Preparation method of 21-(acetyloxy)-17-(1-propionyloxy)-pregn-4-ene-3,20-dione

Abstract

The present invention relates to a method for preparing 21-(acetyloxy)-17-(1-oxopropoxy)-pregnane-4-ene-3,20-dione (VI) having the following formula: Compound (VI) can be used as a precursor for synthesizing the steroid compound clapriston for treating acne.

Description

Technical Field

[0001] The present invention relates to the field of methods for synthesizing pharmaceutically active ingredients, and in particular to a method for preparing 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione on an industrial scale, the compound having the following structural formula (VI):

[0002]

[0003] This is a useful precursor for the synthesis of 21-hydroxy-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione, also known as Clascoterone.

[0004] Clascoterone is a steroidal drug containing a properly formulated pregnane backbone that was recently approved by the U.S. Food and Drug Administration (FDA) for the treatment of acne in pediatric patients 12 years of age and older and in adults. The structural formula of clascoterone is shown below:

[0005] Background Art

[0006] Clapriston was described in US Patent 3,152,154 in 1964. As shown above, this compound is a 17α monoester of a 17α,21-dihydroxy steroid.

[0007] According to the teaching of US Pat. No. 3,152,154, 17-monoesters of 17α,21-dihydroxysteroids can be obtained by acid-catalyzed chemical hydrolysis of the corresponding 17α,21-(1′-alkoxy)1′-pregnane (orthoester) of the following type:

[0008]

[0009] The experimental description reported in US 3,152,154 does not provide any details regarding the reaction yield and the quality of the products obtained.

[0010] The orthoesters described in US 3,152,154 can be prepared according to the procedure described in US Patent 3,147,249. This second patent also does not provide any details regarding the reaction yield and the quality of the product obtained.

[0011] In particular, for the preparation of clapriston, the starting compound for the preparation of the orthoester to be hydrolyzed would be 17,21-dihydroxy-pregn-4-ene-3,20-dione, a compound known as "cortexolone" having the structural formula shown below:

[0012]

[0013] However, on the market, this compound is only available in laboratory quantities and not in the quantities required for industrial production.

[0014] Another possible precursor of clapriston is the compound 17,21-bis(1-oxopropoxy)-pregn-4-ene-3,20-dione, which is a compound having the structural formula (VII) shown below:

[0015]

[0016] Compound (VII) can be prepared according to the method described in patent application WO 2009 / 019138 A2, as shown in "Acylation of 17-hydroxy-20-ketosteroids", R.B. Turner, J. Am. Chem. Soc. 1953, 75, 14, 3489-3492. However, the acid hydrolysis of compound (VII) takes a relatively long time and produces a non-negligible amount of by-products.

[0017] WO 2009 / 019138 A2 also proposes the selective enzymatic hydrolysis of symmetrical diesters, i.e., where the R groups of the two ester groups are identical, using lipase according to the following reaction:

[0018]

[0019] The object of the present invention is to provide a novel intermediate which can be used for the synthesis of clapriston and to provide a method which can synthesize said intermediate on an industrial scale. Summary of the Invention

[0020] The present invention achieves this object. In its first aspect, the present invention relates to a process for the synthesis of the compound 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione of formula (VI):

[0021]

[0022] The compound (VI) can be used as a precursor for the synthesis of clapriston, and the method comprises the following steps:

[0023] a) 17α-hydroxyprogesterone (I) reacts with pyrrolidine to obtain compound (II) 17-hydroxy-3-(1-pyrrolidinyl)pregna-3,5-dien-20-one:

[0024]

[0025] b) Compound (II) is first reacted with hydrochloric acid and then with bromine to obtain intermediate (III), a mixture of (21-chloro / 21-bromo)-17α-hydroxy-3-(1-pyrrolidinium-1-ylidene)-pregn-4-en-20-one chlorides:

[0026]

[0027] c) Intermediate (III) is subjected to alkaline hydrolysis to obtain intermediate (IV), which is a corresponding mixture of 21-chloro / 21-bromo-17α-hydroxypregnane-4-ene-3,20-dione:

[0028]

[0029] d) The intermediate (IV) reacts with acetic acid to obtain compound (V) 21-acetoxy-17α-hydroxypregn-4-ene-3,20-dione:

[0030]

[0031] e) Compound (V) reacts with perchloric acid and propionic anhydride to obtain compound (VI) 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione:

[0032]

[0033] The method of the present invention may further comprise an additional step f): selectively hydrolyzing compound (VI) to obtain clapriston:

[0034]

[0035] Step f) can be performed by chemical or enzymatic routes.

[0036] In a second aspect, the present invention relates to compound (VI) 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione.

[0037] In a third aspect, the present invention relates to obtaining clapriston by enzymatic hydrolysis of compound (VI) in a flow reactor.

[0038] Finally, in a fourth aspect, the present invention relates to clapriston solvated in dimethyl sulfoxide. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The HPLC chromatogram of the compound 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione obtainable by the method of the present invention is shown.

[0040] Figure 2 The XPRD diffraction spectrum of the compound 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione obtainable by the method of the present invention is shown.

[0041] Figure 3 The DSC thermogram of the compound 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione obtainable by the method of the present invention is shown.

[0042] Figure 4 Shown is the XPRD diffraction pattern of dimethyl sulfoxide solvated clapriston and the relative angle data and relative intensity of each peak.

[0043] Figure 5 Shown is the DSC thermogram of clapriston solvated in dimethyl sulfoxide.

[0044] Figure 6 The FT-IR spectrum of clapriston solvated in dimethyl sulfoxide is shown.

