CN116102606B

CN116102606B - Preparation method of brenoside

Info

Publication number: CN116102606B
Application number: CN202211289488.5A
Authority: CN
Inventors: 曾春玲; 陈舟; 胡冬晴; 刘喜荣
Original assignee: Hunan Keyixin Biomedical Co ltd
Current assignee: Hunan Keyixin Biomedical Co ltd
Priority date: 2022-10-20
Filing date: 2022-10-20
Publication date: 2025-01-28
Anticipated expiration: 2042-10-20
Also published as: CN116102606A

Abstract

The present invention provides a method for preparing brinolone, comprising the following steps: mixing a compound SM, a metal, a proton donor, a first organic solvent and liquid ammonia, performing a metal dissolving reduction reaction, and preparing an intermediate 1; mixing the intermediate 1, an oxidant and a second organic solvent, performing an oxidation reaction, and preparing an intermediate 2; mixing the intermediate 2, a reductase and a third organic solvent, performing a reduction reaction, and preparing brinolone. The raw materials required for the preparation method are cheap and readily available, and the obtained product brinolone has the characteristics of high yield, high quality and high selectivity.

Description

Preparation method of breinolone

Technical Field

The invention relates to the technical field of organic chemistry, in particular to a preparation method of breinolone.

Background

3 In 2019, the FDA approved the sabolone product of the company Sage under the trade name Zulresso, a fast-acting neuroactive steroid, which is specific for the treatment of post-partum depression. The structural formula of the buprenolone, which is also named as allopregnanolone or allopregnanolone, is 3 alpha-hydroxy-5 alpha-pregnane-20-ketone, is shown as formula (I), is an artificially synthesized tetrahydroprogesterone analogue, and provides an antidepressant with rapid effect for treating postpartum depression.

The synthesis of the Burenolol is reported in 1934 and is reported by SCHERING company, and the specific synthetic route is shown as (II), wherein 3-hydroxy bisnorcholanic acids is used as a raw material, and the target product is obtained through esterification reaction, addition reaction with a format reagent, hydrolysis reaction and dehydration reaction, and finally through five steps of ozonization reaction, and the reaction is mainly performed to reconstruct a side chain to obtain 20 ketone. However, the raw materials required by the reaction route are not easily available, the yield of the ozonization reaction is low, the yield is not reported in part of the reaction, and the synthesis method has no industrial production and application value.

Another synthetic method of the Burenolone is disclosed in the prior art, the synthetic route is shown as follows, the raw material in III is used for reacting with the potassium tri-sec-butylborohydride, the product is obtained through reduction, but the yield is less than 50%, and the source of the raw material is not described.

Another synthetic route of the Bureolone is described in the prior art, and is shown as IV, wherein the target product is obtained through Mitsunobu reaction, the raw materials are not easy to obtain, and the yield after purification is only 54%.

The method adopts pregnenolone ethyl ester as a raw material in the traditional technology, and the target product is obtained through 4 steps of hydrogenation, hydrolysis, mitsunobu reaction and hydrolysis, wherein the specific route is shown as V. Although the yield is higher, the method is obtained by column chromatography, industrial production cannot be applied, and in addition, the two steps of the reaction are used for degreasing by strong alkali, so that 17-site epimerization is easy to cause, and the purity of the product is difficult to reach the standard.

The synthesis route of the buprenolol disclosed by the traditional technology is shown in (VI), the yield is 51% -104%, wherein the third step uses a ketal thiol protecting group, the reaction yield is low, and acetic acid which is difficult to recycle is used as a solvent. If ethylene glycol is used as the protecting group, 96% yield can be obtained, but reflux using toluene as a solvent is required. The route has the advantages that the 3-position ester group is not easy to generate epimers at the 17-position after the protection of the 20-position carbonyl, but the route also has the defects of longer steps, high requirement on the Mitsunobu reaction operation of the second step, difficult removal of by-product triphenylphosphine oxide, low atom economy of the reaction route and the like.

Disclosure of Invention

Based on the above, the invention provides a preparation method of the breinolone, which has the characteristics of low cost and easy obtainment of raw materials, and the obtained product of the breinolone has the characteristics of high yield, high quality and high selectivity.

