This application claims the priority of the European Patent Application EP22382718.9 filed on 26.07.2022, the European Patent Application EP22383096.9 filed on 14.11.2022, and the European Patent Application EP23382239.4, filed on 14.03.2023.

FIELD OF THE INVENTION

The present invention provides crystal forms of daprodustat, which are cocrystals comprising daprodustat free acid and a daprodustat pharmaceutically acceptable metal salt, wherein the metal salt is an alkali metal salt such as a sodium or a potassium salt. The invention also refers to processes for the preparation of said cocrystals of daprodustat in good yield and high purity, to pharmaceutical compositions containing them and to their use in therapy. The present invention also provides an efficient process for the preparation of daprodustat or a pharmaceutically or veterinary acceptable salt thereof in good yield and high purity suitable for industrial scale applications which involves improved conditions for the preparation of key intermediates.

BACKGROUND OF THE INVENTION

N-[(1 ,3-Dicyclohexylhexahydro-2,4,6-trioxopyrimidin-5-yl)carbonyl ]glycine, also known as daprodustat, compound of formula (I), is an oral hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) first approved in Japan under the trade name Duvroq in the form of film coated tablets in 1 , 2, 4 and 6 mg dosage strengths.

Daprodustat and its tautomer N-[(1 ,3-dicyclohexyl-6-hydroxy-2,4-dioxo-1 ,2,3,4-tetrahydro-5-pyrimidinyl)- carbonyl]glycine were first disclosed in W02007/150011 A2, which describes the preparation of daprodustat by using N, N'-dicyclohexylcarbodiimide (DCC) or N,N'-dicyclohexylurea (DCU) as the starting material. The method involves cyclization of the starting material with malonic acid derivatives to provide 1 ,3-dicyclohexyl barbituric acid, following condensation with ethyl isocyanatoacetate and subsequent ester hydrolysis with aqueous sodium hydroxide in ethanol. DCC is an acute skin irritant in susceptible individuals, and due to its low melting point it is difficult to manipulate and consequently not feasible for industrial application. Alternatively, DCU is reacted with malonyl chloride, which not only is unstable and corrosive but also is an environmentally unfriendly halogenated reagent.

Example 18 of W02007/150011 A2 discloses the preparation of daprodustat from the hydrolysis of an advanced ethyl ester intermediate (i.e., ethyl (1 ,3-dicyclohexyl-6-hydroxy-2,4-dioxo-1 , 2,3,4- tetrahydropyrimidine-5-carbonyl)glycinate) with aqueous sodium hydroxide in ethanol followed by neutralization with hydrochloric acid. Then, daprodustat is isolated from a mixture of diethyl ether and hexane according to Method 1 of said example 18. Alternatively, daprodustat is isolated from acetic acid with hot filtration to remove a small amount of insoluble material according to Method 2 of said example 18. These isolation methods have many disadvantages, for instance, diethyl ether is volatile and explosive, not appropriate for use at industrial scale; hexane is also an inappropriate solvent for use at industrial scale due to its flammability and the safety precautions needed for safe handling; and acetic acid is difficult to remove due to its high boiling point which increases the cost of the process.

CN102432549 A discloses the preparation of key intermediate 1 ,3-dicyclohexyl barbituric acid by reacting DCU with malonic acid in the presence of acetic acid and acetic anhydride. However, the 1 ,3-dicyclohexyl barbituric acid is isolated by solvent evaporation with increases the cost of the process at industrial scale. Daprodustat is neither disclosed nor prepared.

Prior art Annalen der Chemie, Justus Liebigs (1949), 562, 75-136, prepares key ethyl isocyanatoacetate starting from glycine ethyl ester by using phosgene, which is extremely toxic by acute (short-term) inhalation exposure and likely a carcinogen. Daprodustat is neither disclosed nor prepared.

WO2019/052133 A1 discloses solid forms of daprodustat named CS1 and CS9 which have higher purity and lower hygroscopicity compared to the prior art solid form of daprodustat obtained by reproducing the above Method 2 (i.e., from recrystallization in acetic acid). However, the solid forms CS1 and CS9 are isolated from slow evaporation, which is not feasible for industrial scale. According to WO2019/052133 A1 the kinetic solubility in pure water of CS1 is of 0.014 mg/mL and for CS9 is of 0.020 mg/mL, which suggests poor aqueous solubility of either form CS1 or form CS9 of daprodustat.

W02020/102302 A1 discloses crystalline forms of daprodustat named Form 3 and Form 4. Form 3 is prepared by crystallization from ethylbenzene and Form 4 by slurrying daprodustat in diethyl ether. However, it seems that Form 3 corresponds to prior art solid form obtained from the above Method 2, and Form 4 corresponds to prior art solid form obtained from the above Method 1 . Therefore, there is a need in developing solid forms of daprodustat with better solubility to increase the bioavailability, which can be easily manufactured at an industrial scale with low energy and costs. Further, it is also desirable to obtain an efficient and safe process for the preparation of daprodustat in high purity and high yield, which can be easily applied at an industrial scale with low energy and costs and good yields.

SUMMARY OF THE INVENTION

Daprodustat is a poorly soluble drug, and for poorly soluble drugs, it is important to increase solubility to increase the bioavailability of the drug, thereby improving the efficacy and safety of daprodustat.

The present invention refers to crystal forms of daprodustat which are cocrystals comprising daprodustat free acid and a daprodustat pharmaceutically acceptable metal salt, wherein the metal salt is an alkali metal salt such as a sodium or a potassium salt. Particularly, a cocrystal comprising daprodustat free acid and daprodustat sodium salt is referred herein as crystal form N2. Particularly, another cocrystal comprising daprodustat free acid and daprodustat sodium salt is referred herein as crystal form N4. Particularly, a cocrystal comprising daprodustat free acid and daprodustat potassium salt is referred herein as crystal form K2. Particularly, another cocrystal comprising daprodustat free acid and daprodustat potassium salt is referred herein as crystal form K1. Advantageously, these cocrystals of daprodustat have improved properties such as solubility, hygroscopicity and stability as well as manufacturing behaviour (compaction, flowability, filterability etc.) thereby resulting beneficial for the development of medicinal products containing daprodustat.

Thus, the first aspect of the present invention relates to a crystal form of daprodustat which is a cocrystal comprising daprodustat free acid and a daprodustat pharmaceutically acceptable metal salt, wherein the metal salt is an alkali metal salt such as a sodium or a potassium salt.

Particularly, the invention relates to a crystal form N2 of daprodustat having an X-ray powder diffraction pattern comprising peaks at 2theta values of 6.3°±0.2°, 7.4°±0.2°, 7.6°±0.2°, 11.4°±0.2° and 16.2°±0.2°, measured with Ko radiation of copper having an X-ray wavelength of 1 .5406 A at room temperature.

Particularly, the invention relates to a crystal form N4 having an X-ray powder diffraction pattern comprising peaks at 2theta values of 6.4°±0.2°, 7.4°±0.2°, 7.6°±0.2°, 13.4°±0.2° and 16.7°±0.2, measured with Ko radiation of copper having an X-ray wavelength of 1 .5406 A at room temperature.

Particularly, the invention relates to a crystal form K2 having an X-ray powder diffraction pattern comprising peaks at 2theta values of 5.5°±0.2°, 6.3°±0.2°, 7.0°±0.2°, 16.8° ±0.2° and 18.0°±0.2°, measured with Ko radiation of copper having an X-ray wavelength of 1 .5406 A at room temperature. Particularly, the invention relates to a crystal form K1 having an X-ray powder diffraction pattern comprising peaks at 2theta values of 5.4°±0.2°, 7.9°±0.2°, 14.6°±0.2°, 15.6°±0.2° and 17.5°±0.2°, measured with Ko radiation of copper having an X-ray wavelength of 1 .5406 A at room temperature.

The second and third aspects of the invention provide processes for preparing the crystal forms of daprodustat as defined herein.

A fourth aspect of the invention refers to pharmaceutical compositions comprising crystal forms of daprodustat as defined herein, in particular daprodustat in a crystal form selected from the group consisting of crystal form N2, crystal form N4, a crystal form K2, a crystal form K1 , and a combination thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

A fifth aspect refers to crystal forms of daprodustat as defined herein for use in therapy, particularly for the treatment of renal anemia. This aspect can also be formulated as a method of treatment of renal anemia, which comprises administering to a subject in need thereof, including a human, a crystal form of daprodustat as defined herein and one or more pharmaceutically acceptable carriers, diluents or excipients.

It also forms part of the invention the use of a crystal form of daprodustat as defined herein for the manufacture of pharmaceutical composition for the treatment of renal anemia.

The present invention also provides an efficient and environmentally friendly process for manufacturing daprodustat or a salt thereof in good yield and applicable at industrial scale without requiring laborious and unfeasible purification steps, yielding high purity which complies with pharmaceutical standards.

The present inventors found that the hydrolysis of the ester intermediate, ethyl N-[(1 ,3-dicyclohexyl-6- hydroxy-2,4-dioxo-1 ,2,3,4-tetrahydro-5-pyrimidinyl)carbonyl]-glycinate, following the conditions disclosed in example 18 of W02007/150011 A2 (i.e. in the presence of sodium hydroxide in ethanol followed by neutralization with hydrochloric acid) provides daprodustat with significant amounts of inorganic salts such as sodium chloride difficult to separate by conventional purification techniques such as extraction, followed by slurry in water plus an additional crystallization step in acetic acid which requires hot filtration to remove inorganic material.

Advantageously, the salt formed as byproduct from the reaction of a quaternary ammonium hydroxide of formula N[(Ci-C4)alkyl]4OH, preferably tetramethylammonium hydroxide (TMAH), and an acid selected from formic, acetic, monochloroacetic, trichloroacetic and trifluoroacetic acid (TFA) is soluble in the reaction media which enhances the isolation of final daprodustat without requiring laborious and unfeasible purification steps.

Thus, a sixth aspect of the present invention relates to a process for preparing daprodustat of formula (I), or a pharmaceutically or veterinary acceptable salt thereof, which comprises converting a compound of formula (VI), wherein R is (Ci-C )alkyl which is unsubstituted or substituted with one or more substituents independently selected from the group consisting of (Cs-Cejcycloalkyl, heterocycloalkyl, aryl and heteroaryl, particularly, selected from the group consisting of (Cs-Cejcycloalkyl, (C3-Ci2)heterocycloalkyl, (Ce-Ci4)aryl and (C5-Ci2)heteroaryl, preferably R is ethyl, into daprodustat of formula (I) by hydrolysis in the presence of a quaternary ammonium hydroxide of formula N[(Ci-C4)alkyl]4OH, preferably tetramethylammonium hydroxide (TMAH), and a solvent, and optionally converting daprodustat of formula (I) into a pharmaceutically or veterinary acceptable salt thereof.

The compound of formula (VI) may be prepared from 1 ,3-dicyclohexyl barbituric acid of formula (V) which can be obtained by reacting dicyclohexylurea (DCU) with malonic acid. The inventors have found that advantageously, the crystallization of the key intermediate of formula (V) in the presence of an antisolvent, preferably water, avoids the solvent evaporation step for its isolation which increases the cost of the process and the final cost of the medicinal product.

Thus, a seventh aspect of the invention provides a process for preparing key intermediate 1 ,3-dicyclohexyl barbituric acid of formula (V), which comprises the steps of:

(I) reacting dicyclohexylurea (DCU) with malonic acid in the presence of AC2O in acetic acid to provide compound of formula (V),

(II) adding an antisolvent, preferably water, in an amount from 1 to 10 volumes per g of compound (V), preferably from 3 to 6 volumes per g of compound of formula (V),

(ill) optionally, heating the mixture of step (II) at a temperature from 20°C to 85°C, preferably from 20°C to 30°C, and

(iv) isolating the compound of formula (V).

An eighth aspect of the invention refers to a process for preparing daprodustat of formula (I), or a pharmaceutically or veterinary acceptable salt thereof, which comprises: a1) preparing a 1 ,3-dicyclohexyl barbituric acid of formula (V) by a process which comprises the steps of:

(I) reacting dicyclohexylurea (DCU) with malonic acid in the presence of AC2O in acetic acid to provide compound of formula (V),

(II) adding an antisolvent, preferably water, in an amount from 1 to 10 volumes per g of compound (V), preferably from 3 to 6 volumes per g of compound of formula (V),

(ill) optionally, heating the mixture of step (II) at a temperature from 20°C to 85°C, preferably from 20°C to 30°C, and

(iv) isolating the compound of formula (V); a2) reacting the 1 ,3-dicyclohexyl barbituric acid of formula (V) obtained in step (a1) with an isocyanatoacetate of formula (IV), wherein R is (Ci-C )alkyl which is unsubstituted or substituted with one or more substituents independently selected from the group consisting of Cs-Cecycloalkyl, heterocycloalkyl, aryl and heteroaryl, particularly, selected from the group consisting of (Cs-Cejcycloalkyl, (C3-Ci2)heterocycloalkyl, (Ce-Ci4)aryl and (C5-Ci2)heteroaryl, preferably R is ethyl, in the presence of a base and a solvent to provide the compound of formula (VI); wherein R is as defined above, preferably R is ethyl; a3) converting the compound of formula (VI) into daprodustat of formula (I) by hydrolysis in the presence of a base and a solvent; and a4) optionally converting daprodustat of formula (I) into a pharmaceutically or veterinary acceptable salt thereof.

A ninth aspect of the present invention provides a process for preparing a compound of formula (VI), wherein R is (Ci-Cio)alkyl which is unsubstituted or substituted with one or more substituents independently selected from the group consisting of Cs-Cecycloalkyl, heterocycloalkyl, aryl and heteroaryl, particularly, selected from the group consisting of (Cs-Cejcycloalkyl, (C3-Ci2)heterocycloalkyl, (Ce-Ci4)aryl and (C5-Ci2)heteroaryl, preferably R is ethyl, which comprises the steps of: a) converting a formamide of formula (II), into a compound an isocyanoacetate of formula (III), wherein R is as defined above, preferably R is ethyl; b) converting the isocyanoacetate of formula (III) of step (a) into an isocyanatoacetate of formula (IV), wherein R is as defined above, preferably R is ethyl; c) reacting the isocyanatoacetate of formula (IV) obtained in step (b) with a 1 ,3-dicyclohexyl barbituric acid of formula (V), in the presence of a base and a solvent to provide the compound of formula (VI); wherein steps (b) and (c) are carried out in a consecutive manner, i.e. in one-pot manner.

Advantageously, the preparation of key intermediate of formula (VI) following the process according to the ninth aspect, Scheme 1, by reacting 1 ,3-dicyclohexyl barbituric acid of formula (V) with intermediate (IV), which is known to be unstable, in a consecutive manner process (i.e. in a one-pot reaction) starting from intermediate of formula (III) from steps (b) and (c) of the present invention, increases the yield and chemical purity of compound of formula (VI).

