WO2017113489A1 - 巨大戟二萜醇类化合物及其在抗hiv潜伏治疗上的应用 - Google Patents

巨大戟二萜醇类化合物及其在抗hiv潜伏治疗上的应用 Download PDF

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WO2017113489A1
WO2017113489A1 PCT/CN2016/074282 CN2016074282W WO2017113489A1 WO 2017113489 A1 WO2017113489 A1 WO 2017113489A1 CN 2016074282 W CN2016074282 W CN 2016074282W WO 2017113489 A1 WO2017113489 A1 WO 2017113489A1
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compound
formula
substituted
hiv
group
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PCT/CN2016/074282
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English (en)
French (fr)
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张龙飞
王鹏飞
马忠俊
朱焕章
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上海鑫昊生物科技有限公司
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Priority to US16/067,400 priority Critical patent/US10959974B2/en
Priority to EP16880298.1A priority patent/EP3398596A4/en
Publication of WO2017113489A1 publication Critical patent/WO2017113489A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/48—Separation; Purification; Stabilisation; Use of additives
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • 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
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/28—Preparation of carboxylic acid esters by modifying the hydroxylic moiety of the ester, such modification not being an introduction of an ester group
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/02—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
    • C07C69/22—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety
    • C07C69/33—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety esterified with hydroxy compounds having more than three hydroxy groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/72—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 spiro-condensed with carbocyclic rings
    • 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
    • C07D493/10—Spiro-condensed systems

Definitions

  • the present invention relates to the field of medicine, in particular to a large sterol compound and its use in the anti-HIV latent treatment.
  • AIDS Acquired immunodeficiency syndrome
  • WHO statistics there are more than 40 million AIDS patients worldwide, with 5 million new patients each year and about 3 million deaths per year.
  • HAART Highly active antiretroviral therapy
  • People have hoped that HAART can completely eliminate HIV in the body, thus achieving the goal of completely curing AIDS.
  • HIV-1 can lie in resting memory CD4+ T cells, which are produced by the transformation of a small number of HIV-infected activated CD4+ T cells. Its integrated provirus lacks transcriptional activity and, therefore, is not attacked by the immune system and antiretroviral drugs.
  • the molecular mechanism of HIV-1 latently infected cells is generally thought to be the chromatin state at the integration site, the presence of the inhibitory nucleosome nuc-1, the epigenetic modification represented by acetylation, and the host transcription factor such as NF- ⁇ B and viral transcriptional activator Tat and other factors [Coiras, M., MR et al.Understanding HIV-1latency provides clues for the eradication Of long-term reservoirs. Nat. Rev. Microbiol.. 2009., 7: 798-812.].
  • a first aspect of the invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the preparation of a composition or formulation for (a) interfering with HIV virus latency; Activating HIV viruses that have been integrated into the mammalian genome; and/or (c) inducing expression of HIV proviruses in latently infected cells:
  • R 1 is selected from H, substituted or unsubstituted COCH(CH 3 )CH(CH 3 ) 2 , or substituted or unsubstituted OCC 6 H 5 ;
  • R 2 is selected from H, substituted or unsubstituted COCH(CH 3 )CH(CH 3 ) 2 , or substituted or unsubstituted OCC 6 H 5 ;
  • R 3 is selected from H, substituted or unsubstituted COCH(CH 3 )CH(CH 3 ) 2 , or substituted or unsubstituted OCC 6 H 5 ;
  • R 5 is selected from H, COCH 3 , OCH 2 CH 3 , or CO(CH 2 ) 10 CH 3 ;
  • R 9 is selected from the group consisting of CH 2 OR 4 or -C(O)R 10 ; wherein R 4 is selected from the group consisting of H, COCH 3 , OCH 2 CH 3 , CH 2 CH 3 , CO(CH 2 ) 10 CH 3 , Or CO(CH 2 ) 14 CH 3 ; R 10 is selected from the group consisting of H, substituted or unsubstituted C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, a C 1 -C 6 alkoxy group, an -OH group, a C 1 -C 6 ester group, or a combination thereof;
  • any one or both of R 1 and R 2 , R 2 and R 3 , R 3 and R 9 independently form a 5-7 membered substituted or unsubstituted heterocyclic ring, respectively, with an adjacent ring carbon atom.
  • the heterocyclic ring contains 2-3 O atoms and 0-1 heteroatoms selected from N or S;
  • substituted means that H in the group is substituted with one or more substituents selected from the group consisting of halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl.
  • the substituted means substituted with one or more substituents selected from the group consisting of halogen, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, C 2 -C 4 -alkenyl, C 2 -C 4 alkynyl.
  • any one of R 1 and R 2 , R 2 and R 3 , R 3 and R 9 each independently form a 5-7 membered substituted or unsubstituted heterocyclic ring containing 2 - 3 O atoms and 0-1 heteroatoms selected from N or S.
  • any two groups of R 1 and R 2 , R 2 and R 3 , R 3 and R 9 each independently form a 5-7 membered substituted or unsubstituted heterocyclic ring containing 2 - 3 O atoms and 0-1 heteroatoms selected from N or S.
  • the halogen is selected from the group consisting of F, Cl, Br, or I.
  • the compound is an optical isomer or a racemate.
  • the compound has the structure shown in Formula Ic or Formula Id:
  • R 1 , R 2 , R 3 , R 4 and R 5 are as defined in the first aspect of the invention.
  • composition or formulation is also used to (d) treat AIDS; and/or (e) in combination with a repressive viral drug to treat AIDS.
  • the intervening HIV virus latent comprises: inducing HIV proviral expression in a latently infected cell of the HIV virus, and/or activating a latent HIV virus; and/or
  • the treatment of AIDS includes anti-HIV latent treatment.
  • the antiretroviral drug is selected from the group consisting of a reverse transcriptase inhibitor, a protease inhibitor, a co-receptor antagonist, a retroviral integrase inhibitor, a viral adsorption inhibitor, and a specificity.
  • a reverse transcriptase inhibitor a reverse transcriptase inhibitor
  • protease inhibitor a protease inhibitor
  • co-receptor antagonist a co-receptor antagonist
  • retroviral integrase inhibitor a viral adsorption inhibitor
  • a specificity e.g., a specificity.
  • the compound of formula I, or a pharmaceutically acceptable salt thereof is chemically synthesized.
  • the compound of formula I, or a pharmaceutically acceptable salt thereof is extracted from a plant.
  • the extracting comprises extracting using a method selected from the group consisting of solvent extraction, extraction, and chromatography.
  • the plant comprises a Euphorbiaceae plant.
  • the Euphorbiaceae plant includes Euphorbia kansui T.N. Liou ex S. B. Ho.
  • the composition comprises a pharmaceutical composition, a food composition or a nutraceutical composition.
  • the medicament further comprises an additional component selected from the group consisting of an active ingredient of an anti-retroviral virus or an active ingredient that enhances immunity.
  • the pharmaceutical composition comprises (i) a compound of formula I or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.
  • the component (i) is from 0.001 to 99.9% by weight, preferably from 0.1 to 99% by weight, more preferably from 1% to 90% by weight, based on the total weight of the pharmaceutical composition.
  • composition or medicament comprises: an oral preparation and a non-oral preparation.
  • the preparation comprises: a powder, a granule, a capsule, an injection, an elixir, an oral solution, a tablet or a lozenge.
  • the composition is an oral preparation.
  • the composition e.g., a pharmaceutical composition
  • the mammal comprises a mammal having acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • the mammal comprises a human or a non-human mammal.
  • the non-human mammal comprises a rodent such as a mouse or a rat.
  • a second aspect of the invention provides a compound having the structure of formula II, or a pharmaceutically acceptable salt thereof,
  • R 1 is selected from H, or COCH(CH 3 )CH(CH 3 ) 2 , or substituted or unsubstituted OCC 6 H 5 ;
  • R 2 is selected from H, or COCH(CH 3 )CH(CH 3 ) 2 , or substituted or unsubstituted OCC 6 H 5 ;
  • R 3 is selected from H, or COCH(CH 3 )CH(CH 3 ) 2 , or substituted or unsubstituted OCC 6 H 5 ;
  • R 5 is selected from H, COCH 3 , OCH 2 CH 3 , or CO(CH 2 ) 10 CH 3 ;
  • R 9 is selected from CH 2 OR 4 or -C(O)R 10 ; wherein R 4 is selected from H, COCH 3 , CH 2 CH 3 , OCH 2 CH 3 , CO(CH 2 ) 10 CH 3 , or CO(CH 2 ) 14 CH 3 ; and R 10 are selected from the group consisting of H, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 Alkoxy, -OH, C 1 -C 6 ester group, or a combination thereof;
  • any one or both of R 1 and R 2 , R 2 and R 3 , R 3 and R 9 independently form a 5-7 membered substituted or unsubstituted heterocyclic ring, respectively, with an adjacent ring carbon atom.
  • the heterocyclic ring contains 2-3 O atoms and 0-1 heteroatoms selected from N or S;
  • substituted means that H in the group is substituted with one or more substituents selected from the group consisting of halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl;
  • R 1, R 2, R 3 and R 4 are not simultaneously H; and R 1 is COCH (CH 3) CH (CH 3) 2 and R 2 and R 3 are H while, R 4 is OCH 2 CH 3 , CO(CH 2 ) 10 CH 3 , or CO(CH 2 ) 14 CH 3 .
  • R 2 and R 3 form a 5-7 membered substituted or unsubstituted heterocyclic ring with an adjacent ring carbon atom.
  • R 2 and R 3 form a 5-6 membered substituted or unsubstituted heterocyclic ring with an adjacent ring carbon atom.
  • R 2 and R 3 form a 5-membered or unsubstituted heterocyclic ring with an adjacent ring carbon atom.
  • R 1 and R 2 form a 5-7 membered substituted or unsubstituted heterocyclic ring with an adjacent ring carbon atom.
  • R 1 and R 2 form a 5-6 membered substituted or unsubstituted heterocyclic ring with an adjacent ring carbon atom.
  • R 1 and R 2 form a 5-membered or unsubstituted heterocyclic ring with an adjacent ring carbon atom.
  • R 3 and R 9 form a 5-7 membered substituted or unsubstituted heterocyclic ring with an adjacent ring carbon atom.
  • R 3 and R 9 form a 5-6 membered substituted or unsubstituted heterocyclic ring with an adjacent ring carbon atom.
  • R 3 and R 9 form a 6-membered substituted or unsubstituted heterocyclic ring with an adjacent ring carbon atom.
  • the heterocyclic ring is a 5-6 membered heterocyclic ring having 2 oxygen atoms having 1-2 C 1 -C 3 alkyl groups.
  • heterocyclic ring is selected from the group consisting of
  • the "substituted" means that H in the group is substituted with one or more substituents selected from the group consisting of halogen, C 1 -C 4 alkyl, C 1 -C 4 Haloalkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl.
