Polymorphs of Vadadustat and Methods for Preparing the Polymorphs

The present invention relates to crystalline and non-crystalline forms of vadadustat, to processes for their preparation, and to pharmaceutical compositions containing the crystalline or non-crystalline forms.

Background

Vadadustat has the IUPAC name of 2-[[5-(3-chlorophenyl)-3-hydroxypyridine-2- carbonyl]amino]acetic acid and has the chemical structure illustrated below:

Vadadustat has not yet been approved in the United States or in Europe. It is an oral hypoxia- inducible factor prolyl hydroxylase inhibitor (HIF-PHI) in clinical development for the treatment of anemia due to chronic kidney disease (CKD) in dialysis dependent and non-dialysis dependent adult patients (https://akebia.com/research-and-development/; accessed 23 July 2020).

Information about the solid-state properties of a drug substance is important. For example, different forms may have differing solubilities. Also, the handling and stability of a drug substance may depend on the solid form.

Polymorphism may be defined as the ability of a compound to crystallise in more than one distinct crystal species and different crystal arrangements of the same chemical composition are termed polymorphs. Polymorphs of the same compound arise due to differences in the internal arrangement of atoms and have different free energies and therefore different physical properties such as solubility, chemical stability, melting point, density, flow properties, hygroscopicity, bioavailability, and so forth. The compound vadadustat may exist in a number of polymorphic forms and many of these forms may be undesirable for producing pharmaceutically acceptable compositions. This may be for a variety of reasons including lack of stability, high hygroscopicity, low aqueous solubility and difficulty in handing. Definitions

The term "about" or "approximately" means an acceptable error for a particular value as determined by a person of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3 or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of a given value or range. In certain embodiments and with reference to X- ray powder diffraction two-theta peaks, the terms "about" or "approximately" means within ± 0.2 degrees 2Q.

The term "ambient temperature" means one or more room temperatures between about 15 °C to about 30 °C, such as about 15 °C to about 25 °C.

The term "amorphous" describes a solid which is not crystalline i.e. one that has no long-range order in its lattice (see, Oxford Dictionary of Chemistry, 6th Edition, 2008).

The term "anti-solvent" refers to a first solvent which is added to a second solvent to reduce the solubility of a compound in that second solvent. The solubility may be reduced sufficiently such that precipitation of the compound from the first and second solvent combination occurs.

The term "consisting" is closed and excludes additional, unrecited elements or process steps in the claimed invention.

The term "consisting essentially of" is semi-closed and occupies a middle ground between "consisting" and "comprising". "Consisting essentially of" does not exclude additional, unrecited elements or process steps which do not materially affect the essential characteristic(s) of the claimed invention.

The term "comprising" is inclusive or open-ended and does not exclude additional, unrecited elements or process steps in the claimed invention. The term is synonymous with "including but not limited to". The term "comprising" encompasses three alternatives, namely (i) "comprising", (ii) "consisting", and (iii) "consisting essentially of".

The term "crystalline" and related terms used herein, when used to describe a compound, substance, modification, material, component or product, unless otherwise specified, means that the compound, substance, modification, material, component or product is substantially crystalline as determined by X-ray diffraction. See, e.g., Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott, Williams and Wilkins, Baltimore, Md. (2005); The United States Pharmacopeia, 23rd ed., 1843-1844 (1995). The term "molecular complex" is used to denote a crystalline material composed of two or more different components which has a defined single-phase crystal structure. The components are held together by non-covalent bonding, such as hydrogen bonding, ionic bonding, van der Waals interactions, pi-pi interactions, etc. The term "molecular complex" includes salts, cocrystals and salt/co-crystal hybrids.

In one embodiment, the molecular complex is a co-crystal. Without wishing to be bound by theory, it is believed that when the molecular complex is a co-crystal, the co-crystal demonstrates improved physiochemical properties, such as crystallinity, solubility properties and/or modified melting points.

The terms "polymorph," "polymorphic form" or related term herein, refer to a crystal form of one or more molecules of vadadustat, or vadadustat molecular complex, that can exist in two or more forms, as a result of different arrangements or conformations of the molecule(s) in the crystal lattice of the polymorph.

The term "pharmaceutical composition" is intended to encompass a pharmaceutically effective amount of vadadustat of the invention and a pharmaceutically acceptable excipient. As used herein, the term "pharmaceutical compositions" includes pharmaceutical compositions such as tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, or injection preparations.

The term "excipient" refers to a pharmaceutically acceptable organic or inorganic carrier substance. Excipients may be natural or synthetic substances formulated alongside the active ingredient of a medication, included for the purpose of bulking-up formulations that contain potent active ingredients (thus often referred to as "bulking agents," "fillers," or "diluents"), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance, such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life.

The term "overnight" refers to the period of time between the end of one working day to the subsequent working day in which a time frame of about 12 to about 18 hours has elapsed between the end of one procedural step and the instigation of the following step in a procedure.

The term "patient" refers to an animal, preferably a patient, most preferably a human, who has been the object of treatment, observation or experiment. Preferably, the patient has experienced and/or exhibited at least one symptom of the disease or disorder to be treated and/or prevented. Further, a patient may not have exhibited any symptoms of the disorder, disease or condition to be treated and/prevented, but has been deemed by a physician, clinician or other medical professional to be at risk for developing said disorder, disease or condition.

The terms "treat," "treating" and "treatment" refer to the eradication or amelioration of a disease or disorder, or of one or more symptoms associated with the disease or disorder. In certain embodiments, the terms refer to minimizing the spread or worsening of the disease or disorder resulting from the administration of one or more therapeutic agents to a patient with such a disease or disorder. In some embodiments, the terms refer to the administration of a polymorph or molecular complex provided herein, with or without other additional active agents, after the onset of symptoms of a disease.

"Slurry" means a heterogeneous mixture of at least a portion of solid vadadustat in one or more solvents e.g. water. "Slurry" therefore includes a mixture of vadadustat which is partially present as a solid, as well as being partially dissolved in the solvent(s).

Brief Description of the Figures

Certain aspects of the embodiments described herein may be more clearly understood by reference to the drawings, which are intended to illustrate but not limit, the invention, and wherein:

Figure 1 is a representative XRPD pattern of amorphous vadadustat.

Figure 2 is a representative TGA thermogram and a DSC thermogram of amorphous vadadustat.

Figure 3 is a representative XRPD pattern of anhydrous vadadustat.

Figure 4 is a representative TGA thermogram and a DSC thermogram of anhydrous vadadustat.

Figure 5 is a representative IDR dissolution profile for anhydrous vadadustat and the vadadustat urea molecular complex at pH 5.5.

Figure 6 is a representative XRPD pattern of vadadustat urea molecular complex.

Figure 7 is a representative TGA thermogram and a DSC thermogram of vadadustat urea molecular complex.

Figure 8 shows the asymmetric unit of the vadadustat urea molecular complex. Figure 9 illustrates how centrifugal forces are applied to particles in the Speedmixer™. Figure 9A is a view from above showing the base plate and basket. The base plate rotates in a clockwise direction.

Figure 9B is a side view of the base plate and basket.

Figure 9C is a view from above along line A in Figure 9B. The basket rotates in an anti-clockwise direction.

Figure 10 is a representative XRPD pattern of vadadustat crystalline Form CS2.

Figure 11 is a representative TGA thermogram and a DSC thermogram of vadadustat crystalline Form CS2.

Description of the invention Amorphous vadadustat

It has been discovered that vadadustat can be prepared as an amorphous form. The vadadustat polymorph provided by the present invention is useful as an active ingredient in pharmaceutical formulations. In certain embodiments, the amorphous form is purifiable. In certain embodiments and depending on time, temperature and humidity, the amorphous form is stable. In certain embodiments, the amorphous form is easy to isolate and handle. In certain embodiments, the process for preparing the amorphous form is scalable. In certain embodiments, the amorphous form may exhibit a higher solubility as compared to a crystalline form.

The amorphous form described herein may be characterised using a number of methods known to the skilled person in the art, including X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), infrared spectroscopy, Raman spectroscopy, nuclear magnetic resonance (NMR) spectroscopy (including solution and solid- state NMR). The chemical purity may be determined by standard analytical methods, such as thin layer chromatography (TLC), gas chromatography, high performance liquid chromatography (HPLC), and mass spectrometry (MS).

In one aspect, the present invention provides amorphous vadadustat. In one embodiment, the amorphous form may have the X-ray powder diffraction pattern substantially as shown in Figure 1, in which it can be seen that the form has no long-range order in its lattice.

Amorphous vadadustat may have a DSC thermogram comprising an endothermic event with an onset temperature of about 171.5 °C. In one embodiment, the amorphous material may comprise an exothermic event with an onset temperature of about 73.4 °C. In one embodiment, the amorphous material may have a DSC thermogram substantially as shown in Figure 2.

