WO2023006986A1

WO2023006986A1 - Method for preparing and purifying an agent suitable for treating anemia

Info

Publication number: WO2023006986A1
Application number: PCT/EP2022/071427
Authority: WO
Inventors: Thomas Judge; Giuseppina TRUGLIO; Alessandro Restelli
Original assignee: Dipharma Francis S.R.L.
Priority date: 2021-07-30
Filing date: 2022-07-29
Publication date: 2023-02-02

Abstract

The present invention relates to a new process for preparing and purifying N-[(1,3-dicyclohexyl-2,4,6-trioxohexahydropyrimidin-5-yl)carbonyl]glycine, an agent developed for the treatment of anemia, and intermediates thereof.

Description

METHOD FOR PREPARING AND PURIFYING AN AGENT SUITABLE FOR

TREATING ANEMIA

FIELD OF THE INVENTION

The present invention relates to a new process for preparing and purifying N- [(1, 3-di cyclohexyl-2, 4, 6-trioxohexahydropyrimidin-5-yl)carbonyl]glycine, an agent developed for the treatment of anemia, and intermediates thereof. PRIOR ART

/V-[(l,3-Dicyclohexyl-2,4,6-trioxohexahydropyrimidin-5-yl)carbonyl]glycine, also known as GSK1278863, with the following formula (I)

acts as a hypoxia-inducible factor prolyl-hydroxylase inhibitor (HIF-PHI) and thereby prevents degradation of the hypoxia-inducible factor leading to an increase of endogenous production of erythropoietin and stimulating the production of hemoglobin and red blood cells. The compound developed by GlaxoSmithKline (GSK) demonstrated in clinical studies that it was able to improve anemia in hemodialysis patients as well as non-hemodialysis-dependent patients with chronic kidney disease (CKD).

Based on these results, on June 29, 2020, GSK1278863 gained its first global approval in Japan, while phase III clinical studies have been completed in the USA and Europe.

GSK1278863 is known from US 8,324,208, which claims the compound as such and salts thereof.

Example 18 of US 8,324,208 describes two procedures for preparing GSK1278863, but both methods make use of hazardous agents, which use requires high precautions and controls during the manufacturing process.

The need of GSK1278863 at a purity suitable to meet the regulatory requirements has stimulated the search for alternative methods for its preparation, which at the same time have to be efficient, green, robust, safe, cost-effective and amendable to industrial scale manufacturing and purification.

The inventors of the present disclosure have found a new and safe process for the preparation and purification of GSK1278863, which thanks to the high yields, the absence of agents hazardous to the environment and to a minor presence of side products is particularly suitable for an industrial production. This new process, thanks to the particular reaction conditions, provides a highly pure product, which is suitable to meet regulatory requirements required for APIs.

SUMMARY OF THE INVENTION

One embodiment of the invention is directed to a process for preparing a compound of formula (II)

comprising reacting A,A’-dicyclohexylurea of formula (III),

with malonic acid of formula (IV), or a salt thereof, in presence of acetic anhydride.

A further embodiment of the invention is directed to an one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof,

from a compound of formula (II)

A further embodiment of the invention is directed to a process of purification of the compound of formula (I).

DETAILED DESCRIPTION OF THE INVENTION

A first embodiment of the invention is directed to a process for preparing a compound of formula (II),

comprising reacting A,A’-dicyclohexylurea of formula (III),

with malonic acid of formula (IV), or a salt thereof,

0 0

^H<AA>^H

(IV) in presence of acetic anhydride.

According to the present disclosure, by "comprising" herein is meant that additional steps may be taken in the process, which do not substantially change the product produced by the reaction. The term comprising encompasses the terms "consisting of and "consisting essentially of".

A,A’-dicyclohexylurea of formula (III) is a known compound and is commercially available. For instance, it is commercialized by Sigma Aldrich (catalogue number: D80800).

Malonic acid of formula (IV) is a known compound and is commercially available. For instance, it is commercialized by Sigma Aldrich (catalogue number: M1296).

The reaction may be advantageously carried out using about 1.6 to about 0.3 moles of 2 A,A’-dicyclohexylurea of formula (III) per mole of malonic acid of formula (IV).

In one preferred embodiment of the invention, the reaction may be advantageously carried out using about 1.2 to about 0.5 moles of 2 N,N’~ dicyclohexylurea of formula (III) per mole of malonic acid of formula (IV), more preferably from about 1.1 to about 0.6 moles, for instance about 0.7 moles, about 0.8 moles, about 0.9 moles or in equimolar amounts.

According to the present disclosure, a salt of a compound of formula (IV) may be derived from an appropriate base, such as salts of an alkali metal (such as sodium or potassium), an alkaline earth metal (such as calcium or magnesium), ammonium and NR'₄ ⁺, wherein each of R¹, which can be the same or different, is a C1-C6 alkyl.

According to the present disclosure, the term "C1-C6 alkyl" refers to a straight or branched hydrocarbon chain radical, consisting solely of carbon and hydrogen atoms, having from one to six carbon atoms.

In a preferred embodiment of the invention, the "C1-C6 alkyl" group is a linear or branched C1-C4 alkyl group. Examples include methyl, ethyl, n-propyl, /isopropyl, n-butyl, sec-butyl or /er/-butyl.

Acetic anhydride has demonstrated to be a mild activator of malonic acid of formula (IV) and the reaction with /V,A’-dicyclohexylurea of formula (III) proceeds well and with high yields forming a compound of formula (II). In addition, the acetic anhydride allows to avoid hazardous reagents like malonyl dichloride or N,N’~ dicyclohexylcarbodiimide as used in US 8,324,208.

The reaction of malonic acid of formula (IV) with /V,A’-dicyclohexylurea of formula (III) can be carried out in acetic anhydride as sole solvent or in a mixture of acetic anhydride and further solvents.

According to the present disclosure, the term “solvent” herein is meant as a substance capable of dissolving the compounds of the reaction mixture to a sufficient extent to form a homogeneous mixture.

The further solvents in the mixture of acetic anhydride can be selected from a dipolar aprotic solvent, typically dimethylformamide, dimethylacetamide, N- methylpyrrolidone, acetonitrile or dimethylsulfoxide; an acyclic or cyclic ether, for example diethyl ether, methyl tert- butyl ether, or dioxane; a ketone such as methyl ethyl ketone, methyl isobutyl ketone or acetone; an apolar aprotic solvent such as hexane, heptane, toluene or xylene; a C1-C6 alkyl ester of a carboxylic acid, wherein the Ci-C₆ alkyl group is as defined above, for example methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate or butyl acetate; a C1-C6 alkyl carboxylic acid, optionally substituted with one or more halogen atoms and wherein the C1-C6 alkyl group is as defined above, for example acetic acid or propionic; or a mixture of two or more, for example two or three, of the above mentioned solvents.

