures of enantiomers or diastereoisomers, in particular, racemic mixtures. According to a particular embodiment, the compound of formula (I) is in the form of one diastereoisomer of structure (S,S), structure (R,R), structure (S,R), or structure (R,S) as defined above.

[0057] Formula (I) as defined herein encompasses both active compounds, i.e., drugs, and their prodrugs.

[0058] In particular, the term "prodrug" in this invention is R 3 This refers to compounds of formula (I) where the OH group is not present.

[0059] Preferably, the prodrug according to the present invention is R 3 The compound of formula (I) is an O-C1-C6 fatty acid chain, for example, an O-C1-C6 alkyl, particularly O-ethyl, O-isopropyl, or O-terbutyl.

[0060] In the context of this invention, the term "drug" is R 3 This refers to compounds of formula (I) where the OH group is the OH group.

[0061] In general, R 3 It forms an ester group with the adjacent carboxyl group. When converted from a prodrug to a drug, R 3 The ester formed by this ester and an adjacent carboxyl group is generally hydrolyzed in the corresponding carboxylic acid.

[0062] In the context of the present invention, these drugs differ from their corresponding prodrugs in that the ester group present in the prodrug is converted to the corresponding carboxylic acid in the drug. Preferably, the prodrug is R 3 It contains only one ester group formed with an adjacent carboxyl group. Therefore, a drug corresponding to a given prodrug is generally R 3It has the same formula as the above prodrug except for the group R. In other words, in a given prodrug and its drug, substituent R 1 X and R are generally the same. Alternatively, a prodrug may contain two or more ester groups. In such cases, all of these ester groups are converted to carboxylic acids in the corresponding drug.

[0063] Preferably, the compound of formula (I) is in the form of a diastereoisomer of the structure (S,S), as shown in formula (IA): [ka] (In the formula, X, R, R 1 and R 3 (as defined in this disclosure). Respond to.

[0064] In a more preferred embodiment, the compound of formula (IA) has the following enantiomers (I-A'): [ka] (In the formula, X, R, R 1 and R 3 (as defined in this disclosure) That is the case.

[0065] In a preferred embodiment, X is [ka] Preferably, [ka] That is the case.

[0066] In another specific embodiment, X is -NH-CH2-.

[0067] According to the present invention, R is OR 2 That is the case.

[0068] According to some embodiments, R 2This is a C1-C6 fatty acid chain such as a C1-C6 alkyl group, or a C1-C6 alkyl-aryl group, and the above fatty acid chain or alkyl-aryl group may be substituted. 2 If R is a C1-C6 alkyl such as methyl or ethyl, it is particularly unsubstituted (not substituted) or substituted with phenyl. More preferably, 2 These are C1-C6 alkyl groups such as ethyl, or C1-C6 alkyl-aryl groups such as benzyl.

[0069] According to some other embodiments, R 3 is an OH or O-C1-C6 fatty acid chain. More preferably, R 3 These are OH or O-C1-C6 alkyl groups, particularly O-ethyl, O-isopropyl, or O-terbutyl.

[0070] According to some embodiments, R 1 is NR 1a R 1b And here, R 1a H is R 1b is a C1-C6 alkyl group, and the alkyl group is optionally substituted with a C(O)-O-C1-C6 alkyl group or a C(O)-NH-C1-C6 alkyl group. In such embodiments, R 1 The alkyl group may be particularly NH-C1-C6 alkyl, and the alkyl group may be C(O)-O-C1-C6 alkyl or C(O)-NH-C1-C6 alkyl, and may be substituted with C(O)-methyl, C(O)-ethyl, C(O)-isopropyl or C(O)-terbutyl, or C(O)-NH-methyl, C(O)-NH-ethyl, C(O)-NH-isopropyl or C(O)-NH-terbutyl.

[0071] According to a preferred embodiment, R 1 teeth, [ka] Preferably, [ka] That is the case.

[0072] The compound of formula (I) is the compound of the following formula: [ka] (In the formula, R, R 3 , R 5 , R 6 (X and Y are defined in this disclosure) That is the case.

[0073] Preferably, the compound of formula (I) has the following composition (IA a ) compounds: [ka] (In the formula, R, R 3 , R 5 , R 6 (X and Y are defined in this disclosure) That is the case.

[0074] More preferably, the compound of formula (I) has the following structure: formula (I-A' a ) compounds: [ka] (In the formula, R, R 3 , R 5 , R 6 (X and Y are defined in this disclosure) That is the case.

[0075] Y in particular, [ka] It is possible that R 5 The element is preferably an O-C1-C6 alkyl group, such as O-methyl or O-ethyl, particularly O-ethyl.

[0076] Alternatively, Y is -(CH2)m - may be, where m is an integer from 0 to 6, preferably m is 1 or 2, and more preferably m is 2. Y is -(CH2) m In this embodiment, R 5 It is preferably C(O)OH.

[0077] According to some embodiments, in particular, Y is [ka] If R 6 These are O-C1-C6 fatty acid chains, such as O-methyl or O-ethyl, especially O-ethyl.

[0078] According to other specific embodiments, in particular, Y is -(CH2) m -, especially if it is -(CH2)2-, R 6 is NH-CH(R 7 )-(CH2) n -R 8 In such embodiments, R 7 is preferably H, and R 8 n is preferably an aryl such as phenyl, and n is 1, 2, or 3, preferably 1.

[0079] According to a preferred embodiment, R 3 If R is OH, 1 teeth, [ka] Preferably, [ka] That is the case.

[0080] In a preferred embodiment, the compound of formula (I) is the compound of formula (I-A'a) as defined above, where X, R and R 3 As defined above, R 1 teeth, [ka] Preferably, [ka] And here, Y is -(CH2) m - and m is as defined above, in particular m is 2, R 5 is C(O)OH, and R 6 is NH-CH(R 7 )-(CH2) n -R 8 And R 7 H is R 8 is an aryl such as phenyl, and n is 1, 2 or 3, or Y is [ka] And R 5 R is an O-C1-C6 alkyl such as O-methyl or O-ethyl, 6 These are O-C1-C6 fatty acid chains such as O-methyl or O-ethyl.

[0081] In another, more preferred embodiment, the compound of formula (I) is such that X is [ka] And R 1 NR 1a R 1b And here, R 1a is preferably H, and R 1b The alkyl group is preferably a C1-C6 alkyl group such as methyl, ethyl, n-propyl, n-butyl, or t-butyl, and the alkyl group is substituted with C(O)-methyl, C(O)-ethyl, C(O)-isopropyl, or C(O)-terbutyl, or C(O)-NH-methyl, C(O)-NH-ethyl, C(O)-NH-isopropyl, or C(O)-NH-terbutyl, where R is OR 2 And here R 2 The C1-C6 alkyl group is preferably C1-C6 alkyl, particularly ethyl or C1-C6 alkyl, particularly benzyl, and R3 The compound is of formula (I-A') in which is OH or O-C1-C6 alkyl, particularly O-ethyl, O-isopropyl, or O-terbutyl.

[0082] In another, more preferred embodiment, the compound of formula (I) is such that X is [ka] And R 1 NR 1a R 1b And here, R 1a is preferably H, and R 1b The C1-C6 alkyl group is preferably a C1-C6 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, or t-butyl (ter-butyl), preferably ethyl, isopropyl, or ter-butyl, where R is OR 2 And here R 2 is preferably a phenyl-substituted C1-C6 fatty acid chain, and R 3 The compound is of formula (I-A') in which is an O-C1-C6 alkyl, particularly O-ethyl, O-isopropyl, or O-terbutyl.

[0083] In a particular embodiment, X is [ka] If R 1 Preferably, [ka] or NR 1a R 1b And here R 1a H is R 1b This is a C1-C6 alkyl that is either unsubstituted or substituted with C(O)-O-C1-C6 alkyl or C(O)-NH-C1-C6 alkyl. R is OR 2 And here R 2This is a C1-C6 fatty acid chain (the above fatty acid chain is either unsubstituted or substituted with phenyl), or a C1-C6 alkyl-aryl compound. R 3 These are OH or O-C1-C6 fatty acid chains. Y is -(CH2) m -(m=2) or [ka] And, R 5 It is C(O)OH or O-C1-C6 alkyl, R 6 NH-CH(R 7 )-(CH2) n -R 8 (Here, R 7 H is R 8 (where n is an aryl molecule, n is 1, 2, or 3, preferably 1), or an O-C1-C6 fatty acid chain.

[0084] In some preferred embodiments, R 1 teeth [ka] And, R is OR 2 And R 2 These are C1-C6 fatty acid chains, and the above fatty acid chains are either unsubstituted or substituted with phenyl. R 3 These are OH or O-C1-C6 fatty acid chains. Y is -(CH2) m -(m=2) or [ka] And, R 5 It is C(O)OH or O-C1-C6 alkyl, R 6 NH-CH(R 7 )-(CH2) n -R 8(R 7 H is R 8 (where n is an aryl molecule and n is 1, 2, or 3, preferably 1), or an O-C1-C6 fatty acid chain.

