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WO2017070878A1 - C14-位羟基酯化的雷公藤甲素氨基酸衍生物、及其制备方法和应用 - Google Patents

C14-位羟基酯化的雷公藤甲素氨基酸衍生物、及其制备方法和应用 Download PDF

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WO2017070878A1
WO2017070878A1 PCT/CN2015/093161 CN2015093161W WO2017070878A1 WO 2017070878 A1 WO2017070878 A1 WO 2017070878A1 CN 2015093161 W CN2015093161 W CN 2015093161W WO 2017070878 A1 WO2017070878 A1 WO 2017070878A1
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substituted
amino acid
unsubstituted
triptolide
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徐荣臻
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Priority to HK18105555.6A priority Critical patent/HK1246279B/zh
Priority to EP15906944.2A priority patent/EP3248981B1/en
Priority to CA2977559A priority patent/CA2977559C/en
Priority to ES15906944T priority patent/ES2909799T3/es
Priority to JP2017548297A priority patent/JP6827942B2/ja
Priority to DK15906944.2T priority patent/DK3248981T3/da
Priority to US15/551,695 priority patent/US10238623B2/en
Priority to CN201580074254.0A priority patent/CN107207525B/zh
Priority to AU2015413013A priority patent/AU2015413013B2/en
Priority to KR1020177025330A priority patent/KR102452412B1/ko
Priority to PCT/CN2015/093161 priority patent/WO2017070878A1/zh
Publication of WO2017070878A1 publication Critical patent/WO2017070878A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • 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
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/04—Compounds containing oxirane rings containing only hydrogen and carbon atoms in addition to the ring oxygen atoms
    • C07D303/06—Compounds containing oxirane rings containing only hydrogen and carbon atoms in addition to the ring oxygen atoms in which the oxirane rings are condensed with a carbocyclic ring system having three or more relevant rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/22—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains four or more hetero rings

Definitions

  • the invention belongs to the field of natural medicine and medicinal chemistry, and particularly relates to a novel triptolide derivative, in particular to a C14-position hydroxyl esterified triptolide amino acid derivative, a method and a use for preparing the same.
  • Triptolide also known as triptolide, is a kind of three epoxy groups and one ⁇ , ⁇ unsaturated five-membered one isolated from Tripterygium wilfordii.
  • a rosin-type diterpene lactone compound having a unique configuration of a lactone ring structure, having a molecular formula of C 20 H 24 O 6 having a molecular weight of 360.4, and a structural formula represented by the following formula.
  • triptolide has significant biological activities such as anti-tumor, anti-inflammatory, immunosuppressive, and anti-male fertility. Triptolide can not only prolong the survival time of mouse leukemia L615, but also has obvious therapeutic effect on human leukemia. The complete remission rate is 40% (18/45) [Xia Zhilin et al. Pharmacological and clinical research of triptolide. China Journal of Pharmacology 1992, (6) 427-431]. Clinically used to treat psoriasis, rheumatoid arthritis, leukemia, kidney disease, etc.
  • triptolide has high toxicity, its therapeutic window is narrow, and it has great side effects on the digestive system, genitourinary system and blood system. Therefore, clinical development research has been Certain restrictions. Due to the unique chemical structure and biological activity of triptolide, it has attracted great interest from chemical and pharmacologists in various countries. In order to reveal the mechanism of action and structure-activity relationship of triptolide, and to find high-efficiency and low-toxic derivatives, several research groups conducted a systematic study on triptolide and its analogues, and synthesized a series of triptolide derivatives. [Zhang Fan et al. Progress in structural modification of triptolide. Journal of Pharmaceutical Sciences.
  • Minnelide is a water-soluble triptolide prodrug synthesized by scientists at the University of Minnesota in the United States. It can be converted to triptolide in vivo and in vitro. In some animal models, Minnelide showed good anticancer effects and less toxic effects. For example, in a nude mouse model of pancreatic cancer, Minnelide prolongs the survival of mice by reducing the volume of the tumor and reducing the spread of the tumor, and Minnelide does not exhibit significant toxic effects at an effective dose that causes tumor regression.
  • triptolide is entering Phase I clinical trials [Chugh R, et al. A preclinical evaluation of Minnelide as a therapeutic agent against pancreatic cancer. Sci Transl Med. 2012; 4 (156): 156ra 139.].
  • no triptolide or its derivatives have entered Phase II clinical trials. Therefore, researchers still need to further study and explore the safe application of triptolide.
  • Clinically relevant methods as well as new high-efficiency, low-toxic triptolide derivatives.
  • the pharmacokinetic characteristics of triptolide oral and intravenous injections suggest that triptolide has a short elimination half-life, undergoes rapid absorption, distribution, metabolism and elimination in vivo, and plasma concentration and drug concentration of various tissues change with time.
  • One of the objects of the present invention is to provide a novel C14-position hydroxyesterified triptolide amino acid derivative of the formula (I), or a pharmaceutically acceptable adduct or complex thereof, characterized by a single amino acid esterification, a salt, a decomposition product, and a metabolite, and a novel triptolide-esterified triptolide amino acid derivative characterized by dipeptide esterification of the novel C14-position, or a pharmaceutically acceptable adduct thereof, or a compound thereof Substances, salts, decomposition products and metabolites:
  • R 1 is selected from a substituted or unsubstituted C 2 -C 20 alkyl group, a substituted or unsubstituted C 2 -C 20 olefin group, a substituted or unsubstituted C 4 -C 20 conjugated olefin group, a substituted or unsubstituted C 3 -C 7 cycloalkyl or cycloalkenyl group, a substituted or unsubstituted aromatic hydrocarbon group, a substituted or unsubstituted heterocyclic group and an aromatic heterocyclic group, a substituted or unsubstituted amino acid a side chain alkyl group; the amino acid may be racemic or optically pure (left-handed or right-handed); the substituent is selected from the group consisting of halogen, amine group, C 1 -C 6 substituted amine group, nitro group, cyano group, hydroxyl group, C 1 -C 6 alkoxy, fluorenyl, C
  • R 2 and R 3 are selected from H, substituted or unsubstituted C 1 -C 20 alkyl, substituted or unsubstituted C 2 -C 20 alkene, substituted or unsubstituted C 4 -C 20 Co-decene olefin group, substituted or unsubstituted C 3 -C 7 cycloalkyl or cycloalkyl group, substituted or unsubstituted aromatic hydrocarbon group, substituted or unsubstituted heterocyclic group and aromatic heterocyclic group, substituted Or an unsubstituted amino acid side chain alkyl group, the amino acid may be racemic or optically pure (left-handed or right-handed); the substituent is selected from the group consisting of halogen, amine group, C 1 -C 6 substituted amine group, nitro group, Cyano, hydroxy, C 1 -C 6 alkoxy, fluorenyl, C 1 -C 6 alkylthio; R 2 and R
  • R 4 is selected from substituted or unsubstituted C 2 -C 20 alkyl, substituted or unsubstituted C 2 -C 20 alkene, substituted or unsubstituted C 4 -C 20 conjugated alkene group, Substituted or unsubstituted C 3 -C 7 cycloalkyl or cycloalkenyl, substituted or unsubstituted arene, substituted or unsubstituted heterocyclic and aromatic heterocyclic, substituted or unsubstituted amino a side chain alkyl group, the amino acid may be racemic or optically pure (left-handed or right-handed); the substituent is selected from the group consisting of halogen, amine group, C 1 -C 6 substituted amine group, nitro group, cyano group, hydroxyl group, C 1 -C 6 alkoxy, fluorenyl, C 1 -C 6 alkylthio;
  • R 5 and R 6 are selected from H, substituted or unsubstituted C 1 -C 20 alkyl, substituted or unsubstituted C 2 -C 20 alkene, substituted or unsubstituted C 4 -C 20 Co-decene olefin group, substituted or unsubstituted C 3 -C 7 cycloalkyl or cycloalkyl group, substituted or unsubstituted aromatic hydrocarbon group, substituted or unsubstituted heterocyclic group and aromatic heterocyclic group, substituted Or an unsubstituted amino acid side chain alkyl group, the amino acid may be racemic or optically pure and include levorotatory and dextrorotatory; the substituent is selected from the group consisting of halogen, amine group, C 1 -C 6 substituted amine group, Nitro, cyano, hydroxy, C 1 -C 6 alkoxy, fluorenyl, C 1 -C 6 alkylthio.
  • a second object of the present invention is to provide a process for preparing a single amino acid esterification of the present invention, wherein (I) and a dipeptide esterified a compound of the formula (II) C14-position hydroxylated triptolide amino acid derivative, which follows the route below:
  • the single amino acid esterification of the C14-position hydroxyl esterified triptolide amino acid derivative of the formula (I) can be obtained from a triptolide with the corresponding amino acid R 2 R 3 NCHR 1 CO 2 H in a condensing agent or a catalyst. In the presence of a suitable solvent, condensed esterification at a suitable temperature; or it may be condensed and esterified by triptolide and the corresponding amino acid acyl chloride R 2 R 3 NCHR 1 COCl in the presence of an alkaline reagent.
  • amino acid R 2 R 3 NCHR 1 CO 2 H is activated to form an active ester intermediate, and then reacted with triptolide to form a C14-hydroxyesterified triptolide amino acid derivative (I);
  • the amino acid R 2 R 3 NCHR 1 CO 2 H is activated to form a mixed anhydride intermediate, which is then reacted with triptolide to form a C14-hydroxyesterified triptolide amino acid derivative (I).
  • R 1 , R 2 , R 3 , amino acid R 2 R 3 NCHR 1 CO 2 H, R 1 , R 2 , R 3 , amino acid acid chloride R 2 R 3 NCHR 1 COCl, R 1 , R 2 , R 3 , the active ester intermediates R 1 , R 2 , R 3 , and the mixed anhydride intermediates R 1 , R 2 , R 3 are the same as defined above in the formula (I).
  • the present invention also provides a method for preparing a triptolide amino acid derivative esterified with a C14-position hydroxydipeptide of the formula (II) of the present invention: a triptolide amino acid derivative esterified with a C14-position hydroxydipeptide (II)
  • the C14-position hydroxyl-esterified triptolide amino acid derivative (I) can be obtained by the above method, and in the amino acid R 2 R 3 NCHR 1 CO 2 H, R 2 or R 3 is a protecting group.
  • the protecting groups for these amino groups are known to those skilled in the art and can be readily found in literature sources, such as Greene and Wuts, "Protective Groups in Organic Synthesis” Third Edition, John Wiley & Sons Press, New York, NY, 1999. All of them are incorporated herein by reference.
  • the protecting group is then removed to give a derivative of the formula Ia, which is then condensed with the corresponding amino acid R 5 R 6 NCHR 4 CO 2 H in the presence of a condensing agent or catalyst in a suitable solvent at an appropriate temperature.
  • COCl is formed by condensation amidation in the presence of an alkaline reagent; or the amino acid R 5 R 6 NCHR 4 CO 2 H is activated to form an active ester intermediate, and then the triptolide amino acid derivative esterified with a C14-position hydroxyl group ( Ia) reaction formation; the amino acid R 5 R 6 NCHR 4 CO 2 H can also be activated to form a mixed anhydride intermediate, which is then reacted with a C14-hydroxy esterified triptolide amino acid derivative (Ia).
  • R 5 or R 6 is a protecting group, the compound can be further deprotected to give compound IIa.
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 amino acid R 2 R 3 NCHR 1 CO 2 H, R 1 , R 2 , R 3 , amino acid acid chloride R 2 R 3 NCHR 1 in RCO, R 1 , R 2 , R 3 , active ester intermediates R 1 , R 2 , R 3 , and mixed anhydride intermediates R 1 , R 2 , R 3 are as defined above in formula (II) .
  • R 5 R 6 NCHR 4 CO 2 H in R 4 , R 5 , R 6 , amino acid acyl chloride R 5 R 6 NCHR 4 COCl in R 4 , R 5 , R 6 , active ester intermediate R 4 , R 5 , R 6 , and the mixed anhydride intermediates R 4 , R 5 , R 6 are the same as defined above in formula (II).
  • a third object of the present invention is to provide a pharmaceutical composition comprising at least one compound of the present invention, and optionally a pharmaceutically acceptable excipient selected from the group consisting of Formula (I) A triptolide amino acid derivative esterified with a C14-position hydroxy single amino acid ester and a triptolide amino acid derivative esterified with a C14-position hydroxydipeptide of the formula (II).
  • a fourth object of the present invention is to provide a use of a compound of the present invention or a pharmaceutical composition comprising the same for the preparation of a medicament, particularly an antitumor medicament. Accordingly, the invention provides a method of treating a tumor patient comprising administering to a patient in need of treatment a therapeutically effective amount of at least one compound of the invention.
  • the tumor is particularly selected from the group consisting of leukemia, multiple myeloma, lymphoma, liver cancer, gastric cancer, breast cancer, cholangiocarcinoma, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, melanoma, human cervical cancer, glioma, Nasopharyngeal cancer, laryngeal cancer, esophageal cancer, middle ear tumor, prostate cancer, etc.
  • the compound is selected from the group consisting of a triptolide amino acid derivative of a C14-position hydroxy single amino acid of the formula (I) and a triptolide amino acid derivative esterified with a C14-position hydroxydipeptide of the formula (II). .
  • a fifth object of the present invention is to provide a use of a compound of the present invention or a pharmaceutical composition comprising the same for the preparation of a medicament for treating an autoimmune disease.
  • the autoimmune disease is particularly selected from autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and kidney disease, which are associated with abnormal immune function of cells such as T cells or B cells.
  • the compound is selected from the group consisting of a triptolide amino acid derivative of a C14-position hydroxy single amino acid of the formula (I) and a triptolide amino acid derivative esterified with a C14-position hydroxydipeptide of the formula (II). .
  • the present invention relates to a novel amino acid esterification of the general formula (I) and a dipeptide esterification of the novel C14-position hydroxyesterified triptolide Amino acid derivatives or pharmaceutically acceptable adducts, complexes, salts, decomposition products and metabolites thereof:
  • R 1 is selected from substituted or unsubstituted C 2 -C 20 alkyl, substituted or unsubstituted C 2 -C 20 alkene, substituted or unsubstituted C 4 -C 20 conjugated alkene group, substituted or not Substituted C 3 -C 7 cycloalkyl or cycloalkenyl, substituted or unsubstituted arene, substituted or unsubstituted heterocyclic and aromatic heterocyclic, substituted or unsubstituted amino acid side chain alkyl, amino acid Is racemic or optically pure (left-handed or right-handed); substituents are selected from halogen, amine, C 1 -C 6 substituted amine, nitro, cyano, hydroxy, C 1 -C 6 alkane Oxyl, fluorenyl, C 1 -C 6 alkylthio.
  • R 2 , R 3 , R 4 , R 5 and R 6 are selected from H, substituted or unsubstituted C 1 -C 20 alkyl, substituted or unsubstituted C 2 -C 20 alkene, substituted or unsubstituted a C 4 -C 20 conjugated alkene group, a substituted or unsubstituted C 3 -C 7 cycloalkyl or cycloalkenyl group, a substituted or unsubstituted aromatic hydrocarbon group, a substituted or unsubstituted heterocyclic group and an aromatic heterocyclic group a substituted or unsubstituted amino acid side chain alkyl group, the amino acid may be racemic or optically pure (left-handed or right-handed); the substituent is selected from the group consisting of halogen, amine group, C 1 -C 6 substituted amine group, Nitro, cyano, hydroxy, C 1 -C 6 alkoxy, fluorenyl, C 1
  • the invention relates to compounds of Formula I or Formula II wherein R 1 , R 4 are selected from substituted or unsubstituted C 2 -C 20 alkyl. R 1 and R 4 may be the same or different.
  • the invention relates to compounds of Formula I or Formula II wherein R 2 , R 3 , R 5 and R 6 are selected from H, substituted or unsubstituted C 1 -C 20 alkyl. R 2 , R 3 , R 5 and R 6 may be the same or different.
  • the invention relates to compounds of Formula I or Formula II wherein R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are selected from substituted or unsubstituted C 2 -C 20 An alkene-based, substituted or unsubstituted C 4 -C 20 conjugated alkene group.
