Deoxycholic acid intermediates and uses thereof

Abstract

The application discloses a deoxycholic acid intermediate and application thereof, and belongs to the technical field of organic synthesis. The deoxycholic acid intermediate has a structural formula as shown in the specification, wherein R1 is -OH or -OAc; R2 is alpha-OH, beta-OH or =O; and R3 is =O or -CHCH3. In addition, the application further provides application of the deoxycholic acid intermediate in preparation of deoxycholic acid. The intermediate can be used for effectively and safely synthesizing deoxycholic acid.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, specifically to a deoxycholic acid intermediate and its applications. Background Technology

[0002] Deoxycholic acid, chemically known as C64, has the chemical formula C64. 24 H 40 O4, its structural formula is as follows:

[0003]

[0004] Deoxycholic acid, developed by Kythera Biopharmaceuticals, was the world's first topical lipolysis drug approved by the U.S. Food and Drug Administration (FDA) in April 2015. Deoxycholic acid is a free bile acid found in bile, possessing strong surface activity. It can disrupt and dissolve cell membranes, reducing the amount of subcutaneous fat in small, localized areas. In dermatological surgery, it can be used to treat hyperspermia. In products such as face powder, it can remove excess sebum and sweat without causing dryness. In clinical trials, compared to a placebo, deoxycholic acid effectively eliminated subchinial fat and improved overall appearance. Deoxycholic acid can be used to treat and improve moderate to severe subchinial fat protrusion or fullness (double chin) in adults.

[0005] In the development of the plant-derived active pharmaceutical ingredient (API) for this drug, the original patent route (CN 111511755 A) for the total synthesis of deoxycholic acid using 9-hydroxyBN methyl ester as the starting material is shown below:

[0006]

[0007] Furthermore, CN106955287A and CN107011401A mention the following route for the total synthesis of deoxycholic acid using 9-hydroxyAD as the starting material:

[0008]

[0009] Our study found that regardless of whether 9-hydroxyBN methyl ester or 9-hydroxyAD was used as the starting material, the following problems arose:

[0010] 1) The oxidation yield of C12 allyl position is low, generally between 50% and 60%, and the use of chromium oxide or tert-butanol peroxide makes the reagents unsafe and environmentally unfriendly.

[0011] 2) Hydrogenation of the double bond at positions △9 and 11 requires high hydrogen pressure (above 5 MPa), which is dangerous and not conducive to industrial production.

[0012] 3) Five products are generated during the hydrogenation process of the Δ9,11 double bond and C12-ketone. These products are difficult to control and require confirmation using liquid chromatography and NMR. The structures of the five products are as follows:

[0013] Summary of the Invention

[0014] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide a deoxycholic acid intermediate and its application, thereby solving the technical problem of how to effectively and safely synthesize deoxycholic acid in the prior art.

[0015] To achieve the above-mentioned technical objectives, the present invention provides a deoxycholic acid intermediate with the following structural formula: Where R1 is -OH or -OAc; R2 is α-OH, β-OH or =O; R3 is =O or -CHCH3.

[0016] Furthermore, it includes the following compounds:

[0017]

[0018] Furthermore, the present invention also proposes an application of the above-mentioned deoxycholic acid intermediate in the preparation of deoxycholic acid.

[0019] The application further includes the following steps:

[0020] Compound A9 was mixed with palladium on carbon and reacted with hydrogen under pressure at 25–35 °C to obtain compound A10.

[0021] The compound A10 is mixed with dichloromethane and methanol, and then one or more of lithium hydroxide solution, sodium hydroxide solution or potassium hydroxide solution are added at 0-5°C. The mixture is then heated to 45-50°C to react and generate deoxycholic acid.

[0022] In some embodiments, the method further includes: mixing compound A8 with methyl acrylate and cooling to 0–5°C under nitrogen protection, then adding ethylaluminum dichloride solution dropwise, and then generating compound A9 at 25–35°C.

[0023] In some embodiments, the method further includes: mixing compound A6 with tetrahydrofuran, then cooling to -20 to -10°C under nitrogen protection, and continuing to add lithium tri-tert-butoxy-aluminum hydride dropwise to generate compound A7, wherein the structural formula of compound A7 is: ;

[0024] Compound A7 was mixed with acetic anhydride, DMAP and triethylamine, and then refluxed to produce compound A8.

[0025] In some embodiments, the mixture further includes: mixing compound A5 with acetone, then cooling it to -10 to 0°C under nitrogen protection, followed by adding Des Martin oxidant and 4-methylmorpholine oxide to react and generate compound A6.

[0026] In some embodiments, the method further includes: mixing anhydrous tetrahydrofuran and ethyltriphenylphosphine bromide, adding potassium tert-butoxide under nitrogen protection, and then adding compound A4 at 40–45°C to generate compound A5.