[0045] Figure 7 Shown is the XPRD diffraction pattern of methanol-solvated clapriston. DETAILED DESCRIPTION

[0046] The present inventors have found that "asymmetric" diester hydrolysis provides better results than symmetric ester hydrolysis in the production of clapriston.

[0047] In the following description, when the ratio of the amount of solvent to the amount of compound is provided as "volume per weight," the volume of solvent is understood to be measured in milliliters and the weight of the compound is measured in grams. Furthermore, for simplicity or clarity, in some cases the stereochemical configuration of some atoms of the steroidal backbone is not shown in the Figures herein; in these cases, it is understood that the stereochemistry of the molecule corresponds to the natural configuration of the steroidal compound.

[0048] The term "asymmetric" diester refers to the following types of structures:

[0049]

[0050] wherein the alkyl groups R and R' are different.

[0051] After experimental examination, the asymmetric diester showed a more favorable acid hydrolysis behavior than the symmetrical 17,21-bis(1-oxopropoxy)-pregna-4-ene-3,20-dione of formula (II) described in WO 2009 / 019138 A2; in order to avoid confusion with the compound (II) of the present invention (process intermediate 17-hydroxy-3-(1-pyrrolidinyl)pregna-3,5-diene-20-one), the compound (II) of WO 2009 / 019138 A2 will be referred to as compound (VII) in this specification.

[0052] In fact, according to the present inventors' observation, when compound (VI) of the present invention and compound (VII) of WO 2009 / 019138A2 were subjected to acid hydrolysis reaction simultaneously under the same conditions (perchloric acid dissolved in dichloromethane-methanol at 10-12° C.), the reaction of compound (VI) was completed within 37 hours (residual compound (VI) <3%), while compound (VII) required 57 hours to achieve the same result.

[0053] Furthermore, at the end of the reaction, the composition of the mixture was also different, and as shown in the data in Table 1 below, the best results were obtained in terms of clapriston yield when using compound (VI) (the percentage concentrations shown in the table were calculated based on the peak areas in the HPLC test):

[0054] Table 1

[0055]

[0056]

[0057] Apart from residual unreacted reagents, the only by-product present in comparable amounts in both reaction products is a so-called "transposition" by-product, the formation of which cannot be inhibited, as described in the article "Corticosteroid 17α-monoesters from 17α,21-cyclic orthoesters", R. Gardi et al., Tetrahedron Letters (13) 1961, pages 448-451, since it is specific for the reaction product under the reaction conditions and is independent of the starting reactants. The transposition reaction between positions 17 and 21 of steroid compounds is summarized as follows:

[0058]

[0059] According to the reaction mechanism outlined below, a monoester at position 21 having a free hydroxyl group at position 17 is characterized by its instability under acid reaction conditions, resulting in the migration of the acylated group from position 17 to position 21:

[0060]

[0061] In a first aspect, the present invention relates to a method for synthesizing 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione, which comprises the five synthetic steps a) to e) described above.

[0062] Step a) involves reacting compound (I) with pyrrolidine to obtain the corresponding enamine compound 17-hydroxy-3-(1-pyrrolidinyl)pregna-3,5-dien-20-one (II). This step is carried out by preparing a suspension of compound (I) in alcohol, refluxing the suspension, and then adding pyrrolidine. The starting compound (I), 17α-hydroxyprogesterone, is widely available on the market, and does not require the synthesis of an orthoester as a process intermediate.

[0063] The molar amount of pyrrolidine used relative to compound (I) is in excess of 20 to 60%, preferably 40%.

[0064] Alcohols that can be used to prepare the suspension include ethanol, isopropanol, and preferably methanol.

[0065] The reaction mixture is maintained at reflux for 1 to 3 hours, preferably 1.5 to 2.5 hours.

[0066] The obtained compound (II) is isolated from the reaction solvent by crystallization precipitation.

[0067] Step b) involves reacting the enamine (II) first with hydrochloric acid and then with bromine to give the intermediate (III), a mixture of (21-chloro / 21-bromo)-17α-hydroxy-3-(1-pyrrolidinium-1-ylidene)-pregn-4-en-20-one chlorides.

[0068] The reaction is carried out at a temperature of 10 to 40°C, preferably 20 to 30°C.

[0069] The reaction solvent is an alcohol selected from methanol, isopropanol and preferably ethanol. The amount of alcohol used is 15 to 50 volumes, preferably 15 to 30 volumes, relative to the weight of compound (II).

[0070] Hydrochloric acid is used in the form of a 33% (weight) ethanol or isopropanol solution; the amount of the solution used in the reaction is 1 to 3 times the weight of compound (II), preferably 1.5 times.

[0071] The amount of bromine used (in moles) is 1.0 to 3 times, preferably 1.5 times, the molar amount of compound (II).

[0072] Bromine is added as an ethanol solution in a volume ratio of bromine to ethanol of 1:20 to 1:45, preferably 1:25. The ethanolic bromine solution is cooled to -50 to -60°C, preferably -55°C, before adding the solution prepared in the first part of this step. The bromine solution is added over 20 minutes to 2 hours, preferably over 80 minutes to 100 minutes.

[0073] The intermediate (III) obtained at the end of step b) can be crystallized using linear or branched alcohols having 1 to 4 carbon atoms, ethers or mixtures thereof; the preferred solvent for the crystallization of intermediate (III) is methyl tert-butyl ether (MTBE).

