WO2022143501A1 - 氮杂螺环、多环穿心莲内酯类化合物及其制备方法、药物组合物及其应用 - Google Patents

氮杂螺环、多环穿心莲内酯类化合物及其制备方法、药物组合物及其应用 Download PDF

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WO2022143501A1
WO2022143501A1 PCT/CN2021/141536 CN2021141536W WO2022143501A1 WO 2022143501 A1 WO2022143501 A1 WO 2022143501A1 CN 2021141536 W CN2021141536 W CN 2021141536W WO 2022143501 A1 WO2022143501 A1 WO 2022143501A1
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compound
andrographolide
mmol
polycyclic
preparation
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French (fr)
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朱振东
邢琦
车超
黎婷
蒋顶
张家银
张传冰
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
    • C07D405/06—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 linked by a carbon chain containing only aliphatic carbon atoms
    • 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
    • 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
    • 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
    • C07D405/14—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 three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application relates to the field of medicinal chemistry, in particular to an azaspirocyclic and polycyclic andrographolide compound and its preparation method, pharmaceutical composition and its application in antitumor drugs and antiviral drugs.
  • Natural products are of great significance to the field of pharmaceutical research.
  • the diverse structures of natural products and their easy combination with biological macromolecules determine their unparalleled advantages in participating in the physiological process of life. Products play an irreplaceable role in the development of new drugs and are an important source for the discovery of candidate drugs and drug lead structures. So far, more than one-third of the drugs in clinical use are directly derived from natural products or derivatives developed with the active ingredients of natural products as leading compounds.
  • Andrographolide is the main diterpene lactone compound extracted from Andrographis paniculata, is one of the main active ingredients of Chinese medicine Andrographis paniculata, and its structure is shown in the following formula:
  • andrographolide and its derivatives provide protection against a variety of viral infections by effectively enhancing the immune regulation of T cells and stimulating the host immune system to attack viruses. It has been reported that 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), Epstein-Barr virus (EBV) ), human papillomavirus (HPV), human immunodeficiency virus (HIV) and other viruses exhibit broad-spectrum antiviral activity.
  • IAV influenza A virus
  • 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
  • An azaspirocycle, polycyclic andrographolide compound is characterized in that, has following general formula I:
  • -R 1 is -H, -OH, alkoxy or
  • -R 2 is -OH, alkoxy or
  • -R 3 is -OH, alkoxy or
  • -R 4 is alkyl or aryl
  • -R 5 and -R 6 together form the following groups: -R 7 is alkyl or aryl, -R 8 is H or -R 9 is cycloalkyl, benzyl, substituted benzyl, heterocyclyl, C 1 -C 10 straight-chain alkyl or C 3 -C 10 branched-chain alkyl, -R 10 is H;
  • -R 5 and -R 6 together form the following groups: -R 7 is alkyl or aryl, -R 8 is H, -R 10 is H;
  • -R 6 is the following group:
  • -R 8 and -R 5 together form the following group:
  • -R 7 is alkyl or aryl,
  • -R 9 is selected from cycloalkyl, benzyl, substituted benzyl, heterocyclyl, C 1 -C 10 straight chain alkyl and C 3 -C 10 branched chain Any one of the alkyl groups, -R 10 is H;
  • -R 6 , -R 10 and C8, C9 and C11 in the general formula together form the following group: -R 5 and -R 8 together form the following groups: -R 7 is alkyl or aryl.
  • a kind of preparation method of above-mentioned azaspirocycle, polycyclic andrographolide compound comprises the steps:
  • the compound A, the sulfonimide iodide salt and the copper catalyst are added to the second organic solvent to react to obtain the compound B, and the structure of the compound B is:
  • a pharmaceutical composition comprising the above-mentioned azaspirocycle, polycyclic andrographolide compounds or pharmaceutically acceptable salts thereof.
  • This azaspirocyclic, polycyclic andrographolide compound in the attempt of structural modification, broke through the existing modification of hydroxyl groups at positions 3, 19 and 14, and creatively modified the double bonds at positions 8 and 17.
  • Azacyclopropylation was carried out, and on this basis, through the intermolecular 3+2 cycloaddition of azacyclopropyl and acetonitrile, Michael-amination at the C12 position, intramolecular nucleophilic attack of azacyclopropyl ring-opening and nitrogen Heterocyclopropyl group and intramolecular double bond 3+2 cycloaddition to form a series of new amino-substituted polycyclic azaspiro and polycyclic andrographolide compounds, which enhance the biological activity and bioavailability of the compounds and stability.
  • FIG. 1 is a graph showing the activity and cytotoxicity of andrographolide derivative D-1 in Example 89 against novel coronavirus (SARS-CoV-2) in vitro.
  • Figure 2 is a graph showing the activity and cytotoxicity of Chloroquine in Example 89 against the novel coronavirus (SARS-CoV-2) in vitro.
  • FIG. 3 is a graph showing the activity and cytotoxicity of Remdesivir in Example 89 against the novel coronavirus (SARS-CoV-2) in vitro.
  • Structural modification of andrographolide is an important way to discover new antitumor drug skeletons.
  • the structural modification of andrographolide mainly includes: esterification, etherification and oxidation of three hydroxyl groups in different degrees; reduction, epoxidation and epoxidative ring opening of terminal alkenes; conjugated alkenes The reduction, addition and [3+2] cycloaddition of .
  • This application proposes new transformation ideas and strategies, using some novel synthetic methods and technologies to try to transform other positions of andrographolide, or to further modify and transform with andrographolide derivatives as substrates, in order to discover more valuable drug skeleton.
  • -Ac CH3CO- , acetyl; -Me: CH3 , methyl; tBu: tert - butyl; Et: CH3CH2- , ethyl; AcOH: acetic acid; THF: tetrahydrofuran; EtOH : CH3CH 2OH , ethanol.
  • -R 1 is -H, -OH, alkoxy or
  • -R 2 is -OH, alkoxy or
  • -R 3 is -OH, alkoxy or
  • -R 4 is alkyl or aryl
  • -R 5 and -R 6 together form the following groups: -R 7 is alkyl or aryl, -R 8 is H or -R 9 is cycloalkyl, benzyl, substituted benzyl, heterocyclyl, C 1 -C 10 straight-chain alkyl or C 3 -C 10 branched-chain alkyl, -R 10 is H;
  • -R 5 and -R 6 together form the following groups: -R 7 is alkyl or aryl, -R 8 is H, -R 10 is H;
  • -R 6 is the following group:
  • -R 8 and -R 5 together form the following group:
  • -R 7 is alkyl or aryl,
  • -R 9 is selected from cycloalkyl, benzyl, substituted benzyl, heterocyclyl, C 1 -C 10 straight chain alkyl and C 3 -C 10 branched chain Any one of the alkyl groups, -R 10 is H;
  • -R 6 , -R 10 and C8, C9 and C11 in the general formula together form the following group: -R 5 and -R 8 together form the following groups: -R 7 is alkyl or aryl.
  • This azaspirocyclic, polycyclic andrographolide compound in the attempt of structural modification, broke through the existing modification of hydroxyl groups at positions 3, 19 and 14, and creatively modified the double bonds at positions 8 and 17.
  • Azacyclopropylation was carried out, and on this basis, through the intermolecular 3+2 cycloaddition of azacyclopropyl and acetonitrile, Michael-amination at the C12 position, intramolecular nucleophilic attack of azacyclopropyl ring-opening and nitrogen Heterocyclopropyl group and intramolecular double bond 3+2 cycloaddition to form a series of new amino-substituted polycyclic azaspiro and polycyclic andrographolide compounds, which enhance the biological activity and bioavailability of the compounds and stability.
  • -R 1 is -H
  • -R 2 and -R 3 are the same.
  • both -R 2 and -R 3 are -OAc.
  • -R 1 , -R 2 and -R 3 are the same.
  • azaspirocycle and polycyclic andrographolide compounds with general formula I can be divided into the following four categories:
  • the first type a double bond between C12 and C13, a single bond between C13 and C14, -R 8 is a hydrogen group, -R 1 , -R 2 and -R 3 are the same, -R 1 , -R 2 and -R 3 is selected from -OH, alkoxy and In any one, -R 4 is alkyl or aryl; -R 5 and -R 6 together form the following groups: -R 7 is alkyl or aryl; or -R 5 and -R 6 together form the following groups: -R 7 is alkyl or aryl; -R 10 is H.
  • the second category a single bond between C12 and C13, a double bond between C13 and C14, -R 1 is a hydrogen group, -R 2 and -R 3 are the same, -R 2 and -R 3 are selected from -OH, alkoxy and Any one of, -R 4 is an alkyl group or an aryl group, -R 8 is an amino group At the same time -R 5 and -R 6 together form the following group: -R 7 is alkyl or aryl, -R 9 is selected from cycloalkyl, benzyl, substituted benzyl, heterocyclyl, C 1 -C 10 straight chain alkyl and C 3 -C 10 branched chain In any of the alkyl groups, -R 10 is H.
  • the third type a single bond between C12 and C13, a double bond between C13 and C14, -R 1 is a hydrogen group, -R 2 and -R 3 are the same, -R 2 and -R 3 are selected from -OH, alkoxy and In any one, -R 4 is alkyl or aryl; -R 6 is the following group: -R 7 is an alkyl or aryl group, while -R 8 and -R 5 together form the following groups: -R 9 is selected from any one of cycloalkyl, benzyl, substituted benzyl, heterocyclyl, C 1 -C 10 straight-chain alkyl and C 3 -C 10 branched alkyl, -R 10 is H.
  • the fourth category a single bond between C12 and C13, a double bond between C13 and C14, -R 1 is a hydrogen group, -R 2 and -R 3 are the same, -R 2 and -R 3 are selected from -OH, alkoxy and In any one, -R 4 is an alkyl group or an aryl group; -R 6 , -R 10 and C8, C9 and C11 in the general formula together form the following groups: -R 5 and -R 8 together form the following groups: -R 7 is alkyl or aryl.
  • Alkyl refers to a saturated hydrocarbon group containing only C and H atoms, including cycloalkyl and chain alkyl groups, and chain alkyl groups include straight-chain or branched-chain alkyl groups.
  • the alkyl group includes, but is not limited to, -CH 3 and the like.
  • Aryl refers to the general term for a monovalent group left after removing a hydrogen atom from the aromatic nucleus carbon of an aromatic hydrocarbon molecule, which can be a single-ring aryl group or a fused-ring aryl group with 2 to 5 rings, a single-ring aryl group Both aryl and fused-ring aryl groups can be substituted or unsubstituted, and the substituted monocyclic aryl group or substituted fused-ring aryl group contains one, two or more than two substituents on the benzene ring, and the substituents can be, But not limited to, any one of hydroxyl, amino, -X, methyl, ethyl, tert-butyl, -CX 3 , -OCH 3 , -OCH 2 CH 3 , amide group and nitro group, etc., wherein, X is a F, Cl, Br or I atom.
  • aryl groups include, but are not limited to, any of the following.
  • C 1 -C 10 straight-chain 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 2 CH 2 CH 3 , - CH ( CH3 ) 2 , -CH2CH2CH2CH3 or -CH2CH2CH ( CH3 ) 2 etc.
  • 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, which can be Is substituted or unsubstituted, substituted cycloalkyl includes one, two or more substituents, which may be, but not limited to, hydroxyl, amino, -X, methyl, ethyl, -CX 3 , any one of -OCH 3 , -OCH 2 CH 3 and nitro, etc., wherein X is a F, Cl, Br or I atom.
  • the cycloalkyl group may be, but is not limited to, cyclopentyl, cyclohexyl, cycloheptyl, Wait.
  • Benzyl refers to the monovalent group left after removing a hydrogen atom from the methyl carbon in the toluene molecule.
  • the benzene ring of "substituted benzyl” contains one, two or more substituents, which may be, but not limited to, hydroxyl, amino, -X, methyl, ethyl, -CX 3 , Any one of C 3 cycloalkyl, -OCH 3 , -OCH 2 CH 3 , phenyl, and nitro, etc., wherein X is a F, Cl, Br or I atom.
  • the substituted benzyl groups include, but are not limited to, any of the following.
  • Heterocyclic group refers to a cyclic group containing heteroatoms, which can be a monocyclic heterocyclic group of 3 to 12 atoms, a bicyclic heterocyclic group of 7 to 12 atoms, a monocyclic heterocyclic group or a bicyclic heterocyclic group
  • One or more atoms on the ring can be independently and optionally substituted by heteroatoms, which 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
  • the hydrogen atoms on each ring can be replaced by one or more substituents, and the substituents can be, but not limited to, hydroxyl, amine, -X, methyl, ethyl, -CX 3 , -OCH 3 , -OCH Any of 2 CH 3 and nitro, etc., wherein X is a F, Cl, Br or I atom.
  • the heterocyclyl group may include, but is not limited to, imidazolyl, thienyl or furyl, etc.
  • -R 7 is -CH 3
  • -R 9 is
  • a double bond is formed between C12 and C13, a single bond is formed between C13 and C14, and when -R8 is a hydrogen group, -R5 and -R6 together form the following group: -R 10 is H.
  • a single bond is formed between C12 and C13, a double bond is formed between C13 and C14, -R 1 is a hydrogen group, -R 8 is an amine group , -R 5 and -R 6 together form the following groups: -R 10 is H.
  • a single bond is formed between C12 and C13, a double bond is formed between C13 and C14, -R 1 is a hydrogen group, -R 6 is a sulfonamide group , -R 8 and -R 5 together form the following group: -R 10 is H.
  • a single bond is formed between C12 and C13
  • a double bond is formed between C13 and C14
  • -R 1 is a hydrogen group
  • -R 5 and -R 8 together form the following groups:
  • azaspiro, polycyclic andrographolide compounds are one of the compounds with the following structural formula:
  • azaspiro, polycyclic andrographolide compounds can be one of the compounds with the following chemical formula:
  • the azaspirocyclic and polycyclic andrographolide compounds have significantly higher inhibitory effect on tumors than andrographolide.
  • the present application also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned azaspirocycle, polycyclic andrographolide compounds or pharmaceutically acceptable salts thereof.
  • the present application also discloses the application of the azaspirocyclic and polycyclic andrographolide compounds in the preparation of antitumor drugs.
  • the anti-tumor drug is an 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 class of drugs.
  • the present application also discloses the application of the above-mentioned azaspirocyclic and polycyclic andrographolide compounds in the preparation of antiviral drugs.
  • antiviral drugs can be anti-human coronavirus (HCoV-229E) drugs or anti-novel coronavirus (SARS-CoV-2) drugs.
  • HCV-229E anti-human coronavirus
  • SARS-CoV-2 anti-novel coronavirus
  • the application also discloses a preparation method of the above-mentioned azaspirocycle and polycyclic andrographolide compounds, comprising the following steps:
  • R 1 , -R 2 and -R 3 are the same, and R 1 , -R 2 and -R 3 are alkoxy or -R 4 is alkyl or aryl.
  • compound A can be prepared with andrographolide as raw material, and the concrete preparation method is as follows:
  • the acylating agent is esterified with -OH group, and the acylating agent can be acid anhydride or acid halide; acid anhydride and acid halide can be substituted or unsubstituted, and the substituent can be various aliphatic or aromatic substitutions base.
  • the chemical formula of the acid anhydride is: R 4 -C(O)-OC(O)-R 4
  • the chemical formula of the acid halide is R 4 -C(O)-halogen
  • R 4 is an alkyl group or an aryl group
  • the above esterification reaction can be catalyzed using Lewis acids such as anhydrous ZnCl 2 .
  • the first organic solvent can be one, two or more of dichloromethane, tetrahydrofuran, diethyl ether, 1,4-dioxane and toluene, etc., preferably dichloromethane.
  • the reaction is carried out at a temperature of 25°C to 100°C (preferably 50°C) for 2h to 5h.
  • the acylating agent can also be replaced with halogenated hydrocarbons, and the remaining reaction conditions remain unchanged.
  • the halogenated hydrocarbons and the -OH group undergo a condensation reaction.
  • the chemical formula of the halogenated hydrocarbons is: alkyl-halogen.
  • the definitions and interpretations of "radical” and "halogen” apply equally to "alkyl” and "halogen” in this paragraph.
  • Step 2) when in the general formula, there is a double bond between C12 and C13, a single bond between C13 and C14, -R 8
  • the imide salt undergoes a cyclic amination reaction on the double bond at the 8 and 17 positions.
  • the imide salt can be a methanesulfonimide salt or an aromatic sulfonimide salt.
  • the second organic solvent is selected from one, two, three or three of acetonitrile, dichloromethane, tetrahydrofuran, diethyl ether, 1,4-dioxane, toluene, benzene and chloroform more than one;
  • the copper catalyst is selected from one, two or three of cuprous chloride, cuprous iodide and copper trifluoromethanesulfonate, most preferably copper trifluoromethanesulfonate.
  • Step 3) when in the general formula, there is a double bond between C12 and C13, a single bond between C13 and C14, -R 8
  • the synthesis operation includes: adding compound B and copper catalyst obtained in step 2) into an acetonitrile solvent, and reacting under heating conditions to obtain compound C.
  • the copper catalyst is selected from any one of cuprous iodide, cuprous chloride and copper trifluoromethanesulfonate catalyst; the mass of the copper catalyst is 10% to 50% of the mass of compound B, most preferably 20% .
  • Acetonitrile participates in the reaction both as a solvent and as a reactant, and the molar concentration of compound B is 0.005M-0.1M, most preferably 0.01M.
  • the temperature ranges from 25°C to 100°C, most preferably 65°C.
  • Step 4) a single bond between C12 and C13, a double bond between C13 and C14, -R 1 is a hydrogen group,
  • -R 9 is selected from any of cycloalkyl, benzyl, substituted benzyl, heterocyclic, C 1 -C 10 straight-chain alkyl and C 3 -C 10 branched-chain alkyl
  • the synthesis operation includes: adding compound B, amine and basic medium obtained in step 2) into a third organic solvent, and reacting to obtain compound D, and the chemical formula of the amine is R 9 -NH 2
  • -R 9 is selected from -H, cycloalkyl, benzyl, substituted benzyl, aryl, heterocyclic, heteroaryl, C 1 -C 10 straight chain alkyl and C 3 -C 10 Any of branched chain alkyl groups.
  • the amine undergoes an amination substitution reaction at the 12-position.
  • the amine can be various aliphatic or aromatic amines.
  • -R 2 , -R 3 and -R 7 , -R 9 have been introduced in detail in the preceding paragraphs, and will not be repeated here.
  • the third organic solvent is selected from one, two, three or more of dichloromethane, tetrahydrofuran, ether, 1,4-dioxane, toluene, benzene and chloroform ;
  • the alkaline medium is selected from one, two or three of triethylamine, diisopropylethylamine and pyridine, most preferably triethylamine.
  • Step 5) a single bond between C12 and C13, a double bond between C13 and C14, -R 1 is a hydrogen group,
  • -R 9 is selected from any one of cycloalkyl, benzyl, substituted benzyl, heterocyclyl, C 1 -C 10 straight-chain alkyl and C 3 -C 10 branched alkyl, -R
  • the synthesis operation includes: adding the compound D obtained in step 4) and the basic medium into a fourth organic solvent, and reacting under heating conditions to obtain compound E.
  • the amino substituent performs intramolecular nucleophilic attack on the aziridine structural unit, and the three-membered ring is opened and a new six-membered aza ring is formed.
  • -R 2 , -R 3 and -R 7 , -R 9 have been introduced in detail in the preceding paragraphs, and will not be repeated here.
  • the fourth organic solvent is selected from one, two, three or three of acetonitrile, dichloromethane, tetrahydrofuran, ether, 1,4-dioxane, toluene, benzene and chloroform more than one;
  • the alkaline medium is selected from one, two or three of triethylamine, diisopropylethylamine and pyridine, and triethylamine is the most preferred.
  • the temperature ranges from 25°C to 100°C, most preferably 70°C.
  • the synthesis operation includes: adding the compound B obtained in step 2) and the Lewis acid into the fifth organic solvent, and reacting under heating conditions to obtain the compound F.
  • the fifth organic solvent is selected from one, two, three or three of acetonitrile, dichloromethane, methanol, tetrahydrofuran, diethyl ether, 1,4-dioxane, toluene and chloroform more than one species;
  • Lewis acid is selected from one, two or three in indium trifluoromethanesulfonate, scandium trifluoromethanesulfonate, zinc trifluoromethanesulfonate and copper trifluoromethanesulfonate, most preferably trifluoromethane Scandium sulfonate.
  • the temperature ranges from 25°C to 70°C, most preferably 45°C.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • 2,4-difluorobenzenesulfonimide iodonium salt 0.2 mmol, 2 equiv.
  • Cu(OTf) 2 18 mg, 0.05 mmol, 0.5 equiv
  • 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h.
  • the reactant was concentrated, and the product B-2 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 7%.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • 4-chlorobenzenesulfonimide iodonium salt 0.2 mmol, 2 equiv.
  • Cu(OTf ) 2 18 mg, 0.05 mmol, 0.5 equiv
  • 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h.
  • the reactant was concentrated, and the product B-3 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 16%.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • 4-nitrobenzenesulfonimide iodonium salt 0.2 mmol, 2 equiv.
  • Cu ( OTf) 2 18 mg, 0.05 mmol, 0.5 equiv
  • 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h.
  • the reactant was concentrated, and the product B-4 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 8%.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • 4-iodobenzenesulfonimide iodonium salt 0.2 mmol, 2 equiv.
  • Cu(OTf ) 2 18 mg, 0.05 mmol, 0.5 equiv
  • 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h.
  • the reactant was concentrated, and the product B-5 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 22%.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • 4-trifluoromethoxybenzenesulfonimide iodonium salt 0.2 mmol, 2 equiv.
  • Cu(OTf) 2 18 mg, 0.05 mmol, 0.5 equiv
  • 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h.
  • the reactant was concentrated, and the product B-6 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 20%.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • 4-methoxybenzenesulfonimide iodonium salt 0.2 mmol, 2 equiv.
