Use of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate and its solvates for the treatment of motor neuron diseases and neuromuscular junction disorders

Abstract

The present invention relates to the succinate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine and pharma- ceutically acceptable solvates thereof for use in the treatment and / or prevention of motor neuron diseases and neuromuscular junction disorders.

Description

[Technical field]

[0001] The present invention relates to the novel use of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate and pharma- ceutically acceptable solvates thereof in the treatment and / or prevention of motor neuron diseases and neuromuscular junction disorders. [Background technology]

[0002] Motor neuron diseases (MND) and neuromuscular junction disorders (NJD) are both considered neuromuscular disorders that lead to muscle weakness and fatigue that becomes more noticeable over time.

[0003] Motor neurons are nerve cells present in the central nervous system (motor cortex, brainstem, spinal cord...) and the peripheral nervous system responsible for controlling organs such as muscles and glands. Motor neurons are classified as upper or lower motor neurons. Upper motor neurons (UMN) are located in the cerebral cortex and brainstem, where they send signals to interneurons and lower motor neurons (LMN) via glutamatergic neurotransmission. LMNs are located in the spinal cord and innervate skeletal muscle fibers (neuromuscular junctions), where acetylcholine is released to transmit signals across the muscle cell membrane, signaling the muscle to contract or relax. Both UMNs and LMNs are generally essential for voluntary movement, balance, postural coordination, and muscle control.

[0004] Progressive motor neuron degeneration is the hallmark of motor neuron diseases, which ultimately leads to motor neuron apoptosis and fasciculations of the motor unit (the motor neuron - its axon - the neuromuscular junction - the unit that defines the individual muscle fiber it completely innervates) preventing the transmission of the neural inflow that controls the muscle. The death of the motoneuron cell body is the primary process in MND, which occurs in conjunction with the deposition of aggregated proteins in motor neurons and oligodendrocytes, and neuroinflammation. These diseases, which can affect either the UMN or the LMN, and even both, cause muscle weakness, hypotension, slow movement, reflex dysfunction, and muscle atrophy.

[0005] There are many motor neuron diseases, and amyotrophic lateral sclerosis (ALS) is the most common acquired motor neuron disease that can affect UMN and LMN. Sporadic ALS is the most common form of ALS (more than 90% of cases) diagnosed in patients with no known family members affected by the disease. Familial ALS is of genetic origin, passed down through families, and several gene mutations have been identified and implicated. The most common is located in chromosome 9 open reading frame 72 (C9ORF72) and can also affect patients with cognitive impairment, and ALS-FTD is a specific form of ALS in which patients also suffer from frontotemporal dementia (FTD). Amyotrophic lateral sclerosis with Parkinson-dementia complex 1 (ALS-PDC) or Ritiko-Bodig disease is a form of ALS in which patients experience symptoms of ALS, dementia, and Parkinson's disease (PD). Other known genes associated with familial and sporadic ALS include those encoding superoxide dismutase 1 (SOD1), TAR DNA-binding protein of 43 kDa (TDP-43), RNA-binding protein (FUS / TLS: Fused in Sarcoma / Translocated in Sarcoma), and Ubiquilin 2 (UBQLN2). These mutations can cause toxic accumulation of these proteins in motor neurons and astrocytes, thus subsequently leading to neuronal death. TAR DNA-binding protein of 43 kDa (TDP-43) has been shown to accumulate in the cytoplasm of motor neurons in most cases of ALS. TDP-43 is a nuclear RNA-binding protein that is actively shuttled between the nucleus and the cytoplasm and is involved in several aspects of RNA processing. In ALS, TDP-43 is excluded from the nucleus, but such cytoplasmic mislocalization is common in neuronal injury or stress, and TDP-43-positive inclusions may represent secondary pathology in motor neuron disease.

[0006] Other motor neuron diseases can affect either the UMN only, such as primary lateral sclerosis (PLS), or the LMN only, such as progressive muscular atrophy (PMA). Moreover, LMN degeneration and ALS can affect the neuromuscular junction (Dupuis L et al., Curr Drug Targets 2010;11(10):1250-1261 - Gromova A et al., Trends Neurosci. 2020 Sept;43(9):709-724 - Hashizume A et al., J Neurol Neurosurg Psychiatry 2020 Oct;91(10):1085-1091). NJD targets this critical region, blocking nerve impulses that are normally sent to muscles to facilitate movement or their contraction. Myasthenic syndromes (myasthenia gravis and Eaton-Lambert syndrome) affect the efficacy of synaptic transmission, either via immunological or genetic processes. In these pathologies, the absolute number of NMJs remains roughly the same, but their efficacy to trigger muscle action potentials in response to motor neuron stimulation is diminished, leading to fatigable muscle weakness (Gilhus NE et al., Curr Opin Neurol. 2012 Oct;25(5):523-9). In addition, it is recognized that designing treatments to strengthen and stabilize the remaining NMJs, a common final pathway for diseases involving LMN degeneration, would be equally beneficial for ALS, spinal muscular atrophy (SMA) and spinal-bulbar muscular atrophy (SBMA) patients.

[0007] For ALS patients, only one treatment is currently approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Riluzole is an oral drug that blocks glutamate neurotransmission in the CNS. It is believed that these effects may be due in part to the inactivation of voltage-dependent sodium channels in glutamatergic nerve endings. Riluzole also blocks some of the postsynaptic effects of glutamate by noncompetitive blockade of N-methyl-D-aspartate (NMDA) receptors. Still, riluzole has no effect on motor and respiratory function and is not suitable for progressive forms of ALS or other motor neuron diseases.

[0008] Edaravone, an intravenously injected free radical scavenger, has shown efficacy in a small subset of people with ALS in phase 3 clinical trials. The study showed a significantly smaller decline in ALS Functional Rating Scale-Revised scores compared to placebo. To date, there is no indication that edaravone might be effective in a broader population of patients with ALS who do not meet the criteria (Abe et al., Lancet Neurol. 2017, 16(7), 505-512), and the application for marketing authorization from the EMA for the treatment of ALS was withdrawn.

[0009] Other than corticosteroids or immunosuppressants, which can have serious side effects, there are no specific treatments available for neuromuscular junction disorders.

[0010] N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate and solvates thereof, previously disclosed in WO2014 / 102339, are useful for the treatment and / or prevention of neurodegenerative diseases, amyloidopathies, tauopathies and developmental disorders. These compounds are of interest, inter alia, for Alzheimer's disease, Parkinson's disease and tauopathies, and early phase 2 clinical trials are ongoing with N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine disulfate for patients suffering from progressive supranuclear palsy (PSP).

[0011] These salts can modulate tau phosphorylation and increase progranulin (PGRN) neurotrophic factor levels, the deficiency of which is known to accelerate tau deposition and phosphorylation, as demonstrated in human tau-expressing mice (J. Neuropathol. Exp. Neurol. 74, pp. 158-165 (2015)). Thus, these compounds are particularly useful for the treatment of tauopathies such as frontotemporal dementia, and therefore have been proposed specifically to treat ALS-FTD.

Prior technical literature

Chartered Documents

[0012] [Patent Document 1] WO2014 / 102339 [Patent Document 2] WO2006 / 051489

Non-licensed documents

[0013]

Non-patent document 1

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Non-patent document 8

[0014] Limited treatments are available for patients suffering from motor neuron diseases or neuromuscular junction disorders and there remains a need in the art for new chemical entities that may be used in the treatment or prevention of these diseases; therefore, the Applicant has investigated the possibility of using N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate and its solvates for such purposes. [Means for solving the problem]

[0015] The present invention is based on the unexpected finding that the succinate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine has neuroprotective effects on motor neurons and the neuromuscular junction.