  • R 1 , R 2 , R 3 R 4 , R 5 and R 6 may be the same or different.
  • the invention relates to compounds of Formula I or Formula II wherein R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are selected from substituted or unsubstituted C 3 -C 7 A cycloalkyl or cycloalkenyl group.
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 may be the same or different.
  • the invention relates to compounds of Formula I or Formula II wherein R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are selected from substituted or unsubstituted arenes.
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 may be the same or different.
  • the invention relates to compounds of Formula I or Formula II wherein R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are selected from substituted or unsubstituted heterocyclic or aromatic Heterocyclic group.
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 may be the same or different.
  • the invention relates to compounds of Formula I or Formula II wherein R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are selected from substituted or unsubstituted amino acid side chain alkyl groups .
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 may be the same or different.
  • triptolide amino acid derivative of the present invention is shown below. These examples are only intended to further illustrate the invention and are not intended to limit the scope of the invention.
  • the invention particularly preferably exemplifies the following compounds of formula (I):
  • the invention particularly preferably comprises the following compound of formula (II):
  • the present invention relates to compounds of the formula (I) and formula (II) of the invention in the form of their salts, solvates, hydrates, adducts, complexes, polymorphs or prodrugs.
  • C 1 -C 20 alkyl refers to a straight or branched, substituted or unsubstituted alkane group containing from 1 to 20 carbon atoms.
  • Examples of C 1 -C 20 alkane groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl and n-icosane. base.
  • C 2 -C 20 alkyl refers to a straight or branched, substituted or unsubstituted alkane group containing from 2 to 20 carbon atoms.
  • Examples of C 2 -C 20 alkane groups include, but are not limited to, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, n-hexyl and n-icosyl.
  • C 2 -C 20 olefinic group means a straight or branched, substituted or unsubstituted alkene group having 2 to 20 carbon atoms.
  • Examples of C 2 -C 20 alkene groups include, but are not limited to, vinyl, allyl, and eicosyl groups.
  • C 4 -C 20 conjugated alkene group means a straight or branched, substituted or unsubstituted conjugated olefin group having 4 to 20 carbon atoms.
  • Examples of the C 4 -C 20 conjugated alkene group include, but are not limited to, a copolybutadienyl group, a (retinyl) group, and a (9Z)-octadecyl (yl)-9-olefin group.
  • C 3 -C 7 cycloalkyl or cycloalkenyl refers to a hydrocarbyl group of a 3-7 membered monocyclic ring system having a saturated or unsaturated ring, and the C 3 -C 7 cycloalkyl group may be a cyclopropyl group or a cyclobutyl group.
  • Base cyclopentyl, cyclohexyl, cycloheptyl, cyclopropenyl and cyclohexenyl.
  • aromatic hydrocarbon group means an aromatic hydrocarbon group free of hetero atoms, including an aromatic hydrocarbon group, an aromatic hydrocarbon alkyl group and an alkyl aromatic hydrocarbon group.
  • heterocyclic aromatic hydrocarbon group means an aromatic hydrocarbon group containing a hetero atom, and includes a heterocyclic aromatic hydrocarbon group, a heterocyclic arenealkyl group, and an alkylheteroaryl arene group. Heteroatoms refer to nitrogen, oxygen and sulfur.
  • the heterocyclic aromatic hydrocarbon group may contain one or several hetero atoms.
  • amino acid refers to both natural and unnatural amino acids.
  • halogen means fluoro, chloro, bromo or iodo.
  • C 1 -C 6 substituted amine refers to -NC 1 -C 6 alkyl and -NC 3 -C 6 cycloalkyl.
  • C 1 -C 6 alkoxy refers to -OC 1 -C 6 alkyl and -OC 3 -C 6 cycloalkyl.
  • C 1 -C 6 alkylthio refers to -SC 1 -C 6 alkyl and -SC 3 -C 6 cycloalkyl.
  • pharmaceutically acceptable adducts and complexes of the compounds of formula (I) and formula (II) means that the compounds of the invention further interact with other small molecules or biomacromolecules by non-chemical or non-covalent molecules. The product of the combination.
  • the term "pharmaceutically acceptable salts of the compounds of formula (I) and formula (II)” refers to organic acid salts of the compounds of the invention with pharmaceutically acceptable anionic organic acids, which Organic acid salts include, but are not limited to, tosylate, mesylate, malate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, milk Acid salts, alpha-ketoglutarate and alpha-glycerophosphate; also suitable inorganic salts, including but not limited to hydrochlorides, sulfates, nitrates, hydrogencarbonates and carbonates, phosphates, Hydrobromide, hydroiodide, and the like.
  • compositions can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid that provides a pharmaceutically acceptable anion.
  • polymorph refers to the solid crystalline form of a compound of the invention or a complex thereof. Different polymorphs of the same compound may exhibit different physical, chemical and/or spectral properties. Different physical properties include, but are not limited to, stability (eg, for heat or light), compressibility and density (important for formulation and product production), and dissolution rate (which can affect bioabsorbability and availability).
  • Differences in stability can result in chemical reactivity (eg, differential oxidation such that when formulated from one polymorph, the formulation fades faster than when formed from another polymorph) or mechanical properties (eg, when stored) Changes in the kinetically favorable polymorphic tablet granules converted to thermodynamically more stable polymorphs) or both (for example, tablets of one polymorph are more susceptible to breakage at high humidity) .
  • the different physical properties of polymorphs can affect their processing. For example, one polymorph may be more likely to form a solvate than the other or may be more difficult to filter or wash away than the other due to, for example, the shape or size distribution of its particles.
  • hydrate refers to a compound of the invention or a salt thereof, further comprising a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.
  • prodrug refers to a derivative that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide a compound of the invention. Prodrugs undergo this reaction to become active compounds only under biological conditions, or they are active in their unreacted form. Prodrugs can generally be prepared using well-known methods, for example, 1 Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff, ed. 5) and J. Rautio's Prodrugs and Targeted Delivery (2011) 31- 60 (Wiley-VCH, Methods and Principles in Medicinal Chemistry, Vol. 47) and those described in G. Thomas's Fundamentals of Medicinal Chemistry (2003) 195-200 (Wiley).
  • the triptolide derivative of the compound of the present invention has the general formula I (having ten chiral centers, nine chiral centers originating from triptolide, one chiral center derived from amino acids); and formula II (having Eleven chiral centers, nine chiral centers are derived from triptolide, and two chiral centers are derived from the stereochemical structure shown by the structural formula of amino acids.
  • the definitions and conventions of stereochemistry used herein generally follow the McGraw-Hill Dictionary of Chemical Terms (SP Parker, Ed., McGraw-Hill Book Company, New York, 1984); Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (John Wiley & Sons, Inc., New York, 1994).
  • Many organic compounds exist in optically active forms, i.e., they have the ability to rotate planes of plane polarization.
  • treating generally refers to obtaining the desired pharmacological and/or physiological effects.
  • the effect may be prophylactic according to the prevention of the disease or its symptoms in whole or in part; and/or may be therapeutic according to the partial or complete stabilization or cure of the disease and/or side effects due to the disease.
  • treatment encompasses any treatment for a patient's condition, including: (a) preventing a disease or condition in a disease or condition that has not yet been diagnosed; (b) inhibiting the symptoms of the disease, ie preventing Its development; or (c) to alleviate the disease It causes disease or symptom deterioration.
  • the compounds of the present invention can be prepared according to conventional organic chemical synthesis methods.
  • the general preparation method of the compound of the general formula (I) of the present invention is as follows:
  • the single amino acid esterification of the C14-position hydroxyl esterified triptolide amino acid derivative of the formula (I) can be isolated from the naturally extracted triptolide (TPL) and the corresponding amino acid R 2 R 3 NCHR 1 CO 2 H In the presence of a condensing agent or a catalyst, it is condensed and esterified at a suitable temperature in a suitable solvent; it can also be obtained from an alkaline reagent by triptolide and the corresponding amino acid acyl chloride R 2 R 3 NCHR 1 COCl.
  • the amino acid R 2 R 3 NCHR 1 CO 2 H may also be activated to form a mixed anhydride intermediate, which is then reacted with triptolide to form a C14-hydroxyesterified triptolide amino acid derivative (I).
  • R 1 , R 2 , R 3 , amino acid R 2 R 3 NCHR 1 CO 2 H, R 1 , R 2 , R 3 , amino acid acid chloride R 2 R 3 NCHR 1 COCl, R 1 , R 2 , R 3 , the active ester intermediates R 1 , R 2 , R 3 , and the mixed anhydride intermediate R 1 , R 2 , R 3 are the same as defined above in the formula (I).
  • the triptolide amino acid derivative (II) esterified with a C14-position hydroxydipeptide can be obtained by the above method, and the amino acid derivative (I) of the C14-position hydroxyl esterified triptolide is used, and the amino acid R 2 R 3 is used.
  • R 2 or R 3 is a protecting group.
  • the protecting groups for these amino groups are known to those skilled in the art and can be readily found in literature sources, such as Greene and Wuts, "Protective Groups in Organic Synthesis” Third Edition, John Wiley & Sons Press, New York, NY, 1999. All of them are incorporated herein by reference.
  • the protecting group is then removed to give a derivative of the formula Ia, which is then condensed with the corresponding amino acid R 5 R 6 NCHR 4 CO 2 H in the presence of a condensing agent or catalyst in a suitable solvent at an appropriate temperature.
  • Amidated to a C14-position hydroxydipeptide esterified triptolide amino acid derivative (II); a derivative of the formula Ia and a corresponding amino acid acyl chloride R 5 R 6 NCHR 4 may also be obtained by removal of a protecting group.
  • COCl is formed by condensation amidation in the presence of an alkaline reagent; or the amino acid R 5 R 6 NCHR 4 CO 2 H is activated to form an active ester intermediate, and then the triptolide amino acid derivative esterified with a C14-position hydroxyl group ( Ia) reaction formation; the amino acid R 5 R 6 NCHR 4 CO 2 H can also be activated to form a mixed anhydride intermediate, which is then reacted with a C14-hydroxy esterified triptolide amino acid derivative (Ia).
  • R 5 and R 6 are a protecting group, the compound can be further deprotected to give compound IIa.
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 amino acid R 2 R 3 NCHR 1 CO 2 H, R 1 , R 2 , R 3 , amino acid acid chloride R 2 R 3 NCHR 1 in RCO, R 1 , R 2 , R 3 , active ester intermediates R 1 , R 2 , R 3 , and mixed anhydride intermediates R 1 , R 2 , R 3 are as defined above in formula (II) .
  • the above reaction is generally carried out in the presence of an alkali or an alkaline reagent.
  • the base here may be, but not limited to, an organic base.
  • Solvents used include, but are not limited to, aprotic polar solvents such as dichloromethane (DCM), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N -methylpyrrole (NMP) or tetrahydrofuran (THF), and the like.
  • aprotic polar solvents such as dichloromethane (DCM), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N -methylpyrrole (NMP) or tetrahydrofuran (THF), and the like.
  • the reaction temperature of the above reaction is generally from 0 ° C to 50 ° C. It generally varies depending on the starting materials used and the base used.
  • the starting material for the preparation reaction is triptolide (TPL).
  • TPL triptolide
  • Protecting groups are those which, once attached to an active moiety (e.g., a hydroxyl or amino group), prevent these moieties from being interfered by subsequent reactions and which can be removed by conventional methods after the reaction.
  • hydroxy protecting groups include, but are not limited to, alkyl, benzyl, allyl, triphenyl Methyl (ie, triphenylmethyl), acyl (eg, benzoyl, acetyl or HOOC-X"-CO-, X" is an alkylene, alkenylene, cycloalkylene or aroma a silyl group (for example, trimethylsilyl, triethylsilyl and tert-butyldimethylsilyl), an alkoxycarbonyl group, an aminocarbonyl group (for example, dimethylaminocarbonyl, Methyl ethylaminocarbonyl and phenylaminocarbonyl), alkoxymethyl, benzyloxymethyl and alky
  • amino protecting groups include, but are not limited to, alkoxycarbonyl, alkanoyl, aryloxycarbonyl, aryl substituted alkyl, and the like. Hydroxy and amino protecting groups have been discussed in T. W. Greene and P. G.. M. Wuts, Protective Groups in Organic Synthesis, 2nd Edition, John Wiley and Sons (1991). Both the hydroxy group and the amino protecting group can be removed by a conventional method after the reaction.
  • the invention also provides pharmaceutical compositions comprising the compounds of formula I and formula II of the invention.
  • the present invention provides a pharmaceutical composition comprising at least one of the compounds of Formula I or Formula II of the present invention as described above, and optionally a pharmaceutically acceptable excipient.
  • Methods of preparing various pharmaceutical compositions containing a certain amount of active ingredient are known, or will be apparent to those skilled in the art from this disclosure. As described by Remington's Pharmaceutical Sciences, Martin, E. W., ed., Mack Publishing Company, 19th ed. (1995). Methods of preparing the pharmaceutical compositions include the incorporation of suitable pharmaceutical excipients, carriers, diluents and the like.
  • the pharmaceutical preparations of the invention are prepared in a known manner, including conventional methods of mixing, dissolving or lyophilizing.
  • the compounds of the invention may be formulated into pharmaceutical compositions and administered to a patient in a variety of ways suitable for the mode of administration selected, such as orally or parenterally (by intravenous, intramuscular, topical or subcutaneous routes).
  • the compounds of the invention may be administered systemically, for example, orally, in association with a pharmaceutically acceptable carrier such as an inert diluent or an assimilable edible carrier. They can be enclosed in hard or soft shell gelatin capsules and can be compressed into tablets.
  • a pharmaceutically acceptable carrier such as an inert diluent or an assimilable edible carrier. They can be enclosed in hard or soft shell gelatin capsules and can be compressed into tablets.
  • the active compound may be combined with one or more excipients and in the form of swallowable tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. use.
  • Such compositions and preparations should contain at least 0.01% of active compound.
  • the ratio of such compositions and formulations may of course vary and may range from about 0.1% to about 99% by weight of a given unit dosage form.
  • the amount of active compound is such that an effective dosage level can be obtained.
  • Tablets, lozenges, pills, capsules and the like may also contain: a binder such as tragacanth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, Potato starch, alginic acid, etc.; a lubricant such as magnesium stearate; and a sweetener such as sucrose, fructose, lactose or aspartame; or a flavoring agent such as mint, wintergreen or cherry.
  • a binder such as tragacanth, acacia, corn starch or gelatin
  • an excipient such as dicalcium phosphate
  • a disintegrating agent such as corn starch, Potato starch, alginic acid, etc.
  • a lubricant such as magnesium stearate
  • a sweetener such as sucrose, fructose, lactose or aspartame
  • a flavoring agent such as mint, wintergreen or cherry
  • the active compound can also be administered intravenously or intraperitoneally by infusion or injection.
  • An aqueous solution of the active compound or a salt thereof can be prepared, optionally mixed with a non-toxic surfactant.
  • Dispersing agents in glycerol, liquid polyethylene glycols, triacetin and mixtures thereof, and oils can also be prepared. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions of the active ingredient (optionally encapsulated in liposomes) containing the immediate formulation of a suitable injectable or injectable solution or dispersing agent. Or sterile powder. In all cases, the final dosage form must be sterile, liquid, and stable under the conditions of manufacture and storage.
  • the liquid carrier can be a solvent or liquid dispersion medium including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, non-toxic glycerides, and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by liposome formation, by maintaining the desired particle size in the case of a dispersing agent, or by the use of a surfactant.
  • the action of preventing microorganisms can be produced by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents such as sugars, buffers or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use of compositions that delay the absorbent (for example, aluminum monostearate and gelatin).
  • Sterile injectable solutions are prepared by combining the required active compound in a suitable solvent with the various other ingredients enumerated above, followed by filter sterilization.
  • the preferred preparation methods are vacuum drying and lyophilization techniques which result in a powder of the active ingredient plus any additional ingredients present in the previously sterile filtration solution. .
  • Useful solid carriers include comminuted solids (e.g., talc, clay, microcrystalline cellulose, silica, alumina, etc.).
