Compounds and their uses for the treatment of diseases associated with reduced β-galactosidase activity

Abstract

The present invention relates to novel cathepsin B inhibitors that are effective in therapy, in particular in the treatment of diseases associated with a decrease in the activity of β-galactosidase, such as GM-1 gangliosidosis, Morquio syndrome type B, Chediak-Higashi syndrome, galactosialidosis, metachromatic leukodystrophy, Gaucher disease, Alzheimer's disease and traumatic brain injury. The compounds of the present invention are characterized by advantageous properties such as a) good absorption, allowing the compounds to reach the systemic bloodstream after administration to a patient, b) good distribution to the different tissues / organs to be treated, c) proper metabolism, allowing the compounds to be metabolized in the body in a manner that maintains the activity of the compounds and does not generate harmful metabolites, and / or d) proper excretion of the compounds and their metabolites.

Description

[Technical field]

[0001] The present invention relates to novel cathepsin B inhibitors that are effective in therapy, in particular in the treatment of diseases associated with decreased activity of β-galactosidase, such as GM-1 gangliosidosis, Morquio syndrome type B, Chediak-Higashi syndrome, galactosialidosis, metachromatic leukodystrophy, Gaucher disease, Alzheimer's disease and traumatic brain injury. [Background technology]

[0002] Human β-galactosidase (EC 3.2.1.23, also called lactase) is a lysosomal enzyme classified as a member of the glycoside hydrolase family and is present in both animals and plants, as well as in many microorganisms. Its function is to catalyze the hydrolysis of terminal β-D-galactose residues from a variety of substrates, including lactose, oligosaccharides, glycolipids, and glycoproteins. This enzyme is particularly known for its ability to hydrolyze lactose to glucose and galactose.

[0003] Diseases associated with decreased β-galactosidase activity may include two lysosomal storage disorders: GM1-gangliosidosis and mucopolysaccharidosis (MPS) type IVB (also known as Morquio syndrome type B). More specifically, β-gal is known to degrade the glycosphingolipid GM1-ganglioside and the mucopolysaccharide keratan sulfate. Pathogenic mutations in the gene encoding this enzyme may result in: (i) Strong deformation of the catalytic site results in an inactive enzyme. (ii) Small disturbances in the structure within or near the catalytic site result in a decrease in enzyme activity. (iii) Conformational changes that prevent proper folding of the polypeptide, resulting in premature degradation of the enzyme.

[0004] Both of these reduce the amount of functional β-gal in cells and therefore lead to accumulation of the substrate in vivo. Storage of GM1 leads to GM1-gangliosidosis, whereas accumulation of keratan sulfate is associated with Morquio syndrome type B.

[0005] In the specific case of GM1-gangliosidosis, the degradation of GM1 is inhibited due to the reduced activity of β-gal. This leads to the accumulation of the substrate in lysosomes, which undergoes the destruction of its functionality. As the highest concentration of GM1 is present in the CNS, excessive accumulation of GM1 leads to the death of nerve cells, which in turn leads to the progression of neurodegeneration. Due to the devastating effects of GM1-gangliosidosis on the central nervous system, it is essential to find a therapeutic approach that can cross the blood-brain barrier. Since β-gal is used to hydrolyze β-galactosyl residues, its deficiency causes the storage of other substrates such as glycolipid GA1, oligosaccharides derived from glycoproteins, and glycosaminoglycans. Depending on the organ in which these substrates are produced, β-gal deficiency also causes several non-neurological symptoms such as facial dysplasia, impaired bone development (known as osteogenesis imperfecta), or enlargement of both the spleen and liver (a condition called hepatosplenomegaly, which presents with abdominal swelling).

[0006] Several therapeutic strategies have been implemented to treat GM1-gangliosidosis. However, conventional therapies fail to achieve treatment of symptoms in the central nervous system and therefore are ineffective in terms of reducing neurodegeneration. Hematopoietic stem cell therapy (HSCT) has been attempted unsuccessfully in patients with the infantile form (Non-Patent Document 1). Recombinant enzymes have also been used, but they are unable to cross the blood-brain barrier. Substrate suppression therapy (SRT), consisting of the administration of small molecules that partially inhibit the biosynthesis of accumulated substrates, has been attempted in three patients with GM1-gangliosidosis types II and III. In particular, the administration of miglustat made it possible to slow down or even reverse the degenerative progression (Non-Patent Document 2). Gene therapy has been confirmed in both mouse (Non-Patent Document 3) and feline (Non-Patent Document 4) models by intraventricular injection of engineered adeno-associated virus / GLB1 vectors. Many chaperones have been developed so far, with miglustat being the first to be tested. Due to insufficient specificity for GM1-gangliosidosis, other iminosugars have been developed, especially N-octyl-4-epi-β-valienamine (NOEV) and 5N,6S-(N'-butyliminomethylidene)-6-thio-1-deoxygalactonojirimycin (6S-NBI-DGJ), which showed promising results in mouse models (Non-Patent Documents 5 and 6). Unfortunately, chaperone therapy is based on residual activity of misfolded β-gal, and as a consequence, such treatment is completely ineffective when the enzyme is totally inactive.

[0007] Therefore, there is a need to further develop therapeutic strategies targeting GM1-gangliosidosis that can reach the central nervous system and are applicable even when β-gal is totally inactive.

[0008] Morquio syndrome type B (also known as mucopolysaccharidosis type IVB, MPS IVB) is caused by several specific mutations in the GLB1 gene, which codes for β-gal, which catalyzes the breakdown of several mucopolysaccharides. As a result of this defect, keratan sulfate accumulates, mainly causing skeletal dysplasia and frequent respiratory infections. Patients show normal development of neurocognitive functions, but in some advanced stages of the disease, spinal cord compression due to skeletal deformations may occur, causing central nervous system disorders. As well as bone problems, MPS IVB is characterized by several eye symptoms such as corneal opacities, retinopathy or glaucoma, and cardiopulmonary diseases.

[0009] Currently, there is no approved treatment for MPS IVB, and in conjunction with the extreme rarity of the condition, there is also a lack of knowledge about the disease and its clinical manifestations, limiting the possibility of well-designed clinical trials, even if some molecules have shown promising results as pharmaceutical chaperones. As for GM1 gangliosidosis, the only available treatment so far is symptomatic. Therefore, there is an urgent need to develop a curative treatment strategy.

[0010] Other diseases such as Chediak-Higashi Syndrome, galactosialidosis, metachromatic leukodystrophy, Gaucher disease, Alzheimer's disease and traumatic brain injury have been shown to be associated with reduced activity of β-galactosidase.

[0011] One of the important systems affected by many such diseases is the central nervous system. This may be due to the fact that the brain is very vulnerable to LSD, especially GM1-gangliosidosis, due to its limited regenerative capacity and the sensitivity of nerve cells. Damage to the central nervous system is also prominent in the case of Alzheimer's disease and traumatic brain injury. This sensitivity of the central nervous system causes a big problem in terms of treatment, since the entry into the brain is highly protected by the blood-brain barrier, so only small molecules can pass through, and it is impossible to deliver enzymes, for example, into the brain via the bloodstream. Therefore, the treatment of diseases that cause neurological symptoms is a major challenge.

[0012] Among the publications related to the activity of β-galactosidase, it has been described that cathepsin B (catB) has a regulatory role in the maturation and degradation of β-galactosidase (Non-Patent Document 7). These authors showed that inhibition of cathepsin B promotes the activity of β-galactosidase. With a view to developing new therapies for diseases accompanied by reduced or absent β-galactosidase activity, it is desirable to identify new inhibitors of cathepsin B. It would further be beneficial to provide such inhibitors that exhibit properties suitable for use as drugs, i.e. inhibitors that exhibit good chemical stability, cell membrane permeability, solubility, metabolic stability, plasma stability and / or transporter affinity.

[0013] One of the first catB inhibitors to be identified was oxirane E64, a natural epoxysuccinyl dipeptide found in Aspergillus japonicus, which irreversibly and nonselectively inhibits cathepsins (Non-Patent Document 8). The chemical structure of E64 is shown in Table 1.

[0014] Since the discovery of E64, many analogues have been developed, the most prominent representatives of this class of compounds are shown in Table 1 below and are disclosed in the following publications: 9, 10, 11, 12, 13, and 14.

[0015] [Table 1]

[0016] Currently, the only molecule that has demonstrated catB inhibitory properties while exhibiting good absorption, distribution, metabolism, and excretion is E64d, also known as loxstatin. This compound is an ester prodrug of E64c and has been the subject of clinical investigations in Japan as a treatment for muscular dystrophy. Although the study was discontinued due to unclear efficacy, this 3-year trial (including pediatric patients) provided extensive pharmacokinetic (PK) and pharmacodynamic (PD) data while also demonstrating a lack of toxicity in humans (Non-Patent Document 15, Non-Patent Document 16, and Non-Patent Document 17).

[0017] Unfortunately, this compound exhibits poor permeability through the blood-brain barrier, significantly reducing its ability to reach therapeutic targets in the central nervous system (CNS), making it less relevant for pathologies associated with the presence of catB in the brain, such as GM-1 gangliosidosis, Alzheimer's disease and traumatic brain injury.

[0018] Thus, there is a need to develop new compounds that can irreversibly inhibit cat B. Moreover, there is a further need to identify such compounds that can penetrate the blood-brain barrier and thereby reach the central nervous system. [Prior art documents] [Non-patent literature]

[0019] [Non-Patent Document 1] N. Brunetti-Pierri and F. Scaglia, GM1 gangliosidosis: Review of clinical, molecular, and therapeutic aspects, Mol. Genet. Metab., 2008, 94(4), 391-396 [Non-Patent Document 2] F.Deodato et al.,The treatment of juvenile / adult GM1-gangliosidosis with Miglustat may reverse disease progression,Metab.Brain Dis.,2017,32(5),1529-1536 [Non-Patent Document 3] RCBaek et al.,AAV-Mediated Gene Delivery in Adult GM1-Gangliosidosis Mice Corrects Lysosomal Storage in CNS and Improves Survival,PLoS One,2010,5(10),e13468 [Non-Patent Document 4] VJMcCurdy et al.,Sustained normalization of neurological disease after intracranial gene therapy in a feline model,Sci.Transl.Med.,2014,6(231),231-279 [Non-Patent Document 5] Y. Suzuki et al., Therapeutic chaperone effect of N-Octyl 4-Epi-β-valienamine on murine GM1-gangliosidosis, Mol. Genet. Metab., 2012, 106(1), 92-98 [Non-Patent Document 6] T.Takai et al.,A Bicyclic 1-Deoxygalactonojirimycin Derivative as a Novel Pharmacological Chaperone for GM1 Gangliosidosis,Mol.Ther.,2013,21(3),526-532 [Non-Patent Document 7] Y.Okamura-Oho et al.,Maturation and degradation of β-galactosidase in the post-Golgi compartment is regulated by cathepsin B and a non-cysteine ​​protease,FEBS Lett.,1997,419(2-3),231-234

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[0020] In a first aspect, the present invention provides a compound of formula I or formula II, [ka] [ka] During the ceremony, R1 is selected from 3-methylbutyl, 2-methoxyethyl, 2-fluorophenylmethyl, 3-(piperidin-1-yl)propyl and 2-(pyridin-2-yl)ethyl; R2 is selected from hydrogen and methyl; R3 is selected from 2-methylpropyl and 2,2,2-trifluoroethyl; R4 is hydrogen; When R1 is 3-methylbutyl, i. R3 is not 2-methylpropyl, or ii. R3 is 2-methylpropyl and R2 is methyl; The compound is further characterized in that when R3 is 2,2,2-trifluoroethyl, R2 is hydrogen.

[0021] In a second aspect, the present invention provides the compounds of the invention for use in therapy, in particular for the treatment of diseases associated with reduced β-galactosidase activity.

[0022] In a third aspect, the present invention provides a compound of formula I or formula II for use in a method for treating a disease associated with reduced β-galactosidase activity, comprising: [ka] [ka] During the ceremony, R1 is selected from 3-methylbutyl, 2-methoxyethyl, 3-methoxypropyl, 2-fluorophenylmethyl, 3-(piperidin-1-yl)propyl and 2-(pyridin-2-yl)ethyl; R2 is selected from hydrogen and methyl; R3 is selected from 2-methylpropyl and 2,2,2-trifluoroethyl; R4 is hydrogen; When R1 is 3-methylbutyl, i. R3 is not 2-methylpropyl, or ii. R3 is 2-methylpropyl and R2 is methyl; Provided is a compound for use, further characterized in that when R3 is 2,2,2-trifluoroethyl, R2 is hydrogen.

