Preparation method and application of steroid compounds

Abstract

The present invention provides a preparation method and application of a steroidal compound, relating to the technical field of chemical synthesis. The synthesis method of the steroidal compound comprises the following steps: (a) adding compound I, an acidic catalyst, and a brominating agent to a first solvent, and performing a 9,11-bromohydroxylation reaction at 0-10°C to obtain compound II; (b) adding compound II, a debrominating agent, and a carbonyl protecting agent to a second solvent, and performing a rearrangement reaction at 70-80°C to obtain compound III. The present invention provides a novel metal-free, simple-to-operate, and high-yield preparation route.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical synthesis, in particular to a preparation method and application of a steroid compound. Background Art

[0002] Steroid hormone drugs, which contain a steroidal structure in their molecular structure, are widely used clinically and primarily include adrenocortical hormones and sex hormones. Adrenocortical hormones, among others, possess multiple pharmacological effects, including anti-inflammatory, anti-allergic, immune-suppressing, stress-enhancing, anti-endotoxin, and anti-shock properties. They are used clinically to treat numerous conditions and are an essential and important class of drugs.

[0003] Steroidal compounds with an 11-carbonyl-1,4-diene-3,20-dione structure are important intermediates used to prepare prednisone, methylprednisone, prednisone acetate, methylprednisolone acetate, prednisolone, methylprednisolone, and methylprednisolone acetate. Currently reported methods for preparing these compounds generally involve bromohydroxylation of the substrate at positions 9,11, followed by debromination at position 9, oxidation of the hydroxyl group at position 11, and dehydrogenation of the C1,2 position. Related reports are as follows:

[0004] Patent CN 110698528 A discloses a method for preparing a debrominated product of a methylprednisolone intermediate. The substrate is sequentially subjected to a dechlorination reaction, a bromination reaction, a debromination reaction, and an oxidation reaction to obtain the debrominated product of a methylprednisolone intermediate.

[0005] Patent CN 104561217 A discloses a method for synthesizing 6α-methylprednisolone. The substrate undergoes etherification, methylation, hydrogenation, Arthrobacter fermentation dehydrogenation, bromination reaction, and debromination reaction in sequence to produce 6α-methylprednisolone.

[0006] Patent CN111253457A discloses a method for preparing 16α-hydroxyprednisolone, using 21-hydroxypregnan-1,4,9(11),16-tetraene-3,20-dione-21-acetate as a substrate, and sequentially undergoing oxidation reaction, bromohydroxylation reaction, debromination reaction, and alcoholysis reaction to prepare 16α-hydroxyprednisolone.

[0007] CN101760495A discloses a method for preparing a 6α-methylprednisolone intermediate by biodehydrogenation, which uses Arthrobacter simplex to perform C1,2 biodehydrogenation.

[0008] In summary, the commonly used methods for the two-step debromination of steroid compounds at the 9-position and oxidation of the hydroxyl group at the 11-position in existing technologies mostly utilize heavy metals. For example, chromium trichloride is used for the debromination at the 9-position, and chromium trioxide is used for the oxidation of the hydroxyl group at the 11-position. This two-step reaction is cumbersome and uses large amounts of heavy metals, which are increasingly not in line with current environmental requirements. Most existing technologies use Arthrobacter simplex for C1,2-position biological dehydrogenation, but the conversion rate is low. For example, the conversion rate in the examples of CN101760495A is only 70-80%.

[0009] In view of this, the present invention is proposed. Summary of the Invention

[0010] The main purpose of the present invention is to provide a preparation method and application of a steroid compound, in order to at least partially solve at least one of the above technical problems.

[0011] As a first aspect of the present invention, the present invention provides a method for preparing a steroid compound, comprising the following steps: Compound II undergoes a rearrangement reaction under the action of a debrominating agent and a carbonyl protecting agent to obtain Compound III, as shown in the following reaction formula:

[0012]

[0013] Wherein, R1, R2 and R3 shown in the structural formula of the compound of formula II and the compound of formula III are selected independently of each other, and:

[0014] R1=H, methyl or halogen, halogen=F, Cl, Br or I;

[0015] R2=H, OH, OCOR5 or halogen, R5 is an alkyl group with less than three carbon atoms, halogen=F, Cl, Br or I;

[0016] R3=H, OH or methyl;

[0017] Dashed lines represent single or double bonds.

[0018] Furthermore, the debrominating agent is selected from one or more of triethyl orthoformate, trimethyl orthoformate, triethyl orthoacetate, trimethyl orthoacetate, p-toluenesulfonic acid or pyridine hydrobromic acid.

[0019] Furthermore, the carbonyl protecting agent is selected from one or more of ethylene glycol, ethanol or methanol.

[0020] Furthermore, the molar ratio of the compound of formula II to the debrominating agent is 1:(2-4).

[0021] Furthermore, the temperature of the rearrangement reaction is 60-90°C.