[0074] The reaction result is a mixture of intermediates (III) (21-chloro / 21-bromo)-17α-hydroxy-3-(1-pyrrolidinium-1-ylidene)-pregn-4-en-20-one chlorides, which are used as such in the continued synthesis because both products react in the same manner to give the desired 21-acetoxy product; therefore, in this specification, the mixture (III) is represented as a single reaction intermediate.

[0075] The content of the minor component 21-chlorosteroids in the mixture (III) is 5-30%.

[0076] In an alternative embodiment, step b) can be carried out by direct reaction of the enamine (II) with hydrobromic acid, in which case only compound 21-bromo is obtained as intermediate (III).

[0077] Step c) of the method of the present invention comprises alkaline hydrolysis of intermediate (III) to obtain the corresponding 21-chloro / 21-bromo-17α-hydroxypregn-4-ene-3,20-dione mixture; this mixture is also used as it is in the following method reaction, and is therefore represented as a single intermediate, namely intermediate (IV) in this specification.

[0078] The reaction can be carried out in a mixed aqueous solution of acetone, methanol or ethanol, wherein the water content is less than 50% by volume. Preferably, a water / methanol mixture is used, wherein the volume of methanol is greater than 70% of the total volume.

[0079] The base used can be selected from NaHCO3, Na2CO3, KHCO3 or K2CO3; preferably KHCO3 is used in a molar amount greater than 2 times the molar amount of intermediate (III).

[0080] The reaction temperature is between 10°C and the reflux temperature of the mixture; the reaction is preferably maintained between 20°C and 30°C.

[0081] The reaction time is 2 to 16 hours, preferably 4 to 6 hours.

[0082] The obtained intermediate (IV) can be crystallized using solvents such as methyl tert-butyl ether (MTBE), ethyl acetate, acetonitrile, acetone, methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK), linear or branched alcohols having 1 to 4 carbon atoms, or mixtures thereof. For this operation, a 1:1 (v / v) MEK-MeOH mixture is preferably used, employing a heating-cooling technique. This technique is well known to those skilled in the art of organic synthesis and involves heating the product to be purified in the presence of a solvent. The resulting suspension and / or solution is then cooled. The solid product is filtered, while any impurities remain in solution.

[0083] In the next step d), intermediate (IV) is reacted to give compound (V) 21-acetoxy-17α-hydroxypregna-4-ene-3,20-dione.

[0084] The reaction can be carried out in a solvent selected from dimethylformamide (DMF), acetone, methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK), methanol, ethanol, 2-propanol, toluene or a mixture thereof, in the presence of an inorganic base selected from KHCO 3 , NaHCO 3 , K CO 3 , Na CO 3 or an organic base selected from triethylamine (TEA), trimethylamine (TMA) or pyridine, using glacial acetic acid. Preferably, the reaction is carried out in acetone or methyl ethyl ketone (MEK) with glacial acetic acid and triethylamine (TEA). Alternatively, the reaction can be carried out using sodium acetate or potassium acetate.

[0085] The reaction time is 1-24 hours, and the temperature is between 20°C and the reflux temperature of the mixture; preferably, the reaction is carried out at the reflux temperature of the mixture for 4 to 6 hours.

[0086] The obtained compound (V) can be crystallized using a solvent such as methyl tert-butyl ether (MTBE), acetone, methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK), a linear or branched alcohol having 1 to 4 carbon atoms, or a mixture thereof; preferred solvents are methyl ethyl ketone (MEK) and ethanol.

[0087] Finally, step e) of the process of the present invention comprises reacting compound (V) with perchloric acid and propionic anhydride to obtain compound (VI) 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione.

[0088] The reaction is carried out by diluting compound (V) in dichloromethane (DCM) in an amount of 10 to 50 volumes, preferably 25 volumes, relative to the weight of the steroid compound, at a reaction temperature of -25 to +25° C., preferably -25 to -15° C. The reaction time can be 5 to 60 minutes, preferably 5 to 25 minutes.

[0089] The molar ratio of propionic anhydride to the steroid compound is 6:1 to 9:1, preferably 6:1 to 8:1.

[0090] Compound (VI) can be purified by crystallization from ethyl acetate, isopropyl acetate, methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK), acetone, methanol, ethanol, 2-propanol, acetonitrile, toluene, THF or methyl THF.

[0091] In one of its embodiments, the process of the present invention comprises a further step f) which comprises the selective hydrolysis of compound (VI) to give clapriston.

[0092] Step f) can be carried out by acid hydrolysis under conditions similar to those described in US 3152154 for the hydrolysis of the orthoester. For example, the reaction can be carried out under the conditions reported above for the comparison of compound (VI) of the present invention with compound (VII) of the prior art, i.e., using perchloric acid dissolved in dichloromethane-methanol at 10-12° C.; as previously mentioned, under these conditions, the acid hydrolysis of compound (VI) of the present invention requires 37 hours to complete.

[0093] The hydrolysis of compound (VI) can also be accomplished by enzymatic hydrolysis using conventional batch reactor operation or by using flow reactor operation.

[0094] For example, a sample of 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione (VI) was stirred in a multi-necked flask equipped with a mechanical stirrer and a thermometer at LIPOMOD TM In the presence of 34MDP lipase (Biocatalysts, 115 U / mg), the reaction was carried out in toluene / n-butanol at 44-46°C to hydrolyze the product into 21-hydroxy-17-(-1-oxopropoxy)-pregn-4-ene-3,20-dione (clapristone).

[0095] Similarly, but using an easy-Medchem E series flow reactor from Vapourtec, Bury St Edmunds (GB), UK, equipped with a Compound (VI) dissolved in toluene / n-butanol was hydrolyzed to 21-hydroxy-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione (clapristone) using a 435 column (Candida Antarctica lipase B supported on acrylic resin; columns sold by Strem Chemicals GmbH, Bischheim, France).

