WO2024109742A1 - Method for efficiently synthesizing bufadienolides and derivatives thereof - Google Patents

Method for efficiently synthesizing bufadienolides and derivatives thereof Download PDF

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WO2024109742A1
WO2024109742A1 PCT/CN2023/132942 CN2023132942W WO2024109742A1 WO 2024109742 A1 WO2024109742 A1 WO 2024109742A1 CN 2023132942 W CN2023132942 W CN 2023132942W WO 2024109742 A1 WO2024109742 A1 WO 2024109742A1
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compound
formula
preparation
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acid
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林国强
叶文博
赵群飞
张建革
贺庆利
王超
俞少鹏
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上海中医药大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring
    • C07J71/001—Oxiranes
    • C07J71/0021—Oxiranes at position 14(15)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J19/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 by a lactone ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12N9/0042—NADPH-cytochrome P450 reductase (1.6.2.4)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C12N9/0081—Cholesterol monooxygenase (cytochrome P 450scc)(1.14.15.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C12P33/06—Hydroxylating
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/02—Oxidoreductases acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12Y106/02004—NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
    • C12Y114/15006—Cholesterol monooxygenase (side-chain-cleaving) (1.14.15.6), i.e. cytochrome P450scc

Definitions

  • the invention belongs to the technical field of drug synthesis, and relates to a method for preparing active ingredients in precious Chinese medicinal material toad venom, including but not limited to toad venom lactone compounds such as resibufogenin.
  • Toad venom is a traditional precious Chinese medicinal material in my country and is a national second-level protected wild medicinal material species. Traditional medicine believes that it has the effects of detoxification, analgesia, and refreshing. Modern pharmacological studies have shown that toad venom has multiple pharmacological effects such as enhancing myocardial contractility, anti-myocardial ischemia, blood pressure increase, blood pressure decrease, anti-tumor, antibacterial and anti-inflammatory, anesthetic analgesia, and hallucination.
  • toad venom due to the complex production process of toad venom, there are a large number of defective products in its production process, and the bioavailability is low.
  • the content of active ingredients in toad venom from different origins is also quite different, making it difficult to meet fixed standards.
  • the increasingly scarce wildlife resources and low natural abundance also largely restrict the pharmacological research and clinical application of toad venom.
  • toad lactone compounds are basically the same as that of toad venom.
  • the 2020 edition of the Chinese Pharmacopoeia requires that the liquid chromatography of toad venom products must contain characteristic peaks of five toad venom lactone compounds, namely bufalin, cinobufagin, resibufogenin, gamabufalin and bufotaline, and stipulates that the total content of the three toad lactone compounds, namely bufalin, cinobufagin and resibufogenin, must not be less than 7%. Therefore, in order to solve the above problems, it is particularly important to artificially synthesize the chemical components with strong physiological activity in toad venom.
  • the object of the present invention is to provide a method for efficiently synthesizing bufotoxin lactone compounds and derivatives thereof.
  • the first aspect of the present invention provides a method for preparing a bufotoxin lactone compound represented by formula I, the preparation method comprising the following steps:
  • the compound of formula III is obtained by oxidizing androstenedione 4-AD through biological enzymes;
  • the compound of formula III is subjected to chemical semi-synthesis to complete the loading and transformation of the six-membered diene lactone ring to obtain the key intermediate II of toad venom lactone;
  • the key intermediate II is modified in oxidation state to obtain a bufotoxin lactone compound shown in formula I.
  • R 1 , R 2 and R 4 are each independently -H or -OH;
  • R3 is -CH3 or -CHO
  • R5 and R6 are each independently -H, -OH or keto
  • R7 is -H, -OH or oxyacetyl.
  • R 1 , R 2 , R 4 , R 5 , R 6 , and R 7 are all H, and R 3 is methyl.
  • the compound represented by Formula III has the structure represented by Formula 5, and the preparation method comprises: step 3 or steps 2 to 3 or steps 1 to 2 to 3,
  • Step 1 Compound 2 is biocatalytically oxidized by steroidal C14- ⁇ hydroxylase to obtain compound 3;
  • Step 2 Compound 3 is catalytically hydrogenated in an organic solvent in the presence of a hydrogenation catalyst to obtain compound 4;
  • the hydrogenation catalyst is platinum dioxide, palladium carbon, palladium hydroxide, Raney nickel or rhodium/aluminum trioxide;
  • the organic solvent is one or a mixed solvent of two or more selected from pyridine, 4-methylpyridine, tetrahydrofuran, methanol or ethanol;
  • Step 3 Compound 4 is reduced by a reducing agent in an organic solvent to obtain compound 5;
  • the reducing agent is lithium tri-sec-butylborohydride, potassium tri-sec-butylborohydride or diisobutylaluminum hydride;
  • the organic solvent is one or a mixed solvent of two or more selected from tetrahydrofuran, diethyl ether, 1,4-dioxane, ethylene glycol dimethyl ether, benzene or toluene.
  • steroid C14- ⁇ hydroxylase is used for biocatalysis in step 1, and the biocatalysis method includes but is not limited to pure enzyme catalysis, crude enzyme solution catalysis and whole cell conversion.
  • the C14- ⁇ hydroxylase is a complex enzyme, comprising the following two enzymes: (a) a cytochrome P450 enzyme derived from Cochliobolus lunatus, whose amino acid sequence is shown in SEQ ID NO:2; and (b) a cytochrome P450 reductase CPR derived from Cochliobolus lunatus, whose amino acid sequence is shown in SEQ ID No:4.
  • the hydrogenation catalyst in step 2 is palladium hydroxide, 5% by mass of palladium carbon or 10% by mass of palladium carbon, and the organic solvent is pyridine or 4-methylpyridine.
  • the reducing agent in step 3 is potassium tri-sec-butylborohydride or diisobutylaluminum hydride
  • the reaction temperature is -78 to 0° C.
  • the organic solvent is tetrahydrofuran or toluene.
  • R 1 , R 2 , R 4 , R 5 , R 6 , and R 7 are all H, R 3 is methyl, the compound represented by formula III has the structure represented by formula 5, and the compound represented by formula II has the structure represented by formula 10.
  • the preparation method comprises: step 7 or steps 6 to 7 or steps 5 to 6 to 7 or steps 4 to 5 to 6 to 7,
  • Step 4 Add hydrazine hydrate and base to the organic solution of compound 5 for reaction, and then add elemental iodine and base to the crude product for reaction to obtain compound 6;
  • the organic solvent is tetrahydrofuran, ethanol or methanol, or a mixture of two or more thereof;
  • the base is diisopropylethylamine, triethylamine or 4-dimethylaminopyridine;
  • Step 5 Add a transition metal catalyst and a base to compound 6 and compound 7 to react to obtain compound 8;
  • the transition metal catalyst is [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride, tetrakis(triphenylphosphine)palladium, acetone Acid palladium or dichlorobis(triphenylphosphine)palladium;
  • the base is potassium carbonate, potassium phosphate, potassium hydrogen phosphate, potassium tert-butoxide or potassium hydroxide;
  • the reaction solvent is tetrahydrofuran, methanol, water, N,N-dimethylformamide, one or a mixed solvent of two or more thereof;
  • Step 6 adding a hydrogenation homogeneous catalyst to the organic solution of compound 8, and obtaining compound 9 through catalytic hydrogenation reaction;
  • the hydrogenation homogeneous catalyst is triphenylphosphine rhodium chloride, hexafluorophosphate (tricyclohexylphosphine) (1,5-cyclooctadiene) (pyridine) iridium or (1,5-cyclooctadiene) rhodium chloride (I) dimer;
  • the organic solvent used is dichloromethane, benzene or toluene;
  • Step 7 Add an acid to the organic solution of compound 9 to obtain compound 10; the acid is p-toluenesulfonic acid, pyridine toluenesulfonate (PPTS), benzenesulfonic acid, camphorsulfonic acid, perchloric acid, periodic acid, 2% to 98% sulfuric acid or 2% to 36.5% hydrochloric acid; the organic solvent used is 1,4-dioxane, methanol, tetrahydrofuran and water, one or a mixed solvent of two or more.
  • the acid is p-toluenesulfonic acid, pyridine toluenesulfonate (PPTS), benzenesulfonic acid, camphorsulfonic acid, perchloric acid, periodic acid, 2% to 98% sulfuric acid or 2% to 36.5% hydrochloric acid
  • the organic solvent used is 1,4-dioxane, methanol, tetrahydrofuran and water
  • the organic solvent in step 4 is ethanol or tetrahydrofuran, or a mixed solvent of the two; and the base is diisopropylethylamine or triethylamine.
  • the transition metal catalyst in step 5 is [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride or tetrakistriphenylphosphine palladium; the base is potassium phosphate or potassium carbonate; and the solvent is N,N-dimethylformamide or tetrahydrofuran.
  • the hydrogenation homogeneous catalyst in step 6 is (tricyclohexylphosphine) (1,5-cyclooctadiene) (pyridine) iridium hexafluorophosphate or triphenylphosphine rhodium chloride, and the organic solvent is dichloromethane or toluene.
  • the acid in step 7 is dilute hydrochloric acid with a concentration of 3M.
  • R 1 , R 2 , R 4 , R 5 , R 6 , and R 7 are all H, R 3 is methyl, the compound shown in Formula I has the structure shown in Formula 1, and the compound shown in Formula II has the structure shown in Formula 10.
  • the preparation method comprises: step 8,
  • Step 8 Add a halogenating agent and an aqueous perchloric acid solution to the organic solution of compound 10 for reaction.
  • the crude product is directly dissolved in pyridine without purification, and heated at 50-70° C. for reaction for 0.5-2.0 hours to obtain the target compound 1;
  • the organic solvent is one or a mixed solvent of two or more of 1,4-dioxane, tetrahydrofuran, ether, acetone or water;
  • the halogenating agent is N-bromosuccinimide (NBS), N-iodosuccinimide (NIS) or N-chlorosuccinimide (NCS); and the mass fraction of the perchloric acid aqueous solution is 1% to 70%.
