1. A method of treating rheumatoid arthritis in a patient comprising administering a therapeutically effective amount of compound of formula I



Formula (I) t o a patient in need of rheumatoid arthritis treatment wherein the compounds of Formula I are selected from the group consisting of



and wherein the compound of formula (I) are formulated in the form of injections, tablet, or capsule and administered in a therapeutically effective amount to reduce a content of TNF-α, PGE2 and IL-1β.

BACKGROUND OF THE INVENTION

[0001]Technical Field

[0002]The present invention belongs to the field of drug technology, specifically relates to the uses of sesquiterpene lactone compounds as the effective components or pharmaceutical composition in preparing drugs, especially the uses in preparing the drugs to treat rheumatoid arthritis and treat cancers through inhibiting cancer stem cells.

[0003]Description of Related Art

[0004]The rheumatoid arthritis (RA) is a chronic, inflammatory and systematic autoimmune disease. In addition, the tumors greatly threat the health of human. There are approximately two million of cancer patients in China currently and 1.6 million of cases are emerging every year, which is a rather huge number. RA is a progressive and multi-joint inflammatory systemic autoimmune disease, which mainly shows inflammatory hyperplasia of synovium, mononuclear cell infiltration and neovascularization. There is no therapeutic solution and preventive measures for radical cure of these diseases yet and the main drugs prescribed clinically include non-steroidal anti-inflammatory drugs and adrenal cortical hormones, etc. However, these drugs have severe side effects to result in damages to liver and kidney as well as pulmonary fibrosis. Therefore, it is difficult for the patients to adhere to long-term medication. Thus, our purpose is to find an effective and safe medicine for the treatment.

[0005]So far, there is no report concerning the application of the compounds of Formula (I) and their pharmaceutical compositions in preparing drugs to cure rheumatoid arthritis.

[0006]The tumors greatly threat the health of human. There are approximately two million of cancer patients in China currently and 1.6 million of cases are emerging every year, which is a rather huge number. Therefore, the anti-tumor research is a very challenging field, but with great significance to the present life science. The therapeutic methods in the past focus on eradicating and killing cancer cells and the anti-tumor drugs often used clinically are mainly cytotoxic agents so far. However, these anti-cancer drugs have the demerits such as poor selectivity, strong toxic side effects and tending to result in drug resistance, so they are typical double-sided drugs and it is difficult with them to eradicate cancer and a high ratio of some cancers tend to recur. High recurrence rate of malignant tumors is always a challenge annoying the oncologists. More and more studies have demonstrated that there are a small number of tumor stem cells in the tumor cell population, which can amplify the population. They are usually at the slow cycle state with low sensitivity to chemotherapy drugs and are the origins of tumor recurrence. Thus, the discovery of tumor stem cell provides a new target for tumor treatment and the drug research against tumor stem cell provides the possibility to completely heal cancer.

[0007]So far, there is no report concerning the application of the compounds of Formula (I) and their pharmaceutical compositions in treating cancer through inhibiting cancer stem cells.

BRIEF SUMMARY OF THE INVENTION

[0008]The present invention provides the uses of the compounds of Formula (I) in preparing drugs, especially the uses in preparing the drugs to treat rheumatoid arthritis and treat cancers through inhibiting cancer stem cells.

[0009]

[0010]where: R1 and R2 form double bond together or R1 is hydrogen or deuterium, R2 is

[0011]

and the pharmaceutically acceptable salts formed by it and inorganic acid or organic acid, including the quaternary ammonium salts formed with R5Z, R3 and R4 can be the same or different selected from hydrogen, alkyl,cycloalkyl, hydroxy-substituting alkyl, alkenyl, alkynyl, aryl, alkylaryl, arylalkyl, arylalkenyl, arylalkynyl, heterocyclic, trifluoromethyl, polyfluoroalkyl, nitrile group, cyanomethyl, acyl, carbamoyl, sulfonyl, sulfonamide or aryloxyalkyl. R3, R4 and N atom form cyclic structure which is preferably 3-member to 9-member ring, where one or more positions on the ring structure can be replaced by the substituent group including hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, alkylaryl, arylalkyl, arylalkenyl, arylalkynyl or heterocyclic; Z is fluorine, chlorine, bromine, iodine, toluene-p-sulfonate group, mesylate group, benzenesulfonate group or trifluoromethanesulfonate group. R5 is alkyl, cycloalkyl, hydroxy-substituting alkyl, alkenyl, alkynyl, aryl, heterocyclic, aryl-substituting alkyl, arylalkenyl, arylalkynyl, cyano methyl, alkoxy-substituting alkyl or aryloxy substituting alkyl. Inorganic or organic acid can be hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, carbonic acid, boric acid, selenious acid, phosphomolybdic acid, phosphorous acid, sulfurous acid, citric acid, maleic acid, D-malic acid, L- malic acid, DL- malic acid, L- lactic acid, D- lactic acid, DL- lactic acid, oxalic acid, methanesulfonic acid, pentanoic acid, oleic acid, lauric acid, p-toluenesulfonic, 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, phthalic acid, tartaric acid, malonic acid, succinic acid, fumaric acid, glycolic acid, a thiol acid, glycine, sarcosine, sulfonic acid, nicotinic acid, picoline acid, isonicotinic, dichloroacetic acid, benzoic acid or substituted benzoic acid. Or R2 is

[0012]

where X thereof is O or S, R6 is hydrogen, alkyl, cycloalkyl, hydroxy-substituting alkyl, alkenyl, alkynyl, aryl, alkylaryl, arylalkyl, arylalkenyl, arylalkynylp, heterocyclic, trifluoromethyl, polyfluoroalkyl, nitrile group, cyanomethyl, acyl, carbamoyl, sulfonyl, sulfonamide or aryloxyalkyl.

[0013]If there is no bond - - - between R7 and R8, R7 and R8 will form double bond together or R7 is methyl, R8 is hydroxyl or OCOR9, where R9 thereof is alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic.