In one preferred embodiment of the invention, the reaction of malonic acid of formula (IV) with /V,/V’-dicyclohexylurea of formula (III) can be carried out in acetic anhydride as sole solvent. Thus, there is no need to employ solvents as chloroform or tetrahydrofuran (THF) as used in US 8,324,208. Chloroform and tetrahydrofuran are solvents, which according to the ICH Guidelines (ICH Harmonised Tripartite Guideline - Impurities: Guideline for Solvents Q3C(R5)) are part of class 2 solvents, which are toxic solvents that should be avoided.

In a further preferred embodiment of the invention, the reaction can be carried out solely in a mixture of acetic anhydride and acetic acid in absence of any further solvent.

In a further preferred embodiment of the invention, the reaction can be carried out in a mixture of acetic anhydride and acetic acid in presence of one or more further solvents as defined above.

The addition of acetic acid allows avoiding the formation of some impurities and thus obtain the compound of formula (II) at high purity without the need of further purification steps to remove said impurities. Thus, the use of acetic acid allows for instance avoiding an extraction step of the reaction mixture of a compound of formula (III) with a compound formula (IV) with an apolar solvent, such as hexane, heptane or the like.

In addition, the reaction of malonic acid of formula (IV) with N,N’~ dicyclohexylurea of formula (III) in a mixture of acetic anhydride and acetic acid proceeds with very high yields forming a compound of formula (II), wherein the yields are significantly higher than those obtained by the procedures disclosed in US 8,324,208.

Typically, the ratio of the acetic anhydride and acetic acid is between about 1000:1 (volume: volume, v:v) and about 1:100 (v:v)

In a preferred embodiment of the invention, the ratio of the acetic anhydride and acetic acid is between about 100:1 (v:v) and 1:10 (v:v), more preferably between about 10:1 (v:v) and about 3:10 (v:v). Examples of said ratio can be equal or superior to 1:2 (v:v), 1:1 (v:v), 2:1 (v:v), 5: 1 (v:v) or 8:1 (v:v).

In a preferred embodiment of the invention, the ratio of the acetic anhydride and acetic acid is about 1 :1 (v:v).

The reaction may be performed at temperatures from about 0°C to the reflux temperature of the reaction mixture. The reaction may be carried out, for example, at a temperature of about 10°C or above, at a temperature of about 20°C or above, at a temperature of about 30°C or above, or of about 40°C or above, for instance at about room temperature, at about 45°C, at about 50°C, at about 55°C, at about 60°C, at about 65°C, at about 70°C, at about 75°C, at about 80°C, at about 90°C, at about 100°C or at about 110°C.

The reaction time is typically about 0.5 hour to 96 hours, for instance about 1 hour, about 2.5 hours, about 4 hours, about 6 hours, about 9 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, or about 72 hours.

The recovery and the isolation from the obtained mixture of the compound of formula (II), for example as solid, can be achieved by crystallization.

The crystallization can be supported by cooling the reaction mixture, or by concentration by removing the solvent under vacuum, or by seeding with a seed crystal obtained previously, or by performing more than one of the above steps. The crystallization can be further supported by adding a solvent, wherein the compound of formula (II) is poorly soluble.

For instance, the reaction mixture can be cooled down to about 0°C, to about 5°C, to about 10°C, to about 15°C or to room temperature.

The obtained solid can be isolated from the crystallization mixture by filtration or centrifugation according to methods well known to persons skilled in the art. The so obtained compound of formula (II) can be rinsed with a solvent, for instance acetic acid, and can be optionally re-crystallized according to methods well known to persons skilled in the art in order to increase the purity of the product.

At this point, the obtained compound of formula (II) can be further purified by chromatography, for instance by silica gel chromatography.

The compound of formula (II) obtained according to the above procedure can be then further converted into a compound of formula (I), or a salt thereof,

A salt of a compound of formula (I) may be derived from an appropriate base, such as salts of an alkali metal (such as sodium or potassium), an alkaline earth metal (such as calcium or magnesium), ammonium and NR'4⁺, wherein R' is as defined above.

For instance, the compound of formula (I), or a salt thereof,

can be prepared by a process comprising reacting the compound of formula (II) with a compound of formula (V)

wherein R is hydrogen, C1-C6 alkyl, optionally substituted by aryl, or aryl.

According to the present disclosure, "aryl" represents a monocyclic or bicyclic aromatic ring system of, respectively, 6, 9 or 10 carbon atoms, such as benzene, indene and naphthalene. Examples of "aryl" comprise also indan and tetrahydronaphthalene.

In a preferred embodiment of the invention, R is C1-C6 alkyl, wherein C1-C6 alkyl is as defined above, for instance methyl, ethyl, n-propyl, /isopropyl, n-butyl, sec- butyl or /er/-butyl.

In a particularly preferred embodiment of the invention, R is ethyl. In case R is C1-C6 alkyl, optionally substituted by aryl, or aryl, the conversion into the carboxylic acid (R is hydrogen) can be achieved by treatment with a base, wherein the base is typically a hydroxide of an alkali metal or of an alkaline earth metal.

Examples of a hydroxide of an alkali metal or of an alkaline earth metal are sodium hydroxide, potassium hydroxide, magnesium hydroxide or calcium hydroxide.

The conversion of the compound of formula (II) to the compound of formula (I), or a salt thereof, can be carried out in one-pot or a single-vessel.

A further embodiment of the invention is directed to a one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, from a compound of formula (II)

The one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof,

can be carried by a process comprising reacting the compound of formula (II)

with a compound of formula (V) wherein R is as defined above, in presence of a non-nucleophilic organic base or a non-nucleophilic inorganic base and in a solvent selected from an ether, a dipolar aprotic solvent, a polar protic solvent, an ester, a ketone or mixtures thereof, forming a compound of formula (VI)

wherein R is as defined above; and adding first an inorganic base to the reaction mixture comprising the compound of formula (VI), and then a protic acid.

A salt of a compound of formula (I) may be a salt as defined above.

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the non-nucleophilic organic base is typically triethylamine, diAopropylethylamine, N-Ci-Ce alkyl pyrrolidines, A-C1-C6 alkyl morpholine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), pyridine, C1-C6 alkyl pyridines, C1-C6 alkyl piperazines, di-Ci-C₆ alkyl piperazines, wherein "C1-C6 alkyl" is as defined above.

In a preferred embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the non-nucleophilic organic base is triethylamine or diAopropylethylamine

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the non-nucleophilic inorganic base is a base, which has a typical pKa value of about 8 to about 12, for instance of about 9 to about 10.

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the non-nucleophilic inorganic base is typically a carbonate, a hydrogen carbonate, or a /er/-butylate of an alkali metal or of an alkaline earth metal. Examples of non-nucleophilic inorganic bases are lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, magnesium carbonate or calcium carbonate, lithium hydrogen carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, magnesium hydrogen carbonate or calcium hydrogen carbonate, /er/-ButOK, or /er/-ButOLi.

In a preferred embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the non-nucleophilic inorganic base is potassium carbonate (K₂CO₃).