  • Cu (OTf) 2 18 mg, 0.05 mmol, 0.5 equiv
  • 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h.
  • the reactant was concentrated, and the product B-7 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 15%.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • 2-methylbenzenesulfonimide iodonium salt 0.2 mmol, 2 equiv.
  • Cu ( OTf) 2 18 mg, 0.05 mmol, 0.5 equiv
  • 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h.
  • the reactant was concentrated, and the product B-8 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 16%.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • 3-nitrobenzenesulfonimide iodonium salt 0.2 mmol, 2 equiv.
  • Cu ( OTf) 2 18 mg, 0.05 mmol, 0.5 equiv
  • 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h.
  • the reactant was concentrated, and the product B-9 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 15%.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • 4-trifluoromethylbenzenesulfonimide iodonium salt (0.2 mmol, 2 equiv.) were added in sequence, Cu(OTf) 2 (18 mg, 0.05 mmol, 0.5 equiv), and then 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h. After the reaction, the reactant was concentrated, and the product B-10 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 24%.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • 4-fluorobenzenesulfonimide iodonium salt 0.2 mmol, 2 equiv.
  • Cu(OTf ) 2 18 mg, 0.05 mmol, 0.5 equiv
  • 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h.
  • the reactant was concentrated, and the product B-11 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 13%.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • benzenesulfonimide iodonium salt 0.2 mmol, 2 equiv.
  • Cu(OTf) 2 18 mg, 0.05 mmol, 0.5 equiv
  • 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h. After the reaction, the reactant was concentrated, and the product B-12 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 11%.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • methanesulfonimide iodonium salt 0.2 mmol, 2 equiv.
  • Cu(OTf) 2 18 mg, 0.05 mmol, 0.5 equiv
  • 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h. After the reaction, the reactant was concentrated, and the product B-13 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 17%.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • 2-methyl-5-nitrobenzenesulfonimide iodonium salt 0.2 mmol, 2 equiv
  • Cu(OTf) 2 18 mg, 0.05 mmol, 0.5 equiv
  • 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h.
  • the reactant was concentrated, and the product B-14 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 22%.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • 4-acetamidobenzenesulfonimide iodonium salt 0.2 mmol, 2 equiv.
  • Cu ( OTf) 2 18 mg, 0.05 mmol, 0.5 equiv
  • 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h.
  • the reactant was concentrated, and the product B-15 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 5%.
  • Compound A was prepared by referring to the preparation method in Example 1.
  • acetoxy-protected andrographolide A-1 46 mg, 0.1 mmol
  • 4-tert-butylbenzenesulfonimide iodonium salt 0.2 mmol, 2 equiv.
  • Cu (OTf) 2 18 mg, 0.05 mmol, 0.5 equiv
  • 1 mL of acetonitrile was added as a solvent, and the mixture was stirred at room temperature for 7 h.
  • the reactant was concentrated, and the product B-16 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 16%.
  • cyclic aminated andrographolide compound B-1 (20 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were sequentially added, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-1 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 20%.
  • cyclic aminated andrographolide compound B-2 (20 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were sequentially added, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-2 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 37%.
  • cyclic aminated andrographolide compound B-3 (20 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were sequentially added, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-3 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 30%.
  • cyclic aminated andrographolide compound B-4 (20 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were sequentially added, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-4 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 30%.
  • cyclic aminated andrographolide compound B-6 (21 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were sequentially added, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-6 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 35%.
  • cyclic aminated andrographolide compound B-7 (21 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were sequentially added, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-7 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 34%.
  • cyclic aminated andrographolide compound B-8 (20 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were sequentially added, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-8 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 12%.
  • cyclic aminated andrographolide compound B-9 (20 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were sequentially added, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-9 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 20%.
  • cyclic aminated andrographolide compound B-10 (21 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were sequentially added, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-10 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 8%.
  • cyclic aminated andrographolide compound B-11 (20 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were added in sequence, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-11 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 15%.
  • cyclic aminated andrographolide compound B-12 (19 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were sequentially added, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-12 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 10%.
  • cyclic aminated andrographolide compound B-13 (17 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were sequentially added, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-13 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 26%.
  • cyclic aminated andrographolide compound B-14 (21 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were sequentially added, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-14 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 26%.
  • cyclic aminated andrographolide compound B-15 (21 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were sequentially added, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-15 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 22%.
  • cyclic aminated andrographolide compound B-16 (21 mg, 0.03 mmol), Cu(OTf) 2 (1.1 mg, 0.003 mmol, 0.1 equiv) were sequentially added, and 1 mL of acetonitrile was added as a solvent, Stir at 65°C for 12h. After the reaction, the reactant was concentrated, and the product C-16 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 25%.
  • cyclic aminated andrographolide compound B-1 (20 mg, 0.03 mmol), benzylamine (0.09 mmol, 3 equiv.), triethylamine (5.0 ⁇ L, 0.036 mmol, 1.2 equiv.) were sequentially added , and then 200 ⁇ L of dichloromethane was added as a solvent, and the mixture was stirred at room temperature for 3 h. After the reaction, the reactant was concentrated, and the product D-1 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 47%.
  • cyclic aminated andrographolide compound B-1 (20 mg, 0.03 mmol), 2-methylbenzylamine (0.09 mmol, 3 equiv.), and triethylamine (5.0 ⁇ L, 0.036 mmol) were added sequentially , 1.2 equivalents), and then added 200 ⁇ L of dichloromethane as a solvent, and stirred at room temperature for 3 h. After the reaction, the reactant was concentrated, and the product D-6 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 65%.
  • cyclic aminated andrographolide compound B-1 (20 mg, 0.03 mmol), 4-methoxybenzylamine (0.09 mmol, 3 equiv.), and triethylamine (5.0 ⁇ L, 0.036 mmol) were added sequentially mmol, 1.2 equiv), then 200 ⁇ L of dichloromethane was added as a solvent, and the mixture was stirred at room temperature for 3 h. After the reaction, the reactant was concentrated, and the product D-8 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 43%.
  • cyclic aminated andrographolide compound B-1 (20 mg, 0.03 mmol), cyclopropylamine (0.09 mmol, 3 equiv.), and triethylamine (5.0 ⁇ L, 0.036 mmol, 1.2 equiv.) were sequentially added.
  • 200 ⁇ L of dichloromethane was added as a solvent, and the mixture was stirred at room temperature for 3 h.
  • the reactant was concentrated, and the product D-12 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 4%.
  • cyclic aminated andrographolide compound B-1 (20 mg, 0.03 mmol), isopropylamine (0.09 mmol, 3 equiv), and triethylamine (5.0 ⁇ L, 0.036 mmol, 1.2 equiv) were sequentially added , and then 200 ⁇ L of dichloromethane was added as a solvent, and the mixture was stirred at room temperature for 3 h. After the reaction, the reactant was concentrated, and the product D-13 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 4%.
  • cyclic aminated andrographolide compound B-13 (17 mg, 0.03 mmol), benzylamine (0.09 mmol, 3 equiv), triethylamine (7.5 ⁇ L, 0.054 mmol, 1.8 equiv) were added sequentially , and then 200 ⁇ L of dichloromethane was added as a solvent, and the mixture was stirred at room temperature for 3 h. After the reaction, the reactant was concentrated, and the product D-15 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 43%.
  • cyclic aminated andrographolide compound B-13 (17 mg, 0.03 mmol), cyclopropylamine (0.09 mmol, 3 equiv), and triethylamine (7.5 ⁇ L, 0.054 mmol, 1.8 equiv) were added sequentially , and then 200 ⁇ L of dichloromethane was added as a solvent, and the mixture was stirred at room temperature for 3 h. After the reaction, the reactant was concentrated, and the product D-18 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 51%.
  • cyclic aminated andrographolide compound B-13 (17 mg, 0.03 mmol), 4-methoxybenzylamine (0.09 mmol, 3 equiv.), and triethylamine (7.5 ⁇ L, 0.054 mmol) were added sequentially. mmol, 1.8 equiv), then 200 ⁇ L of dichloromethane was added as a solvent, and the mixture was stirred at room temperature for 3 h. After the reaction, the reactant was concentrated, and the product D-20 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 41%.
  • the bisamined andrographolide compound D-1 14 mg, 0.02 mmol was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-1 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 67%.
  • the bisamined andrographolide compound D-2 (14 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-2 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 45%.
  • the bisamined andrographolide compound D-3 (14 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-3 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 65%.
  • the bisamined andrographolide compound D-4 (14 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-4 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 53%.
  • the bisamined andrographolide compound D-5 (15 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-5 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 32%.
  • the bisamined andrographolide compound D-6 (14 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-6 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 33%.
  • the bisamined andrographolide compound D-7 (14 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-7 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 33%.
  • the bisamined andrographolide compound D-8 (14 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-8 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 36%.
  • the bisamined andrographolide compound D-9 (15 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction was completed, the reactant was concentrated, and the product E-9 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 45%.
  • the bisamined andrographolide compound D-10 (13 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-10 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 50%.
  • the bisamined andrographolide compound D-11 (15 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-11 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 33%.
  • the bisamined andrographolide compound D-12 (13 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-12 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 32%.
  • the bisamined andrographolide compound D-13 (13 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-13 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 38%.
  • the bisamined andrographolide compound D-14 (14 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-14 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 32%.
  • the bisamined andrographolide compound D-15 (12 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-15 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 33%.
  • the bisamined andrographolide compound D-16 (12 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-16 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 50%.
  • the bisamined andrographolide compound D-17 (13 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-17 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 43%.
  • the bisamined andrographolide compound D-18 (11 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-18 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 43%.
  • the bisamined andrographolide compound D-19 (13 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction, the reactant was concentrated, and the product E-19 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 33%.
  • the bisamined andrographolide compound D-20 (13 mg, 0.02 mmol) was dissolved in 0.5 mL of acetonitrile solvent, followed by triethylamine (3.3 ⁇ L, 0.024 mmol, 1.2 equiv), 65 Stir at °C for 22h. After the reaction was completed, the reactant was concentrated, and the product E-20 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 45%.
  • the cyclic aminated andrographolide compound B-1 (13 mg, 0.02 mmol) was dissolved in 0.5 mL methanol solvent, and then Sc(OTf) 3 (1 mg, 10 mol%) was added at 40°C. Stir for 24h. After the reaction, the reactant was concentrated, and the product F-1 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 83%.
  • the cyclic aminated andrographolide compound B-2 (13 mg, 0.02 mmol) was dissolved in 0.5 mL methanol solvent, and then Sc(OTf) 3 (1 mg, 10 mol%) was added at 40°C. Stir for 24h. After the reaction, the reactant was concentrated, and the product F-2 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 27%.
  • the cyclic aminated andrographolide compound B-3 (13 mg, 0.02 mmol) was dissolved in 0.5 mL methanol solvent, and then Sc(OTf) 3 (1 mg, 10 mol%) was added at 40°C. Stir for 24h. After the reaction, the reactant was concentrated, and the product F-3 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 16%.
  • the cyclic aminated andrographolide compound B-4 (14 mg, 0.02 mmol) was dissolved in 0.5 mL methanol solvent, and then Sc(OTf) 3 (1 mg, 10 mol%) was added at 40°C. Stir for 24h. After the reaction, the reactant was concentrated, and the product F-4 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 30%.
  • the cyclic aminated andrographolide compound B-5 (15 mg, 0.02 mmol) was dissolved in 0.5 mL methanol solvent, and then Sc(OTf) 3 (1 mg, 10 mol%) was added at 40°C. Stir for 24h. After the reaction, the reactant was concentrated, and the product F-5 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 20%.
  • the cyclic aminated andrographolide compound B-6 (14 mg, 0.02 mmol) was dissolved in 0.5 mL methanol solvent, and then Sc(OTf) 3 (1 mg, 10 mol%) was added at 40°C. Stir for 24h. After the reaction, the reactant was concentrated, and the product F-6 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 47%.
  • the cyclic aminated andrographolide compound B-7 (13 mg, 0.02 mmol) was dissolved in 0.5 mL methanol solvent, and then Sc(OTf) 3 (1 mg, 10 mol%) was added at 40° C. Stir for 24h. After the reaction, the reactant was concentrated, and the product F-7 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 35%.
  • the cyclic aminated andrographolide compound B-9 (14 mg, 0.02 mmol) was dissolved in 0.5 mL methanol solvent, and then Sc(OTf) 3 (1 mg, 10 mol%) was added at 40°C. Stir for 24h. After the reaction was completed, the reactant was concentrated, and the product F-9 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 35%.
  • the cyclic aminated andrographolide compound B-11 (13 mg, 0.02 mmol) was dissolved in 0.5 mL methanol solvent, and then Sc(OTf) 3 (1 mg, 10 mol%) was added at 40°C. Stir for 24h. After the reaction, the reactant was concentrated, and the product F-11 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 30%.
  • the cyclic aminated andrographolide compound B-12 (13 mg, 0.02 mmol) was dissolved in 0.5 mL methanol solvent, and then Sc(OTf) 3 (1 mg, 10 mol%) was added at 40°C. Stir for 24h. After the reaction, the reactant was concentrated, and the product F-12 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 40%.
  • the cyclic aminated andrographolide compound B-13 (11 mg, 0.02 mmol) was dissolved in 0.5 mL methanol solvent, and then Sc(OTf) 3 (1 mg, 10 mol%) was added at 40°C. Stir for 24h. After the reaction, the reactant was concentrated, and the product F-13 was obtained by preparative thin-layer chromatography plate separation and purification with a yield of 51%.
  • the cyclic aminated andrographolide compound B-15 (14 mg, 0.02 mmol) was dissolved in 0.5 mL methanol solvent, and then Sc(OTf) 3 (1 mg, 10 mol%) was added at 40°C. Stir for 24h. After the reaction, the reactant was concentrated, and the product F-15 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 44%.
  • the cyclic aminated andrographolide compound B-16 (14 mg, 0.02 mmol) was dissolved in 0.5 mL methanol solvent, and then Sc(OTf) 3 (1 mg, 10 mol%) was added at 40°C. Stir for 24h. After the reaction, the reactant was concentrated, and the product F-16 was obtained by preparative thin-layer chromatography plate separation and purification, and the yield was 39%.
  • Nasopharyngeal cancer, lung cancer, cervical cancer, breast cancer and liver cancer cells were selected as research objects, and andrographolide was used as a positive control to screen the antitumor activity of andrographolide derivatives.
  • the selected cell lines are: human EBV positive nasopharyngeal carcinoma cell C666-1, non-small cell lung cancer A549, human cervical carcinoma cell HeLa, human breast cancer cell MCF-7 and human hepatoma cell Huh7.
  • the main points of the resazurin experimental method are as follows: tumor cells in logarithmic growth phase are used to inoculate in a 96-well cell culture plate, 100 ⁇ L per well, 7000 cells of C666-1 and 2000 cells of A549, HeLa, MCF7 and Huh 7 are added to each well. , 2 replicate wells were set for each experimental group. After culturing for 24 h, 0.5 ⁇ L of compound in DMSO solution was added to each well to make the final concentration 50 ⁇ M. In addition, the wells with 0.5 ⁇ L of DMSO were set as positive controls, and the wells with only medium were set as negative controls.
  • CPE cytopathic effect
  • MRC5 cells were cultured in EMEM (Sigma) supplemented with 10% fetal bovine serum, 1% double antibody (Hyclone), 1% L-glutamine (Gibco), and 1% non-essential amino acids (Gibco), and supplemented with The EMEM medium containing 5% fetal bovine serum, 1% double antibody, 1% L-glutamine and 1% non-essential amino acids is the experimental medium.
  • MRC5 cells were seeded into 96 microwell plates at a density of 20,000 cells per well, 100 ⁇ l per well and cultured overnight in a 5% CO2, 37°C incubator. The next day, 50 [mu]l of compound was added to each well (double wells), and then 50 [mu]l of diluted virus was added to cell test wells at 200 TCID50 per well, resulting in a final compound concentration of 25 [mu]M.
  • Cell controls (cells, no compound treatment or virus infection), virus controls (cells infected with virus, no compound treatment) and broth controls (broth only) were set up. The final volume of the experimental medium was 200 ⁇ l per well, and the final concentration of DMSO in the medium was 0.5%.
  • Test compounds were prepared as 20 mM stock solutions using DMSO solutions. The initial test concentration of the test compound was 100 ⁇ M, and 10 concentrations were tested, 3-fold serial dilution, and double wells. Remdesivir and Chloroquine were used as control compounds. Control compounds were tested at 10 concentrations, 2-fold serial dilution, double wells, and the initial test concentration was 50 ⁇ M.
  • African green monkey kidney (Vero) cells were obtained from the American Type Culture Collection (ATCC). Cells were cultured in DMEM (WelGene) medium supplemented with 10% fetal bovine serum (Gibco) and 1% dual antibodies (Gibco). DMEM medium supplemented with 2% fetal bovine serum and 1% double antibody was used as the experimental medium.
  • DMEM fetal bovine serum
  • DMEM medium supplemented with 2% fetal bovine serum and 1% double antibody was used as the experimental medium.
