A safe post-treatment process for 11alpha, 17alpha-dihydroxyprogesterone fermentation broth

Abstract

This invention belongs to the technical field of steroid compound preparation, specifically relating to a safe post-processing technology for 11α,17α-dihydroxyprogesterone fermentation broth, comprising four processing steps: 1) extraction; 2) saponification; 3) secondary dissolution for impurity removal; and 4) decolorization. This invention employs a four-step post-processing procedure of extraction-saponification-secondary dissolution for impurity removal-decolorization, which not only improves the yield and purity of 11α,17α-dihydroxyprogesterone, achieving a purity of over 99%, but also solves the problem of high-yield extraction of 11α,17α-dihydroxyprogesterone from fermentation broth. Furthermore, it makes the reaction conditions milder, the operation simpler, safer, and more reliable, making it more suitable for large-scale production.

Description

Technical Field

[0001] This invention belongs to the technical field of steroid compound preparation, specifically relating to a safe post-processing technology for 11α,17α-dihydroxyprogesterone fermentation broth. Background Technology

[0002] Steroid compounds are mainly used in the pharmaceutical field to regulate human metabolism and have shown good efficacy in treating rheumatism, skin diseases, cardiovascular diseases, respiratory diseases, and tumors. Among them, 11α,17α-dihydroxyprogesterone is a common intermediate in steroidal drugs and an important raw material for the synthesis of glucocorticoids such as prednisolone acetate and prednisolone, with a large global market demand.

[0003] Currently, the preparation of 11α,17α-dihydroxyprogesterone usually involves using specific strains to achieve a highly efficient and selective catalytic process for the steroid substrate 17α-hydroxyprogesterone. For example, Bioresource Technology, 2011, 102(20):9368-9373 and Industrial Microbiology, 2011 31(4):40-48 reported the use of Rhizopus nigricans to convert progesterone into C11α-hydroxylated drugs to produce corticosteroids. Another example is the Chinese patent document CN106916872A, which discloses a method for the efficient biotransformation preparation of 11α,17α-dihydroxyprogesterone. This method uses 17α-hydroxyprogesterone as raw material and supplements the carbon source during the biotransformation process to maintain the high activity of the hydroxylase in the mycelium, thereby obtaining a product with a conversion rate of over 90%. The above literature mainly focuses on the microbial transformation of steroidal compounds into 11α-hydroxylated compounds, with low conversion yields. The post-processing procedures of the fermentation products have not been reported in detail.

[0004] Existing technologies include research on post-processing steps for 11α,17α-dihydroxyprogesterone fermentation products, such as the method for preparing Δ1-11α,17α-dihydroxyprogesterone via one-pot continuous fermentation in patent application CN104711311A. This method involves filtering the culture broth to extract the solid mycelial cake, heating it, adding activated carbon, and then refluxing and filtering. The filtrate is concentrated to obtain a crude product, which is then filtered again to obtain Δ1-11α,17α-dihydroxyprogesterone. This solves the problem of low conversion yield caused by the microbial transformation of steroidal compounds to 11α-hydroxylation. However, the existing post-processing steps involve extraction-decolorization-filtration-re-filtration to obtain refined Δ1-11α,17α-dihydroxyprogesterone. Concentrating the mother liquor and then saponifying it can improve the overall yield, but it does not effectively improve the purity, thus affecting the high yield of the fermentation broth extraction. Furthermore, the process is cumbersome and poses certain safety hazards. Therefore, a new technical solution is needed to address these technical problems. Summary of the Invention

[0005] The purpose of this invention is to provide a safe post-processing process for 11α,17α-dihydroxyprogesterone fermentation broth, in order to solve the problems mentioned in the background art, such as the current post-processing steps can only improve the total yield, but the purity cannot be effectively improved, thus affecting the high yield of fermentation broth extraction.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a safe post-treatment process for 11α,17α-dihydroxyprogesterone fermentation broth, comprising the following specific steps:

[0007] S1. Extraction: First, the 11α,17α-dihydroxyprogesterone fermentation broth is added to an organic solvent for batch extraction. Then, distilled water is added and mixed and stirred to remove the low-boiling-point solvent. The volume ratio of the 11α,17α-dihydroxyprogesterone fermentation broth to the organic solvent is 1:0.1 to 0.8. The low-boiling-point solvent is the organic solvent, which is selected from one or a mixture of several of dichloromethane, chloroform, and ethyl acetate.

[0008] S2, Saponification: After S1, add alkali to saponify, filter, and wash the filter cake with water until neutral to obtain a moist solid. The alkali is selected from one or more of sodium hydroxide and potassium hydroxide.

[0009] S3. Secondary dissolution and impurity removal: The wet solid obtained in S2 is added to an organic solvent and water, and the organic solvent is removed by dissolution at 60-90°C. Toluene is then refluxed to remove water. The volume ratio of water to organic solvent is 1-1.5:1, and the organic solvent is selected from one or a mixture of several of methanol, ethyl acetate, acetone, and tetrahydrofuran.

