TWI660945B - Crystalline forms of a prolyl hydroxylase inhibitor - Google Patents

Abstract

The present invention relates to a crystalline solid form of [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid, a method for preparing the same, and a pharmaceutical composition thereof And how to use it.

Description

Crystal morphology of proline hydroxylase inhibitors

The present invention relates to a crystalline solid form of [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid, a method for preparing the same, and a pharmaceutical composition thereof And how to use it.

The compound may exist in one or more crystalline forms. The crystalline form of a drug substance can have different physical properties, including melting point, solubility, dissolution rate, optical and mechanical properties, vapor pressure, hygroscopicity, particle shape, density, and flowability. These attributes can have a direct impact on the processing and / or manufacturing capabilities of the compound as a pharmaceutical product. Crystal morphology can also show different stability and bioavailability. The most stable crystalline form of a pharmaceutical product is usually selected during the development of a pharmaceutical product based on its smallest possibility of conversion to another crystal form and its higher chemical stability. In order to ensure the quality, safety, and efficacy of pharmaceutical products, it is important to choose crystal forms that are stable, reproducibly manufactured, and have favorable physicochemical properties.

As described in US Patent No. 7,323,475, [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid (hereinafter referred to as a compound A) is a potent inhibitor of hypoxia-inducible factor (HIF) proline hydrazone hydroxylase. HIF proline hydroxylase inhibitors can be used to increase the stability and / or activity of HIF, and can be used to treat and prevent HIF-related disorders, including anemia, ischemia, and hypoxia.

By providing the crystal morphology of Compound A, salts, and solvates, the present disclosure meets these and other needs. The present invention also provides an amorphous form of Compound A. The invention also provides a pharmaceutical composition comprising Compound A in an amorphous state or one or more crystalline forms. The invention also provides methods for making amorphous and crystalline solid forms, as well as methods for treating and preventing HIF-related disorders including anemia, ischemia, and hypoxia disorders.

Therefore, one embodiment provided is crystal [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid (Compound A Form A), It is characterized in that the X-ray powder diffraction pattern includes the following peaks: 8.5, 16.2, and 27.4 ° 2θ ± 0.2 ° 2θ.

Another embodiment provided is crystal [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid hemihydrate (Compound A Form B) , Characterized in that the X-ray powder diffraction pattern includes the following peaks: 4.2, 8.3, and 16.6 ° 2θ ± 0.2 ° 2θ.

Another embodiment provided is crystal [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -hexafluoropropan-2-acetate solvate (Compound A, Form C), characterized in that the X-ray powder diffraction pattern includes the following Peaks: 4.5, 13.7, and 16.4 ° 2θ ± 0.2 ° 2θ.

Another embodiment provided is crystal [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid DMSO: water solvate (Compound A Morphology D), characterized in that the X-ray powder diffraction pattern includes the following peaks: 8.4, 8.5, and 16.8 ° 2θ ± 0.2 ° 2θ.

Another embodiment provided is crystal [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid sodium salt (sodium salt of compound A) , Characterized in that the X-ray powder diffraction pattern includes the following peaks: 5.3, 16.0, and 21.6 ° 2θ ± 0.2 ° 2θ.

Yet another embodiment provided is crystal [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid L-spermine (Compound A L-Spermine), characterized in that the X-ray powder diffraction pattern includes the following peaks: 20.8, 21.8, and 25.4 ° 2θ ± 0.2 ° 2θ.

Another embodiment provided is crystal [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid L-lysinate (Compound A L-ionine salt), characterized in that the X-ray powder diffraction pattern includes the following peaks: 19.8, 20.7, and 21.2 ° 2θ ± 0.2 ° 2θ.

Another embodiment provided is crystal [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -ethanolamine salt of acetate (ethanolamine salt of compound A) , Characterized in that the X-ray powder diffraction pattern includes the following peaks: 21.8, 22.7, and 27.1 ° 2θ ± 0.2 ° 2θ.

Another embodiment provided is crystal [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -diethanolamine acetate (diethanolamine of compound A Salt), characterized in that the X-ray powder diffraction pattern includes the following Peaks: 16.9, 23.7, and 25.0 ° 2θ ± 0.2 ° 2θ.

Yet another embodiment provided is crystal [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -trimethylamine acetate (Miki of Compound A Methylamine salt), characterized in that the X-ray powder diffraction pattern includes the following peaks: 10.1, 14.2, and 21.1 ° 2θ ± 0.2 ° 2θ.

Yet another embodiment provided is amorphous [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid (amorphous compound A) .

Yet another embodiment provided is a substantially amorphous [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -potassium acetate (compound A potassium salt).

Another embodiment also provides a pharmaceutical composition comprising Compound A or a salt thereof in a crystalline or amorphous form and a pharmaceutically acceptable excipient.

In addition, the present disclosure provides, in one embodiment, a method for treating, pre-treating, or delaying the onset or exacerbation of a condition mediated at least in part by hypoxia-inducible factor (HIF). The method includes administering to a patient in need thereof a therapeutically effective amount of a compound generally as described above, selected from the group consisting of Compound A Form A, Compound A Form B, Compound A Form C, Compound A Form D, Compound A Sodium Salt, L-spermine salt of compound A, L-lysine salt of compound A, ethanolamine salt of compound A, diethanolamine salt of compound A, trichomemine salt of compound A, amorphous compound A, compound A potassium salt.

Methods are also provided for treating, pre-treating, or delaying the onset or exacerbation of a condition mediated at least in part by erythropoietin (EPO). The method includes administering to a patient in need thereof a therapeutically effective amount of Compounds described above: Compound A Form A, Compound A Form B, Compound A Form C, Compound A Form D, Sodium Salt of Compound A, L-Spermine Salt of Compound A, L-Lysine Acid of Compound A Salt, ethanolamine salt of compound A, diethanolamine salt of compound A, succinylamine salt of compound A, amorphous compound A, potassium salt of compound A.

Methods for treating, pre-treating or delaying the onset or exacerbation of anemia are also provided. The method includes administering to a patient in need thereof a therapeutically effective amount of a compound generally as described above, selected from the group consisting of Compound A Form A, Compound A Form B, Compound A Form C, Compound A Form D, Compound A Sodium Salt, L-spermine salt of compound A, L-lysine salt of compound A, ethanolamine salt of compound A, diethanolamine salt of compound A, trichomemine salt of compound A, amorphous compound A, compound A potassium salt.

Methods of inhibiting HIF hydroxylase activity are also provided. The method includes contacting a HIF hydroxylase with a therapeutically effective amount of a compound generally as described above, selected from the group consisting of Compound A Form A, Compound A Form B, Compound A Form C, Compound A Form D, Compound A Sodium salt, L-spermine salt of compound A, L-lysine salt of compound A, L-lysine salt of compound A, ethanolamine salt of compound A, diethanolamine salt of compound A, Miki of compound A Methylamine salt, amorphous compound A, potassium salt of compound A.

FIG. 1 is an X-ray powder diffraction pattern of Compound A, Form A. FIG.

FIG. 2 is a differential scanning calorimetry (DSC) curve of Compound A Form A. FIG.

FIG. 3 is an X-ray powder diffraction pattern (bottom) of Compound A Form B plotted with an X-ray powder diffraction pattern (Top) of Compound A Form A. FIG.

Figure 4 shows thermogravimetric analysis (TGA) (top) and differential scanning calorimetry (DSC) curves (bottom) of Compound A, Form B.

FIG. 5 is an X-ray powder diffraction pattern (bottom) of Compound A Form C plotted with an X-ray powder diffraction pattern (Top) of Compound A Form A. FIG.

FIG. 6 is a differential scanning calorimetry (DSC) curve (top) and thermogravimetric analysis (TGA) (bottom) of Compound A, Form C. FIG.

FIG. 7 is an X-ray powder diffraction pattern of Compound A, Form D. FIG.

Figure 8 shows thermogravimetric analysis (TGA) (top) and differential scanning calorimetry (DSC) curves (bottom) of Compound A, Form D.

Figure 9 is an X-ray powder diffraction pattern of the sodium salt of Compound A separated (bottom) and at 40 ° C / 75% RH (top).

FIG. 10 is a thermogravimetric analysis (TGA) (top) and a differential scanning calorimetry (DSC) curve (bottom) of a sodium salt of Compound A. FIG.

FIG. 11 is an X-ray powder diffraction pattern of the L-spermine salt of Compound A separated (bottom) and at 40 ° C./75% RH (top).

Figure 12 shows thermogravimetric analysis (TGA) (top) and differential scanning calorimetry (DSC) curves (bottom) of L-spermine salt of compound A.

FIG. 13 is an X-ray powder diffraction pattern of the L-lysine salt of Compound A separated (bottom) and at 40 ° C./75% RH (top).

Figure 14 shows thermogravimetric analysis (TGA) (top) and differential scanning calorimetry (DSC) curves (bottom) of L-lysine salt of compound A.

FIG. 15 shows the X-ray powder diffraction pattern (bottom) of Compound A, Form A, the ethanolamine salt pattern 1 (second bottom) of Compound A, and the pattern 3 of ethanolamine salt of Compound A at 40 ° C / 75% RH (Middle), pattern 2 (subtop) of the ethanolamine salt of compound A and ethanolamine salt pattern 2 (top) of compound A at 40 ° C / 75% RH, isolated.

FIG. 16 is a thermogravimetric analysis (TGA) (top) and a differential scanning calorimetry (DSC) curve (bottom) of an ethanolamine salt of Compound A. FIG.

FIG. 17 is an X-ray powder diffraction pattern (bottom) of Compound A, Form A, pattern 1 (second bottom) of the diethanolamine salt of compound A from acetone, and pattern 1 (second (Top), and ethanolamine salt of compound A at 40 ° C / 75% RH (pattern 2, top).

18 is a thermogravimetric analysis (TGA) (top) and a differential scanning calorimetry (DSC) curve (bottom) of a diethanolamine salt of Compound A. FIG.

FIG. 19 is an X-ray powder diffraction pattern of compound A form A (bottom) and isolated (middle) and the trichomemine salt of compound A at 40 ° C./75% RH (top).

FIG. 20 is a thermogravimetric analysis (TGA) (top) and a differential scanning calorimetry (DSC) curve (bottom) of a tromethamine salt of compound A. FIG.

Figure 21 is an X-ray powder diffraction pattern of the potassium salt of Compound A separated (bottom) and at 40 ° C / 75% RH (top).

Figure 22 shows the thermogravimetric analysis (TGA) (top) and Differential scanning calorimetry (DSC) curve (bottom).

FIG. 23 is an X-ray powder diffraction pattern of the amorphous compound A. FIG.

FIG. 24 shows thermogravimetric analysis (TGA) of Compound A, Form A. FIG.