[0023] In a fourth aspect, the present invention provides a process for producing a compound according to the present invention, comprising: a) A compound of formula III [ka] with a base such as N,N-diisopropylethylamine (DIPEA); b) reacting the deprotonated compound obtained in step a) with 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluoro-phosphate (HATU) in a basic medium; c) reacting the compound obtained in step b) with tetramethylurea (TMU) in a basic medium; d) reacting the compound obtained in step c) with an amine of formula IV in a basic medium [ka] to produce, for example, a dipeptide of formula V [ka] forming a e) Removal of the Boc group from a compound of formula V by reacting such compound with trifluoroacetic acid in an acidic medium to give a compound of formula VI [ka] and f) deprotonating the compound of formula VI with a base such as DIPEA; g) reacting the deprotonated compound obtained in step f) with HATU in a basic medium; h) reacting the compound obtained in step g) with TMU in a basic medium; i) reacting the compound obtained in step h) with a compound of formula VII [ka] to obtain, for example, a compound of formula I; j) optionally hydrolyzing the ester moiety of the compound of formula I to obtain, for example, a compound of formula II; The present invention provides a process including: [Brief description of the drawings]

[0024] [Figure 1]Graph showing inhibition of cathepsin B (catB) activity in pig liver homogenates for VRO006-hydrol, VRO052-hydrol, VRO059_hydrol, VRO042_hydrol, VRO047_hydrol, VRO073_hydrol, VRO109_hydrol and VRO244_hydrol (compounds with suitable inhibitory activity (IC50≦100 nM)). [Diagram 2] Graph showing inhibition of cathepsin B (catB) activity in pig liver homogenates for VRO001_hydrol, VRO035_hydrol, VRO082_hydrol, VRO119_hydrol, VRO243_hydrol, VRO073_hydrol, VRO109_hydrol and VRO244_hydrol (compounds with unfavourable inhibitory activity (IC50>100nM)). [Diagram 3] FIG. 1 is a graph depicting inhibition of human cathepsin B by E64c, VRO052-hydrol and VRO059-hydrol in a whole cell assay. [Figure 4] FIG. 2 depicts the mechanism of formation of a dipeptide in steps a) to d) of the process of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present inventors have advantageously developed novel compounds that are active as inhibitors of cathepsin B and thus are useful in the treatment of diseases associated with reduced β-galactosidase (β-gal) activity. Moreover, the identified compounds have properties that make them particularly suitable for use as pharmaceuticals. In particular, such compounds are characterized by one or more of the following advantageous properties: a) good absorption, allowing the compound to reach the systemic bloodstream after administration to a patient; b) good distribution to the different tissues / organs to be treated; c) proper metabolism of the compound in the body, so as to maintain the compound's activity and not produce harmful metabolic products; and / or d) Proper excretion of the compound and its metabolites.

[0026] The compound of the present invention is further advantageous in that it is suitable for passing through the blood-brain barrier, making it possible to treat diseases that affect the central nervous system.This represents a great improvement over the prior art compound E64d.Furthermore, the compound of the present invention shows a good selectivity for inhibiting catB over other enzymes of the cathepsin family.

[0027] The compounds of the present invention are compounds of formula I or formula II, [ka] [ka] During the ceremony, R1 is selected from 3-methylbutyl, 2-methoxyethyl, 2-fluorophenylmethyl, 3-(piperidin-1-yl)propyl and 2-(pyridin-2-yl)ethyl; R2 is selected from hydrogen and methyl; R3 is selected from 2-methylpropyl and 2,2,2-trifluoroethyl; R4 is hydrogen; When R1 is 3-methylbutyl, i. R3 is not 2-methylpropyl, or ii. R3 is 2-methylpropyl and R2 is methyl; The compound is further characterized in that when R3 is 2,2,2-trifluoroethyl, R2 is hydrogen.

[0028] The structure of the compounds of the present invention consists of two blocks with a dipeptide structure containing both a nitrogen atom and radicals R1, R2 and R4, said dipeptide structure being linked to an epoxy warhead. As in the case of the prior art E64d, the compounds of formula I of the present invention are prodrugs, which are hydrolyzed in the digestive tract upon enteral administration. The ester moiety at the very end of the epoxy head is cleaved to release the free acid. It is in this cleaved form that the compounds are therapeutically active. Thus, the compounds of formula I are advantageously for use as prodrugs, while the compounds of formula II are advantageously for use as therapeutic agents.

[0029] Examples of compounds according to the invention are provided in Table 2 below:

[0030] [Table 2(1)] [Table 2(2)]

[0031] Examples of compounds of formula II according to the present invention are the corresponding compounds provided in the form of the free acid in Table 2. Such compounds include (2S,3S)-3-(((S)-1-((2-2-methoxyethyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxirane-2-carboxylic acid (VRO047_hydrol), (2S,3S)-3-(((S)-1-(isopentyl(methyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxirane-2-carboxylic acid (VRO047_hydrol), Silane-2-carboxylic acid (VRO006_Hydrol), (2S,3S)-3-(((S)-1-((2-fluorobenzyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxirane-2-carboxylic acid (VRO073_Hydrol), (2S,3S)-3-(((S)-4-methyl-1-oxo-1-((3-(piperidin-1-yl)propyl)amino)amino (2S,3S)-3-(((S)-4-methyl-1-oxo-1-((2-(pyridin-2-yl)ethyl)amino)pentan-2-yl)carbamoyl)oxirane-2-carboxylic acid (VRO059_Hydrol), (2S,3S)-3-(((S)-4,4,4-trifluorophenyl)-1-oxo-1-((2-(pyridin-2-yl)ethyl)amino)pentan-2-yl)carbamoyl)oxirane-2-carboxylic acid (VRO052 ... (2S,3S)-3-(((S)-4,4,4-trifluoro-1-((2-fluorobenzyl)amino)-1-oxobutan-2-yl)carbamoyl)oxirane-2-carboxylic acid (VRO244_hydrol).

[0032] The compounds of the invention are advantageously for use in therapy.

[0033] The compounds of the present invention are also advantageous for use in the treatment of diseases associated with reduced β-galactosidase activity. In addition to the compounds according to the present invention listed above, the compounds of formula I or formula II, in which R1 is 3-methoxypropyl and R2, R3 and R4 are as described above, are also for use in the treatment of diseases associated with reduced β-galactosidase activity. Preferably, such a compound of formula I for use in the treatment of diseases associated with reduced β-galactosidase activity is ethyl (2S,3S)-3-(((S)-1-((3-3-methoxypropyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxirane-2-carboxylate (VRO042), having the structure provided below, and such a compound of formula II for use in the treatment of diseases associated with reduced β-galactosidase activity is the free acid counterpart (2S,3S)-3-(((S)-1-((3-3-methoxypropyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxirane-2-carboxylic acid (VRO042_hydrol).

[0034] [ka]

[0035] Various properties of the compounds of formula II make them more suitable to cross the blood-brain barrier than prior art catB inhibitors such as E64b and its known derivatives. In particular, the compounds of the present invention advantageously have the following characteristics: a) a higher lipophilicity, expressed for example as cLogP, which results in better passive penetration of the blood-brain barrier; b) a reduced ability to form hydrogen bonds with other compounds, as a result of a reduction in the number of hydrogen bond donor atoms and / or hydrogen bond acceptor atoms and / or as a result of the formation of intramolecular hydrogen bonds, thereby resulting in improved permeability and reduced affinity for plasma glycoproteins; c) A more peptidomimetic structure resulting in improved metabolic stability and / or enhanced affinity for catB due to better occupancy of the S2 pocket of catB.

[0036] As demonstrated by the present inventors, the above specific radicals R1, R2 and R3 are advantageous in that they improve the drug-likeness and blood-brain barrier crossing ability of the compound of formula II compared to E64c while being active as an inhibitor of catB.It has also been shown that it is essential that R4 is a hydrogen atom.Without wishing to be bound by theory, it is believed that this hydrogen atom is involved in hydrogen bonding with proteins.

[0037] Unlike the hydrogen at R4, the hydrogen at R2 can be replaced with methyl without impairing the inhibitory activity of the compound, as demonstrated in Figure 1, and it can be seen that the ability of VR006_hydrol, which has a methyl at R2, to inhibit isolated catB is similar to that of E64c. This modification of the structure of the prior art makes VRO006_hydrol more lipophilic than E64c (cLogP is about 2) due to the replacement of a hydrogen atom with methyl, and removes one hydrogen donor in the molecule, increasing the topological polar surface area (TPSA) of the molecule by 90 Å. 2 These differences make VRO006_Hydrol more drug-like in nature, better able to enter cells through cell membranes, and better able to cross the blood-brain barrier than E64c.

[0038] The inventors have also identified that bulkier radicals can be advantageously used at the R1 position as enumerated herein (see FIG. 1) without compromising the interaction between the compound and the S3 pocket of catB. This results in improved drug similarity and improved ability to cross the blood-brain barrier over the prior art E64c while maintaining efficient inhibition of catB. This was the case when R1 was 2-methoxyethyl (VRO047_Hydrol) and when R1 was 3-methoxypropyl (VRO042_Hydrol). The same was observed when R1 was 2-(pyridin-2-yl)ethyl (VRO052_Hydrol) or 3-(piperidin-1-yl)propyl (VRO059_Hydrol), since both of these compounds showed almost the same inhibitory activity as E64c against isolated catB. Even the bulky radical 2-(2-fluorophenyl)ethyl (VRO073_hydrol) at R1 position was able to maintain inhibitory activity against isolated catB, and this compound only slightly reduced catB inhibitory activity compared to E64c. This minimal reduction in enzyme inhibition is compensated for by the advantages of drug similarity and the ability to reach the cell interior through the cell membrane and cross the blood-brain barrier.

[0039] However, not all bulky substituents are suitable to maintain sufficient inhibition of catB; for example, we demonstrated that the bulky and highly electronegative 3,3,3-trifluoroethyl significantly reduced the efficiency of VRO035_hydrol in an enzymatic assay against isolated catB (see FIG. 2).

[0040] Particularly preferred radicals at the R1 position are 2-(pyridin-2-yl)ethyl and 3-(pyridin-1-yl)propyl. Indeed, compounds VRO052_hydrol and VRO059_hydrol, which have these radicals at the R1 position, have shown significantly improved inhibitory activity against catB in whole cell assays. Without wishing to be bound by theory, this is believed to be due to the increased lipophilicity of these compounds, which allows them to cross the cell membrane more easily. Therefore, the in vivo efficiency of compounds with these radicals at the R1 position is expected to be higher than that of E64c.

[0041] With regard to R3, which interacts with the S2 pocket of catB, the inventors also achieved improving the drug-likeness of the compound by replacing the 3-methylbutyl present in E64d with various radicals. Most bulky radicals have a negative effect on the inhibitory activity of the compound, such as the pyridinyl moiety (VRO119_hydrol), as demonstrated by FIG. 2. However, the replacement of 3-methylbutyl at R3 with 2,2,2-trifluoroethyl unexpectedly resulted in a significant increase in the inhibitory activity of VRO109_hydrol against catB, as shown in FIG. 1. This trifluoroethyl moiety is further advantageous in that it does not contain oxygen or nitrogen atoms, which have the undesirable property of forming hydrogen bonds with other molecules, and also in that it significantly increases the lipophilicity and drug-likeness of VRO109_hydrol compared to E64c.

[0042] When a suitable radical at R1 position is combined with a suitable radical at R3 position, the compound still exhibits suitable inhibitory activity as shown in Figure 2, and although the compound VRO244_hydrol exhibits only a slightly lower inhibitory activity than the prior art E64c, it is characterized by increased lipophilicity and an increased ability to enter cells and cross the blood-brain barrier, which makes VRO244_hydrol more suitable as a drug than E64c.

[0043] However, it is important not to combine the bulky 2,2,2-trifluoroethyl at R3 with a methyl at R2, since this particular combination robs VRO243_hydrol of inhibitory activity against the isolated enzyme (see Figure 2).

[0044] The advantages of the compounds of formula II identified above make them advantageous prodrugs of the corresponding compounds of formula I.

[0045] Due to their ability to inhibit catB, the compounds of formula I and formula II as described above can be advantageously used in the treatment of diseases associated with reduced β-galactosidase activity, since the regulatory role of catB in the maturation and degradation of β-galactosidase has previously been demonstrated, as explained in detail in the Background section.

[0046] Examples of diseases associated with decreased β-galactosidase activity include GM-1 gangliosidosis, Morquio syndrome type B, Chédiak-Higashi syndrome, galactosialidosis, metachromatic leukodystrophy, Gaucher disease, Alzheimer's disease and traumatic brain injury.Preferably, the compounds of the present invention are for use in the treatment of a disease selected from GM-1 gangliosidosis, Morquio syndrome type B, Chédiak-Higashi syndrome, galactosialidosis, metachromatic leukodystrophy and Gaucher disease, more preferably GM-1 gangliosidosis and Morquio syndrome type B, most preferably GM-1 gangliosidosis.

[0047] In other words, the present invention relates to the use of the compound of the present invention for the manufacture of medicine.Preferably, the present invention relates to the use of the compound of formula I or formula II as above for the manufacture of medicine for the treatment of diseases related to the decrease of β-galactosidase activity, preferably selected from GM-1 gangliosidosis, Morquio syndrome type B, Chédiak-Higashi syndrome, galactosialidosis, metachromatic leukodystrophy, Gaucher disease, Alzheimer's disease and traumatic brain injury, more preferably selected from GM-1 gangliosidosis, Morquio syndrome type B, Chédiak-Higashi syndrome, galactosialidosis, metachromatic leukodystrophy and Gaucher disease, even more preferably selected from GM-1 gangliosidosis and Morquio syndrome type B, most preferably selected from GM-1 gangliosidosis.

[0048] In other words, the present invention relates to a method for treating a disease in a patient in need of such treatment, comprising administering to such a patient a compound of the present invention. The present invention also relates to a method for treating a disease associated with decreased β-galactosidase activity in a patient in need of such treatment, comprising administering to such a patient a compound of formula I or formula II as described above. More preferably, such a disease is selected from GM-1 gangliosidosis, Morquio syndrome type B, Chédiak-Higashi syndrome, galactosialidosis, metachromatic leukodystrophy, Gaucher disease, Alzheimer's disease and traumatic brain injury, more preferably, such a disease is selected from GM-1 gangliosidosis, Morquio syndrome type B, Chédiak-Higashi syndrome, galactosialidosis, metachromatic leukodystrophy and Gaucher disease, even more preferably, such a disease is selected from GM-1 gangliosidosis and Morquio syndrome type B, most preferably, GM-1 gangliosidosis.

[0049] In certain embodiments, the compounds of formula I or formula II are for use in a method of inhibiting cat B. In other words, the present invention relates to the use of compounds of formula I or formula II for inhibiting cat B. In yet other words, the present invention relates to a method of inhibiting cat B in a subject, comprising administering such a compound to the subject.

