1-((2-(2,2,2-trifluoroethoxy)pyridin-4-yl)methyl)urea derivatives as KCNQ enhancers

Abstract

The present application relates to small molecule enhancers of potassium channels (e.g., Kv7 enhancers - also known as KCNQ enhancers), compositions comprising such compounds, and methods of using such compounds to treat amyotrophic lateral sclerosis and other neurological diseases caused by changes in motor neuron excitability, including but not limited to primary lateral sclerosis, pseudobulbar palsy, progressive bulbar palsy, progressive muscular atrophy and epilepsy.

Description

[0001] The present invention belongs to the field of medicine. In particular, the present invention relates to compounds, methods and pharmaceutical compositions for treating amyotrophic lateral sclerosis (ALS) and other nervous system diseases caused by changes in motor neuron excitability, including but not limited to primary lateral sclerosis, pseudobulbar palsy, progressive bulbar palsy, progressive muscular atrophy and epilepsy.

[0002] ALS (sometimes called "Lou Gehrig's Disease") is a fatal neurological disease that affects approximately 1.5-3 in 100,000 people each year. It is characterized by a progressive loss of motor neurons and usually leads to death within 2-3 years of diagnosis. Although research is ongoing, most ALS cases appear to be sporadic, with no known cause.

[0003] ALS patients usually show increased excitability of peripheral and central motor neurons, leading to fasciculation, muscle spasms and spasticity. It is believed that increased neuronal excitability leads to calcium overload and cell death. In fact, motor neuron excitability is negatively correlated with the survival rate of ALS patients (see K. Kanai et al., J. Neurol. Neurosurg. Psychiatry, 83, 734-738 (2012)).

[0004] The study investigated the mechanisms and causes of changes in motor neuron excitability in ALS patients (compared to non-ALS patients). (See K. Kanai et al., Brain, 129, 953-962 (2006)). The results of the study indicate that reduced potassium channel activity in neurons is a major factor in disease-related hyperexcitation (see above). In addition, motor neurons from pluripotent stem cells of ALS patients also exhibit hyperexcitation. (See BJ Wainger et al., Cell Rep., 7, 1-11 (2014)). These stem cell-derived neurons show reduced delayed rectifier potassium currents, and in fact, when the agents retigabine and flupirtine (which are known Kv7 potassium channel enhancers) are added to these stem cell-derived neurons, the hyperexcitability of motor neurons returns to normal and the survival rate of cells in vitro increases (see above). In another study, the Kv7 enhancer retigabine alleviated excitotoxic symptoms and increased survival in an in vitro model of ALS using rat hypoglossal motor neurons (see F. Ghezzi et al., J. Physiol., 596, 2611-2629 (2018)).

[0005] Currently, the only drug approved for the treatment of ALS is riluzole, which has been shown to extend survival by 2-3 months. There is a clear need for more effective, better and longer-lasting treatments.

[0006] The known Kv7 enhancer retigabine acts on the Kv7.2-7.5 (KCNQ2-KCNQ5) potassium channel. It is approved as an adjunctive treatment for patients with drug-resistant epilepsy. It was approved in Europe and the United States in 2011, but was voluntarily withdrawn in 2017. The withdrawal of retigabine from clinical use is believed to be based on a number of tolerability issues, which have led to very limited use of the drug. Tolerability issues include the occurrence of drowsiness and dizziness, which are common and may be mechanism-based, and the less common occurrence of urinary retention and pigmentation changes in the retina and skin. The potential for retinal changes and vision loss that leads to the black box warning on the retigabine label is not thought to be mechanism-based. Urinary retention is likely a result of enhancement of the bladder Kv7.3 / 7.5 channels. However, given its side effects, the development of new Kv7 enhancers is needed.

[0007] Recent studies comparing the effects of riluzole (e.g., an approved ALS treatment) and retigabine on the excitability of motor neurons in ALS patients suggest that enhancers of Kv7 channels may have superior efficacy to riluzole (see M. Kovalchuk et al., Clinical Pharmacology & Therapeutics, 104, 1136-1145 (2018)). Therefore, improved enhancers with better tolerability than retigabine will benefit the clinical treatment of ALS and other hyperexcitability-related disorders. (See B. Kalappa et al., The Journal of Neuroscience, 35(23): 8829–8842) (2015). To date, no agents acting on Kv7 have been approved for the treatment of ALS, and therefore, there is still a need for agents acting on Kv7, such as alternative Kv7 enhancers that provide therapeutic benefits. In addition, it may be beneficial to make such enhancers more selective for Kv7.2 / 7.3 relative to other Kv7 channels. There is also a need for new Kv7 agents that avoid undesirable side effects and that can offer an improved combination of pharmacological properties, including safety, potency, efficacy, and tolerability, particularly for the treatment of peripheral and central motor neuron excitability.

[0008] The present embodiments provide compounds that are potassium channel enhancers (eg, Kv7 enhancers - also known as KCNQ enhancers) that are useful for treating ALS and other neurological diseases caused by changes in motor neuron excitability.

[0009] Specifically, the compound of the following formula (which is labeled as "Formula I") can be used as a Kv7 enhancer:

[0010]

[0011] Where R1 is

[0012]

[0013] Wherein, in Formula I, R2 is H or OH. In addition to the compounds of Formula I, one or more pharmaceutically acceptable salts can be prepared from the compounds of Formula I, and these pharmaceutically acceptable salts can also be prepared and used as Kv7 enhancers.

[0014] In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof is prepared such that R1 is

[0015]

[0016] And R2 is H.

[0017] In other embodiments, the compound of Formula I (or a pharmaceutically acceptable salt thereof) is prepared such that R1 is

[0018]

[0019] and R2 is OH. In addition, in such embodiments, when R2 is OH, the carbon to which the OH group is attached is a stereocenter. Therefore, in some embodiments, the compound of Formula I (or a pharmaceutically acceptable salt) can be a racemic mixture of two enantiomers. In other embodiments, a specific enantiomer can be used, including one of the following enantiomers:

[0020]

[0021]

[0022] Those skilled in the art will understand how to construct embodiments that utilize only one of the aforementioned enantiomers. Other embodiments may be designed as mixtures of different enantiomers having different percentages for each component.

[0023] In other embodiments, the compound of Formula I (or a pharmaceutically acceptable salt thereof) is prepared such that

[0024] R1 is

[0025]

[0026] And R2 is H.

[0027] In other embodiments, the compound of Formula I (or a pharmaceutically acceptable salt thereof) is prepared such that

[0028] R1 is

[0029]

[0030] and R2 is OH. In addition, in such embodiments, when R2 is OH, the carbon to which the OH group is attached is a stereocenter. Therefore, in some embodiments, the compound of Formula I (or a pharmaceutically acceptable salt) can be a racemic mixture of two enantiomers. In other embodiments, a specific enantiomer can be used, including one of the following enantiomers:

[0031]

[0032] Those skilled in the art will understand how to construct embodiments that utilize only one of the aforementioned enantiomers. Other embodiments may be designed as mixtures of different enantiomers having different percentages for each component.

