RU2790174C1 - 1-[[2-(2,2,2-trifluorethoxy)pyride-4-yl]methyl]urea derivatives as kcnq potentiators - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to low-molecular potentiators of potassium channels (such as Kv7 potentiators also called KCNQ potentiators) of the formula

where R1 is

or

, R2 is H or OH, compositions including such compounds, and methods for the use of such compounds for the treatment of amyotrophic lateral sclerosis and other neurological diseases caused by changes in excitability of motor neurons, for example, such as primary lateral sclerosis, pseudobulbar paralysis, progressive bulbar paralysis, progressive muscular atrophy, and epilepsy.

EFFECT: obtainment of compounds for the treatment of neurological diseases.

25 cl, 4 tbl, 3 ex

Description

The present invention relates to the field of medicine. In particular, the present invention relates to compounds, methods and pharmaceutical compositions for the treatment of amyotrophic lateral sclerosis (ALS) 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.

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

Patients with ALS typically have peripheral and central motor neuron hyperexcitability, leading to fasciculations, muscle cramps, and spasticity. It is believed that the increased excitability of neurons leads to calcium overload and cell death. Indeed, motor neuron excitability was negatively correlated with survival in ALS patients (see K. Kanai et. al., J. Neurol. Neurosurg. Psychiatry , 83, 734-738 (2012)).

Research has investigated the mechanism and reason why motor neurons in ALS patients change excitability (compared to non-ALS patients). (See K. Kanai et. al., Brain , 129, 953-962 (2006)). The results of the study showed that the decrease in the activity of potassium channels in neurons is the main factor in the increased excitability associated with the disease (see ibid.). In addition, motor neurons derived from pluripotent stem cells from ALS patients also have increased excitability. (See BJ Wainger et. al., Cell Rep. 7, 1-11 (2014)). These stem cell-derived neurons showed reduced potassium currents, and in fact, when the agents retigabine and flupirtine (known as Kv7 potassium channel potentiators) are added to these stem cell-derived neurons, motor neuron hyperexcitability is normalized and cell survival in vitro increased (see ibid.). In another study, Kv7 potentiating retigabine reduced symptoms of excitotoxicity and increased survival in an in vitro ALS model using rat sublingual motor neurons (see F. Ghezzi et. al., J. Physiol. , 596, 2611-2629 (2018)).

Currently, the only drug approved for the treatment of ALS is riluzole, which has been shown to increase survival by 2-3 months. It is clear that there is a need for more effective, better and longer lasting drugs.

The well-known retigabine, which enhances the action of Kv7, acts on potassium channels Kv7.2-7.5 (KCNQ2-KCNQ5). It has been approved as an add-on therapy in patients with drug-resistant epilepsy. It was approved in Europe and the US in 2011 but was voluntarily withdrawn in 2017. The discontinuation of clinical use of retigabine was thought to be associated with a number of tolerability issues, leading to very limited use of the drug. Tolerability problems include the usual and presumably mechanical occurrence of drowsiness and dizziness, as well as less frequent cases of urinary retention and changes in retinal and skin pigmentation. Retinal changes and the possibility of vision loss resulted in a boxed warning on the retigabine label and is not thought to be related to the mechanism. Urinary retention is most likely the result of potentiation of the Kv7.3/7.5 channels of the bladder. However, given its side effects, there is a need to develop new Kv7 potentiators.

A recent study comparing the effects of riluzole (e.g., an approved treatment for ALS) and retigabine on motor neuron excitability in patients with ALS suggests that Kv7 channel potentiators may be more effective than riluzole (see M. Kovalchuk et. al., Clinical Pharmacology & Therapeutics , 104, 1136-1145 (2018)). Thus, improved potentiators that are better tolerated than retigabine can be used for the clinical treatment of ALS and other irritability disorders. (See B. Kalappa, et al., The Journal of Neuroscience , 35(23):8829-8842) (2015). To date, no Kv7-acting agents have been approved for the treatment of ALS, and thus there remains a need for Kv7-acting agents, such as alternative Kv7 potentiators, that provide a therapeutic effect. In addition, it may be useful for such potentiators to be more selective for Kv7.2/7.3 compared to other Kv7 channels. There is also a need for a new Kv7 agent that avoids unwanted side effects and can provide a combination of improved pharmacological properties, including safety, efficacy, efficacy and tolerability, in particular for the treatment of peripheral and central motor neuron excitability.

The present embodiments provide compounds that are potassium channel potentiators (such as Kv7 potentiators - also referred to as KCNQ potentiators) that can be used in the treatment of ALS and other neurological diseases caused by changes in motor neuron excitability.

In particular, compounds of the following formula (referred to as "FORMULA I") can be used as Kv7 potentiators:

where R1 is

(FORMULA I)

where 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 such pharmaceutically acceptable salts can be prepared and used as Kv7 potentiators.

In some embodiments, compounds of FORMULA I, or pharmaceutically acceptable salts thereof, are prepared such that R1 is

and R2 is H.

In other embodiments, the compounds of FORMULA I (or pharmaceutically acceptable salts thereof) are prepared such that R1 is

and R2 is OH. Moreover, in such embodiments of the invention, when R2 is OH, the carbon to which the OH group is attached is the stereocenter. Accordingly, in some embodiments, the compounds (or pharmaceutically acceptable salts thereof) of FORMULA I may be a racemic mixture of two enantiomers. In other embodiments, a particular enantiomer may be used, including any of the following enantiomers:

Those skilled in the art will understand how to construct embodiments that use only one of the aforementioned enantiomers. Other embodiments of the invention can be designed with mixtures of different enantiomers having different percentages of each component.

In other embodiments, the compounds of FORMULA I (or pharmaceutically acceptable salts thereof) are prepared such that

R1 represents

and R2 is H.

In still further embodiments, the compounds of FORMULA I (or pharmaceutically acceptable salts thereof) are prepared such that

R1 represents

and R2 is OH. Moreover, in such embodiments of the invention, when R2 is OH, the carbon to which the OH group is attached is the stereocenter. Accordingly, in some embodiments, the compounds (or pharmaceutically acceptable salts) of FORMULA I may be a racemic mixture of two enantiomers. In other embodiments, a particular enantiomer may be used, including any of the following enantiomers:

Those skilled in the art will understand how to construct embodiments that use only one of the aforementioned enantionators. Other embodiments can be designed with mixtures of different enantiomers having different percentages of each component.

The present embodiments of the invention further relate to pharmaceutical compositions containing a compound or pharmaceutically acceptable salt of FORMULA I and one or more pharmaceutically acceptable carriers, diluents or excipients. Such pharmaceutical compositions may provide a method for treating a disease. In particular, the present embodiments provide a method of treating a disease caused by changes in motor neuron excitability, comprising administering to a patient in need thereof an effective amount of a FORMULA I compound (or a pharmaceutically acceptable salt thereof). In some particularly preferred embodiments of the invention, the disease caused by changes in motor neuron excitability is ALS.

