RU2787766C2 - Dipeptidyl ketoamide meta-methoxyphenyl derivatives and their use - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a dipeptidyl ketoamide m-methoxyphenyl derivative of the formula II, where R_1a is selected from a group consisting of C₁-C₆ alkyl and C₃-C₆ cycloalkyl, R_2a is selected from a group consisting of H and C₁-C₆ alkyl, R_3a is C₃-C₆ cycloalkyl or methoxy, provided that, when R_3a is methoxy, R_1a is cyclopropyl, and R_2a is H, or its pharmaceutically acceptable salt or stereoisomer, which can be used for the treatment of diseases and conditions associated with increased calpain activity, such as heart damage caused by infarction, ischemia with or without reperfusion, neurodegenerative disorders, malaria, diabetic nephropathy, HIV virus-induced neurotoxicity, malignant tumor, and fibrotic diseases.

EFFECT: obtainment of a dipeptidyl ketoamide m-methoxyphenyl derivative for use in medicine.

16 cl, 6 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The invention relates to dipeptidylketoamide m-methoxyphenyl derivatives and their use in the treatment of diseases and conditions associated with increased activity of calpain, such as heart damage caused by infarction, ischemia with or without reperfusion, neurodegenerative disorders, malaria, diabetic nephropathy, virus-induced neurotoxicity HIV, cancer and fibrotic diseases.

BACKGROUND OF THE INVENTION

Calpains are intracellular proteins belonging to the family of calcium-dependent non-lysosomal cysteine proteases (proteolytic enzymes) widely expressed in mammals and many other organisms. Two main forms, calpain 1 and calpain 2, also known as µ-calpain and m-calpain, have been described, but other isoenzymes of calpain have also been suggested (M.E. Saez et al.; Drug Discovery Today 2006, 11 (19/20), pp. 917-923).

Calpains play an important role in various physiological processes that involve the breakdown of various regulatory proteins (K.K. Wang et al., Trends in Pharmacol. Sci. 1994, 15, pp. 412-419).

Elevated levels of calpain have been measured in various pathophysiological processes, for example: ischemia of the heart, kidneys, lungs, liver or central nervous system, inflammation, muscular dystrophies, eye cataracts, diabetes, HIV-mediated disorders, damage to the central nervous system (for example, brain injury ), Alzheimer's disease, Huntington's disease, Parkinson's disease, multiple sclerosis, etc. (see K.K. Wang, supra) and infectious diseases such as malaria (IM Medana et al., Neuropath and Appl. Neurobiol. 2007, 33, pp. 179-192). An association is believed to exist between these diseases and frequently or persistently elevated intracellular calcium levels. This leads to hyperactivation of calcium-dependent processes and the fact that they cease to be amenable to normal physiological control. Corresponding hyperactivation of calpains can also trigger pathophysiological processes. For this reason, it has been argued that calpain inhibitors can be used to treat these diseases.

Yoshida, Ken Ischi et al. (Jap. Circ. J. 1995, 59 (1), pp. 40-48) described that calpain inhibitors had beneficial effects after cardiac injury caused by ischemia or reperfusion. It has recently been described that calpains are activated during myocardial ischemia-reperfusion and are involved in reperfusion injury, as well as the involvement of calpains in post-infarction remodeling and heart failure. After an acute myocardial infarction, the heart as a whole undergoes a series of structural changes called post-infarction myocardial remodeling, leading to heart failure. Ventricular remodeling includes expansion, hypertrophy, and the formation of a detached collagen scar. Dysregulation of calpain activity plays an important role in reperfusion injury and myocardial remodeling, indicating that calpain inhibition is a potential therapeutic strategy (Ye et al., PLoS ONE, 2015, 10(3), e0120178; Kudo-Sakamoto et al. , Journal Biological Chemistry, 2014, 289(28), pp. 19408-19419). Calpain inhibitors are described by Neuhof et al. (World J Cardiol, 2014, 6(7), 638-652) as a new preventive and therapeutic option for patients with myocardial infarction, revascularization and coronary surgery. The study described by Poncelas et al. (Cardiovascular Research, 2017, 113(8), pp. 950-961) confirms this role for calpains and demonstrates that a pharmacological long-acting calpain inhibitor is a promising therapeutic strategy against adverse post-infarction remodeling.

Calpain inhibitors have also been shown to provide a neuroprotective effect in acute neurodegenerative disorders or ischemias such as those that occur after cerebral stroke (Seung-Chyul Hong et al., Stroke 1994, 25(3), pp. 663-669, and R. T. Bartus et al., Neurological Res. 1995, 17, pp. 249-258).

Calpain inhibitors have also been shown to improve recovery from memory and neuromotor impairment after experimental brain injury (K. E. Saa™an et al., Proc. Natl. Acad. Sci. USA, 1996, 93, pp. 3428-3433 ), and that calpain inhibitors have a protective effect on hypoxia-damaged kidneys (C. L. Edelstein et al., Proc. Natl. Acad. Sci. USA, 1995, 92, pp. 7662-6).

More recent studies have shown that calpastatin (a natural inhibitor of calpain) significantly attenuates the pathophysiological effects of activated calpain in a number of diseases such as a) experimental glomerulonephritis (J. Peltier et al., J A, Soc Nephrol. 2006, 17, pp. 3415-3423 ), b) cardiovascular remodeling in angiotensin II-induced hypertension, c) impaired synaptic transmission in congenital myasthenic slow channel syndrome (Groshong JS et al., J Clin Invest. 2007, 117 (10), pp 2903-2912), d ) excitotoxic DNA fragmentation via mitochondrial cascades (J Takano et al., J Biol Chem. 2005, 280 (16) pp. 16175-16184), and e) necrotic processes in dystrophic muscles (M J Spencer et al., Hum Mol Gen, 2002, 11(21), pp 2645-2655).

Calpains are also known to be associated with Alzheimer's disease (AD) (Nixon R.A., "The calpains in aging and aging-related diseases", Ageing Res Rev. 2003 Oct; 2(4):407-18). Calpain 1 is abnormally upregulated in the brain in AD (Saito K, et al. "Widespread activation of calcium-activated neutral proteinase (calpain) in the brain in Alzheimer's disease: a potential molecular basis for neuronal degeneration", Proc Natl Acad Sci USA. 1993 Apr 1;90(7):2628-32). The level of calpastatin, an endogenous inhibitor of calpains, is significantly reduced in this same neurodegenerative disorder (Nixon R.A., "The calpains in aging and aging-related diseases", Ageing Res Rev. 2003 Oct; 2(4):407-18). Overactivation of calpain through abnormally high calcium levels and clepastatin depletion results in limited cleavage or degradation of key neuronal proteins in AD (Wang KK, "Calpain and caspase: can you tell the difference?", Trends Neurosci. 2000 Jan; 23(1):20 -6). Calpains indirectly modulate proteolytic processing of the amyloid precursor protein (APP), a polypeptide thought to play a fundamental role in AD (Siman R. at al. "Proteolytic processing of beta-amyloid precursor by calpain I", J Neurosci. 1990 Jul; 10(7 ):2400-11).

Other calpain substrates altered in AD include CaM kinase IIα (CaMK-IIα) and PKC, 2 enzymes that regulate APP phosphorylation and influence its metabolism (Wang KK et al., "Development and therapeutic potential of calpain inhibitors" Adv Pharmacol. 1997; 37 (): 117-52); second messenger-related enzymes such as phospholipase C-1, -2, -β3 (Banno Y. et al, "Endogenous cleavage of phospholipase C-beta 3 by agonist-induced activation of calpain in plate humanlets", J Biol Chem. 1995 Mar 3; 270(9):4318-24), and cyclin-dependent kinase 5 (Cdk-5) (Lee MS. et al., "Neurotoxicity induces cleavage of p35 to p25 by calpain", Nature. 2000 May 18 ;405(6784):360-4); transcription factors such as c-Jun, c-Fos, and IκB (Carillo S, "Differential sensitivity of FOS and JUN family members to calpains", Oncogene. 1994 Jun; 9(6):1679-89 and Lin YC, " Activation of NF-kappa B requires proteolysis of the inhibitor I kappa B-alpha: signal-induced phosphorylation of I kappa B-alpha alone does not release active NF-kappa B", Proc Natl Acad Sci U S A. 1995 Jan 17; 92 (2):552-6); and memory-associated cAMP regulatory element-binding protein (CREB) gene (Mbebi C, "Amyloid precursor protein family-induced neuronal death is mediated by impairment of the neuroprotective calcium/calmodulin protein kinase IV-dependent signaling pathway", J Biol Chem. 2002 Jun 7;277(23):20979-90). It has recently been shown that the action of calpain on the GluR1 subunit of AMPA receptors (24), amphiphysin I (25), and the suprachiasmatic nucleus circadian rhythm protein (26) modulates synaptic activity and memory formation.

Accumulating evidence indicates that cognitive impairment in AD begins long before neuronal death, and that signaling between neurons is interrupted early in the disease. The importance of synaptic changes in AD has been confirmed by studies with transgenic mouse models of AD (Sant'Angelo A, "Usefulness of behavioral and electrophysiological studies in transgenic models of Alzheimer's disease", Neurochem Res. 2003 Jul; 28(7):1009-15) and for amyloid β peptide-induced (Aβ-induced) long-term potentiation (LTP) impairment, a widely studied cellular model of learning and memory (Bliss TV. et al., "A synaptic model of memory: long-term potentiation in the hippocampus"; Collingridge GL Nature 1993 Jan 7; 361(6407):31-9).

Moreover, calpains affect the phosphorylation and proteolysis of tau, another protein associated with AD (Wang KK, "Calpain and caspase: can you tell the difference?", Trends Neurosci. 2000 Jan; 23(1):20-6). In addition, the accumulation of phosphorylated tau leads to the formation of so-called neurofibrillary tangles (NFTs), which, together with the well-known amyloid plaques, are a pathological feature of Alzheimer's disease. Similar changes in the tau protein, usually considered an important feature of taupathies, are also seen in other (neuro)degenerative disorders, such as post-stroke disorders, brain inflammation, parkinsonism, normal pressure hydrocephalus, and Creutzfeldt-Jakob disease.

The involvement of calpain in neurodegenerative processes has been demonstrated in transgenic mice using calpastatin, a specific and natural calpain inhibitor (Higuchi et al.; J. Biol. Chem. 2005, 280 (15), pp. 15229-15237). It has been possible with a calpain inhibitor to significantly reduce the clinical signs of autoimmune encephalomyelitis in a mouse model of multiple sclerosis (F. Mokhtarian et al.; J. Neuroimmunology 2006, Vol. 180, pp. 135-146). In addition, it has been shown that calpain inhibitors on the one hand block A@-induced neuronal degeneration (Park et al.; J. Neurosci. 2005, 25, pp. 5365-5375), and, in addition, reduce the release of the precursor protein β -amyloid (β-APP) (J. Higaki et al., Neuron, 1995, I4, pp. 651-659).