Amorphous vadadustat may have a TGA thermogram comprising a mass loss of about 6.6% when heated from about ambient temperature to about 150 °C. In one embodiment, the amorphous material may have a TGA thermogram substantially as shown in Figure 2.

Amorphous vadadustat may be free or substantially free of other polymorphic forms of vadadustat. In certain embodiments, the polymorphic purity of the amorphous material is ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95% or higher. In certain embodiments, the polymorphic purity of the amorphous material is ≥ 95%. In certain embodiments, the polymorphic purity of the amorphous material is ≥ 96%. In certain embodiments, the polymorphic purity of the amorphous material is ≥ 97%. In certain embodiments, the polymorphic purity of the amorphous material is ≥ 98%. In certain embodiments, the polymorphic purity of the amorphous material is ≥ 99%.

The amorphous vadadustat described above may be prepared by a process comprising the steps of:

(a) dissolving vadadustat in a suitable solvent to form a solution of vadadustat;

(b) flash freezing the solution of vadadustat; and

(c) quickly removing the solvent to form the amorphous vadadustat.

The solvent may be any suitable solvent which is capable of producing a clear solution of vadadustat and which can be flash frozen. An example of a suitable solvent includes but is not limited to tetrahydrofuran, water, or a mixture thereof. In one embodiment, the suitable solvent is a mixture of water and tetrahydrofuran, for example, the v/v ratio of water to tetrahydrofuran may be about 1 : about 4 v/v, about 3 : about 7 v/v, about 2 : about 3 v/v, such as about 3 : about 7 v/v.

The w/v ratio of vadadustat to total solvent may be in the range of about 1 g of vadadustat : about 1 to about 50 ml of solvent, such as about 1 g of vadadustat : about 10 to about 30 ml of solvent, for example about 1 g of vadadustat : about 15 to about 25 ml of solvent.

The vadadustat may be dissolved in the solvent at ambient temperature or less. Alternatively, the vadadustat may be dissolved in the solvent at a temperature greater than ambient i.e. greater than 30 °C and below the boiling point of the reaction mixture. The boiling point of the reaction mixture may vary depending on the pressure under which the contacting step is conducted. In one embodiment, the dissolution step is carried out at atmospheric pressure (i.e. 1.0135 x 10 ⁵ Pa). Flash freezing may occur by any suitable means, such as use of a dry ice/acetone bath.

The solvent may be removed by means of lyophilisation (i.e. solvent freeze-drying), spray drying, etc. The solvent is removed at a speed which does not allow the vadadustat to crystallise.

In another aspect, the present invention relates to a pharmaceutical composition comprising amorphous vadadustat as described herein and a pharmaceutically acceptable excipient.

In another aspect, the present invention relates to a method for treating anemia in a patient with chronic kidney disease comprising administering a therapeutically effective amount of amorphous vadadustat as described herein to the patient.

In another aspect, the present invention relates to amorphous vadadustat as described herein for use in treating anemia due to chronic kidney disease.

Anhydrous vadadustat

It has been discovered that vadadustat can be prepared in a well-defined and consistently reproducible anhydrous crystalline form. Moreover, a reliable and scalable method for producing this anhydrous crystalline form has been developed. The vadadustat polymorph provided by the present invention is useful as an active ingredient in pharmaceutical formulations. In certain embodiments, the anhydrous crystalline form is purifiable. In certain embodiments and depending on time, temperature and humidity, the anhydrous crystalline form is stable. In certain embodiments, the anhydrous crystalline form is easy to isolate and handle. In certain embodiments, the process for preparing the anhydrous crystalline form is scalable.

The crystalline form described herein may be characterised using a number of methods known to the skilled person in the art, including single crystal X-ray diffraction, X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), infrared spectroscopy, Raman spectroscopy, nuclear magnetic resonance (NMR) spectroscopy (including solution and solid-state NMR). The chemical purity may be determined by standard analytical methods, such as thin layer chromatography (TLC), gas chromatography, high performance liquid chromatography (HPLC), and mass spectrometry (MS).

In one aspect, the present invention provides a crystalline form of vadadustat which is crystalline vadadustat anhydrate.

The crystalline vadadustat anhydrate may be free or substantially free of other polymorphic forms of vadadustat. In certain embodiments, the polymorphic purity of the anhydrate is ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95% or higher. In certain embodiments, the polymorphic purity of the anhydrate is ≥ 95%. In certain embodiments, the polymorphic purity of the anhydrate is ≥ 96%. In certain embodiments, the polymorphic purity of the anhydrate is ≥ 97%. In certain embodiments, the polymorphic purity of the anhydrate is ≥ 98%. In certain embodiments, the polymorphic purity of the anhydrate is ≥ 99%.

The anhydrate may have an X-ray powder diffraction pattern comprising four or more peaks (for example 4, 5, 6, 7, 8, 9, or 10 peaks) selected from the group consisting of about 4.2,

12.6, 13.6, 15.4, 16.9, 18.1, 19.8, 21.1, 22.1, 23.3, 24.1, 24.9, 25.4, 25.8, 26.6, 27.3, 28.4,

28.7, 29.5, 30.3, 31.2, 32.2, 32.7, 33.8, 34.8, 36.4, 36.9, 38.0, 39.1, and 40.1 degrees two- theta ± 0.2 degrees two-theta. In one embodiment, the anhydrate may have the X-ray powder diffraction pattern substantially as shown in Figure 3.

The anhydrate may have a DSC thermogram comprising an endothermic event with an onset temperature of about 94.5 °C. The DSC thermogram may optionally comprise one or both of two further endothermic events with onset temperatures of about 158.3 °C and about 173 °C. The DSC thermogram may optionally comprise an exothermic event with an onset temperature of about 161.3 °C. The optional endothermic and/or exothermic events may be present depending on the thermodynamic nature of the anhydrate. In one embodiment, the anhydrate may have a DSC thermogram substantially as shown in Figure 4.

The anhydrate may have a TGA thermogram comprising no substantial mass loss when heated from about ambient temperature to about 150 °C. In one embodiment, the anhydrate may have a TGA thermogram substantially as shown in Figure 4.

The crystalline anhydrate vadadustat described above may be prepared by a process comprising the step of treating crystalline vadadustat hydrate under vacuum.

In one embodiment, the crystalline vadadustat hydrate may be Form CS2 as described in EP3549932A.

The vadadustat hydrate may be placed within an enclosed chamber under vacuum at a temperature from about ambient temperature to about 60 °C, for example, about 25 °C, or about 50 °C.

The period of time for which the crystalline vadadustat hydrate is treated under vacuum at the desired temperature is not particularly limiting. In one embodiment, the period of time may be from about 1 minute to about 7 days, for example, about 2 hours. The crystalline vadadustat anhydrate formed by the processes described above may be free or substantially free of other polymorphic forms of vadadustat. In certain embodiments, the polymorphic purity of the anhydrate is ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95% or higher. In certain embodiments, the polymorphic purity of the anhydrate is ≥ 95%. In certain embodiments, the polymorphic purity of the anhydrate is ≥ 96%. In certain embodiments, the polymorphic purity of the anhydrate is ≥ 97%. In certain embodiments, the polymorphic purity of the anhydrate is ≥ 98%. In certain embodiments, the polymorphic purity of the anhydrate is ≥ 99%.

In another aspect, the present invention relates to a pharmaceutical composition comprising crystalline vadadustat anhydrate as described herein and a pharmaceutically acceptable excipient.

In another aspect, the present invention relates to a method for treating anemia in a patient with chronic kidney disease comprising administering a therapeutically effective amount of crystalline vadadustat anhydrate as described herein to the patient.

In another aspect, the present invention relates to crystalline vadadustat anhydrate as described herein for use in treating anemia due to chronic kidney disease.

Vadadustat urea molecular complex

It has been discovered that vadadustat can be prepared in a well-defined and consistently reproducible urea molecular complex. Moreover, a reliable and scalable method for producing this molecular complex has been developed. The vadadustat molecular complex provided by the present invention may be useful as an active ingredient in pharmaceutical formulations. In certain embodiments, the crystalline molecular complex is purifiable. In certain embodiments and depending on time, temperature and humidity, the crystalline molecular complex is stable. In certain embodiments, the crystalline molecular complex is easy to isolate and handle. In certain embodiments, the process for preparing the crystalline molecular complex is scalable.

The crystalline molecular complex described herein may be characterised using a number of methods known to the skilled person in the art, including single crystal X-ray diffraction, X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), infrared spectroscopy, Raman spectroscopy, nuclear magnetic resonance (NMR) spectroscopy (including solution and solid-state NMR). The chemical purity may be determined by standard analytical methods, such as thin layer chromatography (TLC), gas chromatography, high performance liquid chromatography (HPLC), and mass spectrometry (MS). In another aspect, the present invention provides a crystalline molecular complex of vadadustat and urea.

