CN102731456B - Antitumor compound, its preparation method and application - Google Patents
Antitumor compound, its preparation method and application Download PDFInfo
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Abstract
The invention discloses an antitumor compound 2-(1-hydroxy-4-ketone-2, 5-cyclohexadiene)-pyran-4-ketone, its preparation method and an application. The antitumor compound has structural features as shown in the formula 1. The compound has a structure of alpha, beta-nonsaturated ketone, and has a remarkable effect of inhibiting human gynecologic tumor, prostatic neoplasms, digestive system neoplasms and hematological malignancy without influencing a normal hematopoietic system and a metabolic system. In addition, the compound can effectively inhibit the tumor growth of mice bearing H22 tumor but has no influence on the ratio of peripheral white blood cell to peripheral blood granulocyte to lymphocyte, marrow nucleated cells, the ratio of marrow granulocyte series to erythrocyte series, thymus index and spleen index. The synthetic method of the compound is as follows: the antitumor compound is synthesized by six-step reactions with 2-(2-bromomethyl)-1, 3-dioxolane as a raw material.
Description
Technical field
The invention belongs to field of medicaments, relate to antitumor drug, be specifically related to be used for the treatment of the medicine of Mammals gynecological tumor, tumor of prostate, digestive system tumor and hematological system tumor, metabolic system tumour.
Background technology
The mankind's life in malignant tumour serious threat.Show according to Third National residents result, the mortality ratio that China's tumour causes occupies second in all causes of disease, accounts for 17.9%, and sickness rate is the trend rising year by year.Since nearly 20 years, China's tumor mortality rate has risen 29.42%.In middle prime of life crowd, tumour has been listed as and has occupied first of all kinds of causes of the death.Finding effective cancer therapy drug and method, thoroughly capture cancer, is the important topic of world medical circle research.
Chemotherapy is being played the part of very important effect in the research of oncotherapy.Existing most antitumor drug belongs to cytotoxic drug, is mainly to destroy DNA of tumor cell structure, can make tumour patient survival time extend.Traditional chemotherapeutics comprises alkylating agent, platinum complex, and antimetabolite, affects hormone function anticarcinogen etc.Along with clinical long-term a large amount of use, their drawback also constantly manifests:
(1) resistance: cis-platinum is one of the most frequently used classical medicine of current patients with gastric cancer chemotherapy and the external drug sensitive experiment of cancer of the stomach.Its antitumor toxicity and validity obtain certainly.But the generation of cisplatin resistance has reduced the actual efficacy of cis-platinum in recent years, even cause the failure of chemotherapy of gastric cancer, therefore limited cis-platinum clinical application [referring to Jiang Liai. the progress of cancer of the stomach cisplatin resistance mechanism. practical oncology magazine .2010,24:194-197.].5-Fu uridylic is similarly the anticarcinogen of clinical conventional antimetabolic, is widely used in gastrointestinal cancer.The generation of its resistance phenomenon has affected it equally in clinical use [referring to Rita Humeniuk et al.Decreasedlevels of UMP kinase as a mechanism of fluoropyrimidine resistance.Mol Cancer Ther.2009,8:1037-1044.].Tumor drug resistance has number of ways, as cell surface translocator P-gP, MRP etc. transport out intracellular antitumor drug outside born of the same parents, thereby has reduced the effective concentration of born of the same parents' inner cell medicine and can not kill tumour cell.DNA repairs and extremely also can cause tumor drug resistance.Some antitumour drug, if alkylating agent, antimetabolite are directly for DNA, causes DNA damage.The DNA of damage can excise and be repaired or nucleotide excision reparation approach protects tumour cell to avoid the attack of chemotherapeutics by base.In addition detoxification system in cell, apoptosis tolerance and tumor microenvironment all can affect antitumour drug effect [referring to Sun Jie. tumor drug resistance and Reversal Strategy thereof. practical tumour magazine .2010,25:490-493.].
