Preparation method and application of pleuromutilin and derivative thereof

Abstract

The invention discloses a preparation method and application of pleuromutilin and derivatives thereof. The preparation of C7-site substituted, C8-site axial substituted and C8-site exocyclic double bond substituted derivatives of pleuromutilin is realized. The pleuromutilin derivative shows activity which is obviously superior to that of a parent compound pleuromutilin in the aspect of inhibiting proliferation of human cancer cells (such as breast cancer MDA-MB-231 cells and liver cancer HepG2 cells) with high expression of TrxR. Wherein the C8-position axially substituted derivative has particularly outstanding advantages in the aspect of inhibiting TrxR enzyme activity, and compared with a parent compound pleuromutilin and a positive control drug Jinnofen, the TrxR inhibiting efficacy of the C8-position axially substituted derivative is improved to 9 times and 16.7 times respectively.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of grifolan and its derivatives, and particularly relates to a preparation method and application of grifolan and its derivatives. BACKGROUND

[0002] Thioredoxin reductase (TrxR) is a key intracellular selenium-containing homodimeric flavin enzyme, and its core function is to regulate redox balance. Abnormal activity of TrxR is closely related to cell proliferation, apoptosis inhibition and tumor development. Studies have shown that overexpression of TrxR system exists in lung cancer, liver cancer, pancreatic cancer, cervical cancer, malignant pleural mesothelioma, prostate cancer, lymphoma and leukemia, which makes TrxR an ideal intervention target in the field of anti-tumor drug research and development.

[0003] Since the 21st century, the research on inhibitors based on the structure of TrxR has developed rapidly, and can be divided into reversible and irreversible types according to the binding mode with the enzyme. In the reversible inhibitors, ethane selenol which can interact with the high nucleophilic selenium cysteine at the C-terminal of TrxR has entered the phase II clinical research stage, and epicatechin which simulates the structure of reduced coenzyme II (NADPH) has also been promoted to phase II clinical experiment as an ovarian cancer treatment drug. Irreversible inhibitors mainly covalently bind to key residues (cysteine, selenium cysteine) in the catalytic site to play a role, and can be divided into metal complexes containing metal ions and natural products and their derivatives containing Michael acceptor structural units according to the chemical structure. Among them, myricetin, curcumin derivatives, terpenoids such as oregano A, and quinones such as mitomycin C have been reported as irreversible TrxR inhibitors.

[0004] Natural products have become an important source of new drug development due to their structural diversity, complexity and biological activity diversity. About 70% of the drugs currently used in clinical applications are derived from natural products and their derivatives, but only 4% of them are directly used as drugs. Most of them need to be optimized in activity, selectivity, physicochemical properties and pharmacokinetic properties through structural derivatization to meet the requirements of drug development. At the same time, natural products are often limited in source due to low content in nature, difficulty in separation, and insufficient drug properties, which further restricts their clinical application. Therefore, designing an efficient and convenient synthetic route to prepare natural products and derivatizing their skeletons is a key link to promote the research and development of new drugs of natural products.

[0005] Among the TrxR inhibitors of natural products, pleurotin and its homologues derived from mushrooms have attracted much attention due to their unique structures and multiple activities. In 1947, researchers first isolated pleurotin from Hohenbuehelia grisea, and in 1994, they isolated dihydropleurotinic acid and leucopleurotin, the derivatives of pleurotin, from Nematoctonus robustus. All of them have been confirmed to have good anticancer, anti-Gram-positive bacteria, and antipathogenic fungal activities. In 2001, further studies confirmed that pleurotin is a high-activity TrxR inhibitor (IC 50 = 0.17 μM), and it showed significant proliferation inhibition activity on breast cancer cell line MCF-7 (GI 50 = 21.47 μM), leukemia cells (GI 50 = 5.08 μM), etc.

[0006] To break through the limitations of natural sources, researchers have carried out a large number of studies on the total synthesis of pleurotin. In 1989, Hart's group at Ohio State University first reported the total synthesis of pleurotin and dihydropleurotinic acid in J. Am. Chem. Soc. using a linear synthesis strategy. Starting from benzoic acid, dihydropleurotinic acid was obtained in 0.9% overall yield after 25 steps (including the construction of hexahydroindane structure by radical cyclization, the construction of the mother nucleus skeleton by trifluoroboron ether catalyzed Friedel-Crafts reaction, etc.), and further oxidation gave pleurotin. However, this route is long and has extremely low yield, which limits its practicality.

[0007] In 2022, Sorensen's team reported the synthesis of the racemic pentacyclic intermediate of pleurotin in J. Am. Chem. Soc. Starting from commercially available 5-methoxy-1-indanone, an enone intermediate containing A / C rings was constructed through 4 steps, and then B ring was constructed by intermolecular photoenolization / Diels-Alder cycloaddition reaction (yield 57%, dr > 20:1). Subsequently, C5 position epimerization was achieved by photooxidation-reduction reaction (yield 74%, dr = 1:1), and finally the pentacyclic intermediate was obtained through 8 longest linear steps. Although this route shortens the steps, the key C5 position epimerization reaction cannot be scaled up and has low diastereoselectivity, and the target intermediate can only be prepared at a scale of 7.5 milligrams, which is difficult to meet the needs of subsequent research.

[0008] In 2024, the Xuanjun / Ding Hanfeng group published a general synthetic route of pleurotin natural products in J. Am. Chem. Soc. Using commercially available (-)-perilla aldehyde and 1,4-dimethoxybenzene as starting materials, through Johnson-Claisen rearrangement, consecutive borohydride oxidation (2 gram scale, yield 82%, dr at C4 = 13:1), Friedel-Crafts type cyclization dehydration, photooxidation / reduction / enamine / HAT catalytic synergy, etc. Key reactions, 15-16 step reactions were used to realize the divergent total synthesis of four pleurotin sesquiterpene quinone / hydroquinone natural products, which significantly improved the synthesis efficiency and scale. However, this synthetic route has the disadvantages of complex reaction steps, strict experimental conditions, potential risks in raw material supply and quality, and narrow range of product structure derivatization, which limits its industrial application and further study of the structure-activity relationship of pleurotin. SUMMARY

[0009] To solve the above technical problems, the purpose of the present application is to provide a preparation method and application of pleurotin and its derivatives, which realizes the preparation of pleurotin C7 substitution, C8 axial substitution and C8 exocyclic double bond substitution derivatives, and studies the antitumor activity and TrxR inhibitory activity of such derivatives.

[0010] The above-mentioned purpose of the present application is realized by the following technical solutions:

[0011] The first aspect of the present application provides a pleurotin derivative, and the structural formula of the pleurotin derivative is selected from one of the following structural formulas:

[0012]

[0013] The second aspect of the present application provides a preparation method of a pleurotin derivative, comprising the following steps:

[0014] (1) dissolving (named as compound 10) in an organic solvent, adding selenium dioxide, and reacting at 60-100℃, and after the reaction is completed, separating and purifying to obtain (named as compound 35) and (named as compound 36);

[0015] (2) dissolving compound 35 or compound 36 in a mixed solvent of nitrile and ether, adding a cerium-containing oxidizing agent under inert gas protection and at-20℃ to 10℃ to carry out oxidation reaction, after the oxidation reaction is completed, carrying out post-treatment and separation and purification to obtain pleurotin derivative (named as compound 41a) or pleurotin derivative (named as compound 41b).

[0016] Further, in step (1), the organic solvent comprises 1,4-dioxane; the molar ratio of compound 10 to selenium dioxide is 1:(5-15); the amount of the organic solvent used is 1 mmol:(30-50) mL; and the reaction time is 1-4 h.

[0017] Further, in step (1), the separation and purification comprises the steps of concentration under reduced pressure and column chromatography purification.

[0018] Further, in step (1), the preparation method of compound 10 comprises the following steps:

[0019] S1. Under an inert gas atmosphere, at -80 to -70℃, lithium hexamethyldisilazane is reacted with compound 32 (traditionally named compound 33) after adding trifluoromethanesulfonyl aniline and treating after the reaction is completed at 20-40℃, and reacting the intermediate with a palladium-containing catalyst and an organic zinc reagent in a solvent at 80-100℃, and separating and purifying to obtain (traditionally named compound 33) after adding trifluoromethanesulfonyl aniline and treating after the reaction is completed at 20-40℃, and reacting the intermediate with a palladium-containing catalyst and an organic zinc reagent in a solvent at 80-100℃, and separating and purifying to obtain

[0020] S2. Under an inert gas atmosphere, at -30 to -10℃, compound 33 is reduced by a reducing agent, and then oxidized by an oxidizing agent at 20-40℃, and separated and purified to obtain (traditionally named compound 33) after adding trifluoromethanesulfonyl aniline and treating after the reaction is completed at 20-40℃, and reacting the intermediate with a palladium-containing catalyst and an organic zinc reagent in a solvent at 80-100℃, and separating and purifying to obtain

[0021] S3. Compound 34 is treated by hydrofluoric acid-pyridine complex at 40-60℃, and then subjected to Lewis acid catalysis at -30 to -10℃, and separated and purified to obtain compound 10.