[0050] In another particular embodiment, the compound of formula I or formula II is administered to a patient in need thereof in combination with at least one additional compound active for treating one of the diseases associated with the decrease in β-galactosidase as mentioned above. In a preferred embodiment, such additional compound is selected from pharmacological chaperones of β-galactosidase, pharmacological chaperones of glucocerebrosidase, calcium channel blockers and / or glucosylceramide synthase inhibitors, and mixtures thereof. In another preferred embodiment, such additional compound is selected from the group consisting of N-substituted 5-amino-1-hydroxymethyl-cyclopentanetriol, N-octyl-4-epi-β-valienamine (NOEV), ambroxol, cis-(+)-[2-(2-dimethylaminoethyl)-5-(4-methoxyphenyl)-3-oxo-6-thia-2-azabicyclo[5.4.0]undeca-7,9,11-trien-4-yl]ethanoate (diltiazem), [(3S)-1-azabicyclo[2.2.2]octan-3-yl]N-[2-[2-(4-fluorophenyl)-1,3-thiazol-4-yl]propan-2-yl]carbamate (benglustat), imino sugars, and mixtures thereof. More preferably, such imino sugars are preferably selected from the group consisting of N-butyl-deoxygalactonojirimycin (migalastat), 4-epi-isofagomine, 5a-C-pentyl 4-epi-isofagomine, 5a-C-methyl 4-epi-isofagomine, 1,5-dideoxy-1,5-imino-(L)-ribitol (DIR), 5-C-alkyl-imino-L-ribitol, N-(dansylamino)hexylaminocarbohydrate ... is selected from the group consisting of 5N,6S-(N'-butyliminomethylidene)-6-thio-1-deoxygalactonojirimycin (6S-NBI-DGJ), N-nonyl-deoxygalactonojirimycin, N-butyldeoxynojirimycin (miglustat), isofagomine (afegortostat), AZ-3102, and mixtures thereof.

[0051] The compound of formula I of the present invention is preferably administered by enteral administration.Enteral administration refers to any administration means that the compound of formula I is delivered to the digestive tract.Enteral administration of the compound of formula I is advantageous because such compound is hydrolyzed to free acid in the digestive tract.The compound of formula II of the present invention can be administered by any route, and is preferably administered by an administration route that is not enteral administration.

[0052] In a preferred embodiment, the compound of formula I or formula II is administered in the form of a pharmaceutical composition, preferably comprising a pharma- ceutically acceptable carrier, diluent and / or excipient.

[0053] The term "carrier" refers to an organic or inorganic component, of natural or synthetic nature, with which the active ingredient is formulated to facilitate, enhance or enable application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a patient.

[0054] Possible carrier materials for parenteral administration are, for example, sterile water, Ringer's, lactated Ringer's, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes, and especially biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxy-propylene copolymers.

[0055] As used herein, the term "excipient" is intended to indicate any substance that may be present in the compositions described herein and that is not an active ingredient, such as, for example, a carrier, binder, lubricant, thickener, surface active agent, preservative, emulsifier, buffer, flavoring agent, or coloring agent.

[0056] The excipient of the composition can be any pharma- ceutically acceptable excipient, including specific carriers that can target specific cells or tissues. As mentioned above, possible pharmaceutical compositions include those suitable for oral, rectal, topical, transdermal, buccal, sublingual, or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration. Conventional excipients can be used in these formulations according to techniques well known to those skilled in the art. Compositions for parenteral administration are generally physiologically compatible sterile solutions or suspensions, which can be prepared immediately before use from solid or lyophilized forms, if desired. For oral administration, the compositions can be formulated into conventional oral dosage forms, such as tablets, capsules, powders, granules, and liquid preparations, such as syrups, elixirs, and concentrated drops. Non-toxic solid carriers or diluents can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonates, and the like. Compressed tablets also require a binder, which is an agent that imparts cohesiveness to the powder material. For example, starch, gelatin, sugars such as lactose or glucose, and natural or synthetic gums can be used as binders. Disintegrants are also required in tablets to facilitate the disintegration of the tablet. Disintegrants include starch, clay, cellulose, algins, gums, and cross-linked polymers. In addition, tablets also include lubricants and glidants to prevent adhesion of the tablet material to the surface during the manufacturing process and improve the flow properties of the powder material during manufacture. Colloidal silicon dioxide is most commonly used as a glidant, and compounds such as talc or stearic acid are most commonly used as lubricants. For transdermal administration, the composition can be formulated in the form of an ointment, cream, or gel, and suitable penetrants or detergents, such as dimethylsulfoxide, dimethylacetamide, and dimethylformamide, can be used to facilitate penetration. For transmucosal administration, nasal drops, rectal suppositories, or vaginal suppositories can be used. The active compound can be incorporated into any of the known suppository bases by methods known in the art.Examples of such bases include cocoa butter, polyethylene glycol (carbowax), polyethylene sorbitan monostearate, and mixtures thereof with other suitable materials to modify the melting point or dissolution rate.

[0057] The pharmaceutical composition may be formulated to substantially release the active drug immediately upon administration or at any predetermined time or period after administration.

[0058] The compound of formula I or formula II is administered in an effective amount. The term "effective amount" refers to the amount necessary to obtain a physiological effect. The physiological effect may be achieved by a single applied dose or may be achieved by repeated applications. The dosage administered may of course vary depending on known factors such as the physiological characteristics of the particular composition; the age, health and weight of the subject; the nature and extent of the symptoms; the type of concurrent treatment; the frequency of treatment; and the desired effect, and may be adjusted by one skilled in the art. In a particular embodiment, the pharmaceutical composition comprises the compound of formula I or formula II in an amount of 0.1 mg to 5 g, preferably in an amount of 1 mg to 2 g, more preferably in an amount of 10 mg to 1 g. In another particular embodiment, the compound of formula I or formula II is administered in an amount of 0.1 mg to 5 g, preferably in an amount of 1 mg to 2 g, more preferably in an amount of 10 mg to 1 g per day.

[0059] The compounds of the present invention can be produced by a process that involves forming an amide bond between two amino acids to produce a dipeptide structure and coupling the formed dipeptide with an epoxy headgroup.

[0060] The present invention therefore relates to a process for producing the compounds of the present invention, the process comprising: a) A compound of formula II [ka] with a base such as N,N-diisopropylethylamine (DIPEA); b) reacting the deprotonated compound obtained in step a) with 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluoro-phosphate (HATU) in a basic medium; c) reacting the compound obtained in step b) with tetramethylurea (TMU) in a basic medium; d) reacting the compound obtained in step c) with an amine of formula III in a basic medium [ka] to produce, for example, a dipeptide of formula IV [ka] forming a e) Removal of the Boc group from a compound of formula IV by reacting such a compound with trifluoroacetic acid in an acidic medium to give a compound of formula V [ka] and f) deprotonating the compound of formula V with a base such as DIPEA; g) reacting the deprotonated compound obtained in step f) with HATU in a basic medium; h) reacting the compound obtained in step g) with TMU in a basic medium; i) reacting the compound obtained in step h) with a compound of formula VI [ka] and reacting with Includes.

[0061] Steps a) to d) are sub-steps of the first stage of the synthesis of the compounds of the invention, in which the dipeptide portion of the structure is formed. This involves the formation of an amide bond between an acid and a base, commonly referred to as peptide coupling. Many coupling reagents can be used and are well known to those skilled in the art. However, HATU is particularly preferred, since it is relatively tolerant to different types of coupling starting materials. Preferably, such steps are carried out in a basic medium, more preferably at 0° C. The mechanism of amide bond formation mediated by HATU is shown in FIG. 4. It starts with the deprotonation of the acid by a base, preferably DIPEA. The formed carboxylate then attacks the electron-deficient carbon from HATU, thereby forming an unstable O-acyl(tetramethyl)isouronium salt and releasing 7-azabenzotriazole (OAt). The latter immediately attacks the isouronium salt to form tetramethylurea (TMU) and an OAt activated ester. It is this active species that undergoes nucleophilic addition by the amine of formula III. The definitive amide bond is then formed to give the compound of formula IV. Without wishing to be bound by theory, the mechanism is likely via the formation of a seven-membered cyclic transition state that may stabilize the amine during amide bond formation.

[0062] In step e), the Boc protecting group is removed in an acidic medium, preferably containing 25% TFA in DCM. The mechanism starts with the protonation of t-butyl carbamate. This results in the loss of t-butyl cation and the formation of carbamic acid. The carboxylate from TFA then deprotonates the carbamic acid, thereby releasing the free amine and forming one equivalent of carbon dioxide. During the workup, a first acidic extraction can remove some organic impurities, but the amine remains in the aqueous layer as a TFA salt. The free amine can be recovered in the organic layer by basification.

[0063] In steps f) to i), the epoxy head is attached to the deprotected dipeptide structure, in a manner similar to the formation of the dipeptide structure, since it also involves amide bond formation. Compounds of formula I are obtained. Again, various coupling agents well known to those skilled in the art can be used. Such coupling agents include N,N'-dicyclohexylcarbodiimide (DCC) or HATU. However, DCC has been described as causing allergies, so it is preferable to use HATU. HATU has shown the same ability as DCC and even better yields than DCC.

[0064] If it is desired to form a compound of formula II, this process includes one additional step of hydrolyzing the ester moiety of the compound of formula I to release the free acid of formula II.The conditions suitable for such hydrolysis are well known to those skilled in the art.For example, the compound of formula I can be obtained in a basic medium. EXAMPLES

[0065] <Analysis method> 1 H and 13 C NMR and 2D-NMR (COSY, HSQC and HMBC) were obtained on a Bruker Ascend 300 MHz spectrometer. All measurements were performed at room temperature. Chemical shifts (δ, expressed in ppm) were referenced to the solvent peak. All syntheses were followed by UPLC-MS using an ACQUITY UPLC system from Waters coupled to an SQD2 with a UV detector. The column was a UPLC BEH C18 (50×2.1 mm, 1.7 μm), detection was at 214 nm, analysis was performed at 40° C. with a flow rate of 0.5 mL / min, and elution was performed with 0.06% formic acid in water as eluent A and 0.06% formic acid in acetonitrile as eluent B. The gradient was from 5% to 100% B in 5 min, held at 100% B for 2 min, then re-equilibrated to 5% for 30 s.

[0066] All TLC analyses were performed on Merck Silica Gel F254 plates and revealed under both 254 nm and 366 nm, followed by Hanessian staining.

[0067] Example 1: Synthesis of VRO006 First, the dipeptide was formed. In a 25 mL round-bottom flask equipped with a magnetic stirrer, 500 mg of Boc-L-Leu (2.162 mmol, 1.0 equiv.) and 10 mL of DCM were introduced, the resulting colorless solution was stirred at room temperature for 5 min, then cooled to 0-5 °C using an ice bath. 1.051 g of HATU (3.243 mmol, 1.5 equiv.) were added at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Then 312 mg of N,3-dimethylbutan-1-amine hydrochloride (2.270 mmol, 1.05 equiv.) were introduced at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Finally, 1.1 mL of DIPEA (6.485 mmol, 3.0 equiv.) was added dropwise at 0-5 °C. The cloudy slightly yellow mixture was stirred at 0-5 °C for 5 min, then the ice bath was removed and the mixture was stirred at room temperature overnight.

[0068] The reaction mixture, a clear yellowish solution, was transferred to a separatory funnel using 10 mL DCM, washed with 50 mL NaCl 2.6%, then the AP was extracted once with 10 mL DCM. The OP was combined and washed with 50 mL NaCl 2.6%, then the AP was extracted once with 10 mL DCM. The OP was combined and washed once with 50 mL HCl 0.25M, once with 50 mL NaHCO3 10%, and finally with 50 mL NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter, and concentrated to dryness in a rotavapor (40 °C, 850-70 mbar). This material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h to give 766 mg (raw yield: 112.7%) of an orange paste as 362-VRO006-01-001 crude 1#1.

[0069] The crude material was purified by CC using 40 g of silica gel (column with φ=3.2 cm and h=13.5 cm) followed by elution using TLC with heptane-EtOAc (7:3) as eluent; Rf=0.44. The product was eluted with a gradient from pure heptane to heptane-EtOAc (6:4) and the fractions containing the desired product were combined and concentrated to dryness in a rotavapor (40° C., 200-50 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 1 h to give 610 mg (raw yield: 89.7%) of a nearly colorless oil as 362-VRO006-01-001 CC1#1.

[0070] 1 H-NMR(300MHz,CDCl3)δ=5.37-5.11(m,1H,4),4.73-4.54(m,1H,5),3.50-3.23(m,2H,11),3.03(s,2H,10),2.92(s,1H,10),1.55(br.m ,4H,6,7,13),1.42(s,9H,1,2,3),1.40-1.30(m,2H,12),0.98(dd,J=12.7,6.4,6H,8,9,14,15),0.92(dd,J=6.6,2.5,6H,8,9,14,15).

[0071] 13 C NMR(75MHz,CDCl3)δ=48.72(5),48.26(5),46.50(11),43.33(12),42.70(12),37.28( 6),35.73(6),34.83(10),33.61(10),28.29(1,2,3),26.12(7,13),25.92(7,13),24. 56(7,13), 23.46(8,9,14,15), 23.41(8,9,14,15), 22.51(8,9,14,15), 22.47(8,9,14,15), 22.43(8,9,14,15), 22.40(8,9,14,15), 21.75(8,9,14,15), 21.71(8,9,14,15).

