Patent Description:
This disclosure relates to chemical processes for making compounds which are CIC-<NUM> chloride channel inhibitors. As described in <CIT>, CIC-<NUM> chloride channel inhibitors may be useful in the treatment of neuromuscular disorders, such as myasthenia gravis and ALS, or in reversing and/or ameliorating a neuromuscular blockade.

<CIT> relates to CIC-<NUM> chloride channel inhibitors, including compounds of Formula I.

<NPL>) relates to synthesis of some <NUM>-[<NUM>-(<NUM>-phenyl-<NUM>-isoxazolyl)phenoxy methyl]-<NUM>-<NUM>-benzopyran-<NUM>-ones, and discloses preparation of [<NUM>-(<NUM>-phenyl-<NUM>-isoxazolyl)phenoxyl] acetic acid.

In order to develop treatments of neuromuscular disorders, there is a need for CIC-<NUM> chloride channel inhibitors. The present disclosure provides a novel industrially applicable process for the preparation of compounds of Formula I. The compounds of Formula I inhibit CIC-<NUM> ion channels and are capable of restoring neuromuscular transmission, as evidenced by the data generated by investigation of the compound set in biological models described herein. These compounds can thus be used to treat or ameliorate muscle weakness and muscle fatigue in neuromuscular junction disorders caused by disease or by neuromuscular blocking agents.

In one aspect, the disclosure provides a process for the preparation of compounds of Formula I
<CHM>
comprising step a) wherein.

wherein R<NUM> to R<NUM> and n are as defined herein.

The disclosure further relates to novel compounds of Formula II as defined herein.

The terms "C<NUM>-<NUM> alkyl" and "C<NUM>-<NUM> alkyl" refers to a branched or unbranched alkyl group having from one to three or one to five carbon atoms respectively, including but not limited to methyl, ethyl, prop-<NUM>-yl, prop-<NUM>-yl, <NUM>-methyl-prop-<NUM>-yl, <NUM>-methyl-prop-<NUM>-yl, <NUM>,<NUM>-dimethyl-prop-<NUM>-yl, but-<NUM>-yl, but-<NUM>-yl, <NUM>-methyl-but-<NUM>-yl, <NUM>-methyl-but-<NUM>-yl, pent-<NUM>-yl, pent-<NUM>-yl and pent-<NUM>-yl.

The term "C<NUM>-<NUM> alkenyl" refers to a branched or unbranched alkenyl group having from two to five carbon atoms, two of which are connected by a double bond, including but not limited to ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl and isopentenyl.

The term "C<NUM>-<NUM> alkynyl" refers to a branched or unbranched alkynyl group having from two to five carbon atoms, two of which are connected by a triple bond, including but not limited to ethynyl, prop-<NUM>-ynyl, prop-<NUM>-ynyl, but-<NUM>-ynyl, but-<NUM>-ynyl, but-<NUM>-ynyl, buta-<NUM>,<NUM>-diynyl, pent-<NUM>-ynyl, pent-<NUM>-ynyl, pent-<NUM>-ynyl, pent-<NUM>-ynyl, penta-<NUM>,<NUM>-diynyl and penta-<NUM>,<NUM>-diynyl.

The term "C<NUM>-<NUM> cycloalkyl" and "C<NUM>-<NUM> cycloalkyl" refers to a group having three to five or three to six carbon atoms respectively including a monocyclic or bicyclic carbocycle, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The present disclosure provides a novel industrially applicable process for the preparation of compounds of Formula I which are CIC-<NUM> chloride channel inhibitors. The present process allows for better control of impurities, reduces or removes the need for chromatographic steps and has higher yields thereby providing better cost of goods.

Thus, in one aspect, the disclosure provides a process for the preparation of compounds of Formula I
<CHM>
comprising step a) wherein.

In one embodiment, R<NUM> is Cl or Br. In one embodiment, R<NUM> is Cl. In one embodiment, R<NUM> is Br.

