Patent Application
20250073198
The present invention relates to new cathepsin B inhibitors, which are effective in therapy, and in particular in the treatment of diseases associated with an impaired activity of the β-galactosidase, such as GM-1 gangliosidosis, Morquio syndrome type B, Chediak-Higashi Syndrome, Galactosialidosis, Metachromatic leukodystrophy, Gaucher Disease, Alzheimer disease and traumatic brain injury.
The 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 catalyse the hydrolysis of the terminal β-D-galactose residue from various substrates, including lactose, oligosaccharides, glycolipids and glycoproteins, by hydrolysis. This enzyme is especially known for its ability to hydrolyse lactose into glucose and galactose.
Among diseases associated with impaired β-galactosidase activity, one can cite two lysosomal storage diseases: the GM1-gangliosidosis and the 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 on the gene encoding this enzyme may lead to:
Any of those lead to a lower amount of functional β-gal in cells and therefore in an accumulation of substrate in the organism. Storage of GM1 leads to GM1-gangliosidosis while accumulation of keratan sulfate relates to Morquio syndrome type B.
In the particular case of the GM1-gangliosidosis, the reduced activity of β-gal impairs GM1 breakdown. This results in a substrate accumulation within the lysosome, which is experiencing a disruption in its functionality. Since the highest concentration of GM1 is located in the CNS, excessive accumulation of GM1 will lead to the death of neuronal cells, resulting in a neurodegenerative course. The devastating effects of GM1-gangliosidosis on the central nervous system make it an essential need to find therapeutic approaches that are able to cross the blood-brain barrier. Because β-gal is used to hydrolyse the β-galactosyl residue, its deficiency leads to the storage of other substrates, like glycolipids GA1, oligosaccharides from glycoproteins and glycosaminoglycans. Depending on the organ where these substrates are produced, the β-gal defect will also result in some non-neurological manifestations, such as dysmorphic face, disorders in bone development (known as dysostosis) or an enlargement of both the spleen and the liver (a condition called hepatosplenomegaly which shows an enlarged abdomen).
Several therapeutic strategies have been implemented to treat GM1-gangliosidosis. However, conventional therapies fail to achieve treatment of the symptoms in the central nervous system and therefore provide no benefit in terms of reduction of neurodegeneration. Hematopoietic stem cells therapy (HSCT) was unsuccessfully attempted in a patient with infantile form (N. Brunetti-Pierri and F. Scaglia, GM1 gangliosidosis: Review of clinical, molecular, and therapeutic aspects, Mol. Genet. Metab., 2008, 94(4), 391-396). Recombinant enzymes have also been used, but are not able to cross the blood-brain barrier. Substrate reduction therapy (SRT), which consists in administration of small molecules that partially inhibits the biosynthesis of the accumulated substrates, was attempted on three patients with types II and III GM1 gangliosidosis. In particular, the administration of Miglustat made it possible to slow-down the degenerative course, or even to reverse it (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). Gene therapy was conclusive in both mice (R. C. Baek 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) and feline (V. J. McCurdy et al., Sustained normalization of neurological disease after intracranial gene therapy in a feline model, Sci. Transl. Med., 2014, 6(231), 231-279) models through intracerebroventricular injection of an engineered adeno-associated virus/GLB1 vector. Many chaperoned were developed until now, with Migalastat being the first one to be tested. Since it was not specific enough for the GM1-gangliosidosis, other iminosugars were developed, noticeably N-octyl-4-epi-β-valienamine (NOEV) and 5N,6S—(N′-butyliminomethylidene)-6-thio-1-deoxygalactonojirimycin (6S-NBI-DGJ) that showed promising results in the murine model (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 and T. Takai et al., A Bicyclic 1-Deoxygalactonojirimycin Derivative as a Novel Pharmacological Chaperone for GM1 Gangliosidosis, Mol. Ther., 2013, 21(3), 526-532). Unfortunately, chaperone therapy is based on the residual activity of the misfolded β-gal, resulting in a total inefficacy of such treatment in cases where the enzyme is totally inactive.
Therefore, there is need to further develop therapeutic strategies targeting GM1-gangliosidosis, which would allow to reach the central nervous system, and which would be applicable even to cases wherein the β-gal is totally inactive.
The Morquio syndrome type B (also known as mucopolysaccharidosis type IVB, MPS IVB) is caused by some specific mutations in the GLB1 gene encoding for β-gal, which catalyses the degradation of some mucopolysaccharides. This defect results into an accumulation of keratan sulphate that mainly leads to skeletal dysplasia and frequent respiratory infections. Patients exhibit a normal development of their neurocognitive functions, but in some advanced stages of the disease, a spinal cord compression might occur due to the skeletal deformities leading to an impairment of the central nervous system. Beyond bone problems, MPS IVB is characterized by some ocular manifestation, such as corneal clouding, retinopathy or glaucoma, and cardiorespiratory disease.
Not only is there currently no approved treatment for MPS IVB, but also the lack of knowledge of the disease and its clinical manifestations, linked to the extreme rarity of this condition, limits the possibility of a well-designed clinical trial, even if some molecules showed promising results as pharmaceutical chaperones. As for GM1-gangliosidosis, the only available therapy so far is symptomatic. Therefore, there is an acute need for developing curative therapeutic strategies.
Other diseases have been shown to be associated with impaired activity of the β-galactosidase, such as Chediak-Higashi Syndrome, Galactosialidosis, Metachromatic leukodystrophy, Gaucher Disease, Alzheimer disease and traumatic brain injury.
One of the key systems impacted by a number of such diseases is the central nervous system. This might come from the fact that the brain, due to its limited regeneration capacity and neuron sensitivity, is highly vulnerable, namely towards LSDs, and in particular GM1-gangliosidosis. Damages to the central nervous system are also significant in the case of Alzheimer disease and traumatic brain injury. This sensitivity of the central nervous system causes a major problem in terms of treatment because the entry into the brain is highly protected by the blood-brain barrier, which only allows for small molecules to pass through, and makes it impossible, for example, to deliver enzymes in the brain via the bloodstream. The treatment of diseases causing neurological symptoms is therefore a significant challenge.
Among publications related to activity of β-galactosidase, it was described that cathepsin B (catB) had a regulatory role in the maturation and degradation of β-galactosidase (Y. Okamura-Oho et al., Maturation and degradation of β-galactosidase in the post-Golgi compartment are regulated by cathepsin B and a non-cysteine protease, FEBS Lett., 1997, 419(2-3), 231-234). These authors identified that inhibition of cathepsin B promoted the activity of β-galactosidase. In view of developing new therapies for diseases involving a reduced activity of the β-galactosidase or the absence of activity of the β-galactosidase, it would be desired to identify new inhibitors of cathepsin B. It would further be advantageous to provide such inhibitors that exhibit properties making them suitable for use as a drug, namely inhibitors exhibiting good chemical stability, permeability through the cell membrane, solubility, metabolic stability, plasma stability and/or transporters affinity.
One of the first identified catB inhibitor was the oxirane E64, a natural epoxysuccinyl dipeptide found in Aspergillus japonicus that irreversibly and non-selectively inhibits cathepsins (K. Hanada et al., Isolation and Characterization of E-64, a New Thiol Protease Inhibitor, Agric. Biol. Chem., 1978, 42(3), 523-528). The chemical structure of E64 is provided in [Table 1].
Since the discovery of E64, a number of analogues have been developed. The most famous representatives of this class of compounds are provided in [Table 1] below and have been disclosed in the following publications: T. Otsuka et al., WF14865A and B, new cathepsins B and L inhibitors produced by Aphanoascus fulvescens. I. Taxonomy, production, purification and biological properties, J. Antibiot. (Tokyo)., 2000, 53(5), 449-458, A. J. Barrett et al., L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L, Biochem. J., 1982, 201(1), 189-198, M. Murata et al., Novel epoxysuccinyl peptides. Selective inhibitors of cathepsin B, in vitro, FEBS Lett., 1991, 280(2), 307-310, D. J. Buttle and J. Saklatvala, Lysosomal cysteine endopeptidases mediate interleukin 1-stimulated cartilage proteoglycan degradation, Biochem. J., 1992, 287(2), 657-661, D. J. Buttle et al., CA074 methyl ester a proinhibitor for intracellular cathepsin B, Arch. Biochem. Biophys., 1992, 299(2), 377-380, and N. Schaschke et al., Substrate/propeptide-derived endo-epoxysuccinyl peptides as highly potent and selective cathepsin B inhibitors, FEBS Lett., 1998, 421(1), 80-82.
