CN112501235B

CN112501235B - Method for preparing breynolone by using monoalcohol ketone acetate

Info

Publication number: CN112501235B
Application number: CN202011298901.5A
Authority: CN
Inventors: 杨帆; 盖祎; 戴龙华; 汤杰; 汤天天
Original assignee: Shanghai Gelinkai Biotechnology Co ltd; East China Normal University
Current assignee: Shanghai Gelinkai Biotechnology Co ltd; East China Normal University
Priority date: 2020-11-19
Filing date: 2020-11-19
Publication date: 2022-12-02
Anticipated expiration: 2040-11-19
Also published as: CN112501235A

Abstract

The invention discloses a method for preparing medicine breynolone by using monoalcohol ketoacetate, which is used as a raw material to prepare the breynolone by a four-step reaction three-pot method of hydrogenation-hydrolysis, oxidation and enzyme catalytic reduction. In the preparation method, the monoalkenolone acetate serving as the raw material from a natural product is adopted, the 5 alpha-configuration is single, the stereoselectivity of the enzyme-catalyzed reduction of the 3-ketone group in the reaction process is high, and a chemical reagent and a catalyst with higher cost are not needed in the preparation method, so that the defects of generation of a plurality of wastes, harsh reaction conditions and the like in the common preparation method are avoided. The method has mild reaction conditions and simple and convenient operation, and can realize the high-efficiency and low-cost preparation of the medicine, namely the breynolone.

Description

Method for preparing breynolone by using monoalcohol ketone acetate

Technical Field

The invention belongs to the technical field of organic compound preparation, and relates to a method for preparing medicine breynolone from monoalcohol ketone acetate.

Background

Postpartum Depression (PPD) is a significant mental syndrome that occurs in women during puerperium. The 2016 epidemiological data show that 3 hundred million cases of worldwide PPD patients have 7-40% of developed national depression prevalence rate. The pathophysiology of PPD is not fully understood and may be associated with a number of factors, including hormones and genetic factors. Before the approval of brennolone, no treatment was specifically approved for PPD, most of which were chosen for antidepressants, hormones, electroshock and psychological treatments.

Breynolone, chemically known as 5 α -pregnane-3 α -hydroxy-20-one, is the first FDA approved drug to obtain indications for postpartum depression, is a positive allosteric modulator of GABAA receptors, and can correct disorders of the postpartum GABAA receptor system, particularly restore a balance between GABAA receptor and NMDA receptor activity.

AboutThe synthesis of the breynolone is a more classical synthesis method (world intellectual property organization patent WO2009108804, chinese invention patent document CN 103396467A) which takes 3 beta-hydroxy-5-pregnene-20-ketone (short for pregnenolone) as a starting material and utilizes H ₂ The method comprises the steps of reducing 5,6-position double bonds by palladium carbon, further performing Mitsunobu reaction, reversing 3 beta-configuration in the presence of triphenylphosphine, diethyl azodicarboxylate, trifluoroacetic acid and sodium benzoate, further performing reflux hydrolysis in methanol to obtain the breynolone, wherein the reaction cost is high, a lot of wastes are generated in the reaction process, meanwhile, a certain proportion of 5 beta-stereoisomer is always generated in the 5,6-position double bond reduction process, the separation and purification are difficult, and the total yield is reduced. The method reported in j.med.chem.,2005,48 (16), 5203 uses potassium tri-sec-butylborohydride to reduce 3-keto group at-78 ℃ to produce 3 α -hydroxy with a yield of only 63%.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for preparing breynolone by using natural 5 alpha-pregn-16-ene-20-one-3 beta-alcohol acetate (called monoalcohol ketone acetate for short) obtained from sisal hemp extraction hard fiber leftovers as a raw material. In the preparation method, the breynolone is prepared by taking monoalcohol ketoacetate as a raw material and carrying out hydrogenation-hydrolysis, oxidation and enzyme catalytic reduction through a four-step reaction three-pot method.