In other embodiments, a method of treating a disease caused by changes in motor neuron excitability is provided, comprising administering to a patient in need thereof an effective amount of a compound of formula I (or a pharmaceutically acceptable salt thereof). In some particularly preferred embodiments of the invention, the disease caused by changes in motor neuron excitability is ALS.

The present embodiments also relate to compounds of FORMULA I (or pharmaceutically acceptable salts thereof) for use in therapy. More specifically, the present embodiments provide a compound of FORMULA I (or a pharmaceutically acceptable salt thereof) for use in the treatment of a disease caused by changes in motor neuron excitability. In some embodiments, the disease may be ALS.

In addition, the present embodiments of the invention provide the use of a compound of FORMULA I (or pharmaceutically acceptable salts thereof) in the manufacture of a medicament for the treatment of a disease caused by changes in motor neuron excitability. In some such embodiments, the disease is ALS.

«Peripheral nerve hyperexcitability syndromes» Rev Neurosci. 2015;26(2):239-51. Соответственно, указанные в настоящем документе соединения и фармацевтически приемлемые соли можно использовать для лечения одного или нескольких из этих заболеваний.As used herein, the term "disease associated with changes in motor neuron excitability" or "disease caused by changes in motor neuron excitability" includes a disease selected from the group consisting of ALS, primary lateral sclerosis, pseudobulbar palsy, progressive bulbar palsy, epilepsy, and progressive muscle atrophy. These terms also include all diseases listed in A. Verma, et al. , "Atypical Motor Neuron Disease and Related Motor Syndromes", Seminars in Neurology , Volume 21, Number 2, 2001. Such terms also include PNH (peripheral nerve hyperexcitability) disorders. Information on PNH disorders can be found in C.

"Peripheral nerve hyperexcitability syndromes" Rev Neurosci. 2015;26(2):239-51. Accordingly, the compounds and pharmaceutically acceptable salts provided herein may be used to treat one or more of these diseases.

As used interchangeably herein, the terms "patient", "subject" and "individual" refer to a person, more specifically, a patient in need thereof. In some embodiments, the patient is further characterized by the presence of a disease, disorder, or condition (eg, ALS or other disease) in which Kv7 potentiation may be beneficial. In another embodiment of the invention, the patient is further characterized as being at risk for developing the condition described above, or a condition that can be ameliorated by Kv7 potentiation.

An effective amount can be determined by a person skilled in the art using known methods and observing results obtained under similar circumstances. In determining an effective amount for a patient, a number of factors are taken into account, including, but not limited to: size, age, and general health; a specific disease or disorder; the extent or involvement or severity of the disease or disorder; individual patient response; the particular compound being administered; administration mode; characteristics of the bioavailability of the administered drug; the selected dosing regimen; taking concomitant medications; and other relevant circumstances. In some embodiments of the invention, an effective amount provides a clinically significant reduction in the excitability of peripheral and central motor neurons.

The compounds of the present invention are formulated as pharmaceutical compositions, administered by any route that renders the compound bioavailable. Preferably, such compositions are for oral administration. Such pharmaceutical compositions and methods for their preparation are well known in the art (see, for example, Remington: The Science and Practice of Pharmacy, LV Allen, Editor, ^22nd Edition, Pharmaceutical Press, 2012).

The compounds of FORMULA I and their pharmaceutically acceptable salts are particularly useful in the methods of treatment of the invention, with certain configurations being preferred. Such configurations are described in the following list of compounds of the present invention. It is understood that these preferences apply to both the treatments and the compounds of the invention.

Compounds of the present embodiments of the invention (in which R2 is OH) include:

In these compounds, in which R2 is OH, the "planar bond" compounds mentioned above are racemic and/or contain both enantiomers (either in a 50/50 mixture or some other ratio). Compounds with a wedge-shaped and dotted bond above denote a specific enantiomer. Compounds with a "wavy" bond indicate that it is a single enantiomer, but the exact configuration of this single enantiomer is not known, and thus such enantiomers are distinguished by their optical rotation (for example, they rotate light either in (+) or in ( -) direction).

The compounds of the present embodiments also include the following compounds (in which R2 is H):

The above compounds which have an OH group as R2 may be those in which the OH is in the "top" configuration (as indicated by the wedge). Other variants may be developed in which the OH group is in the "bottom" configuration. Those skilled in the art will understand how to prepare this other enantiomer, for example, using a different starting material and/or using other reactions, etc. Such enantiomers have the following structure and are part of the currently claimed embodiments:

Although the present invention covers all individual enantiomers and diastereomers, as well as mixtures of enantiomers of these compounds, including racemates, the compounds listed above and their pharmaceutically acceptable salts are particularly preferred.

The individual enantiomers may be isolated or separated by one of ordinary skill in the art at any convenient point in the synthesis of the compounds of the invention by methods such as selective crystallization techniques, chiral chromatography (see, for example, 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, for example, TA Berger; " Supercritical Fluid Chromatography Primer ", Agilent Technologies, July 2015).

A pharmaceutically acceptable salt of a compound of the invention may be formed, for example, by reacting a suitable free base of a compound of the invention and an appropriate 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, S.M., et al ., "Pharmaceutical Salts", Journal of Pharmaceutical Sciences , 66:1-19, (1977).

The compounds of the present invention, or salts thereof, may be prepared in a variety of ways known to those of ordinary skill in the art, some of which are illustrated in the Schemes, Preparations, and Examples below. The products of each step in the schemes below can be isolated by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In the schemes below, all substituents, unless otherwise indicated, have the meanings previously indicated. The reagents and starting materials are readily available to one of ordinary skill in the art. Without limiting the scope of the invention, the following schemes, preparations and examples are presented to further illustrate aspects of the invention. In addition, one of skill in the art will appreciate that compounds of FORMULA I can be prepared using a starting material or an intermediate with the corresponding desired stereochemical configuration, which can be prepared by one of skill in the art.