According to one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the non-nucleophilic organic base or the non-nucleophilic inorganic base can be added in about equimolar amounts or in defect with respect to the amount of the compound of formula (II), for instance in a range from about 0.01 moles to about 1.2 moles of the base with respect to one mole of the compound of formula (II), or from about 0.05 moles to about 1.10 moles of the base with respect to one mole of the compound of formula (II), or from about 0.075 moles to about 0.20 moles of the base with respect to one mole of the compound of formula (II). For instance, the non-nucleophilic organic base or the non- nucleophilic inorganic base can be added in catalytic quantities, such as at about 0.01, 0.05, 0.075, 0.10, 0.15 or 0.20 moles of the base with respect to one mole of the compound of formula (II).

According to one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the solvent is selected from the group consisting of an ether, a dipolar aprotic solvent, a polar protic solvent, an ester, a ketone or mixtures thereof.

According to one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the ether, which can be an acyclic or cyclic ethereal solvent, is typically a solvent selected for example from anisole, tetrahydrofuran, dioxane or methyl /er/-butyl ether.

According to one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the ether is selected from the group consisting of anisole, tetrahydrofuran, dioxane or methyl /er/-butyl ether.

According to one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the dipolar aprotic solvent is typically a solvent selected for example from dimethylformamide, dimethylacetamide, acetonitrile or dimethylsulfoxide.

According to one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the dipolar aprotic solvent is selected from the group consisting of dimethylformamide, dimethylacetamide, acetonitrile or dimethylsulfoxide.

According to one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the polar protic solvent is typically a solvent selected for example from a linear or branched C₁-C₆ alcohol. According to one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the polar protic solvent is selected from the group consisting of a linear or branched C₁-C₆ alcohol.

Examples of a linear or branched C₁-C₆ alcohol are methanol, ethanol, 1- propanol, 2-propanol, 1 -butanol, 2-butanol or /er/-butanol.

According to one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the ester is typically a solvent selected for example from ethyl acetate, isopropyl acetate or butyl acetate. According to one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the ester is selected from the group consisting of ethyl acetate, isopropyl acetate or butyl acetate.

According to one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the ketone is typically a linear or branched C₃-C₇ ketone, for example acetone, methyl ethyl ketone, or methyl isobutyl ketone.

According to one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the ketone is selected from the group consisting of a linear or branched C₃-C₇ ketone, for example acetone, methyl ethyl ketone, or methyl isobutyl ketone.

Alternatively, the reaction can be carried out in mixtures of two or more, typically two or three, of the above solvents.

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction is carried out in anisole, acetone, acetonitrile, ethanol or mixtures thereof.

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction is carried out in a solvent selected from the group consisting of anisole, acetone, acetonitrile, ethanol or mixtures thereof.

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction is carried out in anisole as sole solvent or in a mixture of anisole and a further solvent or further solvents as defined above. For instance, the reaction can be carried out in anisole and then a linear or branched C₁-C₆ alcohol, such as methanol, ethanol or isopropanol, typically ethanol, is added prior to the addition of the inorganic base.

In a preferred embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction is carried out in anisole, acetone, acetonitrile, ethanol or mixtures thereof.

In a particular preferred embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction is carried out in anisole as sole solvent or in a mixture of anisole and further solvents. For instance, the reaction can be carried out in anisole and then a linear or branched C1-C6 alcohol, such as methanol, ethanol or isopropanol, typically ethanol, is added prior to the addition of the inorganic base.

The two procedures for preparing GSK1278863, or the compound of formula

(I), of Example 18 of US 8,324,208, comprise the reaction of the compound of formula

(II) with ethyl isocyanatoacetate of formula (V) in dichloromethane forming a compound of formula (VI) as defined above and wherein R is ethyl, which is then treated with a 1 M or a 6 M solution of hydrochloric acid (HC1) and isolated after distilling off dichloromethane.

It has been surprisingly found that the choice of certain solvents allow to carry out the one-pot or single-vessel process without any need of a distillation step nor the isolation of the compound of formula (VI). Said simplification and high overall yield makes this procedure particular suitable for a manufacturing process at an industrial scale.

According to the present disclosure, the term “isolation” includes, but is not limited to, an action directed to producing one or more compounds by collecting while and/or after completing a step of the process of the present invention. The term “collection” refers to any action known in the art for this purpose, including, but not limited to, decanting the mother liquor from a solid precipitate to obtain one or more compounds, filtrating the solid precipitate from the reaction mixture to obtain one or more compounds, and also evaporating the liquid medium of the solution or other mixture to obtain a solid, an oil, or any other residue, which includes one or more compounds.

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction is performed without a distillation step of the solvent or solvents.

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction is performed without isolating the compound of formula (VI).

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction is carried out in a solvent or mixture of solvents, wherein the solvent or mixture of solvents does not comprise a chlorinated solvent, for instance wherein the solvent or mixture of solvents does not comprise dichloromethane.

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction is performed without a distillation step of the solvent or solvents and the reaction is carried out in a solvent or mixture of solvents, wherein the solvent or mixture of solvents does not comprise a chlorinated solvent, for instance wherein the solvent or mixture of solvents does not comprise dichloromethane.

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction is performed without a distillation step of the solvent or solvents, without isolating the compound of formula (VI) and wherein the solvent or mixture of solvents does not comprise a chlorinated solvent, for instance wherein the solvent or mixture of solvents does not comprise dichloromethane.

The reaction may be advantageously carried out using about 2.0 to about 0.7 moles of the compound of formula (V) per mole of the compound of formula (II).

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction may be advantageously carried out using about 1.6 to about 0.8 moles of the compound of formula (V) per mole of compound of formula (II), for instance from about 1.5 to about 1.0 moles, from about 1.4 to about 1.1 moles, for instance at 1.1 moles, 1.2 moles, 1.3 moles or 1.4 moles.

In a preferred embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction may be advantageously carried out using about 1.6 to about 0.8 moles of the compound of formula (V) per mole of compound of formula (II), more preferably from about 1.4 to about 1.1 moles, for instance at 1.1 moles, 1.2 moles, 1.3 moles or 1.4 moles.

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction may be carried out at a temperature between about 0°C and the reflux temperature of the reaction mixture, preferably at a temperature between about 10°C and about 150°C. Typically, the reaction can be carried out between about 20°C to about 150°C, between about 30°C to about 150°C, between about 40°C to about 150°C, between about 50°C to about 150°C, between about 60°C to about 150°C, between about 70°C to about 150°C, between about 80°C to about 150°C, between about 90°C to about 150°C, between about 100° C to about 150°C, between about 110°C to about 150°C, between about 120° to about 150°C, between about 130°C to about 150°C, between about 140°C to about 150°C, between about 20°C to about 130°C, between about 30°C to about 130°C, between about 40°C to about 130°C, between about 50°C to about 130°C, between about 60°C to about 130°C, between about 70°C to about 130°C, between about 80°C to about 130°C, between about 90°C to about 130°C, between about 100° C to about 130°C, between about 110°C to about 130°C, between about 120° to about 130°C, between about 20°C to about 120°C, between about 30°C to about 120°C, between about 40°C to about 120°C, between about 50°C to about 120°C, between about 60°C to about 120°C, between about 70°C to about 120°C, between about 80°C to about 120°C, between about 90°C to about 120°C, between about 100° C to about 120°C, between about 110°C to about 120°C, between about 20°C to about 110°C, between about 30°C to about 110°C, between about 40°C to about 110°C, between about 50°C to about 110°C, between about 60°C to about 110°C, between about 70°C to about 110°C, between about 80°C to about 110°C, between about 90°C to about 110°C, between about 100° C to about 110°C, between about 20°C to about 100°C, between about 30°C to about 100°C, between about 40°C to about 100°C, between about 50°C to about 100°C, between about 60°C to about 100°C, between about 70°C to about 100°C, between about 80°C to about 100°C, between about 90°C to about 100°C, or for instance at about 160°C, at 150°C, at about 140°C, at about 130°C, at about 120°C, at about 110°C, at about 100°C, at about 90°C, at about 80°C, at about 70°C, at about 60°C, at about 50°C, at about 40°C, at about 30°C or at about 20°C.

