WO2021228186A1 - 穿心莲内酯类化合物及其制备方法、药物组合物和其在抗肿瘤药物和抗病毒药物中的应用 - Google Patents

穿心莲内酯类化合物及其制备方法、药物组合物和其在抗肿瘤药物和抗病毒药物中的应用 Download PDF

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WO2021228186A1
WO2021228186A1 PCT/CN2021/093539 CN2021093539W WO2021228186A1 WO 2021228186 A1 WO2021228186 A1 WO 2021228186A1 CN 2021093539 W CN2021093539 W CN 2021093539W WO 2021228186 A1 WO2021228186 A1 WO 2021228186A1
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compound
preparation
andrographolide
group
substituted
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PCT/CN2021/093539
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English (en)
French (fr)
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朱振东
车超
蒋顶
邢琦
张家银
黎婷
林光
杨震
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深圳湾实验室
北京大学深圳研究生院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/056—Ortho-condensed systems with two or more oxygen atoms as ring hetero atoms in the oxygen-containing ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • the present invention relates to the field of chemical pharmacy, and more specifically, to an andrographolide compound, a preparation method thereof, a pharmaceutical composition and its application in antineoplastic drugs and antiviral drugs.
  • Natural products are of great significance to the field of pharmaceutical research. So far, more than one-third of the drugs used in clinical applications are directly derived from natural products or derivatives developed based on the active ingredients of natural products as lead compounds.
  • the diverse structure of natural products and their easy combination with biological macromolecules determine their incomparable advantages in participating in the physiological processes of life, which give natural products an irreplaceable important position in the development of new drugs. It is an important source for discovering drug candidates and drug-leading structures.
  • Andrographolide is one of the main diterpene lactone components extracted from the Acanthaceae plant Andrographis, and its structure is shown in the following formula:
  • Andrographolide has shown moderate anti-tumor effects in many in vivo and in vitro models, and can inhibit leukemia cells, liver cancer, breast cancer, lung cancer, prostate cancer, colon cancer, pancreatic cancer, brain cancer, gastric cancer, bone cancer, skin cancer, and The growth of other tumor cells has become a promising and potential anti-tumor drug candidate. Nevertheless, due to the low bioavailability, low water solubility (74 ⁇ g/ml) and relatively weak stability of andrographolide, its anti-tumor application is subject to certain restrictions, and structural modification work has become a solution to its low bioavailability, The main method to enhance clinical efficacy.
  • One of the objectives of the present invention is to provide a new type of andrographolide compound, which can modify the structure of andrographolide, improve the biological activity of andrographolide, and enhance the antitumor and antiviral effects of andrographolide.
  • -R 1 is selected from -H, benzyl, substituted benzyl, aryl, heterocyclyl, heteroaryl, substituted C 1 ⁇ C 10 linear alkyl, substituted C 3 ⁇ C 10 Any one of branched chain alkyl and substituted cycloalkyl;
  • -R 2 and -R 3 are the same, said -R 2 and said -R 3 are selected from any one of -H, methyl, ethyl, propyl and -(O)CR 4 , -R 4 is selected Any one of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, substituted phenyl, furyl, thienyl and pyridyl;
  • -R 5 is -H, alkyl or aryl
  • -R 6 is -H, alkyl or aryl.
  • the second objective of the present invention is to provide a method for preparing the above-mentioned andrographolide compounds, and the technical scheme is as follows:
  • compound A the structure of compound A is:
  • M 1 is alkyl or -(O)CR 4 , -R 4 is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, substituted phenyl, furyl, thienyl And any one of pyridyl;
  • -R 2 and -R 3 are the same, said -R 2 and said -R 3 are selected from any one of -H, methyl, ethyl, propyl and -(O)CR 4 , -R 4 is selected Any one of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, substituted phenyl, furyl, thienyl and pyridyl;
  • -R 2 and -R 3 together form the following groups: -C(R 5 )(R 6 )-, -R 5 is -H, alkyl or aryl, -R 6 is -H, alkyl or aryl base;
  • the compound A, amine and alkaline medium are added to an organic solvent to react to obtain compound B.
  • the chemical formula of the amine is R 1 -NH 2
  • -R 1 is selected from the group consisting of -H, benzyl, and substituted Any one of benzyl, aryl, heterocyclyl, heteroaryl, substituted C 1 to C 10 linear alkyl, substituted C 3 to C 10 branched alkyl, and substituted cycloalkyl
  • the structure of the compound B is:
  • the compound B, the hydrogen atom transfer donor, and the photo-redox catalyst are added to an organic solvent, and the reaction occurs under light irradiation to obtain compound C.
  • the structure of compound C is:
  • the present invention also discloses a pharmaceutical composition comprising the above-mentioned andrographolide compound, and discloses the application of the above-mentioned andrographolide compound and the pharmaceutical composition containing the same in the preparation of antitumor drugs or antiviral drugs.
  • the present invention breaks through the existing modification of the hydroxyl groups at positions 3, 19 and 14 by introducing an amine group at the 12 position and re-bonding the introduced amine group with the C at the 17 position.
  • a new type of 12,17-amino-substituted tricyclic andrographolide compound is formed, which enhances the biological activity of the compound. It has been experimentally verified that the compound has an inhibitory effect on tumor cells and viruses.
  • the structural modification of andrographolide is an important way to discover the framework of new anti-tumor drugs.
  • the structural modification of andrographolide mainly includes: esterification, etherification and oxidation of the three hydroxyl groups in different degrees; reduction, epoxidation and epoxidation of terminal olefins; and conjugated olefins. Reduction, addition and [3+2] cycloaddition etc.
  • the present invention proposes new ideas and strategies for modification, using some novel synthetic methods and technologies to try to modify other positions of andrographolide, or use andrographolide derivatives as substrates for further modification and modification, in order to find more valuable Drug skeleton.
  • -R 1 is selected from -H, benzyl, substituted benzyl, aryl, heterocyclyl, heteroaryl, substituted C 1 ⁇ C 10 linear alkyl, substituted Any one of C 3 to C 10 branched alkyl and substituted cycloalkyl; -R 2 and -R 3 are the same, and -R 2 and -R 3 are selected from -H, methyl, Any one of ethyl, propyl and -(O)CR 4 , -R 4 is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, substituted phenyl, furanyl Any one of, thienyl and pyridyl.
  • R 2 and R 3 together form the following groups: -R 5 is -H, alkyl or aryl; -R 6 is -H, alkyl or aryl. R 5 and R 6 may be the same or different. Specifically, the structure of andrographolide compounds is shown in the following formula:
  • alkyl refers to a saturated hydrocarbon group, which contains only C and H atoms, including cycloalkyl, straight-chain alkyl or branched alkyl, among which, cycloalkyl, straight-chain alkyl or branched alkyl Both can be substituted or unsubstituted.
  • C 1 ⁇ C 10 linear alkyl groups and C 3 ⁇ C 10 branched chain alkyl groups include, but are not limited to, -CH 2 CH(CH 3 ) 2 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH 2 CH 2 CH 3 or -CH 2 CH 2 CH(CH 3 ) 2 etc., substituted C 1 ⁇ C 10 linear alkyl and substituted Substituents on the C 3 -C 10 branched alkyl group are optional.
  • cycloalkyl refers to a monovalent, non-aromatic, saturated or partially unsaturated cyclic alkyl group, which can be a C 3 ⁇ C 12 monocyclic cycloalkyl group or a C 7 ⁇ C 12 bicyclic cycloalkyl group Or a C 7 ⁇ C 12 spirocyclic cycloalkyl group, which may be substituted or unsubstituted.
  • the substituted cycloalkyl group includes one, two or more substituents.
  • the substituents may be, but are not limited to, hydroxyl, Any one of amino, -X, methyl, ethyl, -CX 3 , -OCH 3 , -OCH 2 CH 3 , cyano, ester, carboxy, and nitro, where X is F, Cl , Br or I atom.
  • the cycloalkyl group can be, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, Wait.
  • benzyl refers to the monovalent group remaining after removing a hydrogen atom from the methyl carbon in the toluene molecule.
  • substituted benzyl contains one, two or more substituents on the benzene ring.
  • the substituents can be, but are not limited to, hydroxyl, amino, -X, methyl, ethyl, -CX 3 , C 3 cycloalkyl, -OCH 3 , -OCH 2 CH 3 , phenyl, pyridyl, pyrimidinyl, thienyl, furyl, pyrrolyl, pyrazolyl, thiazolyl, indolyl, benzo Thienyl, benzofuranyl, morpholinyl, piperidinyl, piperazinyl, tetrahydropyrrolyl, cyano, ester, carboxyl, sulfonyl, sulfonylamino, aminosulfonyl, amide, aminoacyl, Any one of a ureido group, a thiour
  • aryl refers to the general term for the monovalent group after removing a hydrogen atom from the aromatic nucleus carbon of the aromatic hydrocarbon molecule. Both the aryl group and the fused ring aryl group can be substituted or unsubstituted.
  • the benzene ring of the substituted monocyclic aryl group or the substituted fused ring aryl group contains one, two or more substituents, and the substituents can be Yes, but not limited to, any one of hydroxyl, amino, -X, methyl, ethyl, -CX 3 , -OCH 3 , -OCH 2 CH 3 and nitro, where X is F, Cl , Br or I atom.
  • the aryl group may include, but is not limited to, phenyl, biphenyl, naphthyl, and the like.
  • heterocyclic group refers to a heteroatom-containing cyclic group, which can be a monocyclic heterocyclic group of 3 to 12 atoms, a bicyclic heterocyclic group of 7 to 12 atoms, or a spiro heterocyclic group of 7 to 12 atoms.
  • One or more atoms in the ring of a cyclic group, a monocyclic heterocyclic group, a bicyclic heterocyclic group or a spirocyclic heterocyclic group can be independently optionally substituted by a heteroatom, and the heteroatom can be N, O, S or P, etc.
  • the ring can be fully saturated or contain one or more degrees of unsaturation, one or more hydrogen atoms on the ring can be replaced by one or more substituents, the substituents can be, but not limited to, hydroxyl, amino , -X, methyl, ethyl, -CX 3 , -OCH 3 , -OCH 2 CH 3 , cyano, ester, carboxy and nitro, etc., where X is F, Cl, Br Or I atom.
  • the heterocyclic group may include, but is not limited to, Imidazolyl, thienyl or furyl, pyridyl, pyrimidinyl, thiapyrrolyl, pyrazolyl, thiazolyl, indolyl, benzothienyl, benzofuranyl, morpholinyl, piperidinyl, piperazine Group, tetrahydropyrrolyl, etc.
  • heteroaryl refers to a monocyclic heterocyclic group containing 1 to 3 benzene ring substituents, a spirocyclic heterocyclic group containing 1 to 3 benzene ring substituents, and 1 to 3 monocyclic heterocyclic groups. Substituent phenyl or spirocyclic heterocyclic group containing 1 to 3 monocyclic heterocyclic substituents.
  • Monocyclic heterocyclic group refers to a monocyclic ring system containing one or more heteroatoms, benzene ring and/or monocyclic ring
  • the heterocyclic group may include one, two or more than two substituents, and the substituents may be, but are not limited to, hydroxyl, amino, -X, methyl, ethyl, -CX 3 , C 3 cycloalkane Any one of group, -OCH 3 , -OCH 2 CH 3 , phenyl, cyano, ester, carboxy, and nitro, wherein X is an F, Cl, Br, or I atom.
  • the heteroaryl group may be, but is not limited to, benzimidazolyl, benzothienyl or benzofuranyl, indole and the like.
  • -R 1 is selected from -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH 2 CH 2 CH 2 CH 3 , -CH 2 CH(CH 3 ) 2 , -CH(CH 3 ) 2 , -CH 2 CH 2 CH(CH 3 ), cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
  • X is an F, Cl, Br or I atom
  • X 1 is an F, Cl, Br or I atom
  • X 2 is an F, Cl, Br or I atom
  • -R 2 and -R 3 are -H or -OAc; or, -R 2 and -R 3 together form the following group: -C(CH 3 ) 2 -or -CH 2 -.
  • the andrographolide compound is specifically a structure of one of the following formulas:
  • the andrographolide compounds of the present invention may also be enantiomers, diastereomers, tautomers, salts, solvates, or isotopically substituted molecules at various positions of the compound represented by the above formula (I).
  • the invention also discloses a preparation method of the above-mentioned andrographolide compound, which comprises the following steps:
  • M 1 is alkyl or -(O)CR 4 , -R 4 is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, substituted phenyl, furyl, thienyl And any one of pyridyl;
  • -R 2 and -R 3 are the same, -R 2 and -R 3 are selected from any one of -H, methyl, ethyl, propyl and -(O)CR 4 , -R 4 is selected from methyl, Any one of ethyl, propyl, isopropyl, butyl, isobutyl, substituted phenyl, furyl, thienyl and pyridyl;
  • -R 5 is -H, alkyl or aryl
  • -R 6 is -H, alkyl or aryl.
  • the chemical formula of the amine is R 1 -NH 2
  • -R 1 is selected from -H, cycloalkyl, benzyl, Any one of substituted benzyl, aryl, heterocyclyl, heteroaryl, C 1 ⁇ C 10 linear alkyl group and C 3 ⁇ C 10 branched chain alkyl group, the structure of compound B is:
  • the amine undergoes an amination substitution reaction on the 12-position.
  • the amine can be various aliphatic amines or aromatic amines.
  • -R 1 , -R 2 and -R 3 have been introduced in detail in the previous paragraphs, so I will not repeat them here.
  • the organic solvent is selected from one, two, three or more of dichloromethane, tetrahydrofuran, diethyl ether, 1,4-dioxane, toluene, benzene and chloroform; alkali
  • the sexual medium is selected from one, two or three of triethylamine, diisopropylethylamine and pyridine, and triethylamine is most preferred.
  • the structure of compound C is:
  • the photo-redox catalyst is selected from any one of metal iridium catalysts, metal rhodium catalysts and organic dye catalysts; the equivalent of the photo-redox catalyst is 1%-10% of the equivalent of compound B, most preferably 2%.
  • the equivalent weight of the hydrogen atom transfer donor is 20% to 100% of the equivalent weight of compound B, most preferably 50%;
  • the hydrogen atom transfer donor is selected from 2,4,6-triisopropylthiophenol, 1,4-bis Any one of diethyl hydrogen-2,6-dimethyl-3,5-pyridinedicarboxylate and 1,4-cyclohexadiene; the wavelength range of light is 300nm-500nm, and the preferred wavelength is 460nm;
  • the organic solvent may be at least one of toluene, benzene, 1,4-dioxane, dichloromethane, chloroform, tetrahydrofuran, and the like, and toluene is preferred.
  • compound A can be prepared using andrographolide as a raw material, and there can be two specific preparation methods, which are as follows:
  • the acylating agent can be acid anhydride or acid halide; acid anhydride and acid halide can be substituted or unsubstituted, and the substituents can be various aliphatic or aromatic substituents .
  • the chemical formula of acid anhydride is: R 4 -C(O)-OC(O)-R 4
  • the chemical formula of acid halide is R 4 -C(O)-halogen
  • -R 4 is selected from methyl, ethyl, Any one of propyl, isopropyl, butyl, isobutyl, substituted phenyl, furanyl, thienyl and pyridyl; the definition and explanation of "halogen" in the preceding paragraphs also apply to this paragraph "halogen".
  • the above-mentioned esterification reaction can be catalyzed by Lewis acid such as anhydrous ZnCl 2.
  • the organic solvent may be one, two or more of dichloromethane, tetrahydrofuran, diethyl ether, 1,4-dioxane, toluene, etc., preferably dichloromethane.
  • the reaction is carried out at a temperature of 25°C-100°C (preferably 50°C) for 2h-5h.
  • the acylating agent can also be replaced by a halogenated hydrocarbon.
  • the remaining reaction conditions remain unchanged.
  • the halogenated hydrocarbon and -OH undergo condensation reaction.
  • the chemical formula of the halogenated hydrocarbon is: alkyl-halogen.
  • the definitions and explanations of "" and “halogen” also apply to "alkyl” and "halogen” in this paragraph.
  • the alkyl group may be a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, and the like.
  • the andrographolide, carbonylation reagent and acid catalyst are added to the reaction medium.
  • a reaction temperature of 60°C ⁇ 100°C preferably 80°C
  • the carbonyl group of the carbonylation reagent and the 3-position and 19-position of andrographolide- OH undergoes condensation reaction to obtain compound D.
  • the carbonylation reagent performs ketal or acetal modification on the -OH at positions 3 and 19.
  • the chemical formula of the carbonylation reagent is R 5 -C(OMe) 2 -R 6 or R 5- CO-R 6 , -R 5 and -R 6 can be independently selected from the following groups: -H, alkyl or aryl.
  • alkyl and aryl in the preceding paragraphs apply equally “Alkyl” and "aryl” in this paragraph. In this way, the structure of the obtained compound D is:
  • the reaction uses a mixed solvent of toluene and dimethyl sulfoxide as the reaction medium, and p-toluenesulfonic acid or concentrated sulfuric acid as the acid catalyst.
  • the equivalent of the acid catalyst is 10%-40% of the equivalent of andrographolide, most preferably 20%; the equivalent weight of the carbonylation reagent is 100%-600% of the equivalent weight of andrographolide, most preferably 400%.
  • compound D is added to an organic solvent, an acylating agent or halogenated hydrocarbon is added, and a basic medium is added, and the acylating agent or halogenated hydrocarbon reacts with the -OH at position 14 of compound D to obtain compound A.
  • the acylating agent is acid anhydride or acid halide;
  • the chemical formula of acid anhydride is: R 4 -C(O)-OC(O)-R 4
  • the chemical formula of acid halide is R 4 -C(O) -Halogen, -R 4 is selected from any one of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, substituted phenyl, furyl, thienyl and pyridyl, halogenated hydrocarbon
  • the chemical formula of is: alkyl-halogen.
  • alkyl may be a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, and the like.
  • the equivalent of the acylating agent or halogenated hydrocarbon is 100% to 600% of the equivalent of compound D;
  • the organic solvent is dichloromethane, tetrahydrofuran, diethyl ether, 1,4-dioxane and toluene
  • the reaction temperature is from room temperature to reflux temperature.
  • compound C can also be used as the structural skeleton to further expand andrographolide derivatives.
  • the present invention also restores the -OH positions 3 and 19, using compound C-II as a substrate, adding a reaction solvent, adding an acid, and a hydrolysis reaction to obtain compound E.
  • the structure of compound E is:
  • the hydrolysis reaction is carried out under acidic conditions, and the reaction solvent is selected from one, two or more of tetrahydrofuran, 1,4-dioxane and water; the acid is selected from hydrochloric acid, p-toluenesulfonic acid, Either sulfuric acid or acetic acid. More preferably, the reaction solvent is tetrahydrofuran and water, the acid is acetic acid, and the volume ratio of acetic acid, tetrahydrofuran and water is (3.5-4.5): (0.5-1.5): (1.5-2.5).
  • the invention also discloses a pharmaceutical composition, which comprises the above-mentioned andrographolide compound.
  • present invention further discloses the application of the above-mentioned andrographolide compound or the above-mentioned pharmaceutical composition in the preparation of antineoplastic drugs.
  • the anti-tumor drug is anti-nasopharyngeal cancer, cervical cancer, liver cancer, breast cancer, lung cancer, prostate cancer, colon cancer, pancreatic cancer, brain cancer, gastric cancer, bone cancer, skin cancer or leukemia drugs.
  • andrographolide and its derivatives can effectively enhance the immune regulation of T cells, stimulate the host immune system to attack viruses, and provide protection against multiple viral infections.
  • andrographolide and its derivatives are effective against influenza A virus (IAV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus type I (HSV-1), and Epstein-Barr virus (EBV).
  • influenza A virus IAV
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • HSV-1 herpes simplex virus type I
  • EBV Epstein-Barr virus
  • HPV human papillomavirus
  • HMV human immunodeficiency virus
  • other viruses show broad-spectrum antiviral activity.
  • the andrographolide compounds of the present invention are also effective against influenza A virus (IAV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus type I (HSV-1), EB virus ( EBV), human papillomavirus (HPV), human immunodeficiency virus (HIV) and other viruses have antiviral activity.