In this regard, calpain inhibitors represent a new therapeutic approach for the treatment of neurodegenerative disorders in general and, in particular, Alzheimer's disease, Parkinson's disease, multiple sclerosis, acute autoimmune encephalitis and Creutzfeldt-Jakob disease.

HIV-induced neurotoxicity has been demonstrated to be mediated by calpain (O'Donnell et al.; J. Neurosci. 2006, 26(3), pp. 981-990) and calpain has been shown to be involved in HIV replication (Teranishi et al.; Biochem Biophys Res Comm 2003, 303 (3), pp. 940-946).

Calpain has also recently been shown to be involved in the development of kidney diseases such as chronic kidney diseases such as diabetic nephropathy. Thus, Y. Shi et al. demonstrated in animal models that the natural calpain inhibitor calpastatin is suppressed during renal ischemia-reperfusion (Am. J. Physiol. Renal Physiol. 2000, 279, pp. 509-517). Moreover, A Dnyanmote et al., Toxicology and Applied Pharmacology 2006, 215, pp. 146-157 showed that inhibition of calpain via calpastatin overexpression reduces the progression of DCVC-induced kidney injury in an acute renal failure model. In addition, Peltier et al. demonstrated that calpain activation and secretion contribute to glomerular damage in experimental glomerulonephritis (J. Am. Soc. Nephrol. 2006, 17, pp. 3415-3423). Calpain inhibitors have also been shown to reduce renal dysfunction and injury induced by renal ischemia-reperfusion, and thus may be useful in increasing renal resistance against renal injury associated with aortic vascular surgery or renal transplantation (P. Chatterjee et al., Biochem Pharmacal 2005, 7, pp. 1121-1131). On this basis, inhibition of calpain can be considered a therapeutic approach suitable for the treatment of kidney diseases, such as chronic kidney disease, for example, diabetic nephropathy.

Calpain has also been identified as a central mediator required for parasitic activity. Parasites such as Plasmodium falciparum and Toxoplasma gondii use host cell calpains to promote exit from the intracellular parasitophoric vacuole and/or host cell membrane. Inhibition of calpain-1 in hypotonic lysed and resealed erythrocytes prevented the release of P. falciparum parasites, which was restored by the addition of purified calpain-1. Likewise, the efficient exit of T. gondii from mammalian fibroblasts was blocked either through small interfering RNA-mediated suppression or through genetic elimination of calpain activity, and could be restored through genetic complementation (D. Greenbaum et al., Science 324, 794 (2009)). Because parasites that do not exit host cells are unable to proliferate, this suggests a strategy for antiparasitic therapeutics. Pharmacological inhibition of calpain has been shown to exhibit antimalarial activity and thus provides a novel antiparasitic strategy for the treatment of diseases caused by protozoan infections such as malaria or toxoplasmosis (Li et al., Mol Biochem Parasitol. 2007; 155(1) : 26-32, Jung et al., Archives of Pharmacal Research (2009), 32(6), 899-906, Chandramohanadas et al., Science (2009), 324, 794).

It has also been described (Leloup and Wells, Expert Opin Ther Targets., 2011, 15(3), 309-323; Storr et al., Nat Rev Cancer., 2011, 11(5), 364-374; Storr et al. , Pathobiology., 2015, 82(3-4), 133-141; Selvakumar and Sharma, Experimental Therapeutic Medicine, 2010, 1, 413-417; Storr et al., Oncotarget, 2016, 30(7), 47927-47937 ; and Guan et al., Proc Amer Assoc Cancer Res., 2005, 46) that calpains, in particular calpain 1 and calpain 2, are involved in a wide variety of commonly occurring malignancies such as breast, colon, and rectal cancers. intestines and leukemia. Calpains 1 and 2 are involved in the development and progression of malignant tumors by allowing transformation, migration and escape of tumor cells, and tumor neovascularization. These reports also mention that numerous tumor cells exhibit abnormally high activity of these calpains. Thus, inhibition of calpain activity can be an effective way to block tumor development by blocking cell transformation and proliferation, as well as tumor vascularization.

Fibrosis refers to the accumulation of extracellular matrix molecules that form scar tissue (collagen-rich extracellular matrix) and is a common feature of fibrotic diseases. Fibrotic diseases can affect any organ such as the kidney, liver, lungs, skin, heart, and eye, causing organ dysfunction and failure and potentially death. In epithelial organs, especially in the lung, liver, skin, and kidney, the replacement of normal cell functional elements by collagen-rich scar tissue and disruption of the architectonics caused by scar contraction are the main factors in the progressive decline in organ function and, as a result, its insufficiency [Friedman et al. Science Translational Medicine, 2013, 5, 167]. Calpain activity has been described as essential for wound healing and scar formation [Nassar et al., PLoS ONE, 2012, 7(5), e37084]. It has also been described that calpains are involved in fibrotic diseases [WO 2017/156074 A1; Buckman et al., Am J Respir Crit Care Med, 2018, 197, A5747; Potz et al., J Nat Sci 2016, 2(9), e218; Ono et al., Nature Reviews Drug Discovery, 2016, 15, 854-876], such as liver fibrosis (alcoholic, viral, autoimmune, metabolic, and hereditary chronic disease), kidney fibrosis (eg, from chronic inflammation, infections, or diabetes type II), pulmonary fibrosis (idiopathic or due to exposure to environmental factors including toxic particles, sarcoidosis, asbestosis, hypersensitivity pneumonitis, bacterial infections including tuberculosis, among others), interstitial fibrosis, systemic scleroderma (an autoimmune disease in which many organs develop fibrosis), macular degeneration (fibrotic disease of the eye), pancreatic fibrosis (due to, for example, alcohol abuse and chronic inflammatory disease of the pancreas), spleen fibrosis (due to sickle cell anemia and other blood disorders), fibrosis of the heart (due to infection, inflammation and hypertrophy), mediastinal Fibrosis, Myelofibrosis, Endomyocardial Fibrosis, Retroperitoneal Fibrosis, Progressive Massive Fibrosis, Nephrogenic Systemic Fibrosis, Fibrotic Complications of Surgery, Especially Surgical Implants, Injection Fibrosis, and Secondary Conditions and Disease Conditions with Fibrosis such as Cirrhosis, Diffuse Parenchymal Lung Disease, Pain Syndrome after vasectomy and arthrofibrosis, among others. A specific calpain inhibitor, BLD-2660, is currently in phase 1 clinical trials against fibrosis. Thus, inhibition of calpain activity may be an effective strategy for managing the course of fibrotic diseases.

WO 2004/078908 A2 and WO 2005/056519 A1 describe calpain inhibitors with the potential to inhibit calpain 1 for the treatment of various diseases, including Alzheimer's disease. In particular, the compound of Example 17 of WO 2005/056519 A1, which has been given the code name SNJ-1945, has been shown to be effective in reducing clinical scores of experimental autoimmune encephalomyelitis (EAE) in vivo (Trager N. et al., "Effects of a novel orally administered calpain inhibitor SNJ-1945 on immunomodulation and neurodegeneration in a murine model of multiples sclerosis", J. Neurochem. 2014 July; 130(2): 268-279).

There remains a need for new strategies to manage diseases or conditions associated with increased calpain activity, such as cardiac injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; malignant tumor; and fibrotic diseases.

SUMMARY OF THE INVENTION

The present inventors have surprisingly discovered that compounds of formula (I) which have a methoxy group at the meta position of the phenyl ring are potent inhibitors of calpain-1. The position of this group is of paramount importance for achieving high calpain-1 inhibition efficacy as shown in the Comparative Examples. Thus, these compounds of formula (I) are useful in the treatment and/or prevention of diseases or conditions associated with increased calpain activity, such as cardiac injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; and malignant tumor.

In a first aspect, the present invention relates to a compound of formula (I):

(I)

where

R ₁ is selected from the group consisting of C ₁ -C ₆ alkyl and C ₃ -C ₆ cycloalkyl,

R ₂ is selected from the group consisting of H and C ₁ -C ₆ alkyl,

R ₃ is selected from the group consisting of C ₁ -C ₆ alkoxy and C ₃ -C ₆ cycloalkyl,

or a pharmaceutically acceptable salt or stereoisomer thereof, for use in the treatment and/or prevention of a disease or condition selected from the group consisting of cardiac injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; malignant tumor; and fibrotic diseases.

In a second aspect, the present invention relates to a pharmaceutical composition comprising a compound of formula (I) as defined in the first aspect, and a pharmaceutically acceptable excipient, for use in the treatment and/or prevention of a disease or condition selected from the group consisting of cardiac injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; malignant tumor; and fibrotic diseases.

In another aspect, the present invention relates to the use of a compound of formula (I) as defined in the first aspect, or a pharmaceutical composition containing said compound and a pharmaceutically acceptable excipient, for the manufacture of a medicament for the treatment and/or prevention of a disease or condition selected from the group, consisting of heart damage caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; malignant tumor; and fibrotic diseases.

In another aspect, the present invention relates to a method of treating and/or preventing a disease or condition selected from the group consisting of cardiac injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; cancer and fibrotic diseases, the method comprising administering to an individual in need thereof a therapeutically effective amount of a compound of formula (I) as defined in the first aspect, or a pharmaceutical composition comprising said compound and a pharmaceutically acceptable excipient.

In the following aspect, the present invention relates to a compound of formula (II):

(I)

where

R _1a is selected from the group consisting of C ₁ -C ₆ alkyl and C ₃ -C ₆ cycloalkyl,

R _2a is selected from the group consisting of H and C ₁ -C ₆ alkyl,

R _3a is C ₃ -C ₆ cycloalkyl,

or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

In another aspect, the present invention relates to a pharmaceutical composition containing a compound of formula (II) as defined in the third aspect, and a pharmaceutically acceptable excipient.

In another aspect, the present invention relates to a compound of formula (II) as defined in the third aspect, or to a pharmaceutical composition, as defined in the fourth aspect, for use in medicine, in particular for the treatment and/or prevention of a disease or condition associated with increased activity of calpain; preferably selected from the group consisting of heart injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; malignant tumor; and fibrotic diseases.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In order to facilitate understanding of the invention, some of the definitions are given.

The term "alkyl", as used herein alone or as part of another group, means a linear or branched saturated monovalent hydrocarbon chain containing the number of carbon atoms indicated in each case, which is usually from one to six carbon atoms, and preferably from one to three. Examples of alkyls are methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, sec-pentyl, t-pentyl, neopentyl, and the like.

The term "alkoxy", as used herein alone or as part of another group, means an alkyl group as defined above bonded through oxygen, i.e. alkyl-O-. Examples of alkoxy include methoxy, ethoxy, isopropoxy, t-butoxy, and the like.

The term "cycloalkyl", as used herein alone or as part of another group, means a monocyclic radical which is saturated or partially saturated, preferably saturated, and which consists only of carbon and hydrogen atoms, and contains the number of carbon atoms specified in each case, which is usually 3 to 6 and preferably 3 to 5. Examples of cycloalkyls are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

The term "salt" is to be understood as any form of a compound according to the present invention wherein said compound is in ionic form, or is charged or conjugated with a counterion (cation or anion), or is in solution. This definition also includes quaternary ammonium salts. In particular, the definition includes pharmaceutically acceptable salts.