[0085] In another specific embodiment, if X = -NH-CH2-, R 1 Preferably NR 1a R 1b And here R 1a H is R 1b This is a C1-C6 alkyl that is either unsubstituted or substituted with C(O)-NH-C1-C6 alkyl. R is OR 2 And here, R 2 It is a C1-C6 alkyl-aryl, R 3 This is the O-C1-C6 fatty acid chain.

[0086] In certain embodiments, the compound of formula (I) according to the present invention is a prodrug as defined above, in particular the following compounds: [ka] They are also selected from pharmaceutically acceptable salts and / or solvates thereof.

[0087] In another specific embodiment, the compounds of formula (I) according to the present invention are drugs as defined above, in particular the following compounds: [ka] or a pharmaceutically acceptable salt and / or solvate thereof.

[0088] In certain preferred embodiments, the compound of formula (I) according to the present invention is the following compound: [ka] JPEG2026509764000049.jpg142164 Selected from the group consisting of pharmaceutically acceptable salts and / or solvates thereof, as well as their conjugates. Method for preparing the compound of formula (I) Compounds of formula (I) as described above, or pharmaceutically acceptable salts and / or solvates thereof, are prepared by the following steps: (a) Compound of formula (II): [ka] Compound of formula (III): [ka] (In the formula, R Z This is R as defined above. 1 (or OH) The process of reacting with: (b) R is OH in some cases Z R as defined above 1 The process of converting It can be obtained by a method that includes [a certain element].

[0089] The compound of formula (III) can be obtained by methods well known to those skilled in the art.

[0090] The reaction between the compound of formula (II) and the compound of formula (III) is, in particular, a peptide coupling reaction as defined above.

[0091] The compound of formula (II) is particularly [ka] That is the case.

[0092] In some cases, additional protection / deprotection and / or functionalization steps, well known to those skilled in the art, may be performed before or after the reaction of the compound of formula (II) and the compound of formula (III) to obtain the compound of formula (I) having the preferred substituents described above.

[0093] In particular, R Z If is OH, the compound obtained from step (a) is [ka] R 1 but [ka] The compound of formula (I) is obtained.

[0094] The one or more peptide couplings performed in the method for preparing the compound of formula (I) are achieved, in particular, in the presence of PyAOP as a coupling agent. Preferably, the base DIEA is also used.

[0095] Peptide coupling can be performed on a solid support, particularly using a resin to which either the amine or acid portion of the reagent is bonded. Such methods are well known to those skilled in the art. A conjugate comprising a compound fragment of formula (I) linked to a biomolecule. The present invention also relates to the use of conjugates comprising fragments of compounds of formula (I) as described above, linked to biomolecules such as peptides, proteins, biomarkers, photolabeling agents such as rhodamine, cyanine derivatives or fluorescein, affinity probes such as biotin, or, but not limited to, E3 ubiquitin ligase recruiters containing thalidomide, VH032, VH101, dBET1, dFKBP12, QCA570, ZNL-02-096 or d9A-2.

[0096] The term "fragment of the compound of formula (I)" refers to a compound of formula (I) in which one end has been modified, for example, by binding to a biomolecule via a linker. Therefore, generally, R 1 The group is modified to enable the above bond. For example, in the conjugate according to the present invention, the fragment of the compound of formula (I) is the following part: [ka] refers to, where [Chemical formula] is a single bond between the fragment and the remaining part of the conjugate, and R, R 3 , X, Y and R 5 are as defined in the present disclosure.

[0097] According to a particular embodiment, the conjugate according to the invention has the following formula (I’): [Chemical formula] [where B is a biomolecule such as a peptide, a protein, a biomarker, for example, a photo-labeling agent, or an E3 ligase recruiter such as thalidomide, L is a linker, R, R 3 , X, Y and R 5 are as defined above] or a pharmaceutically acceptable salt and / or solvate thereof.

[0098] According to a preferred embodiment, the conjugate of formula (I’) has the following structure: [Chemical formula] is a conjugate of formula (I’-A) having

[0099] In particular, the conjugate of formula (I’) has the following structure: [Chemical formula] is a conjugate of formula (I’-A a ) having

[0100] In the conjugate of formula (I’), particularly in the conjugate of formula (I’-Aa), X is preferably [Chemical formula] That is the case.

[0101] R is preferably OR 2 And R 2 The advantageous C1-C6 alkyl groups, particularly ethyl groups, or C1-C6 alkyl-aryl groups, particularly benzyl groups.

[0102] R 3 The ion is preferably OH or O-C1-C6 alkyl, particularly O-ethyl, O-isopropyl, or O-terbutyl.

[0103] Y is -(CH2) m -or [ka] It is possible.

[0104] Y is -(CH2) m -, especially if it is -(CH2)2-, R 5 is preferably C(O)OH, and R 6 Preferably NH-CH2-(CH2) n -R 8 And here R 8 n is preferably an aryl such as phenyl, and n is preferably 1, 2, or 3, especially 1.

[0105] Y [ka] If R 5 Preferably, it is an O-C1-C6 alkyl such as O-ethyl.

[0106] According to some embodiments, the linker L is C1-C 12It corresponds to a divalent group derived from a fatty chain, where one or more methylene units are replaced by a structural linker selected from arylene or fragments -O-, -S-, -C(=O)-, -SO2- or -N(C1-C6 alkyl)-, and the above fatty chain is unsubstituted or substituted with one or more groups selected from halogen, OH, C1-C6 alkyl, and / or C1-C6 alkylaryl groups such as, for example, a benzyl group.

[0107] In one embodiment, the biomolecule is an affinity probe, and the conjugate of formula (I’) is

Chemical formula

[0108] In another embodiment, the biomolecule is a photolabeling agent, and the conjugate of formula (I’) is

Chemical formula

[0109] The present invention also relates to a pharmaceutical composition comprising at least one conjugate as described above, for example, a conjugate of formula (I’), or a pharmaceutically acceptable salt and / or solvate thereof, and at least one pharmaceutically acceptable excipient, for use in the prevention or treatment of heart disorders.

[0110] In some embodiments, the pharmaceutical compositions used according to the present invention are intended for oral or parenteral administration (including, but not limited to, intravenous, intramuscular, intra-arterial, intradermal, subcutaneous, intraperitoneal, intravenous, and intracranial), preferably oral or intravenous administration. Other modes of delivery include, but not limited to, the use of liposomal formulations, intravenous injections, and percutaneous patches. The active ingredient can be administered to animals, preferably mammals including humans, in unit dosage forms mixed with conventional pharmaceutical carriers.

[0111] These compositions may be formulated as applicator sticks, liquids, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols, and delivered transdermally via topical routes. Oral formulations include tablets, pills, powders, capsules, liquids, lozenges, cachets, gels, slurries, and suspensions suitable for oral administration by the patient. Solid formulations include powders, tablets, pills, capsules, cachets, suppositories, and dispersed granules. Liquid formulations include liquids, suspensions, and emulsions, such as water or water / propylene glycol solutions. Compositions may further contain components to provide sustained release and / or comfort. Such components include high molecular weight anionic mucus-mimicking polymers, gelling polysaccharides, and finely powdered drug carrier bases. Compositions may also be delivered as microspheres for sustained release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres that are slowly released subcutaneously; as a biodegradable injectable gel formulation; or as orally administered microspheres. In another embodiment, the composition may be delivered by the use of liposomes that fuse with the cell membrane or are endocytotic, i.e., by liposome-bound receptor ligands (which bind to cell surface membrane protein receptors to induce endocytosis). The use of liposomes allows for the concentration of in vivo delivery of the composition to target cells, particularly if the liposome surface has a target cell-specific receptor ligand or if it is preferentially induced to a specific organ (e.g., the heart).

[0112] Concomitant administration means administering the compound alone or in combination with other active substances such as those disclosed herein (multiple compounds or drugs), simultaneously or sequentially with other active substances such as those disclosed below. Combination therapy or combination The compound of formula (I) can be used in combination with other current standard treatments for heart failure, cardiomyopathy, or myocardial infarction-induced cardiac dysfunction, such as those listed in the 2021 ESC guidelines for the diagnosis and treatment of acute and chronic heart failure (McDonagh TA et al., 2021).

[0113] A heart failure specialist defines the most appropriate combination depending on the patient and their heart condition. Combination therapy with SVC compounds (as dual pharmacophores) may seem intuitively appealing: 1) Concomitant administration with positive inotropic agents having different complementary mechanisms of action (MoA). These include β-agonists (e.g., dobutamine, dopamine, or epinephrine), type 3 phosphodiesterase inhibitors (e.g., milrinone), or alternative positive inotropic agents such as levosimendene, which enhance the total positive inotropic relief in cardiogenic shock without increasing energy requirements.

[0114] 2) Concomitant administration with negative inotropic agents, which should be beneficial but are poorly tolerated due to their negative inotropic effects. This may include concomitant use with β-adrenergic blockers in patients with severe cardiomyopathy or cardiogenic shock, or with myosin inhibitors (such as mavacamten or other emerging drugs).