  • IAV influenza A virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • HSV-1 herpes simplex virus type I
  • EBV EB virus
  • HPV human papillomavirus
  • HAV human immunodeficiency virus
  • SARS-CoV-2 The current outbreak of SARS-CoV-2 is the third recorded spread of animal coronaviruses to humans in the past 20 years, causing a major global epidemic. Tests have verified that the andrographolide compounds of the present invention are also resistant to Coronavirus activity.
  • the compound III-3 was prepared according to the preparation method in Example 1. In an 8ml glass bottle, sequentially add the aminated andrographolide compound III-3 (0.05mmol, 1 equivalent), the photoredox catalyst Ir(ppy) 2 (dtbbpy)]PF 6 (2mol%), 2,4 ,6-Triisopropylthiophenol (6mg, 0.025mmol, 50mol%), then add dried toluene (2ml), change the nitrogen gas 5 times with an oil pump, irradiate with two 40W Kessil KSH150B blue LED lamps, room temperature After stirring for 18 hours, the compound Ia-3 was separated by column chromatography with a yield of 50%.
  • Example 1 Refer to the preparation method in Example 1 to prepare compound III-6.
  • the aminated andrographolide compound III-6 (0.05mmol, 1 equivalent), 9-s-trimethylphenyl-2,7,10-trimethylacridine perchloric acid Salt (6mol%), 2,4,6-triisopropylthiophenol (6mg, 0.025mmol, 50mol%), then add dried toluene (2ml), change nitrogen with oil pump 5 times, use two Irradiated with 40W Kessil KSH150B blue LED lamp, stirred at room temperature for 18 hours, and separated by column chromatography to obtain compound Ia-6 with a yield of 79%.
  • the compound Ia-7 was prepared by referring to the preparation method in Example 6 with a yield of 53%.
  • the compound Ia-8 was prepared by referring to the preparation method in Example 6, with a yield of 53%.
  • the compound Ia-9 was prepared by referring to the preparation method in Example 6, with a yield of 57%.
  • the compound Ia-10 was prepared by referring to the preparation method in Example 6, with a yield of 44%.
  • the compound Ia-11 was prepared by referring to the preparation method in Example 6, and the yield was 47%.
  • the compound I-a-12 was prepared by referring to the preparation method in Example 6 with a yield of 57%.
  • the compound Ia-13 was prepared by referring to the preparation method in Example 6, with a yield of 60%.
  • the compound Ia-14 was prepared by referring to the preparation method in Example 6, and the yield was 57%.
  • the compound Ia-15 was prepared by referring to the preparation method in Example 6, with a yield of 53%.
  • the compound Ia-16 was prepared by referring to the preparation method in Example 6, with a yield of 60%.
  • the compound Ia-17 was prepared by referring to the preparation method in Example 6, and the yield was 53%.
  • the compound Ia-19 was prepared by referring to the preparation method in Example 6, with a yield of 43%.
  • the compound Ia-20 was prepared by referring to the preparation method in Example 6, with a yield of 30%.
  • the compound Ia-21 was prepared with reference to the preparation method in Example 6 with a yield of 50%.
  • the compound Ia-22 was prepared by referring to the preparation method in Example 6 with a yield of 47%.
  • the compound Ia-23 was prepared by referring to the preparation method in Example 6, and the yield was 37%.
  • the compound Ia-24 was prepared by referring to the preparation method in Example 1, with a yield of 47%.
  • the compound Ia-25 was prepared by referring to the preparation method in Example 1, with a yield of 37%.
  • the compound Ia-26 was prepared by referring to the preparation method in Example 1, with a yield of 43%.
  • Andrographolide 1 (10.0g, 28.54mmol) was dissolved in toluene (200ml) and dimethyl sulfoxide (27ml), and then 2,2-dimethoxypropane (14ml) and p-toluenesulfonic acid (catalyzed Equivalent) is added to the reaction system. The temperature was raised to 80 degrees Celsius and the reaction was stirred for 2 hours, and then the system was cooled to room temperature, and the reaction was quenched by adding 7 ml of triethylamine.
  • reaction was extracted with 150 ml of dichloromethane, washed with water, separated, dried with anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and beaten with ether to obtain 8.65 g of off-white solid compound IV-1 with a yield of 78%.
  • the compound VI-1 (30mg, 1.0 equivalent), 9-s-trimethyl-2,7,10-trimethylacridine perchlorate (6mol%) and 2,4,6-triisopropylbenzene -1-thiophenol (0.5 equivalent) was added to a transparent 8*16mm reaction tube with a Teflon stopper. Seal the reaction tube, add 2ml anhydrous toluene, replace with nitrogen more than 3 times, place the reaction tube under two 40W Kessil KSH150B blue lights and stir the reaction, the system gradually turns dark brown, monitor the reaction with a TLC plate until The raw materials react thoroughly. The reaction system was concentrated under reduced pressure and separated with a preparative thin-layer chromatography plate to obtain 20 mg of compound I-b-1 with a yield of 67%.
  • the compound Ib-14 was prepared by referring to the preparation method in Example 27, and the yield was 58%.
  • the compound Ib-16 was prepared by referring to the preparation method in Example 27 with a yield of 57%.
  • Andrographolide 1 (10.0g, 28.54mmol) was dissolved in toluene (200ml) and dimethyl sulfoxide (27ml), and then 2,2-dimethoxypropane (14ml) and p-toluenesulfonic acid (catalyzed Equivalent) is added to the reaction system. The temperature was raised to 80 degrees Celsius and the reaction was stirred for 2 hours, and then the system was cooled to room temperature, and the reaction was quenched by adding 7 ml of triethylamine.
  • reaction was extracted with 150 ml of dichloromethane, washed with water, separated, dried with anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and beaten with ether to obtain 8.65 g of a white solid compound IV-2 with a yield of 78%.
  • the compound Ib-21 was prepared by referring to the preparation method in Example 46 with a yield of 53%.
  • the compound Ib-23 was prepared by referring to the preparation method in Example 46 with a yield of 60%.
  • the compound Ic-3 was prepared by referring to the preparation method in Example 50, and the yield was 93%.
  • the compound Id-6 was prepared according to the preparation method in Example 59.
  • the compound Ia-27 was prepared according to the preparation method in Example 6.
  • Cells such as nasopharyngeal cancer, cervical cancer, liver cancer and leukemia were selected as the research objects, and andrographolide was used as a positive control to screen the anti-tumor activity of andrographolide derivatives.
  • the selected cell lines are: human EBV-positive nasopharyngeal carcinoma cells C666-1 and HONE1-ebv, human cervical cancer cells HeLa, human promyelocytic leukemia cells HL-60, and human liver cancer cells Huh7.
  • the main points of the Resazurin experimental method are as follows: use logarithmic growth phase tumor cells, inoculate them in 96-well cell culture plates, 100 ⁇ L per well, add C666-1 to each well for 7,000, and HONE1-ebv, HeLa, and Huh 7 for 2,000. , HL-60 is 20000, and each experimental group is set with 2 replicate holes. After culturing for 24 hours, 0.5 ⁇ L of compound DMSO solution was added to each well to make the final concentration 50 ⁇ M. In addition, the wells with 0.5 ⁇ L DMSO were set as positive controls, and the wells with medium only were set as negative controls.
  • the cytopathic model was used to evaluate the in vitro anti-coronavirus 229E activity of the compound.
  • MRC5 cells product number CCL-171
  • human coronavirus HCoV 229E product number VR-740
  • ATCC American Type Culture Collection
  • MRC5 cells were cultured in EMEM (Sigma) medium supplemented with 10% fetal bovine serum (Excell), 1% double antibody (Hyclone), 1% L-glutamine (Gibco) and 1% non-essential amino acids (Gibco).
  • EMEM culture medium supplemented with 5% fetal bovine serum, 1% double antibody, 1% L-glutamine and 1% non-essential amino acids was used as the experimental culture medium.
  • MRC5 cells were seeded in a 96 microwell plate at a density of 20,000 cells per well at a density of 100 ⁇ l per well and cultured overnight in a 5% CO 2 , 37°C incubator.
  • 50 ⁇ l of compound was added to each well (double repertoire), and then 50 ⁇ l of diluted virus was added to the cell test well at 200 TCID 50 per well, and the final compound concentration was 25 ⁇ M.
  • Set up cell control (cells, no compound treatment or virus infection), virus control (cells infected with virus, no compound treatment) and culture medium control (only culture medium).
  • the final volume of the experimental culture medium is 200 ⁇ l per well, and the final concentration of DMSO in the culture medium is 0.5%.
  • the cells were cultured in a 5% CO 2 , 35°C incubator for 3 days.
  • Cell viability detection kit CellTiter Glo (Promega) was used to detect cell viability.
  • the cytotoxicity test is the same as the antiviral test, but there is no virus infection.
  • the antiviral activity and cytotoxicity of the compound are represented by the inhibitory rate (%) and cell viability (%) of the compound on the cytopathic effect caused by the virus at a single concentration (25 ⁇ M), respectively. Calculated as follows:
  • Inhibition rate (%) (reading value of test well-average value of virus control)/(average value of cell control-average value of virus control) ⁇ 100.
  • Cell viability (%) (reading value of test well-average value of culture solution control)/(average value of cell control-average value of culture solution control) ⁇ 100.
  • Table 2 shows the inhibitory activity of andrographolide derivatives on human coronavirus HCoV-229E. It can be seen that many compounds show excellent anti-coronavirus activity.

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Abstract

本发明实施例公开了一种穿心莲内酯类化合物及其制备方法、药物组合物和其在抗肿瘤药物和抗病毒药物中的应用，本发明在对穿心莲内酯的结构改造的尝试中，在12位引入胺基，并使引入的胺基和17位的C重新成键，形成了新型的12,17-氨基取代的三环穿心莲内酯类化合物，增强了化合物的生物活性，经实验验证，该类化合物对肿瘤细胞和病毒具有抑制作用。

Description

穿心莲内酯类化合物及其制备方法、药物组合物和其在抗肿瘤药物和抗病毒药物中的应用 技术领域
本发明涉及化学制药领域，更具体地，涉及一种穿心莲内酯类化合物及其制备方法、药物组合物和其在抗肿瘤药物和抗病毒药物中的应用。
背景技术