[0033] The present embodiment further provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt and one or more pharmaceutically acceptable carriers, diluents or excipients. Such pharmaceutical compositions can provide methods for treating diseases. Specifically, the present embodiment provides a method for treating a disease caused by changes in motor neuron excitability, comprising administering an effective amount of a compound of formula I (or a pharmaceutically acceptable salt thereof) to a patient in need. In some particularly preferred embodiments, the disease caused by changes in motor neuron excitability is ALS.

[0034] Other embodiments provide methods for treating diseases caused by changes in motor neuron excitability, comprising administering an effective amount of a compound of Formula I (or a pharmaceutically acceptable salt thereof) to a patient in need thereof. In some particularly preferred embodiments, the disease caused by changes in motor neuron excitability is ALS.

[0035] This embodiment also provides a compound of formula I (or a pharmaceutically acceptable salt thereof) for use in treatment. More specifically, this embodiment provides a compound of formula I (or a pharmaceutically acceptable salt thereof) for use in treating a disease caused by changes in motor neuron excitability. In some embodiments, the disease may be ALS.

[0036] In addition, this embodiment provides the use of a compound of Formula I (or a pharmaceutically acceptable salt thereof) in the preparation of a medicament for treating a disease caused by changes in motor neuron excitability. In some such embodiments, the disease is ALS.

[0037] As used herein, the term "diseases associated with changes in motor neuron excitability" or "diseases caused by changes in motor neuron excitability" includes diseases selected from the group consisting of ALS, primary lateral sclerosis, pseudobulbar palsy, progressive bulbar palsy, epilepsy, and progressive muscular atrophy. These terms also include all diseases listed in A. Verma et al., "Atypical Motor Neuron Disease and Related Motor Syndromes," Seminars in Neurology, Vol. 21, No. 2, 2001. These terms also include PNH (peripheral nerve hyperexcitability) disorders. Information about PNH disorders can be found in C. et al., "Peripheral nerve hyperexcitability syndromes" Rev Neurosci. 2015; 26(2): 239-51. Therefore, the compounds and pharmaceutically acceptable salts herein can be used to treat one or more of these diseases.

[0038] As used interchangeably herein, the terms "patient," "subject," and "individual" refer to a person, more specifically, a patient in need thereof. In certain embodiments, the patient is further characterized by having a disease, disorder, or condition that would benefit from an enhancement of Kv7 (e.g., ALS or another disease). In another embodiment, the patient is further characterized by being at risk of developing the above-mentioned condition or developing a condition that would benefit from an enhancement of Kv7.

[0039] The effective amount can be determined by one skilled in the art by using known techniques and by observing the results obtained under similar circumstances. In determining the effective amount for a patient, many factors are considered, including, but not limited to: their size, age, and general health; the specific disease or disorder involved; the extent or involvement or severity of the disease or disorder; the response of the individual patient; the specific compound administered; the mode of administration; the bioavailability characteristics of the administered formulation; the dosage regimen selected; the use of concomitant medications; and other relevant circumstances. In some embodiments, the effective amount provides a clinically significant reduction in the excitability of peripheral and central motor neurons.

[0040] The compounds of the present invention are formulated into pharmaceutical compositions, which are administered by any route that makes the compounds bioavailable. Preferably, such compositions are used for oral administration. Such pharmaceutical compositions and methods for preparing them are well known in the art (see, for example, Remington: The Science and Practice of Pharmacy, LV Allen, Editor, 22nd edition, Pharmaceutical Press, 2012).

[0041] The compounds of formula I and their pharmaceutically acceptable salts are particularly suitable for the treatment methods of the present invention, and certain configurations are preferred. The compounds of the present invention in the following table describe such configurations. It should be understood that these preferences apply to both the treatment methods and the compounds of the present invention.

[0042] Compounds of this embodiment wherein R2 is OH include:

[0043]

[0044]

[0045] Of these compounds, where R2 is OH, compounds with the above "flat bonds" are racemic and / or both enantiomers exist simultaneously (in a 50 / 50 mixture or some other ratio). Compounds with wedge-shaped bonds and dashed bonds above depict specific enantiomers. Compounds with "wavy" bonds indicate that it is a single enantiomer, but the exact configuration of this single enantiomer is unknown, and therefore, such enantiomers are distinguished by their optical rotation (e.g., whether they rotate light in the (+) or (-) direction).

[0046] Compounds of this embodiment also include the following molecules (wherein R2 is H):

[0047]

[0048] The above compounds with an OH group as R2 may be those with OH in an "up" configuration (as indicated by the wedge projection). Other embodiments may be designed in which the OH group is in a "down" configuration. One skilled in the art will understand how to prepare such other enantiomers, for example by using different starting materials and / or using different reactions, etc. Such enantiomers have the following structures and are part of the presently claimed embodiments:

[0049]

[0050] While the present invention contemplates all individual enantiomers and diastereomers, as well as mixtures of enantiomers of the compounds (including racemates), the above compounds and pharmaceutically acceptable salts thereof are particularly preferred.

[0051] One of ordinary skill in the art can separate or resolve the single enantiomers at any convenient point in the synthesis of the compounds of the invention by methods such as selective crystallization techniques, chiral chromatography (see, e.g., J. Jacques et al., "Enantiomers, Racemates, and Resolutions", John Wiley and Sons, Inc., 1981, and EL Eliel and SH Wilen, "Stereochemistry of Organic Compounds", Wiley-Interscience, 1994), or supercritical fluid chromatography (SFC) (see, e.g., TA Berger; "Supercritical Fluid Chromatography Primer," Agilent Technologies, July 2015).

[0052] Pharmaceutically acceptable salts of the compounds of the present invention can be formed, for example, by reacting a suitable free base of a compound of the present invention with a suitable pharmaceutically acceptable acid in a suitable solvent under standard conditions well known in the art. See, for example, Gould, PL, "Salt selection for basic drugs," International Journal of Pharmaceutics, 33: 201-217 (1986); Bastin, RJ et al. "Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities," Organic Process Research and Development, 4: 427-435 (2000); and Berge, SM et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66: 1-19, (1977).

[0053] The compound of the present invention or its salt can be prepared by various methods known to those of ordinary skill in the art, some of which are described in the following schemes, preparations and examples. The product of each step in the following scheme can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, grinding and crystallization. In the following scheme, unless otherwise indicated, all substituents are as defined above. Reagents and starting materials are readily available to those of ordinary skill in the art. Without limiting the scope of the present invention, the following schemes, preparations and examples are provided to further illustrate aspects of the present invention. In addition, it is understood by those of ordinary skill in the art that the compound of formula I can be prepared by using starting materials or intermediates with corresponding desired stereochemical configurations that can be prepared by those skilled in the art.