FIG. 25 is an X-ray powder diffraction pattern of Compound A Form A (bottom) and isolated (middle) and hydrochloride of Compound A at 40 ° C./75% RH (top).

Figure 26 shows thermogravimetric analysis (TGA) (top) and differential scanning calorimetry (DSC) curves (bottom) of the hydrochloride salt of compound A.

Fig. 27 is an X-ray powder diffraction pattern of the sulfate of Compound A (bottom) and isolated (middle) and at 40 ° C / 75% RH (top).

FIG. 28 is a thermogravimetric analysis (TGA) (top) and a differential scanning calorimetry (DSC) curve (bottom) of the sulfate of the compound A. FIG.

FIG. 29 is an X-ray powder diffraction pattern (bottom), a separated (sub-bottom) and a pattern 1 of a mesylate salt of compound A at 40 ° C./75% RH (middle), and Separated (sub-top) and pattern 2 of the mesylate salt of compound A at 40 ° C / 75% RH (top).

FIG. 30 is a thermogravimetric analysis (TGA) (top) and a differential scanning calorimetry (DSC) curve (bottom) of a mesylate salt of Compound A. FIG.

Figure 31 shows the X-ray powder windings of Compound A Form A (bottom), the isolated ditriethylamine salt (middle) of compound A and the ditriethylamine salt (top) of compound A at 40 ° C / 75% RH. Shoot pattern.

Figure 32 shows thermogravimetric analysis (TGA) (top) and differential scanning calorimetry (DSC) curves (bottom) of the ditriethylamine salt of compound A.

FIG. 33 is an X-ray powder diffraction pattern of Compound A Form A (bottom) and Compound A semi-calcium salt (sub-top) at 40 ° C./75% RH (top).

Figure 34 shows thermogravimetric analysis (TGA) (top) and differential scanning calorimetry (DSC) curves (bottom) of the hemi-calcium salt of compound A.

FIG. 35 is an X-ray powder diffraction pattern of compound A form A (bottom), and a hemimagnesium salt (sub-top) of compound A at 40 ° C./75%RH (top).

FIG. 36 is a differential scanning calorimetry (DSC) curve of a hemi-magnesium salt of Compound A. FIG.

Figure 37 shows the molecular structure of Compound A, Form A.

Compound [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid (compound A) is a hypoxia-inducible factor (HIF) proline hydrazone hydroxylase Effective inhibitor and has the following formula:

The present disclosure provides a crystalline morphology of Compound A, a salt of Compound A, and a solvate of Compound A. The present disclosure also provides an amorphous form of Compound A. The present disclosure also provides a pharmaceutical composition comprising an amorphous or crystalline form of Compound A. The present disclosure also provides methods of preparing amorphous and crystalline solid forms and methods of using them to treat and prevent HIF-related disorders including anemia, ischemia, and hypoxia.

Before going into further detail, the following terms will be defined.

Definition

As used herein, the following terms have the following meanings.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a single compound and multiple different compounds.

The term "about" when used before a numerical label that includes a range, such as temperature, time, amount, and concentration, means an approximation that can vary by ± 10%, ± 5%, or ± 1%.

The term "solvate" refers to a complex formed by the combination of Compound A and a solvent.

The terms "substantially amorphous" and "mostly amorphous" refer to the amorphous compound A where a small amount of the crystalline compound A may be present. In some embodiments, the amount of the crystalline compound A is less than about 10%, or less than about 5%, or less than about 2%, or less than about 1%, or less than about 0.2%, or less than about 0.1%.

"Administering" refers to introducing an agent into a patient. A therapeutic amount can be administered, which can be determined by the treating physician or the like. For Compound A in the crystalline form described herein, the oral route of administration is preferred. Related terms and phrases "administrating" or "administration of" when used in conjunction with a compound or pharmaceutical composition (and grammatical equivalent) refer to direct and / or indirect administration, which Direct application can be made by medical professionals The person administers to the patient or the patient himself, the indirect administration may be a prescribing act. For example, the physician instructs the patient to administer the drug himself and / or to provide the patient with a prescription for administering the drug to the patient. Regardless, administration involves delivering the drug to a patient.

As used herein, "excipients" refers to inert or inactive substances used in the manufacture of pharmaceuticals and includes, without limitation, any use as a binder, disintegrant, coating, compression / encapsulation aid, emulsion or lotion , Lubricants, parenteral injections, sweeteners or flavoring agents, suspensions / gelling agents, or wetting granules. Binders include, for example, carbopol, povidone, xanthan gum, etc .; coatings include, for example, cellulose acetate phthalate, ethyl cellulose, gellan gum, maltodextrin, etc .; compression / encapsulation Adjuvants include, for example, calcium carbonate, glucose, fructose, honey, lactose (dehydrated or monohydrate; optionally combined with aspartame, cellulose or microcrystalline cellulose), starch, sucrose, etc .; disintegrating agents include, for example, Sodium carboxymethylcellulose, gellan gum, sodium carboxymethyl starch, etc .; emulsions or lotions include, for example, maltodextrin, carrageenan, etc .; lubricants include, for example, magnesium stearate, stearic acid, stearic acid Sodium fumarate, etc .; materials used for chewing tablets include, for example, glucose, fructose, lactose (monohydrate, optionally combined with abastian or cellulose), etc .; parenteral injections include, for example, mannitol, povidone, etc. ; Plasticizers include, for example, dibutyl sebacate, polyvinyl acetate phthalate, etc .; suspension / gelling agents include, for example, carrageenan, sodium carboxymethyl starch, xanthan gum, etc .; sweeteners include E.g. Abbastine, glucose, fructose, Sorbitol, sucrose, and the like; and moist granulating agents include, for example, calcium carbonate, maltodextrin, microcrystalline cellulose, and the like.

A "therapeutically effective amount" or "therapeutic amount" means when administered to a patient suffering from a condition The amount of drug or agent at the time of the patient, which will have an expected therapeutic effect, such as reducing, ameliorating, alleviating or eliminating one or more clinical manifestations of the condition the patient is suffering from. The therapeutically effective amount will vary depending on the individual or condition being treated, the weight and age of the individual, the severity of the condition, the particular composition or excipient chosen, the dosage regimen to be followed, All of these factors can be easily determined by those skilled in the art, such as the time of administration, the mode of administration, and the like. The overall therapeutic effect need not be produced by administering one dose, but may only occur after taking a series of doses. Therefore, a therapeutically effective amount may be administered in one or more administrations. For example, without limitation, a therapeutically effective amount in the context of treating anemia refers to a dose of a drug that reduces, ameliorates, alleviates, or eliminates one or more symptoms of anemia in a patient.

"Treatment", "treating", and "treat" are defined as those in which a disease, disorder, or condition is treated with an agent to reduce or ameliorate the disease, disorder, or condition, and / or its symptoms. Harmful or any other undesirable effect. The treatment used herein includes the treatment of a human patient and includes: (a) reducing the risk of developing a condition in a patient who is determined to be predisposed but not yet diagnosed with the condition, (b) preventing the development of the condition, And / or (c) alleviate the condition, ie cause the condition to resolve and / or alleviate one or more symptoms of the condition.

The "XRPD pattern" is an x-y diagram having a diffraction angle (ie, 2θ) on the x-axis and an intensity on the y-axis. The peaks in this pattern can be used to characterize the solid state morphology of the crystal. In the case of any data measurement, there is variability in the XRPD data. Data is often uniquely represented by the diffraction angle of the peak, excluding the intensity of the peak, as the intensity of the peak may be particularly sensitive to sample preparation (For example, particle size, moisture content, solvent content, and better orientation effects affect sensitivity), so samples of the same material prepared under different conditions may produce slightly different patterns; this variability is usually greater than that of the diffraction angle Variability. The variability of the diffraction angle can also be sensitive to sample preparation. Other sources of variability come from instrument parameters and processing of raw X-ray data: different X-ray instrument operations use different parameters and these may result in slightly different XRPD patterns from the same solid state, and similarly, different sets Software processes X-ray data in different ways and this also results in variability. These and other sources of variability are known to those skilled in the pharmaceutical arts. Due to the source of this variability, the variability of ± 0.2 ° 2θ is usually assigned to the diffraction angle in the XRPD pattern.

2. The solid state of compound A

As generally described above, the present disclosure provides a solid form of [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid.

Compound A Form A is characterized in that the X-ray powder diffraction pattern includes peaks at 8.5, 16.2, and 27.4 ° 2θ ± 0.2 ° 2θ. The diffraction pattern includes additional peaks at 12.8, 21.6, and 22.9 ° 2θ ± 0.2 ° 2θ. Form A is also characterized in that its entire X-ray powder diffraction pattern is substantially as shown in FIG. 1.

In some embodiments, Form A is characterized in that its differential scanning calorimetry (DSC) curve includes an endotherm at about 223 ° C. Form A is also characterized in that its entire DSC curve is substantially as shown in FIG. 2.

Compound A Form B is characterized in that the X-ray powder diffraction pattern includes Peaks at 4.2, 8.3, and 16.6 ° 2θ ± 0.2 ° 2θ. The diffraction patterns include additional peaks at 12.5, 14.1, and 17.4 ° 2θ ± 0.2 ° 2θ. Form B is also characterized in that its entire X-ray powder diffraction pattern is substantially as shown in FIG. 3.

In some embodiments, Form B is characterized in that its differential scanning calorimetry (DSC) curve includes an endotherm at about 222 ° C. Form B is also characterized in that its entire DSC curve is substantially as shown in FIG. 4.

Compound A Form C is characterized in that the X-ray powder diffraction pattern includes peaks at 4.5, 13.7, and 16.4 ° 2θ ± 0.2 ° 2θ. The diffraction pattern includes additional peaks at 15.4, 15.5, and 20.6 ° 2θ ± 0.2 ° 2θ. Form C is also characterized in that its entire X-ray powder diffraction pattern is substantially as shown in FIG. 5.

In some embodiments, Form C is characterized in that its differential scanning calorimetry (DSC) curve includes an endotherm at about 222 ° C. Form C is also characterized in that its entire DSC curve is substantially as shown in FIG. 6.

Compound A Form D is characterized in that the X-ray powder diffraction pattern includes peaks at 8.4, 8.5, and 16.8 ° 2θ ± 0.2 ° 2θ. The diffraction patterns include additional peaks at 4.2, 12.6, and 28.4 ° 2θ ± 0.2 ° 2θ. Form D is also characterized in that its entire X-ray powder diffraction pattern is substantially as shown in FIG. 7.

In some embodiments, Form D is characterized in that its differential scanning calorimetry (DSC) curve includes an endotherm at about 222 ° C. Form D is also characterized in that its entire DSC curve is substantially as shown in FIG. 8.