[0115] Accordingly, the present invention also relates to a method for treating a cardiovascular disease selected from heart failure, cardiomyopathy and myocardial infarction-induced cardiac dysfunction in a subject of interest, comprising administering to the subject of interest an effective dose of the compound of formula (I) disclosed above in combination with the current standard treatment for heart failure, cardiomyopathy, or myocardial infarction-induced cardiac dysfunction. In particular, the current standard treatment for heart failure is selected from a positive inotropic agent or a negative inotropic agent, in particular from (i) a positive inotropic agent selected from the group consisting of β-agonists, phosphodiesterase type 3 inhibitors and alternative positive inotropic agents, or (ii) a negative inotropic agent selected from the group consisting of β-adrenergic blockers and myosin inhibitors.

[0116] Another objective of the present invention is, a) A compound of formula (I) as defined in the present invention or a pharmaceutical composition as defined in the present invention, b) Current standard treatment for heart failure, cardiomyopathy, or myocardial infarction-induced cardiac dysfunction It is a combination that includes [the specified element].

[0117] In particular, the current standard treatment for heart failure is selected from either a positive or negative inotropic agent, specifically (i) a positive inotropic agent selected from the group consisting of β-agonists, phosphodiesterase type 3 inhibitors, and alternative positive inotropic agents, or (ii) a negative inotropic agent selected from the group consisting of β-adrenergic blockers and myosin inhibitors. Use of compounds and pharmaceutical compositions in cardiac disorders The compound of formula (I), its pharmaceutically acceptable salts and / or solvates, or the pharmaceutical compositions according to the present invention act as VASH inhibitors, meaning they can inhibit the peptidase activity of VASH that catalyzes the detyrosination of microtubules. This VASH peptidase activity, when dysregulated and especially abnormally elevated, can lead to cardiac disorders, particularly heart failure, cardiomyopathy, and / or myocardial infarction-induced cardiac dysfunction.

[0118] X [ka] The compound of formula (I) is particularly an irreversible VASH inhibitor and covalently binds to the VASH enzyme.

[0119] The present invention relates to compounds of formula (I) according to the present invention, or pharmaceutically acceptable salts and / or solvates thereof, for use as drugs in the prevention and / or treatment of cardiac disorders, particularly heart failure, cardiomyopathy, and / or myocardial infarction-induced cardiac dysfunction.

[0120] In other words, the present invention relates to the use of compounds of formula (I) according to the present invention, or pharmaceutically acceptable salts and / or solvates thereof, for the manufacture of drugs intended in the prevention and / or treatment of cardiac disorders, in particular heart failure, cardiomyopathy, and / or myocardial infarction-induced cardiac dysfunction.

[0121] In other words, the present invention relates to the use of compounds of formula (I) according to the present invention, or pharmaceutically acceptable salts and / or solvates thereof, for the prevention and / or treatment of cardiac disorders, in particular heart failure, cardiomyopathy, and / or myocardial infarction-induced cardiac dysfunction.

[0122] In particular, the present invention relates to the use of compounds of formula (I) according to the present invention, or pharmaceutically acceptable salts and / or solvates thereof, for use in the prevention and / or treatment of heart failure, cardiomyopathy, myocardial infarction-induced cardiac dysfunction, and / or for improving cardiac function in subjects where such use is required.

[0123] In other words, the present invention relates to a method for improving cardiac function in a subject of interest, comprising administering to the subject of interest a compound of formula (I) according to the present invention, or a pharmaceutically acceptable salt and / or solvate thereof.

[0124] In particular, compounds of formula (I) or pharmaceutically acceptable salts and / or solvates improve the dynamics of dysfunctional cardiomyocytes, including increased relaxation rate, increased contractility, and / or reduced viscoelasticity.

[0125] In other words, the present invention relates to a method for treating a cardiovascular disease selected from heart failure, cardiomyopathy, and myocardial infarction-induced cardiac dysfunction in a subject of interest, comprising administering to the subject of interest a compound of formula (I) according to the present invention, or a pharmaceutically acceptable salt and / or solvate thereof.

[0126] In other words, the present invention relates to a method for preventing and / or treating cardiac disorders, particularly heart failure, cardiomyopathy and / or myocardial infarction-induced cardiac dysfunction, comprising administering an effective dose of a compound of formula (I) according to the present invention, or a pharmaceutically acceptable salt and / or solvate thereof, to a subject in need.

[0127] In another aspect, the present invention relates to a pharmaceutical composition according to the present invention for use as a drug in the prevention and / or treatment of cardiac disorders, particularly heart failure, cardiomyopathy and / or myocardial infarction-induced cardiac dysfunction.

[0128] In other words, the present invention relates to the use of pharmaceutical compositions according to the present invention for the manufacture of drugs intended in the prevention and / or treatment of cardiac disorders, particularly heart failure, cardiomyopathy and / or myocardial infarction-induced cardiac dysfunction.

[0129] In other words, the present invention relates to the use of pharmaceutical compositions according to the present invention for the prevention and / or treatment of cardiac disorders, in particular heart failure, cardiomyopathy and / or myocardial infarction-induced cardiac dysfunction.

[0130] In other words, the present invention relates to a method for preventing and / or treating cardiac disorders, particularly heart failure, cardiomyopathy and / or myocardial infarction-induced cardiac dysfunction, comprising administering an effective dose of a pharmaceutical composition according to the present invention to a subject in need.

[0131] In certain embodiments, the subject requiring assistance has heart failure associated with incomplete cardiomyocytes.

[0132] In certain embodiments, the target population has reduced ejection fraction heart failure (HFrEF).

[0133] In certain embodiments, the target population has heart failure with maintained ejection fraction (HFpEF).

[0134] In certain embodiments, the pharmaceutical compositions of the present invention are used to reduce viscoelasticity and / or increase the relaxation rate of cardiomyocytes.

[0135] In some embodiments, the pharmaceutical composition of the present invention is Ca 2+ It is used to increase the relaxation rate of cardiomyocytes via an independent mechanism.

[0136] In certain embodiments, the pharmaceutical compositions of the present invention are used to reduce cardiomyocyte stiffness, improve myocyte relaxation, increase ventricular compliance, and / or reduce diastolic pressure.

[0137] In certain embodiments, the pharmaceutical compositions of the present invention are used to treat patients with diastolic dysfunction.

[0138] In certain embodiments, the pharmaceutical compositions of the present invention are used for the treatment of chronic heart disorders, particularly chronic heart failure or cardiomyopathy.

[0139] In certain embodiments, the pharmaceutical compositions of the present invention are used to treat acute heart failure (AHF) syndromes, including post-myocardial infarction syndrome, post-cardiac surgery syndrome, post-cardiac arrest syndrome, hypertensive crisis conditions, acute symptoms of non-ischemic cardiomyopathy, and acute exacerbations of chronic cardiomyopathy of various etiologies.

[0140] In certain embodiments, the pharmaceutical composition of the present invention is used to treat patients with systolic dysfunction.

[0141] In certain embodiments, the pharmaceutical composition of the present invention is used to prepare pharmaceuticals for use in the treatment of dysfunctional cardiomyocytes.

[0142] In certain embodiments, the pharmaceutical composition of the present invention is used to prepare pharmaceuticals for use in improving the dynamics of failing ventricular cardiomyocytes.

[0143] In certain embodiments, the pharmaceutical composition of the present invention is used to prepare a pharmaceutical for use in reducing the viscoelasticity of cardiomyocytes. Research tool applications Another aspect of the present invention is the use of a conjugate comprising a compound (I) as defined in the present invention, or optionally a labeled biomolecule, linked or unlinked to a fragment of formula (I), as a research tool for research and development activities of cardiac dysfunction or heart failure.

[0144] In some embodiments, the present invention is The role of tubulin detyrosination in cardiac disorders, for example, in vitro methods for studying its onset, invasiveness and progression, and Related kits for performing the above screening assay and method The present invention relates to a compound of formula (I) or its conjugate, for use as a research tool in research and development activities selected from the group consisting of the following.

[0145] These research and development activities, which are illustrative and not limiting examples, use the compound of formula (I) or its conjugate.