The molar ratio of vadadustat to urea may be in the range of about 1 mole of vadadustat : about 0.5 to about 1.5 moles of urea, for example about 1 mole of vadadustat : about 0.8 to about 1.2 moles of urea. In one embodiment, the molar ratio of vadadustat to urea may be about 1 mole of vadadustat : about 1 mole of urea.

The vadadustat urea molecular complex may have an X-ray powder diffraction pattern comprising one or more peaks (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 peaks) selected from the group consisting of about 5.7, 11.4, 12.0, 13.2, 13.5, 13.9, 17.1, 17.3, 18.8, 19.6, 20.6,

20.8, 21.6, 22.5, 23.4, 24.8, 25.2, 25.6, 26.0, 26.3, 26.7, 27.3, 27.9, 28.4, 28.6, 29.0, 29.3,

29.5, 30.1, 30.4, 30.7, 31.7, 32.7, 33.0, 33.6, 34.2, 34.5, 34.8, 35.3, 36.4, 37.2, 37.8, 38.4,

38.8, 39.1, and 39.6 degrees two-theta ± 0.2 degrees two-theta. In one embodiment, the molecular complex may have the X-ray powder diffraction pattern substantially as shown in Figure 6.

The vadadustat urea molecular complex may have a DSC thermogram comprising an endothermic event with an onset temperature of about 162.1 °C. In one embodiment, the molecular complex may have a DSC thermogram substantially as shown in Figure 7.

The molecular complex may have a TGA thermogram comprising about 16.4% mass loss when heated from about ambient temperature to about 240 °C. In one embodiment, the molecular complex may have a TGA thermogram substantially as shown in Figure 7.

The crystalline vadadustat urea molecular complex formed may be free or substantially free of other polymorphic forms of vadadustat. In certain embodiments, the polymorphic purity of the molecular complex is ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95% or higher. In certain embodiments, the polymorphic purity of the molecular complex is ≥ 95%. In certain embodiments, the polymorphic purity of the molecular complex is ≥ 96%. In certain embodiments, the polymorphic purity of the molecular complex is ≥ 97%. In certain embodiments, the polymorphic purity of the molecular complex is ≥ 98%. In certain embodiments, the polymorphic purity of the molecular complex is ≥ 99%.

The vadadustat urea molecular complex described above may be prepared by a process comprising reacting vadadustat and urea using low energy ball milling or low energy grinding.

Urea is present in sufficient quantities to form the desired molecular complex. The molar ratio of vadadustat to urea may be in the range of about 1 mole of vadadustat : about 0.5 to about 1.5 moles of urea, for example about 1 mole of vadadustat : about 0.8 to about 1.2 moles of urea. In one embodiment, the molar ratio of vadadustat to urea may be about 1 mole of vadadustat : about 1 mole of urea.

When low energy ball milling is utilised, the milling process may be controlled by various parameters including the speed at which the milling takes place, the length of milling time and/or the level to which the milling container is filled.

The speed at which the milling takes place may be from about 50 rpm to about 1000 rpm. In one embodiment, the speed may be from about 75 rpm to about 750 rpm. In another embodiment, the speed may be from about 80 rpm to about 650 rpm. In one embodiment, the speed may be about 500 rpm.

Low energy grinding involves shaking the materials within a grinding container. The grinding occurs via the impact and friction of the materials within the container. The process may be controlled by various parameters including the frequency at which the grinding takes place, the length of grinding time and/or the level to which the container is filled.

The frequency at which the grinding takes place may be from about 1 Hz to about 100 Hz. In one embodiment, the frequency may be from about 10 Hz to about 70 Hz. In another embodiment, the frequency may be from about 20 Hz to about 50 Hz. In one embodiment, the frequency may be about 30 Hz.

Regardless of whether milling or grinding is used, milling or grinding media may be used to assist the reaction. In this instance, the incorporation of hard, non-contaminating media can additionally assist in the breakdown of particles where agglomeration has occurred, for example, as a result of the manufacturing process or during transit. Such breakdown of the agglomerates further enhances the reaction of vadadustat with urea. The use of milling/grinding media is well-known within the field of powder processing and materials such as stabilised zirconia and other ceramics are suitable provided they are sufficiently hard or ball bearings e.g. stainless steel ball bearings.

Regardless of whether milling or grinding is used, an improvement in the process can be made by controlling the particle ratio, the size of the milling/grinding media and other parameters as are familiar to the skilled person.

The length of milling or grinding time may be from about 1 minute to about 2 days, for example, about 2 minutes to about 5 hours, such as about 20 minutes to 3 hours, e.g. about 2 hours. The length of the milling or grinding time may be a continuous or aggregate period of time. By "continuous" we mean a period of time without interruption. By "aggregate" we mean the sum or total of more than one periods of time (e.g. 2, 3, 4, 5 or more times). The vadadustat and urea may be contacted at ambient temperature or less. Alternatively, the vadadustat may be contacted with the urea at a temperature greater than ambient i.e. greater than 30 °C and below the boiling point of the reaction mixture. The boiling point of the reaction mixture may vary depending on the pressure under which the contacting step is conducted. In one embodiment, the contacting step is carried out at atmospheric pressure (i.e. 1.0135 x 10 ⁵ Pa).

The process may be carried out in the presence or absence of solvent. In one embodiment, the process is carried out in the absence of solvent.

The process may be carried out in the presence of a solvent such as methanol, ethanol, and/or acetonitrile. The solvent may act to minimise particle welding. The addition of the solvent may be particularly helpful if the vadadustat and/or urea being reacted has agglomerated prior to use, in which case the solvent can assist with breaking down the agglomerates.

The quantity of solvent is not particularly limiting provided there is enough solvent to dissolve, suspend or moisten the vadadustat and/or urea. The w/v ratio of vadadustat to solvent may be in the range from about 1 mg of vadadustat : about 0.01 to about 1.5 μl solvent, such as about 1 mg of vadadustat : about 0.05 to about 1.0 μl solvent, for example about 1 mg of vadadustat : about 0.1 to about 0.75 μl solvent, e.g. about 1 mg of vadadustat : about 0.5 μl solvent. The solvent may be added in one portion or more than one portion (e.g. 2, 3, 4, or 5 portions).

The vadadustat and urea may be contacted with the solvent at ambient temperature or less. Alternatively, the vadadustat may be contacted with the solvent at a temperature greater than ambient i.e. greater than 30 °C and below the boiling point of the reaction mixture. The boiling point of the reaction mixture may vary depending on the pressure under which the contacting step is conducted. In one embodiment, the contacting step is carried out at atmospheric pressure (i.e. 1.0135 x 10 ⁵ Pa).

When the milling or grinding time is applied for an aggregate period of time, the presence or absence of solvent may be changed for each period of time. For example, the process may comprise a first period of time in which the environment is dry (i.e. vadadustat and urea are reacted together optionally with milling media in the absence of solvent), and a second period of time in which the environment is moistened (i.e. "wet") after the addition of solvent.

The vadadustat urea molecular complex is recovered as a crystalline solid. The crystalline molecular complex may be recovered by directly by filtering, decanting or centrifuging. If desired, a proportion of the solvent may be evaporated prior to recovery of the crystalline solid. Alternatively, the vadadustat urea molecular complex described above may be prepared by a process comprising the step of applying dual asymmetric centrifugal forces to a mixture of vadadustat and urea to form the molecular complex.

Urea is present in sufficient quantities to form the desired molecular complex. The molar ratio of vadadustat to urea may be in the range of about 1 mole of vadadustat : about 0.5 to about 1.5 moles of urea, for example about 1 mole of vadadustat : about 0.8 to about 1.2 moles of urea. In one embodiment, the molar ratio of vadadustat to urea may be about 1 mole of vadadustat : about 1 mole of urea.

The vadadustat urea molecular complex is formed using dual asymmetric centrifugal forces. By "dual asymmetric centrifugal forces" we mean that two centrifugal forces, at an angle to each other, are simultaneously applied to the particles. In order to create an efficient mixing environment, the centrifugal forces preferably rotate in opposite directions. The Speed mixer™ by Hauschild (http://www.speedmixer.co.uk/index.php) utilises this dual rotation method whereby the motor of the Speedmixer™ rotates the base plate of the mixing unit in a clockwise direction (see Figure 9A) and the basket is spun in an anti-clockwise direction (see Figures 9B and 9C).

The process may be controlled by various parameters including the rotation speed at which the process takes place, the length of processing time, the level to which the mixing container is filled, the use of milling media and/or the control of the temperature of the components within the milling pot.