[0010] S4, Decolorization: After S3, the solid is dissolved in a lower alcohol, decolorized with activated carbon, filtered, and distilled to remove the lower alcohol, yielding the final product, 11α,17α-dihydroxyprogesterone solid. The lower alcohol is one or more of methanol, ethanol, and isopropanol. The 11α,17α-dihydroxyprogesterone solid is off-white in color and has a purity of ≥99%.

[0011] Compared with the prior art, the beneficial effects of the present invention are:

[0012] 1. This invention employs a four-step post-processing procedure: extraction, saponification, secondary dissolution for impurity removal, and decolorization. This not only improves the yield and purity of 11α,17α-dihydroxyprogesterone, achieving a purity of over 99%, but also solves the problem of high-yield extraction of 11α,17α-dihydroxyprogesterone from fermentation broth. Furthermore, it makes the reaction conditions milder, the operation simpler, safer, and more reliable, making it more suitable for large-scale production.

[0013] 2. This invention uses common extraction and distillation units to make the reaction conditions for the post-treatment of 11α,17α-dihydroxyprogesterone fermentation broth milder, the operation simpler, and safer and more reliable. It effectively improves the post-treatment effect, efficiency and safety of 11α,17α-dihydroxyprogesterone, and ensures that 11α,17α-dihydroxyprogesterone is suitable for large-scale production.

[0014] 3. This invention uses inexpensive and readily available organic solvents, which effectively reduces production costs. The saponification reaction is carried out using alkali, making the post-treatment reaction conditions milder, the operation simpler, and safer and more reliable, thus making it more suitable for large-scale production and ensuring the production effect and efficiency of 11α,17α-dihydroxyprogesterone. Attached Figure Description

[0015] Figure 1 This is the liquid phase spectrum of the 11α,17α-dihydroxyprogesterone product of the present invention. Detailed Implementation

[0016] The following examples are used to further illustrate the content of the present invention and do not limit the application of the present invention. Example 1:

[0017] 40 L of 11α,17α-dihydroxyprogesterone fermentation broth was extracted three times with 24 L of dichloromethane to obtain an extract. The extract was then concentrated under reduced pressure to 5 L, and 10 L of distilled water was added and vigorously mixed. The dichloromethane was removed by distillation. 50 g of sodium hydroxide was then added, and the mixture was stirred for 30 minutes and then filtered. After the filter cake was washed with water until neutral, a moist solid was obtained. 5 L of ethyl acetate and 5 L of water were added and vigorously mixed. At the same time, the temperature was raised to 80 °C and the ethyl acetate was removed by distillation under reduced pressure. The solid was then refluxed with toluene to remove water. Finally, the obtained solid was dissolved in 8 L of methanol, decolorized with activated carbon, filtered, and distilled to remove methanol, yielding a white 11α,17α-dihydroxyprogesterone solid with a purity of 99%. Example 2:

[0018] 40 L of 11α,17α-dihydroxyprogesterone fermentation broth was extracted three times with 15 L of chloroform to obtain an extract. The extract was then concentrated under reduced pressure to 3 L, and 6 L of distilled water was added and vigorously mixed. The chloroform was removed by distillation. 50 g of sodium hydroxide was then added, and the mixture was stirred for 30 minutes and then filtered. After the filter cake was washed with water until neutral, a moist solid was obtained. 5 L of acetone and 6 L of water were added and vigorously mixed. At the same time, the temperature was raised to 60 °C and the acetone was removed by distillation under reduced pressure. The solid was then refluxed with toluene to remove water. Finally, the obtained solid was dissolved in 8 L of ethanol, decolorized with activated carbon, filtered, and the ethanol was removed by distillation to obtain a white 11α,17α-dihydroxyprogesterone solid with a purity of 99%. Example 3:

[0019] 40 L of 11α,17α-dihydroxyprogesterone fermentation broth was extracted three times with a mixed solvent of 10 L dichloromethane and 10 L ethyl acetate to obtain an extract. The extract was then concentrated under reduced pressure to 4 L, and 8 L of distilled water was added and vigorously stirred. Dichloromethane and ethyl acetate were removed by distillation. 60 g of potassium hydroxide was then added, and the mixture was stirred for 20 minutes and then filtered. After the filter cake was washed with water until neutral, a moist solid was obtained. 5 L of tetrahydrofuran and 6 L of water were added and vigorously stirred. At the same time, the temperature was raised to 70 °C and the tetrahydrofuran was removed by distillation under reduced pressure. The solid was then refluxed with toluene to remove water. Finally, the obtained solid was dissolved in a mixed solvent of 9 L methanol and isopropanol, decolorized with activated carbon, filtered, and distilled to remove methanol and isopropanol, yielding a white 11α,17α-dihydroxyprogesterone solid with a purity of 99.14%. Example 4:

[0020] 40 L of 11α,17α-dihydroxyprogesterone fermentation broth was extracted three times with 24 L of dichloromethane to obtain an extract. The extract was then concentrated under reduced pressure to 5 L, and 10 L of distilled water was added and vigorously stirred. The dichloromethane was removed by distillation. 50 g of sodium hydroxide was then added, and the mixture was stirred for 30 minutes before filtration. After the filter cake was washed with water until neutral, a moist solid was obtained. 5 L of methanol and 6 L of water were then added and vigorously stirred. Simultaneously, the temperature was raised to 70 °C, and the methanol was removed by distillation under reduced pressure. The water was then removed by reflux with toluene. Finally, the obtained solid was dissolved in 9 L of a mixed solvent of ethanol and isopropanol, decolorized with activated carbon, filtered, and distilled to remove ethanol and isopropanol, yielding a white 11α,17α-dihydroxyprogesterone solid with a purity of 99.26% (its liquid phase spectrum is shown in Figure 1). Figure 1 (As shown). Example 5:

[0021] 40 L of 11α,17α-dihydroxyprogesterone fermentation broth was extracted three times with 18 L of ethyl acetate to obtain an extract. The extract was then concentrated under reduced pressure to 4 L, and 8 L of distilled water was added and vigorously mixed. The ethyl acetate was then distilled off. 60 g of potassium hydroxide was added, and the mixture was stirred for 20 minutes before filtration. After the filter cake was washed with water until neutral, a moist solid was obtained. 5 L of a mixture of ethyl acetate and methanol and 5 L of water were added and vigorously mixed. At the same time, the temperature was raised to 80 °C, and the ethyl acetate and methanol mixture was distilled off under reduced pressure to remove the solvent. The solid was then refluxed with toluene to remove water. Finally, the obtained solid was dissolved in 8 L of a mixture of methanol and ethanol, decolorized with activated carbon, and then filtered and distilled to remove methanol and ethanol, yielding a white 11α,17α-dihydroxyprogesterone solid with a purity of 99%.

[0022] Comparative Example 1:

[0023] 40 L of 11α,17α-dihydroxyprogesterone fermentation broth was extracted three times with 18 L of ethyl acetate to obtain an extract. The extract was then concentrated under reduced pressure to 4 L, and 8 L of distilled water was added and vigorously mixed and stirred. The ethyl acetate was then distilled off. 60 g of potassium hydroxide was added, and the mixture was stirred for 20 minutes and then filtered. After the filter cake was washed with water until neutral, a moist solid was obtained. 5 L of acetonitrile and 6 L of water were added and vigorously mixed and stirred. At the same time, the temperature was raised to 85 °C and the acetonitrile was removed by distillation under reduced pressure. The solid was then refluxed with toluene to remove water. Finally, the obtained solid was dissolved in 9 L of isopropanol, decolorized with activated carbon, filtered, and distilled off to remove the isopropanol, yielding a white 11α,17α-dihydroxyprogesterone solid with a purity of 96%.

[0024] A comparison of Examples 1-5 with Comparative Example 1 shows that by using the four post-processing steps of extraction, saponification, secondary dissolution for impurity removal, and decolorization, along with the use of an appropriate ratio of organic solvent, the purity of 11α,17α-dihydroxyprogesterone can reach over 99%, thus solving the problem of high-yield extraction of 11α,17α-dihydroxyprogesterone from fermentation broth.

Claims

1. A safe post-treatment process for 11α,17α-dihydroxyprogesterone fermentation broth, characterized in that, The specific steps are as follows: S1. Extraction: First, the 11α,17α-dihydroxyprogesterone fermentation broth is added to an organic solvent for batch extraction. Then, distilled water is added and mixed and stirred to remove the low-boiling-point solvent. The volume ratio of the 11α,17α-dihydroxyprogesterone fermentation broth to the organic solvent is 1:0.1 to 0.

8. The organic solvent is selected from one or a mixture of several of dichloromethane, chloroform, and ethyl acetate. S2, Saponification: After S1, add alkali to saponify, filter, and wash the filter cake with water until neutral to obtain a moist solid. The alkali is selected from one or more of sodium hydroxide and potassium hydroxide. S3. Secondary dissolution and impurity removal: The wet solid obtained in S2 is added to an organic solvent and water, and the organic solvent is removed by dissolution at 60-90°C. Toluene is refluxed to remove water. The volume ratio of water to organic solvent is 1-1.5:

1. The organic solvent is selected from one or a mixture of several of methanol, ethyl acetate, acetone, and tetrahydrofuran. S4, Decolorization: After S3, the solid is dissolved in a lower alcohol, decolorized with activated carbon, filtered, and distilled to remove the lower alcohol, yielding the final product 11α,17α-dihydroxyprogesterone solid, wherein the lower alcohol is one or a mixture of methanol, ethanol, and isopropanol.

2. The safe post-treatment process for 11α,17α-dihydroxyprogesterone fermentation broth according to claim 1, characterized in that, In S1, the low-boiling-point solvent is an organic solvent.

3. The safe post-treatment process for 11α,17α-dihydroxyprogesterone fermentation broth according to claim 1, characterized in that, In S4, the solid 11α,17α-dihydroxyprogesterone is off-white in color and has a purity of ≥99%.

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