[0022] In the present invention, typical but non-limiting reaction temperatures can be, for example, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C or 90°C.

[0023] Furthermore, the method further comprises the following steps: Compound I is subjected to a 9,11-bromohydroxyl reaction under the action of an acidic catalyst and a brominating agent to obtain Compound II, as shown in the following reaction formula:

[0024]

[0025] Furthermore, R1, R2 and R3 shown in the structural formula of the compound of Formula I and the compound of Formula II are selected independently of each other, and:

[0026] R1=H, methyl or halogen, halogen=F, Cl, Br or I;

[0027] R2=H, OH, OCOR5 or halogen, R5 is an alkyl group with less than three carbon atoms, halogen=F, Cl, Br or I;

[0028] R3=H, OH or methyl;

[0029] Dashed lines represent single or double bonds.

[0030] Furthermore, the acidic catalyst is selected from one or more of perchloric acid, fluoroboric acid, trifluoroacetic acid, methanesulfonic acid and sulfuric acid; and the brominating agent is selected from one or more of dibromohydantoin, bromosuccinimide or dibromocyanoacetamide.

[0031] Furthermore, the molar ratio of the compound of formula I to the brominating agent is 1:(0.6-1.5).

[0032] In the present invention, typical but non-limiting molar ratios of the compound of formula I and the brominating agent can be, for example, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5.

[0033] Furthermore, the molar ratio of the compound of formula I to the acidic catalyst is 1:(0.1-0.2).

[0034] In the present invention, typical but non-limiting molar ratios of the compound of formula I and the brominating agent can be, for example, 1:0.12, 1:0.14, 1:0.16, 1:0.18, or 1:0.2.

[0035] As a second aspect of the present invention, the present invention provides a steroid compound prepared by the above preparation method.

[0036] As a third aspect of the present invention, the present invention provides a steroid compound prepared by the above preparation method or use of the above steroid compound in the preparation of 11-carbonyl-1,4-diene-3,20-dione steroid compound.

[0037] As a fourth aspect of the present invention, the present invention provides a method for preparing an 11-carbonyl-1,4-diene-3,20-dione steroid compound, comprising the following steps: Compound III is biotransformed under the action of Nocardia simplex and Streptomyces griseus to obtain Compound IV, as shown in the following reaction formula:

[0038]

[0039] Furthermore, R1, R2, R3 and R4 shown in the structural formula of the compound of formula III and the compound of formula IV are selected independently of each other, and:

[0040] R1=H, methyl or halogen, halogen=F, Cl, Br or I;

[0041] R2=H, OH, OCOR5 or halogen, R5 is an alkyl group with less than three carbon atoms, halogen=F,

[0042] Cl, Br or I;

[0043] R3=H, OH or methyl;

[0044] R4=H, OH or halogen, halogen=F, Cl, Br or I;

[0045] Dashed lines represent single or double bonds.

[0046] It should be noted that when R2 = H, OH or halogen, and halogen = F, Cl, Br or I, R2 is the same as R4;

[0047] When R2=OCOR5 and R5 is an alkyl group with less than three carbon atoms, R2 is different from R4.

[0048] Furthermore, the preparation method of the 11-carbonyl-1,4-diene-3,20-dione steroid compound comprises the following steps:

[0049] Compound III was added to a fermentation medium inoculated with Nocardia simplex and Streptomyces griseus for biotransformation to obtain compound IV.

[0050] Furthermore, in the preparation method of the 11-carbonyl-1,4-diene-3,20-dione steroid compound, the fermentation medium comprises the following components: 4-8 g of glucose, 2-5 g of yeast powder, 2-5 g of peptone, and 1-3 g of potassium dihydrogen phosphate in 1 L of water, the pH of the fermentation medium is 6.8-7.2, the inoculation amount of Nocardia simplex is 5%-15%, the inoculation amount of Streptomyces griseus is 5%-15%, and the temperature of the bioconversion is 25-35°C.

[0051] In the present invention, a typical but non-limiting pH of the fermentation medium may be, for example, 6.8, 6.9, 7.0, 7.1 or 7.2.

[0052] In the present invention, typical but non-limiting inoculum sizes of Nocardia simplex may be, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%.

[0053] In the present invention, typical but non-limiting inoculum sizes of Streptomyces griseus may be, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%.

[0054] In the present invention, typical but non-limiting reaction temperatures may be, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C.

