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WO2009124460A1 - Tripterygium wilfordii diterpene lactone derivatives, their pharmaceutical compositions and their application in inhibiting tumour of reproductive system - Google Patents

Tripterygium wilfordii diterpene lactone derivatives, their pharmaceutical compositions and their application in inhibiting tumour of reproductive system Download PDF

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Publication number
WO2009124460A1
WO2009124460A1 PCT/CN2009/000360 CN2009000360W WO2009124460A1 WO 2009124460 A1 WO2009124460 A1 WO 2009124460A1 CN 2009000360 W CN2009000360 W CN 2009000360W WO 2009124460 A1 WO2009124460 A1 WO 2009124460A1
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Prior art keywords
lldt
pharmaceutically acceptable
tripterygium
configuration
hydrogen
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PCT/CN2009/000360
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French (fr)
Chinese (zh)
Inventor
李援朝
李征
林莉萍
缪泽鸿
任进
李川
甘勇
丁健
冯慧瑾
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中国科学院上海药物研究所
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Publication of WO2009124460A1 publication Critical patent/WO2009124460A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
    • C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
    • C07J73/003—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by oxygen as hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J21/00—Normal steroids containing carbon, hydrogen, halogen or oxygen having an oxygen-containing hetero ring spiro-condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J33/00—Normal steroids having a sulfur-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring

Definitions

  • the present invention relates to the field of medicinal chemistry, and in particular, the present invention relates to a class of tripterygium diterpene lactone derivatives , optical isomers thereof, and pharmaceutically acceptable salts and hydrates thereof, and therapeutically effective doses of triptolide diterpene lactone derivatives, optical isomers thereof, and pharmaceutically acceptable salts and hydrates thereof, and A pharmaceutical composition of a conventional pharmaceutical excipient and a dosage form thereof, and use of the pharmaceutical composition.
  • Tripterygium Wilfordii Hook F also known as yellow vine, belongs to the genus Euonymus genus, and there are four species in China.
  • Tripterygium wilfordii was first collected in the "Shenlong Materia Medica", its main chemical components are diterpenoids, triterpenoids, sesquiterpenes, alkaloids, etc. In the past two decades, studies have shown that it has anti-tumor, anti-inflammatory, immunosuppressive, anti- Fertility, antibacterial and other activities. Tripterygium wilfordii has been used in the treatment of tumors for many years, and one of the active ingredients is triptolide.
  • triptolide In addition to extensive anti-tumor effects, triptolide has also entered clinical trials for the treatment of leukemia, but its use in clinical applications has been limited by the side effects of triptolide.
  • the structural modification of triptolide was mostly limited to the introduction of water-soluble groups, and there was no fundamental change in the main structure. When such prodrugs enter the body, they are still converted to triptolide by hydrolysis or metabolism. It plays a pharmacodynamic role, which determines that they cannot fundamentally improve the side effects of triptolide.
  • most of the anti-tumor efficacy studies of triptolide derivatives are not specific, so the anti-tumor spectrum is not clear enough.
  • the focus of the present invention is on the in-depth study on the structure-activity relationship of triptolide, and the structure is modified and modified differently from the prior art, and a batch of novel structure of tripterygium diterpene lactone derivatives is obtained, wherein One of the most representative structural modifications is the substitution of a chiral epoxy for C14P-0H, which was previously considered to be a necessary group for triptolide activity. In the experimental study, these derivatives have been found to have a good therapeutic effect on tumor diseases, especially tumors of the reproductive system. The preparation of (14S)-14-dehydroxy-14,21-epoxytriptolide has a special effect.
  • C 14 OS0 2 OCH 2 ; w and z respectively represent an oxygen, a hydroxyl group, a halogen, a thiol group attached at the c 12 position and the c 13 position,
  • C 14 XY is a (14S)-14,21-epoxy structure
  • W and Z are oxygen in the a(S) configuration of C 12 and C 13 positions, respectively
  • P is a(R) Hydrogen or hydroxyl group of configuration
  • Q is hydrogen
  • triptolide diterpene lactone derivative, optical isomer thereof, and pharmaceutically acceptable salt and hydrate thereof according to the present invention have the following formula (3) Structure
  • C 14 XY is (14S)-14,21-epoxy structure
  • W is halogen in P(R) configuration
  • Z is hydroxyl group in a(R) configuration
  • P is a(R) structure
  • Q is hydrogen
  • tripterygium diterpene lactone derivative, optical isomer thereof, and pharmaceutically acceptable salt and hydrate thereof according to the present invention have the following formula (4) structure,
  • C 14 XY is (14S)-14,21-epoxy structure
  • W and Z are oxygen in the a(S) configuration of C 12 and C 13 positions, respectively
  • C 5 and C 6 are The carbon-carbon double bond is bonded
  • Q is hydrogen
  • tripterygium diterpene lactone derivative, the optical isomer thereof, and the pharmaceutically acceptable salt and hydrate thereof according to the present invention have the following formula (5) structure
  • C 14 XY is a (14R)-14,21-epoxy structure
  • W and Z are oxygen in the a(S) configuration of C 12 and C 13 positions, respectively, and P is in the a(R) configuration.
  • Hydrogen or a hydroxyl group, Q is hydrogen; or the tripterygium diterpene lactone derivative according to the present invention, an optical isomer thereof, and pharmaceutically acceptable salts and hydrates thereof have the structure represented by the following formula (6),
  • C 14 XY is C 14 (OH )CH 2 OH, C 14 OS(0)OCH 2 or C 14 OS0 2 OCH 2
  • the configuration at the C 14 position is the (R) configuration or the (S) configuration
  • W and Z are oxygen attached to the a(S) configuration at the C 12 and C 13 positions, respectively, P being hydrogen or a hydroxyl group in the a(R) configuration, and Q being hydrogen.
  • the tripterygium diterpene lactone derivative, the optical isomer thereof, and pharmaceutically acceptable salts and hydrates thereof according to the present invention are selected from the group consisting of the following compounds:
  • the tripterygium diterpene lactone derivative, its optical isomer, and pharmaceutically acceptable salts and hydrates thereof according to the present invention are (14S)-14-dehydroxy-14,21-epoxy Triptolide (LLDT-67).
  • the synthesis method of the tripterygium diterpene lactone derivative of the present invention is summarized as follows: The compound represented by the formula (1) is a triptolide (LLDT-1).
  • Reaction Scheme (1) For example, as shown in the reaction scheme (2), using triptolide (LLDT-1) as a starting material, using Tamao Grignard reagent to attack the C 14 -position carbonyl group of triptolide Silicon-containing intermediate (14S)-14P-isopropoxydithiosilyl thioglycolide (LLDT-81), which is simply extracted and concentrated, and directly dissolved in protons without separation and purification.
  • (14S)-14-dehydroxy-14,21-epoxytriptolide can also be obtained by, as shown in the reaction scheme (3), (14S)-14P-hydroxyindole-based triptolide (LLDT-62) was used as a raw material, and the terminal hydroxyl group at the C21 position was acylated with a acylating reagent to obtain (14S)-14P-sulfonyloxyfluorene.
  • the base table triptolide (wherein R is a sulfonyl group) is cyclized under the action of a base to give (14S)-14-dehydroxy-14,21-epoxytriptolide (LLDT-67).
  • the present invention also provides a therapeutically effective amount of the above-described tripterygium diterpenoid derivative, an optical isomer thereof, and pharmaceutically acceptable salts and hydrates thereof, and a conventional pharmaceutical excipient.
  • Pharmaceutical composition In the pharmaceutical composition according to the present invention, the content of the triptolide derivative, its optical isomer, and pharmaceutically acceptable salts and hydrates thereof is from 0.001 to 99.9 wt%.
  • the pharmaceutical composition of the present invention may be in the form of a gastrointestinal administration or a parenteral administration.
  • the gastrointestinal administration can be in the form of a solution, an emulsion, a tablet, a capsule, or the like.
  • the parenteral administration dose may be an injection, including intravenous, intramuscular, subcutaneous, intradermal injection or the like; transdermal administration, such as topical lotion, ointment, patch, etc.; transmucosal Administration, such as sublingual, nasal, rectal, vaginal, ear canal, and the like.
  • transdermal administration such as topical lotion, ointment, patch, etc.
  • transmucosal Administration such as sublingual, nasal, rectal, vaginal, ear canal, and the like.
  • the active component of the pharmaceutical composition of the present invention, the triptolide diterpene lactone derivative, the optical isomer thereof, and the pharmaceutically acceptable salts and hydrates thereof may be used alone or in combination with other antitumor drugs. .
  • the pharmaceutical composition of the present invention may further comprise one or more other antitumor drugs, which are drugs that affect the biosynthesis of tumor cell nucleic acid; drugs that directly destroy tumor cell DNA to prevent their replication; A drug that interferes with the transcription process in DNA; a drug that interferes with mitosis affecting protein synthesis in tumor cells; or a drug that produces antitumor effects by inhibiting cyclooxygenase (COX-2).
  • the drug affecting nucleic acid biosynthesis of tumor cells may be 5-fluorouracil, 6-Mercaptopurine, Methotrexate, Cytarabine and hydroxyurea.
  • the drug that directly destroys the DNA of the tumor cell to prevent its replication may be Chlormethine Hydrochloride, Cytoxam, Thiotepa, Busulfan, Mitochondrial Mitomycin C, Bleomycin, Cisplatin, Omarine (OP), Oxaloplatin, DWA2114R, CI-973, Lobaplatin, Camptothecin Etoposide, etc.;
  • the drug interfering with the transcription process in the DNA of the tumor cell may be actinomycin D, doxorubicin (ADM), idarubicin, and silk Splitting (NVT), ampicillin (mAMSA), etc.
  • the drugs that interfere with mitosis affecting protein synthesis of tumor cells may be, for example, vinblastine (VLB) and vincristine (VCR), carbocine (VRB), paclitaxel (Taxol), Taxotere, L-Door Coolstone K (Harringtonine) and the like; through
  • the triptolide derivative, its optical isomer and its pharmaceutically acceptable salts and hydrates may be administered simultaneously, or sequentially, or separately. medicine.
  • the present invention provides the use of the above pharmaceutical composition for the preparation of a medicament for treating a tumor, and a tumor-associated disease, wherein the pharmaceutically active component is used in an amount of 0.001 to 10 mg/kg.
  • Such administered doses generally produce an effective therapeutic effect.
  • Oral administration of a dose of mg/kg can significantly inhibit the growth of transplanted tumors of human prostate cancer and ovarian cancer mice.
  • the dose to be administered will be adjusted according to the subject to be treated, the age of the subject (months or weeks of age), the general health condition, the route of administration, the sensitivity of the individual to the drug, and the severity of the condition, etc.
  • the subject is administered a therapeutically effective amount of a drug comprising a compound of the invention
  • the pharmaceutical composition of the present invention can be used for treating warm-blooded animals having proliferative diseases such as tumors, including benign tumors and malignant tumors: benign tumors mainly include gliomas, fibroids, lipomas, blood vessels Malignant tumors mainly include malignant tumors such as squamous cell carcinoma, breast cancer, ovarian cancer, etc., which are caused by epithelial tissues, malignant tumors such as fibrosarcoma, osteosarcoma, lymphosarcoma, etc., which are caused by mesoderm tissues, and embryonic cells. Malignant tumors that occur in nerve cells or immature tissues, and malignant tumors that occur from hematopoietic cells.
  • benign tumors mainly include gliomas, fibroids, lipomas, blood vessels
  • malignant tumors mainly include malignant tumors such as squamous cell carcinoma, breast cancer, ovarian cancer, etc., which are caused by epithelial tissues, malignant tumors such as fibrosarcom
  • the “warm-blooded animals” include humans and other animals such as rodents and mammals.
  • the meaning of "proliferative diseases” includes tumors, dysplasia, but not limited to tumors and dysplasia.
  • the pharmaceutical composition of the present invention can also be used for tumor-related diseases, including prostate cancer, human ovarian cancer, glioma, gastric cancer, granulocyte leukemia, colon cancer, breast cancer, adriamycin-sensitive breast cancer, Doxorubicin-resistant tumor diseases such as breast cancer.
  • the pharmaceutical composition of the invention is for the treatment of prostate cancer and ovarian cancer.
  • “Lower” refers to a straight or branched carbon chain with 1 to 6 carbon atoms.
  • Alkoxy may be decyloxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isovaleryloxy Base, tert-pentyloxy, neopentyl
  • the oxy group, 2-nonylbutoxy group, 1,2-dimercaptopropoxy group, 1-ethylpropoxy group, hexyloxy group and the like are preferably a decyloxy group and an ethoxy group.
  • Alkylamino may be guanamine, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, sec-butylamino, tert-butylamino, pentylamino, isoamylamino, tert-amyl a primary lower alkylamine such as an amine group or a neopentylamino group, or a secondary lower alkyl group such as a diammonium group, a diethylamino group, a dipropylamino group, a diisopropylamino group, a dibutylamino group or a diisobutylamino group.
  • Amine groups, preferred among these groups are amidino group, ethylamine group, diammonium group, and diethylamino group.
  • Polar solvent such as ethyl acetate, dioxane, acetone or tert-butanol
  • Aprotic polar solvent means, for example, disulfoxide, hydrazine, hydrazine-dihydrazinamide, dichlorodecane, trichlorodecane, tetrahydrofuran, dioxane or ethylene glycol biguanide;
  • “Proton polar solvent” means, for example, decyl alcohol, ethanol, propanol or tert-butanol;
  • Heating conditions means temperatures above room temperature to the reflux temperature of the solvent
  • “Amidinolation reagent” means, for example, Corey sulfhydryl reagent, Simmons-Smith reagent or diazonium;
  • transacylation reagent means, for example, sulfonyl chloride, ethanesulfonyl chloride, benzoyl chloride, p-toluene chloride, trifluorosulfonyl chloride or trifluoromethanesulfonic anhydride;
  • nucleophile means, for example, hydrogen chloride or pyridine hydrochloride; "Pharmaceutically acceptable salts, specifically, organic acids and aspartame with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, etc.
  • a salt formed with an acidic base such as an acid or a glutamic acid to form an ester, such as a sodium, potassium, calcium, aluminum salt and an ammonium salt, or a salt formed with an organic base such as a guanamine salt, an ethylamine salt or an ethanolamine.
  • Salt etc.
  • an inorganic acid such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid which is esterified with a basic amino acid such as lysine, arginine or ornithine, or with tannic acid, a salt of an organic acid such as acetic acid, picric acid or hydrazine acid.
  • optical isomer includes enantiomers, diastereomers, mixtures of optical isomers, and pure optical isomers.
  • Advantageous Effects The present invention synthesizes a highly efficient and low-toxic Tripterygium divalent lactone derivative, which enables it to be practically used for the treatment of tumor diseases.
  • the results of in vitro drug efficacy showed that among the series of tripterygium diterpene lactone derivatives obtained, four (14S)- 14,21-epoxy structures were substituted for C14P-0H which were previously considered to be active essential groups.
  • the derivatives all showed strong anti-tumor effects, which is undoubtedly a major breakthrough in the direction of structural transformation.
  • FIG. 1 is a graph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human ovarian cancer SK-OV-3 mice.
  • FIG. 2 is a photograph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human ovarian cancer SK-OV-3 mice, wherein the rat tail represents that the transplanted tumor of the living mouse is undetectable.
  • Figure 3 is a graph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human prostate cancer PC-3 mice.
  • Figure 4 is a photograph showing the growth inhibitory effect of LLDT-67 on human prostate cancer PC-3 subcutaneous xenografts in mice.
  • Figure 5 is a graph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human prostate cancer DU-145 mice.
  • FIG. 6 is a photograph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human prostate cancer DU-145 mice.
  • Figure 7 is a graph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human gastric cancer SGC-7901 mice.
  • Fig. 8 is a photograph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human gastric cancer SGC-7901 mice.
  • Figure 9 is a graph showing the growth inhibitory effect of LLDT-67 on human breast cancer MDA-MB-468 subcutaneous xenografts.
  • Figure 10 is a photograph showing the growth inhibitory effect of LLDT-67 on human breast cancer MDA-MB-468 subcutaneous xenografts.
  • Figure 11 is a graph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human liver cancer SMMC-7721 mice.
  • Figure 12 is a photograph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human liver cancer SMMC-7721 mice.
  • the invention is further illustrated by the following examples, but these examples are in no way intended to limit the invention, and the scope of the invention is determined by the claims.
  • the instruments and main experimental materials used in the preparation examples are as follows:
  • LLDT-1 LLDT-67 (CH 3 ) 3 SOI (59.6 mg, 0.27 mmol) and NaH (60%, 9.5 mg, 0.24 mmol) were mixed as a solid, and 2 mL of anhydrous disulfoxide was added under argon. After stirring at room temperature for 20 min, an aqueous solution of triptolide (LLDT-1) (53.7 mg, 0.15 mmol) in anhydrous disulfoxide (2 mL) was added dropwise thereto, and the mixture was stirred. The TLC was used to carry out the conversion of the starting material. The reaction was quenched with EtOAc (EtOAc).
  • LLDT-1 LLDT-67 LLDT-69 (CH 3 ) 3 SOI (374 mg, 1.7 mmol) and -BuOK (174 mg, 1.5 mmol) were dissolved in 6 mL of dry disulfoxide under argon. The system was stirred at room temperature for 20 min. The triptolide (LLDT-1) (360 mg, 1.0 mmol) dissolved in 6 mL of dry disulfoxide solvent was added to the above mixture, and the reaction was stirred at room temperature until the substrate was completely converted, and quenched by adding 10 mL of water. The reaction mixture was washed with ethyl acetate.
  • LLDT-62 Weighed magnesia (108 mg, 4.5 mL) in a reaction flask and replaced the protective reaction system with argon; dissolved isodecyl chlorodecyl silane (0.72 ml, 4.0 mmol) in ⁇ mL In water tetrahydrofuran, take 1 mL of this solution under argon gas to be added to the magnesium cuttings reaction system, and add two drops of 1,2-dibromoethane.
  • the mixture is stirred at 40 °C to initiate the Grignard reaction, and then the remaining silane reagent is The tetrahydrofuran solution was dropwise added to the reaction system, and the addition was completed in 5 minutes, at which time the reaction system was dark gray.
  • the reaction was refluxed for 45 min and then cooled to 0 °C.
  • Triptolide (LLDT-1) (358 mg, 1.0 mL) dissolved in anhydrous tetrahydrofuran was added dropwise to the freshly prepared grid described above. In the reagents.
  • the reaction mixture was stirred at room temperature for 2 hours, and then the mixture was evaporated.
  • the addition product was directly dissolved in a mixed solvent of 5 mL of sterol and 8 mL of tetrahydrofuran at 40 ° C without further purification, and KHC0 3 (416 mg, 4.2 mmol), KF (464 mg, 4.9 mmol) and K. H 2 O 2 (30%, 1.1 mL, 9.71 mmol).
  • LLDT-62 LLDT-80 LLDT-67 (14S)-14P-hydroxyindole table triptolide (0.50 g, 1.3 mmol) was dissolved in dry pyridine (4.0 mL). The solution was cooled to 0.degree. C. and then added M.sub.sub.3 (0.61 mL, 7.7 mmol). The reaction was stirred at room temperature for 10 min, and the solvent was evaporated under reduced pressure. The residue was diluted with water, EtOAc (EtOAc)EtOAc.
  • the concentrated crude product was purified by column chromatography (eluent: CH 2 C1 2 ) to give white solid (5R, 14S)-5-hydroxyl -14-dehydroxy-14,21-epoxytriptolide (LLDT-68) (38.8 mg, 0.1 mmol, yield: 50%) and (14S)- ⁇ 5 , 6 -dehydro-14-de Hydroxy-14,21-epoxytriptolide (LLDT-70) (7.4 mg, 0.02 mmol, yield: 10%).
  • LLDT-77 or LLDT-78 LLDT-79 will (14S, sulfur S)-14a, 21-ethylene glycol cyclic sulfite triptolide (LLDT-77) or (14S, sulfur R)-14a, 21- Ethylene glycol cyclic sulfite triptolide (LLDT-78) (40 mg : 0.092 mmol) was dissolved in 4 mL of acetonitrile, followed by sodium periodate (31 mg, 0.14 mmol), RuCl 3 3H 2 0 ( The reaction mixture was stirred for 15 min.
  • Results 9 compounds in the present invention In the six compounds LLDT-67, LLDT-68, LLDT-69, LLDT-70, LLDT-76, LLDT-78 dose-dependently inhibited in vitro culture Growth of human ovarian cancer SK-OV-3 cells has been shown to have potent anti-tumor effects in vitro. The specific results are shown in Table 1. Table 1. Proliferation inhibitory effect of compounds on human ovarian cancer SK-OV-3 cells Sample SK-OV-3 ic 50 No. 10" 4 M 10" 5 M 10" 6 M 10" 7 M 10" 8 M Evaluation ( ⁇ ⁇ )
  • Human breast cancer MDA-MB-468 cells were cultured in 1640 medium (Gibco, USA) containing 10% fetal calf serum under the culture conditions of 37. C, 5% C0 2 . Tumor cells were inoculated into a 96-well plate at 0.7 xlO 4 /well. After 24 hours, a compound diluted with dimethyl sulfoxide (1 ( ⁇ 2 ⁇ ), physiological saline was added to make the final concentration of each compound in the medium 10_ 4 , 10_ 5 , 10_ 6 , 10" 7 , 10" 8 ⁇ for 72 hours. Discard the medium and fix the cells with 10% cold trichloroacetic acid. Stained with a solution of Sulforhodamine B (SRB).
  • SRB Sulforhodamine B
  • the unbound SRB, Tris dissolved the protein-bound SRB, and the OD value was measured by a microplate reader at a wavelength of 560 nm.
  • the half-inhibitory concentration IC 50 was calculated by the Logit method.
  • Inhibition rate (OD value control well) - OD value administration hole) / OD value control hole ⁇ 100%. Result judgment criteria: Invalid: 10 ⁇ 5 ⁇ ⁇ 50%; Valid: 10 -5 ⁇ ⁇ 50%. According to the inhibition rate of each concentration, use Logit method The half-inhibitory concentration IC 50 was calculated.
  • the tumor cells were seeded in a 96-well plate at a number of 0.4-1.5 x 10 4 cells/well.
  • LLDT-67 diluted with physiological saline was added to prepare the culture.
  • the final concentration of LLDT-67 in the base is 0.008 ⁇ - 40 ⁇ ; the final concentration of dimethyl sulfoxide in the medium is not more than 0.1%.
  • LLDT-67 was treated for 72 hours.
  • SRB method The adherent cells were treated with the SRB method. After 72 hours of drug action, the culture was discarded and the cells were fixed with 10% cold trichloroacetic acid. Stained with a solution of Sulforhodamine B (SRB). The unbound SRB was washed away, and the protein-bound SRB was dissolved in Tris, and the OD value was measured at a wavelength of 560 nm using a microplate reader.
  • MTT method Suspension cells were reduced by tetrazolium salt (MTT).
  • LLDT-67 dose-dependently inhibited the growth of a variety of cultured human tumor cell value, IC 5. There is a strong in vitro antitumor activity between 0.14 ⁇ and 5.99 ⁇ (Table 4). Table 4. Inhibition of LLDT-67 on proliferation of human tumor cells in vitro Cell name Tumor type ⁇ 50 ( ⁇ )
  • SRB method, MTT method Multidrug resistant cells and their parental tumor cells were cultured in RPMI 1640 or DMEM medium (Gibco) containing 10% fetal bovine serum; the culture condition was 37. C, 5% C0 2 o MCF-7, MCF-7/ADR cells were cultured with 1 mM sodium pyruvate and 0.01 mg/ml bovine insulin in addition to RPMI 1640 medium.
  • the drug-resistant cell line was supplemented with 2 ⁇ doxorubicin ( ⁇ 562/ ⁇ 02, MCF-7/ADR cell culture solution) or 1 ⁇ vincristine (KB/VCR cell culture solution) to maintain drug resistance, one week before the experiment. Stop the drug.
  • Tumor cells were seeded in 96-well plates at a number of 0.6-1.5 x 10 4 cells/well, and 24 hours later, LLDT-67, doxorubicin, and vincristine diluted with physiological saline were prepared.
  • Final concentration of LLDT-67 in the medium The degree is 0.008 ⁇ -40 ⁇ , and the final concentration of doxorubicin and vincristine is 0.005 ⁇ -400 ⁇ .
  • the final concentration of dimethyl sulfoxide in the medium does not exceed 0.1%.
  • the drug acts on the cells for 72 hours.
  • the adherent cells were treated with the SRB method.
  • the culture solution was discarded, and the cells were fixed with 10% cold trichloroacetic acid, stained with SRB solution, and then washed with unbound SRB, Tris dissolved and protein-bound SRB, and OD was measured at 560 nm using a microplate reader. value.
  • the suspension cells were treated with the MTT method. After the drug was added for 72 hours, the MTT solution was added, and the culture was continued for 4 hours, and a triple solution (10% SDS-5% isobutanol-0.01 M HCl) was added to the C0 2 incubator overnight.
  • LLDT-67 showed a proliferative growth inhibition effect on drug-resistant cells similar to its corresponding parental cells, and it has anti-multidrug resistance in vitro and may be effective in the treatment of drug-resistant tumors (Table 5). Table 5. Inhibition of LLDT-67 on proliferation of human tumor multidrug resistant cells in vitro
  • RF Resistance Factor
  • EXPERIMENTAL EXAMPLE 6 Growth inhibition of LLDT-67 on subcutaneous xenografts of human ovarian cancer cell line SK-OV-3.
  • Subcutaneous xenograft model of mouse Human ovarian cancer SK-OV-3 cells from Shanghai tumor Hospital; BALB/C mice [SPF (specific-pathogen free)], female, weight: 18 ⁇ 2g, provided by Shanghai Institute of Medicine, Chinese Academy of Sciences, certificate number: SCXK (Shanghai) (2004- 0002).
  • SK-OV-3 cells 5 ⁇ 10 6 / were inoculated subcutaneously into the right axilla of mice, and transplanted tumors were formed and then transferred to mice for 2 generations. Take vigorous growth of the tumor tissue, under sterile conditions, cut into small pieces of about 1 mm 3 with scissors, inoculate the left armpit of the mouse with a trocar, and grow the tumor to about 100-200 mm 3 .
  • T RTV / C RTV xl00% The result criteria were: T/C (%) ⁇ 40%, valid; T/C (%) >40%, invalid.
  • the toxicity of the compound can be preliminarily determined by mouse behavior, secondary stool, and body weight change.
  • LLDT-67 at a dose of 1.0, 2.0, 4.0 mg/kg had a T/C (%) of 50.2%, 12.5%, 2.6%, respectively, and dose-dependently inhibited subcutaneous xenografts in human ovarian cancer SK-OV-3 mice. Growth; In addition, LLDT-67 had no significant toxic side effects on mice at the dose and mode of administration. (See Table 6, Figures 1 and 2). Table 6. Growth inhibition of LLDT-67 on subcutaneous xenografts of human ovarian cancer cells SK-OV-3
  • LLDT-67 was administered orally at 0.5, 1.0, 2.0 mg/kg once daily for 25 days.
  • the positive control mitomycin (MMC) 5 mg/kg was injected into the tail vein and administered once on the first day.
  • the result criteria were: T/C (%) ⁇ 40%, valid; T/C (%) >40%, invalid.
  • the toxicity of the compound can be preliminarily determined by mouse behavior, secondary stool, and body weight change.
  • LLDT-672.0mg/kg H, T/C (%) was 1.63%, which significantly inhibited the growth of human prostate cancer PC-3 mice under subcutaneous transplantation. The inhibition rate was as high as 98.37%, showing a strong anti-tumor. In addition, LLDT-67 has no obvious side effects on mice at the dose and mode of administration. (See Table 7, Figures 3 and 4). Table 7. Growth inhibition of LLDT-67 on human prostate cancer cell line PC-3 subcutaneous xenografts in animals.
  • mice human prostate cancer DU-145 cells were obtained from American ATCC (American Type Culture Collection); BALB/C mice [SPF (specific-pathogen free)], Male, weight: 20 ⁇ 2g, provided by Shanghai Institute of Medicine, Chinese Academy of Sciences, certificate number: SCXK (Shanghai) (2004-0002).
  • SPF specific-pathogen free
  • the tumor-bearing mice were sacrificed, and the subcutaneous tumor tissue was dissected and cut into small pieces of about 1 mm 3 with scissors.
  • the trocars were inoculated into the left axilla of the mice, and the tumors were grown to about 100-200 mm 3 . Randomly grouped (d0).
  • LLDT-67 was administered orally at 0.5, 1.0, 2.0 mg/kg, once a day, 5 days a week for 4 weeks; the positive control drug docetaxel 10 mg/kg, tail vein injection, respectively The drug was administered twice on the first day and the seventh day.