The term "pharmaceutically acceptable salt" embraces salts with a pharmaceutically acceptable acid or base that are synthesized from a parent compound that contains an acidic moiety by adding a pharmaceutically acceptable base, or that are synthesized from a parent compound that contains a basic moiety by adding a pharmaceutically acceptable acid. Pharmaceutically acceptable acids include both inorganic acids, such as hydrochloric, sulfuric, phosphoric, diphosphoric, hydrobromic, hydroiodic, and nitrous acids, as well as organic acids, such as citric, fumaric, maleic, malic, mandelic, ascorbic, oxalic, succinic, tartaric, benzoic, acetic, methanesulfonic (mesylate), ethanesulfonic, benzenesulfonic (besylate), or p-toluenesulfonic (tosylate) acid. Pharmaceutically acceptable bases include alkali metal (eg sodium or potassium) and alkaline earth metal (eg calcium or magnesium) hydroxides, and organic bases such as alkylamines, arylalkylamines and heterocyclic amines. For example, pharmaceutically acceptable salts of the compounds described herein are synthesized from a parent compound that contains a basic or acid moiety by conventional chemical methods. Typically, such salts are obtained, for example, by reacting the free base or free acid forms of these compounds with a stoichiometric amount of the corresponding acid or base, respectively, in water or in an organic solvent, or in a mixture of both.

All stereoisomers of the compounds of the present invention are contemplated, either singly or as mixtures thereof. Racemates, enantiomers or diastereomers can be used as starting materials in the preparation process. When diastereomeric or enantiomeric products are obtained, they can be separated by conventional means, for example, chromatography or functional crystallization.

Unless otherwise indicated, compounds of the invention also include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures, with the exception of replacing a hydrogen atom with deuterium or tritium, or replacing a carbon with ¹³ C- or ¹⁴ C-rich carbon or ¹⁵ N-rich nitrogen, are within the scope of the present invention.

Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. Generally, solvated forms are equivalent to unsolvated forms, and are within the scope of the present invention.

Certain compounds of the present invention may exist in a variety of crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

The term "prevention" as used herein refers to the administration of a compound of the invention at the initial or early stage of a disease or condition, or also to prevent its onset.

The term "treatment" is used to refer to the administration of a compound of the invention to control the progression of a disorder before or after the onset of clinical signs. By controlling the progression of a disorder, it is meant to determine favorable or desired clinical outcomes, including, but not limited to, symptom reduction, reduction in the duration of the disorder, stabilization of the pathological state (in particular, prevention of further deterioration), slowing down the progression of the disorder, improvement of the pathological state, and remission (as a partial , and overall).

The term "individual", as used in the framework of the invention, refers to any animal or person who suffers from one of the diseases or conditions described in the present description. Preferably, the individual is a mammal. The term "mammal" as used herein refers to any species of mammal, including but not limited to domestic and farm animals (cows, horses, pigs, sheep, goats, dogs, cats or rodents), primates and humans. Preferably, the mammal is a human.

The term "heart injury" as used herein refers to any injury to the heart muscle caused by infarction, ischemia with or without reperfusion, such as remodeling.

The term "remodeling" as used herein refers to a group of molecular, cellular and interstitial changes that are clinically manifested by changes in the size, shape and function of the heart as a result of damage to the heart, such as changes in cavity diameter, mass (hypertrophy and atrophy), geometry (thickness and wall of the walls of the heart), scar area, fibrosis and inflammatory infiltrate. Cardiac dysfunction is the main consequence of cardiac remodeling, which forms the pathophysiological basis for the onset and progression of ventricular dysfunction. This interaction begins with genetic changes in response to heart injury with reexpression of fetal genes. Consequently, cellular and molecular changes occur that lead to a gradual deterioration in ventricular function, initially asymptomatic, followed by signs and symptoms of heart failure. Cardiac remodeling is associated with malignant ventricular arrhythmias, including sustained ventricular tachycardia and ventricular fibrillation. During the first hours after occlusion of a coronary artery, disintegration of interfibrillar collagen may occur simultaneously with necrosis of myofibrils. The loss of supporting tissue makes this area more susceptible to stretching and deformity. Due to the loss of necrotic muscle cells and the restructuring of myocytes in the infarcted wall, thinning of the infarcted area and expansion of the cavity occur. This acute expansion of the ventricles, characterized by thinning and elongation of the infarcted area, is called infarct expansion. Infarct expansion increases the likelihood of myocardial rupture and is the anatomical basis for aneurysms.

The term "neurodegenerative disorders" as used herein refers to disorders that lead to progressive degeneration and/or death of neurons. Examples of neurodegenerative disorders are Alzheimer's disease, Parkinson's disease, multiple sclerosis, acute autoimmune encephalitis and Creutzfeldt-Jakob disease.

The term "malignant tumor" or "tumor", as used in the framework of the invention, refers to a broad group of diseases involving unregulated cell growth, which are also called malignant neoplasms. This term is usually applied to a disease characterized by uncontrolled cell division (or increased survival or resistance to apoptosis) and the ability of these cells to invade other adjacent tissues (invasion) and spread to other areas of the body where these cells are not normally present (metastasis), through the lymphatic and blood vessels, circulate in the bloodstream, and then invade normal tissues elsewhere in the body. Depending on whether they can spread through invasion and metastasis, tumors are classified as benign or malignant: benign tumors are tumors that cannot spread through invasion or metastasis, i.e. they grow only locally; while malignant tumors are tumors that are able to spread through invasion and metastasis. Known biological processes associated with cancer include angiogenesis, immune cell infiltration, cell migration and metastasis. Typically, malignant tumors share some of the following common characteristics: maintenance of proliferative signaling, escape from growth suppressors, resistance to cell death, the possibility of replicative immortality, induction of angiogenesis and activation of invasion and eventual metastasis. Malignant tumors invade nearby parts of the body and can also spread to more distant parts of the body through the lymphatic system or bloodstream. Malignant tumors are classified according to the type of cells that are similar to the tumor cells, which are thus considered to be the source of the tumor.

Cancers that can be treated or prevented by the medical applications of the present invention are solid tumors, for example, colon and rectal cancer, breast cancer, lung cancer, pancreatic cancer, laryngeal cancer, tongue cancer, ovarian cancer, prostate cancer. glands, liver cancer, head and neck cancer, esophageal cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, dysgerminoma, embryonic carcinoma, Wilms tumor, cervical cancer, testicular tumor, ovarian carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemanioblastoma, acoustic neuroma, oligodenroglioma, meningioma, neuroblastoma, retinoblastoma and leukemia; preferably breast cancer, colorectal cancer and leukemia.

The term "leukemia", as used herein, refers to a type of malignant tumor of the blood or bone marrow, characterized by an abnormal increase in the level of immature white blood cells, called blasts, and occurring in blood-forming tissue. Leukemia begins in the bone marrow, where developing blood cells, usually developing white blood cells, undergo malignant (cancerous) change. This means that they increase in number in an uncontrolled way, filling up the bone marrow and preventing the normal production of blood cells. The abnormal cells, called blast cells or leukoblasts, have grown in number and eventually leave the bone marrow and travel through the body through the bloodstream. In some cases, these abnormal cells accumulate in various organs, including the lymph nodes, spleen, liver, and central nervous system (brain and spinal cord). There are four main types of leukemia: acute lymphoblastic leukemia, or ALL; acute myeloid leukemia, or AML; chronic lymphocytic leukemia, or CLL; chronic myelogenous leukemia, or CML.

The term "fibrotic disease" as used herein refers to a group of diseases that involve the formation of excessive fibrous connective tissue in an organ or tissue. Fibrosis leads to scarring and thickening of the affected tissue. Fibrosis can occur in many tissues of the body, causing various fibrotic diseases such as cardiac fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, retroperitoneal fibrosis, hypersensitivity pneumonitis, interstitial fibrosis, systemic scleroderma, macular degeneration, pancreatic fibrosis, spleen fibrosis, mediastinal fibrosis, myelofibrosis, endomyocardial fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, fibrotic complications of surgery, chronic allograft vasculopathy and/or chronic organ transplant rejection, ischemia-reperfusion-associated fibrosis, injection fibrosis, cirrhosis, diffuse parenchymal lung disease, pain syndrome after vasectomy and arthrofibrosis. Preferably, the fibrotic disease refers to cardiac fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, or retroperitoneal fibrosis.

Compounds of formula (I)

In a first aspect, the present invention relates to a compound of formula (I)

(I)

where

R ₁ is selected from the group consisting of C ₁ -C ₆ alkyl and C ₃ -C ₆ cycloalkyl,

R ₂ is selected from the group consisting of H and C ₁ -C ₆ alkyl,

R ₃ is selected from the group consisting of C ₁ -C ₆ alkoxy and C ₃ -C ₆ cycloalkyl,

or a pharmaceutically acceptable salt or stereoisomer thereof, for use in the treatment and/or prevention of a disease or condition selected from the group consisting of cardiac injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; malignant tumor; and fibrotic diseases.

In a specific embodiment, R ₁ is selected from the group consisting of C ₁ -C ₃ alkyl and C ₃ -C ₅ cycloalkyl; preferably from the group consisting of methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclobutyl and cyclopentyl; more preferably from the group consisting of methyl, isopropyl and cyclopropyl; even more preferably from the group consisting of isopropyl and cyclopropyl; most preferred is cyclopropyl.

In another specific embodiment, R ₂ is selected from the group consisting of H and C ₁ -C ₃ alkyl; preferably from the group consisting of H, methyl, ethyl, propyl and isopropyl; more preferably from the group consisting of H and methyl; most preferred is H.

In another specific embodiment, R ₃ is selected from the group consisting of C ₁ -C ₃ alkoxy and C ₃ -C ₅ cycloalkyl; preferably from the group consisting of methoxy, ethoxy, propoxy, isopropoxy, cyclopropyl, cyclobutyl and cyclopentyl; even more preferably from the group consisting of methoxy and cyclopropyl; most preferred is cyclopropyl. In another specific embodiment, R ₃ is C ₃ -C ₆ cycloalkyl; preferably C ₃ -C ₅ cycloalkyl; preferably cyclopropyl, cyclobutyl or cyclopentyl; most preferred is cyclopropyl.

In a specific embodiment, R ₁ is selected from the group consisting of C ₁ -C ₃ alkyl and C ₃ -C ₅ cycloalkyl; R ₂ is selected from the group consisting of H and C ₁ -C ₃ alkyl; and R ₃ is selected from the group consisting of C ₁ -C ₃ alkoxy and C ₃ -C ₅ cycloalkyl.

In another specific embodiment, R ₁ is selected from the group consisting of C ₁ -C ₃ alkyl and C ₃ -C ₅ cycloalkyl; R ₂ is selected from the group consisting of H and C ₁ -C ₃ alkyl; and R ₃ is C ₃ -C ₅ cycloalkyl.