  • any one of R 1 and R 2 , R 2 and R 3 , R 3 and R 9 each independently form a 5-7 membered substituted or unsubstituted heterocyclic ring containing 2 - 3 O atoms and 0-1 heteroatoms selected from N or S.
  • any two groups of R 1 and R 2 , R 2 and R 3 , R 3 and R 9 each independently form a 5-7 membered substituted or unsubstituted heterocyclic ring containing 2 - 3 O atoms and 0-1 heteroatoms selected from N or S.
  • the halogen is selected from the group consisting of F, Cl, Br, or I.
  • the compound has the structure shown in Formula IIa or Formula IIb:
  • R 1 , R 2 , R 3 , R 4 , R 5 are as defined in formula II.
  • the compound is selected from the group consisting of:
  • a third aspect of the invention provides a pharmaceutical composition comprising:
  • the compound of formula I has the structure shown in formula II.
  • the antiretroviral drug is selected from the group consisting of a reverse transcriptase inhibitor, a protease inhibitor, a co-receptor antagonist, a retroviral integrase inhibitor, a viral adsorption inhibitor, and a specificity.
  • a reverse transcriptase inhibitor a reverse transcriptase inhibitor
  • protease inhibitor a protease inhibitor
  • co-receptor antagonist a co-receptor antagonist
  • retroviral integrase inhibitor a viral adsorption inhibitor
  • a specificity e.g., a specificity.
  • the weight ratio of the first active ingredient to the second active ingredient is from 0.1% to 99%, preferably from 20% to 80%.
  • the pharmaceutical dosage form is an oral or parenteral dosage form.
  • the oral administration dosage form is a tablet, a powder, a granule or a capsule, or an emulsion or a syrup.
  • the parenteral dosage form is an injection or an injection.
  • the concentration of the compound of formula I (or a compound of formula II) or a pharmaceutically acceptable salt thereof is from 0.001 ug to 1,000,000 ug/ml, preferably from 0.01 ug to 1000 ug/ml, more preferably Ground, 0.1ug-100ug/ml.
  • a fourth aspect of the invention provides a method for non-therapeutic induction of HIV proviral expression in a latently infected HIV virus cell, comprising the steps of: a compound of formula I as defined in the first aspect of the invention or a second aspect of the invention In the presence of a compound of formula II, or a pharmaceutically acceptable salt thereof, the HIV virus latently infected cells are cultured to cause expression of a latent HIV provirus, thereby activating the latent HIV virus.
  • the method further comprises killing or inhibiting the activated HIV virus.
  • a fifth aspect of the invention provides a method of non-therapeutic activation of a latent HIV virus comprising the steps of administering to a subject in need thereof a compound of formula I as defined in the first aspect of the invention or a second aspect of the invention A compound of formula II, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the third aspect of the invention, thereby activating a latent HIV virus.
  • a sixth aspect of the invention provides a method of preparing a pharmaceutical composition for treating AIDS comprising the steps of: a compound of formula I as defined in the first aspect of the invention or a compound of formula II according to the second aspect of the invention, Or a pharmaceutically acceptable salt thereof is mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition.
  • the invention also provides a method of activating a latent HIV virus; and/or a method of treating AIDS, administering to a subject in need thereof a safe and effective amount of Formula I as defined in the first aspect of the invention
  • a compound or a compound of formula II according to the second aspect of the invention, or an acceptable salt thereof, or a pharmaceutical composition according to the third aspect of the invention are also provided.
  • the desired subject is a mammal, such as a human.
  • the administered dose is from 0.1 to 2000 mg, preferably from 1 to 300 mg/day, per day for an adult.
  • a seventh aspect of the invention provides a kit, the kit comprising:
  • the preparation containing the compound of formula I as defined in the first aspect of the invention or the compound of formula II of the second aspect of the invention, or a pharmaceutically acceptable salt thereof, or containing antiretroviral includes capsules, tablets, suppositories, or intravenous injections, respectively.
  • the compound of formula I or the present invention as defined in the first aspect of the invention
  • the concentration of the acceptable salt is from 0.001 ⁇ g to 1,000,000 ⁇ g/ml, preferably from 0.01 ⁇ g to 1000 ⁇ g/ml.
  • the antiretroviral drug comprises a reverse transcriptase inhibitor, a protease inhibitor, a fusion inhibitor, an integrase inhibitor, or a combination thereof.
  • step (II) 5-50 hours after the step (I), preferably 10 to 48 hours, more preferably 15 to 24 hours, administering to the subject a preparation containing an antiretroviral drug; and optionally
  • An eighth aspect of the invention provides a method for non-therapeutic inhibition and/or killing of latent HIV virus in vitro, comprising the steps of:
  • the incubation time in step (i) is from 5 to 50 hours, preferably from 10 to 48 hours, more preferably from 15 to 24 hours.
  • a ninth aspect of the present invention provides a method of treating a latent infection of an HIV virus, comprising the steps of:
  • step (II) 5-50 hours after the step (I), preferably 10 to 48 hours, more preferably 15 to 24 hours, administering to the subject a preparation containing an antiretroviral drug; and optionally ,
  • Figure 1 shows the fluorescence-microscopic observation of the induction of HIV latency by EK-16A (ie 16A);
  • Figure 1 specifically shows the expression of green fluorescence in cells after treatment with J-Lat-A10.6 cells untreated or treated with 10nM EK-16A for 48 hours; no drug group (Figure 1-1 is a white light photo)
  • Figure 1-2 shows the same field fluorescence photo
  • EK-16A treatment group Figure 1-3 is a white light photo
  • Figure 1-4 is the same field green fluorescence photo
  • Figure 2 shows the efficiency of induction of HIV latency by EK-16A (ie, 16A) by flow cytometry
  • Figure 3 shows a comparison of the activation efficiency of latent HIV in J-Lat-A10.6 cells by EK-16A (i.e., 16A) and the comparator Prostratin;
  • Figure 4 is a graph showing the effect of the compounds of Examples 1-7 of the present invention on the activation efficiency of latent HIV in C11 cells;
  • Figure 5 shows the activation of resting blood CD4+ T cells in peripheral blood of AIDS patients by EK-16A (ie 16A) and positive control PHA.
  • the inventors have extensively and intensively studied, and by screening a large number of compounds, it was first discovered that the ingenol compound and its derivative (the compound of formula I, formula II) have anti-HIV latent effects. Specifically, the present inventors used an original active screening method to screen out a large indole alcohol compound and a derivative thereof (the compound of the formula I and the formula II), and can be used as a therapeutic drug against AIDS.
  • the ingenol compound of the present invention and the derivative thereof have the effect of interfering with the latent of HIV, and can be used in combination with the anti-reverse virus to remove the activated latently infected cells, This acceleration lurks The removal of the virus reservoir will provide a new way for the complete cure of AIDS.
  • the present invention has been completed on this basis.
  • substituted or unsubstituted means that the group may be unsubstituted or that H in the group is one or more (eg 1-10, preferably 1-5) More preferably 1-3, optimally, 1-2) substituents are substituted.
  • substituted or “substituted” means that the group has one or more (preferably 1-6, more preferably 1-3) substituents selected from the group consisting of: Halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl.
  • C 1 -C 6 alkyl refers to a straight or branched alkyl group having from 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, iso Butyl, sec-butyl, tert-butyl, or the like.
  • C 2 -C 6 alkenyl refers to a straight or branched alkenyl group having 2 to 6 carbon atoms, such as ethenyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.
  • C 2 -C 6 alkynyl group means a straight or branched alkynyl chain having 2-6 carbon atoms, e.g. ethynyl, propynyl, or the like.
  • C 1 -C 6 haloalkyl refers to a straight or branched alkyl group having 1 to 6 carbon atoms in which hydrogen is substituted by one or more halogens, for example, a halomethyl group. , haloethyl, halopropyl, haloisopropyl, or the like.
  • C 1 -C 6 alkoxy refers to a group having a (C 1 -C 6 alkyl)-O- structure, for example, CH 3 -O-, C 2 H 5 -O- , C 3 H 7 -O-, or the like.
  • C 1 -C 6 ester group refers to a group having a (C 1 -C 6 alkyl)-COO- structure, such as CH 3 COO-, C 2 H 5 COO-, C 3 H 7 COO-, (CH 3 ) 2 CHCOO-, nC 4 H 9 COO-, tC 4 H 9 COO-, or the like.
  • halogen means fluoro, chloro, bromo, or iodo, preferably fluoro and chloro.
  • halo refers to a group substituted by the same or different one or more of the above halogen atoms, and may be partially or fully halogenated, such as trifluoromethyl, pentafluoroethyl, heptafluoro. Isopropyl, or a similar group.
  • the compounds of the invention may contain one or more asymmetric centers and thus occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single diastereomers.
  • Can The asymmetry center of existence depends on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds are included within the scope of the invention.
  • the invention includes all isomeric forms of the compounds.
  • active ingredient of the present invention As used herein, the terms “active ingredient of the present invention”, “ingenuate compound of the present invention and derivatives thereof”, “anti-HIV latent active ingredient of the present invention” and “formula I, formula II of the present invention” "Compound” is used interchangeably and refers to an active ingredient extracted from the genus Euphorbia that has been extracted from a plant of the genus Ganzi, having the effect of inducing proviral expression of HIV latently infected cells to activate the latent virus.
  • the anti-HIV latent active ingredient can be extracted from the genus Carassius, and can be obtained by chemical synthesis.
  • the active ingredient of the present invention has the formula of Formula I:
  • R 1 is selected from H, substituted or unsubstituted COCH(CH 3 )CH(CH 3 ) 2 , or substituted or unsubstituted OCC 6 H 5 ;
  • R 2 is selected from H, substituted or unsubstituted COCH(CH 3 )CH(CH 3 ) 2 , or substituted or unsubstituted OCC 6 H 5 ;
  • R 3 is selected from H, substituted or unsubstituted COCH(CH 3 )CH(CH 3 ) 2 , or substituted or unsubstituted OCC 6 H 5 ;
  • R 5 is selected from H, COCH 3 , OCH 2 CH 3 , or CO(CH 2 ) 10 CH 3 ;
  • R 9 is selected from the group consisting of CH 2 OR 4 or -C(O)R 10 ; wherein R 4 is selected from the group consisting of H, COCH 3 , OCH 2 CH 3 , CH 2 CH 3 , CO(CH 2 ) 10 CH 3 , Or CO(CH 2 ) 14 CH 3 ; R 10 is selected from the group consisting of H, substituted or unsubstituted C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, a C 1 -C 6 alkoxy group, an -OH group, a C 1 -C 6 ester group, or a combination thereof;
  • any one or both of R 1 and R 2 , R 2 and R 3 , R 3 and R 9 independently form a 5-7 membered substituted or unsubstituted heterocyclic ring, respectively, with an adjacent ring carbon atom.
  • the heterocyclic ring contains 2-3 O atoms and 0-1 heteroatoms selected from N or S;
  • substituted means that H in the group is substituted with one or more substituents selected from the group consisting of halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl.