天然产物对药物研究领域有着重要的意义，迄今为止，临床应用的药物中有三分之一以上直接来自天然产物或是以天然产物活性成分为先导化合物发展出来的衍生物。天然产物的多样性结构和易于与生物大分子结合的特点，决定了其在参与生命生理过程中所具有的无可比拟的优势，这些都赋予了天然产物在新药研发中不可替代的重要地位，是发现候选药物和药物先导结构的重要来源。
穿心莲内酯(Andrographo1ide)是从爵床科植物穿心莲中提取得到的主要二萜内酯成分之一，其结构如下式所示：
Figure PCTCN2021093539-appb-000001
药理研究表明，穿心莲内酯及其衍生物具有消炎、抗菌、抗病毒感染、抗肿瘤、抗氧化、抗糖尿病、免疫调节、降血脂、保护心脏、肝以及神经等功效，被誉为天然抗生素药物。因其资源广泛，在动物体内吸收快且无明显的毒副作用，故具有较高的临床药用价值。比如，脱水穿心莲内酯琥珀酸半酯(穿琥宁注射液，炎琥宁注射液)、莲必治注射液、以及穿心莲内酯总酯磺化物(喜炎平注射液)被广泛的在临床使用，用于治疗上呼吸道感染等疾病。
近年来，国内外关于穿心莲内酯在抗肿瘤方面的研究已成为关注的热点。研 究发现，穿心莲内酯及其衍生物通过各种不同的作用机制发挥抗肿瘤作用，其作用机制包括诱导肿瘤细胞凋亡、坏死和自噬、氧化应激诱导细胞死亡、阻滞肿瘤细胞周期、抑制肿瘤新生血管形成、抗炎和免疫系统介导的作用等多方面。穿心莲内酯在许多体内外模型上表现出中等强度的抗肿瘤作用，可以抑制白血病细胞、肝癌、乳腺癌、肺癌、前列腺癌、结肠癌、胰腺癌、脑癌、胃癌、骨癌、皮肤癌以及其它肿瘤细胞的生长，已经成为一类有前景、有潜力的抗肿瘤候选药物。尽管如此，由于穿心莲内酯具有生物利用度低，低水溶性(74μg/ml)以及相对较弱的稳定性，导致其抗肿瘤应用受到一定的限制，结构改造工作成为解决其生物利用度低、增强临床疗效的主要方法。
申请内容
本发明的目的之一为提供一种新型的穿心莲内酯类化合物，对穿心莲内酯的结构进行改造，提高穿心莲内酯的生物活性，增强穿心莲内酯的抗肿瘤和抗病毒药效。
为实现上述目的，本发明的技术方案如下：
一种穿心莲内酯类化合物，其结构如式(Ⅰ)所示：
Figure PCTCN2021093539-appb-000002
其中，-R 1选自-H、苄基、取代的苄基、芳基、杂环基、杂芳基、取代的C 1～C 10的直链烷基、取代的C 3～C 10的支链烷基和取代的环烷基中的任意一种；
-R 2和-R 3相同，所述-R 2和所述-R 3选自-H、甲基、乙基、丙基和-(O)CR 4中的任意一种，-R 4选自甲基、乙基、丙基、异丙基、丁基、异丁基、取代的苯基、呋喃基、噻吩基和吡啶基中的任意一种；
或者-R 2和-R 3一起形成下列基团：
Figure PCTCN2021093539-appb-000003
-R 5为-H、烷基或芳基，-R 6为 -H、烷基或芳基。
本发明的目的之二为提供上述穿心莲内酯类化合物的制备方法，技术方案如下：
提供化合物A，所述化合物A的结构为：
Figure PCTCN2021093539-appb-000004
其中，M 1为烷基或-(O)CR 4，-R 4选自甲基、乙基、丙基、异丙基、丁基、异丁基、取代的苯基、呋喃基、噻吩基和吡啶基中的任意一种；
-R 2和-R 3相同，所述-R 2和所述-R 3选自-H、甲基、乙基、丙基和-(O)CR 4中的任意一种，-R 4选自甲基、乙基、丙基、异丙基、丁基、异丁基、取代的苯基、呋喃基、噻吩基和吡啶基中的任意一种；
或者-R 2和-R 3一起形成下列基团：-C(R 5)(R 6)-，-R 5为-H、烷基或芳基，-R 6为-H、烷基或芳基；
将所述化合物A、胺和碱性介质加入有机溶剂中，发生反应，得到化合物B，所述胺的化学式为R 1-NH 2，-R 1选自选自-H、苄基、取代的苄基、芳基、杂环基、杂芳基、取代的C 1～C 10的直链烷基、取代的C 3～C 10的支链烷基和取代的环烷基中的任意一种，所述化合物B的结构为：
Figure PCTCN2021093539-appb-000005
将所述化合物B、氢原子转移供体和光氧化还原催化剂加入有机溶剂中，在光的照射下发生反应，得到化合物C，所述化合物C的结构为：
Figure PCTCN2021093539-appb-000006
本发明还公开了包括上述穿心莲内酯类化合物的药物组合物，以及公开了上述穿心莲内酯类化合物和包含其的药物组合物在制备抗肿瘤药物中或抗病毒药物中的应用。
实施本发明实施例，将具有如下有益效果：
本发明在结构改造的尝试中，突破了现有的对3位、19位和14位的羟基的改造，在12位引入胺基，并使引入的胺基和17位的C重新成键，形成了新型的12,17-氨基取代的三环穿心莲内酯类化合物，增强了化合物的生物活性，经实验验证，该化合物对肿瘤细胞和病毒具有抑制作用。
具体实施方式
下面将结合具体实施例中对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
对穿心莲内酯进行结构改造是发现新型抗肿瘤药物骨架的重要途径。目前，对穿心莲内酯的结构改造主要包括：对三个羟基不同程度的酯化、醚化以及氧化等；对端基烯烃的还原、环氧化以及环氧化开环等；对共轭烯烃的还原、加成以及[3+2]环加成等。本发明提出了新的改造思路和策略，利用一些新颖的合成方法和技术对穿心莲内酯的其他位置尝试改造，或以穿心莲内酯衍生物为底物进行进一步修饰改造，以期发现更多有价值的药物骨架。
下面简写词的使用贯穿本发明：
-Ac：CH 3CO-，乙酰基
-Me：CH 3，甲基
Bu t：叔丁基
Et：CH 3CH 2-，乙基
AcOH：乙酸
THF：四氢呋喃
EtOH：CH 3CH 2OH，乙醇
本发明的穿心莲内酯类化合物，其结构如式(Ⅰ)所示：
Figure PCTCN2021093539-appb-000007
所述式(Ⅰ)中，-R 1选自-H、苄基、取代的苄基、芳基、杂环基、杂芳基、取代的C 1～C 10的直链烷基、取代的C 3～C 10的支链烷基和取代的环烷基中的任意一种；-R 2和-R 3相同，所述-R 2和所述-R 3选自-H、甲基、乙基、丙基和-(O)CR 4中的任意一种，-R 4选自甲基、乙基、丙基、异丙基、丁基、异丁基、取代的苯基、呋喃基、噻吩基和吡啶基中的任意一种。
或者R 2和R 3一起形成下列基团：
Figure PCTCN2021093539-appb-000008
-R 5为-H、烷基或芳基；-R 6为-H、烷基或芳基。R 5和R 6可以相同，也可以不同。具体的，穿心莲内酯类化合物的结构如下式所示：
Figure PCTCN2021093539-appb-000009
上述的“烷基”是指饱和烃基，只含有C和H两种原子，包括环烷基、直链烷基或支链烷基，其中，环烷基、直链烷基或支链烷基均可以是取代的也可以 是非取代的。
在本发明的具体实施例中，C 1～C 10的直链烷基和C 3～C 10的支链烷基包括，但不限于，-CH 2CH(CH 3) 2、-CH 2CH 2CH 3、-CH(CH 3) 2、-CH 2CH 2CH 2CH 3或-CH 2CH 2CH(CH 3) 2等，取代的C 1～C 10的直链烷基和取代的C 3～C 10的支链烷基上的取代基任选。
上述的“环烷基”是指一价，非芳香族，饱和或部分不饱和环状烷基，可以是C 3～C 12的单环环烷基、C 7～C 12的双环环烷基或C 7～C 12的螺环环烷基，可以是取代的或非取代的，取代的环烷基包括一个、两个或两个以上取代基，取代基可以是，但不限于，羟基、胺基、-X、甲基、乙基、-CX 3、-OCH 3、-OCH 2CH 3、氰基、酯基、羧基和硝基等中的任意一种，其中，X为F、Cl、Br或I原子。在本发明的具体实施例中，环烷基可以是，但不限于，环丙基、环丁基、环戊基、环己基、环庚基、
Figure PCTCN2021093539-appb-000010
Figure PCTCN2021093539-appb-000011
Figure PCTCN2021093539-appb-000012
等。
上述的“苄基”是指甲苯分子中的甲基碳上去掉一个氢原子后剩下的一价基团。
上述的“取代的苄基”的苯环上含有一个、两个或两个以上的取代基，取代 基可以是，但不限于，羟基、胺基、-X、甲基、乙基、-CX 3、C 3的环烷基、-OCH 3、-OCH 2CH 3、苯基、吡啶基、嘧啶基、噻吩基、呋喃基、吡咯基、吡唑基、噻唑基、吲哚基、苯并噻吩基、苯并呋喃基、吗啉基、哌啶基、哌嗪基、四氢吡咯基、氰基、酯基、羧基、磺酰基、磺酰氨基、氨基磺酰基、酰胺基、氨基酰基、脲基、硫脲基、硝基、螺环基或螺环杂环基等中的任意一种，其中，X为F、Cl、Br或I原子。在本发明具体实施例中，取代的苄基包括，但不限于，
Figure PCTCN2021093539-appb-000013
Figure PCTCN2021093539-appb-000014
Figure PCTCN2021093539-appb-000015
Figure PCTCN2021093539-appb-000016
等中的任意一种。
上述的“芳基”是指芳烃分子的芳核碳上去掉一个氢原子后，剩下一价基团的总称，其可以为单环芳基或2～5环的稠环芳基，单环芳基和稠环芳基均可可以为取代或非取代的，取代的单环芳基或取代的稠环芳基的苯环上含有一个、两个或两个以上的取代基，取代基可以是，但不限于，羟基、胺基、-X、甲基、乙基、-CX 3、-OCH 3、-OCH 2CH 3和硝基等中的任意一种，其中，X为F、Cl、Br或I原子。在本发明的具体实施例中，芳基可以包括，但不限于，苯基、联苯基 或萘基等。
上述的“杂环基”是指含有杂原子的环基，可以为3～12个原子的单环杂环基、7～12个原子的双环杂环基或7～12个原子的螺环杂环基，单环杂环基、双环杂环基或螺环杂环基的环上一个或多个原子可以独立任选地被杂原子所取代，杂原子可以是N、O、S或P等，环可以是完全饱和的或包含一个或多个不饱和度，一个或多个环上的氢原子可以被一个或多个取代基所取代，取代基可以是，但不限于，羟基、胺基、-X、甲基、乙基、-CX 3、-OCH 3、-OCH 2CH 3、氰基、酯基、羧基和硝基等中的任意一种，其中，X为F、Cl、Br或I原子。在本发明的具体实施例中，杂环基可以包括，但不限于，
Figure PCTCN2021093539-appb-000017
咪唑基、噻吩基或呋喃基、吡啶基、嘧啶基、噻吡咯基、吡唑基、噻唑基、吲哚基、苯并噻吩基、苯并呋喃基、吗啉基、哌啶基、哌嗪基、四氢吡咯基等。
上述的“杂芳基”是指含有1～3个苯环取代基的单环杂环基、含有1～3个苯环取代基的螺环杂环基、含有1～3个单环杂环取代基的苯基或含有1～3个单环杂环取代基的螺环杂环基，单环杂环基是指包含一个或多个杂原子的单环体系，苯环和/或单环杂环基上可以包括一个、两个或两个以上的取代基，取代基可以是，但不限于，羟基、胺基、-X、甲基、乙基、-CX 3、C 3的环烷基、-OCH 3、-OCH 2CH 3、苯基、氰基、酯基、羧基和硝基等中的任意一种，其中，X为F、Cl、Br或I原子。在本发明的具体实施例中，杂芳基可以是，但不限于，苯并咪唑基、苯并噻吩基或苯并呋喃基、吲哚等。
优选地，-R 1选自-CH 3、-CH 2CH 3、-CH 2CH 2CH 3、-CH 2CH 2CH 2CH 3、-CH 2CH(CH 3) 2、-CH(CH 3) 2、-CH 2CH 2CH(CH 3)、环丙基、环丁基、环戊基、环己基、环庚基、
Figure PCTCN2021093539-appb-000018
Figure PCTCN2021093539-appb-000019
Figure PCTCN2021093539-appb-000020
Figure PCTCN2021093539-appb-000021
苯基、联苯基、萘基、苯并咪唑基、苯并噻吩基、苯并呋喃基、咪唑基、噻吩基、呋喃基、
Figure PCTCN2021093539-appb-000022
Figure PCTCN2021093539-appb-000023
Figure PCTCN2021093539-appb-000024
Figure PCTCN2021093539-appb-000025
中的任意一种；其中，X为F、Cl、Br或I原子，X 1为F、Cl、Br或I原子，X 2为F、Cl、Br或I原子。
优选地，-R 2和-R 3为-H或-OAc；或者，-R 2和-R 3一起形成下列基团：-C(CH 3) 2-或-CH 2-。
在本发明的具体实施例中，穿心莲内酯类化合物具体为下列式中之一的结构：
Figure PCTCN2021093539-appb-000026
Figure PCTCN2021093539-appb-000027
Figure PCTCN2021093539-appb-000028
本发明的穿心莲内酯类化合物还可以是上述式(I)所示化合物的对映体、 非对映体、互变体、盐、溶剂化物或各位置同位素取代形式的分子。
本发明还公开了上述穿心莲内酯类化合物的制备方法，包括以下步骤：
步骤1)：提供化合物A，化合物A的结构为：
Figure PCTCN2021093539-appb-000029
其中，M 1为烷基或-(O)CR 4，-R 4选自甲基、乙基、丙基、异丙基、丁基、异丁基、取代的苯基、呋喃基、噻吩基和吡啶基中的任意一种；
-R 2和-R 3相同，-R 2和-R 3选自-H、甲基、乙基、丙基和-(O)CR 4中的任意一种，-R 4选自甲基、乙基、丙基、异丙基、丁基、异丁基、取代的苯基、呋喃基、噻吩基和吡啶基中的任意一种；
或者-R 2和-R 3一起形成下列基团：
Figure PCTCN2021093539-appb-000030
-R 5为-H、烷基或芳基，-R 6为-H、烷基或芳基。
步骤2)：将化合物A、胺和碱性介质加入有机溶剂中，发生反应，得到化合物B，胺的化学式为R 1-NH 2，-R 1选自-H、环烷基、苄基、取代的苄基、芳基、杂环基、杂芳基、C 1～C 10的直链烷基和C 3～C 10的支链烷基中的任意一种，化合物B的结构为：
Figure PCTCN2021093539-appb-000031
在本步骤中，胺对12位进行氨基化取代反应，具体的，胺可以为各种脂肪胺或芳香胺。-R 1、-R 2和-R 3在前面段落中已详细介绍，在此不一一赘述。
在本步骤中，优选地，有机溶剂选自二氯甲烷、四氢呋喃、乙醚、1,4-二氧六环、甲苯、苯和氯仿中的一种、两种、三种或三种以上；碱性介质选自三乙胺、二异丙基乙基胺和吡啶中的一种、两种或三种，最优选三乙胺。
步骤3)：将化合物B、氢原子转移供体和光氧化还原催化剂加入有机溶剂中，在光的照射下发生反应，得到化合物C，化合物C的结构为：
Figure PCTCN2021093539-appb-000032
在本步骤中，发生分子内胺氢化反应，利用光氧化还原催化剂进行光催化反应。优选地，光氧化还原催化剂选自金属铱催化剂、金属铑催化剂和有机染料类催化剂中的任意一种；光氧化还原催化剂的当量为化合物B的当量的1％～10％，最优选2％。氢原子转移供体的当量为化合物B的当量的20％～100％，最优选50％；氢原子转移供体选自2,4,6-三异丙基苯硫酚、1,4-二氢-2,6-二甲基-3,5-吡啶二羧酸二乙酯和1,4-环己二烯中的任意一种；光的波长范围为300nm～500nm，优选波长为460nm；有机溶剂可以为甲苯、苯、1,4-二氧六环、二氯甲烷、氯仿和四氢呋喃等中的至少一种，优选甲苯。
具体的，化合物A可以以穿心莲内酯为原料来制备，具体的制备方法可以有两种，分别如下：
第一种
将穿心莲内酯加入有机溶剂中，加入酰化剂或卤代烃，加入路易斯酸作为催化剂，酰化剂或卤代烃与穿心莲内酯的3位、19位和14位的-OH发生反应，得到化合物A。
在本步骤中，酰化剂与-OH发生酯化反应，酰化剂可以为酸酐或酰卤；酸酐和酰卤可以为取代或非取代，取代基可以为各种脂肪族或芳香族取代基。具体的，酸酐的化学式为：R 4-C(O)-O-C(O)-R 4，酰卤的化学式为R 4-C(O)-卤素，-R 4选自 甲基、乙基、丙基、异丙基、丁基、异丁基、取代的苯基、呋喃基、噻吩基和吡啶基中的任意一种；前面段落中对“卤素”的定义和解释同样适用本段落中的“卤素”。
上述酯化反应可以使用无水ZnCl 2等路易斯酸进行催化。
有机溶剂可以为二氯甲烷、四氢呋喃、乙醚、1,4-二氧六环和甲苯等中的一种、两种或两种以上，优选二氯甲烷。
优选地，于25℃～100℃温度下反应(优选50℃)，反应2h～5h。
如此，得到的化合物A中3位、19位和14位的取代基-M 1＝-R 2＝-R 3＝-(O)CR 4。
上述过程中，酰化剂还可以用卤代烃代替，其余反应条件不变，卤代烃与-OH发生缩合反应，卤代烃的化学式为：烷基-卤素，前面段落中对“烷基”和“卤素”的定义和解释同样适用本段落中的“烷基”和“卤素”。得到的化合物A中的3位、19位和14位的取代基-M 1＝-R 2＝-R 3＝烷基。具体的，烷基可以为甲基、乙基、丙基、异丙基、丁基、异丁基等。
进一步的，通过上述步骤2)和步骤3)得到化合物C，具体的反应过程如下所示：
Figure PCTCN2021093539-appb-000033
第二种
将穿心莲内酯、羰基化试剂和酸催化剂加入到反应介质中，在60℃～100℃(优选80℃)的反应温度下，羰基化试剂的羰基与穿心莲内酯的3位和19位的-OH发生缩合反应，得到化合物D。
在本步骤中，羰基化试剂对3位和19位的-OH进行缩酮或缩醛的改造，具体的，羰基化试剂的化学式为R 5-C(OMe) 2-R 6或R 5-CO-R 6，-R 5和-R 6可以分别 独立的选自以下基团：-H、烷基或芳基，前面段落中对“烷基”和“芳基”的定义和解释同样适用本段落中的“烷基”和“芳基”。如此，得到的化合物D的结构为：
Figure PCTCN2021093539-appb-000034
优选地，该反应以甲苯和二甲亚砜的混合溶剂为反应介质，以对甲苯磺酸或浓硫酸作酸催化剂，酸催化剂的当量为穿心莲内酯的当量的10％～40％，最优选20％；羰基化试剂的当量为穿心莲内酯的当量的100％～600％，最优选400％。
进一步地，将化合物D加入有机溶剂中，加入酰化剂或卤代烃，加入碱性介质，酰化剂或卤代烃与化合物D的14位的-OH发生反应，得到化合物A。
在本步骤中，具体的，酰化剂为酸酐或酰卤；酸酐的化学式为：R 4-C(O)-O-C(O)-R 4，酰卤的化学式为R 4-C(O)-卤素，-R 4选自甲基、乙基、丙基、异丙基、丁基、异丁基、取代的苯基、呋喃基、噻吩基和吡啶基中的任意一种，卤代烃的化学式为：烷基-卤素，前面段落中对“烷基”、“卤素”和“芳基”的定义和解释同样适用本段落中的“烷基”、“卤素”和“芳基”。具体的，烷基可以为甲基、乙基、丙基、异丙基、丁基、异丁基等。
如此，得到的化合物A中的-M 1＝烷基或-(O)CR 4。
在本步骤中，优选地，酰化剂或卤代烃的当量为化合物D的当量的100％～600％；有机溶剂为二氯甲烷、四氢呋喃、乙醚、1,4-二氧六环和甲苯等中的一种、两种或两种以上，优选二氯甲烷；以三乙胺、二异丙基乙基胺、吡啶等中的一种、两种或两种以上作碱性介质，最优选三乙胺，碱性介质的当量为化合物D的当量的100％～600％。反应温度为室温到回流温度。
进一步的，通过上述步骤2)和步骤3)得到化合物C，具体的反应过程如下所示：
Figure PCTCN2021093539-appb-000035
为了增加上述化合物C的水溶性或生物活性，还可以以化合物C为结构骨架，进一步拓展穿心莲内酯衍生物。
本发明还对3位和19位的-OH进行恢复，以化合物C-II为底物，加入反应溶剂，加入酸，发生水解反应，得到化合物E，化合物E的结构为：
Figure PCTCN2021093539-appb-000036
在本步骤中，在酸性条件下进行水解反应，反应溶剂选自四氢呋喃、1,4-二氧六环和水中的一种、两种或两种以上；酸选自盐酸、对甲苯磺酸、硫酸和醋酸中的任意一种。更优地，反应溶剂为四氢呋喃和水，酸为醋酸，醋酸、四氢呋喃和水的体积比为(3.5～4.5)：(0.5～1.5)：(1.5～2.5)。
本发明还公开了一种药物组合物，包括上述的穿心莲内酯类化合物。
以及本发明进一步公开了上述的穿心莲内酯类化合物或上述的药物组合物在制备抗肿瘤药物中的应用。
优选地，抗肿瘤药物是抗鼻咽癌、宫颈癌、肝癌、乳腺癌、肺癌、前列腺癌、结肠癌、胰腺癌、脑癌、胃癌、骨癌、皮肤癌或白血病类类药物。
除了抗肿瘤的活性外，穿心莲内酯及其衍生物通过有效增强T细胞的免疫调节作用，刺激宿主免疫系统攻击病毒，对多种病毒感染提供保护。据报道，穿心莲内酯及其衍生物对甲型流感病毒(IAV)、乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、I型单纯疱疹病毒(HSV-1)、EB病毒(EBV)、人乳头瘤病毒(HPV)、人类 免疫缺陷病毒(HIV)等病毒表现出广谱的抗病毒活性。因此，本发明的穿心莲内酯类化合物也对甲型流感病毒(IAV)、乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、I型单纯疱疹病毒(HSV-1)、EB病毒(EBV)、人乳头瘤病毒(HPV)、人类免疫缺陷病毒(HIV)等病毒具有抗病毒活性。
目前爆发的SARS-CoV-2是过去20年来有记载的第三次动物冠状病毒对人类的传播，导致了全球性的重大流行病，经试验验证，本发明的穿心莲内酯类化合物还具有抗冠状病毒活性。
以下为具体实施例
实施例1
化合物I-a-1的制备：
在20ml的圆底烧瓶中，加入穿心莲内酯1(1.75g，5mmol)和20ml乙酸酐，搅拌5min之后，再称入催化量的无水ZnCl 2(100mg，10mol％)，升温至50℃，剧烈搅拌4h。悬浮液变得澄清时，降温至室温，加10ml EtOH和5ml水搅拌30min，抽滤得到白色固体II-1(1.9g，收率92％)。
Figure PCTCN2021093539-appb-000037
在10ml的圆底烧瓶中，依次加入乙酰氧基保护的穿心连内酯II-1(190mg，0.4mmol)，4-氟苄胺(0.72mmol，3当量)，三乙胺(0.17ml，1.2mmol，1.2当量)，再加入3ml二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，柱层析分离纯化得到产物III-1，收率28％。
Figure PCTCN2021093539-appb-000038
在8ml的玻璃瓶中，依次加入氨基化的穿心莲内酯化合物III-1(0.05mmol， 1当量)，光氧化还原催化剂Ir(dF(Me)ppy) 2(dtbbpy)]PF 6(2mol％)，2,4,6-三异丙基苯硫酚(6mg，0.025mmol，50mol％)，然后再加入干燥过的甲苯(2ml)，用油泵换氮气5次，用两盏40W Kessil KSH150B blue LED lamp照射，室温搅拌18h，柱层析分离得到化合物I-a-1,收率53％。
化合物I-a-1的结构测定： 1H NMR(500MHz,CDCl 3)δ7.40(s,1H),7.22(dd,J＝8.4,5.6Hz,2H),6.97(t,J＝8.7Hz,2H),4.79(s,2H),4.56(dd,J＝11.1,4.8Hz,1H),4.36(d,J＝11.7Hz,1H),4.12–4.07(m,1H),3.77(d,J＝13.8Hz,1H),3.14(d,J＝9.3Hz,1H),2.92(d,J＝13.8Hz,1H),2.77(dd,J＝11.1,3.2Hz,1H),2.02(d,J＝3.8Hz,6H),1.80–1.42(m,10H),1.16–1.04(m,2H),0.98(s,3H),0.85–0.76(m,5H)； 13C NMR(125MHz,CDCl 3)δ173.26,170.87,170.42,162.85,160.90,145.78,137.22,134.71,134.68,129.78,129.72,115.15,114.98,80.02,70.30,64.96,60.18,59.24,58.51,55.22,53.77,41.18,36.63,35.93,34.90,32.97,31.82,23.78,22.58,21.74,21.12,21.02,14.43；HRMS(m/z)calc.for C 31H 41FNO 6(+)542.2918,found 542.2918。