  • the organic solvent in step 8 is a mixed solvent of 1,4-dioxane and water or acetone and water, and the volume ratio thereof is 12:1 to 5:1; and the halogenating agent is N-bromosuccinimide (NBS) or N-iodosuccinimide (NIS).
  • the second aspect of the present invention provides a bufotoxin lactone compound intermediate 8 having the following formula:
  • the third aspect of the present invention provides a bufotoxin lactone compound intermediate 9 having the following formula:
  • the present invention starts with cheap and readily available androstenedione 4-AD, obtains 14- ⁇ -hydroxy-androstan-17-one compound through biological enzyme oxidation, and then completes the loading of E-ring pyrone by chemical transformation without any protecting group to obtain key intermediates, and then realizes the synthesis of toad venom lactone compounds from this.
  • the method only requires eight steps to synthesize resibufogenin, has a short route, simple operation, and a large reaction scale, and can be used for large-scale preparation of such natural products, with application prospects for industrial production.
  • Figure 1 is a HPLC spectrum.
  • the inventors of the present application have conducted extensive and in-depth research and have provided a method for preparing resifobafugin, an active ingredient in toad venom, using the inexpensive and readily available steroid compound androstenedione (4-AD) as a raw material without introducing a protective group.
  • 4-AD steroid compound androstenedione
  • natural toad lactone compounds can be synthesized in as little as eight steps, and some intermediates can be prepared on a gram scale. This method can promote the widespread application of such compounds in the medical field and alleviate the material scarcity pressure of pharmacological research on active substances in toad venom.
  • the invention provides a steroid compound III with a steroidal mother nucleus 3 ⁇ -hydroxyl group, an AB ring decahydronaphthalene ring cis-configuration, and a C14- ⁇ -configuration hydroxyl group, and a 3- ⁇ -hydroxy-14,15-ene-17-pyrone toad venom-based lactone advanced intermediate II.
  • the two precursors have structures shown in the following structural formulas and are used as intermediates for chemical semi-synthesis of toad lactone compounds.
  • R1, R2 and R4 are -H or -OH; R3 is -CH 3 or -CHO; R5 and R6 are -OH or keto; R7 is -OH or oxyacetyl.
  • the R1, R2, R4-R7 groups of the steroid compound are all H, and R3 is methyl.
  • the invention provides a method for obtaining a large amount of 14 ⁇ -hydroxy-androstane-17-one, a chemical synthesis intermediate of a toad lactone compound, by biocatalysis.
  • Androstenedione (4-AD) is used as a raw material, and C14- ⁇ hydroxylase is used for biocatalysis to complete C14- ⁇ configuration hydroxylation.
  • the biocatalytic method includes but is not limited to pure enzyme catalysis, crude enzyme solution catalysis and whole cell transformation.
  • the present invention provides a cytochrome P450 enzyme and a related P450 reductase (Cytochrome P450 reductase, CPR) which can hydroxylate the C14 position of a steroid nucleus into an ⁇ -configuration hydroxyl group, and the amino acid sequences of the enzymes are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.
  • CPR Cytochrome P450 reductase
  • the present invention optimizes the Pichia pastoris codon preference sequence of the DNA sequence of steroid nucleus C14 ⁇ -hydroxylase and related oxidoreductase (CPR), which is suitable for efficient expression in Pichia pastoris.
  • the DNA sequences after codon optimization are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively.
  • the obtained 14 ⁇ -hydroxy-androstane-17-one can be chemically synthesized into a bufotoxin-based lactone advanced intermediate shown in Formula 1 by the following steps:
  • the obtained bufotoxin-based lactone advanced intermediate can be chemically synthesized into the bufotoxin-based lactone shown in Formula 1 by the following steps:
  • yeast protein expression vector pPICZA-Duet Using pPICZA as template and GACCTTCGTTTGTGCGGCGGCCGCGATCTAACATCCAAAGACGAAAG and TTAACACTAGTCATATGGGATCCATGGTGATGGTGATGATGACCCATGG as primers, DNA fragment Fragment-1 was cloned; DNA fragment Fragment-2 was cloned using primers ATATGACTAGTGTTAACCTGCAGTGAGTTTTAGCCTTAGACATGAC and GCTATGGTGTGTGGGGGGTCTCACTTAATCTTCTGTAC, pPICZA was digested with BamH I, and finally the recombinase from Novazonics was used to reconstruct the plasmid pPICZA-Du et.
  • Pichia pastoris engineered bacteria expressing steroid C14 ⁇ -hydroxylase Synthesize codon-optimized cytochrome P450 (SEQ ID NO: 1) and cytochrome P450 reductase (CPR) (SEQ ID NO: 3) genes from Cochliobolus lunatus.
  • the yeast expression vector pPICZA-Duet was cut with BamHI and SpeI to construct the recombinant vector pPICZA-Duet-CPR.
  • the recombinant vector pPICZA-Duet-CPR was cut with KpnI and XhOI to construct the yeast expression vector pPICZA-Duet-P450-CPR.
  • the yeast expression vector pPICZA-Duet-P450-CPR was introduced into Pichia pastoris KM71H by conventional electroporation to obtain engineered bacteria expressing the corresponding enzymes.
  • yeast engineering bacteria single clones were selected and placed in 5 mL YPD liquid medium (containing 100 ⁇ g/mL Zeocin), cultured at 30°C, 260 rpm for 24 hours.
  • 5 mL of culture was inoculated into 5 mL MGYH glycerol medium, cultured at 30°C, 260 rpm for 24 hours.
  • the conditions for the transformation of C14- ⁇ hydroxylase yeast cells into 4-androstene-3,17-dione were optimized, including the transfer amount of seed culture medium, the concentration of substrate 4-AD and the optimization of conversion time.
  • the optimal conversion conditions were determined as follows: 8% transfer amount, 1.5 g/L 4-AD, and conversion time of 72 h.
  • 1.5 g 4-AD was added to obtain no less than 700 mg of C14- ⁇ hydroxylation product 14- ⁇ -hydroxy-4-androstene-3,17-dione. The results are shown in Figure 1.
  • compound 4 (10.00 g, 32.85 mmol) was dissolved in tetrahydrofuran (100 mL) and heated at -78 °C. Add potassium tri-sec-butylborohydride solution (43mL, 1.0M in THF, 43.00mmol, 1.3eq.) under a bath. React under an ice-water bath. After the conversion of the raw materials is complete, add saturated ammonium chloride solution to quench the reaction. Add dichloromethane to the reaction solution for extraction. After the organic phases are combined, wash with saturated sodium bicarbonate aqueous solution, wash with saturated brine, and dry over anhydrous sodium sulfate. After concentration, the crude product is separated and purified by column chromatography to obtain compound 5 as a white solid (8.19g, 81% yield).
  • compound 5 (3.17 g, 10.34 mmol) was dissolved in ethanol (206 mL), and triethylamine (21 g, 29 mL, 20.0 eq) and hydrazine hydrate (13 g, 12.6 mL, 20.0 eq) were added.
  • the reaction was heated to 50 ° C overnight, and the raw material was completely converted.
  • the crude hydrazone compound was dissolved in tetrahydrofuran (206 mL), and triethylamine (21 g, 21 mL, 20.0 eq) was added.
  • alkenyl iodide 6, borate compound 7 (2.76 g, 12.41 mmol, 1.2 eq), Pd(dppf)Cl 2 (0.750 g, 1.034 mmol, 10 mol%) and potassium phosphate (6.58 g, 31.02, 3.0 eq) were dissolved in N,N-dimethylformamide (23 mL) and heated to 60°C. After the reaction was completed, the mixture was diluted with water, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, the crude product was separated and purified by column chromatography to obtain compound 8 as a white solid (2.28 g, 57% yield).

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Abstract

Disclosed in the present invention is a method for efficiently synthesizing bufadienolides and derivatives thereof. By starting from cheap and easily-available androstenedione 4-AD, the method performs bio-enzyme oxidization on same to obtain 14-α-hydroxy-androstane-17-one compounds, then completes loading of E-ring pyrone by using chemical conversion without any protective group so as to obtain a key intermediate, and achieves synthesis of bufadienolides by starting from the key intermediate. The method synthesizes resibufogenin in just eight operation steps, has simple and short route, simple operations and large reaction scale, can be used for large-scale preparation of such natural products, and has use prospects in industrial production.

Description

一种高效合成蟾毒基内酯类化合物及其衍生物的方法A method for efficiently synthesizing bufotoxin lactone compounds and their derivatives 技术领域Technical Field
本发明属于药物合成技术领域，涉及制备名贵中药材蟾酥中的活性成分包括但不限于脂蟾毒配基(resibufogenin)等蟾毒基内酯类化合物的方法。The invention belongs to the technical field of drug synthesis, and relates to a method for preparing active ingredients in precious Chinese medicinal material toad venom, including but not limited to toad venom lactone compounds such as resibufogenin.
背景技术Background technique
蟾酥是我国传统名贵中药材，属国家二级保护野生药材物种。传统医学认为其具有解毒止痛、开窍醒神的功效。现代药理研究表明，蟾酥具有增强心肌收缩力、抗心肌缺血、升压、降压、抗肿瘤、抑菌抗炎、麻醉止痛，致幻等多种药理作用。然而，由于蟾酥制作工艺复杂，其生产过程中伴有大量的残次品，生物利用较低，不同产地的蟾酥其活性成分含量亦差别较大，难以符合固定的标准。此外，日益稀缺的野生动物资源和较低的天然丰度也在很大程度上制约着蟾酥的药理研究和临床应用。Toad venom is a traditional precious Chinese medicinal material in my country and is a national second-level protected wild medicinal material species. Traditional medicine believes that it has the effects of detoxification, analgesia, and refreshing. Modern pharmacological studies have shown that toad venom has multiple pharmacological effects such as enhancing myocardial contractility, anti-myocardial ischemia, blood pressure increase, blood pressure decrease, anti-tumor, antibacterial and anti-inflammatory, anesthetic analgesia, and hallucination. However, due to the complex production process of toad venom, there are a large number of defective products in its production process, and the bioavailability is low. The content of active ingredients in toad venom from different origins is also quite different, making it difficult to meet fixed standards. In addition, the increasingly scarce wildlife resources and low natural abundance also largely restrict the pharmacological research and clinical application of toad venom.