[0072] The Boc protecting group was then removed as follows: In a 25 mL round bottom flask equipped with a magnetic stirrer, 601 mg of 362-VRO006-01-001 CC1#1 (1.911 mmol, 1.0 equiv.) and 2.1 mL of a 10% HCl solution in EtOAc were introduced and the resulting colorless solution was stirred at room temperature for 3 h 30 min. The reaction mixture was transferred to a separatory funnel using 25 mL of EtOAc and washed with 25 mL of water. The AP was basified to pH>10 using 6 mL of NaOH 25% and then extracted 3 times with 25 mL of EtOAc. The OP was combined and washed using 25 mL of NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter and concentrated to dryness in a rotavapor (40 °C, 250-70 mbar). This material was dried under high vacuum (room temperature, 10-3 mbar) for 1 h to give 371 mg (neat yield: 90.6%) of a nearly colorless oil as 362-VRO006-02-001 crude 1#1, which was used "as is" in the next step without further purification.

[0073] 1 H-NMR(300MHz,CDCl3)δ=3.88-3.57(m,1H,2),3.33(br.m,2H,8),2.99(s,1H,7),2.93(s,1H,7),2.01(b r.s,J=18.9,2H,1),1.95-1.77(m,1H,3'),1.42(br.m,5H,3'',4,9,10),1.09-0.79(m,12H,5,6,11,12).

[0074] Finally, the dipeptide was coupled to the epoxy head. In a 25 mL round-bottom flask equipped with a magnetic stirrer, 282 mg of 362-BB01-03-001 crude 1#1 (1.759 mmol, equiv.) and 5 mL of DCM were introduced, the resulting white suspension was stirred at room temperature for 5 min, and then cooled to 0-5 °C using an ice bath. 814 mg of HATU (2.512 mmol, 1.5 equiv.) were added at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Then 359 mg of 362-VRO006-02-001 crude 1#1 (1.675 mmol, 1.0 equiv.) in solution in 5 mL of DCM was introduced at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Finally, 875 μL of DIPEA (5.024 mmol, 3.0 equiv.) was added dropwise at 0-5 °C. The resulting yellow mixture was stirred at 0-5° C. for 5 min, then the ice bath was removed and the mixture was stirred at room temperature overnight.

[0075] The reaction mixture, a clear yellow solution, was transferred to a separatory funnel using 10 mL DCM and washed with 50 mL NaCl 2.6%, then the AP was extracted once with 10 mL DCM. The OP was combined and washed once with 50 mL NaCl 2.6%, then the AP was extracted once with 10 mL DCM. The OP was combined and washed once with 50 mL HCl 0.25 M, once with 50 mL NaHCO3 10%, and once with 50 mL NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter, and concentrated to dryness in a rotavapor (40 °C, 220-30 mbar). This material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h to give 701 mg (neat yield: 117.4%) of a pale yellowish oil as 362-VRO006-03-001 crude 1#1.

[0076] The crude material was purified by CC using 40 g of silica gel (column with φ=3.2 cm and h=13.5 cm) followed by elution using TLC with heptane-EtOAc (1:1) as eluent; f=0.53. The product was eluted with a gradient from heptane-EtOAc (9:1) to heptane-EtOAc (1:1) and the fractions containing the desired product were combined and concentrated to dryness in a Rotavapor (40 °C, 200-50 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h 30 min to give 334 mg (neat yield: 55.9%) of a colorless oil as 362-VRO006-03-001 CC1#1.

[0077] 1 H-NMR(300MHz,CDCl3)δ=6.84(dd,J=16.2,8.8,1H,5),5.02-4.87(m,1H,6),4.37-4.17(m,2H,2),3.65(dd,J=1.8,0.8,1H,4),3.46(dd,J=1.8,0.9 ,1H,3),3.34(br.m,2H,12),3.04(s,2H,11),2.92(s,1H,11),1.48(br.m ,6H,7,8,13,14),1.31(t,J=7.1,3H,1),1.05-0.86(m,12H,9,10,15,16).

[0078] 13 C-NMR(75MHz,CDCl3)δ=62.20(2),53.88(4),52.81(3),48.28(12),47.06(6),46.72(6),46.64(12) ,42.81(7),42.32(7),37.27(13),35.67(13),34.86(11),33.70(11),26.07(8,14),25.94(8,14),24 .72(9,10,15,16), 23.44(9,10,15,16), 23.41(9,10,15,16), 22.50(9,10,15,16), 22.45(9,10,15,16), 22.38(9,10,15,16), 22.36(9,10,15,16), 21.69(9,10,15,16), 21.60(9,10,15,16), 14.00(1).

[0079] Example 2: Synthesis of VRO047 First, the dipeptide was formed. In a 50 mL round-bottom flask equipped with a magnetic stirrer, 3.232 g of Boc-L-Leu (13.975 mmol, 1.05 equiv.) and 35 mL of DCM were introduced, the resulting cloudy solution was stirred at room temperature for 5 min, and then cooled to 0-5 °C using an ice bath. 6.471 g of HATU (19.946 mmol, 1.5 equiv.) were added at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Then 1157 μL of 2-2-methoxyethylamine (13.309 mmol, 1.0 equiv.) were introduced at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Finally, 4.6 mL of DIPEA (26.618 mmol, 2.0 equiv.) was added dropwise at 0-5 °C. The resulting cloudy yellowish solution was stirred at 0-5 °C for 5 min, then the ice bath was removed and the mixture was stirred at room temperature overnight.

[0080] The reaction mixture, a clear yellow solution, was transferred to a separatory funnel using 10 mL DCM and washed with 50 mL NaCl 2.6%, then the AP was extracted once with 10 mL DCM. The OP was combined and washed once with 50 mL HCl 0.5M, once with 50 mL NaHCO3 10%, and finally with 50 mL NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter, and concentrated to dryness in a rotavapor (40 °C, 850-70 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h 40 min to give 6.986 g (raw yield: 182.0%) of a pale yellow oil as 362-VRO047-01-001 crude 1#1.

[0081] The crude material was purified by CC using 35 g of silica gel (column with φ=3.2 cm and h=11.8 cm) followed by elution using TLC with heptane-EtOAc (1:1) as eluent; Rf=0.46. The product was eluted with a gradient from pure heptane to heptane-EtOAc (1:1) and the fractions containing the desired product were combined and concentrated to dryness in a rotavapor (40° C., 220-50 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 1 h to give 1.267 g (raw yield: 33.0) of a white sticky foam as 362-VRO047-01-001 CC1#1.

[0082] 1 H-NMR(300MHz,CDCl3)δ=6.37(br.s,1H,10),4.88(br.s,1H,4),4.24-3.89(m,1H,5),3.50-3.38(m,4H,11,12),3. 35(s,3H,13),1.76-1.56(m,2H,6'',7),1.56-1.35(m,1H,6'),1.44(s,9H,1,2,3),0.93(dd,J=6.2,1.1,6H,8,9).

[0083] The Boc protecting group was then removed as follows: In a 25 mL round bottom flask equipped with a magnetic stirrer, 1.267 g of 362-VRO047-01-001 CC1#1 (4.393 mmol, equiv.) and 4.4 mL of a 10% HCl solution in EtOAc were introduced and the resulting colorless solution was stirred for 3 h at room temperature. The reaction mixture was transferred to a separatory funnel using 25 mL of EtOAc and washed with 25 mL of water. The AP was basified to pH>10 using 4.5 mL of NaOH 25% and then extracted three times with 25 mL of EtOAc. The OP was combined and washed using 25 mL of NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter and concentrated to dryness in a rotavapor (40°C, 250-70 mbar). This material was dried under high vacuum (room temperature, 10-3 mbar) for 3 h to give 337 mg (40.7% crude yield) of a pale yellow oil as 362-VRO047-02-001 crude 1#1, which was used "as is" in the next step without further purification.

[0084] 1 H-NMR(300MHz,CDCl3)δ=7.49(br.s,1H,7),3.57-3.37(m,5H,2,8,9),3.36(s,3H,10 ),1.83-1.57(m,4H,1,3'',4),1.47-1.28(m,1H,3'),0.95(dd,J=8.4,6.2,6H,5,6).

[0085] Finally, the dipeptide was coupled to the epoxy head. In a 25 mL round-bottom flask equipped with a magnetic stirrer, 290 mg of 362-BB01-03-001 crude 1#1 (1.813 mmol, 1.05 equiv.) and 5 mL of DCM were introduced, the resulting white suspension was stirred at room temperature for 5 min, and then cooled to 0-5 °C using an ice bath. 839 mg of HATU (2.589 mmol, 1.5 equiv.) were added at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Then 325 mg of 362-VRO047-02-001 crude 1#1 (1.726 mmol, 1.0 equiv.) in solution in 5 mL of DCM was introduced at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Finally, 601 μL of DIPEA (3.452 mmol, 2.0 equiv.) was added dropwise at 0-5° C. The resulting mixture was stirred at 0-5° C. for 5 min, then the ice bath was removed and the mixture was stirred at room temperature overnight.

[0086] The reaction mixture, a clear yellow solution, was transferred to a separatory funnel using 10 mL DCM and washed with 50 mL NaCl 5.8%, then the AP was extracted once with 10 mL DCM. The OP was combined and washed once with 50 mL NaCl 5.8%, then the AP was extracted once with 10 mL DCM. The OP was combined and washed once with 50 mL HCl 0.25 M, once with 50 mL NaHCO3 10%, and once with 50 mL NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter, and concentrated to dryness in a rotavapor (40 °C, 220-30 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h to give 560 mg (neat yield: 98.2%) of a yellowish sticky solid as 362-VRO047-03-001 crude 1#1.

[0087] The crude material was purified by CC using 45 g of silica gel (column with φ=3.2 cm and h=15.2 cm) followed by elution using TLC with heptane-EtOAc (3:7) as eluent; f=0.41. The product was eluted with a gradient from heptane-EtOAc (7:3) to heptane-EtOAc (2:8) and the fractions containing the desired product were combined and concentrated to dryness in a Rotavapor (40 °C, 200-40 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h to give 220 mg (neat yield: 38.6%) of a slightly pale yellow solid as 362-VRO047-03-001 CC1#1.

[0088] 1 H-NMR(300MHz,CDCl3)δ=6.58(d,J=8.6,1H,5),6.23(br.s,1H,11),4.41 (td,J=8.5,5.6,1H,6),4.26(qq,J=7.1,3.6,2H,2),3.69(d,J=1.8,1H,4) ,3.51-3.39(m,5H,3,12,13),3.36(s,3H,14),1.56(br.m,J=23.2,13.4, 9.5,5.2,3H,7,8),1.31(t,J=7.1,3H,1),0.93(dd,J=6.1,2.9,6H,9,10).

[0089] 13 C-NMR(75MHz,CDCl3)δ=70.75(13),62.30(2),58.76(14),53.77(4),52.88(3),5 1.16(6),41.51(7),39.26(12),24.76(8),22.78(9,10),22.05(9,10),13.99(1).

[0090] Example 3: Synthesis of VRO052 First, the dipeptide was formed. In a 50 mL round-bottom flask equipped with a magnetic stirrer, 2.235 g of Boc-L-Leu (9.661 mmol, 1.1 equiv.) and 25 mL of DCM were introduced, the resulting suspension was stirred at room temperature for 5 min, then cooled to 0-5 °C using an ice bath. 8.540 g of HATU (26.348 mmol, 3.0 equiv.) were added at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Then 1.1 mL of 2-(2-aminoethyl)pyridine (8.783 mmol, 1.0 equiv.) and 10 mL of DCM (the suspension was too thick due to the amount of HATU) were introduced at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Finally, 7.6 mL of DIPEA (43.914 mmol, 5.0 equiv.) were added dropwise at 0-5 °C. The resulting intense yellow mixture was stirred at 0-5 °C for 5 min, then the ice bath was removed and the mixture was stirred at room temperature overnight.

[0091] The reaction mixture, a brownish orange solution containing white solids, was filtered on a P3 sintered glass filter to remove salts. The filtrate was transferred to a separatory funnel using 5 mL of DCM and washed with 50 mL of NaCl 5.8%, then the AP was extracted once with 10 mL of DCM. This operation was repeated two more times, then the OP was combined and washed once with 50 mL of NaHCO3 10% and finally with 50 mL of NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter and concentrated to dryness in a rotavapor (40 °C, 850-70 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h 30 min to give 8.462 g (raw yield: 287.2%) of a brownish orange oil as 362-VRO052-01-001 crude 1#1.

[0092] An initial attempt at purification by CC was performed using 50 g of silica gel (column with φ=3.2 cm and h=16.9 cm) followed by elution using TLC with DCM containing 1% Et3N as eluent; Rf=0.51. The product was eluted with a gradient from DCM containing 1% Et3N to DCM containing 0.5% MeOH and 1% Et3N, and the fractions containing the desired product were combined and concentrated to dryness in a rotavapor (40° C., 800-50 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 3 h to give 6.666 g (raw yield: 226.3%) of a yellowish oil as 362-VRO052-01-001 CC1#1.

[0093] A second attempt at purification to remove TMU by CC was performed using 55 g of silica gel (column with φ=3.2 cm and h=16.9 cm) followed by elution using TLC with heptane-EtOAc (2:8) and 1% Et3N as eluents; Rf=0.45. The product was eluted with a gradient from heptane-EtOAc (7:3) with 1% Et3N to heptane-EtOAc (1:9) with 1% Et3N, and the fractions containing the desired product were combined and concentrated to dryness in a rotavapor (40° C., 220-50 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h to give 3.228 g (raw yield: 109.6%) of a yellowish oil as 362-VRO052-01-001 CC2#1.