The sodium salt of Compound A is characterized in that the X-ray powder diffraction pattern includes peaks at 5.3, 16.0, and 21.6 ° 2θ ± 0.2 ° 2θ. The diffraction pattern includes additional peaks at 18.7, 19.2, and 24.0 ° 2θ ± 0.2 ° 2θ. The sodium salt of Compound A is also characterized in that its entire X-ray powder diffraction pattern is substantially as shown in Figure 9 Show.

In some embodiments, the sodium salt of Compound A is characterized in that its differential scanning calorimetry (DSC) curve includes an endotherm at about 314 ° C. The sodium salt of Compound A is also characterized in that its entire DSC curve is substantially as shown in FIG. 10.

The L-spermine salt of compound A is characterized in that the X-ray powder diffraction pattern includes peaks at 20.8, 21.8, and 25.4 ° 2θ ± 0.2 ° 2θ. The diffraction pattern includes additional peaks at 22.7, 23.4, and 26.4 ° 2θ ± 0.2 ° 2θ. The L-spermine salt of Compound A is also characterized in that its entire X-ray powder diffraction pattern is substantially as shown in FIG. 11.

In some embodiments, the L-spermine salt of Compound A is characterized in that its differential scanning calorimetry (DSC) curve includes an endotherm at about 210 ° C. The L-spermine salt of Compound A is also characterized in that its entire DSC curve is substantially as shown in FIG. 12.

The L-lysine salt of Compound A is characterized in that the X-ray powder diffraction pattern includes peaks at 19.8, 20.7, and 21.2 ° 2θ ± 0.2 ° 2θ. The diffraction pattern includes additional peaks at 10.2, 16.9, and 18.4 ° 2θ ± 0.2 ° 2θ. The L-lysine salt of Compound A is also characterized in that its entire X-ray powder diffraction pattern is substantially as shown in FIG. 13.

In some embodiments, the L-lysine salt of Compound A is characterized in that its differential scanning calorimetry (DSC) curve includes an endotherm at about 237 ° C. The L-lysine salt of Compound A is also characterized in that its entire DSC curve is substantially as shown in FIG. 14.

The ethanolamine salt of compound A is characterized by an X-ray powder diffraction pattern Includes peaks at 21.8, 22.7, and 27.1 ° 2θ ± 0.2 ° 2θ. The diffraction pattern includes additional peaks at 21.1, 26.2, and 26.6 ° 2θ ± 0.2 ° 2θ. The ethanolamine salt of Compound A is also characterized in that its entire X-ray powder diffraction pattern is substantially as shown in FIG. 15.

In some embodiments, the ethanolamine salt of Compound A is characterized in that its differential scanning calorimetry (DSC) curve includes an endotherm at about 171 ° C. The ethanolamine salt of Compound A is also characterized in that its entire DSC curve is substantially as shown in FIG. 16.

The diethanolamine salt of Compound A is characterized in that the X-ray powder diffraction pattern includes peaks at 16.9, 23.7, and 25.0 ° 2θ ± 0.2 ° 2θ. The diffraction pattern includes additional peaks at 19.6, 22.6, and 26.0 ° 2θ ± 0.2 ° 2θ. The diethanolamine salt of Compound A is also characterized in that its entire X-ray powder diffraction pattern is substantially as shown in FIG. 17.

In some embodiments, the diethanolamine salt of Compound A is characterized in that its differential scanning calorimetry (DSC) curve includes an endotherm at about 150 ° C. The diethanolamine salt of Compound A is also characterized in that its entire DSC curve is substantially as shown in FIG. 18.

The tromethamine salt of compound A is characterized in that the X-ray powder diffraction pattern includes peaks at 10.1, 14.2, and 21.1 ° 2θ ± 0.2 ° 2θ. The diffraction pattern includes additional peaks at 20.1, 25.7, and 28.4 ° 2θ ± 0.2 ° 2θ. The tromethamine salt of compound A is also characterized in that its entire X-ray powder diffraction pattern is substantially as shown in FIG. 19.

In some embodiments, the tromethamine salt of compound A is characterized in that its differential scanning calorimetry (DSC) curve includes an endotherm at about 176 ° C. The tromethamine salt of compound A is also characterized in that its entire DSC curve is substantially as shown in FIG. 20.

Also provided are amorphous [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid (amorphous compound A) and substantially amorphous [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid potassium salt (potassium salt of compound A). The substantially amorphous potassium salt of Compound A is characterized in that its differential scanning calorimetry (DSC) curve includes an endotherm at about 291 ° C (FIG. 22).

As described in the examples below, Form A is the most stable crystalline form of Compound A Forms B, C, and D.

3. Pharmaceutical composition, preparation and route of administration

In one aspect, the present disclosure relates to a pharmaceutical composition comprising [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino group]- One or more crystalline forms of acetic acid (compound A) or a salt thereof and at least one pharmaceutical excipient:

In one embodiment, the pharmaceutical composition comprises a compound selected from the group consisting of compound A, form A, compound A, form B, compound A, form C, compound A, form D, and the sodium salt of compound A, generally as described above. , L-spermine salt of compound A, L-lysine salt of compound A, ethanolamine salt of compound A, diethanolamine salt of compound A, Trimethylamine salt of compound A, amorphous compound A and potassium salt of compound A, and at least one pharmaceutically acceptable excipient.

In one embodiment, the pharmaceutical composition comprises Compound A in Form A. In one embodiment, the pharmaceutical composition comprises Compound A, wherein at least about 85% of Compound A is Form A. In one embodiment, the pharmaceutical composition comprises Compound A, wherein at least about 90% of Compound A is Form A. In one embodiment, the pharmaceutical composition comprises Compound A, wherein at least about 95% of Compound A is Form A. In one embodiment, the pharmaceutical composition comprises Compound A, wherein at least about 99% of Compound A is Form A. In one embodiment, the pharmaceutical composition comprises Compound A, wherein at least about 99.5% of Compound A is Form A. In one embodiment, the pharmaceutical composition comprises Compound A, wherein at least about 99.9% of Compound A is Form A. In one embodiment, the pharmaceutical composition comprises Compound A, wherein at least about 99.99% of Compound A is Form A.

In one embodiment, the pharmaceutical composition further comprises other therapeutic agents selected from the group consisting of vitamin B12, folic acid, ferrous sulfate, recombinant human erythropoietin, and erythrocyte stimulating agent (ESA). In another embodiment, the pharmaceutical composition is formulated for oral delivery. In another embodiment, the pharmaceutical composition is formulated as a tablet or capsule.

The crystalline morphology of the present disclosure can be delivered directly or in the form of a pharmaceutical composition with suitable excipients, as is well known in the art. Various methods of treatment embodied herein may include administering to a subject in need thereof an effective amount of a crystal morphology of the present disclosure, for example, due to, for example, chronic renal failure, diabetes, cancer, AIDS, radiation therapy, chemotherapy, renal dialysis, or Individuals suffering from surgery or at risk for anemia. In one embodiment, the individual is a mammalian individual, and in one embodiment, the individual is a human individual.

The effective amount of the crystalline form can be easily determined by routine experimentation, as is the most effective and convenient route of administration and the most suitable formulation. In one embodiment, the dosage may be from 0.05 mg / kg to about 700 mg / kg per day. Generally, the dosage may be from about 0.1 mg / kg to about 500 mg / kg; from about 0.5 mg / kg to about 250 mg / kg; from about 1 mg / kg to about 100 mg / kg; from about 1 mg / kg To about 10 mg / kg; from about 1 mg / kg to about 5 mg / kg; or from about 1 mg / kg to about 2 mg / kg. For example, the dose may be about 1.0 mg / kg; about 1.2 mg / kg; about 1.5 mg / kg; about 2.0 mg / kg; or about 2.5 mg / kg. Various formulations and drug delivery systems are available in the art (see, eg, Gennaro, A.R., ed. (1995) Remington's Pharmaceutical Sciences).

Suitable routes of administration may include, for example, oral, rectal, transmucosal, intranasal or small intestine administration, and parenteral delivery including intramuscular, subcutaneous, intramedullary and intrathecal, direct intraventricular, intravenous, Intraperitoneal, intranasal or intraocular injection. The crystal morphology or its composition can be administered locally rather than systemically. For example, the crystalline morphology or composition thereof can be delivered via injection or in a targeted drug delivery system, such as a depot formulation or a slow release formulation. In one embodiment, the route of administration is oral.

The pharmaceutical composition of the present disclosure can be manufactured by any method known in the art, such as by conventional mixing, dissolving, granulating, dragee-making Made, ground, emulsified, encapsulated, encapsulated or lyophilized. As noted above, the composition may include one or more pharmaceutically acceptable excipients that assist in processing the active molecule into a formulation for pharmaceutical use.

The appropriate formulation depends on the route of administration chosen. For injection, for example, the composition can be formulated in an aqueous solution, preferably a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal or intranasal administration, penetrants suitable for the barrier to be penetrated are used in the formulation. Such penetrants are generally known in the art. In a preferred embodiment of the present disclosure, the present crystal form is prepared into a formulation for oral administration. For oral administration, it can be easily formulated by combining the crystal form with pharmaceutical excipients well known in the art. Such excipients enable the crystalline morphology of the present disclosure to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by an individual. The crystalline form can also be formulated into rectal compositions, such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glyceride suppositories.

Using solid excipients, optionally, after adding suitable auxiliaries, grinding the resulting mixture and processing the granule mixture to obtain tablets or dragee cores, a pharmaceutical preparation for oral use can be prepared. Suitable excipients are, for example, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose formulations, such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl fiber Cellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, microcrystalline cellulose and / or polyvinylpyrrolidone (PVP or povidone). If needed, disintegrating agents can be added, Such as cross-linked polyvinyl pyrrolidone, agar, croscarmellose sodium or alginic acid or its salt, such as sodium alginate. Wetting agents such as sodium lauryl sulfate or lubricants such as magnesium stearate may also be included.

A suitable coating is provided for the dragee core. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbomer gel, polyethylene glycol and / or titanium dioxide, a lacquer solution and a suitable organic solvent or Solvent mixture. Dyes or pigments can be added to tablets or dragee coatings to identify or describe different combinations of active doses.

Pharmaceutical preparations for oral administration include push-fit capsules made of gelatin, and soft sealed capsules made of gelatin and plasticizers, such as glycerol or sorbitol. Push-on capsules may contain active ingredients which are incorporated with fillers such as lactose, binders such as starch, and / or lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the crystalline form can be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, a stabilizer may be added. All preparations for oral administration should be used in dosages suitable for such administration.

In one embodiment, the crystalline morphology described herein can be applied transdermally, such as through a skin patch, or topically. In one aspect, the transdermal or topical formulation may also contain one or more penetration enhancers or other effectors, including agents that increase the migration of the delivered compound. Transdermal or topical administration may be preferred, for example, where localized delivery is required.

