WO2017140268A1 - 具有降血脂活性的竹柏內酯类化合物，其制备方法及用途 - Google Patents

具有降血脂活性的竹柏內酯类化合物，其制备方法及用途 Download PDF

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WO2017140268A1
WO2017140268A1 PCT/CN2017/073964 CN2017073964W WO2017140268A1 WO 2017140268 A1 WO2017140268 A1 WO 2017140268A1 CN 2017073964 W CN2017073964 W CN 2017073964W WO 2017140268 A1 WO2017140268 A1 WO 2017140268A1
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group
substituted
unsubstituted
compound
alkyl
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PCT/CN2017/073964
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French (fr)
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叶阳
王逸平
姚胜
马艳玲
唐春萍
赵晶
柯昌强
徐文伟
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中国科学院上海药物研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/06—Peri-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
    • C07D493/16—Peri-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
    • C07D493/20—Spiro-condensed systems

Definitions

  • the invention belongs to the field of medicinal chemistry, and more particularly relates to a new class of bantan lactone compounds having hypolipidemic activity, a preparation method thereof and use thereof for drugs for lowering blood fat and atherosclerosis.
  • Atherosclerosis is a chronic inflammatory disease caused by the inflammatory reaction between inflammatory cells and the arterial wall, which is one of the most lethal factors in Western society today.
  • Hypolipidemic therapy is the most effective treatment for atherosclerosis.
  • statins as star drugs in lipid-lowering drugs, occupy a large share in the lipid-lowering market, which is very effective in reducing cardiovascular disease in hyperlipidemic patients. Occurrence and mortality.
  • statins are not effective for all patients with high blood lipids and high risk of atherosclerosis. A large proportion of patients are not sensitive to statin reactions and cannot rely on statins to prevent the development of atherosclerosis. The task of a new type of lipid-lowering drug is imminent.
  • Formula I is selected from one of Formulas I-1, I-2 or I-3:
  • A is selected from the group consisting of NR, O; wherein R is selected from the group consisting of H, C 1 -C 4 acyl, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, substituted or unsubstituted C 6 -C 10 aryl, substituted or unsubstituted C 1 -C 10 heteroaryl;
  • R 1 is selected from the group consisting of H, substituted or unsubstituted C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, substituted or unsubstituted C 1 -C 4 alkyl (eg 2-hydroxyethyl) Base, 2-hydroxymethylethyl), substituted or unsubstituted vinyl;
  • R 2 is selected from the group consisting of: none, H, an oxygen atom, a substituted or unsubstituted C 2 -C 10 ester group, -OR, wherein said R is selected from the group consisting of H, substituted or unsubstituted C 2 -C 10 acyl group, the Fmoc group (Fmoc), or a substituted or unsubstituted C 1 -C 10 alkyl; preferably, R 'is H;
  • R 3 is selected from the group consisting of H, halogen, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, -OH, substituted or unsubstituted C 2 -C 10 ester, -NH-R Wherein R is selected from the group consisting of H, C 1 -C 4 acyl, substituted or unsubstituted C 1 -C 4 alkyl;
  • R 4 is selected from the group consisting of H, halogen, oxygen atom, cyano group, hydroxyl group, carboxyl group, C 1 -C 4 alkoxy group, C 1 -C 4 alkyl group, C 1 -C 4 haloalkyl group, substitution Or an unsubstituted C 2 -C 10 ester group, -OC(O)-R, -NH-R; wherein R is selected from the group consisting of H, C 1 -C 4 acyl, substituted or unsubstituted C a 1 -C 4 alkyl group;
  • R 5 is selected from the group consisting of H, halogen, cyano, hydroxy, -OR, -COOR, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 10 haloalkyl, substituted or not Substituted C 2 -C 30 ester group, substituted or unsubstituted (preferably substituted) C 7 -C 11 aryl-acyl, -NH-R; wherein R is selected from the group consisting of H, C 1 -C 4 is an acyl group, a substituted or unsubstituted alkyl group of C 1 -C 4;
  • R 4 and R 5 together form -R"-OR"-, wherein said R" is none or a substituted or unsubstituted C 1 -C 4 alkylene group;
  • R 6 is selected from the group consisting of H, halogen, oxygen atom, cyano group, carboxyl group, hydroxyl group, -OR, -COOR, substituted or unsubstituted C 1 -C 10 alkyl group, substituted or unsubstituted C 1 -C 10 halogenated alkane a substituted, unsubstituted or unsubstituted C 2 -C 10 ester group, a substituted or unsubstituted (preferably substituted) C 7 -C 11 aryl acyl group, -NH-R; wherein R is selected from the group consisting of H, C 1 -C 4 acyl group, a substituted or unsubstituted C 1 -C 4 alkyl group;
  • R 5 and R 6 together form -R"-OR"-, wherein said R" is none or a substituted or unsubstituted C 1 -C 4 alkylene group;
  • R 7 is selected from the group consisting of H, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl;
  • R 8 is absent, and H, or R 8 and R 2 together form -R"-OR"-, wherein said R" is an alkylene group having no or C 1 -C 4 :
  • R 9 is none, H or R 9 is -OH;
  • substituted means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of a carboxy group, an unsubstituted or halogenated C 1 -C 6 alkyl group, an unsubstituted or halogenated C.
  • a 3- C 6 cycloalkyl group an unsubstituted or halogenated C 2 -C 10 ester group, a halogen, a C 1 -C 10 alkyl-oxy group, an unsubstituted or halogenated C 2 -C 10 acyl group, a hydroxy group, a hydroxy-C 1 -C 10 alkylene group, an unsubstituted or halogenated phenyl group;
  • R 2 , R 5 and R 6 are a group selected from the group consisting of -OR or a substituted or unsubstituted C 2 -C 10 ester group; R 5 and R 6 together form an -OC(CH) 2 -O- structure;
  • each chiral carbon atom may be optionally in the R configuration or the S configuration.
  • the C 2 -C 10 acyl group means a group in which the C 2 -C 10 acid loses a hydroxyl group, and the acid is selected from the group consisting of carboxylic acid, carbonic acid.
  • the carboxylic acid comprises a saturated carboxylic acid, an unsaturated carboxylic acid, or an aromatic carboxylic acid.
  • the R 2 is a C 2 -C 10 aromatic ester group, preferably selected from the group consisting of a benzoyl group or a substituted benzoyl group; wherein the substitution is benzene
  • a substituent selected from the group consisting of halogen, nitro, amino, methyl, methoxy.
  • the R 2 is a carbonate group.
  • R 2 when said R 2 is a hydroxyl group, it further optionally carries a hydroxy protecting group; preferably, said hydroxy protecting group is a protecting group with an acid chloride or a free acid.
  • each of R 1 , R 3 and R 7 is independently a C 1 -C 4 alkyl group.
  • the R 4 is H or a hydroxyl group.
  • the R 5 is H or a hydroxyl group.
  • the R 1 is isopropyl.
  • each of R 3 and R 7 is independently methyl.
  • the compound of formula I has the structure shown in formula Ia below:
  • R 1 is selected from the group consisting of substituted or unsubstituted C 1 -C 4 alkyl, C 1 -C 4 haloalkyl; wherein said substitution refers to one or more hydrogen atoms on the group being C 1 -C 4 ester group substitution;
  • R 2 is -OH, -OR or a substituted or unsubstituted C 2 -C 10 ester group
  • R 7 is selected from the group consisting of C 1 -C 4 alkyl, C 1 -C 4 haloalkyl;
  • the compound of formula I has the structure shown in formula Ib below:
  • R 1 is selected from the group consisting of substituted or unsubstituted C 1 -C 4 alkyl, C 1 -C 4 haloalkyl;
  • R 7 is selected from the group consisting of C 1 -C 4 alkyl, C 1 -C 4 haloalkyl;
  • R 5 is selected from the group consisting of halogen, cyano, hydroxy, C 1 -C 4 alkoxy, -COOR, substituted or unsubstituted C 2 -C 10 ester, substituted or Unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 10 haloalkyl, -NH-R; wherein R is selected from the group consisting of H, C 1 -C 4 acyl, C 1 -C An alkyl group of 4 , a C 1 -C 4 haloalkyl group, a substituted or unsubstituted (preferably substituted) C 7 -C 11 aryl-acyl group.
  • R 6 is selected from the group consisting of halogen, cyano, carboxy, hydroxy, C 1 -C 4 alkoxy, substituted or unsubstituted C 2 -C 10 ester, substituted or unsubstituted a C 1 -C 10 alkyl group, a substituted or unsubstituted C 1 -C 10 haloalkyl group, a substituted or unsubstituted (preferably substituted) C 7 -C 11 aryl-acyl group, -NH-R; wherein R It is selected from the group consisting of H, a C 1 -C 4 acyl group, a C 1 -C 4 alkyl group, and a C 1 -C 4 haloalkyl group.
  • R 2 is selected from the group consisting of O-R', wherein said R' is selected from the group consisting of H, substituted or unsubstituted C 2 -C 10 Acyl, or substituted or unsubstituted C 1 -C 10 alkyl;
  • R 4 is selected from the group consisting of H, halogen, cyano, carboxyl, hydroxy, C 1 -C 4 alkoxy, substituted or unsubstituted C 2 -C 10 ester;
  • R 5 is selected from the group consisting of H, halogen, cyano, hydroxy, C 1 -C 4 alkoxy, -COOR, substituted or unsubstituted C 2 -C 10 ester group, substituted or unsubstituted C 1 - C 10 alkyl, substituted or unsubstituted C 1 -C 10 haloalkyl, -NH-R; wherein R is selected from the group consisting of H, C 1 -C 4 acyl, C 1 -C 4 alkyl, a C 1 -C 4 haloalkyl, substituted or unsubstituted (preferably substituted) C 7 -C 11 aryl-acyl;
  • R 6 is selected from the group consisting of H, halogen, cyano, carboxyl, hydroxy, C 1 -C 4 alkoxy, substituted or unsubstituted C 2 -C 10 ester, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 10 haloalkyl, substituted or unsubstituted (preferably substituted) C 7 -C 11 aryl-acyl, -NH-R; wherein R is selected from the group consisting of H, C 1 -C 4 acyl group, C 1 -C 4 alkyl group, C 1 -C 4 haloalkyl group;
  • substituted means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of a carboxyl group, a phenyl group, a C 3 -C 6 cycloalkyl group, and a C 1 -C 10 ester group.
  • a substituent selected from the group consisting of a carboxyl group, a phenyl group, a C 3 -C 6 cycloalkyl group, and a C 1 -C 10 ester group.
  • Halogen C 1 -C 10 alkyl-oxy group, C 2 -C 10 acyl group, hydroxy group, hydroxy-C 1 -C 10 alkylene group.
  • R 2 , R 4 , R 5 and R 6 are each independently a group selected from the group consisting of: -OR', and the R' is selected from the group consisting of:
  • the A, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are the groups corresponding to the specific compounds in the examples. group.
  • the compound has a structure selected from the group consisting of:
  • LDL low density lipoprotein
  • HDL high density lipoprotein
  • the liver function damage is a high fat-induced liver function damage.
  • the improvement of liver function damage is to reduce the ALT ⁇ AST index.
  • the pharmaceutical composition is further for use in a group selected from the group consisting of:
  • k (non-therapeutic in vitro) upregulates LDLR gene expression levels in hepatocytes.
  • the effective amount of the compound of formula I in the pharmaceutical composition is from 0.1 to 50 mg/kg body weight, preferably from 1 to 20 mg/kg body weight.
  • the pharmaceutical composition is a dosage form selected from the group consisting of an oral dosage form and an injectable dosage form.
  • the dosage form includes tablets, granules, capsules, pills, and the like.
  • kits comprising:
  • the compound of formula I is as described in the first aspect of the invention.
  • the subject of use is a mammal, preferably a human.
  • statin is selected from the group consisting of pravastatin, atorvastatin, or a combination thereof.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are as defined in the first aspect of the invention, and For double or single button;
  • LDL low density lipoprotein
  • HDL high density lipoprotein
  • the subject to be administered is a mammal, preferably a human.
  • the liver function damage is a high fat-induced liver function damage.
  • the improvement of liver function damage is to reduce the ALT ⁇ AST index.
  • the cedar lactone compound has good hypolipidemic activity and is superior in activity to the existing general lipid-lowering compound statins.
  • the compounds can reduce total cholesterol, triglycerides, low density lipoprotein concentrations, and increase high density lipoprotein concentrations in vivo or in vitro, in a time- and dose-dependent manner. Based on the above findings, the inventors completed the present invention.
  • C 1 -C 4 alkyl or “C 1 -C 10 alkyl” as used herein refers to a straight or branched alkyl group having from 1 to 4 or 1 to 10 carbon atoms, such as methyl, Ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.
  • C 3 -C 6 cycloalkyl refers to a cycloalkyl group having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, methylcyclobutyl, cyclopentyl, or the like.
  • C 1 -C 10 acyl or “C 1 -C 4 acyl” refers to a straight or branched alkyl/cycloalkyl group having the form of 0 to 9 or 0 to 3 carbon atoms.
  • Substituents of the /aryl/heteroaryl/alkenyl/alkynyl-carbonyl" structure such as acetyl, propionyl, butyryl, or the like.
  • C 2 -C 30 ester group refers to a group having a structure represented by -OOC-R', wherein R' is a group selected from the group consisting of 1 to 9 carbon atoms: straight A substituent of a chain or branched alkyl, cycloalkyl, alkenyl, alkynyl, aryl or heteroaryl structure, such as an acetyl group, a propionyl group, a butyryl group, or the like.
  • said R' may further be substituted, for example, by one or more substituents selected from the group consisting of halogen, -OH, -COOH, -COO (C 1 -C 6 alkyl), or halo-substituted phenyl, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C 2 -C 6 acyl group, an unsubstituted or halogenated C 1 -C 6 alkyl - hydroxy.
  • substituents selected from the group consisting of halogen, -OH, -COOH, -COO (C 1 -C 6 alkyl), or halo-substituted phenyl, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C 2 -C 6 acyl group, an unsubstituted or halogenated C 1 -C 6 alky
  • C 1 -C 4 alkylene refers to a group formed after the C1 to C4 alkyl group has lost a hydrogen atom as described above, for example, -CH 2 -, -CH 2 -CH 2 -, or the like. .
  • halogen refers to F, Cl, Br and I.
  • C 6 -C 10 aryl refers to an aryl group having 6 to 10 carbon atoms, such as phenyl, naphthyl and the like, which may be substituted or unsubstituted.
  • C 1 -C 10 heteroaryl refers to a heteroaryl group having from 1 to 10 carbon atoms and one or more heteroatoms selected from O, S and/or N, preferably a C2-C8 heteroaryl group.
  • the heteroaryl group can be substituted or unsubstituted.
  • the terms "containing”, “comprising” or “including” mean that the various ingredients may be used together in the mixture or composition of the present invention. Therefore, the terms “consisting essentially of” and “consisting of” are encompassed by the term “contains.”
  • the term "pharmaceutically acceptable” ingredient means a substance which is suitable for use in humans and/or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reaction), that is, a reasonable benefit/risk ratio.
  • the term "effective amount" means an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect.
  • the precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the condition, and the combination of therapeutic and/or therapeutic agents selected for administration. Therefore, it is useless to specify an accurate effective amount in advance. However, for a given condition, routine experimentation can be used to determine the effective amount that the clinician can determine.
  • substituted means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of halogen, -OH, -COOH, -COO (C 1 -C). 6 alkyl), unsubstituted or halogenated phenyl, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C 2 -C 6 acyl, unsubstituted or halogenated C 1 -C 6 Alkyl-hydroxyl.
  • each of the chiral carbon atoms may be optionally in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.
  • compound of the invention refers to a compound of formula I.
  • the term also encompasses various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula I.
  • the term "pharmaceutically acceptable salt” refers to a salt of the compound of the invention formed with an acid or base suitable for use as a medicament.
  • Pharmaceutically acceptable salts include inorganic and organic salts.
  • a preferred class of salts are the salts of the compounds of the invention with acids.
  • Suitable acids for forming salts include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Organic acids such as maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzoic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.
