Method of purification

Abstract

The invention relates to high-purity thymine, a process of purification of thymine, a process for preparing high-purity deoxythymidine, pharmaceutical compositions containing high-purity deoxythymidine and deoxycytidine as well as their use as pharmaceuticals for treatment of mitochondrial depletion syndromes in a mammal.

Description

[0001] METHOD OF PURIFICATION

[0002] FIELD OF THE INVENTION

[0003] The invention relates to high-purity thymine, a process of purification of thymine, a process for preparing high-purity deoxythymidine, pharmaceutical compositions containing high- purity deoxythymidine and deoxycytidine as well as their use as pharmaceuticals for treatment of mitochondrial depletion syndromes in a mammal.

[0004] BACKGROUND OF THE INVENTION

[0005] An investigative product which is currently in clinical development referred to as MT1621 is a combination of

[0006] • deoxycytidine (4-amino-1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2- yl]pyrimidin-2(1 H)-one or“dC”), International Nonproprietary Name (INN): doxecitine and

[0007] • deoxythymidine (1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5- methylpyrimidine-2,4(1 H,3H)-dione or“dT”), INN: doxribtimine.

[0008] MT1621 is a fixed-dose drug product including equal weights of dC and dT and formulated as a powder for oral administration by reconstitution in water or juice. MT1621 provides nucleoside therapy of equal weight ratios of dC and dT to treat mitochondrial depletion syndromes. There is currently no cure for Mitochondria Depletion Syndrome.

[0009] US 2022 / 096514 discloses a method of treating thymidine kinase 2 (TK2) deficiency in a subject by administering deoxycytidine (dC) and deoxythymidine (dT). The method can include administering dC and dT to a subject in doses between 200 mg / kg / day and 600 mg / kg / day for each of dC and dT. In some cases, the method includes performing test administrations with lower dosages of dC and dT, administering adjusted doses after administration of the target doses, or a combination thereof.

[0010] US 2024 / 016938 discloses a method of increasing the oral bioavailability of deoxycytidine (dC) and deoxythymidine (dT) to a patient receiving therapy for a mitochondrial depletion syndrome (MDS) comprising administering to the patient in need thereof, or instructing a patient caregiver to administer, a therapeutically effective amount of dC and dT with food. In frequently used methods known in the art, deoxythymidine (dT) is obtained using thymine as a starting compound, including Shinozuka, Kazuo; Morita, Tsutomu; Hirota, Yoshiki; Sawai, Hiroaki; Chemistry Letters; nb. 11 ; (1991); p. 1941 - 1944. Thymine is one of the four nucleotide bases in the nucleic acid of DNA that are represented by the letters G-C-A-T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nucleobase. Thymine combined with deoxyribose results in the aforementioned nucleoside deoxythymidine, which is synonymous with the term thymidine. Methods for the preparation of thymine are known in the art, one of them is disclosed in Bulletin of the Chemical Society of Japan; vol. 60; nb. 6; (1987); p. 2073 - 2077.

[0011] WO 00 / 02864 relates to precursors for the synthesis of monomeric units useful in the synthesis of polynucleotide analogues. It also discloses a method for the preparation of rac- 2-(N 1-thyminyl)-isopropionic acid wherein thymine is reacted with rac-2-bromopropionic acid in the presence of KOH.

[0012] An HPMC step for separating thymine from phenylalanine is disclosed in JP2010064973.

[0013] The problem underlying the present invention is that usually the preparation of thymine implies a significant impurity which is alpha-hydroxythymine. Alpha-hydroxythymine when processed to obtain deoxythymidine generates the genotoxic impurity alphahydroxythymidine. So far it has been impossible to obtain high-purity thymine, i.e. thymine containing less than 150 ppm of alpha-hydroxythymine.

[0014] Solvent based recrystallizations could not lower the content of alpha-hydroxythymine to less than 150 ppm.

[0015] SUMMARY OF THE INVENTION

[0016] The present invention relates to high-purity thymine, a process of purification of thymine, high-purity deoxythymidine, a process for preparing high-purity deoxythymidine, pharmaceutical compositions containing high-purity deoxythymidine and deoxycytidine as well as their use as pharmaceuticals for treatment of mitochondrial depletion syndromes in a mammal. Further aspects of the invention will become apparent from the detailed specification. DETAILED DESCRIPTION OF THE INVENTION

[0017] A first aspect of the present invention is high-purity thymine, i.e. thymine which contains less than 30 ppm of alpha-hydroxythymine, more preferably less than 20 ppm and most preferably less than 10 ppm. Such purity has never been achieved before. The thymine obtained according to the present invention achieves an overall purity of at least 99.9%, preferably 99.99%.

