Process for the purification of 2-methoxyestradiol, 4-methoxyestradiol, and process intermediates

Abstract

The present invention relates to the field of processes for preparing active pharmaceutical ingredients and, in particular, to an industrial-scale purification process of (17β)-2-methoxy-estra-1,3,5(10)-triene-3,17-diol, also known as 2-methoxyestradiol, from (17β)-4-methoxy-estra-1,3,5(10)-triene-3,17-diol, also known as 4-methoxyestradiol. The invention further relates to intermediates of this process.

Description

Title of the invention: : Process for the purification of 2-methoxyestradiol, 4-methoxyestradiol, and process intermediates FIELD OF INVENTION

[0001] The present invention relates to the field of processes for preparing active ingredients for pharmaceutical use and in particular, according to its first aspect, an industrially applicable process for purifying (17[3]-2-methoxy-estra-l,3,5(10)-triene-3,17-diol, a compound of interest for its antiproliferative characteristics, from (17[3]-4-methoxy-estra-l,3,5(10)-triene-3,17-diol; the two compounds are also known respectively as 2-methoxyestradiol and 4-methoxyestradiol, names by which they are generally referred to in the industry and which will be adopted for the remainder of the description.

[0002] According to a second aspect, the invention relates to intermediates of this process. State of the art

[0003] 2-Methoxyestradiol was described in the article "Demonstration of 2-methoxyestrone and of 2-methoxyestradiol in human pregnancy urine" by VA Fransen, Acta Endocrinologica (1959), vol. 31, pp.603-7.

[0004] The structure of 2-methoxyestradiol is as follows:

[0005] In the documentation, the compound is identified by CAS number 362-07-2.

[0006] 2-Methoxyestradiol is an interesting molecule, particularly with regard to the formation of new blood vessels required by tumor growth (angiogenesis), and induces apoptosis in certain tumor cell lines, as indicated in the article "The impact of endogenous estradiol metabolites on carcinogenesis" by TH Lippert et al., Steroids (2000), vol. 65, pp.357-69.

[0007] The documentation describes numerous methods for the synthesis of 2-methoxyestradiol; one of the most recent is mentioned in the article "Short synthesis of 2-metoxyestradiol and 2-hydroxyestradiol” by PS Kiuru et al., Steroids (2003), vol. 68, pp.373 to 375.

[0008] During this synthesis, an isomer of 2-methoxyestradiol, 4-methoxyestradiol, bearing CAS number 26788-23-8 and whose formula is shown below, is produced:

[0009] 4-Methoxyestradiol is strongly suspected of generating in vivo a carcinogenic and mutagenic metabolite, as indicated in the articles "4-Methoxyestradiol-induced oxidative injuries in human lung epithelial cells" by YH Chen et al., Toxicology and Applied Pharmacology (2007) vol. 220, pp.271 to 277 and "Estradiol 17[3 and its metabolites stimulate cell proliferation and antagonize ascorbic acid-suppressed cell proliferation in human ovarian cancer cells" by H.-H. Li, et al., Reproductive Sciences (2014) vol. 21, pp.102 to 111.

[0010] The formation of high quantities of 4-methoxyestradiol during the process described in the cited article from Steroids of 2003 is in agreement with what is mentioned in the experimental part of the article itself: even though no indication is given regarding the analytical profile of either the crude 2-methoxyestradiol or the 2-methoxyestradiol that can be obtained after chromatographic purification and crystallization, the purification yield of 64% (example 2.1.3 on page 374) is an indication of a low level of initial purity.

[0011] The synthesis process described in this article has also been reproduced several times by the authors of the present invention, by applying minimal variations to the recipes in the various preparations in order to define the optimal reaction parameters, but in all cases it has led to mixtures with a 4-methoxyestradiol content significantly higher than 5% and in some cases even higher than 10%, relative to the 2-methoxyestradiol content, whereas international guidelines in the pharmaceutical field give a maximum limit of content less than or equal to 0.15% for identified impurities and less than or equal to 0.10% for unidentified ones.

[0012] Alternative processes to that of the Steroids article of 2003 are noted, which give lower or even very low levels of 4-methoxyestradiol, but which are complicated and low-yield processes and which nevertheless give rise to significant quantities of other impurities.