Currently, the only molecule that has demonstrated catB inhibition properties, while exhibiting goad absorption, distribution, metabolism, and excretion is E64d, known as Loxistatin. This compound, an ester prodrug of E64c, has been the subject of clinical studies in Japan as a treatment for muscular dystrophies. Despite discontinuing the study due to unclear effectiveness, these 3-year trials (including pediatric patients) yielded extensive pharmacokinetics (PK) and pharmacodynamics (PD) data, while also demonstrating the non-toxicity for human (E. Satoyoshi, Therapeutic Trials on Progressive Muscular Dystrophy, Intern. Med., 1992, 31(7), 841-846, G. Hook et al., Cathepsin B is a New Drug Target for Traumatic Brain Injury Therapeutics: Evidence for E64d as a Promising Lead Drug Candidate, Front. Neurol., 2015, 6, 178 and T. Miyahara et al., Phase I study of EST, a new thiol protease inhibitor. The 1st report: Safety and pharmacokinetics at single administration, Rinsho yakuri/Japanese J. Clin. Pharmacol. Ther., 1985, 16(2), 357-365).
Unfortunately, this compound presents a low penetration of the blood-brain barrier, which significantly reduces the chances of reaching a therapeutic target in the central nervous system (CNS). This makes this compound of low relevance for pathologies associated with the presence of catB in the brain, like GM-1 gangliosidosis, Alzheimer disease and traumatic brain injury.
There is therefore a need to develop new compounds capable of irreversibly inhibiting catB. There is a further need to identify such compounds, which are further capable of reaching the central nervous system by penetrating the blood-brain barrier.
In a first aspect, the invention provides a compound of Formula I or Formula II
In a second aspect, the present invention provides a compound of the invention, for use in therapy, preferably for the treatment of diseases associated with impaired β-galactosidase activity.
In a third aspect, the present invention provides a compound of a compound of Formula I or Formula II
In a fourth aspect, the present invention provides a process for producing a compound according to the invention, comprising the steps of:
The present inventors have advantageously developed new compounds that are active as inhibitors of cathepsin B and therefore beneficial in the treatment of diseases associated with impaired β-galactosidase (β-gal) activity. Furthermore, the identified compounds have properties that make them particularly suitable for use as a medicament. In particular, such compounds are characterized by one or more of the following advantageous properties:
The compounds of the invention are further advantageous in that they are suitable to cross the blood-brain barrier, which makes it possible to treat diseases impacting the central nervous system. This represents a critical improvement, over the prior art compound E64d. Furthermore, the compounds of the invention exhibit good selectivity towards inhibition of catB over other enzymes of the cathepsin family.
The compounds of the present invention are of Formula I or Formula II
The structure of the compounds of the invention consists of two blocks, with a dipeptide structure including both nitrogen atoms and the 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 gastrointestinal tract, upon enteral administration. The ester moiety in the far end of the epoxy warhead is cleaved to release the free acid. It is in this cleaved form that the compounds are therapeutically active. Therefore, the compounds of Formula I are advantageously for use as prodrugs, while compounds of Formula II are advantageously for use as therapeutic agents.
Examples of compounds according to the invention are provided in [Table 2] below:
Examples of compounds of Formula II according to the present invention are the counterparts of the compounds provided in [Table 2], in the form of the free acid. 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 (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)pentan-2-yl)carbamoyl)oxirane-2-carboxylic acid (VRO059_hydrol), (2S,3S)-3-(((S)-4-methyl-1-oxo-1-((2-(pyridin-2-yl)ethyl)amino)pentan-2-yl)carbamoyl)oxirane-2-carboxylic acid (VRO052_hydrol), (2S,3S)-3-(((S)-4,4,4-trifluoro-1-(isopentylamino)-1-oxobutan-2-yl)carbamoyl)oxirane-2-carboxylic acid (VRO109_hydrol) and (2S,3S)-3-(((S)-4,4,4-trifluoro-1-((2-fluorobenzyl)amino)-1-oxobutan-2-yl)carbamoyl)oxirane-2-carboxylic acid (VRO244_hydrol).
The compound of the invention are advantageously for use in therapy.
The compounds of the invention are also advantageously for use in the treatment of a disease associated with impaired β-galactosidase activity. In addition to the compounds according to the present invention recited above, a compound of Formula I or Formula II, wherein R1 is 3-methoxypropyl and wherein R2, R3 and R4 are as described above, is also for use in the treatment of a disease associated with impaired β-galactosidase activity. Preferably such compound of Formula I for use in the treatment of a disease associated with impaired 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 compound of Formula II for use in the treatment of a disease associated with impaired 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_hdrol).
Diverse properties of the compounds of Formula II make them more suitable to cross the blood-brain barrier than the prior art catB inhibitors such as E64b and its known derivatives. In particular, the present compounds are advantageously characterized by
As demonstrated by the present inventors, the specific radicals R1, R2 and R3 described above are advantageous in that they improve the drug-likeness and the ability to cross the blood-brain barrier of the compounds of Formula II compared to E64c, while active as inhibitors of catB. It has also been shown that it is essential that R4 be a hydrogen atom. Without wishing to be being bound by theory, it is believed that this hydrogen atom is involved in a hydrogen-bond with the protein.
Unlike the hydrogen in position R4, the hydrogen in position R2 can be replaced by a methyl without harming the inhibitor activity of the compound, as demonstrated on [
The present inventors have also identified that it was possible to advantageously use more bulky radicals in position R1 such as those recited herein, without harming the interaction between the compound and the S3 pocket of catB (see [
However, not all bulky substituents are suitable for maintaining sufficient inhibition of catB and, for example, the present inventors demonstrated that the bulky and highly electronegative 3,3,3-trifluoroethyl significantly reduced the efficiency of VRO035_hydrol in the enzymatic assay against isolated catB (see [
Particularly preferred radicals in position R1 are 2-(pyridin-2-yl)ethyl and 3-(piperidin-1-yl)propyl. Indeed, compounds VRO052_hydrol and VRO059_hydrol, in which these radicals were present in position R1, exhibited significantly improved inhibitory activity against catB in a whole cell assay. Without wishing to be bound by theory, it is believed that this is due to the increased lipophilicity of these compounds, which more easily cross the cell membrane. The in-vivo efficiency of compounds bearing these radicals in position R1 is thus expected to be higher than that of E64c.
With respect to R3, which interacts with the S2 pocket of catB, the present inventors also achieved the replacement of 3-methylbutyl present in E64d by diverse radicals improving the drug-likeness of the compound. Most bulky radicals negatively impacted the inhibitory activity of the compound, such as for example the pyridinyl moiety (VRO119_hydrol), as demonstrated by [
The combination of a suitable radical in position R1 with a suitable radical in position R3 leads to compounds that still exhibit suitable inhibitory activity, as shown on [
It is however key not to combine the bulky 2,2,2-trifluoroethyl in position R3 with a methyl in position R2, as this specific combination deprives VRO243_hydrol from inhibitory activity on the isolated enzyme (see [
The advantages of the compounds of Formula II identified above make the corresponding compounds of Formula I advantageous prodrugs.
Due to their ability to inhibit catB, the compounds of Formula I and of Formula II, as described above, can advantageously be used in the treatment of a disease associated with impaired β-galactosidase activity. This is due to previously demonstrated regulatory role of catB in the maturation and degradation of 3-galactosidase, as explained in detail in the background section.
Examples of diseases associated with impaired β-galactosidase activity include GM-1 gangliosidosis, Morquio syndrome type B, Chediak-Higashi Syndrome, Galactosialidosis, Metachromatic leukodystrophy, Gaucher Disease, Alzheimer disease and traumatic brain injury. Preferably, the compounds of the invention are for use in the treatment of a disease selected from GM-1 gangliosidosis, Morquio syndrome type B, Chediak-Higashi Syndrome, Galactosialidosis, Metachromatic leukodystrophy and Gaucher Disease, more preferably GM-1 gangliosidosis and Morquio syndrome type B and most preferably GM-1 gangliosidosis.
In other words, the invention relates to the use of a compound of the invention for the manufacture of a medicament. Preferably it relates to the use of a compound of Formula I or of Formula II, as described above, for the manufacture of a medicament for the treatment of a disease associated with impaired β-galactosidase activity, preferably a medicament for the treatment of a disease selected from GM-1 gangliosidosis, Morquio syndrome type B, Chediak-Higashi Syndrome, Galactosialidosis, Metachromatic leukodystrophy, Gaucher Disease, Alzheimer disease and traumatic brain injury, more preferably a disease selected from GM-1 gangliosidosis, Morquio syndrome type B, Chediak-Higashi Syndrome, Galactosialidosis, Metachromatic leukodystrophy and Gaucher Disease, even more preferably a disease selected from GM-1 gangliosidosis and Morquio syndrome type B and most preferably GM-1 gangliosidosis.
