Patent Description:
<NUM>-(aminomethyl)-N-(<NUM>-fluoropyridin-<NUM>-yl)-<NUM>-{<NUM>-[(<NUM>-{[(<NUM>-oxooxolan-<NUM>-yl)methyl]sulfan yl}phenyl)carbamoyl]phenyl}benzamide (Compound A) is an effective Rho-associated protein kinase (ROCK) inhibitor useful in the treatment of fibrotic diseases (see <CIT>). In particular Compound A has been designed to work specifically at the site of fibrosis in the gastrointestinal (GI) tract and to degrade quickly, if absorbed into the bloodstream, through enzyme-mediated metabolism.

Crohn's disease is a chronic inflammatory bowel disease that affects <NUM> people globally and ><NUM>,<NUM> new cases are diagnosed each year. Up to <NUM>% of patients with Crohn's disease can develop significant fibrosis and stricture formation within ten years after diagnosis; this fibrosis associated with Crohn's disease is known as fibrostenotic Crohn's disease.

The current management of fibrotic strictures of the GI tract is primarily surgical as no drugs are specifically approved for fibrosis, which can progress despite intervention with anti-inflammatory therapies.

Relapse rate post surgical intervention is high with ><NUM>% patients requiring further surgery within <NUM> years, many within <NUM> months. Consequently, patients suffer progressive loss of GI function and repeated resections can lead to major health complications such as short bowel syndrome.

Preclinical data has shown that Compound A exhibits strong anti-fibrotic therapeutic effects in multiple animal models of inflammatory bowel disease.

The freebase of Compound A has low stability.

It is an aim of certain embodiments of this invention to provide a stable crystalline form of Compound A. It is an aim of certain embodiments of this invention to provide a crystalline form of Compound A that is more stable than other crystalline forms.

Certain embodiments of this invention satisfy some or all of the above aims.

In a first aspect of the invention is provided the crystalline salt for of the S-enantiomer (S-A) of compound A:
<CHM>
having an enantiomeric excess of <NUM>% or greater, wherein the salt is a hydrochloride salt of a single enantiomer of compound A, characterised in that said crystalline salt form has an XRPD pattern with at least two peaks at 2θ selected from <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>; optionally wherein the crystalline salt form is characterised in that said crystalline salt form has an XRPD pattern with peaks at 2θ <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>.

In a second aspect of the present invention is provided the crystalline salt form of the S-enantiomer (S-A) of the compound A
<CHM>
having an enantiomeric excess of <NUM>% or greater, wherein the salt is a hydrochloride salt of a single enantiomer of compound A, characterised in that said crystalline salt form has an XRPD pattern with at least two peaks at 2θ selected from <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>; optionally wherein the crystalline salt form is characterised in that said crystalline salt form has an XRPD pattern with peaks at 2θ <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>.

In a third aspect of the present invention is provided the crystalline salt form of the S-enantiomer (S-A) of the compound A
<CHM>
having an enantiomeric excess of <NUM>% or greater, wherein the salt is a succinate salt of a single enantiomer of compound A, characterised in that said crystalline salt form has an XRPD pattern with at least two peaks at 2θ selected from <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>; optionally wherein the crystalline salt form is characterised in that said crystalline salt form has an XRPD pattern with peaks at 2θ <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>.

The inventors have found that the S-enantiomer of compound A exhibits lower inhibition of a wide-variety of alternative targets than the R-enantiomer. In particular, the S-enantiomer of compound A exhibits lower inhibition of a wide-variety of kinases other than ROCK than the R-enantiomer. This is particularly surprising as to date the inventors have detected no statistically meaningful difference between the activities of the S-enantiomer and the R-enantiomer of compound A against kinases ROCK1 or ROCK2.

In addition, the S-enantiomer is less active than the R-enantiomer in a significant number of assays that are used to identify undesirable off target activity that may cause adverse effects in humans.

Where the disclosure relates to a pharmaceutically acceptable salt of Compound A, the salt will typically be an acid addition salt. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, <NUM>,<NUM>-naphthalenedisulfonate, <NUM>-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate and trifluoroacetate salts.