  • Useful liquid carriers include water, ethanol or ethylene glycol or a water-ethanol/ethylene glycol mixture, and the compounds of the present invention may be dissolved or dispersed in an effective amount, optionally with the aid of a non-toxic surfactant.
  • Adjuvants such as fragrances
  • additional antimicrobial agents can be added to optimize the properties for a given use.
  • Thickeners can also be used with liquid carriers to form coatable pastes, gels, ointments , soap, etc., used directly on the user's skin.
  • the therapeutic requirements of a compound or an active salt or derivative thereof depend not only on the particular active ingredient selected, but also on the mode of administration, the nature of the condition to be treated, and the age and condition of the patient, ultimately depending on the attending physician or clinical The doctor's decision.
  • unit dosage form is a unit dispersion unit containing a unit dosage unit suitable for administration to humans and other mammalian bodies.
  • the unit dosage form can be a capsule or tablet, or a plurality of capsules or tablets.
  • the amount of unit dose of the active ingredient may vary or be adjusted between about 0.01 mg to about 1000 mg or more, depending on the particular treatment involved.
  • Figure 1 is an immunoblot showing the levels of XPB and Pol II protein in tumor cells and normal cells.
  • Figure 2 is an immunoblot showing the inhibition of XP- and Pol II activity of human THP-1 leukemia cells by 14-D-Valine-TPL.
  • Figure 3 is an immunoblot showing the detection of 14-D-Valine-TPL inhibition of c-myc activity in human K562 leukemia cells.
  • TPL triptolide
  • D-Boc-Valine dextrorotatory (N-Boc) valine
  • DIC N, N'-diisopropylcarbodiimide
  • DMAP 4-dimethylamino Pyridine
  • DCM dichloromethane
  • TFA trifluoroacetic acid
  • the first step thuring triptolide TPL (500 mg, 1.39 mmol, 1.0 eq.), dextrorotatory (N-Boc) valine (1500 mg, 6.91 mmol, 5 eq.) in dichloromethane (20 mL), cooled To 0 ° C. N,N'-diisopropylcarbodiimide (4 mL, 13 mmol, 9.35 eq.) and 4-dimethylaminopyridine (130 mg, 1.07 mmol, 0.8 eq) were added to the mixture at 0 °C.
  • reaction was carried out at 25 ° C for 24 hours, and the reaction mixture was diluted with ethyl acetate, washed with water, and then washed with saturated aqueous ammonium chloride, dried and concentrated to give a crude product. Further purification by preparative silica gel column gave a white solid-D-Boc-Valine-TPL (14-D-Boc-valine-triptolide) (250 mg) in a yield of 32.2%.
  • Step 2 Dissolve the product from the previous step, D-Boc-Valine-TPL (14-dextro-Boc-valine-triptolide) (200 mg) in dichloromethane (15 mL), add 3 mL three Fluoroacetic acid. After the addition was completed, the reaction solution was reacted at 25 ° C for 3.5 hours. The reaction mixture was diluted with methylene chloride and washed with aqueous sodium hydrogen carbonate, then dried and concentrated to give crude. Further purification by preparative high performance liquid chromatography gave a white solid D-Valine-TPL (14-d- valine-triptolide) (120 mg) in a yield of 73%.
  • D-Valine-TPL 14-dextro-valine-triptolide
  • L-Boc-Val L-(N-Boc)proline
  • HATU 2-(7-azobenzene Triazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • DIPEA N,N-diisopropylethylamine
  • DCM dichloromethane
  • TFA trifluoroacetic acid.
  • Step 2 The product of the previous step, 14-L-Boc-Valine-D-Valine-TPL (14-L-(N-Boc)-valine-D-valine-triptolide) (85 mg) ) Dissolved in dichloromethane (5 mL) and 1 mL of trifluoroacetic acid was added dropwise. After the addition was completed, the reaction solution was reacted at 25 ° C for 3.5 hours. The reaction mixture was diluted with methylene chloride and washed with aqueous sodium hydrogen carbonate, then dried and concentrated to give crude. Further purification by preparative high performance liquid chromatography gave 14-L-Valine-D-Valine-TPL (14-L-valine-d-valine-triptolide) (51.5 mg) as a white solid.
  • Compound 14-L-Valine-TPL (14-L-valine-triptolide) was synthesized as in Example 1, and L-Boc-Valine (L-B-N-Boc-valine) was used instead of D-Boc.
  • -Valine-TPL (R-N-Boc-valine.
  • Compound 14-L-Valine-L-Valine-TPL (14-L-valine-L-valine-triptolide) was synthesized as in Example 2, using 14-L-Valine-TPL (14-left-handed) Proline-triptolide, Example 3) instead of 14-D-Valine-TPL (14-d-valine-triptolide, Example 1), with L-Boc-Valine (left-handed-N) -Boc-valine) instead of D-Boc-Valine-TPL (dextrorotatory N-Boc-valine).
  • Compound 14-D-Valine-D-Valine-TPL (14-D-valine-d-valine-triptolide was synthesized as in Example 2, using D-Boc-Valine (D-Boc-Valine) -Boc-valine) instead of L-Boc-Valine-TPL (L-N-Boc-valine).
  • TPL triptolide
  • L-Boc-O-TBDMS-Tyrosine L-(N-Boc-O-tert-butyldimethylsilyl) tyrosine
  • DIC N, N'-diisopropyl Carbodiimide
  • DMAP 4-dimethylaminopyridine
  • DCM dichloromethane
  • TFA trifluoroacetic acid.
  • TBAF tetrabutylammonium fluoride
  • D-Valine-TPL 14-dextro-valine-triptolide
  • L-Boc-Phenylalanine L-(octa)phenylalanine
  • HATU 2-(7-azobenzotriazine) Azole)-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • DIPEA N,N-diisopropylethylamine
  • L-Tyrosine-TPL 14-L-tyrosine-triptolide
  • L-Boc-O-TBDMS-Tyrosine L-(N-Boc-O-tert-butyl) Methylsilyl)tyrosine replaces D-Boc-Valine-TPL (dextrorotatory N-Boc-valine).
  • Example 7 Comparison of the therapeutic window of the compound 14-D-Valine-TPL of the present invention with triptolide (TPL)
  • Leukemia cell lines Sup-B15 (Ph + acute lymphocytic leukemia), CEM (acute lymphocytic leukemia, ALL), Molt-4 (acute lymphocytic leukemia, ALL).
  • Normal blood cell samples from peripheral blood of healthy volunteers.
  • Primary reagent 14-D-Valine-TPL of the invention.
  • Main instruments cell culture incubator, microplate reader
  • the culture broth was a 1640 cell culture medium containing 10% fetal bovine serum.
  • Different concentrations of 14-D-Valine-TPL were added, mixed, and cultured in a carbon dioxide (5% CO 2 ) cell incubator at 37 ° C for 72 hours.
  • the viable cell concentration was then determined by the MTT method.
  • the cell viability of the control group (without compound treatment) was set to 100%, and the cell viability (%) after the action of the compound and the half-growth inhibition concentration (72-hour IC50 value) of the leukemia cells at 72 hours and the therapeutic window index were calculated.
  • Treatment window index normal blood cell IC50 value / leukemia cell IC50 value.
  • a therapeutic window index of >1 indicates a therapeutic window.
  • Compounds are more toxic to tumor cells than normal blood cells, and the larger the therapeutic window, the better the selectivity of the compound for tumor cells.
  • the compound 14-D-Valine-TPL of the present invention has a better therapeutic window as shown in Table 1 below, and triptolide has substantially no therapeutic window.
  • Table 1 shows that triptolide (TPL) has only a small therapeutic window for Molt-4 cells (2.0), while the other three leukemia cells Jurkat, CEM, and Sup-B15 have a therapeutic window of ⁇ 1, respectively, 0.64. 0.93 and 0.94 indicate that triptolide is essentially non-selective to normal cells and tumor cytotoxicity, which is consistent with the results reported in the literature that TPL has essentially no therapeutic window.
  • the 14-D-Valine-TPL of the present invention has a therapeutic window index of 5.54-18.58 for four different types of human leukemia cells, indicating that 14-D-Valine-TPL has a larger therapeutic window for all four leukemias. , wherein the therapeutic window index for Molt-4 cells is as high as 18.58.
  • Example 8 Determination of the activity of the compound 14-D-Valine-TPL of the present invention against leukemia in vitro
  • Leukemia cell lines human KG-1a (acute myeloid leukemia, AML-M0), THP-1 (acute myeloid leukemia, AML-M5) NB4 (acute promyelocytic leukemia, AML), Kasumi-1 (acute marrow) Leukemia M2 type, AML-M2), KG-1 (acute myeloid leukemia, AML), Jurkat (acute lymphocytic leukemia, ALL), H9 (acute lymphocytic leukemia, ALL).
  • Primary reagent 14-D-Valine-TPL of the invention.
  • Main instruments cell culture incubator, microplate reader
  • the culture broth was a 1640 cell culture medium containing 10% fetal bovine serum.
  • Different concentrations of 14-D-Valine-TPL were added, mixed, and cultured in a carbon dioxide (5% CO 2 ) cell incubator at 37 ° C for 72 hours.
  • the viable cell concentration was then determined by the MTT method.
  • the cell viability of the control group (without compound treatment) was set to 100%, and the cell viability (%) after the action of the compound and the half-growth inhibitory concentration of the leukemia cells at 72 hours (72-hour IC 50 value) were calculated.
  • Table 2 shows that 14-D-Valine-TPL of the present invention can induce cell death of human acute myeloid leukemia and acute lymphocytic leukemia and inhibit the growth of these leukemia cells.
  • Example 9 Activity assay of anti-human leukemia in vivo of compound 14-D-Valine-TPL mice of the present invention
  • NOD/SCID mice were purchased from the Shanghai Animal Center of the Chinese Academy of Sciences
  • Leukemia cell lines human KG-1a (acute myeloid leukemia, AML-M0), THP-1 (acute monocytic leukemia, AML-M5), purchased from the ATCC library.
  • the 14-D-Valine-TPL trifluoroacetate salt of the present invention was first dissolved in sterile PBS at a concentration of 10 mg/ml and then diluted with sterile deionized water to the desired working concentration.
  • 7-week-old female NOD/SCID was inoculated subcutaneously in the subcutaneous area of 1 ⁇ 10 7 cells (0.2 ml).
  • the left side was human acute myeloid leukemia KG-1a cells, and the right side was human acute monocytic leukemia THP- 1 cell.
  • the tumor tumors were grown to a length of about 0.5 cm and randomly divided into groups of 3 each.
  • the control group was treated with equal amount of PBS, 14-D-Valine-TPL component 0.2 mg/kg body weight and 0.4 mg/kg body weight, and administered intragastrically twice a day (8 o'clock in the morning and 4 o'clock in the afternoon). , continuous treatment for 14 days.
  • mice were sacrificed, tumor tissues were taken, and the tumor weight was measured.
  • the main organs and tissues such as liver, spleen, heart, lung, large intestine and small intestine were examined.
  • Table 3 and Table 4 show that 14-D-Valine-TPL of the present invention can significantly inhibit the growth of two different types of human acute myeloid leukemia cell lines KG-1a and THP-1 in mice, and has a dose-dependent effect.
  • the 0.4-D-Valine-TPL dose of 0.4 mg per kilogram of body weight can completely abolish the KG-1a and THP-1 xenografts, but the body weight of the mice is not significantly reduced.
  • the mice are dissected to check the heart, lung, liver, spleen and large intestine. There were no obvious abnormalities in the main organs such as the small intestine.
  • Example 10 Determination of anti-human leukemia activity in 14-L-Valine-L-Valine-TPL (14-LLV-TPL) mice
  • NOD/SCID mice were purchased from the Shanghai Animal Center of the Chinese Academy of Sciences
  • Leukemia cell line human KG-1a (acute myeloid leukemia, AML-M0) was purchased from the ATCC library.
  • the 14-LLV-TPL trifluoroacetate salt of the present invention was first dissolved in sterile PBS at a concentration of 10 mg/ml and then diluted with sterile deionized water to the desired working concentration.
  • the main organs and tissues such as liver, spleen, heart, lung, large intestine and small intestine were examined.
  • Table 5 shows that 14-LLV-TPL of the present invention can significantly inhibit the growth of human acute myeloid leukemia cell line KG-1a in mice, and there is a significant dose-dependent effect.
  • Example 11 Determination of anti-human leukemia activity in 14-L-Valine-D-Valine-TPL (14-DLV-TPL) mice
  • NOD/SCID mice were purchased from the Shanghai Animal Center of the Chinese Academy of Sciences
  • Leukemia cell line human KG-1a (acute myeloid leukemia, AML-M0) was purchased from the ATCC library.
  • the 14-DLV-TPL trifluoroacetate salt of the present invention was first dissolved in sterile PBS at a concentration of 10 mg/ml and then diluted with sterile deionized water to the desired working concentration.
  • the main organs and tissues such as liver, spleen, heart, lung, large intestine and small intestine were examined.
  • Table 6 shows that 14-DLV-TPL of the present invention can significantly inhibit the growth of human acute myeloid leukemia cell line KG-1a in mice, and there is a significant dose-dependent effect.
  • Example 12 Determination of anti-human leukemia activity in 14-L-Phenylalanyl-L-tyrosine-TPL (14-LPT-TPL) mice
  • NOD/SCID mice were purchased from the Shanghai Animal Center of the Chinese Academy of Sciences
  • Leukemia cell line human KG-1a (acute myeloid leukemia, AML-M0) was purchased from the ATCC library.
  • the 14-LPT-TPL trifluoroacetate of the present invention was first dissolved in sterile PBS at a concentration of 10 mg/ml and then diluted with sterile deionized water to the desired working concentration.
  • the main organs and tissues such as liver, spleen, heart, lung, large intestine and small intestine were examined.
  • Table 7 shows that 14-LPT-TPL of the present invention can significantly inhibit the growth of human acute myeloid leukemia cell line KG-1a in mice, and there is a significant dose-dependent effect.
  • Example 13 Determination of anti-human leukemia activity in 14-D-Valine-D-Valine-TPL (14-DDV-TPL) mice
  • mice NOD/SCID mice were purchased from the Shanghai Animal Center of the Chinese Academy of Sciences.
  • Leukemia cell line Human K562/ADR (chronic myeloid leukemia, CML) was purchased from the ATCC library.
  • Reagents The 14-DDV-TPL trifluoroacetate salt of the present invention was first dissolved in sterile PBS at a concentration of 10 mg/ml and then diluted with sterile deionized water to the desired working concentration.
  • Main instruments cell culture incubator, microplate reader
  • the main organs and tissues such as liver, spleen, heart, lung, large intestine and small intestine were examined.
  • Table 8 shows that 14-DDV-TPL of the present invention can significantly inhibit the growth of human chronic myeloid leukemia cell line K562/ADR organelle in mice, and there is a significant dose-dependent effect.
  • Example 14 Determination of anti-human leukemia activity in 14-L-Valine-TPL (14-LV-TPL) mice
  • NOD/SCID mice were purchased from the Shanghai Animal Center of the Chinese Academy of Sciences
  • Leukemia cell line Human K562/ADR (chronic myeloid leukemia, CML) was purchased from the ATCC library.
  • the 14-LV-TPL trifluoroacetate salt of the present invention was first dissolved in sterile PBS at a concentration of 10 mg/ml and then diluted with sterile deionized water to the desired working concentration.
  • the main organs and tissues such as liver, spleen, heart, lung, large intestine and small intestine were examined.
  • Table 9 shows that 14-LV-TPL of the present invention can significantly inhibit the growth of human chronic myeloid leukemia cell line K562/ADR organelle in mice, and there is a significant dose-dependent effect.
  • Example 15 Inhibition of T lymphocyte and B lymphocyte activity by 14-D-Valine-TPL in vitro
  • Leukemia cell lines Jurkat (T lymphocytes), H9 (T lymphocytes), CEM (B lymphocytes), and Sup-B15 (B lymphocytes).
  • the culture broth was a 1640 cell culture medium containing 10% fetal bovine serum.
  • Different concentrations of 14-D-Valine-TPL were added, mixed, and cultured in a carbon dioxide (5% CO 2 ) cell incubator at 37 ° C for 72 hours.
