EP2997031A1

EP2997031A1 - Process for the preparation of high purity amorphous pemetrexed disodium and crystalline forms of n-[4-[2-(2-amino-4,7-dihydro-4-oxo-3h-pyrrolo[2,3- d]pyrimidin-5-yl)ethyl]benzoyl]-l-glutamic acid

Info

Publication number: EP2997031A1
Application number: EP14732453.7A
Authority: EP
Inventors: Olga Michalak; Kamil JATCZAK; Wieslaw Pucko; Anna WITKOWSKA; Marta Laszcz; Iwona BUJAK; Aleksandra GROMAN; Marcin Cybulski
Original assignee: Instytut Farmaceutiyczny
Current assignee: Instytut Farmaceutiyczny
Priority date: 2013-05-17
Filing date: 2014-05-16
Publication date: 2016-03-23
Also published as: WO2014185797A1

Abstract

A process for the preparation of high purity amorphous pemetrexed disodium is characterized by reacting N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3- d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid with molar shortage of sodium cations generating compound in regard to carboxyl groups of free acid, under anhydrous conditions. Preferably, in that process pure crystalline Form 1 or Form 2 of N-[4-[2-(2- amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, containing from 27 to 31% of dimethyl sulfoxide, is used.

Description

Process for the preparation of high purity amorphous pemetrexed disodium and crystalline forms of N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3- d] py rimidin-5-yl)ethyl] benzoyl]-L-glutamic acid

Field of the invention

The present invention relates to a process for the preparation of high purity amorphous pemetrexed disodium as well as to the crystalline forms of N-[4-[2-(2- amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, which are used in the preparation thereof. The present invention also discloses the methods of preparation of the new crystalline forms of N-[4-[2-(2-amino-4,7-dihydro-4- oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid.

The amorphous pemetrexed disodium can be used as the active ingredient of the pharmaceutical product in a form of lyophilized powder. Background of the invention

Pemetrexed is an antifolate antineoplastic agent that exerts its action by disrupting folate-dependent metabolic processes essential for cell replication. It works by inhibiting three enzymes used in purine and pyrimidine synthesis de novo - thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARPT). By inhibiting the formation of precursor purine and pyrimidine nucleotides, pemetrexed prevents the formation of DNA and RNA, which are required for the growth and survival of both normal cells and cancer cells.

The pharmaceutical product containing pemetrexed disodium as the active ingredient, indicated for the potential treatment of locally advanced or metastatic non- small cell lung cancer and mesothelioma, is available on the market under brand name ALIMTA®,. It is a sterile lyophilized powder for intravenous infusion. The lyophilizate is to be reconstituted in a sterile physiological salt and further dilution prior to infusion. ALIMTA®, except of pemetrexed disodium equivalent to 100 mg or 500 mg pemetrexed, contains mannitol and optionally hydrochloric acid and/or sodium hydroxide to adjust pH.

Pemetrexed, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin- 5-yl)ethyl]benzoyl]-L-glutamic acid as well as the pharmaceutically accepted salts thereof have been disclosed in the patent application EP 432677 Al .

In the preparative examples of EP 432677 Al, L-glutamic acid derivatives sodium salts have been obtained as the intermediates in the synthesis of the corresponding free acids, isolated in the crystalline form after the basic hydrolysis of appropriate diester followed by neutralization with hydrochloric acid, and then re- crystallized.

A number of pemetrexed disodium crystalline forms and the methods for preparation thereof have been disclosed in the prior art but particular attention has been paid to syntheses of amorphous pemetrexed, because it has been proved to be very convenient in the preparation of the lyophilized pharmaceutical products. The amorphous pemetrexed disodium disclosed in WO 2008/124485 was obtained by removing the solvent from the solution of pemetrexed disodium in water, DMSO, alcohol, ketone, or the mixtures thereof, by the commonly used laboratory techniques, such as evaporation, distillation under vacuum or atmospheric pressure, or spry drying. The amorphous pemetrexed disodium has been characterized by a mass loss upon drying at the level of about 8.268%, as determined by a thermogravimetric analysis method.

In the patent application EP 2072518 Al, a stable amorphous pemetrexed disodium characterized by a bulk density from 0.15 to 0.35 g/mL has been disclosed. It was obtained by lyophilization, drying the solution of pemetrexed disodium in water or the mixture of water and alcohol, in the spray drier or the pneumatic drier.

In WO 2010/028105, methods for preparation of the amorphous pemetrexed disodium have been revealed, comprising first dissolving pemetrexed salt in a solvent, and then precipitating the product by adding an anti-solvent or by heating the solution in alcohol. In EP 1943252 Bl specification, process for preparation of the lyophilized pemetrexed disodium as a pure substance or in a composition with a carrier, directly from diacid or its addition salts without isolation of disodium salt, has been reported. In the said process, pemetrexed diacid or its mono- or di-base-addition salts were contacted with sodium cations generating compound, such as sodium hydroxide, carbonate, phosphate or sulfate in an organic solvent, comprising water or the mixture of water and tert-butanol, dimethyl sulfoxide or 1,4-dioxane. The lyophilizate was obtained by removing the solvent in the drying or freeze-drying processes.

The experimental attempts to produce the amorphous pemetrexed disodium from pemetrexed diacid or its salts other than di-sodium in the reaction with sodium source compound in aqueous medium, did not furnish morphologically homogeneous compound. Leaving aside the mechanism of crystallization process, the authors of the present invention asserted, formation of the amorphous pemetrexed disodium phase has been accompanied by a formation of a substantial amount of its crystalline phase. Depending on the reaction conditions, the content of the crystalline phase in the amorphous phase varies, this phenomenon impedes the validation of the process and as a consequence its implementation into plant technology.

