Solid forms of rock inhibitor

Abstract

To provide a single enantiomer and crystalline forms of a Rho-associated protein kinase (ROCK) inhibitor, useful in the treatment of fibrotic diseases, e.g., fibrostenotic Crohn's disease.SOLUTION: The invention provides the S-enantiomer (S-A) of compound A represented by the structural formula in the figure in a substantially enantiopure form, or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a single enantiomer and crystalline form of 4-(aminomethyl)-N-(3-fluoropyridin-4-yl)-3-{3-[(4-{[(2-oxooxolan-3-yl)methyl]sulfanyl}phenyl)carbamoyl]phenyl}benzamide (Compound A). Compound A is a Rho-associated protein kinase (ROCK) inhibitor that is useful for the treatment of fibrosis, such as fibrotic stricture diseases.

Background Art

[0002] 4-(Aminomethyl)-N-(3-fluoropyridin-4-yl)-3-{3-[(4-{[(2-oxooxolan-3-yl)methyl]sulfanyl}phenyl)carbamoyl]phenyl}benzamide (Compound A) is an effective Rho-associated protein kinase (ROCK) inhibitor that is useful in the treatment of fibrosis (see WO2014 / 118133). In particular, Compound A is designed to act specifically on fibrotic sites in the gastrointestinal (GI) tract and to be rapidly degraded when absorbed into the bloodstream by enzyme-mediated metabolism.

Chemical Formula

[0003] Crohn's disease is a chronic inflammatory bowel disease affecting 1.5 million people worldwide, with more than 70,000 new cases diagnosed each year. Up to 50% of patients with Crohn's disease can develop significant fibrosis and stricture formation within 10 years of diagnosis; this fibrosis associated with Crohn's disease is known as fibrotic stricture disease.

[0004] Since there are no drugs specifically approved for fibrosis, which can progress despite anti-inflammatory therapy interventions, the current management of fibrotic strictures in the GI tract is mainly by surgical procedures.

[0005] The recurrence rate after surgical intervention is high, and more than 50% of patients require further surgery within 10 years, often within 12 months. As a result, patients suffer from a progressive loss of GI function and repeated resections can lead to major health complications such as short bowel syndrome.

[0006] Preclinical data indicate that Compound A exhibits a potent anti-fibrotic therapeutic effect in multiple animal experiments of inflammatory bowel disease.

[0007] The free base form of Compound A has low stability.

[0008] It is an object of certain embodiments of the present invention to provide a stable crystalline form of Compound A. It is an object of certain embodiments of the present invention to provide a crystalline form of Compound A that is more stable than other crystalline forms.

[0009] Certain embodiments of the present invention meet some or all of the above-described objects.

Summary of the Invention

[0010] In a first aspect of the present invention, the S-enantiomer of Compound A (S-A):

Chemical formula

[0011] The inventors have found that the S-enantiomer of compound A exhibits lower inhibition at a greater variety of alternative targets than the R-enantiomer. In particular, the S-enantiomer of compound A exhibits lower inhibition at a greater variety of kinases other than ROCK than the R-enantiomer. This is particularly surprising since to date the inventors have not detected a statistically significant difference between the activities of the S-enantiomer and the R-enantiomer of compound A against the kinase ROCK1 or ROCK2.

[0012] In addition, the S-enantiomer is also less active than the R-enantiomer in a significant number of assays used to identify unwanted off-target activities that may cause side effects in humans.

[0013] When the present invention 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 that form non-toxic salts. Examples include 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, 1,5-naphthalenedisulfonate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, saccharinate, stearate, succinate, tartrate, tosylate, and trifluoroacetate.

[0014] The salt can be selected from hydrochloride and succinate. The inventors have found that the succinate and hydrochloride of Compound A form more stable crystals than the free base. The salt may be succinate. The salt may be hydrochloride. The inventors have found that the crystalline form of the hydrochloride of Compound A is particularly stable.

[0015] In a second aspect of the invention, Compound A:

Chemical formula

[0016] The free base of Compound A has low chemical stability, especially stability against ester hydrolysis. The inventors have found that the salt form of Compound A shows improved stability compared to the free base.

[0017] The present invention relates to a pharmaceutically acceptable salt of Compound A. The salt will typically be an acid addition salt.

[0018] Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include 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, 1,5-naphthalenedisulfonate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / monohydrogenphosphate / dihydrogenphosphate, saccharinate, stearate, succinate, tartrate, tosylate, and trifluoroacetate.

[0019] The salt can be selected from hydrochloride and succinate. The inventors have found that the succinate and hydrochloride of Compound A form more stable crystals than the free base. The salt may be succinate. The salt may be hydrochloride. The inventors have found that the crystal form of the hydrochloride of Compound A is particularly stable.

[0020] The form of the crystalline salt may be a solvate. The form of the crystalline salt may be a hydrate. The form of the crystalline salt may not be a solvate. The form of the crystalline salt may not be a hydrate.

[0021] Compound A contains a single chiral center at the point where the furanone ring is attached to the rest of the molecule. In the crystal form of the first aspect of the present invention, Compound A may be a mixture of two enantiomers. Compound A may be a racemic mixture of two enantiomers. Compound A may be substantially in the form of a single enantiomer.

[0022] The single enantiomer is the S-enantiomer (S-A):

Chem.

[0023] The single enantiomer is the R-enantiomer (R-A):

Chem.

[0024] The term "substantially" may mean that Compound A has an enantiomeric excess of 90% or more. The term "substantially" typically means that Compound A has an enantiomeric excess of 95% or more. It may mean that Compound A has an enantiomeric excess of 98% or more, 99% or more, or 99.5% or more. Throughout this specification, when Compound A is described as being a single enantiomer (either R or S), it shall be taken to mean that it is in a substantially enantiopure form within the meaning of this paragraph unless the enantiomeric excess is otherwise specified.