In one embodiment, R<NUM> is C<NUM>-<NUM> alkyl. In one embodiment, R<NUM> is selected from the group consisting of methyl, ethyl, prop-<NUM>-yl, prop-<NUM>-yl, <NUM>-methyl-prop-<NUM>-yl, <NUM>-methyl-prop-<NUM>-yl, <NUM>,<NUM>-dimethyl-prop-<NUM>-yl, but-<NUM>-yl, but-<NUM>-yl, <NUM>-methyl-but-<NUM>-yl, <NUM>-methyl-but-<NUM>-yl, pent-<NUM>-yl, pent-<NUM>-yl and pent-<NUM>-yl. In one embodiment, R<NUM> is ethyl. In one embodiment, R<NUM> is prop-<NUM>-yl. In one embodiment, R<NUM> is prop-<NUM>-yl. In one embodiment, R<NUM> is but-<NUM>-yl. In one embodiment, R<NUM> is <NUM>-methyl-prop-<NUM>-yl.

In one embodiment, R<NUM> is deuterium. In one embodiment, R<NUM> is F.

In one embodiment, R<NUM> is H. In one embodiment, R<NUM> is C<NUM>-<NUM> alkyl optionally substituted with one or more, identical or different, substituents R<NUM>. In one embodiment, R<NUM> is C<NUM>-<NUM> alkyl substituted with C<NUM>-<NUM> cycloalkyl optionally substituted with one or more, identical or different, substituents R<NUM>. In one embodiment, R<NUM> is Me. In one embodiment, R<NUM> is Et. In one embodiment, R<NUM> is -CH<NUM>F. In one embodiment, R<NUM> is cyclopropyl.

In one embodiment, R<NUM> is C<NUM>-<NUM> alkyl optionally substituted with one or more, identical or different, substituents R<NUM>. In one embodiment, R<NUM> is Me. In one embodiment, R<NUM> is Et. In one embodiment, R<NUM> is <NUM>-methyl-prop-<NUM>-yl.

In one embodiment, R<NUM> is H. In one embodiment, R<NUM> is deuterium. In one embodiment, R<NUM> is C<NUM>-<NUM> alkyl. In one embodiment, R<NUM> is selected from the group consisting of methyl, ethyl, prop-<NUM>-yl, prop-<NUM>-yl, <NUM>-methyl-prop-<NUM>-yl, <NUM>-methyl-prop-<NUM>-yl, <NUM>,<NUM>-dimethyl-prop-<NUM>-yl, but-<NUM>-yl, but-<NUM>-yl, <NUM>-methyl-but-<NUM>-yl, <NUM>-methyl-but-<NUM>-yl, pent-<NUM>-yl, pent-<NUM>-yl and pent-<NUM>-yl.

In one embodiment, R<NUM> is deuterium. In one embodiment, R<NUM> is F. In one embodiment, R<NUM> is C<NUM>-<NUM> cycloalkyl optionally substituted with one or more, identical or different, substituents R<NUM>. In one embodiment, R<NUM> is C<NUM>-<NUM> alkyl substituted with R<NUM>, wherein R<NUM> is C<NUM>-<NUM> cycloalkyl optionally substituted with one or more, identical or different, substituents R<NUM>.

In one embodiment, n is <NUM>. In one embodiment, n is <NUM>. In one embodiment, n is <NUM>. In one embodiment, n is <NUM>.

In one embodiment the compound of Formula II is reacted with an acid. In one embodiment the compound of Formula II is reacted with a concentrated acid. In one embodiment the compound of Formula II is reacted with an acid selected from the group formic acid, acetic acid, propionic acid, butyric acid, benzoic acid, hydrochloric acid and sulfuric acid or a mixture thereof, such as a mixture of formic acid and acetic acid. In one embodiment the acid is acetic acid. In one embodiment, the acid is formic acid. In one embodiment, the acid is concentrated acetic acid. In one embodiment, the acid is concentrated formic acid. In one embodiment, the acid is concentrated acetic acid. In one embodiment, the acid is a mixture of concentrated formic acid and concentrated acetic acid.

In one embodiment, step a) i) is performed at a temperature of between <NUM> and <NUM>, such as between <NUM> and <NUM>, for example between <NUM> and <NUM>.

