Method for synthesizing 7alpha, 11alpha, 22-trihydroxy-23, 24-dinorchol-4-ene-3-ketone by microorganisms

Abstract

The invention relates to the technical field of microorganisms, in particular to a method for synthesizing 7 alpha, 11 alpha, 22-trihydroxy-23, 24-dinorchol-4-ene-3-ketone by microorganisms. The invention provides gibberella fujikuroi GBC-40272-A which is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms, and the preservation number of the gibberella fujikuroi GBC-40272-A is CGMCC (China General Microbiological Culture Collection Center) No.40586. The strain can efficiently convert 4-HBC to synthesize 7 alpha, 11 alpha-dihydroxy-4-HBC, and has the advantages of high conversion rate, high selectivity and high product yield. The method for synthesizing the 7alpha, 11alpha-dihydroxy-4-HBC by utilizing the microorganisms, provided by the invention, is high in conversion rate and short in conversion period, provides an important precursor compound for synthesis of a steroid drug, also provides a new method for synthesis of the steroid drug, and is relatively good in application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a method for synthesizing 7α,11α,22-trihydroxy-23,24-bisnorchol-4-ene-3-one by microorganisms. Background Art

[0002] Steroid drugs are the second largest class of chemical drugs after antibiotics. Due to their anti-inflammatory, anti-tumor, anti-allergic, contraceptive and other effects, they are widely used as contraceptives, as well as for the prevention and treatment of diseases such as cancer, obesity, and diabetes.

[0003] Hydroxylation is one of the important reactions for the functionalization of steroid compounds. This reaction refers to the introduction of a hydroxyl group onto a group of a steroid compound. Hydroxylation can provide intermediates for the chemical synthesis of steroid drugs. Hydroxylating at different sites of steroid drugs can endow them with different physiological functions, thereby obtaining potential steroid drug intermediates. In addition, hydroxylation can increase the polarity of steroid compounds, and thus enhance the biological activity of the compounds. A large number of studies have shown that hydroxylated steroids generally exhibit higher biological activity compared to non-hydroxylated steroids with lower polarity.

[0004] 22-Hydroxy-23,24-bisnorchol-4-ene-3-one (also known as 22-hydroxy-23,24-bisnorchol-4-ene-3-one, 4-HBC), its 1-dehydrogenated product 1,4-HBC, and 9-OH-4-HBC. The side chains of this class of intermediates are not completely degraded and are important precursors for the synthesis of progestins and adrenocortical hormones.

[0005] Traditional steroid drug production processes use diosgenin as a raw material and involve multiple steps of chemical synthesis. There are also reports of directly extracting steroid intermediates or non-steroid compounds from animal and plant tissue fluids and then performing multiple steps of chemical synthesis. The processes of the above methods are complex, have low yields, consume a large amount of energy, and cause serious environmental pollution, and can no longer meet the sustainable development of the steroid drug industry. Preparing steroid compounds through microbial transformation has obvious advantages compared with the above methods. Microbial transformation has high regioselectivity and stereoselectivity, as well as advantages such as strong specificity, mild reaction conditions, short reaction time, high yield, and being green and safe, and is more suitable as an industrial production method for steroid drugs. Hydroxylated products of steroid drug intermediates are commonly found at 7α, 7β, 9α, 14α, and 15α, while hydroxylation at 11α has been rarely studied. Therefore, using microorganisms with high conversion efficiency to hydroxylate 4-HBC to form a novel bioactive steroid compound 7α,11α,22-trihydroxy-23,24-bisnorchol-4-ene-3-one (also known as 7α,11α-dihydroxy-4-HBC) not only provides an important precursor compound for steroid drug synthesis but also opens up a new route for the synthesis of steroid drugs and has important application value. Summary of the Invention

[0006] The present invention provides a microorganism that efficiently converts 22-hydroxy-23,24-bisnorchol-4-ene-3-one to synthesize 7α,11α,22-trihydroxy-23,24-bisnorchol-4-ene-3-one, and a method for synthesizing 7α,11α,22-trihydroxy-23,24-bisnorchol-4-ene-3-one using the microorganism.

[0007] The present invention has screened and obtained Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A and found that this strain can efficiently and highly specifically hydroxylate 22-hydroxy-23,24-bisnorchol-4-ene-3-one at the 7α and 11α sites to synthesize 7α,11α,22-trihydroxy-23,24-bisnorchol-4-ene-3-one, and its reaction formula is as Figure 1 shown. Based on this strain, the present invention further provides a method for synthesizing 7α,11α,22-trihydroxy-23,24-bisnorchol-4-ene-3-one using this strain.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] The present invention provides Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A, which was deposited on May 6, 2023, at the China General Microbiological Culture Collection Center (abbreviated as CGMCC, address: No. 3, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode 100101), and is taxonomically named Gibberella fujikuroi Gibberella fujikuroi, with the deposit number of CGMCC No. 40586.