[0146] The compound of formula (I) above may be bound to, for example, a fluorescent dye, a UV-sensitive dye, HRP, an alkaline phosphatase, or biotin, but are not limited to these. [Brief explanation of the drawing]

[0147] [Figure 1] High-magnification image of immunohistochemical staining for detyrosinated tubulin in healthy myocardial tissue. A single myocardial cell is shown at high magnification. The purple stain is specific detyrosinated tubulin. [Figure 2] Structure of a specific Vash compound (SVC) that acts as a potent inhibitor. [Figure 3] An in vitro detyrosination assay using recombinant human VASH1 (hVASH1) and SVBP complex. An ELISA-based method for measuring detyrosinase activity. Dose-response allows for accurate measurement of the compound's inhibition of the target. Notably, SVC_02 is a prodrug. This compound is inactive in its prodrug form when tested with recombinant enzyme. It contains a specific group that effectively masks a second group essential for binding to the enzyme. [Figure 4] Immunofluorescence staining of wild-type human cells and VASH double knockout cells. SVC_01 treatment induced a background signal comparable to that observed in VASH 2KO cells, indicating involvement of the entire target. Acetylated tubulin levels were unaffected by SVC_01 treatment, and very similar staining patterns and levels were observed under various conditions. [Figure 5] Immunofluorescence for detecting the level of tubulin detyrosination in human cells. A dose-dependent decrease in tubulin detyrosination was observed in human cells after 2 hours of treatment with all SVCs. In-cellulo IC50 measurement of SVC_03 = 10 nM. [Figure 6] An in vitro detyrosination assay using recombinant human enzymes in the absence or presence of SVC_01 or BzlSA (specific carboxypeptidase A inhibitors). Carboxypeptidase A (CPA) hydrolyzes peptide bonds at C-terminal residues with aromatic or aliphatic side chains. No cross-reactivity was observed between different enzymes. The reaction was stopped using SDS-Page Laemli buffer and added to a Western blot using a specific antibody. [Figure 7]This assay uses activated recombinant human rhinovirus (HRV) type 14 3C protease cysteine ​​in the absence (neg) or presence (escalating dose) of SVC_01. HRV is a protease that prefers the non-aromatic residue at P1'. The optimal recognition site for human rhinovirus (HRV) type 14 3C protease is LEVLFQ / GP, with glycine at the P1' position. SVC_01 does not reduce the activity of this cysteine ​​protease. [Figure 8] This assay uses active cathepsin B cysteine ​​protease. The panel shows the activity of cathepsin B in the presence of a commercially available cathepsin B&L inhibitor called FF-FMK (positive control) or in the presence of an increasing dose of SVC_01. Cathepsin B is a cysteine ​​protease that prefers the aromatic residue at the P1' position. Unlike VASH, cathepsin B prefers the aromatic residue at the P1' position, which is considered the best control in terms of specificity. [Figure 9] This assay compares cell viability in human cells exposed to microtubule-binding agents (parthenolide, taxol) and SVC_01. The MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity. This colorimetric quantitative assay is based on the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide, or MTT) to a purple formazan crystal by metabolically active cells. In contrast to microtubule-binding agents that exhibit severe toxicity, SVC_01 does not affect cell proliferation. [Figure 10]Mitochondrial activity assay using the Agilent Seahorse Cell Mito Stress Test Kit in the absence or presence of SVC_01 [20 μM]. This assay is widely recognized as a standard assay for energetically evaluating mitochondrial function. This single assay provides multiple parameters, including basal respiration, ATP-related respiration, maximal and reserve volumes, and non-mitochondrial respiration. This assay allows users to gain insights into the mechanisms of mitochondrial dysfunction and investigate functional differences across cell types, drug candidates, and genetic and biochemical interventions. High-concentration exposure to SVC_01 does not alter mitochondrial activity. [Figure 11] The potassium current mediated by the hERG channel (referred to as the hERG current) was recorded using a patch-clamp method with a whole cell composition. The inhibition rate represents the average percentage calculated from the individual differences in hERG tail current amplitude relative to the extracellular solution (stabilization phase) of each cell. The average inhibition rate at the highest test concentration, i.e., 30 μmol / L, is shown in the table. Six concentration-response curves were created for each compound. [Figure 12] Dose-dependent reduction in detyrosination upon VASH inhibition in primary cardiomyocytes isolated from Sprague Dawley rats. A) Representative Western blot and B) quantification of detyrosinated tubulin and α-tubulin levels after incubation of newly isolated adult rat cardiomyocytes with specified concentrations of compound SVC_01 at 37°C for 2 hours. N=4 Sprague Dawley rats. [Figure 13] Dose-dependent, gradual improvement in systolic and diastolic dynamics during unloaded shortening in primary cardiomyocytes isolated from healthy Sprague Dawley rats upon VASH inhibition. A) resting sarcomere length, B) left ventricular diameter shortening percentage, C) systolic time, and D) diastolic time in isolated healthy rat cardiomyocytes. N=3 Sprague Dawley rats, n=50-60 myocytes per group. One-way ANOVA. [Figure 14]Western blots of cardiomyocytes prepared from Wistar Kyoto (WKY) rats, ZSF1 lean rats, and ZSF1 obese rats. Protein samples were prepared and analyzed by immunoblotting following PAGE separation using the specified antibodies described. [Figure 15] Immunofluorescence images of detyrosinated and tyrosinated tubulin in ZSF-1 obese cardiomyocytes incubated at 37°C for 2 hours in SVC_01 incubation show that detyrosinated microtubules actually disappeared, while the microtubule network was maintained. [Figure 16] Improvement of contraction and relaxation dynamics of HFpEF myocytes during unloaded shortening in primary cardiomyocytes isolated from WKY rats, ZSF1 lean rats, or ZSF1 obese rats upon VASH inhibition or TTL overexpression. A) contraction amplitude, B) contraction time, and C) relaxation time in isolated rat cardiomyocytes. Approximately n=90 myocytes per group from 3 ZSF1 lean or obese rats; n=60 myocytes from 2 WKY rats. Two-way ANOVA with genotype and treatment as factors. [Figure 17] Reduction of lateral stiffness in primary cardiomyocytes isolated from lean or obese ZSF1 rats upon VASH inhibition or TTL overexpression. A) Stiffness (elastic modulus) plotted as a function of nanoindentation rate in lean and obese ZSF1 cardiomyocytes. C) Quantification of Emin (elastic stiffness), Emax (stiffness at maximum indentation velocity), and D) DeltaE (indicator of viscoelasticity) in lean and obese ZSF1 cardiomyocytes. Obese ZSF1 cardiomyocytes have higher viscoelasticity than lean ones, and this viscoelastic stiffness is reduced by overexpression of SVC_01 and TTL. [Figure 18]Reduction of longitudinal stiffness upon VASH inhibition in primary cardiomyocytes isolated from ZSF1 lean or ZSF1 obese rats. A) Diastolic stiffness (elastic modulus at the initial time of maximum strain) when a 10% strain was applied in the long axis direction for 200 ms in isolated cardiomyocytes; B) Steady-state stiffness during strain and retention; and C) Stress relaxation (an indicator of viscoelasticity) in ZSF1 lean and obese cardiomyocytes. ZSF1 obese cardiomyocytes have higher viscoelasticity than lean ones, and this longitudinal viscoelastic stiffness is reduced by SVC_01 treatment. [Figure 19] A) Western blot of myocardial tissue showing effective reduction of detyrosinated tubulin after a single injection of SVC_02 in 8-week-old Sprague Dawley rats. B) Quantification of detyrosination levels (D1 / α-tubulin ratio) in vehicle-treated rats (n=4) and VASHi-treated rats (n=3). [Figure 20] Western blot of myocardial tissue showing effective reduction of detyrosinated tubulin after two intravenous injections of SVC_02 into lean and obese 30-week-old ZSF1 rats. [Figure 21] VASHi rapidly improves diastolic function in HFpEF rats. A) Echocardiographic evaluation of left ventricular diastolic function in ZSF1 lean and obese animals treated with + / -SVC_02. Improvements in the E / A ratio (marker of left ventricular function) and mitral valve deceleration time indicate improved diastolic function and faster ventricular relaxation in ZSF1 obese rats with VASH inhibition. B) Hemodynamic measurements of left ventricular diastolic function in ZSF1 lean and obese animals treated with + / -SVC_02. Improvements in tau (left ventricular diastolic time constant) and dP / dTmin (ventricular relaxation rate) indicate faster ventricular relaxation in ZSF1 obese rats with VASH inhibition. [Figure 22] Transcriptional changes in ZSF1-bound obese rats and lean rats were compared using NanoString, which is a volcano plot of differentially expressed extracellular transcripts and tubulin-related transcripts. [Figure 23]Immunofluorescence imaging for detection of tubulin detyrosination in LV tissue and evaluation of changes in the abundance of detyrosinated tubulin after SVC-02 treatment. [Figure 24] Quantitative analysis of immunofluorescence imaging performed on LV tissue to evaluate changes in microtubule density (SVC-02 as VASHi). [Figure 25] Quantitative analysis of Western blot analysis of myocardial tissue showing effective reduction of detyrosinated tubulin after two intravenous injections of SVC_02 into 30-week-old WKY rats, ZSF1 lean rats, and obese rats. [Figure 26] The donor's patient characteristics. [Figure 27] Mean sarcomere shortening of cardiomyocytes isolated from non-incomplete (NF) and incomplete (HF) human hearts and treated with vehicle (black) and cardiomyocytes treated with SVC-02 (VASHi) (gray). [Figure 28] Relaxation of isolated, incomplete human cardiomyocytes is improved by VASH inhibition (SVC-02). [Figure 29] Hematoxylin and eosin staining showing the normal appearance of various organs in Swiss mice after systemic treatment with SVC-02, compared to control vehicles. [Figure 30] Immunofluorescence for detecting the level of tubulin detyrosineation in human cells. A dose-dependent reduction in tubulin detyrosineation was observed in human cells after 2 hours of treatment with all SVCs. In-cellulo IC50 measurement for SVC_06 = 2.1 nM. [Figure 31] Quantitative analysis of myocardial tissue isolated after oral administration of SVC-06 in Sprague-Dawley rats using Western blot. [Figure 32] In vitro detyrosination assay using recombinant human VASH1 and VASH2 enzymes in the presence of gradually increasing concentrations of SVC_01. Both VASH1 and VASH2 are effectively inhibited by SVC_01. [Figure 33]Human adult cardiomyocytes were treated with either a vehicle or SVC-01 (an inhibitor). The protein lysates were analyzed by Western blotting to assess the levels of tubulin detyrosineation and acetylation. SVC-01 treatment significantly reduced tubulin detyrosineation but had no effect on tubulin acetylation. [Figure 34] Human adult cardiomyocytes were treated with either a vehicle or SVC-01, harvested, and analyzed for mRNA expression of genes involved in the detyrosination-tyrosination cycle. Treatment of human adult cardiomyocytes with SVC-01 did not alter the expression of genes involved in regulating tubulin detyrosination. [Examples]