In a preferred embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction of the compound of formula (II) with the compound of formula (V) may be carried out at temperatures equal to or below about 140°C, for instance at about 130°C, at about 120°C, at about 110°C, at about 100°C, at about 90°C, at about 80°C, at about 70°C, at about 60°C, at about 50°C, at about 40°C or at about room temperature.

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the reaction of a compound of formula (II) with a compound of formula (V) may be advantageously carried out within about 10 minutes to about 240 hours, for example, within about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 144 hours, about 240 hours, and for instance in a range of between about 10 minutes and about 240 hours, in a range of between about 10 minutes and about 144 hours, in a range of between about 10 minutes and about 96 hours, in a range of between about 10 minutes and about 72 hours, in a range of between about 10 minutes and about 48 hours, in a range of between about 10 minutes and about 36 hours, in a range of between about 10 minutes and about 24 hours, in a range of between about 10 minutes and about 12 hours, in a range of between about 10 minutes and about 6 hours, in a range of between about 10 minutes and about 4 hours, in a range of between about 10 minutes and about 3 hours, in a range of between about 10 minutes and about 2 hours, in a range of between about 1 hour and about 240 hours, in a range of between about 1 hour and about 144 hours, in a range of between about 1 hour and about 96 hours, in a range of between about 1 hour and about 72 hours, in a range of between about 1 hour and about 48 hours, in a range of between about 1 hour and about 36 hours, in a range of between about 1 hour and about 24 hours, in a range of between about 1 hour and about 12 hours, in a range of between about 1 hour and about 6 hours, in a range of between about 1 hour and about 4 hours, in a range of between about 1 hour and about 3 hours, in a range of between about 1 hour and about 2 hours, in a range of between about 2 hours and about 240 hours, in a range of between about 2 hours and about 144 hours, in a range of between about 2 hours and about 96 hours, in a range of between about 2 hours and about 72 hours, in a range of between about 2 hours and about 48 hours, in a range of between about 2 hours and about 36 hours, in a range of between about 2 hours and about 24 hours, in a range of between about 2 hours and about 12 hours, in a range of between about 2 hours and about 6 hours, in a range of about between 2 hours and about 4 hours, in a range of between about

2 hours and about 3 hours, in a range of between about 3 hours and about 240 hours, in a range of between about 3 hours and about 144 hours, in a range of between about 3 hours and about 96 hours, in a range of between about 3 hours and about 72 hours, in a range of between about 3 hours and about 48 hours, in a range of between about 3 hours and about 36 hours, in a range of between about 3 hours and about 24 hours, in a range of between about 3 hours and about 12 hours, in a range of between about 3 hours and about 6 hours, in a range of about between 3 hours and about 4 hours, in a range of between about 4 hours and about 240 hours, in a range of between about 4 hours and about 144 hours, in a range of between about 4 hours and about 96 hours, in a range of between about 4 hours and about 72 hours, in a range of between about 4 hours and about 48 hours, in a range of between about 4 hours and about 36 hours, in a range of between about 4 hours and about 24 hours, in a range of between about 4 hours and about 12 hours, in a range of between about 4 hours and about 6 hours, in a range of between about 6 hours and about 240 hours, in a range of between about 6 hours and about 144 hours, in a range of between about 6 hours and about 96 hours, in a range of between about 6 hours and about 72 hours, in a range of between about 6 hours and about 48 hours, in a range of between about 6 hours and about 36 hours, in a range of between about 6 hours and about 24 hours, in a range of between about 6 hours and about 12 hours, in a range of between about 12 hours and about 240 hours, in a range of between about 12 hours and about 144 hours, in a range of between about 12 hours and about 96 hours, in a range of between about 12 hours and about 72 hours, in a range of between about 12 hours and about 48 hours, in a range of between about 12 hours and about 36 hours, in a range of between about 12 hours and about 24 hours, in a range of between about 24 hours and about 240 hours, in a range of between about 24 hours and about 144 hours, in a range of between about 24 hours and about 96 hours, in a range of between about 24 hours and about 72 hours, in a range of between about 24 hours and about 48 hours, in a range of between about 24 hours and about 36 hours, in a range of between about 36 hours and about 240 hours, in a range of between about 36 hours and about 144 hours, in a range of between about 36 hours and about 96 hours, in a range of between about 36 hours and about 72 hours, in a range of between about 36 hours and about 48 hours, in a range of between about 48 hours and about 240 hours, in a range of between about 48 hours and about 144 hours, in a range of between about 48 hours and about 96 hours, in a range of between about 48 hours and about 72 hours, in a range of between about 72 hours and about 240 hours, in a range of between about 72 hours and about 144 hours, in a range of between about 72 hours and about 96 hours, in a range of between about 96 hours and about 240 hours, in a range of between about 96 hours and about 144 hours, in a range of between 144 hours to 240 hours.

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the inorganic base added to the reaction mixture comprising the compound of formula (VI) is typically a hydroxide of an alkali metal or of an alkaline earth metal.

The inorganic base added to the reaction mixture comprising the compound of formula (VI) is for example sodium hydroxide, potassium hydroxide, magnesium hydroxide or calcium hydroxide, preferably sodium hydroxide or potassium hydroxide, more preferably sodium hydroxide.

In a preferred embodiment of the invention, the inorganic base added to the reaction mixture comprising the compound of formula (VI) is sodium hydroxide.

The reaction of the compound of formula (VI) with the inorganic base can be advantageously carried out within about 10 minutes to about 240 hours, for example, within about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours or about 48 hours or in the same ranges as for the reaction of the compound of formula (II) with the compound of formula (V) disclosed above.

The reaction of the compound of formula (VI) with the inorganic base can be carried out at a temperature between about 0°C and the reflux temperature of the reaction mixture, preferably at a temperature between about 10°C and about 150°C, for instance between about 10°C and about 120°C, between about 10°C and about 100°C, between about 20°C and about 80°C, or in the same ranges as for the reaction of the compound of formula (II) with the compound of formula (V) disclosed above.

In a preferred embodiment of the invention, the reaction of the compound of formula (VI) with the inorganic base can be carried out at temperatures equal to or below about 140°C, e.g. at about 130°C, at about 120°C, at about 110°C, at about 100°C, at about 90°C, at about 80°C, at about 70°C, at about 60°C, at about 50°C, at about 40°C or at about room temperature.