  • the tumor volume (TV) l/2xaxb 2 (a, b respectively indicate the length and width).
  • RVTV relative tumor volume
  • the result criteria were: T/C (%) ⁇ 40%, valid; T/C (%) >40%, invalid.
  • the toxicity of the compound can be preliminarily determined by mouse behavior, two stools, and body weight changes.
  • the positive control drug mitomycin (MMC) 5 mg/kg was injected into the tail vein and administered once on the first day. During the administration, the rats were weighed twice a week, and the tumor volume was measured.
  • T/C ( % ) ⁇ 40 %, valid
  • T/C ( % ) >40% invalid
  • the toxicity of the compound can be preliminarily determined by mouse behavior, two stools, and body weight changes.
  • the human breast cancer MDA-MB-468 tumor cells in vitro growth were digested, counted, and inoculated into the underarm of the mice according to lx lO 7 / only, and the tumors were grown to about 100 - 150 mm 3 , and the animals were randomly grouped (d0 ).
  • LLDT-67 was administered orally at 1.0, 2.0, 4.0 mg/kg once daily for 5 times a week for 2.5 weeks.
  • the positive control mitomycin (MMC) 5 mg/kg was injected into the tail vein and administered once on the first day.
  • the rats were weighed twice a week, and the tumor volume was measured.
  • the tumor volume (TV) l/2xaxb 2 (a and b respectively indicate the length and width).
  • the toxicity of the compound can be preliminarily determined by mouse behavior, two stools, and body weight changes.
  • LLDT-67 showed no growth inhibition on human breast cancer MDA-MB-468 subcutaneous xenografts at doses of 1.0, 2.0, and 4.0 mg/kg. In addition, at the dose and mode of administration, LLDT- 67 had no obvious toxic side effects on mice (see Table 10, Figures 9 and 10). Table 10. Growth inhibition of LLDT-67 on human breast cancer cell MDA-MB-468 subcutaneous xenografts in mice
  • LLDT-67 1 mg/kg, po 6 6 19.7 21.0 130 ⁇ 2145 ⁇ 17.9 ⁇ 99.0 > 0.05 l-5/2.5w 36 1227 9.1
  • Experimental Example 11 LLDT-67 was subcutaneously transplanted into human liver cancer cell SMMC-7721 Tumor growth inhibition Methods
  • the subcutaneous xenograft model of mice was used: Human liver cancer SMMC-7721 cells were derived from China Second Military Medical University; BALB/C mice [SPF grade (specific-pathogen free)], female, body weight : 18 ⁇ 2g, provided by Shanghai Institute of Medicine, Chinese Academy of Sciences, certificate number: SCXK (Shanghai) (2004-0002).
  • the tumor-bearing mice were sacrificed.
  • TV tumor volume
  • the relative tumor volume (RTV), RTV V dt /V d , was calculated.
  • V dQ is the tumor volume measured on d Q days
  • V dt is the tumor volume measured on the day of administration dt.
  • the evaluation index of antitumor activity is relative tumor proliferation rate T/C (%)
  • T/C (%) TRTV/CRTV x 100% till Result judgment standard: T/C (%) ⁇ 40%, effective; T/ C (%) >40%, ineffective.
  • the toxicity of the compound can be preliminarily determined by mouse behavior, second stool and weight change.
  • LLDT-67 had no growth inhibitory effect on human liver cancer SMMC-7721 mouse subcutaneous xenografts at doses of 1.0, 2.0, 4.0 mg/kg. In addition, LLDT-67 was administered to mice at the dose and mode of administration. No obvious side effects (see Table 11, Figures 11 and 12). Table 11: Growth inhibition of LLDT-67 on subcutaneous xenografts of human hepatoma cell line SMMC-7721 in mice. Body weight (g) TV (i nm 3 )
  • Experimental Example 12 Preliminary pharmacokinetic study of LLDT-67 was performed using male Sprague-Dawley rats (body weight approximately 250 g) In the pharmacokinetic experiment, 3 rats were orally administered with LLDT-67 (4 mg/kg), and 3 rats were injected with bolus dosing LLDT-67 (2 mg/kg). Blood samples were taken at 0, 5, 15, 30 min, 1, 1.5, 2, 5, and 8 h after administration. Heparin anticoagulated plasma samples, using liquid-quality
  • ICR mice weighing 18-20 grams, were purchased from Sino-British joint venture Sipper-Bikie Laboratory Animal Co., Ltd., Laboratory Animal Production License No.: SCXK( ⁇ ) 2003-0002.
  • LLDT-67 suspension In 0.2% HPMC continuously milling.
  • the experimental animals were divided into three groups of 10 mg/kg, 20 mg/kg and 30 mg/kg, with 4 rats in each group, half male and half female. Mortality and toxicity were observed after single intragastric administration for a total of 14 days. result
  • Tripterygium diterpene lactone derivative of the present invention significantly inhibits various The growth of tumor cells cultured in vitro has a significant dose-dependent inhibition of its IC 5 . Between 0.10 and 5.99 ⁇ , LLDT-67 was shown to have potent in vitro antitumor activity.
  • LLDT-67 has a significant inhibitory effect on the growth of human prostate cancer PC-3 subcutaneous xenografts and human ovarian cancer SK-OV-3 subcutaneous xenografts, and can be somewhat Inhibition of subcutaneous xenograft growth in human prostate cancer DU-145 mice, the growth of subcutaneous xenografts in human gastric cancer SGC-7901 mice was only weakly inhibited at high concentrations (4 mg/kg), but The growth of subcutaneous xenografts of mouse liver cancer SMMC7721 mice and human breast cancer MDA-MB-468 mice had no inhibitory effect. None of the above in vivo pharmacodynamic experiments showed significant toxic side effects.
  • LLDT-67 can specifically inhibit the growth of tumors of the reproductive system. Pharmacokinetic studies of LLDT-67 indicate that it can be administered orally or intravenously. Preliminary acute toxicity experiments showed that LLDT-67 (LD 0 is about 20 mg/kg) is much less toxic than its lead LLDT-2 (LD 5 , about 0.5 mg/kg). All the experimental results show that LLDT-67 has high efficiency, low toxicity and specificity in the treatment of tumor diseases, and has a good application prospect for the treatment of neoplastic tumor diseases including prostate cancer and human ovarian cancer.

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Abstract

The invention discloses Tripterygium wilfordii diterpene lactone derivatives represented by formula (1), their optical isomers and pharmaceutically acceptable salts and hydrates. The invention also provides pharmaceutical compositions and preparations comprising therapeutically effective Tripterygium wilfordii diterpene lactone derivatives represented by formula (1), their optical isomers and pharmaceutically acceptable salts and hydrates, and conventional pharmaceutical auxiliaries. The invention also discloses the application of the pharmaceutical compositions. The invention screens Tripterygium wilfordii diterpene lactone derivatives with good effect and low toxicity and makes them use in preparing medicaments for treating tumor diseases. The pharmaceutical compositions can be formulated to preparations in favor of the absorption by warm blood's tissues and apparatuses and have good application foreground in treating proliferative diseases such as tumors and so on.

Description

雷公藤二萜类内酯衍生物、 其药物组合物及其在抗生殖系统肿瘤 中的应用 技术领域 本发明涉及药物化学领域， 具体地， 本发明涉及一类雷公藤二萜 类内酯衍生物、 其光学异构体及其药学上可接受的盐和水合物及含有 治疗有效剂量的雷公藤二萜类内酯衍生物、 其光学异构体及其药学上 可接受的盐和水合物及常规的药学辅料的药物组合物及其剂型， 以及 该药物组合物的应用。 背景技术 雷公藤 (Tripterygium Wilfordii Hook F)又称黄藤， 属卫矛科雷公藤 属植物， 中国有四种， 其中有三种入药， 分别是主产于长江中下游地 区的雷公藤， 主产于长江流域及西南的昆明山海棠及主产于东北和日 本的东北雷公藤。 雷公藤最早收载于《神龙本草经》， 其主要化学成分 有二萜、 三萜、 倍半萜、 生物碱等， 近二十年来的研究表明它具有抗 肿瘤、 抗炎、 免疫抑制、 抗生育、 抗菌等活性。 雷公藤植物在民间用于治疗肿瘤已有多年历史， 其中活性成分之 一是雷公藤内酯醇。 雷公藤内酯醇除了已得到广泛的抗肿瘤作用研究 外， 还曾经进入临床试验， 用于治疗白血病， 但由于雷公藤内酯醇的 毒副作用， 限制了其在临床中应用。 以往对雷公藤内酯醇的结构改造 大多局限在水溶性基团的引进上， 主体结构上并无根本性改动。 当这 类前药进入体内后， 通过水解或代谢后仍然转变为雷公藤内酯醇在体 内发挥药效作用， 这就决定了它们不可能从根本上改善雷公藤内酯醇 的毒副作用。 同时以往对雷公藤内酯醇衍生物的抗肿瘤药效研究大多 无特异性， 因此抗肿瘤瘤谱不够明确。 本发明的重点在于对雷公藤内酯醇构效关系的深入研究， 对其结 构进行了不同于现有技术的改造和修饰， 获得了一批新型结构的雷公 藤二萜类内酯衍生物， 其中最有代表性的一种结构改造方式是用手性 的环氧代替了以往认为是雷公藤内酯醇活性必需基团的 C14P-0H。 进 理实验研究中发现这些衍生物对于肿瘤疾病， 尤其是生殖系统的肿瘤 具有良好的治疗作用，其中制备的（14S)-14-脱羟基 -14,21-环氧雷公藤内 酯醇的作用尤其明显， 具有高效、 低毒、 特异性强的特点， 具有艮好 的用于治疗肿瘤等增生性疾病的开发前景， 从而完成了本发明。 发明内容 本发明的目的是提供一类雷公藤二萜类内酯衍生物、 其光学异构 体及其药学上可接受的盐和水合物。 本发明的另一目的是提供包括上述雷公藤二萜类内酯衍生物、 其 光学异构体及其药学上可接受的盐和水合物以及常规的药学辅料的药 物组合物及其剂型。 本发明的再一目的是提供上述药物组合物的用途。 根据本发明， 提供以下通式 (1)所示的雷公藤二萜类内酯衍生物、 其光学异构体及其药学上可接受的盐和水合物： FIELD OF THE INVENTION The present invention relates to the field of medicinal chemistry, and in particular, the present invention relates to a class of tripterygium diterpene lactone derivatives , optical isomers thereof, and pharmaceutically acceptable salts and hydrates thereof, and therapeutically effective doses of triptolide diterpene lactone derivatives, optical isomers thereof, and pharmaceutically acceptable salts and hydrates thereof, and A pharmaceutical composition of a conventional pharmaceutical excipient and a dosage form thereof, and use of the pharmaceutical composition. BACKGROUND OF THE INVENTION Tripterygium Wilfordii Hook F, also known as yellow vine, belongs to the genus Euonymus genus, and there are four species in China. Three of them are used as medicines, which are mainly produced in the middle and lower reaches of the Yangtze River. The Yangtze River Valley and the Kunming Mountain Sea Bream in the southwest are mainly produced in the northeast and Japan. Tripterygium wilfordii was first collected in the "Shenlong Materia Medica", its main chemical components are diterpenoids, triterpenoids, sesquiterpenes, alkaloids, etc. In the past two decades, studies have shown that it has anti-tumor, anti-inflammatory, immunosuppressive, anti- Fertility, antibacterial and other activities. Tripterygium wilfordii has been used in the treatment of tumors for many years, and one of the active ingredients is triptolide. In addition to extensive anti-tumor effects, triptolide has also entered clinical trials for the treatment of leukemia, but its use in clinical applications has been limited by the side effects of triptolide. In the past, the structural modification of triptolide was mostly limited to the introduction of water-soluble groups, and there was no fundamental change in the main structure. When such prodrugs enter the body, they are still converted to triptolide by hydrolysis or metabolism. It plays a pharmacodynamic role, which determines that they cannot fundamentally improve the side effects of triptolide. At the same time, most of the anti-tumor efficacy studies of triptolide derivatives are not specific, so the anti-tumor spectrum is not clear enough. The focus of the present invention is on the in-depth study on the structure-activity relationship of triptolide, and the structure is modified and modified differently from the prior art, and a batch of novel structure of tripterygium diterpene lactone derivatives is obtained, wherein One of the most representative structural modifications is the substitution of a chiral epoxy for C14P-0H, which was previously considered to be a necessary group for triptolide activity. In the experimental study, these derivatives have been found to have a good therapeutic effect on tumor diseases, especially tumors of the reproductive system. The preparation of (14S)-14-dehydroxy-14,21-epoxytriptolide has a special effect. Obviously, it has the characteristics of high efficiency, low toxicity, strong specificity, and has a good development prospect for the treatment of proliferative diseases such as tumors, thereby completing the present invention. SUMMARY OF THE INVENTION It is an object of the present invention to provide a class of tripterygium diterpenoid lactone derivatives, optical isomers thereof, and pharmaceutically acceptable salts and hydrates thereof. Another object of the present invention is to provide a pharmaceutical composition comprising the above-described tripterygium diterpene lactone derivative, an optical isomer thereof, and pharmaceutically acceptable salts and hydrates thereof, and a conventional pharmaceutical excipient, and a dosage form thereof. A further object of the invention is to provide the use of the above pharmaceutical compositions. According to the present invention, there is provided a tripterygium diterpene lactone derivative represented by the following formula (1), an optical isomer thereof, and pharmaceutically acceptable salts and hydrates thereof:
Figure imgf000005_0001
Figure imgf000005_0001
(1) 其中， c5和 c6以碳碳单键或碳碳双键相连； 当 C5和 C6以碳碳单键相连时， P和 Q分别表示连接在 C5位和 C6 位上的氢、 氧、 羟基、 卤素、 巯基、 C1〜C6烷氧基、 C1〜C6烷胺基或 C1〜C6烷巯基； 当 C5和 C6以碳碳双键相连时， 此时 C5除与 C4、 C6以及 C10连接 外无其他取代基团， P不代表任何取代基团， C6位上取代基 Q则仅代 表氢原子； (1) wherein, c 5 and c 6 are bonded by a carbon-carbon single bond or a carbon-carbon double bond; when C 5 and C 6 are connected by a single carbon-carbon bond, P and Q are respectively attached to the C 5 position and the C 6 position. Hydrogen, oxygen, hydroxyl, halogen, sulfhydryl, C1 to C6 alkoxy, C1 to C6 alkylamino or C1 to C6 alkyl fluorenyl; when C 5 and C 6 are bonded by a carbon-carbon double bond, then C 5 There are no other substituent groups except for the linkage with C 4 , C 6 and C 10 , P does not represent any substituent group, and the substituent Q at the C 6 position represents only a hydrogen atom;
C14XY表示 C14位处的结构是 C 14C H2。、 C14(OH )CH2OH、 C 14OS(0)OCH2 C 14 XY indicates that the structure at the C 14 position is C 14 CH 2 . , C 14 (OH )CH 2 OH, C 14 OS(0)OCH 2
I I I I
或 C14OS02OCH2 ; w和 z分别表示连接在 c12位和 c13位上的氧、羟基、 卤素、巯基、Or C 14 OS0 2 OCH 2 ; w and z respectively represent an oxygen, a hydroxyl group, a halogen, a thiol group attached at the c 12 position and the c 13 position,
C1〜C6烷氧基、 C1〜C6烷胺基或 C1〜C6烷巯基； 在上式中，连接 X、 Y、 Z、 W、 Ρ和 Q的"—— "代表 "一"或者"…山，，。 根据本发明的雷公藤二萜类内酯衍生物、 其光学异构体及其药学 上可接受的盐和水合物， 其具有以下通式 (2)所示结构， a C1~C6 alkoxy group, a C1~C6 alkylamino group or a C1~C6 alkyl fluorenyl group; in the above formula, "-" which connects X, Y, Z, W, Ρ and Q represents "one" or "... mountain ,,. a tripterygium diterpene lactone derivative, an optical isomer thereof, and a pharmaceutically acceptable salt thereof and hydrate thereof according to the present invention, which has a structure represented by the following formula (2),
Figure imgf000006_0003
Figure imgf000006_0003
Figure imgf000006_0001
Figure imgf000006_0001
(2) 其中， C14XY为（14S)-14,21-环氧结构, W与 Z为分别连接在 C12 和 C13位的 a(S)构型的氧， P为 a(R)构型的氢或羟基， Q为氢； 或者 根据本发明的雷公藤二萜类内酯衍生物、 其光学异构体及其药学 上可接受的盐和水合物具有以下通式 (3)所示结构， (2) where C 14 XY is a (14S)-14,21-epoxy structure, W and Z are oxygen in the a(S) configuration of C 12 and C 13 positions, respectively, and P is a(R) Hydrogen or hydroxyl group of configuration, Q is hydrogen; or the triptolide diterpene lactone derivative, optical isomer thereof, and pharmaceutically acceptable salt and hydrate thereof according to the present invention have the following formula (3) Structure,
Figure imgf000006_0002
Figure imgf000006_0002
(3) 其中， C14XY为（14S)-14,21-环氧结构, W为 P(R)构型的卤素， Z 为 a(R)构型的羟基， P为 a(R)构型的氢或羟基， Q为氢； 或者 根据本发明的雷公藤二萜类内酯衍生物、 其光学异构体及其药学 上可接受的盐和水合物具有以下通式 (4)所示结构， (3) wherein C 14 XY is (14S)-14,21-epoxy structure, W is halogen in P(R) configuration, Z is hydroxyl group in a(R) configuration, and P is a(R) structure Type of hydrogen or hydroxyl group, Q is hydrogen; or the tripterygium diterpene lactone derivative, optical isomer thereof, and pharmaceutically acceptable salt and hydrate thereof according to the present invention have the following formula (4) structure,
Figure imgf000007_0001
Figure imgf000007_0001
(4) 其中， C14XY为（14S)-14,21-环氧结构， W与 Z为分别连接在 C12 和 C13位的 a(S)构型的氧， C5和 C6以碳碳双键相连， Q为氢； 或者 根据本发明的雷公藤二萜类内酯衍生物、 其光学异构体及其药学 上可接受的盐和水合物具有以下通式 (5)所示结构， (4) wherein C 14 XY is (14S)-14,21-epoxy structure, W and Z are oxygen in the a(S) configuration of C 12 and C 13 positions, respectively, and C 5 and C 6 are The carbon-carbon double bond is bonded, and Q is hydrogen; or the tripterygium diterpene lactone derivative, the optical isomer thereof, and the pharmaceutically acceptable salt and hydrate thereof according to the present invention have the following formula (5) structure,
Figure imgf000007_0002
Figure imgf000007_0002
其中， C14XY为（14R)-14,21-环氧结构， W与 Z为分别连接在 C12 和 C13位的 a(S)构型的氧， P为 a(R)构型的氢或羟基， Q为氢； 或者 根据本发明的雷公藤二萜类内酯衍生物、 其光学异构体及其药学 上可接受的盐和水合物具有以下通式 (6)所示结构， Wherein C 14 XY is a (14R)-14,21-epoxy structure, and W and Z are oxygen in the a(S) configuration of C 12 and C 13 positions, respectively, and P is in the a(R) configuration. Hydrogen or a hydroxyl group, Q is hydrogen; or the tripterygium diterpene lactone derivative according to the present invention, an optical isomer thereof, and pharmaceutically acceptable salts and hydrates thereof have the structure represented by the following formula (6),
Figure imgf000008_0001
其中， C14XY为 C14(OH )CH2OH、 C 14OS(0)OCH2或 C14OS02OCH2 , C14位 的构型为 (R)构型或 (S)构型， W与 Z为分别连接在 C12和 C13位的 a(S) 构型的氧， P为 a(R)构型的氢或羟基， Q为氢。 优选地， 根据本发明的雷公藤二萜类内酯衍生物、 其光学异构体 及其药学上可接受的盐和水合物选自由以下化合物组成的组：
Figure imgf000008_0001
Wherein C 14 XY is C 14 (OH )CH 2 OH, C 14 OS(0)OCH 2 or C 14 OS0 2 OCH 2 , and the configuration at the C 14 position is the (R) configuration or the (S) configuration. W and Z are oxygen attached to the a(S) configuration at the C 12 and C 13 positions, respectively, P being hydrogen or a hydroxyl group in the a(R) configuration, and Q being hydrogen. Preferably, the tripterygium diterpene lactone derivative, the optical isomer thereof, and pharmaceutically acceptable salts and hydrates thereof according to the present invention are selected from the group consisting of the following compounds:
(1) (14S)-14-脱羟基 -14,21-环氧雷公藤内酯醇 (1) (1 4 S)-1 4 -dehydroxy-14, 2 1-epoxytriptolide
Figure imgf000008_0002
Figure imgf000008_0002
(2) (14R)- 14-脱羟基 -14,21-环氧雷公藤内酯醇 (2) (14R)- 14-dehydroxy--, 14,21-epoxytriptolide
Figure imgf000008_0003
(3) (5R,l4S)-5a-羟基 -I4-脱羟基 -14,21-环氧雷公藤内酯醇
Figure imgf000008_0003
(3) (5R, l 4 S)-5a-hydroxy-I 4 -dehydroxy-14, 2 1-epoxytriptolide
Figure imgf000009_0001
Figure imgf000009_0001
(4) (12R,13R,14S)-12P-氯 -13α-羟基 -14-脱羟基 -14,21-环氧雷公藤 内酯醇 (4) (12R,13R,14S)-12P-chloro-13α-hydroxy-14-dehydroxy-14,21-epoxytriptolide
Figure imgf000009_0002
Figure imgf000009_0002
(5) (14S)- \5，6-脱氢 -I4-脱羟基 -14,21-环氧雷公藤内酯醇 (5) (1 4 S)- \ 5 , 6 -Dehydro-I 4 -dehydroxy-14, 2 1-epoxytriptolide
Figure imgf000009_0003
Figure imgf000009_0003
(6) (14S)-14P-羟曱基表雷公藤内酯醇 (6) (14S)-14P-hydroxyindole table triptolide
Figure imgf000009_0004
Figure imgf000009_0004
(7) (14S,硫 S)-l4a,2l-乙二醇环亚硫酸酯雷公藤内酯醇
Figure imgf000010_0001
(7) (14S, sulfur S)-l 4 a, 2 l-ethylene glycol cyclic sulfite, triptolide
Figure imgf000010_0001
(8) (MS,硫 R)-l4a,2l-乙二醇环亚硫酸酯雷公藤内酯醇 (8) (MS, sulfur R)-l 4 a, 2 l-ethylene glycol cyclic sulfite, triptolide
Figure imgf000010_0002
Figure imgf000010_0002
(9) (l4S)-l4a,2l-乙二醇环硫酸酯雷公藤内酯醇 (9) (l 4 S)-l 4 a, 2 l-ethylene glycol cyclic sulfate triptolide
Figure imgf000010_0003
更优选地， 根据本发明的雷公藤二萜类内酯衍生物、 其光学异构 体及其药学上可接受的盐和水合物为（14S)-14-脱羟基 -14,21-环氧雷公 藤内酯醇 (LLDT-67)。 本发明的雷公藤二萜类内酯衍生物的合成方法概括如下： 通式 ( 1 )所示的化合物是以雷公藤内酯酮 (LLDT- 1 )为起始物。 例如 , 如反应流程式 (1)所示 , 以雷公藤内酯酮 (LLDT-1)为起始物 , 在非质子极性溶剂中， 于室温条件下， 利用亚曱基化试剂在 C14处形成 S和 R构型的一对环氧化合物（14S)-14-脱羟基 -14,21-环氧雷公藤内酯 醇 (LLDT-67)与（14R)-14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-69);将 (14S)-14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-67)溶解于非质子极性 溶剂中， 于加热条件下， 由二氧化硒氧化得到（5R,14S)-5a-羟基 -14-脱 羟基 -14,21-环氧雷公藤内酯醇 (LLDT-68), 同时得到少量 A5'6-脱氢化合 物（14S)- \5，6-脱氢 -14-脱羟基 -14, 21-环氧雷公藤内酯醇 (LLDT-70); 将 (14S)-14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-67)溶解于非质子极性 溶剂中， 用亲核试剂开环氧得到（12R,13R,14S)-12P-氯 -13α-羟基 -14-脱 羟基 -14,21-环氧雷公藤内酯醇 (LLDT-76)。
Figure imgf000010_0003
More preferably, the tripterygium diterpene lactone derivative, its optical isomer, and pharmaceutically acceptable salts and hydrates thereof according to the present invention are (14S)-14-dehydroxy-14,21-epoxy Triptolide (LLDT-67). The synthesis method of the tripterygium diterpene lactone derivative of the present invention is summarized as follows: The compound represented by the formula (1) is a triptolide (LLDT-1). For example, as shown in the reaction scheme (1), using triptolide (LLDT-1) as a starting material, in an aprotic polar solvent, at room temperature, using a sulfhydrylating reagent at C 14 Formation of a pair of epoxy compounds (14S)-14-dehydroxy-14,21-epoxytriptolide in the S and R configurations Alcohol (LLDT-67) and (14R)-14-dehydroxy-14,21-epoxytriptolide (LLDT-69); (14S)-14-dehydroxy-14,21-epoxydesene The ester alcohol (LLDT-67) is dissolved in an aprotic polar solvent and is oxidized by selenium dioxide to obtain (5R, 14S)-5a-hydroxy-14-dehydroxy-14,21-epoxylidene Ester alcohol (LLDT-68), a small amount of A 5 ' 6 -dehydrogenated compound (14S)- \ 5 , 6 -dehydro-14-dehydroxy-14, 21-epoxytriptolide (LLDT-70) (14S)-14-dehydroxy-14,21-epoxytriptolide (LLDT-67) was dissolved in an aprotic polar solvent, and epoxidized with a nucleophile to obtain (12R, 13R, 14S) )-12P-chloro-13α-hydroxy-14-dehydroxy-14,21-epoxytriptolide (LLDT-76).
Figure imgf000011_0001
反应流程式 (1) 例如 , 如反应流程式 (2)所示 , 以雷公藤内酯酮 (LLDT-1)为起始物 , 用 Tamao格氏试剂进攻雷公藤内酯醇的 C14-位羰基得到含硅的中间体 (14S)-14P-异丙氧基二曱基硅曱基表雷公藤内酯醇 (LLDT-81),该中间体 进行简单萃取浓缩后， 不经分离純化直接溶解于质子极性溶剂与非质 子极性溶剂的混合溶剂中， 氧化水解脱硅得到（14S)-14P-羟曱基表雷公 藤内酯醇（LLDT-62); 进一步以（14S)-14P-羟曱基表雷公藤内酯醇 (LLDT-62)为原料， 在极性溶剂中， 碱性条件下， 利用二氯亚砜将邻二 醇结构转化为五元环亚硫酸酯结构的（14S,硫 S)- 14α,21 -乙二醇环亚硫 酸酯雷公藤内酯醇 (LLDT-77)和（14S,硫 R)-14a,21-乙二醇环亚硫酸酯雷 公藤内酯醇 (LLDT-78); 两者进一步在氧化条件下转化为同一饱和的环 硫酸酯化合物（14S)-14a,21-乙二醇环硫酸酯雷公藤内酯醇 (LLDT-79)。
Figure imgf000011_0001
Reaction Scheme (1) For example, as shown in the reaction scheme (2), using triptolide (LLDT-1) as a starting material, using Tamao Grignard reagent to attack the C 14 -position carbonyl group of triptolide Silicon-containing intermediate (14S)-14P-isopropoxydithiosilyl thioglycolide (LLDT-81), which is simply extracted and concentrated, and directly dissolved in protons without separation and purification. (14S)-14P-hydroxyindole-based triptolide (LLDT-6 2 ) in a mixed solvent of a solvent and an aprotic polar solvent, further to (1 4 S)-1 4 P - Hydroxyindole table triptolide (LLDT-62) as a raw material, in a polar solvent, under alkaline conditions, using dichlorosulfoxide to the adjacent two The alcohol structure is converted to a five-membered cyclic sulfite structure of (14S, sulfur S)-14α, 21-ethylene glycol cyclic sulfite, triptolide (LLDT-77) and (14S, sulfur R)-14a, 21 - ethylene glycol cyclic sulfite triptolide (LLDT-78); both are further converted to the same saturated cyclic sulfate compound (14S)-14a, 21-ethylene glycol cyclic sulfate Tripterygium under oxidative conditions Ester alcohol (LLDT-79).