[0096] In the enzymatic hydrolysis, the enzyme can be used in free form in the reaction mixture, but is preferably used in supported form.

[0097] The reaction can be carried out under static conditions, but is preferably carried out under flowing conditions.

[0098] The reaction temperature is 40 to 80°C, preferably 50 to 70°C.

[0099] The solvent mixture used in the reaction is composed of toluene and linear alcohols, wherein the main component is toluene. The alcohol that can be used is methanol, ethanol, 1-propanol, preferably n-butanol.

[0100] The content of n-butanol in the toluene / n-butanol mixture is calculated relative to the number of moles of compound (VI). 1 to 10 moles, preferably 2.5 to 5 moles, of n-butanol are used per mole of compound (VI).

[0101] In one embodiment of the present invention, clapriston can be obtained as a solvate from dimethyl sulfoxide (DMSO). In the solvate, clapriston and DMSO are present in a 1:1 molar ratio, as determined, for example, by NMR analysis. The solvate obtained from DMSO can be obtained directly from solution after the enzymatic reaction by replacing the reaction solvent with DMSO or by using a solid intermediate composed of a metastable solvate of methanol (as described in Example 11).

[0102] The powder diffraction spectrum (XPRD) of the solvate is as follows Figure 4 As shown, the DSC thermogram is Figure 5 As shown, and FT-IR spectrum as Figure 6 As shown.

[0103] The XPRD diffraction pattern is characterized by two strong doublets, the first doublet of 2θ has reflection angles of 15.71° and 15.79°, and the second doublet of 2θ has reflection angles of 19.61° and 19.71°; other characteristic 2θ peaks in the XPRD diffraction pattern of this solvate are located at 11.38°, 12.74°, 16.50°, 17.78°, 18.39°, 18.76° and 20.06°, respectively; all of these peaks should be considered to include an approximate value of ±0.2° 2θ.

[0104] The DSC thermogram obtained at a heating rate of 10 °C / min in nitrogen showed a single strong endothermic event with a peak at 87.45 °C.

[0105] Once obtained, the solvate of clapriston with DMSO can be recrystallized several times from the solvent until the desired level of purity is obtained. Methods of recrystallization from solvents are well known to those skilled in the art and involve forming a solution of the substance to be purified in the desired solvent by heating the system to a suitable temperature (about 65° C. in the case of DMSO) and then allowing it to cool until the compound solidifies, which can then be recovered using known methods (e.g., filtration).

[0106] Clapriston solvates formed from DMSO are particularly useful in pharmaceutical compositions for topical application or where increased penetration of the active ingredient into body tissues is desired.

[0107] The use of compounds as active ingredients in DMSO-based formulations is described, for example, in US Pat. No. 3,711,602 of 1973, where many of the examples involve steroids.

[0108] The present invention will be further illustrated by the following examples.

[0109] Instruments, methods, and experimental conditions

[0110] NMR: JEOL 400YH (400 MHz) NMR spectrometer; JEOL delta software version 5.1.1; spectra were recorded in deuterated solvents, such as: 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, CD3OD and DMSO-d6.

[0111] TLC:MERCK:TLC silica gel 60F 254 Aluminum sheet 20 x 20 cm, cod. 1.0554.0001.

[0112] TLC staining: 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 adjust the volume to 1 L with H2O. Immerse the plate with the solution and then heat until the product is detected.

[0113] UPLC-MS: UPLC-MS Waters Acquity chromatography system equipped with PDA and QDa detector.

[0114] UPLC-MS method:

[0115]

[0116] XPRD: A D2Phaser diffractometer (version 2) was used, operating in Bragg-Brentano geometry, equipped with a 6-position rotating multipoint sampler. The X-ray source was an X-ray tube with a copper anode, operated at 30 kV and 10 mA. The analytical wavelength used was copper Kα Kβ radiation was filtered through a nickel filter. The X-ray detector was a linear solid-state detector, model LYNXEYE. The sample was mounted in a thin layer on a "zero background" type silicon sample holder. For the DMSO solvate, the diffraction pattern was recorded over a 2θ range of 4.0–40.0°, with increments of 0.016° and a scan rate of 1.0 s / increment, while for the methanol metastable solvate, a scan rate of 0.25 s / increment was used.

[0117] Data were analyzed using DIFFRAC.EVA software (Bruker).

[0118] DSC: Diamond DSC instrument (Perkin Elmer) operated under a nitrogen atmosphere. Samples were prepared in 40 μL aluminum crucibles with lids and sealed using an appropriate press before analysis. Analysis was performed over a range of 25-210°C at a constant heating rate of 10°C / min.

[0119] ATR-FTIR: FTIR Nicolet 6700 spectrophotometer (ThermoFischer Scientific) equipped with ATR Smart iTR (ThermoFischer Scientific) module with diamond crystal. The acquisition was performed by -1 Within 4cm -1 64 scans were performed at a resolution of 100 nm to measure the analytical sample and a blank (measurement without sample), which was acquired before the sample measurement and automatically subtracted from the blank. Spectra were displayed and analyzed using Omnic software (Thermo Fisher Scientific).

[0120] Note

[0121] Unless otherwise stated, the water used in the experimental instructions should be purified water.

[0122] Unless otherwise stated, organic solvents used in the experimental descriptions should be of "technical" grade.

[0123] Unless otherwise stated, reagents and catalysts used in the experimental descriptions were of commercial quality.