  • the viable cell concentration was then determined by the MTT method.
  • the cell viability of the control group (without compound treatment) was set to 100%, and the cell viability (%) after the action of the compound and the half-growth inhibitory concentration of the leukemia cells at 72 hours (72-hour IC 50 value) were calculated.
  • Table 10 shows that 14-D-Valine-TPL of the present invention can significantly inhibit the growth of these T lymphocytes and B lymphocytes.
  • Example 16 Expression of transcription factor TFIIH subunit XPB and RNA polymerase II (Pol II) in different types of tumor cells and normal cells
  • TFIIH subunit XPB xeroderma pigmentosum group B
  • RNA polymerase II RNA polymerase II
  • Leukemia cell lines Nalm6 (lymphoma), Jurkat (acute lymphocytic leukemia), THP-1 (acute monocytic leukemia), KG-1a (acute myeloid leukemia), HL-60 (acute myeloid leukemia), NB4 (Acute promyelocytic leukemia), U937 (acute monocytic leukemia). Normal blood cell samples were obtained from volunteers.
  • Figure 1 shows the results of immunoblotting to detect the expression levels of XPB and Pol II in different types of tumor cells and normal cells. Among them, 1: normal blood cells; 2: Nalm6; 3: Jurkat; 4: THP-1; 5: KG-1a; 6: HL-60; 7: NB4; 8: U937.
  • Example 17 14-D-Valine-TPL inhibits XPB and Pol II activity in human THP-1 leukemia cells
  • XPB and Pol II are key target molecules for the pharmacological action of triptolide [Titov DV, et al. XPB, a subunit of TFIIH, is a target of the natural product triptolide. Nature chemical biology. 2011, 7:182 -188].
  • the present invention uses cell culture techniques and immunoblotting techniques to detect the effect of 14-D-Valine-TPL on the activity of XPB and Pol II in leukemia cells.
  • Leukemia cell line human THP-1 leukemia cell line (acute myeloid leukemia-M5, AML-M5).
  • the well-growth leukemia cells were inoculated into the wells of a 6-well cell culture plate at a density of 1 ⁇ 10 6 /ml.
  • the culture broth was a 1640 cell culture medium containing 10% fetal bovine serum.
  • Different concentrations of 14-D-Valine-TPL were added, mixed, and cultured in a carbon dioxide (5% CO 2 ) cell incubator at 37 ° C for 48 hours. Cellular proteins were then extracted and XPB and Pol II expression levels were detected by immunoblotting.
  • Figure 2 shows the results of 14-D-Valine-TPL inhibition of XPB and Pol II activity in human THP-1 leukemia cells by cell culture technique and immunoblotting
  • Example 18 14-D-Valine-TPL inhibits c-myc oncogene activity in human K562 leukemia cells
  • oncogene c-myc is also one of the key target molecules for triptolide to exert anticancer pharmacological effects [Stéphane Vis conclusions, et al. Triptolide is an inhibitor of RNA polymerase I and II-dependent transcription leading predominantly down-regulation Of short-lived mRNA. Mol Cancer Ther 2009; 8: 2780-2790].
  • the present invention uses cell culture techniques and immunoblotting techniques to examine the effect of 14-D-Valine-TPL on c-myc activity in leukemia cells.
  • Leukemia cell line human K562 leukemia cell line (chronic myeloid leukemia).
  • the well-growth leukemia cells were inoculated into the wells of a 6-well cell culture plate at a density of 1 ⁇ 10 6 /ml.
  • the culture broth was a 1640 cell culture medium containing 10% fetal bovine serum.
  • Different concentrations of 14-D-Valine-TPL were added, mixed, and cultured in a carbon dioxide (5% CO 2 ) cell incubator at 37 ° C for 48 hours. Cellular proteins were then extracted and c-myc protein expression levels were detected by immunoblotting.

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Abstract

本发明属于天然药物及药物化学领域并涉及通式I和通式II的新型雷公藤甲素的酯化氨基酸衍生物或其药学上可接受的加成物、复合物、盐、以及分解和代谢产物，制备这些化合物的方法、包含该化合物的药物组合物及其在制备抗肿瘤、免疫性疾病、XPB或Pol II或致癌基因c-myc异常表达相关疾病药物中的用途。

Description

C14-位羟基酯化的雷公藤甲素氨基酸衍生物、及其制备方法和应用 技术领域
本发明属于天然药物及药物化学领域，具体涉及新型雷公藤甲素衍生物，特别是C14-位羟基酯化的雷公藤甲素氨基酸衍生物、制备这些化合物的方法和用途。
背景技术
雷公藤甲素(Triptolide，TPL)，又叫雷公藤内酯醇，是从卫矛科雷公藤属植物雷公藤中分离到的一种具有3个环氧基团以及一个α，β不饱和五元内酯环结构的构型独特的松香烷型二萜内酯化合物，分子式C20H24O6，分子量360.4，结构式如下式所示。
Figure PCTCN2015093161-appb-000001
已有大量体内和体外研究表明，雷公藤甲素具有显著的抗肿瘤、抗炎、免疫抑制、抗雄性生育等生物活性。雷公藤甲素不仅能显著延长小鼠白血病L615存活时间，而且对人白血病也有明显治疗效果，完全缓解率达40％(18/45)[夏志林等.雷公藤内酯醇的药理和临床研究.中国药理学报1992,(6)427-431]。临床用于治疗银屑病、类风湿性关节炎、白血病、肾病等。但是对雷公藤甲素的毒性研究发现，雷公藤甲素具有很高的毒性，其治疗窗窄，对消化系统、泌尿生殖系统和血液系统等有较大的毒副作用，因此临床开发研究受到了一定限制。由于雷公藤甲素独特的化学结构和生物学活性，引起了各国化学和药理学家的极大兴趣。为揭示雷公藤甲素的作用机制和构效关系，寻找高效低毒的衍生物，多个研究小组对雷公藤甲素以及类似物进行了比较系统的研究，合成了一系列雷公藤甲素衍生物[张凡等.雷公藤内酯醇的结构修饰研究进展.药学学报.2004,39(10)：857-864；邰婷等.雷公藤甲素药代动力学研究进展.药学与临床研究.2012,20(3):229-235]。其中一些已进入临床试验，如Minnelide是由美国明尼苏达大学的科学家合成的一种水溶性雷公藤甲素前药，在体内及体外都可以转化为雷公藤甲素。在一些动物模型中，Minnelide显示出良好的抗癌效果和较小的毒性反应。例如，在胰腺癌裸鼠模型上，Minnelide通过减小肿瘤的体积和减少肿瘤的蔓延，从而延长了小鼠的存活期，并且Minnelide在使肿瘤消退的有效剂量下未表现出明显的毒性作用。Minnelide正在进入I期临床试验阶段[Chugh R,et al.A preclinical evaluation of Minnelide as a therapeutic agent against pancreatic cancer.Sci Transl Med.2012；4(156):156ra139.]。不过遗憾的是，迄今为止，仍未见雷公藤甲素或其衍生物进入II期临床试验。因此，研究者们仍需进一步深入研究，探索雷公藤甲素安全应用于 临床的相关方法以及新的高效低毒雷公藤甲素衍生物。另外，雷公藤甲素口服和静脉注射的药代动力学特征均提示，雷公藤甲素消除半衰期短，在体内经历快速吸收、分布、代谢和消除，血药浓度和各组织药物浓度随时间变化波动大，不具备理想的药动学性质，不利于临床应用，并可能因此增加临床不良反应的发生率。因此，对具有良好药动学特性的新型雷公藤甲素衍生物和相关制剂的探索意义重大[邰婷等.雷公藤甲素药代动力学研究进展.药学与临床研究.2012,20(3):229-235]。
因此，市场仍需具有良好生物活性和药动学特性，毒性较小的雷公藤甲素类药物。迄今，尚未见到具有这些特性的雷公藤甲素衍生物的合成和应用的报道。
发明内容
本发明的目的之一是提供特征为单氨基酸酯化通式为(I)的新型C14-位羟基酯化的雷公藤甲素氨基酸衍生物或其药学上可接受的加成物、复合物，盐、分解产物和代谢产物，以及提供特征为二肽酯化通式为(II)的新型C14-位羟基酯化的雷公藤甲素氨基酸衍生物或其药学上可接受的加成物、复合物，盐、分解产物和代谢产物：
Figure PCTCN2015093161-appb-000002
其中R1选自有取代或无取代的C2-C20烷基、有取代或无取代的C2-C20烯烃基、有取代或无取代的C4-C20的共扼烯烃基、有取代或无取代的C3-C7环烷基或环烯烃基、有取代或无取代的芳烃基、有取代或无取代的杂环基和芳香杂环基、有取代或无取代的氨基酸侧链烷基；氨基酸可以是消旋的，也可以是光学纯(左旋或右旋)；取代基选自卤素，胺基，C1-C6取代胺基，硝基，氰基，羟基，C1-C6烷氧基，巯基，C1-C6烷硫基；
其中R2和R3选自H、有取代或无取代的C1-C20烷基、有取代或无取代的C2-C20烯烃基，有取代或无取代的C4-C20的共扼烯烃基，有取代或无取代的C3-C7环烷基或环烯烃基，有取代或无取代的芳烃基，有取代或无取代的杂环基和芳香杂环基，有取代或无取代的氨基酸侧链烷基，氨基酸可以是消旋的，也可以是光学纯(左旋或右旋)；取代基选自卤素，胺基，C1-C6取代胺基，硝基，氰基，羟基，C1-C6烷氧基，巯基，C1-C6烷硫基；R2和R3可以是Boc，Fmoc，Bn，Cbz等保护基；R2和R3可以相同，也可以不相同。
其中R4选自有取代或无取代的C2-C20烷基、有取代或无取代的C2-C20烯烃基，有取代或无取代的C4-C20的共扼烯烃基，有取代或无取代的C3-C7环烷基或环烯烃基，有取代或无取代的芳烃基，有取代或无取代的杂环基和芳香杂环基，有取代或无取代的氨基酸侧链烷基，氨基酸可以是消旋的，也可以是光学纯(左旋或右旋)；取代基选自卤素，胺基，C1-C6取代胺基，硝基，氰基，羟基，C1-C6 烷氧基，巯基，C1-C6烷硫基；
其中R5和R6选自H、有取代或无取代的C1-C20烷基、有取代或无取代的C2-C20烯烃基，有取代或无取代的C4-C20的共扼烯烃基，有取代或无取代的C3-C7环烷基或环烯烃基，有取代或无取代的芳烃基，有取代或无取代的杂环基和芳香杂环基，有取代或无取代的氨基酸侧链烷基，氨基酸可以是消旋的，也可以是光学纯的而包括左旋的和右旋的；取代基选自卤素，胺基，C1-C6取代胺基，硝基，氰基，羟基，C1-C6烷氧基，巯基，C1-C6烷硫基。R5和R6可以是Boc，Fmoc，Bn,Cbz等保护基；R5和R6可以相同，也可以不相同。
本发明的目的之二是提供制备本发明单氨基酸酯化通式为(I)和二肽酯化通式为(II)C14-位羟基酯化的雷公藤甲素氨基酸衍生物的方法，其按以下工艺路线进行：
Figure PCTCN2015093161-appb-000003
单氨基酸酯化通式为(I)的C14-位羟基酯化的雷公藤甲素氨基酸衍生物可由雷公藤甲素与相应的氨基酸R2R3NCHR1CO2H在有缩合剂或者催化剂的存在下，在合适的溶剂中，适当的温度下经缩合酯化而成；也可以由雷公藤甲素与相应的氨基酸酰氯R2R3NCHR1COCl在碱性试剂存在下，缩合酯化而成；或者将氨基酸R2R3NCHR1CO2H活化生成活性酯中间体，而后与雷公藤甲素反应生成C14-位羟基酯化的雷公藤甲素氨基酸衍生物(I)；也可以将氨基酸R2R3NCHR1CO2H活化生成混合酸酐中间体，而后与雷公藤甲素反应生成C14-位羟基酯化的雷公藤甲素氨基酸衍生物(I)。式(I)中R1、R2、R3、氨基酸R2R3NCHR1CO2H中R1、R2、R3，氨基酸酰氯R2R3NCHR1COCl中R1、R2、R3，活性酯中间体R1、R2、R3，和混合酸酐中间体R1、R2、R3，与上文在式(I)中的定义相同。
