CN111560045A - Method for synthesizing lithocholic acid by taking BA as raw material - Google Patents

Abstract

The invention discloses a method for synthesizing lithocholic acid, which adopts 21-hydroxy-20-methylpregna-4-ene-3-ketone (BA) as a raw material, and synthesizes the lithocholic acid through oxidation reaction, Wittig reaction and reduction reaction. Lithocholic acid. The method for synthesizing lithocholic acid of the invention has the advantages of environmental friendliness, simple steps, few side reactions, high yield, cheap and easy-to-obtain raw materials, and is suitable for industrial production; The problem of large-scale industrial production.

Description

A kind of method for synthesizing lithocholic acid with BA as raw material

technical field

The invention belongs to the field of organic chemistry, in particular to a method for synthesizing lithocholic acid by using 21-hydroxy-20-methylpregna-4-en-3-one (BA) as a raw material.

Background technique

Lithocholic acid (3α-hydroxy-5β-cholanoic acid, formula (4)) is a secondary bile acid, also known as cholelithic acid. important reference value. Lithocholic acid has a wide range of biological activities, such as activating vitamin D receptors (J. Lipid Res. 2005, 46, 46–57.), inhibiting the activity of PTP1B, a target related to the treatment of diabetes (Bioorg.Med.Chem.Lett.2012 , 22, 7237–7242), activation of TGR5 receptor, a relevant target for the treatment of diabetes (J.Med.Chem.2008,51,1831–1841), antibacterial and antifungal (FarmacoSci.1984,39,305–315), Anti-aging activity (Int. J. Mol. Sci. 2014, 15, 16522-16543). In particular, this compound also has anti-tumor activity. In recent years, it was found that lithocholic acid can inhibit the mutation of DNA polymerase β and reduce the probability of tumors arising from DNA polymerase β mutation; in 2011, led by Concordia University, including Lithocholic acid selectively kills several types of cancer cells, such as those found in brain tumors and breast cancer cancer cells, while not affecting normal cells, heralds great prospects for lithocholic acid as a chemotherapeutic agent (Oncotarget 2011, 2, 761-782).

Journal of Biological Chemistry, 1946, 162, 555-563, reported that after methylation with deoxycholic acid, the 3-hydroxyl group was protected with benzoyl chloride, and then the 7-position hydroxy group was reacted with benzoyl chloride, followed by alkaline hydrolysis of the methyl ester. And the benzoyl group on the 3rd position, methyl esterification, high temperature pyrolysis dehydration, alkaline hydrolysis methyl ester, platinum oxide hydrogenation reaction to obtain lithocholic acid. The reaction formula is as follows:

In the process of preparing lithocholic acid by this method, high temperature pyrolysis is required to remove the benzoyl group, the route is cumbersome, the yield is low, and the total yield is only 23.5%, which is not suitable for large-scale production.

In 2016, the patent (CN201610369086.4) applied by the research group of the present invention reported that deoxycholic acid was used as a raw material, and lithocholic acid was synthesized through reactions such as methylation protection, acetic anhydride protection of the 3-hydroxyl group, dehydration, hydrogenation, and hydrolysis. . The reaction formula is as follows:

Although the raw materials are easy to obtain and the yield is high, the total yield is 76.3%, but the route is still relatively long, which limits the industrial application of the route to a certain extent.

The reported lithocholic acid synthetic route not only has the problems of too complicated steps, low yield and high cost, but also the existing synthetic routes reported above all use animal cholic acid deoxycholic acid as the starting material, but due to avian influenza, mad cow disease The emergence of diseases such as Streptococcus suis and African swine fever, people have doubts about the safety of animal-derived raw materials. Therefore, it is of great significance and industrial value to develop a method for the efficient synthesis of lithocholic acid based on plant sources. .

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned defects in the prior art, the present invention uses 21-hydroxy-20-methylpregn-4-en-3-one ((20S)-21-hydroxy-20-methylpregn-4-en-3 -one) as the raw material, through oxidation reaction, Wittig reaction and reduction reaction to synthesize lithocholic acid LCA. The method for synthesizing lithocholic acid of the invention has the advantages of simple steps, environmental friendliness, few side reactions, a yield of 87.4%, cheap and easily available raw materials, and is suitable for industrial production.

The raw material 21-hydroxy-20-methylpregn-4-en-3-one ((20S)-21-hydroxy-20-methylpregn-4-en-3-one) used in the present invention is also called BA ( bisnoralcohol), derived from the fermentation of phytosterols from oil and fat process wastes, is a green raw material of plant origin, with an annual output of 1,000 tons and low price, which can well avoid the infection problems of pathogenic bacteria and viruses in the prior art. .