In another specific embodiment, R ₁ is selected from the group consisting of isopropyl and cyclopropyl; R ₂ is H; and R ₃ is cyclopropyl.

In a specific embodiment, the compound of formula (I) is a compound of formula (Ia):

(Ia)

where R ₁ , R ₂ and R ₃ have the same meaning as in the compound of formula (I), or its stereoisomer or pharmaceutically acceptable salt.

In a particular embodiment, a particular stereoisomer of a compound of formula (I) is a compound of formula (Ib):

(IB)

where R ₁ , R ₂ and R ₃ have the same meaning as in the compound of formula (I), or its pharmaceutically acceptable salt.

In a preferred embodiment, the compound of formula (I) is selected from the group consisting of:

,

,

и

,

,

,

and

,

or a stereoisomer or pharmaceutically acceptable salt thereof.

In a specific embodiment, the compound of formula (Ia) is selected from the group consisting of:

,

,

и

,

,

,

and

,

or a stereoisomer or pharmaceutically acceptable salt thereof.

In a specific embodiment, the compound of formula (Ib) is selected from the group consisting of:

,

,

и

,

,

,

and

,

or their pharmaceutically acceptable salt.

In a specific embodiment, the compound of formula (I) is for use in the treatment and/or prevention of a disease or condition selected from the group consisting of cardiac injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; and malignant tumor.

In a preferred embodiment, the compound of formula (I) is for use in the treatment and/or prevention of cardiac injury caused by infarction, ischemia with or without reperfusion, preferably remodeling after myocardial infarction.

In a specific embodiment, the compound of formula (I) is for use in the treatment and/or prevention of a neurodegenerative disorder selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, acute autoimmune encephalitis, and Creutzfeldt-Jakob disease.

In another specific embodiment, the compound of formula (I) is for use in the treatment and/or prevention of a cancer selected from the group consisting of breast cancer, colon cancer, and leukemia.

In another specific embodiment, the compound of formula (I) is for use in the treatment and/or prevention of a fibrotic disease selected from the group consisting of cardiac fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, and retroperitoneal fibrosis.

In a second aspect, the present invention relates to a pharmaceutical composition comprising a compound of formula (I) as defined in the first aspect, and a pharmaceutically acceptable excipient, for use in the treatment and/or prevention of a disease or condition selected from the group consisting of cardiac injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; malignant tumor; and fibrotic diseases.

In another aspect, the invention relates to the use of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt or stereoisomer thereof, for the manufacture of a medicament for the treatment and/or prevention of a disease or condition selected from the group consisting of cardiac injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; malignant tumor; and fibrotic diseases.

In a further aspect, the present invention relates to a method for treating and/or preventing a disease or condition selected from the group consisting of cardiac injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; malignant tumor; and fibrotic diseases which comprises administering to an individual in need thereof a therapeutically effective amount of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt or stereoisomer thereof.

In specific embodiments of the uses and treatments defined above, the disease or condition is selected from the group consisting of cardiac injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; and malignant tumor.

In other specific embodiments of the uses and treatments defined above, the preferred embodiment of the disease or condition is heart injury caused by infarction, ischemia with or without reperfusion, preferably remodeling after myocardial infarction.

In other specific embodiments of the uses and treatments defined above, the neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, acute autoimmune encephalitis, and Creutzfeldt-Jakob disease.

In other specific embodiments of the uses and treatments defined above, the cancer is selected from the group consisting of breast cancer, colorectal cancer, and leukemia.

In other specific embodiments of the uses and treatments defined above, the fibrotic disease is selected from the group consisting of cardiac fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, and retroperitoneal fibrosis.

Compounds of formula (II)

In a third aspect, the present invention relates to a compound of formula (II):

(II)

where

R _1a is selected from the group consisting of C ₁ -C ₆ alkyl and C ₃ -C ₆ cycloalkyl,

R _2a is selected from the group consisting of H and C ₁ -C ₆ alkyl,

R _3a is C ₃ -C ₆ cycloalkyl,

or a pharmaceutically acceptable salt or stereoisomer thereof.

Compounds of formula (II) are a subgroup of compounds of formula (I).

In a specific embodiment, R _1a is selected from the group consisting of C ₁ -C ₃ alkyl and C ₃ -C ₅ cycloalkyl; preferably from the group consisting of methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclobutyl and cyclopentyl; more preferably from the group consisting of methyl, isopropyl and cyclopropyl; even more preferably from the group consisting of isopropyl and cyclopropyl; most preferred is cyclopropyl.

In another specific embodiment, R _2a is selected from the group consisting of H and C ₁ -C ₃ alkyl; preferably from the group consisting of H, methyl, ethyl, propyl and isopropyl; more preferably from the group consisting of H and methyl; most preferred is H.

In another specific embodiment, R _3a is C ₃ -C ₆ cycloalkyl; preferably C ₃ -C ₅ cycloalkyl; preferably cyclopropyl, cyclobutyl or cyclopentyl; most preferred is cyclopropyl.

In a specific embodiment, R _1a is selected from the group consisting of C ₁ -C ₃ alkyl and C ₃ -C ₅ cycloalkyl; R _2a is selected from the group consisting of H and C ₁ -C ₃ alkyl; and R _3a is C ₃ -C ₅ cycloalkyl.

In another specific embodiment, R _1a is selected from the group consisting of isopropyl and cyclopropyl; R _2a is H; and R _3a is cyclopropyl.

In a specific embodiment, the compound of formula (II) is a compound of formula (IIa):

(IIa)

where R _1a , R _2a and R _3a have the same meaning as in the compounds of formula (II), or its stereoisomer or pharmaceutically acceptable salt.

In a particular embodiment, the particular stereoisomer of a compound of formula (II) is a compound of formula (IIb):

(IIb)

where R _1a , R _2a and R _3a have the same meanings as defined for compounds of formula (II), or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the compound of formula (II) is selected from the group consisting of:

,

и

,

,

and

,

or a stereoisomer or pharmaceutically acceptable salt thereof.

In a specific embodiment, the compound of formula (IIa) is selected from the group consisting of:

,

и

,

,

and

,

or a stereoisomer or pharmaceutically acceptable salt thereof.

In a specific embodiment, the compound of formula (IIb) is selected from the group consisting of:

,

и

,

,

and

,

or their pharmaceutically acceptable salt.

Pharmaceutical compositions

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (II) as defined above, or a stereoisomer or pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

The term "pharmaceutically acceptable excipient" refers to a carrier, diluent or adjuvant which is administered with the active ingredient. Such pharmaceutical excipients may be sterile liquids such as water and oils, including petroleum, animal, vegetable or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Carriers preferably used are water or aqueous saline solutions and aqueous solutions of dextrose and glycerol, in particular for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences", E.W. Martin, 21st Edition, 2005.

The compounds of formula (II) according to the invention can be administered by oral, sublingual, parenteral, subcutaneous, intramuscular, intravenous, transdermal, intranasal, intraocular and/or rectal routes. The compounds may be administered alone or in combination with one or more other compounds of the invention or one or more other drugs.

Pharmaceutical compositions of the present invention may contain compounds of formula (II) in liposomes or microvesicles, and they may take the form of dispersions, solutions, lotions, gels, and the like, including topical preparations.

The definitions described above also apply to pharmaceutical compositions containing a compound of formula (I).

Use of compounds of formula (II)

As explained above, compounds of formula (II) are a subgroup of compounds of formula (I). Thus, the compounds of formula (II) are also calpain-1 inhibitors.

Thus, in a fourth aspect, the present invention relates to compounds of formula (II) for use in medicine, in particular for the treatment and/or prevention of diseases or conditions associated with increased activity of calpain. Preferred diseases or conditions are diseases or conditions selected from the group consisting of cardiac injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; and malignancy such as heart injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; malignant tumor and fibrotic diseases; a more preferred disease or condition is heart injury caused by infarction, ischemia with or without reperfusion, preferably remodeling after myocardial infarction.

In another aspect, the invention relates to the use of a compound of formula (II) as defined above, or a pharmaceutically acceptable salt or stereoisomer thereof, for the manufacture of a medicament for the treatment and/or prevention of a disease or condition associated with increased calpain activity; preferably selected from the group consisting of heart injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; malignant tumor; and fibrotic diseases; more preferably selected from the group consisting of heart injury caused by infarction, ischemia with or without reperfusion, even more preferably remodeling after myocardial infarction.

In a further aspect, the present invention relates to a method for treating and/or preventing a disease or condition associated with increased calpain activity, which comprises administering to an individual in need thereof a therapeutically effective amount of a compound of formula (II) as defined above, or a pharmaceutically acceptable salt thereof. or stereoisomer. Preferably, the disease or condition associated with increased calpain activity is selected from the group consisting of cardiac injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; malignant tumor; and fibrotic diseases; more preferably selected from the group consisting of heart injury caused by infarction, ischemia with or without reperfusion, even more preferably remodeling after myocardial infarction.

In specific embodiments, the disease or condition is selected from the group consisting of heart injury caused by infarction, ischemia with or without reperfusion; neurodegenerative disorders; malaria; diabetic nephropathy; neurotoxicity induced by the HIV virus; and malignant tumor.

In other specific embodiments, the disease or condition is heart injury caused by infarction, ischemia with or without reperfusion, preferably remodeling after myocardial infarction.

In other specific embodiments, the neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, acute autoimmune encephalitis, and Creutzfeldt-Jakob disease.

In other specific embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, and leukemia.

In other specific embodiments, the fibrotic disease is selected from the group consisting of cardiac fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, and retroperitoneal fibrosis.

Process for the preparation of compounds of formula (I) and (II)

Compounds of formula (I) and (II), preferably wherein R ₃ is a cycloalkyl group, can be prepared starting from N-substituted 3-amino-2-hydroxyamide hydrochlorides of formula (VIII) according to the synthesis scheme below:

In the first step, the protected amino acid of formula (VII), HOBt (hydroxybenzotriazole), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and N-substituted 3-amino-2-hydroxyamide hydrochlorides of formula (VIII) are dissolved in a solvent, such as dichloromethane (DCM). DIPEA (N,N-diisopropylethylamine) is then added and the mixture is allowed to react to give a compound of formula (VI). Other amide addition agents are equally effective, such as HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) in the presence of DIPEA in dimethylformamide (DMF) or T3P (propylphosphonic anhydride) in the presence of NEt ₃ in DMF. The compound of formula (VI) is reacted in a solvent such as 1,4-dioxane with a strong acid such as hydrochloric acid or in DCM and treated with trifluoroacetic acid (TFA) to give a compound of formula (V). The compound of formula (III), HOBt, EDC and the compound of formula (V) are dissolved in a solvent such as DCM. DIPEA is then added and the mixture is allowed to react to give a compound of formula (IV). Other amide addition agents are equally effective, such as HATU in the presence of DIPEA in DMF or T3P in the presence of NEt ₃ in DMF. Finally, the compound of formula (IV) is dissolved in solvents such as DCM, DMF, or mixtures thereof, and Dess-Martin periodinan is added to give a compound of formula (I). Other oxidizing agents such as DCC (N, N'-dicyclohexylcarbodiimide) in DMSO (dimethyl sulfoxide) are equally suitable.

