WO2018006804A1 - 孤儿核受体Nur77的配体及其用途 - Google Patents

孤儿核受体Nur77的配体及其用途 Download PDF

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WO2018006804A1
WO2018006804A1 PCT/CN2017/091726 CN2017091726W WO2018006804A1 WO 2018006804 A1 WO2018006804 A1 WO 2018006804A1 CN 2017091726 W CN2017091726 W CN 2017091726W WO 2018006804 A1 WO2018006804 A1 WO 2018006804A1
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cancer cells
alkyl
aryl
nur77
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PCT/CN2017/091726
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English (en)
French (fr)
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张晓坤
林祥志
苏迎
曾志平
胡梦婕
罗强
朱怡
古丽米然⋅阿里同别克
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厦门大学
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Priority to CN201780002454.4A priority Critical patent/CN108026141B/zh
Priority to US16/315,099 priority patent/US10808005B2/en
Priority to JP2019500258A priority patent/JP2019523245A/ja
Priority to EP17823616.2A priority patent/EP3480207A4/en
Publication of WO2018006804A1 publication Critical patent/WO2018006804A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • This application relates to the field of medicine and biology.
  • the present application relates to novel ligands for the orphan nuclear receptor Nur77 and uses thereof.
  • the present application also relates to the use of a compound of formula I-formula V as a ligand for the orphan nuclear receptor Nur77.
  • the present application also relates to the use of a compound of formula I-formula V for the prevention or treatment of a disease associated with the orphan nuclear receptor Nur77.
  • the present application also relates to screening methods for drugs having anti-cancer (especially triple negative breast cancer) activity.
  • the present application also relates to a method for evaluating the diagnosis and treatment effects of triple-negative breast cancer and the like using the nuclear receptor Nur77 as a test index.
  • the orphan nuclear receptor Nur77 also known as NGFIB (nerve growth factor IB) or the orphan nuclear receptor TR3, is a key regulator of the development of cancer, metabolism and inflammatory diseases.
  • NGFIB ner growth factor IB
  • TR3 tumor growth factor 3
  • cytokinins hormones, stress, metabolism, and apoptotic signals.
  • T cell receptor signaling induces Nur77 expression and increases Nur77 expression during T cell receptor-mediated apoptosis (Woronicz JD, Calnan B, et al. Requirement for The Orphan Steroid-Receptor Nur77 in Apoptosis of T-Cell Hybridomas.Nature, 367:277-81,1994;Liu ZG,Smith SW,et al.Apoptotic Signals Delivered through the T-Cell Receptor of a T-Cell Hybrid Require the Immediate-Early Gene Nur77. Nature, 367: 281-4, 1994).
  • Nur77 migrates from the nucleus to the cytoplasm and is localized by a variety of apoptotic signals.
  • mitochondrial localization of Nur77 is associated with the release of cytochrome c (Cytochrome c, Cyt c) and apoptosis.
  • Nur77 which lacks the DNA-binding domain, can be constitutively localized on mitochondria, causing a large amount of cytochrome c to be released from mitochondria, thereby inducing apoptosis (Li Y, Lin BZ, et al.
  • Breast cancer is the "number one killer" of women's health. According to the latest survey data, on average, one woman in the world is diagnosed with breast cancer every three minutes. The incidence and mortality of breast cancer in women in China are also increasing year by year, and it is currently the highest incidence of female malignant tumors. The reason why breast cancer ranks first in the mortality rate of female malignant tumors is its high metastasis. The most typical ones are estrogen receptor (ER), progesterone receptor (PR) and proto-oncogene Her-2. Negative triple-negative breast cancer (TNBC), its greatest feature is strong metastasis, poor patient prognosis, insensitivity to endocrine therapy, and no effective treatment.
  • ER estrogen receptor
  • PR progesterone receptor
  • TNBC Negative triple-negative breast cancer
  • Nur77 as an early response gene, can regulate many key life processes such as cell proliferation, apoptosis, embryonic development, and angiogenesis. Its expression or dysfunction can also lead to a tumor including tumors. A series of diseases. Nur77 is generally expressed in low or no expression in normal tissues, but can be induced in a large amount during the development of cancer and in the course of tumor drug treatment. Its induced expression can mediate the dual effects of cell proliferation and apoptosis, and determine the cell growth. Death, these two opposite phenomena depend on different stimuli and different localization of Nur77 in the cell. Recent studies have shown that Nur77 can specifically bind to its ligand under the stimulation of inflammatory factors such as TNF ⁇ , and exert its anti-inflammatory activity through cytoplasmic translocation to regulate mitochondrial function.
  • TNF ⁇ inflammatory factor
  • the present application establishes a novel screening method for anticancer drugs, that is, targeting Nur77, and searching for a ligand that specifically binds to Nur77 to develop a new compound molecule for treating cancer (such as triple negative breast cancer); Nur77 was established as a test for cancer (eg, triple-negative breast cancer) to assess and diagnose the development of related cancers.
  • terpenoids and steroidal compounds have good antitumor and immunomodulatory activities.
  • the inventors of the present application have discovered a new class of pentacyclic triterpenoids YXY101 and its derivatives, which are capable of binding Nur77 and inhibiting the mTOR signaling pathway, thereby inhibiting tumorigenesis and development.
  • the present application provides promising compounds, and thus, particularly advantageously, the compounds of the invention are capable of acting on cells expressing Nur77. Regulating the newly discovered Nur77-mediated mTOR signaling pathway inventors for the treatment of triple-negative breast cancer and others Cancer (eg liver cancer, cervical cancer, lung cancer, triple positive breast cancer, colorectal cancer or prostate cancer).
  • tautomer refers to a functional group isomer that is produced by the rapid movement of an atom in a molecule at two positions.
  • a typical example of such a tautomer is a keto-enol tautomer.
  • the compounds described herein may exist in tautomeric forms and thus encompass all possible tautomers, and any combination or any mixture thereof.
  • stereoisomer refers to an isomer that is caused by the same order of attachment of atoms or groups of atoms in a molecule, but differs in spatial arrangement.
  • “stereoisomerization” of a compound is classified into conformational and conformational isomerization, and configurational isomerism is also classified into cis-trans isomerization and optical isomerism.
  • “stereoisomer” includes all possible optical isomers and diastereomers, as well as any combination thereof, such as racemates (racemic mixtures), single enantiomeric A conformation, a mixture of diastereomers, a single diastereomer.
  • the compound of the present invention contains an olefinic double bond, it includes a cis isomer and a trans form unless otherwise specified. Isomers, as well as any combination thereof.
  • the term "pharmaceutically acceptable salts” refers to (1) a compound of the present invention, in the presence of acidic functional group (e.g. -COOH, -OH, -SO 3 H, etc.) with a suitable inorganic or organic a salt formed by a cation (base), for example, a salt of a compound of the invention with an alkali metal or alkaline earth metal, an ammonium salt of a compound of the invention, and a salt of a compound of the invention with a nitrogen-containing organic base; and (2) a compound of the invention A salt of a basic functional group (for example, -NH 2 or the like) which is formed with a suitable inorganic or organic anion (acid), for example, a salt of a compound of the present invention with an inorganic acid or an organic carboxylic acid.
  • acidic functional group e.g. -COOH, -OH, -SO 3 H, etc.
  • bases for example, a salt of a compound of the invention with
  • salts of the compounds of the invention include, but are not limited to, alkali metal salts such as sodium, potassium, lithium, and the like; alkaline earth metal salts such as calcium, magnesium, and the like; other metal salts, Such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt, etc.; inorganic alkali salt, such as ammonium salt; organic alkali salt, such as t-octylamine salt, dibenzylamine salt, morpholine salt, Portuguese Glycosylamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, sulfonium salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'- Dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperaz
  • the term "pharmaceutically acceptable ester” refers to an ester which is formed by esterification of an alcohol when a compound of the invention is present; when the compound of the invention has a hydroxyl group, it is organic An ester formed by an esterification reaction of an acid, an inorganic acid, an organic acid salt or the like. The ester can be hydrolyzed to form the corresponding acid or alcohol in the presence of an acid or a base.
  • C1-6 alkyl means a straight or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, or Butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methyl Pentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1, 2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, and the like.
  • C 1-6 alkyl group Preferable examples of the C 1-6 alkyl group include a C 1-5 alkyl group, a C 1-4 alkyl group, and a C 1-3 alkyl group.
  • the "C 1-4 alkyl group” as used in the present invention means a linear or branched alkyl group having 1 to 4 carbon atoms, which includes, but is not limited to, a specific example having 1 to 4 carbon atoms in the above examples. .
  • alkenyl refers to a straight or branched chain hydrocarbon radical containing at least one carbon to carbon double bond, a typical example of which is a C 2-10 alkenyl group, such as a C 2-6 alkene. Base or C 2-4 alkenyl.
  • Specific examples include, but are not limited to, vinyl, propenyl, 2-propenyl, butenyl, 2-butenyl, butadienyl, pentenyl, 2-methyl-butenyl, 3-methyl -butenyl, 1,3-pentadienyl, 1,4-pentadienyl, hexenyl, 2-ethyl-butenyl, 3-methyl-pentenyl, 4-methyl a pentenyl group, a 1,3-hexadienyl group, a 1,4-hexadienyl group, a 1,5-hexadienyl group or the like.
  • alkynyl refers to a straight or branched chain hydrocarbon radical containing at least one carbon to carbon triple bond, a typical example of which is a C 2-10 alkynyl group, for example a C 2-6 alkynyl group or a C. 2-4 alkynyl.
  • Specific examples include, but are not limited to, ethynyl, propynyl, 2-propynyl, butynyl, 2-butynyl, 2-methyl-propynyl, butadiynyl, pentynyl, 2 -methyl-butynyl, 3-methyl-butynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, hexynyl, 2-ethyl-butynyl, 3- Methyl-pentynyl, 4-methyl-pentynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl and the like.
  • cycloalkyl refers to a monocyclic saturated alkyl group, a typical example of which is a 3-8 membered cycloalkyl group, such as 3, 4, 5, 6, 7 or 7 or 8-membered cycloalkyl.
  • 3-8 membered cycloalkyl refers to a cycloalkyl group containing from 3 to 8 carbon atoms. Specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.
  • heterocycloalkyl refers to a cycloalkyl group containing at least 1 up to 4 (eg 1, 2, 3 or 4) heteroatoms selected from N, O and S, wherein
  • cycloalkyl is as described above, and a typical example thereof is a 3-8 membered heterocycloalkyl group such as a 3-, 4-, 5-, 6-, 7- or 8-membered heterocycloalkyl group.
  • 3-8 membered heterocycloalkyl refers to a heterocycloalkyl group containing from 3 to 8 carbon atoms.
  • Oxo 3-8 membered cycloalkyl refers to a 3-8 membered heterocycloalkyl group as defined above, wherein the hetero atom is O.
  • Specific examples include, but are not limited to, epoxyethyl, oxocyclobutyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl and the like.
  • aryl refers to an aromatic group, a typical example of which is a 6-14 membered aryl group, such as a 6-10 membered aryl group.
  • 6-14 membered aryl refers to a monocyclic, bicyclic or polycyclic aromatic group containing from 6 to 14 carbon atoms, including, for example, 6-8 membered aryl groups and 8-14 A fused ring aryl group.
  • the 6-8 membered aryl group means an aryl group having 6 to 8 carbon atoms, such as a phenyl group.
  • the 8- to 14-membered fused ring aryl group means an unsaturated aromatic fused ring formed by having two or more ring-shaped carbon atoms and two or more ring-shaped structures sharing two adjacent carbon atoms. Specific examples include, but are not limited to, naphthalene, anthracene, phenanthrene, and the like.
  • the term "6-10 member aryl” means containing An aromatic group of 6 to 10 carbon atoms, which includes, but is not limited to, an aromatic group having 6 to 10 ring atoms in the above examples.
  • aryl-C 1-6 alkyl refers to a group formed in the manner of an aryl-C 1-6 alkyl group, wherein “aryl” and “C 1-6 alkane”
  • base The definitions of "base” are as described above.
  • C 1-6 alkoxy refers to a group formed in the C 1-6 alkyl-O- form, wherein “C 1-6 alkyl” is as defined above. .
  • C1-6 alkylamino refers to a group formed in the C1-6 alkyl-NH- form, wherein “ C1-6 alkyl” is as defined above.
  • C1-6 alkylthio refers to a group formed in the C1-6 alkyl-S- mode, wherein “ C1-6 alkyl” is as defined above .
  • C 1-6 alkanoyl refers to a group formed in the C 1-5 alkyl-C(O)-form, wherein "C 1-5 alkyl” is as defined above Said.
  • C1-6 alkoxycarbonyl refers to a group formed in the C1-6 alkyl-OC(O)- form, wherein “ C1-6 alkyl” is as defined As mentioned above.
  • C 1-6 alkoxycarbonyl-C 1-6 alkyl refers to a group formed by a C 1-6 alkyl-OC(O)-C 1-6 alkyl group.
  • 3-8 membered cycloalkyl-aminoacyl refers to a group formed in the form of a 3-8 membered cycloalkyl-NHC(O)-, wherein “3-8 membered cycloalkane”
  • base is as described above.
  • halogen includes, for example, a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
  • 6-15 membered heteroaryl refers to an aromatic group containing from 6 to 15 ring atoms and at least one of which is a hetero atom.
  • the 6-15 membered heteroaryl group includes a "5-8 membered heteroaryl group” such as "5-7 membered heteroaryl group", "5-6 membered heteroaryl group” and the like.
  • 5-8 membered heteroaryl include, but are not limited to, furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl , imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, 2-pyridone, 4-pyridone, pyrimidinyl, 1,4-dioxadienyl 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl 4H-1,
  • 6-15 yuan heteroaryl also includes "9-15 yuan thick heteropoly
  • Specific examples include, but are not limited to, benzofuranyl, benzisofuranyl, benzothienyl, fluorenyl, isoindole, benzo Oxazolyl, benzimidazolyl, oxazolyl, benzotriazolyl, quinolyl, 2-quinolinone, 4-quinolinone, 1-isoquinolinone, isoquinolinyl, acridinyl , phenanthryl, benzoxazinyl, pyridazinyl, quinazolinyl, quinoxalinyl, phenolzinyl, acridinyl, fluorenyl, naphthyridinyl, phenazine, phenothiazine, and the like.
  • the term "cell” particularly preferably refers to a cell that expresses Nur77.
  • the compounds of the present invention are capable of specifically binding to Nur77 and functioning as a ligand thereof.
  • the compounds of the invention are capable of acting on cells expressing Nur77. Regulating the Nur77-mediated inflammation and mTOR signaling pathways newly discovered by the inventors.
  • the cell is a triple negative breast cancer and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, triple positive breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • orphan nuclear receptor Nur77 refers to nerve growth factor IB (NGFIB), which is encoded by the NR4A1 gene (Chang C et al. (1989), “Isolation and characterization of human TR3”).
  • Receptor a member of steroid receptor superfamily", J. Steroid Biochem. 34(1-6): 391-5).
  • Nur77 is involved in processes such as cell cycle, inflammation, and apoptosis, and its subcellular localization is associated with cell survival and death (Pei L et al. (2006), “Regulation of macrophage inflammatory gene expression by the orphan nuclear receptor Nur77", Mol . Endocrinol. 20(4): 786-94; Zhang XK (2007), “Targeting Nur77 translocation", Expert Opin. Ther. Targets 11(1): 69-79).
  • Nur77-associated disease refers to a disease whose occurrence and/or progression is associated with the Nur77 signaling pathway. Studies have shown that Nur77 is involved in processes such as cell cycle, inflammation, and apoptosis, and its subcellular localization is associated with cell survival and death (ibid.). In addition, it has been reported that Nur77 can be induced by a variety of stimuli, including physiological stimuli such as fatty acids, prostaglandins, growth factors, inflammatory cytokines, peptide hormones, etc.; and physical stimuli such as magnetic fields, mechanical agitation (shearing forces) , membrane depolarization, etc.
  • physiological stimuli such as fatty acids, prostaglandins, growth factors, inflammatory cytokines, peptide hormones, etc.
  • physical stimuli such as magnetic fields, mechanical agitation (shearing forces) , membrane depolarization, etc.
  • Nur77 has also been shown to be involved in the metastasis of some solid tumors (Ramaswamy S, Ross KN, Lander ES, Golub TR (2003), "A molecular signature of metastasis in primary solid tumors", Nat. Genet. 33(1): 49-54). In addition, Nur77 has been shown to be abnormally expressed in inflamed human synovial tissue, cancer cells, psoriasis patients, atherosclerotic patients, and multiple sclerosis patients.
  • Neur77-associated diseases includes, but is not limited to, inflammation (eg, inflammation associated with atherosclerosis, inflammation associated with obesity, inflammation associated with diabetes, hepatitis, pneumonia, arthritis, and inflammation) Enteropathy), atherosclerosis, obesity, diabetes, psoriasis, multiple sclerosis, and cancer (eg, triple-negative breast cancer).
  • inflammation eg, inflammation associated with atherosclerosis, inflammation associated with obesity, inflammation associated with diabetes, hepatitis, pneumonia, arthritis, and inflammation
  • Enteropathy eg, atherosclerosis, obesity, diabetes, psoriasis, multiple sclerosis
  • cancer eg, triple-negative breast cancer
  • the term "subject" refers to an animal, particularly a mammal, preferably a human.
  • high fat diet means that the amount of fat in a daily intake of an animal (eg, a mammal, such as a human) exceeds the amount of fat required for normal physiological activity of the animal.
  • the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect.
  • a prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease
  • a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such an effective amount is well within the capabilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously. and many more.
  • a compound represented by Formula I-Formula V can target the orphan nuclear receptor Nur77 and function as a ligand thereof.
  • such compounds can be used to inhibit mTOR activity in combination with the orphan nuclear receptor Nur77 for the treatment of cancer associated with the orphan nuclear receptor Nur77 (eg, triple negative breast cancer).
  • the present application relates to the use of a compound, a tautomer, a stereoisomer or a pharmaceutically acceptable salt or ester thereof, of formula I, for use as an orphan nuclear receptor Nur77 a ligand, or a drug used to prepare a ligand for use as an orphan nuclear receptor Nur77:
  • X represents -NH-, -N(R)-, -O-, -CH 2 - or halogen; wherein, when X is a halogen, R 1 is absent;
  • Y When Y is a single bond between the carbon atom to which it is attached, Y represents H, halogen, -OR, -SR or -NRR'; when Y is a double bond between the carbon atom to which it is attached, Y represents O, S or NR;
  • R 1 is absent or represents H, -PO(OR) 2 , C 1-6 alkyl, glycosyl, C 1-6 alkoxycarbonyl-C 1-6 alkyl, 3-8 membered cycloalkyl-ammonia
  • R 2 represents H, D, -PO(OR) 2 , -CONH 2 , -NH 2 , -NHR, -NRR', -NHCOR, -NRCOR, -NHCOOR, -NHCONHR, -NHCONRR', -NRCONHR, -NRCONRR ', -OH, -OR, -OCONHR, -OCONRR', -SH, -SR, -SOR, -SOOR, -SO 2 NHR", nitro, halogen, glycosyl, cyano, trifluoromethyl, C 1-6 alkyl, 3-8 membered cycloalkyl, 3-8 membered heterocycloalkyl, aryl, C 1-6 alkyl substituted aryl, 6-15 membered heteroaryl, alkenyl, alkyne a sulfinyl group, a sulfonic acid or a sulfonate; wherein the C 1-6 al
  • substituents selected from the group consisting of amino, halogen. , hydroxy, oxy, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 alkanoyl, 3-8 membered cycloalkyl (eg cyclopropyl), Oxy 3-8 membered cycloalkyl (eg oxocyclobutyl), cyano, trifluoromethyl, C 1-6 alkoxycarbonyl, C 1-6 alkylamido, ureido, carbamate Base, carboxyl group and aryl group;
  • R 3 and R 4 each independently represent a non-existent or represent H, C 1-6 alkyl, C 1-6 alkanoyl, C 1-6 alkoxycarbonyl, glycosyl, aryl-C 1-6 alkyl Or an aryl group, wherein the C 1-6 alkyl group, the C 1-6 alkanoyl group, the C 1-6 alkoxycarbonyl group, the glycosyl group, the aryl-C 1-6 alkyl group, and the aryl group are unsubstituted or Or a plurality (for example 1, 2, 3 or 4) of substituents selected from the group consisting of halogen, hydroxy, amino, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylamino And a C 1-6 alkanoyl group; preferably, the aryl group is a 6-14 membered aryl group, such as a 6-10 membered aryl group; more preferably a phenyl or naphthyl group;
  • R and R' are each independently selected from H, C 1-6 alkyl, 3-8 membered cycloalkyl, aryl-C 1-6 alkyl or aryl, wherein said C 1-6 alkyl, 3
  • the 8- to 8-membered cycloalkyl, aryl-C 1-6 alkyl and aryl groups are unsubstituted or substituted by one or more (eg 1, 2, 3 or 4) substituents selected from the group consisting of halogens, a hydroxyl group, an amino group, a C 1-6 alkyl group, a C 1-6 alkoxy group, and a C 1-6 alkylamino group;
  • R" represents a C 1-6 alkyl or aryl group (for example a 6-10 membered aryl group, preferably a phenyl group);
  • Virtual real double bond in formula (I) Represents a single bond or a double bond; preferably, ring A contains 0, 1, 2 or 3 carbon-carbon double bonds; ring B contains 0, 1 or 2 carbon-carbon double bonds.
  • X represents -NH -, - N (R) -, - O -, - CH 2 - or halo; wherein when X is halogen, R 1 is absent;
  • Y When Y is a single bond between the carbon atom to which it is attached, Y represents H, halogen, -OR, -SR or -NRR'; when Y is a double bond between the carbon atom to which it is attached, Y represents O, S or NR;
  • R 1 is absent or represents H, -PO(OR) 2 , C 1-6 alkyl, glycosyl, C 1-6 alkoxycarbonyl-C 1-6 alkyl, 3-8 membered cycloalkyl-ammonia
  • R 2 represents H, D, -PO(OR) 2 , -CONH 2 , -NH 2 , -NHR, -NRR', -NHCOR, -NRCOR, -NHCOOR, -NHCONHR, -NHCONRR', -NRCONHR, -NRCONRR ', -OH, -OR, -OCONHR, -OCONRR', -SH, -SR, -SOR, -SOOR, -SO 2 NHR", nitro, halogen, glycosyl, cyano, trifluoromethyl, C 1-6 alkyl, 3-8 membered cycloalkyl, 3-8 membered heterocycloalkyl, aryl, C 1-6 alkyl substituted aryl, 6-15 membered heteroaryl, alkenyl, alkyne a sulfinyl group, a sulfonic acid or a sulfonate; wherein the C 1-6 al
  • substituents selected from the group consisting of amino, halogen. , hydroxy, oxy, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 alkanoyl, 3-8 membered cycloalkyl (eg cyclopropyl), Oxy 3-8 membered cycloalkyl (eg oxocyclobutyl), cyano, trifluoromethyl, C 1-6 alkoxycarbonyl, C 1-6 alkylamido, ureido, carbamate Base, carboxyl group and aryl group;
  • R 3 and R 4 each independently represent a non-existent or represent H, C 1-6 alkyl, C 1-6 alkanoyl, C 1-6 alkoxycarbonyl, glycosyl, aryl-C 1-6 alkyl Or an aryl group, wherein the C 1-6 alkyl group, the C 1-6 alkanoyl group, the C 1-6 alkoxycarbonyl group, the saccharyl group, the aryl-C 1-6 alkyl group and the aryl group are unsubstituted or Or a plurality (for example 1, 2, 3 or 4) of substituents selected from the group consisting of halogen, hydroxy, amino, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylamino And a C 1-6 alkanoyl group; preferably, the aryl group is a 6-14 membered aryl group, such as a 6-10 membered aryl group; more preferably a phenyl or naphthyl group;
  • R and R' are each independently selected from H, C 1-6 alkyl, 3-8 membered cycloalkyl, aryl-C 1-6 alkyl or aryl, wherein said C 1-6 alkyl, 3
  • the 8- to 8-membered cycloalkyl, aryl-C 1-6 alkyl and aryl groups are unsubstituted or substituted by one or more (eg 1, 2, 3 or 4) substituents selected from the group consisting of halogens, a hydroxyl group, an amino group, a C 1-6 alkyl group, a C 1-6 alkoxy group, and a C 1-6 alkylamino group;
  • R" represents a C 1-6 alkyl or aryl group (for example a 6-10 membered aryl group, preferably a phenyl group);
  • Virtual real double bond in formula (I) Represents a single bond or a double bond; preferably, ring A contains 0, 1, 2 or 3 carbon-carbon double bonds; ring B contains 0, 1 or 2 carbon-carbon double bonds.
  • the sulfonate is selected from the group consisting of sodium sulfonate, potassium sulfonate, calcium sulfonate, and magnesium sulfonate.
  • the 6-15 membered heteroaryl is selected from the group consisting of 9-15 membered fused heteroaryl; more preferably, the 6-15 membered heteroaryl is selected from 9-15 membered Benzo-heteroaryl, for example, fluorenyl, benzofuranyl, benzothienyl, benzimidazolyl or quinolyl.
  • Y is a double bond between the carbon atom to which it is attached, and Y represents O.
  • X represents NH
  • Y is a double bond between the carbon atom to which it is attached, and Y represents O
  • R 1 represents one or more (eg 1, 2, 3 or 4)
  • R 2 represents H.
  • X represents O; Y is a double bond between the carbon atom to which it is attached, and Y represents O; R 1 represents H; and R 2 represents H, sulfonate or 6-15.
  • a heteroaryl group preferably, the sulfonate is selected from the group consisting of sodium sulfonate, potassium sulfonate, calcium sulfonate and magnesium sulfonate; preferably, the 6-15 membered heteroaryl is selected from the group consisting of 9-15 yuan thick More preferably, the 6-15 membered heteroaryl group is selected from a 9-15 membered benzofused heteroaryl group, such as an indenyl group, a benzofuranyl group, a benzothienyl group, a benzimidazole or a quinoline. base.
  • X represents O
  • Y is a double bond between the carbon atom to which it is attached, Y represents O
  • R 1 represents a C 1-6 alkyl or aryl-C 1-6 alkyl group. (preferably benzyl); R 2 represents H.
  • R 3 is absent and R 4 represents H. In certain preferred embodiments, R 3 represents H and R 4 is absent. In certain preferred embodiments, both R 3 and R 4 are H.
  • R 3 is absent, R 4 represents H, and ring A and ring B each have two carbon-carbon double bonds.
  • R 3 represents H
  • R 4 is absent, and has 0, 1 or 2 carbon-carbon double bonds in ring A; further preferably, there are 0, 1 or 2 carbons in ring B Carbon double bond.
  • R 3 and R 4 are both H and there are 3 carbon-carbon double bonds in ring A (ie, ring A is a benzene ring); further preferably, ring 0 has 0 or 1 More carbon-carbon double bonds; more preferably, carbon-carbon double bonds between the 7- and 8-position carbon atoms in ring B.
  • neither R 3 nor R 4 is present.
  • the carbon atom at position 2 in ring A is a carbon-oxygen double bond between the O atom to which it is attached, and the carbon atom at the 3-position carbon atom is bonded to the O atom to which it is attached.
  • a carbon-carbon double bond is between the 7 and 8 carbon atoms. In certain preferred embodiments, a carbon-carbon single bond is between the 7 and 8 carbon atoms.
  • Y is a double bond between the carbon atom to which it is attached, and Y represents O.
  • the compound X represents -NH -, - N (R) -, - O -, - CH 2 - or halogen;
  • R represents a C 1-6 alkyl group, or a 3-8 membered a cycloalkyl group (preferably cyclohexyl); wherein, when X is a halogen, R 1 is absent;
  • X represents -NH-, -N(R)-, -O- or halogen (e.g., fluoro); R represents cyclohexyl.
  • R 1 in the compound is absent or represents hydrogen, C 1-4 alkyl, -PO(OR) 2 , monosaccharide, C 1-4 alkoxycarbonyl-C 1 a -4 alkyl group, a 3-6 membered cycloalkyl-amino group, an aryl-C 1-4 alkyl group or an aryl group; wherein the C 1-4 alkyl group, a monosaccharide group, a C 1-4 alkoxy group Carbonyl-C 1-4 alkyl, 3-6 membered cycloalkyl-aminoacyl, aryl-C 1-4 alkyl and aryl are unsubstituted or one or more (eg 1, 2, 3 or 4) Substituted by a substituent selected from the group consisting of halogen, hydroxy, amino, C 1-4 alkyl, C 1-4 alkoxy, C 1-4 alkylamino and C 1-4 alkanoyl; preferably, The aryl
  • R represents a C 1-4 alkyl group.
  • R 1 is absent or represents hydrogen, C 1-4 alkyl, -PO(OR) 2 , glucosyl, C 1-2 alkoxycarbonyl-C 1-2 alkyl, ring Hexyl-aminoacyl, phenyl-C 1-2 alkyl, naphthyl-C 1-2 alkyl, phenyl or naphthyl; wherein the methyl, ethyl, glucosyl, C 1-2 alkoxy Carbonyl-C 1-2 alkyl, cyclohexyl-aminoacyl, phenyl-C 1-2 alkyl, naphthyl-C 1-2 alkyl, phenyl or naphthyl are unsubstituted or one or more For example, 1, 2, 3 or 4) are substituted with a substituent selected from the group consisting of C 1-2 alkyl, C 1-2 alkoxy and C 1-2 alkanoyl;
  • R represents a C 1-4 alkyl group.
  • R 1 is absent or represents hydrogen, C 1-4 alkyl, -PO(OR) 2 , phenyl-C 1-2 alkyl, C 1-2 alkoxycarbonyl-C 1-2 alkyl, 3-6 membered cycloalkyl-aminoacyl;
  • R represents a C 1-3 alkyl group.
  • R 1 is absent or represents hydrogen, methyl, ethyl, -PO(OMe) 2 , -PO(OEt) 2 , -PO(O i Pr) 2 , 2,3, 4,6-Tetraacetoxy- ⁇ -D-glucopyranosyl, EtOCOCH 2 -, cyclohexyl-aminoacyl, benzyl, methoxyphenyl or tert-butylphenyl.
  • R 2 in the compound represents H, D, -OH, -PO(OR) 2 , C 1-6 alkyl, 9-15 membered fused heteroaryl or sulfonate;
  • the C 1-6 alkyl or 6-15 membered heteroaryl is unsubstituted or substituted by one or more (eg 1, 2, 3 or 4) substituents selected from the group consisting of amino, halogen, a hydroxyl group, an oxy group, a C 1-6 alkyl group, a C 1-6 alkoxy group, a C 1-6 alkanoyl group, a cyano group, a trifluoromethyl group and a carboxyl group;
  • R represents H, C 1-6 alkyl or aryl.
  • R 2 represents H, D, -PO(OR) 2 , C 1-4 alkyl, 9-15 membered benzofused heteroaryl or sulfonate; wherein said C 1 -4 alkyl or 9-15 membered benzoheteroaryl is unsubstituted or substituted by one or more (eg 1, 2, 3 or 4) substituents selected from the group consisting of amino, halogen, hydroxy, An oxy group, a C 1-4 alkyl group, a C 1-4 alkoxy group, a C 1-4 alkanoyl group, a cyano group, a trifluoromethyl group and a carboxyl group;
  • R represents H, C 1-4 alkyl or phenyl.
  • R 2 represents H, D, -PO(OR) 2 , C 1-4 alkyl, 9-15 membered benzofused heteroaryl or sulfonate; wherein said C 1 -4 alkyl and 9-15 membered benzofused heteroaryl are unsubstituted or substituted by one or more (eg 1, 2, 3 or 4) substituents selected from the group consisting of amino, halogen, hydroxy, An oxy group, a C 1-4 alkyl group, a C 1-4 alkoxy group, a C 1-4 alkanoyl group, a cyano group, a trifluoromethyl group and a carboxyl group;
  • R represents H, C 1-4 alkyl or phenyl.
  • R 2 represents H, D, -PO(OR) 2 , 2-oxophenyl, decyl or sodium sulfonate; wherein the thiol is unsubstituted or Or a plurality (for example 1, 2, 3 or 4) of substituents selected from the group consisting of amino, fluorine, chlorine, bromine, hydroxy, methyl, methoxy, formyl, cyano, trifluoromethyl And carboxyl groups;
  • R represents H, methyl, ethyl, isopropyl or phenyl.
  • the carbon and carbon double bonds between the 7 and 8 carbon atoms of the compound are preferred embodiments.
  • the compound Y and the carbon atom to which it is attached are carbon-carbon double bonds.
  • the compound Y and the carbon atom to which it is attached are a carbon-carbon single bond.
  • the compound has the structure:
  • R 3 and R 4 each independently represent H, C 1-6 alkyl or C 1-6 alkanoyl
  • R 3 and R 4 each independently represent H, C 1-4 alkyl or C 1-4 alkanoyl;
  • R 3 and R 4 each independently represent H, methyl or butanoyl.
  • the compound has the structure:
  • R 4 represents H, C 1-6 alkanoyl, C 1-6 alkoxycarbonyl or a monosaccharide group substituted by one or more (for example 1, 2, 3 or 4) C 1-6 alkanoyl;
  • R 4 represents H, C 1-4 alkanoyl, C 1-4 alkoxycarbonyl or by one or more (eg 1, 2, 3 or 4) C 1-4 alkanoyl Substituted glucose glycosyl.
  • R 4 represents H or a C 1-2 alkoxycarbonyl group.
  • R 4 represents H, butyryl, ethoxycarbonyl or 2,3,4,6-tetraacetoxy- ⁇ -D-glucopyranose.
  • the compound is selected from the group consisting of:
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77 for inhibition Transcriptional activity of Nur77.
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to inhibit the transcriptional activity of Nur77 in a cell.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77 for inhibition
  • the biological effect of TNF ⁇ is phosphorylation of ⁇ B inhibitory protein (I ⁇ B) kinase ⁇ / ⁇ (IKK ⁇ / ⁇ ), degradation of I ⁇ B ⁇ , nuclear transport of NF- ⁇ B subunit p65, and/ Or activation of NF- ⁇ B.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77 for inhibition mTOR pathway in cells (eg, down-regulation of P-mTOR, p-P70S6K, and/or p-S6 activity).
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77, Prevention or treatment of Nur77-related diseases in subjects in need thereof.
  • the Nur77-associated disease is cancer (eg, the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as an orphan)
  • a ligand for the nuclear receptor Nur77 for use in preventing or treating a disease associated with Nur77 in a subject in need thereof.
  • the disease associated with Nur77 is a cancer (eg, a triple vaginal mammary gland) cancer).
  • the present application relates to a method of inhibiting the transcriptional activity of the orphan nuclear receptor Nur77, comprising: Nur77 with a compound of formula I, a tautomer, a stereoisomer or a pharmaceutically thereof Acceptable salt or ester phase contact steps:
  • the method is for inhibiting the transcriptional activity of Nur77 in a cell.
  • the methods comprise administering to a cell in need thereof an effective amount of the compound to inhibit transcriptional activity of Nur77 in the cell.
  • the cell expresses Nur77.
  • the cell is a triple negative breast cancer cell and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells)
  • the present application relates to a method of inhibiting the biological effects of TNF ⁇ in a cell, comprising administering to a cell in need thereof an effective amount of a compound of formula I, a tautomer thereof, Stereoisomers or pharmaceutically acceptable salts or esters:
  • the biological effect of TNF ⁇ is phosphorylation of ⁇ B inhibitory protein (I ⁇ B) kinase ⁇ / ⁇ (IKK ⁇ / ⁇ ), degradation of I ⁇ B ⁇ , nuclear transport of NF- ⁇ B subunit p65, and/ Or activation of NF- ⁇ B.