[0094] The material recovered from the second CC was re-extracted to remove TMU. The material was transferred to a separatory funnel using 15 mL of EtOAc, then washed 4 times with 50 mL of NaHCO3 10%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter, and concentrated to dryness in a rotavapor (40 °C, 220-40 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 3 h to give 2.416 g (raw yield: 82.0%) of a yellowish oil as 362-VRO052-01-001 crude 2#1.

[0095] 1 H-NMR(300MHz,CDCl3)δ=8.52(dt,J=4.8,1.5,1H,16),7.64(td,J=7.7,1.9,1H,14),7.23-7.12(m,2H,13,15),7.02(br.s,1H,10),5.09-4.77(m,1H,4),4.18-3.92(m,1H,5),3.78-3.56(m,2H,11),3.11-2.91(m,2H,12),1.61(dd,J=16.0,5.9,2H,6’’,7),1.45(d,J=7.4,1H,6’),1.40(s,9H,1,2,3),0.90(d,J=6.1,6H,8,9)。

[0096] 13 C NMR(75MHz,CDCl3)δ=148.61(16),137.04(14),123.65(13,15),121.68(13,15),53.11(5),41.77(6),38.50(11),36.44(12),28.25(1,2,3),24.70(7),22.79(8,9),21.99(8,9)。

[0097] The Boc protecting group was then removed as follows: In a 50 mL round bottom flask equipped with a magnetic stirrer, 2.416 g of 362-VRO052-01-001 CC1#1 (7.202 mmol, equiv.) and 18.1 mL of DCM were introduced, the resulting colorless solution was stirred at room temperature for 15 min, then cooled to 0-5 °C using an ice bath. 6.0 mL of TFA (78.875 mmol, 10.95 equiv.) was added dropwise at 0-5 °C. The reaction mixture was stirred at 0-5 °C for 5 min, then the ice bath was removed and the mixture was stirred at room temperature for 4 h. The reaction mixture was transferred to a separatory funnel using 5 mL of DCM and washed with 50 mL of water. The AP was basified to pH>10 using 20 mL of NaOH 25% and then extracted 3 times with 20 mL of DCM. The OPs were combined and washed using 50 mL of NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter and concentrated to dryness in a rotavapor (40°C, 800-70 mbar). This material was dried under high vacuum (room temperature, 10-3 mbar) for 6 h to give 1.470 g (raw yield: 86.7%) of a pale yellow solid as 362-VRO052-02-001 crude 1#1. This material was used "as is" in the next step without further purification.

[0098] 1 H-NMR(300MHz,CDCl3)δ=8.54(d,J=4.5,1H,13),7.70(br.s,1H,7),7.60(td,J=7.7,1.8,1H,11),7.21-7.08(m,2H,10,12),3.66(q,J=6.4,2H,8) ,3.38(d,J=6.1,1H,2),3.00(t,J=6.6,2H,9),1.74(br.s,2H,1),1.76-1 .51(m,2H,3'',4),1.42-1.22(m,1H,3'),0.91(dd,J=8.5,6.2,6H,5,6).

[0099] Finally, the dipeptide was coupled to the epoxy head. In a 100 mL round-bottom flask equipped with a magnetic stirrer, 970 mg of 362-BB01-03-004 crude 1#1 (6.055 mmol, 1.05 equiv.) and 15 mL of DMF were introduced, the resulting solution was stirred at room temperature for 5 min, and then cooled to 0-5 °C using an ice bath. 5.607 g of HATU (17.299 mmol, 3.0 equiv.) were added at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Then 1.357 g of 362-VRO052-02-001 crude 1#1 (5.766 mmol, 1.0 equiv.) in solution in 15 mL of DMF was introduced at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Finally, 5.0 mL of DIPEA (28.832 mmol, 5.0 equiv.) was added dropwise at 0-5° C. The resulting tan-brownish mixture was stirred at 0-5° C. for 5 min, then the ice bath was removed and the mixture was stirred at room temperature overnight.

[0100] The reaction mixture, a clear dark brown solution, was transferred to a separatory funnel using 30 mL EtOAc and washed with 100 mL NaCl 5.8%, then the AP was extracted 4 times with 30 mL EtOAc. The OPs were combined and washed once with 100 mL NaHCO3 5%, then the AP was extracted once with 20 mL EtOAc. The OPs were combined and washed once with 100 mL NaHCO3 10%, then the AP was extracted once with 20 mL EtOAc. The OPs were combined and the last operation was repeated one more time. The OPs were dried over Na2SO4, filtered on a P3 sintered glass filter and concentrated to dryness in a rotavapor (40 °C, 220-30 mbar). This material was dried under high vacuum (room temperature, 10-3 mbar) for 1 h 30 min to give 2.296 g (neat yield: 105.5%) of a dark brown sticky foam as 362-VRO052-03-002 crude 1#1.

[0101] The crude material was first purified by slurrying with 20 mL of EtOAc. The suspension was stirred at room temperature for about 30 min, then cooled to 0-5° C. and filtered on a P3 sintered glass filter. The filter cake was washed twice with 6 mL of cold EtOAc and then dried under high vacuum (room temperature, 10-3 mbar) for 2 h to give 1.114 g (raw yield: 51.2%) of a slightly beige powder as 362-VRO052-03-002 slurry 1#1.

[0102] This material was repurified by CC using 50 g of silica gel (column with φ=3.2 cm and h=16.9 cm) followed by elution using TLC with pure EtOAc containing 1% Et3N as the eluent; f =0.52. The product was eluted with a gradient from heptane-EtOAc (1:1) to pure EtOAc always containing 1% Et3N, and the fractions containing the desired product were combined and concentrated to dryness in a rotavapor (40 °C, 240-50 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 3 h to give 966 mg (naked yield: 44.4%) of a white solid as 362-VRO052-03-002 CC1#1.

[0103] 1 H-NMR(300MHz,CDCl3)δ=8.55(d,J=4.9,1H,17),7.69(t,J=7.6,1H,16),7.25-7.12( m,3H,11,14,15),6.60(d,J=8.5,1H,5),4.45-4.33(m,1H,6),4.27(qd,J=7.2,3.2,2 H,2),3.70(t,J=6.3,2H,12),3.66(d,J=1.9,1H,4),3.46(d,J=1.9,1H,3),3.03(t,J =6.1,2H,13),1.53(br.m,3H,7,8),1.32(t,J=7.1,3H,1),0.89(d,J=5.8,6H,9,10).

[0104] 13C-NMR(75MHz,CDCl3)δ=147.39(17),138.40(15),124.33(14,16),122.27(14,16),62.26(2),53.86(4), 52.88(3),51.38(6),41.57(7),38.56(12),35.49(13),24.82(8),22.74(9,10),22.01(9,10),14.00(1).

[0105] Example 4: Synthesis of VRO059 First, the dipeptide was formed. In a 50 mL round-bottom flask equipped with a magnetic stirrer, 1.320 g of Boc-L-Leu (5.707 mmol, 1.1 equiv.) and 25 mL of DCM were introduced, the resulting cloudy solution was stirred at room temperature for 5 min, then cooled to 0-5 °C using an ice bath. 5.045 g of HATU (15.565 mmol, 3.0 equiv.) were added at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Then 825 μL of 3-(piperidinyl)propanamine (5.188 mmol, 1.0 equiv.) were introduced at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Finally, 4.5 mL of DIPEA (25.941 mmol, 5.0 equiv.) were added dropwise at 0-5 °C. The resulting yellowish mixture was stirred at 0-5 °C for 5 min, then the ice bath was removed and the mixture was stirred at room temperature overnight.

[0106] The reaction mixture, a brownish-red solution with white solids in suspension, was filtered over a P3 sintered glass filter to remove salts. The filtrate was transferred to a separatory funnel using 5 mL of DCM and washed with 50 mL of NaCl 5.8%, then the AP was extracted once with 10 mL of DCM. The OPs were combined and the previous washes were repeated twice. The OPs were combined and washed once with 50 mL of NaHCO3 10% and finally with 50 mL of NaCl 11.6%. The OPs were dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness in rotavapor (40 °C, 850-70 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h 30 min to give 4.113 g (raw yield: 223.0%) of a brownish-red oil as 362-VRO059-01-002 crude 1#1.

[0107] The crude material was purified by CC using 50 g of silica gel (column with φ=3.2 cm and h=16.9 cm) followed by elution using TLC with DCM containing 1.5% MeOH and 1% Et3N as eluents; Rf=0.56. The product was eluted with a gradient from pure DCM to DCM containing 2% MeOH always containing 1% Et3N, and the fractions containing the desired product were combined and concentrated to dryness in a rotavapor (40° C., 750-60 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h to give 1.557 g (raw yield: 84.4%) of a yellowish oil as 362-VRO059-01-002 CC1#1.

[0108] The material recovered from the CC was re-extracted to remove TMU. The material was transferred to a separatory funnel using 10 mL of EtOAc and then washed twice with 50 mL of NaHCO3 10%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter and concentrated to dryness in a rotavapor (40 °C, 220-40 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 3 h to give 1.181 g (raw yield: 64.0%) of a yellowish viscous material as 362-VRO059-01-001 crude 2#1.

[0109] The re-extracted material was purified by a second CC using 50 g of silica gel (column with φ=3.2 cm and h=16.9 cm) followed by elution using TLC using pure EtOAc with 1% Et3N as eluent; Rf=0.47. The product was eluted with a gradient from heptane-EtOAc (1:1) to pure EtOAc, and the fractions containing the desired product were combined and concentrated to dryness in a rotavapor (40° C., 220-60 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h to give 1.038 g (raw yield: 56.3%) of a pale yellowish solid as 362-VRO059-01-002 CC2#1.

[0110] 1 H-NMR(300MHz,CDCl3)δ=7.63(br.s,1H,10),4.98(d,J=7.7,1H,4),4.10(br.s,1H,5),3.46-3.21(m,2H,11),2.71-2.13(m,6H ,13,14,15),1.79-1.55(m,8H,6'',7,12,16,17),1.54-1.35(m,3H,6',18),1.43(s,9H,1,2,3),0.94(dd,J=6.2,3.5,6H,8,9).

[0111] 13 C NMR(75MHz,CDCl3)δ=58.15(13),54.56(14,15),53.15(5),42.32(6),39.62(11),28.27(1, 2,3),25.84(12,16,17),24.66(7),24.61(12,16,17),24.11(18),23.05(8,9),21.97(8,9).

[0112] The Boc protecting group was then removed as follows: In a 25 mL round bottom flask equipped with a magnetic stirrer, 1.024 g of 362-VRO059-01-001 CC2#1 (2.880 mmol, equiv.) and 7.7 mL of DCM were introduced, the resulting colorless solution was stirred at room temperature for 10 min, then cooled to 0-5 °C using an ice bath. 2.6 mL of TFA (33.430 mmol, 11.61 equiv.) was added dropwise at 0-5 °C. The reaction mixture was stirred at 0-5 °C for 5 min, then the ice bath was removed and the mixture was stirred at room temperature for 4 h. The reaction mixture was transferred to a separatory funnel using 5 mL of DCM and washed with 50 mL of water. The AP was basified to pH > 10 using 8 mL of NaOH 25% and then extracted 3 times with 20 mL of DCM. The OPs were combined and washed with 50 mL of NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter and concentrated to dryness in rotavapor (40°C, 800-70 mbar). This material was dried under high vacuum (room temperature, 10-3 mbar) for 6 h to give 654 mg (88.9% crude yield) of a pale yellowish oil as 362-VRO059-02-002 crude 1#1. This material was used "as is" in the next step without further purification.

[0113] 1 H-NMR(300MHz,CDCl3)δ=7.90(br.s,1H,7),3.44-3.21(m,3H,2,8),2.61-2.26(m,6H,10,11,12),1.66(br.m ,10H,1,3',4,9',9'',13,14),1.51-1.41(m,2H,15),1.40-1.27(m,1H,3''),0.94(dd,J=8.0,6.5,6H,5,6).

[0114] 13 C-NMR(75MHz,CDCl3)δ=57.78(10),54.58(11,12),53.74(2),44.32(3),38.64(8), 25.75(9,13,14),25.59(9,13,14),24.80(4),24.18(15),23.39(5,6),21.37(5,6).

[0115] Finally, the dipeptide was coupled to the epoxy head. In a 25 mL round-bottom flask equipped with a magnetic stirrer, 356 mg of 362-BB01-03-004 crude 1#1 (2.241 mmol, 1.05 equiv.) and 7.5 mL of DMF were introduced, the resulting solution was stirred at room temperature for 5 min, and then cooled to 0-5 °C using an ice bath. 2.075 g of HATU (6.402 mmol, 3.0 equiv.) were added at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Then 545 mg of 362-VRO059-02-002 crude 1#1 (2.134 mmol, 1.0 equiv.) in solution in 7.5 mL of DMF was introduced at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Finally, 1.9 mL of DIPEA (10.699 mmol, 5.0 equiv.) was added dropwise at 0-5° C. The resulting tan-brownish mixture was stirred at 0-5° C. for 5 min, then the ice bath was removed and the mixture was stirred at room temperature overnight.

[0116] The reaction mixture, a clear dark brown solution, is transferred to a separatory funnel using 15 mL EtOAc and washed with 50 mL NaCl 5.8%, then the AP is extracted 4 times with 15 mL EtOAc. The OP is combined and washed 4 times with 50 mL NaHCO3 10%. The OP is dried over Na2SO4, filtered on a P3 sintered glass filter and concentrated to dryness in rotavapor (40 °C, 220-30 mbar). The material is dried under high vacuum (room temperature, 10-3 mbar) for 1 h 30 min to give 1.063 g (raw yield: 125.3%) of a dark brown sticky solid as 362-VRO059-03-003 crude 1#1.