The salt may be selected from a hydrochloride salt and a succinate salt. The inventors have found that succinate and hydrochloride salts of compound A form crystals that are more stable than the freebase. The salt may be a succinate salt. The salt may be a hydrochloride salt. The inventors have found that crystalline forms of the hydrochloride salt of Compound A are particularly stable.

The freebase of Compound A has low chemical stability, particularly with respect to ester hydrolysis. The inventors have found that salt forms of Compound A exhibit improved stability relative to the freebase.

The disclosure relates to a pharmaceutically acceptable salt of Compound A. The salt will typically be an acid addition salt.

Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, <NUM>,<NUM>-naphthalenedisulfonate, <NUM>-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate and trifluoroacetate salts.

The crystalline salt form may be a solvate. The crystalline salt form may be a hydrate. The crystalline salt form may not be a solvate. The crystalline salt form may not be a hydrate.

Compound A contains a single chiral centre, at the point of attachment of the furanone ring to the rest of the molecule. In the crystal forms of the first aspect of the invention, Compound A may be a mixture of the two enantiomers. Compound A may be a racemic mixture of the two enantiomers. Compound A may be substantially in the form of a single enantiomer.

According to the invention, the single enantiomer is the S-enantiomer (S-A):
<CHM>.

In variants of the disclosure, the single enantiomer may be the R-enantiomer (R-A):
<CHM>.

The word 'substantially' may mean that Compound A has an enantiomeric excess of <NUM>% or greater. The word 'substantially' typically means that Compound A has an enantiomeric excess of <NUM>% or greater. It may mean that Compound A has an enantiomeric excess of <NUM>% or greater, <NUM>% or greater or <NUM>% or greater. Throughout this specification, where Compound A is described as being a single enantiomer (either R or S), it is intended to mean that it is in substantially enantiopure form within the meaning of this paragraph, unless an enantiomeric excess is otherwise specified.

The enantiomer may be the faster eluting enantiomer in chiral HPLC performed using a column that comprises silica coated in cellulose-tris(<NUM>,<NUM>-dimethylphenylcarbamate), e.g. a Chiralcel OD-<NUM> column. The enantiomer may be the slower eluting enantiomer in chiral HPLC performed using a column that comprises silica coated in cellulose-tris(<NUM>,<NUM>-dimethylphenylcarbamate), e.g. a Chiralcel OD-<NUM> column. The solvent system used as the mobile phase in the HPLC may be <NUM>% solvent mixture B in solvent mixture A, wherein solvent mixture A is heptane containing <NUM>% diethylamine; and solvent mixture B is a <NUM>:<NUM> mixture of isopropyl alcohol and acetonitrile, also containing <NUM>% diethylamine.

It may be that the salt is a hydrochloride salt of a single enantiomer of compound A, characterised in that said crystalline salt form has an XRPD pattern with at least two peaks at 2θ selected from <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>. It may be that the salt is a hydrochloride salt of a single enantiomer of compound A, characterised in that said crystalline salt form has an XRPD pattern with at least four peaks at 2θ selected from <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>. It may be that the salt is a hydrochloride salt of a single enantiomer of compound A, characterised in that said crystalline salt form has an XRPD pattern with peaks at 2θ selected from <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>. It may be that said crystalline form has an XRPD pattern substantially as shown in <FIG> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>. This crystal form is known in this specification as HCl Salt Form I. This crystal form is a trihydrate. According to the invention, the single enantiomer is the S enantiomer. In variants of the disclosure, the single enantiomer may be the R enantiomer.