Some abbreviations may be used below. These abbreviations mean the following: "ACN" refers to acetonitrile; "Ac" refers to acetyl; "AcOH" refers to acetic acid; "Ac ₂ O" refers to acetic anhydride; "AP5" refers to (2R)-amino-5-phosphonopentanoate; "BDNF" refers to brain-derived neurotrophic factor; "BOC" refers to tert-butoxycarbonyl; "CAS#" refers to a registry number in Chemical Abstracts; "CMAP" refers to the action potential of a compound muscle; "DCM" refers to methylene chloride or dichloromethane; "DIPEA" refers to N,N-diisopropylethylamine; "DMEA" refers to dimethylethylamine; "DMF" refers to N,N-dimethylformamide; "DMSO" refers to 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 mass spectrometry; "Et ₂ O" refers to diethyl ether; "EtOAc" refers to ethyl acetate; "EtOH" refers to ethanol or ethyl alcohol; "h" refers to the hour or 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/MSMS" refers to liquid chromatography with tandem mass spectrometry; "LiHMDS" refers to lithium bis(trimethylsilyl)amide; "KOtBu" refers to potassium tert-butoxide; "Me" refers to methyl; "ms" refers to milliseconds or milliseconds as a unit of time; "MTBE" refers to methyl tert-butyl ether; "min" refers to the minute or minutes; "NaOtBu" refers to sodium t-butoxide; "n-BuLi" refers to n-butyllithium; "NBQX" refers to (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline; "OAc" refers to acetate or acetoxy; "PBS" refers to phosphate buffered saline; "k.t." refers to room temperature; "SCX" refers to strong cation exchange; "SD" refers to standard deviation; "sec" refers to the second or seconds as a unit of time; "SEM" refers to standard error of the mean; "SFC" refers to supercritical fluid chromatography; "TEA" refers to triethylamine; "TFA" refers to trifluoroacetic acid; "THF" refers to tetrahydrofuran; "TMA" refers to trimethylamine; "TMEDA" refers to tetramethylethylenediamine; "Tris" refers to tris(hydroxymethyl)aminomethane or 2 -amino-2-(hydroxymethyl) propane-1,3-diol "[α] _D ²⁰ " refers to the specific optical rotation at 20ºC and 589 nm, where c denotes the concentration in g/ml (usually g/100 ml).

General schemes

Scheme 1

Scheme 1 shows the preparation of compounds of FORMULA I, where R ¹ is defined as described above. Formation of an activated ester of an appropriately substituted amine (or its corresponding salt, e.g. HCl) is well documented in the art using e.g. carbonyldiimidazole, and selective N- versus O-carbonylation (2-ethoxy-4-pyridyl) methanamine (or its corresponding salt form, eg HCl) with said activated intermediate with a suitable organic base can give the desired ureas of the present invention (step 1). The skilled artisan will appreciate that compounds of FORMULA I having stereochemistry can be prepared with a chiral substituted amine to give a single enantiomer, or by chiral resolution of a FORMULA I compound using chiral chromatography methods such as SFC, or using a chiral auxiliary technique such as chiral salt, as is well known in the art.

Scheme 2

Scheme 2 shows the preparation of rac-2-amino-1-[1-(trifluoromethyl)cyclopropyl]ethanol hydrochloride. The preparation of the Weinreb-type amide (step 2) from (trifluoromethyl)cyclopropanecarboxylic acid can be carried out under a variety of conditions well described in the art. Two-step process for the preparation of nitro-1-[1-(trifluoromethyl)cyclopropyl]ethanol by reduction of Weinreb-type amide with standard reducing agents (step 3a) using, for example, lithium aluminum hydride in a suitable organic solvent such as THF or Et ₂ O, followed by isolation aldehyde and addition to the corresponding aldehyde (step 3b) of the nitromethane anion formed in the presence of a strong base such as NaH or KOtBu can give the desired racemic 2-nitro-1-[1-(trifluoromethyl)cyclopropyl]ethanol. The subsequent reduction of the nitro group (step 4) to the corresponding amine can be carried out under a variety of conditions well described in the art, and for ease of use the resulting amine can be converted to a suitable salt form, eg HCl. The skilled artisan will appreciate that a racemic mixture of an amine can be resolved into its two chiral enantiomers using standard techniques well known in the art such as chiral chromatography or preparation using an auxiliary chiral salt.

Scheme 3

Scheme 3 depicts the preparation of the desired (2S)-1-amino-3,3-dimethylbutan-2-ol or its corresponding salt. Kinetic separation of 2-tert-butyloxirane (step 5) using a chiral transition metal catalyst such as Co ²⁺ that has been activated to Co ³⁺ can be achieved based on the literature presented in JACS 9 VOL. 124, NO. 7, 2002 , 1307. The resulting stereochemistry can be assigned depending on the stereochemistry of the chiral catalyst. Therefore, (2S)-2-tert-butyloxirane can be obtained using S,S-(salen)Co ³⁺ OAc (see JACS 9 VOL. 124, NO. 7, 2002 , 1307). In addition, verification of S-stereochemistry can be performed by comparison with the data given in Tetrahedron: Asymmetry 13 (2002) 1209-1217. Stereo-controlled opening of the epoxy ring can be achieved using a nitrogen nucleophile such as NH ₃ in MeOH under conditions well known to one skilled in the art (step 6). The resulting amino alcohol can be converted to the appropriate salt form under conditions well known in the art.

Getting 1

N-methoxy-N-methyl-1-(trifluoromethyl)cyclopropanecarboxamide

Cool 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) to a temperature of 0ºC and a solution of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorane-2,4,6-trioxide (50 wt.% In DMF, 28 ml, 47.5 mmol). The reaction mixture is stirred at room temperature for 20 hours. The reaction mixture is cooled to 0° C. and quenched by pouring into saturated aqueous NH ₄ Cl. The resulting phases are separated and the aqueous layer is extracted with EtOAc. The organic extracts are combined, dried over MgSO ₄ , filtered and evaporated to dryness to give the title compound (4.4 g, 67% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ 3.74 (s, 3H), 3.29 (s, 3H), 1.33-1.25 (m, 4H). ES/MS: m/z 198 [M+H].

Getting 2

(±)-2-nitro-1-[1-(trifluoromethyl)cyclopropyl]ethanol

A 1M solution of lithium aluminum hydride in THF ( ₂₀ ml , 20 mmol) and the resulting mixture was stirred for 1 h. The reaction was quenched by adding dropwise water (0.81 ml), then 2 M aqueous NaOH (0.81 ml) and then more water (2.43 ml). MgSO ₄ (5 g) was added and the resulting mixture was filtered through a pad of diatomaceous earth under minimal vacuum to give what was expected to be a solution of 1-(trifluoromethyl)cyclopropanecarbaldehyde in Et ₂ O. This solution was added dropwise at 0°C to a rapidly stirred solution of nitromethane ( 60 ml, 1.1 mol) and KOtBu (360 mg, 3.17 mmol). After ~1 h, the reaction mixture is warmed to room temperature and stirred overnight. The solvent is decanted from the brown resin 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 (400 MHz, CDCl ₃ ) δ 0.97-0.91 (m, 1H), 1.13-0.99 (m, 3H), 2.66 (d, J=5.1 Hz, 1H), 4.41-4.36 (ddd, J=9.8, 5.1, 2.4 Hz, 1H), 4.59-4.53 (dd, J=9.8, 13.8 Hz, 1H), 4.68 (dd, J=2.4, 13.8 Hz, 1H).