[0025] The enantiomer may be the enantiomer that elutes faster in chiral HPLC carried out using a column coated with silica tris(3,5-dimethylphenylcarbamate), for example, a Chiralcel OD-3 column. The enantiomer may be the enantiomer that elutes slower in chiral HPLC carried out using a column coated with silica tris(3,5-dimethylphenylcarbamate), for example, a Chiralcel OD-3 column. The solvent system used as the mobile phase for HPLC may be 28% solvent mixture B in solvent mixture A, where solvent mixture A is heptane containing 0.05% diethylamine; solvent mixture B is a 4:1 mixture of isopropyl alcohol and acetonitrile, which also contains 0.05% diethylamine.

[0026] The salt is a hydrochloride salt of a single enantiomer of Compound A, and the crystalline salt form is characterized by having an XRPD pattern with at least two peaks at 2θ selected from 16.2 ± 0.2, 22.7 ± 0.2, 23.3 ± 0.2, 24.0 ± 0.2, 24.9 ± 0.2, and 25.4 ± 0.2 when measured using Cu radiation with a K α2 / K α1 ratio of 0.5. The salt is a hydrochloride salt of a single enantiomer of Compound A, and the crystalline salt form is characterized by having an XRPD pattern with at least two peaks at 2θ selected from 16.2 ± 0.2, 22.7 ± 0.2, 23.3 ± 0.2, 24.0 ± 0.2, 24.9 ± 0.2, and 25.4 ± 0.2 when measured using Cu radiation with a K α2 / K α1When measured using Cu radiation with a ratio of α2 / α1 of 0.5, it may be characterized by having an XRPD pattern with at least 4 peaks at 2θ selected from 16.2 ± 0.2, 22.7 ± 0.2, 23.3 ± 0.2, 24.0 ± 0.2, 24.9 ± 0.2, and 25.4 ± 0.2. The salt is the hydrochloride salt of a single enantiomer of Compound A, and the form of the crystalline salt is When measured using Cu radiation with a ratio of α2 / α1 of 0.5, it may be characterized by having an XRPD pattern with peaks at 2θ selected from 16.2 ± 0.2, 22.7 ± 0.2, 23.3 ± 0.2, 24.0 ± 0.2, 24.9 ± 0.2, and 25.4 ± 0.2. The crystalline form is When measured using Cu radiation with a ratio of

[0027] of 0.5, it may have an XRPD pattern substantially as shown in Figure 1. This crystalline form is known herein as Form I of the HCl salt. The single enantiomer may be the S-enantiomer. The single enantiomer may be the R-enantiomer. The salt is the hydrochloride salt of a single enantiomer of Compound A, and the form of the crystalline salt is α2 / α1 When measured using Cu radiation with a ratio of When measured using Cu radiation with a ratio of α2 / α1 of 0.5, it may be characterized by having an XRPD pattern with at least 2 peaks at 2θ selected from 13.6 ± 0.2, 14.4 ± 0.2, 14.5 ± 0.2, 16.2 ± 0.2, and 16.5 ± 0.2. The salt is the hydrochloride salt of a single enantiomer of Compound A, and the form of the crystalline salt is When measured using Cu radiation with a ratio of α2 ​​​​​ / K α1 When measured using Cu radiation with a ratio of / K being 0.5, it may be characterized by having an XRPD pattern with peaks at 2θ selected from 13.6 ± 0.2, 14.4 ± 0.2, 14.5 ± 0.2, 16.2 ± 0.2, and 16.5 ± 0.2. The crystal form is K α2 / K α1 When measured using Cu radiation with a ratio of / K being 0.5, it may have an XRPD pattern substantially as shown in Figure 3. This crystal form is known herein as Form II of the hydrochloride salt. The single enantiomer may be the S-enantiomer. The single enantiomer may be the R-enantiomer.

[0028] The salt is the succinate salt of a single enantiomer of Compound A, and the crystal salt form is K α2 / K α1 When measured using Cu radiation with a ratio of / K being 0.5, it may be characterized by having an XRPD pattern with at least two peaks at 2θ selected from 18.2 ± 0.2, 18.6 ± 0.2, 19.1 ± 0.2, 21.4 ± 0.2, 23.0 ± 0.2, 24.1 ± 0.2, and 25.8 ± 0.2. The salt is the succinate salt of a single enantiomer of Compound A, and the crystal salt form is K α2 / K α1 When measured using Cu radiation with a ratio of / K being 0.5, it may be characterized by having an XRPD pattern with at least four peaks at 2θ selected from 18.2 ± 0.2, 18.6 ± 0.2, 19.1 ± 0.2, 21.4 ± 0.2, 23.0 ± 0.2, 24.1 ± 0.2, and 25.8 ± 0.2. The salt is the succinate salt of a single enantiomer of Compound A, and the crystal salt form is K α2 / K α1It may be characterized by having an XRPD pattern having peaks at 2θ selected from 18.2 ± 0.2, 18.6 ± 0.2, 19.1 ± 0.2, 21.4 ± 0.2, 23.0 ± 0.2, 24.1 ± 0.2 and 25.8 ± 0.2 when measured using Cu radiation with a ratio of K α2 / K α1 It may have an XRPD pattern substantially as shown in Figure 2 when measured using Cu radiation with a ratio of 0.5. This crystal form is known herein as Form I of the succinate. The single enantiomer may be the S - enantiomer. The single enantiomer may be the R - enantiomer.