  • mineral acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid,
  • Organic acids such as maleic acid, lactic acid, malic acid, tartaric acid,
  • Podocarpus nagi is a plant of the genus Podocarpaceae (Podocarpaceae). Its main chemical component is dioxin and it has anti-tumor, anti-bacterial, insecticidal and growth regulation inhibition effects.
  • the inventors discovered for the first time that the cypress lactones were classified.
  • the compound enhances the ability of hepatocytes to take up LDL.
  • In vivo pharmacodynamic evaluation found that the compound can significantly reduce the total cholesterol, triglyceride, low-density lipoprotein and other lipid indexes of golden hamsters with high lipid-induced lipid metabolism disorder, and improve high-density lipoprotein in total cholesterol.
  • the proportion in the liver is improved, and at the same time it can alleviate the formation of fatty liver caused by high-fat diet, so it is a potential lipid-lowering compound.
  • the present inventors carried out a series of structural transformations such as esterification by using the diterpene lactone compounds 2, 3, 4, and 12 separated from the bark, branches, leaves, and fruits of the bamboo cypress as raw materials. , elimination, hydrogenation, etc., to obtain compounds of the formula 2-B1, 3-1 to 3-4, 3-B1 to 3-B10, 7-A1, 12-1 and 4-A and 4-B:
  • R c and R d are as shown in Table 1 below:
  • R a and R b are as shown in Table 2 below:
  • R c and R d are as shown in Table 3 below:
  • LDLR Low-density lipoprotein receptor
  • the expression level of LDLR gene is closely related to plasma cholesterol level.
  • the SRE regulatory site located at the promoter of LDLR gene is regulated by intracellular SREBP1/2, which is closely related to intracellular cholesterol level.
  • SREBP1/2 is closely related to intracellular cholesterol level.
  • SREBP is immobilized on the ER membrane and cannot enter the Golgi activation; when the intracellular cholesterol level is lowered, SREBP is released under the action of cholesterol, and then transferred to the Golgi to be spliced into an activated protein. It enters the nucleus and acts as a transcription factor to regulate the transcription of genes containing SRE, including genes related to cholesterol and fatty acid metabolism such as LDLR and HMG-CoA R.
  • the LDLR gene is regulated by many cytokines and growth factors and hormones such as estrogen, tumor necrosis factor, phorbol ester, and insulin.
  • the statin is an inhibitor of HMG-CoA R. This class of drugs lowers the intracellular cholesterol content by inhibiting the synthesis of cholesterol, thereby up-regulating the intracellular level of LDLR to further enhance its lipid-lowering function. Regulating LDLR levels and function is an important way to regulate blood lipid levels in the body.
  • LDLR gene Another characteristic of the LDLR gene is its unstable nature, which is degraded in a short period of time after synthesis. This process may be related to the 3' end of the LDLR gene itself.
  • the 3' end of the LDLR gene contains an AU-rich region, which is considered to be a characteristic sequence of gene instability, and many RNA-binding proteins can bind to this sequence, causing rapid degradation of mRNA.
  • Berberine has been reported to increase the stability of LDLR gene, increase its gene and protein levels, thereby increasing the function of LDLR and lowering plasma LDL levels. Clinical trials have confirmed that berberine is an effective lipid-lowering factor by regulating LDLR levels. The compound of effect.
  • the bamboo cyanolide compound can up-regulate the gene expression level of the LDLR protein in vivo or in vitro, and increase the stability of the LDLR protein itself, thereby up-regulating the LDLR protein level, thereby promoting hepatocytes for low-density lipoprotein.
  • the bantanide compound has an up-regulation effect on the LDLR protein level, thereby promoting the uptake of low-density lipoprotein by the hepatocytes, thereby lowering the LDL concentration in the blood and achieving the purpose of clearing LDL.
  • the administration of the bamboo cyanolide compound has also improved the liver function damage caused by the high-fat diet, which is expressed as the level of aspartate aminotransferase and alanine aminotransferase in the blood. Improvement.
  • the compound of the present invention has an excellent activity for lowering the LDL content in blood
  • the compound of the present invention and various crystal forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and compounds containing the present invention are mainly
  • the pharmaceutical composition of the active ingredient can be used for the treatment, prevention, and alleviation of diseases caused by excessive levels of LDL in the blood, such as fatty liver.
  • compositions of the present invention comprise a safe or effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier.
  • safe and effective amount it is meant that the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical compositions contain from 1 to 30,000 (active dose range 3-30 mg/kg) mg of the compound/agent of the invention, more preferably from 10 to 2000 mg of the compound/agent of the invention.
  • the "one dose” is one Capsules or tablets.
  • “Pharmaceutically acceptable carrier” means: one or more compatible solid or liquid fillers or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity. By “compatibility” it is meant herein that the components of the composition are capable of intermingling with the compounds of the invention and with each other without significantly reducing the efficacy of the compound.
  • pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid).
  • magnesium stearate magnesium stearate
  • calcium sulfate vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (such as Tween ), a wetting agent (such as sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, a pyrogen-free water, and the like.
  • vegetable oil such as soybean oil, sesame oil, peanut oil, olive oil, etc.
  • polyol such as propylene glycol, glycerin, mannitol, sorbitol, etc.
  • emulsifier such as Tween
  • a wetting agent such as sodium lauryl sulfate
  • a coloring agent such as a flavoring agent, a stabilizer, an antioxidant, a preservative
  • the mode of administration of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration. .
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with: (a) a filler or compatibilizer, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, For example, glycerin; (d) a disintegrant such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent such as paraffin; (f) Absorbing accelerators, for example, quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other materials known in the art. They may contain opacifying agents and the release of the active compound or compound in such compositions may be released in a portion of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric and waxy materials. If necessary, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs.
  • the liquid dosage form may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances.
  • inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethyl
  • compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these and the like.
  • compositions for parenteral injection may comprise a physiologically acceptable sterile aqueous or nonaqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion.
  • Suitable aqueous and nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.
  • Dosage forms for the compounds of the invention for topical administration include ointments, powders, patches, propellants and inhalants.
  • the active ingredient is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, propellants.
  • the compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
  • a safe and effective amount of a compound of the invention is administered to a mammal (e.g., a human) in need of treatment wherein the dosage is a pharmaceutically effective effective dosage, for a 60 kg body weight
  • the dose to be administered is usually from 1 to 2000 mg, preferably from 6 to 600 mg.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc. These are all within the skill of a skilled physician.
  • the bamboo cyanolide compound has been modified to have better hypolipidemic activity than the natural bantan lactone compound (in vitro activity and pravastatin and arva known in the prior art)
  • the statin is equivalent or superior to the above compounds
  • the hypolipidemic activity of the bantan lactone compound of the present invention has a significant time- and dose-dependent manner
  • the bamboo cyanolide compound of the invention can effectively reduce the mechanism of LDL concentration in blood: up-regulating the expression of LDLR gene by stabilizing the stability of LDLR gene, thereby increasing the number of LDLR receptors on the surface of hepatocytes, thereby achieving the purpose of clearing LDL;
  • the compound of the present invention is effective in vitro and in vivo, and has a low active dose, and can exhibit hypolipidemic effect at a dose as low as 3 mg/kg, which is far lower than the reported pravastatin and berberine drugs. Effective dose
  • HDL high-density lipoprotein, a protein beneficial to the body
  • Nuclear magnetic resonance spectrum Varian INOVA 600 nuclear magnetic resonance spectrometer, Bruker AM-500, AM-400, AM-300 nuclear magnetic resonance spectrometer, ⁇ (ppm), with TMS as internal standard;
  • Analytical HPLC Waters 2690 Separate Model, Waters PDA 996 detector coupled Alltch ELSD 2000 detector, Millennium 2000 operating system, Waters RP18 column (5.0 x 125 mm, 5 ⁇ m, Waters), flow rate 1.0 ml/min, CH3CN (Merck, Germany) , H 2 O (Rapex); Jasco HPLC (Chiralcel IA column, 5 ⁇ m, 150 ⁇ 4.6 mm), flow rate 0.6 ml / min, hexane / ethanol (7:3).
  • HPLC-MS Waters 2695 Separate Model, Waters PDA 2998 detector coupled Alltch ELSD 2424 detector, 3100 Ms detector, SunFireTM C-18 column (4.6 x 100 mm, 3.5 ⁇ m, Waters), flow rate 1.0 ml/min, CH3CN (Merck, Germany ), H 2 O (Rapex); Jasco HPLC (Chiralcel IA column, 5 ⁇ m, 150 ⁇ 4.6 mm), flow rate 0.6 ml / min, hexane / ethanol (7: 3).
  • Electrophoresis apparatus and semi-dry transfer tank Bio-Rad Laboratories, Hercules, CA;
  • REVCO carbon dioxide incubator (REVCO, USA);
  • TLC thin layer preparation board HSGF254 is produced by Yantai Chemical Plant.
  • MCI resin CHP20P (75-150 ⁇ m) is produced by Mitsubishi Corporation;
  • Color developer 10% sulfuric acid-vanillin solution, iodine;
  • Plant material bamboo cypress leaves were collected in Tianlin, Guangxi, and bamboo cypress bark was collected from Hainan Qiongzhong. It was collected and identified by Associate Professor Shen Jingui from the Shanghai Institute of Materia Medica. The specimens were kept in the specimen room.
  • DMEM fetal bovine serum
  • FBS fetal bovine serum
  • DiI was purchased from biotuim (Hayward, CA)
  • ⁇ -actin antibody was purchased from Cell Signaling Technology (Beverly, MA)
  • LDLR PCSK9 antibody was purchased from Abcam (Cambridge, United Kingdom); PCR primers were synthesized in Shanghai.
  • the protease inhibitor Cocktail was purchased from Calbiochem (San Diego, CA); the Immobilon-P transfer transfer membrane (PVDF) was purchased from Millipore Corporation (Bedford, MA); the enhanced chemiluminescence reagents (ECL) was purchased from Pierce (Rockford, IL); medical X-ray film was purchased from China Biyuntian Company; TRIzol reagent was purchased from Invitrogen (Carlsbad, CA); M-MLV reverse transcriptase was purchased from Promega (Madison, WI); Real time PCR reaction reagent was purchased from Bio -Rad Laboratories; Golden Gopher High Fat Feed (0.12% Cholesterol, 10% Coconut Oil) was purchased from Shanghai Slex Company.
  • the protein assay kit was purchased from China Biyuntian Company; the liver tissue triglyceride TG and the total cholesterol TC content determination kit were purchased from Shanghai Rongsheng Biotechnology Co., Ltd. Blood index (total cholesterol TC, triglyceride TG, high density lipoprotein HDL, low density lipoprotein LDL, alanine aminotransferase ALT, aspartate aminotransferase AST) assay kit was purchased from Sichuan Mike Bio Company . Other reagents were purchased from Shanghai Sinopharm Group unless otherwise stated.
  • the experimental animals were male Syrian golden hamsters weighing 100 ⁇ 10 g and purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences. The animals were placed in the SPF animal room of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (temperature 23 ⁇ 1 ° C; humidity 60%), natural day/night cycle, and fed with standard food and water. (Experiment approval number: SIMM-AE-2010-10-WYP-01)
  • the bamboo cypress bark (16.2kg) was pulverized with 95% ethanol and extracted three times at room temperature. The extracts were combined and concentrated to an alcohol-free taste, and then transferred to a faucet bottle with petroleum ether, chloroform, ethyl acetate and n-butanol. With water distribution, petroleum ether fraction (95g), chloroform fraction (15g), ethyl acetate fraction (255g), chloroform fraction and ethyl acetate fraction were combined and purified by silica gel column (200-300 mesh, produced by Qingdao Marine Chemical Industry Co., Ltd.).
  • the compound 3 (25 mg, 0.07 mmol) was dissolved in anhydrous pyridine (5 ml), and excess succinic anhydride (25 mg, 3N) was added. The mixture was heated at 70 ° C in an oil bath and stirred at a constant temperature. The reaction was confirmed by TLC, and 2N was added to the reaction mixture. Hydrochloric acid was used to neutralize the pyridine, and the mixture was extracted with ethyl acetate. The residue was purified with EtOAcqqqqqq elut elut elut
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and isobutyryl chloride (0.008 ml, 2.2 N) was added thereto, and the mixture was stirred at room temperature overnight. The reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the mixture to neutralize pyridine. The organic layer was washed with saturated aqueous Na.sub.2, then filtered. The residue was purified by preparative EtOAc EtOAc (EtOAc)
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and n-butyryl (0.008 ml, 2.2 N) was added thereto, and the mixture was stirred at room temperature overnight. The reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the mixture to neutralize pyridine. The organic layer was washed with saturated aqueous Na.sub.2, then filtered. The residue was purified by preparative EtOAc EtOAc (EtOAc)
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and n-hexanoyl chloride (0.012 ml, 1.5 N) was added thereto, and the mixture was stirred at room temperature overnight. The reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to neutralize pyridine. The mixture was extracted with EtOAc. The residue was purified by preparative EtOAc EtOAc (EtOAc)
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and then added to a solution of the pyridine chloride (0.012 ml, 1.1 N). The mixture was stirred at room temperature overnight. The reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to neutralize the pyridine. The mixture was extracted with EtOAc. The residue was purified by preparative EtOAc EtOAc (EtOAc)
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and acetyl chloride (0.008 ml, 3N) was added thereto, and the mixture was stirred at room temperature overnight. The reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the reaction mixture to neutralize the pyridine. The mixture was extracted with EtOAc. The residue was purified by preparative EtOAc EtOAc (EtOAc)
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and cyclopropylcarbonyl chloride (0.008 ml, 1.5 N) was added thereto, and the mixture was stirred at room temperature overnight, and the reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the mixture. The mixture was extracted with EtOAc. EtOAc. The residue was purified by preparative EtOAc (EtOAc) 4-B9: ESIMS m/z: 417.2 [M+H] + , 461.2 [M+HCOO] - .
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and cyclopentanoyl chloride (0.008 ml, 1.2 N) was added thereto, and the mixture was stirred at room temperature overnight. The reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the reaction mixture for neutralization. The pyridine was extracted with EtOAc. EtOAc was evaporated. The residue was purified by preparative EtOAc EtOAc (EtOAc) 4-B10: ESIMS m/z: 445.3 [M+H] + , 489.2 [M+HCOO] - .
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and cyclohexanecarbonyl chloride (0.008 ml, 1.2 N) was added thereto, and the mixture was stirred at room temperature overnight, and the reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the reaction mixture for neutralization.
  • the pyridine was extracted with EtOAc. EtOAc was evaporated. The residue was purified by preparative EtOAc EtOAc (EtOAc) 4-B11: ESIMS m/z: 459.3 [M+H] + , 503.3 [M+HCOO] - .
  • the compound 4 (10 mg, 0.029 mmol) was dissolved in 1 ml of anhydrous pyridine, and cyclopropylcarbonyl chloride (0.008 ml, 3N) was added thereto, and the mixture was stirred at room temperature overnight, and the reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the reaction mixture for neutralization.
  • the pyridine was extracted with EtOAc. EtOAc was evaporated. The residue was purified by preparative EtOAc EtOAc (EtOAc) 4-B13: ESIMS m/z: 485.3 [M+H] + , 529.3 [M+HCOO] - .
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and cyclobutyl carboxylic acid (0.008 ml, 1.5 N) was added, and the mixture was stirred at room temperature. DCC (50 mg, 4.6 N) was added and reacted overnight, and the reaction was confirmed by TLC. 2N dilute hydrochloric acid was added to the reaction mixture to neutralize the pyridine, and the mixture was extracted with ethyl acetate. The residue was purified by preparative EtOAc EtOAc (EtOAc) 4-B14: ESIMS m/z: 513.3 [M+H]+, 557.2 [M+HCOO]-.
