Halogenated allylamine diamine 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 can be used to treat a variety of neuromuscular diseases, such as muscular dystrophy, and neuroinflammatory diseases, including peripheral and central diseases in human subjects, as well as peripheral and central diseases in pets and livestock. In addition, the present invention relates to pharmaceutical compositions containing these compounds, as well as 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 can be used to treat a variety of neuromuscular diseases, such as muscular dystrophy, and neuroinflammatory diseases, including peripheral and central diseases in human subjects, as well as peripheral and central diseases in pets and livestock. In addition, the present invention relates to pharmaceutical compositions containing these compounds and various uses thereof. Background Art

[0002] In most organisms, including humans, two families of mammalian amine oxidases metabolize various monoamines, diamines, and polyamines that are produced endogenously or absorbed from exogenous sources. The first family 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 cofactor. Polyamine oxidase is another FAD-dependent amine oxidase that oxidatively deaminates spermine and spermidine. The second family is copper-dependent and uses other cofactors in addition to FAD, such as oxidized tyrosine residues (abbreviated as TPQ or LTQ). One member of this family is semicarbazide-sensitive amine oxidase (SSAO), also known as primary amine oxidase, plasma amine oxidase, and benzylamine oxidase, and is identical to vascular adhesion protein-1 (VAP-1—described as SSAO / VAP-1 from here on). Diamine oxidase (DAO) and the lysyl oxidase protein family (lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) 1 to 4) also belong to this second family. Pharmacologically, MAO-A is selectively inhibited by clorgyline, MAO-B is selectively inhibited by levopyridine, and SSAO / VAP-1 is selectively inhibited by semicarbazide.

[0003] Monoamine oxidase (MAO) activity is responsible for the oxidative deamination of endogenous amines such as 5-hydroxytryptamine, dopamine, epinephrine, norepinephrine, arylalkylamines, and many amine exogenous substances. Although MAO-A and MAO-B have a common enzymatic role in the metabolism of amines, they have 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 has a higher affinity for substrates such as tyramine, 5-hydroxytryptamine, epinephrine, and norepinephrine, while MAO-B shows preference for dopamine, phenylethylamine, and other trace arylalkylamines. MAO-B is expressed in a variety of organs, including the brain, heart, adipose tissue, pancreas, lungs, kidneys, muscles, and liver.

[0004] Non-selective MAO inhibitors that inhibit both MAO-B and MAO-A have the potential to induce hypertensive crises when dietary tyramine is ingested, so there are certain dietary restrictions (Gardner DM et al., J. Clin. Psychiatry 1996, 57, 99-104). In addition, non-selective inhibitors increase the likelihood of monoamine interactions with other drugs. Therefore, for medical applications, there is a need for MAO-B inhibitors that are highly selective relative to MAO-A.

[0005] SSAO / VAP-1 is an exoenzyme containing a very short cytoplasmic tail, a single transmembrane domain and a large, highly glycosylated extracellular domain containing an active site for amine oxidase activity (Salmi M. and Jalkanen S. Science 1992, 257, 1407-1414). SSAO / VAP-1 also exists in a soluble form and circulates in the plasma of some animals. It has been shown that this form is a cleaved 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 is amine oxidase activity and the second is cell adhesion activity. Both activities are associated with inflammatory processes. Inhibitors of the amine oxidase activity of SSAO / VAP-1 have been found to interfere with leukocyte rolling, adhesion and extravasation and, similar to SSAO / VAP-1 antibodies, exhibit anti-inflammatory properties (Foot JS et al., J. Pharm. Exp. Ther., 2013, 347, 365-374).

[0006] Oxidative deamination of primary monoamines by MAO-B and SSAO / VAP-1 produces ammonia (NH3), aldehydes, and hydrogen peroxide (H2O2), which are agents of established toxicity that can induce significant inflammation through their promiscuous reactivity and oxidative stress. During the SSAO / VAP-1 amine oxidase catalytic cycle, the covalently bound cofactor TPQ is first reduced and then reoxidized by oxygen in the presence of copper to produce ammonia and hydrogen peroxide as byproducts. Similarly, MAO-B, which functions via a single electron transfer process, oxidizes amines to aldehydes and ketones while simultaneously reducing flavin adenine dinucleotide (FAD) to its reduced form, FADH2. The cofactor is then reoxidized by oxygen to produce hydrogen peroxide. It has been hypothesized that excessive concentrations of hydrogen peroxide may be detrimental and may contribute to pathological changes in various inflammatory and neurodegenerative processes ( ME et al., Pharmacol Ther. 1994, 63, 37-122).

[0007] Neuromuscular disease is a term used to describe a variety of conditions in which the underlying pathophysiology of the disease affects muscle function directly, through the degradation of muscle tissue, or indirectly, through effects on the nerves and neuromuscular junctions that control the muscle. Direct effects on muscle can result in muscle wasting and reduced mobility, while indirect central nervous system (CNS)-mediated neuromuscular effects range from occasional spasms to paralysis. Diseases associated with muscle wasting are further classified as muscular dystrophies and include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, and myotonic dystrophy. Indirect neuromuscular diseases are classified as central disorders and include Parkinson's disease, multiple sclerosis, Huntington's disease, and Creutzfeldt-Jakob disease.

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

[0009] DMD is the most prevalent form of muscular dystrophy. DMD is a fatal neuromuscular disorder that is inherited genetically as an X-linked disease and is caused by mutations in the dystrophin gene. Boys are primarily affected, with an incidence of approximately 1 in 3,500 live male births. Muscle degeneration is a hallmark of the disease, with debilitating effects on mobility and daily functioning beginning at a very early age. Cardiac fibrosis and respiratory failure are the most common causes of death in patients with DMD. There is no cure for DMD, and current treatment options focus on symptom relief and management of complications, and are centered on the use of corticosteroids, surgery, ventilation, and physical therapy. Although recent high-profile advances in splicing regulators and gene therapy may allow partial restoration of dystrophin function in a small number of patients, the long-term benefits of such treatments remain unclear and there remains a significant need for alternative or additional treatment options.

[0010] Parkinson's disease is the most common CNS disorder. Parkinson's disease is caused by damage to nerve cells in areas of the brain that produce dopamine, a chemical that is essential for controlling muscles and movements. Parkinson's disease is a progressive, degenerative neurological disorder that causes tremors in the hands, arms, legs, jaws, and face; stiffness or rigidity of the limbs or trunk; slowness of body movements; and postural instability and difficulty walking. Parkinson's disease usually appears later in life, with symptoms starting at age 60, and is thought to affect more than 10 million people worldwide. Currently, treatment can only slow progression and resolve symptoms. First-line treatment includes the use of levodopa (a dopamine precursor that can cross the blood-brain barrier) to increase brain dopamine, and the use of carbidopa and MAO-B inhibitors to reduce the metabolism of levodopa and dopamine. Anticholinergics and medicinal marijuana are also prescribed to alleviate movement disorders.

[0011] MAO-B activity increases muscle and systemic oxidative stress, leading to impaired mitochondrial function and increased reactive oxygen species. Myogenic cell cultures from mdx mice (an animal bred to show the hallmark features of DMD patients and established as a relevant model of human disease) and DMD patients are more sensitive to oxidative stress. This sensitivity is eliminated by treating with an inhibitor of MAO-B (safinamide) in vitro. Pharmacological inhibition of MAO-B has also been shown to be effective in vivo; when given to 3-month-old mdx mice, safinamide reduces myofiber damage and oxidative stress, and improves muscle function (Vitiello L. et al., Frontiers in Physiology (Front. Physiol.) 2018, 9, Article 1087).

[0012] It has been shown that the blockade of SSAO / VAP-1 by enzyme inhibitors has a significant effect in animal models of neutrophil-driven inflammation (Schilter HC et al., Respir. Res. 2015, 16, 42). Unregulated neutrophil activation and activity can lead to the release of excessive myeloperoxidase (MPO), which reacts with hydrogen peroxide to form hypochlorous acid, which causes local tissue damage and further inflammation. Given the coexistence of MAO-B and SSAO / VAP-1 in several organs (heart, lungs, muscles), inhibitors of these two enzymes may be effective in treating diseases involving neutrophils and in the presence of oxidative stress.

[0013] Neutrophils have become the focus of new treatments for muscular dystrophy. Studies in mdx mice have shown that they promote muscle damage; antibody-mediated depletion of host neutrophils in dystrophic mice leads to a delay and significant reduction in skeletal muscle breakdown (Hodgetts S. et al., Neuromuscular Disorders 2006, 16, 591-602). Additionally, it has been shown that neutrophil elastase activity increases in dystrophic mice, and this increase impairs myoblast survival (Arecco, N. et al., Sci. Rep. 2016, 6, No. 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 through 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. Long-term use of these steroids is associated with a high burden of side effects, including immunosuppression, obesity, insulin resistance, and behavioral problems.

[0015] Both MAO activity and SSAO / VAP-1 activity are increased in obese dogs on a high-fat diet (Wenecq, E. et al., Journal of Physiology and Biochemistry (J. Physiol. Biochem.) 2006, 62, 113-124). The substrates of SSAO / VAP-1 have been shown to be fat-soluble, and inhibition of SSAO / VAP-1 can stimulate lipolysis (Romauch, M, Open Biology, 2020, 10, Article 190035). Mice with brain-specific knockout of insulin receptors (NIRKO mice) show brain mitochondrial dysfunction with reduced mitochondrial oxidative activity, increased levels of reactive oxygen species, and increased levels of lipid and protein oxidation in the striatum and nucleus accumbens. NIRKO mice also show increased levels of monoamine oxidase A and B (MAO A and B), resulting in increased dopamine turnover in these regions. Studies in cultured neurons and glial cells indicate that these changes in MAO A and B are a direct consequence of loss of insulin signaling and lead to behavioral impairments (Kleinridders, A. et al., PNAS, 2015, 112, 3463-3468).

[0016] In the diseases related to the reduction of dopamine levels, such as Parkinson's disease, Alzheimer's disease, attention deficit hyperactivity disorder and schizophrenia, the inhibition of MAO-B leads to the increase of dopaminergic neuron activation, thereby alleviating symptoms. Due to the increase of MAO activity and the subsequent formation of H2O2 by MAO-B, the oxidative stress caused by the increase plays a role in the degeneration process related to old age and Alzheimer's disease and Parkinson's disease. Oxidative stress is also considered to be an important factor in the progress of Huntington's disease, wherein it has been shown that MAO-B is upregulated in the basal ganglia and pons of disease patients (Richards G. et al., Brain Res. 2011, 1370, 204-214). Therefore, the MAO-B inhibitors for neuromuscular and neurodegenerative diseases can work by increasing the level of monoamines in the brain and reducing the formation of reactive oxygen species.

[0017] Inflammation associated with SSAO / VAP-1 enzyme activity is also considered an important feature of neuroinflammatory diseases such as Parkinson's disease, Alzheimer's disease and multiple sclerosis, and is also a feature of the pathophysiology that occurs after cerebral infarction / reperfusion events (Aktas, O. et al., Arch Neurol. 2007, 64, 185-189). Excessive SSAO / VAP-1 activity has been independently implicated in these processes (Xu, HL. et al., Journal of Pharmacology and Experimental Therapeutics, 2006, 317, 19-26). Pharmacological inhibition of SSAO / VAP-1 activity has been shown to reduce neutrophil infiltration into the brain and reduce microglial activation in an LPS-induced neuroinflammation model (Becchi et al., Br. J. Pharmacol. 2017, 174, 2302-2317). Endothelial SSAO / VAP-1 is also believed to play a role in cerebral amyloid angiopathy associated with Alzheimer's disease through its effects on cerebrovascular beta amyloid (Aβ) deposition (Sole M. et al., Neurobiology of Ageing 2015, 36, 762), leading to potential blood-brain barrier dysfunction associated with disease progression (Sole M. et al., Biochim. Biophys. Acta. Mol. Basis. Dis. 2019, 1865, 2189-2202). Additionally, SSAO / VAP-1 oxidation of primary amines (such as methylamine to formaldehyde) has been associated with oxidative stress in Alzheimer's disease models (Somfai GM et al., Neurochem Int. 2006, 48, 746-52).

[0018] The enzymatic activity of MAO-B has been shown to play a role in vascular dysfunction. When subjected to pressure overload induced by transverse aortic constriction (TAC), compared with WT mice, MAO-B- / - mice had mitochondrial dysfunction (oxidative stress reduction), reduced fibrosis and apoptosis, and preserved left ventricular function (Sturza A. et al., Hypertension 2013, 62, 140-6 and Kaluderic N. et al., Antioxidants and Redox Signal Transduction 2014, 10, 267-280). SSAO / VAP-1 also has a strong performance in cardiovascular and metabolic diseases, and also has a significant role in inflammation and fibrosis of multiple organs including lungs, liver and kidneys (Salmi M. and Jalkenen S. Antioxidants and Redox Signal Transduction 2017, 30, 314-332).

[0019] MAO-B and SSAO / VAP-1 have significant overlap in several areas of research. They have a common oxidative pathway that can induce local and systemic oxidative stress, which may have an impact on fibrosis, inflammation, and cardiovascular disease. In muscular dystrophy, both targets are present in significant amounts in muscle tissue and appear to play a role in inflammatory pathways and oxidative stress pathways in the progression of DMD, which is the most well-studied of these diseases. The potential beneficial effects of inhibiting these targets in systemic inflammation, fibrosis, and cardiovascular deterioration are also clear. In the CNS, these targets have complementary effects, in which SSAO / VAP-1 can affect the infiltration / migration of leukocytes into the microvasculature, while MAO-B can affect neurodegeneration. Both of these effects are considered to have significant effects on CNS-related neuromuscular diseases, particularly those associated with neuroinflammation, such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis, and further indicate that there is hope for therapeutic intervention in the activity of the two enzymes.