The method for synthesizing lithocholic acid with 21-hydroxy-20-methylpregna-4-en-3-one (BA) raw material provided by the invention comprises the following steps:

Step (a): in the first solvent, the BA represented by the formula (1) undergoes oxidation reaction to obtain the compound of the formula (2);

Step (b): in the second solvent, the compound of formula (2) is subjected to Wittig reaction to obtain the compound of formula (3);

Step (c): in the third solvent, the compound of formula (3) is subjected to reduction reaction to obtain the compound of formula (4) lithocholic acid;

Described reaction process is shown in following reaction formula (i):

In step (a) of the present invention, the oxidation reaction is specifically: in the first solvent, BA represented by formula (1), TEMP O (2,2,6,6-tetramethylpiperidine oxide), Sodium bicarbonate, tetrabutylammonium bromide and oxidant undergo oxidation reaction to obtain the compound of formula (2).

In step (a), the oxidation reaction is carried out under the action of an oxidizing agent, and the oxidizing agent is selected from N-chlorosuccinimide (NCS), N-bromosuccinimide (NBS), 2-iodoacyl One or more of benzoic acid (IBX) and the like; preferably, N-chlorosuccinimide (NCS).

In step (a), the molar ratio of BA, TEMPO, sodium bicarbonate, tetrabutylammonium bromide, and oxidant represented by formula (1) is 1:(0～1):(0～20):(0～1). 1): (1-5); preferably, 1: (0.01-1): (1.35-20): (0.1-1): (1.15-5); more preferably, 1: 0.01: 1.35: 0.1:1.15.

In step (a), the first solvent is selected from one or more of dichloromethane, tetrahydrofuran, toluene, dimethyl sulfoxide, water, etc.; preferably, it is a mixed solvent of dichloromethane and water.

In step (a), the temperature of the oxidation reaction is 0-30°C; preferably, it is 0°C.

In step (a), the oxidation reaction time is 2-8h; preferably, it is 5h.

In a specific embodiment, the synthesis step of the compound of formula (2) includes: BA represented by formula (1) is dissolved in the first solvent, then TEMPO, sodium bicarbonate, tetrabutylammonium bromide, NCS are added to generate Oxidation reaction yields the compound of formula (2).

In step (b), the Wittig reaction is specifically as follows: in the second solvent, the compound of formula (2) and ethoxyformylmethylene triphenylphosphine undergo Wittig reaction to obtain the compound of formula (3).

Wherein, the molar ratio of the compound of formula (2) to ethoxyformylmethylenetriphenylphosphine is 1:(1-5); preferably, it is 1:2.

Wherein, the second solvent is selected from one or more of benzene, toluene, ethyl acetate, tetrahydrofuran, hexane, etc.; preferably, it is toluene.

Wherein, the temperature of the Wittig reaction is 80-130°C; preferably, it is 110°C.

Wherein, the time of the Wittig reaction is 2～8h; preferably, it is 4h.

Or, in step (b), the Wittig reaction is specifically: in the second solvent, the compound of formula (2), sodium hydride and triethyl phosphonoacetate undergo Wittig reaction to obtain the compound of formula (3).

Wherein, the second solvent is selected from one or more of benzene, toluene, ethyl acetate, tetrahydrofuran, hexane, etc.; preferably, it is tetrahydrofuran.

Wherein, the molar ratio of the compound of formula (2), sodium hydride and triethyl phosphonoacetate is 1:(1-5):(1-5); preferably, it is 1:1.5:1.5.

Wherein, the temperature of the Wittig reaction is 0-30°C; preferably, it is 0°C.

Wherein, the time of the Wittig reaction is 2～8h; preferably, it is 4h.

In a specific embodiment, the synthesis step of the compound of formula (3) comprises: compound of formula (2), ethoxyformylmethylene triphenylphosphine or compound of formula (2), sodium hydride, triethyl phosphonoacetate Dissolved in the second solvent, the Wittig reaction takes place to give the compound of formula (3).

In step (c), the reduction reaction is specifically as follows: the compound of formula (3), sodium tert-butoxide, and RaneyNi (RaneyNi) are dissolved in the third solvent, and the H ₂ is replaced and pressurized, and a reduction reaction occurs to obtain the formula (4) the compound lithocholic acid;

Wherein, the molar ratio of the compound of formula (3) and sodium tert-butoxide is 1:(1-5); preferably, it is 1:2.