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and glutaric anhydride (20 mg, 3.1 N) was added thereto, and the mixture was stirred at room temperature overnight. The reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to neutralize pyridine. The mixture was extracted with EtOAc. The residue was purified by preparative EtOAc EtOAc (EtOAc)
  • the compound 4 (10 mg, 0.029 mmol) was dissolved in anhydrous pyridine (1 ml), succinic acid monoethyl ester chloride (0.012 ml, 3N) was added, and the mixture was stirred at room temperature overnight, and the reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the mixture. The mixture was extracted with EtOAc. EtOAc. The residue was purified by preparative EtOAc EtOAc (EtOAc) 4-B18: ESIMS m/z: 605.5 [M+H] + , 649.4 [M+HCOO] - .
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and 2-furoyl chloride (0.010 ml, 1.8 N) was added thereto, and the mixture was stirred at room temperature overnight, and the reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the mixture. The mixture was extracted with EtOAc. EtOAc. The residue was purified by preparative EtOAc EtOAc (EtOAc) 4-B19: ESIMS m/z: 443.3 [M+H] + , 487.2 [M+CH 3 COO] - .
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and 2-thiophenecarbonyl chloride (0.010 ml, 1.6 N) was added thereto, and the mixture was stirred at room temperature overnight. The reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the mixture. The mixture was extracted with EtOAc. EtOAc. The residue was purified by preparative EtOAc EtOAc (EtOAc) 4-B20: ESIMS m/z: 459.3 [M+H] + , 503.2 [M+HCOO] - .
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and 1-benzothiophene-3-carbonyl chloride (12 mg, 1.1 N) was added thereto, and the mixture was stirred at room temperature overnight, and the reaction was confirmed by TLC, and 2N was added to the reaction mixture. Hydrochloric acid was used to neutralize the pyridine, and the mixture was extracted with ethyl acetate. The residue was purified by preparative EtOAc (EtOAc) 4-B22: ESIMS m/z: 509.3 [M+H] + , 553.2 [M+HCOO] - .
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and 4-tert-butylbenzoyl chloride (0.015 ml, 1.3 N) was added thereto, and the mixture was stirred at room temperature overnight, and the reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the reaction mixture.
  • the pyridine was used for the neutralization, and the mixture was extracted with ethyl acetate.
  • the residue was purified by preparative EtOAc (EtOAc) 4-B26: ESIMS m/z: 509.3 [M+H] + , 553.2 [M+HCOO] - .
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and 4-tert-butylbenzoyl chloride (15 mg, 1.3 N) was added thereto, and the mixture was stirred at room temperature overnight, and the reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the reaction mixture.
  • the pyridine was neutralized, extracted with ethyl acetate. EtOAc was evaporated. The residue was purified by preparative EtOAc (EtOAc) 4-B27: ESIMS m/z: 513.3 [M+H] + , 557.2 [M+HCOO] - .
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and 2-chlorobenzoyl chloride (0.010 ml, 1.4 N) was added, and the mixture was stirred at room temperature overnight. The reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the reaction mixture. The pyridine was neutralized, extracted with ethyl acetate. EtOAc (EtOAc m. The residue was purified by preparative EtOAc (EtOAc) 4-B31: ESIMS m/z: 488.3 [M+H] + , 532.2 [M+HCOO] - .
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and 2-fluorobenzoyl chloride (0.010 ml, 1.6 N) was added thereto, and the mixture was stirred at room temperature overnight, and the reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the reaction mixture.
  • the pyridine was neutralized, extracted with ethyl acetate.
  • EtOAc EtOAc m.
  • the residue was purified by preparative EtOAc EtOAc (EtOAc) 4-B32: ESIMS m/z: 471.3 [M+H] + , 515.2 [M+HCOO] - .
  • the compound 4 (10 mg, 0.029 mmol) was dissolved in 1 ml of anhydrous pyridine, and crotonyl chloride (10 mg, 3.3 N) was added thereto, and the mixture was stirred at room temperature overnight. The reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to the reaction mixture to neutralize the pyridine. The mixture was extracted with EtOAc. The residue was purified by preparative EtOAc EtOAc (EtOAc)
  • the compound 4 (20 mg, 0.057 mmol) was dissolved in 1 ml of anhydrous pyridine, and crotonyl chloride (0.010 ml, 1.3 N) was added thereto, and the mixture was stirred at room temperature overnight. The reaction was confirmed by TLC, and 2N diluted hydrochloric acid was added to neutralize pyridine. The mixture was extracted with EtOAc. The residue was purified by preparative EtOAc (EtOAc)
  • the compound 4-MOC (20 mg, 0.035 mmol) was dissolved in 1 ml of anhydrous pyridine, and p-bromobenzoyl chloride (12 mg, 1.5 N) was added and stirred at room temperature overnight. After the reaction was completed, most of the pyridine was removed by distillation under reduced pressure. The residue was dissolved in dichloromethane, and 0.1 ml of diethylamine was added, and the mixture was reacted at room temperature for 0.5 hr. The solvent was evaporated under reduced pressure to give purified crystals (yield: chloroform/methanol 1:1). (18 mg, 97%).
  • the above-mentioned isolated and chemically synthesized compounds (final concentration: 5 ⁇ M) were treated with HepG2 cells deficient in serum for 12 hours, and added with fluorescently labeled low-density lipoprotein (DiI-LDL) at 20 ⁇ g/ml, and incubated at 37 ° C for 4 hours.
  • the cells were gently washed with phosphate buffer (PBS) for 5 times, and the lipid was extracted with isopropyl alcohol, and the fluorescence reading (excitation light: 520 nm; emission light 570 nm) was measured on a microplate reader.
  • the cells were then lysed with 0.2 M sodium hydroxide, the protein content was determined, and the fluorescence/protein values were calculated.
  • the intensity of action +101%-120%; ++121%-140%; +++141%-160%;++++161%-180%;+++++181%-200%;+++ +++>200%.
  • Upregulation of LDLR protein levels may be caused by two reasons: an increase in LDLR gene levels or an increase in the stability of the LDLR protein itself. Based on the above conditions, the expression level of the LDLR gene was examined. Hepatocytes were treated with the compound of the present invention at a concentration of 5 ⁇ M for 24 hours, and total RNA was extracted from the TRIzol reagent, and 3 ⁇ g of RNA was reverse-transcribed into cDNA by M-MLV (reverse transcriptase), and then real-time was performed using specific primers of LDLR. PCR (real-time quantitative PCR), using GAPDH as an internal reference gene to modulate the regulation of the LDLR gene by the compound. The results show that the compounds of the invention significantly increase the expression level of the LDLR gene.
  • the bamboo cyanolide compound exerts an unexpected effect of enhancing LDL uptake in an LDL uptake experiment in vitro.
  • Some of these compounds (such as 4-B19, 4-B29, 4-B31) have very significant effects and can even achieve >200% strength of action. Therefore, experiments have confirmed that the bamboo cyanolide compounds based on the new lipid-lowering target have extremely high clinical application value and potential for new drug development.

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Abstract

提供了一类具有降血脂活性的竹柏內酯类化合物、其制备方法及用途，具体地，所述的化合物具有如式I所示的结构，其中，各基团的定义如说明书中所述。所述化合物具有体内或体外增加低密度脂蛋白摄取的活性，具有作为降血脂药物的潜力。

Description

具有降血脂活性的竹柏內酯类化合物，其制备方法及用途 技术领域
本发明属于药物化学领域，更具体而言，涉及一类新的具有降血脂活性的竹柏內酯类化合物，其制备方法及用于降血脂\动脉粥样硬化的药物中的用途。
背景技术
动脉粥样硬化是因为炎症细胞与动脉壁之间的炎症反应不断恶化而形成的慢性炎症性的疾病，是当今西方社会上致死量最高的因素之一。降血脂疗法是针对动脉粥样硬化最有效的治疗手段，其中他汀类作为降脂药中的明星药物在降脂市场中占据了很大的份额，非常有效的降低了高血脂病人心血管疾病的发生和致死率。然而，他汀类并非对所有的高血脂，和高动脉粥样硬化硬化风险的病人有效果，很大一部分患者对他汀类反应不敏感，不能靠他汀类阻止其动脉粥样硬化的发展进程，发展新型的降血脂药物的任务迫在眉睫。
综上所述，本领域迫切需要提供新的具有降血脂活性的化合物。
发明内容
本发明的目的是提供一类具有降血脂活性的化合物。
本发明的第一方面，提供了一种如下式I所示的化合物，或其药学上可接受的盐：
Figure PCTCN2017073964-appb-000001
其中，式I为选自式I-1，I-2或I-3之一：
Figure PCTCN2017073964-appb-000002
A选自下组：N-R、O；其中，R选自下组：H、C1-C4的酰基、C1-C4的烷基、C1-C4的卤代烷基、取代或未取代的C6-C10芳基、取代或未取代的C1-C10杂芳基；
R1选自下组：H、取代或未取代的C1-C4的烷基、C1-C4的卤代烷基、取代或未取代的C1-C4烷基(如2-羟基乙基，2-羟甲基乙基)、取代或未取代的乙烯基；
R2选自下组：无、H、氧原子、取代或未取代的C2-C10的酯基、-OR，其中，所述的R选自下组：H、取代或未取代的C2-C10的酰基、芴甲氧羰酰基(Fmoc)、或取代或未取代的C1-C10烷基；较佳地，R’为H；
R3选自下组：H、卤素、C1-C4的烷基、C1-C4的卤代烷基、-OH、取代或未取代的C2-C10 的酯基、-NH-R；其中，R选自下组：H、C1-C4的酰基、取代或未取代的C1-C4的烷基；
R4选自下组：H、卤素、氧原子、氰基、羟基、羧基、C1-C4的烷氧基、C1-C4的烷基、C1-C4的卤代烷基、取代或未取代的C2-C10的酯基、-OC(O)-R、-NH-R；其中，R选自下组：H、C1-C4的酰基、取代或未取代的C1-C4的烷基；
R5选自下组：H、卤素、氰基、羟基、-OR、-COOR、取代或未取代C1-C10烷基、取代或未取代的C1-C10卤代烷基、取代或未取代的C2-C30的酯基、取代或未取代(优选为取代)的C7-C11芳基-酰基、-NH-R；其中，R选自下组：H、C1-C4的酰基、取代或未取代的C1-C4的烷基；
或R4和R5共同构成-R”-O-R”-，其中，所述的R”为无，或取代或未取代的C1-C4的亚烷基；
R6选自下组：H、卤素、氧原子、氰基、羧基、羟基、-OR、-COOR、取代或未取代C1-C10烷基、取代或未取代的C1-C10卤代烷基、取代或未取代的C2-C10的酯基、取代或未取代(优选为取代)的C7-C11芳基酰基、-NH-R；其中，R选自下组：H、C1-C4的酰基、取代或未取代的C1-C4的烷基；
或R5和R6共同构成-R”-O-R”-，其中，所述的R”为无，或取代或未取代的C1-C4的亚烷基；
R7选自下组：H、C1-C4的烷基、C1-C4的卤代烷基；
R8为无，H、或R8与R2共同构成-R”-O-R”-，其中，所述的R”为无或C1-C4的亚烷基：
R9为无，H、或R9为-OH；
所述的
Figure PCTCN2017073964-appb-000003
为双键或单键；
所述的“取代”指基团上的一个或多个氢原子被选自下组的取代基取代：羧基、未取代或卤代的C1-C6烷基、未取代或卤代的C3-C6的环烷基、未取代或卤代的C2-C10的酯基、卤素、C1-C10烷基-氧基、未取代或卤代的C2-C10酰基、羟基、羟基-C1-C10的亚烷基、未取代或卤代的苯基；
且当母核为I-1结构时，R2、R5和R6中至少有一个为选自下组的基团：-OR或取代或未取代的C2-C10的酯基；或R5和R6共同形成-OC(CH)2-O-结构；
当母核为I-3结构时，R2、R4和R6中至少有一个为氧原子；
或所述的式I化合物具有以下结构：
Figure PCTCN2017073964-appb-000004
在另一优选例中，在所述化合物中，各手性碳原子可以任选地为R构型或S构型。
在另一优选例中，所述的C2-C10的酰基指C2-C10的酸失去一个羟基形成的基团，且所述的酸选自下组：羧酸、碳酸。