Getting 3

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

To a solution of (±)-2-nitro-1-[1-(trifluoromethyl)cyclopropyl]ethanol (2.9 g, 14.8 mmol) in Et ₂ O (30 ml) at 0ºC was added dropwise a 1 M solution of aluminum hydride lithium in THF (37 ml, 37 mmol) and stir the resulting reaction mixture at room temperature overnight. The mixture is cooled to 0° C. and another 1M solution of lithium aluminum hydride in THF (15 ml, 15 mmol) is added dropwise. The reaction mixture is warmed to room temperature and stirred for 4 hours. The reaction mixture was quenched at 0°C by adding dropwise water (1.96 ml), then 2 M aqueous NaOH (1.96 ml) and then water (5.88 ml). Add MgSO ₄ (5 g), stir the resulting mixture for 10 min and filter through a pad of diatomaceous earth. The filtrate is treated with 4 M HCl in dioxane (20 ml, 80 mmol) and concentrated under reduced pressure. The resulting residue is suspended in Et ₂ O (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].

Alternative way to get 3

Platinum (IV) oxide (242 mg, 1.07 mmol), a solution of (+)-2-nitro-1-[1-(trifluoromethyl)cyclopropyl]ethanol (2.42 g, 11.5 mmol) was placed in a Parr flask in EtOH (24 ml) and acetic acid (4.96 ml). The flask is placed in a hydrogen atmosphere at a pressure of 55 pounds per square inch and shaken for 5 hours at room temperature. The reaction mixture is filtered through a pad of diatomaceous earth and concentrated under reduced pressure. The residue is suspended in 1,4-dioxane (17.2 ml), 4 N HCl in 1,4-dioxane (10 ml, 40 mmol) is added dropwise and stirred for 2 hours. The mixture is filtered, the filter pad is washed with 1,4-dioxane and dried in vacuo 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).

Getting 4 (which gives the connection of example 2)

(±)-1-[2-hydroxy-2-[1-(trifluoromethyl)cyclopropyl]ethyl]-3-[[2-(2,2,2-trifluoroethoxy)-4-pyridyl]methyl]urea

A suspension of [2-(2,2,2-trifluoroethoxy)-4-pyridyl]methanamine hydrochloride [CAS# 2044704-69-8] (200 mg, 0.8 mmol) in DCM (4 mL) was cooled to 0°C, added DIPEA (580 µl, 3.3 mmol) and 1,1'-carbonyldiimidazole (149 mg, 0.9 mmol). 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. Quench with water, dilute with DCM and pass through a hydrophobic frit. The organic layers were evaporated and the resulting residue was purified on silica gel eluting with 0-15% MeOH/DCM to give the title compound (198 mg, 57% yield). ES/MS: m/z =402 [M+H].

Alternative way to get 4

[2-(2,2,2-trifluoroethoxy)-4-pyridyl]methanamine dihydrochloride (500 mg, 1.79 mmol) and DIPEA (1.26 ml, 7.16 mmol) in DCM (10 ml) are stirred to give clear solution. Add 1,1'-carbonyldiimidazole (311 mg, 1.88 mmol) and stir for 30 minutes. (±)-2-Amino-1-[1-(trifluoromethyl)cyclopropyl]ethanol hydrochloride (465 mg, 2.15 mmol) is added and the reaction mixture is stirred at room temperature for 48 hours. Add water, separate the organic phase and dry over sodium sulfate. The organic layers are filtered and evaporated and the resulting residue is purified on silica gel eluting with 0-15% MeOH/DCM to give the title compound (680 mg, 90% yield).

The chiral portion of this molecule can be synthesized using kinetic separation and ring-opening chemistry.

Getting 5

(2S)-1-amino-3,3-dimethyl-butan-2-ol hydrochloride

(2S)-2-tert-Butyloxirane is prepared commercially (CAS# 40102-55-4) or by kinetic optical resolution as described in J. AM. CHEM. SOC. 9vol. 124, NO. 7, 2002 1307, which reports that an epoxide with an R configuration was made with an RR catalyst, hence an epoxide S was made with an SS catalyst:

A mixture of (2S)-2-tert-butyloxirane (107 g, 1.01 mol) and NH ₄ OH (1.3 L, 10.7 mol) in EtOH (427 ml) was heated in a closed vessel at 100ºC for 4 h Cool and concentrate under reduced pressure. The resulting residue was dissolved in DCM (100 ml) at 0° C. and 4 M HCl in dioxane (267 ml, 1.1 mol) was added slowly over 10 minutes until a white precipitate formed. The resulting solid was filtered, washed with cold DCM and dried under suction to give the title compound (103 g, 62.9% yield). ¹ H NMR (300.1 MHz, MeOD): δ 0.94 (s, 9H), 2.76 (dd, J=11.1, 12.6 Hz, 1H), 3.09 (dd, J= 2.5, 12.6 Hz, 1H), 3.41 (dd, J=2.5, 11.1 Hz, 1H). [α] _D ²⁰ =+32.38º (c=0.8 g/100 ml, EtOH). Literature data presented in Tetrahedron: Asymmetry 13 (2002) 1209-1217: [α] _D ²⁰ =+25.9º (c=0.47 g/100 ml, EtOH).

Alternative way to get 5

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

Fill pump A 7 M with a solution of NH ₃ in MeOH. Fill pump B with a solution of (2S)-2-tert-butyloxirane (25 g, 232 mmol) dissolved in 7 M NH ₃ in MeOH (995 ml). The pumps are connected to a 500 ml (OD=1/8 inch) stainless steel tubular reactor in the oven, then a 7 ml (OD=1/16 inch) stainless steel tubular reactor located outside the oven is connected to the outlet to operate as a heat exchanger and connect an EQUILIBAR® back pressure regulator set at 1200 psi downstream of the heat exchanger.

Pump A is used to fill the tubular reactor with 7 M NH ₃ in MeOH at a rate of 5 ml/min. Set the oven temperature to 200ºC. When the tubular reactors are full, switch to pump B at 10 ml/min for 100 minutes and then switch to pump A to deliver 7 M NH ₃ in MeOH at the same flow rate for another 1 hour.

The collected solution was concentrated under reduced pressure at room temperature to give crude (2S)-1-amino-3,3-dimethylbutan-2-ol (24.4 g). The crude product was dissolved in tert-butyl methyl ether (150 ml) and 5.5 M HCl in IPA (46.4 ml, 255 mmol) was added dropwise with vigorous stirring over 5 minutes. The resulting white solid was filtered, washed with MTBE (4 x 25 ml) and dried to give the title compound (23 g, 72% yield).