[0027] In some embodiments, it also includes:

[0028] Compound A3, tetrahydrofuran, and copper nitrate trihydrate were mixed and then heated to 50-55°C for reaction. The mixture was then cooled to below 35°C and hydrogen peroxide was added dropwise. Finally, a saturated Na2EDTA aqueous solution and ethyl acetate were added to generate compound A4.

[0029] In some embodiments, it also includes:

[0030] The starting material 4AD and 4-methoxypyridine were mixed, and then palladium on carbon was added. The mixture was then reacted under hydrogen pressure at 25–35 °C to generate compound A1.

[0031] Compound A1 was mixed with glycerol, and then glucose, 3α-reductase, glucose dehydrogenase, coenzyme NAD and coenzyme NADP were added at 30-35°C. The pH was then adjusted to 7-8 to generate compound A2.

[0032] Toluene, 2-aminomethylpyridine, compound A2 and PTS were mixed, then heated to reflux, and then a water separation reaction was carried out to generate compound A3;

[0033] The structural formula of the starting material 4AD is:

[0034] ;

[0035] The structural formula of compound A1 is:

[0036] ;

[0037] The structural formula of compound A2 is:

[0038] ;

[0039] The structural formula of compound A3 is:

[0040] .

[0041] Compared with existing technologies, the beneficial effects of this invention include: the deoxycholic acid intermediate proposed in this invention enables the synthesis of deoxycholic acid via a novel synthetic route with high yield, avoiding the use of unsafe and environmentally unfriendly reagents such as chromium oxide or tert-butanol peroxide, making it more environmentally friendly and safe. Simultaneously, the use of 3α-reductase selectively reduces C3-keto to 3α-hydroxyl groups, eliminating the presence of the 3β-hydroxy isomer, achieving a yield of nearly 100%, high efficiency, and safety, thus realizing the effective and safe synthesis of deoxycholic acid. Detailed Implementation

[0042] The basic synthetic route of this invention is as follows:

[0043] 1) Starting material 4AD was hydrogenated under conditions of 4-methoxypyridine and 5% palladium on carbon to produce compound A1;

[0044] 2) Compound A1 is selectively reduced to compound A2 by 3α-reductase;

[0045] 3) Compound A2 is reacted with PTS and 2-aminomethylpyridine to form compound A3;

[0046] 4) Compound A3 reacts with copper nitrate, hydrogen peroxide, and disodium ethylenediaminetetraacetate to form compound A4;

[0047] 5) Compound A4 reacts with ethyltriphenylphosphine bromide and potassium tert-butoxide to form compound A5;

[0048] 6) Compound A5 selectively generates compound A6 under the oxidation of Des Martin oxidant and 4-methylmorpholine oxide;

[0049] 7) Compound A6 is selectively converted into compound A7 under the action of lithium tritert-butoxy-aluminum hydride;

[0050] 8) Compound A7 reacts with acetic anhydride, DMAP, and triethylamine to form compound A8;

[0051] 9) Compound A8 reacts with methyl acrylate and ethylaluminum dichloride to form compound A9;

[0052] 10) Compound A9 reacts with ethyl acetate and 10% palladium on carbon to form compound A10;

[0053] 11) Compound A10 reacts with sodium hydroxide to form deoxycholic acid.

[0054] This specific embodiment provides a deoxycholic acid intermediate with the following structural formula: In this context, R1 is -OH or -OAc; R2 is α-OH, β-OH, or =O; and R3 is =O or -CHCH3.

[0055] Furthermore, the deoxycholic acid intermediate includes the following compounds:

[0056]

[0057]

[0058] This specific embodiment also proposes an application of the above-mentioned deoxycholic acid intermediate in the preparation of deoxycholic acid, including the following steps:

[0059] The starting material 4AD and 4-methoxypyridine were mixed, and then palladium on carbon was added. The mixture was then reacted under hydrogen pressure at 25–35 °C to generate compound A1.

[0060] Compound A1 was mixed with glycerol, and then glucose, 3α-reductase, glucose dehydrogenase, coenzyme NAD and coenzyme NADP were added at 30-35°C. The pH was then adjusted to 7-8 to generate compound A2.

[0061] Toluene, 2-aminomethylpyridine, compound A2 and PTS were mixed, then heated to reflux, and then a water separation reaction was carried out to generate compound A3;

[0062] Compound A3, tetrahydrofuran, and copper nitrate trihydrate were mixed and then heated to 50-55°C for reaction. The mixture was then cooled to below 35°C and hydrogen peroxide was added dropwise. Finally, saturated Na2EDTA aqueous solution and ethyl acetate were added to generate compound A4.

[0063] Anhydrous tetrahydrofuran and ethyltriphenylphosphine bromide were mixed, and potassium tert-butoxide was added under nitrogen protection. Then, compound A4 was added at 40-45°C to generate compound A5.

[0064] Compound A5 was mixed with acetone, then cooled to -10 to 0°C under nitrogen protection, and then reacted with Des Martin oxidant and 4-methylmorpholine oxide to generate compound A6.