It may be that the salt is a hydrochloride salt of a single enantiomer of compound A, characterised in that said crystalline salt form has an XRPD pattern with at least two peaks at 2θ selected from <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>. It may be that the salt is a hydrochloride salt of a single enantiomer of compound A, characterised in that said crystalline salt form has an XRPD pattern with at least four peaks at 2θ selected from <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>. It may be that the salt is a hydrochloride salt of a single enantiomer of compound A, characterised in that said crystalline salt form has an XRPD pattern with peaks at 2θ selected from <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>. It may be that said crystalline form has an XRPD pattern substantially as shown in <FIG> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>. This crystal form is known in this specification as HCl Salt Form II. This crystal form is an anhydrate. The single enantiomer may be the S enantiomer. The single enantiomer may be the R enantiomer.

It may be that the salt is a succinate salt of a single enantiomer of compound A, characterised in that said crystalline salt form has an XRPD pattern with at least two peaks at 2θ selected from <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>. It may be that the salt is a succinate salt of a single enantiomer of compound A, characterised in that said crystalline salt form has an XRPD pattern with at least four peaks at 2θ selected from <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>. It may be that the salt is a succinate salt of a single enantiomer of compound A, characterised in that said crystalline salt form has an XRPD pattern with peaks at 2θ selected from <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>. It may be that said crystalline form has an XRPD pattern substantially as shown in <FIG> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>. This crystal form is known in this specification as Succinate Salt Form I. The single enantiomer may be the S enantiomer. The single enantiomer may be the R enantiomer.

HCl Salt Form I, HCl Salt Form II and Succinate Salt Form I are all chemically stable crystalline materials and are more stable than the freebase of compound A. HCl Salt Form I and HCl Salt Form II are more stable than Succinate Salt Form I. HCl Salt Form II is particularly beneficial as it is more crystalline than HCl Salt Form I and also more soluble at low pH.

In a further aspect of the invention is provided a pharmaceutical formulation comprising the crystalline salt form of the first, second or third aspect.

Disclosed herein is the crystalline salt form of the first, second or third aspect or the compound A, or pharmaceutical salt thereof, for use in medical treatment.

In a further aspect of the invention is provided the crystalline salt form of the first, second or third aspect for use in treating a fibrotic disease.

Disclosed herein is a method of treating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the crystalline salt form of the first, second or third aspect or the compound A, or pharmaceutical salt thereof.

Disclosed herein is the use of the crystalline salt form of the first, second or third aspect or the compound A, or pharmaceutical salt thereof, in the manufacture of a medicament for treating a disease.

The disease may be a disease of the gastrointestinal system.

The disease may be an inflammatory bowel disease.

The disease may be Crohn's disease, e.g..

The disease may be a cancer, e.g. colorectal cancer.

The disease may be a skin disease, e.g. keloids or scleroderma.

Disclosed herein is a method of making HCl crystalline salt form II of a single enantiomer of Compound A, the method comprising:.

The solvent may be a mixture of solvents.

The solvent may be a polar aprotic organic solvent. The solvent may comprise a polar aprotic organic solvent. The solvent may be water. The solvent may comprise water. The solvent may be a polar protic organic solvent. The solvent may comprise a polar protic organic solvent.

The solvent may be a mixture of a polar aprotic organic solvent and a solvent selected from a polar protic organic solvent and water. The solvent may be a mixture of a polar aprotic organic solvent and water.

Suitable polar aprotic organic solvents include dimethylsulfoxide (DMSO), acetone, acetonitrile, ethers (e.g. THF, diglyme, ethylene glycol dimethyl ether, t-butylmethyl ether), esters (e.g. ethyl acetate).

Suitable polar protic organic solvents include ethanol, methanol, isopropanol, ethylene glycol.

The solvent may comprise acetone. The solvent is preferably a mixture of acetone and water. The inventors have found that use of a mixture of acetone and water provides a cleaner product than other possible solvent systems.

The proportion of polar aprotic organic solvent (e.g. acetone) to water may be in the range <NUM>:<NUM> to <NUM>:<NUM> acetone:water by volume. The proportion of polar aprotic organic solvent (e.g. acetone) to water may be in the range <NUM>:<NUM> to <NUM>:<NUM> acetone:water by volume. The proportion of polar aprotic organic solvent (e.g. acetone) to water may be in the range <NUM>:<NUM> to <NUM>:<NUM> acetone:water by volume.