  • the compound has the structure shown in Formula Ic or Formula Id:
  • R 1 , R 2 , R 3 , R 4 and R 5 are as defined in the first aspect of the invention.
  • the active ingredient of the present invention has the formula of Formula II:
  • R 1 is selected from H, or COCH(CH 3 )CH(CH 3 ) 2 , or substituted or unsubstituted OCC 6 H 5 ;
  • R 2 is selected from H, or COCH(CH 3 )CH(CH 3 ) 2 , or substituted or unsubstituted OCC 6 H 5 ;
  • R 3 is selected from H, or COCH(CH 3 )CH(CH 3 ) 2 , or substituted or unsubstituted OCC 6 H 5 ;
  • R 5 is selected from H, COCH 3 , OCH 2 CH 3 , or CO(CH 2 ) 10 CH 3 ;
  • R 9 is selected from CH 2 OR 4 or -C(O)R 10 ; wherein R 4 is selected from H, COCH 3 , OCH 2 CH 3 , CH 2 CH 3 , CO(CH 2 ) 10 CH 3 , or CO(CH 2 ) 14 CH 3 ; and R 10 are selected from the group consisting of H, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 Alkoxy, -OH, C 1 -C 6 ester group, or a combination thereof;
  • any one or both of R 1 and R 2 , R 2 and R 3 , R 3 and R 9 independently form a 5-7 membered substituted or unsubstituted heterocyclic ring, respectively, with an adjacent ring carbon atom.
  • the heterocyclic ring contains 2-3 O atoms and 0-1 heteroatoms selected from N or S;
  • substituted means that H in the group is substituted with one or more substituents selected from the group consisting of halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl;
  • R 1, R 2, R 3 and R 4 are not simultaneously H; and R 1 is COCH (CH 3) CH (CH 3) 2 and R 2 and R 3 are H while, R 4 is OCH 2 CH 3 , CO(CH 2 ) 10 CH 3 , or CO(CH 2 ) 14 CH 3 .
  • the compound has the structure shown in Formula IIa or Formula IIb:
  • R 1 , R 2 , R 3 , R 5 are as defined in the second aspect of the invention.
  • the compound is selected from the group consisting of:
  • the present invention adopts the HIV latent cell model for the activity screening test, and the results show that the ingenol compounds and derivatives thereof of the formula I and the formula II have anti-HIV latent effects, and are prepared according to a conventional method using corresponding excipients.
  • the anti-HIV latent effect test was carried out, and it was confirmed that the ingenol compound represented by the formula I and the formula II can cause latent HIV expression in infected cells, and has an effect of interfering with HIV latency.
  • the combination of reverse-transcriptional viral drugs removes latently infected cells that are activated, thereby accelerating the clearance of latent virus reservoirs.
  • the HIV latent cell model involved in the present invention includes: a person carrying a green fluorescent protein gene
  • the T cell 10.6 clone strain (J-Lat-A10.6) was provided by the National Institute of Health Research AIDS Reference Reagents Program and was established by Dr. Eric Verdin of the University of California. The cells are small and the spherical cells are cultured in suspension.
  • Human T cell Jurkat sperm cells were transfected with the HIV-R7/E-/GFP retroviral vector carrying the green fluorescent protein gene (Jordan A, Bisgrove D, Verdin E. HIV reproducibly establishedes a latent infection after acute infection Of T cells in vitro. EMBO J 22: 1868-1877, 2003); another established HIV latent screening model C11 clone obtained by HIV lentivirus-infected T cells by two sorting and activation (Chinese patent: 200810038851. X).
  • composition includes (a) a composition for treating and/or preventing AIDS, (b) a composition for activating a latent HIV virus; further, the composition includes a pharmaceutical composition, a food composition Or a health care product composition.
  • the anti-HIV latent active ingredient of the present invention has an effect of interfering with HIV latency.
  • the expression of a latent HIV virus can be promoted, thereby activating the latent HIV virus.
  • the active ingredients of the present invention may be formulated in a non-toxic, inert, andpharmaceutically acceptable carrier medium.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, orally, intramuscularly, intraperitoneally, intravenously, subcutaneously, intradermally, or topically.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a safe and effective amount of an active ingredient of the invention together with a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, about 1 microgram to 10 mg per kilogram of body weight per day.
  • the glycyrrhizin or derivative thereof is used in an amount of from 0.1 to 2000 mg, preferably from 1 to 300 mg per day for an adult. /day.
  • Oral administration can be carried out into tablets, powders, granules, capsules and the like, and the excipients used may be one or more of starch, lactose, sucrose, mannose, hydroxymethylcellulose, and the like.
  • the disintegrant can be potato starch, One or more of hydroxymethyl cellulose and the like.
  • the binder may be one or more of gum arabic, corn starch, gelatin, dextrin, and the like.
  • the oral preparation may be formulated into an emulsion, a syrup, or the like in addition to the above dosage forms.
  • the non-oral preparation can be prepared as an injection, and can be injected into water for injection, physiological saline or dextrose water, or a certain proportion of ethanol, propanol or ethylene glycol can be added thereto.
  • the active ingredients of the invention are also particularly suitable for use in combination with other anti-HIV drugs.
  • the active ingredient of the present invention can be used in combination with an anti-retroviral drug to remove latently infected cells for use in the treatment of AIDS.
  • a further object of the present invention is to provide a method for preparing an AIDS drug, which comprises using the ingenol compound represented by the formula I and the formula II and a derivative thereof as a pharmaceutical raw material, and using the corresponding excipient, according to The conventional method is prepared into an oral or non-oral preparation, wherein the ingenol compound represented by the formula I and the formula II and the derivative thereof may be used in an amount of 0.1 to 2000 mg, preferably 1 to 300 mg/day, per day for an adult. Take 1 to 5 times a day; the amount and frequency of children should be reduced as appropriate on an adult basis.
  • the present invention also provides a kit (or a medicament for interfering with HIV virus according to the first aspect of the invention), the kit comprising:
  • the preparation containing the ingenol compound represented by Formula I or Formula II and a derivative thereof may be a unit dosage form containing a macroindanol compound and a derivative thereof, the preparation containing the antiretroviral drug It may be a unit dosage form containing an antiretroviral drug.
  • the kit contains at least two unit dosage forms containing an ingenol compound and derivatives thereof and a unit dosage form containing an antiretroviral drug; preferably, each is 4-10.
  • unit dosage form refers to a dosage form required to prepare a composition for single administration for ease of administration, including but not limited to various solid agents (eg, tablets), liquids, capsules, sustained release. Agent.
  • step (II) 5-50 hours after the step (I), preferably 10 to 48 hours, more preferably 15 to 24 hours, administering to the subject a preparation containing an antiretroviral drug; and optionally ,
  • the antiretroviral drug which can be used in the kit of the present invention may be one or more.
  • the antiretroviral drug may be various, and more preferably, it may be well known to those skilled in the art.
  • a combination of antiretroviral cocktail therapies may be used in the kit of the present invention.
  • the invention also provides methods of non-therapeutic and in vivo therapeutic activation in vitro and inhibition of latent HIV virus.
  • the method of in vitro non-therapeutic activation and inhibition of latent HIV virus includes the steps of:
  • the HIV virus latently infected cells are cultured to activate the latent HIV virus.
  • Suitable treatments for the body including the following steps:
  • step (II) 5-50 hours after the step (I), preferably 10 to 48 hours, more preferably 15 to 24 hours, administering to the subject a preparation containing an antiretroviral drug; and optionally ,
  • the ingenol compound and its derivative can be used as an active ingredient to prepare a drug against HIV latent action.
  • E. kansui roots and rhizomes 30Kg were separately extracted twice with 8 times the amount of 95% ethanol, and the resulting extract was recovered to an alcohol-free taste for two hours each time to obtain an extract.
  • the mixture was diluted with water to 3000 mL, extracted three times with an equal volume of dichloromethane, and dichloromethane solvent was recovered to give 101.2 g of dichloromethane extract.
  • the conditions of the silica gel column chromatography were as follows: the mobile phase was a petroleum ether-ethyl acetate solution having a volume ratio of 10 to 2:1.
  • the conditions of the reverse phase chromatography purification were as follows: the octadecyl bonded silica gel was used, and the mobile phase was eluted isocratically in a volume ratio of 93% methanol-water solution, and the compound EK-16A was obtained in 30 minutes. 360 mg), light yellow viscous solid, UV absorption 208 nm.
  • the separation and purification comprises: concentrating the extract, performing silica gel column chromatography, and thin layer detection to obtain the desired fraction, and purifying by reverse phase chromatography to obtain compound EK-16A (ie, 16A), 360 mg.
  • Example 8 The ingenol compound of the present invention and its derivatives efficiently activate HIV expression in latent cells
  • J-Lat-A10.6 or C11 cells were seeded in 96-well plates at 2 x 10E4 cells per well, and 100 ⁇ l of 1640 medium (Gibco) containing 10% FBS (Gibco) was added to each well. After the cells were treated with the compounds of Examples 1-7 for 48 hours, the green fluorescence expression of the cells was observed under a fluorescence microscope, and the cells were collected for flow cytometry analysis, and the proportion of fluorescent cells was analyzed.
  • 1640 medium Gibco
  • FBS FBS
  • J-Lat-A10.6 or C11 cells were seeded in 96-well plates at 2 x 10E4 cells per well, and 100 ⁇ l of 1640 medium (Gibco) containing 10% FBS (Gibco) was added to each well. After the cells were treated with different concentrations of compounds (such as the compounds of Examples 1-7) and Prostratin for 48 hours, the cells were collected for flow cytometry and the proportion of fluorescent cells was analyzed.
  • 1640 medium Gibco
  • FBS FBS
  • the high enzymatic concentration of the ingenol compound and its derivatives ranged from 1-100 nM and had a dose-effect relationship in both cell models.
  • the intensive sterol compound 16A activates the latent HIV-1 with an EC 50 of 3.53 nM and Prostratin of 760 nM; whereas on J-Lat-A10.6 cells, the large sterol compound and its derivatives (e.g. 16A, 16A-4A, 16A- 5A, 16A-6A, 16A-12A, 16A-15A, 16A-17A) the activation of latent HIV-1 is the lowest EC 50 of 4.06nM, and Prostratin of 860nM.
  • the EC50 of the ingenol compounds and their derivatives are more than 200 times lower than that of Prostratin. It can be seen that the ingenol compound and its derivatives are highly active in activating latent HIV-1. High levels of HIV-1 activation can be induced at very low concentrations.
  • the resting CD4+ T cells isolated from the peripheral blood of 5 AIDS patients were treated with 5 ⁇ g/ml PHA (phytohemagglutinin) (positive control) and 0.005 ⁇ M compound 16A for 18h, and then extracted into resting CD4+ T cells. And HIV-1 mRNA in the medium, real-time PCR method was used to detect the change of HIV-1 mRNA expression in resting CD4+ T cells and medium.