[0014]If the bond - - - between R7 and R8 is single bond, R7═R8=methylene.

[0015]R10 is hydrogen or R10 and R8 form single bond.

[0016]R11 is hydrogen or R11 and R13 form single bond or epoxy bond.

[0017]If these is no bond - - - between R12 and R13, R12 and R13 will form double bond together or R12 is hydroxyl or OR14, where R14 thereof is alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, R13 is methyl.

[0018]If the bond - - - between R12 and R13 is single bond, R12═R13=methylene.

[0019]The structural formula (I) is preferably.

[0020]

[0021]The uses of the above-mentioned compounds in preparing the drugs to treat rheumatoid arthritis.

[0022]The uses of the above-mentioned compounds in preparing the adjuvant drugs to treat rheumatoid arthritis.

[0023]The uses of the above-mentioned compounds in preparing the drugs to treat cancers through inhibiting cancer stem cells, where the cancers thereof are preferably acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, skin cancer, breast cancer, ovarian cancer, brain tumor, prostate cancer, head and neck squamous cell carcinoma, laryngeal cancer, pancreatic cancer, retinoblastoma, children hepatoblastoma, liver cancer, malignant melanoma, colorectal cancer, colon cancer, glioma, gastrointestinal tumor, nasopharyngeal carcinoma, brain glioma, gastric cancer, lung adenocarcinoma and lung cancer.

[0024]The uses of the above-mentioned compounds in preparing the adjuvant drugs to treat cancers through inhibiting cancer stem cells, where the cancers thereof are preferably acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, skin cancer, breast cancer, ovarian cancer, brain tumor, prostate cancer, head and neck squamous cell carcinoma, laryngeal cancer, pancreatic cancer, retinoblastoma, children hepatoblastoma, liver cancer, malignant melanoma, colorectal cancer, colon cancer, glioma, gastrointestinal tumor, nasopharyngeal carcinoma, brain glioma, gastric cancer, lung adenocarcinoma and lung cancer.

[0025]The present invention also provides a pharmaceutical composition comprising at least one compound according to any claim of claim 1 to claim 2 as the active component and a pharmaceutically acceptable carrier or other compound(s) to treat rheumatoid arthritis.

[0026]The present invention also provides a pharmaceutical composition comprising at least one compound according to any claim of claim 1 to claim 2 as the active component and a pharmaceutically acceptable carrier or other compound(s) to treat cancer.

[0027]If the compounds according to the present invention are used as drugs, they can be directly used or in the form of pharmaceutical composition. This pharmaceutical composition comprises the compound according to the present invention with a content of 0.1-99%, preferably 0.5-90% and other pharmaceutical carrier and/or excipient which is pharmaceutically acceptable, nontoxic to human and animal and inert, or is a drug combination with other drug(s) to treat rheumatoid arthritis. The composition according to the present invention can be prepared into the form of injection, tablet or capsule, etc.

[0028]Said pharmaceutical carrier or excipient is one or more solids, semi-solids and liquid diluents, fillers and pharmaceutical adjuvants. The pharmaceutical composition according to the present invention is used in the form of dose per unit body weight. The drugs according to the present invention can be delivered in two forms as injection and oral administration, for example, the former can be intravenous and intramuscular injection and the dosage form of the latter can be tablet and capsule.

DETAILED DESCRIPTION OF THE INVENTION

[0029]In order to understand the present invention, the following embodiments are used to further describe the present invention. Nevertheless, it is not to limit the protection scope of the present invention.

[0030]Embodiment 1: Preparation Method of Compound 1-50

[0031]Preparation of Compound 1:

[0032]Dissolve the parthenolide (50 mg, 02 mmol) in 2.5 mL CH2Cl2 and add p-toluenesulfonic acid (5 mg, 0.026 mmol). Place the reaction system at room temperature and stir it overnight. Transfer the reaction solution into the saturated solution of NaHCO3 (10 mL), collect the organic phase and extract the aqueous phase with a small amount of CH2Cl2, then mix the organic phases together and dry with Na2SO4 before filtering it. Distill the organic solvent at reduced temperature with rotary evaporator, then purify it with silica gel column to get Compound 1 (45 mg, yield is 90%). 1H NMR (CDCl3, 400 MHz) δ 6.20 (d, J=3.2 Hz, 1H) 5.49 (d, J=32 Hz, 1H) 3.81 (t, J=10.4 Hz, 1H), 270 (d, J=10.4 Hz, 1H), 2.65-2.62 (m, 2H), 2.40-2.34 (m, 1H), 2.07-2.26 (m, 4H), 1.73-1.86 (m, 2H), 1.68 (s, 3H), 1.36-1.28 (m, 4H); 13C NMR (CDCl3, 100 MHz) δ 169.8, 138.7, 131.7, 130.8, 119.5, 84.1, 80.2, 58.5, 49.5, 382, 34.8, 30.0, 25.7, 23.9, 23.6.

[0033]Common Preparation Method of Compounds 2-20:

[0034]

[0035]Under the protection by nitrogen gas and in ice-water bath, drip the acyl chloride (RCl) into the mixture of Compound 1 (24.8 mg, 0.1 mmol, DMAP (1.25 mg, 0.01 mmol) and trimethylamine (0.12 mL, 12 mmol), once the dripping ends, remove the ice-water bath and stir the system at room temperature until TLC test confirms the reaction ends. Pour the reaction mixture into ice water and extract with ethyl acetate (5 mL×3), wash the organic layer with citric acid solution (20 mL), saturated NaHCO3 (10 mL) and saturated saline solution (10 mL) in turn. Dry the organic phase with anhydrous sodium sulfate and concentrate it under reduced pressure to yield the crude product, then separate it with silica gel column chromatography to get pure product.