Scheme 1 A tenth aspect of the invention refers to a process for preparing daprodustat of formula (I), or a pharmaceutically or veterinary acceptable salt thereof, which comprises: b1) converting a formamide of formula (II), into a compound an isocyanoacetate of formula (III), wherein R is as defined above, preferably R is ethyl; b2) converting the isocyanoacetate of formula (III) of step (b1) into an isocyanatoacetate of formula (IV), wherein R is as defined above, preferably R is ethyl; b3) reacting the isocyanatoacetate of formula (IV) obtained in step (b2) with a 1 ,3-dicyclohexyl barbituric acid of formula (V), in the presence of a base and a solvent to provide the compound of formula (VI) wherein R is as defined above, preferably R is ethyl; wherein steps (b2) and (b3) are carried out in a consecutive manner, i.e. in one-pot manner; b4) converting the compound of formula (VI) into daprodustat of formula (I) by hydrolysis in the presence of a base and a solvent; and b5) optionally converting daprodustat of formula (I) into a pharmaceutically or veterinary acceptable salt thereof.

DEFINITIONS

When describing the compounds and methods of the invention, the following terms have the following meanings, unless otherwise indicated.

The term "about” as used herein refers to a statistically meaningful range of a value. Such a range can lie within experimental error, typical of standard methods used for the measurement and/or determination of a given value or range. In one embodiment, the range is within ±5% of the indicated value. In another embodiment, the range is within ±1% of the indicated value. In yet another embodiment, the range is within ±0.5% of the indicated value.

As used herein, "alkyl” means straight-chain or branched hydrocarbon chain radical containing no unsaturation having from 1 to 10 carbon atoms, represented as (Ci-Cio)alkyl. Such alkyl groups may be selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, straight- or branched- pentyl, straight- or branched- hexyl, straight- or branched-heptyl, straight- or branched-nonyl or straight- or branched- decyl.

The term "cycloalkyl" refers to a non-aromatic, saturated, cyclic hydrocarbon ring containing the specified number of carbon atoms. So, for example, the term "Ca-Cecycloalkyl" refers to a non-aromatic cyclic hydrocarbon ring having from three to six carbon atoms. Exemplary "Ca-Cecycloalkyl" groups useful in the present invention include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The term "heterocycloalkyl" means a non-aromatic heterocyclic ring containing the specified number of ring atoms, e.g., having 3 to 12 carbon atoms, represented as (C3-C12) heterocycloalkyl, being, saturated or having one or more degrees of unsaturation and containing one or more heteroatom substitutions independently selected from 0, S and N. Such a ring may be optionally fused to one or more other "heterocyclic" ring(s) or cycloalkyl ring(s). Examples of "heterocyclic" moieties include, but are not limited to, aziridine, thiirane, oxirane, azetidine, oxetane, thietane, tetrahydrofuran, pyran, 1 ,4-dioxane, 1 ,3-dioxane, piperidine, piperazine, 2,4-piperazinedione, pyrrolidine, imidazolidine, pyrazolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, and the like.

The term "aryl” refers to optionally substituted monocyclic and polycarbocyclic unfused or fused groups having 6 to 14 carbon atoms, represented as (Ce-Cujaryl, and having at least one aromatic ring that complies with Huckel's Rule. Examples of (Ce-Ci4)aryl groups are phenyl, biphenyl, naphthyl, anthracenyl, phenanthrenyl and the like.

The term "heteroaryl” means an optionally substituted aromatic monocyclic ring or polycarbocyclic fused ring system having 5 to 12 carbon atoms, represented as (Cs-C^heteroaryl, wherein at least one ring complies with Huckel's Rule, has the specified number of ring atoms, and that ring contains at least one heteratom independently selected from N, 0 and S. Examples of "(C5-Ci2)heteroaryl" groups include furanyl, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, oxopyridyl, thiadiazolyl, isothiazolyl, pyridinyl, pyridazinyl, pyrazinyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothiophenyl, indolyl and indazolyl.

As used herein, "hydrate” refers to a crystalline form of a molecule that further comprises water incorporated into the crystalline structure. The water molecules in the hydrate may be present in a regular arrangement and/or a non-ordered arrangement. The hydrate may comprise either a stoichiometric or nonstoichiometric amount of the water molecules.

The term "solvate" refers to a crystalline form of a molecule that further comprises solvent molecule/s incorporated into the crystalline structure. When the solvent incorporated in the crystal is water, is called hydrate. The solvent molecules in the solvate may be present in a regular arrangement and/or a non-ordered arrangement. The solvate may comprise either a stoichiometric or nonstoichiometric amount of the solvent molecules. Solvates can exhibit polymorphism.

As used herein, the term "cocrystal" also known as "crystalline molecular complex” refers to a crystalline solid made up of two or more unique chemical species in the same crystal lattice, in a defined stoichiometric ratio, and that possesses distinct physical, crystallographic and spectroscopic properties when compared to the chemical species individually. Present crystal forms are cocrystals which comprise the daprodustat free acid, compound of formula (I), and a daprodustat pharmaceutically acceptable metal salt, wherein the metal is an alkali metal such as sodium or potassium. Cocrystals may be in the form of hydrates or solvates.

A cocrystal is distinct from a "salt" which comprises charged-balanced charged species. The species making up a cocrystal typically are neutral, and are generally held together by weak, freely reversible, non-covalent interactions. The weak interaction is defined as neither ionic bond interaction nor covalent bond interaction, and include hydrogen bonding, van der Waals forces, p-p interactions and halogen bond interactions. Cocrystals can generally be distinguished from salts by the absence of a proton transfer between the chemical species. So, the cocrystals of the present invention hold together by weak interactions without accompanying proton transfer between daprodustat free acid and daprodustat metal salt.

The term "particle size” as used herein in relation to the crystal forms of daprodustat in the form of needles (i.e., acicular morphology) refers to the average length of the needles observed in the microscope images measured with an Environmental Scanning Electron Microscope (ESEM).

As used herein the term "solvent” refers to water or an organic molecule capable of at least partially dissolving another substance (i.e., the solute). Solvents may be liquids at room temperature. Organic solvents may be liquids at room temperature. Suitable organic solvents may be, but are not limited to: hydrocarbon solvents (e.g., n-pentane, n-hexane, n-heptane, n-octane, paraffin, cyclohexane, methylcyclohexane, decahydronaphthalene, mineral oil, crude oils, etc.) which also includes (C6-Ci4)aromatic hydrocarbon solvents (e.g., benzene, toluene, o-xylene, m-xylene, and p-xylene), halogenated (Ci-Ci2)hydrocarbon solvents (e.g., carbon tetrachloride, 1 ,2-dichloroethane, dichloromethane, chloroform, etc.), ester solvents (e.g., ethyl formate, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, ethyl malonate, etc.), ketone solvents (e.g., acetone, methyl ethyl ketone or 2- butanone, methyl isobutyl ketone, cyclohexanone, cyclopentanone, 3-pentanone, etc.), (Ci-Ci2)ether solvents (e.g., diethyl ether, dipropyl ether, diphenyl ether, isopropyl ether, tert-butyl methyl ether, tetrahydrofuran, 1 ,4-dioxane, etc.), amine solvents (e.g., propyl amine, diethylamine, triethylamine, aniline, pyridine), (Ci-Ci2)alcohol solvents (e.g., methanol, ethanol, isopropanol, 1-propanol, 2-methyl-1 -propanol, 1-butanol, 2-butanol, 1-pentanol, 3-methyl-1 -butanol, tert-butanol, 1-octanol, benzylalcohol, phenol, trifluoroethanol, glycerol, ethylene glycol, propylene glycol, m-cresol, etc.), acid solvents (e.g., acetic acid, hexanoic acid, etc.), carbon disulfide, nitrobenzene, N,N-dimethylformamide, N,N, -dimethylacetamide, dimethyl sulfoxide, N-methyl-2-pyrrolidone, acetonitrile, silicone solvents (e.g., silicone oils, polysiloxanes, cyclosilicones). In some embodiments, the solvent may be formed by the combination of two or more solvents. The term "aprotic solvent” as used herein means any molecular solvent which cannot donate H ⁺ , i.e. a compound not having labile hydrogens. Suitable aprotic solvents may be, but are not limited to: hydrocarbon solvents (e.g., n-pentane, n-hexane, n-heptane, n-octane, paraffin, cyclohexane, methylcyclohexane, etc.), halogenated hydrocarbon solvents (e.g., 1 ,2-dichloroethane, dichloromethane, chloroform, etc.), aromatic hydrocarbon solvents (e.g., toluene, o-xylene, m-xylene, and p-xylene, etc), and ether solvents (e.g., diethyl ether, dipropyl ether, diphenyl ether, isopropyl ether, tert-butyl methyl ether, tetrahydrofuran, 2- methyltetrahydrofuran, 1 ,4-dioxane, etc.), acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, or N-methyl-2-pyrrolidone. Preferably, the aprotic solvent is dichloromethane, toluene, or tetrahydrofuran.

The term "(Ci-C3)alcohols” as used herein means methanol, ethanol, isopropanol or 1-propanol.

The term "one-pot" reaction or "consecutive manner” is generally known in the art and refers to a chemical reaction wherein the starting material is converted to the end product of the reaction in a single reaction vessel or container, i.e. there is no intermediary reaction product which is isolated, removed or purified from the reaction vessel. A "one-pot" reaction or "consecutive manner” in its broadest meaning still allows the formation of intermediary products which are, however, further converted to the end-product by addition of further reactants (in situ generation of the intermediate). A "one-pot” reaction or "consecutive manner” also encompasses a reaction in a single reaction vessel where the starting product is converted to the end product through the formation of one or multiple intermediate products that are formed sequentially, even without further addition of a reagent ("multistep" reaction). Thus, a "one-pot" or "consecutive manner” process is characterized by at least two reaction steps carried out without isolation and/or purification of the intermediate product or products, and suitably carried out in a single reaction vessel/container. It will be understood by one of skill in the art that a simple transfer of the whole reaction mass at an intermediate stage, but without isolating and/or purifying the intermediate product, is still a "one-pot" process or "consecutive manner” according to the present invention, not the least because such a process would still achieve the technical advantage associated with a one-pot process in that the intermediate formed in situ does not need to be isolated and/or purified.

The term “hydracid” (aka binary acid) as used herein refers to an inorganic acid in which hydrogen is combined with a second nonmetallic element such as S, F, Cl, Br or I. More preferably the "hydracid” is selected from the group consisting of hydrochloric acid, hydrobromic acid and hydroiodic acid.

The term "inorganic base” refers to hydroxides of an alkali metal such as sodium or potassium and an alkali earth metal such as calcium or magnesium, and mixtures thereof. The term "room temperature” in the context of the present invention refers to a temperature from 15°C to 30°C, preferably from 20°C to 25°C.

As used herein, the term "solvent extraction” refers to the process of separating components of a mixture by using a solvent which possesses greater affinity for one component and may, therefore, separate said one component from at least a second component which is less miscible than said one component with said solvent.

The term "filtration” refers to the act of removing solid particles greater than a predetermined size from a feed comprising a mixture of solid particles and liquid. The expression filtrate refers to the mixture less the solid particles removed by the filtration process. It will be appreciated that this mixture may contain solid particles smaller than the predetermined particle size. The expression filter cake refers to residual solid material remaining on a feed side of a filtration element.

The term "evaporation” refers to the change in state of solvent from liquid to gas and removal of that gas from the reactor. Various solvents may be evaporated during the processes disclosed herein. As known to those of skilled in the art, each solvent may have a different evaporation time and/or temperature.

The term "distillation” refers to the process of separating the component substances from a liquid mixture by selective evaporation and condensation. It may result in essentially complete separation (nearly pure components), or it may be a partial separation that increases the concentration of selected components of the mixture. In either case the process exploits differences in the volatility of mixture's components.

As used herein, the term "slurrying” refers to any process which employs a solvent to wash, suspend or disperse a crude solid product.

The term "phase separation” refers to a solution or mixture having at least two physically distinct regions.

The term "crystallization” refers to any method known to a person skilled in the art such as crystallization from single solvent or combination of solvents by dissolving the compound, optionally at elevated temperature and precipitating the compound by cooling the solution or removing solvent from the solution or both. It further includes methods such as dissolving the compound in a solvent and precipitating it by addition of an "antisolvent” (i.e., a solvent in which the desired compound has low solubility or insolubility, and can be used to precipitate such compound by adding it to a solution in which the compound is dissolved). The terms "conventional isolation techniques” or "purification” as used herein refer to the process of rendering a product clean of foreign elements whereby a purified product can be obtained. The term industrial purification refers to purifications which can be carried out on an industrial scale such as solvent extraction, filtration, slurring, washing, phase separation, distillation, centrifugation or crystallization.

The term "pharmaceutically acceptable metal salt” of daprodustat derived from pharmaceutically acceptable bases include alkali metal such as sodium or potassium.

The term "pharmaceutically acceptable" refers to components which are appropriate for use in pharmaceutical technology for the preparation of compositions for medical use. Each component should be "acceptable" in the sense of being compatible with the other ingredients of the composition. When used in contact with the tissue or organ of humans and animals should not have excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically or veterinary acceptable salt” refers to a salt prepared from a base or acid or their respective conjugated acids or bases, which is acceptable for administration to a patient, such as a mammal. Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. Salts of daprodustat derived from pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases, such as alkyl amines, arylalkyl amines and heterocyclic amines.

BRIEF DESCRIPTION OF THE FIGURES

Examples of the invention are illustrated with the following drawings:

FIG. 1 provides a representative X-ray Powder Diffraction (XRPD) pattern of crystal form N2 of daprodustat. FIG. 2 provides a representative Differential Scanning Calorimetry (DSC) and Thermal Gravimetric Analysis (TGA) plot of crystal form N2 of daprodustat.

FIG. 3 provides a representative ¹ H-RMN plot of crystal form N2 of daprodustat.

FIG. 4 provides a representative infrared absorption spectrum (IR) of crystal form N2 of daprodustat.

FIG. 5 provides a representative X-ray Powder Diffraction (XRPD) pattern of crystal form N4 of daprodustat. FIG. 6 provides a representative Differential Scanning Calorimetry (DSC) and Thermal Gravimetric Analysis (TGA) plot of crystal form N4 of daprodustat.

FIG. 7 provides a representative X-ray Powder Diffraction (XRPD) pattern of crystal form K2 of daprodustat. FIG. 8 provides a representative Differential Scanning Calorimetry (DSC) and Thermal Gravimetric Analysis (TGA) plot of crystal form K2 of daprodustat.

FIG. 9 provides a representative ¹ H-RMN plot of crystal form K2 of daprodustat. FIG. 10 provides a representative infrared absorption spectrum (IR) of crystal form K2 of daprodustat.

FIG. 11 provides a representative micrograph of crystal form N2 of daprodustat obtained by optical microscopy.

FIG. 12 provides a representative micrograph of crystal form K2 of daprodustat obtained by optical microscopy.

FIG. 13 provides a representative micrograph of crystal form N2 of daprodustat obtained by ESEM.

FIG. 14 provides a representative micrograph of crystal form K2 of daprodustat obtained by ESEM.

FIG. 15 provides a representative X-ray Powder Diffraction (XRPD) pattern of crystal form K1 of daprodustat. FIG. 16 provides a representative Differential Scanning Calorimetry (DSC) and Thermal Gravimetric Analysis (TGA) plot of crystal form K1 of daprodustat.