According to the invention, the single enantiomer may be the S enantiomer. The single enantiomer may be the R enantiomer.

Step a) typically comprises stirring the mixture. The mixture may be stirred for greater than <NUM>. The mixture may be stirred for greater than <NUM>. The mixture may be stirred for greater than <NUM>. The mixture may be stirred for less than <NUM>. The mixture may be stirred for less than <NUM>.

Step a) will typically be performed at a temperature in the range <NUM> to <NUM>. Step a) will typically be performed at a temperature in the range <NUM> to <NUM>.

The process may further comprise: step c) drying the HCl salt form II, e.g. under vacuum. Step c) may be carried out at a temperature in the range <NUM> to <NUM>. Step c) may be carried out for more than <NUM>. Step c) may be carried out for less than <NUM>, e.g. less than <NUM>.

The disclosure relates to pharmaceutically acceptable salts of Compound A. For a review on suitable salts, see "<NPL>).

Pharmaceutically acceptable acid addition salts of Compound A may be prepared by one or more of two methods:.

Such reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised.

The term 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term 'hydrate' is employed when said solvent is water.

It is known in the art that an X-ray powder diffraction pattern may be obtained which has one or more measurement errors depending on measurement conditions (such as equipment, sample preparation or machine used). In particular, it is generally known that intensities in an X-ray powder diffraction pattern may fluctuate depending on measurement conditions and sample preparation. For example, persons skilled in the art of X-ray powder diffraction will realise that the relative intensities of peaks may vary according to the orientation of the sample under test and on the type and setting of the instrument used. The skilled person will also realise that the position of reflections can be affected by the precise height at which the sample sits in the diffractometer and the zero calibration of the diffractometer. The surface planarity of the sample may also have a small effect. Hence a person skilled in the art will appreciate that the diffraction pattern data presented herein is not to be construed as absolute and any crystalline form that provides a power diffraction pattern substantially identical to those disclosed herein fall within the scope of the present disclosure (for further information see<NPL>).

The term 'stable' may refer to chemical stability or physical stability. In particular freebase Compound A is chemically unstable whereas the crystal forms of the invention are chemically stable for up to <NUM> days at <NUM> and <NUM>% relative humidity and/or are chemically stable for up to <NUM> days at <NUM> and ambient humidity. Crystal forms of the invention may be chemically stable for up to <NUM> months at <NUM> and <NUM>% relative humidity and/or are chemically stable for up to <NUM> months at <NUM> and <NUM>% relative humidity.

For the above-mentioned compounds of the invention the dosage administered will, of course, vary with the compound employed, the mode of administration, the treatment desired and the disorder indicated. For example, if the compound of the invention is administered orally, then the daily dosage of the compound of the invention may be in the range from <NUM> micrograms per kilogram body weight (µg/kg) to <NUM> milligrams per kilogram body weight (mg/kg).

A crystalline salt form or compound of the invention may be used on their own but will generally be administered in the form of a pharmaceutical composition in which the crystalline salt form or compound, or pharmaceutically acceptable salt thereof, of the invention is in association with a pharmaceutically acceptable adjuvant, diluent or carrier. Conventional procedures for the selection and preparation of suitable pharmaceutical formulations are described in, for example, "<NPL>.

Depending on the mode of administration of the crystalline salt form or compound of the invention, the pharmaceutical composition which is used to administer the crystalline salt form or compound of the invention will preferably comprise from <NUM> to <NUM> %w (per cent by weight) crystalline salt form or compound of the invention, more preferably from <NUM> to <NUM> %w crystalline salt form or compound of the invention, still more preferably from <NUM> to <NUM> %w crystalline salt form or compound of the invention, and even more preferably from <NUM> to <NUM> % crystalline salt form or compound of the invention, all percentages by weight being based on total composition.

The pharmaceutical compositions may be administered topically (e.g. to the skin or eye) in the form, e.g., of creams, gels, lotions, solutions, suspensions, or systemically, e.g. by oral administration in the form of tablets, capsules, syrups, powders or granules; by rectal administration in the form of suppositories; or by inhalation in the form of an aerosol.