活性研究表明，蟾蜍内脂类化合物与蟾酥的生物活性表现基本一致，《中国药典》2020版一部要求蟾酥制品的液相色谱需要含有蟾毒灵(bufalin)，华蟾酥毒基(cinobufagin)，脂蟾毒配基(resibufogenin)，日蟾毒它灵(gamabufalin)和蟾毒它灵(bufotaline)五个蟾毒基内酯类化合物的特征峰，并规定蟾毒灵(bufalin)，华蟾酥毒基(cinobufagin)，脂蟾毒配基(resibufogenin)这三个蟾蜍内酯类化合物的总含量不得少于7％。因此，要解决上述的问题，蟾酥中具有较强生理活性的化学组分进行人工合成就显得尤为重要。Activity studies have shown that the biological activity of toad lactone compounds is basically the same as that of toad venom. The 2020 edition of the Chinese Pharmacopoeia requires that the liquid chromatography of toad venom products must contain characteristic peaks of five toad venom lactone compounds, namely bufalin, cinobufagin, resibufogenin, gamabufalin and bufotaline, and stipulates that the total content of the three toad lactone compounds, namely bufalin, cinobufagin and resibufogenin, must not be less than 7%. Therefore, in order to solve the above problems, it is particularly important to artificially synthesize the chemical components with strong physiological activity in toad venom.
由于蟾酥独特多样的生理活性，其活性成分的人工合成一直是有机合成领域研究的重点(Nat.Prod.Rep.2017,34,361–410；Eur.J.Med.Chem.2020,189,112038)。从上世纪60年代开始，有机合成化学家F.Sondheimer,Petti,Tsai和Wiesner等人从洋地黄毒苷配基，14-dehydrobufalin等强心苷高级天然产物出发实现到蟾酥中的蟾蜍内酯类天然产物，如蟾毒灵，酯蟾毒配基等蟾毒基内酯类化合物的转化；Yoshii,Wiesner,Welzel和Stache等课题组从简单甾体化合物出发实现了复杂蟾蜍内酯类化合物的全合成；Wiesner,Engel,Wicha和Watt等人实现了蟾毒基内酯类化合物衍生物如异位连接的吡喃酮，14-去羟基等化合物的合成。在发明人开展蟾酥合成研究的同时，Inoue等人从简单甾体化合物雄烯二酮出发，利用C16-17位双键环氧化再开环的方式成功实现蟾毒灵等五个复杂蟾毒基内酯类天然产物的全合成(Organic Letters,2020,22,8652–8657)；南京大学俞寿云、戈慧明、谭仁祥等人结合自身发展的雄烯二酮C14-位羟基化反应的特点，在优化了Inoue合成路线部分反应的基础上，实现了五个相同蟾毒基内酯类天然产物的形式合成(ACS Catal.,2022,12,9839–9845)。Due to the unique and diverse physiological activities of toad venom, the artificial synthesis of its active ingredients has always been the focus of research in the field of organic synthesis (Nat. Prod. Rep. 2017, 34, 361–410; Eur. J. Med. Chem. 2020, 189, 112038). Since the 1960s, organic synthetic chemists F. Sondheimer, Petti, Tsai and Wiesner have achieved the transformation of toad lactone natural products in toad venom, such as bufalin and esterbufalin, from advanced natural products of cardiac glycosides such as digitoxin and 14-dehydrobufalin; Yoshii, Wiesner, Welzel and Stache and other research groups have achieved the total synthesis of complex toad lactone compounds from simple steroid compounds; Wiesner, Engel, Wicha and Watt and others have achieved the synthesis of toad lactone derivatives such as ectopically linked pyranone and 14-dehydroxy compounds. While the inventors were conducting research on the synthesis of toad venom, Inoue et al. started from the simple steroid compound androstenedione and successfully achieved the total synthesis of five complex toad venom lactone natural products such as toad venom by epoxidation and ring opening of the double bond at C16-17 (Organic Letters, 2020, 22, 8652–8657); Yu Shouyun, Ge Huiming, Tan Renxiang and others from Nanjing University combined the characteristics of the C14-hydroxylation reaction of androstenedione developed by themselves, and on the basis of optimizing some reactions of Inoue's synthetic route, they achieved the formal synthesis of five identical toad venom lactone natural products (ACS Catal., 2022, 12, 9839–9845).
通过调研上述合成工作不难发现，半合成工作的起始底物基本都是同样珍贵的天然产物，并不具有规模化生产的意义。而从简单甾体出发的全合成路线，除少数 外，大部分甾体本身也并不廉价，而且需要耗费大量步骤进行氧化态的调整，总收率较低，且部分反应条件不利于工业放大，无法满足蟾毒基内酯类化合物生理活性研究的物质需求。Through the investigation of the above-mentioned synthesis work, it is not difficult to find that the starting substrates of semi-synthetic work are basically equally precious natural products, which do not have the significance of large-scale production. In addition, most steroids themselves are not cheap and require a large number of steps to adjust the oxidation state, resulting in a low overall yield. In addition, some reaction conditions are not conducive to industrial scale-up and cannot meet the material requirements for the study of the physiological activity of toad venom lactone compounds.
发明内容Summary of the invention
本发明的目的在于提供一种高效合成蟾毒基内酯类化合物及其衍生物的方法。The object of the present invention is to provide a method for efficiently synthesizing bufotoxin lactone compounds and derivatives thereof.
本发明的第一方面，提供一种式I所示的蟾毒基内酯类化合物的制备方法，所述制备方法包括以下步骤：
The first aspect of the present invention provides a method for preparing a bufotoxin lactone compound represented by formula I, the preparation method comprising the following steps:
由雄烯二酮4-AD经过生物酶氧化得到式III化合物；The compound of formula III is obtained by oxidizing androstenedione 4-AD through biological enzymes;
式III化合物通过化学半合成完成六元二烯内酯环的上载和转化得到蟾毒基内酯类关键中间体II；The compound of formula III is subjected to chemical semi-synthesis to complete the loading and transformation of the six-membered diene lactone ring to obtain the key intermediate II of toad venom lactone;
关键中间体II经过氧化态修饰获得式I所示的蟾毒基内酯类化合物，The key intermediate II is modified in oxidation state to obtain a bufotoxin lactone compound shown in formula I.
式中，R1、R2和R4各自独立地为-H或-OH；In the formula, R 1 , R 2 and R 4 are each independently -H or -OH;
R3为-CH3或-CHO； R3 is -CH3 or -CHO;
R5和R6各自独立地为-H、-OH或酮基； R5 and R6 are each independently -H, -OH or keto;
R7为-H、-OH或氧乙酰基。 R7 is -H, -OH or oxyacetyl.
在另一优选例中，R1、R2、R4、R5、R6、R7都为H，R3为甲基。In another preferred embodiment, R 1 , R 2 , R 4 , R 5 , R 6 , and R 7 are all H, and R 3 is methyl.
在另一优选例中，R1、R2、R4、R5、R6、R7都为H，R3为甲基时，式III所示的化合物具有式5所示的结构，所述制备方法包括：步骤3或步骤2～3或步骤1～2～3，
In another preferred embodiment, when R 1 , R 2 , R 4 , R 5 , R 6 , and R 7 are all H, and R 3 is methyl, the compound represented by Formula III has the structure represented by Formula 5, and the preparation method comprises: step 3 or steps 2 to 3 or steps 1 to 2 to 3,
其中：in:
步骤1：化合物2经甾体C14-α羟化酶生物催化氧化得到化合物3；Step 1: Compound 2 is biocatalytically oxidized by steroidal C14-α hydroxylase to obtain compound 3;
步骤2：在氢化催化剂作用下，化合物3在有机溶剂中经催化加氢得到化合物4；所述的氢化催化剂为二氧化铂、钯碳、氢氧化钯、兰尼镍或铑/三氧化铝；所述的有机溶剂为吡啶、4-甲基吡啶、四氢呋喃、甲醇或乙醇中的一种或两种以上的混合溶剂； Step 2: Compound 3 is catalytically hydrogenated in an organic solvent in the presence of a hydrogenation catalyst to obtain compound 4; the hydrogenation catalyst is platinum dioxide, palladium carbon, palladium hydroxide, Raney nickel or rhodium/aluminum trioxide; the organic solvent is one or a mixed solvent of two or more selected from pyridine, 4-methylpyridine, tetrahydrofuran, methanol or ethanol;
步骤3：化合物4在有机溶剂中通过还原剂还原得到化合物5；所述还原剂为三仲丁基硼氢化锂、三仲丁基硼氢化钾或二异丁基氢化铝；所述有机溶剂为四氢呋喃，乙醚，1,4-二氧六环，乙二醇二甲醚、苯或甲苯中的一种或两种以上的混合溶剂。Step 3: Compound 4 is reduced by a reducing agent in an organic solvent to obtain compound 5; the reducing agent is lithium tri-sec-butylborohydride, potassium tri-sec-butylborohydride or diisobutylaluminum hydride; the organic solvent is one or a mixed solvent of two or more selected from tetrahydrofuran, diethyl ether, 1,4-dioxane, ethylene glycol dimethyl ether, benzene or toluene.
在另一优选例中，步骤1中采用甾体C14-α羟化酶进行生物催化，所述生物催化方法包括但不限于纯酶催化、粗酶液催化和整细胞转化。In another preferred embodiment, steroid C14-α hydroxylase is used for biocatalysis in step 1, and the biocatalysis method includes but is not limited to pure enzyme catalysis, crude enzyme solution catalysis and whole cell conversion.
在另一优选例中，所述的C14-α羟化酶为复合酶，包括以下两种酶：(a)来源于Cochliobolus lunatus的细胞色素P450酶，其氨基酸序列如SEQ ID NO:2所示；和(b)来源于Cochliobolus lunatus的细胞色素P450还原酶CPR，其氨基酸序列如SEQ ID No:4所示。In another preferred embodiment, the C14-α hydroxylase is a complex enzyme, comprising the following two enzymes: (a) a cytochrome P450 enzyme derived from Cochliobolus lunatus, whose amino acid sequence is shown in SEQ ID NO:2; and (b) a cytochrome P450 reductase CPR derived from Cochliobolus lunatus, whose amino acid sequence is shown in SEQ ID No:4.