In still other words, the invention relates to a method of treatment of a disease in a patient in need thereof, comprising administering a compound of the invention to such patient. It also relates to a method of treatment of a disease associated with impaired β-galactosidase activity in a patient in need thereof comprising administering a compound of Formula I or of Formula II, as described above, to such patient. More preferably such disease is selected from GM-1 gangliosidosis, Morquio syndrome type B, Chediak-Higashi Syndrome, Galactosialidosis, Metachromatic leukodystrophy, Gaucher Disease, Alzheimer disease and traumatic brain injury, more preferably such disease is selected from GM-1 gangliosidosis, Morquio syndrome type B, Chediak-Higashi Syndrome, Galactosialidosis, Metachromatic leukodystrophy and Gaucher Disease, even more preferably such disease is selected from GM-1 gangliosidosis and Morquio syndrome type B and most preferably GM-1 gangliosidosis.
In a particular aspect, the compounds of Formula I or of Formula II are for use in a method of inhibiting catB. In other words, the invention relates to the use of a compound of Formula I or of Formula II for inhibiting catB. In still other words, it relates to a method of inhibiting catB in a subject, comprising administration of such compounds to the subject.
In another particular aspect, the compound of Formula I or of Formula II is administered to a patient in need thereof in combination with at least one additional compound that is active to treat one of the diseases associated with impaired β-galactosidase mentioned above. In a preferred aspect, such additional compound is selected from a pharmacological chaperone of the β-galactosidase, a pharmacological chaperone of the glucocerebrosidase, a calcium channel blocker and/or a glucosylceramide synthase inhibitor, and mixtures thereof. In another preferred aspect, such additional compound is selected from the group consisting of N-substituted 5-amino-1-hydroxymethyl-cyclopentanetriols, N-octyl-4-epi-beta-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 (Venglustat), an iminosugar and mixtures thereof. More preferably, such iminosugar is 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)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 (Afegostat), AZ-3102 and mixtures thereof.
The compounds of Formula I of the present invention are preferably administered by enteral administration. By enteral administration, it is intended any means of administration the compound of Formula I is conveyed into the gastrointestinal tract. Enteral administration of the compounds of Formula I is advantageous, as such compounds are hydrolysed to the free acid in the gastrointestinal tract. The compounds of Formula II of the present invention can be administered by any route and are preferably administered by an administration route that is not enteral administration.
In a preferred aspect, the compound of Formula I or of Formula II is administered in the form of a pharmaceutical composition, preferably comprising a pharmaceutically acceptable carrier, diluent and/or excipient.
The term “carrier” refers to an organic or inorganic component, of a natural or synthetic nature, in which the active component is combined in order to facilitate, enhance or enable application. According to the invention, the term “carrier” also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a patient.
Possible carrier substances for parenteral administration are e.g. sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers.
The term “excipient” when used herein is intended to indicate all substances which may be present in a composition described herein and which are not active ingredients such as, e.g., carriers, binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffers, flavoring agents, or colorants.
The excipient of the composition can be any pharmaceutically acceptable excipient, including specific carriers able to target specific cells or tissues. As stated earlier, possible pharmaceutical compositions include those suitable for oral, rectal, topical, transdermal, buccal, sublingual, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. For these formulations, conventional excipients can be used according to techniques well known by those skilled in the art. The compositions for parenteral administration are generally physiologically compatible sterile solutions or suspensions, which can optionally be prepared immediately before use from solid or lyophilized form. For oral administration, the composition 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 may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. For compressed tablets, binders, which are agents, which impart cohesive qualities to powdered materials, are also necessary. For example, starch, gelatine, sugars such as lactose or dextrose, and natural or synthetic gums can be used as binders. Disintegrants are also necessary in the tablets to facilitate break-up of the tablets. Disintegrants include starches, clays, celluloses, algins, gums and cross-linked polymers. Moreover, lubricants and glidants are also included in the tablets to prevent adhesion of the tablet material to surfaces in the manufacturing process and to improve the flow characteristics 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 into ointment, cream or gel form and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and dimethylformamide. For transmucosal administration, nasal sprays, rectal 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 glycols (carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate.
The pharmaceutical composition may be formulated to substantially release the active drug immediately upon administration or at any predetermined time or a time period after administration.
The compound of Formula I or of Formula II is administered in an effective amount. The term “effective amount” refers to an amount necessary to obtain a physiological effect. The physiological effect may be achieved by one application dose or by repeated applications. The dosage administered may, of course, vary depending upon 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 kind of concurrent treatment; the frequency of treatment; and the effect desired and can be adjusted by a person skilled in the art. In a particular embodiment, the pharmaceutical composition comprises the compound of Formula I or of 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 of 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.
The compounds of the invention can be produced by a process comprising forming an amid bond between two amino acids to produce a dipeptide structure and coupling the formed dipeptide with the epoxy warhead.
Therefore, the invention relates to a process for producing the compounds of the invention comprising:
Steps a) to d) are sub-steps of the first stage of the synthesis of the compounds of the invention, wherein the dipeptide part of the structure is formed. This involves the formation of an amide bond between an acid and a base and is commonly called peptide coupling. Many coupling reagents can be used and are well-known to the person skilled in the art. However, HATU is particularly suitable as it is relatively tolerant towards different types of coupling starting materials. Preferably, such steps are performed in basic media and more preferably at 0° C. The mechanism to amide bond formation mediated by HATU is depicted in [
In step e), the Boc protecting group is removed, in an acidic media, preferably with 25% TFA in DCM. The mechanism starts by the protonation of the t-butyl carbamate. This leads to the loss of the t-butyl cation and the formation of a carbamic acid. The carboxylate from TFA will then deprotonate the carbamic acid, thus releasing the free amine and forming one equivalent of carbon dioxide. During the work-up, a first acidic extraction allows to remove some organic impurities while the amine stays in the aqueous layer as a TFA salt. The free amine can be recovered in the organic layer upon basification.
In steps f) to i), the epoxy warhead is bound to the deprotected dipeptide structure. This is performed in a similar way as the formation of the dipeptide structure, since it also involves an amide bond formation. A compound of Formula I is obtained. Again, a variety of coupling agents can be used, which are well-known to the person skilled in the art. Such coupling agents include N,N′-Dicyclohexylcarbodiimide (DCC) or HATU. However, as DCC has been described as being allergenic, it is preferred to use HATU. HATU exhibited the same capacities and even gave better yields than DCC.
In cases wherein it is desired to form a compound of Formula II, the process comprises one further step of hydrolysing the ester moiety of the compound of Formula I, to release the free acid of Formula II. Conditions appropriate for such hydrolysis are well-known to the person skilled in the art. For example the compound of Formula I can be provided in a basic medium.
1H and 13C NMR as well as 2D-NMR (COSY, HSQC and HMBC) were obtained on a Bruker Ascend 300 MHz spectrometer. All measurements were performed at RT. Chemical shifts (δ, expressed in ppm) were referenced against solvent peak. all syntheses were followed by UPLC-MS using an ACQUITY UPLC system from Waters, equipped with a UV detector and coupled to an SQD2. The column was a UPLC BEH C18 (50×2.1 mm, 1.7 μm), the detection was performed at 214 nm, the analyses were run at 40° C. with a flow rate of 0.5 mL/min and elution used 0.06% formic acid in water as eluent A and 0.06% formic acid in acetonitrile as eluant B. The gradient was going from 5 to 100% of B in 5 minutes, staying for 2 minutes at 100% of B, then re-equilibrated to 5% for 30 seconds.
The TLC analysis were all performed on Merck Silica Gel F254 plates and revealed under both 254 and 366 nm and then using hanessian's stain.
Firstly, the dipeptide was formed. In a 25 mL round-bottomed flask, equipped with a magnetic stirrer, 500 mg of Boc-L-Leu (2.162 mmol, 1.0 eq.) and 10 mL of DCM were introduced, the resulting colourless solution was stirred at RT for 5 min. then cooled down to 0-5° C. using an ice-bath. 1.051 g of HATU (3.243 mmol, 1.5 eq.) were added at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Then 312 mg of N,3-dimethylbutan-1-amine hydrochloride (2.270 mmol, 1.05 eq.) were introduced at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Finally, 1.1 mL of DIPEA (6.485 mmol, 3.0 eq.) were added dropwise at 0-5° C. The turbid and slightly yellow mixture was stirred for 5 min at 0-5° C. then the ice-bath was removed and the mixture was stirred O.N. at RT.