Wherein, the mass ratio of the compound of formula (3) to Raney nickel is 1:(0.1-5); preferably, it is 1:1.

Wherein, the third solvent is selected from one or more of isopropanol, n-butanol, ethanol, methanol, tert-butanol, etc.; preferably, it is isopropanol.

Wherein, the pressure of the reduction reaction is 1-10 MPa; preferably, it is 4 MPa.

Wherein, the temperature of the reduction reaction is 40-120°C; preferably, it is 90°C.

Wherein, the time of the reduction reaction is 5-48h; preferably, it is 24h.

The beneficial effects of the present invention include: the preparation method of lithocholic acid of the present invention uses raw material BA as a plant source material, which avoids the infection problem of pathogenic bacteria and viruses, and is cheap and easy to obtain; the synthesis steps of the lithocholic acid are simple and easy to operate. It has few reactions and high yield of 87.4%, is environmentally friendly, and is convenient to realize industrialization and industrialized production; it solves the problems of high synthesis cost, low yield and unsuitable for large-scale industrialized production in the prior art.

Detailed ways

The present invention will be further described in detail with reference to the following specific embodiments. Except for the content specifically mentioned below, the process, conditions, experimental methods, etc. for implementing the present invention are all common knowledge and common knowledge in the field, and the present invention is not particularly limited.

In the following examples, the structure of the compounds was determined by nuclear magnetic resonance; the reagents were mainly provided by Shanghai Sinopharm Chemical Reagent Company; the products were purified mainly by column chromatography, and silica gel (200-300) was produced by Qingdao Ocean Chemical Factory.

Example 1 Preparation of compound of formula (2)

(1-1) BA (10.00 g, 30.26 mmol), TEMPO (47 mg, 0.30 mmol), 100 mL DCM, NaHCO ₃ (3.43 g, 40.85 mmol), tetrabutylammonium bromide (977 mg) were sequentially added to a 500 mL single-necked flask , 3.03 mmol) of H ₂ O (40 mL) solution and NCS (4.65 g, 34.80 mmol) were reacted at 0 °C for 5 h. After TLC detected that the reaction of the raw materials was complete, sodium thiosulfate pentahydrate solution (1.3 g sodium thiosulfate pentahydrate/25 mL H ₂ O) was added, stirred at 10° C. for 20 min, and the liquid was separated. The aqueous phase was washed with dichloromethane (50 mL×3 ) extraction, the organic phases were combined, 120 mL of 1% sodium hydroxide solution was added, stirred for 30 min, the layers were separated, the aqueous phase was back-extracted once with dichloromethane (50 mL), washed with water, and concentrated under reduced pressure to obtain compound 2 (pale yellow solid, 9.50 g, molar yield 95%).

(1-2) BA (5.0 g, 15.13 mmol), IBX (8.5 g, 30.26 mmol), 50 mL of THF and 50 mL of DMSO were sequentially added to a 250 mL single-necked flask, and reacted at room temperature for 5 h. After the reaction was detected by TLC, water was added, filtered with suction, extracted with dichloromethane (50 mL×3), washed with water (50 mL×2), washed with saturated sodium bicarbonate solution (50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, silica gel Purification by column chromatography (PE/EA=3/1, v/v) gave compound 2 (4.9 g, white solid, 98% molar yield). mp: 155-157°C. ¹ H NMR (400 MHz, CDCl ₃ ) δ 9.55 (s, 1H), 5.71 (s, 1H), 2.45-2.23 (m, 5H), 1.99 (t, J=13.7 Hz, 2H), 1.91-1.78(m, 2H), 1.68(t, J=10.2Hz, 2H), 1.43(m, 5H), 1.30-1.19(m, 2H), 1.17(s, 3H), 1.11(d , J=5.5Hz, 3H), 1.06-0.89(m, 3H), 0.75(s, 3H). ¹³ C NMR (100MHz, CDCl ₃ )δ205.00, 199.65, 171.31, 123.99, 55.25, 53.84, 51.04, 49.54, 43.10,39.39,38.68,35.80,35.68,34.06,32.93,32.05,27.11,24.64,21.06,17.48,13.53,12.44.HRMS(ESI):calcdfor C ₂₂ H ₃₂ NaO ₂ [M+Na] ⁺ ,351.2295,found351 .2292.

