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

Abstract

The present invention relates to a process for the preparation of 21-(acetoxy)-17-(1-oxopropoxy)-pregn-4-ene-3, 20-dione (VI) having the following formula: the compound (VI) can be used as a precursor for the synthesis of a steroidal compound, i.e., Claristone, for the treatment of acne, and to a process for the preparation of 21-(acetoxy)-17-(1-oxopropoxy)-pregn-4-ene-3, 20-dione (VI).

Description

[0001] Divisional application

[0002] This application is a divisional application of application number 2021800716293, filed on February 5, 2021, entitled “Preparation method of 21-(acetoxy)-17-(1-propionyloxy)-pregn-4-ene-3,20-dione”. Technical Field

[0003] This invention relates to the field of methods for synthesizing pharmaceutically active ingredients, and particularly to a method for preparing 21-(acetoxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione on an industrial scale, the compound having the following structural formula (VI):

[0004]

[0005] This is a useful precursor for the synthesis of 21-hydroxy-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione (also known as Clascoterone).

[0006] Clascoterone is a steroidal drug containing a properly formulated pregnane backbone, recently approved by the U.S. Food and Drug Administration (FDA) for the treatment of acne in children aged 12 years and older and adults. The structural formula of clascoterone is shown below:

[0007] Background Technology

[0008] Clastrone is described in U.S. Patent 3,152,154, dated 1964. As shown above, this compound is a 17α-monoester of a 17α,21-dihydroxysteroid.

[0009] According to the teachings of US 3,152,154, the 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 types:

[0010]

[0011] The experimental description reported in US 3,152,154 does not provide any details regarding the reaction yield and the quality of the resulting product.

[0012] 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 reaction yield and the quality of the resulting product.

[0013] Specifically, for the preparation of clasone, the starting compound for preparing the orthoester to be hydrolyzed will be 17,21-dihydroxy-pregn-4-ene-3,20-dione, a compound called "cortexolone" with the following structural formula:

[0014]

[0015] However, this compound is only available on the market in laboratory quantities, not in the quantities required for industrial production.

[0016] Another possible precursor to clavone is compound 17,21-bis(1-oxopropoxy)-pregn-4-ene-3,20-dione, which has the following structural formula (VII):

[0017]

[0018] 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”, RBT Burner, J. Am. Chem. Soc. 1953, 75, 14, 3489-3492. However, the acid hydrolysis of compound (VII) requires a relatively long time and produces a non-negligible amount of byproducts.

[0019] WO 2009 / 019138 A2 also recommends using a selective enzymatic hydrolysis of symmetrical diesters using lipases, where two ester groups share the same R group:

[0020]

[0021] The object of this invention is to provide a novel intermediate that can be used to synthesize clastron, and a method for synthesizing said intermediate on an industrial scale. Summary of the Invention

[0022] This invention achieves this objective, and in its first aspect, the invention relates to a method for synthesizing compound 21-(acetoxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione of formula (VI):

[0023]

[0024] The compound (VI) can be used as a precursor for the synthesis of claspone, and the method includes the following steps:

[0025] a) The reaction of 17α-hydroxyprogesterone (I) with pyrrolidine yields compound (II) 17-hydroxy-3-(1-pyrrolyl)pregn-3,5-dien-20-one:

[0026]

[0027] b) Compound (II) reacts first with hydrochloric acid, then with bromine, to give intermediate (III), a mixture of (21-chloro / 21-bromo)-17α-hydroxy-3-(1-pyrrolidone-1-ylidene)-pregn-4-en-20-one chlorides:

[0028]

[0029] c) Alkaline hydrolysis of intermediate (III) yields intermediate (IV), namely, the corresponding mixture of 21-chloro / 21-bromo-17α-hydroxypregn-4-ene-3,20-dione:

[0030]

[0031] d) Intermediate (IV) reacts with acetic acid to give compound (V) 21-acetoxy-17α-hydroxypregn-4-ene-3,20-dione:

[0032]

[0033] e) Compound (V) reacts with perchloric acid and propionic anhydride to give compound (VI) 21-(acetoxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione:

[0034]

[0035] The method of the present invention may further include an additional step f): selectively hydrolyzing compound (VI) to obtain clastoline.

[0036]

[0037] Step f) can be carried out via chemical or enzymatic pathways.

[0038] In a second aspect, the present invention relates to compound (VI)21-(acetoxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione.

[0039] In a third aspect, the present invention relates to the preparation of clastosteroids by enzymatic hydrolysis of compound (VI) in a flow reactor.

[0040] Finally, in a fourth aspect, the present invention relates to dimethyl sulfoxide-solventized clastron. Attached Figure Description

[0041] Figure 1 An HPLC chromatogram of compound 21-(acetoxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione, which can be obtained by the method of the present invention, is shown.