在另一优选例中，步骤2中的氢化催化剂为氢氧化钯、质量分数5％的钯碳或10％的钯碳，所述有机溶剂为吡啶或4-甲基吡啶。In another preferred embodiment, the hydrogenation catalyst in step 2 is palladium hydroxide, 5% by mass of palladium carbon or 10% by mass of palladium carbon, and the organic solvent is pyridine or 4-methylpyridine.
在另一优选例中，步骤3中的还原剂为三仲丁基硼氢化钾或二异丁基氢化铝，反应温度为-78～0℃，有机溶剂为四氢呋喃或甲苯。In another preferred embodiment, the reducing agent in step 3 is potassium tri-sec-butylborohydride or diisobutylaluminum hydride, the reaction temperature is -78 to 0° C., and the organic solvent is tetrahydrofuran or toluene.
在另一优选例中，R1、R2、R4、R5、R6、R7都为H，R3为甲基，式III所示的化合物具有式5所示的结构，式II所示的化合物具有式10所示的结构，所述制备方法包括：步骤7或步骤6～7或步骤5～6～7或步骤4～5～6～7，
In another preferred embodiment, R 1 , R 2 , R 4 , R 5 , R 6 , and R 7 are all H, R 3 is methyl, the compound represented by formula III has the structure represented by formula 5, and the compound represented by formula II has the structure represented by formula 10. The preparation method comprises: step 7 or steps 6 to 7 or steps 5 to 6 to 7 or steps 4 to 5 to 6 to 7,
其中：in:
步骤4：化合物5的有机溶液中加入水合肼和碱反应，粗品中再加入单质碘和碱反应得到化合物6；所述的有机溶剂为四氢呋喃、乙醇或甲醇的一种或两种以上的混合物；所述的碱为二异丙基乙基胺、三乙胺或4-二甲基氨基吡啶；Step 4: Add hydrazine hydrate and base to the organic solution of compound 5 for reaction, and then add elemental iodine and base to the crude product for reaction to obtain compound 6; the organic solvent is tetrahydrofuran, ethanol or methanol, or a mixture of two or more thereof; the base is diisopropylethylamine, triethylamine or 4-dimethylaminopyridine;
步骤5：化合物6和化合物7中加入过渡金属催化剂和碱反应得到化合物8；所述的过渡金属催化剂为[1,1'-双(二苯基膦基)二茂铁]二氯化钯、四(三苯基膦)钯、醋 酸钯或二氯双(三苯基膦)钯；所述碱为碳酸钾、磷酸钾、磷酸氢钾、叔丁醇钾或氢氧化钾等；反应溶剂为四氢呋喃、甲醇、水、N,N-二甲基甲酰胺的一种或两种以上的混合溶剂；Step 5: Add a transition metal catalyst and a base to compound 6 and compound 7 to react to obtain compound 8; the transition metal catalyst is [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride, tetrakis(triphenylphosphine)palladium, acetone Acid palladium or dichlorobis(triphenylphosphine)palladium; the base is potassium carbonate, potassium phosphate, potassium hydrogen phosphate, potassium tert-butoxide or potassium hydroxide; the reaction solvent is tetrahydrofuran, methanol, water, N,N-dimethylformamide, one or a mixed solvent of two or more thereof;
步骤6：化合物8的有机溶液中加入氢化均相催化剂，经催化加氢反应得到化合物9；所述氢化均相催化剂为三苯基膦氯化铑、六氟磷酸(三环已基膦)(1,5-环辛二烯)(吡啶)合铱或(1,5-环辛二烯)氯铑(I)二聚体；采用的有机溶剂为二氯甲烷，苯或甲苯；Step 6: adding a hydrogenation homogeneous catalyst to the organic solution of compound 8, and obtaining compound 9 through catalytic hydrogenation reaction; the hydrogenation homogeneous catalyst is triphenylphosphine rhodium chloride, hexafluorophosphate (tricyclohexylphosphine) (1,5-cyclooctadiene) (pyridine) iridium or (1,5-cyclooctadiene) rhodium chloride (I) dimer; the organic solvent used is dichloromethane, benzene or toluene;
步骤7：化合物9的有机溶液中加入酸得到化合物10；所述的酸为对甲基苯磺酸、吡啶对甲苯磺酸盐(PPTS)、苯磺酸、樟脑磺酸、高氯酸、高碘酸、质量分数2％～98％硫酸或质量分数2～36.5％盐酸；采用的有机溶剂为1,4-二氧六环、甲醇、四氢呋喃和水的一种或两种以上的混合溶剂。Step 7: Add an acid to the organic solution of compound 9 to obtain compound 10; the acid is p-toluenesulfonic acid, pyridine toluenesulfonate (PPTS), benzenesulfonic acid, camphorsulfonic acid, perchloric acid, periodic acid, 2% to 98% sulfuric acid or 2% to 36.5% hydrochloric acid; the organic solvent used is 1,4-dioxane, methanol, tetrahydrofuran and water, one or a mixed solvent of two or more.
在另一优选例中，步骤4中所述的有机溶剂为乙醇或四氢呋喃的一种或两种的混合溶剂；所述的碱为二异丙基乙基胺或三乙胺。In another preferred embodiment, the organic solvent in step 4 is ethanol or tetrahydrofuran, or a mixed solvent of the two; and the base is diisopropylethylamine or triethylamine.
在另一优选例中，步骤5中所述的过渡金属催化剂为[1,1'-双(二苯基膦基)二茂铁]二氯化钯或四三苯基膦钯；所述碱为磷酸钾或碳酸钾；所述溶剂为N,N-二甲基甲酰胺或四氢呋喃。In another preferred embodiment, the transition metal catalyst in step 5 is [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride or tetrakistriphenylphosphine palladium; the base is potassium phosphate or potassium carbonate; and the solvent is N,N-dimethylformamide or tetrahydrofuran.
在另一优选例中，步骤6中所述氢化均相催化剂为六氟磷酸(三环已基膦)(1,5-环辛二烯)(吡啶)合铱或三苯基膦氯化铑，所述有机溶剂为二氯甲烷或甲苯。In another preferred embodiment, the hydrogenation homogeneous catalyst in step 6 is (tricyclohexylphosphine) (1,5-cyclooctadiene) (pyridine) iridium hexafluorophosphate or triphenylphosphine rhodium chloride, and the organic solvent is dichloromethane or toluene.
在另一优选例中，步骤7中所述的酸为浓度为3M的稀盐酸。In another preferred embodiment, the acid in step 7 is dilute hydrochloric acid with a concentration of 3M.
在另一优选例中，R1、R2、R4、R5、R6、R7都为H，R3为甲基，式I所示的化合物具有式1所示的结构，式II所示的化合物具有式10所示的结构，所述制备方法包括：步骤8，
In another preferred embodiment, R 1 , R 2 , R 4 , R 5 , R 6 , and R 7 are all H, R 3 is methyl, the compound shown in Formula I has the structure shown in Formula 1, and the compound shown in Formula II has the structure shown in Formula 10. The preparation method comprises: step 8,
其中，in,
步骤8：化合物10的有机溶液中加入卤化试剂和高氯酸水溶液反应，粗品不经纯化直接溶于吡啶中，在50～70℃之间加热反应0.5～2.0小时得到目标化合物1；Step 8: Add a halogenating agent and an aqueous perchloric acid solution to the organic solution of compound 10 for reaction. The crude product is directly dissolved in pyridine without purification, and heated at 50-70° C. for reaction for 0.5-2.0 hours to obtain the target compound 1;
上述有机溶剂为1,4-二氧六环、四氢呋喃、乙醚、丙酮或水的一种或两种以上的混合溶剂；所述卤化试剂为N-溴代琥珀酰亚胺(NBS)，N-碘代琥珀酰亚胺(NIS)或N-氯代琥珀酰亚胺(NCS)；所述高氯酸水溶液的质量分数为1％～70％。The organic solvent is one or a mixed solvent of two or more of 1,4-dioxane, tetrahydrofuran, ether, acetone or water; the halogenating agent is N-bromosuccinimide (NBS), N-iodosuccinimide (NIS) or N-chlorosuccinimide (NCS); and the mass fraction of the perchloric acid aqueous solution is 1% to 70%.
在另一优选例中，步骤8中所述的有机溶剂为1,4-二氧六环与水或丙酮与水的混合溶剂，其体积比例为12:1～5:1；所述卤化试剂为N-溴代琥珀酰亚胺(NBS)或N-碘代琥珀酰亚胺(NIS)。 In another preferred embodiment, the organic solvent in step 8 is a mixed solvent of 1,4-dioxane and water or acetone and water, and the volume ratio thereof is 12:1 to 5:1; and the halogenating agent is N-bromosuccinimide (NBS) or N-iodosuccinimide (NIS).
本发明的第二方面，提供一种蟾毒基内酯类化合物中间体8，具有下式：
The second aspect of the present invention provides a bufotoxin lactone compound intermediate 8 having the following formula:
本发明的第三方面，提供一种蟾毒基内酯类化合物中间体9，具有下式：
The third aspect of the present invention provides a bufotoxin lactone compound intermediate 9 having the following formula:
本发明从廉价易得的雄烯二酮4-AD出发，经过生物酶氧化得到14-α-羟基-雄甾烷-17-酮化合物，随后在没有任何保护基的情况下，利用化学转化完成E环吡喃酮的上载，得到关键中间体，再由此出发实现蟾毒基内酯类化合物的合成。利用此方法合成脂蟾毒配基仅需八步操作，路线简短，操作简单，反应量级大，可用于大规模制备该类天然产物，具有工业化生产的应用前景The present invention starts with cheap and readily available androstenedione 4-AD, obtains 14-α-hydroxy-androstan-17-one compound through biological enzyme oxidation, and then completes the loading of E-ring pyrone by chemical transformation without any protecting group to obtain key intermediates, and then realizes the synthesis of toad venom lactone compounds from this. The method only requires eight steps to synthesize resibufogenin, has a short route, simple operation, and a large reaction scale, and can be used for large-scale preparation of such natural products, with application prospects for industrial production.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。说明书中所揭示的各个特征，可以被任何提供相同、均等或相似目的的替代性特征取代。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equal or similar purpose. Due to space limitations, they will not be described one by one here.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为HPLC谱图。Figure 1 is a HPLC spectrum.