[0065] Compound A6 was mixed with tetrahydrofuran, and then cooled to -20 to -10°C under nitrogen protection. Lithium tritert-butoxyhydroxide was then added dropwise to generate compound A7, the structural formula of which is:

[0066] Compound A7 was mixed with acetic anhydride, DMAP and triethylamine, and then refluxed to produce compound A8;

[0067] Compound A8 was mixed with methyl acrylate and cooled to 0–5°C under nitrogen protection. Then, ethyl aluminum dichloride solution was added dropwise to generate compound A9 at 25–35°C.

[0068] Compound A9 was mixed with palladium on carbon and reacted with hydrogen under pressure at 25–35 °C to obtain compound A10.

[0069] The compound A10 is mixed with dichloromethane and methanol, and then lithium hydroxide solution, sodium hydroxide solution or potassium hydroxide solution is added at 0-5°C. The mixture is then heated to 45-50°C to react and generate deoxycholic acid.

[0070] The structural formula of the starting material 4AD is:

[0071] ;

[0072] The structural formula of compound A1 is:

[0073] ;

[0074] The structural formula of compound A2 is:

[0075] ;

[0076] The structural formula of compound A3 is:

[0077] .

[0078] The synthesis route is as follows:

[0079]

[0080] This invention primarily uses 4AD as a starting material to synthesize deoxycholic acid, avoiding the high-pressure and hazardous environment involved in the reaction of the Δ9, 11 double bond and C12-ketone. It pioneers a new method for adding the 12β-hydroxyl group, achieving a higher yield—more than 10% higher than the synthesis methods using the other two substrates. It also avoids the use of unsafe and environmentally unfriendly reagents such as chromium oxide or tert-butanol peroxide, making it more environmentally friendly and safer. Furthermore, the use of 3α-reductase selectively reduces C3-ketones to 3α-hydroxyl groups, eliminating the presence of the 3β-hydroxyl isomer. With a yield approaching 100%, this highly efficient and safe method can meet the requirements for stable industrial production.

[0081] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0082] Example 1

[0083] Add 400.0 ml of pyridine to a clean, dry reaction flask, and add 100.0 g of starting material 4AD while stirring. After the system is dissolved, add 10.0 g of 5% palladium on carbon. Maintain the temperature at 25–35 °C and pressurize with hydrogen to 0.2 MPa for 8 hours. Monitor the reaction by TLC until complete. Filter the system to remove the palladium on carbon. Concentrate the organic phase to near dryness in a hot water bath under reduced pressure. Dissolve the organic phase in 400.0 ml of dichloromethane, and add 100.0 ml of 1N hydrochloric acid while stirring for 20 minutes. Allow to stand, separate the layers, wash once with 100.0 ml of water, collect the organic phase, replace with methanol, and then replace with water to ensure no solvent residue. Cool to 0–10 °C, filter, and dry the filter cake to obtain 99.3 g of compound A1. The purity of A1: 5α-H isomer purity = 92.2:7.8.

[0084] Example 2:

[0085] Add 400.0 ml of 4-methylpyridine to a clean, dry reaction flask, and add 100.0 g of starting material 4AD while stirring. After the system is dissolved, add 10.0 g of 5% palladium on carbon. Maintain the temperature at 25–35 °C and pressurize with hydrogen to 0.2 MPa for 8 hours. Monitor the reaction by TLC until complete. Filter the system to remove the palladium on carbon. Concentrate the organic phase to near dryness in a hot water bath under reduced pressure. Dissolve the organic phase in 400.0 ml of dichloromethane, and add 100.0 ml of 1N hydrochloric acid while stirring for 20 minutes. Allow to stand, separate the layers, wash once with 100.0 ml of water, collect the organic phase, replace with methanol, and then replace with water to ensure no solvent residue. Cool to 0–10 °C, filter, and dry the filter cake to obtain 99.0 g of compound A1. The purity of A1: 5α-H isomer purity = 93.3:6.7.

[0086] Example 3:

[0087] Add 400.0 ml of 4-methoxypyridine to a clean, dry reaction flask, and add 100.0 g of starting material 4AD while stirring. After the system is dissolved, add 10.0 g of 5% palladium on carbon. Maintain the temperature at 25–35 °C and pressurize with hydrogen to 0.2 MPa for 8 hours. Monitor the reaction by TLC until complete. Filter the system to remove the palladium on carbon. Concentrate the organic phase to near dryness in a hot water bath under reduced pressure. Dissolve the organic phase in 400.0 ml of dichloromethane, and add 100.0 ml of 1N hydrochloric acid while stirring for 20 minutes. Allow to stand, separate the layers, wash once with 100.0 ml of water, collect the organic phase, replace with methanol, and then replace with water to ensure no solvent residue. Cool to 0–10 °C, filter, and dry the filter cake to obtain 99.1 g of compound A1. The purity of A1:5α-H isomer purity = 98.6:1.4.