[0029] Form I of the HCl salt, Form II of the HCl salt and Form I of the succinate are all chemically stable crystalline materials and are more stable than the free base of Compound A. Form I of the HCl salt and Form II of the HCl salt are more stable than Form I of the succinate. Form II of the HCl salt is particularly beneficial because it is more crystalline than Form I of the HCl salt and is more soluble at low pH.

[0030] In a third aspect of the invention, there is provided a pharmaceutical formulation comprising the crystalline salt form of the first aspect, or Compound A of the second aspect or a pharmaceutically acceptable salt thereof.

[0031] In a fourth aspect of the invention, there is provided the crystalline salt form of the first aspect, or Compound A of the second aspect or a pharmaceutically acceptable salt thereof for use in a medical treatment.

[0032] In a fifth aspect of the invention, there is provided the crystalline salt form of the first aspect, or Compound A of the second aspect or a pharmaceutically acceptable salt thereof for use in the treatment of a disease.

[0033] In a sixth aspect of the invention, there is provided a method for 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 aspect, or Compound A of the second aspect or a pharmaceutically acceptable salt thereof.

[0034] In a seventh aspect of the present invention, there is provided the use of the crystalline salt form of the first aspect, or compound A of the second aspect or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating a disease.

[0035] The disease may be a fibrotic disease.

[0036] The disease may be a gastrointestinal disease.

[0037] The disease may be an inflammatory bowel disease.

[0038] The disease may be Crohn's disease, for example, a fibrotic stricturing disease.

[0039] The disease may be cancer, for example, colorectal cancer.

[0040] The disease may be a skin disease, for example, keloid or scleroderma.

[0041] The disease may be an eye disease.

[0042] In an eighth aspect of the present invention, there is provided a method for producing form II of the HCl crystalline salt of a single enantiomer of compound A, comprising: a) suspending a sample of form I of the HCl crystalline salt of a single enantiomer of compound A in a solvent to form a suspension; b) filtering the suspension to obtain form II of the HCl crystalline salt. A method is provided.

[0043] The solvent may be a mixture of solvents.

[0044] The solvent may be a polar aprotic organic solvent. The solvent may contain a polar aprotic organic solvent. The solvent may be water. The solvent may contain water. The solvent may be a polar protic organic solvent. The solvent may contain a polar protic organic solvent.

[0045] The solvent may be a mixture of an aprotic polar organic solvent and a solvent selected from a protic polar organic solvent and water. The solvent may be a mixture of an aprotic polar organic solvent and water.

[0046] Suitable aprotic polar organic solvents include dimethyl sulfoxide (DMSO), acetone, acetonitrile, ethers (e.g., THF, diglyme, ethylene glycol dimethyl ether, t-butyl-methyl ether), esters (e.g., ethyl acetate).

[0047] Suitable protic polar organic solvents include ethanol, methanol, isopropanol, ethylene glycol.

[0048] The solvent may contain acetone. Preferably, the solvent is a mixture of acetone and water. The inventors have found that by using a mixture of acetone and water, a cleaner product can be obtained than with other possible solvent systems.

[0049] The ratio of the aprotic polar organic solvent (e.g., acetone) to water may be in the range of 1:1 to 50:1 acetone:water by volume. The ratio of the aprotic polar organic solvent (e.g., acetone) to water may be in the range of 10:1 to 30:1 acetone:water by volume. The ratio of the aprotic polar organic solvent (e.g., acetone) to water may be in the range of 15:1 to 25:1 acetone:water by volume.

[0050] The single enantiomer may be the S-enantiomer. The single enantiomer may be the R-enantiomer.

[0051] Step a) typically includes stirring the mixture. The mixture may be stirred for longer than 1 hour. The mixture may be stirred for longer than 5 hours. The mixture may be stirred for longer than 12 hours. The mixture may be stirred for less than 48 hours. The mixture may be stirred for less than 24 hours.

[0052] Step a) will typically be carried out at a temperature in the range of 5°C to 40°C. Step a) will typically be carried out at a temperature in the range of 15°C to 30°C.

[0053] The manufacturing method may further include: Step c) drying the form II of the HCl salt, for example, under vacuum. Step c) may be carried out at a temperature in the range of 30°C to 60°C. Step c) may be carried out for longer than 30 minutes. Step c) may be carried out over less than 6 hours, for example, less than 3 hours.

Brief Description of the Drawings

[0054] Embodiments of the present invention will be further described below with reference to the accompanying drawings:

[0055]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0056] The present invention relates to a pharmaceutically acceptable salt of compound A. For a general review of suitable salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, Weinheim, Germany, 2002).

[0057] The pharmaceutically acceptable acid addition salts of compound A are by one or more of the following two methods: Reacting compound A with the desired acid; By reacting one salt of compound A with a suitable acid or base or converting it to another salt by means of a suitable ion exchange column It can be produced by

[0058] Such reactions are typically carried out in solution. The resulting salt may be precipitated and collected by filtration, or the solvent may be evaporated and recovered. The degree of ionization of the resulting salt may vary from fully ionized to hardly ionized.

[0059] The term "solvate" is used herein to describe a molecular complex comprising a compound of the invention and one or more pharmaceutically acceptable solvent molecules, such as ethanol. The term "hydrate" is utilized when the solvent is water.