The reaction process of the preparation method is shown as the reaction formula (I):

reaction formula (I)

The method comprises the following steps:

(1) Hydrogenation reaction: in an organic solvent, carrying out hydrogenation reaction on monoalcohol ketone acetate, hydrogen and a catalyst under the heating condition, and then carrying out hydrolysis reaction in the presence of inorganic base to obtain 5 alpha-pregnane-3 beta-hydroxy-20-ketone; the reaction process is shown as a reaction formula (1):

reaction formula (1)

(2) And (3) oxidation reaction: dissolving 5 alpha-pregnane-3 beta-hydroxy-20-ketone in an organic solvent, and reacting under controlled temperature in the presence of an oxidant and a catalyst to obtain 5 alpha-pregnane-3,20-diketone; the reaction process is shown as the reaction formula (2):

reaction type (2)

(3) Enzyme-catalyzed reduction reaction: 5 alpha-pregnane-3,20-diketone is subjected to enzyme catalytic reduction reaction in the presence of an organic solvent, alkali, carbonyl reductase and coenzyme to obtain a target product, namely the breynolone; the reaction process is shown as a reaction formula (3):

reaction type (3)

In the step (1), the organic solvent is one or more selected from ethanol, methanol, isopropanol, tetrahydrofuran, toluene, ethyl acetate and the like; preferably, ethanol.

In the step (1), the catalyst is selected from one or more of palladium/carbon, palladium/calcium carbonate, palladium/barium sulfate, active nickel, platinum dioxide and the like; preferably, palladium on carbon.

In the step (1), the mass ratio of the monoalkenolone acetate, hydrogen, the catalyst and the inorganic base is 1: (0.01-0.1): (0.03-0.1): (0.2-0.3); preferably, 1:0.05:0.05:0.23.

in the step (1), the temperature of the hydrogenation reaction is 45-65 ℃; preferably 50 deg.c.

In the step (1), the hydrogenation reaction time is 6-12h; preferably, it is 8h.

In the step (1), the inorganic base is one or more selected from potassium carbonate, lithium carbonate, sodium carbonate, potassium bicarbonate, sodium bicarbonate, potassium hydroxide, sodium hydroxide and the like; preferably, it is potassium carbonate.

In the step (1), the temperature of the hydrolysis reaction is 25-78 ℃; preferably, it is 60 ℃.

In the step (1), the hydrolysis reaction time is 2-5h; preferably, it is 3h.

In the step (2), the organic solvent is one or more selected from dichloromethane, chloroform, acetone, toluene, ethyl acetate, 2-methyltetrahydrofuran and the like; preferably, dichloromethane.

In the step (2), the oxidant is selected from one or more of sodium hypochlorite, chromic oxide, PCC, PDC, sodium dichromate, potassium dichromate and the like; preferably, sodium hypochlorite.

In the step (2), the catalyst is one or more selected from tetramethylpiperidine oxide (TEMPO), 4-hydroxy-tetramethylpiperidine oxide, 4-benzoyloxy-tetramethylpiperidine oxide and the like; preferably, it is tetramethylpiperidine oxide (TEMPO).

In the step (2), the mass ratio of the 5 alpha-pregnane-3 beta-hydroxy-20-ketone to the oxidant to the catalyst is 1: (3.5-4.5): (0.03-0.05); preferably, it is 1:3.75:0.03.

in the step (2), the temperature of the oxidation reaction is 0-25 ℃; preferably 10 deg.c.

In the step (2), the time of the oxidation reaction is 2-5h; preferably, it is 2.5h.

In the step (3), the organic solvent is one or more selected from methyl tert-butyl ether, ethyl acetate, methyl isobutyl ketone, isopropanol, tert-butanol, methanol and the like; preferably, it is methyl tert-butyl ether.

In the step (3), the inorganic base is selected from one or more of sodium hydroxide, potassium carbonate, lithium carbonate, sodium carbonate, potassium bicarbonate, sodium bicarbonate and the like; preferably, sodium hydroxide.