[0010] The present invention provides a microbial preparation, and the microbial preparation contains the Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A described above.

[0011] Preferably, in the microbial preparation, the Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A exists in the form of viable bacteria.

[0012] The microbial preparation can be a liquid preparation or a solid preparation.

[0013] In addition to the Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A, the microbial preparation may further contain excipients allowed in the field of microbial preparations, including but not limited to carriers, lyoprotectants, etc.

[0014] The above-mentioned microbial preparation can be prepared by conventional technical means, with or without adding excipients allowed in the field of microbial preparations.

[0015] The present invention also provides a preparation method of the above-mentioned microbial preparation, and the method includes: the step of culturing Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A. The culturing is preferably carried out at 25-35 °C.

[0016] Based on the function of Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A, the present invention provides the following applications of this strain: The present invention provides the application of the above-mentioned Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A or the microbial preparation in the biotransformation of 22-hydroxy-23,24-bisnorchola-4-en-3-one as a substrate to prepare 7α,11α,22-trihydroxy-23,24-bisnorchola-4-en-3-one.

[0017] The present invention provides the application of the above-mentioned Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A or the microbial preparation in the preparation of a biocatalyst for catalyzing the formation of 7α,11α,22-trihydroxy-23,24-bisnorchola-4-en-3-one from 22-hydroxy-23,24-bisnorchola-4-en-3-one.

[0018] The present invention provides a method for synthesizing 7α,11α,22-trihydroxy-23,24-bisnorchola-4-en-3-one by using microorganisms, and the method includes: using 22-hydroxy-23,24-bisnorchola-4-en-3-one as a substrate, and through the Gibberella fujikuroi ( Gibberella fujikuroiBioconversion of GBC-40272-A gives 7α,11α,22-trihydroxy-23,24-bisnorchola-4-en-3-one.

[0019] The above method includes inoculating Gibberella fujikuroi ( Gibberella fujikuroi )GBC-40272-A into a transformation medium containing 22-hydroxy-23,24-bisnorchola-4-en-3-one for bioconversion.

[0020] Preferably, in the transformation medium, the concentration of 22-hydroxy-23,24-bisnorchola-4-en-3-one is 0.5 - 20 g / L.

[0021] More preferably, in the transformation medium, the concentration of 22-hydroxy-23,24-bisnorchola-4-en-3-one is 1 - 10 g / L; even more preferably 1 - 5 g / L. Even more preferably 1 - 2 g / L.

[0022] For the 22-hydroxy-23,24-bisnorchola-4-en-3-one, organic solvents such as methanol and DMSO can be used as co-solvents.

[0023] Preferably, the transformation medium further comprises the following components: sucrose 25 - 35 g / L, yeast extract 5 - 15 g / L, corn steep liquor 5 - 15 g / L, K2HPO4 1 - 3 g / L, KH2PO4 1 - 2 g / L, MgSO4 0.4 - 0.6 g / L, FeSO4 0.04 - 0.06 g / L.

[0024] The pH of the transformation medium is preferably 6.5 - 7.0.

[0025] Preferably, the temperature of the bioconversion is 25 - 35 °C (more preferably 25 - 30 °C), and / or the pH is 6.5 - 7.0.

[0026] Preferably, the transformation is carried out at 25 - 35 °C and a rotation speed of 180 - 250 r / min for 72 - 120 h.

[0027] Preferably, the inoculation amount of Gibberella fujikuroi ( Gibberella fujikuroi )GBC-40272-A is 5% - 15%. More preferably 8% - 12%.

[0028] Preferably, the seed solution of Gibberella fujikuroi ( Gibberella fujikuroi )GBC-40272-A is inoculated into a transformation medium containing 22-hydroxy-23,24-bisnorchola-4-en-3-one for bioconversion.

[0029] The preparation of the seed solution includes the following steps: taking Gibberella fujikuroi ( Gibberella fujikuroi)Scrape the spores from the fresh PDA slant of GBC-40272-A and inoculate them into the seed medium, and culture for 45 - 52 h to obtain the seed liquid.

[0030] Among them, the seed medium comprises the following components: potato starch 40 - 50 g / L, yeast extract 2 - 4 g / L, corn steep liquor 5 - 15 g / L, CaCO3 2 - 4 g / L, MgSO4 0.4 - 0.6 g / L, FeSO4 0.04 - 0.06 g / L.