[0022] Further, in S1, the palladium-containing catalyst is dichlorobis(triphenylphosphine)palladium(II); the organic zinc reagent is prepared by reacting zinc powder with 2,5-dimethoxybenzyl bromide; the organic solvent comprises tetrahydrofuran; the solvent comprises N,N-dimethylformamide; the molar ratio of lithium hexamethyldisilazane to compound 32 is (3-6):1; and the molar ratio of trifluoromethanesulfonyl aniline to compound 32 is (4-7):1.

[0023] Further, in S2, the reducing agent is diisobutylaluminum hydride; the oxidizing agent is Dess-Martin periodinane; the molar ratio of the reducing agent to compound 33 is (3-6):1; and the molar ratio of the oxidizing agent to the reduced product of compound 33 is (1-2):1.

[0024] Further, in S3, the Lewis acid is boron trifluoride-diethyl ether complex; the molar ratio of the hydrofluoric acid-pyridine complex to compound 34 is (20-40):1; the molar ratio of the hydrofluoric acid-pyridine complex to the acid treatment product of compound 34 is (1-3):1.

[0025] Further, the treatment after each step of reaction includes quenching, extraction, drying, filtration and concentration operations; and the separation and purification method of each step includes silica gel column chromatography, and the eluent is a mixed solvent of ethyl acetate and n-hexane.

[0026] Further, in step (2), the cerium-containing oxidizing agent is cerium ammonium nitrate; the mixed solvent of nitrile and ether is acetonitrile and diethyl ether, and the volume ratio of acetonitrile to diethyl ether is 1:(0.5-2); the inert gas is argon or nitrogen; the molar ratio of compound 35 or compound 36 to the cerium-containing oxidizing agent is 1:(1.5-4), and the use amount ratio of compound 35 or compound 36 to the mixed solvent is 1 mmol:(100-300)mL; and the oxidation reaction time is 15-60 min.

[0027] Further, in step (2), the post-treatment includes: after the reaction solution is diluted with water, dichloromethane is used for extraction, and the organic phase is washed, dried, filtered and concentrated; the washing uses saturated brine; the drying uses anhydrous magnesium sulfate; and the separation and purification is silica gel column chromatography.

[0028] The third aspect of the present application provides a preparation method of a griseofulvin derivative, comprising the following steps:

[0029] Under the atmosphere of inert gas, (compound 11) is heated and reacted with dicyclopentadienyl dimethyl titanium in an organic solvent at 60-100℃, after the reaction is completed, post-treatment and separation and purification are performed to obtain (compound 37); compound 37 is dissolved in a mixed solvent of nitrile and ether, a cerium-containing oxidizing agent is added for oxidation reaction under the protection of inert gas and at-20℃ to 10℃, after the oxidation reaction is completed, post-treatment and separation and purification are performed to obtain a griseofulvin derivative (compound 42).

[0030] Further, the organic solvent includes toluene; the inert gas is nitrogen or argon; the molar ratio of dicyclopentadienyl dimethyl titanium to compound 11 is (4-8):1; the heating reaction time is 1-3h; and the use amount ratio of compound 11 to the organic solvent is 1mmol:(20-40)mL.

[0031] Further, the post-treatment comprises: after the reaction solution is cooled, petroleum ether is added for dilution, the titanium by-product is precipitated by stirring, the precipitate is removed by filtration and rinsing, and the filtrate is concentrated; the filtration is performed by using diatomite; the rinsing agent is petroleum ether; and the separation and purification is performed by using silica gel column chromatography.

[0032] Further, the preparation method of the compound 11 comprises the following steps: after compound 10 is reacted with borane-dimethyl sulfide complex in an organic solvent at 20-40 DEG C under an inert gas atmosphere, sodium hydroxide and hydrogen peroxide are added for oxidation, and an alcohol intermediate is obtained through separation and purification; after the alcohol intermediate is reacted with oxalyl chloride and dimethyl sulfoxide at-80 DEG C to-70 DEG C, triethylamine is added and reacted at 20-40 DEG C, and the compound 11 is obtained through separation and purification.

[0033] Further, in the preparation method of the compound 11, the organic solvent comprises tetrahydrofuran; the molar ratio of the borane-dimethyl sulfide complex to the compound 10 is (4-8):1; the molar ratio of the sodium hydroxide to the compound 10 is (4-8):1; and the molar ratio of the hydrogen peroxide to the compound 10 is (4-8):1.

[0034] Further, in the preparation method of the compound 11, the organic solvent comprises dichloromethane; the molar ratio of the oxalyl chloride to the alcohol intermediate is (1.5-2.5):1; the molar ratio of the dimethyl sulfoxide to the alcohol intermediate is (2-4):1; and the molar ratio of the triethylamine to the alcohol intermediate is (5-7):1.

[0035] The fourth aspect of the present application provides a preparation method of a griseofulvin derivative, comprising the following steps:

[0036] Compound 37 is reacted with borane-dimethyl sulfide complex in an organic solvent at-10 DEG C to 10 DEG C, and the reaction is continued after being warmed to 20-40 DEG C; then sodium hydroxide and hydrogen peroxide are added for oxidation reaction, and after the reaction is completed, griseofulvin derivative (named as compound 38) is obtained through post-treatment and separation and purification; compound 38 is dissolved in a mixed solvent of nitrile and ether, and under the protection of inert gas, a cerium-containing oxidizing agent is added for oxidation reaction at-20 DEG C to 10 DEG C, and after the oxidation reaction is completed, griseofulvin derivative (named as compound 43) is obtained through post-treatment and separation and purification.

[0037] Further, the molar ratio of the borane-dimethyl sulfide complex to compound 37 is (5-8):1; the molar ratio of the sodium hydroxide to compound 37 is (4-7):1; the molar ratio of the hydrogen peroxide to compound 37 is (10-20):1 (calculated based on 30% hydrogen peroxide); and the ratio of the amount of compound 37 to the organic solvent is 1 mmol:(70-100)mL.

[0038] Further, the post-treatment comprises: quenching the reaction solution with water, extracting with an aqueous phase extractant after liquid separation, and washing, drying, filtering and concentrating after combining the organic phases.

[0039] The fifth aspect of the present application provides use of the grifolan derivative of the first aspect in the preparation of an anti-tumor drug.

[0040] The sixth aspect of the present application provides use of the grifolan derivative of the first aspect in the preparation of a TrxR inhibitor.

[0041] The grifolan derivative provided by the present application has anti-tumor activity and TrxR inhibitory activity, and in particular has anti-breast cancer and liver cancer cell proliferation activity.

[0042] The seventh aspect of the present application provides a preparation method of dihydrogrifolanic acid, comprising the following steps:

[0043] P1. Compound 38 is reacted with a hypoiodite oxidizing agent in an organic solvent at 20-40℃, and compound 39 is obtained after separation and purification; (named as compound 39);

[0044] P2. Compound 39 is reacted with 1,8-diazabicyclo[5.4.0]undecane in an organic solvent at 20-40℃, and compound 40 is obtained after separation and purification; (named as compound 40);

[0045] P3. Compound 40 is reacted with sodium chlorite and sodium dihydrogen phosphate in a mixed solvent at -10℃ to 10℃ for 5-15 min, and then the temperature is raised to 20-40℃ for reaction, and compound 22 is obtained after separation and purification; (named as compound 22);

[0046] P4. Compound 22 is reacted with a cerium-containing oxidizing agent in a mixed solvent of nitrile and ether under inert gas protection and at -20℃ to 10℃, and dihydrogrifolanic acid is obtained after separation and purification.

[0047] Further, in P1, the hypoiodite oxidizing agent is Dess-Martin hypoiodite; the organic solvent comprises dichloromethane; the molar ratio of the hypoiodite oxidizing agent to compound 38 is (2-4):1; and the reaction time is 20-40 min.

[0048] Further, in P2, the organic solvent comprises dichloromethane; the 1,8-diazabicyclo[5.4.0]undecane is added twice, and the molar ratio of the single addition amount to compound 39 is (1.5-2.5):1.

[0049] Further, in P3, the mixed solvent is a mixture of tert-butyl alcohol, tetrahydrofuran and 2-methyl-2-butene; the molar ratio of sodium chlorite to compound 40 is (8-12):1; and the molar ratio of sodium dihydrogen phosphate to compound 40 is (8-12):1.

[0050] Further, in P4, the cerium-containing oxidizing agent is cerium ammonium nitrate; the nitrile solvent is acetonitrile, and the ether solvent is diethyl ether; the inert gas is argon or nitrogen; and the molar ratio of the cerium-containing oxidizing agent to compound 22 is (2-3):1.

[0051] Further, the separation and purification method in each step comprises silica gel column chromatography.

[0052] Further, the post-treatment after the reaction of each step comprises filtering, concentrating, extracting, washing and drying operations; wherein the extraction is performed by using ethyl acetate or dichloromethane, the washing is performed by using saturated brine, and the drying is performed by using anhydrous magnesium sulfate or anhydrous sodium sulfate.

[0053] The eighth aspect of the present application provides a preparation method of grifolan.

[0054] The dihydrogrifolanic acid prepared by the preparation method of the seventh aspect is heated with manganese dioxide in an organic solvent at 40-60℃ for 10-14 h, and after the reaction is completed, the post-treatment and separation and purification are performed to obtain the grifolan.