[0148] material and method animal The care and treatment of the animals were approved and carried out in accordance with the standards set forth in the Guide for the Care and Use of Laboratory Animals published by the University of Pennsylvania Institutional Animal Care and Use Committee and the National Institutes of Health. Isolation and culture of rat cardiomyocytes Primary adult ventricular cardiomyocytes were isolated from 6-8 week old Sprague Dawley rats, or 20-30 week old WKY rats, ZSF1 lean rats, and obese rats. Briefly, the hearts of rats anesthetized under isoflurane were removed and back-perfused with collagenase solution on a Langendorff apparatus. Next, the digested hearts were shredded and tritulate using a glass pipette. The resulting supernatant was separated and centrifuged at 300 rpm to isolate cardiomyocytes, which were then resuspended at low density in rat cardiomyocyte medium. The cardiomyocytes were cultured with 25 μmol / L cytochalasin D at 37°C and 5% CO2. Rat cardiomyocyte medium: 1× insulin-transferrin-selenium-X (Gibco 51500056), 1 μg μl -1 Thermo Fisher 115090 medium supplemented with Primosin (Invivogen ant-pm-1), 20 mmol / L HEPES at pH 7.4, and 25 μmol / L cytochalasin D. Contractile force of isolated cardiomyocytes Contractile force was measured using a custom-made cell chamber (IonOptix) attached to an LSM Zeiss 880 inverted confocal microscope, with a 40x or 63x oil 1.4 numerical aperture objective lens and a transmitted light camera (IonOptix MyoCam-S). Experiments were conducted at room temperature, and field stimulation was applied at 1.0 Hz using a cell stimulator (MyoPacer, IonOptix). After pacing for 10–30 seconds to reach a steady state, five traces were recorded and analyzed. Sarcomere length was measured optically by Fourier transform analysis (IonWizard, IonOptix). Western blot For whole-cell protein extraction, isolated rat cardiomyocytes were lysed on ice for 1 hour in RIPA buffer (Cayman #10010263) supplemented with a protease and phosphatase inhibitor cocktail (Cell Signaling #5872S). The supernatant was collected and combined with 4x loading dye (Li-COR #928-40004) supplemented with 10% 2-mercaptoethonol, and boiled for 8 minutes. The resulting lysates were separated on an SDS-PAGE gel, and the proteins were blotted onto a nitrocellulose membrane (Li-COR #926-31902) using miniTrans-Blot Cell (Bio-Rad). The membranes were blocked with Odyssey blocking buffer (TBS) (Li-COR #927-50000) for 1 hour and probed overnight at 4°C with the corresponding primary antibody. The membrane was washed three times with TBS (TBST) containing 0.5% Tween 20, and incubated with the secondary antibody in TBS with added 0.2% Tween 20 at room temperature for 1 hour. The membrane was washed again with TBST (0.5% Tween 20) and imaged using an Odyssey Imager. Image analysis was performed using Image Studio Lite software (LI-COR). All samples were measured in two replicates and analyzed using GAPDH as the baseline. antibody Detyrosinated tubulin; rabbit polyclonal antibody (Abcam ab48389) specifically detecting detyrosinated α-tubulin; or affinity purified antibody (Van der Laan, 2019); Western blot: 1:1,000 dilution. α-tubulin; mouse monoclonal, clone DM1A (Cell Signaling #3873); Western blot: 1:1,000 dilution. GAPDH; Mouse monoclonal (VWR GenScript A01622-40); Western blot: 1:1,000 dilution. Nanoindentation Microscopic mechanical properties were measured using nanoindentation (Piuma; Optics11, Amsterdam, Netherlands) equipped with a Dynamic Dynamics Analysis (DMA) package. Freshly isolated cardiomyocytes were attached to a glass-bottom dish coated with MyoTak (IonOptix) in 1 mM Ca2+ NT solution. A spherical indentation probe with a radius of 4.5 μm and stiffness of 0.1 N / m was used to indent the cardiomyocytes to a depth of 1–2 μm, and then the probe was sinusoidally vibrated at frequencies of 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, and 50 Hz with an amplitude of 1 μm. Using Piuma Data Viewer version 2.0, the DMA indentation depth and cantilever deflection were converted to E' and E'', and the magnitude of the elastic modulus was converted to E 2 =E' 2 +E motif 2 The following was determined. Assuming a Poisson's ratio (n) of 0.5, the average value of E under each experimental condition was calculated as a function of indentation velocity. Low-velocity indentations measure the elastic contribution to stiffness, while high-velocity indentations measure the elastic and viscous contributions. The change in elastic modulus with velocity represents the viscoelasticity of muscle cells and can be fitted to determine the model and magnitude of viscoelastic behavior. Extension and mechanical evaluation of isolated cardiomyocytes Freshly isolated or cultured cardiomyocytes were diluted to a sparse density (approximately 10-fold) in large (22 x 50 mm) glass-bottomed Petri dishes coated with BSA to prevent adhesion of the cardiomyocytes with Normarthilodes solution. A small amount of freshly thawed Myotak® was placed on the dry surface of a 1.5 μL droplet Petri dish and polymerized (2-3 minutes). Once the droplet became "sticky," a laser-etched glass rod (Ion Optix LLC) was lowered into the solution several times to form a thin coating of Myotak, which was then dried for 90 seconds before the probe was immersed in the cell-containing chamber. Subsequently, the cardiomyocytes were adhered to a glass holder by light contact, slightly lifted onto a coverslip, and then subjected to mechanical testing. Using a Myostretcher instrument (Ion Optix LLC), muscle cells were stretched by approximately 10% at intervals of 200 milliseconds (diastolic) or 5 seconds (slow phase), and muscle force and sarcomere length were recorded using Ion Wizard software and the MyoCam imaging system (live FFT). Subsequently, each cell was imaged with transmitted light, its cross-sectional area was determined, and the force was converted into stress. After each protocol, the cell holder was lifted from the solution several times to remove the cells, and further residue was removed using a fiber optic cable. If the cells did not adhere well to the holder, the probe was washed with trypsin / EGTA solution, physically washed with a fiber optic cable, and recoated with Myotak. Immunofluorescence Cells were fixed with 4% PFA (Electron Microscopy Sciences) for 10 minutes, washed three times with PBS, and then permeabilized with 0.1% TritonX-100 at room temperature for 10 minutes. After washing twice with PBS, cells were placed in blocking buffer (1:1 Seablock (Abcam) and 0.1% TritonX-100 (Bio-Rad) in PBS) at room temperature for at least 1 hour, and then labeled with the primary antibody (see below) at 4°C for 24–48 hours. Subsequently, cells were washed three times with TBS, then labeled with the secondary antibody (see below) in TBS at room temperature for 2–4 hours, and finally washed twice with TBS. Stained cells were mounted on #1.5 coverslips with Prolong Diamond Antifade Mountant (Thermo Fisher) for imaging. Alternatively, cells were directly imaged in the chamber with TBS. Imaging was performed using a Zeiss 880 Airyscan confocal microscope operating on an Axiovert Z1 inverted microscope equipped with a Plan-Apochromat 63x oil 1.4 NA objective lens. Image analysis was performed using ZEN Black software for Airyscan processing, which included signal integration from 32 separate sub-resolution detectors within the Airyscan detector, followed by deconvolution of this integrated signal. Transthoracic echocardiography Echocardiographic measurements were obtained using a Vevo2100 Ultrasound System (VisualSonics Inc., Toronto, Ontario, Canada) with an MS250 (13-24 MHz) transducer. Animals were sedated with inhaled isoflurane (2-3%). Parasternal long-axis, parasternal short-axis, apical four-ventricle, and apical two-ventricle views were acquired to assess cardiac structure and function. Diastolic activity was evaluated using pulse wave and tissue Doppler imaging. The protocol used was based on the recommendations of the European Society of Cardiology Working Group on Cardiac Function (Zacchigna S et al. 2021). invasive hemodynamics The animals were induced with isoflurane (5%), then intubated and supported in an unconscious state. The anesthesia plane was maintained with isoflurane (2-3%). A two-French pressure conductance catheter (Transonic Systems Inc., Ithaca, New York, USA) was calibrated using left ventricular volume obtained by echocardiography. Subsequently, the right common carotid artery was transected, and the catheter was advanced retrograde into the left ventricle as described above. Data were acquired using PowerLab and LabChart Pro (ADInstruments) and analyzed offline using LabChart Pro (ADInstruments). Detyrosinated tubulin immunofluorescence Formalin-fixed paraffin-embedded (FFPE) sections on glass slides were deparaffinized by sequential washing with xylene, progressively decreasing concentrations of ethanol (100%, 95%, 75%, and 50%), and then in ddH2O. The slides were placed in 1x Reveal decloaking solution (Biocare Medical, catalog #RV1000M) for antigen retrieval. Antigen retrieval was performed at high pressure for 15 minutes using a pressure cooker (Instant Pot Pro 10-in-1 pressure cooker, 8 Quart, Amazon.com). After cooling, a second antigen retrieval was performed on the slides at high pressure for 15 minutes in 1x Antigen Unmasking solution, citrate base (Vector Laboratories, catalog #H-3300-250) in the pressure cooker. The slides were then washed in ddH2O and then in PBS. The slides were permeabilized with PBS + 0.25% Triton X-100, and then incubated for 1 hour at room temperature in fish serum blocking buffer containing 0.1% Tween 20 (Thermo Scientific, catalog #37527).

