Preparation method of brenoside

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

Technical Field

[0001] The present invention relates to the technical field of organic chemistry, and in particular to a method for preparing brenoside. Background Art

[0002] In March 2019, the FDA approved Sage's brenospermone, trade name Zulresso, a fast-acting neuroactive steroid specifically for the treatment of postpartum depression. Brenolone, also known as allopregnanolone or allopregnanolone, has a chemical name of 3α-hydroxy-5α-pregnane-20-one, and its structural formula is shown in formula (I). It is a synthetic allopregnanolone analog that provides a fast-acting antidepressant to treat postpartum depression.

[0003]

[0004] The synthesis of brenosone was reported in 1934 by SCHERING Company. The specific synthesis route is shown in (II): 3-hydroxy bisnorcholanic acids are used as raw materials, and the target product is obtained through esterification reaction, Grignard reagent addition, hydrolysis and dehydration reaction, and finally ozonation in five steps. The reaction mainly transforms the side chain to obtain 20 ketone. However, the raw materials required for this reaction route are not easy to obtain, and the yield of the ozonation reaction is low. The yield of some reactions is not reported. This synthesis method has no industrial production application value.

[0005]

[0006] Another method for synthesizing brenoside is disclosed in conventional technology. The synthetic route is as follows: the raw material in III is reacted with potassium tri-sec-butylborohydride to obtain a product through reduction, but the yield is less than 50%, and the source of the raw material is not disclosed.

[0007]

[0008] There is also another synthetic route of brenosone recorded in traditional technology, and the synthetic route is shown in IV: the target product is obtained by Mitsunobu reaction, the raw materials are not easy to obtain, and the yield after purification is only 54%.

[0009]

[0010] There is a traditional technology that uses pregnenolone ethyl ester as a raw material and obtains the target product through four steps of hydrogenation, hydrolysis, Mitsunobu reaction and hydrolysis. The specific route is shown in V. Although the yield is relatively high, it is obtained by column chromatography and cannot be applied to industrial production. In addition, two steps of the reaction use strong base for deesterification, which easily leads to 17-position isomerization and makes it difficult to meet the purity of the product.

[0011]

[0012] There is also a traditional technology published brenoside synthesis route as shown in (VI), the yield is 51% to 104%, wherein the third step uses ketalthiocarbamide as a 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 a protecting group, a yield of 96% can be obtained, but toluene is required as a solvent for reflux. The advantage of this route is that after the 20-position carbonyl protection, the alkaline hydrolysis of the 3-position ester group is not easy to cause the 17-position diastereomer to be produced, but this route also has the disadvantages of long steps, high requirements for the operation of the Mitsunobu reaction in the second step, the by-product triphenylphosphine is not easy to remove, and the reaction route has low atom economy.

[0013] Summary of the invention

[0014] Based on this, the present invention provides a method for preparing brenosone, the raw materials of which are cheap and easily available, and the obtained product brenosone has the characteristics of high yield, high quality and high selectivity.

[0015] The present invention is achieved through the following technical solutions.

[0016] A method for preparing brenoside comprises the following steps:

[0017] Compound SM, a metal element, a proton donor, a first organic solvent and liquid ammonia are mixed to carry out a metal dissolving reduction reaction to prepare an intermediate 1; the intermediate 1, an oxidant and a second organic solvent are mixed to carry out an oxidation reaction to prepare an intermediate 2; wherein the structure of the compound SM is shown in formula (1), and the structure of the intermediate 2 is shown in formula (2):

[0018]

[0019] The intermediate 2, the reductase and the third organic solvent are mixed to carry out a reduction reaction to prepare brenolon.

[0020] In one embodiment, the intermediate 1 is a mixture of intermediate 1-1, intermediate 1-2 and intermediate 1-3;

[0021] Wherein, the structure of the intermediate 1-1 is shown in formula (2);

[0022] The structure of the intermediate 1-2 is shown in formula (3):

[0023]

[0024] The structure of the intermediate 1-3 is shown in formula (4):

[0025]

[0026] In one embodiment, the temperature of the dissolved metal reduction reaction is below -40°C, and the time of the dissolved metal reduction reaction is 2h to 3h.