[0054] Certain abbreviations may be used below. The meanings of these abbreviations are as follows: "ACN" means acetonitrile; "Ac" means acetyl; "AcOH" means acetic acid; "Ac2O" means acetic anhydride; "AP5" means (2R)-amino-5-phosphonopentanoate; "BDNF" means brain-derived neurotrophic factor; "BOC" means tert-butyloxycarbonyl; "CAS#" means Chemical Abstracts Registry Number; "CMAP" means compound muscle action potential; "DCM" means methylene chloride or dichloromethane; "DIPEA" means N,N-diisopropylethylamine; "DMEA" means dimethylethylamine; "DMF" means N,N-dimethylformamide; "DMSO" means dimethyl sulfoxide; "D-PBS" refers to Dulbecco's phosphate buffered saline; "EDTA" refers to ethylenediaminetetraacetic acid; "EGTA" refers to ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; "ES / MS" refers to electrospray ionization mass spectrometry; "Et2O" refers to ethyl ether; "EtOAc" refers to ethyl acetate; "EtOH" refers to ethanol or ethyl alcohol; "h" refers to hour / hours; "HEPES" refers to 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; "IPA" refers to isopropanol or isopropyl alcohol; "IPAm" refers to isopropylamine; "iPrOAc" refers to isopropyl acetate; "LC / M SMS refers to liquid chromatography tandem mass spectrometry; LiHMDS refers to lithium bis(trimethylsilyl)amide; KOtBu refers to potassium tert-butoxide; Me refers to methyl; msec refers to time in milliseconds; MTBE refers to methyl tert-butyl ether; min refers to minute / minutes; NaOtBu refers to sodium tert-butoxide; n-BuLi refers to n-butyllithium; NBQX refers to (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline; OAc refers to ester "PBS" means phosphate buffered saline; "RT" means room temperature; "SCX" means strong cation exchange; "SD" means standard deviation; "sec" means time in seconds; "SEM" means standard error of the mean; "SFC" means supercritical fluid chromatography; "TEA" means triethylamine; "TFA" means trifluoroacetic acid; "THF" means tetrahydrofuran; "TMA" means trimethylamine; "TMEDA" means tetramethylethylenediamine; "Tris" means tris(hydroxymethyl)aminomethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol; "[α] D 20 ” refers to the specific rotation at 20°C and 589 nm, where c is the concentration in g / mL (which is usually g / 100 mL).

[0055] General approach

[0056] Solution 1

[0057]

[0058] Scheme 1 illustrates the preparation of compounds of formula I, wherein R 1 As defined above. Activated ester formation of appropriately substituted amines (or their corresponding salts, such as HCl) is well documented in the art, using, for example, carbonyldiimidazole, and selective N- to O-carbonylation of the activated intermediate with (2-ethoxy-4-pyridyl)methylamine (or its corresponding salt form, such as HCl) in the presence of a suitable organic base can produce the desired urea of ​​the present invention (step 1). Those skilled in the art will recognize that compounds of Formula I containing stereochemistry can be prepared by chiral substituted amines to obtain single enantiomers, or by chiral resolution of compounds of Formula I, using chiral chromatographic techniques (e.g., SFC), or by the use of chiral auxiliaries (e.g., chiral salt preparations), which are well known in the art.

[0059] Solution 2

[0060]

[0061] Scheme 2 describes the preparation of racemic-2-amino-1-[1-(trifluoromethyl)-cyclopropyl]ethanol hydrochloride. The preparation of Weinreb-type amides from (trifluoromethyl)cyclopropanecarboxylic acid (step 2) can be accomplished under a variety of conditions well described in the art. A two-step process for the preparation of nitro-1-[1-(trifluoromethyl)cyclopropyl]ethanol: reduction of the Weinreb-type amide (step 3a) with standard reducing agents (using, for example, lithium aluminum hydride in a suitable organic solvent such as THF or Et2O), followed by isolation of the aldehyde and addition of the generated nitromethane anion to the corresponding aldehyde in the presence of a strong base such as NaH or KotBu (step 3b) can afford the desired racemic 2-nitro-1-[1-(trifluoromethyl)cyclopropyl]ethanol. Subsequent reduction of the nitro group (step 4) to the corresponding amine can be accomplished under a variety of conditions well described in the art, and the resulting amine can be converted to a suitable salt form, such as HCl, for ease of use. Those skilled in the art will recognize that racemic mixtures of amines can be resolved into their two chiral enantiomers using standard techniques well known in the art, such as chiral chromatography or preparation using a chiral salt auxiliary.

[0062] Solution 3

[0063]

[0064] Scheme 3 describes the preparation of the necessary (2S)-1-amino-3,3-dimethyl-butan-2-ol or its corresponding salt. Based on the literature data reported in JACS Vol. 9.124, No. 7, 2002, 1307, a chiral transition metal catalyst (e.g., activated to Co 3+ Co 2+ ) is subjected to kinetic resolution of 2-tert-butyl oxirane (step 5). The resulting stereochemistry can be assigned based on the stereochemistry of the chiral catalyst. Thus, (2S)-2-tert-butyl oxirane can be kinetically resolved using S,S-(salen)Co 3+ OAc preparation (see JACS Vol. 9. 124, No. 7, 2002, 1307). In addition, the S-stereochemistry can be verified by comparison with the data reported in Tetrahedron: Asymmetry 13 (2002) 1209-1217. Stereocontrolled epoxide ring opening can be achieved under conditions well known to the skilled person using nitrogen nucleophiles, such as NH3 in MeOH (step 6). The resulting amino alcohol can be converted into a suitable salt form under conditions well known in the art.

[0065] Preparation 1

[0066] N-Methoxy-N-methyl-1-(trifluoromethyl)cyclopropanecarboxamide

[0067]

[0068] A mixture of commercially available 1-(trifluoromethyl)cyclopropanecarboxylic acid [CAS#277756-46-4] (4.8 g, 31.4 mmol) and N, O-dimethylhydroxylamine hydrochloride (4.65 g, 46.7 mmol) in EtOAc (50 mL) was cooled to 0 ° C and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphine-2,4,6-trioxide solution (50% by mass in DMF, 28 mL, 47.5 mmol) was added dropwise through a dropping funnel. The reaction mixture was stirred at room temperature for 20 hours. The reaction mixture was cooled to 0 ° C and quenched by pouring into a saturated NH4Cl aqueous solution. The resulting phases were separated and the aqueous layer was extracted with EtOAc. The organic extracts were combined, dried over MgSO4, filtered, and evaporated to dryness to obtain the title compound. (4.4 g, 67% yield). 1 H NMR (400MHz, CDCl3) δ3.74 (s, 3H), 3.29 (s, 3H), 1.33-1.25 (m, 4H). ES / MS: m / z198 [M+H].