[0055] Furthermore, the preparation method of the 11-carbonyl-1,4-diene-3,20-dione steroid compound comprises the following steps:

[0056] (a) adding compound I, an acidic catalyst and a brominating agent to a first solvent, and performing a 9,11-bromohydroxy reaction at 0-10° C. to obtain compound II;

[0057] (b) adding compound II, a debrominating agent, and a carbonyl protecting agent to a second solvent, and performing a rearrangement reaction at 70-80° C. to obtain compound III;

[0058] (c) Compound III was added to a fermentation medium inoculated with Nocardia simplex and Streptomyces griseus, and bioconversion was carried out at 25-35° C. to obtain Compound IV, as shown in the following reaction formula:

[0059]

[0060] Wherein, R1, R2, R3 and R4 shown in the structural formula of the compound of formula I, the compound of formula II, the compound of formula III and the compound of formula IV are selected independently of each other, and:

[0061] R1=H;

[0062] R2=OH or OAc;

[0063] R3=H or methyl;

[0064] R4=OH;

[0065] Furthermore, in the step (a), the acidic catalyst is selected from perchloric acid, and the brominating agent is selected from dibromohydantoin;

[0066] Furthermore, in the step (b), the debrominating agent is selected from p-toluenesulfonic acid; the carbonyl protecting agent is selected from ethylene glycol;

[0067] Furthermore, in step (c), the fermentation medium comprises the following components: 6 g glucose, 3 g yeast powder, 3 g peptone, and 1.5 g potassium dihydrogen phosphate in 1 L of water; the pH of the fermentation medium is 7; the inoculation amount of Nocardia simplex is 10%, and the inoculation amount of Streptomyces griseus is 10%.

[0068] Furthermore, the Streptomyces griseus and Nocardia simplex in the present invention are obtained by the following method:

[0069] Streptomyces griseus:

[0070] Slant culture medium: 3 g yeast extract, 10 g starch, 10 g agar, 1000 mL water, pH = 7.2, sterilize at 121°C for 15 min, inoculate with bacteria, culture at 28°C for one week, and then inoculate with seed culture medium.

[0071] Seed culture medium: 3 g yeast extract, 10 g glucose, 1000 mL water, pH = 7.2, 180 rpm, 28°C for 24 hours. After sampling and analysis, the qualified ones are added to the fermentation medium.

[0072] Nocardia simplex:

[0073] Slant culture medium: 10 g peptone, 3 g beef extract, 5 g sodium chloride, 15 g agar, 1000 mL water, sterilize at 121°C for 15 min, inoculate with bacteria, culture at 28°C for one week, and then inoculate with seed culture medium.

[0074] Seed culture medium: 6 g glucose, 3 g yeast powder, 3 g peptone, 1.5 g potassium dihydrogen phosphate, pH = 7, 180 rpm, culture at 28°C for 24 hours, take samples and analyze them, and then inoculate them into the fermentation medium.

[0075] Compared with the prior art, the present invention has the following beneficial effects:

[0076] (1) The present invention uses a one-step rearrangement reaction to replace the two-step reactions of the prior art, namely, the debromination reaction at position 9 and the oxidation of the hydroxyl group at position 11. No heavy metals are used in the reaction, thereby providing a new metal-free, easy-to-operate, and high-yield reduction debromination and hydroxyl oxidation technology.

[0077] (2) The present invention uses Nocardia simplex and Streptomyces griseus in combination, so that dehydrogenation and hydrolysis are carried out simultaneously, which has a higher yield, a shorter route and a lower cost than the prior art dehydrogenation using a single bacterial species, such as Arthrobacter simplex or Nocardia simplex. DETAILED DESCRIPTION

[0078] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, conventional conditions were used. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0079] In order to facilitate a clearer understanding of the present invention, the technical solutions of the present invention will be further described below with reference to embodiments.

[0080] Example 1 Preparation of Compound of Formula II

[0081] Example 1-1

[0082]

[0083] Acetone, 1.0 mL of fluoroboric acid, and water were added to a reaction flask, cooled to 0-5°C, and 10 g of compound 1-1A was added to the flask. 6.6 g of dibromohydantoin was added in four portions. The temperature was maintained at 0°C. TLC was monitored until the absence of compound 1-1A was detected. After the reaction was complete, the pH was adjusted to 8 with aqueous sodium sulfite solution. After stabilization, acetic acid was added to adjust the pH to neutral. The acetone was removed by concentration, and the mixture was rinsed with water (1:1) three times. The mixture was diluted with ice water, filtered, washed, and dried to obtain 12.5 g of compound 1-1B, with a yield of 97.8% and an HPLC purity of 99.1%.

[0084] Example 1-2

[0085]

[0086] Acetone, 1.2 mL of methanesulfonic acid, and water were added to a reaction flask, cooled to 0-5°C, and 10 g of compound 1-2A was added to the flask. 3.8 g of dibromocyanoacetamide was added in four portions. The temperature was maintained at 5°C. TLC was monitored until compound 1-2A disappeared. After the reaction was complete, the pH was adjusted to 8 with aqueous sodium sulfite solution. After stabilization, acetic acid was added to adjust the pH to neutral. The acetone was removed by concentration, and the mixture was rinsed with water (1:1) three times, diluted with ice water, filtered, washed, and dried to obtain 12.4 g of compound 1-2B, with a yield of 99.2% and a purity of 98.6% by HPLC.