[0020] Given the shared roles of MAO-B and SSAO / VAP-1 in the above diseases, potent and selective inhibitors of both enzymes (dual inhibitors) could translate into clear and significant therapeutic benefits in these neuromuscular, neuroinflammatory, and neurodegenerative diseases.

[0021] Some known MAO inhibitors also inhibit SSAO / VAP-1 (e.g., the haloallylamine MAO inhibitor Mofegiline shown below (Milczek EM et al. J. Med. Chem. 2008, 51, 8019-8026). Fluoroallylamine inhibitors are also described in US 4454158 as MAO inhibitors.

[0022]

[0023] Other examples structurally related to mfgiline such as LJP1586 are described in WO 2007 / 120528.

[0024]

[0025] Additionally, a series of 2-substituted-3-haloallylamine SSAO / VAP-1 inhibitors, including PXS-4728 and PXS-4681, have been disclosed in patent application WO 2013 / 163675.

[0026]

[0027] In two of these patent applications, the reported enzyme inhibition was more selective for SSAO / VAP-1 than for all other amine oxidases, including MAO-B.

[0028] Recent patent applications describe aryloxy and heteroaryloxy 2-substituted-3-fluoroallylamines 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 are disclosed.

[0029] Further patent applications for SSAO / VAP-1 inhibitors report limited data for MAO-B and MAO-A (WO2018 / 196677, WO 2019 / 101086, WO 2019 / 024924, WO 2020 / 006177, WO 2020 / 063696, WO2020 / 069335 and WO 2021 / 083209). None of the exemplary compounds proposed therein report an IC against the recombinant human form of MAO-B in standard biochemical assays. 50 <100 nM, therefore not considered a potent inhibitor of MAO-B.

[0030] Also disclosed are benzoxazolyl 2-substituted-3-fluoroallylamine SSAO / VAP-1 inhibitors that show potency in inhibiting MAO-B (IC 50 <0.1 μM) and lower inhibitory potency of MAO-A (IC 50 Between 10 and 30 μM) (WO2020 / 069330), such as the examples shown below.

[0031]

[0032] 2-Substituted-3-fluoroallylamine SSAO / VAP-1 inhibitors directly attached to the nitrogen of the five-membered heterocyclic ring have also been described (WO2019 / 180644). Activity against MAO-B and selectivity for MAO-A were reported for some exemplary compounds.

[0033] Relevant to the present invention presented herein, the following compounds have been reported as selective SSAO / VAP-1 inhibitors and data reportedly indicate that they are not potent inhibitors of MAO-B (CN 2019 / 109251166).

[0034] Summary of the invention

[0035] The present invention provides 2-substituted-3-fluoroallylamine compounds, which are potent inhibitors of both SSAO / VAP-1 and MAO-B with particularly high selectivity relative to MAO-A. Surprisingly, modification of the previously described 2-phenoxymethylene-3-fluoroallylamine structure has led to the development of novel compounds, which are potent inhibitors of both the human form of the SSAO / VAP-1 enzyme and the MAO-B enzyme, with high selectivity relative to the inhibition of the MAO-A enzyme, and thus have good safety. In addition, the compounds of the present invention have excellent pharmacokinetic properties and good in vitro safety.

[0036] The present invention describes the synthesis and use of compounds that inhibit the amine oxidase activity of SSAO / VAP-1 and MAO-B, and describes the utility of such inhibitors in treating patients suffering from, but not limited to, neuromuscular disorders.

[0037] The first aspect of the present invention provides a compound of formula I:

[0038]

[0039] or a pharmaceutically acceptable salt, polymorphic form or solvate thereof; wherein:

[0040] R 1 is selected from the group consisting of: hydrogen, fluorine, bromine and methyl; and

[0041] R 2 It is hydrogen or fluorine.

[0042] The second aspect of the present invention provides a pharmaceutical composition comprising a compound according to the first aspect of the present invention or a pharmaceutically acceptable salt or solvate thereof, and at least one pharmaceutically acceptable excipient, carrier or diluent.

[0043] The third aspect of the present invention provides a method for inhibiting the amine oxidase activity of SSAO / VAP-1 and MAO-B in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound according to the first aspect of the present invention or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to the second aspect of the present invention.

[0044] The fourth aspect of the present invention provides a method for treating or preventing a disease or disorder by inhibiting the activity of SSAO / VAP-1 protein and MAO-B protein, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to the first aspect of the present invention or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to the second aspect of the present invention.

[0045] The fifth aspect of the present invention provides use of the compound according to the first aspect of the present invention or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for treating or preventing a disease or disorder by inhibiting the activity of SSAO / VAP-1 protein and MAO-B protein.

[0046] The sixth aspect of the present invention provides a compound according to the first aspect of the present invention or a pharmaceutically acceptable salt or solvate thereof, for use in treating or preventing a disease or disorder by inhibiting the activities of SSAO / VAP-1 protein and MAO-B protein.

[0047] In one embodiment of the methods and uses of the present invention, the disease or condition is ameliorated by selective inhibition of SSAO / VAP-1 and MAO-B relative to MAO-A.

[0048] In one embodiment of the methods and uses of the present invention, the disease is a neuromuscular disease.

[0049] In another embodiment of the present invention, the disease is a neuroinflammatory disease or a neurodegenerative disease.

[0050] Contemplated herein are combination therapies wherein the methods further comprise co-administration of an additional therapeutic agent for the treatment of neuromuscular diseases, neuroinflammatory diseases, and neurodegenerative diseases.

[0051] definition

[0052] The following are some definitions that will aid in understanding the description of the present invention. These are intended as general definitions and should by no means limit the scope of the present invention to only these terms, but are provided for a better understanding of the following description.

[0053] Unless the context requires otherwise or there is a specific description to the contrary, integers, steps or elements of the present invention recited herein are in the singular, and the steps or elements expressly encompass both the singular and the plural of the recited integers, steps or elements.

[0054] Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements, but not the exclusion of any other step or element or integer, or group of elements or integers. Therefore, in the context of this specification, the term "comprising" means "mainly including but not necessarily only including".

[0055] Those skilled in the art will appreciate that the invention described herein may be subject to variations and modifications in addition to those specifically described. It should be understood that the present invention includes all such variations and modifications. The present invention also includes, individually or collectively, all steps, features, compositions and compounds mentioned or indicated in this specification, as well as any and all combinations or any two or more of the steps or features.

[0056] The present invention includes within its scope isotopes of different atoms. Any atom not specifically designated as a particular isotope represents any stable isotope of that atom. Thus, the present disclosure should be understood to include deuterium and tritium isotopes of hydrogen.

[0057] All references cited in this application are specifically incorporated by cross-reference in their entirety. The citation of any such document is not to be construed as an admission that the document forms part of the common general knowledge or belongs to the prior art.

[0058] In the context of this specification, the term "administering" and variants of the term include "administering" and "administration", including contacting, applying, delivering or providing the compound or composition of the present invention to an organism or surface by any appropriate means. In the context of this specification, the term "treatment" refers to any and all uses for remedying a disease state or symptom, preventing the establishment of a disease, or otherwise preventing, hindering, delaying or reversing the progression of a disease or any other form of adverse symptoms.

[0059] In the context of this specification, the term "effective amount" includes within its meaning a sufficient but non-toxic amount of a compound or composition of the present invention sufficient to provide the desired effect. Thus, the term "therapeutically effective amount" includes within its meaning a sufficient but non-toxic amount of a compound or composition of the present invention sufficient to provide the desired therapeutic effect. The exact dosage required varies from subject to subject, depending on a variety of factors, such as the species being treated; the sex, age and general condition of the subject; the severity of the disease being treated; the specific drug being administered; the mode of administration, etc. Therefore, it is not possible to specify an exact "effective amount". However, for any given situation, one of ordinary skill in the art can determine the appropriate "effective amount" using only routine experimentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The pharmacokinetic time course of PXS-5131 against compound 1 in Wistar rats is depicted.

[0061] Figure 2 Depicted is the pharmacodynamic readout of in vivo inhibition of SSAO / VAP-1 in adipose tissue of Sprague Dawley rats following oral administration of PXS-5131 at 0.6 mg / kg.

[0062] Figure 3 Depicted is the pharmacodynamic readout of in vivo inhibition of MAO-B in Sprague Dawley rat brain tissue following oral administration of PXS-5131 at 0.6 mg / kg.

[0063] Figure 4 Depicted is the anti-inflammatory effect of PXS-5131 in BALB / c mice in the carrageenan air pouch model.

[0064] Figure 5 PXS-5131 is depicted to reduce neuronal loss in the turban Wistar rat model of dopaminergic neurodegeneration.

[0065] Figure 6 Depicted that oral administration of PXS-5131 at 6 mg / kg / day helped prevent force loss caused by eccentric contraction in the gastrocnemius (GC) muscle after 4 weeks of dosing in 3 month old mdx mice compared to untreated age-matched mice.

[0066] Figure 7 Depicted are that oral administration of PXS-5131 at 6 mg / kg / day significantly reduced (A) neutrophil influx, (B) macrophage influx, (C) reactive oxygen species, and (D) tibialis anterior muscle fibrosis (as quantified by picrosirius red staining) after 4 weeks of dosing in 3-month-old mdx mice compared to untreated age-matched mice.

[0067] Figure 8 Depicted are MAO-B and SSAO / VAP-1 inhibition in C57BL / 6J mice, heart, muscle, and adipose tissue following oral administration of compound 1 at various doses.

[0068] Fig. 9 Depicted are the activities of MAO-A in heart and muscle tissues of C57BL / 6J mice after oral administration of Compound 1 at various doses.

[0069] Fig.10 Depicted are MAO-B and SSAO / VAP-1 inhibition in mdx mice, muscle, and adipose tissue, respectively, following oral administration of Compound 1.

[0070] Fig.11 The activity of MAO-A in muscle tissue of mdx mice after oral administration of Compound 1 is depicted.

[0071] Fig.12 Depicted are oral administration of Compound 1 (4.8 mg / kg / day) that significantly reduced (A) neutrophil influx, (B) macrophage influx, (C) reactive oxygen species, and (D) diaphragm fibrosis (as quantified by picrosirius red staining) in 3-month-old mdx mice after 30 days of dosing compared to untreated age-matched mice.

[0072] Fig.13 Depicted that oral administration of Compound 1 (4.8 mg / kg / day) significantly reduced cardiac fibrosis (as quantified by picrosirius red staining of the myocardium) after 90 days of dosing in 10 month old mdx mice compared to untreated age-matched mice. DETAILED DESCRIPTION

[0073] The present invention relates to substituted haloallylamine derivatives that can inhibit both SSAO / VAP-1 and MAO-B. In particular, the present invention relates to substituted and unsubstituted 4-((2-(aminomethyl)-3-fluoroallyl)oxy)benzonitrile derivatives.

[0074] In particular, the present invention relates to compounds of formula I:

[0075]

[0076] or a pharmaceutically acceptable salt, polymorphic form or solvate thereof; wherein:

[0077] R 1 is selected from the group consisting of: hydrogen, fluorine, bromine and methyl; and

[0078] R 2 It is hydrogen or fluorine.

[0079] In one embodiment of the compounds of the present invention, R 1 In another embodiment of the compounds of the present invention, R 1 In another embodiment of the compounds of the present invention, R 1 In another embodiment of the compounds of the present invention, R 1 It's methyl.

[0080] In one embodiment of the compounds of the present invention, R 2 In another embodiment of the compounds of the present invention, R 2 It's fluorine.

[0081] In one embodiment of the compounds of the present invention, R 1 is hydrogen and R 2 In another embodiment of the compounds of the present invention, R 1 is fluorine and R 2 In another embodiment of the compounds of the present invention, R 1 is bromine and R 2 In another embodiment of the compounds of the present invention, R 1 is hydrogen and R 2 In another embodiment of the compounds of the present invention, R 1 is methyl and R 2 It's hydrogen.

[0082] In one embodiment, the compounds of the present invention are used as their free base. In another embodiment of the compounds of the present invention, the compounds are used as pharmaceutically acceptable salts. In one embodiment of the compounds of the present invention, the compounds are hydrochloride salts.

[0083] In one embodiment, the compounds of the present invention are potent inhibitors of SSAO / VAP-1 and MAO-B. According to the present invention, the IC 50 Less than 50 nM or less than 40 nM or less than 30 nM or less than 20 nM. In one embodiment, the IC of a potent inhibitor of SSAO / VAP-1 is 50 According to the present invention, the IC of a potent inhibitor of MAO-B is less than 10 nM. 50 Less than 250 nM or less than 200 nM or less than 150 nM or less than 100 nM. In one embodiment, the IC of a potent inhibitor of MAO-B is 50 Less than 50nM.

[0084] In one embodiment, the compounds of the present invention are potent inhibitors of both SSAO / VAP-1 and MAO-B with particularly high selectivity relative to MAO-A. The compounds of the present invention have an IC of greater than 3000 nM, or greater than 10000 nM, or greater than 20000 nM. 50 Inhibits MAO-A. In one embodiment, the compounds of the present invention have an IC of greater than 30000 nM. 50 Inhibits MAO-A.

[0085] The compounds of the present invention are selective inhibitors of SSAO / VAP-1 and MAO-B relative to MAO-A. The determination of the relative selectivity of a given compound for MAO-B inhibition is defined as (MAO-A IC 50 Value / MAO-B IC 50 The relative ratio of (MAO-AIC 50 Value / SSAO / VAP-1IC 50 In another embodiment, for a given compound, (MAO-AIC 50 Value / MAO-B IC 50 In another embodiment, for a given compound, (MAO-AIC 50 Value / SSAO / VAP-1IC 50 value) is at least 300 or at least 600 or at least 3000.

[0086] In the context of the present disclosure, any one or more aspects or embodiments may be combined with any other aspect or embodiment.

[0087] Exemplary compounds according to the present invention include the compounds listed in Table 1:

[0088] Table 1

[0089]

[0090]

[0091] Preparation of compounds of formula I

[0092] The compounds of Formula I 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 do not limit the scope of the claims provided herein.