[0016] Thus, the present invention relates to N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate and solvates thereof for use in the treatment and / or prevention of motor neuron diseases and neuromuscular junction disorders.

[0017] Moreover, the applicant has shown that N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate and its solvates can reduce the abnormal translocation of TDP-43 from the nucleus to the cytoplasm in motor neurons, a pathological feature observed in most ALS cases. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows the XRPD 2θ diffractogram of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sesquisuccinic acid salt Form 1, a crystalline form. [Diagram 2] FIG. 1 shows the XRPD 2θ diffractogram of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine tris-succinic acid salt Form 2, a crystalline form. [Diagram 3] FIG. 1 shows the XRPD 2θ diffractogram of the crystalline form of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sesquisuccinic acid salt hemihydrate (0.5 equivalents of water). [Figure 4] FIG. 1 shows the XRPD 2θ diffractogram of the crystalline form of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sesquisuccinic acid salt monohydrate (1.1 equivalents of water). [Diagram 5] FIG. 1 shows the H-NMR spectrum of Form 1, a crystalline form of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sesquisuccinate (DMSO-d, 500.12 MHz). [Figure 6] FIG. 1 shows the C quantitative NMR spectrum of Form 1, a crystalline form of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sesquisuccinate (DMSO-d, 500.12 MHz). [Figure 7] Effect of incubation of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sesquisuccinate form 1 for 48 h after a 20 min glutamate insult in primary cultures of rat spinal motor neurons on survival (A), neurite network (B) and extranuclear TDP-43 (eTDP43) (C) of MAP-2 positive MN. Results are expressed as percentage of control with mean ± SEM (n=4-6 / group). One-way ANOVA followed by Fisher's PLSD test. [Figure 8]Figure 1. Effect of 24 h preincubation of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sesquisuccinate form 1 prior to a 20 min glutamate insult in primary cultures of rat spinal motor neurons with 24 h post-injury incubation in combination with survival (A), neurite network (B) and extranuclear TDP-43 (eTDP43) (C) in MAP-2 positive MN. Results are expressed as percentage of control, mean ± SEM (n=4-6 / group). One-way ANOVA followed by Fisher's PLSD test. [Figure 9] Figure 1 shows the effect of incubation of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sesquisuccinate form 1 for 48 hours after a 20 minute glutamate insult, compared to riluzole (Ril) and edaravone (Eda), on survival (A), neurite network (B) and extranuclear TDP-43 (eTDP43) (C) of MAP-2 positive MN in primary cultures of SOD1G93A Tg rat spinal motor neurons. Results are expressed as percentage of control with mean + / - SEM (n=5-6). One-way ANOVA followed by Fisher's PLSD test. [Figure 10] Figure 1. Effects of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sesquisuccinate form 1 compared to Riluzole (Ril) on (A) number of NMJs, (B) size of NMJs, and (C) neurite network of NFH(+)MNs in glutamate-lesioned spinal cord explants and myoblast co-cultures. Results are expressed as percentage of control with mean + / - SEM (n=5-6). One-way ANOVA followed by Fisher's PLSD test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Applicant has demonstrated that N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate and pharmaceutically acceptable solvates thereof enhance the survival and integrity of their neurite networks following glutamate-induced injury in wild-type rats and SOD1 G93A The succinate salt has been shown to exert beneficial effects on primary cultures of spinal motor neurons from both tg and tg rats. Furthermore, abnormal translocation of TDP-43 from the nucleus to the cytoplasm of motor neurons is a pathological feature observed in most ALS cases. The results presented in the Examples section show that the succinate salt can also reverse the abnormal translocation of TDP-43 from the nucleus to the cytoplasm under glutamate stress.

[0020] Moreover, N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate and its pharma- ceutically acceptable solvates successfully protected neuromuscular junctions and neurite networks from glutamate stress in cocultures of myoblasts and spinal cord explants.

[0021] All these findings support that N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate and its pharma- ceutically acceptable solvates may be an effective therapy for improving motor neuron diseases such as ALS, SMA, and SBMA, as well as neuromuscular junction disorders such as myasthenia gravis and Eaton-Lambert syndrome.

[0022] Motor neuron diseases and neuromuscular junction disorders include, but are not limited to, non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), neurolathyrism, Konzo, Tay-Sachs disease, Sandhoff disease, progressive muscular atrophy (PMA), monolimbal muscular atrophy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), post-polio syndrome, post-irradiation syndrome, stiff-person syndrome, motor unit disorders resulting from accidents, myasthenia gravis, and Eaton-Lambert syndrome.

[0023] Non-FTD amyotrophic lateral sclerosis designates sporadic or familial forms of amyotrophic lateral sclerosis that are not associated with frontotemporal dementia and includes amyotrophic lateral sclerosis with Parkinson-Dementia Complex 1 (ALS-PDC), familial amyotrophic lateral sclerosis caused by mutations in one of the genes encoding superoxide dismutase 1 (SOD1), TAR DNA-binding protein of 43 kDa (TDP-43), RNA-binding protein (FUS / TLS: Fused in Sarcoma / Translocated in Sarcoma), or ubiquilin 2 (UBQLN2). In ALS, patients with bulbar onset progress more rapidly than those with limb onset or LMN presentation. Recent descriptions of focal variants suggest that some patients have ALS isolated to a single spinal region, including brachial amyotrophic diplegia, leg amyotrophic diplegia, and isolated bulbar palsy, all of which are herein variants of non-FTD amyotrophic lateral sclerosis. Non-FTD ALS also includes respiratory onset ALS, a rare variant that accounts for approximately 3% of all ALS cases, the initial symptom of which is dyspnea with exertion at rest.

[0024] Primary lateral sclerosis (PLS) is a type of motor neuron disease that slowly destroys nerves in the brain, preventing them from activating motor neurons in the spinal cord, which control muscles.

[0025] Hereditary spastic paraplegia (HSP), also known as familial spastic paraplegia (FSP), is caused by slowly degenerating upper motor neurons, causing progressive spasticity and weakness of the legs. This leads to difficulty walking. As the degeneration continues, symptoms worsen, including visual impairment, ataxia, epilepsy, cognitive impairment, peripheral neuropathy, and / or hearing loss.

[0026] Neurolathyrism is caused by toxins derived from consuming large amounts of certain legumes of the genus Lathyrus that contain high concentrations of the glutamate analog neurotoxin β-oxalyl-L-α,β-diaminopropionic acid (ODAP), a mitochondrial poison that leads to excessive cell death, particularly in motor neurons, which may involve the pyramidal tract, causing paralysis characterized by a lack of strength in or inability to move the lower limbs and producing signs of upper motor neuron damage.

[0027] Progressive muscular atrophy (PMA), also known as Duchenne muscular atrophy, is clinically characterized by signs of lower motor neuron dysfunction and can progress to ALS. Symptoms of PMA include atrophy, muscle weakness, lack of reflexes and spasticity, which may be limited to the arms, legs, or both.

[0028] Single-limb muscular atrophy, also known as benign focal muscular atrophy, juvenile partial atrophy, and Hirayama disease, is a rare benign lower motor neuron disorder characterized by muscle weakness and wasting in the distal upper limbs during adolescence, followed by spontaneous cessation of progression and stabilization of symptoms.