[0069] Preparation 2

[0070] (±)-2-Nitro-1-[1-(trifluoromethyl)cyclopropyl]ethanol

[0071]

[0072] A 1M solution of lithium aluminum hydride in THF (20 mL, 20 mmol) was added dropwise to a 0°C solution of N-methoxy-N-methyl-1-(trifluoromethyl)cyclopropanecarboxamide (4.3 g, 20.9 mmol) in Et2O (50 mL), and the resulting mixture was stirred for 1 hour. The reaction was quenched by dropwise addition of water (0.81 mL), followed by a 2M aqueous NaOH solution (0.81 mL) and additional water (2.43 mL). MgSO4 (5 g) was added and the resulting mixture was filtered through a pad of celite at minimum vacuum to obtain what is believed to be a solution of 1-(trifluoromethyl)cyclopropanecarboxaldehyde in Et2O. This solution was added dropwise to a rapidly stirred solution of nitromethane (60 mL, 1.1 mol) and KOtBu (360 mg, 3.17 mmol) at 0°C. After approximately 1 hour, the reaction was warmed to room temperature and stirred overnight. The solvent was decanted from the brown gum in the flask and concentrated under reduced pressure. The resulting residue was purified on silica gel eluting with 0-10% MeOH / DCM to give the title compound as a colorless oil (2.9 g, 67% yield). 1 H NMR(400MHz, CDCl3)δ 0.97-0.91(m,1H),1.13-0.99(m,3H),2.66(d,J=5.1Hz,1H),4.41-4.36(ddd,J=9.8 ,5.1,2.4Hz,1H),4.59-4.53(dd,J=9.8,13.8Hz,1H),4.68(dd,J=2.4,13.8Hz,1H).

[0073] Preparation 3

[0074] (±)-2-Amino-1-[1-(trifluoromethyl)cyclopropyl]ethanol hydrochloride

[0075]

[0076] A 1M solution of lithium aluminum hydride in THF (37 mL, 37 mmol) was added dropwise to a 0°C solution of (±)-2-nitro-1-[1-(trifluoromethyl)cyclopropyl]ethanol (2.9 g, 14.8 mmol) in Et2O (30 mL), and the resulting reaction mixture was stirred to room temperature overnight. The mixture was cooled to 0°C, and additional 1M solution of lithium aluminum hydride in THF (15 mL, 15 mmol) was added dropwise. The reaction mixture was warmed to room temperature and stirred for 4 hours. The reaction mixture was quenched at 0°C by dropwise addition of water (1.96 mL), then 2M aqueous NaOH solution (1.96 mL), then water (5.88 mL). MgSO4 (5 g) was added, the resulting mixture was stirred for 10 min, and filtered through a bed of celite. The filtrate was treated with 4M HCl in dioxane (20 mL, 80 mmol) and concentrated under reduced pressure. The resulting residue was suspended in Et2O (50 mL) with rapid stirring. The resulting white solid was isolated by filtration to give the title compound (1.52 g, 47% yield). ES / MS: m / z=170 [M+H].

[0077] Alternative method for preparation 3

[0078] To a PARR flask was added a solution of platinum (IV) oxide (242 mg, 1.07 mmol), (+)-2-nitro-1-[1-(trifluoromethyl)cyclopropyl]ethanol (2.42 g, 11.5 mmol) in EtOH (24 mL) and acetic acid (4.96 mL). The flask was placed under a hydrogen atmosphere at 55 psi and shaken at room temperature for 5 hours. The reaction mixture was filtered through a celite bed and concentrated under reduced pressure. The residue was slurried in 1,4-dioxane (17.2 mL) and 4N HCl in 1,4-dioxane (10 mL, 40 mmol) was added dropwise and stirred for 2 hours. The mixture was filtered and the filter cake was washed with 1,4-dioxane and dried under vacuum for 15 minutes. The resulting solid was dried in a vacuum oven at 45°C for 3 hours to give the title compound (2.21 g, 80% yield).

[0079] Preparation 4 (which gave Example 2)

[0080] (±)-1-[2-Hydroxy-2-[1-(trifluoromethyl)cyclopropyl]ethyl]-3-[[2-(2,2,2-trifluoroethoxy)-4-pyridinyl]methyl]urea

[0081]

[0082] A suspension of [2-(2,2,2-trifluoroethoxy)-4-pyridinyl]methylamine hydrochloride [CAS#2044704-69-8] (200 mg, 0.8 mmol) in DCM (4 mL) was cooled to 0 ° C, and DIPEA (580 μL, 3.3 mmol) and 1,1'-carbonyldiimidazole (149 mg, 0.9 mmol) were added. The resulting reaction mixture was vigorously stirred at 0 ° C for 15 minutes and (±)-2-amino-1-[1-(trifluoromethyl)cyclopropyl]ethanol hydrochloride (232 mg, 1.01 mmol) was added. The resulting mixture was stirred at room temperature overnight. It was quenched by adding water, diluted with DCM, and passed through a hydrophobic frit. The organic layer was evaporated and the resulting residue was purified on silica gel, eluted with 0-15% MeOH / DCM to obtain the title compound (198 mg, 57% yield). ES / MS: m / z=402[M+H].

[0083] Alternative method for preparation of 4

[0084] [2-(2,2,2-trifluoroethoxy)-4-pyridinyl]methylamine dihydrochloride (500 mg, 1.79 mmol) and DIPEA (1.26 mL, 7.16 mmol) in DCM (10 mL) were stirred to obtain a clear solution. 1,1'-Carbonyldiimidazole (311 mg, 1.88 mmol) was added and stirred for 30 minutes. (±)-2-Amino-1-[1-(trifluoromethyl)cyclopropyl]ethanol hydrochloride (465 mg, 2.15 mmol) was added and the reaction mixture was stirred at room temperature for 48 h. Water was added, the organic phase was separated and dried over sodium sulfate. The organics were filtered and evaporated, and the resulting residue was purified on silica gel, eluting with 0-15% MeOH / DCM to obtain the title compound (680 mg, 90% yield).

[0085] The chiral portion of the molecule can be synthesized using kinetic resolution and ring-opening chemistry.

[0086] Preparation 5

[0087] (2S)-1-Amino-3,3-dimethyl-butan-2-ol hydrochloride

[0088]

[0089] (2S)-2-tert-butyl oxirane was obtained commercially (CAS# 40102-55-4) or by kinetic resolution as reported in J.AM.CHEM.SOC. Vol. 9, No. 7, 2002, 1307, wherein it was reported that the R-configured epoxide was obtained from an RR catalyst and thus the S-epoxide was obtained from an SS catalyst:

[0090]

[0091] In a sealed container, a mixture of (2S)-2-tert-butyl oxirane (107 g, 1.01 moles) and NH4OH (1.3 L, 10.7 moles) in EtOH (427 mL) was heated at 100 ° C for 4 hours. Cool and concentrate under reduced pressure. The resulting residue was dissolved in DCM (100 mL) at 0 ° C, and a 4M solution of HCl in dioxane (267 mL, 1.1 moles) was slowly added within 10 minutes, while a white precipitate formed. The resulting solid was filtered, washed with cold DCM, and dried by vacuum suction to obtain the title compound (103 g; 62.9% yield). 1 H NMR (300.1MHz, MeOD): d 0.94 (s, 9H), 2.76 (dd, J=11.1, 12.6Hz, 1H), 3.09 (dd, J=2.5, 12.6Hz, 1H), 3.41 (dd, J=2.5, 11.1Hz, 1H).[α] D 20 = +32.38° (c = 0.8 g / 100 mL, EtOH). Literature data reported in Tetrahedron: Asymmetry 13 (2002) 1209–1217 [α] D 20 =+25.9°(c=0.47g / 100mL, EtOH).