The invention is realized by the following technical scheme.

A preparation method of breinolone, comprising the following steps:

Mixing a compound SM, a metal simple substance, a proton donor, a first organic solvent and liquid ammonia, and carrying out a dissolved metal reduction reaction to prepare an intermediate 1, mixing the intermediate 1, an oxidant and a second organic solvent, and carrying out an oxidation reaction to prepare an intermediate 2, wherein the structure of the compound SM is shown as a formula (1), and the structure of the intermediate 2 is shown as a formula (2):

And mixing the intermediate 2, reductase and a third organic solvent, and carrying out reduction reaction to prepare the breinolone.

In one embodiment, intermediate 1 is intermediate 1-1, a mixture of intermediate 1-2 and intermediate 1-3;

wherein the structure of the intermediate 1-1 is shown as a formula (2);

the structure of the intermediate 1-2 is shown as a formula (3):

the structure of the intermediate 1-3 is shown as a formula (4):

In one embodiment, the temperature of the dissolved metal reduction reaction is below-40 ℃ and the time of the dissolved metal reduction reaction is 2-3 hours.

In one embodiment, the elemental metal satisfies one or more of the following conditions:

(1) The metal simple substance is selected from one or more of metallic lithium and metallic sodium;

(2) The molar ratio of the metal simple substance to the compound SM is (5-20): 1.

In one embodiment, the proton donor satisfies one or more of the following conditions:

(1) The proton donor is selected from one or more of tertiary butanol and isopropanol;

(2) The volume mass ratio of the proton donor to the compound SM is (0.5 mL-2 mL) 1g.

In one embodiment, the first organic solvent satisfies one or more of the following conditions:

(1) The first organic solvent is selected from one or more of tetrahydrofuran and diethyl ether;

(2) The volume mass ratio of the first organic solvent to the compound SM is (8 mL-30 mL) 1g;

(3) The volume mass ratio of the first organic solvent to the liquid ammonia is (1 mL-2 mL) 1g.

In one embodiment, the reductase is a 3α -hydroxysteroid dehydrogenase.

In one embodiment, the second organic solvent is selected from one or more of dichloromethane, dimethyl sulfoxide, acetonitrile and acetic acid;

the third organic solvent is selected from one or more of alcohol and dimethyl sulfoxide.

In one embodiment, the conditions for the reduction reaction include a temperature of 28 ℃ to 35 ℃ and a pH of 6.5 to 8.

In one embodiment, the oxidizing agent is selected from one or more of pyridine chlorochromate, 2-iodoxybenzoic acid, and sodium hypochlorite.

Compared with the prior art, the preparation method of the breinolone has the following beneficial effects:

The invention takes progesterone as a starting material, effectively controls the configuration of 5-position hydrogen through dissolving metal reduction reaction to obtain a mixture intermediate 1, enables various intermediates of the mixture intermediate 1 to be converted into an intermediate 2 with a single structure through oxidation reaction without separation and purification, and finally reduces the intermediate 2 into 3 alpha-hydroxyl selectively under the catalysis of reductase to obtain the high-yield, high-quality and high-selectivity breinolone.

Furthermore, the method has the advantages of low-cost and easily-obtained initial raw materials, few byproducts and impurities, short process route, simple operation, no need of adopting dangerous processes such as high-pressure hydrogenation and the like, and suitability for industrial production.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of 5 alpha pregna-3, 20-dione provided by an embodiment of the present invention;

FIG. 2 is a nuclear magnetic carbon spectrum of 5 alpha pregna-3, 20-dione provided by an embodiment of the present invention;

FIG. 3 is a nuclear magnetic hydrogen spectrum of the breinolone provided by the embodiment of the invention;

Fig. 4 is a nuclear magnetic carbon spectrum of the breinolone provided by the embodiment of the invention;

Fig. 5 is a process route diagram of the preparation process of the breinolone provided by the invention.

Detailed Description

In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. The drawings illustrate preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise. In the description of the present invention, the meaning of "several" means at least one, such as one, two, etc., unless specifically defined otherwise.