In one embodiment of the one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof, the protic acid can be a mineral acid or an organic acid.

A mineral acid can be, for example, selected from the group comprising sulfuric acid, phosphoric acid and a hydrohalic acid, for example hydrochloric acid.

An organic acid can be, for example, selected from the group comprising a sulfonic acid, typically camphorsulfonic acid, para-toluene sulfonic acid, methanesulfonic acid or trifluoromethanesulfonic acid; an aryl -carboxylic acid, typically benzoic acid; and a C1-C6 alkyl -carboxylic acid, wherein the C1-C6 alkyl group may be linear or branched, optionally substituted by one or more halogen atoms, such as one to three chlorine or fluorine atoms, such as acetic acid or trifluoroacetic acid.

In a preferred embodiment of the invention, the protic acid is hydrochloric acid or acetic acid.

The treatment with the protic acid can be advantageously carried out within about 10 minutes to about 96 hours, for example, within about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours or about 48 hours, at a temperature between about 0°C and the reflux temperature of the reaction mixture, preferably at a temperature between about 10°C and about 150°C, for instance between about 10°C and about 120°C, between about 10°C and about 100°C, between about 20°C and about 80°C or at temperatures equal to or below about 140°C, e.g. at about 130°C, at about 120°C, at about 110°C, at about 100°C, at about 90°C, at about 80°C, at about 70°C, at about 60°C, at about 50°C, at about 40°C or at about room temperature, or in the same ranges as for the reaction of the compound of formula (II) with the compound of formula (V) disclosed above.

The herein disclosed process allows obtaining the compound of formula (I) at high yields, which generally exceed 90% of the purified compound of formula (I) from the compound of formula (II). The yields of the herein disclosed process are higher than the yields obtained in US 8,324,208. In specific, US 8,324,208 reports a yield of 66% in Method 18.1b) and about 72% in Method 2b+c) (a yield of 92% for obtaining the ethyl ester of formula (VI) from the compound of formula (II) and a yield of 78% for obtaining /V-[(l,3-dicyclohexyl-2,4,6-trioxohexahydropyrimidin-5- yl)carbonyl]glycine of formula (I) from the ethyl ester of formula (VI) is reported).

It has been surprisingly found that the reaction mixture comprising the compounds of formula (II) and of formula (V) is particularly soluble in warm anisole, for instance even at room temperature or above. This excellent solubility allows reducing the solvent volumes, which is particularly advantageous for a large scale synthesis, where volumes of the solvents could become an issue. At the same time, the use of a lower amount of the solvent results to be more sustainable.

The compound of formula (II) can be for instance prepared according to the procedures disclosed above.

For instance, the compound of formula (II) can be prepared from N,N’~ dicyclohexylurea of formula (III),

For instance, the compound of formula (II) can be prepared from N,N’- dicyclohexylurea of formula (III), and malonic acid of formula (IV), or a salt thereof,

in presence of acetic anhydride.

The reaction A,A’-dicyclohexylurea of formula (III) and malonic acid of formula (IV), or a salt thereof, can be carried out as described above.

The compound of formula (V) is a known compound, it can be prepared by known methods and/or is commercially available. For instance, the compound of formula (V), wherein R is ethyl or butyl, is commercialized by Sigma Aldrich (catalogue number 238627 for the ethyl ester and 482889 for the butyl ester).

The reaction mixture comprising the compound of formula (I) may be purified by known methods. For example, the compound of formula (I) can be isolated from the reaction mixture by filtration. At this point, the isolated compound of formula (I) can be dried, optionally under reduced pressure.

The isolated compound of formula (I) can be further purified by chromatography, for instance by silica gel chromatography.

The isolated compound of formula (I) may be recrystallized to further increase the degree of purity, for instance according to the methods described in US 8,324,208.

If the case, the isolated compound of formula (I) can be purified by chromatography and by recrystallization.

However, it has been observed that the content of the compound of formula (VI), wherein R is C1-C6 alkyl, optionally substituted by aryl, after hydrolysis of the compound of formula (VI) with a base, as described above, is about 2% or about 1% (measured by HPLC at 254 nm) or less. The inventors have performed conventional crystallization purification procedures, for example in glacial acetic acid as described in US 8,324,208, or in isopropanol, but these purification methods have not allowed to reduce the content of the formula (VI), wherein R is C1-C6 alkyl, optionally substituted by aryl, in such a way to meet the regulatory requirements required for APIs.

It has been surprisingly found that the content of the impurity of formula (VI) can be significantly reduced by treating the reaction mixture comprising the compound of formula (I) and the impurity of formula (VI) with an inorganic base in a linear or branched Ci-C₆ alcohol or a mixture of water and a linear or branched C1-C6 alcohol.

Therefore, an additional embodiment of the invention is directed to a process for the purification of a compound of formula (I), or a salt thereof,

comprising treating a reaction mixture comprising the compound of formula (I), or a salt thereof,

with a content equal to or greater than 98% or equal to or greater than 99%, for instance between about 98.0 to 99.5%, as measured by HPLC at 254 nm, and the compound of formula (VI)

wherein R is C1-C6 alkyl, optionally substituted by an aryl, with a content equal to or less than 2% or equal to or less than 1%, for instance between about 2.0 to about 0.3% or between about 1.0 to about 0.5%, as measured by HPLC at 254 nm, with an inorganic base in a linear or branched C1-C6 alcohol or in a mixture of water and a linear or branched C1-C6 alcohol, then adding a protic acid to the mixture; and finally isolating the compound of formula (I), or a salt thereof, with a purity greater than about 99.8%, for instance between about 99.8% and 100.0% as measured by HPLC at 254 nm, and wherein the content of the compound of formula (VI) is less than about 0.2%, preferably less than about 0.1%, or less than about 0.07%, for instance present in a percentage between 0.1% and 0.00001% (100 ppb), or in a percentage between 0.05% and 0.0001% (1 ppm).

In a preferred embodiment, R is ethyl.

The inorganic base used in the process for the purification of a compound of formula (I), or a salt thereof, is typically a hydroxide of an alkali metal or of an alkaline earth metal.

The inorganic base is for example sodium hydroxide, potassium hydroxide, magnesium hydroxide or calcium hydroxide, preferably sodium hydroxide or potassium hydroxide, more preferably sodium hydroxide.

Examples of a linear or branched C1-C6 alcohol are methanol, ethanol, 1 -propanol, 2-propanol, 1 -butanol, 2-butanol or /er/-butanol, preferably methanol, ethanol, 1 -propanol, 2-propanol, more preferably ethanol.

The purification of the compound of formula (I) from the impurity of the formula (VI) can be carried out at a temperature between about 0°C and the reflux temperature of the reaction mixture.