[0117] The crude material was purified by CC using 50 g of silica gel (column with φ=3.2 cm and h=16.9 cm) followed by elution using TLC using DCM containing 1% MeOH and 1% Et3N as eluent; f=0.36. The product was eluted with a gradient from pure DCM to DCM with 1% MeOH always containing 1% Et3N, fractions containing the desired product were combined and concentrated to dryness in a rotavapor (40 °C, 800-50 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h to give 397 mg (neat yield: 46.8%) of a viscous brownish orange solid as 362-VRO059-03-003 CC1#1.

[0118] This material was repurified by CC using 50 g of silica gel (column with φ=3.2 cm and h=16.9 cm) followed by elution using TLC using heptane-acetone (3:7) containing 1% Et3N as the eluent; f =0.57. The product was eluted with a gradient from heptane-acetone (8:2) to heptane-acetone (3:7) always containing 1% Et3N, and the fractions containing the desired product were combined and concentrated to dryness in Rotavapor (40°C, 320-50 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 1 h to give 116 mg (raw yield: 17.4%) of a brownish-beige solid as 362-VRO059-03-003 CC2#1. This material was re-extracted. It was transferred to a separatory funnel using 20 mL EtOAc and 20 mL water, then HCl 1M was added to bring the pH to 1. The AP was basified to pH 9 and extracted 3 times with 20 mL EtOAc. The OP was combined, dried over Na2SO4, filtered on a P3 sintered glass filter and concentrated to dryness in Rotavapor (40°C, 220-30 mbar). This material was dried under high vacuum (room temperature, 10-3 mbar) for 3 h to give 111 mg (neat yield: 13.7%) of a viscous orange-yellow solid as 362-VRO059-03-003 Extract 1#1.

[0119] 1H-NMR(300MHz,CDCl3)δ=8.17(t,J=5.0,1H,11),6.69(d,J=8.5,1H,5),4.43 -4.31(m,1H,6),4.25(qq,J=7.1,3.6,2H,2),3.67(d,J=1.9,1H,4),3.46(d, J=1.9,1H,3),3.44-3.23(m,2H,12),2.73-2.30(m,6H,14,15,16),1.63(br. m,11H,7,8,13,17,18,19),1.30(t,J=7.1,3H,1),0.93(d,J=5.3,6H,9,10).

[0120] 13 C-NMR(75MHz,CDCl3)δ=62.20(2),58.25(14),54.47(15,16),53.89(4),52.84(3),51.43(6),42.09(7),40.01(12), 25.79(13,17,18,19),24.76(8),23.98(13,17,18,19),23.87(13,17,18,19),22.95(9,10),22.02(9,10),13.99(1).

[0121] Example 5: Synthesis of VRO073 First, the dipeptide was formed. In a 25 mL round-bottom flask equipped with a magnetic stirrer, 970 mg of Boc-L-Leu (4.195 mmol, 1.05 equiv.) and 10 mL of DCM were introduced, the resulting cloudy solution was stirred at room temperature for 5 min, then cooled to 0-5 °C using an ice bath. 1.943 g of HATU (5.993 mmol, 1.5 equiv.) were added at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Then 457 μL of 2-fluorobenzylamine (3.995 mmol, 1.0 equiv.) were introduced at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Finally, 1.4 mL of DIPEA (7.991 mmol, 2.0 equiv.) were added dropwise at 0-5 °C. The resulting cloudy intense yellow solution was stirred at 0-5 °C for 5 min, then the ice bath was removed and the mixture was stirred at room temperature overnight.

[0122] The reaction mixture, a slightly cloudy yellow solution, was transferred to a separatory funnel using 10 mL DCM and washed with 50 mL NaCl 5.8%, then the AP was extracted once with 10 mL DCM. This operation was repeated two more times, then the OP was combined and washed once with 50 mL NaHCO3 10% and finally with 50 mL NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter and concentrated to dryness in a rotavapor (40 °C, 850-70 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 3 h to give 1.841 g (raw yield: 136.2%) of a beige-yellow sticky solid as 362-VRO073-01-001 crude 1#1.

[0123] The crude material was purified by CC using 45 g of silica gel (column with φ=3.2 cm and h=15.2 cm) followed by elution using TLC with heptane-EtOAc (7:3) as eluent; Rf=0.49. The product was eluted with a gradient from pure heptane to heptane-EtOAc (6:4) and the fractions containing the desired product were combined and concentrated to dryness in a rotavapor (40° C., 220-50 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h to give 1.261 g (raw yield: 93.3%) of a white foam as 362-VRO073-01-001 CC1#1.

[0124] 1 H-NMR(300MHz,CDCl3)δ=7.31(td,J=7.8,1.6,1H,14),7.27-7.18(m,1H,12) ,7.14-6.96(m,2H,13,15),6.54(br.s,1H,10),4.85(s,1H,4),4.85(br.s,J =6.8,1H),4.48(d,J=4.6,2H,11),4.10(br.s,1H,5),1.77-1.57(m,2H,6',7 ),1.55-1.45(m,1H,6''),1.41(s,9H,1,2,3),0.92(dd,J=6.3,2.9,6H,8,9).

[0125] 13C NMR(75MHz,CDCl3)δ=129.95(12,14),129.23(12,14),129.12(12,14),124.24(13),124.19(13),115.41(15), 115.13(15),53.04(5),40.90(6),37.40(11),37.34(11),28.18(1,2,3),24.68(7),22.85(8,9),21.92(8,9).

[0126] The Boc protecting group was then removed as follows: In a 25 mL round bottom flask equipped with a magnetic stirrer, 1.249 g of 362-VRO073-01-001 CC1#1 (3.691 mmol, 1.0 equiv.) and 9.4 mL of DCM were introduced, the resulting colorless solution was stirred at room temperature for 10 min, then cooled to 0-5 °C using an ice bath. 3.1 mL of TFA (40.48 mmol, 10.97 equiv.) was added dropwise at 0-5 °C. The reaction mixture was stirred at 0-5 °C for 5 min, then the ice bath was removed and the mixture was stirred at room temperature for 2 h 30 min. The reaction mixture was transferred to a separatory funnel using 25 mL of DCM and washed with 25 mL of water. The AP was basified to pH > 10 using 3 mL of NaOH 25% and then extracted 3 times with 25 mL of DCM. The OPs were combined and washed using 25 mL of NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter and concentrated to dryness in a Rotavapor (40°C, 800-70 mbar). This material was dried under high vacuum (room temperature, 10-3 mbar) for 1 h to give 465 mg (52.9% crude yield) of a nearly colorless oil as 362-VRO073-02-001 crude 1#1. This material was used "as is" in the next step without further purification.

[0127] 1H-NMR(300MHz,CDCl3)δ=7.67(br.s,1H,7),7.32(td,J=7.7,1.7,1H,11),7.29-7.18(m,1H,9),7.15-6.98(m,2H,10,12),4.49( d,J=6.1,2H,8),3.42(dd,J=9.9,3.4,1H,2),1.83-1.62(m,2H,3'',4),1.57-1.21(m,3H,1,3'),0.94(dd,J=8.7,6.0,6H,5,6).

[0128] Finally, the dipeptide was coupled to the epoxy head. In a 25 mL round-bottom flask equipped with a magnetic stirrer, 320 mg of 362-BB01-03-001 crude 1#1 (1.996 mmol, 1.05 equiv.) and 5 mL of DCM were introduced, the resulting white suspension was stirred at room temperature for 5 min, and then cooled to 0-5 °C using an ice bath. 924 mg of HATU (2.851 mmol, 1.5 equiv.) were added at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Then 453 mg of 362-VRO073-02-001 crude 1#1 (1.901 mmol, 1.0 equiv.) in solution in 5 mL of DCM were introduced at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Finally, 993 μL of DIPEA (5.703 mmol, 3.0 equiv.) was added dropwise at 0–5° C. The resulting yellowish mixture was stirred at 0–5° C. for 5 min, then the ice bath was removed and the mixture was stirred at room temperature overnight.

[0129] The reaction mixture, a yellowish orange solution, was transferred to a separatory funnel using 10 mL DCM and washed with 50 mL NaCl 2.6%, then the AP was extracted once with 10 mL DCM. The OP was combined and washed once with 50 mL NaCl 2.6%, then the AP was extracted once with 10 mL DCM. The OP was combined and washed with 50 mL NaCl 5.8% and 5 mL HCl 1M, resulting in an emulsion that needed to be filtered through Celite. The filtrate was transferred again to a separatory funnel using 10 mL DCM and washed once with 50 mL NaHCO3 10% and once with 50 mL NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter and concentrated to dryness in a rotavapor (40 °C, 220-30 mbar). This material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h to give 835 mg (crude yield: 115.5%) of a pasty orange solid as 362-VRO073-03-001 crude 1#1.

[0130] The crude material was purified by CC using 45 g of silica gel (column with φ=3.2 cm and h=15.2 cm) followed by elution using TLC with heptane-EtOAc (1:1) as eluent; f =0.50. The product was eluted with a gradient from pure heptane to heptane-EtOAc (2:8) and the fractions containing the desired product were combined and concentrated to dryness in a Rotavapor (40 °C, 200-50 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 3 h to give 110 mg (raw yield: 15.2%) of a white solid as 362-VRO073-03-001 CC1#1.

[0131] 1H-NMR(300MHz,CDCl3)δ=7.36-7.21(m,2H,13,15),7.17-6.99(m,2H,14,16),6.51(d,J=8 .6,1H,5),6.35(t,J=5.5,1H,11),4.48(dd,J=5.8,2.5,2H,12),4.41(td,J=8.5,5.8,1H,6 ),4.27(qq,J=6.9,3.6,2H,2),3.66(d,J=1.9,1H,4),3.44(d,J=1.9,1H,3),1.75-1.60(m ,1H,7''),1.60-1.44(m,2H,7',8),1.31(t,J=7.1,3H,1),0.90(dd,J=6.2,4.5,6H,9,10).

[0132] Example 6: Synthesis of VRO109 First, the dipeptide was formed. In a 25 mL round-bottom flask equipped with a magnetic stirrer, 190 mg of (S)-Boc-2-amino-4,4,4-trifluoro-butyric acid (0.739 mmol, 1.0 equiv.) and 10 mL of DCM were introduced, the resulting mixture was stirred at room temperature for 5 min, then cooled to 0-5 °C using an ice bath. 359 mg of HATU (1.108 mmol, 1.5 equiv.) were added at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Then 94 μL of IAA (0.813 mmol, 1.1 equiv.) were introduced at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Finally, 257 μL of DIPEA (1.477 mmol, 2.0 equiv.) were added dropwise at 0-5 °C. The resulting slightly pale yellow mixture was stirred at 0-5 °C for 5 min, then the ice bath was removed and the mixture was stirred at room temperature overnight.

[0133] The reaction mixture, a yellowish orange solution, was concentrated to dryness in a rotavapor (40° C., 750-400 mbar). The residue was transferred to a separatory funnel using 20 mL of EtOAc and washed with 50 mL of NaHCO3 5%, then the AP was extracted twice with 15 mL of EtOAc. The OP was combined and washed twice with 50 mL of NaHCO3 5%, once with 50 mL of NaHCO3 10%, and once with 50 mL of NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter, and concentrated to dryness in a rotavapor (40° C., 220-30 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 6 h to give 224 mg (raw yield: 92.9%) of an off-white crystallized solid as 362-VRO109-01-001 crude 1#1.

[0134] The crude material was purified by CC using 50 g of silica gel (column with φ=3.2 cm and h=16.9 cm) followed by elution using TLC with heptane-EtOAc (7:3) as eluent; Rf=0.46. The product was eluted with a gradient from pure heptane to heptane-EtOAc (6:4) and the fractions containing the desired product were combined and concentrated to dryness in a rotavapor (40° C., 220-50 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 4 h to give 204 mg (raw yield: 84.6%) of a white foam as 362-109-01-001 CC1#1.

[0135] 1 H-NMR(300MHz,CDCl3)δ=6.26(br.s,1H,7),4.97(d,J=6.6,1H,4),4.49-4.26(m,1H,5),3.39-3.16(m,2H,8),2.93-2.69(m, 1H,6'),2.62-2.37(m,1H,6''),1.69-1.51(m,1H,10),1.45(s,9H,1,2,3),1.39(q,J=7.3,2H,9),0.91(d,J=6.6,6H,11,12).

[0136] 13C NMR (75MHz, CDCl3) δ=49.48(5),38.12(9),38.00(8),28.15(1,2,3),25.67(10),22.32(11,12).

[0137] The Boc protecting group was then removed as follows: In a 10 mL round bottom flask equipped with a magnetic stirrer, 201 mg of 362-VRO109-01-001 CC1#1 (0.616 mmol, equiv.) and 5 mL of DCM were introduced, the resulting colorless solution was stirred at room temperature for 10 min, then cooled to 0-5 °C using an ice bath. 1.7 mL of TFA (22.200 mmol, 36.045 equiv.) was added dropwise at 0-5 °C. The reaction mixture was stirred at 0-5 °C for 5 min, then the ice bath was removed and the mixture was stirred at room temperature for 1 h. The reaction mixture was transferred to a separatory funnel using 25 mL of DCM and washed with 50 mL of water. The AP was basified to pH > 10 using 4 mL of NaOH 25% and then extracted three times with 15 mL of DCM. The OPs were combined, dried over Na2SO4, filtered on a P3 sintered glass filter, and concentrated to dryness in a Rotavapor (40°C, 750-400 mbar). This material was dried under high vacuum (room temperature, 10-3 mbar) for 6 h to give 256 mg (neat yield: 96.6%) of a nearly colorless oil as 362-VRO109-02-001 crude 1#1. This material was used "as is" in the next step without further purification.