[0027] In one embodiment, the metal element satisfies one or more of the following conditions:

[0028] (1) The metal element is selected from one or more of metal lithium and metal sodium;

[0029] (2) The molar ratio of the metal element to the compound SM is (5-20):1.

[0030] In one embodiment, the proton donor satisfies one or more of the following conditions:

[0031] (1) The proton donor is selected from one or more of tert-butyl alcohol and isopropyl alcohol;

[0032] (2) The volume mass ratio of the proton donor to the compound SM is (0.5 mL to 2 mL): 1 g.

[0033] In one embodiment, the first organic solvent satisfies one or more of the following conditions:

[0034] (1) the first organic solvent is selected from one or more of tetrahydrofuran and diethyl ether;

[0035] (2) The volume mass ratio of the first organic solvent to the compound SM is (8 mL to 30 mL): 1 g;

[0036] (3) The volume mass ratio of the first organic solvent to liquid ammonia is (1 mL to 2 mL): 1 g.

[0037] In one embodiment, the reductase is 3α-hydroxysteroid dehydrogenase.

[0038] In one embodiment, the second organic solvent is selected from one or more of dichloromethane, dimethyl sulfoxide, acetonitrile and acetic acid;

[0039] The third organic solvent is selected from one or more of alcohol and dimethyl sulfoxide.

[0040] In one embodiment, the conditions for the reduction reaction include: a temperature of 28° C. to 35° C. and a pH value of 6.5 to 8.

[0041] In one embodiment, the oxidant is selected from one or more of pyridinium chlorochromate, 2-iodoxybenzoic acid and sodium hypochlorite.

[0042] Compared with the prior art, the preparation method of brucenolide of the present invention has the following beneficial effects:

[0043] The present invention uses progesterone as a starting material, and effectively controls the configuration of hydrogen at position 5 through a dissolving metal reduction reaction to obtain a mixture intermediate 1. Then, through an oxidation reaction, multiple intermediates in the mixture intermediate 1 can be converted into an intermediate 2 with a single structure without separation and purification. Finally, under the catalytic conditions of a reductase, the intermediate 2 is selectively reduced to a 3α-hydroxyl group, thereby finally obtaining brexanolone with high yield, quality and selectivity.

[0044] Furthermore, the starting raw materials used in the present invention are cheap and easily available, with few by-products and impurities, a short process route, and simple operation, and no dangerous processes such as high-pressure hydrogenation are required, so the present invention is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The H NMR spectrum of 5α-pregnane-3,20-dione provided in the embodiments of the present invention;

[0046] Figure 2 The NMR carbon spectrum of 5α-pregnane-3,20-dione provided in the embodiments of the present invention;

[0047] Figure 3 The H NMR spectrum of Brenolone provided in the embodiments of the present invention;

[0048] Figure 4 The NMR carbon spectrum of Bruynolone provided in the embodiments of the present invention;

[0049] Figure 5 The present invention provides a process map for the preparation of brenoside. DETAILED DESCRIPTION

[0050] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0051] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the invention, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. In the description of the present invention, the meaning of "several" is at least one, such as one, two, etc., unless otherwise clearly and specifically defined.

[0052] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0053] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0054] Unless otherwise specified, all percentages, fractions and ratios are calculated by the total mass of the composition of the present invention. Unless otherwise specified, all masses of the listed ingredients are given to the content of active substances, so they do not include solvents or by-products that may be contained in commercially available materials. The term "mass percentage content" herein can be represented by the symbol "%". Unless otherwise specified, all molecular weights in this article are weight average molecular weights expressed in Daltons. Unless otherwise specified, all formulations and tests in this article occur in an environment of 25°C. "Including", "including", "containing", "containing", "having" or other variants herein are intended to cover non-closed inclusions, and these terms are not distinguished. The term "comprising" means that other steps and ingredients that do not affect the final result can be added. The composition and method / process of the present invention include, consist of and are essentially composed of the necessary elements and restrictions described herein and any additional or optional ingredients, components, steps or restrictions described herein. The terms "efficacy", "performance", "effect" and "efficacy" are not distinguished between herein.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0056] Combination Figure 5 As shown, the present invention provides a method for preparing brenoside, comprising the following steps:

[0057] Compound SM, a metal element, a proton donor, a first organic solvent and liquid ammonia are mixed to carry out a dissolved metal reduction reaction to prepare an intermediate 1; intermediate 1, an oxidant and a second organic solvent are mixed to carry out an oxidation reaction to prepare an intermediate 2; wherein the structure of compound SM is shown in formula (1), and the structure of intermediate 2 is shown in formula (2):

[0058]

[0059]

[0060] The intermediate 2, the reductase and the third organic solvent are mixed to carry out a reduction reaction to prepare brenolon.

[0061] In a specific example, intermediate 1 is a mixture of intermediate 1-1, intermediate 1-2 and intermediate 1-3;

[0062] Wherein, the structure of intermediate 1-1 is shown in formula (2);

[0063] The structure of intermediate 1-2 is shown in formula (3):

[0064]

[0065] The structure of intermediate 1-3 is shown in formula (4):

[0066]

[0067] It can be understood that compound SM is progesterone, also known as pregnane-4-ene-3,20-dione, which is commercially available in large quantities at a low cost.

[0068] The difficulty in reducing the raw material compound SM to intermediate 1 is that the 5-position hydrogen is in α configuration after olefin reduction. SM will be mainly 5β through hydrogenation reaction, and it is difficult to separate the 5α configuration isomer alone, and the yield is very low. The present invention prepares the 5α isomer through a dissolving metal reduction reaction, and the reduction product is a mixture, mainly including intermediates 1-1, 1-2 and 1-3. Among them, intermediate 1-1 is the next reaction product intermediate 2: 5α-pregnane-3,20-dione, and the configuration of the 3-position and 20-position hydroxyl groups of intermediates 1-2 and 1-3 is not important because 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 the starting material SM, the product will contain more 5β isomers of intermediate 2, the yield is low, and the isomers have similar polarity and are difficult to purify.

[0069] In a specific example, the temperature of the dissolved metal reduction reaction is below -40°C, and the time of the dissolved metal reduction reaction is 2h to 3h.

[0070] In a more specific example, the temperature of the dissolved metal reduction reaction is -50°C to -40°C.

[0071] It can be understood that in the present invention, the temperature of the dissolved metal reduction reaction includes but is not limited to -40°C, -41°C, -42°C, -43°C, -44°C, -45°C, -46°C, -47°C, -48°C, -49°C, and -50°C; 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, and 3h.

[0072] In a specific example, the metal element is selected from one or more of metal lithium and metal sodium.

[0073] In a specific example, the molar ratio of the metal element to the compound SM is (5-20):1.

[0074] It can be understood that in the present invention, the molar ratio of the metal element to the 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, and 20:1.

[0075] In a specific example, the proton donor is selected from one or more of tert-butyl alcohol and isopropyl alcohol.

[0076] In a specific example, the volume mass ratio of the proton donor to the compound SM is (0.5 mL to 2 mL): 1 g.

[0077] It can be understood that in the present invention, the volume mass ratio of the proton donor to the 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, and 2mL:1g.

[0078] In a specific example, the first organic solvent is selected from one or more of tetrahydrofuran and diethyl ether.

[0079] In a specific example, the volume mass ratio of the first organic solvent to the compound SM is (8 mL to 30 mL): 1 g.

[0080] It can be understood that in the present invention, the volume 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, and 30mL:1g.

[0081] In a specific example, the volume mass ratio of the first organic solvent to liquid ammonia is (1 mL to 2 mL): 1 g.

[0082] It can be understood that in the present invention, the volume 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, and 2mL:1g.

[0083] In a specific example, the dissolving metal reduction reaction further includes a quenching reaction after completion.

[0084] More specifically, the reagent used to quench the reaction is selected from one or more of ethanol, methanol, isoprene and solid ammonium chloride.