The dual asymmetric centrifugal forces may be applied for a continuous period of time. By "continuous" we mean a period of time without interruption. The period of time may be from about 1 second to about 10 minutes, such as about 5 seconds to about 5 minutes, for example, about 10 seconds to about 200 seconds e.g. 2 minutes.

Alternatively, the dual asymmetric centrifugal forces may be applied for an aggregate period of time. By "aggregate" we mean the sum or total of more than one periods of time (e.g. 2, 3, 4, 5 or more times). The advantage of applying the centrifugal forces in a stepwise manner is that excessive heating of the particles can be avoided. The dual asymmetric centrifugal forces may be applied for an aggregate period of about 1 second to about 20 minutes, for example about 30 seconds to about 15 minutes and such as about 10 seconds to about 10 minutes e.g. 6 minutes. In one embodiment, the dual asymmetric centrifugal forces are applied in a stepwise manner with periods of cooling therebetween. In another embodiment, the dual asymmetric centrifugal forces may be applied in a stepwise manner at one or more different speeds. The speed of the dual asymmetric centrifugal forces may be from about 200 rpm to about 4000 rpm. In one embodiment, the speed may be from about 300 rpm to about 3750 rpm, for example about 500 rpm to about 3500 rpm. In one embodiment, the speed may be about 3500 rpm. In another embodiment, the speed may be about 2300 rpm.

The level to which the mixing container is filled is determined by various factors which will be apparent to the skilled person. These factors include the apparent density of the vadadustat and urea, the volume of the mixing container and the weight restrictions imposed on the mixer itself.

Milling media as described above may be used to assist the reaction. In certain embodiments, the dual asymmetric centrifugal forces may be applied in a stepwise manner in which milling media may be used for some, but not all, periods of time.

The process may be carried out in the presence or absence of solvent. In one embodiment, the process is carried out in the absence of solvent.

The process may be carried out in the presence of a solvent such as methanol, ethanol, and/or acetonitrile. The solvent may act to minimise particle welding. The addition of the solvent may be particularly helpful if the vadadustat and/or urea being reacted has agglomerated prior to use, in which case the solvent can assist with breaking down the agglomerates.

When the dual asymmetric centrifugal forces are applied for an aggregate period of time, the presence or absence of solvent may be changed for each period of time. For example, the process may comprise a first period of time in which the environment is dry (i.e. vadadustat and urea are reacted together optionally with milling media in the absence of solvent), and a second period of time in which the environment is moistened (i.e. "wet") after the addition of solvent.

The vadadustat urea molecular complex is recovered as a crystalline solid. The crystalline molecular complex may be recovered by directly by filtering, decanting or centrifuging. If desired, a proportion of the solvent (if present) may be evaporated prior to recovery of the crystalline solid.

Howsoever the crystalline molecular complex is recovered, the separated molecular complex may be dried. Drying may be performed using known methods, for example, at temperatures in the range of about 10 °C to about 60 °C, such as about 20 °C to about 40 °C, for example, ambient temperature under vacuum (for example about 1 mbar to about 30 mbar) for about 1 hour to about 24 hours. Alternatively, the crystalline molecular complex may be left to dry under ambient temperature naturally i.e. without the active application of vacuum. It is preferred that the drying conditions are maintained below the point at which the molecular complex degrades and so when the molecular complex is known to degrade within the temperature or pressure ranges given above, the drying conditions should be maintained below the degradation temperature or vacuum.

The crystalline vadadustat urea molecular complex described above may be prepared by a process comprising the steps of:

(a) contacting vadadustat and urea with a solvent selected from the group consisting of methanol, ethanol, acetonitrile, and a mixture thereof; and

(b) recovering vadadustat urea molecular complex as a crystalline solid.

Urea may be utilised as a solid, or as a solution in a solvent (e.g. methanol and/or ethanol).

In one embodiment, step (a) may comprise the steps of:

(a1) contacting vadadustat with a solvent selected from the group consisting of methanol, ethanol, acetonitrile, and a mixture thereof; and (a2) adding urea to the solution or suspension of vadadustat.

In another embodiment, step (a) may comprise the step of:

(a1') contacting a solid admixture of vadadustat and urea with a solvent selected from the group consisting of methanol, ethanol, acetonitrile, and a mixture thereof to form a solution or suspension.

The quantity of the solvent is not particularly limiting provided there is enough solvent (a) to dissolve the vadadustat and form a solution, or suspend the vadadustat, and/or (b) to dissolve the urea and form a solution, or suspend the urea. The w/v ratio of vadadustat to solvent may be in the range of about 1 mg of vadadustat : about 1 to about 1000 μl of solvent, such as about 1 mg of vadadustat : about 1 to about 500 μl of solvent, for example about 1 mg of vadadustat : about 1 to about 150 μl of solvent, e.g. about 1 mg of vadadustat : about 10 to about 60 mI of solvent. In one embodiment, the w/v ratio of vadadustat to solvent may be about 1 mg of vadadustat : about 15 to about 50 mI of solvent.

The vadadustat and urea may be contacted with the solvent at ambient temperature or less. In one embodiment, the contacting step may be carried out at one or more temperatures in the range of ≥ about 0 °C to about ≤ 25 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 1 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 2 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 3 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 4 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 5 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≤ about 20 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≤ about 15 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≤ about 10 °C. In one embodiment, the contacting step is carried out at one or more temperatures in the range of ≥ about 0 °C to ≤ about 10 °C, for example, about 5 °C. In one embodiment, the contacting step may be carried out at about ambient temperature e.g. about 25 °C.

Alternatively, the vadadustat and urea may be contacted with the solvent at a temperature greater than ambient i.e. greater than 30 °C and below the boiling point of the reaction mixture. The boiling point of the reaction mixture may vary depending on the pressure under which the contacting step is conducted. In one embodiment, the contacting step is carried out at atmospheric pressure (i.e. 1.0135 x 10 ⁵ Pa). In one embodiment, the contacting step may be carried out at one or more temperatures in the range of ≥ about 40 °C to about ≤ 60 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 41 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 42 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 43 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 44 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 45 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 46 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 47 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 48 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 49 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≥ about 50 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≤ about 59 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≤ about 58 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≤ about 57 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≤ about 56 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≤ about 55 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≤ about 54 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≤ about 53 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≤ about 52 °C. In some embodiments, the contacting step is carried out at one or more temperatures ≤ about 51 °C. In one embodiment, the contacting step is carried out at one or more temperatures in the range of ≥ about 45 °C to ≤ about 55 °C. In one embodiment, the contacting step is carried out at a temperature of about 50 °C. The dissolution or suspension of vadadustat and/or urea may be encouraged through the use of an aid such as stirring, shaking and/or sonication. Additional solvent may be added to aid the dissolution or suspension of the vadadustat and/or urea.

When urea is inputted into the reaction as a solid, the w/v ratio of urea to solvent may be in the range of about 1 mg of urea : about 1 to about 1000 μl of solvent, such as about 1 mg of urea : about 1 to about 500 mI of solvent, for example about 1 mg of urea : about 50 to about 350 mI of solvent, e.g. about 1 mg of urea : about 75 to about 250 mI of solvent.

The period of time for which the mixture of vadadustat, urea and solvent is treated at the desired temperature is not particularly limiting. In one embodiment, the period of time may be from about 1 minute to about 24 hours, for example, about 1 hour.

After combining the vadadustat, urea and solvent, the reaction mixture may be treated for a period of time at ambient temperature or less as described above.

The solution or suspension may then be cooled such that the resulting solution or suspension has a temperature below that of the solution or suspension of step (a), (a2), or (a1'). The rate of cooling may be from about 0.05 °C/minute to about 2 °C/minute, such as about 0.1 °C/minute to about 1.5 °C/minute, for example about 0.1 °C/minute or 0.5 °C/minute. A suspension may eventually be observed on cooling a solution of the reaction mixture.

The solution or suspension may be cooled to ambient temperature or a temperature of less than ambient temperature. In one embodiment, the solution or suspension may be cooled to one or more temperatures in the range of ≥ about 0 °C to about ≤ 20 °C. In some embodiments, the solution or suspension is cooled to one or more temperatures ≥ about 1 °C. In some embodiments, the solution or suspension is cooled to one or more temperatures ≥ about 2 °C. In some embodiments, the solution or suspension is cooled to one or more temperatures ≥ about 3 °C. In some embodiments, the solution or suspension is cooled to one or more temperatures ≥ about 4 °C. In some embodiments, the solution or suspension is cooled to one or more temperatures ≥ about 5 °C. In some embodiments, the solution or suspension is cooled to one or more temperatures ≤ about 15 °C. In some embodiments, the solution or suspension is cooled to one or more temperatures ≤ about 14 °C. In some embodiments, the solution or suspension is cooled to one or more temperatures ≤ about 13 °C. In some embodiments, the solution or suspension is cooled to one or more temperatures ≤ about 12 °C. In some embodiments, the solution or suspension may be cooled to one or more temperatures ≤ about 11 °C. In some embodiments, the solution or suspension is cooled to one or more temperatures ≤ about 10 °C. In one embodiment, the solution or suspension is cooled to one or more temperatures in the range of about 5°C to about 10 °C, for example, about 5 °C. The reaction mixture may be left for a further period of time, e.g. about 1 minute to about 10 days at the desired temperature.

