Benzylidene aminoguanidine derivatives as NR2B selective NMDA receptor antagonists and therapeutic uses thereof

Abstract

The present invention relates to benzylidene aminoguanidine derivatives as NR2B selective NMDA receptor antagonists and therapeutic uses thereof.

Description

TECHNICAL FIELD

[0001] The present invention relates to compounds having potential therapeutic use by preventing or treating a disease, disorder or medical condition mediated by N-methyl-D-aspartate (NMDA) receptor activity involving NR2B. BACKGROUND

[0002] N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors permeable to Ca 2+ , Na + and K + . NMDARs are essential for physiological synaptic plasticity of the developing and mature CNS. NMDARs are multi-subunit complexes associated with NR1, NR2 and, rarely, NR3 subunits. Most NMDARs are tetrameric complexes composed of two NR1 subunits and two NR2 subunits; hexameric complexes containing NR1 / NR2 / NR3 have also been found. NR1 is encoded by a single gene with at least eight different splice variants, NR2 is encoded by four different genes NR2A (GRIN2A), NR2B (GRIN2B), NR2C (GRIN2C) and NR2D (GRIN2D); two NR3 genes originate from NR3A (GRIN3A) and NR3B (GRIN3B) subunits. To be activated, NMDARs require the binding of glutamate through the NR2 subunit, the binding of glycine through the NR1 subunit and the removal of Mg 2+ block by membrane depolarization. The binding of glutamate to the NR2 subunit determines the duration of channel opening and desensitization process.

[0003] NMDARs containing different NR2 subunits have different pharmacological and kinetic properties. While the NR1 subunit is expressed in almost all neurons and at all developmental stages of the brain, the NR2 subunit genes exhibit different regional and developmental expression patterns. The NR2A subunit is widely expressed in the adult mammalian brain, while the expression of NR2B is restricted to the cortex, hippocampus, striatum, amygdala, ventral thalamic nuclei, olfactory bulb and dorsal horn of the spinal cord, the NR2C subunit is expressed in the cerebellum and NR2D in the midbrain. Outside the central nervous system, NMDARs are also present in Schwann cells.

[0004] NMDARs have been a major target for neurology drug development because preclinical studies have provided substantial evidence for their role in many cell and animal models of neurological diseases. NMDARs are best known for their role in excitotoxicity, a pathological process in which excessive release of glutamate leads to over-activation of NMDARs, resulting in massive influx of extracellular Ca 2+ into the cell, followed by intracellular Ca 2+Concentrations rise to pathological levels. Intracellular Ca 2+ The elevation of intracellular Ca2+levels can further lead to a cascade of downstream neurotoxicity, resulting in increased formation of reactive oxygen species (ROS) and activation of caspase-dependent and caspase-independent cell death, with mitochondria playing a key role. This process is implicated in acute ischemic stroke and traumatic brain injury. Glutamate excitotoxicity also contributes, at least in part, to neuronal loss in chronic neurodegenerative diseases, including Alzheimer’s disease (AD) and other dementias, Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), and possibly multiple sclerosis (MS) and prion diseases. Overactive excitotoxic pathways are also observed in epilepsy and neuropathic pain.

[0005] The first generation of NMDAR antagonists developed in the 1980-1990s bind to the agonist binding domain (i.e., the glycine or glutamate binding site) or the pore channel. They showed preclinical efficacy in different indications (e.g., excitotoxic neurodegeneration, neuropathic pain, ischemia-induced neurodegeneration, depression...), but most of the compounds, except memantine, were abandoned due to their broad spectrum and lack of subunit specificity, due to unacceptable side effects (e.g., hallucinations, memory and motor deficits...). Glycine binding site competitive antagonists have little receptor subunit selectivity, as expected, since these compounds target the binding site located on NR1, which is present in all receptor subtypes. NMDAR pore blockers generally fail to discriminate between NMDAR subtypes, and the NMDAR pore channel blockers dizocilpine (MK-801) and phencyclidine (PCP) induce psychotic and negative symptoms and cognitive impairment in healthy individuals that are similar to those present in schizophrenia and exacerbate these symptoms in schizophrenic patients, thus limiting the widespread use of this class of drugs. MK-801 has also been shown to cause brain damage in experimental rats. The channel blockers ketamine and dextromethorphan have been reported to alleviate symptoms of various neurological diseases by antagonizing NMDARs by binding to a site within the channel pore, but these drugs produce unacceptable side effects, including hallucinations, restlessness, and cognitive and motor dysfunction, at analgesic doses. Memantine is the only NMDAR pore blocker compound approved for Alzheimer’s disease; its noncompetitive, low-affinity mechanism of action allows for blocking of excess NMDAR activation by glutamate production while allowing for normal activation of the NMDAR channel.

[0006] Subunit-selective NMDAR antagonists appear to have greatly improved side effects compared to broad-spectrum antagonists. Thus, NR2B-selective antagonists have been the focus of intensive research and development over the past few years, because of the NR2B-containing receptor organization and subcellular localization, and their contribution to pathological processes associated with glutamatergic pathway hyperexcitability. For example, in the adult spinal cord, NR2B expression is restricted to the 2nd layer of the dorsal horn, a region that receives major sensory afferents from nociceptors and thermoreceptors. The localized positioning of NR2B-containing receptors in this region can partially explain why NR2B-selective antagonists, such as Bimoclomol and its related structures (i.e. traxoprodil / CP101,606 and Ro25-6981), have analgesic effects. Thus, the therapeutic potential of NR2B-selective antagonists has been well established (Mony et al. British J Pharmacol 2009; 157: 1301-1317; Chazot P Current Medicinal Chemistry, 2004, 11, 389-396 389).

[0007] However, NR2B-selective antagonists have not been developed into approved drugs. Bimoclomol, the most promising NR2B negative allosteric modulator, exhibits poor oral bioavailability and limitations due to its inhibition of GIRK channels and interactions with alpha 1 adrenergic, serotonin, and sigma receptors. Despite initial promising results, the development of traxoprodil for the treatment of chronic pain, PD, major depression has been discontinued due to apparent dissociative side effects. Although well-tolerated, Rislenemdaz (also known as CERC-301 and MK-0657) did not provide a clinically meaningful improvement in motor function in patients with moderate Parkinson’s disease. In 2011, a phase II clinical trial of EVT-101 for major depressive disorder was terminated early due to a clinical hold issued by the US Food and Drug Administration (NCT01128452). In 2021, MIJ821 was the only NR2B-selective antagonist evaluated in a phase II clinical trial for treatment-resistant depression (NCT03756129).

[0008] Thus, there remains a need for novel NMDAR antagonists that target the NR2B receptor subunit.

[0009] Some benzylidene guanidine derivatives of formula (I) are known in the literature. Compound 2 (-2,6-dichlorobenzylidene) hydrazinecarboxamide, also known as clonidine, is an alpha-2-type alpha adrenergic receptor agonist that has been marketed as an antihypertensive drug.

[0010]

[0011] It has also been reported for its therapeutic potential in several other areas. It has been suggested that clorgyline has anti-prion activity through its anti-PFAR activity (D. Tribouillard-Tanvier et al. 2008 PLoS One 3, e1981); it has also been reported for its activity in preventing protein misfolding based on its inhibitory activity on the PP1c / PPP1 R15A phosphatase complex. Based on its effect on protein misfolding, clorgyline was studied in a randomized phase 2 study in ALS patients. Clorgyline was described to reduce NMDA-induced current and NMDA-induced cytosolic Ca 2+ stress (Ruiz et al. Int. J. Mol. Sci. 2020, 21, 6088).

[0012] The approximate derivative 2-(2-chlorobenzylidene)hydrazinecarboxamide, known as icerguastat, IFB-088 or sephin1, but without hypotensive activity, also exhibits PP1c / PPP1 R15A phosphatase complex inhibitory activity, preventing protein misfolding. This compound shows therapeutic potential in the treatment of Charcot-Marie-Tooth (CMT) disease and ALS. IFB-088 / icerguastat was shown to reduce NMDA-induced cytosolic Ca 2+ stress (Ruiz et al. Int. J. Mol. Sci. 2020, 21, 6088). Ring et al. evaluated the ability of IFB-088 and certain benzylidene guanidine derivatives of formula (I) to inhibit NMDARs (Bioorganic Medicinal Chem. 2013 (21) 1764-1774), achieved by evaluating their ability to displace radiolabeled MK-801; these compounds showed high IC 50 .

[0013] Further benzylidene guanidine compounds and their therapeutic applications related to protein misfolding stress can be found in EP2943467, WO2016 / 001389, WO2016 / 001390 or WO2017 / 021216. EP109465 (CNRS) discloses clorgyline derivatives as PFAR ligands for the treatment of prion diseases. WO2002 / 011715 (Melacure) discloses benzylidene guanidine compounds as melanocortin receptor ligands for the treatment of diseases.

[0014] WO2005 / 031000 (Acadia Pharmaceuticals) discloses benzylidene guanidine compounds as neuropeptide FF receptor 2 agonists for the treatment of neuropathic pain.

[0015] In order to exploit NMDA receptor antagonists as possible therapeutic approaches, it is necessary to develop new NR2B selective negative allosteric modulators, in order to reduce the risk of side effects and to be able to target both the central and peripheral nervous system. The prior art publications do not describe benzaldehyde guanidine derivatives of formula (I) with the action of NR2B subunit selective NMDA receptor antagonists, which do not induce psychoses and negative symptoms and cognitive impairment, similar to those present in schizophrenia. This is the object of the present application.

[0016] Furthermore, in order to exploit NMDA receptor antagonists as possible therapeutic approaches, it is necessary to develop new NR2B selective negative allosteric modulators with good oral bioavailability, able to cross the blood-brain barrier and target both the central and peripheral nervous system. The prior art NR2B subunit NMDAR antagonists, such as benzamide, have such limitations that do not occur in the benzaldehyde guanidine derivatives of formula (I) of the present application. This is the object of the present application. SUMMARY

[0017] NR2B subunit selective NMDA antagonism can be achieved by compounds that specifically bind to and act on the allosteric modulatory site of the NR2B subunit containing the receptor. This binding site can be characterized by substitution (binding) studies with specific radioligands, such as 125 I]-benzamide [J. Neurochem., 61, 120-126 (1993)] or 3 H]-Ro25,6981 [J. Neurochem., 70, 2147-2155 (1998)].

[0018] Surprisingly, it was found that the benzaldehyde guanidine derivatives of formula (I) of the present application are functional antagonists of the NMDA receptor, which target the NMDA receptor mainly by binding to or in the proximity of the benzamide binding site on the NR2B subunit, rather than by binding to the pore channel, as MK801, as shown in Ring et al., 2013 (Ring et al., Bioorganic Medicinal Chem. 2013 (21) 1764-1774).

[0019] The benzaldehyde guanidine derivatives of formula (I) of the present application inhibit the influx of calcium by antagonizing the NR2B subunit containing the NMDAR, thus protecting the cell from glutamate-induced excitotoxicity. Therefore, they are considered selective NR2B subunit antagonists or NR2B negative allosteric modulators. Therefore, the benzaldehyde guanidine derivatives of formula (I) of the present application can inhibit the downstream neurotoxic cascade and, for example, are able to reduce the formation of reactive oxygen species (ROS).

[0020] The present benzylidene guanidine derivatives of formula (I) do not have the limitations of the NMDAR antagonists of the prior art and do not cause the psychoses and negative symptoms as well as cognitive impairment present in schizophrenia.

[0021] The present invention relates to benzylidene guanidine derivatives of formula (I) for the treatment of NMDA receptor related diseases and disorders involving NMDA receptor subunit 2B (NR2B) or disorders of glutamate homeostasis. The present benzylidene guanidine derivatives of formula (I) are a new therapy for the treatment of acute and chronic glutamate hyperenergized states.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] Abbreviations

[0024] AD Alzheimer's disease ALS amyotrophic lateral sclerosis CMAP compound muscle action potential CMT Charcot-Marie-Tooth disease CNS central nervous system HD Huntington's disease

[0025] MS multiple sclerosis

[0026] NMDA N-methyl-D-aspartate NMDAR N-methyl-D-aspartate receptor PNS peripheral nervous system PBA pseudobulbar affect PD Parkinson's disease

[0027] ROS reactive oxygen species

[0028] SCA spinocerebellar ataxia

[0029] Definitions

[0030] The following terms, as used herein, have the following meanings, unless expressly stated otherwise.

[0031] The term "alkyl" as used herein includes both saturated straight chain alkyl and branched alkyl groups, which can or can not be substituted (mono- or poly-). Preferably, the alkyl group is a C 1-20 alkyl, more preferably C 1-15 alkyl, more preferably C 1-12 alkyl, more preferably C 1-6 alkyl, more preferably C 1-3 alkyl. Particularly preferred alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl and hexyl. Preferably, the alkyl group is unsubstituted. Unless otherwise indicated, the term "alkoxy" refers to a moiety of the structure -O-alkyl, wherein alkyl is as defined above.

[0032] The term "aryl" as used herein refers to a C 6-12Aryl. Typical examples include phenyl and naphthalene, and the like. Suitable substituents include one or more R 10 The term "aryloxy" means a moiety of the structure -O-aryl, where aryl is as defined above, unless otherwise specified.

[0033] The term "excitotoxicity" refers to the pathological process of glutamate or glutamate and its analogs, both excitatory neurotransmitters, over-activation, leading to neuronal damage and destruction or neurotoxicity. These neurotransmitters activate neuronal excitatory receptors, such as NMDA and AMPA receptors. These excitotoxins, such as NMDA (N-methyl-D-aspartate) and kainic acid, or glutamate at too high a concentration (i.e., glutamate hyperenergized state), by binding to these receptors, cause a large influx of calcium ions into the cell. Glutamate hyperenergized state can also result from interference with glutamate re-uptake by glutamate transporters, such as the excitatory amino acid transporter (EAAT) family and the vesicular glutamate transporter (VGLUT) family. Reduced expression of calcium buffers can also decrease the cell's ability to handle high Ca2+ levels in the cytoplasm after glutamate activates NMDA receptors. Ca 2+ In turn, activates a variety of enzymes, including phospholipase C, endonucleases, and proteases, such as calpains. These enzymes then degrade cellular structures: cytoskeleton, cell membrane, DNA, leading to neurotoxicity. The increase in intracellular Ca 2+ The increase in intracellular Ca2+ concentration also leads to an increase in reactive oxygen species formation, which will cause damage to multiple organelles and processes, ultimately disrupting normal physiological functioning. This pathophysiological mechanism is implicated in a variety of neurological diseases, such as spinal cord trauma, brain trauma, stroke, acquired deafness (ototoxicity caused by excessive exposure to noise), neurodegenerative diseases of the central nervous system, such as MS, AD, ALS, PD, HD, epilepsy, and fibromyalgia.

[0034] The term "NR2B" refers to the glutamate NMDA receptor subunit epsilon-2, also known as N-methyl D-aspartate receptor subtype 2B (NMDAR2B or NR2B), which is a protein that in humans is encoded by the GRIN2B gene.

[0035] The term "halo" or "halogen" means chlorine, bromine, iodine, or fluorine. The term "haloalkyl" means an alkyl radical having one or more hydrogen atoms replaced by a halogen atom.

[0036] The term "hydroxyl" refers to the functional group (—OH).

[0037] The term "treatment or prevention" as used herein in the treatment or prevention of a disease generally relates to therapy and therapy in both humans and animals (e.g., in veterinary applications) in which there can be an intended therapeutic effect, e.g., inhibition of the progression of the disease, including reduction in the rate of progression, cessation in the rate of progression, alleviation of symptoms of the disease, amelioration of the disease, cure of the disease. Treatment as a prophylactic measure (i.e., prevention) is also included, e.g., use in a patient who does not yet have the disease but who is at risk of developing the disease.

[0038] Herein, the term "treatment" includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease, substantially ameliorating a clinical symptom of a disease or disorder, or substantially increasing the survival of a patient. The term "prevention" refers to preventing or delaying the onset and / or appearance of symptoms of a neurodegenerative disease. For example, treatment or prevention of neuropathic pain includes: preventing the onset of neuropathic pain, inhibiting the progression of neuropathic pain, reducing the rate of progression of neuropathic pain, reducing the incidence of neuropathic pain, reducing the severity of neuropathic pain, alleviating one or more symptoms of neuropathic pain, ameliorating neuropathic pain, cure of neuropathic pain, and the like.

[0039] The term "therapeutically effective amount" or "effective amount" as used herein means the amount of a compound or a material, composition or dosage form comprising a compound that, when administered to a patient for a desired treatment, is effective to achieve a desired therapeutic result in a manner consistent with a reasonable benefit / risk ratio.

[0040] The term "patient" or "subject" refers to an animal, such as a mammal, including, but not limited to, humans. Thus, the methods and uses disclosed herein can be useful in human therapy and veterinary applications. In one embodiment, the patient is a mammal. In another embodiment, the patient is a human.

[0041] "e.g." has the same meaning as "for example, but not limited to". Likewise, "including" has the same meaning as "including, but not limited to" and "comprising" has the same meaning as "comprising, but not limited to".

[0042] The terms "psychotic symptoms" or "negative symptoms" have the same meaning. Psychotic symptoms include hallucinations, delusions (i.e., false beliefs that do not go away even when proven to be false), disorganized thinking. It can also include disorganized or incoherent speech, bizarre and potentially dangerous behavior, slow or unusual movements, loss of interest in activities, loss of interest in personal hygiene, problems at school or work and in interpersonal relationships, apathy, an attitude of unexpressed emotion, mood swings or other mood symptoms, such as depression or mania.

[0043] Psychosis is a mental abnormality condition that exhibits psychotic symptoms; psychosis has several different causes, including psychiatric illnesses such as schizophrenia or schizoaffective disorder, bipolar disorder, sensory deprivation, and in rare cases, major depressive disorder (psychotic depression). Other causes include trauma, lack of sleep, certain illnesses, certain medications, and drugs such as cannabis, hallucinogens, and stimulants.