[0029] Spinal muscular atrophy (SMA) is a debilitating disease that affects motor nerve cells in the spinal cord, taking away people's ability to walk, eat, or breathe. SMA is caused by a mutation in the survival motor neuron gene 1 (SMN1), which is critical for the function of nerves that control muscles. Without it, nerve cells cannot function properly and eventually die, leading to wasting and sometimes fatal muscle weakness.

[0030] Spinal-bulbar muscular atrophy (SBMA), also known as spinal and bulbar muscular atrophy and Kennedy disease, is an inherited disorder in which loss of motor neurons affects voluntary muscle movement, particularly the facial and swallowing muscles, as well as the muscles of the arms and legs, especially those closest to the center of the body.

[0031] Progressive bulbar palsy (PBP) involves both upper and lower motor movements. This form of MND often causes difficulty in speaking or swallowing. When the lower motor neurons are affected, the tongue tends to atrophy with visible fasciculations and reduced mobility. This results in a rather nasal speech. When the upper motor neurons are affected, the tongue tends to be spastic, causing dysarthria, a speech mechanism difficulty.

[0032] Stiff-person syndrome, also known as stiff-person spectrum disorder, is a neurological disorder that affects the brain and spinal cord, causing trunk shaking and limb stiffness, painful muscle spasms, task-specific phobia, exaggerated startle response, and ankylosing deformities such as fixed lumbar hyperlordosis.

[0033] "Accident-induced motor unit damage", as used herein, refers to damage to motor units caused by nerve lesions following an accident, for example, a domestic or traffic accident.

[0034] Myasthenia gravis is an autoimmune disorder that impairs acetylcholine transmission at the neuromuscular junction, i.e., antibodies that attack acetylcholine receptors, resulting in muscle weakness. The most common symptom of myasthenia gravis is weak ptosis, which causes double vision and excessive weakness of the affected muscles after the weak eye muscles are used.

[0035] Eaton-Lambert syndrome is an autoimmune disease in which antibodies block the release of the neurotransmitter acetylcholine receptors at the neuromuscular junction. It causes muscle weakness that tends to begin in the hip and thigh muscles and then typically spreads to the shoulder muscles, then down the arms and legs to the hands and feet. The nerves that connect the head, face, eyes, nose, muscles, and ears to the brain (cranial nerves) are the last to be affected.

[0036] In one embodiment, the succinate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine is represented by formula I

[0037] [ka]

[0038] wherein x is 1 to 4, preferably x is 1.4 to 3.1, more preferably x is 1.4 to 1.6, even more preferably x is about 1.5, and even more preferably x is 1.5. It is of the following.

[0039] In other words, the succinate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine contains 1 to 4 equivalents, preferably 1.4 to 3.1 equivalents, more preferably 1.4 to 1.6 equivalents, even more preferably about 1.5 equivalents, and even more preferably 1.5 equivalents of succinate per molecule of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine.

[0040] In one preferred embodiment, the succinate salt of formula I is the sesquisuccinate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine.

[0041] In one embodiment, the preferred succinate salt is a compound of formula I, where x is from 2.9 to 3.1, preferably where x is about 3, and more preferably where x is 3.

[0042] In another preferred embodiment, the succinate salt of formula I is the tris-succinate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine.

[0043] In certain embodiments, the succinate salt of formula I is in the form of a pharma- ceutically acceptable solvate, preferably a hydrate. The stoichiometry of the solvate is between 0.4 and 2, preferably between 0.4 and 1.2, more preferably between 0.5 and 1.1, even more preferably about 0.5 or about 1.1, even more preferably 0.5 or 1.1 molecules of solvate per molecule of succinate salt of formula I.

[0044] In one embodiment, the succinate salt of formula I is N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sesquisuccinate hemihydrate.

[0045] In another embodiment, the succinate salt of formula I is N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sesquisuccinate monohydrate.

[0046] In one embodiment, one particularly preferred succinate salt is the compound of formula I in a crystalline form having an X-ray powder diffraction (XRPD) pattern comprising peaks at diffraction angles 2θ=3.8°±0.2°, 10.3°±0.2°, 12.4°±0.2°, 16.2°±0.2°, 17.9°±0.2°, 19.8°±0.2°, 20.4°±0.2°, 23.8°±0.2°, and 26.7°±0.2°, where x is 1.5, when illuminated with a CuKα source. This specific crystalline form is referred to throughout this application as "Form 1" and its XRPD diffractogram, recorded on a PANalytical Expert Pro using a PIXcel detector (128 channel), is shown in Figure 1.

[0047] Another preferred succinate salt is the compound of formula I, wherein x is 3, in a crystalline form having an X-ray powder diffraction (XRPD) pattern comprising peaks at diffraction angles 2θ=5.3°±0.2°, 15.0°±0.2°, 15.3°±0.2°, 16.8°±0.2°, 17.9°±0.2°, 20.5°±0.2°, 21.2°±0.2°, 23.9°±0.2°, 24.3°±0.2°, and 26.6°±0.2° when illuminated with a CuKα source. This specific crystalline form is referred to throughout this application as "Form 2" and its XRPD diffractogram, recorded on a PANalytical Expert Pro using a PIXcel detector (128 channel), is shown in FIG. 2.

[0048] Another preferred succinate salt is the hemihydrate of the compound of formula I, in crystalline form, having an X-ray powder diffraction (XRPD) pattern comprising peaks at diffraction angles 2θ=3.8°±0.2°, 4.4°±0.2°, 9.2°±0.2°, 10.6°±0.2°, 13.8°±0.2°, 17.7°±0.2°, 19.9°±0.2°, 21.6°±0.2° and 23.6°±0.2°, where x is 1.5, when irradiated with a CuKα source. Its XRPD diffractogram, recorded on a PANalytical Expert Pro using a PIXcel detector (128 channel), is shown in FIG. 3.

[0049] Another preferred succinate salt is the monohydrate of the compound of formula I in crystalline form having an X-ray powder diffraction (XRPD) pattern comprising peaks at diffraction angles 2θ=4.2°±0.2°, 4.4°±0.2°, 9.6°±0.2°, 10.6°±0.2°, 15.0°±0.2°, 16.3°±0.2°, 17.4°±0.2°, 19.7°±0.2°, 22.8°±0.2°, 29.1 and 29.7°±0.2°, where x is 1.5, when irradiated with a CuKα source. Its XRPD diffractogram recorded on a PANalytical Expert Pro using a PIXcel detector (128 channel) is shown in FIG. 4.

[0050] Therefore, N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate and pharma- ceutically acceptable solvates are useful as medicaments, in particular for treating or preventing motor neuron diseases, neuromuscular junction disorders, and all diseases in which abnormal translocation of TDP-43 from the nucleus to the cytoplasm in motor neurons is observed.

[0051] The present invention therefore also relates to the succinate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine as defined herein, or a pharma- ceutically acceptable solvate thereof, for use in treating and / or preventing motor neuron diseases and neuromuscular junction disorders, in particular selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), neurolathyrism, Konzo, Tay-Sachs disease, Sandhoff disease, progressive muscular atrophy (PMA), monolimbal muscular atrophy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), post-polio syndrome, post-irradiation syndrome, stiff-person syndrome, motor unit disorders resulting from accidents, myasthenia gravis, Eaton-Lambert syndrome. Preferably, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), progressive muscular atrophy (PMA), single limb muscular atrophy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), myasthenia gravis, Eaton-Lambert syndrome, motor unit disorder caused by accident. More preferably, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP). Even more preferably, the disease is non-FTD amyotrophic lateral sclerosis.