  • PHA phytohemagglutinin
  • cells treated with known HIV-infected latent pools were treated with compound 16A and then antiretroviral drugs were administered, and the number of viral latent pools treated with compound 16A was found to be significantly lower than the number of untreated viral latent pools.

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Abstract

本发明提供了巨大戟二萜醇类化合物及其在制备抗HIV潜伏的药物中的应用。具体地，本发明提供了巨大戟二萜醇类化合物或其药学上可接受的盐的制药用途，(a)干预HIV病毒潜伏；(b)激活已整合于哺乳动物基因组中的HIV病毒；和/或(c)诱导潜伏于感染细胞中HIV前病毒进行表达。本发明化合物还可与抗逆传录病毒药物联合使用，加速潜伏病毒储藏库的清除。

Description

巨大戟二萜醇类化合物及其在抗HIV潜伏治疗上的应用 技术领域
本发明涉及医药领域，具体地巨大戟二萜醇类化合物及其在抗HIV潜伏治疗上的应用。
背景技术
获得性免疫缺陷综合征(AIDS)是由HIV感染引起的一种严重危害人们生命健康的传染性疾病。据WHO统计全球艾滋病患者已超过4000万，每年新增患者500万，而每年死亡约为300万。目前，艾滋病临床治疗方法主要是高效抗逆传录病毒疗法(Highly active antiretroviral therapy,HAART)，该疗法不仅有效控制HIV复制，并且能重建AIDS患者的免疫功能，为AIDS的治疗打开了希望之门。人们曾寄希望于凭借HAART完全清除体内的HIV，从而达到彻底治愈AIDS的目的。但随后的实践证明，虽然HAART可以最大限度地抑制患者体内病毒复制，使血浆病毒载量(virus load)降低至现有常规检测方法测不出的水平，但感染者体内仍有病毒持续存在，一旦停止药物治疗,病毒载量又会反弹到治疗前水平(Ho,D.D.Toward HIV eradication or remission:the tasks ahead.Science，1998.280:1866–1867.)。HIV难以在体内被完全清除的一个重要原因是HIV-1能够潜伏在静息的记忆CD4+T细胞中，该潜伏感染细胞是由一小部分HIV感染的活化CD4+T细胞转化而产生的,其整合的前病毒缺乏转录活性，因而,不会被免疫系统和抗逆转录酶病毒的药物攻击。尽管感染个体携带潜伏感染细胞数量较少,但衰减率是如此之慢，以至于欲在个体生存期内仅靠HAART治疗将其彻底清除是不可能的.因此,HIV潜伏感染的静息CD4+T细胞是构成机体内病毒储藏库(reservoir)的主要部分，同时也是目前临床治疗不能彻底清除HIV的巨大障碍[Finzi,D.et al.Latent infection of CD4+T cells provides a mechanism for lifelong persistence of HIV-1,even in patients on effective combination therapy.Nature Med.1999,5,512–517]。一般认为HIV-1潜伏感染细胞形成的分子机制与整合位点处的染色质状态,抑制性核小体nuc-1的存在,以乙酰化为代表的表观遗传修饰,宿主转录因子如NF-κB以及病毒转录活化因子Tat等因素有关[Coiras,M.,M.R.et al.Understanding HIV-1latency provides clues for the eradication  of long-term reservoirs.Nat.Rev.Microbiol..2009.,7:798-812.]。据此机制，有研究提出了清除潜伏病毒储存库的治疗策略，即试图通过药物诱导HIV潜伏感染细胞的前病毒表达，使其潜伏病毒再次激活，同时结合高效抗逆传录病毒疗法及在人免疫系统作用下，来杀死激活的潜伏感染的细胞，以此加速病毒库的清除(Richman et al.The Challenge of Finding a Cure for HIV Infection,Science，2009，1304，323)。尽管，该策略已在临床上已有几个治疗方案，但其结果仍未尽人意，不是激活剂无效，就是虽有效但其毒副作用大，而在国内，至今仍未有自主知识产权的抗艾滋病新药上市。因而，研发具有自主知识产权、安全、有效的、价廉的能清除HIV-1病毒储存库的新型干预药物具有十分重要的意义。
中药作为我国所特有的医药资源，以其种类繁多、临床应用广泛、作用温和、副作用少等优点而越来越受到现代医药学的关注。
发明内容
本发明的目的是提供一种具有抗HIV潜伏作用的药物，尤其涉及巨大戟二萜醇类化合物及其衍生物在抗HIV潜伏治疗上的应用。
本发明第一方面提供了一种式I化合物或其药学上可接受的盐的用途，用于制备一组合物或制剂，所述组合物或制剂用于(a)干预HIV病毒潜伏；(b)激活已整合于哺乳动物基因组中的HIV病毒；和/或(c)诱导潜伏于感染细胞中HIV前病毒进行表达：
Figure PCTCN2016074282-appb-000001
式中，
R1选自H、取代或未取代的COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
R2选自H、取代或未取代的COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
R3选自H、取代或未取代的COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
R5选自H、COCH3、OCH2CH3、或CO(CH2)10CH3；
R9选自下组：CH2OR4或-C(O)R10；其中，R4选自H、COCH3、OCH2CH3、CH2CH3、CO(CH2)10CH3、或CO(CH2)14CH3；R10选自下组：H、取代或未取代的C1-C6烷基、C2-C6链烯基、C2-C6链炔基、C1-C6烷氧基、-OH、C1-C6酯基、或其组合；
或者，R1和R2、R2和R3、R3和R9的任意一组或二组分别独立地与相邻的环碳原子形成5-7元取代或未取代的杂环，所述杂环含有2-3个O原子以及0-1个选自N或S的杂原子；
其中，所述“取代的”是指基团中的H被选自下组的一个或多个取代基所取代：卤素、C1-C6烷基、C1-C6卤代烷基、C2-C6链烯基、C2-C6链炔基。
在另一优选例中，所述取代的是指被选自下组的一个或多个取代基所取代：卤素、C1-C4烷基、C1-C4卤代烷基、C2-C4链烯基、C2-C4链炔基。
在另一优选例中，R1和R2、R2和R3、R3和R9的任意一组分别独立地形成5-7元取代或未取代的杂环，所述杂环含有2-3个O原子以及0-1个选自N或S的杂原子。
在另一优选例中，R1和R2、R2和R3、R3和R9的任意二组分别独立地形成5-7元取代或未取代的杂环，所述杂环含有2-3个O原子以及0-1个选自N或S的杂原子。
在另一优选例中，所述卤素选自下组：F、Cl、Br、或I。
在另一优选例中，所述化合物为光学异构体或外消旋体。
在另一优选例中，所述化合物具有式Ic或式Id所示的结构：
Figure PCTCN2016074282-appb-000002
其中，R1、R2、R3、R4、R5的定义如本发明第一方面所述。
在另一优选例中，所述的组合物或制剂还用于(d)治疗艾滋病；和/或(e)与抗逆传录病毒药物联用从而治疗艾滋病。
在另一优选例中，所述的干预HIV病毒潜伏包括：诱导HIV病毒潜伏感染细胞中HIV前病毒表达、和/或激活潜伏的HIV病毒；和/或
所述的治疗艾滋病包括抗HIV潜伏治疗。
在另一优选例中，所述抗逆转录病毒药物选自下组：逆转录酶抑制剂、蛋白酶抑制剂、辅助受体拮抗剂、逆转录病毒整合酶抑制剂、病毒吸附抑制剂、特异性病毒转录抑制剂、细胞周期蛋白依赖性激酶抑制剂、抗HIV抗体、或其组合。
在另一优选例中，所述式I化合物、或其药学上可接受的盐为化学合成的。
在另一优选例中，所述式I化合物、或其药学上可接受的盐是提取自植物。
在另一优选例中，所述的提取包括采用选自下组的方法进行提取：溶剂提取法、萃取法、和色谱法。
在另一优选例中，所述的植物包括大戟科植物。
在另一优选例中，所述大戟科植物包括甘遂(Euphorbia kansui T.N.Liou ex S.B.Ho)。
在另一优选例中，组合物包括药物组合物、食品组合物或保健品组合物。
在另一优选例中，所述的药物还包括选自下组的额外组分：抗逆传录病毒的活性成分或增强免疫力的活性成分。
在另一优选例中，所述的药物组合物含有(i)式I所示的化合物或其药学上可接受的盐；以及(ii)药学上可接受的载体。
在另一优选例中，所述组分(i)占所述药物组合物总重量的0.001-99.9wt％,较佳地0.1-99wt％，更佳地1％-90wt％。
在另一优选例中，所述的组合物或药物包括：口服制剂和非口服制剂。
在另一优选例中，所述的制剂包括：粉剂、颗粒剂、胶囊剂、注射剂、酊剂、口服液、片剂或含片。
在另一优选例中，所述组合物为口服制剂。
在另一优选例中，所述的组合物(如药物组合物)通过以下方式施用于哺乳动物：口服、静脉注射、或局部注射。
在另一优选例中，所述哺乳动物包括患有获得性免疫缺陷综合征(AIDS)的哺乳动物。
在另一优选例中，所述哺乳动物包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物，如小鼠、大鼠。
本发明第二方面提供了一种具有式II所示结构的化合物、或其药学上可接受的盐，
Figure PCTCN2016074282-appb-000003
式中，
R1选自H、或COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
R2选自H、或COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
R3选自H、或COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
R5选自H、COCH3、OCH2CH3、或CO(CH2)10CH3；