[0087] Examples 1-3

[0088]

[0089] Acetone, 1.4 mL of perchloric acid, and water were added to a reaction flask, cooled to 0-5°C, and 10 g of compound 1-3A was added to the flask. A total of 5.8 g of dibromohydantoin was added in four portions, maintaining the temperature at 3°C. TLC monitoring indicated the absence of compound 1-3A. After the reaction was complete, the pH was adjusted to 8 with aqueous sodium sulfite solution. After stabilization, acetic acid was added to adjust the pH to neutral. The acetone was removed by concentration, and the mixture was rinsed with water (1x) three times. The mixture was diluted with ice water, filtered, washed, and dried to obtain 12.3 g of compound 1-3B, with a yield of 99.4% and a purity of 99.6% by HPLC.

[0090] Examples 1-4

[0091]

[0092] Acetone, 1.6 mL of trifluoroacetic acid, and water were added to a reaction flask, cooled to 0-5°C, and 10 g of compound 1-4A was added to the flask. 7.4 g of bromosuccinimide was added in four portions. The temperature was maintained at 2°C. TLC was monitored until the absence of compound 1-4A. After the reaction was complete, the pH was adjusted to 8 with aqueous sodium sulfite solution. After stabilization, acetic acid was added to adjust the pH to neutral. The acetone was removed by concentration, and the mixture was rinsed with water (1:1) three times, diluted with ice water, filtered, washed with water, and dried to obtain 12.5 g of compound 1-4B, with a yield of 98.3% and a purity of 98.4% by HPLC.

[0093] Examples 1-5

[0094]

[0095] Acetone, 1.8 mL of sulfuric acid, and water were added to a reaction flask, cooled to 0-5°C, and 10 g of compound 1-5A was added to the flask. 5.4 g of dibromohydantoin was added in four portions. The temperature was maintained at 5°C. TLC was monitored until the absence of compound 1-5A was detected. After the reaction was complete, the pH was adjusted to 8 with aqueous sodium sulfite solution. After stabilization, acetic acid was added to adjust the pH to neutral. The acetone was removed by concentration, and the mixture was rinsed with water (1:1) three times. The mixture was diluted with ice water, filtered, washed, and dried to obtain 12.0 g of compound 1-5B, with a yield of 97.7% and an HPLC purity of 99.4%.

[0096] Examples 1-6

[0097]

[0098] Acetone, 2.0 mL of trifluoroacetic acid, and water were added to a reaction flask, cooled to 0-5°C, and 10 g of compound 1-6A was added to the flask. 7.1 g of bromosuccinimide was added in four portions. The temperature was maintained at 0°C. TLC was monitored until the absence of compound 1-6A. After the reaction was complete, the pH was adjusted to 8 with aqueous sodium sulfite solution. After stabilization, acetic acid was added to adjust the pH to neutral. The acetone was removed by concentration, and the mixture was rinsed with water (1:1) three times, diluted with ice water, filtered, washed with water, and dried to obtain 12.2 g of compound 1-6B, with a yield of 97.2% and a purity of 99.0% by HPLC.

[0099] Examples 1-7

[0100]

[0101] Acetone, 1.4 mL of perchloric acid, and water were added to a reaction flask, cooled to 0-5°C, and 10 g of compound 1-7A was added to the flask. 5.7 g of dibromohydantoin was added in four portions. The temperature was maintained at 3°C. TLC was monitored until the absence of compound 1-7A was detected. After the reaction was complete, the pH was adjusted to 8 with aqueous sodium sulfite solution. After stabilization, acetic acid was added to adjust the pH to neutral. The acetone was removed by concentration, and the mixture was rinsed with water (1:1) three times. The mixture was diluted with ice water, filtered, washed, and dried to obtain 12.2 g of compound 1-7B, with a yield of 98.0% and a purity of 98.4% by HPLC.

[0102] Examples 1-8

[0103]

[0104] Acetone, 1.6 mL of trifluoroacetic acid, and water were added to a reaction flask, cooled to 0-5°C, and 10 g of compound 1-8A was added to the flask. 7.5 g of bromosuccinimide was added in four portions. The temperature was maintained at 2°C. TLC was monitored until the absence of compound 1-8A. After the reaction was complete, the pH was adjusted to 8 with aqueous sodium sulfite solution. After stabilization, acetic acid was added to adjust the pH to neutral. The acetone was removed by concentration, and the mixture was rinsed with water (1:1) three times, diluted with ice water, filtered, washed with water, and dried to obtain 12.4 g of compound 1-8B, with a yield of 97.1% and a purity of 98.9% by HPLC.

[0105] Examples 1-9

[0106]

[0107] Acetone, 1.8 mL of methanesulfonic acid, and water were added to a reaction flask, cooled to 0-5°C, and 10 g of compound 1-9A was added to the flask. 5.2 g of dibromocyanoacetamide was added in four portions. The temperature was maintained at 5°C and monitored by TLC until compound 1-9A was obtained. After the reaction was complete, the pH was adjusted to 8 with aqueous sodium sulfite solution. After stabilization, acetic acid was added to adjust the pH to neutral. The acetone was removed by concentration, and the mixture was rinsed with water (1:1) three times. The mixture was diluted with ice water, filtered, washed, and dried to obtain 12.2 g of compound 1-9B, with a yield of 96.9% and a purity of 96.9% by HPLC.