  • the method is for inhibiting TNF ⁇ -induced I ⁇ B ⁇ degradation.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the present application relates to a method of inhibiting the mTOR signaling pathway comprising administering to a cell in need thereof an effective amount of a compound, a tautomer, a stereoisomer thereof, or a compound of Formula I, or Pharmaceutically acceptable salts or esters:
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to downregulate P-mTOR, p-P70S6K and/ in cells Or the activity of p-S6.
  • the cell expresses Nur77.
  • the cell is three Yin breast cancer cells and other cancer cells (such as liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells or prostate cancer cells)
  • the present application relates to a method of preventing or treating a disease associated with Nur77 comprising administering to a subject in need thereof an effective amount of a compound of formula I, a tautomer thereof , a stereoisomer or a pharmaceutically acceptable salt or ester:
  • the Nur77-associated disease is cancer (eg, triple-negative breast cancer).
  • the application relates to the use of a compound, a tautomer, a stereoisomer or a pharmaceutically acceptable salt or ester thereof, according to formula IV, for use as a ligand for the orphan nuclear receptor Nur77 Or a drug for the preparation of a ligand for use as an orphan nuclear receptor Nur77:
  • X represents NH, O or CH 2 ;
  • Y represents O, S or NR
  • R 5 and R 6 each independently represent a H or C 1-6 alkyl group
  • R 7 When R 7 and a carbon atom to which it is bonded are a single bond, R 7 represents OH, and when R 7 and a carbon atom to which it is bonded are a double bond, R 7 represents O;
  • R 8 represents H or C 1-6 alkyl
  • Virtual real double bond in formula (IV) Represents a single button or a double button.
  • the compound is selected from the group consisting of:
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77 for inhibition Transcriptional activity of Nur77.
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to inhibit the transcriptional activity of Nur77 in a cell.
  • the cell expresses Nur77.
  • the cell is a triple negative breast cancer cell and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells)
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77 for inhibition
  • the biological effect of TNF ⁇ is phosphorylation of ⁇ B inhibitory protein (I ⁇ B) kinase ⁇ / ⁇ (IKK ⁇ / ⁇ ), degradation of I ⁇ B ⁇ , nuclear transport of NF- ⁇ B subunit p65, and/ Or activation of NF- ⁇ B.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77 for inhibition mTOR pathway in cells (eg, down-regulation of P-mTOR, p-P70S6K, and/or p-S6 activity).
  • the cell expresses Nur77.
  • the cell is a triple negative breast cancer cell and other cancer cells (eg, Such as liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells or prostate cancer cells).
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77, Prevention or treatment of Nur77-related diseases in subjects in need thereof.
  • the Nur77-associated disease is cancer (eg, triple-negative breast cancer).
  • the present application relates to a method of inhibiting the transcriptional activity of the orphan nuclear receptor Nur77, comprising: Nur77 with a compound of formula IV, a tautomer, a stereoisomer or a pharmaceutically thereof Acceptable salt or ester phase contact steps:
  • X represents NH, O or CH 2 ;
  • Y represents O, S or NR
  • R 5 and R 6 each independently represent a H or C 1-6 alkyl group
  • R 7 When R 7 and a carbon atom to which it is bonded are a single bond, R 7 represents OH, and when R 7 and the carbon atom to which it is bonded are a double bond, R 7 represents O;
  • R 8 represents H or C 1-6 alkyl
  • Virtual real double bond in formula (IV) Represents a single button or a double button.
  • the compound is selected from the group consisting of:
  • the method is for inhibiting the transcriptional activity of Nur77 in a cell.
  • the methods comprise administering to a cell in need thereof an effective amount of the compound to inhibit transcriptional activity of Nur77 in the cell.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the present application relates to a method of inhibiting the biological effects of TNF ⁇ in a cell, comprising administering to a cell in need thereof an effective amount of a compound, a tautomer thereof, of the formula IV, Stereoisomers or pharmaceutically acceptable salts or esters:
  • X represents NH, O or CH 2 ;
  • Y represents O, S or NR
  • R 5 and R 6 each independently represent a H or C 1-6 alkyl group
  • R 7 When R 7 and a carbon atom to which it is bonded are a single bond, R 7 represents OH, and when R 7 and the carbon atom to which it is bonded are a double bond, R 7 represents O;
  • R 8 represents H or C 1-6 alkyl
  • Virtual real double bond in formula (IV) Represents a single button or a double button.
  • the compound is selected from the group consisting of:
  • the biological effect of TNF ⁇ is phosphorylation of ⁇ B inhibitory protein (I ⁇ B) kinase ⁇ / ⁇ (IKK ⁇ / ⁇ ), degradation of I ⁇ B ⁇ , nuclear transport of NF- ⁇ B subunit p65, and/ Or activation of NF- ⁇ B.
  • the method is for inhibiting TNF ⁇ -induced I ⁇ B ⁇ degradation.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the present application relates to a method of inhibiting the mTOR pathway comprising administering to a cell in need thereof an effective amount of a compound, a tautomer, a stereoisomer or a pharmaceutically thereof, as shown in Formula IV.
  • X represents NH, O or CH 2 ;
  • Y represents O, S or NR
  • R 5 and R 6 each independently represent a H or C 1-6 alkyl group
  • R 7 When R 7 and a carbon atom to which it is bonded are a single bond, R 7 represents OH, and when R 7 and the carbon atom to which it is bonded are a double bond, R 7 represents O;
  • R 8 represents H or C 1-6 alkyl
  • Virtual real double bond in formula (IV) Represents a single button or a double button.
  • the compound is selected from the group consisting of:
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to down regulate P-mTOR, p-P70S6K and/or p -S6 activity.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the present application relates to a method of preventing or treating a disease associated with Nur77 comprising administering to a subject in need thereof an effective amount of a compound of formula IV, a tautomer thereof, , a stereoisomer or a pharmaceutically acceptable salt or ester:
  • X represents NH, O or CH 2 ;
  • Y represents O, S or NR
  • R 5 and R 6 each independently represent a H or C 1-6 alkyl group
  • R 7 When R 7 and a carbon atom to which it is bonded are a single bond, R 7 represents OH, and when R 7 and the carbon atom to which it is bonded are a double bond, R 7 represents O;
  • R 8 represents H or C 1-6 alkyl
  • Virtual real double bond in formula (IV) Represents a single button or a double button.
  • the compound is selected from the group consisting of:
  • the Nur77-associated disease is cancer (eg, triple-negative breast cancer).
  • the application relates to the use of a compound, a tautomer, a stereoisomer or a pharmaceutically acceptable salt or ester thereof, which is used as a ligand for the orphan nuclear receptor Nur77, or for preparation Drugs used as ligands for the orphan nuclear receptor Nur77:
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77 for inhibition Transcriptional activity of Nur77.
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to inhibit the transcriptional activity of Nur77 in a cell.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable is used as a ligand for the orphan nuclear receptor Nur77 for inhibiting the biological effects of TNF ⁇ in cells.
  • the biological effect of TNF ⁇ is phosphorylation of ⁇ B inhibitory protein (I ⁇ B) kinase ⁇ / ⁇ (IKK ⁇ / ⁇ ), degradation of I ⁇ B ⁇ , nuclear transport of NF- ⁇ B subunit p65, and/ Or activation of NF- ⁇ B.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77 for inhibition mTOR pathway in cells (eg, downregulation of P-mTOR, p-P70S6 and/or p-S6 activity).
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77, Prevention or treatment of Nur77-related diseases in subjects in need thereof.
  • the Nur77-associated disease is cancer (eg, triple-negative breast cancer).
  • the present application relates to a method of inhibiting the transcriptional activity of the orphan nuclear receptor Nur77, comprising: Nur77 with the following compounds, tautomers, stereoisomers or pharmaceutically acceptable salts thereof or The steps of contacting the ester phase:
  • the method is for inhibiting the transcriptional activity of Nur77 in a cell.
  • the methods comprise administering to a cell in need thereof an effective amount of the compound to inhibit transcriptional activity of Nur77 in the cell.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the present application relates to a method of inhibiting the biological effects of TNF ⁇ in a cell, comprising administering to a cell in need thereof an effective amount of a compound shown below, a tautomer thereof, a stereoisomer Construct or pharmaceutically acceptable salt or ester:
  • the biological effect of TNF ⁇ is phosphorylation of ⁇ B inhibitory protein (I ⁇ B) kinase ⁇ / ⁇ (IKK ⁇ / ⁇ ), degradation of I ⁇ B ⁇ , nuclear transport of NF- ⁇ B subunit p65, and/ Or activation of NF- ⁇ B.
  • the method is for inhibiting TNF ⁇ -induced I ⁇ B ⁇ degradation.
  • the cell expresses Nur77.
  • the cell is a cancer cell (eg, a liver cancer cell, a cervical cancer cell, a lung cancer cell, a breast cancer cell, a colorectal cancer cell, or a prostate cancer cell).
  • the present application relates to a method of inhibiting the mTOR pathway comprising administering to a cell in need thereof an effective amount of a compound shown below, a tautomer thereof, a stereoisomer or a pharmaceutically acceptable Accepted salt or ester:
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to down regulate P-mTOR, p-P70S6K and/or p -S6 activity.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the present application relates to a method of preventing or treating a disease associated with Nur77 comprising administering to a subject in need thereof an effective amount of a compound shown below, a tautomer thereof, a stereo Isomer or pharmaceutically acceptable salt or ester:
  • the Nur77-associated disease is cancer (eg, triple-negative breast cancer).
  • the present application relates to a compound, a tautomer, a stereoisomer or a pharmaceutically acceptable salt or ester thereof as shown below:
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-6 alkyl and aryl;
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-4 alkyl, phenyl and naphthyl;
  • R 9 and R 10 are each independently selected from the group consisting of methyl, ethyl and isopropyl.
  • the compound is selected from the group consisting of:
  • the present application relates to the use of a compound, a tautomer, a stereoisomer or a pharmaceutically acceptable salt or ester thereof, according to Formula V, for use as a ligand for the orphan nuclear receptor Nur77 Or a drug for the preparation of a ligand for use as an orphan nuclear receptor Nur77:
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-6 alkyl and aryl;
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-4 alkyl, phenyl and naphthyl;
  • R 9 and R 10 are each independently selected from the group consisting of methyl, ethyl and isopropyl.
  • the compound is selected from the group consisting of:
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77, Inhibition of the transcriptional activity of Nur77.
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to inhibit transcription of Nur77 in vivo, in vitro or ex vivo active.
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to inhibit the transcriptional activity of Nur77 in a cell.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77, Inhibition of the biological effects of TNF ⁇ in cells.
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to inhibit TNF ⁇ in cells in vivo, in vitro or ex vivo Biological effects.
  • the biological effect of TNF ⁇ is phosphorylation of ⁇ B inhibitory protein (I ⁇ B) kinase ⁇ / ⁇ (IKK ⁇ / ⁇ ), degradation of I ⁇ B ⁇ , nuclear transport of NF- ⁇ B subunit p65, and / or activation of NF- ⁇ B.
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to inhibit TNF[alpha]-induced I ⁇ B[alpha] degradation.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77 for inhibition mTOR pathway in cells (eg, downregulation of P-mTOR, p-P70S6 and/or p-S6 activity).
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77, A disease associated with Nur77 is prevented or treated in a subject in need thereof.
  • the disease associated with Nur77 is cancer.
  • the cancer is selected from the group consisting of liver cancer, cervical cancer, lung cancer, and breast cancer.
  • the cancer is triple negative breast cancer (ie, breast cancer with negative estrogen receptor (ER), progesterone receptor (PR), and proto-oncogene Her-2).
  • the medicament is for use in treating cancer and comprises the compound, a tautomer, a stereoisomer or a pharmaceutically acceptable salt or ester thereof, and TNF ⁇ .
  • the present application relates to a method of inhibiting the transcriptional activity of the orphan nuclear receptor Nur77, comprising: Nur77 and a compound of Formula V, a tautomer, a stereoisomer thereof or a pharmaceutically acceptable The steps of contacting the salt or ester:
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-6 alkyl and aryl;
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-4 alkyl, phenyl and naphthyl;
  • R 9 and R 10 are each independently selected from the group consisting of methyl, ethyl and isopropyl.
  • the compound is selected from the group consisting of:
  • the method is for inhibiting the transcriptional activity of Nur77 in a cell.
  • the methods comprise administering to a cell in need thereof an effective amount of the compound to inhibit transcriptional activity of Nur77 in the cell.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the present application relates to a method of inhibiting the biological effects of TNF ⁇ in a cell, comprising administering to a cell in need thereof an effective amount of a compound of formula V, a tautomer thereof, a stereo Isomer or pharmaceutically acceptable salt or ester:
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-6 alkyl and aryl.
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-4 alkyl, phenyl, and naphthyl.
  • R 9 and R 10 are each independently selected from the group consisting of methyl, ethyl, and isopropyl.
  • the compound is selected from the group consisting of:
  • the biological effect of TNF ⁇ is phosphorylation of ⁇ B inhibitory protein (I ⁇ B) kinase ⁇ / ⁇ (IKK ⁇ / ⁇ ), degradation of I ⁇ B ⁇ , nuclear transport of NF- ⁇ B subunit p65, and/ Or activation of NF- ⁇ B.
  • the method is for inhibiting TNF ⁇ -induced I ⁇ B ⁇ degradation.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the present application relates to a method of inhibiting the mTOR pathway comprising administering to a cell in need thereof an effective amount of a compound of formula V, a tautomer, a stereoisomer or a pharmaceutically thereof Acceptable salts or esters:
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-6 alkyl and aryl.
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-4 alkyl, phenyl, and naphthyl.
  • R 9 and R 10 are each independently selected from the group consisting of methyl, ethyl, and isopropyl.
  • the compound is selected from the group consisting of:
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to down regulate P-mTOR, p-P70S6K and/or p -S6 activity.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the present application relates to a method of preventing or treating a disease associated with Nur77, comprising administering to a subject in need thereof an effective amount of a compound of formula V, a tautomer thereof, Stereoisomer or Pharmaceutically acceptable salts or esters:
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-6 alkyl and aryl.
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-4 alkyl, phenyl, and naphthyl.
  • R 9 and R 10 are each independently selected from the group consisting of methyl, ethyl, and isopropyl.
  • the compound is selected from the group consisting of:
  • the Nur77-associated disease is cancer (eg, triple-negative breast cancer).
  • the application relates to the following compounds, tautomers, stereoisomers or pharmaceutically acceptable salts or esters thereof:
  • the application relates to the following compounds, tautomers, stereoisomers or pharmaceutically acceptable
  • a salt or ester accepted for use as a ligand for the orphan nuclear receptor Nur77 or for the preparation of a ligand for use as a ligand for the orphan nuclear receptor Nur77:
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77, Inhibition of the transcriptional activity of Nur77.
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to inhibit transcription of Nur77 in vivo, in vitro or ex vivo active.
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to inhibit the transcriptional activity of Nur77 in a cell.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77, Inhibition of the biological effects of TNF ⁇ in cells.
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to inhibit TNF ⁇ in cells in vivo, in vitro or ex vivo Biological effects.
  • the biological effect of TNF ⁇ is phosphorylation of ⁇ B inhibitory protein (I ⁇ B) kinase ⁇ / ⁇ (IKK ⁇ / ⁇ ), degradation of I ⁇ B ⁇ , nuclear transport of NF- ⁇ B subunit p65, and / or activation of NF- ⁇ B.
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to inhibit TNF[alpha]-induced I ⁇ B[alpha] degradation.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77 for inhibition mTOR pathway in cells (eg, down-regulation of P-mTOR, p-P70S6, and/or p-S6 activity).
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used as a ligand for the orphan nuclear receptor Nur77, A disease associated with Nur77 is prevented or treated in a subject in need thereof.
  • the disease associated with Nur77 is cancer.
  • the cancer is selected from the group consisting of liver cancer, cervical cancer, lung cancer, and breast cancer.
  • the cancer is triple negative breast cancer (ie, breast cancer with negative estrogen receptor (ER), progesterone receptor (PR), and proto-oncogene Her-2).
  • the medicament is for use in treating cancer and comprises the compound, a tautomer, a stereoisomer or a pharmaceutically acceptable salt or ester thereof, and TNF ⁇ .
  • the present application relates to a method of inhibiting the transcriptional activity of the orphan nuclear receptor Nur77, comprising: Nur77 with the following compounds, tautomers, stereoisomers or pharmaceutically acceptable salts thereof or The steps of contacting the ester phase:
  • the method is for inhibiting the transcriptional activity of Nur77 in a cell.
  • the methods comprise administering to a cell in need thereof an effective amount of the compound to inhibit transcriptional activity of Nur77 in the cell.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the present application relates to a method of inhibiting the biological effects of TNF ⁇ in a cell, comprising administering to a cell in need thereof an effective amount of the following compound, tautomer, stereoisomer or Pharmaceutically acceptable salts or esters:
  • the biological effect of TNF ⁇ is phosphorylation of ⁇ B inhibitory protein (I ⁇ B) kinase ⁇ / ⁇ (IKK ⁇ / ⁇ ), degradation of I ⁇ B ⁇ , nuclear transport of NF- ⁇ B subunit p65, and/ Or activation of NF- ⁇ B.
  • the method is for inhibiting TNF ⁇ -induced I ⁇ B ⁇ degradation.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the present application relates to a method of inhibiting the mTOR pathway comprising administering to a cell in need thereof an effective amount of the following compound, tautomer, stereoisomer or pharmaceutically acceptable salt thereof Or ester:
  • the compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester or the drug is used to down regulate P-mTOR, p-P70S6K and/or p -S6 activity.
  • the cell expresses Nur77.
  • the cells are triple negative breast cancer cells and other cancer cells (eg, liver cancer cells, cervical cancer cells, lung cancer cells, Sanyang breast cancer cells, colorectal cancer cells, or prostate cancer cells).
  • the present application relates to a method of preventing or treating a disease associated with Nur77, comprising An effective amount of the following compound, its tautomer, stereoisomer or pharmaceutically acceptable salt or ester is administered to a subject in need thereof:
  • the Nur77-associated disease is cancer (eg, triple-negative breast cancer).
  • the present application relates to a method of screening a drug having anticancer activity, comprising the steps of:
  • a compound represented by Formula I-Formula V can target the orphan nuclear receptor Nur77 and function as a ligand thereof.
  • such compounds can be used to reduce or inhibit the activity (eg, transcriptional activity) of the orphan nuclear receptor Nur77.
  • the inhibition of mTOR signaling pathway can be used as an effective indicator for screening anticancer drugs.
  • the present inventors have found that compounds which inhibit the mTOR signaling pathway by Nur77 have potential anticancer activity.
  • the present application also provides a screening method for a drug having anticancer (especially triple negative breast cancer) activity.
  • the compounds of the present application are also useful for the prevention and treatment of diseases associated with the orphan nuclear receptor Nur77, such as cancer (eg, triple negative breast cancer). Therefore, the present invention provides a new, effective, specific binding to Nur77 Ligands, which can be used to develop new therapies for the treatment of cancer, such as triple-negative breast cancer.
  • cancer eg, triple negative breast cancer
  • the molecular biology experimental methods and immunoassays used in the present invention are basically referred to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method described in FMAusubel et al., Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes according to the conditions recommended by the product manufacturer.
  • the reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.
  • Figure 1 shows the screening of compounds that bind to Nur77 using the Biacore T200 instrument.
  • the results of the experiment for detecting the binding of YXY101 to Nur77-LBD using a Biacore T200 instrument are shown, wherein red dots represent compound YXY101; blue dots represent control compounds.
  • the results showed that the compound YXY101 was able to bind to Nur77-LBD.
  • Figure 2A shows the chemical structural formula of tripterine (compound YXY101).
  • Figure 2B shows the results of experiments with further binding of different concentrations of YXY101 (0.04 ⁇ M, 0.08 ⁇ M, 0.16 ⁇ M, 0.32 ⁇ M, 0.64 ⁇ M) to Nur77-LBD.
  • the results showed that the dissociation constant (Kd) of the compound YXY101 combined with Nur77-LBD was 292 nM;
  • 2C shows experimental results of detecting the binding of YXY101 to Nur77-LBD by circular dichroism spectroscopy, wherein the red curve represents YXY101+Nur77-LBD; the blue curve represents Nur77-LBD.
  • the results showed that the compound YXY101 could change the CD spectrum of Nur77-LBD. This indicates that the compound YXY101 can bind to Nur77-LBD.
  • 2D shows the results of an experiment for detecting the binding of YXY101 to Nur77-LBD by HPLC, wherein the red curve represents YXY101+Nur77-LBD; the purple curve represents YXY101+RXR ⁇ -LBD.
  • the results showed that the compound YXY101 was able to combine with Nur77-LBD to form a complex, but did not bind to RXR ⁇ -LBD.
  • Figure 2E shows the detection of the binding of YXY101 to Nur77-LBD using the dual luciferase reporter system. Test results. The results showed that the compound YXY101 inhibited the transcriptional activation of Nur77, but had no significant effect on the transcriptional activation of the glucocorticoid receptor (GR). This indicates that the compound YXY101 is capable of binding to Nur77-LBD and inhibiting its transcriptional activity; and, the compound YXY101 does not bind to GR.
  • GR glucocorticoid receptor
  • Figure 2F shows the molecular docking of YXY101 with Nur77.
  • Molecular docking results show that YXY101 binds to the known hydrophobic grooves on the surface of the Nur77 protein mainly by hydrophobic interaction.
  • Figures 3A-3B show the results of immunoblot analysis of I ⁇ B ⁇ and phosphorylated IKK ⁇ / ⁇ in cells treated with different concentrations of YXY101 and TNF ⁇ .
  • Figure 3C shows the results of immunofluorescence staining of cells treated with YXY101 and TNF ⁇ (Scale bar: 20 ⁇ m).
  • Figure 3D shows the results of analysis of NF- ⁇ B activity of cells treated with YXY101 and TNF ⁇ , wherein **P < 0.01, ***P < 0.001 (T test).
  • Figure 3E shows the results of immunoblot analysis of I ⁇ B ⁇ and phosphorylated IKK ⁇ / ⁇ in different cancer cell lines treated with different concentrations of YXY101 and TNF ⁇ .
  • FIG. 3F shows the results of immunoblot analysis of I ⁇ B ⁇ in HepG2 cells treated with different compounds and stimulated with TNF ⁇ .
  • 4A-4B show the results of immunoblot analysis of Nur77, RXR ⁇ and I ⁇ B ⁇ in HepG2 cells transfected with different siRNAs and treated with different concentrations of YXY101 and TNF ⁇ .
  • Figure 4C shows the results of immunoblot analysis of I ⁇ B ⁇ in MEF cells and Nur77-/-MEF cells treated with different concentrations of YXY101 and TNF ⁇ .
  • Figure 4D shows the results of immunofluorescence staining of MEF cells and Nur77-/-MEF cells treated with YXY101 and TNF ⁇ (Scale bar: 10 ⁇ m).
  • Figure 4E shows the results of immunoblot analysis of I ⁇ B ⁇ in MEF cells and Nur77-/-MEF cells treated with different concentrations of XS0284 and TNF ⁇ .
  • Figure 5A shows the proliferation ratio of different breast cancer cells (MDA-MB-231; MDA-MB-468; BT549; SKBR3; T47D and MCF-7) versus YXY101 concentration, and the IC50 value of YXY101; -MB-231, MDA-MB-468, BT549 and SKBR3 are triple negative breast cancer cells, in red
  • the curve indicates; T47D and MCF-7 are three positive breast cancer cells, indicated by a blue curve.
  • Figure 5B shows the results of immunoblot analysis of parp and ER in MDA-MB-231 and MCF-7 cells treated with different concentrations of YXY101 (0 ⁇ M, 0.25 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 2 ⁇ M, or 4 ⁇ M).
  • Figure 5C shows the results of immunoblot analysis of parp and p-mTOR in MDA-MB-231 treated with different concentrations of YXY101 (0 ⁇ M, 2 ⁇ M, or 4 ⁇ M) for 12 h or 24 h.
  • Figure 5D shows the results of immunoblot analysis of parp in MDA-MB-231 cells treated with YXY101 in combination with TNF ⁇ at different times (1 h, 6 h or 12 h).
  • Figure 5E shows the concentration profile of Hela cells versus YXY101. The results show that the IC50 value of YXY101 for wild-type Hela cells is 1.189 ⁇ M, while the IC50 for Nur77 knock-out Hela cells is 58.166 ⁇ M.
  • Figure 6 shows the results of immunohistochemical staining of tumor tissues of nude mice of each group.
  • the left panel of Figure 6A shows the tumors formed in each group of nude mice; the middle panel shows the RTV of the tumors formed in each group of nude mice; the right panel shows the weight of the tumors formed in each group of nude mice.
  • Fig. 7A shows the overall condition of MMTV-PYVT mice at 11 weeks, 13 weeks or 17 weeks after intragastric administration of 2 mg/kg of YXY101 two weeks.
  • Fig. 7B shows the results of HE staining and immunohistochemical staining of tumor tissues of MMTV-PYVT mice from 11 weeks, 13 weeks or 17 weeks after intragastric administration of 2 mg/kg of YXY101 two weeks.
  • Figure 7C shows the weight, morphology, HE staining and immunohistochemical staining of lung tissue from MMTV-PYVT mice from 11 weeks, 13 weeks or 17 weeks after intragastric administration of 2 mg/kg of YXY101 for two weeks. Analysis results.
  • Figure 8A shows the survival curves of wild-type mmtv-PyMT mice, Nur77 knockout mmtv-PyMT mice within 60 days.
  • Figure 8B shows the statistical results of tumor weight comparison at 11 weeks, 13 weeks, and 17 weeks in wild type mmtv-PyMT mice, Nur77 knockout mmtv-PyMT mice.
  • Figure 8C shows a comparison of tumor appearance morphological size at 11 weeks, 13 weeks, and 17 weeks in wild type mmtv-PyMT mice and Nur77 knockout mmtv-PyMT mice.
  • Figure 8D shows the effect of detecting YXY101 on the survival rate of mmtv-PyMT mice at the animal level.
  • Figure 9A shows the treatment of MB-MDA-231 breast cancer cells with TNF ⁇ under starvation conditions, using immunoblotting The technique of the trace detects the result of p-P70S6K.
  • Figure 9B shows the results of treatment of SKBR3 breast cancer cells with TNF ⁇ under starvation conditions using immunoblotting techniques to detect p-P70S6K.
  • Figure 9C shows that wild-type and Nur77 knockout MEF cells were treated with TNF ⁇ under starvation conditions for different time (30 min, 2 h) or different concentrations (20 ng/ml/, 40 ng/ml) and detected by immunoblotting.
  • Figure 10A shows the results of treatment of MB-MDA-231 breast cancer cells with TNF ⁇ (20 ng/ml), YXY101 (1 ⁇ M, 2 ⁇ M, 4 ⁇ M) under starvation conditions, and detection of p-mTOR and p-P70S6K by immunoblotting. .
  • Figure 10B shows that the Flag empty plasmid or the Flag-Nur77 plasmid was transfected in MB-MDA-231 breast cancer cells, and treated with TNF ⁇ , YXY101 for 3 h, 6 h under starvation conditions, and p-mTOR, p was detected by immunoblotting. -P70S6K, Nur77 results.
  • Figure 10C shows the results of silencing Nur77 treatment of MB-MDA-231 cells and treatment with TNF ⁇ , YXY101 under starvation conditions, and detection of p-P70S6K and Nur77 by immunoblotting.
  • Figure 10D shows the results of treatment of wild-type or Nur77 knock-out MEF cells with TNF ⁇ , YXY101 (0.5 Mm, 1 ⁇ M), and the results of immunoblotting using p-S6, p-mTOR, and p62.
  • Figure 11A shows the proliferation ratio of different breast cancer cells (MDA-MB-231 and MCF-7) versus XS0503 concentration, and the IC50 value of XS0503.
  • Figure 11B shows the results of IC50 analysis of MDA-MB-231 cells treated with YXY101 derivatives XS0077, XS0335, XS0419, XS0474, XS0488.
  • Figure 11C shows BT474, ZR-75-1, BT549, HCC1937, HS578T, MCF-7, T47D treated with YXY101 derivatives XS0284, XS0285, XS0394, XS0418, XS0419, XS0474, XS0486, XS0462, XS0491, XS0492, XS0488.
  • the inhibition rate analysis results of these seven breast cancer cells.
  • Figure 12A shows the use of YXY101 (4 ⁇ M) and different YXY101 derivatives (4 ⁇ M) XS0394, XS0395, XS0419, XS0420, XS0421, XS0284, XS0335, XS0488, XS0491, XS0492, XS0418, XS0502, XS0503, XS0504, XS0506, XS0507, XS0508, XS0077 and TNF ⁇ (20ng/ml) Results of immunoblot analysis of PARP in treated MDA-MB-231 cells.
  • Figure 12B shows YXY101 (2 ⁇ M) and different YXY101 derivatives (2 ⁇ M) XS0284, XS0285, XS0335, XS0394, XS0418, XS0419, XS0454, XS0455, XS0462, XS0473, in the case of starvation treatment with serum-free medium for 10 h.
  • Figure 12C shows P-mTOR, P-p70S6K, P-S6 and I ⁇ B ⁇ in MDA-MB-231 cells treated with YXY101 (2 ⁇ M) and YXY101 derivatives XS0455 (1, 2, 4 ⁇ M) and TNF (20 ng/ml). The results of immunoblot analysis.
  • Figure 13 shows the results of experiments comparing the binding of different compounds (XS0418, XS0419, XS0474, XS0394, XS0492, XS0491, XS0488) to Nur77-LBD using a Biacore T200 instrument.
  • the dissociation constant (Kd) of the compound XS0418 bound to Nur77-LBD was 3.67 ⁇ M; the dissociation constant (Kd) of the compound XS0419 bound to Nur77-LBD was 404 nM; the dissociation constant of the binding of the compound XS0474 to Nur77-LBD ( Kd) was 2.60 ⁇ M; the dissociation constant (Kd) of compound XS0394 and Nur77-LBD was 1.26 ⁇ M; the dissociation constant (Kd) of compound XS0492 and Nur77-LBD was 1.30 ⁇ M; and compound XS0491 was combined with Nur77-LBD.
  • the dissociation constant (Kd) was 0.336 ⁇ M; the dissociation constant (Kd) of the compound XS0488 in combination with Nur77-LBD was 1.28 ⁇ M.
  • Figure 14 is a treatment of LFD feeding and HFD feeding with WT mice and PYVT mice, respectively, and gavage of YXY101 and its derivative XS0284.
  • Figure 14A shows the morphological appearance characteristics of mice in different experimental groups under different treatment conditions.
  • Figure 14B compares the morphological characteristics of the above mouse tumors.
  • Figure 14C shows the change in body weight of the above mice during the 12-day period of intragastric administration.
  • Figure 14D shows the statistical significance of the change in body weight of the above mice before and after treatment.
  • Figure 15A shows a comparison of the morphology of tumors after treatment with YXY101 and its derivative XS0284 in WT and Nur77 knockout mice of PYVT.
  • Figure 15B shows the statistical results of tumor tissue weight in the above mice.
  • Figure 16 shows the acute toxicity model of mice induced by intragastric or intraperitoneal injection of 200 mg/kg of YXY101 and its derivative XS0284.
  • the intragastric or intraperitoneal injection of YXY101 and XS0284 200 mg/kg was observed. After 12 hours, the histomorphology of the heart, liver, small intestine, white fat and kidney of each group and the results of HE staining were observed.
  • Figure 17 is a graph showing the mode of action of computer-assisted simulation of the binding of Nur77 to its ligand.
  • YXY101 The binding of YXY101 to Nur77 was determined by surface plasmon resonance. Briefly, 50 [mu]g of purified Nur77 ligand binding domain (Nur77-LBD) protein was coupled to Biacore's CM5 chip; binding of YXY101 (20 [mu]m) to Nur77-LBD was then determined by a Biacore T200 instrument. Unrelated compounds were used as controls. The measurement results are shown in Fig. 1.
  • Figure 1 shows the results of an experiment in which YXY101 was combined with Nur77-LBD using a Biacore T200 instrument, wherein red dots represent compound YXY101; blue dots represent control compounds. The results showed that the compound YXY101 was able to bind to Nur77-LBD.
  • the dissociation constant (Kd) of YXY101 and Nur77 was determined by surface plasmon resonance. Briefly, binding of different concentrations of YXY101 (0.04 ⁇ M, 0.08 ⁇ M, 0.16 ⁇ M, 0.32 ⁇ M, 0.64 ⁇ M) to Nur77-LBD was determined using a Biacore T200 instrument. The measurement results are shown in Fig. 2B.
  • YXY101 and Nur77-LBD were further analyzed using circular dichroism spectroscopy (CD). Briefly, YXY101 (1 ml, 1 mg/ml) was added to a phosphate buffer (10 ⁇ m, pH 7.4) of Nur77-LBD protein (1 ml, 1 mg/ml) and incubated at 4 ° C for 3 h. Subsequently, 0.7 ml of the solution was taken and detected using a Jasco J-810 spectropolarimeter. CD spectra from 190 nm to 260 nm were recorded. A separate Nur77-LBD solution (i.e., no compound YXY101 was added) was used as a control. The results of the detection are shown in Figure 2C.
  • YXY101 and Nur77-LBD were further analyzed using high performance liquid chromatography (HPLC). Briefly, YXY101 (600 uL, 0.1 mg/ml) was incubated with purified Nur77-LBD protein (5 ml, 1 mg/ml). After incubation for 3 h at 4 °C, the complex of YXY101 and Nur77-LBD was captured with Ni beads. Subsequently, the complex was dissociated using chloroform, and YXY101 in the dissociated product was extracted.
  • HPLC high performance liquid chromatography
  • YXY101 in the extracted product was detected using an HPLC spectrometer (Shimadzu LC 20A, Japan), wherein the column used was an ODS column (5 um, 4.6*250 mm), and the mobile phase was acetonitrile containing 0.2% H 3 PO 4 .
  • the aqueous solution has a detection wavelength of 425 nm.
  • the above experiment was repeated using YXY101 and RXR ⁇ -LBD (ligand binding domain of retinoid X receptor ⁇ ), and used as a control. The results of the detection are shown in Figure 2D.
  • YXY101 and Nur77-LBD were further analyzed using a dual luciferase reporter assay.
  • the experiment was repeated using YXY101 with a glucocorticoid receptor (GR) for use as a control.
  • GR glucocorticoid receptor
  • the compound YXY101 inhibited the transcriptional activation of Nur77, but had no significant effect on the transcriptional activation of the glucocorticoid receptor (GR). This indicates that the compound YXY101 is capable of binding to Nur77-LBD and inhibiting its transcriptional activity; and, the compound YXY101 does not bind to GR.
  • YXY101 and Nur77 (PDB code: 4JGV).
  • the conformation of YXY101 was constructed by the Lamarck genetic algorithm.
  • the lattice center is selected in the reported THPN coordinates (-12.08, 18.29, -4.233), and the grid size is set to 40*40*40 (X, Y, Z) grid points, each grid The dot spacing is 0.375A.
  • HepG2 cells were treated with different concentrations of YXY101 (0 ⁇ M, 0.25 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 2 ⁇ M or 4 ⁇ M) for 1 hour and treated with TNF ⁇ (0 ng/mL or 20 ng/mL) for 30 minutes. Subsequently, I ⁇ B ⁇ and phosphorylated IKK ⁇ / ⁇ in the cells were detected by Western Blotting. The results are shown in Figures 3A-3B.
  • HepG2 cells were treated with YXY101 (0 or 1 ⁇ M) for 1 hour and then treated with TNF ⁇ (20 ng/mL) for 30 minutes. Subsequently, the NF- ⁇ B subunit p65 in the cells was detected by immunofluorescence staining. Untreated cells were used as controls. The results are shown in Figure 3C.
  • Figure 3C shows the results of immunofluorescence staining of HepG2 cells treated with YXY101 and TNF ⁇ (Scalebar: 20 ⁇ m). The results in Figure 3C show that TNF ⁇ is able to induce nuclear translocation of the NF- ⁇ B subunit p65 in cells; whereas YXY101 is able to inhibit TNF ⁇ -induced p65 nuclear translocation.