[0042] Figure 2 The XPRD diffraction spectrum of compound 21-(acetoxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione, which can be obtained by the method of the present invention, is shown.

[0043] Figure 3 The DSC thermograph of compound 21-(acetoxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione, which can be obtained by the method of the present invention, is shown.

[0044] Figure 4 The XPRD diffraction pattern of dimethyl sulfoxide solvated claspone is shown, along with the relative angle data and relative intensity of each peak.

[0045] Figure 5 The DSC thermogram of dimethyl sulfoxide solvated claspone is shown.

[0046] Figure 6 The FT-IR spectrum of dimethyl sulfoxide solvated clastosone is shown.

[0047] Figure 7 The XPRD diffraction pattern of methanol-solventized clastodone is shown. Detailed Implementation

[0048] The inventors have discovered that in the production of clastoline, "asymmetric" diester hydrolysis provides better results than symmetric diester hydrolysis.

[0049] In the following description, when the ratio of solvent quantity to compound quantity is provided as "volume per weight," the volume of solvent is understood to be measured in milliliters and the weight of compound in grams. Furthermore, for simplicity or clarity, in some cases, the stereochemical configurations of some atoms in the steroidal compound skeleton are not shown in the figures herein; in these cases, the stereochemistry of the molecule can be understood to correspond to the native configuration of the steroidal compound.

[0050] The term "asymmetric" diester refers to the following types of structures:

[0051]

[0052] The alkyl groups R and R' are different.

[0053] Experimental examination revealed that the asymmetric diester exhibited more favorable acid hydrolysis behavior than the symmetric 17,21-bis(1-oxopropoxy)-pregn-4-ene-3,20-dione of formula (II) as described in WO 2009 / 019138 A2; to avoid confusion with compound (II) of the present invention (method intermediate 17-hydroxy-3-(1-pyrrolidinyl)pregn-3,5-diene-20one), compound (II) of WO 2009 / 019138A2 will be referred to as compound (VII) in this specification.

[0054] In fact, according to the inventors' observations, when compound (VI) of the present invention and compound (VII) of WO 2009 / 019138A2 were subjected to acid hydrolysis reaction (perchloric acid dissolved in dichloromethane-methanol at 10-12°C) under the same conditions, 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.

[0055] Furthermore, the composition of the mixture differed at the end of the reaction, and as shown in Table 1 below, the best results for clastoline yield were obtained when using compound (VI) (the percentage concentrations shown in the table are calculated based on the peak area from the HPLC test):

[0056] Table 1

[0057]

[0058]

[0059] Aside from residual unreacted reagents, the only byproduct present in considerable amounts in both reaction products is a byproduct known as “transposition,” as described in the article “Corticosteroid 17α-monoesters from 17α,21-cyclic orthoesters,” R. Gardi et al., Tetrahedron Letters (13) 1961, pages 448-451. Its formation cannot be inhibited because it is specific to the reaction products under the reaction conditions and is independent of the initial reactants. The transposition reactions between positions 17 and 21 of the steroid compound are summarized below:

[0060]

[0061] According to the reaction mechanism outlined below, a monoester having a free hydroxyl group at position 17 and a hydroxyl group at position 21 is characterized by its instability under acidic conditions, leading to the migration of the acylated group from position 17 to position 21:

[0062]

[0063] In a first aspect, the present invention relates to a method for synthesizing 21-(acetoxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione, said method comprising the five synthetic steps a) to e) described above.

[0064] Step a) involves reacting compound (I) with pyrrolidine to yield the corresponding enamine compound 17-hydroxy-3-(1-pyrrolyl)pregn-3,5-dien-20-one (II), which is carried out as follows: a suspension of compound (I) in an alcohol is prepared, the suspension is refluxed, and then pyrrolidine is added. The starting compound (I), 17α-hydroxyprogesterone, is widely available on the market and does not require a synthetic orthoester as a method intermediate.

[0065] The molar amount of pyrrolidine is 20 to 60% excess relative to compound (I), preferably 40%.

[0066] Alcohols that can be used to prepare suspensions include ethanol and isopropanol, with methanol being preferred.

[0067] The reaction mixture is kept under reflux for 1 to 3 hours, preferably 1.5 to 2.5 hours.

[0068] Compound (II) was obtained by crystallization precipitation from the reaction solvent.

[0069] Step b) involves reacting enamine (II) first with hydrochloric acid and then with bromine to give intermediate (III), which is a mixture of chlorides of (21-chloro / 21-bromo)-17α-hydroxy-3-(1-pyrrolidone-1-ylidene)-pregn-4-en-20-one.