In the step (3), the carbonyl reductase is the carbonyl reductase RH, and the protein sequence of the carbonyl reductase RH is shown in SEQ ID NO. 1: <xnotran> marlegkvaivtgaaqgmgaatarlfvhegarvvlgdvleekgralaaelgdaaiftpldvsdesswesavavavdrfggldilvnnagvmhwapiedldvarterlldvnvlgnllgakavvpimkkagrgvivnissvdglrgvnglaaytaskwavrgltkalayelgpagirvcsvhpggvdttlgnpgglvgddlqskyvgvplqrigesediaratlfvasdeasyisgaelavdggwsagtyypglpgtppalmpn. </xnotran>

In the step (3), the coenzyme is selected from NAD ⁺ 、NADP ⁺ One or more of NADH, NADPH; preferably, NAD ⁺ 。

In the step (3), the mass ratio of the 5 alpha-pregnane-3,20-diketone to the alkali to the reductase to the coenzyme is 1: (0.12-0.15): (0.4-1): (0.0004-0.001); preferably, 1:0.13:0.5:0.00045.

in the step (2), the temperature of the enzyme catalytic reduction reaction is 25-35 ℃; preferably, it is 30 ℃.

In the step (2), the time of the enzyme catalytic reduction reaction is 12-30h; preferably 24h.

The invention has the beneficial effects that:

1) The natural product monoalcohol ketone acetate ester which is obtained by extracting hard fiber leftover resources from the plant sisal hemp and recycling is taken as a raw material, the 5 alpha-configuration is single, the defect of stereoisomer existing in the reduction of 4-or 5-position double bond in the existing method is overcome, the operation of separating isomer is not needed, and the loss and the cost improvement caused by separation are avoided.

2) The reaction stereoselectivity of the enzyme-catalyzed reduction of the 3-keto group is high, the 3 alpha-hydroxyl structure is obtained in a single configuration, a chemical reagent and a catalyst with higher cost are not needed, and the defects of generation of a plurality of wastes and harsh reaction conditions in a common preparation method are avoided.

The method has mild reaction conditions and simple and convenient operation, and can realize the efficient, low-cost and environment-friendly preparation of the medicine, namely the breynolone.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited. (all the results obtained under the optimized experimental conditions are shown in the examples, and have potential application value).

EXAMPLE 1 preparation of alpha-pregnane-3 beta-hydroxy-20-one

Adding 50g of monoalkenyl alcohol ketone acetate, 500g of ethanol and 2.5g of 5% palladium/carbon into a 1L hydrogenation tank, replacing five times by nitrogen, stirring and heating to 50 +/-5 ℃, controlling the temperature of the system to be 50 +/-5 ℃, the pressure to be 0.005-0.01 MPa, carrying out hydrogenation reaction for 6-12h, and detecting the reaction end point by TLC. Replacing three times with nitrogen, filtering, rinsing with ethanol, adding 11.5g of potassium carbonate and 10g of water into filtrate, heating to 60 +/-5 ℃, carrying out heat preservation reaction for 2-5h, adjusting the pH to be neutral by using 10% diluted hydrochloric acid, concentrating the ethanol under reduced pressure to obtain a large amount of crystals, cooling to 0 +/-5 ℃, crystallizing for 12-24 h, filtering, rinsing with glacial ethanol, drying the solids in a hot air circulation oven at 75 ℃ for 6-12h, and obtaining 42.2g of white crystals of 5 alpha-pregnane-3 beta-hydroxy-20-ketone with the yield of 95%. ¹ H NMR(CDCl ₃ ,400MHz):δ3.63-3.57(m,1H),2.52(dd,J＝9.0,8.8Hz,1H),2.19-2.11(m,1H),2.11(s,3H),2.01-1.99(m,1H),1.83-1.63(m,5H),1.46-1.10(m,13H),1.03-0.88(m,2H),0.81(s,3H),0.72-0.65(m,1H),0.60(s,3H)。