[0092] Alternative method for preparation of 5

[0093] Prepare two ISCO 2-1000ml syringe pumps labeled A and B:

[0094] Pump A was filled with a solution of 7M NH3 in MeOH. Pump B was filled with a solution of (2S)-2-tert-butyloxirane (25 g, 232 mmol) dissolved in a solution of 7M NH3 in MeOH (995 mL). The pump was connected to a 500 mL stainless steel tube reactor (OD = 1 / 8") in the oven and then to the outlet, a 7 mL stainless steel tube reactor (OD = 1 / 16") outside the oven as a heat exchanger, and after the heat exchanger was connected a 1200 psi Back pressure regulator.

[0095] Using pump A, fill the tubular reactor with 7M NH3 in MeOH at 5 mL / min. Set the oven temperature to 200°C. Once the tubular reactor is filled, switch to pump B at 10 mL / min for 100 min, then switch to pump A to deliver 7M NH3 in MeOH at the same flow rate for an additional 1 h.

[0096] The collected solution was concentrated under reduced pressure at room temperature to give crude (2S)-1-amino-3,3-dimethyl-butan-2-ol (24.4 g). The crude material was dissolved in tert-butyl methyl ether (150 mL) and a solution of 5.5 M HCl in IPA (46.4 mL, 255 mmol) was added dropwise over 5 minutes with vigorous stirring. The resulting white solid was filtered, washed with MTBE (4x25 mL), and dried to give the title compound (23 g, 72% yield).

[0097] Preparation 6

[0098] [2-(2,2,2-Trifluoroethoxy)-4-pyridyl]methylamine dihydrochloride

[0099]

[0100] 2-(2,2,2-trifluoroethoxy)pyridine-4-carbonitrile [CAS#618446-30-3] (82.5 g, 367 mmol) was stirred in EtOH (500 mL), the solution was decanted from the undissolved solid, and the solid was washed with EtOH (3x50 mL) each time the decanted solution was removed. The EtOH solution was combined, diluted with additional EtOH (150 mL), and a concentrated aqueous HCl solution (125 mL) was added. A slurry of 10% palladium on carbon (3.7 g) in about 10 mL of EtOH was added. The resulting reaction mixture was shaken overnight at room temperature under an atmosphere of H2 at 60 psi. The mixture was filtered through a diatomaceous earth pad, washed with EtOH, and the filtrate was concentrated under reduced pressure to leave a white solid. The resulting solid was slurried in MTBE for 30 min at 45 ° C, the mixture was cooled to room temperature, and filtered to obtain a solid. The solid was dissolved in water (400 mL) and extracted twice with MTBE (400 then 200 mL). The aqueous phase was concentrated under reduced pressure to give a cream solid, which was slurried in THF (100 mL) and filtered. The filter cake was washed with THF (2×30 ml) and vacuum dried to give the title compound (46.16 g, 44% yield). The filtrate was further concentrated under reduced pressure and dried in a vacuum oven overnight. The resulting solid was slurried in THF (50 mL) for 30 min and filtered to give another batch of the title compound (34 g, 32.5% yield). ES / MS: m / z 307 [M+H]. Chloride analysis (IC) showed a molar ratio of 2:1 chloride: parent.

[0101] Example 1

[0102] 1-[(2S)-2-Hydroxy-3,3-dimethyl-butyl]-3-[[2-(2,2,2-trifluoroethoxy)-4-pyridinyl]methyl]urea

[0103]

[0104] [2-(2,2,2-trifluoroethoxy)-4-pyridinyl]methylamine hydrochloride (8.06 g, 33.2 mmol) was stirred in DCM (50 mL) and DIPEA (29 mL, 166 mmol) was added. The resulting mixture was stirred for 5 min and 1,1'-carbonyldiimidazole (5.7 g, 33.2 mmol) was added. The mixture was stirred for 10 min, and (2S)-1-amino-3,3-dimethyl-butan-2-ol hydrochloride (5 g, 32.5 mmol) was added, and the resulting reaction mixture was stirred for a weekend. The reaction mixture was washed with water, the organic phase was separated, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography, eluted with 0 to 10% MeOH in DCM, and the title compound (8.16 g, 72% yield) was obtained after solvent evaporation.

[0105] The material was combined with another batch of the title compound (4.37 g) prepared as described above and recrystallized from iPrOAc (45 ml) to give 11.29 g of the title compound. ES / MS: m / z 350 [M+H]. [α] D 20 =+29.845° (c=0.2g / 100mL, MeOH).

[0106] Example 2

[0107] Chiral separation of (+)-1-[2-hydroxy-2-[1-(trifluoromethyl)cyclopropyl]ethyl]-3-[[2-(2,2,2-trifluoroethoxy)-4-pyridinyl]methyl]urea and (-)1-[2-hydroxy-2-[1-(trifluoromethyl)cyclopropyl]ethyl]-3-[[2-(2,2,2-trifluoroethoxy)-4-pyridinyl]methyl]urea

[0108]

[0109] use Chiral purification of (±)-1-[2-hydroxy-2-[1-(trifluoromethyl)cyclopropyl]ethyl]-3-[[2-(2,2,2-trifluoroethoxy)-4-pyridinyl]methyl]urea (680 mg) was performed by SFC on an AD-H (250x30 mm, 5μ) column at 35°C, 100 bar, eluting with 92:8 CO2 / ethanol and 0.2% N,N-dimethylethylamine @152 mL / min and monitoring at 220 nm. The fractions were evaporated and dried in a vacuum oven at 45°C to give:

[0110] Enantiomer 1 (first eluting peak, 285.4 mg): (-)-1-[2-hydroxy-2-[1-(trifluoromethyl)cyclopropyl]ethyl]-3-[[2-(2,2,2-trifluoroethoxy)-4-pyridinyl]methyl]urea; [α] D 20 =-21.0°(c=0.20g / 100mL, MeOH);

[0111] Enantiomer 2 (second eluting peak, 289.5 mg). Enantiomer 2 was subjected to a second SFC purification using the above method; the fractions were evaporated and dried in a vacuum oven at 45 °C to give (+)-1-[2-hydroxy-2-[1-(trifluoromethyl)cyclopropyl]ethyl]-3-[[2-(2,2,2-trifluoroethoxy)-4-pyridyl]methyl]urea (236 mg); [α] D 20 =+15.0°(c=0.20g / 100mL, MeOH).