[0070] The reaction is carried out at a temperature of 10 to 40°C, preferably 20 to 30°C.

[0071] The reaction solvent is an alcohol selected from methanol, isopropanol, and preferably ethanol. The amount of alcohol used is 15 to 50 volumes relative to the weight of compound (II), preferably 15 to 30 volumes.

[0072] Hydrochloric acid is used in the form of a 33% (by weight) solution of ethanol or isopropanol; the amount of this solution used in the reaction is 1 to 3 times the weight of compound (II), preferably 1.5 times.

[0073] The amount of bromine used (in moles) is 1.0 to 3 times the molar amount of compound (II), preferably 1.5 times.

[0074] Bromine is added in the form of an ethanol solution, with a bromine-to-ethanol volume ratio of 1:20 to 1:45, preferably 1:25. Before adding the solution prepared in the first part of this step, the bromine ethanol solution is cooled to -50 to -60°C, preferably -55°C. The bromine solution is added over 20 minutes to 2 hours, preferably over 80 minutes to 100 minutes.

[0075] The intermediate (III) obtained at the end of step b) can be crystallized using a straight-chain or branched alcohol, ether or mixture thereof with 1-4 carbon atoms; the preferred solvent for crystallizing intermediate (III) is methyl tert-butyl ether (MTBE).

[0076] The reaction result is a mixture of intermediate (III) (21-chloro / 21-bromo)-17α-hydroxy-3-(1-pyrrolidone-1-ylidene)-pregn-4-en-20-one chloride, which is used as is in the continuation of the synthesis because both products react in the same manner to give the desired 21-acetoxy product; therefore, in this specification, mixture (III) is represented as a single reaction intermediate.

[0077] The minor component of mixture (III) is 21-chloro-steroidal compounds, which account for 5-30%.

[0078] In an alternative embodiment, step b) can be carried out by reacting enamine (II) directly with hydrobromic acid, in which case only compound 21-bromine as intermediate (III) is obtained.

[0079] Step c) of the method of the present invention includes 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 is in the following method reaction, and therefore is referred to as a single intermediate, namely intermediate (IV), in this specification.

[0080] 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. A water / methanol mixture is preferred, wherein the volume of methanol is greater than 70% of the total volume.

[0081] The alkali used can be selected from NaHCO3, Na2CO3, KHCO3 or K2CO3; KHCO3 is preferred, and its molar amount is more than twice the molar amount of intermediate (III).

[0082] 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.

[0083] The reaction time is 2 to 16 hours, preferably 4 to 6 hours.

[0084] 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), straight-chain or branched alcohols with 1-4 carbon atoms, or mixtures thereof; for this operation, a 1:1 (v / v) MEK-MeOH mixture is preferred, and the process is carried out using 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.

[0085] In the next step d), intermediate (IV) reacts to give compound (V) 21-acetoxy-17α-hydroxypregn-4-ene-3,20-dione.

[0086] 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 mixtures thereof, in the presence of an inorganic base selected from KHCO3, NaHCO3, K2CO3, or Na2CO3, or an organic base selected from triethylamine (TEA), trimethylamine (TMA), or pyridine, using glacial acetic acid. Preferably, it 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.

[0087] 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.

[0088] The obtained compound (V) can be crystallized using solvents such as methyl tert-butyl ether (MTBE), acetone, methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK), straight-chain or branched alcohols of 1-4 carbon atoms or mixtures thereof; preferred solvents are methyl ethyl ketone (MEK) and ethanol.

[0089] Finally, step e) of the method of the present invention includes reacting compound (V) with perchloric acid and propionic anhydride to obtain compound (VI) 21-(acetoxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione.

[0090] The reaction is carried out by diluting compound (V) in dichloromethane (DCM), wherein the amount of DCM used is 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.

[0091] The molar ratio of propionic anhydride to steroidal compound is 6:1 to 9:1, preferably 6:1 to 8:1.

[0092] 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.

[0093] In one embodiment, the method of the present invention includes another step f), which includes the selective hydrolysis of compound (VI) to obtain clastosteroids.

[0094] Step f) can be carried out by acid hydrolysis under conditions similar to those described in US 3152154 for the hydrolysis of orthoester esters. For example, the reaction can be carried out under the conditions reported above for comparing the compound (VI) of the present invention and the compound (VII) of the known art, i.e., at 10-12°C with perchloric acid dissolved in dichloromethane-methanol; as previously mentioned, under these conditions, the acid hydrolysis of the compound (VI) of the present invention requires 37 hours to complete.

[0095] The hydrolysis of compound (VI) can also be accomplished by enzymatic hydrolysis using conventional batch reactor operation or by using flow reactor operation.