实施例2
化合物I-a-2的制备：
Figure PCTCN2021093539-appb-000039
参照实施例1中的制备方法制备化合物III-2。在8ml的玻璃瓶中，依次加入氨基化的穿心莲内酯化合物III-2(0.05mmol，1当量)，光氧化还原催化剂Ir(dFppy) 2(dtbbpy)]PF 6(2mol％)，2,4,6-三异丙基苯硫酚(6mg，0.025mmol，50mol％)，然后再加入干燥过的甲苯(2ml)，用油泵换氮气5次，用两盏40W Kessil KSH150B blue LED lamp照射，室温搅拌18h，柱层析分离得到化合物I-a-2,收率23％。
化合物I-a-2的结构测定： 1H NMR(500MHz,CDCl 3)δ7.39(s,1H),7.28–7.26(m,1H),7.22(t,J＝7.8Hz,2H),7.17(d,J＝7.0Hz,1H),4.79(s,2H),4.57(dd,J＝11.1,4.6Hz,1H),4.37(d,J＝11.7Hz,1H),4.11(d,J＝11.8Hz,1H),3.78(d,J＝14.0Hz,1H),3.15(d,J＝9.6Hz,1H),2.93(d,J＝14.0Hz,1H),2.76(dd,J＝11.1,3.2Hz,1H),2.03(d,J＝4.3Hz,6H),1.78–1.38(m,10H),1.17–1.03(m,2H),0.99(s,3H),0.85(s,5H)； 13C NMR(125MHz,CDCl 3)δ173.36,170.99,170.53,145.91,137.17,132.59,129.65,129.07,128.48,128.26,125.33,80.05,77.33,77.07,76.82,70.40,65.02,60.27,59.25,58.61,55.22,53.74,41.19,36.64,35.96,34.91,32.99,31.82,23.80,22.63,21.76,21.21,21.11,14.47；HRMS(m/z)calc.for C 31H 41ClNO 6(+)558.2622,found 558.2620。
实施例3
化合物I-a-3的制备：
Figure PCTCN2021093539-appb-000040
参照实施例1中的制备方法制备化合物III-3。在8ml的玻璃瓶中，依次加入氨基化的穿心莲内酯化合物III-3(0.05mmol，1当量)，光氧化还原催化剂Ir(ppy) 2(dtbbpy)]PF 6(2mol％)，2,4,6-三异丙基苯硫酚(6mg，0.025mmol，50mol％)，然后再加入干燥过的甲苯(2ml)，用油泵换氮气5次，用两盏40W Kessil KSH150B blue LED lamp照射，室温搅拌18h，柱层析分离得到化合物I-a-3,收率50％。
化合物I-a-3的结构测定： 1H NMR(300MHz,CDCl 3)δ7.41(d,J＝8.2Hz,3H),7.16(d,J＝8.2Hz,2H),4.79(s,2H),4.57(dd,J＝10.1,5.6Hz,1H),4.37(d,J＝11.7Hz,1H),4.14–4.07(m,1H),3.76(d,J＝14.0Hz,1H),3.15(d,J＝10.1Hz,1H),2.92(d,J＝14.0Hz,1H),2.76(d,J＝8.3Hz,1H),2.03(d,J＝2.8Hz,6H),1.82 –1.36(m,10H),1.17–1.02(m,2H),0.98(s,3H),0.88–0.71(m,5H)； 13C NMR(75MHz,CDCl 3)δ173.56,171.21,170.74,146.14,138.34,137.31,131.63,130.24,120.86,80.22,77.58,77.26,76.94,70.62,65.22,60.44,59.42,58.82,55.39,53.90,41.37,36.82,36.15,35.07,33.16,31.99,23.98,22.82,21.94,21.41,21.32,14.66；HRMS(m/z)calc.for C 31H 41BrNO 6(+)602.2117,found 602.2110。
实施例4
化合物I-a-4的制备：
Figure PCTCN2021093539-appb-000041
参照实施例1中的制备方法制备化合物III-4。在8ml的玻璃瓶中，依次加入氨基化的穿心莲内酯化合物III-4(0.05mmol，1当量)，光氧化还原催化剂Ir(dF(CF 3)ppy) 2(dtbbpy)]PF 6(6mol％)，2,4,6-三异丙基苯硫酚(6mg，0.025mmol，50mol％)，然后再加入干燥过的甲苯(2ml)，用油泵换氮气5次，用两盏40W Kessil KSH150B blue LED lamp照射，室温搅拌18h，柱层析分离得到化合物I-a-4,收率52％。
化合物I-a-4的结构测定： 1H NMR(300MHz,CDCl 3)δ7.55(d,J＝8.1Hz,2H),7.41(d,J＝7.4Hz,3H),4.79(s,2H),4.57(dd,J＝10.2,5.6Hz,1H),4.37(d,J＝11.7Hz,1H),4.11(d,J＝11.6Hz,1H),3.86(d,J＝14.3Hz,1H),3.19(d,J＝9.7Hz,1H),3.03(d,J＝14.4Hz,1H),2.76(dd,J＝10.7,2.5Hz,1H),2.03(d,J＝2.2Hz,6H),1.87–1.34(m,10H),1.21–1.12(m,1H),1.12–1.04(m,1H),0.99(s,3H),0.91–0.75(m,5H)； 13C NMR(75MHz,CDCl 3)δ173.53,171.21,170.75,146.17,143.71,137.31,129.64,129.26,128.63,128.45,125.50,125.46,80.22,77.58,77.26,76.94,70.60,65.22,60.61,59.44,59.03,55.40,53.90,41.37,36.82,36.16,35.10,33.15,31.98,23.98,22.82,21.94,21.41,21.31,14.66；HRMS(m/z)calc.for C 32H 41F 3NO 6 (+)592.2886,found 592.2880。
实施例5
化合物I-a-5的制备：
Figure PCTCN2021093539-appb-000042
参照实施例1中的制备方法制备化合物III-5。在8ml的玻璃瓶中，依次加入氨基化的穿心莲内酯化合物III-5(0.05mmol，1当量)，光氧化还原催化剂9-均三甲基苯基-10-三甲基吖啶四氟硼酸盐(6mol％)，2,4,6-三异丙基苯硫酚(6mg，0.025mmol，50mol％)，然后再加入干燥过的甲苯(2ml)，用油泵换氮气5次，用两盏40W Kessil KSH150B blue LED lamp照射，室温搅拌18h，柱层析分离得到化合物I-a-5,收率40％。
化合物I-a-5的结构测定： 1H NMR(300MHz,CDCl 3)δ8.16(d,J＝8.6Hz,2H),7.48(d,J＝8.5Hz,2H),7.40(s,1H),4.80(s,2H),4.57(dd,J＝10.0,5.5Hz,1H),4.37(d,J＝11.7Hz,1H),4.12(d,J＝11.7Hz,1H),3.89(d,J＝14.7Hz,1H),3.22(d,J＝9.4Hz,1H),3.09(d,J＝14.9Hz,1H),2.77–2.64(m,1H),2.03(d,J＝1.9Hz,6H),1.74–1.57(m,10H),1.08(d,J＝11.0Hz,2H),0.99(s,3H),0.87(s,5H)； 13C NMR(75MHz,CDCl 3)δ173.41,171.23,170.78,147.51,147.33,146.34,137.22,129.01,123.87,80.22,77.59,77.27,76.95,70.60,65.23,60.78,59.50,58.92,55.41,53.92,41.39,36.84,36.19,35.14,33.07,31.98,23.99,22.84,21.94,21.44,21.34,14.69；HRMS(m/z)calc.for C 31H 41FN 2O 8(+)569.2863,found 569.2857。
实施例6
化合物I-a-6的制备：
Figure PCTCN2021093539-appb-000043
参照实施例1中的制备方法制备化合物III-6。在8ml的玻璃瓶中，依次加入氨基化的穿心莲内酯化合物III-6(0.05mmol，1当量)，9-均三甲基苯基-2,7,10-三甲基吖啶高氯酸盐(6mol％)，2,4,6-三异丙基苯硫酚(6mg，0.025mmol，50mol％)，然后再加入干燥过的甲苯(2ml)，用油泵换氮气5次，用两盏40W Kessil KSH150B blue LED lamp照射，室温搅拌18h，柱层析分离得到化合物I-a-6,收率79％。
化合物I-a-6的结构测定： 1H NMR(300MHz,CDCl 3)δ7.42(s,1H),7.37–7.18(m,5H),4.78(s,2H),4.57(dd,J＝10.2,5.6Hz,1H),4.36(d,J＝11.7Hz,1H),4.10(d,J＝12.0Hz,1H),3.82(s,1H),3.15(d,J＝9.9Hz,1H),2.96(d,J＝13.9Hz,1H),2.87–2.73(m,1H),2.02(d,J＝2.3Hz,6H),1.60(qdd,J＝26.7,12.8,2.8Hz,10H),1.11(dd,J＝27.1,7.5Hz,2H),0.98(s,3H),0.88–0.74(m,5H)； 13C NMR(75MHz,CDCl 3)δ173.65,171.15,170.68,146.01,139.28,137.42,130.11,128.50,127.09,80.21,77.58,77.26,76.94,70.59,65.18,60.47,59.48,59.41,55.37,53.89,41.34,36.79,36.11,35.06,33.23,31.99,29.96,29.88,29.51,27.40,23.97,22.79,21.92,21.38,21.28,21.24,14.63；HRMS(m/z)calc.for C 31H 42NO 6(+)524.3012,found 524.3004。
实施例7
化合物I-a-7的制备：
Figure PCTCN2021093539-appb-000044
参照实施例6中的制备方法制备化合物I-a-7，收率53％。化合物I-a-7的结 构测定： 1H NMR(500MHz,CDCl 3)δ7.42(s,1H),7.15(d,J＝7.9Hz,2H),7.10(d,J＝7.8Hz,2H),4.79(s,2H),4.57(dd,J＝11.2,4.8Hz,1H),4.37(d,J＝11.7Hz,1H),4.11(d,J＝11.7Hz,1H),3.80(d,J＝13.7Hz,1H),3.14(d,J＝10.3Hz,1H),2.94(d,J＝13.8Hz,1H),2.83(dd,J＝11.1,2.7Hz,1H),2.32(s,3H),2.02(d,J＝3.7Hz,6H),1.80–1.40(m,10H),1.09(dd,J＝21.0,19.2Hz,2H),0.98(s,3H),0.85–0.76(m,5H)； 13C NMR(125MHz,CDCl 3)δ173.64,171.14,170.68,145.93,137.53,136.73,136.06,129.22,128.54,80.29,77.51,77.26,77.01,70.59,65.22,60.41,59.41,59.19,55.47,53.99,41.43,36.87,36.17,35.13,33.29,32.06,29.91,24.03,22.83,21.99,21.38,21.29,14.67,14.31；HRMS(m/z)calc.for C 32H 44NO 6(+)538.3169,found 538.3159。
实施例8
化合物I-a-8的制备：
Figure PCTCN2021093539-appb-000045
参照实施例6中的制备方法制备化合物I-a-8，收率53％。化合物I-a-8的结构测定： 1H NMR(300MHz,CDCl 3)δ7.44(s,1H),7.16(d,J＝8.5Hz,2H),6.83(d,J＝8.6Hz,2H),4.80(s,2H),4.56(dd,J＝10.0,5.6Hz,1H),4.36(d,J＝11.7Hz,1H),4.09(d,J＝11.8Hz,1H),3.78(s,4H),3.12(d,J＝9.7Hz,1H),2.92(d,J＝13.6Hz,1H),2.81(d,J＝8.3Hz,1H),2.02(d,J＝2.5Hz,6H),1.80–1.37(m,10H),1.17–1.03(m,2H),0.97(s,3H),0.86–0.71(m,5H)； 13C NMR(75MHz,CDCl 3)δ173.85,171.21,170.73,158.81,146.14,137.33,130.79,129.81,113.86,77.68,77.26,76.83,65.18,60.13,59.26,58.74,55.47,55.34,53.85,41.32,36.77,36.09,34.98,33.19,32.01,23.96,22.78,21.92,21.41,21.31,14.63；HRMS(m/z)calc.for C 32H 44FNO 7(+)554.3118,found 554.3110。
实施例9
化合物I-a-9的制备：
Figure PCTCN2021093539-appb-000046
参照实施例6中的制备方法制备化合物I-a-9，收率57％。化合物I-a-9的结构测定： 1H NMR(300MHz,CDCl 3)δ7.26(s,1H),7.11(d,J＝8.1Hz,2H),6.75(d,J＝8.4Hz,2H),4.84(s,2H),4.56(dd,J＝10.0,5.8Hz,1H),4.36(d,J＝11.7Hz,1H),4.13–4.07(m,1H),3.79(s,1H),3.00(m,4H),2.03(d,J＝2.6Hz,6H),1.84–1.38(m,10H),1.15-1.02(m,2H),0.98(s,3H),0.86-0.76(m,5H). 13C NMR(125MHz,CDCl 3)δ172.92,171.04,170.59,152.64,135.92,125.06,124.16,115.29,105.83,80.05,67.11,66.68,65.03,60.41,59.10,58.54,55.18,41.18,36.62,35.96,33.63,31.85,29.73,23.78,22.62,21.74,21.21,21.11,14.43；HRMS(m/z)calc.for C 31H 43NO 7(+)540.2961 found 540.2956。
实施例10
化合物I-a-10的制备：
Figure PCTCN2021093539-appb-000047
参照实施例6中的制备方法制备化合物I-a-10，收率44％。化合物I-a-10的结构测定： 1H NMR(500MHz,CDCl 3)δ7.42(s,1H),7.32(d,J＝8.2Hz,2H),7.20(d,J＝8.1Hz,2H),4.77(d,J＝4.4Hz,2H),4.57(dd,J＝11.3,4.7Hz,1H),4.37(d,J＝11.7Hz,1H),4.12(d,J＝11.7Hz,1H),3.81(d,J＝13.9Hz,1H),3.15(d,J＝10.1Hz,1H),2.96(d,J＝13.9Hz,1H),2.87(dd,J＝11.1,2.8Hz,1H),2.03(d,J＝2.9Hz, 6H),1.82–1.50(m,10H),1.31(s,9H),1.08(d,J＝11.1Hz,2H),0.99(s,3H),0.86–0.78(m,5H)； 13C NMR(125MHz,CDCl 3)δ173.66,171.14,170.68,150.08,145.91,137.60,136.19,128.21,125.44,77.52,77.26,77.01,60.56,59.43,59.13,55.50,54.00,41.46,36.90,36.21,35.18,34.70,33.34,32.10,31.64,24.06,22.86,22.02,21.39,21.28,14.69；HRMS(m/z)calc.for C 35H 50NO 6(+)580.3638 found 580.3629。
实施例11
化合物I-a-11的制备：
Figure PCTCN2021093539-appb-000048
参照实施例6中的制备方法制备化合物I-a-11，收率47％。化合物I-a-11的结构测定： 1H NMR(500MHz,CDCl 3)δ7.43(d,J＝7.1Hz,1H),7.36(s,1H),7.14(dt,J＝16.7,7.1Hz,3H),4.75(d,J＝8.5Hz,2H),4.59(dd,J＝11.3,4.9Hz,1H),4.38(d,J＝11.7Hz,1H),4.16–4.11(m,1H),3.68(d,J＝14.5Hz,1H),3.19(d,J＝10.3Hz,1H),3.07–2.98(m,1H),2.83(d,J＝9.6Hz,1H),2.24(s,3H),2.03(d,J＝3.5Hz,6H),1.79–1.58(m,10H),1.09(d,J＝10.7Hz,2H),1.00(s,3H),0.91–0.85(m,5H)； 13C NMR(125MHz,CDCl 3)δ173.83,171.22,170.75,145.98,137.46,136.64,130.35,128.41,126.82,126.07,77.58,77.26,76.94,70.61,65.28,60.82,59.83,57.26,55.44,54.00,41.39,36.85,36.17,35.18,33.42,32.04,24.02,22.84,21.98,21.43,21.33,19.60,14.68；HRMS(m/z)calc.for C 32H 44NO 6(+)538.3169 found 538.3176。
实施例12
化合物I-a-12的制备：
Figure PCTCN2021093539-appb-000049
参照实施例6中的制备方法制备化合物I-a-12，收率57％。化合物I-a-12的结构测定：
1H NMR(300MHz,CDCl 3)δ7.48(s,1H),7.34(t,J＝7.4Hz,1H),7.24–7.15(m,1H),7.09(t,J＝7.1Hz,1H),7.04–6.94(m,1H),4.83(s,2H),4.57(dd,J＝10.3,5.4Hz,1H),4.36(d,J＝11.7Hz,1H),4.10(d,J＝11.8Hz,1H),3.81(d,J＝13.8Hz,1H),3.12(dd,J＝22.3,12.2Hz,2H),2.85(dd,J＝11.0,2.8Hz,1H),2.02(d,J＝1.8Hz,6H),1.83–1.37(m,10H),1.11(d,J＝11.4Hz,2H),0.98(s,3H),0.86–0.75(m,5H)； 13C NMR(75MHz,CDCl 3)δ173.59,171.11,170.65,162.54,160.58,146.14,137.15,131.26,128.88,124.12,115.61,115.44,80.25,77.51,77.26,77.01,70.63,65.20,60.37,59.58,55.44,53.86,52.68,41.42,36.85,36.16,35.06,33.31,32.07,24.02,22.83,21.97,21.37,21.27,14.65；HRMS(m/z)calc.for C 31H 41FNO 6(+)542.2918 found 542.2910。
实施例13
化合物I-a-13的制备：
Figure PCTCN2021093539-appb-000050
参照实施例6中的制备方法制备化合物I-a-13，收率60％。化合物I-a-13的结构测定： 1H NMR(400MHz,CDCl 3)δ7.41(s,1H),7.21(t,J＝8.1Hz,1H),6.86(d,J＝7.0Hz,2H),6.77(d,J＝7.5Hz,1H),4.78(s,2H),4.57(dd,J＝10.8,5.0Hz,1H),4.37(d,J＝11.7Hz,1H),4.12(d,J＝11.6Hz,1H),3.81(s,3H),3.16(d,J＝10.1 Hz,1H),2.96(d,J＝14.0Hz,1H),2.85(d,J＝8.5Hz,1H),2.03(d,J＝3.7Hz,6H),1.69–1.50(m,10H),1.16–1.03(m,2H),0.99(s,3H),0.85(s,5H)； 13C NMR(100MHz,CDCl 3)δ173.41,171.21,170.73,159.94,145.97,141.17,137.48,129.54,120.82,114.31,112.09,80.27,77.58,77.46,77.26,76.94,70.63,65.26,60.64,59.36,55.47,53.94,41.40,36.85,36.17,35.13,33.31,32.05,29.70,24.02,22.84,21.99,21.42,21.33,14.68；HRMS(m/z)calc.for C 32H 44NO 7(+)554.3118 found 554.3118。
实施例14
化合物I-a-14的制备：
Figure PCTCN2021093539-appb-000051
参照实施例6中的制备方法制备化合物I-a-14，收率57％。化合物I-a-14的结构测定： 1H NMR(300MHz,CDCl 3)δ7.42(s,1H),7.30(s,1H),7.21(d,J＝5.7Hz,2H),7.17–7.10(m,1H),4.80(s,2H),4.58(d,J＝9.6Hz,1H),4.37(d,J＝11.7Hz,1H),4.11(d,J＝11.6Hz,1H),3.80(d,J＝14.1Hz,1H),3.16(d,J＝10.1Hz,1H),2.93(d,J＝14.1Hz,1H),2.78(d,J＝8.2Hz,1H),2.03(d,J＝3.0Hz,6H),1.84–1.34(m,10H),1.10(dd,J＝17.6,16.0Hz,2H),0.99(s,3H),0.90–0.76(m,5H)； 13C NMR(75MHz,CDCl 3)δ173.61,171.21,170.74,146.13,141.65,137.31,134.51,129.81,128.35,127.34,126.66,80.23,77.58,77.26,76.94,70.64,65.25,60.56,59.38,58.96,55.39,53.90,41.36,36.82,36.16,35.04,33.20,32.00,23.98,22.83,21.95,21.41,21.32,14.66；HRMS(m/z)calc.for C 31H 41ClNO 6(+)558.2622 found 558.2618。
实施例15
化合物I-a-15的制备：
Figure PCTCN2021093539-appb-000052