  • the NF- ⁇ B reporter gene was transfected into HEK-293T cells and then treated with YXY101 (0 or 1 ⁇ M) and TNF ⁇ (20 ng/mL). Subsequently, NF- ⁇ B activity in the cells was examined. Untreated cells were used as controls. The results are shown in Figure 3D.
  • Figure 3D shows the results of analysis of NF- ⁇ B activity of cells treated with YXY101 and TNF ⁇ , wherein **P < 0.01, ***P < 0.001 (T test).
  • the results in Figure 4D show that TNF ⁇ is able to induce transcriptional activation of NF- ⁇ B in cells; whereas YXY101 is able to inhibit TNF ⁇ -induced NF- ⁇ B transcriptional activation.
  • a variety of cancer cell lines (LO2, SMMC-7721, QGY-7703, HeLa, H460) were treated with different concentrations of YXY101 (0 ⁇ M, 1 ⁇ M or 4 ⁇ M) for 1 hour and then treated with TNF ⁇ (0 ng/mL or 20 ng/mL). minute. Subsequently, I ⁇ B ⁇ and phosphorylated IKK ⁇ / ⁇ in the cells were detected by Western Blotting. The results are shown in Figure 3E.
  • TNF ⁇ was able to induce phosphorylation of IKK ⁇ / ⁇ and degradation of I ⁇ B ⁇ in various cell lines, while YXY101 was able to inhibit TNF ⁇ -induced phosphorylation of IKK ⁇ / ⁇ and degradation of I ⁇ B ⁇ .
  • HepG2 cells were also used for the experiments. Briefly, HepG2 cells were treated with various concentrations of various compounds (YXY101, XS0284, and XS0287) for the indicated times and then treated with TNF ⁇ (0 ng/mL or 20 ng/mL) for 30 minutes. Subsequently, I ⁇ B ⁇ in the cells was detected by Western Blotting. The results are shown in Figure 3F.
  • Figure 3F shows that YXY101 and its derivatives XS0284 and XS0287 are capable of inhibiting TNF ⁇ -induced degradation of I ⁇ B ⁇ in HepG2 cells.
  • YXY101 and its derivatives XS0284 and XS0287 are capable of inhibiting various biological effects of TNF ⁇ in cells, including phosphorylation of IKK ⁇ / ⁇ phosphorylation, degradation of I ⁇ B ⁇ , and NF- ⁇ B subunit p65 Nuclear transport, and transcriptional activation of NF- ⁇ B.
  • Control siRNA, Nur77 siRNA or RXR ⁇ siRNA were transfected into HepG2 cells. Subsequently, HepG2 cells were treated with different concentrations of YXY101 (0 ⁇ M, 1 ⁇ M or 4 ⁇ M) for 1 hour and then treated with TNF ⁇ (0 ng/mL or 20 ng/mL) for 30 minutes. Subsequently, Nur77, RXR ⁇ and I ⁇ B ⁇ in the cells were detected by Western Blotting. The results are shown in Figures 4A-4B.
  • FIGS. 4A-4B show the results of immunoblot analysis of Nur77, RXR ⁇ and I ⁇ B ⁇ in HepG2 cells transfected with different siRNAs and treated with different concentrations of YXY101 and TNF ⁇ .
  • the results showed that Nur77 siRNA effectively inhibited/knockout the expression of Nur77 in cells; and, RXR ⁇ siRNA effectively inhibited/knockout the expression of RXR ⁇ in cells; control siRNA did not affect the normal expression of Nur77 and RXR ⁇ .
  • MEF cells and Nur77-/-MEF cells i.e., MEF cells not expressing Nur77.
  • MEF cells and Nur77-/-MEF cells were treated with different concentrations of YXY101 (0 ⁇ M or 1 ⁇ M) for 1 hour and treated with TNF ⁇ (0 ng/mL or 20 ng/mL) for 30 minutes.
  • TNF ⁇ 0. ng/mL or 20 ng/mL
  • MEF cells and Nur77-/-MEF cells were treated with YXY101 (0 or 1 ⁇ M) for 1 hour and then treated with TNF ⁇ (20 ng/mL) for 30 minutes. Subsequently, the NF- ⁇ B subunit p65 in the cells was detected by immunofluorescence staining. Untreated cells were used as controls. The results are shown in Figure 4D.
  • Figure 4D shows the results of immunofluorescence staining of MEF cells and Nur77-/-MEF cells treated with YXY101 and TNF ⁇ (Scale bar: 10 ⁇ m).
  • the results showed that YXY101 was able to inhibit TNF ⁇ -induced p65 nuclear translocation in MEF cells expressing Nur77; however, in Nur77-/-MEF cells, YXY101 lost the ability to inhibit p65 entry into the nucleus.
  • Figure 4E shows the results of immunoblot analysis of I ⁇ B ⁇ in MEF cells and Nur77-/-MEF cells treated with different concentrations of XS0284 and TNF ⁇ .
  • the results showed that XS0284 was able to inhibit TNF ⁇ -induced degradation of I ⁇ B ⁇ in MEF cells expressing Nur77; however, in Nur77-/-MEF cells, XS0284 lost the ability to inhibit I ⁇ B ⁇ degradation.
  • Example 4 YXY101 has significant tumor suppressor activity, which is particularly sensitive to triple negative breast cancer, and this biological function is dependent on Nur77.
  • MDA-MB-231 Different breast cancer cells (MDA-MB-231) were treated with different concentrations of YXY101 (1 ⁇ M, 1.3 ⁇ M, 1.6 ⁇ M, 1.9 ⁇ M, 2.2 ⁇ M, 2.5 ⁇ M, 2.8 ⁇ M, 3.1 ⁇ M, 3.4 ⁇ M, 3.7 ⁇ M, 4.0 ⁇ M).
  • MDA-MB-468; BT549; SKBR3; T47D and MCF-7) were treated for 72 h and the proliferation ratio of breast cancer cells was determined.
  • a curve of the cancer cell proliferation ratio against the concentration of YXY101 was plotted, and the IC50 of YXY101 was determined. The results are shown in Figure 5A.
  • Figure 5A shows the proliferation ratio of different breast cancer cells (MDA-MB-231; MDA-MB-468; BT549; SKBR3; T47D and MCF-7) versus YXY101 concentration, and the IC50 value of YXY101; -MB-231, MDA-MB-468, BT549 and SKBR3 are triple negative breast cancer cells, indicated by a red curve; T47D and MCF-7 are three positive breast cancer cells, indicated by a blue curve.
  • YXY101 The ability to inhibit the proliferation of triple-negative breast cancer cells was significantly stronger than its ability to inhibit the proliferation of triple-positive breast cancer cells; among them, YXY101 inhibited the proliferation of MDA-MB-231, MDA-MB-468, BT549 and SKBR3 with IC50 of 1.204 and 0.187, respectively. , 0.245 and 2.646 ⁇ M, well below their IC50 for inhibition of T47D and MCF-7 proliferation (74.465 ⁇ M and 11.498 ⁇ M, respectively). This suggests that YXY101 can be used particularly advantageously for the treatment of triple negative breast cancer.
  • Figure 5E shows that the IC50 value of YXY101 in wild-type Hela cells is 1.189 ⁇ M, while the IC50 in Nur77 knock-out Hela cells is 58.166 ⁇ M.
  • the results indicate that the tumor suppressive effect of YXY101 is dependent on Nur77, plus 1 and Figure 2B show that YXY101 specifically binds to Nur77, and the tumor suppressing activity of YXY101 is closely related to Nur77.
  • MDA-MB-231 and MCF-7 cells were treated with different concentrations of YXY101 (0 ⁇ M, 0.25 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 2 ⁇ M, or 4 ⁇ M), followed by detection of parp and ER in the cells by WB. The results are shown in Figure 5B.
  • Figure 5B shows the results of immunoblot analysis of parp and ER in MDA-MB-231 and MCF-7 cells treated with different concentrations of YXY101 (0 ⁇ M, 0.25 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 2 ⁇ M, or 4 ⁇ M). The results showed that YXY101 induced parp cleavage in MDA-MB-231, ie, apoptotic phenomenon.
  • MDA-MB-231 was treated with different concentrations of YXY101 (0 ⁇ M, 2 ⁇ M, or 4 ⁇ M) for different times (12 h or 24 h), followed by detection of parp and p-mTOR in the cells by WB. The results are shown in Figure 5C.
  • Figure 5C shows the results of immunoblot analysis of parp and p-mTOR in MDA-MB-231 treated with different concentrations of YXY101 (0 ⁇ M, 2 ⁇ M, or 4 ⁇ M) for 12 h or 24 h.
  • the results showed that YXY101 induced apoptosis in MDA-MB-231 in a time- and concentration-dependent manner.
  • MDA-MB-231 cells were also treated with YXY101 in combination with TNF ⁇ for different times (1 h, 6 h or 12 h), followed by detection of parp in the cells by WB. The results are shown in Figure 5D.
  • Figure 5D shows the results of immunoblot analysis of parp in MDA-MB-231 cells treated with YXY101 in combination with TNF ⁇ at different times (1 h, 6 h or 12 h). The results showed that in MDA-MB-231, the combination of YXY101 and TNF ⁇ induced stronger apoptosis.
  • MDA-MB-231 cells BALB/c (nu/nu) nude mice, weighing 18-20g, female, SPF animal house, feed, drinking water, animal cages, litter are autoclaved The litter is changed every two days and strictly aseptic. Under sterile conditions, collect the cells to be inoculated in the logarithmic growth phase, wash them with serum-free medium, count the number of viable cells >95% under an inverted microscope, adjust the cell concentration to 1 ⁇ 10 6 /ml, and resuspend the tumor cells. Cell suspensions were prepared in PBS. Each nude mouse was inoculated with 0.2 ml of the above cell suspension on the right side of the arm, and the tumor growth was measured periodically.
  • the drug-administered group When the diameter of the transplanted tumor of nude mice reached about 0.5 cm, the nude mice without hemorrhage, necrosis and infection were selected for experiment, the nude mice were weighed, the tumor diameter was measured, and grouped, 6 nude mice in each group, the experimental group The rats were administered with TNF ⁇ (120 ⁇ 10 4 U/kg), YXY101 (2 mg/kg), and the combination group, and the blank control group was given the same amount of physiological saline. TNF ⁇ was administered by intratumoral injection and administered every other day; YXY101 was administered intragastrically and administered daily. The nude mice were sacrificed 6 hours after the last administration, and the tumor weight was called.
  • nude mice During the treatment, the food intake and body weight of the nude mice were not significantly reduced, the activity was normal, and no symptoms such as loose hair and diarrhea appeared. At the end of the treatment, no death occurred in each group of nude mice. After the nude mice were sacrificed, the autopsy showed clear tumor boundaries, uneven surface, tough texture, localized blood vessels, and necrosis in the central area of some tumors. No metastatic lesions were observed in all groups of nude mice. No obvious changes in appearance were found in the heart, liver, spleen, lung, kidney and other organs of nude mice.
  • the tumor volume was calculated based on the measurement results, and the tumor growth curve was plotted with the time as the abscissa and the tumor volume as the ordinate.
  • the anti-tumor activity evaluation index was the relative tumor proliferation rate T/C (%): wherein T, the RTV of the treatment group; C, the RTV of the negative control group. Efficacy evaluation criteria: T/C%>40% was ineffective; T/C% ⁇ 40% and p ⁇ 0.05 was effective.
  • the experimental results are shown in Fig. 6.
  • the left panel of Figure 6A shows the tumors formed in each group of nude mice; the middle panel shows the RTV of the tumors formed in each group of nude mice; the right panel shows the weight of the tumors formed in each group of nude mice.
  • the results in Fig. 6A show that tumors were produced in nude mice after inoculation with MDA-MB-231 cells; and, YXY101 has a good tumor suppressing effect; and the combination of YXY101 and TNF ⁇ induces a stronger tumor suppressing effect. .
  • Figure 6B shows the results of immunohistochemical staining of tumor tissues of nude mice of each group.
  • the results of Fig. 6B show that YXY101 can induce caspase3 cleavage in tumor tissues and promote apoptosis of tumor cells; and the combined administration of YXY101 and TNF ⁇ can induce stronger apoptosis of tumor cells.
  • MMTV-PYVT breast cancer transgenic mice 9-week-old, female, SPF animal housing, feed, drinking water, animal cages, litter are autoclaved, changing litter every two days, strictly Bacterial operation.
  • mice Female MMTV-PYVT breast cancer transgenic mice were housed under the conditions of temperature 23 ⁇ 1°C, humidity: 40-60%, natural light, free drinking water, and free access to common feed. Thirty-six mice with a long tumor in the chest were randomly divided into three groups, and each group was subdivided into a control group and a YXY101 treatment group.
  • Control group 9-week-old mice were given normal saline before the feed was given at 7:00 pm every day;
  • YXY101 group 9-week-old mice were intragastrically administered once daily at 7:00 pm, at a dose of 2 mg/kg.
  • Control group 11-week-old mice were given normal saline before the feed was given at 7:00 pm every day;
  • Group YXY101 11-week-old mice were intragastrically administered once daily at 7:00 pm, at a dose of 2 mg/kg.
  • Control group 15 weeks old mice were given normal saline before the feed at 7:00 pm every day;
  • YXY101 group 15 weeks old mice were given a single dose of 2 mg/kg before the feed was given at 7:00 every night.
  • Fig. 7A shows the overall condition of MMTV-PYVT mice at 11 weeks, 13 weeks or 17 weeks after intragastric administration of 2 mg/kg of YXY101 two weeks.
  • the results in Figure 7A show that YXY101 inhibits the progression of breast cancer tumors.
  • Fig. 7B shows the results of HE staining and immunohistochemical staining of tumor tissues of MMTV-PYVT mice from 11 weeks, 13 weeks or 17 weeks after intragastric administration of 2 mg/kg of YXY101 two weeks.
  • the results in Figure 7B show that YXY101 can inhibit the proliferation of breast cancer tumors.
  • Figure 7C shows the weight, morphology, HE staining and immunohistochemical staining of lung tissue from MMTV-PYVT mice from 11 weeks, 13 weeks or 17 weeks after intragastric administration of 2 mg/kg of YXY101 for two weeks. Analysis results. The results of Figure 7C show that YXY101 can inhibit lung metastasis caused by breast cancer.
  • Figure 8A shows the survival curve of wild-type mmtv-PyMT mice and Nur77 knock-out mmtv-PyMT mice at 60 days in the animal level
  • Figure 8D shows the detection of YXY101 versus mmtv- at the animal level.
  • the effect of PyMT mouse survival The results showed that the survival rate of mice after knockout of Nur77 was significantly improved, and the results predicted that Nur77 was significantly associated with the development of breast cancer.
  • Figure 8B shows the statistical results of the tumor tissue weight of the above two mice. The results show that the weight of breast cancer tissue can be significantly decreased after knocking out Nur77, and Figure 8C also shows the comparison of the appearance and morphology of these tumor tissues. Clearly comparing the knockout Nur77 mice with smaller and smoother tumor tissue. The above results indicate that Nur77 plays a crucial role in the development of breast cancer.
  • Nur77 we have creatively targeted Nur77 and used SPR to screen for YXY101, a drug molecule that specifically binds to Nur77, which has significant tumor suppressor activity, especially for triple-negative breast cancer.
  • the therapeutic activity for breast cancer is dependent on Nur77.
  • This compound is a very promising active molecule for the treatment of triple-negative breast cancer, and this screening method is an effective way to find a drug molecule that specifically binds to Nur77 and targets a compound of triple-negative breast cancer.
  • Example 7 Inflammation can induce tumor production to some extent, and this process is related to Nur77.
  • 9A-9B show that after treatment of MB-MDA-231 and SKBR3 breast cancer cells with TNF ⁇ for 1 h, 3 h, and 6 h under starvation conditions, p-P70S6K was detected by immunoblotting, and the results showed that induction of TNF ⁇ was observed.
  • the lower p-P70S6K is up-regulated and time-dependent.
  • the results indicate that the mTOR signaling pathway is activated in an inflammatory environment, which may be closely related to tumorigenesis.
  • Figure 9C shows that wild-type or Nur77 knockout MEF cells were treated with TNF ⁇ concentration gradient or time gradient under starvation conditions, and p-mTOR, p-P70S6K, and p-S6 were detected by immunoblotting, and the results showed that TNF ⁇ pairs Activation of the mTOR signaling pathway is regulated by time and concentration, and such activation is dependent on Nur77.
  • Nur77 is not only important for the development of breast cancer, but also can be used as a target for the treatment of breast cancer. Therefore, Nur77 is a valuable test for the development of breast cancer. Biochemical indicators used to assess breast cancer progression and evaluate breast cancer treatment strategies.
  • Example 8 YXY101 is able to significantly inhibit mTOR activity, thereby inhibiting tumors, a process that relies on Nur77
  • This part of the experiment simulates the tumor environment, that is, TNF ⁇ , a cytokine that causes acute inflammation, to simulate tumorigenesis.
  • This method can provide an experimental basis for screening drugs for tumor suppression.
  • Nur77-dependent compounds that inhibit the mTOR signaling pathway are screened to find a targeted drug effective for the treatment of triple-negative breast cancer.
  • Figure 10A shows the results of treatment of MB-MDA-231 breast cancer cells with TNF ⁇ (20 ng/ml), YXY101 (1 ⁇ M, 2 ⁇ M, 4 ⁇ M) under starvation conditions, and detection of p-mTOR and p-P70S6K by immunoblotting. .
  • the results showed that YXY101 significantly down-regulated p-mTOR and p-P70S6K activated by TNF ⁇ , suggesting that inhibition of mTOR activity may be its intrinsic mechanism of tumor suppression.
  • Figure 10B shows that the Flag empty plasmid or the Flag-Nur77 plasmid was transfected in MB-MDA-231 breast cancer cells, and treated with TNF ⁇ , YXY101 for 3 h, 6 h under starvation conditions, and p-mTOR, p was detected by immunoblotting.
  • -P70S6K, Nur77 results.
  • Figure 10C shows the results of silencing Nur77 treatment of MB-MDA-231 cells and treatment with TNF ⁇ , YXY101 under starvation conditions, and detection of p-P70S6K and Nur77 by immunoblotting.
  • Figure 10D shows the use TNF ⁇ , YXY101 (0.5Mm, 1 ⁇ M) were used to treat wild-type or Nur77 knockout MEF cells, and the results of p-S6, p-mTOR, and p62 were detected by immunoblotting. Combining the results of Figures 10B-10D, the two ways of overexpressing and knocking out Nur77 simultaneously indicate that the activity of YXY101 to down-regulate mTOR activity is dependent on Nur77.
  • the inhibition of mTOR signaling pathway can be used as an effective indicator for screening anticancer drugs.
  • compounds with strong mTOR signaling pathway inhibited by Nur77 have potential anticancer activity.
  • YXY101 derivative XS0503 (0.16 ⁇ M, 0.31 ⁇ M, 0.625 ⁇ M, 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 10.0 ⁇ M) for 24 h, and The proliferation ratio of breast cancer cells was determined. A curve of the cancer cell proliferation ratio against the concentration of XS0503 was plotted, and the IC50 of YXY101 was determined. The results are shown in Figure 11A.
  • Figure 11A shows the proliferation ratio of different breast cancer cells (MDA-MB-231 and MCF-7) versus XS0503 concentration, and the IC50 value of XS0503; wherein MDA-MB-231 is represented by a red curve; MCF-7 For three positive breast cancer cells, it is represented by a blue curve.
  • MDA-MB-231 is represented by a red curve
  • MCF-7 For three positive breast cancer cells, it is represented by a blue curve.
  • the results showed that XS0503 inhibited the proliferation of triple-negative breast cancer cells significantly more than its ability to inhibit the proliferation of triple-positive breast cancer cells.
  • the IC50 of XS0503 inhibiting MDA-MB-231 proliferation was 1.19 ⁇ M, which was lower than its inhibitory MCF-7.
  • Proliferation IC50 (2.65 ⁇ M) This suggests that YXY101 can be used particularly advantageously for the treatment of triple negative breast cancer.
  • MDA-MB-231 was treated with different YXY101 derivatives XS0077, XS0335, XS0419, XS0474, XS0488 (0.16 ⁇ M, 0.31 ⁇ M, 0.625 ⁇ M, 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 10.0 ⁇ M), followed by measurement of MDA-MB. -231 proliferation ratio. The results are shown in Figure 11B.
  • Figure 11C shows BT474, ZR-75-1, BT549, HCC1937, HS578T, MCF-7, T47D treated with YXY101 derivatives XS0284, XS0285, XS0394, XS0418, XS0419, XS0474, XS0486, XS0462, XS0491, XS0492, XS0488.
  • the inhibition rate analysis results of these seven breast cancer cells.
  • YXY101 (4 ⁇ M) and different YXY101 derivatives (4 ⁇ M) XS0394, XS0395, XS0419, XS0420, XS0421, XS0284, XS0335, XS0488, XS0491, XS0492, XS0418, XS0502, XS0503, XS0504, XS0506, XS0507, XS0508
  • the red font-labeled derivative induced PARP cleavage in the triple-negative breast cancer MDA-MB-231 cells more significantly than YXY101, including XS0418, XS0419, XS0492, XS0508, and XS0077.
  • Figure 12B shows YXY101 (2 ⁇ M) and different YXY101 derivatives (2 ⁇ M) XS0284, XS0285, XS0335, XS0394, XS0418, XS0419, XS0454, XS0455, XS0462, XS0473, in the case of starvation treatment with serum-free medium for 10 h.
  • Figure 12C shows P-mTOR, P-p70S6K, P-S6 and I ⁇ B ⁇ in MDA-MB-231 cells treated with YXY101 (2 ⁇ M) and YXY101 derivatives XS0455 (1, 2, 4 ⁇ M) and TNF ⁇ (20 ng/ml).
  • YXY101 (2 ⁇ M
  • YXY101 derivatives XS0455 (1, 2, 4 ⁇ M) and TNF ⁇ (20 ng/ml).
  • the results of immunoblot analysis The results showed that XS0455 was able to adjust P-mTOR, P-p70S6K, and P-S6 in a concentration-dependent manner, without significantly causing PARP cleavage and I ⁇ B ⁇ degradation.
  • XS0455 can selectively down-regulate P-mTOR, P-p70S6K, and P-S6, but it does not significantly cause PARP cleavage and I ⁇ B ⁇ degradation. This compound is expected to become a new specific anti-cancer drug molecule.
  • Figure 13 shows the results of experiments comparing the binding of different compounds (XS0418, XS0419, XS0474, XS0394, XS0492, XS0491, XS0488) to Nur77-LBD using a Biacore T200 instrument.
  • Example 11 XS0284 is more effective in treating high fat-induced breast cancer than YXY101
  • YXY101 series derivatives (such as XS0284) can also inhibit tumor development, and even tumor suppression is better than YXY101.
  • FIGS 15A-15B show two PYVT mice, wild type and Nur77 knockout type, administered YXY101 (5 mg/kg) and XS0284 (10 mg/kg) for two weeks by gavage, respectively.
  • YXY101 and XS0284 were effective in inhibiting tumors in wild-type mice, but neither of the two compounds could exert an inhibitory effect in Nur77-deficient mice.
  • Example 13 The acute toxic side effect of XS0284 is lower than YXY101
  • a mouse acute toxicity model induced by intragastric injection of 200 mg/kg of YXY101 and its derivative XS0284 or intraperitoneal injection of 20 mg/kg of YXY101 and its derivative XS0284 was used.
  • the food intake, drinking water, spontaneous activity, mental state, limb activity, bowel mass, hair gloss, etc. of each group were observed, and detailed records may appear.
  • Figure 16A shows the gavage group: 1) The cardiomyocytes of the blank group have clear boundaries, many nuclei and obvious nuclei, and the myocardial fibers are arranged neatly and clearly; the number of hepatic sinus in the liver tissue is clear and clear, and the hepatic lobule boundary is obvious; The intestinal tissue is clearly defined, the cells are full, the intestinal villi are arranged neatly and the boundaries are obvious; the adipose tissue cells are clearly defined and arranged neatly.
  • the myocardial cells and myocardial fibers of the YXY101 group were disorderly arranged; the hepatic lobule boundary was blurred, the cytoplasm was reduced, and most of the hepatocytes died; the intestinal tissue was swollen, the cell death increased, the inflammatory reaction was obvious, and the intestinal villi were found.
  • the arrangement is disordered; the adipose tissue cells are disorderly arranged and individual fibers are broken.
  • the XS0284 group had relatively clear cardiac cardiomyocyte boundaries, and the myocardial fibers were relatively neatly arranged with obvious boundaries.
  • the hepatocytes in the liver tissue had obvious boundaries and clear structure; the intestinal tissue was clear, the cells were full, and the intestinal villi were arranged. It is neat and has obvious boundaries; the adipose tissue cells have clear boundaries, neatly arranged, and reduced fiber breakage. It can be concluded that the derivative XS0284 group showed a weaker toxic effect on the liver and small intestine than YXY101.
  • Figure 16B shows the intraperitoneal injection group.
  • the cardiomyocytes of the blank group have clear boundaries, many nuclei and obvious nuclei, and the myocardial fibers are arranged neatly and clearly; the number of hepatic sinusoids in the liver tissue is clear and clear, and the hepatic lobule boundary is obvious; The cell boundaries are clear and well-arranged; the glomeruli of the kidney tissue are clearly defined, the cells are full, the cells are neatly arranged, and the boundaries are obvious.
  • the myocardial cells and myocardial fibers of the YXY101 group were disorderly arranged; the hepatic lobule boundary was basically obvious, and the hepatic cells were normal; but the adipose tissue cells were disorderly arranged and a large number of inflammatory cells infiltrated; The boundary of the ball is blurred, and there are a large number of inflammatory cells infiltrating, and the arrangement is not neat.
  • the cardiomyocytes of XS0284 group have relatively clear boundary, and the myocardial fibers are relatively neatly arranged and the boundaries are obvious.
  • the liver cells have obvious boundary and clear structure; the adipose tissue cells have clear boundaries, neatly arranged, and fiber breakage. Reduced; renal tissue glomerular boundaries are clear, cells are full, with a small amount of inflammatory cell infiltration. It can be concluded that the XS0284 group of the intraperitoneal injection group showed a weaker toxicity to kidney and fat than YXY101.
  • Nur77 is the target. Virtual screening of marine natural products resulted in some well-bound compounds, as shown below.
  • the steps of computer-assisted virtual screening in this example are as follows: download protein eutectic structure PDB ID: 3V3Q, protein pretreatment, hydrogenation, removal of crystal water molecules and small molecules of glycerol in crystal structure; ligands selected, ligands Center, 12angstrom size to build the grid grid file; run Glide docking, select the pre-built grid grid file and the processed Seaweed Metabolism Database for docking; according to the docking score and the interaction between the compound and the protein receptor, The docking results were comprehensively evaluated.
  • An example of the mode of action for the simulated combination is shown in Figure 17.
  • the present invention also synthesizes the following compounds:
  • the compound YXY101 (50 mg, 0.11 mmol) was weighed into a 25 ml reaction flask, and 4 ml of acetone was added thereto to stir and dissolve, and then a drop of concentrated hydrochloric acid was added as a catalyst, and the reaction was carried out at room temperature for 12 hours. The reaction was quenched, and the solvent was evaporated, evaporated, evaporated, evaporated
  • the compound YXY101 (50 mg, 0.11 mmol) was stirred and dissolved in 2 mL of deuterated methanol solvent, followed by addition.
  • Sodium borohydride (44 mg, 1.1 mmol) was added, and the mixture was stirred at room temperature for 30 min. 1 mol/L HCl (1 mL) was quenched, 9 mL of pure water was added, and extracted with dichloromethane (3 mL each time). The organic layer was collected and dried over anhydrous sodium sulfate.
  • the compound XS0419 (50.1 mg) was obtained as a white solid.
  • tripterine 135.2 mg, 0.3 mmol was stirred and dissolved in 2 mL of DMF, followed by sodium hydrogencarbonate (138.6 mg, 1.65 mmol), and ethyl bromide (234 ⁇ L, 0.15 mmol) was stirred at room temperature for 12 hours.
  • the reaction was quenched with 1 mol/L HCl (1 mL), 9 mL of pure water was added, and extracted with ethyl acetate three times (15 mL each time), the organic layer was collected, dried over anhydrous sodium sulfate, and the organic solvent ethyl acetate was removed by distillation under reduced pressure.
  • the ester was obtained as an orange-red solid mixture.
  • scutellarin 250 mg, 0.54 mmol was dissolved in 20 mL of tetrahydrofuran, followed by LiAlH 4 (1.2 mL, 1.1 mmol), stirred at room temperature for 2 h, and quenched with 10 mL of deionized water, 1 mol/L HCl (5 mL) The mixture was acidified and extracted with ethyl acetate (3 mL). The organic layer was collected and dried over anhydrous sodium sulfate, and the organic solvent ethyl acetate was removed by distillation under reduced pressure, and the crude product was mixed with orange-yellow solid.
  • the present invention also synthesizes the following compounds:
  • the preparation method is exemplified by XS0439: Compound YXY101 (100 mg, 0.22 mmol) was dissolved in dichloromethane (4 mL) under stirring. 7-Methoxy substituted hydrazine (65.3 mg, 0.44 mmol) was added, and then aluminum trichloride hexahydrate (5.3 mg, 0.022 mmol) was added, and the reaction was stirred at room temperature for 5 hours. The reaction was stopped, and deionized water (15 mL) was added to the reaction mixture and extracted three times with ethyl acetate.
  • compound XS0486 Taking compound XS0486 as an example: firstly, compound YXY101 (50 mg, 0.11 mmol) was dissolved in dioxane (600 ⁇ L), triethylamine (150 ⁇ L, 0.33 mmol) was added, and then the reaction bottle was added with dioxane (100 ⁇ L).
  • the compound YXY101 (50 mg, 0.11 mmol) was weighed into a 50 ml round bottom flask, dissolved in 2 ml of DCM, transferred to -78 ° C, stirred, and then subjected to DAST (150 ul, 10 eq), and reacted at -78 ° C for 1 h.
  • the reaction mixture was poured directly into a large amount of ice to stop the reaction.
  • the aqueous phase was extracted three times with DCM.
  • the organic phase was combined and dried over anhydrous Na 2 SO 4 EtOAc.
  • the system was separated and purified by silica gel column chromatography to give an orange-red solid.
  • the compound YXY101 (50 mg, 0.11 mmol) was weighed into a 25 ml thick-walled pressure tube, and tetrabutylammonium bromide (TBAB, 17.5 mg, 0.05 mmol) was added thereto, and dissolved in 2 ml of dichloromethane (DCM). Then, 5% NaOH (180 ⁇ l) was added dropwise, and the reaction was carried out for 30 min at room temperature, and then transferred to an oil bath at 50 ° C, and 2,3,4,6-tetraacetoxy- ⁇ -D-glucopyran bromide was added dropwise.
  • TBAB tetrabutylammonium bromide
  • the compound-dichloromethane solution (57 mg-1 ml, 0.138 mmol) was reacted at 50 ° C for 12 h, the reaction was stopped, a large amount of water and saturated brine were added, and the mixture was extracted with DCM three times, and the organic phases were combined and dried over anhydrous Na 2 SO 4 The organic phase was concentrated under reduced pressure and purified with silicagel eluting
  • reaction solution was poured into a large amount of ice directly stop the reaction, the aqueous phase extracted with DCM three times, the combined organic phases were washed with saturated NaHCO 3 The organic phase was washed, dried over anhydrous Na 2 SO 4 the organic phase, the organic phase was concentrated under reduced pressure, neutralized with acetic acid
  • XS0077 50 mg, 0.11 mmol was weighed into a 25 ml thick-walled pressure tube, and tetrabutylammonium bromide (TBAB, 17.5 mg, 0.05 mmol) was added thereto, and dissolved in 2 ml of dichloromethane (DCM). Then add 5% NaOH (180 ⁇ L) dropwise, react at room temperature for 30 min, transfer to 50 ° C oil bath, add 2,3,4,6-tetraacetoxy- ⁇ -D-glucopyran bromide dropwise.