The reaction mixture, a clear yellowish solution was transferred into a separating funnel using 10 mL of DCM and washed with 50 mL of NaCl 2.6%, then AP was extracted once with 10 mL of DCM. OPs were combined and washed with 50 mL of NaCl 2.6%, then AP was extracted once with 10 mL of DCM. OPs were combined, washed once with 50 mL of HCl 0.25 M, once with 50 mL of NaHCO3 10% and finally with 50 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 850-70 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h to yield 766 mg (raw yield: 112.7%) of an orange pasty material as 362-VRO006-01-001 crude1 #1.
The crude material was purified by CC using 40 g of silica gel (column with ø=3.2 cm and h=13.5 cm) and elution followed using TLC using heptane-EtOAc (7:3) as eluant; Rf=0.44. The product was eluted with a gradient from pure heptane to heptane-EtOAc (6:4), fractions containing the desired product were combined and concentrated to dryness into the rotavapor (40° C., 200-50 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 1h to yield 610 mg (raw yield: 89.7%) of an almost colourless oil as 362-VRO006-01-001 CC1 #1.
1H-NMR (300 MHz, 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).
13C NMR (75 MHz, 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).
The Boc protecting group was then removed as follows. In a 25 mL round-bottomed flask, equipped with a magnetic stirrer, 601 mg of 362-VRO006-01-001 CC1 #1 (1.911 mmol, 1.0 eq.) and 2.1 mL of a solution of 10% HCl in EtOAc were introduced, the resulting colourless solution was stirred at RT for 3h30. The reaction mixture was transferred into a separating funnel using 25 mL of EtOAc and washed with 25 mL of water. AP was basified using 6 mL of NaOH 25% to reach pH>10, then extracted 3 times with 25 mL of EtOAc. OPs were combined and washed using 25 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 250-70 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 1h to yield 371 mg (raw yield: 90.6%) of an almost colourless oil as 362-VRO006-02-001 crude1 #1. This material was used “as it” without further purification in the next step.
1H-NMR (300 MHz, 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 (br. 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).
Finally, the dipeptide was coupled to the epoxy warhead. In a 25 mL round-bottomed flask, equipped with a magnetic stirrer, 282 mg of 362-BB01-03-001 crude1 #1 (1.759 mmol, eq.) and 5 mL of DCM were introduced, the resulting white suspension was stirred at RT for 5 min. then cooled down to 0-5° C. using an ice-bath. 814 mg of HATU (2.512 mmol, 1.5 eq.) were added at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Then 359 mg of 362-VRO006-02-001 crude1 #1 (1.675 mmol, 1.0 eq.) in solution in 5 mL of DCM were introduced at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Finally, 875 μL of DIPEA (5.024 mmol, 3.0 eq.) were added dropwise at 0-5° C. The resulting yellow mixture was stirred for 5 min at 0-5° C. then the ice-bath was removed and the mixture was stirred O.N. at RT.
The reaction mixture, a clear yellow solution, was transferred into a separating funnel using 10 mL of DCM and washed with 50 mL of NaCl 2.6%, then AP was extracted once with 10 mL of DCM. OPs were combined, and washed once with 50 mL NaCl 2.6%, then AP was extracted once with 10 mL of DCM. OPs were combined and washed once with 50 mL of HCl 0.25 M, once with 50 mL of NaHCO3 10% and once with 50 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 220-30 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h to yield 701 mg (raw yield: 117.4%) of a pale yellowish oil as 362-VRO006-03-001 crude1 #1.
The crude material was purified by CC using 40 g of silica gel (column with ø=3.2 cm and h=13.5 cm) and elution followed using TLC using heptane-EtOAc (1:1) as eluant; Rf=0.53. The product was eluted with a gradient from heptane-EtOAc (9:1) to heptane-EtOAc (1:1), fractions containing the desired product were combined and concentrated to dryness into the rotavapor (40° C., 200-50 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h30 to yield 334 mg (raw yield: 55.9%) of a colourless oil as 362-VRO006-03-001 CC1 #1.
1H-NMR (300 MHz, 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).
13C-NMR (75 MHz, 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).
Firstly, the dipeptide was formed. In a 50 mL round-bottomed flask, equipped with a magnetic stirrer, 3.232 g of Boc-L-Leu (13.975 mmol, 1.05 eq.) and 35 mL of DCM were introduced, the resulting turbid solution was stirred at RT for 5 min. then cooled down to 0-5° C. using an ice-bath. 6.471 g of HATU (19.946 mmol, 1.5 eq.) were added at 0-5° C. and the reaction mixture was stirred for 5 minutes a this temperature. Then 1157 μL of 2-2-methoxyethylamine (13.309 mmol, 1.0 eq.) were introduced at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Finally, 4.6 mL of DIPEA (26.618 mmol, 2.0 eq.) were added dropwise at 0-5° C. The resulting turbid and yellowish solution was stirred for 5 min at 0-5° C. then the ice-bath was removed and the mixture was stirred O.N. at RT.
The reaction mixture, a clear yellow solution, was transferred into a separating funnel using 10 mL of DCM and washed with 50 mL of NaCl 2.6%, then AP was extracted once with 10 mL of DCM. OPs were combined and washed once with 50 mL of HCl 0.5 M, once with 50 mL of NaHCO3 10% and finally with 50 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 850-70 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h40 to yield 6.986 g (raw yield: 182.0%) of a pale yellow oil as 362-VRO047-01-001 crude1 #1.
The crude material was purified by CC using 35 g of silica gel (column with ø=3.2 cm and h=11.8 cm) and elution followed using TLC using heptane-EtOAc (1:1) as eluant; Rf=0.46. The product was eluted with a gradient from pure heptane to heptane-EtOAc (1:1), fractions containing the desired product were combined and concentrated to dryness into the rotavapor (40° C., 220-50 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 1h to yield 1.267 g (raw yield: 33.0) of a white sticky foam as 362-VRO047-01-001 CC1 #1.
1H-NMR (300 MHz, 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).
Then the Boc protecting group was removed as follows. In a 25 mL round-bottomed flask, equipped with a magnetic stirrer, 1.267 g of 362-VRO047-01-001 CC1 #1 (4.393 mmol, eq.) and 4.4 mL of a solution of 10% HCl in EtOAc were introduced, the resulting colourless solution was stirred at RT for 3 h. The reaction mixture was transferred into a separating funnel using 25 mL of EtOAc and washed with 25 mL of water. AP was basified using 4.5 mL of NaOH 25% to reach pH>10, then extracted 3 times with 25 mL of EtOAc. OPs were combined and washed using 25 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 250-70 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 3h to yield 337 mg (raw yield: 40.7%) of a pale yellow oil as 362-VRO047-02-001 crude1 #1. This material was used “as it” without further purification in the next step.
1H-NMR (300 MHz, 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).
Finally, the dipeptide was coupled to the epoxy warhead. In a 25 mL round-bottomed flask, equipped with a magnetic stirrer, 290 mg of 362-BB01-03-001 crude1 #1 (1.813 mmol, 1.05 eq.) and 5 mL of DCM were introduced, the resulting white suspension was stirred at RT for 5 min. then cooled down to 0-5° C. using an ice-bath. 839 mg of HATU (2.589 mmol, 1.5 eq.) were added at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Then 325 mg of 362-VRO047-02-001 crude1 #1 (1.726 mmol, 1.0 eq.) in solution in 5 mL of DCM were introduced at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Finally, 601 μL of DIPEA (3.452 mmol, 2.0 eq.) were added dropwise at 0-5° C. The resulting mixture was stirred for 5 min at 0-5° C. then the ice-bath was removed and the mixture was stirred O.N. at RT.
The reaction mixture, a clear yellow solution, was transferred into a separating funnel using 10 mL of DCM and washed with 50 mL of NaCl 5.8%, then AP was extracted once with 10 mL of DCM. OPs were combined, and washed once with 50 mL NaCl 5.8%, then AP was extracted once with 10 mL of DCM. OPs were combined and washed once with 50 mL of HCl 0.25 M, once with 50 mL of NaHCO3 10% and once with 50 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 220-30 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h to yield 560 mg (raw yield: 98.2%) of a yellowish viscous solid as 362-VRO047-03-001 crude1 #1.
The crude material was purified by CC using 45 g of silica gel (column with ø=3.2 cm and h=15.2 cm) and elution followed using TLC using heptane-EtOAc (3:7) as eluant; Rf=0.41. The product was eluted with a gradient from heptane-EtOAc (7:3) to heptane-EtOAc (2:8), fractions containing the desired product were combined and concentrated to dryness into the rotavapor (40° C., 200-40 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h to yield 220 mg (raw yield: 38.6%) of a slightly pale yellow solid as 362-VRO047-03-001 CC1 #1.
1H-NMR (300 MHz, 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).