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Abstract

一种具有抗肿瘤、抗病毒活性的氮杂螺环、多环穿心莲内酯类化合物、其制备方法，以及包括该氮杂螺环、多环穿心莲内酯类化合物的药物组合物及其应用。所述氮杂螺环、多环穿心莲内酯类化合物，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，创造性的对8，17位双键进行氮杂环丙化，并在此基础上通过氮杂环丙基与乙腈分子间3+2环加成、C12位Michael-胺化、分子内亲核进攻氮杂环丙基开环及氮杂环丙基与分子内双键3+2环加成，形成了一系列氨基取代的多环氮杂螺环、多环穿心莲内酯类化合物，增强了化合物的生物活性、生物利用度和稳定性。所述化合物可用于制备抗肿瘤和抗病毒药物。

Description

氮杂螺环、多环穿心莲内酯类化合物及其制备方法、药物组合物及其应用 技术领域
本申请涉及药物化学领域，特别是涉及一种氮杂螺环、多环穿心莲内酯类化合物及其制备方法、药物组合物及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，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。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。
穿心莲内酯(Andrographolide)、为爵床科植物穿心莲中提取得到的主要二萜内酯类化合物，是中药穿心莲的主要有效成分之一，其结构如下式所示：
Figure PCTCN2021141536-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、免疫调节、治疗心脑血管疾病、保肝利胆等作用。由于其对细菌性、病毒性上呼吸道感染及痢疾有特殊功效，被誉为天然抗生素药物。目前在中国及亚洲其他国家广泛应用在炎症相关性疾病的治疗，如喉炎、上呼吸道感染及风湿性关节炎。
近年来，随着穿心莲内酯药理作用研究的不断深入，其临床应用得到不断 拓展。研究发现，穿心莲内酯及其衍生物在抗肿瘤方面展现了诱人的应用前景，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，已经成为一类有前景、有潜力的抗肿瘤候选药物。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。
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、抗病毒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
基于此，有必要提供一种具有改善的药代动力学特性、具有更强抗肿瘤、抗病毒活性的氮杂螺环、多环穿心莲内酯类化合物。
此外，还有必要提供一种上述氮杂螺环、多环穿心莲内酯类化合物的制备方法、包括该氮杂螺环、多环穿心莲内酯类化合物的药物组合物及其应用。
一种氮杂螺环、多环穿心莲内酯类化合物，其特征在于，具有如下通式I：
Figure PCTCN2021141536-appb-000002
其中，C12和C13之间成双键且C13和C14之间成单键，或者C12和C13之间成单键且C13和C14之间成双键；
-R 1为-H、-OH、烷氧基或
Figure PCTCN2021141536-appb-000003
-R 2为-OH、烷氧基或
Figure PCTCN2021141536-appb-000004
-R 3为-OH、烷氧基或
Figure PCTCN2021141536-appb-000005
-R 4为烷基或芳基；
-R 5和-R 6一起形成下列基团：
Figure PCTCN2021141536-appb-000006
-R 7为烷基或芳基，-R 8为H或
Figure PCTCN2021141536-appb-000007
-R 9为环烷基、苄基、取代的苄基、杂环基、C 1～C 10的直链烷基或C 3～C 10的支链烷基，-R 10为H；
或者-R 5和-R 6一起形成下列基团：
Figure PCTCN2021141536-appb-000008
-R 7为烷基或芳基，-R 8为H，-R 10为H；
或者，-R 6为下列基团：
Figure PCTCN2021141536-appb-000009
同时-R 8和-R 5共同形成下列基团：
Figure PCTCN2021141536-appb-000010
-R 7为烷基或芳基，-R 9选自环烷基、苄基、取代的苄基、杂环基、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，-R 10为H；
或者，-R 6、-R 10及通式中的C8、C9和C11共同形成下列基团：
Figure PCTCN2021141536-appb-000011
-R 5和-R 8共同形成下列基团：
Figure PCTCN2021141536-appb-000012
-R 7为烷基或芳基。
一种上述的氮杂螺环、多环穿心莲内酯类化合物的制备方法，包括如下步骤：
提供化合物A，所述化合物A的结构为：
Figure PCTCN2021141536-appb-000013
将所述化合物A、磺酰亚胺碘盐和铜催化剂加入第二有机溶剂中，发生反应，得到所述化合物B，所述化合物B的结构为：
Figure PCTCN2021141536-appb-000014
一种药物组合物，包括上述的氮杂螺环、多环穿心莲内酯类化合物或其药学上可接受的盐。
一种上述的氮杂螺环、多环穿心莲内酯类化合物在制备抗肿瘤药物中的应用。
一种上述的氮杂螺环、多环穿心莲内酯类化合物在制备抗病毒药物中的应用。
这种氮杂螺环、多环穿心莲内酯类化合物，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，创造性的对8，17位双键进行氮杂环丙化，并在此基础上通过氮杂环丙基与乙腈分子间3+2环加成、C12位Michael-胺化、分子内亲核进攻氮杂环丙基开环及氮杂环丙基与分子内双键3+2环加成，形成了一系列新型的氨基取代的多环氮杂螺环、多环穿心莲内酯类化合物，增强了化合物的生物活性、生物利用度和稳定性。
结合具体实施例部分的实验数据，这种氮杂螺环、多环穿心莲内酯类化合物的体外抗肿瘤、抗病毒活性较穿心莲内酯具有显著提高。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
其中：
图1为实施例89中穿心莲内酯衍生物D-1体外抗新型冠状病毒(SARS-CoV-2)的活性和细胞毒性曲线图。
图2为实施例89中氯喹(Chloroquine)体外抗新型冠状病毒(SARS-CoV-2)的活性和细胞毒性曲线图。
图3为实施例89中瑞德西韦(Remdesivir)体外抗新型冠状病毒(SARS-CoV-2)的活性和细胞毒性曲线图。
具体实施方式
为了便于理解本申请，下面将参照相关附图对本申请进行更全面的描述。附图中给出了本申请的较佳实施例。但是，本申请可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本申请的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请。
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。本申请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申请：
-Ac：CH 3CO-，乙酰基；-Me：CH 3，甲基；tBu：叔丁基；Et：CH 3CH 2-，乙基；AcOH：乙酸；THF：四氢呋喃；EtOH：CH 3CH 2OH，乙醇。
本申请公开了一种氮杂螺环、多环穿心莲内酯类化合物，具有如下通式I：
Figure PCTCN2021141536-appb-000015
其中，C12和C13之间成双键且C13和C14之间成单键，或者C12和C13之间成单键且C13和C14之间成双键；
-R 1为-H、-OH、烷氧基或
Figure PCTCN2021141536-appb-000016
-R 2为-OH、烷氧基或
Figure PCTCN2021141536-appb-000017
-R 3为-OH、烷氧基或
Figure PCTCN2021141536-appb-000018
-R 4为烷基或芳基；
-R 5和-R 6一起形成下列基团：
Figure PCTCN2021141536-appb-000019
-R 7为烷基或芳基，-R 8为H或
Figure PCTCN2021141536-appb-000020
-R 9为环烷基、苄基、取代的苄基、杂环基、C 1～C 10的直链烷基或C 3～C 10的支链烷基，-R 10为H；
或者-R 5和-R 6一起形成下列基团：
Figure PCTCN2021141536-appb-000021
-R 7为烷基或芳基，-R 8为H，-R 10为H；
或者，-R 6为下列基团：
Figure PCTCN2021141536-appb-000022
同时-R 8和-R 5共同形成下列基团：
Figure PCTCN2021141536-appb-000023
-R 7为烷基或芳基，-R 9选自环烷基、苄基、取代的苄基、杂环基、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，-R 10为H；
或者，-R 6、-R 10及通式中的C8、C9和C11共同形成下列基团：
Figure PCTCN2021141536-appb-000024
-R 5和-R 8共同形成下列基团：
Figure PCTCN2021141536-appb-000025
-R 7为烷基或芳基。
这种氮杂螺环、多环穿心莲内酯类化合物，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，创造性的对8，17位双键进行氮杂环丙化，并在此基础上通过氮杂环丙基与乙腈分子间3+2环加成、C12位Michael-胺化、分子内亲核进攻氮杂环丙基开环及氮杂环丙基与分子内双键3+2环加成，形成了一系列新型的氨基取代的多环氮杂螺环、多环穿心莲内酯类化合物，增强了化合物的生物活性、生物利用度和稳定性。
结合具体实施例部分的实验数据，这种氮杂螺环、多环穿心莲内酯类化合物的体外抗肿瘤、抗病毒活性较穿心莲内酯具有显著提高。
在一个优选的实施例中，-R 1为-H，-R 2和-R 3相同。
更优选的，-R 2和-R 3均为-OAc。
在一个优选的实施例中，-R 1、-R 2和-R 3相同。
具体来说，上述具有通式I的氮杂螺环、多环穿心莲内酯类化合物，可以分为如下四类：
第一类：C12和C13之间成双键，C13和C14之间成单键，-R 8为氢基，-R 1,-R 2和-R 3相同，-R 1，-R 2和-R 3选自-OH、烷氧基和
Figure PCTCN2021141536-appb-000026
中的任意一种，-R 4为烷基或芳基；-R 5和-R 6一起形成下列基团：
Figure PCTCN2021141536-appb-000027
-R 7为烷基或芳基；或者 -R 5和-R 6一起形成下列基团：
Figure PCTCN2021141536-appb-000028
-R 7为烷基或芳基；-R 10为H。
第二类：C12和C13之间成单键，C13和C14之间成双键，-R 1为氢基，-R 2和-R 3相同，-R 2和-R 3选自-OH、烷氧基和
Figure PCTCN2021141536-appb-000029
中的任意一种，-R 4为烷基或芳基，-R 8为胺基
Figure PCTCN2021141536-appb-000030
同时-R 5和-R 6共同形成下列基团：
Figure PCTCN2021141536-appb-000031
-R 7为烷基或芳基，-R 9选自环烷基、苄基、取代的苄基、杂环基、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，-R 10为H。
第三类：C12和C13之间成单键，C13和C14之间成双键，-R 1为氢基，-R 2和-R 3相同，-R 2和-R 3选自-OH、烷氧基和
Figure PCTCN2021141536-appb-000032
中的任意一种，-R 4为烷基或芳基；-R 6为下列基团：
Figure PCTCN2021141536-appb-000033
-R 7为烷基或芳基，同时-R 8和-R 5共同形成下列基团：
Figure PCTCN2021141536-appb-000034
-R 9选自环烷基、苄基、取代的苄基、杂环基、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，-R 10为H。
第四类：C12和C13之间成单键，C13，C14之间成双键，-R 1为氢基，-R 2和-R 3相同，-R 2和-R 3选自-OH、烷氧基和
Figure PCTCN2021141536-appb-000035
中的任意一种，-R 4为烷基或芳基；-R 6、-R 10及通式中的C8、C9和C11共同形成下列基团：
Figure PCTCN2021141536-appb-000036
-R 5和-R 8共同形成下列基团：
Figure PCTCN2021141536-appb-000037
-R 7为烷基或芳基。
本申请中，上述出现的各种基团和取代基均满足如下定义。
“烷基”是指饱和烃基，只含有C和H两种原子，包括环烷基和链烷基，链烷基包括直链烷基或支链烷基。在本申请的具体实施例中，烷基包括，但不限于-CH 3等。
“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、叔丁基、-CX 3、-OCH 3、-OCH 2CH 3、酰胺基和硝基等中的任意一种，其中，X为F、Cl、Br或I原子。
具体来说，芳基包括，但不限于，
Figure PCTCN2021141536-appb-000038
Figure PCTCN2021141536-appb-000039
Figure PCTCN2021141536-appb-000040
等中的任意一种。
具体来说，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。
“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的单环环烷基或C 7～C 12的双环环烷基，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和硝基等中的任意一种，其中，X为F、Cl、Br或I原子。在本申请的具体实施例中，环烷基可以是，但不限于，环戊基、环己基、环庚基、
Figure PCTCN2021141536-appb-000041
等。
“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。
“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、和硝基等中的任意一种，其中，X为F、Cl、Br或I原子。在本申请具体实施例中，取代的苄基包括，但不限于，
Figure PCTCN2021141536-appb-000042
Figure PCTCN2021141536-appb-000043
Figure PCTCN2021141536-appb-000044
等中的任意一种。
“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，单环杂环基或双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和硝基等中的任意一种，其中，X为F、Cl、Br或I原子。在本申请的具体实施例中，杂环基可以包括，但不限于，
Figure PCTCN2021141536-appb-000045
咪唑基、噻吩基或呋喃基等。
在一个优选的实施例中，-R 7为-CH 3、
Figure PCTCN2021141536-appb-000046
Figure PCTCN2021141536-appb-000047
在一个优选的实施例中，-R 9为
Figure PCTCN2021141536-appb-000048
Figure PCTCN2021141536-appb-000049
在一个优选的实施例中，C12和C13之间成双键，C13和C14之间成单键，-R 8为氢基时，-R 5和-R 6共同形成下列基团：
Figure PCTCN2021141536-appb-000050
-R 10为H。
在一个优选的实施例中，C12和C13之间成单键，C13和C14之间成双键，-R 1为氢基，-R 8为胺基
Figure PCTCN2021141536-appb-000051
时，-R 5和-R 6共同形成下列基团：
Figure PCTCN2021141536-appb-000052
-R 10为H。
在一个优选的实施例中，C12和C13之间成单键，C13和C14之间成双键，-R 1为氢基，-R 6为磺酰胺基
Figure PCTCN2021141536-appb-000053
时，-R 8和-R 5共同形成下列基团：
Figure PCTCN2021141536-appb-000054
-R 10为H。
在一个优选的实施例中，C12和C13之间成单键，C13和C14之间成双键，-R 1为氢基，-R 6、-R 10及通式中的C8、C9和C11共同形成下列基团：
Figure PCTCN2021141536-appb-000055
-R 5和-R 8共同形成下列基团：
Figure PCTCN2021141536-appb-000056
具体来说，氮杂螺环、多环穿心莲内酯类化合物为具有如下结构式的化合物之一：
Figure PCTCN2021141536-appb-000057
其中，各个取代基的定义参照前文所述。
更具体的，氮杂螺环、多环穿心莲内酯类化合物可以为具有如下化学式的化合物之一：
Figure PCTCN2021141536-appb-000058
Figure PCTCN2021141536-appb-000059
Figure PCTCN2021141536-appb-000060
Figure PCTCN2021141536-appb-000061
Figure PCTCN2021141536-appb-000062
Figure PCTCN2021141536-appb-000063
结合具体实施例部分的实验数据，这种氮杂螺环、多环穿心莲内酯类化合物对肿瘤的抑制作用较穿心莲内酯具有显著提高。
基于此，本申请还公开了一种药物组合物，包括上述的氮杂螺环、多环穿心莲内酯类化合物或其药学上可接受的盐。
此外，本申请还公开了上述氮杂螺环、多环穿心莲内酯类化合物在制备抗肿瘤药物中的应用。
具体来说，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。
结合具体实施例部分的实验数据，这种氮杂螺环、多环穿心莲内酯类化合物的抗病毒活性较穿心莲内酯具有显著提高。
基于此，本申请还公开了上述的氮杂螺环、多环穿心莲内酯类化合物在制备抗病毒药物中的应用。
具体来说，抗病毒药物可以抗人冠状病毒(HCoV-229E)药物或抗新型冠状病毒(SARS-CoV-2)药物。
本申请还公开了具有上述氮杂螺环、多环穿心莲内酯类化合物的制备方法，包括如下步骤：
步骤1)：提供化合物A，化合物A的结构为：
Figure PCTCN2021141536-appb-000064
其中，R 1，-R 2和-R 3相同，R 1，-R 2和-R 3为烷氧基或
Figure PCTCN2021141536-appb-000065
-R 4为烷基或芳基。
具体的，化合物A可以以穿心莲内酯为原料来制备，具体的制备方法如下：
将穿心莲内酯加入第一有机溶剂中，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基发生反应，得到化合物A。
在本反应中，酰化剂与-OH基发生酯化反应，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为烷基或芳基；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”。
上述酯化反应可以使用无水ZnCl 2等路易斯酸进行催化。
第一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。
优选的，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
Figure PCTCN2021141536-appb-000066
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基发生缩合反应，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-R 1＝-R 2＝-R 3＝烷氧基。
步骤2)：当通式中C12和C13之间成双键，C13和C14之间成单键，-R 8
Figure PCTCN2021141536-appb-000067
芳基，-R 10为H时，合成操作包括：将化合物A、磺酰亚胺碘盐和铜催化剂加入第二有机溶剂中，发生反应，得到目标化合物B，磺酰亚胺碘盐的化学式为R 7SO 2N＝IPh，-R 7选自甲基、芳基中的任意一种。
化合物B的结构为：
Figure PCTCN2021141536-appb-000068
结合上述步骤1)和步骤2)得到化合物B，具体的反应过程如下所示：
Figure PCTCN2021141536-appb-000069
在本反应中，亚胺盐对8，17位双键进行环胺化反应。具体的，亚胺盐可以为甲磺酰亚胺盐或芳香磺酰亚胺盐。-R 1、-R 2、-R 3和-R 7在前面段落中已详细介绍，在此不一一赘述。
在本反应中，优选的，第二有机溶剂选自乙腈，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；铜催化剂选自氯化亚铜、碘化亚铜和三氟甲磺酸铜中的一种、两种或三种，最优选三氟甲磺酸铜。
步骤3)：当通式中C12和C13之间成双键，C13和C14之间成单键，-R 8
Figure PCTCN2021141536-appb-000070
基或芳基，-R10为H时，合成操作包括：将步骤2)中所得化合物B、铜催化剂加入乙腈溶剂中，在加热条件下发生反应，得到化合物C。
化合物C的结构为：
Figure PCTCN2021141536-appb-000071
结合上述步骤1)、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 PCTCN2021141536-appb-000072
在本步骤中，发生分子间3+2环加成反应，利用铜催化剂进行环加成反应。
优选的，铜催化剂选自碘化亚铜、氯化亚铜和三氟甲磺酸铜催化剂中的任意一种；铜催化剂的质量为化合物B的质量的10％～50％，最优选20％。乙腈既作为溶剂也作为反应物参与反应，化合物B的摩尔浓度为0.005M～0.1M，最优选0.01M。温度范围为25℃～100℃，最优选65℃。
步骤4)：C12和C13之间成单键，C13和C14之间成双键，-R 1为氢基，
Figure PCTCN2021141536-appb-000073
基或芳基，-R 9选自环烷基、苄基、取代的苄基、杂环基、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，-R 10为H时，合成操作包括：将步骤2)中所得化合物B、胺和碱性介质加入第三有机溶剂中，发生反应，得到化合物D，胺的化学式为R 9-NH 2，-R 9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D的结构为：
Figure PCTCN2021141536-appb-000074
结合上述步骤1)、2)和步骤4)得到化合物D，具体的反应过程如下所示：
Figure PCTCN2021141536-appb-000075
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 2、-R 3和-R 7、-R 9在前面段落中已详细介绍，在此不一一赘述。
在本步骤中，优选的，第三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5)：C12和C13之间成单键，C13和C14之间成双键，-R 1为氢基，
Figure PCTCN2021141536-appb-000076
下列基团：
Figure PCTCN2021141536-appb-000077
-R 9选自环烷基、苄基、取代的苄基、杂环基、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，-R 10为H时，合成操作包括：将步骤4)所得化合物D和碱性介质加入第四有机溶剂中，在加热条件下发生反应，得到化合物E。
化合物E的结构为：
Figure PCTCN2021141536-appb-000078
结合上述步骤1)、步骤2)、步骤4)和步骤5)得到化合物E，具体的反应过程如下所示：
Figure PCTCN2021141536-appb-000079
在本步骤中，胺基取代基对氮杂环丙烷结构单元进行分子内亲核进攻，三元环开环的同时形成新的氮杂六元环。-R 2、-R 3和-R 7、-R 9在前面段落中已详细介绍，在此不一一赘述。
在本步骤中，优选的，第四有机溶剂选自乙腈、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。温度范围为25℃～100℃，最优选70℃。
Figure PCTCN2021141536-appb-000080
和-R 8共同形成下列基团：
Figure PCTCN2021141536-appb-000081
-R 7为烷基或芳基时，合成操作包括：将步骤2)所得化合物B和路易斯酸加入第五有机溶剂中，在加热条件下发生反应，得到化合物F。
化合物F的结构为：
Figure PCTCN2021141536-appb-000082
结合上述步骤1)、2)和步骤6)得到化合物F，具体的反应过程如下所示：
Figure PCTCN2021141536-appb-000083
在本步骤中，发生脱氧及双键的重排，所得到的脱氧穿心莲内酯衍生物中间体进一步进行分子内3+2环加成生成多并环化合物。-R 2、-R 3和-R 7在前面段落中已详细介绍，在此不一一赘述。
在本步骤中，优选的，第五有机溶剂选自乙腈、二氯甲烷、甲醇、四氢呋喃、乙醚、1,4-二氧六环、甲苯和氯仿中的一种、两种、三种或三种以上；路易斯酸选自三氟甲磺酸铟、三氟甲磺酸钪、三氟甲磺酸锌和三氟甲磺酸铜中的一种、两种或三种，最优选三氟甲磺酸钪。温度范围为25℃～70℃，最优选45℃。
以下为具体实施例。
实施例1
化合物B-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A(1.9g，收率92％)。
Figure PCTCN2021141536-appb-000084
在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A(46mg，0.1mmol)，TsN＝IPh(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物B-1，收率39％。
Figure PCTCN2021141536-appb-000085
化合物B-1的结构测定： 1H NMR(500MHz,CDCl 3)δ7.80(d,J＝8.2Hz,2H),7.32(d,J＝8.2Hz,2H),7.08(d,J＝7.0Hz,1H),5.77(d,J＝5.7Hz,1H),4.56(t,J＝8.1Hz,1H),4.44(dd,J＝11.1,5.8Hz,1H),4.34(d,J＝11.8Hz,1H),4.17–4.11(m,2H),2.64(s,1H),2.43(s,3H),2.37(dd,J＝10.0,3.3Hz,1H),2.21–2.12(m,2H),2.07(d,J＝3.8Hz,3H),2.05(s,3H),2.03(s,1H),1.94–1.88(m,2H),1.87(s,3H),1.71(ddd,J＝16.3,11.7,3.6Hz,5H),1.33–1.27(m,2H),1.06(s,3H),0.86(s,3H).HRMS(m/z)calc.forC33H43NNaO10S:[M+Na] +,668.2500；found 668.2501.
实施例2
化合物B-2的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，2,4-二氟苯磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物B-2，收率7％。
Figure PCTCN2021141536-appb-000086
化合物B-2的结构测定： 1H NMR(400MHz,CDCl 3)δ7.93(td,J＝8.6,6.1Hz,1H),7.07–7.00(m,1H),6.99–6.88(m,2H),5.74(d,J＝5.9Hz,1H),4.58(dd,J＝11.8,4.6Hz,1H),4.42(dd,J＝11.2,6.0Hz,1H),4.34(d,J＝11.8Hz,1H),4.17(d,J＝11.8Hz,1H),4.07(dd,J＝11.2,1.7Hz,1H),2.85(s,1H),2.33–2.28(m,1H),2.25–2.18(m,1H),2.08(s,3H),2.05(s,6H),2.00(d,J＝3.0Hz,1H),1.95–1.90(m,1H),1.78–1.68(m,7H),1.37–1.32(m,2H),1.07(s,3H),0.89(s,3H).HRMS(m/z)calc.forC32H39F2NNaO10S:[M+Na] +,690.2155；found 690.2156.
实施例3
化合物B-3的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，4-氯苯磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分 离纯化得到产物B-3，收率16％。
Figure PCTCN2021141536-appb-000087
化合物B-3的结构测定： 1H NMR(400MHz,CDCl 3)δ7.86(d,J＝8.7Hz,2H),7.50(d,J＝8.7Hz,2H),7.07(dd,J＝9.7,3.8Hz,1H),5.76(d,J＝5.6Hz,1H),4.58(dd,J＝11.8,4.4Hz,1H),4.45(dd,J＝11.1,5.7Hz,1H),4.34(d,J＝11.8Hz,1H),4.19–4.13(m,2H),2.65(s,1H),2.33(dt,J＝12.9,3.3Hz,1H),2.26–2.18(m,2H),2.07(s,3H),2.05(s,3H),1.96(d,J＝9.7Hz,1H),1.87(s,3H),1.80–1.63(m,7H),1.35–1.27(m,2H),1.06(s,3H),0.86(s,3H).HRMS(m/z)calc.forC32H40ClNNaO10S:[M+Na] +,688.1954；found 688.1956.
实施例4
化合物B-4的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，4-硝基苯磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物B-4，收率8％。
Figure PCTCN2021141536-appb-000088
化合物B-4的结构测定： 1H NMR(400MHz,CDCl 3)δ8.35(d,J＝9.0Hz,2H),8.11(d,J＝9.0Hz,2H),7.03(td,J＝6.8,1.2Hz,1H),5.75(d,J＝5.6Hz, 1H),4.57(dd,J＝11.7,4.1Hz,1H),4.43(dd,J＝11.2,5.7Hz,1H),4.32(d,J＝11.8Hz,1H),4.17–4.10(m,2H),2.73(s,1H),2.26(ddd,J＝9.2,7.5,2.8Hz,3H),2.06(s,3H),2.04(s,3H),2.01–1.97(m,1H),1.86(s,3H),1.85–1.61(m,7H),1.37–1.27(m,2H),1.05(s,3H),0.86(s,3H).HRMS(m/z)calc.forC32H40N2NaO12S:[M+Na] +,699.2194；found 699.2195.
实施例5
化合物B-5的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，4-碘苯磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物B-5，收率22％。
Figure PCTCN2021141536-appb-000089
化合物B-5的结构测定： 1H NMR(400MHz,CDCl 3)δ7.88(d,J＝8.6Hz,2H),7.62(d,J＝8.6Hz,2H),7.06(dd,J＝9.6,3.8Hz,1H),5.75(d,J＝5.7Hz,1H),4.57(dd,J＝11.9,4.4Hz,1H),4.45(dd,J＝11.1,5.7Hz,1H),4.33(d,J＝11.8Hz,1H),4.18–4.15(m,1H),4.13(dd,J＝5.6,2.2Hz,1H),2.64(s,1H),2.33–2.16(m,4H),2.06(s,3H),2.04(s,3H),1.96(d,J＝9.5Hz,1H),1.86(s,3H),1.79–1.62(m,6H),1.35–1.28(m,2H),1.05(s,3H),0.85(s,3H).HRMS(m/z)calc.forC32H40INNaO10S:[M+Na] +,780.1310；found 780.1312.