[0108] Example 2 Preparation of Formula III Compound

[0109] Example 2-1

[0110]

[0111] 100 mL of ethanol, 10 g of compound 1-1B, and 4.8 g of trimethyl orthoformate were added to a reaction flask, heated to 60°C, stirred at this temperature for 2-3 h, cooled to 30-35°C, added with 10 times the amount of water, cooled to below 5°C, and adjusted to pH 1-2 by adding hydrochloric acid. The mixture was stirred for crystallization for 1 hour, filtered, and the filter cake was washed with water and dried to obtain 8.1 g of compound 1-1C with a yield of 99.0% and an HPLC purity of 98.8%.

[0112] Example 2-2

[0113]

[0114] 100 mL of ethylene glycol, 10 g of compound 1-2B, and 13.2 g of pyridine hydrobromide were added to a reaction flask, heated to 70°C, stirred at this temperature for 2-3 h, cooled to 30-35°C, added with 10 times the amount of water, cooled to below 5°C, and adjusted to pH 1-2 by adding hydrochloric acid. The mixture was stirred for 1 hour to allow crystallization, filtered, and the filter cake was washed with water and dried to obtain 8.2 g of compound 1-2C with a yield of 98.5% and an HPLC purity of 99.0%.

[0115] Example 2-3

[0116]

[0117] 100 mL of ethylene glycol, 10 g of compound 1-3B, and 14.6 g of dibenzoyl peroxide were added to a reaction flask, heated to 75°C, stirred at this temperature for 2-3 h, cooled to 30-35°C, added with 10 times the amount of water, cooled to below 5°C, and adjusted to pH 1-2 by adding hydrochloric acid. The mixture was stirred for crystallization for 1 hour, filtered, and the filter cake was washed with water and dried to obtain 8.6 g of compound 1-3C with a yield of 99.2% and an HPLC purity of 99.3%.

[0118] Examples 2-4

[0119]

[0120] 100 mL of methanol, 10 g of compound 1-4B, and 10.6 g of dibenzoyl peroxide were added to a reaction flask, heated to 80°C, stirred at this temperature for 2-3 h, cooled to 30-35°C, added with 10 times the amount of water, cooled to below 5°C, and adjusted to pH 1-2 by adding hydrochloric acid. The mixture was stirred for 1 hour to allow crystallization, filtered, and the filter cake was washed with water and dried to obtain 8.37 g of compound 1-4C with a yield of 97.6% and an HPLC purity of 98.3%.

[0121] Examples 2-5

[0122]

[0123] 100 mL of ethylene glycol, 10 g of compound 1-5B, and 13.8 g of dibenzoyl peroxide were added to a reaction flask, heated to 90°C, stirred at this temperature for 2-3 h, cooled to 30-35°C, added with 10 times the amount of water, cooled to below 5°C, and adjusted to pH 1-2 by adding hydrochloric acid. The mixture was stirred for crystallization for 1 hour, filtered, and the filter cake was washed with water and dried to obtain 8.1 g of compound 1-5C with a yield of 96.1% and an HPLC purity of 99.1%.

[0124] Examples 2-6

[0125]

[0126] 100 mL of ethanol, 10 g of compound 1-6B, and 10.3 g of triethyl orthoacetate were added to a reaction flask, heated to 60°C, stirred at this temperature for 2-3 h, cooled to 30-35°C, added with 10 times the amount of water, cooled to below 5°C, and adjusted to pH 1-2 by adding hydrochloric acid. The mixture was stirred for 1 hour to allow crystallization, filtered, and the filter cake was washed with water and dried to obtain 8.3 g of compound 1-6C with a yield of 95.9% and an HPLC purity of 97.4%.

[0127] Examples 2-7

[0128]

[0129] 100 mL of ethylene glycol, 10 g of compound 1-7B, and 10.4 g of p-toluenesulfonic acid were added to a reaction flask, heated to 60°C, stirred at this temperature for 2-3 h, cooled to 30-35°C, added with 10 times the amount of water, cooled to below 5°C, and adjusted to pH 1-2 by adding hydrochloric acid. The mixture was stirred for 1 hour to allow crystallization, filtered, and the filter cake was washed with water and dried to obtain 8.5 g of compound 1-7C with a yield of 98.0% and an HPLC purity of 97.5%.

[0130] Examples 2-8

[0131]

[0132] 100 mL of ethylene glycol, 10 g of compound 1-8B, and 14.1 g of pyridine hydrobromide were added to a reaction flask, heated to 70°C, stirred at this temperature for 2-3 h, cooled to 30-35°C, added with 10 times the amount of water, cooled to below 5°C, and adjusted to pH 1-2 by adding hydrochloric acid. The mixture was stirred for 1 hour to allow crystallization, filtered, and the filter cake was washed with water and dried to obtain 8.3 g of compound 1-8C with a yield of 97.3% and an HPLC purity of 98.0%.