本发明也提供制备本发明通式(II)的C14-位羟基二肽酯化的雷公藤甲素氨基酸衍生物的方法：C14-位羟基二肽酯化的雷公藤甲素氨基酸衍生物(II)可按上述方法，先制取C14-位羟基酯化的雷公藤 甲素氨基酸衍生物(I)，所用氨基酸R2R3NCHR1CO2H中，R2或R3为保护基。这些氨基的保护基是本领域技术人员已知的，并且能容易地在文献资源中找到，如Greene和Wuts的《Protective Groups in Organic Synthesis》第三版，John Wiley&Sons出版社，纽约，NY，1999，在此通过引用将其全部结合到本说明书中。而后脱除保护基得到通式为Ia的衍生物，再与相应的氨基酸R5R6NCHR4CO2H在有缩合剂或者催化剂的存在下，在合适的溶剂中，适当的温度下经缩合酰胺化而成C14-位羟基二肽酯化的雷公藤甲素氨基酸衍生物(II)；也可以由脱除保护基得到通式为Ia的衍生物与相应的氨基酸酰氯R5R6NCHR4COCl在碱性试剂存在下，缩合酰胺化而成；或者将氨基酸R5R6NCHR4CO2H活化生成活性酯中间体，而后与C14-位羟基酯化的雷公藤甲素氨基酸衍生物(Ia)反应生成；也可以将氨基酸R5R6NCHR4CO2H活化生成混合酸酐中间体，而后与C14-位羟基酯化的雷公藤甲素氨基酸衍生物(Ia)反应生成。当R5或R6为保护基时，可进一步脱保护基得到化合物IIa。式(II)中R1,R2,R3,R4,R5和R6、氨基酸R2R3NCHR1CO2H中R1、R2、R3，氨基酸酰氯R2R3NCHR1COCl中R1、R2、R3，活性酯中间体R1、R2、R3，和混合酸酐中间体R1、R2、R3与上文在式(II)中的定义相同。氨基酸R5R6NCHR4CO2H中R4、R5、R6，氨基酸酰氯R5R6NCHR4COCl中R4、R5、R6，活性酯中间体R4、R5、R6，和混合酸酐中间体R4、R5、R6与上文在式(II)中的定义相同。
本发明的目的之三是提供一种药物组合物，所述药物组合物包括至少一种本发明化合物，和任选的药学上可以接受的赋形剂，所述化合物选自通式(I)的C14-位羟基单氨基酸酯化的雷公藤甲素氨基酸衍生物和通式(II)的C14-位羟基二肽酯化的雷公藤甲素氨基酸衍生物。
本发明的目的之四是提供本发明化合物或包含该化合物的药物组合物在制备药物、特别是抗肿瘤药物中的用途。相应地，本发明提供一种治疗肿瘤患者的方法，包括给予需要治疗的患者治疗有效量的至少一种本发明的化合物。所述肿瘤特别选自白血病、多发性骨髓瘤、淋巴瘤、肝癌、胃癌、乳腺癌、胆管细胞癌、胰腺癌、肺癌、大肠癌、骨肉瘤、黑色素瘤、人宫颈癌、神经胶质瘤、鼻咽癌、喉癌、食管癌、中耳肿瘤、前列腺癌等。所述化合物选自通式(I)的C14-位羟基单氨基酸酯化的雷公藤甲素氨基酸衍生物和通式(II)的C14-位羟基二肽酯化的雷公藤甲素氨基酸衍生物。
本发明的目的之五是提供本发明化合物或包含该化合物的药物组合物在制备治疗自身免疫性疾病药物中的用途。所述自身免疫性疾病特别选自T细胞或B细胞等免疫细胞功能异常相关的自身免疫性疾病如类风湿性关节炎、系统性红斑狼疮、银屑病、肾病等。所述化合物选自通式(I)的C14-位羟基单氨基酸酯化的雷公藤甲素氨基酸衍生物和通式(II)的C14-位羟基二肽酯化的雷公藤甲素氨基酸衍生物。
具体实施方式
本发明涉及单氨基酸酯化通式为(I)和二肽酯化通式为(II)的新型C14-位羟基酯化的雷公藤甲素 氨基酸衍生物或其药学上可接受的加成物，复合物，盐、分解产物和代谢产物：
Figure PCTCN2015093161-appb-000004
其中R1选自取代或无取代的C2-C20烷基、取代或无取代的C2-C20烯烃基，取代或无取代的C4-C20的共扼烯烃基，取代或无取代的C3-C7环烷基或环烯烃基，取代或无取代的芳烃基，取代或无取代的杂环基和芳香杂环基，取代或无取代的氨基酸侧链烷基，氨基酸可以是消旋的，也可以是光学纯的(左旋或右旋)；取代基选自卤素，胺基，C1-C6取代胺基，硝基，氰基，羟基，C1-C6烷氧基，巯基，C1-C6烷硫基。
其中R2,R3,R4,R5和R6选自H、取代或无取代的C1-C20烷基、取代或无取代的C2-C20烯烃基，取代或无取代的C4-C20的共扼烯烃基，取代或无取代的C3-C7环烷基或环烯烃基，取代或无取代的芳烃基，取代或无取代的杂环基和芳香杂环基，取代或无取代的氨基酸侧链烷基，氨基酸可以是消旋的，也可以是光学纯的(左旋或右旋)；取代基选自卤素，胺基，C1-C6取代胺基，硝基，氰基，羟基，C1-C6烷氧基，巯基，C1-C6烷硫基。R2和R3,R5和R6可以是Boc，Fmoc，Bn,Cbz等保护基；R2和R3可以相同，也可以不相同。R5和R6可以相同，也可以不相同。
在一种实施方式中，本发明涉及通式I或通式II的化合物，其中R1,R4,选自取代或无取代的C2-C20烷基。R1和R4可以相同，也可以不相同。
在一种实施方式中，本发明涉及通式I或通式II的化合物，其中R2,R3,R5和R6选自H、取代或无取代的C1-C20烷基。R2,R3,R5和R6可以相同，也可以不相同。
在一种实施方式中，本发明涉及通式I或通式II的化合物，其中R1,R2,R3,R4,R5和R6选自取代或无取代的C2-C20烯烃基、取代或无取代的C4-C20的共扼烯烃基。R1,R2,R3R4,R5和R6可以相同，也可以不相同。
在一种实施方式中，本发明涉及通式I或通式II的化合物，其中R1,R2,R3,R4,R5和R6选自取代或无取代的C3-C7环烷基或环烯烃基。R1,R2,R3,R4,R5和R6可以相同，也可以不相同。
在一种实施方式中，本发明涉及通式I或通式II的化合物，其中R1,R2,R3,R4,R5和R6选自取代或无取代的芳烃基。R1,R2,R3,R4,R5和R6可以相同，也可以不相同。
在一种实施方式中，本发明涉及通式I或通式II的化合物，其中R1,R2,R3,R4,R5和R6选自取代或无取代的杂环基或芳香杂环基。R1,R2,R3,R4,R5和R6可以相同，也可以不相同。
在一种实施方式中，本发明涉及通式I或通式II的化合物，其中R1,R2,R3,R4,R5和R6选自取代或无取代的氨基酸侧链烷基。R1,R2,R3,R4,R5和R6可以相同，也可以不相同。
本发明的部分雷公藤甲素氨基酸衍生物如下所示。这些实施例举只对本发明做进一步说明，并不对本发明的范围构成任何限制。
Figure PCTCN2015093161-appb-000005
上面所列化合物的部分数据如下表所示：
Figure PCTCN2015093161-appb-000006
在另一种实施方式中，本发明特别优选如下的通式(I)化合物：
14-D-Valine-TPL(14-右旋缬氨酸-雷公藤甲素)
Figure PCTCN2015093161-appb-000007
14-L-Valine-TPL(14-左旋缬氨酸-雷公藤甲素)
Figure PCTCN2015093161-appb-000008
在另一种实施方式中，本发明特别优选如下的通式(II)化合物：
14-D-Valine-D-Valine-TPL(14-右旋缬氨酸-右旋缬氨酸-雷公藤甲素)
Figure PCTCN2015093161-appb-000009
14-L-Valine-L-Valine-TPL(14-左旋缬氨酸-左旋缬氨酸-雷公藤甲素)
Figure PCTCN2015093161-appb-000010
14-L-Phenylalanyl-L-tyrosine-TPL(14-左旋苯丙氨酸-左旋酪氨酸-雷公藤甲素)
Figure PCTCN2015093161-appb-000011
14-L-Valine-D-Valine-TPL(14-左旋缬氨酸-右旋缬氨酸-雷公藤甲素)
Figure PCTCN2015093161-appb-000012
本发明涉及以下形式的本发明的通式(I)和通式(II)化合物：它们的盐、溶剂化物、水合物、加成物、复合物、多晶型物或前药。
如本文所使用，术语“C1-C20烷基”是指含有1-20个碳原子的直链或支链、取代或无取代的烷烃基。C1-C20烷烃基的例子包括但不限于甲基、乙基、正丙基、异丙基、正丁基,异丁基,叔丁基、正戊基、正己基和正二十烷基。
术语“C2-C20烷基”是指含有2-20个碳原子的直链或支链、取代或无取代的烷烃基。C2-C20烷烃基的例子包括但不限于乙基、正丙基、异丙基、正丁基,异丁基,叔丁基、正戊基、正己基和正二十烷基。
术语“C2-C20烯烃基”是指含有2-20个碳原子的直链或支链、取代或无取代的烯烃基。C2-C20烯烃基的例子包括但不限于乙烯基、烯丙基、和二十烯烃基。
术语“C4-C20共扼烯烃基”是指含有4-20个碳原子的直链或支链、取代或无取代的共扼烯烃基。C4-C20共扼烯烃基的例子包括但不限于共扼丁二烯烃基、(视黄酰)基和(9Z)-十八(酰)-9-烯烃基。
术语“C3-C7环烷基或环烯烃基”是指具有饱和或不饱和环的3-7元单环系统的烃基，C3-C7环烷基可以为环丙基、环丁基、环戊基、环己基、环庚基、环丙烯基和环烯己基。
术语“芳烃基”是指无杂原子的芳烃基，包括芳烃基，芳烃基烷基和烷基芳烃基。
术语“杂环芳烃基”是指含有杂原子的芳烃基，包括杂环芳烃基，杂环芳烃基烷基和烷基杂环芳烃基。杂原子是指氮，氧和硫。杂环芳烃基可含有一个或同时含有数个杂原子。
术语“氨基酸”是指天然氨基酸和非天然氨基酸。
术语“卤素”是指氟、氯、溴或碘。
术语“C1-C6取代胺基”是指-N-C1-C6烷基和-N-C3-C6环烷基。
术语“C1-C6烷氧基”是指-O-C1-C6烷基和-O-C3-C6环烷基。
术语“C1-C6烷硫基”是指-S-C1-C6烷基和-S-C3-C6环烷基。
术语“式(I)和式(II)化合物的药学上可以接受的加成物和复合物”是指本发明的化合物进一步与其他小分子或生物大分子以非化学键或者非共价分子间力结合的产物。
如本文所使用，术语“通式(I)和通式(II)化合物的药学上可以接受的盐”是指本发明的化合物与药学上可以接受的阴离子的有机酸形成的有机酸盐，这些有机酸盐包括但不限于甲苯磺酸盐、甲磺酸盐、苹果酸盐、醋酸盐、柠檬酸盐、丙二酸盐、酒石酸盐、琥珀酸盐、苯甲酸盐、抗坏血酸盐、乳酸盐、α-酮戊二酸盐和α-甘油磷酸盐；也可形成合适的无机盐，包括但不限于盐酸盐、硫酸盐、硝酸盐、碳酸氢盐和碳酸盐、磷酸盐、氢溴酸盐、氢碘酸盐等。
药学上可以接受的盐可使用本领域熟知的标准程序获得，例如，通过将足量的碱性化合物和提供药学上可以接受的阴离子的合适的酸反应生成。
如本文所使用，术语“多晶型物”是指本发明的化合物或其复合物的固体晶体形式。相同化合物的不同的多晶型物可以显示不同的物理、化学和/或光谱性质。不同的物理性质包括但不限于稳定性(例如，对热或光)、可压缩性和密度(对于配制制剂和产品生产是重要的)和溶解速率(其可以影响生物吸收度和利用度)。稳定性的不同会造成化学反应性(例如差异氧化，使得当由一种多晶型物构成时比由另一多晶型物构成时剂型更快地褪色)或机械性能(例如，储存时作为动力学有利的多晶型物的片剂碎末转化成热力学更加稳定的多晶型物)或两者(例如，一种多晶型物的片剂在高潮湿度时更加容易破碎)中的变化。多晶型物的不同的物理性质可以影响它们的加工。例如，一种多晶型物可能比另一种更可能形成溶剂化物或可能比另一种更加难以过滤或洗去杂质，这是由于例如其颗粒的形状或大小分布。
如本文所使用，术语“水合物”是指本发明的化合物或其盐，其进一步包含通过非共价分子间力结合的化学计量的或非化学计量的量的水。
如本文所使用，除非另外说明，术语“前药”是指可以在生物学条件(体外或体内)下水解、氧化或进行其他反应以提供本发明的化合物的衍生物。前药仅在生物学条件下经过该反应成为活性化合物，或者它们在它们不反应的形式中具有活性。通常可以使用公知的方法制备前药，例如1 Burger's Medicinal Chemistry and Drug Discovery(1995)172-178,949-982(Manfred E.Wolff编,第5版)和J.Rautio’s Prodrugs and Targeted Delivery(2011)31-60(Wiley-VCH,Methods and Principles in Medicinal Chemistry第47卷)和G.Thomas’s Fundamentals of Medicinal Chemistry(2003)195-200(Wiley)中描述的那些方法。
本发明化合物中雷公藤甲素酯衍生物具有通式I(有十个手性中心，九个手性中心源于雷公藤甲素，一个手性中心源于氨基酸)；和通式II(有十一个手性中心，九个手性中心源于雷公藤甲素，二个手性中心源于氨基酸)结构式所显示的立体化学结构。本文使用的立体化学的定义和约定一般遵循McGraw-Hill Dictionary of Chemical Terms(S.P.Parker,Ed.,McGraw-Hill Book Company,New York,1984)；Eliel,E.和Wilen,S.,Stereochemistry of Organic Compounds(John Wiley&Sons,Inc.,New York,1994)。许多有机化合物以光学活性形式存在，即它们具有旋转平面偏光的平面的能力。
本文使用的术语“治疗”一般是指获得需要的药理和/或生理效应。该效应根据完全或部分地预防疾病或其症状，可以是预防性的；和/或根据部分或完全稳定或治愈疾病和/或由于疾病产生的副作用，可以是治疗性的。本文使用的“治疗”涵盖了对患者疾病的任何治疗，包括：(a)预防疾病或症状但还没诊断出患病的患者所发生的疾病或症状；(b)抑制疾病的症状，即阻止其发展；或(c)缓解疾病的症 状，即导致疾病或症状退化。
本发明的化合物可以按照常规的有机化学合成方法制备。例如，本发明的通式(I)化合物的通用制备方法如下：
Figure PCTCN2015093161-appb-000013
单氨基酸酯化通式为(I)的C14-位羟基酯化的雷公藤甲素氨基酸衍生物可由天然提取分离的雷公藤甲素(TPL)与相应的氨基酸R2R3NCHR1CO2H在有缩合剂或者催化剂的存在下，在合适的溶剂中，适当的温度下经缩合酯化而成；也可以由雷公藤甲素与相应的氨基酸酰氯R2R3NCHR1COCl在碱性试剂存在下，缩合酯化而成；或者将氨基酸R2R3NCHR1CO2H活化生成活性酯中间体，而后与雷公藤甲素反应生成C14-位羟基酯化的雷公藤甲素氨基酸衍生物(I)；也可以将氨基酸R2R3NCHR1CO2H活化生成混合酸酐中间体，而后与雷公藤甲素反应生成C14-位羟基酯化的雷公藤甲素氨基酸衍生物(I)。式(I)中R1、R2、R3、氨基酸R2R3NCHR1CO2H中R1、R2、R3，氨基酸酰氯R2R3NCHR1COCl中R1、R2、R3，活性酯中间体R1、R2、R3，和混合酸酐中间R1、R2、R3、与上文在式(I)中的定义相同。
本发明的通式(II)化合物的通用制备方法如下：
Figure PCTCN2015093161-appb-000014
C14-位羟基二肽酯化的雷公藤甲素氨基酸衍生物(II)可按上述方法，先制取C14-位羟基酯化的雷公藤甲素氨基酸衍生物(I)，所用氨基酸R2 R3NCHR1CO2H中，R2或R3为保护基。这些氨基的保护 基是本领域技术人员已知的，并且能容易地在文献资源中找到，如Greene和Wuts的《Protective Groups in Organic Synthesis》第三版，John Wiley&Sons出版社，纽约，NY，1999，在此通过引用将其全部结合到本文中。而后脱除保护基得到通式为Ia的衍生物，再与相应的氨基酸R5R6NCHR4CO2H在有缩合剂或者催化剂的存在下，在合适的溶剂中，适当的温度下经缩合酰胺化而成C14-位羟基二肽酯化的雷公藤甲素氨基酸衍生物(II)；也可以由脱除保护基得到通式为Ia的衍生物与相应的氨基酸酰氯R5R6NCHR4COCl在碱性试剂存在下，缩合酰胺化而成；或者将氨基酸R5R6NCHR4CO2H活化生成活性酯中间体，而后与C14-位羟基酯化的雷公藤甲素氨基酸衍生物(Ia)反应生成；也可以将氨基酸R5R6NCHR4CO2H活化生成混合酸酐中间体，而后与C14-位羟基酯化的雷公藤甲素氨基酸衍生物(Ia)反应生成。当R5和R6为保护基时，可进一步脱保护基得到化合物IIa。式(II)中R1,R2,R3,R4,R5和R6、氨基酸R2R3NCHR1CO2H中R1、R2、R3，氨基酸酰氯R2R3NCHR1COCl中R1、R2、R3，活性酯中间体R1、R2、R3，和混合酸酐中间体R1、R2、R3与上文在式(II)中的定义相同。氨基酸R5R6NCHR4CO2H中R4、R5、R6，氨基酸酰氯R5R6NCHR4COCl中R4、R5、R6，活性酯中间体R4、R5、R6，和混合酸酐中间体R4、R5、R6与上文在式(II)中的定义相同。
上述反应一般在有碱或碱性试剂存在下进行。这里碱可以是但不局限于有机碱。例如二异丙基乙基胺，三乙胺，二甲基胺基吡啶。