具体实施方式Detailed ways
本申请的发明人经过广泛而深入的研究，提供一种蟾酥中活性成分脂蟾毒配基的制备方法，以价廉易得的甾体化合物雄烯二酮(4-AD)为原料，在不引入保护基的 情况下，通过生物-化学接力策略，最短历经八步转化即可合成天然蟾蜍内酯类化合物，部分中间体可以克级规模制备。本方法可以促进该类化合物在医药领域的广泛应用，缓解蟾酥中活性物质药理研究的物料稀缺压力。The inventors of the present application have conducted extensive and in-depth research and have provided a method for preparing resifobafugin, an active ingredient in toad venom, using the inexpensive and readily available steroid compound androstenedione (4-AD) as a raw material without introducing a protective group. Under these circumstances, through the biochemical relay strategy, natural toad lactone compounds can be synthesized in as little as eight steps, and some intermediates can be prepared on a gram scale. This method can promote the widespread application of such compounds in the medical field and alleviate the material scarcity pressure of pharmacological research on active substances in toad venom.
制备方法Preparation
本发明提供了一种甾体母核3β羟基、AB环十氢萘环顺式构型、C14-α构型羟基的甾体化合物III和3-β-羟基-14,15-烯-17-吡喃酮蟾毒基类内酯高级中间体II，所述两种前体具有以下结构式所示的结构，用于作为化学半合成蟾蜍内酯类化合物的中间体。
The invention provides a steroid compound III with a steroidal mother nucleus 3β-hydroxyl group, an AB ring decahydronaphthalene ring cis-configuration, and a C14-α-configuration hydroxyl group, and a 3-β-hydroxy-14,15-ene-17-pyrone toad venom-based lactone advanced intermediate II. The two precursors have structures shown in the following structural formulas and are used as intermediates for chemical semi-synthesis of toad lactone compounds.
式中，R1、R2和R4为-H或-OH；R3为-CH3或-CHO；R5和R6为-OH或酮基；R7为-OH或氧乙酰基。In the formula, R1, R2 and R4 are -H or -OH; R3 is -CH 3 or -CHO; R5 and R6 are -OH or keto; R7 is -OH or oxyacetyl.
在另一优选例中，所述甾体化合物R1、R2、R4-R7基团都为H，R3为甲基。In another preferred embodiment, the R1, R2, R4-R7 groups of the steroid compound are all H, and R3 is methyl.
本发明提供了一种生物催化大量获得蟾蜍内酯化合物化学合成中间体14α-羟基-雄甾烷-17-酮的方法，以雄烯二酮(4-AD)为原料，采用C14-α羟化酶生物催化完成C14-α构型羟基化。The invention provides a method for obtaining a large amount of 14α-hydroxy-androstane-17-one, a chemical synthesis intermediate of a toad lactone compound, by biocatalysis. Androstenedione (4-AD) is used as a raw material, and C14-α hydroxylase is used for biocatalysis to complete C14-α configuration hydroxylation.
所述生物催化方法，包括但不仅限于纯酶催化、粗酶液催化和整细胞转化。The biocatalytic method includes but is not limited to pure enzyme catalysis, crude enzyme solution catalysis and whole cell transformation.
本发明提供了一种将甾体母核C14位羟基化成α构型羟基的细胞色素P450酶及相关P450还原酶(Cytochrome P450 reductase，CPR)，所述酶的氨基酸序列分别如SEQ ID NO:2、SEQ ID NO:4所示。The present invention provides a cytochrome P450 enzyme and a related P450 reductase (Cytochrome P450 reductase, CPR) which can hydroxylate the C14 position of a steroid nucleus into an α-configuration hydroxyl group, and the amino acid sequences of the enzymes are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.
本发明对甾体母核C14位α羟基化酶及相关的氧化还原酶(CPR)的DNA序列进行毕赤酵母密码子偏爱序列优化，适合毕赤酵母高效表达，密码子优化后的DNA序列分别如SEQ ID NO:1、SEQ ID NO:3所示。The present invention optimizes the Pichia pastoris codon preference sequence of the DNA sequence of steroid nucleus C14 α-hydroxylase and related oxidoreductase (CPR), which is suitable for efficient expression in Pichia pastoris. The DNA sequences after codon optimization are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively.
本发明中，获得的14α-羟基-雄甾烷-17-酮可以通过以下步骤化学合成式1所示的蟾毒基类内酯高级中间体：
In the present invention, the obtained 14α-hydroxy-androstane-17-one can be chemically synthesized into a bufotoxin-based lactone advanced intermediate shown in Formula 1 by the following steps:
本发明中，获得的蟾毒基类内酯高级中间体可以通过以下步骤化学合成式1所示的蟾毒基类内酯：
In the present invention, the obtained bufotoxin-based lactone advanced intermediate can be chemically synthesized into the bufotoxin-based lactone shown in Formula 1 by the following steps:
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.
序列：
sequence:
SEQ ID NO:1
SEQ ID NO:1
SEQ ID NO:2

SEQ ID NO:2

SEQ ID NO:3
SEQ ID NO:3
SEQ ID NO:4

SEQ ID NO:4

实施例1化合物3β,14α-双羟基-5β-雄甾烷-17-酮的化学合成方法Example 1 Chemical Synthesis of Compound 3β,14α-Dihydroxy-5β-androstane-17-one
步骤1：生物合成部分Step 1: Biosynthesis
构建酵母蛋白表达载体pPICZA‐Duet:以pPICZA为模板，以GACCTTCGTTTGTGCGGCGGCCGCGATCTAACATCCAAAGACGAAAG、TTAACACTAGTCATATGGGATCCATGGTGATGGTGATGATGACCCATGG为引物克隆出DNA片段Fragment‐1； 以ATATGACTAGTGTTAACCTGCAGTGAGTTTTAGCCTTAGACATGAC、GCTATGGTGTGTGGGGGGTCTCACTTAATCTTCTGTAC为引物克隆出DNA片段Fragment‐2，用BamH I酶切pPICZA，最后用诺唯赞公司的重组酶重组构建质粒pPICZA‐Du et。Construction of yeast protein expression vector pPICZA-Duet: Using pPICZA as template and GACCTTCGTTTGTGCGGCGGCCGCGATCTAACATCCAAAGACGAAAG and TTAACACTAGTCATATGGGATCCATGGTGATGGTGATGATGACCCATGG as primers, DNA fragment Fragment-1 was cloned; DNA fragment Fragment-2 was cloned using primers ATATGACTAGTGTTAACCTGCAGTGAGTTTTAGCCTTAGACATGAC and GCTATGGTGTGTGGGGGGTCTCACTTAATCTTCTGTAC, pPICZA was digested with BamH I, and finally the recombinase from Novazonics was used to reconstruct the plasmid pPICZA-Du et.
构建表达甾体C14α-羟化酶的毕赤酵母工程菌：合成密码子优化后的来源于Cochliobolus lunatus的细胞色素P450(SEQ ID NO:1)和细胞色素P450还原酶(cytochrome P450reductase,CPR)(SEQ ID NO:3)基因。先用BamHI和SpeI酶切酵母表达载体pPICZA‐Duet，构建重组载体pPICZA‐Duet‐CPR，再用KpnI和XhOI酶切重组载体pPICZA‐Duet‐CPR，构建酵母表达载体pPICZA‐Duet‐P450‐CPR，将酵母表达载体pPICZA‐Duet‐P450‐CPR按照常规电转方法导入毕赤酵母KM71H，获得表达相应酶的工程菌。Construction of Pichia pastoris engineered bacteria expressing steroid C14α-hydroxylase: Synthesize codon-optimized cytochrome P450 (SEQ ID NO: 1) and cytochrome P450 reductase (CPR) (SEQ ID NO: 3) genes from Cochliobolus lunatus. First, the yeast expression vector pPICZA-Duet was cut with BamHI and SpeI to construct the recombinant vector pPICZA-Duet-CPR. Then, the recombinant vector pPICZA-Duet-CPR was cut with KpnI and XhOI to construct the yeast expression vector pPICZA-Duet-P450-CPR. The yeast expression vector pPICZA-Duet-P450-CPR was introduced into Pichia pastoris KM71H by conventional electroporation to obtain engineered bacteria expressing the corresponding enzymes.
酵母工程菌阳性克隆的筛选及细胞转化条件的优化：挑5～10个酵母工程菌单克隆于5mL YPD液体培养基中(含100μg/mL的Zeocin)，30℃，260rpm，培养24小时。将0.5mL培养物接入5mL MGYH甘油培养基，30℃，260rpm，培养24h。室温，2500g离心10min，在超净台中更换至5mL MMH甲醇培养基(含甲醇0.5％)，加入底物4‐AD，30℃，260rpm，每24小时加0.5％甲醇，总共摇了3天，取200μL菌液用等体积乙酸乙酯萃取两次，氮吹，用200μL甲醇复溶，用Shimadzu 8040LCMS检测，C14-α羟化酶的酵母工程菌能细胞催化4‐AD生产相应的产物14-α‐羟基‐4-雄烯‐3,17‐二酮。对C14-α羟化酶酵母工程菌细胞转化4-雄烯‐3,17‐二酮的条件进行优化：包括种子培养液转接量、底物4-AD的浓度和转化时间的优化，确定最优转化条件为：8％转接量、1.5g/L 4-AD，转化72h，每发酵1L C14-α羟化酶酵母工程菌，投入1.5g 4-AD可获得不低于700mg C14-α羟基化产物14-α‐羟基‐4雄烯‐3,17‐二酮，结果如图1所示。Screening of positive clones of yeast engineering bacteria and optimization of cell transformation conditions: 5 to 10 yeast engineering bacteria single clones were selected and placed in 5 mL YPD liquid medium (containing 100 μg/mL Zeocin), cultured at 30°C, 260 rpm for 24 hours. 0.5 mL of culture was inoculated into 5 mL MGYH glycerol medium, cultured at 30°C, 260 rpm for 24 hours. Centrifuged at room temperature, 2500 g for 10 min, replaced with 5 mL MMH methanol medium (containing 0.5% methanol) in a clean bench, added substrate 4-AD, 30°C, 260 rpm, added 0.5% methanol every 24 hours, and shaken for a total of 3 days. 200 μL of bacterial solution was extracted twice with an equal volume of ethyl acetate, nitrogen blown, and re-dissolved with 200 μL methanol. The yeast engineering bacteria with C14-α hydroxylase were able to catalyze 4-AD to produce the corresponding product 14-α-hydroxy-4-androstene-3,17-dione. The conditions for the transformation of C14-α hydroxylase yeast cells into 4-androstene-3,17-dione were optimized, including the transfer amount of seed culture medium, the concentration of substrate 4-AD and the optimization of conversion time. The optimal conversion conditions were determined as follows: 8% transfer amount, 1.5 g/L 4-AD, and conversion time of 72 h. For every 1 L of C14-α hydroxylase yeast fermentation, 1.5 g 4-AD was added to obtain no less than 700 mg of C14-α hydroxylation product 14-α-hydroxy-4-androstene-3,17-dione. The results are shown in Figure 1.