[0052] In other words, the present invention also provides methods of treating and / or preventing motor neuron diseases or neuromuscular junction disorders, particularly those cited above and embodiments thereof, comprising administering to a patient in need thereof a pharma- ceutical effective amount of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate or a pharma- ceutical acceptable solvate thereof as described herein. In certain embodiments, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP).

[0053] In other words, the present invention also provides the use of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate or a pharma- ceutically acceptable solvate thereof as described herein, in particular in the above cited and embodiments thereof, for the manufacture of a medicament for treating and / or preventing a motor neuron disease or a neuromuscular junction disorder. In certain embodiments, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP).

[0054] In one particular embodiment, the invention also relates to N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate or a pharma- ceutically acceptable solvate thereof, as defined herein, for use in delaying the onset in a patient of motor neuron diseases and neuromuscular junction disorders, particularly selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), neurolathyrism, Konzo, Tay-Sachs disease, Sandhoff disease, progressive muscular atrophy (PMA), single limb muscular atrophy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), post-polio syndrome, post-irradiation syndrome, stiff-person syndrome, motor unit disorders resulting from accidents, myasthenia gravis, Eaton-Lambert syndrome. Preferably, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), progressive muscular atrophy (PMA), single limb muscular atrophy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), myasthenia gravis, Eaton-Lambert syndrome, motor unit disorder caused by accident. More preferably, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP). Even more preferably, the disease is non-FTD amyotrophic lateral sclerosis.

[0055] In other words, the present invention provides methods for delaying the onset of a motor neuron disease or neuromuscular junction disorder in a patient, in particular those cited above and embodiments thereof, comprising administering to a patient in need thereof a pharma- ceutical effective amount of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate or a pharma- ceutical acceptable solvate thereof. In certain embodiments, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP).

[0056] In other words, the present invention also provides the use of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate or a pharma- ceutically acceptable solvate thereof as described herein, in particular in the above cited and embodiments thereof, for the manufacture of a medicament for delaying the onset of a motor neuron disease or neuromuscular junction disorder in a patient. In certain embodiments, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP).

[0057] According to a further feature of the present invention, there is provided a method for reducing abnormal translocation of TDP43 from the nucleus to the cytoplasm in motor neurons in a patient, preferably a warm-blooded animal, even more preferably a human, in need of such treatment, comprising administering to the patient an effective amount of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate or a pharma- ceutically acceptable solvate thereof.

[0058] According to one embodiment, succinate salts of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine and their pharmaceutical acceptable solvates may be administered as part of a combination therapy. Thus, within the scope of the present invention are embodiments including the co-administration of compositions and medicaments containing, in addition to the succinate salts of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine or their pharmaceutical acceptable solvates as the active ingredient, additional therapeutic agents and / or active ingredients. Such multiple drug regimens are often referred to as "combination therapy" and may be used in the treatment and / or prevention of any motor neuron disease or neuromuscular junction disorder. The use of such combinations of therapeutic agents is particularly appropriate for the treatment of the above-mentioned motor neuron diseases in patients in need of treatment or at risk of becoming such patients.

[0059] In addition to the requirements of therapeutic efficacy that may necessitate the use of an active agent in addition to the succinate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine or a pharma- ceutically acceptable solvate thereof, there may be additional arguments that impose or strongly recommend the use of a drug combination with an active ingredient that represents an adjunct therapy, i.e., that complements and supplements the function performed by the succinate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine or a pharma- ceutically acceptable solvate thereof. Suitable supplemental therapeutic agents for use for adjunctive treatment purposes include drugs that treat diseases or conditions that are directly caused by or indirectly associated with motor neuron degeneration, neuromuscular junction degeneration and / or pathological accumulation of TDP-43 in the cytoplasm of motor neurons, instead of directly treating and / or preventing such diseases or conditions that are mediated by or related to said degeneration.

[0060] According to a further feature of the invention, N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate salt, a pharma- ceutically acceptable solvate thereof, may be used in combination therapy with other drugs used to treat motor neuron diseases such as ALS, SMA, SBMA and neuromuscular junction disorders such as myasthenia gravis and Eaton-Lambert syndrome. More particularly, the compounds of formula I and pharma- ceutically acceptable solvates thereof may be used as adjunctive therapy in combination with inhibitors of glucosylceramide degradation such as riluzole, edaravone, pyridostigmine, ambroxol and conduritol B epoxide, acetylcholine release inducers such as guanidine, corticosteroids such as prednisolone, anti-seizure drugs such as carbamazepine and phenytoin, or drugs for the treatment of ALS currently in clinical trials as disclosed in Van Eijk et al., Current opinion in neurology 2020, 33(5), p. 655.

[0061] Thus, the methods of treatment and pharmaceutical compositions of the present invention may use N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate salt or a pharma- ceutically acceptable solvate thereof in monotherapy for motor neuron diseases and neuromuscular junction disorders. However, the methods and compositions may also be used in polytherapy, where one or more N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate salts or pharma- ceutically acceptable solvates thereof are co-administered in combination with one or more other therapeutic agents.

[0062] In the above-mentioned embodiments, the combination of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate or a pharma- ceutically acceptable solvate thereof and other therapeutically active agents may be administered, in terms of dosage form, either separately or in conjunction with each other, and in terms of their time of administration, either sequentially or simultaneously. Thus, administration of one component agent may be prior to, concurrently with, or subsequent to administration of the other component agent.

[0063] Generally, for pharmaceutical use, the succinate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine or a pharma- ceutically acceptable solvate thereof may be formulated as a pharmaceutical composition comprising at least one N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine succinate salt or a pharma- ceutically acceptable solvate thereof and at least one pharma- ceutically acceptable carrier, excipient, additive and / or adjuvant, and optionally one or more further therapeutic agents and / or active ingredients.

[0064] By way of non-limiting example, the pharmaceutical composition may be in a suitable form for oral administration, parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), topical administration, by inhalation, by skin patch, by implant, by suppository, etc. Such suitable dosage forms - which may be solid, semi-solid or liquid depending on the mode of administration - and methods for use in their preparation and carriers, excipients and additives will be apparent to those skilled in the art; see the latest edition of Remington's Pharmaceutical Sciences. The pharmaceutical composition may be formulated in solid form and redissolved or suspended before use. A preferred pharmaceutical composition of the succinate salt or solvate thereof of the present invention is a solid dosage form suitable for oral administration.

[0065] Some preferred, but non-limiting examples of dosage forms include tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, lotions, soft and hard gelatin capsules, suppositories, drops, sterile injectable solutions for administration as a bolus and / or for continuous administration and sterile packaged powders (usually reconstituted before use), which may be combined with carriers, additives and excipients that are themselves suitable for such formulations, For example, it may be formulated with lactose, dextrose, sucrose, sorbitol, mannitol, starch, agar, gum acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, (sterile) water, methylcellulose, methyl and propyl hydroxybenzoate, talc, magnesium stearate, edible oils, vegetable oils and mineral oils or suitable mixtures thereof. Particularly preferred dosage forms of the succinate salt and its solvates of the present invention are soft and hard capsules, especially soft and hard gelatin capsules, preferably hard gelatin capsules, formulated with at least one microcrystalline cellulose additive, especially Avicel® PH101. The pharmaceutical compositions may optionally contain other substances commonly used in pharmaceutical formulations, such as, for example, lubricants, wetting agents, emulsifying and suspending agents, dispersing agents, disintegrating agents, stabilizing agents, isotonic agents, extenders, fillers, preservatives, sweetening agents, flavoring agents, flavorings, coloring agents, antibacterial and / or antifungal agents, such as, for example, parabens, chlorobutanol, phenol, sorbic acid, dispensing agents, flow regulators, release agents, etc. The compositions may be formulated so as to provide quick, sustained or delayed release of the active compounds contained therein.