In one embodiment, the reaction time of step a) i) is between <NUM> hours and <NUM> hours, such as between <NUM> and <NUM> hours, for example between <NUM> and <NUM> hours.

In one embodiment, the amount of acid used in step a) i) is between <NUM> per gram of starting material and <NUM> per gram of starting material.

In one embodiment the compound of Formula II is reacted with a base.

In one embodiment, the compound of Formula I is isolated from the reaction mixture by adding water and extracting the product into an organic solvent. In one embodiment, the organic solvent used for the extraction is selected from the list consisting of toluene, ethyl acetate, isopropyl acetate and tert-butyl methyl ether. In one embodiment, the organic solvent used for the extraction is tert-butyl methyl ether.

In one embodiment, the compound of Formula I is purified by crystallisation from an organic solvent. In one embodiment, the compound of Formula I is purified by crystallisation from an organic solvent is selected from the list consisting of toluene, ethyl acetate, isopropyl acetate and tert-butyl methyl ether. In one embodiment, the compound of Formula I is purified by crystallisation from toluene.

In one embodiment, the process further comprises step b) wherein.

In one embodiment, the solvent in step b) i) is dimethylformamide (DMF). In one embodiment, the compound of formula IV is selected from the group consisting of ethyl vinyl ether, n-butyl vinyl ether and tert-butyl vinyl ether. In one embodiment, the compound of formula IV is n-butyl vinyl ether. In one embodiment, the base in step b) ii) is a trialkylamine. In one embodiment, the base in step b) ii) is selected from the group consisting of trimethylamine, triethylamine, triisopropylamine and N,N-diisopropylethylamine. In one embodiment, the base in step b) ii) is triethylamine.

In one embodiment, the process further comprises step c) wherein.

In one embodiment, the organic solvent in step c) i) is an alcohol. In one embodiment, the organic solvent in step c) i) is an alcohol selected from the group consisting of methanol, ethanol, propanol, n-butanol and tert-butanol. In one embodiment, the base in step c) ii) is selected from the group consisting of pyridine, trimethylamine, triethylamine, triisopropylamine and N,N-diisopropylethylamine.

In one embodiment, the process further comprises step d) wherein.

wherein R<NUM> to R<NUM> and n are as defined herein and LG is a leaving group.

In one embodiment, the leaving group is selected from the group consisting of tosylate, mesylate, triflate, nosylate, brosylate, bromide, iodide and chloride. In one embodiment, the leaving group (LG) of Formula VI is tosylate.

In one embodiment, the base in step d) ii) is an inorganic base. In one embodiment, the base in step d) ii) is an inorganic base selected from the group consisting of lithium carbonate, sodium carbonate, potassium carbonate, caesium carbonate, lithium hydrogencarbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, lithium hydroxide, sodium hydroxide and potassium hydroxide.

In one embodiment, the organic solvent is selected from the group consisting of pentane, hexane, heptane, toluene, xylene, dichloromethane, tetrahydrofuran and acetonitrile.

In one embodiment, the process comprises step a) as outlined in Scheme <NUM>. In one embodiment, the process comprises steps a) and b) as outlined in Scheme <NUM>. In one embodiment, the process comprises steps a), b) and c) as outlined in Scheme <NUM>. In one embodiment, the process comprises steps a), b), c), and d) as outlined in Scheme <NUM>.

In one embodiment, the compound of Formula I is an inhibitor of the CIC-<NUM> chloride ion channel.

In one embodiment, the compound of Formula I is at least <NUM>% pure, such as at least <NUM> % pure, such as at least <NUM> % pure, such as at least <NUM> % pure, such as at least <NUM> % pure, such as at least <NUM> % pure.

In certain embodiments, the compound or the compound for use according to the present disclosure can have ><NUM>% enantiomeric excess. In certain embodiments, the compound or the compound for use according to the present disclosure can have ><NUM>% e.

In one embodiment, the compound of Formula I is selected from the group consisting of:.

In one aspect, the disclosure relates to a process for the preparation of a pharmaceutical composition comprising the steps of:.