Example 2 preparation of alpha-pregnane-3,20-dione

Adding 16g of water into a 500mL three-neck flask, sequentially adding 2g of sodium bicarbonate, 200g of dichloromethane and 40g of the hydro-hydrolysate 5 alpha-pregnane-3 beta-hydroxy-20-one in the previous step under stirring, stirring for complete dissolution, cooling, and adding 1.2g of tetramethyl piperidine oxide (TEMPO) when the internal temperature of the reaction solution is 10 ℃. 150g of 10% sodium hypochlorite solution is uniformly dripped, the dripping time is controlled to be 1-2 h, and the dripping temperature is controlled to be 6-15 ℃. After the dropwise addition, the reaction is carried out for 2-5h at the temperature of 10-15 ℃, sampling TLC analysis is carried out to analyze that the raw materials are completely reacted, 30g of 20% sodium bicarbonate solution is added after the reaction is finished to quench the reaction, the mixture is stirred for 10 min, standing and layering are carried out (emulsion layer separation and aqueous phase extraction), the aqueous phase is extracted by dichloromethane, stirred for 30min once, standing and layering are carried out, the dichloromethane organic phase is combined, washed by 80g 3 of saturated sodium chloride solution and stirred for 10 min for three times, and standing and layering are carried out. Concentrating dichloromethane to a large amount of crystals under the reduced pressure condition of an organic phase, carrying the crystals twice by water until the dichloromethane is completely concentrated, adding 200g of water, stirring for 20 minutes, performing suction filtration, and drying filter cakes by hot-air circulation at 75 ℃ for 6-12 hours to obtain yellowish to off-white powder 5 alpha-pregnant powderStanol-3,20-dione 39.2g, 99% yield. ¹ H NMR(CDCl ₃ ,400MHz):δ2.56-2.51(m,1H),2.44-1.96(m,10H),1.74-1.13(m,13H),1.02(s,3H),1.00-0.89(m,1H),0.82-0.77(m,1H),0.63(s,3H)。

Example 3 preparation of alpha-pregnane-3 alpha-hydroxy-20-one (breynolone)

BL21 (DE 3)/pET 24a-RH strain was inoculated into a test tube containing 4mL of LB medium (yeast extract 5g/L, tryptone 10g/L, sodium chloride 10 g/L), cultured overnight at 37 ℃, the culture broth was inoculated into a flask containing 1L of TB medium (yeast extract 24g/L, tryptone 12g/L, dipotassium hydrogenphosphate trihydrate 16.4g/L, potassium dihydrogenphosphate 2.3g/L, glycerol 5 g/L), cultured with shaking at 37 ℃ until OD =3.0, induced by addition of 0.2mM isopropyl-. Beta. -D-thiogalactoside (IPTG), and further cultured overnight with cooling to 25 ℃. Centrifuging at 3500 rpm for 20 min, collecting precipitate to obtain RH thallus, adding 3 times volume of deionized water into the RH thallus, ultrasonically disrupting the thallus, centrifuging at 8000rpm for 30min, and collecting the supernatant to obtain RH enzyme solution. GDH enzyme solution was obtained in the same manner using BL21 (DE 3)/pET 24a-GDH strain.