  • TBAB tetrabutylammonium bromide

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Abstract

提供如式I所示的化合物用作孤儿核受体Nur77的配体的用途、用于预防或治疗与孤儿核受体Nur77相关的疾病的用途。

Description

孤儿核受体Nur77的配体及其用途 技术领域
本申请涉及医药领域和生物领域。具体而言，本申请涉及孤儿核受体Nur77的新型配体及其用途。本申请还涉及如式I-式V所示的化合物用作孤儿核受体Nur77的配体的用途。本申请还涉及如式I-式V所示的化合物用于预防或治疗与孤儿核受体Nur77相关的疾病的用途。本申请还涉及具有抗癌(特别是三阴性乳腺癌)活性的药物的筛选方法。本申请还涉及以核受体Nur77为检验指标，评估三阴性乳腺癌等相关疾病诊疗效果的方法。
背景技术
孤儿核受体Nur77，其也被称为NGFIB(神经生长因子IB)或孤儿核受体TR3，是癌症、新陈代谢和炎症性疾病发生发展过程中一个的关键调节子。作为一类即刻早期应答基因，Nur77在大量细胞过程中起着重要作用，包括由细胞分裂素、激素、压力、代谢和凋亡信号等不同刺激引起的细胞生存、凋亡、炎症和自噬过程(Pei L等人(2006)，″Regulation of macrophage inflammatory gene expression by the orphan nuclear receptor Nur77″，Mol.Endocrinol.20(4)：786-94；Zhang XK(2007)，″Targeting Nur77 translocation″，Expert Opin.Ther.Targets 11(1)：69-79)。Nur77的死亡作用最早于1994年发现，T细胞受体信号能够诱导Nur77表达，在T细胞受体所介导的细胞凋亡过程中Nur77的表达增加(Woronicz JD，Calnan B，et al.Requirement for the Orphan Steroid-Receptor Nur77 in Apoptosis of T-Cell Hybridomas.Nature，367：277-81，1994；Liu ZG，Smith SW，et al.Apoptotic Signals Delivered through the T-Cell Receptor of a T-Cell Hybrid Require the Immediate-Early Gene Nur77.Nature，367：281-4，1994)。在研究类维甲酸化合物AHPN(也叫CD437)时，我们发现了Nur77在肿瘤细胞凋亡中的作用，研究表明，在多种凋亡信号的刺激下，Nur77从细胞核迁移到细胞质，并定位于线粒体上，Nur77的线粒体定位同细胞色素c(Cytochrome c，Cyt c)的释放及细胞凋亡相关。缺失DNA结合结构域的Nur77能够 组成型地定位于线粒体上，使大量细胞色素c被从线粒体释放出来，从而诱导细胞凋亡(Li Y，Lin BZ，et al.Molecular determinants of AHPN(CD437)-induced growth arrest and apoptosis in human lung cancer cell lines.Mol Cell Biol，1998，18(8)；4719-4731；Li H，Kolluri SK，et al.Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3，Science，2000，289(5482)；1159-1164；Lin B，Kolluri SK，et al.Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3，Cell，2004，16；527-540)Nur77转位到细胞质中，定位于线粒体是其促凋亡的重要条件，因此，许多药物通过Nur77的线粒体定位诱导肿瘤及其它重大疾病细胞的细胞凋亡，从而Nur77线粒体定位将是开发新型药物的一个有效靶点。
乳腺癌是女性健康的“头号杀手”。最新调查数据显示，平均每3分钟世界上就有一位妇女被诊断为乳腺癌，我国女性乳腺癌的发病率和死亡率也是逐年上升，目前已位居女性恶性肿瘤发病率之首。而乳腺癌作为女性恶性肿瘤死亡率排名第一位的原因在于其高转移性，其中最典型的是雌激素受体(ER)、孕激素受体(PR)和原癌基因Her-2均为阴性的三阴性乳腺癌(TNBC)，其最大的特征就是转移性强、患者预后较差，对内分泌治疗不敏感，并尚无有效治疗药物。前期研究发现，Nur77作为一个早期反应基因，能够调节如细胞的增殖、凋亡、胚胎发育、血管生成在内的很多关键的生命活动过程，其表达或功能异常也会导致包括肿瘤在内的一系列疾病。在正常组织中Nur77一般表达很低或不表达，但在癌症发生发展过程以及肿瘤药物治疗过程中可被大量诱导，它的诱导表达可介导细胞增殖和凋亡双重作用，决定细胞的生与死，这两种截然相反的现象取决于不同的刺激和Nur77在细胞内不同的定位。近期研究又表明，在TNFα等炎症因子刺激下，Nur77可以特异性结合其配体，通过胞质移位调控线粒体功能而发挥其抗炎活性。
而在前期调研中，我们发现乳腺癌特别是三阴性乳腺癌的发生发展同样始终伴随着炎症反应及mTOR信号通路的高度激活，慢性炎症同样是乳腺癌发生发展的重要基础，并且也有许多对其他肿瘤有效的非类固醇类消炎药，也被拿来用于乳腺癌的防治。因此，基于Nur77易被炎症因子调控并对乳腺癌关键的调控作用，寻找有效的、特异性结合Nur77的配体，以开发用于治疗三阴性乳腺癌的药物有着更为重要的现实意义。 因此，本申请建立新型抗癌药物筛选方法，即以Nur77为靶点，寻找有效的、特异性结合Nur77的配体，以开发用于治疗癌症(如三阴乳腺癌)的新化合物分子；并且建立以Nur77作为癌症(如三阴性乳腺癌)检验指标的方法来评估和诊断相关癌症的发生发展。
海洋生态环境由于与陆地差异较大，因此，导致海洋生物体内小分子代谢物的生物合成途径也迥然不同。因此，海洋天然产物拥有大量结构独特的化合物并具有许多各种各样的生物学活性。比如，萜类和甾体化合物等具有较好的抗肿瘤以及免疫调节活性等。本申请的发明人通过大量的实验研究发现了一类新的五环三萜类化合物YXY101及其衍生物，其能够结合Nur77，抑制mTOR信号通路从而抑制肿瘤发生发展。本申请提供了有前景的化合物，因此，特别有利地，本发明化合物能够作用于表达Nur77的细胞.调控发明人新近发现的Nur77所介导的mTOR信号通路，用于治疗三阴性乳腺癌及其他癌症(例如肝癌，宫颈癌，肺癌、三阳性乳腺癌、结直肠癌或前列腺癌)。
发明内容
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“互变异构体”是指，因分子中某一原子在两个位置迅速移动而产生的官能团异构体。此类互变异构体的典型实例为酮-烯醇互变异构体。本发明所述的化合物可以以互变异构体形式存在，并因此涵盖所有可能的互变异构体，及其任何组合或任何混合物。
如本文中所使用的，术语“立体异构体”是指，分子中原子或原子团的连接次序相同，但空间排列不同而引起的异构体。在本申请中，化合物的“立体异构”分为构象和构型异构，而构型异构还分为顺反异构和旋光异构。因此，在本申请中，“立体异构体”包括所有可能的光学异构体和非对映异构体，以及其任何组合，例如外消旋体(外消旋混合物)，单一对映异构体，非对映异构体混合物，单一非对映异构体。例如，当本发明所述的化合物含有烯烃双键时，除非特别说明，否则其包括顺式异构体和反式 异构体，以及其任何组合。
如本文中所使用的，术语“药学上可接受的盐”是指，(1)本发明化合物中存在的酸性官能团(例如-COOH、-OH、-SO3H等)与适当的无机或者有机阳离子(碱)形成的盐，例如本发明化合物与碱金属或碱土金属形成的盐、本发明化合物的铵盐，和本发明化合物与含氮有机碱形成的盐；以及(2)本发明化合物中存在的碱性官能团(例如-NH2等)与适当的无机或者有机阴离子(酸)形成的盐，例如本发明化合物与无机酸或有机羧酸形成的盐。
因此，本发明化合物的“药学上可接受的盐”包括但不限于，碱金属盐，如钠盐、钾盐、锂盐等；碱土金属盐，如钙盐、镁盐等；其他金属盐，如铝盐、铁盐、锌盐、铜盐、镍盐、钴盐等；无机碱盐，如铵盐；有机碱盐，如叔辛基胺盐、二苄基胺盐、吗啉盐、葡糖胺盐、苯基甘氨酸烷基酯盐、乙二胺盐、N-甲基葡糖胺盐、胍盐、二乙胺盐、三乙胺盐、二环己基胺盐、N，N’-二苄基乙二胺盐、氯普鲁卡因盐、普鲁卡因盐、二乙醇胺盐、N-苄基-苯乙基胺盐、哌嗪盐、四甲基胺盐、三(羟甲基)氨基甲烷盐；氢卤酸盐，如氢氟酸盐、盐酸盐、氢溴酸盐、氢碘酸盐等；无机酸盐，如硝酸盐、高氯酸盐、硫酸盐、磷酸盐等；低级烷磺酸盐，如甲磺酸盐、三氟甲磺酸盐、乙磺酸盐等；芳基磺酸盐，如苯磺酸盐、对苯磺酸盐等；有机酸盐，如醋酸盐、苹果酸盐、富马酸盐、琥珀酸盐、柠檬酸盐、酒石酸盐、草酸盐、马来酸盐等；氨基酸盐，如甘氨酸盐、三甲基甘氨酸盐、精氨酸盐、鸟氨酸盐、谷氨酸盐、天冬氨酸盐等。
如本文中所使用的，术语“药学上可接受的酯”是指，当本发明化合物存在羧基时，其与醇发生酯化反应而形成的酯；当本发明化合物存在羟基时，其与有机酸、无机酸、有机酸盐等发生酯化反应而形成的酯。酯在酸或者碱存在的条件下，可以发生水解反应生成相应的酸或醇。
如本文中所使用的，术语“C1-6烷基”表示直链或支链的含有1-6个碳原子的烷基，如甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、异戊基、2-甲基丁基、新戊基、1-乙基丙基、正己基、异己基、3-甲基戊基、2-甲基戊基、1-甲基戊基、3，3-二甲基丁基、2，2-二甲基丁基、1，1-二甲基丁基、1，2-二甲基丁基、1，3-二甲基丁基、2，3-二甲基丁基、2-乙基丁基、1，2-二甲基丙基等。C1-6烷基的优选实例包括C1-5烷基、C1-4烷基、C1-3烷基。本发明所述的“C1-4烷基”表示直链或支链的含有 1-4个碳原子的烷基，其包括但不限于上述实例中的含有1-4个碳原子的具体实例。
如本文中所使用的，术语“链烯基”是指含有至少一个碳碳双键的直链或支链烃基，其典型的实例为C2-10链烯基，例如C2-6链烯基或C2-4链烯基。具体的实例包括但不限于：乙烯基、丙烯基、2-丙烯基、丁烯基、2-丁烯基、丁二烯基、戊烯基、2-甲基-丁烯基、3-甲基-丁烯基、1，3-戊二烯基、1，4-戊二烯基、己烯基、2-乙基-丁烯基、3-甲基-戊烯基、4-甲基-戊烯基、1，3-己二烯基、1，4-己二烯基、1，5-己二烯基等。
如本文中所使用的，术语“炔基”是指至少含有一个碳碳三键的直链或支链烃基，其典型的实例为C2-10炔基，例如C2-6炔基或C2-4炔基。具体的实例包括但不限于：乙炔基、丙炔基、2-丙炔基、丁炔基、2-丁炔基、2-甲基-丙炔基、丁二炔基、戊炔基、2-甲基-丁炔基、3-甲基-丁炔基、1，3-戊二炔基、1，4-戊二炔基、己炔基、2-乙基-丁炔基、3-甲基-戊炔基、4-甲基-戊炔基、1，3-己二炔基、1，4-己二炔基、1，5-己二炔基等。
如本文中所使用的，术语“环烷基”是指单环饱和烷基，其典型的实例为3-8元环烷基，例如3元、4元、5元、6元、7元或8元环烷基。如本文中所使用的，术语“3-8元环烷基”是指含有3-8个碳原子的环烷基。具体的实例包括但不限于：环丙基、环丁基、环戊基、环己基、环庚基、环辛基等。
如本文中所使用的，术语“杂环烷基”是指含有至少1个至多4个(例如1、2、3或4个)选自N、O和S的杂原子的环烷基，其中“环烷基”的定义如前文所述，其典型的实例为3-8元杂环烷基，例如3元、4元、5元、6元、7元或8元杂环烷基。如本文中所使用的，术语“3-8元杂环烷基”是指含有3-8个碳原子的杂环烷基。如本文中所使用的“氧代3-8元环烷基”是指所述杂原子为O的如前文所定义的3-8元杂环烷基。具体的实例包括但不限于：环氧乙基、氧代环丁基、吡咯烷基、四氢呋喃基、哌啶基、哌嗪基、吗啉基、硫吗啉基等。
如本文中所使用的，术语“芳基”是指芳香族基团，其典型实例为6-14元芳基，例如6-10元芳基。如本文中所使用的，术语“6-14元芳基”是指含有6-14个碳原子的单环、双环或多环芳香族基团，包括例如6-8元芳基和8-14元稠环芳基。6-8元芳基是指含有6-8个碳原子的芳基，例如苯基等。8-14元稠环芳基是指含有8-14个环碳原子、由两个或两个以上环状结构彼此共用两个相邻的碳原子所形成的不饱和的具有芳香性的稠环基团，具体实例包括但不仅限于：萘、蒽、菲等。术语“6-10元芳基”是指含有 6-10个碳原子的芳香族基团，其包括但不限于上述实例中环原子个数为6-10个的芳香族基团。
如本文中所使用的，术语“芳基-C1-6烷基”是指以芳基-C1-6烷基-方式形成的基团，其中“芳基”和“C1-6烷基”的定义各自如前文所述。
如本文中所使用的，术语“C1-6烷氧基”是指以C1-6烷基-O-方式形成的基团，其中“C1-6烷基”的定义如前文所述。
如本文中所使用的，术语“C1-6烷氨基”是指以C1-6烷基-NH-方式形成的基团，其中“C1-6烷基”的定义如前文所述。
如本文中所使用的，术语“C1-6烷硫基”是指以C1-6烷基-S-方式形成的基团，其中“C1-6烷基”的定义如前文所述。
如本文中所使用的，术语“C1-6烷酰基”是指以C1-5烷基-C(O)-方式形成的基团，其中“C1-5烷基”的定义如前文所述。
如本文中所使用的，术语“C1-6烷氧羰基”是指以C1-6烷基-O-C(O)-方式形成的基团，其中“C1-6烷基”的定义如前文所述。
如本文中所使用的，术语“C1-6烷氧羰基-C1-6烷基”是指以C1-6烷基-O-C(O)-C1-6烷基-方式形成的基团，其中“C1-6烷基”的定义如前文所述。
如本文中所使用的，术语“3-8元环烷基-氨酰基”是指以3-8元环烷基-NHC(O)-方式形成的基团，其中“3-8元环烷基”的定义如前文所述。
如本文中所使用的，术语“卤素”包括例如氟原子、氯原子、溴原子和碘原子。
如本文中所使用的，术语“6-15元杂芳基”是指含有6-15个环原子且其中至少一个为杂原子的、具有芳香性的基团。6-15元杂芳基包括“5-8元杂芳基”，例如“5-7元杂芳基”、“5-6元杂芳基”等。“5-8元杂芳基”具体实例包括但不限于，呋喃基、噻吩基、吡咯基、噻唑基、异噻唑基、噻二唑基、噁唑基、异噁唑基、噁二唑基、咪唑基、吡唑基、1，2，3-三唑基、1，2，4-三唑基、1，2，3-噁二唑基、1，2，4-噁二唑基、1，2，5-噁二唑基、1，3，4-噁二唑基、吡啶基、2-吡啶酮、4-吡啶酮、嘧啶基、1，4-二氧杂环己二烯基、2H-1，2-噁嗪基、4H-1，2-噁嗪基、6H-1，2-噁嗪基、4H-1，3-噁嗪基、6H-1，3-噁嗪基、4H-1，4-噁嗪基、哒嗪基、吡嗪基、1，2，3-三嗪基、1，3，5-三嗪基、1，2，4，5-四嗪基、氮杂环庚三烯基、1，3-二氮杂环庚三烯基、氮杂环辛四烯基等。6-15元杂芳基还包括“9-15元稠杂芳 基”(例如9-15元苯并稠杂芳基)，其具体实例包括但不限于：苯并呋喃基、苯并异呋喃基、苯并噻吩基、吲哚基、异吲哚、苯并噁唑基、苯并咪唑基、吲唑基、苯并三唑基、喹啉基、2-喹啉酮、4-喹啉酮、1-异喹啉酮、异喹啉基、吖啶基、菲啶基、苯并哒嗪基、酞嗪基、喹唑啉基、喹喔啉基、酚嗪基、喋啶基、嘌呤基、萘啶基、吩嗪、吩噻嗪等。
如本文中所使用的，术语“细胞”特别优选地是指，表达Nur77的细胞。本发明化合物能够与Nur77特异性结合，并作为其配体发挥作用。因此，特别有利地，本发明化合物能够作用于表达Nur77的细胞.调控发明人新近发现的Nur77所介导的炎症及mTOR信号通路。在某些优选的实施方案中，所述细胞为三阴性乳腺癌及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳性乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
如本文中所使用的，术语“孤儿核受体Nur77”或“Nur77”是指，神经生长因子IB(NGFIB)，其由NR4A1基因编码(Chang C等(1989)，″Isolation and characterization of human TR3 receptor：a member of steroid receptor superfamily″，J.Steroid Biochem.34(1-6)：391-5)。Nur77参与细胞周期、炎症和细胞凋亡等过程，并且其亚细胞定位与细胞的存活和死亡相关(Pei L等人(2006)，″Regulation of macrophage inflammatory gene expression by the orphan nuclear receptor Nur77″，Mol.Endocrinol.20(4)：786-94；Zhang XK(2007)，″Targeting Nur77 translocation″，Expert Opin.Ther.Targets 11(1)：69-79)。
如本文中所使用的，术语“与Nur77相关的疾病”是指，其发生和/或进展与Nur77信号通路相关的疾病。研究已显示，Nur77参与细胞周期、炎症和细胞凋亡等过程，并且其亚细胞定位与细胞的存活和死亡相关(同上)。此外，还已报道，Nur77可被多种刺激诱导，包括生理刺激，例如脂肪酸，前列腺素，生长因子，炎性细胞因子，肽激素等；以及物理刺激，例如磁场，机械搅拌(剪切力)，膜去极化等(Maxwell MA，Muscat GE(2006)，″The NR4A subgroup：immediate early response genes with pleiotropic physiological roles″，Nucl Recept Signal 4：e002)。另外，还已显示，Nur77参与一些实体肿瘤的转移(Ramaswamy S，Ross KN，Lander ES，Golub TR(2003)，″A molecular signature of metastasis in primary solid tumors″，Nat.Genet.33(1)： 49-54)。此外，还已显示，Nur77在发炎的人体滑膜组织、癌细胞、牛皮癣患者、动脉粥样硬化患者、多发性硬化患者中异常表达。因此，术语“与Nur77相关的疾病”包括但不限于，炎症(例如，与动脉粥样硬化相关的炎症，与肥胖症相关的炎症，与糖尿病相关的炎症，肝炎，肺炎，关节炎和炎症性肠病)、动脉粥样硬化、肥胖症、糖尿病、牛皮癣、多发性硬化和癌症(例如三阴乳腺癌)。
如本文中所使用的，术语“受试者”是指动物，特别是哺乳动物，优选人。
如本文中所使用的，术语“高脂肪膳食”是指，动物体(例如哺乳动物，例如人)日常摄入的膳食中的脂肪含量超出了动物体正常生理活动所需的脂肪量。
如本文中所使用的，术语“有效量”是指，足以获得或至少部分获得期望的效果的量。例如，预防有效量是指，足以预防，阻止，或延迟疾病的发生的量；治疗有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度，患者自己的免疫系统的总体状态，患者的一般情况例如年龄、体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
在本申请中，发明人通过大量的研究发现，如式I-式V所示的化合物(例如化合物YXY101)能够靶向孤儿核受体Nur77，并作为其配体发挥作用。例如，此类化合物可用于与孤儿核受体Nur77结合而抑制mTOR的活性，用于治疗与孤儿核受体Nur77相关的癌症(例如三阴乳腺癌)。
因此，在第一个方面，本申请涉及如式I所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯的用途，其用作孤儿核受体Nur77的配体，或者用于制备用作孤儿核受体Nur77的配体的药物：
Figure PCTCN2017091726-appb-000001
其中，
X代表-NH-、-N(R)-、-O-、-CH2-或卤素；其中，当X为卤素时，R1不存在；
当Y和与之相连的碳原子之间为单键时，Y代表H、卤素、-OR、-SR或-NRR’；当Y和与之相连的碳原子之间为双键时，Y代表O、S或NR；
R1不存在，或代表H、-PO(OR)2、C1-6烷基、糖基、C1-6烷氧羰基-C1-6烷基、3-8元环烷基-氨酰基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、糖基、C1-6烷氧羰基-C1-6烷基、3-8元环烷基-氨酰基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基、C1-6烷氨基和C1-6烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
R2代表H、D、-PO(OR)2、-CONH2、-NH2、-NHR、-NRR’、-NHCOR、-NRCOR、-NHCOOR、-NHCONHR、-NHCONRR’、-NRCONHR、-NRCONRR’、-OH、-OR、-OCONHR、-OCONRR’、-SH、-SR、-SOR、-SOOR、-SO2NHR”、硝基、卤素、糖基、氰基、三氟甲基、C1-6烷基、3-8元环烷基、3-8元杂环烷基、芳基、C1-6烷基取代的芳基、6-15元杂芳基、链烯基、炔基、亚磺基、磺酸或磺酸盐；其中所述C1-6烷基、3-8元环烷基、3-8元杂环烷基、芳基、C1-6烷基取代的芳基、6-15元杂芳基、链烯基和炔基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：氨基、卤素、羟基、氧基、C1-6烷基、C1-6烷氧基、C1-6烷硫基、C1-6烷酰基、3-8元环烷基(例如环丙基)、氧代3-8元环烷基(例如氧代环丁基)、氰基、三氟甲基、C1-6烷氧羰基、C1-6烷基酰胺基、脲基、氨基甲酸酯基、羧基以及芳基；
R3和R4各自独立地表示不存在，或代表H、C1-6烷基、C1-6烷酰基、C1-6烷氧羰基、糖基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、C1-6烷酰基、C1-6烷氧羰基、糖基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基、C1-6烷氨基和C1-6烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
R和R’各自独立地选自H、C1-6烷基、3-8元环烷基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、3-8元环烷基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、 2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基和C1-6烷氨基；
R”代表C1-6烷基或芳基(例如6-10元芳基，优选苯基)；
式(I)中的虚实双键
Figure PCTCN2017091726-appb-000002
代表单键或者双键；优选地，环A包含0、1、2或3个碳碳双键；环B包含0、1或2个碳碳双键。
在某些优选实施方案中，X代表-NH-、-N(R)-、-O-、-CH2-或卤素；其中，当X为卤素时，R1不存在；
当Y和与之相连的碳原子之间为单键时，Y代表H、卤素、-OR、-SR或-NRR’；当Y和与之相连的碳原子之间为双键时，Y代表O、S或NR；
R1不存在，或代表H、-PO(OR)2、C1-6烷基、糖基、C1-6烷氧羰基-C1-6烷基、3-8元环烷基-氨酰基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、糖基、C1-6烷氧羰基-C1-6烷基、3-8元环烷基-氨酰基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基、C1-6烷氨基和C1-6烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
R2代表H、D、-PO(OR)2、-CONH2、-NH2、-NHR、-NRR’、-NHCOR、-NRCOR、-NHCOOR、-NHCONHR、-NHCONRR’、-NRCONHR、-NRCONRR’、-OH、-OR、-OCONHR、-OCONRR’、-SH、-SR、-SOR、-SOOR、-SO2NHR”、硝基、卤素、糖基、氰基、三氟甲基、C1-6烷基、3-8元环烷基、3-8元杂环烷基、芳基、C1-6烷基取代的芳基、6-15元杂芳基、链烯基、炔基、亚磺基、磺酸或磺酸盐；其中所述C1-6烷基、3-8元环烷基、3-8元杂环烷基、芳基、C1-6烷基取代的芳基、6-15元杂芳基、链烯基和炔基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：氨基、卤素、羟基、氧基、C1-6烷基、C1-6烷氧基、C1-6烷硫基、C1-6烷酰基、3-8元环烷基(例如环丙基)、氧代3-8元环烷基(例如氧代环丁基)、氰基、三氟甲基、C1-6烷氧羰基、C1-6烷基酰胺基、脲基、氨基甲酸酯基、羧基以及芳基；
R3和R4各自独立地表示不存在，或代表H、C1-6烷基、C1-6烷酰基、C1-6烷氧羰基、糖基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、C1-6烷酰基、C1-6烷氧羰基、 糖基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基、C1-6烷氨基和C1-6烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
R和R’各自独立地选自H、C1-6烷基、3-8元环烷基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、3-8元环烷基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基和C1-6烷氨基；
R”代表C1-6烷基或芳基(例如6-10元芳基，优选苯基)；
式(I)中的虚实双键
Figure PCTCN2017091726-appb-000003
代表单键或者双键；优选地，环A包含0、1、2或3个碳碳双键；环B包含0、1或2个碳碳双键。
在本发明的某些优选实施方案中，所述磺酸盐选自磺酸钠、磺酸钾、磺酸钙和磺酸镁。
在本发明的某些优选实施方案中，所述6-15元杂芳基选自9-15元稠杂芳基；更优选地，所述6-15元杂芳基选自9-15元苯并稠杂芳基，例如吲哚基、苯并呋喃基、苯并噻吩基、苯并咪唑基或喹啉基。
在某些优选的实施方案中，Y和与之相连的碳原子之间为双键，且Y代表O。
在某些优选的实施方案中，X代表NH；Y和与之相连的碳原子之间为双键，且Y代表O；R1代表被一个或多个(例如1、2、3或4个)选自下述的取代基取代的芳基：C1-6烷基和C1-6烷氧基；优选地，所述芳基为苯基或萘基；R2代表H。
在某些优选的实施方案中，X代表O；Y和与之相连的碳原子之间为双键，且Y代表O；R1代表H；R2代表H、磺酸盐或6-15元杂芳基；优选地，所述磺酸盐选自磺酸钠、磺酸钾、磺酸钙和磺酸镁；优选地，所述6-15元杂芳基选自9-15元稠杂芳基；更优选地，所述6-15元杂芳基选自9-15元苯并稠杂芳基，例如吲哚基、苯并呋喃基、苯并噻吩基、苯并咪唑或喹啉基。
在某些优选的实施方案中，X代表O；Y和与之相连的碳原子之间为双键，Y代表O；R1代表C1-6烷基或芳基-C1-6烷基(优选苄基)；R2代表H。
在某些优选的实施方案中，R3不存在，且R4代表H。在某些优选的实施方案中，R3代表H，且R4不存在。在某些优选的实施方案中，R3和R4均为H。
在某些优选的实施方案中，R3不存在，R4代表H，且环A和环B中各有两个碳碳双键。
在某些优选的实施方案中，R3代表H，R4不存在，且环A中有0、1或2个碳碳双键；进一步优选地，环B中有0、1或2个碳碳双键。
在某些优选的实施方案中，R3和R4均为H，且环A中有3个碳碳双键(即，环A为苯环)；进一步优选地，环B中有0或1个碳碳双键；更优选地，环B中7位和8位碳原子之间为碳碳双键。
在某些优选的实施方案中，R3和R4均不存在。在此类实施方案中，环A中2位碳原子与其所连接的O原子之间为碳氧双键，并且3位碳原子与其所连接的O原子之间为碳氧双键。
在某些优选的实施方案中，7位和8位碳原子之间为碳碳双键。在某些优选的实施方案中，7位和8位碳原子之间为碳碳单键。
在某些优选实施方案中，Y和与之相连的碳原子之间为双键，且Y代表O。
在某些优选实施方案中，所述化合物中的X代表-NH-、-N(R)-、-O-、-CH2-或卤素；R代表C1-6烷基或3-8元环烷基(优选环己基)；其中，当X为卤素时，R1不存在；
在某些优选实施方案中，X代表-NH-、-N(R)-、-O-或卤素(例如氟)；R代表环己基。
在某些优选实施方案中，所述化合物中的R1不存在，或代表氢、C1-4烷基、-PO(OR)2、单糖基、C1-4烷氧羰基-C1-4烷基、3-6元环烷基-氨酰基、芳基-C1-4烷基或芳基；其中，所述C1-4烷基、单糖基、C1-4烷氧羰基-C1-4烷基、3-6元环烷基-氨酰基、芳基-C1-4烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-4烷基、C1-4烷氧基、C1-4烷氨基和C1-4烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
R代表C1-4烷基。
在某些优选实施方案中，R1不存在，或代表氢、C1-4烷基、-PO(OR)2、葡萄糖基、C1-2烷氧羰基-C1-2烷基、环己基-氨酰基、苯基-C1-2烷基、萘基-C1-2烷基、苯基或萘基；其中，所述甲基、乙基、葡萄糖基、C1-2烷氧羰基-C1-2烷基、环己基-氨酰基、苯基-C1-2 烷基、萘基-C1-2烷基、苯基或萘基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：C1-2烷基、C1-2烷氧基和C1-2烷酰基；
R代表C1-4烷基。
在某些优选实施方案中，R1不存在，或代表氢、C1-4烷基、-PO(OR)2、苯基-C1-2烷基、C1-2烷氧羰基-C1-2烷基、3-6元环烷基-氨酰基；
R代表C1-3烷基。
在某些优选实施方案中，R1不存在，或代表氢、甲基、乙基、-PO(OMe)2、-PO(OEt)2、-PO(OiPr)2、2，3，4，6-四乙酰氧基-α-D-吡喃葡萄糖基、EtOCOCH2-、环己基-氨酰基、苄基、甲氧基苯基或叔丁基苯基。
在某些优选实施方案中，所述化合物中的R2代表H、D、-OH、-PO(OR)2、C1-6烷基、9-15元稠杂芳基或磺酸盐；其中所述C1-6烷基或6-15元杂芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：氨基、卤素、羟基、氧基、C1-6烷基、C1-6烷氧基、C1-6烷酰基、氰基、三氟甲基和羧基；
R代表H、C1-6烷基或芳基。
在某些优选实施方案中，R2代表H、D、-PO(OR)2、C1-4烷基、9-15元苯并稠杂芳基或磺酸盐；其中，所述C1-4烷基或9-15元苯并稠杂芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：氨基、卤素、羟基、氧基、C1-4烷基、C1-4烷氧基、C1-4烷酰基、氰基、三氟甲基和羧基；
R代表H、C1-4烷基或苯基。
在某些优选实施方案中，R2代表H、D、-PO(OR)2、C1-4烷基、9-15元苯并稠杂芳基或磺酸盐；其中，所述C1-4烷基和9-15元苯并稠杂芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：氨基、卤素、羟基、氧基、C1-4烷基、C1-4烷氧基、C1-4烷酰基、氰基、三氟甲基和羧基；
R代表H、C1-4烷基或苯基。
在某些优选实施方案中，R2代表H、D、-PO(OR)2、2-氧代苯基、吲哚基或磺酸钠；其中，所述吲哚基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：氨基、氟、氯、溴、羟基、甲基、甲氧基、甲酰基、氰基、三氟甲基和羧基；
R代表H、甲基、乙基、异丙基或苯基。
在某些优选实施方案中，所述化合物的7位和8位碳原子之间为碳碳双键。
在某些优选实施方案中，所述化合物Y和与之相连的碳原子之间为碳碳双键。
在某些优选实施方案中，所述化合物Y和与之相连的碳原子之间为碳碳单键。
在某些优选实施方案中，所述化合物具有如下结构：
Figure PCTCN2017091726-appb-000004
其中，R3和R4各自独立地代表H、C1-6烷基或C1-6烷酰基；
优选地，R3和R4各自独立地代表H、C1-4烷基或C1-4烷酰基；
优选地，R3和R4各自独立地代表H、甲基或丁酰基。
在某些优选实施方案中，所述化合物具有如下结构：
Figure PCTCN2017091726-appb-000005
其中，R4代表H、C1-6烷酰基、C1-6烷氧羰基或被一个或多个(例如1、2、3或4个)C1-6烷酰基取代的单糖基；
在某些优选实施方案中，R4代表H、C1-4烷酰基、C1-4烷氧羰基或被一个或多个(例如1、2、3或4个)C1-4烷酰基取代的葡萄糖糖基。
在某些优选实施方案中，R4代表H或C1-2烷氧羰基。
在某些优选实施方案中，R4代表H、丁酰基、乙氧羰基或2，3，4，6-四乙酰氧基-α-D-吡喃葡萄糖糖基。
在某些优选实施方案中，所述化合物选自下列化合物：
Figure PCTCN2017091726-appb-000006
Figure PCTCN2017091726-appb-000007
Figure PCTCN2017091726-appb-000008
Figure PCTCN2017091726-appb-000009
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制Nur77的转录活性。在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于抑制Nur77在细胞中的转录活性。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制TNFα在细胞中的生物学效应。在某些优选实施方案中，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制细胞中mTOR通路(例如下调P-mTOR、p-P70S6K和/或p-S6活性)。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于在有此需要的受试者中预防或治疗与Nur77相关的疾病。
在某些优选实施方案中，所述与Nur77相关的疾病为癌症(例如所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于在有此需要的受试者中预防或治疗与Nur77相关的疾病。在某些优选实施方案中，所述与Nur77相关的疾病为癌症(例如三阴乳腺癌)。
在另一个方面，本申请涉及一种抑制孤儿核受体Nur77的转录活性的方法，其包括，将Nur77与如式I所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯相接触的步骤：
Figure PCTCN2017091726-appb-000010
其中，各原子和取代基的定义如本申请第一方面所述。
在某些优选实施方案中，所述方法用于抑制Nur77在细胞中的转录活性。在某些优选实施方案中，所述方法包括，给有此需要的细胞施用有效量的所述化合物，从而抑制Nur77在细胞中的转录活性。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)
在另一个方面，本申请涉及一种抑制TNFα在细胞中的生物学效应的方法，其包括，给有此需要的细胞施用有效量的如式I所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000011
其中各原子和取代基的定义如本申请第一方面所述。
在某些优选实施方案中，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活。在某些优选实施方案中，所述方法用于抑制TNFα诱导的IκBα降解。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在另一个方面，本申请涉及一种抑制mTOR信号通路的方法，其包括，给有此需要的细胞施用有效量的如式I所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000012
其中个原子和取代基的定义如不申请第一方面所述。
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于下调细胞中P-mTOR、p-P70S6K和/或p-S6的活性。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三 阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)
在另一个方面，本申请涉及一种预防或治疗与Nur77相关的疾病的方法，其包括，给有此需要的受试者施用有效量的如式I所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000013
其中各原子和取代基的定义如本申请第一方面所述。
在某些优选实施方案中，所述与Nur77相关的疾病为癌症(例如三阴乳腺癌)。
在另一个方面，本申请涉及如式IV所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯的用途，其用作孤儿核受体Nur77的配体，或者用于制备用作孤儿核受体Nur77的配体的药物：
Figure PCTCN2017091726-appb-000014
其中，X代表NH、O或CH2；
Y代表O、S或NR；
R5和R6各自独立地代表代表H或C1-6烷基；
当R7和与之相连的碳原子之间为单键时，R7代表OH，当R7和与之相连的碳原子 之间为双键时，R7代表O；
R8代表H或C1-6烷基；
式(IV)中的虚实双键
Figure PCTCN2017091726-appb-000015
代表单键或者双键。
在某些优选实施方案中，所述化合物选自下列化合物：
Figure PCTCN2017091726-appb-000016
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制Nur77的转录活性。在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于抑制Nur77在细胞中的转录活性。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制TNFα在细胞中的生物学效应。在某些优选实施方案中，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制细胞中mTOR通路(例如下调P-mTOR、p-P70S6K和/或p-S6活性)。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例 如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于在有此需要的受试者中预防或治疗与Nur77相关的疾病。在某些优选实施方案中，所述与Nur77相关的疾病为癌症(例如三阴乳腺癌)。
在另一个方面，本申请涉及一种抑制孤儿核受体Nur77的转录活性的方法，其包括，将Nur77与如式IV所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯相接触的步骤：
Figure PCTCN2017091726-appb-000017
其中，X代表NH、O或CH2；
Y代表O、S或NR；
R5和R6各自独立地代表代表H或C1-6烷基；
当R7和与之相连的碳原子之间为单键时，R7代表OH，当R7和与之相连的碳原子之间为双键时，R7代表O；
R8代表H或C1-6烷基；
式(IV)中的虚实双键
Figure PCTCN2017091726-appb-000018
代表单键或者双键。
在某些优选实施方案中，所述化合物选自下列化合物：
Figure PCTCN2017091726-appb-000019
Figure PCTCN2017091726-appb-000020