[0013] Patent application WO 01 / 14405 A2 describes in great detail the chromatographic purification of 2-methoxyestradiol from a series of impurities including 4-methoxyestradiol, by the use of silica gel and a mixture of organic solvents with a predominantly chloroform base as the eluent; the preferred embodiment is shown on page 16 of the text. In particular, 2-methoxyestradiol is purified from 4-methoxyestradiol (Table 1 of Example 2 on page 18) but already from a good level of purity, by the use of silica gel in quantities equal to 171 times the mass of the mixture to be purified and with the use of 2.5 L of eluent mixture, which in the example is equivalent to a ratio between volume and eluent and mass of sample equal to 0.714 L / g;If we relate these values ​​to an industrial scale, for the purification of a 50 kg batch of 2-methoxyestradiol, 8.5 t of silica gel eluted by 35,700 L of solvent mixture would be required; if, in addition, we consider the elution times, the solvent evaporation times to recover the product, the analytical support for controlling the fractions, and the flow of the silica gel and solvent removed, it becomes clear that this purification methodology has no use for batch applications in industrial production, due to the difficulty of the chromatographic technique, the quantities of materials involved, and the execution times.

[0014] The authors investigated possible and alternative purification methods, including chromatographic methods (but less expensive than that of WO 01 / 14405 A2), using crystallization or combinations of these techniques. The experiment was carried out on 2-methoxyestradiol samples with a 4-methoxyestradiol content greater than 5%, obtained according to the method described in the cited article in Steroids from 2003.With regard to chromatographic processes, various mixtures of eluents composed of alcohols, chlorinated solvents, ketones and ethers were tested, such as various dichloromethane / methanol mixtures with a mass ratio of silica gel to product between 50:1 and 200:1, a dichloromethane / acetone mixture of 98:2 with a mass ratio of silica gel to product of 100:1, a chloroform / methanol mixture of 99:1 with a mass ratio of silica gel to product of 200:1 and diisopropyl ether-ethyl ether mixtures with a gradient from 70:30 to 0:100 and a mass ratio of silica gel to product of 200:1.

[0015] As regards crystallizations, these were tested by using various pure solvents or mixtures thereof;In particular, the crystallization of the pure solvents methanol, acetonitrile, isopropyl acetate, ethyl acetate, acetone, methyltetrahydrofuran, methyl ethyl ketone (MEK), ethanol, isopropanol, toluene, methyl tert-butyl ether (MTBE), diisopropyl ether, chloroform, dichloromethane (DCM) and acetic acid was tested, as well as among the mixtures DCM / methanol, MTBE / tetrahydrofuran (tetrahydrofuran is usually abbreviated as THF), MTBE / 1,4-dioxane, MTBE / 1-pentanol, MTBE / 2-methoxytetrahydropyranyl (tetrahydropyranyl is usually abbreviated as THP), chloroform / 1-pentanol, chloroform / 2-methoxy-THP, chloroform / 3-methyl-1-butanol, acetonitrile / THF, acetonitrile / THF / 3-methyl-1-butanol, methanol / THF, methanol / THF / 3-methyl-1-butanol, acetonitrile / THF / 1,2-dimethoxyethane, acetonitrile / THF / 2-ethoxyethanol, chloroform / THF, chloroform / methanol, dichloromethane / acetonitrile, dichloromethane / ethyl acetate and water / dimethylformamide / methanol. ;

[0016] In any case, negative results are obtained: the best result was the reduction of the 4-methoxyestradiol content to around 5% by crystallization with MTBE; however, successive crystallizations with MTBE did not lead to further improvements in the 4-methoxyestradiol content.

[0017] It has therefore not been possible, despite the extensive experimental testing campaign, to identify a chromatographic (flash or gravimetric) crystallization technique or an inherent combination that would allow the objective of a 4-methoxyestradiol content of less than 0.15% to be achieved, as required by international guidelines.