Figure imgf000012_0001
反应流程式 (2) 此外， （14S)-14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-67)也可通 过如下方式获得， 如反应流程式 (3)所示， 以（14S)-14P-羟曱基表雷公藤 内酯醇 (LLDT-62)为原料， 用碩酰化试剂将 C21位的末端羟基碩酰化后 得到（14S)-14P-磺酰氧基曱基表雷公藤内酯醇 (其中 R为磺酰基)， 在碱 的作用下合环得到（14S)-14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-67)。
Figure imgf000013_0001
Figure imgf000012_0001
Reaction Scheme (2) Further, (14S)-14-dehydroxy-14,21-epoxytriptolide (LLDT-67) can also be obtained by, as shown in the reaction scheme (3), (14S)-14P-hydroxyindole-based triptolide (LLDT-62) was used as a raw material, and the terminal hydroxyl group at the C21 position was acylated with a acylating reagent to obtain (14S)-14P-sulfonyloxyfluorene. The base table triptolide (wherein R is a sulfonyl group) is cyclized under the action of a base to give (14S)-14-dehydroxy-14,21-epoxytriptolide (LLDT-67).
Figure imgf000013_0001
反应流程式 (3) 另外， 本发明也提供含有治疗有效剂量的上述雷公藤二萜类内酯 衍生物、 其光学异构体及其药学上可接受的盐和水合物以及常规的药 学辅料的药物组合物。 在根据本发明的药物组合物中， 所述雷公藤内酯醇衍生物、 其光 学异构体及其药学上可接受的盐和水合物的含量为 0.001 ~ 99.9wt% 。 本发明的药物组合物的剂型可以为经胃肠道给药的剂型或非经胃 肠道给药剂型。 所述经胃肠道给药的剂型可以为溶液剂、 乳剂、 片剂、 胶嚢剂等。 所述的非胃肠道给药剂量可以为注射剂， 包括静脉注射、 肌肉注射、 皮下注射、 皮内注射等注射途径； 经皮肤给药， 如外用洗 剂、 软膏剂、 贴剂等； 经粘膜给药， 如舌下、 鼻腔、 直肠、 阴道、 耳 道等。 本发明的药物组合物的活性组分雷公藤二萜类内酯衍生物、 其光 学异构体及其药学上可接受的盐和水合物可以被单一用药， 也可以与 其它抗肿瘤药物联合治疗。 因此， 本发明的药物组合物可以进一步包 括一种或多种其它抗肿瘤药物， 所述抗肿瘤药物为影响肿瘤细胞核酸 生物合成的药物； 直接破坏肿瘤细胞 DNA阻止其复制的药物； 嵌入肿 瘤细胞 DNA中干扰转录过程的药物；干扰有丝分裂影响肿瘤细胞蛋白 质合成的药物； 或通过抑制环氧化酶 (COX-2)产生抗肿瘤作用的药物。 其中，所述影响肿瘤细胞核酸生物合成的药物可以为 5 -氟尿嘧啶 (5 - Fluorouracil), 疏噪呤 (6-Mercaptopurine)、 曱氨蝶呤 (Methotrexate)、 阿 糖胞苷 (Cytarabine)和羟基脲 (Hydroxyurea)等； 所述直接破坏肿瘤细胞 DNA阻止其复制的药物可以为氮芥 (Chlormethine Hydrochloride)、环磷 酰胺（Cytoxam)、 噻替派（Thiotepa)、 白消安（Busulfan)、 丝裂霉素 C(Mitomycin C), 博来霉素 (Bleomycin), 顺铂 (Cisplatin)、 奥马铂 (OP)、 草酸铂 (Oxaloplatin)、 DWA2114R, CI-973、 洛铂 (Lobaplatin)、 喜树碱 (Camptothecin), 依托泊苷 (Etoposide)等； 所述嵌入肿瘤细胞 DNA中干 扰转录过程的药物可以为放线菌素 D(Actinomycin D)、 阿霉素 (ADM)、 依达比星 (Idarubicin)以及丝裂蒽醌 (NVT)、安丫啶 (mAMSA)等； 所述干 扰有丝分裂影响肿瘤细胞蛋白质合成的药物可以为如长春碱 (VLB)和 长春新碱 (VCR)、碳长春碱 (VRB)、 紫杉醇 (Taxol)、 紫杉特尔（Taxotere)、 L -门
Figure imgf000014_0001
酷石威 (Harringtonine)等； 所 过抑制环氧化酶 (COX-2)产生抗肿瘤作用的药物可以为阿司匹林 (Aspirin)、 p引味美辛 (Indomethaein)、 布洛芬 (Spansule Capsulae Ibuprofeni) , 尼美舒利 (Nimesulide)、 塞来昔布 (Celecoxib)、 罗非昔布 (Rofecoxib)等。 药物联合治疗时， 雷公藤内酯醇衍生物、 其光学异构体及其药学 上可接受的盐和水合物与其它化疗药物可以是同时给药、 可以是序贯 给药， 也可以是分开给药。 此外， 本发明还提供了上述药物组合物在制备用于治疗肿瘤、 以 及与肿瘤相关的疾病的药物中的应用， 其中， 药物活性组分以 0.001 ~ 10mg/kg的量被使用。这样的给药剂量通常能产生有效的治疗效果。如 (14S)-14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-67)以 1.0、 2.0、 4.0 mg/kg的剂量口服给药能明显抑制人前列腺癌、卵巢癌棵小鼠移植瘤的 生长。但是，给药剂量将根据治疗对象、受治疗者的年龄 (月龄或周龄)、 一般健康状况、 给药途径、 个体对药物的敏感性以及病情的严重程度 等作相应的调整， 以对受治疗者施用含本发明化合物治疗有效量的药 Reaction Scheme (3) Further, the present invention also provides a therapeutically effective amount of the above-described tripterygium diterpenoid derivative, an optical isomer thereof, and pharmaceutically acceptable salts and hydrates thereof, and a conventional pharmaceutical excipient. Pharmaceutical composition. In the pharmaceutical composition according to the present invention, the content of the triptolide derivative, its optical isomer, and pharmaceutically acceptable salts and hydrates thereof is from 0.001 to 99.9 wt%. The pharmaceutical composition of the present invention may be in the form of a gastrointestinal administration or a parenteral administration. The gastrointestinal administration can be in the form of a solution, an emulsion, a tablet, a capsule, or the like. The parenteral administration dose may be an injection, including intravenous, intramuscular, subcutaneous, intradermal injection or the like; transdermal administration, such as topical lotion, ointment, patch, etc.; transmucosal Administration, such as sublingual, nasal, rectal, vaginal, ear canal, and the like. The active component of the pharmaceutical composition of the present invention, the triptolide diterpene lactone derivative, the optical isomer thereof, and the pharmaceutically acceptable salts and hydrates thereof, may be used alone or in combination with other antitumor drugs. . Therefore, the pharmaceutical composition of the present invention may further comprise one or more other antitumor drugs, which are drugs that affect the biosynthesis of tumor cell nucleic acid; drugs that directly destroy tumor cell DNA to prevent their replication; A drug that interferes with the transcription process in DNA; a drug that interferes with mitosis affecting protein synthesis in tumor cells; or a drug that produces antitumor effects by inhibiting cyclooxygenase (COX-2). Wherein, the drug affecting nucleic acid biosynthesis of tumor cells may be 5-fluorouracil, 6-Mercaptopurine, Methotrexate, Cytarabine and hydroxyurea. (Hydroxyurea), etc.; the drug that directly destroys the DNA of the tumor cell to prevent its replication may be Chlormethine Hydrochloride, Cytoxam, Thiotepa, Busulfan, Mitochondrial Mitomycin C, Bleomycin, Cisplatin, Omarine (OP), Oxaloplatin, DWA2114R, CI-973, Lobaplatin, Camptothecin Etoposide, etc.; the drug interfering with the transcription process in the DNA of the tumor cell may be actinomycin D, doxorubicin (ADM), idarubicin, and silk Splitting (NVT), ampicillin (mAMSA), etc.; the drugs that interfere with mitosis affecting protein synthesis of tumor cells may be, for example, vinblastine (VLB) and vincristine (VCR), carbocine (VRB), paclitaxel (Taxol), Taxotere, L-Door
Figure imgf000014_0001
Coolstone K (Harringtonine) and the like; through the inhibition of cyclooxygenase (COX-2) produced antitumor drugs can be aspirin (Aspirin), p lead delicious oct (Indomethaein), ibuprofen (Spansule Capsulae Ibuprofeni), Nimesulide, Celecoxib, Rofecoxib, and the like. In the combination therapy, the triptolide derivative, its optical isomer and its pharmaceutically acceptable salts and hydrates may be administered simultaneously, or sequentially, or separately. medicine. Further, the present invention provides the use of the above pharmaceutical composition for the preparation of a medicament for treating a tumor, and a tumor-associated disease, wherein the pharmaceutically active component is used in an amount of 0.001 to 10 mg/kg. Such administered doses generally produce an effective therapeutic effect. Such as (14S)-14-dehydroxy-14,21-epoxytriptolide (LLDT-67) to 1.0, 2.0, 4.0 Oral administration of a dose of mg/kg can significantly inhibit the growth of transplanted tumors of human prostate cancer and ovarian cancer mice. However, the dose to be administered will be adjusted according to the subject to be treated, the age of the subject (months or weeks of age), the general health condition, the route of administration, the sensitivity of the individual to the drug, and the severity of the condition, etc. The subject is administered a therapeutically effective amount of a drug comprising a compound of the invention
本发明的药物组合物可以用于治疗患有肿瘤等增生性疾病的温血 动物， 所述"肿瘤"包括良性肿瘤和恶性肿瘤： 良性肿瘤主要有神经胶 质瘤、 纤维瘤、 脂肪瘤、 血管瘤等； 恶性肿瘤主要包括由上皮组织发 生的恶性肿瘤如鳞状上皮癌、 乳腺癌、 卵巢癌等， 由中胚层组织发生 的恶性肿瘤如纤维肉瘤、 骨肉瘤、 淋巴肉瘤等， 以及由胚胎细胞、 神 经细胞或未成熟组织发生的恶性肿瘤和由造血细胞发生的恶性肿瘤 等。 所述"温血动物"包括人和其它动物， 如啮齿类动物和哺乳类动物。 所述"增生性疾病"的含义包括肿瘤、 不典型增生， 但不局限于肿瘤和 不典型增生。 本发明的药物组合物还可以用于与肿瘤相关的疾病， 包括前列腺 癌、 人卵巢癌、 神经胶质瘤、 胃癌、 粒细胞性白血病、 结肠癌、 乳腺 癌、 阿霉素敏感的乳腺癌、 阿霉素抗药的乳腺癌等肿瘤疾病。 优选地， 本发明的药物组合物用于治疗前列腺癌和卵巢癌。 本发明中所使用的一些术语具有如下意义： The pharmaceutical composition of the present invention can be used for treating warm-blooded animals having proliferative diseases such as tumors, including benign tumors and malignant tumors: benign tumors mainly include gliomas, fibroids, lipomas, blood vessels Malignant tumors mainly include malignant tumors such as squamous cell carcinoma, breast cancer, ovarian cancer, etc., which are caused by epithelial tissues, malignant tumors such as fibrosarcoma, osteosarcoma, lymphosarcoma, etc., which are caused by mesoderm tissues, and embryonic cells. Malignant tumors that occur in nerve cells or immature tissues, and malignant tumors that occur from hematopoietic cells. The "warm-blooded animals" include humans and other animals such as rodents and mammals. The meaning of "proliferative diseases" includes tumors, dysplasia, but not limited to tumors and dysplasia. The pharmaceutical composition of the present invention can also be used for tumor-related diseases, including prostate cancer, human ovarian cancer, glioma, gastric cancer, granulocyte leukemia, colon cancer, breast cancer, adriamycin-sensitive breast cancer, Doxorubicin-resistant tumor diseases such as breast cancer. Preferably, the pharmaceutical composition of the invention is for the treatment of prostate cancer and ovarian cancer. Some of the terms used in the present invention have the following meanings:
"低级，，指碳原子数为 1 ~ 6个的直链或支链碳链。 "Lower, refers to a straight or branched carbon chain with 1 to 6 carbon atoms.
"烷氧基"可为曱氧基、 乙氧基、 丙氧基、 异丙氧基、 丁氧基、 异 丁氧基、 仲丁氧基、 叔丁氧基、 戊氧基、 异戊氧基、 叔戊氧基、 新戊 氧基、 2 -曱基丁氧基、 1 , 2 -二曱基丙氧基、 1 -乙基丙氧基、 己氧 基等， 以曱氧基和乙氧基为佳。 "Alkoxy" may be decyloxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isovaleryloxy Base, tert-pentyloxy, neopentyl The oxy group, 2-nonylbutoxy group, 1,2-dimercaptopropoxy group, 1-ethylpropoxy group, hexyloxy group and the like are preferably a decyloxy group and an ethoxy group.
"烷胺基"可为曱胺基、 乙胺基、 丙胺基、 异丙胺基、 丁胺基、 异 丁胺基、 仲丁胺基、 叔丁胺基、 戊胺基、 异戊胺基、 叔戊胺基、 新戊 胺基等一级低级烷胺基， 或二曱胺基、 二乙胺基、 二丙胺基、 二异丙 胺基、 二丁胺基、 二异丁胺基等二级低级烷胺基， 这些基团中较好的 有曱胺基、 乙胺基、 二曱胺基、 二乙胺基。 "Alkylamino" may be guanamine, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, sec-butylamino, tert-butylamino, pentylamino, isoamylamino, tert-amyl a primary lower alkylamine such as an amine group or a neopentylamino group, or a secondary lower alkyl group such as a diammonium group, a diethylamino group, a dipropylamino group, a diisopropylamino group, a dibutylamino group or a diisobutylamino group. Amine groups, preferred among these groups are amidino group, ethylamine group, diammonium group, and diethylamino group.
"烷巯基"可为曱巯基、 乙巯基、 丙巯基、 异丙巯基、 丁巯基、 异 丁巯基、 仲丁巯基、 叔丁巯基、 戊巯基、 异戊巯基、 叔戊巯基、 新戊 巯基、 2 -曱基丁巯基、 1 , 2 -二曱基丙巯基、 1 -乙基丙巯基、 己巯 基等， 以曱巯基和乙巯基为佳。 "Alkyl fluorenyl" may be fluorenyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, tert-amyl, neopentyl, 2 - mercapto fluorenyl, 1,2-dimercaptopropyl, 1-ethylpropenyl, hexyl, etc., preferably fluorenyl and acetyl.
"极性溶剂，，为例如乙酸乙酯、 二氧六环、 丙酮或叔丁醇等； "Polar solvent, such as ethyl acetate, dioxane, acetone or tert-butanol;
"非质子极性溶剂"指例如二曱亚砜、 Ν,Ν-二曱基曱酰胺、 二氯曱 烷、 三氯曱烷、 四氢呋喃、 二氧六环或乙二醇双曱醚等； "Aprotic polar solvent" means, for example, disulfoxide, hydrazine, hydrazine-dihydrazinamide, dichlorodecane, trichlorodecane, tetrahydrofuran, dioxane or ethylene glycol biguanide;
"质子极性溶剂"指例如曱醇、 乙醇、 丙醇或叔丁醇等； "Proton polar solvent" means, for example, decyl alcohol, ethanol, propanol or tert-butanol;
"加热条件"指室温以上至溶剂回流温度； "Heating conditions" means temperatures above room temperature to the reflux temperature of the solvent;
"亚曱基化试剂"指例如 Corey亚曱基化试剂、 Simmons-Smith试剂 或重氮曱烷等； "Amidinolation reagent" means, for example, Corey sulfhydryl reagent, Simmons-Smith reagent or diazonium;
"橫酰化试剂"指例如曱磺酰氯、 乙磺酰氯、 苯橫酰氯、 对曱苯橫 酰氯、 三氟曱磺酰氯或三氟曱磺酸酐等； The "transacylation reagent" means, for example, sulfonyl chloride, ethanesulfonyl chloride, benzoyl chloride, p-toluene chloride, trifluorosulfonyl chloride or trifluoromethanesulfonic anhydride;
"亲核试剂" 指例如氯化氢或吡啶盐酸盐等； "药学上可接受的盐，，具体的可列举与丙酸、 草酸、 丙二酸、 琥珀 酸、 富马酸、 马来酸、 乳酸、 苹果酸、 酒石酸、 柠檬酸等有机酸和天 冬氨酸、 谷氨酸等酸性氨基酸形成酯后再与无机碱形成的盐， 如钠、 钾、 钙、 铝盐和铵盐， 或与有机碱形成的盐， 如曱胺盐、 乙胺盐、 乙 醇胺盐等， 或与赖氨酸、 精氨酸、 鸟氨酸等碱性氨基酸形成酯后的盐 酸、 氢溴酸、 氢氟酸、 硫酸、 硝酸、 磷酸等无机酸的盐， 或与曱酸、 乙酸、 苦味酸、 曱橫酸等有机酸的盐。 "nucleophile" means, for example, hydrogen chloride or pyridine hydrochloride; "Pharmaceutically acceptable salts, specifically, organic acids and aspartame with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, etc. A salt formed with an acidic base such as an acid or a glutamic acid to form an ester, such as a sodium, potassium, calcium, aluminum salt and an ammonium salt, or a salt formed with an organic base such as a guanamine salt, an ethylamine salt or an ethanolamine. Salt, etc., or a salt of an inorganic acid such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid which is esterified with a basic amino acid such as lysine, arginine or ornithine, or with tannic acid, a salt of an organic acid such as acetic acid, picric acid or hydrazine acid.
"光学异构体 "的含义包括对映异构体、 非对映异构体、 光学异构 体的混合物及純光学异构体。 有益效果 本发明合成了高效、 低毒的雷公藤二萜类内酯衍生物， 使其能够 实际地用于肿瘤疾病的治疗。 体外药效结果显示， 所获得的一系列雷 公藤二萜类内酯衍生物中， 用（14S)- 14,21-环氧结构代替了以往认为是 活性必需基团的 C14P-0H 的 4 个衍生物均表现出了较强的抗肿瘤作 用， 这无疑是结构改造方向上的一种重大突破。 急性毒性实验结果表 明本发明中的雷公藤二萜类内酯衍生物（14S)-14-脱羟基 -14,21-环氧雷 公藤内酯醇 (LLDT-67), 0%死亡剂量 LD。高达 20mg/kg, 而其先导化合 物雷公藤内酯醇的半数致死剂量 LD5Q则仅为 0.5mg/kg,因此，（14S)-14- 脱羟基 -14,21-环氧雷公藤内酯醇的毒性远低于雷公藤内酯醇 (LLDT-2), 使得可安全使用的剂量范围大为提高。 从体内药效结果可 知， （14S)-14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-67)对生殖系统肿 瘤具有明显的抑制作用， 且表现出了高度的特异性， 因而更有利于降 低其毒副作用。 本发明的药物组合物能够制成有利于温血动物的组织、 器官吸收利用的制剂形式， 其在治疗肿瘤等增生性疾病方面具有良好 的应用前景。 附图说明 图 1为说明 LLDT-67对人卵巢癌 SK-OV-3棵小鼠皮下移植瘤的生 长抑制作用的曲线图。 图 2为说明 LLDT-67对人卵巢癌 SK-OV-3棵小鼠皮下移植瘤的生 长抑制作用的照片， 其中鼠尾代表该成活鼠移植瘤已不可检出。 图 3为说明 LLDT-67对人前列腺癌 PC-3棵小鼠皮下移植瘤的生长 抑制作用的曲线图。 图 4为说明 LLDT-67对人前列腺癌 PC-3棵小鼠皮下移植瘤的生长 抑制作用的照片。 图 5为说明 LLDT-67对人前列腺癌 DU-145棵小鼠皮下移植瘤的 生长抑制作用的曲线图。 图 6为说明 LLDT-67对人前列腺癌 DU-145棵小鼠皮下移植瘤的 生长抑制作用照片。 图 7为说明 LLDT-67对人胃癌 SGC-7901棵小鼠皮下移植瘤的生 长抑制作用的曲线图。 图 8为说明 LLDT-67对人胃癌 SGC-7901棵小鼠皮下移植瘤的生 长抑制作用的照片。 图 9为说明 LLDT-67对人乳腺癌 MDA-MB-468棵小鼠皮下移植瘤 的生长抑制作用的曲线图。 图 10为说明 LLDT-67对人乳腺癌 MDA-MB-468棵小鼠皮下移植 瘤的生长抑制作用的照片。 图 11为说明 LLDT-67对人肝癌 SMMC-7721棵小鼠皮下移植瘤的 生长抑制作用的曲线图。 图 12为说明 LLDT-67对人肝癌 SMMC-7721棵小鼠皮下移植瘤的 生长抑制作用的照片。 具体实施方式 下面结合实例对本发明进行进一步阐述， 但这些实施例绝不是对 本发明的任何限制， 本发明的范围由权利要求决定。 制备实施例 所用仪器及主要实验材料如下： The meaning of "optical isomer" includes enantiomers, diastereomers, mixtures of optical isomers, and pure optical isomers. Advantageous Effects The present invention synthesizes a highly efficient and low-toxic Tripterygium divalent lactone derivative, which enables it to be practically used for the treatment of tumor diseases. The results of in vitro drug efficacy showed that among the series of tripterygium diterpene lactone derivatives obtained, four (14S)- 14,21-epoxy structures were substituted for C14P-0H which were previously considered to be active essential groups. The derivatives all showed strong anti-tumor effects, which is undoubtedly a major breakthrough in the direction of structural transformation. The results of the acute toxicity test showed that the tripterygium diterpene lactone derivative (14S)-14-dehydroxy-14,21-epoxytriptolide (LLDT-67), 0% dead dose LD in the present invention. Up to 20mg/kg, and the LD 5Q of its lead compound triptolide is only 0.5mg/kg, therefore, the toxicity of (14S)-14-dehydroxy-14,21-epoxytriptolide Far less than triptolide (LLDT-2), the range of doses that can be safely used is greatly increased. From the results of in vivo efficacy, (14S)-14-dehydroxy-14,21-epoxytriptolide (LLDT-67) has a significant inhibitory effect on reproductive system tumors and shows a high degree of specificity. Therefore, it is more conducive to reducing its toxic side effects. The pharmaceutical composition of the present invention can be made into a tissue that is beneficial to warm-blooded animals, A form of preparation for organ absorption and utilization, which has a good application prospect in the treatment of proliferative diseases such as tumors. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human ovarian cancer SK-OV-3 mice. Fig. 2 is a photograph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human ovarian cancer SK-OV-3 mice, wherein the rat tail represents that the transplanted tumor of the living mouse is undetectable. Figure 3 is a graph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human prostate cancer PC-3 mice. Figure 4 is a photograph showing the growth inhibitory effect of LLDT-67 on human prostate cancer PC-3 subcutaneous xenografts in mice. Figure 5 is a graph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human prostate cancer DU-145 mice. Fig. 6 is a photograph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human prostate cancer DU-145 mice. Figure 7 is a graph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human gastric cancer SGC-7901 mice. Fig. 8 is a photograph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human gastric cancer SGC-7901 mice. Figure 9 is a graph showing the growth inhibitory effect of LLDT-67 on human breast cancer MDA-MB-468 subcutaneous xenografts. Figure 10 is a photograph showing the growth inhibitory effect of LLDT-67 on human breast cancer MDA-MB-468 subcutaneous xenografts. Figure 11 is a graph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human liver cancer SMMC-7721 mice. Figure 12 is a photograph showing the growth inhibitory effect of LLDT-67 on subcutaneous xenografts of human liver cancer SMMC-7721 mice. The invention is further illustrated by the following examples, but these examples are in no way intended to limit the invention, and the scope of the invention is determined by the claims. The instruments and main experimental materials used in the preparation examples are as follows:
Fisher- Johns显微熔点仪 (温度未经校正）， JASCODIP-181旋光仪， Perkin-Elmer599B型和 Nicole Magan750型红外光谱仪， BrukerAM-400 型和 Varian Mercury plus-400型核磁共振仪， MAT-711和 MAT-95型质 谱仪， H及 200-300 目柱层析硅胶 (青岛海洋化工厂）， HSGF254 TLC 板 (；烟台市化工研究院：)。 制备实施例 1 (14S)-14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-67) Fisher-Johns micro melting point instrument (temperature uncorrected), JASCODIP-181 polarimeter, Perkin-Elmer 599B and Nicole Magan 750 infrared spectrometer, Bruker AM-400 and Varian Mercury plus-400 NMR, MAT-711 and MAT-95 mass spectrometer, H and 200-300 mesh column chromatography silica gel (Qingdao Ocean Chemical Plant), HSGF254 TLC plate (; Yantai Institute of Chemical Industry:). Preparation Example 1 (14S)-14-dehydroxy-14,21-epoxytriptolide (LLDT-67)
Figure imgf000019_0001
LLDT-1 LLDT-67 将 (CH3)3SOI (59.6 mg, 0.27 mmol)和 NaH (60%, 9.5 mg, 0.24 mmol) 以固体形式混合， 氩气保护下加入 2 mL无水二曱亚砜， 室温搅拌 20 min, 向其中滴加溶有雷公藤内酯酮 (LLDT-1) (53.7 mg, 0.15 mmol)的无 水二曱亚砜溶液 (2 mL),搅拌反应。 TLC检测原料转化完全，加入 3 mL 水淬灭反应， 乙酸乙酯萃取， 有机层用水、 饱和食盐水洗涤， 无水硫 酸钠干燥， 减压蒸除溶剂， 所得粗产物经柱层析純化 (洗脱剂： 乙酸乙 酯:环己烷 =1 :5), 立体专一性地得到白色固体 (14S)-14-脱羟基 -14,21-环 氧雷公藤内酯醇 (LLDT-67) (47.5 mg, 0.13 mmol, 产率： 85%)。 lR NMR (CDC13, 300 MHz) δ 4.67 (s, 2 H), 3.88 (d, J = 2.9 Hz, 1 H), 3.52 (d, J = 3.1 Hz, 1 H), 3.40 (d, J = 5.5 Hz, 1 H), 2.84 (d, J = 5.2 Hz, 1 H); 2.80-2.69 (m, 2 H), 2.37-2.25 (m, 1 H), 2.21-2.05 (m, 2 H), 1.91-1.80 (m, 2 H), 1.57 (dd, J= 12.4, 4.9 Hz, 1 H), 1.29-1.17 (m, 1 H), 1.06 (s, 3 H), 0.86 (d, J = 6.8 Hz, 3 H), 0.80 (d, J= 6.8 Hz, 3 H); 13C NMR (CDC13, 100 MHz) δ 173.2 (C), 160.2 (C), 125.2 (C), 69.9 (CH2), 65.2 (C), 65.0 (C), 58.4 (C), 56.8 (CH), 55.9 (CH), 55.6 (C), 54.1 (CH), 47.9 (CH2), 40.4 (CH): 35.6 (C), 30.2 (CH2), 23.4 (CH2), 23.2 (CH), 19.8 (CH3), 17.6 (CH3), 17.0 (CH2), 13.5 (CH3); IR (KBr) 3427, 3016, 2981, 2929, 1745, 1674, 1442, 1022 cm"1; MS (EI, 70 eV) m/z (%) 373 ([M+l]+, 2), 372 (M+, 1), 357 (5), 343 (21), 91 (100); HRMS (EI) calcd. for C21H2406 372.1573, found 372.1578; Anal. (C21H2406) calcd. C 67.73, H 6.50, found C 67.71, H 6.51 ; [a]2 D° -138 (c 0.109, CHC13); mp 245-246 °C . 制备实施例 2 (14S)-14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-67) 和（ 14R)- 14-脱羟基- 14,21 -环氧雷公藤内酯醇 (LLDT-69)
Figure imgf000021_0001
Figure imgf000019_0001
LLDT-1 LLDT-67 (CH 3 ) 3 SOI (59.6 mg, 0.27 mmol) and NaH (60%, 9.5 mg, 0.24 mmol) were mixed as a solid, and 2 mL of anhydrous disulfoxide was added under argon. After stirring at room temperature for 20 min, an aqueous solution of triptolide (LLDT-1) (53.7 mg, 0.15 mmol) in anhydrous disulfoxide (2 mL) was added dropwise thereto, and the mixture was stirred. The TLC was used to carry out the conversion of the starting material. The reaction was quenched with EtOAc (EtOAc). Detachment: Ethyl acetate: cyclohexane = 1:5), stereospecifically obtained as a white solid (14S)-14-dehydroxy-14,21-epoxytriptolide (LLDT-67) (47.5 Mg, 0.13 mmol, yield: 85%). lR NMR (CDC1 3 , 300 MHz) δ 4.67 (s, 2 H), 3.88 (d, J = 2.9 Hz, 1 H), 3.52 (d, J = 3.1 Hz, 1 H), 3.40 (d, J = 5.5 Hz, 1 H), 2.84 (d, J = 5.2 Hz, 1 H); 2.80-2.69 (m, 2 H), 2.37-2.25 (m, 1 H), 2.21-2.05 (m, 2 H), 1.91-1.80 (m, 2 H), 1.57 (dd, J= 12.4, 4.9 Hz, 1 H), 1.29-1.17 (m, 1 H), 1.06 (s, 3 H), 0.86 (d, J = 6.8 Hz, 3 H), 0.80 (d, J = 6.8 Hz, 3 H); 13 C NMR (CDC1 3 , 100 MHz) δ 173.2 (C), 160.2 (C), 125.2 (C), 69.9 (CH 2 ) , 65.2 (C), 65.0 (C), 58.4 (C), 56.8 (CH), 55.9 (CH), 55.6 (C), 54.1 (CH), 47.9 (CH 2 ), 40.4 (CH) : 35.6 (C ), 30.2 (CH 2 ), 23.4 (CH 2 ), 23.2 (CH), 19.8 (CH 3 ), 17.6 (CH 3 ), 17.0 (CH 2 ), 13.5 (CH 3 ); IR (KBr) 3427, 3016 , 2981, 2929, 1745, 1674, 1442, 1022 cm"1; MS (EI, 70 eV) m/z (%) 373 ([M+l] + , 2), 372 (M + , 1), 357 (5), 343 (21), 91 (100); HRMS (EI) calcd. for C 21 H 24 0 6 372.1573, found 372.1578; Anal. (C 21 H 24 0 6 ) calcd. C 67.73, H 6.50, found C 67.71, H 6.51; [ a] 2 D ° -138 (c 0.109, CHC1 3);. EXAMPLE 2 (14S) mp 245-246 ° C preparation -14- off -14,21- epoxy group triptolide (LLDT-67) and (14R) - 14- dehydroxylation - 14,21 - epoxy triptolide (LLDT-69)
Figure imgf000021_0001
LLDT-1 LLDT-67 LLDT-69 氩气保护下将 (CH3)3SOI (374mg,1.7mmol)和 -BuOK (174 mg,1.5 mmol)溶于 6 mL干燥的二曱亚砜中，该混合体系于室温下搅拌 20 min。 将溶解于 6 mL干燥二曱亚砜溶剂中的雷公藤内酯酮 (LLDT-1) (360 mg, 1.0 mmol)加入上述混合物中， 室温下搅拌反应至底物转化完全， 加入 10 mL水淬灭反应， 乙酸乙酯萃取， 有机层用水、 饱和食盐水洗涤， 无水硫酸钠干燥。 减压蒸出溶剂后将粗产物经柱层析純化 (洗脱剂： 乙 酸乙酯:环己烷 =1 :5)。 所得白色固体用半制备 HPLC分离得到一对差向 异构体（14S)-14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-67) (0.196 g, 0.53 mmol, 产率： 53%) 和（14R)-14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-69) (0.126 g, 0.34 mmol, 产率： 34%)。 LLDT-1 LLDT-67 LLDT-69 (CH 3 ) 3 SOI (374 mg, 1.7 mmol) and -BuOK (174 mg, 1.5 mmol) were dissolved in 6 mL of dry disulfoxide under argon. The system was stirred at room temperature for 20 min. The triptolide (LLDT-1) (360 mg, 1.0 mmol) dissolved in 6 mL of dry disulfoxide solvent was added to the above mixture, and the reaction was stirred at room temperature until the substrate was completely converted, and quenched by adding 10 mL of water. The reaction mixture was washed with ethyl acetate. After distilling off the solvent under reduced pressure, the crude product was purified by column chromatography (eluent: ethyl acetate: cyclohexane = 1:5). The obtained white solid was separated by semi-preparative HPLC to give a pair of epimer (14S)-14-dehydroxy-14,21-epoxy-tratolide (LLDT-67) (0.196 g, 0.53 mmol, yield: 53%) and (14R)-14-dehydroxy-14,21-epoxytriptolide (LLDT-69) (0.126 g, 0.34 mmol, yield: 34%).