[0044] The term "cognitive disorder" as used herein refers to a description of a person's condition in which they have difficulties in memory or concentration; they can also have difficulties in speaking or understanding, difficulty in recognizing people, places or things.

[0045] The term "over-activation of NMDA receptors" as used herein refers to an abnormally high signaling activity of NMDARs on CNS and / or PNS cells (e.g. neurons, astrocytes, oligodendrocytes, Schwann cells) or cells outside the nervous system (e.g. kidney, bone...).

[0046] Example description

[0047] The present application, as described herein, relates to certain benzylidene guanidine compounds, which are negative allosteric modulators of the NMDAR NR2B subunit, as well as pharmaceutical compositions comprising these compounds, for the treatment or prevention of therapeutic indications.

[0048] According to a first object, the present application is directed to a method of selectively inhibiting the subunit 2B (NR2B) of the N-methyl-D-aspartate (NMDA) receptor in a cell having a NMDA receptor comprising the subunit 2B (NR2B) of the NMDA receptor, the method comprising treating the cell with an effective amount of a compound of general formula (I):

[0049]

[0050] and the (Z) and / or (E) isomers thereof, or the tautomers thereof, or the pharmaceutically acceptable salts thereof,

[0051] in which: R1, R2, R3, R4, R5are independently hydrogen, deuterium, halogen, halogenated alkyl, alkyl, alkoxy, hydroxyl, aryl or aryloxy,

[0052] thereby reducing the neuroprotective effect of excitotoxic NMDA receptor activity.

[0053] According to a preferred embodiment, the cell is selected from the group consisting of neurons, motor neurons, sensory neurons, Schwann cells, oligodendrocytes, astrocytes, Purkinje cells, parathyroid, pulmonary artery smooth muscle cells, cardiac cells, bone cells, kidney cells

[0054] According to one embodiment, the effect of reducing excitotoxic NMDA receptor activity in cells is advantageously reduced by lowering the intracellular Ca 2+ concentration.

[0055] According to alternative or cumulative embodiments, the effect of reducing excitotoxic NMDA receptor activity in cells is reduced by lowering the reactive oxygen species (ROS) concentration.

[0056] According to a further object, there is provided a method of selectively inhibiting subunit 2B (NR2B) of the N-methyl-D-aspartate (NMDA) receptor in a subject having an NMDA receptor comprising subunit 2B (NR2B), the method comprising administering an effective amount of a compound of general formula (I)

[0057]

[0058] and (Z) and / or (E) isomers thereof, or tautomers thereof, or pharmaceutically acceptable salts thereof,

[0059] wherein: R1, R2, R3, R4, R5are independently hydrogen, deuterium, halogen, haloalkyl, alkyl, alkoxy, hydroxyl, aryl or aryloxy.

[0060] in said subject in need thereof.

[0061] According to a further further object, there is also provided a method for preventing or treating a disease, disorder or medical condition caused by over-activation of the N-methyl-D-aspartate (NMDA) receptor containing subunit 2B (NR2B), by selectively targeting the NR2B subunit of said NMDA receptor, wherein the method comprises administering to a subject in need thereof an effective amount of a compound of general formula (I):

[0062]

[0063] and (Z) and / or (E) isomers thereof, or tautomers thereof, or pharmaceutically acceptable salts thereof,

[0064] wherein: R1, R2, R3, R4, R5are independently hydrogen, deuterium, halogen, haloalkyl, alkyl, alkoxy, hydroxyl, aryl or aryloxy.

[0065] According to alternative or cumulative embodiments, the over-activation of the N-methyl-D-aspartate (NMDA) receptor containing NR2B is triggered by glutamate dyshomeostasis or acute or chronic glutamate hyperenergization.

[0066] According to one embodiment, the disease, disorder or medical condition can be selected from the group consisting of:

[0067] (a) depression or depressive disorder, major depressive disorder, treatment resistant major depressive disorder, postpartum depression, bipolar depression;

[0068] (b) anxiety, obsessive-compulsive disorder, generalized anxiety disorder, agoraphobia with panic attacks, panic disorder, post-traumatic stress disorder, social anxiety disorder;

[0069] (c) autism or autism spectrum disorder, Asperger syndrome or pervasive developmental disorder not otherwise specified (PDD-NOS);

[0070] (d) epilepsy, seizure disorder;

[0071] (e) migraine, chronic tension-type headache (CTTH), migraine with allodynia, chronic headache;

[0072] (f) brain dysfunction selected from the group consisting of fragile X syndrome, tuberous sclerosis, Down syndrome and other forms of intellectual disability;

[0073] (g) withdrawal syndromes, such as from alcohol, opioids or cocaine;

[0074] (h) pain, hyperalgesia, nociception, acute pain, chronic pain or cancer-related pain;

[0075] (i) pain associated with excitotoxicity, preferably with glutamate excitotoxicity, and / or with dysregulation of glutamatergic neurotransmission;

[0076] (j) neuropathic pain;

[0077] (k) pseudobulbar affect (PBA).

[0078] (l) movement disorder;

[0079] (m) amyotrophic lateral sclerosis (ALS) or bulbar onset amyotrophic lateral sclerosis;

[0080] (n) Charcot-Marie-Tooth disease;

[0081] (o) multiple sclerosis (MS);

[0082] (p) Parkinson's disease, atypical parkinsonism (e.g. progressive supranuclear palsy);

[0083] (q) Alzheimer's disease (AD), dementia, frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD).

[0084] (r) Huntington's disease (HD);

[0085] (s) focal brain injury caused by trauma, tumor or stroke;

[0086] (t) brain or spinal cord injury, peripheral nervous system injury, cerebral ischemia, head or neuronal trauma, neuronal hemorrhage, neuronal ischemia, reperfusion injury, neuronal injury;

[0087] (u) neuronal exposure to toxic substances, methamphetamine-induced neurotoxicity;

[0088] (v) stroke, cardiogenic shock, coronary artery bypass graft (CABG) surgery related neurological injury;

[0089] (w) idiopathic pulmonary fibrosis (IPF) and chronic cough;

[0090] (x) spinocerebellar ataxia and Friedreich's ataxia;

[0091] (y) kidney injury, acute kidney injury (AKI), ischemia-reperfusion induced AKI, hyperparathyroidism associated with chronic kidney disease (CKD), glomerulosclerosis and homocysteine-induced glomerulosclerosis.

[0092] (z) bone disease, bone fracture, bone injury, bone defect conditions associated with post-traumatic bone surgery, post-prosthetic joint replacement, post-orthopedic bone surgery, post-dental surgery, bone chemotherapy treatment, bone radiotherapy treatment, osteoporosis, Paget's disease, achondroplasia, osteochondritis, hyperparathyroidism, osteogenesis imperfecta, congenital hypophosphatasia, fibrous lesions, fibrous dysplasia, multiple myeloma, abnormal bone turnover, osteolytic bone disease, osteomalacia, and periodontal disease and symptoms thereof.

[0093] According to one embodiment, the neuropathic pain is selected from the group consisting of peripheral neuropathic pain; central neuropathic pain; chronic neuropathic pain; refractory neuropathic pain; neuropathic pain associated with metabolic dysfunction, including for example diabetes and prediabetes; neuropathic pain associated with diabetes; neuropathic pain associated with prediabetes; neuropathic pain associated with painful polyneuropathy; neuropathic pain associated with painful polyneuropathy of diabetes, including for example diabetic peripheral neuropathy; neuropathic pain associated with painful diabetic polyneuropathy; neuropathic pain associated with post-herpetic neuralgia; neuropathic pain associated with trigeminal neuralgia; neuropathic pain associated with occipital neuralgia; neuropathic pain associated with painful radiculopathy, including for example lumbar and cervical painful radiculopathy; neuropathic pain associated with infectious diseases, including for example shingles, HIV infection, Lyme disease, diphtheria, and leprosy; neuropathic pain associated with liver or kidney disease, including for example chronic liver or kidney disorders, including for example liver disease, liver failure, kidney disease, and kidney failure; neuropathic pain associated with immune or inflammatory diseases, including for example Guillain-Barre syndrome and Miller-Fisher syndrome, rheumatoid arthritis, lupus, systemic lupus erythematosus, Sjogren’s syndrome, and celiac disease; neuropathic pain associated with hereditary neuropathies or channelopathies, including for example hereditary erythromelalgia, paroxymal extreme pain disorder, and Charcot-Marie-Tooth disease (CMT); neuropathic pain associated with small-fiber sensory neuropathy; neuropathic pain associated with thyroid hormone disorders, including for example hypothyroidism; neuropathic pain associated with stroke; neuropathic pain associated with cancer, including for example lymphoma and multiple myeloma; neuropathic pain associated with chemotherapy, for example cancer chemotherapy; neuropathic pain associated with peripheral nerve injury pain; post-traumatic neuropathic pain associated with nerve injury; neuropathic pain associated with post-traumatic neuropathy; neuropathic pain associated with spinal cord injury, including for example trauma-induced spinal cord injury, for example road traffic accidents; neuropathic pain associated with traumatic peripheral nerve injury; neuropathic pain associated with postoperative neuropathy (for example postoperative neuropathic pain); neuropathic pain following surgery, including for example neuropathic pain following nerve surgery, including for example spinal cord surgery; neuropathic pain associated with fibromyalgia; neuropathic pain associated with lower back pain; neuropathic pain associated with carpal tunnel syndrome; neuropathic pain associated with causalgia; neuropathic pain associated with reflex sympathetic dystrophy (RSD); neuropathic pain associated with complex regional pain syndrome (CRPS), including for example type 1 and type 2; neuropathic pain associated with amputation; neuropathic pain associated with neurodegenerative diseases, for example amyotrophic lateral sclerosis and Parkinson’s disease; neuropathic pain associated with stroke, including for example central post-stroke pain; neuropathic pain associated with syringomyelia;Neuropathic pain associated with demyelinating diseases, including, for example, multiple sclerosis, transverse myelitis and neuromyelitis optica; or idiopathic neuropathic pain.

[0094] According to one embodiment, the method of the application prevents, treats or alleviates pseudobulbar affect (PBA) or a symptom thereof in a subject selected from a patient having Parkinson's disease (PD) or atypical Parkinsonism (e.g. progressive supranuclear palsy), amyotrophic lateral sclerosis (ALS), bulbar onset ALS, spinal onset ALS, primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), frontotemporal spectrum disorder, multiple sclerosis (MS), Alzheimer's disease (AD), dementia, Alzheimer's disease dementia agitation, frontotemporal dementia (FTD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), a tumor, stroke, traumatic brain injury (e.g. focal brain injury caused by trauma).

[0095] According to one embodiment, the method of the application prevents, treats or alleviates depression or a symptom thereof in a subject selected from a patient having Parkinson's disease (PD) or atypical Parkinsonism (e.g. progressive supranuclear palsy) Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), bulbar onset ALS, spinal onset ALS, primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), frontotemporal spectrum disorder, multiple sclerosis (MS), CMT.

[0096] According to one embodiment, the method of the application prevents, treats or alleviates movement disorder or a symptom thereof in a subject selected from a patient having Parkinson's disease (PD) or atypical Parkinsonism (e.g. progressive supranuclear palsy).

[0097] According to one embodiment, the method of the application does not simultaneously involve side effects selected from psychotomimetic effects, cognitive impairment and symptoms associated with schizophrenia.

[0098] According to one embodiment, in the above formula (I):

[0099] R1, R3 and R5 are independently selected from H, CI, F, Br and OH;

[0100] R2 = R4 = H.

[0101] According to one preferred embodiment, the compound is selected from the group consisting of:

[0102] 2-(2,6-dichlorobenzylidene)hydrazinecarboxamide

[0103] 2-(2-chlorobenzylidene)hydrazinecarboxamide

[0104] 2-(2-chloro-4-fluorobenzylidene)hydrazinecarboxamide

[0105] 2-(2-chloro-6-fluorobenzylidene)hydrazinecarboxamide

[0106] 2-(2-bromobenzylidene)hydrazinecarboxamide

[0107] 2-(2-fluorobenzylidene)hydrazinecarboxamide

[0108] 2-(2,4-difluorobenzylidene)hydrazinecarboxamide

[0109] 2-(2,6-difluorobenzylidene)hydrazinecarboxamide acetate

[0110] 2-(2,4-dichlorobenzylidene)hydrazinecarboxamide acetate

[0111] 2-(2,3-dichlorobenzylidene)hydrazinecarboxamide

[0112] 2-(2,3,4-trichlorobenzylidene)hydrazinecarboxamide

[0113] 2-(3,4,5-trichlorobenzylidene)hydrazinecarboxamide

[0114] 2-(2,4,6-trifluorobenzylidene)hydrazinecarboxamide acetate

[0115] 2-(2,4,5-trifluorobenzylidene)hydrazinecarboxamide

[0116] 2-(2,6-difluoro-4-chlorobenzylidene)hydrazinecarboxamide

[0117] 2-(2,4-dichloro-3-fluorobenzylidene)hydrazinecarboxamide

[0118] 2-(2-chloro-,4,6-difluorobenzylidene)hydrazinecarboxamide

[0119] 2-(2-chloro-,4,5-difluorobenzylidene)hydrazinecarboxamide

[0120] 2-(2-chloro-4-hydroxybenzylidene)hydrazinecarboxamide

[0121] 2-(2-chloro-3-methylbenzylidene)hydrazinecarboxamide

[0122] 2-(2-chloro-4-methylbenzylidene)hydrazinecarboxamide

[0123] 2-(2-chloro-5-methylbenzylidene)hydrazinecarboxamide

[0124] 2-(2,4-dichloro-6-fluorobenzylidene)hydrazinecarboxamide

[0125] 2-(2,6-dichloro-4-fluorobenzylidene)hydrazinecarboxamide

[0126] 2-(2,3-dichloro-4-fluorobenzylidene)hydrazinecarboxamide

[0127] 2-(2-chloro-3,5-difluorobenzylidene)hydrazinecarboxamide

[0128] 2-(3,4-dichloro-6-fluorobenzylidene)hydrazinecarboxamide

[0129] 2-(3,5-dichloro-4-fluorobenzylidene)hydrazinecarboxamide

[0130] 2-(2,4-dichloro-5-fluorobenzylidene)hydrazinecarboxamide

[0131] 2-(2,3,5-trichlorobenzylidene)hydrazinecarboxamide

[0132] 2-(3,4,5-trifluorobenzylidene)hydrazinecarboxamide

[0133] 2-(2,3,4-trifluorobenzylidene)hydrazinecarboxamide

[0134] and the (Z) and / or (E) isomers thereof, or the tautomers thereof, or the pharmaceutically acceptable salts thereof.

[0135] More preferably, the compound of formula (I) can be selected from:

[0136]

[0137] and the (Z) and / or (E) isomers thereof, or the tautomers thereof, or the pharmaceutically acceptable salts thereof.

[0138] More preferably, the compound of formula (I) can be selected from:

[0139] - the (Z) isomer of the compound 2 of the following formula:

[0140]

[0141] and the (Z) and / or (E) isomers thereof, or the tautomers thereof, or the pharmaceutically acceptable salts thereof; and

[0142] - the (Z) isomer of the compound 1 of the following formula:

[0143]

[0144] In another embodiment, the present application relates to a compound of general formula (I):

[0145]

[0146] and the (Z) and / or (E) isomers thereof, or the tautomers thereof, or the pharmaceutically acceptable salts thereof,

[0147] wherein: R1, R2, R3, R4, R5are independently hydrogen, deuterium, halogen, haloalkyl, alkyl, alkoxy, hydroxyl, aryl or aryloxy,

[0148] for use in the treatment of a disease, disorder or medical condition mediated by NR2B containing NMDA receptor activity, such as the diseases, disorders or medical conditions described in the present patent application.

[0149] According to another object, the present application relates to the following compounds selected from the group per se:

[0150] 2-(2,4,6-trifluorobenzylidene)hydrazinecarboximidate acetate

[0151] 2-(2,6-difluoro-4-chlorobenzylidene)hydrazinecarboximidate

[0152] 2-(2-chloro-, 4,6-difluorobenzylidene)hydrazinecarboximidate

[0153] 2-(2-chloro-4-hydroxybenzylidene)hydrazinecarboximidate

[0154] 2-(2,4-dichloro-6-fluorobenzylidene)hydrazinecarboximidate

[0155] 2-(2,6-dichloro-4-fluorobenzylidene)hydrazinecarboximidate

[0156] 2-(2-chloro-3,5-difluorobenzylidene)hydrazinecarboximidate

[0157] and the (Z) and / or (E) isomers thereof, or the tautomers thereof, or the pharmaceutically acceptable salts thereof.

[0158] Without being bound by theory, certain compounds of formula (I) can be respectively a prodrug or an active metabolite.

[0159] Both metabolites and prodrugs are part of the present application.

[0160] In one embodiment, the compounds of formula (I) wherein: R1, R2, R4, R5are independently hydrogen, deuterium, halogen, haloalkyl, alkyl, alkoxy, hydroxyl, aryl or aryloxy, and wherein R3is hydrogen or deuterium, can be a prodrug, and the compounds of formula (I) wherein R3is hydroxyl, can be an active metabolite.

[0161] In particular, 2-(2-chlorobenzylidene)hydrazinecarboximidate can be a prodrug of its active metabolite 2-(2-chloro-4-hydroxybenzylidene)hydrazinecarboximidate.