R9选自CH2OR4、或-C(O)R10；式中，R4选自H、COCH3、CH2CH3、OCH2CH3、CO(CH2)10CH3、或CO(CH2)14CH3；以及R10选自下组：H、C1-C6烷基、C2-C6链烯基、C2-C6链炔基、C1-C6烷氧基、-OH、C1-C6酯基、或其组合；
或者，R1和R2、R2和R3、R3和R9的任意一组或二组分别独立地与相邻的环碳原子形成5-7元取代或未取代的杂环，所述杂环含有2-3个O原子以及0-1个选自N或S的杂原子；
其中，所述“取代的”是指基团中的H被选自下组的一个或多个取代基所取代：卤素、C1-C6烷基、C1-C6卤代烷基、C2-C6链烯基、C2-C6链炔基；
附加条件是，R1、R2、R3和R4不同时为H；并且R1为COCH(CH3)CH(CH3)2且R2和R3都为H时，R4为OCH2CH3、CO(CH2)10CH3、或CO(CH2)14CH3。
在另一优选例中，R2和R3与相邻的环碳原子形成5-7元取代或未取代的杂环。
在另一优选例中，R2和R3与相邻的环碳原子形成5-6元取代或未取代的杂环。
在另一优选例中，R2和R3与相邻的环碳原子形成5元取代或未取代的杂环。
在另一优选例中，R1和R2与相邻的环碳原子形成5-7元取代或未取代的杂环。
在另一优选例中，R1和R2与相邻的环碳原子形成5-6元取代或未取代的杂环。
在另一优选例中，R1和R2与相邻的环碳原子形成5元取代或未取代的杂环。
在另一优选例中，R3和R9与相邻的环碳原子形成5-7元取代或未取代的杂环。
在另一优选例中，R3和R9与相邻的环碳原子形成5-6元取代或未取代的杂环。
在另一优选例中，R3和R9与相邻的环碳原子形成6元取代或未取代的杂环。
在另一优选例中，所述的杂环为具有1-2个C1-C3烷基的含2个氧原子的5-6 元杂环。
在另一优选例中，所述的杂环选自下组：
Figure PCTCN2016074282-appb-000004
在另一优选例中，所述“取代的”是指基团中的H被选自下组的一个或多个取代基所取代：卤素、C1-C4烷基、C1-C4卤代烷基、C2-C4链烯基、C2-C4链炔基。
在另一优选例中，R1和R2、R2和R3、R3和R9的任意一组分别独立地形成5-7元取代或未取代的杂环，所述杂环含有2-3个O原子以及0-1个选自N或S的杂原子。
在另一优选例中，R1和R2、R2和R3、R3和R9的任意二组分别独立地形成5-7元取代或未取代的杂环，所述杂环含有2-3个O原子以及0-1个选自N或S的杂原子。
在另一优选例中，所述卤素选自下组：F、Cl、Br、或I。
在另一优选例中，所述化合物具有式IIa或式IIb所示的结构：
Figure PCTCN2016074282-appb-000005
R1、R2、R3、R4、R5如式II中所定义。
在另一优选例中，所述化合物选自下组：
Figure PCTCN2016074282-appb-000006
Figure PCTCN2016074282-appb-000007
本发明第三方面提供了一种药物组合物，包括：
(a1)用于干预HIV病毒潜伏的第一活性成分，所述第一活性成分为式I化合物或其可接受的盐；和
(a2)用于抑制HIV病毒复制的第二活性成分，所述第二活性成分为抗逆转录病毒药物；和
(b)药学上可接受的载体；
其中式I化合物的定义如本发明第一方面所述。
在另一优选例中，所述的式I化合物具有式II所示的结构。
在另一优选例中，所述抗逆转录病毒药物选自下组：逆转录酶抑制剂、蛋白酶抑制剂、辅助受体拮抗剂、逆转录病毒整合酶抑制剂、病毒吸附抑制剂、特异性病毒转录抑制剂、细胞周期蛋白依赖性激酶抑制剂、抗HIV抗体、或其组合。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为0.1％-99％，较佳地，20％-80％。
在另一优选例中，所述的药物剂型为口服给药或非口服给药剂型。
在另一优选例中，所述的口服给药剂型是片剂、散剂、颗粒剂或胶囊剂，或乳剂或糖浆剂。
在另一优选例中，所述的非口服给药剂型是注射剂或针剂。
在另一优选例中，所述的式I化合物(或式II化合物)或其药学上可接受的盐的浓度为0.001ug-1000000ug/ml，较佳地为0.01ug-1000ug/ml，更佳地，0.1ug-100ug/ml。
本发明第四方面提供了一种体外非治疗性诱导HIV病毒潜伏感染细胞中HIV前病毒表达的方法，包括步骤：在如本发明第一方面中定义的式I化合物或本发明第二方面所述的式II化合物、或其药学上可接受的盐的存在下，培养所述的HIV病毒潜伏感染细胞中，使得潜伏的HIV前病毒表达，从而激活潜伏的HIV病毒。
在另一优选例中，所述的方法还包括：对被激活的HIV病毒进行杀灭或抑制。
本发明第五方面提供了一种非治疗性的激活潜伏的HIV病毒的方法，包括步骤：给需要的对象施用如本发明第一方面中定义的式I化合物或本发明第二方面所述的式II化合物、或其药学上可接受的盐或本发明第三方面所述的药物组合物，从而激活潜伏的HIV病毒。
本发明第六方面提供了一种制备用于治疗艾滋病的药物组合物的方法，包括步骤：将如本发明第一方面中定义的式I化合物或本发明第二方面所述的式II化合物、或其药学上可接受的盐与药学上可接受的载体混合，从而形成药物组合物。
在另一优选例中，本发明还提供了一种激活潜伏的HIV病毒的方法；和/或治疗艾滋病的方法，向需要的对象施用安全有效量的如本发明第一方面中定义的式I化合物或本发明第二方面所述的式II化合物、或其可接受的盐或本发明第三方面所述的药物组合物。
在另一优选例中，所述需要的对象为哺乳动物，例如人。
在另一优选例中，所述施用的剂量为成人每日0.1～2000mg，优选1～300mg/天。
本发明第七方面提供了一种药盒，所述的药盒含有：
一含有如本发明第一方面中定义的式I化合物或本发明第二方面所述的式II化合物、或其药学上可接受的盐的制剂；
一含有抗逆转录病毒药物的制剂；和
说明书。
在另一优选例中，所述含有如本发明第一方面中定义的式I化合物或本发明第二方面所述的式II化合物、或其药学上可接受的盐的制剂或含有抗逆转录病毒的制剂的剂型分别包括胶囊、片剂、栓剂、或静脉注射剂。
在另一优选例中，所述含有如本发明第一方面中定义的式I化合物或本发 明第二方面所述的式II化合物、或其可接受的盐的制剂中，所述如本发明第一方面中定义的式I化合物或本发明第二方面所述的式II化合物、或其可接受的盐的浓度为0.001μg-1000000μg/ml，较佳地，较佳地为0.01μg-1000μg/ml。
在另一优选例中，所述抗逆转录病毒药物包括反转录酶抑制剂、蛋白酶抑制剂、融合抑制剂、整合酶抑制剂或其组合。
在另一优选例中，所述的说明书中记载了如下的使用方法：
(I)给需要的对象施用含有如本发明第一方面中定义的式I化合物或本发明第二方面所述的式II化合物、或其可接受的盐的制剂；
(II)步骤(I)后的5-50小时,较佳地为10-48小时，更佳地为15-24小时，给所述对象施用含有抗逆转录病毒药物的制剂；和任选的
(III)重复步骤(I)-(II)。
本发明第八方面提供了一种体外非治疗性的抑制和/或杀灭潜伏HIV病毒的方法，包括步骤：
(i)在如本发明第一方面中定义的式I化合物或本发明第二方面所述的式II化合物、或其药学上可接受的盐存在下，培养被HIV病毒潜伏感染的细胞，从而获得含经激活的HIV病毒的细胞；
(ii)向(I)中含经激活的HIV病毒的细胞中加入抗逆转录病毒药物，从而抑制和/或HIV病毒。
在另一优选例中，步骤(i)中的培养时间为5-50小时,较佳地为10-48小时，更佳地为15-24小时。
本发明第九方面提供了一种治疗HIV病毒潜伏感染的方法，包括步骤：
(I)给需要的对象施用含有如本发明第一方面中定义的式I化合物或如本发明第二方面所述的式II化合物、或其药学上可接受的盐的制剂；
(II)步骤(I)后的5-50小时,较佳地为10-48小时，更佳地为15-24小时，给所述对象施用含有抗逆转录病毒药物的制剂；和任选的，
(III)重复步骤(I)-(II)；
从而治疗HIV病毒潜伏感染。
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。
附图说明
图1显示了荧光显微镜观察EK-16A(即16A)对HIV潜伏的诱导激活；
其中，图1具体显示了J-Lat-A10.6细胞未处理或经10nM EK-16A处理48h后，荧光显微镜观察细胞内的绿色荧光表达情况；未加药物组(图1-1为白光照片，图1-2为同一视野荧光照片)，EK-16A处理组(图1-3为白光照片，图1-4为同一视野绿色荧光照片)。
图2显示了流式细胞术检测EK-16A(即16A)对HIV潜伏的诱导激活效率；
其中，图2具体显示了J-Lat-A10.6细胞未处理(MOCK组)或经10nM EK-16A处理48h后，流式细胞术分析得到的荧光细胞所占比例。
图3显示了EK-16A(即16A)和对照药Prostratin对J-Lat-A10.6细胞中潜伏HIV的激活效率的比较；
其中，流式细胞术检测不同浓度EK-16A和Prostratin对J-Lat-A10.6细胞系中潜伏HIV的激活效果，并绘制浓度依赖曲线。所有数据结果皆为通过3次独立实验得到数据的平均值。
图4显示了本发明实施例1-7的化合物对C11细胞中潜伏HIV的激活效率的影响；
其中，流式细胞术检测不同浓度的实施例1-7的化合物对C11细胞系中潜伏HIV的激活效果，并绘制浓度依赖曲线。所有数据结果皆为通过3次独立实验得到数据的平均值。
图5显示了EK-16A(即16A)和阳性对照PHA对艾滋病病人外周血静息CD4+T细胞的激活作用。
具体实施方式
本发明人经过广泛而深入的研究，通过筛选大量的化合物，首次意外发现巨大戟二萜醇类化合物及其衍生物(式I、式II化合物)具有抗HIV潜伏的作用。具体地，本发明人采用独创的活性筛选方法筛选出巨大戟二萜醇类化合物及其衍生物(式I、式II化合物)，可作为抗艾滋病的治疗药物。本发明的巨大戟二萜醇类化合物及其衍生物(式I、式II化合物)具有干预HIV潜伏的作用，与抗逆传录病毒药物联合使用，可清除被激活的潜伏感染的细胞，以此加速潜伏 病毒储藏库的清除，将为艾滋病的彻底治愈提供新的途径。在此基础上完成了本发明。
基团定义
如本文所用，术语“取代或未取代的”指所述基团可以是未取代的，或者所述基团中的H被一个或多个(如1-10个，较佳地1-5个，更佳地1-3个，最佳地，1-2个)取代基所取代。