[0124] Example 1

[0125] This example relates to step a) of the process of the invention, from 17α-hydroxyprogesterone (I) to 17-hydroxy-3-(1-pyrrolidinyl)pregna-3,5-diene-20-one (II):

[0126]

[0127] 148.1 g of 17α-hydroxyprogesterone (I) were suspended in 740 ml of methanol. The suspension was heated to reflux (65° C.) and no solid dissolution was observed. 52.4 ml of pyrrolidine were added dropwise: complete dissolution of the starting product was observed and the enamine (II) was almost immediately reprecipitated. The mixture was stirred at this temperature for 2 hours. It was then cooled, first to room temperature and then to 0° C. for 1 hour. It was filtered through a Buchner funnel and washed with 200 ml of cold ethanol. The solid was dried under vacuum at 25° C. for 10 hours to give 166.2 g of compound (II) as an off-white solid.

[0128] 17-α-Hydroxy-Progesterone (I) Analysis:

[0129] 1 H-NMR, CDCl3: 5.74 (1H, s, H-4); 2.77 (1H, s, OH-17); 2.72-2.65 (1H, m); 2.47-2.26 (4H, m); 2.29 (3H, s, H-21); 2.06-2.01 (1H, m); 1. 90-1.81 (2H, m); 1.77-1.56 (7H, m); 1.46-1.33 (3H, m); 1.19 (3H, s, H-19); 1.17-1.07 (1H, m); 1.02-0.95 (1H, m); 0.77 (3H, s, H-18).

[0130] MS:331(M + +1).

[0131] Analysis of compound (II):

[0132] 1 H-NMR, CDCl3: 5.07-5.06 (1H, m, H-6); 4.78 (1H, s, H-4); 3.15-3.12 (4H, m, N- CH2); 2.74 (1H, br, OH); 2.71-2.65 (1H, m); 2.33-2.29 (2H, m); 2.28 (3H, s, H-21); 2.21-2.15 (1H, m); 1.91-1.56 (12H, m); 1.47-1.24 (4H, m); 1.11-1.05 (1H, m); 1.01 (3H, s, H-19); 0.76 (3H, s, H-18).

[0133] Example 2

[0134] This example relates to step b) of the process according to the invention, from the enamine (II) to the intermediate (III), the mixture (21-chloro / 21-bromo)-17α-hydroxy-3-(1-pyrrolidinium-1-ylidene)-pregn-4-en-20-one chloride:

[0135]

[0136] 83 g of compound (II) obtained in the previous example was suspended in 1660 ml of ethanol at 20-25°C under a nitrogen atmosphere. 125.6 g of 33% w / w HCl in ethanol was added; complete dissolution was observed. A previously prepared ethanolic solution of bromine (16.6 ml of dibromine dissolved in 415 ml of ethanol) cooled to -55°C was then added dropwise over approximately 90 minutes. A precipitate was observed near the end of the addition. After the addition was complete, the mixture was stirred at 20-25°C for approximately 1 hour and monitored by TLC: the starting material had almost completely disappeared. The solvent was removed by rotary evaporation under vacuum at 45°C, followed by stripping three times with MTBE (450 ml each), leaving a total volume of approximately 330 ml. The mixture was cooled to 0°C and maintained under stirring for 1 hour. The mixture was then filtered through a Buchner funnel and washed with cold MTBE. The product was dried under vacuum at 45°C for 2 hours to yield 106.6 g of intermediate (III) as a white powder.

[0137] Analysis of intermediate (III):

[0138] 1 H-NMR, DMSO: 6.51 (1H, s, H-4); 5.62 (1H, br, OH-17); 4.60 (1H, A part of AB system, J AB =15 Hz, H-21); 4.37 (1H, B part of AB system, J AB =15Hz, H-21); 3.98-3.78 (4H, m, N- CH2); 2.89-2.74 (2H, m); 2.61-2.55 (3H, m); 2.05-1.19 (16H, m); 1.12 (3H, s, H-19); 1.09-0.99 (1H, m); 0.95-0.89 (1H, m); 0.56 (3H, s, H-18).

[0139] In the spectrum, the following peaks belonging to the 21-chloro derivative of the imino group are also present: 5.59 (1H, br, OH-17); 4.79 (1H, A part of the AB system, J AB =17 Hz, H-21); 4.48 (1H, B part of AB system, J AB =17Hz, H-21).

[0140] MS:462,464(M + +1) 21-bromo;

[0141] MS:418,420(M + +1) 21-Chloro.

[0142] Example 3

[0143] This example relates to step c) of the process according to the invention, from intermediate (III) to intermediate (IV), namely the mixture 21-chloro / 21-bromo-17α-hydroxypregna-4-ene-3,20-dione:

[0144]

[0145] 105.5 g of intermediate (III) obtained in the previous example was dissolved in 1582 ml of methanol. Aqueous potassium bicarbonate solution (114.5 g of KHCO₃ dissolved in 458 g of water) was added, and the mixture was stirred at 25°C for approximately 5 hours. TLC indicated a complete reaction. 2000 ml of water was added, and the mixture was stirred for 30 minutes. The mixture was filtered through a Buchner funnel and washed with 500 ml of water.

[0146] The product was dried in a vacuum oven at 50° C. for 16 hours to obtain 75.1 g of an off-white solid, which was recrystallized from 225 ml of a 1:1 (v / v) MEK-methanol mixture to obtain 70.2 g of intermediate (IV) as an off-white solid.