According to the European Medicines Agency guidelines authorized by International Conference on Harmonization, Harmonized Tripartite Guideline. Q3A(R2), Impurities in new drug substances, and Q3C(R4), Impurities: Guideline for Residual Solvents, approved in Geneva in 2006, active pharmaceutical ingredient must fall into line with the particular specifications regarding purity. That means, the content of impurities and residual solvents cannot exceed acceptable limits. For the pharmaceutical substances, which are not disclosed in monographs of the European Pharmacopoeia, acceptable level of a single identified impurity is <0.15% and of an unidentified impurity is <0.10%.

According to the literature survey, pemetrexed disodium can be obtained, in general, in the reaction of pemetrexed free diacid or the acid addition salt of its ester, such as p-toluenesulfonic acid addition salt, with stoichiometric amount or molar excess of sodium source compound, such as sodium hydroxide, carbonate, phosphate or sulfate in aqueous media or in a water miscible solvent. To initiate a precipitation of the amorphous product, an addition of the precipitation initiating solvent is necessary. However, obtaining the amorphous pemetrexed disodium product complying with the requirements regarding pharmaceutical purity as well as amorphous homogeneity (i.e. free of crystalline forms admixtures) following this approach is very troublesome. In our unpublished Polish patent application P-403942, a process for the preparation of the amorphous pemetrexed disodium of high homogeneity, deprived of crystalline phase admixtures, which meets the purity requirements for pharmaceutical substances, has been disclosed. That product resulted from the reaction of N-[4-[2-(2- amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, herein referred to as pemetrexed diacid, with the sodium cations generating compound under anhydrous conditions.

N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5- yl)ethyl]benzoyl]-L-glutamic acid and its chemical purity is crucial to obtain pemetrexed disodium of pharmaceutical purity. In view of the literature revue, pemetrexed diacid can crystallize in different crystalline forms.

In WO 2008/021405 specification, pemetrexed diacid crystalline forms A, B, C, D, E, F, and G have been dislosed.

Pemetrexed diacid forms A and B crystallize as hydrates, containing 7.7% and 2.5 - 3.9% of water, respectively, by adjusting pH to 3.0 - 4.5 of the solution of pemetrexed disodium in water or in the mixture of water and the water miscible organic solvent.

Pemetrexed diacid crystalline forms C, D and E are obtained as solvates with DMSO or DMF, when precipitating from the mixtures of DMSO/water/methanol, DMF/water/methanol, or DMF/ethanol.

Pemetrexed diacid crystalline form F is obtained from the salt of pemetrexed diethyl ester with p-toluenesulfonic acid, which is subjected to basic hydrolysis, and adjusting pH of the solution to about 3.9 - 4.1. Anhydrous pemetrexed diacid crystalline form G is formed upon drying form B at the temperature of about 160°C - 200°C.

In the International patent application WO 2008/124485, the preparation of pemetrexed diacid crystalline forms A and B has been disclosed. The process embraced hydrolysis of pemetrexed diethyl ester salt with p-toluenesulfonic acid under basic conditions, then adjustment of pH to about 3.0 of the solution consisting of water/ethanol or isopropanol/water.

In EP 2351755 A4 specification, the preparation of three pemetrexed diacid polymorphic forms H, I, and J has been disclosed. Crystalline form H was obtained, when pH of the solution, containing disodium salt in the mixture of water and water miscible solvent, was brought to about 1.5 - 2.5. The group of the water miscible solvents comprised alcohols, preferably ethanol, acetonitrile, THF, dimethyl ethylene glycol and acetone.

Pemetrexed diacid crystallized as form I from the aqueous solution of pemetrexed salt at the concentration below 0.07 mol/L when adjusting pH of the solution to about 2.0 - 3.0, while crystalline form J was obtained from the aqueous solution of pemetrexed salt at the concentration higher than 0.07 mol/L when pH of the solution was brought to about 2.0 - 4.0.

On account of easy formation of solvates and hydrates of pemetrexed diacid, the authors of the present invention faced the dilemma of selecting appropriate solvents, from which the crystalline form of pemetrexed diacid of high purity and strictly defined chemical composition, suitable to produce the final pemetrexed disodium product of the pharmaceutical purity.

The stability studies performed under standard and accelerated conditions, proved susceptibility of the amorphous pemetrexed disodium towards UV radiation, oxidative conditions and air oxygen. The stress testing carried out using hydrogen peroxide (3%), and basic or acidic conditions, showed substance degradation. The aforementioned results indicate the limited stability of the amorphous pemetrexed disodium under certain reaction conditions, which may cause obtaining the final product of demanded high purity difficult. When scaling-up the process described in the examples of the Polish patent application P-403942, using high chemical purity pemetrexed diacid under anhydrous conditions, amorphous pemetrexed disodium of moderate purity has been obtained. The purification of that product in the final step has not been successful. This outcome results from the presence of substantial amount of impurity in the reaction product, detected by high performance liquid chromatography (HPLC) at RRT 1.03 at the level of 0.27 - 0.30%. This contamination affects the quality of the final product and limits applicability of pemetrexed disodium obtained by this method as the active pharmaceutical ingredient. In addition, when scaling-up the process, the difficulties may be encountered in regard of removing the most common solvents such as ethanol, propanol, toluene or acetone, from amorphous pemetrexed disodium to the level accepted for the pharmaceutical substance.