In a preferred embodiment, the purification of the compound of formula (I) from the impurity of the formula (VI) can be carried out at temperatures between about 0°C and about 60°C, between about 10°C and about 60°C, between about 20°C and about 60°C, between about 30°C and about 60°C, between about 40°C and about 60°C, between about 50°C and about 60°C, between about 0°C and about 50°C, between about 10°C and about 50°C, between about 20°C and about 50°C, between about 30°C and about 50°C, between about 40°C and about 50°C, between about 0°C and about 40°C, between about 10°C and about 40°C, between about 20°C and about 40°C, between about 30°C and about 40°C, between about 0°C and about 30°C, between about 10°C and about 30°C, between about 20°C and about 30°C, between about 0°C and about 20°C, between about 10°C and about 20°C, or between about 0°C and about 10°C, for example at 10°C, 20°C, 30°C, 40°C or at

50°C. Then, the compound of formula (I) can be isolated by treating the reaction mixture with a protic acid, which leads to the precipitation of the pure product of formula (I).

A protic acid used in the process for the purification of a compound of formula (I), or a salt thereof, is for example a mineral acid or an organic acid as defined above, preferably a mineral acid. For example, the mineral acid may be hydrochloric acid.

The treatment with the protic acid can be advantageously carried out within about 10 minutes to about 96 hours, for example, within about 1 hour, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, or within about 48 hours, at a temperature between about 0°C and the reflux temperature of the reaction mixture, preferably at a temperature between about 10°C and about 80°C, for example at about 70°C, about 60°C, about 50°C, about 40°C or at about room temperature.

It has been surprisingly found that the compound of formula (I) prepared according to the process of the present invention has a chemical purity, evaluated by HPLC at 254 nm, equal to or greater than 99.8% (Area %), preferably equal to or greater than 99.9%, for instance between about 99.8% and 100.0% as measured by HPLC at 254 nm, and wherein the content of each impurity, for instance of the compound of formula (VI) measured by HPLC at 254 nm is less than about 0.2%, preferably less than about 0.1%, or less than about 0.07%, equal to or less than 0.05%, equal to or less than 0.03%, equal to or less than 0.01%, equal to or less than 0.005%, equal to or less than 0.001%, or equal to or less than 0.0005%, for instance present in a percentage between 0.1% and 0.00001% (100 ppb), or in a percentage between 0.05% and 0.0001% (1 ppm).

For example, the reaction mixture comprising the compound of formula (I), or a salt thereof,

with a content equal to or greater than 98% or equal to or greater than 99%, for instance between about 98.0% and 99.5%, as measured by HPLC at 254 nm, and the compound of formula (VI)

(VI); wherein R is C1-C6 alkyl, optionally substituted by aryl; with a content equal to or less than 2% or equal to or less than 1%, for instance between about 2.0 to about 0.3% or between about 1.0 to about 0.5%, as measured by HPLC at 254 nm, can be obtained by reacting a compound of formula (II)

with a compound of formula (V)

wherein R is C1-C6 alkyl, optionally substituted by aryl; or aryl, as disclosed above. The following examples further illustrate, but do not limit, the disclosure. Example 1 - Synthesis of l,3-Dicyclohexylpyrimidine-2,4,6(lH,3H,5H)- trione of Formula (II)

Dicyclohexylurea of formula (III) (101 g, 432 mmol) and malonic acid of formula (IV) (59.1 g, 568 mmol) are suspended in a mixture of acetic anhydride (590 mL) and acetic acid (500 mL) with mechanical stirring and internal temperature probe under a nitrogen blanket. Stirring is started and the reaction is warmed in a 50°C bath overnight. The reaction is then allowed to cool to room temperature as the product precipitated from the reaction solution. The internal temperature remained 3°C cooler than the bath temperature during the course of the reaction. The white solid is filtered off and the flask rinsed with acetic acid (80 mL). The filter cake is then rinsed with additional acetic acid (100 mL) and dried overnight by pulling dry air through the filter cake providing 104 g of l,3-dicyclohexylpyrimidine-2,4,6(lH,3H,5H)-trione of formula (II) (83% yield) as a white solid. MS (ESI)⁺ m/z [M + H] calcd for C16H24N2O3 : 293.2; found 293.2.

Example 2 - Synthesis of l,3-Dicyclohexylpyrimidine-2,4,6(lH,3H,5H)- trione of Formula (II)

The procedure of Example 1 is repeated starting from a suspension of 330 mg (1.41 mmol) of dicyclohexylurea of formula (III) in 2 mL of acetic anhydride neat. The reaction mixture is heated to 50°C for 6 hours, then cooled down to room temperature. After stirring at room temperature overnight, the formed precipitate of l,3-dicyclohexylpyrimidine-2,4,6(lH,3H,5H)-trione of formula (II) is filtered off and rinsed with acetic acid and hexane providing 1,3-dicyclohexylpyrimidine- 2,4,6(lH,3H,5H)-trione of formula (II) with a yield of 71%.

Example 3 - Synthesis of /V-[(l,3-dicyclohexyl-2,4,6- trioxohexahydropyrimidin-5-yl)carbonyl]glycine of Formula (I) l,3-Dicyclohexylpyrimidine-2,4,6(lH,3H,5H)-trione of formula (II) (20.12 g, 64.7 mmol) is dispersed in anisole (77.3 mL) in a 70°C bath with mechanical stirring. Triethylamine (1.17 mL, 8.41 mmol) is added, followed by ethyl isocyanatoacetate of formula (V) (9.93 mL, 84.1 mmol). A clear pale yellow solution quickly results and the reaction is stirred for 90 minutes. Ethanol (96 mL) is added and a pale yellow precipitate forms. Stirring is continued and sodium hydroxide (7.76 g, 194 mmol) is added. The precipitate quickly dissolves and the reaction clears. The reaction is stirred for 90 minutes. Acetic acid (80 mL) is added with vigorous stirring. The slurry is stirred for another 90 minutes and the heating bath is turned off. The reaction is stirred overnight at room temperature. The white slurry is filtered off and rinsed with ethanol (40 mL). The white solid obtained is dried in a vacuum oven at 80°C overnight resulting in 28.7 g of dry crude product. The crude product is transferred to a 500 mL flask and recrystallized from acetic acid (250 mL) using a mechanical stirrer and a bath temperature of 120°C until completely dissolved taking approximately 1 hour, then stirred as a clear solution for an additional 30 minutes. Heating is then stopped and the pure product starts to crystallize as the solution cools near 100°C. Stirring is continued as the mixture cools to room temperature over approximately 4 hours. The slurry is filtered off and washed with additional acetic acid. The product is dried on the frit by slowly pulling air through the filter cake with occasional stirring until dry, resulting in 23.40 g (92%) of A-[(l,3-di cyclohexyl-2, 4, 6-trioxohexahydropyrimidin- 5-yl)carbonyl]glycine of formula (I) as a white powder. MS (ESI)⁺ m/z [M + H] calcd for C19H27N3O6: 394.2; found 394.2. Combustion analysis theory: C, 58.00; H, 6.92; N, 10.68. Found: C, 57.86, H, 6.77, N, 10.71.