[0112] Example 3

[0113]

[0114] 1-[[2-(2,2,2-trifluoroethoxy)-4-pyridinyl]methyl]-3-[2-[1-(trifluoromethyl)cyclopropyl]ethyl]urea

[0115] In a round bottom flask, 2-[1-(trifluoromethyl)cyclopropyl]ethylamine hydrochloride (500 mg, 2.6 mmol; see WO2013 / 134252, but also commercially available [CAS#: 1454690-80-2]) was stirred in DCM (5 mL) and DIPEA (1.4 mL, 8 mmol) was added. When a clear solution was obtained, 1,1'-carbonyldiimidazole (428 mg, 2.6 mmol) was added and the resulting mixture was stirred at room temperature for 30 min. [2-(2,2,2-trifluoroethoxy)-4-pyridinyl]methylamine dihydrochloride (810 mg, 2.9 mmol) was added in one portion and stirred at room temperature for 30 min. The reaction mixture was warmed to 40 °C for 3 hours and stirred at room temperature for 16 h. The reaction mixture was transferred to a microwave vial and heated at 100 °C for 30 min. The resulting mixture was purified by silica gel chromatography eluting with 0-10% MeOH in DCM to give the title compound (892 mg, 88% yield). ES / MS: m / z 386 [M+H]

[0116] Alternative method to Example 3

[0117] Commercially available 2-[1-(trifluoromethyl)cyclopropyl]ethylamine hydrochloride (758 mg, 0.4 mmol, CAS#561297-93-6), DCM (2 mL), DIPEA (698 μL, 0.4 mmol) and 1,1'-carbonyldiimidazole (649 mg, 0.4 mmol) were added to a microwave reaction vessel. Shake at room temperature for 5 h. A 0.5 M prepared solution of commercially available [2-(2,2,2-trifluoroethoxy)-4-pyridyl]methylamine (CAS#1454690-80-2; alternatively, see WO 2013 / 134252) in DCM (0.8 mL, 0.4 mmol) was added at room temperature. The resulting mixture was heated in a microwave at 100 °C for 30 min. The reaction mixture was decanted into a larger round-bottom flask and diluted with water (5 mL) and DCM (5 mL). The mixture was stirred at room temperature for 15 min and passed through a hydrophobic frit to separate the organic phase. The filtrate was concentrated under reduced pressure and the resulting residue was purified by high pH reverse phase chromatography using a 75x30 mm C18 column, 5μ particle size, 110A, with C18 15x30mm protected AXIA column, eluted with 23-57% ACN in 10mM NH4HCO3 (pH~10) (containing 5% MeOH as aqueous phase) to give 1-[[2-(2,2,2-trifluoroethoxy)-4-pyridinyl]methyl]-3-[2-[1-(trifluoromethyl)cyclopropyl]ethyl]urea (90.6 mg, 59% yield). ES / MS: m / z 386 [M+H]

[0118] Determination

[0119] Bioassay data showing that compounds of the present embodiments are active in enhancing Kv7 are provided below.

[0120] Determination #1

[0121] Optopatch excitability assay of Example 1 in motor neurons from ALS patient iPSC lines

[0122] Altered excitability of cortical and lower motor neurons is an important factor in ALS pathophysiology (see, e.g., K. Kanai et al., J Neurol Neurosurg Psychiatry, 83, 734-738 (2012); P. Menon et al., Eur J Neurol., 24, 816-824 (2017)). All-optical electrophysiology ("Optopatch") was used to assess the excitability of cultured motor neurons from iPSC lines of two ALS patients with different pathogenic mutations.

[0123] Cell culture: The two iPSC lines were derived from a patient with a TARDBP pathogenic mutation and a patient with a C9orf72 pathogenic repeat expansion mutation, respectively. Motor neurons were generated from these lines by a 2D differentiation method adapted from a dual SMAD inhibitory neuronal patterning protocol containing motor neuron morphogens (see SM Chambers et al., Nat Biotechnol., 27, 275–280 (2009)). Differentiation was verified by visual inspection, karyotyping, and staining for β-III-tubulin and nuclear motor neuron marker ISL1. Neurons were cultured on a monolayer of mouse glia in mTeSR (Stem Cell Technologies) supplemented with 10 ng / mL BDNF. 48 hours before imaging, 100 nM trans-retinal was added to the culture medium.

[0124] Transduction with Optopatch vector: At 15 days in vitro, cultured motor neurons were transduced with lentiviral vectors to drive the co-expression of the actuator CheRiff-mOrange2 and the voltage indicator QuasAr3-Citrine (for details, see DR Hochbaum et al., Nat. Methods, 11, 825-833 (2014)). 48 hours before imaging, 100 nM trans-retinal was added to the culture medium.

[0125] Solution: Brainphys containing 3 mM potassium TM Recordings were performed in imaging buffer. The gap junction blocker 2-aminoethoxydiphenylborate (50 μM) was added to eliminate electrical coupling between cells, and NBQX (10 μM), gabazine (20 μM), and AP5 (25 μM) were used to block synaptic transmission.

[0126] Optopatch recordings: Five days after transduction, Optopatch imaging was performed on a custom-built ultra-widefield fluorescence microscope at room temperature. Motor neurons were excited with a red laser (200 W / cm 2 ; 635 nm) to monitor the changes in QuasAr fluorescence and excited with a blue LED (0-127 mW / cm 2 , 470nm) irradiation to depolarize the cell membrane with CheRiff. Use a customized blue light stimulation scheme, which consists of: i) 2 seconds of red illumination for monitoring spontaneous activity only, ii) 5x500 milliseconds of increasing blue light intensity and iii) 2x2 seconds of linearly increasing blue light ramps, each with a different maximum blue intensity. Optopatch data are recorded at a 1kHz frame rate using a Hamamatsu ORCA-Flash 4.0sCMOS camera. The field of view size is 4mm x0.5mm. Custom control software written in MATLAB is used to control the illumination scheme and record all movies. In order to examine the acute effects of Kv7.2 / 7.3 enhancers, neurons were incubated with test compounds for 15min before imaging.

[0127] Data analysis: Image segmentation analysis was performed using temporal principal component analysis and spatial-temporal independent component analysis to isolate individual neurons. Action potentials were found using a spike finding algorithm, and data for the effects of compounds on average firing frequency, adaptability, base strength, and action potential waveform shape were analyzed by comparison with vehicle controls (for details, see CA Werley et al., Curr. Protoc. Pharmacol., 78, 11.20.1–11.20.24. (2017)).

[0128] According to the above scheme, the main effect of Example 1 is the response of action potential firing frequency to low-intensity blue light illumination. 2 The compound reduced the action potential frequency in a concentration-dependent manner at a blue LED illumination intensity of 1:1.

[0129] Fitting a 4-parameter logistic equation to the data can be used to determine the potency of the effect of Example 1 on action potential frequency (EC 50 ). The results are shown in Table 1 for two separate differentiation efforts performed on lines from patients carrying TARDBP and C9orf72 mutations, respectively. The effects observed were qualitatively similar, but more potent than those seen with the known Kv7.2 / 7.3 enhancer flupirtine, demonstrating the potential utility of Example 1 for treating ALS by reducing the excitability of motor neurons from patients.

[0130] Table 1: Inhibition of excitability of motor neurons from ALS patients (expressed as mean values ​​(95% confidence intervals))

[0131]

[0132] Assay #2

[0133] Regulation of Kv7.2 / 7.3 conductance by the Kv7 enhancer in a mammalian expression system

[0134] The potency and efficacy of Kv7 enhancers were assessed by automated electrophysiology on the IonWorks Barracuda (Molecular Devices) platform using the population patch clamp mode of the instrument.