(2) untoward reaction: chemotherapy is as the primary treatment means of malignant tumour, lethality is strong, quick result, but its untoward reaction is also more obvious, especially cytotoxic drug selectivity is not strong, in inhibition or killing tumor cell, also human normal cell, tissue are caused to infringement in various degree.Common as bone marrow depression, immunosuppression, cardiac damage, peripheral neuropathy, stomatocace etc. [referring to Pan Xiangrong. the Chinese medicine recent developments that chemotherapy of tumors untoward reaction is sent out. Medical review .2010,16:2358-2360.], severe patient can cause the exhaustion of tissue organ function even can cause because toxicity is irreversible death.These untoward reactions not only directly affect patient's life quality, also can hinder the smooth enforcement of chemotherapy regimen.Wherein bone marrow depression untoward reaction is the most common.Clinical manifestation is oligoleukocythemia and thrombopenia, and severe patient is also shown in anaemia.Anthracycline, taxanes, vinca, camptothecin, platinum medicine, and the conventional antitumor drug such as mitomycin, gemcitabine, endoxan, ifosfamide, methotrexate, mustargen all shows obvious bone marrow inhibition [referring to Zhang Yanhua, Liu Hong. the serious adverse reaction of antitumor drug and control. pharmacovigilance .2010,7:40-43.].
Finding the little antitumor drug of side effect is an important topic of current tumour medicine research.
Summary of the invention
Task of the present invention is to provide a kind of antineoplastic compound, and the preparation method of this compound is provided simultaneously.
This antineoplastic compound provided by the invention is compound 2-(1-hydroxyl-4-ketone-2, the 5-cyclohexadiene)-pyrans-4-ketone having with following formula I structure.
The preparation method of antineoplastic compound provided by the invention comprises the following steps:
(1) get 2-(2-bromotrifluoromethane)-1,3-dioxolanes and Sodium Nitrite obtain 2-(2-nitro-ethyl)-1,3-dioxolanes after nitration reaction purifying;
(2) get 2-(2-nitro-ethyl)-1,3-dioxolanes and 4-acetylenylbenzene methyl ether, under the condition existing at phenylcarbimide, carry out 1, after 3-Dipolar Cycloaddition purifying, obtain 3-(1,3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole;
(3) get 3-(1,3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole, after the ring-opening reaction of hexacarbonylmolybdenum purifying, obtain (2Z)-3-amino-4-(1,3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone;
(4) get (2Z)-3-amino-4-(1,3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone carries out cyclization and obtains 2-(4-p-methoxy-phenyl)-pyrans-4-ketone under acidic conditions;
(5) get 2-(4-p-methoxy-phenyl)-pyrans-4-ketone, at BBr
3effect under carry out demethylating reaction and obtain 2-(4-hydroxy phenyl)-pyrans-4-ketone;
(6) get 2-(4-hydroxy phenyl)-pyrans-4-ketone carries out oxidizing reaction and obtains 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone under the effect of oxygenant and catalyzer.
Described in above-mentioned steps (1), get 2-(2-bromotrifluoromethane)-1, 3-dioxolanes and Sodium Nitrite, after nitration reaction purifying, obtain 2-(2-nitro-ethyl)-1, the concrete grammar of 3-dioxolanes is: get 2-(2-bromotrifluoromethane)-1, 3-dioxolanes, Sodium Nitrite and Phloroglucinol are dissolved in organic solvent, mix, stirring at room temperature, reclaim solvent, carry out column chromatography purification by sherwood oil/acetone purification on normal-phase silica gel and obtain 2-(2-nitro-ethyl)-1, 3-dioxolanes, described organic solvent is DMSO, DMF, benzene, toluene or ether.
Described in above-mentioned steps (2), get 2-(2-nitro-ethyl)-1, 3-dioxolanes and 4-acetylenylbenzene methyl ether, under the condition existing at phenylcarbimide, carry out 1, after 3-Dipolar Cycloaddition purifying, obtain 3-(1, 3-dioxolanes-2-methylene radical) concrete grammar of-5-(4-p-methoxy-phenyl)-isoxzzole is: get 2-(2-nitro-ethyl)-1, 3-dioxolanes and 4-acetylenylbenzene methyl ether are dissolved in organic solvent, mix, add subsequently triethylamine and phenylcarbimide, this mixture reflux was cooled to room temperature after 12-18 hour, add water, stir, diatomite filtration, then be extracted with ethyl acetate, combined ethyl acetate extraction liquid, reclaim solvent, product obtains 3-(1 with cyclohexane/ethyl acetate purification on normal-phase silica gel column chromatography purification, 3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole, described organic solvent is benzene, toluene, DMSO, DMF or ether.