For oral administration the crystalline salt form or compound of the invention may be admixed with an adjuvant or a carrier, for example, lactose, saccharose, sorbitol, mannitol; a starch, for example, potato starch, corn starch or amylopectin; a cellulose derivative; a binder, for example, gelatine or polyvinylpyrrolidone; and/or a lubricant, for example, magnesium stearate, calcium stearate, polyethylene glycol, a wax, paraffin, and the like, and then compressed into tablets. If coated tablets are required, the cores, prepared as described above, may be coated with a concentrated sugar solution which may contain, for example, gum arabic, gelatine, talcum and titanium dioxide. Alternatively, the tablet may be coated with a suitable polymer dissolved in a readily volatile organic solvent.

For the preparation of soft gelatine capsules, the crystalline salt form or compound of the invention may be admixed with, for example, a vegetable oil or polyethylene glycol. Hard gelatine capsules may contain granules of the salt form using either the above-mentioned excipients for tablets. Also liquid or semisolid formulations of the crystalline salt form or compound of the invention may be filled into hard gelatine capsules. Liquid preparations for oral application may be in the form of syrups or suspensions, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally such liquid preparations may contain colouring agents, flavouring agents, sweetening agents (such as saccharine), preservative agents and/or carboxymethylcellulose as a thickening agent or other excipients known to those skilled in art.

The size of the dose for therapeutic purposes of crystalline salt form or compound of the invention will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well-known principles of medicine.

Dosage levels, dose frequency, and treatment durations of crystalline salt form or compound of the invention are expected to differ depending on the formulation and clinical indication, age, and co-morbid medical conditions of the patient.

The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

<CHM>
To a suspension of compound <NUM> (<NUM>, <NUM> mmol, <NUM> eq) in THF (tetrahydrofuran; <NUM>) was added TEA (triethylamine; <NUM>, <NUM> mmol, <NUM>, <NUM> eq) and compound <NUM> (<NUM>, <NUM> mol, <NUM> eq) at <NUM> under N<NUM>. The mixture was stirred at <NUM> under N<NUM> for <NUM> hrs. LCMS showed compound <NUM> was consumed and desired MS (RT = <NUM>) was detected. The reaction was filtered and the solid was washed with petroleum ether (<NUM>) to wash off the dark colour. The filter cake was concentrated to give racemic compound <NUM> (<NUM>, <NUM> mol, <NUM>% yield, <NUM>% purity) as a yellow solid.

<CHM>
To a solution of racemic compound <NUM> (<NUM>, <NUM> mmol, <NUM>% purity, <NUM> eq) in MeOH (<NUM>), THF (<NUM>) and DMA (dimethylacetamide; <NUM>) was Pd/C (<NUM>, <NUM> mmol, <NUM>% purity, <NUM> eq) at <NUM> under N<NUM>. The suspension was degassed and purged with H<NUM> (<NUM> Psi) for three times. The mixture was heated to <NUM> and stirred under H<NUM> (<NUM> Psi) at <NUM> for <NUM> hrs. TLC (Petroleum ether: Ethyl acetate = <NUM>: <NUM>) showed compound <NUM> (Rf = <NUM>) was consumed and one new spot (Rf =<NUM>) was detected. Three batches of the reaction was filtered and concentrated in vacuum to leave only DMA which would not come off on the rotary evaporator. This was quenched into H<NUM>O (<NUM>) and filtered, then the filter cake was concentrated to give compound <NUM> (<NUM>, <NUM> mol, <NUM>% yield, <NUM>% purity) as an off-white solid.

The two enantiomers of compound <NUM> were separated using super critical fluid chromatography (SFC) with a chiral column.