实施例6
化合物B-6的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，4-三氟甲氧基苯磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物B-6，收率20％。
Figure PCTCN2021141536-appb-000090
化合物B-6的结构测定： 1H NMR(400MHz,CDCl 3)δ7.97(d,J＝8.9Hz,2H),7.34(d,J＝8.1Hz,2H),7.08(t,J＝6.1Hz,1H),5.75(d,J＝5.6Hz,1H),4.58(dd,J＝11.9,4.4Hz,1H),4.42(dd,J＝11.2,5.7Hz,1H),4.33(d,J＝11.8Hz,1H),4.18–4.10(m,2H),2.65(s,1H),2.31(dd,J＝8.7,4.2Hz,1H),2.26(s,1H),2.21(dd,J＝7.7,1.9Hz,1H),2.07(s,3H),2.05(s,3H),2.01(dd,J＝12.2,3.7Hz,2H),1.90(dd,J＝17.0,7.7Hz,1H),1.80(s,3H),1.78–1.64(m,5H),1.37–1.27(m,2H),1.06(s,3H),0.86(s,3H).HRMS(m/z)calc.for C33H40F3NNaO11S:[M+Na] +,738.2166；found 738.2167.
实施例7
化合物B-7的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，4-甲氧基苯磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物B-7，收率15％。
Figure PCTCN2021141536-appb-000091
化合物B-7的结构测定： 1H NMR(400MHz,CDCl 3)δ7.84(d,J＝8.9Hz,2H),6.99(d,J＝8.9Hz,2H),5.77(d,J＝5.7Hz,1H),4.56(dd,J＝11.8,4.3Hz,1H),4.44(dd,J＝11.1,5.8Hz,1H),4.34(d,J＝11.8Hz,1H),4.17–4.11(m,2H),3.87(s,3H),2.62(s,1H),2.35(dt,J＝12.9,3.1Hz,1H),2.24–2.12(m,3H),2.07(s,3H),2.04(s,3H),2.03(d,J＝2.7Hz,2H),1.92(d,J＝8.5Hz,2H),1.88(s,3H),1.71(ddd,J＝17.3,11.1,3.9Hz,5H),1.32–1.28(m,1H),1.05(s,3H),0.86(s,3H).HRMS(m/z)calc.forC33H43NNaO11S:[M+Na] +,684.2449；found 684.2450.
实施例8
化合物B-8的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，2-甲基苯磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物B-8，收率16％。
Figure PCTCN2021141536-appb-000092
化合物B-8的结构测定： 1H NMR(500MHz,CDCl 3)δ7.95(dd,J＝7.9,1.2Hz,1H),7.47(td,J＝7.5,1.3Hz,1H),7.32(dd,J＝15.6,7.7Hz,2H),6.99–6.93(m,1H),5.78(d,J＝5.9Hz,1H),4.55(dd,J＝11.8,4.6Hz,1H),4.46(dd,J＝ 11.2,6.1Hz,1H),4.33(d,J＝11.8Hz,1H),4.15(d,J＝9.6Hz,1H),4.12(dd,J＝4.5,2.6Hz,1H),2.77(s,1H),2.67(s,3H),2.38(dt,J＝13.6,3.4Hz,1H),2.29–2.21(m,2H),2.07(s,3H),2.04(s,3H),2.01(d,J＝6.2Hz,1H),1.97–1.94(m,1H),1.93(s,3H),1.77–1.70(m,3H),1.69–1.64(m,2H),1.62–1.57(m,1H),1.32(dd,J＝12.6,2.2Hz,1H),1.25–1.20(m,1H),1.05(s,3H),0.87(s,3H).HRMS(m/z)calc.forC33H43NNaO10S:[M+Na] +,668.2500；found 668.2502.
实施例9
化合物B-9的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，3-硝基苯磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物B-9，收率15％。
Figure PCTCN2021141536-appb-000093
化合物B-9的结构测定： 1H NMR(500MHz,CDCl 3)δ8.63(t,J＝1.9Hz,1H),8.44(ddd,J＝8.2,2.2,0.9Hz,1H),8.35–8.31(m,1H),7.80(t,J＝8.0Hz,1H),7.03–6.97(m,1H),5.74(d,J＝5.7Hz,1H),4.58(dd,J＝11.8,4.3Hz,1H),4.42(dd,J＝11.2,5.8Hz,1H),4.34(d,J＝11.8Hz,1H),4.16(d,J＝11.8Hz,1H),4.10(dd,J＝11.0,1.6Hz,1H),2.77(s,1H),2.31(dd,J＝10.0,3.9Hz,2H),2.28–2.21(m,1H),2.10(d,J＝1.9Hz,1H),2.08(s,3H),2.05(s,3H),2.02(dd,J＝7.9,5.8Hz,1H),1.89(s,3H),1.88–1.81(m,1H),1.78–1.69(m,5H),1.34(dd,J＝12.6,2.2Hz,1H),1.29(dd,J＝10.2,6.6Hz,1H),1.07(s,3H),0.88(s,3H). HRMS(m/z)calc.forC 32H 40N 2O 12SNa:[M+Na] +,699.2194；found 699.2195.
实施例10
化合物B-10的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，4-三氟甲基苯磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物B-10，收率24％。
Figure PCTCN2021141536-appb-000094
化合物B-10的结构测定： 1H NMR(300MHz,CDCl 3)δ8.04(d,J＝8.2Hz,2H),7.78(d,J＝8.3Hz,2H),7.06(t,J＝6.2Hz,1H),5.73(d,J＝5.6Hz,1H),4.57(dd,J＝11.6,4.5Hz,1H),4.42(dd,J＝11.2,5.7Hz,1H),4.32(d,J＝11.8Hz,1H),4.11(dd,J＝17.8,6.6Hz,2H),2.67(s,1H),2.34–2.16(m,4H),2.06(s,3H),2.04(s,3H),1.97(d,J＝3.5Hz,1H),1.91–1.84(m,1H),1.80(s,3H),1.77–1.62(m,5H),1.36–1.27(m,2H),1.05(s,3H),0.85(s,3H).HRMS(m/z)calc.forC33H40F3NNaO10S:[M+Na] +,722.2217；found 722.2220.
实施例11
化合物B-11的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，4-氟苯磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL 乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物B-11，收率13％。
Figure PCTCN2021141536-appb-000095
化合物B-11的结构测定： 1H NMR(300MHz,CDCl 3)δ8.01–7.85(m,2H),7.25–7.15(m,2H),7.07(t,J＝6.0Hz,1H),5.76(d,J＝5.6Hz,1H),4.57(dd,J＝11.5,4.5Hz,1H),4.43(dd,J＝11.2,5.7Hz,1H),4.33(d,J＝11.8Hz,1H),4.20–4.16(m,1H),4.15–4.11(m,1H),2.64(s,1H),2.33(dd,J＝12.9,3.3Hz,1H),2.26(s,1H),2.22–2.10(m,2H),2.07(s,3H),2.04(s,3H),1.93(dd,J＝13.4,8.5Hz,2H),1.85(s,3H),1.78–1.69(m,4H),1.66(d,J＝2.9Hz,1H),1.34–1.26(m,2H),1.05(s,3H),0.86(s,3H).HRMS(m/z)calc.forC32H40FNNaO10S:[M+Na] +,672.2249；found 672.2252.
实施例12
化合物B-12的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，苯磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物B-12，收率11％。
Figure PCTCN2021141536-appb-000096
化合物B-12的结构测定： 1H NMR(300MHz,CDCl 3)δ7.95–7.88(m,2H),7.55(ddd,J＝14.6,8.9,4.8Hz,5H),7.08(t,J＝6.1Hz,1H),5.75(d,J＝5.6Hz,1H),4.57(dd,J＝11.4,4.9Hz,1H),4.42(dd,J＝11.1,5.7Hz,1H),4.34(d,J＝11.9Hz,1H),4.12(dd,J＝17.4,6.5Hz,2H),2.65(s,1H),2.17(d,J＝6.5Hz,1H),2.07(s,3H),2.05(s,3H),2.03(s,2H),1.96–1.88(m,2H),1.82(s,3H),1.73(d,J＝13.2Hz,4H),1.28(s,2H),1.06(s,3H),0.86(s,3H).HRMS(m/z)calc.forC32H41NNaO10S:[M+Na] +,654.2343；found654.2340.
实施例13
化合物B-13的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，甲磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物B-13，收率17％。
Figure PCTCN2021141536-appb-000097
化合物B-13的结构测定： 1H NMR(300MHz,CDCl 3)δ7.15(t,J＝5.9Hz,1H),5.86(d,J＝5.6Hz,1H),4.56(dd,J＝11.3,4.4Hz,1H),4.49(dd,J＝11.2,5.7Hz,1H),4.33(d,J＝11.8Hz,1H),4.27(dd,J＝11.3,1.3Hz,1H),4.15(d,J＝11.9Hz,1H),3.02(s,3H),2.61(s,1H),2.23(dd,J＝12.1,8.3Hz,2H),2.12(s,3H),2.06(s,3H),2.05(s,3H),2.00(d,J＝4.5Hz,2H),1.79(s,2H),1.64(dd,J＝10.7,7.4Hz,4H),1.35–1.28(m,3H),1.04(s,3H),0.89(s,3H).HRMS(m/z)calc.forC27H39NNaO10S:[M+Na] +,592.2187；found 592.2189.
实施例14
化合物B-14的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，2-甲基-5-硝基苯磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物B-14，收率22％。
Figure PCTCN2021141536-appb-000098
化合物B-14的结构测定： 1H NMR(500MHz,CDCl 3)δ8.77(d,J＝2.4Hz,1H),8.32(dd,J＝8.4,2.4Hz,1H),7.53(d,J＝8.5Hz,1H),6.92–6.86(m,1H),5.78(d,J＝6.0Hz,1H),4.56(dd,J＝11.7,4.5Hz,1H),4.47(dd,J＝11.2,6.0Hz,1H),4.34(d,J＝11.8Hz,1H),4.19–4.13(m,2H),2.88(s,1H),2.81(s,3H),2.36–2.32(m,1H),2.24–2.15(m,1H),2.09(s,3H),2.06(s,3H),2.01(s,3H),1.99–1.90(m,2H),1.79–1.63(m,6H),1.38–1.30(m,3H),1.08(s,3H),0.90(s,3H).HRMS(m/z)calc.forC33H42N2O12SNa:[M+Na] +,713.2351；found 713.2352.
实施例15
化合物B-15的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，4-乙酰氨基苯磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层 色谱板分离纯化得到产物B-15，收率5％。
Figure PCTCN2021141536-appb-000099
化合物B-15的结构测定： 1H NMR(400MHz,CDCl 3)δ8.04(s,1H),7.84(d,J＝8.8Hz,2H),7.69(d,J＝8.7Hz,2H),7.04(t,J＝5.9Hz,1H),5.77(d,J＝5.7Hz,1H),4.56(dd,J＝11.8,4.3Hz,1H),4.45(dd,J＝11.1,5.8Hz,1H),4.34(d,J＝11.8Hz,1H),4.15(d,J＝11.5Hz,2H),2.65(s,1H),2.35(dd,J＝9.9,3.2Hz,1H),2.21(s,3H),2.17(s,1H),2.08(s,3H),2.05(s,3H),2.03(s,1H),1.90(s,3H),1.88–1.82(m,1H),1.79–1.59(m,7H),1.41–1.28(m,2H),1.05(s,3H),0.86(s,3H).HRMS(m/z)calc.forC34H44N2O11SNa:[M+Na] +,711.2558；found 711.2560.
实施例16
化合物B-16的制备：
参照实施例1中的制备方法制备化合物A。在8mL的样品瓶中，依次加入乙酰氧基保护的穿心连内酯A-1(46mg，0.1mmol)，4-叔丁基苯磺酰亚胺碘盐(0.2mmol，2当量)，Cu(OTf) 2(18mg，0.05mmol，0.5当量)，再加入1mL乙腈作为溶剂，室温搅拌7h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物B-16，收率16％。
Figure PCTCN2021141536-appb-000100
化合物B-16的结构测定： 1H NMR(500MHz,CDCl 3)δ7.82(d,J＝8.6Hz,2H),7.52(d,J＝8.7Hz,2H),7.11(dd,J＝9.9,3.7Hz,1H),5.75(d,J＝5.7Hz, 1H),4.58(dd,J＝11.8,4.3Hz,1H),4.42(dd,J＝11.1,5.8Hz,1H),4.34(d,J＝11.8Hz,1H),4.16(d,J＝11.8Hz,1H),4.08(dd,J＝11.1,1.4Hz,1H),2.64(s,1H),2.36(dt,J＝15.7,4.6Hz,1H),2.25(dd,J＝14.1,3.3Hz,1H),2.20–2.15(m,1H),2.07(s,3H),2.05(s,3H),2.04–2.02(m,1H),2.00–1.96(m,1H),1.92(dd,J＝16.7,7.6Hz,1H),1.80(s,3H),1.78–1.62(m,7H),1.33(s,9H),1.06(s,3H),0.86(s,3H).HRMS(m/z)calc.forC36H49NO10SNa:[M+Na] +,710.2969；found 710.2970.
实施例17
化合物C-1的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-1，收率20％。
Figure PCTCN2021141536-appb-000101
化合物C-1的结构测定： 1H NMR(400MHz,CDCl 3)δ7.73(d,J＝8.3Hz,2H),7.36(d,J＝8.1Hz,2H),7.10–7.02(m,1H),5.78(d,J＝5.9Hz,1H),4.51(td,J＝11.6,5.5Hz,2H),4.31(d,J＝11.8Hz,1H),4.23(dd,J＝11.2,1.7Hz,1H),4.15–4.05(m,2H),3.56(d,J＝10.4Hz,1H),3.39(d,J＝10.4Hz,1H),2.45(s,3H),2.19(s,3H),2.15(s,3H),2.07(s,3H),2.03(s,3H),1.78(t,J＝5.7Hz,1H),1.72–1.64(m,3H),1.37–1.29(m,3H),1.15–1.06(m,4H),0.98(s,3H),0.81(s,3H).HRMS(m/z)calc.forC35H47N2O10S:[M+H] +,687.2946；found 687.2919.
实施例18
化合物C-2的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-2(20mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-2，收率37％。
Figure PCTCN2021141536-appb-000102
化合物C-2的结构测定： 1H NMR(400MHz,CDCl 3)δ8.01(td,J＝8.5,6.0Hz,1H),7.17–7.07(m,2H),7.01(dd,J＝10.3,2.1Hz,1H),5.82(d,J＝5.8Hz,1H),4.54(td,J＝11.0,5.5Hz,2H),4.34–4.23(m,2H),4.13–4.07(m,1H),3.68(d,J＝10.3Hz,1H),3.54(d,J＝10.3Hz,1H),2.38–2.30(m,1H),2.18(s,3H),2.13(s,3H),2.07(s,3H),2.06(d,J＝3.0Hz,1H),2.05(s,3H),1.86(t,J＝5.5Hz,1H),1.74(s,3H),1.46–1.37(m,3H),1.23–1.15(m,3H),1.02(s,3H),0.83(s,3H).HRMS(m/z)calc.for C34H43F2N2O10S:[M+H] +,709.2601；found 709.2602.
实施例19
化合物C-3的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-3(20mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-3，收率30％。
Figure PCTCN2021141536-appb-000103
化合物C-3的结构测定： 1H NMR(500MHz,CDCl 3)δ7.80(d,J＝8.7Hz,2H),7.57(d,J＝8.7Hz,2H),7.07(t,J＝6.6Hz,1H),5.80(d,J＝5.8Hz,1H),4.62–4.42(m,2H),4.32(d,J＝11.8Hz,1H),4.25(dd,J＝11.2,1.6Hz,1H),4.08(dd,J＝14.8,9.2Hz,1H),3.56(d,J＝10.3Hz,1H),3.40(d,J＝10.2Hz,1H),2.33–2.25(m,1H),2.22(s,3H),2.16(s,3H),2.08(s,3H),2.06(d,J＝9.9Hz,2H),2.04(s,3H),1.81(s,1H),1.75–1.68(m,3H),1.60(s,2H),1.39–1.36(m,1H),1.16(t,J＝11.0Hz,2H),1.00(s,3H),0.82(s,3H).HRMS(m/z)calc.forC 34H 44ClN 2O 10S:[M+H] +,707.2400；found 707.2400.
实施例20
化合物C-4的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-4(20mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-4，收率30％。
Figure PCTCN2021141536-appb-000104
化合物C-4的结构测定： 1H NMR(300MHz,CDCl 3)δ8.44(d,J＝8.9Hz,2H),8.06(d,J＝8.9Hz,2H),7.05(t,J＝7.3Hz,1H),5.79(d,J＝5.7Hz,1H),4.56–4.45(m,2H),4.28(ddd,J＝12.8,11.5,4.8Hz,2H),4.08(d,J＝11.8Hz,1H),3.60(d,J＝10.3Hz,1H),3.42(d,J＝10.4Hz,1H),2.32(ddd,J＝15.3,7.6, 5.3Hz,1H),2.22(s,3H),2.14(s,3H),2.08(s,3H),2.04(s,3H),1.82(t,J＝5.7Hz,1H),1.77–1.66(m,4H),1.39(dd,J＝14.6,6.8Hz,2H),1.22–1.08(m,4H),0.99(s,3H),0.82(s,3H).HRMS(m/z)calc.forC34H43N3NaO12S:[M+Na] +,740.2460；found 740.2462.
实施例21
化合物C-5的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-5(23mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-5，收率44％。
Figure PCTCN2021141536-appb-000105
化合物C-5的结构测定： 1H NMR(400MHz,CDCl 3)δ7.95(d,J＝8.6Hz,2H),7.57(d,J＝8.6Hz,2H),7.08(t,J＝6.7Hz,1H),5.80(d,J＝5.8Hz,1H),4.52(ddd,J＝14.1,11.5,5.4Hz,2H),4.34–4.23(m,2H),4.09(d,J＝11.8Hz,1H),3.57(d,J＝10.3Hz,1H),3.40(d,J＝10.3Hz,1H),2.34–2.23(m,1H),2.21(s,3H),2.17(s,3H),2.09(s,3H),2.05(s,3H),1.81(t,J＝5.7Hz,1H),1.76–1.67(m,4H),1.38(dd,J＝23.2,12.4Hz,3H),1.21–1.13(m,3H),1.01(s,3H),0.83(s,3H).HRMS(m/z)calc.forC34H43IN2NaO10S:[M+Na] +,821.1575；found 821.1576.
实施例22
化合物C-6的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-6(21mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-6，收率35％。
Figure PCTCN2021141536-appb-000106
化合物C-6的结构测定： 1H NMR(500MHz,CDCl 3)δ7.93(d,J＝8.8Hz,2H),7.42(d,J＝8.2Hz,2H),7.06(s,1H),5.80(d,J＝5.5Hz,1H),4.52(ddd,J＝19.5,11.6,5.4Hz,2H),4.32(d,J＝11.8Hz,1H),4.25(dd,J＝11.2,1.5Hz,1H),4.09(d,J＝11.8Hz,1H),3.58(d,J＝10.3Hz,1H),3.41(s,1H),2.25–2.21(m,2H),2.16(s,3H),2.08(s,3H),2.04(s,3H),1.77–1.65(m,5H),1.38(s,2H),1.12(ddd,J＝37.7,28.0,12.2Hz,6H),1.00(s,3H),0.83(s,3H).HRMS(m/z)calc.forC 35H 44F 3N 2O 11S:[M+H] +,757.2612；found 757.2614.
实施例23
化合物C-7的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-7(21mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-7，收率34％。
Figure PCTCN2021141536-appb-000107
化合物C-7的结构测定： 1H NMR(300MHz,CDCl 3)δ7.79(d,J＝9.0Hz, 2H),7.03(d,J＝8.9Hz,2H),5.79(d,J＝5.4Hz,1H),4.56–4.47(m,2H),4.31(d,J＝11.7Hz,1H),4.23(dd,J＝11.3,1.7Hz,1H),4.11(s,1H),3.90(s,3H),3.56(d,J＝10.9Hz,1H),3.40(d,J＝10.4Hz,1H),2.16(s,3H),2.08(s,3H),2.05(s,3H),2.04–2.02(m,3H),2.00(d,J＝1.3Hz,1H),1.82–1.75(m,3H),1.39–1.30(m,6H),0.99(s,3H),0.81(s,3H).HRMS(m/z)calc.forC35H47N2O11S:[M+H] +,703.2895；found703.2894.
实施例24
化合物C-8的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-8(20mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-8，收率12％。
Figure PCTCN2021141536-appb-000108
化合物C-8的结构测定： 1H NMR(500MHz,CDCl 3)δ7.94(d,J＝8.0Hz,1H),7.55(t,J＝7.0Hz,1H),7.38(d,J＝7.7Hz,1H),7.17(t,J＝6.7Hz,1H),5.85(d,J＝5.9Hz,1H),4.55(dd,J＝11.2,5.9Hz,2H),4.32–4.24(m,2H),4.09(d,J＝11.8Hz,1H),3.65(d,J＝10.0Hz,1H),3.38(d,J＝10.0Hz,1H),2.64(s,3H),2.36–2.30(m,1H),2.25–2.21(m,1H),2.18(s,3H),2.11(s,3H),2.05(d,J＝3.7Hz,6H),1.86(s,1H),1.76–1.66(m,5H),1.45(dd,J＝18.1,5.9Hz,2H),1.17(dd,J＝18.3,9.0Hz,3H),1.01(s,3H),0.81(s,3H).HRMS(m/z)calc.forC35H47N2O10S:[M+H] +,687.2946；found 687.2947.