[0096] For example, a sample of 21-(acetoxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione (VI) was tested in a multi-necked flask equipped with a mechanical stirrer and a thermometer in LIPOMOD. TM In the presence of 34MDP lipase (Biocatalysts, 115 U / mg), it reacts in toluene / n-butanol at 44-46 °C to hydrolyze to 21-hydroxy-17-(-1-oxopropoxy)-pregn-4-ene-3,20-dione (clastron).

[0097] Similarly, but operated using the easy-Medchem E series flow reactor from Bury St Edmunds (GB) Vaportec, UK, which is equipped with a packed reactor. A 435 column (Candida Antarctica lipase B supported on acrylic resin; column sold by Bischheim Strem Chemicals, AG, France) hydrolyzed compound (VI) dissolved in toluene / n-butanol to 21-hydroxy-17-(-1-oxopropoxy)-pregn-4-ene-3,20-dione (clastron).

[0098] In enzymatic hydrolysis, the enzyme can be used in free form in the reaction mixture, but it is preferred to use it in loaded form.

[0099] The reaction can be carried out under static conditions, but is preferably carried out under flowing conditions.

[0100] The reaction temperature is 40 to 80°C, preferably 50 to 70°C.

[0101] The solvent mixture used in the reaction consists of toluene and a straight-chain alcohol, with toluene being the main component. Methanol, ethanol, 1-propanol, and preferably n-butanol can be used.

[0102] The content of n-butanol in the toluene / n-butanol mixture is calculated relative to the moles of compound (VI). One to ten moles, preferably 2.5 to 5 moles, of n-butanol are used per mole of compound (VI).

[0103] In one embodiment of the invention, the solvate clastoline can be obtained from dimethyl sulfoxide (DMSO). In the solvate, clastoline 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 using DMSO instead of the reaction solvent or by using a solid intermediate consisting of a metastable solvate of methanol (as described in Example 11).

[0104] The powder diffraction (XPRD) spectrum of the solvate is as follows: Figure 4 As shown, the DSC thermogram is as follows Figure 5 As shown, and the FT-IR spectrum as shown Figure 6 As shown.

[0105] 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 these peaks should be considered to include an approximation of ±0.2°2θ.

[0106] The DSC thermogram obtained in nitrogen at a heating rate of 10 °C / min showed a single strong endothermic event with a peak at 87.45 °C.

[0107] Once obtained, the solvate of clastolide with DMSO can be recrystallized from the solvent several times until the desired level of purity is achieved. Methods for recrystallization from the solvent are well known to those skilled in the art and involve heating the system to a suitable temperature (approximately 65°C in the case of DMSO) to form a solution of the substance to be purified in the desired solvent, then cooling it until the compound solidifies, which can then be recovered using known methods (e.g., filtration).

[0108] Clarithone solvates formed from DMSO are particularly useful in pharmaceutical compositions for topical application or in cases where increased penetration of the active ingredient into body tissues is required.

[0109] The use of compounds as active ingredients in DMSO-based formulations is described, for example, in U.S. Patent 3,711,602 of 1973, in which many examples relate to steroidal compounds.

[0110] The present invention will be further illustrated by the following embodiments.

[0111] Instruments, methods and experimental conditions

[0112] NMR: JEOL 400YH (400MHz) NMR spectrometer; JEOLδ software version 5.1.1; spectra were recorded in deuterated solvents, e.g., chloroform-d, D 99.8%, with 0.1% (v / v) tetramethylsilane (TMS) as an internal standard; and chloroform-d, “100%”, D 99.96%, with 0.03% (v / v) TMS, CD3OD and DMSO-d6.

[0113] TLC:MERCK:TLC Silicone 60F 254 Aluminum plate, 20 x 20 cm, COD. 1.0554.0001.

[0114] TLC staining: Cerium phosphomolybdate: Dissolve 25 g of phosphomolybdic acid and 10 g of cerium sulfate (IV) in 600 ml of H2O. Add 60 ml of 98% H2SO4 and adjust the volume to 1 L with H2O. Impregnate the plate with the solution and then heat until the product is detected.

[0115] UPLC-MS: UPLC-MS Waters Acquity chromatography system, equipped with PDA and QDa detectors.