[0147] Analysis of intermediate (IV):

[0148] 1 H-NMR, DMSO-d6: 5.63 (1H, s, H-4); 5.56 (1H, s, OH-17); 4.58 (1H, A part of AB system, J AB=15Hz, H-21); 4.35 (1H, B part of AB system, J AB =15Hz, H-21); 2.60-2.53 (1H, m); 2.45-2.36 (2H, m); 2.26-2.13 (2H, m); 1.99-1.94 (1H, m); 1. 84-1.18 (11H, m); 1.14 (3H, s, H-19); 1.05-0.94 (1H, m); 0.92-0.85 (1H, m); 0.56 (3H, s, H-18).

[0149] In the spectrum, the following peaks belonging to the 21-chloro derivative are also present: 5.54 (1H, s, OH-17); 4.77 (1H, A part of the AB system, J AB =17 Hz, H-21); 4.46 (1H, B part of AB system, J AB =17Hz, H-21).

[0150] MS:409,411(M + +1)21-bromo; 365,367 (M + +1) 21-Chloro.

[0151] Example 4

[0152] This example relates to step d) of the process of the invention, obtaining compound (V) 21-acetoxy-17α-hydroxypregn-4-ene-3,20-dione from intermediate (IV):

[0153]

[0154] 70g of the intermediate (IV) obtained in the previous embodiment was suspended in 2100ml of acetone under a nitrogen stream. 190.8ml of TEA and 39.2ml of glacial acetic acid were added and heated to reflux (58°C). No clear solution was observed. After 5 hours, the reaction was essentially complete. The solvent was removed by rotary evaporator, and the residue was absorbed by water (650ml) and DCM (450ml), and the layers were separated. The aqueous layer was extracted again with DCM (100ml), and the combined organic layers were washed with water (2x400ml). The solvent was removed by rotary evaporator, and 400ml of MEK was added. The solvent was removed by rotary evaporator until a paste was obtained. 400ml of MEK was added and the operation was repeated. 400ml of MEK was added and the solvent was removed until a mixture of about 350ml in volume was obtained. The mixture was cooled to 0°C and kept for 1 hour, and filtered through a Buchner funnel and washed with cold MEK (80ml). The product was dried in a vacuum oven at 45° C. to obtain 59.1 g of compound (V) as a white solid.

[0155] Analysis of compound (V):

[0156] 1 H-NMR, CDCl3: 5.73 (1H, s, H-4); 5.08 (1H, A part of AB system, J AB =17 Hz, H-21); 4.87 (1H, B part of AB system, J AB =17Hz, H-21); 2.76-2.69 (1H, m); 2.72 (1H, s, OH-17); 2.48-2.26 (4H, m); 2.17 (3H, s, CO- CH3 ); 2.07-2.01 (1H, m); 1.90-1.33 (11H, m); 1.19 (3H, s, H-19); 1.15-1.04 (1H, m); 1.01-0.94 (1H, m); 0.72 (3H, s, H-18).

[0157] MS:389(M + +1).

[0158] Example 5

[0159] This example relates to step e) of the method of the present invention, from compound (V) to the target compound (VI) of the present invention, 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione:

[0160]

[0161] 28.8g of compound (V) obtained in the previous embodiment was dissolved in 720ml DCM under a nitrogen stream. 71.1ml of propionic anhydride was added and the mixture was cooled to -20°C. 7.3ml of 70% by weight HClO4 aqueous solution was added and an exothermic reaction was observed at -20 to -15°C. The mixture was stirred at -20°C for 10 minutes. After the reaction was complete, the reaction mixture was poured into 650ml of NaHCO3 saturated aqueous solution and stirred for 30 minutes. The layers were separated and the aqueous layer was extracted again with 100ml of DCM. The combined organic layers were washed with water (2x300 ml). DCM was removed under vacuum by a rotary evaporator until a paste was obtained. 350ml of heptane was added and the solvent was removed until a paste was obtained. 350ml of heptane was added and the solvent was distilled until a mixture with a residual volume of approximately 290ml was obtained. The mixture was stirred at 25°C for 1 hour and filtered through a Buchner funnel, washing with heptane. The product was dried in a vacuum oven at 45°C to give 32.3 g of an off-white solid (Compound (VI).

[0162] Analysis of compound (VI):

[0163] 1 H-NMR, CDCl3: 5.75 (1H, s, H-4); 4.89 (1H, A part of AB system, J AB =16 Hz, H-21); 4.63 (1H, B part of AB system, J AB =17Hz, H-21); 2.88-2.81 (1H, m); 2.49-2.27 (6H, m); 2.17 (3H, s, CO- CH3 ); 2.08-2.03 (1H, m); 1.95-1.60 (9H, m); 1.53-1.34 (2H, m); 1.20 (3H, s, H-19); 1.17-1.10 (1H, m); 1.16 (3H, t, J = 7Hz, CH2- CH3 ); 1.07-0.99 (1H, m); 0.76 (3H, s, H-18).

[0164] MS:445(M + +1).

[0165] HPLC (purity): 99%, chromatogram as shown Figure 1 shown.

[0166] DSC and XPRD analysis were also performed on the samples under the above test conditions; the results of the two tests are shown in Figure 2. Figure 2 and Figure 3 shown.

[0167] Example 6

[0168] This example relates to the hydrolysis of compound (VI) of the present invention to clapriston using a supported enzyme in a flow reactor.

[0169]

[0170] The method was performed using a Vapourtec easy-Medchem E-series flow reactor, in which a tubular reactor was supplied filled with 845 mg of 435 (acrylic resin-supported Candida Antarctica lipase B).

[0171] 14.28 g of compound (VI) was dissolved in 1000 ml of toluene in a bottle designed to be connected to a flow reactor; 7.5 ml of n-butanol was added and stirred until dissolved. The solution was passed through a tubular reactor filled with enzyme, maintained at 60°C, and a flow rate of 0.1 ml / min.