实施例25
化合物C-9的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-9(20mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-9，收率20％。
Figure PCTCN2021141536-appb-000109
化合物C-9的结构测定： 1H NMR(300MHz,CDCl 3)δ8.69(t,J＝1.9Hz,1H),8.55–8.48(m,1H),8.19(d,J＝8.1Hz,1H),7.85(t,J＝8.0Hz,1H),7.05(t,J＝7.6Hz,1H),5.79(d,J＝5.7Hz,1H),4.49(dd,J＝11.3,5.9Hz,1H),4.34–4.22(m,3H),4.10(d,J＝11.8Hz,1H),3.65(d,J＝10.8Hz,1H),3.48(d,J＝10.4Hz,1H),2.31(ddd,J＝12.7,6.3,3.6Hz,1H),2.22(s,3H),2.16(s,3H),2.09(s,3H),2.04(s,3H),1.82(t,J＝5.8Hz,1H),1.75–1.64(m,5H),1.46(d,J＝7.5Hz,1H),1.39–1.35(m,1H),1.18–1.08(m,3H),0.99(s,3H),0.85(s,3H).HRMS(m/z)calc.forC34H43N3NaO12S:[M+Na] +,740.2460；found 740.2460.
实施例26
化合物C-10的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-10(21mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-10，收率8％。
Figure PCTCN2021141536-appb-000110
化合物C-10的结构测定： 1H NMR(300MHz,CDCl 3)δ8.03(d,J＝8.1Hz,2H),7.74(d,J＝8.3Hz,2H),6.81(t,J＝6.0Hz,1H),5.91(t,J＝7.4Hz,1H),4.61(dd,J＝11.3,6.2Hz,1H),4.27(td,J＝9.9,5.0Hz,3H),4.15(d,J＝12.2Hz,1H),3.84(dd,J＝14.4,5.3Hz,1H),3.69(dd,J＝14.5,4.5Hz,1H),3.16(dd,J＝18.3,6.0Hz,1H),2.95(dd,J＝18.0,7.5Hz,1H),2.40(s,3H),2.09(s,3H),2.06(s,3H),2.04(s,3H),1.87(d,J＝13.6Hz,2H),1.74(s,2H),1.68(d,J＝2.0Hz,2H),1.58–1.43(m,2H),1.18(dd,J＝13.5,7.6Hz,2H),1.02(s,3H),0.98(s,3H).HRMS(m/z)calc.for C35H43F3N2NaO10S:[M+Na]+,763.2483；found 763.2485.
实施例27
化合物C-11的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-11(20mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-11，收率15％。
Figure PCTCN2021141536-appb-000111
化合物C-11的结构测定： 1H NMR(300MHz,CDCl 3)δ7.90(ddd,J＝9.0,5.7,3.2Hz,2H),7.18–7.08(m,2H),6.94(s,1H),6.79(d,J＝6.1Hz,1H),5.89(dd,J＝14.1,6.0Hz,1H),4.76(d,J＝30.6Hz,1H),4.63–4.43(m,2H),4.31–4.22(m,2H),4.16–4.07(m,1H),3.87–3.62(m,1H),3.04(ddd,J＝25.4,18.2, 6.8Hz,1H),2.45(dd,J＝17.2,12.0Hz,1H),2.37(d,J＝4.5Hz,2H),2.24–2.13(m,1H),2.05(dd,J＝14.3,8.0Hz,12H),1.97–1.86(m,1H),1.73(s,1H),1.69–1.55(m,3H),1.51–1.39(m,1H),1.22(s,1H),0.99(d,J＝13.5Hz,3H),0.77(d,J＝28.4Hz,3H).HRMS(m/z)calc.forC34H43FN2NaO10S:[M+Na] +,713.2515；found 713.2516.
实施例28
化合物C-12的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-12(19mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-12，收率10％。
Figure PCTCN2021141536-appb-000112
化合物C-12的结构测定： 1H NMR(300MHz,CDCl 3)δ7.89–7.82(m,2H),7.69–7.56(m,3H),7.06(t,J＝6.9Hz,1H),5.78(d,J＝5.8Hz,1H),4.56–4.45(m,2H),4.33–4.20(m,2H),4.08(d,J＝11.7Hz,1H),3.55(d,J＝10.4Hz,1H),3.39(d,J＝10.5Hz,1H),2.22(s,3H),2.16(s,3H),2.07(s,3H),2.03(s,3H),1.80–1.68(m,6H),1.58(s,1H),1.33(d,J＝7.1Hz,1H),1.18–1.03(m,4H),0.98(s,3H),0.81(s,3H).HRMS(m/z)calc.forC34H44N2NaO10S:[M+Na] +,695.2609；found695.2610.
实施例29
化合物C-13的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-13(17mg， 0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-13，收率26％。
Figure PCTCN2021141536-appb-000113
化合物C-13的结构测定： 1H NMR(400MHz,CDCl 3)δ7.17(t,J＝6.7Hz,1H),5.86(d,J＝5.8Hz,1H),4.54(ddd,J＝23.4,11.5,5.4Hz,2H),4.35(d,J＝11.8Hz,1H),4.27(dd,J＝11.2,1.6Hz,1H),4.16–4.04(m,2H),3.60(d,J＝10.2Hz,1H),3.55(d,J＝10.1Hz,1H),3.06(s,3H),2.45–2.36(m,1H),2.20(s,3H),2.15(s,3H),2.09(s,3H),2.06(s,3H),1.91–1.84(m,2H),1.61(dd,J＝15.0,7.3Hz,4H),1.55(s,2H),1.21(s,2H),1.05(s,3H),0.87(s,3H).HRMS(m/z)calc.forC29H43N2O10S:[M+H] +,611.2633；found 611.2634.
实施例30
化合物C-14的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-14(21mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-14，收率26％。
Figure PCTCN2021141536-appb-000114
化合物C-14的结构测定： 1H NMR(400MHz,CDCl 3)δ8.67(d,J＝2.2Hz,1H),8.39(dd,J＝8.3,2.4Hz,1H),7.62(d,J＝8.5Hz,1H),7.18(t,J＝6.7Hz, 1H),5.86(d,J＝5.8Hz,1H),4.60–4.51(m,2H),4.33–4.26(m,2H),4.09(d,J＝11.8Hz,1H),3.71(d,J＝10.0Hz,1H),3.48(d,J＝10.2Hz,1H),2.78(s,3H),2.43–2.33(m,1H),2.31–2.22(m,1H),2.18(s,3H),2.05(s,6H),1.98–1.87(m,2H),1.82–1.72(m,6H),1.58(d,J＝22.5Hz,3H),1.21(s,2H),1.02(s,3H),0.83(s,3H).HRMS(m/z)calc.forC35H45N3NaO12S:[M+Na] +,754.2616；found 754.2617.
实施例31
化合物C-15的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-15(21mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-15，收率22％。
Figure PCTCN2021141536-appb-000115
化合物C-15的结构测定： 1H NMR(400MHz,CDCl 3)δ7.82(d,J＝8.8Hz,2H),7.77(t,J＝6.0Hz,2H),7.54(d,J＝4.9Hz,1H),7.05(s,1H),5.80(d,J＝5.4Hz,1H),4.53(dd,J＝11.3,6.3Hz,2H),4.33(d,J＝11.7Hz,1H),4.24(d,J＝11.3Hz,1H),4.10(d,J＝11.7Hz,1H),3.57(s,1H),3.45(s,1H),2.25(d,J＝3.6Hz,3H),2.22(dd,J＝5.8,2.8Hz,2H),2.17(s,3H),2.09(s,3H),2.05(d,J＝4.0Hz,6H),1.74–1.66(m,4H),1.42–1.37(m,2H),1.13(dd,J＝11.9,7.4Hz,4H),1.00(s,3H),0.83(s,3H).HRMS(m/z)calc.forC36H48N3O11S:[M+H] +,730.3004；found 730.3007.
实施例32
化合物C-16的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-16(21mg，0.03mmol)，Cu(OTf) 2(1.1mg，0.003mmol，0.1当量)，再加入1mL乙腈作为溶剂，65℃条件下搅拌12h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物C-16，收率25％。
Figure PCTCN2021141536-appb-000116
化合物C-16的结构测定： 1H NMR(500MHz,CDCl 3)δ7.78(d,J＝8.6Hz,2H),7.59(d,J＝8.6Hz,2H),7.04(t,J＝7.2Hz,1H),5.80(d,J＝5.8Hz,1H),4.57–4.48(m,2H),4.32(d,J＝11.8Hz,1H),4.24(dd,J＝11.2,1.7Hz,1H),4.10(d,J＝11.8Hz,1H),3.61(d,J＝10.4Hz,1H),3.43(d,J＝10.2Hz,1H),2.22(s,3H),2.17(s,3H),2.08(s,3H),2.04(s,3H),2.03–1.99(m,1H),1.80(d,J＝5.5Hz,1H),1.72(s,1H),1.41–1.37(m,1H),1.35(s,9H),1.30(s,2H),1.23–1.04(m,6H),0.99(s,3H),0.83(s,3H).HRMS(m/z)calc.forC38H52N2O10S:[M+H] +,729.3415；found 729.3416.
实施例33
化合物D-1的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，苄胺(0.09mmol，3当量)，三乙胺(5.0μL，0.036mmol，1.2当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-1，收率47％。
Figure PCTCN2021141536-appb-000117
化合物D-1的结构测定： 1H NMR(400MHz,CDCl 3)δ7.83(d,J＝8.2Hz,2H),7.41(s,1H),7.39–7.27(m,7H),4.83(dd,J＝40.0,18.1Hz,2H),4.49(dd,J＝11.8,4.7Hz,1H),4.27(d,J＝11.8Hz,1H),4.11(d,J＝11.8Hz,1H),3.71(d,J＝13.2Hz,1H),3.43(d,J＝13.3Hz,1H),2.59(s,1H),2.42(s,3H),2.35(dd,J＝9.7,6.5Hz,1H),2.15(s,1H),2.05(s,6H),2.00–1.91(m,2H),1.86(s,2H),1.69–1.56(m,4H),1.47(dd,J＝14.8,5.8Hz,1H),1.29(d,J＝6.0Hz,2H),1.01(s,3H),0.90(dd,J＝14.5,6.0Hz,2H),0.74(s,3H).HRMS(m/z)calc.forC38H49N2O8S:[M+H] +,693.3204；found 693.3204.
实施例34
化合物D-2的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，4-氟苄胺(0.09mmol，3当量)，三乙胺(5.0μL，0.036mmol，1.2当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-2，收率55％。
Figure PCTCN2021141536-appb-000118
化合物D-2的结构测定： 1H NMR(300MHz,CDCl 3)δ7.78(d,J＝8.3Hz,2H),7.42(s,1H),7.27(s,1H),7.26–7.20(m,3H),7.02(t,J＝8.7Hz,2H),4.96–4.73(m,2H),4.49(dd,J＝11.7,4.7Hz,1H),4.27(d,J＝11.8Hz,1H),4.11(d,J＝ 11.8Hz,1H),3.63(d,J＝13.2Hz,1H),3.55(t,J＝7.0Hz,1H),3.41(d,J＝13.2Hz,1H),2.60(s,1H),2.41(s,3H),2.36(d,J＝13.2Hz,1H),2.26(s,1H),2.22–2.12(m,1H),2.05(d,J＝2.0Hz,6H),1.95(s,2H),1.71(dd,J＝11.2,6.7Hz,2H),1.59(dd,J＝12.4,3.5Hz,2H),1.48(dd,J＝13.6,8.0Hz,1H),1.34–1.28(m,2H),1.01(s,3H),0.92(dd,J＝12.3,6.0Hz,2H),0.74(s,3H).HRMS(m/z)calc.forC38H47FN2NaO8S:[M+Na] +,733.2929；found 733.2903.
实施例35
化合物D-3的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，2-噻吩甲胺(0.09mmol，3当量)，三乙胺(5.0μL，0.036mmol，1.2当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-3，收率58％。
Figure PCTCN2021141536-appb-000119
化合物D-3的结构测定： 1H NMR(500MHz,CDCl 3)δ7.81(d,J＝8.2Hz,2H),7.39(s,1H),7.29(d,J＝8.2Hz,2H),7.22(d,J＝5.0Hz,1H),6.98(dd,J＝5.0,3.5Hz,1H),6.91(d,J＝3.3Hz,1H),4.82(dd,J＝42.1,18.0Hz,2H),4.53(dd,J＝11.9,4.6Hz,1H),4.28(d,J＝11.8Hz,1H),4.12(d,J＝11.8Hz,1H),3.84(d,J＝14.2Hz,1H),3.70(d,J＝14.2Hz,1H),3.57(t,J＝7.1Hz,1H),2.60(s,1H),2.42(s,3H),2.14(s,1H),2.05(d,J＝3.3Hz,6H),1.99(d,J＝15.0Hz,1H),1.86(d,J＝13.6Hz,2H),1.72–1.60(m,4H),1.49(dd,J＝14.2,6.5Hz,1H),1.43(d,J＝4.0Hz,1H),1.12(d,J＝10.8Hz,1H),1.02(s,3H),0.96–0.87(m,3H),0.76(s,3H).HRMS(m/z)calc.forC36H46N2NaO8S2:[M+Na] +,721.2588；found 721.2573.
实施例36
化合物D-4的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，2-呋喃甲胺(0.09mmol，3当量)，三乙胺(5.0μL，0.036mmol，1.2当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-4，收率57％。
Figure PCTCN2021141536-appb-000120
化合物D-4的结构测定： 1H NMR(400MHz,CDCl 3)δ7.84(d,J＝8.3Hz,2H),7.39–7.35(m,2H),7.32(d,J＝8.1Hz,2H),6.32(dd,J＝3.1,1.9Hz,1H),6.14(d,J＝3.0Hz,1H),4.79(dt,J＝18.2,9.7Hz,2H),4.55(dd,J＝11.9,4.7Hz,1H),4.29(d,J＝11.8Hz,1H),4.13(d,J＝11.7Hz,1H),3.62–3.56(m,2H),2.60(s,1H),2.43(s,3H),2.15(t,J＝12.1Hz,1H),2.06(s,3H),2.05(s,3H),2.02(s,1H),1.91–1.82(m,3H),1.67(ddd,J＝29.2,12.3,5.8Hz,5H),1.56–1.49(m,1H),1.39(d,J＝4.9Hz,1H),1.20–1.14(m,1H),1.02(s,3H),0.95–0.89(m,2H),0.77(s,3H).HRMS(m/z)calc.forC36H46N2NaO9S:[M+Na] +,705.2816；found 705.2798.
实施例37
化合物D-5的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，4-三氟甲基苄胺(0.09mmol，3当量)，三乙胺(5.0μL，0.036mmol，1.2当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-5，收率58％。
Figure PCTCN2021141536-appb-000121
化合物D-5的结构测定： 1H NMR(500MHz,CDCl 3)δ7.77(d,J＝8.2Hz,2H),7.58(d,J＝8.1Hz,2H),7.40(d,J＝4.0Hz,2H),7.38(s,1H),7.22(d,J＝8.1Hz,2H),4.84(dd,J＝44.0,17.8Hz,2H),4.49(dd,J＝12.0,4.5Hz,1H),4.27(d,J＝11.8Hz,1H),4.11(d,J＝7.4Hz,1H),3.68(d,J＝13.7Hz,1H),3.55(t,J＝7.0Hz,1H),3.51(d,J＝13.7Hz,1H),2.38(s,4H),2.12(d,J＝12.4Hz,1H),2.05–2.03(m,7H),1.98(s,1H),1.82(s,1H),1.73–1.58(m,4H),1.50(dd,J＝13.3,6.6Hz,1H),1.35(d,J＝5.0Hz,1H),1.07(d,J＝13.1Hz,1H),1.00(s,3H),0.96–0.88(m,2H),0.75(s,3H).；HRMS(ESI)exact mass calculated for[M+H] +(C 39H 48F 3N 2O 8S)requires m/z 761.3083,found m/z761.3076,difference-0.15ppm.
实施例38
化合物D-6的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，2-甲基苄胺(0.09mmol，3当量)，三乙胺(5.0μL，0.036mmol，1.2当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-6，收率65％。
Figure PCTCN2021141536-appb-000122
化合物D-6的结构测定： 1H NMR(500MHz,CDCl 3)δ7.75(d,J＝8.2Hz,2H),7.38(s,1H),7.24(d,J＝8.1Hz,2H),7.21–7.13(m,4H),4.89–4.75(m, 2H),4.51(dd,J＝11.9,4.6Hz,1H),4.27(d,J＝11.8Hz,1H),4.11(d,J＝11.6Hz,1H),3.62(d,J＝13.0Hz,1H),3.56(t,J＝6.5Hz,1H),3.49(d,J＝13.0Hz,1H),2.40(s,3H),2.36(d,J＝12.7Hz,1H),2.28(s,3H),2.15(dd,J＝15.2,4.8Hz,1H),2.04(d,J＝2.4Hz,7H),1.97(s,1H),1.84–1.79(m,2H),1.67–1.52(m,4H),1.51–1.45(m,1H),1.11(d,J＝10.9Hz,1H),1.01(s,3H),0.83(s,2H),0.75(s,3H).；HRMS(ESI)exact mass calculated for[M+H] +(C 39H 51N 2O 8S)requires m/z 707.3366,found m/z707.3360,difference-0.01ppm.
实施例39
化合物D-7的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，2-氟苄胺(0.09mmol，3当量)，三乙胺(5.0μL，0.036mmol，1.2当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-7，收率68％。
Figure PCTCN2021141536-appb-000123
化合物D-7的结构测定： 1H NMR(500MHz,CDCl 3)δ7.78(d,J＝8.3Hz,2H),7.42(s,1H),7.33(td,J＝7.6,1.5Hz,1H),7.27(s,1H),7.25(s,2H),7.12(td,J＝7.5,1.0Hz,1H),7.05(dd,J＝9.8,8.4Hz,1H),4.86(d,J＝18.1Hz,1H),4.74(dd,J＝18.1,1.1Hz,1H),4.50(dd,J＝11.9,4.6Hz,1H),4.27(d,J＝11.8Hz,1H),4.11(d,J＝11.5Hz,1H),3.68(d,J＝13.5Hz,1H),3.59(d,J＝13.5Hz,1H),3.53(t,J＝7.0Hz,1H),2.40(s,3H),2.36(d,J＝9.8Hz,1H),2.15(t,J＝11.9Hz,1H),2.04(d,J＝2.4Hz,7H),1.98(d,J＝15.3Hz,1H),1.83(d,J＝10.3Hz,1H),1.70–1.56(m,4H),1.54–1.46(m,1H),1.38(d,J＝3.7Hz,1H),1.08(dd,J＝12.6, 2.0Hz,1H),1.00(s,3H),0.88–0.78(m,2H),0.75(s,3H).；HRMS(ESI)exact mass calculated for[M+H] +(C 38H 48FN 2O 8S)requires m/z 711.3115,found m/z711.3110,difference-0.06ppm.
实施例40
化合物D-8的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，4-甲氧基苄胺(0.09mmol，3当量)，三乙胺(5.0μL，0.036mmol，1.2当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-8，收率43％。
Figure PCTCN2021141536-appb-000124
化合物D-8的结构测定： 1H NMR(300MHz,CDCl 3)δ7.83(d,J＝8.3Hz,2H),7.43(s,1H),7.30(s,2H),7.22(d,J＝8.5Hz,2H),6.90(d,J＝8.6Hz,2H),4.82(q,J＝18.0Hz,2H),4.44(dd,J＝11.7,4.6Hz,1H),4.25(d,J＝11.8Hz,1H),4.09(d,J＝11.7Hz,1H),3.81(s,3H),3.69(d,J＝13.2Hz,1H),3.47(t,J＝6.9Hz,1H),3.33(d,J＝13.2Hz,1H),2.42(s,4H),2.37–2.28(m,1H),2.04(d,J＝1.3Hz,7H),1.93(s,1H),1.84(s,2H),1.61(dd,J＝23.1,10.0Hz,4H),1.42(dd,J＝13.9,6.0Hz,1H),1.19(d,J＝3.0Hz,1H),1.00(s,3H),0.87–0.77(m,2H),0.71(s,3H).；HRMS(ESI)exact mass calculated for[M+H] +(C 39H 51N 2O 9S)requires m/z 723.3315,found m/z723.3308,difference-0.10ppm.
实施例41
化合物D-9的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，3-氯-4-氟苄胺(0.09mmol，3当量)，三乙胺(5.0μL，0.036mmol，1.2当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-9，收率59％。
Figure PCTCN2021141536-appb-000125
化合物D-9的结构测定： 1H NMR(300MHz,CDCl 3)δ7.78(d,J＝8.2Hz,2H),7.42(s,1H),7.33(d,J＝6.9Hz,1H),7.27(s,1H),7.25(s,1H),7.09(d,J＝7.3Hz,2H),4.86(q,J＝18.5Hz,2H),4.50(dd,J＝11.7,4.6Hz,1H),4.28(d,J＝11.8Hz,1H),4.11(d,J＝11.6Hz,1H),3.60(d,J＝13.5Hz,1H),3.50(t,J＝6.9Hz,1H),3.38(d,J＝13.5Hz,1H),2.40(s,3H),2.36(s,1H),2.24–2.13(m,1H),2.05(d,J＝2.8Hz,7H),1.98(s,1H),1.79(s,1H),1.64(dd,J＝21.5,8.0Hz,4H),1.47(t,J＝6.5Hz,1H),1.34(d,J＝4.4Hz,1H),1.10(s,1H),1.02(s,3H),0.91–0.84(m,2H),0.75(s,3H).；HRMS(ESI)exact mass calculated for[M+H] +(C 38H 47ClFN 2O 8S)requires m/z 745.2726,found m/z745.2721,difference-0.07ppm.