[0036]The yield of Compound 2 is 62.8%. 1H NMR(400 MHz, CDCl3z)δ 6.13(1H, d, J=3.1 Hz), 5.40(1H, d, J=2.8 Hz) 3.72(1H, t, J=10.2 Hz) 3.08(1H, d, J=10.1 Hz) 2.65˜2.59(1H, m) 2.41˜2.35(2H, m), 2.21˜2.13(4H, m), 2.04˜2.00(1H, m), 1.97(3H, s), 1.91˜1.83(1H, m), 1.65(3H, s), 1.48(3H, s); 13C NMR(100 MHz, CDCl3)δ 169.6, 169.3, 138.4, 130.6, 129.4, 117.8, 87.7, 82.0, 55.6, 49.1, 35.4, 33.9, 29.4, 24.9, 232, 21.5, 17.8.

[0037]The yield of Compound 3 is 70.0%, 1H NMR (400 MHz, CDCl3) δ6.20 (1H, d, J=3.1 Hz), 5.47 (1H, d, J=2.8 Hz), 3.79 (1H, t, J=10.1 Hz), 3.13 (1H, d, J=10.2 Hz), 2.71˜2.66 (1H, m), 2.49˜2.42 (2H, m), 2.38˜2.30 (2H, m), 2.28˜2.26 (4H, m), 2.11˜2.08 (1H, dd, J=13.8, 1.8 Hz), 1.97˜1.89 (1H, m), 1.72 (3H, s), 1.57 (6H, d, J=9.0 Hz); 13C NMR (100 MHz, CDCl3) δ173.8, 170.2, 139.5, 131.5, 130.4, 118.6, 88.4, 83.0, 56.6, 50.1, 36.5, 34.9, 30.4, 28.7, 25.9, 24.1, 18.8, 9.1.

[0038]The yield of Compound 4 is 30.0%, 1H NMR (400 MHz, CDCl3)δ6.22 (1H, d, J=2.7 Hz), 5.49 (1H, d, J=2.1 Hz), 3.81 (1H, t, J=10.1 Hz), 3.16 (1H, d, J=103 Hz), 2.52˜2.44 (2H, m), 236˜2.23 (6H, m), 2.13˜2.10 (1H, m), 1.99˜1.91 (1H, m), 1.74 (3H, s), 1.70˜1.68 (1H, m), 1.65 (3H, s), 1.57 (2H, s), 0.98 (3H, t, J=73 Hz); 13C NMR (100 MHz, CDCl3)δ173.2, 170.3, 139.5, 131.6, 130.5, 118.7, 88.5, 83.0, 56.8, 50.2, 37.4, 36.6, 35.0, 30.5, 25.9, 242, 18.8, 18.5, 13.6.

[0039]The yield of Compound 5 is 37.6%, 1H NMR (400 MHz, CDCl3) δ6.19 (1, d, J=3.1 Hz), 5.47 (1H, d, J=2.9 Hz), 3.78 (1H, t, J=10.1 Hz), 3.13 (1H, d, J=10.0 Hz), 2.72˜2.66 (1H, m), 2.49˜2.42 (2H, m), 2.37˜2.31 (1H, m), 2.30˜2.26 (4H, m), 2.12˜2.08 (1H, dd, J=13.7, 2.2 Hz), 1.97˜1.88 (1H, m), 1.75 (3H, s), 1.64˜1.56 (2H, m), 1.55 (3H, s), 1.40˜131 (3H, m), 0.92 (3H, t, J=73 Hz); 13C NMR (100 MHz, CDCl3) δ173.4, 1702, 139.5, 131.6, 130.4, 118.7, 88.5, 83.0, 56.8, 50.2, 36.5, 352, 34.9, 30.5, 27.1, 25.9, 24.1, 222, 18.8, 13.8.

[0040]The yield of Compound 6 is 26.8%, 1H NMR (400 MHz, CDCl3) δ6.21 (1H, d, J=3.0 Hz), 5.48 (1H, d, J=2.8 Hz), 3.81 (1H, t, J=10.1 Hz), 3.15 (1H, d, J=10.3 Hz), 2.74˜2.68 (1H, m), 2.57˜2.44 (3H, m), 2.30˜229 (3H, m), 2.14˜2.10 (1H, dd, J=13.7, 2.1 Hz), 1.98˜1.90 (1H, m), 1.74 (3H, s), 1.66 (1H, s), 1.56 (3H, s), 1.21˜1.17 (6H, m); 13C NMR (100 MHz, CDCl3) δ176.6, 170.2, 139.6, 131.5, 130.4, 118.6, 882, 83.0, 56.8, 50.1, 36.5, 35.0, 34.7, 30.5, 26.0, 24.2, 19.0, 18.9, 18.7.

[0041]The yield of Compound 7 is 47.8% 1H NMR (400 MHz, CDCl3) δ6.19 (1H, d, J=3.2 Hz), 5.46 (1H, d, J=2.9 Hz), 3.78 (1H, t, J=10.1 Hz), 3.13 (1H, d, J=10.1 Hz), 2.71˜2.66 (1H, m), 2.50˜2.42 (2H, m), 2.28˜2.26 (3H, m), 2.20˜2.07 (4H, m), 1.97˜1.89 (1H, m), 1.72˜1.69 (4H, m), 1.56 (3H, s), 0.97 (6H, d, J=5.9 Hz); 13C NMR (100 MHz, CDCl3) δ172.7, 170.2, 139.5, 131.6, 130.5, 118.7, 88.5, 83.0, 56.8, 502, 44.6, 36.6, 35.0, 30.5, 25.9, 25.8, 24.2, 22.4, 22.3, 18.8.