All the said obstacles have been circumvented in the process according to the present invention, due to: a) changing the proportion of the reagents used in the process of pemetrexed disodium formation, b) using pemetrexed diacid of a well defined crystalline form and strictly established chemical composition, c) removing the residual solvents by final maceration of the amorphous pemetrexed disodium in an alkane type aprotic solvent and subsequent drying the amorphous solid.

Summary of the invention

One aspect of the present invention is the process for preparation of high purity amorphous pemetrexed disodium in the reaction of N-[4-[2-(2-amino-4,7-dihydro-4- oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)etyhl]benzoyl]-L-glutamic acid with sodium cations generating compound, characterized by the use of the molar shortage of sodium cations generating compound in respect to carboxyl groups of N-[4-[2-(2-amino-4,7- dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic free acid, under anhydrous conditions. Preferably, in the process according to the invention, 2% molar shortage of sodium cations generating compound to one carboxyl group of N-[4-[2-(2-amino-4,7~ dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid is used.

Another aspect of the invention is the crystalline Form 1 of N-[4-[2-(2-amino- 4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, characterized by X-ray powder diffraction pattern (XRPD), recorded on the diffractometer equipped with the copper anode of Koci λ = 1,54056 A wave length at the measurement range from 7 to 30°, having the peaks at 2Θ angles about 9,33; 13,42; 16,99; 17,73; 18,44; 19,30; 28,28; 30,98 ± 0,2°. The other aspect of the invention is the crystalline Form 2 of N-[4-[2-(2-amino-

4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, characterized by X-ray powder diffraction pattern (XRPD), recorded on the diffractometer equipped with the copper anode of Kct_\ λ = 1,54056 A wave length at the measurement range from 7 to 30°, having the peaks at 2Θ angles about 7,39; 9,41; 11,17; 11,68; 19,72; 23,68; 27,73 ± 0,2°.

Another aspect of the invention is the use of the crystalline form of N-[4-[2-(2- amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, selected from Form 1 and Form 2, to produce the amorphous pemetrexed disodium of purity above 99.7%. Description of the Figures

Fig. 1 represents X-ray powder diffraction pattern (XRPD) of the crystalline Form 1 of N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]- L-glutamic acid.

Fig. 2 represents DSC profile of the crystalline Form 1 of N-[4-[2-(2-amino-4,7- dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, obtained by differential scanning calorimetry.

Fig. 3 represents the thermal characteristics of the crystalline Form 1 of N-[4-[2-(2- amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, obtained by the thermogravimetric analysis (TGA). Fig. 4. represents DSC the profile of the crystalline Form 1 of N-[4-[2-(2-amino-4,7- dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, obtained by the differential scanning calorimetry with heating-cooling loop.

Fig. 5 represents X-ray powder diffraction pattern (XRPD) of the crystalline Form 2 of N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]- L-glutamic acid.

Fig. 6 represents DSC profile of the crystalline Form 2 of N-[4-[2-(2-amino-4,7- dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, obtained by the differential scanning calorimetry. Fig. 7 represents the thermal characteristics of the crystalline Form 2 of N-[4-[2-(2- amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, obtained by the thermogravimetric analysis (TGA).

Fig. 8 represents X-ray powder diffraction pattern (XRPD) of the amorphous pemetrexed disodium. Fig. 9 represents DSC curve of the amorphous pemetrexed disodium, obtained by the differential scanning calorimetry.

Fig. 10 represents the thermal characteristics of the amorphous pemetrexed disodium.

Detailed description of the invention

The strategy disclosed in the present invention is based on the observation, that when the molar shortage of sodium methanolate to diacid in synthesis of pemetrexed disodium is used, the main impurity detected by HPLC analysis at 1.03 RRT is not formed, whereas the use of even small (eg. 10%) molar excess of sodium methanolate to diacid, results in the formation of substantial amounts, reaching 0.27-0.30%, of this impurity (the comparative example 3). Preparation of the high purity amorphous pemetrexed disodium in the reaction of N-

[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L- glutamic acid with the sodium cations generating compound, according to the present invention embraces: a ) reacting N- [4- [2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3 -d]pyrimidin-5- yl)ethyl]benzoyl]-L-glutamic acid with the molar shortage of sodium cations generating compound to carboxyl groups of N-[4-[2-(2-ammo-4,7-dihydro-4-oxo-3H-pyrrolo[2,3- d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid under anhydrous conditions, b) optionally, adding anti-solvent to precipitate the product of the reaction, c) isolating the crude amorphous pemetrexed disodium, d) suspending the crude amorphous pemetrexed disodium in an anhydrous alcohol solvent and stirring the suspension at ambient temperature, e) isolating and drying the amorphous product, f) suspending and stirring the product in an alkane type aprotic solvent to remove the residual solvents, and g) isolating and drying the final product.

As used herein, the term 'high purity amorphous pemetrexed disodium' refers to the substance free of other polymorphic and pseudo-polymorphic forms at amounts detectable by routinely used analytical methods, such as X-ray powder diffraction and infra-red absorption, that means containing below 2%, preferably below 1% of other crystalline forms. In addition, high purity amorphous pemetrexed disodium is to be characterized by chemical purity above 99.7%, determined by high performance liquid chromatography (HPLC). According to the present invention, the starting N-[4-[2-(2-amino-4,7-dihydro-4- oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, herein referred to as pemetrexed diacid, can be obtained following the synthetic pathway disclosed, among the others, in EP 589720 A2 and EP 1212325 Al, as depicted in Scheme 1. This process is based on coupling 4-[2-(2-amino-4-oxo-3,7-dihydro-3H-pyrrolo[2,3- d]pyrimidin-5-yl)ethyl]benzoic acid, activated by 4-chloro-2,4-dimethoxy-l,3,5-triazine (CDMT), with ethyl L-glutamate hydrochloride in the presence of N-methylmorpholine, then purifying the product as an acid addition salt with p-toluenesulfonic acid.