[0018] The objective of the present invention is to provide a methodology for purifying the mixture composed of 2-methoxyestradiol as the main contaminant, which is applicable in an industrial-scale process. Summary of the invention

[0019] This objective is achieved by the present invention, which relates to a process for purifying 2-methoxyestradiol from 4-methoxyestradiol and comprises the following steps: a. Reaction of a (N-3) mixture comprising 2-methoxyestradiol and 4-methoxyestradiol with:

[0020] a') a carboxylic acid selected from formic acid, acetic acid and acid propanoic acid or

[0021] a”) the derivative of a carboxylic acid selected from an ether, an anhydride, a chloride and an acyl bromide,

[0022] to obtain - in case a') an N-2' mixture comprising the corresponding monoesters in position 17 in the steroid skeleton: ................ - in case a”) an N-2” mixture comprising the diesters corresponding to positions 3 and 17 of the steroid skeleton: where X represents an O-alkyl or an O-aryl in the case of esters, OCO-alkyl in the case of anhydrides and Cl or Br in the case of acyl chlorides and bromides; b) separation for crystallization of the N-1' or N1" derivative from the N-2' or N-2" mixture: c) hydrolysis of the N-l' or Nl derivative to obtain 2-methoxyestradiol: NJ” According to its second aspect, the invention relates to the compounds: - 2-methoxy-3-hydroxyestra-l,3,5(10)-triene-17[3-yl] formate, whose formula is as follows: - 2-methoxy-3-hydroxyestra-l,3,5(10)-triene-17[3-yl] acetate, whose formula is as follows: - 2-methoxy-3-hydroxyestra-l,3,5(10)-triene-17[3-yl] propanoate, whose formula is as follows: - 2-methoxy-3-hydroxyestra-l,3,5(10)-triene-17[3-yl] dibenzoate, whose formula is as follows: - 2-methoxy-3-hydroxyestra-l,3,5(10)-triene-17[3-yl] difuroate, whose formula is as follows: Detailed description of the invention

[0023] During the process of the invention, certain mixtures of reactants or products in solvents are in the form of suspensions or solutions in the various phases of the process itself; the term "mixture" is therefore used hereafter to indicate either a suspension or a solution.

[0024] According to its first aspect, the invention relates to a process for purifying 2-methoxyestradiol from 4-methoxyestradiol, which makes it possible to reduce the content of 4-methoxyestradiol to values ​​less than 0.15% relative to the content of 2-methoxyestradiol (% HPLC surface).

[0025] The process of the invention comprises the aforementioned steps a) to c).

[0026] During step a), a mixture, N-3, is formed comprising 2-methoxyestradiol and 4-methoxyestradiol and a compound capable of esterifying one or both of the hydroxyl groups of the two diols.

[0027] This step can be carried out according to the two alternative modalities a') and a”) mentioned above.

[0028] According to modality a'), esterification is carried out with an acid chosen from formic acid, acetic acid and propanoic acid; according to this modality, the acid itself acts as a solvent.

[0029] Formic or acetic acid is preferred.

[0030] The N-3 mixture first comprises a suspension of 2-methoxyestradiol and 4-methoxyestradiol in acid; the two compounds are then solubilized, forming a solution. The esterification reaction takes place in the solution thus formed; the authors were surprised to observe that, by operating according to this method, the esterification is completely regioselective and the reaction yields exclusively the esters at position 17 of the steroid skeleton.

[0031] The acid used for the formation of the N-3 mixture can be pure or diluted with water; it is preferable to use acid with a degree of purity greater than or equal to 95%, or better yet greater than or equal to 98%.

[0032] The ratio between the volume of acid in mL and the mass in grams of the N-3 mixture is between 4 and 8, preferably between 5.5 and 6.5.

[0033] This step is carried out at a temperature between 10 and 90 °C, preferably between 20 and 80 °C.

[0034] To facilitate the esterification reaction, it is possible, but not necessary according to modality a'), to use para-toluenesulfonic acid as a catalyst.

[0035] The authors observed that after the formation of the N-3 mixture, achieved by adjusting the mass ratios between acid and 2-methoxyestradiol / 4-methoxyestradiol and within the aforementioned temperature ranges, spontaneous crystallization and selective precipitation of the 17-ester of 2-methoxyestradiol occur at this stage, forming a new suspension while the 17-ester of 4-methoxyestradiol remains in solution. The esterification reaction of 2-methoxyestradiol and 4-methoxyestradiol and the selective precipitation of the 17-ester of 2-methoxyestradiol proceed simultaneously during step a').