[0042]The yield of Compound 8 is 13.9% 1H NMR (400 MHz, CDCl3) δ6.20 (1H, d, J=2.9 Hz), 5.47 (1H, d, J=2.4 Hz), 4.07˜3.95 (2H, q, J=16.4 Hz), 3.79 (1H, t, J=10.1 Hz), 3.47 (3H, s), 3.19 (1H, d, J=10.1 Hz), 2.71˜2.66 (1H, m), 2.51˜2.44 (2H, m), 2.27 (3H, s), 2.11˜2.08 (1H, dd, J=12.5, 0.7 Hz), 2.04˜1.95 (1H, m), 1.72 (3H, s) 1.63 (1H, s), 1.58 (3H, s); 13C NMR (100 MHz, CDCl3) δ170.2, 169.7, 139.3, 131.9, 130.0, 118.9, 89.6, 82.9, 70.3, 59.3, 56.4, 50.0, 36.4, 35.0, 30.4, 25.9, 242, 18.9.

[0043]The yield of Compound 9 is 56.2%, 1H NMR (400 MHz, CDCl3) δ6.23 (1H, d, J=33 Hz), 5.50 (1H, d, J=3.1 Hz), 4.14˜4.05 (2H, m), 3.81 (1H, t, J=10.1 Hz), 3.20 (1H, d, J=10.0 Hz), 2.74˜2.68 (1H, m), 2.54˜2.47 (2H, m), 2.30˜2.29 (3H, m), 2.15˜2.11 (1H, dd, J=13.8, 2.3 Hz), 2.07˜1.97 (1H, m), 1.75 (3H, s), 1.62 (4H, s); 13C NMR (100 MHz, CDCl3) δ170.1, 166.4, 139.2, 132.1, 129.8, 119.0, 90.8, 82.8, 56.4, 50.1, 42.0, 363, 35.0, 303, 25.9, 242, 18.8.

[0044]The yield of Compound 10 is 20.7%, 1H NMR (400 MHz, CDCl3) δ6.23 (1H, d, J=3.3 Hz), 5.96 (1H, s), 5.51 (1H, d, J=3.0 Hz), 3.81 (1H, t, J=10.1 Hz), 3.21 (1H, d, J=10.1 Hz), 2.76˜2.70 (1H, m), 2.56˜2.48 (2H, m), 2.30 (3H, s), 2.15˜2.11 (1H, dd, J=13.8, 2.3 Hz), 2.07˜1.99 (1H, m), 1.75 (3H, s), 1.66 (4H, s); 13C NMR (100 MHz, CDCl3) δ169.9, 163.4, 139.2, 132.4, 129.5, 119.0, 923, 82.5, 65.2, 56.5, 50.1, 36.0, 34.9, 302, 25.9, 24.1, 18.6.

[0045]The yield of Compound 11 is 46.5% 1H NMR (400 MHz, CDCl3) δ6.22 (1H, d, J=3.3 Hz), 5.49 (1H, d, J=3.0 Hz), 3.86 (2H, s), 3.80 (1H, J=10.1 Hz), 3.18 (1H, d, J=9.9 Hz), 2.74˜2.68 (1H, m), 2.53˜2.46 (2H, m), 230˜2.29 (3H, m), 2.14˜2.10 (1H, dd, J=13.8, 2.3 Hz), 2.06˜1.94 (1H, m), 1.74 (3H, s), 1.71 (1H, s), 1.60 (3H, s); 13C NMR (100 MHz, CDCl3) δ170.1, 166.3, 139.3, 132.1, 129.9, 118.9, 90.8, 82.8, 56.5, 50.1, 36.2, 34.9, 303, 27.7, 25.9, 24.2, 18.7.

[0046]The yield of Compound 12 is 25.8% 1H NMR (400 MHz, CDCl3) δ6.22 (1H, d, J=3.2 Hz), 5.49 (1H, d, J=2.8 Hz), 3.81 (1H, t, J=102 Hz), 3.47 (2H, t, J=6.5 Hz), 3.15 (1H, d, J=10.0 Hz), 2.73˜2.68 (1H, m), 2.51˜2.44 (2H, m), 2.40˜2.32 (2H, m), 2.30 (2H, d, J=6.4 Hz), 2.14˜2.10 (1H, dd, J=13.7, 1.9 Hz), 1.99˜1.91 (3H, m), 1.83˜1.78 (2H, m), 1.74 (3H, s), 1.60 (3H, s), 1.57 (2H, s); 13C NMR (100 MHz, CDCl3) δ172.5, 170.2, 139.4, 131.7, 130.2, 118.8, 88.75, 83.0, 56.7, 50.1, 36.5, 35.0, 34.5, 33.5, 32.0, 30.5, 25.9, 24.2, 23.6, 18.8.

[0047]The yield of Compound 13 is 47.6% 1H NMR (400 MHz, CDCl3) δ6.22 (1H, d, J=3.3 Hz), 5.49 (1H, d, J=3.0 Hz), 3.81 (1H, t, J=102 Hz), 3.37˜3.33 (2H, m), 3.16 (1H, d, J=10.1 Hz), 2.74˜2.68 (1H, m), 2.51˜2.45 (2H, m), 2.41˜233 (2H, m), 2.31˜2.29 (3H, m), 2.14˜2.10 (1H, dd, J=13.7, 2.2 Hz), 2.00˜1.92 (1H, m), 1.74 (3H, s), 1.73˜1.66 (4H, m), 1.62 (1H, s), 1.57 (3H, s); 13C NMR (100 MHz, CDCl3) δ171.5, 169.2, 138.4, 130.7, 129.2, 117,7, 87.7, 82.0, 55.7, 50.1, 49.1, 35.5, 34.0, 33.9, 29.4, 272, 24.9, 23.1, 212, 17.8.