In step (b), the vadadustat urea molecular complex is recovered as a crystalline solid. The crystalline molecular complex may be recovered directly by filtering, decanting or centrifuging. If desired, the suspension may be mobilised with additional portions of the solvent (e.g. methanol, ethanol and/or acetontrile) prior to recovery of the crystalline solid. Alternatively, a proportion or substantially all of the solvent may be evaporated prior to recovery of the crystalline solid.

Howsoever the crystalline molecular complex is recovered, the separated molecular complex may be washed with solvent (e.g. methanol, ethanol and/or acetontrile) and dried. Drying may be performed using known methods, for example, at temperatures in the range of about 10 °C to about 60 °C, such as about 20 °C to about 40 °C, for example, ambient temperature under vacuum (for example about 1 mbar to about 30 mbar) for about 1 hour to about 24 hours. Alternatively, the crystalline molecular complex may be left to dry under ambient temperature naturally i.e. without the active application of vacuum. It is preferred that the drying conditions are maintained below the point at which the molecular complex degrades and so when the molecular complex is known to degrade within the temperature or pressure ranges given above, the drying conditions should be maintained below the degradation temperature or vacuum.

Steps (a) → (b), (a1) → (a2) → (b), and (a1') → (b) may be carried out one or more times (e.g. 1, 2, 3, 4 or 5 times). When steps (a) → (b), (a1) → (a2) → (b), and (a1') → (b) are carried out more than once (e.g. 2, 3, 4 or 5 times), one or more of the steps may be optionally seeded as appropriate with crystalline vadadustat urea molecular complex (which was previously prepared and isolated by a method described herein).

In another aspect, the present invention relates to a pharmaceutical composition comprising crystalline vadadustat urea molecular complex as described herein and a pharmaceutically acceptable excipient.

In another aspect, the present invention relates to a method for treating anemia in a patient with chronic kidney disease comprising administering a therapeutically effective amount of crystalline vadadustat urea molecular complex as described herein to the patient.

In another aspect, the present invention relates to crystalline vadadustat urea molecular complex as described herein for use in treating anemia due to chronic kidney disease. Crystalline vadadustat Form CS2

EP3549932A describes crystalline vadadustat Form CS2. EP3549932A is incorporated herein by reference in its entirety for all purposes.

The crystalline vadadustat Form CS2 may have an X-ray powder diffraction pattern comprising one or more peaks (for example 1, 2, 3, 4, 5, 6, 7, 8, or 9 peaks) selected from the group consisting of about 10.9, 12.6, 13.4, 14.1, 15.0, 16.1, 18.3, 20.0, and 22.0 degrees two-theta ± 0.2 degrees two-theta. The crystalline vadadustat Form CS2 may have an X-ray powder diffraction pattern having peaks selected from the group consisting of about 3.6, 10.9, 12.6, 13.4, 14.1, 14.6, 15.0, 16.1, 17.3, 18.3, 20.0, 21.5, 22.0, 24.5, 24.9, 25.4, 25.8, 26.7, 27.0, 27.9, 28.5, 29.1, 29.6, 30.3, 30.7, 31.3, 31.7, 32.6, 33.1, 34.9, 35.0, 35.2, 36.6, 37.9, 38.1, 39.2, 39.7, 40.8, and 41.1 degrees two-theta ± 0.2 degrees two-theta. In one embodiment, Form CS2 may have the X-ray powder diffraction pattern substantially as shown in Figure 10.

Form CS2 may have a DSC thermogram comprising an endothermic event with an onset temperatures of about 84.9 °C. The DSC thermogram may further optionally comprise one or both of two endothermic events with onset temperatures of about 65.6 °C, and about 172.3 °C. Form CS2 may have a DSC thermogram substantially as shown in Figure 11.

Form CS2 may have a TGA thermogram comprising about 5.3% weight loss when heated from about ambient temperature to about 100 °C. Form CS2 may have a TGA thermogram substantially as shown in Figure 11.

In another aspect, the present invention provides a process for preparing crystalline vadadustat Form CS2 as described in EP3549932A, the process comprising the steps of:

(a) adding a solution of vadadustat in a solvent selected from the group consisting of methyl tetrahydrofuran, dimethoxyethane, and a mixture thereof to water; and

(b) recovering vadadustat Form CS2 as a crystalline solid.

Vadadustat is dissolved in an ethereal solvent selected from the group consisting of methyl tetrahydrofuran (e.g. 2- or 3-methyl tetrahydrofuran), dimethoxyethane (e.g. 1,1- or 1,2- dimethoxyethane), and a mixture thereof to form a solution. In one embodiment, the solvent is 2-methyl tetrahydrofuran. In another embodiment, the solvent is 1,2-dimethoxyethane.

The process is a reverse anti-solvent addition process in which a solution of vadadustat is added to water. The quantity of the solvent is not particularly limiting provided there is enough solvent to dissolve the vadadustat and form a solution. The w/v ratio of vadadustat to the first solvent may be in the range of about 1 mg of vadadustat : about 1 to about 100 μl of solvent, such as about 1 mg of vadadustat : about 1 to about 50 mI of solvent, for example about 1 mg of vadadustat : about 1 to about 20 mI of solvent e.g. about 1 mg of vadadustat : about 1 to about 10 mI of solvent.

The vadadustat may be dissolved in the first solvent at ambient temperature or less. In one embodiment, the dissolving step may be carried out at one or more temperatures in the range of ≥ about 0 °C to about ≤ 25 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 1 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 2 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 3 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 4 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 5 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≤ about 20 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≤ about 15 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≤ about 10 °C. In one embodiment, the dissolving step is carried out at one or more temperatures in the range of ≥ about 0 °C to ≤ about 10 °C, for example, about 5 °C. In one embodiment, the dissolving step may be carried out at about ambient temperature e.g. about 25 °C.

Alternatively, the vadadustat may be dissolved in the solvent at a temperature greater than ambient i.e. greater than 30 °C and below the boiling point of the reaction mixture. The boiling point of the reaction mixture may vary depending on the pressure under which the contacting step is conducted. In one embodiment, the dissolving step is carried out at atmospheric pressure (i.e. 1.0135 x 10 ⁵ Pa). In one embodiment, the dissolving step may be carried out at one or more temperatures in the range of ≥ about 40 °C to about ≤ 60 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 41 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 42 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 43 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 44 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 45 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 46 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 47 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 48 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 49 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≥ about 50 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≤ about 59 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≤ about 58 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≤ about 57 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≤ about 56 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≤ about 55 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≤ about 54 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≤ about 53 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≤ about 52 °C. In some embodiments, the dissolving step is carried out at one or more temperatures ≤ about 51 °C. In one embodiment, the dissolving step is carried out at one or more temperatures in the range of ≥ about 45 °C to ≤ about 55 °C. In one embodiment, the dissolving step is carried out at a temperature of about 50 °C.

The dissolution of vadadustat may be encouraged through the use of an aid such as stirring, shaking and/or sonication. Additional solvent may be added to aid the dissolution of the vadadustat.

The period of time for which the mixture of vadadustat and solvent is treated at the desired temperature is not particularly limiting. In one embodiment, the period of time may be from about 1 minute to about 24 hours, for example, about 5 minutes.

The solution of vadadustat may be optionally filtered before it is added to the water.

The water may be at maintained at ambient temperature before the addition of the vadadustat solution. Alternatively, the water may be cooled and maintained at a temperature less than ambient temperature before the addition of the vadadustat solution. In one embodiment, the water may be cooled and maintained at one or more temperatures in the range of ≥ about 0 °C to about ≤ 25 °C. In some embodiments the water may be cooled and maintained at one or more temperatures ≥ about 1 °C. In some embodiments, the water may be cooled and maintained at one or more temperatures ≥ about 2 °C. In some embodiments, the water may be cooled and maintained at one or more temperatures ≥ about 3 °C. In some embodiments, the water may be cooled and maintained at one or more temperatures ≥ about 4 °C. In some embodiments, the water may be cooled and maintained at one or more temperatures ≥ about 5 °C. In some embodiments, the water may be cooled and maintained at one or more temperatures ≤ about 20 °C. In some embodiments, the water may be cooled and maintained at one or more temperatures ≤ about 17 °C. In some embodiments, the water may be cooled and maintained at one or more temperatures ≤ about 15 °C. In one embodiment, the water may be cooled and maintained at one or more temperatures in the range of ≥ about 10 °C to ≤ about 20 °C, for example, about 15 °C.