[0036] Precipitation for crystallization of 2-methoxyestradiol 17-ester, compound N-1', occurs from the N-2' mixture without the need for the addition of solvent, which lowers the solubility. It is also possible to induce precipitation of 2-methoxyestradiol 17-ester by adding a small amount of the same compound (crystallization nuclei or seed) obtained during a previous preparation; however, it is preferable to wait for the spontaneous crystallization of 2-methoxyestradiol 17-ester, N-1', from the reaction mixture.

[0037] If we operate according to modality a”), the esterification is carried out with a derivative of an acid chosen from an ester, an anhydride, a chloride or an acyl bromide.

[0038] The general composition of the reactive mixture of step a”) comprises 2-methoxyestradiol (containing varying percentages of 4-methoxyestradiol, N-3 mixture), an organic solvent, an organic base, 4-dimethylaminopyridine (4-DMAP) as a catalyst and the carboxylic acid derivative.

[0039] As derivatives of carboxylic acids, acyl bromides are preferably used and even better, acyl chlorides.

[0040] The acyl chloride is selected from furoyl chloride and benzoyl chloride, with a degree of purity greater than or equal to 95%, or better greater than or equal to 98%.

[0041] The organic solvent, inert under the reaction conditions, is chosen from: tetrahydrofuran, dichloromethane, chloroform, hexane, heptane, dimethylformamide and dimethylacetamide. Dichloromethane and tetrahydrofuran are preferred.

[0042] The organic base is chosen from triethylamine, trimethylamine, pyridine; triethylamine is preferably used.

[0043] Step a”) is carried out at a temperature between 10 and 50 °C, preferably between 20 and 30 °C.

[0044] The ratio between the volume of solvent in millilitres and the mass of the N-3 mixture in grams is between 10 and 25, preferably between 15 and 20.

[0045] The ratio between the volume of the solvent and that of the base is between 3 and 6.5, preferably between 4 and 5.5.

[0046] The volume of the solvent is at least 10 times that of the acyl chloride used.

[0047] The ratio between the mass of the N-3 mixture and that of the catalyst (4-DMAP) is between 20 and 30, preferably between 23 and 27.

[0048] The reactive mixture of step a”) is heated under reflux for a time interval of between 12 and 36 hours, preferably between 16 and 30 hours during which, unlike what occurs in the regioselective reaction of step a'), the esterification reaction occurs on the two hydroxyl groups at positions 3 and 17 of the steroid skeleton.

[0049] Step b) is carried out in different ways depending on whether one starts from the N-2' or N-2 mixture”.

[0050] In the first case, in step b), the 17-ester of 2-methoxyestradiol (N-1' derivative) is separated from the N-2' mixture. The recovery of the N-1' derivative can be carried out by known methods, for example by centrifugation or preferably by simple filtration.

[0051] In the second case, in step b), the separation of the 3,17-diester Nl” from the N-2’’ mixture occurs by crystallization from methyl tert-butyl ether (MTBE) after preparation of the reaction mixture itself.

[0052] It is also possible to induce the precipitation of 3,17-diester of 2-methoxyestradiol by adding a small amount of the same compound (crystallization seeds) obtained during a previous preparation; spontaneous crystallization of 3,17-diester of 2-methoxyestradiol, Nl”, from MTBE is preferably expected.

[0053] Finally, in step c), the intermediate N-l' or Nl” is hydrolyzed to give 2-methoxyestradiol.

[0054] Even in the case of step c), the operational modalities vary slightly in the case where the hydrolysis takes place on the intermediate N-l' or Nl”.

[0055] The hydrolysis of 17-2-methoxyestradiol (N-l') ester is carried out in an alcoholic solvent selected from methanol, ethanol, and isopropanol, pure or mixed together, anhydrous or in the presence of water. Pure methanol with a water content of less than 5% by volume is preferred, and ideally, pure anhydrous methanol is used.

[0056] The base used for the hydrolysis reaction is chosen from lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate and cesium carbonate; sodium hydroxide is preferred.

[0057] The reaction temperature is between 15 and 45 °C, preferably between 20 and 40 °C.

[0058] The amount of base used is such that the pH of the solution is between 8 and 14, preferably between 11 and 13.

[0059] The hydrolysis of 3,17-2-methoxyestradiol (Nl”) diester is carried out in a solvent selected from tetrahydrofuran, methanol, ethanol, and isopropanol, pure or mixed together, anhydrous or in the presence of water. Tetrahydrofuran and methanol, pure or mixed together, are preferred.