参照实施例6中的制备方法制备化合物I-a-15，收率53％。化合物I-a-15的结构测定： 1H NMR(400MHz,CDCl 3)δ7.50(s,1H),7.35(d,J＝1.1Hz,1H),6.28(dd,J＝3.1,1.9Hz,1H),6.13(d,J＝3.0Hz,1H),4.82(s,2H),4.57(s,1H),4.36(d,J＝11.7Hz,1H),4.10(d,J＝11.6Hz,1H),3.69(d,J＝14.5Hz,1H),3.28(d,J＝14.5Hz,1H),3.09(d,J＝9.7Hz,1H),2.92(dd,J＝11.3,3.4Hz,1H),2.02(d,J＝6.3Hz,6H),1.77–1.38(m,10H),1.06(d,J＝12.6Hz,2H),0.98(s,3H),0.83(s,3H),0.80–0.67(m,2H)； 13C NMR(100MHz,CDCl 3)δ173.60,171.20,170.74,146.44,142.38,130.34,110.38,108.92,80.23,77.51,77.26,77.01,60.65,58.65,55.40,53.74,51.81,41.40,36.82,36.15,35.08,33.07,32.06,24.00,22.83,21.97,21.42,21.33,14.66；HRMS(m/z)calc.for C 29H 40NO 7(+)514.2805 found 514.2800。
实施例16
化合物I-a-16的制备：
Figure PCTCN2021093539-appb-000053
参照实施例6中的制备方法制备化合物I-a-16，收率60％。化合物I-a-16的结构测定： 1H NMR(300MHz,CDCl 3)δ7.51(s,1H),7.20(d,J＝4.9Hz,1H),6.93(dd,J＝5.0,3.5Hz,1H),6.84(d,J＝3.2Hz,1H),4.82(s,2H),4.56(dd,J＝10.1,5.7Hz,1H),4.36(d,J＝11.7Hz,1H),4.14–4.08(m,1H),3.89(d,J＝14.5Hz,1H),3.36(d,J＝14.5Hz,1H),3.17(d,J＝9.6Hz,1H),2.95(dd,J＝11.1,3.3Hz,1H),2.03(d,J＝3.5Hz,6H),1.83–1.42(m,10H),1.09(s,2H),0.98(s,3H),0.87–0.73(m,5H)； 13C NMR(75MHz,CDCl 3)δ173.65,171.21,170.73,146.09,142.71,137.10,126.91,125.65,124.84,80.23,77.58,77.47,77.26,76.94,70.74,65.23,60.42,58.72,55.39,53.93,53.82,41.38,36.82,36.15,35.13,33.33,32.02,23.99,22.83,21.95,21.42,21.32,14.65；HRMS(m/z)calc.for C 29H 40NO 6S(+)530.2576 found 530.2574。
实施例17
化合物I-a-17的制备：
Figure PCTCN2021093539-appb-000054
参照实施例6中的制备方法制备化合物I-a-17，收率53％。化合物I-a-17的结构测定：. 1H NMR(300MHz,CDCl 3)δ8.14–8.06(m,1H),7.88–7.80(m,1H),7.74(d,J＝8.1Hz,1H),7.61(d,J＝6.9Hz,1H),7.52–7.39(m,3H),7.37(s,1H),4.64(dt,J＝16.0,11.9Hz,3H),4.38(d,J＝11.7Hz,1H),4.12(dd,J＝13.3,5.6Hz,2H),3.55(d,J＝14.6Hz,1H),3.28(d,J＝9.9Hz,1H),2.87(dd,J＝11.1,2.6Hz,1H),2.03(s,6H),1.86–1.42(m,10H),1.11(s,2H),0.99(s,3H),0.87(s,5H)； 13C NMR(75MHz,CDCl 3)δ173.82,171.22,170.75,146.02,137.38,135.01,133.90,132.13,128.80,127.65,126.01,125.97,125.83,125.73,123.98,80.27,77.58,77.26,76.94,70.57,65.27,61.21,60.10,57.76,55.42,54.01,41.38,36.85,36.17,35.20,33.41,32.01,24.01,22.83,21.96,21.42,21.32,14.70；HRMS(m/z)calc.for C 35H 44NO 6(+)574.3169 found 574.3138。
实施例18
化合物I-a-18的制备：
Figure PCTCN2021093539-appb-000055
参照实施例6中的制备方法制备化合物I-a-18，收率60％。化合物I-a-18的结构测定： 1H NMR(300MHz,CDCl 3)δ7.41(s,1H),7.34(d,J＝6.7Hz,1H),7.09(dd,J＝13.6,6.6Hz,2H),4.81(s,2H),4.58(d,J＝9.2Hz,1H),4.37(d,J＝11.6Hz,1H),4.11(d,J＝11.5Hz,1H),3.75(d,J＝13.9Hz,1H),3.15(d,J＝10.1Hz,1H),2.89(d,J＝14.0Hz,1H),2.74(d,J＝9.0Hz,1H),2.03(s,6H),1.64(m,J＝48.5,31.1,19.0Hz,10H),1.08(d,J＝12.1Hz,2H),0.99(s,3H),0.86(s,5H)； 13C NMR(75MHz,CDCl 3)δ173.52,171.21,170.75,158.51,156.05,146.15,137.29,136.50,130.26,128.10,128.03,121.12,120.95,116.65,116.44,80.22,77.58,77.27,76.95,70.62,65.23,60.42,59.39,58.34,55.39,53.92,41.36,36.82,36.16,35.05,33.14,32.00,23.98,22.83,21.94,21.41,21.32,14.66；HRMS(m/z)calc.for C 31H 40ClFNO 6(+)576.2528 found 576.2525。
实施例19
化合物I-a-19的制备：
Figure PCTCN2021093539-appb-000056
参照实施例6中的制备方法制备化合物I-a-19，收率43％。化合物I-a-19的结构测定： 1H NMR(400MHz,CDCl 3)δ7.47(s,1H),4.87–4.78(m,2H),4.57(dd,J＝10.8,5.3Hz,1H),4.37(d,J＝11.7Hz,1H),4.12(d,J＝11.7Hz,1H),3.16–3.02(m,2H),2.51(dd,J＝11.6,5.4Hz,1H),2.03(d,J＝8.3Hz,8H),1.71–1.42(m,10H), 1.13–1.04(m,2H),0.99(s,3H),0.83(d,J＝16.0Hz,10H)； 13C NMR(100MHz,CDCl 3)δ173.66,171.18,170.73,139.16,114.38,80.20,77.51,77.26,77.00,70.71,65.20,59.95,59.12,57.07,55.38,53.79,41.38,36.79,36.15,34.80,32.73,32.11,23.97,22.82,21.99,21.41,21.33,19.26,14.61,12.04；HRMS(m/z)calc.for C 27H 42NO 66(+)476.3012 found 476.3005。
实施例20
化合物I-a-20的制备：
Figure PCTCN2021093539-appb-000057
参照实施例6中的制备方法制备化合物I-a-20，收率30％。化合物I-a-20的结构测定： 1H NMR(300MHz,CDCl 3)δ7.66(s,1H),4.95–4.76(m,2H),4.65–4.48(m,1H),4.37(d,J＝11.7Hz,1H),4.12(d,J＝11.6Hz,1H),3.51(d,J＝10.2Hz,1H),3.02(dd,J＝39.3,8.4Hz,2H),2.04(d,J＝6.4Hz,7H),1.71(ddd,J＝16.5,10.0,4.6Hz,10H),1.16(d,J＝6.6Hz,3H),1.10(s,2H),1.00(s,3H),0.98–0.93(m,3H),0.91–0.77(m,5H)； 13C NMR(75MHz,CDCl 3)δ173.16,171.13,170.77,149.90,139.99,80.11,77.51,77.26,77.01,70.86,65.24,56.39,55.36,53.91,51.02,41.37,36.80,36.27,32.05,29.94,29.52,27.51,23.94,22.91,21.97,21.41,21.33,14.58；HRMS(m/z)calc.for C 27H 42NO 6(+)476.3012 found 476.3012。
实施例21
化合物I-a-21的制备：
Figure PCTCN2021093539-appb-000058
参照实施例6中的制备方法制备化合物I-a-21，收率50％。化合物I-a-21的结构测定： 1H NMR(300MHz,CDCl 3)δ7.41(s,1H),4.93–4.78(m,2H),4.56(dd,J＝10.1,5.9Hz,1H),4.37(d,J＝11.7Hz,1H),4.11(dd,J＝9.5,2.2Hz,1H),3.11–2.99(m,2H),2.59–2.46(m,1H),2.03(d,J＝6.4Hz,8H),1.80–1.32(m,13H),1.14–1.04(m,2H),0.99(s,3H),0.86(d,J＝7.3Hz,4H),0.85–0.74(m,5H)； 13C NMR(75MHz,CDCl 3)δ173.70,171.19,170.73,146.57,136.77,77.58,77.26,76.94,70.64,65.18,60.12,59.24,55.39,55.06,53.85,41.38,36.79,36.13,35.01,32.93,32.15,28.53,23.98,22.81,22.00,21.41,21.33,20.87,14.62,14.21；HRMS(m/z)calc.for C 28H 44NO 6(+)490.3169 found 490.3164。
实施例22
化合物I-a-22的制备：
Figure PCTCN2021093539-appb-000059
参照实施例6中的制备方法制备化合物I-a-22，收率47％。化合物I-a-22的结构测定： 1H NMR(400MHz,CDCl 3)δ7.32(s,1H),4.91–4.71(m,2H),4.57(dd,J＝10.7,5.3Hz,1H),4.37(d,J＝11.7Hz,1H),4.12(d,J＝11.7Hz,1H),2.97(dd,J＝18.2,8.9Hz,2H),2.10(t,J＝12.0Hz,1H),2.03(d,J＝7.6Hz,6H),1.81–1.43(m,12H),1.12–1.04(m,2H),0.99(s,3H),0.85(d,J＝8.7Hz,8H),0.78(d,J＝6.3Hz,4H)； 13C NMR(100MHz,CDCl 3)δ173.92,171.22,170.73,146.18,137.79,80.28,77.58,77.26,76.94,70.54,65.24,63.57,60.65,59.84,55.43,53.81,41.40,36.80,36.11,35.10,33.46,32.24,25.92,24.00,22.83,22.03,21.51,21.43,21.34,20.80,14.66；HRMS(m/z)calc.for C 28H 44NO 6(+)490.3169 found 490.3164。
实施例23
化合物I-a-23的制备：
Figure PCTCN2021093539-appb-000060
参照实施例6中的制备方法制备化合物I-a-23，收率37％。化合物I-a-23的结构测定： 1H NMR(400MHz,CDCl 3)δ7.43(s,1H),4.91–4.75(m,2H),4.56(dd,J＝10.7,5.4Hz,1H),4.36(d,J＝11.7Hz,1H),4.17–4.09(m,1H),3.16–3.01(m,2H),2.62–2.52(m,1H),2.10–1.97(m,8H),1.77–1.39(m,11H),1.38–1.28(m,2H),1.16–1.05(m,2H),0.99(s,3H),0.88–0.72(m,11H)； 13C NMR(100MHz,CDCl 3)δ173.55,171.07,170.64,146.42,136.39,80.24,77.51,77.26,77.01,70.62,65.17,60.56,60.11,59.27,55.46,53.90,53.55,41.45,36.85,36.18,35.21,34.99,32.83,32.15,29.90,26.72,24.01,23.06,22.83,22.59,22.03,21.35,21.27,14.63；HRMS(m/z)calc.for C 29H 46NO 6(+)504.3325 found 504.3323。
实施例24
化合物I-a-24的制备：
Figure PCTCN2021093539-appb-000061
参照实施例1中的制备方法制备化合物I-a-24，收率47％。化合物I-a-24的结构测定： 1H NMR(500MHz,MeOD)δ7.73(s,1H),4.94(dd,J＝3.6,1.4Hz,2H),4.57(dd,J＝11.6,4.6Hz,1H),4.46(d,J＝11.7Hz,1H),4.14(d,J＝11.7Hz,1H),3.55(d,J＝10.5Hz,1H),3.44–3.33(m,1H),3.03(dd,J＝11.5,3.4Hz,1H),2.27–2.10(m,1H),2.03(d,J＝8.8Hz,6H),1.83–1.47(m,18H),1.21(d,J＝13.8Hz,2H),1.03(s,3H),0.98–0.85(m,5H)； 13C NMR(125MHz,MeOD)δ173.83,171.37,170.93,151.14,139.40,80.12,71.09,64.53,62.01,58.10,54.80,53.11,51.59,48.11, 47.94,47.77,47.60,47.43,47.26,47.09,41.01,36.10,35.61,34.02,31.32,28.22,24.27,23.96,23.36,22.18,21.55,21.47,19.68,19.59,13.28；HRMS(m/z)calc.for C 29H 44NO 6(+)502.3169 found 502.3163。
实施例25
化合物I-a-25的制备：
Figure PCTCN2021093539-appb-000062
参照实施例1中的制备方法制备化合物I-a-25，收率37％。化合物I-a-25的结构测定： 1H NMR(300MHz,CDCl 3)δ7.39(s,1H),4.85(d,J＝6.2Hz,2H),4.66–4.49(m,1H),4.38(d,J＝11.7Hz,1H),4.11(d,J＝11.7Hz,1H),3.47(d,J＝9.5Hz,1H),2.88(d,J＝10.3Hz,1H),2.42(s,1H),2.04(d,J＝5.9Hz,6H),1.75–1.46(m,21H),1.11–1.05(m,2H),1.00(s,4H),0.86(s,5H)； 13C NMR(100MHz,CDCl 3)δ171.54,171.10,170.79,146.49,133.32,80.01,77.56,77.25,76.93,75.48,65.32,63.69,55.26,54.67,54.01,52.72,41.29,36.79,36.28,34.40,32.17,31.88,29.93,29.55,24.40,23.86,22.93,21.92,21.41,21.32,14.53,14.30；HRMS(m/z)calc.for C 30H 46NO 6(+)516.3325 found 516.3326。
实施例26
化合物I-a-26的制备：
Figure PCTCN2021093539-appb-000063
参照实施例1中的制备方法制备化合物I-a-26，收率43％。化合物I-a-26的结构测定： 1H NMR(500MHz,MeOD)δ7.73(s,1H),5.01–4.90(m,2H),4.57(dd,J＝11.6,4.6Hz,1H),4.46(d,J＝11.7Hz,1H),4.14(d,J＝11.7Hz,1H),3.66(s,1H), 3.03(d,J＝9.1Hz,1H),2.81(s,1H),2.31–2.09(m,1H),2.03(d,J＝8.5Hz,6H),1.91–1.37(m,20H),1.28–1.08(m,4H),1.02(s,3H),0.98–0.82(m,5H)； 13C NMR(125MHz,MeOD)δ173.78,171.39,170.94,148.73,143.94,80.13,75.62,71.16,64.52,55.97,54.82,53.23,51.88,41.01,36.09,35.63,34.22,32.57,31.30,27.45,27.27,25.10,24.47,23.36,21.54,21.45,19.67,19.59,13.32；HRMS(m/z)calc.for C 31H 49NO 6(+)530.3482 found 530.3474。
实施例27
化合物I-b-1的制备：
Figure PCTCN2021093539-appb-000064
将穿心莲内酯1(10.0g,28.54mmol)溶于甲苯(200ml)和二甲基亚砜(27ml)中，然后将2,2-二甲氧基丙烷(14ml)和对甲苯磺酸(催化当量)加入反应体系中。升温至80摄氏度搅拌反应2小时，然后将体系降至室温，加入7ml三乙胺淬灭反应。用150ml二氯甲烷萃取反应，水洗，分液，用无水硫酸钠干燥，减压蒸干，用乙醚打浆得到8.65g类白色固体化合物IV-1，收率78％。
Figure PCTCN2021093539-appb-000065
将化合物IV-1(8.65g,22.17mmol)、乙酸酐(11.31g,110.86mmol)、三乙胺(11.22g,110.85mmol)和二氯甲烷(250ml)加入反应瓶中，升温到45摄氏度回流反应2小时。反应完后，降至室温，加入饱和碳酸氢钠水溶液淬灭反应直至没有气泡产生，然后用二氯甲烷萃取三次。有机相用水洗3次、盐水洗3次，分液后用无水硫酸钠干燥有机相。抽滤，减压浓缩后用柱层析(5～25％EA/PE)分离纯化，得到白色固体化合物V-1(8.52g,89％)。 1H NMR(400MHz,CDCl 3)δ7.03(dd,J＝ 6.9,1.6Hz,1H),5.93(d,J＝6.1Hz,1H),4.90(s,1H),4.64–4.46(m,2H),4.24(dd,J＝11.2,1.9Hz,1H),3.96(d,J＝11.6Hz,1H),3.50(dd,J＝8.5,3.9Hz,1H),3.18(d,J＝11.6Hz,1H),2.55–2.35(m,3H),2.12(s,3H),2.09–1.92(m,2H),1.92–1.67(m,4H),1.43(s,3H),1.37(s,3H),1.34–1.24(m,3H),1.20(s,3H),0.96(s,3H).
Figure PCTCN2021093539-appb-000066
将化合物V-1(100mg,0.23ml)、苄胺(0.42mmol)、三乙胺(70mg,0.69mmol)和二氯甲烷(3ml)加入反应瓶中，在室温下搅拌反应3小时。反应完毕后，加入饱和碳酸氢钠水溶液淬灭反应，用二氯甲烷萃取3次，分液，有机相用水和盐水各洗一次。有机相用无水硫酸钠干燥，抽滤，减压浓缩后柱层析(5-30％EA/PE)纯化，得到42mg化合物VI-1，收率38％。
Figure PCTCN2021093539-appb-000067
将化合物VI-1(30mg,1.0当量)、9-均三甲基-2,7,10-三甲基吖啶高氯酸盐(6mol％)和2,4,6-三异丙基苯-1-硫酚(0.5当量)加入透明的带特氟塞的8*16mm反应管中。将反应管密闭，加入2ml无水甲苯，用氮气置换3次以上，将反应管置于两个40W Kessil KSH150B的蓝光灯照射下搅拌反应，体系逐渐变为深棕色，用TLC板监测反应，直至原料反应彻底。将反应体系减压浓缩，用制备级薄层色谱板分离得到20mg化合物I-b-1，收率67％。
化合物I-b-1的结构测定： 1H NMR(300MHz,CDCl 3)δ7.44(s,1H),7.7-7.32(m,5H),4.82(s,2H),3.99(d,J＝11.7Hz,1H),3.84(d,J＝13.8Hz,1H),3.44-3.50(m,1H),3.17-3.21(m,2H),3.95(d,J＝14.1Hz,1H),2.83(d,J＝8.4Hz,1H),1.93-2.02(m,2H),1.59-1.86(m,7H),1.43(s,3H),1.37(s,3H),1.17(s,3H),1.03(s,3H),0.76-1.03(m,4H)； 13CNMR(75MHz,CDCl 3)δ173.52,145.67,137.22,130.10,128.31,126.90, 98.91,70.44,63.94,60.40,59.36,59.26,53.63,52.34,37.53,35.21,34.09,33.24,31.37,29.29,27.32,26.10,25.49,24.73,20.39,15.93,14.10；HRMS(m/z)calc.for C 30H 42NO 4(+)480.3108,found 480.3111。
实施例28
化合物I-b-2的制备：
Figure PCTCN2021093539-appb-000068
参照实施例27中的制备方法制备化合物I-b-2，收率62％。化合物I-b-2的结构测定： 1H NMR(400MHz,CDCl 3)δ7.41(s,1H),7.22-7.26(m,2H),6.97-7.01(m,2H),4.82(s,2H),4.00(d,J＝11.6Hz,1H),3.78(d,J＝13.6Hz,1H),3.44-3.48(m,1H),3.13-3.20(m,2H),2.93(d,J＝14.0Hz,1H),2.79(d,J＝13.6Hz,1H),1.94-2.02(m,2H),1.80(d,J＝12.8Hz,1H),1.70-1.74(m,1H),1.58-1.70(m,5H),1.43(s,3H),1.37(s,3H),1.17(s,3H),1.03(s,3H),0.81-1.01(m,4H)； 13CNMR(100MHz,CDCl 3)δ173.44,145.67,137.18,134.51,129.84,115.19,114.98,98.92,70.40,63.94,60.28,59.36,58.50,53.65,52.33,37.53,35.20,34.08,33.18,31.37,29.66,27.30,26.10,25.49,24.72,20.38,15.92；HRMS(m/z)calc.for C 30H 41FNO 4(+)498.3014,found 498.3001.