Getting 6

[2-(2,2,2-trifluoroethoxy)-4-pyridyl]methanamine dihydrochloride

In EtOH (500 ml) stir 2-(2,2,2-trifluoroethoxy)pyridine-4-carbonitrile [CAS# 618446-30-3] 82.5 g, 367 mmol), decanted from the undissolved solid solution and solid washed with EtOH (3×50 ml), each time decanting the solution. Solutions in EtOH are combined, diluted with more EtOH (150 ml) and conc. aqueous HCl solution (125 ml). Add a suspension of 10% palladium on carbon (3.7 g) in about 10 ml of EtOH. The resulting reaction mixture was shaken under H ₂ at 60 psi. inch at room temperature overnight. The mixture is filtered through a pad of diatomaceous earth, washed with EtOH, and the filtrate is concentrated under reduced pressure to give a white solid. The resulting solid is suspended in MTBE at 45°C for 30 minutes, the mixture is cooled to room temperature and filtered to obtain a solid. The solid is dissolved in water (400 ml) and extracted twice with MTBE (400, then 200 ml). The aqueous phase is concentrated under reduced pressure to give a cream-colored solid, which is suspended in THF (100 ml) and filtered. The filter cake was washed with THF (2 x 30 mL) and dried in vacuo to give the title compound (46.16 g, 44% yield). The filtrate is further concentrated under reduced pressure and dried in a vacuum oven overnight. The resulting solid was suspended in THF (50 ml) for 30 minutes and filtered to give an additional crop of the title compound (34 g, 32.5% yield). ES/MS: m/z 307 [M+H]. Chloride ion (IC) analysis indicated a chloride ion:parent ion molar ratio of 2:1.

Example 1

1-[(2S)-2-hydroxy-3,3-dimethylbutyl]-3-[[2-(2,2,2-trifluoroethoxy)-4-pyridyl]methyl]urea

[2-(2,2,2-trifluoroethoxy)-4-pyridyl]methanamine 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 minutes and 1,1'-carbonyldiimidazole (5.7 g, 33.2 mmol) was added. The mixture was stirred for 10 minutes 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 over the weekend. Wash the reaction mixture with water, separate the organic phase and concentrate under reduced pressure. The resulting residue was purified by silica gel flash column chromatography eluting with 0-10% MeOH in DCM to give the title compound after solvent evaporation (8.16 g, 72% yield).

The product was combined with another batch of the title compound (4.37 g) prepared as 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 \u003d + 29.845º (c \u003d 0.2 g / 100 ml, MeOH).

Example 2

(+)-1-[2-hydroxy-2-[1-(trifluoromethyl)cyclopropyl]ethyl]-3-[[2-(2,2,2-trifluoroethoxy)-4-pyridyl]methyl]urea and (- )1-[2-hydroxy-2-[1-(trifluoromethyl)cyclopropyl]ethyl]-3-[[2-(2,2,2-trifluoroethoxy)-4-pyridyl]methyl]urea via chiral resolution

(±)-1-[2-Hydroxy-2-[1(trifluoromethyl)cyclopropyl]ethyl]-3-[[2-(2,2,2-trifluoroethoxy)-4-pyridyl]methyl]urea (680 mg) purified with chiral SFC using a CHIRALPAK® AD-H column (250×30 mm, 5 μ) at 35ºC, 100 bar, eluting with 92:8 CO ₂ /ethanol with 0.2% N,N-dimethylethylamine 152 ml/min and detection at 220 nm, the fractions are evaporated and dried in a vacuum oven at 45ºC, obtaining:

enantiomer 1 (1st elution peak, 285.4 mg): (-)-1-[2-hydroxy-2-[1-(trifluoromethyl)cyclopropyl]ethyl]-3-[[2-(2.2, 2-trifluoroethoxy)-4-pyridyl]methyl]urea; [α] _D ²⁰ =-21.0º (c=0.20 g/100 ml, MeOH);

enantiomer 2 (2nd elution peak, 289.5 mg). Enantiomer 2 is subjected to SFC purification a second time using the method described above; fractions are evaporated and dried in a vacuum oven at 45ºC to obtain (+)-1-[2-hydroxy-2-[1-(trifluoromethyl)cyclopropyl]-ethyl]-3-[[2-(2,2,2-trifluoroethoxy )-4-pyridyl]methyl]urea (236 mg); [α] _D ²⁰ \u003d + 15.0º (c \u003d 0.20 g / 100 ml, MeOH).

Example 3

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

In a round bottom flask, 2-[1-(trifluoromethyl)cyclopropyl]ethanamine hydrochloride (500 mg, 2.6 mmol; see WO 2013/134252, but also commercially available [CAS#: 1454690-80-2]) in DCM is stirred (5 ml) and add DIPEA (1.4 ml, 8 mmol). When a clear solution formed, 1,1'-carbonyldiimidazole (428 mg, 2.6 mmol) was added and the resulting mixture was stirred at room temperature for 30 minutes. [2-(2,2,2-Trifluoroethoxy)-4-pyridyl]methanamine dihydrochloride (810 mg, 2.9 mmol) was added in one portion and stirred at room temperature for 30 minutes. The reaction mixture was heated at 40° C. for 3 hours and stirred at room temperature for 16 hours. The reaction mixture is transferred to a microwave oven and heated at 100°C for 30 minutes. The resulting mixture was purified by silica gel chromatography eluting with 0-10% MeOH in DCM to give the title compound (892 mg (892 mg, 88% yield). ES/MS: m/z 386 [M+H]

Alternative way for example 3

Commercially available 2-[1-(trifluoromethyl)cyclopropyl]ethanamide 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). Shake at room temperature for 5 hours. A prepared 0.5 M solution of commercially available [2-(2,2,2-trifluoroethoxy)-4-pyridyl]methanamine (CAS# 1454690-80-2 alternatively, see WO 2013/134252) in DCM is added at room temperature ( 0.8 ml, 0.4 mmol). The resulting mixture is heated in a microwave oven at 100ºC for 30 minutes. The reaction mixture was poured into a larger round bottom flask and diluted with water (5 ml) and DCM (5 ml). Stirred for 15 min at room temperature and passed through a hydrophobic frit to separate the organic phase. Concentrate the filtrate under reduced pressure and purify the resulting residue by high pH reverse phase chromatography using a PHENOMENEX® GEMINI®-NX C18 75×30 mm column, particle size 5 µm, 110A, AXIA column with GEMINI®-NX C18 15×30 mm protection, eluting with 23-57% ACN in 10 mM NH ₄ HCO ₃ (pH ~10) containing 5% MeOH as an aqueous phase to give 1-[[2-(2,2,2-trifluoroethoxy)-4- pyridyl]methyl]-3-[2-[1-(trifluoromethyl)cyclopropyl]ethyl]urea (90.6 mg, 59% yield). ES/MS: m/z 386 [M+H]

Analyzes

Bioassay data showing that the compounds of the present embodiment are active in potentiating Kv7 are shown below.