In one aspect, the present disclosure is directed to a compound of Formula II as defined herein.

In one embodiment, the compound of Formula II is selected from the group consisting of:.

This disclosure is also directed, in part, to pharmaceutical compositions comprising compounds of Formula I prepared by the disclosed processes. In one embodiment, a compound of Formula I prepared by the above process may be included in pharmaceutical compositions. These compositions may also comprise one or more conventional pharmaceutically acceptable carriers. The compositions may comprise further active ingredients/agents or other components to increase the efficiency of the composition.

Thus, another aspect of the disclosure is a process for preparing a pharmaceutical composition comprising a compound of Formula I, characterized in that the compound of Formula I is prepared by a process according to the present invention.

A pharmaceutical composition comprising a compound of Formula I of the disclosure and a pharmaceutically acceptable carrier constitutes another aspect of the invention.

<NUM>H-NMR spectra were recorded either on a Jeol LA400 (<NUM>) spectrometer and were calibrated using residual nondeuterated solvent as internal reference (<NUM> ppm for CHCl<NUM>).

Equipment: Agilent <NUM> Infinity series LC (High Pressure Degasser, Binary Pump, Autosampler and Column Oven) with Agilent <NUM> DAD detector scanning from <NUM> to <NUM>. Mass detection was afforded with API <NUM> mass spectrometer (electrospray).

Column: Agilent Poroshell <NUM> EC-C18 (<NUM>, <NUM> × <NUM>).

Conditions: <NUM>% v/v Formic acid in water [eluent A]; MeCN [eluent B]; Flow rate <NUM>/min and <NUM> minutes equilibration time between samples.

Compounds were analysed using a Waters ACQUITY ultra-performance convergence chromatography (UPC2) system equipped with a binary solvent delivery pump, an auto-sampler, a column oven (CM-<NUM>), a back-pressure regulator, and a diode array detector.

Conditions: <NUM>, <NUM>/min, isocratic <NUM>:<NUM> EtOH:CO<NUM> (<NUM>% v/v TFA), <NUM> BarG.

(S)-<NUM>-(<NUM>-Bromo-<NUM>-(isoxazol-<NUM>-yl)phenoxy)propanoic acid B. <NUM> was prepared using the schematic shown below.

<NUM>-Bromo-<NUM>-hydroxybenzaldehyde B. <NUM> (<NUM>, <NUM> mmol) was added to a stirred solution of methyl (R)-<NUM>-(tosyloxy)propanoate B. <NUM> (<NUM>, <NUM> mmol) in hexane (<NUM>) and the resulting mixture was heated to <NUM> until dissolution. Potassium carbonate (<NUM>, <NUM> mmol) was added to the reaction mixture and it was stirred at <NUM> for <NUM> and at room temperature for <NUM>.

Solvent was removed in vacuo and the residue was partitioned between water (<NUM>) and ethyl acetate (<NUM>). The aqueous layer was extracted with more ethyl acetate. The organic layers were combined together and were washed with water (2x300 mL) and brine, dried over sodium sulfate, filtered and concentrated under reduced pressure in order to afford a pale yellow oil. This crude material was purified via silica gel flash column chromatography using heptane:ethyl acetate <NUM>-<NUM>% as solvent system in order to afford the desired product, methyl (S)-<NUM>-(<NUM>-bromo-<NUM>-formylphenoxy)propanoate B. <NUM>, as a colourless oil, which solidified overnight (<NUM>, <NUM>% yield).

<NUM> NMR (<NUM>, CDCl<NUM>) δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>). LC/MS (Agilent) m/z <NUM> (M+H)+ at <NUM> mins.

Hydroxyamine hydrochloride (<NUM>, <NUM> mmol) was added under nitrogen to a stirred solution of methyl (S)-<NUM>-(<NUM>-bromo-<NUM>-formylphenoxy)propanoate B. <NUM> (<NUM>, <NUM> mmol) in methanol (<NUM>), previously cooled to -<NUM>. Then, pyridine (<NUM>, <NUM> mmol) was added dropwise to the mixture and it was stirred at that temperature for <NUM>. The reaction mixture was stored at <NUM> overnight.