1H NMR(600MHz,CDCl3)δ＝5.75(s,1H),2.51–2.44(m,2H),2.44–2.34(m,4H),2.08–2.02(m,1H),2.01–1.89(m,3H),1.87–1.82(m,1H),1.81–1.73(m,2H),1.68–1.63(m,1H),1.63–1.58(m,1H),1.56–1.49(m,2H),1.47–1.37(m,2H),1.22(s,3H),1.05(s,3H)ppm。 1 H NMR (600 MHz, CDCl 3 ) δ＝5.75(s,1H),2.51–2.44(m,2H),2.44–2.34(m,4H),2.08–2.02(m,1H),2.01–1.89(m,3H),1.87–1.82(m,1H),1.81–1.73(m,2H),1.68–1.63(m,1H),1.63–1.58(m,1H),1.56–1.49(m,2H),1.47–1.37(m,2H),1.22(s,3H),1.05(s,3H)ppm.
步骤2：合成化合物4Step 2: Synthesis of compound 4
化合物3(10.30g,34.06mmol)溶于4-甲基吡啶(114mL)，加入10％Pd/C(2.06g)。氢气氛围下反应，原料转化完全后，加入硅藻土抽滤。滤液加入1.2M稀盐酸水溶液洗涤，乙酸乙酯萃取。饱和氯化钠水溶液洗涤，无水硫酸钠干燥。浓缩后粗品经柱层析分离纯化得到化合物4白色固体(8.50g,82％yield)。Compound 3 (10.30 g, 34.06 mmol) was dissolved in 4-methylpyridine (114 mL) and 10% Pd/C (2.06 g) was added. The reaction was carried out under a hydrogen atmosphere. After the raw material was completely converted, diatomaceous earth was added for suction filtration. The filtrate was washed with 1.2 M dilute hydrochloric acid aqueous solution and extracted with ethyl acetate. The filtrate was washed with saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate. After concentration, the crude product was separated and purified by column chromatography to obtain compound 4 as a white solid (8.50 g, 82% yield).
1H NMR(600MHz,CDCl3):δ＝2.69(t,J＝13.8Hz,1H),2.46-2.33(m,3H),2.21–2.09(m,2H),2.07–2.01(m,2H),1.98–1.81(m,6H),1.61–1.54(m,2H),1.50–1.33(m,5H),1.05(s,3H),1.01(s,3H)ppm。 1 H NMR (600 MHz, CDCl 3 ): δ=2.69 (t, J=13.8 Hz, 1H), 2.46-2.33 (m, 3H), 2.21-2.09 (m, 2H), 2.07-2.01 (m, 2H), 1.98-1.81 (m, 6H), 1.61-1.54 (m, 2H), 1.50-1.33 (m, 5H), 1.05 (s, 3H), 1.01 (s, 3H) ppm.
步骤3：合成化合物5Step 3: Synthesis of compound 5
氮气保护下将化合物4(10.00g,32.85mmol)溶于四氢呋喃(100mL)，-78℃ 浴下加入三仲丁基硼氢化钾溶液(43mL,1.0M in THF,43.00mmol,1.3eq.)。冰水浴下反应，原料转化完全后滴加饱和氯化铵溶液淬灭反应。反应液加入二氯甲烷萃取，合并有机相后饱和碳酸氢钠水溶液洗涤，饱和食盐水洗涤，无水硫酸钠干燥。浓缩后粗品经柱层析分离纯化得到化合物5白色固体(8.19g,81％yield)。Under nitrogen protection, compound 4 (10.00 g, 32.85 mmol) was dissolved in tetrahydrofuran (100 mL) and heated at -78 °C. Add potassium tri-sec-butylborohydride solution (43mL, 1.0M in THF, 43.00mmol, 1.3eq.) under a bath. React under an ice-water bath. After the conversion of the raw materials is complete, add saturated ammonium chloride solution to quench the reaction. Add dichloromethane to the reaction solution for extraction. After the organic phases are combined, wash with saturated sodium bicarbonate aqueous solution, wash with saturated brine, and dry over anhydrous sodium sulfate. After concentration, the crude product is separated and purified by column chromatography to obtain compound 5 as a white solid (8.19g, 81% yield).
1H NMR(600MHz,CDCl3):δ＝4.15–4.11(m,1H),2.45–2.32(m,2H),2.01–1.93(m,2H),1.93–1.86(m,2H),1.84–1.72(m,3H),1.64–1.46(m,8H),1.43–1.34(m,2H),1.30–1.23(m,2H),1.00(s,3H),0.99(s,3H)ppm。 1 H NMR (600 MHz, CDCl 3 ): δ＝4.15–4.11 (m, 1H), 2.45–2.32 (m, 2H), 2.01–1.93 (m, 2H), 1.93–1.86 (m, 2H), 1.84–1.72 (m, 3H), 1.64–1.46 (m, 8H), 1.43–1.34 (m, 2H), 1.30–1.23 (m, 2H), 1.00 (s, 3H), 0.99 (s, 3H) ppm.
实施例2化合物14,15-双键-17-吡喃酮蟾毒基类内酯的化学合成方法Example 2 Chemical Synthesis Method of Compound 14,15-Double Bond-17-Pyrone Toad Toxin-Based Lactone
步骤4：合成化合物6Step 4: Synthesis of compound 6
氮气保护下将化合物5(3.17g,10.34mmol)溶于乙醇(206mL)，加入三乙胺(21g,29mL,20.0eq)和水合肼(13g,12.6mL,20.0eq)。加热至50℃反应过夜，原料转化完全。减压浓缩除去反应溶剂后，将粗品腙化合物溶于四氢呋喃(206mL)，加入三乙胺(21g,21mL,20.0eq)。冰水浴下滴加碘(7.88g,3.0eq)的四氢呋喃溶液。反应结束后硫代硫酸钠饱和水溶液淬灭，乙酸乙酯萃取后饱和食盐水洗涤，无水硫酸钠干燥。浓缩后粗品直接投下一步。Under nitrogen protection, compound 5 (3.17 g, 10.34 mmol) was dissolved in ethanol (206 mL), and triethylamine (21 g, 29 mL, 20.0 eq) and hydrazine hydrate (13 g, 12.6 mL, 20.0 eq) were added. The reaction was heated to 50 ° C overnight, and the raw material was completely converted. After the reaction solvent was removed by concentration under reduced pressure, the crude hydrazone compound was dissolved in tetrahydrofuran (206 mL), and triethylamine (21 g, 21 mL, 20.0 eq) was added. A tetrahydrofuran solution of iodine (7.88 g, 3.0 eq) was added dropwise under an ice-water bath. After the reaction was completed, saturated aqueous sodium thiosulfate was used to quench, ethyl acetate was extracted, and then washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the crude product was directly used for the next step.
步骤5：合成化合物8Step 5: Synthesis of compound 8
氮气保护下将烯基碘6、硼酸酯化合物7(2.76g,12.41mmol,1.2eq)、Pd(dppf)Cl2(0.750g,1.034mmol,10mol％)和磷酸钾(6.58g,31.02,3.0eq)溶于N,N-二甲基甲酰胺(23mL)，加热至60℃。反应结束后加水稀释后，乙酸乙酯萃取，饱和食盐水洗涤，无水硫酸钠干燥。浓缩后粗品经柱层析分离纯化得到化合物8白色固体(2.28g,57％yield)。Under nitrogen protection, alkenyl iodide 6, borate compound 7 (2.76 g, 12.41 mmol, 1.2 eq), Pd(dppf)Cl 2 (0.750 g, 1.034 mmol, 10 mol%) and potassium phosphate (6.58 g, 31.02, 3.0 eq) were dissolved in N,N-dimethylformamide (23 mL) and heated to 60°C. After the reaction was completed, the mixture was diluted with water, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, the crude product was separated and purified by column chromatography to obtain compound 8 as a white solid (2.28 g, 57% yield).
1H NMR(600MHz,CDCl3):δ7.54–7.53(m,1H),7.46–7.42(m,1H),6.36–6.32(m,1H),5.89–5.86(m,1H),4.15–4.11(m,1H),2.45–2.39(m,1H),2.34–2.27(m,1H),2.20–2.14(m,2H),2.11–2.04(m,2H),1.99–1.89(m,2H),1.79–1.73(m,1H),1.67–1.53(m,2H),1.52–1.46(m,4H),1.26–1.17(m,3H),1.05(s,3H),1.01(s,3H)ppm。 1 H NMR (600 MHz, CDCl 3 ): δ7.54–7.53 (m, 1H), 7.46–7.42 (m, 1H), 6.36–6.32 (m, 1H), 5.89–5.86 (m, 1H), 4.15–4.11 (m, 1H), 2.45–2.39 (m, 1H), 2.34–2.27 (m, 1H), 2.20–2.14 (m, 2H), 2.11–2.04 (m, 2H), 1.99–1.89 (m, 2H), 1.79–1.73 (m, 1H), 1.67–1.53 (m, 2H), 1.52–1.46 (m, 4H), 1.26–1.17 (m, 3H), 1.05 (s, 3H), 1.01 (s, 3H) ppm.