<CHM>
To a solution of compound <NUM> (<NPL>) (<NUM>, <NUM> mmol, <NUM>% purity, <NUM> eq) in DCM (<NUM>) was added compound R-<NUM> (<NUM>, <NUM> mmol, <NUM>% purity, <NUM> eq) and DMAP (dimethylaminopyridine; <NUM>, <NUM> mmol, <NUM> eq), TEA (<NUM>, <NUM> mmol, <NUM>, <NUM> eq) at <NUM> under N<NUM>. T<NUM>P (Propylphosphonic anhydride; <NUM>, <NUM> mmol, <NUM>, <NUM>% purity, <NUM> eq) was added to the mixture at <NUM> and the mixture was stirred at <NUM> for <NUM> hrs. LCMS showed compound <NUM> was consumed and desired MS (RT = <NUM>) was detected. The reaction was diluted with DCM (<NUM>) and washed with saturated NaHCO<NUM> solution (<NUM> * <NUM>). The organic layer was dried over Na<NUM>SO<NUM>, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO<NUM>, Petroleum ether: Ethyl acetate = <NUM>: <NUM> to <NUM>: <NUM>, Petroleum ether: Ethyl acetate = <NUM>: <NUM>, Rf = <NUM>) to give compound R-<NUM> (<NUM>, <NUM> mmol, <NUM>% yield, <NUM>% purity) as a light yellow solid.

<CHM>
To a solution of compound R-<NUM> (<NUM>, <NUM> mmol, <NUM>% purity, <NUM> eq) in DCM (dichloromethane; <NUM>) and dioxane (<NUM>) was added dropwise HCl/dioxane (<NUM>, <NUM>, <NUM> eq) at <NUM>. The mixture was stirred at <NUM> for <NUM> hrs. LCMS showed compound R-<NUM> was consumed and desired MS (RT = <NUM>) was detected. The reaction mixture was cooled to <NUM> and stirred at <NUM> for <NUM> hrs. The mixture was concentrated under reduced pressure to give a residue. The residue was diluted with deionized H<NUM>O (<NUM>) and adjusted pH to <NUM> with sat. NaHCO<NUM> solution (<NUM>), filtered and filter cake was concentrated. The crude product from both reactions was combined and triturated with deionized H<NUM>O (<NUM>) at <NUM> for <NUM> hrs, then filtered and filter cake was concentrated to give R-A HCl (<NUM>, <NUM> mmol, <NUM>% yield, <NUM>% purity, HCl) as a light yellow solid.

<CHM>
To a solution of compound <NUM> (<NPL>) (<NUM>, <NUM> mmol, <NUM>% purity, <NUM> eq) in DCM (<NUM>) was added compound S-<NUM> (<NUM>, <NUM> mmol, <NUM>% purity, <NUM> eq) and DMAP (<NUM>, <NUM> mmol, <NUM> eq), TEA (<NUM>, <NUM> mmol, <NUM>, <NUM> eq) at <NUM> under N<NUM>. T<NUM>P (<NUM>, <NUM> mmol, <NUM>, <NUM>% purity, <NUM> eq) was added the mixture at <NUM> and the mixture was stirred at <NUM> for <NUM> hrs. LCMS showed compound <NUM> was consumed and desired MS (RT = <NUM>) was detected. The reaction was diluted with DCM (<NUM>) and washed with saturated NaHCO<NUM> solution (<NUM> * <NUM>). The organic layer was dried over Na<NUM>SO<NUM>, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO<NUM>, Petroleum ether: Ethyl acetate = <NUM>: <NUM> to <NUM>: <NUM>, Petroleum ether: Ethyl acetate = <NUM>: <NUM>, Rf = <NUM>) to give compound S-<NUM> (<NUM>, <NUM> mmol, <NUM>% yield, <NUM>% purity) as a light yellow solid.