[0066] The pharmaceutical compositions of the present invention are preferably in unit dosage form and may be suitably packaged, for example, in boxes, blisters, vials, bottles, sachets, ampoules, or any other suitable single-dose or multi-dose holders or containers (which may be appropriately labeled), optionally with one or more leaflets containing product information and / or instructions for use. Generally, such unit dosages will contain between 0.05 and 1000 mg, usually between 1 and 500 mg, of at least one compound of the present invention, for example about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage.

[0067] Typically, depending on the condition to be prevented or treated and the route of administration, the active compounds of the invention will usually be administered at between 0.01 and 100 mg per kilogram, more often between 0.1 and 50 mg, for example between 1 and 25 mg, for example about 0.5, 1, 2, 5, 10, 15, 20 or 25 mg per kilogram of patient body weight per day, which may be administered as a single daily dose, divided into one or more daily doses, or essentially continuously, for example using an intravenous infusion.

[0068] All references to the compounds of the invention, and in particular to compounds of formula I, include references to solvates, in particular hydrates, multi-component complexes and liquid crystals thereof.

[0069] Compounds disclosed throughout this application were named using ChemDraw® Ultra version 11.0 (CambridgeSoft, Cambridge, Mass., USA).

[0070] N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine free base may be obtained as disclosed in WO2006 / 051489. The succinate salt and its solvates may be prepared according to techniques known in the art, such as those involving precipitation, crystallization, recrystallization, lyophilization, phase transfer or ion exchange resins.

[0071] definition The following definitions and explanations are for terms as they are used throughout the entire application, including both the specification, figures, and claims.

[0072] Unless otherwise stated, any reference herein to a compound of the invention includes the compound of formula I per se and its pharma- ceutically acceptable solvates. All references to the compound of formula I include a reference to its pharma- ceutically acceptable solvates and all crystalline forms thereof.

[0073] The term "administration" or variants thereof (e.g., "administering") means providing an active agent or ingredient (e.g., N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine), alone or as part of a pharma- ceutically acceptable composition, to a patient whose condition, symptom, or disease is to be treated or prevented.

[0074] The term "human" refers to subjects of both genders and at any stage of development (i.e., neonates, infants, juveniles, adolescents, adults).

[0075] The term "patient" refers to a warm-blooded animal, more preferably a human, awaiting or receiving medical care or who is / will be the subject of a medical procedure.

[0076] "Pharmaceutically acceptable" means that the components of a pharmaceutical composition are compatible with each other and not harmful to the patient.

[0077] The terms "prevent", "preventing" and "prevention", as used herein, refer to a method of delaying or precluding the onset of a condition or disease and / or its attendant symptoms, barring a patient from acquiring a condition or disease, or reducing a patient's risk of acquiring a condition or disease.

[0078] The expression "reducing" as used herein refers to a partial reduction or a complete reduction.

[0079] The term "sesqui" as used herein refers to 1.5 equivalents, and a sesquisuccinate salt of a compound means its succinic acid addition salt in which succinic acid is present in a 3:2 ratio compared to the free base form of the compound.

[0080] The term "solvate" is used herein to describe a compound of the present invention that contains stoichiometric or substoichiometric amounts of one or more pharma- ceutically acceptable solvent molecules, such as ethanol. The term "hydrate" is used when said solvent is water. The pharma-ceutically acceptable solvent molecules may be co-crystallized with the compound of the present invention and / or may be present in and / or adsorbed to the solid crystalline and / or amorphous phases thereof.

[0081] The term "therapeutically effective amount" (or more simply "effective amount"), as used herein, means an amount of an active agent or ingredient (e.g., N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) that is sufficient to achieve the desired therapeutic or protective effect in the patient to which it is administered.

[0082] The terms "treat", "treating" and "treatment" as used herein are meant to include alleviating, attenuating or arresting a condition or disease and / or its attendant symptoms.

[0083] The present invention will be better understood with reference to the following examples, which are intended to represent specific embodiments of the invention and are not intended as limiting the scope of the invention.

[0084] Chemical Examples The following abbreviations are used throughout this application: °C: degrees Celsius, DMSO: dimethyl sulfoxide, δ: NMR chemical shift expressed in ppm, eq: equivalent, EDTA: ethylenediaminetetraacetic acid, eq.: equivalent, g: grams, h: hours, HPLC: high performance liquid chromatography, Hz: hertz, L: liter, M: mol / L, mM: mmol / L, μM: μmol / L, Me: methyl, MEK: methyl ethyl ketone, mg: milligram, MHz: megahertz, min: minute, mL: milliliter, mm: millimeter, mol: mole, mmol: millimole, μmol: micromole, nm: nanometer, NMR: nuclear magnetic resonance, PBS: phosphate buffer solution, ppm: parts per million, RH: relative humidity, RPM: revolutions per minute, rt: retention time, RT: room temperature (approximately 15-25 °C), s: seconds, TFA: trifluoroacetic acid, UV: ultraviolet, v: volume, XRPD: X-ray powder diffraction.

[0085] All temperatures reported are expressed in degrees Celsius (° C.) and all reactions were conducted at room temperature (RT) unless otherwise noted.

[0086] Experimental preparations or purification procedures used in the present invention, unless specific details are described, are presumed to be known to those skilled in the art and are described in standard reference manuals such as: i) Gordon, AJ; Ford, RA "The Chemist's Companion - A Handbook of Practical Data, Techniques, and References", Wiley: New York, 1972; ii) Vogel, Textbook of Practical Organic Chemistry, Pearson Prentice Hall: London, 1989; iii) P. Heinrich Stahl and Camille G. Wernuth "Handbook of Pharmaceutical Salts", Wiley VCH.

[0087] HPLC analysis. Equipment: Dionex Ultimate 3000 Column: Supelco Ascentis Express C18 15 x 4.6 mm 2.7 μm Column temperature: 50℃ Autosampler temperature: ambient UV wavelength: 275nm Injection volume: 20μL Flow rate: 1mL / min Mobile phase A: 0.1% TFA in water Mobile phase B: 0.1% TFA in acetonitrile Gradient Program:

[0088] [Table 1]

[0089] NMR analysis NMR experiments were performed on a Bruker AVIIIHD spectrometer equipped with a DCH cryoprobe operating at 500.12 MHz for proton and carbon. Experiments were performed in deuterated DMSO and samples were prepared to approximately 10 mM concentration. Further analysis was occasionally performed in D2O and / or methanol-d4. Chemical shifts are expressed in parts per million (ppm, δ units). Coupling constants are expressed in Hz. Abbreviations representing the multiplicities observed in NMR spectra are as follows: s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), br (broad line).

[0090] X-ray powder diffraction (XRPD) XRPD analysis was performed on a PANalytical Expert Pro using a PIXcel detector (128 channels) scanning samples between 3 and 35°2θ. The material was gently ground to release any aggregates and loaded into a multi-well plate with a Mylar polymer film to support the sample. The multi-well plate was then placed into the diffractometer and analyzed using Cu Kα radiation (α1λ=1.54060Å; α2=1.54443Å; β=1.39225Å; α1:α2 ratio=0.5) running in transmission mode (step width 0.0130°2θ, step time 18.87 s) using a 40 kV / 40 mA generator setting. Data were visualized and images were generated using the HiScore Plus 4.7 desktop application (PANalytical, 2017).