Any crystalline forms that provide X-ray diffraction patterns substantially identical to those disclosed in the accompanying Figures fall within the scope of the present invention. The ability to ascertain substantial identities of X-ray diffraction patterns is within the purview of one of ordinary skill in the art.

DETAILED DESCRIPTION OF THE INVENTION

According to the first aspect, the invention relates to a crystal form of daprodustat which is a cocrystal comprising daprodustat free acid and a daprodustat pharmaceutically acceptable metal salt, wherein the metal salt is an alkali metal salt.

In certain embodiments, the molar ratio of daprodustat free acid to daprodustat metal salt in the cocrystal is of from 1 :3 to 3:1 , preferably from 1 :2 to 2:1, more preferably is about 1 :1. In certain embodiments, the molar ratio of daprodustat free acid and daprodustat metal salt in the cocrystal is about 2:1. In preferred embodiments, the molar ratio of daprodustat free acid and daprodustat metal salt in the cocrystal is about 1 : 1.

In a particular embodiment, the invention relates to a crystal form of daprodustat which is a cocrystal comprising daprodustat free acid and daprodustat sodium salt.

In an embodiment, the invention relates to a crystal form of daprodustat which is a cocrystal comprising daprodustat free acid and daprodustat sodium salt referred as form N2, which has an X-ray powder diffraction pattern comprising peaks at 2theta values of 6.3°±0.2°, 7.4°±0.2°, 7.6°±0.2°, 11 .4°±0.2° and 16.2°±0.2°, measured with Ko radiation of copper having an X-ray wavelength of 1.5406 A at room temperature. In an embodiment, the crystal form N2 of daprodustat further comprises peaks at 2theta values of 13.0°±0.2°, 13.5°±0.2°, 14.5°±0.2°, 14.8°±0.2° and 15.2°±0.2°, measured with Ko radiation of copper having an X-ray wavelength of 1 .5406 A at room temperature. In an embodiment, the crystal form N2 of daprodustat is characterized by XRPD peaks located at one, two, three, four, five, six, seven, eight, nine or ten of the following approximate positions: 6.3, 7.4, 7.6, 11.4, 13.0, 13.5, 14.5, 14.8, 15.2, 16.2 ±0.2 degrees 2Theta measured with Ko radiation of copper having an X-ray wavelength of 1 .5406 A at room temperature. In an embodiment, the crystal form N2 of daprodustat is characterized by an XRPD pattern which matches the pattern exhibited in FIG. 1 . In an embodiment, the crystal form N2 of daprodustat is characterized by an XRPD pattern having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 peaks matching peaks in the representative crystal form N2 of daprodustat pattern provided in Table 1.

Table 1

In certain embodiments, the crystal form N2 of daprodustat is characterized by thermal analysis. A representative DSC plot for crystal form N2 is shown in FIG. 2. In certain embodiments, the crystal form N2 is characterized by a DSC plot comprising an endothermic event with a first onset temperature at about 115°C. In certain embodiments, the crystal form N2 is characterized by a DSC plot further comprising a second endothermic event with an onset temperature at about 190°C. In certain embodiments, the crystal form N2 is characterized by a DSC plot further comprising a third endothermic event with an onset temperature at about 201 °C. In certain embodiments, the crystal form N2 is characterized by a DSC plot further comprising a fourth endothermic event with an onset temperature at about 228°C.

A representative TGA plot for crystal form N2 of daprodustat is also shown in FIG. 2. In certain embodiments, the crystal form N2 of daprodustat is characterized by a TGA plot comprising a mass loss of about 4.4%, of the total mass of the sample upon heating from about 35°C to about 150°C. In certain embodiments, the crystal form N2 of daprodustat contains water or other solvent in the crystal lattice. In certain embodiments, the crystal form N2 of daprodustat contains water in the crystal lattice. In certain embodiments, the crystal form N2 of daprodustat contains water in an amount from 0.2% to 10%, preferably from 2% to 8%, more preferably from 4% to 6%, by weight with respect to the total weight of the crystal. In certain embodiments, the crystal form N2 of daprodustat is a hydrate. In certain embodiments, the crystal form N2 of daprodustat is a hydrate which contains an amount of water of about 4.4% as measured by TGA analysis.

In certain embodiments, the XRPD pattern of the crystal form N2 of daprodustat is substantially unchanged following the adsorption/desorption analysis. In certain embodiments, the crystal form N2 is stable with respect to humidity. In certain embodiments, the crystal form N2 of daprodustat is slightly hygroscopic according to European Pharmacopoeia 6.0 (5.11).

In certain embodiments, the crystal form N2 of daprodustat is characterized by proton nuclear magnetic resonance ( ¹ H-NMR) as shown in FIG. 3. In certain embodiments, the crystal form N2 of daprodustat is characterized by having a proton NMR spectrum with signals 5 (ppm) at 10.13, 4.66, 3.96, 2.31, 1.77, 1.57, and 1.20. In certain embodiments, the crystal form N2 of daprodustat is characterized by having a proton NMR spectrum with signals at: 1 .20 ppm (m, 6H), 1 .57 ppm (m, 6H), 1 .77 ppm (m, 4H), 2.31 ppm (m, 4H), 3.96 ppm (d, 2H, J=6 Hz), 4.66 ppm (m, 2H), and 10.13 ppm (t, 1 H, J=6 Hz).

In certain embodiments, the crystal form N2 of daprodustat is characterized by an infrared absorption spectrum (FTIR) which has a spectrum shown in FIG. 4, and has peaks at the following wavenumbers (cm ¹ ): 691 , 761 , 789, 840, 895, 924, 966, 1000, 1024, 1057, 1132, 1178, 1237, 1266, 1306, 1338, 1379, 1420, 1444, 1479, 1522, 1586, 1670, 1716, 2849, 2914, 2930, 2973, 3233, 3522, and 3649. Although the wavenumbers (cm ¹ ) may contain an error of ±0.5% according to the measuring apparatus, measurement conditions, etc., such a level of error is within an acceptable range in the present invention.

In certain embodiments, the molar ratio of daprodustat free acid to daprodustat sodium salt in the crystal form N2 is of from 1 :3 to 3:1 , preferably from 1 :2 to 2:1, more preferably is about 1 :1. In certain embodiments, the molar ratio of daprodustat free acid and daprodustat sodium salt in the crystal form N2 is about 2: 1. In preferred embodiments, the molar ratio of daprodustat free acid and daprodustat sodium salt in the crystal form N2 is about 1 :1.

In certain embodiments, the crystal form N2 of daprodustat is characterized by its stability profile. In certain embodiments, the crystal form N2 of daprodustat material is stable, e.g., its XRPD pattern remains substantially unchanged, upon exposure to elevated temperature, upon exposure to elevated humidity, upon exposure to one or more solvents, and/or upon grinding or milling and compression.

In certain embodiments, the crystal form N2 of daprodustat keeps its form unchanged in a dry environment at room temperature, at 45°C and at 60°C for at least one month. In certain embodiments, the crystal form N2 of daprodustat keeps its form unchanged under accelerated conditions at 40°C and 75% RH (Relative Humidity) for at least one month or for at least two months.

In certain embodiments, the crystal form N2 of daprodustat keeps its form unchanged upon slurry in water at room temperature for at least one hour. In other words, the crystal form N2 of daprodustat does not undergo dissociation (i.e., phase separation into their individual constituents) in the presence of water.

In a particular embodiment, the invention relates to a crystal form of daprodustat which is a cocrystal comprising daprodustat free acid and daprodustat sodium salt referred as form N4 which has an X-ray powder diffraction (XRPD) pattern comprising peaks at 2theta values of 6.4°±0.2°, 7.4°±0.2°, 7.6°±0.2°, 13.4° ±0.2° and 16.7°±0.2, measured with Ko radiation of copper having an X-ray wavelength of 1.5406 A at room temperature.

In an embodiment, the crystal form N4 of daprodustat further comprises peaks at 2theta values of 15.0°±0.2°, 26.9°±0.2°, 27.5°±0.2°, 27.7°±0.2° and 28.2°±0.2°, measured with Ko radiation of copper having an X-ray wavelength of 1 .5406 A at room temperature.

In an embodiment, the crystal form N4 of daprodustat is characterized by XRPD peaks located at one, two, three, four, five, six, seven, eight, nine or ten of the following approximate positions: 6.4, 7.4, 7.6, 13.4, 15.0, 16.7, 26.9, 27.5, 27.7 and 28.2 ±0.2 degrees 2Theta.

In certain embodiments, the crystal form N4 is characterized by an XRPD pattern which matches the pattern exhibited in FIG.5. In an embodiment, the crystal form N4 of daprodustat is characterized by an XRPD pattern having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 peaks matching peaks in the representative crystal form N4 of daprodustat pattern provided in Table 2. Table 2

In certain embodiments, a representative DSC/TGA plots for crystal form N4 is shown in FIG. 6. In certain embodiments, the crystal form N4 of daprodustat is characterized by a DSC plot comprising an endothermic event with a first onset temperature at about 107°C. In certain embodiments, form N4 is characterized by a DSC plot further comprising a second endothermic event with an onset temperature at about 190°C. In certain embodiments, form N4 is characterized by a DSC plot further comprising a third endothermic event with an onset temperature at about 200°C. In certain embodiments, form N4 is characterized by a DSC plot further comprising a fourth endothermic event with an onset temperature at about 228°C.ln certain embodiments, the crystal form N4 of daprodustat is characterized by a TGA plot comprising a mass loss of about 4.0%, of the total mass of the sample upon heating from about 35°C to about 150°C. In certain embodiments, the crystal form N4 of daprodustat contains water or other solvents in the crystal lattice. In certain embodiments, the crystal form N4 of daprodustat contains water in the crystal lattice. In certain embodiments, the crystal form N4 of daprodustat contains water in an amount of from 0.2% to 10%, preferably from 0.2% to 5%, more preferably from 2% to 5%, even more preferably from 3.9% to 4.5% by weight with respect to the total weight of the crystal. In certain embodiments, the crystal form N4 of daprodustat is a hydrate. In certain embodiments, the crystal form N4 of daprodustat is a hydrate which contains an amount of water of about 4.0% as measured by TGA analysis.

In certain embodiments, the XRPD pattern of the crystal form N4 of daprodustat is substantially unchanged following the adsorption/desorption analysis. In certain embodiments, the crystal form N4 is stable with respect to humidity. In certain embodiments, the crystal form N4 of daprodustat is slightly hygroscopic according to European Pharmacopoeia 6.0 (5.11). In certain embodiments, the water content in the crystal form N4 of daprodustat is lower than the water content in the crystal form N2 of daprodustat.

In certain embodiments, the molar ratio of daprodustat free acid to daprodustat sodium salt in the crystal form N4 is of from 1 :3 to 3:1, preferably from 1 :2 to 2:1 , more preferably is about 1 :1. In certain embodiments, the molar ratio of daprodustat free acid and daprodustat sodium salt in the crystal form N4 is about 2: 1. In preferred embodiments, the molar ratio of daprodustat free acid and daprodustat sodium salt in the crystal form N4 is about 1 :1.

In certain embodiments, the crystal form N4 of daprodustat is characterized by its stability profile. In certain embodiments, the crystal form N4 of daprodustat material is stable, e.g., its XRPD pattern remains substantially unchanged, upon exposure to elevated temperature, upon exposure to elevated humidity, upon exposure to one or more solvents, and/or upon grinding or milling and compression.

In a particular embodiment, the invention relates to a crystal form of daprodustat which is a cocrystal comprising daprodustat free acid and daprodustat potassium salt.

In an embodiment, the invention relates to a crystal form of daprodustat which is a cocrystal comprising daprodustat free acid and daprodustat potassium salt referred as form K2, which has an X-ray powder diffraction (XRPD) pattern comprising peaks at 2theta values of 5.5°±0.2°, 6.3°±0.2°, 7.0°±0.2°, 16.8°±0.2° and 18.0°±0.2°, measured with Ko radiation of copper having an X-ray wavelength of 1.5406 A at room temperature.

In an embodiment, the crystal form K2 of daprodustat further comprises peaks at 2theta values of 7.8°±0.2°, 11.8°±0.2°, 12.7°±0.2°, 13.7°±0.2° and 27.6°±0.2°, measured with Ko radiation of copper having an X- ray wavelength of 1 .5406 A at room temperature.

In an embodiment, the crystal form K2 of daprodustat is characterized by XRPD peaks located at one, two, three, four, five, six, seven, eight, nine or ten of the following approximate positions: 5.5, 6.3, 7.0, 7.8, 11.8, 12.7, 13.7, 16.8, 18.0, and 27.6 ±0.2 degrees 2Theta.

In an embodiment, the crystal form K2 of daprodustat is characterized by an XRPD pattern which matches the pattern exhibited in FIG. 7. In an embodiment, the crystal form K2 of daprodustat is characterized by an XRPD pattern having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 peaks matching peaks in the representative crystal form K2 of daprodustat pattern provided in Table 3. Table 3

In certain embodiments, the crystal form K2 of daprodustat is characterized by thermal analysis. A representative DSC plot for the crystal form K2 is shown in FIG. 8. In certain embodiments, the crystal form K2 is characterized by a DSC plot comprising a first endothermic event with an onset temperature at about 33°C. In certain embodiments, the crystal form K2 is characterized by a DSC plot further comprising a second endothermic event with an onset temperature at about 248°C.

A representative TGA plot for the crystal form K2 of daprodustat is also shown in FIG. 8. In certain embodiments, the crystal form K2 of daprodustat is characterized by a TGA plot comprising a mass loss of about 5.4%, of the total mass of the sample upon heating from about 30°C to about 130°C. In certain embodiments, the crystal form K2 of daprodustat contains water or other solvent in the crystal lattice. In certain embodiments, the crystal form K2 of daprodustat contains water in the crystal lattice. In certain embodiments, the crystal form K2 of daprodustat contains water in an amount from 0.2% to 10%, preferably from 2% to 8%, more preferably from 4% to 6% by weight with respect to the total weight of the crystal. In certain embodiments, the crystal form K2 of daprodustat is a hydrate. In certain embodiments, the crystal form K2 of daprodustat is a hydrate which contains an amount of water of about 5.4% as measured by TGA analysis. In certain embodiments, the XRPD pattern of the crystal form K2 of daprodustat is substantially unchanged following the adsorption/desorption analysis. In certain embodiments, the crystal form K2 is stable with respect to humidity.

In certain embodiments, the crystal form K2 of daprodustat is characterized by proton nuclear magnetic resonance ( ¹ H-NMR) as shown in FIG. 9. In certain embodiments, the crystal form K2 of daprodustat is characterized by having a proton NMR spectrum with signals 5 (ppm) at 10.08, 4.65, 3.93, 2.30, 1.77, 1.57, and 1.20. In certain embodiments, the crystal form K2 of daprodustat is characterized by having a proton NMR spectrum with signals at: 1.20 ppm (m, 6H), 1.57 ppm (m, 6H), 1.77 ppm (m, 4H), 2.30 ppm (m, 4H), 3.93 ppm (d, 2H, J=6 Hz), 4.65 ppm (m, 2H), and 10.08 ppm (t, 1 H, J=6 Hz).