Analysis #1

Optopatch Excitability Analysis for Example 1 in Motor Neurons Derived from ALS Patient iPSC Lines

Altered excitability of cortical neurons and lower motor neurons is an important factor in the pathophysiology of ALS (see, for example, 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 evaluate the excitability of cultured motor neurons derived from iPSC lines of two ALS patients with various pathogenic mutations.

Cell Culture: Two iPSC lines were obtained from one patient with a pathogenic mutation in TARDBP and one patient with a pathogenic repeat spread mutation in C9orf72 , respectively. Motor neurons were generated from these lines using a 2D differentiation method adapted from the SMADS dual inhibition neuronal patterning protocol. (See SM Chambers, et. al. , Nat Biotechnol. , 27, 275-280 (2009)) by incorporating motor neuron morphogens. Differentiation was confirmed by visual inspection, karyotyping, and staining for beta-III-tubulin and nuclear motor neuron marker ISL1. Neurons were cultured on a mouse glia monolayer in mTeSR (Stem Cell Technologies) supplemented with 10 ng/ml BDNF. Forty-eight hours prior to imaging, 100 nM transretinal was added to the medium.

Transduction with Optopatch vectors: After 15 days in vitro , cultured motor neurons were transduced with a lentiviral vector to drive co-expression of the CheRiff-mOrange2 actuator and the QuasAr3-Citrine strain indicator (for details, see DR Hochbaum, et. al., Nat. Methods , 11, 825-833 (2014)). Forty-eight hours prior to imaging, 100 nM transretinal was added to the medium.

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

Optopatch recordings: Five days after transduction, Optopatch imaging was performed on a dedicated ultra wide field fluorescence microscope at room temperature. Motor neurons were exposed to red laser excitation (200 W/ ^cm2 ; 635 nm) to track changes in QuasAr fluorescence and excitation with a blue light emitting diode (0-127 mW/ ^cm2 , 470 nm) to depolarize the cell membrane using CheRiff. A customized blue light stimulus protocol was used, consisting of: i) 2 s red light only to control spontaneous activity, 2) 5 x 500 ms blue light intensity ramps, and iii) 2 x 2 s linearly increasing blue light values, each with another maximum intensity of blue. Optopatch data was recorded using a Hamamatsu ORCA-Flash 4.0 sCMOS camera at a frame rate of 1 kHz. The size of the field of view is 4 mm × 0.5 mm. Custom control software written in MATLAB was used to control the lighting protocols and record all the scenes. To study the acute effects of Kv7.2/7.3 potentiators, neurons were incubated with test compound for 15 minutes prior to imaging.

Data analysis: Image segmentation analysis was performed using temporal principal component analysis and spatiotemporal independent component analysis to isolate individual neurons. A peak search algorithm was used to search for action potentials, and the data was analyzed for complex effects on mean arousal level, adaptation, reobase, and action potential waveform by comparison with a solvent control (for details, see CA Werley, et. al., Curr. Protoc. Pharmacol. , 78, 11,20,1-11,20,24 (2017)).

In accordance with the protocol described above, the main effect of the compound of example 1 was the level of excitation of the action potential in response to low-intensity blue light illumination. At a blue LED intensity of 5.1 mW/cm ² , the compound reduced the frequency of the action potential in a concentration-dependent manner.

Fitting a 4-parameter logistic equation to the data can be used to determine the effectiveness (EC ₅₀ ) of the effect of the compound of example 1 on the frequency of the action potential. The results are shown in Table 1 for two separate attempts to differentiate each of the lines derived from patients carrying TARDBP and C9orf72 mutations. The observed effects are qualitatively similar to, but stronger than those observed with the known Kv7.2/7.3 potentiator flupirtine, demonstrating the potential utility of the compound of Example 1 in the treatment of ALS by reducing the excitability of the patient's motor neurons.

Table 1: Excitability suppression of motor neurons obtained from ALS patients (presented as mean value (95% confidence

TARDBP C9orf72 Diff 1 Diff 2 Diff 1 Diff 2 Compound
according to example 1
EC ₅₀ (µM) 0.15
(0.05, 0.25) 0.15
(0.06, 0.24) 0.17
(0.09, 0.25) 0.20
(0.14, 0.25) Flupirtine
EC ₅₀ (µM) 1.15
(0.65, 1.66) 0.84
(0.48, 1.21) 1.66
(1.31, 2.01) 1.35
(0.82, 1.88)

Analysis #2

Modulation of Kv7.2/7.3 Conductivity by Kv7 Potentiators in the Mammalian Expression System

The activity and efficacy of Kv7 potentiators was assessed using automated electrophysiology on the IonWorks Barracuda platform (Molecular Devices) using the instrument's population-clamp mode.

Cell Culture: HEK293 cells stably expressing hKv7.2 (when induced with tetracycline) and hKv7.3 (Catalogue # CT6147, Charles River) were used for these studies. Cells were maintained in Dulbecco's Modified Eagle's Medium/Ham's F-12 Nutrient Formula (Sigma-Aldrich) supplemented with 5% tetracycline-screened fetal bovine serum (Sigma-Aldrich), 15 mM HEPES, 500 μg/ml G418, 100 U/mL penicillin, 100 µg/mL streptomycin, 29.2 mg/mL L-glutamine, 100 µg/mL Zeocin, and 5 µg/mL blasticidin. Expression of hKv7.2 was induced by adding 1 μg/ml doxycycline 24 h before recording.

Cells were cultured in Corning T-150 flasks to 85%-95% confluence. At the beginning of the experiment, the cells were washed once with D-PBS without calcium and magnesium, and then dissociated by incubation in 3 ml of 0.25% trypsin for 8 minutes at 37ºC. The cells were resuspended in the medium, triturated gently, and centrifuged for 4 min at 1000 rpm. Cells were resuspended in external solution to a final concentration of 2.5-3.5 M cells/ml.