在某些优选实施方案中，所述方法用于抑制Nur77在细胞中的转录活性。在某些优选实施方案中，所述方法包括，给有此需要的细胞施用有效量的所述化合物，从而抑制Nur77在细胞中的转录活性。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在另一个方面，本申请涉及一种抑制TNFα在细胞中的生物学效应的方法，其包括，给有此需要的细胞施用有效量的如式IV所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000021
其中，X代表NH、O或CH2；
Y代表O、S或NR；
R5和R6各自独立地代表代表H或C1-6烷基；
当R7和与之相连的碳原子之间为单键时，R7代表OH，当R7和与之相连的碳原子之间为双键时，R7代表O；
R8代表H或C1-6烷基；
式(IV)中的虚实双键
Figure PCTCN2017091726-appb-000022
代表单键或者双键。
在某些优选实施方案中，所述化合物选自下列化合物：
Figure PCTCN2017091726-appb-000023
在某些优选实施方案中，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活。在某些优选实施方案中，所述方法用于抑制TNFα诱导的IκBα降解。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在另一个方面，本申请涉及一种抑制mTOR通路的方法，其包括，给有此需要的细胞施用有效量的如式IV所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000024
其中，X代表NH、O或CH2；
Y代表O、S或NR；
R5和R6各自独立地代表代表H或C1-6烷基；
当R7和与之相连的碳原子之间为单键时，R7代表OH，当R7和与之相连的碳原子之间为双键时，R7代表O；
R8代表H或C1-6烷基；
式(IV)中的虚实双键
Figure PCTCN2017091726-appb-000025
代表单键或者双键。
在某些优选实施方案中，所述化合物选自下列化合物：
Figure PCTCN2017091726-appb-000026
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于下调P-mTOR、p-P70S6K和/或p-S6活性。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在另一个方面，本申请涉及一种预防或治疗与Nur77相关的疾病的方法，其包括，给有此需要的受试者施用有效量的如式IV所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000027
其中，X代表NH、O或CH2；
Y代表O、S或NR；
R5和R6各自独立地代表代表H或C1-6烷基；
当R7和与之相连的碳原子之间为单键时，R7代表OH，当R7和与之相连的碳原子之间为双键时，R7代表O；
R8代表H或C1-6烷基；
式(IV)中的虚实双键
Figure PCTCN2017091726-appb-000028
代表单键或者双键。
在某些优选实施方案中，所述化合物选自下列化合物：
Figure PCTCN2017091726-appb-000029
在某些优选实施方案中，所述与Nur77相关的疾病为癌症(例如三阴乳腺癌)。
在另一个方面，本申请涉及下列化合物、其互变异构体、立体异构体或药学上可接受的盐或酯的用途，其用作孤儿核受体Nur77的配体，或者用于制备用作孤儿核受体Nur77的配体的药物：
Figure PCTCN2017091726-appb-000030
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制Nur77的转录活性。在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于抑制Nur77在细胞中的转录活性。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接 受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制TNFα在细胞中的生物学效应。在某些优选实施方案中，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制细胞中mTOR通路(例如下调P-mTOR、p-P70S6和/或p-S6活性)。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于在有此需要的受试者中预防或治疗与Nur77相关的疾病。在某些优选实施方案中，所述与Nur77相关的疾病为癌症(例如三阴乳腺癌)。
在另一个方面，本申请涉及一种抑制孤儿核受体Nur77的转录活性的方法，其包括，将Nur77与下列化合物、其互变异构体、立体异构体或药学上可接受的盐或酯相接触的步骤：
Figure PCTCN2017091726-appb-000031
在某些优选实施方案中，所述方法用于抑制Nur77在细胞中的转录活性。在某些优选实施方案中，所述方法包括，给有此需要的细胞施用有效量的所述化合物，从而抑制Nur77在细胞中的转录活性。在某些优选实施方案中，所述细胞表达Nur77。在 某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在另一个方面，本申请涉及一种抑制TNFα在细胞中的生物学效应的方法，其包括，给有此需要的细胞施用有效量的下列所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000032
在某些优选实施方案中，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活。在某些优选实施方案中，所述方法用于抑制TNFα诱导的IκBα降解。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在另一个方面，本申请涉及一种抑制mTOR通路的方法，其包括，给有此需要的细胞施用有效量的下列所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000033
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于下调P-mTOR、p-P70S6K和/或p-S6活性。在某些优选 实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在另一个方面，本申请涉及一种预防或治疗与Nur77相关的疾病的方法，其包括，给有此需要的受试者施用有效量的如下所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000034
在某些优选实施方案中，所述与Nur77相关的疾病为癌症(例如三阴乳腺癌)。
在另一个方面，本申请涉及如下所示化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000035
其中，
R9和R10各自独立地选自氢、C1-6烷基和芳基；
优选地，R9和R10各自独立地选自氢、C1-4烷基、苯基和萘基；
优选地，R9和R10各自独立地选自甲基、乙基和异丙基。
在某些优选实施方案中，所述化合物选自下列化合物：
Figure PCTCN2017091726-appb-000036
在另一个方面，本申请涉及如式V所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯的用途，其用作孤儿核受体Nur77的配体，或者用于制备用作孤儿核受体Nur77的配体的药物：
Figure PCTCN2017091726-appb-000037
其中，
R9和R10各自独立地选自氢、C1-6烷基和芳基；
优选地，R9和R10各自独立地选自氢、C1-4烷基、苯基和萘基；
优选地，R9和R10各自独立地选自甲基、乙基和异丙基。
在某些优选实施方案中，所述化合物选自下列化合物：
Figure PCTCN2017091726-appb-000038
在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制Nur77的转录活性。在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于在体内、体外或离体抑制Nur77的转录活性。在某些 优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于抑制Nur77在细胞中的转录活性。在某些优选的实施方案中，所述细胞表达Nur77。在某些优选的实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制TNFα在细胞中的生物学效应。在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于在体内、体外或离体抑制TNFα在细胞中的生物学效应。在某些优选的实施方案中，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活。在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于抑制TNFα诱导的IκBα降解。在某些优选的实施方案中，所述细胞表达Nur77。在某些优选的实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制细胞中mTOR通路(例如下调P-mTOR、p-P70S6和/或p-S6活性)。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于在有此需要的受试者中预防或治疗与Nur77相关的疾病。
在某些优选的实施方案中，所述与Nur77相关的疾病为癌症。在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于在患有癌症的受试者中抑制癌细胞的增殖和/或转移和/或促进癌细胞的凋亡。 在某些优选的实施方案中，所述癌症选自肝癌，宫颈癌，肺癌、和乳腺癌。在某些优选的实施方案中，所述癌症为三阴性乳腺癌(即，雌激素受体(ER)、孕激素受体(PR)和原癌基因Her-2均为阴性的乳腺癌)。在某些优选的实施方案中，所述药物用于治疗癌症，并且包含所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯，以及TNFα。
在另一个方面，本申请涉及一种抑制孤儿核受体Nur77的转录活性的方法，其包括，将Nur77与式V所示化合物、其互变异构体、立体异构体或药学上可接受的盐或酯相接触的步骤：
Figure PCTCN2017091726-appb-000039
其中，
R9和R10各自独立地选自氢、C1-6烷基和芳基；
优选地，R9和R10各自独立地选自氢、C1-4烷基、苯基和萘基；
优选地，R9和R10各自独立地选自甲基、乙基和异丙基。
在某些优选实施方案中，所述化合物选自下列化合物：
Figure PCTCN2017091726-appb-000040
在某些优选实施方案中，所述方法用于抑制Nur77在细胞中的转录活性。在某些优选实施方案中，所述方法包括，给有此需要的细胞施用有效量的所述化合物，从而抑制Nur77在细胞中的转录活性。在某些优选实施方案中，所述细胞表达Nur77。在 某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在另一个方面，本申请涉及一种抑制TNFα在细胞中的生物学效应的方法，其包括，给有此需要的细胞施用有效量的式V所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000041
其中，
R9和R10各自独立地选自氢、C1-6烷基和芳基。
在某些优选实施方案中，R9和R10各自独立地选自氢、C1-4烷基、苯基和萘基。
在某些优选实施方案中，R9和R10各自独立地选自甲基、乙基和异丙基。
在某些优选实施方案中，所述化合物选自下列化合物：
Figure PCTCN2017091726-appb-000042
在某些优选实施方案中，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活。在某些优选实施方案中，所述方法用于抑制TNFα诱导的IκBα降解。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在另一个方面，本申请涉及一种抑制mTOR通路的方法，其包括，给有此需要的细胞施用有效量的式V所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000043
其中，
R9和R10各自独立地选自氢、C1-6烷基和芳基。
在某些优选实施方案中，R9和R10各自独立地选自氢、C1-4烷基、苯基和萘基。
在某些优选实施方案中，R9和R10各自独立地选自甲基、乙基和异丙基。
在某些优选实施方案中，所述化合物选自下列化合物：
Figure PCTCN2017091726-appb-000044
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于下调P-mTOR、p-P70S6K和/或p-S6活性。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在另一个方面，本申请涉及一种预防或治疗与Nur77相关的疾病的方法，其包括，给有此需要的受试者施用有效量的式V所示的化合物、其互变异构体、立体异构体或 药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000045
其中，
R9和R10各自独立地选自氢、C1-6烷基和芳基。
在某些优选实施方案中，R9和R10各自独立地选自氢、C1-4烷基、苯基和萘基。
在某些优选实施方案中，R9和R10各自独立地选自甲基、乙基和异丙基。
在某些优选实施方案中，所述化合物选自下列化合物：
Figure PCTCN2017091726-appb-000046
在某些优选实施方案中，所述与Nur77相关的疾病为癌症(例如三阴乳腺癌)。
在另一个方面，本申请涉及下列化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000047
在另一个方面，本申请涉及下列化合物、其互变异构体、立体异构体或药学上可 接受的盐或酯的用途，其用作孤儿核受体Nur77的配体，或者用于制备用作孤儿核受体Nur77的配体的药物：
Figure PCTCN2017091726-appb-000048
在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制Nur77的转录活性。在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于在体内、体外或离体抑制Nur77的转录活性。在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于抑制Nur77在细胞中的转录活性。在某些优选的实施方案中，所述细胞表达Nur77。在某些优选的实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制TNFα在细胞中的生物学效应。在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于在体内、体外或离体抑制TNFα在细胞中的生物学效应。在某些优选的实施方案中，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活。在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于抑制TNFα诱导的IκBα降解。在某些优选的实施方案中，所述细胞表达Nur77。在某些优选的实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制细胞中mTOR通路 (例如下调P-mTOR、p-P70S6和/或p-S6活性)。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于在有此需要的受试者中预防或治疗与Nur77相关的疾病。在某些优选的实施方案中，所述与Nur77相关的疾病为癌症。在某些优选的实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于在患有癌症的受试者中抑制癌细胞的增殖和/或转移和/或促进癌细胞的凋亡。在某些优选的实施方案中，所述癌症选自肝癌，宫颈癌，肺癌、和乳腺癌。在某些优选的实施方案中，所述癌症为三阴性乳腺癌(即，雌激素受体(ER)、孕激素受体(PR)和原癌基因Her-2均为阴性的乳腺癌)。在某些优选的实施方案中，所述药物用于治疗癌症，并且包含所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯，以及TNFα。
在另一个方面，本申请涉及一种抑制孤儿核受体Nur77的转录活性的方法，其包括，将Nur77与下列化合物、其互变异构体、立体异构体或药学上可接受的盐或酯相接触的步骤：
Figure PCTCN2017091726-appb-000049
在某些优选实施方案中，所述方法用于抑制Nur77在细胞中的转录活性。在某些优选实施方案中，所述方法包括，给有此需要的细胞施用有效量的所述化合物，从而抑制Nur77在细胞中的转录活性。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在另一个方面，本申请涉及一种抑制TNFα在细胞中的生物学效应的方法，其包括，给有此需要的细胞施用有效量的下列化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000050
在某些优选实施方案中，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活。在某些优选实施方案中，所述方法用于抑制TNFα诱导的IκBα降解。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在另一个方面，本申请涉及一种抑制mTOR通路的方法，其包括，给有此需要的细胞施用有效量的下列化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000051
在某些优选实施方案中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于下调P-mTOR、p-P70S6K和/或p-S6活性。在某些优选实施方案中，所述细胞表达Nur77。在某些优选实施方案中，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
在另一个方面，本申请涉及一种预防或治疗与Nur77相关的疾病的方法，其包括， 给有此需要的受试者施用有效量的下列化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
Figure PCTCN2017091726-appb-000052
在某些优选实施方案中，所述与Nur77相关的疾病为癌症(例如三阴乳腺癌)。
在另一个方面，本申请涉及一种具有抗癌活性的药物的筛选方法，其包括以下步骤：
(1)提供表达孤儿核受体Nur77的癌细胞(例如三阴乳腺癌细胞)，设立阴性对照(即不表达Nur77)；
(2)将所述细胞用TNFα和候选试剂处理；
(3)检测(例如通过免疫印迹法进行检测)处理后的细胞中P-mTOR、p-P70S6K和p-S6的水平；
(4)比较所述细胞与阴性对照细胞中相应蛋白的水平，如有至少一种蛋白水平降低即判断所述候选试剂具有抗癌活性。
发明的有益效果
(1)本发明人首次发现，如式I-式V所示的化合物能够靶向孤儿核受体Nur77，并作为其配体发挥作用。例如，此类化合物可用于降低或抑制孤儿核受体Nur77的活性(例如转录活性)。
(2)mTOR信号通路的抑制作用可作为筛选抗癌药物的有效指标。本发明人发现，通过Nur77抑制mTOR信号通路强的化合物具有潜在的抗癌活性。由此，本申请还提供了一种具有抗癌(特别是三阴性乳腺癌)活性的药物的筛选方法。
(3)本申请的化合物还可用于预防和治疗与孤儿核受体Nur77相关的疾病，例如癌症(例如三阴乳腺癌)。因此，本发明提供了新的、有效的、特异性结合Nur77的 配体，其可用于开发治疗癌症(例如三阴乳腺癌)的新疗法。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley & Sons，Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
附图说明
图1显示了用Biacore T200仪器筛选能与Nur77结合的化合物。如图显示了用Biacore T200仪器检测YXY101与Nur77-LBD的结合的实验结果，其中，红色点代表化合物YXY101；蓝色点代表对照化合物。结果显示，化合物YXY101能够与Nur77-LBD结合。
图2A显示了雷公藤红素(化合物YXY101)的化学结构式。
图2B显示了进一步用不同浓度的YXY101(0.04μM，0.08μM，0.16μM，0.32μM，0.64μM)与Nur77-LBD的结合的实验结果。结果显示，化合物YXY101与Nur77-LBD结合的解离常数(Kd)为292nM；
图2C显示了用圆二色光谱法检测YXY101与Nur77-LBD的结合的实验结果，其中，红色曲线代表YXY101+Nur77-LBD；蓝色曲线代表Nur77-LBD。结果显示，化合物YXY101能改变Nur77-LBD的CD光谱。这表明，化合物YXY101能够与Nur77-LBD结合。
图2D显示了用HPLC法检测YXY101与Nur77-LBD的结合的实验结果，其中，红色曲线代表YXY101+Nur77-LBD；紫色曲线代表YXY101+RXRα-LBD。结果显示，化合物YXY101能够与Nur77-LBD结合形成复合体，但不与RXRα-LBD结合。
图2E显示了用双荧光素酶报告基因系统检测YXY101与Nur77-LBD的结合的实 验结果。结果显示，化合物YXY101能抑制Nur77的转录激活功能，但对糖皮质激素受体(GR)的转录激活功能无明显作用。这表明，化合物YXY101能够与Nur77-LBD结合，并抑制其转录活性；并且，化合物YXY101不与GR结合。
图2F显示了YXY101与Nur77的分子对接。分子对接结果显示，YXY101主要通过疏水作用结合到Nur77蛋白表面的已知的疏水凹槽。
图3A-3B显示了经不同浓度的YXY101和TNFα处理的细胞中IκBα和磷酸化的IKKα/β的免疫印迹分析结果。
图3C显示了经YXY101和TNFα处理的细胞的免疫荧光染色结果(Scale bar：20μm)。
图3D显示了经YXY101和TNFα处理的细胞的NF-κB活性的分析结果，其中，＊＊P＜0.01，＊＊＊P＜0.001(T检验)。
图3E显示了经不同浓度的YXY101和TNFα处理的不同癌细胞系中IκBα和磷酸化的IKKα/β的免疫印迹分析结果。
图3F显示了经不同化合物处理、且接受TNFα刺激的HepG2细胞中IκBα的免疫印迹分析结果。
图4A-4B显示了转染了不同siRNA、且经不同浓度的YXY101和TNFα处理的HepG2细胞中Nur77、RXRα和IκBα的免疫印迹分析结果。
图4C显示了经不同浓度的YXY101和TNFα处理的MEF细胞和Nur77-/-MEF细胞中IκBα的免疫印迹分析结果。
图4D显示了经YXY101和TNFα处理的MEF细胞和Nur77-/-MEF细胞的免疫荧光染色结果(Scale bar：10μm)。
图4E显示了经不同浓度的XS0284和TNFα处理的MEF细胞和Nur77-/-MEF细胞中IκBα的免疫印迹分析结果。
图5A显示了不同的乳腺癌细胞(MDA-MB-231；MDA-MB-468；BT549；SKBR3；T47D和MCF-7)的增殖比率对YXY101浓度的曲线，以及YXY101的IC50值；其中，MDA-MB-231，MDA-MB-468，BT549和SKBR3为三阴性乳腺癌细胞，用红色 曲线表示；T47D和MCF-7为三阳性乳腺癌细胞，用蓝色曲线表示。
图5B显示了用不同浓度的YXY101(0μM，0.25μM，0.5μM，1μM，2μM，或4μM)处理的MDA-MB-231和MCF-7细胞中parp和ER的免疫印迹分析结果。
图5C显示了用不同浓度的YXY101(0μM，2μM，或4μM)处理12h或24h的MDA-MB-231中parp和p-mTOR的免疫印迹分析结果。
图5D显示，用YXY101与TNFα联合处理不同时间(1h，6h或12h)的MDA-MB-231细胞中parp的免疫印迹分析结果。
图5E显示了Hela细胞的增殖比率对YXY101的浓度曲线，结果显示，YXY101对野生型Hela细胞的IC50值为1.189μM，而在Nur77敲除的Hela细胞中的IC50为58.166μM。
图6显示了各组裸鼠的肿瘤组织的免疫组化染色结果。图6A的左图显示了各组裸鼠中形成的肿瘤；中间图显示了各组裸鼠中形成的肿瘤的RTV；右图显示了各组裸鼠中形成的肿瘤的重量。
图7A显示了，在灌胃给药2mg/kg的YXY101两周后，11周，13周或17周的MMTV-PYVT小鼠的整体情况。
图7B显示了，在灌胃给药2mg/kg的YXY101两周后，来自11周，13周或17周的MMTV-PYVT小鼠的肿瘤组织的HE染色和免疫组化染色的结果。
图7C显示了，在灌胃给药2mg/kg的YXY101两周后，来自11周，13周或17周的MMTV-PYVT小鼠的肺组织的重量、形态、HE染色和免疫组化染色的分析结果。
图8A显示了野生型mmtv-PyMT小鼠、敲除Nur77的mmtv-PyMT小鼠在60天内的存活曲线。
图8B显示了野生型mmtv-PyMT小鼠、敲除Nur77的mmtv-PyMT小鼠在11周、13周、17周时的肿瘤重量对比的统计学结果。
图8C显示了野生型mmtv-PyMT小鼠、敲除Nur77的mmtv-PyMT小鼠在11周、13周、17周时的肿瘤外观形态大小比较。
图8D显示了在动物水平上检测YXY101对mmtv-PyMT小鼠存活率的影响。
图9A显示了在饥饿条件下用TNFα处理MB-MDA-231乳腺癌细胞，运用免疫印 迹的技术检测p-P70S6K的结果。
图9B显示了在饥饿条件下用TNFα处理SKBR3乳腺癌细胞，运用免疫印迹的技术检测p-P70S6K的结果。
图9C显示了在饥饿条件下用TNFα处理野生型和Nur77敲除型的MEF细胞不同时间(30min、2h)或不同浓度(20ng/ml/、40ng/ml)，并运用免疫印迹的技术检测p-mTOR、p-P70S6K、p-S6的结果。
图10A显示了在饥饿条件下用TNFα(20ng/ml)、YXY101(1μM、2μM、4μM)处理MB-MDA-231乳腺癌细胞，并运用免疫印迹的技术检测p-mTOR、p-P70S6K的结果。
图10B显示了在MB-MDA-231乳腺癌细胞中外转Flag空载质粒或者Flag-Nur77质粒，并在饥饿条件下用TNFα、YXY101处理3h、6h，运用免疫印迹的技术检测p-mTOR、p-P70S6K、Nur77的结果。
图10C显示了对MB-MDA-231细胞进行沉默Nur77处理，并在饥饿条件下用TNFα、YXY101处理，运用免疫印迹的技术检测p-P70S6K、Nur77的结果。
图10D显示了用TNFα、YXY101(0.5Mm、1μM)处理野生型或者Nur77敲除型的MEF细胞，运用免疫印迹的技术检测p-S6、p-mTOR、p62的结果。
图11A显示了不同的乳腺癌细胞(MDA-MB-231和MCF-7)的增殖比率对XS0503浓度的曲线，以及XS0503的IC50值
图11B显示了用YXY101衍生物XS0077、XS0335、XS0419、XS0474、XS0488处理的MDA-MB-231细胞的IC50分析结果。
图11C显示了用YXY101衍生物XS0284、XS0285、XS0394、XS0418、XS0419、XS0474、XS0486、XS0462、XS0491、XS0492、XS0488处理的BT474、ZR-75-1、BT549、HCC1937、HS578T、MCF-7、T47D这七株乳腺癌细胞的抑制率分析结果。
图12A显示了用YXY101(4μM)和不同的YXY101衍生物(4μM)XS0394、XS0395、XS0419、XS0420、XS0421、XS0284、XS0335、XS0488、XS0491、XS0492、XS0418、XS0502、XS0503、XS0504、XS0506、XS0507、XS0508、XS0077以及TNFα(20ng/ml) 处理的MDA-MB-231细胞中PARP的免疫印迹分析结果。
图12B显示了在使用无血清培养基饥饿处理10h情况下，用YXY101(2μM)和不同的YXY101衍生物(2μM)XS0284、XS0285、XS0335、XS0394、XS0418、XS0419、XS0454、XS0455、XS0462、XS0473、XS0474、XS0480、XS0486、XS0488、XS0491、XS0492以及TNFα(20ng/ml)处理的MDA-MB-231细胞中P-mTOR、P-p70S6K、P-S6的免疫印迹分析结果。
图12C显示了用YXY101(2μM)和YXY101衍生物XS0455(1、2、4μM)以及TNF(20ng/ml)处理的MDA-MB-231细胞中P-mTOR、P-p70S6K、P-S6和IκBα的免疫印迹分析结果。
图13显示了用Biacore T200仪器检测不同化合物(XS0418、XS0419、XS0474、XS0394、XS0492、XS0491、XS0488)与Nur77-LBD的结合的实验结果。结果显示，化合物XS0418与Nur77-LBD结合的解离常数(Kd)为3.67μM；化合物XS0419与Nur77-LBD结合的解离常数(Kd)为404nM；化合物XS0474与Nur77-LBD结合的解离常数(Kd)为2.60μM；化合物XS0394与Nur77-LBD结合的解离常数(Kd)为1.26μM；化合物XS0492与Nur77-LBD结合的解离常数(Kd)为1.30μM；化合物XS0491与Nur77-LBD结合的解离常数(Kd)为0.336μM；化合物XS0488与Nur77-LBD结合的解离常数(Kd)为1.28μM。
图14是使用WT小鼠和PYVT小鼠，分别给予LFD饲养和HFD饲养，并灌胃YXY101及其衍生物XS0284两个化合物进行处理。图14A显示了不同处理条件下，不同实验组老鼠的形态外观特征。图14B对比了以上老鼠肿瘤的形态大小特征。图14C显示了上述老鼠在灌胃给药的12天时间里的体重变化曲线。图14D显示了上述老鼠在处理前后的体重变化的统计学意义。
图15A显示了在PYVT的WT和Nur77敲除的小鼠中，进行灌胃YXY101及其衍生物XS0284处理后，肿瘤的形态大小的比较。
图15B显示了上述小鼠中肿瘤组织重量的统计学结果。
图16显示了采用灌胃或腹腔注射200mg/kg的YXY101及其衍生物XS0284一次后诱发的小鼠急性毒性模型后，观察灌胃或腹腔注射YXY101和XS0284(200mg/kg) 12个小时后，分别观察各组小鼠心脏、肝脏、小肠、白色脂肪和肾脏的组织形态以及HE染色的结果。
图17显示了利用计算机辅助模拟Nur77与其配体结合的作用模式图。
实施例1.化合物YXY101的表征
(1)表面等离子共振术(SPR)
通过表面等离子共振术来测定YXY101与Nur77的结合。简言之，将50μg经纯化的Nur77配体结合域(Nur77-LBD)蛋白与Biacore的CM5芯片偶联；随后通过Biacore T200仪器来测定YXY101(20μm)与Nur77-LBD的结合。将无关化合物用作对照。测定结果示于图1中。
图1显示了用Biacore T200仪器检测YXY101与Nur77-LBD的结合的实验结果，其中，红色点代表化合物YXY101；蓝色点代表对照化合物。结果显示，化合物YXY101能够与Nur77-LBD结合。
(2)YXY101与Nur77的结合动力学
通过表面等离子共振术来测定YXY101与Nur77的解离常数(Kd)。简言之，使用Biacore T200仪器来测定不同浓度的YXY101(0.04μM，0.08μM，0.16μM，0.32μM，0.64μM)与Nur77-LBD的结合。测定结果示于图2B中。
结果显示，化合物YXY101与Nur77-LBD结合的解离常数(Kd)为292nM。
(3)圆二色光谱法(CD)
使用圆二色光谱法(CD)对YXY101与Nur77-LBD的结合作进一步分析。简言之，将YXY101(1ml，1mg/ml)加入Nur77-LBD蛋白(1ml，1mg/ml)的磷酸盐缓冲液(10μm，PH7.4)中，并在4℃下孵育3h。随后，取0.7ml的溶液，并用Jasco J-810分光偏振计进行检测。记录从190nm到260nm的CD光谱。将单独的Nur77-LBD溶液(即，未添加化合物YXY101)用作对照。检测结果示于图2C中。
结果显示，化合物YXY101能改变Nur77-LBD的CD光谱。这表明，化合物YXY101能够与Nur77-LBD结合。
(4)高效液相色谱(HPLC)分析
使用高效液相色谱(HPLC)对YXY101与Nur77-LBD的结合作进一步分析。简言之，将YXY101(600uL，0.1mg/ml)与经纯化的Nur77-LBD蛋白(5ml，1mg/ml)共同孵育。在4℃条件下孵育3h后，用Ni beads捕获YXY101和Nur77-LBD的复合体。随后，使用氯仿将该复合体解离，并将解离产物中的YXY101萃取出来。然后，使用HPLC分光仪(Shimadzu LC 20A，Japan)来检测萃取产物中的YXY101，其中，所使用的柱子为ODS柱(5um，4.6＊250mm)，流动相为含有0.2％H3PO4的乙腈水溶液，检测波长为425nm。另外，还使用YXY101与RXRα-LBD(维甲类X受体α的配体结合域)重复上述实验，用作对照。检测结果示于图2D中。
结果显示，化合物YXY101能够与Nur77-LBD结合形成复合体，但不与RXRα-LBD结合。
(5)双荧光素酶报告基因检测法
使用双荧光素酶报告基因检测法对YXY101与Nur77-LBD的结合作进一步分析。另外，还使用YXY101与糖皮质激素受体(GR)来重复该实验，用作对照。实验结果示于图2E中。
结果显示，化合物YXY101能抑制Nur77的转录激活功能，但对糖皮质激素受体(GR)的转录激活功能无明显作用。这表明，化合物YXY101能够与Nur77-LBD结合，并抑制其转录活性；并且，化合物YXY101不与GR结合。
(6)分子模拟
使用AutoDock V4.2.来完成YXY101与Nur77(PDB code：4JGV)的对接。YXY101的构象由拉马克遗传算法构建。在Nur77的晶体结构中，格中心被选择在已报道的THPN坐标(-12.08，18.29，-4.233)，网格尺寸设置为40＊40＊40(X，Y，Z)格点，每个格点的间隔为0.375A。
在分子对接中，应用标准对接方案：随机放置个体数量为150；能量评估的最大数量为250万；基因突变的比率为0.02；交叉比率为0.8；个体进行局部搜索的概率为0.06；波的下界是0.01；分子可视化使用版本0.99的PyMOL。结果示于图2F中。
分子对接结果显示，YXY101主要通过疏水作用结合到Nur77蛋白表面的已知的疏水凹槽。
实施例2.YXY101抑制TNFα的生物学效应
HepG2细胞用不同浓度的YXY101(0μM，0.25μM，0.5μM，1μM，2μM或4μM)处理1小时，再用TNFα(0ng/mL或20ng/mL)处理30分钟。随后，用免疫印迹法(Western Bloting)检测细胞中的IκBα和磷酸化的IKKα/β。结果示于图3A-3B中。
图3A-3B的结果显示，TNFα能够在细胞中诱导IKKα/β的磷酸化以及IκBα的降解；而YXY101则能够抑制TNFα诱导的IKKα/β的磷酸化和IκBα的降解。
HepG2细胞用YXY101(0或1μM)处理1小时，然后用TNFα(20ng/mL)处理30分钟。随后，用免疫荧光染色法检测细胞中的NF-κB亚基p65。未经处理的细胞用作对照。结果示于图3C中。
图3C显示了经YXY101和TNFα处理的HepG2细胞的免疫荧光染色结果(Scalebar：20μm)。图3C的结果显示，TNFα能够在细胞中诱导NF-κB亚基p65的入核转运；而YXY101则能够抑制TNFα诱导的p65入核转运。
将NF-κB的报告基因转染入HEK-293T细胞，然后用YXY101(0或1μM)和TNFα(20ng/mL)进行处理。随后，检测细胞中的NF-κB活性。未经处理的细胞用作对照。结果示于图3D中。
图3D显示了经YXY101和TNFα处理的细胞的NF-κB活性的分析结果，其中，＊＊P＜0.01，＊＊＊P＜0.001(T检验)。图4D的结果显示，TNFα能够在细胞中诱导NF-κB的转录激活；而YXY101则能够抑制TNFα诱导的NF-κB转录激活。
多种癌细胞系(LO2，SMMC-7721，QGY-7703，HeLa，H460)用不同浓度的YXY101(0μM，1μM或4μM)处理1小时，再用TNFα(0ng/mL或20ng/mL)处理30分钟。随后，用免疫印迹法(Western Bloting)检测细胞中的IκBα和磷酸化的IKKα/β。结果示于图3E中。
结果显示，TNFα能够在各种细胞系中诱导IKKα/β的磷酸化以及IκBα的降解；而YXY101则能够抑制TNFα诱导的IKKα/β的磷酸化和IκBα的降解。
另外，还使用HepG2细胞来进行实验。简言之，HepG2细胞用指定浓度的各种化合物(YXY101、XS0284和XS0287)处理指定的时间，然后再用TNFα(0ng/mL或20ng/mL)处理30分钟。随后，用免疫印迹法(Western Bloting)检测细胞中的IκBα。结果示于图3F中。
图3F显示在HepG2细胞中，YXY101及其衍生物XS0284和XS0287能够抑制TNFα诱导的IκBα的降解。
图3A-3F的结果表明，YXY101及其衍生物XS0284和XS0287能够抑制TNFα在细胞中的各种生物学效应，包括IKKα/β的磷酸化的磷酸化、IκBα的降解、NF-κB亚基p65的入核转运、和NF-κB的转录激活。
实施例3.YXY101对TNFα生物学效应的抑制作用由Nur77介导
将对照SiRNA、Nur77 SiRNA或RXRα SiRNA转染入HepG2细胞。随后，HepG2细胞用不同浓度的YXY101(0μM，1μM或4μM)处理1小时，再用TNFα(0ng/mL或20ng/mL)处理30分钟。随后，用免疫印迹法(Western Bloting)检测细胞中的Nur77、RXRα和IκBα。结果示于图4A-4B中。
图4A-4B显示了转染了不同siRNA、且经不同浓度的YXY101和TNFα处理的HepG2细胞中Nur77、RXRα和IκBα的免疫印迹分析结果。结果显示，Nur77 SiRNA有效抑制/敲除了Nur77在细胞中的表达；并且，RXRα SiRNA有效抑制/敲除了RXRα在细胞中的表达；对照SiRNA则不影响Nur77和RXRα的正常表达。进一步，图5A-5B的结果显示，在表达Nur77的细胞中，YXY101均能够抑制TNFα诱导的IκBα的降解；然而，当Nur77的表达被敲除时，YXY101丧失了抑制IκBα降解的能力。这些结果表明，YXY101对TNFα生物学效应的抑制作用是由Nur77介导的。
还使用MEF细胞和Nur77-/-MEF细胞(即，不表达Nur77的MEF细胞)验证了上述实验结果。简言之，MEF细胞和Nur77-/-MEF细胞用不同浓度的YXY101(0μM或1μM)处理1小时，再用TNFα(0ng/mL或20ng/mL)处理30分钟。随后，用免疫印迹法(Western Bloting)检测细胞中的IκBα。结果示于图4C中。
结果显示，在表达Nur77的MEF细胞中，YXY101均能够抑制TNFα诱导的IκBα 的降解；然而，在Nur77-/-MEF细胞中，YXY101丧失了抑制IκBα降解的能力。这些结果表明，YXY101对TNFα生物学效应的抑制作用是由Nur77介导的。
