Haloarylamine dual amine oxidase inhibitors

Abstract

The present invention relates to novel compounds capable of inhibiting semicarbazide-sensitive amine oxidase (SSAO / VAP-1) and monoamine oxidase B (MAO-B). These compounds are useful for the treatment of various neuromuscular and neuroinflammatory diseases, including muscular dystrophies, including peripheral and central disorders, in humans as well as pets and livestock. Furthermore, the present invention relates to pharmaceutical compositions containing these compounds and various uses thereof.

Description

[Technical field]

[0001] The present invention relates to novel compounds capable of inhibiting semicarbazide-sensitive amine oxidase (SSAO / VAP-1) and monoamine oxidase B (MAO-B). These compounds are useful for the treatment of various neuromuscular diseases, such as muscular dystrophies, neuroinflammatory diseases, including peripheral and central disorders, in human subjects as well as in pets and livestock. Furthermore, the present invention relates to pharmaceutical compositions containing these compounds and various uses thereof. [Background technology]

[0002] In most organisms, including humans, two groups of mammalian amine oxidases metabolize a variety of mono-, di-, and polyamines that are produced endogenously or absorbed from exogenous sources. The first group includes monoamine oxidases (MAO-A and MAO-B), which are present in the mitochondria of most cell types and use covalently bound flavin adenine dinucleotide (FAD) as a coenzyme. Polyamine oxidases are other FAD-dependent amine oxidases that oxidatively deaminate spermine and spermidine. The second group is copper-dependent and uses other cofactors besides FAD, such as oxidized tyrosine residues (TPQ or LTQ). One member of this group is semicarbazide-sensitive amine oxidase (SSAO), also known as primary amine oxidase, plasma amine oxidase, and benzylamine oxidase, which is identical to vascular adhesion protein-1 (VAP-1, hereafter referred to as SSAO / VAP-1). The diamine oxidase (DAO) and lysyl oxidase family of proteins (lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) 1-4) also belong to this second group. Pharmacologically, MAO-A is selectively inhibited by clorgyline, MAO-B by L-deprenyl, and SSAO / VAP-1 by semicarbazide.

[0003] Monoamine oxidase (MAO) activity is involved in the oxidative deamination of endogenous amines such as serotonin, dopamine, adrenaline, noradrenaline, arylalkylamines, and many amine xenobiotics. MAO-A and MAO-B share a common enzymatic role in the metabolism of amines, but they share only 70% sequence similarity, are encoded by different genes (Bach AW et al., Proc. Natl. Acad. Sci. USA 1988, 85, 4934-4938), and are distributed differently in the body. MAO-A shows high affinity for substrates such as tyramine, serotonin, adrenaline, and noradrenaline, while MAO-B acts preferentially on dopamine, phenylethylamine, and trace amounts of arylalkylamines. MAO-B is expressed in multiple organs, including the brain, heart, adipose tissue, pancreas, lung, kidney, muscle, and liver.

[0004] Nonselective MAO inhibitors, which inhibit both MAO-B and MAO-A, may induce hypertensive episodes when dietary tyramine is ingested, and therefore certain dietary restrictions are imposed (Gardner D. M. et al., J. Clin. Psychiatry, 1996, 57, 99-104). Furthermore, nonselective inhibitors increase the possibility of monoamine interactions with other drugs. Therefore, for pharmaceutical applications, MAO-B inhibitors with high selectivity over MAO-A are needed.

[0005] SSAO / VAP-1 is an ectoenzyme that contains a very short cytoplasmic tail, a single transmembrane domain, and a large, highly glycosylated extracellular domain that contains an active site for amine oxidase activity (Salmi M. & Jalkanen S. Science 1992, 257, 1407-1414). SSAO / VAP-1 is a It also exists as a soluble form circulating in the plasma of animals. This form has been shown to be a cleavage product of membrane-bound SSAO / VAP-1 (Stolen CM et al., Circ. Res. 2004, 95, 50-59). SSAO / VAP-1 is highly expressed in adipose tissue, lung, liver, aorta and muscle tissue. SSAO / VAP-1 appears to have two physiological functions, the first being amine oxidase activity and the second being cell adhesion activity. These two activities are related to inflammatory processes. Inhibitors of the amine oxidase activity of SSAO / VAP-1 have been found to disrupt leukocyte rolling, adhesion and extravasation and exhibit anti-inflammatory properties, similar to SSAO / VAP-1 antibodies (Foot JS et al., J.Pharm.Exp.Ther, 2013, 347, 365-374).

[0006] Oxidative deamination of primary monoamines by both MAO-B and SSAO / VAP-1 produces ammonia (NH3), aldehydes and hydrogen peroxide (H2O2), toxic agents that can cause significant inflammation through their promiscuous reactivity and oxidative stress. In the catalytic cycle of SSAO / VAP-1 amine oxidase, the covalently bound coenzyme TPQ is first reduced and then reoxidized by oxygen in the presence of copper, producing ammonia and hydrogen peroxide as by-products. Similarly, MAO-B oxidizes amines to aldehydes and ketones through a one-electron transfer process, while simultaneously reducing flavin adenine dinucleotide (FAD) to the reduced form FADH2. This coenzyme is then reoxidized by oxygen to produce hydrogen peroxide. Excessive concentrations of hydrogen peroxide are harmful and can lead to the pathology of various inflammatory and neurodegenerative processes (Gotz ME, et al., Pharm. acol Ther.1994, 63, 37-122).

[0007] Neuromuscular disease is a term used to describe a variety of disorders in which muscle function is affected by the underlying pathophysiology, either directly through the breakdown of muscle tissue or indirectly by affecting the nerves and neuromuscular junctions that control the muscles. Direct effects on muscles result in muscle wasting and reduced motor skills, while indirect effects on neuromuscular function via the central nervous system (CNS) cause symptoms ranging from occasional spasticity to paralysis. Diseases involving muscle wasting are further classified as muscular dystrophies, including Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, and myotonic muscular dystrophy. Indirect neuromuscular diseases are classified as central disorders, including Parkinson's disease, multiple sclerosis, Huntington's disease, and Creutzfeldt-Jakob disease.

[0008] Neuroinflammatory diseases is a term used to describe a variety of disorders in which immune responses modulate the nervous system, leading to chronic inflammation. This inflammation can lead to neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, prion diseases, motor neuron diseases, Huntington's disease, spinocerebellar ataxia, and spinal muscular atrophy. Moreover, neuroinflammation is also an underlying cause of neurological diseases such as epilepsy, stroke, and brain trauma.

[0009] DMD is the most common form of muscular dystrophy. It is a fatal neuromuscular disorder that is inherited genetically as an X-linked disease and is caused by mutations in the dystrophin gene. It mainly affects boys, with an incidence of 1 in 3,500 male births. It is characterized by muscle degeneration, which impairs athletic ability and daily life from a young age. The most common causes of death in DMD are myocardial fibrosis and respiratory failure. There is no cure for DMD, and current treatments focus on symptom relief and management of complications, including corticosteroids, surgery, mechanical ventilation, and physical therapy. Recent advances in splicing modulators and gene therapy have partially restored dystrophin function in some patients, but the long-term effects of such treatments are still unknown, and alternative or additional treatment options are still needed.

[0010] Parkinson's disease is the most common CNS disorder. It results from damage to nerve cells in the area of ​​the brain that produces dopamine, a chemical essential for muscle and movement control. It is a progressive degenerative neurological condition that causes tremors in the hands, arms, legs, jaw, and face, stiffness and rigidity of the limbs and trunk, slowness of body movements, unstable posture, and difficulty walking. It generally develops after the age of 60, and it is said that more than 10 million people worldwide are affected. Currently, treatments can only slow the progression or treat the symptoms. The first choice of medication is levodopa (a precursor of dopamine that can cross the blood-brain barrier), which increases the amount of dopamine in the brain, and carbidopa and MAO-B inhibitors, which suppress the metabolism of levodopa and dopamine. Anticholinergic drugs and medicinal cannabis are also prescribed to reduce dyskinesias.

[0011] MAO-B activity increases oxidative stress in muscle and throughout the body, leading to impaired mitochondrial function and increased reactive oxygen species. Myogenic cell cultures obtained from both mdx mice (animals bred to exhibit characteristic characteristics of DMD patients and established as a relevant model of the human disease) and DMD patients are more sensitive to oxidative stress. This sensitivity was abolished by treatment with an inhibitor of MAO-B (safinamide) in vitro. Pharmacological inhibition of MAO-B has also been shown to be effective in vivo, where safinamide reduced muscle fiber damage and oxidative stress and improved muscle function when administered to 3-month-old mdx mice (Vitiello L. et al., Front. Physiol. 2018, 9, Article 1087).

[0012] Blocking SSAO / VAP-1 with enzyme inhibitors has been shown to be highly effective in animal models of neutrophil-induced inflammation (Schilter HC et al., Respir. Res. 2015, 16, 42). Unregulated neutrophil activation and activity releases excess myeloperoxidase (MPO), which reacts with hydrogen peroxide to form hypochlorous acid, causing local tissue damage and further inflammation. Since MAO-B and SSAO / VAP-1 are localized in several organs (heart, lungs, muscles), inhibitors of both enzymes may be effective in treating diseases involving neutrophils and oxidative stress.

[0013] Neutrophils have been the focus of attention as a new therapeutic approach for the treatment of muscular dystrophies. Studies in mdx mice suggest that they promote muscle pathology; antibody-mediated depletion of host neutrophils significantly delayed or reduced skeletal muscle destruction in a mouse model of muscular dystrophy (Hodgetts S. et al., Neuromuscular Disorders 2006, 16, 591-602). Furthermore, it has been shown that neutrophil elastase activity is elevated in a mouse model of muscular dystrophy, and this elevation compromises myoblast survival (Arecco, N. et al., Sci. Rep. 2016, 6, Article 24708). SSAO / VAP-1 is upregulated in muscle donor tissue from DMD patients, and pharmacological inhibition of SSAO / VAP-1 activity in vivo reduces inflammation in mdx mice (WO 2015 / 189534; WO 2017 / 017414), further supporting the theory that neutrophils, recruited at least in part by SSAO-mediated activity, are involved in the progression of muscular dystrophy.

[0014] The current standard of care for patients with DMD is the use of glucocorticoids such as prednisolone and deflazacort, but long-term use of these steroids comes with heavy side effects, including immunosuppression, obesity, insulin resistance and behavioral problems.

[0015] In obese dogs fed a high-fat diet, both MAO and SSAO / VAP-1 activity was increased (Wenecq, E. et al., J. Physiol. Biochem. 2006, 62, 113-124). Substrates of SSAO / VAP-1 have been shown to have lipolytic activity, and inhibiting SSAO / VAP-1 promotes lipolysis (Romauch, M., Open Biology, 2020, 10, Article 190035). Mice with brain-specific knockout of insulin receptor (NIRKO mice) show brain mitochondrial dysfunction with reduced mitochondrial oxidative activity, increased ROS, and elevated levels of lipid and protein oxidation in the striatum and nucleus accumbens. NIRKO mice also have elevated levels of monoamine oxidase A and B (MAO A and B), leading to increased metabolic reversal of dopamine in these regions. Studies in cultured neuronal and glial cells indicate that these changes in MAO A and B directly lead to a loss of insulin signaling and thus to behavioral deficits (Kleinridders, A et al., PNAS, 2015, 112, 3463-3468).

[0016] In diseases associated with reduced dopamine levels, such as Parkinson's disease, Alzheimer's disease, attention deficit hyperactivity disorder, and schizophrenia, inhibition of MAO-B leads to greater activation of dopaminergic neurons, thereby alleviating symptoms. Oxidative stress due to increased MAO activity and the associated increased production of H2O2 by MAO-B are involved in degenerative processes associated with aging, Alzheimer's disease, and Parkinson's disease. Oxidative stress has also been recognized as an important factor in the progression of Huntington's disease, and patients with this disease have been shown to have increased expression of MAO-B in the basal ganglia and cerebral cortex (Richards G. et al., Brain Res. 2011, 1370, 204-214). Thus, MAO-B inhibitors used in neuromuscular and neurodegenerative diseases may act by increasing monoamine levels in the brain while simultaneously suppressing the production of reactive oxygen species.

[0017] Inflammation associated with SSAO / VAP-1 enzymatic activity is also considered to be a key feature of neuroinflammatory diseases such as Parkinson's disease, Alzheimer's disease and multiple sclerosis, and is a feature of the pathophysiology occurring after cerebral occlusion / reperfusion events (Aktas, O. et al., Arch Neurol. 2007, 64, 185-189). Excessive activity of SSAO / VAP-1 is independently involved in these processes (Xu, H. L. et al., J. Pharm. Exp. Ther. 2006, 317, 19-26). Pharmacological inhibition of SSAO / VAP-1 activity has been shown to reduce neutrophil infiltration into the brain and suppress microglial activation in an LPS-induced neuroinflammation model (Becchi et al., Br. J. Pharmacol. 2017, 174, 2302-2317). In addition, endothelial SSAO / VAP-1 is involved in cerebral amyloid angiopathy associated with Alzheimer's disease by acting on cerebrovascular amyloid beta (Aβ) deposition (Sole M. et al., Neurobiology of Ageing 2015, 36, 762), which is thought to lead to an underlying blood-brain barrier dysfunction associated with disease progression (Sole M. et al., Biochim. Biophys. Acta. Mol. Basis. Dis. 2019, 1865, 2189-2202). Furthermore, SSAO / VAP-1 oxidation of primary amines (e.g., methylamine to formaldehyde) has been linked to oxidative stress in models of Alzheimer's disease (Somfai GM et al., Neurochem Int. 2006, 48, 746-52). .

[0018] MAO-B enzymatic activity has been implicated in vascular dysfunction. MAO-B- / - mice exhibit mitochondrial dysfunction (reduced oxidative stress), reduced fibrosis and apoptosis, and preserved left ventricular function when subjected to pressure overload induced by transverse aortic constriction (TAC) compared to WT mice (Sturza A. et al., Hypertension 2013, 62, 140-6 and Kaluderic N. et al., Antioxid. Redox Signal. 2014, 10, 267-280). SSA O / VAP-1 is deeply involved in inflammation and fibrosis in multiple organs, including the lungs, liver, and kidneys, and is also strongly involved in cardiovascular and metabolic diseases (Salmi M. & Jalkenen S. Antioxid. & Redox Signal. 2017, 30, 314-332).

[0019] MAO-B and SSAO / VAP-1 have a large overlap in several areas of research. They share a common oxidative pathway that can induce local and systemic oxidative stress, which is involved in fibrosis, inflammation, and cardiovascular disease. In muscular dystrophies, both are abundant in muscle tissue and appear to be involved in both inflammatory and oxidative stress pathways in the progression of DMD, the most well-studied of these diseases. It is also clear that inhibiting these targets could have beneficial effects on systemic inflammation, fibrosis, and cardiovascular deterioration. In the CNS, SSAO / VAP-1 plays complementary roles in influencing leukocyte infiltration / migration into microvasculature, while MAO-B influences neurodegeneration. Both of these actions are likely to have a major impact on neuromuscular disorders related to the CNS, especially those with neuroinflammation, such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis, and further raise the hope of therapeutic intervention of both enzyme activities.

[0020] Considering the complexity of the actions of MAO-B and SSAO / VAP-1 in the aforementioned diseases, potent and selective inhibitors of both these enzymes (dual inhibitors) could provide clear and significant therapeutic advantages in these neuromuscular, neuroinflammatory and neurodegenerative diseases.

[0021] Some known MAO inhibitors also inhibit SSAO / VAP-1 (e.g., the haloarylamine MAO inhibitor mofegiline exemplified below (Milczek EM et al., J. Med. Chem. 2008, 51, 8019-8026)). Fluoroarylamine inhibitors are also described as MAO inhibitors in U.S. Pat. No. 4,454,158. [ka]

[0022] Other examples structurally related to mofegiline, such as LJP1586, are described in WO 2007 / 120528. [ka]

[0023] Additionally, a series of 2-substituted-3-haloarylamine SSAO / VAP-1 inhibitors are disclosed in WO 2013 / 163675, including PXS-4728 and PXS-4681. [ka]

[0024] In both of these patent applications, the enzyme inhibition reported was selective for SSAO / VAP-1 over all other amine oxidases, including MAO-B.