Described in above-mentioned steps (3), get 3-(1, 3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole, after the ring-opening reaction of hexacarbonylmolybdenum purifying, obtain (2Z)-3-amino-4-(1, 3-dioxolanes-2-) concrete grammar of-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone is: get 3-(1, 3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole is dissolved in acetonitrile/water mixed solvent, then add hexacarbonylmolybdenum, mixture reflux 3-5 hour, reclaim solvent, cyclohexane/ethyl acetate purification on normal-phase silica gel column chromatography purification obtains (2Z)-3-amino-4-(1, 3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone.
Described in above-mentioned steps (4), get (2Z)-3-amino-4-(1, 3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone, under acidic conditions, carrying out the concrete grammar that cyclization obtains 2-(4-p-methoxy-phenyl)-pyrans-4-ketone is: get (2Z)-3-amino-4-(1, 3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone is dissolved in the mixed organic solvents of formic acid/acetic acid, after reflux 12-18 hour, be cooled to room temperature, after adding water, be extracted with ethyl acetate, sodium hydroxide solution is washed till neutrality, after reclaiming solvent, use cyclohexane/ethyl acetate purification on normal-phase silica gel column chromatography purification, obtain 2-(4-p-methoxy-phenyl)-pyrans-4-ketone.
Described in above-mentioned steps (5), get 2-(4-p-methoxy-phenyl)-pyrans-4-ketone, at BBr
3effect under carry out the concrete grammar that demethylating reaction obtains 2-(4-hydroxy phenyl)-pyrans-4 ketone and be: under-75 DEG C or 0 DEG C of condition, get 2-(4-p-methoxy-phenyl)-pyrans-4-ketone and be dissolved in methylene dichloride, add BBr
3, under mixture room temperature, stir 6-8 hour, add frozen water, ethyl acetate extraction, merges organic layer, obtains 2-(4-hydroxy phenyl)-pyrans-4 ketone.
2-(4-hydroxy phenyl)-pyrans-4 ketone of getting described in above-mentioned steps (6) carries out oxidizing reaction and obtains 2-(1-hydroxyl-4-ketone-2 under the effect of oxygenant and catalyzer, 5-cyclohexadiene)-pyrans-4-ketone concrete grammar is: gets 2-(4-hydroxy phenyl)-pyrans-4 ketone and is dissolved in the mixed solvent of acetonitrile and water, add oxygenant and catalyzer, stirring at room temperature 3-6 hour, methylene chloride/methanol purification on normal-phase silica gel column purification for product, obtain 2-(1-hydroxyl-4-ketone-2, 5-cyclohexadiene)-pyrans-4-ketone, described oxygenant is [two (trifluoroacetyl oxygen base) iodine] benzene (PIFA) or iodobenzene diethyl ester (PIA), described catalyzer is 2, 2, 6, 6-tetramethyl--1-piperidone (TEMPO), described acetonitrile and the mixed solvent of water are that the ratio that is 9: 1 by volume by acetonitrile and water mixes.
The present invention adopts 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone carries out in vitro tests (in vitro) to gynecological tumor cell, prostate tumor cells, digestive system tumor cell, hematological system tumor cell, blood system normal cell, metabolic system normal cell, confirms that this compound has selectivity anticancer activity.Tumor-bearing mice is carried out to experiment (in vivo) in antitumor and hematopoiesis, immunologic function body simultaneously, confirm that this compound can effectively suppress tumor-bearing mice tumor growth and not affect hematopoiesis and immunologic function.