The reaction mixture was warmed to room temperature and was poured into water (<NUM>). This solution was extracted with ethyl acetate (2x400 mL). The organic layers were combined together and were washed with water (2x300 mL) and brine, dried over sodium sulfate, filtered and concentrated under reduced pressure in order to afford the desired product, methyl (S,E)-<NUM>-(<NUM>-bromo-<NUM>-((hydroxyimino)-methyl)phenoxy)propanoate B. <NUM>, as a white solid (<NUM>, quantitative yield).

N-Chlorosuccinimide (<NUM>, <NUM> mmol) was added to a solution of methyl (S,E)-<NUM>-(<NUM>-bromo-<NUM>-((hydroxyimino)methyl)phenoxy)propanoate B. <NUM> (<NUM>, <NUM> mmol) in dimethylformamide (<NUM>) at room temperature, followed by the addition of a <NUM> solution of hydrochloric acid (<NUM> aqueous). The resulting reaction mixture was stirred at room temperature for <NUM>. After that time, the mixture was cooled to <NUM> and triethylamine (<NUM>, <NUM> mmol) was added to it, followed by the addition of butyl vinyl ether (<NUM>, <NUM> mmol). The reaction mixture was stirred at room temperature for <NUM>.

The reaction mixture was poured into water (<NUM>), acidified with a <NUM> solution of hydrochloric acid to pH <NUM> and it was extracted with tert-butylmethyl ether (2x350 mL). The organic layers were combined together and they were washed with water and brine, dried over sodium sulfate, filtered and concentrated under reduced pressure in order to afford the desired product, methyl (<NUM>)-<NUM>-(<NUM>-bromo-<NUM>-(<NUM>-butoxy-<NUM>,<NUM>-dihydroisoxazol-<NUM>-yl)phenoxy)propanoate B. <NUM> (a ca. <NUM>:<NUM> mixture of diastereoisomers), as a yellow oil (<NUM>, <NUM>% yield).

<NUM> NMR (<NUM>, CDCI3) δ <NUM> and <NUM> (two d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> and <NUM> (two d, J = <NUM>, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> and <NUM> (two s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> and <NUM> (two dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> and <NUM> (two t, J = <NUM>, <NUM>). LC/MS (Agilent) m/z <NUM> (M+H)+ at <NUM> mins.

A solution of ethyl (<NUM>)-<NUM>-(<NUM>-bromo-<NUM>-(<NUM>-butoxy-<NUM>,<NUM>-dihydroisoxazol-<NUM>-yl)phenoxy)propanoate B. <NUM> (<NUM>, <NUM> mmol) in aqueous formic acid (<NUM>%) (<NUM>, <NUM> mol) was heated at <NUM> for <NUM>.

The reaction mixture was cooled to room temperature and solvent was removed in vacuo. The crude material was azeotroped with acetonitrile and dried in vacuo for <NUM> at <NUM>. The isolated oil was partitioned between tert-butylmethyl ether and water. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure in order to give a yellow oil. This oil was triturated in dichloromethane (<NUM>) and hexane (<NUM>) and the isolated solid was dried overnight in the vacuum oven in order to afford the desired product, (<NUM>)-<NUM>-(<NUM>-bromo-<NUM>-(isoxazol-<NUM>-yl)phenoxy)propanoic acid B. <NUM>, as a beige solid (<NUM>, <NUM>% yield).

<NUM> NMR (<NUM>, CDCl<NUM>) δ <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>). LC/MS (Agilent) m/z <NUM> (M+H)+ at <NUM> mins.

The overall yield was <NUM>% starting from <NUM>-bromo-<NUM>-hydroxybenzaldehyde B. <NUM> and the product was ><NUM>% pure as judged by LC/MS and <NUM> NMR.

Chiral SCF method: (S)-enantiomer <NUM> mins; (R)-enantiomer <NUM> mins.