[0055] Further, the organic solvent is acetonitrile; the molar ratio of the manganese dioxide to the dihydrogrifolanic acid is (8-12):1; and the amount ratio of the dihydrogrifolanic acid to the organic solvent is 1 mmol:(400-600) mL.

[0056] Further, the post-treatment comprises filtering the reaction liquid through a filter medium, washing the filter residue with an organic solvent, collecting the filtrate and concentrating; and the separation and purification is silica gel column chromatography.

[0057] The grifolan, dihydrogrifolanic acid and grifolan derivative provided by the present application can all be used as anti-tumor drug lead compounds.

[0058] Advantages of the present application:

[0059] 1. The present application provides a class of antitumor activity of grifolan derivatives, specifically including C7 position substitution, C8 position ring outside double bond substitution and C8 position axial substitution of grifolan derivatives. The present application is obtained by selective allylic oxidation reaction, which can be substituted at C7 position of grifolan derivatives (compound 41a, compound 41b); methylene reaction is used to generate olefin structure, and C8 position ring outside double bond substitution of grifolan derivatives (compound 42) is obtained; then hydrogen boronization-oxidation reaction is further prepared to obtain C8 position axial substitution of grifolan derivatives (compound 43) - the above key structure modification is based on the five-ring mother nucleus skeleton of grifolan.

[0060] 2. The activity test results of the present application show that the grifolan derivatives of this kind have significantly better activity than the parent compound (-)-grifolan in inhibiting the proliferation of human cancer cells (such as breast cancer MDA-MB-231 cells, liver cancer HepG2 cells) with high expression of TrxR. Among them, the derivative substituted at C8 position has the most prominent advantage in inhibiting TrxR enzyme activity, and the inhibitory effect on TrxR is 9 times and 16.7 times higher than that of the parent compound (-)-grifolan and the positive control drug, respectively. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 The total reaction equation diagram of each reaction in the embodiments of the present application is shown. DETAILED DESCRIPTION

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0063] The present application will be further described below with reference to the drawings and specific embodiments, so that those skilled in the art can better understand the present application and implement it, but the embodiments are not limiting to the present application.

[0064] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified, and can be used directly without additional pretreatment; for solvents that need to be anhydrous, the solvent anhydrous treatment method known in the art can be used without additional special treatment steps.

[0065] In this embodiment of the invention, the term "concentration" specifically refers to the operation of removing solvent by rotary evaporation under reduced pressure conditions using a water pump; all experimental apparatus involved in the method of this invention are standard apparatuses conventionally used in the field of organic synthesis. For the purification of the crude product obtained from the reaction, well-known separation and purification methods in the art, such as recrystallization, distillation, or column chromatography, can be selected based on the physicochemical properties of the target product, such as solubility, boiling point, and polarity, to obtain a high-purity target product.

[0066] Compound 10 in the following examples The preparation method includes the following steps:

[0067] (1)

[0068] Under a nitrogen atmosphere, lithium hexamethyldisilazane (LHMDS, 21.6 mL, 28.08 mmol, 1.3 M n-hexane solution, 4.8 equivalents) was added dropwise to compound 32 over 5 minutes at -78 °C. 2.88 g (5.85 mmol, 1.0 equivalent) of anhydrous tetrahydrofuran (THF, 58 mL) was added to a stirred solution. The reaction mixture was stirred at this temperature for 1 hour, followed by the addition of trifluoromethanesulfonyl aniline (PhNTf2, 11.7 g, 32.76 mmol, 5.6 equivalent) dissolved in anhydrous THF (50 mL). The reaction mixture was immediately brought to room temperature and stirred overnight at room temperature, then quenched with saturated ammonium chloride aqueous solution (100 mL). The aqueous phase was extracted with ethyl acetate (100 mL × 2), and the combined organic phases were dried over anhydrous magnesium sulfate (MgSO4), filtered, and concentrated. The resulting residue was used directly in the next reaction step without purification.

[0069] Zinc powder (3.3 g, 50.3 mmol, 4.3 equiv) was suspended in THF (10 mL) under nitrogen atmosphere, 1,2-dibromoethane (20 μί) was added at 80 °C, after refluxing for 10 min, it was cooled to room temperature, 2,5-dimethoxybenzyl bromide (2.7 g, 11.7 mmol, 1.0 equiv) dissolved in THF (20 mL) was added. After stirring for 2 h, Pd(PPh3)2Cl2 (821 mg, 1.17 mmol, 0.1 equiv) and the above residue (5.85 mmol, 0.5 equiv) dissolved in N,N-dimethylformamide (DMF, 50 mL) were added. The reaction mixture was warmed to 90 °C, after 2 h, saturated aqueous ammonium chloride (50 mL) was added. The organic layer was separated, the aqueous phase was extracted with ethyl acetate (100 mL x 3), the combined organic phase was washed with saturated brine (200 mL), dried over anhydrous sodium sulfate (Na2S04) and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (eluent: ethyl acetate / n-hexane = 1 : 15) to give colorless oil 33 (i.e. compound 33) as 2.57 g, 70% overall yield for two steps). 2.57 g, 70% overall yield for two steps).

[0070] Analytical data of colorless oil 33 (i.e. compound 33) are as follows: TLC: Rf= 0.35 (Si02, petroleum ether / EtOAc 10: 1); f = 0.35 (Si02, petroleum ether / EtOAc 10: 1); 1 H NMR (400 MHz, CDC13) δ 7.67-7.64 (m, 4H), 7.44-7.35 (m, 6H), 6.76 (d, J = 8.4 Hz, 1H), 6.70-6.66 (m, 2H), 5.19 (s, 1H), 3.75 (s, 3H), 3.74 (s, 3H), 3.54-3.43 (m, 2H), 3.46 (s, 3H), 3.32-3.24 (m, 2H), 2.49-2.23 (m, 3H), 2.16-1.97 (m, 3H), 1.92-1.82 (m, 2H), 1.62-1.52 (m, 1H), 1.41-1.35 (m, 1H), 1.32-1.24 (m, 1H), 1.09 (d, J = 6.4 Hz, 3H), 1.05 (s, 9H); 13 C NMR (100 MHz, CDC13) δ

[0071] 175.7, 153.3, 151.9, 138.7, 135.6, 135.57, 134.1, 134.0, 129.5, 129.4, 127.5, 125.6, 117.1, 111.8, 111.2, 67.8, 56.3, 55.6, 55.5, 51.4, 51.2, 41.5, 38.4, 33.3, 33.1, 28.2, 27.4, 26.8, 21.9, 19.3, 18.0; FTIR (Neat): v = 2921, 2854, 1719, 1499, 1464, 1277, 1223, 1049, 1028, 821, 803, 701 cm -1 HRMS (ESI) m / z: [M + Na] + calcd for C 39 H 50 O5NaSi, 649.3325; found 649.3324. [a] D 23 = +49.0 (c = 0.1, CHCI3).

[0072] (2)

[0073] Compound 33 (1.13 g, 1.80 mmol, 1.0 eq) was dissolved in dichloromethane (DCM, 95 mL) under nitrogen atmosphere, and diisobutylaluminum hydride (DIBAL-H, 5.4 mL, 8.10 mmol, 1.5 M in toluene, 4.5 eq) was added dropwise with stirring at -20 °C. The reaction mixture was continued to stir at the same temperature for 1 h. Then, the reaction was quenched by the addition of saturated aqueous sodium potassium tartrate solution (100 mL), and the aqueous phase was extracted with dichloromethane (100 mL x 3) after separation. The combined organic phase was washed with saturated brine (300 mL), dried over anhydrous magnesium sulfate (MgS04), filtered and concentrated. The residue was used in the next step without purification.

[0074] The above residue (1.80 mmol, 1.0 eq) was dissolved in dichloromethane (35 mL) with stirring at room temperature, and Dess-Martin periodinane (DMP, 1.15 g, 2.7 mmol, 1.5 eq) was added. After the reaction mixture was stirred at room temperature for 30 min, it was filtered through silica gel and concentrated under reduced pressure. The residue was purified by flash chromatography on a silica gel column (eluent: ethyl acetate / n-hexane = 1:15) to give compound 34 (980 mg, 91% overall yield for two steps) as a colorless oil.