[0149] Sections were co-stained with mouse monoclonal (B-5-1-2) antibody against α-tubulin (Sigma-Aldrich, catalog #T5168; 1:50) and rabbit polyclonal detyrosinated α-tubulin antibody (ab48389 Lot #GR3425171; 1:50) in blocking buffer at room temperature for 2 days. After washing the sections in PBS containing 0.1% Tween 20 (PBST), they were incubated in PBST with Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody and Alexa Fluor 568-conjugated goat anti-mouse secondary antibody, along with Alexa Fluor 488-conjugated WGA (Invitrogen catalog #W11261; final concentration 25 μg / mL), at room temperature for 2 days. Next, the sections were stained with Hoechst 33342 trihydrochloride trihydrate (Invitrogen catalog #H3570, final concentration in PBS 10 μg / mL) and mounted using Prolong Diamond Antifade Mountant (Invitrogen catalog #P36961).

[0150] Longitudinal cardiomyocytes within the cardiomyocyte were identified. A 4-channel Z-stack (4 × 500 nm slice) of the cardiomyocyte region was imaged at an 18.3 nm × 18.3 nm pixel size using a Zeiss Airyscan 880 microscope with a 63x oil immersion objective lens (1.4 NA). Background was subtracted from the images, and a maximum intensity projection was created using FIJI (NIH). Rectangular ROIs were drawn in a blinded manner within the cytoplasm of individual cardiomyocytes, excluding the nucleus and perinuclear region, using the WGA and Hoechst channels. Images of α-tubulin and detyrosinized microtubules were extracted from the ROIs. A classification model for each channel was trained using a preliminary set of α-tubulin and detyrosinized microtubule images with a trainable Weka Segmentation FIJI plugin. The final classification model was applied to all images in the dataset for each channel to generate a binary map of the microtubule network. Microtubule density was calculated as the area coverage of the binary map within each ROI. Cathepsin B assay The screening kit manufactured by Merck (ref: MAK200) was used according to the manufacturer's recommendations. Nanostring mRNA analysis Two custom NanoString coding sets [one for tubulin-related transcripts (tubulin isoforms, modifying enzymes, and microtubule-related proteins), and the other for heart failure and extracellular matrix-related transcripts], along with three housekeeping genes (Gapdh, Rpl4, and Tbp), were designed as described above with the assistance of NanoString Technologies. Nineteen isolated mRNAs were assessed for concentration (>20 ng / uL) and quality (OD260 / 230 >1.8) using a BioTek Synergy 4 microplate reader. 200–300 ng of total RNA were sent to the Wistar Institute core and processed using NanoString Technologies' nCounter Custom CNV assay to determine the levels of target tubulin-related mRNA. RNA quantities were then measured using an nCounter digital analyzer according to the manufacturer's protocol, and barcode counts were compiled into files for analysis using nSolver Analysis software (NanoString 4.0). An internal negative control included in NanoString Prep was used to subtract the background, and transcript levels that were quantitatively below the background level were excluded from the analysis. Next, the data were normalized to the geometric mean of housekeeping genes (Gapdh, Rpl4, and Tbp) using the nSolver package and exported for statistical analysis. Significance was assessed using an uncorrected paired t-test with OriginPro2019. Human cardiomyocyte tissue procurement Failure-causing human hearts were procured at the University of Pennsylvania Hospital during orthotopic heart transplants, with the prior informed consent of all participants. Healthy hearts were obtained from deceased donors during organ donation. Consent to use donor heart tissue for research was obtained from close relatives. In all cases, the heart was stopped on-site using ice-cold myocardial resuscitation solution and transported to the laboratory on wet ice. Isolation of human muscle cells Once the heart was removed, it was transported from the operating room to the laboratory in a state of cold myocardial palsy. Myocardial cells were isolated using known methods. A wedge-shaped tissue apex was excised from the left ventricular free wall, and a catheter was placed in the left anterior descending artery. The cannula was washed with cold saline to evaluate wedge perfusion, and major vessels were ligated. The myocardium was then perfused for 10 minutes with warm, non-circulating Krebs-Henseleit buffer (KHB) solution (12.5 mM glucose, 5.4 mM KCl, 1 mM lactate, 1.2 mM MgSO4, 130 mM NaCl, 1.2 mM NaH2PO4, 25 mM NaHCO3, 2 mM sodium pyruvate, 20 mM BDM, and 10 mM taurine, pH 7.4). Subsequently, the myocardium was perfused for 5 minutes with circulating KHB and type II collagenase (294 units / mL) (Worthington Biochemical Corp., Lakewood, NJ, USA), and calcium was slowly reintroduced to a final concentration of 1 mM. The collagenase and calcium-supplemented KHB was recirculated for 25-30 minutes depending on the rate of myocardial digestion. Then, the tissue was removed from the cannula, rinsed with KHB supplemented with 1% bovine serum albumin (BSA) to halt digestion, and shredded. The cell suspension was then filtered through a 280 μM nylon mesh, centrifuged (25 x g, 2 minutes), and resuspended in standard Tyrode's solution. After treating the cells with VASH inhibitor (VASHi) (1 mM) or vehicle (DMSO), they were incubated in an incubator for 4 hours. Contractile force of human muscle cells Contractility was evaluated as described above. After 4 hours of incubation with VASHi or vehicle, cardiomyocytes were transferred to a 35 mm glass-bottom dish. Contractility data were collected at 37°C. Cardiomyocytes were paced at 0.5 Hz using a Myopacifier (IonOptix MYP100) with a custom-made carbon electrode lowered into the glass-bottom dish. Cells were paced for 10–15 seconds until steady-state contraction was achieved, after which data were recorded using high-speed video imaging with a Nikon PU-2000 inverted microscope (IonWizard, IonOptix) equipped with a 40x objective lens. A minimum of 5 contractions were recorded for each muscle cell and analyzed offline. Results and Discussion Validated targets in heart failure treatment Proteomic profiling of myocardial tissue taken from a cohort of HCM patients who underwent septal resection for symptomatic LVOTO revealed that many of the abnormalities in HCM patients were similar regardless of the presence or absence of identified sarcomere protein mutations (Schuldt M et al. Circ Heart Fail. 2021; 14:39-55). However, a notable exception was the abnormality of cytoskeletal proteins, particularly the amount of detyrosinated α-tubulin, which was far greater in HCM patients and in patients with known sarcomere gene mutations than in patients without confirmed mutations. This particular finding is noteworthy because recent studies have shown that this specific posttranslational modification, detyrosination of α-tubulin, has significant effects on the stability and density of the cardiomyocyte cytoskeleton and on cellular biomechanics. The increased specific detyrosination and associated changes in the cardiomyocyte microtubule network are causally related to increased stiffness and viscoelasticity, which reduces contractility and delays both contraction and relaxation. Microtubules undergo extensive changes as part of adaptive and pathological cardiac remodeling, resulting in various derivative effects on the structure and function of cardiomyocytes. Distribution of tubulin detyrosine in myocardial tissue Therefore, the inventors attempted to represent the distribution of tubulin detyrosinization in the myocardial tissue of healthy animals (Figure 1). Using an affinity-purified antibody that specifically detects detyrosinized α-tubulin (Van der Laan et al., 2019), immunohistochemical staining of formaldehyde-embedded left ventricular cardiac tissue was optimized. Although microtubule densification has been demonstrated to cause hypertrophy and rigidity (Cheng G. et al., 2008), healthy ventricular cardiomyocytes exhibit a typical homogeneous staining pattern with detyrosinized microtubules in both longitudinal and transverse directions, as previously shown in isolated cardiomyocytes (Robison et al. 2016). Notably, an increase in staining level in the outer layer of cardiac tissue was also observed. Design and synthesis of specific VASH compounds (SVCs)To date, no small molecules have been designed to inhibit VASH, the enzyme responsible for the detyrosination of tubulin. Therefore, various compounds were designed based on the enzyme's native substrate. Extensive analysis of the human proteome was conducted to identify human proteins encoding the -EEY sequence at the C-terminus. As a result, only three proteins, including α-tubulin, met the criteria. Therefore, five compounds were developed that share a common tyrosine molecule in their structure, which is essential for specificity (Figure 2). The inventors designed prodrugs that are inactive to the therapeutic target in an in vitro detyrosination assay using recombinant human VASH1 protein (Figure 3) and its native substrate. A SAR database containing the new chemical entities was constructed using computer-aided drug discovery and repeated cycles of medical post-processing and subsequent ELISA (in vitro) and HCS (in cellulo) tests. The potency and selectivity of these compounds will be evaluated within the drug specifications for further development. The first prototype inhibitor (Epo-Y) exhibits dose-dependent inhibition of detyrosinase activity. However, a newly developed specific VASH compound called SVC_01 (SVC) exhibits approximately 100-fold superior inhibition compared to Epo-Y. The prodrug SVC_02 is inactive to the therapeutic target, and presentation of SVC_02 to the reactive mixture does not induce a decrease in activity. Next, the activity of compound SVC_01 was assayed in human cell lines to further demonstrate permeability and cellular efficacy. After exposing human cells to the SVC_01 compound for 2 hours, they were fixed, and labeling of detyrosinated tubulin and another post-translational modification called acetylation was analyzed by specific immunofluorescence (Figure 4). Staining levels were compared to human double knockout VASH cell lines that did not have detectable levels of detyrosinated tubulin. Our specific VASH compound SVC_01 completely suppressed the level of detyrosination of tubulin in human cells without affecting the acetylation level, further demonstrating a specific approach. Despite its specificity, SVC_01 was used at micromolar concentrations to obtain complete inhibition. To improve the intracellular IC50 of SVC, we designed a compound with higher cell permeability.As a result, SVC_02 and SVC_03 were obtained, both of which acted as prodrugs and were ineffective against recombinant VASH1 enzyme but highly effective in human cells (low nanomolar IC50 (Figure 5)). Since the substrate is quite unique in the human proteome (ending in -EEY), specificity analysis was performed by selecting different cysteine ​​proteases and testing their activity in the presence of SVC. The protease activity was selected based on different proteolytic mechanisms.