[0162] According to another object, the present application relates to a method for preventing or treating a disease, disorder or medical condition caused by over-activation of the N-methyl-D-aspartate (NMDA) receptor containing subunit 2B (NR2B) by selectively targeting the NR2B subunit of the NMDA receptor, wherein the method comprises:

[0163] (a) administering to a subject in need thereof an effective amount of a compound of general formula (I):

[0164]

[0165] and (Z) and / or (E) isomers thereof, or tautomers thereof, or pharmaceutically acceptable salts thereof, and wherein: R1, R2, R4, R5are independently hydrogen, deuterium, halogen, haloalkyl, alkyl, alkoxy, hydroxyl, aryl or aryloxy, and wherein R3is hydrogen or deuterium;

[0166] (b) inhibiting the N-methyl-D-aspartate (NMDA) receptor containing subunit 2B (NR2B) in the subject by selectively targeting the NR2B subunit of the NMDA receptor using a compound of general formula (I), wherein R3is hydroxyl, and wherein the compound of general formula (I) wherein R3is hydroxyl, is a metabolite of the compound administered in step (a). According to a preferred embodiment, the compound administered at step (a) is 2-(2-chlorobenzylidene(hydrazinecarboxamide and the metabolite of the compound is 2-(2-chloro-4-hydroxybenzylidene)hydrazinecarboxamide.

[0167] According to another object, the present application relates to a method for preventing or treating a disease, disorder or medical condition caused by over-activation of the N-methyl-D-aspartate (NMDA) receptor containing subunit 2B (NR2B) by selectively targeting the NR2B subunit of the NMDA receptor, wherein the method comprises administering to a subject in need thereof an effective amount of a compound of general formula (I):

[0168]

[0169] and (Z) and / or (E) isomers thereof, or tautomers thereof, or pharmaceutically acceptable salts thereof, wherein: R1, R2, R4, R5are independently hydrogen, deuterium, halogen, haloalkyl, alkyl, alkoxy, hydroxyl, aryl or aryloxy, and R3is hydroxyl.

[0170] According to a preferred embodiment, the compound is 2-(2-chloro-4-hydroxybenzylidene)hydrazinecarboxamide.

[0171] Therapeutic indications

[0172] Applicants have demonstrated that the compounds of the present application have potential therapeutic applications in the treatment and / or prevention of diseases, disorders or medical conditions mediated by NR2B-containing NMDA receptor activity. The diseases, disorders or medical conditions of the present application will be described in more detail below.

[0173] Unless otherwise specified, the present application is preferably related to human patients.

[0174] Neurodegeneration

[0175] According to one embodiment, the above-mentioned diseases, disorders or medical conditions mediated by NR2B-containing NMDA receptor activity are caused by excitotoxicity mediated by NMDA receptors.

[0176] According to a second embodiment, the above-mentioned diseases, disorders or medical conditions mediated by NR2B-containing NMDA receptor activity are a neurological disease in which glutamate-mediated excitotoxicity is associated with neuronal cell death.

[0177] In one embodiment, the present application relates to the inhibition of ion channels, such as calcium channels, which are activated by exogenous chemicals or endogenous chemicals, such as glutamate, leading to neurodegeneration.

[0178] In another embodiment, the present application relates to the treatment, slowing, reducing, decreasing and / or prevention of neurodegeneration.

[0179] The present application relates to the reduction of neuronal cell damage associated with glutamate excitotoxicity, wherein said glutamate excitotoxicity is mediated by overstimulation of NMDA receptors, said glutamate excitotoxicity being associated with a disease, disorder or medical condition mediated by NR2B-containing NMDA receptor activity, such as the diseases, disorders or medical conditions described in the present patent application.

[0180] The present invention relates to reducing neuronal cell damage associated with glutamate excitotoxicity, wherein the glutamate excitotoxicity is over-stimulation of NMDA receptor mediated by glutamate re-uptake interference of glutamate transporters (e.g. the excitatory amino acid transporter (EAAT) family and the vesicular glutamate transporter (VGLUT) family), wherein the glutamate excitotoxicity is associated with a disease, disorder or medical condition mediated by NR2B containing NMDA receptor activity, such as described in the present patent application. For example, loss of the Na+-dependent glutamate transporter EAAT2 is suspected to be associated with neurodegenerative diseases, such as Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, ALS - Parkinson's syndrome dementia syndrome (Glenn Lin et al., Glutamate transporter EAAT2: a new target for the treatment of neurodegenerative diseases, Future Med Chem. 2012 Sep; 4(13): 1689-1700).

[0181] depression

[0182] Depression is a mental illness characterized by a low mood and aversion to activity, affecting millions of people worldwide. People with depression can have feelings of sadness, hopelessness, and suicidal thoughts. Existing antidepressants targeting serotonin and norepinephrine neurotransmission induce adverse side effects and only produce therapeutic effects after long-term administration. Several NMDA receptor antagonists for the treatment of depression have been demonstrated to have similar antidepressant effects in different animal models and in the clinic (Ates-Alagoz Z and Adejare A. NMDA receptor antagonists for treatment of depression. Pharmaceuticals. 2013; 6: 480-499). NR2B-specific NMDAR antagonists have been shown to have antidepressant effects (Henter ID et al., Glutamatergic modulators of depression. Harv. Rev. Psychiatry 2018; 26(6); 307-319).

[0183] According to a preferred embodiment, the present application relates to a method for preventing or treating a disease, disorder or medical condition mediated by NR2B-containing NMDA receptor activity, wherein the method comprises administering to a subject in need of such treatment an effective amount of a compound of general formula (I) and its (Z) and / or (E) isomers, or a tautomer or a pharmaceutically acceptable salt thereof, wherein the disease, disorder or medical condition is selected from the group consisting of depression or depressive disorder, major depressive disorder, treatment-resistant major depressive disorder, postpartum depression, bipolar depression and suicidal ideation.

[0184] In one embodiment, the depression is major depressive disorder.

[0185] In one embodiment, the depression is treatment-resistant major depressive disorder.

[0186] In one embodiment, the depression is postpartum depression.

[0187] In one embodiment, the depression is bipolar depression.

[0188] Seizure disorders, epilepsy and brain dysfunction

[0189] Epilepsy is a group of nervous system disorders caused by abnormal electrical activity in the brain, characterized by recurrent seizures ranging from brief to long periods of vigorous shaking. NMDAR NR2B subunits were shown to contribute to pathological and biochemical events associated with epilepsy (Zhu X. et al., NMDA receptor NR2B subunits contribute to PTZ-kindling-induced hippocampal astrocytosis and oxidative stress. Brain Res. 2015 Brief Report).

[0190] According to a preferred embodiment, the present application relates to a method for preventing or treating a disease, disorder or medical condition mediated by NR2B-containing NMDA receptor activity, selected from the group consisting of seizure disorders, epilepsy, Lennox-Gastaut syndrome, Sturge-Weber syndrome, tuberous sclerosis and infantile spasm syndrome (ISS).

[0191] In one embodiment, the seizure disorder is epilepsy.

[0192] In one embodiment, the seizure disorder is Lennox-Gastaut syndrome.

[0193] In one embodiment, the seizure disorder is Sturge-Weber syndrome.

[0194] The present application also relates to a method of preventing or treating a brain dysfunction mediated by NR2B-containing NMDA receptor activity, in particular wherein the brain dysfunction is selected from the group consisting of fragile X syndrome, Down's syndrome, Alzheimer's disease (AD) and other forms of mental retardation.

[0195] Anxiety disorders

[0196] Anxiety and trauma-related disorders, including post-traumatic stress disorder, are associated with excessive fear responses, often including the inability to extinguish learned fear, increased avoidance behavior, and alterations in cognition and emotion. NMDARs are involved in the regulation of these fear-related behaviors (Radulovic J. et al., N-Methyl D-Aspartate Receptor subunit signaling in fear extinction. Psychopharmacology. 2019; 236(1): 239-250).

[0197] According to a preferred embodiment, the present application relates to a method of preventing or treating a disease, disorder or medical condition mediated by NR2B-containing NMDA receptor activity, selected from the group consisting of anxiety disorders, obsessive-compulsive disorder, generalized anxiety disorder, agoraphobia with panic disorder, panic disorder, social anxiety disorder and post-traumatic stress disorder. In a preferred embodiment, the anxiety disorder is post-traumatic stress disorder (PTSD). In another embodiment, the present application relates to a method of preventing, treating or alleviating anxiety symptoms, wherein the subject is selected from a group consisting of patients with amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, atypical Parkinsonism, Huntington's disease, Alzheimer's disease, Shy-Drager syndrome.

[0198] Autism spectrum disorders Autism spectrum disorders (ASD) refer to a variety of diseases characterized by social deficits and repetitive behaviors, speech and non-verbal communication. ASD is associated with abnormal imbalance and abnormalities of neuronal excitatory and inhibitory synapses. NMDAR dysfunction is associated with ASD (Lee E.J. et al., NMDA receptor dysfunction in autism spectrum disorders. Curr Opin Pharmacol. 2015; 20: 8-13).

[0199] According to one preferred embodiment, the present application relates to a method of preventing or treating a disease, disorder or medical condition mediated by NR2B containing NMDA receptor activity selected from the group consisting of autism spectrum disorder, autism, Asperger syndrome, childhood disintegrative disorder, pervasive developmental disorder not otherwise specified (PDD-NOS) and Rett syndrome. In one embodiment, the autism spectrum disorder is autism.

[0200] In one embodiment, the autism spectrum disorder is Asperger syndrome.

[0201] In one embodiment, the autism spectrum disorder is childhood disintegrative disorder.

[0202] In one embodiment, the autism spectrum disorder is Rett syndrome.

[0203] In one embodiment, the autism spectrum disorder is PDD-NOS.

[0204] Migraine

[0205] Migraine is a common health condition, usually characterized by moderate or severe headaches, which feel like a throbbing pain on one side of the head. Many people also experience symptoms such as feeling unwell, being sick and increased sensitivity to light or sound. There are several types of migraine, including (i) migraine with aura, where there are specific warning signs before the migraine starts, such as seeing flashing lights; (ii) migraine without aura (the most common type, where there are no specific warning signs of a migraine); (iii) migraine with no headache, also known as silent migraine, where the aura or other migraine symptoms occur, but the headache does not develop. Migraine cannot be cured, but there are many treatments that can help to reduce the symptoms, including painkillers (such as paracetamol, ibuprofen), triptans and anti-emetics.

[0206] Recent developments suggest that specific inhibition of GluN2B containing NMDARs can be effective in preventing migraine (Crivellaro G. et al. Specific activation of GluN1-N2B NMDA receptors underlies facilitation of cortical spreading depression in a genetic mouse model of migraine with reduced astrocytic glutamate clearance. Neurobiology of Disease 2021 156: 105419).

[0207] According to one preferred embodiment, the present application relates to a method of preventing or treating a disease, disorder or medical condition mediated by NR2B-containing NMDA receptor activity selected from the group consisting of migraine, migraine with aura, migraine without aura, silent migraine, silent migraine, retinal migraine, migraine with unusual pain, familial hemiplegic migraine, chronic headache, chronic tension-type headache (CTTH).

[0208] In one embodiment, the migraine is migraine with aura.

[0209] In one embodiment, the migraine is migraine without aura.

[0210] In one embodiment, the migraine is silent migraine.

[0211] In one embodiment, the migraine is migraine with unusual pain.

[0212] In one embodiment, the migraine is familial hemiplegic migraine.

[0213] In one embodiment, the migraine is retinal migraine.

[0214] In one embodiment, the migraine is chronic headache.

[0215] Drug poisoning and withdrawal

[0216] Abuse of drugs (e.g. alcohol, cocaine, opioids, etc.) exerts certain effects on the central nervous system by affecting glutamatergic transmission, in particular through NMDARs (Landa L. Implication of NMDA receptors in behavioural sensitization to psychostimulants: a short review. Eur J Pharmacol. 2014; 5:730: 77-81). The NR2B subunit of NMDA receptors has been identified as a possible central modulator of many addictive behaviours, such as alcohol dependence (Nagy J. The NR2B subunit of NMDA receptor: a potential target for the treatment of alcohol dependence. Curr Drug Targets CNS Neurol Disord. 2004 Jun;3(3): 169-79), cocaine dependence (Smaga I. et al. Enhancement of the GluN2B subunit of glutamatergic NMDA receptors in rat brain areas after cocaine abstinence. J Psychopharmacol. 2021; 35(10): 1226-1239).

[0217] According to one preferred embodiment, the present application relates to a method for preventing or treating a disease, disorder or medical condition mediated by NR2B-containing NMDA receptor activity, selected from the group consisting of drug intoxication, drug withdrawal syndrome, addictive behaviour associated with abuse of drugs, suppression of dependence on abuse of drugs, habit or addiction; the drug being selected from the group consisting of alcohol, nicotine, cannabis, opioids, phencyclidine, amphetamines (e.g. methamphetamine) and cocaine, barbiturates (e.g. pentobarbital) and benzodiazepines, such as temazepam, diazepam and flunitrazepam. Prescription opioids include morphine, methadone, oxycodone, hydrocodone acetaminophen, pseudoephedrine hydrocodone, hydromorphone, fentanyl, codeine, methadone, oxymorphone hydrochloride, meperidine, tramadol, carfentanyl, buprenorphine. Illicit opioids such as heroin.

[0218] Pain

[0219] Neuropathic pain is pain resulting from an injury or disease affecting the somatosensory system; it occurs after nerve and spinal cord injuries and in certain diseases, and can lead to weakness and a decrease in quality of life in patients. Neuropathic pain affects 7-8% of the European population. Neuropathic pain can be caused by diseases of the peripheral nervous system (PNS) or the central nervous system (CNS). Thus, neuropathic pain can be divided into peripheral neuropathic pain, central neuropathic pain or mixed (peripheral and central) neuropathic pain. For example, CNS pain is present in spinal cord injury, multiple sclerosis and certain strokes, while PNS pain is usually present in peripheral neuropathies caused by diabetes, metabolic disorders, viral infections (shingles, HIV...), nutritional deficiencies, toxins, distant manifestations of malignancies, immune-mediated diseases and body trauma of the nerve trunk. Neuropathic pain is also common in cancer, this pain being either a direct consequence of the cancer on the peripheral nerves (e.g. tumor compression), or a side effect of chemotherapy (chemotherapy-induced peripheral neuropathy), radiation damage or surgery.

[0220] The involvement of NR2B-containing NMDARs in neuropathic pain has been demonstrated (Aiyer et al., Clin J Pain. A systematic review of NMDA receptor antagonists for treatment of neuropathic pain in clinical practice. 2017; Qu et al., Exp. Neurology. Role of the spinal cord NR2B-containing NMDA receptors in the development of neuropathic pain 2009; 215:298-307).

[0221] According to one preferred embodiment, the present application relates to a method for preventing or treating a disease, disorder or medical condition mediated by NR2B-containing NMDA receptor activity, selected from the group consisting of pain, hyperalgesia, nociception, acute pain, chronic pain, cancer-related pain and neuropathic pain.

[0222] According to one preferred embodiment, the disease, disorder or medical condition is selected from the group consisting of pain, hyperalgesia, nociception, acute pain, chronic pain (e.g. chronic pain after a bone fracture), cancer-related pain (e.g. cancer-induced bone pain and neuropathic pain).

[0223] In one embodiment, the pain is hyperalgesia. According to a preferred embodiment, the hyperalgesia is selected from the group comprising opioid-induced hyperalgesia, hyperalgesia induced by other analgesics, preferably analgesics acting on glutamate neurotransmission, or hyperalgesia induced by chemotherapy drugs or any other drugs.

[0224] According to a preferred embodiment, the present application relates to a method for preventing, treating or alleviating a symptom of pain, wherein the subject is selected from the group consisting of a patient suffering from neuropathic pain.

[0225] In one embodiment, the neuropathic pain is peripheral neuropathic pain.

[0226] In one embodiment, the neuropathic pain is central neuropathic pain.

[0227] In one embodiment, the neuropathic pain is chronic neuropathic pain.

[0228] In one embodiment, the neuropathic pain is refractory neuropathic pain.

[0229] In one embodiment, the neuropathic pain is neuropathic pain associated with metabolic dysfunction, such as diabetes and prediabetes.

[0230] In one embodiment, the neuropathic pain is neuropathic pain associated with painful polyneuropathy.

[0231] In one embodiment, the neuropathic pain is neuropathic pain associated with diabetic neuropathy, including for example diabetic peripheral neuropathy.

[0232] In one embodiment, the neuropathic pain is neuropathic pain associated with post-herpetic neuralgia, trigeminal neuralgia or occipital neuralgia.

[0233] In one embodiment, the neuropathic pain is associated with painful radiculopathy, for example including lumbar and cervical radiculopathy.

[0234] In one embodiment, the neuropathic pain is neuropathic pain associated with infectious diseases, for example shingles, HIV infection, Lyme disease, diphtheria and leprosy.

[0235] In one embodiment, the neuropathic pain is neuropathic pain associated with liver or kidney diseases, for example including chronic liver or kidney diseases, including liver diseases, liver failure, kidney diseases and kidney failure.

[0236] In one embodiment, the neuropathic pain is neuropathic pain associated with immune or inflammatory diseases, for example including Guillain-Barre syndrome, rheumatoid arthritis, lupus, Sjogren's syndrome and celiac disease.

[0237] In one embodiment, the neuropathic pain is neuropathic pain associated with a genetic neuropathy or channelopathy, including hereditary erythromelalgia, paroxysmal extreme pain disorder, and Charcot-Marie-Tooth disease (CMT).

[0238] In one embodiment, the neuropathic pain is neuropathic pain associated with small fiber sensory neuropathy.

[0239] In one embodiment, the neuropathic pain is neuropathic pain associated with thyroid hormone dysregulation (e.g., hypothyroidism).

[0240] In one embodiment, the neuropathic pain is neuropathic pain associated with stroke.

[0241] In one embodiment, the neuropathic pain is neuropathic pain associated with cancer, e.g., lymphoma and multiple myeloma. In one embodiment, the neuropathic pain is neuropathic pain associated with chemotherapy (e.g., cancer chemotherapy or chemotherapy-induced peripheral neuropathy).