Solutions: External solution consisted of (in mM): 140 NaCl, 5 KCl, 2 CaCl ₂ , 1 MgCl ₂ , 10 HEPES, 10 glucose, pH 7.4. The internal solution consisted of (in mM): 90 K-gluconate, 40 KCl, 3.2 EGTA, 3.2 MgCl ₂ , 5 HEPES, pH 7.25. Membrane perforating amphotericin B was prepared daily as a stock solution of 27 mg/mL in DMSO and then added to the internal solution to a final concentration of 0.1 mg/mL. Dilutions of test substances were prepared in 384-well plates from 10 mM DMSO stock solutions and diluted using acoustic dosing (Labcyte ECHO®) such that final DMSO concentrations were 0.1%.

Electrophysiological recordings: Resuspended cells were placed in an IONWORKS® BARRACUDATM (IWB) instrument, external solution was added to a 384-well patch plate, and a hole test was performed to determine blocked wells and bias voltages. The cells were then added to the plate using the instrument (9 μl per well). Before introducing the amphotericin perforating agent into the internal solution, two seal tests were performed and approximately 8 minutes were allowed for electrical access, which was confirmed by a third seal test. The command voltage protocol consisted of a family of 1 second voltage steps from -80 mV to +40 mV from a holding potential of -80 mV and was applied up to (baseline) and 6 minutes after addition of the test sample.

Data Analysis: Data were collected and leaks subtracted using IWB software. The current amplitudes for the last 10 ms of each voltage step were averaged and exported. Further analysis was performed using Microsoft Excel and GraphPad Prism. The current amplitude was converted to conductivity (G) by the following formula: G=I/(V-Ek) where I=current, V=step potential, Ek=reverse potential of potassium (-84 mV). The electrical conductivity in the presence of the test sample was normalized to the original conductivity at +40 mV for the same well. The conductance-voltage (GV) curves corresponded to the Boltzmann equation y=bottom+(top-bottom)/(1+EXP((V _m -V _0.5 /k)).

The test sample-mediated shift at the midpoint of the conductance curve (V _0.5 ) is shown in Table 2 for the compounds of Examples 1-3.

Table 2: Voltage control difference at half maximum conductivity in the presence of different concentrations of test compound.

Voltage shift (ΔV _0.5 ) Connection according to example 1 Connection according to example 2 Connection according to example 3 Concentration (µM) Average (n=9) SD Average (n=2) SD Average (n=4) SD 10 -38.0 3.7 -25.8 6.8 -21.9 1.3 3.33 -28.0 5.1 -23.0 4.5 -23.9 1.5 1.11 -13.2 3.2 -10.6 6.6 -21.9 1.8 0.37 -3.5 4.4 -1.9 4.1 -19.6 4.5 0.12 0.0 3.4 2.9 3.3 -11.0 2.4 0.04 1.3 2.4 7.4 5.2 -4.4 2.1 0.01 1.6 2.7 4.1 3.3 -1.8 0.5 0.005 3.0 2.9 5.0 1.8 1.9 1.8 0.0015 2.0 2.9 4.1 3.2 0.1 1.3 0.0005 2.6 4.4 6.1 0 3.9 1.4

(SD above refers to standard deviation).

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

Table 3: Activity and efficiency of Kv7.2/7.3 potentiators

Connection according to example 1 Connection according to example 2 Connection according to example 3 EC ₅₀ (µM) 2.0 1.1 0.10 Maximum shift -44 -28 -23 Incline 1.2 1.3 1.1

Analysis #3

Tracking thresholds to measure the effect of the compound of Example 1 at 3.10 and 30 mg/kg IP on peripheral nerve excitability

Threshold tracking is a non-invasive technique that measures the excitability properties of peripheral axons, providing information about their membrane potential and ion channel function.

Method

Used 16 male Wistar rats weighing 307-446 g from the company Charles River. Animals were housed in groups with standard housing conditions (4 per cage, from 07:00 to 19:00, light phase, constant temperature (21ºC) and humidity, free access to food and water, and environmental enrichment).

Rats were anesthetized with isoflurane (2-2.5%, O ₂ at 0.5 L/min) and then placed supine on a heating pad to keep the tail temperature above 32°C. The placement of the ring electrodes was marked and the marked areas of the tail were scraped with a blade to remove hair and top skin. These areas were washed with water and dried to ensure good conductivity between the skin and sticky electrodes.

A sticky ring stimulating electrode (+, anode) was wrapped around the foot. A second stimulating electrode with a sticky ring (-, cathode) was wound 1.5 cm from the base of the tail. The recording electrode-needle was placed slightly off-center at the top of the tail, at a distance of 6 cm from the stimulating cathode at the base of the tail. A reference needle electrode was placed almost off-center at the top of the tail, at a distance of 2 cm from the recording electrode. A sticky ground electrode was wrapped around the tail 2 cm proximal to the recording electrode.

Study protocol

Launch the Multitrack program (description below) and Spike.

Record 15 minutes of stable baseline

After 15 minutes, dose the compound of Example 1 or HEC.

Take a drop of blood for PK in the following cases:

10 minutes after dosing

20 minutes after dosing

30 minutes after dosing

40 minutes after dosing

45 minutes after IP (intraperitoneal) administration of the compound of Example 1, a dose of XE-991 (XE-991 is a commercially available compound that blocks the potentiating effects of KCNQ 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.

(XE-991 is administered only after the compound of example 1 at a dose of 30 mg/kg)

Take a drop of blood for PK in the following cases:

10 minutes after dosing (55 minutes after administration of the compound of example 1)

20 minutes after dosing (65 minutes after administration of the compound of example 1)

30 minutes after dosing (75 minutes after administration of the compound of example 1)

40 minutes after dosing (85 minutes after administration of the compound of example 1)

In a threshold tracking assay to measure peripheral axonal excitability in rat studies, the compound was administered intraperitoneally at 3, 10, or 30 mg/kg using a mixture of 1% hydroxyethylcellulose:0.25% polysorbate 80:0.05% defoamer:purified water ( HEC). Dry blood spots (DBS) were collected at about 10, 20, 30 and 40 minutes post-dose. The DBS samples were dried at room temperature for about 2 hours. Brain samples were taken at the final time point and frozen until analysis. DBS samples were shipped and stored at room temperature.

A stock solution of the compound of Example 1 at a concentration of 1 mg/mL was prepared and serially diluted with pooled rat blood to obtain standards ranging from 1 to 10,000 ng/mL. Blood was added to blank DBS cards to create standards. One 3 mm DBS standard or sample punch was added to a 96-well plate and 180 μl of internal standard (IS) in 1:1 ACN:MeOH was added. Shake for 45 minutes, dilute the extraction solution with water twice and analyze by LC/MSMS to analyze drug concentration.

Brain samples were homogenized using 1.14 ml MeOH:H ₂ O (2:8). Standards were prepared by adding the stock solution to a series of blank brain homogenates ranging from 5 to 50,000 ng/mL. 25 µl of standard or sample was pipetted into a 96-well plate, 180 µl of internal standard (IS) in 1:1 ACN:MeOH was added and mixed. Samples were centrifuged at 4000 rpm at 4°C for 10 min. The supernatant was diluted 15 times with water and analyzed by LC/MSMS.