[0151] The selection criteria are detailed below: - A protease that has been shown to exhibit carboxypeptidase activity against α-tubulin. Carboxypeptidase A hydrolyzes peptide bonds at C-terminal residues with aromatic or aliphatic side chains (Figure 6). - A cysteine ​​protease that prefers the non-aromatic residue at the P1' position. The optimal recognition site for human rhinovirus (HRV) type 14 3C protease is LEVLFQ / GP, which has glycine at the P1' position (Figure 7). - A cysteine ​​protease that prefers aromatic residues at the P1' position. Similar to VASH, cathepsin B prefers aromatic residues at the P1' position. This is considered to be the best control for the inventors in terms of specificity (Figure 8). None of these proteases were inhibited in the presence of high micromolar SVCs, further supporting the specificity of these compounds for their therapeutic target, VASH1. SVC has a superior safety profile compared to microtubule-binding agents. Next, safety pharmacology experiments were conducted to directly compare the cytotoxicity of SVC in human cells. For comparison, a microtubule-binding agent containing parthenolide, previously referred to as a detyrosination inhibitor (Fonrose et al., 2007), was used. This compound was subsequently shown to disrupt microtubules by nonspecifically binding to tubulin in its molecular structure (Hotta T. et al., 2021). When using human cell lines, both parthenol and taxol were used, resulting in significant problems with cell viability in the micromolar concentration range (Figure 9). On the other hand, with SVC_01, no toxicity was observed even after exposure for several days, even at a high concentration of 100 micromoles. Mitochondrial health was assayed using the Seahorse XF analyzer. The Seahorse XF analyzer measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of living cells in multiwell plates, and evaluates major cellular functions such as mitochondrial respiration and glycolysis. The XF analyzer performs compound addition and mixing, label-free analytical detection, and automated OCR and ECAR measurements in real time. No mitochondrial defects were detected at a concentration of 20 micromolars of our VASH inhibitor (Figure 10). With the expectation of applying the active pharmaceutical ingredient (API) to the heart, hERG analysis was also performed using SVC. hERG (human ether-a-go-go related gene) encodes inward-rectifying voltage-gated potassium channels (IKr) involved in cardiac repolarization. No potential cardiotoxicity was observed in dose-response analysis in the presence of SVC_03 (Figure 12). Demonstration of concepts and efficacy in preclinical models For efficacy testing, primary cardiomyocytes isolated from Sprague Dawley rats were administered SVC_01. As previously observed in human cells (Figures 4 and 5), dose-dependent inhibition of tubulin detyrosination was reduced by nearly 80% after incubation with SVC_01 for 2 hours (Figure 12). This finding further supports the idea that VASH is the major detyrosinase in these cell types and that our newly designed specific VASH compound is highly effective in binding to the target. Next, functional studies were conducted by measuring key cardiomyocyte parameters, including resting left ventricular diameter shortening, systolic time, and relaxation time. We observed dose-dependent and statistically significant improvements in systolic and relaxation times in the presence of SVC_01 (Figure 13). The ZSF1 rat model is a hybrid rat obtained by mating a ZDF female rat with an SHHF male rat. This model is an established HFpEF rat model. Obese ZSF1 (O-ZSF1) hybrid rats are compound heterozygotes possessing two different mutant alleles encoding a defective leptin receptor, resulting in loss of receptor function. Consequently, these animals exhibit increased food intake and develop prominent features of obesity, diabetes, and consequently, HFpEF. ZSF1 rats inheriting two wild-type alleles or only one parental mutant allele have normal leptin signaling, balanced food intake and energy expenditure, normal body weight, and do not develop disease traits (L-ZSF1). The inventors performed Western blot analysis on cardiomyocytes prepared from Wistar Kyoto (WKY) rats, lean ZSF1 rats, and obese ZSF1 rats (Figure 14). Consistent with previously published data on the roles of microtubule densification and tubulin detyrosination, increased total tubulin and detyrosinated tubulin were observed in the cardiomyocytes of obese ZSF1 rats compared to lean and WKY controls. Cardiomyocytes isolated from ZSF1 rats incubated with SVC_01 showed a significant reduction in tubulin detyrosination (Figure 15).Furthermore, treatment with our VASH inhibitor improved the functional parameters of lesional cardiomyocytes isolated from 30-week-old obese ZSF1 rats. After 2 hours of treatment with this compound, both contractile and diastolic dynamics improved. The beneficial effect of this treatment was similar to that observed in adenovirus-induced cardiomyocytes overexpressing TTL, a reverse enzyme responsible for retyrosination and thus reducing detyrosination (Figure 16). This functional improvement was statistically significant, and importantly, cardiomyocytes isolated from obese rats showed significant functional impairment when treated with the vehicle. Similar results were observed in the assessment of lateral stiffness. Indeed, both VASHi treatment and TTL overexpression resulted in significant and potent functional improvements in the lateral and longitudinal stiffness of cardiomyocytes (Figures 17, 18). To investigate the efficacy of a treatment that reduces clinically relevant cardiac outcomes, Sprague dawley rats were injected with the compound (dose 6.6 mg / kg) via tail vein, and cardiac tissue was collected for Western blot analysis. Eight hours after intravenous injection, a significant reduction in tubulin detyrosine formation was observed in myocardial tissue (Figure 19). The same administration route was used in the ZSF1 rat model, where the therapeutic target was bound to cardiac tissue. As a result, eight hours after intravenous injection of SVC_02, tubulin detyrosine formation was reduced by approximately 60% in both lean and obese animals (Figure 20). The main results were a significant decrease in Tau, mitral valve deceleration time, and a significant downward trend in LVDEP after acute IV injection of VASHi (Figure 21).

[0152] In response to stress loading, ZSF1 obese rats exhibit decreased peak exercise tolerance and a rapid, velocity-dependent increase in filling pressure. SVC reduced microtubule detyrosine in the hearts of ZSF1 obese rats. This resulted in faster relaxation, as evidenced by significant improvements in mitral deceleration time (A) and left ventricular relaxation time constant (TAU) (B), a tendency toward decreased spontaneous LVEDP, and mitigation of induced LVEDP increases. Taken together, these results demonstrate that ZSF1 rats are a valid model for HFpEF, and more importantly, reducing microtubule detyrosine using SVC has therapeutic potential to alleviate relaxation impairment in HFpEF, making it an effective strategy for the prevention or treatment of heart failure.

[0153] Nanostring analysis was performed using a custom gene expression panel to investigate whether extracellular matrix (ECM) or microtubule network-related transcripts changed in accordance with increased myocardial sclerosis and microtubule network stabilization. Among extracellular matrix proteins, Vcan, Fn1, and Col1a1 were significantly increased in obese isolated cardiomyocytes compared to lean ZSF1 rats (Figure 22). Among microtubule network proteins and regulators, Tubb2a, Vash1, Hdac6, and Map1a were significantly increased in obese ZSF1 rats compared to lean ZSF1 rats.