[0085] It can be understood that the ratio of each substance in the mixture intermediate 1 is mainly affected by the amount of proton donor and the quenching method: using more proton donors during the dissolved metal reduction reaction or using a protic reagent to quench the reaction will reduce the ratio of 1-1 and increase the ratio of 1-3.

[0086] In one specific example, the reductase is 3α-hydroxysteroid dehydrogenase.

[0087] The difficulty of selectively reducing intermediate 2 to brucenolide lies in the selectivity of 3-carbonyl and 20-carbonyl, and the configuration control of 3α-hydroxyl after reduction. The reducing agent used in the traditional chemical method is sodium borohydride or potassium borohydride, which reacts with intermediate 2 to obtain dicarbonyl reduced impurities and 3β isomers. It is difficult to separate the 3α configuration isomer alone, and the yield is very low. The present invention uses 3α-hydroxysteroid dehydrogenase to highly selectively reduce intermediate 2 to the target product brucenolide under suitable conditions. Among them, the enzyme can be used in the form of purified enzyme or enzyme solution after cell disruption.

[0088] In a specific example, the third organic solvent is selected from one or more of alcohol and dimethyl sulfoxide.

[0089] Preferably, the alcohol is tert-butyl alcohol.

[0090] In a specific example, the conditions for the reduction reaction include: a temperature of 28° C. to 35° C. and a pH value of 6.5 to 8.

[0091] It can be understood that in the present invention, the temperature of the reduction reaction includes but is not limited to 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, and 35°C; the pH value 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, and 8.

[0092] In a specific example, the pH value is adjusted using an alkali. Preferably, the alkali is an aqueous sodium hydroxide solution. More specifically, the mass concentration of the aqueous sodium hydroxide solution is 2% to 10%.

[0093] In a specific example, the conditions for the reduction reaction further include the addition of glucose, glucose dehydrogenase bacteria and oxidized coenzyme.

[0094] In a specific example, the oxidant is selected from one or more of pyridinium chlorochromate, 2-iodoxybenzoic acid and sodium hypochlorite.

[0095] In a specific example, the second organic solvent is selected from one or more of dichloromethane, dimethyl sulfoxide, acetonitrile and acetic acid.

[0096] In a specific example, the oxidant is pyridinium chlorochromate, and the second organic solvent is dichloromethane. More specifically, the intermediate 2 is dissolved in dichloromethane and then pyridinium chlorochromate is added.

[0097] In a specific example, the oxidant is 2-iodoxybenzoic acid, and the second organic solvent is dimethyl sulfoxide. More specifically, the intermediate 2 is dissolved in dimethyl sulfoxide and then 2-iodoxybenzoic acid is added.

[0098] In a specific example, the oxidant is sodium hypochlorite, and the second organic solvent is a mixed solvent of acetonitrile and acetic acid. More specifically, after the intermediate 2 is dissolved in the mixed solvent of acetonitrile and acetic acid, the sodium hypochlorite solution is added dropwise.

[0099] In a specific example, the oxidation reaction further includes a quenching reaction after completion.

[0100] In a specific example, the preparation of intermediate 2 further includes the following purification steps: beating or recrystallization.

[0101] The preparation method of brexanolone of the present invention is further described in detail below in conjunction with specific examples. The raw materials used in the following examples are all commercially available products unless otherwise specified.

[0102] In the embodiments of the present invention, progesterone is produced by the applicant, and the 3α enzyme is the abbreviation of 3α-hydroxysteroid dehydrogenase, which is constructed by the applicant and comes from Comamonas testosteroni.