Preferably, pemetrexed diethyl ester p-toluenesulfonate is subjected to hydrolysis upon sodium hydroxide aqueous solution at ambient temperature for 2 h, yielding the expected reaction product. To the reaction mixture alcohol solvent, such as ethanol, is added at ambient temperature, pH of the post-reaction mixture is adjusted to about 3.0 - 3.5, then the solution is heated at 70°C, and after cooling down to ambient temperature the solid pemetrexed diacid precipitates.

The amorphous pemetrexed disodium of expected pharmaceutical purity can be obtained when the pemetrexed diacid of chemical purity above 99% is used.

The purity of pemetrexed diacid is crucial for the preparation of the amorphous pemetrexed disodium of demanded purity, therefore pemetrexed diacid usually requires the additional crystallization(s) to increase its purity prior its use in the following steps of synthesis.

Pemetrexed diacid can be easily re-crystallized, dissolving the solid in aprotic solvent, such as dimethyl sulfoxide (DMSO), dimethyloformamide (DMF) or N- methylpyrrolidon, and then precipitating the solid by the addition of a polar anti-solvent selected form the group of alcohol solvents, preferably ethanol (EtOH). Pemetrexed diacid can be isolated as any optional crystalline form.

Preferably, according to the present invention, pemetrexed diacid is subjected to one or more crystallizations, preferably two crystallizations, in the mixture of DMSO/EtOH.

It has been discovered that under these conditions, two crystalline forms, 1 or 2, of pemetrexed diacid can be obtained and none of them corresponds to the crystalline forms described in the prior art.

Crystalline Form 1 is obtained, when to pemetrexed diacid dissolved in dimethyl sulfoxide ethanol is added as the anti-solvent, and EtOH/DMSO volume ratio is maintained from about 2.0 to 4.0. Anti-solvent is added to that solution dropwise or one- time at the temperature range of 40 - 55°C, furnishing crystalline product precipitation. After bringing down the temperature of the reaction mixture to ambient, the crystalline solid is filtered off, washed with ethanol, and air dried in an air flow drier at 40-45 °C to the constant mass. According to the same manner the second crystallization is performed in DMSO/EtOH, yielding pemetrexed diacid of chemical purity above 99.7% (HPLC). The formation of the crystalline Form 1 also takes place after water addition to the reaction mixture at the amount not higher than 10% of the total volume of other solvents used (DMSO and EtOH).

Crystalline Form 2 is obtained analogously, when to pemetrexed diacid dissolved in dimethyl sulfoxide, ethanol is added as anti-solvent at higher EtOH/DMSO volume ratio, from about 4.2 do 6.0.

Crystalline Form 1 is thermodynamically more stable than form 2.

The crystalline Form 1 of pemetrexed diacid is characterized by X-ray powder diffraction pattern (XRPD), recorded on the diffractometer equipped with the copper anode of Κ ι λ = 1,54056 A wave length at the measurement range from 7 to 30°, having the peaks at 2Θ angles about 9.33; 13.42; 16.99; 17.73; 18.44; 19.30; 28.28; 30.98 ± 0.2°.

The crystalline Form 1 of pemetrexed diacid is characterized by X-ray powder diffraction pattern recorded on diffractometer equipped with a copper anode of Κα λ = 1,54056 A wave length, represented as relative intensities of diffraction peaks I/Io, diffraction angles 2Θ and interplanar distances d, with scanning range from 3 to 40°, scanning rate 0,5 min and step size 0,02°. The data are collected in Table 1 :

Table 1.

2Θ, Π d, [A] I Io, [%]

9.33 9.470 38

12.23 7.229 2

12.80 6.908 8

13.42 6.592 2

14.44 6.129 4

15.36 5.762 1

16.99 5.216 7

17.73 4.998 27

18.44 4.807 42 18.73 4.734 44

19.30 4.595 5

19.96 4.445 11

20.52 4.326 74

21.47 4.135 5

21.96 4.045 9

22.31 3.981 17

22.49 3.950 26

22.76 3.903 21

22.97 3.869 19

23.49 3.784 100

24.29 3.661 12

24.86 3.579 10

25.86 3.442 17

26.18 3.401 20

26.63 3.345 17

27.09 3.289 15

28.28 3.154 16

28.99 3.078 13

29.31 3.045 12

29.96 2.980 10

30.16 2.961 7

30.98 2.884 8

34.09 2.628 9

34.50 2.598 8 36.53 2.458

An exemplary X-ray powder diffraction pattern of pemetrexed diacid crystalline Form 1 is presented in Fig. 1.

DSC profile of pemetrexed diacid crystalline Form 1, obtained by the differential scanning calorimetry, depicted in Fig. 2, is characterized by a broad endothermic effect, which comes from residual solvents evaporation, at the temperature range from about 30 to 120°C. The endothermic peak measured as 'onset' at the temperature 135.28°C and the value of determined enthalpy: 98.20 J/g, results from the substance melting. The jagged base line, which appears after melting effect, is the result of decomposition of the compound. It is assumed, within this temperature range evaporation of adsorbed DMSO (boiling point 189°C) takes place.