N-substituted 3-amino-2-hydroxyamide hydrochlorides of formula (VIII) can be prepared starting from protected amino aldehydes of formula (XIII) according to the synthesis scheme shown below:

In the first step, the protected amino aldehydes of formula (XIII) are dissolved in a solvent such as 1,4-dioxane and sodium bisulfite is added, and then an aqueous solution of potassium cyanide is added to give a compound of formula (XII). The compound of formula (XII) is dissolved in a concentrated aqueous acid solution such as concentrated hydrochloric acid and refluxed to give a compound of formula (XI). An aqueous solution of a compound of formula (XI) is adjusted to an alkaline pH (preferably in the range of 10-12), for example with sodium hydroxide, and Boc ₂ O (di-tert-butyl dicarbonate) is added. After complete conversion, the mixture is acidified with eg KHSO ₄ and the compound of formula (X) is extracted using a water-immiscible solvent such as ethyl acetate. The compound of formula (X), HOBt and EDC are dissolved in a solvent such as anhydrous DCM. DIPEA and an amine of formula (XV) are then added and the mixture is allowed to react for 8-24 hours to give a compound of formula (VIII). It is also possible to use T3P or HATU instead of EDC and HOBt with good results. The compound of formula (IX) is reacted in a solvent such as 1,4-dioxane with a strong acid such as hydrochloric acid to give a compound of formula (VIII).

In an alternative synthetic route, which is also illustrated in the previous scheme, the protected aminoaldehydes of formula (XIII) are dissolved in a solvent such as anhydrous DCM and an acid such as acetic acid and an isocyanide compound of formula (XIV) are added and allowed to react under an inert atmosphere, such as an argon atmosphere, at room temperature. The solvent was then removed and the resulting compound was extracted with ethyl acetate and washed with saturated sodium bicarbonate aqueous solution. The product is then dissolved in a mixture of THF (tetrahydrofuran) and MeOH (methanol) and treated with aqueous lithium hydroxide to give a compound of formula (IX). Then, the compound of formula (IX) is reacted as described above in a solvent such as 1,4-dioxane with a strong acid such as hydrochloric acid to obtain a compound of formula (VIII).

Compounds of formula (I), where R ₃ represents an alkoxy group, can be obtained starting from the hydrochlorides of N-substituted 3-amino-2-hydroxyamides of formula (VIII), according to the synthesis scheme below:

In the first step, the protected amino acid of formula (VII), HOBt and the compound of formula (XXII) are dissolved in a solvent such as dichloromethane (DCM). DIPEA is then added and the mixture is allowed to react to give a compound of formula (XXI). Other amide addition agents are equally effective, such as HATU in the presence of DIPEA in DMF or T3P in the presence of NEt ₃ in DMF. The compound of formula (XXI) is reacted in a solvent such as 1,4-dioxane with a strong acid such as hydrochloric acid or in DCM and treated with trifluoroacetic acid (TFA) to give a compound of formula (XX). The compound of formula (III), HOBt, EDC and the compound of formula (XX) are dissolved in a solvent such as DCM. DIPEA is then added and the mixture is allowed to react to give a compound of formula (XIX). Other amide addition agents are equally effective, such as HATU in the presence of DIPEA in DMF or T3P in the presence of NEt ₃ in DMF. The compound of formula (XIX) is then treated with LiOH in THF/MeOH/water and then treated with an aqueous acid such as HCl to give a compound of formula (XVIII). The compound of formula (XVII), HOBt, EDC and the compound of formula (XVIII) are dissolved in a solvent such as DCM. DIPEA is then added and the mixture is allowed to react to give a compound of formula (XVI). Other amide addition agents are equally effective, such as HATU in the presence of DIPEA in DMF or T3P in the presence of NEt ₃ in DMF. Finally, the compound of formula (XVI) is dissolved in a solvent such as DCM, DMF, or a mixture thereof, and Dess-Martin periodinan is added to give a compound of formula (I). Other oxidizing agents such as DCC (N, N'-dicyclohexylcarbodiimide) in DMSO (dimethyl sulfoxide) are equally suitable.

The compound of formula (XXII) can be obtained from the compound of formula (X), the synthesis of which is described above, by treatment with oxalyl chloride in methanol, as shown in the synthesis scheme below:

The starting compounds of formulas (XVII), (XIII), (XIV), (XV), (VII) and (III) are either commercially available or can be obtained by methods described in the literature.

The following examples are only illustrative of certain embodiments of the invention and should not be considered limiting in any way.

Examples

Abbreviations

The examples use the following abbreviations:

Boc: tert-butoxycarbonyl

conc.: concentrate

Boc ₂ O: di-tert-butyl dicarbonate

DCM: dichloromethane

DIPEA: N,N-diisopropylethylamine

DMSO: dimethyl sulfoxide

EDC HCl: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

EtOAc: ethyl acetate

HBTU: N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate

HCl: hydrochloric acid

HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HOBt: hydroxybenzotriazole

LC-MS: liquid chromatography-mass spectrometry

Leu-OH: leucine

MeOH: methanol

Min: minutes

Phe-OH: phenylalanine

Saturation: saturated

T3P: propylphosphonic anhydride

TBME: tert-butyl methyl ether

THF: tetrahydrofuran

t _R : holding time

Val-OH: valine

Materials and methods

LC-MS : Products according to the examples were characterized using liquid chromatography coupled with mass spectrometry (LC-MS). HPLC-MS analysis was performed as follows: in an Alliance HT 2795 (Waters) chromatograph equipped with a 2996 photodiode array detector and coupled to an LC/MS 3100 mass detector. -3.5 µm) and using mixtures of 10 mM aqueous NH ₄ CO ₃ pH=9 (A) and acetonitrile (B) as eluents at 50°C and a flow rate of 1.6 ml/min with the following elution conditions : 5% to 100% B in 3.5 min. The detector was set to positive electrospray mode (ESI+) in the mass range 100-700. Cone voltage 10 V. Source T: 120°C. Desolvation T: 350°C.

Reagents : 2-methoxybenzoic acid (Sigma Aldrich); 3-methoxybenzoic acid (Sigma Aldrich); 4-methoxybenzoic acid (Sigma Aldrich); acetic acid (VWR); Boc-L-Phe-OH (Fluorochem); Boc-L-Leu-OH (Fluorochem); Boc-L-Val-OH (Fluorochem); cyclopropylamine (Sigma Aldrich); cyclopropyl isocyanide (Fluorochem); 15% Dess-Martin periodinan solution in DCM (Acros); EDC (Iris Biotech); HBTU (Iris Biotech); 4 N HCl in 1,4-dioxane (TCI Europe Organic Chem); HOBt (Carbosynth); LiOH H ₂ O (Sigma Aldrich); 1.0 M lithium aluminum hydride in tetrahydrofuran (Sigma Aldrich); N,O-dimethylhydroxylamine hydrochloride (Fluorochem); (S)-2-(Boc-amino)-3-cyclopropylpropionic acid (Fluorochem).

Solvents : DCM (Scharlab); ethanol (Panreac); EtOAc (Scharlab); hexane (Scharlab); MeOH (Scharlab); TBME (SDS); THF (Panreac).

Synthesis of intermediates

Intermediate 1: (S)-tert-Butyl(1-(methoxy(methyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate

To a solution of Boc-L-Phe-OH (10.0 g, 37.7 mmol, 1.0 eq.) in DCM (150 mL) was added N,O-dimethylhydroxylamine hydrochloride (4.0 g, 41.1 mmol , 1.1 eq.), HBTU (III) (15.7 g, 41.1 mmol, 1.1 eq.) and DIPEA (20 ml, 113.0 mmol, 3.0 eq.). The reaction mixture was stirred for 1 hour at room temperature. The volatiles were then removed in vacuo and the crude mixture was extracted with EtOAc and washed with saturated NaHCO ₃ solution (2×200 ml). The crude material was purified by flash chromatography (ISCO Rf) using hexane/EtOAc as eluents, from 0% to 40% in EtOAc, the product was eluted at 35%. 11.6 g (37.7 mmol, 100% yield) were obtained.

LC-MS: t _R =2.58 min; m/z=309

Intermediate 2: Synthesis of (S)-tert-butyl (1-oxo-3-phenylpropan-2-yl)carbamate

A solution of lithium aluminum hydride 1.0 M in tetrahydrofuran (37.7 ml, 37.7 mmol, 1.0 eq.) was added to a solution of intermediate 1 (11.6 g, 37.7 mmol, 1.0 eq.) in THF (250 ml) chilled to 0°C. The mixture was stirred overnight at room temperature. The reaction mixture was diluted with EtOAc and the solvent was evaporated. The crude material was purified by silica gel chromatography using hexane/EtOAc as solvents, 0% to 60% in EtOAc, the product eluted at 25%. 6.7 g of the desired product was obtained (26.9 mmol, 68% yield).

LC-MS: t _R =2.48 min; m/z=250

Intermediate 3: Synthesis of tert-butyl ((2S)-4-(cyclopropylamino)-3-hydroxy-4-oxo-1-phenylbutan-2-yl)carbamate a

Acetic acid (0.35 ml, 8.0 mmol, 2.0 eq.) and cyclopropyl isocyanide (0.30 ml, 4.4 mmol, 1.1 eq.) were added to a solution of intermediate 2 (1.0 g, 4.0 mmol, 1.0 eq.) in DCM (20 ml) and the mixture was stirred at rt. After 15 min, a mixture of THF/MeOH/H ₂ O (7/5/3) was added to the mixture, followed by LiOH·H ₂ O (0.67 g, 16.0 mmol, 4 eq.). After 15 min the solvents were removed in vacuo. The product was extracted into EtOAc and the solution was washed with sat. NaHCO ₃ solution and purified by flash chromatography (ISCO Rf) using hexane/hexane:ethanol (8:2) as solvents, from 0% to 50% in hexane:ethanol (8:2), the product was eluted at 25% . 0.31 g of product was obtained (yield 23%).

LC-MS: t _R =2.32 min; m/z=335

Intermediate 4: Synthesis of (3S)-3-amino-N-cyclopropyl-2-hydroxy-4-phenylbutanamide hydrochloride

A solution of 4N HCl in 1,4-dioxane (1 ml, 4.1 mmol, 4 eq.) was added to intermediate 3 (0.31 g, 0.9 mmol, 1.0 eq.). The mixture was stirred at room temperature for 1 hour and then evaporated to dryness. This product was used for the next step without further purification.