[experimental data]
One, synthetic the compounds of this invention 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone:
(1) 2-(2-bromotrifluoromethane)-1, 3-dioxolanes and Sodium Nitrite obtain yellow liquid material after nitration reaction purifying, be judged as 2-(2-nitro-ethyl)-1 with reference to pertinent literature, 3-dioxolanes [referring to: Robyn L.Crumbie et al. α-Nitroketones and esters from acylimidazoles.J.Org.Chem.1982, 47:4040-4045.], concrete grammar is: 2-(2-bromotrifluoromethane)-1, 3-dioxolanes, Sodium Nitrite and Phloroglucinol are dissolved in organic solvent, mix, stirring at room temperature, reclaim solvent, carry out column chromatography purification by sherwood oil/acetone purification on normal-phase silica gel and obtain 2-(2-nitro-ethyl)-1, 3-dioxolanes.Organic solvent can be DMSO, DMF, benzene, toluene, ether.2-(2-nitro-ethyl)-1, the structure of 3-dioxolanes is shown in Fig. 4;
(2) 2-(2-nitro-ethyl)-1, under the condition that 3-dioxolanes and 4-acetylenylbenzene methyl ether exist at phenylcarbimide, carry out 1, after 3-Dipolar Cycloaddition purifying, obtain yellow liquid material, be judged as 3-(1 with reference to pertinent literature, 3-dioxolanes-2-methylene radical) and-5-(4-p-methoxy-phenyl)-isoxzzole [referring to: Chun-Sing Li, Edith Lacasse.Synthesis ofpyran-4-ones fromisoxazoles.Tetrahedron Letters.2002, 43:3565-3568.], concrete grammar is: get 2-(2-nitro-ethyl)-1, 3-dioxolanes and 4-acetylenylbenzene methyl ether are dissolved in organic solvent, mix, add subsequently triethylamine and phenylcarbimide, this mixture reflux was cooled to room temperature after 12-18 hour, add water, stir, diatomite filtration, then be extracted with ethyl acetate, combined ethyl acetate extraction liquid, reclaim solvent, product obtains 3-(1 with cyclohexane/ethyl acetate purification on normal-phase silica gel column chromatography purification, 3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole.Organic solvent can be benzene, toluene, DMSO, DMF, ether.The structure of 3-(1,3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole is shown in Fig. 4.
(3) 3-(1, 3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole obtains yellow solid matter after the ring-opening reaction of hexacarbonylmolybdenum purifying, be judged as (2Z)-3-amino-4-(1 with reference to pertinent literature, 3-dioxolanes-2-) and-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone [referring to: Chun-Sing Li, Edith Lacasse.Synthesis of pyran-4-ones from isoxazoles.Tetrahedron Letters.2002, 43:3565-3568.], concrete grammar is: get 3-(1, 3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole is dissolved in acetonitrile/water mixed solvent, then add hexacarbonylmolybdenum, mixture reflux 3-5 hour, reclaim solvent, cyclohexane/ethyl acetate purification on normal-phase silica gel column chromatography purification obtains (2Z)-3-amino-4-(1, 3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone.(2Z) structure of-3-amino-4-(1,3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone is shown in Fig. 4.
(4) (2Z)-3-amino-4-(1, 3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone carries out cyclization at acidic conditions and obtains yellow solid matter, be judged as 2-(4-p-methoxy-phenyl)-pyrans-4-ketone [referring to Chun-Sing Li with reference to pertinent literature, Edith Lacasse.Synthesis of pyran-4-ones from isoxazoles.Tetrahedron Letters.2002, 43:3565-3568.], concrete grammar is: get (2Z)-3-amino-4-(1, 3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone is dissolved in the mixed organic solvents of formic acid/acetic acid, after reflux 12-18 hour, be cooled to room temperature, after adding water, be extracted with ethyl acetate, sodium hydroxide solution is washed till neutrality, after reclaiming solvent, use cyclohexane/ethyl acetate purification on normal-phase silica gel column chromatography purification, obtain 2-(4-p-methoxy-phenyl)-pyrans-4-ketone.The structure of 2-(4-p-methoxy-phenyl)-pyrans-4-ketone is shown in Fig. 4.
(5) 2-(4-p-methoxy-phenyl)-pyrans-4-ketone is at BBr
3effect under carry out demethylating reaction and obtain yellow solid matter, with reference to pertinent literature be judged as 2-(4-hydroxy phenyl)-pyrans-4 ketone [referring to Song Yanling, wait .3, the synthetic method of 5-dihydroxyl bibenzyl is improved. Chinese pharmaceutical chemistry magazine .2008,18:129-130,134; Zhuo Guanglan, complete synthesis Deng. natural product (E)-trans-resveratrol. Chinese pharmaceutical chemistry magazine .2002,12:152-154], concrete grammar is: under-75 DEG C or 0 DEG C of condition, get 2-(4-p-methoxy-phenyl)-pyrans-4-ketone and be dissolved in methylene dichloride, add BBr
3, under mixture room temperature, stir 6-8 hour, add frozen water, ethyl acetate extraction, merges organic layer, obtains 2-(4-hydroxy phenyl)-pyrans-4-ketone.The structure of 2-(4-hydroxy phenyl)-pyrans-4-ketone is shown in Fig. 4.