[0172] The reaction solution samples were collected and analyzed by UPLC-MS to monitor the progress of clapriston conversion.

[0173] From the data shown in the table below, it can be seen that the efficiency of the enzyme remains unchanged even after more than 100 hours of constant flow reaction.

[0174]

[0175]

[0176] Example 7

[0177] This example involves the hydrolysis of compound (VI) to clapriston using a supported enzyme in a conventional sealed reactor.

[0178]

[0179] In a 100 ml glass reactor, 250 mg of 21-acetoxy-17α-propoxyprogesterone (VI) was dissolved in 17.5 ml of toluene and 250 mg of 435 (Candida Antarctica lipase B supported on acrylic resin) was added, and finally 257 μl of n-butanol was added. The mixture was stirred and heated to 60° C., and the progress of the reaction was monitored over time by UPLC analysis.

[0180] After stirring for 14 hours and 30 minutes, the contents of residual compound (VI) and clapriston in the reaction mixture were calculated based on the area of ​​each peak in the UPLC chromatogram to be 0.75% and 96%, respectively.

[0181] Example 8

[0182] This example relates to the enzymatic hydrolysis of compound (VI) according to the invention to clapriston, in comparison with the enzymatic hydrolysis of the symmetrical diester 17,21-bis(1-oxopropoxy)-pregn-4-ene-3,20-dione (VII) described in WO 2009 / 019138 operated in a conventional reactor.

[0183]

[0184] In a 50 ml glass flask, 250 mg of 21-acetoxy-17α-propoxyprogesterone (Compound (VI), UPLC purity = 99.7%) was dissolved in 17.5 ml of toluene; 250 mg of 435 (Candida Antarctica lipase B supported on acrylic resin) was added, and finally 257 μl of di-n-butanol was added. The mixture was stirred and heated to 60° C., and the progress of the reaction was monitored over time by UPLC analysis.

[0185] The experiment was repeated using 17,21-dipropoxy-17α-progesterone (Compound (VII), UPLC purity = 99.5%) under the same conditions except that 258 mg of Compound (VII) was used due to its higher molecular weight than Compound (VI).

[0186] The progress of the clapriston conversion was monitored by UPLC-MS analysis and the results obtained are reported in the table below (clapriston is represented in the table as CLA). It can be seen that compound (VI) hydrolyzes faster than compound (VII).

[0187]

[0188] Example 9

[0189] This example is concerned with obtaining clapriston solvated with DMSO.

[0190] 445 ml of the solution obtained at the end of the reaction in Example 6 was concentrated at 50° C. under reduced pressure until 14.3 g of solution was obtained. 6.2 ml of dimethyl sulfoxide was then added, and evaporation was continued at 50° C. under reduced pressure until a solution in which the solvent consisted of at least 99% dimethyl sulfoxide was obtained (GC control). The solution was then stirred at 20-25° C. for 16 hours to obtain a solid precipitate, which was filtered and subjected to wet XPRD analysis. The obtained diffraction pattern is shown in FIG. Figure 4 The main peaks of the diffraction pattern characterized by 2θ (±0.2°) angular position and relative intensity are shown in the following table:

[0191] 2θ(°) strength(%) 11.38 47.3 12.74 51.8 15.71 96.2 15.79 100.0 16.50 32.4 17.78 65.5 18.39 48.3 18.76 25.4 19.61 79.2 19.71 86.4 20.06 40.3

[0192] The wet solid (3.5 g) was then dissolved in 3.5 ml of dimethyl sulfoxide at 60° C. with stirring, and the solution was then cooled to 25° C. over approximately 1 hour and stirred for 4 hours.

[0193] The precipitated solid separated by filtration was dried under reduced pressure at 40°C for 16 hours (2.6 g of white solid) and subjected to XPRD, DSC, FT-IR, 1 H-NMR (CDCl3) analysis. The XPRD diffraction pattern of the dry product was the same as that of the wet product ( Figure 4 ); DSC thermogram and FT-IR spectrum are as follows Figure 5 and Figure 6 As shown ( Figure 5 The DSC thermogram shows an effective range from 25°C to 155°C.) NMR spectroscopy showed that the solid was a solvate of clapriston and dimethyl sulfoxide in a 1:1 molar ratio.

[0194] HPLC purity: >99%.

[0195] Example 10

[0196] This example relates to the enzymatic hydrolysis of compound (VI) of the invention to clapriston, compared with the enzymatic hydrolysis of the symmetrical diester 17,21-bis(1-oxopropoxy)-pregn-4-ene-3,20-dione (VII) by enzymes supported on an inert material in a flow reactor.

[0197] 1.01 g of compound (VII) (purity 99.5%) was dissolved in 68.5 ml of toluene, 1.01 ml of n-butanol was added, and the mixture was stirred until dissolved.

[0198] The solution thus obtained was passed through a pre-filled 1.068 g A 435-well tubular reactor was operated at a flow rate of 0.134 ml / min and a residence time of 19.1 minutes at 60° C. The instrument used was a Vapourtec easy-Medchem E-series.

[0199] Under the same conditions, the operation was carried out in a similar manner using a solution of compound (VI) (purity 99.6%), also in toluene and n-butanol.

[0200] The reaction solution samples were collected and analyzed by UPLC-MS to monitor the progress of clapriston conversion.

[0201] The UPLC peak area ratio of the unreacted compound (VII) to the unreacted compound (VI) was 1.64.

[0202] Example 11

[0203] This example concerns the preparation of clapriston solvated in DMSO from its metastable solvate in methanol.