[0075] ​Analytical data for colorless oil 34 (i.e., compound 34) are as follows: TLC: Rf= 0.41 (Si02, petroleum ether / EtOAc 10: 1); 1 H NMR (400 MHz, CDC13) δ 9.66 (d, J = 1.2 Hz, 1H), 7.66 - 7.63 (m, 4H), 7.44 - 7.36 (m, 6H), 6.76 (d, J = 8.8 Hz, 1H), 6.70 (dd, J = 8.4, 2.8 Hz, 1H), 6.62 (d, J = 3.2 Hz, 1H), 5.30 (s, 1H), 3.75 (s, 3H), 3.71 (s, 3H), 3.52 - 3.42 (m, 2H), 3.16 - 3.00 (m, 2H), 2.42 - 2.31 (m, 2H), 2.23 - 2.01 (m, 4H), 1.97 - 1.92 (m, 1H), 1.86 - 1.78 (m, 1H), 1.51 - 1.33 (m, 2H), 1.27 - 1.20 (m, 1H), 1.09 (d, J = 6.4 Hz, 3H), 1.05 (s, 9H); 13 C NMR (100 MHz, CDC13) δ 201.3, 153.3, 151.8, 135.6, 135.5, 134.8, 133.9, 133.8, 129.5, 129.1, 127.5, 127.3, 117.3, 111.6, 111.3, 67.6, 61.4, 56.1, 55.6, 50.3, 41.6, 38.2, 32.5, 28.4, 27.6, 27.4, 26.9, 21.2, 19.3, 18.2; FTIR (Neat): v = 2920, 2851, 1707, 1499, 1469, 1225, 1109, 1075, 1050, 1028, 998, 739, 701 cm"1; HRMS (ESI) m / z: [M + Na]+calcd for C 38 H 48 O4NaSi, 619.3220; found 619.3216. [a]D23= +115.0 (c = 0.1, CHCl3).

[0076] (3)

[0077] Compound 34 (1.18 g, 1.98 mmol, 1.0 eq) was dissolved in anhydrous tetrahydrofuran / methanol (39.6 mL, THF / MeOH = 1 : 1) and hydrofluoric acid (HF, 5.4 mL, 59.4 mmol, 65% content in pyridine, 30 eq) was added slowly with stirring at room temperature. After the reaction mixture was stirred at 50 °C for 5 h, it was quenched by the addition of saturated aqueous sodium bicarbonate solution at 0 °C, extracted with ethyl acetate, and the combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate (Na2S04), filtered, and concentrated under reduced pressure. The residue was used in the next step without purification.

[0078] The above residue (1.98 mmol, 1.0 eq) was dissolved in toluene (198 mL) and boron trifluoride diethyl ether complex (BF3·Et20, 499 μL, 3.96 mmol, 2 eq) was added dropwise with stirring at -20 °C. The reaction mixture was stirred at this temperature for 40 min and then quenched by the addition of saturated aqueous sodium bicarbonate solution (200 mL). The aqueous phase was extracted with ethyl acetate (200 mL x 3), and the combined organic layers were washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (eluent: ethyl acetate / n-hexane = 1 :40) to give white foamy solid 10 (610 mg, 91% overall yield for two steps).

[0079] Analytical data for white foamy solid 10 (i.e., compound 10) are as follows: TLC: Rf= 0.47 (Si02, petroleum ether / EtOAc 10: 1); f = 0.47 (Si02, petroleum ether / EtOAc 10: 1); 1 H NMR (400 MHz, CDC13) δ 6.67 (s, 2H), 5.37 (br s, 1H), 4.63 (s, 1H), 3.99 (dd, J = 12.0, 8.4 Hz, 1H), 3.78 (s, 3H), 3.77 (s, 3H), 3.51 (dd, J = 12.4, 6.8 Hz, 1H), 3.31 (d, J = 16.8 Hz, 1H), 3.13 (d, J = 16.8 Hz, 1H), 2.25 - 1.71 (m, 10H), 1.41 - 1.33 (m, 1H), 0.96 (d, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDC13) δ 154.2, 150.2, 140.0, 129.6, 128.2, 119.5, 109.3, 108.8, 79.2, 74.2, 56.2, 55.7, 49.4, 49.1, 45.4, 32.2, 31.7, 31.6, 27.1, 25.6, 21.1, 19.7; FTIR (Neat): v = 2918, 2848, 1462, 1434, 1255, 1115, 1104, 1080, 1053, 1026, 983, 789, 777, 711 cm -1 ; HRMS (ESI) m / z: [M + H] + calcd for C 22 H 29 O3, 341.2116; found 341.2109. [a] D 23 = +41.4 (c = 0.2, CHCl3).

[0080] The preparation method of compound 11 in the following examples includes the following steps:

[0081] (1)

[0082] Compound 10 (496 mg, 1.46 mmol, 1.0 equivalent) was dissolved in tetrahydrofuran (THF, 30 mL) under nitrogen atmosphere at 0°C, and borane-dimethyl sulfide complex (BH3-Me2S, 876 μL, 8.76 mmol, 10 M solution in THF, 6.0 equivalent) was added dropwise. The reaction mixture was raised to room temperature and stirred for 12 hours, and then aqueous sodium hydroxide (1.46 mL, 8.76 mmol, 6 N, 6.0 equivalent) and 30% hydrogen peroxide (H2O2, 895 μL, 8.76 mmol, 6.0 equivalent) were added successively. The reaction mixture was continuously stirred at room temperature for 30 minutes. Subsequently, water (25 mL) was added for quenching, and the aqueous phase was extracted with ethyl acetate (25 mL x 3) after separation. The combined organic layer was washed with saturated brine (50 mL), dried over anhydrous magnesium sulfate (MgSO4), filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (eluent: ethyl acetate / n-hexane = 1:7) to obtain an alcohol (435 mg, 83%) as a white solid.

[0083] (2) Oxaloyl chloride (191 μL, 2.24 mmol, 2.0 equivalent) was dissolved in anhydrous dichloromethane (DCM, 22 mL) at -78 °C, and dimethyl sulfoxide (DMSO, 231 μL, 3.25 mmol, 2.9 equivalent) was added dropwise over 25 minutes. After 20 minutes, an anhydrous DCM solution (10 mL) of the above-mentioned white solid alcohol (401 mg, 1.12 mmol, 1.0 equivalent) was added at -78 °C. The reaction mixture was stirred at this temperature for 45 minutes, and then triethylamine (934 μL, 6.72 mmol, 6.0 equivalent) was added. The reaction system was then brought to room temperature and stirred for 2 hours. After the reaction was completed, water (50 mL) was added to quench the reaction, and the mixture was extracted with dichloromethane (50 mL × 2). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous magnesium sulfate (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified by rapid silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1:15) to give a white solid 11 (341 mg, overall yield of two steps: 71%).

[0084] The analytical data for white solid 11 (i.e., compound 11) are as follows: TLC:R f =0.32(SiO2,petroleumether / EtOAc 5:1); 1 H NMR (400MHz, CDCl3) δ6.64(q,J=8.6Hz,2H),4.38(s,1H),3.98(dd,J=12.8,8.4Hz,1H),3.78(s,3H),3.74(s,3H),3.55(dd,J= 12.4,5.6Hz,1H),3.19(d,J=17.6Hz,1H),2.67(d,J=5.2Hz,1H),2.49-1.81(m,11H),1.49-1.41(m,1H),0.99(d,J=6.8Hz,3H); 13 C NMR (100MHz, CDCl3) δ210.3,153.6,150.5,126.1,125.3,108.9,108.6,74.6,74.1,56.0,55.7,55.6,51.1,51.0 ,45.4,40.2,34.6,32.5,26.3,22.5,21.2,20.2; FTIR(Neat):ν=2956,2922,2852,1463,1259,1087,1017,798cm -1 HRMS(ESI)m / z:[M+H] + calcd for C 22 H 29 O4,357.2066; found 357.2304.[α]D 23 = +38.5 (c = 0.1, CHCl3).

[0085] Example 1

[0086] Grifolan derivative A method for preparing grifolan derivative (Compound 41a) and grifolan derivative (Compound 41b) comprises the following steps:

[0087] (1)

[0088] Compound 10 (24 mg, 0.07 mmol, 1.0 equiv) was dissolved in 1,4-dioxane (3.6 mL), and selenium dioxide (SeO2, 78 mg, 0.7 mmol, 10 equiv) was added. After the reaction mixture was stirred at 80 °C for 2 h, it was concentrated under reduced pressure. The resulting residue was purified by flash chromatography on a silica gel column (eluent: ethyl acetate / n-hexane = 1:5) to give compound 35 (3 mg, 12%) and compound 36 (14 mg, 56%) as amorphous white solids.

[0089] Analytical data of compound 35 are as follows: TLC: Rf= 0.12 (SiO2, petroleum ether / EtOAc 8:1); f = 0.12 (SiO2, petroleum ether / EtOAc 8:1); 1 HNMR (400 MHz, CDCl3) δ 6.68 (s, 2H), 5.38 (s, 1H), 4.67 (s, 1H), 4.43-4.39 (m, 1H), 3.99 (dd, J = 12.4, 8.4 Hz, 1H), 3.78 (s, 3H), 3.77 (s, 3H), 3.50 (dd, J = 12.0, 6.8 Hz, 1H), 3.35 (d, J = 16.8 Hz, 1H), 3.16 (d, J = 16.8 Hz, 1H), 2.25-2.10 (m, 4H), 1.98-1.88 (m, 2H), 1.76-1.64 (m, 2H), 1.39-1.32 (m, 1H), 0.97 (d, J = 7.2 Hz, 3H); 13 ​​​CNMR (100 MHz, CDC13) δ 154.1, 150.2, 142.7, 128.6, 127.7, 124.0, 109.4, 108.9, 79.0, 74.0, 70.1, 56.2, 55.7, 49.7, 47.2, 45.4, 32.2, 32.1, 31.3, 30.6, 25.2, 21.0; FTIR (Neat): v = 3395, 2921, 2852, 1728, 1686, 1646, 1467, 1408, 1378, 1263, 1184, 1081, 767, 719 cm -1 HRMS (ESI) m / z: [M + H] + calcd for C 22 H 29 O4, 357.2066; found 357.2049. [a] D 23 = +34.00 (c = 0.05, CHCl3).