For administration by inhalation, use a suitable propellant, such as dichlorodifluoromethyl, in the form of an aerosol spray presentation from a pressurized pack or nebulizer Alkane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or any other suitable gas, conveniently delivers the crystal morphology used in this disclosure. In the case of pressurized aerosols, an appropriate dosage unit can be determined by providing a valve to deliver a metered amount. It can be configured for use in inhalers or blowers such as gelatin capsules and kits. These usually comprise a powder mixture of a crystalline morphology with a suitable powder base such as lactose or starch.

Compositions formulated for parenteral administration by injection, such as by bolus injection or continuous infusion, can be presented in unit dosage form, such as in an ampoule or multi-dose container, with the addition of a preservative. The composition may take the form of, for example, a suspension, solution or emulsion in an oily or aqueous carrier, and may contain formulations such as suspending, stabilizing and / or dispersing agents. Parenteral formulations include other compositions in aqueous or water-soluble form.

Crystalline suspensions can also be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil and synthetic fatty acid esters such as ethyl oleate or triglycerides or liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspending agent may also contain suitable stabilizers or substances that increase the solubility of the crystal form to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable carrier such as sterile pyrogen-free water before use.

As mentioned above, the currently disclosed composition can also be formulated as a depot formulation. Such long acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the current crystalline morphology can be combined with suitable polymers or hydrophobic materials (e.g., as Emulsions in acceptable oils) or ion exchange resins, or formulated as poorly soluble derivatives, for example as poorly soluble salts.

For any composition used in the various treatment methods embodied herein, various techniques known in the art can be used to initially estimate the therapeutically effective dose. For example, in a cell culture assay, a dose can be formulated in animal models to achieve a circulating concentration range that includes in cell culture as determined IC _50. For example, using data obtained from cell culture analysis and non-human animal studies, a dosage range suitable for a human individual can be determined. In one embodiment, the regularly administered dose may be from 0.05 mg / kg to about 700 mg / kg. The dosage administered may be once a day, once every other day, one Monday, two, three times, or other appropriate intervals, which can easily be determined by a capable medical practitioner. Usually, the dose administered is 2 or 3 times a week. Generally, the dosage may be from about 0.1 mg / kg to about 500 mg / kg; from about 0.5 mg / kg to about 250 mg / kg; from about 1 mg / kg to about 100 mg / kg; from about 1 mg / kg To about 10 mg / kg; from about 1 mg / kg to about 5 mg / kg; or from about 1 mg / kg to about 2 mg / kg. For example, the dose may be about 1.0 mg / kg; about 1.2 mg / kg; about 1.5 mg / kg; about 2.0 mg / kg; or about 2.5 mg / kg.

A therapeutically effective dose of a compound refers to the amount of the compound that results in an improvement in symptoms or prolonged survival in an individual. Toxicity and therapeutic efficacy of the molecule can be determined by standard pharmaceutical procedure by the in cell cultures or experimental animals, e.g., by determining the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅₀ (treatment at 50% of the population Effective dose). The dose ratio of toxic to therapeutic effects is the therapeutic index and it can be expressed as LD ₅₀ / ED ₅₀ ratio. Compounds that exhibit high therapeutic indices are preferred.

Preferably, the dose falls within a range of circulating concentrations including having little or no toxicity to ED _50. The dosage can vary within this range, depending on the dosage form employed and the route of administration used. The precise formulation, route of administration, and dosage should be selected according to the characteristics of the individual's condition according to methods known in the art.

The dose and interval can be adjusted individually to provide an active fraction plasma level sufficient to adjust the required parameters, such as endogenous erythropoietin plasma level, ie, the minimum effective concentration (MEC). The MEC will vary for each compound, but can be estimated from, for example, in vitro data. The dose required to obtain MEC will depend on individual characteristics and the route of administration. A treatment regimen that maintains a plasma level above the MEC by about 10-90% of the treatment duration, preferably about 30-90% of the treatment duration, and most preferably between 50-90% of the treatment duration, should be administered Compound or composition thereof. In the case of local administration or selective absorption, the effective local concentration of the drug may not correlate with plasma concentration. Alternatively, adjustments to desired parameters, such as stimulating endogenous erythropoietin, may be achieved by: 1) administering a loading dose, followed by a maintenance dose, and 2) applying an induction dose to quickly achieve the desired within the target range Parameters, such as erythropoietin levels, followed by low maintenance doses to maintain, for example, blood cell volume ratios within the desired target range, or 3) repeated intermittent dose administration.

The amount of compound or composition administered will, of course, depend on a number of factors, including the sex, age, and weight of the individual being treated, the severity of the pain, the mode of administration, and the judgment of the prescribing physician.

If desired, the composition of the invention can be presented in a package or dispenser device containing one or more unit dosage forms, said unit dosage forms comprising the active ingredient. Such a bag or device may, for example, contain metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by an instruction manual. Compositions comprising the crystalline morphology of the present disclosure formulated in compatible pharmaceutical excipients may also be prepared or placed in suitable containers and labeled for the treatment of the conditions indicated. Suitable conditions identified on the label may include treatment of a condition, disorder, or disease, with anemia being a major indication.

4. How to use

One aspect of the present disclosure provides one or more crystalline or amorphous [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid (Compound A ) Or a composition comprising one or more crystalline or amorphous compound A, or a solvate or salt thereof, for use in the manufacture of a medicament for the treatment of various conditions or disorders described herein. It also provides methods of using a crystalline or amorphous morphology or a composition or medicament to treat, pretreat or delay the worsening or onset of various conditions or disorders described herein. In one embodiment, the crystal form of Compound A used in the method is Form A. In one embodiment, the crystal form of Compound A used in the method is Form B, Form C, or Form D. As used in this section "Methods of Use" and corresponding method claims, "Compound" refers to Compound A or its solvate or salt in crystalline or amorphous form.

In one embodiment, at least about 85% of the compounds used in the method The compound is Form A of Compound A. In one embodiment, at least about 90% of the compounds used in the method are Compound A, Form A. In one embodiment, at least about 95% of the compounds used in the method are Compound A, Form A. In one embodiment, at least about 99% of the compounds used in the method are Compound A, Form A. In one embodiment, at least about 99.5% of the compounds used in the method are Compound A, Form A. In one embodiment, at least about 99.99% of the compounds used in the method are Compound A, Form A.

The drug or composition can be used to regulate the stability and / or activity of HIF, thereby initiating HIF-regulated gene expression. The crystalline or amorphous form or its composition or medicament can be used in methods to treat, pretreat, or delay the worsening or onset of HIF-related diseases, including but not limited to anemia, ischemia, and hypoxic conditions. In various embodiments, the crystalline or amorphous morphology or its composition or drug. In another embodiment, the crystalline or amorphous form, or composition or drug thereof, is administered to a patient who is diagnosed with a condition associated with the development of chronic ischemia, such as cardiogenic cirrhosis, macular Degeneration, pulmonary embolism, acute respiratory failure, neonatal respiratory distress syndrome, and congestive heart failure. In another embodiment, the crystalline or amorphous form or its composition or medicament is administered immediately after the wound or injury. In other embodiments, based on threatened conditions, such as hypertension, diabetes, arterial occlusive disease, chronic venous insufficiency, Raynaud's disease, chronic skin ulcers, cirrhosis, congestive heart failure, and systemic sclerosis, Crystalline or amorphous Or its composition or medicament is administered to an individual. In other embodiments, the crystalline or amorphous form or a composition or medicament thereof can be administered to pretreat the individual to reduce or prevent the development of tissue damage associated with ischemia or hypoxia.

The crystalline or amorphous form or its composition or drug can also be used to increase endogenous erythropoietin (EPO). The crystalline or amorphous form or its composition or medicament can be administered to prevent, pretreat, or treat EPO-related disorders, including, for example, disorders related to anemia and nervous system disorders. Conditions associated with anemia include disorders such as acute or chronic kidney disease, diabetes, cancer, ulcers, viruses such as HIV, bacterial or parasitic infections; inflammation and the like. Anemia conditions may also include those associated with procedures or treatments, including, for example, radiation therapy, chemotherapy, dialysis, and surgery. Anemia-related disorders also include abnormal hemoglobin and / or red blood cells, as found in disorders such as small red blood cell anemia, hypoproteinemia, anemia, and the like.

The present disclosure also relates to the use of crystalline or amorphous forms or compositions or drugs thereof to treat, pretreat, or delay the onset of a disorder associated with a disorder selected from the group consisting of: anemia disorders; neurological disorders and / or injuries, including Stroke, trauma, epilepsy, and neurodegenerative diseases; myocardial ischemia, including but not limited to myocardial infarction and congestive heart failure; liver ischemia, including but not limited to cardiogenic cirrhosis; kidney ischemia, including but not limited to acute kidney disease Failure and chronic renal failure; peripheral vascular disorders, ulcers, burns and chronic wounds; pulmonary embolism; and ischemia-reperfusion injury.

The present disclosure also relates to inhibiting at least one modified hypoxia-inducible factor alpha subunit. Method of hydroxylase activity in situ. The hydroxylase of HIF may be proline hydroxylase, which includes but is not limited to EGLN1, EGLN2, and EGLN3 (also referred to as PHD2, PHD1, and PHD3, respectively), and is made by Taylor (2001, Gene 275: 125-132) The descriptions were characterized by Aravind and Koonin (2001, Genome Biol 2: RESEARCH0007), Epstein et al. (2001, Cell 107: 43-54), and Bruick and McKnight (2001, Science 294: 1337-1340). The method includes contacting the enzyme with an inhibitory effective amount of one or more Compound A in a crystalline or amorphous form. In some embodiments, the HIF hydroxylase is asparagine hydroxylase or proline hydroxylase. In other embodiments, the HIF hydroxylase is a factor that inhibits HIF, human EGLN1, EGLN2, or EGLN3.

Although the present disclosure has been described in connection with specific implementation aspects and examples, it is apparent to those skilled in the art in view of the technology and the present disclosure that equivalents of the specifically disclosed materials and methods are also applicable to the present disclosure. Published; and is intended to include such equivalents within the scope of the appended patent applications.

Examples

Unless otherwise stated, the following abbreviations used throughout the specification have the following definitions:

X-ray powder diffraction (XRPD)

X-ray powder was collected on a Bruker AXS C2 GADDS diffractometer using copper (Cu) Kα radiation (40kV, 40 mA), automatic XYZ stage, laser video microscope for automatic sample positioning, and HiStar two-dimensional area detector. Shoot pattern. X-ray optics consist of 0.3 mm pinholes The collimator consists of a single Gobel multilayer mirror. A weekly performance check was performed using the certified standard NIST 1976 Corundum (flatbed).