另外，将MEF细胞和Nur77-/-MEF细胞用YXY101(0或1μM)处理1小时，然后用TNFα(20ng/mL)处理30分钟。随后，用免疫荧光染色法检测细胞中的NF-κB亚基p65。未经处理的细胞用作对照。结果示于图4D中。
图4D显示了经YXY101和TNFα处理的MEF细胞和Nur77-/-MEF细胞的免疫荧光染色结果(Scale bar：10μm)。结果显示，在表达Nur77的MEF细胞中，YXY101能够抑制TNFα诱导的p65入核转运；然而，在Nur77-/-MEF细胞中，YXY101丧失了抑制p65入核的能力。这些结果表明，YXY101对TNFα生物学效应的抑制作用是由Nur77介导的。
图4E显示了经不同浓度的XS0284和TNFα处理的MEF细胞和Nur77-/-MEF细胞中IκBα的免疫印迹分析结果。结果显示，在表达Nur77的MEF细胞中，XS0284能够抑制TNFα诱导的IκBα的降解；然而，在Nur77-/-MEF细胞中，XS0284丧失了抑制IκBα降解的能力。这些结果表明，XS0284对TNFα生物学效应的抑制作用是由Nur77介导的。
实施例4.YXY101具有明显抑制肿瘤的活性，其中对三阴性乳腺癌尤为敏感，此生物学功能依赖于Nur77
用不同浓度的YXY101(1μM，1.3μM，1.6μM，1.9μM，2.2μM，2.5μM，2.8μM，3.1μM，3.4μM，3.7μM，4.0μM)处理不同的乳腺癌细胞(MDA-MB-231；MDA-MB-468；BT549；SKBR3；T47D和MCF-7)处理72h，并测定乳腺癌细胞的增殖比率。绘制癌细胞增殖比率对YXY101浓度的曲线，并测定YXY101的IC50。结果示于图5A中。
图5A显示了不同的乳腺癌细胞(MDA-MB-231；MDA-MB-468；BT549；SKBR3；T47D和MCF-7)的增殖比率对YXY101浓度的曲线，以及YXY101的IC50值；其中，MDA-MB-231，MDA-MB-468，BT549和SKBR3为三阴性乳腺癌细胞，用红色曲线表示；T47D和MCF-7为三阳性乳腺癌细胞，用蓝色曲线表示。结果显示，YXY101 抑制三阴性乳腺癌细胞增殖的能力显著强于其抑制三阳性乳腺癌细胞增殖的能力；其中，YXY101抑制MDA-MB-231，MDA-MB-468，BT549和SKBR3增殖的IC50分别为1.204、0.187、0.245和2.646μM，远远低于其抑制T47D和MCF-7增殖的IC50(分别为74.465μM和11.498μM)。这表明YXY101可特别有利地用于治疗三阴性乳腺癌。
图5E显示了YXY101在野生型的Hela细胞中的IC50值为1.189μM，而在Nur77敲除的Hela细胞中的IC50为58.166μM，结果表示YXY101发挥的肿瘤抑制作用是依赖于Nur77的，加之图1和图2B显示的YXY101能特异性结合Nur77，YXY101的肿瘤抑制活性与Nur77息息相关。
进一步，用不同浓度的YXY101(0μM，0.25μM，0.5μM，1μM，2μM，或4μM)处理MDA-MB-231和MCF-7细胞，随后通过WB检测细胞中的parp和ER。结果示于图5B中。
图5B显示了用不同浓度的YXY101(0μM，0.25μM，0.5μM，1μM，2μM，或4μM)处理的MDA-MB-231和MCF-7细胞中parp和ER的免疫印迹分析结果。结果显示，YXY101可以在MDA-MB-231中诱导parp切割，即凋亡现象产生。
进一步，用不同浓度的YXY101(0μM，2μM，或4μM)处理MDA-MB-231不同时间(12h或24h)，随后通过WB检测细胞中的parp和p-mTOR。结果示于图5C中。
图5C显示了用不同浓度的YXY101(0μM，2μM，或4μM)处理12h或24h的MDA-MB-231中parp和p-mTOR的免疫印迹分析结果。结果显示，在MDA-MB-231中，YXY101诱导凋亡是时间依赖性和浓度依赖性。
此外，还用YXY101与TNFα联合处理MDA-MB-231细胞不同时间(1h，6h或12h)，随后通过WB检测细胞中的parp。结果示于图5D中。
图5D显示，用YXY101与TNFα联合处理不同时间(1h，6h或12h)的MDA-MB-231细胞中parp的免疫印迹分析结果。结果显示，在MDA-MB-231中，YXY101与TNFα的联合给药能诱导更强的细胞凋亡。
实施例5.YXY101对乳腺癌肿瘤的增殖的抑制作用
在本部分，我们通过裸鼠移植瘤实验检测YXY101单独或与TNFα联合用药时对三阴性乳腺癌的抗肿瘤作用。
1)实验动物和试剂
MDA-MB-231细胞细胞，BALB/c(nu/nu)裸鼠，体重18-20g，雌性，SPF级动物房中饲养，饲料、饮用水、动物笼具、垫料均经高压灭菌处理，每两天更换垫料，严格无菌操作。无菌条件下，收集对数生长期的待接种细胞，用无血清培养液洗涤，倒置显微镜下计数活细胞数＞95％，调整细胞浓度为1×106/ml，将肿瘤细胞重悬于PBS中制成细胞悬液。每只裸鼠右侧腋下接种0.2ml上述细胞悬液，定期观察测量肿瘤生长情况。
2)分组及给药
给药组：当裸鼠移植瘤直径达到约0.5cm时，选择无出血、坏死、感染的裸鼠进行实验，给裸鼠称重，测量瘤径，分组，每组6只裸鼠，实验组分别按TNFα(120×104U/kg)、YXY101(2mg/kg)、联合用药组给药，空白对照组给予等量的生理盐水。TNFα采用瘤内注射给药，隔天给药；YXY101灌胃给药，每天给药。末次给药6hr后处死裸鼠，称瘤重。
治疗过程中，裸鼠进食量及体重无明显减少，活动度正常，无松毛、腹泻等中毒症状出现。治疗结束时，各组裸鼠均未出现死亡。裸鼠处死后，尸体解剖可见肿瘤边界清楚，表面凹凸不平，质地较坚韧，局部血管明显扩张，有的瘤块中心区可出现坏死。各组裸鼠均未见转移病灶，各处理组裸鼠的心、肝、脾、肺、肾等脏器未发现明显外观改变。
3)观察指标
肿瘤生长曲线的绘制：
肿瘤体积(tumor volume，TV)的计算公式为：V＝1/2×a×b×c；其中，a、b、c分别表示长宽高。根据测量结果计算出肿瘤体积，以时间为横坐标，肿瘤体积为纵坐标绘制肿瘤生长曲线。
抗肿瘤活性评价指标为相对肿瘤增殖率T/C(％)：其中T，治疗组的RTV；C，阴性对照组的RTV。疗效评价标准：T/C％＞40％为无效；T/C％≤40％并且p＜0.05为有效。
实验结果示于图6中。图6A的左图显示了各组裸鼠中形成的肿瘤；中间图显示了各组裸鼠中形成的肿瘤的RTV；右图显示了各组裸鼠中形成的肿瘤的重量。图6A的结果显示，在接种MDA-MB-231细胞后，裸鼠产生了肿瘤；并且，YXY101有良好的抑制肿瘤的作用；并且YXY101与TNFα的联合给药能诱导更强的抑制肿瘤的作用。
图6B显示了各组裸鼠的肿瘤组织的免疫组化染色结果。图6B的结果显示，YXY101能够在肿瘤组织内诱导caspase3的切割，促进肿瘤细胞的凋亡；并且YXY101与TNFα的联合给药能诱导更强的肿瘤细胞的凋亡。
实施例6.YXY101对乳腺癌肿瘤的增殖与转移的抑制作用
1)实验动物和试剂
MMTV-PYVT乳腺癌转基因小鼠，9周龄大小，雌性，SPF级动物房中饲养，饲料、饮用水、动物笼具、垫料均经高压灭菌处理，每两天更换垫料，严格无菌操作。
2)分组及给药
雌性MMTV-PYVT乳腺癌转基因小鼠，在温度23±1℃，湿度：40-60％，自然光照，自由饮水，自由进食普通饲料的条件下饲养。选择胸部刚开始长肿瘤的36只小鼠，随机分为3大组，每大组再分为对照组和YXY101处理组。
按照下述方法给药：
11wk时间点
对照组：取9周龄小鼠于每天晚上7点给予饲料前灌胃生理盐水；
YXY101组：取9周龄小鼠于每天晚上7点给予饲料前灌胃一次，剂量2mg/kg。
13k时间点
对照组：取11周龄小鼠于每天晚上7点给予饲料前灌胃生理盐水；
YXY101组：取11周龄小鼠于每天晚上7点给予饲料前灌胃一次，剂量2mg/kg。
17k时间点
对照组：取15周龄小鼠于每天晚上7点给予饲料前灌胃生理盐水；
YXY101组：取15周龄小鼠于每天晚上7点给予饲料前灌胃一次，剂量2mg/kg。
按照上述方法连续给药2周后，对于每一只动物，摘眼球取血，移取上清测定血清炎症指标；取边界清楚，表面凹凸不平，质地较坚韧的肿瘤组织进行常规处理及包 埋，用于免疫染色。
实验结果示于图7和图8中。图7A显示了，在灌胃给药2mg/kg的YXY101两周后，11周，13周或17周的MMTV-PYVT小鼠的整体情况。图7A的结果显示，YXY101能抑制乳腺癌肿瘤的进展。
图7B显示了，在灌胃给药2mg/kg的YXY101两周后，来自11周，13周或17周的MMTV-PYVT小鼠的肿瘤组织的HE染色和免疫组化染色的结果。图7B的结果显示，YXY101可以抑制乳腺癌肿瘤的增殖。
图7C显示了，在灌胃给药2mg/kg的YXY101两周后，来自11周，13周或17周的MMTV-PYVT小鼠的肺组织的重量、形态、HE染色和免疫组化染色的分析结果。图7C的结果显示，YXY101可以抑制乳腺癌引起的肺转移。
图7A-7C的这些实验结果表明，YXY101能够抑制乳腺癌肿瘤的增殖与转移。
图8A显示了，在动物水平上，野生型的mmtv-PyMT小鼠和Nur77敲除的mmtv-PyMT小鼠在60天内的存活率曲线，图8D显示了，在动物水平上检测YXY101对mmtv-PyMT小鼠存活率的影响。结果表明，敲除了Nur77后小鼠的存活率显著提高，该结果预示着Nur77与乳腺癌的发生发展有显著地关联。图8B显示了上述两种小鼠的肿瘤组织重量的统计学结果，结果显示，敲除了Nur77后，乳腺癌瘤组织的重量能够明显下降，图8C也显示了这些瘤组织的外观形态比较，能够清晰地比较出敲除Nur77小鼠的瘤组织更小也更平滑。以上结果均表明Nur77在乳腺癌的发生发展过程中起着至关重要的作用。
综上所述，我们创新性地以Nur77为靶点，运用SPR手段筛选得到能够特异性结合Nur77的药物分子YXY101，其具有显著的抑制肿瘤活性，特别是对于三阴性乳腺癌非常敏感，而这种对于乳腺癌的治疗活性是依赖于Nur77的。该化合物是非常具有前景的治疗三阴乳腺癌的活性分子，而这种筛选方法是一种发现特异性结合Nur77并靶向治疗三阴性乳腺癌化合物药物分子的有效方式。
实施例7.炎症能一定程度上诱导肿瘤产生，该过程与Nur77相关
在前期调研中，我们发现乳腺癌特别是三阴性乳腺癌的发生发展同样始终伴随着 炎症反应及mTOR信号通路的高度激活，慢性炎症同样是乳腺癌发生发展的重要基础，并且也有许多对其他肿瘤有效的非类固醇类消炎药，也被拿来用于乳腺癌的防治。
图9A-9B显示，在饥饿条件下用TNFα处理MB-MDA-231、SKBR3这两种乳腺癌细胞1h、3h、6h后，运用免疫印迹的手段检测p-P70S6K，结果显示，在TNFα的诱导下p-P70S6K能够上调，并具有时间依赖性，结果表明在炎症环境下，mTOR信号通路被激活，这可能与肿瘤的发生密切相关。图9C显示，在饥饿条件下用TNFα浓度梯度或时间梯度地处理野生型或Nur77敲除型的MEF细胞，用免疫印迹手段检测p-mTOR、p-P70S6K、p-S6，结果显示，TNFα对mTOR信号通路的激活受时间和浓度调控，并且此种激活是依赖于Nur77的。
结合实施例6和实施例7，Nur77不仅对于乳腺癌的发生发展而言至关重要，同时也能作为药物的靶点来治疗乳腺癌，故Nur77是一种具有价值的检测乳腺癌发生发展的生化指标，用以评估乳腺癌进程、评估乳腺癌的治疗策略等。
实施例8.YXY101能够显著抑制mTOR活性，进而抑制肿瘤，该过程依赖于Nur77
本部分实验模拟肿瘤内环境，即以TNFα这个引起急性炎症的细胞因子来模拟肿瘤发生，这种方法能够为之后筛选抑制肿瘤药物提供实验基础。通过这种方法筛选Nur77依赖性的抑制mTOR信号通路的化合物，从而找到有效治疗三阴性乳腺癌的靶向性药物。
图10A显示了在饥饿条件下用TNFα(20ng/ml)、YXY101(1μM、2μM、4μM)处理MB-MDA-231乳腺癌细胞，并运用免疫印迹的技术检测p-mTOR、p-P70S6K的结果。结果表明，YXY101能显著下调由TNFα所激活的p-mTOR、p-P70S6K，预示着抑制mTOR的活性可能是其抑制肿瘤的内在机理。图10B显示了在MB-MDA-231乳腺癌细胞中外转Flag空载质粒或者Flag-Nur77质粒，并在饥饿条件下用TNFα、YXY101处理3h、6h，运用免疫印迹的技术检测p-mTOR、p-P70S6K、Nur77的结果。图10C显示了对MB-MDA-231细胞进行沉默Nur77处理，并在饥饿条件下用TNFα、YXY101处理，运用免疫印迹的技术检测p-P70S6K、Nur77的结果。图10D显示了用 TNFα、YXY101(0.5Mm、1μM)处理野生型或者Nur77敲除型的MEF细胞，运用免疫印迹的技术检测p-S6、p-mTOR、p62的结果。综合图10B-10D的结果，从过表达和敲除Nur77的两种方式同时表明YXY101下调mTOR活性的活性是依赖于Nur77的。
综上，mTOR信号通路的抑制作用可作为筛选抗癌药物的有效指标，通过这种方法筛选出通过Nur77抑制mTOR信号通路强的化合物具有潜在的抗癌活性。
实施例9.YXY101衍生物对三阴性乳腺癌细胞的促凋亡作用
用不同浓度的YXY101衍生物XS0503(0.16μM，0.31μM，0.625μM，1.25μM，2.5μM，5μM，10.0μM)处理不同的乳腺癌细胞(MDA-MB-231和MCF-7)处理24h，并测定乳腺癌细胞的增殖比率。绘制癌细胞增殖比率对XS0503浓度的曲线，并测定YXY101的IC50。结果示于图11A中。
图11A显示了不同的乳腺癌细胞(MDA-MB-231和MCF-7)的增殖比率对XS0503浓度的曲线，以及XS0503的IC50值；其中，MDA-MB-231用红色曲线表示；MCF-7为三阳性乳腺癌细胞，用蓝色曲线表示。结果显示，XS0503抑制三阴性乳腺癌细胞增殖的能力显著强于其抑制三阳性乳腺癌细胞增殖的能力；其中，XS0503抑制MDA-MB-231增殖的IC50为1.19μM，低于其抑制MCF-7增殖的IC50(2.65μM)。这表明YXY101可特别有利地用于治疗三阴性乳腺癌。
进一步，用不同的YXY101衍生物XS0077、XS0335、XS0419、XS0474、XS0488(0.16μM，0.31μM，0.625μM，1.25μM，2.5μM，5μM，10.0μM)处理MDA-MB-231，随后测定MDA-MB-231的增殖比率。结果示于图11B中。结果显示，XS0077、XS0335、XS0419、XS0474、XS0488抑制MDA-MB-231增殖的IC50分别为1.84μM，1.88μM，1.27μM，2.93μM，1.90μM。
图11C显示了用YXY101衍生物XS0284、XS0285、XS0394、XS0418、XS0419、XS0474、XS0486、XS0462、XS0491、XS0492、XS0488处理的BT474、ZR-75-1、BT549、HCC1937、HS578T、MCF-7、T47D这七株乳腺癌细胞的抑制率分析结果。结果显示，以上衍生物对不同的乳腺癌细胞均有一定的抑制作用，其中XS0284、XS0285、XS0418、 XS0419、XS0462、XS0488对多种乳腺癌细胞的抑制作用接近甚至强于YXY101。
进一步的，用YXY101(4μM)和不同的YXY101衍生物(4μM)XS0394、XS0395、XS0419、XS0420、XS0421、XS0284、XS0335、XS0488、XS0491、XS0492、XS0418、XS0502、XS0503、XS0504、XS0506、XS0507、XS0508、XS0077以及TNFα(20ng/ml)处理的MDA-MB-231细胞中PARP的免疫印迹分析。结果如图12A中表明，红色字体标记的衍生物相比YXY101而言，更加显著地诱导三阴性乳腺癌MDA-MB-231细胞中PARP切割，包括XS0418、XS0419、XS0492、XS0508、XS0077。
图12B显示了在使用无血清培养基饥饿处理10h情况下，用YXY101(2μM)和不同的YXY101衍生物(2μM)XS0284、XS0285、XS0335、XS0394、XS0418、XS0419、XS0454、XS0455、XS0462、XS0473、XS0474、XS0480、XS0486、XS0488、XS0491、XS0492以及TNFα(20ng/ml)处理的MDA-MB-231细胞中P-mTOR、P-p70S6K、P-S6的免疫印迹分析结果。红色字体标记的衍生物相比YXY101而言，更加显著地降低P-mTOR、P-p70S6K、P-S6，包括XS0335、XS0394、XS0418、XS0419、XS0455、XS0474、XS0486、XS0491、XS0492。
图12C显示了用YXY101(2μM)和YXY101衍生物XS0455(1、2、4μM)以及TNFα(20ng/ml)处理的MDA-MB-231细胞中P-mTOR、P-p70S6K、P-S6和IκBα的免疫印迹分析结果。结果显示XS0455能够浓度依赖地下调P-mTOR、P-p70S6K、P-S6，而不会明显引起PARP切割和IκBα的降解逆转。结合前期数据，XS0455能够选择性下调P-mTOR、P-p70S6K、P-S6，但不会明显引起PARP切割和IκBα的降解逆转，该化合物有望成为新型专一性抗癌的药物分子。
实施例10.表面等离子共振术(SPR)对其他化合物的表征
根据实施例1和2中所描述的方法，使用Biacore T200仪器，通过SPR来检测多种YXY101衍生物物(XS0418、XS0419、XS0474、XS0394、XS0492、XS0491、XS0488)与Nur77-LBD的结合。结果示于图13中。
图13显示了用Biacore T200仪器检测不同化合物(XS0418、XS0419、XS0474、XS0394、XS0492、XS0491、XS0488)与Nur77-LBD的结合的实验结果。结果显示， 化合物XS0418与Nur77-LBD结合的解离常数(Kd)为3.67μM；化合物XS0419与Nur77-LBD结合的解离常数(Kd)为404nM；化合物XS0474与Nur77-LBD结合的解离常数(Kd)为2.60μM；化合物XS0394与Nur77-LBD结合的解离常数(Kd)为1.26μM；化合物XS0492与Nur77-LBD结合的解离常数(Kd)为1.30μM；化合物XS0491与Nur77-LBD结合的解离常数(Kd)为0.336μM；化合物XS0488与Nur77-LBD结合的解离常数(Kd)为1.28μM。
实施例11.XS0284对高脂诱导的乳腺癌的治疗作用强于YXY101
为了证明YXY101衍生物也能抑制乳腺癌，我们选取其中抑制P-mTOR、P-p70S6K、P-S6效果明显的XS0284进行验证。图14A-14D的结果显示，无论是低脂条件或是高脂条件饲养下，YXY101和XS0284均能明显抑制乳腺癌的发展，其中在低脂饲养条件下，XS0284的抑制乳腺癌的作用接近于YXY101，而在高脂饲养条件下，XS0284的抑制作用强于YXY101，以上结论可从瘤组织大小和小鼠体重变化等方面体现。
这些结果表明，YXY101的系列衍生物(例如XS0284)亦能抑制肿瘤的发展，甚至肿瘤抑制的效果优于YXY101。
实施例12.YXY101及其衍生物的抑制肿瘤作用是依赖于Nur77的
为了验证YXY101及其衍生物发挥抑制肿瘤作用与Nur77的依赖关系，我们选取其中抑制P-mTOR、P-p70S6K、P-S6效果明显的YXY101及其衍生物XS0284进行验证。图15A-15B显示了两种PYVT小鼠，野生型和Nur77敲除型，分别灌胃给药YXY101(5mg/kg)、XS0284(10mg/kg)两周。通过对比各实验组瘤组织大小，结果表明，在野生型小鼠中，YXY101以及XS0284均能有效抑制肿瘤，但在Nur77缺失小鼠中，两种化合物均不能发挥出抑制作用。
以上结果表明，YXY101及其衍生物发挥其肿瘤抑制作用是依赖于Nur77的。
实施例13.XS0284的药物急性毒副作用低于YXY101
本实施例采用，一次性灌胃注射200mg/kg的YXY101及其衍生物XS0284或腹腔注射20mg/kg的YXY101及其衍生物XS0284后诱发的小鼠急性毒性模型。在本实施例中，灌胃或腹腔注射YXY101和XS0284后，分别观察各组小鼠进食量、饮水、自发活动能力、精神状态、四肢活动、大小便质量、毛发光泽等，并且详细记录可能出现的一些毒性反应化及开始和消失时间点。组织病理学观察心脏、肝脏、小肠、白色脂肪和肾脏的组织形态。
实验结果如图16所示。图16A显示了灌胃组：1)空白组小鼠的心脏心肌细胞界限清晰，胞核多且明显，心肌纤维排列整齐，界限明显；肝组织中肝窦数量多且清楚，肝小叶界限明显；肠组织界限清晰，细胞饱满，肠绒毛排列整齐、界限明显；脂肪组织细胞界限清晰、排列整齐。2)与空白组比，YXY101组小鼠的心肌细胞和心肌纤维排列杂乱；肝小叶界限模糊，胞浆减少，肝细胞多数出现死亡；肠组织肿胀明显，细胞死亡增多，炎症反应明显，肠绒毛排列紊乱；脂肪组织细胞排列杂乱，个别纤维断裂。3)与空白组比，XS0284组小鼠的心脏心肌细胞界限相对清晰，心肌纤维相对排列整齐，界限明显；肝组织中肝细胞界限明显，结构清晰；肠组织界限清晰，细胞饱满，肠绒毛排列整齐、界限明显；脂肪组织细胞界限清晰、排列整齐，纤维断裂减少。由此可以得出衍生物XS0284组显示对肝脏和小肠的毒性作用比YXY101更弱。
图16B显示了腹腔注射组1)空白组小鼠的心脏心肌细胞界限清晰，胞核多且明显，心肌纤维排列整齐，界限明显；肝组织中肝窦数量多且清楚，肝小叶界限明显；脂肪组织细胞界限清晰、排列整齐；肾脏组织肾小球界限清晰，细胞饱满，排列整齐、界限明显。2)与空白组比，YXY101组小鼠的心肌细胞和心肌纤维排列杂乱；肝小叶界限基本明显，肝细胞饱满形态正常；但脂肪组织细胞排列杂乱，且有大量炎性细胞浸润；肾脏组织肾小球界限模糊，且有大量炎性细胞浸润，排列不整齐。3)与空白组比，XS0284组小鼠的心脏心肌细胞界限相对清晰，心肌纤维相对排列整齐，界限明显；肝组织中肝细胞界限明显，结构清晰；脂肪组织细胞界限清晰、排列整齐，纤维断裂减少；肾脏组织肾小球界限清晰，细胞饱满，有少量炎性细胞浸润。由此可以得出腹腔注射组衍生物XS0284组显示对肾脏和脂肪的毒性作用比YXY101更弱。
这些结果表明YXY101的系列衍生物，例如XS0284具有比YXY101更低的动物 急性毒作用。进一步提示着这系列化合物具有比YXY101更强的靶向性和专一性，以及这系列YXY101衍生物作为开发成更安全的抗癌药的可能。
实施例14.
利用计算机辅助分子对接技术，以Nur77为靶点。对海洋天然产物进行虚拟筛选从中获得一些结合较好的化合物，具体如下所示。
Figure PCTCN2017091726-appb-000053
该实例中利用计算机辅助进行虚拟筛选的步骤如下：下载蛋白共晶结构PDB ID：3V3Q，蛋白预处理，加氢，去除结晶水分子和晶体结构中甘油小分子；选中配体，以配体为中心，12angstrom的大小构建对接grid格点文件；运行Glide docking，选中事先构建的grid格点文件和处理好的Seaweed Metabolism Database进行对接；根据对接打分和化合物与蛋白受体之间的相互作用，对对接结果进行综合评价。模拟结合的作用模式图示例见图17。
化合物XS0077的制备
Figure PCTCN2017091726-appb-000054
首先将化合物YXY101(50mg，0.11mmol)搅拌溶解于2mL DMF中，随后加入碳酸氢钠(56mg，0.66mmol)，碘甲烷(42μL，0.66mmol)室温搅拌反应12小时。1mol/L HCl(1mL)淬灭反应，加入9mL纯水，用乙酸乙酯萃取3次(每次5mL)，收集有机层，无水硫酸钠干燥后，用减压蒸馏法除去有机溶剂乙酸乙酯，得到橘红色固体混合粗品。以正己烷和乙酸乙酯(hexane/ethyl acetate＝10∶1)为洗脱剂，300-400目硅胶填充柱层析分离，得到橘红色固体产物。
1H NMR(600MHz，DMSO-d6)δppm 0.44(s，3H)，0.91(d，J＝14.31Hz，1H)，1.07(s，3H)，1.12(s，3H)，1.21(s，3H)，1.30-1.35(m，1H)，1.38(s，3H)，1.41-1.46(m，1H)，1.50-1.59(m，3H)，1.61-1.72(m，4H)，1.78-1.86(m，1H)，1.95(td，J＝13.98，3.76Hz，1H)，2.06(d，J＝14.12Hz，1H)，2.09(s，3H)，2.17-2.22(m，1H)，2.31(d，J＝15.77Hz，1H)，3.48(s，3H)，6.35(d，J＝7.15Hz，1H)，6.39(d，J＝1.28Hz，1H)，7.07(dd，J＝7.15，1.10Hz，1H)，8.72(s，1H).
13C NMR(151MHz，DMSO-d6)δppm 10.10，17.96，21.41，28.08，29.19，29.42，30.12，30.34，31.34，32.23，32.91，34.39，36.02，37.83，38.8，39.83，41.99，43.64，44.48，51.44，117.26，118.05，120.18，126.89，133.13，146.42，162.94，167.80，177.93，177.96.
实施例15.化合物XS0284的制备
Figure PCTCN2017091726-appb-000055
在氮气环境下，在搅拌条件下，将化合物YXY101(50mg，0.11mmol)溶于甲醇(2.5mL)。加入亚硫酸氢钠水溶液(14mg，0.13mmol，溶解于1mL水中)，并于室温下反应3小时。反应后，用旋转蒸发仪浓缩反应体系得无色晶体。用甲醇/水对无色晶体进行重结晶，所获得的产物经真空干燥得呈白色固体的化合物XS0284(56.6mg)。
1H NMR(600MHz，DMSO-d6)δppm 0.59(s，3H)0.86(d，J＝12.10Hz，1H)1.05(s，3H)1.09(s，3H)1.18(s，3H)1.43-1.60(m，6H)1.62(s，3H)1.78-1.85(m，1H)1.93-2.04(m，3H)2.21(s，3H)2.32(d，J＝15.22Hz，1H)4.48(d，J＝6.24Hz，1H)5.81(d，J＝6.60Hz，1H)6.58(s，1H)7.61(br.s.，1H)8.81(br.s.，1H).
13C NMR(151MHz，DMSO-d6)δppm 13.57，18.46，21.64，29.04，29.98，30.47，30.60，30.64，31.94，32.92，34.80，35.11，36.90，36.96，37.90，38.07，43.95，44.39，60.19，108.96，118.87，123.26，124.39，140.89，141.93，144.02，150.07，180.00.
实施例16.化合物XS0285的制备
Figure PCTCN2017091726-appb-000056
在搅拌条件下，将化合物YXY101(50mg，0.11mmol)溶于N，N-二甲基甲酰胺(2mL)。加入碳酸氢钠(50.4mg，0.6mmol)，然后加入苄基溴(0.17mg，20μL)，并于室温下搅拌反应24小时。停止反应，向反应体系中加入去离子水(15mL)，并用 乙酸乙酯萃取3次。合并乙酸乙酯层，用饱和NaCl水溶液洗涤3次，用无水Na2SO4干燥，用旋转蒸发仪浓缩得粗产物(深红棕色油状物)。粗产物经快速柱层析法(乙酸乙酯∶正己烷)分离纯化，真空干燥得呈红色固体的化合物XS-0285(44mg)。
1H NMR(600MHz，CHLOROFORM-d)δppm 0.50(s，3H)0.97(d，J＝13.75Hz，1H)1.09(s，3H)1.21(s，3H)1.22-1.25(m，3H)1.25-1.28(m，1H)1.41(s，3H)1.47-1.58(m，3H)1.58-1.72(m，5H)1.87(d，J＝6.05Hz，1H)1.99-2.11(m，3H)2.21(d，J＝1.65Hz，3H)2.24(d，J＝14.12Hz，1H)2.44(d，J＝15.77Hz，1H)4.93(d，J＝12.29Hz，1H)5.02(d，J＝12.47Hz，1H)6.32(d，J＝7.15Hz，1H)6.49(s，1H)7.01(d，J＝6.97Hz，1H)7.27-7.30(m，2H)7.30-7.36(m，3H).
13C NMR(151MHz，CHLOROFORM-d)δppm 10.29，18.53，21.58，28.61，29.53，29.90，30.55，30.76，31.57，32.76，33.27，34.69，36.35，38.25，39.43，40.44，42.93，44.25，45.03，66.32，117.36，118.12，119.62，127.38，128.24，128.32，128.64，134.24，135.68，146.06，164.79，170.27，177.95，178.36.
实施例17.化合物XS0335的制备
Figure PCTCN2017091726-appb-000057
在搅拌条件下，将化合物YXY101(50mg，0.11mmol)溶于二氯甲烷(1mL)。加入钯碳(5mg)，再加入二氯甲烷(1mL)，持续通入氢气，并在室温下搅拌反应24小时。停止反应，向反应体系中加入去离子水(15mL)，并用乙酸乙酯萃取3次。合并乙酸乙酯层，然后用饱和NaCl洗3次，用无水Na2SO4干燥，用旋转蒸发仪浓缩得粗产物(无色油状物)。粗产物经快速柱层析法(乙酸乙酯∶正己烷)分离纯化，真空干燥得呈白色固体的化合物XS-0335(46mg)。
1H NMR(600MHz，METHANOL-d4)δppm 0.93-1.00(m，2H)1.03(s，3H)1.11 (s，3H)1.19(t，J＝3.48Hz，4H)1.26(s，3H)1.41-1.45(m，6H)1.47-1.55(m，2H)1.57(d，J＝8.25Hz，1H)1.59-1.68(m，2H)1.83-1.98(m，3H)2.06-2.11(m，4H)2.11-2.20(m，4H)2.43(d，J＝15.77Hz，1H)2.68(d，J＝14.67Hz，1H)6.67(s，1H).
13C NMR(151MHz，METHANOL-d4)δppm 11.94，18.51，20.54，26.82，28.66，30.74，31.15，31.57，31.61，32.02，32.09，32.32，33.63，34.65，37.09，37.56，38.53，39.45，39.85，41.69，58.12，58.48，112.24，121.74，129.36，141.47，142.39，144.19，182.95.
实施例18.化合物XS0366，XS0434-XS0438，XS0440，XS0441，XS0443，XS0463和XS0464的制备
Figure PCTCN2017091726-appb-000058
以XS0366为例，在搅拌条件下，将化合物YXY101(100mg，0.22mmol)溶于二氯甲烷(4mL)。加入吲哚(52mg，0.44mmol)，再加入六水合三氯化铝(5.3mg，0.022mmol)，并在室温下搅拌反应5小时。停止反应，向反应体系中加入去离子水(15mL)，并用乙酸乙酯萃取3次。合并乙酸乙酯层，然后用饱和NaCl洗3次，用无水Na2SO4干燥，用旋转蒸发仪浓缩得粗产物(棕色油状物)。粗产物经快速柱层析法(乙酸乙酯∶正己烷)分离纯化，真空干燥得呈紫红色固体的化合物XS0366(122.2mg)。
1H NMR(600MHz，CHLOROFORM-d)δppm 0.73(br.s.，3H)0.87-0.90(m，1H)0.95-1.00(m，3H)1.01(br.s.，3H)1.14(br.s.，3H)1.19(t，J＝7.06Hz，1H)1.25-1.27(m，1H)1.34(br.s.，3H)1.42-1.57(m，4H)1.57-1.76(m，4H)1.90(s，3H)1.99-2.07(m，2H)2.10-2.17(m，1H)2.40(d，J＝15.04Hz，1H)4.90(d，J＝5.69Hz，1H) 6.21(d，J＝6.24Hz，1H)6.23(br.s.，1H)6.79(br.s.，1H)7.11(t，J＝7.43Hz，1H)7.16(t，J＝7.43Hz，1H)7.28(d，J＝7.89Hz，1H)7.75(d，J＝7.70Hz，1H)7.88(br.s.，1H).
13C NMR(151MHz，CHLOROFORM-d)δppm 11.53，18.84，21.93，28.89，29.63，29.75，30.40，30.54，30.72，31.55，32.84，34.62，35.50，36.74，36.94，37.76，40.35，43.62，44.28，108.93，111.30，119.09，119.28，120.23，121.55，121.58，121.67，127.10，127.84，136.49，139.92，142.17，142.83，147.48，184.30.
根据上述方法，本发明还合成了下述化合物：
Figure PCTCN2017091726-appb-000059
实施例19.化合物XS0395的制备
Figure PCTCN2017091726-appb-000060
称取化合物YXY101(50mg，0.11mmol)于25ml反应瓶中，加入4ml丙酮搅拌溶解，再加入一滴浓盐酸作催化剂，室温条件下反应12h。反应停止，直接将反应液减压浓缩溶剂，用乙酸乙酯∶正己烷＝4∶1体系经硅胶柱层析分离纯化，得白色固体，产率51％。
1H NMR(600MHz，DMSO-d6)δppm 0.62(s，3H)，0.82-1.86(m，1H)，1.04(s，3H)，1.09(s，3H)，1.15(s，3H)，1.25-1.38(m，4H)，1.39(s，3H)，1.42-1.50(m，2H)，1.55-1.71(m，4H)，1.77(td，J＝13.9，6.1Hz，1H)，1.93-2.02(m，2H)，2.03(s，3H)，2.09(s，3H)，2.27-2.36(m，2H)，2.67(dd，J＝16.2，2.7Hz，1H)，3.71-3.78(m，1H)，5.72(d，J＝6.4Hz，1H)，6.63(s，1H)，7.91(s，1H)，8.94(s，1H)，12.06(br.s.，1H).
13C NMR(151MHz，DMSO-d6)δppm 11.64，18.43，22.63，29.01，29.90，30.36，30.47，30.62，30.77，31.44，31.87，32.88，32.92，34.94，35.60，36.79，36.85，36.98，37.74，39.87，43.74，44.36，51.72，109.06，120.00，122.04，126.53，140.46，141.62，143.85，149.91，179.96.208.01.
实施例20.化合物XS0419的制备
Figure PCTCN2017091726-appb-000061
将化合物YXY101(50mg，0.11mmol)搅拌溶解于2mL氘代甲醇溶剂中，随后加 入硼氢化钠(44mg，1.1mmol)，室温搅拌反应30min。1mol/L HCl(1mL)猝灭反应，加入9mL纯水，用二氯甲烷萃取三次(每次5mL)，收集有机层，无水硫酸钠干燥后，用减压蒸馏快速除去有机溶剂二氯甲烷，得到白色固体的化合物XS0419(50.1mg)。
1H NMR(600MHz，DMSO-d6)ppm 0.66(s，3H)0.85(d，J＝13.9Hz，1H)1.05(s，3H)1.11(s，3H)1.17(s，3H)1.22(s，3H)1.29(ddd，J＝13.7，4.4Hz，1H)1.36-1.41(m，1H)1.43-1.51(m，3H)1.53-1.59(m，1H)1.59-1.63(m，1H)1.63-1.69(m，1H)1.79(ddd，J＝13.8，6.5Hz，1H)1.86(ddd，J＝13.8，5.0Hz，1H)1.94-2.00(m，2H)2.01(s，3H)2.04(d，J＝13.6Hz，1H)2.34(d，J＝15.6Hz，1H)2.91(dd，J＝20.0，1.5Hz，1H)3.18(dd，J＝20.5，6.2Hz，1H)5.72(dd，J＝6.1，1.8Hz，1H)6.61(s，1H)7.82(s，1H)8.80(s，1H)12.05(br.s.，1H).
13C NMR(151MHz，DMSO-d6)ppm 11.53，18.10，22.70，27.30，28.45，29.46，29.77，30.08，30.19，31.39，32.44，34.06，34.09，34.40，36.07，36.56，37.14，39.44，43.25，43.83，108.17，117.67，120.10，123.10，139.35，140.56，143.11，149.22，179.51.
实施例21.化合物XS0462的制备
Figure PCTCN2017091726-appb-000062
首先将雷公藤红素(135.2mg，0.3mmol)搅拌溶解于2mL DMF中，随后加入碳酸氢钠(138.6mg，1.65mmol)，溴乙烷(234μL，0.15mmol)室温搅拌反应12小时。1mol/L HCl(1mL)淬灭反应，加入9mL纯水，用乙酸乙酯萃取3次(每次15mL)，收集有机层，无水硫酸钠干燥后，用减压蒸馏法除去有机溶剂乙酸乙酯，得到橘红色固体混合粗品。以正己烷和乙酸乙酯(hexane/ethyl acetate＝10∶1)为洗脱剂，300-400目硅胶填充柱层析分离，得到橘红色固体产物。
1H NMR(600MHz，DMSO-d6)δppm 0.47(s，3H)，0.91(d，J＝14.1Hz，1H)，1.07(s， 3H)，1.11(s，3H)，1.12-1.15(m，3H)，1.21(s，3H)，1.31-1.36(m，1H)，1.38(s，3H)，1.41-1.46(m，1H)，1.52-1.58(m，3H)，1.61-1.71(m，4H)，1.78-1.87(m，1H)，1.90-1.99(m，1H)，2.03-2.08(m，1H)，2.09(s，3H)，2.21(d，J＝11.2Hz，1H)，2.34(d，J＝15.6Hz，1H)，3.91(m，2H)，6.35(d，J＝7.2Hz，1H)，6.39(s，1H)，7.05-7.10(m，1H)，8.73(s，1H).
13C NMR(151MHz，DMSO-d6)δppm 10.53，14.28，18.50，21.84，28.55，29.57，29.75，30.58，31.76，32.76，33.32，34.78，36.43，38.20，39.27，40.13，40.44，42.43，44.04，44.93，60.33，117.74，118.51，120.56，127.29，133.67，146.86，163.40，168.31，177.83，178.41.
实施例22.化合物XS0474的制备
Figure PCTCN2017091726-appb-000063
将化合物YXY101(50mg，0.11mmol)搅拌溶解于2mL氘代甲醇溶剂中，随后加入硼氘化钠(48.4mg，1.1mmol)，室温搅拌反应30min。1mol/L HCl(1mL)猝灭反应，加入9mL纯水，用二氯甲烷萃取三次(每次5mL)，收集有机层，无水硫酸钠干燥后，用减压蒸馏快速除去有机溶剂二氯甲烷，得到白色固体的化合物XS0474(50.2mg)。
1H NMR(600MHz，DMSO-d6)ppm 0.66(s，3H)0.85(d，J＝12.84Hz，1H)1.04(s，3H)1.10(s，3H)1.17(s，3H)1.22(s，3H)1.29(td，J＝13.66，4.40Hz，1H)1.35-1.41(m，1H)1.43-1.52(m，3H)1.53-1.58(m，1H)1.58-1.63(m，1H)1.63-1.69(m，1H)1.79(td，J＝13.66，6.60Hz，1H)1.86(td，J＝13.71，5.04Hz，1H)1.94-1.99(m，2H)2.01(s，3H)2.04(d，J＝12.10Hz，1H)2.34(d，J＝15.59Hz，1H)3.16(d，J＝6.05Hz，1H)5.71(d，J＝6.24Hz，1H)6.61(s，1H)7.82(s，1H)8.83(s，1H)12.03(br.s.，1H).