[0025] Recent patent applications have described aryloxy and heteroaryloxy 2-substituted-3-fluoroarylamines as SSAO / VAP-1 inhibitors (WO 2018 / 233633, WO 2018 / 151985, WO 2019 / 129213, WO 2019 / 241751 and WO 2020 / 063854). No MAO-A or MAO-B inhibition data were disclosed.

[0026] Related SSAO / VAP-1 inhibitor patent applications further report limited data on both MAO-B and MAO-A (WO 2018 / 196677, WO 2019 / 101086, WO 2019 / 024924, WO 2020 / 006177, WO 2020 / 063696, WO 2020 / 069335, WO 2021 / 083209). Exemplary compounds shown therein include All of the compounds had IC50 values ​​of <100 nM against the recombinant human form of MAO-B in standard biochemical assays. 50 have not been reported and therefore they would not be considered to be potent inhibitors of MAO-B.

[0027] The potency (IC) of benzoxazole-based 2-substituted-3-fluoroarylamine SSAO / VAP-1 inhibitors to inhibit MAO-B is shown in the following examples: 50 <0.1 μM) and lower MAO-A inhibitory potency (IC 50 It has also been disclosed that the antibody shows a potent agonist activity of 10 to 30 μM (International Publication No. WO 2020 / 069330). [ka]

[0028] 2-Substituted-3-fluoroarylamine SSAO / VAP-1 inhibitors directly bonded to the nitrogen of a 5-membered heterocycle have also been described (WO 2019 / 180644). Activity against MAO-B and selectivity against MAO-A have been reported for several of the example compounds.

[0029] Related to the invention presented herein, the following compounds have been reported as selective SSAO / VAP-1 inhibitors, and data have been reported showing that they are not potent inhibitors of MAO-B (China Patent Application Publication No. 2019 / 109251166): [ka] Summary of the Invention [Problem to be solved by the invention]

[0030] The present invention provides 2-substituted-3-fluoroallylamine compounds that are potent inhibitors of both SSAO / VAP-1 and MAO-B, and have high selectivity for MAO-A in particular. Surprisingly, modification of the 2-phenoxymethylene-3-fluoroallylamine structure described above has led to the development of novel compounds that are potent inhibitors of both human SSAO / VAP-1 and MAO-B enzymes, with high selectivity for inhibition of MAO-A enzyme, and a resulting desirable safety profile. Furthermore, the compounds of the present invention have excellent pharmacokinetic properties and a desirable safety profile in vitro. [Means for solving the problem]

[0031] The present invention describes the synthesis and use of compounds that inhibit the amine oxidase activity of SSAO / VAP-1 and MAO-B, and the utility of such inhibitors to treat patients suffering from, but not limited to, neuromuscular disorders.

[0032] A first aspect of the present invention relates to a compound of formula 1: [ka] or a pharma- ceutically acceptable salt, polymorph, or solvate thereof, R 1 is selected from the group consisting of hydrogen, fluorine, bromine and methyl; and R 2 is hydrogen or fluorine; The present invention provides a compound or a pharma- ceutically acceptable salt, polymorph or solvate thereof.

[0033] A second aspect of the invention provides a pharmaceutical composition comprising a compound according to the first aspect of the invention, or a pharma- ceutically acceptable salt or solvate thereof, and at least one pharma- ceutically acceptable excipient, carrier or diluent.

[0034] A third aspect of the invention provides a method of inhibiting the amine oxidase activity of SSAO / VAP-1 and MAO-B in a subject in need thereof, comprising administering to the subject an effective amount of a compound according to the first aspect of the invention, or a pharma- ceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to the second aspect of the invention.

[0035] A fourth aspect of the present invention is a method for treating or preventing a disease or disorder by inhibiting the activity of the SSAO / VAP-1 protein and the MAO-B protein, comprising the steps of: The present invention provides a method for treating a rheumatoid arthritis, comprising administering to a subject in need thereof an effective amount of a compound according to the first aspect of the invention, or a pharma- ceutical salt or solvate thereof, or a pharmaceutical composition according to the second aspect of the invention.

[0036] A fifth aspect of the present invention provides the use of a compound according to the first aspect of the present invention, or a pharma- ceutical acceptable salt or solvate thereof, for the manufacture of a medicament for treating or preventing a disease or disorder by inhibiting the activity of the SSAO / VAP-1 protein and the MAO-B protein.

[0037] A sixth aspect of the present invention provides a compound according to the first aspect of the present invention, or a pharma- ceutically acceptable salt or solvate thereof, for use in treating or preventing a disease or disorder by inhibiting said activity of said SSAO / VAP-1 protein and said MAO-B protein.

[0038] In one embodiment of the methods and uses of the present invention, said disease or disorder is ameliorated by said selective inhibition of SSAO / VAP-1 and MAO-B versus MAO-A.

[0039] In one embodiment of the methods and uses of the present invention, the disease is a neuromuscular disease.

[0040] In another embodiment of the invention, the disease is a neuroinflammatory or neurodegenerative disease.

[0041] It is contemplated herein that the method is a combination therapy further comprising co-administration of an additional therapeutic agent used in said treatment of neuromuscular, neuroinflammatory and neurodegenerative diseases.

[0042] definition Below are some definitions that may be helpful in understanding the description of the present invention. These are intended to be general definitions and should in no way limit the scope of the present invention to only these terms, but are presented for a better understanding of the following description.

[0043] Unless otherwise indicated by context or specifically stated to the contrary, integers, steps, or elements of the invention herein described as singular integers, steps, or elements explicitly encompass both the singular and plural forms of the described integer, step, or element.

[0044] Throughout this specification, unless the context dictates otherwise, the word "comprises" or variations of the term "comprises" or "comprising" will be understood to mean the inclusion of a stated step or element or integer or group of steps or elements or integers, but not the exclusion of other steps or elements or integers or groups of elements or integers. Thus, in the context of this specification, the term "comprises" means "including primarily, but not necessarily only."

[0045] Those skilled in the art will appreciate that the invention described herein may be susceptible to variations and modifications other than those specifically described. The invention is understood to encompass all such variations and modifications. The invention also includes all steps, features, compositions and compounds referred to or shown in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0046] The present invention includes within its scope different isotopes of atoms. Any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom. Thus, in this disclosure, deuterium and tritium isotopes of hydrogen should be understood to be included.

[0047] All documents cited in this application are specifically incorporated herein by cross-reference in their entirety. The reference to such a document should not be construed as an admission that the document forms part of the general knowledge or is prior art.

[0048] In the context of this specification, the term "administering" and variations thereof, including "administer" and "administration," include contacting, applying, delivering or providing a compound or composition of the present invention to an organism or surface by any suitable means. In the context of this specification, the term "treatment" refers to any and all uses that ameliorate a disease state or symptom, prevent the establishment of a disease, or otherwise prevent, hinder, slow, or reverse the progression of a disease or other undesirable symptom in any manner.

[0049] In the context of this specification, the term "effective amount" includes within its meaning an amount of a compound or composition of the present invention that is sufficient to produce the desired effect but is non-toxic. Thus, the term "therapeutically effective amount" includes within its meaning an amount of a compound or composition of the present invention that is sufficient to produce the desired therapeutic effect but is non-toxic. The exact amount required will vary from subject to subject, depending on factors such as the species being treated, the sex, age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, the mode of administration, and the like. Thus, an exact "effective amount" cannot be specified. However, in any given case, one of ordinary skill in the art can determine the appropriate "effective amount" through only routine experimentation. [Brief description of the drawings]

[0050] [Figure 1] FIG. 1 shows the pharmacokinetic time course of PXS-5131 versus Compound 1 in Wistar rats. [Diagram 2] FIG. 2 shows the in vivo pharmacokinetic readout of SSAO / VAP-1 inhibition in adipose tissue of Sprague-Dawley rats following oral administration of PXS-5131 at 0.6 mg / kg. [Diagram 3] FIG. 3 shows the in vivo pharmacokinetic readout of MAO-B inhibition in brain tissue of Sprague-Dawley rats following oral administration of PXS-5131 at 0.6 mg / kg. [Figure 4] Figure 4 shows the anti-inflammatory effect of PXS-5131 in the carrageenan air pouch model in BALB / c mice. [Diagram 5] FIG. 5 shows that PXS-5131 reduces neuronal loss in the Hooded Spot Wistar rat model of dopaminergic neurodegeneration. [Figure 6] FIG. 6 shows that oral administration of 6 mg / kg / day PXS-5131 for 4 weeks to 3-month-old mdx mice helps prevent force loss due to eccentric contractions in the gastrocnemius (GC) muscle compared to untreated age-matched mice. [Figure 7] FIG. 7 shows that oral administration of PXS-5131 at 6 mg / kg / day for 4 weeks to 3-month-old mdx mice significantly reduced (A) neutrophil influx, (B) macrophage influx, (C) reactive oxygen species, and (D) fibrosis (quantified by picrosirius red staining) in the tibialis anterior muscle compared to untreated mice of the same age. [Figure 8] FIG. 8 shows MAO-B and SSAO / VAP-1 inhibition in heart, muscle and adipose tissue of C57BL / 6J mice after oral administration of Compound 1 at various doses. [Figure 9] FIG. 9 shows the activity of MAO-A from cardiac and muscle tissues of C57BL / 6J mice after oral administration of various doses of Compound 1. [Figure 10] FIG. 10 shows MAO-B and SSAO / VAP-1 inhibition in muscle and adipose tissue, respectively, of mdx mice after oral administration of Compound 1. [Figure 11] FIG. 11 shows the activity of MAO-A from mdx mouse muscle tissue after oral administration of Compound 1. [Figure 12] FIG. 12 shows that oral administration of compound 1 (4.8 mg / kg / day) for 30 days to 3-month-old mdx mice significantly reduced (A) neutrophil influx, (B) macrophage influx, (C) reactive oxygen species, and (D) fibrosis (quantified by picrosirius red staining) in the diaphragm muscle compared to untreated mice of the same age. [Figure 13] FIG. 13 shows that administration of compound 1 (4.8 mg / kg / day) for 90 days to 10-month-old mdx mice significantly reduced cardiac fibrosis (quantified by picrosirius red staining of the myocardium) compared to untreated age-matched mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] The present invention relates to substituted haloarylamine derivatives capable of inhibiting both SSAO / VAP-1 and MAO-B. In particular, the present invention relates to substituted and unsubstituted 4-((2-(aminomethyl)-3-fluoroaryl)oxy)benzonitrile derivatives.

[0052] In particular, the present invention relates to a compound of formula 1: [ka] or a pharma- ceutically acceptable salt, polymorph, or solvate thereof, R 1 is selected from the group consisting of hydrogen, fluorine, bromine and methyl; and R 2 is hydrogen or fluorine; or a pharma- ceutically acceptable salt, polymorph or solvate thereof.

[0053] In one embodiment of the compound of the present invention, R 1 is hydrogen. In another embodiment of the compound of the present invention, R 1 is fluorine. In a further embodiment of the compound of the invention, R 1 is bromine. In another embodiment of the compound of the present invention, R 1 is methyl.

[0054] In one embodiment of the compound of the invention, R 2 is hydrogen. In another embodiment of the compound of the present invention, R 2 is fluorine.

[0055] In one embodiment of the compound of the present invention, R 1 is hydrogen, and R 2 is hydrogen. In another embodiment of the compound of the present invention, R 1 is fluorine, R 2 is hydrogen. In a further embodiment of the compound of the invention, R 1 is bromine, R 2 is hydrogen. In another embodiment of the compound of the present invention, R 1is hydrogen, and R 2 is fluorine. In a further embodiment of the compound of the invention, R 1 is methyl, R 2 is hydrogen.

[0056] In one embodiment, the compound of the invention is used as its free base. In another embodiment, the compound of the invention is used as a pharma- ceutically acceptable salt. In one embodiment, the compound of the invention is the hydrochloride salt.

[0057] In one embodiment, the compounds of the invention are potent inhibitors of SSAO / VAP-1 and MAO-B. According to the invention, the IC 50 In one embodiment, the IC of a potent inhibitor of SSAO / VAP-1 is less than 50 nM, or less than 40 nM, or less than 30 nM, or less than 20 nM. 50 is less than 10 nM According to the present invention, the IC of potent inhibitors of MAO-B 50 In one embodiment, the IC of a potent inhibitor of MAO-B is less than 250 nM, or less than 200 nM, or less than 150 nM, or less than 100 nM. 50 is less than 50 nM.

[0058] In one embodiment, the compounds of the invention are potent inhibitors of both SSAO / VAP-1 and MAO-B, with particular selectivity for MAO-A. The compounds of the invention have IC values ​​of greater than 3000 nM, or greater than 10000 nM, or greater than 20000 nM. 50 In one embodiment, the compounds of the present invention have an IC of greater than 30,000 nM. 50 It inhibits MAO-A.

[0059] The compounds of the present invention are selective inhibitors of SSAO / VAP-1 and MAO-B versus MAO-A. The relative selectivity of a given compound to inhibit MAO-B is determined by the IC of MAO-A. 50 Value / IC of MAO-B 50The relative ratio of MAO-A to MAO-C is at least 12. 50 Value / SSAO / VAP-1 IC 50 In another embodiment, the relative ratio of (IC value of MAO-A) for a given compound is at least 60. 50 Value / IC of MAO-B 50 In another embodiment, the relative ratio of (MAO-A IC value) for a given compound is at least 120, or at least 600. 50 Value / SSAO / VAP-1 IC 50 The relative ratio of the α, β, β and β values ​​is at least 300, or at least 600, or at least 3,000.

[0060] In the context of the present disclosure, any one or more aspect(s) or embodiment(s) may be combined with any other aspect(s) or embodiment(s).

[0061] Exemplary compounds according to the present invention include those set forth in Table 1.

[0062] [Table 1]

[0063] Preparation of Compounds of Formula 1 The compounds of formula 1 described herein are synthesized using standard synthetic chemistry techniques using methods known in the art. The methods described are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0064] Standard synthetic chemistry techniques are covered in Advanced Organic Chemistry, 8th Edition by March, John Wiley and Sons Standard procedures for the use of protecting groups to temporarily protect functional groups such as alcohols, amines, and carboxylic acids are described, for example, in Protecting Groups in Organic Synthesis, 5th Edition, John Wiley and Sons Inc.

[0065] Alternative reaction conditions used for the chemical transformations described herein may vary in solvents, reaction temperatures, reaction times, and chemical reagents. Unless otherwise noted, starting materials used in chemical syntheses and biological applications are commercially available.

[0066] In General Schemes 1 and 2, P is a group used to protect the nitrogen functionality. Examples of P include carboxylates such as tert-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (FMOC) and benzyloxycarbonyl (CBZ) groups. In some embodiments, hydroxybenzonitrile of formula II (Scheme 1) is treated with allyl bromide of formula III in the presence of a suitable base using a suitable polar solvent at a temperature between 0° C. and 40° C. for several hours (Method A). In some embodiments, the base is potassium carbonate (K2CO3) or alternatively cesium carbonate (Cs2CO3). In another embodiment, the base is sodium hydride (NaH). In some embodiments, the solvent is dimethylformamide (DMF) or alternatively acetone. In another embodiment, the solvent is acetonitrile. Following standard extraction and purification procedures, the product described by formula IV can be obtained in good yield and purity. [ka]

[0067] In some embodiments, fluorobenzonitriles of formula V (Scheme 2) are treated with allylic alcohols of formula VI in the presence of a suitable base and in a suitable solvent at temperatures between 0° C. and 40° C. for several minutes (Method C). In one embodiment, the base is NaH. In another embodiment, the solvent is tetrahydrofuran (THF) or alternatively DMF. Following standard extraction and purification procedures, the product of formula IV can be obtained in good yield and purity. [ka]

[0068] There are many well-established chemical procedures for deprotecting the compound described by formula IV to the compound described by formula I (Method B; Schemes 1 and 2). For example, when P is a Boc protecting group, the compound described by formula IV can be treated with an acidic reagent such as dry hydrogen chloride in a solvent such as diethyl ether or dioxane to prepare the hydrochloride salt of the compound described by formula I. In another embodiment, the acidic reagent is trifluoroacetic acid (TFA). In one embodiment, the compound of formula I can be used as a free base. Generally, the free base compound is converted to an acid addition salt for ease of handling and to improve chemical stability. Examples of acid addition salts include, but are not limited to, hydrochloride, hydrobromide, 2,2,2-trifluoroacetate, and methanesulfonate.