[0103] The gene sequence of 3α-hydroxysteroid dehydrogenase is shown below (SEQ ID NO.1):

[0104] 1 atgtccatca tcgtgataag cggctgcgcc accggcattg gtgcggctac gcgcaaggtc

[0105] 61 ctggaggcgg ccggtcacca gatcgtaggc atcgatatac gcgatgcgga agtgattgcc

[0106] 121 gatctctcga cggccgaagg tcgaaagcag gcgattgccg atgtactggc gaagtgcagc

[0107] 181 aagggcatgg acggcctggt gctgtgcgcc ggcctgggac cgcagaccaa ggtgcttggc

[0108] 241 aatgtggttt cggtcaatta ttttggcgcg accgagctga tggatgcctt tttgccagcg

[0109] 301 ctgaaaaaag gccatcagcc cgcagccgtc gtcatctcgt ccgtggcttc cgcgcatctg

[0110] 361 gcttttgaca agaacccact ggcgctggca ctggaagccg gcgaggaagc caaggcccgc

[0111] 421 gccattgtcg aacatgcggg agagcagggc ggaaatctgg cctatgcggg cagcaagaat

[0112] 481 gctttgacgg tggctgtgcg caaacgcgcc gccgcctggg gcgaggctgg cgtgcgcctg

[0113] 541 aacaccatcg cccccggtgc aaccgagact cccttgctgc aggcgggcct gcaggacccg

[0114] 601 cgctatggcg aatccattgc caagttcgtt cctcccatgg gccgccgtgc cgagccgtcc

[0115] 661 gagatggcgt cggtcatcgc ctttttgatg agcccggccg caagctatgt gcatggcgcg

[0116] 721 cagatcgtca ttgatggcgg cattgatgcg gtgatgcgcc cgacacagtt c

[0117] The protein sequence of 3α-hydroxysteroid dehydrogenase is shown below (SEQ ID NO.2):

[0118] 1 msiivisgca tgigaatrkv leaaghqivg idirdaevia dlstaegrkq aiadvlakcs

[0119] 61 kgmdglvlca glgpqtkvlg nvvsvnyfga telmdaflpa lkkghqpaav vissvasahl

[0120] 121 afdknplala leageeakar aivehageqg gnlayagskn altvavrkra aawgeagvrl

[0121] 181 ntiapgatet pllqaglqdp rygesiakfv ppmgrraeps emasviaflm spaasyvhga

[0122] 241 qividggida vmrptqf

[0123] Glucose dehydrogenase was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Coenzyme I (NAD+): oxidized nicotinamide adenine dinucleotide was purchased from Shanghai Yueteng Biotechnology Co., Ltd. The remaining reagents were commercially available.

[0124] For the sake of simplicity, the reaction feed ratio in the present invention is often expressed as a weight-to-volume ratio. Unless otherwise specified, the weight-to-volume ratio refers to the ratio of the weight of the reaction raw materials to the weight or volume of the reaction reagents.

[0125] Example 1

[0126] This embodiment provides a method for preparing a mixture 1 (intermediate 1), which is as follows:

[0127] In a 500ml three-necked flask, 10g (31.8mmol) of progesterone was added, 150ml of tetrahydrofuran and 20ml of tert-butanol were measured, and the mixture was cooled to -70°C, stirred, and about 80g of ammonia gas was introduced. 2.5g (357mmol) of lithium bars were added, and the temperature was naturally raised after the addition to keep the temperature below -40°C (-50°C to -40°C). After the reaction was completed for 3h, TLC showed that the raw material was completely converted and the product had no fluorescence. 20ml of ethanol was added to quench the lithium. After the ammonia was completely driven out, a large amount of water was added, and then sodium chloride and ethyl acetate were added to make the stratification more obvious. The liquids were separated, the organic layer was concentrated, 150ml of dichloromethane and 100ml of water were added again, extracted, separated, and the organic layer was spin-dried to obtain a yellow viscous solid, which was dried to obtain 9.14g of mixture 1, which was directly used in the next step without purification.