[0060] It is known in the art that X-ray powder diffraction patterns can be obtained with one or more measurement errors depending on the measurement conditions (such as the apparatus used, sample preparation or machinery, etc.). In particular, it is well known that the intensity of X-ray powder diffraction patterns can vary depending on the measurement conditions and sample preparation. For example, one skilled in the art in the field of X-ray powder diffraction patterns will understand that the relative intensity of the peaks can vary depending on the orientation of the sample and the type and settings of the apparatus used. One skilled in the art will also understand that the exact height at which the sample is placed on the diffractometer and the zero calibration of the diffractometer can affect the position of the reflections. The flatness of the surface of the sample may also have a significant impact. Thus, one skilled in the art will understand that the diffraction pattern data presented herein should not be construed as absolute, and that any crystalline form that provides a pattern substantially identical to the powder diffraction pattern disclosed herein is within the scope of this disclosure (for further information, see Jenkins, R & Snyder, R.L., "Introduction to X-Ray Powder Diffractometry", John Wiley & Sons, 1996).

[0061] The term "stable" may refer to chemical stability or physical stability. In particular, while the free base compound A is chemically unstable, the crystalline form of the present invention is chemically stable up to 7 days at 40 °C and 75% relative humidity, and / or chemically stable up to 7 days at 60 °C and ambient humidity. The crystalline form of the present invention may be chemically stable up to 3 months at 40 °C and 75% relative humidity, and / or chemically stable up to 3 months at 25 °C and 60% relative humidity.

[0062] For the above-described compounds of the present invention, the dosage administered will, of course, vary depending on the compound utilized, the mode of administration, the desired treatment, and the disorder to which it is applied. For example, when the compound of the present invention is administered orally, the daily dosage of the compound of the present invention may then range from 0.01 micrograms (μg / kg) per kilogram of body weight to 100 milligrams (mg / kg) per kilogram of body weight.

[0063] The crystalline salt form or compound of the present invention may be used by itself, but generally, the crystalline salt form or compound of the present invention, or a pharmaceutically acceptable salt thereof, will be administered in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable adjuvant, diluent or carrier. Conventional procedures for selecting and manufacturing suitable pharmaceutical formulations are described, for example, in "Pharmaceuticals - The Science of Dosage Form Designs", M.E. Aulton, Churchill Livingstone, 1988.

[0064] Depending on the form of the crystalline salt of the present invention or the mode of administration of the compound, the pharmaceutical composition used to administer the crystalline salt of the present invention or the compound preferably contains 0.05 to 99% w (weight percent) of the crystalline salt of the present invention or the compound, more preferably 0.05 to 80% w of the crystalline salt of the present invention or the compound, still more preferably 0.10 to 70% w of the crystalline salt of the present invention or the compound, and even more preferably 0.10 to 50% w of the crystalline salt of the present invention or the compound, where all weight percents will be based on the total amount of the composition.

[0065] The pharmaceutical composition may be administered topically (e.g., to the skin or eye) in the form of, for example, a cream, gel, lotion, solution, suspension, or may be administered orally in the form of, for example, tablets, capsules, syrups, powders or granules; rectally in the form of suppositories, or systemically by inhalation in the form of an aerosol.

[0066] In the case of oral administration, the crystalline salt form or compound of the present invention may be admixed with adjuvants or carriers such as lactose, saccharose, sorbitol, mannitol; starches such as potato starch, corn starch or amylopectin; cellulose derivatives; binders such as gelatin or polyvinylpyrrolidone; and / or lubricants such as magnesium stearate, calcium stearate, polyethylene glycol, wax, paraffin, etc., and then tableted. If tablet coating is required, the core produced above may be coated with a sugar concentrate that may contain, for example, gum arabic, gelatin, talc and titanium dioxide. Alternatively, the tablets may be coated with a suitable polymer dissolved in a highly volatile organic solvent.

[0067] When manufacturing soft gelatin capsules, the crystalline salt form or compound of the present invention may be admixed with, for example, vegetable oil or polyethylene glycol. Hard gelatin capsules may contain granules in the form of a salt using any of the excipients for tablets described above. Also, liquid or semi-solid formulations of the crystalline salt form or compound of the present invention may be filled into hard gelatin capsules. Liquid formulations for oral application may be in the form of syrups or suspensions, the remainder being a mixture of sugar and ethanol, water, glycerol and polyethylene glycol. Such liquid formulations may optionally contain colorants, flavors, sweeteners (such as saccharin), preservatives and / or carboxymethyl cellulose as a thickening agent, or other excipients known to those skilled in the art.

[0068] The size of the dose for therapeutic purposes of the crystalline salt form or compound of the present invention will, of course, vary according to the nature and severity of the symptoms, the age and sex of the animal or patient, and the route of administration, in accordance with well-known principles of medicine.

[0069] The dosage levels, dosing frequencies, and treatment durations of the crystalline salt form or compound of the present invention are thought to vary according to the formulation and the patient's clinical indications, age, and co-existing medical conditions.

[0070] Throughout the description of this specification and the claims, the words "comprising" and "containing" and their variants mean "including but not limited to", and they do not exclude (and do not exclude) other parts, additives, components, integers or steps. Throughout the description of this specification and the claims, unless otherwise specified in the context, the singular form includes the plural form. In particular, when an indefinite article is used, this specification should be understood to intend the plural form as well as the singular form, unless otherwise specified in the context.

[0071] Features, integers, characteristics, compounds, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are to be understood as applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All features disclosed in this specification (including any appended claims, abstract and drawings) and / or all of the steps of any methods or processes so disclosed may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any appended claims, abstract and drawings) or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0072] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification and which are open to public inspection with this specification, and the contents of all such papers and documents are hereby incorporated herein by reference.