The water may be optionally seeded with crystalline vadadustat Form CS2, which was previously prepared and isolated either by a method described herein or in as described in EP3549932A. The quantity of water is not particularly limiting provided that on addition of the vadadustat solution, crystalline Form CS2 precipitates. Typically, it is desirable that sufficient water is present to cause the precipitation of crystalline Form CS2 and, while excess water is not generally required, it does not appear to adversely affect the formation of Form CS2. The w/v of vadadustat to water may be in the range of about lg of vadadustat : about 25 ml to about 200 ml of water, such as about lg of vadadustat : about 50 ml to about 150 ml of water, for example about lg of vadadustat : about 60 ml to about 100 ml of water.

The v/v ratio of solvent : water may be in the range of about 1 ml solvent : about 10 to about 50 ml water, such as about 1 ml solvent : about 15 to about 30 ml solvent, such as about 1 ml solvent : about 20 to about 25 ml water. In one embodiment, the v/v ratio of solvent : water may be about 1 ml solvent : about 20 ml water. In another embodiment, the v/v ratio of solvent : water may be about 1 ml solvent : about 25 ml water.

The solution of vadadustat may be added at a controlled rate to the water. The controlled rate of addition does not include bulk addition in which the solution of vadadustat is added to the water in a single portion. The rate of addition may be any suitable rate capable of producing a suspension. The rate of addition may be adapted as appropriate by various parameters including the quantity and concentration of the solution of vadadustat to be added, the scale of the reaction, the size of the reaction vessels, and the length of processing time. In one embodiment, the solution of vadadustat is added to the water dropwise.

The controlled addition may take from about 5 minutes to about 60 minutes, such as about 10 to about 45 minutes, for example about 15 to about 30 minutes.

The reaction mixture may be optionally stirred during the controlled addition. Stirring may be continued for a further period of time after the addition is complete e.g. for about 1 hour to about 5 days, such as about 2 days.

The resulting suspension may be cooled. The rate of cooling may be from about 0.05 °C/minute to about 2 °C/minute, such as about 0.1 °C/minute to about 1.5 °C/minute, for example about 0.1 °C/minute or 0.5 °C/minute.

The suspension may be cooled to ambient temperature or a temperature of less than ambient temperature. In one embodiment, the suspension may be cooled to one or more temperatures in the range of ≥ about 0 °C to about ≤ 20 °C. In some embodiments, the suspension is cooled to one or more temperatures ≥ about 1 °C. In some embodiments, the s suspension is cooled to one or more temperatures ≥ about 2 °C. In some embodiments, the suspension is cooled to one or more temperatures ≥ about 3 °C. In some embodiments, the suspension is cooled to one or more temperatures ≥ about 4 °C. In some embodiments, the suspension is cooled to one or more temperatures ≥ about 5 °C. In some embodiments, the suspension is cooled to one or more temperatures ≤ about 15 °C. In some embodiments, the suspension is cooled to one or more temperatures ≤ about 14 °C. In some embodiments, the suspension is cooled to one or more temperatures ≤ about 13 °C. In some embodiments, the suspension is cooled to one or more temperatures ≤ about 12 °C. In some embodiments, the suspension may be cooled to one or more temperatures ≤ about 11 °C. In some embodiments, the suspension is cooled to one or more temperatures ≤ about 10 °C. In one embodiment, the suspension is cooled to one or more temperatures in the range of about 5°C to about 10 °C.

The suspension may be left for a further period of time, e.g. about 1 minute to about 10 days (such as about 2 days) at the desired temperature. The suspension may be optionally stirred during this time.

The crystalline vadadustat Form CS2 is recovered as a crystalline solid. The crystalline solid may be recovered directly by filtering, decanting or centrifuging. If desired, the suspension may be mobilised with additional portions of the solvent (e.g. water) prior to recovery of the crystalline solid. Alternatively, a proportion or substantially all of the solvent may be evaporated prior to recovery of the crystalline solid.

Howsoever the crystalline solid is recovered, the separated solid may be washed with solvent (e.g. water) and dried. Drying may be performed using known methods, for example, at temperatures in the range of about 10 °C to about 60 °C, such as about 20 °C to about 40 °C, for example, ambient temperature under vacuum (for example about 1 mbar to about 30 mbar) for about 1 hour to about 24 hours. Alternatively, the crystalline solid may be left to dry under ambient temperature naturally i.e. without the active application of vacuum. It is preferred that the drying conditions are maintained below the point at which the crystalline solid degrades and so when the crystalline solid is known to degrade within the temperature or pressure ranges given above, the drying conditions should be maintained below the degradation temperature or vacuum.

In yet another aspect, crystalline vadadustat Form CS2 as described in EP3549932A may be prepared by shaking a slurry of amorphous vadadustat as described above in water for a period of time and at a temperature effective to form the crystalline vadadustat Form CS2.

The quantity of water is not particularly limiting provided sufficient water is used to form a slurry which is capable of being stirred but not so much water that the amorphous vadadustat and/or crystalline Form CS2 dissolves. The w/v of amorphous vadadustat to water may be in the range of about 1 mg of vadadustat : about 1 μl to about 20 μl of water, such as about 1 mg of vadadustat : about 5 μl to about 15 mI of water, for example about 1 mg of vadadustat : about 7 mI to about 12 mI of water e.g. about 1 mg of vadadustat : about 10 μl of water. The reaction mixture may be shaken at ambient temperature or less. In one embodiment, the reaction mixture may be shaken at one or more temperatures in the range of ≥ about 0 °C to about ≤ 25 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≥ about 1 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≥ about 2 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≥ about 3 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≥ about 4 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≥ about 5 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≤ about 20 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≤ about 15 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≤ about 10 °C. In one embodiment, the reaction mixture may be shaken at one or more temperatures in the range of ≥ about 0 °C to ≤ about 10 °C, for example, about 5 °C. In one embodiment, the reaction mixture may be shaken at about ambient temperature e.g. about 25 °C.

Alternatively, the reaction mixture may be shaken at a temperature greater than ambient i.e. greater than 30 °C and below the boiling point of the reaction mixture. The boiling point of the reaction mixture may vary depending on the pressure under which the step is conducted. In one embodiment, the reaction mixture may be shaken at atmospheric pressure (i.e. 1.0135 x 10 ⁵ Pa). In one embodiment, the reaction mixture may be shaken at one or more temperatures in the range of ≥ about 40 °C to about ≤ 60 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≥ about 41 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≥ about 42 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≥ about 43 °C. In some embodiments, the reaction mixture may be shaken at at one or more temperatures ≥ about 44 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≥ about 45 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≥ about 46 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≥ about 47 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≥ about 48 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≥ about 49 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≥ about 50 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≤ about 59 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≤ about 58 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≤ about 57 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≤ about 56 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≤ about 55 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≤ about 54 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≤ about 53 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≤ about 52 °C. In some embodiments, the reaction mixture may be shaken at one or more temperatures ≤ about 51 °C. In one embodiment, the reaction mixture may be shaken at one or more temperatures in the range of ≥ about 45 °C to ≤ about 55 °C. In one embodiment, the reaction mixture may be shaken at a temperature of about 50 °C.

The period of time for which the mixture of vadadustat and water is shaken at the desired temperature is not particularly limiting. In one embodiment, the period of time may be from about 1 hour to about 10 days, for example, about 6 days.

The crystalline vadadustat Form CS2 is recovered as a crystalline solid. The crystalline solid may be recovered directly by filtering, decanting or centrifuging. If desired, the suspension may be mobilised with additional portions of the solvent (e.g. water) prior to recovery of the crystalline solid. Alternatively, a proportion or substantially all of the solvent may be evaporated prior to recovery of the crystalline solid.

Howsoever the crystalline solid is recovered, the separated solid may be washed with solvent (e.g. water) and dried. Drying may be performed using known methods, for example, at temperatures in the range of about 10 °C to about 60 °C, such as about 20 °C to about 40 °C, for example, ambient temperature under vacuum (for example about 1 mbar to about 30 mbar) for about 1 hour to about 24 hours. Alternatively, the crystalline solid may be left to dry under ambient temperature naturally i.e. without the active application of vacuum. It is preferred that the drying conditions are maintained below the point at which the crystalline solid degrades and so when the crystalline solid is known to degrade within the temperature or pressure ranges given above, the drying conditions should be maintained below the degradation temperature or vacuum.