13C-NMR (75 MHz, CDCl3) δ=70.75 (13), 62.30 (2), 58.76 (14), 53.77 (4), 52.88 (3), 51.16 (6), 41.51 (7), 39.26 (12), 24.76 (8), 22.78 (9, 10), 22.05 (9, 10), 13.99 (1).
Firstly, the dipeptide was formed. In a 50 mL round-bottomed flask, equipped with a magnetic stirrer, 2.235 g of Boc-L-Leu (9.661 mmol, 1.1 eq.) and 25 mL of DCM were introduced, the resulting suspension was stirred at RT for 5 min. then cooled down to 0-5° C. using an ice-bath. 8.540 g of HATU (26.348 mmol, 3.0 eq.) were added at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Then 1.1 mL of 2-(2-aminoethyl)pyridine (8.783 mmol, 1.0 eq.) and 10 mL of DCM (due to the amount of HATU the suspension was too thick) were introduced at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Finally, 7.6 mL of DIPEA (43.914 mmol, 5.0 eq.) were added dropwise at 0-5° C. The resulting strong yellow mixture was stirred for 5 min at 0-5° C. then the ice-bath was removed and the mixture was stirred O.N. at RT.
The reaction mixture, a brown-orange solution with a white solid, was filtered over a P3 sintered glass filter to remove the salt. The filtrate was transferred into a separating funnel using 5 mL of DCM and washed with 50 mL of NaCl 5.8%, then AP was extracted once with 10 mL of DCM. This operation was repeated 2 more times, then OPs were combined and washed once with 50 mL of NaHCO3 10% and finally with 50 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 850-70 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h30 to yield 8.462 g (raw yield: 287.2%) of a brown-orange oily material as 362-VRO052-01-001 crude1 #1.
A first attempt of purification by CC was performed using 50 g of silica gel (column with ø=3.2 cm and h=16.9 cm) and elution followed using TLC using DCM with 1% Et3N as eluant; Rf=0.51. The product was eluted with a gradient from DCM with 1% Et3N to DCM with 0.5% of MeOH and 1% Et3N, fractions containing the desired product were combined and concentrated to dryness into the rotavapor (40° C., 800-50 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 3h to yield 6.666 g (raw yield: 226.3%) of a yellowish oily material as 362-VRO052-01-001 CC1 #1.
A second attempt of purification to remove TMU by CC was performed using 55 g of silica gel (column with ø=3.2 cm and h=16.9 cm) and elution followed using TLC using heptane-EtOAc (2:8) and 1% Et3N as eluant; 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, fractions con-taining the desired product were combined and concentrated to dryness into the rotavapor (40° C., 220-50 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h to yield 3.228 g (raw yield: 109.6%) of a yellowish oily material as 362-VRO052-01-001 CC2 #1.
In order to get rid from TMU, the material recovered from the 2nd CC was re-extracted. The material was transferred into a separating funnel using 15 mL of EtOAc, then washed 4 times with 50 mL of NaHCO3 10%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 220-40 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 3h to yield 2.416 g (raw yield: 82.0%) of a yellowish oily material as 362-VRO052-01-001 crude2 #1.
1H-NMR (300 MHz, 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).
13C NMR (75 MHz, 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).
Then the Boc protecting group was removed as follows. In a 50 mL round-bottomed flask, equipped with a magnetic stirrer, 2.416 g of 362-VRO052-01-001 CC1 #1 (7.202 mmol, eq.) and 18.1 mL of DCM were introduced, the resulting colourless solution was stirred at RT for 15 min then cooled down to 0-5° C. using an ice-bath. 6.0 mL of TFA (78.875 mmol, 10.95 eq.) were added dropwise at 0-5° C. The reaction mixture was stirred for 5 min. at 0-5° C., then the ice-bath was removed and the mixture was stirred for 4 h at RT. The reaction mixture was transferred into a separating funnel using 5 mL of DCM and washed with 50 mL of water. AP was basified using 20 mL of NaOH 25% to reach pH>10, then extracted 3 times with 20 mL of DCM. OPs were combined and washed using 50 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concen-trated to dryness into the rotavapor (40° C., 800-70 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 6h to yield 1.470 g (raw yield: 86.7%) of a pale yellow solid as 362-VRO052-02-001 crude1 #1. This material was used “as is” without further purification in the next step.
1H-NMR (300 MHz, 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).
Finally, the dipeptide was coupled to the epoxy warhead. In a 100 mL round-bottomed flask, equipped with a magnetic stirrer, 970 mg of 362-BB01-03-004 crude1 #1 (6.055 mmol, 1.05 eq.) and 15 mL of DMF were introduced, the resulting solution was stirred at RT for 5 min. then cooled down to 0-5° C. using an ice-bath. 5.607 g of HATU (17.299 mmol, 3.0 eq.) were added at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Then 1.357 g of 362-VRO052-02-001 crude1 #1 (5.766 mmol, 1.0 eq.) in solution in 15 mL of DMF were introduced at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Finally, 5.0 mL of DIPEA (28.832 mmol, 5.0 eq.) were added dropwise at 0-5° C. The resulting yellow-brownish mixture was stirred for 5 min at 0-5° C. then the ice-bath was removed and the mixture was stirred O.N. at RT.
The reaction mixture, a clear dark brown solution, was transferred into a separating funnel using 30 mL of EtOAc and washed with 100 mL of NaCl 5.8%, then AP was extracted 4 times with 30 mL of EtOAc. OPs were combined, and washed once with 100 mL NaHCO3 5%, then AP was extracted once with 20 mL of EtOAc. OPs were combined and washed once with 100 mL NaHCO3 10%, then AP was extracted once with 20 mL of EtOAc. OPs were combined and the last operation was repeated one more time. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 220-30 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 1h30 to yield 2.296 g (raw yield: 105.5%) of a dark brown sticky foam as 362-VRO052-03-002 crude1 #1.
The crude material was firstly purified through a slurry using 20 mL of EtOAc. The suspension was stirred at RT for around 30 min, then cooled down to 0-5° C. and filtered over a P3 sintered glass filter. The filter cake was washed twice with 6 mL of cold EtOAc, then dried under high vacuum (RT, 10-3 mbar) for 2h to yield 1.114 g (raw yield: 51.2%%) of a slightly beige powder as 362-VRO052-03-002 slurry1 #1.
This material was re-purified by CC using 50 g of silica gel (column with 0=3.2 cm and h=16.9 cm) and elution followed using TLC using pure EtOAc with 1% Et3N as eluant; Rf=0.52. The product was eluted with a gradient from heptane-EtOAc (1:1) to pure EtOAc always with 1% Et3N, fractions containing the desired product were combined and concentrated to dryness into the rotavapor (40° C., 240-50 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 3h to yield 966 mg (raw yield: 44.4%) of a white solid as 362-VRO052-03-002 CC1 #1.
1H-NMR (300 MHz, 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, 2H, 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).
13C-NMR (75 MHz, 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).
Firstly, the dipeptide was formed. In a 50 mL round-bottomed flask, equipped with a magnetic stirrer, 1.320 g of Boc-L-Leu (5.707 mmol, 1.1 eq.) and 25 mL of DCM were introduced, the resulting turbid solution was stirred at RT for 5 min. then cooled down to 0-5° C. using an ice-bath. 5.045 g of HATU (15.565 mmol, 3.0 eq.) were added at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Then 825 μL of 3-(piperdinyl)propanamine (5.188 mmol, 1.0 eq.) were introduced at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Finally, 4.5 mL of DIPEA (25.941 mmol, 5.0 eq.) were added dropwise at 0-5° C. The resulting yellowish mixture was stirred for 5 min at 0-5° C. then the ice-bath was removed and the mixture was stirred O.N. at RT.
The reaction mixture, a brown-red solution with a white solid in suspension, was filtered over a P3 sintered glass filter to remove the salts. The filtrate was transferred into a separating funnel using 5 mL of DCM and washed with 50 mL of NaCl 5.8%, then AP was extracted once with 10 mL of DCM. OPs were combined and the previous washing were repeated 2 times. OPs were combined and washed once with 50 mL of NaHCO3 10% and finally with 50 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 850-70 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h30 to yield 4.113 g (raw yield: 223.0%) of a brown-red oily material as 362-VRO059-01-002 crude1 #1.
The crude material was purified by CC using 50 g of silica gel (column with ø=3.2 cm and h=16.9 cm) and elution followed using TLC using DCM with 1.5% of MeOH and 1% Et3N as eluant; Rf=0.56. The product was eluted with a gradient from pure DCM to DCM with 2% MeOH always with 1% Et3N, fractions containing the desired product were combined and concentrated to dryness into the rotavapor (40° C., 750-60 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h to yield 1.557 g (raw yield: 84.4%) of a yellowish oil as 362-VRO059-01-002 CC1 #1.