The words "preferably," "more preferably," and the like in the present invention refer to embodiments of the invention that may provide certain benefits in some instances. However, other embodiments may be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values for the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

All percentages, fractions and ratios are calculated on the total mass of the composition of the invention, unless otherwise indicated. All of the mass of the ingredients listed, unless otherwise indicated, are given to the active substance content and therefore they do not include solvents or by-products that may be included in commercially available materials. The term "mass percent" herein may be represented by the symbol "%". All molecular weights herein are weight average molecular weights expressed in daltons, unless indicated otherwise. All formulations and testing herein occurred in a 25 ℃ environment unless otherwise indicated. The terms "comprising," "including," "containing," "having," or other variations thereof herein are intended to cover a non-closed inclusion, without distinguishing between them. The term "comprising" means that other steps and ingredients may be added that do not affect the end result. The compositions and methods/processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as additional or optional ingredients, components, steps, or limitations of any of the embodiments described herein. The terms "efficacy," "performance," "effect," "efficacy" are not differentiated herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The invention provides a preparation method of breinolone, which is shown in fig. 5, and comprises the following steps:

And mixing the intermediate 2, reductase and a third organic solvent for reduction reaction to prepare the breinolone.

In one specific example, intermediate 1 is intermediate 1-1, a mixture of intermediate 1-2 and intermediate 1-3;

wherein the structure of the intermediate 1-1 is shown as a formula (2);

the structure of the intermediate 1-2 is shown as a formula (3):

The structure of the intermediate 1-3 is shown as a formula (4):

it will be appreciated that compound SM is a progestin, also known as pregn-4-ene-3, 20-dione, which is commercially available in large quantities at low cost.

The difficulty with the reduction of starting compound SM to intermediate 1 is that the hydrogen at the 5-position is in the alpha configuration after olefin reduction. SM will be predominantly 5β by hydrogenation, separation of the 5α configurational isomer alone is difficult and yields are low. The invention prepares 5 alpha isomer through dissolving metal reduction reaction, and the reduction product is a mixture and mainly comprises intermediates 1-1, 1-2 and 1-3. Wherein intermediate 1-1 is the next reaction product intermediate 2:5α pregna-3, 20-dione, the configuration of the hydroxyl groups at the 3-and 20-positions of intermediates 1-2 and 1-3 is not important, as the hydroxyl groups will be oxidized in the next reaction. Although pure intermediate 1-1 or intermediate 2 can also be obtained by direct hydrogenation of starting material SM, the product will contain more 5 β isomer of intermediate 2, in lower yields, and with close polarity of the isomers, difficult purification.

In a specific example, the temperature of the dissolved metal reduction reaction is below-40 ℃, and the time of the dissolved metal reduction reaction is 2-3 hours.

In a more specific example, the temperature of the dissolved metal reduction reaction is-50 ℃ to-40 ℃.

It is understood that in the present invention, the temperature of the dissolved metal reduction reaction includes, but is not limited to, -40 ℃, -41 ℃, -42 ℃, -43 ℃, -44 ℃, -45 ℃, -46 ℃, -47 ℃, -48 ℃, -49 ℃, -50 ℃, and the time of the dissolved metal reduction reaction includes, but is not limited to, 2h, 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h.

In a specific example, the elemental metal is selected from one or more of metallic lithium and metallic sodium.

In a specific example, the molar ratio of the metal element to the compound SM is (5 to 20): 1.

It is understood that in the present invention, the molar ratio of elemental metal to compound SM includes, but is not limited to, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1.

In a specific example, the proton donor is selected from one or more of t-butanol and isopropanol.

In a specific example, the volume/mass ratio of the proton donor to the compound SM is (0.5 mL-2 mL): 1g.

It will be appreciated that in the present invention the volume to mass ratio of proton donor to compound SM includes, but is not limited to 0.5mL:1g、0.6mL:1g、0.7mL:1g、0.8mL:1g、0.9mL:1g、1mL:1g、1.1mL:1g、1.2mL:1g、1.3mL:1g、1.4mL:1g、1.5mL:1g、1.6mL:1g、1.7mL:1g、1.8mL:1g、1.9mL:1g、2mL:1g.