[0048]The yield of Compound 14 is 78.2%, 1H NMR (400 MHz, CDCl3) δ6.47˜6.37 (1H, dd, J=17.3, 1.3 Hz), 6.21 (1H, d, J=33 Hz), 6.13˜6.06 (1H, m), 5.80˜5.77 (1H, dd, J=10.3, 1.3 Hz), 5.48 (1H, d, J=3.1 Hz), 3.82 (1H, J=10.1 Hz), 3.15 (1H, d, J=10.1 Hz), 2.73˜2.67 (1H, m), 2.58˜2.44 (2H, m), 2.29˜2.27 (3H, m), 2.12˜2.08 (1H, dd, J=13.7, 2.3 Hz), 2.00˜1.92 (1H, m), 1.73 (3H, s), 1.59 (4H, s); 13C NMR (100 MHz, CDCl3) δ170.3, 165.5, 139.4, 131.7, 130.2, 130.1, 130.0, 118.8, 88.8, 83.0, 57.1, 50.1, 36.5, 35.0, 30.5, 25.9, 242, 18.6.

[0049]The yield of Compound 15 is 13.5%, 1H NMR (400 MHz, CDCl3) δ6.22 (1H, d, J=3.3 Hz), 5.49 (1H, d, J=3.0 Hz), 5.20 (1H, d, J=5.0 Hz), 5.16 (1H, s), 3.81 (1H, t, J=10.1 Hz), 3.16 (1H, d, J=8.8 Hz), 3.12˜3.09 (2H, m), 2.74˜2.68 (1H, m), 2.51˜2.44 (2H, m), 230˜2.28 (4H, m), 2.14˜2.10 (1H, dd, J=13.7, 2.1 Hz), 2.00˜1.92 (1H, m), 1.88˜1.86 (1H, dd, J=6.9, 1.3 Hz), 1.74 (3H, s), 1.59 (1H, s), 1.58 (3H, s); 13C NMR (100 MHz, CDCl3) δ171.0, 170.2, 139.5, 131.7, 130.7, 1303, 118.7, 118.2, 89.0, 83.0, 56.7, 50.1, 402, 36.5, 35.0, 30.5, 25.9, 24.2, 18.8.

[0050]The yield of Compound 16 is 26.6%, 1H NMR (400 MHz, CDCl3) δ6.20 (1H, d, J=3.2 Hz), 5.68 (1H, s), 5.47 (H, d, J=2.8 Hz), 3.81 (1H, t, J=10.2 Hz), 3.15 (1H, d, J=10.0 Hz), 2.73˜2.67 (1H, m), 2.57˜2.43 (2H, m), 2.28˜2.27 (3H, m), 2.15 (3H, s), 2.12˜2.08 (1H, dd, J=13.9, 2.1 Hz), 2.00˜1.92 (2H, m), 1.87 (3H, s), 1.73 (3H, s), 1.59 (3H, s); 13C NMR (100 MHz, CDCl3) δ170.3, 166.2, 155.4, 139.5, 131.5, 130.6, 118.7, 117.7, 882, 83.1, 57.0, 503, 36.8, 34.9, 30.6, 27.4, 25.9, 242, 20.1, 18.9.

[0051]The yield of Compound 17 is 23.3%, 1H NMR (400 MHz, CDCl3) δ7.76 (1H, d, J=16.0 Hz), 7.59˜7.57 (2H, m), 7.39 (3H, d, J=5.0 Hz), 6.45 (1H, d, J=16.0 Hz), 6.24 (1H, d, J=3.1 Hz), 5.51 (1H, d, J=2.7 Hz), 3.88 (1H, t, J=10.2 Hz), 3.20 (1H, d, J=10.0 Hz), 2.78˜2.73 (1H, m), 2.65˜2.61 (1H, m), 2.55˜2.48 (1H, m), 2.31 (3H, s), 2.16 (1H, d, J=13.5 Hz), 2.07˜1.98 (1H, q), 1.75 (3H, s), 1.63 (4H, s); 13C NMR (100 MHz, CDCl3) δ170.4, 166.4, 144.5, 139.5, 134.7, 131.6, 130.0, 129.9, 128.8, 128.2, 119.7, 118.8, 88.7, 83.1, 573, 50.0, 36.7, 35.0, 30.6, 26.0, 24.2, 18.6.

[0052]The yield of Compound 18 is 53.3% 1H NMR (400 MHz, CDCl3) δ 6.18 (1H, d, J=3.2 Hz), 5.87˜5.80 (1H, m), 5.46 (1H, d, J=3.0 Hz), 5.09 (1H, d, J=16.9 Hz), 5.00 (1H, d, J=9.6 Hz), 3.80˜3.74 (1H, m), 3.12 (1H, d, J=7.4 Hz), 2.68 (1H, s), 2.45˜2.40 (3H, m), 2.37 (4H, s), 2.25 (3H, s), 2.11˜2.07 (1H, m), 1,95˜1.89 (1H, m), 1.71 (3H, s), 1.54 (3H, d, J=3.8 Hz): 13C NMR (100 MHz, CDCl3) δ172.4, 170.2, 139.5, 136.9, 131.6, 1303, 118.7, 1153, 88.7, 82.9, 56.7, 50.1, 36.5, 35.0, 34.6, 30.4, 29.0, 25.9, 24.2, 18.8.

[0053]The yield of Compound 19 is 65.2%, 1H NMR (400 MHz, CDCl3) δ6.21 (1H, d, J=3.1 Hz), 5.49 (1H, d, J=2.7 Hz), 3.82 (1H, t, J=10.0 Hz), 3.15 (1H, d, J=93 Hz), 2.91˜2.82 (3H, m), 2.14˜2.10 (1H, m), 2.04˜2.01 (3H, m), 1.97˜1.87 (8H, m), 1.83˜1.75 (6H, m), 1.63 (1H, s); 13C NMR (100 MHz, CDCl3) δ172.4, 170.2, 139.4, 131.7, 130.2, 118.8, 88.8, 83.6, 83.0, 69.0, 56.7, 50.1, 36.5, 34.9, 34.1, 30.5, 25.9, 242, 23.7, 18.8, 17.8.