[0060] The base used for the hydrolysis reaction is chosen from lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate and cesium carbonate; sodium hydroxide is preferred.

[0061] The reaction temperature is between 20 °C and the reflux temperature of the reaction mixture.

[0062] The amount of base used is such that the pH of the solution is between 8 and 14, preferably between 11 and 13.

[0063] According to its second aspect, the invention relates to the following intermediate compounds of the process: - 2-methoxy-3-hydroxyestra-l,3,5(10)-triene-17[3-yl] formate, whose formula is as follows: - 2-methoxy-3-hydroxyestra-l,3,5(10)-triene-17[3-yl] acetate, whose formula is as follows: - 2-methoxy-3-hydroxyestra-l,3,5(10)-triene-17[3-yl] propionate, whose formula is as follows: - 2-methoxy-3-hydroxyestra-l,3,5(10)-triene-17[3-yl] dibenzoate, whose formula is as follows: - 2-methoxy-3-hydroxyestra-l,3,5(10)-triene-17[3-yl] difuroate, whose formula is as follows:

[0064] The invention will then be illustrated by the following examples.

[0065] instruments, methods and experimental conditions

[0066] NMR

[0067] JEOL 400 YH (400 MHz) NMR spectrometer; JEOL Delta v5.1.1 software;

[0068] Spectra recorded in deuterium solvents such as: chloroform-d, D 99.8%, containing 0.1% (v / v) tetramethylsilane (TMS) as an internal standard; and chloroform-d, "100%", D 99.96%, containing 0.03% (v / v) of TMS and DMSO-d6.

[0069] SM

[0070] WATERS SYNAPT G2-Si QTof Spectrometer

[0071] Negative ionization

[0072] Ionization takes place by ESI (electron spray ionization).

[0073] HPLC

[0074] HPLC Agilent model 1260 Infinity II

[0075] UV-DAD Detector

[0076] Mobile phases: water / acetonitrile / methanol 55:40:5 (v / v)

[0077] Chromatography conditions:

[0078] Column: Supelco Ascetis Express C18, 150 x 4.6 mm, 2.7 pm

[0079] Flow rate: 1.0 mL / minute

[0080] Detector: UV at 204 and 208 nm

[0081] Injection volume: 10 pL

[0082] Temperature: 25 °C.

[0083] UPLC

[0084] Chromatographic system: Waters Acquity UPLC;

[0085] Detector: Acquity UPLC PDA and X detector (X = 220 nm)

[0086] Column: Acquity UPLC BEH C18 1.7 pm (2.1 x 50 mm), T = 35°C

[0087] Solvent A: acetonitrile and 0.01% formic acid

[0088] Solvent B: water and 0.01% formic acid

[0089] Gradient: Time (min) Flow rate (mL / min) %A %B 1 0.0 0.5 30 70 2 0.5 0.5 30 70 3 4.0 0.5 90 10 4 4.1 0.5 30 70 5 5.5 0.5 30 70

[0090] Chromatographic purity (Ph Eur):

[0091] The chromatograms obtained by the "HPLC Titration" test, carried out by liquid chromatography under the following conditions defined in Ph Eur 2.2.29:

[0092] Solvent: acetonitrile / Water 70:30 v / v

[0093] Reference solution A: Dissolve 20 mg of 2-methoxyestradiol Working STD in 14 mL of acetonitrile. Dilute to 20 mL with water. Prepare this solution in duplicate.

[0094] Sample solution: Dissolve 20 mg of product in 14 mL of acetonitrile, dilute to 20 mL with water. Prepare this solution in duplicate.

[0095] Reference solution B: Take 1 mL of the sample solution and dilute to 100 mL with solvent. From this solution, dilute 1 to 10 mL with solvent.

[0096] Identify the impurities according to their relative retention times: Name RRT RT 17[3-estradiol 0.80 4.4 4-methoxy-estradiol 0.87 4.8 2-methoxy-estradiol 1 5.5

[0097] Calculate the impurity content at 204 nm, according to the formula: Cu% = (ru / rs) x 0.1 where Cu% represents the content of each impurity ru represents the response zone of each impurity rs represents the response zone of 2-methoxy-estradiol in reference solution B.

[0098] For the calculation of the total impurities, all impurities less than 0.05% will be eliminated.