In certain embodiments, the crystal form K2 of daprodustat is characterized by an infrared absorption spectrum (FTIR) which has a spectrum shown in FIG. 10, and has peaks at the following wavenumbers (cm ¹ ): 693, 740, 762, 790, 882, 894, 918, 996, 1054, 1134, 1187, 1235, 1258, 1302, 1340, 1378, 1446, 1472, 1506, 1522, 1589, 1669, 2655, 2852, 2935, 2977, 3180, 3477, and 3546. Although the wavenumbers (cm ¹ ) may contain an error of ±0.5% according to the measuring apparatus, measurement conditions, etc., such a level of error is within an acceptable range in the present invention.

In certain embodiments, the molar ratio of daprodustat free acid to daprodustat potassium salt in the crystal form K2 is of from 1 :3 to 3: 1 , preferably from 1 :2 to 2: 1 , more preferably is 1 : 1. In certain embodiments, the molar ratio of daprodustat free acid and daprodustat potassium salt in the crystal form K2 is about 2: 1. In preferred embodiments, the molar ratio of daprodustat free acid and daprodustat potassium salt in the crystal form K2 is about 1 :1.

In certain embodiments, the crystal form K2 of daprodustat is characterized by its stability profile. In certain embodiments, the crystal form K2 of daprodustat material is stable, e.g., its XRPD pattern remains substantially unchanged, upon exposure to elevated temperature, upon exposure to elevated humidity, upon exposure to one or more solvents, and/or upon grinding or milling and compression.

In certain embodiments, the crystal form K2 of daprodustat keeps its form unchanged in a dry environment at room temperature, at 45°C and at 60°C for at least one month. In certain embodiments, the crystal form K2 of daprodustat keeps its form unchanged under accelerated conditions at 40°C and 75% RH (Relative Humidity) for at least one month or for at least two months.

In certain embodiments, the crystal form K2 of daprodustat keeps its form unchanged in a dry environment under vacuum at 45°C and at 60°C least 12 hours (i.e., overnight). In certain embodiments, the crystal form K2 of daprodustat keeps its form unchanged upon slurry in water at room temperature for at least one hour. In other words, the crystal form K2 of daprodustat does not undergo dissociation (i.e., phase separation into their individual constituents) in the presence of water.

In certain embodiments, a sample of the crystal form N2 of daprodustat comprises particles having needles (acicular) morphology. In certain embodiments, a sample of the crystal form N2 of daprodustat comprises acicular crystals as shown in FIG. 11. In certain embodiments, the particles of the crystal form N2 of daprodustat have a size of less than about 300 microns, less than about 250 microns, less than about 200 microns, less than about 150 microns, less than about 100 microns, less than about 50 microns, less than about 25 microns or less than about 10 microns. In certain embodiments, the particles of the crystal form N2 of daprodustat have a size between 100 to 5 microns, between 80 to 5 microns, between 60 to 5 microns, between 40 to 5 microns or between 30 to 5 microns. In certain embodiments, the particles of the crystal form N2 of daprodustat comprises acicular crystals having a size as shown in FIG. 13.

In certain embodiments, a sample of the crystal form N4 of daprodustat comprises particles having needles (acicular) morphology. In certain embodiments, the particles of the crystal form N4 of daprodustat have a size of less than about 300 microns, less than about 250 microns, less than about 200 microns, less than about 150 microns, less than about 100 microns, less than about 50 microns, less than about 25 microns or less than about 10 microns. In certain embodiments, the particles of the crystal form N4 of daprodustat have a size between 100 to 5 microns, between 80 to 5 microns, between 60 to 5 microns, between 40 to 5 microns, or between 30 to 5 microns.

In certain embodiments, a sample of the crystal form K2 of daprodustat comprises particles having needles (acicular) morphology. In certain embodiments, a sample of the crystal form K2 of daprodustat comprises acicular crystals as shown in FIG. 12. In certain embodiments, the particles of the crystal form K2 of daprodustat have a size of less than about 300 microns, less than about 250 microns, less than about 200 microns, less than about 150 microns, less than about 100 microns, less than about 50 microns, less than about 25 microns or less than about 10 microns. In certain embodiments, the particles of the crystal form N2 of daprodustat have a size between 120 to 10 microns, between 90 to 10 microns, between 60 to 10 microns, or between 30 to 10 microns. In certain embodiments, the particles of the crystal form K2 of daprodustat comprises acicular crystals having a size as shown in FIG. 14.

In an embodiment, the invention relates to a crystal form of daprodustat which is a cocrystal comprising daprodustat free acid and daprodustat potassium salt referred as form K1 , which has an X-ray powder diffraction (XRPD) pattern comprising peaks at 2theta values of 5.4°±0.2°, 7.9°±0.2°, 14.6°±0.2°, 15.6°±0.2° and 17.5°±0.2°, measured with Ko radiation of copper having an X-ray wavelength of 1.5406 A at room temperature.

In an embodiment, the crystal form K1 of daprodustat further comprises peaks at 2theta values of 10.2°±0.2°, 11.3°±0.2°, 12.1 °±0.2°, 15.8°±0.2° and 16.2°±0.2°, measured with Ko radiation of copper having an X-ray wavelength of 1 .5406 A at room temperature.

In an embodiment, the crystal form K1 of daprodustat is characterized by XRPD peaks located at one, two, three, four, five, six, seven, eight, nine or ten of the following approximate positions: 5.4, 7.9, 10.2, 11.3, 12.1 , 14.6, 15.6, 15.8, 16.2, and 17.5 ±0.2 degrees 2Theta.

In an embodiment, the crystal form K1 of daprodustat is characterized by an XRPD pattern which matches the pattern exhibited in FIG. 15. In an embodiment, the crystal form K1 of daprodustat is characterized by an XRPD pattern having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 peaks matching peaks in the representative crystal form K1 of daprodustat pattern provided in Table 4.

Table 4

In certain embodiments, the crystal form K1 of daprodustat is characterized by thermal analysis. A representative DSC plot for the crystal form K1 is shown in FIG. 16. In certain embodiments, the crystal form K1 is characterized by a DSC plot comprising a first endothermic event with an onset temperature at about 56°C. In certain embodiments, the crystal form K1 is characterized by a DSC plot further comprising an exothermic event with an onset temperature at about 202°C. In certain embodiments, the crystal form K1 is characterized by a DSC plot further comprising two overlapped endothermic events with onset temperatures at about 251 °C and about 254°C, respectively.

A representative TGA plot for the crystal form K1 of daprodustat is also shown in FIG. 16. In certain embodiments, the crystal form K1 of daprodustat is characterized by a TGA plot comprising a mass loss of about 13.2%, of the total mass of the sample upon heating from about 40°C to about 130°C. In certain embodiments, the crystal form K1 of daprodustat contains water or other solvent in the crystal lattice. In certain embodiments, the crystal form K1 of daprodustat contains water in the crystal lattice. In certain embodiments, the crystal form K1 of daprodustat is a hydrate. In certain embodiments, the crystal form K1 of daprodustat is a hydrate which contains an amount of water between 10% and 14% as measured by TGA analysis.

In certain embodiments, the molar ratio of daprodustat free acid to daprodustat potassium salt in the crystal form KI is of from 1 :3 to 3:1 , preferably from 1 :2 to 2:1, more preferably is about 1 :1. In certain embodiments, the molar ratio of daprodustat free acid and daprodustat potassium salt in the crystal form K1 is about 2:1. In preferred embodiments, the molar ratio of daprodustat free acid and daprodustat potassium salt in the crystal form K1 is about 1 :1.

In certain embodiments, a sample of the crystal form K1 of daprodustat comprises particles having needles (acicular) morphology. In certain embodiments, the particles of the crystal form K1 of daprodustat have a size of less than about 300 microns, less than about 250 microns, less than about 200 microns, less than about 150 microns, less than about 100 microns, less than about 50 microns, less than about 25 microns or less than about 10 microns. In certain embodiments, the particles of the crystal form K1 of daprodustat have a size between 120 to 10 microns, between 90 to 10 microns, between 60 to 10 microns, or between 30 to 10 microns.

The invention further encompasses the crystal form N2, the crystal form N4, the crystal form K2 and the crystal form K1 of daprodustat in pure form or when admixed with other materials, for example, other polymorphs, solvates or remaining reaction solvents or side products. Particularly, the invention also refers to mixtures comprising crystal form of N2 with crystal form N4 thereof. Particularly, the invention also refers to mixtures of crystal form of K2 with crystal form K1 thereof.

According to the second aspect, the invention provides a process for the preparation of crystal forms of daprodustat as defined above, comprising the following steps: a) providing daprodustat free acid in a solvent or a mixture of solvents, b) optionally, heating the mixture of step (a) at a suitable temperature, preferably from 70°C to 100°C, more preferably from 75°C to 95°C, c) adding a source of a pharmaceutically acceptable metal inorganic base, preferably an alkali metal inorganic base, particularly a pharmaceutically acceptable metal hydroxide, more preferably in the form of an alkali metal hydroxide, in an amount of about 0.5 molar equivalents with respect to daprodustat free acid of step (a), d) optionally, cooling the solution resulting from step (c) to room temperature, and e) isolating the crystal form of daprodustat.

Daprodustat free acid used in (a) may be in solvate, hydrate, anhydrous, crystalline form, or non-crystalline form thereof. Preferably, daprodustat free acid is in crystalline form, even more preferably daprodustat free acid used in step (a) is in anhydrous form. Daprodustat solvate used in step (a) may be a diethyl ether solvate as prepared in Ex. 18 Method 1 of W02007/150011 A2.

In certain embodiments, daprodustat solvate is methyl tert-butyl ether solvate, dioxane solvate, tetrahydrofuran solvate, methanol solvate, ethanol solvate, isopropanol solvate, tert-butanol solvate, dimethylformamide (DMF) solvate or dimethylsulfoxide (DMSO) solvate, all prepared in Table 5 of example 1.

Suitable solvent used in step (a) may be selected from the group of (C6-Ci4)aromatic hydrocarbon solvents such as toluene, o-xylene, m-xylene, and p-xylene; halogenated (Ci-Ci2)hydrocarbon solvents such as 1 ,2- dichloroethane, dichloromethane, chloroform; (Ci-Ci2)ether solvents such as diethyl ether, dipropyl ether, diphenyl ether, isopropyl ether, tert-butyl methyl ether, tetrahydrofuran, 1 ,4-dioxane; (Ci-Ci2)alcohol solvents such as methanol, ethanol, isopropanol, 1-propanol, 2-methyl-1 -propanol, 1-butanol, 2-butanol, 1- pentanol, 3-methyl-1 -butanol, tert-butanol, 1 -octanol, benzylalcohol, phenol, trifluoroethanol, glycerol, ethylene glycol, propylene glycol, m-cresol; nitrobenzene; N,N-dimethylformamide; N,N,- dimethylacetamide; N-methyl-2-pyrrolidone; or acetonitrile. In some embodiments, the solvent may be formed by the combination of two or more solvents. In certain embodiments, the solvent used in step (a) is selected from the group consisting of (Ci-Cejalcohol, acetonitrile, dichloromethane (DCM), toluene, tetrahydrofuran, and a combination thereof.

In certain embodiments, the solvent used in step (a) is a mixture of water and another solvent selected from the group consisting of (Ci-C6)alcohol, acetonitrile, dichloromethane (DCM), toluene, and tetrahydrofuran. In certain embodiments, the solvent of step (a) is mixture of a (Ci-Ce)alcohol or acetonitrile with water. In preferred embodiments, the solvent used in step (a) is a mixture of acetonitrile and water. In preferred embodiments, the solvent used in step (a) is a mixture of acetonitrile and water in a ratio from 5: 1 (v/v) to 15:1 (v/v), preferably from 8: 1 (v/v) to 12:1 (v/v), more preferably acetonitrile and water in a ratio of 9:1 (v/v).

In certain embodiments, the pharmaceutically acceptable metal inorganic base, particularly a metal hydroxide, is an alkali metal inorganic base, selected from a sodium and a potassium inorganic base. In certain embodiments, the source of the pharmaceutically acceptable metal hydroxide of step (b) is in the form of an alkali metal hydroxide. In certain embodiments, the alkali metal hydroxide is selected from sodium hydroxide and potassium hydroxide. In certain embodiments, the pharmaceutically acceptable metal inorganic base is sodium to provide the cocrystal of daprodustat and daprodustat sodium salt. In certain embodiments, the pharmaceutically acceptable metal inorganic base is potassium to provide the cocrystal of daprodustat and daprodustat potassium salt.

In certain embodiments, step (b) is carried out to provide a solution. In certain embodiments, step (d) is carried out to induce the crystallization of the crystal form. In certain embodiments, seeding with the desired crystal form of daprodustat is used to control the crystallization before step (d) is carried out. In a preferred embodiment, steps (b) and (d) are carried out. In a more preferred embodiment, steps (b) and (d) are carried out, and seeding with the desired crystal form of daprodustat is used before step (d) is carried out.

In one embodiment, in the process for the preparation of the crystal form of daprodustat according to the second aspect, daprodustat free acid of step (a) is prepared by the process as defined in the sixth aspect, or alternatively by the process as defined in any of the eighth aspect, or alternatively by the process as defined in the tenth aspect.

Alternatively, according to the third aspect, the preparation of crystal forms of daprodustat which are cocrystals comprising daprodustat free acid and a daprodustat pharmaceutically acceptable metal salt, wherein the metal salt is an alkali metal salt such as a sodium or a potassium salt, comprises the steps of:

I. providing an ester intermediate of formula (VI), wherein R is (Ci-C )alkyl which is unsubstituted or substituted with one or more substituents independently selected from the group consisting of (Cs-Cejcycloalkyl, (C3-Ci2)heterocycloalkyl, (Ce-Ci4)ary I and (Cs-C^heteroaryl, preferably R is ethyl, in a solvent or a mixture of solvents, ii. optionally, heating the mixture of step (i) at a suitable temperature, preferably from room temperature to 100°C, more preferably from 30°C to 75°C, iii. adding a source of a pharmaceutically acceptable metal inorganic base, preferably an alkali metal inorganic base, particularly a pharmaceutically acceptable metal hydroxide-salt, , more preferably in the form of an alkali metal hydroxide, in an amount from 1.0 to 1.2, or alternatively from 1.0 to 2.2, preferably from 2.0 to 2.2 molar equivalents with respect to the ester intermediate of step (i) to form the corresponding daprodustat alkali metal of formula (la), wherein M is an alkali metal such as sodium or potassium, iv. adding an acid, preferably a hydracid such as hydrochloric acid, hydrobromic acid or hydroiodic acid, more preferably hydrochloric acid, in an amount from 0.5 to 0.7, or alternatively from 0.5 to 1 .7, preferably from 1 .5 to 1 .7 molar equivalents to the mixture of step (iii), v. optionally, cooling the mixture resulting from step iv) to room temperature, and vi. isolating the crystal form of daprodustat.