实施例42
化合物D-10的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，异丁胺(0.09mmol，3当量)，三乙胺(5.0μL，0.036mmol，1.2当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-10，收率43％。
Figure PCTCN2021141536-appb-000126
化合物D-10的结构测定： 1H NMR(300MHz,CDCl 3)δ7.83(d,J＝8.3Hz,2H),7.41(s,1H),7.30(d,J＝8.1Hz,2H),4.99–4.79(m,2H),4.56(dd,J＝11.6,4.9Hz,1H),4.29(d,J＝11.8Hz,1H),4.13(d,J＝11.8Hz,1H),3.61–3.50(m,1H),2.43(s,3H),2.36(s,2H),2.17(dd,J＝11.1,5.8Hz,2H),2.05(d,J＝3.7Hz,7H),2.00(s,1H),1.88(d,J＝13.6Hz,1H),1.74(s,2H),1.66–1.53(m,4H),1.50(d,J＝7.8Hz,1H),0.99(s,4H),0.95(dd,J＝13.9,3.0Hz,2H),0.84(d,J＝1.7Hz,3H),0.82(d,J＝1.6Hz,3H),0.78(s,3H).；HRMS(ESI)exact mass calculated for[M+H] +(C 35H 51N 2O 8S)requires m/z 659.3366,found m/z659.3359,difference-0.11ppm.
实施例43
化合物D-11的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，3-氯苄胺(0.09mmol，3当量)，三乙胺(5.0μL，0.036mmol，1.2当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-11，收率46％。
Figure PCTCN2021141536-appb-000127
化合物D-11的结构测定： 1H NMR(300MHz,CDCl 3)δ7.81(d,J＝8.2Hz,2H),7.41(s,1H),7.29(d,J＝4.4Hz,5H),7.11(dd,J＝5.5,3.1Hz,1H),4.84(q,J ＝17.9Hz,2H),4.48(dd,J＝11.4,5.0Hz,1H),4.27(d,J＝11.8Hz,1H),4.10(d,J＝12.1Hz,1H),3.67(d,J＝13.6Hz,1H),3.51–3.42(m,1H),3.41(s,1H),2.41(s,3H),2.35(s,1H),2.17(d,J＝17.9Hz,1H),2.04(d,J＝1.5Hz,7H),1.97(s,1H),1.87(s,1H),1.71–1.55(m,4H),1.45(dd,J＝15.1,5.3Hz,1H),1.32(s,1H),1.07(s,1H),1.02(s,3H),0.90(dd,J＝11.9,5.1Hz,2H),0.73(s,3H).；HRMS(ESI)exact mass calculated for[M+H] +(C 38H 48ClN 2O 8S)requires m/z 727.2820,found m/z727.2814,difference-0.08ppm.
实施例44
化合物D-12的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，环丙胺(0.09mmol，3当量)，三乙胺(5.0μL，0.036mmol，1.2当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-12，收率4％。
Figure PCTCN2021141536-appb-000128
化合物D-12的结构测定： 1H NMR(300MHz,CDCl 3)δ7.82(d,J＝8.2Hz,2H),7.46(s,1H),7.32(d,J＝8.1Hz,2H),4.90(q,J＝17.3Hz,2H),4.55(dd,J＝11.5,4.9Hz,1H),4.28(d,J＝11.8Hz,1H),4.12(d,J＝11.7Hz,1H),3.76(dd,J＝9.4,5.8Hz,1H),2.44(s,3H),2.37(s,1H),2.13(d,J＝12.9Hz,1H),2.04(d,J＝3.2Hz,7H),1.98(s,1H),1.86(s,2H),1.69–1.41(m,6H),1.15(d,J＝9.8Hz,1H),1.01(s,3H),0.94–0.88(m,2H),0.77(s,3H),0.36–0.20(m,3H),0.09–0.01(m,1H).；HRMS(ESI)exact mass calculated for[M+H] +(C 34H 47N 2O 8S)requires m/z 643.3053,found m/z643.3049,difference-0.08ppm.
实施例45
化合物D-13的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，异丙胺(0.09mmol，3当量)，三乙胺(5.0μL，0.036mmol，1.2当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-13，收率4％。
Figure PCTCN2021141536-appb-000129
化合物D-13的结构测定： 1H NMR(300MHz,CDCl 3)δ7.83(d,J＝8.3Hz,2H),7.45(s,1H),7.29(d,J＝8.1Hz,2H),5.01–4.79(m,2H),4.56(dd,J＝11.7,4.8Hz,1H),4.29(d,J＝11.8Hz,1H),4.13(d,J＝11.8Hz,1H),3.79–3.68(m,1H),2.55(dt,J＝12.4,6.2Hz,1H),2.42(s,3H),2.39(s,1H),2.21–2.10(m,1H),2.05(d,J＝3.6Hz,7H),1.90(d,J＝13.5Hz,1H),1.75(s,3H),1.70–1.54(m,4H),1.48(d,J＝7.5Hz,1H),1.19(d,J＝12.6Hz,1H),1.01(s,3H),0.93(d,J＝6.3Hz,4H),0.83(d,J＝6.1Hz,3H),0.79(s,3H)；HRMS(ESI)exact mass calculated for[M+H] +(C 34H 49N 2O 8S)requires m/z 645.3210,found m/z645.3205,difference 0.13ppm.
实施例46
化合物D-14的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-1(20mg，0.03mmol)，4-羟基苄胺(0.09mmol，3当量)，三乙胺(5.0μL，0.036mmol，1.2当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓 缩反应物，制备薄层色谱板分离纯化得到产物D-14，收率40％。
Figure PCTCN2021141536-appb-000130
化合物D-14的结构测定： 1H NMR(300MHz,CDCl 3)δ7.83(d,J＝8.2Hz,2H),7.35(d,J＝8.0Hz,2H),6.99(d,J＝9.1Hz,1H),6.87(dd,J＝18.6,8.0Hz,3H),6.59(d,J＝8.4Hz,2H),4.62–4.25(m,4H),4.21(d,J＝11.9Hz,1H),3.64(dd,J＝15.8,5.3Hz,2H),3.26(d,J＝13.4Hz,1H),3.07–2.85(m,1H),2.65(d,J＝11.8Hz,1H),2.43(s,3H),2.06(d,J＝5.0Hz,6H),1.99(d,J＝11.1Hz,1H),1.86–1.74(m,2H),1.63(s,4H),1.42(d,J＝11.9Hz,2H),1.06–1.00(m,1H),0.94(s,3H),0.86(s,3H),0.81(d,J＝8.7Hz,1H),0.63–0.46(m,1H)；HRMS(ESI)exact mass calculated for[M+Na] +(C 38H 48N 2NaO 9S)requires m/z 731.2978,found m/z 731.2973,difference 0.04ppm.
实施例47
化合物D-15的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-13(17mg，0.03mmol)，苄胺(0.09mmol，3当量)，三乙胺(7.5μL，0.054mmol，1.8当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-15，收率43％。
Figure PCTCN2021141536-appb-000131
化合物D-15的结构测定： 1H NMR(400MHz,CDCl 3)δ7.40(s,1H),7.37– 7.30(m,4H),7.29–7.26(m,1H),4.90–4.80(m,2H),4.48(dd,J＝11.7,4.7Hz,1H),4.26(d,J＝11.8Hz,1H),4.09(d,J＝11.8Hz,1H),3.81(d,J＝13.5Hz,1H),3.55(t,J＝7.1Hz,1H),3.48(d,J＝13.5Hz,1H),2.77(s,3H),2.16–2.08(m,2H),2.03(s,7H),2.00–1.96(m,1H),1.91(d,J＝13.9Hz,1H),1.81(d,J＝13.5Hz,1H),1.67–1.52(m,4H),1.33(d,J＝4.9Hz,1H),1.04(s,1H),1.02–0.99(m,1H),0.97(s,3H),0.88–0.81(m,2H),0.76(s,3H).；HRMS(ESI)exact mass calculated for[M+H] +(C 32H 45N 2O 8S)requires m/z 617.2897,found m/z617.2893,difference 0.31ppm.
实施例48
化合物D-16的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-13(17mg，0.03mmol)，呋喃甲胺(0.09mmol，3当量)，三乙胺(7.5μL，0.054mmol，1.8当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-16，收率12％。
Figure PCTCN2021141536-appb-000132
化合物D-16的结构测定： 1H NMR(300MHz,CDCl 3)δ7.39(d,J＝17.3Hz,2H),6.37–6.26(m,1H),6.19(d,J＝3.0Hz,1H),4.97–4.78(m,2H),4.54(dd,J＝11.5,5.0Hz,1H),4.28(d,J＝11.8Hz,1H),4.12(d,J＝11.7Hz,1H),3.77(d,J＝14.6Hz,1H),3.62(dd,J＝16.7,10.7Hz,2H),2.95(s,3H),2.31–2.10(m,2H),2.04(s,7H),1.93–1.80(m,2H),1.68(s,3H),1.56(s,1H),1.42(d,J＝5.3Hz,1H),1.13(d,J＝2.0Hz,1H),1.05(d,J＝4.8Hz,1H),1.00(s,3H),0.92–0.84(m,2H),0.79(s,3H).；HRMS(ESI)exact mass calculated for[M+H] + (C 30H 43N 2O 9S)requires m/z 607.2689,found m/z607.2684,difference 0.21ppm.
实施例49
化合物D-17的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-13(17mg，0.03mmol)，3-氯-4-氟苄胺(0.09mmol，3当量)，三乙胺(7.5μL，0.054mmol，1.8当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-17，收率45％。
Figure PCTCN2021141536-appb-000133
化合物D-17的结构测定： 1H NMR(300MHz,CDCl 3)δ7.45(dd,J＝7.1,1.8Hz,1H),7.40(s,1H),7.20–7.13(m,1H),7.10(t,J＝8.5Hz,1H),4.94–4.81(m,2H),4.50(dd,J＝11.7,4.8Hz,1H),4.28(d,J＝11.8Hz,1H),4.10(d,J＝12.1Hz,1H),3.74(d,J＝13.8Hz,1H),3.48(dd,J＝17.6,10.4Hz,2H),2.86(s,3H),2.20–2.07(m,2H),2.04(d,J＝1.0Hz,7H),1.93(s,1H),1.84(d,J＝13.3Hz,1H),1.69–1.51(m,4H),1.38(d,J＝5.1Hz,1H),1.10(s,1H),1.06–1.02(m,1H),1.00(s,3H),0.92–0.84(m,2H),0.78(s,3H).；HRMS(ESI)exact mass calculated for[M+H] +(C 32H 43ClFN 2O 8S)requires m/z 669.2413,found m/z 669.2409,difference 0.28ppm.
实施例50
化合物D-18的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-13(17mg，0.03mmol)，环丙胺(0.09mmol，3当量)，三乙胺(7.5μL，0.054mmol，1.8 当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-18，收率51％。
Figure PCTCN2021141536-appb-000134
化合物D-18的结构测定： 1H NMR(300MHz,CDCl 3)δ7.55(s,1H),5.06–4.86(m,2H),4.54(dd,J＝11.4,5.1Hz,1H),4.28(d,J＝11.8Hz,1H),4.11(d,J＝11.8Hz,1H),3.93–3.80(m,1H),3.12(s,3H),2.19(d,J＝15.9Hz,3H),2.04(d,J＝1.4Hz,7H),2.01–1.96(m,2H),1.82(d,J＝13.2Hz,1H),1.71–1.63(m,3H),1.46(d,J＝7.6Hz,1H),1.19–1.14(m,2H),1.00(s,3H),0.92(dd,J＝8.3,4.1Hz,2H),0.80(s,3H),0.59–0.37(m,4H).；HRMS(ESI)exact mass calculated for[M+H] +(C 28H 43N 2O 8S)requires m/z 567.2740,found m/z567.2737,difference 0.38ppm.
实施例51
化合物D-19的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-13(17mg，0.03mmol)，4-氟苄胺(0.09mmol，3当量)，三乙胺(7.5μL，0.054mmol，1.8当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-19，收率42％。
Figure PCTCN2021141536-appb-000135
化合物D-19的结构测定: 1H NMR(300MHz,CDCl 3)δ7.41(s,1H),7.35– 7.27(m,2H),7.02(t,J＝8.6Hz,2H),4.97–4.78(m,2H),4.50(dd,J＝11.7,4.7Hz,1H),4.27(d,J＝11.8Hz,1H),4.10(d,J＝11.8Hz,1H),3.75(d,J＝13.3Hz,1H),3.55(t,J＝7.0Hz,1H),3.47(d,J＝13.4Hz,1H),2.82(s,3H),2.18–2.11(m,1H),2.04(s,8H),1.92–1.77(m,2H),1.71–1.52(m,4H),1.37–1.32(m,1H),1.05(dd,J＝12.6,5.5Hz,2H),0.99(s,3H),0.93–0.84(m,2H),0.77(s,3H).；HRMS(ESI)exact mass calculated for[M+H] +(C 32H 44FN 2O 8S)requires m/z 635.2802,found m/z635.2796,difference-0.14ppm.
实施例52
化合物D-20的制备：
在8mL的样品瓶中，依次加入环氨基化的穿心莲内酯化合物B-13(17mg，0.03mmol)，4-甲氧基苄胺(0.09mmol，3当量)，三乙胺(7.5μL，0.054mmol，1.8当量)，再加入200μL二氯甲烷作为溶剂，室温搅拌3h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物D-20，收率41％。
Figure PCTCN2021141536-appb-000136
化合物D-20的结构测定: 1H NMR(300MHz,CDCl 3)δ7.50(s,1H),7.28(s,1H),7.25(s,1H),6.89(d,J＝8.6Hz,2H),4.98–4.79(m,2H),4.45(dd,J＝11.7,4.7Hz,1H),4.26(d,J＝11.8Hz,1H),4.11(d,J＝7.0Hz,1H),3.83–3.76(m,4H),3.55(t,J＝6.9Hz,1H),3.42(d,J＝13.3Hz,1H),2.87(s,3H),2.16–2.09(m,1H),2.04(d,J＝2.3Hz,8H),1.93–1.78(m,2H),1.75–1.45(m,5H),1.32(s,1H),1.03–0.99(m,1H),0.98(s,3H),0.91–0.80(m,2H),0.75(s,3H).；HRMS(ESI)exact mass calculated for[M+H] +(C 33H 47N 2O 9S)requires m/z 647.3002,found m/z647.2999,difference 0.29ppm.
实施例53
化合物E-1的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-1(14mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-1，收率67％。
Figure PCTCN2021141536-appb-000137
化合物E-1的结构测定： 1H NMR(300MHz,CDCl 3)δ7.84(d,J＝8.2Hz,2H),7.34(d,J＝8.1Hz,2H),7.09(dd,J＝5.1,1.7Hz,3H),6.95(dd,J＝6.8,2.5Hz,3H),6.88(s,1H),4.46(d,J＝8.0Hz,1H),4.40(s,1H),4.24(s,1H),4.21–4.16(m,1H),3.75–3.62(m,2H),3.32(d,J＝13.6Hz,1H),2.98(dd,J＝11.6,9.8Hz,1H),2.61(d,J＝11.8Hz,1H),2.43(s,3H),2.06(d,J＝5.5Hz,6H),1.96(s,2H),1.89(s,1H),1.82(s,1H),1.68–1.61(m,3H),1.54–1.46(m,2H),1.43(s,1H),1.16(d,J＝3.7Hz,1H),1.12–1.06(m,2H),0.94(s,3H),0.88(s,3H).HRMS(m/z)calc.forC38H49N2O8S:[M+H] +,693.3204；found 693.3204.
实施例54
化合物E-2的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-2(14mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-2，收率45％。
Figure PCTCN2021141536-appb-000138
化合物E-2的结构测定： 1H NMR(300MHz,CDCl 3)δ7.78(d,J＝8.3Hz,2H),7.42(s,1H),7.27(s,1H),7.26–7.20(m,3H),7.02(t,J＝8.7Hz,2H),4.96–4.73(m,2H),4.49(dd,J＝11.7,4.7Hz,1H),4.27(d,J＝11.8Hz,1H),4.11(d,J＝11.8Hz,1H),3.63(d,J＝13.2Hz,1H),3.55(t,J＝7.0Hz,1H),3.41(d,J＝13.2Hz,1H),2.60(s,1H),2.41(s,3H),2.36(d,J＝13.2Hz,1H),2.26(s,1H),2.22–2.12(m,1H),2.05(d,J＝2.0Hz,6H),1.95(s,2H),1.71(dd,J＝11.2,6.7Hz,2H),1.59(dd,J＝12.4,3.5Hz,2H),1.48(dd,J＝13.6,8.0Hz,1H),1.34–1.28(m,2H),1.01(s,3H),0.92(dd,J＝12.3,6.0Hz,2H),0.74(s,3H).HRMS(m/z)calc.forC38H47FN2NaO8S:[M+Na] +,733.2929；found 733.2903.
实施例55
化合物E-3的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-3(14mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-3，收率65％。
Figure PCTCN2021141536-appb-000139
化合物E-3的结构测定： 1H NMR(300MHz,CDCl 3)δ7.79(d,J＝8.3Hz,2H),7.32(d,J＝8.0Hz,2H),7.13(s,1H),6.99(dd,J＝5.1,1.1Hz,1H),6.74(dd, J＝5.1,3.5Hz,1H),6.67(d,J＝2.7Hz,1H),6.48(d,J＝9.7Hz,1H),4.56(s,2H),4.43(dd,J＝14.0,8.5Hz,2H),4.21(d,J＝11.9Hz,1H),3.87(d,J＝14.9Hz,1H),3.69(dd,J＝11.1,6.5Hz,1H),3.59(d,J＝14.9Hz,1H),3.02–2.84(m,1H),2.54(d,J＝11.9Hz,1H),2.43(s,3H),2.05(d,J＝4.5Hz,6H),1.95(t,J＝5.5Hz,1H),1.75(ddd,J＝18.1,14.1,7.8Hz,4H),1.52(ddd,J＝17.3,11.4,4.4Hz,6H),1.06(t,J＝7.6Hz,1H),0.93(s,3H),0.88(s,3H).HRMS(m/z)calc.forC36H46N2NaO8S2:[M+Na] +,721.2588；found 721.2573.
实施例56
化合物E-4的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-4(14mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-4，收率53％。
Figure PCTCN2021141536-appb-000140
化合物E-4的结构测定： 1H NMR(300MHz,CDCl 3)δ7.81(d,J＝8.2Hz,2H),7.33(d,J＝8.0Hz,2H),7.20–7.11(m,2H),6.39(d,J＝9.5Hz,1H),6.14(dd,J＝3.1,1.9Hz,1H),5.94(d,J＝3.1Hz,1H),4.60(s,2H),4.43(dd,J＝14.0,8.2Hz,2H),4.21(d,J＝11.9Hz,1H),3.66(dd,J＝11.0,6.6Hz,1H),3.58(d,J＝14.9Hz,1H),3.40(d,J＝14.8Hz,1H),2.99–2.83(m,1H),2.52(d,J＝11.8Hz,1H),2.43(s,3H),2.05(d,J＝3.3Hz,6H),1.96(s,2H),1.89(s,1H),1.77(s,2H),1.53–1.47(m,2H),1.34(s,1H),1.10(dd,J＝12.1,3.5Hz,4H),0.93(s,3H),0.87(s,3H).HRMS(m/z)calc.forC36H47N2O9S:[M+H] +,683.2997；found 683.2993.
实施例57
化合物E-5的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-5(15mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-5，收率32％。
Figure PCTCN2021141536-appb-000141
化合物E-5的结构测定： 1H NMR(500MHz,CDCl 3)δ7.83(d,J＝8.2Hz,2H),7.34(d,J＝8.0Hz,2H),7.28(d,J＝8.2Hz,2H),7.09(d,J＝8.0Hz,2H),7.03(d,J＝9.5Hz,1H),6.98(s,1H),4.51(dd,J＝18.3,1.4Hz,1H),4.48–4.44(m,1H),4.42(d,J＝11.9Hz,1H),4.30–4.20(m,2H),3.76(d,J＝14.0Hz,1H),3.69(dd,J＝11.1,6.8Hz,1H),3.40(d,J＝14.0Hz,1H),2.99(dd,J＝11.6,9.8Hz,1H),2.50(d,J＝11.8Hz,1H),2.43(s,3H),2.13(dd,J＝18.2,6.9Hz,1H),2.06(d,J＝9.9Hz,6H),1.89–1.76(m,2H),1.62(s,4H),1.49–1.40(m,2H),1.10(t,J＝9.8Hz,1H),0.94(s,3H),0.89(s,3H),0.88(s,1H),0.74–0.63(m,1H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 39H 47F 3N 2NaO 8S)requires m/z 783.2903,found m/z783.2897,difference 0.08ppm.
实施例58
化合物E-6的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-6(14mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2 当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-6，收率33％。
Figure PCTCN2021141536-appb-000142
化合物E-6的结构测定： 1H NMR(500MHz,CDCl 3)δ7.85(d,J＝8.2Hz,2H),7.34(d,J＝8.0Hz,2H),7.06(d,J＝9.4Hz,1H),6.99(dd,J＝5.4,3.4Hz,1H),6.98–6.92(m,3H),6.71(s,1H),4.48–4.40(m,2H),4.31(dd,J＝17.9,1.5Hz,1H),4.23(d,J＝11.9Hz,1H),4.05(dd,J＝18.0,1.6Hz,1H),3.68(d,J＝13.1Hz,1H),3.64(dd,J＝11.0,7.0Hz,1H),3.42(d,J＝13.0Hz,1H),3.08–2.99(m,1H),2.87(d,J＝11.9Hz,1H),2.43(s,3H),2.14(s,3H),2.06(d,J＝9.1Hz,6H),2.03–1.99(m,1H),1.94–1.86(m,1H),1.86–1.78(m,1H),1.61(s,3H),1.53–1.43(m,3H),1.08–1.04(m,1H),0.95(s,3H),0.89(s,3H),0.82(s,1H),0.67–0.56(m,1H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 39H 50N 2NaO 8S)requires m/z 729.3186,found m/z 729.3180,difference-0.08ppm.