Samples and standards were analyzed using a Sciex API 4000 Triple Quadrupole Mass Spectrometer (Sciex, Division of MDS Inc., Toronto, Canada) connected to a Shimadzu HPLC system (LC-IOAD, Shimadzu Corporation) and a Gilson 215 autosampler. Samples (0.01 ml) was loaded onto a 5 µm Betasil C-18, 20 x 2.1 mm Javelin HPLC column (Thermo Electron Corp. Cat#70105-022106) and eluted with a gradient. Chromatographic conditions included mobile phase A consisting of water/1M NH ₄ HCO ₃ (2000:10, v/v) and mobile phase B consisting of MeOH/1M NH ₄ HCO ₃ (2000:10, v/v). vol.), which passed through a 2.5-minute gradient at a flow rate of 1.5 ml/min. For mass spectrometric detection, positive ion turbo mode and ion source temperature of 740ºC were used. Quantification was performed using multiple reaction monitoring (MRM) on the following transitions: Example 1 compound (m/z 350.2 to m/z 233.0) and analog internal standard (m/z 263.1 to m/ z 148.1). Linear regression plots of compounds for peak area to internal standard ratios versus drug concentrations are obtained using the quadratic factor 1/x ² .

The internal standard analog used is 2-(dimethylamino)-N-pentyl-3-phenylpropanamide, 2,2,2-trifluoroacetic acid salt (1:1) and has the following structure:

This analog internal standard was purchased from Syncom, a Dutch company with address Kadijk 3, 9747 AT Groningen, The Netherlands.

Drug binding to plasma proteins and rat brain homogenates was determined using an in vitro dialysis method after adding the drug to these matrices and incubating for 4.5 hours at 37° C. subjected to orbital shaking. The assay was performed using a dialysis micro balance device and dialysis membrane strips (MWCO 12-14k). A sample at time 0 was taken after the protein matrix and samples were taken from both the protein side and the membrane buffer side after 4.5 hours of incubation. The starting product was quantified by LC-MSMS both at time 0 and at 45 minutes. The unbound fraction was calculated by dividing the concentration on the buffer side by the concentration on the protein side. Percent recovery was also calculated by dividing the sum of the buffer and protein chambers by the time 0 concentration at 4.5 hours. The concentration of unbound compound was calculated using the total concentration*unbound fraction.

Results: The effect of the compound of Example 1 on the absolute threshold is shown in Table 4.

Table 4

Compound Dose Unbound exposure in blood, nM (+SEM) Absolute tracking threshold for 40% CMAP, % of baseline (+SEM) Connection according to example 1 3 mg/kg (IP) 145 (±90) 121 (±10) Connection according to example 1 10 mg/kg (IP) 193 (±50) 140 (±8) Connection according to example 1 30 mg/kg (IP) 352 (±245) 160 (±18)

CMAP = complex muscle action potential.

(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 7 March 2018; accepted 13 April 2018 online pre-published 00 month 2018 doi:10.1002/cpt.1096, CLINICAL PHARMACOLOGY & THERAPEUTICS , VOLUME 00 NUMBER 00, MONTH 2018 and J. Fleckenstein et al., "Activation of axonal Kv7 channels in human peripheral nerve by flupirtine but not placebo - therapeutic potential for peripheral neuropathies: results of a randomized controlled trial”, Journal of Translational Medicine 2013, 11:34).

There is a significant difference in time and time-treatment effect relationship (two-tailed analysis of variance RM; time effect F(26,312)=13.18, p=<0.0001; time-treatment effect relationship F(78,312)=2.888 , p<0.0001 Bonferroni's multiple comparison test showed that the compound of Example 1 at 30mg/kg significantly increased the absolute threshold (decrease in excitability) compared to vehicle XE-991 was able to reverse this increase (increase excitability).

Claims (50)

1. Compound formula

where R1 is

or

and where R2 is H or OH, or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, where R1 is

or a pharmaceutically acceptable salt thereof. 3. A compound according to claim 2, wherein R2 is OH, or a pharmaceutically acceptable salt thereof. 4. The compound according to claim 3 of the formula

where the compound includes a single enantiomer that has a (+) optical rotation in methanol, or a pharmaceutically acceptable salt thereof. 5. The compound according to claim 3 of the formula

wherein the compound comprises a single enantiomer that has (-) optical rotation in methanol, or a pharmaceutically acceptable salt thereof. 6. A compound according to claim 2, wherein R2 is H, or a pharmaceutically acceptable salt thereof. 7. The compound according to claim 1, where R1 is

or a pharmaceutically acceptable salt thereof. 8. A compound according to claim 7 wherein R2 is H, or a pharmaceutically acceptable salt thereof. 9. A compound according to claim 7 wherein R2 is OH, or a pharmaceutically acceptable salt thereof. 10. The compound according to claim 9 of the formula

or a pharmaceutically acceptable salt thereof. 11. The compound according to claim 9 of the formula

or a pharmaceutically acceptable salt thereof. 12. A pharmaceutical composition for the treatment of a disease caused by changes in motor neuron excitability, comprising an effective amount of a compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents or excipients. 13. A method of treating a disease caused by changes in motor neuron excitability, comprising administering to a patient in need thereof an effective amount of a compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof. 14. The method of claim 13 wherein the disease caused by changes in motor neuron excitability is amyotrophic lateral sclerosis. 15. A method for treating a disease caused by changes in motor neuron excitability, comprising administering to a patient in need thereof a pharmaceutical composition according to claim 12. 16. The method of claim 15, wherein the disease caused by changes in motor neuron excitability is amyotrophic lateral sclerosis. 17. A compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, for use in therapy. 18. A compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, for the treatment of a disease caused by changes in motor neuron excitability. 19. The compound of claim 18, or a pharmaceutically acceptable salt thereof, wherein the disease caused by changes in motor neuron excitability is amyotrophic lateral sclerosis. 20. The use of a compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a disease caused by changes in motor neuron excitability. 21. The use of a compound according to claim 20, or a pharmaceutically acceptable salt thereof, wherein the disease caused by changes in motor neuron excitability is amyotrophic lateral sclerosis. 22. A method of preparing a pharmaceutical composition, comprising mixing a compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients. 23. Compound Formula

or a pharmaceutically acceptable salt thereof. 24. Compound Formula

where the compound includes a single enantiomer that has a (+) optical rotation in methanol, or a pharmaceutically acceptable salt thereof. 25. Compound Formula

or a pharmaceutically acceptable salt thereof.