[0031] The pH of the transformation medium is preferably 6.5 - 7.0.

[0032] The temperature of the seed culture is preferably 25 - 35 °C.

[0033] The above method further comprises: after the transformation is completed, collecting the transformation liquid and extracting 7α,11α,22 - trihydroxy - 23,24 - bisnorchola - 4 - en - 3 - one therefrom.

[0034] The present invention also provides the application of 7α,11α,22 - trihydroxy - 23,24 - bisnorchola - 4 - en - 3 - one in the preparation of anti - inflammatory drugs.

[0035] The beneficial effects of the present invention at least include: Gibberella fujikuroi Gibberella fujikuroi )CGMCC No.40586 can efficiently transform 4 - HBC to synthesize 7α,11α - dihydroxy - 4 - HBC, having the advantages of high conversion rate, high selectivity, and high product yield. 7α,11α - dihydroxy - 4 - HBC has anti - inflammatory activity and can be used as an intermediate for synthesizing steroid drugs or for developing anti - inflammatory drugs.

[0036] The method provided by the present invention for hydroxylating 4 - HBC by Gibberella fujikuroi Gibberella fujikuroi )CGMCC No.40586 to synthesize the novel steroid compound 7α,11α - dihydroxy - 4 - HBC with biological activity has a high conversion rate (exceeding 65%) and a short conversion period (not exceeding 120 h). This method has the advantages of simple operation, low cost, and high yield. It not only provides an important precursor compound for steroid drug synthesis, but also provides a new method for the synthesis and development of steroid drugs, having good application prospects and economic value. Description of the Drawings

[0037] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0038] Figure 1 It is the reaction formula for the conversion of 4-HBC to synthesize 7α, 11α-dihydroxy-4-HBC in the invention content.

[0039] Figure 2 It is the key HMBC correlation of the conversion product of Gibberella fujikuroi CGMCC No. 40586 in Example 2 of the present invention.

[0040] Figure 3 It is the HMBC spectrum of the conversion product of Gibberella fujikuroi CGMCC No. 40586 in Example 2 of the present invention.

[0041] Figure 4 It is the NOESY spectrum of the conversion product of Gibberella fujikuroi CGMCC No. 40586 in Example 2 of the present invention. Detailed implementation manners

[0042] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts belong to the scope of protection of the present invention.

[0043] Unless otherwise specified, the chemical reagents used in the following examples are all conventional commercially available reagents, and the technical means used are conventional means well known to those skilled in the art.

[0044] The medium formulations used in the following examples are as follows: Potato medium (g / L): Potato 200.00, Glucose 20.00. After peeling and cutting the potato into pieces, weigh it, add distilled water and boil for 0.5 h, then use double-layer gauze to filter out the residue, add glucose to dissolve and make up the volume, and the pH is natural. Add agar (20.00 g / L) to the medium to obtain PDA medium.

[0045] Seed medium (g / L): Potato starch 45.00, Yeast extract 3.00, Corn steep liquor 10.00, CaCO3 3.00, MgSO4 0.50, FeSO4 0.05. Stir and dissolve a small amount of distilled water in potato starch, gelatinize it, and then add other components to dissolve in turn. Adjust the pH to 6.5 after making up the volume, and dispense into 50 mL / 250 mL conical flasks.

[0046] Transformation medium (g / L): sucrose 30.00, yeast extract 10.00, corn steep liquor 10.00, K2HPO4 2.00, KH2PO4 1.60, MgSO4 0.50, FeSO4 0.05. After volume fixation, adjust the pH to 6.5 and dispense into 250 mL conical flasks at 50 mL per flask.

[0047] The above media were autoclaved at 68.95 kPa and 115.0 °C for 30 min.

[0048] Example 1 Obtaining of a microorganism for transforming 4-HBC to synthesize 7α, 11α-dihydroxy-4-HBC In the present invention, a microorganism capable of transforming 4-HBC to synthesize 7α, 11α-dihydroxy-4-HBC was obtained. After identification, it was Gibberella fujikuroi ( Gibberella fujikuroi ), and it was named GBC-40272-A. Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A was deposited at the China General Microbiological Culture Collection Center (abbreviation: CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101) on May 6, 2023. Its taxonomic name was Gibberella fujikuroi Gibberella fujikuroi , and the deposit number was CGMCC No. 40586. Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A in the present invention is also referred to as Gibberella fujikuroi CGMCC No. 40586.