[0093] Standard synthetic chemistry techniques are described, but not limited to, in March's Advanced Organic Chemistry, 8th edition, John Wiley and Sons Inc. Standard methods for temporarily protecting functional groups such as alcohols, amines, and carboxylic acids using protecting groups are described, for example, in Protecting Groups in Organic Synthesis, 5th edition, John Wiley and Sons Inc.

[0094] Alternative reaction conditions for the chemical transformations described herein may be employed, such as variations in solvents, reaction temperatures, reaction times, and different chemical reagents. Unless otherwise stated, starting materials for chemical syntheses and biological applications are available from commercial sources.

[0095] In General Scheme 1 and General Scheme 2, P is a group for protecting nitrogen functionality. Examples of P are carbonates, such as tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (FMOC) and benzyloxycarbonyl (CBZ) groups. In some embodiments, at a temperature between 0°C and 40°C, the hydroxybenzonitrile of formula II (Scheme 1) is treated with allyl bromide of formula III for several hours (Method A) using a suitable polar solvent in the presence of a suitable base. 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. According to standard extraction and purification methods, the product described by formula IV can be obtained with good yield and purity.

[0096]

[0097] In some embodiments, fluorobenzonitrile of formula V (Scheme 2) is treated with allyl alcohol of formula VI using a suitable solvent in the presence of a suitable base for several minutes at a temperature between 0°C and 40°C (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 methods, the product described by formula IV can be obtained in good yield and purity.

[0098]

[0099] There are many well-established chemical procedures for deprotecting the compound described by Formula IV to the compound described by Formula I (Method B; Scheme 1 and Scheme 2). For example, if P is a Boc protecting group, the compound described by Formula IV can be treated with an acidic reagent such as anhydrous hydrogen chloride in a solvent such as diethyl ether or dioxane to provide the compound described by Formula I as a hydrochloride salt. 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. Typically, the free base compound is converted to an acid addition salt for ease of handling and improved chemical stability. Examples of acid addition salts include, but are not limited to, hydrochlorides, hydrobromides, 2,2,2-trifluoroacetates, and mesylate salts.

[0100] Therapeutic Uses

[0101] The present invention provides methods of inhibiting membrane-bound SSAO / VAP-1 and soluble SSAO / VAP-1 using the compounds described by Formula I. In addition, the present invention provides methods of inhibiting MAO-B using the compounds described by Formula I. The relative inhibitory potency of the compounds can be determined by the amount required to inhibit the amine oxidase activity of SSAO / VAP-1 and MAO-B in a variety of ways, for example, in in vitro assays with recombinant human proteins or with recombinant non-human enzymes, in cell assays expressing normal rodent enzymes, in cell assays that have been transfected with human proteins, in vivo tests in rodents and other mammalian species, etc.

[0102] Also disclosed are methods of using the compounds described by Formula I to inhibit SSAO / VAP-1 and MAO-B in patients suffering from neuromuscular diseases, as well as methods of treating symptoms associated with neuromuscular diseases.

[0103] Therefore, in one aspect, 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, the method comprising administering to the subject an effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof.

[0104] In another aspect, the present invention relates to a method for treating or preventing a disease or disorder by inhibiting the activity of SSAO / VAP-1 protein and MAO-B protein, the method comprising administering a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof or a pharmaceutical composition thereof to a subject in need thereof. In one embodiment, the present invention relates to a method for treating a disease or disorder by inhibiting the activity of SSAO / VAP-1 protein and MAO-B protein. In another embodiment, the present invention relates to a method for preventing a disease or disorder by inhibiting the activity of SSAO / VAP-1 protein and MAO-B protein.

[0105] In yet another aspect, the invention relates to a method of treating a disease or condition 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 pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof.

[0106] 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: a neuromuscular disorder, a neurodegenerative disease, or a neuroinflammatory disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof. In one embodiment, the present invention is directed to treating a disease or disorder selected from the group consisting of: a neuromuscular disorder, a neurodegenerative disease, or a neuroinflammatory disease. In another embodiment, the present invention is directed to preventing a disease or disorder selected from the group consisting of: a neuromuscular disorder, a neurodegenerative disease, or a neuroinflammatory disease.

[0107] Another embodiment of the present invention is a method of treatment, wherein the disease or condition is ameliorated by selective inhibition of SSAO / VAP-1 and MAO-B relative to MAO-A. By selectively inhibiting SSAO / VAP-1 and MAO-B relative to MAO-A, the compounds of the present invention provide a good safety profile.

[0108] A further aspect of the present invention provides use of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for treating or preventing a disease or disorder by inhibiting the activity of SSAO / VAP-1 protein and MAO-B protein.

[0109] Another aspect of the present invention provides the use of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for treating or preventing a disease or disorder selected from the group consisting of a neuromuscular disorder, a neurodegenerative disease or a neuroinflammatory disease.

[0110] The above methods and uses are applicable to situations where the disease is a neuromuscular disease. As used herein, "neuromuscular disease or disease" covers conditions that directly impair muscle function, i.e., pathological changes in voluntary muscles, or conditions that indirectly impair muscle function, i.e., pathological changes in nerves or neuromuscular junctions. This specification covers a wide range of diseases, including muscular dystrophy, 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 and congenital muscular dystrophy; there are also indirect central diseases 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, 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, muscular dystrophy is Duchenne muscular dystrophy (DMD).

[0111] The term "neuromuscular disease" may also be used to refer to motor neuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy, ion channelopathies, myopathies, mitochondrial diseases such as Friedreich's ataxia, neuromuscular junction diseases such as myasthenia gravis, and peripheral nerve diseases such as Charcot-Marie-Tooth disease.

[0112] The dual benefits of SSAO / VAP-1 and MAO-B inhibition have the potential to treat inflammatory diseases associated with oxidative stress.

[0113] As used herein, "inflammation or inflammatory disorder" encompasses a variety of indications, including arthritis (including juvenile rheumatoid arthritis), Crohn's disease, ulcerative colitis, inflammatory bowel disease (e.g., irritable bowel syndrome), psoriasis, asthma, lung inflammation, COPD, bronchiectasis, skin inflammation, eye disease, contact dermatitis, liver inflammation, autoimmune liver disease, autoimmune hepatitis, primary biliary cirrhosis, sclerosing cholangitis, autoimmune cholangitis, alcoholic liver disease, atherosclerosis, chronic heart failure, congestive heart failure, ischemic disease, stroke and its complications, inflammation caused by bacterial infection, such as that experienced in sepsis, acute lung injury and cystic fibrosis, myocardial infarction and its complications, inflammatory cell destruction after stroke, synovitis and systemic inflammatory sepsis.

[0114] Diseases associated with the term "oxidative stress" encompass conditions in which an increase in reactive oxygen species is consistently associated with disease progression or directly associated with disease symptoms, and include many different diseases, including cancer, muscular dystrophy, Alzheimer's disease, Parkinson's disease, diabetes, cardiovascular disorders such as high blood pressure, atherosclerosis and stroke, inflammatory diseases, 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.

[0115] In one embodiment of the methods and uses of the present invention, the disease is an inflammatory disorder selected from a neurological disease, a metabolic disease and a fibrotic disease.

[0116] The above methods and uses are also applicable to the case where the disease is a neurological disease or disorder. As used herein, "neurological disease or disorder" covers neuroinflammatory diseases and neurodegenerative diseases, and encompasses a variety of indications, including 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 apolipoprotein E4 alleles. In one embodiment of the methods and uses 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.

[0117] Other examples of neurological disorders include psychiatric disorders, including depression, anxiety disorders, panic attacks, social phobia, schizophrenia, anorexia, and depressive mood disorders.

[0118] Further examples of neurological disorders include withdrawal syndromes induced by alcohol, nicotine and other addictive drugs.

[0119] The above methods and uses are also applicable to the case where the disease is a metabolic disorder or disease. "Metabolic disorder or disease" as used herein encompasses a variety of indications, including such diseases, such as obesity, type 1 diabetes and type 2 diabetes, hypertension, atherosclerosis, high blood pressure, chronic kidney disease, dyslipidemia, heart disease, metabolic syndrome, insulin resistance, diabetic nephropathy, glomerulosclerosis, diabetic retinopathy, choroidal neovascularization and cardiovascular complications caused therefrom.

[0120] According to another aspect of the present invention, a method of treating patients with type 1 diabetes and type 2 diabetes and diabetes-related diseases with a dual inhibitor of SSAO / VAP-1 and MAO-B is provided. Diabetes-related diseases contemplated herein for treatment include diabetic nephropathy, diabetic macular edema, glomerulosclerosis, diabetic retinopathy, choroidal neovascularization, and cardiovascular complications caused therefrom.

[0121] The above method and purposes are also applicable to the situation that the disease is fibrosis. " fibrosis " adopted herein includes such diseases, such as cystic fibrosis, idiopathic pulmonary fibrosis, renal fibrosis, chronic obstructive pulmonary disease, pleural fibrosis and radiation pulmonary fibrosis, non-diabetic renal fibrosis, pancreatic fibrosis, scleroderma, connective tissue disease, scar, skin fibrosis, fibrosis related to organ transplantation, stenosis, liver fibrosis, including alcoholic liver disease, fatty liver disease, non-alcoholic steatohepatitis, acute hepatitis and chronic hepatitis, biliary cirrhosis, primary sclerosing cholangitis, hepatocellular carcinoma and toxic liver injury, and other diseases that excessive fibrosis contributes to disease pathological changes. In one embodiment, fibrosis is cardiac fibrosis.

[0122] Drugs and / or therapeutic preparations

[0123] In another embodiment of the present invention, there is provided a composition comprising a compound of formula I and at least one pharmaceutically acceptable excipient, carrier or diluent thereof. The compound of formula I may also exist as a suitable salt, including a pharmaceutically acceptable salt.

[0124] The phrase "pharmaceutically acceptable carrier" refers to any carrier known to those skilled in the art to be suitable for a particular mode of administration. Additionally, the compound may be formulated as the sole pharmaceutically active ingredient in a composition, or may be combined with other active ingredients.

[0125] The phrase "pharmaceutically acceptable salt" refers to any salt preparation suitable for pharmaceutical use. Pharmaceutically acceptable salts are those salts which are, within the scope of sound medical judgment, suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit / risk ratio.

[0126] Pharmaceutically acceptable salts are well known in the art and include acid addition salts and basic salts. Half salts of acids and bases can also be formed. Pharmaceutically acceptable salts include amine salts of inorganic acids (e.g., hydrochlorides, hydrobromides, sulfates, etc.); and amine salts of organic acids (e.g., formate, acetate, lactate, malate, tartrate, citrate, ascorbate, succinate, maleate, butyrate, valerate, fumarate, etc.).

[0127] For compounds of Formula I having basic sites, suitable pharmaceutically acceptable salts may be acid addition salts. For example, suitable pharmaceutically acceptable salts of such compounds may be prepared by mixing a pharmaceutically 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 invention.

[0128] Pharmaceutically acceptable salts are described in detail in S. M. Berge et al., J. Pharmaceutical Sciences, 1977, 66: 1-19. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base functional group 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, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, 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, and the like. Suitable base salts are formed from bases which form non-toxic salts.

[0129] Pharmaceutically acceptable salts of compounds of Formula I can be prepared by methods known to those skilled in the art, including, for example:

[0130] i. by reacting a compound of formula I with a desired acid or base;

[0131] ii. by removing an acid- or base-labile protecting group from a suitable precursor of a compound of formula I, or by ring-opening a suitable cyclic precursor such as a lactone or lactam using the desired acid or base; or

[0132] iii. Converting one salt of a compound of formula I into another salt by reaction with a suitable acid or base or by passing through a suitable ion exchange column.

[0133] The above reactions (i)-(iii) are usually carried out in solution. The resulting salt may be precipitated and collected by filtration, or may be recovered by evaporating the solvent. The degree of ionization in the resulting salt may vary from completely ionized to almost non-ionized.

[0134] Therefore, for example, suitable pharmaceutically acceptable salts of compounds according to the present invention can be prepared by mixing pharmaceutically acceptable acids 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 compounds of the present invention. Therefore, suitable pharmaceutically acceptable salts of compounds of the present invention include acid addition salts.

[0135] The compounds of the present invention may exist in the form of non-solvates and solvates. As used herein, the term "solvate" is used to describe a molecular complex comprising a compound of the present invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules (e.g., ethanol). When the solvent is water, the term "hydrate" is used.

[0136] The compositions herein include one or more compounds provided herein. In one embodiment, the compound is formulated into a suitable pharmaceutical preparation, such as a solution, suspension, tablet, cream, gel, dispersible tablet, pill, capsule, powder, sustained-release formulation or elixir, for oral administration or in a sterile solution or suspension for parenteral administration, as well as transdermal patches and dry powder inhalers. In one embodiment, the above-mentioned compounds are formulated into pharmaceutical compositions using techniques and methods well known in the art (see, for example, Ansel "Introduction to Pharmaceutical Dosage Forms", Fourth Edition, 1985, 126).

[0137] In the composition, effective concentrations of one or more compounds or pharmaceutically acceptable derivatives thereof are mixed with a suitable pharmaceutical carrier. As described above, prior to formulation, the compound may be derivatized as a corresponding salt, ester, enol ether or ester, acetal, ketal, orthoester, hemiacetal, hemiketal, acid, base, solvate, hydrate or prodrug. The concentration of the compound in the composition is effective to deliver an amount to treat, prevent or ameliorate one or more symptoms of the disease or disorder to be treated after administration.

[0138] In one embodiment, the composition is formulated for single-dose administration. To formulate the composition, the weight fraction of the compound is dissolved, suspended, dispersed or otherwise mixed in a selected carrier at an effective concentration to alleviate, prevent or improve one or more symptoms.

[0139] The active compound is contained in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically effective effect without producing adverse side effects in the patient being treated. The therapeutically effective concentration can be determined empirically by testing the compound in the in vitro system and in vivo system described herein and in PCT publication WO04 / 018997, and then extrapolating the human dosage therefrom.