[0242] In one embodiment, the neuropathic pain is neuropathic pain associated with peripheral nerve injury pain.

[0243] In one embodiment, the neuropathic pain is neuropathic pain associated with post-traumatic nerve injury.

[0244] In one embodiment, the neuropathic pain is neuropathic pain associated with post-traumatic neuropathy.

[0245] In one embodiment, the neuropathic pain is neuropathic pain associated with spinal cord injury, e.g., including spinal cord injury resulting from trauma, e.g., road traffic accident.

[0246] In one embodiment, the neuropathic pain is neuropathic pain associated with traumatic peripheral nerve injury.

[0247] In one embodiment, the neuropathic pain is neuropathic pain associated with post-surgical neuropathy (e.g., post-surgical neuropathic pain).

[0248] In one embodiment, the neuropathic pain is post-surgical neuropathic pain, e.g., including neuropathic pain following nerve surgery (including spinal cord surgery).

[0249] In one embodiment, the neuropathic pain is neuropathic pain associated with fibromyalgia.

[0250] In one embodiment, the neuropathic pain is neuropathic pain associated with lower back pain.

[0251] In one embodiment, the neuropathic pain is neuropathic pain associated with carpal tunnel syndrome.

[0252] In one embodiment, the neuropathic pain is neuropathic pain associated with causalgia.

[0253] In one embodiment, the neuropathic pain is neuropathic pain associated with reflex sympathetic dystrophy.

[0254] In one embodiment, the neuropathic pain is neuropathic pain associated with complex regional pain syndrome (CRPS), e.g., including type 1 and type 2.

[0255] In one embodiment, the neuropathic pain is neuropathic pain associated with amputation.

[0256] In one embodiment, the neuropathic pain is neuropathic pain associated with neurodegenerative diseases, e.g., Parkinson's disease and amyotrophic lateral sclerosis.

[0257] In one embodiment, the neuropathic pain is neuropathic pain associated with stroke, including, e.g., central post-stroke pain.

[0258] In one embodiment, the neuropathic pain is neuropathic pain associated with syringomyelia.

[0259] In one embodiment, the neuropathic pain is neuropathic pain associated with demyelinating diseases, e.g., multiple sclerosis, transverse myelitis and neuromyelitis optica.

[0260] In one embodiment, the neuropathic pain is idiopathic neuropathic pain.

[0261] According to a preferred embodiment, the present application relates to the prevention, treatment or alleviation of the symptoms of pain in a subject having ALS in need of such treatment.

[0262] According to a preferred embodiment, the present application relates to the prevention, treatment or alleviation of the symptoms of pain in a subject having CMT in need of such treatment.

[0263] According to a preferred embodiment, the present application relates to the prevention, treatment or alleviation of the symptoms of hyperalgesia in a subject having CMT in need of such treatment.

[0264] According to a preferred embodiment, the present application relates to the prevention, treatment or alleviation of neuropathic pain caused by hyperalgesia in a CMT patient, preferably a CMT1A patient.

[0265] According to a preferred embodiment, the present application relates to the prevention, treatment or alleviation of the symptoms of pain in a subject having MS in need of such treatment.

[0266] According to a preferred embodiment, the present application relates to preventing, treating or alleviating the symptoms of pain in a subject suffering from PD in need of such treatment.

[0267] Pseudobulbar affect (PBA)

[0268] Pseudobulbar affect (PBA) is a condition affecting people with certain neurological diseases or brain injuries that causes involuntary, sudden and frequent crying and / or laughing that is out of proportion to the emotion experienced. PBA is also known as pathological laughing and crying, emotional lability, emotional incontinence, emotionalism and involuntary emotional expression disorder. PBA occurs in neurological diseases such as ALS, MS, dementia, PD, atypical parkinsonism (e.g. progressive supranuclear palsy), traumatic focal brain injury, tumors and stroke (estimated between 11% to 52%). PBA affects up to 49% of ALS patients and is more common in patients with bulbar onset of the disease (Gallagher et al., Pathological Laughter and crying in ALS: a search for their origin. Acta Neurol. Scand. 1989; 80(2): 114-117). Recent studies suggest that the lifetime prevalence of PBA in Alzheimer's disease is about 10% to 74%, in multiple sclerosis patients 10% and is associated with more severe cognitive, physical and neurological disability (Schiffer et al., Review of pseudobulbar affect including a novel and potential therapy. J. Neuropsychiatry Clin Neurosci. 2005; 17(4): 447-454). PBA can have a major impact on the quality of life of patients.

[0269] Dextromethorphan is a non-competitive NMDAR antagonist and its use in combination with quinidine sulfate (Q) is the only treatment for PBA approved by the US Food and Drug Administration and the EMA.

[0270] According to a preferred embodiment, the present application relates to a method of preventing, treating or alleviating pseudobulbar affect (PBA) or a symptom thereof in a subject in need of such treatment, wherein said subject is selected from the group consisting of:

[0271] a) amyotrophic lateral sclerosis (ALS), bulbar onset ALS, spinal onset ALS, primary lateral sclerosis (PLS), progressive muscular atrophy (PMA) or frontotemporal spectrum disorder; or

[0272] b) multiple sclerosis (MS); or

[0273] c) atypical Parkinsonism (progressive supranuclear palsy (PSP)), frontotemporal dementia (FTD) and corticobasal degeneration (CBD);

[0274] d) Alzheimer’s disease, or dementia, or Alzheimer’s disease dementia agitation; or

[0275] e) focal brain injury caused by trauma, tumor or stroke.

[0276] Dementia and Alzheimer’s Disease (AD)

[0277] Dementia is a general term for memory loss, language, problem solving and other thinking abilities severe enough to interfere with daily life. AD is the most common cause of dementia. Memantine is a low-affinity NMDAR channel blocker that has been used to treat moderate to severe AD (Liu J. The Role of NMDA Receptors in Alzheimer’s Disease. Front Neurosci. 2019; 13:43). The Tau mutation A152T (hTauAT) is a risk factor for frontotemporal dementia (FTD) spectrum disorders, including progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), which leads to excitotoxicity mediated by NMDA receptors containing NR2B due to enhanced extracellular glutamate (Decker JM. The Tau / A152T mutation, a risk factor for frontotemporal-spectrum disorders, leads to NR2B receptor-mediated excitotoxicity. EMBO Reports (2016) 17:552-569)

[0278] According to one preferred embodiment, the present application relates to a method of preventing or treating a disease, disorder or medical condition mediated by NR2B containing NMDA receptor activity, wherein the disease, disorder or medical condition is selected from the group consisting of Alzheimer’s disease, dementia, frontotemporal dementia (FTD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD).

[0279] Amyotrophic Lateral Sclerosis (ALS)

[0280] Amyotrophic lateral sclerosis is a fatal neurodegenerative disease with striking phenotypic heterogeneity, the cause of which is the selective loss of upper and lower motor neurons (Grad et al. Clinical Spectrum of Amyotrophic Lateral Sclerosis (ALS), Cold Spring Harb Perspect Med. August 2017; 7(8): a024117). There is compelling evidence that both direct and indirect toxicity of glutamate contributes to the pathology of motor neuron degeneration. Memantine, a non-competitive NMDAR antagonist, has been shown to protect neurons from NMDA or glutamate-induced toxicity in vitro and in animal models of ALS (Wang et al. Memantine prolongs survival in an amyotrophic lateral sclerosis mouse model. Eur J Neurosc. 2005; 22: 2376-2380).

[0281] According to one preferred embodiment, the present application relates to the prevention or treatment of a disease, disorder or medical condition mediated by NR2B-containing NMDA receptor activity, selected from the group consisting of ALS, and its multiple phenotypes, such as bulbar onset ALS, spinal onset ALS, primary lateral sclerosis (PLS), progressive muscular atrophy (PMA) and frontotemporal spectrum disorder.

[0282] Multiple sclerosis (MS)

[0283] Multiple sclerosis is a neurodegenerative disease caused by an autoimmune reaction against myelin in the central nervous system. Glutamate excitotoxicity is a pathophysiological process that is believed to play a role in the pathophysiology of multiple sclerosis, such as myelin breakdown, blood brain barrier disruption, neurovascular damage, cell death and axonal degeneration. Non-selective NMDAR antagonists, such as memantine and MK-801, and the selective antagonist of NR2B-containing NMDARs, Ro25-6981, can be effective in modulating disease in animal models of MS (Farjam et al. Inhibition of NR2B-containing N-Methyl-D-Aspartate Receptors (NMDARs) in experimental autoimmune encephalomyelitis, a model of multiple sclerosis Iran J Pharm Res. 2014 Spring; 13(2): 695-705.). According to a preferred embodiment, the present application relates to the prevention or treatment of a disease, disorder or medical condition mediated by NR2B-containing NMDA receptor activity, selected from the group consisting of multiple sclerosis and optic neuritis.

[0284] Dyskinesia and Parkinson’s disease (PD)

[0285] The term dyskinesia is used to describe unexpected, involuntary and uncontrollable movements, including jerking, jerking, twisting or simple restlessness. Dyskinesia is a symptom of several medical conditions, which are distinguished by their underlying cause. For example, in PD, dyskinesia is associated with long-term use of certain medications, including levodopa. Less commonly, dyskinesia also occurs when levodopa is first effective or gradually disappears, which is known as “diphasic dyskinesia”. Non-selective NMDAR antagonists, such as MK-801, have been shown to be effective in modulating levodopa-induced dyskinesia (Wang XS et al. Modulation of CaMKIIa-GluN2B interaction in levodopa-induced dyskinesia in 6-OHDA-lesioned Parkinson’s rats. Biomed Pharmacother 2018 107: 769-776). Patients with Huntington’s disease (HD) also exhibit dyskinesia, known as chorea.

[0286] According to a preferred embodiment, the present application relates to a method of preventing, treating or alleviating a movement disorder or chorea in a subject selected from the group consisting of patients suffering from Parkinson's disease, atypical Parkinsonism and Huntington's disease. Atypical Parkinsonism includes multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), frontotemporal lobar degeneration (FTLD), Lewy body dementia (DLB).

[0287] According to a preferred embodiment, the movement disorder is levodopa-induced dyskinesia. According to a preferred embodiment, the chorea is Huntington's chorea or HD. In this context, movement disorder and chorea have the same meaning.

[0288] Ataxia

[0289] Spinocerebellar ataxias (SCA) are a group of heterogeneous inherited autosomal dominant diseases and are the leading cause of cerebellar ataxia. Although SCAs are associated with mutations in various genomic loci, they share common pathogenic pathways (Perkins et al. Cerebellar ataxias: β-III spectrin’s interactions suggest common pathogenic pathways. J. Physiol. 2016, 594, 4661-4676). Impaired expression and / or function of glutamate transporters, excitatory amino acid transporters, in particular EAAT1 and EAAT4, are one of the important factors leading to Purkinje cell degeneration in SCAs (Kasumu et al. Deranged calcium signaling in Purkinje cells and pathogenesis in spinocerebellar ataxia 2 (SCA2) and other ataxias, Cerebellum 2012 11(3):630-639). Defective calcium buffering can also be a direct or indirect triggering event (Maltecca et al. Purkinje neuron Ca2+ influx reduction rescues ataxia in SCA28 model).

[0290] Friedreich's ataxia (FA) is one of the most common ataxias and is caused by mutations in the FXN gene; the dynamic balance of calcium is also impaired in FA.

[0291] In a randomized, double-blind, placebo-controlled trial in patients with ataxia for different etiologies (e.g. Friedreich’s ataxia and SCA), the antiglutamatergic compound riluzole was identified to improve ataxia symptoms (Ristori et al. Riluzole in cerebellar ataxia: a randomized, double-blind, placebo-controlled pilot trial. Neurology 2010 74(10) 839-45; Romano et al. Riluzole in patients with hereditary cerebellar ataxia: a randomised, double-blind, placebo-controlled trial, Lancet Neurol 2015; 14:985-91).

[0292] According to one preferred embodiment, the present application relates to a method of preventing or treating a disease, disorder or medical condition mediated by NR2B containing NMDA receptor activity selected from the group consisting of SCA1, SCA2, SCA3, SCA5, SCA6, SCA7, SCA14, SCA15, SCA16, SCA28, spastic ataxia 5 (SPAX5), episodic ataxia 6 (EA6) and Friedreich’s ataxia.

[0293] According to one preferred embodiment, the ataxia is Friedreich’s ataxia.

[0294] According to one preferred embodiment, the ataxia is spinocerebellar ataxia SCA28 or SPAX5.

[0295] Neuronal injury, stroke

[0296] To respond to PNS injury, Schwann cells dedifferentiate and acquire the ability to migrate and proliferate. Activated Schwann cells have functions necessary for nerve repair, including phagocytosis of debris, secretion of trophic factors and deposition of provisional extracellular matrix proteins. In rat models, NMDAR NR1 and NR2B subunits are expressed in Schwann cells and are upregulated in the sciatic nerve after crush injury.

[0297] These results define NMDARs as Schwann cell signaling receptors for protein ligands and major regulators of Schwann cell physiology, which can be particularly important in peripheral nervous system (PNS) injury.

[0298] According to one preferred embodiment, the present application relates to a method for preventing or treating a disease, disorder or medical condition mediated by NR2B-containing NMDA receptor activity, selected from the group consisting of:

[0299] a) brain or spinal cord injury, PNS injury, cerebral ischemia, head or neuronal trauma, neuronal hemorrhage, neuronal ischemia, reperfusion injury, neuronal injury, polyneuropathy; or

[0300] b) neuronal exposure to toxic substances, methamphetamine-induced neurotoxicity; or

[0301] c) stroke, cardiogenic shock, coronary artery bypass graft (CABG) surgery related neurological injury.

[0302] According to one preferred embodiment, the neuronal injury is a polyneuropathy selected from the group consisting of diabetic neuropathy, alcohol-induced polyneuropathy, chemotherapy-induced neuropathy and other toxic neuropathies, immune-mediated neuropathies (e.g. Guillain-Barre syndrome, chronic inflammatory radiculoneuropathy) and hereditary neuropathies, such as Charcot-Marie-Tooth disease.

[0303] Kidney injury

[0304] NMDARs are expressed in the renal cortex and medulla, playing a role in the regulation of renal blood flow, glomerular filtration, proximal tubule reabsorption and urine concentration in the medullary collecting duct (Valdivielso et al. Glutamate-Gated NMDA Receptors: Insights into the Function and Signaling in the Kidney Biomolecules 2020, 10, 1051). Prolonged activation of NMDARs induces Ca 2+The influx and oxidative stress, which can lead to glomerulosclerosis, for example in hyperhomocysteinemia (Dryer S. Glutamate receptors in the kidney, Nephrol Dial Transplant. 2015 Oct; 30(10): 1630-8). Acute kidney injury (AKI), formerly known as acute renal failure, is a clinical syndrome characterized by a sudden decrease in kidney function, measured by an increase in creatinine or a decrease in urine output. One of the main causes of AKI is renal ischemia-reperfusion injury, which is associated with a reduction in the supply of oxygen and nutrients, leading to apoptosis and necrosis of renal tubular cells and subsequent impairment of kidney function. AKI is associated with activation of NMDARs and oxidative stress. Various NMDAR antagonists can attenuate ischemia-reperfusion-induced AKI and reduce oxidative stress, suggesting that antagonism of various allosteric sites of NMDARs has a beneficial effect on IR-induced AKI (Pundir et al. Effect of modulating the allosteric sites of N-methyl-D-aspartate receptors in ischemia reperfusion induced acute kidney injury, J Surg Res 2013; 183: 668-77).

[0305] According to one preferred embodiment, the present application relates to a method for preventing or treating a disease, disorder or medical condition mediated by NR2B-containing NMDA receptor activity, selected from the group consisting of: kidney injury, preferably from acute kidney injury, ischemia-reperfusion-induced AKI, parathyroid hyperfunction associated with chronic kidney disease (CKD), glomerulosclerosis and hyperhomocysteinemia-induced glomerulosclerosis.

[0306] Myocardial pathogenesis

[0307] It is believed that NMDAR activation plays an important role in myocardial pathogenesis; it promotes ventricular arrhythmias, oxidative stress and autophagy of cardiomyocytes (Bozic and Valdivielso, The potential of targeting NMDA receptors outside the CNS, Expert Opin Ther Targets 2014 19(3)).

[0308] According to one preferred embodiment, the present application relates to a method of preventing or treating a disease, disorder or medical condition mediated by NR2B containing NMDA receptor activity selected from the group consisting of: myocardial pathology, reperfusion-induced cardiac arrhythmias, myocardial cardiac arrhythmias, ventricular cardiac arrhythmias, ventricular tachycardia, bradycardia.

[0309] Osteopathy

[0310] Glutamate release by chondrocytes, osteoclasts and osteoblasts plays an important role in the regulation of bone remodeling through the activation of glutamate transporters, metabolic and ionotropic receptors expressed in bone cells (Itzstein et al., Molecular identification of NMDA glutamate receptors expressed in bone cells, 2001 J. Cell Biochem., 82, 134-144.). Pharmacological inhibition of osteoclast NMDARs leads to a decrease in bone resorption in vitro, and load-induced bone mass increase is associated with a decrease in osteoclast NMDARs in vivo. This suggests that NMDAR antagonists can be beneficial for osteoporosis. According to one preferred embodiment, the present application relates to a method for preventing, treating or alleviating osteopenia or osteoporosis or a symptom thereof in a subject in need of such treatment. According to one embodiment, the subject is selected from the group consisting of: a woman and a postmenopausal woman.

[0311] ALS patients, especially women, have a deteriorated bone health status compared to healthy people, and a high incidence of bone fractures is found in ALS patients (Sato et al., Etidronate for fracture prevention in amyotrophic lateral sclerosis: a randomized controlled trial. Bone. 2006 Nov;39:1080-6).