In an embodiment in the above process, step (iii) comprises adding a source of a pharmaceutically acceptable metal inorganic base, particularly a pharmaceutically acceptable metal hydroxide, preferably an alkali metal inorganic base, more preferably in the form of an alkali metal hydroxide, in an amount from 1 .0 to 2.2, preferably from 2.0 to 2.2 molar equivalents with respect to the ester intermediate of formula (VI), wherein R is as defined above, of step (I); and step (iv) comprises adding an acid, preferably a hydracid such as hydrochloric acid, hydrobromic acid or hydroiodic acid, more preferably hydrochloric acid, in an amount from 0.5 to 1 .7, preferably from 1 .5 to 1 .7 molar equivalents to the mixture of step (iii). Advantageously, the process for the preparation of crystal forms of daprodustat as defined herein starting from ester intermediate of formula (VI), wherein R is as defined above, is reproducible and does not generate an excess of inorganic impurities difficult to remove from final product.

Ester intermediate used in step (i) as starting material may be prepared as described in W02007/150011 A2, particularly compound VI wherein R is ethyl, i.e. N-[(1 ,3-dicyclohexyl-6-hydroxy-2,4-dioxo-1 , 2,3,4- tetrahydro-5-pyrimidinyl)carbonyl]glycine as prepared in Example 18.

Suitable solvent used in step (i) may be selected from the group of water, (Ci-Ci2)alcohol solvents such as methanol, ethanol, isopropanol, 1-propanol, 2-methyl- 1-propanol, 1-butanol, 2-butanol, 1-pentanol, 3- methyl-1 -butanol, tert-butanol, 1 -octanol, benzylalcohol, phenol, trifluoroethanol, glycerol, ethylene glycol, propylene glycol, m-cresol; (Ci-Ci2)ether solvents such as diethyl ether, tert-butyldimethyl ether, tetrahydrofuran and dioxane; nitrobenzene; N,N-dimethylformamide; N,N, -dimethylacetamide; N-methyl-2- pyrrolidone; and acetonitrile. In certain embodiments, the solvent may be formed by the combination of two or more solvents. In certain embodiments, the solvent used in step (i) is a (Ci-Ci2)alcohol or acetonitrile. In certain embodiments, the solvent of step (i) is methanol, ethanol, isopropanol, 1-propanol or acetonitrile.

In certain embodiments, the solvent of step (i) is a mixture of a (Ci-Ci2)alcohol or acetonitrile with water. In certain embodiments, the (Ci-Ci2)alcohol is selected from methanol, ethanol, isopropanol and 1-propanol. In certain embodiments, the solvent used in step (i) is a mixture of ethanol or acetonitrile with water. In certain embodiments, the solvent used in step (i) is a mixture of ethanol or acetonitrile with water in a ratio from 5:1 (v/v) to 15:1 (v/v), preferably from 8:1 (v/v) to 12:1 (v/v), more preferably acetonitrile and water in a ratio of 9: 1 (v/v).

In certain embodiments, the pharmaceutically acceptable metal salt of step (iii) is an alkali metal salt selected from a sodium and a potassium salt. In certain embodiments, the source of the pharmaceutically acceptable metal inorganic base, particularly a metal hydroxide, of step (iii) is in the form of an alkali metal hydroxide. In certain embodiments, the alkali metal hydroxide is selected from sodium hydroxide and potassium hydroxide. In an embodiment, metal hydroxide of step (iii) is added as an aqueous solution. In certain embodiments, the pharmaceutically acceptable metal salt of step (iii) is sodium to provide the cocrystal of daprodustat free acid and daprodustat sodium salt. In certain embodiments, the pharmaceutically acceptable metal salt of step (iii) is potassium to provide the cocrystal of daprodustat free acid and daprodustat potassium salt. In certain embodiments, the step (iii) further comprises the substeps of: (iii.1) removing completely or partially the solvent or mixture of solvents of step (I) by evaporation or distillation, and (iii.2) adding a different solvent or mixture of solvents to provide a solution or a suspension.

In certain embodiments, in substep (iii.1) the solvent is a (Ci-Ci2)alcohol and is removed partially or completely by evaporation or distillation, and in substep (iii.2) the solvent is a mixture of acetonitrile and water in a ratio from 5:1 (v/v) to 15:1 (v/v), preferably from 8:1 (v/v) to 12:1 (v/v), more preferably acetonitrile and water in a ratio of 9:1 (v/v) to provide a solution or a suspension of the corresponding daprodustat alkali metal of formula (la), wherein M is an alkali metal such as sodium or potassium.

Suitable acid used in step (iv) is an inorganic or an organic acid. Suitable acids are those which have a pKa (relative to water) below 5, preferably below 3, more preferably below 1. Preferably, the acid has a pKa (relative to water) from -10 to 5, preferably from -10 to 3, more preferably from -10 to 1 . Examples of suitable acids include but are not limited to hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, formic acid, acetic acid, dichloroacetic acid, methanesulfonic acid, p-toluenesulfonic acid and camphorsulfonic acid. Preferably, the acid used in step (iv) is a hydracid such as hydrochloric, hydrobromic or hydroiodic acid, which may be present either as gas or as an aqueous solution or generated in situ, for example from an alkylsilyl halogenide in the presence of a protic solvent. More preferably, the acid used in step (iv) is hydrochloric acid in aqueous solution.

In certain embodiments, step (ii) is carried out to provide a solution. In certain embodiments, step (v) is carried out to induce the crystallization of the crystal form. In certain embodiments, seeding with the desired crystal form of daprodustat is used to control the crystallization before step (v) is carried out. In certain embodiments, seeding with the desired crystal form of daprodustat is carried out before or during step (iv). In certain embodiments, seeding with the desired crystal form of daprodustat is carried out as many times as necessary to control the desired crystal form of daprodustat. In a preferred embodiment, steps (ii) and (v) are carried out. In a more preferred embodiment, steps (ii) and (v) are carried out, and seeding with the desired crystal form of daprodustat is used before step (v) is carried out. In a preferred embodiment, steps (ii) and (v) are carried out, and seeding with the desired crystal form of daprodustat is used before step (v), or before or during step (iv) are carried out. In certain embodiments, hot filtration of the resulting mixture of step (iii) may be carried out to remove any insoluble material.

In certain embodiments, the present invention refers to the potassium salt of daprodustat of formula (la) wherein M is potassium. In certain embodiments, the potassium salt of daprodustat of formula (la) wherein M is potassium is in solvate, hydrate, crystalline form, or non-crystalline form thereof. In one embodiment, in the process for the preparation of the crystal form of daprodustat according to the third aspect, wherein the ester intermediate of formula (VI) is prepared by the process as defined in the ninth aspect.

In certain embodiments, the preparation of crystal form N2 of daprodustat which is a cocrystal comprising daprodustat free acid and a daprodustat sodium salt, comprises the steps of: i. providing an ester intermediate of formula (VI), wherein R is (Ci-C )alkyl which is unsubstituted or substituted with one or more substituents independently selected from the group consisting of (Cs-Cejcycloalkyl, (C3-Ci2)heterocycloalkyl, (Ce-Ci4)ary I and (Cs-C^heteroaryl, preferably R is ethyl, in a solvent or a mixture of solvents, ii. optionally, heating the mixture of step (i) at a suitable temperature, preferably from room temperature to 100°C, more preferably from 30°C to 75°C, iii. adding a source of an alkali metal which is sodium, preferably in the form of an alkali metal hydroxide which is sodium hydroxide, in an amount from 1 .0 to 1 .2, or alternatively from 1 .0 to 2.2, preferably from 2.0 to 2.2 molar equivalents with respect to the ester intermediate of step (i) to form the corresponding daprodustat sodium salt, iv. adding an acid, preferably a hydracid such as hydrochloric acid, hydrobromic acid or hydroiodic acid, more preferably hydrochloric acid, in an amount from 0.5 to 0.7, or alternatively from 0.5 to 1 .7, preferably from 1 .5 to 1 .7 molar equivalents to the mixture of step (iii), v. optionally, cooling the mixture resulting from step iv) to room temperature, and vi. isolating the crystal form of daprodustat.

In an embodiment in the above process, step (iii) comprises adding a source of a pharmaceutically acceptable metal inorganic base, particularly a pharmaceutically acceptable metal hydroxide, preferably an alkali metal inorganic base, more preferably in the form of an alkali metal hydroxide, in an amount from 1 .0 to 2.2, preferably from 2.0 to 2.2 molar equivalents with respect to the ester intermediate of formula (VI), wherein R is as defined above, of step (i); and step (iv) comprises adding an acid, preferably a hydracid such as hydrochloric acid, hydrobromic acid or hydroiodic acid, more preferably hydrochloric acid, in an amount from 0.5 to 1 .7, preferably from 1 .5 to 1 .7 molar equivalents to the mixture of step (iii).

In certain embodiments, steps (ii) and (v) as defined above are carried out. In certain embodiments, seeding with crystal form N2 of daprodustat is used to control the crystallization before step (v) is carried out.

In certain embodiments, the step (iii) further comprises the substeps of: (iii.1) removing completely or partially the solvent or mixture of solvents of step (i) by evaporation or distillation, and (iii.2) adding a different solvent or mixture of solvents to provide a solution or a suspension.

In certain embodiments, in substep (iii.1) the solvent is a (Ci-Ci2)alcohol and is removed partially or completely by evaporation or distillation, and in substep (iii.2) the solvent is a mixture of acetonitrile and water in a ratio from 5:1 (v/v) to 15:1 (v/v), preferably from 8:1 (v/v) to 12:1 (v/v), more preferably acetonitrile and water in a ratio of 9:1 (v/v) to provide a solution or a suspension of the corresponding daprodustat alkali metal of formula (la), wherein M is sodium.

In certain embodiments, the preparation of crystal form K2 of daprodustat which is a cocrystal comprising daprodustat free acid and a daprodustat potassium salt, comprises the steps of: i. providing an ester intermediate of formula (VI), wherein R is (Ci-C )alkyl which is unsubstituted or substituted with one or more substituents independently selected from the group consisting of (Cs-Cejcycloalkyl, (C3-Ci2)heterocycloalkyl, (Ce-Ci4)ary I and (Cs-C^heteroaryl, preferably R is ethyl, in a solvent or a mixture of solvents, ii. optionally, heating the mixture of step (i) at a suitable temperature, preferably from room temperature to 100°C, more preferably from 30°C to 75°C, iii. adding a source of an alkali metal which is potassium, preferably in the form of an alkali metal hydroxide which is potassium hydroxide, in an amount from 1 .0 to 1 .2, or alternatively from 1 .0 to 2.2, preferably from 2.0 to 2.2 molar equivalents with respect to the ester intermediate of step (i) to form the corresponding daprodustat potassium salt, iv. adding an acid, preferably a hydracid such as hydrochloric acid, hydrobromic acid or hydroiodic acid, more preferably hydrochloric acid, in an amount from 0.5 to 0.7, or alternatively from 0.5 to 1 .7, preferably from 1 .5 to 1.7 molar equivalents to the mixture of step (ill), v. optionally, cooling the mixture resulting from step iv) to room temperature, and vi. isolating the crystal form of daprodustat.

In an embodiment in the above process, step (ill) comprises adding a source of a pharmaceutically acceptable metal inorganic base, particularly a pharmaceutically acceptable metal hydroxide, preferably an alkali metal inorganic base, more preferably in the form of an alkali metal hydroxide, in an amount from 1 .0 to 2.2, preferably from 2.0 to 2.2 molar equivalents with respect to the ester intermediate of formula (VI), wherein R is as defined above, of step (I); and step (iv) comprises adding an acid, preferably a hydracid such as hydrochloric acid, hydrobromic acid or hydroiodic acid, more preferably hydrochloric acid, in an amount from 0.5 to 1 .7, preferably from 1 .5 to 1 .7 molar equivalents to the mixture of step (ill).

In certain embodiments, steps (II) and (v) as defined above are carried out. In certain embodiments, seeding with crystal form K2 of daprodustat is used to control the crystallization before step (v) is carried out.

In certain embodiments, the step (ill) further comprises the substeps of: (iii.1) removing completely or partially the solvent or mixture of solvents of step (I) by evaporation or distillation, and (ill.2) adding a different solvent or mixture of solvents to provide a solution or a suspension.

In certain embodiments, in substep (iii.1) the solvent is a (Ci-Ci2)alcohol and is removed partially or completely by evaporation or distillation, and in substep (ill.2) the solvent is a mixture of acetonitrile and water in a ratio from 5:1 (v/v) to 15:1 (v/v), preferably from 8:1 (v/v) to 12:1 (v/v), more preferably acetonitrile and water in a ratio of 9:1 (v/v) to provide a solution or a suspension of the corresponding daprodustat alkali metal of formula (la), wherein M potassium.

It also forms part of the invention, a process for the preparation of crystal form K1 of daprodustat which is a cocrystal comprising daprodustat free acid and a daprodustat potassium salt, the process comprising the steps of:

I. providing an ester intermediate of formula (VI), wherein R is (Ci-C )alkyl which is unsubstituted or substituted with one or more substituents independently selected from the group consisting of (Cs-Cejcycloalkyl, (C3-Ci2)heterocycloalkyl, (Ce-Ci4)ary I and (Cs-C^heteroaryl, preferably R is ethyl, in a solvent or a mixture of solvents ii. optionally, heating the mixture of step (i) at a suitable temperature, preferably from room temperature to 100°C, more preferably from 30°C to 75°C, iii. adding a source of an alkali metal which is potassium, preferably in the form of an alkali metal hydroxide which is potassium hydroxide, in an amount from 1 .0 to 1 .2, or alternatively from 1 .0 to 2.2, preferably from 2.0 to 2.2 molar equivalents with respect to the ester intermediate of step (i) to form the corresponding daprodustat potassium salt, iv. adding an acid, preferably a hydracid such as hydrochloric acid, hydrobromic acid or hydroiodic acid, more preferably hydrochloric acid, in an amount from 0.5 to 0.7, or alternatively from 0.5 to 1 .7, preferably from 1 .5 to 1 .7 molar equivalents to the mixture of step (iii), v. optionally, cooling the mixture resulting from step iv) to room temperature, and vi. isolating the crystal form of daprodustat.

In an embodiment in the above process, step (iii) comprises adding a source of a pharmaceutically acceptable metal inorganic base, particularly a pharmaceutically acceptable metal hydroxide, preferably an alkali metal inorganic base, more preferably in the form of an alkali metal hydroxide, in an amount from 1 .0 to 2.2, preferably from 2.0 to 2.2 molar equivalents with respect to the ester intermediate of formula (VI), wherein R is as defined above, of step (I); and step (iv) comprises adding an acid, preferably a hydracid such as hydrochloric acid, hydrobromic acid or hydroiodic acid, more preferably hydrochloric acid, in an amount from 0.5 to 1 .7, preferably from 1 .5 to 1 .7 molar equivalents to the mixture of step (iii).

In certain embodiments, steps (ii) and (v) as defined above are carried out. In certain embodiments, seeding with crystal form K1 of daprodustat is used to control the crystallization before step (v) is carried out. In certain embodiments, the step (iii) further comprises the substeps of: (iii.1) removing completely or partially the solvent or mixture of solvents of step (I) by evaporation or distillation, and (iii.2) adding a different solvent or mixture of solvents to provide a solution or a suspension.