上述反应一般在溶剂中进行。使用的溶剂包括但不局限于非质子极性溶剂，例如二氯甲烷(DCM),二甲基亚砜(DMSO)、二甲基甲酰胺(DMF),二甲基乙酰胺(DMA),N-甲基吡咯(NMP)或四氢呋喃(THF)等。
上述反应的反应温度一般为0℃至50℃。一般随所用的反应原料以及碱的不同而变化。
制备反应的原料是雷公藤甲素(TPL)。该原料是由天然产物提取分离而得到，可以在市场上购买获得。
制备反应的有机酸、有机酸酐或有机酰氯全部可以在市场上购买获得。
常规的化学转换可用于实施本发明。本领域的技术人员可以决定用于这些化学转换的适当的化学试剂、溶剂、保护基和反应条件。相关信息描述于R.Larock，Comprehensive Organic Transformations，VCH出版商(1989)；T.W.Greene和P.G.M.Wuts，Protective Groups in Organic Synthesis，第3版，John Wiley and Sons(1999)；L.Fieser和M.Fieser，Fieser and Fieser’s Reagents for Organic Synthesis，John Wiley and Sons(1994)；及L.Paquette编辑的Encyclopedia of Reagents for Organic Synthesis，John Wiley and Sons(1995)及其后来的版本。
保护基指那些一旦连接活性部分(例如，羟基或氨基)，防止这些部分被后来的反应干扰并可在反应后通过常规的方法去除的基团。羟基保护基的例子包括但不限于，烷基、苯甲基、烯丙基、三苯 甲基(即，三苯基甲基)、酰基(例如，苯甲酰基、乙酰基或HOOC-X”-CO-，X”为亚烷基、亚链烯基、亚环烷基或亚芳基)、甲硅烷基(例如，三甲基甲硅烷基、三乙基甲硅烷基和叔丁基二甲基甲硅烷基)、烷氧基羰基、氨基羰基(例如，二甲基氨基羰基、甲基乙氨基羰基和苯基氨基羰基)、烷氧甲基、苯甲氧甲基和烷基巯甲基。氨基保护基的例子包括但不限于，烷氧基羰基、烷酰基、芳氧基羰基、芳基取代的烷基等。羟基和氨基保护基已在T.W.Greene和P.G..M.Wuts，Protective Groups in Organic Synthesis，第2版，John Wiley and Sons(1991)中讨论。羟基和氨基保护基都可在反应后通过常规的方法去除。
本发明还提供了包含本发明通式I和式II化合物的药物组合物。
本发明提供了这样的药物组合物，其中包含至少一种如上所述的本发明的式I或式II的化合物，和任选的药学上可以接受的赋形剂。
制备各种含有一定量的活性成分的药物组合物的方法是已知的，或根据本发明的公开内容对于本领域技术人员是显而易见的。如Remington’s Pharmaceutical Sciences,Martin,E.W.,ed.,Mack Publishing Company,19th ed.(1995)所述。制备所述药物组合物的方法包括掺入适当的药学赋形剂、载体、稀释剂等。
以已知的方法制造本发明的药物制剂，包括常规的混合、溶解或冻干方法。
本发明的化合物可以制成药物组合物，并向患者以适于选定的施用方式的各种途径施用，例如口服或肠胃外(通过静脉内、肌内、局部或皮下途径)。
因此，本发明的化合物结合药学上可以接受的载体(如惰性稀释剂或可同化的可食用的载体)可以全身施用，例如，口服。它们可以封闭在硬或软壳的明胶胶囊中，可以压为片剂。对于口服治疗施用，活性化合物可以结合一种或多种赋形剂，并以可吞咽的片剂、颊含片剂、含片、胶囊剂、酏剂、悬浮剂、糖浆、圆片等的形式使用。这种组合物和制剂应该包含至少0.01％的活性化合物。这种组合物和制剂的比例当然可以变化，可以占给定的单位剂型重量的大约0.1％至大约99％。在这种治疗有用的组合物中，活性化合物的量使得能够获得有效剂量水平。
片剂、含片、丸剂、胶囊剂等也可以包含：粘合剂，如黄蓍胶、阿拉伯胶、玉米淀粉或明胶；赋形剂，如磷酸氢二钙；崩解剂，如玉米淀粉、马铃薯淀粉、藻酸等；润滑剂，如硬脂酸镁；和甜味剂，如蔗糖、果糖、乳糖或阿司帕坦；或调味剂，如薄荷、冬青油或樱桃香味。当单位剂型是胶囊时，除了上面类型的材料，它还可以包含液体载体，如植物油或聚乙二醇。各种其他材料可以存在，作为包衣，或以其他方式改变固体单位剂型的物理形式。例如，片剂、丸剂或胶囊剂可以用明胶、蜡、虫胶或糖等包衣。糖浆或酏剂可以包含活性化合物，蔗糖或果糖作为甜味剂，对羟苯甲酸甲酯或对羟苯甲酸丙酯作为防腐剂，染料和调味剂(如樱桃香料或桔子香料)。当然，用于制备任何 单位剂型的任何材料应该是药学上可以接受的且以应用的量基本上无毒。此外，活性化合物可以掺入缓释制剂和缓释装置中。
活性化合物也可以通过输注或注射来静脉内或腹膜内施用。可以制备活性化合物或其盐的水溶液，任选地混和无毒的表面活性剂。也可以制备在甘油、液体聚乙二醇、甘油三乙酸酯及其混合物以及油中的分散剂。在普通的储存和使用条件下，这些制剂包含防腐剂以防止微生物生长。
适于注射或输注的药物剂型可以包括包含适于无菌的可注射或可输注的溶液或分散剂的即时制剂的活性成分(任选封装在脂质体中)的无菌水溶液或分散剂或无菌粉末。在所有情况下，最终的剂型在生产和储存条件下必须是无菌的、液体的和稳定的。液体载体可以是溶剂或液体分散介质，包括，例如水、乙醇、多元醇(例如，甘油、丙二醇、液体聚乙二醇等)、植物油、无毒的甘油酯及其合适的混合物。可以维持合适的流动性，例如，通过脂质体的形成，通过在分散剂的情况下维持所需的粒子大小，或通过表面活性剂的使用。可以通过各种抗细菌剂和抗真菌剂(如对羟苯甲酸酯、氯丁醇、苯酚、山梨酸、硫柳汞等)产生预防微生物的作用。在许多情况下，优选包括等渗剂，如糖、缓冲剂或氯化钠。通过使用延缓吸收剂的组合物(例如，单硬脂酸铝和明胶)可以产生可注射的组合物的延长吸收。
通过将合适的溶剂中的需要量的活性化合物与需要的上面列举的各种其他成分结合，然后进行过滤灭菌，制备无菌可注射溶液。在用于制备无菌注射溶液的无菌粉末的情况下，优选的制备方法是真空干燥和冷冻干燥技术，这会产生活性成分加上任何另外需要的以前无菌过滤溶液中存在的成分的粉末。
有用的固体载体包括粉碎的固体(如滑石、粘土、微晶纤维素、二氧化硅、氧化铝等)。有用的液体载体包括水、乙醇或乙二醇或水-乙醇/乙二醇混合物，本发明的化合物可以任选在无毒的表面活性剂的帮助下以有效含量溶解或分散在其中。可以加入佐剂(如香味)和另外的抗微生物剂来优化对于给定用途的性质。
增稠剂(如合成的聚合物、脂肪酸、脂肪酸盐和酯、脂肪醇、改性纤维素或改性无机材料)也可和液体载体用于形成可涂覆的糊剂、凝胶、软膏、肥皂等，直接用于使用者的皮肤上。
化合物或其活性盐或衍生物的治疗需要量，不仅取决于选择的特定的活性成分，而且取决于施药方式、待治疗的疾病的本质和患者的年龄和状态，最终取决于在场医师或临床医生的决定。
上述制剂可以以单位剂型存在，该单位剂型是含有单位剂量的物理分散单元，适于向人体和其它哺乳动物体给药。单位剂型可以是胶囊或片剂，或是很多胶囊或片剂。根据所涉及的具体治疗，活性成分的单位剂量的量可以在大约0.01毫克到大约1000毫克或更多之间进行变化或调整。
在下列实施例中，将更加具体地解释本发明。但应理解，下列实施例旨在说明本发明而不对本 发明的范围构成任何限制。
附图说明
图1为检测肿瘤细胞和正常细胞XPB和Pol II蛋白水平的免疫印迹图。
图2为检测14-D-Valine-TPL抑制人THP-1白血病细胞XPB和Pol II活性的免疫印迹图。
图3为检测14-D-Valine-TPL抑制人K562白血病细胞c-myc活性的免疫印迹图。
具体实施方式
以下实施例中所用的化学原料均为商购获得或通过本领域熟知的合成方法获得。
实施例1：化合物14-D-Valine-TPL(14-右旋缬氨酸-雷公藤甲素)的合成与鉴定
Figure PCTCN2015093161-appb-000015
式中，TPL：雷公藤甲素；D-Boc-Valine：右旋(N-Boc)缬氨酸；DIC：N,N'-二异丙基碳二亚胺；DMAP：4-二甲氨基吡啶；DCM：二氯甲烷；TFA:三氟乙酸。
第一步：将雷公藤甲素TPL(500mg,1.39mmol,1.0eq.),右旋(N-Boc)缬氨酸(1500mg,6.91mmol,5eq.)溶于二氯甲烷(20mL)，冷却到0℃.在0℃下向混合溶液中加入N,N'-二异丙基碳二亚胺(4mL,13mmol,9.35eq.)和4-二甲氨基吡啶(130mg，1.07mmol,0.8eq.)在25℃条件下反应24小时，反应液用乙酸乙酯稀释后用水洗，然后再用饱和氯化铵洗，干燥，浓缩，得到粗产品。再通过制备硅胶柱纯化，产生白色固体-D-Boc-Valine-TPL(14-右旋-Boc-缬氨酸-雷公藤甲素)(250mg)，收率为32.2％。
第二步：将上一步的产品，D-Boc-Valine-TPL(14-右旋-Boc-缬氨酸-雷公藤甲素)(200mg)溶于二氯甲烷(15mL)，滴加3mL三氟乙酸。加完后，反应液在25℃条件下反应3.5小时。反应液用二氯甲烷稀释后用碳酸氢钠水溶液洗，然后干燥，浓缩，得到粗产品。再通过制备高效液相色谱法纯化，产生白色固体D-Valine-TPL(14-右旋-缬氨酸-雷公藤甲素)(120mg)，收率为73％。
LC-MS:保留时间:0.83min(UV220：97.8％),m/z:460.28(M+H).
1H NMR(400MHz,CDCl3)δ5.11(s,1H),4.78–4.62(m,2H),4.01(s,1H),3.86(d,J＝2.7Hz,1H),3.58(d,J＝2.4Hz,1H),3.52(d,J＝4.8Hz,1H),2.72(d,J＝11.9Hz,1H),2.47(s,1H),2.33(d,J＝16.3 Hz,1H),2.26–2.08(m,2H),1.94(dd,J＝36.2,22.1Hz,2H),1.58(dd,J＝12.0,4.5Hz,1H),1.23(d,J＝5.6Hz,4H),1.16(d,J＝4.8Hz,3H),1.03(s,3H),0.93(d,J＝6.4Hz,3H),0.81(d,J＝6.5Hz,3H).
实施例2：化合物14-L-Valine-D-Valine-TPL(14-左旋缬氨酸-右旋缬氨酸-雷公藤甲素)的合成与鉴定
Figure PCTCN2015093161-appb-000016
式中，D-Valine-TPL：14-右旋-缬氨酸-雷公藤甲素；L-Boc-Val：左旋(N-Boc)缬氨酸；HATU：2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯；DIPEA：N,N-二异丙基乙胺；DCM：二氯甲烷；TFA:三氟乙酸.
第一步：将D-Valine-TPL(14-右旋-缬氨酸-雷公藤甲素)(110mg，实施例1),左旋(N-Boc)缬氨酸(1.2eq.)溶于二氯甲烷(10mL)，向混合溶液中加入HATU(2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯,1.5eq.)；DIPEA(N,N-二异丙基乙胺,2eq.)在25℃条件下反应半小时，反应液用1M碳酸钾溶液洗，干燥，浓缩，得到粗产品。再通过制备硅胶柱纯化，产生白色固体14-L-Boc-Valine-D-Valine-TPL(14-左旋(N-Boc)-缬氨酸-右旋缬氨酸-雷公藤甲素(85mg)。
第二步：将上一步的产品，14-L-Boc-Valine-D-Valine-TPL(14-左旋(N-Boc)-缬氨酸-右旋缬氨酸-雷公藤甲素)(85mg)溶于二氯甲烷(5mL)，滴加1mL三氟乙酸。加完后，反应液在25℃条件下反应3.5小时。反应液用二氯甲烷稀释后用碳酸氢钠水溶液洗，然后干燥，浓缩，得到粗产品。再通过制备高效液相色谱法纯化，产生白色固体14-L-Valine-D-Valine-TPL(14-左旋缬氨酸-右旋缬氨酸-雷公藤甲素)(51.5mg)。
LC-MS:保留时间:0.96min(UV220：97.7％),m/z:559.44(M+H).
1H NMR(400MHz,CDCl3)δ7.52(d,J＝7.8Hz,1H),5.60(b,3H),5.06(s,1H),4.80–4.65(m,2H),4.58(dd,J＝7.8,4.0Hz,1H),4.20(s,1H),3.88(d,J＝2.7Hz,1H),3.57(d,J＝2.2Hz,1H),3.50(d,J＝ 5.3Hz,1H),2.72(d,J＝11.7Hz,1H),2.47–2.09(m,5H),1.92(dd,J＝17.9,10.2Hz,2H),1.57(dd,J＝11.5,5.1Hz,1H),1.25(dd,J＝12.2,5.5Hz,2H),1.13–1.04(m,11H),1.02(s,3H),0.92(t,J＝8.4Hz,3H),0.83(d,J＝6.7Hz,3H).
实施例3：化合物14-L-Valine-TPL(14-左旋缬氨酸-雷公藤甲素)的合成与鉴定
化合物14-L-Valine-TPL(14-左旋缬氨酸-雷公藤甲素)按实施例1的方法合成，用L-Boc-Valine(左旋-N-Boc-缬氨酸)代替D-Boc-Valine-TPL(右旋N-Boc-缬氨酸。
LC-MS:保留时间:0.96min(UV220,100％),m/z:460.28(M+H)
1H NMR(300MHz,CDCl3)δ5.10(d,J＝5.4Hz,1H),4.73–4.60(m,2H),3.99(s,1H),3.91–3.79(m,1H),3.63–3.44(m,2H),2.67(s,1H),2.55–1.79(m,7H),1.65–1.47(m,1H),1.31–1.09(m,7H),1.01(s,3H),0.97–0.86(m,3H),0.85–0.72(m,3H).
实施例4：化合物14-L-Valine-L-Valine-TPL(14-左旋缬氨酸-左旋缬氨酸-雷公藤甲素)的合成与鉴定
化合物14-L-Valine-L-Valine-TPL(14-左旋缬氨酸-左旋缬氨酸-雷公藤甲素)按实施例2的方法合成，用14-L-Valine-TPL(14-左旋缬氨酸-雷公藤甲素，实施例3)代替14-D-Valine-TPL(14-右旋缬氨酸-雷公藤甲素，实施例1)，用L-Boc-Valine(左旋-N-Boc-缬氨酸)代替D-Boc-Valine-TPL(右旋N-Boc-缬氨酸)。
LC-MS:保留时间:1.78min(ELSD：99.6％),m/z:559.50(M+H).
1H NMR(400MHz,CDCl3)δ7.83(s,1H),5.08(d,J＝7.5Hz,1H),4.75–4.65(m,2H),4.57(dd,J＝8.7,4.7Hz,1H),3.84(dd,J＝5.8,3.2Hz,1H),3.53(ddd,J＝17.4,11.7,4.1Hz,2H),2.70(s,1H),2.43–2.27(m,3H),2.23–2.12(m,2H),2.04–1.88(m,2H),1.67–1.54(m,1H),1.30–1.20(m,1H),1.12–0.99(m,10H),0.95(t,J＝7.5Hz,5H),0.90–0.77(m,3H).
实施例5：化合物14-D-Valine-D-Valine-TPL(14-右旋缬氨酸-右旋缬氨酸-雷公藤甲素)的合成与鉴定
化合物14-D-Valine-D-Valine-TPL(14-右旋缬氨酸-右旋缬氨酸-雷公藤甲素按实施例2的方法合成，用D-Boc-Valine(右旋-N-Boc-缬氨酸)代替L-Boc-Valine-TPL(左旋N-Boc-缬氨酸)。
LC-MS:保留时间:1.95min(ELSD：98.0％),m/z:559.50(M+H).
1H NMR(400MHz,CDCl3)δ7.81(d,J＝8.8Hz,1H),5.08(s,1H),4.69(s,2H),4.61(dd,J＝8.8,4.5Hz,1H),3.84(d,J＝3.2Hz,1H),3.57(d,J＝2.7Hz,1H),3.49(d,J＝5.8Hz,2H),2.74–2.68(m,1H),2.39–2.27(m,3H),2.22–2.10(m,2H),1.94(ddd,J＝25.7,16.2,10.6Hz,2H),1.59(dd,J＝12.4,4.6Hz,1H),1.28–1.20(m,1H),1.06(dt,J＝12.6,6.1Hz,12H),0.95(dd,J＝12.7,6.9Hz,6H),0.85(d,J＝6.9Hz, 3H).