[0054]The yield of Compound 20 is 24.0%, 1H NMR (400 MHz, CDCl3) δ7.32˜7.18 (5H, m), 6.22 (1H, d, J=33 Hz), 5.49 (1H, d, J=3.0 Hz), 3.80 (1H, t, J=10.1 Hz), 3.12 (1H, d, J=10.1 Hz), 2.99 (2H, t, J=7.9 Hz), 2.73˜2.59 (3H, m), 2.49˜2.43 (2H, m), 230˜2.28 (3H, m), 2.14˜2.10 (1H, dd, J=13.8, 2.3 Hz), 1.96˜1.87 (1H, m), 1.74 (3H, s), 1.65 (1H, s), 1.56 (3H, s): 13C NMR (100 MHz, CDCl3) δ172.3, 170.2, 140.8, 139.5, 131.6, 130.4, 128.4, 126.0, 118.7, 88.8, 83.0, 56.7, 50.1, 36.8, 36.5, 35.0, 31.0, 30.5, 26.0, 24.2, 18.8.

[0055]Synthesis of Compound 21:

[0056]

[0057]In one dry and clean reaction flask of 10 mL, add 5-acetylenic acid and Compound 19 (200 mg, 0.584 mmol), 6-azido-1-n-hexanol (125.43 mg, 0.876 mmol), copper sulfate hydrate (145.80 mg, 0.584 mmol) and sodium ascorbate (462.78 mg, 2.336 mmol), then add the mixture solution of distilled water and tert-butyl alcohol (1:2) (3 mL) to dissolve them to homogeneous phase and then stir it for 2 hours at room temperature. Remove partial solvent with rotary evaporator and extract the remaining reaction solution mixture with ethyl acetate, then collect the organic phase and dry with anhydrous sodium sulfate and filter at reduced pressure. Dry the organic phase with rotary evaporator and separate it with silica column chromatography to get Compound 21 (120.0 mg) with a yield of 61.3%. 1H NMR (400 MHz, CDCl3) δ7.52 (1H, s), 6.20 (1H, d, J=3.3 Hz), 5.49 (1H, d, J=3.0 Hz), 4.34 (2H, t, J=7.1 Hz), 3.81 (1H, t, J=10.2 Hz), 3.63 (2H, t, J=6.4 Hz), 3.14 (1H, d, J=10.1 Hz), 2.81˜2.76 (2H, m), 2.74˜2.67 (1H, m), 2.51˜2.43 (2H, m), 239˜231 (2H, m), 2.29˜2.27 (4H, m), 2.13˜2.09 (1H, dd, J=13.8, 2.3 Hz), 2.05˜2.00 (3H, m), 1.97˜1.88 (5H, m), 1.72 (4H, s), 1.60˜1.53 (6H, m); 13C NMR (100 MHz, CDCl3) δ172.6, 170.2, 147.1, 139.4, 131.7, 130.0, 1213, 118.8, 88.6, 83.1, 622, 56.7, 50.0, 49.9, 36.4, 34.9, 34.7, 32.3, 30.4, 30.1, 26.1, 25.8, 25.1, 24.8, 24.7, 24.1, 18.8.

[0058]Preparation of Compound 22:

[0059]

[0060]Mix dimethylamine hydrochloride (1.5 g, 18 mmol) and K2CO3 (5.0 g, 36 mmol) and then add them into 100 ml CH2Cl2 to stir 15 minutes, then filter under pressure and directly add into Compound 5 (300 mg, 12 mmol) and stir for 3 h at room temperature. Remove the solvent at reduced pressure and dissolve it with a few amount of CH2Cl2, wash it three times rapidly with water and dry with Na2SO4 before filer it, then remove the CH2Cl2 at reduced pressure to obtain the crude product—dimethyl amine intermediate. Dissolve it again with a few amount of CH2Cl2 and add the dilute hydrochloric acid solution (equivalent to dimethyl amine intermediate) with stirring where the pH value of aqueous solution shall be tested during stirring and the dripping of hydrochloric acid solution shall be stopped when it becomes 4-5. Collect the aqueous phase and dry it through freezing to get Compound 22.

[0061]Dimethyl amine intermediate: 1H NMR (CDCl3, 400 MHz) δ 3.76 (t, J=10.0 Hz, 1H), 2.96 (s, 1H), 2.49-2.67 (m, 3H), 2.28-2.34 (m, 1H), 2.30-2.34 (m, 2H) 2.18 (s, 6H), 2.09 (br s, 2H), 1.96 (d, J=11.2 Hz, 1H) 1.67-1.73 (m, 2H) 1.60 (s, 3H), 1.22 (br s, 3H), 1.18 (br s, 2H); 13C NMR (CDCl3, 300 MHz) δ 177.0, 131.8, 131.3, 84.0, 80.2, 58.3, 58.1, 50.9, 46.0, 44.6,38.4, 353, 30.0, 272, 23.7, 22.8.

[0062]Compound 22: [α]D20=−42.0 (c=10, H2O); IR (KBr): 3334, 2927, 2856, 1767, 1467, 992, 967, 874, 831, 719, 669, 626, 504 cm-1; 1H NMR (D2O, 400 MHz) δ 4.14 (t, J=10.3 Hz, 1H), 3.51 (q, J=12.6 Hz, 1H), 3.40 (dd, J=13.3, 2.9 Hz, 1H), 3.18-3.04 (m, 1H), 2.96 (d, J=10.6 Hz, 6H), 2.67 (d, J=10.2 Hz, 1H), 2.37 (dd, J=16.2, 8.1 Hz, 1H), 2.27-2.05 (m, 4H), 1.87 (d, J=12.9 Hz, 1H), 1.73 (dd, J=19.5, 11.7 Hz, 2H), 1.66 (s, 3H), 1.46-131 (m, 2H), 1.26 (s, 3H) 13C NMR (CDCl3, 100 MHz) δ 178.4, 132.6, 131.4, 85.1, 80.7, 56.9, 55.6, 49.9, 45.1, 42.3, 41.5, 39.2, 34.4, 29.5, 25.9, 23.2, 21.4. HRMS calcd for C17H27NO3 [M+H]+ 294.1991, found 294.2069.