[0128] Example 2

[0129] This embodiment provides a method for preparing compound 2 (intermediate 2), which is as follows:

[0130] 9.14 g of the mixture 1 synthesized in Example 1 was dissolved in 180 ml of dichloromethane, stirred at room temperature, 10 g of silica gel was added, and then 17.1 g (79.5 mmol) of PCC was added in batches. After 36 h of reaction, TLC showed that the conversion was basically complete. The reaction solution was passed through a silica gel column to remove the chromium reagent. The organic solvent was spin-dried to obtain a brown solid. After beating with isopropyl ether, 7.25 g of a white solid of compound 2 was obtained. The nuclear magnetic structure was identified as 5α-pregnane-3,20-dione, with a two-step yield of 72%. Its nuclear magnetic hydrogen spectrum is as follows Figure 1 As shown in the NMR carbon spectrum Figure 2 shown. 1 HNMR(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). 13 C NMR(101MHz,Chloroform-d)δ211.83,209.48,63.74,56.46,53.68,46.67,44.65,44.17,3 8.94,38.54,38.13,35.69,35.37,31.65,31.51,28.82,24.42,22.84,21.44,13.44,11.46.

[0131] Example 3

[0132] This embodiment provides a method for preparing brucerolide, which is as follows:

[0133] In a 250ml three-necked flask, add 5α-pregnane-3,20-dione (7.0g), tert-butyl alcohol (50ml), drinking water (30ml), 3.5g monohydrate glucose and 0.05g oxidized NAD+ in sequence, stir for 5min, slowly adjust the pH to 6.5-7.5 with 10% liquid alkali, adjust the temperature to 28-32°C, add 3α-steroid dehydrogenase solution (4.2ml) and glucose dehydrogenase bacterial solution (5ml) to start timing reaction. In the middle process, slowly add 5% liquid alkali to control the pH to 6.5-7.5. After about 3 hours, the pH value no longer changes, and the sample TLC test shows that there is no substrate residue.

[0134] The reaction solution was vacuum concentrated at 65-70°C until there was no tert-butyl alcohol, water (100 ml) was added, the temperature was lowered to 0°C and stirred for 1 hour, the system was filtered, and 8.3 g of crude protein was obtained. The crude product was added with acetone (50 ml) and heated to 50°C to dissolve, the protein was filtered out, the filtrate was concentrated to a small volume, filtered, and dried to obtain 6.5 g of white solid, with a yield of 92.8%. It was confirmed to be brexanolone by nuclear magnetic resonance, and its nuclear magnetic hydrogen spectrum was as follows Figure 3 As shown in the NMR carbon spectrum Figure 4 shown. 1 H 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). 13 C 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.

[0135] Example 4

[0136] This embodiment provides a method for preparing a mixture 1 (intermediate 1), which is as follows:

[0137] Ammonia gas was introduced into a 500 ml three-necked flask at -60°C with stirring. After the liquid ammonia weighed about 50 g, 1.5 g (214 mmol) of metallic lithium was added in batches. The system turned dark blue. An organic solution containing 10 g (31.8 mmol) of progesterone, 8 ml of tert-butyl alcohol (83.8 mmol) and 80 ml of tetrahydrofuran was then added dropwise. The temperature was naturally raised after the addition to keep the temperature below -40°C (-50°C to -40°C). After two hours of reaction, TLC showed that the raw material was completely converted and the product had no fluorescence. 15 ml of ethanol was added to quench the lithium. After the ammonia was completely driven out, most of the organic solvent was removed by concentration. 300 ml of water and 300 ml of dichloromethane were added, and the liquid was extracted and separated. The organic layer was dried and then spin-dried to obtain a light yellow oil, which was directly put into the next step without purification.

[0138] Example 5

[0139] This embodiment provides a method for preparing compound 2 (intermediate 2), which is as follows:

[0140] The mixture 1 prepared in Example 4 was dissolved in a mixed solvent of 100 ml of acetonitrile and 20 ml of acetic acid, and after dissolution, it was placed in an ice bath, and 53 ml of sodium hypochlorite aqueous solution (7.5% concentration, about 57.7 mmol) was slowly added dropwise. During the reaction, a white solid gradually precipitated. After 4 hours of reaction, the conversion was basically complete. After sodium thiosulfate was added to quench the reaction, most of the acetonitrile was removed by concentration, 100 ml of water was added for water precipitation, and the mixture was filtered off with suction. After drying, the mixture was slurried twice with 100 ml of isopropyl ether to obtain 8.81 g of the product, and the two-step yield was 87.5%.