The beam divergence, the effective size of the X-ray beam on the sample, is approximately 4 mm. Using θ-θ continuous scanning mode, it has a sample-detector distance of 20 cm, which provides an effective 2θ range of 3.2 ° -29.7 °. Typically, the sample is exposed to an x-ray beam for 120 seconds. The software used for data collection was GADDS of WNT4.1.16, and the data was analyzed and presented using Diffrac Plus EVA v11.0.0.2 or v13.0.0.2.

Alternatively, use copper Kα radiation (40 kV, 40 mA), θ-2θ goniometer and V4 divergence and receiving slit, germanium (Ge) monochromator, and Lynxeye detector to collect X-rays on a Bruker D8 diffractometer Powder diffraction pattern. The performance of the instrument was checked using a certified Corundum standard (NIST 1976). The software used for data collection was Diffrac Plus XRD Commander v2.5.0 and the data was analyzed and presented using Diffrac Plus EVA v11.0.0.2 or v13.0.0.2.

Using the received powder, the sample was run as a flat specimen at ambient conditions. The sample was lightly packed into a cavity cut into polished, zero-background (510) silicon wafers. During analysis, the sample rotates in its own plane. Details of data collection are: ‧Angle range: 2 to 42 ° 2θ

‧Step size: 0.05 ° 2θ

‧Collection time: 0.5 s / step

‧Analysis duration: 7min

Differential scanning calorimetry (DSC)

DSC will be collected on a TA instrument Q2000 equipped with a 50-position autosampler. Use sapphire for thermal capacity calibration and qualified indium for energy and temperature calibration. Generally, 0.5-3 mg of each sample was heated from 25 ° C to 300 ° C at 10 ° C / min in a pinhole aluminum pan. A dry nitrogen purge was maintained on the sample at 50 ml / min. DSC was used to adjust the temperature using a basic heating rate of 2 ° C / min and a temperature adjustment parameter of ± 0.318 ° C (amplitude) every 60 seconds (period). The instrument control software was Q series Advantage v2.8.0.392 and Thermal Advantage v4.8.3, and the data was analyzed using Universal Analysis v4.4A.

Alternatively, DSC data will be collected on a Mettler DSC 823 e equipped with a 34-bit autosampler. Use qualified indium to calibrate the energy and temperature of the instrument. Typically, 0.5-3 mg of each sample is heated from 25 ° C to 300 ° C or from 25 ° C to 320 ° C in a pinhole aluminum pan at 10 ° C / min. A nitrogen purge was maintained on the sample at 50 ml / min. The instrument control and data analysis software is STARe v9.20.

Thermogravimetric analysis (TGA)

TGA data was collected on a Mettler TGNSDT A 851 e equipped with a 34-bit autosampler. Temperature calibration of the instrument with qualified indium. Generally, 1-30 mg of each sample is loaded into a pre-weighed aluminum crucible and heated from ambient temperature to 350 ° C at 10 ° C / min. A nitrogen purge was maintained on the sample at 50 ml / min. The instrument control and data analysis software is STARe v9.20.

Example 1. Preparation of Compound A Form A method

Crystals [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid (Compound A Form A) were prepared by the following method.

Method I

The crystalline compound A form A (see Example 1, Method I) was used in this method. 15 mg of crystalline material was used while each solvent was added in increments until a clean solution was obtained or until 50 volumes (750 μL) of solvent were added. After each solvent was added, the samples were ultrasonicated for 5 seconds. When insoluble, the slurry was stirred between 25 ° C and 50 ° C (4 h at each temperature) at 500 rpm for a period from 16 hours to six days. Any resulting solution was then evaporated at room temperature. The solids obtained from this experiment were analyzed by XRPD.

Each of the following solvents used in Method I above provides Form A: acetic acid, acetone, acetophenone, benzonitrile, benzyl alcohol, butyronitrile, chlorobenzene, cyclohexanone, 1,2-dichlorobenzene, 1, 2-dichloroethane, dimethoxyethane, dimethylacetamide, DMSO, 1,4-dioxane, ethylene glycol, EtoAc, formamidine, hexafluorobenzene, hexane, IPA, IPA: 10% water, iPrOAc, MeCN, MEK, MIBK, nitromethane, perfluorohexane, propionitrile, cyclobutane, tertiary butyl methyl ether, tertiary butanol, tetrahydronaphthalene, THF and toluene.

Using Method I, hexafluoropropan-2-ol, methanol and ethanol did not provide Form A.

Method II

Crystalline Compound A Form A (see Example 1, Method VIII) was used in this method. 15 mg of crystalline material and 30 volumes (450 μL) of solvent were used, with the exception of 5 volumes of DMSO and DMA. The slurry was ultrasonically treated for 5 seconds. The slurry was stirred at 5 ° C and 500 rpm for a period of six days. Any resulting solution was then allowed to evaporate at room temperature. Solid obtained by XRPD line.

Each of the following solvents used in Method II above provides Form A: benzonitrile, cyclobutane, formamidine, tetralin, acetophenone, benzyl alcohol, ethylene glycol, 1,2-dichlorobenzene, Chlorobenzene, cyclohexanone, butyronitrile, acetic acid, nitromethane, propionitrile, dimethoxyethane, 1,2-dichloroethane, hexafluorobenzene, tertiary butanol, hexane, and perfluorohexane alkyl.

Using Method II, hexafluoropropan-2-ol did not provide Form A.

Method III

Crystalline Compound A Form A (see Example 1, Method VIII) was used in this method. Method 3 is essentially as described in Method II above, except that the slurry is stirred at 50 ° C and 500 rpm for a period of six days. The obtained solid was analyzed by XRPD.

Each of the following solvents used in Method 3 above provides Form A: benzonitrile, cyclobutane, formamidine, tetralin, acetophenone, benzyl alcohol, ethylene glycol, 1,2-dichlorobenzene, Chlorobenzene, cyclohexanone, butyronitrile, acetic acid, nitromethane, propionitrile, dimethoxyethane, 1,2-dichloroethane, hexafluorobenzene, tertiary Butanol, hexane and perfluorohexane.

Using method III, dimethylacetamide, tertiary butyl methyl ether and hexafluoropropan-2-ol did not provide Form A.

Method IV

The crystalline compound A form B (see Example 2) was used in this method. 15 mg of crystalline material and 30 volumes (450 μL) of solvent were used, with the exception of 5 volumes of DMSO and DMA. The slurry was ultrasonically treated for 5 seconds. The slurry was stirred at 500 rpm between 25 ° C and 50 ° C (4 hours at each temperature) for six days. Any resulting solution was then left to evaporate quickly at room temperature. The solids were analyzed by XRPD.

Each of the following solvents used in Method IV above provides Form A: benzonitrile, cyclobutane, formamidine, tetralin, acetophenone, benzyl alcohol, ethylene glycol, 1,2-dichlorobenzene, Chlorobenzene, cyclohexanone, butyronitrile, acetic acid, tertiary butyl methyl ether, nitromethane, propionitrile, dimethoxyethane, 1,2-dichloroethane, hexafluorobenzene, tertiary butanol, Hexane, perfluorohexane and hexafluoropropan-2-ol.

Method V

Crystalline Compound A Form A (see Example 1, Method VIII) was used in this method. Before adding 10 volumes (200 μL) of solvent, 20 mg of crystal material was dissolved in tetrahydrofuran (410 μL), with the exception of using 5 volumes of DMSO and DMA. The slurry was stirred at 500 rpm between 25 ° C and 50 ° C (4 hours at each temperature) for 48 hours. Make in hot / Any solution obtained after the cold cycle was evaporated at room temperature. The obtained solid was analyzed by XRPD.

Each of the following solvents used in Method V above provides Form A: benzonitrile, cyclobutane, formamidine, tetralin, acetophenone, benzyl alcohol, ethylene glycol, DMSO, 1,2-dichloro Benzene, chlorobenzene, cyclohexanone, butyronitrile, acetic acid, tertiary butyl methyl ether, propionitrile, dimethoxyethane, 1,2-dichloroethane, hexafluorobenzene, tertiary butanol, and hexane.

Using method V, nitromethane, hexafluoropropan-2-ol and perfluorohexane did not provide Form A.

Method VI

Crystalline Compound A Form A (30 mg, see Example 1, Method VIII) was dissolved in 10 mL of acetone. This solution was subjected to rapid solvent evaporation on a rotary evaporator (40 ° C, 35-50 Torr). 12.85 mg of the resulting material was used with 10 volumes (128.5 μL) of solvent, with the exception of using 5 volumes of DMSO and DMA. The slurry was subjected to ultrasonic treatment for 5 seconds. The slurry was stirred at 500 rpm between 25 ° C and 50 ° C (8-hour cycle) for a period of 6 days. Any resulting solution was then allowed to evaporate at room temperature. The obtained solid was analyzed by XRPD.

Each of the following solvents used in Method VI above provides Form A: benzonitrile, cyclobutane, formamidine, tetralin, acetophenone, benzyl alcohol, ethylene glycol, DMSO, 1,2-dichloro Benzene, chlorobenzene, butyronitrile, acetic acid, tertiary butyl methyl ether, nitromethane, propionitrile, dimethoxyethane, 1,2-dichloroethane, hexafluorobenzene, tertiary butanol and hexane.

Using Method VI, cyclohexanone, hexafluoropropan-2-ol and perfluorohexane did not provide Form A.

Method VII

Crystalline Compound A Form A (see Example 1, Method VIII) was used in this method. 30 mg was suspended in 7 volumes of solvent (10% water). The slurry was ultrasonically treated for 5 seconds. The slurry was stirred at 500 rpm between 25 ° C and 50 ° C (8-hour cycle) for a period of 4 days. The obtained solid was analyzed by XRPD.

Each of the following solvents used in the above method VII provides Form A: acetone, acetonitrile, ethanol, methanol, 2-methyl-tetrahydrofuran, and isopropanol.

Method VIII

An aqueous sodium hydroxide solution was slowly added to the stirred suspension of Compound A in water over a temperature range (10 ° C to 90 ° C). The aqueous acetic acid solution was then slowly added over a temperature range (10 ° C to 90 ° C) and the mixture was stirred. The solid was filtered, washed with water, and dried under vacuum to constant weight. A white to pale yellow crystalline solid Compound A Form A was obtained.

data

The XRPD pattern of Compound A Form A is shown in Figure 1, and the peaks in the XRPD pattern and their related intensities are shown in Table 1 below.

The results of differential scanning calorimetry and thermogravimetric analysis of Compound A Form A are shown in Figures 2 and 24, respectively. Thermogravimetric analysis showed a negligible weight loss of about 0.4% between 25 ° C and 225 ° C, followed by a steady weight loss above 225 ° C, indicating the material sublimation or decomposition at these temperatures (Figure 24). Differential scanning calorimetry analysis of Compound A Form A showed a very shallow exothermic line in the range of about 80-190 ° C, followed by a sharp endothermic line (maximum peak) at about 224.3 ° C. The sharp endothermic line corresponds to the melting of the material, as determined by a hotstage microscopy.