[0140] The concentration of the active compound in the pharmaceutical composition will depend on the absorption, distribution, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosing regimen and amount administered, and other factors known to those skilled in the art.

[0141] In one embodiment, the therapeutically effective dose should produce 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 should provide a dosage of about 0.001 mg to about 2000 mg of the compound per kilogram of body weight per day. The pharmaceutical dosage unit form is prepared to provide about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, 1000 mg or 2000 mg per dosage unit form, and in one embodiment, a combination of about 10 mg to about 500 mg of the active ingredient or essential ingredients is provided.

[0142] Administration can be carried out at intervals of several minutes, hours, days, weeks, months or years, or can be carried out continuously during any of these periods. Suitable dosage is in the range of about 0.1ng per kg of body weight to 1g per kg of body weight per dosage. Dosage is preferably in the range of 1 μg to 1g per kg of body weight per dosage, such as in the range of 1mg to 1g per kg of body weight per dosage. Suitably, dosage is in the range of 1 μg to 500mg per kg of body weight per dosage, such as 1 μg to 200mg per kg of body weight per dosage, or 1 μg to 100mg per kg of body weight per dosage. Other suitable dosages can be in the range of 1mg to 250mg per kg of body weight, including 1mg to 10, 20, 50 or 100mg per kg of body weight per dosage or 10 μg to 100mg per kg of body weight per dosage.

[0143] Appropriate dosage and dosing regimen can be determined by the attending physician, and may depend on the particular condition being treated; the severity of the condition; and the general health, age, and weight of the subject.

[0144] In cases where the compound exhibits insufficient solubility, methods for solubilizing the compound may be used. Such methods are known to those skilled in the art and include, but are not limited to, the use of co-solvents such as dimethyl sulfoxide (DMSO); the use of surfactants such as Dissolved in aqueous sodium bicarbonate solution; Formulated into nanoparticles, etc. Derivatives of the compounds, such as prodrugs of the compounds, can also be used to formulate effective pharmaceutical compositions.

[0145] After mixing or adding the compounds, the resulting mixture can be a solution, suspension, emulsion, etc. 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 is sufficient to improve the symptoms of the disease, disorder or condition being treated and can be determined empirically.

[0146] Pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms (such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, oral solutions or suspensions, and oil-water emulsions containing appropriate amounts of compounds or pharmaceutically acceptable derivatives thereof). In one embodiment, pharmaceutical therapeutically active compounds and their derivatives are formulated and administered in unit dosage forms or multiple dosage forms. The active ingredient can be administered once or divided into many smaller doses, administered once at intervals. As used herein, unit dosage forms refer to physically discrete units suitable for human and animal subjects, and are packaged separately as known in the art. Each unit dose contains a predetermined amount of therapeutically active compounds sufficient to produce the desired therapeutic effect, and the required pharmaceutical carrier, vehicle, or diluent. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. Unit dosage forms can be administered in fractions or multiples thereof. Multiple dosage forms are multiple identical unit dosage forms packaged in a single container, administered in separate unit dosage forms. Examples of multiple dosage forms include vials, tablet or capsule bottles, or pint or gallon bottles. Thus, a multiple dosage form is a plurality of unit doses that are not separated in packaging.

[0147] Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 15th edition, 1975.

[0148] Dosage forms or compositions containing active ingredients in the range of 0.005% to 100% (weight %) with the remainder consisting of non-toxic carriers can be prepared. Methods for preparing these compositions are known to those skilled in the art. Contemplated compositions may contain 0.001%-100% (weight %) active ingredients, in one embodiment 0.1-95% (weight %), and in another embodiment 75-85% (weight %).

[0149] Mode of administration

[0150] Convenient modes of administration include injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, topical creams or gels or powders, 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 effects 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.

[0151] Oral administration composition

[0152] Oral pharmaceutical dosage forms can be solid, gel or liquid. Solid dosage forms are tablets, capsules, granules and bulk powders. Types of oral tablets include compressed, chewable lozenges and tablets that can be enteric coated, sugar coated or film coated. Capsules can be hard or soft gelatin capsules, while granules and powders can be provided in non-effervescent or effervescent form in combination with other ingredients known to those skilled in the art.

[0153] Solid compositions for oral administration

[0154] 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 properties: binders, lubricants; diluents; glidants; disintegrants; colorants; sweeteners; flavorings; wetting agents; emetic coatings; and film coatings. Examples of binders include microcrystalline cellulose, tragacanth, glucose solution, acacia gum, gelatin solution, molasses, polyvinylpyrrolidone, povidone, crospovidone, sucrose and starch paste. Lubricants include talc, starch, magnesium stearate or calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrants include cross-linked sodium carboxymethylcellulose, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Colorants include, for example, any approved certified water-soluble FD and C dyes, mixtures thereof; and water-insoluble FD and C dyes suspended on hydrated alumina. Sweeteners include sucrose, lactose, mannitol and artificial sweeteners (such as saccharin) and any number of spray-dried spices. Flavoring agents include natural flavors extracted from plants such as fruits, and synthetic mixtures of compounds that produce pleasant sensations such as, but not limited to, mint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Emetic coatings include fatty acids, fats, waxes, shellac, ammoniated shellac, and cellulose acetate phthalate. Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate.

[0155] The compound or its pharmaceutically acceptable derivative can be provided in the form of a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated into 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 ingredients.

[0156] When the dosage unit form is a capsule, it may contain, in addition to materials of the types described above, a liquid carrier such as a fatty oil. Additionally, the dosage unit form may contain various other materials that modify the physical form of the dosage unit, such as sugar and other enteric coatings. The compound may also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum, or the like. In addition to the active compound, a syrup may contain sucrose as a sweetener and certain preservatives, dyes, coloring agents, and flavoring agents.

[0157] The active substance can also be mixed with other active substances that do not impair the desired effect or with substances that supplement the desired effect, such as antacids, H2 blockers and diuretics. The active ingredient is a compound described herein or a pharmaceutically acceptable derivative thereof. Higher concentrations, up to about 98% by weight of the active ingredient, can be included.

[0158] In all embodiments, tablets and capsules may be coated as known to those skilled in the art to modify or sustain the dissolution of the active ingredient. Thus, for example, they may be coated with conventional enteric coatings such as phenyl salicylate, waxes and cellulose acetate phthalate.

[0159] Oral administration of liquid compositions

[0160] 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 oil-in-water or water-in-oil.

[0161] Pharmaceutically administrable liquid 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 such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, etc., to form a solution or suspension. If necessary, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting agents, emulsifiers, solubilizers, pH buffers, etc., for example acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate and other such agents.

[0162] Elixirs are clear, sweetened, aqueous or hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of sugars such as sucrose and may contain preservatives. Emulsions are two-phase systems in which one liquid is dispersed in another liquid in the form of globules. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifiers, and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used in non-effervescent granules reconstituted into liquid oral dosage forms include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable substances used in effervescent granules will be reconstituted into liquid oral dosage forms, including organic acids and a source of carbon dioxide. Coloring agents and flavoring agents are used in all of the above dosage forms.

[0163] Solvents include glycerine, sorbitol, ethanol and syrup. Examples of preservatives include glycerine, methyl and propyl parabens, benzoic acid, sodium benzoate and ethanol. Examples of non-aqueous liquids used in emulsions include mineral oil and cottonseed oil. Examples of emulsifiers include gelatin, gum arabic, gum tragacanth, bentonite and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, gum tragacanth, magnesium aluminum silicate and gum arabic. Sweeteners include sucrose, syrup, glycerine and artificial sweeteners such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic acids include citric acid and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Colorants include any approved certified water-soluble FD and C dyes and mixtures thereof. Flavoring agents include natural flavorings extracted from plants, such as fruits, and synthetic mixtures of compounds that produce a pleasant mouthfeel.

[0164] For solid dosage forms, in one embodiment, solutions or suspensions, such as propylene carbonate, vegetable oils or triglycerides, are encapsulated in gelatin capsules. For liquid dosage forms, for example, polyethylene glycol solutions can be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier (e.g., water) to allow for easy measurement of administration.

[0165] Alternatively, liquid or semisolid oral preparations can be prepared by dissolving or dispersing the active compound or salt in vegetable oils, ethylene glycol, triglycerides, propylene glycol esters (e.g., propylene carbonate), and other such carriers and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those listed in U.S. Pat. Nos. RE28,819 and 4,358,603. Briefly, such formulations include, but are not limited to, formulations containing dialkylated mono- or polyalkylene glycols of the compounds provided herein, including, but not limited to, 1,2-dimethoxymethane, diethylene glycol dimethyl ether, triethylene glycol dimethyl ether, tetraethylene glycol dimethyl ether, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, wherein 350, 550 and 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.

[0166] Injections, solutions and emulsions

[0167] It is also contemplated herein that parenteral administration characterized by subcutaneous, intramuscular or intravenous injection in one embodiment. Injections can be prepared in conventional forms, can be liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquids before injection, or emulsions. Injections, 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 also contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

[0168] Implantation of a slow-release or sustained-release system is also contemplated herein, thereby maintaining a constant dosage level. Briefly, the compounds provided herein are dispersed in a solid internal matrix, e.g., 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, carbonated silicone copolymers, hydrophilic polymers such as hydrogels of acrylic acid and methacrylate, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate, the The cross-linked partially hydrolyzed polyvinyl acetate is surrounded by an outer polymer membrane, for example, polyethylene, polypropylene, ethylene / propylene copolymers, ethylene / ethyl acrylate copolymers, ethylene / vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, chloroprene rubber, chlorinated polyethylene, polyvinyl chloride, copolymers of vinyl chloride and vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene / vinyl alcohol copolymers, ethylene / vinyl acetate / vinyl alcohol terpolymers and ethylene / vinyloxyethanol copolymers that are insoluble in body fluids. In the release rate controlling step, the compound diffuses through the outer polymer membrane. The percentage of active compound contained in such parenteral compositions is highly dependent on its specific properties, as well as the activity of the compound and the needs of the subject.

[0169] Parenteral administration of the composition includes intravenous, subcutaneous and intramuscular administration. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products (such as lyophilized powders, including subcutaneous tablets) that are combined with solvents before use, sterile suspensions ready for injection, sterile dry insoluble products that are combined with excipients before use, and sterile emulsions. The solution can be aqueous or non-aqueous.

[0170] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

[0171] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, sequestering or chelating agents and other pharmaceutically acceptable substances.

[0172] Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils, olive oil, cottonseed oil, corn oil, sesame oil and peanut oil of plant origin. Antibacterial agents of antibacterial or antibacterial concentrations must be added to parenteral products packaged in multi-dose containers, which include phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspensions and dispersants include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifiers include polysorbate 80 ( 80). Metal ion sequestering agents or chelators include EDTA. Pharmaceutical carriers also include ethanol, polyethylene glycol and propylene glycol for water-miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid to adjust pH.

[0173] The concentration of the pharmaceutically active compound is adjusted so that the injection provides an effective amount to produce the desired pharmacological effect. As known in the art, the exact dose depends on the age, weight and condition of the patient or animal.

[0174] The unit dose parenteral preparation is packaged in an ampoule, a vial or a syringe with a needle. As is known and practiced in the art, all preparations for parenteral administration must be sterile.

[0175] Illustratively, intravenous or intraarterial infusion of a sterile aqueous solution containing the active compound is an effective mode of administration.Another embodiment is a sterile aqueous or oily solution or suspension containing the active substance for injection as required to produce the desired pharmacological effect.

[0176] Injections 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 higher of the active compound relative to the treated tissue, and in certain embodiments, greater than 1% w / w of the active compound.

[0177] The compound may be suspended in a micronized or other suitable form or may be 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 is sufficient to alleviate the symptoms of the condition and can be determined empirically.

[0178] Topical application

[0179] The topical mixture is prepared as described for topical and systemic administration. The resulting mixture can be a solution, suspension, emulsion, etc., and formulated into a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, rinse, spray, suppository, bandage, skin patch or any other preparation suitable for topical administration.

[0180] The compound or its pharmaceutically acceptable derivative can be formulated as an aerosol for topical application (such as by inhalation). These preparations for respiratory administration can be in the form of an aerosol or solution for sprays, or in the form of a fine powder for insufflation, which can be used alone or in combination with an inert carrier (such as lactose). In this case, in one embodiment, the diameter of the particles of the preparation is less than 50 microns, and in one embodiment less than 10 microns.

[0181] The compound can be formulated for topical or local (local or topical) application, such as in the form of gels, creams and lotions for topical application to the skin and mucous membranes, such as in the eye, and applied to the eye or for intracranial or intraspinal application. Topical administration is contemplated for transdermal delivery, also for ocular or mucosal administration, or for inhalation therapy. Nasal solutions of the active compound can also be administered alone or in combination with other pharmaceutically acceptable excipients.

[0182] These solutions, particularly those intended for ophthalmic use, can be formulated with appropriate salts as 0.01% to 10% (volume %) isotonic solutions at a pH of about 5-7.

[0183] Other routes of administration Composition

[0184] Other routes of administration are also contemplated herein, such as transdermal patches, including iontophoresis and electrophoresis devices, vaginal and rectal administration.

[0185] Transdermal patches including iontophoresis and electrophoresis devices are well known to those skilled in the art. For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules, and tablets with systemic effects. Rectal suppositories used herein refer to solids for insertion into the rectum, which melt or soften at body temperature, releasing one or more pharmacological or therapeutically active ingredients. The pharmaceutically acceptable substances used in rectal suppositories are alkalis or vehicles and agents that increase the melting point. Examples of alkalis include a suitable mixture of monoglycerides, diglycerides, and triglycerides of the glycerols of the cocoa butter (cocoa butter), glycerol-gelatin, carbohydrates (polyoxyethylene glycol), and fatty acids. Various combinations of alkalis can be used. Agents that increase the melting point of suppositories include acetate and wax. Rectal suppositories can be prepared by compression methods or by molding. In one embodiment, the weight of the rectal suppository is about 2 to 3 gm.