[0312] According to one preferred embodiment, the present application relates to a method for preventing, treating or alleviating osteopenia or osteoporosis or a symptom thereof in a subject selected from the group consisting of: a patient suffering from ALS, bulbar onset ALS, spinal onset ALS, primary lateral sclerosis (PLS), progressive muscular atrophy (PMA) and frontotemporal spectrum disorder.

[0313] According to one embodiment, the present application relates to a method for enhancing bone formation, increasing bone density, stimulating osteoblast differentiation or inhibiting osteoclast differentiation in a subject in need thereof, comprising administering to said mammal an effective amount of a compound of general formula (I):

[0314]

[0315] and (Z) and / or (E) isomers thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof,

[0316] wherein: R1, R2, R3, R4, R5are independently hydrogen, deuterium, halogen, haloalkyl, alkyl, alkoxy, hydroxyl, aryl, or aryloxy, wherein the subject has (a) a bone fracture, bone injury, or bone defect condition associated with post-traumatic bone surgery, post-prosthetic joint replacement, post-orthopedic bone surgery, post-dental surgery, bone chemotherapy treatment or bone radiation treatment and / or (b) a bone remodeling disorder selected from the group consisting of osteoporosis, Paget's disease, achondroplasia, osteochondritis, hyperparathyroidism, osteogenesis imperfecta, congenital hypophosphatasia, fibrous dysplasia, fibrous structural dysplasia, multiple myeloma, abnormal bone turnover, osteolytic bone disease, osteomalacia, and periodontal disease

[0317] Idiopathic pulmonary fibrosis (IPF) and chronic cough IPF is a serious chronic disease that affects the tissue around the air sacs or alveoli in the lungs. The most common symptoms of IPF are shortness of breath and cough. The NR2B-selective NMDAR antagonist anatabine was shown to be effective in a phase 2a study in patients with IPF (Algernon Pharmaceuticals, Vancouver, British Columbia).

[0318] According to one preferred embodiment, the present application relates to the prevention or treatment of a disease, disorder or medical condition mediated by NR2B-containing NMDA receptor activity, selected from idiopathic pulmonary fibrosis (IPF) and chronic cough.

[0319] Treatment and prevention

[0320] As described above, the present application relates to certain compounds and pharmaceutical compositions comprising these compounds, which are useful in the treatment or prevention of a disease, disorder or medical condition, as described herein.

[0321] For example, the compounds can be administered at or shortly after diagnosis or discovery of the disease, disorder or medical condition, to prevent or lessen the development of the disease, disorder or medical condition. Alternatively, the compounds can be administered during the disease, disorder or medical condition.

[0322] Dosing

[0323] The compounds of the present application can be used for oral, rectal, nasal, intrabronchial, topical (including buccal, sublingual, and intraocular), vaginal or parenteral administration (including subcutaneous, intramuscular, intravenous, intra-arterial and intradermal), intraperitoneal or intrathecal administration. The formulations are preferably those suitable for oral administration. The formulations can conveniently be presented in unit dosage form, e.g. in the form of discrete portions containing a unit or a fraction of a unit dose. For example, the formulations can be in the form of tablets and sustained release capsules, and can be prepared by any of the methods known in the pharmaceutical art.

[0324] Dose

[0325] A person of ordinary skill in the art can readily determine an appropriate dose of a compound of the present application for administration to a subject. Typically, a physician will determine the actual dosage which will be appropriate in individual cases. This will depend on a variety of factors including the activity of the particular compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health condition, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition being treated and the individual undergoing therapy. The dosages disclosed herein are exemplary of the active amounts of the compounds of the present application that can be used in the treatment of something. Of course, individual requirements vary depending on the condition being treated, the stage and severity of such condition, and the age, body weight, general health condition, sex, diet, mode and time of administration, rate of excretion, drug combination, and the like, of the individual being treated. Deviations from the preferred dosage are deemed to be within the scope of the present application.

[0326] According to the present application, an amount of a compound of the present application can be administered to target a particular condition or disease. Of course, this dosage will be further adjusted depending on the type of administration of the compound. For example, for an "effective amount" for acute treatment, parenteral administration of the combination of the present application is preferred. The precise dosage and optimal route of administration are readily determined by one of ordinary skill in the art through comparison of the drug's blood levels to the concentration required to produce a therapeutic effect.

[0327] The compounds of the present application can also be administered to a patient in such a way that the concentration of the drug is sufficient to achieve one or more of the therapeutic indications disclosed herein.

[0328] When the compounds of the present application are administered according to the present application, it is not expected that unacceptable toxicological effects will occur. The compounds of the present application can have good bioavailability, which can be tested in one of several bioassay methods to determine the concentration of the compound that must have a given pharmacological effect.

[0329] Salts

[0330] The compounds of the present application can exist in the form of salts, in particular pharmaceutically and veterinarily acceptable salts.

[0331] Pharmaceutically acceptable salts of the compounds of the present application include the appropriate acid addition or base salts thereof. A review of suitable pharmaceutical salts can be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed by strong inorganic acids, such as mineral acids, for example hydrohalic acids, such as hydrochloric, hydrobromic and hydroiodic acid, sulfuric acid, phosphoric acid, sulfates, bisulfates, hemisulfates, thiocyanates, persulfates and sulfonic acids; strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (for example, by halogen), such as acetic acid; saturated or unsaturated dicarboxylic acids, for example, oxalic, malonic, succinic, maleic, fumaric, phthalic or terephthalic acid; with hydroxycarboxylic acids, for example, ascorbic, glycolic, lactic, malic, tartaric or citric acid; with amino acids, for example, aspartic or glutamic acid; with benzoic acids; or with organic sulfonic acids, for example, (C1-C4)-alkyl or arylsulfonic acids which are unsubstituted or substituted (for example, by halogen), such as methanesulfonic acid or p-toluenesulfonic acid. Pharmaceutically or veterinarily unacceptable salts can still be of value as intermediates.

[0332] For example, preferred salts include acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanoate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmitoate, pectinoate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, lactobionate, polypropionate, camphorate, undecanoate and succinate, organic sulfonic acids, such as methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, camphorsulfonate, 2-naphthalenesulfonate, benzenesulfonate, p-chlorobenzenesulfonate and p-toluenesulfonate; and inorganic acids, such as hydrochloride, hydrobromide, hydroiodide, bisulfate, bisulfite, bisulfate, hemisulfate, thiocyanate, persulfate, phosphoric acid and sulfonic acid.

[0333] Tautomers

[0334] In all aspects of the application discussed above, the present application includes all tautomers of the compounds of the present application, as appropriate. Those skilled in the art will recognize compounds having tautomeric properties. The corresponding tautomers can be isolated / prepared by known methods.

[0335] The compounds of formula (I) can include tautomeric forms of the following formulae:

[0336]

[0337] For example, tautomeric forms of Compound 1 (guanabenz) are:

[0338]

[0339] As another example, the tautomers of compound 2 are:

[0340]

[0341] Geometric isomers

[0342] Certain compounds of the present application can exist in geometric isomeric forms. They can have one or more geometric centers and thus can exist in two or more geometric forms. The double bond between the benzylidene and guanidine moieties (-HC=N- bond) enables the compounds of formula (I) to exist as E- or Z-isomers. For certain compounds, there is a high thermal isomerization barrier between the two isomers; thus, spontaneous isomerization of compound 1 (guanabenz) in solid and solution states is almost negligible (Xie et al., J Pharma Biomed Analysis. LC-MS / MS determination of guanabenz E / Z isomers and its applications to in vitro and in vivo DMPK profiling studies 2021, 205). By way of example, the geometric isomeric forms of compound 1 are:

[0343]

[0344] (Deepika K. et al., Crystal Growth & Design. Geometrical Isomerism in Guanabenz Free Base Synthesis, Characterization, Crystal Structure and Theoretical Studies 2019). The present application contemplates the use of all individual stereoisomers and geometric isomers of these inhibitors and mixtures thereof. The term as used in the claims includes these forms, provided that the above-mentioned forms retain the appropriate functional activity (although not necessarily to the same extent).

[0345] The present application also includes all suitable isotopic variations of the agent or a pharmaceutically acceptable salt thereof. An isotopic variation of the agent or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be present in the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, such as2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, 18 F and 36 Cl. Certain isotopic variants of the agent, and pharmaceutically acceptable salts thereof, for example, containing a positron-emitting isotope (e.g., 3 H or 14 C) or a non-radioactive isotope (e.g., 13 C) are useful in drug and / or substrate tissue distribution studies. Tritium (i.e., 3 H) and carbon-14 (i.e., 14 C) isotopes are particularly preferred because of their ease of preparation and detectability. Substitution with isotopes such as deuterium (i.e., 2 H) may afford certain therapeutic advantages due to greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence can be preferred in some circumstances. For example, the application includes compounds of general formula (I) wherein any hydrogen atom has been replaced by a deuterium atom. Isotopic variants of agents of the application, and pharmaceutically acceptable salts thereof, can generally be prepared by conventional procedures using appropriate isotopic variants of suitable reagents.

[0346] Formulations

[0347] For use according to the application, the compounds or physiologically acceptable salts or other physiologically functional derivatives described herein can be provided as pharmaceutical formulations including the compound or physiologically acceptable salt or other physiologically functional derivative thereof, together with one or more pharmaceutically acceptable carriers therefore, and optionally other therapeutic and / or prophylactic ingredients. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject in need thereof. The pharmaceutical composition can be for use in humans or animals, in human and veterinary medicine.

[0348] Examples of such excipients suitable for use in the various forms of pharmaceutical compositions described herein can be found in the Handbook of Pharmaceutical Excipients, 2ndEdition, Edited by A Wade and PJ Weller (1994).

[0349] Acceptable therapeutic carriers or diluents for the pharmaceutical compositions are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.

[0350] The pharmaceutical carrier, excipient or diluent can be chosen according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions can include any suitable binder, lubricant, suspending agent, coating agent, solubilizing agent, buffer, flavoring agent, surface active agent, thickening agent, preservative (including antioxidant), etc., and substances that are included to make the formulation isotonic with the blood of the intended recipient, in addition to, or in place of, the carrier, excipient or diluent.

[0351] Examples of suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, lactose such as anhydrous, free-flowing, beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose and polyethylene glycol.

[0352] Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical compositions. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents can also be used.

[0353] The pharmaceutical formulations include those suitable for oral, topical (including buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular, and intravenous) administration, nasal, intraocular, and pulmonary (e.g., inhalation) administration. The formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. All methods include the step of bringing the active compound(s) into association with the carrier or diluent and, where necessary, the step of bringing the product into a desired form.

[0354] For pharmaceutical formulations that are suitable for oral administration in which the carrier is a solid, it is preferable to present the active agent in a unit dosage form, such as, for example, a pill, capsule, or tablet, each containing a predetermined amount of the active agent. Tablets can be made by compression or molding, the pharmaceutical compositions utilizing one or more excipients. Compressed tablets can be prepared by compressing, in a suitable machine, the active agent in a free-flowing form, such as, for example, a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface-active, or dispersing agent. Molded tablets can be made by molding, in a suitable machine, the active agent in the form of a soft mass, using an inert liquid diluent. Tablets can optionally be coated or scored for ease of breaking. Capsules are made by preparing the active agent, alone or in combination with one or more excipients, and then encasing it in a hard or soft shell, which is then sealed. Sachets, which contain the active agent in the form of a powder or granules, are made by sealing, in a suitable manner, the active agent, with or without the addition of an excipient, in a small bag. The active agent can also be presented as a dispersion, such as, for example, for suspension in water or for application to the food of the subject prior to administration. Granules can be packaged, such as, for example, in sachets. Pharmaceutical formulations that are suitable for oral administration in which the carrier is a liquid can be presented as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion, or as a water-in-oil liquid emulsion.

[0355] The pharmaceutical formulations of the present application can be presented as discrete units, such as, for example, capsules, gelatin, drops, pouches, pills, or tablets, each containing a predetermined amount of the active agent; as a powder or granules; as a solution, emulsion, or suspension of the active agent in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; or as a pill, and the like. For oral administration compositions, such as tablets and capsules, the term "acceptable carrier" includes such diluents as binders, such as syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose, and starch; fillers and carriers, such as corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and alginic acid; and lubricants, such as magnesium stearate, sodium stearate, and other metallic stearates, glycerol stearate, stearic acid, silicone fluid, talc, waxes, oils, and colloidal silica. Flavoring agents, such as peppermint, oil of wintergreen, cherry flavoring, and the like, can also be used. It can be desirable to add a coloring agent to make the dosage form readily identifiable. Tablets can also be coated by methods well known in the art.

[0356] Tablets can optionally be coated or scored and formulated to provide slow or controlled release of the active agent.

[0357] Other pharmaceutical formulations suitable for oral administration include lozenges containing the active agent, usually sucrose and acacia or tragacanth; pastilles containing the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes containing the active agent in a suitable liquid carrier.

[0358] Oral formulations include controlled release dosage forms, such as tablets, wherein the active compound is formulated with an appropriate release controlling matrix, or coated with a suitable release controlling film.

[0359] Other forms of administration include solutions or emulsions which can be injected intravenously, intra-arterially, intrathecally, subcutaneously, intradermally, intraperitoneally, intraocularly, topically, peri-ocularly or intramuscularly, made from sterile or sterilisable solutions. Injectable forms generally contain from 10 to 1000 mg of active ingredient, preferably between 10 and 250 mg.

[0360] The pharmaceutical compositions of the present application can also be in the form of a suppository, pessary, suspension, emulsion, lotion, ointment, cream, gel, spray, solution, or dusting powder.

[0361] Another method of transdermal administration is the use of a skin patch.

[0362] Pharmaceutical formulations adapted for parenteral administration include aqueous or oil suspensions, or emulsions, as well as sterile liquid solutions, including suspensions of the active compounds in aqueous or non-aqueous vehicles, or diluents.

[0363] Injectable preparations can be adapted for bolus injection or continuous infusion. Alternatively, the active compound can be in powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0364] The active compound can also be formulated in a depot 5 form, to be administered by injection or implantation (e.g., subcutaneously or intramuscularly). For example, the depot 10 form can comprise a suitable polymer or hydrophobic material or ion exchange resin.

[0365] Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M, and preferably 0.05 M, phosphate buffers, or 0.8% saline. Additionally, such pharmaceutically acceptable carriers can be aqueous, non-aqueous, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, lactated Ringer's, or fixed oils. Preservatives and other additives can also be present such as, for example, anti-bacterial agents, anti-oxidants, chelating agents, inert gases, and the like. According to another aspect of the present application, there is provided a process for the preparation of the above pharmaceutical or veterinary compositions, comprising bringing the active compound into association with the carrier by means of an excipient.

[0366] Generally, the methods of preparing formulations are those conventionally used for compounding pharmaceutically acceptable carriers with active agent. The present application extends to methods of preparing a pharmaceutical composition comprising combining a compound of Formula (I) with a pharmaceutically or veterinarily acceptable carrier or vehicle. BRIEF DESCRIPTION OF DRAWINGS

[0367] The present application is further described with reference to the following figures, in which:

[0368] Figure 1 Reduction of intracellular calcium flux after treatment with different concentrations of Compound 1 (A), 2 (B) and 3 (C) in a recombinant NMDAR expressing HEK293 cell line stimulated with glutamate. Data shown have been normalized to the maximum and minimum responses observed in the presence of control ligand (MK-801) and vehicle (y-axis), respectively, and the corresponding compound concentrations (in nM) are plotted on a log10 scale (x-axis). Figure 1 Figure 1 Figure 1 Reduction of intracellular calcium flux after treatment with different concentrations of Compound 1 (A), 2 (B) and 3 (C) in a recombinant NMDAR expressing HEK293 cell line stimulated with glutamate. Data shown have been normalized to the maximum and minimum responses observed in the presence of control ligand (MK-801) and vehicle (y-axis), respectively, and the corresponding compound concentrations (in nM) are plotted on a log10 scale (x-axis).

[0369] Figure 2 Reduction of intracellular calcium flux after treatment with different concentrations of Compound 1 (A), 2 (B) and 3 (C) in a recombinant NMDAR expressing HEK293 cell line stimulated with glutamate. Data shown have been normalized to the maximum and minimum responses observed in the presence of control ligand (MK-801) and vehicle (y-axis), respectively, and the corresponding compound concentrations (in nM) are plotted on a log10 scale (x-axis). Figure 2 Figure 2 Figure 2 Reduction of intracellular calcium flux after treatment with different concentrations of Compound 1 (A), 2 (B) and 3 (C) in a recombinant NMDAR expressing HEK293 cell line stimulated with glutamate. Data shown have been normalized to the maximum and minimum responses observed in the presence of control ligand (MK-801) and vehicle (y-axis), respectively, and the corresponding compound concentrations (in nM) are plotted on a log10 scale (x-axis). Figure 2 Figure 2 Figure 2 Reduction of intracellular calcium flux after treatment with different concentrations of Compound 1 (A), 2 (B) and 3 (C) in a recombinant NMDAR expressing HEK293 cell line stimulated with glutamate. Data shown have been normalized to the maximum and minimum responses observed in the presence of control ligand (MK-801) and vehicle (y-axis), respectively, and the corresponding compound concentrations (in nM) are plotted on a log10 scale (x-axis). Figure 2 Figure 2 Figure 2 Reduction of intracellular calcium flux after treatment with different concentrations of Compound 1 (A), 2 (B) and 3 (C) in a recombinant NMDAR expressing HEK293 cell line stimulated with glutamate. Data shown have been normalized to the maximum and minimum responses observed in the presence of control ligand (MK-801) and vehicle (y-axis), respectively, and the corresponding compound concentrations (in nM) are plotted on a log10 scale (x-axis). Figure 2

[0370] Reduction of intracellular calcium flux after treatment with different concentrations of Compound 1 (A), 2 (B) and 3 (C) in a recombinant NMDAR expressing HEK293 cell line stimulated with glutamate. Data shown have been normalized to the maximum and minimum responses observed in the presence of control ligand (MK-801) and vehicle (y-axis), respectively, and the corresponding compound concentrations (in nM) are plotted on a log10 scale (x-axis). Figure 3

[0371] Reduction of intracellular calcium flux after treatment with different concentrations of Compound 1 (A), 2 (B) and 3 (C) in a recombinant NMDAR expressing HEK293 cell line stimulated with glutamate. Data shown have been normalized to the maximum and minimum responses observed in the presence of control ligand (MK-801) and vehicle (y-axis), respectively, and the corresponding compound concentrations (in nM) are plotted on a log10 scale (x-axis). Figure 4 ​​​​​​​​The effects of compound 2 on sciatic nerve regeneration after mechanical stress were assessed by electromyography and histological analysis. AB: Electromyography (EMG) profile after sciatic nerve injury. Latency (ms) and amplitude (mV) of CMAP were recorded in the gastrocnemius muscle after sciatic nerve stimulation. Results are expressed as mean + / - SEM (n = 10⁻¹¹ / group). Dashed line: solvent; plain line: compound 2 (3 mg / kg, twice daily).