DMF (<NUM>) was added to methyl (S,E)-<NUM>-(<NUM>-bromo-<NUM>-((hydroxyimino)methyl)phenoxy)propanoate B. <NUM> (<NUM>) and the temperature adjusted to <NUM>. N-chlorosuccinimide (<NUM>, <NUM> eq) was added and the reaction was stirred for <NUM> minutes at <NUM>-<NUM>. N-chlorosuccinimide (4x <NUM>, 4x <NUM> eq) was added in <NUM> equal portions every <NUM> minutes keeping the temperature between <NUM>-<NUM> and the reaction was stirred for <NUM> minutes at <NUM>-<NUM>. The reaction was cooled to <NUM>-<NUM> and butyl vinyl ether (<NUM>, <NUM> eq) was added followed by triethylamine (<NUM>, <NUM> eq) dropwise at <NUM>-<NUM> over <NUM> hour. The reaction was stirred at <NUM>-<NUM> over <NUM> hours then water (<NUM>) was added over <NUM> minutes. The product was extracted with tert-butyl methyl ether (2x <NUM>) and the organic phase washed with <NUM>% w/w brine (2x <NUM>), dried over magnesium sulfate, filtered and concentrated to give methyl (<NUM>)-<NUM>-(<NUM>-bromo-<NUM>-(<NUM>-butoxy-<NUM>,<NUM>-dihydroisoxazol-<NUM>-yl)phenoxy)propanoate B. <NUM> as a ca. <NUM>:<NUM> mixture of diastereoisomers (<NUM>, quantitative) as an amber oil.

(<NUM>)-<NUM>-(<NUM>-bromo-<NUM>-(<NUM>-butoxy-<NUM>,<NUM>-dihydroisoxazol-<NUM>-yl)phenoxy)propanoate B. <NUM> (<NUM>) was dissolved in acetic acid (<NUM>) and water (<NUM>) was added at <NUM>-<NUM>. The reaction was heated to reflux (ca. <NUM>) for <NUM> hours or until in process control showed that the reaction was complete. The reaction was cooled to <NUM>-<NUM> and water (<NUM>) was added. The product was extracted with tert-butyl methyl ether (<NUM> then <NUM>) and the combined organic phases washed with <NUM>% w/w brine (3x <NUM>). The organic phase was distilled leaving ca. <NUM> volumes then toluene (<NUM>) was added. The organic phase was distilled leaving ca. <NUM> volumes then toluene (<NUM>) was added. The organic phase was distilled leaving ca. <NUM> volumes then cooled to <NUM>-<NUM> and filtered. The organic phase was cooled to <NUM>-<NUM>, a seed crystal was added. The solution was stirred at <NUM>-<NUM> for <NUM> minutes, cooled to <NUM>-<NUM> over <NUM> hours, stirred at <NUM>-<NUM> for <NUM> hours, then filtered washing toluene (<NUM>) and n-heptane (2x <NUM>). The solid was dried at <NUM> until no further change in mass giving (<NUM>)-<NUM>-(<NUM>-bromo-<NUM>-(isoxazol-<NUM>-yl)phenoxy)propanoic acid B. <NUM> as a beige solid (<NUM>, <NUM>%) in ><NUM>% purity as determined by LC/MS and <NUM> NMR and ><NUM>% enantiomeric excess as determined by chiral HPLC.

The investigatory goal of these experiments was to evaluate how compounds affect the open probability and current amplitude of human CIC-<NUM> channels expressed in CHO cells. Experiments were performed using an automated patch clamp system that allowed high throughput testing of whole cell patches together with both intracellular and extracellular addition of compound.

Automated whole cell patch clamp experiments were performed with the Qpatch <NUM> system (Sophion Bioscience, Ballerup, Denmark) at room temperature. Data acquisition and analysis were performed in the Qassay software (ver. <NUM>, Odense).