Embodiments and/or optional features of the invention have been described above. Any aspect of the invention may be combined with any other aspect of the invention, unless the context demands otherwise. Any of the embodiments or optional features of any aspect may be combined, singly or in combination, with any aspect of the invention, unless the context demands otherwise.

The invention will now be described further by reference to the following examples, which are intended to illustrate but not limit, the scope of the invention.

Examples

Abbreviations API active pharmaceutical ingredient

DMSO dimethyl sulfoxide rpm revolutions per minute

RH relative humidity

RT room temperature

THF tetrahydrofuran

Instrument and Methodology Details

1.1 X-ray Powder Diffraction (XRPD)

1.1.1 Bruker AXS D8 Advance

XRPD diffractograms were collected on a Bruker D8 diffractometer using Cu Ka radiation (40 kV, 40 mA) and a Q-2Q goniometer fitted with a Ge monochromator. The incident beam passes through a 2.0 mm divergence slit followed by a 0.2 mm anti-scatter slit and knife edge. The diffracted beam passes through an 8.0 mm receiving slit with 2.5° Soller slits followed by the Lynxeye Detector. The software used for data collection and analysis was Diffrac Plus XRD Commander and Diffrac Plus EVA respectively.

Samples were run under ambient conditions as flat plate specimens using powder as received. The sample was prepared on a polished, zero-background (510) silicon wafer by gently pressing onto the flat surface or packed into a cut cavity. The sample was rotated in its own plane.

The details of the standard data collection method are:

• Angular range: 2 to 42° 2Q

• Step size: 0.05° 2Q

• Collection time: 0.5 s/step (total collection time: 6.40 min)

1.1.2 PANalytical Empyrean

XRPD diffractograms were collected on a PANalytical Empyrean diffractometer using Cu Ka radiation (45 kV, 40 mA) in transmission geometry. A 0.5° slit, 4 mm mask and 0.04 rad Soller slits with a focusing mirror were used on the incident beam. A PIXcel ³⁰ detector, placed on the diffracted beam, was fitted with a receiving slit and 0.04 rad Soller slits. The software used for data collection was X'Pert Data Collector using X'Pert Operator Interface. The data were analysed and presented using Diffrac Plus EVA or HighScore Plus.

Non-ambient conditions (XRPD under Vacuum)

XRPD diffractograms were collected on a PANalytical Empyrean diffractometer using Cu Ka radiation (45 kV, 40 mA) in reflection geometry. The instrument was fitted with an Anton Paar CHC plus ⁺ stage fitted with graphite/Kapton windows, equipped with air cooling coupled and a low vacuum pump system using an Edwards RV3 pump. A programmable divergence slit (in automatic mode), with a 10 mm fixed incident beam mask, Ni filter and 0.04 rad Soller slits were used on the incident beam. A PIXcel ³⁰ detector, placed on the diffracted beam, was fitted with a programmable anti-scatter slit (in automatic mode) and 0.04 rad Soller slits.

The software used for data collection was X'Pert Data Collector and the data analysed and presented using Diffrac Plus EVA or Highscore Plus.

For experiments under vacuum the samples were prepared and analysed in an Anton Paar chromed sample holder. The sample was held isothermally at 25 °C whilst the chamber was under vacuum.

1.2 Differential Scanning Calorimetry (DSC)

1.2.1 TA Instruments Q2000

DSC data were collected on a TA Instruments Q2000 equipped with a 50 position auto-sampler. Typically, 0.5 - 3 mg of each sample, in a pin-holed aluminium pan, was heated at 10 °C/min from 25 °C to 220 °C. A purge of dry nitrogen at 50 ml/min was maintained over the sample.

The instrument control software was Advantage for Q Series and Thermal Advantage and the data were analysed using Universal Analysis or TRIOS.

1.2.2 TA Instruments Discovery DSC

DSC data were collected on a TA Instruments Discovery DSC equipped with a 50 position autosampler. Typically, 0.5 - 3 mg of each sample, in a pin-holed aluminium pan, was heated at 10 °C/min from 25 °C to 200 °C. A purge of dry nitrogen at 50 ml/min was maintained over the sample.

The instrument control software was TRIOS and the data were analysed using TRIOS or Universal Analysis.

1.3 Thermo-Gravimetric Analysis (TGA)

1.3.1 TA Instruments Discovery TGA

TGA data were collected on a TA Instruments Discovery TGA, equipped with a 25 position autosampler. Typically, 5 - 10 mg of each sample was loaded onto a pre-tared aluminium DSC pan and heated at 10 °C/min from ambient temperature to 350 °C. A nitrogen purge at 25 ml/min was maintained over the sample.

The instrument control software was TRIOS and the data were analysed using TRIOS or Universal Analysis. 1.4 Four Hour Solubility

Sufficient sample was suspended in 1.0 ml media for a maximum anticipated concentration of ca. 10 mg/ml of the free form of the compound. The resulting suspensions were then shaken at 25 °C/ 750 rpm for 4 hours. The pH of the sample solutions was checked after 1 hour to ensure that the desired pH was maintained throughout (± 0.2).

After equilibration, the appearance was noted, and the final pH of the saturated solution was measured. Samples were then filtered through a glass 'C' fibre filter (particle retention size 1.2 μm).

The samples suspended in pH 6.8 and pH 7.5 buffer were diluted 10 times with appropriate buffers, all other samples were analysed undiluted.

Quantitation was by HPLC with reference to a standard solution of approximately 0.15 mg/ml. Different volumes of the standard, diluted and undiluted sample solutions were injected. The solubility was calculated using the peak areas determined by integration of the peak found at the same retention time as the principal peak in the standard injection.

HPLC method for solubility measurements

Analysis was performed on an Agilent HP1100/ Infinity II 1260 series system equipped with a diode array detector and using OpenLAB software. Preparation of solubility media

1.5 Sirius inForm

Data were collected on a Sirius inForm instrument fitted with a dual UV Dip Probe attachment and Ag/AgCI combination pH electrode. The electrode was calibrated using the four plus parameters derived from a blank titration. The base titrant was standardised by titration with TRIS. 0.5 M HCI and NaOH aqueous solutions were used as the acid and base titrants respectively for the testing. Stirring was facilitated by a dual overhead stirrer to allow thorough mixing within the vessel, and media was introduced via a capillary bundle attached to a dispensing bank comprised of six precision dispensing units. A Peltier heating jacket was used to maintain the temperature of the titration vessel. Discs were introduced to the vessel via the tablet picker housed in the probe arm, after the desired temperature of the media had been reached. Sirius inForm Assay Design, Control and Refine software were used to design, run and refine data respectively.

1.5.1 Molar Extinction Coefficient (MEC)

The reference sample was prepared as a 10.0 mM stock solution in DMSO. Two MEC data sets were collected using 50 μl and 150 μl aliquot additions of the DMSO stock, respectively. Each MEC assay was performed as a double titration, from pH 2.0 - 12.0 (low to high). Titration one was carried out under aqueous conditions in 40 ml media (36 ml ISA water, 4 ml 0.1 M acetate phosphate buffer). 20 ml ISA water was added in titration two, to build a multi-point MEC calibration. UV Spectra were collected after each addition of the DMSO stock over the physiological pH range, in order to capture any change in UV associated with a changing pH environment, using a 5 mm path length probe. The two data sets were then compiled into a multiset and imported into the dissolution data files in order to convert the UV absorbance measured to concentration. The concentration range for UV data collected was 11.5 - 53.9 pm.

1.5.2 Intrinsic Dissolution Rate (IDR)

Ca. 8 to 24 mg of the sample was compressed in a 3 mm disc recess, under 100 kg for 2 minutes, with greaseproof paper on the compression base, to form non-disintegrating discs. The discs were then plugged with a bung so that only one surface was exposed to the media during analysis and transferred to the Sirius inForm dissolution apparatus. Analysis was performed at 37 °C in 40 ml media (36 ml ISA water, 4 ml 0.1 M acetate phosphate buffer). Dissolution data were collected over 3 pH sectors (pH 5.5, 6.5 8i 7.4) for a total of 1.5 hours - 30 minutes per sector, with UV spectra collected every 30 seconds. A stir speed of 100 rpm was used with a 5 mm path length probe. The IDR was calculated based on the surface area of the 3 mm disc recess used (7.07 mm ² surface area).

Preparation of dissolution media Amorphous Vadadustat

Example 1

Vadadustat (approx. 1.00 g) was dissolved in a solvent mixture of 3:7 v/v water : THF (20 vol, 20 ml). The solution was frozen in dry ice/acetone bath and then lyophilised overnight. The resulting material was analysed by XRPD and identified as amorphous vadadustat.