实施例59
化合物E-7的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-7(14mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-7，收率33％。
Figure PCTCN2021141536-appb-000143
化合物E-7的结构测定： 1H NMR(500MHz,CDCl 3)δ7.84(d,J＝8.2Hz,2H),7.35(d,J＝8.0Hz,2H),7.12–7.05(m,1H),7.03(s,1H),6.96–6.90(m,2H),6.86–6.79(m,1H),6.76(d,J＝9.5Hz,1H),4.48–4.37(m,3H),4.23(dd,J＝12.8,6.8Hz,2H),3.72–3.64(m,1H),3.61(d,J＝13.5Hz,1H),3.48(d,J＝13.6Hz,1H),3.00(dd,J＝11.8,9.8Hz,1H),2.73(d,J＝11.9Hz,1H),2.43(s,3H),2.06(d,J＝8.0Hz,7H),1.99(d,J＝13.4Hz,1H),1.86–1.75(m,2H),1.63(s,4H),1.49–1.40(m,2H),1.07(t,J＝9.5Hz,1H),0.94(s,3H),0.89(s,3H),0.86(dd,J＝11.1,7.5Hz,1H),0.68–0.54(m,1H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 38H 47FN 2NaO 8S)requires m/z 733.2935,found m/z733.2929,difference 0.08ppm.
实施例60
化合物E-8的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-8(14mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-8，收率36％。
Figure PCTCN2021141536-appb-000144
化合物E-8的结构测定： 1H NMR(300MHz,CDCl 3)δ7.83(d,J＝8.2Hz, 2H),7.34(d,J＝8.0Hz,2H),6.95(d,J＝9.1Hz,1H),6.93–6.82(m,3H),6.62(d,J＝8.5Hz,2H),4.56–4.30(m,4H),4.23(t,J＝11.0Hz,1H),3.74(s,3H),3.64(dd,J＝11.8,5.8Hz,2H),3.28(d,J＝13.4Hz,1H),3.04–2.87(m,1H),2.60(d,J＝11.9Hz,1H),2.43(s,3H),2.06(d,J＝5.4Hz,7H),1.84–1.74(m,2H),1.61(s,4H),1.46(s,2H),1.05(s,1H),0.94(s,3H),0.87(s,3H),0.84–0.75(m,1H),0.67–0.51(m,1H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 39H 50N 2NaO 9S)requires m/z 745.3135,found m/z745.3130,difference 0.08ppm.
实施例61
化合物E-9的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-9(15mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-9，收率45％。
Figure PCTCN2021141536-appb-000145
化合物E-9的结构测定： 1H NMR(400MHz,CDCl 3)δ7.81(d,J＝7.8Hz,2H),7.34(d,J＝7.9Hz,2H),7.03(d,J＝10.8Hz,2H),6.93–6.81(m,2H),6.78(t,J＝8.5Hz,1H),4.58(d,J＝18.2Hz,1H),4.50–4.34(m,3H),4.22(d,J＝11.7Hz,1H),3.65(d,J＝14.4Hz,2H),3.29(d,J＝13.8Hz,1H),2.98(t,J＝10.7Hz,1H),2.49(d,J＝11.7Hz,1H),2.43(s,3H),2.12(d,J＝12.1Hz,1H),2.06(d,J＝8.0Hz,7H),1.86–1.77(m,2H),1.62(s,4H),1.46(d,J＝10.6Hz,2H),1.10–1.05(m,1H),0.94(s,3H),0.88(s,3H),0.83(s,1H),0.67(dd,J＝22.7,7.5Hz,1H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 38H 46ClFN 2NaO 8S) requires m/z 767.2545,found m/z767.2539,difference 0.07ppm.
实施例62
化合物E-10的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-10(13mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-10，收率50％。
Figure PCTCN2021141536-appb-000146
化合物E-10的结构测定： 1H NMR(400MHz,CDCl 3)δ7.80(d,J＝8.2Hz,2H),7.30(d,J＝8.1Hz,2H),7.24(s,1H),6.74(d,J＝9.7Hz,1H),4.78(s,2H),4.43(dd,J＝17.1,10.1Hz,2H),4.19(d,J＝11.8Hz,1H),3.54(dd,J＝11.1,6.6Hz,1H),3.00–2.93(m,1H),2.69(d,J＝11.6Hz,1H),2.42(s,3H),2.34–2.28(m,1H),2.21(d,J＝7.6Hz,1H),2.04(t,J＝7.2Hz,7H),1.95(dd,J＝13.7,7.4Hz,2H),1.81–1.70(m,3H),1.48–1.42(m,3H),1.05(t,J＝9.2Hz,2H),0.94(s,3H),0.88(d,J＝3.2Hz,2H),0.84(s,4H),0.59(dd,J＝6.3,2.3Hz,6H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 35H 50N 2NaO 8S)requires m/z 681.3186,found m/z681.3180,difference 0.07ppm.
实施例63
化合物E-11的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-11(15mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2 当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-11，收率33％。
Figure PCTCN2021141536-appb-000147
化合物E-11的结构测定： 1H NMR(400MHz,CDCl 3)δ7.84(d,J＝8.1Hz,2H),7.34(d,J＝8.1Hz,2H),7.12–7.00(m,2H),6.97(d,J＝4.3Hz,2H),6.89(dd,J＝19.8,8.4Hz,2H),4.56–4.36(m,3H),4.26(dd,J＝29.3,15.0Hz,2H),3.68(dd,J＝17.7,10.3Hz,2H),3.29(d,J＝13.7Hz,1H),3.07–2.91(m,1H),2.62(d,J＝11.9Hz,1H),2.43(s,3H),2.06(d,J＝7.4Hz,7H),1.80(dd,J＝11.5,6.8Hz,2H),1.61(s,3H),1.53(d,J＝11.0Hz,1H),1.48–1.42(m,2H),1.08(d,J＝9.8Hz,1H),0.94(s,3H),0.88(s,3H),0.81(dd,J＝20.9,6.7Hz,1H),0.71–0.56(m,1H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 38H 47ClN 2NaO 8S)requires m/z 749.2639,found m/z749.2634,difference 0.06ppm.
实施例64
化合物E-12的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-12(13mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-12，收率32％。
Figure PCTCN2021141536-appb-000148
化合物E-12的结构测定： 1H NMR(400MHz,CDCl 3)δ7.80(d,J＝8.2Hz,2H),7.32(d,J＝8.1Hz,2H),7.28(s,1H),6.54(d,J＝8.9Hz,1H),4.93–4.71(m,2H),4.43(dd,J＝14.1,7.0Hz,2H),4.20(d,J＝11.9Hz,1H),3.74(dd,J＝11.4,6.6Hz,1H),3.04–2.87(m,1H),2.79(d,J＝11.5Hz,1H),2.42(s,3H),2.05(d,J＝3.4Hz,6H),2.02(s,1H),1.88–1.71(m,2H),1.60(s,4H),1.54(d,J＝6.9Hz,1H),1.40(dd,J＝26.1,12.3Hz,2H),1.13–1.05(m,1H),0.95(s,3H),0.87(s,4H),0.67–0.56(m,1H),0.14(t,J＝6.3Hz,2H),-0.08–-0.21(m,2H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 34H 46N 2NaO 8S)requires m/z 665.2873,found m/z665.2868,difference 0.14ppm.
实施例65
化合物E-13的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-13(13mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-13，收率38％。
Figure PCTCN2021141536-appb-000149
化合物E-13的结构测定： 1H NMR(400MHz,CDCl 3)δ7.84(d,J＝8.2Hz,2H),7.31(d,J＝8.0Hz,2H),7.25(s,1H),7.02(d,J＝9.1Hz,1H),4.86–4.71(m, 2H),4.47–4.36(m,2H),4.19(d,J＝11.9Hz,1H),4.05(dd,J＝10.6,6.8Hz,1H),3.02–2.90(m,1H),2.89–2.79(m,1H),2.67(d,J＝11.8Hz,1H),2.41(s,3H),2.26–2.18(m,1H),2.05(d,J＝3.3Hz,6H),2.00(s,1H),1.59(s,4H),1.46–1.36(m,3H),1.08–1.01(m,1H),0.98(d,J＝6.7Hz,3H),0.93(s,3H),0.88(t,J＝5.1Hz,2H),0.83(s,3H),0.66(d,J＝6.9Hz,3H),0.62–0.53(m,1H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 34H 48N 2NaO 8S)requires m/z 667.3029,found m/z 667.3025,difference 0.20ppm.
实施例66
化合物E-14的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-14(14mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-14，收率32％。
Figure PCTCN2021141536-appb-000150
化合物E-14的结构测定： 1H NMR(300MHz,CDCl 3)δ7.83(d,J＝8.2Hz,2H),7.35(d,J＝8.0Hz,2H),6.99(d,J＝9.1Hz,1H),6.87(dd,J＝18.6,8.0Hz,3H),6.59(d,J＝8.4Hz,2H),4.62–4.25(m,4H),4.21(d,J＝11.9Hz,1H),3.64(dd,J＝15.8,5.3Hz,2H),3.26(d,J＝13.4Hz,1H),3.07–2.85(m,1H),2.65(d,J＝11.8Hz,1H),2.43(s,3H),2.06(d,J＝5.0Hz,6H),1.99(d,J＝11.1Hz,1H),1.86–1.74(m,2H),1.63(s,4H),1.42(d,J＝11.9Hz,2H),1.06–1.00(m,1H),0.94(s,3H),0.86(s,3H),0.81(d,J＝8.7Hz,1H),0.63–0.46(m,1H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 38H 48N 2NaO 9S)requires m/z 731.2978, found m/z731.2973,difference 0.04ppm.
实施例67
化合物E-15的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-15(12mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-15，收率33％。
Figure PCTCN2021141536-appb-000151
化合物E-15的结构测定： 1H NMR(400MHz,CDCl 3)δ7.23(d,J＝7.2Hz,2H),7.21–7.15(m,3H),7.08(s,1H),6.67(d,J＝9.9Hz,1H),4.63–4.51(m,2H),4.49–4.37(m,2H),4.29(d,J＝11.9Hz,1H),3.86–3.69(m,2H),3.47(d,J＝13.8Hz,1H),3.00(t,J＝10.5Hz,1H),2.86(s,3H),2.49(d,J＝11.2Hz,1H),2.26–2.14(m,1H),2.08(s,3H),2.03(s,3H),1.98(d,J＝10.4Hz,2H),1.92–1.81(m,1H),1.76–1.69(m,2H),1.62(s,3H),1.32(d,J＝9.5Hz,2H),1.18–1.12(m,1H),0.97(d,J＝6.7Hz,6H),0.83–0.75(m,1H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 32H 44N 2NaO 8S)requires m/z 639.2716,found m/z639.2712,difference 0.28ppm.
实施例68
化合物E-15的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-16(12mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2 当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-16，收率50％。
Figure PCTCN2021141536-appb-000152
化合物E-16的结构测定： 1H NMR(300MHz,CDCl 3)δ7.29(d,J＝1.1Hz,1H),7.22(s,1H),6.22(dt,J＝13.0,6.5Hz,2H),6.04(d,J＝3.1Hz,1H),4.63(s,2H),4.60–4.50(m,1H),4.45(d,J＝11.9Hz,1H),4.26(d,J＝11.9Hz,1H),3.81–3.67(m,2H),3.50(d,J＝15.0Hz,1H),3.06(t,J＝10.5Hz,1H),2.92(s,3H),2.61(d,J＝11.2Hz,1H),2.07(s,3H),2.03(s,3H),1.99–1.90(m,2H),1.91–1.79(m,3H),1.74–1.66(m,4H),1.32(d,J＝8.8Hz,1H),1.19–1.09(m,1H),0.95(d,J＝2.8Hz,6H),0.90–0.79(m,1H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 30H 42N 2NaO 9S)requires m/z 629.2509,found m/z629.2504,difference 0.19ppm.
实施例69
化合物E-17的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-17(13mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-17，收率43％。
Figure PCTCN2021141536-appb-000153
化合物E-17的结构测定： 1H NMR(300MHz,CDCl 3)δ7.21(dd,J＝7.0,1.8Hz,1H),7.16(s,1H),7.11–7.07(m,1H),7.03(t,J＝8.5Hz,1H),6.77(d,J＝9.8Hz,1H),4.74–4.51(m,3H),4.45(d,J＝11.9Hz,1H),4.28(d,J＝12.0Hz,1H),3.79–3.73(m,1H),3.41(d,J＝14.0Hz,1H),3.16–3.03(m,1H),2.96(s,3H),2.51(d,J＝11.3Hz,1H),2.30–2.17(m,1H),2.08(s,3H),2.04(s,3H),2.01–1.79(m,4H),1.73(d,J＝7.7Hz,2H),1.59–1.49(m,2H),1.16–1.10(m,2H),0.96(s,6H),0.86–0.81(m,2H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 32H 42ClFN 2NaO 8S)requires m/z 691.2232,found m/z 691.2226,difference 0.29ppm.
实施例70
化合物E-18的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-18(11mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-18，收率43％。
Figure PCTCN2021141536-appb-000154
化合物E-18的结构测定： 1H NMR(400MHz,CDCl 3)δ7.56(s,1H),6.99(s,1H),4.96–4.72(m,3H),4.61–4.50(m,2H),4.35–4.22(m,2H),4.18–4.02(m,2H),3.12(s,3H),2.20–2.16(m,2H),2.09–2.04(m,8H),1.79(dd,J＝19.2,10.9Hz,3H),1.19(s,2H),0.99(s,3H),0.84(d,J＝9.1Hz,2H),0.79(s,3H),0.63–0.35(m,5H).HRMS(ESI)exact mass calculated for[M+Na] +(C 28H 42N 2O 8SNa)requires m/z 589.2560,found m/z 589.2554,difference-0.06ppm.
实施例71
化合物E-19的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-19(13mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物E-19，收率33％。
Figure PCTCN2021141536-appb-000155
化合物E-19的结构测定： 1H NMR(400MHz,CDCl 3)δ7.20–7.14(m,2H),7.08(s,1H),6.93(t,J＝8.6Hz,2H),6.73(d,J＝9.9Hz,1H),4.64–4.52(m,2H),4.48–4.46(m,1H),4.44(s,1H),4.28(d,J＝11.9Hz,1H),3.83–3.76(m,1H),3.44(d,J＝13.8Hz,1H),3.05–2.99(m,1H),2.91(s,3H),2.52(d,J＝11.2Hz,1H),2.30–2.12(m,1H),2.08(s,3H),2.03(s,3H),1.97(d,J＝10.6Hz,2H),1.92–1.81(m,1H),1.76–1.70(m,2H),1.56(d,J＝7.6Hz,1H),1.36–1.30(m,2H),1.18–1.09(m,2H),0.96(d,J＝2.7Hz,6H),0.83–0.72(m,2H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 32H 43FN 2NaO 8S)requires m/z 657.2622,found m/z657.2616,difference 0.05ppm.
实施例72
化合物E-20的制备：
在8mL的样品瓶中，将双氨基化的穿心莲内酯化合物D-20(13mg，0.02mmol)溶解在0.5mL乙腈溶剂中，再加入三乙胺(3.3μL，0.024mmol，1.2当量)，65℃下搅拌22h。反应结束后，浓缩反应物，制备薄层色谱板分离纯 化得到产物E-20，收率45％。
Figure PCTCN2021141536-appb-000156
化合物E-20的结构测定： 1H NMR(400MHz,CDCl 3)δ7.12–7.04(m,3H),6.76(d,J＝8.6Hz,2H),6.70(d,J＝9.7Hz,1H),4.63–4.53(m,2H),4.50–4.45(m,1H),4.44(s,1H),4.28(d,J＝11.9Hz,1H),3.75(s,4H),3.41(d,J＝13.7Hz,1H),2.99(t,J＝10.4Hz,1H),2.88(s,3H),2.51(d,J＝11.1Hz,1H),2.18(dd,J＝24.6,11.0Hz,1H),2.07(s,3H),2.03(s,3H),1.99–1.91(m,2H),1.90–1.82(m,1H),1.73(d,J＝6.8Hz,1H),1.59(d,J＝7.9Hz,2H),1.38–1.31(m,2H),1.17–1.09(m,2H),0.96(d,J＝3.5Hz,6H),0.84–0.77(m,2H).；HRMS(ESI)exact mass calculated for[M+Na] +(C 33H 46N 2NaO 9S)requires m/z 669.2822,found m/z669.2817,difference 0.08ppm.
实施例73
化合物F-1的制备：
在8mL的样品瓶中，将环氨基化的穿心莲内酯化合物B-1(13mg，0.02mmol)溶解在0.5mL甲醇溶剂中，再加入Sc(OTf) 3(1mg,10mol％)，40℃下搅拌24h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物F-1，收率83％。
Figure PCTCN2021141536-appb-000157
化合物F-1的结构测定： 1H NMR(400MHz,CDCl 3)δ7.71(d,J＝8.2Hz,2H),7.46(d,J＝1.4Hz,1H),7.30(d,J＝8.1Hz,2H),5.42(s,1H),4.85(dd,J＝6.9,5.1Hz,1H),4.35(dd,J＝19.1,7.0Hz,3H),4.11(d,J＝11.8Hz,1H),3.93(dd,J＝17.3,1.9Hz,1H),2.49(s,3H),2.17–2.09(m,1H),2.04(d,J＝3.0Hz,6H),2.03(s,1H),2.00–1.90(m,1H),1.75–1.67(m,1H),1.62–1.54(m,2H),1.28(s,2H),1.20–1.09(m,1H),0.93(s,3H),0.79(dd,J＝11.9,5.0Hz,1H),0.75(s,1H),0.62(s,3H),0.48(d,J＝14.3Hz,1H).HRMS(m/z)calc.forC31H39NNaO8S:[M+Na] +,608.2289；found 608.2283.
实施例74
化合物F-2的制备：
在8mL的样品瓶中，将环氨基化的穿心莲内酯化合物B-2(13mg，0.02mmol)溶解在0.5mL甲醇溶剂中，再加入Sc(OTf) 3(1mg,10mol％)，40℃下搅拌24h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物F-2，收率27％。
Figure PCTCN2021141536-appb-000158
化合物F-2的结构测定： 1H NMR(300MHz,CDCl 3)δ7.88–7.78(m,1H),7.40(s,1H),7.03–6.93(m,2H),5.40(s,1H),4.79(d,J＝7.1Hz,2H),4.67(dd,J＝11.1,6.5Hz,1H),4.49(dd,J＝11.2,5.0Hz,1H),4.38(d,J＝11.9Hz,1H),4.25(d,J＝17.8Hz,1H),4.14(d,J＝11.9Hz,2H),2.12(d,J＝6.3Hz,1H),2.05(d,J＝2.0Hz,6H),1.80(dd,J＝13.3,3.1Hz,1H),1.73–1.64(m,3H),1.47–1.36(m,2H),1.09(dd,J＝11.3,5.5Hz,3H),0.95(s,3H),0.70(s,3H).HRMS(m/z)calc.forC30H35F2NO8SNa:[M+Na] +,630.1944；found 630.1945.
实施例75
化合物F-3的制备：
在8mL的样品瓶中，将环氨基化的穿心莲内酯化合物B-3(13mg，0.02mmol)溶解在0.5mL甲醇溶剂中，再加入Sc(OTf) 3(1mg,10mol％)，40℃下搅拌24h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物F-3，收率16％。
Figure PCTCN2021141536-appb-000159
化合物F-3的结构测定： 1H NMR(500MHz,CDCl 3)δ7.77(d,J＝8.6Hz,2H),7.48(d,J＝8.6Hz,2H),7.45(d,J＝1.4Hz,1H),5.42(s,1H),4.91–4.79(m,2H),4.36–4.28(m,2H),4.13(d,J＝11.9Hz,1H),3.98(d,J＝15.1Hz,1H),2.13(ddd,J＝13.2,6.3,2.7Hz,1H),2.05(d,J＝1.5Hz,6H),1.99(dd,J＝28.5,13.2Hz,2H),1.75–1.71(m,1H),1.68–1.64(m,1H),1.47–1.42(m,1H),1.33(dd,J＝14.2,6.7Hz,2H),0.97(s,3H),0.91–0.80(m,4H),0.64(s,3H)；HRMS(m/z)calc.for C30H36ClNNaO8S:[M+Na] +,628.1742；found 628.1744.
实施例76
化合物F-4的制备：
在8mL的样品瓶中，将环氨基化的穿心莲内酯化合物B-4(14mg，0.02mmol)溶解在0.5mL甲醇溶剂中，再加入Sc(OTf) 3(1mg,10mol％)，40℃下搅拌24h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物F-4，收率30％。
Figure PCTCN2021141536-appb-000160
化合物F-4的结构测定： 1H NMR(400MHz,CDCl 3)δ8.34(d,J＝8.9Hz,2H),8.00(d,J＝8.9Hz,2H),7.45(d,J＝1.2Hz,1H),5.49(s,1H),4.84(q,J＝18.3Hz,3H),4.34(dd,J＝14.4,5.0Hz,3H),4.14–4.08(m,2H),2.27–2.20(m,1H),2.10(dd,J＝6.2,3.1Hz,1H),2.04(d,J＝3.2Hz,6H),1.73(dd,J＝14.3,4.8Hz,3H),1.46–1.32(m,5H),0.91(s,3H),0.67(s,3H)；HRMS(m/z)calc.for C30H36N2NaO10S:[M+Na] +,639.1983；found 639.1984.
实施例77
化合物F-5的制备：
在8mL的样品瓶中，将环氨基化的穿心莲内酯化合物B-5(15mg，0.02mmol)溶解在0.5mL甲醇溶剂中，再加入Sc(OTf) 3(1mg,10mol％)，40℃下搅拌24h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物F-5，收率20％。
Figure PCTCN2021141536-appb-000161
化合物F-5的结构测定： 1H NMR(500MHz,CDCl 3)δ7.87(d,J＝8.6Hz,2H),7.54(d,J＝8.6Hz,2H),7.45(d,J＝1.4Hz,1H),5.42(s,1H),4.86(dt,J＝29.9,17.3Hz,2H),4.36–4.28(m,2H),4.13(d,J＝11.9Hz,1H),3.96(d,J＝17.6Hz,1H),2.15–2.09(m,1H),2.05(d,J＝1.7Hz,6H),1.75–1.65(m,3H),1.48– 1.42(m,1H),1.24–1.16(m,2H),1.03(s,3H),0.97(t,J＝7.4Hz,1H),0.91–0.76(m,4H),0.64(s,3H)；HRMS(m/z)calc.for C30H36INNaO8S:[M+Na] +,720.1099；found 720.1100.