[0116] UPLC-MS method:

[0117] Column: Waters Acquity BEH C18, 2.1(ID) x 50(L) mm, 7μm;

[0118] Flow rate: 0.8 ml / min;

[0119] Mobile phase: Acetonitrile / water ratio of 1:1 (0-0.5') to 9:1 (0.5'-2.5');

[0120] pH adjuster: 0.01% formic acid;

[0121] Ultraviolet detector: 244nm;

[0122] XPRD: The D2Phaser diffractometer (version 2) operates in Bragg-Brentano geometry and is equipped with a 6-position rotating multi-spot sampler. The X-ray source is an X-ray tube with a copper anode, operating at 30 kV and 10 mA. The analytical wavelength used is copper Kα. Kβ radiation was filtered by a nickel filter. The X-ray detector was a linear solid-state detector, model LYNXEYE. The sample was layered in thin sheets on a silicon sample holder of the "zero background" type. In the case of DMSO solvate, the diffraction pattern was recorded in the range of 4.0–40.0° at 2θ angles in increments of 0.016° at a scan rate of 1.0 s / increment, while in the case of methanol metastable solvate, a scan rate of 0.25 s / increment was used.

[0123] The data was analyzed using DIFFRAC.EVA software (Bruker).

[0124] DSC: Diamond DSC instrument (PerkinElmer Instruments Ltd.), operated under a nitrogen atmosphere. Samples were prepared in 40 μL aluminum crucibles with lids and sealed using a suitable press before analysis. Analysis was performed in the range of 25–210 °C at a constant heating rate of 10 °C / min.

[0125] ATR-FTIR: FTIR Nicolet 6700 spectrophotometer (ThermoFischer Scientific), equipped with an ATR Smart iTR (ThermoFischer Scientific) module with a diamond crystal. Data collection was performed at 4000-650 cm⁻¹. -1 Within a range of 4cm -1 The system performed 64 scans at a resolution for measuring and analyzing both the sample and blank (measurements were performed in the absence of a sample). The blank was acquired before sample measurements and automatically subtracted from them. Spectral display and analysis were performed using Omnic software (Thermo Fisher Scientific).

[0126] Note

[0127] Unless otherwise stated, the water used in the experimental instructions should be pure water.

[0128] Unless otherwise stated, the organic solvents used in the experimental instructions should be of "technical" grade.

[0129] Unless otherwise stated, reagents and catalysts used in the experimental instructions should be of commercial quality.

[0130] Example 1

[0131] This embodiment relates to step a) of the method of the present invention, from 17α-hydroxyprogesterone (I) to 17-hydroxy-3-(1-pyrrolidinyl)pregn-3,5-diene-20 one (II):

[0132]

[0133] 148.1 g of 17α-hydroxyprogesterone (I) was 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 was added dropwise: the starting product was observed to dissolve completely, and the enamine (II) almost immediately redeposited. The mixture was stirred at this temperature for 2 hours. It was then cooled, first to room temperature, and then to 0 °C and held for 1 hour. The mixture 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 a grayish-white solid.

[0134] 17-α-hydroxyprogesterone (I) analysis:

[0135] 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).

[0136] MS:331(M + +1).

[0137] Analysis of compound (II):

[0138] 1H-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).

[0139] Example 2

[0140] This example relates to step b) of the method of the present invention, from enamine (II) to intermediate (III), namely the mixture (21-chloro / 21-bromo)-17α-hydroxy-3-(1-pyrrolidone-1-ylidene)-pregn-4-en-20-one chloride:

[0141]

[0142] Compound (II) obtained in the previous example was suspended in 1660 ml of ethanol at 20–25 °C under a nitrogen atmosphere. A solution of 125.6 g of HCl in ethanol (33% w / w) was added; complete dissolution was observed. Then, a pre-prepared ethanol solution of bromine (16.6 ml of dibromine dissolved in 415 ml of ethanol) cooled to -55 °C was added dropwise over approximately 90 minutes. Precipitation 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 almost completely disappeared. The solvent was removed by rotary evaporation under vacuum at 45 °C, followed by three strippings with MTBE (450 ml each time), leaving approximately 330 ml of total volume on the last stripping. The mixture was cooled to 0 °C and kept stirred for 1 hour. It was then filtered through a Buchner funnel and washed with cold MTBE. The product was dried at 45 °C under vacuum for 2 hours to give 106.6 g of intermediate (III) as a white powder.

[0143] Intermediate (III) analysis:

[0144] 1 ¹H-NMR, DMSO: 6.51 (¹H, s, H⁻⁴); 5.62 (¹H, br, OH⁻¹⁷); 4.60 (¹H, A portion of the AB system, J⁻¹) AB =15Hz, 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).

[0145] The spectrum also shows the following peaks belonging to the 21-chloro derivatives of imino groups: 5.59 (1H, br, OH-17); 4.79 (1H, A part of the AB system, J...). AB =17Hz, H-21); 4.48 (1H, B part of AB system, J AB =17Hz, H-21).

[0146] MS:462, 464 (M) + +1)21-bromine;

[0147] MS: 418, 420 (M) + +1)21-Chlorine.