[0063]Preparation of Compound 23:

[0064]

[0065]Mix dimethylamine hydrochloride (1.5 g, 18 mmol) and K2CO3 (5.0 g, 36 mmol) and then add them into 10 ml CH2Cl2 to stir 15 minutes, then filler under pressure and directly add into Compound 5 (300 mg, 1.2 mmol) and stir for 3 h at room temperature. Remove the solvent at reduced pressure and dissolve it with a few amount of CH2Cl2, then wash it three times rapidly with water and dry with Na2SO4 before filter it. Remove the CH2Cl2 at reduced pressure to yield the crude product—dimethyl amine intermediate, then dissolve it again with a few amount of CH2Cl2 and add fumaric acid (equivalent to dimethyl amine intermediate) with stirring. Then concentrate and dry it to get Compound 23. 1H NMR (DMSO, 400 MHz) δ 6.58 (s, 2H), 3.80 (t, J=10.3 Hz, 1H), 2.64 (s, 3H), 2.49-2.53 (m, 3H), 2.26-2.27 (m, 1H), 2.23 (s, 6H), 1.96-2.10 (m, 6H), 1.60 (s, 3H), 1.57-1.59 (m, 2H), 1.23-1.25 (m, 1H), 1.15 (s, 3H); 13C NMR (CDCl3, 100 MHz) 177.8, 167.6, 1352, 133.7, 131.4, 83.4, 8031, 58.0, 57.1, 51.7, 45.2, 43.6, 41.0, 353, 302, 27.0, 242, 232.

[0066]Preparation of Compound 24:

[0067]Dissolve Compound 1 (628 mg, 12.5 mmol) in 35 ml CH2Cl2 and then add m-CPBA (680 mg, 4.0 mmol). Place and stir the reaction system at room temperature and monitor the reaction with TCL. After the raw materials disappear, pour the reaction mixture into 5% NaHCO3 (60 mL) and wash the organic phase with water (20 ml), then collect the organic phase and dry it with Na2SO4 before filer it, then dry it through rotary evaporation to get the crude product, then purity it with silica gel column to get Compound 24. 1H NMR (CDCl3, 400 MHz) δ 6.17 (d, J=32 Hz, 1H), 5.47 (d, J=2.8 Hz, 11H), 4.04 (t, J=10.8 Hz, 1H, 2.36-2.20 (m, 4H), 2.03-1.08 (m, 4H), 1.68-1.62 (m, 1H), 1.46 (s, 3H), 1.46 (d, J=12.8 Hz, 1H), 1.29 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 169.5, 137.9, 119.5, 81.7, 79.5, 69.7, 62.1, 55.3, 49.2, 37.2, 33.2, 29.3, 23.1, 23.0, 21.8.

[0068]Preparation of Compound 25:

[0069]According to the paper “Hongquan Yin, Xiulan Qi, Huiming Hua, Yuehu Pei, Study on Chemical Ingredients of Aplotaxis Auriculata, Chin J Med Chem, 2005, Vol 15, No. 4, P217-220”, it is produced through separating and purifying from aplotaxis auriculata.

[0070]Preparation of Compound 26 and Compound 30:

[0071]

[0072]Stir the methanol solution of Compound 25 (150 mg, 0.65 mmol) and sodium methoxide (20 mg, 0.37 mmol) at 30° C. for 10 hours. After TLC test is finished, pour the reaction solution into ice water, extract it with ethyl acetate four times and mix the layers of ethyl acetate, then wash it with 5% hydrochloric acid, saturated NaHCO3 aqueous solution and saturated saline solution in turn. Dry with anhydrous magnesium sulfate, then filter, concentrate and purify it with silica gel column to get the methoxylation product (136 mg, 80%).

[0073]1H-NMR (400 MHz, CDCl3) δ 5.19 (d, J=1.6 Hz, 1H) 5.03 (d, J=1.6 Hz, 1H), 4.87 (s, 1H), 4.76 (s, 1H, 3.93 (t, J=9.2 Hz, 1H), 3.70 (dd, J=4.4, 9.8 Hz, H), 3.63 (dd, J=3.2, 9.8 Hz, 1H), 3.37 (s, 3H), 2.90 (m, 1H), 2.83 (m, 1H), 2.51 (m, 2H), 2.49 (m, 1H), 2.44 (m, 1H), 2.38 (m, 1H), 2.19 (m, 1H), 2.07 (m, 1H), 1.95 (m, 1H), 1.86 (m, 1H), 1.32 (m, 1H); 13C-NMR (100 MHz, CDCl3) δ 175.7, 151.8, 149.9, 111.5, 108.8, 85.3, 68.9, 59.0, 51.7, 47.7, 46.9, 43.9, 37.7, 32.5, 32.4, 30.1.

[0074]Add Zn—Cu alloy (812 mg) and anhydrous ether (4 mL) in a dual-port bottle connected with condenser pipe and dry pipe, then add one granule of iodine and stir it until the color of iodine disappears. Add the ether solution (2 mL) of methoxylation product (262 mg, 1 mmol) and diiodomethane (0.8 mL, 10 mmol) and then reflux it for 72 hours. After TLC shows the reaction is completed, pour out the ether solution and wash the residual solid two times with ether (3 mL), mix the ether layers and wash it respectively with saturated NH4Cl aqueous solution and water (10 mL) then dry it with anhydrous sodium sulfate before filter and concentrate. Purify it with chromatography column to get the product which is white solid Compound 30 (206 mg, 71%). 1H-NMR (400 MHz, CDCl3) δ 4.20 (m, 1H), 3.65 (dd, J=3.9, 9.9 Hz, 1H), 3.59 (dd, J=3.0, 9.9 Hz, 1H), 3.34 (s, 1H), 2.30-2.40 (m, 3H), 1.92-2.07 (m, 2H), 1.15-1.70 (m, 7H), 0.85 (m, 1H), 0.70 (m, 1H), 0.50 (m, 1H), 0.40 (m, 1H), 0.16-0.36 (m, 4H); 13C-NMR (100 MHz, CDCl3) δ 176.4, 83.9, 69.2, 59.2, 51.5, 47.9, 473, 443, 36.2, 35.9, 29.0, 273, 26.7, 18.5, 12.5, 10.4, 10.0, 8.8; ESI-HRMS m/z: 291.1964 [M+H].