[0090] Analytical data for compound 36 are as follows: M.p. = 163.8 - 170.2 °C. TLC: R f = 0.18 (SiO2, petroleum ether / EtOAc 8: 1); 1 H NMR (400 MHz, CDC13) δ 6.68 (s, 2H), 5.57 (d, J = 4.8 Hz, 1H), 4.54 (s, 1H), 4.33 (t, J = 4.4 Hz, 1H), 3.98 (dd, J = 12.4, 8.4 Hz, 1H), 3.77 (s, 6H), 3.54 (dd, J = 12.4, 6.8 Hz, 1H), 3.37 (d, J = 16.8 Hz, 1H), 3.18 (d, J = 16.8 Hz, 1H), 2.27 - 1.96 (m, 6H), 1.89 - 1.77 (m, 2H), 1.45 - 1.37 (m, 1H), 0.98 (d, J = 6.8 Hz, 3H); 13 CNMR (100 MHz, CDC13) δ 154.1, 150.2, 146.0, 128.5, 127.7, 120.8, 109.5, 108.9, 78.3, 74.3, 66.5, 56.3, 55.7, 50.0, 44.9, 43.7, 32.4, 31.6, 31.1, 29.5, 25.7, 21.1; FTIR (Neat): v = 3359, 2920, 2851, 1659, 1633, 1470, 1433, 1377, 1254, 1190, 1081, 799, 714 cm -1HRMS (ESI) m / z: [M+H] + calcd for C 22 H 29 O4,357.2066; found 357.2049. [a] D 23 = +81.50 (c = 0.1, CHCI3).

[0091] (2) Compound 35 (0.03 mmol, 1.0 equiv) was dissolved in a mixture solution of acetonitrile (3.0 mL) and diethyl ether (3.0 mL) under argon protection and ice-salt bath cooling. Ammonium cerium (IV) nitrate (CAN, 41 mg, 0.075 mmol, 2.5 equiv) was added slowly, and the dropping time was about 3 min. The resulting yellow solution was stirred at 0 °C for 30 min. The light yellow solution was diluted with water (10 mL) and extracted with dichloromethane (15 mL x 2). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous magnesium sulfate (MgS04), filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel to give pleuromutilin derivative (compound 41a, 2 mg, 73%).

[0092] Analytical data of compound 41a are as follows: TLC: R f = 0.32 (Si02, petroleum ether / EtOAc 3: 1); 1 HNMR (400 MHz, CDCI3) δ 6.68 (q, J = 10.4 Hz, 2H), 5.45 (s, 1H), 4.54 (s, 1H), 4.46-4.42 (m, 1H), 3.97 (dd, J = 12.4, 8.4 Hz, 1H), 3.51 (dd, J = 12.4, 6.4 Hz, 1H), 3.14-3.00 (m, 2H), 2.25-2.03 (m, 4H), 1.94-1.88 (m, 2H), 1.79-1.70 (m, 1H), 1.68-1.61 (m, 1H), 1.36-1.28 (m, 1H), 0.97 (d, J = 7.2 Hz, 3H); 13 C NMR (100 MHz, CDCI3) δ 186.8, 186.4, 143.4, 142.8, 138.9, 137.8, 135.1, 126.6, 78.3, 75.0, 69.8, 49.5, 46.8, 44.9, 32.3, 31.6, 30.6, 30.4, 25.0, 20.9; FTIR (Neat): v = 2921, 2852, 1646, 1461, 1188, 1080, 762, 704 cm -1HRMS (ESI) m / z: [M + H] + calcd for C 20 H 23 O4,327.1596;found 327.1599.[α] D 23 = +46.00 (c = 0.05, CHCI3).

[0093] (3) Compound 36 (0.03 mmol, 1.0 equiv) was dissolved in a mixture solution of acetonitrile (3.0 mL) and diethyl ether (3.0 mL) under ice-salt bath cooling with argon protection. Ammonium cerium nitrate (CAN, 41 mg, 0.075 mmol, 2.5 equiv) was added slowly, and the dropping time was about 3 minutes. The resulting yellow solution was stirred at 0 °C for 30 minutes. The light yellow solution was diluted with water (10 mL) and extracted with dichloromethane (15 mL x 2). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous magnesium sulfate (MgS04), filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel to give the pleuromutilin derivative (compound 41b, 12 mg, 94%).

[0094] Analytical data of compound 41b are as follows: TLC: R f = 0.21 (Si02, petroleum ether / EtOAc 3:1); 1 HNMR (400 MHz, CDCI3) δ 6.68 (q, J = 10.4 Hz, 2H), 5.63 (d, J = 4.4 Hz, 1H), 4.38-4.36 (m, 2H), 3.95 (dd, J = 12.4, 8.4 Hz, 1H), 3.56 (dd, J = 12.8, 6.0 Hz, 1H), 3.12-3.00 (m, 2H), 2.30-2.24 (m, 1H), 2.19-2.12 (m, 2H), 2.05-1.80 (m, 5H), 1.41-1.35 (m, 1H), 0.98 (d, J = 7.2 Hz, 3H); 13 CNMR (100 MHz, CDCI3) δ 186.7, 186.4, 143.3, 142.9, 142.4, 137.8, 135.1, 123.4, 77.9, 75.5, 66.0, 49.7, 44.4, 43.4, 32.6, 31.0, 30.7, 29.4, 25.5, 21.0; FTIR (Neat): v = 3360, 2920, 2851, 1654, 1469, 1294, 1094, 1018, 797, 702 cm -1HRMS (ESI) m / z: [M+H] + calcd for C 20 H 23 O4,327.1596;found 327.1596.[α] D 23 = +12.50 (c = 0.1, CHCl3).

[0095] Example 2

[0096] Griseofulvin derivatives The method for preparing compound 42 comprises the following steps:

[0097] (1)

[0098] Compound 11 (45 mg, 0.126 mmol, 1.0 equivalent) was dissolved in anhydrous toluene (3 mL) under a nitrogen atmosphere, and then dicyclopentadienyl dimethyl titanium (Cp2TiMe2, 0.9 M solution in THF / toluene, 0.84 mL, 0.756 mmol, 6.0 equivalents) was added at room temperature. The reaction mixture was stirred vigorously at 80 °C for 2 hours. After cooling to room temperature, petroleum ether (6 mL) was added to the reaction system to dilute it to precipitate the titanium by-product generated, and the resulting suspension was continuously stirred for 1 hour. The orange precipitate generated was removed by filtration through diatomite, and then rinsed with petroleum ether. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1:40) to obtain compound 37 as a light yellow oil (29 mg, 64%).

[0099] The analytical data of compound 37 (i.e., compound 37) as a light yellow oil are as follows: 1 H NMR (400 MHz, CDCl3) δ 6.65 (q, J = 8.8 Hz, 2H), 4.55 (br s, 1H), 4.37 (s, 1H), 4.06 (br s, 1H), 3.95 (dd, J = 12.4, 8.4 Hz, 1H), 3.78 (s, 3H), 3.76 (s, 3H), 3.51 (dd, J = 12.4, 5.6 Hz, 1H), 3.00 (d, J = 17.6 Hz, 1H), 2.73 (dd, J = 18.0, 5.2 Hz, 1H), 2.41-2.37 (m, 1H), 2.31-2.30 (m, 1H), 2.24-2.19 (m, 1H), 2.15-2.09 (m, 4H), 1.92-1.82 (m, 3H), 1.80-1.70 (m, 2H), 0.96 (d, J = 6.8 Hz, 3H); 13 ​C NMR (100 MHz, CDC13) δ 153.9, 150.4, 147.2, 126.40, 126.37, 108.64, 108.62, 107.7, 74.7, 74.6, 56.0, 55.8, 52.6, 49.0, 47.7, 46.0, 36.1, 34.4, 32.5, 25.8, 24.3, 23.5, 21.3; FTIR (Neat): v = 3358, 2921, 2852, 1735, 1718, 1647, 1458, 1260, 1095, 1023, 801 cm -1 ; HRMS (ESI) m / z: [M + Na] + calcd for C 23 H 30 O3, 377.2093; found 377.2089. [a] D 23 = +48.8 (c = 0.6, CHCl3).

[0100] (2) Compound 37 (0.03 mmol, 1.0 equiv) was dissolved in a mixture of acetonitrile (3.0 mL) and diethyl ether (3.0 mL) under argon protection and cooled in an ice-salt bath. Cerium ammonium nitrate (CAN, 41 mg, 0.075 mmol, 2.5 equiv) was added slowly, and the dropwise addition time was about 3 min. The resulting yellow solution was stirred at 0 °C for 30 min. The light yellow solution was diluted with water (10 mL) and extracted with dichloromethane (15 mL x 2). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous magnesium sulfate (MgS04), filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel to give the pleuromutilin derivative (compound 42, 7 mg, 64%).