In a specific example, the first organic solvent is selected from one or more of tetrahydrofuran and diethyl ether.

In a specific example, the volume/mass ratio of the first organic solvent to the compound SM is (8 mL-30 mL): 1g.

It will be appreciated that in the present invention the volume to mass ratio of the first organic solvent to the compound SM includes, but is not limited to 8mL:1g、9mL:1g、10mL:1g、11mL:1g、12mL:1g、13mL:1g、14mL:1g、15mL:1g、20mL:1g、25mL:1g、26mL:1g、27mL:1g、28mL:1g、29mL:1g、30mL:1g.

In a specific example, the volume/mass ratio of the first organic solvent to the liquid ammonia is (1 mL-2 mL) 1g.

It will be appreciated that, in the present invention, the volumetric mass ratio of the first organic solvent to liquid ammonia includes, but is not limited to, 1mL:1g, 1.1mL:1g, 1.2mL:1g, 1.3mL:1g, 1.4mL:1g, 1.5mL:1g, 1.6mL:1g, 1.7mL:1g, 1.8mL:1g, 1.9mL:1g, 2mL:1g.

In a specific example, the quenching reaction is also included after the dissolved metal reduction reaction is completed.

More specifically, the quenching reaction employs a reagent selected from one or more of ethanol, methanol, isoprene, and solid ammonium chloride.

It will be appreciated that the ratio of each species of mixture intermediate 1 is primarily affected by the amount of proton donor and the manner of quenching, either by using more proton donor during the dissolution metal reduction reaction or by using a protic reagent to quench the reaction will decrease the ratio of 1-1 and increase the ratio of 1-3.

In a specific example, the reductase is a3α -hydroxysteroid dehydrogenase.

The difficulty in the selective reduction of intermediate 2 to breinolone is the selectivity of the 3-carbonyl and 20-carbonyl groups, and the configuration control of the 3 alpha-hydroxy groups after reduction. The traditional chemical method adopts sodium borohydride or potassium borohydride as a reducing reagent, and the reducing reagent reacts with the intermediate 2 to obtain dicarbonyl reducing impurities and 3 beta isomer, so that 3 alpha configuration isomer is difficult to separate independently, and the yield is low. The invention reduces the intermediate 2 into the target product, namely the breinolone, with high selectivity under proper conditions by 3 alpha-hydroxysteroid dehydrogenase. The form of the enzyme may be purified enzyme or enzyme solution after cell disruption.

In a specific example, the third organic solvent is selected from one or more of an alcohol and dimethyl sulfoxide.

Preferably, the alcohol is t-butanol.

In a specific example, the conditions of the reduction reaction include a temperature of 28-35 ℃ and a pH of 6.5-8.

It is understood that in the present invention, the temperature of the reduction reaction includes, but is not limited to, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃,35 ℃, and the pH of the reduction reaction includes, but is not limited to, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.

In one specific example, the pH is adjusted with a base. Preferably, the base is an aqueous sodium hydroxide solution. More specifically, the mass concentration of the sodium hydroxide aqueous solution is 2% -10%.

In a specific example, the conditions for the reduction reaction further include the addition of glucose, glucose dehydrogenase bacteria and oxidized coenzyme.

In a specific example, the oxidizing agent is selected from one or more of pyridine chlorochromate, 2-iodoxybenzoic acid, and sodium hypochlorite.

In a specific example, the second organic solvent is selected from one or more of dichloromethane, dimethyl sulfoxide, acetonitrile, and acetic acid.

In a specific example, the oxidizing agent is pyridine chlorochromate and the second organic solvent is dichloromethane. More specifically, pyridine chlorochromate was added after intermediate 2 was dissolved in methylene chloride.

In a specific example, the oxidizing agent is 2-iodoxybenzoic acid and the second organic solvent is dimethyl sulfoxide. More specifically, intermediate 2 was dissolved in dimethyl sulfoxide and 2-iodoxybenzoic acid was added.