[0099] TLC MERCK: TLC silica gel 60 F254 Aluminum foil 20 x 20 cm, code 1.0554.0001.

[0100] TLC Revealers Cerium phosphomolybdate: Dissolve 25 g of phosphomolybdic acid and 10 g of cerium(IV) sulfate in 600 mL of H₂O. Add 60 mL of 98% H₂SO₄ and bring the solution to 1 L with H₂O. Impregnate the plate with the solution and then heat it until the products are collected. REMARKS

[0101] Water used in experimental descriptions is to be understood as pure water, unless otherwise stated.

[0102] Organic solvents used in experimental descriptions are to be understood as "technical" use, unless otherwise stated.

[0103] Reagents used in the experimental descriptions are to be understood to be of commercial grade, unless otherwise stated. EXAMPLE 1

[0104] This example corresponds to the process of the invention carried out using a carboxylic acid in the esterification step.

[0105] Step a')

[0106] 2-Methoxy-estradiol (33.8 g, previously crystallized with MTBE) with a 4-Methoxy-estradiol content of 4.7% is suspended in a reactor with 202.8 mL of formic acid, under nitrogen, at 25 ± 5 °C.

[0107] The suspension thus formed is stirred. After 35 minutes, the complete dissolution of the initial solid material is observed. After one hour and 10 minutes, the formation of a new solid material in suspension is observed. After 2 hours, the state of the reaction is checked by UPLC: 2-methoxy-estradiol < 1%.

[0108] Step b)

[0109] The suspension obtained is filtered, the solid material obtained (2-methoxy-estradiol formate) is washed on a Buchner flask with formic acid (34 mL) and summarily dried, also on a Buchner flask (wet solid material).

[0110] A sample of solid material thus obtained, after drying under reduced pressure, is analyzed by 'H-NMR and by mass spectrometry.

[0111] 'H-NMR (400 MHz, DMSO-d6): 8.62 (1H, s, Ar-OH); 8.26 (1H, s, CHO); 6.76 (1H, s, Ar-H), 6.44 (1H, s, Ar-H); 4.68 (1H, t, H-17, J=8.5 Hz); 3.71 (3H, s, OCH3); 2.73-2.57 (2H, m); 2.34-2.23 (1H, m); 2.20-2.08 (2H, m); 1.83-1.74 (2H, m); 1.73-1.63 (1H, m); 1.59-1.48 (1H, m); 1.44-1.14 (6H, m); 0.80 (3H, s, H-18).

[0112] Mass (ESI): m / z = M+-l = 329.

[0113] Step c)

[0114] The solid material obtained is loaded into a reactor with 6.7 g of sodium hydroxide pellets.

[0115] Methanol (369 mL) is added and, after stirring, the initial solid material is completely dissolved after 15 minutes. The internal temperature rises from 25 to 32 °C, and the measured pH is >12.

[0116] After an additional 30 minutes of stirring, complete dissolution of the solid material is observed and the state of the reaction is checked by UPLC: complete conversion, 2-methoxyestradiol formate < 1%.

[0117] An aqueous solution of ammonium chloride (26 g in 1300 mL of H2O) is added to the reaction mixture.

[0118] The hydroalcoholic mixture obtained is then stirred at 25 ± 5 °C for 30 minutes and then from 0 to 5 °C for 1 h.

[0119] The formation of a precipitate is observed which is filtered on a Buchner filter and washed with water on the filter.

[0120] The solid material obtained is then dried under study at 55 °C for 16 h under reduced pressure.

[0121] 23.9 g of 2-methoxy-estradiol (white solid with yellowish reflections) is obtained with a 4-methoxy-estradiol content of less than 0.05%. EXAMPLE 2

[0122] This example corresponds to the process of the invention carried out using a second carboxylic acid in the esterification step. Step a')

[0123] 2-Methoxy-estradiol (8 g) containing 3.92% of 4-methoxy-estradiol (HPLC) is suspended in 48 mL of glacial acetic acid in the presence of 453 mg of PTSA in a nitrogen-filled flask at 25 ± 5 °C.

[0124] The suspension is stirred and heated at 70 °C for 16 hours: after 2 hours, the solid matter is completely dissolved. After 16 hours, the reaction is monitored by UPLC: 2-methoxy-estradiol < 1%.