[0101] Analytical data of compound 42 are as follows: TLC: R f = 0.65 (Si02, petroleum ether / EtOAc 4: 1); 1HNMR (400 MHz, CDC13) δ 6.67 (q, J = 10.0 Hz, 2H), 4.68 (s, 1H), 4.23-4.20 (m, 2H), 3.94 (dd, J = 12.8, 8.4 Hz, 1H), 3.55 (dd, J = 12.8, 4.8 Hz, 1H), 2.83 (d, J = 19.6 Hz, 1H), 2.49-2.42 (m, 2H), 2.33-2.32 (m, 1H), 2.17-2.06 (m, 3H), 1.99-1.93 (m, 1H), 1.89-1.73 (m, 5H), 1.33-1.25 (m, 1H), 0.97 (d, J = 7.2 Hz, 3H); 13 C NMR (100 MHz, CDC13) δ 187.3, 186.7, 146.1, 141.1, 140.9, 137.8, 135.3, 107.6, 75.9, 74.3, 52.1, 48.8, 46.7, 45.5, 35.8, 33.9, 32.7, 25.6, 24.2, 23.0, 21.3; FTIR (Neat): v = 2921, 2850, 2201, 2193, 1656, 1297, 1119, 1005, 837, 794, 708 cm -1 HRMS (ESI) m / z: [M + H] + calcd for C 21 H 25 O3, 325.1804; found 325.1811. [a] D 23 = +90.50 (c = 0.1, CHCl3).

[0102] Example 3

[0103] Griseofulvin derivatives The method for preparing (Compound 43) comprises the following steps:

[0104] (1)

[0105] Compound 37 (21 mg, 0.059 mmol, 1.0 equiv) was dissolved in tetrahydrofuran (THF, 5 mL) at 0 °C, and borane-dimethyl sulfide complex (BH3-Me2S, 40 μL, 0.4 mmol, 10 M solution in THF, 6.8 equiv) was added dropwise. The reaction mixture was warmed to room temperature and stirred for 12 h. Subsequently, aqueous sodium hydroxide (NaOH, 55 μL, 0.33 mmol, 6 N, 5.6 equiv) and 30% hydrogen peroxide (H2O2, 110 μL) were added. The reaction mixture was stirred at room temperature for another 30 min. After quenching with water, the mixture was partitioned and the aqueous phase was extracted with ethyl acetate. After combining the organic layers, they were washed with saturated brine, dried over anhydrous magnesium sulfate (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (eluent: ethyl acetate / n-hexane = 1:7) to give white solid 38 (20 mg, 90%). 20 mg, 90%).

[0106] Analytical data of white solid 38 (i.e. compound 38) are as follows: 1 H NMR (400 MHz, CDC13) δ 6.65 (s, 2H), 4.45 (s, 1H), 3.98 (dd, J = 12.4, 9.2 Hz, 1H), 3.79 (s, 3H), 3.75 (s, 3H), 3.40 (dd, J = 12.0, 7.6 Hz, 1H), 2.98 (t, J = 8.8 Hz, 1H), 2.88-2.83 (m, 2H), 3.40 (d, J = 18.0 Hz, 1H), 2.24-2.17 (m, 2H), 2.11-2.08 (m, 2H), 2.02 (t, J = 6.0 Hz, 1H), 1.81-1.46 (m, 9H), 1.20-1.13 (m, 1H), 0.93 (d, J = 6.8 Hz, 3H); 13 C NMR (100 MHz, CDC13) δ 153.6, 150.2, 127.4, 108.9, 108.7, 73.8, 73.5, 63.1, 56.1, 55.7, 53.9, 47.1, 46.4, 44.5, 42.9, 36.6, 31.8, 28.1, 25.8, 25.2, 21.0, 18.9; FTIR (Neat): v = 3362, 2920, 2851, 1713, 1658, 1633, 1475, 1434, 1254, 1122, 1093, 1077, 1046, 985, 799 cm -1 ; HRMS (ESI) m / z: [M+H] + calcd for C 23 H 33 O4, 373.2379; found 373.2379. [a]D 23 = +25.2 (c = 0.5, CHCl3).

[0107] (2) Compound 38 (0.03 mmol, 1.0 equiv) was dissolved in a mixture solution of acetonitrile (3.0 mL) and diethyl ether (3.0 mL) under ice-salt bath cooling with argon protection. Ammonium cerium (IV) nitrate (CAN, 41 mg, 0.075 mmol, 2.5 equiv) was added slowly, and the dropping time was about 3 minutes. The resulting yellow solution was stirred at 0 °C for 30 minutes. The light yellow solution was diluted with water (10 mL) and extracted with dichloromethane (15 mL x 2). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous magnesium sulfate (MgSO4), filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel to give the pleuromutilin derivative (compound 43, 5 mg, 54%).

[0108] Analytical data of compound 43 were as follows: TLC: R f = 0.12 (SiO2, petroleum ether / EtOAc 4:1); 1 HNMR (400 MHz, CDCl3) δ 6.68 (q, J = 10.0 Hz, 2H), 4.38 (s, 1H), 3.98 (dd, J = 12.0, 8.4 Hz, 1H), 3.39 (dd, J = 12.0, 7.2 Hz, 1H), 3.21-3.18 (m, 2H), 2.63-2.49 (m, 2H), 2.23-2.16 (m, 1H), 2.13-2.02 (m, 4H), 1.86-1.63 (m, 4H), 1.60-1.55 (m, 3H), 1.53-1.42 (m, 1H), 0.93 (d, J = 7.2 Hz, 3H); 13 C NMR (100 MHz, CDCl3) δ 187.1, 186.6, 143.0, 141.7, 137.7, 135.5, 74.6, 72.4, 62.9, 53.4, 47.0, 45.9, 43.2, 42.3, 36.2, 31.7, 28.2, 25.3, 25.0, 20.9, 19.0; FTIR (Neat): v = 3392, 2919, 2850, 1652, 1459, 1295, 1120, 967, 796, 720 cm -1 HRMS (ESI) m / z: [M + H]+calcd for C + H 21 H 27 O4, 343.1909; found 343.1919. [a] D23 = +51.00 (c = 0.1, CHCl3).

[0109] Example 4

[0110] A dihydropyrrolizic acid The preparation method includes the following steps:

[0111] (1)

[0112] Compound 38 ( 20 mg (0.054 mmol, 1.0 equivalent) was dissolved in dichloromethane (DCM, 2 mL), and Dess-Martin periodane (DMP, 31 mg, 0.162 mmol, 3.0 equivalent) was added with stirring at room temperature. The reaction mixture was stirred at room temperature for 30 minutes, filtered through diatomaceous earth, and concentrated under reduced pressure. The residue was purified by rapid silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1:20) to give a colorless oil 39 ( 18mg, 90%.

[0113] The analytical data for colorless oily substance 39 (i.e., compound 39) are as follows: 1 H NMR (400MHz, CDCl3) δ8.90 (s, 1H), 6.66 (q, J = 8.8Hz, 2H), 4.32 (s, 1H), 3.96 (dd, J = 12.0, 8.4Hz, 1H) ,3.77(s,3H),3.76(s,3H),3.41(dd,J=12.4,7.2Hz,1H),3.03(d,J=3.6Hz,2H),2.42(t,J=6.4Hz,1 H),2.29-2.24(m,2H),2.22-2.16(m,2H),2.13-2.09(m,1H),2.04-1.94(m,2H),1.85-1.78(m,1H), 1.71-1.70(m,1H),1.57-1.55(m,1H),1.42-1.36(m,1H),1.22-1.14(m,1H),0.93(d,J=7.2Hz,3H); 13C NMR (100 MHz, CDC13) δ 204.1, 154.0, 149.9, 126.9, 126.1, 109.33, 109.25, 74.1, 72.9, 56.0, 55.7, 53.0, 52.0, 47.6, 46.4, 46.0, 35.6, 31.9, 25.8, 25.7, 25.1, 21.1, 20.4; FTIR (Neat): v = 2924, 2853, 1714, 1671, 1477, 1463, 1436, 1256, 1186, 1122, 1099, 1078, 1045 cm -1 ; HRMS (ESI) m / z: [M + H] + calcd for C 23 H 30 O4, 371.2222; found 371.2222. [a] D 23 = +28.0 (c = 0.4, CHCl3).

[0114] (2)

[0115] Compound 39 (10 mg, 0.0274 mmol, 1.0 equiv) was dissolved in dichloromethane (DCM, 7.2 mL) and DBU (8 μL, 0.0548 mmol, 2.0 equiv) was added with stirring at room temperature. After the reaction mixture was stirred at room temperature for 12 hours, 2.0 equiv of DBU was added again. The resulting reaction mixture was continued to stir at room temperature for 24 hours, and then concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (eluent: ethyl acetate / n-hexane = 1:30) to give white foamy solid 40 (7 mg, 70%).