In a specific example, the oxidizing agent is sodium hypochlorite and the second organic solvent is a mixed solvent of acetonitrile and acetic acid. More specifically, intermediate 2 was dissolved in a mixed solvent of acetonitrile and acetic acid, and then sodium hypochlorite solution was added dropwise.

In a specific example, the quenching reaction is also included after the oxidation reaction is completed.

In a specific example, intermediate 2 is prepared further comprising a purification step of beating or recrystallisation.

The preparation method of the breinolone of the present invention is described in further detail below with reference to specific examples. The raw materials used in the following examples are all commercially available products unless otherwise specified.

In the embodiment of the application, progesterone is self-produced by the applicant, and the 3 alpha enzyme is 3 alpha-hydroxysteroid dehydrogenase for short, which is self-constructed by the inventor and comes from testosterone comamonas (Comamonas testosteroni)

The gene sequence of 3α -hydroxysteroid dehydrogenase is shown below (SEQ ID NO. 1):

1 atgtccatca tcgtgataag cggctgcgcc accggcattg gtgcggctac gcgcaaggtc

61 ctggaggcgg ccggtcacca gatcgtaggc atcgatatac gcgatgcgga agtgattgcc

121 gatctctcga cggccgaagg tcgaaagcag gcgattgccg atgtactggc gaagtgcagc

181 aagggcatgg acggcctggt gctgtgcgcc ggcctgggac cgcagaccaa ggtgcttggc

241 aatgtggttt cggtcaatta ttttggcgcg accgagctga tggatgcctt tttgccagcg

301 ctgaaaaaag gccatcagcc cgcagccgtc gtcatctcgt ccgtggcttc cgcgcatctg

361 gcttttgaca agaacccact ggcgctggca ctggaagccg gcgaggaagc caaggcccgc

421 gccattgtcg aacatgcggg agagcagggc ggaaatctgg cctatgcggg cagcaagaat

481 gctttgacgg tggctgtgcg caaacgcgcc gccgcctggg gcgaggctgg cgtgcgcctg

541 aacaccatcg cccccggtgc aaccgagact cccttgctgc aggcgggcct gcaggacccg

601 cgctatggcg aatccattgc caagttcgtt cctcccatgg gccgccgtgc cgagccgtcc

661 gagatggcgt cggtcatcgc ctttttgatg agcccggccg caagctatgt gcatggcgcg

721 cagatcgtca ttgatggcgg cattgatgcg gtgatgcgcc cgacacagtt c

The protein sequence of 3α -hydroxysteroid dehydrogenase is shown below (SEQ ID NO. 2):

1 msiivisgca tgigaatrkv leaaghqivg idirdaevia dlstaegrkq aiadvlakcs

61 kgmdglvlca glgpqtkvlg nvvsvnyfga telmdaflpa lkkghqpaav vissvasahl

121 afdknplala leageeakar aivehageqg gnlayagskn altvavrkra aawgeagvrl

181 ntiapgatet pllqaglqdp rygesiakfv ppmgrraeps emasviaflm spaasyvhga

241 qividggida vmrptqf

Glucose dehydrogenase, available from Shanghai Meilin Biochemical technologies Co. Coenzyme I (NAD+), oxidized nicotinamide adenine dinucleotide, available from Shanghai Biotechnology Co., ltd. The remaining reagents were all commercially available.

For the sake of simplicity, the reaction charge ratio in the present invention is often expressed as a weight-to-volume ratio, which refers to the ratio of the weight of the reaction raw materials to the weight or volume of the reaction reagent unless otherwise specified.

Example 1

This example provides a process for the preparation of mixture 1 (intermediate 1), which is specifically as follows:

10g (31.8 mmol) of progesterone is added into a 500ml three-port bottle, 150ml of tetrahydrofuran and 20ml of tertiary butanol are measured, the mixture is cooled to-70 ℃, stirring is carried out, ammonia gas is introduced into the mixture about 80g, 2.5g (357 mmol) of lithium strips are added, natural heating is carried out after the addition, the temperature is kept below-40 ℃ to minus 50 ℃ and minus 40 ℃, after 3 hours of reaction, TLC shows complete conversion of raw materials, the product is non-fluorescent, 20ml of ethanol is added to quench lithium, a large amount of water is added after complete ammonia removal, sodium chloride and ethyl acetate are added to make the delamination more obvious, the separated liquid is concentrated, 150ml of dichloromethane and 100ml of water are added again, extraction and separated liquid are carried out, the organic layer is dried by spin-drying to obtain yellow viscous solid, and 9.14g of mixture 1 is obtained after the reaction is directly used for the next reaction without purification.