In the exemplary TG curve of pemetrexed diacid crystalline Form 1 (Fig. 3, full line) at the temperature range of 30 - 230°C, a significant weight loss of 30.77 % is observed. This weight loss is consistent with the total water (0.49 %) and DMSO (30.36 %) content. The comparison of TGA, SDTA (dotted line, Single Differential Thermal Analysis) and DTG (broken line, first derivative of TGA curve) indicates, the endothermic effect on STDC curve shown at about 134°C results from the substance melting. The broad endothermic effect on DTG curve at temperature range of 100 - 230°C represents mainly evaporation of adsorbed DMSO.

To confirm the chemical structure of pemetrexed diacid crystalline Form 1 additional DSC experiment was performed, using the loop comprising heating the sample up to 140°C, cooling to 0°C and further heating up to 300°C (Fig. 4). During the second heating at temperature range of 118 - 132°C two very small endothermic effects have been observed. The lack of reappearance of the endothermic peak at the temperature 140°C during the second heating cycle indicates substance decomposition, which is the prove that crystalline form 1 is the polymorph, but not pemetrexed diacid solvate with DMSO.

HPLC analysis of the impurities profile of pemetrexed diacid crystalline form 1 of starting and heated at 140 and 150°C for 1 h samples, proved pemetrexed diacid decomposition at 140°C and further degradation at 150°C. The results are presented in Table 3 of the Example 1. Pemetrexed diacid crystalline Form 2 is characterized by X-ray powder diffraction pattern (XRPD), recorded on the diffractometer equipped with the copper anode of Koi! λ = 1,54056 A wave length at the measurement range from 7 to 30°, having the peaks at 2Θ angles about 7.39; 9.41 ; 11.17; 11.68; 19.72; 23.68; 27.73 ± 0.2°.

The crystalline Form 2 of pemetrexed diacid is characterized by X-ray powder diffraction pattern recorded on diffractometer equipped with a copper anode of Koc λ = 1,54056 A wave length, represented as relative intensities of diffraction peaks I/Io, diffraction angles 2Θ and interplanar distances d, with scanning range from 3 to 40°, scanning rate 0,5°/min and step size 0,02°. The data are collected in Table 2:

Table 2.

2Θ, Π d, [A] I Io, [%]

5.82 15.170 2

7.41 11.922 10

9.44 9.361 58

11.01 8.031 12

11.21 7.885 16

11.69 7.564 6

12.70 6.966 14

14.14 6.257 4

14.85 5.960 5

15.44 5.735 5

17.55 5.049 22

18.18 4.875 44

18.92 4.687 40

19.72 4.499 73

19.94 4.450 49

20.52 4.324 99 21.73 4.086 25

22.52 3.945 28

22.87 3.886 41

23.73 3.747 100

24.30 3.660 78

25.17 3.536 23

25.48 3.493 21

26.24 3.394 17

26.78 3.326 15

27.20 3.276 17

27.74 3.213 20

28.55 3.124 19

29.08 3.068 19

29.94 2.982 16

30.68 2.912 26

31.59 2.830 11

34.11 2.626 15

34.81 2.575 8

35.80 2.506 14

36.30 2.473 10

38.43 2.340 10

The exemplary X-ray powder diffraction pattern of pemetrexed diacid crystalline Form 2 is presented in Fig. 5.

DSC profile of pemetrexed diacid crystalline Form 2, obtained by the differential scanning calorimetry. depicted in Fig. 6, is characterized by the broad endothermic effect at the temperature range of about 30 to 120°C, which is attributed to the evaporation of the residual solvents, and the endothermic peak at 124,17°C measured as „onset" of -85,67 J/g enthalpy determined, which indicates the melting. The jagged base line shown after the melting effect represents substance decomposition. Supposedly, at this temperature range evaporation of adsorbed DMSO (boiling point 189°C) also takes place.

In TG curve (Fig. 7, full line), the significant weight loss of 29.03 % at temperature range from 30 to 230°C is observed. This weight loss is consistent with total water (0.40 %) and DMSO (27.85 %) content. The comparison of TGA, SDTA (dotted line) and DTG (broken line, first derivative of TGA curve) indicates the endothermic effect on SDTA curve at the temperature about 122°C, which corresponds to melting. The broad endothermic effect in DTG curve at temperature range of 100 - 220°C is mainly attributed to the evaporation of adsorbed DMSO.

The new pemetrexed diacid crystalline forms 1 and 2, due to their well defined chemical structure and DMSO content, which varies from 27 to 31%, are very useful substrates for the preparation of high purity pemetrexed disodium. To attain the expected result in the further synthetic steps, the content of DMSO molecules in the crystalline form should be considered, while calculating exact amount of sodium generating compound, for example sodium methanolate, in particular when molar shortage of this reagent in regard to pemetrexed diacid is used. The exact content of pemetrexed free diacid in its crystalline forms 1 or 2 obtained according to the present invention can be determined by potentiometric alkacymetric titration of carboxyl groups or titration of the primary amine groups with perchloric acid. The potentiometric titration and gas chromatography analysis (GC) are useful tools to determine the content of DMSO; both analytical methods provide consistent results of the pemetrexed diacid content determination.

The synthesis of the amorphous pemetrexed disodium is performed according to the following manner.

Preparation of the amorphous pemetrexed disodium is accomplished in the reaction of N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5- yl)ethyl]benzoyl]-L-glutamic acid with the sodium cations generating compound, under anhydrous conditions. According to the present invention, the sodium cations generating compound is used at the amount below the molar ratio in regard to carboxyl groups of reacting N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5- yl)ethyl]benzoyl]-L-glutamic acid.