To evoke CIC-<NUM> currents in whole cell patches, the membrane potential was initially stepped from a holding potential of -<NUM> mV to +<NUM> mV for <NUM> and then to various test voltages (sweeps) ranging from +<NUM> mV to -<NUM> mV in steps of <NUM> mV for <NUM>. To obtain tail currents, the membrane potential was stepped to -<NUM> mV after each test voltage for <NUM> and then relaxed to -<NUM> mV for <NUM> sec between sweeps (<FIG>). I/V relationships for whole cell instant and steady state current amplitudes were obtained by plotting average current densities at the beginning and at the end of the <NUM> step against the membrane potential (<FIG>).

In order to determine the relative overall open probability (P<NUM>), the instantaneous tail currents were normalized to the maximal tail current obtained following the most positive voltage step and plotted against the test voltage. Plots of normalized tail currents from each whole cell patch were then fitted to a Boltzmann function allowing determination of half activation voltages (V<NUM>/<NUM>, <FIG>).

For automated patch clamp experiments extracellular solutions contained: <NUM> CaCl<NUM>, <NUM> MgCl<NUM>, <NUM> HEPES, <NUM> KCI, <NUM> NaCl, <NUM> Glucose, pH adjusted to <NUM> with NaOH (<NUM>). Osmolality adjusted to -<NUM> using sucrose.

Intracellular solutions contained: <NUM> CsF, <NUM> CsCI, <NUM>/<NUM> KOH/EGTA, <NUM> HEPES, <NUM> NaCl, pH adjusted to <NUM> with NaOH (<NUM>). Osmolality adjusted to -<NUM> mOsm using sucrose.

Cells used in patch clamp experiments were Chinese hamster ovary cells (CHO) constitutively expressing human CIC-<NUM> channels. The amino acid sequence encoded by the cDNA used to create this cell line was identical to the translated sequence for GenBank accession number NM_000083. Cells were produced by Charles River (Catalogue CT6175, Cleveland OH, USA) in a cryopreserved format. Experiments were performed on the cells directly after thawing (<NUM> × <NUM><NUM> cells used in each experiment).

To evaluate the compound effect on CIC-<NUM>, when applied directly to the intracellular side of the cell membrane, the half activation voltage, V<NUM>/<NUM>, was determined from whole cell patches with compound added to the intracellular solution and then compared to V<NUM>/<NUM> determined from control cell patches with only vehicle added to the intracellular solution. Additionally, the effect of extracellular added compound was evaluated by determine V<NUM>/<NUM> and steady state current amplitudes before and after exchanging the extracellular solution to contain compound.

The difference in half activation voltage of CIC-<NUM> channels, ΔV<NUM>/<NUM>, was determined as the difference between the cell patches treated intracellularly with compound and control cells patches and is reported in Table <NUM> below. A positive shift in ΔV<NUM>/<NUM> is reflecting CIC-<NUM> channel inhibition by the tested compound. P-values of <<NUM> is considered significant.

Isometric hindlimb force was measured in <NUM>-week old female Lewis rats in the presence and absence of compound.

Rats were placed under anesthesia with isoflurane (<NUM>-<NUM>%), intubated and subsequently connected to a micro ventilator (Microvent <NUM>, Hallowell EMC, US). Two stimulation electrodes were inserted through the skin to stimulate the sciatic nerve. A small incision was made proximal to the ankle, to expose the Achilles tendon, which was tied by cotton string, and connected to a force transducer (Fort250, World Precision Instruments) with adjustable position (Vernier control). The Achilles tendon was then cut distal to the attached cotton string. The rat was placed on a heated pad, and to prevent movement artefacts from contraction of the ankle dorsiflexors, the foot was fixated by tape on a footplate.