A hot stage microscope of Compound A, Form A shows a material below its melting point Minor changes in materials. Some changes in birefringence were recorded in the range of about 150-200 ° C. The sample melted in a temperature range of about 218.5-222.4 ° C.

The moisture adsorption data for Compound A Form A shows a negligible weight gain, obtaining a weight gain of about 0.2% relative humidity between 5% and 95%, which is lost upon desorption. Smaller moisture adsorption of compound A form A indicates a kinetic non-absorbent material.

Example 2. Preparation of Compound A Form B method

The crystalline compound A form B was provided by lyophilizing form A in a mixture of 1,4-dioxane: water (2: 1). 20 mg of crystalline compound A form B was dissolved in 20 volumes of 1,4-dioxane, and then 20 volumes of co-solvent was added. The solvent system was allowed to evaporate in a fume hood at room temperature. The solids obtained from this experiment were analyzed by XRPD.

Each of the following cosolvents used in the method described above provides Form B. 1,4-dioxane: water (1: 1), 1,4-dioxane: water (1: 1), 1,4-dioxane: methanol (1: 1), 1,4-dioxane Dioxane: ethanol, 1,4-dioxane: acetone (1: 1), 1,4-dioxane: tetrahydrofuran (1: 1), and 1,4-dioxane: heptane (1: 1) .

data

The XRPD pattern of Compound A Form B is shown in FIG. 3, and the peaks in the XRPD pattern and their related intensities are shown in Table 2 below. After the sample was stored at 25 ° C / 96% RH for 12 days, the crystal pattern changed. And return to form A.

Except for water, no residual solvents were observed. A weight loss of 2.8% w / w in the TGA thermogram between room temperature and 90 ° C indicates the presence of 0.5 equivalents of water (theoretical 2.5% w / w) (Figure 4). DSC thermograms show endothermic events related to weight loss (Figure 4). A strong endothermic event occurred at 222.3 ° C (-127.8 J / g), which matched the melting of Form A.

Collect high-resolution XRPD data for more than one month. After a full month (32 days) at ambient temperature, the sample (morphology B) returned almost completely to anhydrous form A.

Example 3. Preparation of Compound A Form C method

Following the procedures described in Methods I, II, III, and IV of Example 1, using hexafluoroprop-2-ol as a solvent, crystals were prepared [(4-hydroxy-1-methyl-7-phenoxy- Isoquinoline-3-carbonyl) -amino] -hexafluoropropan-2-ol acetate solvate (Compound A Form C).

data

The XRPD pattern C of the compound A form C is shown in FIG. 5, and the peak in the XRPD pattern and its related intensity are shown in Table 3 below.

The residual solvent was observed by proton nuclear magnetic resonance, and it was identified as hexafluoropropan-2-ol. Thermal analysis was also performed on this sample (Figure 6). A weight loss of 7.8% w / w in the TGA thermogram between room temperature and 130 ° C indicates the presence of 1/6 equivalents of hexafluoropropan-2-ol in the sample (theoretical 7.36% w / w). The DSC thermogram showed an endothermic event related to weight loss, followed by a small exothermic event (ca. 130 ° C) (Figure 6). A strong endothermic event occurred at 222.2 ° C (-17.9 J / g), which matched the melting of Form A. In summary, the hexafluoropropan-2-ol-separated material is a metastable solvate under ambient conditions and is converted to Form A.

Example 4. Preparation of Compound A Form D method

THF / DMSO (20 Volume THF / 5 volume DMSO) slowly prepared crystals [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid DMSO: water solvate (Compound A Form D).

data

The XRPD pattern of Compound A Form D is shown in FIG. 8, and the peaks in the XRPD pattern and their related intensities are shown in Table 4 below. When dried at ambient temperature, the sample (Form D) was converted to Form A.

Residual solvent was observed by proton nuclear magnetic resonance, and it was identified as DMSO. The TGA and DSC thermograms are shown in Figure 8. TGA shows a first weight loss of 18.5% (a combination of water and DMSO) between 40-150 ° C and a second weight loss (possible DMSO) between 170-220 ° C. Due to the loss of water and DMSO, two broad endotherms at 37.6 ° C and 90.4 ° C are possible. Due to the melting of Form A, a small endotherm was observed at 222.0 ° C.

Example 5. Preparation of salts of compound A method

The following salt was prepared using crystalline Compound A, Form A. Compound A Form A (50 mg per experiment) was dissolved in acetone or tetrahydrofuran (50 volumes, 2.1 ml) at 50 ° C. The solution is treated with 1.1 mol eq. Of the corresponding counter ion (for example, 1.0 M aqueous sodium hydroxide, potassium hydroxide or hydrochloric acid). The temperature was maintained at 50 ° C for 20 minutes, and then cooled to 0 ° C at 0.1 ° C / min while stirring. After 20 h at 0 ° C, the solid was filtered, air-dried for 10 min, and analyzed by appropriate techniques.

data Compound A sodium salt

The XRPD pattern of the sodium salt of compound A is shown in FIG. 9, and the peaks in the XRPD pattern and their related intensities are shown in Table 5 below.

By ion chromatography (Metrohm761 Compact IC, IC Net software v2.3), the stoichiometry (ionic compound A: counter ion) was determined to be 1: 1. The TGA and DSC thermograms are shown in Figure 10. The TGA thermogram shows a weight loss of 11.5% between 40-90 ° C. The DSC thermogram showed a wide endotherm at 64.1 ° C, followed by two exotherms at 150.5 ° C and 190.3 ° C and a sharp melt at 313.6 ° C. The purity was determined to be about 99.6%.

Compound A potassium salt

By ion chromatography (Metrohm761 Compact IC, IC Net software v2.3), the stoichiometry (ionic compound A: counter ion) was determined to be 1: 1. The XRPD pattern of the potassium salt of Compound A is shown in FIG. 21. As observed in the figure, the potassium salt is substantially amorphous. Thermal analysis indicated a possible loss of water, followed by a recrystallization event to produce a non-solvated crystal morphology that melted at 291 ° C (Figure 22).

L-spermine salt of compound A

The XRPD pattern of the L-spermine salt of compound A is shown in FIG. 11, and the peaks in the XRPD pattern and their related intensities are shown in Table 6 below.

By NMR, the stoichiometry (ionic compound A: counter ion) was determined to be about 1: 1. The TGA and DSC thermograms are shown in Figure 12. The TGA thermogram shows a weight loss of 4.5% between 40-100 ° C. The DSC thermogram showed two broad endotherms at 79.6 ° C and 143.3 ° C, an exotherm at 172.5 ° C, and then an endotherm at 210.1 ° C. The purity was determined to be about 99.5%.

L-lysine salt of compound A

The XRPD pattern of the L-lysine salt of Compound A is shown in Figure 13, And the peaks in the XRPD pattern and their related intensities are shown in Table 7 below.

By NMR, the stoichiometry (ionic compound A: counter ion) was determined to be about 1: 1. The TGA and DSC thermograms are shown in Figure 14. The TGA thermogram shows a weight loss of 12.1% between 235-270 ° C. The DSC thermogram showed a sharp melt at 230.7 ° C and a wide endotherm at 237.1 ° C, and the purity was determined to be about 99.6%.

Ethanolamine salt of compound A

The XRPD pattern of the ethanolamine salt of Compound A is shown in FIG. 15, and the peaks in the XRPD pattern and their related intensities are shown in Tables 8, 9 and 10 below. Pattern 1 was observed from acetone, pattern 2 was observed from tetrahydrofuran, and pattern 3 was observed at 40 ° C / 75% RH.

For tetrahydrofuran and acetone, the stoichiometry (ionic compound A: counter ion) was determined to be about 1: 1 by NMR. The TGA and DSC thermograms of the ethanolamine salt of the compound A derived from acetone are shown in FIG. 16. The TGA thermogram showed a 10.1% weight loss (0.8 equivalents of ethanolamine) between 155-250 ° C. The DSC thermogram showed a sharp melt at 171.4 ° C and a wide endotherm at 186.0 ° C. The purity was determined to be about 99.0%. For the ethanolamine salt of compound A derived from tetrahydrofuran, the TGA thermogram is 155- A weight loss of 10.1% (0.8 equivalents of ethanolamine) was shown between 250 ° C, and the DSC thermogram showed a sharp melt at 172.4 ° C and a wide endotherm at 185.5 ° C. The purity was determined to be about 99.1%.

Diethanolamine salt of compound A

The XRPD pattern of the diethanolamine salt of Compound A is shown in FIG. 17, and the peaks in the XRPD pattern and their related intensities are shown in Tables 11 and 12 below. Pattern 1 was observed from acetone, and pattern 2 was observed at 40 ° C / 75% RH.

By NMR, the stoichiometry (ionic compound A: counter ion) was determined to be about 1: 1. The TGA and DSC thermograms are shown in Figure 18. The TGA thermogram shows a weight loss of 12.6% between 155-250 ° C. The DSC thermogram showed a sharp melt at 150.2 ° C and a broad endotherm at 172.2 ° C. The purity was determined to be about 99.7%.

Trimethylamine salt of compound A

The XRPD pattern of the tromethamine salt of compound A is shown in FIG. 19, The peaks in the XRPD pattern and their related intensities are shown in Table 13 below.

By NMR, the stoichiometry (ionic compound A: counter ion) was determined to be about 1: 1. The TGA and DSC thermograms are shown in Figure 20. The TGA thermogram shows a weight loss of 11.0% between 180-260 ° C. The DSC thermogram showed a sharp melt at 176.5 ° C and a broad endotherm at 182.6 ° C. The purity was determined to be about 99.7%.

Compound A Hydrochloride

The XRPD pattern of the hydrochloride of compound A is shown in FIG. 25, and the peaks in the XRPD pattern and their related intensities are shown in Table 14 below.

By ion chromatography, the stoichiometry (ionic compound A: counter ion) was determined to be about 1: 1. The TGA and DSC thermograms are shown in Figure 26. The TGA thermogram showed a 6.5% weight loss between 100-170 ° C and a second weight loss of 3.4% between 185-210 ° C. DSC thermograms show two small endotherms at 154.3 and 201.6 ° C, and at 223.0 ° C Melt sharply. The purity was determined to be about 99.1%.

Compound A sulfate

The XRPD pattern of the sulfate salt of compound A is shown in FIG. 27 and is a mixture of salt and form A. The peaks in the XRPD pattern and their relative intensities are shown in Table 15 below.

The TGA and DSC thermograms are shown in Figure 28. The TGA thermogram showed a 6.5% weight loss between 10-110 ° C and a second weight loss of 27.4% between 180-280 ° C. DSC thermograms show three small endotherms associated with the first weight loss at 31.8, 55.7, and 91.0. And due to decomposition it showed a large, broad endotherm at 201.4 ° C.