[0116] Analytical data of white foamy solid 40 (i.e. compound 40) are as follows: 1 H NMR (400 MHz, CDC13) δ 9.50 (d, J = 3.2 Hz, 1H), 6.67 (s, 2H), 4.58 (s, 1H), 4.01 (dd, J = 12.0, 8.4 Hz, 1H), 3.80 (s, 3H), 3.71 (s, 3H), 3.44 (dd, J = 12.0, 7.2 Hz, 1H), 2.79-2.69 (m, 2H), 2.27-1.59 (m, 11H), 1.49-1.38 (m, 1H), 1.23-1.20 (m, 1H), 0.95 (d, J = 7.2 Hz, 3H); 13 ​C NMR (100 MHz, CDC13) δ 205.0, 153.7, 151.0, 125.7, 124.9, 109.3, 108.9, 73.9, 72.6, 56.0, 55.8, 52.1, 48.5, 46.7, 46.2, 43.3, 34.8, 31.8, 26.6, 25.0, 24.0, 21.5, 20.9; FTIR (Neat): v = 2964, 2935, 2903, 2868, 1712, 1699, 1538, 1228, 1210, 1110, 1044, 1018, 710 cm -1 ; HRMS (ESI) m / z: [M + H] + calcd for C 23 H 31 O4, 371.2222; found 371.2224. [a] D 23 = +14.6 (c = 0.4, CHCI3).

[0117] (3)

[0118] Compound 40 (7 mg, 0.019 mmol, 1.0 equiv) was dissolved in tert-butanol (0.3 mL), tetrahydrofuran (THF, 5 mL) and 2-methyl-2-butene (60 μί) under ice bath, sodium chlorite (NaCI02, 14 mg, 0.190 mmol, 10.0 equiv) in water was added slowly, followed by sodium dihydrogen phosphate (NaH2P04, 23 mg, 0.190 mmol, 10.0 equiv) in water, dropwise with a time of about 15 min. After stirring at 0 °C for about 10 min, the reaction mixture was warmed to room temperature and stirred for 1 h. The reaction mixture was diluted with water (1 mL) and extracted with ethyl acetate (1 mL x 3). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (eluent: ethyl acetate / n-hexane = 1 :3) to give 22 (5 mg, 68%) as a white foamy solid.

[0119] Analytical data of white foamy solid 22 (i.e. compound 22) are as follows: TLC: R f = 0.11 (Si02, petroleum ether / EtOAc 3: 1); 1H NMR (400 MHz, CDC13) δ 6.67 (t, 9.4, 2H), 4.56 (s, 1H), 4.00 (dd, J = 12.4, 8.8 Hz, 1H), 3.80 (s, 3H), 3.73 (s, 3H), 3.43 (dd, J = 12.4, 7.2 Hz, 1H), 2.72 (d, J = 3.2 Hz, 2H), 2.23 - 1.58 (m, 12H), 1.25 - 1.20 (m, 1H), 0.94 (d, J = 7.2 Hz, 3H); 13 C NMR (100 MHz, CDC13) δ 180.3, 153.7, 151.0, 125.43, 125.38, 109.5, 108.8, 73.9, 72.6, 56.0, 52.0, 46.7, 46.2, 44.9, 42.5, 34.9, 31.8, 30.4, 25.0, 24.5, 21.8, 21.0; FTIR (Neat): v 3360, 3190, 2920, 2851, 1656, 1633, 1470, 1423, 1411, 1255, 1085 cm -1 ; HRMS (ESI) m / z: [M + H] + calcd for C 23 H 31 O5, 387.2171; found 387.2164. [a] D 23 = +4.6 (c = 0.2, CHCl3).

[0120] (4)

[0121] Compound 22 (5 mg, 0.013 mmol, 1.0 equiv) was dissolved in a solution of acetonitrile (2.0 mL) and diethyl ether (0.8 mL) under argon protection, and cooled in an ice-salt bath. Cerium ammonium nitrate (CAN, 18 mg, 0.033 mmol, 2.5 equiv) was added slowly, and the dropwise addition time was about 3 minutes. The resulting yellow solution was stirred at 0 °C for 30 minutes. After dilution with water (4 mL), the light yellow solution was extracted with dichloromethane (12 mL x 2). After the combined organic layers were washed with saturated brine (10 mL), anhydrous magnesium sulfate (MgS04) was added for drying and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (eluent: ethyl acetate / n-hexane = 2:1) to give the quinonic acid (4 mg, 87%) as a light yellow solid.

[0122] The analytical data of the light yellow solid quinonic acid (i.e., dihydro- grifolanonic acid) are as follows: TLC: Rf= 0.13 (Si02, petroleum ether / EtOAc 1:2); IR (KBr): v 3400, 2926, 2855, 1730, 1658, 1630, 1510, 1450, 1410, 1370, 1330, 1280, 1250, 1220, 1180, 1150, 1080, 1040, 1020, 980, 950, 920, 860, 830, 800, 770, 740, 700 cm f = 0.13 (Si02, petroleum ether / EtOAc 1:2); IR (KBr): v 3400, 2926, 2855, 1730, 1658, 1630, 1510, 1450, 1410, 1370, 1330, 1280, 1250, 1220, 1180, 1150, 1080, 1040, 1020, 980, 950, 920, 860, 830, 800, 770, 740, 700 cm1 H NMR (400 MHz, CDC13) δ 6.69 (dd, J = 12.0, 10.0 Hz, 2H), 4.44 (br s, 1H), 3.99 (dd, J = 12.4, 8.4 Hz, 1H), 3.46 (dd, J = 12.4, 6.4 Hz, 1H), 2.58-2.45 (m, 2H), 2.26-2.00 (m, 6H), 1.81-1.59 (m, 6H), 1.22-1.18 (m, 1H), 0.95 (d, J = 7.2 Hz, 3H); 13 CNMR (100 MHz, CDC13) δ 187.0, 186.6, 179.1, 140.6, 140.4, 137.7, 135.7, 75.0, 72.0, 51.6, 46.4, 45.8, 43.5, 42.6, 34.4, 31.9, 30.2, 24.8, 24.4, 21.7, 20.9; FTIR (Neat): v = 2921, 2852, 1657, 1632, 1467, 1261, 1119, 1100 cm -1 ; HRMS (ESI) m / z: [M + H] + calcd for C 21 H 25 O5,357.1702; found 357.1696. [a] D 23 = +106 (c = 0.2, ACN).

[0123] Example 5

[0124] A preparation method of grifolan , comprising the following steps:

[0125]

[0126] Dihydrogrifolanic acid prepared in Example 4 (2 mg, 0.006 mmol, 1.0 equivalent) was dissolved in acetonitrile (3.0 mL), and activated manganese dioxide (5 mg, 0.06 mmol, 10.0 equivalent) was added. The reaction mixture was heated to 50°C and stirred for 12 hours. The reaction solution was filtered through a nylon membrane filter (0.22 μm, 13 mm) and rinsed with acetonitrile (3.0 mL). The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column flash chromatography (eluent: ethyl acetate / n-hexane = 1:4) to obtain grifolan as a light yellow amorphous solid (1.1 mg, 55%).

[0127] The analytical data of grifolan as a light yellow amorphous solid are as follows: TLC: R f= 0.20 (SiO2, petroleum ether / EtOAc 3:1); 1 H NMR (400 MHz, CDC13) δ 6.78 (q, J = 10.4 Hz, 2H), 5.47 (dd, J = 6.8, 1.6 Hz, 1H), 4.49 (br s, 1H), 4.02 (dd, J = 12.4, 8.8 Hz, 1H), 3.35 (dd, J = 12.0, 8.8 Hz, 1H), 2.36 (dd, J = 14.4, 6.8 Hz, 1H), 2.29-2.23 (m, 2H), 2.15-2.11 (m, 1H), 2.06 (ddd, J = 12.0, 8.4, 3.2 Hz, 1H), 1.96-1.74 (m, 4H), 1.69 (qd, J = 13.2, 4.0 Hz, 1H), 1.55 (m, 1H), 1.39 (qd, J = 12, 4 Hz, 1H), 1.17 (td, J = 11.6, 6.4 Hz, 1H), 0.93 (d, J = 6.8 Hz, 3H); 13 C NMR (100 MHz, CDC13) δ 186.7, 185.3, 174.6, 143.8, 138.0, 136.0 (C*2), 73.8, 71.3, 69.4, 52.0, 48.7, 47.2, 44.5, 39.5, 32.7, 30.5, 24.8, 23.9, 22.5, 20.1; FTIR (Neat): v = 2922, 2853, 1782, 1661, 1456, 1259, 1087, 1016, 797 cm -1 ; HRMS (ESI) m / z: [M + Na] + calcd for C 21 H 22 O5Na, 377.1365; found 377.1360. [a] D 23 = -14 (c = 0.1, CHCl3).

[0128] The total reaction equation of each reaction in the embodiments of the present application is shown in Figure 1 .

[0129] Test Example

[0130] The anti-proliferative activity and thioredoxin reductase 1 (TrxR1) inhibitory activity of the grifolan prepared in Example 5, the dihydrogrifolan acid prepared in Example 4, and the grifolan derivatives (compounds 41a, 41b, 42, 43) prepared in Examples 1-3 were evaluated through cell experiments and enzyme activity experiments.

