Method for purifying 2-methoxyestradiol from 4-methoxyestradiol and intermediate thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of processes for preparing pharmaceutically active ingredients and, in particular, in its first aspect, to an industrially applicable process for purifying (17β)-2-methoxy-estrast-1,3,5(10)-triene-3,17-diol from (17β)-4-methoxy-estrast-1,3,5(10)-triene-3,17-diol, the target compound having known antiproliferative properties; these two compounds are also known by the names 2-methoxyestradiol and 4-methoxyestradiol, respectively, as they are commonly referred to in the art and which will be adopted in the following description.

[0002] In its second aspect, the invention relates to intermediates of this process. Background Art

[0003] 2-Methoxyestradiol has been described in the article “Demonstration of 2-methoxyestrone and of 2-methoxyestradiol in human pregnancy urine”, VA Frandsen, Acta Endocrinologica (1959), vol. 31, pages 603-7.

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

[0005]

[0006] In the literature, the CAS number of this compound is 362-07-2.

[0007] 2-Methoxyestradiol is a particular target molecule because it blocks the formation of new blood vessels (angiogenesis) required for tumor growth and induces apoptosis in some tumor cell lines, as reported in the article “The impact of endo genousestradiol metabolites on carcino genesis”, TH Lippert et al., Steroids (2000), vol. 65, pages 357-69.

[0008] Many methods for the synthesis of 2-methoxyestradiol are described in the literature; the most recent one is reported in the article “Short synthesis of 2-methoxyestradiol and 2-hydroxyestradiol”, PS Skiuru et al., Steroids (2003), vol. 68, pages 373-375.

[0009] In this synthesis, an isomer of 2-methoxyestradiol, 4-methoxyestradiol, was produced, which has a CAS number of 26788-23-8 and a molecular formula reported as follows:

[0010]

[0011] 4-Methoxyestradiol is strongly suspected of producing carcinogenic and mutagenic metabolites in vivo, as reported in the articles “4-Methoxyestradiol-induced oxidative injuries in human lung epithelial cells”, YH Chen et al., Toxicology and Applied Pharmacology (2007) vol. 220, pages 271-277 and “Estradiol 17β and its metabolites stimulate cell proliferation and anta gonize ascorbic acid-suppressed cell proliferation in human ovarian cancer cells”, H.-H. Li, et al., Reproductive Sciences (2014) vol. 21, pages 102-111.

[0012] In the process described in the mentioned 2003 Steroids article, the formation of significant amounts of 4-methoxyestradiol is consistent with that reported in the experimental section of the article itself: Although no indication is given about the analytical profile of the crude 2-methoxyestradiol or of the 2-methoxyestradiol obtainable after chromatographic purification and crystallization, the 64% purification yield (Example 2.1.3 on page 374) suggests that the initial purity level was low.

[0013] The inventors have also repeated the synthesis method described in this article several times, making minimal changes to the recipe in various preparations in order to determine the optimal reaction parameters, but in all cases resulted in a mixture with a 4-methoxyestradiol content far higher than 5%, in some cases even higher than 10%, relative to the content of 2-methoxyestradiol, while international guidelines in the pharmaceutical field set as maximum thresholds a content of less than or equal to 0.15% for identified impurities and less than or equal to 0.10% for unidentified impurities.

[0014] Alternative processes to the Steroids article of 2003 are known which lead to lower or even very low 4-methoxyestradiol contents, but they are complex and low-yielding processes and still produce large amounts of other impurities.

[0015] Patent application WO 01 / 14405 A2 describes in great detail the chromatographic purification of 2-methoxyestradiol from a range of impurities including 4-methoxyestradiol using silica gel and an organic solvent mixture based mainly on chloroform as eluent; see preferred embodiments on page 16 of the text. In particular, 2-methoxyestradiol was purified from 4-methoxyestradiol (Table 1 on page 18 and Example 2), but starting from an already good level of purity, an amount of silica gel equal to 171 times the weight of the mixture to be purified was used, and 2.5 L of an eluent mixture was used, which in the example corresponds to a ratio of eluent volume to sample weight equal to 0.714 L / g; reporting these values ​​on an industrial scale, 8.5 tons of silica gel would have to be eluted with 35,700 L of a solvent mixture to purify a 50 kg batch of 2-methoxyestradiol; if, in addition, one takes into account the elution time, the solvent evaporation time for the recovery of the product, the analytical support for the fraction control and the disposal of the spent silica gel and solvent, it is obvious that this purification method is not useful for application to industrial production batches due to the tediousness of the chromatographic technique, the amount of material to be used and the execution time.

[0016] The inventors investigated possible alternative purification methods, chromatography (but without the tedium of WO 01 / 14405 A2), crystallization or a combination of these techniques. The experiments were carried out on 2-methoxyestradiol samples with a 4-methoxyestradiol content of more than 5%, obtained by the method of the mentioned Steroids article of 2003. As for the chromatography methods, various eluent mixtures consisting of alcohols, chlorinated solvents, ketones and ethers have been tried, such as various dichloromethane / methanol mixtures in a weight ratio of silica gel to product of 50:1 to 200:1, dichloromethane / acetone mixtures 98:2 in a weight ratio of silica gel to product of 100:1, chloroform / methanol mixtures 99:1 in a weight ratio of silica gel to product of 200:1, and diisopropyl ether-ethyl ether mixtures in a gradient from 70:30 to 0:100 in a weight ratio of silica gel to product of 200:1.

[0017] For crystallization, various solvents were attempted to be used in pure form or in mixtures with each other; specifically, crystallization was attempted from the following 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, and from the following mixtures: DCM / methanol, MTBE / tetrahydrofuran (tetrahydrofuran is usually abbreviated as THF), MTBE / 1,4-dioxane, MTBE / 1-pentanol, MTBE / 2-methoxy- Tetrahydropyranyl (tetrahydropyranyl is often 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.

[0018] Negative results were obtained in all cases: the best result was a reduction of the 4-methoxyestradiol content to about 5% by crystallization from MTBE; however, subsequent crystallization from MTBE did not lead to a further improvement in the 4-methoxyestradiol content.

[0019] Therefore, despite an extensive experimental testing campaign, it has not been possible to identify techniques of chromatography type (rapid and gravimetric), crystallization or a combination thereof that would allow reaching the target 4-methoxyestradiol content of less than 0.15% required by international guidelines.

[0020] The object of the present invention is to provide a process for purifying a mixture consisting of 2-methoxyestradiol and 4-methoxyestradiol as the main contaminant, which process has practical applicability in an industrial-scale process. Summary of the invention

[0021] This object is achieved by the present invention, which relates to a method for purifying 2-methoxyestradiol from 4-methoxyestradiol, comprising the following steps:

[0022] a) reacting a mixture comprising 2-methoxyestradiol and 4-methoxyestradiol (N-3) with:

[0023] a') a carboxylic acid selected from the group consisting of formic acid, acetic acid and propionic acid, or

[0024] a″) a carboxylic acid derivative selected from the group consisting of esters, anhydrides, acid chlorides and acid bromides,

[0025] get:

[0026] - In case a'), a mixture N-2' comprising the corresponding monoester at position 17 of the steroid backbone:

[0027]

[0028] - In case a"), the mixture N-2" comprises the corresponding diesters in positions 3 and 17 of the steroid backbone:

[0029]

[0030] wherein X is O-alkyl or O-aryl in the case of esters, OCO-alkyl in the case of anhydrides, Cl or Br in the case of acid chlorides and bromides;

[0031] b) isolating the derivative N-1' or the derivative N-1" from the mixture N-2' or the mixture N-2", respectively, by crystallization:

[0032]

[0033] c) hydrolyzing the derivative N-1' or the derivative N-1", to produce 2-methoxyestradiol:

[0034]

[0035] In its second aspect, the present invention relates to the compound 2-methoxy-3-hydroxyestrast-1,3,5(10)-triene-17β-carboxylate, which has the following formula:

[0036]

[0037] and 2-methoxy-3-hydroxyestra-1,3,5(10)-triene-3,17β-yl difuroate, having the following formula:

[0038] DETAILED DESCRIPTION

[0039] In the process of the invention, the mixture of some reagents or products in the solvent is in the form of a suspension or solution in the various steps of the process itself; the term "mixture" is used hereinafter to denote both suspensions and solutions.

[0040] In its first aspect, the present invention relates to a process for purifying 2-methoxyestradiol from 4-methoxyestradiol, which process allows reducing the 4-methoxyestradiol content to values ​​below 0.15% (HPLC area %) relative to the 2-methoxyestradiol content.

[0041] The method of the present invention comprises the above steps a) to c).

[0042] In step a), a mixture N-3 is formed, which comprises 2-methoxyestradiol and 4-methoxyestradiol and a compound capable of esterifying one or both hydroxyl groups of these two diols.

[0043] This step can be performed according to the two optional modes a') and a") mentioned above.

[0044] According to mode a'), the esterification is carried out with an acid selected from formic acid, acetic acid and propionic acid; in this mode, the acid itself serves as solvent.

[0045] Preference is given to using formic acid or acetic acid.

[0046] Initially, mixture N-3 consists of a suspension of 2-methoxyestradiol and 4-methoxyestradiol in an acid; these two compounds are then dissolved to form a solution. In the solution thus formed, an esterification reaction takes place; the inventors surprisingly observed that, by operating according to this mode, the esterification is completely regioselective and, as a result of the reaction, only the ester in position 17 of the steroid skeleton is obtained.

[0047] The acid used to form mixture N-3 can be pure or diluted with water; preferably, an acid having a purity higher than or equal to 95%, more preferably higher than or equal to 98% is used.

[0048] The ratio of milliliters of acid to grams of N-3 mixture is from 4 to 8, preferably from 5.5 to 6.5.

[0049] This step is performed at a temperature of 10°C to 90°C, preferably 20°C to 80°C.

[0050] In order to promote the esterification reaction, in mode a'), p-toluenesulfonic acid may be used as a catalyst, but it is not necessary.

[0051] The inventors have observed that by adopting this weight ratio between acid and 2-methoxyestradiol / 4-methoxyestradiol and forming the N-3 mixture within the above temperature range, in this step, the 17-ester of 2-methoxyestradiol undergoes spontaneous crystallization and selective precipitation to form 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 are carried out simultaneously in step a').

[0052] The 17-ester of 2-methoxyestradiol (i.e., compound N-1') is precipitated from the mixture N-2' by crystallization without the addition of any solvent having the function of reducing its solubility. It is also possible to induce the precipitation of the 17-ester of 2-methoxyestradiol by adding a small amount of the same compound obtained in a previous preparation (crystallization germs or seeds); however, it is preferred to wait for the 17-ester of 2-methoxyestradiol (i.e., N-1') to spontaneously crystallize from the reaction mixture.

[0053] By operating according to mode a"), the esterification is carried out with an acid derivative chosen from esters, anhydrides, acid chlorides or acid bromides.

[0054] The general composition of the reagent mixture of step a") includes 2-methoxyestradiol (containing a variable percentage of 4-methoxyestradiol, mixture N-3), an organic solvent, an organic base, 4-dimethylaminopyridine (4-DMAP) as a catalyst and a carboxylic acid derivative.

[0055] Acid bromides and even more preferably acid chlorides are preferably used as carboxylic acid derivatives.

[0056] The acyl chloride is selected from furoyl chloride and benzoyl chloride, and has a purity higher than or equal to 95%, more preferably higher than or equal to 98%.

[0057] The organic solvent inert to the reaction conditions is selected from the group consisting of tetrahydrofuran, dichloromethane, chloroform, hexane, heptane, dimethylformamide, dimethylacetamide. Dichloromethane and tetrahydrofuran are preferably used.

[0058] The organic base is selected from triethylamine, trimethylamine, and pyridine; preferably triethylamine is used.

[0059] Step a") is carried out at a temperature of 10 to 50°C, preferably 20 to 30°C.

[0060] The ratio of the volume of solvent in milliliters to the weight of the N-3 mixture in grams is 10 to 25, preferably 15 to 20.

[0061] The ratio of the volume of the solvent to the volume of the base is 3 to 6.5, preferably 4 to 5.5.

[0062] The volume of solvent is at least 10 times the volume of the acid chloride used.

[0063] The ratio of the weight of the N-3 mixture to the weight of the catalyst (4-DMAP) is 20 to 30, preferably 23 to 27.

[0064] The reagent mixture of step a") is heated and refluxed for 12-36 hours, preferably 16-30 hours, during which, unlike the regioselective reaction of step a'), esterification occurs at the hydroxyl groups at positions 3 and 17 of the steroid skeleton.

[0065] Step b) is carried out according to different modes, depending on whether it starts from mixture N-2' or N-2".

[0066] In the first case, in step b), the 17-ester of 2-methoxyestradiol (derivative N-1') is separated from the mixture N-2'. The recovery of the derivative N-1' can be carried out by known methods, for example by centrifugation or preferably by simple filtration.

[0067] In the second case, in step b), the separation of the 3,17-diester N-1″ from the mixture N-2″ is carried out by crystallization from methyl tert-butyl ether (MTBE) after work-up of the reaction mixture itself.

[0068] The precipitation of the 3,17-diester of 2-methoxyestradiol can also be induced by adding a small amount of the same compound obtained in a previous preparation (crystallization germs or seeds); however, it is preferred to wait for the 3,17-diester of 2-methoxyestradiol (N-1") to crystallize spontaneously from MTBE.

[0069] Finally, in step c), the intermediate N-1' or N-1" is hydrolyzed to give 2-methoxyestradiol.

[0070] Even in the case of step c), in the case where the hydrolysis is carried out on intermediate N-1′ or intermediate N-1″, the mode of operation is slightly different.

[0071] The hydrolysis of 17-ester of 2-methoxyestradiol (N-1') is carried out in an alcohol solvent selected from methanol, ethanol and isopropanol, which is pure or mixed with each other, anhydrous or in the presence of water. Preferably, pure methanol with a water content of less than 5% by volume is used, and more preferably pure anhydrous methanol is used.

[0072] The base used for the hydrolysis reaction is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate and cesium carbonate; preferably sodium hydroxide is used.

[0073] The reaction temperature is 15 to 45°C, preferably 20 to 40°C.

[0074] The amount of base used is such that the pH of the solution is between 8 and 14, preferably between 11 and 13.

[0075] The hydrolysis of the 3,17-diester of 2-methoxyestradiol (N-1") is carried out in a solvent selected from tetrahydrofuran, methanol, ethanol and isopropanol, either pure or mixed with one another, anhydrous or in the presence of water. Preferably, tetrahydrofuran and methanol are used, either pure or mixed with one another.

[0076] The base used for the hydrolysis reaction is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate and cesium carbonate; preferably sodium hydroxide is used.

[0077] The reaction temperature is between 20°C and the reflux temperature of the reaction mixture.

[0078] The amount of base used is such that the pH of the solution is between 8 and 14, preferably between 11 and 13.

[0079] In its second aspect, the present invention relates to the following intermediate compounds of this process:

[0080] -2-methoxy-3-hydroxyestrast-1,3,5(10)-triene-17β-carboxylate, which has the following formula:

[0081]

[0082] -2-methoxy-3-hydroxyestra-1,3,5(10)-triene-3,17β-difurancarboxylate, which has the following formula:

[0083]

[0084] The present invention will be further illustrated by the following examples.

[0085] Experimental apparatus, methods and conditions

[0086] NMR

[0087] NMR spectrometer JEOL 400YH (400 MHz); JEOL Delta software v 5.1.1;

[0088] In deuterated solvents (e.g., chloroform-d, D 99.8%, containing 0.1% (v / v) tetramethylsilane (TMS) as internal standard; and chloroform-d, “100%”, D 99.96%, containing 0.03% (v / v) TMS and DMSO-d 6 ) to record the spectra.

[0089] MS

[0090] WATERS SYNAPT G2-Si QTof Spectrometer

[0091] Negative ionization

[0092] Ionization is done in ESI (electron spray ionization)

[0093] HPLC

[0094] Agilent HPLC 1260 Infinity II

[0095] UV-DAD Detector

[0096] Mobile phase: water / acetonitrile / methanol 55:40:5 (v / v)

[0097] Chromatographic conditions:

[0098] Column: Supelco Ascetis Express C18, 150 x 4.6 mm, 2.7 μm

[0099] Flow rate: 1.0mL / min

[0100] Detector: UV at 204nm and 280nm

[0101] Injection volume: 10 μL

[0102] Temperature: 25℃

[0103] UPLC

[0104] Chromatographic system: Waters Acquity UPLC;

[0105] Detector: Acquity UPLC PDA and λ detector (λ=220nm)

[0106] Column: Acquity UPLC BEH C18 1.7μm (2.1×50mm), T=35℃

[0107] Solvent A: Acetonitrile 0.01% Formic Acid

[0108] Solvent B: Water 0.01% Formic Acid

[0109] gradient:

[0110] 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

[0111] Chromatographic purity (European Pharmacopoeia (Ph Eur)):

[0112] Chromatogram obtained using the "HPLC titration" test performed by liquid chromatography under the following conditions defined in the European Pharmacopoeia 2.2.29:

[0113] Solvent: acetonitrile / water 70:30 v / v.

[0114] Reference solution A: Dissolve 20 mg of 2-methoxy-estradiol working standard solution (STD) in 14 mL of acetonitrile. Dilute to 20 mL with water. Prepare this solution in duplicate.

[0115] Sample solution: Dissolve 20 mg of product in 14 mL of acetonitrile and dilute to 20 mL with water. Prepare this solution in duplicate.

[0116] Reference solution B: Take 1 mL of the sample solution and dilute it to 100 mL with solvent. Dilute 1 mL of the solution to 10 mL with solvent.

[0117] Identification of impurities based on relative retention times:

[0118]

[0119] The impurity content at 204nm is calculated according to the following formula:

[0120] C u % = (r u / r s )×0.1

[0121] in:

[0122] C u % is the content of each impurity

[0123] r u is the response area of ​​each impurity

[0124] r s is the response area of ​​2-methoxy-estradiol in reference solution B.

[0125] For the calculation of total impurities, all impurities below 0.05% were discarded.

[0126] TLC

[0127] METCK: TLC Silica Gel 60F 254 Aluminium sheet 20 x 20 cm, code 1.0554.0001.

[0128] TLC detector

[0129] Cerium phosphomolybdate: Dissolve 25 g phosphomolybdic acid and 10 g cerium (IV) sulfate in 600 mL of H 2 O. Add 60 mL 98% HO 2 SO 4 , use H 2 O and bring to 1 L. Dip a test strip into the solution and heat until product is detected.

[0130] Remark

[0131] Unless otherwise stated, water used in the experimental descriptions is to be understood as pure water.

[0132] Unless otherwise stated, the organic solvents used in the experimental descriptions are understood to be of "technical" grade.

[0133] Unless otherwise stated, reagents used in the experimental descriptions are understood to be of commercial quality.

[0134] Example 1

[0135] This example relates to the process of the invention carried out by using a carboxylic acid in the esterification step.

[0136] Step a')

[0137] 2-Methoxyestradiol (33.8 g, previously crystallized from MTBE) containing 4.7% of 4-methoxyestradiol was suspended in a reactor containing 202.8 mL of formic acid at 25°C ± 5°C under nitrogen.

[0138] The suspension thus formed was placed under stirring. After 35 minutes, complete dissolution of the initial solid was observed. After 1 hour and 10 minutes, the formation of new solids in the suspension was observed. After 2 hours, the reaction status was checked by UPLC: 2-methoxyestradiol <1%.

[0139] Step b)

[0140] The suspension obtained was filtered and the solid obtained (formate of 2-methoxyestradiol) was washed with formic acid (34 mL) on a buchner funnel and always dried quickly on the buchner funnel (wet solid).

[0141] The solid sample thus obtained was dried under reduced pressure and 1 H-NMR and mass spectrometry were performed for analysis.

[0142] 1 H-NMR (400MHz, 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=8,5Hz); 3.71(3H,s,OCH) 3 );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).

[0143] Mass (ESI): m / z = M + -1=329.

[0144] Step c)

[0145] The wet solid obtained was charged into a reactor together with 6.7 g of pelletized sodium hydroxide.

[0146] Methanol (369 mL) was added and stirred, and after 15 minutes, the initial solid was observed to be completely dissolved. The internal temperature rose from 25°C to 32°C, and the measured pH was ≥12.

[0147] After stirring for an additional 30 minutes, complete dissolution of the solid was observed and the reaction status was monitored by UPLC: complete conversion, <1% formate of 2-methoxyestradiol.

[0148] To the reaction mixture was added aqueous ammonium chloride (26 g in 1300 mL H 2 O in).

[0149] The resulting hydroalcoholic mixture was then stirred at 25°C ± 5°C for 30 minutes and then at 0-5°C for 1 hour.

[0150] The formation of a precipitate was observed, which was filtered on a Buchner funnel and washed with water on the filter.

[0151] The solid obtained was then oven-dried at 55° C. under reduced pressure for 16 hours.

[0152] 23.9 g of 2-methoxyestradiol (white solid with yellow reflection) were obtained, wherein the content of 4-methoxyestradiol was less than 0.05%.

[0153] Example 2

[0154] This example relates to the process of the invention carried out by using a second carboxylic acid in the esterification step.

[0155] Step a')

[0156] 2-Methoxyestradiol (8 g) containing 3.92% (HPLC) of 4-methoxyestradiol was suspended in 48 mL of glacial acetic acid containing 453 mg of PTSA in a flask at 25°C ± 5°C under nitrogen.

[0157] The suspension was stirred and heated to 70 °C for 16 h: after 2 h, the solid was completely dissolved. After 16 h, the reaction was monitored by UPLC: 2-methoxyestradiol <1%.

[0158] The reaction mixture was cooled to 25°C; reprecipitation was observed and the mixture was kept stirring for 1 hour.

[0159] Step b)

[0160] The solid was filtered and washed with acetic acid (8 mL) and dried in vacuo at 45 °C to give 5.85 g of powder.

[0161] Step c)

[0162] Charge 5.35 g of the solid obtained in step b) and 931 mg of sodium hydroxide into a round-bottom flask.

[0163] Add methanol (53 mL) and stir the mixture at 20 < T (°C) < 25 °C for 3 hours. Monitor the hydrolysis by UPLC: complete conversion.

[0164] Prepare an aqueous ammonium chloride solution (2.6 g in 54 mL H 2 O).

[0165] Add the aqueous solution to the reaction mixture and stir the resulting hydroalcoholic mixture at 25 °C ± 5 °C for 30 minutes, then at 0 - 5 °C for 1 hour.

[0166] Filter the precipitate and wash it with water (65 mL).

[0167] Dry the solid at 50 °C for 16 hours to obtain 4.6 g of 2-methoxyestradiol, in which the presence of 4-methoxyestradiol is not detected.

[0168] Example 3

[0169] This example relates to the method of the present invention using a carboxylic acid derivative in the esterification step.

[0170] Step a'')

[0171] In a flask under nitrogen with the temperature maintained at 25 °C ± 5 °C, suspend 8 g of 2-methoxyestradiol containing 3.92% (HPLC) of 4-methoxyestradiol, 29.5 mL of triethylamine, and 0.32 g of 4-dimethylaminopyridine (DMAP) in 160 mL of dichloromethane in the flask under nitrogen. No complete dissolution of the solid is observed.

[0172] Cool the mixture to 0 °C and add 13.5 mL of benzoyl chloride dropwise. Raise the temperature to 13 °C and observe the formation of a precipitate.

[0173] Then heat, reflux, and stir the mixture for 16 hours. Then monitor the reaction by UPLC: 2-methoxyestradiol < 1%.

[0174] Step b)

[0175] Cool the mixture obtained in step a'') to 20 °C and pour it into 120 mL of ice-cold 1 M hydrochloric acid, keeping T < 20 °C. Then stir the mixture for 20 minutes. Separate the layers and extract the aqueous phase with dichloromethane (30 mL). Wash the organic phase with an aqueous NaHCO 3 solution and then with water (pH ≥ 7).

[0176] The organic layer was evaporated in vacuo to give an oil. MTBE (240 mL) was added and the system was evaporated until 60 mL of a suspension containing a solid was obtained, which was cooled and filtered, and washed with MTBE at a temperature of 0-5° C. The solid was dried in vacuo at 50° C. to give 9.7 g of a white powder.

[0177] Step c)

[0178] 300 mg of the solid obtained in step b) and 70 mg of sodium hydroxide were suspended in methanol (6 mL) and THF (1.5 mL) and heated at reflux for 6 h (a clear solution was obtained); the reaction was then monitored by UPLC: 2-methoxyestradiol 3,17 dibenzoate <1%.

[0179] The reaction mixture was cooled to room temperature, poured into an aqueous ammonium chloride solution (5 g, 20 mL), stirred at room temperature for 20 minutes, then stirred at 0° C. for 1 hour, the formation of solids was observed, the solids were filtered and washed with water. The solids were dried under vacuum at 45° C. to obtain 120 mg of 2-methoxyestradiol, of which 4-methoxyestradiol was not detected in HPLC analysis.

[0180] Example 4

[0181] This example relates to the process of the invention carried out by using a second carboxylic acid derivative in the esterification step.

[0182] Step a)

[0183] In a flask maintained at 25°C ± 5°C under nitrogen, 8 g of 2-methoxyestradiol containing 3.92% (HPLC) of 4-methoxyestradiol, 29.5 mL of triethylamine and 0.32 g of 4-dimethylaminopyridine (DMAP) were dissolved in 120 mL of THF.

[0184] The mixture was cooled to 0°C and 11.4 mL of furanyl chloride was added dropwise. The temperature was raised to 15°C and the formation of a precipitate was observed.

[0185] The mixture was then heated, refluxed and stirred for 24 hours. The reaction was then monitored by UPLC: 2-methoxyestradiol <1%.

[0186] Step b)

[0187] The mixture obtained in step a") was cooled to 20°C and poured into 120 mL of ice-cold 1 M hydrochloric acid, keeping T < 20°C. Then 80 mL of isopropyl acetate was added and the mixture was stirred for 20 minutes. The layers were separated and the aqueous phase was extracted with isopropyl acetate (2×50 mL). The organic phases were combined and washed with NaHCO 3 The solution was washed with water (pH ≥ 7).

[0188] The organic layer was evaporated in vacuo to give a yellow solid (18.6 g). The product was suspended in MTBE (54 mL), stirred at 20°C for 20 minutes, and then cooled to 0°C. After 1 hour, the solid was filtered and washed with MTBE at a temperature of 0-5°C. The solid was dried in vacuo at 50°C to give 10.27 g of a white powder.

[0189] Step c)

[0190] 200 mg of the solid obtained in step b) and 50 mg of sodium hydroxide were suspended in methanol (3 mL) and THF (1 mL) and stirred at 25° C. for 1.5 hours to obtain a clear solution; the reaction was then monitored by UPLC: 2-methoxyestradiol 3,17difuroyl <1%.

[0191] The reaction mixture was poured into an aqueous ammonium chloride solution (3.5 g, 10 mL), stirred at room temperature for 20 minutes, then stirred at 0° C. for 1 hour, the formation of solids was observed, the solids were filtered and washed with water. The solids were dried in vacuo at 50° C. to obtain 90 mg of 2-methoxyestradiol, in which 4-methoxyestradiol was not detected (HPLC).

Claims

1. A method for purifying 2-methoxyestradiol from 4-methoxyestradiol, comprising the following steps: a) reacting a mixture comprising 2-methoxyestradiol and 4-methoxyestradiol (N-3) with: a') a carboxylic acid selected from the group consisting of formic acid, acetic acid and propionic acid, or a″) a carboxylic acid derivative selected from the group consisting of esters, anhydrides, acid chlorides and acid bromides, get: - In case a'), a mixture N-2' comprising the corresponding monoester at position 17 of the steroid backbone: - In case a"), the mixture N-2" comprises the corresponding diesters in positions 3 and 17 of the steroid backbone: wherein X is O-alkyl or O-aryl in the case of esters, OCO-alkyl in the case of anhydrides, Cl or Br in the case of acid chlorides and bromides; b) isolating the derivative N-1' or the derivative N-1" from the mixture N-2' or the mixture N-2", respectively, by crystallization: c) hydrolyzing the derivative N-1' or the derivative N-1", to produce 2-methoxyestradiol:

2. The process according to claim 1, wherein in step a'), the acid is selected from formic acid and acetic acid.

3. The process according to any one of claims 1 or 2, wherein in step a') the acid used serves as a solvent.

4. The process according to any one of the preceding claims, wherein in step a') the ratio of milliliters of acid to grams of N-3 mixture is from 4 to 8 and step a') is carried out at a temperature of from 10°C to 90°C.

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

6. The method according to claim 1, wherein step a") is performed using a reagent mixture (N-3) comprising 2-methoxyestradiol and 4-methoxyestradiol, an organic solvent, an organic base, 4-dimethylaminopyridine and the carboxylic acid derivative.

7. The method according to claim 6, wherein the carboxylic acid derivative is selected from the group consisting of acid chlorides and acid bromides.

8. The method of claim 7, wherein the acyl chloride is selected from benzoyl chloride and furanoyl chloride.

9. The process according to any one of claims 6 to 8, wherein step a") is carried out at a temperature of 10°C to 50°C, the ratio of the volume of solvent in milliliters to the weight of the N-3 mixture in grams is 10 to 25, the ratio of the volume of solvent to the volume of the organic base is 3 to 6.5, the volume of solvent is at least 10 times the volume of the acid chloride used, and the ratio of the weight of the mixture N-3 to the weight of the 4-dimethylaminopyridine catalyst is 20 to 30.

10. The intermediate of the method according to claim 1, selected from: -2-methoxy-3-hydroxyestrast-1,3,5(10)-triene-17β-carboxylate, which has the following formula: -2-methoxy-3-hydroxyestra-1,3,5(10)-triene-3,17β-difurancarboxylate, which has the following formula:

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