[0186] Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substances and by the same methods as for formulations for oral administration.

[0187] Target preparation

[0188] The compounds provided herein or their pharmaceutically 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 well known to those skilled in the art. All such targeting methods are contemplated herein for use in the compositions of the present invention.

[0189] Use with other drugs

[0190] According to another aspect of the present invention, it is contemplated that the compounds of Formula I described herein can be administered in combination with drugs considered by those skilled in the art to be current standards of care to subjects in need thereof for conditions of interest. Such combinations provide one or more advantages to the subject, for example, a reduction in the required dose to achieve similar benefits, obtaining the desired relief in less time, etc.

[0191] Compounds according to the present invention can be used together with other drugs as part of a treatment regimen. For example, in order to treat a specific disease or condition, it may be necessary to use a combination of active compounds. Therefore, within the scope of the present invention, two or more pharmaceutical compositions can be combined in the form of a kit suitable for co-administered compositions, wherein at least one contains a compound of formula (I) according to the present invention.

[0192] In one embodiment of the methods of the invention, the compound of formula I may be administered with a second therapeutic agent. In one embodiment, the second therapeutic agent is selected from the group consisting of an anti-inflammatory agent, an anti-hypertensive agent, an anti-fibrotic agent, an anti-angiogenic agent, and an immunosuppressant.

[0193] In one embodiment of the method of the invention, the compound of Formula I can be administered with a second therapeutic agent selected from the group consisting of a steroidal anti-inflammatory agent such as prednisolone and deflazacort, or a non-steroidal anti-inflammatory agent such as aspirin, paracetamol, ibuprofen, naproxen, indomethacin, celecoxib, and diclofenac.

[0194] In another embodiment of the methods of the invention, the compound of Formula I can 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 angiotensin converting enzyme inhibitors, such as benazepril, captopril, enalapril, and fosinopril.

[0195] In another embodiment of the methods of the invention, the compound of Formula I can be administered with a second therapeutic agent selected from the group consisting of pirfenidone and nintedanib.

[0196] In another embodiment of the methods of the invention, the compound of Formula I can be administered with a second therapeutic agent selected from the group consisting of axitinib, bevacizumab, everolimus, lenalidomide, regorafenib, sorafenib, sunitinib, thalidomide, vandetanib and zivaflibercept.

[0197] In another embodiment of the method of the invention, the compound of Formula I can be administered with a second therapeutic agent selected from the group consisting of: glucocorticoids such as prednisone, dexamethasone and hydrocortisone; cytostatics such as cyclophosphamide, azathioprine, dactinomycin and methotrexate; antibodies that directly act on the immune response such as basiliximab; asiliximab, daclizumab, and muromonab; agents that act on immunophilins, such as cyclosporine, rapamycin, tacrolimus, and everolimus; and other immunosuppressive agents, including fingolimod, interferon therapy, infliximab, etanercept, and adalimumab.

[0198] In one embodiment of the method of the invention, the compound of formula I can be administered with a second therapeutic agent. In one embodiment, the second therapeutic agent is selected from drugs considered to be useful for treating muscular dystrophy, including: prednisone, deflazacort, ataluren, myostatin, eteplirsen, golodirsen, casimersen, idebenone, pamrevlumab, edasalonexent, givinostat, rimeporide, and ifetroban.

[0199] When two or more active ingredients are administered in combination, the active ingredients may be administered simultaneously, sequentially or separately. In one embodiment, the compound of formula I is administered in combination with a second therapeutic agent simultaneously. 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.

[0200] The invention will now be described in more detail by way of example only, with reference to the following non-limiting examples. The examples are intended to illustrate the invention and should not be construed as limiting the generality of the disclosure of the specification throughout the specification.

[0201] For the purposes of this specification, the following abbreviations have the designated meanings:

[0202] rt = room temperature

[0203] min = minutes

[0204] h = hours

[0205] NaHMDS = Sodium Hexamethyldisilazide

[0206] THF = Tetrahydrofuran

[0207] DMF = dimethylformamide

[0208] NaO t Bu = Sodium tert-butoxide

[0209] TFA = trifluoroacetic acid

[0210] DIBAL = diisobutylaluminum hydride

[0211] MTBE = methyl tert-butyl ether

[0212] DIPEA = diisopropylethylamine

[0213] LC-MS = Liquid Chromatography-Mass Spectrometry

[0214] rpm = revolutions per minute

[0215] NMR = Nuclear Magnetic Resonance

[0216] DMSO = dimethyl sulfoxide

[0217] Boc=tert-butyloxycarbonyl

[0218] HPLC = High Performance Liquid Chromatography (also known as High Pressure Liquid Chromatography)

[0219] rac. = racemic

[0220] TLC = Thin Layer Chromatography

[0221] Experimental: General Methods

[0222] All commercially available solvents and reagents were used as is. Where appropriate, the reaction was carried out under an argon atmosphere. The reaction was monitored using reversed phase conditions using analytical thin layer chromatography (TLC) or analytical liquid chromatography-mass spectrometry (LC-MS) recorded on a Shimadzu LC-MS 2020 instrument or an Agilent LC-MS 1200 instrument. If necessary, column chromatography or preparative HPLC was used to purify intermediates and final compounds. Normal phase column chromatography was carried out on silica gel or pre-packed silica gel cartridges using a flash chromatography system (CombiFlash Rf200, Teledyne Isco system, the United States) under medium pressure. Using a flash chromatography system ( X2) Reverse phase column chromatography was performed on a pre-packed C18 cartridge under low pressure. The eluent was monitored by ultraviolet light (λ=254 / 280 nm). 1 H-NMR spectroscopy and 19 F-NMR spectra were recorded using a Bruker 300MHz NMR spectrometer or a Bruker Avance III plus 400MHz NMR spectrometer. Chemical shifts (δ) were reported as parts per million (ppm) relative to tetramethylsilane (TMS; internal standard). The following abbreviations are used for multiplicity: s = singlet; br s = wide singlet; d = doublet; t = triplet; q = quartet; p = quintet; m = multiplet; and br m = wide multiplet. Low-resolution mass spectra (MS) were obtained as electrospray-atmospheric pressure ionization (ES-API) mass spectra on Shimadzu LC-MS 2020 or Agilent LC-MS 1200 instruments. All animal experiments were performed in accordance with institutional guidelines and approved by local ethics committees.

[0223] Example 1

[0224] Preparation of tert-butyl (E)-(2-(bromomethyl)-3-fluoroallyl)carbamate (Intermediate 6)

[0225]

[0226] Step A: 3-(2,5-dimethyl-1 H Preparation of tert-butyl pyrrol-1-yl) propionate (Intermediate 1)

[0227]

[0228] β-Alanine tert-butyl ester hydrochloride (75 kg), 2,5-hexanedione (97.5 L) and potassium carbonate (75 kg) were added sequentially to a container containing acetonitrile (754 L) under nitrogen. The resulting mixture was heated to 75 ° C within 2 hours and then stirred for another 4 hours. The reaction mixture was cooled to 55 ° C and then concentrated in vacuo to about 290 L. Water (1200 L) was then added to the residue at 55 ° C. The mixture was stirred at 55 ° C for 1 hour and then cooled to 10 ° C within 2 hours. Stirring was continued at 10 ° C for 10 hours. The slurry was then centrifuged and the liquid phase was removed. The resulting "filter cake" was rinsed with additional water (150 L × 3), centrifuged and the liquid phase was removed after each rinse cycle. After the filtrate was finally removed, the wet "filter cake" was dried under vacuum at 40 ° C for 24 hours to obtain intermediate 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).

[0229] Step B: 2-(Difluoromethyl)-2-((2,5-dimethyl-1 H -pyrrol-1-yl)methyl)malonic acid 1-(tert-butyl) Preparation of 3-ethyl ester (Intermediate 2)

[0230]

[0231] This process was carried out on two batches of intermediate 1 (39.5 kg x 2). These batches were combined at a later stage.

[0232] NaHMDS (2.0 M; 245 kg) was added to a vessel (A) containing THF (277 L) under nitrogen over 20 minutes. The resulting mixture was cooled to -58°C. Intermediate 1 (39.5 kg) was added to a vessel (B) containing THF (198 L) at -50°C under nitrogen. 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 then added to vessel (A) over 1 hour, during which an exotherm was observed. The resulting mixture was stirred at -58°C for 1 hour. NaO was added thereto over 15 minutes. tBu (15.3 kg). Then difluorochloromethane (17.8 kg) was slowly added at -20°C. The mixture was stirred at -30°C for 2 hours. Then additional difluorochloromethane (7.00 kg) was slowly charged into the container at -20°C. Stirring was continued for another 2 hours at -20°C. Water (356 L) was charged into the reaction vessel, 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 and the mixture was stirred for 1 hour. The organic layer was separated and concentrated in vacuo. The residue was dissolved in n-heptane (584.8 kg) and silica gel was added. Stirring was continued for 30 minutes and then by Filtration was performed and washed with n-heptane (434 kg). The combined filtrates were concentrated in vacuo and the residue thus obtained was recrystallized from EtOH:H2O (1:1) to give the title compound, Intermediate 2, as a light 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).

[0233] Step C: E )-2-((2,5-dimethyl-1 H -pyrrol-1-yl)methyl)-3-fluoroacrylate ethyl ester (Intermediate 3) preparation

[0234]

[0235] Intermediate 2 (91 kg) was added to a reaction vessel (A) containing TFA (279 L) at 10 ° C under nitrogen. The resulting mixture was heated to 35 ° C, stirred for 13 hours, and then cooled to 5 ° C. Triethylamine (620 L) was added to the reaction vessel (B), followed by n-heptane (532 L). The resulting solution was cooled to 5 ° C. The contents of the reactor (A) were then transferred to the reactor (B) within 2 hours. The resulting mixture in the reactor vessel (B) was heated to 40 ° C and continued to stir 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 sodium chloride (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 dissolved in toluene (520 L).After further concentration to 650 to 750 L, the resulting crude Intermediate 3 solution 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).

[0236] Step D: E )-2-((2,5-dimethyl-1 H -pyrrol-1-yl)methyl)-3-fluoroprop-2-en-1-ol (Intermediate 4) Preparation

[0237]

[0238] Diisobutylaluminum hydride (DIBAL) (1.5M in toluene; 199.2kg) was added dropwise to a solution of intermediate 3 prepared in the previous step in toluene (520L) at -55°C under nitrogen over 4 hours. The resulting mixture was stirred at -45°C for 1 hour. IPC analysis thereafter indicated incomplete conversion. Additional amounts of DIBAL (1.5M in toluene; 15kg) were added dropwise over 40 minutes at -55°C. The mixture was then stirred for another 30 minutes at -45°C and then warmed to 10°C. To quench the reaction, a cold (0°C) solution of citric acid (64kg) in water (320L) was slowly added over 2 hours. The organic phase was separated and washed with two solutions of NaCl (32kg) in water (160L). Silica gel (32kg) was added to the organic phase, and the resulting slurry was stirred for 30 minutes. 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 about 50 L. n-heptane (319 L) was added to the residue, and the resulting mixture was cooled to 0 ° C and stirred for 10 hours. The solid was then filtered and dried to give intermediate 4 (11.4 kg, 24% over two steps) as an off-white solid. 1 H-NMR (400MHz, CDCl3) δppm: 6.70 (d, J = 83.4Hz, 1H), 5.80 (s, 2H), 4.63 (t, J = 1.2Hz, 2H), 3.76 (t, J = 4.0Hz, 2H), 2.23 (s, 6H).

[0239] Step E: E Preparation of tert-butyl (3-fluoro-2-(hydroxymethyl)allyl)carbamate (Intermediate 5)

[0240]

[0241] A solution of potassium hydroxide (1.20 kg) in water (10.0 L) was added to a reaction vessel containing intermediate 4 (2.0 kg), hydroxylamine hydrochloride (3.80 kg) and water (8.00 L). The resulting mixture was heated to reflux and stirred for 18 hours. After cooling to room temperature, 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 minutes. Acetone (9.1 L) was added dropwise at 20 ° C, and the mixture was stirred for 2 hours. Finally, a solution of di-tert-butyl dicarbonate (2.37 kg) in MTBE (10 L) was added, and stirring was continued for another 18 hours at 15 ° C. The organic layer was then separated and washed with an aqueous NaOH solution (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 minutes. The mixture was then filtered and the filter "cake" was washed with MTBE (10 L). The filtrate was concentrated in vacuo to afford Intermediate 5 (2.0 kg, 89%). 1 H-NMR (400MHz, CDCl3) δppm: 6.78 (d, J = 81.2Hz, 1H), 4.86 (br.s, 1H), 4.00–3.96 (m, 4H), 1.45 (s, 9H).

[0242] Step F: E Preparation of tert-butyl (2-(bromomethyl)-3-fluoroallyl)carbamate (Intermediate 6)

[0243]

[0244] A solution of methanesulfonic anhydride (5.78kg) in THF (20.0L) was added to a container containing intermediate 5 (4.0kg), DIPEA (10.2L) and THF (40.0L) at 5°C. The resulting mixture was stirred for 30 minutes. Lithium bromide (8.47kg) was added thereto and stirring was continued for 2 hours at 5°C. Water (20.0L) was then added dropwise to the reaction mixture. The organic layer was separated and concentrated to about 8.0L. Ethyl acetate (24L) and water (20L) were added thereto. The organic layer was separated again and concentrated to about 12L. Heptane (40L) and silica gel (2.0kg) 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 (20L). The filtrate was concentrated in vacuo to dryness. Additional n-heptane was added and stirring was continued for 1 hour at 5°C. The resulting solid was collected by filtration and dried in vacuo at 30°C to afford Intermediate 6 (3.20 kg, 61%). 1H-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).