在另一优选例中，所述的羧酸包括饱和羧酸、不饱和羧酸、或芳香族羧酸。
在另一优选例中，所述的R2为C2-C10的芳香酯基，优选地选自下组：苯甲酰基、或取代的苯甲酰基；其中，所述的取代是指苯甲酰基上的一个或多个氢原子被选自下组的取代基取代：卤素、硝基、氨基、甲基、甲氧基。
在另一优选例中，所述的R2为碳酸酯基。
在另一优选例中，当所述的R2为羟基时，还任选地带有羟基保护基；较佳地，所述的羟 基保护基为带酰氯或者游离酸的保护基。
在另一优选例中，所述的R1、R3和R7各自独立地为C1-C4的烷基。
在另一优选例中，所述的R4为H或羟基。
在另一优选例中，所述的R5为H或羟基。
在另一优选例中，所述的R1为异丙基。
在另一优选例中，所述的R3和R7各自独立地为甲基。
在另一优选例中，所述的式I化合物具有以下式Ia所示的结构：
Figure PCTCN2017073964-appb-000005
式中，
R1选自下组：取代或未取代的C1-C4的烷基、C1-C4的卤代烷基；其中，所述的取代指基团上的一个或多个氢原子被C1-C4的酯基取代；
R2为-OH、-OR或取代或未取代的C2-C10的酯基；
R7选自下组：C1-C4的烷基、C1-C4的卤代烷基；
其余各基团的定义如本发明第一方面中所述。
在另一优选例中，所述的式I化合物具有以下式Ib所示的结构：
Figure PCTCN2017073964-appb-000006
式中，
R1选自下组：取代或未取代的C1-C4的烷基、C1-C4的卤代烷基；
R2与R8共同构成-O-；
R7选自下组：C1-C4的烷基、C1-C4的卤代烷基；
其余各基团的定义如本发明第一方面中所述。
在另一优选例中，R5选自下组：卤素、氰基、羟基、C1-C4的烷氧基、-COOR、取代或未取代的C2-C10的酯基、取代或未取代C1-C10烷基、取代或未取代的C1-C10卤代烷基、-NH-R；其中，R选自下组：H、C1-C4的酰基、C1-C4的烷基、C1-C4的卤代烷基、取代或未取代(优选为取代)的C7-C11芳基-酰基。
在另一优选例中，R6选自下组：卤素、氰基、羧基、羟基、C1-C4的烷氧基、取代或未取代的C2-C10的酯基、取代或未取代C1-C10烷基、取代或未取代的C1-C10卤代烷基、取代或未取代(优选为取代)的C7-C11芳基-酰基、-NH-R；其中，R选自下组：H、C1-C4的酰基、C1-C4的烷基、C1-C4的卤代烷基。
在另一优选例中，在另一优选例中，R2选自下组：O-R’，其中，所述的R’选自下组：H、取代或未取代的C2-C10的酰基、或取代或未取代的C1-C10的烷基；
R4选自下组：H、卤素、氰基、羧基、羟基、C1-C4的烷氧基、取代或未取代的C2-C10的 酯基；
R5选自下组：H、卤素、氰基、羟基、C1-C4的烷氧基、-COOR、取代或未取代的C2-C10的酯基、取代或未取代C1-C10烷基、取代或未取代的C1-C10卤代烷基、-NH-R；其中，R选自下组：H、C1-C4的酰基、C1-C4的烷基、C1-C4的卤代烷基、取代或未取代(优选为取代)的C7-C11芳基-酰基；
R6选自下组：H、卤素、氰基、羧基、羟基、C1-C4的烷氧基、取代或未取代的C2-C10的酯基、取代或未取代C1-C10烷基、取代或未取代的C1-C10卤代烷基、取代或未取代(优选为取代)的C7-C11芳基-酰基、-NH-R；其中，R选自下组：H、C1-C4的酰基、C1-C4的烷基、C1-C4的卤代烷基；
所述的“取代”指基团上的一个或多个氢原子被选自下组的取代基取代：羧基、苯基、C3-C6的环烷基、C1-C10的酯基、卤素、C1-C10烷基-氧基、C2-C10酰基、羟基、羟基-C1-C10的亚烷基。
在另一优选例中，所述的R2、R4、R5和R6各自独立地为选自下组的基团：-OR’，且所述的R’选自下组：
Figure PCTCN2017073964-appb-000007
在另一优选例中，所述的A、R1、R2、R3、R4、R5、R6、R7、R8和R9为如实施例中各具体化合物所对应的基团。
在另一优选例中，所述的化合物具有选自下组的结构：
Figure PCTCN2017073964-appb-000008
Figure PCTCN2017073964-appb-000009
或所述的化合物为如式3B、4A或4B所示，其中，Ra、Rb、Rc和Rd的定义如表1、表2和表3中所述：
Figure PCTCN2017073964-appb-000010
本发明的第二方面，提供了一种如本发明第一方面所述的化合物用于选自下组的一种或多种用途：
a)制备用于降血脂的药物组合物；
b)制备用于降低低密度脂蛋白(LDL)含量的药物组合物；
c)制备用于稳定LDLR基因稳定性的药物组合物；
d)制备用于上调LDLR基因表达的药物组合物；
e)制备用于增加肝细胞表面LDLR受体数量的药物组合物；
f)制备用于减少LDLR受体降解的药物组合物；
g)制备用于降低血液中TC和/或TG浓度的药物组合物；
h)制备用于增加血液中高密度脂蛋白(HDL)浓度的药物组合物；
i)制备用于改善肝功能损伤的药物组合物。
在另一优选例中，所述的肝功能损伤为高脂诱导的肝功能损伤。
在另一优选例中，所述的改善肝功能损伤为降低ALT\AST指标。
在另一优选例中，所述的药物组合物还用于选自下组的用途：
j)(体外非治疗性地)增加肝细胞对LDL的摄取率；
k)(体外非治疗性地)上调肝细胞LDLR基因表达水平。
在另一优选例中，所述的药物组合物中，所述式I化合物的有效剂量为0.1-50mg/kg体重，较佳地为1-20mg/kg体重。
在另一优选例中，所述的药物组合物为选自下组的剂型：口服剂型、注射剂型。
在另一优选例中，所述的剂型包括片剂、颗粒剂、胶囊剂、丸剂等。
本发明的第三方面，提供了一种药盒，所述药盒含有：
(i)第一容器，以及装于该第一容器中的活性成分(a)式I化合物；或含有活性成分(a)的药物；
(ii)第二容器，以及装于该第二容器中的活性成分(b)他汀类药物，或其药学上可接受的盐；或含有活性成分(b)的药物；以及
(iii)说明书，所述说明书中记载了联合给予活性成分(a)和活性成分(b)从而降低使用对象体内低密度脂蛋白含量的说明。
在另一优选例中，所述的式I化合物如本发明第一方面中所述。
在另一优选例中，所述的使用对象为哺乳动物，较佳地为人。
在另一优选例中，所述的他汀类药物选自下组：普伐他汀、阿伐他汀，或其组合。
本发明的第四方面，提供了一种如下式I所示的化合物，或其药学上可接受的盐的用途：
Figure PCTCN2017073964-appb-000011
其中，A、R1、R2、R3、R4、R5、R6、R7、R8和R9等定义如本发明第一方面中所述，并且所述的
Figure PCTCN2017073964-appb-000012
为双键或单键；
其中，所述的化合物被用于选自下组的用途：
a)降低施用对象的血脂水平；
b)降低施用对象低密度脂蛋白(LDL)含量；
c)稳定施用对象LDLR基因稳定性；
d)上调施用对象LDLR基因表达；
e)增加施用对象肝细胞表面LDLR受体数量；
f)减少施用对象LDLR受体降解；
g)降低施用对象血液中TC和/或TG浓度；
h)增加施用对象血液中高密度脂蛋白(HDL)浓度；
i)改善施用对象肝功能损伤。
在另一优选例中，所述的施用对象为哺乳动物，较佳地为人。
在另一优选例中，所述的肝功能损伤为高脂诱导的肝功能损伤。
在另一优选例中，所述的改善肝功能损伤为降低ALT\AST指标。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
本发明人经过长期而深入的研究，意外地发现，竹柏内酯类化合物具有良好的降血脂活性，且活性优于现有通用的降血脂化合物他汀类药物。所述的化合物可以时间依赖性及剂量依赖性地在体内或体外降低总胆固醇、甘油三酯、低密度脂蛋白浓度，并增加高密度脂蛋白浓度。基于上述发现，发明人完成了本发明。
术语
如本文所用，术语“C1-C4烷基”或“C1-C10烷基”指具有1～4个或1-10个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基，或类似基团。
术语“C3-C6环烷基”指具有3～6个碳原子的环烷基，例如环丙基、环丁基、甲基环丁基、环戊基，或类似基团。
如本文所用，术语“C1-C10酰基”或“C1-C4酰基”指形如“具有0～9个或0-3个碳原子的直链或支链烷基/环烷基/芳基/杂芳基/烯基/炔基-羰基”结构的取代基，如乙酰基、丙酰基、丁酰基，或类似基团。
术语“C2-C30酯基”指形如-OOC-R'所示结构的基团，其中，所述的R'为具有1～9个碳原子的选自下组的基团：直链或支链烷基、环烷基、烯基、炔基、芳基或杂芳基结构的取代基，如乙酰基、丙酰基、丁酰基，或类似基团。其中，所述的R'还可以进一步被取代，例如被选自下组的一个或多个取代基所取代：卤素、-OH、-COOH、-COO(C1-C6烷基)、未取代或卤代的苯基、未取代或卤代的C1-C6烷基、未取代或卤代的C2-C6酰基、未取代或卤代的C1-C6烷基-羟基。
术语“C1-C4亚烷基”指如上文所述的C1～C4烷基失去一个氢原子之后形成的基团，例如-CH2-、-CH2-CH2-，或类似基团。
术语“卤素”指F、Cl、Br和I。
术语“C6-C10芳基”指具有6-10个碳原子的芳基，例如苯基、萘基等，所述的芳基可以是取代或未取代的。
术语“C1-C10杂芳基”指具有1-10个碳原子和一个或多个选自O、S和/或N的杂原子的杂芳基，优选C2-C8杂芳基。所述的杂芳基可以是取代或未取代的。
本发明中，术语“含有”、“包含”或“包括”表示各种成分可一起应用于本发明的混合物或组合物中。因此，术语“主要由...组成”和“由...组成”包含在术语“含有”中。
本发明中，术语“药学上可接受的”成分是指适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)，即有合理的效益/风险比的物质。
本发明中，术语“有效量”指治疗剂治疗、缓解或预防目标疾病或状况的量，或是表现出可检测的治疗或预防效果的量。对于某一对象的精确有效量取决于该对象的体型和健康状况、病症的性质和程度、以及选择给予的治疗剂和/或治疗剂的组合。因此，预先指定准确的有效量是没用的。然而，对于某给定的状况而言，可以用常规实验来确定该有效量，临床医师是能够判断出来的。
在本文中，除特别说明之处，术语“取代”指基团上的一个或多个氢原子被选自下组的取代基取代：卤素、-OH、-COOH、-COO(C1-C6烷基)、未取代或卤代的苯基、未取代或卤代的C1-C6烷基、未取代或卤代的C2-C6酰基、未取代或卤代的C1-C6烷基-羟基。
除非特别说明，本发明中，所有出现的化合物均意在包括所有可能的光学异构体，如单一手性的化合物，或各种不同手性化合物的混合物(即外消旋体)。本发明的所有化合物之中，各手性碳原子可以任选地为R构型或S构型，或R构型和S构型的混合物。
如本文所用，术语“本发明化合物”指式I所示的化合物。该术语还包括及式I化合物的各种晶型形式、药学上可接受的盐、水合物或溶剂合物。
如本文所用，术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸，甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等有机酸；以及天冬氨酸、谷氨酸等酸性氨基酸。
基于竹柏内酯母核的化合物
竹柏(Podocarpus nagi)为罗汉松科(Podocarpaceae)罗汉松属植物，主要化学成分为降二萜双内酯，具有抗肿瘤、抗菌、杀虫、生长调节抑制等作用。本发明人首次发现，竹柏内酯类化 合物可以增强肝细胞摄取低密度脂蛋白的能力。体内药效学评价发现所述的化合物可以非常显著的降低高脂诱导脂质代谢紊乱的金黄地鼠的总胆固醇，甘油三酯，低密度脂蛋白等血脂指标，提高高密度脂蛋白在总胆固醇中的比例，改善其肝功能，同时可以缓解高脂饮食引起的脂肪肝形成，因此是一种潜在的降脂化合物。
具体地，本发明人以从竹柏树皮、树枝、树叶和果实中分离得到的降二萜内酯类化合物2、3、4和12等为原料，进行了一系列结构改造，经过如酯化，消除，氢化等步骤，得到式2-B1、3-1～3-4、3-B1～3-B10、7-A1、12-1和4-A以及4-B化合物：
Figure PCTCN2017073964-appb-000013
其中Rc和Rd如下表1中所示：
表1
化合物编号 Rc取代基 Rd取代基 ESI-MS m/z[M+H]+
3-B1 H A1 391.2
3-B2 H A2 405.1
3-B3 H A8 433.2
3-B4 A1 A1 433.2
3-B5 A2 A2 461.2
3-B6 C1 C1 485.2
3-B7 C2 C2 513.4
3-B8 D1 D1 449.2
3-B9 D3 D3 463.2
3-B10 P1 P2 557.2
Figure PCTCN2017073964-appb-000014
其中Ra和Rb如下表2中所示：
表2
化合物编号 Ra取代基 Rb取代基 ESI-MS m/z[M+H]+
4-A1 Me H 363.2
4-A2 H Me 363.2
4-A3 Me Me 377.2
4-A4 Et H 377.2
4-A5 H Et 377.2
4-A6 Et Et 405.3
4-A7 H -CH2-CH＝CH2 389.2
4-A8 -CH2-CH＝CH2 -CH2-CH＝CH2 429.3
Figure PCTCN2017073964-appb-000015
其中Rc和Rd如下表3中所示：
表3
化合物编号 Rc取代基 Rd取代基 ESI-MS m/z[M+H]+
4-B1 A1 H 391.2
4-B2 A2 H 405.3
4-B3 A3 H 419.3
4-B4 A4 H 419.3
4-B5 A5 H 447.4
4-B6 A6 H 503.4
4-B7 A1 A1 433.2
4-B8 A5 A5 545.4
4-B9 C1 H 417.2
4-B10 C3 H 445.3
4-B11 C4 H 459.3
4-B12 C5 H 487.4
4-B13 C1 C1 485.3
4-B14 C2 C2 513.3
4-B15 D1 H 449.3
4-B16 D3 H 463.3
4-B17 D2 H 477.4
4-B18 D2 D2 605.5
4-B19 H1 H 443.3
4-B20 H2 H 459.3
4-B21 H3 H 488.2
4-B22 H4 H 509.3
4-B23 H5 H 471.3
4-B24 H6 H 550.3
4-B25 P1 H 453.3
4-B26 P2 H 509.3
4-B27 P3 H 513.3
4-B28 P4 H 479.3
4-B29 P5 H 481.3
4-B30 P1 P1 557.3
4-B31 P6 H 488.3
4-B32 P7 H 471.3
4-B33 P8 H 488.3
4-B34 P9 H 532.3
4-B35 P11 H 532.3
4-B36 P10 H 488.3
4-B37 P6 P6 666.3
4-B38 V1 H 417.3
4-B39 V2 V2 485.3
4-B40 A7 H 467.3
4-B41 H A1 391.2
4-B42 H A2 419.3
4-B43 H A3 419.3
4-B44 H P11 531.3
其中，上述的反应可以由本领域技术人员结合本领域的现有技术，根据实际反应条件进 行设计，本发明实施例中提供了一些示例性的方法(见实施例2-8中详述)。
竹柏内酯类化合物降低LDL浓度的用途
低密度脂蛋白受体(LDLR)是肝脏回收摄取低密度脂蛋白LDL的主要受体，于1974年被发现，并开始广泛开始其结构和功能方面的相关研究。LDLR由839个氨基酸残基构成，合成于粗面内质网，经过剪接和糖基化修饰后转运至细胞膜上的大小约160KD胞膜糖蛋白。LDLR基因突变是遗传性高血脂症的主要原因之一。
LDLR基因的表达水平和血浆胆固醇水平密切相关，位于LDLR基因启动子部位的SRE调节位点受细胞内SREBP1/2的调节，而后者是与胞内胆固醇水平密切相关。但细胞内胆固醇水平高时，SREBP被固定于ER膜上，不能进入高尔基体激活；当细胞内胆固醇水平降低时，SREBP在胆固醇的作用下被释放，随后转移到高尔基体内被剪接成活化蛋白，进入细胞核内，作为转录因子调节含有SRE的基因转录，包括LDLR，HMG-CoA R等胆固醇和脂肪酸代谢相关的基因。除了胆固醇水平调节，LDLR基因还受许多细胞因子和生长因子和激素的调节，如雌激素，肿瘤坏死因子，佛波酯，胰岛素等。他汀类药物是HMG-CoA R的抑制剂，本类药物通过抑制胆固醇的合成，降低细胞内胆固醇的含量，从而负反馈型上调LDLR的细胞内水平，达到进一步增强其降脂功能的作用。调节LDLR水平和功能是调节机体血脂水平的一个重要途径。
LDLR基因另外一个特点是其基因具有不稳定的特性，基因合成后在很短的时间内即降解。这个过程可能与LDLR基因本身的3′端序列有关。LDLR基因3′端含有AU富集区，此区被看做是基因不稳定的一个特征性序列，很多RNA结合蛋白可以与此序列结合，从而引起mRNA的迅速降解。小檗碱被报道可以增加LDLR基因的稳定性，提高其基因和蛋白水平，从而增加LDLR的功能，降低血浆LDL水平，经过临床试验已证实小檗碱是一个有效的通过调节LDLR水平发挥降脂效果的化合物。
本申请中，所述的竹柏内酯类化合物可以在体内或体外上调LDLR蛋白的基因表达水平，同时增加LDLR蛋白本身的稳定性，从而使得LDLR蛋白水平上调，进而促进肝细胞对于低密度脂蛋白的摄取。在体内实验中，所述的竹柏内酯类化合物对LDLR蛋白水平有上调作用，进而促进肝细胞对于低密度脂蛋白的摄取，从而降低血液中LDL浓度，达到清除LDL的目的。
除此之外，本发明人发现，在体内实验中，除了LDL水平得到下降之外，受试动物的血清总胆固醇(TC)水平，甘油三酯(TG)水平也同样得到下调。同时，高密度脂蛋白在总胆固醇中的比例显著提高。这说明竹柏内酯类化合物对于高脂诱导的脂肪肝形成具有减缓作用。
另外，在体内实验中，给予竹柏内酯类化合物对于高脂饮食导致的肝功能损伤也产生了一定的改善，具体表现为血液中天冬氨酸氨基转移酶、丙氨酸氨基转移酶指标水平的改善。
药物组合物和施用方法
由于本发明化合物具有优异的降低血液中LDL含量的活性，因此本发明化合物及其各种晶型，药学上可接受的无机或有机盐，水合物或溶剂合物，以及含有本发明化合物为主要活性成分的药物组合物可用于治疗、预防以及缓解由于血液中LDL含量过高导致的疾病，如脂肪肝等。
本发明的药物组合物包含安全有效量范围内的本发明化合物或其药理上可接受的盐及药理上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-3000(活性剂量范围3-30mg/kg)mg本发明化合物/剂，更佳地，含有10-2000mg本发明化合物/剂。较佳地，所述的“一剂”为一个 胶囊或药片。
“药学上可以接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温
Figure PCTCN2017073964-appb-000016
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、瘤内、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明化合物可以单独给药，或者与其他药学上可接受的化合物联合给药。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选6～600mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这 些都是熟练医师技能范围之内的。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
与现有技术相比，本发明的主要优点包括：
1.对竹柏内酯类化合物进行了改造，使其具有相较于天然竹柏内酯类化合物而言更好的降血脂活性(体外活性与现有技术中已知的药物普伐他汀和阿伐他汀相当，或优于上述化合物)；
2.本发明的竹柏内酯化合物的降血脂活性具有明显的时间依赖性和剂量依赖性；
3.本发明的竹柏内酯化合物可以有效降低血液中LDL浓度作用机制：通过稳定LDLR基因的稳定性上调LDLR基因的表达，从而增加肝细胞表面LDLR受体数量，达到清除LDL的目的；
4.本发明化合物的体外、体内活性均有效，活性剂量低，可以在低达3mg/kg的剂量下就表现出降血脂效果，远低于与已报道的普伐他汀和小檗碱类药物起效剂量；
5.除了降低LDL外，还能降低血液中TC、TG浓度，并增加HDL(高密度脂蛋白，对机体有利的一种蛋白)浓度；
6.除了降低血脂指标外，对高脂诱导的肝功能损伤有明显的改善功能，能够显著降低ALT\AST(衡量肝功能指标)。
仪器和器材
此外光谱UV：SHIMADIU UV-2550和Beckman DU-7紫外可见光光谱仪；
红外光谱IR：Perkin-Elmer 577型红外分光光度仪；
低分辨质谱LR-EIMS：Finnigan MAT-95；
高分辨质谱HR-EIMS：Kratos 1H spectrometer；
核磁共振谱：Varian INOVA 600型核磁共振仪，Bruker AM-500，AM-400，AM-300型核磁共振仪，δ(ppm)，以TMS为内标；
LC-MS：Agilent 1100液相偶联Bruker esquire质谱仪；
分析型HPLC：Waters 2690Separate Model,Waters PDA 996检测器偶联Alltch ELSD 2000检测器，Millennium 2000操作系统，Waters RP18column(5.0×125mm,5μm,Waters)，流速1.0ml/min，CH3CN(Merck,Germany)，H2O(乐百氏)；Jasco HPLC(Chiralcel IA column,5μm,150×4.6mm),流速0.6ml/min,hexane/ethanol(7:3).