[0135] Cell culture: HEK293 cells stably expressing hKv7.2 (under tetracycline induction) and hKv7.3 (Catalog No. CT6147, Charles River) were used for these studies. Cells were maintained in Dulbecco's modified Eagle's medium / nutrient mixture Ham's F-12 (Sigma-Aldrich) supplemented with 5% tetracycline-selected fetal bovine serum (Sigma-Aldrich), 15mM HEPES, 500μg / mL G418, 100U / mL penicillin, 100μg / mL streptomycin, 29.2mg / mL L-glutamine, 100μg / mL zeocin, and 5μg / mL blasticidin. Expression of hKv7.2 was induced by adding 1μg / mL doxycycline 24 hours before recording.

[0136] Cells were cultured in Corning T-150 flasks to a confluency of 85%-95%. At the beginning of the experiment, cells were washed once with D-PBS without calcium and magnesium and then incubated in 3 ml of 0.25% trypsin at 37°C for 8 minutes to dissociate. Cells were resuspended in culture medium, gently triturated and centrifuged at 1,000 rpm for 4 minutes. Cells were resuspended in external solution to a final concentration of 2.5-3.5M cells / mL.

[0137] Solutions: The external solution consisted of (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.4. The internal solution consisted of (in mM): 90 K-gluconate, 40 KCl, 3.2 EGTA, 3.2 MgCl2, 5 HEPES, pH 7.25. The membrane permeable agent amphotericin B was prepared daily as a 27 mg / mL DMSO stock solution and then added to the internal solution to a final concentration of 0.1 mg / mL. Test article dilutions were prepared in 384-well plates using 10 mM DMSO stock solutions and dispensed using acoustic dispensing (Labcyte ) was diluted to a final DMSO concentration of 0.1%.

[0138] Electrophysiological recordings: The resuspended cells were placed in BARRACUDA TM (IWB) instrument, external solution is added to 384-well patch plate (patch plate), and hole test is carried out to determine the hole and offset voltage of blocking. Then cells are added to the patch plate (9 μ L per hole) by instrument. Before the penetrant amphotericin is introduced into the internal solution, two sealing tests are carried out, and approximately 8 minutes are allowed to obtain electrical access (electrical access), which is verified by the third sealing test. The command voltage scheme is composed of a 1 second voltage step from -80mV to +40mV, a clamping potential from -80mV, and is applied before adding the test article (baseline) and after adding 6 minutes.

[0139] Data analysis: Data were acquired using IWB software and leakage was subtracted. The current amplitude during the last 10 milliseconds of each voltage step was averaged and exported. Further analysis was performed using Microsoft Excel and GraphPad Prism. The current amplitude was converted to conductance (G) by the following formula: G = I / (V-Ek), where I = current, V = step potential, Ek = potassium reversal potential (-84mV). The conductance in the presence of the test article was normalized to the baseline conductance of the same well at +40mV. The conductance-voltage (GV) curve conforms to the Boltzmann equation y = Bottom + (Top-Bottom) / (1 + EXP ((Vm-V0.5 / k)).

[0140] The shifts in the midpoints (V0.5) of the conductance curves mediated by the test articles of Examples 1-3 are shown in Table 2.

[0141] Table 2: Difference in voltage from control in the maximum half conductance in the presence of different concentrations of test compounds.

[0142]

[0143]

[0144] (The SD above refers to standard deviation).

[0145] Fitting a 4-parameter logistic equation to the data can be used to determine the potency (EC50) and efficacy (maximum excursion) of each test compound. The results of Examples 1-3 are shown in Table 3.

[0146] Table 3: Potency and efficacy of Kv7.2 / 7.3 enhancers

[0147] Example 1 Example 2 Example 3 <![CDATA[EC 50 (μM)]]> 2.0 1.1 0.10 Maximum offset -44 -28 -23 Slope 1.2 1.3 1.1

[0148] Determination #3

[0149] Threshold tracing to measure the effect of 3, 10 and 30 mg / kg IP (intraperitoneal) of Example 1 on peripheral nerve excitability

[0150] Threshold tracing is a non-invasive technique that allows the measurement of the excitatory properties of peripheral axons by providing information about their membrane potential and ion channel function.

[0151] method

[0152] Sixteen male Wistar rats from Charles River, weighing between 307-446 g, were used. The animals were group housed under standard housing conditions (4 per cage, 07:00h to 19:00h light phase, constant temperature (21°C) and humidity, and free access to food and water, as well as environmental enrichment).

[0153] Rats were anesthetized with isoflurane (2-2.5%, 0.5 L / min of O2) and then placed on their backs on a heating pad to keep the tail temperature above 32°C. The locations of the ring electrodes were marked, and the marked parts of the tail were shaved with a blade to remove hair and superficial skin. These sites were washed with water and dried to allow good conduction between the skin and the adhesive electrodes.

[0154] Wrap an adhesive ring stimulating electrode (+ve anode) around the rat's foot. Wrap a second adhesive ring stimulating electrode (-ve cathode) 1.5 cm from the base of the tail. Place a needle recording electrode near the center of the top of the rat's tail, 6 cm away from the stimulating cathode at the base of the tail. Place a needle reference electrode near the center of the top of the rat's tail, 2 cm away from the recording electrode. Wrap an adhesive ground electrode around the tail 2 cm proximal to the recording electrode.

[0155] Study plan

[0156] Start the Multitrack program (described below) and Spike.

[0157] Record a stable baseline for 15 minutes

[0158] 15 minutes later, the drug was administered with Example 1 or HEC

[0159] Blood samples were collected for PK at the following time points:

[0160] ο10 minutes after administration

[0161] ο20 minutes after administration

[0162] ο 30 minutes after administration

[0163] ο40 minutes after administration

[0164] 45 minutes after IP (intraperitoneal) administration of Example 1, XE-991 (XE-991 is a commercially available compound that blocks KCNQ enhancer effects in an in vivo formalin assay—see Y. Zheng et al., “Activation of peripheral KCNQ channels relieves gout pain,” Pain, 156 (2015) 1025–1035; and R. Zaczek, et al., “Two New Potent Neurotransmitter Release Enhancers, 10,10-Bis(4-Pyridinylmethyl)-9(10H)-Anthracenone and 10,10-Bis(2-Fluoro-4-Pyridinylmethyl)-9(10H)-Anthracenone: Comparison to Linopirdine” The Journal of Pharmacology and Experimental Therapeutics, 285: 724-730, 1998) was administered.

[0165] o (XE-991 was administered at 30 mg / kg only after Example 1)

[0166] Blood samples were collected for PK at the following time points:

[0167] 10 minutes after administration (55 minutes after administration in Example 1)

[0168] 20 minutes after administration (65 minutes after administration in Example 1)

[0169] 30 minutes after administration (75 minutes after administration in Example 1)

[0170] 40 minutes after administration (85 minutes after administration in Example 1)

[0171] In a threshold tracing assay measuring the excitatory properties of peripheral axon studies in rats, compounds were administered IP at 3, 10, or 30 mg / kg using a 1% hydroxyethylcellulose: 0.25% polysorbate 80: 0.05% antifoam: purified water (HEC) formulation. Dried blood spots (DBS) were collected approximately 10, 20, 30, and finally 40 minutes after dosing. DBS samples were dried at room temperature for approximately 2 hours. Brain samples were obtained at the final time point and frozen until analysis. DBS samples were shipped and stored at room temperature.