[0372] CF: Histological analysis of peripheral nerves after sciatic nerve injury. Results are expressed as mean ± SEM (n = 4-5 / group). White bars: contralateral solvent treatment; black bars: ipsilateral solvent treatment; basal bars: ipsilateral compound 2 (3 mg / kg, twice daily) treatment. (C) Myelin sheath thickness as a percentage of the contralateral nerve; (D) Percentage of myelin sheath axons of each sciatic nerve compared to the contralateral nerve; (E) Myelin sheath thickness in micrometers; (F) Images of sciatic nerve sections.

[0373] Figure 5 This study demonstrates the effect of compound 2 (3 mg / kg, once daily) on heat hypersensitivity in PMP22 transgenic rats (a model of CMT1A) treated for 16 weeks using a hot plate test (52°C). Four-week-old WT and CMT1A rats were orally administered either the solvent or compound 2 at a dose of 3 mg / kg for 16 weeks (n = 8–12 rats per condition). CMT1A and WT rats were placed in glass cylinders on a hot plate at 52°C. Delays in raising, shaking, or licking paws were recorded. Data are presented in seconds and are expressed as mean + SEM. *p < 0.05. Comparison with Kruskal-Wallis' CMT1A solvent, followed by Dunn's post-test. White bars: solvent treatment in WT rats; black bars: solvent treatment in PMP22 transgenic rats; background stripes: PMP22 transgenic rats treated with compound 2 (2.29 mg / kg, once daily).

[0374] Figure 6 Compound 2 was shown to increase the activity of primary motor neurons under glutamate stress. Figure 6 Compound 2 (A) at 100 nM and 500 nM enhanced the activity of primary motor neurons in wild-type rats after 20 minutes of treatment with 5 μM glutamate. Figure 6 Compound 2 (B) from 10 nM to 5 μM can increase the concentration of SOD1. G93A Viability of primary motor neurons in transgenic rats after 20 minutes of treatment with 5 μM glutamate. Data are expressed as a percentage of control and as mean - / + SEM. White bars: solvent-treated; black bars: glutamate-only treated; background bars: glutamate-stimulated and treated with compound 2 (different concentrations).

[0375] Figure 7 Primary SOD1 was displayed G93A The reduction of reactive oxygen species (ROS) in transgenic motor neurons was observed with stimulation by 5 μM glutamate treatment for 20 minutes, followed by treatment with compound 2 at 100 and 500 nM. Data are presented as a percentage of control and as mean ± / + SEM values. White bars: solvent-treated; black bars: glutamate-treated only; understripped bars: glutamate stimulation and treatment with compound 2 (at different concentrations).

[0376] Figure 8 This shows that intracellular calcium flux (ICF) decreased after treatment with different concentrations of compound 2 in primary cortical neurons stimulated by NMDA. Figure 8 A) and reactive oxygen species concentration ( Figure 8 The decrease of B) in the middle.

[0377] White bars: solvent treatment; black bars: NMDA treatment only; bottom bars: NMDA stimulation and treatment with compound 2 (different concentrations); gray bars: NMDA stimulation and treatment with 5 μM benzylpenicillin.

[0378] Figure 9 The protein translation rate in SH-SYY cells stimulated with carotenoids in the presence of compound 2 or its hydroxylated metabolite compound 19 is shown. Representative images of newly synthesized proteins (labeled with puromycin) in SH-SY5Y cells stimulated with carotenoids, treated with the solvent, compound 2 (10 μM) (A), or compound 19 (10 μM) (B). A graphical representation of the percentage of puromycin-labeled proteins in carotenoid-treated cells, using the solvent, compound 2 (C) (n = 8 independent experiments), and compound 19 (D) (n = 6 independent experiments).

[0379] Figure 10 The cell viability of HeLa cells stimulated with streptovirin after treatment with compound 2 or its hydroxylated metabolite compound 19 is shown. The percentage change in cell viability after streptovirin treatment is graphically represented, with solvent-treated cells having a fixed viability of 0%. A - Compound 2 (n = 8 independent experiments) and B - or Compound 19 (n = 6 independent experiments). Detailed Implementation

[0380] The invention is further described with reference to the following non-limiting embodiments.

[0381] Example 1: Chemical Synthesis of Compound 19

[0382] The compounds can be prepared by applying and adapting the procedures disclosed in EP2943467, WO2016 / 001389, WO2016 / 001390 or WO2017 / 021216.

[0383] For example, the preparation of 2-(2-chloro-4-hydroxybenzylidene)hydrazinecarboxamide is as follows:

[0384]

[0385] To a solution of 2-chloro-4-hydroxybenzaldehyde (2.0 g, 1 eq.) in ethanol (30 ml) at 25 °C was added, sequentially, aminoguanidine hydrochloride (1 eq.) and sodium acetate (1 eq.). The resulting reaction mixture was heated at 80 °C for the next ~6 hours. The reaction completion was monitored on TLC using dichloromethane / methanol (9 / 1) as the mobile phase. After completion of the reaction, the reaction mixture was allowed to cool to 25 °C and then poured into a saturated solution of NaHC03(100 ml). The resulting precipitate was filtered off under vacuum and washed with water (30 ml). The resulting solid material was triturated with diethyl ether (2 x 25 ml) and dried under vacuum to provide 2.1 g of 2-(2-chloro-4-hydroxybenzylidene)hydrazinecarboxamide.

[0386] More specifically, the following compounds have been synthesised:

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394] Example 2: Assessment of central nervous system (CNS) activity of a single oral dose of compound 2 as the acetate salt (compound 2A) in rats

[0395] The objective of this study was to evaluate the potential effects of a single oral administration of Compound 2 on the central nervous system activity in conscious rats. The FOB (Functional Observation Battery) allows the detection of central nervous system dysfunction by clinical observation and measurement of the response to different stimuli, thus predicting the safety of the test compound on the central nervous system. This test is adapted from the method described by Mattson J.L. et al. (1996, J. Am. Coll. Toxicol., 15, 239).

[0396] Methods

[0397] A total of 32 male rats (Sprague-Dawley) were assigned to four groups (n = 8 animals per group) and received a single oral administration of vehicle (sterile saline solution) or test article (Compound 2 as acetate salt, 2A) at dose levels of 1, 3 or 9 mg / kg (corresponding to Compound 2 as free base). The animals were not fasted or deprived of water prior to treatment. The allocation of the treatment groups was performed by computerized randomization stratified according to the body weight of the animals. A functional observation was performed on all animals before and approximately 1, 3 and 6 hours after administration. The following parameters were evaluated and scored: touch escape, piloerection, coat appearance, salivation, lacrimation, pupil size (presence of miosis or mydriasis), exophthalmos, reaction to handling, grooming, palpebral closure, tremors, convulsions, wakefulness (hypoactivity and hyperactivity), ataxia, hypotonia, gait, posture, stereotypy, behavior, respiration, defecation, urination. The following measurements, reflexes and reactions were recorded: touch response, visual stimulus, pupillary reflex, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, forelimb grip strength. The study was performed by CiToxLAB France (BP 563 - 27005 Evreux, France).

[0398] Results

[0399] No clinical signs possibly related to Compound 2 treatment were observed during the functional observation evaluation. No neurological, autonomic or behavioral changes possibly related to treatment with Compound 2 at concentrations of 1, 3 or 9 mg / kg were observed during the FOB evaluation. Under the experimental conditions of this study, a single oral administration of Compound 2 had no effect on CNS activity up to 9 mg / kg.

[0400] Example 3: Safety profile of benzylidene aminoguanidine derivatives of formula (I) in humans

[0401] The clinical safety of three benzylidene aminoguanidine derivatives of formula (I) has been evaluated in healthy human volunteers; they were free of psychotic and negative symptoms, and also free of cognitive impairment.

[0402] Compound 1 (2-(2,6-dichlorobenzylidene)hydrazinecarboxamide / chloroguanide)

[0403] In both the control and open-label treatment trials, adverse events that occurred with Compound 1 (i.e., guanabenz) were not similar to adverse events in schizophrenia. Reported side effects of Compound 1 were drowsiness, dry mouth, dizziness, and weakness; cardiovascular side effects were rare except for a decrease in heart rate (Holmes et al., Guanabenz, A review of its pharmacodynamic properties and therapeutic efficacy in hypertension. Drugs (1983) 26:212-229).

[0404] Compound 2 [2-(2-chlorobenzylidene)hydrazinecarboxamide / IFB-088 / icerguastate] and compound 19 [2- (2-chloro-4-hydroxybenzylidene)hydrazinecarboxamide ]。

[0405] Compound 2 and Compound 19 are the major human metabolites of Compound 2, with similar levels of human exposure following repeated dosing of Compound 2.

[0406] The tolerability and pharmacokinetic profile of Compound 2 and 19 have been evaluated in a randomized, double-blind, placebo-controlled single ascending dose and multiple ascending doses (NCT03610334 with results). Compound 2 was dosed as the acetate salt (Compound 2A). Tolerability was good at all tested doses, with no serious adverse events. Adverse events that occurred with Compound 2 and 19 were not similar to adverse events in schizophrenia. No clinically significant abnormalities were reported for vital signs (heart rate, blood pressure) and laboratory parameters (blood, liver, or kidney function). There were no reports of clinically significant Compound 2 and 19-related hypotension and dizziness.

[0407] Example 4: NMDAR (NR1A / NR2B) human glutamate ion channel cell-based antagonist Ca 2+ flux assay

[0408] Materials and methods

[0409] NMDAR (1A / 2B) human glutamate ion channel cell-based antagonist Ca 2+ flux assay has been performed at DiscoverX (DiscoverX Inc.) (assay N° ITEM 87-1002-1544AN). Briefly, Hek293 cells stably transfected to express NMDAR subunits 1A / 2B were seeded into 384-well microplates and incubated at 37°C. Cells were loaded with a dye prior to testing. EC 80At the specified concentration, in the presence of the NMDAR antagonist (MK-801), the benzylidene aminoguanidine derivative of formula (I) was added to cells. Cells were further incubated at 37°C for 30–60 min, and the compound activity in terms of calcium flux was measured on a FLIPR Tetra (MDS).

[0410] Results

[0411] The control compound MK-801 blocked Ca 2+ The flux showed antagonistic activity against NMDAR (NR1A / NR2B) in the experiment, IC50 50 =69 nM. Compounds 1, 2, and 3 inhibit intracellular Ca2+ by antagonizing the NMDAR subunit NR1A / NR2B. 2+ flux ( Figure 1 ), and the IC shown in the measurement 50 The values ​​were 625 nM (A), 1156 nM (B), and 702 nM (C), respectively.

[0412] Example 5: NMDAR Radiolabeled Ligand Substitution Assay

[0413] Materials and methods

[0414] To elucidate the mechanism of action by which compounds 1, 2, and 3 exhibit NMDAR antagonist activity, we evaluated their ability to substitute radiolabeled NMDAR ligands. The following radiolabeled ligands were used:

[0415] -MDL-105,519, a potent and selective glycine antagonist that binds to the NMDAR NR1 subunit;

[0416] -MK-801, a non-competitive antagonist that binds within the ion channels of NMDAR;

[0417] -CGP-39653, a potent and selective glutamate antagonist that binds to the NMDAR NR2 subunit;

[0418] β-Benzylpiperazine is an NR2B selective negative allosteric modulator that binds near the polyamine site.

[0419] Assays were performed at Eurofins (Assay Ref. 232910 / 233010 / 234000 / Safety Screen 44 Panel) according to previously published methods (Siegel BW et al., (1996) Eur J Pharmacol. 312(3):357-365; Javitt DC and Zukin SR (1989) Interaction of [3H]MK-801 with multiple states of the N-methyl-D-aspartate receptor complex of rat brain. Proc Natl Acad Sci U S A. 86(2):740-744; Reynolds IJ et al. (1987) 3H-labeled MK-801 binding to the excitatory amino acid receptor complex from rat brain is enhanced by glycine. Proc Natl Acad Sci U S A. 84(21):7744-7748; Sills MA et al., [“H]CGP39653: a new N-methyl-D-aspartate antagonist radioligand with nanomolar affinity in rat brain. Eur. J. Pharmacol. 1991; 192: 19-24; Schoemaker HA & Lang SZ, Binding of 3 H]-ifenprodil, a novel NMDA antagonist to a polyamine-sensitive site in the rat cerebral cortex. Eur J Pharmacol. 1990; 176(2):249-250).

[0420] Results

[0421] Under the test conditions, compounds 1, 2 and 3 were unable to displace the radiolabeled ligands MDL-105,519, CGP-39653 and MK-801. Therefore, the compounds cannot provide their NMDA antagonist activity by binding to the glycine and glutamate sites and the pore channel, respectively.

[0422] Compounds 1, 2, and 3 can replace the radiolabeled ligand benzylpyrrol, and showed 400 nM in the assay, respectively. Figure 2 (A) 620nM Figure 2 (B) and 250nM ( Figure 2 IC in C) 50 Compounds 10, 13, 15, 17, 19, 23, and 26 can replace the radiolabeled ligand benzylpiperazine and showed a yield of 150 nM in the assay. Figure 2 D in the middle), 1390nM ( Figure 2 E in), 830nM ( Figure 2 (F in the middle), 350nM ( Figure 2 (G in) 2670nM ( Figure 2 H in), 3060nM ( Figure 2 I) and 310nM ( Figure 2 The IC50 of J in [the text is missing]. Therefore, these compounds exhibit NMDA antagonist activity, which is mediated by binding to or near the benzylosinol binding site on the NR2B subunit.

[0423] Example 6: Effect of the benzylidene aminoguanidine derivative of formula (I) on peripheral motor axon regeneration in a mouse model of sciatic nerve injury.

[0424] Sciatic nerve injury in rodents is used to simulate peripheral nerve regeneration. Sciatic nerve injury, also known as axonal transection, refers to axonal breakage due to mechanical damage without interrupting the connective tissue and basilar membrane tubules of Schwann cells (SCs). Following injury, the distal portion of the axon enters a programmed degenerative process called Wallerian degeneration. Wallerian degeneration is characterized by axonal breakage, associated with macrophage infiltration to clear debris and phenotypic switching of SCs. SCs play a crucial role in peripheral nerve regeneration because they coordinate with macrophages to clear debris, attract and guide axonal budding, and ultimately form a new myelin sheath to ensure the proper transmission of electrical signals from neurons.

[0425] Materials and methods

[0426] Sciatic nerve injury

[0427] Six-week-old male Swiss (CD-1) mice were provided by Janvier Labs and housed in an animal facility with a diurnal reverse cycle. Sciatic nerve injury was performed as previously described (Henriques et al., 2017 Scientific report; Bouscary et al., 2019 Frontiers in Pharmacology). Mice were anesthetized with ketamine chloride hydrate (100 mg / kg) and xylazine (10 mg / kg) and then placed on a heating pad. The skin was incised to expose the sciatic nerve in the mid-thigh and injured with fine forceps to ensure peripheral damage. The nerve was compressed twice with hemostatic forceps (1.5 mm wide; Koenig, Strasbourg, France), with a 90-degree rotation between each compression. The skin incision was sutured, allowing the mice to recover in isolation until the anesthesia ended. The hind limb opposite the lesion served as an uninjured control. Analgesia was induced preoperatively and over the following days with buprenorphine (0.1 mg / kg). The surgery caused nerve degeneration within two weeks, followed by localized neuroinflammation that lasted for up to four weeks. Nerve function gradually recovered over 4-5 weeks following the mechanical injury.

[0428] treat

[0429] Solvent: Salt water (0.9% sodium chloride in water)

[0430] Dosage: Compound 2: 3 mg / kg, twice daily;

[0431] Compound 3: 1.5 mg / kg, once daily, via oral (oral gavage).

[0432] Dosage frequency: Compound 2 twice daily, Compound 3 once daily, starting from the day of injury.

[0433] Electromyography

[0434] Electromyography (EMG) was used on days 0, 7, and postoperative days 14 and 21 to measure nerve fiber function on both the ipsilateral and contralateral sides. Mice were anesthetized intraperitoneally with ketamine chloride hydrate (100 mg / kg) and xylazine (10 mg / kg). A stimulating needle electrode was inserted into the sciatic nerve notch, and a recording needle electrode was inserted into the gastrocnemius muscle. Reference and ground electrodes were inserted into the lower back and the base of the paws of the animals. Compound muscle action potentials (CMAPs) were measured: more precisely, the amplitude (mV) and latency (ms) of the action potentials were recorded in the gastrocnemius muscle after stimulation of the sciatic nerve. The sciatic nerve was stimulated with a single pulse of 0.2 ms, with a maximum intensity of 12.8 mA. Reference values ​​for latency were less than or equal to 1 ms, and reference values ​​for amplitude were between 40 mA and 60 mA.