[0091] Solvents and reagents were purchased and used as received from commercial suppliers unless otherwise specified.

[0092] Example 1 Form 1: Synthesis and characterization of the crystalline form of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sesquisuccinate 514.6 mg of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine was weighed out into a 20 mL scintillation vial. 10 mL of methyl ethyl ketone (99% purity) was added to the vial, which was stirred at 40° C. using a temperature control block, stir plate, and magnetic needle. A clear solution was not observed, so an additional 5 mL of MEK was added to aid dissolution at 40° C. A clear solution was obtained, and the clear solution at 40° C. was polish filtered using a PTFE filter (0.45 micron) and syringe. The filtered solution was added back into a clean 20 mL scintillation vial and reheated to 40° C. 142.5 mg of succinic acid (1 molar equivalent) was added to the vial. A clear solution was initially observed. After stirring at 40° C. for 30 min, crystallization of the material was observed. The experiment was temperature cycled between 40° C. and 5° C. in 3-hour cycles over 20 h. The slurry was filtered using a Buchner flask and funnel using grade 1 Whatman filter paper (pore size=11 μm) under vacuum at 5° C. The wet cake was washed with 2.5 mL of chilled (5° C.) MEK and dried under reduced pressure at 40° C. for 2 h. The dried isolated solid was analyzed by XRPD and HPLC. The filtered mother liquor was analyzed by HPLC for concentration.

[0093] An isolated yield of 78.5% was achieved and, based on the mother liquor concentration, an HPLC yield of 93.5% was determined (see Table 1).

[0094] [Table 2]

[0095] HPLC indicated 99.2% purity; rt = 10.65 min. The XRPD 2θ diffractogram showed that the material was crystalline (FIG. 1) and contained peaks at diffraction angles of 2θ=3.8°±0.2° (100%), 10.3°±0.2° (11.3%), 12.4°±0.2° (6.8%), 16.2°±0.2° (23.5%), 17.9°±0.2° (11.9%), 19.8°±0.2° (10.2%), 20.4°±0.2° (13.0%), 23.8°±0.2° (23.3%) and 26.7°±0.2° (9.8%) when illuminated with a CuKα light source.

[0096] Another batch of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine, sesquisuccinate was prepared and further characterized by HPLC, XRPD and NMR. HPLC indicated a purity of 98.4%. The XRPD 2θ diffractogram was identical to that of the previously described batch. In DMSO-d6 1 H and quantitative 13 The C NMR spectra are presented in Figures 5 and 6, respectively. 1 H NMR spectrum (500.12 MHz - DMSO-d6): δ (ppm): 7.13 (2H dd J1 = 6 Hz, J2 = 3 Hz), 6.87 (2H dd J1 = 6 Hz, J2 = 3 Hz), 6.71 (1H br s), 3.30 (2H t J = 7 Hz), 2.42 (6H m, 2.33 4H dt J1 = 16 Hz, J2=7 Hz), 2.04 (4H d J = 7 Hz), 1.74 (2H quintet J = 7 Hz), 1.67 (2H septet J = 7 Hz), 1.52 (2H quintet J = 7Hz), 0.85 (12H d J = 7 Hz) ppm. quantitative 13C NMR spectrum (121.16 MHz - DMSO-d6): δ (ppm): 174.3, 156.0, 119.5, 111.9, 63.9 56.1, 55.6, 53.0 (d), 29.8, 26.8, 26.6, 24.2, 21.3. Quantitative 13 C NMR confirmed the stoichiometry and successful formation of the sesquisuccinate salt.

[0097] Biological Examples Example 1 Effects of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine)sesquisuccinate form 1 on glutamate-injured rat primary motoneurons Primary culture of spinal motor neurons Rat spinal motor neurons (MNs) were cultured as previously described by Boussicault et al., J Neurosci Res. 2020 Dec;98(12), 2435-50 and Wang et al., Hum. Mol. Genet. 2013 Dec;1;22(23), 4706-19. Pregnant female rats (Wistar rats; Janvier Labs France) at day 14 of gestation were sacrificed using deep anesthesia with a CO2 chamber followed by cervical dislocation. Embryos (E14) were then removed from the uterus and immediately placed in ice-cold L15 Leibovitz medium supplemented with 2% penicillin (10,000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1% bovine serum albumin (BSA). Spinal cords were removed and placed in ice-cold Leibovitz medium (L15). Spinal cords were treated with trypsin-ethylenediaminetetraacetic acid (EDTA) solution at a final concentration of 0.05% trypsin and 0.02% EDTA for 20 min at 37°C. Dissociation was stopped by the addition of Dulbecco's modified Eagle medium (DMEM) supplemented with 4.5 g / l glucose containing DNase I grade II (final concentration 0.5 mg / mL) and 10% fetal calf serum (FCS). Cells were mechanically dissociated by three forced passages with the tip of a 10 mL pipette. Cells were then centrifuged at 515 x g for 10 min at +4°C. The supernatant was discarded and the pellet was resuspended in a defined culture medium consisting of neurobasal medium supplemented with a 2% solution of B27 supplement, 2 mmol / l L-glutamine, 2% PS solution and 10 ng / mL brain-derived neurotrophic factor (BDNF). Viable cells were counted using the trypan blue exclusion test on a Neubauer cytometer. The cells were seeded at a density of 20,000 cells per well in a 96-well plate precoated with poly-L-lysine and cultured in an air (95%)-CO2 (5%) incubator at 37°C. The medium was changed every 2 days.

[0098] Compound treatment-48 hour incubation On day 13 of culture, glutamate was added for 20 min to a final concentration of 5 μM diluted in control medium in the presence of sesquisuccinate form 1. After 20 min, glutamate was washed out and fresh culture medium supplemented with sesquisuccinate form 1 was added for 48 h.

[0099] Endpoint Assessment After 15 days of culture (48 hours after glutamate insult), cell culture supernatants were collected and spinal MNs were fixed with a cold solution of ethanol (95%) and acetic acid (5%) for 5 minutes at -20°C for immunostaining. Cells were washed twice in PBS. Cell membranes were permeabilized and non-specific sites were blocked with a solution of PBS containing 0.1% saponin and 1% FCS for 15 minutes at room temperature. Cells were then incubated for 2 hours with: a) Mouse monoclonal antibody anti-microtubule associated protein 2 (MAP-2) at a dilution of 1 / 400 in PBS containing 1% fetal bovine serum and 0.1% saponin. This antibody specifically binds to MAP-2 present in the cell bodies and neurites of all MNs. This antibody was revealed with Alexa Fluor 488 goat anti-mouse IgG at a dilution of 1 / 400 in PBS containing 1% FCS, 0.1% saponin for 1 h at room temperature. b) Rabbit polyclonal antibody anti-TDP43 at a dilution of 1 / 100 in PBS containing 1% fetal bovine serum and 0.1% saponin. Cytoplasmic localization of TDP43 was investigated. The antibody was revealed with Alexa Fluor 568 goat anti-rabbit IgG at a dilution of 1 / 400 in PBS containing 1% FCS, 0.1% saponin for 1 h at room temperature. Neuronal nuclei were counterstained with a fluorescent dye (Hoechst).