实施例6：化合物14-L-Phenylalanyl-L-tyrosine-TPL(14-左旋苯丙氨酸-左旋酪氨酸-雷公藤甲素)的合成与鉴定
式中，TPL：雷公藤甲素；L-Boc-O-TBDMS-Tyrosine：左旋(N-Boc-O-叔丁基二甲基硅)酪氨酸；DIC：N,N'-二异丙基碳二亚胺；DMAP：4-二甲氨基吡啶；DCM：二氯甲烷；TFA:三氟乙酸.TBAF：四丁基氟化铵；
D-Valine-TPL：14-右旋-缬氨酸-雷公藤甲素；L-Boc-Phenylalanine：左旋(N-Boc)苯丙氨酸；HATU：2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯；DIPEA：N,N-二异丙基乙胺；
化合物L-Tyrosine-TPL(14-左旋酪氨酸-雷公藤甲素)按实施例1的方法合成，用L-Boc-O-TBDMS-Tyrosine：左旋(N-Boc-O-叔丁基二甲基硅)酪氨酸代替D-Boc-Valine-TPL(右旋N-Boc-缬氨酸)。
化合物14-L-Phenylalanyl-L-tyrosine-TPL(14-左旋苯丙氨酸-左旋酪氨酸-雷公藤甲素按实施例2的方法合成，用化合物L-Tyrosine-TPL(14-左旋酪氨酸-雷公藤甲素)代替14-D-Valine-TPL(14-右旋缬氨酸-雷公藤甲素，实施例1)，用L-Boc-Phenylalanine(左旋(N-Boc)苯丙氨酸)代替D-Boc-Valine-TPL(右旋N-Boc-缬氨酸)。
LC-MS:保留时间:1.06min(UV220：97.8.0％),m/z:671.56(M+H).
1H NMR(400MHz,DMSO)δ9.34(s,0H),9.17(s,0H),8.07(d,J＝15.1Hz,1H),7.42–7.14(m,2H), 7.13–7.03(m,1H),6.89(d,J＝7.6Hz,0H),6.74–6.62(m,1H),5.01(d,J＝6.3Hz,0H),4.82(d,J＝17.7Hz,1H),4.64–4.58(m,0H),4.03(d,J＝3.2Hz,1H),3.94(d,J＝3.2Hz,0H),3.25(d,J＝14.2Hz,0H),3.15(s,0H),3.05(dd,J＝13.9,6.2Hz,0H),2.94–2.77(m,1H),2.64(s,1H),2.31–2.07(m,1H),1.92(d,J＝24.5Hz,1H),1.87–1.72(m,1H),1.66–1.53(m,1H),1.38–1.17(m,1H),1.04(d,J＝6.8Hz,0H),0.97–0.88(m,2H),0.80(dd,J＝6.7,3.8Hz,1H),0.65(d,J＝6.8Hz,3H).
实施例7：本发明的化合物14-D-Valine-TPL与雷公藤甲素(TPL)治疗窗比较
(1)实验材料
白血病细胞株：Sup-B15(Ph+急性淋巴细胞白血病)、CEM(急性淋巴细胞白血病，ALL)、Molt-4(急性淋巴细胞白血病，ALL)。正常血细胞样本：来自健康志愿者外周血。
主要试剂：本发明的14-D-Valine-TPL。主要仪器：细胞培养箱，酶标仪
(2)实验方法
取生长良好的白血病细胞或正常人血细胞5000个，接种到96孔细胞培养板孔内。培养液为含10％胎牛血清的1640细胞培养液。加入不同浓度的14-D-Valine-TPL，混匀后，置于二氧化碳(5％CO2)细胞培养箱37℃培养72小时。然后用MTT法测定活细胞浓度。在本实验中对照组(不加化合物处理)细胞活力设为100％，并计算出化合物作用后细胞活力(％)和72小时白血病细胞半数生长抑制浓度(72小时IC50值)以及治疗窗指数。治疗窗指数＝正常血细胞IC50值/白血病细胞IC50值。治疗窗指数<或＝1表明无治疗窗，化合物对正常细胞和肿瘤细胞毒性一样，无选择性。治疗窗指数>1表明有治疗窗，与正常血细胞相比，化合物对肿瘤细胞毒性更大，治疗窗越大，表明化合物对肿瘤细胞选择性越好。
(3)实验结果
实验结果如下表1所示本发明的化合物14-D-Valine-TPL具有较好的治疗窗，而雷公藤甲素基本没有治疗窗。表1显示雷公藤甲素(TPL)仅对Molt-4细胞有较小的治疗窗(2.0)，而对其它3种白血病细胞Jurkat、CEM和Sup-B15治疗窗均<1，分别为0.64、0.93和0.94，表明雷公藤甲素对正常细胞和肿瘤细胞毒性基本无选择性，这与文献报道的TPL基本无治疗窗的结果是一致的。相反，本发明的14-D-Valine-TPL对已检测的4种不同类型人白血病细胞治疗窗指数为5.54-18.58，表明14-D-Valine-TPL对4种白血病均有较大的治疗窗，其中对Molt-4细胞的治疗窗指数高达18.58。
表1 14-D-Valine-TPL与雷公藤甲素(TPL)治疗窗比较
  14-D-Valine-TPL   TPL  
白血病细胞系 IC50(nM/L) 治疗窗 IC50 治疗窗
Jurkat 36.301 5.54 33.829 0.64
CEM 25.488 7.89 23.286 0.93
Molt-4 10.824 18.58 10.718 2.03
Sup-B15 20.674 9.73 23.153 0.94
正常血细胞        
No1 202.706   29.289  
No2 198.826   20.772  
No3 205.784   9.383  
No4 199.835   8.968  
No5 213.719   20.072  
No6 189.872   20.445  
No7 203.546   19.515  
No8 184.509   9.710  
No9 210.835   57.443  
合计 201.07   21.733  
实施例8：本发明的化合物14-D-Valine-TPL体外抗白血病的活性测定
(1)实验材料
白血病细胞株：人KG-1a(急性髓系白血病、AML-M0)、THP-1(急性髓系白血病、AML-M5)NB4(急性早幼粒细胞白血病，AML)、Kasumi-1(急性髓系白血病M2型，AML-M2)、KG-1(急性髓系白血病，AML)、Jurkat(急性淋巴细胞白血病，ALL)、H9(急性淋巴细胞白血病，ALL)。
主要试剂：本发明的14-D-Valine-TPL。主要仪器：细胞培养箱，酶标仪
(2)实验方法
取生长良好的白血病细胞5000个，接种到96孔细胞培养板孔内。培养液为含10％胎牛血清的1640细胞培养液。加入不同浓度的14-D-Valine-TPL，混匀后，置于二氧化碳(5％CO2)细胞培养箱37℃培养72小时。然后用MTT法测定活细胞浓度。在本实验中对照组(不加化合物处理)细胞活力设为100％，并计算出化合物作用后细胞活力(％)和72小时白血病细胞半数生长抑制浓度(72小时IC50值)。
(3)实验结果
实验结果见表2。表2显示本发明的14-D-Valine-TPL能诱导人急性髓系白血病和急性淋巴细胞白血病细胞死亡和抑制这些白血病细胞生长。
表2本发明衍生物对不同类型白血病细胞生长抑制作用
  14-D-Val-TPL 14-L-Val-TPL 14-L-Val-14-L-Val-TPL
细胞系 IC50(nM/L) IC50(nM/L) IC50(nM/L)
KG-1 49.0 8.86  
KG-1a 31.0   20
THP-1 16.0 5.0  
HL-60 65.8    
U937 60.0    
Jurkat 36.301    
CEM 25.488    
Molt-4 10.824    
Sup-B15 20.674    
实施例9：本发明的化合物14-D-Valine-TPL小鼠体内抗人白血病的活性测定
(1)实验材料
实验动物：NOD/SCID小鼠购于中科院上海动物中心
白血病细胞株：人KG-1a(急性髓系白血病，AML-M0)、THP-1(急性单核细胞白血病，AML-M5)，购于ATCC库。
试剂：本发明的14-D-Valine-TPL三氟乙酸盐先用无菌PBS溶解，浓度为10mg/ml，然后再用无菌去离子水稀释至所需工作浓度。
主要仪器：细胞培养箱，酶标仪
(2)实验方法
取7周龄雌性NOD/SCID，以1X107细胞(0.2ml)每只小鼠的量接种在其腋下皮下，左边为人急性髓系白血病KG-1a细胞，右边为人急性单核细胞白血病THP-1细胞。待肿瘤瘤经长至0.5cm左右，随机分组，每组3只。对照组用等量PBS，14-D-Valine-TPL组分0.2mg/kg体重和0.4mg/kg体重两组，每天灌胃给药2次(上午8点钟和下午4点钟各一次)，连续治疗14天。共观察30天，期间测量小鼠瘤经、体重、活动、饮食等情况。实验结束时，处死小鼠，取肿瘤组织，称瘤重，检查小鼠肝、脾、心、肺、大肠、小肠等主要器官和组织。
(3)实验结果
实验结果见表3与表4。
表3 14-D-Valine-TPL(DV)对小鼠体内人急性髓系白血病KG-1a细胞生长影响
Figure PCTCN2015093161-appb-000018
Figure PCTCN2015093161-appb-000019
表4 14-D-Valine-TPL(DV)对小鼠体内人急性髓系白血病THP-1细胞生长影响
Figure PCTCN2015093161-appb-000020
注：DV0.2：0.2mg/kg；DV0.4：0.4mg/kg。
表3、表4显示本发明的14-D-Valine-TPL能显著抑制两种不同类型人急性髓系白血病细胞系KG-1a和THP-1在小鼠体内生长，并存在明显剂量依赖效应。每公斤体重0.4mg剂量的14-D-Valine-TPL能使KG-1a和THP-1移植瘤彻底消退，而小鼠体重未见明显减轻，小鼠解剖检查心脏、肺、肝脏、脾脏、大肠，小肠等主要器官未见明显异常。这些结果表明，本发明的14-D-Valine-TPL不仅具有明显抗不同类型人急性髓系白血病活性，而且未见明显毒性反应。
实施例10：14-L-Valine-L-Valine-TPL(14-LLV-TPL)小鼠体内抗人白血病的活性测定
(1)实验材料
实验动物：NOD/SCID小鼠购于中科院上海动物中心
白血病细胞株：人KG-1a(急性髓系白血病，AML-M0)购于ATCC库。
试剂：本发明的14-LLV-TPL三氟乙酸盐先用无菌PBS溶解，浓度为10mg/ml，然后再用无菌去离子水稀释至所需工作浓度。
主要仪器：细胞培养箱，酶标仪
(2)实验方法
取7周龄雌性NOD/SCID，以1X107细胞(0.2ml)每只小鼠的量接种在其腋下皮下，左边为人急性髓系白血病KG-1a细胞。待肿瘤瘤经长至50-100mm3左右，随机分组，每组3只。对照组用等量PBS，TPL-LLV组分0.23mg/kg体重和0.46mg/kg体重两组，每天灌胃给药2次(上午8点钟和下午4点钟各一次)，连续治疗14天。共观察30天，期间测量小鼠瘤经、体重、活动、饮食等情况。实验结束时，处死小鼠，取肿瘤组织，称瘤重，检查小鼠肝、脾、心、肺、大肠、小肠等主要器官和组织。
(3)实验结果
实验结果见表5。表5显示本发明的14-LLV-TPL能显著抑制人急性髓系白血病细胞系KG-1a在小鼠体内生长，并存在明显剂量依赖效应。
表5 14-LLV-TPL对小鼠体内人急性髓系白血病KG-1a细胞生长影响
Figure PCTCN2015093161-appb-000021
实施例11：14-L-Valine-D-Valine-TPL(14-DLV-TPL)小鼠体内抗人白血病的活性测定
(1)实验材料
实验动物：NOD/SCID小鼠购于中科院上海动物中心
白血病细胞株：人KG-1a(急性髓系白血病，AML-M0)购于ATCC库。
试剂：本发明的14-DLV-TPL三氟乙酸盐先用无菌PBS溶解，浓度为10mg/ml，然后再用无菌去离子水稀释至所需工作浓度。
主要仪器：细胞培养箱，酶标仪
(2)实验方法
取7周龄雌性NOD/SCID，以1X107细胞(0.2ml)每只小鼠的量接种在其腋下皮下，左边为人急性髓系白血病KG-1a细胞。待肿瘤瘤经长至50-100mm3左右，随机分组，每组3只。对照组用等量PBS，TPL-DLV组分0.23mg/kg体重和0.46mg/kg体重两组，每天灌胃给药2次(上午8点钟和下午4点钟各一次)，连续治疗14天。共观察30天，期间测量小鼠瘤经、体重、活动、饮食等情况。实验结束时，处死小鼠，取肿瘤组织，称瘤重，检查小鼠肝、脾、心、肺、大肠、小肠等主要器官和组织。
(3)实验结果
实验结果见表6。表6显示本发明的14-DLV-TPL能显著抑制人急性髓系白血病细胞系KG-1a在小鼠体内生长，并存在明显剂量依赖效应。
表6 14-DLV-TPL对小鼠体内人急性髓系白血病KG-1a细胞生长影响
Figure PCTCN2015093161-appb-000022
实施例12：14-L-Phenylalanyl-L-tyrosine-TPL(14-LPT-TPL)小鼠体内抗人白血病的活性测定
(1)实验材料
实验动物：NOD/SCID小鼠购于中科院上海动物中心
白血病细胞株：人KG-1a(急性髓系白血病，AML-M0)购于ATCC库。
试剂：本发明的14-LPT-TPL三氟乙酸盐先用无菌PBS溶解，浓度为10mg/ml，然后再用无菌去离子水稀释至所需工作浓度。
主要仪器：细胞培养箱，酶标仪
(2)实验方法
取7周龄雌性NOD/SCID，以1X107细胞(0.2ml)每只小鼠的量接种在其腋下皮下，左边为人急性髓系白血病KG-1a细胞。待肿瘤瘤经长至50-100mm3左右，随机分组，每组3只。对照组用等量PBS，TPL-DLV组分0.32mg/kg体重和0.64mg/kg体重两组，每天灌胃给药2次(上午8点钟和下午4点钟各一次)，连续治疗14天。共观察30天，期间测量小鼠瘤经、体重、活动、饮食等情况。实验结束时，处死小鼠，取肿瘤组织，称瘤重，检查小鼠肝、脾、心、肺、大肠、小肠等主要器官和组织。
(3)实验结果
实验结果见表7。表7显示本发明的14-LPT-TPL能显著抑制人急性髓系白血病细胞系KG-1a在小鼠体内生长，并存在明显剂量依赖效应。
表7 14-LPT-TPL对小鼠体内人急性髓系白血病KG-1a细胞生长影响
Figure PCTCN2015093161-appb-000023
实施例13：14-D-Valine-D-Valine-TPL(14-DDV-TPL)小鼠体内抗人白血病的活性测定
(1)实验材料
实验动物：NOD/SCID小鼠购于中科院上海动物中心。白血病细胞株：人K562/ADR(慢性髓系白血病，CML)购于ATCC库。试剂：本发明的14-DDV-TPL三氟乙酸盐先用无菌PBS溶解，浓度为10mg/ml，然后再用无菌去离子水稀释至所需工作浓度。主要仪器：细胞培养箱，酶标仪
(2)实验方法
取7周龄雌性NOD/SCID，以1X107细胞(0.2ml)每只小鼠的量接种在其腋下皮下，左边为人K562/ADR细胞。待肿瘤瘤经长至50-100mm3左右，随机分组，每组3只。对照组用等量PBS，TPL-DDV组分0.23mg/kg体重和0.46mg/kg体重两组，每天灌胃给药2次(上午8点钟和下午4点钟各一次)，连续治疗14天。共观察30天，期间测量小鼠瘤经、体重、活动、饮食等情况。实验结束时，处死小鼠，取肿瘤组织，称瘤重，检查小鼠肝、脾、心、肺、大肠、小肠等主要器官和组织。