( 14R)- 14-脱羟基- 14,21 -环氧雷公藤内酯醇 (LLDT-69)： (14R)- 14-dehydroxy- 14,21-epoxytriptolide (LLDT-69):
¾ NMR (CDC13, 300 MHz) δ 4.79 (dt, J = 16.8, 2.6 Hz, 1 H), 4.60 (dd, J= 16.8, 2.8 Hz, 1 H), 3.81 (d, J= 3.1 Hz, 1 H), 3.52 (d, J= 3.1 Hz, 1 H), 3.38 (d, J= 4.2 Hz, 1 H), 2.89 (d, J= 4.7 Hz, 1 H), 2.85 (d, J= 4.7 Hz, 1 H), 2.81-2.72 (m, 1 H), 2.39-2.27 (m, 1 H), 2.17 (dt, J = 14.3, 4.2 Hz, 1 H), 2.11-1.94 (m, 1 H), 1.94-1.81 (m, 2 H), 1.47-1.29 (m, 2 H), 0.96 (s, 3 H), 0.89 (d, J = 6.8 Hz, 3 H), 0.77 (d, J = 6.8 Hz, 3 H); 13C NMR (CDC13, 100 MHz) δ 173.2 (C), 161.6 (C), 125.4 (C), 70.8 (CH2), 65.9 (C), 64.6 (C): 58.3 (C), 57.2 (CH), 56.2 (CH), 55.9 (C), 54.7 (CH), 48.2 (CH2), 36.9 (CH) 36.1 (C), 28.4 (CH2), 26.9 (CH2), 23.0 (CH), 21.7 (CH3), 20.2 (CH3), 17.3 (CH3), 16.6 (CH2); IR (KBr) 3433, 2947, 2929, 1768, 1755, 1689, 1442 cm"1; MS (EI, 70 eV) m/z (%) 372 (M+, 1), 357 (9), 343 (18), 325 (34), 259 (100); HRMS (EI) calcd. for C21H2406 372.1573, found 372.1586; [a]2 D° -156 (c 0.135, CHC13); mp 177-179 °C . 制备实施例 3 (14S)-14P-羟曱基表雷公藤内酯醇 (LLDT-62) 3⁄4 NMR (CDC1 3 , 300 MHz) δ 4.79 (dt, J = 16.8, 2.6 Hz, 1 H), 4.60 (dd, J= 16.8, 2.8 Hz, 1 H), 3.81 (d, J= 3.1 Hz, 1 H), 3.52 (d, J = 3.1 Hz, 1 H), 3.38 (d, J = 4.2 Hz, 1 H), 2.89 (d, J = 4.7 Hz, 1 H), 2.85 (d, J = 4.7 Hz , 1 H), 2.81-2.72 (m, 1 H), 2.39-2.27 (m, 1 H), 2.17 (dt, J = 14.3, 4.2 Hz, 1 H), 2.11-1.94 (m, 1 H), 1.94-1.81 (m, 2 H), 1.47-1.29 (m, 2 H), 0.96 (s, 3 H), 0.89 (d, J = 6.8 Hz, 3 H), 0.77 (d, J = 6.8 Hz, 3 H); 13 C NMR (CDC1 3 , 100 MHz) δ 173.2 (C), 161.6 (C), 125.4 (C), 70.8 (CH 2 ), 65.9 (C), 64.6 (C) : 58.3 (C) , 57.2 (CH), 56.2 (CH), 55.9 (C), 54.7 (CH), 48.2 (CH 2 ), 36.9 (CH) 36.1 (C), 28.4 (CH 2 ), 26.9 (CH 2 ), 23.0 (CH), 21.7 (CH 3 ), 20.2 (CH 3 ), 17.3 (CH 3 ), 16.6 (CH 2 ); IR (KBr) 3433, 2947, 2929, 1768, 1755, 1689, 1442 cm"1; MS (EI, 70 eV) m/z (%) 372 (M + , 1), 357 (9), 343 (18), 325 ( 34), 259 (100); HRMS (EI) calcd. for C 21 H 24 0 6 372.1573, found 372.1586; [a] 2 D ° -156 (c 0.135, CHC1 3 ); mp 177-179 ° C . Preparation Example 3 (1 4 S)-1 4 P-hydroxyindole-based triptolide (LLDT-6 2 )
Figure imgf000022_0001
Figure imgf000022_0001
LLDT-1 LLDT-81 LLDT-1 LLDT-81
Figure imgf000022_0002
Figure imgf000022_0002
LLDT-62 在反应瓶中称量镁屑（108 mg, 4.5 mL), 氩气置换保护反应体系； 将异丙氧基二曱基氯曱基硅烷 (0.72 ml, 4.0 mmol)溶于 Ί mL无水四氢 呋喃中， 氩气保护下取 lmL该溶液滴加入镁屑反应体系中， 并加入两 滴 1,2-二溴乙烷， 40 °C搅拌下引发格氏反应， 随后将剩余的硅烷试剂的 四氢呋喃溶液逐滴滴入反应体系中， 5 min滴加完毕， 此时反应体系呈 暗灰色。 回流该反应体系 45 min , 随后冷却至 0 °C。 将溶于无水四氢呋 喃的雷公藤内酯酮 (LLDT-1) (358 mg, 1.0 mL)滴加入上述新鲜制备的格 氏试剂中。 室温下搅拌反应 2 小时， 饱和氯化铵溶液淬灭反应， 乙酸 乙酯萃取， 有机层用饱和食盐水洗涤， 无水硫酸钠干燥， 减压蒸出溶 剂得一单一加成产物。 该加成产物不经純化直接于 40°C下溶于 5 mL 曱醇和 8 mL四氢呋喃的混合溶剂中 ,搅拌下依次加入 KHC03 (416 mg, 4.2 mmol)、KF(464 mg,4.9 mmol)和 H2O2 (30%, 1.1 mL, 9.71 mmol)。40°C 下反应， TLC (环己烷： 乙酸乙酯 = 1 : 1)检查原料消失后， 硫代硫酸钠水 溶液 (50%)中止反应。 反应混合物用乙酸乙酯萃取， 有机层用饱和食盐 水洗涤， 无水硫酸钠干燥。 浓缩后的粗产物经柱层析純化 (洗脱剂： 乙 酸乙酯：环己烷 =1 :3)得白色固体（ 14S)- 14β-羟曱基表雷公藤内酯醇 (LLDT-62) (273 mg, 0.70 mmol, 产率： 70%)。 lR NMR (CDC13, 300 MHz) δ 4.67 (s, 2 H), 4.26 (d, J= 11.8 Hz, 1 H)， 3.87-3.80 (m, 2 H), 3.64 (ά, J = 11.5 Hz, 1 H), 3.46 (ά, J = 3.3 Hz, 1 H), 2.76-2.64 (m, 1 H), 2.45 (sept., J = 6.9 Hz, 1 H), 2.37-2.25 (m, 1 H), 2.23-2.04 (m, 2 H), 1.89 (t, J = 14.1 Hz, 1 H), 1.55 (dd, J = 12.6, 5.2 Hz, 1 H), 1.25-1.13 (m, 1 H), 1.07 (s, 3 H), 0.91 (d, J = 6.9 Hz, 3 H), 0.89 (d, J = 6.9 Hz, 3 H); 13C NMR (CDC13, 100 MHz) δ 173.2 (C), 160.2 (C), 125.4 (C), 74.4 (C), 70.0 (CH2), 67.5 (C), 65.3 (C), 65.2 (CH2), 65.0 (C), 56.5 (CH), 56.1 (CH), 54.4 (CH), 40.3 (CH), 36.0 (C), 30.1 (CH2), 25.5 (CH), 23.4 (CH2), 20.9 (CH3), 18.6 (CH3), 17.1 (CH2), 13.7 (CH3); IR (KBr) 3415， 3361, 2966, 2927, 2875, 1755, 1724, 1672, 1439, 1074, 1018 cm"1; MS (EI, 70 eV) m/z (%) 391 ([M+l]+, 2), 372 (1), 71 (100); HRMS (EI) calcd. for C2iH2707 (M+H)+ 391.1757, found 391.1752; [a]2 D° -116 (c 0.085, CHC13); mp 237-239 °C . 制备实施例 4 (14S)-14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-67) LLDT-62 Weighed magnesia (108 mg, 4.5 mL) in a reaction flask and replaced the protective reaction system with argon; dissolved isodecyl chlorodecyl silane (0.72 ml, 4.0 mmol) in Ί mL In water tetrahydrofuran, take 1 mL of this solution under argon gas to be added to the magnesium cuttings reaction system, and add two drops of 1,2-dibromoethane. The mixture is stirred at 40 °C to initiate the Grignard reaction, and then the remaining silane reagent is The tetrahydrofuran solution was dropwise added to the reaction system, and the addition was completed in 5 minutes, at which time the reaction system was dark gray. The reaction was refluxed for 45 min and then cooled to 0 °C. Triptolide (LLDT-1) (358 mg, 1.0 mL) dissolved in anhydrous tetrahydrofuran was added dropwise to the freshly prepared grid described above. In the reagents. The reaction mixture was stirred at room temperature for 2 hours, and then the mixture was evaporated. The addition product was directly dissolved in a mixed solvent of 5 mL of sterol and 8 mL of tetrahydrofuran at 40 ° C without further purification, and KHC0 3 (416 mg, 4.2 mmol), KF (464 mg, 4.9 mmol) and K. H 2 O 2 (30%, 1.1 mL, 9.71 mmol). The reaction was carried out at 40 ° C, TLC (cyclohexane: ethyl acetate = 1 : 1). After the disappearance of the starting material, the aqueous sodium thiosulfate solution (50%) was quenched. The reaction mixture was extracted with EtOAc. The concentrated crude product was purified by column chromatography (eluent: ethyl acetate: cyclohexane = 1:3) to afford white solid (14S) - 14β-hydroxyindole-based triptolide (LLDT-62) ( 273 mg, 0.70 mmol, Yield: 70%). lR NMR (CDC1 3 , 300 MHz) δ 4.67 (s, 2 H), 4.26 (d, J = 11.8 Hz, 1 H), 3.87-3.80 (m, 2 H), 3.64 (ά, J = 11.5 Hz, 1 H), 3.46 (ά, J = 3.3 Hz, 1 H), 2.76-2.64 (m, 1 H), 2.45 (sept., J = 6.9 Hz, 1 H), 2.37-2.25 (m, 1 H) , 2.23-2.04 (m, 2 H), 1.89 (t, J = 14.1 Hz, 1 H), 1.55 (dd, J = 12.6, 5.2 Hz, 1 H), 1.25-1.13 (m, 1 H), 1.07 (s, 3 H), 0.91 (d, J = 6.9 Hz, 3 H), 0.89 (d, J = 6.9 Hz, 3 H); 13 C NMR (CDC1 3 , 100 MHz) δ 173.2 (C), 160.2 (C), 125.4 (C), 74.4 (C), 70.0 (CH 2 ), 67.5 (C), 65.3 (C), 65.2 (CH 2 ), 65.0 (C), 56.5 (CH), 56.1 (CH) , 54.4 (CH), 40.3 (CH), 36.0 (C), 30.1 (CH 2 ), 25.5 (CH), 23.4 (CH 2 ), 20.9 (CH 3 ), 18.6 (CH 3 ), 17.1 (CH 2 ) , 13.7 (CH 3 ); IR (KBr) 3415, 3361, 2966, 2927, 2875, 1755, 1724, 1672, 1439, 1074, 1018 cm"1; MS (EI, 70 eV) m/z (%) 391 ([M+l] + , 2), 372 (1), 71 (100); HRMS (EI) calcd. for C 2 iH 27 0 7 (M+H) + 391.1757, found 391.1752; [a] 2 D °-116 (c 0.085, CHC1 3 ); mp 237-239 ° C. Preparation Example 4 (14S)-14-dehydroxy-14,21-epoxylide Ester alcohol (LLDT-67)
Figure imgf000024_0001
Figure imgf000024_0001
LLDT-62 LLDT-80 LLDT-67 将 (14S)-14P-羟曱基表雷公藤内酯醇 (LLDT-62) (0.50 g, 1.3 mmol) 溶于干燥的吡啶中（4.0 mL)。 将该溶液冷却至 0°C后滴加入 MsCl (0.61 mL, 7.7 mmol)。 室温下搅拌反应 10 min, 减压蒸出溶剂。 残留物加水 稀释， 乙酸乙酯萃取， 有机层用水、 饱和食盐水洗涤， 无水硫酸钠干 燥。 浓缩所得粗产品经柱层析純化 (洗脱剂： 乙酸乙酯:环己烷 =1 :2)得到 一无色油状物（14S)-14-C-曱橫酰氧曱基表雷公藤内酯醇 (LLDT-80) (0.390 g, 0.85 mmol, 产率： 50%)。 将（14S)-14-C-曱磺酰氧曱基表雷公藤内酯醇 (LLDT-80) (0.504 g, 1.1 mmol) 溶解于 20 mL曱醇溶剂中， 加入 K2C03 (1.33 g, 9.6 mmol)。 室温下搅拌反应 20 min后减压蒸出溶剂， 残留物加水稀释， 乙酸乙酯 萃取， 饱和食盐水洗涤， 无水硫酸钠干燥。 粗产品经柱层析純化 (洗脱 剂： 乙酸乙酯:环己烷 =1 :5)得到白色固体 (14S)-14-脱羟基 -14,21-环氧雷 公藤内酯醇 (LLDT-67) (307 mg, 0.82 mmol, 产率： 65% )。 LLDT-62 LLDT-80 LLDT-67 (14S)-14P-hydroxyindole table triptolide (LLDT-62) (0.50 g, 1.3 mmol) was dissolved in dry pyridine (4.0 mL). The solution was cooled to 0.degree. C. and then added M.sub.sub.3 (0.61 mL, 7.7 mmol). The reaction was stirred at room temperature for 10 min, and the solvent was evaporated under reduced pressure. The residue was diluted with water, EtOAc (EtOAc)EtOAc. The obtained crude product was purified by column chromatography (eluent: ethyl acetate: hexanes: 1:1) to give a colorless oil (14S)-14-C-purine acyloxy hydrazide. Alcohol (LLDT-80) (0.390 g, 0.85 mmol, yield: 50%). Dissolve (1 4 S)-1 4 -C-nonylsulfonyloxyindolyl tartanol (LLDT-80) (0.504 g, 1.1 mmol) in 20 mL of methanol solvent and add K 2 C0 3 ( 1.33 g, 9.6 mmol). The reaction mixture was stirred at room temperature for 20 min. The crude product was purified by column chromatography (eluent: ethyl acetate: hexanes: 1:1) to afford white solid (14S)-14-dehydroxy-14,21-epoxy-ditinolide (LLDT-67) (307 mg, 0.82 mmol, yield: 65%).
(14S)-14-C-曱磺酰氧曱基表雷公藤内酯醇 (LLDT-80): lR NMR (CDC13, 300 MHz) δ 4.70-4.57 (m, 4 H), 3.80 (d, J= 3.2 Hz, 1 H), 3.77 (d, J= 5.6 Hz, 1 H), 3.47 (d, J= 3.2 Hz, 1 H), 3.10 (s, 3 H), 2.77-2.67 (m, 1 H), 2.54 (sept, J= 6.9 Hz, 1 H), 2.37-2.26 (m, 1 H), 2.25-2.06 (m, 1 H), 1.91 (dd, J= 14.8, 13.4 Hz, 1 H), 1.54 (dd, J= 12.9, 5.1 Hz, 1 H), 1.28-1.13 (m, 2 H), 1.07 (s, 3 H), 0.92 (d, J= 7.7 Hz, 3 H), 0.89 (d, J= 7.7 Hz, 3 H); 13C NMR (CDC13, 100 MHz) (5 173.2 (C), 160.1 (C), 125.4 (C), 74.5 (C), 72.0 (CH2), 69.9 (CH2), 67.8 (C), 65.1 (C), 63.3 (C), 55.9 (CH), 55.4 (CH), 54.0 (CH), 40.5 (CH), 37.5 (CH), 36.0 (C), 29.7 (CH2), 25.6 (CH3), 23.5 (CH2), 20.5 (C¾), 18.5 (CH3), 17.1 (CH2), 13.5 (CH3); IR (KBr) 3483, 3024, 2972, 2937, 1749, 1674, 1446, 1354, 1174 cm"1; MS (EI, 70 eV) mlz (%) 468 (M+), 450 (3), 432 (1), 407 (1), 354 (17), 111 (100); HRMS (EI) calcd. for C22H28S09468.1454, found 468.1457; [a]¾3 -114 (c 0.530, Acetone). 制备实施例 5 (5R,14S)-5a-羟基 -14-脱羟基 -14,21-环氧雷公藤内酯 醇 (LLDT-68 )和（148)-/ 5，6-脱氢 -14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-70) (1 4 S)-1 4 -C-sulfonyloxycarbonyl-based triptolide (LLDT-80): lR NMR (CDC1 3 , 300 MHz) δ 4.70-4.57 (m, 4 H), 3.80 ( d, J = 3.2 Hz, 1 H), 3.77 (d, J = 5.6 Hz, 1 H), 3.47 (d, J = 3.2 Hz, 1 H), 3.10 (s, 3 H), 2.77-2.67 (m , 1 H), 2.54 (sept, J= 6.9 Hz, 1 H), 2.37-2.26 (m, 1 H), 2.25-2.06 (m, 1 H), 1.91 (dd, J= 14.8, 13.4 Hz, 1 H), 1.54 (dd, J= 12.9, 5.1 Hz, 1 H), 1.28-1.13 (m, 2 H), 1.07 (s, 3 H), 0.92 (d, J = 7.7 Hz, 3 H), 0.89 (d, J = 7.7 Hz, 3 H) ; 13 C NMR (CDC1 3 , 100 MHz) (5 173.2 (C), 160.1 (C), 125.4 (C), 74.5 (C), 72.0 (CH 2 ), 69.9 (CH 2 ), 67.8 (C), 65.1 (C), 63.3 (C), 55.9 (CH), 55.4 (CH), 54.0 (CH), 40.5 (CH), 37.5 (CH), 36.0 (C), 29.7 (CH 2 ), 25.6 (CH 3 ), 23.5 (CH 2 ), 20.5 (C3⁄4), 18.5 (CH 3 ), 17.1 (CH 2 ), 13.5 (CH 3 ); IR (KBr) 3483, 3024, 2972, 2937, 1749, 1674, 1446, 1354 , 1174 cm"1; MS (EI, 70 eV) mlz (%) 468 (M + ), 450 (3), 432 (1), 407 (1), 354 (17), 111 (100); HRMS ( EI) calcd. for C 22 H 28 S0 9 468.1454, found 468.1457; [a] 3⁄4 3 -114 (c 0.530, Acetone). Preparation Example 5 (5R, 14S)-5a-hydroxy-14-dehydroxy-14 , 21-epoxytriptolide (LLDT-68) and (148)-/ 5 , 6 -dehydro-14-dehydroxy-14,21-epoxytriptolide (LLDT-70)
Figure imgf000025_0001
Figure imgf000025_0001
LLDT-67 LLDT-68 LLDT-70 将 (14S)-14-脱羟基 -14,21-环氧雷公藤内酯醇 （LLDT-67)溶解于 8 mL 1,4-二氧六环中， 加入 Se02 (111 mg, 1.0 mmol)。反应体系在回流状 态下反应 24 h, 随后停止反应， 将反应体系冷却至室温， 硅藻土过滤， 乙酸乙酯洗涤滤渣， 减压除去溶剂。 残留物用乙酸乙酯和饱和碳酸钠 溶液处理， 有机层用水、 饱和食盐水洗涤， 无水硫酸钠干燥。 浓缩后 的粗产品经柱层析純化 (洗脱剂： CH2C12)得到白色固体 (5R,14S)-5-羟基 -14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-68) (38.8 mg, 0.1 mmol, 产 率： 50%)和（14S)- \5，6-脱氢 -14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-70) (7.4 mg, 0.02 mmol, 产率： 10%)。 LLDT-67 LLDT-68 LLDT-70 Dissolve (14S)-14-dehydroxy-14,21-epoxytriptolide (LLDT-67) in 8 mL of 1,4-dioxane and add Se0 2 (111 mg, 1.0 mmol). The reaction system was reacted under reflux for 24 h, then the reaction was stopped, the reaction system was cooled to room temperature, filtered over Celite, and the residue was washed with ethyl acetate. The residue was treated with ethyl acetate and aq. The concentrated crude product was purified by column chromatography (eluent: CH 2 C1 2 ) to give white solid (5R, 14S)-5-hydroxyl -14-dehydroxy-14,21-epoxytriptolide (LLDT-68) (38.8 mg, 0.1 mmol, yield: 50%) and (14S)- \ 5 , 6 -dehydro-14-de Hydroxy-14,21-epoxytriptolide (LLDT-70) (7.4 mg, 0.02 mmol, yield: 10%).
(5R,14S)-5a-羟基 -14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-68 ): lR NMR (CDC13, 300 MHz) δ 4.92 (dt, J = 17.1, 3.1 Hz, 1 H), 4.71 (dd, J = 17.1, 3.9 Hz, 1 H), 3.92(d, J = 3.3 Hz, 1 H), 3.63 (d, J = 3.3 Hz,l H), 3.40 (d, J= 4.8 Hz, 1 H), 2.86 (d, J= 5.1 Hz, 1 H), 2.79 (d, J= 5.1 Hz, 1 H), 2.41-2.04 (m, 4 H), 1.95-1.74 (m, 2 H), 1.29 (dd, J = 12.7, 4.9 Hz, 1 H), 1.12 (s, 3 H), 0.87 (d, J = 6.8 Hz, 3 H), 0.84 (d, J = 6.8 Hz, 3 H); 13C NMR (CDC13, 100 MHz) δ 173.2 (C), 159.0 (C), 128.0 (C), 72.3 (C), 68.7 (CH2), 65.8 (C), 63.2 (C), 58.4 (C), 56.4 (CH), 55.5 (C), 55.0 (CH), 54.5 (CH), 48.0 (C), 40.6 (C), 31.0 (CH2), 24.6 (CH2), 23.4 (CH), 19.7 (CH3), 17.8 (CH3), 17.4 (CH2), 16.2 (CH3); IR (KBr) 3479, 2956, 1736, 1668, 1442, 1037 cm"1; MS (EI, 70 eV) mlz (%) 389 ([M+l]+, 2), 373 (2), 359 (100), 341 (18); HRMS (EI) calcd. for C21H2507 (M+H)+ 389.1601, found 389.1606; [a]2 D° -120 (c 0.223, CHC13); mp 233-235 °C . (5R,14S)-5a-hydroxy-14-dehydroxy-14,21-epoxytriptolide (LLDT-68): lR NMR (CDC1 3 , 300 MHz) δ 4.92 (dt, J = 17.1, 3.1 Hz, 1 H), 4.71 (dd, J = 17.1, 3.9 Hz, 1 H), 3.92 (d, J = 3.3 Hz, 1 H), 3.63 (d, J = 3.3 Hz, l H), 3.40 (d , J= 4.8 Hz, 1 H), 2.86 (d, J= 5.1 Hz, 1 H), 2.79 (d, J= 5.1 Hz, 1 H), 2.41-2.04 (m, 4 H), 1.95-1.74 ( m, 2 H), 1.29 (dd, J = 12.7, 4.9 Hz, 1 H), 1.12 (s, 3 H), 0.87 (d, J = 6.8 Hz, 3 H), 0.84 (d, J = 6.8 Hz) , 3 H); 13 C NMR (CDC1 3 , 100 MHz) δ 173.2 (C), 159.0 (C), 128.0 (C), 72.3 (C), 68.7 (CH 2 ), 65.8 (C), 63.2 (C ), 58.4 (C), 56.4 (CH), 55.5 (C), 55.0 (CH), 54.5 (CH), 48.0 (C), 40.6 (C), 31.0 (CH 2 ), 24.6 (CH 2 ), 23.4 (CH), 19.7 (CH 3 ), 17.8 (CH 3 ), 17.4 (CH 2 ), 16.2 (CH 3 ); IR (KBr) 3479, 2956, 1736, 1668, 1442, 1037 cm"1; MS (EI , 70 eV) mlz (%) 389 ([M+l] + , 2), 373 (2), 359 (100), 341 (18); HRMS (EI) calcd. for C 21 H 25 0 7 (M +H) + 389.1601, found 389.1606; [a] 2 D ° -120 (c 0.223, CHC1 3 ); mp 233-235 °C .