[0069] therapeutic use The present invention provides methods of using compounds described by formula I to inhibit membrane-bound and soluble SSAO / VAP-1. Additionally, the present invention provides methods of using compounds described by formula I to inhibit MAO-B. The relative inhibitory potencies of the compounds can be determined from the amounts required to inhibit the amine oxidase activity of SSAO / VAP-1 and MAO-B in a variety of ways, such as in vitro assays using recombinant human proteins or recombinant non-human enzymes, in cell assays expressing normal rodent enzymes, in cell assays transfected with human proteins, in vivo tests in rodents and other mammalian species, etc.

[0070] The present invention also discloses methods of using compounds of formula I to inhibit SSAO / VAP-1 and MAO-B in patients suffering from neuromuscular disorders, as well as methods of treating symptoms associated with such disorders.

[0071] Thus, in one embodiment, the present invention relates to a method of inhibiting the amine oxidase activity of SSAO / VAP-1 and MAO-B in a subject in need thereof, comprising administering to said subject an effective amount of a compound of formula 1, or a pharma- ceutical acceptable salt or solvate thereof, or a pharmaceutical composition thereof.

[0072] In another aspect, the present invention relates to a method for treating or preventing a disease or disorder by inhibiting the activity of the SSAO / VAP-1 protein and the MAO-B protein, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula 1, or a pharma- ceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof. In one embodiment, the present invention relates to a method for treating a disease or disorder by inhibiting the activity of the SSAO / VAP-1 protein and the MAO-B protein. In another embodiment, the present invention relates to a method for preventing a disease or disorder by inhibiting the activity of the SSAO / VAP-1 protein and the MAO-B protein.

[0073] In yet another aspect, the present invention relates to a method for treating a disease or disorder modulated by SSAO / VAP-1 and MAO-B, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, or a pharma- ceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof.

[0074] In a further aspect, the present invention relates to a method for treating or preventing a disease or disorder selected from the group consisting of neuromuscular disorders, neurodegenerative disorders, and neuroinflammatory disorders, said method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, or a pharma- ceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof. In one embodiment, the present invention relates to the treatment of a disease or disorder selected from the group consisting of neuromuscular disorders, neurodegenerative disorders, and neuroinflammatory disorders. In another embodiment, the present invention relates to the treatment of a disease or disorder selected from the group consisting of neuromuscular disorders, neurodegenerative disorders, and neuroinflammatory disorders. The present invention relates to preventing a disease or disorder selected from the group consisting of inflammatory diseases.

[0075] Another embodiment of the invention is a method of treatment, wherein said disease or disorder is ameliorated by selective inhibition of SSAO / VAP-1 and MAO-B versus MAO-A. By selectively inhibiting SSAO / VAP-1 and MAO-B versus MAO-A, the compounds of the invention provide a favorable safety profile.

[0076] In a further aspect of the invention, there is provided the use of a compound of formula I, or a pharma- ceutically acceptable salt or solvate thereof, for the manufacture of a medicament for treating or preventing a disease or disorder by inhibiting said activity of said SSAO / VAP-1 protein and said MAO-B protein.

[0077] In another aspect of the invention, there is provided the use of a compound of formula I, or a pharma- ceutically acceptable salt or solvate thereof, for the manufacture of a medicament for treating or preventing a disease or disorder selected from the group consisting of neuromuscular disorders, neurodegenerative diseases and neuroinflammatory diseases.

[0078] The above method and use are applicable when the disease is a neuromuscular disorder. As used herein, the term "neuromuscular disorder or disease" refers to disorders that impair the function of voluntary muscles, either directly through pathology of the voluntary muscles or indirectly through pathology of the nerves or neuromuscular junction. It covers a wide range of diseases, including muscular dystrophies, such as Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophies, and indirect central disorders, such as Parkinson's disease, multiple sclerosis, Huntington's disease, and Creutzfeldt-Jakob disease. In one embodiment of the methods and uses of the present invention, the muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy and myotonic dystrophy. In one embodiment of the methods and uses of the present invention, the muscular dystrophy is Duchenne muscular dystrophy (DMD).

[0079] The term "neuromuscular disease" can also refer to motor neuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy, ion channelopathies, myopathies, mitochondrial diseases such as Friedreich's ataxia, diseases of the neuromuscular junction such as myasthenia gravis, and peripheral nerve diseases such as Charcot-Marie-Tooth disease.

[0080] The dual benefit of inhibiting SSAO / VAP-1 and MAO-B is the potential for treating inflammatory diseases associated with oxidative stress.

[0081] As used herein, "inflammation or inflammatory disorder" encompasses a wide variety of indications including arthritis (including juvenile rheumatoid arthritis), Crohn's disease, ulcerative colitis, inflammatory bowel disease (e.g., irritable bowel disease), psoriasis, asthma, pneumonia, COPD, bronchiectasis, dermatitis, eye disease, contact dermatitis, hepatitis, chronic liver disease, autoimmune hepatitis, primary biliary cirrhosis, sclerosing cholangitis, autoimmune cholangitis, alcoholic hepatitis, atherosclerosis, chronic heart failure, congestive heart failure, ischemic disease, stroke and its complications, sepsis, acute lung injury, and inflammation due to bacterial infection experienced in cystic fibrosis, myocardial infarction and its complications, inflammatory cell destruction after stroke, synovitis, and systemic inflammatory sepsis.

[0082] The term "oxidative stress" refers to diseases in which an increase in reactive oxygen species is directly related to the progression or symptoms of the disease, including cancer, muscular dystrophy, Alzheimer's disease, Parkinson's disease, diabetes, high blood pressure, cardiovascular diseases such as atherosclerosis and stroke, inflammation, and the like. These include many different disorders including neurological disorders, chronic fatigue syndrome, fragile x syndrome, depression, attention deficit hyperactivity disorder, autism, Asperger's syndrome, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, asthma and male infertility.

[0083] In one embodiment of the methods and uses of the present invention, the disease is an inflammatory disease selected from a neurological disease, a metabolic disease and a fibrotic disease.

[0084] The above method and use are also applicable when the disease is a neurological disease or disorder. As used herein, "neurological disease or disorder" refers to both neuroinflammatory and neurodegenerative diseases, and includes various indications such as stroke, Alzheimer's disease, familial Alzheimer's disease, Parkinson's disease, senile dementia, vascular dementia, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Down's syndrome, and homozygotes of the apolipoprotein E4 allele. In one embodiment of the method and use of the present invention, the neurodegenerative or neuroinflammatory disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, myasthenia gravis, and Charcot-Marie-Tooth disease.

[0085] Other examples of neurological disorders include psychiatric disorders, such as depression, anxiety, panic attacks, social phobia, schizophrenia, anorexia, and depressive mood disorders.

[0086] Further examples of neurological disorders include withdrawal syndromes induced by alcohol, nicotine and other addictive drugs.

[0087] The above-mentioned method and use are also applicable when the disease is a metabolic disorder or disease. As used herein, "metabolic disorder or disease" encompasses a wide variety of indications, including obesity, type 1 and type 2 diabetes, hypertension, atherosclerosis, hypertension, chronic kidney disease, dyslipidemia, heart disease, metabolic syndrome, insulin resistance, diabetic nephropathy, glomerulosclerosis, diabetic retinopathy, choroidal neovascularization, and cardiovascular complications resulting therefrom.

[0088] In accordance with yet another aspect of the present invention, there is provided a method of treating patients with type 1 and type 2 diabetes and diabetes-related conditions with a dual SSAO / VAP-1 and MAO-B inhibitor, including diabetic nephropathy, diabetic macular edema, glomerulosclerosis, diabetic retinopathy, choroidal neovascularization, and cardiovascular complications resulting therefrom.

[0089] The above-mentioned methods and uses are also applicable when the disease is fibrosis. As used herein, "fibrosis" includes cystic fibrosis, idiopathic pulmonary fibrosis, renal fibrosis, chronic obstructive pulmonary disease, pleural fibrosis and radiation-induced pulmonary fibrosis, non-diabetic renal fibrosis, pancreatic fibrosis, scleroderma, connective tissue disease, scarring, skin fibrosis, fibrosis associated with organ transplantation, stenosis, hepatic fibrosis including alcoholic liver disease, fatty liver disease, non-alcoholic steatohepatitis, acute and chronic hepatitis, biliary cirrhosis, primary sclerosing cholangitis, hepatocellular carcinoma and toxic liver injury, and other diseases in which excessive fibrosis contributes to disease pathology. In one embodiment, the fibrosis is myocardial fibrosis.

[0090] Pharmaceutical and / or Therapeutic Formulations In another embodiment of the present invention, there is provided a composition comprising a compound of formula I and at least one pharma- ceutically acceptable excipient, carrier or diluent thereof. The compound(s) of formula I may also be present as a suitable salt, including a pharma- ceutically acceptable salt.

[0091] The term "pharmaceutical acceptable carrier" refers to a carrier that is suitable for a particular mode of administration and is understood by those of skill in the art to be suitable for such a mode. The term "carrier" refers to any of the carriers known in the art. Additionally, the compounds may be formulated as the only pharma- ceutical active ingredient in the composition or may be formulated with other active ingredients.

[0092] The term "pharmaceutical acceptable salt" refers to any salt preparation that is suitable for use in pharmaceutical applications. A pharmaceutical acceptable salt means a salt that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., commensurate with a reasonable benefit / risk assessment.

[0093] Pharmaceutically acceptable salts include acid addition salts and base salts, as is well known in the art. Hemi-salts of acids and bases can also be formed. Pharmaceutically acceptable salts include amine salts of mineral acids (e.g., hydrochloride, hydrobromide, sulfate, etc.); and amine salts of organic acids (e.g., formate, acetate, lactate, malate, tartrate, citrate, ascorbate, succinate, maleate, butyrate, valerate, fumarate, etc.).

[0094] For compounds of formula I that have a basic moiety, suitable pharma-ceutically acceptable salts may be acid addition salts.For example, suitable pharma-ceutically acceptable salts of such compounds can be prepared by mixing a pharma-ceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid with the compound of the present invention.

[0095] SMBerge et al. provide a detailed description of pharma- ceutically acceptable salts in J. Pharmaceutical Sciences. 1977, 66:1-19. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or can be prepared separately by reacting the free base function with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, cyclopentanepropionate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxybutanol ... Examples of suitable base salts include s-ethanesulfonate, lactobionate, lactate, laurate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. Suitable base salts are formed from bases which form non-toxic salts.

[0096] Pharmaceutically acceptable salts of compounds of formula 1 can be prepared by methods known to those skilled in the art, including, for example, the following. i. by reacting a compound of formula 1 with a desired acid or base; ii. by removing an acid- or base-labile protecting group from a suitable precursor of a compound of formula 1, or by ring-opening a suitable cyclic precursor, such as a lactone or lactam, with a desired acid or base; or iii. By converting one salt of a compound of formula 1 to another salt by reaction with an appropriate acid or base, or by using an appropriate ion exchange column.

[0097] The above reactions (i) to (iii) are usually carried out in a solution. The resulting salt may be precipitated and collected by filtration or by evaporating the solvent. The degree of ionization in the resulting salt may vary from completely ionized to almost non-ionized.

[0098] Thus, for example, suitable pharma- ceutically acceptable salts of the compounds of the present invention can be prepared by mixing a pharma- ceutically acceptable acid, such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid or citric acid, with a compound of the present invention. Thus, suitable pharma-ceutically acceptable salts of the compounds of the present invention include acid addition salts.

[0099] The compounds of the present invention can exist in both unsolvated and solvated forms.The term "solvate" is used herein to describe a molecular complex that comprises the compounds of the present invention and a stoichiometric amount of one or more pharma-ceutically acceptable solvent molecules, such as ethanol.The term "hydrate" is employed when the solvent is water.

[0100] In the present specification, the composition comprises one or more compounds provided herein.In one embodiment, the compound is formulated into suitable pharmaceutical preparations, such as solution, suspension, tablet, cream, gel, dispersible tablet, pill, capsule, powder, sustained release formulation or elixir, for oral administration or as sterile solution or suspension for parenteral administration, and is formulated into transdermal patch formulation and dry powder inhalant.In one embodiment, the compound is formulated into pharmaceutical composition by using techniques and procedures well known in the art (for example, see Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126).

[0101] In the composition, the effective concentration of one or more compounds or their pharma- ceutically acceptable derivatives is mixed with a suitable pharmaceutical carrier.The compound may be derivatized as the corresponding salt, ester, enol ether or ester, acetal, ketal, orthoester, hemiacetal, hemiketal, acid, base, solvate, hydrate or prodrug before formulation as described above.The concentration of the compound in the composition is effective to deliver the amount that treats, prevents or improves one or more symptoms of the disease or disorder to be treated when administered.

[0102] In one embodiment, the composition is formulated for single administration by dissolving, suspending, dispersing or otherwise mixing a portion by weight of the compound in a carrier to provide an effective concentration to alleviate, prevent or ameliorate one or more symptoms being treated.

[0103] The active compound is contained in a pharma- ceutically acceptable carrier in an amount sufficient to exert a therapeutically acceptable effect without undesirable side effects on the treated patient. The therapeutically effective concentration can be empirically determined by testing the compound in the in vitro and in vivo systems described herein and in WO 04 / 018997, and the dosage for humans can be extrapolated therefrom.

[0104] The concentration of active compound in the pharmaceutical composition will depend on the degree of absorption, distribution, inactivation and excretion rates of the active compound, the physicochemical properties of the compound, the administration schedule and amount administered, as well as other factors known to those of skill in the art.

[0105] In one embodiment, a therapeutically effective dosage should provide a serum concentration of the active ingredient of about 0.1 ng / mL to about 50-100 μg / mL. In another embodiment, the pharmaceutical composition desirably provides a dosage of about 0.001 mg to about 2000 mg of compound per kg of body weight per day. Pharmaceutical dosage units may be from about 0.01 mg, 0.1 mg, or 1 mg to about 500 mg, 1000 mg, or 2000 mg of active ingredient or essential ingredients combined per dosage unit form, and in one embodiment, from about 10 mg to about Prepare a final volume of 500 mg.

[0106] Administration may be at intervals of minutes, hours, days, weeks, months or years, or may be continuous over any one of these time periods. Suitable dosages are within the range of about 0.1 ng / kg body weight to 1 g / kg body weight per administration. Suitably, the dosage is within the range of 1 μg to 1 g / kg body weight per administration, for example, within the range of 1 mg to 1 g / kg body weight per administration. Suitably, the dosage is within the range of 1 μg to 500 mg / kg body weight per administration, for example, within the range of 1 μg to 200 mg / kg body weight per administration, or within the range of 1 μg to 100 mg / kg body weight per administration. Other suitable dosages may be within the range of 1 mg to 250 mg / kg body weight, for example, within the range of 1 mg to 10, 20, 50 or 100 mg / kg body weight per administration, or within the range of 10 μg to 100 mg / kg body weight per administration.

[0107] Appropriate dosages and administration regimens will be determined by the attending physician and will depend on the particular condition being treated, the severity of the condition, and the general health, age, and weight of the subject.