[0088] Example 4:

[0089] Add 400.0 ml of 1-methylimidazole to a clean, dry reaction flask, and add 100.0 g of starting material 4AD while stirring. After the system is dissolved, add 10.0 g of 5% palladium on carbon. Maintain the temperature at 25–35 °C and pressurize with hydrogen to 0.2 MPa for 8 hours. Monitor the reaction by TLC until complete. Filter the system to remove the palladium on carbon. Concentrate the organic phase to near dryness in a hot water bath under reduced pressure. Dissolve the organic phase in 400.0 ml of dichloromethane, and add 100.0 ml of 1N hydrochloric acid while stirring for 20 minutes. Allow to stand, separate the layers, wash once with 100.0 ml of water, collect the organic phase, replace with methanol, and then replace with water to ensure no solvent residue. Cool to 0–10 °C, filter, and dry the filter cake to obtain 99.5 g of compound A1. The purity of A1: 5α-H isomer purity = 93.5:6.5.

[0090] The specific details of the raw materials and products in Examples 1-4 are shown in Table 1.

[0091] Table 1. Details of the raw materials and products in Examples 1-4

[0092]

[0093] The comparison in Table 1 confirms that the starting material 4AD, under the action of 4-methoxypyridine, can control the 5α-H isomer to within 2%, and the purity of compound A1 is about 99%. The compound A1 obtained by this method has high purity, low impurities, high yield, and significantly reduced cost, which is conducive to stable industrial production.

[0094] NMR data for compound A1: 1 H NMR (400MHz, CDCl3): δ=2.69 (dd, J=15.1, 13.3Hz, 1H), 2.48 (ddd, J=19.2, 8.8, 1.0Hz, 1H), 2. 33(td, J=14.6, 5.3Hz, 1H), 2.24-1.81(m, 9H), 1.76-1.13(m, 10H), 1.07(s, 3H), 0.91(s, 3H).

[0095] Example 5

[0096] Add 200.0 ml of water to a clean reaction flask, and while stirring, add 98.0 g of compound A1 (purity: 98.6%) and 700.0 ml of glycerol. After compound A1 is completely dissolved, adjust the reaction temperature to 30–35 °C, and add 120.0 g of glucose, 30.0 g of 3α-reductase, 30.0 g of glucose dehydrogenase, 2.0 g of coenzyme I (NAD), and 2.0 g of coenzyme II (NADP). After stirring evenly, adjust the pH to 7–8 with 2 mol / L sodium hydroxide solution and react for 3 hours, monitoring the concentration of compound A1 by HPLC until it is below 0.1%. After the reaction is complete, raise the temperature of the system to 70–80 °C and stir for 40 min. Filter while hot, collect the filtrate, and reheat the filter cake to 70–80 °C with 100.0 ml of water, stirring for 40 min. Combine the filtrates after hot filtration. The filtrate was concentrated under reduced pressure at 50–60°C to remove glycerol, then cooled to 0–10°C. 1N hydrochloric acid was slowly added dropwise to adjust the pH to 6–7, resulting in the precipitation of a large amount of solid. The mixture was kept at this temperature and stirred for 40 minutes, then filtered. The filter cake was rinsed with a small amount of tap water and dried in a 50°C hot air circulating oven until it met the required standard. 97.7 g of compound A2 was obtained, with a purity of 98.3%.

[0097] NMR data of compound A2: 1H NMR (400MHz, CDCl3): δ=3.65-3.62(m, 1H, 3-H), 0.95(s, 3H, 19-CH3), 0.85(s, 3H, 18-CH3).

[0098] The reaction formula for this embodiment is as follows:

[0099] .

[0100] Example 6

[0101] In a clean, dry reaction flask, 800.0 ml of toluene and 200.0 ml of 2-aminomethylpyridine were added. While stirring, 96.0 g of compound A2 and 9.6 g of PTS were added. The system was heated to reflux and subjected to water separation using a water separator for 8 hours. The reaction was monitored by TLC until complete. The system was cooled to approximately 60°C and concentrated under reduced pressure until no liquid flowed out. 700.0 ml of ethyl acetate was added, and the mixture was washed three times with 250.0 ml of water each time. The organic phase was collected, dried with 20.0 g of anhydrous magnesium sulfate until it met the acceptable standard, filtered, and the filtrate was depressurized to a viscous state. 600.0 ml of petroleum ether was added, and the system was heated to approximately 50°C and maintained at this temperature for 1 hour. The mixture was filtered while hot, and the filter cake was dried until it met the acceptable standard, yielding 106.5 g of compound A3 with a purity of 94.5%.