[0073] Examples Example 1 - Formation of racemic compound 4 Preparation of compound 3

Chemical formula

[0074] LCMS: Product: RT = 0.592 min, m / z = 254.0 (M + H) + 1 H NMR: 400 MHz, DMSO δ / ppm: 8.15 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H), 4.35 - 4.30 (m, 1H), 4.20 - 4.15 (m, 1H), 3.54 (dd, J = 4.8, 4.4 Hz, 1H), 3.31 - 3.26 (m, 1H), 3.12 - 3.05 (m, 1H), 2.39 - 2.34 (m, 1H), 2.11 - 2.00 (m, 1H)

[0075] Preparation of compound 4 -

Chemical formula

[0076] LCMS: Product: RT = 0.260 minutes, m / z = 224.1 (M+H) + HPLC: Product: RT = 0.585 minutes, purity 99.5% under 220 nm SFC: (racemate), Product: RT = 0.926 minutes, Product: Rt = 1.507 minutes 1 1H NMR: 400 MHz, DMSO δ / ppm: 7.13 (d, J = 8.8 Hz, 2H), 6.53 (d, J = 8.4 Hz, 2H), 5.29 (s, 2H), 4.31 - 4.26 (m, 1H), 4.17 - 4.11 (m, 1H), 3.08 (dd, J = 3.6, 3.6 Hz, 1H), 2.83 - 2.77 (m, 1H), 2.76 - 2.68 (m, 1H), 2.38 - 2.30 (m, 1H), 2.06 - 1.96 (m, 1H)

[0077] Example 2 - Separation of Enantiomers of Compound 4 The two enantiomers of Compound 4 were separated by supercritical fluid chromatography (SFC) using a chiral column.

Chemical formula

[0078] SFC Column Conditions: Isocratic

Table 1

[0079] Compound 4 (332 g, 1.48 mol, purity 99.5%) was purified by preparative SFC (column: DAICEL CHIRALPAK AD (250 mm * 50 mm, 10 μm); mobile phase: [Neu-MeOH]; B%: 50% - 50%, 5.6; for 2880 minutes), and R-4 (150 g, 666 mmol, yield 45.0%, purity 99.1%) was obtained as a yellow solid, and S-4 (162 g, crude) was obtained as a yellow solid. The crude S-4 was triturated with petroleum ether:ethyl acetate = 3:1 (1.62 L) at 25 °C for 2 hours, and S-4 (132 g, 591 mmol, yield 40.0%, purity 99.8%) was obtained as a bright yellow solid.

[0080] R-4 LCMS: Product: RT = 0.663 minutes, m / z = 224.0 (M + H) + HPLC: Product: RT = 1.419 minutes, purity 99.1% under 220 nm SFC: (Homochiral), Product: RT = 0.912 minutes, ee%: 99.4% 1 1H NMR: 400 MHz, DMSO δ / ppm: 7.13 (d, J = 8.8 Hz, 2H), 6.53 (d, J = 8.4 Hz, 2H), 5.29 (s, 2H), 4.31 - 4.26 (m, 1H), 4.17 - 4.11 (m, 1H), 3.08 (dd, J = 3.6, 3.6 Hz, 1H), 2.83 - 2.77 (m, 1H), 2.76 - 2.68 (m, 1H), 2.38 - 2.30 (m, 1H), 2.06 - 1.96 (m, 1H)

[0081] S-4 LCMS: Product: RT = 0.656 minutes, m / z = 224.0 (M + H) + HPLC: Product: RT = 1.358 minutes, purity 99.8% under 220 nm SFC: (Homochiral), Product: RT = 1.491 minutes, ee%: 99.7% 11H NMR: 400 MHz, DMSO δ / ppm: 7.13 (d, J = 8.4 Hz, 2H), 6.53 (d, J = 8.8 Hz, 2H), 5.29 (s, 2H), 4.31 - 4.26 (m, 1H), 4.17 - 4.11 (m, 1H), 3.08 (dd, J = 3.6, 3.6 Hz, 1H), 2.83 - 2.77 (m, 1H), 2.75 - 2.68 (m, 1H), 2.38 - 2.30 (m, 1H), 2.06 - 1.96 (m, 1H)

[0082] Example 3 - Conversion of R-4 to R-A Preparation of Compound R-6 [Chemical Structure] To a solution of Compound 5 (Boland et al., Bioorg. Med. Chem. Lett. 2013, 6442 - 6446) (141 g, 294 mmol, purity 97.4%, 1.00 equiv) in DCM (1.41 L) were added Compound R-4 (69.7 g, 309 mmol, purity 99.1%, 1.05 equiv), DMAP (dimethylaminopyridine; 7.19 g, 58.9 mmol, 0.200 equiv), and TEA (89.4 g, 883 mmol, 123 mL, 3.00 equiv) at 20 °C under N2. T3P (anhydrous propylphosphonic acid; 281 g, 442 mmol, 263 mL, purity 50.0%, 1.50 equiv) was added to the mixture at 20 °C, and the mixture was stirred at 20 °C for 3 hours. LCMS indicated that Compound 5 was consumed and the desired MS (RT = 0.922 min) was detected. The reaction mixture was diluted with DCM (1.40 L) and washed with saturated NaHCO3 solution (1.40 L x 3). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 - 0:1, petroleum ether:ethyl acetate = 0:1, R f = 0.50) to give Compound R-6 (195 g, 289 mmol, yield 98.0%, purity 99.3%) as a light yellow solid.