(14S)-△ 5，6-脱氢- 14-脱羟基- 14,21 -环氧雷公藤内酯醇 (LLDT-70)： lR NMR (CDC13, 300 MHz) δ 6.03 (d, J = 3.7 Hz, 1 H), 4.95 (d, J = 16.0 Hz, 1 H), 4.83 (dd, J = 16.0, 2.5 Hz, 1 H), 3.86 (d, J = 3.0 Hz, 1 H), 3.56-3.48 (m, 2 H), 2.92 (ά, J = 4.9 Hz, 1 H), 2.86 (d, J = 4.9 Hz, 1 H),(14S)-△ 5 , 6 -Dehydro- 14-dehydroxy- 14,21-epoxy-dragonone (LLDT-70): lR NMR (CDC1 3 , 300 MHz) δ 6.03 (d, J = 3.7 Hz, 1 H), 4.95 (d, J = 16.0 Hz, 1 H), 4.83 (dd, J = 16.0, 2.5 Hz, 1 H), 3.86 (d, J = 3.0 Hz, 1 H), 3.56-3.48 (m, 2 H), 2.92 (ά, J = 4.9 Hz, 1 H), 2.86 (d, J = 4.9 Hz, 1 H),
2.51- 2.40 (m, 1 H), 2.36-2.21 (m, 1 H), 1.90 (sept., J = 6.8 Hz, 1 H),2.51- 2.40 (m, 1 H), 2.36-2.21 (m, 1 H), 1.90 (sept., J = 6.8 Hz, 1 H),
1.52- 1.34 (m, 2 H), 1.29 (s, 3 H), 0.92 (d, J= 6.8 Hz, 3 H), 0.78 (d, J= 6.8 Hz, 3 H); 13C NMR (CDC13, 100 MHz) δ 173.0 (C), 153.1 (C), 140.4 (C), 126.9 (C), 121.8 (CH), 68.9 (CH2), 65.1 (C), 64.4 (C), 61.3 (C), 56.6 (CH), 55.8 (C), 54.2 (CH), 53.7 (CH), 48.4 (CH2), 37.2 (C), 30.3 (CH2), 23.1 (CH), 22.8 (CH3), 20.3 (CH3), 17.2 (CH3), 17.1 (CH2); IR (KBr) 3433, 2979, 2927, 1747, 1657, 1358, 1024 cm"1; MS (EI, 70 eV) m/z (%) 370 (M+， 13), 355 (22), 341 (91), 327 (95), 115 (100); HRMS (EI) calcd. for C21H2206 370.1416, found 370.1404; [a]2 D° -443 (c 0.174, Acetone); mp 205-207 °C . 制备实施例 6 (12R,13R,14S)-12P-氯 -13a-羟基 -14-脱羟基 -14,21-环 氧雷公藤内酯醇 (LLDT-76) 1.52- 1.34 (m, 2 H), 1.29 (s, 3 H), 0.92 (d, J = 6.8 Hz, 3 H), 0.78 (d, J = 6.8 Hz, 3 H); 13 C NMR (CDC1 3 , 100 MHz) δ 173.0 (C), 153.1 (C), 140.4 (C), 126.9 (C), 121.8 (CH), 68.9 (CH 2 ), 65.1 (C), 64.4 (C), 61.3 (C), 56.6 (CH), 55.8 (C), 54.2 (CH), 53.7 (CH) , 48.4 (CH 2 ), 37.2 (C), 30.3 (CH 2 ), 23.1 (CH), 22.8 (CH 3 ), 20.3 (CH 3 ), 17.2 (CH 3 ), 17.1 (CH 2 ); IR (KBr 3433, 2979, 2927, 1747, 1657, 1358, 1024 cm"1; MS (EI, 70 eV) m/z (%) 370 (M + , 13), 355 (22), 341 (91), 327 (95), 115 (100); HRMS (EI) calcd. for C 21 H 22 0 6 370.1416, found 370.1404; [a] 2 D ° -443 (c 0.174, Acetone); mp 205-207 ° C . Example 6 (12R,13R,14S)-12P-chloro-13a-hydroxy-14-dehydroxy-14,21-epoxytriptolide (LLDT-76)
Figure imgf000027_0001
Figure imgf000027_0001
LLDT-67 LLDT-76 将 (14S)-14-脱羟基 -14,21-环氧雷公藤内酯醇 （LLDT-67)溶解于 6 mL丙酮中，加入 6 mL盐酸溶液 (1.67%, 2.7 mmol)。混合物在回流状态 下反应 7 h。 减压蒸出溶剂后所得残留物用水处理， 乙酸乙酯萃取， 有 机层用水、 饱和食盐水洗涤， 无水硫酸钠干燥， 浓缩后的粗产品经柱 层析純化（洗脱剂：乙酸乙酯:环己烷 =1 :3)得到白色固体 (12R,13R,14S)- 12β-氯 -13a-羟基 -14-脱羟基 -14,21-环氧雷公藤内酯醇 (LLDT-76) (17.5 mg, 0.043 mmol, 产率： 39%)。 lR NMR (CDC13, 300 MHz) δ 4.69 (s, 2 H), 4.16 (d, J = 5.7 Hz, 1 H), 3.75 (d, J= 5.7 Hz, 1 H), 3.46 (d, J= 6.0 Hz, 1 H), 2.93-2.83 (m, 2 H), 2.78 (d, J= 5.1 Hz, 1 H), 2.36-2.25 (m, 1 H), 2.21-2.04 (m, 2 H), 1.94-1.74 (m, 2 H), 1.59 (dd, J = 12.3, 4.7 Hz, 1 H), 1.35-1.21 (m, 1 H), 1.03 (s, 3 H), 0.99 (d, J= 2.2 Hz, 3 H), 0.96 (d, J= 2.2 Hz, 3 H); 13C NMR (Acetone-d6, 100 MHz) δ 173.7 (C), 162.2 (C), 124.6 (C), 76.6 (C), 70.7 (CH2), 69.9 (C): 60.3 (CH), 59.8 (C), 58.3 (CH), 58.2 (C), 58.2 (CH), 48.0 (CH2), 40.2 (CH): 35.8 (C), 31.3 (CH2), 28.8 (CH), 23.1 (CH2), 18.0 (CH3), 17.4 (CH2), 16.3 (CH3), 13.9 (CH3); IR (KBr) 3462, 2933, 2252, 1743, 1674, 1439, 1003 cm"1; MS (EI, 70 eV) m/z (%) 408 (M+, 9), 390 (3), 373 (8), 365 (100); HRMS (EI) calcd. for C21H25C106408.1339, found 408.1339; [a]2 D 5 -97.9 (c 1.15, Acetone); mp 174-175 °C . 制备实施例 7 (14S,硫 S)-14a,21-乙二醇环亚硫酸酯雷公藤内酯醇 (LLDT-77)和（14S,硫 R)-14a,21-乙二醇环亚硫酸酯雷公藤内酯醇 (LLDT-78) LLDT-67 LLDT-76 Dissolve (14S)-14-dehydroxy-14,21-epoxytriptolide (LLDT-67) in 6 mL of acetone and add 6 mL of hydrochloric acid solution (1.67%, 2.7 mmol) . The mixture was reacted under reflux for 7 h. The solvent was evaporated under reduced pressure. EtOAc (EtOAc m. :cyclohexane = 1:3) to give a white solid (12R, 13R, 14S) - 12β-chloro-13a-hydroxy-14-dehydroxy-14,21-epoxytriptolide (LLDT-76) (17.5 Mg, 0.043 mmol, yield: 39%). lR NMR (CDC1 3 , 300 MHz) δ 4.69 (s, 2 H), 4.16 (d, J = 5.7 Hz, 1 H), 3.75 (d, J = 5.7 Hz, 1 H), 3.46 (d, J= 6.0 Hz, 1 H), 2.93-2.83 (m, 2 H), 2.78 (d, J = 5.1 Hz, 1 H), 2.36-2.25 (m, 1 H), 2.21-2.04 (m, 2 H), 1.94-1.74 (m, 2 H), 1.59 (dd, J = 12.3, 4.7 Hz, 1 H), 1.35-1.21 (m, 1 H), 1.03 (s, 3 H), 0.99 (d, J = 2.2 Hz, 3 H), 0.96 (d, J = 2.2 Hz, 3 H) 13 C NMR (Acetone-d 6 , 100 MHz) δ 173.7 (C), 162.2 (C), 124.6 (C), 76.6 (C), 70.7 (CH 2 ), 69.9 (C) : 60.3 (CH), 59.8 (C), 58.3 (CH), 58.2 (C), 58.2 (CH), 48.0 (CH 2 ), 40.2 (CH) : 35.8 (C), 31.3 (CH 2 ), 28.8 (CH), 23.1 (CH) 2 ), 18.0 (CH 3 ), 17.4 (CH 2 ), 16.3 (CH 3 ), 13.9 (CH 3 ); IR (KBr) 3462, 2933, 2252, 1743, 1674, 1439, 1003 cm"1; MS ( EI, 70 eV) m/z (%) 408 (M + , 9), 390 (3), 373 (8), 365 (100); HRMS (EI) calcd. for C 21 H 25 C10 6 408.1339, found 408.1339; [a] 2 D 5 -97.9 (c 1.15, Acetone); mp 174-175 ° C. Preparation Example 7 (14S, sulfur S)-14a, 21-ethylene glycol cyclic sulfite, triptolide (LLDT-77) and (14S, sulfur R)-14a, 21-ethylene glycol cyclic sulfite triptolide (LLDT-78)
Figure imgf000028_0001
Figure imgf000028_0001
LLDT-62 LLDT-77 LLDT-78 将 (14S)-14P-羟曱基表雷公藤内酯醇 (LLDT-62) (78 mg, 0.2 mmol) 溶解于 6 mL无水 CH2C12中，滴加干燥的 Et3N (0.21 mL, 1.6 mmol)。 反 应体系冷却到 0°C , 在氩气保护下将 SOC12(0.3 mL, 1.2 mmol)小心加入 反应体系中， 搅拌反应 2 h后， 加入水中止反应， CH2C12萃取， 有机 层用水、 饱和食盐水洗涤， 无水硫酸钠干燥， 浓缩后的粗产品经柱层 析分离純化（洗脱剂： 乙酸乙酯：环己烷 =1 :5)得到白色固体（14S,硫 S)-14a,21-乙二醇环亚硫酸酯雷公藤内酯醇 (LLDT-77) (39.2 mg, 0.09 mmol,产率： 43%)和 (14S,硫 R)-14,21-乙二醇环亚硫酸酯雷公藤内酯醇 (LLDT-78) (30.5 mg, 0.07 mmol, 产率： 36%)。 LLDT-62 LLDT-77 LLDT-78 Dissolve (14S)-14P-hydroxyindole-based triptolide (LLDT-62) (78 mg, 0.2 mmol) in 6 mL of anhydrous CH 2 C1 2 Dry Et 3 N (0.21 mL, 1.6 mmol). The reaction system was cooled to 0 ° C. Under the protection of argon, SOC1 2 (0.3 mL, 1.2 mmol) was carefully added to the reaction system. After stirring for 2 h, the reaction was added to water, CH 2 C1 2 was extracted, and the organic layer was washed with water. The mixture was washed with saturated brine and dried over anhydrous sodium sulfate. EtOAc EtOAc EtOAc S)-14a, 21-ethylene glycol cyclic sulfite triptolide (LLDT-77) (39.2 mg, 0.09 mmol, yield: 43%) and (14S, sulfur R)-14,21-ethane Alcohol cyclic sulfite triptolide (LLDT-78) (30.5 mg, 0.07 mmol, yield: 36%).
(14S,硫 S)-14a,21-乙二醇环亚硫酸酯雷公藤内酯醇 (LLDT-77): lR NMR (CDC13, 300 MHz) δ 4.93 (d, J= 9.6 Hz, 1 H), 4.68 (s, 2 H), 4.58 (d, J = 9.6 Hz, 1 H), 3.84 (d, J = 3.0 Hz, 1 H), 3.71 (d, J = 5.7 Hz, 1 H), 3.61 (d, J = 2.9 Hz, 1 H), 2.78-2.67 (m, 1 H), 2.61 (sept, J = 6.9 Hz, 1 H), 2.38-2.26 (m, 1 H), 2.25-2.05 (m, 2 H), 1.91 (t, J = 14.1 Hz, 1 H), 1.54 (dd, J = 12.6, 4.5 Hz, 1 H), 1.26-1.14 (m, 1 H), 1.07 (s, 3 H), 0.95 (d, J = 6.9 Hz, 3 H), 0.93 (d, J = 6.9 Hz, 3 H); 13C NMR (CDC13, 100 MHz) δ 173.1 (C), 159.8 (C), 125.5 (C), 91.9 (C), 74.1 (CH2), 69.9 (CH2), 65.1 (C), 64.9 (C), 61.7 (C), 56.5 (CH), 55.6 (CH), 55.5 (CH), 40.4 (CH), 35.8 (C), 30.0 (CH2), 25.7 (CH), 23.3 (CH2), 20.6 (CH3), 18.7 (CH3), 17.1 (CH2), 13.5 (CH3); IR (KBr) 3475, 2972, 2933, 2875, 1745, 1680, 1441, 1219, 1018 cm"1; MS (EI, 70 eV) m/z (%) 436 (M+, 2), 407 (1), 393 (6), 241 (100); HRMS (EI) calcd. for C21H24S08 436.1192, found 436.1199; [a]2 D 5 -97.0 (c 0.400, Acetone); mp 269-271 °C . (14S, sulfur S)-14a, 21-ethylene glycol cyclic sulfite, triptolide (LLDT-77): lR NMR (CDC1 3 , 300 MHz) δ 4.93 (d, J = 9.6 Hz, 1 H) , 4.68 (s, 2 H), 4.58 (d, J = 9.6 Hz, 1 H), 3.84 (d, J = 3.0 Hz, 1 H), 3.71 (d, J = 5.7 Hz, 1 H), 3.61 ( d, J = 2.9 Hz, 1 H), 2.78-2.67 (m, 1 H), 2.61 (sept, J = 6.9 Hz, 1 H), 2.38-2.26 (m, 1 H), 2.25-2.05 (m, 2 H), 1.91 (t, J = 14.1 Hz, 1 H), 1.54 (dd, J = 12.6, 4.5 Hz, 1 H), 1.26-1.14 (m, 1 H), 1.07 (s, 3 H), 0.95 (d, J = 6.9 Hz, 3 H), 0.93 (d, J = 6.9 Hz, 3 H); 13 C NMR (CDC1 3 , 100 MHz) δ 173.1 (C), 159.8 (C), 125.5 (C ), 91.9 (C), 74.1 (CH 2 ), 69.9 (CH 2 ), 65.1 (C), 64.9 (C), 61.7 (C), 56.5 (CH), 55.6 (CH), 55.5 (CH), 40.4 (CH), 35.8 (C), 30.0 (CH 2 ), 25.7 (CH), 23.3 (CH 2 ), 20.6 (CH 3 ), 18.7 (CH 3 ), 17.1 (CH 2 ), 13.5 (CH 3 ); IR (KBr) 3475, 2972, 2933, 2875, 1745, 1680, 1441, 1219, 1018 cm"1; MS (EI, 70 eV) m/z (%) 436 (M + , 2), 407 (1) , 393 (6), 241 (100); HRMS (EI) calcd. for C 21 H 24 S0 8 436.1192, found 436.1199; [a] 2 D 5 -97.0 (c 0.400, Acet Mp 269-271 °C .
(14S,硫 R)-14a,21-乙二醇环亚硫酸酯雷公藤内酯醇 (LLDT-78): lR NMR (CDC13, 300 MHz) δ 4.80 (d, J= 9.6 Hz, 1 H), 4.68 (s, 2 H), 4.58 (d, J = 9.6 Hz, 1 H), 3.84 (d, J = 3.0 Hz, 1 H), 3.79 (d, J = 5.6 Hz, 1 H), 3.53 (d, J = 3.0 Hz, 1 H), 2.80-2.68 (m, 1 H), 2.38-2.26 (m, 1 H), 2.24-2.02 (m, 3 H), 1.96 (t, J = 14.1 Hz, 1 H), 1.54 (dd, J= 12.6, 5.1 Hz, 1 H), 1.28-1.15 (m, 1 H), 1.10 (s, 3 H), 0.92 (d, J= 6.8 Hz, 3 H), 0.90 (d, J = 6.8 Hz, 3 H); "C NMR (CDC13, 100 MHz) δ 173.1 (C), 159.9 (C), 125.3 (C), 92.5 (C), 74.1 (CH2), 69.9 (CH2), 66.5 (C), 65.0 (C), 61.0 (C), 58.3 (CH), 55.4 (CH), 54.7 (CH), 40.4 (CH), 35.7 (C), 30.2 (CH2), 25.7 (CH), 23.3 (CH2), 20.5 (CH3), 18.2 (CH3), 17.0 (CH2), 13.4 (CH3); IR (KBr) 3435， 2970, 2933, 2877, 2254, 1755, 1674, 1444, 1346, 1223, 1020 cm_1;MS (EI, 70 eV) m/z (%) 436 (M+, 7), 421 (9), 71 (100); HRMS (EI) calcd. for C2iH24S08 436.1192, found 436.1182; [a]2 D 5 -96.9 (c 0.425, Acetone); mp 160- 162 °C . 制备实施例 8 (14S)-14a,21-乙二醇环硫酸酯雷公藤内酯醇 (LLDT-79) (14S, sulfur R)-14a, 21-ethylene glycol cyclic sulfite, triptolide (LLDT-78): lR NMR (CDC1 3 , 300 MHz) δ 4.80 (d, J = 9.6 Hz, 1 H) , 4.68 (s, 2 H), 4.58 (d, J = 9.6 Hz, 1 H), 3.84 (d, J = 3.0 Hz, 1 H), 3.79 (d, J = 5.6 Hz, 1 H), 3.53 ( d, J = 3.0 Hz, 1 H), 2.80-2.68 (m, 1 H), 2.38-2.26 (m, 1 H), 2.24-2.02 (m, 3 H), 1.96 (t, J = 14.1 Hz, 1 H), 1.54 (dd, J = 12.6, 5.1 Hz, 1 H), 1.28-1.15 (m, 1 H), 1.10 (s, 3 H), 0.92 (d, J = 6.8 Hz, 3 H), 0.90 (d, J = 6.8 Hz, 3 H); "C NMR (CDC1 3 , 100 MHz) δ 173.1 (C), 159.9 (C), 125.3 (C), 92.5 (C), 74.1 (CH 2 ), 69.9 (CH 2 ), 66.5 (C), 65.0 (C), 61.0 (C), 58.3 (CH), 55.4 (CH), 54.7 (CH), 40.4 (CH), 35.7 (C), 30.2 (CH 2 ), 25.7 (CH) , 23.3 (CH 2 ), 20.5 (CH 3 ), 18.2 (CH 3 ), 17.0 (CH 2 ), 13.4 (CH 3 ); IR (KBr) 3435, 2970, 2933, 2877, 2254, 1755, 1674, 1444 , 1346, 1223, 1020 cm _1 ;MS (EI, 70 eV) m/z (%) 436 (M + , 7), 421 (9), 71 (100); HRMS (EI) calcd. for C 2 iH 24 S0 8 436.1192, found 436.1182; [a] 2 D 5 -96.9 (c 0.425, Acetone); mp 160-162 ° C. Preparation Example 8 (14S)-14a, 21-ethylene glycol cyclic sulfate Tripterygium Ester alcohol (LLDT-79)
Figure imgf000030_0001
Figure imgf000030_0001
LLDT-77或 LLDT-78 LLDT-79 将（14S,硫 S)- 14a,21-乙二醇环亚硫酸酯雷公藤内酯醇 (LLDT-77) 或（14S,硫 R)-14a,21-乙二醇环亚硫酸酯雷公藤内酯醇 (LLDT-78) (40 mg: 0.092 mmol)溶解于 4 mL乙腈中，依次加入高碘酸钠 (31 mg, 0.14 mmol), RuCl3 3H20 (6 mg, 0.028 mmol)和 1 mL水， 室温下搅拌反应 15 min, 减压蒸出溶剂， 浓缩物用水稀释后用乙酸乙酯萃取， 水、 饱和食盐水 洗涤有机相，无水硫酸钠干燥，浓缩后的粗产品经柱层析純化 (洗脱剂： 乙酸乙酯:环己烷 =1 :4)得到白色固体 (14S)-14a,21-乙二醇环硫酸酯雷公 藤内酯醇 (LLDT-79) (36 mg, 0.079 mmol, 产率： 86%)。 lR NMR (CDC13, 300 MHz) δ 4.92 (ά, J = 10.0 Hz, 1 H), 4.72-2.63 (m, 3 H), 3.87 (d, J = 3.0 Hz, 1 H), 3.83 (d, J = 5.6 Hz, 1 H), 3.65 (d, J = 3.0 Hz, 1 H), 2.79-2.68 (m, 1 H), 2.51 (sept, J= 6.8 Hz, 1 H), 2.39-2.07 (m 3 H), 1.98 (t, J= 14.2 Hz, 1 H), 1.53 (dd, J = 12.6, 5.2 Hz, 1 H), 1.29-1.14 (m, 1 H), 1.09 (s, 3 H), 0.99 (d, J= 6.8 Hz, 3 H), 0.97 (d, J= 6.8 Hz, 3 H); 13C NMR (CDC13, 100 MHz) δ 172.9 (C), 159.4 (C), 125.6 (C), 91.3 (C),LLDT-77 or LLDT-78 LLDT-79 will (14S, sulfur S)-14a, 21-ethylene glycol cyclic sulfite triptolide (LLDT-77) or (14S, sulfur R)-14a, 21- Ethylene glycol cyclic sulfite triptolide (LLDT-78) (40 mg : 0.092 mmol) was dissolved in 4 mL of acetonitrile, followed by sodium periodate (31 mg, 0.14 mmol), RuCl 3 3H 2 0 ( The reaction mixture was stirred for 15 min. The concentrated crude product was purified by column chromatography (eluent: ethyl acetate: hexanes: 1:1) to afford white solid (14S)-14a, 21-ethylene glycol cyclosulphate triptolide (LLDT) -79) (36 mg, 0.079 mmol, yield: 86%). lR NMR (CDC1 3 , 300 MHz) δ 4.92 (ά, J = 10.0 Hz, 1 H), 4.72-2.63 (m, 3 H), 3.87 (d, J = 3.0 Hz, 1 H), 3.83 (d, J = 5.6 Hz, 1 H), 3.65 (d, J = 3.0 Hz, 1 H), 2.79-2.68 ( m, 1 H), 2.51 (sept, J = 6.8 Hz, 1 H), 2.39-2.07 (m 3 H), 1.98 (t, J = 14.2 Hz, 1 H), 1.53 (dd, J = 12.6, 5.2 Hz, 1 H), 1.29-1.14 (m, 1 H), 1.09 (s, 3 H), 0.99 (d, J = 6.8 Hz, 3 H), 0.97 (d, J = 6.8 Hz, 3 H); 13 C NMR (CDC1 3 , 100 MHz) δ 172.9 (C), 159.4 (C), 125.6 (C), 91.3 (C),
73.4 (CH2), 69.9 (CH2), 66.3 (C), 65.2 (C), 61.9 (C), 58.3 (CH), 55.7 (CH),73.4 (CH 2 ), 69.9 (CH 2 ), 66.3 (C), 65.2 (C), 61.9 (C), 58.3 (CH), 55.7 (CH),
55.5 (CH), 40.3 (CH), 35.7 (C), 30.2 (CH2), 25.5 (CH), 23.2 (CH2), 20.4 (CH3), 18.3 (CH3), 17.0 (CH2), 13.4 (CH3); IR (KBr) 3442, 2970, 2941, 1749, 1676, 1441, 1394, 1217, 1001 cm"1; MS (EI, 70 eV) m/z (%) 452 (M+ : 8), 437 (10), 423 (10), 111 (100); HRMS (EI) calcd. for C21H24S09 55.5 (CH), 40.3 (CH), 35.7 (C), 30.2 (CH 2 ), 25.5 (CH), 23.2 (CH 2 ), 20.4 (CH 3 ), 18.3 (CH 3 ), 17.0 (CH 2 ), 13.4 (CH 3 ); IR (KBr) 3442, 2970, 2941, 1749, 1676, 1441, 1394, 1217, 1001 cm"1; MS (EI, 70 eV) m/z (%) 452 (M + : 8 ), 437 (10), 423 (10), 111 (100); HRMS (EI) calcd. for C 21 H 24 S0 9
452.1141, found 452.1169; [α]έ3-107 (c 0.275, CHC13); mp 205-207 °C . 制备实施例 9 含有 LLDT-67的适用于人类应用的几种代表性剂型 (l) LLDT-67口服制剂： 剂或口服液 452.1141, found 452.1169; [α]έ 3 -107 (c 0.275, CHC1 3 ); mp 205-207 ° C . Preparation Example 9 Several representative dosage forms (LL) LLDT for human applications containing LLDT-67 -67 oral preparation: agent or oral solution
口服液：  Oral solution:
LLDT-67 4mg/ml 亚油酸乙酯 l% w/w) 乙二醇乙醚 17% (w/w) LLDT-67 4mg/ml ethyl linoleate l% w/w) ethylene glycol ether 17% (w/w)
Cremophor EL 52% (w/w) (聚氧乙烯蓖麻油） Cremophor EL 52% (w/w) (polyoxyethylene castor oil)
辛^/癸酸甘油酯 30% (w/w) 使用： 以适量水稀释后口服 Xin / glyceryl citrate 30% (w / w) Use: Oral diluted with appropriate amount of water
LLDT-67 4mg/ml 亚油酸乙酯 LLDT-67 4mg/ml ethyl linoleate
乙二醇乙醚
Figure imgf000032_0001
Glycol ether
Figure imgf000032_0001
Cremophor EL 48%  Cremophor EL 48%
(聚氧乙烯蓖麻油）  (polyoxyethylene castor oil)
辛 ^/癸酸甘油酯 28% (w/w) 软胶嚢剂， 口服液各组分范围Xin^/capric glyceride 28% (w/w) soft gelatin, oral liquid component range
LLDT-67 4mg/ml 亚油酸乙酯 0.5-10% (w/w) 乙二醇乙醚
Figure imgf000032_0002
LLDT-67 4mg/ml ethyl linoleate 0.5-10% (w/w) ethylene glycol ether
Figure imgf000032_0002
Cremophor EL 35〜60% (w/w) Cremophor EL 35~60% (w/w)
(聚氧乙烯蓖麻油） (polyoxyethylene castor oil)
辛 ^/癸酸甘油酯 20〜50% (w/w)Xin^/capric glyceride 20~50% (w/w)
(2) LLDT-67注射用针剂 (2) LLDT-67 injection injection
注射液 1： Injection 1:
LLDT-67 0.5mg/ml 15% (w/w) 磷脂 PL-100M 2% (w/w)
Figure imgf000033_0001
LLDT-67 0.5mg/ml 15% (w/w) phospholipid PL-100M 2% (w/w)
Figure imgf000033_0001
注射用水 80.5% (w/w) 注射液 1各组分范围: Water for injection 80.5% (w/w) Injection 1 Range of components:
LLDT-67 0.5mg/ml
Figure imgf000033_0002
LLDT-67 0.5mg/ml
Figure imgf000033_0002
磷脂 PL-100M 0.5 Phospholipids PL-100M 0.5
0.5〜3% (w/w) 注射用水 加至 lOOg 注射液 2:  0.5~3% (w/w) water for injection to lOOg injection 2:
LLDT-67 2mg/ml 丙二醇 2% (w/w) LLDT-67 2mg/ml Propylene Glycol 2% (w/w)
Cremophor EL 18% (w/w)Cremophor EL 18% (w/w)
(聚氧乙烯蓖麻油) (polyoxyethylene castor oil)
生理盐水 80% (w/w) 注射液 2各组分范围: Saline 80% (w/w) Injection 2 Range of components:
LLDT-67 2mg/ml 丙二醇 1〜10% (w/w)LLDT-67 2mg/ml Propylene glycol 1~10% (w/w)
Cremophor EL 5-25% (w/w) (聚氧乙烯蓖麻油） Cremophor EL 5-25% (w/w) (polyoxyethylene castor oil)
生理盐水 加至 100g (3) LLDT-67软會剂 Normal saline added to 100g (3) LLDT-67 softener
LLDT-67 4mg/g 硬脂 S史甘油酯 3.5% (w/w) 硬脂酸 12% w/w) 液体石蜡 6% (w/w) 白凡士林 l% (w/w) 羊毛脂 5% (w/w) 蒸偖水加至 100g  LLDT-67 4mg/g Stearin S-Glyceryl ester 3.5% (w/w) Stearic acid 12% w/w) Liquid paraffin 6% (w/w) White Vaseline l% (w/w) Lanolin 5% (w/w) steamed water added to 100g
LLDT-67软膏剂各组分范围  LLDT-67 ointment range of each component
LLDT-67 4mg/g 硬脂酸甘油酉 〜 6% (w/w) 硬脂酸 8-20% (w/w) 液体石蜡 3-10% (w/w) 白凡士林 0.5-4% (w/w) 羊毛脂 2〜8% (w/w) 蒸馏水加至 lOOg 药效学评价实施例 以下实施例中， 受试样品由本发明化学合成实施例提供， 以先导 化合物雷公藤内酯醇 (LLDT-2)作为阳性对照。 实验实施例 1 本发明的 9 个化合物对体外培养的人卵巢癌 SK-OV-3细胞的增殖抑制作用。 方法 人卵巢癌 SK-OV-3 细胞用含 10 %胎牛血清的 DMEM 培养基 (Gibco, 美国）培养， 培养条件为 37。C、 5% C02。 肿瘤细胞 0.7 xlO4/ 孔接种于 96-孔板， 24小时后，加入贮备液为二曱基亚砜配制（ 1(Τ2Μ )、 生理盐水稀释的各化合物， 使培养基中终浓度为 ιο-4、 ιο-5、 ιο-6、 ιο-7、LLDT-67 4mg/g glyceryl stearate~ 6% (w/w) 8-20% stearic acid (w/w) 3-10% liquid paraffin (w/w) white petrolatum 0.5-4% (w /w) Lanolin 2 to 8% (w/w) Distilled water added to 100 g Pharmacodynamic evaluation Example In the following examples, the test sample was provided by the chemical synthesis example of the present invention, and the lead compound triptolide (LLDT-2) was used. As a positive control. Experimental Example 1 The inhibitory effects of the nine compounds of the present invention on the proliferation of human ovarian cancer SK-OV-3 cells cultured in vitro. Methods Human ovarian cancer SK-OV-3 cells were cultured in DMEM medium (Gibco, USA) containing 10% fetal calf serum under the culture conditions of 37. C, 5% C0 2 . Tumor cells were inoculated into a 96-well plate at 0.7 xlO 4 /well. After 24 hours, the stock solution was added to the solution of dimercaptosulfoxide (1 (Τ 2 Μ ), diluted with physiological saline, so that the final concentration in the medium was Ιο- 4 , ιο- 5 , ιο- 6 , ιο- 7 ,
10"8 Μ, 作用 72小时。 弃去培养液， 用 10%冷三氯醋酸固定细胞。 用 磺酰罗丹明 B(Sulforhodamine B,SRB)溶液染色。 洗去未结合 SRB后， Tris溶解与蛋白结合的 SRB, 酶标仪在 560nm波长下测定 OD值。 按 下列公式计算细胞生长抑制率：抑制率 =(OD值对, ¾孔 - OD值给《孔) /OD值 对照孔 xl00% 。 结果判定标准： 无效： 10-5M<50% ； 有效： 10-5M≥50%。 根据各浓度的抑制率， 釆用 Logit法计算半数抑制浓度 IC5。。 结果 在本发明的 9 个化合物中， 6 个化合物 LLDT-67、 LLDT-68、 LLDT-69、 LLDT-70、 LLDT-76、 LLDT-78剂量依赖性地抑制体外培养 的人卵巢癌 SK-OV-3细胞生长， 显示其具有有效的体外抗肿瘤作用。 具体结果见表 1。 表 1.化合物对人卵巢癌 SK-OV-3细胞的增殖抑制作用 样品 SK-OV-3 ic50 编号 10"4M 10"5 M 10"6 M 10"7 M 10"8 M 评价 ( μ Μ )10" 8 Μ, for 72 hours. Discard the medium and fix the cells with 10% cold trichloroacetic acid. Stain with Sulforhodamine B (SRB) solution. Wash away the unbound SRB, Tris dissolves with protein. The bound SRB and the microplate reader were used to measure the OD value at a wavelength of 560 nm. The cell growth inhibition rate was calculated according to the following formula: inhibition rate = (OD value pair, 3⁄4 hole-OD value to "hole" / OD value control hole x l00%. Judging criteria: Invalid: 10 -5 M<50% ; Valid: 10 -5 M ≥ 50%. According to the inhibition rate of each concentration, the half-inhibitory concentration IC 5 is calculated by the Logit method. Results 9 compounds in the present invention In the six compounds LLDT-67, LLDT-68, LLDT-69, LLDT-70, LLDT-76, LLDT-78 dose-dependently inhibited in vitro culture Growth of human ovarian cancer SK-OV-3 cells has been shown to have potent anti-tumor effects in vitro. The specific results are shown in Table 1. Table 1. Proliferation inhibitory effect of compounds on human ovarian cancer SK-OV-3 cells Sample SK-OV-3 ic 50 No. 10" 4 M 10" 5 M 10" 6 M 10" 7 M 10" 8 M Evaluation (μ Μ )
LLDT-2 96.1 94.6 93.4 93.2 52.9 有效 0.009LLDT-2 96.1 94.6 93.4 93.2 52.9 valid 0.009
LLDT-62 58.7 0 0 0 0 无效 LLDT-62 58.7 0 0 0 0 is invalid
LLDT-67 92.3 93.3 93.5 43.1 0 有效 0.101 LLDT-67 92.3 93.3 93.5 43.1 0 Valid 0.101
LLDT-68 94.4 94.3 5.3 0 0 有效 1.864LLDT-68 94.4 94.3 5.3 0 0 Effective 1.864
LLDT-69 94.6 94.0 27.3 1.0 0 有效 1.560LLDT-69 94.6 94.0 27.3 1.0 0 Valid 1.560
LLDT-70 93.9 92.4 90.1 5.9 8.6 有效 0.574LLDT-70 93.9 92.4 90.1 5.9 8.6 Valid 0.574
LLDT-76 91.0 92.3 91.4 27.8 3.5 有效 0.143LLDT-76 91.0 92.3 91.4 27.8 3.5 Valid 0.143
LLDT-77 50.9 0 0 0 0 无效 LLDT-77 50.9 0 0 0 0 is invalid
LLDT-78 93.6 92.3 0 0 0 有效 5.102 LLDT-78 93.6 92.3 0 0 0 Effective 5.102
LLDT-79 83.7 0.4 0 0 0.0 无效 实验实施例 2 本发明的 9 个化合物对体外培养的人乳腺癌 MDA-MB-468细胞的增殖抑制作用。 方法 LLDT-79 83.7 0.4 0 0 0.0 Ineffective Experimental Example 2 Inhibition of proliferation of human breast cancer MDA-MB-468 cells cultured in vitro by the nine compounds of the present invention. Method
人乳腺癌 MDA-MB-468 细胞用含 10 %胎牛血清的 1640培养基 (Gibco, 美国）培养， 培养条件为 37。C、 5% C02。 肿瘤细胞 0.7 xlO4/ 孔接种于 96-孔板， 24小时后， 加入用二曱基亚砜配制（1(Τ2Μ )、 生理 盐水稀释的化合物， 使培养基中各化合物的终浓度为 10_4、 10_5、 10_6、 10"7, 10"8Μ,作用 72小时。 弃去培养液， 用 10%冷三氯醋酸固定细胞。 用磺酰罗丹明 B(Sulforhodamine B,SRB)溶液染色。 洗去未结合 SRB , Tris溶解与蛋白结合的 SRB, 用酶标仪在 560nm波长下测定 OD值。 按下列公式计算细胞生长抑制率： 抑制率 = (OD值对照孔 - OD值给药 孔) / OD值对照孔 χ100% 。 结果判定标准： 无效： 10·5Μ<50%; 有效： 10-5Μ≥50%。 Human breast cancer MDA-MB-468 cells were cultured in 1640 medium (Gibco, USA) containing 10% fetal calf serum under the culture conditions of 37. C, 5% C0 2 . Tumor cells were inoculated into a 96-well plate at 0.7 xlO 4 /well. After 24 hours, a compound diluted with dimethyl sulfoxide (1 (Τ 2 Μ ), physiological saline was added to make the final concentration of each compound in the medium 10_ 4 , 10_ 5 , 10_ 6 , 10" 7 , 10" 8 Μ for 72 hours. Discard the medium and fix the cells with 10% cold trichloroacetic acid. Stained with a solution of Sulforhodamine B (SRB). The unbound SRB, Tris dissolved the protein-bound SRB, and the OD value was measured by a microplate reader at a wavelength of 560 nm. The cell growth inhibition rate was calculated according to the following formula: Inhibition rate = (OD value control well-OD value administration well) / OD value control well 100%. Result judgment criteria: Invalid: 10· 5 Μ<50%; Valid: 10 -5 Μ≥50%.