HPLC-MS：Waters 2695Separate Model,Waters PDA 2998检测器偶联Alltch ELSD 2424检测器，3100Ms detector，SunFireTM C-18column(4.6×100mm,3.5μm,Waters)，流速1.0ml/min，CH3CN(Merck,Germany)，H2O(乐百氏)；Jasco HPLC(Chiralcel IA column,5μm,150×4.6mm),流速0.6ml/min,hexane/ethanol(7:3).
制备型HPLC：Varian SD1instrument,Varians 320单波长检测器，C18column(220×25nm,10μm,Waters)，流速15ml/min,CH3CN(Merck,Germany)，H2O(乐百氏)；
SpectraMax M2e多功能酶标仪(美国分子仪器公司)；
电泳仪和半干电转印槽(Bio-Rad Laboratories,Hercules,CA)；
PCR仪(Bio-Rad Laboratories,Hercules,CA)；
台式冷冻离心机(德国Hettich公司)；
752C紫外可见分光光度计(上海第三分析仪器)；
DK-8B电热恒温水槽(上海精宏实验设备有限公司)；
REVCO二氧化碳培养箱(美国REVCO公司)；
试剂与材料
柱层析硅胶：100-200目，200-300目硅胶和硅胶H均为青岛海洋化工厂生产；
TLC薄层制备板：HSGF254为烟台化工厂生产。
MCI树脂：CHP20P(75-150μm)为三菱公司生产；
葡聚糖凝胶Sephadex LH-20：Pharmacia Biotech AB，Uppsala，Sweden。
显色剂：10％硫酸-香兰醛溶液，碘；
植物材料：竹柏枝叶采于广西田林，竹柏树皮采于海南琼中，由上海药物研究所标本室沈金贵副教授采集并鉴定，标本保存于本所标本室。
DMEM、胎牛血清(fetal bovine serum，FBS)购自Gibco-BRL(Grand Island,NY)；DiI购自biotuim(Hayward,CA)；β-actin抗体购自Cell Signaling Technology(Beverly,MA)；LDLR,PCSK9抗体购自Abcam(Cambridge,United Kingdom)；PCR引物在上海生工合成。蛋白酶抑制剂Cocktail购自Calbiochem(San Diego,CA)；Immobilon-P transfer转移膜(PVDF)购自Millipore Corporation(Bedford,MA)；增强化学发光试剂(Enhanced chemiluminescence reagents，ECL)购自Pierce(Rockford,IL)；医用X-光胶片购自中国碧云天公司；TRIzol reagent购自Invitrogen(Carlsbad,CA)；M-MLV反转录酶购自Promega(Madison,WI)；Real time PCR反应试剂购自Bio-Rad Laboratories；金黄地鼠高脂饲料(0.12％胆固醇，10％椰子油)购自上海斯莱克斯公司。蛋白测定试剂盒购自中国碧云天公司；肝脏组织甘油三酯TG，总胆固醇TC含量测定试剂盒购自上海荣盛生物科技公司。血液指标(总胆固醇TC，甘油三酯TG，高密度脂蛋白HDL，低密度脂蛋白LDL，丙氨酸氨基转移酶ALT，天冬氨酸氨基转移酶AST)测定试剂盒购自四川迈克生物公司。其他试剂除特殊说明外均购自上海国药集团。
实验动物为雄性叙利亚金黄地鼠，体重100±10g，购自中国科学院上海实验动物中心。动物放置于中国科学院上海药物研究所SPF动物房(温度23±1℃；湿度60％)，自然昼/夜循环，用标准的食物和水饲养。(实验批准号：SIMM-AE-2010-10-WYP-01)
根据文献报道的方法从竹柏枝叶中得到化合物2(3-deoxynagilactone)、3(nagilactone A)、4(1-deoxy-2-hydroxy-nagilactone A)、7(nagilactone B)、12(nagilactone E)并作为原料进一步进行结构修饰。
实施例1化合物2、3、4、7、12的提取分离方法
竹柏树皮(16.2kg)打粉后用95％乙醇浸泡，室温提取三次，合并提取液并浓缩至无醇味，然后转移至龙头瓶中，分别用石油醚、氯仿、乙酸乙酯、正丁醇与水分配，得到石油醚部位(95g)、氯仿部位(15g)、乙酸乙酯部位(255g)，氯仿部位和乙酸乙酯部位合并，硅胶柱纯化(200-300目，青岛海洋化工生产)，用氯仿：甲醇系统200：1，100：1，50：1，30：1，20：1，10：1，5：1，丙酮洗脱，TLC检测后合并得到12个馏分，馏分2通过凝胶柱层析(氯仿/甲醇1：1)，再通过硅胶柱层析(氯仿/丙酮50：1-10：1)得到化合物2(188mg)，12(209mg)。馏分6经过硅胶和凝胶柱层析以及Pre-HPLC制备分离分别得到化合物3(1.2g)和4(2.1g)，馏分7和8合并经MCI和硅胶柱层析得到化合物7(380mg)。
竹柏枝叶(30kg)粉碎，用95％乙醇浸泡三次，浓缩提取液至无醇味，然后依次用石油醚、 二氯甲烷、乙酸乙酯和正丁醇分别与水分配，得到二氯甲烷部位(175g)和乙酸乙酯部位(105g)，氯仿和乙酸乙酯部位分别经硅胶、凝胶、MCI、Pre-HPLC等柱层析得到化合物2(800mg)，3(1.2g)，4(2.1g)，7(1.1g)。
化合物的特性如下：
3-Deoxynagilactone(2)
白色粉末，MF:C19H22O6,ESIMS m/z 347.1[M+H]+,391.3[M+HCOO]-；1H NMR(300MHz,CDCl3):δ5.90(s,1H,H-11),5.29(d,J＝8.9Hz,1H,H-7),4.98(dd,J＝8.7,6.0Hz,1H,H-6),3.50(m,1H,H-2),3.25(d,J＝6.8Hz,1H,H-15),3.23(d,J＝2.3Hz,1H,H-1),2.44(d,J＝14.5,2.3Hz,1H,H-3a),1.85(d,J＝14.4Hz,1H,H-3b),1.71(d,J＝5.7Hz,1H,H-5),1.55(s,3H,H-20),1.51(s,3H,H-18),1.32(d,J＝6.6Hz,3H,H-16),1.25(d,J＝6.6Hz,3H,H-17).
Nagilactone A(3)
白色粉末，MF:C19H24O6.ESIMS m/z:349.2[M+H]+,719.3[2M+Na]+.393.2[M+HCOO]-,1H NMR(300MHz,Pyr):δ1.26(d,J＝6.6Hz,3H),1.30(d,J＝6.6Hz,3H),1.33(s,3H,H-18),1.55(m,1H),1.81(d,J＝5.9Hz,1H,H-5),2.01(s,3H,H-17),2.55(m,1H),3.50(m,1H,H-15),4.18(m,1H,H-1),5.17(dd,J＝5.7,8.6Hz,1H,H-6),5.67(d,J＝8.2Hz,1H,H-7),7.38(s,1H,H-11).
1-Deoxy-2-hydroxy-nagilactone A(4)
白色粉末，MF:C19H24O6.ESIMS m/z:349.2[M+H]+,719.3[2M+Na]+,393.7[M+HCOO]-,1H NMR(300MHz,Pyr):δ1.26(d,J＝6.7Hz,3H,H-17),1.33(d,J＝6.7Hz,3H,H-16),1.45(s,3H,H-18),1.84(s,3H,H-20),1.82(d,J＝5.3Hz,H-5),2.05(dd,J＝13.3,6.5Hz,1H),2.24(dd,J＝13.7,4.9Hz,1H),2.53(m,1H),2.69(t,J＝13.1Hz,1H),3.48(m,1H,H-15),4.35(br m,1H,H-2),5.11(dd,J＝6.5,8.5Hz,1H,H-6),5.63(d,J＝8.4Hz,1H,H-7),6.33(s,1H,H-11),7.80(brs,1H).
Nagilactone B(7)
白色粉末，MF:C19H24O7,ESIMS m/z:365.2[M+H]+,751.4[2M+Na]+,363.3[M-H]-,727.5[2M-H]-,1H NMR(300MHz,Pyr):δ7.04(s,1H,H-11),5.66(d,J＝8.7Hz,1H,H-7),5.18(m,1H,H-6),4.33(m,2H),3.50(m,1H,H-15),2.76(t,J＝12.5Hz,1H),2.13(d,J＝17.1Hz,1H),2.03(s,3H,H-20),1.92(d,J＝6.8Hz,1H,H-5),1.48(s,3H,H-18),1.33(d,J＝6.8Hz,3H,H-16),1.27(d,J＝6.7Hz,3H,H-17).
Nagilactone E(12)
白色粉末，MF:C19H24O6,ESIMS m/z:349.1[M+H]+,719.3[2M+Na]+,347.0[M-H]-.1H NMR(300MHz,CDCl3):δ5.97(s,1H,H-11),4.99(d,J＝3.7Hz,1H,H-6),4.42(d,J＝3.8Hz,1H,H-14),3.97(s,1H,H-7),3.67(m,1H,H-3),3.47(d,J＝10.0Hz,1H),2.17(m,1H),1.93–1.72(m,3H),1.59(s,3H),1.52(s,3H),1.24(d,J＝8.1Hz,3H),1.11(dd,J＝6.7,4.5Hz,6H).
实施例2化合物2-B1的合成
取化合物2(30mg，0.08mmol)溶于无水吡啶(5ml)，加入过量的醋酸酐(0.02ml，2N)，室温搅拌过夜，TLC检测反应确认结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以P-TLC纯化(氯仿/甲醇＝50/1(V/V))得到白色固体粉末2-B1(25mg，74％)。2-B1:MF:C21H24O7.MW:388.ESIMS m/z:389.2[M+H]+,447.2[M+CH3COO]+.1H NMR (300MHz,CDCl3):δ6.28(d,J＝9.6Hz,1H,H-7),5.93(s,1H,H-11),5.06(m,1H),3.21(s,1H),2.93(m,1H),2.45(m,1H),2.15(s,3H,Ac),1.87(m,1H),1.72(s,1H),1.52(s,3H),1.44(s,3H),1.29–1.18(m,6H,H-16,17).
实施例3化合物3-1～3-4的合成
实验1：化合物3-1和3-2的合成
取化合物3(30mg，0.09mmol)，加入Pd/C(10％)催化剂5mg，加入甲醇10ml，室温搅拌，通入H2，反应过夜，TLC检测反应确认结束，将反应液中的不溶物过滤，滤液浓缩，LC/MS检测有2个主要峰，用P-HPLC制备，得白色固体3-1(14mg,46％)和3-2(8mg,28％)。
3-1:C19H28O6.ESIMS m/z:353.1[M+H]+,375.1[M+Na]+,705.4[2M+H]+,737.4[2M+Na]+.411.1[M+CH3COO]-,HRESIMS m/z:375.1763[M+Na]+(calcd for C19H28O6Na,375.1784),1H NMR(300MHz,CDCl3):δ4.69(s,1H),3.89(1H),3.42(m,1H),3.09(1H),2.71–2.31(m,3H),2.25(s,1H),2.09(1H),1.78(m,1H),1.60(m,1H),1.30(s,3H),1.25(s,2H),1.16(m,3H),1.10(s,2H),0.94(d,J＝6.8Hz,2H).
3-2:C19H28O5.ESIMS m/z:337.1[M+H]+,335.1[M-H]-,335.1[M-H]-,HRESIMS m/z:359.1850[M+Na]+(calcd for C19H28O5Na,359.1834),1H NMR (300MHz,CDCl3):δ3.99–3.89(m,1H),3.07(m,1H),2.86(m,1H),2.70(m,1H),2.47–2.29(m,2H),2.29–2.15(m,1H),2.10(m,1H),1.92(s,1H),1.79–1.57(m,3H),1.52(d,J＝11.6Hz,1H),1.26(s,3H),1.20(m,1H),1.07(s,3H),0.91(d,J＝6.4Hz,3H),0.80(d,J＝6.4Hz,3H).
实验2：化合物3-3的和合成
取化合物3(16mg，0.04mmol)，加入甲醇5ml(干燥)，加入过量的硼氢化钠(30mg)，室温搅拌过夜，TLC检测反应确认结束，反应液浓缩，氯仿萃取，萃取液用纯水洗涤，无水硫酸钠干燥，浓缩后P-TLC纯化得白色固体3-3(4mg,30％)。
3-3:C19H24O5.MW:332.ESIMS m/z:333.3[M+H]+,687.4[2M+Na]+,331.1[M-H]-,HRESIMS m/z:355.1529[M+Na]+(calcd for C19H24O5Na,359.1521),1H NMR(300MHz,CDCl3):δ7.53(s,1H,H-11),5.11(t,J＝5.4Hz,2H),4.02(d,J＝5.3Hz,1H),2.57(m,5H),2.28(d,J＝5.8Hz,1H),2.17(s,1H),2.12(m,1H),1.93(d,J＝7.9Hz,1H),1.60(s,3H),1.49(s,3H),1.25(s,3H),1.08(d,J＝6.7Hz,3H),0.99(d,J＝6.7Hz,3H).
实验3：化合物3-4的合成
取化合物3(20mg，0.06mmol)，加入干燥二氯甲烷5ml和3A分子筛，室温搅拌，加入PCC(100mg，8N)，TLC监测反应，2h后将溶液中的不溶物过滤，滤液浓缩，经硅胶柱层析纯化后得白色固体3-4(10mg，48％)。
3-4:C19H22O7.ESIMS m/z:385.2[M+Na]+,747.3[2M+Na]+,361.4[M-H]-,723.6[2M-H]-,HRESIMS m/z:385.1251[M+Na]+(calcd for C19H22O7Na,385.1263),1H NMR(300MHz,CDCl3):δ6.29(s,1H,H-11),6.09(d,J＝4.0Hz,1H),5.04(t,J＝4.7Hz,1H),4.52(d,J＝9.0Hz,1H),4.02(dd,J＝14.7,8.4Hz,1H),2.62(s,2H),2.47–2.30(m,2H),2.17(s,1H),2.07–1.96(m,3H),1.85(m,2H),1.80–1.68(m,2H),1.66–1.47(m,2H),1.30(d,J＝6.8Hz,3H),1.27(d,J＝6.2Hz,3H),1.25(s,3H),1.21(s,3H),1.07(d,J＝6.6Hz,3H),0.93(d,J＝6.7Hz,3H).13C NMR(100MHz,MeOD):δ209.8s,209.6s,183.2s,166.0s,153.4s,125.6d,104.1s,74.2d,56.9d,50.7d,46.6s,44.2s,38.2d,37.3t,29.5t,26.3q,24.0q,18.5q,17.7q.