[0172] Prepare a 1-mg / mL stock solution of Example 1 and serially dilute it into pooled rat blood to prepare standards ranging from 1 to 10000 ng / mL. Add blood to a blank DBS card to prepare standards. Add a 3 mm long DBS standard or sample to a 96-well plate, then add 180 μL of internal standard (IS) in 1:1 ACN:MeOH. Shake for 45 min, dilute the extract 2-fold with water, and analyze drug concentration by LC / MSMS.

[0173] Brain samples were homogenized using 1.14 mL of MeOH:H2O (2:8). Standards were prepared by spiking the stock solution into a series of blank brain homogenates ranging from 5 to 50,000 ng / mL. 25 μL of standard or sample was transferred into a 96-well plate and 180 μL of internal standard (IS) in 1:1 ACN:MeOH was added and mixed. Samples were centrifuged at 4000 RPM for 10 minutes at 4°C. The supernatant was diluted 15-fold with water and analyzed by LC / MSMS.

[0174] Sample and standard use Sciex API 4000 triple quadrupole mass spectrometer (Sciex, Division of MDS Inc., Toronto, Canada) and Shimadzu HPLC system (LC-IOAD, Shimadzu Corporation) and Gilson215 automatic sampler coupling to analyze.Sample (0.01mL) is injected into the HPLC column of 5-μm Betasil C-18, 20x2.1mmJavelin (Thermo Electron Corp.Cat#70105-022106), and uses gradient elution.Chromatographic conditions include water / 1MNH4HCO3 of mobile phase A (2000:10, v / v) and MeOH / 1M N NH4HCO3 of mobile phase B (2000:10, v / v), it is with 2.5-min gradient, the flow rate of 1.5mL / min runs.Positive ion mode with turbo spray and 740 ℃ ion source temperature are used for mass spectrometry detection. Multiple reaction monitoring (MRM) was used for quantification in the following transitions: Multiple reaction monitoring (MRM) was used for quantification in the following transitions: Example 1 (m / z 350.2 to m / z 233.0) and simulated internal standard (m / z 263.1 to m / z 148.1). The linear regression plot of the compound to internal standard peak area ratio versus drug concentration was given by 1 / x2 quadratically. The linear regression plot of the compound to internal standard peak area ratio versus drug concentration was given by 1 / x2 quadratically. 2 The quadratic result is obtained.

[0175] The simulated internal standard used was 2-(dimethylamino)-N-pentyl-3-phenyl-propionamide 2,2,2-trifluoroacetic acid (1:1) and has the following structure:

[0176]

[0177] The simulated internal standard was purchased from Syncom, a Dutch company with an address of Kadijk 3, 9747AT Groningen, The Netherlands.

[0178] Binding of drugs to rat plasma proteins and brain homogenates was determined using an in vitro dialysis method after spiking the drugs into these matrices and incubating at 37°C for 4.5 h with orbital shaking. The assays were performed using an HT dialysis microbalance apparatus and dialysis membrane strips (MWCO 12–14k). Samples were collected at time 0 after the protein matrix and from both the protein and buffer sides of the membrane after 4.5 h of incubation. The matrix was quantified by LC-MSMS at time points 0 and 45 min. The unbound fraction was calculated by dividing the concentration on the buffer side by the concentration on the protein side. The percent recovery could also be calculated by dividing the sum of the buffer and protein compartments by the time 0 concentration after 4.5 h. The unbound compound concentration was calculated using total concentration*unbound fraction.

[0179] Results: The effect of Example 1 on the absolute threshold is shown in Table 4

[0180] Table 4

[0181]

[0182] CMAP = Compound muscle action potential

[0183] (For more information on this assay, see R. Sittl et al., “The Kv7 potassium channel activator flupirtine affects clinical excitability parameters of myelinated axons in isolated rat sural nerve,” Journal of the Peripheral Nervous System 15:63–72 (2010); M. Kovalchuk et al., “Acute Effects of Riluzole and Retigabine on Axonal Excitability in Patients With Amyotrophic Lateral Sclerosis: A Randomized, Double-Blind, Placebo-Controlled, Crossover Trial,” received March 7, 2018; accepted April 13, 2018; advance online publication 00Month 2018. doi:10.1002 / cpt.1096, CLINICALPHARMACOLOGY&THERAPEUTICS, Volume 00, Number 00, MONTH 2018; and J. Fleckenstein et al. "Activation of axonal Kv7channels in human peripheral nerve by flupirtine but not placebo-therapeutic potential for peripheral neuropathies: results of arandomised controlled trial," Journal of Translational Medicine 2013,11:34.).

[0184] There were significant differences between time and time*treatment interactions (two-way RM ANOVA; time effect F(26,312)=13.18, p=<0.0001; time*treatment interaction F(78,312)=2.888, p<0.0001. Bonferroni multiple comparisons test showed that Example 1 at 30 mg / kg significantly increased absolute threshold (decreased excitability) compared to vehicle. XE-991 was able to reverse this increase (increased excitability).

Claims

1. A compound of the following formula or a pharmaceutically acceptable salt thereof: Where R1 is And wherein R2 is H or OH.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is:

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein R2 is OH.

4. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein the compound is of the following formula: The compound described therein is a single enantiomer having an optical rotation of (+) when measured at 20°C and 589 nm at a concentration of 0.20 g / 100 mL in methanol.

5. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein the compound is of the following formula: The compound described therein is a single enantiomer having an optical rotation of (-) when measured at 20°C and 589 nm at a concentration of 0.20 g / 100 mL in methanol.

6. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein R2 is H.

7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is 8. The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein R2 is H.

9. The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein R2 is OH.

10. The compound according to claim 9 or a pharmaceutically acceptable salt thereof, wherein the compound is of the following formula:

11. The compound according to claim 9 or a pharmaceutically acceptable salt thereof, wherein the compound is of the following formula:

12. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is of the following formula:

13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

14. Use of a compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a disease caused by changes in motor neuron excitability, wherein the disease is selected from ALS, primary lateral sclerosis, pseudobulbar palsy, progressive bulbar palsy, epilepsy, progressive muscular atrophy, and peripheral nerve hyperexcitability disorder.

15. The use according to claim 14, wherein the disease caused by changes in motor neuron excitability is ALS.

16. Use of the pharmaceutical composition of claim 13 in the preparation of a medicament for treating a disease caused by changes in motor neuron excitability, wherein the disease is selected from ALS, primary lateral sclerosis, pseudobulbar palsy, progressive bulbar palsy, epilepsy, progressive muscular atrophy, and peripheral nerve hyperexcitability disorder.

17. The use according to claim 16, wherein the disease caused by changes in motor neuron excitability is ALS.

18. A process for preparing a pharmaceutical composition comprising mixing a compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable carriers or excipients.

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