[0100] For each condition, 30 pictures per well (representative of the entire well) were automatically taken using Image Express (Molecular Devices) at 20x magnification. All images were taken under the same conditions (exposure time, gain and laser intensity). Analysis of the different endpoints was performed automatically using a custom module editor (Molecular Devices). The following morphological criteria were used to distinguish motor neurons (MAP-2) from interneurons (stained MAP-2): a cell body diameter >15 mm and the presence of at least three neuritic processes (Ferraiuolo et al., Brain 2011:134;2627-2641).

[0101] The endpoints were: - MN viability (MAP-2 staining count of MN) - Neurite network of MN (MAP-2 staining, total neurite length expressed in μm) - Cytoplasmic TDP43 (extranuclear, eTDP43) in MAP-2 positive MNs (overlap between MAP-2 and cytoplasmic TDP-43 in μm 2 Results are expressed as the ratio of duplicated MAP-2 / TDP-43 per number of MN.

[0102] statistical analysis All values ​​are expressed as mean + / - SEM. Statistical analysis was performed using one-way ANOVA followed by Fisher's PLSD test. All tests were performed using GraphPad Prism software version 9 and statistical significance was set at *p<0.05, **p<0.001, ***p<0.001 and ****p<0.0001.

[0103] result - Motor neuron survival: As expected, glutamate intoxication significantly reduced cell survival compared to the control group (mean: 58%; Fig. 7A). Several doses of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sesquisuccinate form 1 (from 30 nM to 300 nM) showed a positive and significant effect on survival compared to the glutamate condition. The maximum effect was obtained at a dose of 30 nM (mean: 79%). - Neuritic network integrity: Glutamate significantly reduced the neurite network (mean: 62%, FIG. 7B). Application of several doses of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sesquisuccinate form 1 (30 nM to 300 nM) was able to improve the neurite network after glutamate insult, with a maximal effect at 100 nM (mean: 77%). - Extranuclear TDP-43: Glutamate application significantly increased the abnormal cytoplasmic TDP-43 signal (mean: 135% compared to control; Figure 7C). Sesquisuccinate form 1 at concentrations from 30 to 300 nM was able to significantly reduce the accumulation of TDP-43 in the cytoplasm, up to 17% at 300 nM concentration.

[0104] Example 2 Effects of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine)sesquisuccinate form 1 pretreatment on rat primary motor neurons injured by 24-hour glutamate pretreatment-injury-24-hour treatment Primary culture of spinal motor neurons The culture was carried out as described in Example 1.

[0105] Compound treatment - 24 h preincubation + 24 h incubation On day 12 of culture, primary motor neurons were treated for 24 hours with sesquisuccinate form 1. On day 13 of culture, glutamate was added for 20 minutes to a final concentration of 5 μM diluted in control medium in the presence of sesquisuccinate form 1. After 20 minutes, glutamate was washed out and fresh culture medium plus sesquisuccinate form 1 was added for an additional 24 hours.

[0106] Endpoint Assessment Endpoint assessments were performed as described in Example 1.

[0107] statistical analysis Statistical analyses were performed as described in Example 1.

[0108] result - Motor neuron survival: As expected, glutamate significantly reduced cell survival compared to the control group (mean: 62%; FIG. 8A). Several doses of sesquisuccinate form 1 (from 30 nM to 300 nM) displayed a positive and significant effect on survival compared to the glutamate condition. The maximum effect was obtained with a dose of 300 nM sesquisuccinate form 1 (mean survival: 79%). - Neuritic network integrity: Glutamate strongly impaired the neurite network of spinal motor neurons (mean: 60%; Figure 8B). All doses of sesquisuccinate form 1 from 30 nM to 300 nM were able to protect the neurite network from glutamate damage, with a maximal effect at 300 nM (mean: 81%). - Extranuclear TDP-43: Glutamate application significantly increased the cytoplasmic TDP43 signal (mean: 139% compared to control, Figure 8C). Sesquisuccinate form 1 (doses from 30 nM to 300 nM) was also able to reduce the aberrant distribution of TDP-43 by up to 14%.

[0109] Example 3 Effect of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sesquisuccinate form 1 on rat primary SOD1 motoneuron maturation after glutamate insult Rats overexpressing human superoxide dismutase 1 (SOD1) with a mutation known to cause human familial ALS (e.g., SOD1 G93A Genetic rodent models, including rats, have been used to study ALS pathogenesis. G93A ALS rat models expressing mutant forms of ALS exhibit features that closely recapitulate the clinical and histopathological features of the human disease (Nagai et al., The Journal of Neuroscience, 1 Dec. 2001, 21(23):9246-9254). In human or rodent studies (SOD1 model), MN loss is preceded by increased excitability. Increased neuronal excitability correlates with structural changes in dendritic arbors and spines, such that dendritic atrophy and spine loss in medium spiny neurons (MSNs) of the striatum and lower MNs in the lumbar spinal cord are observed (Ferrucci et al., Neurobiology of Disease 37 (2010) 370-383; Avossa et al., Neuroscience 138 (2006) 1179-1194).

[0110] SOD1 G93A Genotyping of embryos for motor neurons Females (Sprague-Dawley, Janvier) were transfected with Tg SOD1 mice at Neuro-Sys (Gardanne, France). G93A The embryos were mated with male rats (Taconic Bioscience, US). Pregnant females on day 14 of gestation were used for culture. On the day of dissection, a piece of each embryo head (approximately 3 mm) was cut using a new scalpel. 3) into a 2 mL DNase-free tube. DNA was extracted with SYBR Green Extract-N-Amp Tissue PCR Kit (Sigma Aldrich). Briefly, 120 μL of extraction solution was placed on each piece of embryo head. They were then incubated at room temperature for 10 minutes. At the end of this incubation period, the heads were incubated at 95°C for 5 minutes. Immediately after this final incubation, 100 μL of neutralization solution was added and each DNA extract was diluted 1 / 40 and stored at +4°C until use. A genomic fragment with human SOD1 primers (5'-CATCAGCCCTAATCCATCTGA-3' SEQ ID NO:1; 5'-CGCGACTAACAATCAAAGTGA-3' SEQ ID NO:2) was used to amplify SOD1 G93A The gene was determined. SOD1 primer was diluted to 3 μM in sterile ultrapure water. Briefly, the mix for PCR was prepared with ultrapure water (4 μL per sample), 3 μM primer (2 μL per sample) and master mix (10 μL per sample). 16 μL of PCR mix was added to each well in a PCR 96-well plate. 4 μL of each diluted DNA was added according to the plan deposit.

[0111] RT-PCR was carried out using a CFX96 Biorad RT-PCR system using the following program: - Initial denaturation: 95℃-20 seconds - 45 cycles: 95℃-10 sec, 65℃-10 sec, 72℃-30 sec - Melting curve: 95℃-15 sec, 64℃-1 min, 90℃-30 sec, 60℃-15 sec Amplification plots and melting curves were analyzed by Biorad software.

[0112] The results for each sample were compared with the negative control (ultrapure water) and the positive control (Tg SOD1 G93A DNA from the embryos) and a conclusion was made regarding the genotype of each embryo (WT or Tg).

[0113] Spinal cord SOD1 G93A Primary culture of motor neurons Rat spinal motor neurons were cultured as described by Boussicault et al., J Neurosci Res. 2020 Dec;98(12), 2435-50 and Wang et al., Hum. Mol. Genet., 2013 Dec. 1;22(23), 4706-19. Pregnant female rats on day 14 of gestation were sacrificed by cervical dislocation. Culture was performed as described in Example 1.