[0083] LCMS: Product: RT = 0.922 min, m / z = 671.3 (M + H) + SFC: (Homochiral), Product: RT = 4.269 minutes, ee%: 100% 1 1H NMR: 400 MHz, DMSO δ / ppm: 10.5 (s, 1H), 10.37 (s, 1H), 8.59 (d, J = 2.4 Hz, 1H), 8.39 (d, J = 5.6 Hz, 1H), 8.02 - 8.01 (m, 3H), 7.93 - 7.90 (m, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.70 - 7.64 (m, 2H), 7.57 (d, J = 8.0 Hz, 1H), 7.51 (t, J = 6.0 Hz, 1H), 7.42 (d, J = 8.8 Hz, 2H), 4.33 - 4.28 (m, 1H), 4.19 - 4.12 (m, 3H), 3.08 - 3.02 (m, 1H), 2.93 - 2.85 (m, 1H), 2.38 - 2.31 (m, 1H), 2.09 - 2.01 (m, 1H), 1.37 (s, 9H), 1.22 (s, 1H)

[0084] Production of R-4

Chemical Structure

[0085] LCMS: Product: RT = 0.773 minutes, m / z = 571.3 (M+H) + HPLC: Product: RT = 7.942 minutes, purity 99.1% at 220 nm Chiral NP-HPLC, Product: Rt = 13.573 minutes, ee%: 100% Chiral NP-HPLC conditions:

Table 2

[0086] EA-CHNS: C: 54.0%, H: 5.36%, N: 7.98%, S: 4.59% 1 H NMR: 400 MHz, DMSO δ / ppm: 10.7 (s, 1H), 10.56 (s, 1H), 8.60 (d, J = 2.8 Hz, 1H), 5.54 (s, 2H), 8.40 (d, J = 5.2 Hz, 1H), 8.18 (s, 1H), 8.11 (dd, J = 2.0, 2.0 Hz, 1H), 8.07 - 8.05 (m, 2H), 7.91 - 7.88 (m, 2H), 7.85 (d, J = 8.8 Hz, 2H), 7.72 - 7.67 (m, 2H), 7.41 (d, J = 8.8 Hz, 2H), 4.33 - 4.28 (m, 1H), 4.18 - 4.12 (m, 1H), 4.06 (s, 2H), 3.32 (s, 1H), 3.08 - 3.02 (m, 1H), 2.91 - 2.87 (m, 1H), 2.38 - 2.31 (m, 1H), 2.09 - 2.02 (m, 1H)

[0087] Example 4 - Conversion of S-4 to S-A Production of Compound S-6

Chemical Structure

[0088] LCMS: Product: RT = 0.917 min, m / z = 671.3 (M+H) + HPLC: Product: RT = 2.119 min, purity 98.9% under 220 nm SFC: (Homochiral), Product: Rt = 5.810 min, ee%: 100% 11H NMR: 400 MHz, DMSO δ / ppm: 10.5 (s, 1H), 10.4 (s, 1H), 8.59 (d, J = 2.4 Hz, 1H), 8.39 (d, J = 5.6 Hz, 1H), 8.02 - 8.01 (m, 3H), 7.93 - 7.90 (m, 2H), 7.79 (d, J = 8.8 Hz, 2H), 7.70 - 7.64 (m, 2H), 7.58 (d, J = 8.0 Hz, 1H), 7.51 (t, J = 6.0 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 4.33 - 4.28 (m, 1H), 4.19 - 4.12 (m, 3H), 3.08 - 3.02 (m, 1H), 2.93 - 2.85 (m, 1H), 2.38 - 2.32 (m, 1H), 2.09 - 2.01 (m, 1H), 1.38 (s, 9H), 1.22 (s, 1H)

[0089] Preparation of S-A [Chemical Structure] To a solution of compound S-6 (98.3 g, 145 mmol, purity 98.9%, 1.00 equivalent) in DCM (983 mL) and dioxane (983 mL), HCl / dioxane (4 M, 363 mL, 10.0 equivalents) was added dropwise at 35 °C. The mixture was stirred at 35 °C for 2 hours. LCMS indicated that compound S-6 was consumed and the desired MS (RT = 0.774 minutes) was detected. Two batches of the reaction mixture were cooled to 25 °C and stirred at 25 °C for 12 hours. The mixture was concentrated under reduced pressure to obtain a residue. The residue was diluted with deionized H2O (2.00 L), the pH was adjusted to 7 with saturated NaHCO3 solution (310 mL), and the mixture was filtered and the filter cake was concentrated. The crude products from both reactions were combined, triturated with deionized H2O (1.50 L) at 25 °C for 12 hours, then filtered and the filter cake was concentrated to obtain S-A·HCl (112 g, 182 mmol, yield 62.8%, purity 98.8%, HCl) as a bright yellow solid.

[0090] LCMS: Product: RT = 0.774 minutes, m / z = 571.3 (M + H) + Chiral NP-HPLC, Product: Rt = 11.407 minutes, ee%: 100% Chiral NP-HPLC is the same as described in Example 3 EA-CHNS: C: 56.7%, H: 5.39%, N: 8.35%, S: 4.72% 1 H NMR: 400 MHz, DMSO δ / ppm: 10.7 (s, 1H), 10.5 (s, 1H), 8.60 (d, J = 2.8 Hz, 1H), 8.41 - 8.40 (m, 3H), 8.16 (s, 1H), 8.11 (dd, J = 2.0, 1.6 Hz, 1H), 8.08 - 8.06 (m, 2H), 7.91 - 7.87 (m, 2H), 7.85 (d, J = 8.8 Hz, 2H), 7.72 - 7.67 (m, 2H), 7.41 (d, J = 8.8 Hz, 2H), 4.33 - 4.28 (m, 1H), 4.18 - 4.12 (m, 1H), 4.06 (s, 2H), 3.36 (s, 1H), 3.08 - 3.02 (m, 1H), 2.91 - 2.87 (m, 1H), 2.37 - 2.31 (m, 1H), 2.07 - 2.02 (m, 1H)

[0091] This method directly forms Form I of S-A·HCl salt. For Form I of S-A·HCl salt, XRPD was performed under the following conditions:

[0092] XRPD Conditions The XRPD diffraction pattern was collected using an X-ray diffractometer. The sample was prepared by lightly pressing it onto a flat surface on a zero-background silicon wafer. The parameters of XRPD diffraction are shown below.