[0049] Example 2 Biocatalytic synthesis of 7α, 11α-dihydroxy-4-HBC by Gibberella fujikuroi CGMCC No. 40586 Use Gibberella fujikuroi CGMCC No. 40586 for biocatalytic synthesis of 7α, 11α-dihydroxy-4-HBC. The specific method is as follows: 1. Preparation of seed liquid Take a fresh PDA slant of Gibberella fujikuroi CGMCC No. 40586, scrape two loops of spores using a sterile inoculation loop, and inoculate them into the seed medium. Incubate at 28 °C and 220 r / min for 48 h to obtain the seed liquid.

[0050] 2. Biotransformation Accurately weigh 4-HBC and place it in a sterilized centrifuge tube. Add methanol to fully dissolve it and prepare a substrate stock solution with a concentration of 50 mg / mL. Using aseptic operation, pipette the stock solution into the transformation medium to make the final concentration of the substrate (4-HBC) 1 g / L. Inoculate the seed solution from step 1 into the transformation medium at an inoculation amount of 10% (v / v). Additionally, set up a substrate control group and a strain blank control group. Incubate at 28 °C and 220 r / min for 120 h to obtain the transformation solution.

[0051] 3. Treatment of the transformation product Pour the transformation solution obtained in step 2 into a centrifuge tube, add ethyl acetate to balance the volume, and centrifuge at 3000 rpm for 10 min. After centrifugation, pour the supernatant into a separatory funnel. Add an equal volume of ethyl acetate to the centrifuge tube, tighten the tube cap, and wash the bacterial cells thoroughly. Then pour the organic phase into the separatory funnel, shake well, and let it stand for 15 min. Repeat the above extraction process 3 times. Combine the upper organic phases and perform rotary evaporation under reduced pressure. After the liquid is completely evaporated, add 5 mL of methanol to redissolve it into an Eppendorf tube, and add an appropriate amount of anhydrous magnesium sulfate to absorb water and dry it, thus obtaining the sample of the transformation product to be tested.

[0052] 4. Identification of the transformation product Dissolve the product sample prepared in step 3 in deuterated chloroform and perform mass spectrometry (MS), nuclear magnetic resonance (NMR), HMBC, HMQC, and NOESY tests to analyze the structure of the product.

[0053] Based on the MS(ESI) m / z 363.25 in the mass spectrometry results, the relative molecular mass of the product is calculated to be 362.25. Compared with the relative molecular mass of the substrate 4-HBC (C 22 H 34 O2) which is 330.50, it has increased by 32. It is speculated that 2 oxygen atoms have been added, and the molecular formula is C 22 H 34 O4.

[0054] According to the analysis of the nuclear magnetic resonance spectrum 1 In the 1H NMR (600 MHz, CDCl3) spectrum, two methyl groups attached to quaternary carbons are given δ H 0.80 (3H, s), 1.36 (3H, s), one methyl group attached to a tertiary carbon δ H 1.08 (3H, d, J = 6.6 Hz), one proton signal on a double bond δ H 5.81 (1H, s), one hydroxymethyl group attached to a tertiary carbon δ H 3.39 (1H, dd, J= 10.6, 3.2 Hz), 3.64 (1H, dd, J = 10.5, 6.6 Hz), suggesting the characteristic proton signals of 4-HBC. 13 The 13C NMR (150 MHz, CDCl3) spectrum showed a total of 22 carbon signals. Combining the analysis of the HSQC spectrum, it was inferred that the compound included 3 methyl groups ( δ C 13.4, 16.8, 18.3), 7 methylene groups ( δ C 67.7, 42.9, 42.3, 35.0, 34.2, 28.4, 26.9), 7 tertiary carbons ( δ C 74.1, 69.2, 56.5, 54.5, 51.3, 38.5, 37.2) and 2 quaternary carbons ( δ C 39.4, 44.0), 1 carbonyl carbon ( δ C 200.0), 2 double bond carbons ( δ C 167.2, 125.4).

[0055] By comparing the 1H and 1 13C NMR data of the transformation product (Table 1) with the spectrum of 4-HBC, it could be seen that the two compounds were extremely similar, with the same parent nucleus. The difference was that the transformation product had two fewer methylene carbon signals than 4-HBC and had 13 74.1 (C-7) and 69.2 (C-11) more. This indicated that the 7th and 11th positions of the transformation product might be substituted by hydroxyl groups. In the HMBC spectrum, although there were no relevant carbon signals between H-7 and H-11, δ C 2.53 (H-6) and δ H 74.1 (C-7), δ C 1.58 (H-8) and δ H 74.1 (C-7) correlation confirmed the substitution of the 7th position by a hydroxyl group; δ C 1.14 (H-9) and δ H 69.2 (C-11), δ C 1.28 (H-12) and δ H 69.2 (C-11), δ CThe relevance of 69.2 (C-11) also confirmed that 11 positions were substituted by hydroxyl groups, as shown in Figure 2 and Figure 3 .