13C NMR(151MHz，DMSO-d6)ppm 11.60，18.15，22.74，26.98，28.52，29.51，29.83，30.14，30.25，31.45，32.50，34.15，34.21，34.46，36.14，36.62，37.21，39.49，43.31，43.89， 108.23，117.68，120.20，123.11，139.45，140.60，143.18，149.32，179.59.
实施例23.化合物XS0503的制备
Figure PCTCN2017091726-appb-000064
首先将扁蒴藤素(250mg，0.54mmol)溶解于20mL四氢呋喃中，随后加入LiAlH4(1.2mL，1.1mmol)，室温搅拌反应2h，加入10mL去离子水猝灭反应，1mol/L HCl(5mL)酸化，用乙酸乙酯萃取3次(每次15mL)，收集有机层，无水硫酸钠干燥后，用减压蒸馏法除去有机溶剂乙酸乙酯，得到橙黄色固体混合粗品。以正己烷和乙酸乙酯(hexane/ethyl acetate＝2∶1)为洗脱剂，300-400目硅胶填充柱层析分离，得到橙黄色固体产物。
1H NMR(600MHz，DMSO-d6)ppm 0.76(s，3H)0.83-0.87(m，1H)0.89(s，3H)1.11(s，3H)1.21(s，3H)1.23-1.25(m，1H)1.26(s，3H)1.27-1.34(m，2H)1.48(dd，J＝6.7，3.9Hz，1H)1.50-1.54(m，1H)1.55-1.59(m，1H)1.60(d，J＝5.0Hz，1H)1.64(dd，J＝9.2，4.6Hz，2H)1.65-1.69(m，2H)1.69-1.72(m，1H)1.91(d，J＝5.3Hz，1H)1.93-1.97(m，1H)2.02(s，3H)2.92(d，J＝19.4Hz，1H)2.96(dd，J＝10.3，4.8Hz，1H)3.15-3.20(m，1H)3.21(t，J＝6.0Hz，1H)4.44(t，J＝5.0Hz，1H)5.73(dd，J＝6.1，1.7Hz，1H)6.61(s，1H)7.80(s，1H)8.78(s，1H).
13C NMR(151MHz，CHLOROFORM-d)ppm 11.49，19.35，25.65，27.80，28.10，28.75，29.36，30.31，30.45，30.57，32.27，32.89，33.58，34.24，36.55，36.82，36.95，37.68，42.91，43.15，71.82，108.42，118.15，120.42，125.27，139.96，141.09，141.87，151.04.
实施例24.化合物XS0508的制备
Figure PCTCN2017091726-appb-000065
称取XS0077(50mg，0.11mmol)于50ml圆底瓶中，加入3ml甲醇溶解，置换气体，氮气保护，加入亚硫酸氢钠溶液(28mg NaHSO3-1ml H2O，0.26mmol)，室温下氮气保护反应3h，停止反应，减压蒸馏浓缩溶剂，加入吡啶溶解残留固体，抽滤，减压浓缩得白色固体，产率90％。
1H NMR(600MHz，DMSO-d6)δppm 0.57(s，3H)1.07(s，3H)1.12-1.14(m，1H)1.14-1.16(m，1H)1.17(s，3H)1.21(s，3H)1.22-1.23(m，2H)1.24-1.28(m，4H)1.28-1.32(m，2H)1.38(s，3H)1.40-1.46(m，2H)1.47-1.52(m，2H)1.57(d，J＝6.79Hz，1H)1.59-1.63(m，4H)1.64-1.72(m，6H)1.75-1.83(m，4H)1.92-1.96(m，1H)1.97-2.01(m，1H)2.03-2.07(m，1H)2.09(s，3H)2.18(d，J＝10.82Hz，2H)2.73(d，J＝15.04Hz，1H)3.40-3.49(m，1H)3.80-3.89(m，1H)6.36(d，J＝7.34Hz，1H)6.40(d，J＝0.92Hz，1H)7.07(dd，J＝6.97，0.92Hz，1H)7.73(d，J＝8.07Hz，1H)8.71(s，1H).
13C NMR(151MHz，DMSO-d6)δppm 10.08，18.32，21.83，24.68，24.75，25.13，25.29，25.63，25.67，28.38，28.59，29.91，30.13，31.02，31.08，31.39，31.82，31.91，31.93，32.36，33.10，36.02，36.13，37.67，38.99，42.16，42.81，44.45，44.49，50.05，54.77，117.23，117.93，120.04，126.75，133.25，146.43，153.97，163.09，168.74，175.44，177.83.
实施例25.化合物XS0536的制备
Figure PCTCN2017091726-appb-000066
称取XS0077(150mg，0.32mmol)于厚壁耐压管中，加入碳酸钾(220mg，1.6mmol)，加入4ml丙酮室温下搅拌溶解样品，再加入100μL硫酸二甲酯溶液，转移至70℃油浴加热8h。加入1mol/L HCl淬灭，加水，乙酸乙酯萃取3次，无水硫酸钠干燥，旋转蒸发仪浓缩溶剂，用乙酸乙酯∶正己烷＝1∶20体系经硅胶柱层析分离纯化得到纯品，白色固体，产率为21％。
1H NMR(600MHz，CHLOROFORM-d)d ppm 0.61(s，3H)0.94(d，J＝13.75Hz，1H)1.08(s，3H)1.17(s，3H)1.22(s，3H)1.34(s，3H)1.40(dd，J＝14.12，4.22Hz，1H)1.44(dd，J＝14.40，2.84Hz，1H)1.51-1.57(m，2H)1.57-1.63(m，1H)1.63-1.69(m，2H)1.73(d，J＝11.37Hz，1H)1.85(td，J＝13.71，6.33Hz，1H)2.01-2.07(m，1H)2.07-2.13(m，2H)2.17(s，3H)2.18-2.23(m，1H)2.44(d，J＝15.59Hz，1H)3.02(d，J＝19.99Hz，1H)3.28(dd，J＝20.00，5.87Hz，1H)3.54(s，3H)3.77(s，3H)3.87(s，3H)5.77(d，J＝4.58Hz，1H)6.78(s，1H).
13C NMR(151MHz，CHLOROFORM-d)d ppm 11.77，18.28，22.73，27.85，28.85，29.87，30.29，30.50，30.85，31.55，32.88，34.16，34.51，34.79，36.83，37.15，37.52，40.45，43.69，44.33，51.47，55.88，60.30，106.30，117.63，125.49，127.80，144.62，144.80，149.13，150.89，179.05.
实施例26.化合物XS0286的制备
Figure PCTCN2017091726-appb-000067
在搅拌条件下，将化合物YXY101(50mg，0.11mmol)溶于N，N-二甲基甲酰胺(2mL)。加入催化剂EDCI(85.4mg，0.55mmol)和HOBT(74.3mg，0.55mmol)，搅拌使其溶解；加入对叔丁基苯胺(49.2mg，0.33mmol)，并在室温下搅拌反应36小时。停止反应，向反应体系中加入去离子水(15mL)，并用乙酸乙酯萃取3次。 合并乙酸乙酯层，然后用饱和NaCl洗3次，用无水Na2SO4干燥，用旋转蒸发仪浓缩得粗产物(暗红色油状物)。粗产物经快速柱层析法(乙酸乙酯∶正己烷)分离纯化，真空干燥得呈暗红色固体的化合物XS0286(10mg)。
1H NMR(600MHz，CHLOROFORM-d)δppm 0.64(s，3H)1.08(d，J＝13.20Hz，1H)1.15(s，3H)1.24-1.27(m，6H)1.40-1.42(m，4H)1.48-1.57(m，3H)1.63(m，5H)1.65-1.79(m，5H)1.85(d，J＝11.92Hz，1H)1.91(d，J＝6.24Hz，1H)1.98-2.06(m，3H)2.08(d，J＝12.10Hz，2H)2.18(d，J＝1.65Hz，3H)2.54(d，J＝15.77Hz，1H)6.29(d，J＝6.79Hz，1H)6.45(s，1H)6.92-7.00(m，2H)7.30-7.34(m，2H)7.34-7.37(m，2H)7.37-7.41(m，1H).
13C NMR(151MHz，CHLOROFORM-d)δppm 10.21，18.49，21.75，28.58，29.63，29.69，30.41，30.83，31.33，33.24，33.73，34.35，34.98，36.32，38.06，39.38，41.10，42.96，44.47，45.11，76.81，77.02，77.23，116.99，117.95，119.53，119.81，125.89，127.38，133.95，135.11，146.01，147.26，164.68，170.02，175.83，178.38.
实施例27.化合物XS0287的制备
Figure PCTCN2017091726-appb-000068
在搅拌条件下，将化合物YXY101(50mg，0.11mmol)溶于N，N-二甲基甲酰胺(2mL)。加入催化剂EDCI(85.4mg，0.55mmol)和HOBT(74.3mg，0.55mmol)，搅拌使其溶解；加入对甲氧基苯胺(40.6mg，0.33mmol)，并在室温下搅拌反应36小时。停止反应，向反应体系中加入去离子水(15mL)，并用乙酸乙酯萃取3次。合并乙酸乙酯层，然后用饱和NaCl洗3次，用无水Na2SO4干燥，用旋转蒸发仪浓缩得粗产物(暗红色油状物)。粗产物经快速柱层析法(乙酸乙酯∶正己烷)分离纯化，真空干燥得呈暗红色固体的化合物XS-0287(20mg)。
1H NMR(600MHz，CHLOROFORM-d)δppm 0.53(s，3H)1.05(d，J＝13.94Hz，1H)1.13(s，3H)1.21(s，3H)1.24(s，3H)1.26(s，3H)1.35(s，3H)1.48-1.54(m，2H)1.58(d，J＝7.34Hz，2H)1.64-1.72(m，2H)1.78-1.89(m，3H)2.03-2.11(m，2H)2.18(s，3H)2.55(d，J＝15.77Hz，1H)3.79(s，3H)6.25(d，J＝7.15Hz，1H)6.34(s，1H)6.91(m，J＝8.99Hz，2H)6.96(d，J＝7.15Hz，1H)7.40(m，J＝8.80Hz，2H)7.48(s，1H).
13C NMR(151MHz，CHLOROFORM-d)δppm 10.23，18.30，21.75，28.46，29.39，29.70，30.30，30.79，31.52，32.86，33.81，34.89，36.21，37.99，39.28，40.96，42.96，44.43，45.12，55.48，114.21，117.18，117.82，119.38，121.99，127.34，130.98，134.06，146.06，156.41，164.80，170.23，175.88，178.42.
实施例28.化合物XS0394的制备
Figure PCTCN2017091726-appb-000069
在搅拌条件下，将化合物YXY101(50mg，0.11mmol)和三乙胺(37μL)溶于重蒸四氢呋喃中(3.0mL，0.26mmol)，然后冷却至低温下-30℃，10分钟后加入逐滴加入氯甲酸乙酯(22μL，0.23mmol)加入至反应液中。-30℃搅拌反应12小时后，停止反应，砂芯漏斗过滤除去不溶物，四氢呋喃洗涤，得黄色滤液。向黄色滤液中加入10mL纯水，用乙酸乙酯萃取3次(每次15mL)，合并有机层，将有机层用饱和食盐水洗三次(每次30mL)，有机相无水硫酸钠干燥后，用减压蒸馏法除去有机溶剂，得到橘黄色油状液体混合粗品。以正己烷和乙酸乙酯(hexane/ethyl acetate＝10∶1)为洗脱剂，300-400目硅胶填充柱层析分离，得到黄色固体产物。
1H NMR(600MHz，DMSO-d6)ppm 1.05-1.07(m，1H)1.08(s，3H)1.09-1.12(m，1H)1.18(s，3H)1.23(s，3H)1.27(t，J＝7.06Hz，3H)1.28-1.31(m，1H)1.41(s，3H)1.43-1.50(m，4H)1.53(dd，J＝12.10，5.69Hz，1H)1.59(d，J＝7.89Hz，1H)1.65(d，J＝10.64Hz， 2H)1.70-1.78(m，2H)1.79-1.85(m，1H)1.90(td，J＝13.98，3.39Hz，1H)1.95-2.01(m，1H)2.01-2.06(m，1H)2.15(s，3H)2.26(d，J＝15.22Hz，1H)4.21(q，J＝1.00Hz，2H)6.30(d，J＝7.15Hz，1H)6.40(d，J＝0.73Hz，1H)7.29(dd，J＝7.52，0.73Hz，1H)12.07(br.s，1H).
13C NMR(151MHz，CHLOROFORM-d)ppm 11.17，14.09，19.00，21.96，28.60，29.35，29.36，30.47，30.67，31.49，32.67，33.38，34.53，36.20，38.24，39.13，40.16，42.88，44.05，45.34，65.05，117.76，117.78，122.70，126.08，133.37，142.54，152.62，163.30，172.96，183.86.183.90.
实施例29.化合物XS0418的制备
Figure PCTCN2017091726-appb-000070
首先将化合物YXY101(50mg，0.11mmol)搅拌溶解于2mL四氢呋喃中，随后加入碳酸钾(15.2mg，0.11mmol)，溴乙酸乙酯(18.37mg，0.11mmol)溶于1mL四氢呋喃中，然后逐滴加入反应液，室温搅拌反应4小时。1mol/L HCl(1mL)淬灭反应，加入9mL纯水，用乙酸乙酯萃取3次(每次15mL)，收集有机层，无水硫酸钠干燥后，用减压蒸馏法除去有机溶剂乙酸乙酯，得到橘红色固体混合粗品。以正己烷和乙酸乙酯(hexane/ethyl acetate＝10∶1)为洗脱剂，300-400目硅胶填充柱层析分离，得到橘红色固体产物。
1H NMR(600MHz，CHLOROFORM-d)ppm 0.53(s，3H)0.99(d，J＝14.31Hz，1H)1.11(s，3H)1.25(t，J＝7.20Hz，3H)1.27(s，3H)1.28(s，3H)1.39-1.44(m，1H)1.45(s，3H)1.51(dd，J＝14.95，4.13Hz，1H)1.54-1.58(m，1H)1.60(d，J＝7.89Hz，1H)1.63-1.67(m，1H)1.67-1.71(m，1H)1.75(dd，J＝15.96，8.07Hz，1H)1.80-1.82(m，1H)1.83-1.85(m，1H)1.85-1.92(m，1H)2.06(td，J＝14.12，3.85Hz，1H)2.13-2.18(m，1H)2.21 (s，3H)2.25(d，J＝14.31Hz，1H)2.48(d，J＝15.96Hz，1H)4.19(q，J＝7.15Hz，2H)4.41(d，J＝15.77Hz，1H)4.54(d，J＝15.96Hz，1H)6.35(d，J＝7.15Hz，1H)6.53(d，J＝1.10Hz，1H)7.02(dd，J＝7.06，1.19Hz，1H)
13C NMR(151MHz，CHLOROFORM-d)ppm 10.23，14.04，18.57，21.61，28.59，29.57，29.74，30.47，30.63，31.55，32.56，33.50，34.67，36.31，38.24，39.39，40.40，42.91，44.19，45.01，60.52，61.28，117.15，118.13，119.52，127.37，134.09，145.98，164.74，167.70，170.03，177.53，178.31
实施例30.化合物XS0421，XS0457，XS0473和XS0493的制备
Figure PCTCN2017091726-appb-000071
以XS0421合成为例：称取化合物YXY101(50mg，0.11mmol)于50ml密封管中，加入亚磷酸二甲酯(123mg，1.1mmol)、六水合氯化铝2.7mg(0.1eq)，加入2mL DCM溶解，密封，室温条件下反应6h。反应停止时，加入饱和NaCl淬灭，乙酸乙酯萃取三次，合并有机相，无水Na2SO4干燥，将有机相减压浓缩溶剂，用乙酸乙酯∶正己烷＝1∶2体系经硅胶柱层析分离纯化，得白色固体，产率55％。
1H NMR(600MHz，DMSO-d6)ppm 0.61(3H，s)0.86(1H，d，J＝12.84Hz)1.06(3H，s)1.10(3H，s)1.19(3H，s)1.22-1.27(1H，m)1.27-1.32(1H，m)1.40(1H，d，J＝4.95Hz)1.43(1H，d，J＝5.32Hz)1.49(1H，d，J＝8.07Hz)1.51-1.55(1H，m)1.57(3H，s)1.59-1.60(1H，m)1.60-1.64(1H，m)1.76-1.85(1H，m)1.93-1.97(1H，m)1.99(1H，d，J＝2.20Hz)2.00-2.02(1H，m)2.02-2.06(1H，m)2.13(3H，s)2.32(1H，d，J＝15.59Hz)3.49(3H，d，J＝8.80Hz)3.51(3H，d，J＝8.99Hz)4.19(1H，dd，J＝23.66，6.24Hz)5.64(1H， dd，J＝6.33，3.21Hz)6.66(1H，s)7.97(1H，s)9.04(1H，br.s.)12.04(1H，s).
13C NMR(151MHz，DMSO-d6)ppm 12.60，14.00(1C，s)18.04，21.58(1C，d，J＝6.60Hz)22.11，28.78，29.48，30.02(1C，d，J＝13.20Hz)30.18，31.00，31.45，32.42，33.49，33.57，34.57，36.42，37.43，37.89，38.79，43.88，52.31(1C，d，J＝6.60Hz)52.81(1C，d，J＝6.60Hz)109.32，114.64(1C，d，J＝12.10Hz)117.90(1C，d，J＝7.70Hz)121.35(1C，d，J＝4.40Hz)140.50(1C，d，J＝6.60Hz)141.05(1C，d，J＝3.30Hz)144.08(1C，d，J＝3.30Hz)150.62(1C，d，J＝12.10Hz)179.51.
根据上述制备方法，本发明还合成了以下化合物：
Figure PCTCN2017091726-appb-000072
实施例31.化合物XS0439，XS0442，XS0444-XS0449，XS0478-XS0480，XS0487和XS0490的制备
Figure PCTCN2017091726-appb-000073
制备方法以XS0439为例：在搅拌条件下，将化合物YXY101(100mg，0.22mmol)溶于二氯甲烷(4mL)。加入7-甲氧基取代吲哚(65.3mg，0.44mmol)，再加入六水合三氯化铝(5.3mg，0.022mmol)，并在室温下搅拌反应5小时。停止反应，向反应体系中加入去离子水(15mL)，并用乙酸乙酯萃取3次。合并乙酸乙酯层，然后用饱和NaCl洗3次，用无水Na2SO4干燥，用旋转蒸发仪浓缩得粗产物(棕色油状物)。粗产物经快速柱层析法(乙酸乙酯∶正己烷＝1∶4)分离纯化，真空干燥得呈紫红色固体的化合物。
1H-NMR(DMSO-d6)δppm 0.71(s，3H)，0.86(d，J＝9.9Hz，1H)，0.96(s，3H)，1.01(s，3H)，1.10(s，3H)，1.22-1.29(m，2H)，1.32(s，3H)，1.34-1.40(m，2H)，1.45(d，J＝8.1Hz，1H)，1.50-1.61(m，3H)，1.62-1.75(m，2H)，1.79(s，3H)，1.96-2.08(m，3H)，2.34(d，J＝15.4Hz，1H)，3.88(s，3H)，4.79(d，J＝5.9Hz，1H)，6.12(d，J＝6.2Hz，1H)，6.18(s，1H)，6.62(d，J＝7.7Hz，1H)，6.73(s，1H)，6.90(t，J＝7.9Hz，1H)，7.22(d，J＝7.9Hz，1H)，7.88(br.s.，1H)，8.99(br.s.，1H)，10.69(s，1H)，12.04(br.s.，1H).
13C-NMR(DMSO-d6)δppm 11.9，18.5，22.3，29.1，29.9，30.4，30.5，30.6，31.8，32.9，34.9，35.4，35.5，35.6，36.8，36.9，37.8，43.5，44.3，55.4，101.8，108.8，112.2，119.2，119.8，121.1，121.6，122.6，126.3，126.7，128.5，140.8，141.4，144.0，146.6，147.0，180.0.
实施例32.化合物XS0486，XS0491和XS0492的制备
Figure PCTCN2017091726-appb-000074
以化合物XS0486为例：首先将化合物YXY101(50mg，0.11mmol)溶于二氧六环(600μL)中，加入三乙胺(150μL，0.33mmol)，再加入二氧六环(100μL)洗涤反应瓶壁上残留，冷却至0℃，然后将亚磷酸二甲酯(110mg，1.1mmol)溶解于50μL四氯化碳中，在将溶解后的亚磷酸二甲酯慢慢滴入到溶有雷公藤红素的反应液中，0℃搅拌反应12h，加入10mL冰冷的去离子水，10mL饱和氯化铵溶液猝灭反应，用乙酸乙酯萃取3次(每次15mL)，收集有机层，无水硫酸钠干燥后，用减压蒸馏法除去有机溶剂乙酸乙酯，得到红色固体混合粗品。以正己烷和乙酸乙酯(hexane/ethyl acetate＝1∶1)为洗脱剂，300-400目硅胶填充柱层析快速分离，得到红色固体产物。
1H NMR(600MHz，DMSO-d6)δppm 0.60(s，3H)0.96(d，J＝13.57Hz，1H)1.08(s，3H)1.23(s，3H)1.24(s，3H)1.38(s，3H)1.41-1.48(m，2H)1.55-1.60(m，3H)1.61-1.67(m，2H)1.69-1.74(m，1H)1.78(dd，J＝16.14，7.89Hz，1H)1.81-1.88(m，1H)1.95(td， J＝14.12，3.85Hz，1H)2.02(d，J＝13.94Hz，1H)2.09(s，3H)2.21-2.24(m，1H)2.27(d，J＝15.96Hz，1H)3.74(dd，J＝11.55，5.14Hz，6H)6.36(d，J＝7.15Hz，1H)6.39(d，J＝1.28Hz，1H)7.08(dd，J＝6.97，1.10Hz，1H)8.75(s，1H).
13C NMR(151MHz，DMSO-d6)δppm 10.07，18.53，21.43，28.03，29.09，29.24，29.71，30.07，31.21，31.46，32.76，33.92，35.85，37.71，38.78，41.23(d，J＝5.50Hz，)41.97，43.37，44.45，55.15(dd，J＝17.61，5.50Hz，2C)117.24，118.13，120.12，126.89，133.12，146.44，162.82，167.43，172.21(d，J＝11.00Hz)177.98.
实施例33.化合物XS0488的制备
Figure PCTCN2017091726-appb-000075
首先将化合物XS0077(80mg，0.17mmol)溶于四氢呋喃(4mL)中，加入三乙胺(370μL，2.5mmol)，再加入4-DMAP(24.7mg，0.22mmol)，搅拌均匀，加入正丁酰氯(113μL，1.1mmol)，室温搅拌反应30min，加入10mL饱和氯化铵溶液猝灭反应，用乙酸乙酯萃取3次(每次15mL)，收集有机层，无水硫酸钠干燥后，用减压蒸馏法除去有机溶剂乙酸乙酯，得到红色固体混合粗品。以正己烷和乙酸乙酯(hexane/ethyl acetate＝4∶1)为洗脱剂，300-400目硅胶填充柱层析快速分离，得到明黄色固体产物。
1H NMR(600MHz，DMSO-d6)ppm 0.46-0.51(m，3H)0.91(d，J＝1.00Hz，1H)0.95-1.01(m，3H)1.08(s，3H)1.12(s，3H)1.23(s，3H)1.36(ddd，J＝13.80，4.20Hz，1H)1.42(s，3H)1.44-1.48(m，1H)1.54-1.60(m，3H)1.63(s，2H)1.66-1.68(m，2H)1.70(d，J＝9.17Hz，2H)1.83(dd，J＝13.57，7.70Hz，1H)1.93-1.99(m，1H)2.06(d，J＝13.94Hz，1H)2.10(s，3H)2.22(d，J＝7.34Hz，1H)2.32(d，J＝15.59Hz，1H)2.55(t，J＝7.15Hz，2H)3.49(s，3H)6.38(s，1H)6.41(d，J＝7.15Hz，1H)7.31(d，J＝6.97Hz，1H).
13C NMR(151MHz，DMSO-d6)ppm 13.41，18.05，21.59，28.06，29.11，29.42，30.11， 30.16，30.32，31.32，32.21，32.31，33.06，34.36，34.91，36.00，37.90，38.77，39.83，42.22，43.64，44.85，51.48，117.99，122.13，125.21，133.52，136.69，142.26，162.81，170.65，171.21，176.21，177.91.
实施例34.化合物XS0506的制备
Figure PCTCN2017091726-appb-000076
称取化合物YXY101(50mg，0.11mmol)于50ml圆底瓶中，加入二环己基碳二亚胺(23mg，0.11mmol)、葡萄糖(24mg，0.11mmol)，加入2ml DCM溶解，室温反应12h。加入大量水淬灭，乙酸乙酯萃取三次，合并有机相，无水Na2SO4干燥，将有机相减压浓缩溶剂，用乙酸乙酯∶正己烷＝1∶4体系经硅胶柱层析分离纯化，得橙红色固体，产率42％。
1H NMR(600MHz，DMSO-d6)δppm 0.57(s，3H)1.07(s，3H)1.12-1.14(m，1H)1.14-1.16(m，1H)1.17(s，3H)1.21(s，3H)1.22-1.23(m，2H)1.24-1.28(m，4H)1.28-1.32(m，2H)1.38(s，3H)1.40-1.46(m，2H)1.47-1.52(m，2H)1.57(d，J＝6.79Hz，1H)1.59-1.63(m，4H)1.64-1.72(m，6H)1.75-1.83(m，4H)1.92-1.96(m，1H)1.97-2.01(m，1H)2.03-2.07(m，1H)2.09(s，3H)2.18(d，J＝10.82Hz，2H)2.73(d，J＝15.04Hz，1H)3.40-3.49(m，1H)3.80-3.89(m，1H)6.36(d，J＝7.34Hz，1H)6.40(d，J＝0.92Hz，1H)7.07(dd，J＝6.97，0.92Hz，1H)7.73(d，J＝8.07Hz，1H)8.71(s，1H).
13C NMR(151MHz，DMSO-d6)δppm 10.08，18.32，21.83，24.68，24.75，25.13，25.29，25.63，25.67，28.38，28.59，29.91，30.13，31.02，31.08，31.39，31.82，31.91，31.93，32.36，33.10，36.02，36.13，37.67，38.99，42.16，42.81，44.45，44.49，50.05，54.77，117.23，117.93， 120.04，126.75，133.25，146.43，153.97，163.09，168.74，175.44，177.83.
实施例35.化合物XS0507的制备
Figure PCTCN2017091726-appb-000077
称取化合物YXY101(50mg，0.11mmol)于50ml圆底瓶中，加入2ml DCM溶解，转移至-78℃搅拌，加入DAST(150ul，10eq)，-78℃反应1h。将反应液直接倒入大量冰中停止反应，加入DCM萃取水相三次，合并有机相，无水Na2SO4干燥，将有机相减压浓缩溶剂，用乙酸乙酯∶正己烷＝1∶4体系经硅胶柱层析分离纯化，得橙红色固体，产率58％。
1H NMR(600MHz，DMSO-d6)δppm 0.56(s，3H)0.98(d，J＝11.92Hz，1H)1.08(s，3H)1.22(s，3H)1.29(s，3H)1.38(s，3H)1.43-1.47(m，1H)1.48-1.52(m，1H)1.55-1.59(m，2H)1.60-1.64(m，1H)1.67(d，J＝4.03Hz，1H)1.71(d，J＝3.85Hz，1H)1.80(dd，J＝16.41，8.16Hz，1H)1.83-1.87(m，1H)1.88-1.92(m，1H)1.96(d，J＝17.61Hz，1H)1.95-1.95(m，1H)2.09(s，3H)2.20(d，J＝1.83Hz，2H)6.35(d，J＝7.34Hz，1H)6.38(d，J＝1.28Hz，1H)7.06(dd，J＝6.97，1.28Hz，1H)8.73(s，1H).
13C NMR(151MHz，DMSO-d6)δppm 10.07，18.85，21.41，27.89，29.26，29.33，29.46，29.94，31.16，32.71，33.79，35.78，37.87，38.74，40.04，40.22，41.89，43.26，44.33，117.21，118.14，120.15，126.99，132.88，146.41，162.78，167.39(d，J＝418.15Hz，1C)，166.71，177.97.
实施例36.化合物XS0509的制备
Figure PCTCN2017091726-appb-000078
称取化合物YXY101(50mg，0.11mmol)于25ml厚壁耐压管中，加入四丁基溴化铵(Tetrabutylammonium bromide，TBAB，17.5mg，0.05mmol)，加入2ml二氯甲烷(dichloromethane，DCM)溶解，再逐滴加入5％NaOH(180μl)，室温条件下反应30min，再转移至50℃油浴下，逐滴加入2，3，4，6-四乙酰氧基-α-D吡喃葡萄糖溴化物-二氯甲烷溶液(57mg-1ml，0.138mmol)，50℃反应12h，停止反应，加入大量水和饱和食盐水，加入DCM萃取3次，合并有机相，无水Na2SO4干燥，将有机相减压浓缩，用乙酸乙酯∶正己烷＝4∶1体系经硅胶层析柱进行纯化，得黄色固体，产率25％。
1H NMR(600MHz，DMSO-d6)δppm 0.51(s，3H)0.95(d，J＝13.57Hz，1H)1.08(d，J＝6.24Hz，6H)1.22(s，3H)1.23(br.s.，1H)1.38(s，3H)1.41(d，J＝4.22Hz，1H)1.46(d，J＝11.92Hz，1H)1.56(d，J＝7.52Hz，2H)1.58(s，3H)1.60-1.63(m，1H)1.64(d，J＝4.95Hz，2H)1.66-1.69(m，1H)1.81-1.87(m，1H)1.91(s，3H)1.94(d，J＝3.67Hz，1H)1.97(s，3H)2.00-2.04(m，1H)2.08(s，3H)2.12(s，3H)2.19(d，J＝8.80Hz，1H)2.31(d，J＝15.59Hz，1H)3.85-3.90(m，1H)3.92-3.96(m，1H)4.30(dd，J＝8.16，4.13Hz，1H)5.05(dd，J＝10.09，8.44Hz，1H)5.23(d，J＝3.48Hz，1H)5.29(dd，J＝10.45，3.67Hz，1H)5.83(d，J＝8.25Hz，1H)6.36(d，J＝7.15Hz，1H)6.37(d，J＝0.92Hz，1H)7.06(d，J＝6.97Hz，1H)8.71(s，1H).
13C NMR(151MHz，DMSO-d6)δppm 10.06，18.63，19.97，20.31，20.42，21.57，28.11，28.59，29.22，29.97，30.07，31.31，32.62，32.84，34.57，35.93，37.76，38.52，40.05，40.24，41.97，43.46，44.54，61.76，67.35，67.85，69.78，71.21，91.64，117.29，118.08，120.11，126.93，133.07，146.36，162.86，167.78，169.24，169.41，169.57，169.97，176.51，177.93.
实施例37.化合物XS0514，XS0515的制备
Figure PCTCN2017091726-appb-000079
以合成XS0514为例：称取XS0419(50mg，0.11mmol)于50ml圆底瓶中，加入2ml DCM溶解，转移至-78℃搅拌，加入二乙胺基三氟化硫(Diethylaminosulfurtrifluoride，DAST，150ul，10eq)，-78℃反应1h。将反应液直接倒入大量冰中停止反应，加入DCM萃取水相三次，合并有机相，用饱和NaHCO3洗涤有机相，无水Na2SO4干燥有机相，将有机相减压浓缩，用乙酸乙酯∶正己烷＝1∶8体系经硅胶柱层析进行分离纯化，得橙红色固体，产率58％。
1H NMR(600MHz，DMSO-d6)δppm 0.61(s，3H)0.95(d，J＝14.49Hz，1H)1.06(s，3H)1.18(s，3H)1.23(s，3H)1.28(s，3H)1.41(d，J＝14.86Hz，2H)1.47(dd，J＝13.85，4.31Hz，2H)1.54(d，J＝7.70Hz，1H)1.58-1.63(m，2H)1.74-1.84(m，2H)1.88(d，J＝6.05Hz，1H)1.94(d，J＝10.64Hz，1H)1.98(d，J＝10.09Hz，1H)2.01(s，3H)2.07(d，J＝17.97Hz，1H)2.24(d，J＝15.96Hz，1H)2.89-2.94(m，1H)3.16-3.21(m，1H)5.74(d，J＝4.77Hz，1H)6.61(s，1H)7.81(br.s.，1H)8.78(br.s.，1H).
13C NMR(151MHz，DMSO-d6)δppm 11.56，18.66，22.57，27.30，28.27，29.21，29.54，29.98，30.02，30.08，31.18，33.69，33.99，34.13，36.05，36.27，37.14，40.25，43.12，43.42，108.21，118.07，120.13，123.02，139.23，140.61，143.17，148.76，167.64(d，J＝380.73Hz，1C).
实施例38.化合物XS0516的制备
Figure PCTCN2017091726-appb-000080
称取XS0077(50mg，0.11mmol)于25ml厚壁耐压管中，加入四丁基溴化铵(Tetrabutylammonium bromide，TBAB，17.5mg，0.05mmol)，加入2ml二氯甲烷(dichloromethane，DCM)溶解，再逐滴加入5％NaOH(180μL)，室温条件下反应30min，再转移至50℃油浴下，逐滴加入2，3，4，6-四乙酰氧基-α-D吡喃葡萄糖溴化物-二氯甲烷溶液(57mg-1ml，0.138mmol)，50℃反应12h，停止反应，加入大量水和饱和食盐水，加入DCM萃取3次，合并有机相，无水Na2SO4干燥，将有机相减压浓缩，用乙酸乙酯∶正己烷＝4∶1体系经硅胶层析柱进行纯化，得黄色固体，产率25％。
1H NMR(600MHz，CHLOROFORM-d)δppm 0.53(s，3H)0.97(d，J＝14.31Hz，1H)1.10(s，3H)1.18(s，3H)1.26(s，3H)1.38(td，J＝14.03，4.58Hz，1H)1.47(s，3H)1.50(dd，J＝15.59，4.58Hz，1H)1.53-1.56(m，1H)1.58(d，J＝8.25Hz，1H)1.62-1.65(m，2H)1.66(d，J＝5.32Hz，1H)1.67-1.72(m，2H)1.76-1.80(m，1H)1.82-1.87(m，1H)1.87-1.91(m，1H)1.98(s，3H)2.01(s，3H)2.10-2.14(m，1H)2.15(s，3H)2.18(s，3H)2.23(s，3H)2.42(d，J＝15.77Hz，1H)3.55(s，3H)3.86(t，J＝7.34Hz，1H)4.06(dd，J＝11.10，7.61Hz，1H)4.16(dd，J＝11.10，6.14Hz，1H)5.11(dd，J＝10.36，3.58Hz，1H)5.31(d，J＝7.89Hz，1H)5.38-5.43(m，2H)6.31(d，J＝7.15Hz，1H)6.39(d，J＝1.10Hz，1H)7.02(dd，J＝7.06，1.01Hz，1H).
13C NMR(151MHz，CHLOROFORM-d)δppm 11.54，18.33，20.61，20.70，21.02，21.86，28.57，29.57，29.85，30.52，30.82，31.55，32.65，33.67，34.70，36.34，38.20，39.24，40.37，42.30，44.27，45.07，51.54，60.81，66.90，69.09，70.49，70.84，100.37，117.93，123.33，126.92，134.32，134.96，145.71，162.50，170.07，170.33，170.35，170.37，170.61，178.68，179.14.