[0141] Example 6

[0142] This embodiment provides a method for preparing brucerolide, which is as follows:

[0143] In a 100ml three-necked flask, add 5α-pregnane-3,20-dione (5.0g), DMSO (20ml), drinking water (20ml), 2.5g monohydrate glucose and 0.03g oxidized NAD+ in sequence, stir for 5min, slowly adjust the pH to 6.5-7.5 with 10% liquid alkali, adjust the temperature to 28-32°C, add 3α-steroid dehydrogenase solution (3.2ml) and glucose dehydrogenase bacterial solution (4.0ml) to start the timing reaction. In the middle process, slowly add 5% liquid alkali to control the pH to 6.5-7.5. After about 4 hours, the pH value no longer changes, and the sample TLC test shows that there is no substrate residue.

[0144] The reaction solution was concentrated under vacuum at 80°C until it became thick, water (500 ml) was added, the temperature was lowered to 5°C and stirred for 1 hour, the system was filtered, and 5.3 g of crude protein was obtained. Ethyl acetate (70 ml) and 0.5 g of activated carbon were added to the crude product and the temperature was raised to 60°C to dissolve, diatomaceous earth was applied and filtered, the filtrate was concentrated to a small volume and filtered, and 4.8 g of white solid was obtained by drying, with a yield of 86.0%.

[0145] Comparative Example 1

[0146] This comparative example provides a method for preparing compound 2 (intermediate 2), which is as follows:

[0147] Weigh 15g of progesterone, add 300ml of ethyl acetate to dissolve, then add 1.5g of 5% palladium carbon, empty the air in the reaction bottle, connect the reaction bottle with a hydrogen balloon and react overnight. TLC shows that the fluorescence disappears and two products are produced, namely 5α-pregnant-3,20-dione (compound 2) and 5β-pregnant-3,20-dione, of which 5α-pregnant-3,20-dione has poor solubility and most of it has precipitated. Filter by suction, wash the filter residue with a small amount of ethyl acetate, and the mother liquor is 5β-pregnant-3,20-dione containing a small amount of 5α isomer. Scrape out the filter residue, add 100ml of dichloromethane, stir and filter to remove palladium carbon, spin dry the dichloro solution to obtain 3.2g of crude 5α-pregnant-3,20-dione, and beat with isopropyl ether to obtain 3.0g of fine compound 2, with a yield of 29.8%.

[0148] Comparative Example 2

[0149] This comparative example provides a preparation method of intermediate 1, which is as follows:

[0150] In a 500ml three-necked flask, add 10g (31.8mmol) of progesterone, measure 150ml of tetrahydrofuran, 20ml of tert-butanol, cool to -70°C, stir, introduce about 30g of ammonia, add 2.5g (357mmol) of lithium bar, and naturally heat up after the addition to keep the temperature below -40°C (-50°C to -40°C). After reacting for 3h, TLC shows that the raw material is not completely converted, and a large number of other fluorescent products are produced. Therefore, the 4-olefin is not completely reduced, which will be converted into the initial raw material progesterone in the next oxidation reaction, which will lead to a significant decrease in yield and difficulty in separation.

[0151] Comparative Example 3

[0152] This comparative example provides a method for preparing brenoside, which is as follows:

[0153] Weigh 5.0g of compound 2, add 100mL toluene to dissolve, add 5.0g aluminum isopropoxide and 7.5mL isopropanol, and stir for 20min at 80°C. TLC (P:E=2:1) ​​shows that most of the raw materials have been converted, and two main products with increased polarity are produced. 2.2g of product is obtained by column chromatography, with a yield of 43.7%.

[0154] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0155] The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in detail, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to interpret the contents of the claims.

Claims

1. A method for preparing brenoside, characterized in that: The steps include: 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 brenoside according to claim 1, characterized in that: The time for the dissolved metal reduction reaction is 2h~3h.

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

1.

5. The method for preparing brenoside according to claim 1, characterized in that: 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 brenoside 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 brenoside according to claim 1, characterized in that: 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 brenoside 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: temperature of 28°C to 35°C and pH of 6.5 to 8.

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