[0148] Example 3

[0149] This example relates to step c) of the method of the present invention, from intermediate (III) to intermediate (IV), namely the mixture 21-chloro / 21-bromo-17α-hydroxypregn-4-ene-3,20-dione:

[0150]

[0151] 105.5 g of intermediate (III) obtained in the previous examples was dissolved in 1582 ml of methanol; an aqueous solution of potassium bicarbonate (114.5 g of KHCO3 dissolved in 458 g of water) was added, and the mixture was stirred at 25 °C for about 5 hours; complete reaction was detected by TLC. 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.

[0152] The product was vacuum dried in an oven at 50°C for 16 hours to obtain 75.1 g of grayish-white solid. It was recrystallized from 225 ml of a 1:1 (v / v) MEK-methanol mixture to obtain 70.2 g of grayish-white solid intermediate (IV).

[0153] Intermediate (IV) analysis:

[0154] 1¹H-NMR, DMSO-d6: 5.63 (¹H, s, H-4); 5.56 (¹H, s, OH-17); 4.58 (¹H, A portion 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).

[0155] The spectrum also shows the following peaks belonging to the 21-chloro derivative: 5.54 (1H, s, OH-17); 4.77 (1H, A part of the AB system, J... AB =17Hz, H-21); 4.46 (1H, B part of AB system, J AB =17Hz, H-21).

[0156] MS:409, 411(M) + +1)21-bromo; 365, 367 (M) + +1)21-Chlorine.

[0157] Example 4

[0158] This embodiment relates to step d) of the method of the present invention, obtaining compound (V) 21-acetoxy-17α-hydroxypregn-4-ene-3,20-dione from intermediate (IV):

[0159]

[0160] 70 g of the intermediate (IV) obtained in the previous example was suspended in 2100 ml of acetone under a nitrogen stream. 190.8 ml of TEA and 39.2 ml of glacial acetic acid were added, and the mixture was heated to reflux (58°C). A clear solution was never observed. After 5 hours, the reaction was essentially complete. The solvent was removed by rotary evaporation, and the residue was absorbed with water (650 ml) and DCM (450 ml), and the layers were separated. The aqueous layer was re-extracted with DCM (100 ml), and the confluent organic layers were washed with water (2 x 400 ml). The solvent was removed by rotary evaporation, and 400 ml of MEK was added. The solvent was removed by rotary evaporation until a paste was obtained. This process was repeated with an additional 400 ml of MEK. 400 ml of MEK was added, and the solvent was removed until a mixture of approximately 350 ml in volume was obtained. The mixture was cooled to 0°C and held for 1 hour, then filtered through a Buchner funnel and washed with cold MEK (80 ml). The product was dried under vacuum in an oven at 45°C to obtain 59.1 g of compound (V) as a white solid.

[0161] Analysis of compound (V):

[0162] 1 H-NMR, CDCl3: 5.73 (1H, s, H-4); 5.08 (1H, A portion of AB system, J AB =17Hz, 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).

[0163] MS:389(M + +1).

[0164] Example 5

[0165] This embodiment relates to step e) of the method of the present invention, from compound (V) to the target compound (VI) of the present invention, 21-(acetoxy)-17-(1-oxopropoxy)-pregn-4-ene-3,20-dione:

[0166]

[0167] 28.8 g of compound (V) obtained in the previous example was dissolved in 720 ml of DCM under a nitrogen stream. 71.1 ml of propionic anhydride was added, and the mixture was cooled to -20 °C. 7.3 ml of a 70 wt% aqueous solution of HClO4 was added, and exothermic reaction was observed between -20 and -15 °C. The mixture was stirred at -20 °C for 10 minutes. Once the reaction was complete, the reaction mixture was poured into 650 ml of a saturated aqueous solution of NaHCO3 and stirred for 30 minutes. The layers were separated, and the aqueous layer was re-extracted with 100 ml of DCM. The combined organic layers were washed with water (2 x 300 ml). DCM was removed under vacuum using a rotary evaporator until a paste was obtained. 350 ml of heptane was added to remove the solvent until a paste was obtained. This process was repeated with an additional 350 ml of heptane. Finally, 350 ml of heptane was added, and the solvent was distilled off until a mixture with a residual volume of approximately 290 ml was obtained. The mixture was stirred at 25°C for 1 hour and filtered through a Buchner funnel, then washed with heptane. The product was dried under vacuum in an oven at 45°C to give 32.3 g of a grayish-white solid (compound (VI)).

[0168] Analysis of compound (VI):

[0169] 1 H-NMR, CDCl3: 5.75 (1H, s, H-4); 4.89 (1H, A portion of AB system, J AB =16Hz, 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).

[0170] MS:445(M + +1).

[0171] HPLC (purity): 99%, chromatogram as shown Figure 1 As shown.