实施例78
化合物F-6的制备：
在8mL的样品瓶中，将环氨基化的穿心莲内酯化合物B-6(14mg，0.02mmol)溶解在0.5mL甲醇溶剂中，再加入Sc(OTf) 3(1mg,10mol％)，40℃下搅拌24h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物F-6，收率47％。
Figure PCTCN2021141536-appb-000162
化合物F-6的结构测定： 1H NMR(500MHz,CDCl 3)δ7.93(d,J＝8.8Hz,2H),7.42(d,J＝8.2Hz,2H),7.06(s,1H),5.80(d,J＝5.5Hz,1H),4.52(ddd,J＝19.5,11.6,5.4Hz,2H),4.32(d,J＝11.8Hz,1H),4.25(dd,J＝11.2,1.5Hz,1H),4.09(d,J＝11.8Hz,1H),3.58(d,J＝10.3Hz,1H),3.41(s,1H),2.25–2.21(m,2H),2.16(s,3H),2.08(s,3H),2.04(s,3H),1.77–1.65(m,5H),1.38(s,2H),1.12(ddd,J＝37.7,28.0,12.2Hz,6H),1.00(s,3H),0.83(s,3H)；HRMS(m/z)calc.for C 35H 44F 3N 2O 11S:[M+H] +,757.2612；found 757.2614.
实施例79
化合物F-7的制备：
在8mL的样品瓶中，将环氨基化的穿心莲内酯化合物B-7(13mg，0.02mmol)溶解在0.5mL甲醇溶剂中，再加入Sc(OTf) 3(1mg,10mol％)，40℃ 下搅拌24h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物F-7，收率35％。
Figure PCTCN2021141536-appb-000163
化合物F-7的结构测定： 1H NMR(300MHz,CDCl 3)δ7.75(d,J＝8.9Hz,2H),7.46(d,J＝1.5Hz,1H),6.95(d,J＝8.9Hz,2H),5.40(s,1H),4.84(d,J＝6.3Hz,2H),4.39–4.27(m,4H),4.11(dd,J＝9.2,5.2Hz,2H),3.92(s,3H),2.05–2.03(m,8H),1.73(d,J＝7.8Hz,2H),1.44(d,J＝8.0Hz,1H),0.93(s,3H),0.88–0.75(m,5H),0.62(s,3H)；HRMS(m/z)calc.for C 31H 39NO 9SNa:[M+Na] +,624.2238；found 624.2238.
实施例80
化合物F-9的制备：
在8mL的样品瓶中，将环氨基化的穿心莲内酯化合物B-9(14mg，0.02mmol)溶解在0.5mL甲醇溶剂中，再加入Sc(OTf) 3(1mg,10mol％)，40℃下搅拌24h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物F-9，收率35％。
Figure PCTCN2021141536-appb-000164
化合物F-9的结构测定： 1H NMR(300MHz,CDCl 3)δ8.64(t,J＝1.9Hz,1H),8.48(dd,J＝8.2,1.2Hz,1H),8.17(d,J＝7.8Hz,1H),7.77(t,J＝8.0Hz, 1H),7.49(d,J＝1.3Hz,1H),5.61(s,1H),4.87(d,J＝1.6Hz,2H),4.35–4.30(m,2H),4.11(dd,J＝9.5,7.0Hz,2H),2.25–2.19(m,1H),2.17(s,3H),2.04(s,3H),2.02–1.97(m,2H),1.67(s,2H),1.33(s,3H),0.88(s,3H),0.83–0.70(m,4H),0.65(s,3H).HRMS(m/z)calc.for C30H36N2NaO10S:[M+Na] +,639.1983；found 639.1984.
实施例81
化合物F-10的制备：
在8mL的样品瓶中，将环氨基化的穿心莲内酯化合物B-10(14mg，0.02mmol)溶解在0.5mL甲醇溶剂中，再加入Sc(OTf) 3(1mg,10mol％)，40℃下搅拌24h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物F-10，收率29％。
Figure PCTCN2021141536-appb-000165
化合物F-10的结构测定： 1H NMR(300MHz,CDCl 3)δ7.96(d,J＝8.2Hz,2H),7.77(d,J＝8.3Hz,2H),7.45(d,J＝1.3Hz,1H),5.43(s,1H),4.93–4.77(m,2H),4.37–4.27(m,3H),4.11(d,J＝11.9Hz,1H),4.02(d,J＝15.9Hz,1H),2.26–2.18(m,1H),2.13–2.06(m,2H),2.03(s,6H),1.76–1.65(m,3H),1.44–1.40(m,1H),0.90(s,3H),0.85–0.70(m,4H),0.63(s,3H).HRMS(m/z)calc.for C31H36F3NNaO8S:[M+Na] +,662.2006；found 662.2008.
实施例82
化合物F-11的制备：
在8mL的样品瓶中，将环氨基化的穿心莲内酯化合物B-11(13mg，0.02 mmol)溶解在0.5mL甲醇溶剂中，再加入Sc(OTf) 3(1mg,10mol％)，40℃下搅拌24h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物F-11，收率30％。
Figure PCTCN2021141536-appb-000166
化合物F-11的结构测定： 1H NMR(300MHz,CDCl 3)δ7.85–7.81(m,2H),7.44(d,J＝1.4Hz,1H),7.16(d,J＝8.5Hz,2H),5.43(s,1H),4.91–4.76(m,2H),4.30(dd,J＝24.9,12.2Hz,3H),4.12(d,J＝11.9Hz,1H),3.98(d,J＝17.1Hz,1H),2.25–2.19(m,1H),2.04(d,J＝1.5Hz,6H),1.99(d,J＝6.7Hz,1H),1.76–1.65(m,4H),1.45–1.40(m,1H),0.94(s,3H),0.90–0.84(m,4H),0.65(s,3H)；HRMS(m/z)calc.for C30H36FNNaO8S:[M+Na] +,612.2038；found 612.2040.
实施例83
化合物F-12的制备：
在8mL的样品瓶中，将环氨基化的穿心莲内酯化合物B-12(13mg，0.02mmol)溶解在0.5mL甲醇溶剂中，再加入Sc(OTf) 3(1mg,10mol％)，40℃下搅拌24h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物F-12，收率40％。
Figure PCTCN2021141536-appb-000167
化合物F-12的结构测定： 1H NMR(400MHz,CDCl 3)δ7.84–7.81(m,2H), 7.65(t,J＝7.5Hz,1H),7.50(t,J＝7.8Hz,2H),7.46(d,J＝1.4Hz,1H),5.44(s,1H),4.85(dd,J＝7.4,5.5Hz,2H),4.40–4.31(m,4H),4.11(d,J＝11.9Hz,1H),3.97(d,J＝19.4Hz,1H),2.15–2.09(m,2H),2.04(d,J＝2.6Hz,6H),1.73–1.68(m,2H),1.37(d,J＝11.2Hz,2H),1.20(d,J＝11.9Hz,3H),1.11–1.06(m,1H),0.92(s,3H),0.63(s,3H)；HRMS(m/z)calc.for C30H37NO8SNa:[M+Na] +,594.2132；found 594.2134.
实施例84
化合物F-13的制备：
在8mL的样品瓶中，将环氨基化的穿心莲内酯化合物B-13(11mg，0.02mmol)溶解在0.5mL甲醇溶剂中，再加入Sc(OTf) 3(1mg,10mol％)，40℃下搅拌24h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物F-13，收率51％。
Figure PCTCN2021141536-appb-000168
化合物F-13的结构测定： 1H NMR(300MHz,CDCl 3)δ7.41(d,J＝1.3Hz,1H),5.58(s,1H),4.83(dd,J＝10.4,1.6Hz,3H),4.65–4.59(m,1H),4.43(d,J＝11.8Hz,1H),4.32–4.27(m,1H),4.24–4.16(m,2H),2.85(s,3H),2.07–2.05(m,6H),1.92(d,J＝13.8Hz,3H),1.76(dd,J＝14.7,4.5Hz,5H),1.52(d,J＝5.3Hz,2H),1.01(s,3H),0.79(s,3H).HRMS(m/z)calc.for C25H35NNaO8S:[M+Na] +,532.1976；found 532.1976.
实施例85
化合物F-15的制备：
在8mL的样品瓶中，将环氨基化的穿心莲内酯化合物B-15(14mg，0.02mmol)溶解在0.5mL甲醇溶剂中，再加入Sc(OTf) 3(1mg,10mol％)，40℃下搅拌24h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物F-15，收率44％。
Figure PCTCN2021141536-appb-000169
化合物F-15的结构测定： 1H NMR(400MHz,CDCl 3)δ7.86(s,1H),7.78(d,J＝8.8Hz,2H),7.71(d,J＝8.8Hz,2H),7.48(d,J＝1.4Hz,1H),5.44(s,1H),4.86(d,J＝10.4Hz,2H),4.41–4.31(m,4H),4.11–4.06(m,1H),3.90(d,J＝17.5Hz,1H),2.25(s,1H),2.23(s,3H),2.15(dd,J＝13.7,4.0Hz,1H),2.06(d,J＝1.5Hz,3H),2.04(s,3H),2.03(s,1H),1.93(s,1H),1.73(d,J＝13.5Hz,1H),1.56(d,J＝3.9Hz,1H),1.41(dd,J＝14.7,7.1Hz,1H),1.16–1.05(m,2H),0.96(t,J＝6.5Hz,1H),0.91(s,3H),0.62(s,3H)；HRMS(m/z)calc.for C32H40N2O9SNa:[M+Na] +,651.2347；found 651.2348.
实施例86
化合物F-16的制备：
在8mL的样品瓶中，将环氨基化的穿心莲内酯化合物B-16(14mg，0.02mmol)溶解在0.5mL甲醇溶剂中，再加入Sc(OTf) 3(1mg,10mol％)，40℃下搅拌24h。反应结束后，浓缩反应物，制备薄层色谱板分离纯化得到产物F-16，收率39％。
Figure PCTCN2021141536-appb-000170
化合物F-16的结构测定： 1H NMR(400MHz,CDCl 3)δ7.78–7.73(m,2H),7.54–7.50(m,2H),7.48(d,J＝1.4Hz,1H),5.48(s,1H),4.95–4.78(m,2H),4.46–4.32(m,4H),4.14(d,J＝11.8Hz,1H),3.96(d,J＝17.5Hz,1H),2.18–2.09(m,2H),2.05(d,J＝4.0Hz,6H),1.97(d,J＝16.2Hz,1H),1.73(dt,J＝6.5,3.1Hz,1H),1.59(dd,J＝13.0,6.3Hz,3H),1.40(s,9H),1.26(d,J＝11.3Hz,2H),1.22–1.15(m,1H),0.94(s,3H),0.65(s,3H)；HRMS(m/z)calc.forC34H45NO8SNa:[M+Na] +,650.2758；found 650.2760.
实施例87
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、肺癌、宫颈癌、乳腺癌以及肝癌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。选取的细胞系分别为：人EBV阳性鼻咽癌细胞C666-1、非小细胞肺癌A549、人宫颈癌细胞HeLa、人乳腺癌细胞MCF-7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，A549、HeLa、MCF7和Huh 7为2000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。
表1：穿心莲内酯衍生物对肿瘤细胞系的抗增殖活性
Figure PCTCN2021141536-appb-000171
Figure PCTCN2021141536-appb-000172
Figure PCTCN2021141536-appb-000173
其中，肿瘤细胞抑制活性：肿瘤抑制率>80％＝++++；80％>肿瘤抑制率>60％＝+++，60％>肿瘤抑制率>30％＝++，肿瘤抑制率<30％＝+，ND表示没有测试该细胞系。
由表1可以看出，这种氮杂螺环、多环穿心莲内酯类化合物表现出优良的抗肿瘤活性，部分化合物对肿瘤的抑制作用较穿心莲内酯具有显著提高。
实施例88
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细胞和人冠状病毒HCoV 229E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，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，并且添加了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％CO2、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200TCID50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％CO2、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。代表性的穿心莲内酯衍生物对人冠状病毒HCoV-229E的抑制活性见下表2。
表2：穿心莲内酯衍生物体外抗人冠状病毒HCoV-229E的活性
Figure PCTCN2021141536-appb-000174
Figure PCTCN2021141536-appb-000175
其中，25微摩尔浓度下对人冠状病毒HCoV-229E的抑制活性：抑制率>70％＝++++；70％>抑制率>50％＝+++；50％>抑制率>30％＝++；肿瘤抑制率<30％＝+。
由表2可以看出，穿心莲内酯衍生物中部分化合物表现出较好的对人冠状病毒HCoV-229E的抑制活性，其活性明显高于阳性对照穿心莲内酯。
实施例89
应用细胞免疫荧光实验评价化合物体外抗新型冠状病毒活性
受试化合物使用DMSO溶液配置为20mM储存液。测试化合物起始测试浓度为100μM，测试10个浓度，3倍梯度稀释，双复孔。瑞德西韦(Remdesivir)和氯喹(Chloroquine)用作对照化合物。对照化合物测试10个浓度，2倍梯度稀释，双复孔，起始测试浓度为50μM。
非洲绿猴肾(Vero)细胞来自美国典型培养物保藏中心(ATCC)。细胞于添加了10％胎牛血清(Gibco)和1％双抗(Gibco)的DMEM(WelGene)培养液中培养。添加了2％胎牛血清和1％双抗的DMEM培养液用作实验培养液。新型冠状病毒βCoV/KOR/KCDC03/2020株由韩国疾控中心(Korea Centers for Disease Control and Prevention，KCDC)提供，序列号NCCP43326。
Vero细胞经胰酶消化后，用实验培养液稀释后使用自动分液器加入到384孔细胞测试板中，每孔25μl，12,000个细胞。细胞于5％CO2、37℃培养箱中培养过夜。第二天，化合物使用DMSO进行稀释，然后使用液体工作站将稀释的化合物加入测试细胞孔中。然后每孔加入25μl实验培养液稀释后SARS-CoV-2病毒，MOI＝0.0125。设置细胞对照(细胞，无化合物处理或病毒感染)和无化合物处理对照(细胞感染病毒，无化合物处理，加入0.5％DMSO)。每孔细胞培养液终体积为50μl。细胞于5％CO2、37℃培养箱中继续培养24小时。病毒感染24小时后，每孔加入17μl 16％多聚甲醛。然后于室温放置30分钟，吸去上清，使用DPBS洗板二次。每孔加入25μl 0.25％TritonX-100，室温放置20分钟；吸去0.25％TritonX-100，使用DPBS洗板二次。每孔加入25μl稀释后的一抗(1:3000倍稀释)，37℃孵育1个小时，吸去一抗，使用DPBS洗板二次，每孔加入25μl稀释后的二抗Alexa Fluor 488标记的羊抗兔 IgG(1:2000倍稀释)和2.5μg/ml(1:4000倍稀释)的Hoechst 33342，37℃孵育1个小时。吸去二抗和Hoechst，使用DPBS洗板二次，使用高内涵成像分析仪Operetta读板，仪器设定：488/405emission,20倍物镜，每个孔5个视野。
使用Columbus软件定量分析高内涵成像分析仪读板所得的图片中的细胞总数(Hoechst染色细胞数量)和新型冠状病毒感染细胞数(Alexa Fluor 488标记的细胞数量)。感染细胞比例和细胞总数数据用于化合物抗病毒活性和细胞毒性分析，结果见下表3，图1、图2和图3。
表3 穿心莲内酯衍生物体外抗新型冠状病毒(SARS-CoV-2)的活性和细胞毒性
序号 化合物编号 EC 50(μM) CC 50(μM)
1 D-1 2.8 97.9
2 Chloroquine 4.3 >150
3 Remdesivir 5.8 >50
结合表3、图1、图2以及图3，可以看出，穿心莲内酯衍生物D-1表现出很强的体外抗新型冠状病毒(SARS-CoV-2)的活性，其活性明显优于对照药物瑞德西韦(Remdesivir)和氯喹(Chloroquine)；此外，D-1表现出较低的细胞毒性，毒性介于瑞德西韦和氯喹之间。
以上所揭露的仅为本申请较佳实施例而已，当然不能以此来限定本申请之权利范围，因此依本申请权利要求所作的等同变化，仍属本申请所涵盖的范围。

Claims (13)

  1. 一种氮杂螺环、多环穿心莲内酯类化合物，其特征在于，具有如下通式I：
    Figure PCTCN2021141536-appb-100001
    其中，C12和C13之间成双键且C13和C14之间成单键，或者C12和C13之间成单键且C13和C14之间成双键；
    -R 1为-H、-OH、烷氧基或
    Figure PCTCN2021141536-appb-100002
    -R 2为-OH、烷氧基或
    Figure PCTCN2021141536-appb-100003
    -R 3为-OH、烷氧基或
    Figure PCTCN2021141536-appb-100004
    -R 4为烷基或芳基；
    -R 5和-R 6一起形成下列基团：
    Figure PCTCN2021141536-appb-100005
    -R 7为烷基或芳基，-R 8为H或
    Figure PCTCN2021141536-appb-100006
    -R 9为环烷基、苄基、取代的苄基、杂环基、C 1～C 10的直链烷基或C 3～C 10的支链烷基，-R 10为H；
    或者-R 5和-R 6一起形成下列基团：
    Figure PCTCN2021141536-appb-100007
    -R 7为烷基或芳基，-R 8为H，-R 10为H；
    或者，-R 6为下列基团：
    Figure PCTCN2021141536-appb-100008
    同时-R 8和-R 5共同形成下列基团：
    Figure PCTCN2021141536-appb-100009
    -R 7为烷基或芳基，-R 9选自环烷基、苄基、取代的苄基、杂环基、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，-R 10为H；
    或者，-R 6、-R 10及通式中的C8、C9和C11共同形成下列基团：
    Figure PCTCN2021141536-appb-100010
    -R 5和-R 8共同形成下列基团：
    Figure PCTCN2021141536-appb-100011
    -R 7为烷基或芳基。
  2. 根据权利要求1所述的氮杂螺环、多环穿心莲内酯类化合物，其特征在于，-R 1为-H，-R 2和-R 3相同；
    或者，-R 1、-R 2和-R 3相同。
  3. 根据权利要求1所述的氮杂螺环、多环穿心莲内酯类化合物，其特征在于，-R 7为-CH 3、
    Figure PCTCN2021141536-appb-100012
    Figure PCTCN2021141536-appb-100013
    -R 9为
    Figure PCTCN2021141536-appb-100014
    Figure PCTCN2021141536-appb-100015
  4. 根据权利要求1～3中任意一项所述的氮杂螺环、多环穿心莲内酯类化合物，其特征在于，所述氮杂螺环、多环穿心莲内酯类化合物为具有如下结构式的化合物之一：
    Figure PCTCN2021141536-appb-100016
    Figure PCTCN2021141536-appb-100017
  5. 根据权利要求1所述的氮杂螺环、多环穿心莲内酯类化合物，其特征在于，所述氮杂螺环、多环穿心莲内酯类化合物为具有如下化学式的化合物之一：
    Figure PCTCN2021141536-appb-100018
    Figure PCTCN2021141536-appb-100019
    Figure PCTCN2021141536-appb-100020
    Figure PCTCN2021141536-appb-100021
    Figure PCTCN2021141536-appb-100022
    Figure PCTCN2021141536-appb-100023
  6. 一种权利要求4所述的氮杂螺环、多环穿心莲内酯类化合物的制备方法，其特征在于，包括如下步骤：
    提供化合物A，所述化合物A的结构为：
    Figure PCTCN2021141536-appb-100024
    将所述化合物A、磺酰亚胺碘盐和铜催化剂加入第二有机溶剂中，发生反应，得到所述化合物B，所述化合物B的结构为：
    Figure PCTCN2021141536-appb-100025
  7. 根据权利要求6所述的氮杂螺环、多环穿心莲内酯类化合物的制备方法，其特征在于，还包括如下步骤：
    将穿心莲内酯加入第一有机溶剂中，加入酰化剂或卤代烃，加入路易斯酸作为催化剂，反应后得到所述化合物A；
    或者，将所述化合物B、铜催化剂加入乙腈溶剂中，在加热条件下发生反应，得到所述化合物C，所述化合物C的结构为：
    Figure PCTCN2021141536-appb-100026
    或者，将所述化合物B、胺和碱性介质加入第三有机溶剂中，发生反应，得到所述化合物D，所述化合物D的结构为：
    Figure PCTCN2021141536-appb-100027
    或者，将所述化合物B和路易斯酸加入第五有机溶剂中，在加热条件下发生反应，得到化合物F，所述化合物F的结构为：
    Figure PCTCN2021141536-appb-100028
  8. 根据权利要求7所述的氮杂螺环、多环穿心莲内酯类化合物的制备方法，其特征在于，还包括如下步骤：
    将化合物D和碱性介质加入第四有机溶剂中，在加热条件下发生反应， 得到化合物E，所述化合物E的结构为：
    Figure PCTCN2021141536-appb-100029
  9. 一种药物组合物，其特征在于，包括权利要求1～5中任意一项所述的氮杂螺环、多环穿心莲内酯类化合物或其药学上可接受的盐。
  10. 权利要求1～5中任意一项所述的氮杂螺环、多环穿心莲内酯类化合物在制备抗肿瘤药物中的应用。
  11. 根据权利要求10所述的应用，其特征在于，所述抗肿瘤药物为抗鼻咽癌、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
  12. 权利要求1～5中任意一项所述的氮杂螺环、多环穿心莲内酯类化合物在制备抗病毒药物中的应用。
  13. 根据权利要求12所述的应用，其特征在于，所述抗病毒药物为抗人冠状病毒药物或抗新型冠状病毒药物。
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