如本文所用，所述的“取代”或“取代的”指所述基团具有一个或多个(较佳地1-6个，更佳地1-3个)选自下组的取代基：卤素、C1-C6烷基、C1-C6卤代烷基、C2-C6链烯基、C2-C6链炔基。
如本文所用，术语“C1-C6烷基”是指具有1-6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。
如本文所用，术语“C2-C6链烯基”指具有2-6个碳原子的直链或支链的烯基，例如乙烯基、烯丙基、1-丙烯基、异丙烯基、1-丁烯基、2-丁烯基、或类似基团。
如本文所用，术语“C2-C6链炔基”是指具有2-6个碳原子的直链或支链的炔基，例如乙炔基、丙炔基、或类似基团。
如本文所用，术语“C1-C6卤代烷基”是指氢被1个或1个以上的卤素取代的具有1-6个碳原子的直链或支链烷基，例如，卤代甲基、卤代乙基、卤代丙基、卤代异丙基、或类似基团。
如本文所用，术语“C1-C6烷氧基”是指具有(C1-C6烷基)-O-结构的基团，例如，CH3-O-、C2H5-O-、C3H7-O-、或类似基团。
如本文所用，术语“C1-C6酯基”是指具有(C1-C6烷基)-COO-结构的基团，例如CH3COO-、C2H5COO-、C3H7COO-、(CH3)2CHCOO-、nC4H9COO-、tC4H9COO-、或类似基团。
如本文所用，术语“卤素”是指氟、氯、溴、或碘，优选氟和氯。
如本文所用，术语“卤代的”指被相同或不同的一个或多个上述卤原子取代的基团，可以部分卤代或全部卤代，例如三氟甲基、五氟乙基、七氟异丙基、或类似基团。
本发明的化合物可以含有一个或多个不对称中心，并因此以消旋体、外消旋混合物、单一对映体、非对映异构体化合物和单一非对映体的形式出现。可 以存在的不对称中心，取决于分子上各种取代基的性质。每个这种不对称中心将独立地产生两个旋光异构体，并且所有可能的旋光异构体和非对映体混合物和纯或部分纯的化合物包括在本发明的范围之内。本发明包括化合物的所有异构形式。
活性成分
本文所用，术语“本发明的活性成分”、“本发明的巨大戟二萜醇类化合物及其衍生物”、“本发明的抗HIV潜伏的活性成分”和“本发明的式I、式II化合物”可互换使用，都指提取自甘遂属植物的、具有诱导HIV潜伏感染细胞的前病毒表达从而使潜伏病毒激活的作用的提取自大戟属植物的活性成分。
在本发明中，抗HIV潜伏的活性成分可用甘遂属植物为原料进行提取，可以通过化学合成的方式获得。
在本发明中，本发明的活性成分具有式I所示的通式：
Figure PCTCN2016074282-appb-000008
式中，
R1选自H、取代或未取代的COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
R2选自H、取代或未取代的COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
R3选自H、取代或未取代的COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
R5选自H、COCH3、OCH2CH3、或CO(CH2)10CH3；
R9选自下组：CH2OR4或-C(O)R10；其中，R4选自H、COCH3、OCH2CH3、CH2CH3、CO(CH2)10CH3、或CO(CH2)14CH3；R10选自下组：H、取代或未取代的C1-C6烷基、C2-C6链烯基、C2-C6链炔基、C1-C6烷氧基、-OH、C1-C6酯基、或其组合；
或者，R1和R2、R2和R3、R3和R9的任意一组或二组分别独立地与相邻的环碳原子形成5-7元取代或未取代的杂环，所述杂环含有2-3个O原子以及0-1个选自N或S的杂原子；
其中，所述“取代的”是指基团中的H被选自下组的一个或多个取代基所取代：卤素、C1-C6烷基、C1-C6卤代烷基、C2-C6链烯基、C2-C6链炔基。
在一优选实施方式中，所述化合物具有式Ic或式Id所示的结构：
Figure PCTCN2016074282-appb-000009
其中，R1、R2、R3、R4、R5的定义如本发明第一方面所述。
在本发明中，本发明的活性成分具有式II所示的通式：
Figure PCTCN2016074282-appb-000010
式中，
R1选自H、或COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
R2选自H、或COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
R3选自H、或COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
R5选自H、COCH3、OCH2CH3、或CO(CH2)10CH3；
R9选自CH2OR4、或-C(O)R10；式中，R4选自H、COCH3、OCH2CH3、CH2CH3、CO(CH2)10CH3、或CO(CH2)14CH3；以及R10选自下组：H、C1-C6烷基、C2-C6链烯基、C2-C6链炔基、C1-C6烷氧基、-OH、C1-C6酯基、或其组合；
或者，R1和R2、R2和R3、R3和R9的任意一组或二组分别独立地与相邻的环碳原子形成5-7元取代或未取代的杂环，所述杂环含有2-3个O原子以及0-1个选自N或S的杂原子；
其中，所述“取代的”是指基团中的H被选自下组的一个或多个取代基所取代：卤素、C1-C6烷基、C1-C6卤代烷基、C2-C6链烯基、C2-C6链炔基；
附加条件是，R1、R2、R3和R4不同时为H；并且R1为COCH(CH3)CH(CH3)2且R2和R3都为H时，R4为OCH2CH3、CO(CH2)10CH3、或CO(CH2)14CH3。
在一优选实施方式中，所述化合物具有式IIa或式IIb所示的结构：
Figure PCTCN2016074282-appb-000011
R1、R2、R3、R5的定义如本发明第二方面所述。
在一优选实施方式中，所述化合物选自下组：
Figure PCTCN2016074282-appb-000012
活性筛选试验
本发明采用HIV潜伏细胞模型进行活性筛选试验，结果显示式I、式II所示的巨大戟二萜醇化合物及其衍生物具有抗HIV潜伏作用，并用相应的赋型剂，按照常规的方法制成适用剂型，进行抗HIV潜伏作用试验，结果证实，所述的式I、式II所示的巨大戟二萜醇化合物能使感染细胞内潜伏的HIV表达，具有干预HIV潜伏的作用，与抗逆传录病毒药物联合使用，可清除被激活的潜伏感染的细胞，以此加速潜伏病毒储藏库的清除。
本发明涉及的HIV潜伏细胞模型，包括：一个携带绿色荧光蛋白基因的人 T细胞10.6克隆株克隆株(J-Lat-A10.6)，由美国国家健康卫生研究院AIDS参考试剂计划部提供，由加洲大学Eric Verdin博士建立。该细胞小，球形细胞被悬浮培养。由携带绿色荧光蛋白基因HIV-R7/E-/GFP反转录病毒载体转染人T细胞Jurkat精细胞分选而来(Jordan A,Bisgrove D,Verdin E.HIV reproducibly establishes a latent infection after acute infection of T cells in vitro.EMBO J 22:1868-1877,2003)；另一个建立的HIV潜伏筛选模型C11克隆，由HIV慢病毒感染T细胞经2次分选和激活而获得(中国专利：200810038851.X)。
组合物和施用方法
如本文所用，术语“组合物”包括(a)治疗和/或预防艾滋病的组合物，(b)激活潜伏的HIV病毒的组合物；此外，所述的组合物包括药物组合物、食品组合物或保健品组合物。
本发明的抗HIV潜伏的活性成分具有干预HIV潜伏的作用。因此，当在治疗上施用或给予本发明的抗HIV潜伏的活性成分时，可促进潜伏的HIV病毒的表达，进而激活潜伏的HIV病毒。通常，可将本发明的活性成分配制于无毒的、惰性的和药学上可接受的载体介质。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：口服、肌内、腹膜内、静脉内、皮下、皮内、或局部给药。
本发明还提供了一种药物组合物，它含有安全有效量的本发明的活性成分以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。诸如片剂和胶囊之类的药物组合物，可通过常规方法进行制备。药物组合物如针剂、溶液、片剂和胶囊宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克-10毫克/千克体重，优选地，甘草利酮或其衍生物的用量可以为：成年人每日0.1～2000mg，优选1～300mg/天。
作为预防和抗艾滋病药物，可以制成口服和非口服制剂。口服给药可制成片剂、散剂、颗粒剂、胶囊剂等常用剂型，所用的赋型剂可以为淀粉、乳糖、蔗糖、甘露糖、羟甲基纤维素等中的一种或几种。崩解剂可以为马铃薯淀粉、 羟甲基纤维素等中的一种或几种。粘合剂可以为阿拉伯胶、玉米淀粉、明胶、糊精等中的一种或几种。口服制剂除上述剂型外，还可以制成乳剂、糖浆剂等。
非口服制剂可以制成注射剂，可以与注射用水、生理盐水、葡萄糖水制成注射剂，也可以在其中加入一定比例的乙醇、丙醇、乙二醇等。
此外，本发明的活性成分还特别适合与其他抗HIV的药物联用。尤其是本发明的活性成分可与抗逆传录病毒药物联合使用，清除潜伏感染的细胞，从而用于艾滋病的治疗。
本发明的进一步目的是提供一种治疗艾滋病药物的制备方法，采用所述的式I、式II所示的巨大戟二萜醇化合物及其衍生物为药物原料，用相应的赋型剂，按照常规的方法制成口服和非口服制剂，其中式I、式II所示的巨大戟二萜醇化合物及其衍生物的用量可以为：成年人每日0.1～2000mg，优选1～300mg/天，每日服用1～5次；儿童的用量和次数需在成人的基础上酌情递减。
药盒
本发明还提供了一种药盒(或本发明第一方面所述干预HIV病毒潜伏的药物)，所述的药盒含有：
一含有式I或式II所示的巨大戟二萜醇化合物及其衍生物的制剂；
一含有抗逆转录病毒药物的制剂；和
说明书。
所述含有式I或式II所示的巨大戟二萜醇化合物及其衍生物的制剂可以是含有巨大戟二萜醇化合物及其衍生物的单元剂型，所述含有抗逆转录病毒药物的制剂可以是含有抗逆转录病毒药物的单元剂型。
药盒中装有至少两个含有巨大戟二萜醇化合物及其衍生物的单元剂型和含有抗逆转录病毒药物的单元剂型；较佳地，各为4-10个。
如本文所用，术语“单元剂型”是指为了服用方便，将组合物制备成单次服用所需的剂型，包括但不限于各种固体剂(如片剂)、液体剂、胶囊剂、缓释剂。