Compound A mesylate

The XRPD pattern of the mesylate salt of Compound A is shown in FIG. 29, and the peaks in the XRPD pattern and their related intensities are shown in Tables 16 and 17 below. Pattern 1 was observed from acetone, and pattern 2 was observed from tetrahydrofuran. Pattern 1 returned to Form A at 40 ° C / 75% RH, and Pattern 2 returned to the mixture of Form A and Pattern 1 at 40 ° C / 75% RH.

For tetrahydrofuran and acetone, the stoichiometry (ionic compound A: counter ion) was determined to be about 1: 1 by NMR. The TGA thermogram and DSC thermogram of the mesylate salt of compound A derived from tetrahydrofuran are shown in FIG. 30. The TGA thermogram shows a 2.1% weight loss between 40-100 ° C. The DSC thermogram showed a small endotherm at 153.5 ° C and a sharp endotherm at 166.9 ° C. The purity was determined to be about 99.3%. For acetone derived The TGA thermogram of the mesylate salt of Compound A showed no weight loss until the sample was degraded at about 180 ° C, and the DSC thermogram showed an endotherm due to the sample melting at 144.5 ° C. The purity was determined to be about 98.9%.

Example 6. Preparation of Amorphous A

The amorphous compound A was obtained as follows. The crystalline compound A form A (500 mg) was dissolved in tetrahydrofuran (1.5 mL) at room temperature. The solution was filtered to remove any residual crystalline material. The solvent was removed by rapid evaporation in a rotary evaporator. A small portion of the obtained solid was checked by XRPD. Alternatively, Compound A Form A from a 1,4-dioxane: water (2: 1 v / v) mixture by lyophilization, or by packing and sealing Compound A in a capillary, and placing the sample at about 240 Melted on a Kofler hot stage for 1 minute and cooled at ambient temperature to obtain amorphous compound A. The XRPD of the amorphous compound A is shown in FIG. 23.

Example 7. Preparation of Ditriethylamine of Compound A method

The crystalline compound A form A (50 mg) was dissolved in acetone (50 volumes) at 50 ° C. The solution was treated with 2.1 mol eq. Of triethylamine (TEA). The temperature was maintained at 50 ° C for 20 minutes, and then cooled to 0 ° C at 0.1 ° C / min while stirring. After 72 h at 0 ° C, the solid was filtered, air-dried for 5 min and analyzed by XRPD. The solution was set to evaporate slowly under ambient conditions.

data

The XRPD pattern of the double TEA salt of compound A is shown in FIG. 31, and the peaks in the XRPD pattern and their related intensities are shown in Table 18 below.

The TGA and DSC thermograms are shown in Figure 32. TEA was separated into double salts, however, after storage at 40 ° C / 75% RH for one week, the salts began to dissociate into Form A.

Example 8. Preparation of hemi-calcium and magnesium salts method

Calcium and magnesium salts of compound A were prepared from sodium salts by ion exchange. Compound A (450 mg) was dissolved in acetone (50 volumes, 22.5 ml) at 50 ° C. The solution was treated with 1.1 mol eq. Of sodium hydroxide (1M in water). A suspension formed after the addition, which was then cooled to 0 ° C at 0.1 ° C / min. After 48 h at 0 ° C, the solid was filtered, air-dried for 10 min, and analyzed by XRPD. The sodium salt (50 mg) was dissolved in methanol (20 volumes, 1 ml) at room temperature. The solution was heated to 50 ° C and treated with the corresponding counter ion (1M methanol solution). The mixture was cooled to 0 ° C at 0.1 ° C / min. After 24 h at 0 ° C, the solid was filtered, air dried for 5 min and analyzed by XRPD (first batch). The liquid was stored and set to evaporate slowly under ambient conditions to provide a second batch. The materials isolated from the Ca ⁺² and Mg ⁺² half-salt experiments crystallized after one week incubation at 40 ° C / 75% RH.

data

The XRPD patterns of the hemi-calcium and magnesium salts of Compound A are shown in Figure 33 and Figure 35, respectively. The peaks in the XRPD pattern and their associated intensities are shown in Tables 19 and 20 below.

DSC thermograms of the hemi-calcium and magnesium salts of Compound A are shown in Figure 34 and Figure 36, respectively.

Example 9. Compound A Form A Single Crystal

Single crystals are grown from acetone of sufficient quality for structural determination by single crystal X-ray diffraction.

The structural solution is obtained by a direct method, and the full matrix least squares correction is performed on F ² with a weighted w ^-1 = σ2 ( F _o ² ) + (0.0697 P ) ² + (0.3149 P ), where P = ( F _o ² +2 F _c ² ) / 3, an anisotropic displacement parameter, using empirical absorption correction of spherical harmonics, is implemented in the SCALE3 ABSPACK scaling algorithm. For all data, the final wR ² = {Σ [ w ( F _o ² -2 F _c ² ) ² ] / Σ [ w ( F _o ² ) ² ] ^½ } = 0.1376, for those with F _o > 4σ ( F _o The F value of the 2496 reflection, usually R ₁ = 0.0467, for all data and 248 parameters, S = 1.045. Final Δ / σ (max), 0.000, Δ / σ (average), 0.000. The final difference is mapped between +0.211 and -0.318 eÅ ^-3 .

Figure 37 shows a molecular view of Compound A Form A according to the crystal structure showing the numbering scheme used. Anisotropic atomic displacement ellipsoids of non-hydrogen atoms are shown at a 50% probability level. Hydrogen atoms are displayed with an arbitrarily small radius.

Example 10. Preparation of Compound A a) 5-phenoxyphthalide

The reactor was charged with DMF (68 Kg) and stirring was started. The reactor was then filled with phenol (51 Kg), acetone (8 Kg), 5-bromophthalide (85 Kg), copper bromide (9 Kg), and potassium carbonate (77 Kg). The mixture was heated above 85 ° C and held until the reaction was complete, then cooled. Add water. The solid was filtered and washed with water. The solid was dissolved in dichloromethane, washed with aqueous HCl, and then with water. The solvent was removed under pressure and methanol was added. The mixture was stirred and filtered. The solid was washed with methanol and dried in an oven to give 5-phenoxyphthalide (yield: 72%, HPLC: 99.6%).

b) methyl 2-chloromethyl-4-phenoxybenzoate

Toluene (24 Kg) was charged into the reactor and stirring was started. The reactor was then charged with 5-phenoxyphthalide (56 Kg), thionyl chloride (41 Kg), trimethyl borate (1 Kg), dichlorotriphenylphosphorane (2.5 Kg), and potassium carbonate (77 Kg). The mixture was heated to reflux until the reaction was complete, and then the solvent was removed, leaving 2-chloromethyl-4-phenoxybenzidine chloride. Fill with methanol and heat the mixture over 50 ° C until the reaction is complete. The solvent was removed and replaced with DMF. In the next step, a solution of the product methyl 2-chloromethyl-4-phenoxybenzoate in DMF (HPLC: 85%) was used directly.

c) Methyl 4-hydroxy-7-phenoxyisoquinoline-3-carboxylate (1a)

A solution of methyl 2-chloromethyl-4-phenoxybenzoate in DMF (about 68 Kg) was charged into the reactor and stirring was started. The reactor was then charged with methyl p-toluenesulfonylglycinate (66 Kg), potassium carbonate (60 Kg) and sodium iodide (4 Kg). The mixture was heated to at least 50 ° C until the reaction was complete. Cool the mixture. A methanol solution of sodium methoxide was added, and the mixture was stirred until the reaction was completed. Acetic acid and water were added, the mixture was stirred, filtered and washed with water. The solid was purified with acetone powder and dried in the oven, resulting in 1a (yield from step b): 58%; HPLC: 99.4%). ¹ H NMR (200 MHz, DMSO-d6) δ 11.60 (s, 1 H), 8.74 (s, 1 H), 8.32 (d, J = 9.0 Hz, 1 H), 7.60 (dd, J = 2.3 and 9.0 Hz, 1 H) 7.49 (m, 3 H), 7.24 (m, 3 H), 3.96 (s, 3 H); MS-(+)-ion M + 1 = 296.09.

d) 1-((dimethylamino) methyl) -4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester (1b)

1a (29.5 g) and acetic acid (44.3 g ± 5%) were charged into the flask and then stirred. Slowly add bis-dimethylaminomethane (12.8 g ± 2%). The mixture was heated to 55 ± 5 ° C and held until the reaction was complete. The reaction products were estimated by MS, HPLC and ¹ H NMR. ¹ H NMR (200 MHz, DMSO-d6) δ 11.7 (s, 1 H), 8.38 (d, J = 9.0 Hz, 1 H), 7.61 (dd, J = 9.0, 2.7 Hz, 1 H), 7.49 (m, 3 H), 7.21 (m, 3 H), 5.34 (s, 2H), 3.97 (s, 3 H), 1.98 (s, 3 H); MS-(+)-ion M + 1 = 368.12 .

e) 1-((Ethyloxy) methyl) -4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester (1c)

The solution of 1b from a) above was cooled to below 25 ° C, at which time acetic anhydride (28.6 g ± 3.5%) was added to keep the temperature below 50 ° C. The resulting mixture was heated to 100 ± 5 ° C until the reaction was completed.

The solutions from 1c and 1d above were cooled to below 65 ± 5 ° C. Water (250 mL) was added slowly. The mixture was then cooled to below 20 ± 5 ° C and filtered. The moist cake was washed with water (3 x 50 mL) and added to a new flask. Dichloromethane (90 mL) and water (30 mL) were added, and the resulting mixture was stirred. The dichloromethane layer was separated and estimated by HPLC.