实施例29
化合物I-b-3的制备：
Figure PCTCN2021093539-appb-000069
参照实施例27中的制备方法制备化合物I-b-3，收率53％。化合物I-b-3的结构测定：H NMR(300MHz,CDCl3)δ7.27(s,1H),7.14(d,J＝8.4Hz,2H),6.78 (d,J＝7.8Hz,2H),4.87(s,2H),4.00(d,J＝11.7Hz,1H),3.82-3.92(m,1H),3.35-3.58(m,2H),3.22-3.32(m,1H),3.17(d,J＝11.6Hz,2H),2.90(d,J＝7.8Hz,1H),1.86-2.05(m,2H),1.83(d,J＝13.2Hz,1H),1.79–1.47(m,7H),1.42(s,3H),1.37(s,3H),1.17(s,3H),1.04(s,3H),0.59-1.02(m,6H)； 13C NMR(100MHz,CDCl 3)δ173.59,155.70,130.40,129.75,115.35,98.92,70.74,65.75,63.80,59.23,58.52,53.21,52.13,37.42,35.09,34.61,33.92,31.13,29.57,29.19,27.19,25.98,25.38,22.56,20.22,15.76,15.11,14.00；HRMS(m/z)calc.for C 30H 42NO 5(+)496.3057,found 496.3058。
实施例30
化合物I-b-4的制备：
Figure PCTCN2021093539-appb-000070
参照实施例27中的制备方法制备化合物I-b-4，收率40％。化合物I-b-4的结构测定：1H NMR(400MHz,CDCl3)δ7.50–7.35(m,3H),7.17(d,J＝8.0Hz,2H),4.83(d,J＝9.7Hz,2H),4.01(d,J＝11.7Hz,1H),3.78(d,J＝13.7Hz,1H),3.46(dd,J＝9.0,4.1Hz,1H),3.30–3.04(m,1H),2.93(d,J＝14.2Hz,1H),2.77(d,J＝8.8Hz,1H),2.06–1.89(m,2H),1.82(d,J＝13.0Hz,1H),1.70(dd,J＝11.2,6.8Hz,3H),1.65–1.49(m,6H),1.43(s,3H),1.37(s,3H),1.17(s,3H),1.03(s,3H),0.97(dd,J＝8.2,4.8Hz,2H),0.93–0.73(m,4H)； 13CNMR(100MHz,CDCl 3)δ173.32,145.97,137.16,131.48,131.28,129.89,98.83,70.30,63.85,60.30,59.26,58.52,53.55,52.24,37.45,35.11,33.98,33.09,31.80,31.26,29.57,29.19,27.19,26.01,25.40,24.62,22.57,20.29,15.83,14.00；HRMS(m/z)calc.for C 30H 41BrNO 4(+)558.2213,found 558.2213。
实施例31
化合物I-b-5的制备：
Figure PCTCN2021093539-appb-000071
参照实施例27中的制备方法制备化合物I-b-5，收率66％。化合物I-b-5的结构测定： 1H NMR(400MHz,CDCl 3)δ7.47(s,1H),7.21(d,J＝4.8Hz,1H),6.97–6.89(m,1H),6.85(s,1H),4.84(s,2H),4.02(d,J＝11.6Hz,1H),3.90(d,J＝14.4Hz,1H),3.46(dd,J＝9.1,4.1Hz,1H),3.37(d,J＝14.5Hz,1H),3.18(t,J＝9.7Hz,2H),2.96(dd,J＝11.0,2.8Hz,1H),2.04–1.87(m,1H),1.84(d,J＝12.8Hz,1H),1.78–1.51(m,7H),1.43(s,3H),1.37(s,3H),1.18(s,3H),1.02(s,3H),0.95–0.64(m,4H)； 13CNMR(100MHz,CDCl 3)δ173.49,145.66,142.53,137.92,126.67,125.37,124.57,98.92,70.53,63.95,60.40,58.68,53.72,53.64,52.34,37.54,35.31,35.26,34.10,33.32,31.38,29.68,27.33,26.11,25.50,24.73,20.39,15.93；HRMS(m/z)calc.for C 28H 40NO 4S(+)486.2673,found 486.2673。
实施例32
化合物I-b-6的制备：
Figure PCTCN2021093539-appb-000072
参照实施例27中的制备方法制备化合物I-b-6，收率67％。化合物I-b-6的结构测定： 1H NMR(400MHz,CDCl 3)δ7.55(t,J＝8.8Hz,2H),7.42(d,J＝8.8Hz,3H),4.94–4.66(m,2H),4.01(d,J＝11.6Hz,1H),3.88(d,J＝14.4Hz,1H),3.47(dd,J＝9.0,4.1Hz,1H),3.19(d,J＝11.5Hz,1H),3.05(d,J＝14.4Hz,1H),2.77(d,J＝8.6Hz,1H),2.09–1.90(m,1H),1.84(d,J＝12.8Hz,1H),1.78–1.49(m,6H),1.43(s,3H),1.37(s,3H),1.18(s,3H),1.04(s,3H),1.03–0.97(m,1H),0.96–0.70(m,3H)； 13CNMR(100MHz,CDCl 3)δ177.47,173.39,145.73,144.06,137.14,128.39,125.22,98.95,70.39,63.96,60.58,59.38,58.82,53.64,52.32,37.56,35.29,35.22,34.07, 33.20,31.34,29.77,27.27,26.11,25.50,24.71,20.38,15.94；HRMS(m/z)calc.for C 31H 41F 3NO 4(+)548.2982,found 548.2982。
实施例33
化合物I-b-7的制备：
Figure PCTCN2021093539-appb-000073
参照实施例27中的制备方法制备化合物I-b-7，收率57％。化合物I-b-7的结构测定： 1H NMR(400MHz,CDCl 3)δ7.44(s,1H),7.19(d,J＝8.3Hz,2H),6.85(d,J＝8.6Hz,2H),4.83(s,2H),4.02(d,J＝11.5Hz,1H),3.80(s,3H),3.77(s,1H),3.46(dd,J＝9.1,4.1Hz,1H),3.19(d,J＝11.6Hz,1H),3.13(d,J＝11.7Hz,1H),2.93(d,J＝14.5Hz,1H),2.83(d,J＝9.5Hz,1H),2.10–1.88(m,2H),1.82(d,J＝12.8Hz,1H),1.78–1.61(m,7H),1.43(s,3H),1.37(s,3H),1.18(s,3H),1.03(s,3H),0.98-1.01(m,1H),0.96–0.68(m,4H)； 13CNMR(100MHz,CDCl 3)δ173.52,159.05,145.78,129.89,129.86,129.61,129.59,113.70,98.91,70.47,60.23,59.26,58.59,55.25,53.63,52.34,37.53,35.21,34.10,33.37,32.46,31.39,29.67,29.30,27.33,27.21,26.10,25.49,24.73,22.93,20.39,15.93,14.34；HRMS(m/z)calc.for C 31H 44NO 5(+)510.3214,found 510.3209。
实施例34
化合物I-b-8的制备：
Figure PCTCN2021093539-appb-000074
参照实施例27中的制备方法制备化合物I-b-8，收率63％。化合物I-b-8的结构测定： 1H NMR(400MHz,CDCl 3)δ7.44(s,1H),7.33(d,J＝8.2Hz,2H),7.21 (d,J＝8.0Hz,2H),4.92–4.68(m,2H),4.02(d,J＝11.6Hz,1H),3.82(d,J＝13.9Hz,1H),3.46(dd,J＝9.1,4.1Hz,1H),3.24–3.08(m,2H),2.97(d,J＝14.0Hz,1H),2.88(d,J＝8.2Hz,1H),2.11–1.91(m,2H),1.84(d,J＝13.2Hz,1H),1.78–1.63(m,6H),1.43(s,3H),1.36(s,3H),1.32(s,9H),1.18(s,3H),1.03(s,3H),0.99(s,1H),0.95–0.65(m,4H)； 13CNMR(100MHz,CDCl 3)δ173.92,145.84,137.54,136.08,127.97,125.20,98.91,70.43,63.96,60.42,59.29,58.92,55.57,53.63,52.36,37.54,35.22,34.44,34.10,33.37,31.36,29.68,29.39,27.34,26.11,25.50,24.74,22.74,20.40,15.94；HRMS(m/z)calc.for C 34H 50NO 4(+)536.3734,found 536.3735。
实施例35
化合物I-b-9的制备：
Figure PCTCN2021093539-appb-000075
参照实施例27中的制备方法制备化合物I-b-9，收率70％。化合物I-b-9的结构测定： 1H NMR(400MHz,CDCl 3)δ8.17(d,J＝8.7Hz,2H),7.49(d,J＝8.6Hz,2H),7.41(s,1H),4.91–4.71(m,2H),4.02(d,J＝11.6Hz,1H),3.90(d,J＝14.9Hz,1H),3.47(dd,J＝9.1,4.1Hz,1H),3.21(t,J＝10.1Hz,1H),3.10(d,J＝14.9Hz,1H),2.76–2.65(m,1H),2.07–1.89(m,2H),1.84(d,J＝13.0Hz,1H),1.58-1.72(m,6H),1.43(s,3H),1.37(s,3H),1.18(s,3H),1.05(s,3H),1.01(d,J＝10.9Hz,1H),0.95–0.74(m,4H)； 13CNMR(100MHz,CDCl 3)δ173.34,147.31,147.02,145.95,137.03,128.77,123.62,114.06,98.98,70.43,63.96,60.73,59.42,58.71,53.65,53.46,52.30,37.57,35.31,35.23,34.07,33.12,31.32,29.70,27.30,26.12,25.51,24.71,20.37,15.97；HRMS(m/z)calc.for C 30H 41N 2O 6(+)525.2959,found 525.2951。
实施例36
化合物I-b-10的制备：
Figure PCTCN2021093539-appb-000076
参照实施例27中的制备方法制备化合物I-b-10，收率70％。化合物I-b-10的结构测定： 1H NMR(400MHz,CDCl 3)δ7.41(s,1H),7.35(d,J＝5.3Hz,1H),7.16–7.10(m,1H),7.07(t,J＝8.6Hz,1H),4.95–4.73(m,2H),4.02(d,J＝11.6Hz,1H),3.77(d,J＝14.3Hz,1H),3.47(dd,J＝9.0,4.0Hz,1H),3.24–3.12(m,2H),2.91(d,J＝14.1Hz,1H),2.76(d,J＝7.7Hz,1H),2.06–1.88(m,2H),1.82(d,J＝12.8Hz,1H),1.57-1.72(m,6H),1.43(s,3H),1.37(s,3H),1.18(s,3H),1.04(s,3H),1.00(d,J＝10.8Hz,1H),0.82-0.88(m,4H)； 13CNMR(100MHz,CDCl 3)δ173.38,145.70,137.15,136.60,130.02,127.85,122.61,116.41,98.94,70.41,63.95,60.40,59.34,58.14,53.66,52.32,37.55,35.22,34.08,33.18,31.36,29.67,27.29,26.12,25.50,24.72,20.38,15.94；HRMS(m/z)calc.for C 30H 40ClFNO4(+)532.2624,found 532.2624。
实施例37
化合物I-b-11的制备：
Figure PCTCN2021093539-appb-000077
参照实施例27中的制备方法制备化合物I-b-11，收率67％。化合物I-b-11的结构测定： 1H NMR(400MHz,CDCl 3)δ7.44(s,1H),7.17(d,J＝7.9Hz,2H),7.12(d,J＝7.9Hz,2H),4.82(s,2H),4.02(d,J＝11.6Hz,1H),3.82(d,J＝13.6Hz,1H),3.46(dd,J＝9.1,4.1Hz,1H),3.19(d,J＝8.9Hz,1H),3.14(d,J＝10.2Hz,1H),2.94(d,J＝13.7Hz,1H),2.84(d,J＝8.2Hz,1H),2.34(s,3H),2.08–1.90(m,2H), 1.78–1.67(m,2H),1.66–1.60(m,5H),1.43(s,3H),1.37(s,3H),1.17(s,3H),1.02(s,3H),0.82-0.98(m,5H)； 13CNMR(100MHz,CDCl 3)δ173.45,145.49,137.80,136.59,136.00,128.89,128.22,98.80,70.35,63.84,60.28,59.21,58.84,53.53,52.26,37.43,35.11,34.00,33.13,31.27,29.57,29.37,27.24,26.00,25.39,24.64,20.97,20.30,15.83；HRMS(m/z)calc.for C 31H 44NO 4(+)494.3265,found 494.3270。
实施例38
化合物I-b-12的制备：
Figure PCTCN2021093539-appb-000078
参照实施例27中的制备方法制备化合物I-b-12，收率70％。化合物I-b-12的结构测定： 1H NMR(400MHz,CDCl 3)δ7.52(d,J＝6.7Hz,1H),7.35(s,1H),7.21–7.12(m,2H),7.03–6.96(m,1H),4.83–4.69(m,2H),4.03(d,J＝11.7Hz,1H),3.82(d,J＝14.8Hz,1H),3.47(dd,J＝9.1,4.1Hz,1H),3.36(d,J＝14.7Hz,1H),3.22(t,J＝12.4Hz,2H),2.90–2.85(m,1H),2.08–1.81(m,3H),1.80–1.61(m,8H),1.44(s,3H),1.38(s,3H),1.19(s,3H),1.04(s,3H),1.02(d,J＝12.8Hz,1H),0.93–0.78(m,6H),0.70–0.59(m,1H),0.57–0.47(m,1H)； 13CNMR(100MHz,CDCl 3)δ173.67,145.42,141.00,138.73,137.35,127.64,126.54,125.79,125.62,98.89,70.41,63.95,60.94,59.85,56.37,53.75,52.44,37.54,35.36,35.25,34.16,33.39,31.40,29.68,27.38,26.14,25.54,24.80,20.42,15.93,12.69,7.44,6.60；HRMS(m/z)calc.for C 33H 46NO 4(+)520.3421,found 520.3414.
实施例39:化合物I-b-13的制备：
Figure PCTCN2021093539-appb-000079
参照实施例27中的制备方法制备化合物I-b-13，收率60％。化合物I-b-13的结构测定： 1H NMR(400MHz,CDCl 3)δ8.16–8.06(m,1H),7.85(dd,J＝6.0,3.4Hz,1H),7.75(d,J＝8.2Hz,1H),7.63(d,J＝6.9Hz,1H),7.55–7.40(m,3H),7.37(s,1H),4.71(dt,J＝45.9,9.8Hz,2H),4.14(d,J＝14.5Hz,1H),4.02(d,J＝11.6Hz,1H),3.57(d,J＝14.6Hz,1H),3.48(dd,J＝9.0,4.1Hz,1H),3.29(d,J＝9.2Hz,1H),3.19(d,J＝11.5Hz,1H),2.89(dd,J＝11.1,2.9Hz,1H),1.95-2.05(m,2H),1.87(d,J＝12.8Hz,1H),1.78–1.68(m,2H),1.69–1.60(m,5H),1.44(s,3H),1.38(s,3H),1.18(s,3H),1.05(s,3H),1.02(d,J＝11.3Hz,1H),0.94–0.79(m,4H)； 13CNMR(100MHz,CDCl 3)δ173.68,145.57,137.22,134.84,133.65,131.87,128.56,127.36,125.75,125.66,125.57,125.50,123.70,98.91,70.35,63.96,61.23,60.06,57.55,53.75,52.42,37.54,35.39,35.25,34.16,33.45,31.38,29.68,27.36,26.13,25.54,24.80,20.40,15.95；HRMS(m/z)calc.for C 34H 44NO 4(+)530.3265,found 530.3269。
实施例40
化合物I-b-14的制备：
Figure PCTCN2021093539-appb-000080
参照实施例27中的制备方法制备化合物I-b-14，收率58％。化合物I-b-14的结构测定： 1H NMR(400MHz,CDCl 3)δ7.63(d,J＝8.2Hz,2H),7.39(s,1H),7.05(d,J＝8.2Hz,2H),4.87–4.73(m,2H),4.01(d,J＝11.6Hz,1H),3.77(d,J＝14.2Hz,1H),3.46(dd,J＝9.0,4.2Hz,1H),3.17(dd,J＝16.1,10.7Hz,2H),2.92(d,J＝14.1Hz,1H),2.77(d,J＝8.1Hz,1H),2.09–1.89(m,2H),1.82(d,J＝12.8Hz,1H),1.71(dd,J＝13.3,5.0Hz,2H),1.67–1.61(m,6H),1.43(s,3H),1.37(s,3H),1.22(dd,J＝11.6,4.4Hz,2H),1.18(s,3H),1.13(dd,J＝8.5,5.4Hz,1H),1.03(s,3H),1.00(d,J＝13.0Hz,1H),0.93–0.71(m,4H)； 13CNMR(100MHz,CDCl 3)δ173.42,145.65, 138.85,137.36,137.19,130.28,98.93,92.05,70.40,63.95,60.45,59.35,58.71,53.65,52.33,37.55,35.29,35.21,34.08,33.21,31.35,29.68,27.30,26.11,25.50,24.72,20.39,15.94；HRMS(m/z)calc.for C 30H 41INO 4(+)606.2075,found 606.2076。
实施例41
化合物I-b-15的制备：
Figure PCTCN2021093539-appb-000081
参照实施例27中的制备方法制备化合物I-b-15，收率40％。化合物I-b-15的结构测定： 1H NMR(400MHz,CDCl 3)δ7.42(s,1H),7.32(s,1H),7.23(d,J＝6.1Hz,2H),7.15(d,J＝6.2Hz,1H),4.88–4.72(m,2H),4.02(d,J＝11.6Hz,1H),3.82(d,J＝14.1Hz,1H),3.47(dd,J＝9.0,4.1Hz,1H),3.18(t,J＝12.1Hz,2H),2.94(d,J＝14.1Hz,1H),2.79(d,J＝7.9Hz,1H),1.95-2.05(m,2H),1.83(d,J＝12.9Hz,1H),1.78–1.67(m,1H),1.67–1.60(m,6H),1.43(s,3H),1.37(s,3H),1.18(s,3H),1.04(s,3H),1.01(d,J＝12.7Hz,1H),0.95–0.71(m,4H)； 13CNMR(100MHz,CDCl 3)δ173.47,145.66,141.46,137.19,134.27,129.56,128.09,127.08,126.40,98.92,70.42,63.95,60.56,59.35,58.79,53.65,52.35,37.55,35.23,34.09,33.25,31.37,29.68,27.31,26.12,25.51,24.73,20.39,15.94；HRMS(m/z)calc.for C 30H 41ClNO 4(+)514.2719,found 514.2724。
实施例42
化合物I-b-16的制备：
Figure PCTCN2021093539-appb-000082
参照实施例27中的制备方法制备化合物I-b-16，收率57％。化合物I-b-16 的结构测定： 1H NMR(400MHz,CDCl 3)δ7.50(s,1H),7.36(s,1H),6.29(s,1H),6.14(s,1H),4.85(s,2H),4.01(d,J＝11.7Hz,1H),3.71(d,J＝14.7Hz,1H),3.45(dd,J＝8.8,4.1Hz,1H),3.27(d,J＝14.2Hz,1H),3.18(d,J＝11.6Hz,1H),3.09(d,J＝11.4Hz,1H),2.92(d,J＝10.3Hz,1H),2.12–1.98(m,1H),1.94(dd,J＝13.8,5.1Hz,1H),1.86–1.75(m,2H),1.62-1.57(m,4H),1.42(s,3H),1.36(s,3H),1.17(s,3H),1.13-1.10(m,2H),1.01(s,3H),0.97–0.91(m,1H),0.91–0.82(m,2H),0.78(t,J＝10.1Hz,1H)； 13CNMR(100MHz,CDCl 3)δ173.44,145.99,142.15,129.89,110.12,108.98,98.93,70.54,63.94,60.55,58.60,53.46,52.27,51.53,37.55,35.18,34.03,33.02,31.40,29.68,27.29,26.08,25.43,24.66,20.41,15.97；HRMS(m/z)calc.for C 28H 40NO 5(+)470.2901,found 470.2906。
实施例43
化合物I-b-17的制备：
Figure PCTCN2021093539-appb-000083