In order to get rid of TMU, the material recovered from the CC was re-extracted. The material was transferred into a separating funnel using 10 mL of EtOAc, then washed 2 times with 50 mL of NaHCO3 10%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 220-40 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 3h to yield 1.181 g (raw yield: 64.0%) of a yellowish viscous material as 362-VRO059-01-001 crude2 #1.
The re-extracted material was purified by a 2nd CC using 50 g of silica gel (column with ø=3.2 cm and h=16.9 cm) and elution followed using TLC using pure EtOAc with 1% of Et3N as eluant; Rf=0.47. The product was eluted with a gradient from heptane-EtOAc (1:1) to pure EtOAc, fractions containing the desired product were combined and concentrated to dryness into the rotavapor (40° C., 220-60 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h to yield 1.038 g (raw yield: 56.3%) of a pale yellowish solid as 362-VRO059-01-002 CC2 #1.
1H-NMR (300 MHz, 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).
13C NMR (75 MHz, 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).
Then the Boc protecting group was removed as follows. In a 25 mL round-bottomed flask, equipped with a magnetic stirrer, 1.024 g of 362-VRO059-01-001 CC2 #1 (2.880 mmol, eq.) and 7.7 mL of DCM were introduced, the resulting colourless solution was stirred at RT for 10 min then cooled down to 0-5° C. using an ice-bath. 2.6 mL of TFA (33.430 mmol, 11.61 eq.) were added dropwise at 0-5° C. The reaction mixture was stirred for 5 min. at 0-5° C., then the ice-bath was removed and the mixture was stirred for 4 h at RT. The reaction mixture was transferred into a separating funnel using 5 mL of DCM and washed with 50 mL of water. AP was basified using 8 mL of NaOH 25% to reach pH>10, then extracted 3 times with 20 mL of DCM. OPs were combined and washed using 50 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 800-70 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 6h to yield 654 mg (raw yield: 88.9%) of a pale yellowish oil as 362-VRO059-02-002 crude1 #1. This material was used “as it” without further purification in the next step.
1H-NMR (300 MHz, 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).
13C-NMR (75 MHz, 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).
Finally, the dipeptide was coupled to the epoxy warhead. In a 25 mL round-bottomed flask, equipped with a magnetic stirrer, 356 mg of 362-BB01-03-004 crude1 #1 (2.241 mmol, 1.05 eq.) and 7.5 mL of DMF were introduced, the resulting solution was stirred at RT for 5 min. then cooled down to 0-5° C. using an ice-bath. 2.075 g of HATU (6.402 mmol, 3.0 eq.) were added at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Then 545 mg of 362-VRO059-02-002 crude1 #1 (2.134 mmol, 1.0 eq.) in solution in 7.5 mL of DMF were introduced at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Finally, 1.9 mL of DIPEA (10.699 mmol, 5.0 eq.) were added dropwise at 0-5° C. The resulting yellow-brownish mixture was stirred for 5 min at 0-5° C. then the ice-bath was removed and the mixture was stirred O.N. at RT.
The reaction mixture, a clear dark brown solution, was transferred into a separating funnel using 15 mL of EtOAc and washed with 50 mL of NaCl 5.8%, then AP is extracted 4 times with 15 mL of EtOAc. OPs are combined and washed 4 times with 50 mL NaHCO3 10%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 220-30 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 1h30 to yield 1.063 g (raw yield: 125.3%) of a dark brown sticky solid as 362-VRO059-03-003 crude1 #1.
The crude material was purified by CC using 50 g of silica gel (column with ø=3.2 cm and h=16.9 cm) and elution followed using TLC using DCM containing 1% MeOH and 1% Et3N as eluant; Rf=0.36. The product was eluted with a gradient from pure DCM to DCM with 1% MeOH always with 1% Et3N, fractions containing the desired product were combined and concentrated to dryness into the rotavapor (40° C., 800-50 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h to yield 397 mg (raw yield: 46.8%) of a viscous brown-orange solid as 362-VRO059-03-003 CC1 #1.
This material was re-purified by CC using 50 g of silica gel (column with ø=3.2 cm and h=16.9 cm) and elution followed using TLC using heptane-acetone (3:7) with 1% Et3N as eluant; Rf=0.57. The product was eluted with a gradient from heptane-acetone (8:2) to heptane-acetone (3:7) always with 1% Et3N, fractions containing the desired product were combined and concentrated to dryness into the rotavapor (40° C., 320-50 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 1h to yield 116 mg (raw yield: 17.4%) of a brown-beige solid as 362-VRO059-03-003 CC2 #1.
This material was re-extracted; it was transferred into a separating funnel using 20 mL of EtOAc and 20 mL of water, then HCl 1M was added to reach pH 1. AP was the basified to pH 9 and extracted 3 times with 20 mL of EtOAc. OPs were combined and was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 220-30 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 3h to yield 111 mg (raw yield: 13.7%) of a viscous orange-yellow solid as 362-VRO059-03-003 extrac1 #1.
1H-NMR (300 MHz, 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).
13C-NMR (75 MHz, 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).
Firstly, the dipeptide was formed. In a 25 mL round-bottomed flask, equipped with a magnetic stirrer, 970 mg of Boc-L-Leu (4.195 mmol, 1.05 eq.) and 10 mL of DCM were introduced, the resulting turbid solution was stirred at RT for 5 min. then cooled down to 0-5° C. using an ice-bath. 1.943 g of HATU (5.993 mmol, 1.5 eq.) were added at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Then 457 μL of 2-fluorobenzylamine (3.995 mmol, 1.0 eq.) were introduced at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Finally, 1.4 mL of DIPEA (7.991 mmol, 2.0 eq.) were added dropwise at 0-5° C. The resulting turbid and strong yellow solution was stirred for 5 min at 0-5° C. then the ice-bath was removed and the mixture was stirred O.N. at RT.
The reaction mixture, a slightly turbid yellow solution, was transferred into a separating funnel using 10 mL of DCM and washed with 50 mL of NaCl 5.8%, then AP was extracted once with 10 mL of DCM. This operation was repeated 2 more times, then OPs were combined and washed once with 50 mL of NaHCO3 10% and finally with 50 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 850-70 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 3h to yield 1.841 g (raw yield: 136.2%) of a beige-yellow viscous solid as 362-VRO073-01-001 crude1 #1.
The crude material was purified by CC using 45 g of silica gel (column with ø=3.2 cm and h=15.2 cm) and elution followed using TLC using heptane-EtOAc (7:3) as eluant; Rf=0.49. The product was eluted with a gradient from pure heptane to heptane-EtOAc (6:4), fractions containing the desired product were combined and concentrated to dryness into the rotavapor (40° C., 220-50 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h to yield 1.261 g (raw yield: 93.3%) of a white foam as 362-VRO073-01-001 CC1 #1.
1H-NMR (300 MHz, 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).
13C NMR (75 MHz, 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).
Then the Boc protecting group was removed as follows. In a 25 mL round-bottomed flask, equipped with a magnetic stirrer, 1.249 g of 362-VRO073-01-001 CC1 #1 (3.691 mmol, 1.0 eq.) and 9.4 mL of DCM were introduced, the resulting colourless solution was stirred at RT for 10 min then cooled down to 0-5° C. using an ice-bath. 3.1 mL of TFA (40.48 mmol, 10.97 eq.) were added dropwise at 0-5° C. The reaction mixture was stirred for 5 min. at 0-5° C., then the ice-bath was removed and the mixture was stirred for 2h30 at RT. The reaction mixture was transferred into a separating funnel using 25 mL of DCM and washed with 25 mL of water. AP was basified using 3 mL of NaOH 25% to reach pH>10, then extracted 3 times with 25 mL of DCM. OPs were combined and washed using 25 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 800-70 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 1h to yield 465 mg (raw yield: 52.9%) of an almost colourless oil as 362-VRO073-02-001 crude1 #1. This material was used “as it” without further purification in the next step.
1H-NMR (300 MHz, 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).
Finally, the dipeptide was coupled to the epoxy warhead. In a 25 mL round-bottomed flask, equipped with a magnetic stirrer, 320 mg of 362-BB01-03-001 crude1 #1 (1.996 mmol, 1.05 eq.) and 5 mL of DCM were introduced, the resulting white suspension was stirred at RT for 5 min. then cooled down to 0-5° C. using an ice-bath. 924 mg of HATU (2.851 mmol, 1.5 eq.) were added at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Then 453 mg of 362-VRO073-02-001 crude1 #1 (1.901 mmol, 1.0 eq.) in solution in 5 mL of DCM were introduced at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Finally, 993 μL of DIPEA (5.703 mmol, 3.0 eq.) were added dropwise at 0-5° C. The resulting yellowish mixture was stirred for 5 min at 0-5° C. then the ice-bath was removed and the mixture was stirred O.N. at RT.