[0204] 632 ml of the solution obtained at the end of the reaction in Example 6 were distilled at 50° C. under reduced pressure until the weight reached 9 g.

[0205] The solution was taken up in methanol and concentrated three times under reduced pressure at 50° C. (26.4 ml of methanol were used in each dilution / concentration cycle) to ensure that there was always a solution.

[0206] 8.8 ml of methanol were added, and the solution was stirred at 20-25°C for 30 minutes and then at 4°C for 6 hours.

[0207] The suspension thus obtained was filtered to give a white solid, whose XPRD diffractogram was immediately recorded (wet product) and which showed a solid phase different from any known form. Figure 7 The main peaks of the diffraction pattern characterized by 2θ angle position and relative intensity are shown in the following table:

[0208] 2θ(°) strength(%) 5.74 100.0 6.02 58.6 11.49 25.2 11.63 11.4 11.74 13.1 15.67 24.4 17.24 10.4

[0209] The wet solid was quickly dissolved in 7 ml of dimethyl sulfoxide.

[0210] The solution thus obtained was distilled under reduced pressure at 50°C to remove any methanol residues, and then stirred at room temperature for 4 hours.

[0211] The suspension thus obtained was filtered and the solid was dried in a vacuum oven at 40°C for 16 hours.

[0212] 2.3 g of a white solid was obtained, the XPRD diffraction pattern of which corresponded to Figure 4 Shown is the diffraction pattern of clapriston solvated in dimethyl sulfoxide.

[0213] Example 12

[0214] This example is concerned with obtaining clapriston solvated with DMSO.

[0215] 3100 ml of the reaction solution containing approximately 40 g of clapriston, obtained in the same manner as described in Example 6, was concentrated at 50° C. under reduced pressure until 144.2 g of solution was obtained. 40 ml of DMSO was added, and the solution was further concentrated at 50° C. under reduced pressure until a final weight of 84.9 g was obtained. The solution was heated to 65° C., cooled to 20° C. over approximately 1 hour, and stirred for 22 hours (solid precipitation was observed). After filtration, the wet product was dried under reduced pressure at 40° C. for 20 hours to obtain 34.3 g of DMSO-solvated clapriston (white solid, UPLC purity = 99.19%).

[0216] DMSO solvated clapriston (34.3 g) was further purified by recrystallization. The solvate was mixed with 23.4 ml of DMSO. The suspension was heated to 65° C. and stirred for 10 minutes, then cooled to 20° C. over 1 hour and stirred at 20° C. for 22 hours. The suspension was filtered and the wet solid was dried under reduced pressure in a 40° C. oven for 20 hours to obtain DMSO solvated clapriston as a white solid with a UPLC purity of 99.70%.

[0217] Example 13

[0218] This example involves obtaining DMSO-solvated clapriston using seed crystal initiation.

[0219] 3350 ml of the reaction solution containing 43 g of clapriston, obtained in the same manner as described in Example 6, was concentrated at 50° C. under reduced pressure until 93.2 g of solution was obtained. 43 ml of DMSO was added, and distillation was continued under reduced pressure to a final weight of 92.5 g. The solution was heated to 65° C., stirred for 10 minutes, and then cooled to 50° C. over approximately 15 minutes. 0.23 g of DMSO-solvated clapriston, obtained by the procedure described in Example 12, was added and stirred for 10 minutes. The suspension was cooled to 20° C. over 1 hour and then stirred at 20° C. for 18 hours. The solid obtained was filtered and then dried in an oven at 40° C. under reduced pressure for 20 hours to obtain 43.4 g of DMSO-solvated clapriston (white solid, UPLC purity = 99.41%).

[0220] Also in this case, as in Example 12, the obtained DMSO-solvated clapristone can be recrystallized from DMSO until the desired level of purity is obtained.

Claims

1. A method for synthesizing the compound of formula (VI) 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione: The following steps are involved: a) 17α-hydroxyprogesterone (I) reacts with pyrrolidine to obtain compound (II) 17-hydroxy-3-(1-pyrrolidinyl)pregna-3,5-dien-20-one: b) Compound (II) is first reacted with hydrochloric acid and then with bromine to obtain intermediate (III), a mixture of (21-chloro / 21-bromo)-17α-hydroxy-3-(1-pyrrolidinium-1-ylidene)-pregn-4-en-20-one chlorides: c) Intermediate (III) is subjected to alkaline hydrolysis to obtain intermediate (IV), i.e., the corresponding mixture of (21-chloro / 21-bromo)-17α-hydroxypregnane-4-ene-3,20-dione: d) The intermediate (IV) reacts with acetic acid to obtain compound (V) 21-acetoxy-17α-hydroxypregn-4-ene-3,20-dione: e) Compound (V) reacts with perchloric acid and propionic anhydride to obtain compound (VI) 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione:

2. The method according to claim 1, further comprising step f): selectively hydrolyzing the compound 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione of formula (VI) to obtain the compound 21-hydroxy-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione (clapristone).

3. The process according to claim 2, wherein step f) is performed by acid hydrolysis.

4. The method according to claim 2, wherein step f) is performed by enzymatic hydrolysis.

5. The method according to claim 4, wherein the enzymatic hydrolysis is performed using supported lipase as a reactant.

6. A process according to claim 4 or 5, wherein the operation is carried out in a flow reactor.

7. The process according to claim 6, wherein the operation is carried out in the presence of toluene and alcohol.

8. The method of claim 7, wherein the alcohol is n-butanol.

9. Compound 21-(acetyloxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione (VI):

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