根据各浓度的抑制率， 釆用 Logit法计算半数抑制浓度 IC50。 结果 在本发明的 9 个化合物中， 5 个化合物 LLDT-67、 LLDT-68、 LLDT-69、 LLDT-70、 LLDT-76剂量依赖性地抑制体外培养的人乳腺癌 MDA-MB-468肿瘤细胞的生长， 显示其具有有效的体外抗肿瘤作用。 具体结果见表 2。 Based on the inhibition rate of each concentration, the half-inhibitory concentration IC 50 was calculated by the Logit method. Results Among the 9 compounds of the present invention, 5 compounds LLDT-67, LLDT-68, LLDT-69, LLDT-70, LLDT-76 dose-dependently inhibit human breast cancer MDA-MB-468 tumor cells cultured in vitro. The growth showed that it has an effective anti-tumor effect in vitro. The specific results are shown in Table 2.
表 2.化合物对人乳腺癌 MDA-MB-468细胞的体外增殖抑制作用 样品 MDA-MB-468 ic50 Table 2. Inhibition of in vitro proliferation of compounds against human breast cancer MDA-MB-468 cells Sample MDA-MB-468 ic 50
评价  Evaluation
编号 10"4M 10"5 M 10"6 M 10"7 M 10"8 M ( μ Μ) No. 10" 4 M 10" 5 M 10" 6 M 10" 7 M 10" 8 M ( μ Μ)
LLDT-2 94.8 93.6 93.0 93.1 31.5 有效 0.012  LLDT-2 94.8 93.6 93.0 93.1 31.5 Valid 0.012
LLDT-62 60.0 3.2 0 0 0 无效  LLDT-62 60.0 3.2 0 0 0 Invalid
LLDT-67 94.8 94.0 92.9 25.0 7.0 有效 0.176  LLDT-67 94.8 94.0 92.9 25.0 7.0 Valid 0.176
LLDT-68 95.8 90.8 25.7 17.7 17.6 有效 2.369  LLDT-68 95.8 90.8 25.7 17.7 17.6 Valid 2.369
LLDT-69 94.1 93.9 11.0 1.6 1.5 有效 1.918  LLDT-69 94.1 93.9 11.0 1.6 1.5 Valid 1.918
LLDT-70 94.3 92.3 44.0 6.2 8.0 有效 1.138  LLDT-70 94.3 92.3 44.0 6.2 8.0 Valid 1.138
LLDT-76 92.9 91.8 90.7 17.9 4.7 有效 0.207  LLDT-76 92.9 91.8 90.7 17.9 4.7 Effective 0.207
LLDT-77 70.7 4.1 0.4 0 0.2 无效  LLDT-77 70.7 4.1 0.4 0 0.2 is invalid
LLDT-78 91.7 46.8 4.2 0.6 1.8 无效  LLDT-78 91.7 46.8 4.2 0.6 1.8 is invalid
LLDT-79 73.4 24.5 1.8 2.4 1.3 无效 实验实施例 3 本发明的 9个化合物对体外培养的人前列腺癌 PC-3 细胞的增殖抑制作用。 方法 人前列腺癌 PC-3细胞用含 10 %胎牛血清的 F-12培养基 (Gibco, 美国）培养， 培养条件为 37。C、 5% C02。 肿瘤细胞 0.7 xlO4/孔接种于 96-孔板， 24小时后， 加入用二曱基亚砜配制 （1(Τ2Μ )、 生理盐水稀释 的化合物，使培养基中各化合物的终浓度为 10-4、 10-5、 10-6、 10-7、 10"8Μ, 作用 72小时。 弃去培养液， 用 10%冷三氯醋酸固定细胞。 用磺酰罗丹 明 B(Sulforhodamine B,SRB)溶液染色。 洗去未结合 SRB, Tris溶解与 蛋白结合的 SRB, 用酶标仪在 560nm波长下测定 OD值。 最后用以下 列公式计算细胞生长抑制率： 抑制率 = (OD值对照孔 - OD值给药孔) / OD 值对照孔 χ100% 。 结果判定标准： 无效： 10·5Μ<50%; 有效： 10-5Μ≥50%。 根据各浓度的抑制率， 釆用 Logit法计算半数抑制浓度 IC50。 结果 在本发明的 9 个化合物中， 6 个化合物 LLDT-67、 LLDT-68、 LLDT-69、 LLDT-70、 LLDT-76、 LLDT-78剂量依赖性地抑制体外培养 的人前列腺癌 PC-3肿瘤细胞的生长，显示其具有有效的体外抗肿瘤作 用。 具体结果见表 3。 表 3.化合物对人前列腺癌 PC-3细胞的增殖抑制作用 样品 PC-3 IC50 LLDT-79 73.4 24.5 1.8 2.4 1.3 invalid Experimental Example 3 The inhibitory effects of the nine compounds of the present invention on proliferation of human prostate cancer PC-3 cells cultured in vitro. Methods Human prostate cancer PC-3 cells were cultured in F-12 medium (Gibco, USA) containing 10% fetal bovine serum under the culture conditions of 37. C, 5% C0 2 . Tumor cells were inoculated into a 96-well plate at 0.7 xlO 4 /well. After 24 hours, a compound diluted with dimethyl sulfoxide (1 (Τ 2 Μ ), physiological saline was added to make the final concentration of each compound in the medium 10- 4 , 10- 5 , 10- 6 , 10- 7 , 10" 8 Μ, for 72 hours. Discard the culture solution and fix the cells with 10% cold trichloroacetic acid. Use Sulfuryl Rhodamine B (Sulforhodamine B, SRB) solution staining. Washing unbound SRB, Tris dissolves protein-bound SRB, and measured the OD value at 560 nm using a microplate reader. Finally, the cell growth inhibition rate was calculated by the following formula: Inhibition rate = (OD value control well) - OD value administration hole) / OD value control hole χ 100%. Result judgment criteria: Invalid: 10· 5 Μ <50%; Valid: 10 -5 Μ ≥ 50%. According to the inhibition rate of each concentration, use Logit method The half-inhibitory concentration IC 50 was calculated. Results Among the 9 compounds of the present invention, 6 compounds LLDT-67, LLDT-68, LLDT-69, LLDT-70, LLDT-76, LLDT-78 inhibited in vitro culture in a dose-dependent manner. The growth of human prostate cancer PC-3 tumor cells has been shown to have potent anti-tumor effects in vitro. The results are shown in Table 3. Table 3. Inhibition of proliferation of compounds against human prostate cancer PC-3 cells Sample PC-3 IC 50
评价  Evaluation
编号 10-4M 10"5 M 10"6 M 10"7M 10"8M ( μΜ ) LLDT-2 70.3 68.9 66.5 67.1 13.5 有效 0.022 No. 10 -4 M 10" 5 M 10" 6 M 10" 7 M 10" 8 M ( μΜ ) LLDT-2 70.3 68.9 66.5 67.1 13.5 Valid 0.022
LLDT-62 38.9 6.1 0 0 4.6 无效  LLDT-62 38.9 6.1 0 0 4.6 Invalid
LLDT-67 69.6 67.1 66.3 19.7 0 有效 0.266  LLDT-67 69.6 67.1 66.3 19.7 0 effective 0.266
LLDT-68 67.7 55.7 4.9 0 0 有效 7.340  LLDT-68 67.7 55.7 4.9 0 0 Effective 7.340
LLDT-69 69.3 67.7 15.4 0 0 有效 2.701  LLDT-69 69.3 67.7 15.4 0 0 Valid 2.701
LLDT-70 69.7 63.0 36.0 0 0.1 有效 1.803  LLDT-70 69.7 63.0 36.0 0 0.1 Valid 1.803
LLDT-76 69.9 63.4 60.4 14.3 0 有效 0.472  LLDT-76 69.9 63.4 60.4 14.3 0 effective 0.472
LLDT-77 60.3 16.8 3.8 4.3 5.3 无效 20.013 LLDT-77 60.3 16.8 3.8 4.3 5.3 Invalid 20.013
LLDT-78 70.8 57.9 9.5 0 0 有效 6.515 LLDT-78 70.8 57.9 9.5 0 0 Effective 6.515
LLDT-79 53.8 18.6 0 0 4.1 无效 13.934 实验实施例 4 LLDT-67对人肿瘤细胞的体外增殖抑制作用 方法  LLDT-79 53.8 18.6 0 0 4.1 Invalid 13.934 Experimental Example 4 Inhibition of in vitro proliferation of human tumor cells by LLDT-67 Method
SRB法， MTT法： SRB method, MTT method:
RPMI1640, DMEM、 F12或 5A培养基 (Gibco), 内含 10 %胎牛血 清， 根据肿瘤细胞生长需要加以选择， 对肿瘤细胞进行培养； 培养条 件为 37。C、 5% C02 o 按照 0.4-1.5 xlO4 细胞 /孔的数目接种肿瘤细胞于 96-孔板， 24小时后 ,加入用二曱基亚砜配制、生理盐水稀释的 LLDT-67, 使培养基中 LLDT-67的终浓度为 0.008 μΜ - 40 μΜ; 培养基中二曱基 亚砜的终浓度不超过 0.1%。 LLDT-67处理 72小时。 RPMI1640, DMEM, F12 or 5A medium (Gibco) containing 10% fetal bovine serum, selected according to the growth of tumor cells, cultured tumor cells; culture condition is 37. C, 5% C0 2 o The tumor cells were seeded in a 96-well plate at a number of 0.4-1.5 x 10 4 cells/well. After 24 hours, LLDT-67 diluted with physiological saline was added to prepare the culture. The final concentration of LLDT-67 in the base is 0.008 μΜ - 40 μΜ; the final concentration of dimethyl sulfoxide in the medium is not more than 0.1%. LLDT-67 was treated for 72 hours.
SRB法： 贴壁细胞釆用 SRB法。 药物作用 72小时后弃去培养液， 用 10% 冷三氯醋酸固定细胞。用碩酰罗丹明 B(Sulforhodamine B,SRB) 溶液染色。 洗去未结合 SRB, 用 Tris溶解与蛋白结合的 SRB, 用酶标 仪在 560 nm波长下测定 OD值。 MTT 法： 悬浮细胞釆用四氮唑盐 （MTT )还原法。 药物作用 72 小时后加 MTT液， 继续培养 4小时， 加入三联液（ 10% SDS-5% 异丁 醇 -0.01 M HCl ), C02培养箱中过夜。 用酶标仪在 570 nm波长下测定 OD值。 釆用下列公式计算细胞增殖生长抑制率： 抑制率 = (OD 值对照孔― OD值给药孔) / OD值对照孔 χ 100% 根据各浓度的抑制率， 釆用 Logit法计算半数抑制浓度 IC50。 结果 SRB method: The adherent cells were treated with the SRB method. After 72 hours of drug action, the culture was discarded and the cells were fixed with 10% cold trichloroacetic acid. Stained with a solution of Sulforhodamine B (SRB). The unbound SRB was washed away, and the protein-bound SRB was dissolved in Tris, and the OD value was measured at a wavelength of 560 nm using a microplate reader. MTT method: Suspension cells were reduced by tetrazolium salt (MTT). After the drug was applied for 72 hours, MTT solution was added, and the culture was continued for 4 hours, and a triple solution (10% SDS-5% isobutanol-0.01 M HCl) was added, and the mixture was kept in a C0 2 incubator overnight. The OD value was measured at a wavelength of 570 nm using a microplate reader.计算 Calculate the cell proliferation inhibition rate using the following formula: Inhibition rate = (OD value control well - OD value administration hole) / OD value control hole χ 100% According to the inhibition rate of each concentration, Log Calculate the half inhibition concentration IC by Logit method 50 . result
LLDT-67 剂量依赖性地抑制多种体外培养人肿瘤细胞的增值生 长， IC5。在 0.14 μΜ - 5.99 μΜ之间， 有艮强的体外抗肿瘤活性（表 4 )。 表 4. LLDT-67对人肿瘤细胞的体外增殖抑制作用 细胞名称 肿瘤类型 Ι 50(μΜ) LLDT-67 dose-dependently inhibited the growth of a variety of cultured human tumor cell value, IC 5. There is a strong in vitro antitumor activity between 0.14 μΜ and 5.99 μΜ (Table 4). Table 4. Inhibition of LLDT-67 on proliferation of human tumor cells in vitro Cell name Tumor type Ι 50 (μΜ)
Molt-4 T-淋巴性人白血病细胞株 0.25  Molt-4 T-lymphoid human leukemia cell line 0.25
HL-60 急性粒细胞人白血病细胞株 0.27  HL-60 acute granulocyte human leukemia cell line 0.27
A549 肺癌 0.70  A549 lung cancer 0.70
SGC-7901 胃癌 0.18  SGC-7901 Gastric Cancer 0.18
MKN-28 5.63  MKN-28 5.63
MKN-45 胃癌 5.99  MKN-45 gastric cancer 5.99
Bel-7402 肝癌 0.29  Bel-7402 Liver cancer 0.29
SMMC-7721 肝癌 0.33  SMMC-7721 Liver cancer 0.33
786-0 0.39  786-0 0.39
HCT-116 结肠癌 0.22 SW1116 结肠癌 1.13 HCT-116 colon cancer 0.22 SW1116 Colon Cancer 1.13
HCT-15 结肠癌 0.58  HCT-15 colon cancer 0.58
T-24 膀胱癌 0.14  T-24 Bladder Cancer 0.14
MDA-MB-231 乳腺癌 0.28  MDA-MB-231 Breast Cancer 0.28
MDA-MB-468 乳腺癌 0.28  MDA-MB-468 Breast Cancer 0.28
MCF-7 乳腺癌 0.21  MCF-7 breast cancer 0.21
DU-145 前列腺癌 0.26  DU-145 prostate cancer 0.26
PC-3 前列腺癌 0.52  PC-3 prostate cancer 0.52
HO-8910 卵巢癌 0.54  HO-8910 Ovarian Cancer 0.54
SKOV-3 卵巢癌 0.16  SKOV-3 ovarian cancer 0.16
Rh30 人横紋肌肉瘤 0.37 实验实施例 5 LLDT-67对人肿瘤耐药细胞的体外增殖抑制作用 方法  Rh30 human rhabdomyosarcoma 0.37 Experimental Example 5 Inhibition of in vitro proliferation of human tumor resistant cells by LLDT-67
SRB法， MTT法： 用含 10 % 胎牛血清的 RPMI 1640或 DMEM培养基 (Gibco)培养多 药耐药细胞及其亲本株肿瘤细胞；培养条件为 37。C、 5% C02 o MCF-7 , MCF-7/ADR细胞的培养液中除 RPMI 1640培养液外，还含 1 mM丙酮 酸钠和 0.01 mg/ml牛胰岛素。 耐药细胞株在培养液中加入 2 μΜ 阿霉 素（Κ562/Α02 、MCF-7/ADR 细胞培液）或 1 μΜ的长春新碱（ KB/VCR 细胞培液）维持耐药， 实验前一周停药。 按照 0.6-1.5 xlO4细胞 /孔的数 目接种肿瘤细胞于 96-孔板， 24小时后， 加入用二曱基亚砜配制、 生理 盐水稀释的 LLDT-67、 阿霉素、 长春新碱。 培养基中 LLDT-67的终浓 度为 0.008 μΜ -40 μΜ,阿霉素及长春新碱的终浓度 0.005 μΜ -400 μΜ。 培养基中二曱基亚砜的终浓度不超过 0.1%。 药物作用细胞 72小时。 贴壁性细胞釆用 SRB法。药物作用 72 小时后弃去培养液，用 10% 冷三氯醋酸固定细胞， SRB溶液染色， 然后洗去未结合 SRB , Tris溶 解与蛋白结合的 SRB, 用酶标仪在 560 nm波长下测定 OD值。 悬浮性细胞釆用 MTT法。 药物作用 72小时后加入 MTT液， 继续 培养 4小时， 加入三联液（ 10% SDS-5% 异丁醇 -0.01 M HC1 ), C02培 养箱中过夜。 用酶标仪在 570 nm波长下测定 OD值。 釆用下列公式计算细胞增殖生长抑制率： 抑制率= (OD值对照孔 - OD值给药扎)/ OD值对照孔 X 100% 。 根据各浓度抑制率， 釆用 Logit法计算半数抑制浓度 IC50。 结果 SRB method, MTT method: Multidrug resistant cells and their parental tumor cells were cultured in RPMI 1640 or DMEM medium (Gibco) containing 10% fetal bovine serum; the culture condition was 37. C, 5% C0 2 o MCF-7, MCF-7/ADR cells were cultured with 1 mM sodium pyruvate and 0.01 mg/ml bovine insulin in addition to RPMI 1640 medium. The drug-resistant cell line was supplemented with 2 μΜ doxorubicin (Κ562/Α02, MCF-7/ADR cell culture solution) or 1 μΜ vincristine (KB/VCR cell culture solution) to maintain drug resistance, one week before the experiment. Stop the drug. Tumor cells were seeded in 96-well plates at a number of 0.6-1.5 x 10 4 cells/well, and 24 hours later, LLDT-67, doxorubicin, and vincristine diluted with physiological saline were prepared. Final concentration of LLDT-67 in the medium The degree is 0.008 μΜ -40 μΜ, and the final concentration of doxorubicin and vincristine is 0.005 μΜ -400 μΜ. The final concentration of dimethyl sulfoxide in the medium does not exceed 0.1%. The drug acts on the cells for 72 hours. The adherent cells were treated with the SRB method. After 72 hours of drug administration, the culture solution was discarded, and the cells were fixed with 10% cold trichloroacetic acid, stained with SRB solution, and then washed with unbound SRB, Tris dissolved and protein-bound SRB, and OD was measured at 560 nm using a microplate reader. value. The suspension cells were treated with the MTT method. After the drug was added for 72 hours, the MTT solution was added, and the culture was continued for 4 hours, and a triple solution (10% SDS-5% isobutanol-0.01 M HCl) was added to the C0 2 incubator overnight. The OD value was measured at a wavelength of 570 nm using a microplate reader.细胞 Calculate the cell proliferation inhibition rate using the following formula: Inhibition rate = (OD value control hole-OD value administration) / OD value control hole X 100%. According to the respective concentration inhibition rates, the half-inhibitory concentration IC 50 was calculated by the Logit method. result
LLDT-67 对耐药细胞显示出和其相应亲本细胞相近的细胞增殖生 长抑制作用，有体外抗多药耐药作用，可能对耐药肿瘤的治疗有效（表 5 )。 表 5. LLDT-67对人肿瘤多药耐药细胞的体外增殖抑制作用 LLDT-67 showed a proliferative growth inhibition effect on drug-resistant cells similar to its corresponding parental cells, and it has anti-multidrug resistance in vitro and may be effective in the treatment of drug-resistant tumors (Table 5). Table 5. Inhibition of LLDT-67 on proliferation of human tumor multidrug resistant cells in vitro
LLDT-67 Adr VCR 肿瘤类型 LLDT-67 Adr VCR Tumor Type
Ι (μΜ) RF Κ 50 ( μΜ) RF IC50( μΜ) RF Ι (μΜ) RF Κ 50 ( μΜ) RF IC 50 ( μΜ) RF
K562 白血病细 包阿 0.56 0.61 NT NT 霉素敏感株 1.09 83.46 K562 leukemia fine package 0.56 0.61 NT NTmycin sensitive strain 1.09 83.46
K562/A02 白血病细 包阿 0.80 58.74 NT K562/A02 Leukemia fine package A 0.80 58.74 NT
霉素耐药株 KB 口腔鳞癌长春 0.49 NT 0.01 新碱敏感株 0.69 NT 223Drug resistant strain KB Oral Squamous Cell Carcinoma Changchun 0.49 NT 0.01 Neobase Sensitive Plant 0.69 NT 223
KB/VCR 口腔鳞癌长春 0.34 NT 2.23 KB/VCR Oral Squamous Cell Carcinoma Changchun 0.34 NT 2.23
新碱耐药株  Neobase resistant strain
MCF-7 乳腺癌阿霉素 0.43 0.36 NT  MCF-7 Breast Cancer Doxorubicin 0.43 0.36 NT
敏感株 1.86 129.67 NT Sensitive strain 1.86 129.67 NT
MCF-7/Adr 乳腺癌阿霉素 0.80 46.68 NT MCF-7/Adr Breast Cancer Doxorubicin 0.80 46.68 NT
耐药株 注： NT, not tested, 未检测； RF, Resistance Factor, 耐药因子， 评价化合物抗耐 药作用的指标， 计算公式为 RF=IC5。（耐药细胞株） /IC5。 (敏感亲本细胞株)。 实验实施例 6 LLDT-67对人卵巢癌细胞 SK-OV-3棵小鼠皮下移 植瘤的生长抑制作用 方法 釆用棵小鼠皮下移植瘤模型： 人卵巢癌 SK-OV-3细胞来自上海肿瘤医院； BALB/C棵小鼠 [SPF 级（specific-pathogen free,无特定病原体级） ] , 雌性， 体重： 18±2g,由 中科院上海药物所提供，合格证编号： SCXK(沪）(2004-0002)。 SK-OV-3 细胞 5χ106/只接种于棵小鼠右侧腋窝皮下， 形成移植瘤后再在棵小鼠 体内传 2代后使用。 取生长旺盛的瘤组织， 在无菌条件下， 用剪刀剪 成 1 mm3左右的小块， 用套管针接种于棵小鼠左侧腋窝皮下， 待肿瘤 生长至 100-200 mm3左右，将动物随机分组 (d0)。 LLDT-67按 1.0、 2.0、 4.0 mg/kg, 口服给药，每天 1次，连续 21 d。阳性对照丝裂霉素（ MMC ) 5 mg/kg, 尾静脉注射， 于第 1天给药 1次。 给药过程中， 每周两次称 鼠重， 测量瘤体积， 肿瘤体积 (tumor volume , TV) = l/2xaxb2 ( a、 b分 别表示长、 宽）。 根据测量的结果计算出相对肿瘤体积（relative tumor volume, RTV ), RTV = Vdt/Vd。（ Vd。为 d。天测量时肿瘤体积， Vdt为给 药 dt天测量时肿瘤体积）。 抗肿瘤活性的评价指标为相对肿瘤体积比 T/C ( % ), T/C ( % ) =Drug-resistant strains Note: NT, not tested, untested; RF, Resistance Factor, drug resistance factor, an index for evaluating the resistance of a compound to a drug, calculated as RF = IC 5 . (drug resistant cell line) / IC 5 . (sensitive parental cell line). EXPERIMENTAL EXAMPLE 6 Growth inhibition of LLDT-67 on subcutaneous xenografts of human ovarian cancer cell line SK-OV-3. Subcutaneous xenograft model of mouse: Human ovarian cancer SK-OV-3 cells from Shanghai tumor Hospital; BALB/C mice [SPF (specific-pathogen free)], female, weight: 18±2g, provided by Shanghai Institute of Medicine, Chinese Academy of Sciences, certificate number: SCXK (Shanghai) (2004- 0002). SK-OV-3 cells 5χ10 6 / were inoculated subcutaneously into the right axilla of mice, and transplanted tumors were formed and then transferred to mice for 2 generations. Take vigorous growth of the tumor tissue, under sterile conditions, cut into small pieces of about 1 mm 3 with scissors, inoculate the left armpit of the mouse with a trocar, and grow the tumor to about 100-200 mm 3 . Animals were randomly grouped (d0). LLDT-67 was administered orally at 1.0, 2.0, 4.0 mg/kg once daily for 21 days. The positive control mitomycin (MMC) 5 mg/kg was injected into the tail vein and administered once on the first day. During the administration, weigh twice a week Rat weight, measuring tumor volume, tumor volume (TV) = l/2xaxb 2 (a, b respectively indicate length, width). Based on the measured results, the relative tumor volume (RTV), RTV = V dt /V d , was calculated. (V d . is d. The tumor volume at the time of measurement, V dt is the tumor volume measured on the day of administration dt). The anti-tumor activity was evaluated as the relative tumor volume ratio T/C (%), T/C (%) =
TRTV/ CRTVxl00%。 结果判定标准： T/C ( % ) <40% , 有效； T/C ( % ) >40%, 无效。 T RTV / C RTV xl00%. The result criteria were: T/C (%) <40%, valid; T/C (%) >40%, invalid.