本发明药盒所述的说明书中记载了如下的使用方法：
(I)给需要的对象施用含有巨大戟二萜醇化合物及其衍生物的制剂；
(II)步骤(I)后的5-50小时,较佳地为10-48小时，更佳地为15-24小时，给所述对象施用含有抗逆转录病毒药物的制剂；和任选的，
(III)重复步骤(I)-(II)。
此外，可用于本发明药盒的抗逆转录病毒药物可以为一种或多种，优选地，所述抗逆转录病毒药物可以为多种，更优选地，可以为本领域技术人员所熟知的抗逆转录病毒鸡尾酒疗法的药物组合。
激活并抑制潜伏HIV病毒的方法
本发明还提供了体外非治疗性的以及体内治疗性的激活并抑制潜伏HIV病毒的方法。
其中，体外非治疗性的激活并抑制潜伏HIV病毒(或诱导HIV病毒潜伏感染细胞中HIV前病毒表达)的方法包括步骤：
在本发明的巨大戟二萜醇化合物及其衍生物的存在下，培养所述的HIV病毒潜伏感染细胞中，从而激活潜伏的HIV病毒。
当在该步骤后，向激活了潜伏HIV病毒的培养体系中再加入抗逆转录病毒药物，则获得了本发明抑制和/或杀灭潜伏HIV病毒的方法。
适用于体内的治疗方法，包括以下步骤：
(I)给需要的对象施用含有本发明的巨大戟二萜醇化合物及其衍生物的制剂；
(II)步骤(I)后的5-50小时,较佳地为10-48小时，更佳地为15-24小时，给所述对象施用含有抗逆转录病毒药物的制剂；和任选的，
(III)重复步骤(I)-(II)。
本发明的主要优点包括：
1.经过大量筛选，首次发现能够显著干预HIV潜伏作用的巨大戟二萜醇化合物的衍生物。
2.提出了巨大戟二萜醇化合物的新用途，所述的巨大戟二萜醇化合物在低浓度时即能明显使感染细胞内潜伏的HIV表达。
3.所述的巨大戟二萜醇化合物及其衍生物可作为活性成分，制备抗HIV潜伏作用的药物。
4.可进一步制备治疗艾滋病药物，将为艾滋病的彻底治愈提供新的途径。
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，否则百分比和份数是重量百分比和重量份数。
实施例1本发明的巨大戟二萜醇化合物EK-16A(即16A)的制备
甘遂(E.kansui)根及根茎30Kg,分别用8倍量的95％乙醇回流提取两次，每次两小时，将所得提取物回收至无醇味，得浸膏。加水稀释至3000mL,用等体积的二氯甲烷萃取三次，回收二氯甲烷溶剂，得二氯甲烷萃取物101.2g。
所述的硅胶柱层析的条件：采用的流动相为体积比为10～2：1的石油醚-乙酸乙酯溶液。所述的反相色谱纯化的条件：采用的填料为十八烷基键合硅胶，采用的流动相为体积比为93％甲醇-水的溶液等度洗脱，30分钟得化合物EK-16A(360mg)，淡黄色粘稠状固体，紫外吸收208nm。
所述的分离纯化包括：将萃取液浓缩后进行硅胶柱层析，薄层检识得到所需要的馏分，再通过反相色谱纯化，得到化合物EK-16A(即16A)，360mg。
Figure PCTCN2016074282-appb-000013
1H NMR(CDCl3):6.02(m 1H H-1),6.02(m 1H H-7),5.43(m 1H H-3),4.15(s 2H H-20),4.07(dd 1H H-8),4.05(s 1H H-5),3.58(s 1H 4-OH),2.71(dd 1H H-12)，2.61(m 1H H-11)，1.78(s 3H Me-19)，1.19(s 3H Me-17)，1.05(s 3H Me-16)，0.96(d 3H Me-18)，3-R(2.32m 1H；1.91m 1H；0.92d 3H；0.95d 3H；1.13d 3H),13-R(2.18t 2H；1.55m 2H；1.25s-(CH2)8-；0.87t 3H)。
实施例2本发明的巨大戟二萜醇化合物的衍生物16A-4A的制备
将3-O-(2，3-二甲基丁酰基)-13-O-十二烷酰基巨大戟二萜醇(3-O-(2，3-dimethylbutanoyl)-13-O-dodecanoyl ingenol)(30mg，0.046mmol)溶于5％KOH的MeOH的(3ml)中，室温搅拌3h后，(DCM/MeOH＝30/1，原料Rf＝0.5，硫酸乙醇显黑色，产物Rf＝0.2，硫酸乙醇显黑色)。TLC显示原料反应完全。乙酸乙酯和水萃取反应体系，有机相经水、饱和的碳酸氢钠水溶液、饱和食盐 水洗，无水硫酸钠干燥。滤液减压浓缩后的淡黄色粘稠物20mg。滤液减压浓缩后经硅胶柱层析分离(DCM/MeOH＝50/1)，反相制备液相进行纯化。以等度甲醇/水85％进行洗脱，37分钟得化合物化合物16A-4A(13mg)，淡黄色粘稠状固体，紫外吸收206nm。
Figure PCTCN2016074282-appb-000014
1H NMR(CDCl3):6.03(d 1H H-7),5.93(dd 1H H-1),4.43(s 1H H-3),4.16(ABq 1H H-20),4.16(ABq 1H H-20),4.12(s(OH)1H),4.06(1H H-8),3.82(s 1H H-5),3.33(br(OH)1H 5-OH),3.35(br(OH)1H 4-OH),2.72(dd 1H),2.43(m 1H H-11),2.24(dd 1H H-12),1.86(d 3H Me-19),1.22(s,3H,Me-17),1.14(d 1H H-14),1.07(s 3H Me-16),0.92(d 3H Me-18),2.20(t 2H),1.55(m2H),1.25(s-(CH2)8-),0.88(t 3H),3.67(s(OH)1H)。
实施例3本发明的巨大戟二萜醇化合物的衍生物16A-5A的制备
将化合物16A(40mg,0.04mmol)溶于二氯甲烷(5ml)，加入三乙胺，冰浴下滴加乙酰氯(0.05ml)的二氯甲烷溶液，常温下搅拌0.5h，TLC点板跟踪，DCM/MeOH＝80/1，原料Rf＝0.4，硫酸乙醇显黑色，产物Rf1＝0.7，硫酸乙醇显黑色，原料点消失后，反应完全。将冰水混合物(10ml)倒入烧瓶内猝灭反应，并用二氯甲烷萃取(3×10ml)。合并有机相，依次用NaHCO3、无水Na2SO4洗涤有机相并干燥，滤液减压浓缩后经硅胶柱层析分离(DCM/MeOH＝200/1)制得化合物16A-5A，即得到黄色油状化合物。
Figure PCTCN2016074282-appb-000015
1H NMR(CDCl3):6.10(d 1H H-7),6.02(d 1H H-1),5.45(s 1H H-3),4.75(d 1H H-20),4.48(d 1H H-20),4.05(dd 1H H-8),3.89(d 1H-5),3.55(d 1H 4-OH),3.50(s 1H 5-OH),2.58(m 1H H-11),2.06(s 3H 20R),1.79(d 3H Me-19),1.43(m 1H H-14),1.19(s 3H Me-17),1.07(d 3H Me-16), 0.98(d 3H Me-18),3-R(2.32m 1H；1.92m 1H；0.92d 3H；0.97d 3H；1.14d 3H),13-R(2.20t 2H；1.56m 2H；1.25s-(CH2)8–；0.88t 3H)。
实施例4本发明的巨大戟二萜醇化合物的衍生物16A-6A的制备
将16A-4A溶于二氯甲烷(10ml)，加入三乙胺，冰浴下滴加乙酰氯(0.05ml)的二氯甲烷溶液，常温下搅拌0.5h，TLC点板跟踪，DCM/MeOH＝80/1，原料Rf＝0.4，硫酸乙醇显黑色，产物Rf1＝0.7，硫酸乙醇显黑色，原料点消失后，反应完全。将冰水混合物(10ml)倒入烧瓶内猝灭反应，并用乙酸乙酯萃取(3×10ml)。合并有机相，依次用NaHCO3、无水Na2SO4洗涤有机相并干燥，滤液减压浓缩后经硅胶柱层析分离(DCM/MeOH＝80/1)制得化合物16A-6A，即得到橘红色油状化合物。
Figure PCTCN2016074282-appb-000016
1H NMR(CDCl3):6.09(dd 1H H-7),5.93(dd 1H H-1),4.71(d 1H H-20),4.52(d 1H H-20),4.45(dd 1H H-5),4.01(dd 1H H-8),3.70(d 1H 4-OH),2.72(dd 1H H-12),2.25(d 1H H-12),2.06(s 3H 20R),1.86(dd 3H Me-19),1.77(m 1H H-14),1.22(s 3H Me-17),1.07(s 3H Me-16),0.97(d 3H M-18),2.20(t 2H；1.55m 2H；1.25s–(CH2)8–；0.88t 3H)。
实施例5本发明的巨大戟二萜醇化合物的衍生物16A-12A的制备
将化合物16A(60mg，0.046mmol)溶于丙酮(6ml)中，缓慢滴加浓硫酸(60ul)，室温搅拌过夜后，(DCM/MeOH＝50/1，原料Rf＝0.2，产物1Rf1＝0.8)TLC显示原料反应完全。向反应液中缓慢滴加饱碳酸氢钠淬灭反应。二氯甲烷萃取反应体系，有机相经水、饱和的碳酸氢钠水溶液、饱和食盐水洗，无水硫酸钠干燥。滤液减压浓缩后得到油状化合物，经硅胶柱层析分离(DCM/MeOH＝80/1)制得的化合物合并。反相制备液相进行纯化。以等度甲醇/水95％进行洗脱，24分钟得化合物16A-12A(2.3mg)，淡黄色粘稠状固体，紫外吸收235nm。
Figure PCTCN2016074282-appb-000017
1H NMR(CDCl3):6.15(d J＝5.11H H-1),5.99(d J＝5.11H H-7),5.33(s1H H-3),4.34(s 1H H-5),4.12(dd 1H H-8),2.80(m 1H H-11),2.67(dd J＝3.2,13.41H H-12a),2.35(m 1H C-2′),2.31(dd J＝5.5,11.51H H-12b)，2.20(t J＝7.52H C-2″),1.92(m 1H C-3′),1.79(d J＝1.53H Me-19),1.55(m2H C-11″),1.23(s 3H Me-17),1.10(d J＝6.93H C-5′),1.08(s 3H Me-16),1.00(d J＝6.93H C-6′),0.93(d J＝6.93H C-4′),0.90(d J＝7.23H Me-18),0.88(t J＝7.23H C-12″),1.25(s–(CH2)8)–,1.35(s 3H),1.48(s3H).