[0102] The reaction in this embodiment is as follows:

[0103]

[0104] NMR data for compound A3: H 1 NMR (400MHz, CDCl3): δ=8.52 (d, J=4.1Hz, 1H), 7.65 (dt, J=1.7, 7.7Hz, 1H), 7.42 (d, J=7.8Hz, 1H), 7.15-7.11 (m, 1H), 4.61-4.58 (m, 2H), 3.66-3.61 (m, 1H), 2.52-1.18 (m, 22H), 0.94 (s, 3H, 19-CH3), 0.88 (s, 3H, 18-CH3).

[0105] Example 7

[0106] In a clean, dry reaction flask, 800.0 ml of ethyl acetate and 200.0 ml of 2-aminomethylpyridine were added. While stirring, 96.0 g of compound A2 and 9.6 g of PTS were added. The system was heated to reflux and subjected to water separation using a separatory apparatus for 12 h. The reaction was monitored by TLC until complete. The system was cooled to room temperature and washed three times with 250.0 ml of water each time. The organic phase was collected, dried with 20.0 g of anhydrous magnesium sulfate until it met the acceptable standard, filtered, and the filtrate was depressurized to a viscous state. 600.0 ml of petroleum ether was added, and the system was heated to approximately 50 °C and maintained at this temperature for 1 h. The mixture was filtered while hot, and the filter cake was dried until it met the acceptable standard, yielding 107.5 g of compound A3 with a purity of 94.0%.

[0107] Example 8

[0108] In a clean, dry reaction flask, 700.0 ml of n-heptane and 200.0 ml of 2-aminomethylpyridine were added. While stirring, 96.0 g of compound A2 and 9.6 g of PTS were added. The system was heated to reflux and subjected to water separation using a water separator for 10 hours. The reaction was monitored by TLC until complete. The system was cooled to approximately 60°C and concentrated under reduced pressure until no liquid flowed out. 700.0 ml of ethyl acetate was added, and the mixture was washed three times with 250.0 ml of water each time. The organic phase was collected, dried with 20.0 g of anhydrous magnesium sulfate until it met the acceptable standard, filtered, and the filtrate was depressurized to a viscous state. 600.0 ml of petroleum ether was added, and the system was heated to approximately 50°C and maintained at this temperature for 1 hour. The mixture was filtered while hot, and the filter cake was dried until it met the acceptable standard, yielding 108.1 g of compound A3 with a purity of 98.0%.

[0109] The specific details of the raw materials and products in Examples 6-8 are shown in Table 2.

[0110] Table 2. Details of raw materials and products in Examples 6-8

[0111] project Feed amount (g) Product dry weight (g) A3 purity (%) Weight yield (%) Example 6 96.0 106.5 94.5％ 110.9％ Example 7 96.0 107.5 94.0％ 112.0％ Example 8 96.0 108.1 98.3％ 112.6％

[0112] By comparing Table 2, it was confirmed that the reaction time varies for solvents with different boiling points. The higher the boiling point, the more difficult it is to concentrate completely. The longer the concentration time, the easier it is for the product to deteriorate into raw materials. At the same time, the water-removing effect of the solvent affects the degree of reaction. Ethyl acetate has a poor water-removing effect and a poor degree of reaction.

[0113] Example 9

[0114] Add 1600.0 ml of tetrahydrofuran to a clean, dry reaction flask, then add 106.0 g of compound A3 (purity: 98.3%) and 74.0 g of copper nitrate trihydrate while stirring. Stir at room temperature for 45 minutes, then heat to 50°C and stir for 30 minutes. Cool to room temperature and add 160.0 ml of 30% hydrogen peroxide solution dropwise. After the addition is complete, stir at room temperature for 9 hours. Add saturated Na2EDTA aqueous solution (233.0 g dissolved in 1100.0 ml of water) and 1300.0 ml of ethyl acetate. Continue stirring at room temperature for 24 hours. Allow to stand and separate the liquids. Wash the organic phase twice with 1100.0 ml of 1N dilute hydrochloric acid each time. The aqueous phases were combined and extracted three times with 220.0 ml of ethyl acetate each time. The organic phases were combined, and 22.0 g of anhydrous magnesium sulfate was added and dried until qualified. The mixture was filtered, and the filtrate was depressurized to near dryness. 350.0 ml of isopropyl ether was added, and the mixture was kept warm and stirred for 1 hour. The mixture was filtered, and the filter cake was dried until qualified to obtain 66.0 g of compound A4 with a purity of 97.7%.

[0115] The reaction equation is as follows:

[0116]

[0117] NMR data for compound A4: 1 H NMR (400MHz, CDCl3): δ=3.84-3.79(m,1H), 3.71-3.64(m,1H), 2.54-2.45(m,1H), 2.21-2.02(m,1H)2.00-1.02(m,18H),0.99(s,3H,19-CH3),0.96(s,3H,18-CH3).