[0093] Parameters of XRPD Test [Table 3]

[0094] XRPD of Form I of S-A·HCl salt (see also Figure 1) [Table 4]

[0095] Example 5 - Formation of Form I of S-A Succinate Formation of Form I of S-A Free Base: Approximately 100 mg of Form I of S-A·HCl salt was suspended in 1 mL of acetonitrile at room temperature (24 °C). After stirring for 15 minutes, a highly viscous suspension was observed, and then 6 equivalents of aqueous NaHCO3 solution (84 mg of NaHCO3 dissolved in 0.8 mL of water) was added. After stirring for 35 minutes, a suspension was observed. 2 mL of acetonitrile and 10 mL of water were added, and then, when stirred for 10 minutes, an oil-containing solid precipitated. Finally, after stirring at room temperature for 15 hours, a suspension was obtained. The solid was collected by filtration and dried under vacuum at 40 °C for about 3 hours to obtain Form I of S-A free base.

[0096] Formation of Form I of S-A Succinate: Approximately 400 mg of Form I of S-A free base and 1.1 equivalents of succinic acid (91 mg) were added to 8 mL of MEK (methyl ethyl ketone) at room temperature (about 24 °C). The mixture was stirred at room temperature for 4 hours, then the solid was collected by filtration, washed with MEK, and dried under vacuum conditions at 40 °C for 24 hours to obtain Form I of S-A succinate.

[0097] XRPD was performed on Form I of S-A succinate. The conditions were the same as those described for the XRPD analysis of Form I of the HCl salt in Example 4. XRPD Peaks of Form I of Succinate (see also Figure 2)

Table 5

[0098] Example 6 - Formation of Form II of S-A·HCl Salt Approximately 600 mg of Form I of S-A·HCl salt was suspended in 20 mL of acetone / water (19 / 1, v / v) at room temperature and stirred for about 16 hours. The solid was collected by filtration and dried under vacuum at 40 °C for about 2 hours. Approximately 545 mg of Form II of the HCl salt was produced in a 91% yield. XRPD was performed on the S-A·HCl salt in Form II. The conditions were the same as those described for the XRPD analysis of the HCl salt in Form I in Example 4.

[0099] XRPD peaks of the S-A·HCl salt in Form II (see also Figure 3) [Table 6]

[0100] Example 7 - Stability test data Method: Approximately 10 mg of the free base in Form I, the HCl salt in Form II, the succinate salt in Form I, and the received HCl salt in Form I were each placed at 60 °C / sealed system and 40 °C / 75% RH (open system). Samples were prepared in duplicate for each condition. On day 0 and day 7, the samples were analyzed by HPLC and XRPD respectively to check the purity and crystal form.

[0101] The results are shown in the following table. [Table 7]

[0102] All three salt forms are more stable than the free base compound A. The two HCl salt forms are more stable than the succinate salt form.

[0103] The long-term stability of the HCl salt in Form II was also tested under the following conditions: · 25 °C ± 2 °C, 60% RH ± 5% RH · 40 °C ± 2 °C, 75% RH ± 5% RH

[0104] Samples were prepared in duplicate for each condition. On day 0 and after 1 month and 3 months, the samples were analyzed by HPLC and XRPD respectively to check the purity and crystal form.

[0105] The results are shown in the following table. Stability of Form II of the HCl salt at 25°C ± 2°C, 60% RH ± 5% RH [Table 8]

[0106] Stability of Form II of the HCl salt at 40°C ± 2°C, 75% RH ± 5% RH [Table 9]

[0107] As described above, Form II of the HCl salt is stable for up to 3 months even under accelerated conditions.

[0108] Example 8 - Solubility test data Method: Approximately 15 mg of the sample was weighed and placed in each sample vial, and then 3.0 mL of water, simulated gastric fluid (SGF), fasted state simulated intestinal fluid (FaSSIF), or fed state simulated intestinal fluid (FeSSIF) medium was added. The samples were prepared in triplicate for each medium. The suspension was continuously shaken at 37°C for up to 24 hours. At 0.5, 2, and 24 hours, the suspension was filtered and the filtrate was analyzed by HPLC.

[0109] The results are shown in the following table. [Table 10]

[0110] Form II of the HCl salt and Form I of the succinate salt were more soluble in SGF than Form I of the HCl salt.

[0111] Example 9 - Activity of R- and S-Compound A against kinases other than ROCK The HCl salts of the S-enantiomer and R-enantiomer of Compound A were tested against the kinases in the panel.

[0112] The compound was received as a powder and resuspended in 10 mM DMSO stock. The compound was tested in a 10-dose IC50 mode with serial 3-fold dilutions starting from 10 μM. The control compound, staurosporine, was tested in a 10-dose IC50 mode with serial 4-fold dilutions starting from 20 μM. The reactions were carried out at the Km (Michaelis constant) ATP concentration (shown in the table).