[0133] Examples 2-9

[0134]

[0135] 100 mL of methanol, 10 g of compound 1-9B, and 9.4 g of triethyl orthoformate were added to a reaction flask, heated to 75°C, stirred at this temperature for 2-3 h, cooled to 30-35°C, added with 10 times the amount of water, cooled to below 5°C, and adjusted to pH 1-2 by adding hydrochloric acid. The mixture was stirred for 1 hour to allow crystallization, filtered, and the filter cake was washed with water and dried to obtain 8.4 g of compound 1-9C with a yield of 97.2% and an HPLC purity of 96.8%.

[0136] Example 2-10

[0137] This example differs from Example 2-3 in that 14.6 g of the debrominating agent dibenzoyl peroxide is replaced with 8.9 g of triethyl orthoformate. Other conditions remain the same. After post-treatment, 8.2 g of compound 1-3C is obtained with a yield of 94.8% and an HPLC purity of 94.1%.

[0138] Example 2-11

[0139] This example differs from Example 2-3 in that 14.6 g of dibenzoyl peroxide as the debrominating agent is replaced with 9.6 g of pyridine hydrobromide. Other conditions remain the same. After post-treatment, 8.3 g of compound 1-3C is obtained with a yield of 95.2% and an HPLC purity of 95.7%.

[0140] Example 2-12

[0141] The difference between this example and example 2-3 is that the reaction temperature is replaced by 70° C. at 75° C., and the other conditions are the same. After post-treatment, 8.2 g of compound 1-3C is obtained with a yield of 93.9% and an HPLC purity of 93.8%.

[0142] Example 2-13

[0143] The difference between this example and example 2-3 is that the reaction temperature of 75°C is replaced by 80°C. The other conditions are the same. After post-treatment, 8.3 g of compound 1-3C is obtained with a yield of 95.7% and an HPLC purity of 95.6%.

[0144] Example 3 Preparation of Formula IV Compound

[0145] Example 3

[0146] Streptomyces griseus:

[0147] Slant culture medium: 3 g yeast extract, 10 g starch, 10 g agar, 1000 mL water, pH = 7.2, sterilize at 121°C for 15 min, inoculate with bacteria, culture at 28°C for one week, and then inoculate with seed culture medium.

[0148] Seed culture medium: 3 g yeast extract, 10 g glucose, 1000 mL water, pH = 7.2, 180 rpm, 28°C for 24 hours. After sampling and analysis, the qualified ones are added to the fermentation medium.

[0149] Nocardia simplex:

[0150] Slant culture medium: 10 g peptone, 3 g beef extract, 5 g sodium chloride, 15 g agar, 1000 mL water, sterilize at 121°C for 15 min, inoculate with bacteria, culture at 28°C for one week, and then inoculate with seed culture medium.

[0151] Seed culture medium: 6 g glucose, 3 g yeast powder, 3 g peptone, 1.5 g potassium dihydrogen phosphate, pH = 7, 180 rpm, culture at 28°C for 24 hours, take samples and analyze them, and then inoculate them into the fermentation medium.

[0152] Example 3-1

[0153]

[0154] Qualified seed cultures of the two strains were inoculated with 5% inoculum of Nocardia simplex and 5% inoculum of Streptomyces griseus in a fermentation medium. The fermentation medium consisted of 4 g of glucose, 2 g of yeast extract, 2 g of peptone, and 0.5 g of potassium dihydrogen phosphate per liter of water. The culture was incubated at 180 rpm for 24 hours at a pH of 6.8. Samples were collected for analysis. If qualified, 10 g of compound 1-1C (d90 ≤ 20 μm) was added, and ethanol was added. The mixture was incubated at 200 rpm at 25°C for 48 hours. Samples were collected and tested for qualified conversion. The fermentation broth was filtered to obtain a filter cake, which was then extracted and purified to yield 8.0 g of compound 1-1D, with a yield of 99.1% and a purity of 98.5% by HPLC.

[0155] Example 3-2

[0156]

[0157] Qualified seed cultures of the two strains were taken, and Nocardia simplex was inoculated at a 7.5% inoculum rate into a fermentation medium, while Streptomyces griseus was inoculated at a 7.5% inoculum rate into a fermentation medium. The fermentation medium composition was: 5 g glucose, 2.5 g yeast extract, 2.5 g peptone, and 1.0 g potassium dihydrogen phosphate per liter of water. The culture was incubated at 180 rpm for 24 hours at a pH of 6.9. Samples were taken for analysis. If qualified, 10 g of compound 1-2C (d90 ≤ 20 μm) was added. Ethanol was added, and the mixture was incubated at 200 rpm at 26°C for 48 hours. Samples were then collected and tested for qualified conversion. The fermentation broth was filtered to obtain a filter cake, which was then extracted and purified to obtain 7.3 g of compound 1-2D, with a yield of 98.1% and a purity of 99.0% by HPLC.