实施例4：化合物3-B1～3-B10的合成
实验1：化合物3-B1和3-B4的合成
取化合物3(70mg，0.2mmol)溶于无水吡啶(5ml)，加入过量的醋酸酐(0.04ml，2N)，室温搅拌过夜，TLC检测反应确认结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以Pre-TLC纯 化(氯仿/甲醇＝50/1(V/V))得到白色固体粉末3-B1(20mg，26％)和3-B4(41mg，47％)。
3-B1：MF:C21H26O7.ESIMS m/z:391.2[M+H]+,389.1[M-H]-,449.2[M+CH3COO]-.1H NMR(300MHz,CDCl3):δ6.80(s,1H,H-11),6.30(d,J＝9.1Hz,1H,H-7),5.08(dd,J＝9.1,5.5Hz,1H,H-6),4.01(s,1H,H-1),2.96(dt,J＝13.2,6.8Hz,1H,H-15),2.44–2.26(m,1H),2.16(d,J＝8.6Hz,3H,Ac),1.99–1.85(m,1H),1.82(d,J＝5.6Hz,1H),1.74(dd,J＝14.9,7.0Hz,1H),1.62–1.50(m,1H),1.35(s,3H,H-20),1.30(s,3H,H-19),1.24(t,J＝5.9Hz,6H,H-16,17).
3-B4:MF:C23H28O8.ESIMS m/z:433.2[M+H]+,455.2[M+Na]+.1H NMR(300MHz,CDCl3):δ6.32(d,J＝9.2Hz,1H,H-7),5.85(s,1H,H-11),5.13–4.99(m,2H,H-1,6),2.96(m,1H,H-15),2.30(dd,J＝14.3,4.9Hz,1H),2.17(s,3H,Ac),2.14(s,3H,Ac),2.06(d,J＝7.0Hz,1H),1.88(d,J＝5.9Hz,1H),1.80–1.65(m,1H),1.45(s,3H,H-18),1.32(s,3H,H-19),1.23(t,J＝6.7Hz,6H,H-16,17).
实验2：化合物3-B2和3-B5的合成
取化合物3(22mg，0.06mmol)溶于无水吡啶(5ml)，加入过量的丙酸酐(0.02ml，2N)，油浴加热40℃，恒温搅拌，TLC检测反应确认结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以P-TLC纯化(氯仿/甲醇＝50/1(V/V))得到白色固体粉末3-B2(8mg，28％)和3-B5(16mg,63％)。
3-B2:MF:C22H28O7.ESIMS m/z:405.1[M+H]+,427.1[M+Na]+,831.3[2M+Na]+,463.2[M+CH3COO]+,HRESIMS m/z:427.1707[M+Na]+(calcd for C22H28O7Na,427.1733),1H NMR(300MHz,CDCl3):δ6.80(s,1H,H-11),6.32(d,J＝9.1Hz,1H,H-7),5.08(dd,J＝9.1,5.5Hz,1H,H-6),3.99(m,1H,H-1),2.95(dt,J＝13.7,6.9Hz,1H,H-15),2.55–2.28(m,3H),2.10(d,J＝4.3Hz,1H),1.91(td,J＝12.7,6.3Hz,1H),1.81(d,J＝5.5Hz,1H),1.73(dd,J＝17.6,9.2Hz,1H),1.66(s,1H),1.62–1.48(m,1H),1.34(s,3H),1.25(s,3H),1.22(s,3H),1.18(s,3H).
3-B5:MF:C25H32O8.ESIMS m/z:461.2[M+H]+,943.4[2M+Na]+,HRESIMS m/z:483.2004[M+Na]+(calcd for C25H32O8Na,483.1995),1H NMR(300MHz,CDCl3):δ6.35(d,J＝9.2Hz,1H,H-7),5.84(s,1H,H-11),5.07(m,2H),2.96(m,1H,H-15),2.53–2.24(m,5H),2.06(m,1H),1.89(d,J＝5.9Hz,1H),1.71(d,J＝15.5Hz,2H),1.46(s,3H),1.32(s,3H),1.28–1.13(m,9H).
实验3：化合物3-B3的合成
取化合物3(17mg，0.05mmol)溶于无水吡啶(5ml)，加入过量的特戊酸酐(0.03ml，1.6N)，油浴加热50℃，恒温和搅拌，TLC检测反应确认结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以P-TLC纯化(氯仿/甲醇＝50/1(V/V))得到白色固体粉末3-B3(13mg，62％)。
3-B3:MF:C24H32O7.ESIMS m/z:433.2[M+H]+,455.2[M+Na]+,887.4[2M+Na]+,431.1[M-H]-,491.2[M+CH3COO]-,HRESIMS m/z:455.1930[M+Na]+(calcd for C24H32O7Na,455.2046),1H NMR(300MHz,CDCl3):δ6.79(s,1H,H-11),6.28(d,J＝9.2Hz,1H,H-7),5.08(dd,J＝9.1,5.5Hz,1H,H-6),4.00(t,J＝6.5Hz,1H,H-1),2.92(dt,J＝13.6,7.0Hz,1H,H-15),2.43–2.25(m,9H),1.90(m,1H),1.82(m,1H),1.75(d,J＝7.5Hz,1H),1.52(m,2H),1.35(s,3H),1.28(s,3H),1.25(s,9H),1.23(m,9H).
实验4：化合物3-B6的合成
取化合物3(18mg，0.06mmol)溶于无水二氯甲烷(5ml)，加入DMAP(10mg，1.6N)和环丙基甲酸(0.02ml，4N)，室温搅拌，加入DCC(50mg，4.7N)，TLC检测反应确认结束，先将反应中生成的沉淀过滤，滤液浓缩后以硅胶纯化(氯仿/甲醇＝50/1(V/V))得到白色固体粉末3-B6(12mg，48％)。
3-B6:C27H32O8.ESIMS m/z:485.2[M+H]+,507.1[M+Na]+,HRESIMS m/z:507.1996[M+Na]+(calcd for C27H32O8Na,359.1521),1H NMR(300MHz,CDCl3):δ6.37(d,J＝9.0Hz,1H,H-7),5.87(s,1H,H-11),5.07(m,2H),4.09–3.96(m,1H),3.53–3.37(m,1H),2.97(m,1H,H-15),2.32(m,1H),2.13–1.95(m,1H),1.87(d,J＝5.8Hz,1H),1.81–1.53(m,1H),1.48(s,2H),1.31(s,3H),1.22(t,J＝5.8Hz,3H),1.07(d,J＝11.2Hz,6H),0.97(d,J＝7.9Hz,4H).
实验5：化合物3-B7的合成
取化合物3(32mg，0.09mmol)溶于无水吡啶(5ml)，加入环丁基甲酰氯(0.03ml，3N)，油浴加热50℃，恒温搅拌过夜，TLC检测反应确认结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以P-TLC纯化(氯仿/甲醇＝50/1(V/V))得到白色固体粉末3-B7(21mg，45％)。
3-B7:C29H36O8.ESIMS m/z:513.4[M+H]+,1047.6[2M+Na]+,557.8[M+HCOO]-,HRESIMS m/z:535.2296[M+Na]+(calcd for C29H36O8Na,535.2308),1H NMR(300MHz,CDCl3):δ6.32(d,J＝9.1Hz,1H,H-7),5.83(s,1H,H-11),5.15–4.99(m,2H,H-6,H-1),3.20(dd,J＝16.9,8.3Hz,2H),3.03–2.85(m,1H),2.32(dd,J＝24.1,15.2Hz,9H),2.15–1.77(m,8H),1.76–1.51(m,3H),1.45(s,3H),1.32(s,3H),1.22(dd,J＝11.1,6.8Hz,6H).
实验6：化合物3-B8的合成
取化合物3(25mg，0.07mmol)溶于无水吡啶(5ml)，加入过量的琥珀酸酐(25mg，3N)，油浴加热70℃，恒温搅拌，TLC检测反应确认结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以硅胶纯化(氯仿/甲醇＝20/1(V/V))得到白色固体粉末3-B8(30mg，93％)。
3-B8:C23H28O9.ESIMS m/z:449.2[M+H]+,471.2[M+Na]+,919.5[2M+Na]+,895.8[2M-H]-,HRESIMS m/z:471.1617[M+Na]+(calcd for C23H28O9Na,471.1631),1H NMR(300MHz,CDCl3):δ6.78(s,1H,H-11),6.31(d,J＝9.2Hz,1H,H-7),5.08(dd,J＝8.9,5.5Hz,1H,H-6),4.00(t,J＝6.4Hz,1H,H-1),2.93(dd,J＝13.2,6.6Hz,1H,H-15),2.81–2.61(m,4H),2.42–2.24(m,1H),1.91(dd,J＝14.2,6.7Hz,1H),1.81(d,J＝5.4Hz,1H),1.76(d,J＝6.8Hz,1H),1.62–1.45(m,1H),1.47-1.34(m,1H),1.31(s,3H),1.28(s,3H),1.22(d,J＝2.8Hz,3H),1.21(s,3H).
实验7：化合物3-B9的合成
取化合物3(21mg，0.06mmol)溶于无水吡啶(5ml)，加入过量的琥珀酸酐(25mg，3N)，油浴加热70℃，恒温搅拌，TLC检测反应确认结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以硅胶纯化(氯仿/甲醇＝20/1(V/V))得到白色固体粉末3-B9(17mg，61％)。
3-B9:C24H30O9.ESIMS m/z:485.3[M+Na]+,947.5[2M+Na]+.923.8[2M-H]-,HRESIMS m/z:485.1794[M+Na]+(calcd for C24H30O9Na,485.1788),1H NMR(300MHz,CDCl3):δ6.80(s,1H,H-11),6.31(d,J＝8.9Hz,1H,H-7),5.08(m,1H,H-6),3.99(s,1H,H-1),2.94(m,1H),2.33(m,1H),2.17(s,1H),1.98(m,3H),1.81(d,J＝5.1Hz,1H),1.55(d,J＝6.9Hz,1H),1.33(s,3H),1.29(s,3H),1.22(t,J＝6.8Hz,6H).
实验8：化合物3-B10的合成
取化合物3(20mg，0.06mmol)溶于无水吡啶(5ml)，加入苯甲酰氯(0.03ml，3N)，室温搅拌，TLC检测反应确认结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以P-TLC纯化(氯仿/甲醇＝50/1(V/V))得到白色固体粉末3-B10(28mg，88％)。
3-B10:C33H32O8,ESIMS m/z:557.2[M+H]+,1135.6[2M+Na]+,601.6[M+HCOO]-, HRESIMS m/z:579.1982[M+Na]+(calcd for C33H32O8Na,579.1995),1H NMR(300MHz,CDCl3):δ8.04(t,J＝7.4Hz,4H),7.69–7.53(m,2H),7.47(t,J＝7.6Hz,4H),6.63(d,J＝9.3Hz,1H,H-7),5.99(s,1H,H-11),5.37(t,J＝5.8Hz,1H,H-1),5.27(dd,J＝9.0,5.8Hz,1H,H-6),3.05(dt,J＝13.5,6.7Hz,1H,H-15),2.45–2.30(m,1H),2.29–2.13(m,1H),2.05(d,J＝5.7Hz,1H),1.87(m,1H),1.77(s,3H),1.72–1.54(m,2H),1.38(s,3H),1.25(d,J＝6.6Hz,3H),1.12(d,J＝6.7Hz,3H).
实施例5化合物4-A1～4-A8的合成
实验1:4-A1和4-A2的合成
化合物4(35mg,0.1mmol)和硫酸二甲酯(15mg,0.12mmol)溶解于2ml的无水DMF中，加入1.2当量的NaH(3mg,0.12mmol)，反应液于室温下搅拌过夜。TLC检测确认反应结束，反应液中加入30ml饱和的NH4Cl溶液，用20ml的乙酸乙酯萃取三次。有机相合并，无水硫酸镁干燥、减压蒸馏除去溶剂。残余物经过Pre-TLC(二氯甲烷-丙酮20:1)纯化，即得化合物4-A1(15mg,41％)和4-A2(13mg,36％)。
4-A1:白色固体，MF:C20H26O6,ESIMS m/z:363.2[M+H]+,1H NMR(600MHz,Pyr-d5):δ1.26(d,J＝6.8Hz,3H,H-17),1.33(d,J＝6.8Hz,3H,H-16),1.44(s,3H,H-18),1.76(s,3H,H-20),1.81(d,J＝6.5Hz,1H,H-5),1.86(m,1H,H-1),2.17(m,1H,H-3),2.26(dd,J＝13.4,9.0Hz,1H,H-1),2.42(dd,J＝13.7,12.2Hz,1H,H-3),3.36(s,3H,H-21),3.48(m,1H,H-15),3.67(m,1H,H-2),5.12(dd,J＝8.3,6.6Hz,1H,H-6),5.64(dd,J＝8.4,2.9Hz,1H,H-7),6.29(s,1H,H-11),7.86(d,J＝2.8Hz,1H).
4-A2:白色固体，MF:C20H26O6,ESI MS m/z 363.2[M+H]+,1H NMR(500MHz,Pyr-d5):δ1.24(d,J＝7.0Hz,3H,H-17),1.26(d,J＝7.0Hz,3H,H-16),1.41(s,3H,H-18),1.62(s,3H,H-20),1.80(d,J＝6.3Hz,1H,H-5),2.01(dd,J＝13.4,6.7Hz,1H,H-1),2.22(dd,J＝13.6,5.0Hz,1H,H-3),2.48(dd,J＝13.4,6.0Hz,1H,H-1),2.63(t,J＝13.0Hz,1H,H-3),3.22(m,1H,H-15),3.60(s,3H,H-21),4.33(m,1H,H-2),4.97(d,J＝8.5Hz,1H,H-7),5.16(dd,J＝8.4,6.3Hz,1H,H-6),6.29(s,1H,H-11),6.71(d,J＝5.0Hz,1H).
实验3:4-A3的合成
化合物4(35mg,0.1mmol)和硫酸二甲酯(126mg,1.0mmol)溶解于2ml的无水DMF中，加入10当量的NaH(24mg,1.0mmol)，反应液于室温下搅拌过夜。TLC检测确认反应结束，反应液中加入50ml饱和的NH4Cl溶液，用20ml的乙酸乙酯萃取三次。有机相合并，无水硫酸镁干燥、减压蒸馏除去溶剂。残余物经过Sephadex LH-20(氯仿-甲醇1:1)纯化，即得化合物4-A3(17mg,77％)。
4-A3:白色固体，MF:C21H28O6.ESIMS m/z:377.2[M+H]+,1H NMR(500MHz,Pyr-d5):δ1.24(d,J＝3.5Hz,3H,H-17),1.25(d,J＝3.4Hz,3H,H-16),1.40(s,3H,H-18),1.54(s,3H,H-20),1.73(d,J＝6.3Hz,1H,H-5),1.83(dd,J＝13.5,6.1Hz,1H,H-1),2.12(dd,J＝13.9,5.5Hz,1H,H-3),2.21(dd,J＝13.6,8.8Hz,1H,H-1),2.35(dd,J＝13.8,9.2Hz,1H,H-3),3.22(m,1H,H-15),3.34(s,3H,H-21),3.59(s,3H,H-22),3.63(m,1H,H-2),4.97(d,J＝8.5Hz,1H,H-7),5.17(dd,J＝8.4,6.3Hz,1H,H-6),6.25(brs,1H,H-11).
实验4:4-A4和4-A5的合成
化合物4(35mg,0.1mmol)和硫酸二乙酯(18.5mg,0.12mmol)溶解于2ml的无水DMF中，加入1.2当量的NaH(3mg,0.12mmol)，反应液于室温下搅拌过夜。TLC检测确认反应结束，反应液中加入30ml饱和的NH4Cl溶液，用20ml的乙酸乙酯萃取三次。 有机相合并，无水硫酸镁干燥、减压蒸馏除去溶剂。残余物经过Pre-TLC(二氯甲烷-丙酮25:1)纯化，即得化合物4-A4(16mg,43％)和4-A5(17mg,45％)。4-A4:ESI MS m/z377.2[M+H]+；4-A5:ESI MS m/z 377.2[M+H]+.