Example 2

Vadadustat (2.00 g) was dissolved in a solvent mixture of 3:7 v/v water : THF (20 vol, 40 ml). The solution was frozen in dry ice/acetone bath and then lyophilised overnight. The resulting material was analysed by XRPD and identified as amorphous vadadustat.

Example 3

Vadadustat (1.25 g) was dissolved in a solvent mixture of 3:7 v/v water : THF (25 vol, 20 ml). The solution was transferred into HPLC vials (600 μl, approx. 30 mg). The vials of API solution were flash frozen in dry ice/acetone bath and then lyophilised overnight.

Characterisation of amorphous vadadustat

Figure 1 shows a representative XRPD pattern of amorphous vadadustat. Amorphous vadadustat was also characterised by TGA and DSC analysis (see Figure 2).

Anhydrous Vadadustat Example 4

Vadadustat crystalline form CS2 (as described in EP3549932A) (prepared according to Example 14) was placed under vacuum and analysed by XRPD. After 1.5 hours the material had fully converted to anhydrous vadadustat. The resulting material was analysed by XRPD and identified as anhydrous vadadustat.

Example 5

Vadadustat crystalline form CS2 (as described in EP3549932A) (prepared according to Example 14) was held isothermally at 50 °C under nitrogen for 1 hour in a TGA. The resulting material was analysed by XRPD and identified as anhydrous vadadustat.

Example 6

Vadadustat crystalline form CS2 (as described in EP3549932A) (1.00 g, prepared according to Example 14) was placed at 50 °C under vacuum for 1.5 hours. The resulting material was analysed by XRPD and identified as anhydrous vadadustat.

Characterisation of anhydrous vadadustat

Figure 3 shows a representative XRPD pattern of anhydrous vadadustat. The following table provides an XRPD peak list for the anhydrate:

Anhydrous vadadustat was also characterised by TGA and DSC analysis (see Figure 4). The solubility and the IDR of the anhydrous vadadustat were determined and the results are shown in the table below:

The IDR dissolution profile for anhydrous vadadustat at pH 5.5 is shown in Figure 5.

Vadadustat Urea Molecular Complex 1 mol ea Example 7

Vadadustat (30 mg) and urea (1 mol eq., 5 mg) were wetted with methanol (15 μl). Two stainless steel 3 mm grinding balls were added to the vial and the sample ground for 2 hours at 500 rpm in a planetary mill. After grinding the sample was left uncapped to dry the solid before analysis. The resulting material was analysed by XRPD and identified as vadadustat urea molecular complex 1 mol eq.

Example 8

Vadadustat (30 mg) and urea (1 mol eq., 6 mg) were wetted with ethanol (15 μl). Two stainless steel 3 mm grinding balls were added to the vial and the sample ground for 2 hours at 500 rpm in a planetary mill. After grinding the sample was left uncapped to dry the solid before analysis. The resulting material was analysed by XRPD and identified as vadadustat urea molecular complex 1 mol eq.

Example 9

Vadadustat (30 mg) and urea (1 mol eq., 6 mg) were wetted with acetonitrile (15 mI). Two stainless steel 3 mm grinding balls were added to the vial and the sample ground for 2 hours at 500 rpm in a planetary mill. After grinding the sample was left uncapped to dry the solid before analysis. The resulting material was analysed by XRPD and identified as vadadustat urea molecular complex 1 mol eq.

Example 10

Vadadustat (30 mg) and urea (1 mol eq., 6 mg) were dissolved in methanol (20 vol, 600 mI) and heated at 50 °C for 1 hour. The solution was cooled to 5 °C at 0.1 °C/min. The resulting material was analysed by XRPD and identified as vadadustat urea molecular complex 1 mol eq.

Example 11

Vadadustat (30 mg) and urea (1 mol eq., 6 mg) were dissolved in ethanol (40 vol, 1200 mI) and heated at 50 °C for 1 hour. The solution was cooled to 5 °C at 0.1 °C/min. The resulting material was analysed by XRPD and identified as vadadustat urea molecular complex 1 mol eq.

Example 12

Vadadustat (30 mg) and urea (1 mol eq., 6 mg) were suspended in acetonitrile (50 vol, 1500 mI) and heated at 50 °C for 1 hour. The solution was cooled to 5 °C at 0.1 °C/min. The resulting material was analysed by XRPD and identified as vadadustat urea molecular complex 1 mol eq.

Example 13

Vadadustat (1.00 g) and urea (1 mol eq., 200 mg) were dissolved in methanol (15 vol, 15 ml) and heated at 50 °C. The solution was cooled to 5 °C at 0.1 °C/min. The resulting suspension was filtered and dried under suction. The resulting material was analysed by XRPD and identified as vadadustat urea molecular complex 1 mol eq. Characterisation of vadadustat urea molecular complex 1 mol ea

Figure 6 shows a representative XRPD pattern of vadadustat urea molecular complex 1 mole eq. The following table provides an XRPD peak list for the molecular complex:

The molecular complex was also characterised by TGA and DSC analysis (see Figure 7).

Single crystals of the molecular complex were grown from the mother liquors of Example 13. The single crystal parameters for the molecular complex as determined by SCXRD are:

Crystal system Triclinic Space group P-1 Unit cell dimensions a = 7.0713(2) A o = 92.671(3)° b = 7.2727(2) A b = 91.455(2)° c = 15.5671(6) A y = 99.046(2)°

Volume 789.33(4) A ³

Figure 8 shows the asymmetric unit of the vadadustat urea molecular complex. The figure provides confirmation of the nature of the molecular complex in that the ratio of urea : vadadustat is 1: 1.

The molecular complex was stored under 40 °C/75% RH and 25 °C/97% RH, and samples analysed by XRPD after 7 and 42 days. XRPD analysis confirmed no changed in form. The solubility and the IDR of the molecular complex were determined and the results are shown in the table below:

The IDR dissolution profile for the vadadustat urea molecular complex at pH 5.5 is shown in Figure 5. Crystalline form CS2 fas described in EP3549932A1

Example 14 (not according to the invention)

Vadadustat (2.00 g) was dissolved in DMSO (5 vol, 10 ml) at 90 °C before cooling to 25 °C. The solution was filtered and transferred to a 1 litre bottle. Seeded water (900 ml, seeded with CS2 prepared according to Example 15) was added to the API solution. The resulting suspensions was left to stir at RT overnight before filtering. The resulting material was analysed by XRPD and identified as vadadustat crystalline form CS2 as described in EP3549932A (to Crystal Pharmaceutical (Suzhou) Co. Ltd.)

Example 15 (not according to the invention)

Vadadustat (30 mg) was dissolved in DMSO (5 vol, 150 μl) at RT and then heated to 90 °C. Water (15 ml) at RT was added to the API solution. The resulting suspensions was left to stir at RT overnight before filtering. The resulting material was analysed by XRPD and identified as vadadustat crystalline form CS2 as described in EP3549932A.

Example 16 (according to the invention)

Reverse Anti-Solvent Addition with 2-Methyl THF and Water

Vadadustat (200 mg) was dissolved in 2-methyl THF (3 vol, 600 mI) at 50 °C. Water (12 ml) was cooled to 15 °C and seeded (prepared according to Example 14Error! Reference source not found.)- The API solution was filtered and added dropwise to the seeded water whilst stirring. The resulting suspension was cooled to 5 °C at 0.1 °C/min overnight and left to stir at 5 °C for 2 days before filtering. The resulting material was analysed by XRPD and identified as vadadustat crystalline form CS2 as described in EP3549932A.

Example 17 (according to the invention)

Reverse Anti-Solvent Addition with 1,2-Dimethoxyethane and Water

Vadadustat (200 mg) was dissolved in 1,2-dimethoxyethane (4 vol, 800 mI) at 50 °C. Water (20 ml) was cooled to 15 °C and seeded (prepared according to Example 14). The API solution was filtered and added dropwise to the seeded water whilst stirring. The resulting suspension was cooled to 5 °C at 0.1 °C/min overnight and left to stir at 5 °C for 2 days before filtering. The resulting material was analysed by XRPD and identified as vadadustat crystalline form CS2 as described in EP3549932A.

Example 18 (according to the invention)

Slurry of Amorphous Vadadustat in Water

Amorphous vadadustat (approx. 30 mg, prepared according to Example 3) was slurried in water (300 mI, 10 vol) and shaken at 50 °C for 6 days. The resulting suspension was filtered and dried under suction before analysis. The resulting material was analysed by XRPD and identified as vadadustat crystalline form CS2 as described in EP3549932A. Characterisation of vadadustat crystalline form CS2

Figure 10 shows a representative XRPD pattern of vadadustat crystalline form CS2 prepared according to Example 16. The following table provides an XRPD peak list for the crystalline form:

Vadadustat crystalline Form CS2 was also characterised by TGA and DSC analysis (see Figure

11).