The preparation of the compound of the second formula (3)

(2-1) Compound 2 (1.0 g, 3.04 mmol), ethoxyformylmethylene triphenylphosphine (2.12 g, 6.08 mmol) and toluene (15 mL) were sequentially added to a 100 mL single-necked flask, and the reaction was refluxed for 4 h. TLC detected the completion of the reaction, concentrated under reduced pressure, and purified by silica gel column chromatography (PE/EA=3/1, v/v) to obtain compound 3 (1.15 g, white solid, molar yield 98%).

(2-2) Sodium hydride (913 mg, 22.83 mmol) and 50 mL of THF were successively added to a 250 mL single-necked flask, and after stirring for 15 min, triethyl phosphonoacetate (4.5 mL, 22.83 mmol), compound 2 (5.00 g, 15.22 mmol) were successively added. ) and reacted at 0 °C for 4 h. After TLC detected that the reaction of the raw materials was complete, the mixture was concentrated under reduced pressure and slurried with methanol to obtain compound 3 (white solid, 5.76 g, molar yield 95%).

(2-3) Compound 2 (1.0 g, 3.04 mmol), ethoxyformylmethylene triphenylphosphine (2.12 g, 6.08 mmol) and THF (15 mL) were sequentially added to a 100 mL single-necked flask, and the reaction was refluxed for 4 h. TLC detected the completion of the reaction, concentrated under reduced pressure, and purified by silica gel column chromatography (PE/EA=3/1, v/v) to obtain compound 3 (1.10 g, white solid, molar yield 94%).

(2-4) Sodium hydride (913 mg, 22.83 mmol) and 50 mL of toluene were successively added to a 250 mL single-necked flask, and after stirring for 15 min, triethyl phosphonoacetate (4.5 mL, 22.83 mmol) was added successively, compound 2 (5.00 g, 15.22 mmol), and reacted at 0 °C for 4 h. After TLC detected the reaction of the raw materials, the mixture was concentrated under reduced pressure and slurried with methanol to obtain compound 3 (white solid, 5.64 g, 93% molar yield). mp: 160-162°C. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.81 (dd, J=15.3, 9.0 Hz, 1H), 5.71 (d, J=13.4 Hz, 2H), 4.24-4.09 (m, 2H), 2.45-2.21(m, 5H), 2.00(d, J=12.6Hz, 2H), 1.80(m, 1H), 1.76-1.33(m, 7H), 1.26(m, 6H), 1.17(s , 3H), 1.08(d, J=6.2Hz, 3H), 1.05-0.86(m, 3H), 0.73(s, 3H). ¹³ C NMR (100MHz, CDCl ₃ )δ199.70, 171.51, 167.16, 154.56, 123.94 HRMS calc ₂₆ H ₃₈ NaO ₃ [M+Na] ⁺ , 421.2713, found421.2708.

The preparation of the compound of embodiment three formula (4)

(3-1) Compound 3 (1.0g, 2.51mmol), 20mL isopropanol, 1.0g Raney Ni, H ₂ (4.0MPa), sodium tert-butoxide (482mg, 5.02mmol), 90 mL were successively added to the autoclave. The reaction was carried out at °C for 48 hours. After TLC detected the completion of the reaction, acetic acid was added to adjust the pH to 5, and celite was suction filtered. The filtrate was concentrated under reduced pressure, dissolved in ethyl acetate (30 mL), washed with water and saturated sodium chloride solution in turn, dried over anhydrous sodium sulfate, and dried under reduced pressure. Concentration and column chromatography (PE:EA=2:1) gave lithocholic acid (841 mg, white solid, 89% molar yield).

(3-2) Compound 3 (1.0g, 2.51mmol), 20mL isopropanol, sodium tert-butoxide (482mg, 5.02mmol) and 1.0g Raney Ni were successively added to the autoclave, and H ₂ was replaced and pressurized to 4.0MPa, react at 90°C for 24h. After TLC detected that the reaction of the raw materials was complete, acetic acid was added to adjust the pH to 5, suction filtration was performed using celite, the filtrate was concentrated under reduced pressure, dissolved in ethyl acetate (30 mL), washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure , column chromatography (PE:EA=2:1) to obtain lithocholic acid (white solid, 860 mg, yield 91%). mp: 185-186.5°C. ¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.93(s, 1H), 4.42(d, J=3.6Hz, 1H), 3.41-3.33(m, 1H), 2.27- 2.18(m, 1H), 2.14-2.05(m, 1H), 1.92(d, J=10.8Hz, 1H), 1.83-1.74(m, 2H), 1.71-1.64(m, 2H), 1.62-1.57( m,1H),1.56-1.46(m,2H),1.39-1.29(m,7H),1.25-1.12(m,6H),1.11-1.00(m,4H),0.94-0.88(m,1H), 0.87(t, J=3.1Hz, 6H), 0.61(s, 3H). ¹³ C NMR (125MHz, DMSO-d ₆ )δ175.28, 70.32, 56.51, 56.01, 42.73, 42.01, 40.44, 40.15, 36.74, 35.85,35.62,35.30,34.66,31.18,31.14,30.83,28.18,27.37,26.63,24.31,23.73,20.89,18.58,12.32.HRMS(ESI):calcd for C ₂₄ H ₄₀ NaO ₄ [M+Na] ⁺ , 399.2870,found399.2885.