实施例39.化合物XS0534的制备
Figure PCTCN2017091726-appb-000081
称取XS0077(150mg，0.32mmol)溶于4mL丙酮中，搅拌溶解，加入无水碳酸钾(180mg，1.3mmol)，搅拌加入硫酸二甲酯(95μL，0.96mmol)，油浴70℃反应12h；用1mol/L的HCl猝灭反应，并调节PH＝7，乙酸乙酯萃取三次，合并有机相，无水Na2SO4干燥，将有机相减压浓缩溶剂，用乙酸乙酯∶正己烷＝1∶10体系经硅胶柱层析分离纯化，得白色固体，产率24.3％。
1H NMR(600MHz，DMSO-d6)δppm 0.51(s，3H)0.87-0.92(m，1H)1.06(s，3H)1.12(s，3H)1.18(s，3H)1.27(s，3H)1.32-1.37(m，1H)1.37-1.42(m，1H)1.47-1.52(m，3H)1.55-1.61(m，2H)1.65(dd，J＝15.68，8.16Hz，1H)1.80(td，J＝13.43，7.24Hz，1H)1.84-1.90(m，1H)1.97(td，J＝13.75，4.03Hz，1H)2.02(s，3H)2.06(d，J＝13.57Hz，1H)2.12-2.18(m，1H)2.33(d，J＝15.77Hz，1H)2.95(dd，J＝20.17，1.40Hz，1H)3.21(dd，J＝20.72，6.24Hz，1H)3.46-3.50(m，3H)3.77(s，3H)5.72(dd，J＝6.42，1.28Hz，1H)6.72(s，1H)8.10(s，1H).
13C NMR(151MHz，DMSO-d6)δppm 11.35，17.69，22.41，27.31，28.41，29.42，29.90，30.13，30.25，31.36，32.37，34.01，34.21，34.36，36.41，36.49，37.11，39.85，43.23，43.80，51.37，55.82，105.29，117.48，119.79，124.58，139.03，141.52，145.83，148.73，178.04.
实施例40.化合物XS0455，XS0504的制备
Figure PCTCN2017091726-appb-000082
以XS0455为例：首先将化合物YXY101(40mg，0.09mmol)搅拌溶解于1.2mL THF中，随后加入钐粉(70mg，0.36mmol)，对甲基苯磺酸(67mg，0.36mmol)，室温搅拌反应4小时。砂芯漏斗过滤催化剂钐粉以及不溶物，用乙酸乙酯洗涤3次(每次10mL)，收集滤液，无水硫酸钠干燥后，用减压蒸馏法除去有机溶剂乙酸乙酯和四氢呋喃，得到黄褐色固体混合粗品。以正己烷和乙酸乙酯(hexane/ethyl acetate＝2∶1)为洗脱剂，300-400目硅胶填充柱层析分离，得到黄褐色固体产物。
1H NMR(600MHz，DMSO-d6)δppm 0.82(1H，d，J＝13.39Hz)0.89(3H，s)0.94(3H，br.s.)0.94(3H，br.s.)1.02(3H，s)1.13(3H，s)1.22(1H，d，J＝15.96Hz)1.29-1.34(1H，m)1.36(1H，dd，J＝13.39，3.12Hz)1.63-1.76(4H，m)1.92(1H，ddd，J＝13.60，4.00Hz)1.96-2.00(1H，m)2.02(1H，d，J＝6.97Hz)2.06(3H，s)2.12(2H，dd，J＝13.66，7.06Hz)5.41(1H，d，J＝5.87Hz)6.21(1H，d，J＝9.90Hz)6.31(1H，d，J＝9.90Hz)6.56(1H，s)8.03(1H，s)9.20(1H，s)12.03(1H，br.s).
13C NMR(151MHz，DMSO-d6)δppm 11.18，18.81，19.25，22.49，23.39，29.25，30.51，30.75，31.24，32.68，32.82，36.79，37.26，38.37，40.04，40.24，43.38，46.31，108.52，119.50，119.71，121.02，122.84，128.56，137.37，142.00，142.94，144.16，179.82.
实施例41.化合物XS0420的制备
Figure PCTCN2017091726-appb-000083
称取化合物YXY101(50mg，0.11mmol)于50ml圆底瓶中，加入PTSA(100mg，过量)，加入甲苯2mL，室温搅拌6h。加入饱和NaHCO3溶液淬灭，加入乙酸乙酯萃取3次，合并有机相，无水Na2SO4干燥，将有机相减压浓缩溶剂，用乙酸乙酯∶正己烷＝1∶4体系经硅胶柱层析分离纯化，得灰白色固体，产率70％。
1H NMR(600MHz，DMSO-d6)δppm 1.03(s，3H)1.06(s，3H)1.07-1.11(m，1H)1.15(s，3H)1.18(s，3H)1.21-1.24(m，1H)1.28-1.32(m，1H)1.37(td，J＝13.75，5.32Hz，1H)1.46(dd，J＝8.62，4.03Hz，2H)1.48-1.50(m，2H)1.51-1.55(m，1H)1.58(dd，J＝12.93，1.93Hz，1H)1.83-1.90(m，3H)2.33(t，J＝1.00Hz，1H)2.36(s，3H)2.38(s，3H)2.80-2.92(m，2H)2.80-2.92(m，2H)7.06(d，J＝8.62Hz，1H)7.18(s，1H)7.49(d，J＝8.44Hz，1H)8.48(br.s.，1H)9.92(s，1H).
13C NMR(151MHz，DMSO-d6)δppm 10.96，19.45，21.73，21.95，23.51，24.03，25.45，30.89，30.96，34.59，35.08，36.41，37.98，38.30，40.71，41.70，43.50，99.01，102.91，115.74，120.73，126.07，127.17，127.30，129.03，132.36，143.60，146.12，175.68.
实施例42.化合物XS0502的制备
Figure PCTCN2017091726-appb-000084
称取化合物XS0077(50mg，0.11mmol)于50ml圆底瓶中，加入SeO2(200mg，过量)，加入二氧六环2mL，55℃搅拌12h。加入去离子水淬灭反应，加入乙酸乙酯萃取3次，合并有机相，无水Na2SO4干燥，将有机相减压浓缩溶剂，用乙酸乙酯∶正己烷＝1∶10体系经硅胶柱层析分离纯化，得灰白色固体，产率16.8％。
1H NMR(600MHz，DMSO-d6)δppm 0.75(s，3H)0.93-0.97(m，1H)1.10(s，3H)1.21(s，3H)1.22(s，3H)1.40(td，J＝14.08，4.13Hz，1H)1.49(dt，J＝14.53，4.65Hz，1H)1.71-1.79(m，2H)1.79-1.82(m，1H)1.82-1.88(m，1H)2.07-2.10(m，1H)2.10-2.12(m，1H)2.13(s，3H)2.29(d，J＝14.31Hz，1H)2.46(t，J＝4.77Hz，1H)2.54(s，3H)3.50(s，3H)6.20(d，J＝9.54Hz，1H)6.45(d，J＝9.54Hz，1H)7.11(s，1H)9.44(s，1H).
13C NMR(151MHz，DMSO-d6)δppm 11.88，17.45，19.70，21.88，26.78，29.04，30.34，30.42，30.47，31.02，34.19，36.47，40.05，40.43，41.84，46.15，51.58，122.97，125.72，125.78，129.24，136.20，139.22，142.11，142.43，144.61，146.30，176.92，178.25，181.38.
尽管本发明的具体实施方式已经得到详细的描述，根据已经公开的所有教导，本领域技术人员可以对本发明技术方案的细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (53)

  1. 如式I所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯的用途，其用作孤儿核受体Nur77的配体，或者用于制备用作孤儿核受体Nur77的配体的药物：
    Figure PCTCN2017091726-appb-100001
    其中，X代表-NH-、-N(R)-、-O-、-CH2-或卤素；其中，当X为卤素时，R1不存在；
    当Y和与之相连的碳原子之间为单键时，Y代表H、卤素、-OR、-SR或-NRR’；当Y和与之相连的碳原子之间为双键时，Y代表O、S或NR；
    R1不存在，或代表H、-PO(OR)2、C1-6烷基、糖基、C1-6烷氧羰基-C1-6烷基、3-8元环烷基-氨酰基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、糖基、C1-6烷氧羰基-C1-6烷基、3-8元环烷基-氨酰基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基、C1-6烷氨基和C1-6烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
    R2代表H、D、-PO(OR)2、-CONH2、-NH2、-NHR、-NRR’、-NHCOR、-NRCOR、-NHCOOR、-NHCONHR、-NHCONRR’、-NRCONHR、-NRCONRR’、-OH、-OR、-OCONHR、-OCONRR’、-SH、-SR、-SOR、-SOOR、-SO2NHR”、硝基、卤素、糖基、氰基、三氟甲基、C1-6烷基、3-8元环烷基、3-8元杂环烷基、芳基、C1-6烷基取代的芳基、6-15元杂芳基、链烯基、炔基、亚磺基、磺酸或磺酸盐；其中所述C1-6烷基、3-8元环烷基、3-8元杂环烷基、芳基、C1-6烷基取代的芳基、6-15元杂芳基、链烯基和炔基未被取 代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：氨基、卤素、羟基、氧基、C1-6烷基、C1-6烷氧基、C1-6烷硫基、C1-6烷酰基、3-8元环烷基(例如环丙基)、氧代3-8元环烷基(例如氧代环丁基)、氰基、三氟甲基、C1-6烷氧羰基、C1-6烷基酰胺基、脲基、氨基甲酸酯基、羧基以及芳基；
    R3和R4各自独立地表示不存在，或代表H、C1-6烷基、C1-6烷酰基、C1-6烷氧羰基、糖基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、C1-6烷酰基、C1-6烷氧羰基、糖基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基、C1-6烷氨基和C1-6烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
    R和R’各自独立地选自H、C1-6烷基、3-8元环烷基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、3-8元环烷基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基和C1-6烷氨基；
    R”代表C1-6烷基或芳基(例如6-10元芳基，优选苯基)；
    式(I)中的虚实双键
    Figure PCTCN2017091726-appb-100002
    代表单键或者双键；优选地，环A包含0、1、2或3个碳碳双键；环B包含0、1或2个碳碳双键。
  2. 权利要求1的用途，其中，Y和与之相连的碳原子之间为双键，且Y代表O。
  3. 权利要求1或2的用途，其中，所述化合物中的X代表-NH-、-N(R)-、-O-、-CH2-或卤素；R代表C1-6烷基或3-8元环烷基(优选环己基)；其中，当X为卤素时，R1不存在；
    优选地，X代表-NH-、-N(R)-、-O-或卤素；R代表环己基。
  4. 权利要求1-3任一项的用途，其中，所述化合物中的R1不存在，或代表氢、C1-4烷基、-PO(OR)2、单糖基、C1-4烷氧羰基-C1-4烷基、3-6元环烷基-氨酰基、芳基-C1-4烷基或芳基；其中，所述C1-4烷基、单糖基、C1-4烷氧羰基-C1-4烷基、3-6元环烷基-氨酰基、芳基-C1-4烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自 下述的取代基取代：卤素、羟基、氨基、C1-4烷基、C1-4烷氧基、C1-4烷氨基和C1-4烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
    R代表C1-4烷基；
    优选地，R1不存在，或代表氢、C1-4烷基、-PO(OR)2、葡萄糖基、C1-2烷氧羰基-C1-2烷基、环己基-氨酰基、苯基-C1-2烷基、萘基-C1-2烷基、苯基或萘基；其中，所述甲基、乙基、葡萄糖基、C1-2烷氧羰基-C1-2烷基、环己基-氨酰基、苯基-C1-2烷基、萘基-C1-2烷基、苯基或萘基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：C1-2烷基、C1-2烷氧基和C1-2烷酰基；
    R代表C1-4烷基；
    优选地，R1不存在，或代表氢、C1-4烷基、-PO(OR)2、苯基-C1-2烷基、C1-2烷氧羰基-C1-2烷基、3-6元环烷基-氨酰基；
    R代表C1-3烷基；
    优选地，R1不存在，或代表氢、甲基、乙基、-PO(OMe)2、-PO(OEt)2、-PO(OiPr)2、2，3，4，6-四乙酰氧基-α-D-吡喃葡萄糖基、EtOCOCH2-、环己基-氨酰基、苄基、甲氧基苯基或叔丁基苯基。
  5. 权利要求1-4任一项的用途，其中，所述化合物中的R2代表H、D、-OH、-PO(OR)2、C1-6烷基、9-15元稠杂芳基或磺酸盐；其中所述C1-6烷基或6-15元杂芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：氨基、卤素、羟基、氧基、C1-6烷基、C1-6烷氧基、C1-6烷酰基、氰基、三氟甲基和羧基；
    R代表H、C1-6烷基或芳基；
    优选地，R2代表H、D、-PO(OR)2、C1-4烷基、9-15元苯并稠杂芳基或磺酸盐；其中，所述C1-4烷基或9-15元苯并稠杂芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：氨基、卤素、羟基、氧基、C1-4烷基、C1-4烷氧基、C1-4烷酰基、氰基、三氟甲基和羧基；
    R代表H、C1-4烷基或苯基；
    优选地，R2代表H、D、-PO(OR)2、C1-4烷基、9-15元苯并稠杂芳基或磺酸盐；其中，所述C1-4烷基和9-15元苯并稠杂芳基未被取代或被一个或多个(例如1、2、3或4 个)选自下述的取代基取代：氨基、卤素、羟基、氧基、C1-4烷基、C1-4烷氧基、C1-4烷酰基、氰基、三氟甲基和羧基；
    R代表H、C1-4烷基或苯基；
    优选地，R2代表H、D、-PO(OR)2、2-氧代苯基、吲哚基或磺酸钠；其中，所述吲哚基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：氨基、氟、氯、溴、羟基、甲基、甲氧基、甲酰基、氰基、三氟甲基和羧基；
    R代表H、甲基、乙基、异丙基或苯基。
  6. 权利要求1-5任一项的用途，其中，所述化合物的7位和8位碳原子之间为碳碳双键。
  7. 权利要求1-6任一项的用途，其中，所述化合物Y和与之相连的碳原子之间为碳碳双键。
  8. 权利要求1-6任一项的用途，其中，所述化合物Y和与之相连的碳原子之间为碳碳单键。
  9. 权利要求1-8任一项的用途，其中，所述化合物具有如下结构：
    Figure PCTCN2017091726-appb-100003
    其中，R3和R4各自独立地代表H、C1-6烷基或C1-6烷酰基；
    优选地，R3和R4各自独立地代表H、C1-4烷基或C1-4烷酰基；
    优选地，R3和R4各自独立地代表H、甲基或丁酰基。
  10. 权利要求1-8任一项的用途，其中，所述化合物具有如下结构：
    Figure PCTCN2017091726-appb-100004
    其中，R4代表H、C1-6烷酰基、C1-6烷氧羰基或被一个或多个(例如1、2、3或4个)C1-6烷酰基取代的单糖基；
    优选地，R4代表H、C1-4烷酰基、C1-4烷氧羰基或被一个或多个(例如1、2、3或4个)C1-4烷酰基取代的葡萄糖糖基；
    优选地，R4代表H或C1-2烷氧羰基；
    优选地，R4代表H、丁酰基、乙氧羰基或2，3，4，6-四乙酰氧基-α-D-吡喃葡萄糖糖基。
  11. 权利要求1的用途，其中，所述化合物选自下列化合物：
    Figure PCTCN2017091726-appb-100005
    Figure PCTCN2017091726-appb-100006
    Figure PCTCN2017091726-appb-100007
    Figure PCTCN2017091726-appb-100008
  12. 权利要求1-11任一项的用途，其中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制Nur77的转录活性；
    优选地，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于抑制Nur77在细胞中的转录活性；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  13. 权利要求1-11任一项的用途，其中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制TNFα在细胞中的生物学效应；
    优选地，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  14. 权利要求1-11任一项的用途，其中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制细胞中mTOR通路(例如下调P-mTOR、p-P70S6K和/或p-S6活性)；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  15. 权利要求1-11任一项的用途，其中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于在有此 需要的受试者中预防或治疗与Nur77相关的疾病；
    优选地，所述与Nur77相关的疾病为癌症(例如三阴乳腺癌)。
  16. 一种抑制孤儿核受体Nur77的转录活性的方法，其包括，将Nur77与如式I所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯相接触的步骤：
    Figure PCTCN2017091726-appb-100009
    其中，X代表-NH-、-N(R)-、-O-、-CH2-或卤素；其中，当X为卤素时，R1不存在；
    当Y和与之相连的碳原子之间为单键时，Y代表H、卤素、-OR、-SR或-NRR’；当Y和与之相连的碳原子之间为双键时，Y代表O、S或NR；
    R1不存在，或代表H、-PO(OR)2、C1-6烷基、糖基、C1-6烷氧羰基-C1-6烷基、3-8元环烷基-氨酰基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、糖基、C1-6烷氧羰基-C1-6烷基、3-8元环烷基-氨酰基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基、C1-6烷氨基和C1-6烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
    R2代表H、D、-PO(OR)2、-CONH2、-NH2、-NHR、-NRR’、-NHCOR、-NRCOR、-NHCOOR、-NHCONHR、-NHCONRR’、-NRCONHR、-NRCONRR’、-OH、-OR、-OCONHR、-OCONRR’、-SH、-SR、-SOR、-SOOR、-SO2NHR”、硝基、卤素、糖基、氰基、三氟甲基、C1-6烷基、3-8元环烷基、3-8元杂环烷基、芳基、C1-6烷基取代的芳基、6-15元杂芳基、链烯基、炔基、亚磺基、磺酸或磺酸盐；其中所述C1-6烷基、3-8元环烷基、3-8元杂环烷基、芳基、C1-6烷基取代的芳基、6-15元杂芳基、链烯基和炔基未被取 代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：氨基、卤素、羟基、氧基、C1-6烷基、C1-6烷氧基、C1-6烷硫基、C1-6烷酰基、3-8元环烷基(例如环丙基)、氧代3-8元环烷基(例如氧代环丁基)、氰基、三氟甲基、C1-6烷氧羰基、C1-6烷基酰胺基、脲基、氨基甲酸酯基、羧基以及芳基；
    R3和R4各自独立地表示不存在，或代表H、C1-6烷基、C1-6烷酰基、C1-6烷氧羰基、糖基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、C1-6烷酰基、C1-6烷氧羰基、糖基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基、C1-6烷氨基和C1-6烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
    R和R’各自独立地选自H、C1-6烷基、3-8元环烷基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、3-8元环烷基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基和C1-6烷氨基；
    R”代表C1-6烷基或芳基(例如6-10元芳基，优选苯基)；
    式(I)中的虚实双键
    Figure PCTCN2017091726-appb-100010
    代表单键或者双键；优选地，环A包含0、1、2或3个碳碳双键；环B包含0、1或2个碳碳双键。
  17. 权利要求16的方法，其中，式I所示的化合物如权利要求2-11任一项中所定义；
    优选地，所述方法用于抑制Nur77在细胞中的转录活性；
    优选地，所述方法包括，给有此需要的细胞施用有效量的所述化合物，从而抑制Nur77在细胞中的转录活性；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  18. 一种抑制TNFα在细胞中的生物学效应的方法，其包括，给有此需要的细胞施用有效量的如式I所示的化合物、其互变异构体、立体异构体或药学上可接受的盐 或酯：
    Figure PCTCN2017091726-appb-100011
    其中，X代表-NH-、-N(R)-、-O-、-CH2-或卤素；其中，当X为卤素时，R1不存在；
    当Y和与之相连的碳原子之间为单键时，Y代表H、卤素、-OR、-SR或-NRR’；当Y和与之相连的碳原子之间为双键时，Y代表O、S或NR；
    R1不存在，或代表H、-PO(OR)2、C1-6烷基、糖基、C1-6烷氧羰基-C1-6烷基、3-8元环烷基-氨酰基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、糖基、C1-6烷氧羰基-C1-6烷基、3-8元环烷基-氨酰基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基、C1-6烷氨基和C1-6烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
    R2代表H、D、-PO(OR)2、-CONH2、-NH2、-NHR、-NRR”、-NHCOR、-NRCOR、-NHCOOR、-NHCONHR、-NHCONRR’、-NRCONHR、-NRCONRR’、-OH、-OR、-OCONHR、-OCONRR’、-SH、-SR、-SOR、-SOOR、-SO2NHR”、硝基、卤素、糖基、氰基、三氟甲基、C1-6烷基、3-8元环烷基、3-8元杂环烷基、芳基、C1-6烷基取代的芳基、6-15元杂芳基、链烯基、炔基、亚磺基、磺酸或磺酸盐；其中所述C1-6烷基、3-8元环烷基、3-8元杂环烷基、芳基、C1-6烷基取代的芳基、6-15元杂芳基、链烯基和炔基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：氨基、卤素、羟基、氧基、C1-6烷基、C1-6烷氧基、C1-6烷硫基、C1-6烷酰基、3-8元环烷基(例如环丙基)、氧代3-8元环烷基(例如氧代环丁基)、氰基、三氟甲基、C1-6烷氧羰基、C1-6烷基酰胺基、脲基、氨基甲酸酯基、羧基以及芳基；
    R3和R4各自独立地表示不存在，或代表H、C1-6烷基、C1-6烷酰基、C1-6烷氧羰基、糖基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、C1-6烷酰基、C1-6烷氧羰基、糖基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基、C1-6烷氨基和C1-6烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
    R和R’各自独立地选自H、C1-6烷基、3-8元环烷基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、3-8元环烷基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基和C1-6烷氨基；
    R”代表C1-6烷基或芳基(例如6-10元芳基，优选苯基)；
    式(I)中的虚实双键
    Figure PCTCN2017091726-appb-100012
    代表单键或者双键；优选地，环A包含0、1、2或3个碳碳双键；环B包含0、1或2个碳碳双键。
  19. 权利要求18的方法，其中，式I所示的化合物如权利要求2-11任一项中所定义；
    优选地，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活；
    优选地，所述方法用于抑制TNFα诱导的IκBα降解；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  20. 一种抑制mTOR信号通路的方法，其包括，给有此需要的细胞施用有效量的如式I所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
    Figure PCTCN2017091726-appb-100013
    其中，X代表-NH-、-N(R)-、-O-、-CH2-或卤素；其中，当X为卤素时，R1不存在；
    当Y和与之相连的碳原子之间为单键时，Y代表H、卤素、-OR、-SR或-NRR’；当Y和与之相连的碳原子之间为双键时，Y代表O、S或NR；
    R1不存在，或代表H、-PO(OR)2、C1-6烷基、糖基、C1-6烷氧羰基-C1-6烷基、3-8元环烷基-氨酰基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、糖基、C1-6烷氧羰基-C1-6烷基、3-8元环烷基-氨酰基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基、C1-6烷氨基和C1-6烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
    R2代表H、D、-PO(OR)2、-CONH2、-NH2、-NHR、-NRR’、-NHCOR、-NRCOR、-NHCOOR、-NHCONHR、-NHCONRR’、-NRCONHR、-NRCONRR’、-OH、-OR、-OCONHR、-OCONRR’、-SH、-SR、-SOR、-SOOR、-SO2NHR”、硝基、卤素、糖基、氰基、三氟甲基、C1-6烷基、3-8元环烷基、3-8元杂环烷基、芳基、C1-6烷基取代的芳基、6-15元杂芳基、链烯基、炔基、亚磺基、磺酸或磺酸盐；其中所述C1-6烷基、3-8元环烷基、3-8元杂环烷基、芳基、C1-6烷基取代的芳基、6-15元杂芳基、链烯基和炔基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：氨基、卤素、羟基、氧基、C1-6烷基、C1-6烷氧基、C1-6烷硫基、C1-6烷酰基、3-8元环烷基(例如环丙基)、氧代3-8元环烷基(例如氧代环丁基)、氰基、三氟甲基、C1-6烷氧羰基、C1-6烷基酰胺基、脲基、氨基甲酸酯基、羧基以及芳基；
    R3和R4各自独立地表示不存在，或代表H、C1-6烷基、C1-6烷酰基、C1-6烷氧羰 基、糖基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、C1-6烷酰基、C1-6烷氧羰基、糖基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基、C1-6烷氨基和C1-6烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
    R和R’各自独立地选自H、C1-6烷基、3-8元环烷基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、3-8元环烷基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基和C1-6烷氨基；
    R”代表C1-6烷基或芳基(例如6-10元芳基，优选苯基)；
    式(I)中的虚实双键
    Figure PCTCN2017091726-appb-100014
    代表单键或者双键；优选地，环A包含0、1、2或3个碳碳双键；环B包含0、1或2个碳碳双键。
  21. 权利要求20的方法，其中，式I所示的化合物如权利要求2-11任一项中所定义；
    优选地，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于下调细胞中P-mTOR、p-P70S6K和/或p-S6的活性；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  22. 一种预防或治疗与Nur77相关的疾病的方法，其包括，给有此需要的受试者施用有效量的如式I所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
    Figure PCTCN2017091726-appb-100015
    其中，X代表-NH-、-N(R)-、-O-、-CH2-或卤素；其中，当X为卤素时，R1不存在；
    当Y和与之相连的碳原子之间为单键时，Y代表H、卤素、-OR、-SR或-NRR’；当Y和与之相连的碳原子之间为双键时，Y代表O、S或NR；
    R1不存在，或代表H、-PO(OR)2、C1-6烷基、糖基、C1-6烷氧羰基-C1-6烷基、3-8元环烷基-氨酰基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、糖基、C1-6烷氧羰基-C1-6烷基、3-8元环烷基-氨酰基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基、C1-6烷氨基和C1-6烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
    R2代表H、D、-PO(OR)2、-CONH2、-NH2、-NHR、-NRR’、-NHCOR、-NRCOR、-NHCOOR、-NHCONHR、-NHCONRR’、-NRCONHR、-NRCONRR’、-OH、-OR、-OCONHR、-OCONRR’、-SH、-SR、-SOR、-SOOR、-SO2NHR”、硝基、卤素、糖基、氰基、三氟甲基、C1-6烷基、3-8元环烷基、3-8元杂环烷基、芳基、C1-6烷基取代的芳基、6-15元杂芳基、链烯基、炔基、亚磺基、磺酸或磺酸盐；其中所述C1-6烷基、3-8元环烷基、3-8元杂环烷基、芳基、C1-6烷基取代的芳基、6-15元杂芳基、链烯基和炔基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：氨基、卤素、羟基、氧基、C1-6烷基、C1-6烷氧基、C1-6烷硫基、C1-6烷酰基、3-8元环烷基(例如环丙基)、氧代3-8元环烷基(例如氧代环丁基)、氰基、三氟甲基、C1-6烷氧羰基、C1-6烷基酰胺基、脲基、氨基甲酸酯基、羧基以及芳基；
    R3和R4各自独立地表示不存在，或代表H、C1-6烷基、C1-6烷酰基、C1-6烷氧羰 基、糖基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、C1-6烷酰基、C1-6烷氧羰基、糖基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基、C1-6烷氨基和C1-6烷酰基；优选地，所述芳基为6-14元芳基，例如6-10元芳基；更优选地为苯基或萘基；
    R和R’各自独立地选自H、C1-6烷基、3-8元环烷基、芳基-C1-6烷基或芳基，其中所述C1-6烷基、3-8元环烷基、芳基-C1-6烷基和芳基未被取代或被一个或多个(例如1、2、3或4个)选自下述的取代基取代：卤素、羟基、氨基、C1-6烷基、C1-6烷氧基和C1-6烷氨基；
    R”代表C1-6烷基或芳基(例如6-10元芳基，优选苯基)；
    式(I)中的虚实双键
    Figure PCTCN2017091726-appb-100016
    代表单键或者双键；优选地，环A包含0、1、2或3个碳碳双键；环B包含0、1或2个碳碳双键。
  23. 权利要求22的方法，其中，式I所示的化合物如权利要求2-11任一项中所定义；
    优选地，所述与Nur77相关的疾病为癌症(例如三阴乳腺癌)。
  24. 如式IV所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯的用途，其用作孤儿核受体Nur77的配体，或者用于制备用作孤儿核受体Nur77的配体的药物：
    Figure PCTCN2017091726-appb-100017
    其中，X代表NH、O或CH2；
    Y代表O、S或NR；
    R5和R6各自独立地代表代表H或C1-6烷基；
    当R7和与之相连的碳原子之间为单键时，R7代表OH，当R7和与之相连的碳原子之间为双键时，R7代表O；
    R8代表H或C1-6烷基；
    式(IV)中的虚实双键
    Figure PCTCN2017091726-appb-100018
    代表单键或者双键。
  25. 权利要求24的用途，其中所述化合物选自下列化合物：
    Figure PCTCN2017091726-appb-100019
  26. 权利要求24或25的用途，其中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制Nur77的转录活性；
    优选地，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于抑制Nur77在细胞中的转录活性；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  27. 权利要求24或25的用途，其中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制TNFα在细胞中的生物学效应；
    优选地，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  28. 权利要求24或25的用途，其中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制细胞中mTOR通路(例如下调P-mTOR、p-P70S6K和/或p-S6活性)；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  29. 权利要求25或26的用途，其中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于在有此需要的受试者中预防或治疗与Nur77相关的疾病；
    优选地，所述与Nur77相关的疾病为癌症(例如三阴乳腺癌)。
  30. 一种抑制孤儿核受体Nur77的转录活性的方法，其包括，将Nur77与如式IV所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯相接触的步骤：
    Figure PCTCN2017091726-appb-100020
    其中，X代表NH、O或CH2；
    Y代表O、S或NR；
    R5和R6各自独立地代表代表H或C1-6烷基；
    当R7和与之相连的碳原子之间为单键时，R7代表OH，当R7和与之相连的碳原子之间为双键时，R7代表O；
    R8代表H或C1-6烷基；
    式(IV)中的虚实双键
    Figure PCTCN2017091726-appb-100021
    代表单键或者双键。
  31. 权利要求30的方法，其中，式IV所示的化合物如权利要求25中所定义；
    优选地，所述方法用于抑制Nur77在细胞中的转录活性；
    优选地，所述方法包括，给有此需要的细胞施用有效量的所述化合物，从而抑制Nur77在细胞中的转录活性；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  32. 一种抑制TNFα在细胞中的生物学效应的方法，其包括，给有此需要的细胞施用有效量的如式IV所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
    Figure PCTCN2017091726-appb-100022
    其中，X代表NH、O或CH2；
    Y代表O、S或NR；
    R5和R6各自独立地代表代表H或C1-6烷基；
    当R7和与之相连的碳原子之间为单键时，R7代表OH，当R7和与之相连的碳原子之间为双键时，R7代表O；
    R8代表H或C1-6烷基；
    式(IV)中的虚实双键
    Figure PCTCN2017091726-appb-100023
    代表单键或者双键。
  33. 权利要求32的方法，其中，式I所示的化合物如权利要求26中所定义；
    优选地，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活；
    优选地，所述方法用于抑制TNFα诱导的IκBα降解；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  34. 一种抑制mTOR通路的方法，其包括，给有此需要的细胞施用有效量的如式IV所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
    Figure PCTCN2017091726-appb-100024
    其中，X代表NH、O或CH2；
    Y代表O、S或NR；
    R5和R6各自独立地代表代表H或C1-6烷基；
    当R7和与之相连的碳原子之间为单键时，R7代表OH，当R7和与之相连的碳原子之间为双键时，R7代表O；
    R8代表H或C1-6烷基；
    式(IV)中的虚实双键
    Figure PCTCN2017091726-appb-100025
    代表单键或者双键。
  35. 权利要求34的方法，其中，式I所示的化合物如权利要求25中所定义；
    优选地，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于下调P-mTOR、p-P70S6K和/或p-S6活性；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  36. 一种预防或治疗与Nur77相关的疾病的方法，其包括，给有此需要的受试者施用有效量的如式IV所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
    Figure PCTCN2017091726-appb-100026
    其中，X代表NH、O或CH2；
    Y代表O、S或NR；
    R5和R6各自独立地代表代表H或C1-6烷基；
    当R7和与之相连的碳原子之间为单键时，R7代表OH，当R7和与之相连的碳原子之间为双键时，R7代表O；
    R8代表H或C1-6烷基；
    式(IV)中的虚实双键
    Figure PCTCN2017091726-appb-100027
    代表单键或者双键。
  37. 权利要求36的方法，其中，式IV所示的化合物如权利要求25中所定义；
    优选地，所述与Nur77相关的疾病为癌症(例如三阴乳腺癌)。
  38. 下列化合物、其互变异构体、立体异构体或药学上可接受的盐或酯的用途，其用作孤儿核受体Nur77的配体，或者用于制备用作孤儿核受体Nur77的配体的药物：
    Figure PCTCN2017091726-appb-100028
  39. 权利要求38的用途，其中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制Nur77的转录活性；
    优选地，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于抑制Nur77在细胞中的转录活性；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  40. 权利要求38的用途，其中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制TNFα在细胞中的生物学效应；
    优选地，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  41. 权利要求38的用途，其中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于抑制细胞中mTOR通路(例如下调P-mTOR、p-P70S6和/或p-S6活性)；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  42. 权利要求38的用途，其中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用作孤儿核受体Nur77的配体，用于在有此需要的受试者中预防或治疗与Nur77相关的疾病；
    优选地，所述与Nur77相关的疾病为癌症(例如三阴乳腺癌)。
  43. 一种抑制孤儿核受体Nur77的转录活性的方法，其包括，将Nur77与下列化合物、其互变异构体、立体异构体或药学上可接受的盐或酯相接触的步骤：
    Figure PCTCN2017091726-appb-100029
  44. 权利要求43的方法，其中，所述方法用于抑制Nur77在细胞中的转录活性；
    优选地，所述方法包括，给有此需要的细胞施用有效量的所述化合物，从而抑制Nur77在细胞中的转录活性；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  45. 一种抑制TNFα在细胞中的生物学效应的方法，其包括，给有此需要的细胞施用有效量的下列所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
    Figure PCTCN2017091726-appb-100030
  46. 权利要求45的方法，其中，TNFα的生物学效应为κB抑制蛋白(IκB)激酶α/β(IKKα/β)的磷酸化，IκBα的降解，NF-κB亚基p65的入核转运，和/或NF-κB的激活；
    优选地，所述方法用于抑制TNFα诱导的IκBα降解；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  47. 一种抑制mTOR通路的方法，其包括，给有此需要的细胞施用有效量的下列所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
    Figure PCTCN2017091726-appb-100031
  48. 权利要求47的方法，其中，所述化合物、其互变异构体、立体异构体或药学上可接受的盐或酯或者所述药物用于下调P-mTOR、p-P70S6K和/或p-S6活性；
    优选地，所述细胞表达Nur77；
    优选地，所述细胞为三阴乳腺癌细胞及其他癌细胞(例如肝癌细胞，宫颈癌细胞，肺癌细胞、三阳乳腺癌细胞、结直肠癌细胞或前列腺癌细胞)。
  49. 一种预防或治疗与Nur77相关的疾病的方法，其包括，给有此需要的受试者施用有效量的如下所示的化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
    Figure PCTCN2017091726-appb-100032
  50. 权利要求49的方法，其中所述与Nur77相关的疾病为癌症(例如三阴乳腺癌)。
  51. 如下所示化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
    Figure PCTCN2017091726-appb-100033
    其中，
    R9和R10各自独立地选自氢、C1-6烷基和芳基；
    优选地，R9和R10各自独立地选自氢、C1-4烷基、苯基和萘基；
    优选地，R9和R10各自独立地选自甲基、乙基和异丙基。
  52. 下列化合物、其互变异构体、立体异构体或药学上可接受的盐或酯：
    Figure PCTCN2017091726-appb-100034
  53. 一种具有抗癌活性的药物的筛选方法，其包括以下步骤：
    (1)提供表达孤儿核受体Nur77的癌细胞(例如三阴乳腺癌细胞)，设立阴性对照(即不表达Nur77)；
    (2)将所述细胞用TNFα和候选试剂处理；
    (3)检测(例如通过免疫印迹法进行检测)处理后的细胞中P-mTOR、p-P70S6K和p-S6的水平；
    (4)比较所述细胞与阴性对照细胞中相应蛋白的水平，如有至少一种蛋白水平降低即判断所述候选试剂具有抗癌活性。
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