LC-MS: t _R =1.50 min; m/z=235

Intermediate 5: tert-butyl ((2S)-1-(((2S)-4-(cyclopropylamino)-3-hydroxy-4-oxo-1-phenylbutan-2-yl)amino)-4-methyl-1 -oxopentan-2-yl)carbamate

A solution of intermediate 4 (251 mg, 0.9 mmol, 1.0 eq.) and DIPEA (0.6 ml, 3.7 mmol, 4 eq.) in DCM (5 ml) was added to a solution of Boc-L-Leu -OH (300 mg, 1.2 mmol, 1.2 eq.), EDC (249 mg, 1.3 mmol, 1.5 eq.) and HOBt (199 mg, 1.3 mmol, 1.5 eq. ) in DCM (5 ml). The reaction mixture was stirred for 1 hour at room temperature. Volatiles were removed in vacuo. The crude mixture was extracted with EtOAc and washed with sat. NaHCO ₃ solution (2×10 ml) and purified by flash chromatography (ISCO Rf) using hexane/TBME as eluents, from 0% to 80% in TBME, the product eluted at 60%. 298 mg of product was obtained (0.7 mmol, 72% yield).

LC-MS (Method A): t _R =2.60 min; m/z=448.

Intermediate 6: (2S)-2-amino-N-((2S)-4-(cyclopropylamino)-3-hydroxy-4-oxo-1-phenylbutan-2-yl)-4-methylpentanamide hydrochloride

A solution of HCl (4 N) in 1,4-dioxane (3.3 ml, 13.2 mmol, 20 eq.) was added to intermediate 5 (298 mg, 0.6 mmol, 1.0 eq.). The mixture was stirred at room temperature for 1 hour and then evaporated to dryness. The product was used for the next step without further purification.

LC-MS: t _R = 1.92-2.03 min; m/z=348.

Intermediate 7: N-((2S)-1-(((2S)-4-(cyclopropylamino)-3-hydroxy-4-oxo-1-phenylbutan-2-yl)amino)-4-methyl-1- oxopentan-2-yl)-3-methoxybenzamide

A solution of intermediate 6 (115 mg, 0.3 mmol, 1.0 eq.) and DIPEA (231 μl, 1.3 mmol, 4.0 eq.) in DCM (5 ml) was added to a solution of 3-methoxybenzoic acid ( 60.4 mg, 0.4 mmol, 1.2 eq.), EDC (108 mg, 0.6 mmol, 1.7 eq.) and HOBt (86 mg, 0.6 mmol, 1.5 eq.) in DCM (4 ml). The reaction mixture was stirred for 1 hour at room temperature. Volatiles were removed in vacuo and the crude mixture was extracted with EtOAc and washed with sat. NaHCO ₃ solution (2×20 ml). The material was purified by flash chromatography (ISCO Rf) using hexane/hexane:ethanol (8:2) as eluents, from 0% to 60% in hexane:ethanol (8:2), the product was eluted at 40%. Received 100 mg of product (0.2 mmol, 100%).

LC-MS: t _R =2.47 min; m/z=482.

Intermediate 8: Synthesis of tert-butyl ((2S)-1-((4-(cyclopropylamino)-3-hydroxy-4-oxo-1-phenylbutan-2-yl)amino)-3-methyl-1-oxobutan- 2-yl)carbamate

A solution of intermediate 4 (1.06 g, 3.91 mmol, 1.0 eq.) and DIPEA (2.74 ml, 15.66 mmol, 4 eq.) in DCM (5 ml) was added to a solution of Boc-L -Val-OH (1.28 g, 5.87 mmol, 1.5 eq.), EDC (1.13 g, 5.87 mmol, 1.5 eq.) and HOBt (0.9 g, 5, 87 mmol, 1.5 eq.) in DCM (5 ml). The reaction mixture was stirred for 45 minutes at room temperature and then the volatiles were removed in vacuo. The crude mixture was extracted with EtOAc and washed with sat. NaHCO ₃ solution. The crude material was purified by flash chromatography (ISCO Rf) using DCM/DCM:methanol (8:2) as eluents, from 0% to 50% in DCM:methanol (8:2), the product was eluted at 15%. 600 mg of product was obtained (1.384 mmol, 35% yield).

LC-MS: t _R =2.47 min; m/z=434

Intermediate 9: Synthesis of 3-((S)-2-amino-3-methylbutanamido)-N-cyclopropyl-2-hydroxy-4-phenylbutanamide hydrochloride

A solution of 4 N HCl in 1,4-dioxane (6.9 ml, 27.7 mmol, 20 eq.) was added to intermediate 8 (600 mg, 1.384 mmol, 1.0 eq.). The mixture was stirred at room temperature for 1 hour and then evaporated to dryness. The product was used for the next step without further purification.

LC-MS: t _R = 1.77-1.88 min; m/z=334

Intermediate 10: Synthesis of N-((2S)-1-((4-(cyclopropylamino)-3-hydroxy-4-oxo-1-phenylbutan-2-yl)amino)-3-methyl-1-oxobutan-2 -yl)-3-methoxybenzamide

A solution of intermediate 9 (461 mg, 0.3 mmol, 1.0 eq.) and DIPEA (0.97 ml, 5.53 mmol, 4.0 eq.) in DCM (9 ml) was added to a solution of 3-methoxybenzoic acids (316 mg, 2.07 mmol, 1.5 eq.), EDC (398 mg, 2.07 mmol, 1.5 eq.) and HOBt (318 mg, 2.07 mmol, 1.5 eq.) in DCM (9 ml). The reaction mixture was stirred for 1 hour at room temperature and then the volatiles were removed in vacuo. The crude mixture was extracted with EtOAc and washed with sat. NaHCO ₃ solution. The crude material was purified by flash chromatography (ISCO Rf) using DCM/DCM:methanol (8:2) as eluents, 0% to 20% in DCM:methanol (8:2), the product was eluted at 15%. 470 mg of product (1 mmol, 73%) was obtained.

LC-MS: t _R =2.32 min; m/z=468.

Intermediate 11: Synthesis of tert-butyl ((2S)-3-cyclopropyl-1-((4-(cyclopropylamino)-3-hydroxy-4-oxo-1-phenylbutan-2-yl)amino)-1-oxopropan- 2-yl)carbamate

A solution of intermediate 4 (530 mg, 1.96 mmol, 1.0 eq.) and DIPEA (1.37 ml, 7.83 mmol, 4 eq.) in DCM (3 ml) was added to a solution of (S)-2 -(Boc-amino)-3-cyclopropylpropionic acid (673 mg, 2.94 mmol, 1.5 eq.), EDC (563 mg, 2.94 mmol, 1.5 eq.) and HOBt (450 mg, 2 .94 mmol, 1.5 eq.) in DCM (2 ml). The reaction mixture was stirred for one and a half hours at room temperature. Volatiles were removed in vacuo and the crude mixture was extracted with EtOAc and washed with sat. NaHCO ₃ solution. The crude mixture was purified by flash chromatography (ISCO Rf) using DCM/DCM:methanol (8:2) as eluents, 0% to 20% in DCM:methanol (8:2), the product was eluted at 12%. 430 mg of product was obtained (0.96 mmol, 49% yield).

LC-MS: t _R =2.5 min; m/z=446

Intermediate 12: Synthesis of 3-((S)-2-amino-3-cyclopropylpropanamido)-N-cyclopropyl-2-hydroxy-4-phenylbutanamide hydrochloride

A solution of 4 N HCl in 1,4-dioxane (4.83 ml, 19.3 mmol, 20 eq.) was added to intermediate 11 (430 mg, 0.96 mmol, 1.0 eq.). The mixture was stirred at room temperature for 1 hour and then evaporated to dryness. The product was used for the next step without further purification.

LC-MS (Method A): tR=1.82-1.92 min; m/z=346

Intermediate 13: Synthesis of N-((2S)-3-cyclopropyl-1-((4-(cyclopropylamino)-3-hydroxy-4-oxo-1-phenylbutan-2-yl)amino)-1-oxopropan-2 -yl)-3-methoxybenzamide

A solution of intermediate 12 (333 mg, 0.3 mmol, 1.0 eq.) and DIPEA (0.67 ml, 3.86 mmol, 4.0 eq.) in DCM (6 ml) was added to a solution of 3-methoxybenzoic acids (220 mg, 1.45 mmol, 1.5 eq.), EDC (277 mg, 1.45 mmol, 1.5 eq.) and HOBt (221 mg, 1.45 mmol, 1.5 eq.) in DCM (6 ml). The reaction mixture was stirred for 1 hour at room temperature and then the volatiles were removed in vacuo. The crude mixture was extracted with EtOAc and washed with sat. NaHCO ₃ solution. The crude material was purified by flash chromatography (ISCO Rf) using DCM/DCM:methanol (8:2) as eluents, from 0% to 50% in DCM:methanol (8:2), the product was eluted at 38%. 440 mg of product (0.88 mmol, 91%) was obtained.

LC-MS: t _R =2.37 min; m/z=480

Intermediate 14: Synthesis of tert-butyl ((2S)-1-cyano-1-hydroxy-3-phenylpropan-2-yl)carbamate

Sodium bisulfate (11.2 g, 107.0 mmol, 4 eq.) was added to a solution of intermediate 2 (6.7 g, 26.9 mmol, 1.0 eq.) in 1,4-dioxane (150 ml) at 0°C. The reaction mixture was stirred at 0° C. for 10 minutes and potassium cyanide (7.0 g, 107.0 mmol, 4 eq.) dissolved in water (45 ml) was added. The reaction mixture was stirred overnight at room temperature. The organic solvent was evaporated, water and EtOAc were added and the layers were separated. The combined organic layers were washed with sat. aq. NaHCO ₃ , dried and concentrated under reduced pressure. 7.0 g of product was obtained (23.5 mmol, 94% yield).

LC-MS: t _R =2.83 min; m/z=277

Intermediate 15: Synthesis of (3S)-3-((tert-butoxycarbonyl)amino)-2-hydroxy-4-phenylbutanoic acid

Intermediate 14 (6.5 g, 23.5 mmol, 1.0 eq.) was dissolved in 20 ml conc. HCl and boiled for 1 hour. The crude mixture was then cooled and washed with DCM. 10 N NaOH was added to the aqueous layer until pH 11 and washed with DCM. Boc ₂ O (5.6 g, 25.8 mmol, 1.1 eq.) was added to the aqueous layer. The mixture was stirred at room temperature overnight. After complete conversion, the mixture was acidified with KHSO ₄ to pH 2 and extracted with EtOAc. The organic layer was evaporated and the product was used for the next step without further purification. 5.1 g (17.2 mmol, 73% yield, 2 steps) were obtained.

LC-MS: t _R =1.62 min; m/z=296

Intermediate 16: Synthesis of (3S)-methyl 3-amino-2-hydroxy-4-phenylbutanoate hydrochloride

Oxalyl chloride (6 ml, 69.9 mmol, 4.0 eq.) was added slowly to a solution of intermediate 15 (5.1 g, 17.26 mmol, 1.0 eq.) in MeOH (300 ml) at 0°C . The mixture was stirred overnight at room temperature. The crude mixture was evaporated and then co-evaporated with MeOH and the product was used for the next step without further purification. 4.2 g (17.26 mmol, quantitative yield) was obtained.

LC-MS: t _R = 1.58-1.65 min; m/z=210.