[0245] Example 2

[0246] Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)benzonitrile hydrochloride (Compound 1)

[0247]

[0248] Step G-1: E Preparation of tert-butyl )-(2-((4-cyanophenoxy)methyl)-3-fluoroallyl)carbamate

[0249]

[0250] Potassium carbonate (2.78 g) and intermediate 6 (2.70 g) were added sequentially to a stirred solution of 4-hydroxybenzonitrile (1.20 g) in acetonitrile (25.0 mL) at room temperature. The resulting suspension was stirred at room temperature overnight. The reaction mixture was stirred by Filtration was performed to remove inorganics and the filtrate was concentrated in vacuo to give a white solid. The solid was then crystallized from ether / pentane to give tert-butyl (E)-(2-((4-cyanophenoxy)methyl)-3-fluoroallyl)carbamate (2.81 g, 91%) as a white solid.

[0251] Step G-2: E Alternative preparation of tert-butyl (2-((4-cyanophenoxy)methyl)-3-fluoroallyl)carbamate Preparation

[0252] Potassium carbonate (4.05kg) and intermediate 6 (5.1kg) are added successively to a stirred solution of 4-hydroxybenzonitrile (2.7kg) in DMSO (10.15L) at room temperature. The resulting suspension is stirred for 4 hours. Water (50.75L) is added and the mixture is stirred for 1 hour. The product is filtered and washed with water (10L). The crude product is dissolved in DMSO (15L). Water (60L) is slowly added and the mixture is stirred for 1 hour. The product is filtered, washed with water (10L) and dried at 55°C. (E)-tert-butyl (2-((4-cyanophenoxy)methyl)-3-fluoroallyl)carbamate (5.1kg, 89%) is obtained as a white solid.

[0253] 1H-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] + .

[0254] Step H-1: E )-4-((2-(aminomethyl)-3-fluoroallyl)oxy)benzonitrile hydrochloride (Compound 1) preparation

[0255]

[0256] TFA (40.0 mL) was added to a stirred solution of (E)-(2-((4-cyanophenoxy)methyl)-3-fluoroallyl)carbamate (2.75 g) in CH2Cl2 (40.0 mL) at room temperature. The resulting solution was stirred at room temperature for 2 hours. LC-MS analysis thereafter indicated complete consumption of the starting material. The reaction mixture was concentrated in vacuo, and the residual TFA was removed by coevaporation with ethyl acetate (20 mL×2) to give an off-white solid. The solid was dissolved in ethyl acetate (10.0 mL) and HCl (2.0 M in ether; 40.0 mL) was added to immediately precipitate a white solid. After stirring for 15 minutes, the solid was collected by filtration and dried under high vacuum to give a 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.

[0257] Step H-2: E )-4-((2-(aminomethyl)-3-fluoroallyl)oxy)benzonitrile hydrochloride (Compound 1) Alternative preparation

[0258] Ethyl acetate (25.5 L) containing 6 M HCl was added 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 room temperature. The resulting solution was stirred at room temperature for 2 hours. LC-MS analysis thereafter indicated 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 hours. The product was filtered and 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 to 5 ° C and the solid was filtered. The product was dried in vacuo at 45 ° C to give compound 1 (3.3 kg, 76%) as a white solid with approximately 7% moisture.

[0259] 1 H 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] + .

[0260] Example 3

[0261] Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-2-fluorobenzonitrile hydrochloride (Compound 2)

[0262]

[0263] Step I: E Preparation of tert-butyl )-(2-((4-cyano-3-fluorophenoxy)methyl)-3-fluoroallyl)carbamate Preparation

[0264]

[0265] Cesium carbonate (1.17g) is added to a stirred solution of 2-fluoro-4-hydroxybenzonitrile (411mg) and intermediate 6 (804mg) in DMF (2.0mL). The resulting mixture is stirred for 4 hours, after which LC-MS analysis indicates that the starting material is completely consumed. Water (20mL) is then added, and the resulting suspension is ultrasonically treated and stirred for 10 minutes. The solid is collected by filtration, washed with water, and then dried at 60°C for 1 hour to obtain (E)-(2-((4-cyano-3-fluorophenoxy)methyl)-3-fluoroallyl)t-butyl carbamate (888mg, 91%), which is 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] + .

[0266] Step J: E )-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-2-fluorobenzonitrile hydrochloride (Compound 2) Preparation

[0267]

[0268] TFA (0.50 mL) was added to a stirred solution of (E)-(2-((4-cyano-3-fluorophenoxy)methyl)-3-fluoroallyl)carbamate (50.0 mg) in CH2Cl2 (3.0 mL). The resulting mixture was stirred for 2 hours, after which LC-MS analysis indicated that the starting material was completely consumed. The reaction mixture was concentrated to dryness, and the remaining TFA was removed under high vacuum. After the residue was dissolved in ethyl acetate (3.0 mL), HCl (2.0 M in diethyl ether; 77 μL) was added. The mixture was stirred at room temperature for 30 minutes, during which time solids precipitated. The reaction mixture was transferred to a vial and the solid was centrifuged in a centrifuge (4000 rpm, 4.0 minutes). The supernatant was decanted, and the solid was washed by adding ethyl acetate. After a short ultrasonic treatment, the vial was returned to the centrifuge, and the solid was centrifuged. The supernatant was removed and the solid "cake" was 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] + .

[0269] Example 4

[0270] Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-2-bromobenzonitrile hydrochloride (Compound 3)

[0271]

[0272] Step K: E Preparation of tert-butyl )-(2-((3-bromo-4-cyanophenoxy)methyl)-3-fluoroallyl)carbamate Preparation

[0273]

[0274] NaH (60% in paraffin oil; 120 mg) was added to a stirred solution of intermediate 5 (573 mg) in DMF (5.0 mL) at 0°C and stirred for 30 minutes. A solution of 2-bromo-4-fluorobenzonitrile (500 mg) in DMF (5.0 mL) was then added dropwise at 0°C. Stirring was continued at room temperature for 2 hours. The reaction mixture was cooled to 0°C and quenched with saturated NH4Cl aqueous solution. The product was extracted with ethyl acetate (20 mL×3), and the combined organic layers were dried over Na2SO4 and then 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] + .

[0275] Step L: E )-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-2-bromobenzonitrile hydrochloride (Compound 3) Preparation

[0276]

[0277] HCl (4.0 M in 1,4-dioxane; 2.0 mL) was added to a stirred solution of (E)-tert-butyl(2-((3-bromo-4-cyanophenoxy)methyl)-3-fluoroallyl)carbamate (100 mg) in 1,4-dioxane (2.0 mL) at 0°C. The resulting mixture was stirred at room temperature for 3 hours. 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. 1 H-NMR (methanol-d4, 400MHz) δppm: 7.76 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 2.4 Hz, 1H), 7.38-7.16 (m, 2H), 4.72 (d, J = 2.8 Hz, 2H), 3.84 (d, J = 2.0 Hz, 2H); MS MS: 285.0 [M+H] + .

[0278] Example 5

[0279] Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-3-fluorobenzonitrile hydrochloride (Compound 4)

[0280]

[0281] Step M: E Preparation of tert-butyl )-(2-((4-cyano-2-fluorophenoxy)methyl)-3-fluoroallyl)carbamate Preparation

[0282]

[0283] Potassium carbonate (206 mg) and intermediate 6 (200 mg) were added sequentially to a stirred solution of 3-fluoro-4-hydroxybenzonitrile (112 mg) in acetonitrile (4.0 mL) at room temperature. The reaction mixture was stirred by The inorganics were removed by plug filtration and the filtrate was concentrated in vacuo to afford 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] + .

[0284] Step N: Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-3-fluorobenzonitrile hydrochloride (Compound 4)

[0285]

[0286] TFA (10.0 mL) was added to a stirred solution of (E)-(2-((4-cyano-2-fluorophenoxy)methyl)-3-fluoroallyl)carbamic acid tert-butyl ester (230 mg) in CH2Cl2 (10.0 mL) at room temperature. The resulting solution was stirred at room temperature for 2 hours. LC-MS analysis thereafter indicated complete consumption of the starting material. The reaction mixture was concentrated in vacuo, and the remaining TFA was removed by coevaporation with ethyl acetate (20 mL×2) to give an off-white solid. The solid was dissolved in ethyl acetate (10.0 mL) and HCl (2.0 M in ether; 5.00 mL) was added to immediately precipitate a white solid. After stirring for 15 minutes, the contents of the reaction vessel were transferred to a vial. The solid was then centrifuged in a centrifuge (4000 rpm, 2 minutes, 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%). 1H-NMR (methanol-d4, 300MHz) δppm: 7.65–7.56 (m, 2H), 7.36 (t, J = 8.6 Hz, 1H), 7.30 (dt, 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] + .

[0287] Example 6

[0288] Preparation of (E)-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-2-methylbenzonitrile hydrochloride (Compound 5)

[0289]

[0290] Step O: Preparation of tert-butyl (E)-(2-((4-cyano-3-methylphenoxy)methyl)-3-fluoroallyl)carbamate

[0291]

[0292] A mixture of 4-hydroxy-2-methylbenzonitrile (109mg), intermediate 6 (200mg) and potassium carbonate (206mg) in acetonitrile (4.0mL) was stirred at room temperature overnight. LCMS analysis showed that the conversion was complete. The reaction mixture was diluted with CH2Cl2 (20mL), filtered, and then concentrated in vacuo to give (E)-(2-((4-cyano-3-methylphenoxy)methyl)-3-fluoroallyl)carbamic acid tert-butyl ester (240mg, 100%) as a pale solid. The material can be carried out 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] + .

[0293] Step P: E )-4-((2-(aminomethyl)-3-fluoroallyl)oxy)-2-methylbenzonitrile hydrochloride (compound 5) Preparation

[0294]

[0295] TFA (10.0 mL) was added to a stirred solution of (E)-(2-((4-cyano-3-methylphenoxy)methyl)-3-fluoroallyl)carbamic acid tert-butyl ester (240 mg) in CH2Cl2 (10 mL) at room temperature. The resulting solution was stirred at room temperature for 2 hours. LC-MS analysis thereafter indicated complete consumption of the starting material. The reaction mixture was concentrated in vacuo, and the residual TFA was removed by coevaporation with CH2Cl2 (20 mL×2) to give a light yellow oil. The solid was dissolved in ethyl acetate (10.0 mL) and HCl (2.0 M in ether; 1.00 mL) was added to immediately precipitate a white solid. After stirring for 15 minutes, 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 = 8.6 Hz, 1H), 7.27 (dt, J = 81.0, 0.9 Hz, 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] + .

[0296] Comparative Example 7

[0297] Preparation of (-)-(E)-2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroprop-2-en-1-amine hydrochloride (PXS-5131)

[0298]

[0299] Step Q: Preparation of 2,3-dihydrobenzofuran-2-carbonyl chloride

[0300]

[0301] Oxalyl chloride (125 mL) was added dropwise to a stirred solution of 2,3-dihydrobenzofuran-2-carboxylic acid (78.0 g) and DMF (1.0 mL) in CH2Cl2 (780 mL) at 0°C. 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. The material was not characterized and used immediately in the following steps.

[0302] Step R: Preparation of 2-diazo-1-(2,3-dihydrobenzofuran-2-yl)ethanone

[0303]

[0304] A solution of crude 2,3-dihydrobenzofuran-2-carbonyl chloride in THF / CH3CN (1:1; 780 mL) was added dropwise under N2 to a cold (0°C) stirred solution of (trimethylsilyl)diazomethane (2M in ether; 546 mL) in THF / CH3CN (1:1; 780 mL). The resulting mixture was stirred at 0°C for 1 hour. The reaction mixture was then evaporated in vacuo to give 2-diazo-1-(2,3-dihydrobenzofuran-2-yl)ethanone (92.6 g) as a crude brown oil. The 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).

[0305] Step S: Preparation of 2-bromo-1-(2,3-dihydrobenzofuran-2-yl)ethanone

[0306]

[0307] HBr / AcOH (48%, 216mL) was added dropwise to a stirred solution of crude 2-diazo-1-(2,3-dihydrobenzofuran-2-yl)ethanone (92.6g) in CH2Cl2 (926mL) at 0°C. The resulting mixture was stirred at this temperature for 15 minutes. The reaction mixture was diluted with water (800mL), and the aqueous layer was extracted with CH2Cl2 (300mL). The combined organics were washed with saturated NaHCO3 aqueous solution (600mL×3) and then with brine (800mL). After drying over Na2SO4, the solvent was removed in vacuo to give 2-bromo-1-(2,3-dihydrobenzofuran-2-yl)ethanone (98.0g) as a crude brown oil. The material was immediately subjected to the subsequent steps 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).

[0308] Step T: Preparation of 2-azido-1-(2,3-dihydrobenzofuran-2-yl)ethanone

[0309]

[0310] NaN3 (132g) was added to a stirred solution of crude 2-bromo-1-(2,3-dihydrobenzofuran-2-yl)ethanone (98.0g) in acetone (926mL) at 0°C. The resulting mixture was stirred at this temperature for 3 hours. The reaction mixture was partitioned between water (1L) and ethyl acetate (800mL), and the aqueous layer was extracted with additional ethyl acetate (800mL×2). The combined organics were washed with brine (1L), dried over Na2SO4, and then concentrated in vacuo to give 2-azido-1-(2,3-dihydrobenzofuran-2-yl)ethanone (92g) as a crude brown oil. The material was carried to the subsequent steps without purification. 1 H-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).

[0311] Step U: Preparation of tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-2-oxoethylcarbamate

[0312]

[0313] Di-tert-butyl dicarbonate (135g) and Pd / C (10%; 9.10g, 50% wet) are added to a solution of crude azido-1-(2,3-dihydrobenzofuran-2-yl)ethanone (91.0g) in ethyl acetate (910mL). The resulting suspension is stirred overnight at room temperature under an atmosphere of H2. The catalyst is removed by filtration, and the filtrate is evaporated in vacuo. The crude material is purified on silica gel and eluted with petroleum ether / ethyl acetate (20: 1) to give tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-2-oxoethylcarbamate (39.0g, 33%) as a yellow solid. 1H-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).