[0108] If the solubility of the compound is not sufficient, methods can be used to solubilize it. Such methods include, but are not limited to, methods known to those skilled in the art, such as using cosolvents such as dimethylsulfoxide (DMSO), using surfactants such as TWEEN®, dissolving in aqueous sodium bicarbonate, formulating the compound of interest as nanoparticles, etc. In addition, derivatives such as prodrugs of the compound can also be used to formulate effective pharmaceutical compositions.

[0109] Upon mixing or addition of the compound, the resulting mixture may be a solution, suspension, emulsion, etc. The form of the resulting mixture will depend on many factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration will be a concentration sufficient to ameliorate the symptoms of the disease, disorder, or condition being treated, and can be empirically determined.

[0110] The pharmaceutical compositions are administered to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, as well as oil-water emulsions containing an appropriate amount of the compound or its pharma- ceutically acceptable derivatives. In one embodiment, the pharma- ceutical and therapeutically active compounds and derivatives thereof are formulated and administered in unit dosage forms or multiple dosage forms. The active ingredient may be administered at once or may be divided into multiple smaller doses to be administered at intervals of time. As used herein, unit dosage forms refer to physically separate units suitable for administration to human and animal subjects, and are individually packaged as known in the art. Each unit dosage contains a predetermined amount of the therapeutically active compound sufficient to produce the desired therapeutic effect, together with the relevant pharmaceutical carrier, vehicle or diluent as required. Examples of unit dosage forms include ampoules, syringes, and individually packaged tablets or capsules. The unit dosage forms may be administered separately or in multiples of their portions. A multiple-dose form is a plurality of identical unit dosage forms packaged in a single container and administered as segregated unit dosage forms. Examples of multiple-dose forms include vials or bottles of tablets or capsules, or bottles of pint or gallon size. Thus, a multiple-dose form is a dosage form in which a plurality of unit dosage forms are packaged together in one package.

[0111] Actual methods for preparing such dosage forms will be known, or apparent, to those skilled in the art; see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., See 15th Edition, 1975.

[0112] Dosage forms or compositions can be prepared containing active ingredient in the range of 0.005% to 100% (by weight), with the remainder consisting of non-toxic carriers. Methods for preparing these compositions are known to those skilled in the art. Contemplated compositions may contain 0.001% to 100% (by weight), active ingredient, in one embodiment 0.1 to 95% (by weight), and in another embodiment 75 to 85% (by weight).

[0113] Mode of administration Convenient modes of administration include injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, topical cream or gel or powder, vaginal or rectal administration. Depending on the route of administration, the formulation and / or compound may be coated with a material to protect the compound from the action of enzymes, acids and other natural conditions that may inactivate the therapeutic activity of the compound. The compound may also be administered parenterally or intraperitoneally.

[0114] Composition for oral administration Oral pharmaceutical dosage forms are either solid, gel or liquid.Solid dosage forms are tablets, capsules, granules and bulk powders.Types of oral tablets include compressed, chewable lozenges and tablet tablets, which may be enteric coated, sugar coated or film coated.Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form in combination with other ingredients known to those skilled in the art.

[0115] Solid Compositions for Oral Administration In certain embodiments, the formulation is a solid dosage form, in one embodiment, a capsule or tablet. Tablets, pills, capsules, lozenges, etc. may contain one or more of the following ingredients, or compounds of similar nature: for example, binders; lubricants; diluents; glidants; disintegrants; colorants; sweeteners; flavorings; wetting agents; emetic coatings; and film coatings. Examples of binders include microcrystalline cellulose, tragacanth gum, glucose solution, acacia mucilage, gelatin solution, molasses, polyvinylpyrrolidine, povidone, crospovidone, sucrose, and starch paste. Lubricants include, for example, talc, starch, magnesium or calcium stearate, liposomes, stearic acid, and the like. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol, dicalcium phosphate, and the like. Lubricants include, for example, colloidal silicon dioxide, and the like, but are not limited thereto. Disintegrants include, for example, croscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar, and carboxymethylcellulose. Coloring agents include, for example, any of the approved water-soluble FD and C dyes, mixtures thereof; and water-insoluble FD and C dyes suspended on alumina hydrate. Sweeteners include sucrose, lactose, mannitol, and artificial sweeteners such as saccharin, and any number of spray-dried flavors. Flavoring agents include, but are not limited to, natural flavors extracted from plants such as fruits, and synthetic blends of compounds that produce a pleasant sensation, such as peppermint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, polyoxyethylene laural ether, and the like. Emetic coating agents include fatty acids, oils, waxes, shellac, ammoniated shellac, cellulose acetate phthalate, and the like. Film coating agents include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate.

[0116] The compound or a pharma- ceutically acceptable derivative thereof can be provided in a composition in such a way that it is protected from the acidic environment of the stomach. For example, the compound or a pharma- ceutical acceptable derivative thereof can be formulated in a composition that is enclosed in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition can also be formulated in combination with an antacid or other such ingredient.

[0117] When the dosage unit form is a capsule, it can contain liquid carriers such as fatty oils in addition to the above-mentioned materials.Furthermore, dosage unit form can contain various other materials that change the physical form of the dosage unit, such as coatings of sugar and other enteric agents.The compound can also be administered as a component of elixirs, suspensions, syrups, wafers, sprinkles, chewing gums, etc.Syrups can contain sucrose as a sweetener, and certain preservatives, dyes and colorings, and flavorings, in addition to the active compounds.

[0118] The active substances can also be mixed with other active substances which do not impair the desired action, or with substances which supplement the desired action, such as antacids, H2 blockers, diuretics, etc. The active material is a compound described herein or a pharma- ceutically acceptable derivative thereof. Higher concentrations, up to about 98% by weight of the active ingredient, may also be included.

[0119] In all embodiments, tablets and capsules may be coated with coatings known to those skilled in the art to control or maintain the solubility of the active ingredient therein. Thus, tablets and capsules may be coated with conventional enteric digestible coatings such as, for example, phenyl salicylate, waxes and cellulose acetate phthalate.

[0120] Liquid Compositions for Oral Administration Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

[0121] Liquid pharma- ceutically administrable compositions can be prepared, for example, by dissolving, dispersing, or otherwise mixing the active compound, as defined above, and optional pharmaceutical adjuvants in a carrier, for example, water, saline, aqueous dextrose, glycerol, glycol, ethanol, and the like, thereby forming a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents, and the like, for example, acetic acid, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and the like.

[0122] Elixirs are clear, sweetened, aqueous or hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, such as sucrose, and may contain preservatives. Emulsions are two-phase systems in which one liquid is dispersed in the other liquid in the form of small globules. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifiers, and preservatives. Suspensions use pharma- ceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used in non-effervescent granules to be reconstituted into liquid oral dosage forms include diluents, sweeteners, wetting agents, etc. Pharmaceutically acceptable substances used in effervescent granules to be reconstituted into liquid oral dosage forms include organic acids and carbon dioxide sources. Coloring and flavoring agents are used in all of the above dosage forms.

[0123] Examples of solvents include glycerin, sorbitol, ethyl alcohol, starch syrup, etc. Examples of preservatives include glycerin, methylparaben, propylparaben, benzoic acid, sodium benzoate, ethanol, etc. Examples of non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil. Examples of emulsifiers include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate, etc. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, veegum, acacia, etc. Sweetening agents include sucrose, starch syrup, glycerin, artificial sweeteners such as saccharin, etc. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, polyoxyethylene lauryl ether, etc. Organic acids include citric acid, tartaric acid, etc. Carbon dioxide gas sources include sodium bicarbonate, sodium carbonate, etc. Coloring agents include any of the certified water soluble FD and C dyes, and mixtures thereof.Flavoring agents include natural flavors extracted from fruits and other plants, and synthetic blends of compounds which produce a pleasant taste sensation.

[0124] For a solid dosage form, the solution or suspension, for example in propylene carbonate, vegetable oils or triglycerides, is in one embodiment encapsulated in a gelatin capsule. For a liquid dosage form, the solution, for example in a polyethylene glycol, may be diluted with a sufficient quantity of a pharma- ceutically acceptable liquid carrier, for example water, to be easily measured for administration.

[0125] Alternatively, liquid or semisolid oral formulations can be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions with hard or soft gelatin capsule shells. Other useful formulations include those described in U.S. Patents RE28,819 and 4,358,603. Briefly, such formulations include those comprising a compound provided herein, a dialkylated mono- or polyalkylene glycol (e.g., but not limited to, 1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether (where 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol), and one or more antioxidants, such as, but not limited to, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarin, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.

[0126] Injections, solutions and emulsions In one embodiment herein characterized by injection, parenteral administration is intended to include either subcutaneous, intramuscular or intravenous. Injectables can be prepared in conventional forms, for example, as liquid solutions or suspensions, as solid forms suitable for dissolving or suspending in liquid prior to injection, or as emulsions. Injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical composition to be administered may contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other such agents, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0127] Also contemplated herein is implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained. Briefly, the compounds provided herein include those that are dispersed in a solid internal matrix, such as hydrophilic polymers such as polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyacetic acid, and the like. These include compounds that are coated on the outside with, for example, polyethylene, polypropylene, ethylene / propylene copolymer, ethylene / ethyl acrylate copolymer, ethylene / vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, neoprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl acetate and vinyl chloride copolymer, vinylidene chloride, ethylene, propylene, ionomeric polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene / vinyl alcohol copolymer, ethylene / vinyl acetate / vinyl alcohol terpolymer, ethylene / vinyloxyethanol copolymer (insoluble in body fluids). The compound diffuses through the outer polymeric membrane in a release rate controlled step. The percentage of active compound contained in such parenteral compositions is largely determined by its specific nature, and the activity of the compound and the needs of the subject.

[0128] Parenteral administration of the composition includes intravenous administration, subcutaneous administration and intramuscular administration. Formulations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products ready to be combined with a solvent immediately before use, including subcutaneous injections, such as lyophilized powders, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle immediately before use, and sterile emulsions. The solutions can be either aqueous or non-aqueous.

[0129] If administered intravenously, suitable carriers include saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0130] Pharmaceutically acceptable carriers for parenteral formulations include aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering and chelating agents, and other pharma- ceutically acceptable substances.

[0131] Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose injection, lactated Ringer's injection, etc. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, olive oil, cottonseed oil, corn oil, sesame oil, peanut oil, etc. Parenteral preparations filled in multi-dose containers must be supplemented with bacteriostatic or bactericidal concentrations of antibacterial agents, including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, p-hydroxybenzoic acid methyl and propyl esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonicity agents include sodium chloride, dextrose, etc. Buffers include phosphates, citrates, etc. Antioxidants include sodium bisulfate, etc. Local anesthetics include procaine hydrochloride, etc. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, etc. Examples of emulsifiers include polysorbate 80 (TWEEN® 80). Examples of sequestering or chelating agents include EDTA. Examples of pharmaceutical carriers include water-miscible vehicles such as ethyl alcohol, polyethylene glycol, and propylene glycol; and pH adjusters such as sodium hydroxide, hydrochloric acid, citric acid, and lactic acid.

[0132] The concentration of the pharma- ceutical active compound is adjusted to provide an effective amount that produces the desired pharmacological effect upon injection. The exact dose will vary according to the age, weight and condition of the patient or animal as is known in the art.

[0133] Unit dose parenteral preparations are packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration must be sterile, as is known in the art and can be used.

[0134] Intravenous or intraarterial infusion of a sterile aqueous solution containing an active compound is one example of an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing the active material, injected as needed to produce the desired pharmacological effect.

[0135] Injectables are designed for local and systemic administration. In one embodiment, a therapeutically effective dose is formulated to contain a concentration of at least about 0.1% w / w to about 90% w / w or more of the active compound in the treated tissue(s), and in certain embodiments, greater than 1% w / w.

[0136] The compound may be suspended in micronized or other suitable form, or derivatized to produce a more soluble active product, or to produce a prodrug. The form of the resulting mixture depends on many factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration must be sufficient to ameliorate symptoms, but can be empirically determined.

[0137] Topical administration Mixtures for topical administration are prepared in the same manner as described for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, irrigation, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.

[0138] The compound or its pharma- ceutically acceptable derivatives can be formulated as aerosols for topical application, such as by inhalation. These formulations for administration to the respiratory tract can be used in the form of aerosols or solutions for nebulizers, or as fine powders for insufflation, alone or in combination with inert carriers such as lactose. In such cases, the diameter of the particles of the formulation will be less than 50 microns in one embodiment, and less than 10 microns in one embodiment.

[0139] The compounds can be formulated for topical or local application, for example, in the form of gels, creams and lotions, for topical application to the skin and mucous membranes, and for ocular or intrathoracic or intraspinal application. Topical administration is intended for transdermal delivery, as well as for administration to the eyes or mucous membranes, or for inhalation therapy. The active compounds can also be prepared and administered as nasal solutions, alone or in combination with other pharma- ceutically acceptable excipients.

[0140] These solutions, particularly those intended for ophthalmic use, may be formulated as 0.01% to 10% (by volume) isotonic solutions (pH about 5-7) containing appropriate salts.

[0141] Compositions for other routes of administration Other routes of administration are also contemplated herein, such as transdermal patches, including iontophoresis and electrophoretic devices, vaginal and rectal administration.

[0142] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those skilled in the art. For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules, and systemic effect tablets. Rectal suppositories are used herein to mean solid bodies for rectal insertion that melt or soften at body temperature to release one or more pharmacologically or therapeutically active ingredients. The pharma-ceutically acceptable substances utilized in rectal suppositories are bases or vehicles and agents to increase their melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol), and suitable mixtures of mono-, di-, and triglycerides of fatty acids. Various bases may also be used in combination. Suppository melting point elevating agents include spermaceti, wax, and the like. Rectal suppositories can be prepared by either compression or molding. The weight of a rectal suppository is, in one embodiment, about 2 to 3 gm.

[0143] Tablets and capsules for rectal administration are manufactured using the same pharma- ceutically acceptable substance and by the same methods as formulations for oral administration.

[0144] Targeted formulations The compounds provided herein or their pharma- ceutically acceptable derivatives can also be formulated to target specific tissues, receptors or other areas of the body of the subject to be treated.Many such targeting methods are known to those skilled in the art.All such targeting methods are contemplated herein for use in the defensive purposes of the present compositions.

[0145] Coadministration with other drugs In accordance with another aspect of the present invention, it is contemplated that the compounds of formula 1 described herein may be administered to a subject in need thereof in combination with drugs that are currently considered by those of skill in the art to be standard of care for the condition of interest, such that such combination provides one or more benefits to the subject, such as, for example, administering a lower dose to achieve the same effect or achieving the desired relief in a shorter period of time.

[0146] The compound of the present invention can be administered together with other drugs as part of a treatment regimen.For example, when it is intended to treat a particular disease or condition, it may be desirable to administer active compounds in combination.Therefore, it is within the scope of the present invention to combine two or more pharmaceutical compositions that contain at least one compound of formula (I) of the present invention into a kit form suitable for co-administration of the compositions.

[0147] In one embodiment of the method of the invention, the compound of formula I may be administered in combination with a second therapeutic agent, hi one embodiment, the second therapeutic agent is selected from the group consisting of anti-inflammatory agents, anti-hypertensive agents, anti-fibrotic agents, anti-angiogenic agents, and immunosuppressants.

[0148] In one embodiment of the method of the invention, the compound of formula I may be administered with a second therapeutic agent selected from the group consisting of steroidal anti-inflammatory agents, such as prednisolone and deflazacort, and non-steroidal anti-inflammatory agents, such as aspirin, paracetamol, ibuprofen, naproxen, indomethacin, celecoxib, and diclofenac.

[0149] In another embodiment of the method of the present invention, the compound of formula I may be administered with a second therapeutic agent selected from the group consisting of calcium channel blockers, such as verapamil, nisoldipine, and amlodipine; angiotensin II receptor antagonists, such as irbesartan, losartan, telmisartan, and valsartan; and inhibitors of angiotensin-converting enzyme, such as benazepril, captopril, enalapril, and fosinopril.