Intermediate 17: Synthesis of methyl 3-((S)-2-((tert-butoxycarbonyl)amino)-3-cyclopropylpropanamido)-2-hydroxy-4-phenylbutanoate

A solution of intermediate 16 (3 g, 12.21 mmol, 1.0 eq.) and DIPEA (8.53 ml, 48.8 mmol, 4 eq.) in DCM (15 ml) was added to a solution of (S)-2 -(Boc-amino)-3-cyclopropylpropionic acid (2.8 g, 12.21 mmol, 1 eq.), EDC (3.51 g, 18.31 mmol, 1.5 eq.) and HOBt (2, 8 g, 18.31 mmol, 1.5 eq) in DCM (10 ml). The reaction mixture was stirred for 1 hour at room temperature. Volatiles were removed in vacuo. The crude mixture was extracted with EtOAc and washed with sat. NaHCO ₃ solution. The crude product was purified by flash chromatography (Biotage) using DCM/DCM:methanol (8:2) as eluents, 0% to 30% in DCM:methanol (8:2), the product was eluted at 20%. 2.2 g of product was obtained (5.23 mmol, 43% yield).

LC-MS: t _R =2.64 min; m/z=421.

Intermediate 18: Synthesis of methyl 3-((S)-2-amino-3-cyclopropylpropanamido)-2-hydroxy-4-phenylbutanoate hydrochloride

A solution of 4 N HCl in 1,4-dioxane (26.2 ml, 105 mmol, 20 eq.) was added to intermediate 17 (2.2 g, 5.23 mmol, 1.0 eq.). The mixture was stirred at room temperature for 1 hour and then evaporated to dryness. The product was used for the next step without further purification.

LC-MS: t _R =1.78 min; m/z=321.

Intermediate 19: Synthesis of methyl 3-((S)-3-cyclopropyl-2-(3-methoxybenzamido)propanamido)-2-hydroxy-4-phenylbutanoate

A solution of intermediate 18 (1.87 g, 5.23 mmol, 1.0 eq.) and DIPEA (3.66 ml, 3.86 mmol, 4.0 eq.) in DCM (10 ml) was added to a solution of 3 -methoxybenzoic acid (0.955 g, 6.28 mmol, 1.2 eq.), EDC (1.5 g, 7.85 mmol, 1.5 eq.) and HOBt (1.2 g, 7.85 mmol, 1.5 eq.) in DCM (10 ml). The reaction mixture was stirred for 1 hour at room temperature and then the volatiles were removed in vacuo. The crude mixture was extracted with EtOAc and washed with sat. NaHCO ₃ solution. The crude product was purified by flash chromatography (Biotage) using DCM/DCM:methanol (8:2) as eluents, from 0% to 50% in DCM:methanol (8:2), the product was eluted at 25%. 1 g of product was obtained (2.2 mmol, 42%).

LC-MS: t _R =2.47 min; m/z=455.

Intermediate 20: Synthesis of 3-((S)-3-cyclopropyl-2-(3-methoxybenzamido)propanamido)-2-hydroxy-4-phenylbutanoic acid

LiOH H ₂ O (111 mg, 2.64 mmol, 3.0 eq.) was added to a solution of intermediate 19 (400 mg, 0.88 mmol, 1.0 eq.) dissolved in 13.5 ml of THF /MeOH/H ₂ O (5/3/1). After complete conversion, the solvents were removed in vacuo. The crude material was dissolved in 1 M HCl and extracted with DCM until all intermediate 20 was extracted into the organic layer. The organic layers were evaporated to dryness. Intermediate 20 was used for the next step without further purification.

LC-MS: t _R =1.85 min; m/z=441.

Intermediate 21: Synthesis of N-((2S)-3-cyclopropyl-1-((3-hydroxy-4-(methoxyamino)-4-oxo-1-phenylbutan-2-yl)amino)-1-oxopropan-2 -yl)-3-methoxybenzamide

EDC (307 mg, 1.6 mmol, 1.5 eq.) and HOBt (245 mg, 1.6 mmol, 1.5 eq.) and DIPEA (0.75 ml, 4.27 mmol, 4.0 eq.). The reaction mixture was stirred for 1 hour at room temperature. Thereafter, 0.1 ml DIPEA was added and the reaction mixture was stirred overnight at room temperature. Added 0.5 equiv. both EDC and HOBt, and stirring was continued for 2 hours at room temperature. Volatiles were removed in vacuo. The crude mixture was extracted with EtOAc and washed with sat. NaHCO ₃ solution and 5% aqueous citric acid solution. The crude product was purified by flash chromatography (Biotage) using DCM/DCM:methanol (8:2) as eluents, from 0% to 50% in DCM:methanol (8:2), the product was eluted at 20%. 170 mg of product was obtained (0.37 mmol, 35% yield).

LC-MS: t _R =2.18 min; m/z=470

Intermediate 22: N-((2S)-1-(((2S)-4-(cyclopropylamino)-3-hydroxy-4-oxo-1-phenylbutan-2-yl)amino)-4-methyl-1- oxopentan-2-yl)-4-methoxybenzamide

A solution of intermediate 6 (200 mg, 0.6 mmol, 1.0 eq.) and DIPEA (392 μl, 2.3 mmol, 4.0 eq.) in DCM (6 ml) was added to a solution of 4-methoxybenzoic acid ( 105 mg, 0.7 mmol, 1.2 eq), EDC (188 mg, 1.0 mmol, 1.7 eq) and HOBt (150 mg, 1.0 mmol, 1.7 eq) in DCM (6 ml). The reaction mixture was stirred for 1 hour at room temperature and the volatiles were removed in vacuo. The crude material was extracted with EtOAc and washed with sat. NaHCO ₃ solution (3×10 ml) and 5% aqueous citric acid solution (3×10 ml). The crude mixture was purified by flash chromatography (ISCO Rf) using hexane/hexane:ethanol (8:2) as eluents, from 0% to 100% in hexane:ethanol (8:2), the product was eluted at 35%. 115 mg of product (0.2 mmol, 41%) was obtained.

LC-MS: t _R= 2.35 min; m/z=482

Intermediate 23: N-((2S)-1-(((2S)-4-(cyclopropylamino)-3-hydroxy-4-oxo-1-phenylbutan-2-yl)amino)-4-methyl-1- oxopentan-2-yl)-2-methoxybenzamide

A solution of intermediate 6 (200 mg, 0.5 mmol, 1.0 eq.) and DIPEA (350 μl, 2.0 mmol, 4.0 eq.) in DCM (6 ml) was added to a solution of 2-methoxybenzoic acid ( 95 mg, 0.6 mmol, 1.2 eq), EDC (169 mg, 0.9 mmol, 1.7 eq) and HOBt (135 mg, 0.9 mmol, 1.7 eq) in DCM (5 ml). The reaction mixture was stirred for 1 hour at room temperature and then the volatiles were removed in vacuo. The crude mixture was extracted with EtOAc and washed with sat. NaHCO ₃ solution (3×10 ml) and 5% aqueous citric acid solution (3×10 ml). The crude product was purified by flash chromatography (ISCO Rf) using hexane/hexane:ethanol (8:2) as eluents, from 0% to 100% in hexane:ethanol (8:2), the product was eluted at 35%. 250 mg (0.5 mmol, 100%) were obtained.

LC-MS: t _{R =} 2.47 min; m/z=482.

Preparation of Compounds of Examples and Comparative Examples

Example 1: N-((S)-1-(((S)-4-(cyclopropylamino)-3,4-dioxo-1-phenylbutan-2-yl)amino)-4-methyl-1-oxopentan-2 -yl)-3-methoxybenzamide

5 μl of water was added to a 15% solution of Dess-Martin periodinan in DCM (640 μl, 0.3 mmol, 1.5 eq.). The mixture was then added to a solution of intermediate 7 (100 mg, 0.2 mmol, 1.0 mmol) in DCM (4 ml). The mixture was stirred for 15 minutes at room temperature and then the volatiles were removed in vacuo. The crude mixture was extracted with EtOAc and washed with sat. NaHCO ₃ solution (2×10 ml). The crude product was purified by flash chromatography (ISCO Rf) using hexane/hexane:ethanol (8:2) as solvents, from 0% to 60% in hexane:ethanol (8:2), the product was eluted at 30% in hexane: ethanol (8:2). 47.6 mg of product was obtained (0.1 mmol, 47% yield).

LC-MS: t _R =2.63 min; m/z=480.

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ 8.75 (dd, J =26.3, 5.1 Hz, 1H), 8.38 (dd, J =18.9, 8.1 Hz , 1H), 8.33 (d, J = 7.7 Hz, 1H), 7.49-7.31 (m, 3H), 7.27-7.15 (m, 5H), 7.09 ( m, 1H), 5.22-5.13 (m, 1H), 4.54 (m, 1H), 3.80 (s, 3H), 3.13 (m, 1H), 2.88-2 .77 (m, 1H), 2.76-2.70 (m, 1H), 1.67-1.55 (m, 1H), 1.53-1.43 (m, 1H), 1.29 (m, 1H), 0.91-0.78 (m, 6H), 0.65 (m, 2H), 0.61-0.53 (m, 2H).

Example 2 Synthesis of N-((S)-1-((4-(cyclopropylamino)-3,4-dioxo-1-phenylbutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl) -3-methoxybenzamide

A 15% solution of Dess-Martin periodinan in DCM (3.13 ml, 1.51 mmol, 1.5 eq.) was added to a solution of intermediate 10 (470 mg, 1 mmol, 1.0 eq.) dissolved in the previously prepared DCM solution (10 ml) and water (5 µl). The mixture was stirred for 15 minutes at room temperature. After that, 1.5 eq. 15% Dess-Martin periodinan solution and the mixture was stirred for an additional 15 minutes at room temperature. Volatiles were removed in vacuo and the crude mixture was extracted with EtOAc and washed with 1 M NaOH solution. The crude product was triturated with hexane and filtered. 352 mg of product was obtained (0.76 mmol, 76% yield).

LC-MS: t _R =2.52 min; m/z=466

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ 0.61 (s, 2H), 0.61-0.68 (m, 2H), 0.73 (dd, J =23.0, 6, 7 Hz, 3H), 0.87 (dd, J =6.7, 1.8 Hz, 3H), 1.92-2.10 (m, 1H), 2.68-2.85 (m, 2H ), 3.07-3.18 (m, 1H), 3.81 (d, J = 1.1 Hz, 3H), 4.32 (td, J = 8.3, 5.6 Hz, 1H) , 5.18-5.28 (m, 1H), 7.10 (dddd, J =8.0, 2.8, 1.9, 1.1 Hz, 1H), 7.15-7.25 ( m, 5H), 7.34-7.46 (m, 3H), 8.15 (dd, J =11.8, 9.0 Hz, 1H), 8.45 (dd, J =14.4, 7.2 Hz, 1H), 8.76 (dd, J =21.8, 5.1 Hz, 1H).