The organic layer was added to the flask and cooled 5 ± 5 ° C. Morpholine was added and the mixture was stirred until the reaction was complete. The solvent was replaced with a mixture of acetone / methanol. After cooling, compound 1c precipitated and was filtered, washed and dried in an oven (yield: 81%, HPLC:> 99.7%). ¹ H NMR (200 MHz, DMSO-d6) δ 11.6 (S, 1 H), 8.31 (d, J = 9.0 Hz, 1 H), 7.87 (d, J = 2.3 Hz, 1 H), 7.49 (m 3H), 7.24 (m, 3H), 3.95 (s, 3H), 3.68 (s, 2H), 2.08 (s, 6H); MS-(+)-ion M + 1 = 357.17.

f) 4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxylic acid methyl ester (1e)

1c (16.0 g), Pd / C (2.08 g), anhydrous sodium carbonate (2.56 g), and ethyl acetate (120 mL) were charged to the reactor. The flask was vacuum purged with nitrogen (3 X) and vacuum purged with hydrogen (3 X). The flask was then pressurized with hydrogen and stirred at about 60 ° C until the reaction was completed. The flask was cooled to 20-25 ° C, the pressure was released to the environment, the headspace was flushed three times with nitrogen, and the mixture was filtered. The filtrate was concentrated. Methanol was added. Stir and then cool the mixture. The product was precipitated and filtered and dried in an oven (yield: 90%, HPLC: 99.7%).

g) [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid (Compound A)

Fill 1e (30.92 g), glycine (22.52 g), methanol (155 mL), sodium methoxide solution (64.81 g) into a pressure flask and seal it (as an alternative, sodium glycine is used instead of glycine And sodium methoxide). The reaction was heated to about 110 ° C until the reaction was complete. The mixture was cooled, filtered, washed with methanol, dried under vacuum, dissolved in water and washed with ethyl acetate. The ethyl acetate was removed, and a solution of acetic acid (18.0 g) was added to the resulting aqueous layer. The suspension was stirred at room temperature, filtered, the solid was washed with water (3 × 30 mL), cold acetone (5-10 ° C, 2 × 20 mL), and dried under vacuum to obtain compound A (yield: 86.1%, HPLC: 99.8%).

Example 11. Biological test

The solid forms provided herein can be used to inhibit HIF hydroxylase activity, thereby increasing the stability and / or activity of hypoxia-inducible factor (HIF), and can be used to treat and prevent hypoxia-induced factor-related disorders and disorders (see For example, U.S. Patent No. 7,323,475, U.S. Patent Application Publication No. 2007/0004627, U.S. Patent Application Publication No. 2006/0276477, and U.S. Patent Application Publication No. 2007/0259960, incorporated herein by reference).

The biological activity of the solid form provided herein can be evaluated using any conventionally known methods. In a specific embodiment, cells derived from animal tissues, preferably human tissues, when stimulated by a compound of the invention, can express erythropoietin and are cultured for the production of endogenous proteins in vitro. Cells contemplated for use in this method include, but are not limited to, cells derived from liver, hematopoietic, kidney, and neural tissue.

Cell culture techniques are generally available in the prior art and include maintenance Any method to maintain cell viability and promote endogenous protein expression. Cells are typically cultured in growth media optimized for cell growth, viability, and protein yield. The cells can be in suspension or attached to a matrix, and the medium can be supplied in a batch feed or continuous flow regime. The compound of the present invention is added to the culture medium in an amount that stimulates erythropoietin production without affecting cell viability. The erythropoietin produced by the cells is secreted into the culture medium. The culture medium is then collected and erythropoietin is purified using methods known to those skilled in the art (see, eg, Lai et al. (1987) US Patent No. 4,667,016; and Egrie (1985) US Patent No. 4,558,006).

Suitable assay methods are well known in the art. The following is given as an example and not as a limitation.

Cell-based HIFα Stability Analysis

Human cells derived from various tissues (for example, Hep3B cells from liver cell tissues) were individually seeded into 35 mm culture dishes and grown in a standard medium at 37 ° C, 20% O ₂ , and 5% CO ₂ For example, DMEM (Dalbock's Modified Eagle's Medium), 10% FBS (fetal bovine serum). When the cell layer reached confluence, the medium was replaced with OPTI-MEM medium (Invitrogen Life Technologies, Carlsbad, CA), and the cell layer was warmed at 37 ° C in 20% O ₂ , 5% CO ₂ Incubate for about 24 hours. Compound A or 0.013% DMSO (dimethylarsine) was then added to the existing medium and the incubation was continued overnight.

After incubation, the medium was removed, centrifuged and stored for analysis (see cell-based VEGF and EPO assays below). In cold phosphate buffered saline (PBS) Cells were washed twice and then in 1 mL of 10 mMTris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 0.5% IGEPAL (Sigma-Aldrich, St. Louis, Missouri), and a protease inhibitor mix (Roche Molecular Biochemicals) And lysed on ice for 15min. The cell lysate was centrifuged at 3000 xg for 5 min at 4 ° C, and the cytoplasmic fraction (supernatant) was collected. Nuclei (particles) were resuspended and lysed in 100 μL of 20 mM HEPES (pH 7.2), 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol and protease mixture (Roche Molecular Biochemicals), and lysed at 13,000xg Centrifuge at 4 ° C for 5 min, and collect the nuclear protein fraction (supernatant).

The collected nuclear protein fractions were analyzed for HIF-1α using a QUANTIKINE immunoassay (R & D Systems, Inc., Minneapolis MN) according to the manufacturer's instructions.

Cell-based EPO analysis

At 25,000 cells per well, Hep3B cells (human liver cancer cells from ATCC, cat # HB-8064) were seeded in 96-well plates. The next day, the cells were washed once with DMEM (Cellgro, cat # 10-013-CM) + 0.5% fetal bovine serum (Cellgro, cat # 35-010-CV), and mixed with compounds or vehicles of various concentrations (vehicle) The control (0.15% DMSO) was incubated in DMEM + 0.5% fetal bovine serum for 72 hours. A cell-free culture supernatant was generated by transferring to a 96-well plate at the bottom of the cone and centrifuging at 2000 rpm for 5 min. Human EPO ELISA kit (R & D Systems, cat # DEP 00) was used to quantify the EPO of the supernatant.

The EPO values of the compounds reported herein (eg, Table 22) are the measured values of cells plus compound minus the value of the vehicle control used for the same cell preparation. The EPO value of the vehicle control used for cell preparation varied between 0-12.5 mIU / mL.

HIF-PH determination

Ketoglutarate α- [1- ¹⁴ C] -sodium salt, α-ketoglutarate sodium salt, and HPLC purified peptides were obtained from, for example, Perkin-Elmer (Wellesley MA), Sigma-Aldrich ( (Sigma-Aldrich) and SynPep Corp (Doublin CA) and other commercial sources. The peptide used for the assay is the HIFα fragment as described above or disclosed in International Publication WO2005 / 118836, which is incorporated herein by reference. For example, the HIF peptide used in the HIF-PH assay is [methoxycoumarin] -DLDLEALAPYIPADDDFQL-amidamine. HIF-PH, such as HIF-PH2 (also known as EGLN1 or PHD2), is expressed in, for example, insect Hi5 cells, and is partially purified, for example, by a SP ion exchange chromatography column. Enzyme activity was measured by capturing ¹⁴ CO ₂ using the assay described by Kivirikko and Myllyla (1982, Methods Enzymol , 82: 245-304). The assay reaction included 50 mM HEPES (pH 7.4), 100 μM α-ketoglutarate sodium salt, 0.30 μ Ci / mL α-ketoglutarate α- [1- ¹⁴ C] sodium salt, 40 μM FeSO ₄ , 1 mM ascorbate, 1541.8 units / mL catalase, with or without 50 μM peptide matrix, and compounds of the invention at various concentrations. The reaction was started by adding HIF-PH enzyme.

Peptide-dependent percentage turnover was calculated by subtracting the lack of matrix peptide percent turnover from the percent turnover of matrix peptide present. Inhibitor is used at a concentration of a given peptide - dependent percent turnover Percent inhibition is calculated and IC _50. IC ₅₀ values _were calculated for each inhibitor using the GraFit software (Erithacus Software Ltd., Surrey UK) . The results are summarized in Table 22.

Table 21 below is used to demonstrate the pharmacological effects of Compound A. By inhibiting HIF proline hydroxylase (such as PHD2, also known as EGLN1), compound A stabilizes HIFα, which thus binds HIFβ to form an active transcription factor that increases participation in response to hypoxia and ischemia The manifestations of many genes of the disorder, including erythropoietin (EPO). Therefore, Compound A is useful for the prevention, pretreatment, or treatment of HIF and or EPO-related disorders, including anemia, ischemia, and hypoxia.

* Compared with DMSO-only control, cellular EPO measured with 30 μM compound in DMSO

Claims (25)

A crystal [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl) -amino] -acetic acid (compound A form A), characterized by X-ray powder diffraction The graph includes the following peaks: 8.5, 12.8, 16.2, 21.6, 22.9, and 27.4 ° 2θ ± 0.2 ° 2θ. The compound A form A according to claim 1, wherein the diffraction pattern is substantially as shown in FIG. 1. The compound A form A according to claim 1, wherein a differential scanning calorimetry (DSC) curve includes an endotherm at about 223 ° C. The compound A form A according to claim 3, wherein the DSC curve is substantially as shown in FIG. 2. A pharmaceutical composition comprising the compound A form A according to claim 1 and a pharmaceutically acceptable excipient. The pharmaceutical composition according to claim 5, wherein at least 85% of Compound A is Form A. The pharmaceutical composition according to claim 5, wherein at least 90% of Compound A is Form A. The pharmaceutical composition according to claim 5, wherein at least 95% of Compound A is Form A. The pharmaceutical composition according to claim 5, wherein at least 99% of the compound A is in the form A. The pharmaceutical composition according to claim 5, wherein at least 99.5% of Compound A is Form A. The pharmaceutical composition according to claim 5, wherein at least 99.9% of Compound A is Form A. The pharmaceutical composition according to claim 5, wherein at least 99.99% of Compound A is Form A. Use of Compound A, Form A according to claim 1 in the manufacture of a medicament for treating, pretreating or delaying the onset or aggravation of a condition at least partially caused by hypoxia-inducible factor (HIF) in a patient in need . Use according to claim 13, wherein the at least partially HIF-mediated condition is tissue damage associated with ischemia or hypoxia. Use according to claim 14, wherein the ischemia is associated with an acute event selected from the group consisting of myocardial infarction, pulmonary embolism, intestinal infarction, chronic renal failure, ischemic stroke, and renal ischemia-reperfusion injury. Use according to claim 14, wherein the ischemia is associated with a chronic event selected from the group consisting of cardiac sclerosis, transient ischemic attack, macular degeneration, peripheral arterial disease, and congestive heart failure. A compound A form A according to claim 1 for use in the manufacture of a medicament for treating, pretreating or delaying the onset or worsening of a condition at least partially erythropoietin (EPO) mediated in a patient in need. A compound A form A according to claim 1 for use in the manufacture of a medicament for treating, pretreating or delaying the onset or worsening of anemia in a patient in need. Use according to claim 13, 17 or 18, wherein at least 85% of the compound is compound A form A. Use according to claim 13, 17 or 18, wherein at least 90% of the compound is compound A form A. Use according to claim 13, 17 or 18, wherein at least 95% of the compound is compound A form A. Use according to claim 13, 17 or 18, wherein at least 99% of the compound is compound A form A. Use according to claim 13, 17 or 18, wherein at least 99.5% of the compound is compound A form A. Use according to claim 13, 17 or 18, wherein at least 99.9% of the compound is compound A form A. Use according to claim 13, 17 or 18, wherein at least 99.99% of the compound is compound A form A.