[0118] Example 10

[0119] Add 850.0 ml of acetone and 850.0 ml of methanol to a clean, dry reaction flask. Under nitrogen protection, add 106.0 g of compound A3 (purity: 98.3%), 130.0 g of copper trifluoromethanesulfonate, and 106.0 g of sodium vitamin C while stirring at room temperature. After the addition is complete, maintain the temperature and stir for 5–10 minutes. Insert an oxygen tube below the liquid surface of the reaction system and purge with pure oxygen for 30 minutes. While maintaining the oxygen supply, raise the temperature of the reaction system to 50–55°C and maintain the temperature and purge for 5 hours. The reaction system was cooled to room temperature, and saturated Na2EDTA aqueous solution (233.0 g dissolved in 1100.0 ml water) and 1300.0 ml ethyl acetate were added. The mixture was stirred at room temperature for 3 hours, allowed to stand, and separated. The aqueous phase was extracted three times with 220.0 ml ethyl acetate each time. The organic phases were combined, and 22.0 g of anhydrous magnesium sulfate was added to the organic phase. The mixture was dried to the acceptable level, filtered, and the filtrate was depressurized to near dryness. 350.0 ml isopropyl ether was added, and the mixture was stirred at the specified temperature for 1 hour. The mixture was filtered, and the filter cake was dried to the acceptable level to obtain 60.7 g of compound A4 with a purity of 96.2%.

[0120] The specific details of the raw materials and products in Examples 9-10 are shown in Table 3.

[0121] Table 3 details the raw materials and products of Examples 9-10.

[0122] project Feed amount (g) Product dry weight (g) A4 purity (%) Weight yield (%) Example 9 106.0 66.0 97.7％ 62.26％ Example 10 106.0 60.7 96.2％ 57.26％

[0123] Example 11

[0124] At room temperature, 650.0 ml of anhydrous tetrahydrofuran and 200.0 g of ethyltriphenylphosphine bromide were added to a clean, dry reaction flask. Under nitrogen protection, 55.0 g of potassium tert-butoxide was added, and the mixture was stirred at this temperature for 1 h. The system was then heated to 40–45 °C and stirred at this temperature for 30 min. 65.0 g of compound A4 (purity: 97.7%) was added, and the reaction was continued at this temperature for 4 h. The reaction was monitored by TLC until complete. The reaction system was cooled to room temperature and then slowly added to 300.0 ml of ice water. The mixture was separated, and the aqueous phase was extracted three times with 100.0 ml of ethyl acetate each time. The organic phases were combined, dried with 15.0 g of anhydrous magnesium sulfate until acceptable, filtered, and concentrated under reduced pressure. 200.0 ml of methyl tert-butyl ether was added, and the mixture was stirred at room temperature for 1 h. The mixture was filtered, and the filter cake was dried until acceptable, yielding 62.3 g of compound A5 with a purity of 98.6%.

[0125] The reaction equation is as follows:

[0126]

[0127] NMR data for compound A5: 1H NMR (400MHz, CDCl3): δ=5.20-5.14(m,1H), 3.84-3.79(m,1H), 3.71-3.64(m,1H), 2.06-1.01(m,23H), 0.99(s,3H,19-CH3), 0.96(s,3H,18-CH3).

[0128] Example 12

[0129] Add 600.0 ml of acetone to a clean, dry reaction flask, and add 62.0 g of compound A5 (purity: 98.6%) while stirring. Under nitrogen protection, lower the temperature to -10 to 0 °C, and slowly add 80.0 g of Des Martin oxidant and 2.8 g of 4-methylmorpholine oxide. Insulate the reaction temperature for 6 h, and monitor the reaction until complete by TLC. Control the temperature below 10 °C, and slowly add 1800 ml of sodium thiosulfate solution (1 mol / L). After the addition is complete, control the temperature at 0 to 10 °C and stir for 1 h. Filter, and dry the filter cake to the acceptable level to obtain 56.5 g of compound A6 (purity: 98.7%).

[0130] The reaction equation is as follows:

[0131]

[0132] NMR data for compound A6: 1 H NMR (400MHz, CDCl3): δ = 5.51-5.41 (m, 1H), 3.84-3.79 (m, 1H), 2.11-1.01 (m, 23H), 0.98 (s, 3H, 19-CH3), 0.96 (s, 3H, 18-CH3).

[0133] Example 13

[0134] Add 560.0 ml of tetrahydrofuran and 56.0 g of compound A6 (purity: 98.7%) to a clean, dry reaction flask. Under nitrogen protection, cool the system to -20 to -10 °C, and slowly add 190.0 ml of a 1 mol / L THF solution of lithium tri-tert-butoxy-aluminum hydride. After the addition is complete, maintain the reaction temperature for 22 h, and monitor the reaction until complete by TLC. Slowly add 840.0 ml of 4 mol / L hydrochloric acid at a temperature below 10 °C, allow to stand, and separate the layers. Extract the aqueous phase three times with 150.0 ml of dichloromethane each time. Combine the organic phases and concentrate under reduced pressure, replacing with n-heptane, until a viscous state is reached. Add 150.0 ml of n-heptane, heat to 50–55 °C, and stir for 30 minutes. Then cool to 10–15 °C and stir for 60 minutes. Filter, and dry the filter cake to obtain compound A7 (46.1 g) with a purity of 99.0%.