另外， 可通过小鼠行为学、 二便及体重变化初步判定化合物的毒性情 况。 结果  In addition, the toxicity of the compound can be preliminarily determined by mouse behavior, secondary stool, and body weight change. Result
LLDT-67在 1.0、 2.0、 4.0 mg/kg剂量时 T/C ( % )分别为 50.2%、 12.5%、 2.6%, 剂量依赖性地抑制人卵巢癌 SK-OV-3 棵小鼠皮下移植 瘤生长； 另外， 在所用剂量及给药方式时， LLDT-67 对棵小鼠无明显 的毒副作用。 （见表 6、 图 1和 2)。 表 6. LLDT-67对人卵巢癌细胞 SK-OV-3棵小鼠皮下移植瘤的生长抑 LLDT-67 at a dose of 1.0, 2.0, 4.0 mg/kg had a T/C (%) of 50.2%, 12.5%, 2.6%, respectively, and dose-dependently inhibited subcutaneous xenografts in human ovarian cancer SK-OV-3 mice. Growth; In addition, LLDT-67 had no significant toxic side effects on mice at the dose and mode of administration. (See Table 6, Figures 1 and 2). Table 6. Growth inhibition of LLDT-67 on subcutaneous xenografts of human ovarian cancer cells SK-OV-3
制作用 剂量、 动物数 体重 (g) TV (mm3) T/C 组别 RTV P值 给药方式 开始 最后 开始 最后 do d2i (%) 溶剂 0.4ml/只 P.o 12 12 19.8 18.6 142 1370 10 对照 l-7/3w ± 44 ± 640 ± 4.9 Dosage, animal weight (g) TV (mm 3 ) T/C group RTV P value administration mode start last start last do d 2 i (%) solvent 0.4ml / Po 12 12 19.8 18.6 142 1370 10 Control l-7/3w ± 44 ± 640 ± 4.9
MMC 5 mg/kg, i.v 6 6 20.3 19.2 137 642 4.8 46.9 <0.05 dl/3w ±29 士 165 士 1.4 MMC 5 mg/kg, i.v 6 6 20.3 19.2 137 642 4.8 46.9 <0.05 dl/3w ±29 士 165 士 1.4
LLDT-67 1 mg/kg, P.o 6 6 20.3 17.3 140 696 5.1 50.2 <0.05 l-7/3w . ± 42 ± 361 ± 2.8 LLDT-67 1 mg/kg, Po 6 6 20.3 17.3 140 696 5.1 50.2 <0.05 L-7/3w . ± 42 ± 361 ± 2.8
LLDT-67 2mg/kg, P.o 6 6 21.2 19.8 144 195 1.3 12.5 <0.01 l-7/3w . ± 26 ± 178 ± 0.9 LLDT-67 2mg/kg, P.o 6 6 21.2 19.8 144 195 1.3 12.5 <0.01 l-7/3w . ± 26 ± 178 ± 0.9
LLDT-67 4 mg/kg, P.o 6 6 20.3 141 38 0.23 LLDT-67 4 mg/kg, P.o 6 6 20.3 141 38 0.23
2.6 <0.01 l-7/3w . 20.8 ± 42 ± 50 ±0.3 实验实施例 7 LLDT-67对人前列腺癌细胞 PC-3棵小鼠皮下移植 瘤的生长抑制作用 方法 釆用棵小鼠皮下移植瘤模型： 人前列腺癌 PC-3 细胞来自 日本， BALB/C 棵小鼠 [SPF 级 ( specific-pathogen free,无特定病原体级） ] , 雄性， 体重： 20士 2g, 由 中科院上海药物所提供， 合格证编号： SCXK (沪）（2004-0002)。 将体 外生长旺盛的人前列腺癌 PC-3细胞消化， 计数， 按 lxlO6/只接种于棵 小鼠腋下， 待肿瘤生长至 100 - 150 mm3左右， 将动物随机分组 (d0)。 2.6 <0.01 l-7/3w . 20.8 ± 42 ± 50 ±0.3 Experimental Example 7 Growth inhibition of LLDT-67 on human prostate cancer cell line PC-3 in mice subcutaneously transplanted tumors Model: Human prostate cancer PC-3 cells from Japan, BALB/C mice [SPF (specific-pathogen free)], male, weight: 20 ± 2 g, provided by Shanghai Institute of Traditional Chinese Medicine, qualified Certificate number: SCXK (Shanghai) (2004-0002). The human prostate cancer PC-3 cells vigorously grown in vitro were digested, counted, and inoculated into the mice under lxlO 6 /only, and the tumors were grown to about 100 - 150 mm 3 , and the animals were randomly divided into groups (d0).
LLDT-67按 0.5、 1.0、 2.0 mg/kg, 口服给药， 每天 1次， 连续 25 d。 阳 性对照丝裂霉素 （MMC ) 5 mg/kg, 尾静脉注射， 于第 1天给药 1次。  LLDT-67 was administered orally at 0.5, 1.0, 2.0 mg/kg once daily for 25 days. The positive control mitomycin (MMC) 5 mg/kg was injected into the tail vein and administered once on the first day.
给药过程中，每周两次称鼠重，测量瘤体积，肿瘤体积 (tumor volume ,TV) = l/2xaxb2 ( a、 b分别表示长、 宽）。 根据测量的结果计算出相对肿瘤 体积（relative tumor volume, RTV ), RTV = Vdt/Vd0 ( VdQ为 do天测量 时肿瘤体积， Vdt为给药 dt天测量时的肿瘤体积）。 抗肿瘤活性的评价指标为相对肿瘤体积比 T/C ( % ), T/C ( % ) = TRTV/ CRTVx l00%。 结果判定标准： T/C (%) <40% , 有效； T/C (%) >40%, 无效。 During the administration, the rats were weighed twice a week, and the tumor volume was measured. The tumor volume (TV) = l/2xaxb 2 (a and b respectively indicate the length and width). Based on the measured results, the relative tumor volume (RTV) was calculated, RTV = V dt /V d0 (V d Q is the tumor volume measured on do days, and V dt is the tumor volume measured on the dt day of administration). The anti-tumor activity was evaluated as the relative tumor volume ratio T/C (%), T/C (%) = T RTV / C RTV x l00%. The result criteria were: T/C (%) <40%, valid; T/C (%) >40%, invalid.
另外， 可通过小鼠行为学、 二便及体重变化初步判定化合物的毒性情 况。 结果  In addition, the toxicity of the compound can be preliminarily determined by mouse behavior, secondary stool, and body weight change. Result
LLDT-672.0mg/kg H, T/C (%)为 1.63%, 能够显著抑制人前列 腺癌 PC-3 棵小鼠移皮下移植瘤的生长， 抑制率高达 98.37%, 显示出 非常强的抗肿瘤作用； 另外， 在所用剂量及给药方式下， LLDT-67 对 棵小鼠无明显的毒副作用。 （见表 7、 图 3和 4)。 表 7. LLDT-67对人前列腺癌细胞 PC-3棵小鼠皮下移植瘤的生长抑制作用 动物数 体重 (g) TV( mm3) T/C 组别 剂量、给药方式■ - RTV P值 开始 最后 开始 最后 do d2i (%) 溶剂 0.4ml/只， p.o 6 6 21.5 18.0 99 621 6.5 对照 l-7x3.5w ±20 ±170 ±2.3 LLDT-672.0mg/kg H, T/C (%) was 1.63%, which significantly inhibited the growth of human prostate cancer PC-3 mice under subcutaneous transplantation. The inhibition rate was as high as 98.37%, showing a strong anti-tumor. In addition, LLDT-67 has no obvious side effects on mice at the dose and mode of administration. (See Table 7, Figures 3 and 4). Table 7. Growth inhibition of LLDT-67 on human prostate cancer cell line PC-3 subcutaneous xenografts in animals. Body weight ( g ) TV (mm 3 ) T/C group dose, mode of administration ■ - RTV P value Start last and finally do d 2 i (%) Solvent 0.4ml / only, po 6 6 21.5 18.0 99 621 6.5 Control l-7x3.5w ±20 ±170 ±2.3
MMC 5mg/kg, i.v 6 6 20.5 18.8 98 337 3.6 <0.05 MMC 5mg/kg, i.v 6 6 20.5 18.8 98 337 3.6 <0.05
55.6 dlx3.5w ±25 ±105 ±1.5  55.6 dlx3.5w ±25 ±105 ±1.5
LLDT-67 2mg/kg, p.o 6 6 22.5 21.8 99 10.13 0.11 1.63 <0.01 l-7x3.5w ±30 ±6.9 ±0.06 LLDT-67 2mg/kg, p.o 6 6 22.5 21.8 99 10.13 0.11 1.63 <0.01 l-7x3.5w ±30 ±6.9 ±0.06
LLDT-67 lmg/kg, p.o 6 6 21.7 19.0 98 411 4.3 LLDT-67 lmg/kg, p.o 6 6 21.7 19.0 98 411 4.3
67.0 >0.05 l-7x3.5w ±31 ±213 ±2.2  67.0 >0.05 l-7x3.5w ±31 ±213 ±2.2
LLDT-67 0.5mg/kg, p.o 6 6 22.2 19.0 96 741 8.8 LLDT-67 0.5mg/kg, p.o 6 6 22.2 19.0 96 741 8.8
134.9 >0.05 l-7x3.5w ±47 士 147 ±4.3 l o 实验实施例 8 LLDT-67对人前列腺癌细胞 DU- 145棵小鼠皮下移  134.9 >0.05 l-7x3.5w ±47 ± 147 ±4.3 l o Experimental Example 8 LLDT-67 on human prostate cancer cells DU- 145 mice subcutaneously
植瘤的生长抑制作用 方法 釆用棵小鼠皮下移植瘤模型： 人前列腺癌 DU-145 细胞来自美国 ATCC(American Type Culture Collection); BALB/C棵小鼠 [SPF级（ specific-pathogen free,无特定病原 体级） ] , 雄性， 体重： 20±2g, 由中科院上海药物所提供， 合格证编 号： SCXK (沪）（2004-0002)。 处死荷瘤小鼠， 解剖取出皮下肿瘤组织， 用剪刀剪成 1 mm3左右的小块， 用套管针接种于小鼠左侧腋窝皮下， 待肿瘤生长至 100-200 mm3左右， 将动物随机分组 (d0)。 LLDT-67按 0.5、 1.0、 2.0 mg/kg, 口服给药， 每天 1次， 每周 5天， 连续给药 4周； 阳性对照药多西他赛 10 mg/kg, 尾静脉注射， 分别于第 1天、 第 7天 给药 2次。给药过程中，每周 2次称鼠重，测量瘤体积，肿瘤体积 (tumor volume ,TV)= l/2xaxb2 ( a、 b分别表示长、 宽）。 根据测量的结果计算 出相对肿瘤体积 （relative tumor volume, RTV ), RTV = Vdt/Vd0 ( Vd0 为 dQ天测量时肿瘤体积， Vdt为给药 dt天测量时的肿瘤体积）。 抗肿瘤活性的评价指标为相对肿瘤增殖率 T/C ( % ), T/C ( % ) = TRTV/ CRTVxl00%。 结果判定标准： T/C ( % ) <40%, 有效； T/C ( % ) >40%, 无效。 另外， 可通过小鼠行为学、 二便及体重变化初步判定化合物的毒性情 况。 结果 Growth inhibition of tumor Methods The mouse subcutaneous xenograft model was used: human prostate cancer DU-145 cells were obtained from American ATCC (American Type Culture Collection); BALB/C mice [SPF (specific-pathogen free)], Male, weight: 20±2g, provided by Shanghai Institute of Medicine, Chinese Academy of Sciences, certificate number: SCXK (Shanghai) (2004-0002). The tumor-bearing mice were sacrificed, and the subcutaneous tumor tissue was dissected and cut into small pieces of about 1 mm 3 with scissors. The trocars were inoculated into the left axilla of the mice, and the tumors were grown to about 100-200 mm 3 . Randomly grouped (d0). LLDT-67 was administered orally at 0.5, 1.0, 2.0 mg/kg, once a day, 5 days a week for 4 weeks; the positive control drug docetaxel 10 mg/kg, tail vein injection, respectively The drug was administered twice on the first day and the seventh day. During the administration, the rats were weighed twice a week, and the tumor volume was measured. The tumor volume (TV) = l/2xaxb 2 (a, b respectively indicate the length and width). Based on the measured results, the relative tumor volume (RTV), RTV = V dt / V d0 (V d0 is the tumor volume measured at d Q days, and V dt is the tumor volume measured at the time of administration dt). The evaluation index of antitumor activity was relative tumor growth rate T/C (%), T/C (%) = T RTV / C RTV x l00%. The result criteria were: T/C (%) <40%, valid; T/C (%) >40%, invalid. In addition, the toxicity of the compound can be preliminarily determined by mouse behavior, two stools, and body weight changes. result
LLDT-67 1.0、 2.0 mg/kg给药时，其 T/C( % )分别为 56.4%、 50.4% , 显示一定程度地抑制人前列腺癌 DU-145棵小鼠皮下移植瘤生长作用； 另外， 在所用剂量及给药方式下， LLDT-67 对棵小鼠无明显的毒副作 用。 （见表 8、 图 5和 6)。 表 8. LLDT-67对人前列腺癌细胞 DU-145棵小鼠皮下移植瘤的生长抑制作用 动物数 体重 (g) TV (m Lm3) T/C 组别 剂量、 给药方式 - RTV P值 开始 最后 开始 最后 do d28 (%) 溶剂 0.4ml/ 只 p.o 6 6 22.0 24.7 112 1098 7.1 对照 士 16 ± 405 ± 2.6 多西他赛 10mg/kg,dl i.v 6 6 23.2 24.0 108 340 3.3 47.1 <0.0 、 d5x4w ± 29 ± 100 士 1.0 When administered with LLDT-67 1.0 and 2.0 mg/kg, the T/C (%) was 56.4% and 50.4%, respectively, indicating a certain degree of inhibition of the growth of subcutaneous xenografts in human prostate cancer DU-145 mice; In addition, LLDT-67 had no significant toxic side effects on mice at the dose and mode of administration. (See Table 8, Figures 5 and 6). Table 8. Growth inhibition of LLDT-67 on human prostate cancer cell line DU-145 subcutaneous xenografts in animals. Body weight ( g ) TV (m Lm 3 ) T/C group dose, mode of administration - RTV P value Start last and last do d 2 8 (%) Solvent 0.4ml / po 6 6 22.0 24.7 112 1098 7.1 Control ± 16 ± 405 ± 2.6 Docetaxel 10mg / kg, dl iv 6 6 23.2 24.0 108 340 3.3 47.1 < 0.0, d5x4w ± 29 ± 100 ± 1.0
LLDT-67 2mg/kg, p.o 6 6 18.7 24.7 107 381 3.5 50.4 <0.0 l-5x4w 士 16 士 147 士 1.5 LLDT-67 2mg/kg, p.o 6 6 18.7 24.7 107 381 3.5 50.4 <0.0 l-5x4w ± 16 147 士 1.5
LLDT-67 lmg/kg, p.o 6 6 21.3 23.2 109 414 4.1 LLDT-67 lmg/kg, p.o 6 6 21.3 23.2 109 414 4.1
56.4 <0.0 l-5x4w ± 47 ± 105 ± 0.9  56.4 <0.0 l-5x4w ± 47 ± 105 ± 0.9
LLDT-67 0.5mg/kg, p.o 6 6 22.7 26.5 110 701 6.6 LLDT-67 0.5mg/kg, p.o 6 6 22.7 26.5 110 701 6.6
92.5 >0.0 l-5x4w ± 28 ± 266 ± 2.7 实验实施例 9 LLDT-67对人胃癌细胞 SGC-7901棵小鼠皮下移植 瘤的生长抑制作用 方法 釆用棵小鼠皮下移植瘤模型： 人胃癌 SGC-7901细胞来自上海市第六人民医院； BALB/C棵小鼠  92.5 >0.0 l-5x4w ± 28 ± 266 ± 2.7 Experimental Example 9 Growth inhibition effect of LLDT-67 on subcutaneous xenografts of human gastric cancer cell SGC-7901 in mice Subcutaneous xenograft model of mouse: Human gastric cancer SGC -7901 cells from Shanghai Sixth People's Hospital; BALB/C mice
[SPF级（specific-pathogen free,无特定病原体级）] ,雌性，体重： 18士 2g, 由中科院上海药物所提供， 合格证编号： SCXK (沪）（2004-0002)。 处 死荷瘤小鼠， 解剖取出皮下肿瘤组织， 用剪刀剪成 1 mm3左右的小块， 用套管针接种于小鼠左侧腋窝皮下， 待肿瘤生长至 100-200 mm3左右， 将动物随机分组 (d0)。 LLDT-67按 1.0、 2.0、 4.0 mg/kg, 口服给药， 每 天 1次， 每周 5次， 连续 3周。 阳性对照药丝裂霉素 ( MMC ) 5 mg/kg, 尾静脉注射， 于第 1天给药 1次。 给药过程中， 每周两次称鼠重， 测 量瘤体积，肿瘤体积 (tumor volume ,TV) = l/2 axb2 ( a、 b分另1 J表示长、 宽）。 根据测量的结果计算出相对肿瘤体积 （relative tumor volume, RTV ), RTV = Vdt/VdQ ( VdQ为 dQ天测量时肿瘤体积， Vdt为给药 dt天测 量时的肿瘤体积）。 抗肿瘤活性的评价指标为相对肿瘤增殖率 T/C( % ), T/C( % )= TRTV/ CRTVX100%。 结果判定标准： T/C ( % ) <40 %, 有效； T/C ( % ) >40%, 无效。 另外， 可通过小鼠行为学、 二便及体重变化初步判定化合物的毒性情 况。 结果 [SPF level (specific-pathogen free)], female, weight: 18 ± 2g, provided by the Shanghai Institute of Medicine, Chinese Academy of Sciences, certificate number: SCXK (Shanghai) (2004-0002). The tumor-bearing mice were sacrificed, and the subcutaneous tumor tissue was dissected and cut into small pieces of about 1 mm 3 with scissors. The mice were inoculated subcutaneously into the left axilla of the mouse with a trocar. The tumors were grown to a size of about 100-200 mm 3 and the animals were randomly divided into groups (d0). LLDT-67 was administered orally at 1.0, 2.0, 4.0 mg/kg once a day for 5 times a week for 3 weeks. The positive control drug mitomycin (MMC) 5 mg/kg was injected into the tail vein and administered once on the first day. During the administration, the rats were weighed twice a week, and the tumor volume was measured. The tumor volume (TV) = l/2 axb 2 (a, b is 1 J for long and wide). Based on the measured results, the relative tumor volume (RTV), RTV = V dt / V dQ (V dQ is the tumor volume measured at d Q days, and V dt is the tumor volume measured at the time of administration dt). The evaluation index of antitumor activity is relative tumor growth rate T/C (%), T/C (%) = T RTV / C RTV X100%. The result criteria were: T/C ( % ) <40 %, valid; T/C ( % ) >40%, invalid. In addition, the toxicity of the compound can be preliminarily determined by mouse behavior, two stools, and body weight changes. result
LLDT-67 4.0 mg/kg给药时， 其 T/C ( % )为 35.0%, 能够抑制人胃 癌 SGC-7901棵小鼠皮下移植瘤的生长； 另外，在所用剂量及给药方式 下， LLDT-67 对棵小鼠无明显的毒副作用 （见表 9、 图 7和 8)。 表 9. LLDT-67对人胃癌细胞 SGC-7901棵小鼠皮下移植瘤的生长 抑制作用 动物数 体重 (g) TV (mm3) When LLDT-67 was administered at 4.0 mg/kg, its T/C (%) was 35.0%, which inhibited the growth of subcutaneous xenografts in human gastric cancer SGC-7901 mice. In addition, at the dose and mode of administration, LLDT -67 had no significant toxic side effects on mice (see Table 9, Figures 7 and 8). Table 9. Growth inhibition of LLDT-67 on subcutaneous xenografts of human gastric cancer cells SGC-7901 in mice. Body weight (g) TV (mm 3 )
剂量、 T/C 组别 开 最 开 最 dQ d21 RTV P值 给药方式 （％) Dosage, T/C group, the most open d Q d 21 RTV P value administration method (%)
始 后 始 后 溶剂 0.4ml/只 po 12 12 18.7 22.5 153 1643 11.1 对照 l-5/3w ± 47 ± 689 ± 4.7 After the beginning and after the solvent 0.4ml / only po 12 12 18.7 22.5 153 1643 11.1 Control l-5/3w ± 47 ± 689 ± 4.7
MMC 5 mg/kg, iv 6 6 18.5 20.5 157 364 2.3 21.0 <0.05 dl/4.5w ±12 士 192 士 1.2 MMC 5 mg/kg, iv 6 6 18.5 20.5 157 364 2.3 21.0 <0.05 dl/4.5w ±12 士 192 士 1.2
LLDT-67 4mg/kg, po 6 6 20.0 21.6 153 547 3.9 35.0 <0.05 l-5/3w 7 ±65 ± 230 士 1.9 LLDT-67 4mg/kg, po 6 6 20.0 21.6 153 547 3.9 35.0 <0.05 l-5/3w 7 ±65 ± 230 ± 1.9
LLDT-67 2mg/kg, po 6 6 18.3 21.3 160 1177 7.5 68.0 >0.05 l-5/3w ± 35 ± 312 士 1.8 LLDT-67 2mg/kg, po 6 6 18.3 21.3 160 1177 7.5 68.0 >0.05 l-5/3w ± 35 ± 312 ± 1.8
LLDT-67 1 mg/kg, po 6 6 19.3 22.8 157 1473 10.0 LLDT-67 1 mg/kg, po 6 6 19.3 22.8 157 1473 10.0
91.0 >0.05 l-5/3w ± 42 ± 411 ±4.0 实验实施例 10 LLDT-67对人乳腺癌细胞 MDA-MB-468棵小鼠 皮下移植瘤的生长抑制作用 方法 釆用棵小鼠皮下移植瘤模型： 人乳腺癌 MDA-MB-468 购自美国 ATCC(American Type Culture Collection); BALB/C棵小鼠 [SPF级（ specific-pathogen free,无特定病原 体级） ] , 雌性， 体重： 18±2g, 由中科院上海药物所提供， 合格证编 号： SCXK(沪）(2004-0002)。将体外生长旺盛的人乳腺癌 MDA-MB-468 肿瘤细胞消化， 计数， 按 l x lO7/只接种于棵小鼠腋下， 待肿瘤生长至 100 - 150 mm3左右， 将动物随机分组 (d0)。 LLDT-67按 1.0、 2.0、 4.0 mg/kg, 口服给药， 每天 1次， 每周 5次， 连续 2.5周。 阳性对照丝裂 霉素 ( MMC ) 5 mg/kg, 尾静脉注射， 于第 1天给药 1次。 给药过程中， 每周两次称鼠重，测量瘤体积，肿瘤体积 (tumor volume , TV) = l/2xaxb2 ( a、b分别表示长、宽）。根据测量的结果计算出相对肿瘤体积（relative tumor volume, RTV ), RTV = Vdt/Vd0 ( Vd0为 d。天测量时肿瘤体积， Vdt为给药 dt天测量时的肿瘤体积）。 抗肿瘤活性的评价指标为相对肿瘤增殖率 T/C ( % ), T/C ( % ) = TRTV/ CRTVxl00%。 结果判定标准： T/C ( % ) <40%, 有效； T/C ( % ) >40%, 无效。 91.0 >0.05 l-5/3w ± 42 ± 411 ±4.0 Experimental Example 10 Growth inhibition effect of LLDT-67 on human breast cancer cell MDA-MB-468 subcutaneous xenografts in mice Model: Human breast cancer MDA-MB-468 was purchased from American ATCC (American Type Culture Collection); BALB/C mice [SPF (specific-pathogen free)], female, body weight: 18±2g , provided by the Shanghai Institute of Medicine, Chinese Academy of Sciences, certificate number: SCXK (Shanghai) (2004-0002). The human breast cancer MDA-MB-468 tumor cells in vitro growth were digested, counted, and inoculated into the underarm of the mice according to lx lO 7 / only, and the tumors were grown to about 100 - 150 mm 3 , and the animals were randomly grouped (d0 ). LLDT-67 was administered orally at 1.0, 2.0, 4.0 mg/kg once daily for 5 times a week for 2.5 weeks. The positive control mitomycin (MMC) 5 mg/kg was injected into the tail vein and administered once on the first day. During the administration, the rats were weighed twice a week, and the tumor volume was measured. The tumor volume (TV) = l/2xaxb 2 (a and b respectively indicate the length and width). Calculate the relative tumor volume based on the measured results (relative Tumor volume, RTV ), RTV = V dt /V d0 (V d0 is d. Tumor volume at day measurement, V dt is the tumor volume measured at dt day of administration). The evaluation index of antitumor activity was relative tumor growth rate T/C (%), T/C (%) = T RTV / C RTV x l00%. The result criteria were: T/C (%) <40%, valid; T/C (%) >40%, invalid.
另外， 可通过小鼠行为学、 二便及体重变化初步判定化合物的毒性情 In addition, the toxicity of the compound can be preliminarily determined by mouse behavior, two stools, and body weight changes.