步骤6：合成化合物9Step 6: Synthesis of compound 9
将化合物8(2.22g,5.77mmol)溶于二氯甲烷(190mL)，加入六氟磷酸(三环已基膦)(1,5-环辛二烯)(吡啶)合铱(0.694g,0.865mml,15mol％)。抽换氢气，室温下反应。硅藻土抽滤，二氯甲烷洗涤滤饼。浓缩滤液后粗品经柱层析分离纯化得到化合物9白色固体(2.05g,92％yield)。Compound 8 (2.22 g, 5.77 mmol) was dissolved in dichloromethane (190 mL), and hexafluorophosphate (tricyclohexylphosphine) (1,5-cyclooctadiene) (pyridine) iridium (0.694 g, 0.865 mml, 15 mol%) was added. The hydrogen was replaced and the reaction was carried out at room temperature. The mixture was filtered through diatomaceous earth and the filter cake was washed with dichloromethane. After the filtrate was concentrated, the crude product was separated and purified by column chromatography to obtain compound 9 as a white solid (2.05 g, 92% yield).
1H NMR(600MHz,CDCl3):δ＝7.30–7.26(m,2H),6.31–6.27(m,1H),4.13(s,1H),3.13(t,J＝9.3Hz,1H),2.05–1.89(m,4H),1.83–1.68(m,5H),1.67–1.57(m,2H),1.53–1.40(m,5H),1.35(dd,J＝13.3,2.6Hz,2H),1.30–1.21(m,3H),0.98(s,3H),0.64(s,3H)ppm。 1 H NMR (600 MHz, CDCl 3 ): δ＝7.30–7.26 (m, 2H), 6.31–6.27 (m, 1H), 4.13 (s, 1H), 3.13 (t, J＝9.3 Hz, 1H), 2.05–1.89 (m, 4H), 1.83–1.68 (m, 5H), 1.67–1.57 (m, 2H), 1.53–1.40 (m, 5H), 1.35 (dd, J＝13.3, 2.6 Hz, 2H), 1.30–1.21 (m, 3H), 0.98 (s, 3H), 0.64 (s, 3H) ppm.
步骤7：合成化合物10Step 7: Synthesis of compound 10
化合物9(600mg,1.55mmol)溶于二氧六环(155mL)，加入盐酸(3.0mL,3.0 M，水溶液)，室温搅拌。原料转化完全后加水淬灭，乙酸乙酯萃取。饱和氯化钠洗涤，无水硫酸钠干燥。浓缩后粗品经柱层析分离纯化得到化合物10白色固体(418mg,73％yield)。Compound 9 (600 mg, 1.55 mmol) was dissolved in dioxane (155 mL), and hydrochloric acid (3.0 mL, 3.0 M, aqueous solution), stirred at room temperature. After the conversion of the raw material was complete, water was added to quench, and extracted with ethyl acetate. Washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. After concentration, the crude product was separated and purified by column chromatography to obtain compound 10 as a white solid (418 mg, 73% yield).
1H NMR(600MHz,Chloroform-d)δ＝7.32(dd,J＝9.6,2.5Hz,1H),7.30(s,1H),6.30(d,J＝9.4Hz,1H),5.25–5.22(m,1H),4.12–4.09(m,1H),2.73(t,J＝9.4Hz,1H),2.43–2.36(m,2H),2.05(d,J＝7.7Hz,1H),2.00–1.91(m,2H),1.85–1.80(m,1H),1.79–1.73(m,1H),1.70–1.64(m,1H),1.59–1.23(m,12H),0.97(s,3H),0.71(s,3H)ppm。 1 H NMR (600 MHz, Chloroform-d) δ＝7.32(dd, J＝9.6,2.5 Hz, 1H),7.30(s, 1H),6.30(d, J＝9.4 Hz, 1H),5.25–5.22(m, 1H),4.12–4.09(m, 1H),2.73(t, J＝9.4 Hz, 1H),2.43–2.36(m, 2H),2.05(d, J＝7.7 Hz, 1H),2.00–1.91(m, 2H),1.85–1.80(m, 1H),1.79–1.73(m, 1H),1.70–1.64(m, 1H),1.59–1.23(m, 12H),0.97(s, 3H),0.71(s, 3H)ppm.
实施例3目标化合物脂蟾毒配基的化学合成方法Example 3 Chemical Synthesis Method of Target Compound Resifobafugin
将化合物10(360mg,0.977mmol)溶于丙酮和水的混合溶液(98mL，丙酮/水＝9:1v/v)中，加入N-溴代琥珀酰亚胺(127mg,1.08mmol,1.1eq.)和质量分数1％高氯酸水溶液(0.98mL)。室温搅拌1小时。缓慢滴加饱和碳酸氢钠水溶液和饱和硫代硫酸钠水溶液淬灭，乙酸乙酯萃取，饱和食盐水洗涤，无水硫酸钠干燥。浓缩后粗品不经纯化，直接溶于吡啶(9.8mL)，在60℃下反应2小时。浓缩后粗品经柱层析分离纯化(石油醚/EtOAc＝2:1)得到化合物1白色固体(190mg,收率51％)。核磁氢谱与标准谱图对比，结果一致。Compound 10 (360 mg, 0.977 mmol) was dissolved in a mixed solution of acetone and water (98 mL, acetone/water = 9:1 v/v), and N-bromosuccinimide (127 mg, 1.08 mmol, 1.1 eq.) and a 1% perchloric acid aqueous solution (0.98 mL) were added. Stir at room temperature for 1 hour. Saturated sodium bicarbonate aqueous solution and saturated sodium thiosulfate aqueous solution were slowly added dropwise to quench, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, the crude product was directly dissolved in pyridine (9.8 mL) without purification and reacted at 60 ° C for 2 hours. After concentration, the crude product was separated and purified by column chromatography (petroleum ether/EtOAc = 2:1) to obtain compound 1 as a white solid (190 mg, yield 51%). The H NMR spectrum was compared with the standard spectrum, and the results were consistent.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。 All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims (10)

  1. 一种式I所示的蟾毒基内酯类化合物的制备方法，特征在于，所述制备方法包括以下步骤：
    A method for preparing a bufotoxin lactone compound represented by formula I, characterized in that the preparation method comprises the following steps:
    由雄烯二酮4-AD经过生物酶氧化得到式III化合物；The compound of formula III is obtained by oxidizing androstenedione 4-AD through biological enzymes;
    式III化合物通过化学半合成完成六元二烯内酯环的上载和转化得到蟾毒基内酯类关键中间体II；The compound of formula III is subjected to chemical semi-synthesis to complete the loading and transformation of the six-membered diene lactone ring to obtain the key intermediate II of toad venom lactone;
    关键中间体II经过氧化态修饰获得式I所示的蟾毒基内酯类化合物，The key intermediate II is modified in oxidation state to obtain a bufotoxin lactone compound shown in formula I.
    式中，R1、R2和R4各自独立地为-H或-OH；In the formula, R 1 , R 2 and R 4 are each independently -H or -OH;
    R3为-CH3或-CHO； R3 is -CH3 or -CHO;
    R5和R6各自独立地为-H、-OH或酮基； R5 and R6 are each independently -H, -OH or keto;
    R7为-H、-OH或氧乙酰基。 R7 is -H, -OH or oxyacetyl.
  2. 如权利要求1所述的制备方法，其特征在于，R1、R2、R4、R5、R6、R7都为H，R3为甲基。The preparation method according to claim 1, characterized in that R 1 , R 2 , R 4 , R 5 , R 6 , and R 7 are all H, and R 3 is methyl.
  3. 如权利要求1所述的制备方法，其特征在于，R1、R2、R4、R5、R6、R7都为H，R3为甲基时，式III所示的化合物具有式5所示的结构，所述制备方法包括：步骤3或步骤2～3或步骤1～2～3，
    The preparation method according to claim 1, characterized in that when R 1 , R 2 , R 4 , R 5 , R 6 , and R 7 are all H, and R 3 is methyl, the compound represented by formula III has the structure represented by formula 5, and the preparation method comprises: step 3 or steps 2 to 3 or steps 1 to 2 to 3,
    其中：in:
    步骤1：化合物2经甾体C14-α羟化酶生物催化氧化得到化合物3；Step 1: Compound 2 is biocatalytically oxidized by steroidal C14-α hydroxylase to obtain compound 3;
    步骤2：在氢化催化剂作用下，化合物3在有机溶剂中经催化加氢得到化合物4；所述的氢化催化剂为二氧化铂、钯碳、氢氧化钯、兰尼镍或铑/三氧化铝；所述的有机溶剂为吡啶、4-甲基吡啶、四氢呋喃、甲醇或乙醇中的一种或两种以上的混合溶剂；Step 2: Compound 3 is catalytically hydrogenated in an organic solvent in the presence of a hydrogenation catalyst to obtain compound 4; the hydrogenation catalyst is platinum dioxide, palladium carbon, palladium hydroxide, Raney nickel or rhodium/aluminum trioxide; the organic solvent is one or a mixed solvent of two or more selected from pyridine, 4-methylpyridine, tetrahydrofuran, methanol or ethanol;
    步骤3：化合物4在有机溶剂中通过还原剂还原得到化合物5；所述还原剂为三仲丁基硼氢化锂、三仲丁基硼氢化钾或二异丁基氢化铝；所述有机溶剂为四氢呋喃， 乙醚，1,4-二氧六环，乙二醇二甲醚、苯或甲苯中的一种或两种以上的混合溶剂。Step 3: Compound 4 is reduced by a reducing agent in an organic solvent to obtain compound 5; the reducing agent is lithium tri-sec-butylborohydride, potassium tri-sec-butylborohydride or diisobutylaluminum hydride; the organic solvent is tetrahydrofuran, One or a mixed solvent of two or more of diethyl ether, 1,4-dioxane, ethylene glycol dimethyl ether, benzene or toluene.