[0075]Dissolve Compound 30 (0.023 g, 0.080 mmol) in acetonitrile (0.52 mL), then add 4M NaOH aqueous solution (0.11 mL) and reflux 5 hours, after TLC test shows the reaction is completed, cool it to room temperature before add 10% hydrochloric acid to adjust the pH value to 3. Add ethyl acetate (20 mL) and wash twice with water (2×20 mL), then extract the aqueous phase twice with ethyl acetate(2×20 mL), mix the organic layers and dry it with anhydrous sodium sulfate, then filter and concentrate it before separate with chromatography column to get the product which is white solid 26 (0.019 g, 94%).

[0076]

[0077]Molecular formula: C17H22O2

[0078]Molecular weight: 258

[0079]Form: white amorphous powder

[0080]Spectrum data:

[0081]1H-NMR (400 MHz, CDCl3) δ 6.17 (d, J=3.2 Hz, 1H), 5.45 (d, J=3.2 Hz, 1H), 4.24 (dd, J=8.8, 10.8 Hz, 1H), 2.87 (m, 1H), 2.20 (m, 1H), 2.07 (m, 1H), 1.95 (dd, J=8.8, 10.4 Hz, 1H), 1.71 (m, 2H), 1.37-1.61 (m, 5H), 0.98 (m, 1H), 0.64 (m, 1H), 0.49 (m, 1H), 0.43 (m, 1H), 0.27-0.37 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 169.4, 139.4, 118.5, 82.5, 51.6, 47.4, 442, 34.2, 32.8, 26.4, 26.2, 25.3, 17.4, 11.1, 10.7, 10.5, 7.8; ESI-HRMS m/z: 259.1692 [M+H].

[0082]Preparation of Compound 27:

[0083]

[0084]Suspend dimethylamine hydrochloride (245 mg) in 11 mL CH2Cl2 solution, then add K2CO3 (380 mg) and stir 0.5 hour, then add Compound 26 (42 mg) and reflux 4 hours, then separate with silica gel column (petroleum ether ethyl acetate:=70:30) to get the oily Compound 27 with the yield: 78%

[0085]Molecular formula: C19H29NO2

[0086]Molecular weight: 303

[0087]Form: oily liquid

[0088]Spectrum data:

[0089]1H-NMR (400 MHz, CDCl3) δ4.19 (dd, J=9.5, 102 Hz, 1H), 2.66 (dd, J=12.8, 4.8 Hz, 1H), 2.49 (dd, J=12.8, 6.8 Hz, 1H), 2.25-2.38 (m, 2H), 2.21 (s, 6H), 1.98-2.12 (m, 2H), 1.80-1.84 (m, 1H), 1.68 (m, 1H), 1.27-1.48 (m, 5H), 1.11 (m, 1H), 1.01 (m, 1H), 0.69 (m, 1H), 0.46 (m, 1H), 0.18-137 (m, 4H), 0.11 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 181.3, 82.8, 58.0, 50.8, 46.4, 46.0, 45.0, 44.6, 35.2, 34.9, 28.2, 26.3, 25.7, 17.5, 11.5, 9.6, 92, 7.9; ESI-HRMS m/z: 304.2273 [M+H].

[0090]Synthesis Method of Compound 28:

[0091]

[0092]Dissolve Compound 27 (38 mg) in 2 mL CH2Cl2. Slowly add 0.05 M dilute hydrochloric acid with stirring until the pH value becomes 4. Separate the aqueous phase, wash once with CH2Cl2. Freeze, and dry the aqueous phase to get Compound 28 (31 mg) with a yield of 73%.

[0093]Molecular formula: C19H30NO2Cl

[0094]Molecular weight: 339

[0095]Form: White solid

[0096]Spectrum data:

[0097]1H-NMR (400 MHz, D2O) δ 4.42 (m, 1H), 331-337 (m, 1H), 3.21-3.25 (n, 1H), 2.92 (m, 1H), 2.82 (s, 3H), 2.80 (s, 3H), 2.11-2.26 (m, 2H), 1.94 (m, 1H), 1.79 (m, 11H), 1.39-1.58 (m, 5H), 1.23 (m, 1H), 1.13 (m, 1H), 0.69 (m, 1H), 0.46 (m, 1H, 0.18-1.33 (m, 4H), 0.11 (m, 2H); 13C NMR (100 MHz, D2O) 178.2, 85.8, 56.0, 51.0, 47.2, 45.9, 44.8, 42.5, 42.2, 35.7, 35.2, 27.5, 272, 26.6, 183, 12.2, 103, 9.8, 8.6; ESI-HRMS m/z: 304.2271 [M+H].

[0098]Synthesis of Compound 29:

[0099]

[0100]Add 20 mg Compound 26 into the reaction flask and dissolve it with 1 mL THF, then add 86 mg proline ethyl ester hydrochloride and 0.2 mL DBU, stir for 24 hours before purify it with chromatography column to get Compound 29 (12 mg) with a yield: 39%.

[0101]Molecular formula: C24H35NO4

[0102]Molecular weight: 401

[0103]Form: colorless oily compound

[0104]Spectrum data:

[0105]1H NMR (400 MHz, CDCl3): δ 4.10-4.22 (m, 3H) 3.31 (dd, J=8.8, 5.2 Hz, 1H), 3.05 (dd, J=1