In certain embodiments, in substep (iii.1) the solvent is a (Ci-Ci2)alcohol and is removed partially or completely by evaporation or distillation, and in substep (iii.2) the solvent is a mixture of acetonitrile and water in a ratio from 5:1 (v/v) to 15:1 (v/v), preferably from 8:1 (v/v) to 12:1 (v/v), more preferably acetonitrile and water in a ratio of 9:1 (v/v) to provide a solution or a suspension of the corresponding daprodustat alkali metal of formula (la), wherein M potassium.

It also forms part of the invention, a process for the preparation of a crystal form N4 of daprodustat which comprises drying the crystal form N2 of daprodustat under vacuum and isolating the crystal form N4 of daprodustat. In certain embodiments, the process for the preparation of a crystal form N4 comprises drying the crystal form N2 of daprodustat under vacuum (about 12 mbar) at a temperature from room temperature to 75°C, preferably from 45°C to 60°C, for at least 6 hours, preferably for at least 12 hours, and isolating the crystal form N4 of daprodustat. In certain embodiments, the process for the preparation of a crystal form N4 comprises drying the crystal form N2 of daprodustat under vacuum (about 12 mbar) at a temperature from 45°C to 60°C for at least 6 hours, preferably for at least 12 hours, and isolating the crystal form N4 of daprodustat.

The above processes may further provide the crystal form N2, the crystal form N4, the crystal form K2 and the crystal form K1 of daprodustat in pure form or when admixed with other materials, for example, other polymorphs, solvates or remaining reaction solvents or side products. Particularly, the above processes may provide mixtures comprising crystal form of N2 with crystal form N4 thereof. Particularly, the above processes may provide mixtures of crystal form of K2 with crystal form K1 thereof.

According to the fourth aspect of the invention, the pharmaceutical compositions comprising crystal forms of daprodustat selected from the group consisting of crystal form N2, crystal form N4, a crystal form K2, a crystal form K1 , and a combination thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients may further contain disintegrants, glidants, lubricants, binders, colorants, and combinations thereof. In an embodiment, the pharmaceutical compositions comprising crystal forms of daprodustat selected from the group consisting of crystal form N2, crystal form N4, a crystal form K2, a crystal form K1 may be tablets, dispersible tablets or granules suitable for oral use.

According to the fifth aspect, the crystal forms of daprodustat selected from the group consisting of crystal form N2, crystal form N4, a crystal form K2, a crystal form K1 , and a combination thereof may be particularly indicated for the treatment of symptomatic anaemia associated with chronic kidney disease (CKD), in adults on chronic maintenance dialysis or in adults not in dialysis.

As mentioned above a sixth aspect of the present invention relates to a process for preparing daprodustat of formula (I), or a salt thereof which comprises hydrolysing a compound of formula (VI) in the presence of a quaternary ammonium hydroxide of formula N[(Ci-C4)alkyl]4OH, preferably tetramethylammonium hydroxide (TMAH), and a solvent, and optionally converting daprodustat of formula (I) into a pharmaceutically or veterinary acceptable salt thereof.

In an embodiment, the amount of the quaternary ammonium hydroxide, preferably tetramethylammonium hydroxide (TMAH), is in a molar ratio from 1 : 1 to 4: 1 of base to compound of formula (VI), preferably from 2:1 to 3: 1 of base to compound of formula (VI).

In an embodiment, suitable solvents of the sixth aspect are selected from the group consisting of a (Ci- Cajalcohols, preferably methanol, ethanol or isopropanol; water; and mixtures thereof.

In an embodiment, the solvent is a mixture of a (Ci-C3)alcohols, preferably methanol, ethanol or isopropanol, and water in a volume ratio from 10: 1 to 1 :2, preferably from 5:1 to 1 :2, even more preferably from 10: 1 to 5:1.

In an embodiment of the sixth aspect after the hydrolysis reaction has been finished an acid selected from formic, acetic, monochloroacetic, trichloroacetic and trifluoroacetic acid (TFA), preferably trifluoroacetic acid (TFA), is added.

In an embodiment, the amount of acid, preferably trifluoroacetic acid (TFA), is in a molar ratio from 1 : 1 to 4:1 of acid to compound of formula (VI), preferably from 2:1 to 3: 1 of base to compound of formula (VI).

In an embodiment, daprodustat is further purified by slurrying or crystallizing it in an organic solvent selected from alcohols, preferably (Ci-C3)alcohols, more preferably methanol, ethanol or isopropanol, more preferably in ethanol, or an acid solvent such as acetic acid. In an embodiment, daprodustat is purified by crystallization in acetic acid.

In an embodiment of the sixth aspect, the compound (VI) is obtained by reacting a compound of formula (V) with an isocyanatoacetate of formula (IV), in the presence of a base and a solvent, wherein the compound of formula (V) is preferably obtained according to the process of the seventh aspect. In an embodiment of the sixth aspect, the compound (VI) is obtained from a compound of formula (II), preferably according to the process of the fourth aspect.

In an embodiment of the sixth aspect, the process further comprises the preparation of a crystal form of daprodustat as defined above by the process as defined in the second aspect of the invention.

The seventh aspect of the invention provides a process for preparing key intermediate 1 ,3-dicyclohexyl barbituric acid of formula (V), which comprises the steps of:

(i) reacting dicyclohexylurea (DCU) with malonic acid in the presence of AC2O in acetic acid to provide compound of formula (V),

(ii) adding an antisolvent, preferably water, in an amount from 1 to 10 volumes per g of compound (V), preferably from 3 to 6 volumes per g of compound of formula (V),

(iii) optionally, heating the mixture of step (ii) at a temperature from 20°C to 85°C, preferably from 20°C to 30°C, and

(iv) isolating the compound of formula (V).

In an embodiment of the seventh aspect, the compound (V) is further converted into daprodustat by reacting it with an isocyanatoacetate of formula (IV), in the presence of a base and a solvent to provide the compound of formula (VI); and hydrolyzing compound of formula (VI) in the presence of a base and a solvent, preferably according to the process of the sixth aspect, or alternatively using NaOH or KOH instead of a quaternary ammonium hydroxide, followed by neutralization with an acid such as hydrochloric acid.

In an embodiment of the seventh aspect of the invention, the amount of malonic acid in step (i) to dicyclohexylurea (DCU) is in a molar ratio from 2:1 to 1 :1 , preferably from 1.5:1 to 1.1 :1.

In an embodiment, the amount of AC2O used in step (i) to dicyclohexylurea (DCU) is in a molar ratio from 4:1 to 2: 1 , preferably from 3: 1 to 2:1.

In an embodiment, the mixture of step (i) is heated at a temperature from 70°C to 95°C, preferably from 85°C to 90°C.

In an embodiment, the amount of antisolvent, preferably water, of step (ii) is from 1 to 10 volumes per g of compound (V), preferably from 3 to 6 volumes per g of compound of formula (V).

In an embodiment, the mixture of compound of formula (V) and the antisolvent, preferably water, of step (ii) is heated at a temperature from 20°C to 85°C, preferably from 20°C to 30°C as the yield increases. In an embodiment, compound of formula (V) is further purified by crystallization or slurrying it in an organic solvent selected from (Ci-C3)alcohols, preferably methanol, ethanol or isopropanol, more preferably in isopropanol.

In an embodiment of the eighth aspect, the process further comprises the preparation of a crystal form of daprodustat as defined above by the process as defined in the second aspect of the invention.

A ninth aspect of the present invention provides a process for preparing a compound of formula (VI), wherein R is (Ci-Cio)alkyl which is unsubstituted or substituted with one or more substituents independently selected from the group consisting of Cs-Cecycloalkyl, heterocycloalkyl, aryl and heteroaryl, particularly, selected from the group consisting of (Cs-Cejcycloalkyl, (C3-Ci2)heterocycloalkyl, (Ce-Ci4)aryl and (Cs-C^heteroaryl, preferably R is ethyl, which comprises the steps of: a) converting a formamide of formula (II), into a compound an isocyanoacetate of formula (III), wherein R is as defined above, preferably R is ethyl; b) converting the isocyanoacetate of formula (III) of step (a) into an isocyanatoacetate of formula (IV), wherein R is as defined above, preferably R is ethyl; c) reacting the isocyanatoacetate of formula (IV) obtained in step (b) with a 1 ,3-dicyclohexyl barbituric acid of formula (V), in the presence of a base and a solvent to provide the compound of formula (VI); wherein steps (b) and (c) are carried out in a consecutive manner.

In an embodiment, the formamide of formula (II) is prepared from glycine ethyl ester hydrochloride in the presence of trimethyl orthoformate or triethyl orthoformate. In an embodiment, the formamide of formula (II) is prepared from glycine ethyl ester hydrochloride in the presence of methyl formate or ethyl formate and a base, preferably an organic base such as TEA. In an embodiment, the formamide of formula (II) is alternatively prepared from glycine ethyl ester in the presence of methyl formate. In an embodiment, the formamide of formula (II) is prepared from glycine ethyl ester hydrochloride in the presence of ethyl formate and a base, preferably an organic base such as TEA.

In an embodiment, the reaction mixture may be heated at a temperature from 20°C to 85°C, preferably from 35°C to 85°C, more preferably from 45°C to 85°C, for the time sufficient to complete the reaction (e.g., from 4 to 48 h).

In an embodiment, the organic salt (e.g., triethylamine hydrochlrohide) formed in the preparation of formamide of formula (II) when using methyl formate or ethyl formate and a base, preferably an organic base such as TEA, may be removed by adding an organic solvent, preferably from (C6-Ci4)aromatic hydrocarbon solvent such as toluene. In an embodiment, the formamide of formula (II) is purified by fractioned distillation. In an embodiment, the formamide of formula (II) is used in the next step without further purification.

In an embodiment of the ninth aspect, step (a) is carried out in the presence of a dehydrating agent and a base. Examples of dehydrating agent include but are not limited to phosgene, phosphorus oxychloride (POCh), p-toluensulfonic chloride, triphenylphosphine (PPh3)/iodine (I2), Burgess reagent, Appel reagent, or trifluoromethyl sulfonic acid anhydride. Preferably, the dehydrating agent is POCI3. Suitable base may be an organic base selected from ammonia derivatives, such as diethylamine, triethylamine (TEA), N,N- dicyclohexylmethylamine, N,N-dicyclohexylamine, and N, N-diisopropylethylamine (DIPEA), and heterocyclic bases such as pyridine, and diazabicycloundecene (DBU), and mixtures thereof. Preferably, the base is TEA and DIPEA.

In an embodiment, the isocyanoacetate of formula (III) is used in the next step (b) without further purification.

In an embodiment, step (b) is carried out in the presence of dimethylsulfoxide (DMSO) and trifluoroacetic anhydride (TFAA). Alternatively, step (b) is carried out using mercuric oxide, lead tetraacetate, ozone, halogen- or acid-catalyzed oxidations by dimethyl sulfoxide (DMSO) and pyridine N-oxide.

In an embodiment, the temperature used in step (b) is from -20°C to -40°C, preferably about -30°C.

In an embodiment, for the preparation of compound (IV) a solution of DMSO in a solvent is added to a solution of compound (III) and trifluoroacetic anhydride in a solvent.

In an embodiment, in step (c) the amount of the isocyanatoacetate of formula (IV), preferably wherein R is ethyl, to compound of formula (V) is in a molar ratio from 1 :1 to 2:1 , preferably from 1 :1 to 1.5: 1 , more preferably from 1 :1 to 1.2:1.

In an embodiment, step (c) is carried out in the presence of a base and a solvent to provide the compound of formula (VI). A suitable base may be an organic base including ammonia derivatives, such as diethylamine, triethylamine (TEA), N,N-dicyclohexyl-methylamine, N,N-dicyclohexylamine, and N,N- diisopropylethylamine (DIPEA), and heterocyclic bases such as pyridine, diazabicycloundecene (DBU) and mixtures thereof. Preferably, the base used in step (c) is an organic base selected from TEA or DIPEA. In an embodiment, the amount of base may be from 1 .0 to 3.0, preferably from 1 .1 to 2.5, more preferably from 1 .1 to 1 .5 molar equivalents with respect to compound of formula (V). A suitable solvent for steps (a), (b) and (c) may be an aprotic organic solvent. In an embodiment, the aprotic solvent may be selected from halogenated hydrocarbon solvents (e.g., 1 ,2-dichloroethane, dichloromethane, chloroform, etc.), aromatic hydrocarbon solvents (e.g., toluene, o-xylene, m-xylene, and p-xylene), and ether solvents (e.g., diethyl ether, dipropyl ether, diphenyl ether, isopropyl ether, tert-butyl methyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, etc.). Preferably, the solvent is dichloromethane, toluene or tetrahydrofuran.

In an embodiment, the 1 ,3-dicyclohexyl barbituric acid of formula (V) is added to the isocyanatoacetate of formula (IV). In an embodiment, compound of formula (V) is added in solid form or dissolved in solution or suspended in an aprotic solvent with or without the presence of a base. In an embodiment, a solution of isocyanatoacetate of formula (IV) in solution of an aprotic solvent is added to a solution or a suspension of compound of formula (V) in an aprotic solvent. In a preferred embodiment, a solution of compound of formula (V) in an aprotic is added to a solution of isocyanatoacetate of formula (IV) in an aprotic solvent. The aprotic solvent is as defined above for step (c). Preferably, the aprotic solvent is dichloromethane, toluene or tetrahydrofuran.

In an embodiment, for the preparation of compound (VI), compound (V) is added in solution of an aprotic solvent, preferably DCM, with or without base. Additionally, the reaction medium of compound (IV) is added into another vessel containing compound (V), with or without base and with or without an aprotic solvent.

In an embodiment of the ninth aspect, the compound (VI) is further converted into daprodustat by hydrolysis in the presence of a base and a solvent, preferably according to the process of the sixth aspect, or alternatively using NaOH or KOH, instead of a quaternary ammonium hydroxide, followed by neutralization with an acid such as hydrochloric acid.

In an embodiment of the ninth aspect, the process further comprises the preparation of a crystal form of daprodustat as defined above by the process as defined in the third aspect of the invention.

In an embodiment, the invention relates to a process for preparing daprodustat of formula (I), or a pharmaceutically or veterinary acceptable salt thereof, which comprises:

1) converting a formamide of formula (II), wherein R is (Ci-C )alkyl which is unsubstituted or substituted with one or more substituents independently selected from the group consisting of (Cs-Cejcycloalkyl, heterocycloalkyl, aryl and heteroaryl, particularly, selected from the group consisting of (Cs-Cejcycloalkyl, (C3-Ci2)heterocycloalkyl, (Ce-Ci4)aryl and (C5- Ci2)heteroaryl, preferably R is ethyl; into a compound an isocyanoacetate of formula (III), wherein R is as defined above, preferably R is ethyl;

2) converting the isocyanoacetate of formula (III) of step (1) into an isocyanatoacetate of formula (IV), wherein R is as defined above, preferably R is ethyl;

3) preparing 1 ,3-dicyclohexyl barbituric acid of formula (V), by a process which comprises the steps of:

(i) reacting dicyclohexylurea (DCU) with malonic acid in the presence of AC2O in acetic acid to provide compound of formula (V),

(ii) adding an antisolvent, preferably water, in an amount from 1 to 10 volumes per g of compound (V), preferably from 3 to 6 volumes per g of compound of formula (V),

(ill) optionally, heating the mixture of step (II) at a temperature from 20°C to 85°C, preferably from 20°C to 30°C, and

(iv) isolating the compound of formula (V);

4) reacting the isocyanatoacetate of formula (IV) obtained in step (2) with the 1 ,3-dicyclohexyl barbituric acid of formula (V) obtained in step (3), in the presence of a base and a solvent to provide the compound of formula (VI) wherein R is (Ci-C )alkyl which is unsubstituted or substituted with one or more substituents independently selected from the group consisting of (Cs-Cejcycloalkyl, heterocycloalkyl, aryl and heteroaryl, particularly, selected from the group consisting of (Cs-Cejcycloalkyl, (C3-Ci2)heterocycloalkyl, (Ce-Ci4)aryl and (C5-Ci2)heteroaryl, preferably R is ethyl; wherein steps (2) and (4) are carried out in a consecutive manner;

5) converting a compound of formula (VI) into daprodustat of formula (I) by hydrolysis in the presence of a quaternary ammonium hydroxide of formula N[(Ci-C4)alkyl]4OH, preferably tetramethylammonium hydroxide (TMAH), and a solvent, and optionally converting daprodustat of formula (I) into a pharmaceutically or veterinary acceptable salt thereof.