Example 2

This example provides a process for the preparation of compound 2 (intermediate 2), which is specifically as follows:

9.14g of the mixture 1 synthesized in example 1 was dissolved in 180ml of methylene chloride, stirred at room temperature, 10g of silica gel was added, 17.1g (79.5 mmol) of PCC was added again, TLC showed substantially complete conversion after 36h of reaction, the reaction solution was passed through a column of silica gel to remove the chromium reagent, the organic solvent was dried by spin to give a tan solid, after beating with isopropyl ether, 7.25g of a white solid of compound 2 was obtained, the nuclear magnetic structure was identified as 5α pregna-3, 20-dione, the two-step yield was 72%, the nuclear magnetic hydrogen spectrum was as shown in FIG. 1, and the nuclear magnetic carbon spectrum was as shown in FIG. 2 .¹HNMR(400MHz,Chloroform-d)δ2.51(t,J＝8.9Hz,1H),2.43–2.21(m,3H),2.20–2.12(m,1H),2.10(s,3H),2.07–1.96(m,2H),1.75–1.59(m,4H),1.59–1.10(m,10H),1.00(s,3H),0.99–0.86(m,1H),0.77(ddd,J＝12.8,10.4,4.1Hz,1H),0.62(s,3H).¹³C NMR(101MHz,Chloroform-d)δ211.83,209.48,63.74,56.46,53.68,46.67,44.65,44.17,38.94,38.54,38.13,35.69,35.37,31.65,31.51,28.82,24.42,22.84,21.44,13.44,11.46.

Example 3

The embodiment provides a preparation method of breinolone, which comprises the following steps:

To a 250ml three-necked flask, 5. Alpha. Pregna-3, 20-dione (7.0 g), t-butanol (50 ml), drinking water (30 ml), 3.5g of glucose monohydrate and 0.05g of NAD+ oxide were sequentially added, and after stirring for 5min, pH was slowly adjusted to 6.5-7.5 with 10% aqueous alkali, the temperature was adjusted to 28-32℃and 3. Alpha. -steroid dehydrogenase solution (4.2 ml) and glucose dehydrogenase bacterial solution (5 ml) were added to start the time-lapse reaction. In the middle process, 5% liquid alkali is slowly added dropwise to control the pH value to be 6.5-7.5. After about 3 hours, the pH value did not change any more and no substrate remained as measured by sampling TLC.

The reaction solution was concentrated in vacuo at 65-70℃until no tert-butanol was present, water (100 ml) was added, and the mixture was stirred for 1 hour at 0℃and the system was filtered to give 8.3g of crude protein-containing product. Adding acetone (50 ml) into the crude product, heating to 50deg.C for dissolving, filtering to remove protein, concentrating the filtrate to small volume, filtering, oven drying to obtain white solid 6.5g with 92.8% yield, and determining to obtain Bureolone by nuclear magnetism, wherein the nuclear magnetic hydrogen spectrum is shown in figure 3, and the nuclear magnetic carbon spectrum is shown in figure 4 .¹H NMR(400MHz,Chloroform-d)δ4.02(t,J＝2.8Hz,1H),2.50(t,J＝8.9Hz,1H),2.18–2.10(m,1H),2.09(s,3H),1.98(dt,J＝11.9,3.3Hz,1H),1.72–1.08(m,19H),0.93(qd,J＝12.0,5.8Hz,1H),0.83–0.77(m,1H),0.76(s,3H),0.58(s,3H).¹³C NMR(101MHz,Chloroform-d)δ209.77,66.51,63.85,56.79,54.22,44.27,39.11,39.11,36.13,35.87,35.50,32.21,31.96,31.54,29.01,28.45,24.38,22.79,20.80,13.47,11.18.