[0135] The reaction equation is as follows:

[0136]

[0137] NMR data for compound A7: 1 H NMR (400MHz, CDCl3): δ = 5.20-5.14 (m, 1H), 3.81-3.66 (m, 2H), 2.06-1.01 (m, 23H), 0.99 (s, 3H, 19-CH3), 0.95 (s, 3H, 18-CH3).

[0138] Example 14

[0139] Add 270.0 ml of chloroform to a clean, dry reaction flask, and while stirring, add 45.0 g of compound A7 (purity: 99.0%), 4.5 g of DMAP, 90.0 ml of triethylamine, and 90.0 ml of acetic anhydride. Heat the system to reflux for 5 hours, and monitor the reaction until complete by TLC. Cool the system to 10–20 °C, and slowly add 200.0 ml of methanol dropwise while maintaining a temperature T ≤ 30 °C. After the addition is complete, concentrate the system to a viscous state, replace with methanol, and finally concentrate again until approximately 100 ml remains. Cool to 0–10 °C, filter, and dry the filter cake to obtain 53.3 g of compound A8 with a purity of 99.3%.

[0140] The reaction equation is as follows:

[0141]

[0142] NMR data for compound A8: 1 H NMR (400MHz, CDCl3): δ = 5.23-5.18 (m, 1H), 4.73 (m, 1H), 2.18 (s, 6H), 2.11-0.91 (m, 24H), 0.93 (s, 3H, 19-CH3), 0.87 (s, 3H, 18-CH3).

[0143] Example 15

[0144] Add 500.0 ml of dichloromethane to a clean, dry reaction flask. While stirring, add 50.0 g of compound A8 (purity: 99.3%) and 35.0 ml of methyl acrylate. Under nitrogen protection, lower the temperature to 0–5 °C. Slowly add 250.0 ml of 1.8 mol / L ethylaluminum dichlorotoluene solution dropwise. After the addition is complete, raise the temperature to 25–35 °C and stir for 20 h. Monitor the reaction by TLC until complete to obtain compound A9. Slowly add the system to 1000.0 ml of ice water. Separate the layers. Extract the aqueous phase three times with 100.0 ml of dichloromethane each time. Combine the organic phases and dry them with 15.0 g of anhydrous magnesium sulfate until acceptable. Filter the solution and concentrate the filtrate to near dryness under reduced pressure. Replace with ethyl acetate and concentrate the dichloromethane completely to a viscous state. Add 500.0 ml of ethyl acetate for later use.

[0145] The reaction equation is as follows:

[0146]

[0147] NMR data for compound A9: 1 H NMR (400MHz, CDCl3): δ=5.23-5.18(m, 1H), 4.73(m, 1H), 3.66(s, 3H), 3.45-3.32(m ,1H), 2.04(s,6H), 2.11-0.91(m,26H), 0.94(s,3H,19-CH3), 0.82(s,3H,18-CH3).

[0148] Example 16

[0149] 5.0 g of 10% palladium on carbon was added to the prepared compound A9 system. The system was kept at 25–35 °C and pressurized with hydrogen to 0.6 MPa for 4 h. The reaction was monitored by HPLC until complete. The palladium on carbon was removed by filtration, and the filter cake was washed with a small amount of ethyl acetate. The filtrate was concentrated under reduced pressure to a viscous state, cooled to 0–10 °C, filtered, and the filter cake was dried to meet the requirements, yielding 56.8 g of compound A10 with a purity of 99.6%.

[0150] The reaction equation is as follows:

[0151]

[0152] NMR data for compound A10: 1H NMR (400MHz, CDCl3): δ=5.07 (d, 1H, J=2.8Hz), 4.70 (m, 1H), 3.66 (s, 3H), 2.34-2.31 (m, 1H), 2.22- 2.20 (m, 1H), 2.10 (s, 3H), 2.03 (s, 3H), 1.88-1.10 (m, 24H), 0.90 (s, 3H), 0.81 (s, 3H), 0.79 (s, 3H).

[0153] Example 17

[0154] In a clean, dry reaction flask, add 110.0 ml of dichloromethane and 220.0 ml of methanol. While stirring, add 55.0 g of compound A10 (purity: 99.6%). Cool the system to 0–5 °C and slowly add sodium hydroxide solution (28.0 g sodium hydroxide dissolved in 56.0 ml water). After the addition is complete, heat the system to 45–50 °C and maintain the temperature for 3 hours. Monitor the reaction by TLC until complete. Concentrate the system under reduced pressure until no dichloromethane or methanol remains, then replace with water. After concentration, cool the system to 10–20 °C and extract once with 220.0 ml of dichloromethane. Separate the layers. Adjust the pH of the aqueous phase to 1–2 with 2N hydrochloric acid, stir for 30 minutes, filter, and dry the filter cake to obtain 41.5 g of deoxycholic acid with a purity of 99.8%.