[0435] Tissue collection and histology

[0436] On day 21, mice were deeply anesthetized with ketamine chlorhydrate (100 mg / kg) and xylazine (10 mg / kg) and injected with cold PBS (3 min). The contralateral and ipsilateral tibial anterior nerve and sciatic nerve were collected. Sciatic nerves (n=5 per group, cryopreserved for future analysis) were fixed overnight with 4% glutaraldehyde and kept in 0.02% PBS azide at +4°C until use. Nerves were fixed for 1 hour in 1% osmium tetroxide in phosphate buffer, dehydrated in a succession of alcohol solutions and then embedded in Epon. The embedded tissue was placed at +60°C during the 3 days of polymerization. Transversal sections (1.5 microns thick) were generated using a microtome, stained with toluidine blue / carmine for 30 seconds and then dehydrated and mounted in Eukitt. Images were acquired using a confocal laser scanning microscope. Morphological analysis was automatically performed using MetaXpress (Molecular device) (one section per animal, four different areas). The following endpoint parameters were determined (i) number of myelinated axons, (ii) myelin thickness and G coefficient (ratio axon / fiber diameter).

[0437] Results

[0438] Effect of compound 3 on functional recovery

[0439] After nerve injury, spontaneous toe extension on the ipsilateral paw is lost due to denervation of the hindlimb muscles. Recovery usually occurs after 10 days post injury, as shown in the vehicle group in Figure 3 Compound 3 (median survival of 8 days) was faster than the control group, indicating an improved recovery.

[0440] Effect of compound 2 on electromyographic profile after nerve injury

[0441] Muscle denervation and demyelination lead to severe impairment of the compound muscle action potential (CMAP) detected by electromyography. CMAP latency and amplitude were analyzed. Latency is defined as the time (in milliseconds) between stimulation and the beginning of the action potential (negative phase of CMAP) (A in Figure 4 Amplitude, in mV, depends on the number of motor axons responding to the stimulation (B in Figure 4 At day 21 post injury, latency was significantly decreased in mice treated with compound 2 (3 mg / kg, twice a day) compared to the control group / compression group. At this time, compound 2 (3 mg / kg, twice a day) also increased the amplitude of the signal, indicating an improved regeneration. These results show that compound 2 (3 mg / kg, twice a day) reduces axonal degeneration and improves regeneration after nerve injury.

[0442] Effect of compound 2 on axonal myelination

[0443] Sciatic nerve injury results in loss of myelinated axons Figure 4 and reduced myelin thickness Figure 4 C-E-F). Given the diameter of the axons, the G ratio is an index that provides information on myelin thickness. Compound 2 did not significantly increase the number of myelinated axons, however, at 3 mg / kg twice daily, it had a strong effect on myelin thickness of myelinated axons Figure 4 C-D). Assessment of the G ratio confirmed the positive effect of compound 2 (3 mg / kg twice daily) Figure 4 E). Histology showed that compound 2 (3 mg / kg twice daily) improved the myelin status of peripheral axons after injury Figure 4 C-D-E-F).

[0444] These results indicate that compound 2 (3 mg / kg twice daily) reduces axonal degeneration by preventing myelin degeneration and improving regeneration after nerve injury. Overall, these results indicate that compound 2 supports axonal and myelin regeneration in a model of peripheral motor injury.

[0445] Example 7: Hot plate test.

[0446] Materials and methods

[0447] Transgenic rats overexpressing the PMP22 gene are an established model of Charcot-Marie-Tooth disease subtype 1A (CMT1A). This model has demonstrated behavioral and neuromuscular dysfunction (Sereda et al., Neuron. 1996; 16: 1049-1060). CMT1A transgenic rats were treated with compound 2 starting at 4 weeks of age for 16 weeks (3 months). Treatment was administered orally once daily. The hot plate test was performed after 16 weeks of treatment. The animals were placed in a glass cylinder on a hot plate, the temperature of which was adjusted to 52°C (hot). The latency to lift, shake or lick the paw was recorded. The cut-off time was set to 45 seconds.

[0448] Results

[0449] The recorded hyperalgesia (first sign / reaction) at 52°C was recognized as such since the mean latency of the untreated transgenic CMT1A rats was 10.78 seconds, faster than the latency of 16.02 seconds measured for wild-type rats; the latter latency was derived from published data. The CMT1A transgenic rats reacted to the painful stimulus (i.e. nociceptive response to heat) faster than normal, corresponding to hyperalgesia (i.e. overreaction to painful stimuli).

[0450] Oral administration of Compound 2 acetate salt for 3 months (equivalent to 2.29 mg / kg / day of Compound 2 as free base) restored normal pain response latency in CMT1A transgenic rats, thus correcting the hyperalgesia symptoms Figure 5 ). This result is promising for the treatment of hyperalgesia and neuropathic pain caused by hyperalgesia, such as in CMT patients.

[0451] Example 8: Effect of benzylidene aminoguanidine derivatives of formula (I) on the survival of primary rat motor neurons stimulated with glutamate. G93A Example 8: Effect of benzylidene aminoguanidine derivatives of formula (I) on the survival of primary rat motor neurons stimulated with glutamate.

[0452] Materials and methods

[0453] Example 8: Effect of benzylidene aminoguanidine derivatives of formula (I) on the survival of primary rat motor neurons stimulated with glutamate. G93A Spinal cord motor neurons (MNs) of wild-type rats (WT) and SOD1 G93A transgenic rats were cultured as described by Boussicault et al., 2020 and Wang et al., 2013. Briefly, female rats at 14 days of gestation were killed using deep anesthesia with a CO2 chamber and cervical dislocation. Then, fetuses (E14) were removed from the uterus and immediately placed in ice-cold L15 Leibovitz medium containing 2% of a penicillin (10,000 U / mL), streptomycin (10 mg / mL) solution (PS) and 1% of bovine serum albumin (BSA). The spinal cord was excised and placed in ice-cold Leibovitz (L15) medium. The spinal cord was treated with a trypsin-EDTA solution at 37°C for 20 minutes with a final concentration of 0.05% trypsin and 0.02% EDTA. The dissociation was stopped by adding Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g / L of glucose with secondary DNAse I (final concentration of 0.5 mg / ml) and 10% fetal calf serum (FCS). Mechanical dissociation was performed by three forced passages through a 10 mL pipette tip. Then, centrifugation was performed at 515 x g for 10 minutes at 4°C. The supernatant was discarded and the pellet was resuspended in a specific medium consisting of neural basal medium containing 2% of B27 supplement solution, 2 mM of L-glutamine, 2% of PS solution and 10 ng / mL of brain-derived neurotrophic factor (BDNF). Live cells were counted in a Neubauer cytometer using the trypan blue exclusion test. These cells were seeded in a 96-well plate pre-coated with poly-L-lysine at a density of 20,000 per well and incubated in an air (95%) - carbon dioxide (5%) incubator at 37°C. The medium was changed every 2 days. After 13 days of culture, motor neurons were injured with glutamate.

[0454] On day 13 of the culture, compound 2 was applied 1 hour before the application of glutamate. Glutamate was added to a final concentration of 5 μΜ, diluted in control medium still in the presence of the compound for 20 minutes. After 20 minutes, glutamate was washed away and fresh medium with compound 2 was added for another 24 hours.

[0455] After 24 hours of glutamate application, the supernatant was discarded and cells were fixed for immunostaining with a cold solution of ethanol (95%) and acetic acid (5%) for 5 minutes at -20°C. Cells were washed twice in PBS and then permeabilized, blocking non-specific sites for 15 minutes at room temperature using a PBS solution containing 0.1% saponin and 1% FCS. Cells were then incubated for 2 hours with mouse monoclonal antibody anti-microtubule associated protein 2 (MAP-2) at a dilution of 1 / 400 in PBS containing 1% fetal calf serum and 0.1% saponin. The antibody was revealed using Alexa Fluor 488 goat anti-mouse IgG in PBS at a dilution of 1 / 400, containing 1% FCS and 0.1% saponin, for 1 hour at room temperature. For each case, 30 pictures per well (representing the area of the whole well) were automatically taken using an ImageXpress (Molecular Devices) with a magnification of 20x. All images were generated using the same acquisition parameters. From the images, the analysis was directly automatically performed by ImageXpress (Molecular Devices). According to the images, the analysis was directly automatically performed by ImageXpress (Molecular Devices). According to the images, the analysis was directly automatically performed by

[0456] Results

[0457] In this in vitro glutamate excitotoxicity assay, nM concentrations of compound 2 increased the survival of primary motor neurons of WT (A) or SOD1 (B) transgenic rats stimulated by 5 μΜ glutamate for 20 minutes. In WT rat motor neurons, 100 nM and 500 nM of compound 2 increased the survival of motor neurons stimulated by 5 μΜ glutamate for 20 minutes (A). In SOD1 (B) rat motor neurons, 10 nM to 5 μΜ of compound 2 increased the survival of primary motor neurons stimulated by 5 μΜ glutamate (B). Figure 6 G93A Figure 6 G93A Figure 6 G93A Figure 6

[0458] Example 9: Effect of benzylidene aminoguanidine derivatives of formula (I) on the production of reactive oxygen species by glutamate-stimulated primary rat motor neurons. ​​​​​

[0459] Materials and methods

[0460] Rat spinal cord motor neurons (MN) were cultured as described in example 8. Four hours after the application of glutamate, the cell culture supernatant was discarded. Live cells were incubated with MitoSOX TM Red (marker of mitochondrially produced ROS) for 10 minutes at 37°C. MitoSOX TM The reagent is cell permeant and shows fluorescence once oxidized by superoxide. Then, cells were incubated for 2 hours with mouse monoclonal antibody anti microtubule associated protein 2 (MAP-2) at a dilution of 1 / 400 in PBS containing 1% fetal calf serum and 0.1% saponin. The antibody was revealed with Alexa Fluor 488 goat anti mouse IgG at a dilution of 1 / 400 in PBS containing 1% FCS and 0.1% saponin, for 1 hour at room temperature. Cell nuclei were counterstained with the fluorescent dye Hoechst (sigma).

[0461] For each case, 30 pictures per well (representative of the whole well area) were automatically taken using an ImageXpress (Molecular Devices) at a magnification of 20x. All images were acquired by using the same acquisition parameters. From the images, analysis was directly automatically performed by (Molecular Devices). The amount of ROS in MAP-2 positive neurons (in pm2, MAP-2 and mitochondria ROS overlap) was measured. 2

[0462] Results

[0463] In SOD1 G93A transgenic rat motor neurons, 100 nM and 500 nM of compound 2 reduced the amount of ROS produced by motor neurons stimulated for 20 minutes with 5 mM of glutamate (p < 0.05). Figure 7

[0464] Example 10: Effect of benzylidene aminoguanidine derivatives of formula (I) on calcium flux and reactive oxygen species production in NMDA-stimulated wild type primary cortical neurons.

[0465] Materials and methods

[0466] ​​Rat cortical neurons were cultured as described by Callizot et al. in 2013. Briefly, 15-day pregnant female rats (Wistar) were killed using deep anesthesia with a CO2chamber and cervical dislocation. Then, fetuses were collected and immediately placed in ice-cold L15 Leibovitz medium containing 2% of a penicillin (10,000 U / mL), streptomycin (10 mg / mL) solution (PS) and 1% of bovine serum albumin (BSA). The cortex was treated with a trypsin-EDTA solution at 37°C for 20 minutes with a final concentration of 0.05% trypsin and 0.02% EDTA. The dissociation was stopped by adding Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g / L of glucose, with secondary DNAse I (final concentration of 0.5 mg / mL) and 10% fetal calf serum (FCS). Mechanical dissociation was performed by three forced passages through a 10 mL pipette tip. Then, centrifugation was performed at 515 x g for 10 minutes at 4°C. The supernatant was discarded and the pellet was resuspended in a specific medium consisting of Neurobasal medium containing 2% of B27 supplement solution, 2 mmol / L of L-glutamine, 2% of PS solution and 10 ng / mL of brain-derived neurotrophic factor (BDNF). Live cells were counted using trypan blue exclusion test in a Neubauer cytometer. These cells were seeded in a 96-well plate pre-coated with poly-L-lysine at a density of 25,000 per well and will be incubated in an air (95%)-carbon dioxide (5%) incubator at 37°C. On day 15 of culture, the compounds were dissolved in the culture medium. The primary cortical neurons were incubated with the compounds for 60 minutes before NMDA exposure. After 60 minutes of incubation with the compounds, NMDA was added to a final concentration of 30 μΜ, diluted in control medium in the presence of the compounds for 1 hour. Then, Viable cells MitoSOX TM Red (mitochondrial Specificity ROS marker) was incubated for 10 minutes at 37°C. MitoSOX TMThe reagent is cell-penetrating and exhibits fluorescence upon oxidation with superoxide. Cells were then washed twice with heated PBS and fixed using a cold solution of ethanol (95%) and acetic acid (5%) at -20°C for 5 minutes. At room temperature, a PBS solution containing 0.1% saponin and 1% FBS was allowed to penetrate the cell membrane, blocking non-specific binding sites for 15 minutes. Cultures were then incubated with a mouse monoclonal antibody against microtubule-associated protein 2 (MAP-2) at a dilution of 1 / 400 in PBS containing 1% FBS and 0.1% saponin. This antibody was then developed using Alexa Fluor 488 goat anti-mouse IgG at a dilution of 1 / 800 in PBS containing 1% FBS and 0.1% saponin, and the reaction was carried out for 1 hour at room temperature.

[0467] Finally, use The ROS level in cortical neurons was quantified. For each condition, using... (Molecular Devices) automatically captured 20 images (representing the entire well region) at 20x magnification, using the same acquisition parameters (488 nM MAP-2 staining (green) fluorescence reading and 568 nM mitochondrial-generated ROS (red) fluorescence reading). Based on the images, [the data was analyzed / distributed]. (Molecular Devices) performs direct and automated analysis, specifically quantifying the ROS content in MAP-2 stained cortical neurons.

[0468] On day 15 of culture, one hour before compound 2 administration, cells were incubated with Fluo 4AM (4 μM) at 37°C for 2 hours. One hour after the application of compound 2, NMDA (30 μM) was applied to the cells. Intracellular Ca2+ was measured immediately after NMDA administration using a Glomax device. 2+ The level (in total cells) was measured every 3 minutes for 1 hour.

[0469] Results

[0470] Compound 2 reduced calcium flux within cortical neurons of WT rats stimulated with 30 μM NMDA. Figure 8 Compound 2 (A) exhibits activity from nanomolar to micromolar concentrations. Nanomolar concentrations of compound 2 also reduced ROS production in cortical neurons of WT rats stimulated with 30 μM NMDA. Figure 8 (B in the text). In this experimental setting, compound 2 at a concentration of 1 μM had the same inhibitory effect on calcium influx or ROS generation as 5 μM benzylpenicillin.

[0471] Example 11: Assessment of protein translation rate in SH-SY5Y stimulated with thapsigargin in the presence of Compound 2 or Compound 19

[0472] Compounds of Formula (I), including Compound 2, were shown to be inhibitors of the PPP1R15A-PP1 interaction (see WO2014 / 108520 and WO2016 / 001389), inducing a protective effect against protein misfolding stress and accumulation of misfolded proteins. Compound 2 selectively disrupts the stress-induced dephosphorylation of the translation initiation factor 2 (eIF2a) a subunit by binding to the regulatory subunit of the protein phosphatase 1, PPP1R15A (GADD34), thus protecting cells from the lethal accumulation of misfolded proteins. Compound 2 sets the protein translation rate in stressed cells to a level that can be controlled by available chaperones, thus restoring protein homeostasis. The aim of this study was to determine whether Compound 19, a hydroxylated metabolite of Compound 2, also has a protective effect against protein misfolding stress and accumulation of misfolded proteins.

[0473] Methods: SH-SY5Y cells (600,000 cells / well) were plated in 6-well plates. Forty-eight hours after seeding, cells were treated with 10 mM of Compound in the presence of 1 mM of thapsigargin for 1 hour, 2 hours, 3 hours, 4 hours and 6 hours. At the end of each time point, puromycin (5 pg / mL; Sigma Aldrich) was added to the culture medium for 15 minutes at 37 degrees Celsius. After labelling, cells were washed with ice-cold PBS and snap-frozen. Cells were then lysed in 100 microliters of RIPA buffer (Sigma Aldrich) containing protease and phosphatase inhibitors (Sigma Aldrich). 10 pg of protein lysate was separated on a Bolt 4-12% gradient gel. Proteins were then transferred to nitrocellulose membranes and then incubated with total protein staining (Licor), anti-puromycin antibody (clone 12D10, Millipore), p-eIF2a antibody (#3398, Cell Signalling) and total eIF2a antibody (#2103, Cell Signalling). Near-infrared fluorescence imager (CLX, Licor) analysed and quantified the newly synthesised proteins. Protein translation rate was estimated by comparing the quantification of puromycin-labelled proteins to the 0 hour time point. Statistical analysis was performed on GraphPad Prism software. Two-way ANOVA was performed followed by Sidak’s multiple comparison test.

[0474] Results:

[0475] Compared with carotenoid-treated cells, compound 2 (10 μM) increased the phosphorylation level of eIF2α in carotenoid-stimulated SH-SY5Y cells. The increased eIF2α phosphorylation was associated with a decrease in newly synthesized puromycin-tagged proteins, suggesting that compound 2 (10 μM) prolonged protein translational attenuation. Figure 9 Compound 19 is one of the major hydroxylated metabolites of compound 2 in animals, including humans. Compound 19 (10 μM) did not alter the phosphorylation level of eIF2α in SH-SY5Y cells stimulated with carotenoids compared to solvent-treated cells. Compound 19 (10 μM) did not alter the protein translation rate in carotenoid-stimulated cells compared to solvent-treated cells. Figure 9 (B, D in the middle).