Example 4

Introducing ammonia gas into a 500ml three-port bottle at the temperature of 60 ℃ below zero, stirring, adding 1.5g (214 mmol) of metallic lithium in batches after the liquid ammonia weighs about 50g, enabling the system to turn deep blue, dropwise adding an organic solution containing 10g (31.8 mmol) of progesterone, 8ml of tertiary butanol (83.8 mmol) and 80ml of tetrahydrofuran, heating naturally after the addition, keeping the temperature below-40 ℃ for two hours (-50 ℃ to-40 ℃), after the reaction, TLC shows that the raw materials are completely converted, the product is non-fluorescent, adding 15ml of ethanol to quench lithium, concentrating after ammonia removal is complete, removing most of organic solvent, adding 300ml of water and 300ml of dichloromethane, extracting and separating liquid, drying an organic layer, and directly putting into the next step without purification.

Example 5

The mixture 1 prepared in example 4 was dissolved in a mixed solvent of 100ml of acetonitrile and 20ml of acetic acid, and after dissolution, placed in an ice bath, 53ml of aqueous sodium hypochlorite solution (7.5% strength, about 57.7 mmol) was slowly added dropwise, a white solid gradually precipitated during the reaction, the reaction was substantially completely converted after 4 hours, the reaction was quenched by adding sodium thiosulfate, most of acetonitrile was removed by concentration, water was added by 100ml for water precipitation, suction filtration, and after drying, pulping with 100ml of isopropyl ether twice, 8.81g of the product was obtained, and the yield in two steps was 87.5%.

Example 6

To a 100ml three-necked flask, 5. Alpha. Pregna-3, 20-dione (5.0 g), DMSO (20 ml), drinking water (20 ml), 2.5g of glucose monohydrate and 0.03g of NAD+ oxide were sequentially added, and after stirring for 5min, pH was slowly adjusted to 6.5-7.5 with 10% aqueous alkali, the temperature was adjusted to 28-32℃and 3. Alpha. -steroid dehydrogenase solution (3.2 ml) and glucose dehydrogenase bacterial solution (4.0 ml) were added to start the time-lapse reaction. In the middle process, 5% liquid alkali is slowly added dropwise to control the pH value to be 6.5-7.5. After about 4 hours, the pH value did not change any more and no substrate remained as measured by sampling TLC.

The reaction solution was concentrated to a thick state in vacuo at 80℃and water (500 ml) was added thereto, and the mixture was stirred for 1 hour at 5℃and filtered to obtain 5.3g of crude protein-containing product. Ethyl acetate (70 ml) and 0.5g of active carbon are added into the crude product, the mixture is heated to 60 ℃ for dissolution, diatomite is paved for filtration, the filtrate is concentrated to a small volume for filtration, and the white solid is obtained by drying 4.8g, and the yield is 86.0%.

Comparative example 1

This comparative example provides a process for the preparation of compound 2 (intermediate 2), specifically as follows:

15g of progesterone is weighed, 300ml of ethyl acetate is added for dissolution, 1.5g of 5% palladium-carbon is added, the reaction flask is emptied, the reaction flask is connected with a hydrogen balloon for reaction overnight, TLC shows that fluorescence disappears, and two products are produced, namely 5 alpha pregna-3, 20-dione (compound 2) and 5 beta pregna-3, 20-dione, wherein 5 alpha pregna-3, 20-dione has poor solubility and most of the 5 alpha pregna-3, 20-dione is separated out. Filtering, washing filter residue with a small amount of ethyl acetate, and obtaining mother liquor which is 5 beta pregna-3, 20-diketone containing a small amount of 5 alpha isomer. Scraping filter residues, adding 100ml of dichloromethane, stirring, filtering to remove palladium carbon, spin-drying a dichloro solution to obtain 3.2g of 5 alpha pregna-3, 20-diketone crude product, pulping with isopropyl ether to obtain 3.0g of compound 2 refined product, and the yield is 29.8%.