[0018] A second aspect of the present invention is a method for preparing high-purity thymine, i.e. thymine which contains less than 30 ppm of alpha-hydroxythymine, more preferably less than 20 ppm and most preferably less than 10 ppm of alpha-hydroxythymine.

[0019] The key step for the purification of thymine according to the present invention involves the use of a limited selection of alkaline agents : sodium carbonate, sodium bicarbonate, sodium hydroxide or mixtures thereof. The most preferred alkaline agent used in step i) is sodium carbonate.

[0020] The preferred method according to the present invention involves the following steps : i. Mixing the thymine (which typically forms a suspension in water) to be purified with an adequate amount of an alkaline agent selected from sodium carbonate, sodium bicarbonate, sodium hydroxide or mixtures thereof, and generate a solution, e.g. by heating; preferably the solution is kept under heating for between 1-24 hours. ii. The solution is then cooled and neutralized to pH 0-10, preferably 3.0-9.0, more preferably 7.0-8.0. iii. Filtration of the suspension, rinsing the cake and drying the solid.

[0021] In step i) the mixing of the thymine to be purified is done with an adequate amount (10-20 Vol) of an aqueous solution of alkaline agent (0.3-1.0 mol / L) selected from sodium carbonate, sodium bicarbonate, sodium hydroxide or mixtures thereof, and generate a solution, e.g. by heating; preferably the solution is kept under heating (80°C to reflux) for between 1-24 hours. (pH range 9-10.5).

[0022] With the cooling of the solution in step ii), a crystallization of thymine is triggered. Most preferably, the solution of step i) is kept under heating for 12-24 h, preferably for 18-24 h, most preferably for 24 hours. The high purity thymine is obtained avoiding a costly chromatographic step.

[0023] A third aspect of the present invention is high-purity deoxythymidine which is obtained from high-purity thymine, i.e. thymine which contains less than 30 ppm of alpha-hydroxythymine, more preferably less than 20 ppm and most preferably less than 10 ppm of alpha- hydroxythymine. The high purity high-purity deoxythymidine is thus obtained from high purity thymine, thus avoiding a costly chromatographic step.

[0024] High-purity deoxythymidine contains less than 30 ppm of alpha-hydroxythymidine, more preferably less than 20 ppm and most preferably less than 10 ppm.

[0025] Deoxythymidine is obtained through methods known in the art, in order to obtain high purity deoxythymidine, high-purity thymine according to the present invention is used.

[0026] Another aspect of the present invention is a pharmaceutical composition containing high-purity deoxythymidine obtained according to the present invention and deoxycytidine. The drug product is formulated with excipients as a solid white powder suitable for reconstitution in water or apple juice, for oral administration as nucleoside therapy. Excipients used in the drug product formulation are chemically inert, including glidants, lubricants and filling agents. Preferably, colloidal silica anhydrous (silicon dioxide) is added as a glidant, and magnesium stearate is added as a lubricant to aid in the mechanical filling of unit dose packages.

[0027] EXAMPLES

[0028] The following examples illustrate how high purity thymine and thymidine may be prepared. They are provided for illustrative purposes only and are not intended, nor should they be construed, as limiting the invention in any manner. Those skilled in the art will appreciate that routine variations and modifications of the following examples can be made without exceeding the spirit or scope of the invention.

[0029] EXAMPLES

[0030] Abbreviations / recurrent reagents g: gram h: Hour HPLC: High Pressure Liquid Chromatography

[0031] L: liter

[0032] M: molar (mol per liter) mL: milliliter nm: nanometer ppm :part per million v:v :volume:volume vol: L / kg