[0172] DSC and XPRD analyses were also performed on the samples under the above test conditions; the results of the two tests are as follows: Figure 2 and Figure 3 As shown.

[0173] Example 6

[0174] This embodiment relates to the use of a loaded enzyme in a flow reactor to hydrolyze the compound (VI) of the present invention into clastosteroids.

[0175]

[0176] This method was carried out using a Vaportec easy-Medchem E-series flow reactor, wherein the supplied tubular reactor was filled with 845 mg 435 (Acrylic resin-supported Candida Antarctica lipase B).

[0177] 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 then passed through a tubular reactor filled with the enzyme and kept at a constant temperature of 60 °C at a flow rate of 0.1 ml / min.

[0178] Samples of the reaction solution were collected, and the progress of claspone conversion was monitored by UPLC-MS analysis.

[0179] As can be seen from the data in the table below, the enzyme efficiency remains unchanged even after more than 100 hours of constant-flow reaction.

[0180]

[0181]

[0182] Example 7

[0183] This embodiment relates to the use of a loaded enzyme in a conventional sealed reactor to hydrolyze compound (VI) into clastosteroids.

[0184]

[0185] In a 100 ml glass reactor, 250 mg of 21-acetoxy-17α-propoxyprogesterone (VI) was dissolved in 17.5 ml of toluene, and then 250 mg of [unclear text - possibly a typo, should be removed] was added. 435 (acrylic resin-supported Candida Antarctica lipase B) was added, followed by 257 μl of n-butanol. The mixture was stirred and heated to 60 °C, and the reaction progress was monitored by UPLC analysis.

[0186] After stirring for 14 hours and 30 minutes, the contents of residual compound (VI) and clastoline in the reaction mixture were calculated to be 0.75% and 96% respectively, based on the area of ​​each peak in the UPLC chromatogram.

[0187] Example 8

[0188] This embodiment relates to the enzymatic hydrolysis of compound (VI) of the present invention to clastoone, and compares it 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, which is operated in a conventional reactor.

[0189]

[0190] 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; then 250 mg of [the solution was added]. 435 (acrylic resin-supported Candida Antarctica lipase B) was added, followed by 257 μl of di-n-butanol. The mixture was stirred and heated to 60 °C, and the reaction progress was monitored by UPLC analysis.

[0191] The experiment was repeated under the same conditions using 17,21-dipropoxy-17α-progesterone (compound (VII), UPLC purity = 99.5%), except that 258 mg of compound (VII) was used because compound (VII) has a higher molecular weight than compound (VI).

[0192] The progress of controlled claspone conversion was analyzed by UPLC-MS, and the results are reported in the table below (claspone is represented as CLA in the table). It can be seen that the hydrolysis of compound (VI) is faster than that of compound (VII).

[0193]

[0194] Example 9

[0195] This embodiment relates to obtaining clastosone solvated using DMSO.

[0196] The 445 mL solution obtained at the end of the reaction in Example 6 was concentrated at 50 °C and under reduced pressure until 14.3 g of solution was obtained. Then, 6.2 mL of dimethyl sulfoxide was added, and evaporation continued at 50 °C and under reduced pressure until a solution was obtained in which the solvent consisted of at least 99% dimethyl sulfoxide (GC controlled). The solution was then stirred at 20–25 °C for 16 hours to obtain a solid precipitate. After filtration, the solid precipitate was subjected to wet XPRD analysis. The obtained diffraction pattern is shown below. Figure 4 As shown in the figure; the main peaks of the diffraction pattern characterized by 2θ (±0.2°) angular position and relative intensity are listed in the table below:

[0197] 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

[0198] The wet solid (3.5 g) was then dissolved in 3.5 ml of dimethyl sulfoxide at 60 °C with stirring. The solution was then cooled to 25 °C over approximately 1 hour and stirred for 4 hours.

[0199] The precipitated solid separated by filtration was dried under reduced pressure at 40°C for 16 hours (2.6 g white solid), and subjected to XPRD, DSC, FT-IR, and other analytical methods. 1 1H-NMR (CDCl3) analysis. The XPRD diffraction pattern of the dried product is the same as that of the wet product. Figure 4 The DSC thermogram and FT-IR spectrum are shown below. Figure 5 and Figure 6 As shown ( Figure 5 The effective range of the DSC thermal spectrum is shown from 25 °C to 155 °C. NMR spectroscopy shows that the solid is a solvate of claspone and dimethyl sulfoxide in a molar ratio of 1:1.

[0200] HPLC purity: >99%.

[0201] Example 10

[0202] This embodiment relates to the enzymatic hydrolysis of compound (VI) of the present invention to clastoone, and compares it with the enzymatic hydrolysis of the symmetric diester 17,21-bis(1-oxopropoxy)-pregn-4-ene-3,20-dione (VII) operated in a flow reactor by an enzyme loaded on an inert material.