<CHM>
To a solution of compound S-<NUM> (<NUM>, <NUM> mmol, <NUM>% purity, <NUM> eq) in DCM (<NUM>) and dioxane (<NUM>) was added dropwise HCl/dioxane (<NUM>, <NUM>, <NUM> eq) at <NUM>. The mixture was stirred at <NUM> for <NUM> hrs. LCMS showed compound S-<NUM> was consumed and desired MS (RT = <NUM>) was detected. Two batches of the reaction mixture was cooled to <NUM> and stirred at <NUM> for <NUM> hrs. The mixture was concentrated under reduced pressure to give a residue. The residue was diluted with deionized H<NUM>O (<NUM>) and adjusted pH to <NUM> with sat. NaHCO<NUM> solution (<NUM>), filtered and filter cake was concentrated. The crude product from both reactions was combined and triturated with deionized H<NUM>O (<NUM>) at <NUM> for <NUM> hrs, then filtered and filter cake was concentrated to give S-A HCl (<NUM>, <NUM> mmol, <NUM>% yield, <NUM>% purity, HCl) as a light yellow solid.

This method directly forms S-A HCl Salt Form I. XRPD was carried out on S-A HCl Salt Form I using the following conditions:.

XRPD diffractograms were collected with an X-ray diffractometer. The sample was prepared on a zero-background silicon wafer by gently pressing onto the flat surface. The parameters of XRPD diffraction are given below.

Formation of S-A Freebase Form I: Around <NUM> of S-A HCl Salt Form I was suspended in <NUM> of acetonitrile at room temperature (<NUM>). A thick suspension was observed after stirring for <NUM> minutes, then <NUM> eq. of NaHCO<NUM> aqueous solution (<NUM> of NaHCO<NUM> was dissolved in <NUM> of water) was added. After stirring for <NUM> minutes, a suspension was observed. And <NUM> of acetonitrile and <NUM> of water was added, then a solid mixed with oil was precipitated after stirring for <NUM> minutes. Finally, a suspension was obtained after stirring for <NUM> at RT. The solid was collected by filtration and dried at <NUM> under vacuum for ~<NUM> to give S-A Freebase Form I.

Formation of S-A Succinate Form I: About <NUM> of S-A Freebase Form I and <NUM> eq. of succinic acid (<NUM>) were added into <NUM> of MEK (methylethyl ketone) at room temperature (~<NUM>). The mixture was stirred at room temperature for <NUM>, and then solid was collected by filtration and washed by MEK, and dried under vacuum condition at <NUM> for <NUM> to provide S-A Succinate Salt Form I.

XRPD was carried out on S-A Succinate Salt Form I. Conditions were the same as those described in Example <NUM> for the XRPD analysis of HCl Salt form I.

About <NUM> of S-A HCl Salt Form I was suspended in <NUM> of acetone/water (<NUM>/<NUM>, v/v) at room temperature, and stirred for about <NUM>. Solids were collected by filtration, dried under vacuum at <NUM> for ~<NUM>. Around <NUM> of HCl Salt Form II was prepared with yield of <NUM>%.

XRPD was carried out on S-A HCl Salt Form II. Conditions were the same as those described in Example <NUM> for the XRPD analysis of HCl Salt form I.

About <NUM> of freebase Form I, HCl salt Form II, Succinate Form I and received HCl salt Form I were placed at <NUM>/capped and <NUM>/<NUM>% RH (open), respectively. Samples were prepared in duplicate for each condition. At day <NUM> and day <NUM>, the samples were analyzed by HPLC and XRPD to check the purity and crystal form, respectively.

The results are provided in the following table.

All three salt forms are more stable than freebase compound A. The two HCl salt forms are more stable than the succinate salt form.

The longer term stability on HCl salt form II was also tested under the following conditions:.

Samples were prepared in duplicate for each condition. At day <NUM> and at <NUM> month, and <NUM> months, the samples were analyzed by HPLC and XRPD to check the purity and crystal form, respectively.

The results are provided in the following tables.

As can be seen, HCl salt form II is stable for up to <NUM> months even under accelerated conditions.

About <NUM> of sample was weighed into each sample vial and then <NUM> of water, simulated gastric fluid (SGF), Fasted State Simulated Intestinal Fluid (FaSSIF) or Fed State Simulated Intestinal Fluid (FeSSIF) media was added. Samples were prepared in triplicate for each media. The suspensions were kept shaking at <NUM> for up to <NUM>. At <NUM>, <NUM> and <NUM>, suspensions were filtered and the filtrate was analyzed by HPLC.