[0033] TLC: Thin Layer Chromatography

[0034] Example 1 : Preparation of high-purity thymine a. Charging the reactor with 1.5 L of water at room temperature b. Stirring and adding 106 g of sodium carbonate c. 100 g of thymine to be purified is added: a white suspension at room temperature is obtained d. Heating the jacket of a reactor to -100°C : target temperature of reactor mass is about 90°C e. At 90°C a solution is obtained. If needed, this is the point where a clarification can be performed by hot filtration into another reactor f. Maintaining the temperature of 90°C at least overnight and monitor the residual Hydroxy-Thymine by HPLC area ratio versus Thymine peak at 254nm. Typically 24h to go from 1000ppm to Not Detectable(<10ppm). Typically at this stage pH is around 11. g. the reaction mixture is cooled to 70°C: crystallization will spontaneously start. h. the pH is adjusted to 7.0-8.0 by slow addition of 6M hydrochloric acid (-170 mL): crystallization will be increasingly visible. There is a CO2 gas exhaust during neutralization of sodium carbonate, but no foaming observed. i. After 1 hour and stable pH of about 7.0-8.0, the reaction mixture is cooled down to 20°C in between 1 to 6 hours and stirred for about 3 hours. j. Filtration of the suspension : the cake is rinsed with 3*100 mL fresh water. pH of washings liquors to be adjusted to about 7.0-8.0 at last washing. If pH is beyond that range, more rinsing is performed. k. the solid is dried on filter, then in vacuum drying oven at 50°C / 20 mbar until constant weight. Expected yield: 90-95% (reference experiment: 92.7%); the residual hydroxy- Thymine impurity in the final product is determined by HPLC to be undetectable, which is below 10ppm.

[0035] Example 2 : Preparation of high-purity thymidine

[0036] The preparation of thymidine consists in three chemical steps

[0037] Preparation of 1-chloro-2-deoxy-3’,5’-bis-(O-p-chlorobenzoyl) D-ribofuranose a. 143.5 g of 2-desoxyribose and 1 ,7L of methanol are charged to a reaction vessel. b. 5.7 g of acetyl chloride is added to the mixture, while maintaining an internal reaction temperature below 5°C. The resulting mixture is stirred for at least 3 hours at 5 °C. c. The temperature is adjusted to 20°C d. After the reaction is judged to be complete, 57 g of sodium bicarbonate are added and the resulting mixture is stirred at least 30 minutes at 20 °C. e. The slurry is filtered and the salt cake is washed with methanol. f. The filtrate and the wash are combined and concentrated. g. 0.3L of toluene are charged and the resulting mixture is stirred and concentrated under reduced pressure. This step is repeated. h. 1.4 L of toluene are charged and then after stirring for about 30 minutes, approximately 324 g of triethylamine is charged. i. 393 g of p-chlorobenzoyl chloride are added while maintaining the internal temperature at not more than 60°C and the resulting mixture is adjusted to 60°C and stirred for at least 3 hours. j. After the reaction conversion is judged to be complete the mixture is cooled to 20°, about 0.7 L of purified water are added and the mixture is stirred for at least 30 minutes. k. The resulting solids are filtered, washed with about 0.3 L of toluene and the filtrate and wash are combined. l. The layers are separated and the aqueous layer is discarded. m. Approximately 300 g of sodium sulfate anhydrous are added into the organic layer. n. The resulting slurry is stirred for at least 30 minutes, filtered and the salt cake is washed with 0.3 L of toluene. o. The filtrate and wash are combined and concentrated under reduced pressure and p. The resulting mixture is cooled to 0°C and stirred for at least 1 hour. q. The resulting slurry is filtered and the filtrate is concentrated. r. 885 g of acetyl chloride and 565 g of tert-butyl methyl ether are charged to the concentrate and the resulting mixture is cooled to 0°C. s. A pre-made solution of methanol 0.1 L and tert-butyl methyl ether 85 g is added while maintaining an internal reaction temperature below 20°C t. The mixture is stirred at 15°C for at least 30 hours. u. After the reaction is judged to be complete, the temperature is cooled to 0°C and stirred for at least 1 hour. v. The resulting slurry is filtered and the product cake is washed with cooled tert-butyl methyl ether. w. The product cake is collected and dried at not more than 30°C of external temperature for at least 12 hours. Expected yield: 70%