The reaction mixture, a yellow-orange solution, was transferred into a separating funnel using 10 mL of DCM and washed with 50 mL of NaCl 2.6%, then AP was extracted once with 10 mL of DCM. OPs were combined, and washed once with 50 mL NaCl 2.6%, then AP was extracted once with 10 mL of DCM. OPs were combined and washed with 50 mL of NaCl 5.8% and 5 mL of HCl 1M, which resulted in an emulsion that had to be filtered on celite. The filtrate was transferred again into the separating funnel using 10 mL of DCM and washed once with 50 mL of NaHCO3 10% and once with 50 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 220-30 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h to yield 835 mg (raw yield: 115.5%) of a pasty orange solid as 362-VRO073-03-001 crude1 #1.
The crude material was purified by CC using 45 g of silica gel (column with ø=3.2 cm and h=15.2 cm) and elution followed using TLC using heptane-EtOAc (1:1) as eluant; Rf=0.50. The product was eluted with a gradient from pure heptane to heptane-EtOAc (2:8), fractions containing the desired product were combined and concentrated to dryness into the rotavapor (40° C., 200-50 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 3h to yield 110 mg (raw yield: 15.2%) of a white solid as 362-VRO073-03-001 CC1 #1.
1H-NMR (300 MHz, 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).
Firstly, the dipeptide was formed. In a 25 mL round-bottomed flask, equipped with a magnetic stirrer, 190 mg of (S)-Boc-2-amino-4,4,4-trifluoro-butyric acid (0.739 mmol, 1.0 eq.) and 10 mL of DCM were introduced, the resulting mixture was stirred at RT for 5 min. then cooled down to 0-5° C. using an ice-bath. 359 mg of HATU (1.108 mmol, 1.5 eq.) were added at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Then 94 μL of IAA (0.813 mmol, 1.1 eq.) were introduced at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Finally, 257 μL of DIPEA (1.477 mmol, 2.0 eq.) were added dropwise at 0-5° C. The resulting slightly pale yellow mixture was stirred for 5 min at 0-5° C. then the ice-bath was removed and the mixture was stirred O.N. at RT.
The reaction mixture, a yellow-orange solution, was concentrated to dryness into the rotavapor (40° C., 750-400 mbar). The residue was transferred into a separating funnel using 20 mL of EtOAc and washed with 50 mL of NaHCO3 5%, then AP was extracted 2 times with 15 mL of EtOAc. OPs were combined, and washed 2 times with 50 mL NaHCO3 5%, once with 50 mL of NaHCO3 10% and once with 50 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 220-30 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 6h to yield 224 mg (raw yield: 92.9%) of an off-white crystallized solid as 362-VRO109-01-001 crude1 #1.
The crude material was purified by CC using 50 g of silica gel (column with ø=3.2 cm and h=16.9 cm) and elution followed using TLC using heptane-EtOAc (7:3) as eluant; Rf=0.46. The product was eluted with a gradient from pure heptane to heptane-EtOAc (6:4), fractions containing the desired product were combined and concentrated to dryness into the rotavapor (40° C., 220-50 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 4h to yield 204 mg (raw yield: 84.6%) of a white foam as 362-109-01-001 CC1 #1.
1H-NMR (300 MHz, 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).
13C NMR (75 MHz, CDCl3) δ=49.48 (5), 38.12 (9), 38.00 (8), 28.15 (1, 2, 3), 25.67 (10), 22.32 (11, 12).
Then the Boc protecting group was removed as follows. In a 10 mL round-bottomed flask, equipped with a magnetic stirrer, 201 mg of 362-VRO109-01-001 CC1 #1 (0.616 mmol, eq.) and 5 mL of DCM were introduced, the resulting colourless solution was stirred at RT for 10 min then cooled down to 0-5° C. using an ice-bath. 1.7 mL of TFA (22.200 mmol, 36.045 eq.) were added dropwise at 0-5° C. The reaction mixture was stirred for 5 min. at 0-5° C., then the ice-bath was removed and the mixture was stirred for 1 h at RT. The reaction mixture was transferred into a separating funnel using 25 mL of DCM and washed with 50 mL of water. AP was basified using 4 mL of NaOH 25% to reach pH>10, then extracted 3 times with 15 mL of DCM. OPs were combined and dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 750-400 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 6h to yield 256 mg (raw yield: 96.6%) of an almost colourless oil as 362-VRO109-02-001 crude1 #1. This material was used “as it” without further purification in the next step.
1H-NMR (300 MHz, 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).
13C-NMR (75 MHz, 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).
Finally, the dipeptide was coupled to the epoxy warhead. In a 25 mL round-bottomed flask, equipped with a magnetic stirrer, 115 mg of 362-BB01-03-004 crude1 #1 (0.720 mmol, eq.) and 5 mL of DMF were introduced, the resulting solution was stirred at RT for 5 min. then cooled down to 0-5° C. using an ice-bath. 318 mg of HATU (0.981 mmol, 1.5 eq.) were added at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Then 148 mg of 362-VRO109-02-001 crude1 #1 (0.654 mmol, 1.0 eq.) in solution in 5 mL of DMF were introduced at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Finally, 228 μL of DIPEA (1.308 mmol, 2.0 eq.) were added dropwise at 0-5° C. The resulting yellow mixture was stirred for 5 min at 0-5° C. then the ice-bath was removed and the mixture was stirred O.N. at RT.
The reaction mixture, an orange solution, was s transferred into a separating funnel using 20 mL of EtOAc and washed with 50 mL of NaHCO3 5%, then AP was extracted 2 times with 15 mL of EtOAc. OPs were combined, and washed 3 times with 50 mL NaHCO3 5% and once with 50 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 220-140 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 6h to yield 237 mg (raw yield: 102.5%) of a very pale orange solid as 362-VRO109-03-001 crude1 #1.
The crude material was purified through a slurry using 5 mL of EtOAc-heptane (1:1). The suspension was stirred at RT for 20 min then cooled down to 0-5° C. and stirred for 15 min. The suspension was filtered over an ultrafiltration unit equipped with a nylon membrane (0.45 μm), the filter cake was washed 3 times with cold EtOAc-heptane (1:9), then dried under high vacuum (RT, 10-3 mbar) for 3h to yield 175 mg (raw yield: 72.6%) of a white powder as 362-VRO109-03-001 slurry1 #1.
1H-NMR (300 MHz, 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, 11), 1.40 (q, J=7.2, 2H, 10), 1.32 (t, J=7.2, 3H, 1), 1.25 (d, J=1.8, OH), 0.91 (d, J=6.6, 6H, 12, 13).
Firstly, the dipeptide was formed. In a 25 mL round-bottomed flask, equipped with a magnetic stirrer, 190 mg of Boc-(S)-2-amino-4,4,4-butyric acid (0.739 mmol, 1.0 eq.) and 10 mL of DCM were introduced, the resulting mixture was stirred at RT for 5 min. then cooled down to 0-5° C. using an ice-bath. 359 mg of HATU (1.108 mmol, 1.5 eq.) were added at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Then 93 μL of 2-fluorobenzylamine (0.813 mmol, 1.1 eq.) were introduced at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Finally, 257 μL of DIPEA (1.477 mmol, 2.0 eq.) were added dropwise at 0-5° C. The resulting slightly pale yellow mixture was stirred for 5 min at 0-5° C. then the ice-bath was removed and the mixture was stirred O.N. at RT.
The reaction mixture, a clear yellow solution, was concentrated to dryness into the rotavapor (40° C., 750-400 mbar). The residue was transferred into a separating funnel using 20 mL of EtOAc and washed with 50 mL of NaHCO3 5%, then AP was extracted 2 times with 15 mL of EtOAc. OPs were combined, and washed 2 times with 50 mL NaHCO3 5%, once with 50 mL of NaHCO3 10% and once with 50 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 220-140 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 6h to yield 279 mg (raw yield: 103.7%) of an orange solid as 362-VRO244-01-001 crude1 #1.
The crude material was purified by CC using 50 g of silica gel (column with ø=3.2 cm and h=16.9 cm) and elution followed using TLC using heptane-acetone (7:3) as eluant; Rf=0.45. The product was eluted with a gradient from pure heptane to heptane-acetone (6:4), fractions containing the desired product were combined and concentrated to dryness into the rotavapor (40° C., 350-50 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h to yield 259 mg (raw yield: 96.2%) of a slightly pale yellow solid as 362-VRO243-01-001 CC1 #1.