[0056] Table 1 1 H (600 MHz) and 13 C NMR (150 MHz) data (CDCl3)

[0057] The relative configuration of the conversion product was determined by the NOESY spectrum, as shown in Figure 4 . The presence of NOE correlations between H-11 / H-18, H-11 / H-19, and H-7 / H-9 indicated a configuration of 7α, 11α.

[0058] Based on the above results, the product obtained by the conversion of 4-HBC by Gibberella fujikuroi CGMCC No.40586 was 7α, 11α-dihydroxy-4-HBC.

[0059] The results of the biocatalysis experiment showed that the conversion yield (i.e., the product yield) of the biocatalysis of 4-HBC by Gibberella fujikuroi CGMCC No.40586 to synthesize 7α, 11α-dihydroxy-4-HBC could reach 66%.

[0060] Example 3 Pharmacological effects of 7α, 11α-dihydroxy-4-HBC Using the conventional LPS-induced RAW264.7 cell model in the art, the in vitro anti-inflammatory activity of 7α, 11α-dihydroxy-4-HBC prepared in Example 2 above was evaluated. A blank control group, an LPS model group (treated with LPS), a positive control group (treated with LPS + 50 µM L-arginine), and an experimental group (treated with LPS + 50 µM 7α,11α-dihydroxy-4-HBC) were set up respectively. The cell viability and NO production of each group were detected. Through experimental determination, compared with the blank control group, at a concentration of 50 µM 7α,11α-dihydroxy-4-HBC, the cell viability was 95.52%. The detection results of NO production showed that compared with the LPS model group, the NO production inhibition rate of the positive control group was 55.83%, and the NO production inhibition rate of the experimental group reached 58.21%, indicating that 7α, 11α-dihydroxy-4-HBC has a strong inhibitory effect on NO production in the LPS-induced RAW264.7 cell inflammation model and can be used as a candidate compound for the treatment of inflammation.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Gibberella fujikuroi ( Gibberella fujikuroi ), GBC-40272-A, characterized in that It is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC No. 40586.

2. A microbial preparation, characterized in that, The microbial agent contains Gibberella fujikuroi Gibberella fujikuroi ) GBC-40272-A as claimed in claim 1.

3. Use of Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A or the microbial preparation according to claim 2 in the biotransformation of 22-hydroxy-23,24-bisnorchol-4-en-3-one as a substrate to prepare 7α,11α,22-trihydroxy-23,24-bisnorchol-4-en-3-one.

4. Use of Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A or the microbial preparation according to claim 2 in the preparation of a biocatalyst for catalyzing the formation of 7α,11α,22-trihydroxy-23,24-bisnorchol-4-en-3-one from 22-hydroxy-23,24-bisnorchol-4-en-3-one.

5. A method for synthesizing 7α,11α,22-trihydroxy-23,24-bisnorchol-4-en-3-one using microorganisms, characterized in that, The method includes: using 22-hydroxy-23,24-bisnorchola-4-en-3-one as a substrate, and performing biotransformation with Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A to obtain 7α,11α,22-trihydroxy-23,24-bisnorchola-4-en-3-one.

6. The method according to claim 5, wherein The method includes: inoculating Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A into a transformation medium containing 22-hydroxy-23,24-bisnorchol-4-en-3-one for biotransformation.

7. The method according to claim 6, wherein In the conversion medium, the concentration of 22-hydroxy-23,24-dinorchola-4-en-3-one is 0.5 - 20 g / L.

8. The method according to claim 6, wherein The conversion medium further comprises the following components: sucrose 25 - 35 g / L, yeast extract 5 - 15 g / L, corn steep liquor 5 - 15 g / L, K2HPO4 1 - 3 g / L, KH2PO4 1 - 2 g / L, MgSO4 0.4 - 0.6 g / L, FeSO4 0.04 - 0.06 g / L; And / or, the temperature of the bioconversion is 25 - 35 °C, and / or, the pH is 6.5 - 7.

0.

9. The method according to any one of claims 6 to 8, characterized in that, The inoculation amount of Gibberella fujikuroi ( Gibberella fujikuroi ) GBC-40272-A is 5% - 15%. Use of 10.7α,11α,22-trihydroxy-23,24-dinorchola-4-en-3-one in the preparation of anti-inflammatory drugs.

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