[0314] Step V: E )-tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate and ( Z )-Uncle Preparation of tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate

[0315]

[0316] NaHMDS (1.0M in THF; 246mL) was slowly added to a stirred solution of fluoromethyl (triphenylphosphine) tetrafluoroborate (75.6g) in THF (910mL) at -25°C under N2. The mixture was stirred at -25°C for 30 minutes and then cooled to -78°C. A solution of tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-2-oxoethylcarbamate (45.6g) in THF (364mL) was then added to the reaction mixture. After all were added, stirring was continued at -78°C for 1 hour. The reaction mixture was partitioned between water (1.2L) and ethyl acetate (1.2L), and the aqueous layer was extracted with additional ethyl acetate (800mL×2). The combined organics were washed with brine (1.5L), dried over Na2SO4, and then concentrated in vacuo. Initial purification of the crude material was performed on silica gel eluting with petroleum ether / ethyl acetate (30:1 to 10:1) to afford a mixture of racemic (E)-tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate and racemic (Z)-tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate (1:1; 9.70 g). The mixture was then subjected to preparative HPLC purification to afford tert-butyl (E)-2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate (4.10 g, 9%) and tert-butyl (Z)-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(br .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).

[0317] Step W: Racemization (E )-tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate Separation of two enantiomers

[0318]

[0319] 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 eluted with n-hexane / isopropanol / diethylamine (95:5:0.1; v / v / v) at an isocratic flow of 1 mL / min to give each enantiomer of (E)-tert-butyl 2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate (Peak 1 (6.10 minutes), 2.30 g; Peak 2 (9.40 minutes), 2.32 g).

[0320] Step X: (-)-(E )-2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroprop-2-en-1-amine hydrochloride (PXS- Preparation of 5131)

[0321]

[0322] HCl (2.0 M in ether; 1.0 mL) was added at room temperature to a stirred solution of the enantiomer represented by peak 2 containing purified (E)-2-(2,3-dihydrobenzofuran-2-yl)-3-fluoroallylcarbamate (30 mg) in methanol (1.0 mL). The resulting mixture was stirred at room temperature for 2 hours. All volatiles were removed in vacuo and the resulting residue was dissolved in ethyl acetate (0.5 mL). After addition of ether (2.0 mL), a white precipitate was obtained. The contents were transferred to a vial and centrifuged in a centrifuge (4000 rpm, 4 minutes) to give a solid "filter cake". After decanting the supernatant, more ether was added and (after a brief sonication) the vial was returned to the centrifuge. This centrifugation / washing process 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 methanol solution; 1 H-NMR (300MHz; methanol-d4) δppm: 3.19 (dd, J = 15.9, 9.1 Hz, 1H), 3.41 (dd, J 15.9,9.2Hz,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: Deprotection of peak 1 in step W yields [α] of similar magnitude but opposite sign D Compounds]

[0323] Comparative Example 8

[0324] The following compounds listed in Table 2 are for comparison purposes only. Except for Compound 6 and Compound 14, these compounds can be synthesized using methods similar to those described herein.

[0325] These compounds can be prepared from commercially available 4-hydroxybenzonitrile according to the procedure described in Foot JS et al., J. Pharm. Exp. Ther., 2013, 347, 365-374.

[0326] Table 2.

[0327]

[0328]

[0329] Example 9

[0330] Methods for determining the ability of compounds of formula I to inhibit human recombinant SSAO / VAP-1

[0331] The human recombinant SSAO / VAP-1 amine oxidase activity was determined using the method described for monoamine oxidase, copper-containing amine oxidase and related enzymes (Holt A. and Palcic M., "Nature-Laboratory Manual (Nat. Protoc.)" 2006, 1, 2498-2505). Briefly, a cloned cDNA template corresponding to residues 34-763 of human SSAO / VAP-1 and combined with a mouse Ig kappa (κ) signal sequence, an N-terminal flag epitope tag and a tobacco etch virus (TEV) cleavage site was assembled by Geneart AG in a mammalian expression vector (pLO-CMV). This vector containing human SSAO / VAP-1 residues was transfected into the CHO-K1 glycosylation mutant cell line Lec 8. A clone stably expressing human SSAO / VAP-1 was isolated and cultured on a large scale. Active human SSAO / VAP-1 was purified and recovered using immunoaffinity chromatography. This was used as a source of SSAO / VAP-1 activity. A high-throughput colorimetric assay was developed using a 96- or 384-well format. Briefly, 50 μL of 0.1 M sodium phosphate buffer (pH 7.4) containing purified human SSAO / VAP-1 (0.25 μg / mL) was added to each well in a standard 96-well plate assay. The test compound was dissolved in DMSO and tested in a concentration response curve (CRC) with 4-11 data points, usually in the micromolar or nanomolar range, after incubation with human SSAO / VAP-1 for 30 minutes at 37°C. After incubation for 30 minutes, 50 μL of a reaction mixture containing 600 μM benzylamine (Sigma Aldrich), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U / mL horseradish peroxidase (Sigma Aldrich) prepared in 0.1 M sodium phosphate buffer (pH 7.4) 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 for each well was calculated using MARS data analysis software (BMG Labtech), and this value was used to derive the IC 50 The ability of the compounds of Formula I to inhibit SSAO / VAP-1 is shown in Table 3.

[0332] Example 10

[0333] Methods for determining the ability of compounds of formula I to inhibit human recombinant MAO-B

[0334] The ability of the compounds of the 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 performed in a similar manner to human SSAO / VAP-1 (Example 9), except that 100 μM of the substrate benzylamine was used and the assay was performed after an incubation time of 2 hours. The ability of the compounds of Formula I to inhibit MAO-B is shown in Table 3.

[0335] Embodiment 11

[0336] Methods for determining the ability of compounds of formula I to inhibit human recombinant MAO-A

[0337] The selectivity of the compounds of the present invention was tested by measuring their ability to inhibit MAO-A activity in vitro using recombinant human MAO-A (0.003 mg / mL; Sigma Aldrich). The assay was performed in a similar manner to human SSAO / VAP-1 (Example 9), except that 100 μM of the substrate tyramine was used 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.

[0338]

[0339] Table 3*

[0340]

[0341]

[0342] A1=<10nM A2=<50nM A3=>30000nM B1=10–500nM B2=50–500nM B3=>3000nM-30000nM C1=>500–1000nM C2=>500–1000nM C3=300–3000nM D1=>1000nM D2=>1000nM D3=<300nM

[0343] *For the purposes of the present invention, the IC values ​​of potent inhibitors of SSAO / VAP-1 under the assay conditions described in Examples 9 and 10 are 50 Less than 10 nM (A1), while the IC of the potent inhibitor of MAO-B 50 Less than 50 nM (A2). To be considered selective relative to MAO-A, under the assay conditions described in Example 11, IC 50 Must be greater than 3000nM (A3 and B3).

[0344] As shown in Table 3, under the described assay conditions, the compounds of the invention are potent inhibitors of human recombinant SSAO / VAP-1 and MAO-B in vitro. Importantly, the compounds of the invention have no activity against recombinant human MAO-A, which is an important off-target effect. Surprisingly, this feature is highly dependent on the nature and position of the substituents on the phenyl ring.

[0345] Example 12

[0346] Pharmacokinetic parameters of compound 1 and PXS-5131

[0347] Pharmacokinetic (PK) studies were conducted at Sundia MediTech Co. Ltd., Shanghai. Compound 1 or PXS-5131 was administered orally to Wistar rats weighing approximately 280 g at 3 mg / kg. Blood samples were collected by tail vein sampling at different times between 15 minutes and 8 hours. Plasma was analyzed by high performance liquid chromatography-tandem mass spectrometry using Shimadzu LC30AD HPLC and QTRAP 5500 mass spectrometer. The time course and basic PK parameters thus obtained are summarized in Figure 1 middle.

[0348] Embodiment 13

[0349] Method for determining the ability of compounds of Formula I to inhibit rat adipose tissue SSAO / VAP-1 and rat brain tissue MAO-B Law

[0350] Sprague Dawley rats were orally administered the compounds under investigation and sacrificed at various time points, and gonadal fat and brain tissues were collected.

[0351] 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 then centrifuged at 2000 rpm for 5 minutes at 4°C, and the resulting supernatant fraction (minus the lipid top layer) was used for the assay. SSAO / VAP-1 activity was determined by using a nonspecific MAO inhibitor (e.g., pargyline) to determine a high signal and a mixture of a nonspecific MAO inhibitor and a SSAO / VAP-1 inhibitor (e.g., semicarbazide) to determine a low signal.

[0352] Brain tissue is homogenized in ice-cold Tris buffer (10mM Tris.HCl, 0.5mM EDTA, sucrose 250mM, pH 7.4). The homogenate is then centrifuged at 4°C for 3 minutes at 4,000rpm. The resulting supernatant is transferred and centrifuged again at 4°C for 3 minutes at 4,000rpm. The process is repeated for the last time, this time at 10,000rpm for 8 minutes. The supernatant is discarded, and the resulting precipitate containing the mitochondrial extract is resuspended in 0.1M sodium phosphate buffer solution (1:3 ratio). MAO-B activity is determined by using specific MAO-A inhibitors (e.g., clorgyline) and SSAO / VAP-1 inhibitors (e.g., semicarbazide) to determine high signals and assay. Low signals are determined for adipose tissue assays.

[0353] The activity of the treated samples was analyzed using the fluorescence method described in Example 9 for SSAO / VAP-1 activity and in Example 10 for MAO-B.

[0354] Embodiment 14

[0355] Description of the dual inhibitor PXS-5131

[0356] PXS-5131 is a prototype dual inhibitor of both MAO-B and SSAO / VAP-1. The compound has a pharmacokinetic profile inferior to that of compound 1. Oral administration of a single dose of compound PXS-5131 to Sprague Dawley rats in vivo results in long-term inhibition of SSAO / VAP-1 and MAO-B, as Figure 2 and Figure 3 As shown. Figure 2 As shown, a single dose of PXS-5131 inhibited SSAO / VAP-1 enzyme activity for more than 24 hours. Figure 3 As shown, a single dose of PXS-5131 inhibited MAO-B enzyme activity for more than 24 hours. PXS-5131 was used to evaluate these combination effects in the following in vivo models.

[0357] Embodiment 15

[0358] Efficacy of the dual inhibitor PXS-5131 in the carrageenan air pouch model

[0359] On day 0, the nape of BALB / c mice was shaved, anesthetized, and 6 mL of sterile air (0.2 μm, Sartorius, catalog number 16532) was injected subcutaneously using a 23G×11 / 2 inch needle fixed to a 6 mL syringe. The injection site was sealed with flexible collodion (Macron, catalog number 4580-02). On day 3, an additional 3 mL of sterile air was injected into the neck.

[0360] 3% lambda carrageenan was prepared by dissolving 1.8 g lambda carrageenan (FLUKA, Cat. No. 22049, Batch No. 1408463) in 60 mL hot deionized water. Once dissolved, the solution was cooled to room temperature and the volume was made up to 60 mL with deionized water.

[0361] 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% lambda carrageenan directly into the air pouch using a 23G x 2 inch needle mounted on a 1 mL syringe. Sham-operated animals were administered 1 mL of deionized water instead of lambda carrageenan solution.

[0362] Four hours after the 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 were immediately removed using an 18G × 1-inch needle mounted on a 5-mL syringe, and the exudate volume was recorded. An aliquot of the exudate was taken for a total leukocyte count; a second aliquot of the exudate was taken for a leukocyte differential count.

[0363] The anti-inflammatory effect of PXS-5131 was shown in Figure 4 Injection of carrageenan caused an increase in inflammation, which was reduced by a single administration of PXS-5131, as seen by a reduction in both exudate volume and neutrophil counts in the air pouches. *p<0.05, **p<0.01, ***p<0.001 compared to carrageenan treated mice (black bars).

[0364] Compound 1 was also outlined in this model.

[0365] Example 16

[0366] Efficacy of the dual inhibitor PXS-5131 in a neuroinflammation model induced by lipopolysaccharide (LPS) in the substantia nigra

[0367] Hooded Wistar male rats weighing between 300 and 400 g were obtained from Adelaide Laboratory Animal Services.On day 0, animals received 2 mg / kg of PXS-5131 or sterile saline (vehicle) via intraperitoneal injection (ip) 1 hour before and 5 hours after LPS infusion.

[0368] Dopaminergic neuron counts after unilateral LPS infusion

[0369] Unilateral Infusion: Rats receiving unilateral injection of 2 μL of 1.5 mg / mL LPS (O55:B5, Sigma) solution into the substantia nigra were anesthetized with isoflurane and placed in a stereotaxic frame. For all rats, a hole was drilled in the skull over the appropriate target structure according to the following coordinates: -5.3 mm posterior to bregma; ±2.0 mm lateral to bregma; -8.0 mm ventral to bregma. All rats received a unilateral injection of LPS, with the other cerebral hemisphere left intact. Animals received daily intraperitoneal injections of PXS-5131 (2 mg / kg) or vehicle for two weeks.

[0370] Immunohistochemistry procedures: Two weeks after injury, rats were transcardially perfused with PBS followed by PFA 4%. The brains were postfixed in PFA 4%, and 30 μm coronal sections containing the substantia nigra were collected and washed with 3× 0.01 M phosphate-buffered saline (PBS), pH 7.4 for 10 min. Dopaminergic neurons were stained with anti-tyrosine hydroxylase (TH) antibody 1:10,000 and visualized using nickel-enhanced 3,3'-diaminobenzidine (DAB) and counted manually. The values ​​for the sections were averaged for each animal and are shown in Figure 5 middle.