Muscle contractile properties were assessed by applying an electrical current (under supramaximal voltage conditions) to the nerve and recording the force generated by the muscle. The muscle was stretched until maximal force was obtained, when assessed by <NUM> stimulation. Isometric force was measured every <NUM> seconds at <NUM> (Twitch), <NUM> pulses, and at every <NUM> minutes at <NUM> (Tetanic) for <NUM> second (<NUM> pulses). This stimulation pattern was employed throughout the experiment, except in a few cases where <NUM> stimulation was replaced by <NUM> (<NUM> pulses). Neuromuscular transmission was partially inhibited by constant infusion of Cisatracurium (Nimbex, GlaxoSmithKline) at a concentration of <NUM>/kg at an adjustable infusion speed, adjusted individually for each animal to obtain a level of inhibition of ca. <NUM>% of the forced generated at <NUM> stimulation on the <NUM>th pulse. When the level of neuromuscular inhibition was stable, the test article was injected i. at the chosen concentration. The effect of test article was assessed on its ability to increase force generated from the stimulation pattern applied. The effect was assessed in the ability to increase force per se (tetanic, <NUM>, stimulation), and the ratio between individual twitch peaks (<NUM> stimulation). The effect was monitored for at least <NUM> hour after injection of test article. In addition, the time from injection of test article to maximal effect on force (both twitch and tetanic) was noted and the time for the effect to disappear (return to baseline), if possible. When appropriate the infusion of neuromuscular blocking agent was ceased, with the stimulation pattern continued, and the return of force to control levels was monitored. Animals were sacrificed by cervical dislocation while still fully sedated.

(<NUM>)-<NUM>-[<NUM>-bromo-<NUM>-fluoro-<NUM>-(<NUM>,<NUM>-oxazol-<NUM>-yl)phenoxy]propanoic acid was dosed <NUM>/kg i. The average increase in tetanic force was <NUM>% (<NUM> experiments). <NUM>-[<NUM>-bromo-<NUM>-(<NUM>,<NUM>-oxazol-<NUM>-yl)phenoxy]acetic acid was dosed <NUM>/kg i. The average increase in tetanic force was <NUM>% (<NUM> experiments).

Claim 1:
A process for the preparation of compounds of Formula I
<CHM>
comprising step a) wherein
i) a compound of Formula II is reacted with an acid or base
<CHM>
and
ii) a compound of Formula I is isolated from the reaction mixture,
wherein
- R<NUM> is selected from the group consisting of H, F, Cl, Br and I;
- R<NUM> is selected from the group consisting of C<NUM>-<NUM> alkyl and C<NUM>-<NUM> cycloalkyl
- R<NUM> is selected from the group consisting of deuterium, F, Cl, Br and I;
- R<NUM> is selected from the group consisting of H, deuterium, C<NUM>-<NUM> alkyl, C<NUM>-<NUM> alkenyl, C<NUM>-<NUM> alkynyl, C<NUM>-<NUM> cycloalkyl and C<NUM> cycloalkenyl, each of which may be optionally substituted with one or more, identical or different, substituents R<NUM>;
- R<NUM> is selected from the group consisting of C<NUM>-<NUM> alkyl optionally substituted with one or more, identical or different, substituents R<NUM>, C<NUM>-<NUM> alkenyl, C<NUM>-<NUM> alkynyl, C<NUM>-<NUM> cycloalkyl optionally substituted with one or more, identical or different, substituents R<NUM>, phenyl optionally substituted with one or more, identical or different, substituents R<NUM> and benzyl optionally substituted with one or more, identical or different, substituents R<NUM>;
- R<NUM> is selected from the group consisting of H, deuterium C<NUM>-<NUM> alkyl and C<NUM>-<NUM> cycloalkyl;
- R<NUM> is independently selected from the group consisting of deuterium, tritium, F, Cl, Br, I, CN, isocyanide, C<NUM>-<NUM> cycloalkyl optionally substituted with one or more, identical or different, substituents R<NUM>, O-C<NUM>-<NUM> alkyl optionally substituted with one or more, identical or different, substituents R<NUM>, S-C<NUM>-<NUM> alkyl optionally substituted with one or more, identical or different, substituents R<NUM>, CH<NUM>-O-C<NUM>-<NUM> alkyl optionally substituted with one or more, identical or different, substituents R<NUM> and CH<NUM>-S-C<NUM>-<NUM> alkyl optionally substituted with one or more, identical or different, substituents R<NUM>;
- R<NUM> is independently selected from the group consisting of deuterium and F;
- R<NUM> is independently selected from the group consisting of deuterium, methoxy, nitro, cyano, Cl, Br, I and F; and
- n is an integer <NUM>, <NUM>, <NUM> or <NUM>.