[0038] Glycosylation step: preparation of 1-chloro-2-deoxy-3’,5’-bis-(O-p-chlorobenzoyl) D- ribofuranose i. 221g, of thymine obtained according to Example 1 , containing less than 30 ppm of alpha-hydroxythymine, more preferably less than 20 ppm and most preferably less than 10 ppm, 1 ,50g of ammonium sulfate and about 600 g, of bis(trimethylsilyl)amine (HMDS) are charged to a reaction vessel, the mixture is heated above 110°C ii. After dissolution, the mixture is cooled to rt and 2.5 L of methylene chloride (MC) are added. iii. 335g of trimethylsilyl trifluoromethanesulfonate are added while maintaining the temperature below 20°C. iv. 299 g of 1-chloro-2-deoxy-3’,5’-bis-(O-p-chlorobenzoyl) D-ribofuranose is added and the mixture is stirred for at least 4 hours at 0 °C v. The reaction mixture is transferred to a vessel containing a mixture of about 1 ,8L of 0.7N aqueous sodium hydroxide solution and about 2.4 L of methylene chloride, and stirred. vi. The biphasic mixture is stirred for at least 30 minutes, heated to 35°C, stirred for at least 30 minutes, then allowed to settle for at least 30 minutes and the layers are separated. The organic layer is held. vii. 1 ,8L of methylene chloride and about 2.4L of 1 N aqueous sodium hydroxide solution are added to the aqueous layer and the temperature is adjusted to 35°C. The biphasic mixture is stirred for at least 30 minutes, then allowed to settle for at least 15 minutes and the layers are separated. viii. The organic layers are combined, 3L of purified water are added and the temperature is adjusted to 30°C. The biphasic mixture is stirred for at least 20 minutes, then allowed to settle for at least 20 minutes and the layers are separated. ix. 3L of purified water are added and the temperature is adjusted to 30°C. The biphasic mixture is stirred for at least 20 minutes, then allowed to settle for at least 20 minutes and the layers are separated. x. The organic layer is polish filtered and then concentrated. xi. 3L of acetone are added to the concentrate and the solution is stirred for at least 5 minutes. xii. 0.75L of purified water are added and the resulting mixture is heated to 60°C and maintained for at least 1 hour. xiii. The stirring mixture is cooled to 5°C and maintained for at least 30 minutes. xiv. The resulting slurry is filtered and the product cake is washed with a cooled aqueous acetone solution of purified water 50:50 v:v. xv. The product cake is collected and dried at not more than 70°C of external temperature for at least 12 hours. Expected yield: 80% xvi. Preparation of thymidine xvii. 1-chloro-2-deoxy-3’,5’-bis-(O-p-chlorobenzoyl) D-ribofuranose (310g) and 2.8 L of methanol are charged to a reaction vessel. xviii. Approximately 257 g of ammonia (gas) are added to the solution while maintaining the internal temperature below 35°C. The resulting mixture is heated to 35°C and stirred for at least 8 hours. xix. The reaction mixture is concentrated. xx. 2.2 L of acetone are added to and the resulting mixture is concentrated. This process step is repeated. xxi. 3.1 L of acetone are added and the resulting mixture is heated to 50°C to 60°C and stirred for at least 1 hour. xxii. The mixture is cooled to 5°C and stirred for at least 30 minutes. xxiii. The resulting slurry is filtered, and the product cake is washed with acetone. xxiv. The wet product cake is charged into a reaction vessel with about 1.5 L of acetone xxv. The mixture is heated to above 50°C and stirred for at least 1 hour. xxvi. The mixture is cooled to 0°C to 5°C and stirred for at least 30 minutes. xxvii. The resulting slurry is filtered and the product cake is washed with cooled acetone. xxviii. The wet product cake is collected and dried at an external temperature of not more than 70°C. Expected yield: 79%

Claims

CLAIMS1. A process of purification of thymine, comprising the following steps of : i. mixing the thymine to be purified with an adequate amount of an alkaline agent selected from sodium carbonate, sodium bicarbonate, sodium hydroxide or mixtures thereof, in such a way to generate a solution; ii. cooling and neutralizing the solution to pH 0-10, iii. filtering the suspension, rinsing the cake and drying the solid purified thymine.

2. A process of purification according to claim 1 , wherein the solution of thymine and the alkaline agent of step i) is heated to about 90°C and the pH is adjusted beyond 10.

3. A process of purification according to claim 1 or 2, wherein the solution in step i) is generated by heating for a period of 1-24 hours.

4. A process of purification according to claim 3, wherein the heating period is between 18- 24 hours.

5. A process of purification according to any of claim 1 to 4, wherein the neutralizing adjustment ii) is performed to obtain a pH ranging between about 7.0-8.0.

6. A process of purification according to any of claim 1 to 5, wherein the cooling step ii) is adjusted to below 80°C, preferably below 70°C.

7. A process of purification according to any of claim 1 to 6, wherein the alkaline agent is sodium carbonate.

8. High-purity thymine obtainable through a method according to claims 1 to 7, containing less than 30 ppm of alpha-hydroxythymine impurity, more preferably less than 20 ppm and most preferably less than 10 ppm.

9. High-purity deoxythymidine obtainable from high-purity thymine according to claim 8.

10. A fixed-dose pharmaceutical composition comprising a first therapeutic agent which is high-purity deoxythymidine according to claim 9 and a second therapeutic agent which is deoxycytidine as well as chemically inert excipients.

11. Use of a pharmaceutical composition of claim 10 for the treatment of mitochondrial depletion syndromes in a mammal.