Preferably, the sodium cations generating compound is used at 2% molar shortage in regard to one carboxyl group of N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H- pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid.

The sodium cations generating compound is selected from the group comprising sodium hydroxide, carbonate or alkoxide, preferably sodium alkoxide, more preferably sodium methoxide. The anhydrous reaction conditions are maintained due to the use of alcohol solvent, such as methanol, ethanol or isopropanol.

The yield of the amorphous pemetrexed disodium isolated from the post-reaction mixture can be increased by adding the anti-solvent to the reaction mixture.

Preferably, the anti-solvent is selected from the group of alcohol solvents comprising ethanol, isopropanol, n-butanol, terf-butanol; acetonitrile; acetone; ethers, including, diisopropyl ether, tert-b tyl methyl ether, dioxane, tetrahydrofurane; chloroalkanes, such as chloroform or methylene dichloride. Most preferably, alcohol solvents, especially ethanol, isopropanol or n-butanol, are used.

After the anti-solvent addition the reaction mixture is stirred at ambient temperature until the product starts precipitating. The amorphous pemetrexed disodium is isolated by the standard procedures, for example, filtrating, decanting or solvent evaporating, and then washed with the proper solvent, preferably, the alcohol.

If required, to obtain the final product of increased purity and free of the residual solvents, the amorphous pemetrexed disodium can be purified, preferably, by maceration, i.e. stirring the suspension in a small amount of the solvent. The volume-weight ratio of the solvent mixture in respect to the mass of the crude pemetrexed disodium used in maceration, ranges from 2: 1 to 10: 1, preferably, it is about 3:1 (v/w), thus it is significantly smaller in comparison with the solvents ratio used in the standard crystallization.

The crude amorphous solid of pemetrexed disodium is suspended in the alcohol, preferably, in methanol or ethanol, upon stirring. Stirring is continued at ambient temperature for 1 to 24 h, preferably for 3 h. The solid amorphous pemetrexed disodium, separated and dried, is subjected to the second maceration in the aprotic solvent, preferably selected from the group comprising the alkanes, such as pentane, heptane, hexane, or cyclohexane. Stirring the suspension is continued for 1 to 24 h, preferably for 3 h. The term 'ambient temperature' refers to the temperature within the range from

15 to 30°C, for example 20 - 25°C.

The amorphous pemetrexed disodium product obtained according to the present invention is characterized by broad background resulting from incoherent dispersion of amorphous substance, which is observed in X-ray powder diffraction pattern (XRPD) recorded on a diffractometer equipped with a copper anode of radiation wavelength Kai λ = 1.54056 A, with the scanning range from 3 to 40^°, the scanning rate 0,5°/min and the step size 0,02°, as depicted on Fig. 8.

In TGA curve of the amorphous pemetrexed disodium (Fig. 9, full line), obtained under the dynamic heating regime ranging from 30°C to 300°C at the heating rate 10°C/min, the mass loss at the temperature range of about 30°C-220°C is observed. The comparative analysis of TGA and SDTA curves indicates that this effect corresponds to the solvents evaporation. The loss of mass accounting for 7.40% is slightly higher than 6.18% water content measured by coulometric titration. This difference can be attributed to the presence of other solvents, which were used in the synthetic process. The DSC profile of the amorphous pemetrexed disodium, obtained by the differential scanning calorimetry at the dynamic heating from 25 to 300°C at the heating rate 10°C/min, depicted in Fig. 10, is characterized by two endothermic and one exothermic peak. The first broad endothermic peak, which appears at the temperature range of 30-200°C results from the evaporation of adsorbed solvents. The second endothermic peak at about 234°C is the effect of substance melting, and the exothermic peak at about 251 °C represents substance decomposition.

The present invention provides the process with the use of pemetrexed diacid crystalline forms 1 or 2, which are the polymorphic forms of pemetrexed diacid and DMSO of the well established composition that enable the preparation of the stable amorphous pemetrexed disodium, free of other crystalline forms inclusions, characterized by high pharmaceutical purity level above 99.7%, having the content of the single impurities below 0,1% and residual solvents below the level approved for active pharmaceutical ingredients.

The present invention is illustrated by the following examples.

Examples

Analytical methods Liquid chromatography (HPLC)

Purity of the compounds was determined by High Performance Liquid Chromatography with the use of apparatus equipped with a steady flow pump, thermostated columns, PDA detector and Empower software.

Measurement parameters: column Gemini CI 8, 3 μπι, 150 x 4.6 mm, phase A: K₂HP0₄ (4 g/1, pH=5,2) in concentration gradient 95% - 25%, phase B: CH₃CN in concentration gradient 5% - 75%, 0.9 ml/min, R_t = 23,9 min. X-Ray powder diffraction patterns were recorded on X-ray powder diffractometer MiniFlex type by Rigaku, using CuKa radiation of λ = 1,54056A wave length with the following parameters:

■ Scanning range 2Θ: from 3 to 40°

^■ Scanning rate Δω: 0,5°/min ^■ Step size: 0,02°

■ Temperature of measurement: ambient temperature

■ Detector: scintillating counter.