[0125] The reaction mixture is cooled to 25 °C; reprecipitation is observed and the mixture is kept under stirring for 1 h. Step b)

[0126] The solid material is filtered and washed with acetic acid (8 mL) and dried under vacuum at 45 °C to obtain 5.85 g of powder. Step c)

[0127] 5.35 g of the solid material obtained in step b) and 931 mg of sodium hydroxide are loaded into a round-bottom flask.

[0128] Methanol (53 mL) is added and the mixture is stirred for 3 h at 20 and 25 °C. Hydrolysis is monitored by UPLC: complete conversion.

[0129] An aqueous solution of ammonium chloride is prepared (2.6 g in 54 mL of H2O).

[0130] The aqueous solution is added to the reaction mixture and the resulting hydroalcoholic mixture is stirred at 25 ± 5 °C for 30 minutes and then between 0 and 5 °C for 1 h.

[0131] The precipitate is filtered and washed with water (65 mL).

[0132] The solid material is dried in an oven at 50 °C for 16 h, yielding 4.6 g of 2- methoxy-estradiol where no presence of 4-methoxy-estradiol is detected. EXAMPLE 3

[0133] This example corresponds to the process of the invention carried out by using a derivative of a carboxylic acid in the esterification step. Step a”)

[0134] In a nitrogen-filled flask, maintaining a temperature of 25 ± 5 °C, 8 g of 2-methoxy-estradiol containing 3.92% (HPLC) of 4-methoxyestradiol, 29.5 mL of triethylamine, and 0.32 g of 4-dimethylaminopyridine (DMAP) were mixed in a nitrogen-filled flask. Complete dissolution of the solids was not observed.

[0135] The mixture is cooled to 0 °C and 13.5 mL of benzoyl chloride is added dropwise. The temperature reaches 13 °C and a precipitate is observed to form.

[0136] The mixture is then heated under reflux and stirred for 16 h. The reaction is then monitored by UPLC: 2-methoxy-estradiol < 1%. Step b)

[0137] The mixture obtained in step a”) is cooled to 20 °C and poured over 120 mL of 1 M hydrochloric acid, with ice cooling to maintain a temperature below 20 °C. The mixture is then stirred for 20 minutes. The phases are separated, and the aqueous phase is extracted with dichloromethane (30 mL). The organic phases are washed with an aqueous solution of NaHCO3 and then with water (pH > 7).

[0138] The organic phase is vaporized under vacuum to give an oil. MTBE (240 mL) is added and the system is subjected to evaporation until 60 mL of a suspension containing a solid material is obtained. This suspension is cooled and filtered by washing with MTBE at a temperature between 0 and 5 °C. The solid material is dried at 50 °C under vacuum to obtain 9.7 g of white powder. Step c)

[0139] 300 mg of the solid material obtained in step b) and 70 mg of sodium hydroxide are suspended in the presence of methanol (6 mL) and THF (1.5 mL) and heated under reflux for 6 h (to obtain a clear solution); the reaction is then followed by UPLC: 2-methoxy-estradiol 3,17-dibenzoate < 1%.

[0140] The reaction mixture is cooled to room temperature and poured into an aqueous ammonium chloride solution (5 g in 20 mL) and stirred for 20 minutes at room temperature and then for 1 h at 0 °C, forming a solid that is filtered and washed with water. The solid is dried at 45 °C under vacuum to obtain 120 mg of 2-methoxyestradiol, the analysis of which by HPLC reveals no 4-methoxyestradiol. EXAMPLE 4

[0141] This example corresponds to the process of the invention carried out by using a second derivative of a carboxylic acid in the esterification step. Step a”)

[0142] In a nitrogen-filled flask maintained at 25 ± 5 °C, 8 g of 2-methoxy-estradiol containing 3.92% (HPLC) of 4-methoxy-estradiol, 29.5 mL of triethylamine and 0.32 g of 4-dimethylaminopyridine (DMAP) are dissolved in 120 mL of THF;

[0143] The mixture is cooled to 0 °C and 11.4 mL of furoyl chloride is added dropwise. The temperature rises to 15 °C and a precipitate is observed to form.