[0150] In another embodiment of the method of the present invention, the compound of formula I is administered in combination with pirfenidone and nintedanib. The therapeutic agent may be administered in combination with a second therapeutic agent selected from the group consisting of:

[0151] In another embodiment of the method of the present invention, the compound of formula I may be administered with a second therapeutic agent selected from the group consisting of axitinib, bevacizumab, everolimus, lenalidomide, regorafenib, sorafenib, sunitinib, thalidomide, vandetanib and zivaflibercept.

[0152] In another embodiment of the method of the present invention, the compound of formula I may be administered with a second therapeutic agent selected from the group consisting of glucocorticoids such as prednisone, dexamethasone, and hydrocortisone; intravenous agents such as cyclophosphamide, azathioprine, dactinomycin, and methotrexate; antibodies acting directly on the immune response such as basiliximab, daclizumab, muromonab; drugs acting on immunophilins such as cyclosporine, rapamycin, tacrolimus, everolimus; and other immunosuppressants such as fingolimod, interferon therapy, infliximab, etanercept, and adalimumab.

[0153] In one embodiment of the method of the invention, the compound of formula I may be administered with a second therapeutic agent, hi one embodiment, the second therapeutic agent is selected from agents believed to be useful in the treatment of muscular dystrophies, including prednisone, deflazacort, ataluren, myostatin, eteplirsen, golodirsen, casimersen, idebenone, panrevlumab, edasalonexent, divinostat, rimeporide, and ifetroban.

[0154] When two or more active ingredients are co-administered, the active ingredients can be administered simultaneously, sequentially or separately. In one embodiment, the compound of formula I is co-administered simultaneously with the second therapeutic agent. In another embodiment, the compound of formula I and the second therapeutic agent are administered sequentially. In a further embodiment, the compound of formula I and the second therapeutic agent are administered separately.

[0155] The invention will now be described in more detail, by way of example only, with reference to the following non-limiting examples, which are intended to serve to illustrate the invention and should not be construed as limiting the generality of the disclosure throughout the specification.

[0156] The abbreviations used in this specification have the following meanings: rt=room temperature min=minutes h=time NaHMDS = sodium hexamethyldisilazide THF = tetrahydrofuran DMF = Dimethylformamide NaOtBu = sodium t-butoxide TFA = trifluoroacetic acid DIBAL = diisobutylaluminum hydride MTBE = Methyl tertiary butyl ether DIPEA = diisopropylethylamine LC-MS = Liquid Chromatography-Mass Spectrometer rpm = revolutions per minute NMR=nuclear magnetic resonance DMSO = dimethyl sulfoxide Boc = tert-butyloxycarbonyl HPLC = High Performance Liquid Chromatography (also known as High Pressure Liquid Chromatography) rac. = racemic TLC = Thin Layer Chromatography EXAMPLES

[0157] Experiment: General Method All commercially available solvents and reagents were used as received. Where necessary, reactions were carried out under an argon atmosphere. Reactions were monitored by analytical thin layer chromatography (TLC) or analytical liquid chromatography-mass spectrometry (LC-MS) using either a Shimadzu LC-MS 2020 instrument or an Agilent LC-MS 1200 instrument using reversed phase conditions. Purification of intermediates and final compounds was performed using column chromatography or preparative HPLC where necessary. Normal phase column chromatography was performed using silica gel or packed silica gel cartridges at medium pressure using a flash chromatography system (CombiFlash Rf200, Teledyne Isco systems, USA). For reversed phase column chromatography, packed C18 cartridges were used at low pressure using a flash chromatography system (Reveleris® X2). Eluents were monitored with UV light (λ=254 / 280 nm). 1 H-NMR and 19 F-NMR spectra were recorded using a Bruker 300 MHz NMR spectrometer or a Bruker Avance III plus 400 MHz NMR spectrometer. Chemical shifts (δ) were expressed as parts per million (ppm) relative to tetramethylsilane (TMS; internal standard). The following abbreviations were used for multiplicities: s = singlet; br s = broad singlet; d = doublet; t = triplet; q = quartet; p = pentet; m = multiplet; and br m = broad multiplet. Low-resolution mass spectra (MS) were obtained as electrospray-atmospheric pressure ionization (ES-API) mass spectra using a Shimadzu LC-MS 2020 or an Agilent LC-MS 1200 instrument. All animal experiments were performed in compliance with the relevant institutional guidelines and with the approval of the local ethical committee.

[0158] Example 1 Preparation of tert-butyl (E)-(2-(bromomethyl)-3-fluoroallyl)carbamate (Int-6) [ka] Procedure A: Preparation of tert-butyl 3-(2,5-dimethyl-1H-pyrrol-1-yl)propanoate (Int-1) [ka]

[0159] To a vessel charged with acetonitrile (754 L) under nitrogen were sequentially added β-alanine tert-butyl ester hydrochloride (75 kg), 2,5-hexanedione (97.5 L), and potassium carbonate (75 kg). The resulting mixture was heated to 75° C. over 2 hours and stirred for an additional 4 hours. The reaction mixture was cooled to 55° C. and concentrated in vacuo to ∼290 L, followed by addition of 10 mL of 100 mL of acetonitrile. Water (1200 L) was added to the residue at 55° C. The mixture was stirred at 55° C. for 1 h and then cooled to 10° C. over 2 h. Stirring was continued at 10° C. for 10 h. The resulting "cake" was washed with more water (150 L×3) and centrifuged to remove the liquid phase after each washing cycle. After final removal of the filtrate, the wet "cake" was dried under vacuum at 40° C. for 24 h to give Int-1 (79.4 kg, 85%). 1 H-NMR (300MHz, CDCl3) δppm: 5.78 (s, 2H), 4.02-4.07 (m, 2H), 2.50-2.56 (m, 2H), 2.25 (s, 6H), 1.47 (s, 9H).

[0160] Procedure B. Preparation of 1-(tert-butyl) 3-ethyl 2-(difluoromethyl)-2-((2,5-dimethyl-1H-pyrrol-1-yl)methyl)malonate (Int-2) [ka] This process was carried out on two batches of Int-1 (39.5 kg x 2), which were later combined.

[0161] To vessel (A) charged with THF (277 L) under nitrogen, NaHMDS (2.0M; 245 kg) was added over 20 minutes. The resulting mixture was cooled to -58°C. To vessel (B) charged with THF (198 L) at -50°C under nitrogen, Int-1 (39.5 kg) was added. The resulting mixture was stirred at -50°C for 25 minutes. The contents of vessel (B) were then transferred to vessel (A) at -58°C over 30 minutes. Ethyl chloroformate (17.4 L) was added to vessel (A) over 1 hour, during which an exotherm was observed. The resulting mixture was stirred at -58°C for 1 hour, to which NaOtBu (15.3 kg) was added over 15 minutes. Chlorodifluoromethane (17.8 kg) was then added slowly at -20°C. The mixture was stirred at -30°C for 2 hours. The vessel was then slowly charged with additional chlorodifluoromethane (7.00 kg) at -20°C. Stirring was continued for another 2 hours at -20°C. The reaction vessel was charged with water (356 L) followed by citric acid (79 kg). The resulting mixture was stirred for 30 minutes and the organic layer was separated. NaCl solution (10%, 342.4 kg) was added, the mixture was stirred for 1 hour, the organic layer was separated and concentrated under vacuum. The residue was dissolved in n-heptane (584.8 kg) and silica gel was added. After stirring for 30 minutes, it was filtered through Celite® and washed with n-heptane (434 kg). The combined filtrate was concentrated in vacuum and the residue thus obtained was recrystallized from EtOH:H2O (1:1) to give the title compound Int 2 as a pale yellow solid (91.0 kg, 74.5%). 1 H-NMR(300MHz,CDCl3)δppm:6.29(t,J=54.6Hz,1H),.72(s,2H),4.56-4.69(m,2H),4 .12-4.23(m,1H),3.98-4.08(m,1H),2.20(s,6H),1.36(s,9H),1.23(t,J=7.2Hz,3H).

[0162] Procedure C: Preparation of ethyl (E)-2-((2,5-dimethyl-1H-pyrrol-1-yl)methyl)-3-fluoroacrylate (Int-3) [ka] Int-2 (91 kg) was added to a reactor (A) charged with TFA (279 L) at 10° C. under nitrogen. The resulting mixture was heated to 35° C. and stirred for 13 hours, then cooled to 5° C. Triethylamine (620 L) was added to a reactor (B) followed by n-heptane (532 L). The resulting solution was cooled to 5° C. The contents of reactor (A) were then transferred to reactor (B) over 2 hours, and the resulting mixture in reactor (B) was heated to 40° C. and stirring was continued for 10 hours. The reaction mixture was cooled to 10° C. and then washed with water (910 L). The organic phase was separated and washed with a solution of citric acid (91 kg) in water (455 L), followed by a solution of NaCl (73 kg) in water (410 L). Silica gel (91 kg) was added to the organic phase, and the resulting slurry was stirred for 30 minutes. The slurry was then filtered and the filter "cake" was washed with n-heptane. The filtrate was concentrated in vacuo and the resulting residue was taken up in toluene (520 L). After further concentration to 650-750 L, the resulting solution of crude Int-3 was used in the subsequent step. 1 H-NMR (300MHz, CDCl3) δppm:7.60(d,J=80.1Hz,1H),5.77(s,2H),4.67-4.68(m,2H),4.20(q,J=7.2Hz,2H),2.26(s,6H),1.27(t,J=7.2Hz,3H).

[0163] Procedure D. Preparation of (E)-2-((2,5-dimethyl-1H-pyrrol-1-yl)methyl)-3-fluoroprop-2-en-1-ol (Int-4) [ka] To the solution of Int-3 in toluene (520 L) prepared in the previous step, diisobutylaluminum hydride (DIBAL) (1.5 M in toluene; 199.2 kg) was added dropwise over 4 h at −55° C. under nitrogen, and the resulting mixture was stirred at −45° C. for 1 h. IP C analysis confirmed incomplete conversion. An additional amount of DIBAL (1.5M in toluene; 15 kg) was added dropwise over 40 min at -55°C. The mixture was then stirred at -45°C for an additional 30 min before warming to 10°C. To quench the reaction, a cold (0°C) solution of citric acid (64 kg) in water (320 L) was added slowly over 2 h, the organic phase was separated and washed with a binary solution of NaCl (32 kg) in water (160 L). Silica gel (32 kg) was added to the organic phase and the resulting slurry was stirred for 30 min. The slurry was filtered through a pad of silica gel (160 kg) and the filter "cake" was washed with toluene (296 L). The filtrate was then concentrated to ∼50 L. n-Heptane (319 L) was added to the residue and the resulting mixture was cooled to 0°C and stirred for 10 h. The solid was then filtered and dried to give Int-4 (11.4 kg, 24% over two steps) as an off-white solid. 1 H-NMR (400MHz, CDCl3) δppm: 6.70 (d, J = 83.4 Hz, 1H), 5.80 (s, 2H), 4.63 (t, J = 1.2 Hz, 2H), 3.76 (t, J = 4.0 Hz, 2H), 2.23 (s, 6H).

[0164] Procedure E: Preparation of tert-butyl (E)-(3-fluoro-2-(hydroxymethyl)allyl)carbamate (Int-5) [ka] To a reaction vessel containing Int-4 (2.0 kg), hydroxylamine hydrochloride (3.80 kg) and water (8.00 L) was added a solution of potassium hydroxide (1.20 kg) in water (10.0 L). The resulting mixture was heated to reflux and stirred for 18 h. After cooling to rt, the reaction mixture was filtered and the filter "cake" was washed with water (6.0 L). Sodium carbonate (3.46 kg) was then added to the filtrate and the resulting mixture was stirred for 30 min. Acetone (9.1 L) was added dropwise at 20° C. and the mixture was stirred for 2 h. Finally, a solution of di-tert-butyl dicarbonate (2.37 kg) in MTBE (10 L) was added and stirring was continued for another 18 h at 15° C. The organic layer was then separated and washed with aqueous NaOH (1.0 M; 16 L×4). Silica gel (2.0 kg) was added to the organic layer and the resulting slurry was stirred for 30 min. The mixture was then filtered and the filter "cake" was washed with MTBE (10 L). The filtrate was concentrated in vacuo to give Int-5 (2.0 kg, 89%). 1 H-NMR (400MHz, CDCl3) δppm: 6.78 (d, J = 81.2 Hz, 1H), 4.86 (br.s, 1H), 4.00-3.96 (m, 4H), 1.45 (s, 9H).

[0165] Procedure F: Preparation of tert-butyl (E)-(2-(bromomethyl)-3-fluoroallyl)carbamate (Int-6) [ka] To a vessel charged with Int-5 (4.0 kg), DIPEA (10.2 L) and THF (40.0 L) at 5° C. was added a solution of methanesulfonic anhydride (5.78 kg) in THF (20.0 L). The resulting mixture was stirred for 30 minutes. To this was added lithium bromide (8.47 kg) and stirring was continued at 5° C. for 2 hours. Water (20.0 L) was then added dropwise to the reaction mixture. The organic layer was separated and concentrated to 8.0 L. To this was added ethyl acetate (24 L) and water (20 L). The organic layer was again separated and concentrated to 12 L. Heptane (40 L) and silica gel (2.0 kg) were added and the resulting slurry was stirred for 30 minutes. The slurry was then filtered and the filter “cake” was washed with n-heptane (20 L). The filtrate was concentrated to dryness under vacuum. More n-heptane was added and stirring was continued at 5° C. for 1 hour. The resulting solid was collected by filtration and dried under vacuum at 30° C. to give Int-6 (3.20 kg, 61%). 1 H-NMR (400MHz, CDCl3) δppm: 6.78(d,J=81.2Hz,1H), 4.68(br.s,1H), 4.00(d,J=4.4Hz,2H), 3.96(d,J=3.2Hz,2H), 1.45(s,9H).

[0166] Example 2 Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)benzonitrile hydrochloride (Compound 1) [ka]

[0167] Procedure G-1: Preparation of tert-butyl (E)-(2-((4-cyanophenoxy)methyl)-3-fluoroallyl)carbamate [ka] To a stirred solution of 4-hydroxybenzonitrile (1.20 g) in acetonitrile (25.0 mL) at rt, potassium carbonate (2.78 g) and Int-6 (2.70 g) were added sequentially. The resulting suspension was stirred overnight at rt. The reaction mixture was filtered through a plug of Celite® to remove inorganics and the filtrate was concentrated in vacuo to give a white solid. This solid was crystallized from diethyl ether / pentane to give tert-butyl (E)-(2-((4-cyanatophenoxy)methyl)-3-fluoroallyl)carbamate (2.81 g, 91%) as a white solid.

[0168] Procedure G-2: Alternative preparation of tert-butyl (E)-(2-((4-cyanophenoxy)methyl)-3-fluoroallyl)carbamate To a stirred solution of 4-hydroxybenzonitrile (2.7 kg) in DMSO (10.15 L) at rt, potassium carbonate (4.05 kg) and Int-6 (5.1 kg) were added sequentially. The resulting suspension was stirred for 4 h. Water (50.75 L) was added and stirred for 1 h. The product was filtered and washed with water (10 L). The crude product was dissolved in DMSO (15 L). Water (60 L) was added slowly and the mixture was stirred for 1 h. The product was filtered, washed with water (10 L) and dried at 55° C. to give tert-butyl (E)-(2-((4-cyanophenoxy)methyl)-3-fluoroallyl)carbamate (5.1 kg, 89%) as a white solid.

[0169] 1 H-NMR(300MHz,CDCl3)δppm:7.65-7.58(m,2H),7.02-6.95(m,2H),6.78(dp,J=81.6,1.1 Hz,1H),4.52(dd,J=3.7,1.2Hz,2H),4.05-3.98(m,2H),1.42(s,9H);MS:207.0 "M-Boc+H" + .

[0170] Procedure H-1: Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)benzonitrile hydrochloride (compound 1) [ka] tert-Butyl (E)-(2-((4-cyanophenyl)phenyl)propanediol in CH2Cl2 (40.0 mL) To a stirred solution of (phenoxy)methyl)-3-fluoroallyl)carbamate (2.75 g) at room temperature was added TFA (40.0 mL). The resulting solution was stirred at rt for 2 h. After this time, LC-MS analysis showed complete consumption of the starting material. The reaction mixture was concentrated in vacuo and the residual TFA was removed by azeotroping with ethyl acetate (20 mL x 2) to give an off-white solid. This solid was dissolved in ethyl acetate (10.0 mL) and hydrochloric acid (2.0 M in diethyl ether; 40.0 mL) was added, at which time a white solid precipitated immediately. After stirring for 15 min, the solid was collected by filtration and dried under high vacuum to give the crude product (2.11 g). The crude material (1.75 g) was crystallized from hot isopropanol / methanol (17.5 mL / 3.50 mL) to give compound 1 (1.26 g, 72%) as a white solid.

[0171] Procedure H-2: Alternative preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)benzonitrile hydrochloride (compound 1) To a stirred solution of tert-butyl (E)-(2-((4-cyanophenoxy)methyl)-3-fluoroallyl)carbamate (5.1 kg) in ethyl acetate (25.5 L) at rt was added 6 M HCl in ethyl acetate (25.5 L). The resulting solution was stirred at rt for 2 h. After this time LC-MS analysis showed complete consumption of the starting material. The precipitated product was filtered and washed with ethyl acetate (10 L). The crude product was suspended in ethyl acetate (40 L) and the mixture was stirred for 4 h. The product was filtered, washed with ethyl acetate (10 L) and dried at 60 °C. The solid was dissolved in water (16 L) and the mixture was heated to 55 °C. The mixture was cooled to 0-5 °C and the solid was filtered. The product was dried under vacuum at 45 °C to give compound 1 (3.3 kg, 76%) as a white solid containing approximately 7% moisture.

[0172] 1H-NMR(500MHz,DMSO-d6)δppm:8.36(s,3H),7.86-7.72(m,2H),7.32(d,J=81.9Hz,1H),7.19-7.09(m,2H),4.74(d,J=3.3Hz,2H),3.57(d,J=2.0Hz,2H). MS:207.0 "M+H"+.

[0173] Example 3 Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-2-fluorobenzonitrile hydrochloride (compound 2) [ka]

[0174] Procedure I: Preparation of tert-butyl (E)-(2-((4-cyano-3-fluorophenoxy)methyl)-3-fluoroallyl)carbamate [ka] To a stirred solution of 2-fluoro-4-hydroxybenzonitrile (411 mg) and Int-6 (804 mg) in DMF (2.0 mL) was added cesium carbonate (1.17 g). The resulting mixture was stirred for 4 h after which LC-MS analysis showed complete consumption of the starting material. Water (20 mL) was then added and the resulting suspension was sonicated and stirred for 10 min. The solid was collected by filtration, washed with water, and dried at 60° C. for 1 h to yield tert-butyl (E)-(2-((4-cyano-3-fluorophenoxy)methyl)-3-fluoroallyl)carbamate (888 mg, 91%) as a white powder. 1 H-NMR(300MHz,CDCl3)δppm:7.55(dd,J=8.6,7.5Hz,1H),6.83-6.72(m,2H),6.78(dp,J=81.0,1.1H z,1H),4.51(dd,J=3.7,1.2Hz,2H),4.01(ddd,J=6.3,2.5,1.0Hz,2H),1.43(s,9H);MS:325.0 "M+H" + .

[0175] Procedure J: Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-2-fluorobenzonitrile hydrochloride (compound 2) [ka] To a stirred solution of tert-butyl (E)-(2-((4-cyano-3-fluorophenoxy)methyl)-3-fluoroallyl)carbamate (50.0 mg) in CHCl (3.0 mL) was added TFA (0.50 mL). The resulting mixture was stirred for 2 h, after which LC-MS analysis showed complete consumption of the starting material. The reaction mixture was concentrated to dryness and residual TFA was removed under high vacuum. The residue was dissolved in ethyl acetate (3.0 mL) followed by addition of HCl (2.0 M in diethyl ether; 77 μL). The mixture was stirred at rt for 30 min during which time a solid precipitated. The reaction mixture was transferred to a vial and the solid was centrifuged in a centrifuge (4000 rpm, 4.0 min). The supernatant was decanted and the solid was washed with the addition of ethyl acetate. After brief sonication, the vial was placed back in the centrifuge and the solid was centrifuged. The supernatant was removed and the solid "cake" was then dried under high vacuum to give compound 2 (36 mg, 90%) as a white powder. 1 H-NMR(300MHz,DMSO-d6)δppm:8.35(s,3H),7.88(dd,J=8.8,7.9Hz,1H),7.36(d,J=81.0Hz,1H),7.3 2-7.17(m,1H),7.05(dd,J=8.8,2.4Hz,1H),4.78(dd,J=3.5,1.1Hz,2H),3.60(s,2H);MS:225.0 "M+H" + .

[0176] Example 4 Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-2-bromobenzonitrile hydrochloride (compound 3) [ka]

[0177] Procedure K: Preparation of tert-butyl (E)-(2-((3-bromo-4-cyanophenoxy)methyl)-3-fluoroallyl)carbamate [ka] To a stirred solution of Int-5 (573 mg) in DMF (5.0 mL) was added NaH (60% in paraffin oil; 120 mg) at 0° C. and stirred at rt for 30 min. Then, a solution of 2-bromo-4-fluorobenzonitrile (500 mg) in DMF (5.0 mL) was added dropwise at 0° C. Stirring at rt was continued for 2 h. The reaction mixture was cooled to 0° C. and quenched with saturated aqueous NH4Cl solution. The product was extracted with ethyl acetate (20 mL×3), the combined organic layers were dried over Na2SO4, and concentrated in vacuo to give tert-butyl (E)-(2-((3-bromo-4-cyanophenoxy)methyl)-3-fluoroallyl)carbamate (750 mg, 82%). MS: 385.0 “M+H” + .

[0178] Procedure L: Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-2-bromobenzonitrile hydrochloride (compound 3) [ka] To a stirred solution of tert-butyl (E)-(2-((3-bromo-4-cyanophenoxy)methyl)-3-fluoroallyl)carbamate (100 mg) in 1,4-dioxane (2.0 mL) at 0° C. was added HCl (4.0 M in 1,4-dioxane; 2.0 mL). The resulting mixture was stirred at rt for 3 h. The reaction mixture was concentrated in vacuo and the residue was triturated with ethyl acetate. The resulting solid was isolated and dried to give compound 3 (58.6 mg, 79%) as a white solid. 1H-NMR(Methanol-d4,400MHz)δppm:7.76(d,J=8.0Hz,1H),7.47(d,J=2.4Hz,1H) ,7.38-7.16(m,2H),4.72(d,J=2.8Hz,2H),3.84(d,J=2.0Hz,2H);MS:285.0 "M+H" + .

[0179] Example 5 Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-3-fluorobenzonitrile hydrochloride (compound 4) [ka]

[0180] Procedure M: Preparation of tert-butyl (E)-(2-((4-cyano-2-fluorophenoxy)methyl)-3-fluoroallyl)carbamate [ka] To a stirred solution of 3-fluoro-4-hydroxybenzonitrile (112 mg) in acetonitrile (4.0 mL) at rt was added potassium carbonate (206 mg) and Int-6 (200 mg) sequentially. The reaction mixture was filtered through a plug of Celite® to remove inorganics and the filtrate was concentrated in vacuo to give tert-butyl (E)-(2-((4-cyano-2-fluorophenoxy)methyl)-3-fluoroallyl)carbamate (230 mg, 95%) as a white solid. This material was used in the next step without purification. 1 H-NMR(300MHz, CDCl3)δppm:7.47-7.37(m,2H),7.06(t,J=8.3Hz,1H),6.80(dp,J=81.4,1.1H z,1H),4.63-4.57(m,2H),4.03(ddd,J=6.3,2.5,1.0Hz,2H),1.42(s,9H);MS:225.0 "M-Boc+H" + .

[0181] Procedure N: Preparation of E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-3-fluorobenzonitrile hydrochloride (compound 4) [ka] To a stirred solution of tert-butyl (E)-(2-((4-cyano-2-fluorophenoxy)methyl)-3-fluoroallyl)carbamate (230 mg) in CHCl (10.0 mL) at room temperature was added TFA (10.0 mL). The resulting solution was stirred at rt for 2 h. After this time, LC-MS analysis confirmed complete consumption of the starting material. The reaction mixture was concentrated in vacuo and residual TFA was removed by azeotroping with ethyl acetate (20 mL×2) to give an off-white solid. This solid was dissolved in ethyl acetate (10.0 mL) and HCl (2.0 M in diethyl ether; 5.00 mL) was added, at which time a white solid precipitated immediately. After stirring for 15 min, the contents of the reaction vessel were transferred to a vial. The solid was spun in a centrifuge (4000 rpm, 2 min, 20° C.). The supernatant was carefully decanted and the solid “cake” was washed with additional ethyl acetate. The solid was centrifuged again and the supernatant was decanted. The solid "cake" was dried under high vacuum to give compound 4 (181 mg, 98%). 1 H-NMR(Methanol-d4,300MHz)δppm:7.65-7.56(m,2H),7.36(t,J=8.6Hz,1H),7.30(d t,J=81.2,0.9Hz,1H),4.80(dd,J=3.5,1.1Hz,2H),3.90-3.82(m,2H);MS:225.0 "M+H" + .

[0182] Example 6 Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-2-methylbenzonitrile hydrochloride (compound 5) [ka]

[0183] Procedure O: Preparation of tert-butyl (E)-(2-((4-cyano-3-methylphenoxy)methyl)-3-fluoroallyl)carbamate [ka] A mixture of 4-hydroxy-2-methylbenzonitrile (109 mg), Int-6 (200 mg) and potassium carbonate (206 mg) in acetonitrile (4.0 mL) was stirred at rt overnight. LCMS analysis after that time confirmed complete conversion. The reaction mixture was diluted with CH2Cl2 (20 mL), filtered and concentrated in vacuo to give tert-butyl (E)-(2-((4-cyano-3-methylphenoxy)methyl)-3-fluoroallyl)carbamate (240 mg, 100%) as an off-white solid. This material was carried on to the next step without further purification. 1 H-NMR(300MHz,CDCl3)δppm:7.50(d,J=8.5Hz,1H),6.84-6.73(m,2H),6.75(dt,J=81.4,1 .1Hz,1H),4.50-4.43(m,2H),4.01-3.93(m,2H),1.99(s,3H),1.39(s,9H);MS:321.0 "M+H" + .

[0184] Procedure P: Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-2-methylbenzonitrile hydrochloride (Compound 5) [ka] To a stirred solution of tert-butyl (E)-(2-((4-cyano-3-methylphenoxy)methyl)-3-fluoroallyl)carbamate (240 mg) in CHCl (10 mL) at room temperature was added TFA (10.0 mL). The resulting solution was stirred at rt for 2 h. After this time, LC-MS analysis confirmed complete consumption of the starting material. The reaction mixture was concentrated in vacuo and residual TFA was removed by azeotroping with CHCl (20 mL×2) to give a pale yellow oil. This solid was dissolved in ethyl acetate (10.0 mL) and HCl (2.0 M in diethyl ether; 1.00 mL) was added, at which time a white solid precipitated out. After stirring for 15 min, the solid was filtered and dried under high vacuum to give compound 5 (175 mg, 91%) as a white solid. 1 H-NMR(Methanol-d4,300MHz)δppm:7.63(d,J=8.6Hz,1H),7.27(dt,J=81.0,0.9Hz,1H),7.08-7.04(m,1H) ,6.99(ddd,J=8.6,2.6,0.6Hz,1H),4.70(dd,J=3.6,1.1Hz,2H),3.84(d,J=2.1Hz,2H),2.53(d,J=0.7Hz,3H );MS:221.0 "M+H" + .

[0185] Comparative Example 7 Preparation of (-)(E)-2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroprop-2-en-1-amine hydrochloride (PXS-5131) [ka]

[0186] Procedure Q: Preparation of 2,3-dihydrobenzofuran-2-carbonyl chloride [ka] To a stirred solution of 2,3-dihydrobenzofuran-2-carboxylic acid (78.0 g) and DMF (1.0 mL) in CHCl (780 mL) at 0° C., oxalyl chloride (125 mL) was added dropwise. The progress of the reaction was monitored by TLC. After complete consumption of the starting material, the reaction mixture was concentrated in vacuo to give 2,3-dihydrobenzofuran-2-carbonyl chloride (92.0 g) as a crude red oil. This material was not characterized and was used immediately in the next procedure.

[0187] Procedure R: Preparation of 2-diazo-1-(2,3-dihydrobenzofuran-2-yl)ethanone [ka] To a cold (0° C.) stirred solution of (trimethylsilyl)diazomethane (2 M in diethyl ether; 546 mL) in THF / CH3CN (1:1; 780 mL) under N2 was added dropwise a solution of crude 2,3-dihydrobenzofuran-2-carbonyl chloride in THF / CH3CN (1:1; 780 mL). After stirring the resulting mixture at 0° C. for 1 h, the reaction mixture was evaporated in vacuo to give 2-diazo-1-(2,3-dihydrobenzofuran-2-yl)ethanone (92.6 g) as a crude brown oil. This material was used immediately in the subsequent step without purification. 1 H-NMR(400MHz,CDCl3)δppm:3.24(dd,J=16.4,5.6Hz,1H),3.39(dd,J=16.4,10.4Hz,1H),4.95(dd,J= 10.8,5.6Hz,1H),5.67(s,1H),6.70(d,J=8.0Hz,1H),6.75(dd,J=6.9,6.8Hz,1H),6.98-7.04(m,2H).

[0188] Procedure S: Preparation of 2-bromo-1-(2,3-dihydrobenzofuran-2-yl)ethanone [ka] To a stirred solution of crude 2-diazo-1-(2,3-dihydrobenzofuran-2-yl)ethanone (92.6 g) in CHCl (926 mL) at 0° C., HBr / AcOH (48%, 216 mL) was added dropwise. The resulting mixture was allowed to stir at this temperature for 15 min. The reaction mixture was diluted with water (800 mL) and the aqueous layer was extracted with CHCl (300 mL). The combined organics were washed with saturated aqueous NaHCO (600 mL×3) followed by brine (800 mL). After drying over NaSO, the solvent was removed in vacuo to give 2-bromo-1-(2,3-dihydrobenzofuran-2-yl)ethanone (98.0 g) as a crude brown oil. This material was immediately carried forward to the next step without purification. 1 H-NMR(400MHz,CDCl3)δppm:3.41(dd,J=16.0,6.4Hz,1H),3.59(dd,J=16.0,10.8H,1Hz),4.18(d,J=14.4Hz,1H),4.35 (d,J=14.0Hz,1H),5.30(dd,J=12.4,6.4Hz,1H),6.88(d,J=8.0Hz,1H),6.92(dd,J=7.4,7.4Hz,1H),7.15-7.20(m,2H).

[0189] Procedure T: Preparation of 2-azido-1-(2,3-dihydrobenzofuran-2-yl)ethanone [ka] To a stirred solution of crude 2-bromo-1-(2,3-dihydrobenzofuran-2-yl)ethanone (98.0 g) in acetone (926 mL) at 0° C. was added NaN3 (132 g). The resulting mixture was stirred at this temperature for 3 h. The reaction mixture was partitioned between water (1 L) and ethyl acetate (800 mL) and the aqueous layer was further extracted with ethyl acetate (800 mL×2). The combined organics were washed with brine (1 L), dried over Na2SO4, and then concentrated in vacuo to give 2-azido-1-(2,3-dihydrobenzofuran-2-yl)ethanone (92 g) as a crude brown oil. This material was used in the subsequent step without purification. 1H-NMR(400MHz,CDCl3)δppm:3.44(dd,J=16.4,6.0Hz,1H),3.54(dd,J=16.0,11.2Hz,1H),4.19(d,J=19.2Hz,1H),4.38 (d,J=19.2Hz,1H),5.17(dd,J=11.2,6.0Hz,1H),6.88(d,J=8.0Hz,1H),6.93(dd,J=8.0,7.9Hz,1H),7.15-7.26(m,2H).