The procedure of Example 3 allows to obtain A-[(l,3-dicyclohexyl-2,4,6- trioxohexahydropyrimidin-5-yl)carbonyl]glycine of formula (I) with a yield of 92%, which is higher than the yields obtained in US 8,324,208. In specific, US 8,324,208 reports a yield of 66% in Method 18.1b) and about 72% in Method 2b+c) (a yield of 92% for obtaining the ethyl ester of formula (VI) from the compound of formula (II) and a yield of 78% for obtaining A-[(l,3-dicyclohexyl-2,4,6- trioxohexahydropyrimidin-5-yl)carbonyl]glycine of formula (I) from the ethyl ester of formula (VI) is reported). Example 4 - Synthesis of /V-[(l,3-dicyclohexyl-2,4,6- trioxohexahydropyrimidin-5-yl)carbonyl]glycine of Formula (I) l,3-Dicyclohexylpyrimidine-2,4,6(lH,3H,5H)-trione of formula (II) (277.4 mg, 0.8918 mmol) is slurried in ethanol (77.3 mL). Ethyl isocyanatoacetate of formula (V) (0.1211 mL, 1.026 mmol) is added, followed by triethylamine (0.1367 mL, 0.9810 mmol). Then further ethyl isocyanatoacetate of formula (V) (0.082 mL, 0.714 mmol) and 2 mL of ethanol is added. After over 2 hours stirring at room temperature, 1.070 mL of an aqueous 2.5 M solution of sodium hydroxide (2.676 mmol) are added dropwise providing a clear pale yellow solution. Then, 2 mL a 2.0 M of hydrogen chloride solution in water (4 mmol) is added slowly until the mixture becomes acidic to pH paper and a cream-colored precipitate is formed. The precipitate is then filtered off, rinsed with 2 mL ethanol, washed with water and finally dried providing 326 mg (92.9%) of N-[( 1,3 -di cyclohexyl-2, 4, 6-trioxohexahy dropyrimidin-5- yl)carbonyl]glycine of formula (I) as a white solid.

The procedure of Example 4 allows to obtain /V-[(l,3-dicyclohexyl-2,4,6- trioxohexahydropyrimidin-5-yl)carbonyl]glycine of formula (I) with a yield of 92.9%, which is higher than the yields obtained in US 8,324,208. In specific, US 8,324,208 reports a yield of 66% in Method 18.1b) and about 72% in Method 2b+c) (a yield of 92% for obtaining the ethyl ester of formula (VI) from the compound of formula (II) and a yield of 78% for obtaining A-[(l,3-dicyclohexyl-2,4,6- trioxohexahydropyrimidin-5-yl)carbonyl]glycine of formula (I) from the ethyl ester of formula (VI) is reported).

Example 5 - Synthesis of /V-[(l,3-dicyclohexyl-2,4,6- trioxohexahydropyrimidin-5-yl)carbonyl]glycine of Formula (I) l,3-Dicyclohexylpyrimidine-2,4,6(lH,3H,5H)-trione of formula (II) (308.6 mg, 0.9922 mmol) is slurried in acetone (2.66 mL) in a round bottom flask with magnetic bar at room temperature. Potassium carbonate (137.1 mg, 0.9922 mmol) is added, followed by ethyl isocyanatoacetate of formula (V) (0.1169 mL, 1.042 mmol). The pinkish slurry is then stirred at room temperature. The HPLC analysis at 210 nm after one hour demonstrates a conversion into ethyl /V-[(l,3-dicyclohexyl-2,4,6- trioxohexahydropyrimidin-5-yl)carbonyl]glycine of formula (VI) of 97% and about 3% of the starting material. MS (ESI)⁺ m/z [M + H] calcd for C21H31N3O6: 422.2; found 422.3; MS (ESI) m/z [M - H] found 420.4. The so obtained ethyl /V-[(l,3- dicyclohexyl-2,4,6-trioxohexahydropyrimidin-5-yl)carbonyl]glycine of formula (VI) is then converted into /V-[(l,3-dicyclohexyl-2,4,6-trioxohexahydropyrimidin-5- yl)carbonyl]glycine of formula (I) according to the procedures disclosed in Examples 3 or 4.

Example 6 - Synthesis of /V-[(l,3-dicyclohexyl-2,4,6- trioxohexahydropyrimidin-5-yl)carbonyl]glycine of Formula (I) l,3-Dicyclohexylpyrimidine-2,4,6(lH,3H,5H)-trione of formula (II) (311 mg, 1.00 mmol) is dissolved in acetonitrile (1.9 mL) in a round bottom flask with magnetic bar at room temperature. Potassium carbonate (280 mg, 2.0 mmol) is added, followed by ethyl isocyanatoacetate of formula (V) (0.112 mL, 1.00 mmol). The pinkish slurry is then stirred at 35°C. The HPLC analysis at 210 nm after one hour demonstrates a conversion into ethyl /V-[(l,3-dicyclohexyl-2,4,6-trioxohexahydropyrimidin-5- yl)carbonyl]glycine of formula (VI) of 100% and no starting material is detected. MS (ESI)⁺ m/z [M + H] calcd for C21H31N3O6: 422.2; found 422.3; MS (ESI) m/z [M - H] found 420.4. The so obtained ethyl /V-[(l,3-dicyclohexyl-2,4,6- trioxohexahydropyrimidin-5-yl)carbonyl]glycine of formula (VI) is then converted into iV-[(l, 3 -di cyclohexyl -2, 4, 6-trioxohexahydropyrimidin-5-yl)carbonyl]gly cine of formula (I) according to the procedures disclosed in Examples 3 or 4.

Example 7 - Purification of /V-[(l,3-dicyclohexyl-2,4,6-trioxo- hexahydropyrimidin-5-yl)carbonyl]glycine of Formula (I)

13 g (33 mmol) of a mixture comprising /V-[(l,3-dicyclohexyl-2,4,6-trioxo- hexahydropyrimidin-5-yl)carbonyl]glycine of formula (I) of 99.08% (determined by HPLC at 254 nm) and A-[(l ,3-dicyclohexyl-2,4,6-trioxohexahydropyrimidin-5- yl)carbonyl]glycine of formula (VI) with a content of 0.89% (determined by HPLC at 254 nm), ethanol (65 mL), water (130 mL) and 9.8 mL of NaOH 30% are loaded under nitrogen atmosphere into a 500ml reactor, equipped with a magnetic stirrer, keeping the temperature between 20°C and 25°C. The mixture is stirred until a clear solution is formed. After further 30 minutes, HC1 37% is added over 30 minutes to the mixture keeping the temperature between 20°C and 25°C until reaching a pH value of 2 and the product starts to precipitate. The mixture is then heated up to between 45°C and 50°C, kept at this temperature for 30 minutes and then cooled down to a temperature between 20° C and 25°C. The precipitate is filtered off, washed with 13 mL of ethanol and dried in a vacuum stove at 80°C for 8 hours obtaining 12,2 g of /V-[(l,3-dicyclohexyl-2,4,6-trioxo-hexahydropyrimidin-5- yl)carbonyl]glycine of formula (I) (94% yield) with a purity of 99.87% (HPLC detector at 254 nm), and wherein the content of /V-[(l,3-dicyclohexyl-2,4,6- trioxohexahydropyrimidin-5-yl)carbonyl]glycine of formula (VI) is 0.07% (measured by HPLC at 254 nm).