[0158] Example 3-3

[0159]

[0160] Qualified seed cultures of the two strains were taken, and Nocardia simplex was inoculated at a 10% inoculum rate into a fermentation medium, while Streptomyces griseus was inoculated at a 10% inoculum rate into a fermentation medium. The fermentation medium consisted of 6 g of glucose, 3 g of yeast extract, 3 g of peptone, and 1.5 g of potassium dihydrogen phosphate in 1 L of water. The culture was incubated at 180 rpm for 24 hours at a pH of 7.0. Samples were taken for analysis. If qualified, 10 g of compound 1-3C (d90 ≤ 20 μm) was added, and ethanol was added. The mixture was incubated at 200 rpm at 28°C for 48 hours, and samples were collected. After qualified conversion, the fermentation broth was filtered to obtain a filter cake, which was extracted and purified to obtain 7.5 g of compound 1-3D, with a yield of 99.6% and a purity of 99.5% by HPLC.

[0161] Examples 3-4

[0162]

[0163] Qualified seed cultures of the two strains were taken, and Nocardia simplex was inoculated at a 12.5% ​​inoculum rate into a fermentation medium, while Streptomyces griseus was inoculated at a 12.5% ​​inoculum rate into a fermentation medium. The fermentation medium composition was: 7 g glucose, 3.5 g yeast extract, 3.5 g peptone, and 2.0 g potassium dihydrogen phosphate per liter of water. The culture was incubated at 180 rpm for 24 hours at a pH of 7.1. Samples were taken for analysis. If qualified, 10 g of compound 1-4C (d90 ≤ 20 μm) was added. Ethanol was added, and the mixture was incubated at 200 rpm at 30°C for 48 hours. Samples were then collected and tested for qualified conversion. The fermentation broth was filtered to obtain a filter cake, which was then extracted and purified to obtain 8.1 g of compound 1-4D, with a yield of 98.7% and a purity of 97.9% by HPLC.

[0164] Examples 3-5

[0165]

[0166] Qualified seed cultures of the two strains were taken, and Nocardia simplex was inoculated at a 10% inoculum rate into a fermentation medium, while Streptomyces griseus was inoculated at a 10% inoculum rate into a fermentation medium. The fermentation medium consisted of 8 g of glucose, 4 g of yeast extract, 4 g of peptone, and 2.5 g of potassium dihydrogen phosphate in 1 L of water. The culture was incubated at 180 rpm for 24 hours, with a pH of 7.2. Samples were taken for analysis. If qualified, 10 g of compound 1-5C (d90 ≤ 20 μm) was added, and ethanol was added. The mixture was incubated at 200 rpm at 33°C for 48 hours, and samples were collected. After qualified conversion, the fermentation broth was filtered to obtain a filter cake, which was extracted and purified to obtain 7.0 g of compound 1-5D, with a yield of 99.2% and a purity of 99.3% by HPLC.

[0167] Examples 3-6

[0168]

[0169] Qualified seed cultures of the two strains were taken, and Nocardia simplex was inoculated at a 15% inoculum rate into a fermentation medium, while Streptomyces griseus was inoculated at a 15% inoculum rate into a fermentation medium. The fermentation medium consisted of 4 g of glucose, 5 g of yeast extract, 5 g of peptone, and 3.0 g of potassium dihydrogen phosphate in 1 L of water. The culture was incubated at 180 rpm for 24 hours, with a pH of 7.0. Samples were taken for analysis. If qualified, 10 g of compound 1-6C (d90 ≤ 20 μm) was added, and ethanol was added. The mixture was incubated at 200 rpm at 35°C for 48 hours, and samples were collected. After qualified conversion, the fermentation broth was filtered to obtain a filter cake, which was then extracted and purified to obtain 8.2 g of compound 1-6D, with a yield of 99.0% and a purity of 99.4% by HPLC.

[0170] Examples 3-7

[0171] The difference between this example and Example 3-3 is that the inoculation of Nocardia simplex at a 10% inoculation rate and the inoculation of Streptomyces griseus at a 10% inoculation rate into the fermentation medium are replaced by the inoculation of Nocardia simplex at a 15% inoculation rate and the inoculation of Streptomyces griseus at a 5% inoculation rate into the fermentation medium, and the other conditions are the same. After post-treatment, 7.2 g of compound 1-3D was obtained with a yield of 96.5% and an HPLC purity of 93.2%.

[0172] Examples 3-8

[0173] The difference between this example and example 3-3 is that pH=7.0 is replaced by pH=6.8. The other conditions are the same. After post-treatment, 7.2 g of compound 1-3D is obtained with a yield of 95.6% and an HPLC purity of 95.2%.