  4. 如权利要求1所述的制备方法，其特征在于，R1、R2、R4、R5、R6、R7都为H，R3为甲基，式III所示的化合物具有式5所示的结构，式II所示的化合物具有式10所示的结构，所述制备方法包括：步骤7或步骤6～7或步骤5～6～7或步骤4～5～6～7，
    The preparation method according to claim 1, characterized in that R 1 , R 2 , R 4 , R 5 , R 6 , and R 7 are all H, R 3 is methyl, the compound shown in formula III has the structure shown in formula 5, and the compound shown in formula II has the structure shown in formula 10, and the preparation method comprises: step 7 or steps 6 to 7 or steps 5 to 6 to 7 or steps 4 to 5 to 6 to 7,
    其中：in:
    步骤4：化合物5的有机溶液中加入水合肼和碱反应，粗品中再加入单质碘和碱反应得到化合物6；所述的有机溶剂为四氢呋喃、乙醇或甲醇的一种或两种以上的混合物；所述的碱为二异丙基乙基胺、三乙胺或4-二甲基氨基吡啶；Step 4: Add hydrazine hydrate and base to the organic solution of compound 5 for reaction, and then add elemental iodine and base to the crude product for reaction to obtain compound 6; the organic solvent is tetrahydrofuran, ethanol or methanol, or a mixture of two or more thereof; the base is diisopropylethylamine, triethylamine or 4-dimethylaminopyridine;
    步骤5：化合物6和化合物7中加入过渡金属催化剂和碱反应得到化合物8；所述的过渡金属催化剂为[1,1'-双(二苯基膦基)二茂铁]二氯化钯、四(三苯基膦)钯、醋酸钯或二氯双(三苯基膦)钯；所述碱为碳酸钾、磷酸钾、磷酸氢钾、叔丁醇钾或氢氧化钾等；反应溶剂为四氢呋喃、甲醇、水、N,N-二甲基甲酰胺的一种或两种以上的混合溶剂；Step 5: Add a transition metal catalyst and a base to compound 6 and compound 7 to react to obtain compound 8; the transition metal catalyst is [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride, tetrakis(triphenylphosphine)palladium, palladium acetate or dichlorobis(triphenylphosphine)palladium; the base is potassium carbonate, potassium phosphate, potassium hydrogen phosphate, potassium tert-butoxide or potassium hydroxide; the reaction solvent is tetrahydrofuran, methanol, water, N,N-dimethylformamide, one or a mixed solvent of two or more thereof;
    步骤6：化合物8的有机溶液中加入氢化均相催化剂，经催化加氢反应得到化合物9；所述氢化均相催化剂为三苯基膦氯化铑、六氟磷酸(三环已基膦)(1,5-环辛二烯)(吡啶)合铱或(1,5-环辛二烯)氯铑(I)二聚体；采用的有机溶剂为二氯甲烷，苯或甲苯；Step 6: adding a hydrogenation homogeneous catalyst to the organic solution of compound 8, and obtaining compound 9 through catalytic hydrogenation reaction; the hydrogenation homogeneous catalyst is triphenylphosphine rhodium chloride, hexafluorophosphate (tricyclohexylphosphine) (1,5-cyclooctadiene) (pyridine) iridium or (1,5-cyclooctadiene) rhodium chloride (I) dimer; the organic solvent used is dichloromethane, benzene or toluene;
    步骤7：化合物9的有机溶液中加入酸得到化合物10；所述的酸为对甲基苯磺酸、吡啶对甲苯磺酸盐(PPTS)、苯磺酸、樟脑磺酸、高氯酸、高碘酸、质量分数2％～98％硫酸或质量分数2～36.5％盐酸；采用的有机溶剂为1,4-二氧六环、甲醇、四氢呋喃和水的一种或两种以上的混合溶剂。Step 7: Add an acid to the organic solution of compound 9 to obtain compound 10; the acid is p-toluenesulfonic acid, pyridine toluenesulfonate (PPTS), benzenesulfonic acid, camphorsulfonic acid, perchloric acid, periodic acid, 2% to 98% sulfuric acid or 2% to 36.5% hydrochloric acid; the organic solvent used is 1,4-dioxane, methanol, tetrahydrofuran and water, one or a mixed solvent of two or more.
  5. 如权利要求1所述的制备方法，其特征在于，R1、R2、R4、R5、R6、R7都 为H，R3为甲基，式I所示的化合物具有式1所示的结构，式II所示的化合物具有式10所示的结构，所述制备方法包括：步骤8，
    The preparation method according to claim 1, characterized in that R 1 , R 2 , R 4 , R 5 , R 6 , and R 7 are is H, R 3 is methyl, the compound shown in formula I has the structure shown in formula 1, and the compound shown in formula II has the structure shown in formula 10. The preparation method comprises: step 8,
    其中，in,
    步骤8：化合物10的有机溶液中加入卤化试剂和高氯酸水溶液反应，粗品不经纯化直接溶于吡啶中，在50～70℃之间加热反应0.5～2.0小时得到目标化合物1；Step 8: Add a halogenating agent and an aqueous perchloric acid solution to the organic solution of compound 10 for reaction. The crude product is directly dissolved in pyridine without purification, and heated at 50-70° C. for reaction for 0.5-2.0 hours to obtain the target compound 1;
    上述有机溶剂为1,4-二氧六环、四氢呋喃、乙醚、丙酮或水的一种或两种以上的混合溶剂；所述卤化试剂为N-溴代琥珀酰亚胺(NBS)，N-碘代琥珀酰亚胺(NIS)或N-氯代琥珀酰亚胺(NCS)；所述高氯酸水溶液的质量分数为1％～70％。The organic solvent is one or a mixed solvent of two or more of 1,4-dioxane, tetrahydrofuran, ether, acetone or water; the halogenating agent is N-bromosuccinimide (NBS), N-iodosuccinimide (NIS) or N-chlorosuccinimide (NCS); and the mass fraction of the perchloric acid aqueous solution is 1% to 70%.
  6. 如权利要求3所述的制备方法，其特征在于，采用甾体C14-α羟化酶进行生物催化，所述生物催化方法包括但不限于纯酶催化、粗酶液催化和整细胞转化；优选的，所述的C14-α羟化酶为复合酶，包括以下两种酶：(a)来源于Cochliobolus lunatus的细胞色素P450酶，其氨基酸序列如SEQ ID NO:2所示；和(b)来源于Cochliobolus lunatus的细胞色素P450还原酶CPR，其氨基酸序列如SEQ ID No:4所示。The preparation method as described in claim 3 is characterized in that steroid C14-α hydroxylase is used for biocatalysis, and the biocatalysis method includes but is not limited to pure enzyme catalysis, crude enzyme solution catalysis and whole cell conversion; preferably, the C14-α hydroxylase is a complex enzyme, including the following two enzymes: (a) cytochrome P450 enzyme derived from Cochliobolus lunatus, whose amino acid sequence is shown in SEQ ID NO:2; and (b) cytochrome P450 reductase CPR derived from Cochliobolus lunatus, whose amino acid sequence is shown in SEQ ID No:4.
  7. 如权利要求3所述的制备方法，其特征在于，所述制备方法具有以下一个或多个特征：The preparation method according to claim 3, characterized in that the preparation method has one or more of the following characteristics:
    步骤2中的氢化催化剂为氢氧化钯、质量分数5％的钯碳或10％的钯碳，所述有机溶剂为吡啶或4-甲基吡啶；The hydrogenation catalyst in step 2 is palladium hydroxide, 5% palladium carbon or 10% palladium carbon by mass, and the organic solvent is pyridine or 4-methylpyridine;
    步骤3中的还原剂为三仲丁基硼氢化钾或二异丁基氢化铝，反应温度为-78～0℃，有机溶剂为四氢呋喃或甲苯。The reducing agent in step 3 is potassium tri-sec-butylborohydride or diisobutylaluminum hydride, the reaction temperature is -78 to 0° C., and the organic solvent is tetrahydrofuran or toluene.
  8. 如权利要求4所述的制备方法，其特征在于，所述制备方法具有以下一个或多个特征：The preparation method according to claim 4, characterized in that the preparation method has one or more of the following characteristics:
    步骤4中所述的有机溶剂为乙醇或四氢呋喃的一种或两种的混合溶剂；所述的碱为二异丙基乙基胺或三乙胺；The organic solvent in step 4 is ethanol or tetrahydrofuran or a mixed solvent of the two; the base is diisopropylethylamine or triethylamine;
    步骤5中所述的过渡金属催化剂为[1,1'-双(二苯基膦基)二茂铁]二氯化钯或四三苯基膦钯；所述碱为磷酸钾或碳酸钾；所述溶剂为N,N-二甲基甲酰胺或四氢呋喃；The transition metal catalyst in step 5 is [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride or tetrakistriphenylphosphine palladium; the base is potassium phosphate or potassium carbonate; the solvent is N,N-dimethylformamide or tetrahydrofuran;
    步骤6中所述氢化均相催化剂为六氟磷酸(三环已基膦)(1,5-环辛二烯)(吡啶)合铱或三苯基膦氯化铑，所述有机溶剂为二氯甲烷或甲苯；In step 6, the hydrogenation homogeneous catalyst is hexafluorophosphate (tricyclohexylphosphine) (1,5-cyclooctadiene) (pyridine) iridium or triphenylphosphine rhodium chloride, and the organic solvent is dichloromethane or toluene;
    步骤7中所述的酸为浓度为3M的稀盐酸。 The acid described in step 7 is dilute hydrochloric acid with a concentration of 3M.
  9. 如权利要求5所述的制备方法，其特征在于，所述制备方法具有以下一个或多个特征：The preparation method according to claim 5, characterized in that the preparation method has one or more of the following characteristics:
    步骤8中所述的有机溶剂为1,4-二氧六环与水或丙酮与水的混合溶剂，其体积比例为12:1～5:1；The organic solvent in step 8 is a mixed solvent of 1,4-dioxane and water or acetone and water, and the volume ratio thereof is 12:1 to 5:1;
    步骤8中所述卤化试剂为N-溴代琥珀酰亚胺(NBS)或N-碘代琥珀酰亚胺(NIS)。The halogenating agent in step 8 is N-bromosuccinimide (NBS) or N-iodosuccinimide (NIS).
  10. 一种蟾毒基内酯类化合物中间体，具有下式：
    A toad venom lactone compound intermediate having the following formula:
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