^■S Differential scanning calorimetry (DSC) measurements were performed in the furnace sample chamber DSC822^e by Mettler Toledo, under the following conditions:

^■ Melting pot: aluminum, 40 yiL capacity,

^■ Purge gas: N₂, flow rate 60 mL/min, ^■ Measurement conditions: the samples were heated under dynamic regime from 25 to 300°C at heating rate 10°C/min,

Thermogravimetric measurements (TGA) were performed in the furnace sample chamber TGA/SDTA85 l^e by Mettler Toledo, under following conditions:

^■ Melting pot: aluminum, 40 μΐ, capacity,

^■ Purge gas: N₂, flow rate 60 mL/min,

^■ Measurement conditions: the samples were heated under dynamic regime from 30 to 300°C at heating rate 10°C/min,

Water content measurements

Water content was determined by Karl Fischer volumetric titration following Ph. Eur. 2.5.12 procedures on 907 Titrando by Methrom. Gas Chromatography (GC)

Measurements of dimethyl sulfoxide (DMSO) and ethanol residues using GC analysis were performed on the gas chromatograph equipped with a flame ionization detector. GC analysis parameters for DMSO residue measurements: column DB-WAX (30 m x 0,32 mm), column temperature 100°C (10°C/min) - 220°C (1 min), injector 240° C, carrier gas - nitrogen (50 kPa), split 20 : 1, detector: 260° C, hydrogen 40 mL/min, air 400 mL/min, injection Ιμί.

GC analysis parameters for ethanol residue measurements: column DB-624 (60 m x 0,32 mm), column temperature 100°C (6 min, 40°C/min) -» 240°C (5 min), injector 240° C, carrier gas - nitrogen (100 kPa), split 5 : 1, sensitivity -5, detector 260° C, hydrogen 45 mL/min, air 450 mL/min.

Headspace conditions

Oven: 95° C, Needle: 110° C, Transfer line: 120° C, Column, Injection: 140 kPa

Thermostat: 30 min, Pressurize: 1 min, Inject: 0,05 min, Withdraw: 0,2 min Example 1

Preparation of pemetrexed diacid Form 1 from pemetrexed diethyl ester p- toluenesulfonate

a) Hydrolysis of pemetrexed diethyl ester 7-toluenesulfonate to obtain crude pemetrexed diacid

Pemetrexed diethyl ester -toluenesulfonate (922 g, 1.406 mol) was treated with 1.5 M NaOH aqueous solution (3688 mL), the mixture was stirred at ambient temperature for 2 h. Ethanol was added (4167 mL), pH of the reaction mixture was adjusted to about 3.0 adding 1.5 M HCl aqueous solution (3520 mL), the resulting mixture was heated at 70-75 °C. After cooling down the solution to ambient temperature, the solid was filtered off and washed with H₂0-EtOH (1 :1, v/v) (2 x 1845 mL). The solid was dried in an air flow drier at 40-45°C. Pemetrexed diacid was obtained in 590 g yield (HPLC 99.10%). b) Crystallization of crude pemetrexed diacid

The crude pemetrexed diacid (590 g, 1.38 mmol, HPLC 99.10%) was dissolved in DMSO (1263 mL) at 45-55°C. Ethanol (4794 mL) was added and the mixture was stirred for about 1 h. The solid was filtered off, washed with ethanol (3 x 2766 mL), air dried and dried in an air flow dryer at 40-45 °C to the constant mass (700 g, HPLC 99.48%).

Water content: 0.49%. HPLC: 99.45%. GC: DMSO-30.36%, EtOH<LOQ.

Purity of the starting and heated (140 and 150°C, 1 h) samples of Form 1 was determined by HPLC. The HPLC analysis of impurities profile indicates, pemetrexed diacid Form 1 decomposes upon heating at 140°C, and at 150°C further degradation was detected.

Example 2

Preparation of pemetrexed diacid Form 2 from pemetrexed disodium salt a) Transformation of pemetrexed disodium salt into pemetrexed diacid

Pemetrexed disodium salt (278 g, 0.590 mol) was dissolved in water (1800 mL).

Ethanol (2145 mL) was added and pH of the reaction mixture was adjusted to about 3.0 with 1.5 M HCl aqueous solution (930 mL). The solution was heated at 70-75°C. After cooling down to ambient temperature, the solid was filtered off and washed with H₂0- EtOH (1 :1, v/v) (2 x 300 mL). The solid was dried in an air flow dryer at 40-45°C. Pemetrexed diacid was obtained in 211 g yield (HPLC 99.19%). b) Crystallization of the crude pemetrexed diacid

Crystallization I

The crude pemetrexed diacid (211 g, HPLC 99.19%) was dissolved in DMSO (443 mL) at 45-55°C for 1 h. Ethanol (1683 mL) was added and stirring was continued for about lh. The solid was filtered off, washed with ethanol (3x 323 mL) and dried in an air flow dryer at 40-45°C (230 g, HPLC 99.39%).

Crystallization II

The crude pemetrexed diacid (216 g, HPLC 99.39%) was dissolved in DMSO (550 mL) at 45-55 °C for 1 h. Ethanol was added (2300 mL) and stirring was continued for about lh. The solid was filtered off, washed with ethanol (2 x 500 mL) and dried in an air flow dryer at 40-45°C (176 g, HPLC 99.52 %, pemetrexed diacid content 78.6%). Water content: 0.40%. HPLC: 99.48%. GC: DMSO-27.85%, EtOH<LOQ. Example 3 (comparative)

Preparation of the amorphous pemetrexed disodium using 10% molar excess of sodium methanolate to one -COOH group of pemetrexed diacid To the solution of MeONa (9.70 g, 0,1796 mol) in MeOH (500 niL) cooled to 0- 5°C, pemetrexed diacid (50 g, HPLC 99.75%, pemetrexed diacid content 69.79 %) was added. The resulting solution was stirred at 5-15°C for 45 min under inert gas atmosphere. Methyl tert-butyl ether (MTBE) (500 mL) was added, stirring was continued for 30 min, then the solid was filtered off and washed with MTBE-methanol (1 :1, v/v) (2 x 100 mL) and cold methanol (1 x 160 mL). The solid was dried to the constant mass in a rotary vacuum vaporator (water bath temperature 25-30°C/10-15 mbar) for 4 h. 34.7 g of pemetrexed disodium was obtained (99.48% HPLC, unidentified impurity RRT(1.03): 0.27%).