[0476] Example 12: Evaluation of HeLa cell viability stimulated by streptovirin in the presence of compound 2 or compound 19

[0477] The aim of this study was to evaluate the effects of compounds 2 and 19 on protecting HeLa cells from streptovirin-induced cell death.

[0478] method:

[0479] HeLa cells were preserved in Eagle's Minimum Essential Medium (EMEM) supplemented with glutamine, sodium pyruvate, non-essential amino acids, penicillin, and streptomycin containing 10% fetal bovine serum (FBS) at 37°C under a 5% carbon dioxide atmosphere. The day before treatment, cells were seeded in 96-well plates at a density of 1500 cells / well. ER stimulation was induced by adding 1 μg / mL streptovirin (Sigma-Aldrich) and test compounds (0.1–10 μM). Forty-eight hours after streptovirin treatment, WST-8 reduction to methyl ether was measured using a Cell Counting Kit-8 (Sigma) as recommended by the supplier. The condition was assessed to evaluate cell viability. Cell viability was determined by the presence of streptoviral agents in cells treated with streptoviral agents. Calculated by comparing with simulated treated cells as a percentage. Cell viability was calculated based on raw data, as shown below:

[0480] Survival rate percentage = (DO 样本 -DO 空白 ) / (Average DO 溶媒 -DO 空白 )*100

[0481] The percentage variation in cell viability after streptovirin treatment was normalized, while the viability of stimulated cells after vector treatment was fixed at 0%.

[0482] Statistical analysis was performed using GraphPad Prism software. When the data failed the D'Agostino & Pearson normality test, a one-way ANOVA was performed first, followed by Friedman's multiple comparison test. When the data passed the D'Agostino & Pearson normality test, Dunnett's multiple comparison test was then performed.

[0483] result:

[0484] Compared to 0 μM of compound 2 in streptoviral-stimulated HeLa cells, compound 2 increased cell viability in a dose-dependent manner. Figure 10 Compound 19 did not improve the cell viability of HeLa cells treated with streptovirin 1 μg / mL for 48 hours (A in the text). Figure 10 (B in the middle).

[0485] Compound 2, but not compound 19, selectively disrupts stress-induced dephosphorylation of the α subunit of translation initiation factor 2 (eIF2α) by binding to the regulatory subunit PPP1R15A (GADD34) of protein phosphatase 1, thereby protecting cells from the lethal accumulation of misfolded proteins. Compound 2, but not compound 19, restores protein homeostasis by setting the translation rate in stimulated cells to a level controllable by available companion molecules.

Claims

1. A compound of general formula (I): and its (Z) and / or (E) isomers, or tautomers thereof, or pharmaceutically acceptable salts thereof, in: R1, R2, R3, R4, and R5 are independently hydrogen, deuterium, halogen, haloalkyl, alkyl, alkoxy, hydroxyl, aryl, or aryloxy. This is used to selectively inhibit the N-methyl-D-aspartate (NMDA) receptor subunit 2B (NR2B) in cells containing the NMDA receptor subunit 2B (NR2B) within the subject. This reduces the neuroprotective effect of excitotoxic NMDA receptor activity.

2. The compound for use according to claim 1, wherein by reducing intracellular calcium... 2+ The concentration reduces the activity of excitotoxic NMDA receptors in cells.

3. The compound for use according to claim 1 or 2, wherein the effect of reducing the activity of excitotoxic NMDA receptors in cells is achieved by reducing the concentration of reactive oxygen species.

4. A compound of general formula (I): and its (Z) and / or (E) isomers, or tautomers thereof, or pharmaceutically acceptable salts thereof, in: R1, R2, R3, R4, and R5 are independently hydrogen, deuterium, halogen, haloalkyl, alkyl, alkoxy, hydroxyl, aryl, or aryloxy. This is used to selectively inhibit N-methyl-D-aspartate (NMDA) receptor subunit 2B (NR2B) in subjects with NMDA receptors containing NMDA receptor subunit 2B (NR2B).

5. A compound of general formula (I): and its (Z) and / or (E) isomers, or tautomers thereof, or pharmaceutically acceptable salts thereof, in: R1, R2, R3, R4, and R5 are independently hydrogen, deuterium, halogen, haloalkyl, alkyl, alkoxy, hydroxyl, aryl, or aryloxy. This is used to prevent or treat diseases, disorders, or medical conditions caused by overactivation of N-methyl-D-aspartate (NMDA) receptors containing subunit 2B (NR2B) by selectively targeting the NR2B subunit of the NMDA receptor in subjects.

6. The compound for use according to claim 5, wherein the disease, disorder, or medical condition is selected from: (a) Depression or depressive disorder, major depressive disorder, medication-resistant major depressive disorder, postpartum depression, bipolar disorder; (b) Anxiety disorder, obsessive-compulsive disorder, generalized anxiety disorder, agoraphobia with panic disorder, panic disorder, post-traumatic stress disorder, social anxiety disorder; (c) Autism or autism spectrum disorder, Asperger's syndrome, or pervasive developmental disorder unclassified (PDD-NOS); (d) Epilepsy, epileptic seizure disorder; (e) Migraine, chronic tension-type headache (CTTH), migraine with abnormal pain, chronic headache; (f) Brain dysfunction, selected from Fragile X syndrome, tuberous sclerosis, Down syndrome and other forms of intellectual disability; (g) Withdrawal syndrome, such as from alcohol, opioids or cocaine; (h) Pain, hyperalgesia, nociception, acute pain, chronic pain, or cancer-related pain; (i) Pain associated with excitotoxicity, preferably with glutamate excitotoxicity, and / or with dysregulation of glutamatergic neurotransmission; (j) Neuropathic pain; (k) Pseudobulbar effect (PBA). (l) Movement disorders; (m) Amyotrophic lateral sclerosis (ALS) or medullary-onset amyotrophic lateral sclerosis; (n) Charcot-Marie-Duss disease (CMT); (o) Multiple sclerosis (MS); (p) Parkinson's disease, atypical Parkinson's disease (e.g., progressive supranuclear palsy); (q) Alzheimer's disease (AD), dementia, frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). (r) Huntington's disease (HD); (s) Focal brain injury caused by trauma, tumor or stroke; (t) Brain or spinal cord injury, peripheral nervous system injury, cerebral ischemia, head or neuronal trauma, neuronal hemorrhage, neuronal ischemia, reperfusion injury, neuronal injury; (u) Neuronal exposure to toxic substances, methamphetamine-induced neurotoxicity; (v) Neurological damage associated with stroke, cardiogenic shock, or coronary artery bypass grafting (CABG) surgery; (w) Idiopathic pulmonary fibrosis (IPF) and chronic cough (x) Spinocerebellar ataxia and Friedreich ataxia; (y) Kidney injury, acute kidney injury (AKI), ischemia-reperfusion induced AKI, hyperparathyroidism associated with chronic kidney disease (CKD), glomerulosclerosis, and glomerulosclerosis induced by hyperhomocysteinemia. (z) Bone diseases, fractures, bone injuries, bone defects associated with post-traumatic bone surgery, post-prosthetic joint replacement, post-orthopedic surgery, post-dental surgery, bone chemotherapy, bone radiotherapy, osteoporosis, Paget's disease, achondroplasia, osteochondritis, hyperparathyroidism, osteogenesis imperfecta, congenital hypophosphatase, fibromatosis, fibrous dysplasia, multiple myeloma, bone turnover disorder, osteolytic osteopathy, osteomalacia, and periodontal disease. And its symptoms.

7. The compound of claim 6, wherein the neuropathic pain is selected from the group consisting of: peripheral neuropathic pain; central neuropathic pain; chronic neuropathic pain; refractory neuropathic pain; neuropathic pain associated with metabolic dysfunction, including, for example, diabetes and prediabetes; neuropathic pain associated with diabetes; neuropathic pain associated with prediabetes; neuropathic pain associated with painful polyneuropathy; neuropathic pain associated with painful polydiabetes, including, for example, diabetic peripheral neuropathy; neuropathic pain associated with painful diabetic polyneuropathy; neuropathic pain associated with postherpetic neuralgia; neuropathic pain associated with trigeminal neuralgia; and neuropathic pain associated with occipital neuralgia. Neuropathic pain; neuropathic pain associated with painful radiculopathy, including, for example, painful radiculopathy of the lumbar and cervical spine; neuropathic pain associated with infectious diseases, including, for example, herpes zoster, HIV infection, Lyme disease, diphtheria, and leprosy; neuropathic pain associated with liver or kidney disease, including, for example, chronic liver or kidney disorders, including, for example, liver disease, liver failure, kidney disease, and kidney failure; neuropathic pain associated with immune or inflammatory diseases, including, for example, Guillain-Barré syndrome and Miller-Fischer syndrome, rheumatoid arthritis, lupus, systemic lupus erythematosus, Sjögren's syndrome, and celiac disease; neuropathic pain associated with hereditary neuropathy or channel disease, including, for example, hereditary erythromelalgia, paroxysmal extreme pain syndrome. And Charcot-Malidoux disease (CMT); neuropathic pain associated with small fiber sensory neuropathy; neuropathic pain associated with thyroid hormone disorders, including, for example, hypothyroidism; neuropathic pain associated with stroke; neuropathic pain associated with cancer, including, for example, lymphoma and multiple myeloma; neuropathic pain associated with chemotherapy, such as cancer chemotherapy; neuropathic pain associated with peripheral nerve injury; post-traumatic neuropathic pain associated with nerve injury; neuropathic pain associated with post-traumatic neuropathy; neuropathic pain associated with spinal cord injury, including, for example, traumatic spinal cord injury, such as road traffic accidents; neuropathic pain associated with traumatic peripheral nerve injury; neuropathic pain associated with postoperative neuropathy (e.g.) Postoperative neuropathic pain; neuropathic pain following surgery, including, for example, neuropathic pain following neurosurgery, including, for example, spinal cord surgery; neuropathic pain associated with fibromyalgia; neuropathic pain associated with lower back pain; neuropathic pain associated with carpal tunnel syndrome; neuropathic pain associated with burning pain; neuropathic pain associated with sympathetic reflex dystrophy (RSD); neuropathic pain associated with complex regional pain syndromes (CRPS), including, for example, types 1 and 2; neuropathic pain associated with amputation; neuropathic pain associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis and Parkinson's disease; neuropathic pain associated with stroke, including, for example, post-stroke central pain; neuropathic pain associated with syringomyelia;Neuropathic pain associated with demyelinating diseases, including, for example, multiple sclerosis, transverse myelitis, and neuromyelitis optica; or idiopathic neuropathic pain.

8. The compound for use according to any one of the preceding claims, which prevents, treats or alleviates pseudobulbar effect (PBA) or its symptoms in a subject selected from patients with the following diseases: Parkinson's disease (PD) or atypical Parkinson's disease (e.g., progressive supranuclear palsy), amyotrophic lateral sclerosis (ALS), medullary-onset ALS, spinal-onset ALS, primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), frontotemporal spectrum disorder, multiple sclerosis (MS), Alzheimer's disease (AD), dementia, Alzheimer's dementia agitation, frontotemporal dementia (FTD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), tumors, stroke, traumatic brain injury.

9. The compound for use according to any one of the preceding claims, which prevents, treats or alleviates depression or its symptoms in a subject selected from patients with the following diseases: Parkinson's disease (PD) or atypical Parkinson's disease (e.g., progressive supranuclear palsy), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), medullary-onset ALS, spinal-onset ALS, primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), frontotemporal spectrum disorder, multiple sclerosis (MS), and Charcot-Marie-Duss disease (CMT).

10. The compound for use according to any one of the preceding claims, which prevents, treats or alleviates movement disorders or their symptoms in a subject selected from patients with Parkinson's disease (PD), atypical Parkinson's disease (e.g., progressive supranuclear palsy), or Huntington's disease (HD).

11. The compound for use according to any one of the preceding claims, for the prevention, treatment or relief of diseases and disorders associated with glutamate homeostasis or acute or chronic hyperglutamate states.

12. The compound for use according to any one of the preceding claims, which does not simultaneously involve side effects selected from psychotic effects, cognitive impairment, and symptoms associated with schizophrenia.

13. The compound for use according to any one of the preceding claims, wherein, In formula (I), R1, R2, R4, and R5 are independently hydrogen, deuterium, halogen, haloalkyl, alkyl, alkoxy, hydroxyl, aryl, or aryloxy, and R3 is hydroxyl.

14. The compound for use according to any one of the preceding claims, wherein, In formula (I), R1, R2, R4, and R5 are independently hydrogen, deuterium, halogen, haloalkyl, alkyl, alkoxy, hydroxyl, aryl, or aryloxy, and R3 is hydrogen or deuterium.

15. The compound for use according to any one of the preceding claims, wherein, In equation (I): R1, R3, and R5 are independently selected from H, Cl, F, Br, and OH; R2 = R4 = H.

16. A compound for use according to any one of the preceding claims, wherein the compound of formula (I) is selected from the list consisting of: 2-(2,6-Dichlorobenzyl)hydrazinoimide 2-(2-Chlorobenzyl)hydrazinoimide 2-(2-chloro-4-fluorobenzyl)hydrazinoimide 2-(2-chloro-6-fluorobenzyl)hydrazinoimide 2-(2-bromobenzyl)hydrazinoimide 2-(2-Fluorobenzymyl)hydrazinoimide 2-(2,4-Difluorobenzyl)hydrazinoimide 2-(2,6-Difluorobenzyl)hydrazinoimide acetate 2-(2,4-Dichlorobenzyl)hydrazinoimide acetate 2-(2,3-Dichlorobenzyl)hydrazinoimide 2-(2,3,4-trichlorobenzyl)hydrazinoimide 2-(3,4,5-trichlorobenzyl)hydrazinoimide 2-(2,4,6-trifluorobenzyl)hydrazinoimide acetate 2-(2,4,5-trifluorobenzyl)hydrazinoimide 2-(2,6-Difluoro-4-chlorobenzyl)hydrazinoimide 2-(2,4-Dichloro-3-fluorobenzyl)hydrazinoimide 2-(2-chloro-,4,6-difluorobenzyl)hydrazinoimide 2-(2-chloro-,4,5-difluorobenzyl)hydrazinoimide 2-(2-chloro-4-hydroxybenzylidene)hydrazinoimide 2-(2-chloro-3-methylbenzyl)hydrazinoimide 2-(2-chloro-4-methylbenzyl)hydrazinoimide 2-(2-chloro-5-methylbenzyl)hydrazinoimide 2-(2,4-Dichloro-6-fluorobenzyl)hydrazinoimide 2-(2,6-Dichloro-4-fluorobenzyl)hydrazinoimide 2-(2,3-Dichloro-4-fluorobenzyl)hydrazinoimide 2-(2-chloro-3,5-difluorobenzyl)hydrazinoimide 2-(3,4-Dichloro-6-fluorobenzyl)hydrazinoimide 2-(3,5-Dichloro-4-fluorobenzyl)hydrazinoimide 2-(2,4-Dichloro-5-fluorobenzyl)hydrazinoimide 2-(2,3,5-trichlorobenzyl)hydrazinoimide 2-(3,4,5-trifluorobenzyl)hydrazinoimide 2-(2,3,4-trifluorobenzyl)hydrazinoimide And its (Z) and / or (E) isomers, or tautomers thereof, or pharmaceutically acceptable salts thereof.

17. The compound for use according to any one of the preceding claims, wherein the compound of formula (I) is selected from: And its (Z) and / or (E) isomers, or tautomers thereof, or pharmaceutically acceptable salts thereof.

18. The compound for use according to any one of the preceding claims, wherein the compound of formula (I) is compound 2 of the following formula: And its (Z) and / or (E) isomers, or tautomers thereof, or pharmaceutically acceptable salts thereof.

19. A compound for use according to any one of the preceding claims, wherein the compound of formula (I) is a (Z) isomer of compound 1:

20. The compound for use according to any one of the preceding claims, wherein the subject is a human.

21. A compound selected from the group consisting of: 2-(2,4,6-trifluorobenzyl)hydrazinoimide acetate 2-(2,6-Difluoro-4-chlorobenzyl)hydrazinoimide 2-(2-chloro-,4,6-difluorobenzyl)hydrazinoimide 2-(2-chloro-4-hydroxybenzylidene)hydrazinoimide 2-(2,4-Dichloro-6-fluorobenzyl)hydrazinoimide 2-(2,6-Dichloro-4-fluorobenzyl)hydrazinoimide 2-(2-Dichloro-3,5-difluorobenzyl)hydrazinoimide And its (Z) and / or (E) isomers, or tautomers thereof, or pharmaceutically acceptable salts thereof.

22. A method for preventing or treating diseases, disorders, or medical conditions caused by overactivation of N-methyl-D-aspartate (NMDA) receptors containing subunit 2B (NR2B) by selectively targeting the NR2B subunit of the NMDA receptor, wherein the method comprises: (a) Administer an effective amount of a compound of general formula (I) to the subject in need: And its (Z) and / or (E) isomers, or tautomers thereof, or pharmaceutically acceptable salts thereof, wherein: R1, R2, R4, R5 are independently hydrogen, deuterium, halogen, haloalkyl, alkyl, alkoxy, hydroxyl, aryl or aryloxy, and wherein R3 is hydrogen or deuterium; (b) Inhibiting the N-methyl-D-aspartate (NMDA) receptor containing subunit 2B (NR2B) in the subject by selectively targeting the NR2B subunit of the NMDA receptor with a compound of general formula (I), wherein R3 is a hydroxyl group, and wherein the compound of general formula (I), wherein R3 is a hydroxyl group, is a metabolite of the compound given in step (a).

23. The method of claim 22, wherein the compound given in step (a) is 2-(2-chlorobenzyl)hydrazidecarboximide, and the metabolite of the compound is 2-(2-chloro-4-hydroxybenzyl)hydrazidecarboximide.

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