[0138] 1 H-NMR(300MHz,DMSO-d6-d6)δ=8.07(t,J=5.3,1H,4),3.48(dd,J=7.5,5.4,1H,2),3.19-2.97(m,2H,5),2.78-2.53 (m,1H,3''),2.49-2.24(m,1H,3'),1.56(hept,J=6.7,1H,7),1.30(q,J=7.2,2H,6),0.86(dd,J=6.6,0.6,6H,8,9).

[0139] 13C-NMR (75MHz, DMSO-d6) δ=49.20(2),49.16(2),37.62(6),37.46(3),37.11(3),36.57(5),24.78(7),22.07(8,9),22.05(8,9).

[0140] Finally, the dipeptide was coupled to the epoxy head. In a 25 mL round-bottom flask equipped with a magnetic stirrer, 115 mg of 362-BB01-03-004 crude 1#1 (0.720 mmol, equiv.) and 5 mL of DMF were introduced, the resulting solution was stirred at room temperature for 5 min, and then cooled to 0-5 °C using an ice bath. 318 mg of HATU (0.981 mmol, 1.5 equiv.) were added at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Then 148 mg of 362-VRO109-02-001 crude 1#1 (0.654 mmol, 1.0 equiv.) in solution in 5 mL of DMF was introduced at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Finally, 228 μL of DIPEA (1.308 mmol, 2.0 equiv.) was added dropwise at 0-5 °C. The resulting yellow mixture was stirred at 0-5° C. for 5 min, then the ice bath was removed and the mixture was stirred at room temperature overnight.

[0141] The reaction mixture, an orange solution, was transferred to a separatory funnel using 20 mL EtOAc and washed with 50 mL NaHCO 35%, then the AP was extracted twice with 15 mL EtOAc. The OP was combined and washed three times with 50 mL NaHCO 35% and once with 50 mL NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter and concentrated to dryness in a rotavapor (40 °C, 220-140 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 6 h to give 237 mg (raw yield: 102.5%) of a very pale orange solid as 362-VRO109-03-001 crude 1#1.

[0142] The crude material was purified by slurrying with 5 mL of EtOAc-heptane (1:1). The suspension was stirred at room temperature for 20 min, then cooled to 0-5° C. and stirred for 15 min. The suspension was filtered through an ultrafiltration apparatus equipped with a nylon membrane (0.45 μm), and the filter cake was washed three times with cold EtOAc-heptane (1:9), then dried under high vacuum (room temperature, 10-3 mbar) for 3 h to give 175 mg (raw yield: 72.6%) of a white powder as 362-VRO109-03-001 slurry 1#1.

[0143] 1 H-NMR(300MHz,CDCl3)δ=6.59(d,J=8.6,1H,5),6.18(s,1H,8),4.70(td,J=8.9,4.8,1H,6),4.28 (qd,J=7.1,2.5,2H,2),3.72(d,J=1.9,1H,4),3.45(d,J=1.8,1H,3),3.27(tdd,J=7.4,5.8,4.7, 2H,9),2.74(ddt,J=16.4,10.7,5.3,1H,7'),2.65-2.40(m,1H,7''),1.60(dt,J=13.4,7.0,2H,1 1),1.40(q,J=7.2,2H,10),1.32(t,J=7.2,3H,1),1.25(d,J=1.8,0H),0.91(d,J=6.6,6H,12,13).

[0144] Example 7: Synthesis of VRO244 First, the dipeptide was formed. In a 25 mL round-bottom flask equipped with a magnetic stirrer, 190 mg of Boc-(S)-2-amino-4,4,4-butyric acid (0.739 mmol, 1.0 equiv.) and 10 mL of DCM were introduced, the resulting mixture was stirred at room temperature for 5 min, then cooled to 0-5 °C using an ice bath. 359 mg of HATU (1.108 mmol, 1.5 equiv.) were added at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Then 93 μL of 2-fluorobenzylamine (0.813 mmol, 1.1 equiv.) were introduced at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Finally, 257 μL of DIPEA (1.477 mmol, 2.0 equiv.) were added dropwise at 0-5 °C. The resulting slightly pale yellow mixture was stirred at 0-5 °C for 5 min, then the ice bath was removed and the mixture was stirred at room temperature overnight.

[0145] The reaction mixture, a clear yellow solution, was concentrated to dryness in a rotavapor (40° C., 750-400 mbar). The residue was transferred to a separatory funnel using 20 mL of EtOAc and washed with 50 mL of NaHCO3 5%, then the AP was extracted twice with 15 mL of EtOAc. The OP was combined and washed twice with 50 mL of NaHCO3 5%, once with 50 mL of NaHCO3 10%, and once with 50 mL of NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter, and concentrated to dryness in a rotavapor (40° C., 220-140 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 6 h to give 279 mg (raw yield: 103.7%) of an orange solid as 362-VRO244-01-001 crude 1#1.

[0146] The crude material was purified by CC using 50 g of silica gel (column with φ=3.2 cm and h=16.9 cm) followed by elution using TLC with heptane-acetone (7:3) as eluent; Rf=0.45. The product was eluted with a gradient from pure heptane to heptane-acetone (6:4) and the fractions containing the desired product were combined and concentrated to dryness in a rotavapor (40° C., 350-50 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h to give 259 mg (raw yield: 96.2%) of a slightly pale yellow solid as 362-VRO243-01-001 CC1#1.

[0147] 1 H-NMR(300MHz,CDCl3)δ=7.36-7.20(m,2H,9,11),7.14-7.06(m,1H,10),7.09-6.98(m,1H,12),6.73(br.s,1H,7 ),4.95(br.s,1H,4),4.61-4.31(m,3H,5,8),2.96-2.72(m,1H,6'),2.65-2.38(m,1H,6''),1.42(s,9H,1,2,3).

[0148] 13 C NMR(75MHz,CDCl3)δ=130.01(9,11),129.53(9,11),129.41(9,11),124.33,(10),124. 28(10),115.54(12),115.26(12),49.56(5),37.81(6,8),37.76(6,8),28.10(1,2,3).

[0149] The Boc protecting group was then removed as follows: In a 10 mL round bottom flask equipped with a magnetic stirrer, 252 mg of 362-VRO244-01-001 CC1#1 (0.692 mmol, 1.0 equiv.) and 5 mL of DCM were introduced, the resulting colorless solution was stirred at room temperature for 10 min, then cooled to 0-5 °C using an ice bath. 1.7 mL of TFA (22.200 mmol, 32.10 equiv.) was added dropwise at 0-5 °C. The reaction mixture was stirred at 0-5 °C for 5 min, then the ice bath was removed and the mixture was stirred at room temperature for 1 h. The reaction mixture was transferred to a separatory funnel using 25 mL of DCM and washed with 50 mL of water. The AP was basified to pH > 10 using 4 mL of NaOH 25% and then extracted three times with 15 mL of DCM. The OPs were combined, dried over Na2SO4, filtered on a P3 sintered glass filter, and concentrated to dryness in a Rotavapor (40°C, 750-400 mbar). This material was dried under high vacuum (room temperature, 10-3 mbar) for 4 h to give 172 mg (neat yield: 94.1%) of a nearly colorless oil as 362-VRO244-02-001 crude 1#1. This material was used "as is" in the next step without further purification.

[0150] 1 H-NMR(300MHz,DMSO-d6)δ=8.62(t,J=4.7,1H,4),7.39-7.23(m,2H,6,8),7.22-7.05(m,2H,7,9),4.34(d ,J=5.9,2H,5),3.58(dd,J=7.8,5.1,1H,2),3.03(s,2H,1),2.82-2.58(m,1H,3''),2.48-2.30(m,1H,3').

[0151] 13 C-NMR(75MHz,DMSO-d6)δ=129.26(8),129.21(8),128.69(6),128.58(6),124.00(7),123.95(7),114.8 6(9),114.58(9),49.29(2),49.25(2),37.78(3),37.43(3),37.08(3),36.73(3),35.85(5),35.79(5).

[0152] Finally, the dipeptide was coupled to the epoxy head. In a 25 mL round-bottom flask equipped with a magnetic stirrer, 111 mg of 362-BB01-03-005 crude 1#1 (0.695 mmol, equiv.) and 5 mL of DMF were introduced, the resulting DMF was stirred at room temperature for 5 min, and then cooled to 0-5 °C using an ice bath. 307 mg of HATU (0.948 mmol, 1.5 equiv.) were added at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Then 167 mg of 362-VRO244-02-001 crude 1#1 (0.632 mmol, 1.0 equiv.) in solution in 5 mL of DMF was introduced at 0-5 °C, and the reaction mixture was stirred at this temperature for 5 min. Finally, 220 μL of DIPEA (1.264 mmol, 2.0 equiv.) was added dropwise at 0-5 °C. The resulting yellow mixture was stirred at 0-5° C. for 5 min, then the ice bath was removed and the mixture was stirred at room temperature overnight.

[0153] The reaction mixture, an orange solution, was transferred to a separatory funnel using 20 mL EtOAc and washed with 50 mL NaHCO 35%, then the AP was extracted twice with 15 mL EtOAc. The OP was combined and washed three times with 50 mL NaHCO 35% and once with 50 mL NaCl 11.6%. The OP was dried over Na2SO4, filtered on a P3 sintered glass filter and concentrated to dryness in a rotavapor (40 °C, 220-30 mbar). The material was dried under high vacuum (room temperature, 10-3 mbar) for 2 h to give 274 mg (raw yield: 106.7%) of a brownish orange solid as 362-VRO244-03-001 crude 1#1.

[0154] The crude material was purified by slurrying with 3 mL of EtOAc-heptane (2:1). The granular suspension was stirred at 40° C. for 10 min, then at room temperature for 15 min, and finally cooled to 0-5° C. using an ice bath before filtering. The suspension was filtered through an ultrafiltration apparatus equipped with a nylon membrane (0.45 μm), the filter cake was washed and triturated five times with 1 mL of cold EtOAc-heptane (1:1), and then dried under high vacuum (room temperature, 10-3 mbar) for 2 h to give 131 mg (raw yield: 51.0%) of off-white granules as 362-VRO244-03-001 slurry 1#1.

[0155] 1 H-NMR(300MHz,DMSO-d6)δ=8.89(d,J=8.6,1H,5),8.76(t,J=5.8,1H,8), 7.38-7.24(m,2H,10,12),7.24-7.11(m,2H,11,13),4.70(td,J=9.0,4.0 ,1H,6),4.33(d,J=5.8,2H,9),4.27-4.10(m,2H,2),3.69(d,J=1.8,1H,4 ),3.57(d,J=1.8,1H,3),2.91-2.55(br.m,2H,7),1.23(t,J=7.1,3H,1).

[0156] 13 C-NMR(75MHz,DMSO-d6)δ=129.08(10,12),129.02(10,12),128.76(10,12),128.65(10,12),124.02(11),114.92( 13),114.64(13),61.37(2),52.87(4),51.09(3),46.83(6),35.99(9),35.93(9),34.39(7),34.05(7),13.63(1).

[0157] Example 8: In vitro enzyme activity assay for isolated enzymes The ability of compounds VRO006_hydrol, VRO047_hydrol, VRO052_hydrol, VRO059_hydrol, VRO073_hydrol, VRO109_hydrol, VRO244_hydrol, VRO001_hydrol, VRO082_hydrol, VRO035_hydrol, VRO119_hydrol and VRO243_hydrol to inhibit isolated cathepsin B was evaluated in an enzyme assay and compared with the inhibitory activity of prior art E64c. In this assay, compounds VRO006, VRO042, VRO047, VRO052, VRO059, VRO073, VRO109, VRO244, VRO001, VRO082, VRO035, VRO119, and VRO243 were used, and the assay was performed in the presence of NaOH to mimic the ester cleavage that occurs in the gastrointestinal tract and release the free acid (VROxxx_hydrol compound) from the prodrug (corresponding VROxxx compound). The chemical structures of compounds VRO006, VRO042, VRO047, VRO052, VRO059, VRO073, VRO109, and VRO244 and their chemical names are shown in Table 2 above. The structures of VRO001, VRO082, VRO035, VRO119, and VRO243 are shown in Table 3 below. Cathepsin B was derived from porcine liver homogenate.

[0158] [Table 3]

[0159] The enzyme assay was performed as follows: homogenized porcine liver samples (75 mg fresh tissue per sample) were incubated with Z-FR-AMC (benzyloxycarbonyl-L-arginyl-L-arginine-4-methylcoumaryl-7-amide) at 50 mM in 1 M sodium acetate (pH 6) supplemented with 2.5 mM EDTA, 2.5 mM DTT and 0.1% Triton X-100, and graded concentrations of test substances (0.1 nM to 50 mM) for 1 h at 37°C. Stock solutions of test substances were 20 mM in DMSO. Fluorescence was measured at 445 nm using 365 nm as the excitation wavelength.

[0160] The compounds of the present invention VRO006_hydrol, VRO047_hydrol, VRO052_hydrol, VRO059_hydrol, VRO073_hydrol, VRO109_hydrol and VRO244_hydrol showed suitable inhibitory activity with IC50 of about 100 nM or less (see FIG. 1), whereas the comparative compounds VRO001_hydrol, VRO082_hydrol, VRO035_hydrol, VRO119_hydrol and VRO243_hydrol did not effectively inhibit catB with IC50 of more than 100, as can be seen from FIG. 2.

[0161] This assay demonstrated that the hydrogen from the amide at the center of the molecule is important for enzyme affinity and is probably involved in H-bonding with the protein. Indeed, VRO001_hydrol was proven unable to inhibit catB. On the other hand, the hydrogen on the amide next to the isoamylamine moiety is not involved in binding, since the activity from VRO006 is quite similar to that of E64c.