[0190] Procedure U: Preparation of tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-2-oxoethylcarbamate [ka] To a solution of crude azido-1-(2,3-dihydrobenzofuran-2-yl)ethanone (91.0 g) in ethyl acetate (910 mL) was charged di-tert-butyl dicarbonate (135 g) and Pd / C (10%; 9.10 g, 50% wet). The resulting suspension was stirred overnight at rt under H2 atmosphere. The catalyst was removed by filtration and the filtrate was evaporated in vacuo. The crude material was purified on silica gel eluting with petroleum ether / ethyl acetate (20:1) to give tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-2-oxoethylcarbamate (39.0 g, 33%) as a yellow solid. 1 H-NMR(400MHz,CDCl3)δppm:1.43(s,9H),3.36(dd,J=16.0,6.4Hz,1H),3.53(dd,J=16.0,10.8Hz,1H),4.22(dd,J=21.2,4.8Hz,1H) ,4.41(dd,J=20.4,4.8Hz,1H),5.16(dd,J=11.2,6.4Hz,1H),6.87(d,J=8.0Hz,1H),6.90(dd,J=7.6,7.2Hz,1H),7.14-7.18(m,2H).

[0191] Procedure V: Preparation of (E)-tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate and (Z)-tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate [ka] To a stirred solution of fluoromethyl(triphenylphosphonium)tetrafluoroborate (75.6 g) in THF (910 mL) at −25° C. under N2 was added NaHMDS (1.0 M in THF; 246 mL) slowly at −25° C. The mixture was stirred at −25° C. for 30 min and then cooled to −78° C. To the reaction mixture was then added a solution of tert-butyl 2(2,3-dihydrobenzofuran-2-yl)-2-oxoethylcarbamate (45.6 g) in THF (364 mL). After complete addition, stirring was continued at −78° C. for 1 h. The reaction mixture was partitioned between water (1.2 L) and ethyl acetate (1.2 L) and the aqueous layer was further extracted with ethyl acetate (800 mL×2). The combined organics were washed with brine (1.5 L), dried over Na2SO4, and concentrated under vacuum. The crude material was initially purified on silica gel, eluting with petroleum ether / ethyl acetate (30:1→10:1) to give a mixture of rac.(E)-tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate and rac.(Z)-tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate (1:1, 9.70 g). This mixture was then subjected to preparative HPLC purification to give (E)-tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate (4.10 g, 9%) and (Z)-tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate (3.60 g, 8%). (E)-tert-Butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate: 1H-NMR(300MHz,CDCl3)δppm:1.42(s,9H),3.18(dd,J=15.7,8.5Hz,1H),3.39(dd,J= 15.7,9.4Hz,1H),3.93(dd,J=14.3,3.1Hz,1H),4.05(dd,J=14.9,5.3Hz,1H),4.75(b r.s,1H),5.23(dd,J=9.4,9.4Hz,1H),6.79(d,J=82.7Hz,1H),6.80(d,J=8.0Hz,1H)6 .88(ddd,J=7.5,7.5,1.0Hz,1H),7.14(dd,J=8.0,7.9Hz,1H),7.18(d,J=7.5Hz,1H).

[0192] Procedure W: Separation of the two enantiomers of racemic (E)-tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate [ka] Racemic (E)-tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate (5.00 g) was separated by chiral chromatography using a CHIRALCEL OZ-H column and running n-hexane / iso-propyl alcohol / diethylamine (95:5:0.1; v / v / v) at 1 mL / min to give the enantiomers of (E)-tert-butyl 2(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate (peak 1 (6.10 min), 2.30 g; peak 2 (9.40 min), 2.32 g).

[0193] Procedure X: Preparation of (-)-(E)-2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroprop-2-en-1-amine hydrochloride (PXS-5131) [ka] To a stirred solution of purified (E)-tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate (30 mg) in methanol (1.0 mL) at rt of the enantiomer represented by peak 2, HCl (2.0 M in diethyl ether; 1.0 mL) was added. The resulting mixture was stirred at rt for 2 h. All volatiles were removed in vacuo and the resulting residue was dissolved in ethyl acetate (0.5 mL). After addition of diethyl ether (2.0 mL), a white precipitate formed. The contents were transferred to a vial and centrifuged in a centrifuge (4000 rpm, 4 min) to obtain a solid "cake". After decanting the supernatant, more diethyl ether was added and (after brief sonication) the vial was placed back in the centrifuge. This centrifugation and washing step was repeated a total of three times. The solid thus obtained was dried under high vacuum to give PXS-5131 (13 mg, 62%). [α] D =-26.02°, c=11.2 mg / mL (in methanol); 1 H-NMR(300MHz,Methanol-d4)δppm:3.19(dd,J=15.9,9.1Hz,1H),3.41(dd,J15.9,9 .2Hz,1H),3.72(dd,J=14.0,2.6Hz,1H),3.84(ddd,J=13.6,2.1,0.5Hz,1H),5.36(dd ,J=9.1,9.1Hz,1H),6.83(d,J=8.0Hz,1H),6.90(ddd,J=7.5,7.5,0.9Hz,1H),7.15( dd,J=7.6,7.6Hz,1H),7.22(d,J=80.9Hz,1H),7.24(d,J=7.4Hz,1H);MS:194.0 "M+H" + [Note: When peak 1 is deprotected in step W, [α] D Compounds with the same magnitude but opposite sign were obtained.

[0194] Comparative Example 8 The following compounds listed in Table 2 are included for comparative purposes only and can be synthesized using methods similar to those described herein, with the exception of Compound 6 and Compound 14. These compounds can be prepared from commercially available 4-hydroxybenzonitriles according to the procedure described in Foot JS et al., J. Pharm. Exp. Ther. 2013, 347, 365-374.

[0195] [Table 2]

[0196] Example 9 Methods for determining the ability of compounds of formula I to inhibit human recombinant SSAO / VAP-1

[0197] Human recombinant SSAO / VAP-1 amine oxidase activity was assayed using monoamine oxidase The activity of the human SSAO / VAP-1 was determined as described for copper-containing amine oxidases and related enzymes (Holt A. and Palcic M., Nat. Protoc. 2006, 1, 2498-2505). Briefly, a cloned cDNA template corresponding to residues 34-763 of human SSAO / VAP-1 and incorporating a mouse Ig kappa (κ) signal sequence, an N-terminal Flag epitope tag, and a Tobacco Etch Virus (TEV) cleavage site was incorporated into a mammalian expression vector (pLO-CMV) from Geneart AG. This vector containing the human SSAO / VAP-1 residues was transfected into the CHO-K1 glycomutant cell line, Lec 8. A clone stably expressing human SSAO / VAP-1 was isolated and cultured on a large scale. The active human SSAO / VAP-1 was purified and collected by immunoaffinity chromatography and used as a source of SSAO / VAP-1 activity. A high-throughput colorimetric assay was developed using a 96- or 384-well format. Briefly, in a standard 96-well plate assay, 50 μL of purified human SSAO / VAP-1 (0.25 μg / mL) in 0.1 M sodium phosphate buffer (pH 7.4) was added to each well. Test compounds were dissolved in DMSO and incubated with human SSAO / VAP-1 for 30 min at 37°C, followed by testing to obtain concentration-response curves (CRCs) with 4-11 data points in the typical micromolar or nanomolar range. After 30 min of incubation, 600 μM benzylamine (Sigma Aldrich), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U / mL of 1,2-dichlorophenyl 1,3 ... 50 μL of reaction mixture containing horseradish peroxidase (Sigma Aldrich) was added to the corresponding wells. Fluorescence units (RFU) were read every 2.5 min for 30 min at 37 °C with excitation at 565 nm and emission at 590 nm (Optima; BMG LABTECH). The kinetic slope of each well was calculated using MARS data analysis software (BMG LABTECH), and this value was used to calculate the IC 50Values ​​(Dotmatics) were estimated. The ability of compounds of formula I to inhibit SSAO / VAP-1 is shown in Table 3.

[0198] Example 10 Methods for determining the ability of compounds of formula I to inhibit human recombinant MAO B

[0199] The ability of the compounds of the present invention to inhibit MAO-B activity in vitro was tested. Recombinant human MAO-B (0.06 mg / mL; Sigma Aldrich) was used as a source of MAO-B enzyme activity. The assay was carried out in a similar manner to human SSAO / VAP-1 (Example 9), except that the substrate benzylamine was used at 100 μM and the assay was carried out after an incubation time of 2 hours. The ability of the compounds of formula I to inhibit MAO-B is shown in Table 3.

[0200] Example 11 Methods for determining the ability of compounds of formula I to inhibit human recombinant MAO A

[0201] The selectivity of the compounds of the present invention was tested by determining their ability to inhibit MAO-A activity in vitro using recombinant human MAO-A (0.003 mg / mL; Sigma Aldrich). The procedure was similar to that for human SSAO / VAP-1 (Example 9), except that the substrate tyramine was used at 100 μM instead of benzylamine, and the assay was performed after an incubation time of 2 hours. The ability of the compounds of formula I to inhibit MAO-A is shown in Table 3. [ka]

[0202] [Table 3]

[0203] As shown in Table 3, the compounds of the invention are potent inhibitors of both human recombinant SSAO / VAP-1 and MAO-B in vitro under the assay conditions described. Importantly, the compounds of the invention are devoid of activity against recombinant human MAO-A, an important off-target. Surprisingly, this profile is highly dependent on the nature and position of the substituents on the phenyl ring.

[0204] Example 12 Pharmacokinetic parameters of compound 1 and PXS-5131

[0205] Pharmacokinetic (PK) studies were performed at Sundia MediTech Co. Ltd. (Shanghai). Either compound 1 or PXS-5131 was administered orally at 3 mg / kg to Wistar rats weighing approximately 280 g. Blood samples were collected via tail vein sampling at intervals between 15 min and 8 h. Plasma was analyzed using high performance liquid chromatography-tandem mass spectrometry with a Shimadzu LC30AD HPLC and QTRAP 5500 mass spectrometer. The time course and basic PK parameters thus obtained are outlined in Figure 1.

[0206] Example 13 Methods for determining the ability of compounds of formula I to inhibit rat adipose tissue SSAO / VAP-1 and rat brain tissue MAO-B

[0207] Sprague-Dawley rats were orally dosed with the compounds of interest and sacrificed at various time points to harvest gonadal fat and brain tissue.

[0208] Adipose tissue was homogenized in ice-cold HES buffer (20 mM HEPES, 1 mM EDTA, sucrose 250 mM, protease and phosphatase inhibitors, pH 7.4). The homogenate was centrifuged at 2000 rpm for 5 min at 4°C, and the resulting supernatant (minus the lipid upper layer) was used for the assay. SSAO / VAP-1 activity was determined using a nonspecific MAO inhibitor (e.g., pargyline) for high signals and a mixture of a nonspecific MAO inhibitor and a SSAO / VAP-1 inhibitor (e.g., semicarbazide) for low signals.

[0209] Brain tissue was homogenized in ice-cold Tris buffer (10 mM Tris·HCl, 0.5 mM EDTA, sucrose 250 mM, pH 7.4). The homogenate was then centrifuged at 4,000 rpm for 3 min at 4°C. The resulting supernatant was decanted and centrifuged again at 4,000 rpm for 3 min at 4°C. This step was repeated one last time, this time at 10,000 rpm for 8 min. The supernatant was discarded and the resulting pellet containing the mitochondrial extract was resuspended in 0.1 M sodium phosphate buffer (1:3 ratio). MAO-B activity was assayed using specific MAO-A inhibitors (e.g. clorgyline) and SSAO / VAP-1 inhibitors (e.g. semicarbazide) for high signals and similar to the adipose tissue assay for low signals.

[0210] The activity of the treated samples was analyzed using the fluorometric methods described in Example 9 for SSAO / VAP-1 activity and in Example 10 for MAO-B.

[0211] Example 14 Description of dual inhibitor PXS-5131

[0212] PXS-5131 is a prototype of both MAO-B and SSAO / VAP-1. PXS-5131 is a potent inhibitor of SSAO / VAP-1 and MAO-B. The pharmacokinetic profile of this compound is inferior to that of compound 1. A single dose of PXS-5131 administered orally to Sprague-Dawley rats inhibits SSAO / VAP-1 and MAO-B in vivo for a long period of time, as shown in Figures 2 and 3. As depicted in Figure 2, a single dose of PXS-5131 inhibits SSAO / VAP-1 enzyme activity for more than 24 hours. As shown in Figure 3, a single dose of PXS-5131 inhibits MAO-B enzyme activity for more than 24 hours. To evaluate these combined effects, PXS-5131 was used in the following in vivo model.

[0213] Example 15 Efficacy of the dual inhibitor PXS-5131 in the carrageenan-air pouch model

[0214] On day 0, BALB / c mice were shaved at the nape, anesthetized, and injected subcutaneously with 6 mL of sterile air (0.2 μm, Sartorius, Cat. No. 16532) using a 23G×1.5 inch needle attached to a 6 mL syringe. The injection site was sealed with flexible collodion (Macron, Cat. 4580-02). On day 3, an additional 3 mL of sterile air was injected into the neck.

[0215] 3% λ-carrageenan was prepared by dissolving 1.8 g of λ-carrageenan (FLUKA, Cat. 22049, Lot 1408463) in 60 mL of hot deionized water, then cooling to room temperature and making up the volume to 60 mL with deionized water.

[0216] On day 6, mice were administered PXS-5131 in PBS (vehicle) or PBS alone by oral gavage. One hour after administration, mice were injected with 1 mL of 3% λ-carrageenan directly into the air pouch using a 23G×2 inch needle attached to a 1 mL syringe. Sham animals received 1 mL of deionized water instead of the λ-carrageenan solution.

[0217] Four hours after carrageenan / deionized water injection, mice were anesthetized and 3 mL of 10 U / mL heparinized saline was injected into the air pouch. The air pouch was gently massaged and the contents immediately removed with an 18G x 1 inch needle attached to a 5 mL syringe, and the volume of exudate was recorded. An aliquot of the exudate was taken for measurement of total white blood cell count; a second aliquot was taken for measurement of differential white blood cell count.

[0218] The anti-inflammatory effect of PXS-5131 is shown in Figure 4. Although carrageenan injection increased inflammation, a single application of PXS-5131 reduced inflammation compared to carrageenan-treated mice (black bars), as evidenced by a reduction in both the amount of exudate and the number of neutrophils in the air pouch ( * p<0.05, ** p<0.01, *** p<0.001).

[0219] Compound 1 has also been profiled in this model.

[0220] Example 16 Efficacy of the dual inhibitor PXS-5131 in an intranigral lipopolysaccharide (LPS)-induced neuroinflammatory model

[0221] Male Hooded Wistar rats weighing 300-400 g were obtained from Adelaide Laboratory Animal Services, Inc. On day 0, rats were administered 2 mg / kg PXS-5131 or sterile saline (vehicle) via intraperitoneal injection (ip) both 1 h before and 5 h after LPS injection.

[0222] Number of dopaminergic neurons after unilateral administration of LPS Unilateral injection: Rats were unilaterally injected with 2 μL of a 1.5 mg / mL solution of LPS (O55:B5, Sigma) in the substantia nigra. They were anesthetized with isofluorane and placed in a stereotaxic frame. For all rats, a hole was drilled in the skull over the target structure, appropriately positioned on the coordinates (-5.3 mm posterior; ±2.0 mm lateral; -8.0 mm ventral from bregma). All rats received a unilateral injection of LPS, leaving the other hemisphere intact. The animals were administered either PXS-5131 (2 mg / kg) or vehicle ip daily for 2 weeks.