参照实施例27中的制备方法制备化合物I-b-17，收率16％。化合物I-b-17的结构测定： 1H NMR(400MHz,CDCl 3)δ7.33(s,1H),4.91–4.76(m,2H),4.03(d,J＝11.6Hz,1H),3.46(dd,J＝8.9,4.0Hz,1H),3.20(d,J＝11.6Hz,1H),2.98(dd,J＝20.1,9.3Hz,2H),2.12(s,1H),2.06–1.89(m,3H),1.85–1.66(m,6H),1.65–1.47(m,4H),1.43(s,3H),1.37(s,3H),1.19(s,3H),1.15–1.08(m,2H),1.02(s,3H),0.99–0.93(m,2H),0.87(d,J＝6.8Hz,6H),0.79(d,J＝5.1Hz,4H). 13CNMR(100MHz,CDCl 3)δ173.74,145.82,129.86,98.90,70.34,63.95,63.42,60.61,59.78,53.70,52.96,37.54,35.55,34.09,31.90,31.59,29.67,29.30,27.28,26.12,25.57,24.76,21.98,20.53,15.91,14.10；HRMS(m/z)calc.for C 27H 44NO 4(+)446.3265,found 446.3268。
实施例44
化合物I-b-18的制备：
Figure PCTCN2021093539-appb-000084
参照实施例27中的制备方法制备化合物I-b-18，收率36％。化合物I-b-18的结构测定：H NMR(300MHz,MeOD)δ7.84(s,1H),5.00(s,2H),4.06(d,J＝11.6Hz,1H),3.82(d,J＝7.4Hz,1H),3.50(dd,J＝8.6,3.9Hz,1H),3.22(d,J＝11.7Hz,1H),2.94(d,J＝12.3Hz,1H),2.68(t,J＝12.1Hz,1H),2.58–2.29(m,2H),2.00(dd,J＝13.1,5.1Hz,2H),1.94–1.75(m,5H),1.75–1.60(m,5H),1.58–1.48(m,3H),1.41(s,3H),1.33(s,3H),1.19(s,3H),1.13(s,3H),0.91(dd,J＝12.4,5.8Hz,9H)； 13CNMR(100MHz,CDCl 3)δ172.95,129.60,128.49,99.90,99.11,76.36,71.09,63.83,58.75,52.91,52.05,37.60,35.27,33.95,33.47,30.98,29.47,29.24,27.08,26.23,26.05,25.40,24.48,22.57,22.03,20.14,15.91；HRMS(m/z)calc.for C 28H 46NO 4(+)460.3421,found 460.3422。
实施例45
化合物I-b-19的制备：
Figure PCTCN2021093539-appb-000085
参照实施例27中的制备方法制备化合物I-b-19，收率50％。化合物I-b-19的结构测定： 1H NMR(400MHz,CDCl 3)δ7.26(s,1H),4.99–4.76(m,2H),4.01(d,J＝11.6Hz,1H),3.47(dd,J＝8.7,3.7Hz,2H),3.34–3.23(m,1H),3.20(d,J＝11.6Hz,1H),2.89–2.65(m,1H),2.44(dd,J＝27.2,7.9Hz,2H),2.12–1.91(m,4H),1.88–1.67(m,5H),1.65–1.49(m,4H),1.42(s,3H),1.37(s,3H),1.19(s,3H),1.09(s,3H), 0.90(dd,J＝14.3,7.1Hz,9H)； 13CNMR(100MHz,CDCl 3)δ173.11,129.86,129.12,99.08,76.43,70.98,63.87,59.01,54.40,53.03,52.07,37.60,35.90,35.25,33.95,31.49,30.13,29.68,29.29,27.16,26.08,25.40,24.51,22.66,20.26,15.93,14.10,13.68；HRMS(m/z)calc.for C 27H 44NO 4(+)446.3265,found 446.3269。
实施例46
化合物I-b-20的制备：
Figure PCTCN2021093539-appb-000086
将穿心莲内酯1(10.0g,28.54mmol)溶于甲苯(200ml)和二甲基亚砜(27ml)中，然后将2,2-二甲氧基丙烷(14ml)和对甲苯磺酸(催化当量)加入反应体系中。升温至80摄氏度搅拌反应2小时，然后将体系降至室温，加入7ml三乙胺淬灭反应。用150ml二氯甲烷萃取反应，水洗，分液，用无水硫酸钠干燥，减压蒸干，用乙醚打浆得到8.65g类白色固体化合物IV-2，收率78％。
Figure PCTCN2021093539-appb-000087
将化合物IV-2(8.65g,22.17mmol)、乙酸酐(11.31g,110.86mmol)、三乙胺(11.22g,110.85mmol)和二氯甲烷(250ml)加入反应瓶中，升温到45摄氏度回流反应2小时。反应完后，降至室温，加入饱和碳酸氢钠水溶液淬灭反应直至没有气泡产生，然后用二氯甲烷萃取三次。有机相用水洗3次、盐水洗3次，分液后用无水硫酸钠干燥有机相。抽滤，减压浓缩后用柱层析(5～25％EA/PE)分离纯化，得到白色固体化合物V-2(8.52g,89％)。
Figure PCTCN2021093539-appb-000088
将化合物V-2(100mg,0.23ml)、苄胺(0.42mmol)、三乙胺(70mg,0.69mmol)和二氯甲烷(3ml)加入反应瓶中，在室温下搅拌反应3小时。反应完毕后，加入饱和碳酸氢钠水溶液淬灭反应，用二氯甲烷萃取3次，分液，有机相用水和盐水各洗一次。有机相用无水硫酸钠干燥，抽滤，减压浓缩后柱层析(5-30％EA/PE)纯化，得到42mg化合物VI-20，收率38％。
Figure PCTCN2021093539-appb-000089
将化合物VI-10(30mg,1.0当量)、9-均三甲基-2,7,10-三甲基吖啶高氯酸盐(6mol％)和2,4,6-三异丙基苯-1-硫酚(0.5当量)加入透明的带特氟塞的8*16mm反应管中。将反应管密闭，加入2ml无水甲苯，用氮气置换3次以上，将反应管置于两个40W Kessil KSH150B的蓝光灯照射下搅拌反应，体系逐渐变为深棕色，用TLC板监测反应，直至原料反应彻底。将反应体系减压浓缩，用制备级薄层色谱板分离得到17mg化合物I-b-20，收率56％。
化合物I-b-20的结构测定： 1H NMR(400MHz,CDCl 3)δ7.42(s,1H),7.31(dd,J＝11.7,4.7Hz,4H),7.23(d,J＝6.5Hz,1H),4.93(d,J＝6.3Hz,1H),4.83(d,J＝6.5Hz,1H),4.80(s,2H),4.03(d,J＝11.4Hz,1H),3.86(d,J＝13.9Hz,1H),3.51–3.36(m,2H),3.16(d,J＝10.1Hz,1H),2.97(d,J＝13.9Hz,1H),2.84(dd,J＝10.7,2.7Hz,1H),2.30–2.17(m,1H),1.91–1.80(m,1H),1.79–1.72(m,1H),1.63(d,J＝10.5Hz,4H),1.37(s,3H),1.22–1.14(m,2H),1.05(td,J＝13.3,2.8Hz,1H),0.95(d,J＝12.8Hz,1H),0.91(d,J＝4.7Hz,3H),0.89–0.68(m,3H)； 13CNMR(100MHz,CDCl 3)δ173.47,145.64,139.01,137.27,128.30,126.90,87.66,79.62,70.41,69.28, 60.40,59.27,54.79,53.41,37.48,35.82,35.44,34.79,33.05,31.40,29.68,25.75,20.42,19.92,15.10；HRMS(m/z)calc.for C 28H 38NO 4(+)452.2795,found 452.2792。
实施例47
化合物I-b-21的制备：
Figure PCTCN2021093539-appb-000090
参照实施例46中的制备方法制备化合物I-b-21，收率53％。化合物I-b-21的结构测定： 1H NMR(400MHz,CDCl 3)δ7.51(s,1H),7.21(d,J＝4.9Hz,1H),6.94(dd,J＝5.0,3.5Hz,1H),6.84(d,J＝3.2Hz,1H),4.93(d,J＝6.3Hz,1H),4.84(s,2H),4.81(d,J＝6.4Hz,1H),4.03(d,J＝11.4Hz,1H),3.90(d,J＝14.4Hz,1H),3.47–3.40(m,2H),3.36(d,J＝14.5Hz,1H),3.17(d,J＝9.4Hz,1H),2.96(dd,J＝11.2,3.4Hz,1H),2.29–2.19(m,1H),1.88–1.80(m,1H),1.78–1.70(m,2H),1.69–1.63(m,3H),1.58-1.54(m,1H),1.37(s,3H),1.21–1.08(m,3H),1.04(td,J＝13.4,3.0Hz,1H),0.97–0.93(m,1H),0.91(s,3H),0.90–0.73(m,3H)； 13CNMR(100MHz,CDCl 3)δ173.43,145.65,142.52,136.95,126.66,125.35,124.55,87.66,79.61,70.51,69.28,60.34,58.53,54.78,53.73,53.32,37.48,35.82,35.44,34.84,33.11,31.40,29.68,25.75,20.40,19.91,15.09；HRMS(m/z)calc.for C 26H 36NO 4S(+)458.2360,found 458.2357。
实施例48
化合物I-b-22的制备：
Figure PCTCN2021093539-appb-000091
参照实施例46中的制备方法制备化合物I-b-22，收率60％。化合物I-b-22 的结构测定： 1H NMR(400MHz,CDCl 3)δ7.49(s,1H),7.36(d,J＝1.1Hz,1H),6.30(dd,J＝3.0,1.9Hz,1H),6.14(d,J＝2.9Hz,1H),4.93(d,J＝6.3Hz,1H),4.85(s,2H),4.80(d,J＝6.1Hz,1H),4.02(d,J＝11.4Hz,1H),3.71(d,J＝14.5Hz,1H),3.44(dd,J＝12.2,5.5Hz,2H),3.26(d,J＝14.5Hz,1H),3.09(d,J＝10.2Hz,1H),2.92(dd,J＝11.1,3.2Hz,1H),2.28–2.20(m,1H),2.07–1.92(m,1H),1.80(dd,J＝12.8,3.0Hz,1H),1.77–1.69(m,3H),1.61–1.52(m,2H),1.37(s,3H),1.23–1.08(m,3H),1.04(dd,J＝13.6,2.8Hz,1H),1.00–0.91(m,2H),0.89(s,3H),0.81–0.65(m,2H)； 13CNMR(100MHz,CDCl 3)δ151.46,145.91,142.10,136.51,110.10,87.66,79.61,70.51,69.26,60.54,58.46,54.76,53.22,51.59,37.47,35.80,35.43,34.78,32.83,31.42,29.67,25.75,20.39,19.93,15.06；HRMS(m/z)calc.for C 26H 36NO 5(+)442.2588,found 442.2590。
实施例49
化合物I-b-23的制备：
Figure PCTCN2021093539-appb-000092
参照实施例46中的制备方法制备化合物I-b-23，收率60％。化合物I-b-23的结构测定： 1H NMR(400MHz,CDCl 3)δ7.41(s,1H),7.24(dd,J＝8.3,5.6Hz,2H),6.99(t,J＝8.7Hz,2H),4.93(d,J＝6.3Hz,1H),4.82(s,2H),4.80(s,1H),4.03(d,J＝11.4Hz,1H),3.79(d,J＝13.8Hz,1H),3.49–3.39(m,2H),3.15(d,J＝9.4Hz,1H),2.94(d,J＝13.8Hz,1H),2.78(dd,J＝10.6,2.4Hz,1H),2.28–2.19(m,1H),1.85–1.77(m,1H),1.74(dt,J＝13.3,3.4Hz,1H),1.62(t,J＝15.0Hz,7H),1.37(s,3H),1.23–1.10(m,3H),1.08–1.02(m,1H),0.95(d,J＝11.0Hz,1H),0.91(d,J＝4.7Hz,3H),0.89–0.67(m,3H)； 13CNMR(100MHz,CDCl 3)δ173.37,160.63,145.70,137.20,134.54,129.76,115.19,114.98,87.65,79.60,70.38,69.26,60.23,59.23,58.50,54.79,53.42,37.47,35.81,35.44,34.77,32.97,31.40,29.67,25.74,20.41,19.91, 15.08；HRMS(m/z)calc.for C 28H 37FNO 4(+)470.2701,found 470.2701。
实施例50
化合物I-c-1的制备：
Figure PCTCN2021093539-appb-000093
将I-b-1(0.03mmol，1.0当量),AcOH、THF和H 2O(AcOH:THF:H 2O＝4:1:2(v/v))的混合溶剂2毫升加入反应管内，在45摄氏度搅拌反应2小时。降至室温，减压浓缩溶剂后用制备级薄层色谱板分离纯化得到7mg淡黄色固体化合物I-c-1，收率53％。 1H NMR(400MHz,CDCl 3)δ7.42(s,1H),7.37–7.28(m,4H),7.25-7.24(m,1H),4.80(s,2H),4.21(d,J＝11.1Hz,1H),3.85(d,J＝14.0Hz,1H),3.45(dd,J＝11.8,3.9Hz,1H),3.32(d,J＝10.7Hz,1H),3.15(d,J＝11.0Hz,1H),2.97(d,J＝13.4Hz,1H),2.84(d,J＝11.2Hz,2H),2.02(d,J＝5.3Hz,1H),1.86–1.76(m,2H),1.76–1.64(m,4H),1.21(s,3H),1.19–1.01(m,3H),0.96(d,J＝10.9Hz,1H),0.87(dt,J＝7.3,5.2Hz,3H),0.78(s,3H)； 13C NMR(100MHz,CDCl 3)δ173.54,131.49,129.91,128.55,128.38,128.30,80.76,70.53,64.22,59.22,55.05,53.59,42.77,36.53,35.79,31.57,29.68,29.30,27.69,27.19,22.67,22.51,20.84,14.99,14.10.HRMS(m/z)calc.for C 27H 38NO 4(+)440.2795,found 440.2796。
实施例51
化合物I-c-2的制备：
Figure PCTCN2021093539-appb-000094
参照实施例50中的制备方法制备化合物I-c-2，收率53％。化合物I-c-2的结构测定： 1H NMR(300MHz,CDCl3)δ7.26(s,1H),7.23(d,J＝8.1Hz,2H),6.87 (d,J＝8.4Hz,2H),4.89(s,2H),4.19(d,J＝11.0Hz,1H),4.10–3.83(m,1H),3.81(d,J＝3.6Hz,3H),3.7–3.55(m,1H),3.47–3.25(m,3H),2.93(s,1H),2.16–1.94(m,2H),1.88–1.68(m,6H),1.62(s,2H),1.20(s,3H),1.11–1.02(m,2H),0.97(d,J＝6.8Hz,2H),0.89(t,J＝8.9Hz,4H),0.80(s,3H)； 13C NMR(100MHz,CDCl 3)δ173.40,129.94,114.22,114.17,114.07,114.00,80.60,70.82,64.15,58.99,55.28,54.98,53.41,42.70,36.50,35.80,31.90,31.41,29.67,27.61,22.53,20.73,14.97,14.10；HRMS(m/z)calc.for C 28H 40NO 5(+)470.2901,found 470.2903。
实施例52
化合物I-c-3的制备：
Figure PCTCN2021093539-appb-000095
参照实施例50中的制备方法制备化合物I-c-3，收率93％。化合物I-c-3的结构测定： 1H NMR(400MHz,CDCl 3)δ7.40(s,1H),7.34(d,J＝7.3Hz,1H),7.17–7.10(m,1H),7.06(t,J＝8.6Hz,1H),4.82(s,2H),4.21(d,J＝11.1Hz,1H),3.76(d,J＝14.1Hz,1H),3.45(dd,J＝11.1,4.6Hz,1H),3.32(d,J＝11.1Hz,1H),3.14(d,J＝10.2Hz,1H),2.90(d,J＝14.0Hz,1H),2.75(d,J＝8.2Hz,1H),2.07(d,J＝16.1Hz,1H),1.87–1.76(m,2H),1.76–1.62(m,8H),1.22(s,3H),1.19–1.00(m,3H),0.97(d,J＝10.6Hz,1H),0.92–0.82(m,2H),0.79(s,3H)； 13C NMR(100MHz,CDCl 3)δ173.37,155.72,145.77,136.93,129.96,127.79,120.79,116.32,116.11,80.57,70.38,65.73,64.12,60.15,59.05,57.97,54.95,53.54,42.61,36.41,35.68,34.57,32.94,31.47,29.57,27.54,22.45,20.73,15.13,14.90.HRMS(m/z)calc.for C 27H 36ClFNO 4(+)492.2311,found 492.2321.
实施例53
化合物I-c-4的制备：
Figure PCTCN2021093539-appb-000096
参照实施例50中的制备方法制备化合物I-c-4，收率94％。化合物I-c-4的结构测定： 1H NMR(400MHz,CDCl 3)δ7.52(s,1H),7.21(t,J＝5.6Hz,1H),6.97–6.91(m,1H),6.85(s,1H),4.84(s,2H),4.21(d,J＝11.1Hz,1H),3.90(d,J＝13.1Hz,1H),3.48–3.42(m,1H),3.38(d,J＝14.2Hz,1H),3.32(d,J＝11.1Hz,1H),3.16(d,J＝10.5Hz,1H),2.96(d,J＝8.4Hz,1H),2.01(d,J＝5.7Hz,1H),1.80(dd,J＝16.0,6.5Hz,4H),1.75–1.62(m,7H),1.21(s,3H),1.14–1.00(m,3H),0.96(d,J＝12.4Hz,1H),0.91–0.83(m,2H),0.78(s,3H). 13C NMR(100MHz,CDCl 3)δ173.51,145.76,136.64,126.65,125.54,124.45,80.71,70.48,64.17,60.12,58.33,54.96,53.45,42.59,36.36,35.67,34.55,33.07,31.42,29.57,27.56,22.44,20.75,14.89.HRMS(m/z)calc.for C 25H 36NO 4S(+)446.2360,found 446.2351。
实施例54
化合物I-c-5的制备：
Figure PCTCN2021093539-appb-000097
参照实施例50中的制备方法制备化合物I-c-5，收率66％。化合物I-c-5的结构测定： 1H NMR(400MHz,CDCl 3)δ7.50(s,1H),7.36(s,1H),6.30(s,1H),6.15(s,1H),4.91–4.75(m,2H),4.20(d,J＝11.1Hz,1H),3.71(d,J＝14.9Hz,1H),3.44(dd,J＝11.3,4.4Hz,1H),3.30(t,J＝14.8Hz,2H),3.08(d,J＝10.0Hz,1H),2.93(d,J＝11.5Hz,1H),2.06–1.98(m,1H),1.81–1.64(m,9H),1.22(s,3H),1.05(ddd,J＝13.0,10.2,6.2Hz,3H),0.96(d,J＝10.6Hz,1H),0.91–0.83(m,3H),0.76(s,3H), 0.73(d,J＝7.6Hz,1H). 13C NMR(125MHz,CDCl 3)δ173.52,142.19,129.93,110.16,100.82,80.81,70.61,64.25,60.50,58.45,55.06,53.47,51.57,42.80,36.53,35.79,34.73,31.65,29.72,29.34,27.71,22.63,20.89,15.01,14.15；HRMS(m/z)calc.for C 25H 36NO 5(+)430.2588,found 430.2591。
实施例55
化合物I-d-1的制备：
Figure PCTCN2021093539-appb-000098
在25ml的圆底烧瓶中加入穿心莲内酯衍生物I-a-5(300mg,0.53mmol,1eq,见实施例5)，锌粉(540mg,8.4mmol,16eq)，溶剂氯仿10ml，滴加1ml醋酸，室温搅拌反应30min，TLC检测反应，待反应结束，垫硅藻土抽滤，滤液用饱和碳酸氢钠溶液洗涤，二氯甲烷萃取，合并有机相，无水硫酸钠干燥后浓缩，柱层析(hexane/ethyl acetate 1:3)分离得到120mg化合物I-d-1,收率43％。
实施例56
化合物I-d-2的制备：
Figure PCTCN2021093539-appb-000099
在8ml的圆底烧瓶中，加入化合物I-d-1(20mg，0.04mmol,1eq)，三乙胺(30μl,0.2mmol,5eq)，1ml二氯甲烷，降温至0℃，加入丙烯酰氯(9μl，0.6mmol，1.5eq)，在该条件下反应30min。柱层析分离得到化合物I-d-2,收率53％。
化合物I-d-2的结构测定：1H NMR(300MHz,CDCl3)δ7.71(s,1H),7.52(s,4H),6.44(d,J＝16.8Hz,1H),6.26(dd,J＝16.5,10.1Hz,1H),5.78(d,J＝9.9Hz,1H),4.83(s,2H),4.57(d,J＝8.9Hz,1H),4.49–4.23(m,1H),4.11(d,J＝11.6Hz, 1H),3.85(s,1H),3.19(s,1H),3.02(s,1H),2.84(s,1H),2.34(s,2H),2.03(d,J＝2.0Hz,6H),1.81–1.56(m,6H),1.44(d,J＝8.1Hz,2H),1.06(d,J＝12.2Hz,2H),0.98(s,3H),0.86(s,5H)；HRMS(m/z)calc.for C34H45N2O7(+)593.3227,found 593.3223.