Comparative example 2

This comparative example provides a process for the preparation of intermediate 1, which is specifically as follows:

10g (31.8 mmol) of progesterone is added into a 500ml three-necked flask, 150ml of tetrahydrofuran and 20ml of tertiary butanol are measured, the mixture is cooled to-70 ℃, stirring is carried out, about 30g of ammonia gas is introduced, 2.5g (357 mmol) of lithium strips are added, natural heating is carried out after the addition, the temperature is kept below-40 ℃ (-50 ℃ to-40 ℃), after 3 hours of reaction, TLC shows that the raw materials are not completely converted, and more other fluorescent products are generated, so that 4-olefin is not completely reduced, the 4-olefin is converted into the original raw material progesterone in the next oxidation reaction, and the yield is obviously reduced and the separation is difficult.

Comparative example 3

The comparative example provides a preparation method of the breinolone, which comprises the following steps:

5.0g of Compound 2 was weighed, 100mL of toluene was added for dissolution, 5.0g of aluminum isopropoxide and 7.5mL of isopropanol were added, and stirring was performed at 80℃for 20 minutes. TLC (P: e=2:1) showed that the vast majority of the starting material had been converted, with two major products of increasing polarity being produced, and passed through the column to give 2.2g of product in 43.7% yield.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. It should be understood that, based on the technical solutions provided by the present invention, those skilled in the art may obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims

1. A method for preparing brenoside, characterized in that it comprises the following steps:

Compound SM, a metal element, a proton donor, a first organic solvent and liquid ammonia are mixed to carry out a dissolving metal reduction reaction to prepare an intermediate 1; the temperature of the dissolving metal reduction reaction is below -40°C, the metal element is selected from one or more of metal lithium and metal sodium, the proton donor is selected from one or more of tert-butyl alcohol and isopropanol, and the first organic solvent is selected from one or more of tetrahydrofuran and diethyl ether,

The intermediate 1, the oxidant and the second organic solvent are mixed to carry out an oxidation reaction to prepare the intermediate 2; wherein the oxidant is selected from one or more of pyridinium chlorochromate, 2-iodoxybenzoic acid and sodium hypochlorite; the structure of the compound SM is shown in formula (1), and the structure of the intermediate 2 is shown in formula (2):

Formula (1);

Formula (2);

The intermediate 2, the reductase and the third organic solvent are mixed to carry out a reduction reaction to prepare brenoside; the reductase is 3α-hydroxysteroid dehydrogenase.

2. The method for preparing brenoside according to claim 1, characterized in that the intermediate 1 is a mixture of intermediate 1-1, intermediate 1-2 and intermediate 1-3;

Wherein, the structure of the intermediate 1-1 is shown in formula (2);

The structure of the intermediate 1-2 is shown in formula (3):

Formula (3);

The structure of the intermediate 1-3 is shown in formula (4):

Formula (4).

3. The method for preparing brunosone according to claim 1, wherein the time for the dissolving metal reduction reaction is 2 h to 3 h.

4. The method for preparing bruneolone according to claim 1, wherein the molar ratio of the metal element to the compound SM is (5-20):1.

5. The method for preparing brucenolide according to claim 1, wherein the volume mass ratio of the proton donor to the compound SM is (0.5 mL~2 mL): 1 g.

6. The method for preparing brucenolide according to claim 1, characterized in that the volume mass ratio of the first organic solvent to the compound SM is (8 mL~30 mL): 1 g.

7. The method for preparing brucenolide according to claim 1, wherein the volume mass ratio of the first organic solvent to liquid ammonia is (1 mL~2 mL): 1 g.

8. The method for preparing brenoside according to any one of claims 1 to 7, characterized in that the second organic solvent is selected from one or more of dichloromethane, dimethyl sulfoxide, acetonitrile and acetic acid.

9. The method for preparing brexanolone according to any one of claims 1 to 7, characterized in that the third organic solvent is selected from one or more of alcohol and dimethyl sulfoxide.

10. The method for preparing brenoside according to any one of claims 1 to 7, characterized in that the conditions for the reduction reaction include: a temperature of 28°C to 35°C and a pH value of 6.5 to 8.