13C NMR(CDCl3):127.7(C-1),132.2(C-2),82.6(C-3),83.1(C-4),70.9(C-5),138.8(C-6),43.9(C-8),205.9(C-9),92.3(C-10),38.3(C-11),35.7(C12),70.0(C-13),29.7(C14),31.7(C-15),23.5(C-16),17.8(C-17),18.7(C-18),16.8(C-19),66.9(C20),177.0(C-1′),46.7(C-2′),31.7(C-3′),21.4(C-4′),20.0(C-5′),15.0(C-′),175.0(C-1″),35.3(C-2″),28.8(C-3″),30.6(C-4″),30.5(C-5″),30.3(C-6″),30.2(C-7″),30.1(C-8″),30.1(C-9″),32.8(C-10″),23.6(C-11″),14.2(C-12″),111.9(C-1″′),28.6(C-2″′),52.57(C-2″′)
实施例6本发明的巨大戟二萜醇化合物的衍生物16A-15A的制备
将过硫酸氢钾复合盐(oxone)(47.6mg，77.5mmol)、四甲基哌啶(tempo)(0.12mg，0.8mmol)、CaCl2(0.86mg，7.8mmol)加入到溶有16A(50mg，77.5mmol)的DCM溶液中，室温搅拌2h后，TLC点板监测原料已反应完全，DCM/MeOH＝80/1,有两个新点生成，Rf原料＝0.2,产物Rf1＝0.4，产物Rf2＝0.8。过滤除去无机盐，浓缩有机相。反相制备液相进行纯化。以等度甲醇/水95％进行洗脱，47分钟得化合物16A-15A(1.8mg)，淡黄色粘稠状固体，紫外吸收226nm。
Figure PCTCN2016074282-appb-000018
1H NMR(CDCl3):9.34(s 1H H-20),6.50(s 1H H-7),6.14(s 1H H-1),5.25(s 1H H-3),4.29(d 1H H-8),3.57(s 1H 5-OH),3.57(s 1H 4-OH), 3.89(t 1H H-5),2.72(dd 1H H-12),2.39(dd 1H H-12),2.44(m 1H H-11),1.79(s 3H Me-19),1.28(s 3H Me-17),1.20(d 1H H-14),1.09(s 3H Me-16),1.01(d 3H Me-18),3-R(2.35m 1H；1.93m 1H；0.94d 3H；0.97d 3H；1.16d 3H),13-R(2.21t 2H；1.55m 2H；1.25s–(CH2)8–；0.88t 3H)。
实施例7本发明的巨大戟二萜醇化合物的衍生物16A-17A的制备
将溴乙烷(10mg，93mmol)，碳酸钾(16mg，116.25mmol)加入到溶有(50mg，77.5mmol)16A的乙腈溶液中，70℃搅拌过夜后，TLC点板监测原料反应完全，DCM/MeOH＝60/1,有一个新点生成，原料Rf＝0.3,产物Rf＝0.25。减压旋干除去有机溶剂，加入少量水后乙酸乙酯萃取，有机相依次用饱和食盐水洗涤，无水硫酸钠干燥后浓缩。反相制备液相进行纯化。以等度甲醇/水90％进行洗脱，37分钟得化合物16A-17A(3.8mg)，淡黄色粘稠状固体，紫外吸收226nm。
Figure PCTCN2016074282-appb-000019
1H NMR(CDCl3):6.20(s 1H H-7),5.51(s 1H H-1),5.33(s 1H H-3),4.79(s 1H H-5),4.18(s 1H H-8),2.67(dd 1H H-12),2.35(m 1H H-11),1.88(s 3H Me-19),1.80(s 2H H-20),1.35(s 3H Me-17),1.12(s 3H Me-16),0.84(d 3H Me-18),13-R(2.20t 2H；1.55m 2H；1.25s–(CH2)8；0.88t3H)。
实施例8本发明的巨大戟二萜醇化合物及其衍生物高效激活潜伏细胞中HIV的表达
按每孔2×10E4个细胞将J-Lat-A10.6或C11细胞种植于96孔板，每孔加入100μl含10％FBS(Gibco)的1640培养基(Gibco)。细胞经实施例1-7的化合物处理48h后，在荧光显微镜下观察细胞绿色荧光表达情况，并收集细胞进行流式细胞术检测，分析荧光细胞所占比例。
结果如图1和图2所示。结果显示,未加诱导剂处理的HIV潜伏感染细胞，其荧光阳性的细胞比例仅有约为1-2％的背景激活(图1-1和图1-2)。经10nM的EK-16A(即16A)处理,细胞模型中表达绿色荧光的细胞比例明显增加；其中，J-Lat-A10.6细胞经实施例1的化合物处理后绿色荧光阳性的细胞比例高 达90％(图1-3，图1-4，图2)，C11细胞中绿色荧光阳性的细胞比例也有近80％。
实施例1-7的化合物处理后的活性数据如表1所示。
表1
Figure PCTCN2016074282-appb-000020
结果显示，与对照组相比，实施例1-7化合物处理后，HIV病毒的激活率增加了32-45倍。
结果表明，巨大戟二萜醇化合物及其衍生物对HIV潜伏感染细胞中的潜伏病毒具有高效的激活作用。
实施例9巨大戟二萜醇化合物及其衍生物与PKC激活剂Prostratin对潜伏HIV-1激活效率的比较
按每孔2×10E4个细胞将J-Lat-A10.6或C11细胞种植于96孔板，每孔加入100μl含10％FBS(Gibco)的1640培养基(Gibco)。细胞分别经不同浓度的化合物(如实施例1－7的化合物)和Prostratin处理48h后，收集细胞进行流式细胞术检测，分析荧光细胞所占比例。
结果如图3及图4所示。
结果显示，巨大戟二萜醇化合物及其衍生物的较好激活浓度范围为1-100nM，且在两种细胞模型上均具有剂量效应关系。在C11细胞上，巨大戟二萜醇化合物16A激活潜伏HIV-1的EC50为3.53nM，而Prostratin为760nM；而在J-Lat-A10.6细胞上，巨大戟二萜醇化合物及其衍生物(如16A、16A－4A、16A－5A、16A-6A、16A-12A、16A-15A、16A-17A)激活潜伏HIV-1的最低EC50 为4.06nM，而Prostratin为860nM。
在两种潜伏细胞系上，巨大戟二萜醇化合物及其衍生物的EC50均比Prostratin低200倍以上，可见巨大戟二萜醇化合物及其衍生物对于激活潜伏HIV-1的活性极高，在极低的浓度下就能诱导HIV-1高水平的激活。
实施例10巨大戟二萜醇化合物对外周血静息CD4+T细胞的激活作用
分别用5μg/ml PHA(植物血凝素)(阳性对照)和0.005μM化合物16A处理从5例艾滋病病人外周血分离得到的静息CD4+T细胞18h后，抽提静息CD4+T细胞内及培养基中HIV-1mRNA，用real time PCR方法分别检测静息CD4+T细胞内及培养基中HIV-1mRNA表达量的变化。
结果如图5所示。结果显示，经化合物16A处理后的细胞内激活的HIV-mRNA倍数显著高于对照以及PHA处理的细胞，可见化合物16A有效地激活了潜伏的HIV病毒。而在所有的实验病例中，大部分的患者均可以观察到潜伏的HIV病毒的有效激活。
此外，将已知HIV感染潜伏库的细胞经化合物16A处理后，再施用抗逆转录病毒药物，可以发现经化合物16A处理的病毒潜伏库的数量显著低于未处理的病毒潜伏库数量。
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. 一种式I化合物或其药学上可接受的盐的用途，其特征在于，用于制备一组合物或制剂，所述组合物或制剂用于(a)干预HIV病毒潜伏；(b)激活已整合于哺乳动物基因组中的HIV病毒；和/或(c)诱导潜伏于感染细胞中HIV前病毒进行表达：
    Figure PCTCN2016074282-appb-100001
    式中，
    R1选自H、取代或未取代的COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
    R2选自H、取代或未取代的COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
    R3选自H、取代或未取代的COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
    R5选自H、COCH3、OCH2CH3、或CO(CH2)10CH3；
    R9选自下组：CH2OR4或-C(O)R10；其中，R4选自H、COCH3、OCH2CH3、CH2CH3、CO(CH2)10CH3、或CO(CH2)14CH3；R10选自下组：H、取代或未取代的C1-C6烷基、C2-C6链烯基、C2-C6链炔基、C1-C6烷氧基、-OH、C1-C6酯基、或其组合；
    或者，R1和R2、R2和R3、R3和R9的任意一组或二组分别独立地与相邻的环碳原子形成5-7元取代或未取代的杂环，所述杂环含有2-3个O原子以及0-1个选自N或S的杂原子；
    其中，所述“取代的”是指基团中的H被选自下组的一个或多个取代基所取代：卤素、C1-C6烷基、C1-C6卤代烷基、C2-C6链烯基、C2-C6链炔基。
  2. 一种具有式II所示结构的化合物、或其药学上可接受的盐，
    Figure PCTCN2016074282-appb-100002
    式中，
    R1选自H、或COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
    R2选自H、或COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
    R3选自H、或COCH(CH3)CH(CH3)2、或取代或未取代的OCC6H5；
    R5选自H、COCH3、OCH2CH3、或CO(CH2)10CH3；
    R9选自CH2OR4、或-C(O)R10；式中，R4选自H、COCH3、CH2CH3、OCH2CH3、CO(CH2)10CH3、或CO(CH2)14CH3；以及R10选自下组：H、C1-C6烷基、C2-C6链烯基、C2-C6链炔基、C1-C6烷氧基、-OH、C1-C6酯基、或其组合；
    或者，R1和R2、R2和R3、R3和R9的任意一组或二组分别独立地与相邻的环碳原子形成5-7元取代或未取代的杂环，所述杂环含有2-3个O原子以及0-1个选自N或S的杂原子；
    其中，所述“取代的”是指基团中的H被选自下组的一个或多个取代基所取代：卤素、C1-C6烷基、C1-C6卤代烷基、C2-C6链烯基、C2-C6链炔基；
    附加条件是，R1、R2、R3和R4不同时为H；并且R1为COCH(CH3)CH(CH3)2且R2和R3都为H时，R4为OCH2CH3、CO(CH2)10CH3、或CO(CH2)14CH3。
  3. 如权利要求2所述的化合物、或其药学上可接受的盐，其特征在于，所述化合物具有式IIa或式IIb所示的结构：
    Figure PCTCN2016074282-appb-100003
    R1、R2、R3、R4、R5如式II中所定义。
  4. 一种药物组合物，其特征在于，包括：
    (a1)用于干预HIV病毒潜伏的第一活性成分，所述第一活性成分为式I化合物或其可接受的盐；和
    (a2)用于抑制HIV病毒复制的第二活性成分，所述第二活性成分为抗逆转录病毒药物；和
    (b)药学上可接受的载体；
    其中式I化合物的定义如权利要求1所述。
  5. 一种体外非治疗性诱导HIV病毒潜伏感染细胞中HIV前病毒表达的方 法，包括步骤：在如权利要求1中定义的式I化合物或权利要求2所述的式II化合物、或其药学上可接受的盐的存在下，培养所述的HIV病毒潜伏感染细胞中，使得潜伏的HIV前病毒表达，从而激活潜伏的HIV病毒。
  6. 一种非治疗性的激活潜伏的HIV病毒的方法，包括步骤：给需要的对象施用如权利要求1中定义的式I化合物或权利要求2所述的式II化合物、或其药学上可接受的盐或权利要求4所述的药物组合物，从而激活潜伏的HIV病毒。
  7. 一种制备用于治疗艾滋病的药物组合物的方法，包括步骤：将如权利要求1中定义的式I化合物或权利要求2所述的式II化合物、或其药学上可接受的盐与药学上可接受的载体混合，从而形成药物组合物。
  8. 一种药盒，所述的药盒含有：
    一含有如权利要求1中定义的式I化合物或权利要求2所述的式II化合物、或其药学上可接受的盐的制剂；
    一含有抗逆转录病毒药物的制剂；和
    说明书。
  9. 一种体外非治疗性的抑制和/或杀灭潜伏HIV病毒的方法，包括步骤：
    (i)在如权利要求1中定义的式I化合物或权利要求2所述的式II化合物、或其药学上可接受的盐存在下，培养被HIV病毒潜伏感染的细胞，从而获得含经激活的HIV病毒的细胞；
    (ii)向(I)中含经激活的HIV病毒的细胞中加入抗逆转录病毒药物，从而抑制和/或HIV病毒。
  10. 一种治疗HIV病毒潜伏感染的方法，包括步骤：
    (I)给需要的对象施用含有如权利要求1中定义的式I化合物或如权利要求2所述的式II化合物、或其药学上可接受的盐的制剂；
    (II)步骤(I)后的5-50小时,较佳地为10-48小时，更佳地为15-24小时，给所述对象施用含有抗逆转录病毒药物的制剂；和任选的，
    (III)重复步骤(I)-(II)；
    从而治疗HIV病毒潜伏感染。
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