结果 Result
LLDT-67在 1.0、2.0、4.0 mg/kg的剂量时对人乳腺癌 MDA-MB-468 棵小鼠皮下移植瘤未显示出生长抑制作用； 另外， 在所用剂量及给药 方式下， LLDT-67 对棵小鼠无明显的毒副作用 （见表 10、 图 9和 10)。 表 10. LLDT-67对人乳腺癌细胞 MDA-MB-468棵小鼠皮下移植瘤 的生长抑制作用 LLDT-67 showed no growth inhibition on human breast cancer MDA-MB-468 subcutaneous xenografts at doses of 1.0, 2.0, and 4.0 mg/kg. In addition, at the dose and mode of administration, LLDT- 67 had no obvious toxic side effects on mice (see Table 10, Figures 9 and 10). Table 10. Growth inhibition of LLDT-67 on human breast cancer cell MDA-MB-468 subcutaneous xenografts in mice
动物数 体重 g) TV (mmJ) Animal weight g) TV (mm J )
组别 剂量、 给药 开 最 开 最 do d n RTV T/C P值 方式 始 后 始 后 (%) 溶剂对 0.4ml/ po 12 12 19.5 20.4 131士 2213士 17.1士  Group Dosage, Dosage Open Most Do d n RTV T/C P Value Method Start After Start (%) Solvent Pair 0.4ml/ po 12 12 19.5 20.4 131 ± 2213 ± 17.1
■o  ■o
、 29 648 4.5 l-5/2.5w  , 29 648 4.5 l-5/2.5w
MMC 5mg/kg, iv 6 6 19.3 18.5 130士 1509士 11.9士 70.0 >0.05 l/2.5w 19 328 3.8  MMC 5mg/kg, iv 6 6 19.3 18.5 130 士 1509士 11.9士 70.0 >0.05 l/2.5w 19 328 3.8
LLDT-67 4mg/kg, po 6 6 19.8 19.2 134士 2663士 19.6士 115.0 >0.05 l-5/2.5w 22 119 5.6  LLDT-67 4mg/kg, po 6 6 19.8 19.2 134 士 2663士 19.6士 115.0 >0.05 l-5/2.5w 22 119 5.6
LLDT-67 2mg/kg, po 6 6 19.5 20.3 136士 2268士 16.8士 98.0 >0.05 l-5/2.5w 28 336 2.0  LLDT-67 2mg/kg, po 6 6 19.5 20.3 136 士 2268士 16.8士 98.0 >0.05 l-5/2.5w 28 336 2.0
LLDT-67 1 mg/kg, po 6 6 19.7 21.0 130士 2145士 17.0士 99.0 >0.05 l-5/2.5w 36 1227 9.1 实验实施例 11 LLDT-67对人肝癌细胞 SMMC-7721棵小鼠皮下 移植瘤的生长抑制作用 方法 釆用棵小鼠皮下移植瘤模型： 人肝癌 SMMC-7721 细胞源自中国第二军医大学； BALB/C棵小 鼠 [SPF级（ specific-pathogen free,无特定病原体级）]，雌性，体重： 18±2g, 由中科院上海药物所提供， 合格证编号： SCXK (沪）（2004-0002)。 处 死荷瘤小鼠， 解剖取出皮下肿瘤组织， 用剪刀剪成 1 mm3左右的小块， 用套管针接种于小鼠左侧腋窝皮下， 待肿瘤生长至 100-150mm3左右， 将动物随机分组 (d0)。 LLDT-67 1.0、 2.0、 4.0 mg/kg, 口服给药,每天 1 次， 每周 5 d, 连续 3周； 阳性对照丝裂霉素 （MMC ) 5 mg/kg, 静脉 注射， 于第 1 天给药 1 次。 每周 2 次称鼠重并测量瘤体积 (tumor volume , TV), TV = l/2xaxb2, 其中 a、 b分别表示长、 宽。 根据测量的 结果，计算相对肿瘤体积（relative tumor volume, RTV ), RTV = Vdt/Vd。 ( VdQ为 dQ天测量时肿瘤体积， Vdt为给药 dt天测量时的肿瘤体积）。 抗肿瘤活性的评价指标为相对肿瘤增殖率 T/C ( % ), T/C ( % ) = TRTV/CRTVx100%„ 结果判定标准： T/C ( % ) <40%, 有效； T/C ( % ) >40%, 无效。 另外， 可通过小鼠行为学、 二便及体重变化初步判定化合物毒性情况。 结果 LLDT-67 1 mg/kg, po 6 6 19.7 21.0 130 ± 2145 ± 17.9 ± 99.0 > 0.05 l-5/2.5w 36 1227 9.1 Experimental Example 11 LLDT-67 was subcutaneously transplanted into human liver cancer cell SMMC-7721 Tumor growth inhibition Methods The subcutaneous xenograft model of mice was used: Human liver cancer SMMC-7721 cells were derived from China Second Military Medical University; BALB/C mice [SPF grade (specific-pathogen free)], female, body weight : 18±2g, provided by Shanghai Institute of Medicine, Chinese Academy of Sciences, certificate number: SCXK (Shanghai) (2004-0002). The tumor-bearing mice were sacrificed. The subcutaneous tumor tissues were dissected and cut into small pieces of about 1 mm 3 with scissors. They were inoculated subcutaneously into the left axilla of the mice with a trocar. The tumors were grown to about 100-150 mm 3 and the animals were randomized. Group (d0). LLDT-67 1.0, 2.0, 4.0 mg/kg, orally, once daily, 5 days per week for 3 weeks; positive control mitomycin (MMC) 5 mg/kg, intravenously, on day 1 Dosing once. The rats were weighed twice a week and measured for tumor volume (TV), TV = l/2xaxb 2 , where a and b represent length and width, respectively. Based on the measured results, the relative tumor volume (RTV), RTV = V dt /V d , was calculated. (V dQ is the tumor volume measured on d Q days, and V dt is the tumor volume measured on the day of administration dt). The evaluation index of antitumor activity is relative tumor proliferation rate T/C (%), T/C (%) = TRTV/CRTV x 100% „ Result judgment standard: T/C (%) <40%, effective; T/ C (%) >40%, ineffective. In addition, the toxicity of the compound can be preliminarily determined by mouse behavior, second stool and weight change.
LLDT-67在 1.0 、 2.0、 4.0 mg/kg的剂量时对人肝癌 SMMC-7721 棵小鼠皮下移植瘤无生长抑制作用； 另外， 在所用剂量及给药方式下， LLDT-67 对棵小鼠无明显的毒副作用 （见表 11、 图 11和 12)。 表 11 : LLDT-67对人肝癌细胞 SMMC-7721棵小鼠皮下移植瘤的 生长抑制作用 动物数 体重 (g) TV (i nm3) LLDT-67 had no growth inhibitory effect on human liver cancer SMMC-7721 mouse subcutaneous xenografts at doses of 1.0, 2.0, 4.0 mg/kg. In addition, LLDT-67 was administered to mice at the dose and mode of administration. No obvious side effects (see Table 11, Figures 11 and 12). Table 11: Growth inhibition of LLDT-67 on subcutaneous xenografts of human hepatoma cell line SMMC-7721 in mice. Body weight (g) TV (i nm 3 )
剂量、给药方 · T/C 组别 开 最 开 最 RTV P值 式 do d2i (%) Dosage, dosage, T/C group, the most open RTV P value do d 2 i (%)
始 后 始 后  After the beginning and after
0.4ml/只 115士 1158士 10.7士 0.4ml / only 115 1158 ± 10.7
溶剂对照 po 12 12 20.9 25.8 Solvent control po 12 12 20.9 25.8
l-5/3w 31 256 3.1  L-5/3w 31 256 3.1
5 g/kg, 111士 472士 4.2士 5 g/kg, 111 士 472士 4.2士
MMC iv 6 6 21.8 23.7 39.0 <0.05 dl/3w 14 189 1.4  MMC iv 6 6 21.8 23.7 39.0 <0.05 dl/3w 14 189 1.4
4mg/kg, 111士 1166士 10.4士 4mg/kg, 111士 1166士 10.4士
LLDT-67 po 6 6 20.5 21.8 98.0 >0.05 l-5/3w 19 413 3.0  LLDT-67 po 6 6 20.5 21.8 98.0 >0.05 l-5/3w 19 413 3.0
2mg/kg, 110士 1566士 14.6士 2mg/kg, 110 士 1566士 14.6士
LLDT-67 po 6 6 18.3 25.7 137.0 >0.05 l-5/3w 13 380 5.2  LLDT-67 po 6 6 18.3 25.7 137.0 >0.05 l-5/3w 13 380 5.2
1 mg/kg, 114士 1641士 15.1±7 1 mg/kg, 114 ± 1641 ± 15.1 ± 7
LLDT-67 po 6 6 20.3 25.8 142.0 >0.05 l-5/3w 20 566 .1 实验实施例 12 LLDT-67的初步药物代谢动力学研究 方法 釆用雄性 Sprague-Dawley大鼠 （体重约 250 g )开展药代动力学实 验，其中 3只大鼠灌胃口服 LLDT-67( 4 mg/kg ) ,另外 3只大鼠静注（ bolus dosing ) LLDT-67 ( 2 mg/kg )。 给药后按 0、 5、 15、 30 min、 1、 1.5、 2、 5、 及 8 h的时间点釆血样， 肝素抗凝得血浆样品， 应用液相 -质语  LLDT-67 po 6 6 20.3 25.8 142.0 >0.05 l-5/3w 20 566 .1 Experimental Example 12 Preliminary pharmacokinetic study of LLDT-67 was performed using male Sprague-Dawley rats (body weight approximately 250 g) In the pharmacokinetic experiment, 3 rats were orally administered with LLDT-67 (4 mg/kg), and 3 rats were injected with bolus dosing LLDT-67 (2 mg/kg). Blood samples were taken at 0, 5, 15, 30 min, 1, 1.5, 2, 5, and 8 h after administration. Heparin anticoagulated plasma samples, using liquid-quality
( LC/MS )联用技术测定血浆中的 LLDT-67的浓度，釆用非房室模型方 法计算相关药动学参数 (所得参数见表 12)。 表 12 大鼠给药 LLDT-67后的药动学参数（用非房室模型分析） (LC/MS) was used to determine the concentration of LLDT-67 in plasma, and the pharmacokinetic parameters were calculated using a non-compartmental model method (see Table 12 for the parameters obtained). Table 12 Pharmacokinetic parameters after administration of LLDT-67 in rats (analysis by non-compartmental model)
Figure imgf000054_0001
Figure imgf000054_0001
结果 大鼠上进行的药代动力学研究结果表明， 口服给药 (4mg/kg)后， LLDT-67在胃肠道中吸收较快， 15 min达峰。 其平均总暴露水平 ^t/C 为 59.3 ng，h/mL。 LLDT-67 的大鼠平均口服生物利用度约为 7%。 静脉 注射给药后， LLDT-67 在大鼠的稳态表观分布体积为 2.14-2.60 L/kg; 血浆总清除率为 4.424.89 L/h/kg。 静脉注射给药后， LLDT-67 在大鼠 体内的消除半衰期约为 0.5 h。 LLDT-67可进行口服给药或静注给药。 实验实施例 13 LLDT-67初步急性毒性研究 方法  Results The pharmacokinetic study performed on rats showed that LLDT-67 absorbed faster in the gastrointestinal tract after oral administration (4 mg/kg) and peaked at 15 min. The average total exposure level ^t/C was 59.3 ng, h/mL. The average oral bioavailability of rats in LLDT-67 was approximately 7%. After intravenous administration, the steady-state apparent volume of LLDT-67 in rats was 2.14-2.60 L/kg; the total plasma clearance was 4.424.89 L/h/kg. After intravenous administration, the elimination half-life of LLDT-67 in rats was approximately 0.5 h. LLDT-67 can be administered orally or intravenously. Experimental Example 13 LLDT-67 preliminary acute toxicity study method
ICR小鼠，体重 18-20克， 购自中英合资西普尔 -必凯实验动物有 限公司， 实验动物生产许可证号： SCXK(^)2003-0002。 LLDT-67混悬 于 0.2% HPMC中 (续碾磨）。 实验动物分为 10 mg/kg、 20 mg/kg、 30 mg/kg三个给药组， 每组 4只， 雌、 雄各半。 单次灌胃给药后观察死亡 率及毒性情况， 共观察 14天。 结果 ICR mice, weighing 18-20 grams, were purchased from Sino-British joint venture Sipper-Bikie Laboratory Animal Co., Ltd., Laboratory Animal Production License No.: SCXK(^) 2003-0002. LLDT-67 suspension In 0.2% HPMC (continued milling). The experimental animals were divided into three groups of 10 mg/kg, 20 mg/kg and 30 mg/kg, with 4 rats in each group, half male and half female. Mortality and toxicity were observed after single intragastric administration for a total of 14 days. result
10 mg/kg和 20 mg/kg的给药组， 未见小鼠死亡， 30 mg/kg的给药 组， 死亡率为 25% ( 1/4, 第 5天死亡）。在本实验条件下， LLDT-67 0% 死亡剂量（LD。）为 20 mg/kg。 In the 10 mg/kg and 20 mg/kg administration groups, no mice died, and the 30 mg/kg administration group had a mortality rate of 25% (1/4, died on the 5th day). Under the experimental conditions, the 0% mortality dose (LD.) of LLDT-67 was 20 mg/kg.
产业上利用的可能性 本发明的雷公藤二萜类内酯衍生物，特别是 (14S)-14-脱羟基 -14,21- 环氧雷公藤内酯醇 (LLDT-67), 明显抑制多种体外培养的肿瘤细胞的生 长， 抑制作用具有明显的剂量依赖性， 其 IC5。在 0.10-5.99 μΜ之间， 显示 LLDT-67具有很强的体外抗肿瘤活性。 进一步的体内药效实验证 明 LLDT-67 对于人前列腺癌 PC-3 棵小鼠皮下移植瘤和人卵巢癌 SK-OV-3 棵小鼠皮下移植瘤的生长具有明显的抑制作用， 并能一定程 度地抑制人前列腺癌 DU-145 棵小鼠皮下移植瘤生长， 对人胃癌 SGC-7901棵小鼠皮下移植瘤的生长则仅在高浓度（4mg/kg )下有较弱 的抑制作用， 而对人肝癌 SMMC7721 棵小鼠皮下移植瘤和人乳腺癌 MDA-MB-468棵小鼠皮下移植瘤的生长则基本无抑制作用。 上述体内 药效实验中均未表现出明显的毒副反应。 表明 LLDT-67能够特异性地 抑制生殖系统肿瘤的生长。 对 LLDT-67的药物代谢动力学研究表明其 可进行口服或静脉注射给药。 初步急性毒性实验表明 LLDT-67 ( LD0 约为 20 mg/kg )毒性远远低于其先导化和物 LLDT-2( LD5。为 0.5 mg/kg 左右 )。 所有的实验结果均表明 LLDT-67在治疗肿瘤疾病方面具有高效、 低毒、 特异性强的特点， 具有良好的用于治疗包括前列腺癌、 人卵巢 癌的等生殖系统肿瘤疾病的应用前景。 Industrial Applicability The Tripterygium diterpene lactone derivative of the present invention, particularly (14S)-14-dehydroxy-14,21-epoxytriptolide (LLDT-67), significantly inhibits various The growth of tumor cells cultured in vitro has a significant dose-dependent inhibition of its IC 5 . Between 0.10 and 5.99 μΜ, LLDT-67 was shown to have potent in vitro antitumor activity. Further in vivo pharmacodynamic experiments have demonstrated that LLDT-67 has a significant inhibitory effect on the growth of human prostate cancer PC-3 subcutaneous xenografts and human ovarian cancer SK-OV-3 subcutaneous xenografts, and can be somewhat Inhibition of subcutaneous xenograft growth in human prostate cancer DU-145 mice, the growth of subcutaneous xenografts in human gastric cancer SGC-7901 mice was only weakly inhibited at high concentrations (4 mg/kg), but The growth of subcutaneous xenografts of mouse liver cancer SMMC7721 mice and human breast cancer MDA-MB-468 mice had no inhibitory effect. None of the above in vivo pharmacodynamic experiments showed significant toxic side effects. It is shown that LLDT-67 can specifically inhibit the growth of tumors of the reproductive system. Pharmacokinetic studies of LLDT-67 indicate that it can be administered orally or intravenously. Preliminary acute toxicity experiments showed that LLDT-67 (LD 0 is about 20 mg/kg) is much less toxic than its lead LLDT-2 (LD 5 , about 0.5 mg/kg). All the experimental results show that LLDT-67 has high efficiency, low toxicity and specificity in the treatment of tumor diseases, and has a good application prospect for the treatment of neoplastic tumor diseases including prostate cancer and human ovarian cancer.

Claims

权 利 要 求 Rights request
1、 通式 (1)所示的雷公藤二萜类内酯衍生物、 其光学异构体及其药 学上可接受的盐和水合物： 1. A tripterygium diterpene lactone derivative represented by the formula (1), an optical isomer thereof, and a pharmaceutically acceptable salt thereof and a hydrate thereof:
Figure imgf000057_0001
Figure imgf000057_0001
(1) 其中， c5和 c6以碳碳单键或碳碳双键相连； 当 C5和 C6以碳碳单键相连时， P和 Q分别表示连接在 C5位和 C6 位上的氢、 氧、 羟基、 卤素、 巯基、 C1〜C6烷氧基、 C1〜C6烷胺基或 C1〜C6烷巯基； 当 C5和 C6以碳碳双键相连时， 此时 C5除与 C4、 C6以及 C10连接 外无其他取代基团， P不代表任何取代基团， C6位上取代基 Q则仅代 表氢原子； (1) wherein, c 5 and c 6 are bonded by a carbon-carbon single bond or a carbon-carbon double bond; when C 5 and C 6 are connected by a single carbon-carbon bond, P and Q are respectively attached to the C 5 position and the C 6 position. Hydrogen, oxygen, hydroxyl, halogen, sulfhydryl, C1 to C6 alkoxy, C1 to C6 alkylamino or C1 to C6 alkyl fluorenyl; when C 5 and C 6 are bonded by a carbon-carbon double bond, then C 5 There are no other substituent groups except for the linkage with C 4 , C 6 and C 10 , P does not represent any substituent group, and the substituent Q at the C 6 position represents only a hydrogen atom;
C14XY表示 C14位处的结构是 C 14CH20、 C14(OH )CH2OH、 C 14OS(0)OCH2 C 14 XY indicates that the structure at the C 14 position is C 14CH 2 0, C 14 (OH )CH 2 OH, C 14 OS(0)OCH 2
I I I I
或 C14OS02OCH2 ; w和 z分别表示连接在 c12位和 c13位上的氧、羟基、 卤素、巯基、Or C 14 OS0 2 OCH 2 ; w and z respectively represent an oxygen, a hydroxyl group, a halogen, a thiol group attached at the c 12 position and the c 13 position,
C1〜C6烷氧基、 C1〜C6烷胺基或 C1〜C6烷巯基； 在上式中，连接 X、 Y Z W Ρ和 Q的' '代表 "―"或者' a C1-C6 alkoxy group, a C1 to C6 alkylamino group or a C1 to C6 alkyl fluorenyl group; In the above formula, the connection of X, YZW Ρ and Q 'represents' - "or"
2、 如权利要求 1所述的雷公藤二萜类内酯衍生物、 其光学异构体 及其药学上可接受的盐和水合物， 其具有以下通式 (2)所示结构， The tripterygium diterpene lactone derivative according to claim 1, an optical isomer thereof, and a pharmaceutically acceptable salt thereof and hydrate thereof, which has a structure represented by the following formula (2),
Figure imgf000058_0003
Figure imgf000058_0003
Figure imgf000058_0001
Figure imgf000058_0001
(2) 其中， C14XY为（14S)-14,21-环氧结构, W与 Z为分别连接在 C12 和 C13位的 a(S)构型的氧， P为 a(R)构型的氢或羟基， Q为氢。 (2) where C 14 XY is a (14S)-14,21-epoxy structure, W and Z are oxygen in the a(S) configuration of C 12 and C 13 positions, respectively, and P is a(R) Hydrogen or hydroxyl in configuration, Q is hydrogen.
3、 如权利要求 1所述的雷公藤二萜类内酯衍生物、 其光学异构体 及其药学上可接受的盐和水合物， 其具有以下通式 (3)所示结构， The tripterygium diterpene lactone derivative according to claim 1, an optical isomer thereof, and a pharmaceutically acceptable salt thereof and hydrate thereof, which has a structure represented by the following formula (3),
Figure imgf000058_0002
Figure imgf000058_0002
(3) 其中， C14XY为（14S)-14,21-环氧结构, W为 P(R)构型的卤素， Z 为 a(R)构型的羟基， P为 a(R)构型的氢或羟基， Q为氢。 4、 如权利要求 1所述的雷公藤二萜类内酯衍生物、 其光学异构体 及其药学上可接受的盐和水合物， 其具有以下通式 (4)所示结构， (3) wherein C 14 XY is (14S)-14,21-epoxy structure, W is halogen in P(R) configuration, Z is hydroxyl group in a(R) configuration, and P is a(R) structure Type of hydrogen or hydroxyl, Q is hydrogen. The tripterygium diterpene lactone derivative according to claim 1, an optical isomer thereof, and a pharmaceutically acceptable salt thereof and hydrate thereof, which has a structure represented by the following formula (4),
Figure imgf000059_0001
Figure imgf000059_0001
(4) 其中， C14XY为（14S)-14,21-环氧结构， W与 Z为分别连接在 C 和 C 13位的 a(S)构型的氧， C5和 C6以碳碳双键相连， Q为氢。 (4) where, C 14 XY is (14S) -14,21- epoxy structure, W and Z are connected to C 13 and C at position a (S) configuration oxo, C 5 and C 6 to C The carbon double bonds are connected and Q is hydrogen.
5、 如权利要求 1所述的雷公藤二萜类内酯衍生物、 其光学异构体 及其药学上可接受的盐和水合物， 其具有以下通式 (5)所示结构， The tripterygium diterpenoid lactone derivative according to claim 1, an optical isomer thereof, and a pharmaceutically acceptable salt thereof and hydrate thereof, which has a structure represented by the following formula (5),
Figure imgf000059_0002
Figure imgf000059_0002
(5) 其中， C14XY为（14R)-14,21-环氧结构， W与 Z为分别连接在 C 和 C13位的 a(S)构型的氧， P为 a(R)构型的氢或羟基， Q为氢。 (5) where, C 14 XY of (14R) -14,21- epoxy structure, W and Z are connected to C 13 and C at position a (S) -configuration oxygen, P is a (R) configuration Type of hydrogen or hydroxyl, Q is hydrogen.
6、 如权利要求 1所述的雷公藤二萜类内酯衍生物、 其光学异构体 及其药学上可接受的盐和水合物， 其具有以下通式 (6)所示结构， The tripterygium diterpenoid lactone derivative according to claim 1, an optical isomer thereof, and a pharmaceutically acceptable salt thereof and hydrate thereof, which has a structure represented by the following formula (6),
Figure imgf000060_0001
Figure imgf000060_0001
其中， C14XY为 C14(0H )CH20H、 C 14OS(0)OCH2或 C14OS02OCH2 , C14位 的构型为 (R)构型或 (S)构型， W与 Z为分别连接在 C12和 C13位的 a(S) 构型的氧， P为 a(R)构型的氢或羟基， Q为氢。 Wherein C 14 XY is C 14(0H )CH 2 0H, C 14 OS(0)OCH 2 or C 14 OS0 2 OCH 2 , and the configuration at the C 14 position is the (R) configuration or the (S) configuration. W and Z are oxygen attached to the a(S) configuration at the C 12 and C 13 positions, respectively, P being hydrogen or a hydroxyl group in the a(R) configuration, and Q being hydrogen.
7、 如权利要求 1所述的雷公藤二萜类内酯衍生物、 其光学异构体 及其药学上可接受的盐和水合物， 其为选自由以下化合物组成的组中 的一个： The tripterygium diterpene lactone derivative according to claim 1, an optical isomer thereof, and a pharmaceutically acceptable salt and hydrate thereof, which is one selected from the group consisting of:
(14S)-14—脱羟基 -14,21-环氧雷公藤内酯醇 (1 4 S)-1 4 —dehydroxy-14, 2 1-epoxytriptolide
Figure imgf000060_0002
Figure imgf000060_0002
(14R)-14-脱羟基 -14,21-环氧雷公藤内酯醇
Figure imgf000061_0001
(14R)-14-dehydroxy-14,21-epoxytriptolide
Figure imgf000061_0001
(5R,14S)-5a-羟基 -14-脱羟基 -14,21-环氧雷公藤内酯醇
Figure imgf000061_0002
(5R,14S)-5a-hydroxy-14-dehydroxy-14,21-epoxytriptolide
Figure imgf000061_0002
(14S)- \5，6-脱氢 -14-脱羟基 -14,21-环氧雷公藤内酯醇
Figure imgf000061_0003
(14S)- \ 5 , 6 -Dehydro-14-dehydroxy-14,21-epoxytriptolide
Figure imgf000061_0003
(14S)-14P-羟曱基表雷公藤内酯醇 (14S)-14P-hydroxyindole table triptolide
Figure imgf000062_0001
Figure imgf000062_0002
Figure imgf000062_0001
Figure imgf000062_0002
(14S,硫 R)-14a,21-乙二醇环亚硫酸酯雷公藤内酯醇 (14S, sulfur R)-14a, 21-ethylene glycol cyclic sulfite, triptolide
Figure imgf000062_0003
Figure imgf000062_0003
(14S)-14α,21 -乙二醇环硫酸酯雷公藤内酯醇 (14S)-14α,21-ethylene glycol cyclic sulfate triptolide
Figure imgf000062_0004
Figure imgf000062_0004
8、 如权利要求 1所述的雷公藤二萜类内酯衍生物、 其光学异构体 及其药学上可接受的盐和水合物， 其特征在于， 其为（14S)-14-脱羟基 -14,21-环氧雷公藤内酯醇。 The tripterygium diterpene lactone derivative according to claim 1, an optical isomer thereof, and pharmaceutically acceptable salts and hydrates thereof, which are (14S)-14-dehydroxyl -14,21-epoxytriptolide.
9、 一种药物组合物， 其特征在于， 含有治疗有效剂量的权利要求9. A pharmaceutical composition characterized by a therapeutically effective dose
1所述的雷公藤二萜类内酯衍生物、其光学异构体及其药学上可接受的 盐和水合物以及常规的药学辅料。 A tripterygium diterpene lactone derivative, an optical isomer thereof, and pharmaceutically acceptable salts and hydrates thereof, and a conventional pharmaceutical excipient.
10、 如权利要求 9所述的药物组合物， 其特征在于， 所述雷公藤 内酯醇衍生物、 其光学异构体及其药学上可接受的盐和水合物的含量 为 0.001 ~ 99.9wt %„ The pharmaceutical composition according to claim 9, wherein the triptolide derivative, the optical isomer thereof, and a pharmaceutically acceptable salt and hydrate thereof are contained in an amount of 0.001 to 99.9 wt%. „
11、 如权利要求 9所述的药物组合物， 其特征在于， 该药物组合 物进一步包括一种或多种选自影响肿瘤细胞核酸生物合成的药物； 直 接破坏肿瘤细胞 DNA阻止其复制的药物； 嵌入肿瘤细胞 DNA中干扰 转录过程的药物； 干扰有丝分裂影响肿瘤细胞蛋白质合成的药物或通 过抑制环氧化酶产生抗肿瘤作用的药物的抗肿瘤药物。 The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition further comprises one or more selected from the group consisting of a drug that affects nucleic acid biosynthesis of a tumor cell; a drug that directly destroys tumor cell DNA to prevent its replication; A drug that interferes with the transcription process in the DNA of a tumor cell; a drug that interferes with mitosis affecting protein synthesis of a tumor cell or an antitumor drug that produces an antitumor effect by a cyclooxygenase.
12、 如权利要求 9〜11所述的药物组合物， 其特征在于， 所述组合 物被制成经胃肠道给药的剂型或非经胃肠道给药剂型。 The pharmaceutical composition according to any one of claims 9 to 11, wherein the composition is formulated into a gastrointestinal administration form or a parenteral administration form.
13、 权利要求 9〜11任一项所述的药物组合物在制备用于治疗人体 生殖系统肿瘤的药物中的应用。 Use of the pharmaceutical composition according to any one of claims 9 to 11 for the preparation of a medicament for treating a tumor of the human reproductive system.
14、 如权利要求 13所述的应用， 其特征在于， 所述人体生殖系统 肿瘤为前列腺癌或卵巢癌。 14. The use according to claim 13, wherein the human reproductive system tumor is prostate cancer or ovarian cancer.
15、 如权利要求 13所述的应用， 其特征在于， 所述药物组合物中 雷公藤内酯醇衍生物、 其光学异构体及其药学上可接受的盐以 0.001 ~ 10mg/kg的量被使用。 The use according to claim 13, wherein the triptolide derivative, the optical isomer thereof and the pharmaceutically acceptable salt thereof in the pharmaceutical composition are in an amount of 0.001 to 10 mg/kg. use.
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