[0203] Dissolve 1.01 g of compound (VII) (99.5% purity) in 68.5 ml of toluene, add 1.01 ml of n-butanol, and stir the mixture until dissolved.

[0204] The resulting solution was pre-filled with 1.068g of solution. A tubular reactor of size 435 was operated at a flow rate of 0.134 ml / min and held at 60°C for 19.1 minutes. The instrument used was a Vaportec easy-Medchem E-series.

[0205] Under the same conditions, a solution of compound (VI) (99.6% purity) was used, and the operation was carried out in a similar manner in toluene and n-butanol.

[0206] Samples of the reaction solution were collected, and the progress of claspone conversion was monitored by UPLC-MS analysis.

[0207] The ratio of the UPLC peak areas of unreacted compound (VII) to unreacted compound (VI) was 1.64.

[0208] Example 11

[0209] This embodiment relates to obtaining clastosone solvated with DMSO from a metastable methanol solvate.

[0210] The 632 ml solution obtained at the end of the reaction in Example 6 was distilled at 50°C under reduced pressure until the weight reached 9 g.

[0211] The solution was absorbed by methanol and concentrated three times under reduced pressure and at 50°C (using 26.4 ml of methanol for each dilution / concentration cycle) to ensure that there was always solution available.

[0212] Add 8.8 ml of methanol, stir the solution at 20-25°C for 30 minutes, and then stir at 4°C for 6 hours.

[0213] The resulting suspension was filtered to obtain a white solid, and its XPRD diffraction pattern (wet product) was immediately recorded. The XPRD diffraction pattern showed a solid phase unlike any known form. The XPRD diffraction pattern of this compound is shown below. Figure 7 As shown; the list of main peaks in the diffraction pattern characterized by 2θ angular position and relative intensity is shown in the table below:

[0214] 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

[0215] The wet solid was rapidly dissolved in 7 ml of dimethyl sulfoxide.

[0216] The resulting solution was distilled under reduced pressure at 50°C to remove any methanol residue, and then stirred at room temperature for 4 hours.

[0217] The resulting suspension was filtered, and the solids were dried in a vacuum oven at 40°C for 16 hours.

[0218] 2.3 g of white solid was obtained, and its XPRD diffraction pattern corresponds to Figure 4 The diffraction pattern of dimethyl sulfoxide solvated clastosone is shown.

[0219] Example 12

[0220] This embodiment relates to obtaining clastosone solvated using DMSO.

[0221] A 3100 mL reaction solution containing approximately 40 g of clastoline, obtained in the same manner as described in Example 6, was concentrated at 50 °C under reduced pressure until a solution of 144.2 g 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 (a solid precipitate 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-solventized clastoline (white solid, UPLC purity = 99.19%).

[0222] Clarithone solvated in DMSO (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 min, then cooled to 20 °C over 1 hour and stirred at 20 °C for 22 h. The suspension was filtered, and the wet solid was dried under reduced pressure in an oven at 40 °C for 20 h to obtain DMSO solvated clathone as a white solid with a UPLC purity of 99.70%.

[0223] Example 13

[0224] This embodiment involves obtaining DMSO-solvated clastoone using seed initiation.

[0225] The 3350 ml reaction solution containing 43 g clastoline, obtained in the same manner as described in Example 6, was concentrated at 50 °C and under reduced pressure until a solution of 93.2 g was obtained. 43 ml of DMSO was added, and distillation was continued under reduced pressure until a final weight of 92.5 g was obtained. 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-solventized clastoline obtained according to the procedure described in Example 12 was added, and the mixture was stirred for 10 minutes. The suspension was cooled to 20 °C over 1 hour and then stirred at 20 °C for 18 hours. The obtained solid was filtered and then dried under reduced pressure in an oven at 40 °C for 20 hours to obtain 43.4 g of DMSO-solventized clastoline (white solid, UPLC purity = 99.41%).

[0226] In the same case, as in Example 12, the DMSO-solventized clastoline obtained can be recrystallized from DMSO until the desired level of purity is obtained.

Claims

1. A solvate of claspone and dimethyl sulfoxide, characterized in that, The XPRD diffraction pattern shows peaks at 2θ of 15.71°, 15.79°, 19.61°, and 19.71° ± 0.2°.

2. The solvate according to claim 1, wherein the molar ratio of clastosone to dimethyl sulfoxide is 1:

1.

3. The solvate according to any one of claims 1 or 2, which is used as a pharmaceutical.

4. A pharmaceutical composition comprising the solvate of any one of claims 1 or 2 and a pharmaceutically acceptable excipient.

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