1H-NMR (300 MHz, 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).
13C NMR (75 MHz, 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).
Then the Boc protecting group was removed as follows. In a 10 mL round-bottomed flask, equipped with a magnetic stirrer, 252 mg of 362-VRO244-01-001 CC1 #1 (0.692 mmol, 1.0 eq.) and 5 mL of DCM were introduced, the resulting colourless solution was stirred at RT for 10 min then cooled down to 0-5° C. using an ice-bath. 1.7 mL of TFA (22.200 mmol, 32.10 eq.) were added dropwise at 0-5° C. The reaction mixture was stirred for 5 min. at 0-5° C., then the ice-bath was removed and the mixture was stirred for 1 h at RT. The reaction mixture was transferred into a separating funnel using 25 mL of DCM and washed with 50 mL of water. AP was basified using 4 mL of NaOH 25% to reach pH>10, then extracted 3 times with 15 mL of DCM. OPs were combined and dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 750-400 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 4h to yield 172 mg (raw yield: 94.1%) of an almost colourless oil as 362-VRO244-02-001 crude1 #1. This material was used “as it” without further purification in the next step.
1H-NMR (300 MHz, 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′).
13C-NMR (75 MHz, DMSO-d6) δ=129.26 (8), 129.21 (8), 128.69 (6), 128.58 (6), 124.00 (7), 123.95 (7), 114.86 (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).
Finally, the dipeptide was coupled to the epoxy warhead. In a 25 mL round-bottomed flask, equipped with a magnetic stirrer, 111 mg of 362-BB01-03-005 crude1 #1 (0.695 mmol, eq.) and 5 mL of DMF were introduced, the resulting DMF was stirred at RT for 5 min. then cooled down to 0-5° C. using an ice-bath. 307 mg of HATU (0.948 mmol, 1.5 eq.) were added at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Then 167 mg of 362-VRO244-02-001 crude1 #1 (0.632 mmol, 1.0 eq.) in solution in 5 mL of DMF were introduced at 0-5° C. and the reaction mixture was stirred for 5 min at this temperature. Finally, 220 μL of DIPEA (1.264 mmol, 2.0 eq.) were added dropwise at 0-5° C. The resulting yellow mixture was stirred for 5 min at 0-5° C. then the ice-bath was removed and the mixture was stirred O.N. at RT.
The reaction mixture, an orange solution, was transferred into a separating funnel using 20 mL of EtOAc and washed with 50 mL of NaHCO3 5%, then AP was extracted 2 times with 15 mL of EtOAc. OPs were combined, and washed 3 times with 50 mL NaHCO3 5% and once with 50 mL of NaCl 11.6%. OP was dried over Na2SO4, filtered over a P3 sintered glass filter and concentrated to dryness into the rotavapor (40° C., 220-30 mbar). The material was dried under high vacuum (RT, 10-3 mbar) for 2h to yield 274 mg (raw yield: 106.7%) of a brown-orange solid as 362-VRO244-03-001 crude1 #1.
The crude material was purified through a slurry using 3 mL of EtOAc-heptane (2:1). The granular suspension was stirred at 40° C. for 10 min, then at RT for 15 min and finally cooled down to 0-5° C. using an ice-bath before filtration. The suspen-sion was filtered over an ultrafiltration unit equipped with a nylon membrane (0.45 μm), the filter cake was washed and triturated 5 times with 1 mL of cold EtOAc-heptane (1:1), then dried under high vacuum (RT, 10-3 mbar) for 2h to yield 131 mg (raw yield: 51.0%) of off-white granules as 362-VRO244-03-001 slurry1 #1.
1H-NMR (300 MHz, 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).
13C-NMR (75 MHz, 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).
The ability of the 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 assessed in an enzymatic assay and compared to the inhibitory activity of the prior art E64c. In this assay, the 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, in order to simulate the ester cleavage that occurs within the gastrointestinal tract, and to release the free acid (VROxxx_hydrol compound) from the prodrug (corresponding VROxxx compound). The chemical structures of compounds VRO016, VRO042, VRO047, VRO052, VRO059, VRO073, VRO19 and VRO244 and their chemical names are provided in [Table 2] above. The structures of VRO001, VRO082, VRO035, VRO119 and VRO243 are provided in [Table 3] below. The cathepsin B was from pork liver homogenate.
The enzymatic assay was performed as follows. Samples of homogenized pork liver (75 mg of fresh tissue per sample) were incubated for 1 hour at 37° C. with Z—FR-AMC (benzyloxycarbonyl-L-arginyl-L-arginine-4-methylcoumaryl-7-amide) at 50 mM in 1 M sodium acetate pH 6.0, supplemented with 2.5 mM EDTA, 2.5 mM DTT and 0.1% Triton X-100, and graded concentrations of test substances (between 0.1 nM and 50 mM). The stock solutions of test substances were at 20 mM in DMSO. Fluorescence was determined at 445 nm using 365 nm as the excitation wavelength.
The compounds of the invention VRO006_hydrol, VRO047_hydrol, VRO052_hydrol, VRO059_hydrol, VRO073_hydrol, VRO109_hydrol and VRO244_hydrol exhibited a suitable inhibitory activity with an IC50 of about 100 nM or less (see [
This assay allowed to demonstrate that the hydrogen from the amide in the centre of the molecule is crucial for the enzymatic affinity and is probably involved in an H-bond with the protein. Indeed, VRO001_hydrol proved unable to inhibit catB. On the other hand, the hydrogen on the amide next to the isoamylamine moiety is not involved in the binding since the activity from VRO006 is quite similar to the one of E64c.
All modifications made on R1 were well tolerated. The methoxy compound VRO047_hydrol showed almost the same activity as E64c. The same applies for VRO052_hydrol and VRO059_hydrol as well as for VRO073_hydrol. Even with bulky aromatic moieties, like in VRO052_hydrol and VRO073_hydrol, the activity is only slightly reduced. However, the tolerance of the S3 pocket of catB has limits. Indeed, the bulky and highly electronegative —CF3 moiety present in VRO035_hydrol was not accepted and the inhibitory activity was lost for this compound.
Concerning R3, which binds to the S2 pocket of catB, the tolerance was much lower. Even small changes, like the introduction of an ether in VRO082_hydrol did not allow to maintain the inhibitory activity. The bulkiness from the pyridinyl moiety added in VRO119_hydrol was also not tolerated. However, one candidate created the surprise, especially considering the results obtained with other moieties in position R3. VRO109_hydrol includes a trifluoro moiety instead of the leucine present in E64c. Adding such a trifluoro moiety in position R1 resulted in loss of the inhibitory activity, whereas several other moieties, and even bulky ones managed to keep such activity. Now, surprisingly it was demonstrated that VRO109_hydrol exhibits an inhibitory activity that is increased compared to E64c. From all the candidates tested herein VRO109_hydrol is the only one to exhibit a small but significant increase in inhibitory activity compared to the prior art E64c. The trifluoro moiety is particularly advantageous, as it is not susceptible of generating a hydrogen-bond and significantly increases the lipophilicity of the compound compared to E64c.
The assay performed with VRO244_hydrol provides evidence that the trifluoro moiety in position R3 is compatible with a bulky moiety in position R1 (in this case 2-fluorophenylmethyl).
Individually, the replacement of leucine by a trifluoro moiety and the methylation of the amide close to isoamylamine moiety showed promising results with respect to both activity and drug-likeness, as explained above. However, their combination in VRO243_hydrol implied a drastic loss of activity. This certainly results from the 3D conformation of the enzyme's catalytic site and how the trifluoro moiety is positioned into it. This particular 3D layout is probably incompatible with the one obtained when the nitrogen is methylated.
To confirm the results obtained on the isolated catB (see Example 9), the inhibitory activity of compounds of the invention on catB was assessed in a whole cell assay. In this case the free acid (hydrolyzed) form of the compounds of the invention was used.
Various concentration (50, 10, 2, 0.4, 0.08, 0.016, 0.0032 et 0 mM) of test substances were placed in 96 well plate and 50,000 HEK293T cells were added to each well. The plates were then incubated at 37° C. for one hour. The plates were centrifuged for 7 minutes at 2000 rpm, and the supernatant was removed. 100 ml of freshly prepared Z—FR-AMC substrate containing 1 M sodium acetate pH 6.0, 2.5 mM EDTA, 2.5 mM DTT, 0.1% Triton X-100 and Z—FR-AMC 20 mM. Following a 1-hour incubation, fluorescence was determined at 445 nm using 365 nm as the excitation wavelength.
As shown in [
| Number | Date | Country | Kind |
|---|---|---|---|
| 22151412.8 | Jan 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2022/062740 | 12/23/2022 | WO |