In an embodiment, after the hydrolysis reaction of step (5) has been finished an acid selected from formic, acetic, monochloroacetic, trichloroacetic and trifluoroacetic acid (TFA), preferably trifluoroacetic acid (TFA) is added.

In an embodiment, daprodustat is further purified by slurrying in an organic solvent selected from alcohols, preferably (Ci-C3)alcohols, more preferably methanol, ethanol or isopropanol, more preferably in ethanol. In another embodiment, daprodustat is further purified by crystallizing it in an acid solvent, preferably acetic acid.

In an embodiment of the tenth aspect, the process further comprises the preparation of a crystal form of daprodustat as defined above by the process as defined in the second aspect of the invention.

In the following, the present invention is further illustrated by examples. They should in no case be interpreted as a limitation of the scope of the invention as defined in the claims. Unless indicated otherwise, all indications of percentage are by weight and temperatures are in degrees Celsius.

EXAMPLES

GENERAL METHODS

X-Ray Powder Diffraction (XRPD)

Sample preparation: Approximately 20 mg of non-manipulated sample were prepared in standard sample holders using two foils of polyacetate.

Data acquisition: Powder diffraction pattern was acquired on a Bruker D8 Advance Series 2Theta/Theta powder diffraction system using CuKo 1 -radiation in transmission geometry. The system is equipped with a VANTEC-1 single photon counting PSD, a Germanium monochromator, a ninety positions auto changer sample stage, fixed divergence slits and a radial seller. Programs used: Data collection with DIFFRAC plus XRD Commander V.2.5.1 , and evaluation with HighScore Plus V.4.9.

Measurement conditions: The samples were measured at room temperature in a range from 4° to 40° in 20 in a 0.5 hours measurement using an angular step of 0.049° and a time per step of 2787 s.

The skilled person can appreciate that powder X-ray diffraction pattern may be obtained with a measurement error that is dependent upon the measurement conditions employed. It is generally known that intensities in an X-ray diffraction pattern may fluctuate depending upon measurement conditions employed and the relative intensity values can e.g., vary by ±30%. It should be further understood that relative intensities may also vary depending upon experimental conditions and, accordingly, the exact order of intensity should not be taken into account. Additionally, a measurement error of diffraction angle for a conventional X-ray diffraction pattern is typically about ± 0.2 degrees 2Theta, and such degree of measurement error should be taken into account as pertaining to the aforementioned diffraction angles. Consequently, it is to be understood that the crystal forms of the present invention are not limited to the crystalline forms that provide X-ray diffraction patterns completely identical to the X-ray diffraction patterns depicted in the accompanying figures. Differential Scanning Calorimetry (DSC)

Sample preparation: Approximately 1-4 mg of sample were weighed (using a MX5 Mettler Toledo microbalance) into 40 pL aluminium crucibles with a pinhole lid.

Data acquisition: DSC analyses were recorded in a Mettler Toledo DSC822e calorimeter. Programs used: Data collection and evaluation with software STARe.

Measurement conditions: The samples were heated under dry nitrogen (flow rate: 50 mL/min) at 10 °C/min from 30 to 300 °C.

Thermal Gravimetric Analysis (TGA)

Sample preparation: Approximately 1-4 mg of sample were weighed (using a MX5 Mettler Toledo microbalance) into 40 L aluminium crucibles with a pinhole lid.

Data acquisition: Thermogravimetric analyses were recorded in a Mettler Toledo TGA/DSC 3+ with a balance XP1 type. Programs used: Data collection and evaluation with software STARe.

Measurement conditions: The samples were heated under dry nitrogen (flow rate: 10 mL/min) at 10 °C/min from 30 to 300 °C.

Proton nuclear magnetic resonance ( ¹ H-NMR)

Sample preparation: Approximately 2-5 mg of sample were dissolved in 0.7 mL of deuterated solvent (dimethylsulfoxide-d6).

Data acquisition: Proton nuclear magnetic resonance analyses were recorded in a Bruker 300 NMR spectrometer, equipped with a z gradient 5 mm BBC (Broadband Observe) probe with ATM and an automatic autosampler.

Measurement conditions: The samples were analyzed at room temperature.

Fourier Transformed Infrared spectroscopy analysis (FTIR)

The FTIR spectra were recorded using an Agilent Technologies Cary 630 FTIR spectrometer, equipped with an Agilent Diamond single reflection ATR system, a mid-infrared source as the excitation source and a DTGS detector. The spectra were acquired in 32 scans at a resolution of 4 cm ¹ in the range of 4000- 650 cm’ ¹ .

Dynamic Vapor Sorption Analysis (DVS)

Sample preparation: Approximately 5-10 mg of sample were weighed (using a MX5 Mettler Toledo microbalance) into 150 pL platinum crucibles without lid.

Data acquisition: The experiments were performed in a Mettler Toledo TGA I DSC 1 LF instrument equipped with a LF SDTA FRS2 sensor and coupled with a Modular Humidity Generator MHG 32. Data collection and evaluation was done with STARe software. Measurement conditions: The samples were analysed following a humidity cycle from 5%RH up to 90%RH and down to 5%RH in 10% steps of 60 minutes at 25°C (RH: Relative Humidity).

Optical Microscopy

Sample preparation: Crystals of the sample were immersed in perfluorinated oil.

Equipment description: Zeiss Stemi SV 11 stereomicroscope has a variable amplification in the range between 15x and 154x and is equipped with cross coupled polarization filters (Carl Zeiss Pol 45517 and 455174) and a I/2 filter (Carl Zeiss Lambda 455172). For sample illumination, a transmission cold light source Zeiss KL2500 LCD is used. The size of the crystals can be measured using a standard microscale (5+100/100 mm, Carl Zeiss 474026).

Environmental Scanning Electron Microscopy (ESEM)

Sample preparation: The samples were mounted on an aluminium stub using a double adhesive carbon conductive tab.

Equipment: Environmental Scanning Electron Microscopy from FEI company, model Quanta 600 with attached EDX (Energy Dispersive X-ray Spectroscopy) from Oxford Instruments. For the samples morphology images secondary electrons were collected. The working conditions are: Working Distance (WD) = 9.9-10.3 mm; Accelerating Voltage (HV) = 20 kV; Magnification (Mag) = variable (1000x to 4000x).

Atomic absorption spectrometry (AAS)

Equipment: ICP-OES, Optima 8000 Perkin Elmer

Measurement conditions: wavelength for sodium: 589,592 nm; wavelength for potassium: 766,490 nm Sample preparation: 25 mg of a sample, 3 mL of concentrated HNO3 and 1 mL of H2O2 were digested in a digester ultraWAVE Milestone. The temperature was heated at 100°C in 5 min, at 170°C in 10 min, and finally at 240°C in 10 min. The sample was kept at this final temperature for 15 min. Then, the sample was diluted in 25 mL of an aqueous solution of 2% of HNO3 followed by a second dilution of 1 mL in 20 mL of an aqueous solution of 2% of HNO3.

Example 1 : Synthesis of daprodustat solvates

Various daprodustat solvates (Form A, Form B, Form C, Form D, Form E, Form F, Form G and Form H) were prepared as shown in Table 5. All solids obtained herein were crystalline solvates of daprodustat as characterized by XRPD and DSC/TGA analysis.

Table 5

V: Volume

General methods:

Evaporation: A sample of daprodustat obtained by reproducing the Method 2 of W02007/150011 A2 (50 mg) was dissolved in the minimum quantity of solvent and evaporated at ambient conditions until a solid was obtained.

Cooling crystallization: A sample of daprodustat obtained by reproducing the Method 2 of WC2007/150011 A2 (50 mg) was dissolved in the minimum quantity of solvent at 75°C in a solvent and the obtained solution was slowly cooled down to room temperature or 4°C to induce crystallization.

Slurry: A sample of daprodustat obtained by reproducing the Method 2 of WC2007/150011 A2 (50 mg) was suspended in a solvent at 75°C, and the obtained suspension was stirred at this temperature for 2 hours and cooled down to room temperature.

Example 2: Synthesis of cocrystal of daprodustat sodium salt and daprodustat free acid

A sample of daprodustat obtained by reproducing the Method 2 of W02007/150011 A2 (1 g, 2.54 mmol) was suspended in a mixture of acetonitrile and water (9:1 respectively, 55 mL) at 95°C. To the resulting solution, an aqueous solution of sodium hydroxide (1 M, 0.5 equiv., 1.28 mL) was added dropwise, and the obtained suspension was left to cool down to room temperature. The resulting solid was filtered off, dried under vacuum (2 mbar, 40°C, 3 hours).

XRPD (FIG. 1): Crystalline. DSC (FIG. 2): Endothermic peaks with onset temperatures at 115°C (-63 J/g), 190°C (-11 J/g), 202°C (-2 J/g) and 228°C (-39 J/g).

TGA (FIG. 2): Weight loss of 4.4% between 33 and 150°C (probable loss of water; 2 theoretical equivalents: 4.3%). Decomposition starting at about 230°C.

¹ H-NMR (300 MHz, DMSO, FIG. 3): 5 (ppm): 10.13 (t, 1 H, J=6 Hz), 4.66 (m, 2H), 3.96 (d, 2H, J=6 Hz), 2.31 (m, 4H), 1.77 (m, 4H), 1.57 (m, 6H), 1.20 (m, 6H). No residual solvents detected.

FTIR: v (cm ¹ , FIG. 4): 691 , 761 , 789, 840, 895, 924, 966, 1000, 1024, 1057, 1132, 1178, 1237, 1266, 1306, 1338, 1379, 1420, 1444, 1479, 1522, 1586, 1670, 1716, 2849, 2914, 2930, 2973, 3233, 3522, 3649.

DVS: Weight increase of about 0.4% between 10% and 80% Relative Humidity (slightly hygroscopic). No changes observed in the XRPD before and after the analysis.

AAS: 2.9 % Na (which corresponds to a 1 : 1 molar ratio of daprodustat free acid and daprodustat sodium salt)

Example 3: Synthesis of cocrystal of daprodustat potassium salt and daprodustat free acid

A sample of daprodustat obtained by reproducing the Method 2 of W02007/150011 A2 (1 g, 2.54 mmol) was suspended in a mixture of acetonitrile and water (9:1 respectively, 55 mL) at 95°C. To the resulting solution, an aqueous solution of potassium hydroxide (1 M, 0.5 equiv., 1.28 mL) was added dropwise, and the obtained suspension was left to cool down to room temperature. The resulting solid was filtered off, dried under vacuum (2 mbar, 40°C, 3 hours).

XRPD (FIG. 7): Crystalline.

DSC (FIG. 8): Endothermic peaks with onset temperatures at 33°C (-87 J/g) and 248°C (-46 J/g).

TGA (FIG. 8): Weight loss of 5.4% between 27 and 125°C (probable loss of water; 2.5 theoretical equivalents: 5.2%). Decomposition starting at about 250°C.

¹ H-NMR (300 MHz, DMSO, FIG. 9): 5 (ppm): 10.08 (t, 1 H, J=6 Hz), 4.65 (m, 2H), 3.93 (d, 2H, J=6 Hz), 2.30 (m, 4H), 1.77 (m, 4H), 1.57 (m, 6H), 1.20 (m, 6H). No residual solvents detected.

FTIR: v (cm ¹ , FIG. 10): 693, 740, 762, 790, 882, 894, 918, 996, 1054, 1134, 1187, 1235, 1258, 1302, 1340, 1378, 1446, 1472, 1506, 1522, 1589, 1669, 2655, 2852, 2935, 2977, 3180, 3477, 3546.

DVS: Weight increase of about 3.9% between 10% and 80% Relative Humidity (hygroscopic). No changes observed in the XRPD before and after the analysis.

AAS: 4.6 % K (which corresponds to a 1 :1 molar ratio of daprodustat free acid and daprodustat potassium salt)

Example 4: Temperature stability study

Samples of crystals N2 and K2 were stored in closed vials at different temperatures (T), for instance at 45°C and at 60°C. After some time, solids were separated and analysed by XRPD analysis. Results are gathered in Table 6. Table 6

Example 5: Solubility study

Solubility test method according to the Chinese Pharmacopoeia was used and different pHs of different organs in human body were considered: FeSSIF (Fed state simulated intestinal fluids, pH=5.0), FaSSIF (Fasted state simulated intestinal fluids, pH=6.5) and pure water. Prior art form CS1, crystal form N2 and crystal form K2 of daprodustat were suspended in the different mediums at 25°C to obtain saturated solutions. The solutions were sampled at fixed time points (1 h, 4 hours and 24 hours). Concentrations values in mg/mL were measured by HPLC. The results are listed in Table 7.

Table 7

High Performance Liquid Chromatography (HPLC):

Equipment: Agilent Technologies 1200 series

Column: Zorbax C18 (50mm x2.1) 1.8pm

Eluent: ACN/H2O 6:4

Flow rate: 0.5 ml/min

Wavelength: 266nm

Injection volume: 5 pL

As from the results obtained in Table 7, the crystal form N2 of daprodustat showed improved solubility at pH=6.5 and pure water compared to prior art form CS1. Also, the crystal form K2 of daprodustat showed improved solubility at pH=5.0, pH=6.5 and pure water compared to prior art form CS1 .

Example 6: Drying stability study

Samples of crystals N2 and K2 were dried overnight in vacuum (12 mbar) at different temperatures (T) in a vacuum drying oven. The resulting solids were analyzed by XRPD. Results are gathered in Table 8. Table 8

As shown in table 8, the crystal form K2 of daprodustat kept its form unchanged by XRPD analysis. The crystal form N2 of daprodustat transformed to the crystal form N4 characterized by XRPD, DSC/TGA and DVS analysis as shown below.

XRPD (FIG. 5 and Table 2): Crystalline (very similar to crystal form N2).

DSC (FIG. 6): Endothermic peaks with onset temperatures at 107°C (-62 J/g), 190°C (-6 J/g), 200°C (-2 J/g) and 229°C (-38 J/g).

TGA (FIG. 6): Weight loss of 4.0% between 39°C and 141°C (probable loss of water; 2 theoretical equivalents: 4.3%). Decomposition starting at about 220°C.