Example 4

Preparation of the amorphous pemetrexed disodium salt using 2% molar shortage of sodium methanolate to one -COOH group of pemetrexed diacid

To the solution of MeONa (18.03 g, 0.333 mol) in MeOH (1000 mL) cooled to 0-5°C, pemetrexed diacid (100 g, HPLC 99.68%, diacid content 72.81 %) was added. The resulting solution was stirred at 4 - 6°C for 1 h under inert gas atmosphere. Unconsumed pemetrexed diacid was removed by filtration and to the clear filtrate EtOH (1000 mL) was added. After 30 min of stirring at 20-25°C the solid was filtered off, washed with EtOH (1 x 200 mL) and dried in vacuum rotary vaporator for 1.5 h.

The crude amorphous solid of pemetrexed disodium (HPLC 99.61 %) was macerated in MeOH (250 mL) at ambient temperature for 3 h. The solid was filtered off, washed with cold MeOH (1 x 100 mL) and dried in vacuum rotary vaporator.

The amorphous pemetrexed disodium was macerated in cyclohexane (200 mL) at ambient temperature for 3 h. The solid was filtered off, washed with cyclohexane (75 mL) and dried to the constant mass in vacuum rotary vaporator (water bath temperature 25-40°C/10-15 mbar) for 4 h. 61.74 g of pemetrexed disodium was obtained (HPLC 99.71 %, GC: MeOH 108 ppm, EtOH 197 ppm, cyclohexane 35 ppm).

Claims

1. A process for the preparation of high purity amorphous pemetrexed disodium, in the reaction of N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin- 5-yl)ethyl]benzoyl]-L-glutamic acid with sodium cations generating compound, characterized by that the process comprises: a ) reacting N- [4-[2-(2-amino-4,7-dihydro-4-oxo-3 H-pyrrolo[2,3 -d]pyrimidin- 5-yl)ethyl]benzoyl]-L-glutamic acid with the molar shortage of sodium cations generating compound in regard to carboxyl groups of N-[4-[2-(2- amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]- L-glutamic acid under anhydrous conditions,

b) optionally, adding an anti-solvent to precipitate the product as an amorphous solid,

c) separating the crude amorphous pemetrexed disodium, d) suspending the crude amorphous pemetrexed disodium in an anhydrous alcohol solvent and stirring the suspension at ambient temperature, e) isolating and drying the amorphous solid product, f) suspending and stirring the product in an alkane type aprotic solvent to remove residual solvents, and g) isolating and drying the final product.

2. The process according to Claim 1, wherein 2% molar shortage of sodium cations generating compound in regard to one carboxyl group of N-[4-[2-(2-amino-4,7- dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid is used.

3. The process according to Claim 1, wherein sodium cations generating compound is sodium hydroxide, carbonate or alkoxide.

4. The process according to Claim 3, wherein the sodium cations source compound is sodium alkoxide.

5. The process according to Claim 4, wherein, the sodium cations source compound is sodium methanolate.

6. The process according to Claim 1, wherein in step a) the reaction is carried out in an alcohol solvent, such as methanol, anhydrous ethanol or isopropanol.

7. The process according to any one of the Claims 1 - 6, wherein in step a) the reaction is carried out in methanol, using sodium methanolate as the sodium cations generating compound.

8. The process according to Claim 1, wherein in step b) to the post-reaction mixture the anti-solvent is added, selected from the group comprising alcohols, acetonitrile, acetone, ethers and haloalkanes.

9. The process according to Claim 8, wherein the anti-solvent used in step b) is an alcohol solvent, preferably ethanol, isopropanol or n-butanol.

10. The process according to Claim 1, wherein the solvent used in step d) is methanol.

11. The process according to Claim 1, wherein the alkane type solvent used in step f) is pentane, heptane, hexane or cycloheksane.

12. The process according to Claim 1, wherein in step a) the crystalline Form 1 or Form 2 of N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5- yl)ethyl]benzoyl]-L-glutamic acid, containing from 27 to 31% of dimethyl sulfoxide, is used.

13. A crystalline Form 1 of N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3- d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, characterized by X-ray powder diffraction pattern (XRPD), recorded on the diffractometer equipped with the copper anode of Kcci λ = 1,54056 A wave length at the measurement range from 7 to 30°, having the peaks at 2Θ angles about 9.33; 13.42; 16.99; 17.73; 18.44; 19.30; 28.28; 30.98 ± 0.2°.

14. A crystalline Form 2 of N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3- d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, characterized by X-ray powder diffraction pattern (XRPD), recorded on the diffractometer equipped with the copper anode of Kcci λ = 1,54056 A wave length at the measurement range from 7 to 30°, having the peaks at 2Θ angles about 7.39; 9.41; 11.17; 11.68; 19.72; 23.68; 27.73 ± 0.2°.

15. The use of the crystalline form of N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H- pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, selected from Form 1 and Form 2, for the preparation of the amorphous pemetrexed disodium of purity above 99.7%.