Example 8 - Purification of /V-[(l,3-dicyclohexyl-2,4,6-trioxo- hexahydropyrimidin-5-yl)carbonyl]glycine of Formula (I) - Crystallization from isopropanol (comparative example)

1.0 g (2.5 mmol) of a mixture comprising /V-[(l,3-dicyclohexyl-2,4,6-trioxo- hexahydropyrimidin-5-yl)carbonyl]glycine of formula (I) of 99.08% (determined by HPLC at 254 nm) and ethyl A-[( l ,3-di cyclohexyl-2, 4, 6-trioxohexahydropyrimidin-5- yl)carbonyl]glycine of formula (VI) with a content of 0.89% (determined by HPLC at 254 nm) are suspended in 60 mL of isopropanol. The mixture is heated to reflux and stirred until complete dissolution of the solid. The slightly opalescent solution is filtered and the solution is heated again to reflux. Then, the solution is allowed to cool down to 20°C within 3 hours and kept at 20°C for another 16 hours. The formed precipitate is filtered off, the solid washed with 2 mL of isopropanol and dried in a vacuum stove at 80°C. The obtained solid contains A-[( l ,3-di cyclohexyl-2, 4, 6-trioxo-hexahydropyrimidin-5- yl)carbonyl]glycine of formula (I) of 98.99% and ethyl A-[( l ,3-dicyclohexyl-2,4,6- trioxohexahydropyrimidin-5-yl)carbonyl]glycine of formula (VI) with a content of 0.93% (evaluated by HPLC at 254 nm). Consequently, a conventional crystallization procedure in isopropanol demonstrates that a crystallization from isopropanol does not allow to increase the purity of the desired product.

Claims

1 A one-pot or single-vessel process for preparing a compound of formula (I), or a salt thereof,

comprising reacting the compound of formula (II)

with a compound of formula (V)

wherein R is hydrogen, C1-C6 alkyl, optionally substituted by aryl, or aryl, in presence of a non-nucleophilic organic base or a non-nucleophilic inorganic base and in a solvent selected from an ether, a dipolar aprotic solvent, a polar protic solvent, an ester, a ketone or mixtures thereof, forming a compound of formula (VI) (VI); wherein R is as defined above, and adding first an inorganic base to the reaction mixture comprising the compound of formula (VI), and then a protic acid.

2. The process according to claim 1, wherein the non-nucleophilic organic base is typically triethylamine, di/sopropylethylamine, N-Ci-Ce alkyl pyrrolidines, N- Ci- Ce alkyl morpholine, diazabicycloundecene, pyridine, C1-C6 alkyl pyridines, C1-C6 alkyl piperazines, di-Ci-C₆ alkyl piperazines, and the non-nucleophilic inorganic base is typically a carbonate, a hydrogen carbonate, or a /er/-butylate of an alkali metal or of an alkaline earth metal.

3. The process according to claims 1 or 2 without any distillation step.

4. The process according to claims 1 to 3 without isolating the compound of formula (VI).

5. The process according to claims 1 to 4, wherein the reaction is carried out in a solvent or mixture of solvents, wherein the solvent or mixture of solvents does not comprise a chlorinated solvent.

6. The process according to claims 1 to 4 wherein the reaction is carried out in a solvent or mixture of solvents, wherein the solvent or mixture of solvents does not comprise dichloromethane.

7. The process according to claim 1 to 6, wherein the ether is typically selected from an ether, for example anisole, tetrahydrofuran, dioxane or methyl tert-bvAy\ ether; the dipolar aprotic solvent is typically selected for example from dimethylformamide, dimethylacetamide, acetonitrile or dimethylsulfoxide; the polar protic solvent is typically a solvent selected for example from a linear or branched Ci- Ce alcohol; the ester is typically selected for example from ethyl acetate, isopropyl acetate or butyl acetate; and the ketone is typically a linear or branched C3-C7 ketone, for example acetone, methyl ethyl ketone, or methyl isobutyl ketone.

8. The process according to claims 1 to 6, wherein the reaction is carried out in anisole, acetone, acetonitrile, ethanol or mixtures thereof.

9. The process according to claims 1 to 6, wherein the reaction is carried out in anisole.

10. The process according to claims 1 to 9, wherein the compound of formula (II)

is prepared from /V,/V’-dicyclohexylurea of formula (III),

11. The process according to claims 1 to 10, comprising reacting N,N’~ dicyclohexylurea of formula (III),

with malonic acid of formula (IV), or a salt thereof,

0 0

^HΌ^L ^H

(IV) in presence of acetic anhydride.

12. The process according to claim 11, wherein the reaction of the compound of formula (III) with the compound of formula (IV) is carried out in a mixture of acetic anhydride and acetic acid.

13. The process according to claim 12, wherein the ratio of the acetic anhydride and acetic acid is about 1 : 1 (v:v).

14. A process for purifying a compound of formula (I), or a salt thereof,

(VI); wherein R is C1-C6 alkyl, optionally substituted by aryl, with a content equal to or less than 2% or equal to or less than 1%, for instance between about 2.0 to about 0.3% or between about 1.0 to about 0.5%, as measured by HPLC at 254 nm, with an inorganic base in a linear or branched C1-C6 alcohol or in a mixture of water and a linear or branched C1-C6 alcohol; then adding a protic acid to the mixture; and finally isolating the compound of formula (I), or a salt thereof, with a purity greater than about 99.8%, for instance between about 99.8% and 100.0% as measured by HPLC at 254 nm, and wherein the content of the compound of formula (VI) is less than about 0.2%, preferably less than about 0.1%, or equal to or less than about 0.07%, for instance present in a percentage between 0.1% and 0.00001% (100 ppb), or in a percentage between 0.05% and 0.0001% (1 ppm).

15. The process according to claim 14, wherein R is ethyl.

16. The process according to claims 14 or 15, wherein the inorganic base is sodium hydroxide, potassium hydroxide, magnesium hydroxide or calcium hydroxide, preferably sodium hydroxide or potassium hydroxide, more preferably sodium hydroxide.

17. The process according to claims 14 to 16, wherein the linear or branched C1-C6 alcohol is methanol, ethanol, 1 -propanol, 2-propanol, 1 -butanol, 2-butanol or /er/-butanol, preferably methanol, ethanol, 1 -propanol, 2-propanol, more preferably ethanol.

18. The process according to claims 14 to 17, wherein the reaction mixture as defined in claim 14, is obtained by a process carried out in the same reaction vessel, thus by carrying out a “one-pot reaction” according to claims 1 to 13.

19. The compound of formula (I) as defined in claim 1 with a chemical purity, evaluated by HPLC at 254 nm, greater than about 99.8%, for instance between about 99.8% and 100.0% and wherein the content of the compound of formula (VI) as defined in claim 1 and , evaluated by HPLC at 254 nm is less than about 0.2%, preferably less than about 0.1%, more preferably equal to or less than about 0.07%, for instance present in a percentage between 0.1% and 0.00001% (100 ppb), or in in a percentage between 0.05% and 0.0001% (1 ppm).