HCl Salt Form II and Succinate Salt Form I were more soluble in SGF than HCl Salt Form I.

The HCl salts of S enantiomer and the R enantiomer of compound A were tested against a panel of kinases.

Compounds were received as powder and resuspended to <NUM> DMSO stock. Compounds were tested in <NUM>-dose IC50 mode with a <NUM>-fold serial dilution starting at <NUM>. Control compound, staurosporine, was tested in <NUM>-dose IC50 mode with <NUM>-fold serial dilution starting at <NUM>. Reactions were carried out at Km (Michaelis constant) ATP concentration (as indicated in the table).

Reaction Conditions: Buffer Conditions: <NUM> HEPES ((<NUM>-(<NUM>-hydroxyethyl)-<NUM>-piperazineethanesulfonic acid; pH <NUM>), <NUM> MgCl<NUM>, <NUM> EGTA (Ethyleneglycol- bis(β-aminoethyl)-N,N,N',N'-tetraacetic Acid), <NUM>% Brij35, <NUM>/ml BSA (Bovine serum albumin), <NUM> Na<NUM>VO<NUM>, <NUM> DTT (Dithiothreitol), <NUM>% DMSO. Reaction Procedure: <NUM>. Prepare indicated substrate in freshly prepared reaction buffer. Deliver required cofactors to the substrate solution above. Deliver indicated kinase into the substrate solution and mix gently. Deliver compounds in DMSO into the kinase reaction mixture utilizing acoustic technology (Echo550). Deliver <NUM>P-ATP (specific activity <NUM>µCi/µl final) into the reaction mixture to initiate the reaction. Incubate kinase reaction for <NUM> minutes at room temperature. Reactions are spotted onto P81 ion exchange paper (Whatman # <NUM>-<NUM>). Wash filters extensively in <NUM>% phosphoric acid. Measure the radioactive phosphorylated substrate remaining on the filter paper. Data Analysis: Kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions. IC50 values and curve fits were obtained using Prism4 Software (GraphPad). The results are provided in the following table.

The S-enantiomer of compound A is less active than the R-enantiomer against six out of the seven off-target kinases. The R-enantiomer of compound A is less active than the S-enantiomer against just one out of the seven off-target kinases. This is particularly surprising as to date the inventors have detected no statistically meaningful difference between the activities of the S-enantiomer and the R-enantiomer of compound A against kinases ROCK1 or ROCK2.

The use of in vitro pharmacological screening against a diverse range of targets (receptors, ion channels, enzymes and transporters) is used to identify undesirable off-target activity that may cause adverse drug reactions in humans. Screening panels (e.g. SafetyScreen44™ Panel, Eurofins) comprise a selection of off-targets linked with known issues in humans that could hinder or halt the development of drug candidates or market withdrawal if discovered after a drug is approved. The HCl salts of the S enantiomer and the R enantiomer of compound A were tested against a panel of such targets.

Typical adverse drug reactions associated with these off-targets has been summarised (<NPL>).

Antagonism: increase in inflammation; decrease in bone mass.

The tests were carried out according to the methods described in the following tables and indicated literature references.

The S-enantiomer of compound A is less active than the R-enantiomer in seven out of the thirteen assays. The R-enantiomer of compound A is less active than the S-enantiomer in just one of the thirteen assays.

Claim 1:
The crystalline salt form of the S-enantiomer (S-A) of the compound A:
<CHM>
having an enantiomeric excess of <NUM>% or greater, wherein the salt is a hydrochloride salt of a single enantiomer of compound A, characterised in that said crystalline salt form has an XRPD pattern with at least two peaks at 2θ selected from <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>; optionally wherein the crystalline salt form is characterised in that said crystalline salt form has an XRPD pattern with peaks at 2θ <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM>, <NUM>± <NUM> and <NUM>± <NUM> when measured using Cu radiation with a Kα2 / Kα1 ratio of <NUM>.