DVS: Weight increase of about 0.5% between 10% and 80% Relative Humidity (slightly hygroscopic). No changes observed in the XRPD before and after the analysis.

Example 7: Slurry experiments

Samples of crystals N2 and K2 were suspended in water (100 mg in 1 mL) and stirred for 1 hour at room temperature. After that, the resulting solids were separated and analysed by XRPD. Crystal form N2 kept its form unchanged as confirmed by XRPD analysis. Crystal form K2 kept its form unchanged as confirmed by XRPD analysis.

Samples of crystals N2 and K2 were suspended in water (100 mg in 1 mL) and stirred for 30 min and for 60 min at three different temperatures of 40°C, of 60°C and of 80°C. Crystal form N2 kept its form unchanged in all experiments as confirmed by XRPD analysis. Crystal form K2 kept its form unchanged in all experiments as confirmed by XRPD analysis.

Example 8: Synthesis of crystal form N2 of daprodustat from intermediate compound (VI) wherein R is ethyl

Ethyl (1 ,3-dicyclohexyl-2,4,6-trioxohexahydropyrimidine-5-carbonyl)g lycinate (5.0 g, 11.9 mmol, 1.0 eq.) was suspended in ethanol (20 mL). An 1 M aqueous solution of NaOH (23.8 mL, 23.8 mmol, 2.0 equiv.) was added and stirred at 20-25 °C for 2 h. The solvent was distilled off at reduced pressure at 40 °C. Acetonitrile (25 mL) was added, the medium was heated at 40 °C until a solution was obtained and then the solvent was distilled off at reduced pressure at 40 °C. This operation was repeated once. Acetonitrile (248 mL) and water (10 mL) were added. To the obtained solution an 1 M aqueous solution of HCI (11.9 mL, 11.9 mmol, 1.0 equiv.) was added at 20-25 °C. The white solution was seeded with crystal form N2 of daprodustat (as prepared in example 2). Then, an 1 M aqueous solution of HCI (6.0 mL, 6.0 mmol, 0.5 equiv.) was added at 20-25 °C. The white solution was seeded with crystal form N2 of daprodustat (as prepared in example 2) and stirred at 20-25 °C for 20 h. The white solid was filtered at reduced pressure. The obtained white solid was suspended in water (100 mL) at 20-25 °C for 1 hour. The white suspension was filtered and washed with water (2x25 mL) at reduced pressure. The obtained white solid was suspended once again in water (100 mL) at 20-25 °C for 1 hour. The white solid was filtered, washed with water (2x25 mL) at reduced pressure and dried at reduced pressure at 40 °C for 4 h (3.3g, 7.8 mmol, Yield: 66%) XRPD: Crystal form N2 of daprodustat.

Example 9: Synthesis of crystal form K1 of daprodustat from intermediate compound (VI) wherein R is ethyl

Ethyl (1 ,3-dicyclohexyl-2,4,6-trioxohexahydropyrimidine-5-carbonyl)g lycinate (5.0 g, 11.9 mmol, 1.0 equiv.) was suspended in ethanol (20 mL). An 1 M aqueous solution of KOH (23.8 mL, 23.8 mmol, 2.0 equiv.) was added and stirred at 20-25 °C for 2 h. The solvent was distilled off at reduced pressure at 40 °C. Acetonitrile (25 mL) was added, the medium was heated at 40 °C until a solution was obtained and then the solvent is distilled off at reduced pressure at 40 °C. This operation was repeated once. Acetonitrile (248 mL) and water (10 mL) were added. To the obtained solution an 1 M aqueous solution of HCI (11.9 mL, 11.9 mmol, 1.0 equiv.) was added at 20-25 °C. The white solution was seeded with crystal form K2 of daprodustat. Then, an 1 M aqueous solution of HCI (6.0 mL, 6.0 mmol, 0.5 equiv.) was added at 20-25 °C. The white solution was seeded with crystal form K2 of daprodustat (as prepared in example 3) and stirred at 20-25 °C for 20 h. The white solid was filtered at reduced pressure. The obtained white solid was suspended in water (100 mL) at 20-25 °C for 1 h. The white suspension was filtered and washed with water (2x25 mL) at reduced pressure. The obtained white solid was suspended once again in water (100 mL) at 20-25 °C for 1 h. The white solid was filtered, washed with water (2x25 mL) at reduced pressure and dried at reduced pressure at 40 °C for 4 h (3.1 g, 7.0 mmol, Yield: 60%).

XRPD (FIG. 15 and Table 4): Crystalline crystal form K1 of daprodustat.

DSC (FIG. 16): Endothermic peaks with onset temperatures at 56°C (-142 J/g) and 251 °C (-76 J/g). TGA (FIG. 16): Weight loss of 13.2% between 40°C and 130°C. Decomposition starting at about 250°C. KF: 11.57%

Crystal form K1 of daprodustat obtained above was dried under vacuum at room temperature to provide crystal form K2 of daprodustat.

Example 10: Synthesis of 1,3-dicyclohexylpyrimidine-2,4,6(1 H,3H,5H)-trione (Compound V)

1 ,3-dicyclohexylurea (DCU) (175.0 g, 780.0 mmol, 1.0 equiv.) and malonic acid (97.4 g, 936.0 mmol, 1.2 equiv.) were stirred with acetic acid (700 mL) at 20-25 °C. To the resulting white suspension, acetic anhydride (199.1 g, 184.0 mL, 1.950 mol, 2.5 eq.) was added at 20-25 °C. The resulting mixture was heated at 80-85 °C and stirred at this temperature for 5 h. Then, the system was cooled down to 20 25 °C and water (700 mL) was added. The resulting suspension was stirred at 20-25 °C for 16 h. The white solid was separated by filtration at reduced pressure at 20-25 °C and washed with a 1 :1 mixture of acetic acid and water (2 x 85 mL), water (175 mL) and isopropanol (175 mL). The resulting solid was slurried from isopropanol (1300 mL) at 80 °C and stirred 10 min at this temperature. Then, the mixture was cooled to 20- 25 °C and stirred at this temperature for 16 h and then stirred for 1 h at 0-5 °C. The white solid was separated by filtration at reduced pressure at 0-5 °C, washed with ice-cooled isopropanol (2x100 mL) and dried at 40- 45 °C.

Yield: 70% (158.6 g, 542.5 mmol)

Purity by HPLC: 99.54%

Example 11 : Synthesis of ethyl formylglycinate (Compound II)

Ethyl glycinate hydrochloride (100.0 g, 716.4 mmol, 1.0 equiv.) was mixed with trimethyl orthoformate (300 mL, 291.0 g, 3.8 equiv.) at 20-25 °C. The resulting white suspension was heated at reflux temperature and stirred at this temperature for 4 h. Then, the solvent was distilled at reduced pressure at 40-75 °C. The brownish residue obtained was purified by fractioned distillation at reduced pressure to give a clear oil. Yield: 86% (80.8 g, 616.2 mmol) Purity by HPLC: 98.23%

Example 12: Synthesis of ethyl formylglycinate (Compound II)

Ethyl glycinate hydrochloride (25.0 g, 179 mmol, 1.0 equiv.) was mixed with ethyl formate (75.0 mL, 69.1 g, 932 mmol, 5.2 equiv.) and triethylamine (26.2 mL, 19,0 g, 188 mmol, 1 ,05 equiv.) at 20-25 °C. The resulting white suspension was heated at reflux temperature and stirred at this temperature for 24 h. The reaction medium was cooled down to 20-25 °C and the white solid was filtered off and washed with ethyl formate. The solvent of the filtrate was distilled at reduced pressure at 40 °C. The residue obtained was mixed with toluene (100 mL) and the resulting mixture was stirred for 1 h at 20-25 °C. The white solid was filtered off and washed with toluene. Then, the solvent of the filtrate was distilled at reduced pressure at 40 °C. The brownish oil obtained was purified by distillation at reduced pressure to give a clear oil.

Yield: 81 % (19.0 g, 145 mmol).

Purity by HPLC: 99,69%

Example 13: Synthesis of ethyl (1,3-dicyclohexyl-6-hydroxy-2,4-dioxo-1,2,3,4-tetrahydropyri midine- 5-carbonyl)glycinate (Compound VI)

A solution of ethyl formyl glycinate (compound II, 48-0 g, 366.0 mmol, 1.0 equiv.) and triethylamine (92.6 g, 128 mL, 915.1 mmol, 2.5 equiv.) in dichloromethane (288 mL) was cooled to 0-5 °C. A solution of phosphorus oxychloride (61.7 g, 37.5 mL, 403 mmol, 1.1 equiv.) in dichloromethane (96 mL) was slowly added keeping the temperature of the system under 5 °C. The resulting mixture was stirred at 0-5 °C for 1 h. At that point a 10% aqueous solution of potassium carbonate (384 mL) was added at a temperature under 25 °C. Then the mixture was stirred at 20-25 °C for 1 h. The phases were separated and the aqueous phase was extracted with dichloromethane (96 mL). The organic phases were joined and washed with a saturated aqueous solution of sodium bicarbonate (96 mL) and water (96 mL). The solvent of the organic phase was distilled at reduced pressure to yield a reddish oil, corresponding to ethyl 2-isocyanoacetate (compound III). This oil was used in the next step without further purification.

A solution of the reddish oil (compound III) and DMSO (35.7 g, 32.5 mL, 457.5 mmol, 1.25 equiv.) in dichloromethane (240 mL) was cooled between -30 °C and -35 °C and a solution of trifluoroacetic anhydride (7.7 g, 5.2 mL, 36.6 mmol, 0.10 equiv.) in dichloromethane (48 mL) was slowly added. The system was stirred at -35 °C for 30 min. Ethyl 2-isocyanatoacetate (compound IV) was formed in the reaction medium.

At this point, 1 ,3-dicyclohexylpyrimidine-2,4,6(1 H,3H,5H)-trione (compound V) (95.8 g, 327.7 mmol, 0.94 equiv.) and diisopropylethylamine (90.1 g, 121 mL, 697.1 mmol, 2.0 equiv.) were added. The resulting solution was stirred at 20-25 °C for 16 h. Then, a 3N aqueous solution of hydrochloric acid (300 mL) was added and the mixture was stirred at 20-25 °C for 15 min. The phases were separated, and the aqueous phase was extracted with dichloromethane (200 mL). The organic phases were joined and washed with a saturated aqueous solution of sodium bicarbonate (200 mL) and with water (200 mL). The solvent of the organic phase was distilled at reduced pressure and swapped with isopropanol (1056 mL) to yield a yellowish suspension, which was heated at reflux temperature to yield a solution that was cooled to 20-25 °C. The obtained suspension was stirred at this temperature for 1 h. The white solid was separated by filtration at reduced pressure at 20-25 °C and washed with isopropanol (2 x 100 mL). The white solid was recrystallized from IPA (1000 mL), filtered off and dried at 40-45 °C.

Yield: 70% (96.7 g, 229.4 mmol)

Purity by HPLC: 99.66%

Example 14: Synthesis of ethyl (1,3-dicyclohexyl-6-hydroxy-2,4-dioxo-1, 2,3,4- tetrahydropyrimidine-5-carbonyl)glycinate (Compound VI)

A solution of ethyl formylglycinate (compound II, 35.0 g, 267 mmol, 1.0 equiv.) and triethylamine (59.4 g, 81.8 mL, 587 mmol, 2.2 equiv.) in dichloromethane (210 mL) was cooled to 0-5 °C. A solution of phosphorus oxychloride (45.0 g, 27.4 mL, 294 mmol, 1.1 equiv.) in dichloromethane (70 mL) was slowly added keeping the temperature of the reaction medium under 5 °C. The resulting mixture was stirred at 0-5 °C for 2 h. At that point, a 10% aqueous solution of potassium carbonate (350 mL) was added at a temperature under 25 °C. Then the mixture was stirred at 20-25 °C for 1 h. The phases were separated and the aqueous phase was extracted twice with dichloromethane (2x70 mL). The organic phases were joined and washed with water (70 mL) and brine (70 mL). The solvent of the organic phase was distilled at reduced pressure to yield a reddish oil, corresponding to ethyl 2-isocyanoacetate 30 (compound III). This oil was used in the next step without further purification.

A mixture of DMSO (24.0 g, 21.8 mL, 308 mmol, 1.2 equiv.) in dichloromethane (55 mL) was cooled to a temperature between -30 °C and -35 °C. Trifluoroacetic anhydride (5.38 g, 3.62 mL, 25.6 mmol, 0.10 equiv.) was slowly added at this temperature. Then, a solution of the reddish oil previously obtained (compound III) in dichloromethane (50 mL) was slowly added keeping the temperature between -30 °C and -35 °C. The solution was stirred at this temperature for 30 min. Ethyl 2-isocyanatoacetate (compound IV) was formed in the reaction medium.

At this point, a solution of 1 ,3-dicyclohexylpyrimidine-2,4,6(1 H,3H,5H)-trione (compound V) (59.9 g, 205 mmol, 0.8 equiv.) and diisopropylethylamine (36.4 g, 49.1 mL, 282 mmol, 1.1 equiv.) in dichloromethane (120 mL) was added, keeping the temperature below 0 °C. The resulting solution was stirred at 20-25 °C for 4 h. Then, a solution of HCI 37% (50 mL) in water (450 mL) was added and the mixture was stirred at 20- 25 °C for 15 min. The phases were separated, and the aqueous phase was extracted with dichloromethane (70 mL). The organic phases were joined and washed with water (70 mL) and a saturated aqueous solution of sodium bicarbonate (70 mL). The solvent of the organic phase was distilled at reduced pressure and swapped with isopropanol (700 mL) to yield a yellowish suspension, which was heated at reflux temperature to yield a solution that was cooled to 20-25 °C. The obtained suspension was stirred at this temperature for 16 h. The white solid was separated by filtration at 20-25 °C and washed with isopropanol (3x35 mL). The white solid was recrystallized from IPA (6600 mL), filtered and dried at 40-45 °C.

Yield: 74% (63.5 g, 151 mmol)

Purity by HPLC: 99.50%

Example 15: Synthesis of (1,3-dicyclohexyl-6-hydroxy-2,4-dioxo-1,2,3,4-tetrahydropyri midine-5- carbonyl)glycine (Compound I)

To a suspension of ethyl (1 ,3-dicyclohexyl-6-hydroxy-2,4-dioxo-1 ,2,3,4-tetrahydro-pyrimidine-5- carbonyl)glycinate (compound VI as obtained above) (5.0 g, 11.9 mmol, 1.0 equiv.) in ethanol (60 mL) cooled to 0-5 °C was added 25% of an aqueous solution of tetramethylammonium hydroxide (9.0 mL, 26 mmol, 2.2 equiv.) at 0-5 °C. The resulting mixture was heated at 20-25 °C for 1 h. Afterwards, trifluoroacetic acid (3.0 g, 2.0 mL, 26 mmol, 2.2 equiv.) was added and the resulting white suspension was stirred at 20-25 °C for 1 h. The solid was filtered, washed with ethanol (2 x 5 mL) and dried at 40-45 °C.

Yield 90 % (4.2 g, 10.7 mmol)

Purity by HPLC: 99.93%