The protection content of the present invention is not limited to the above embodiments. Variations and advantages that can occur to those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the appended claims are the scope of protection.

Claims (10)

1. A method for synthesizing lithocholic acid, comprising the steps of:

step (a): in a first solvent, using BA shown in a formula (1) as a raw material to carry out oxidation reaction to obtain a compound shown in a formula (2);

step (b): in a second solvent, carrying out a Wittig reaction on the compound shown in the formula (2) to obtain a compound shown in the formula (3);

step (c): in a third solvent, carrying out reduction reaction on the compound of the formula (3) to obtain lithocholic acid of the compound of the formula (4);

the reaction process is shown as a reaction formula (i):

2. the method according to claim 1, wherein in step (a), the oxidation reaction is specifically: in the first solvent, BA shown in a formula (1), TEMPO, sodium bicarbonate, tetrabutylammonium bromide and an oxidizing agent are subjected to oxidation reaction to obtain a compound shown in a formula (2).

3. The method according to claim 2, wherein the molar ratio of BA, TEMPO, sodium bicarbonate, tetrabutylammonium bromide and the oxidizing agent represented by formula (1) is 1: (0-1): (0-20): (0-1): (1-5); and/or the oxidant is selected from one or more of N-chlorosuccinimide NCS, N-bromosuccinimide NBS and 2-iodosylbenzoic acid IBX; and/or, the first solvent is selected from one or more of dichloromethane, tetrahydrofuran, toluene, dimethyl sulfoxide and water; and/or the temperature of the oxidation reaction is 0-30 ℃; and/or the time of the oxidation reaction is 2-8 h.

4. The method as claimed in claim 1, wherein in step (b), the Wittig reaction is specifically: in the second solvent, the compound of the formula (2) and carbethoxy methylene triphenylphosphine are subjected to a Wittig reaction to obtain a compound of the formula (3).

5. The process according to claim 4, wherein the compound of formula (2) is present in a molar ratio of 1: (1-5); and/or the second solvent is selected from one or more of benzene, toluene, ethyl acetate, tetrahydrofuran and hexane; and/or the temperature of the Wittig reaction is 80-130 ℃; and/or the Wittig reaction time is 2-8 h.

6. The method as claimed in claim 1, wherein in step (b), the Wittig reaction is specifically: in the second solvent, carrying out Wittig reaction on the compound shown in the formula (2), sodium hydride and triethyl phosphonoacetate to obtain the compound shown in the formula (3).

7. The method of claim 6, wherein the second solvent is selected from one or more of benzene, toluene, ethyl acetate, tetrahydrofuran, hexane; and/or the molar ratio of the compound of formula (2), sodium hydride and triethyl phosphonoacetate is 1: (1-5): (1-5); and/or the temperature of the Wittig reaction is 0-30 ℃; and/or the Wittig reaction time is 2-8 h.

8. The method according to claim 1, wherein in step (c), the reduction reaction is specifically: the compound of formula (3), sodium tert-butoxide, Raney's nickel are dissolved in the third solvent, H₂Pressurizing after replacement, and carrying out reduction reaction to obtain lithocholic acid.

9. The process of claim 8, wherein the compound of formula (3) is present in a molar ratio of sodium tert-butoxide to 1: (1-5); and/or the mass ratio of the compound of the formula (3) to the Raney nickel is 1: (0.1-5).

10. The method of claim 8, wherein the third solvent is selected from one or more of isopropanol, n-butanol, ethanol, methanol, t-butanol; and/or the pressure intensity of the reduction reaction is 1-10 MPa; and/or the temperature of the reduction reaction is 40-120 ℃; and/or the time of the reduction reaction is 5-48 h.