(3)实验结果
实验结果见表8。表8显示本发明的14-DDV-TPL能显著抑制人慢性髓系白血病细胞系K562/ADR细胞器在小鼠体内生长，并存在明显剂量依赖效应。
表8 14-DDV-TPL对小鼠体内人慢性髓系白血病K562/ADR细胞生长影响
Figure PCTCN2015093161-appb-000024
实施例14：14-L-Valine-TPL(14-LV-TPL)小鼠体内抗人白血病的活性测定
(1)实验材料
实验动物：NOD/SCID小鼠购于中科院上海动物中心
白血病细胞株：人K562/ADR(慢性髓系白血病，CML)购于ATCC库。
试剂：本发明的14-LV-TPL三氟乙酸盐先用无菌PBS溶解，浓度为10mg/ml，然后再用无菌去离子水稀释至所需工作浓度。
主要仪器：细胞培养箱，酶标仪
(2)实验方法
取7周龄雌性NOD/SCID，以1X107细胞(0.2ml)每只小鼠的量接种在其腋下皮下，左边为人K562/ADR细胞。待肿瘤瘤经长至50-100mm3左右，随机分组，每组3只。对照组用等量PBS，TPL-LV组分0.2mg/kg体重和0.4mg/kg体重两组，每天灌胃给药2次(上午8点钟和下午4点钟各一次)，连续治疗14天。共观察30天，期间测量小鼠瘤经、体重、活动、饮食等情况。实验结束时，处死小鼠，取肿瘤组织，称瘤重，检查小鼠肝、脾、心、肺、大肠、小肠等主要器官和组织。
(3)实验结果
实验结果见表9。表9显示本发明的14-LV-TPL能显著抑制人慢性髓系白血病细胞系K562/ADR细胞器在小鼠体内生长，并存在明显剂量依赖效应。
表9 14-LV-TPL对小鼠体内人慢性髓系白血病K562/ADR细胞生长影响
Figure PCTCN2015093161-appb-000025
实施例15：14-D-Valine-TPL体外抑制T淋巴细胞和B淋巴细胞活性测定
(1)实验材料
白血病细胞株：Jurkat(T淋巴细胞)、H9(T淋巴细胞)、CEM(B淋巴细胞)和Sup-B15(B淋巴细胞)。
主要试剂：本发明的14-D-Valine-TPL
主要仪器：细胞培养箱，酶标仪
(2)实验方法
取生长良好的淋巴细胞5000个，接种到96孔细胞培养板孔内。培养液为含10％胎牛血清的1640细胞培养液。加入不同浓度的14-D-Valine-TPL，混匀后，置于二氧化碳(5％CO2)细胞培养箱37℃ 培养72小时。然后用MTT法测定活细胞浓度。在本实验中对照组(不加化合物处理)细胞活力设为100％，并计算出化合物作用后细胞活力(％)和72小时白血病细胞半数生长抑制浓度(72小时IC50值)。
(3)实验结果
实验结果见表10。表10显示本发明的14-D-Valine-TPL能显著抑制这些T淋巴细胞和B淋巴细胞生长。
表10 14-D-Valine-TPL对T/B淋巴细胞增殖抑制作用
  14-D-Valine-TPL
免疫细胞系 IC50(nM/L)
Jurkat(T淋巴细胞) 36.301
CEM(B淋巴细胞) 25.488
Molt-4(T淋巴细胞) 10.824
Sup-B15(B淋巴细胞) 20.674
实施例16：转录因子TFIIH亚单位XPB和RNA聚合酶II(Pol II)在不同类型肿瘤细胞及正常细胞中的表达
已有文献报道转录因子TFIIH亚单位XPB(着色性干皮病B组，xeroderma pigmentosum group B)和RNA聚合酶II(Pol II)是雷公藤甲素发挥药理作用的关键靶分子[Titov DV,et al.XPB,a subunit of TFIIH,is a target of the natural product triptolide.Nature chemical biology.2011,7:182-188]。为了解肿瘤细胞和正常细胞中XPB和Pol II表达水平是否不一样，本发明应用免疫印迹技术检测XPB和Pol II在不同类型肿瘤细胞和正常细胞中的蛋白质水平。
(1)实验材料
白血病细胞株：Nalm6(淋巴瘤)、Jurkat(急性淋巴细胞白血病)、THP-1(急性单核细胞白血病)、KG-1a(急性髓系白血病)、HL-60(急性髓系白血病)、NB4(急性早幼粒细胞白血病),U937(急性单核细胞白血病)。正常血细胞样本来自志愿者。
(2)实验方法采用常规免疫印迹技术。
按常规法提取白血病细胞和正常血细胞样本细胞蛋白，经SDS-PAGE蛋白电泳分离蛋白，然后转印到NC膜上，按常规法进行一抗和二抗孵育、显色、曝光等实验步骤。GAPDH作为内参蛋白。结果：如图1显示XPB和PolII蛋白质在多数肿瘤细胞中呈高表达状态，而在正常血细胞中低表达或不表达。这一结果表明XPB和PolII异常高表达相关疾病可能是雷公藤甲素及其衍生物的用药适应症。
图1为应用免疫印迹技术检测XPB和Pol II在不同类型肿瘤细胞及正常细胞中的表达水平结果。其中，1：正常血细胞；2：Nalm6；3：Jurkat；4：THP-1；5：KG-1a；6：HL-60；7：NB4；8：U937。
实施例17：14-D-Valine-TPL抑制人THP-1白血病细胞XPB和Pol II活性
已知XPB和Pol II是雷公藤甲素发挥药理作用的关键靶分子[Titov DV,et al.XPB,a subunit of TFIIH,is a target of the natural product triptolide.Nature chemical biology.2011,7:182-188]。为了解本发明的化合物是否具有抑制这两个靶分子的活性，本发明应用细胞培养技术和免疫印迹技术检测14-D-Valine-TPL对白血病细胞XPB和Pol II活性影响。
(1)实验材料
白血病细胞株：人THP-1白血病细胞株(急性髓系白血病-M5，AML-M5)。
试剂：14-D-Valine-TPL
(2)实验方法
取生长良好的白血病细胞接种到6孔细胞培养板孔内，密度为1×106/ml。培养液为含10％胎牛血清的1640细胞培养液。加入不同浓度的14-D-Valine-TPL，混匀后，置于二氧化碳(5％CO2)细胞培养箱37℃培养48小时。然后提取细胞蛋白，用免疫印迹检测XPB和Pol II表达水平。
(3)实验结果
实验结果见图2。14-D-Valine-TPL呈剂量依赖方式下调XPB和Pol II水平。160nM浓度14-D-Valine-TPL即可见明显抑制THP-1细胞XPB和Pol II水平。这些结果表明本发明的14-D-Valine-TPL化合物能抑制XPB和Pol II活性，可用于XPB和Pol II异常高表达相关疾病。
图2为应用细胞培养技术和免疫印迹技术检测14-D-Valine-TPL抑制人THP-1白血病细胞XPB和Pol II活性结果
实施例18：14-D-Valine-TPL抑制人K562白血病细胞c-myc致癌基因活性
已知致癌基因c-myc也是雷公藤甲素发挥抗癌药理作用的关键靶分子之一[Stéphane Vispé,,et al.Triptolide is an inhibitor of RNA polymerase I and II-dependent transcription leading predominantly to down-regulation of short-lived mRNA.Mol Cancer Ther 2009；8:2780-2790]。为了解本发明的化合物是否具有抑制c-myc的活性，本发明应用细胞培养技术和免疫印迹技术检测14-D-Valine-TPL对白血病细胞c-myc活性影响。
(1)实验材料
白血病细胞株：人K562白血病细胞株(慢性髓系白血病急变)。
试剂：14-D-Valine-TPL
(2)实验方法
取生长良好的白血病细胞接种到6孔细胞培养板孔内，密度为1×106/ml。培养液为含10％胎牛血清的1640细胞培养液。加入不同浓度的14-D-Valine-TPL，混匀后，置于二氧化碳(5％CO2)细胞培养箱37℃培养48小时。然后提取细胞蛋白，用免疫印迹检测c-myc蛋白表达水平。
(3)实验结果
实验结果见图3。14-D-Valine-TPL呈剂量依赖方式下调c-myc蛋白水平。80nM浓度14-D-Valine-TPL即可见明显抑制K562细胞c-myc水平。这一结果表明，本发明14-D-Valine-TPL化合物能抑制肿瘤细胞致癌基因c-myc，可用于c-myc异常高表达相关疾病。

Claims (23)

  1. 通式(I)和通式(II)的C14-位羟基酯化的雷公藤甲素氨基酸衍生物，
    Figure PCTCN2015093161-appb-100001
    其中R1和R4选自有取代或无取代的C2-C20烷基、有取代或无取代的C2-C20烯烃基，有取代或无取代的C4-C20的共扼烯烃基，有取代或无取代的C3-C7环烷基或环烯烃基，有取代或无取代的芳烃基，有取代或无取代的杂环基和芳香杂环基，有取代或无取代的氨基酸侧链烷基；氨基酸可以是消旋的，也可以是光学纯(左旋或右旋)的；取代基选自卤素，胺基，C1-C6取代胺基，硝基，氰基，羟基，C1-C6烷氧基，巯基，C1-C6烷硫基；
    其中R2,R3,R5和R6选自H、有取代或无取代的C1-C20烷基、有取代或无取代的C2-C20烯烃基，有取代或无取代的C4-C20的共扼烯烃基，有取代或无取代的C3-C7环烷基或环烯烃基，有取代或无取代的芳烃基，有取代或无取代的杂环基和芳香杂环基，有取代或无取代的氨基酸侧链烷基，氨基酸可以是消旋的，也可以是光学纯(左旋或右旋)的；取代基选自卤素，胺基，C1-C6取代胺基，硝基，氰基，羟基，C1-C6烷氧基，巯基，C1-C6烷硫基。R2和R3,R5和R6可以是BOC，Fmoc，Bn等保护基。R2和R3相同或不相同。R5和R6相同或不相同。
  2. 根据权利要求1所述的C14-位羟基酯化的雷公藤甲素氨基酸衍生物的药学上可接受的加成物或者复合物。
  3. 根据权利要求1所述的C14-位羟基酯化的雷公藤甲素氨基酸衍生物的药学上可接受的盐。
  4. 根据权利要求1所述的C14-位羟基酯化的雷公藤甲素氨基酸衍生物的分解和代谢产物。
  5. 根据权利要求1的雷公藤甲素的C14-位羟基酯化的雷公藤甲素氨基酸衍生物的药学上可接受的加成物、复合物、盐、以及分解和代谢产物，其中R1和R4选自有取代或无取代的C2-C20烷基、有取代或无取代的C2-C20烯烃基，取代或无取代的C4-C20的共扼烯烃基，有取代或无取代的C3-C7环烷基或环烯烃基，有取代或无取代的芳烃基，有取代或无取代的杂环基和芳香杂环基，有取代或无取代的氨基酸侧链烷基，氨基酸可以是消旋的，也可以是光学纯(左旋或右旋)；取代基选自卤素，胺基，C1-C6取代胺基，硝基，氰基，羟基，C1-C6烷氧基，巯基，C1-C6烷硫基；R2和R3,R5和R6是或不是保护基；R2和R3相同或不相同；R5和R6相同或不相同；其中R2,R3,R5和R6选自H、有取代或无取代的C1-C20烷基、有取代或无取代的C2-C20烯烃基，取代或无取代的C4-C20的共扼烯烃 基，有取代或无取代的C3-C7环烷基或环烯烃基，有取代或无取代的芳烃基，有取代或无取代的杂环基和芳香杂环基，有取代或无取代的氨基酸侧链烷基，氨基酸可以是消旋的，也可以是光学纯(左旋或右旋)的；取代基选自卤素，胺基，C1-C6取代胺基，硝基，氰基，羟基，C1-C6烷氧基，巯基，C1-C6烷硫基。
  6. 根据权利要求1-4任一项的C14-位羟基酯化的雷公藤甲素氨基酸衍生物或其药学上可接受的加成物、复合物、盐、以及分解和代谢产物，其中R1和R2选自取代或无取代的芳烃基；取代基选自卤素，胺基，C1-C6取代胺基，硝基，氰基，羟基，C1-C6烷氧基，巯基，C1-C6烷硫基；取代基可选择一个或数个同时存在。
  7. 根据权利要求1的C14-位羟基酯化的雷公藤甲素氨基酸衍生物，其特征在于它选自下述化合物：
    Figure PCTCN2015093161-appb-100002
  8. 一种制备通式(I)化合物的方法，其特征在于它包括以下步骤：
    Figure PCTCN2015093161-appb-100003
  9. 一种制备通式(II)化合物的通用制备方法如下，其特征在于它包括以下步骤：
    Figure PCTCN2015093161-appb-100004
  10. 一种药物组合物，其中包含权利要求1-7中任一项的C14-位羟基酯化的雷公藤甲素氨基酸衍生物或其药学上可接受的加成物、复合物、盐、以及分解和代谢产物和任选的药学上可以接受的赋形剂。
  11. 权利要求1-7中的任一项的C14-位羟基酯化的雷公藤甲素氨基酸衍生物或其药学上可接受的加成物、复合物、盐、以及分解和代谢产物在制备抗肿瘤药物中的用途。
  12. 权利要求1-7任一项的C14-位羟基酯化的雷公藤甲素氨基酸衍生物或其药学上可接受的加成物、复合物、盐、以及分解和代谢产物作为抗肿瘤治疗剂的用途。
  13. 一种治疗肿瘤患者的方法，包括给予需要治疗的患者治疗有效量的根据权利要求1-7任一项的C14-位羟基酯化的雷公藤甲素氨基酸衍生物或其药学上可接受的加成物、复合物、盐、以及分解和代谢产物。
  14. 根据权利要求11或12所述的用途，其特征在于，所述肿瘤选自白血病、多发性骨髓瘤、淋巴瘤、肝癌、胃癌、乳腺癌、胆管细胞癌、胰腺癌、肺癌、大肠癌、骨肉瘤、人宫颈癌、神经胶质瘤、鼻咽癌、喉癌、食管癌、中耳肿瘤、黑色素瘤和前列腺癌。
  15. 根据权利要求13所述的方法，其特征在于，所述肿瘤选自白血病、多发性骨髓瘤、淋巴瘤、肝癌、胃癌、乳腺癌、胆管细胞癌、胰腺癌、肺癌、大肠癌、骨肉瘤、人宫颈癌、神经胶质瘤、鼻咽癌、喉癌、食管癌、中耳肿瘤、黑色素瘤和前列腺癌。
  16. 根据权利要求1-7中的任一项的C14-位羟基酯化的雷公藤甲素氨基酸衍生物或其药学上可接受的加成物、复合物、盐、以及分解和代谢产物在制备抗免疫性疾病药物中的用途。
  17. 根据权利要求16的用途，其特征在于，所述免疫性疾病选自与免疫细胞功能异常相关的自 身免疫性疾病。
  18. 根据权利要求16的用途，其特征在于，所述免疫性疾病选自与T细胞或B细胞功能异常相关的自身免疫性疾病.
  19. 根据权利要求16的用途，其特征在于，所述免疫性疾病的选择范围包括类风湿性关节炎、系统性红斑狼疮、银屑病、肾病。
  20. 根据权利要求1-7中的任一项的C14-位羟基酯化的雷公藤甲素氨基酸衍生物或其药学上可接受的加成物、复合物、盐、以及分解和代谢产物在制备XPB或Pol II异常表达相关疾病药物中的用途。
  21. 根据权利要求20的用途，其特征在于，所述XPB或Pol II异常表达相关疾病选自与XPB或Pol II过表达相关的肿瘤。
  22. 根据权利要求1-7中的任一项的C14-位羟基酯化的雷公藤甲素氨基酸衍生物或其药学上可接受的加成物、复合物、盐、以及分解和代谢产物在制备致癌基因c-myc异常表达相关疾病药物中的用途。
  23. 根据权利要求22的用途，其特征在于，所述c-myc异常表达相关疾病选自与c-myc过表达相关的肿瘤。
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