[0174] Examples 3-9

[0175] The difference between this example and example 3-3 is that pH=7.0 is replaced by pH=7.2. Other conditions are the same. After post-treatment, 7.1 g of compound 1-3D is obtained with a yield of 94.6% and an HPLC purity of 96.2%.

[0176] Examples 3-10

[0177] This example differs from Example 3-3 in that the components of the fermentation medium are replaced with 4 g of glucose, 2 g of yeast powder, 2 g of peptone, and 1.0 g of potassium dihydrogen phosphate in 1 L of water instead of 6 g of glucose, 3 g of yeast powder, 3 g of peptone, and 1.5 g of potassium dihydrogen phosphate in 1 L of water. All other conditions remain the same. After post-treatment, 7.2 g of compound 1-3D was obtained with a yield of 95.7% and an HPLC purity of 94.9%.

[0178] Comparative Example 1

[0179] The difference between this example and Example 3-3 is that the inoculation of Nocardia simplex at a 10% inoculum into the fermentation medium and the inoculation of Streptomyces griseus at a 10% inoculum into the fermentation medium are replaced by the inoculation of Nocardia simplex at a 10% inoculum into the fermentation medium. The other conditions are the same. After post-treatment, 7.1 g of compound 1-3D was obtained with a yield of 95.4% and an HPLC purity of 94.8%.

[0180] Comparative Example 2

[0181] This example differs from Example 3-3 in that the inoculation of Nocardia simplex at a 10% inoculum into the fermentation medium and the inoculation of Streptomyces griseus at a 10% inoculum into the fermentation medium are replaced by the inoculation of only Streptomyces griseus at a 10% inoculum into the fermentation medium. Other conditions are the same. After post-treatment, 7.0 g of compound 1-3D was obtained with a yield of 93.9% and an HPLC purity of 95.5%.

[0182] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a 11-carbonyl-1,4-diene-3,20-dione steroid compound, characterized in that: The following steps are involved: (a) Compound I undergoes a 9,11-bromohydroxyl reaction in the presence of an acidic catalyst and a brominating agent to obtain Compound II; (b) Compound II is biotransformed under the action of Nocardia simplex and Streptomyces griseus to obtain Compound III, as shown in the following reaction formula: Wherein, R1, R2, R3 and R4 shown in the structural formula of the compound of formula I, the compound of formula II and the compound of formula III are selected independently of each other, and: R1=H, methyl or halogen, halogen=F; R2=OH or OCOR5, R5 is an alkyl group with less than three carbon atoms; R3=H, OH or methyl; R4=OH; 2. The method for preparing a steroid compound according to claim 1, wherein The acidic catalyst is selected from one or more of perchloric acid, fluoroboric acid, trifluoroacetic acid, methanesulfonic acid and sulfuric acid.

3. The method for preparing a steroid compound according to claim 1, wherein: The brominating agent is selected from one or more of dibromohydantoin, bromosuccinimide or dibromocyanoacetamide.

4. The method for preparing the 11-carbonyl-1,4-diene-3,20-dione steroid compound according to claim 1, characterized in that: The following steps are involved: Compound II was added into a fermentation medium inoculated with Nocardia simplex and Streptomyces griseus for biotransformation to obtain Compound III.

5. The method for preparing the 11-carbonyl-1,4-diene-3,20-dione steroid compound according to claim 4, characterized in that: The fermentation medium contains the following components: 1L of water contains 4-8g of glucose, 2-5g of yeast powder, 2-5g of peptone, and 1-3g of potassium dihydrogen phosphate; The pH of the fermentation medium is 6.8-7.2; The inoculation amount of Nocardia simplex is 5%-15%, and the inoculation amount of Streptomyces griseus is 5%-15%; The temperature of the bioconversion is 25-35°C.

6. The method for preparing the 11-carbonyl-1,4-diene-3,20-dione steroid compound according to claim 1, comprising the following steps: (a) adding compound I to a first solvent in the presence of an acidic catalyst and a brominating agent, and performing a 9,11-bromohydroxy reaction at 0-10° C. to obtain compound II; (b) adding compound II to a fermentation medium inoculated with Nocardia simplex and Streptomyces griseus, and carrying out biotransformation at 25-35° C. to obtain compound III; in, R1, R2, R3 and R4 shown in the structural formula of the compound of formula I, the compound of formula II and the compound of formula III are selected independently of each other, and: R1=H, methyl or halogen, halogen=F; R2=OH or OCOR5, R5 is an alkyl group with less than three carbon atoms; R3=H, OH or methyl; R4=OH; In the step (a), the acidic catalyst is selected from perchloric acid, and the brominating agent is selected from dibromohydantoin; In step (b), the fermentation medium comprises the following components: 6 grams of glucose, 3 grams of yeast powder, 3 grams of peptone, and 1.5 grams of potassium dihydrogen phosphate in 1 L of water; the pH of the fermentation medium is 7; the inoculation amount of the Nocardia simplex is 10%, and the inoculation amount of the Streptomyces griseus is 10%.

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