[0162] All modifications made in R1 were well tolerated. The methoxy compound VRO047_hydrol showed almost the same activity as E64c, as did VRO052_hydrol and VRO059_hydrol, as well as VRO073_hydrol. The activity was only slightly decreased with the bulky aromatic moiety, as in VRO052_hydrol and VRO073_hydrol. However, the S3 pocket of catB has a limited tolerance. Indeed, the bulky and highly electronegative -CF3 moiety present in VRO035_hydrol was not accepted, and the inhibitory activity of this compound was lost.

[0163] Concerning R3, which binds to the S2 pocket of catB, the tolerance was rather low. Even slight changes such as the introduction of an ether into VRO082_hydrol could not maintain the inhibitory activity. The bulk from the pyridinyl moiety added to VRO119_hydrol was also not tolerated. However, one candidate brought a surprise, especially considering the results obtained with other moieties at the R3 position. VRO109_hydrol contains a trifluoro moiety instead of the leucine present in E64c. Addition of such a trifluoro moiety at the R1 position caused a loss of inhibitory activity, while some other moieties, and even bulky moieties, managed to maintain such activity. Here, surprisingly, it was demonstrated that VRO109_hydrol shows increased inhibitory activity compared to E64c. Of all the candidates tested here, only VRO109_hydrol showed a small but significant increase in inhibitory activity compared to the prior art E64c. The trifluoro moiety is particularly advantageous because it is less likely to generate hydrogen bonds and significantly increases the lipophilicity of the compound compared to E64c.

[0164] Assays performed with VRO244_hydrol provide evidence that the trifluoro moiety at the R3 position is compatible with a bulky moiety at the R1 position (in this case, 2-fluorophenylmethyl).

[0165] Individually, the replacement of leucine with a trifluoro moiety and the methylation of the amide close to the isoamylamine moiety showed promising results in terms of both activity and drug mimicry, as explained above. However, their combination in VRO243_Hydrol showed a significant loss of activity. This is surely due to the 3D conformation of the catalytic site of the enzyme and how the trifluoro moiety is positioned there. This particular 3D layout is probably incompatible with the one obtained when the nitrogen is methylated.

[0166] Example 10: In vitro whole cell enzyme activity assay To confirm the results obtained with isolated catB (see Example 9), the inhibitory activity of the compounds of the invention against catB was evaluated in a whole cell assay, in which the free acid (hydrolyzed) form of the compounds of the invention was used.

[0167] Various concentrations of test substances (50, 10, 2, 0.4, 0.08, 0.016, 0.0032, and 0 mM) were placed in a 96-well plate, and 50'000 HEK293T cells were added to each well. The plate was then incubated at 37°C for 1 hour. The plate was centrifuged at 2000 rpm for 7 minutes, and the supernatant was removed. 100 ml of freshly prepared Z-FR-AMC substrate contained 1 M sodium acetate (pH 6.0), 2.5 mM EDTA, 2.5 mM DTT, 0.1% Triton X-100, and 20 mM Z-FR-AMC. After 1 hour of incubation, the fluorescence was measured at 445 nm using 365 nm as the excitation wavelength.

[0168] As shown in Figure 3, the inhibitory activity of VRO052_hydrol and VRO059_hydrol was confirmed in the whole cell assay. Such activity was closer to that of E64c than that of the isolated enzyme assay. Although the activity of VRO052_hydrol and VRO059_hydrol was slightly lower than that of E64c in the assay with isolated enzyme, these compounds show similar inhibitory activity to that of E64c in the whole cell assay. This improvement in the relative inhibitory activity of VRO052_hydrol and VRO059_hydrol is believed to be due to the fact that the compounds of the present invention are more lipophilic and therefore more easily enter the cells by crossing the cell membrane than the compounds of the prior art. Thus, this test confirms that the improved lipophilicity compensates for the slight decrease in inhibitory activity with the isolated enzyme. Based on these findings, it is expected that VRO052_hydrol and VRO059_hydrol will show similar inhibitory activity to E64d in vivo.

Claims

1. A compound of formula I or formula II, 【Chemistry 1】 【Chemistry 2】 During the ceremony, R １ is selected from 3-methylbutyl, 2-methoxyethyl, 2-fluorophenylmethyl, 3-(piperidin-1-yl)propyl, and 2-(pyridin-2-yl)ethyl; R ２ is selected from hydrogen and methyl; R ３ is selected from 2-methylpropyl and 2,2,2-trifluoroethyl; R ４ is hydrogen, The compound is R １ is 3-methylbutyl, i.R ３ is not 2-methylpropyl, or ii. R ３ is 2-methylpropyl, and R ２ is methyl; The compound is R ３ is 2,2,2-trifluoroethyl, R ２ is hydrogen.

2. R １ The compound of claim 1, wherein is selected from 3-methylbutyl, 3-(piperidin-1-yl)propyl, and 2-(pyridin-2-yl)ethyl.

3. R １ is 3-(piperidin-1-yl)propyl or 2-(pyridin-2-yl)ethyl, and R ３ is 2-methylpropyl, or R １ is 3-methylbutyl, and R ３ The compound of claim 1, wherein is 2,2,2-trifluoroethyl.

4. The compound of formula I is ethyl (2S,3S)-3-(((S)-1-(isopentyl(methyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxirane-2-carboxylate, ethyl (2S,3S)-3-(((S)-4-methyl-1-oxo-1-((2-(pyridin-2-yl)ethyl)amino)pentan-2-yl)carbamoyl)oxirane-2-carboxylate, ethyl (2S,3S)-3-(((S)-4-methyl-1-oxo-1-((3-(piperidine-1- ethyl (2S,3S)-3-(((S)-1-((2-2-methoxyethyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxirane-2-carboxylate, ethyl (2S,3S)-3-(((S)-1-((2-fluorobenzyl ... ((S)-4,4,4-trifluoro-1-(isopentylamino)-1-oxobutan-2-yl)carbamoyl)oxirane-2-carboxylate and ethyl (2S,3S)-3-(((S)-4,4,4-trifluoro-1-((2-fluorobenzyl)amino)-1-oxobutan-2-yl)carbamoyl)oxirane-2-carboxylate, preferably ethyl (2S,3S)-3-(((S)-4-methyl-1-oxo-1-((2-(pyridin-2-yl)ethyl )amino)pentan-2-yl)carbamoyl)oxirane-2-carboxylate, ethyl (2S,3S)-3-(((S)-4-methyl-1-oxo-1-((3-(piperidin-1-yl)propyl)amino)pentan-2-yl)carbamoyl)oxirane-2-carboxylate and ethyl (2S,3S)-3-(((S)-4,4,4-trifluoro-1-(isopentylamino)-1-oxobutan-2-yl)carbamoyl)oxirane-2-carboxylate; or the compound of formula II is (2S,3S)-3-(((S)-1-(isopentyl(methyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxirane-2-carboxylic acid, (2S,3S)-3-(((S)-4-methyl-1-oxo-1-((2-(pyridin-2-yl)ethyl)amino)pentan-2-yl)carbamoyl)oxirane-2-carboxylic acid, (2S,3S)-3-(((S)-4-methyl-1-oxo-1-((3-(piperidine-1- (2S,3S)-3-(((S)-1-((2-2-methoxyethyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxirane-2-carboxylic acid, (2S,3S)-3-(((S)-1-((2-fluorobenzyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxirane-2-carboxylic acid, (2S,3S)-3-(((S)-4, 4,4-trifluoro-1-(isopentylamino)-1-oxobutan-2-yl)carbamoyl)oxirane-2-carboxylic acid and (2S,3S)-3-(((S)-4,4,4-trifluoro-1-((2-fluorobenzyl)amino)-1-oxobutan-2-yl)carbamoyl)oxirane-2-carboxylic acid, preferably (2S,3S)-3-(((S)-4-methyl-1-oxo-1-((2-(pyridin-2-yl)ethyl)amino)pentan-2-yl 2. The compound of claim 1, wherein the compound is selected from the group consisting of (2S,3S)-3-(((S)-4-methyl-1-oxo-1-((3-(piperidin-1-yl)propyl)amino)pentan-2-yl)carbamoyl)oxirane-2-carboxylic acid, (2S,3S)-3-(((S)-4-methyl-1-oxo-1-((3-(piperidin-1-yl)propyl)amino)pentan-2-yl)carbamoyl)oxirane-2-carboxylic acid, and (2S,3S)-3-(((S)-4,4,4-trifluoro-1-(isopentylamino)-1-oxobutan-2-yl)carbamoyl)oxirane-2-carboxylic acid.

5. A compound according to any one of claims 1 to 4 for use in therapy.

6. 1. A compound of Formula I or Formula II for use in a method for treating a disease associated with decreased β-galactosidase activity, comprising: 【Transformation 3】 【Chemistry 4】 During the ceremony, R １ is selected from 3-methylbutyl, 2-methoxyethyl, 3-methoxypropyl, 2-fluorophenylmethyl, 3-(piperidin-1-yl)propyl, and 2-(pyridin-2-yl)ethyl; R ２ is selected from hydrogen and methyl; R ３ is selected from 2-methylpropyl and 2,2,2-trifluoroethyl; R ４ is hydrogen, The compound is R １ is 3-methylbutyl, i.R ３ is not 2-methylpropyl, or ii. R ３ is 2-methylpropyl, and R ２ is methyl; The compound is R ３ is 2,2,2-trifluoroethyl, R ２ is hydrogen.

7. 7. The compound for use according to claim 6, wherein the compound of formula I or formula II is as defined in any one of claims 2 to 4, or wherein the compound of formula I is ethyl (2S,3S)-3-(((S)-1-((3-3-methoxypropyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxirane-2-carboxylate and the compound of formula II is (2S,3S)-3-(((S)-1-((3-3-methoxypropyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxirane-2-carboxylic acid.

8. 7. The compound for use according to claim 6, wherein the disease associated with decreased β-galactosidase activity is selected from GM-1 gangliosidosis, Morquio syndrome type B, Chediak-Higashi syndrome, galactosialidosis, metachromatic leukodystrophy, Gaucher disease, Alzheimer's disease and traumatic brain injury.

9. 9. The compound for use according to claim 8, wherein the disease associated with decreased β-galactosidase activity is selected from GM-1 gangliosidosis, Morquio syndrome type B, Chediak-Higashi syndrome, galactosialidosis, metachromatic leukodystrophy and Gaucher disease.

10. 7. The compound for use according to claim 6, wherein said compound is for use in a method for treating a disease associated with decreased β-galactosidase activity by inhibiting the activity of cathepsin B.

11. 7. The compound for use according to claim 6, wherein said compound is a compound of formula I and said compound is administered enterally to a patient in need thereof.

12. 7. The compound for use according to claim 6, wherein the compound is a compound of formula II and the compound is administered to a patient in need thereof by a route of administration that is not enteral administration.

13. 7. The compound for use of claim 6, wherein the compound of formula I or the compound of formula II is administered to a patient in need thereof in combination with at least one additional compound selected from a pharmacological chaperone of β-galactosidase, a pharmacological chaperone of glucocerebrosidase, a calcium channel blocker, a glucosylceramide synthase inhibitor, and mixtures thereof.

14. the at least one additional compound is selected from the group consisting of N-substituted 5-amino-1-hydroxymethyl-cyclopentanetriol, N-octyl-4-epi-β-valienamine (NOEV), ambroxol, cis-(+)-[2-(2-dimethylaminoethyl)-5-(4-methoxyphenyl)-3-oxo-6-thia-2-azabicyclo[5.4.0]undeca-7,9,11-trien-4-yl]ethanoate (diltiazem), [(3S)-1-azabicyclo[2.2.2]octan-3-yl]N-[2-[2-(4-fluorophenyl)-1,3-thiazol-4-yl]propan-2-yl]carbamate (benglustat), iminosugars, and mixtures thereof; and such iminosugars are preferably selected from the group consisting of N-butyl-deoxygalactonojirimycin.

14. The compound for use according to claim 13, selected from the group consisting of (miglustat), 4-epi-isofagomine, 5a-C-pentyl 4-epi-isofagomine, 5a-C-methyl 4-epi-isofagomine, 1,5-dideoxy-1,5-imino-(L)-ribitol (DIR), 5-C-alkyl-imino-L-ribitol, N-(dansylamino)hexylaminocarbonylpentyl-1,5-dideoxy-1,5-imino-D-galactitol, 5N,6S-(N'-butyliminomethylidene)-6-thio-1-deoxygalactonojirimycin (6S-NBI-DGJ), N-nonyl-deoxygalactonojirimycin, N-butyldeoxynojirimycin (miglustat), isofagomine (afegorstat), AZ-3102 and mixtures thereof.

15. A process for producing a compound according to any one of claims 1 to 4, comprising: a) a compound of formula III 【Transformation 5】 with a base such as N,N-diisopropylethylamine (DIPEA); b) reacting the deprotonated compound obtained in step a) with 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) in a basic medium; c) reacting the compound obtained in step b) with tetramethylurea (TMU) in a basic medium; d) reacting the compound obtained in step c) with an amine of formula IV in a basic medium 【Transformation 6】 to produce, for example, a dipeptide of formula V 【Transformation 7】 forming a e) Removal of the Boc group from a compound of formula V by reacting such a compound with trifluoroacetic acid in an acidic medium to give a compound of formula VI 【Transformation 8】 and f) deprotonating the compound of formula VI with a base such as DIPEA; g) reacting the deprotonated compound obtained in step f) with HATU in a basic medium; h) reacting the compound obtained in step g) with TMU in a basic medium; i) reacting the compound obtained in step h) with a compound of formula VII 【Chemistry 9】 to obtain, for example, a compound of formula I; j) optionally hydrolyzing the ester moiety of said compound of formula I to obtain, for example, a compound of formula II; The process includes:

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