[0155] Example 18

[0156] In a clean, dry reaction flask, add 110.0 ml of dichloromethane and 220.0 ml of methanol. While stirring, add 55.0 g of compound A10 (purity: 99.6%). Cool the system to 0–5 °C and slowly add potassium hydroxide solution (39.0 g potassium hydroxide dissolved in 56.0 ml water). After the addition is complete, heat the system to 45–50 °C and maintain the temperature for 2 hours. Monitor the reaction by TLC until complete. Concentrate the system under reduced pressure until no dichloromethane or methanol remains, then replace with water. After concentration, cool the system to 10–20 °C and extract once with 220.0 ml of dichloromethane. Separate the layers. Adjust the pH of the aqueous phase to 1–2 with 2N hydrochloric acid, maintain the temperature and stir for 30 minutes, filter, and dry the filter cake to obtain 41.6 g of deoxycholic acid with a purity of 99.8%.

[0157] Example 19

[0158] In a clean, dry reaction flask, add 110.0 ml of dichloromethane and 220.0 ml of methanol. While stirring, add 55.0 g of compound A10 (purity: 99.6%). Cool the system to 0–5 °C and slowly add lithium hydroxide solution (20.0 g of lithium hydroxide dissolved in 56.0 ml of water). After the addition is complete, heat the system to 45–50 °C and maintain the temperature for 5 hours. Monitor the reaction by TLC until complete. Concentrate the system under reduced pressure until no dichloromethane or methanol remains, then replace with water. After concentration, cool the system to 10–20 °C and extract once with 220.0 ml of dichloromethane. Separate the layers. Adjust the pH of the aqueous phase to 1–2 with 2N hydrochloric acid, maintain the temperature and stir for 30 minutes, filter, and dry the filter cake to obtain 41.0 g of deoxycholic acid with a purity of 99.6%.

[0159] NMR data for deoxycholic acid: 1H NMR (400MHz, CDCl3): δ=3.94(t, J=2.7Hz, 1H), 3.58-3.45(m, 1H), 2.41-2.11(m, 2H), 1.99-1.71(m, 7H), 1.67-1.04(m, 19H), 0.99(d, J=6.4Hz, 3H), 0.92(s, 3H), 0.70(s, 3H).

[0160] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing deoxycholic acid, characterized in that, Includes the following steps: Anhydrous tetrahydrofuran and ethyltriphenylphosphine bromide were mixed, and potassium tert-butoxide was added under nitrogen protection. Then, compound A4 was added at 40-45°C to generate compound A5. Compound A5 was mixed with acetone, then cooled to -10 to 0°C under nitrogen protection, and then reacted with Des Martin oxidant and 4-methylmorpholine oxide to generate compound A6. Compound A6 was mixed with tetrahydrofuran, and then cooled to -20 to -10°C under nitrogen protection. Lithium tritert-butoxyhydroxide was then added dropwise to generate compound A7, the structural formula of which is: ; Compound A7 was mixed with acetic anhydride, DMAP and triethylamine, and then refluxed to produce compound A8; Compound A8 was mixed with methyl acrylate and cooled to 0–5°C under nitrogen protection. Then, ethyl aluminum dichloride solution was added dropwise, and compound A9 was generated at 25–35°C. Compound A9 was mixed with palladium on carbon and reacted with hydrogen under pressure at 25–35 °C to obtain compound A10. The compound A10 is mixed with dichloromethane and methanol, and then one or more of lithium hydroxide solution, sodium hydroxide solution or potassium hydroxide solution are added at 0-5°C. The mixture is then heated to 45-50°C to react and generate deoxycholic acid. The synthetic route is as follows: 。 2. The preparation method according to claim 1, characterized in that, Also includes: Compound A3, tetrahydrofuran, and copper nitrate trihydrate were mixed and then heated to 50-55°C for reaction. The mixture was then cooled to below 35°C and hydrogen peroxide was added dropwise. Finally, a saturated aqueous solution of Na2EDTA and ethyl acetate were added to generate compound A4.

3. The preparation method according to claim 2, characterized in that, Also includes: The starting material 4AD and 4-methoxypyridine were mixed, and then palladium on carbon was added. The mixture was then reacted under hydrogen pressure at 25–35 °C to generate compound A1. Compound A1 was mixed with glycerol, and then glucose, 3α-reductase, glucose dehydrogenase, coenzyme NAD and coenzyme NADP were added at 30-35°C. The pH was then adjusted to 7-8 to generate compound A2. Toluene, 2-aminomethylpyridine, compound A2 and PTS were mixed, then heated to reflux, and then a water separation reaction was carried out to generate compound A3; The structural formula of the starting material 4AD is: ； The structural formula of compound A1 is: ； The structural formula of compound A2 is: ； The structural formula of compound A3 is: 。

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