[0223] Immunohistochemical procedure: Two weeks after the lesion, rats were perfused transcardially with PBS and then with PFA 4%. Brains were postfixed with PFA 4% and 30 μm coronal sections containing the substantia nigra were taken and washed 3× for 10 min in 0.01 M phosphate-buffered saline, pH 7.4. (PBS). Dopaminergic neurons were stained with anti-tyrosine hydroxylase (TH) antibody 1:10000, visualized with nickel-enhanced 3,3'-diaminobenzidine (DAB), and the number of neurons was counted by visual inspection. Values ​​across sections were averaged for each animal. The results are shown in Figure 5.

[0224] As shown in Figure 5, unilateral injection of LPS into the substantia nigra of the brain results in a dramatic loss of dopaminergic neurons compared to the intact control hemisphere. Daily ip administration of PXS-5131 at 2 mg / kg for 2 weeks significantly prevented the neuronal loss compared to the intact control hemisphere, t-test * p<0.05.

[0225] Example 17 Efficacy of dual inhibitor PXS-5131 in a mouse model of Duchenne muscular dystrophy

[0226] mdx mice (C57BL / 10ScSn-Dmdmdx / J) were obtained from Jackson Laboratories, Maine, USA. Three-month-old animals were administered PXS-5131 (6 mg / kg) daily, mixed with chow for 30 days. Untreated age-matched animals served as controls. The drug was administered using small, palatable gelatin pellets prepared with 20% sucralose plus food flavoring and 10% gelatin (Zhang L. Protoc. Exch. 2011, 236). Animals were preconditioned to recognize and eat gelatin by a routine of exposing each individual to inactive gelatin for 3 days in the absence of other food after an overnight fast. Palatability was further improved by dipping the pellets in corn oil containing traces of almond oil and dipping crushed food pellets in breadcrumbs.

[0227] In vivo gastrocnemius muscle function was assessed as described by Blaauw B. et al., Hum. Mol. Genet. 2008, 17, 3686-3696. Mice were anesthetized, electrodes were placed on both sides of the sciatic nerve, and the common peroneal nerve was cut. Muscle torque generation was measured using a lever system (Aurora Scientific 305B). A lever arm of 2.1 mm was used for all groups, as there were no significant differences in body weight between groups. Eccentric contractions were performed by moving the foot backwards at a speed of 40 mm / s while stimulating the gastrocnemius at a frequency (100 Hz) sufficient to induce complete tetanic fusion. Contractions were repeated once every 20 seconds to avoid inducing fatigue (see Figure 6).

[0228] Figure 6 demonstrates that oral administration of PXS-5131 at 6 mg / kg / day helps prevent loss of force from eccentric contractions in the gastrocnemius (GC) muscle after 4 weeks of oral administration to 3-month-old mice compared to untreated age-matched mice. *p<0.05.

[0229] After the force measurements described above, the animals were sacrificed by cervical dislocation, and the skeletal muscles and the heart (excluding the atria) were harvested, flash frozen in isopentane precooled with liquid nitrogen, and stored at -80°C. 12 μm sections were made from limb muscles (tibialis anterior) using a cryotome to produce multiple serial slides containing sections at least at three different levels. Transverse sections of the heart were made using the same procedure.

[0230] The amount of neutrophil and macrophage infiltration was measured by immunofluorescence staining of serial sections of skeletal muscle prepared as described above using anti-Ly6G and anti-F4 / 80 antibodies (for neutrophils and macrophages, AbCam, ab25377 and ab6640, respectively). The amount of infiltration was evaluated as the percentage of phosphor-positive pixels relative to the total area of ​​the section using Image-J software (Figures 7A and B).

[0231] The amount of reactive oxygen species (ROS) present in skeletal muscle tissue sections was assessed using dihydroethidium (DHE, Sigma) as a fluorescent probe in freshly cut tissue sections as described in Menazza et al., Hum. Mol. Genet. 2010, 19, 4207-4215. In the presence of ROS, DHE is oxidized to form ethidium, which can then bind to nuclear DNA and emit red fluorescence. The amount of this red fluorescence was measured in at least 20 random fields per section using Metafluor software (Leica), and data were averaged per field (Figure 7C).

[0232] The percentage of fibrous tissue in skeletal muscles was measured by picrosirius red staining. Sections were treated with xylene for 10 min, 100% ethanol for 2 min, 90% ethanol for 2 min, 70% ethanol for 2 min, distilled water for 2 min, and stained with 0.02% picrosirius red (Sigma, 365548) in a saturated aqueous solution of picric acid for 1 min before being fast-immersed twice in acetic acid washes (30 mL glacial acetic acid in 200 mL water). Sections were dehydrated in ascending alcohol concentrations (reverse as above) and treated in xylene for 10 min before mounting with Eukitt (Sigma, cat.no 03989). Overall images of at least three sections per muscle sample were then acquired using a bright-field microscope and then analyzed with Image-J software to measure the percentage of fibrotic (red) area over the total surface (Figure 7D).

[0233] As shown in Figure 7 (A-D), oral administration of 6 mg / kg / day PXS-5131 after 4 weeks of treatment in 3-month-old mdx mice significantly reduced (A) neutrophil influx, (B) macrophage influx, (C) reactive oxygen species, and (D) fibrosis (quantified by picrosirius red staining) in the tibialis anterior muscle compared to untreated age-matched mice. ****p<0.0001 compared to untreated mice.

[0234] Example 18 Methods to determine the ability of compounds of formula I to inhibit adipose tissue SSAO / VAP-1, muscle and heart tissue MAO-B, and muscle and heart tissue MAO-A in both C57BL / 6J and mdx mice (C57BL / 10ScSn-Dmdmdx / J).

[0235] Mice were orally dosed with the test compound and sacrificed at various time points to harvest abdominal fat, muscle and heart tissues.

[0236] SSAO / VAP-1 activity in adipose tissue was measured as described in Example 13.

[0237] Skeletal muscles were pulverized in liquid nitrogen and lysed in ice-cold HEPES-buffered medium (20 mM HEPES, 0.8% w / v free fatty acid bovine serum albumin (BSA), 75 mM sucrose, 10 mM The cells were suspended in 0.1 M EDTA, 215 mM D-mannitol, pH 7.4. After homogenization, the cells were centrifuged at 2,500 rpm for 10 min at 4°C. The supernatant was decanted and centrifuged at 10,000 rpm for 8 min. The supernatant was discarded and the resulting pellet containing the mitochondrial extract was resuspended in 0.1 M sodium phosphate buffer (1:3 ratio).

[0238] The myocardium was washed three times with PBS solution on ice, and then homogenates and extracts were prepared as described for skeletal muscle.

[0239] MAO-B activity was assayed using specific MAO-A inhibitors (e.g., clorgyline) and SSAO / VAP-1 inhibitors (e.g., semicarbazide) for high signals and similar to the adipose tissue assay (Example 13) for low signals.

[0240] MAO-A activity was assayed using the selective MAO-B inhibitor mofegiline (at 30 nM) for high signals and a mixture of a specific MAO-A inhibitor (clorgyline) and mofegiline (at 30 nM) for low signals.

[0241] The activity of the treated samples was analyzed using the fluorometric methods described in Example 9 for SSAO / VAP-1 activity, Example 10 for MAO-B activity, and Example 11 for MAO-A activity (Figures 8 and 9).

[0242] As depicted in Figure 8, a single dose of Compound 1 shows a dose-dependent decrease in both SSAO / VAP-1 and MAO-B activity 4 hours after administration in C57BL / 6J mice. Inhibition of enzyme activity is still evident at 24 hours. Compared to vehicle-treated mice, * p<0.05, ** p<0.01 and *** p<0.001.

[0243] As depicted in FIG. 9, a single dose of Compound 1 had no inhibitory effect on cardiac and muscular MAO-A activity 4 and 24 hours after administration to C57BL / 6J mice.

[0244] A similar inhibitory profile was observed when compound 1 was administered to mdx mice (C57BL / 10ScSn-Dmdmdx / J) at a dose of 4.8 mg / kg / day for 3 days.

[0245] As depicted in Figure 10, a single dose of Compound 1 completely suppresses adipose tissue SSAO / VAP-1 and muscle MAO-B activity in C57BL / 10ScSn-Dmdmdx / J mice. *p<0.05 and ***p<0.001 compared to vehicle-treated mice.

[0246] As depicted in FIG. 11, a single dose of Compound 1 had no inhibitory effect on muscle MAO-A after administration in C57BL / 10ScSn-Dmdmdx / J mice.

[0247] Example 19 Efficacy of the dual inhibitor compound 1 in a mouse model of Duchenne muscular dystrophy

[0248] Three-month-old mdx mice (C57BL / 10ScSn-Dmdmdx / J) were administered Compound 1 daily (ranging from 1 to 6 mg / kg) in drinking water for 30 days. Untreated age-matched animals served as controls. A second cohort of 10-month-old mice Compound 1 (range 1-6 mg / kg) was administered daily in drinking water for 90 days. Untreated age-matched animals served as controls.

[0249] After the end of each treatment period, animals were sacrificed by cervical dislocation, and skeletal muscles and hearts (excluding atria) were harvested, flash frozen in isopentane precooled with liquid nitrogen, and stored at -80°C. 12 μm sections were made from diaphragm muscles with a cryotome, and multiple serial slides containing at least three different levels of sections were prepared. Transverse sections of the heart were made using the same procedure.

[0250] The amount of infiltration of neutrophils and macrophages, the amount of reactive oxygen species (ROS) present, and the percentage of fibrotic tissue were determined as in Example 17.

[0251] As shown in FIG. 12 (AD), oral administration of 4.8 mg / kg / day of compound 1 for 30 days in 3-month-old mdx mice significantly reduced (A) neutrophil influx, (B) macrophage influx, (C) reactive oxygen species, and (D) fibrosis (quantified by picrosirius red staining) in the diaphragm muscle compared to untreated age-matched mice. **** p<0.0001.

[0252] As shown in Figure 13, oral administration of Compound 1 at 4.8 mg / kg / day significantly reduces cardiac fibrosis (quantified by picrosirius red staining of the myocardium) after 90 days of treatment in 10-month-old mdx mice compared to untreated age-matched mice. **** p<0.0001.

[0253] Example 20 Standardized commercial methods (such as those from Eurofins Panlabs Inc. (https: / / www.eurofinsdiscoveryservices.com / services / in-vitro-assays / ) or Evotec SE (https: / / www.evotec.com / en / execute / integrated-pre-clinical-development / adme) Assessment of the metabolic stability profile of compound 1 using ELISA kits (available through a contract research organization at

[0254] The in vivo metabolic potential was assessed in vitro using the following assays.

[0255] Liver microsome stability (human, rat, dog; in the presence of NADPH).

[0256] Hepatocyte stability (human, rat, dog)

[0257] Plasma stability (human, rat, dog)

[0258] Whole blood stability (human, rat)

[0259] Stability in simulated gastric fluid (10μM test product, pH 1.2)

[0260] Identification of metabolites in human hepatocytes

[0261] Compound 1 was found to have favorable metabolic stability in all assays.

[0262] Example 21 Standardized commercial methods (such as those from Eurofins Panlabs Inc (https: / / www.eurofinsdiscoveryservices.com / services / in-vitro-assays / ) or Evotec SE (https: / / www.evotec.com / en / execute / integrated-pre-clinical-development / adme)) are available. Assessment of the off-target profile of compound 1 using ELISA kits (available through contract research organizations)

[0263] Potential in vivo off-target promiscuity was assessed in vitro using the following assay.

[0264] Inhibition of cytochrome P450 activity (30 μM single concentration; 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4)

[0265] Eurofins Discovery Safety Screen® Panel (44 enzymes, receptors, ion channels, transporters) (Inhibition rate at 10 μM concentration)

[0266] Eurofins Transporter Protein Screen Panel (13 common transporters (inhibition rate at 10 μM concentration))

[0267] Aptuit Cardiac Ion Channel Panel (7 targets (automated patch clamp assay; concentrations 0-300 µM)).

[0268] Compound 1 was found to have a good off-target profile in all assays.

[0269] Example 22 Evaluation of the cytotoxicity profile of compound 1 in a miniaturized bacterial reverse mutagenicity screening assay.

[0270] The mutagenic potential of compound 1 was confirmed by testing it in the presence and absence of metabolic activation using a compact revertant mutagenicity assay with Salmonella Typhi TA98, TA100, and E. coli WP2 uvrA pKM101. Test articles were formulated in dimethyl sulfoxide (DMSO) at a nominal concentration of 50 mg / mL, and serial dilutions were made from this stock solution. Final dose concentrations were 1000, 500, 250, 125, 62.5, 31.3, 15.6, and 7.81 μg / well (test article). Metabolic activation was provided by using an S9 mixture containing 10% v / v Aroclor-1254-induced rat liver S9 (Regensys System reagent, Moltox, Boone, NC).

[0271] Compound 1 did not induce any responses indicative of potential mutagenicity in this assay.

[0272] Example 23 Assessment of the phospholipidosis-inducing potential of compound 1 in a cell-based fluorescent assay

[0273] Compound 1 was tested for its potential to induce phospholipidosis in vivo at concentrations up to 100 μM according to the methods outlined by Fujimura et al. (Experimental and Toxicologic Pathology. 2007, 58, 375-382).

[0274] Compound 1 had no effect on cell phenotype or cell viability in this assay.

[0275] Example 24 Stability of Compound 1 in Grape Syrup Formulations at Room Temperature

[0276] Methyl parahydroxybenzoate (126.2 mg), propyl parahydroxybenzoate (20.0 mg), monobasic sodium phosphate (1%, 1.0 g) and sucrose (16.56 g) were dissolved in 100 mL purified water, vortexed for 10 min, then sonicated for 30 min and filtered through a 0.45 μm filter. The pH was adjusted to 5 with 0.5 M NaOH. Grape flavor (LorAnnOils, 0180-0800; 1%, 0.5 mL) was added to the resulting solution (50 mL), followed by 50 mg of compound 1 to a final concentration of 1 mg / mL. The solution was divided into two 25 mL portions, one of which was frozen and the second was left at room temperature (rt) in a sealed Falcone tube.

[0277] As shown in Table 4, no discernible decomposition was observed in Compound 1 after 91 days at room temperature. [Table 4]

Claims

1. Compound of Formula 1: 【Chemistry 1】 or a pharma- ceutically acceptable salt, polymorph, or solvate thereof, R １ is selected from the group consisting of hydrogen, fluorine, bromine and methyl; and R ２ is hydrogen or fluorine; The compound or a pharma- ceutically acceptable salt, polymorph or solvate thereof.

2. The compound according to claim 1, selected from the group consisting of the following compounds: 【Table 1】 or a pharma- ceutically acceptable salt or solvate thereof.

3. 3. A pharmaceutical composition comprising a compound according to claim 1 or 2, or a pharma- ceutically acceptable salt or solvate thereof, and at least one pharma- ceutically acceptable excipient, carrier or diluent.

4. The pharmaceutical composition of claim 3 for inhibiting the amine oxidase activity of semicarbazide-sensitive amine oxidase / vascular adhesion protein-1 (SSAO / VAP-1) and monoamine oxidase B (MAO-B) in a subject in need thereof.

5. The pharmaceutical composition of claim 3 for treating or preventing a disease or disorder by inhibiting the activity of the SSAO / VAP-1 protein and the MAO-B protein.

6. 6. The pharmaceutical composition of claim 5, wherein the disease or disorder is ameliorated by the selective inhibition of SSAO / VAP-1 and MAO-B over MAO-A.

7. The pharmaceutical composition according to claim 5 or 6, wherein the disease is a neuromuscular disorder.

8. The pharmaceutical composition of claim 7, wherein the neuromuscular disorder is a muscular dystrophy.

9. 9. The pharmaceutical composition of claim 8, wherein the muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy and myotonic dystrophy.

10. The pharmaceutical composition according to claim 5 or 6, wherein the disease is a neurodegenerative disease or a neuroinflammatory disease.

11. 11. The pharmaceutical composition of claim 10, wherein the neurodegenerative or neuroinflammatory disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, myasthenia gravis, and Charcot-Marie-Tooth disease.

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