[0144] The mixture is then heated under reflux and stirred for 24 h. The reaction is then monitored by UPLC: 2-methoxy-estradiol < 1%. Step b)

[0145] The mixture obtained in step a”) is cooled to 20 °C and poured onto 120 mL of ice-cooled 1 M hydrochloric acid, maintaining a temperature below 20 °C. 80 mL of isopropyl acetate is then added, and the mixture is stirred for 20 minutes. The phases are separated, and the aqueous phase is extracted with isopropyl acetate (2 x 50 mL). The organic phases are recombined and washed with an aqueous solution of NaHCO3 followed by water (pH > 7).

[0146] The organic phase is vaporized under vacuum, yielding a yellow solid (18.6 g). The product is suspended in MTBE (54 mL), stirred at 20 °C for 20 minutes, and then cooled to 0 °C. After one hour, the solid is filtered and washed with MTBE at a temperature between 0 and 5 °C. The solid is dried at 50 °C under vacuum, yielding 10.27 g of white powder. Step c)

[0147] 200 mg of the solid material obtained in step b) and 50 mg of sodium hydroxide are suspended with methanol (3 mL) and with THF (1 mL) and stirred at 25 °C for 1.5 h to give a clear solution; the reaction is then followed by UPLC: 2-methoxy-estradiol 3,17-difuroyl < 1 %.

[0148] The reaction mixture is poured into an aqueous solution of ammonium chloride (3.5 g in 10 mL) and stirred for 20 minutes at room temperature and then for 1 h at 0 °C, forming a solid which is filtered and then washed with water. The solid is dried at 50 °C under vacuum to give 90 mg of 2-methoxyestradiol in which no 4-methoxyestradiol is detectable (HPLC).

Claims

1. Demands Process for the purification of 2-methoxyestradiol from 4-methoxyestradiol, comprising the following steps: a) reaction of a mixture (N-3) comprising 2-methoxyestradiol and 4-methoxyestradiol with: a') a carboxylic acid selected from formic acid, acetic acid and propionic acid or a”) the derivative of a carboxylic acid chosen from an ester, an anhydride, a chloride and an acyl bromide, which gives: - in case a'), an N-2' mixture comprising the corresponding monoesters at position 17 of the steroid skeleton: - in case a”), an N-2” mixture comprising the diesters corresponding to positions 3 and 17 of the steroid skeleton: where X represents O-alkyl or O-aryl in the case of esters, OCO-alkyl in the case of anhydrides and Cl or Br in the case of acyl chlorides and bromides; b) separation for crystallization of the derivative N-1' or N1" respectively from the mixture N-2' or N-2'': N-2' c) hydrolysis of the N-l' or Nl derivative to obtain 2-methoxyestradiol:

2.

3.

4. A method according to claim 1, wherein in step a'), the acid is chosen between formic acid and acetic acid. A process according to any one of claims 1 or 2, wherein in step a'), the acid used acts as a solvent. A method according to any one of the preceding claims, wherein in step a'), the ratio between the volume in mL of acid and the mass in grams of the N-3 mixture is between 4 and 8 and where said step a') is carried out at a temperature between 10 and 90 °C.

5. A process according to any one of the preceding claims, wherein in step a'), para-toluenesulfonic acid is used as a catalyst.

6. A process according to claim 1, wherein step a”) is carried out with a reactive mixture (N-3) comprising 2-methoxyestradiol and 4-methoxyestradiol, an organic solvent, an organic base, 4-dimethylaminopyridine and said derivative of a carboxylic acid.

7. A method according to claim 6, wherein said derivative of a carboxylic acid is selected from acyl chlorides and bromides.

8. A method according to claim 7, wherein said acyl chloride is selected from benzoyl chloride and furoyl chloride.

9. A process according to claim 8, wherein step a”) is carried out at a temperature between 10 and 50 °C, where the ratio of the volume of solvent in mL to the mass of the N-3 mixture in grams is between 10 and 25, where the ratio of the volume of solvent to that of the organic base is between 3 and 6.5, where the volume of solvent is at least 10 times that of the acyl chloride used and where the ratio of the mass of the N-3 mixture to that of the 4-dimethylaminopyridine catalyst is between 20 and 30.

10. An intermediate of the process of claim 1, selected from: - 2-methoxy-3-hydroxyestra-1,3,5(10)-triene-17[3-yl] formate, having the following formula: O H 5 | | K | A HO - 2-methoxy-3-hydroxyestra-1,3,5(10)-triene-17[3-yl] difuroate, having the following formula: