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    • 专利摘要:
    Allopregnanolone purification method. The present invention relates to a method for the preparation of allopregnanolone with a high degree of purity, which eventually occurs through the formation of 3 α - trifluoroacetoxy-5 α -pregnan-20-one. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2748924A1
    申请号:ES201930751
    申请日:2019-08-22
    公开日:2020-03-18
    发明作者:Enrico Torregrossa；Marco Brusasca；Cristina Manfrotto
    申请人:Farmabios SpA；
    IPC主号:
    专利说明:

    [0001]
    [0002]
    [0003]
    [0004] The present invention relates to a process for the preparation of allopregnanolone, more particularly for the preparation of allopregnanolone with a high degree of purity.
    [0005]
    [0006]
    [0007]
    [0008] with the chemical name 1- (3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta [a] phenanthren-17-yl) ethanone.
    [0009]
    [0010] Allopregnanolone is a positive allosteric modulator of yaminobutyric acid (GABA) activity at the GABAa ionotropic receptor. In addition, it also acts as an inhibitor of L-type voltage-dependent calcium channels (L-VGCC).
    [0011]
    [0012] Allopregnanolone is currently being studied for the treatment of different pathologies such as epilepsy, postpartum depression and post-traumatic stress disorder (PTSD).
    [0013]
    [0014] Background of the Invention
    [0015]
    [0016] Various procedures for the preparation of allopregnanolone are known in the literature.
    [0017]
    [0018] The conversion of 3p-hydroxy-5a-pregnan-20-one (isopregnanolone) to 3a-hydroxy-5apregnan-20-one (allopregnanolone) through the Mitsunobu reaction and subsequent basic hydrolysis is described, for example, in J Med. Chem 1990, 33, 1572-1581. However, the resulting product must undergo a laborious purification phase consisting of crystallization from ethyl acetate / aqueous ethanol, column chromatography with 2% acetone from dichloromethane, and finally crystallization from aqueous ethanol.
    [0019] International patent application WO 2009/108804 describes a process consisting of the catalytic hydrogenation of pregnenolone to give isopregnanolone, purified by crystallization from hexane / ethyl acetate, which is then subjected to a Mitsunobu reaction and hydrolysis to produce allopregnanolone, purified by column chromatography with ethyl acetate in hexane (0-35%).
    [0020]
    [0021] Finally, in Collect. Czech. Chem. Commun. 2009, 74, 643-650, the conversion of isopregnanolone into the formic ester of allopregnanolone through the Mitsunobu reaction is described. The resulting ester intermediate is subjected to petroleum ether / acetone column chromatography (98: 2) and subsequently to crystallization from acetone. After basic hydrolysis, allopregnanolone is purified by crystallization from acetone / ethyl acetate.
    [0022]
    [0023] All procedures known in the art provide for a laborious purification phase that includes the use of at least one chromatography and / or chromatography followed by crystallization. Furthermore, purification of allopregnanolone according to crystallizations known in the art does not allow to effectively remove impurities and reaction by-products.
    [0024]
    [0025] However, since the product is for use in therapy, a procedure is required that allows allopurinolone to be obtained with a pharmaceutical grade purity and, at the same time, that it is easily industrially applicable.
    [0026]
    [0027] Therefore, a need still exists for an improved process for the preparation of allopregnanolone that overcomes all the drawbacks of the known procedures. In particular, there is a need for an improved method for the purification of allopregnanolone, for example, a method that does not contemplate laborious multi-stage purification and / or the use of chromatography, which is not time consuming, costly and disadvantageous with respect to performance.
    [0028]
    [0029] Summary of the invention
    [0030] A first object of the present invention is a method for the purification of allopregnanolone comprising the crystallization of allopregnanolone from acetonitrile.
    [0031]
    [0032] Another object of the present invention is a process for the preparation of allopregnanolone that is carried out through the formation, and the subsequent purification, of 3a-trifluoroacetoxy-5a-pregnan-20-one (hereinafter also called 3a allopregnanolone trifluoroacetate).
    [0033]
    [0034] Allopregnanolone 3a-trifluoroacetate is another object of the invention.
    [0035]
    [0036] Definitions
    [0037]
    [0038] All terms used in the present description, unless otherwise indicated, are to be understood in their common meaning as known in the art.
    [0039]
    [0040] The term "pharmaceutical grade purity", within the scope of the present invention, means that the product has a purity suitable for use as a medicine.
    [0041]
    [0042] The annotation "V" preceded by a number means how many times, in terms of volume, the quantity of one substance exceeds the given quantity of another substance. For example, given 7.5 g of allopregnanolone 3a-trifluoroacetate, adding 10 V of methanol means adding 75 ml of methanol.
    [0043]
    [0044] The term "approximately" encompasses the range of experimental error that can occur in a measurement. In particular, when a value is referenced, it means the given value plus or minus 5%, and when a range is referenced, it means the external values plus or minus 5%.
    [0045]
    [0046] Brief description of the figures
    [0047]
    [0048] Figure 1: XRPD spectrum of allopregnanolone obtained according to the invention. Figure 2: 1H NMR spectrum of allopregnanolone obtained according to the invention.
    [0049] Figure 3: 13C NMR spectrum of allopregnanolone obtained according to the invention.
    [0050] Figure 4: HPLC chromatogram of allopregnanolone obtained according to the invention.
    [0051] Figure 5: XRPD spectrum of allopregnanolone 3a-trifluoroacetate obtained according to the invention.
    [0052] Figure 6: 1H NMR spectrum of the allopregnanolone 3a-trifluoroacetate obtained according to the invention.
    [0053] Figure 7: 13C NMR spectrum of allopregnanolone 3a-trifluoroacetate obtained according to the invention.
    [0054] Figure 8: HPLC chromatogram of allopregnanolone obtained by crystallization from aqueous ethanol.
    [0055]
    [0056] Detailed description of the invention
    [0057]
    [0058] Starting from isopregnanolone and following the procedure described in WO 2009/108804, it was possible to isolate allopregnanolone. However, chromatography as the final stage of purification does not allow the process to be applied on an industrial scale. Furthermore, the isolated product shows a content of triphenylphosphine oxide (TPPO), as well as other unknown impurities, in amounts much higher than the allowed ICH limit of 0.10% (Table 1).
    [0059]
    [0060] Table 1
    [0061]
    [0062]
    [0063]
    [0064]
    [0065] in which% of A (or% of area) refers to the percentage of concentration of the compound with respect to the total in the injected solution and is obtained by the following formula:
    [0066]
    [0067] % of A = [Area under the peak curve (signal of interest) / sum of total areas]%
    [0068]
    [0069] TPPO, a by-product of the Mitsunobu reaction derived from triphenylphosphine oxidation, is a potentially toxic impurity that can significantly affect the quality and safety of the final product. In fact, to purify the product (allopregnanolone) or its precursors, such as, for example, its 3-formyl derivative, and to eliminate the by-products of the Mitsunobu reaction, including TPPO, the procedures known in the literature always use chromatography .
    [0070]
    [0071] In an attempt to obtain allopregnanolone according to the ICH specifications (i.e. unknown impurities <0.10% and known impurities <0.15% according to HPLC analysis) without using chromatographic techniques, the inventors of the present invention have carried out various crystallization experiments with solvents or solvent mixtures known in the literature such as, for example, aqueous ethanol, acetone / ethyl acetate, hexane / ethyl acetate, ethyl acetate / ethanol, etc. (see Example 1). However, some impurities, particularly TPPO, proved to be difficult to remove. It should be noted that, in the experiments carried out with said solvents or solvent mixtures (BE experiments, Example 1), even the later recrystallization from the same or from another solvent did not allow to increase the purity of the final product.
    [0072]
    [0073] After extensive experimentation, the inventors have surprisingly discovered that crystallization of allopregnanolone from acetonitrile allows the final product to be obtained according to ICH specifications and therefore substantially free (<< 0.10% A; << <0.10% w / w) of TPPO.
    [0074]
    [0075] Therefore, a first object of the present invention is a method for the purification of allopregnanolone comprising the crystallization of allopregnanolone from acetonitrile.
    [0076]
    [0077] Said purification can be carried out according to the methods known in the art, in particular, by cold-heat crystallization in which the allopregnanolone is first dissolved in hot acetonitrile to facilitate and accelerate the dissolution, and then the resulting solution it is cooled to achieve product precipitation.
    [0078]
    [0079] In one embodiment, the method for purification of allopregnanolone of the present invention comprises:
    [0080]
    [0081] - dissolving allopregnanolone in acetonitrile at a warm temperature;
    [0082] - cooling of the solution; Y
    [0083] - the separation of the precipitate.
    [0084]
    [0085] In particular, allopregnanolone dissolves in about 5-20 V, preferably in about 10 V, of acetonitrile and the resulting mixture is heated to a temperature between 70 ° C and 90 ° C, preferably between 75 ° C ± 5 ° C and 85 ° C ± 5 ° C, more preferably at about 80 ° C. During heating, the mixture can be kept under stirring, for example, for about 15 ± 5 minutes or, if necessary, for 30 ± 5 minutes until product dissolution is complete.
    [0086]
    [0087] The solution is then allowed to cool preferably at a temperature between -2 ° C and 25 ° C, more preferably at 0 ° C ± 2 ° C, thus achieving the precipitation of allopregnanolone crystals. During cooling, the mixture can be kept under stirring for a sufficient time to ensure the formation of allopregnanolone crystals. In one embodiment, the solution is kept under stirring for approximately 30 ± 5 minutes.
    [0088] The separation of the allopregnanolone crystals, obtained according to the process of the present invention, can be carried out according to any method known in the art, including, but not limited to, vacuum filtration, gravity filtration , distillation, centrifugation, slow evaporation and the like. In a preferred embodiment, allopregnanolone is isolated by vacuum filtration. The crystalline solid is washed with approximately 1 V cold acetonitrile (5 ° C ± 2 ° C) and dried at a temperature between 30 ° C and 50 ° C, preferably at approximately 45 ° C, for a period of time suitable for bring residual solvents below ICH limits. A suitable period of time can be, for example, between 10 and 20 hours, preferably approximately 16 hours. Drying can be carried out in accordance with methods known in the art including, but not limited to, Vacuum Oven, Rotavapor®, Air Drying Chamber, Static Bed Dryer, Fluid Bed Dryer, Spray Dryer and the like. Preferably, drying is carried out by vacuum static bed drying at 45 ° C ± 5 ° C.
    [0089]
    [0090] In a preferred embodiment, the crystallization of allopregnanolone can be followed by recrystallization from the same or a different solvent. Preferably, the recrystallization is carried out from acetonitrile under the same conditions indicated above.
    [0091]
    [0092] In a particularly preferred embodiment, the process for the purification of allopregnanolone, according to the present invention, comprises:
    [0093]
    [0094] - the solution of allopregnanolone in acetonitrile at a temperature of approximately 85 ° C ± 5 ° C, being kept under stirring for approximately 15 minutes;
    [0095] - cooling the solution to about 0 ° C while keeping it under stirring for about 30 minutes;
    [0096] - separation of the precipitate by vacuum filtration;
    [0097] - washing the precipitate with acetonitrile;
    [0098] - vacuum drying at a temperature of about 40 ° C for about 16 hours;
    [0099] and optionally
    [0100] - recrystallization of the precipitate from acetonitrile.
    [0101]
    [0102] It should also be noted that the process of the present invention allows allopregnanolone to be obtained in pharmaceutical grade purity and also in crystalline form (also referred to herein as Form X).
    [0103] Allopregnanolone, obtained according to the present invention, has been characterized by X-ray powder diffractometry (XRPD) and its TPPO content has been determined by high-performance liquid chromatography (HPLC).
    [0104]
    [0105] More particularly, the allopregnanolone obtained according to the present invention shows, in the XRPD diffractogram (Figure 1), at least three of the following characterization peaks: 7.25, 8.87, 9.58, 11.43, 14, 41, 14.77, 15.73, 17.78, 18.16, 18.60 and 19.98 ± 0.2 degrees 2-0.
    [0106]
    [0107] The HPLC chromatogram (Figure 4) shows that allopregnanolone, obtained according to the present invention, has much less than 0.10% TPPO (retention time related to allopregnanolone rRT = approximately 0.59).
    [0108]
    [0109] Although the invention has been described in detail, it should be noted that the only essential feature of the present method for purification of allopregnanolone is the crystallization solvent, which is acetonitrile.
    [0110]
    [0111] For example, it has been observed that, even by changing the duration of the hot / cold cycles, allopregnanolone is obtained according to the desired specifications.
    [0112]
    [0113] Only the use of acetonitrile to give allopregnanolone in crystalline form X allowed to develop an improved procedure for the purification of allopregnanolone.
    [0114]
    [0115] Without being limited to any specific theory, the inventors of the present invention believe that this surprising result is due to the peculiar solubility properties of both allopregnanolone and TPPO in acetonitrile. In fact, the product crystallizes from acetonitrile with high performance and complete removal of triphenylphosphine oxide that remains in solution also after cooling of the solution. It should be noted that no solvent or solvent mixture known in the literature can achieve this dual function.
    [0116]
    [0117] Allopregnanolone can be obtained by any method known in the art.
    [0118]
    [0119] However, in an attempt to further increase the purity of the final product, the inventors have also developed a process for the preparation of allopregnanolone.
    [0120] Therefore, a further object of the present invention is a method for the purification of allopregnanolone further comprising the preparation of allopregnanolone by Mitsunobu reaction of a compound of formula
    [0121]
    [0122]
    [0123]
    [0124]
    [0125] to give a compound of formula
    [0126]
    [0127]
    [0128]
    [0129]
    [0130] In particular, isopregnanolone (III) is reacted with trifluoroacetic acid in the presence of azodicarboxylate, triphenylphosphine and sodium benzoate in a suitable reaction solvent, preferably tetrahydrofuran.
    [0131]
    [0132] The azodicarboxylate can be selected from the group consisting of diethylazodicarboxylate (DEAD), diisopropylazodicarboxylate (DIAD) and di-tert-butyl azodicarboxylate (DBAD), and is preferably DEAD solid or dissolved in a suitable solvent, in particular toluene.
    [0133]
    [0134] The intermediate of formula (II), which is allopregnanolone 3a-trifluoroacetate or 3atrifluoroacetoxy-5a-pregnan-20-one, is a further object of the present invention.
    [0135]
    [0136] It should be noted that the conversion of 3a-hydroxy-5a-pregnan-20-one to 3p-trifluoroacetoxy-5apregnan-20-one through the Mitsunobu reaction is known in the literature (Varasi et al. "A Revised Mechanism for the Mitsunobu Reaction "). However, the C3 of the resulting compound has an inverted configuration with respect to the intermediate of the present invention and is still isolated and purified by chromatography.
    [0137]
    [0138] After extensive experimentation, the inventors of the present invention have discovered that the isolation and purification of allopregnanolone 3a-trifluoroacetate Filtration of a solution of the compound of formula (II) in diisopropyl ether (DIPE) followed by recrystallization from isopropyl alcohol (IPA) can advantageously influence the purity of the final product, allopregnanolone.
    [0139]
    [0140] Therefore, a further object of the present invention is a method for the purification of allopregnanolone comprising the filtration of a solution of the compound of formula (II) in diisopropyl ether and its recrystallization from isopropyl alcohol.
    [0141]
    [0142] In particular, the allopregnanolone (II) 3a-trifluoroacetate dissolves in about 2-10 V, preferably about 6 V, of diisopropyl ether. To promote dissolution, the mixture can be kept under stirring for a suitable period of time, for example, for about 30 minutes, until a precipitate (TPPO) is obtained. The solid precipitate is removed according to filtration techniques known in the art, such as, for example, gravity filtration and vacuum filtration. The filtrate, which is the solution of allopregnanolone 3a-trifluoroacetate in diisopropyl ether, is concentrated and then recovered with approximately 2-10 V, preferably approximately 6 V, of isopropyl alcohol.
    [0143]
    [0144] The resulting mixture is heated to a temperature between 60 ° C and 90 ° C, preferably between 70 ° C and 80 ° C, more preferably to about 75 ° C. While heating, the mixture can be kept under stirring, for example, for about 15 ± 5 minutes or, if necessary, for 30 ± 5 minutes until product dissolution is complete.
    [0145]
    [0146] The solution is preferably allowed to cool to a temperature between -2 ° C and 25 ° C, more preferably at 0 ° C, to obtain the precipitation of crystals of allopregnanolone 3a-trifluoroacetate. While cooling, the mixture can be kept under stirring for long enough to ensure the formation of allopregnanolone 3a-trifluoroacetate crystals. In one embodiment, the solution is kept under stirring for approximately 30 ± 5 minutes.
    [0147]
    [0148] Separation of the allopregnanolone 3a-trifluoroacetate crystals obtained according to the process of the present invention can be carried out according to any of the methods known in the art, including, but not limited to, vacuum filtration, filtration by gravity, distillation, centrifugation, slow evaporation and the like. In a preferred embodiment, allopregnanolone 3a-trifluoroacetate is isolated by vacuum filtration. The crystalline solid is washed with approximately 1 V of alcohol cold isopropyl (5 ° C ± 2 ° C) and dries at a temperature of 30 ° C to 50 ° C, preferably at about 20-25 ° C, for a suitable period of time. Drying can be performed according to methods known in the art including, but not limited to, vacuum oven, Rotavapor®, air drying chamber, static bed dryer, fluid bed dryer, spray dryer, and the like. . In a preferred embodiment, drying is performed by vacuum static bed drying at 45 ° C ± 5 ° C.
    [0149]
    [0150] Without limiting themselves to any specific theory, the inventors of the present invention surprisingly found that allopregnanolone 3a-trifluoroacetate is completely soluble in diisopropyl ether and TPPO is insoluble, while allopregnanolone 3a-trifluoroacetate crystallizes with high yields in isopropyl alcohol and TPPO is completely soluble.
    [0151]
    [0152] The allopregnanolone 3a-trifluoroacetate, obtained according to the present invention, has been characterized by X-ray powder diffractometry (XRPD).
    [0153]
    [0154] More particularly, the allopregnanolone 3a-trifluoroacetate obtained according to the present invention shows, on the XRPD diffractogram (Figure 5), at least three of the following characteristic peaks: 5.93, 11.91, 12.76, 16 , 79, 17.46, 17.89, 18.27, 20.11, 21.45 and 23.93 ± 0.2 degrees 2-0.
    [0155]
    [0156] Allopregnanolone 3a-trifluoroacetate (II), isolated and purified according to the procedure described above, is then converted to allopregnanolone (I) by hydrolysis reaction in the presence of a base, preferably sodium hydroxide, even more preferably hydroxide of 28% w / v sodium, in a suitable solvent, preferably methanol, and purified by crystallization from acetonitrile according to the first object of the invention.
    [0157]
    [0158] Surprisingly, the resulting allopregnanolone has a pharmaceutical grade purity and, more particularly, a TPPO content of less than 0.05% in A (see Table 2).
    [0159]
    [0160] fable 2
    [0161]
    [0162]
    [0163]
    [0164]
    [0165] * n.d. = not detectable
    [0166] Furthermore, by correcting (dividing)% of A by the appropriate relative response factor k (where k = (A tppo * [Alopregnanolone] / AAlopregnanolone * [TPPO] = 129), the value% of A, representative of the concentration TPPO residual decreases further:
    [0167] 0.05% / 129 = 0.0004% (<5 ppm)
    [0168]
    [0169] In other words, TPPO is practically undetectable (<5 ppm) in allopregnanolone obtained according to the present invention.
    [0170] Therefore, in a particularly preferred embodiment, the method for purification of allopregnanolone of the present invention comprises:
    [0171]
    [0172] - the Mitsunobu reaction of isopregnanolone (111) to give allophagnanolone 3a-trifluoroacetate (II);
    [0173] - filtration of a solution of allopregnanolone (II) 3a-trifluoroacetate in diisopropyl ether followed by crystallization from isopropyl alcohol;
    [0174] - the hydrolysis reaction of 3a-trifluoroacetate of allopregnanolone (II) to give allopregnanolone (I); Y
    [0175] - the crystallization of allopregnanolone (I) from acetonitrile.
    [0176]
    [0177] Even if the present invention has been described in its characterizing features, the changes and equivalents that are apparent to those skilled in the field are included in the present invention. Hereinafter, the present invention will be illustrated by some examples which are for illustrative purposes only and are not intended to limit the scope of the invention.
    [0178]
    [0179] Examples
    [0180]
    [0181] The melting point of allopregnanolone (I) obtained according to the method of the present invention was experimentally determined by the Mettler MP90 melting point system model to give a range of 174-175 ° C.
    [0182]
    [0183] The optical rotation was determined by the Jasco P2000 polarimeter and was obtained as [a] D 96 ° (c = 0.5, chloroform), in which "c" is the concentration expressed in g / 100 ml and the chloroform is the solvent in which the measurement was made.
    [0184] 1H and 13C NMR were obtained using a Bruker AV spectrometer (300MHz) at 25 ° C observing 1H and 13C at 300.13 and 75.47 MHz, respectively. Chemical shifts are expressed as ppm related to tetramethylsilane and the spectra were obtained by dissolving the sample in CDCh.
    [0185]
    [0186] Allopregnanolone powder X-ray diffraction spectra (XRPD) were performed with a Bruker D5005 diffractometer, using Cu Ka radiation (A = 1.5418A), equipped with a scintillation detector and a curved graphite monochromator on the beam diffracted. The samples were gently milled in an agate mortar to obtain a fine powder and to disintegrate eventual particle agglomerates. Data was collected at room temperature on a low monocrystalline silicon bottom sample holder. Detection: 20 degrees, angular range measurement from 3 ° to 40 °, 0.03 ° step and 4s / step count time.
    [0187]
    [0188] Allopregnanolone 3a-trifluoroacetate powder X-ray diffraction spectra (XRPD) were performed with a Bruker D8 diffractometer, using Cu Ka radiation, equipped with a scintillation detector and a curved graphite monochromator on the diffracted beam. The samples were gently ground in an agate mortar to obtain a fine powder and to separate eventual particle agglomerates. Data was collected at room temperature on a low monocrystalline silicon bottom sample holder. Detection: 20 degrees, angular range measurement from 3 ° to 35 ° (20), 0.04 ° step and 2s / step count time.
    [0189]
    [0190] HPLC chromatograms were obtained using Agilent 1100 Series and Agilent 1200 Series equipment and injecting 10 ^ l of solution into a Kinetex XB C18; 150 x 4.6 mm; Column of 5 ^ m. The sample was gradient eluted with a mobile phase composed of a water / acetonitrile mixture.
    [0191]
    [0192] The compounds were finally analyzed by applying a wavelength equal to 210 nm.
    [0193]
    [0194] Mass analyzes were performed using a Varian 500MS kit at ESI +.
    [0195]
    [0196] Example 1 (comparative)
    [0197]
    [0198] Crystallization tests in allopognanolone strongly doped with TPPO (10% w / w) The results are reported in Table 3:
    [0199] Table 3
    [0200]
    [0201]
    [0202]
    [0203]
    [0204] B-E tests result in allopregnanolone containing approximately 50% of TPPO impurities (rRT = 0.59) as shown, for example, in Figure 8.
    [0205]
    [0206] The comparative example demonstrates that acetonitrile, which differs from all other solvents or solvent mixtures, has the unexpected advantage of providing allopregnanolone with higher yield and purity.
    [0207]
    [0208] Example 2
    [0209]
    [0210] Preparation of allopregnanolone 3a-trifluoroacetate
    [0211]
    [0212] 15 g of isopregnanolone and 150 ml (10 V) of tetrahydrofuran were charged into a reaction flask at room temperature. The resulting suspension was stirred under an inert atmosphere (N 2 ). Then, 26.51 ml of diethylazodicarboxylate (DEAD, 40% by weight in toluene) and 4.46 ml of trifluoroacetic acid were added at a temperature of approximately 5 ° C ± 3 ° C. Subsequently, 15.3 g of triphenylphosphine was added, the reaction mixture was kept under stirring for approximately 10 minutes and the temperature was raised to 25 ° C ± 2 ° C. Then 8.4 g of sodium benzoate was added and the mixture was kept under stirring for about 2 hours. The solvent was removed by vacuum distillation to 4 V of residual solvent.
    [0213]
    [0214] Subsequently, 90 ml (6 V) of dichloromethane were added, the temperature was raised to approximately 30 ° C and then 90 ml (6 V) of water was added and the mixture was kept under stirring for 10 ± 5 minutes. The phases were separated and the collected organic phases were treated first with a saturated solution of NaHCO 3 (90 ml, 6 V), then with 1N HCl (90 ml, 6 V), and finally with water (90 ml, 6 V). The organic phases were concentrated by distillation under vacuum.
    [0215] Example 3
    [0216]
    [0217] Purification of allopregnanolone 3a-trifluoroacetate
    [0218]
    [0219] Allopregnanolone 3a-trifluoroacetate was dissolved in 6V diisopropyl ether. The mixture was kept under stirring for approximately 30 minutes until a precipitate was obtained. The precipitated solid was removed by vacuum filtration. The filtrate was concentrated and collected with 6V isopropyl alcohol. The mixture was heated to 75 ° C and kept under stirring for approximately 15 minutes. Subsequently, the mixture was cooled to 0 ° C and kept under stirring for approximately 30 minutes. The precipitate was filtered and washed with 15 ml (1 V) of cold isopropyl alcohol. The filtrate was then dried under vacuum at room temperature to give allopregnanolone 3a-trifluoroacetate (7.5 g).
    [0220]
    [0221] 1H NMR: 4.03 (1H, m, H-3a); 2.53 (1H, m, H-17); 2.11 (3H, s, -COCH 3 ); 0.78 (3H, s, methyl-19); 0.60 (3H, s, methyl-18); 1.00 - 2.20 (m, 22H).
    [0222]
    [0223] 13C NMR: 11.2 (CH 3 ); 13.3 (CH 3 ); 20.7 (CH 2 ); 22.7 (CH 2 ); 24.2 (CH 2 ); 25.6 (CH 2 ); 27.9 (CH 2 ); 31.4 (CH 3 ); 31.5 (CH 2 ); 32.2 (CH 2 ); 32.4 (CH 2 ); 35.3 (CH); 35.6; 38.8 (CH 2 ); 39.7 (CH); 44.1; 53.7 (CH); 56.5 (CH); 63.6 (CH-17); 75.6 (C-3); 114.0 (q, jc-F = 285 Hz, -CF); 156.8 (q, jc-F = 41 Hz, -COOCF 3 ); 209.6 (C-20).
    [0224]
    [0225] MS-ESI positive m / z 415.3 [M H] +.
    [0226]
    [0227] Example 4
    [0228]
    [0229] Preparation of allopregnanolone
    [0230]
    [0231] 7.5 g of allopregnanolone 3a-trifluoroacetate and 75 ml (10 V) of MeOH were charged into a reaction flask at room temperature. The resulting suspension was kept under stirring under an inert atmosphere (N 2 ) and cooled to 5 ° C ± 3 ° C. Subsequently, 3 g of 28% w / v NaOH was added and the reaction mixture was kept under stirring for 10 ± 5 minutes. The reaction mixture was worked up by extraction into dichloromethane / water (6V / 10V) while keeping under constant stirring for 10 ± 5 minutes. The phases were separated and concentrated by distillation in vacuo.
    [0232] Example 5
    [0233]
    [0234] Allopregnanolone purification
    [0235]
    [0236] Acetonitrile (10 V) was charged into a reaction flask, the mixture was heated to 85 ° C ± 5 ° C and kept under stirring for approximately 15 minutes. The mixture was then cooled to 0 ° C and kept under stirring for approximately 30 minutes. The precipitate was isolated by filtration under reduced pressure and washed with 7.5 ml (1 V) of cold acetonitrile. Finally, the filtrate was dried under vacuum at 45 ° C for approximately 16 hours to give allopregnanolone (5.0 g).
    [0237]
    [0238] 1H NMR: 4.03 (1H, m, H-3a); 2.53 (1H, m, H-17); 2.11 (3H, s, -COCH 3 ); 1.57 (1H, s, exchange with D 2 O, OH); 0.78 (3H, s, methyl-19); 0.60 (3H, s, methyl-18); 1.00 - 2.20 (m, 22H).
    [0239]
    [0240] 13C NMR: 11.1 (CH 3 ); 13.3 (CH 3 ); 20.7 (CH 2 ); 22.6 (CH 2 ); 24.2 (CH 2 ); 28.3 (CH 2 ); 28.8 (CH 2 ); 31.4 (CH 3 ); 31.8 (CH 2 ); 32.1 (CH 2 ); 35.3 (CH); 35.7 (CH 2 ); 36.0; 38.9 (CH); 38.9 (CH 2 ); 44.1; 54.1 (CH); 56.7 (CH); 63.7 (CH-17); 66.3 (CH-3); 209.6 (C-20).
    [0241]
    [0242] MS-ESI positive m / z 319.2 [MH] +; 301.1 [MH 2 OH] +
    权利要求:
    Claims (10)
    [1]
    1. A method for the purification of allopregnanolone of formula (I)

    [2]
    2. The method according to claim 1, which comprises recrystallization of allopregnanolone from acetonitrile.
    [3]
    3. The method according to claim 1, further comprising the preparation of allopregnanolone by Mitsunobu reaction of a compound of formula

    [4]
    4. The method according to claim 3, wherein the Mitsunobu reaction occurs by reaction of the compound of formula (III) with trifluoroacetic acid in the presence of an azodicarboxylate, triphenylphosphine and sodium benzoate, in a suitable reaction solvent , preferably tetrahydrofuran.
    [5]
    5. The method according to claim 4, wherein the azodicarboxylate is selected from the group consisting of diethylazodicarboxylate, diisopropyl azodicarboxylate and di-tert-butyl azodicarboxylate, preferably diethylazodicarboxylate.
    [6]
    The method according to one or more of claims 3-5, further comprising filtering a solution of the compound of formula (II) in diisopropyl ether and recrystallizing it from isopropyl alcohol.
    [7]
    7. The method according to claim 3, wherein said preparation further comprises converting the compound of formula (II) to the compound of formula (I) by hydrolysis reaction in the presence of a base, preferably sodium hydroxide in a suitable solvent, preferably methanol.
    [8]
    8. Alopregnanolone, obtained according to any of the preceding claims, which has a content of triphenylphosphine oxide (TPPO) less than 0.05% of area.
    [9]
    9. Alopregnanolone obtained according to any of the preceding claims, showing, on the XRPD diffractogram, at least three of the following characterization peaks: 7.25, 8.87, 9.58, 11.43, 14, 41, 14.77, 15.73, 17.78, 18.16, 18.60 and 19.98 ± 0.2 degrees 20.
    [10]
    10. The compound of formula (II).
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    同族专利:
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    US20200087341A1|2020-03-19|
    US10711030B2|2020-07-14|
    ES2748924B2|2020-10-05|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2009108804A2|2008-02-26|2009-09-03|Emory University|Steroid analogues for neuroprotection|
    US3013031A|1958-07-14|1961-12-12|Merck & Co Inc|9-16-pregnadiene-20-one compounds through 17 and 21 brominated intermediates|
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