[0113] Reaction conditions: Buffer conditions: 20 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; pH 7.5), 10 mM MgCl2, 1 mM EGTA (ethylene glycol-bis(β-aminoethyl)-N,N,N’,N’-tetraacetic acid), 0.01% Brij35, 0.02 mg / ml BSA (bovine serum albumin), 0.1 mM Na3VO4, 2 mM DTT (dithiothreitol), 1% DMSO; Reaction procedure: 1. Prepare the indicated substrate in freshly made reaction buffer. 2. Supply the necessary coenzymes to the above substrate solution. 3. Supply the indicated kinase to the substrate solution and mix gently. 4. Supply the compound in DMSO to the kinase reaction mixture using an acoustic technique (Echo550). 5. 33 Supply P-ATP (specific activity 0.01 μCi / μl final) to the reaction mixture to initiate the reaction. 6. Incubate the kinase reaction at room temperature for 120 minutes. 7. Spot the reaction on P81 ion exchange paper (Whatman # 3698-915). 8. Wash the filter thoroughly with 0.75% phosphoric acid. 9. Measure the radioactive phosphorylated substrate remaining on the filter paper. Data analysis: Kinase activity data are expressed as the percentage of kinase activity remaining in the test sample compared to the vehicle (dimethyl sulfoxide) reaction. IC 50 values and curve fits were obtained using Prism4 software (GraphPad).

[0114] The results are shown in the following table.

Table 11

[0115] The S-enantiomer of Compound A is less active than the R-enantiomer against 6 out of 7 off-target kinases. The R-enantiomer of Compound A is less active than the S-enantiomer against only 1 out of 7 off-target kinases. This is particularly surprising since the inventors have not detected a statistically significant difference between the activities of the S- and R-enantiomers of Compound A against the kinases ROCK1 or ROCK2 to date.

[0116] Example 10 - Activity of R- and S-Compound A against the CEREP panel The use of in vitro pharmacological screening against various targets (receptors, ion channels, enzymes and transporters) is used to identify unwanted off-target activities that may cause adverse drug reactions in humans. Screening panels (e.g., the SafetyScreen44® panel, Eurofins) include the selection of off-targets associated with known issues in humans that, if discovered after drug approval, could prevent or abort the development of a drug candidate or lead to withdrawal from the market. The HCl salts of the S- and R-enantiomers of Compound A were tested against a series of such targets.

[0117] Typical drug adverse reactions associated with these off-targets are summarized (Bowes et al., Nature Reviews Drug Discovery 2012, 11, 909).

[0118] G-protein coupled receptor Cannabinoid receptor CB1 Agonist action / activation: Euphoria and dysphoria; anxiety; memory impairment and decreased concentration; analgesia; hypothermia Antagonist action: Improvement in weight loss; vomiting; depression Cannabinoid receptor CB2 Antagonistic effect: Increase in inflammatory effect; Decrease in bone mass Muscarinic acetylcholine receptor M1 Activating effect: Promotion of spasm; Increase in gastric acid secretion; Hypertension; Tachycardia; Hyperthermia Antagonistic effect: Decrease in cognitive function; Decrease in gastric acid secretion; Blurred vision Muscarinic acetylcholine receptor M2 Activating effect: Decrease in heart rate; Reflex; Increase in blood pressure; Negative chronotropic and inotropic effects; Decrease in cardiac conduction; Decrease in cardiac action potential duration Antagonistic effect: Tachycardia; Bronchoconstriction; Tremor μ-opioid receptor (mu-MOP) Activating effect: Sedation; Decrease in gastrointestinal motility; Miosis; Tendency to abuse; Respiratory depression; Miosis; Hypothermia Antagonistic effect: Hyperactivity of gastrointestinal motility; Indigestion; Flatulence

[0119] Enzyme Monoamine oxidase A (MAO) Inhibitory effect: Increase in blood pressure when combined with amines such as tyramine; Possibility of drug-drug interaction; Dizziness; Sleep disorder; Nausea

[0120] Transporter Uptake of norepinephrine transporter Inhibitory effect: Increase in heart rate; Increase in blood pressure; Increase in locomotor activity; Constipation; Possibility of abuse Uptake of serotonin transporter Inhibitory effect: Hyperactivity of gastrointestinal motility; Decrease in upper gastrointestinal transit; Decrease in plasma renin; Increase in other serotonin-mediated effects; Insomnia; Anxiety; Nausea; Sexual dysfunction

[0121] The tests were carried out according to the methods described in the following table and the indicated references. CEREP panel:

Table 12

[0122]

Table 13

Table 14

[0123] 1. Cesura, A.M. et al., (1990), Mol. Pharmacol., 37:358-366 2. Felder, C.C. et al., (1995), Mol. Pharmacol., 48:443-450 3. Sur, C. et al., (2003), Proc. Natl. Acad. Sci. U.S.A., 100:13674-13679 4. Michal, P. et al., (2001), Brit.J. Pharmacol., 132:1217-1228 5. Wang,J.B. et al., (1994), FEBS Lett., 338:217-222 6. Perovic, S. and Muller, W.E.G., (1995), Arzneim-Forsch. Drug Res., 45:1145-1148 7. Verrico C. et al., (2007), Psychopharmacology, 189, 489-503

[0124] The results are shown in the following table.

Table 15

Table 16

[0125] The S-enantiomer of Compound A is less active than the R-enantiomer in 7 out of 13 assays. The R-enantiomer of Compound A is less active than the S-enantiomer in only 1 out of 13 assays. When this data for the CEREP panel is combined with the data provided in Example 9 for the kinase panel, the S-enantiomer is shown to be more selective than the R-enantiomer against a wide variety of off-target proteins and is less likely to be associated with undesirable toxicities and side effects that limit efficacy and tolerability in humans.

Claims

[Claim 1] An invention as described in the claims, specification, or drawings.

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