实施例57
化合物I-d-3的制备：
Figure PCTCN2021093539-appb-000100
在8ml的圆底烧瓶中，加入化合物I-d-1(20mg，0.04mmol,1eq)，三乙胺(30μl,0.2mmol,5eq)，1ml二氯甲烷，降温至0℃，滴加甲磺酰氯(8μl,0.6mmol,1.5eq)，在该条件下反应30min。柱层析分离得到化合物I-d-3，收率47％。
化合物I-d-3的结构测定：1H NMR(300MHz,CDCl3)δ7.43(d,J＝7.8Hz,2H),7.31(s,1H),7.22(d,J＝8.2Hz,2H),4.81(s,2H),4.67–4.50(m,1H),4.37(d,J＝11.7Hz,1H),4.10(d,J＝12.1Hz,1H),3.79(d,J＝13.7Hz,1H),3.14(d,J＝8.1Hz,1H),2.94(d,J＝13.1Hz,1H),2.81(d,J＝9.0Hz,1H),2.17(s,1H),2.03(d,J＝3.0Hz,7H),1.83–1.50(m,6H),1.50–1.36(m,2H),1.11(d,J＝16.0Hz,2H),0.98(s,3H),0.85(s,5H)；HRMS(m/z)calc.for C32H45N2O8S(+)617.2897,found 617.2892。
实施例58
化合物I-d-4的制备：
Figure PCTCN2021093539-appb-000101
在8ml的圆底烧瓶中，加入化合物I-d-1(20mg,0.04mmol,1eq)，加1ml二氯甲烷，滴加正丁基异氰酸酯(10μl，0.6mmol，1.5eq)，在该条件下反应30 min，柱层析分离得到化合物I-d-4，收率52％。
化合物I-d-4的结构测定：1H NMR(300MHz,CDCl3)δ7.21(d,J＝7.0Hz,4H),7.18(s,1H),6.82(s,1H),4.82(s,2H),4.66–4.48(m,1H),4.46–4.23(m,2H),4.10(d,J＝11.7Hz,1H),3.78(s,1H),3.23(dd,J＝12.7,6.8Hz,3H),3.02(s,1H),2.84(d,J＝9.0Hz,1H),2.41–2.15(m,1H),2.03(d,J＝3.3Hz,6H),1.68(m,,6H),1.57–1.42(m,4H),1.41–1.25(m,2H),1.16–1.00(m,2H),0.98(s,3H),0.91(t,J＝7.2Hz,3H),0.85(m,5H)；HRMS(m/z)calc.for C36H52N3O7(+)638.3805,found 638.3802。
实施例59
化合物I-d-5的制备：
Figure PCTCN2021093539-appb-000102
在室温条件下，在8ml的烧瓶中，加入化合物I-d-1(30mg，0.055mmol、1eq)，炔酸(20mg，0.07mmol，1.2eq)，HATU(25mg，0.07mmol，1.2eq)，DIPEA(21mg，0.17mmol,3eq)，DMF(1ml)，搅拌过夜，反应结束之后，用5ml乙酸乙酯稀释，用饱和食盐水3ml洗三遍，旋干。薄层色谱制备板分离得到化合物I-d-5，收率32％。
化合物I-d-5的结构测定：1H NMR(300MHz,CDCl3)δ7.54(d,J＝8.4Hz,2H),7.48(s,1H),7.23(d,J＝8.4Hz,2H),4.82(s,2H),4.56(dd,J＝10.1,5.5Hz,1H),4.36(d,J＝11.7Hz,1H),4.17(d,J＝2.4Hz,2H),4.12(s,2H),4.08(s,1H),3.82(d,J＝13.7Hz,1H),3.77–3.62(m,12H),3.20(s,1H),2.88(s,1H),2.86(d,J＝10.5Hz,2H),2.43(t,J＝2.4Hz,1H),2.03(d,J＝3.1Hz,6H),1.84–1.58(m,6H),1.50–1.37(m,2H),1.17–1.01(m,2H),0.98(s,2H),0.85(s,3H),0.85(m,5H)；HRMS(m/z)calc.for C42H59N2O11(+)767.4119,found 767.4113。
实施例60
化合物I-d-6的制备：
Figure PCTCN2021093539-appb-000103
参照实施例59中的制备方法制备化合物I-d-6。化合物I-d-6的结构测定： 1H NMR(400MHz,CD 2Cl 2)δ7.47(s,1H),7.45(d,J＝1.6Hz,2H),7.41(s,1H),7.25(d,J＝8.3Hz,2H),4.80(d,J＝0.9Hz,2H),4.56(dt,J＝21.0,10.4Hz,1H),4.37(d,J＝11.7Hz,1H),4.11(d,J＝11.7Hz,1H),3.82(d,J＝13.7Hz,1H),3.12(d,J＝9.1Hz,1H),2.97–2.87(m,1H),2.80(dd,J＝11.1,3.3Hz,1H),2.62–2.56(m,4H),2.10(d,J＝2.4Hz,1H),2.02(s,6H),1.82–1.53(m,12H),1.53–1.42(m,2H),1.27–1.15(m,2H),1.12(dd,J＝12.4,2.0Hz,1H),1.00(s,3H),0.95–0.89(m,1H),0.88(s,3H),0.84–0.78(m,1H)；HRMS(m/z)calc.for C36H46N2O7(+)619.3377,found 619.3378。
实施例61
化合物I-a-27的制备：
Figure PCTCN2021093539-appb-000104
参照实施例6中的制备方法制备化合物I-a-27。化合物I-a-27的结构测定： 1H NMR(400MHz,CDCl 3)δ7.41(s,1H),7.20(d,J＝8.5Hz,2H),6.92(d,J＝8.6Hz,2H),4.80(s,2H),4.68(d,J＝2.3Hz,2H),4.58(dd,J＝10.8,4.9Hz,1H),4.38(d,J＝11.7Hz,1H),4.12(d,J＝11.7Hz,1H),3.80(t,J＝16.0Hz,1H),3.14(d,J＝9.8Hz,1H),2.93(d,J＝13.7Hz,1H),2.82(dd,J＝11.0,2.9Hz,1H),2.52(t,J＝2.3Hz,1H),2.04(d,J＝3.1Hz,6H),1.79(d,J＝12.8Hz,1H),1.75–1.38(m,12H),1.21 (dd,J＝14.8,9.2Hz,2H),1.08(d,J＝10.8Hz,1H),0.99(s,3H),0.89(t,J＝5.6Hz,1H),0.85(s,3H),0.79(d,J＝12.0Hz,1H)；HRMS(m/z)calc.for C34H43NO7(+)578.3112，found 578.3112。
对照例62
穿心莲内酯衍生物对肿瘤细胞系的抗增殖活性
选取鼻咽癌、宫颈癌、肝癌以及白血病等细胞作为研究对象，以穿心莲内酯为阳性对照，筛选穿心莲内酯衍生物的抗肿瘤活性。选取的的细胞系分别为：人EBV阳性鼻咽癌细胞C666-1和HONE1-ebv、人宫颈癌细胞HeLa、人早幼粒白血病细胞HL-60和人肝癌细胞Huh7。
Resazurin实验方法的要点如下：采用对数生长期的肿瘤细胞，接种于96孔细胞培养板中，每孔100μL，每孔加入C666-1为7000个，HONE1-ebv、HeLa、Huh 7为2000个，HL-60为20000个，每个实验组设置2个复孔。培养24h后，每孔加入0.5μL化合物的DMSO溶液使其终浓度为50μM。此外，设置加0.5μL DMSO的孔为阳性对照，只加培养基的孔为阴性对照。化合物孵育72h后，加入10μL Resazurin的PBS溶液使其终浓度为100μM，37℃孵育3h，在摇床上震荡均匀后，用酶标仪检测荧光强度，激发光为560nm，发射光为590nm，抑制率计算公式为Inhition％＝100％-(F-F blank)/(F Ctrl-F blank)×100％，其中F为加化合物孔的荧光强度，F blank为阴性对照孔读数，F Ctrl为阳性孔荧光数值。测试结果见表1，从初步的筛选结果来看，氮杂三环穿心莲内酯衍生物中不少化合物表现出优良的抗肿瘤活性，部分化合物活性明显高于阳性对照穿心莲内酯。
表1：穿心莲内酯衍生物对肿瘤细胞系的抗增殖活性
Figure PCTCN2021093539-appb-000105
Figure PCTCN2021093539-appb-000106
Figure PCTCN2021093539-appb-000107
肿瘤细胞抑制活性：肿瘤抑制率>70％＝++++；70％>肿瘤抑制率>50％＝+++；50％>肿瘤抑制率>30％＝++；肿瘤抑制率<30％＝+；ND表示没有测试该细胞系。
对照例63
应用细胞病变模型评价化合物体外抗冠状病毒229E活性。
运用细胞病变效应(CPE)实验，检测化合物体外抗人普通冠状病毒(HCoV)229E活性，同时检测化合物对MRC5细胞的毒性。MRC5细胞(货号CCL-171)和人冠状病毒HCoV 229E(货号VR-740)购自美国典型培养物保藏中心(ATCC)。MRC5细胞使用添加了10％胎牛血清(Excell)，1％双抗(Hyclone)，1％L-谷氨酰胺(Gibco)和1％非必需氨基酸(Gibco)的EMEM(Sigma)培养液培养。添加了5％胎牛血清，1％双抗，1％L-谷氨酰胺和1％非必需氨基酸的EMEM培养液为实验培养液。
MRC5细胞以每孔20,000个细胞，每孔100μl的密度接种到96微孔板中并于5％CO 2、37℃培养箱中培养过夜。第二天，每孔加入50μl的化合物(双复孔)，随后将50μl稀释好的病毒以每孔200 TCID 50加入细胞测试孔，最终化合物的浓度为25μM。设置细胞对照(细胞，无化合物处理或病毒感染)，病毒对照(细胞感染病毒，无化合物处理)和培养液对照(只有培养液)。实验培养液终体积为每孔200μl，培养液中DMSO的终浓度为0.5％。细胞于5％CO 2、35℃ 培养箱中继续培养3天。使用细胞活力检测试剂盒CellTiter Glo(Promega)检测细胞活力。细胞毒性实验与抗病毒实验条件相同，但无病毒感染。
化合物的抗病毒活性和细胞毒性分别由在单一浓度下(25μM)的化合物对病毒引起的细胞病变效应的抑制率(％)和细胞活率(％)表示。计算公式如下：
抑制率(％)＝(测试孔读值-病毒对照平均值)/(细胞对照平均值-病毒对照平均值)×100。
细胞活率(％)＝(测试孔读值-培养液对照平均值)/(细胞对照平均值-培养液对照平均值)×100。
归一化的抑制率(％)＝抑制率(％)-[1-细胞活率(％)]。
表2给出了穿心莲内酯衍生物对人冠状病毒HCoV-229E的抑制活性，可见，不少化合物表现出优良的抗冠状病毒活性。
表2：穿心莲内酯衍生物对人冠状病毒HCoV-229E的抑制活性
Figure PCTCN2021093539-appb-000108
Figure PCTCN2021093539-appb-000109
表2中，25微摩尔浓度下对人冠状病毒HCoV-229E的抑制活性：归一化的抑制率>80％＝++++；80％>抑制率>60％＝+++；60％>抑制率>30％＝++；肿瘤抑制率<30％＝+。
以上所揭露的仅为本发明较佳实施例而已，当然不能以此来限定本发明之权利范围，因此依本发明权利要求所作的等同变化，仍属本发明所涵盖的范围。

Claims (16)

  1. 一种穿心莲内酯类化合物，其特征在于，其结构如式(Ⅰ)所示：
    Figure PCTCN2021093539-appb-100001
    其中，-R 1选自-H、苄基、取代的苄基、芳基、杂环基、杂芳基、取代的C 1～C 10的直链烷基、取代的C 3～C 10的支链烷基和取代的环烷基中的任意一种；
    -R 2和-R 3相同，所述-R 2和所述-R 3选自-H、甲基、乙基、丙基和-(O)CR 4中的任意一种，-R 4选自甲基、乙基、丙基、异丙基、丁基、异丁基、取代的苯基、呋喃基、噻吩基和吡啶基中的任意一种；
    或者-R 2和-R 3一起形成下列基团：
    Figure PCTCN2021093539-appb-100002
    -R 5为-H、烷基或芳基，-R 6为-H、烷基或芳基。
  2. 根据权利要求1所述的穿心莲内酯类化合物，其特征在于，
    所述取代的苄基的苯环上含有一个、两个或两个以上的取代基，所述取代基选自羟基、胺基、-X、甲基、乙基、-CX 3、C 3～C 12的单环环烷基、-OCH 3、-OCH 2CH 3、苯基、吡啶基、嘧啶基、噻吩基、呋喃基、吡咯基、吡唑基、噻唑基、吲哚基、苯并噻吩基、苯并呋喃基、吗啉基、哌啶基、哌嗪基、四氢吡咯基、氰基、酯基、羧基、磺酰基、磺酰氨基、氨基磺酰基、酰胺基、氨基酰基、脲基、硫脲基和硝基中的任意一种，其中，X为F、Cl、Br或I原子；
    所述取代的环烷基为C 3～C 12的单环环烷基、C 7～C 12的双环环烷基或C 7～C 12的螺环环烷基的环上含有一个、两个或两个以上的取代基；
    所述芳基为单环芳基或2～5环的稠环芳基；
    所述杂环基为3～12个原子的单环杂环基、7～12个原子的双环杂环基或7～12个原子的螺环杂环基；
    所述杂芳基为含有1～3个苯环取代基的单环杂环基、含有1～3个苯环取代基的螺环杂环基、含有1～3个单环杂环取代基的苯基或含有1～3个单环杂环取代基的螺环杂环基；
    所述取代的C 1～C 10的直链烷基为C 1～C 10的直链烷基上含有一个、两个或两个以上的取代基；
    所述取代的C 3～C 10的支链烷基为C 3～C 10的支链烷基上含有一个、两个或两个以上的取代基；
    所述烷基为环烷基、直链烷基或支链烷基。
  3. 根据权利要求2所述的穿心莲内酯类化合物，其特征在于，
    所述-R 1选自-CH 3、-CH 2CH 3、-CH 2CH 2CH 3、-CH 2CH 2CH 2CH 3、-CH 2CH(CH 3) 2、-CH(CH 3) 2、-CH 2CH 2CH(CH 3)、环丙基、环丁基、环戊基、环己基、环庚基、
    Figure PCTCN2021093539-appb-100003
    Figure PCTCN2021093539-appb-100004
    苯基、联苯基、萘基、苯并咪唑基、苯并噻吩基、苯并呋喃基、咪 唑基、噻吩基、呋喃基、
    Figure PCTCN2021093539-appb-100005
    Figure PCTCN2021093539-appb-100006
    Figure PCTCN2021093539-appb-100007
    Figure PCTCN2021093539-appb-100008
    Figure PCTCN2021093539-appb-100009
    中的任意一种；其中，X为F、Cl、Br或I原子，X 1为F、Cl、Br或I原子，X 2为F、Cl、Br或I原子。
  4. 根据权利要求3所述的穿心莲内酯类化合物，其特征在于，
    所述-R 2和所述-R 3为-H或-OAc；
    或者，所述-R 2和所述-R 3一起形成下列基团：-C(CH 3) 2-或-CH 2-。
  5. 根据权利要求1所述的穿心莲内酯类化合物，其特征在于，为下列式中之一的结构：
    Figure PCTCN2021093539-appb-100010
    Figure PCTCN2021093539-appb-100011
    Figure PCTCN2021093539-appb-100012
    Figure PCTCN2021093539-appb-100013
  6. 根据权利要求1所述的穿心莲内酯类化合物，其特征在于，还包括式(I)所示化合物的对映体、非对映体、互变体、盐、溶剂化物或各位置同位素取代形式的分子。
  7. 一种如权利要求1～6任意一种所述的穿心莲内酯类化合物的制备方法，其特征在于，包括以下步骤：
    提供化合物A，所述化合物A的结构为：
    Figure PCTCN2021093539-appb-100014
    其中，M 1为烷基或-(O)CR 4，-R 4选自甲基、乙基、丙基、异丙基、丁基、异丁基、取代的苯基、呋喃基、噻吩基和吡啶基中的任意一种；
    -R 2和-R 3相同，所述-R 2和所述-R 3选自-H、甲基、乙基、丙基和-(O)CR 4中 的任意一种，-R 4选自甲基、乙基、丙基、异丙基、丁基、异丁基、取代的苯基、呋喃基、噻吩基和吡啶基中的任意一种；
    或者-R 2和-R 3一起形成下列基团：
    Figure PCTCN2021093539-appb-100015
    -R 5为-H、烷基或芳基，-R 6为-H、烷基或芳基；
    将所述化合物A、胺和碱性介质加入有机溶剂中，发生反应，得到化合物B，所述胺的化学式为R 1-NH 2，-R 1选自-H、苄基、取代的苄基、芳基、杂环基、杂芳基、取代的C 1～C 10的直链烷基、取代的C 3～C 10的支链烷基和取代的环烷基中的任意一种，所述化合物B的结构为：
    Figure PCTCN2021093539-appb-100016
    将所述化合物B、氢原子转移供体和光氧化还原催化剂加入有机溶剂中，在光的照射下发生反应，得到化合物C，所述化合物C的结构为：
    Figure PCTCN2021093539-appb-100017
  8. 根据权利要求7所述的制备方法，其特征在于，
    所述有机溶剂选自二氯甲烷、四氢呋喃、乙醚、1,4-二氧六环、甲苯、苯和氯仿中的一种、两种、三种或三种以上；
    所述碱性介质选自三乙胺、二异丙基乙基胺和吡啶中的一种、两种或三种；
    所述氢原子转移供体的当量为所述化合物B的当量的20％～100％；所述氢原子转移供体选自2,4,6-三异丙基苯硫酚、1,4-二氢-2,6-二甲基-3,5-吡啶二羧酸二乙酯和1,4-环己二烯中的任意一种；
    所述光氧化还原催化剂的当量为所述化合物B的当量的1％～10％；所述光氧化还原催化剂选自金属铱催化剂、金属铑催化剂和有机染料类催化剂中的任意一种；
    所述光的波长范围为300nm～500nm。
  9. 根据权利要求7或8所述的制备方法，其特征在于，所述化合物A的制备方法包括以下步骤：
    将穿心莲内酯加入所述有机溶剂中，加入酰化剂或卤代烃，加入路易斯酸作为催化剂，所述酰化剂或卤代烃与所述穿心莲内酯的3位、19位和14位的-OH发生反应，得到所述化合物A；
    所述酰化剂为酸酐或酰卤；所述酸酐的化学式为：R 4-C(O)-O-C(O)-R 4，所述酰卤的化学式为R 4-C(O)-卤素，所述-R 4选自甲基、乙基、丙基、异丙基、丁基、异丁基、取代的苯基、呋喃基、噻吩基和吡啶基中的任意一种；所述卤代烃的化学式为：烷基-卤素。
  10. 根据权利要求7或8所述的制备方法，其特征在于，所述化合物A的制备方法包括以下步骤：
    将穿心莲内酯、羰基化试剂和酸催化剂加入到反应介质中，在60℃～100℃的反应温度下，所述羰基化试剂的羰基与所述穿心莲内酯的3位和19位的-OH发生缩合反应，得到化合物D，所述羰基化试剂的化学式为R 5-C(OMe) 2-R 6或R 5-CO-R 6，其中，-R 5为-H、烷基或芳基；-R 6为-H、烷基或芳基，所述化合物D的结构为：
    Figure PCTCN2021093539-appb-100018
    将所述化合物D加入所述有机溶剂中，加入酰化剂或卤代烃，加入所述碱性介质，所述酰化剂或卤代烃与所述化合物D的14位的-OH发生反应，得到所述化合物A，所述酰化剂为酸酐或酰卤；所述酸酐的化学式为：R 4-C(O)-O-C(O)-R 4，所述酰卤的化学式为R 4-C(O)-卤素，所述R 4选自甲基、乙基、丙基、异丙基、丁基、异丁基、取代的苯基、呋喃基、噻吩基和吡啶基中的任意一种；所述卤代烃的化学式为：烷基-卤素。
  11. 根据权利要求10所述的制备方法，其特征在于，
    所述反应介质为甲苯和二甲亚砜的混合溶剂；
    所述酸催化剂为对甲苯磺酸或浓硫酸；
    所述化合物A的制备方法中，所述酸催化剂的当量为所述穿心莲内酯的当量的10％～40％；所述羰基化试剂的当量为所述穿心莲内酯的当量的100％～600％；所述酰化剂或卤代烃的当量为所述化合物D的当量的100％～600％；所述碱性介质的当量为所述化合物D的当量的100％～600％。
  12. 根据权利要求10所述的制备方法，其特征在于，得到所述化合物C后，还包括以下步骤：
    将所述化合物C加入反应溶剂中，加入酸，发生水解反应，得到化合物E，所述化合物E的结构为：
    Figure PCTCN2021093539-appb-100019
  13. 根据权利要求12所述的制备方法，其特征在于，所述反应溶剂选自四氢呋喃、1,4-二氧六环和水中的一种、两种或两种以上；
    所述酸选自盐酸、对甲苯磺酸、硫酸和醋酸中的任意一种。
  14. 一种药物组合物，其特征在于，包括权利要求1～6任意一项所述的穿心莲内酯类化合物。
  15. 权利要求1～6任意一项所述的穿心莲内酯类化合物或权利要求14所述的药物组合物在制备抗肿瘤药物中或抗病毒药物中的应用。
  16. 根据权利要求15所述的应用，其特征在于，所述抗肿瘤药物是抗鼻咽癌、宫颈癌、肝癌、乳腺癌、肺癌、前列腺癌、结肠癌、胰腺癌、脑癌、胃癌、骨癌、皮肤癌或白血病类药物；
    所述抗病毒药物是抗冠状病毒、抗甲型流感病毒、抗乙型肝炎病毒、抗丙型肝炎病毒、抗I型单纯疱疹病毒、抗EB病毒、抗人乳头瘤病毒或抗人类免疫缺陷病毒类药物。
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