Example 3: Synthesis of N-((S)-3-cyclopropyl-1-((4-(cyclopropylamino)-3,4-dioxo-1-phenylbutan-2-yl)amino)-1-oxopropan-2-yl) -3-methoxybenzamide

A 15% solution of Dess-Martin periodinan in DCM (2.73 ml, 1.31 mmol, 1.5 eq) was added to a solution of intermediate 13 (420 mg, 0.88 mmol, 1.0 eq) dissolved in 8 ml of pre-prepared DCM solution (10 ml) and 5 µl of water. The mixture was stirred for 15 minutes at room temperature. After that, 1.5 eq. 15% Dess-Martin periodinan solution and the mixture was stirred for an additional 15 minutes at room temperature. Volatiles were removed in vacuo and the crude mixture was extracted with EtOAc and washed with 1 M NaOH solution. The crude product was triturated with hexane and filtered. 274 mg of product was obtained (0.57 mmol, 65% yield).

LC-MS: t _R =2.57 min; m/z=478

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ -0.01-0.20 (m, 2H), 0.25-0.43 (m, 1H), 0.54-0.70 (m , 4H), 0.70-0.79 (m, 1H), 1.18-1.35 (m, 1H), 1.47-1.68 (m, 2H), 2.69-2.88 (m, 2H), 3.12 (td, J =13.5, 4.2 Hz, 1H), 3.80 (d, J =1.2 Hz, 3H), 4.55 (td, J = 8.7, 5.1 Hz, 1H), 5.20 (dtd, J =9.0, 7.8, 4.1 Hz, 1H), 7.08-7.12 (m, 1H), 7 .16-7.25 (m, 5H), 7.34-7.46 (m, 3H), 8.29-8.45 (m, 2H), 8.75 (dd, J =27.7, 5.1 Hz, 1H).

Example 4: Synthesis of N-((S)-3-cyclopropyl-1-((4-(methoxyamino)-3,4-dioxo-1-phenylbutan-2-yl)amino)-1-oxopropan-2-yl) -3-methoxybenzamide

A 15% solution of Dess-Martin periodinan in DCM (0.827 ml, 0.4 mmol, 1.5 eq.) was added in 4 portions to a solution of intermediate 21 (170 mg, 0.36 mmol, 1.0 eq.) in DMSO ( 2.5 ml), stirring the mixture for 10 minutes between additions. The mixture was stirred for 2 hours at room temperature and then the volatiles were removed in vacuo. The crude mixture was extracted with EtOAc and washed with 1 M NaOH. The volatiles were evaporated and the crude mixture was purified by reverse phase chromatography (Biotage) using H ₂ O/acetonitrile as solvents, 0% to 100% in acetonitrile, the product eluted at 20%. 14.2 mg of product was obtained (0.03 mmol, 8% yield).

LC-MS: t _R =2.03 min; m/z=468

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ -0.04-0.23 (m, 1H), 0.34 (ddd, J =17.7, 9.1, 5.5 Hz, 1H ), 1.43-1.68 (m, 1H), 2.52 (d, J =1.9 Hz, 1H), 2.67-2.83 (m, 1H), 3.10-3, 22 (m, 2H), 3.65 (d, J =13.1 Hz, 2H), 3.79 (d, J =2.2 Hz, 3H), 4.53 (tt, J =9.0 , 4.6 Hz, 1H), 5.32 (s, 1H), 6.95-7.33 (m, 6H), 7.33-7.46 (m, 3H), 8.09 (d, J = 72.5 Hz, 1H), 8.34 (dd, J = 26.3, 8.3 Hz, 1H).

Comparative Example 5: N-((S)-1-(((S)-4-(cyclopropylamino)-3,4-dioxo-1-phenylbutan-2-yl)amino)-4-methyl-1-oxopentan- 2-yl)-4-methoxybenzamide

5 μl of water was added to a 15% solution of Dess-Martin periodinan in DCM (744 μl, 0.3 mmol, 1.5 eq.). The mixture was then added to a solution of intermediate 22 (115 mg, 0.2 mmol, 1.0 eq.) in DCM (5 ml). The mixture was stirred for 15 minutes at room temperature. Volatiles were removed in vacuo. The crude material was extracted with EtOAc and washed with sat. NaHCO ₃ solution (2×10 ml). The crude material was purified by flash chromatography (ISCO Rf) using hexane/hexane:ethanol (8:2) as solvents, 0% to 30% in hexane:ethanol (8:2), the product eluted at 20%. 31.2 mg (0.2 mmol, 27%) was obtained.

LC-MS: t _R =2.60 min; m/z=480

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ 8.74 (dd, J =25.5, 5.1 Hz, 1H), 8.33 (dd, J =34.8, 7.4 Hz , 1H), 8.19 (dd, J =14.2, 8.4 Hz, 1H), 7.88-7.82 (m, 2H), 7.24-7.14 (m, 5H), 7.03-6.95(m, 2H), 5.17(m, 1H), 4.52(m, 1H), 3.81(s, 3H), 3.12(m, 1H), 2 .91-2.74(m, 1H), 2.74-2.65(m, 1H), 1.60(m, 1H), 1.48(m, 1H), 1.30(m, 1H ), 0.92-0.78 (m, 6H), 0.68-0.61 (m, 2H), 0.58 (m, 2H).

Comparative Example 6: N-((S)-1-(((S)-4-(cyclopropylamino)-3,4-dioxo-1-phenylbutan-2-yl)amino)-4-methyl-1-oxopentan- 2-yl)-2-methoxybenzamide

5 μl of water was added to a 15% solution of Dess-Martin periodinan in DCM (1.6 ml, 0.7 mmol, 1.5 eq.). The mixture was then added to a solution of intermediate 23 (250 mg, 0.5 mmol, 1.0 eq.) in DCM (10 ml). The mixture was stirred for 15 minutes at room temperature. Volatiles were removed in vacuo and the crude mixture was extracted with EtOAc and washed with sat. NaHCO ₃ solution (2×10 ml). The crude product was purified by flash chromatography (ISCO Rf) using hexane/hexane:ethanol (8:2) as solvents, 0% to 30% in hexane:ethanol (8:2), the product eluted at 20%. 89.2 mg of product (0.2 mmol, 35%) was obtained.

LC-MS: t _R =2.68 min; m/z=480

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ 8.78 (dd, J =19.7, 5.1 Hz, 1H), 8.51 (dd, J =32.3, 7.5 Hz , 1H), 8.26 (dd, J = 8.2, 6.1 Hz, 1H), 7.79 (m, 1H), 7.49 (m, 1H), 7.25-7.21 ( m, 4H), 7.20-7.13 (m, 2H), 7.05 (m, 1H), 5.24-5.17 (m, 1H), 4.63-4.55 (m, 1H), 3.86 (s, 3H), 3.15 (m, 1H), 2.85-2.75 (m, 1H), 2.75-2.70 (m, 1H), 1.66 -1.46(m, 2H), 1.36(m, 1H), 0.91-0.77(m, 6H), 0.67-0.62(m, 2H), 0.61-0 .55 (m, 2H).

biological data

Assay to determine the ability to inhibit calpain-1

Calpain-1 activity was measured using the Calpain-Glo Protease kit from Promega. The analysis was performed in 384-well black plates with a transparent bottom. 5 µl of the compound in 10 mM HEPES and 1 mM aqueous EDTA were incubated with 15 µl of a 16 ng/ml calpain-1 solution (Sigma Aldrich) for 2 minutes, and then 20 µl of the Calpain-GloT™ Reagent provided by in a set. The luminescence induced by calpain-1 induced substrate lysis was measured on a Hamamatsu FDSS 7000 reader and the data was calculated from the maximum luminescence peak. Calpain inhibition was calculated by the formula:

% inhibition = (LT-LB)*100/(LC-LB);

where LT is the luminescence observed for the test compound, LB is the luminescence obtained from an empty well without calpain-1, and LC is the luminescence observed in control wells containing calpain 1 in the absence of inhibitor.

All compounds were tested using at least 6 concentrations to allow calculation of IC50 (the concentration that inhibits calpain-1 activity by 50%).

In each case, the analysis included calpastatin and ALLN as control inhibitors.

Compound Calpain-1 inhibitory activity, IC ₅₀ (nM) Example 1 0.03 Example 2 2.2 Example 3 0.008 Example 4 7.1 Comparative Example 5 255 Comparative Example 6 110

Claims (34)

1. Compound of formula (II):

(II) where R _1a is selected from the group consisting of C ₁ -C ₆ alkyl and C ₃ -C ₆ cycloalkyl, R _2a is selected from the group consisting of H and C ₁ -C ₆ alkyl, R _3a is C ₃ -C ₆ cycloalkyl or methoxy, with the proviso that when R _3a is methoxy, R _1a is cyclopropyl, and R _2a is H, or a pharmaceutically acceptable salt or stereoisomer thereof. 2. The compound of formula (II) according to claim 1, where R _1a is selected from the group consisting of C ₁ -C ₃ alkyl and C ₃ -C ₅ cycloalkyl, R _2a is selected from the group consisting of H and C ₁ -C ₃ alkyl, R _3a is C ₃ -C ₅ cycloalkyl or methoxy, with the proviso that when R _3a is methoxy, R _1a is cyclopropyl, and R _2a is H. 3. A compound of formula (II) according to any one of claims 1 or 2, wherein R _1a is selected from the group consisting of methyl, isopropyl and cyclopropyl. 4. A compound of formula (II) according to any one of claims 1 or 2, wherein R _2a is selected from the group consisting of H and methyl. 5. A compound of formula (II) according to any one of claims 1 or 2, wherein R _3a is cyclopropyl. 6. A compound of formula (II) according to any one of claims 1 or 2, which is selected from the group consisting of:

,

,

and

, or a stereoisomer or pharmaceutically acceptable salt thereof. 7. A compound of formula (II) according to any one of claims 1 or 2, which is selected from the group consisting of:

,

,

and

, or a stereoisomer or pharmaceutically acceptable salt thereof. 8. A compound of formula (II) according to any one of claims 1 or 2, which is selected from the group consisting of:

,

,

and

, or their pharmaceutically acceptable salt. 9. Pharmaceutical composition for the treatment and/or prevention of diseases or conditions susceptible to treatment by inhibition of calpain 1, containing a compound of formula (II) according to any one of claims 1 to 8, and a pharmaceutically acceptable excipient. 10. A method of treating and/or preventing a disease or condition susceptible to treatment by inhibition of calpain 1; wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a compound of formula (II) according to any one of claims 1-8. 11. The method of claim 10 wherein the disease or condition is heart injury caused by infarction, ischemia with or without reperfusion. 12. The method of claim 11 wherein the damage to the heart is remodeling after myocardial infarction. 13. The method of claim 10 wherein the disease or condition is selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, acute autoimmune encephalitis, and Creutzfeldt-Jakob disease. 14. The method of claim 10 wherein the disease or condition is selected from the group consisting of breast cancer, colorectal cancer, and leukemia. 15. The method of claim 10 wherein the disease or condition is selected from the group consisting of cardiac fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, and retroperitoneal fibrosis. 16. The method according to any one of claims 10-15 for the treatment and/or prevention of a disease or condition susceptible to treatment with inhibition of calpain 1; wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 9.