Adding 100g of 5 alpha-pregnane-3,20-diketone, 80g of dextrose monohydrate, 10g of monopotassium phosphate, 210g of methyl tert-butyl ether and 1000g of purified water into a 3L fermentation tank, fully stirring, heating to 30 ℃, adjusting the pH to be 7.0 by using sodium hydroxide, 25g of GDH enzyme liquid, 23g of RH enzyme liquid and 0.045g of nicotinamide adenine dinucleotide, controlling the pH to be 7.5 +/-0.2 by using a sodium hydroxide solution in the reaction process, keeping the temperature to be 25-30 ℃, and stirring for reacting for 12-30 hours. The conversion of substrate was 97% by HPLC. After the conversion was completed, the conversion solution was extracted three times using 2000mL of dichloromethane, the obtained dichloromethane layer was washed three times with 150g × 3 saturated saline, dried over anhydrous sodium sulfate, filtered, and after dichloromethane was evaporated to dryness, 240g of methanol was added to recrystallize, filter, and dry to obtain 89g of 5 α -pregnane-3 α -hydroxy-20-one with a yield of 88.0%. ¹ HNMR(CDCl ₃ ,400MHz):δ4.05(s,1H),2.53(dd,J＝9.1,8.2Hz,1H),2.17-2.11(m,1H),2.11(s,3H),2.02-1.99(m,1H),1.69-1.12(m,19H),1.01-0.93(m,1H),0.84-0.78(m,1H),0.78(s,3H),0.60(s,3H)。 ¹³ C NMR(CDCl ₃ ,100MHz):δ209.82,66.46,63.84,56.77,54.19,44.27,39.09,39.07,36.10,35.84,35.48,32.19,31.95,31.54,28.98,28.44,24.37,22.76,20.79,13.47,11.18。HRMS(ESI):[M+Na] ⁺ (C ₂₁ H ₃₄ NaO ₂ ) Calcd 341.2457, found 341.2449.

The method takes easily-obtained natural products as raw materials to efficiently prepare the breynolone, has high stereoselectivity of products, does not need to use chemical reagents and catalysts with higher cost, and provides a foundation for further improving the effect of the medicine breynolone in guaranteeing human health.

The protection content of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.

SEQUENCE LISTING

<110> university of east China, shanghai Geling Kai Biotech Co., ltd

<120> method for preparing breynolone by using monoalcohol ketone acetate

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 263

<212> PRT

<213> Artificial sequence

<400> 1

Met Ala Arg Leu Glu Gly Lys Val Ala Ile Val Thr Gly Ala Ala Gln

1 5 10 15

Gly Met Gly Ala Ala Thr Ala Arg Leu Phe Val His Glu Gly Ala Arg

20 25 30

Val Val Leu Gly Asp Val Leu Glu Glu Lys Gly Arg Ala Leu Ala Ala

35 40 45

Glu Leu Gly Asp Ala Ala Ile Phe Thr Pro Leu Asp Val Ser Asp Glu

50 55 60

Ser Ser Trp Glu Ser Ala Val Ala Val Ala Val Asp Arg Phe Gly Gly

65 70 75 80

Leu Asp Ile Leu Val Asn Asn Ala Gly Val Met His Trp Ala Pro Ile

85 90 95

Glu Asp Leu Asp Val Ala Arg Thr Glu Arg Leu Leu Asp Val Asn Val

100 105 110

Leu Gly Asn Leu Leu Gly Ala Lys Ala Val Val Pro Ile Met Lys Lys

115 120 125

Ala Gly Arg Gly Val Ile Val Asn Ile Ser Ser Val Asp Gly Leu Arg

130 135 140

Gly Val Asn Gly Leu Ala Ala Tyr Thr Ala Ser Lys Trp Ala Val Arg

145 150 155 160

Gly Leu Thr Lys Ala Leu Ala Tyr Glu Leu Gly Pro Ala Gly Ile Arg

165 170 175

Val Cys Ser Val His Pro Gly Gly Val Asp Thr Thr Leu Gly Asn Pro

180 185 190

Gly Gly Leu Val Gly Asp Asp Leu Gln Ser Lys Tyr Val Gly Val Pro

195 200 205

Leu Gln Arg Ile Gly Glu Ser Glu Asp Ile Ala Arg Ala Thr Leu Phe

210 215 220

Val Ala Ser Asp Glu Ala Ser Tyr Ile Ser Gly Ala Glu Leu Ala Val

225 230 235 240

Asp Gly Gly Trp Ser Ala Gly Thr Tyr Tyr Pro Gly Leu Pro Gly Thr

245 250 255

Pro Pro Ala Leu Met Pro Asn

260

Claims

1. A method for preparing breynolone by using monoalkenolone acetate is characterized in that the reaction process of the method is shown as a reaction formula (I):

the method comprises the following specific steps:

(1) Hydrogenation reaction: in an organic solvent, mono-enol ketone acetate, hydrogen and a catalyst are subjected to hydrogenation reaction under the heating condition, and then hydrolysis reaction is carried out in the presence of inorganic base to obtain 5 alpha-pregnane-3 beta-hydroxy-20-ketone; the catalyst is selected from one or more of palladium/carbon, palladium/calcium carbonate, palladium/barium sulfate, active nickel and platinum dioxide; the reaction process is shown as a reaction formula (1):

(2) And (3) oxidation reaction: dissolving the 5 alpha-pregnane-3 beta-hydroxy-20-ketone obtained in the step (1) in an organic solvent, and controlling the temperature to react in the presence of an oxidant and a catalyst to obtain 5 alpha-pregnane-3,20-diketone; the catalyst is one or more selected from tetramethylpiperidine oxide TEMPO, 4-hydroxy-tetramethylpiperidine oxide and 4-benzoyloxy-tetramethylpiperidine oxide; the reaction process is shown as the reaction formula (2):

(3) Enzyme-catalyzed reduction reaction: carrying out enzyme catalytic reduction reaction on the 5 alpha-pregnane-3,20-diketone obtained in the step (2) in the presence of an organic solvent, alkali, reductase and coenzyme to obtain a target product, namely the breynolne; the reductase is carbonyl reductase RH, and the protein sequence of the carbonyl reductase RH is shown in SEQ ID NO. 1; the alkali is selected from one or more of sodium hydroxide, potassium carbonate, lithium carbonate, sodium carbonate, potassium bicarbonate and sodium bicarbonate; the temperature of the enzyme catalytic reduction reaction is 25-35 ℃; the reaction process is shown as a reaction formula (3):

2. the method according to claim 1, wherein in the step (1), the organic solvent is one or more selected from ethanol, methanol, isopropanol, tetrahydrofuran, toluene and ethyl acetate; and/or the inorganic base is selected from one or more of potassium carbonate, lithium carbonate, sodium carbonate, potassium bicarbonate, sodium bicarbonate, potassium hydroxide and sodium hydroxide.

3. The method of claim 1, wherein in the step (1), the monoalkenolone acetate, the hydrogen gas, the catalyst and the inorganic base are mixed in a mass ratio of 1: (0.01-0.1): (0.03-0.1): (0.2-0.3).

4. The method of claim 1, wherein in step (1), the temperature of the hydrogenation reaction is 45-65 ℃; and/or the temperature of the hydrolysis reaction is 25-78 ℃.

5. The method according to claim 1, wherein in the step (2), the organic solvent is one or more selected from dichloromethane, chloroform, acetone, toluene, ethyl acetate and 2-methyltetrahydrofuran; and/or the oxidant is selected from one or more of sodium hypochlorite, dichromium trioxide, PCC, PDC, sodium dichromate and potassium dichromate.

6. The method according to claim 1, wherein in step (2), the mass ratio of the 5 α -pregnane-3 β -hydroxy-20-one to the oxidizing agent to the catalyst is 1: (3.5-4.5): (0.03-0.05); and/or the temperature of the oxidation reaction is 0-25 ℃.

7. The method of claim 1, wherein in step (3), the organic solvent is selected from one or more of methyl tert-butyl ether, ethyl acetate, methyl isobutyl ketone, isopropanol, tert-butanol, and methanol.

8. The method of claim 1, wherein in step (3), the coenzyme is selected from the group consisting of NAD ⁺ 、NADP ⁺ NADH, NADPH.

9. The method of claim 1, wherein in step (3), the 5 α -pregnane-3,20-dione, base, reductase and coenzyme are present in a mass ratio of 1: (0.12-0.15): (0.4-1): (0.0004-0.001).