[0154] Intravenous delivery of SVC-02 significantly reduced the amount of detyrosinated tubulin in the myocardium of ZSF1 rats (Figure 20). This reduction was also visualized by super-resolution immunofluorescence imaging of fixed LV tissue sections from obese animals treated with SVC-02 as both vehicle and VASHi (Figure 23). As expected, obese animals treated with VASHi (SVC-02) showed a significantly reduced density of detyrosinated microtubules compared to those treated with vehicle (Figure 24). Furthermore, the amount of detyrosinated tubulin in obese rats was three times higher than in WKY controls and lean controls (Figure 25), confirming that the detyrosination level was reduced to a lower level after treatment with SVC-02.

[0155] Cardiomyocytes were isolated from failing (N=4) and non-failing (N=3) human hearts and treated with a vehicle or VASHi (SVC-02) to evaluate the bioactivity of small molecule compounds in primary human cardiomyocytes. Patient attributes are detailed in the table (Figure 26). Waveform data from all non-failing and failing patients are summarized (Figure 27). SVC-02 shortened the time to 50% relaxation, similar to in vivo observations using isolated cardiomyocytes and obese rats (Figure 28). Notably, SVC-02 did not significantly alter contractility, contractile velocity, or resting sarcomere length in failing human cardiomyocytes.

[0156] To further confirm the safety profile of the compound in animal models, Swiss mice were administered SVC-02, and various organ structures, including the kidneys, heart, and lungs, were analyzed by immunohistological description. Treatment with the specific VASH inhibitor (SVC-02) did not alter organ structure (Figure 29) and did not result in significant tissue damage.

[0157] Further explanation of the specific VASH compound revealed that SVC-06 showed an extremely low IC50 (2.1 nM) using a high-content screening method in cells (Figure 30). Furthermore, oral administration of SVC-06 to Sprague-Dawley rats significantly reduced the level of tubulin detyrosination in myocardial tissue. Western blot signal quantification showed that a single administration of SVC-06 reduced the level of tubulin detyrosination in Sprague-Dawley rats by more than 40% after oral administration (Figure 31).

[0158] The specificity of SVC-01 was assayed using both human VASH1 and VASH2 recombinant enzymes. SVC-01 was found to be effective in inhibiting the detyrosination activity of both enzymes (Figure 32). Furthermore, when adult cardiomyocytes were treated with SVC-01, tubulin detyrosination was significantly reduced while other post-translational modifications such as acetylation remained unchanged (Figure 33). These results demonstrate the target specificity and the mechanism of action of the developed compound. Finally, to ensure that there was no impact on the regulation of other genes involved in the detyrosination-tyrosination cycle, the gene expression of all relevant genes was analyzed in adult human cardiomyocytes after SVC-02 treatment. No changes were observed in the expression of VASH1, VASH2, MATCAP, TTL, or SVBP genes between vehicle controls and SVC-02 treated cells (Figure 34). References [Table 1] JPEG2026509764000067.jpg87169

Claims

1. Compounds of formula (I) for use in the prevention and / or treatment of heart failure, cardiomyopathy, myocardial infarction-induced cardiac dysfunction in the target population, and / or for the improvement of cardiac function: 【Chemistry 1】 [In the formula, X is, 【Chemistry 2】 or -NH-CH ２ - and R １ teeth, 【Transformation 3】 or NR １ａ R １ｂ where R １ａ is H and R １ｂ is unsubstituted or C(O)-O-C １ -C ６ alkyl or C(O)-NH-C １ -C ６ alkyl-substituted C １ -C ６ alkyl, R stands for O-R ２ And here, R ２ C １ -C ６ Fatty acid chains (the said fatty acid chains are sometimes substituted) or C １ -C ６ It is an alkyl-aryl, R ３ is OH or O-C １ -C ６ It is a fatty acid chain, Y is - (CH ２ ) ｍ - (m=2) or 【Chemistry 4】 And, R ５ This is C(O)OH or O-C １ -C ６ It is alkyl, R ６ NH-CH(R ７ )-(CH ２ ) ｎ -R ８ (Here, R ７ H is R ８ (where n is an aryl compound, and n is 1, 2, or 3, preferably 1), or O-C １ -C ６ [It is a fatty acid chain.] or a pharmaceutically acceptable salt and / or solvate thereof.

2. A compound of formula (I) for use according to claim 1, formulated in a pharmaceutical composition with pharmaceutically acceptable excipients.

3. A compound of formula (I) for use according to claim 2, wherein the pharmaceutical composition is intended for oral or parenteral administration.

4. In the compound of formula (I), X 【Transformation 5】 And, R １ but 【Transformation 6】 or NR １ａ R １ｂ And here, R １ａ H is R １ｂ is either unsubstituted or C(O)-O-C １ -C ６ Alkyl or C(O)-NH-C １ -C ６ C substituted with alkyl １ -C ６ It is alkyl, R stands for O-R ２ And here, R ２ C １ -C ６ Fatty acids (the fatty acids are either unsubstituted or substituted with phenyl), or C １ -C ６ It is an alkyl-aryl, R ３ is OH or O-C １ -C ６ It is a fatty acid chain, Y is - (CH ２ ) ｍ - (m=2) or 【Transformation 7】 And, R ５ This is C(O)OH or O-C １ -C ６ It is alkyl, R ６ NH-CH(R ７ )-(CH ２ ) ｎ -R ８ (Here, R ７ H is R ８ (where n is an aryl compound, and n is 1, 2, or 3, preferably 1), or O-C １ -C ６ It is a fatty acid chain. A compound of formula (I) for use according to any one of claims 1 to 3.

5. In the compound of formula (I), X-NH-CH ２ - R １ NR １ａ R １ｂ And here, R １ａ H is R １ｂ is C(O)-NH-C １ -C ６ C substituted with alkyl １ - It is C alkyl, R stands for O-R ２ And here, R ２ is C １ -C ６ It is an alkyl-aryl, R ３ O-C １ -C ６ It is a fatty acid chain. A compound of formula (I) for use according to any one of claims 1 to 3.

6. In the compound of formula (I), R １ teeth, 【Transformation 8】 And, R is O-R ２ where R ２ is C １ -C ６ fatty chain, and the fatty chain is unsubstituted or substituted with phenyl R ３ is OH or O-C １ -C ６ It is a fatty acid chain, Y is - (CH ２ ) ｍ - (m=2) or 【Chemistry 9】 And, R ５ is COOH or O—C １ —C ６ alkyl, and R ６ NH-CH(R ７ )-(CH ２ ) ｎ -R ８ (Here, R ７ H is R ８ (where is an aryl and n is 1, 2 or 3, preferably 1) or O-C １ -C ６ It is a fatty acid chain. A compound of formula (I) for use in the applications described in claim 4.

7. The compound of formula (I) is the following compound: 【Chemistry 10】 【change】 Compounds of formula (I), pharmaceutically acceptable salts and / or solvates thereof, and conjugates thereof, for use in the applications described in any one of claims 1 to 6, selected from among them.

8. A compound of formula (I) for use according to any one of claims 1 to 7, which improves the dynamics of a dysfunctional cardiomyocyte, including increasing the relaxation rate, increasing the contractile force, and / or decreasing the viscoelasticity.

9. A compound of formula (I) for use according to any one of claims 1 to 8, wherein the subject requiring the compound has impaired ejection fraction heart failure (HFrEF).

10. A compound of formula (I) for use according to any one of claims 1 to 8, wherein the subject requiring the compound has heart failure with maintained ejection fraction (HFpEF).

11. A compound of formula (I) for use according to any one of claims 1 to 10, wherein the subject requiring the treatment is in combination with a currently standard therapeutic agent for heart failure, cardiomyopathy, or myocardial infarction-induced cardiac dysfunction.

12. a) A compound of formula (I) according to any one of claims 1, 4 to 7, or a pharmaceutical composition according to claim 2 or 3, and b) Current standard treatments for heart failure, cardiomyopathy, or myocardial infarction-induced cardiac dysfunction A combination that includes [the specified element].

13. In particular: - In vitro methods for studying the role of tubulin detyrosine in cardiac disorders, e.g., its onset, invasiveness, and progression, and - Related kits for performing the above screening assay and method Use of a conjugate comprising a compound (I) according to any one of claims 1 to 7, or optionally a labeled biomolecule, linked to or unlinked to a fragment of formula (I), as a research tool for research and development activities of cardiac dysfunction or heart failure selected from the group consisting of the above.

14. The use of a conjugate according to claim 13, comprising a fragment of a compound of formula (I) according to any one of claims 1 to 7, linked to a biomolecule, selected from peptides; proteins; biomarkers; photolabeling agents such as rhodamine, coumarin derivatives, cyanine derivatives, or fluorescein; affinity probes such as biotin; or E3 ubiquitin ligase recruiters such as thalidomide, VH032, VH101, dBET1, dFKBP12, QCA570, ZNL-02-096, or d9A-2.

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