[0114] Compound treatment-48 hour incubation On day 13, the culture medium was removed and the cultures were exposed to test compounds: N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sesquisuccinate Form 1 compared to riluzole and edaravone, and glutamate (5 μM) in defined medium for 20 minutes at 37° C. After glutamate exposure, the cultures were washed with defined medium at 37° C. and then placed in fresh culture medium containing the test compounds, riluzole or edaravone, for an additional 48 hours.

[0115] Endpoint Assessment -Endpoint assessments were performed as described in Example 1.

[0116] statistical analysis Statistical analyses were performed as described in Example 1.

[0117] result Glutamate insult significantly reduced the number of MNs, the length of their neurite network, and triggered an abnormal distribution of TDP-43 into the cytoplasm (see Figure 9 ).

[0118] N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sesquisuccinate form 1 at doses of 3 nM to 100 nM was neuroprotective. The protective effect on neuronal survival and on the distribution of TDP-43 was dose-dependent. At a dose of 100 nM, the protective effect of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sesquisuccinate form 1 was comparable to that of riluzole or edaravone.

[0119] Example 4 Effects of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sesquisuccinate form 1 on glutamate-injured spinal cord / muscle neuromuscular junction. Cultivation of human myoblasts and rat spinal cord explants All experiments were performed in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals and in compliance with current European Union regulations (Directive 2010 / 63 / EU).

[0120] Human muscle cell lines were established from dissociated cells (22000 cells per well). They were plated in 0.1% water on gelatin-coated 48-well plates and grown in a growth medium consisting of a mix of 62% MEM medium and 25% M199 medium supplemented with 2 mM glutamine, 10 μg / mL human insulin, 10 ng / mL human recombinant epidermal growth factor (EGF), 2 ng / mL human recombinant basic fibroblast growth factor (bFGF), 10% fetal calf serum (FCS) and 2% penicillin 10.000 U / mL and streptomycin 10.000 μg / mL (PS). The medium was changed every 2 days.

[0121] Five days after the start of the culture, immediately after the fusion of satellite cells, whole transverse slices of spinal cord with four attached dorsal root ganglia (DRG) taken from 13-day-old Wistar rat embryos (Janvier Labs, France) were placed on the muscle monolayer (one explant per well in the central region). The presence of DRG is necessary to achieve a good innervation ratio. Innervated cultures were maintained in a mixed (67% / 25%) medium composed of MEM and medium 199 supplemented with 5% FCS, 5 μg / mL insulin, 2 mM glutamine and 2% PS. After 24 hours of coculture, the outgrowth of neurites from the spinal cord explants is usually observed. These neurites contacted myotubes and induced the formation of neuromuscular junctions, and the first contractions were observed about 8 days after coculture. Soon afterwards, innervated muscle fibers located in close proximity to the spinal cord explants were virtually continuously contracting. Innervated fibers were morphologically and spatially distinct from, and easily distinguished from, non-innervated ones. Plates were maintained at 37°C in a humidified incubator in an air (95%)-CO2 (5%) atmosphere.

[0122] Compound treatment-48 hour incubation On day 27 (co-culture), co-cultures were incubated with N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sesquisuccinate form 1 or riluzole in the presence of glutamate (60 μM final concentration diluted in control medium) for 20 min.

[0123] After 20 min of glutamate intoxication, the supernatant was removed and fresh culture medium supplemented with N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sesquisuccinate form 1 or with riluzole was added for an additional 48 h.

[0124] Endpoint Assessment immunostaining After 48 h of intoxication, cells were incubated with 500 nM α-bungarotoxin coupled with Alexa 488 for 15 min in culture medium at 37° C. to detect motor endplates. After washing twice in PBS, cells were fixed with a solution of 4% paraformaldehyde in PBS, pH=7.3, for 20 min at room temperature.

[0125] The cells were washed twice in PBS and permeabilized with a solution of 0.1% saponin and 1% FCS in PBS for 15 min at room temperature to block non-specific sites.

[0126] The co-cultures were then incubated with mouse monoclonal anti-neurofilament 200KD antibody (NFH) at a dilution of 1 / 400 in PBS containing 1% FCS, 0.1% saponin for 2 hours at room temperature. Motor neuron neurites and axons were stained with antibody against NFH. The antibody was revealed with Alexa Fluor 568 goat anti-mouse IgG at a dilution of 1 / 400 in PBS containing 1% FCS, 0.1% saponin for 1 hour at room temperature. Neuronal nuclei were labeled with Hoechst solution, a nuclear fluorescent marker at 1 μg / ml in the same solution.

[0127] The analysis of the different endpoints was performed automatically. One co-culture was performed (6 wells per condition). For each condition, 20 pictures per well were taken automatically using Image Express (Molecular Devices) at 10x magnification. All images were taken under the same conditions.

[0128] The following endpoints were measured automatically: (1) The number of NMJs, (2) Average size of NMJ (μm 2 NMJ area at (3) Total neurite length (μm) (= innervation network).

[0129] statistical analysis Statistical analyses were performed as described in Example 1.

[0130] result As shown in FIG. 10, application of glutamate (60 μM, 20 min) resulted in a significant reduction in the number (A) and total area (B) of NMJs in culture. The area of ​​NMJs indicates the degree of clustering of their subunits. Functional NMJs are larger than non-innervated NMJs. In addition, injury led to a significant loss of MN neurites (C). Sesquisuccinate form 1 (300 nM) displayed a neuroprotective effect in a dose-dependent manner, as it increased the number and total area of ​​NMJs as well as the total MN neurite network. The protective effect of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sesquisuccinate form 1 was comparable to that of riluzole.

Claims

1. 2. The succinate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine and pharmaceutically acceptable solvates thereof for use in treating and / or preventing motor neuron diseases and neuromuscular junction disorders selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), neurolathyrism, Konzo, Tay-Sachs disease, Sandhoff disease, progressive muscular atrophy (PMA), monolimbal muscular atrophy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), post-polio syndrome, post-irradiation syndrome, stiff-person syndrome, motor unit disorders resulting from accidents, myasthenia gravis, Eaton-Lambert syndrome.

2. 2. The succinate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine and pharmaceutically acceptable solvates thereof for use in delaying the onset in a patient of a motor neuron disease and neuromuscular junction disorder selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), neurolathyrism, Konzo, Tay-Sachs disease, Sandhoff disease, progressive muscular atrophy (PMA), monolimbal muscular atrophy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), post-polio syndrome, post-irradiation syndrome, stiff-person syndrome, motor unit disorder resulting from accidents, myasthenia gravis, Eaton-Lambert syndrome.

3. Formula I 【Chemistry 1】 [wherein x is 1 to 4] 3. The succinate salt for use according to claim 1 or 2, having the formula:

4. 4. The succinate salt and its pharma- ceutically acceptable solvates for use according to claim 3, characterized in that x is about 1.

5.

5. 5. The succinate salt for use according to claim 4, wherein the succinate salt is N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sesquisuccinate.

6. 6. The succinate salt for use according to any one of claims 1 to 5, wherein the motor neuron disease and neuromuscular junction disorder is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), progressive muscular atrophy (PMA), single limb muscular atrophy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), myasthenia gravis, Eaton-Lambert syndrome, motor unit disorders resulting from accidents.

7. 7. The succinate salt for use according to claim 6, wherein the motor neuron disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP).

8. 8. The succinate salt for use according to claim 7, wherein the motor neuron disease is non-FTD amyotrophic lateral sclerosis.

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