实验5:4-A6的合成
化合物4(35mg,0.1mmol)和硫酸二甲酯(154mg,1.0mmol)溶解于2ml的无水DMF中，加入10当量的NaH(24mg,1.0mmol)，反应液于室温下搅拌过夜。TLC检测确认反应结束，反应液中加入50ml饱和的NH4Cl溶液，用20ml的乙酸乙酯萃取三次。有机相合并，无水硫酸镁干燥、减压蒸馏除去溶剂。残余物经过Sephadex LH-20(氯仿-甲醇1:1)纯化，即得化合物4-A6(32mg,79％)。4-A6:ESI MS m/z 405.3[M+H]+.
实验6:4-A7和4-A8的合成
化合物4(35mg,0.1mmol)和3-溴基-1-丙烯(18.2mg,0.15mmol)溶解于2ml的无水DMF中，加入1.5当量的NaH(4mg,0.15mmol)，反应液于室温下搅拌过夜。TLC检测确认反应结束，反应液中加入30ml饱和的NH4Cl溶液，用20ml的乙酸乙酯萃取三次。有机相合并，无水硫酸镁干燥、减压蒸馏除去溶剂。残余物经过制备型HPLC纯化，即得化合物4-A7(12mg,31％)和4-A8(20mg,47％)。
4-A7:白色固体，WF：C22H28O6,ESI MS m/z 389.2[M+H]+；1H NMR(600MHz,Pyr-d5):δ1.25(d,J＝6.7Hz,3H,H-17),1.28(d,J＝6.7Hz,3H,H-16),1.41(s,3H,H-18),1.65(s,3H,H-20),1.81(t,J＝3.0Hz,1H,H-5),2.02(dd,J＝13.4,6.8Hz,1H,H-1),2.22(dd,J＝13.6,5.0Hz,1H,H-3),2.49(dd,J＝13.5,9.0Hz,1H,H-1),2.63(t J＝13.0Hz,1H,H-3),3.25(m,1H,H-15),4.23(ddt,J＝12.5,6.2,1.4Hz,1H,H-21),4.33(m,1H,H-2),4.61(ddt,J＝12.5,5.2,1.5Hz,1H,H-21),5.15-5.18(3H,H-6,H-7,H-23),5.36(dq,J＝17.2,1.7Hz,1H,H-23),6.04(m,1H,H-22),6.31(s,1H,H-11),6.72(brs,1H).
4-A8:白色固体，MF:C25H32O6,ESI MS m/z 429.9[M+H]+,1H NMR(600MHz,Pyr-d5):δ1.24(d,J＝3.5Hz,3H,H-17),1.26(d,J＝3.5Hz,3H,H-16),1.41(s,3H,H-18),1.59(s,3H,H-20),1.78(d,J＝4.0Hz,1H,H-5),1.87(dd,J＝13.5,6.2Hz,1H,H-1),2.13(dd,J＝13.8,5.2Hz,1H,H-3),2.26(dd,J＝13.6,8.9Hz,1H,H-1),2.40(dd,J＝13.7,8.0Hz,1H,H-3),3.25(m,1H,H-15),3.80(m,1H,H-2),4.10(m,2H,H-21),4.23(ddt,J＝12.5,6.0,1.4Hz,1H,H-24),4.59(ddt,J＝12.5,6.0,1.5Hz,1H,H-24),5.15-5.19(3H,H-6,H-7,H-26),5.22(dq,J＝10.6,1.4Hz,1H,H-23),5.36(dq,J＝17.3,1.8Hz,1H,H-26),5.44(dq,J＝17.2,1.8Hz,1H,H-23),6.05(m,2H,H-22,H-25),6.28(s,1H,H-11).
实施例6化合物4-B1～4-B44的合成
实验1:化合物4-B1的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶(1ml)，加入乙酰氯(0.004ml,1.5eq.)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B1(6mg,27％)。
4-B1:ESIMS m/z:391.2[M+H]+,435.2[M+HCOO]-.MF:C21H26O7.MW:390.ESIMS m/z:391.2[M+H]+,435.2[M+HCOO]-.1H NMR(500MHz,CDCl3):δ5.94(s,1H,H-11),5.31(d,J＝9.1Hz,1H,H-7),5.08(m,1H,H-2),4.96(dd,J＝8.3,6.6Hz,1H,H-6),3.25(m,1H,H-15),2.40(dd,J＝13.9,9.5Hz,1H),2.32(t,J＝11.7Hz,1H),2.09(s,3H),2.00(dd,J＝13.9,5.8Hz,1H),1.89(d,J＝6.6Hz,1H,H-5),1.77(dd,J＝13.9,6.0Hz,1H), 1.51(s,3H,H-20),1.45(s,3H,H-19),1.33(d,J＝6.8Hz,3H,H-16),1.25(d,J＝6.8Hz,3H,H-17).
13C NMR(126MHz,CDCl3):δ180.1,171.6,170.6,164.5,162.1,109.0,106.5,73.8,66.6,60.3,48.6,42.4,40.1,36.8,32.6,29.6,26.8,24.3,21.3,20.9,20.4.
实验2:化合物4-B2的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加丙酰氯(0.008ml,2.6eq.)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B2(5mg,22％)。
4-B2:ESIMS m/z:405.3[M+H]+,449.1[M+HCOO]-.
实验3:化合物4-B3的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入异丁酰氯(0.008ml,2.2N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B3(12mg,50％)。
4-B3:ESIMS m/z:419.3[M+H]+,463.3[M+HCOO]-.
实验4:化合物4-B4的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入正丁酰(0.008ml,2.2N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B4(8mg,34％)。
4-B4:ESIMS m/z:419.3[M+H]+,463.3[M+HCOO]-.
实验5:化合物4-B5的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入正己酰氯(0.012ml,1.5N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B5(18mg,71％)。
4-B5:ESIMS m/z:447.4[M+H]+,491.2[M+HCOO]-.
实验6:化合物4-B6的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入葵酰氯(0.012ml,1.1N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B6(8mg,28％)。
4-B6:ESIMS m/z:503.4[M+H]+,547.3[M+HCOO]-.
实验7:化合物4-B7的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入乙酰氯(0.008ml,3N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B7(13mg,53％)。
4-B7:ESIMS m/z:433.2[M+H]+,477.2[M+HCOO]-.
实验8:化合物4-B8的合成
取化合物4(10mg,0.029mmol)溶于无水吡啶1ml，加入正己酰氯(0.06ml,1.5N)，室温 搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B8(8mg,51％)。
4-B8:ESIMS m/z:545.4[M+H]+,590.1[M+HCOO]-.
实验9:化合物4-B9的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入环丙基甲酰氯(0.008ml,1.5N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B9(12mg,51％)。4-B9:ESIMS m/z:417.2[M+H]+,461.2[M+HCOO]-.
实验10:化合物4-B10的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入环戊甲酰氯(0.008ml,1.2N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B10(10mg,40％)。4-B10:ESIMS m/z:445.3[M+H]+,489.2[M+HCOO]-.
实验11:化合物4-B11的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入环己甲酰氯(0.008ml,1.2N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B11(10mg,40％)。4-B11:ESIMS m/z:459.3[M+H]+,503.3[M+HCOO]-.
实验12:化合物4-B12的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入3-环己基丙酰氯(0.008ml,1.2N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B12(8mg,29％)。4-B12:ESIMS m/z:487.4[M+H]+,531.3[M+CH3COO]-.
实验13:化合物4-B13的合成
取化合物4(10mg,0.029mmol)溶于无水吡啶1ml，加入环丙基甲酰氯(0.008ml,3N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B13(7mg,50％)。4-B13:ESIMS m/z:485.3[M+H]+,529.3[M+HCOO]-.
实验14:化合物4-B14的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入环丁基甲酸(0.008ml,1.5N)，室温搅拌，加入DCC(50mg，4.6N)，反应过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B14(17mg,58％)。4-B14:ESIMS m/z:513.3[M+H]+,557.2[M+HCOO]-.
实验15:化合物4-B15的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入丁二酸酐(20mg,3.5N)，室 温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B15(10mg,39％)。
4-B15:ESIMS m/z:449.3[M+H]+,447.2[M+HCOO]-.
实验16:化合物4-B16的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入戊二酸酐(20mg,3.1N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B16(7mg,27％)。
4-B16:ESIMS m/z:463.3[M+H]+,461.2[M+HCOO]-.
实验17:化合物4-B17的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入琥珀酸单乙酯酰氯(0.012ml,1.5N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B17(10mg,37％)。4-B17:MF:C25H32O9.MW:476.ESIMS m/z:477.4[M+H]+,521.3[M+HCOO]-.
实验18:化合物4-B18的合成
取化合物4(10mg,0.029mmol)溶于无水吡啶1ml，加入琥珀酸单乙酯酰氯(0.012ml,3N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B18(17mg,97％)。4-B18:ESIMS m/z:605.5[M+H]+,649.4[M+HCOO]-.
实验19:化合物4-B19的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入2-呋喃甲酰氯(0.010ml,1.8N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B19(12mg,48％)。4-B19:ESIMS m/z:443.3[M+H]+,487.2[M+CH3COO]-.
实验20:化合物4-B20的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入2-噻吩甲酰氯(0.010ml,1.6N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B20(10mg,38％)。4-B20:ESIMS m/z:459.3[M+H]+,503.2[M+HCOO]-.
实验21:化合物4-B21的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入2-氯烟酰氯(12mg,1.2N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B21(14mg,55％)。
4-B21:ESIMS m/z:488.2[M+H]+,532.2[M+HCOO]-.
实验22:化合物4-B22的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入1-苯并噻吩-3-羰酰氯(12mg, 1.1N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B22(13mg,45％)。4-B22:ESIMS m/z:509.3[M+H]+,553.2[M+HCOO]-.
实验23:化合物4-B23的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入1,5-二甲基-1H-吡唑-3-羰酰氯(10mg,1.1N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B23(9mg,34％)。4-B23:ESIMS m/z:471.3[M+H]+,515.2[M+HCOO]-.
实验24:化合物4-B24的合成
取化合物B(20mg,0.057mmol)溶于无水吡啶(1ml)，加入4-甲基-2-苯基-1,3-噻唑-5-碳酰氯(15mg,1.2N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B24(10mg,35％)。4-B24:ESIMS m/z:550.3[M+H]+,594.2[M+HCOO]-.
实验25:化合物4-B25的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入苯甲酰氯(0.010ml,1.5N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B25(13mg,51％)。4-B25:ESIMS m/z:453.3[M+H]+,497.2[M+HCOO]-.
实验26:化合物4-B26的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入4-叔丁基苯甲酰氯(0.015ml,1.3N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B26(14mg,48％)。4-B26:ESIMS m/z:509.3[M+H]+,553.2[M+HCOO]-.
实验27:化合物4-B27的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入4-叔丁基苯甲酰氯(15mg,1.3N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B27(11mg,38％)。4-B27:ESIMS m/z:513.3[M+H]+,557.2[M+HCOO]-.
实验28:化合物4-B28的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入反式肉桂酰氯(10mg,1.1N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B28(10mg,37％)。4-B28:ESIMS m/z:479.3[M+H]+,523.2[M+HCOO]-.
实验29:化合物4-B29的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入3-苯基丙酰氯(0.015ml,1.3 N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B29(12mg,44％)。4-B29:ESIMS m/z:481.3[M+H]+,525.2[M+HCOO]-.
实验30:化合物4-B30的合成
取化合物4(10mg,0.029mmol)溶于无水吡啶1ml，加入苯甲酰氯(0.005ml,1.5N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B30(7mg,51％)。
4-B30:ESIMS m/z:557.3[M+H]+,601.2[M+HCOO]-.
实验31:化合物4-B31的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入2-氯苯甲酰氯(0.010ml,1.4N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B31(11mg,40％)。4-B31:ESIMS m/z:488.3[M+H]+,532.2[M+HCOO]-.
实验32:化合物4-B32的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入2-氟苯甲酰氯(0.010ml,1.6N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B32(10mg,40％)。4-B32:ESIMS m/z:471.3[M+H]+,515.2[M+HCOO]-.
实验33:化合物4-B33的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入3-氯苯甲酰氯(0.010ml,1.4N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末B33(11mg,40％)。4-B33:ESIMS m/z:488.3[M+H]+,532.2[M+HCOO]-.
实验34:化合物4-B34的合成
取化合物4(20mg,0.057mmo)溶于无水吡啶1ml，加入3-溴苯甲酰氯(0.010ml,1.3N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B34(11mg,36％)。4-B34:ESIMS m/z:532.3[M+H]+,576.2[M+HCOO]-.
实验35:化合物4-B35的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入4-溴苯甲酰氯(15mg,1.2N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B35(11mg,36％)。4-B35:ESIMS m/z:532.3[M+H]+,576.2[M+HCOO]-.
实验36:化合物4-B36的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入4-氯苯甲酰氯(0.010ml,1.4 N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B36(11mg,40％)。4-B36:ESIMS m/z:488.3[M+H]+,532.2[M+HCOO]-.
实验37:化合物4-B37的合成
取化合物4(10mg,0.029mmol)溶于无水吡啶1ml，加入2-氯苯甲酰氯(0.005ml,1.4N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B37(8mg,44％)。4-B37:ESIMS m/z:666.3[M+H]+,670.2[M+HCOO]-.
实验38:化合物4-B38的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入2-甲基丙烯酰氯(0.010ml,1.8N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B38(10mg,42％)。4-B38:ESIMS m/z:417.3[M+H]+,461.2[M+HCOO]-.
实验39:化合物4-B39的合成
取化合物4(10mg,0.029mmol)溶于无水吡啶1ml，加入巴豆酰氯(10mg,3.3N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B39(12mg,86％)。
4-B39:ESIMS m/z:485.3[M+H]+,529.2[M+HCOO]-.
实验40:化合物4-B40的合成
取化合物4(20mg,0.057mmol)溶于无水吡啶1ml，加入巴豆酰氯(0.010ml,1.3N)，室温搅拌过夜，TLC检测确认反应结束，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以制备HPLC纯化，得到白色固体粉末4-B40(8mg,30％)。
4-B40:ESIMS m/z:467.3[M+H]+,511.2[M+HCOO]-.
实验41:化合物4-B41的合成
取化合物4(300mg,0.86mmol)溶于无水吡啶2ml，加入芴甲氧羰酰氯(300mg,1.4N)，室温搅拌过夜，反应结束后，反应液中加入2N稀盐酸用于中和吡啶，加入乙酸乙酯萃取，有机层以饱和NaCl、纯水洗涤，无水硫酸钠干燥，过滤，浓缩。残余物以Sephadex LH-20纯化(氯仿/甲醇＝1/1(V/V))，得到黄色固体粉末4-MOC(205mg,42％)。
取化合物4-MOC(20mg,0.035mmol)溶于无水吡啶1ml，加入乙酰氯(0.008ml,3.0N)，室温搅拌过夜，反应结束后，减压蒸馏去除大部分吡啶，得到的残余物溶于二氯甲烷，加入二乙胺(0.1ml)，室温反应0.5h，减压蒸馏去除溶剂，得到残余物以Sephadex LH-20纯化(氯仿/甲醇＝1/1(V/V))，得到黄色固体粉末4-B41(10mg,73％)。4-B41:ESIMS m/z:391.2[M+H]+,435.2[M+HCOO]-.
实验42:化合物4-B42的合成
取化合物4-MOC(20mg,0.035mmol)溶于无水吡啶1ml，加入异丁酰氯(0.008ml,2.2N)，室温搅拌过夜，反应结束后，减压蒸馏去除大部分吡啶，得到的残余物溶于二氯甲烷，加入二乙胺(0.1ml)，室温反应0.5h，减压蒸馏去除溶剂，得到残余物以Sephadex LH-20纯化(氯仿 /甲醇＝1/1(V/V))，得到黄色固体粉末4-B42(10mg,68％)。4-B42:ESIMS m/z:419.3[M+H]+,463.3[M+HCOO]-.