[0131] The method for testing the anti-proliferative activity is as follows: (1) Cell culture: breast cancer cell line MDA-MB-231 (ATCC) and liver cancer cell line HepG2 (ATCC) with high expression of TrxR1 are cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% (v / v) fetal bovine serum (FBS, Gibco) and 1% (v / v) penicillin / streptomycin mixture (VWR) at 37°C, 5% CO2; all cell lines are used within 1-35 passages, and mycoplasma detection is performed every 3 months. (2) Anti-proliferation experiment: 24 hours before the experiment, cells are seeded in a 96-well plate (Corning) at a density of 3000 cells per well; then different concentrations of grifolan, dihydrogrifolan or grifolan derivatives are added for 72 hours. CCK-8 method is used to evaluate cell proliferation: 10 μL of CCK-8 reagent (APExBio) is added to each well, and after incubation at 37°C for 2 hours, the absorbance (OD) at 450 nm is measured using a multifunctional enzyme label instrument (VICTOR Nivo, PerkinElmer); the percentage of absorbance of each concentration group relative to the control group represents the cell survival rate, the experiment is set in triplicate, and the IC 50 value is calculated by normalizing the cell growth curve using GraphPad Prism 9 software, with auranofin as a positive control.

[0132] The method for testing the activity of TrxR1 in vitro is as follows: the DTNB method is used to determine the inhibitory activity of grifolan, dihydrogrifolan or grifolan derivatives on TrxR1: TrxR1_C647S / U648C (1.2 μM) reduced with 200 μM NADPH and different concentrations of grifolan, dihydrogrifolan or grifolan derivatives in TE buffer (50 mM Tris-HCl, 1 mM EDTA, pH 8.0) are incubated in a 96-well plate at 37°C for 2 hours (total volume of mixture 50 μL); then 200 μM NADPH and 6 mM DTNB in TE buffer (100 μL) are added, and the absorbance at 412 nm is measured using a Multiskan spectrophotometer (Thermo); after the inhibition is completed (120 minutes), the TrxR1 enzyme activity is calculated, and auranofin is also used as a positive control.

[0133] The test results are shown in Table 1:

[0134] Table 1 Anti-proliferative activity data of grifolan, dihydrogrifolan and grifolan derivatives and inhibitory activity data on isolated TrxR1

[0135]

[0136] Note: a Data from CCK-8 method (anti-proliferative activity detection) and DTNB method (in vitro TrxR1 activity detection), results expressed as mean ± standard deviation (SD), n = 3 (i.e. 3 independent experiments).

[0137] As can be seen from Table 1, the C7 / C8 substituted grifolan derivatives (compounds 41b, 42, 43) have significantly stronger inhibitory activity on tumor cells than grifolan. Among them, the C8 position containing exocyclic double bond grifolan derivative (compound 42) and the C8 position substituted with axial hydroxyl grifolan derivative (compound 43) have particularly prominent anti-proliferative activity in MDA-MB-231 breast cancer cells and HepG2 hepatoma cells: in MDA-MB-231 cells, the IC 50 values of compound 42 and compound 43 are 4.65 μM and 4.09 μM, respectively, which are much lower than the 39.32 μM of grifolan; in HepG2 cells, the IC 50 values of compound 42 and compound 43 are further reduced to 2.06 μM and 1.42 μM, which are significantly better than the 5.76 μM of grifolan.

[0138] Further, the DTNB method was used to determine the inhibitory activity of grifolan and soluble grifolan derivatives (compound 41b, compound 43) on purified TrxR1. The results show that after incubating reduced rat recombinant TrxR1 with the above grifolan and soluble grifolan derivatives, strong enzyme inhibition can be produced: the IC 50 of grifolan is 0.99 μM, the IC 50 of grifolan derivative (compound 41b) is reduced to 0.23 μM, and the IC 50 of grifolan derivative (compound 43) is only 0.11 μM; the IC 50 of auranofin, the positive control of TrxR inhibitor, is 1.84 μM. Therefore, the inhibitory activity of grifolan derivative (compound 43) on TrxR1 is 9 times that of the parent natural product grifolan and 16.7 times that of the positive control auranofin.

[0139] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Those skilled in the art should understand that on the basis of the above description, other different forms of changes or variations can also be made. Here, it is not necessary and impossible to exhaust all the embodiments. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A derivative of grifolan, characterized in that, The structural formula of the grifolan derivative is selected from one of the following structural formulas:

2. A method for preparing the grifolan derivative according to claim 1, characterized by, comprising the following steps: (1) adding dissolved in organic solvent, adding selenium dioxide, and carrying out the reaction at 60-100°C, and after the reaction is completed, separating and purifying to obtain (2) adding a cerium-containing oxidizing agent to the mixture to perform an oxidation reaction under inert gas protection at -20 to 10°C The oxidation reaction is completed, and the griseofulvin derivative is obtained by post-treatment and separation and purification or the griseofulvin derivative 3. The production method according to claim 2, characterized by, In step (1), the process for the preparation of a compound of formula (I) comprises the steps of: S1. Lithium hexamethyldisilazane is added to a solution of the compound of formula (I) in an inert solvent at -80 to -70°C under an inert gas atmosphere, and the reaction is allowed to proceed at -80 to -70°C for 1 to 2 hours. After the reaction in an organic solvent, trifluoromethanesulfonyl aniline is added, and after the reaction is completed at 20 to 40°C, the intermediate is obtained by treatment; the intermediate is reacted with a palladium-containing catalyst and an organic zinc reagent in a solvent at 80 to 100°C, and the product is obtained by separation and purification. S2. under inert gas atmosphere, at -30 to -10 °C, is added dropwise to a solution of After reduction by the reducing agent, oxidation by the oxidizing agent at 20-40 °C, separation and purification to obtain S3. at 40-60 °C After treatment with hydrofluoric acid-pyridine complex, and then reaction catalyzed by Lewis acid at -30 to -10 °C, the product was obtained by separation and purification 4. A method for preparing the grifolan derivative according to claim 1, characterized by, comprising the following steps: In an inert gas atmosphere, the compound of formula (I) is dissolved in a mixed solvent of nitrile and ether, and then a cerium-containing oxidizing agent is added for oxidation reaction under the protection of inert gas at -20 to 10°C. After the oxidation reaction is completed, the gray side fungus derivative is obtained by post-treatment and separation and purification. and heated to react with dicyclopentadienyl dimethyl titanium in an organic solvent at 60-100°C. After the reaction is completed, the gray side fungus derivative is obtained by post-treatment and separation and purification. and dissolved in a mixed solvent of nitrile and ether. Under the protection of inert gas at -20 to 10°C, a cerium-containing oxidizing agent is added for oxidation reaction. After the oxidation reaction is completed, the gray side fungus derivative is obtained by post-treatment and separation and purification. ​ 5. The preparation method according to claim 4, characterized in that, The The method of preparing the compound of Formula (I) comprises the steps of: Under an inert gas atmosphere, After reacting with a borane-dimethyl sulfide complex in an organic solvent at 20-40°C, sodium hydroxide and hydrogen peroxide are added for oxidation, followed by separation and purification to obtain an alcohol intermediate. The alcohol intermediate is then reacted with oxalyl chloride and dimethyl sulfoxide at -80°C to -70°C, followed by the addition of triethylamine and reaction at 20-40°C, followed by separation and purification to obtain...

6. A method for preparing the grifolan derivative according to claim 1, characterized by, comprising the following steps: At -10℃ to 10℃, The reaction with the borane-dimethyl sulfide complex in an organic solvent was continued at 20-40°C; subsequently, sodium hydroxide and hydrogen peroxide were added for oxidation. After the reaction was complete, the product was obtained through post-treatment and purification. Will Dissolved in a mixed solvent of nitrile and ether, under inert gas protection at -20°C to 10°C, an oxidation reaction is carried out by adding a cerium-containing oxidant. After the oxidation reaction is completed, post-treatment and separation purification are performed to obtain the pleurotin derivative.

7. Use of the grifolan derivative of claim 1 in the preparation of an antitumor drug.

8. Use of the grifolan derivative of claim 1 in the preparation of a TrxR inhibitor.

9. A method for preparing dihydroagaric acid, characterized by, comprising the following steps: P1. The compound of formula (I) is prepared by the process comprising: with a hypervalent iodine oxidizing agent in an organic solvent at 20-40 °C, and isolated and purified to obtain P2. The method of P1, wherein the compound of formula (I) is prepared by the process of P2. with 1,8-diazabicyclo[5.4.0]undecane in an organic solvent at 20-40 °C, and isolating and purifying P3. will be After reacting with sodium chlorite and sodium dihydrogen phosphate in a mixed solvent at -10 to 10 °C for 5-15 min, the temperature is raised to 20-40 °C for reaction, and then separated and purified to obtain P4. Under inert gas protection and at -20 to 10 °C, the compound of P3 is reacted with a cerium-containing oxidizing agent in a mixed solvent of a nitrile and an ether, and dihydro-grisolic acid is obtained by separation and purification ​ 10. A method of preparing grifolan, characterized by, comprising the following steps: The dihydropyrrolidone obtained by the preparation method according to claim 9 The reaction was carried out with manganese dioxide in an organic solvent at 40-60°C for 10-14 hours. After the reaction was completed, the pleurotin was obtained through post-processing and purification.

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