[0371] like Figure 5 As shown, unilateral injection of LPS into the substantia nigra induced a significant loss of dopaminergic neurons compared to the intact control hemisphere. PXS-5131 administered intraperitoneally daily at 2 mg / kg for 2 weeks provided significant protection against loss compared to the intact control hemisphere, t-test *p<0.05.

[0372] Embodiment 17

[0373] Efficacy of the dual inhibitor PXS-5131 in a mouse model of Duchenne muscular dystrophy

[0374] mdx mice (C57BL / 10ScSn-Dmdmdx / J) were obtained from Jackson Laboratories, Maine, US. Animals aged 3 months received daily doses of PXS-5131 (6 mg / kg) administered in food for 30 days. Untreated, age-matched animals were used as controls. The drug was administered using small, palatable gelatin pellets made from 10% gelatin and 20% sucralose plus food flavoring (Zhang L. Protoc. Exch. 2011, 236). Animals were preconditioned to recognize and routinely consume gelatin for three days, a three-day routine that included fasting overnight, followed by exposure of each animal to gelatin without active ingredients in the absence of other food. Palatability was further improved by soaking the pellets in corn oil containing trace amounts of almond oil and then sprinkling bread crumbs with crushed food pellets.

[0375] The gastrocnemius muscle was evaluated for in vivo muscle function as described in Blaauw B. et al., Hum. Mol. Genet. 2008, 17, 3686-3696. The mice were anesthetized and electrodes were placed on either side of the sciatic nerve while the common peroneal nerve was cut. Muscle torque generation was measured using a lever system (Aurora Scientific 305B). All groups used a 2.1 mm lever arm because no significant differences in body weight were observed between the groups. Eccentric contractions were performed by moving the foot backward at a speed of 40 mm / s while the gastrocnemius muscle was stimulated at a frequency (100 Hz) sufficient to induce complete tetanic fusion. The contraction was repeated every 20 seconds to avoid inducing fatigue (see Figure 6 ).

[0376] Figure 6 PXS-5131 orally administered at 6 mg / kg / day helps prevent force loss caused by eccentric contraction in the gastrocnemius (GC) muscle compared to untreated age-matched mice after 4 weeks of dosing in 3-month-old mice. *p<0.05.

[0377] After the above force measurements, animals were sacrificed by cervical dislocation and skeletal muscle and heart (minus atria) were collected, snap frozen in isopentane precooled with liquid nitrogen and stored at -80°C. 12 μm sections from limb muscle (tibialis anterior) were prepared with a cryostat; multiple serial slides were prepared, containing sections at least three different levels. Heart cross sections were prepared using the same procedure.

[0378] The amount of neutrophil and macrophage infiltration was determined in serial sections of skeletal muscle prepared as described above, fixed, and then stained via immunofluorescence with anti-Ly6G and anti-F4 / 80 antibodies (AbCam, ab25377, and ab6640 for neutrophils and macrophages, respectively). The amount of infiltration was assessed as the percentage of fluorophore-positive pixels to the total area of ​​the section using Image-J software ( Figure 7 A and Figure 7 B).

[0379] The amount of reactive oxygen species (ROS) was assessed in freshly cut tissue sections using dihydroethidium (DHE, Sigma) as a fluorescent probe as described in Menazza et al., Human Molecular Genetics 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. Metafluor software (Leica) was then used to measure the amount of red fluorescence from at least 20 random fields per section, and the data for each field were averaged ( Figure 7 C).

[0380] The percentage of fibrotic tissue in skeletal muscle was determined by Sirius red staining in skeletal muscle. The sections were treated with xylene for 10 minutes, 100% ethanol for 2 minutes, 90% ethanol for 2 minutes, 70% ethanol for 2 minutes, distilled water for 2 minutes, and stained with a saturated picric acid aqueous solution containing 0.02% Sirius red (Sigma, 365548) for 1 minute, followed by rapid immersion in acetic acid wash solution (200 mL of water containing 30 mL of glacial acetic acid) twice. The sections were dehydrated at increasing alcohol concentrations (opposite to the above) and treated in xylene for 10 minutes, followed by sealing with Eukitt (Sigma, catalog number 03989). Full images of at least three sections of each muscle sample were then acquired using a bright field microscope and analyzed using Image-J software to measure the percentage of fibrotic (red) area relative to the total surface ( Figure 7 D).

[0381] like Figure 7 As shown in (A to D), oral administration of PXS-5131 at 6 mg / kg / day significantly reduced (A) neutrophil influx, (B) macrophage influx, (C) reactive oxygen species, and (D) tibialis anterior muscle fibrosis (as quantified by picrosirius red staining) in 3-month-old mdx mice after 4 weeks of dosing compared to untreated age-matched mice. ****p<0.0001 compared to untreated mice.

[0382] Example 18

[0383] The compound of formula I was tested in C57BL / 6J mice and mdx Inhibition of lipid metabolism in mice (C57BL / 10ScSn-Dmdmdx / J) Methods for detecting the ability of adipose tissue SSAO / VAP-1, muscle and cardiac tissue MAO-B, and muscle and cardiac tissue MAO-A

[0384] Mice were orally administered the compounds under study and sacrificed at various time points, and abdominal fat, muscle, and heart tissues were collected.

[0385] SSAO / VAP-1 activity in adipose tissue was determined as described in Example 13.

[0386] Skeletal muscle was pulverized in liquid nitrogen and then suspended in ice-cold HEPES buffer medium (20 mM HEPES, 0.8% w / v free fatty acid bovine serum albumin (BSA), 75 mM sucrose, 10 mM EDTA, 215 mM D mannitol, pH 7.4). Homogenized and then centrifuged at 2,500 rpm for 10 minutes at 4°C. The supernatant was transferred and centrifuged at 10,000 rpm for 8 minutes. The supernatant was discarded and the resulting pellet containing the mitochondrial extract was resuspended in 0.1 M sodium phosphate buffer solution (1:3 ratio).

[0387] Myocardium was washed three times with PBS solution on ice, and then homogenates and extracts were prepared as described for skeletal muscle.

[0388] MAO-B activity is determined by determining a high signal using a specific MAO-A inhibitor (eg, clorgyline) and a SSAO / VAP-1 inhibitor (eg, semicarbazide). A low signal is determined on adipose tissue (Example 13).

[0389] MAO-A activity was determined by using the selective MAO-B inhibitor mfgiline (30 nM) to determine the high signal and a mixture of the specific MAO-A inhibitor (clorgyline) and mfgiline (30 nM) to determine the low signal.

[0390] The activity of the treated samples was analyzed using the fluorescence method described in Example 9 for SSAO / VAP-1 activity, Example 10 for MAO-B activity, and Example 11 for MAO-A activity ( Figure 8 and Fig. 9 ).

[0391] like Figure 8 As shown, a single dose of Compound 1 provided a dose-related reduction in SSAO / VAP-1 and MAO-B activity 4 hours after administration in C57BL / 6J mice. Inhibition of enzyme activity was still evident at the 24 hour time point. *p<0.05, **p<0.01 and ***p<0.001 compared to vehicle-treated mice.

[0392] like Fig. 9 As shown, a single dose of Compound 1 had no inhibitory effect on cardiac and muscle MAO-A activity at 4 and 24 hours after administration in C57BL / 6J mice.

[0393] 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 over 3 days.

[0394] like Fig.10As shown, a single dose of Compound 1 completely inhibited 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.

[0395] like Fig.11 As shown, a single dose of compound 1 had no inhibitory effect on muscle MAO-A after administration in C57BL / 10ScSn-Dmdmdx / J mice.

[0396] Example 19

[0397] Efficacy of dual inhibitor compound 1 in a mouse model of Duchenne muscular dystrophy

[0398] Three-month-old mdx mice (C57BL / 10ScSn-Dmdmdx / J) received daily doses of Compound 1 (in the range of 1 to 6 mg / kg) administered in drinking water for 30 days. Untreated, age-matched animals were used as controls. A second group of 10-month-old mice received daily doses of Compound 1 (in the range of 1 to 6 mg / kg) administered in drinking water for 90 days. Untreated, age-matched animals were used as controls.

[0399] At the end of each treatment period, animals were sacrificed by cervical dislocation, and skeletal muscle and heart (minus atria) were collected, snap-frozen in isopentane precooled with liquid nitrogen and stored at -80°C. 12 μm sections from the diaphragm were prepared using a cryostat; multiple serial slides were prepared, containing sections at least three different levels. Heart cross-sections were prepared using the same procedure.

[0400] The amount of neutrophil and macrophage infiltration, the presence of reactive oxygen species (ROS), and the percentage of fibrotic tissue were determined as described in Example 17.

[0401] like Fig.12 As shown in (A to D), oral administration of Compound 1 at 4.8 mg / kg / day significantly reduced (A) neutrophil influx, (B) macrophage influx, (C) reactive oxygen species, and (D) diaphragm fibrosis (as quantified by picrosirius red staining) in 3-month-old mdx mice after 30 days of dosing compared to untreated age-matched mice. ****p<0.0001 compared to untreated mice.

[0402] like Fig.13As shown, oral administration of Compound 1 at 4.8 mg / kg / day significantly reduced cardiac fibrosis (as quantified by picrosirius red staining of the myocardium) in 10 month old mdx mice after 90 days of dosing compared to untreated age-matched mice. ****p<0.0001 compared to untreated mice.

[0403] Example 20

[0404] Standardized commercially available methods (available through contract research organizations such as Eurofins) were used. (Eurofins Panlabs Inc.) (https: / / www.eurofinsdiscoveryservices.com / services / in-vitro-assays / ) or Evotec SE (https: / / www.evotec.com / en / Metabolic stability profile of compound 1 Evaluate.

[0405] The potential for in vivo metabolism was evaluated in vitro using the following assays:

[0406] Liver microsomal stability (human, rat, dog; in the presence of NADPH)

[0407] Hepatocyte stability (human, rat, dog)

[0408] Plasma stability (human, rat, dog)

[0409] Whole blood stability (human, rat)

[0410] Stability in simulated gastric fluid (10 μM test sample, pH 1.2)

[0411] Metabolite identification in human hepatocytes

[0412] Compound 1 was found to have good metabolic stability in all assays.

[0413] Example 21

[0414] Standardized commercially available methods (available through contract research organizations such as Eurofins) were used. (EurofinsPanlabsInc.)(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 off-target profile of compound 1.

[0415] The potential for in vivo off-target promiscuity was evaluated in vitro using the following assays:

[0416] Inhibition of cytochrome P450 activity (30 μM single concentration; 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4)

[0417] Eurofins Discovery Safety Screen of 44 enzymes, receptors, ion channels and transporters Panel (Percent Inhibition at 10 μM Concentration)

[0418] Eurotransporter screening panel of 13 common transporters (percent inhibition at 10 μM concentration)

[0419] Apuit Cardiac Ion Channel Panel of 7 Targets (Automated Patch Clamp Assay; Concentrations 0 to 300 μM)

[0420] Compound 1 was found to have a good off-target profile in all assays.

[0421] Example 22

[0422] Evaluation of the cytotoxicity profile of compound 1 in a small bacterial reverse mutation screening assay

[0423] The mutagenic potential of compound 1 was tested in a small reverse mutation assay using Salmonella typhimurium strains TA98, TA100 and Escherichia coli WP2 uvrApKM101 in the presence and absence of a metabolic activation system. The test article was prepared in dimethyl sulfoxide (DMSO) at a nominal concentration of 50 mg / mL, and serial dilutions were prepared from this stock solution. The final dosing concentration of the test article was 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 μg / well. Metabolic activation was provided using an S9 mixture containing 10% v / v Aroclor-1254-induced rat liver S9 (Regensys System reagents, Moltox, Boone, NC).

[0424] Compound 1 did not induce responses indicative of mutagenic potential in this assay.

[0425] Example 23

[0426] Evaluation of the potential of compound 1 to induce phospholipidosis in a cell-based fluorescence assay

[0427] Compound 1 was tested in vitro for its potential to induce phospholipidosis at concentrations up to 100 μM according to the method outlined by Fujimura et al. (Experimental and Toxicologic Pathology, 2007, 58, 375-382).

[0428] Compound 1 had no effect on cell phenotype or cell viability in this assay.

[0429] Example 24

[0430] Stability of Compound 1 in Glucose Syrup Formulation at Room Temperature

[0431] Methylparaben (126.2mg), propylparaben (20.0mg), sodium dihydrogen phosphate (1%, 1.0g) and sucrose (16.56g) were dissolved in 100mL purified water, vortexed for 10 minutes, then ultrasonicated for 30 minutes, and then filtered through a 0.45μm filter. 0.5M NaOH was used to adjust the pH to pH 5. Grape flavor (LorAnnOils, 0180-0800; 1%, 0.5mL) was added to 50mL of the resulting solution, followed by the addition of 50mg of compound 1 to obtain a final concentration of 1mg / mL. Divided into two portions, each 25mL, one frozen, and one placed in a sealed falcon tube at room temperature (rt).

[0432] As shown in Table 4, no significant degradation of Compound 1 was observed after 91 days at room temperature.

[0433] Table 4

[0434]

Claims

1. A compound of formula I: or a pharmaceutically acceptable salt thereof; wherein: R 1 is selected from the group consisting of: hydrogen, fluorine, bromine and methyl; and R 2 It is hydrogen or fluorine.

2. The compound according to claim 1, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

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

4. Use of the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating or preventing a disease or disorder by inhibiting the activities of SSAO / VAP-1 protein and MAO-B protein.

5. The use according to claim 4, wherein the disease or condition is ameliorated by selective inhibition of SSAO / VAP-1 and MAO-B relative to MAO-A.

6. The use according to claim 4, wherein the disease is a neuromuscular disorder.

7. The use according to claim 6, wherein the neuromuscular disease is muscular dystrophy.

8. Use according to claim 7, wherein the muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy and myotonic dystrophy.

9. The use according to claim 4, wherein the disease is a neurodegenerative or neuroinflammatory disease.

10. The use according to claim 9, 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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