实验43:化合物4-B43的合成
取化合物4-MOC(20mg,0.035mmol)溶于无水吡啶1ml，加入正丁酰氯(0.008ml,2.2N)，室温搅拌过夜，反应结束后，减压蒸馏去除大部分吡啶，得到的残余物溶于二氯甲烷，加入二乙胺0.1ml，室温反应0.5h，减压蒸馏去除溶剂，得到残余物以Sephadex LH-20纯化(氯仿/甲醇1:1)，得到黄色固体粉末4-B43(6mg,41％)。4-B43:ESIMS m/z:419.3[M+H]+,463.3[M+HCOO]-.
实验44:化合物4-B44的合成
取化合物4-MOC(20mg,0.035mmol)溶于无水吡啶1ml，加入对溴苯甲酰氯(12mg,1.5N)，室温搅拌过夜，反应结束后，减压蒸馏去除大部分吡啶，得到的残余物溶于二氯甲烷，加入二乙胺0.1ml，室温反应0.5h，减压蒸馏去除溶剂，得到残余物以Sephadex LH-20纯化(氯仿/甲醇1:1)，得到黄色固体粉末4-B44(18mg,97％)。
4-B44:ESIMS m/z:531.3,533.2,[M+H]+,575.2,577.1[M+HCOO]-.
实施例7化合物7-A1的合成
化合物7(20mg,0.055mmol)溶解于20ml的丙酮中，加入对甲苯磺酸—水合物(10mg,0.052mmol)，反应液于室温下搅拌过夜。TLC检测确认反应结束，反应液中加入30ml饱和的NaHCO3溶液，用20ml的乙酸乙酯萃取三次。有机相合并，无水硫酸镁干燥、减压蒸馏除去溶剂。残余物经过Sephadex LH-20(氯仿-甲醇1:1)纯化，即得化合物7-A1(17mg,77％)。
7-A1:白色固体，MF:C22H28O7,ESI MS m/z 405.2[M+H]+,13C NMR(125MHz,Pyr-d5):δ182.0(C-19),170.4(C-9),165.6(C-14),162.9(C-12),111.9(C-20),109.8(C-8),108.6(C-11),79.7(C-6),75.4(C-1),73.1(C-2),60.6(C-7),48.9(C-5),42.4(C-4),41.7(C-10),34.8(C-3),30.1(C-15),26.8and 25.3(C-21and C-22),24.4(C-18),21.2(C-17),10.7(C-16),18.8(C-20).
实施例8化合物12-1的合成
取化合物12(75mg，0.22mmol)，加入干燥二氯甲烷15ml和3A分子筛，室温搅拌，加入PCC(150mg，3N)，TLC监测反应，7h后将溶液中的不溶物过滤，滤液浓缩，经硅胶柱层析纯化后得白色固体12-1(61mg，81％)。
12-1:C19H22O6.ESIMS m/z:347.1[M+H]+,369.1[M+Na]+345.1[M+H]+,HRESIMS m/z:369.1319[M+Na]+(calcd for C19H22O6Na,369.1314),1H NMR(300MHz,CDCl3):δ6.11(s,1H,H-11),4.89(d,J＝4.4Hz,1H,H-7),4.48(d,J＝3.4Hz,1H),4.03(s),2.70(m,2H),2.42(d,J＝4.5Hz,1H),2.11(m,3H),2.12(m,1H),1.54(s,3H),1.14(m,9H),0.90(s,1H).
实施例9化合物活性测试
9.1竹柏内酯类化合物活性测试
前述分离以及化学合成的各化合物(终浓度为5μM)处理脱脂血清饥饿12小时的HepG2细胞24小时，加入荧光标记的低密度脂蛋白(DiI-LDL)20μg/ml，37℃孵育4小时，用磷酸盐缓冲液(PBS)轻轻洗细胞5次后用异丙醇提取脂质，于酶标仪上测定荧光读数(激发光：520nm；发射光570nm)。然后用0.2M氢氧化钠裂解细胞，测定蛋白含量，计算出荧光/蛋白的数值。 实验结果见表4，多个化合物中在5μM浓度下对低密度脂蛋白LDL摄取起到显著增强作用。对结构改造得的新化合物，也表现出HepG2肝细胞低密度脂蛋白摄取有增强作用(表4)。
表4.部分化合物对肝细胞低密度脂蛋白摄取的增强作用
  低密度脂蛋白摄取率/DMSO(％) STD(％)
Con.(DMSO,阴性对照) 100 6.89
普伐他汀 119 1.7
2 +++ 6.6
3 +++ 0.91
4 +++ 8.47
ZBM30(7) +++ 0.03
12 +++ 10.4
3-3 + 2.8
12-1 ++ 8.6
7-A1 ++ 0.18
4-B1 + 0.0499
4-B2 ++++ 0.0517
4-B3 +++ 0.0421
4-B4 ++ 0.02
4-B5 ++ 0.0929
4-B7 + 0.0412
4-B9 ++++ 0.1685
4-B10 ++ 0.0132
4-B11 +++ 0.0248
4-B12 ++ 0.0414
4-B14 ++ 0.044
4-B15 ++++ 0.0046
4-B16 + 0.011
4-B17 +++ 0.0893
4-B18 + 0.0698
4-B19 ++++++ 0.0596
4-B20 +++ 0.0175
4-B23 ++++ 0.0693
4-B25 +++ 0.0759
4-B26 +++ 0.0104
4-B27 + 0.1009
4-B28 +++ 0.0569
4-B29 ++++++ 0.1679
4-B31 +++++ 0.0998
4-B32 ++++ 0.0361
4-B33 + 0.0506
4-B34 ++++ 0.0137
4-B35 + 0.0689
4-B36 ++ 0.0354
4-B37 ++ 0.08
4-B38 ++ 0.078
4-B40 ++++ 0.0349
4-B43 + 0.0602
4-B44 + 0.0602
作用强度+101％-120％；++121％-140％；+++141％-160％；++++161％-180％；+++++181％-200％；++++++>200％.
9.2竹柏内酯类化合物促进肝细胞低密度脂蛋白(LDL)摄取活性机制研究化合物上调低密度脂蛋白受体(LDLR)基因表达水平
LDLR蛋白水平上调可能是由两方面原因引起的：LDLR基因水平上升或LDLR蛋白本身的稳定性增加。基于以上的条件，检测了LDLR基因的表达水平。用本发明化合物在5μM浓度下处理肝细胞24小时，TRIzol试剂提取细胞总RNA，取3μg RNA用M-MLV(逆转录酶)反转录成cDNA,再用LDLR的特异性引物进行real-time PCR(实时定量PCR)，以GAPDH作为内参照基因比对化合物对LDLR基因的调节作用。结果显示，本发明化合物显著性提高了LDLR基因的表达水平。
实验中用到的基因引物序列及PCR条件见表5。
表5.引物序列及PCR反应条件
Figure PCTCN2017073964-appb-000017
综上所述，竹柏内酯类化合物在体外的LDL摄取实验中发挥了出乎意料的增强LDL摄取的效果。其中部分化合物(如4-B19、4-B29、4-B31)效果非常显著，甚至可以达到>200％的作用强度。因此，实验证实，基于新的降脂靶点的竹柏内酯类化合物具有极高的临床应用价值和新药开发潜力。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种如下式I所示的化合物，或其药学上可接受的盐：
    Figure PCTCN2017073964-appb-100001
    其中，式I为选自式I-1，I-2或I-3之一：
    Figure PCTCN2017073964-appb-100002
    A选自下组：N-R、O；其中，R选自下组：H、C1-C4的酰基、C1-C4的烷基、C1-C4的卤代烷基、取代或未取代的C6-C10芳基、取代或未取代的C1-C10杂芳基；
    R1选自下组：H、取代或未取代的C1-C4的烷基、C1-C4的卤代烷基、取代或未取代的C1-C4烷基(如2-羟基乙基，2-羟甲基乙基)、取代或未取代的乙烯基；
    R2选自下组：无、H、氧原子、取代或未取代的C2-C10的酯基、-OR，其中，所述的R选自下组：H、取代或未取代的C2-C10的酰基、芴甲氧羰酰基(Fmoc)、或取代或未取代的C1-C10烷基；较佳地，R’为H；
    R3选自下组：H、卤素、C1-C4的烷基、C1-C4的卤代烷基、-OH、取代或未取代的C2-C10的酯基、-NH-R；其中，R选自下组：H、C1-C4的酰基、取代或未取代的C1-C4的烷基；
    R4选自下组：H、卤素、氧原子、氰基、羟基、羧基、C1-C4的烷氧基、C1-C4的烷基、C1-C4的卤代烷基、取代或未取代的C2-C10的酯基、-OC(O)-R、-NH-R；其中，R选自下组：H、C1-C4的酰基、取代或未取代的C1-C4的烷基；
    R5选自下组：H、卤素、氰基、羟基、-OR、-COOR、取代或未取代C1-C10烷基、取代或未取代的C1-C10卤代烷基、取代或未取代的C2-C30的酯基、取代或未取代(优选为取代)的C7-C11芳基-酰基、-NH-R；其中，R选自下组：H、C1-C4的酰基、取代或未取代的C1-C4的烷基；
    或R4和R5共同构成-R”-O-R”-，其中，所述的R”为无，或取代或未取代的C1-C4的亚烷基；
    R6选自下组：H、卤素、氧原子、氰基、羧基、羟基、-OR、-COOR、取代或未取代C1-C10烷基、取代或未取代的C1-C10卤代烷基、取代或未取代的C2-C10的酯基、取代或未取代(优选为取代)的C7-C11芳基酰基、-NH-R；其中，R选自下组：H、C1-C4的酰基、取代或未取代的C1-C4的烷基；
    或R5和R6共同构成-R”-O-R”-，其中，所述的R”为无，或取代或未取代的C1-C4的亚烷基；
    R7选自下组：H、C1-C4的烷基、C1-C4的卤代烷基；
    R8为无，H、或R8与R2共同构成-R”-O-R”-，其中，所述的R”为无或C1-C4的亚烷基：
    R9为无，H、或R9为-OH；
    所述的
    Figure PCTCN2017073964-appb-100003
    为双键或单键；
    所述的“取代”指基团上的一个或多个氢原子被选自下组的取代基取代：羧基、未取代或卤代的C1-C6烷基、未取代或卤代的C3-C6的环烷基、未取代或卤代的C2-C10的酯基、卤素、C1-C10烷基-氧基、未取代或卤代的C2-C10酰基、羟基、羟基-C1-C10的亚烷基、未取代或卤代的苯基；
    且当母核为I-1结构时，R2、R5和R6中至少有一个为选自下组的基团：-OR或取代或未取代的C2-C10的酯基；或R5和R6共同形成-OC(CH)2-O-结构；
    当母核为I-3结构时，R2、R4和R6中至少有一个为氧原子；
    或所述的式I化合物具有以下结构：
    Figure PCTCN2017073964-appb-100004
  2. 如权利要求1所述的化合物，其特征在于，所述的式I化合物具有以下式Ia所示的结构：
    Figure PCTCN2017073964-appb-100005
    式中，
    R1选自下组：取代或未取代的C1-C4的烷基、C1-C4的卤代烷基；其中，所述的取代指基团上的一个或多个氢原子被C1-C4的酯基取代；
    R2为-OH、-OR或取代或未取代的C2-C10的酯基；
    R7选自下组：C1-C4的烷基、C1-C4的卤代烷基；
    其余各基团的定义如权利要求1中所述。
  3. 如权利要求1所述的化合物，其特征在于，所述的式I化合物具有以下式Ib所示的结构：
    Figure PCTCN2017073964-appb-100006
    式中，
    R1选自下组：取代或未取代的C1-C4的烷基、C1-C4的卤代烷基；
    R2与R8共同构成-O-；
    R7选自下组：C1-C4的烷基、C1-C4的卤代烷基；
    其余各基团的定义如权利要求1中所述。
  4. 如权利要求1-3任一所述的化合物，其特征在于，
    R2选自下组：O-R’，其中，所述的R’选自下组：H、取代或未取代的C2-C10的酰基、或取代或未取代的C1-C10的烷基；
    R4选自下组：H、卤素、氰基、羧基、羟基、C1-C4的烷氧基、取代或未取代的C2-C10的酯基；
    R5选自下组：H、卤素、氰基、羟基、C1-C4的烷氧基、-COOR、取代或未取代的C2-C10的酯基、取代或未取代C1-C10烷基、取代或未取代的C1-C10卤代烷基、-NH-R；其中，R选自下组：H、C1-C4的酰基、C1-C4的烷基、C1-C4的卤代烷基、取代或未取代(优选为取代)的C7-C11芳基-酰基；
    R6选自下组：H、卤素、氰基、羧基、羟基、C1-C4的烷氧基、取代或未取代的C2-C10的酯基、取代或未取代C1-C10烷基、取代或未取代的C1-C10卤代烷基、取代或未取代(优选为取代)的C7-C11芳基-酰基、-NH-R；其中，R选自下组：H、C1-C4的酰基、C1-C4的烷基、C1-C4的卤代烷基；
    所述的“取代”指基团上的一个或多个氢原子被选自下组的取代基取代：羧基、苯基、C3-C6的环烷基、C1-C10的酯基、卤素、C1-C10烷基-氧基、C2-C10酰基、羟基、羟基-C1-C10的亚烷基。
  5. 如权利要求1所述的化合物，其特征在于，所述的R2、R4、R5和R6各自独立地为选自下组的基团：-OR’，且所述的R’选自下组：
    Figure PCTCN2017073964-appb-100007
  6. 如权利要求1所述的化合物，其特征在于，所述的化合物具有选自下组的结构：
    Figure PCTCN2017073964-appb-100008
    或所述的化合物为如式3B、4A或4B所示，其中，Ra、Rb、Rc和Rd的定义如表1、表2和表3中所述：
    Figure PCTCN2017073964-appb-100009
    表1
    Figure PCTCN2017073964-appb-100010
    表2
    化合物编号 Ra Rb
    4-A1 Me H 4-A2 H Me 4-A3 Me Me 4-A4 Et H 4-A5 H Et 4-A6 Et Et 4-A7 H -CH2-CH＝CH2 4-A8 -CH2-CH＝CH2 -CH2-CH＝CH2
    Figure PCTCN2017073964-appb-100011
    表3
    Figure PCTCN2017073964-appb-100012
    Figure PCTCN2017073964-appb-100013
  7. 如权利要求1所述的化合物的用途，其特征在于，用于选自下组的一种或多种用途：
    a)制备用于降血脂的药物组合物；
    b)制备用于降低低密度脂蛋白(LDL)含量的药物组合物；
    c)制备用于稳定LDLR基因稳定性的药物组合物；
    d)制备用于上调LDLR基因表达的药物组合物；
    e)制备用于增加肝细胞表面LDLR受体数量的药物组合物；
    f)制备用于减少LDLR受体降解的药物组合物；
    g)制备用于降低血液中TC和/或TG浓度的药物组合物；
    h)制备用于增加血液中高密度脂蛋白(HDL)浓度的药物组合物；
    i)制备用于改善肝功能损伤的药物组合物。
  8. 如权利要求1所述的用途，其特征在于，所述的药物组合物还用于选自下组的用途：
    j)(体外非治疗性地)增加肝细胞对LDL的摄取率；
    k)(体外非治疗性地)上调肝细胞LDLR基因表达水平。
  9. 如权利要求1所述的用途，其特征在于，所述的药物组合物为选自下组的剂型：口服剂型、注射剂型。
  10. 一种药盒，其特征在于，所述药盒含有：
    (i)第一容器，以及装于该第一容器中的活性成分(a)式I化合物；或含有活性成分(a)的药物；
    (ii)第二容器，以及装于该第二容器中的活性成分(b)他汀类药物，或其药学上可接受的盐；或含有活性成分(b)的药物；以及
    (iii)说明书，所述说明书中记载了联合给予活性成分(a)和活性成分(b)从而降低使用对象体内低密度脂蛋白含量的说明。
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