(6) 2-(4-hydroxy phenyl)-pyrans-4-ketone carries out oxidizing reaction and obtains yellow solid matter under the effect of oxygenant and catalyzer, oxygenant is [two (trifluoroacetyl oxygen base) iodine] benzene (PIFA) or iodobenzene diethyl ester (PIA), catalyzer is 2,2,6,6-tetramethyl--1-piperidone (TEMPO).Concrete grammar is: get) 2-(4-hydroxy phenyl)-pyrans-4-ketone is dissolved in acetonitrile/water (9: 1) mixed solvent, add oxygenant and catalyzer, stirring at room temperature 3-6 hour, product obtains yellow solid matter with methylene chloride/methanol purification on normal-phase silica gel column purification.Oxygenant is [two (trifluoroacetyl oxygen base) iodine] benzene (PIFA) or iodobenzene diethyl ester (PIA), and catalyzer is 2,2,6,6-tetramethyl--1-piperidone (TEMPO).
The yellow solid matter that above-mentioned steps (6) is obtained is tested, the MS of this yellow solid matter, and UV,
13c-NMR,
1h-NMR data are as follows:
Yellow solid: UV
: 252.5nm, ESI-MS (m/z): 203[M-1]
-,
1h-NMR (400MHz, in DMSO): δ 8.10 (1H, d, J=6.0Hz, H-6), δ 6.96 (2H, d, J=10.0Hz, H-2 ', 6 '), δ 6.30 (2H, d, J=10.0Hz, H-3 ', 5 '), δ 6.54 (1H, d, J=2.4Hz, H-3), δ 6.29 (1H, dd, J=6.0,2.4Hz, H-5),
13c NMR (100MHz, in DMSO): δ 185.3 (C-4 '), δ 178.2 (C-4), δ 167.1 (C-2), δ 156.9 (C-6), δ 148.4 (C-2 ', 6 '), δ 129.1 (C-3 ', 5 '), δ 116.9 (C-3), δ 113.5 (C-5), δ 69.0 (C-1 ') (see Fig. 1, 2, 3), be judged as 2-(1-hydroxyl-4-ketone-2 with reference to pertinent literature, 5-cyclohexadiene) and-pyrans-4-ketone [referring to: An-Shen Lin et al.First total synthesis ofprotoapigenone and its analogues as potent cytotoxic agents.J.Med.Chem.2007, 50:3921-2927.], 2-(1-hydroxyl-4-ketone-2, 5-cyclohexadiene)-pyrans-4-ketone structure as shown in Equation 1.
Two, carry out antitumor pharmacological experiment with the synthetic 2-of the present invention (1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone.
The tumour cell that carries out anticancer experiment in vitro for compound 2-provided by the invention (1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone comprises humanized's gynecological tumor cell, prostate tumor cells, digestive system tumor cell and hematological system tumor cell.Gynecological tumor cell comprises breast carcinoma cell strain MDA-MB-231, MCF-7 and Ovarian Cancer Cells SKOV3; Prostate tumor cells strain is PC-3; Digestive system tumor cell comprises hepatoma cell strain HepG2, pancreas cancer cell strain PANC-1; Hematological system tumor comprises myeloma cell strain RPMI8226, myeloma cell strain U266.Synthetic 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone compound can suppress above-mentioned tumor cell proliferation, and especially to gynaecology's system tumor, tumor of prostate and digestive system tumor are more responsive, and inhibition is better than former apigenin.
The normal cell that carries out cellulotoxic experiment for compound 2-provided by the invention (1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone is blood system normal cell PBMC and metabolic system normal cell human embryonic kidney cell HEK 293.Synthetic 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone compound to the more former apigenin of normal blood system restraining effect a little less than, show that it is less on the impact of normal blood system and metabolic system.
The Mammals that carries out anti-tumor in vivo experiment for compound 2-provided by the invention (1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone is H22 tumor-bearing mice.(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone compound has obvious restraining effect to the tumor growth of tumor-bearing mice to synthetic 2-, and inhibition is better than former apigenin.
The Mammals that carries out the system evaluation of body inner blood for compound 2-provided by the invention (1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone is H22 tumor-bearing mice.Synthetic 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone compound is to tumor-bearing mice peripheral white blood cell, Peripheral blood cells is counted lymphocyte number ratio, bone marrow nucleated cell number, the red ratio of medullary cell grain all do not make significant difference, and former apigenin has remarkably influenced to These parameters, show synthetic 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone compound do not affect mammiferous hemopoietic function, and former apigenin suppresses mammiferous hemopoietic function.
The Mammals that carries out vivo immuning system evaluation for compound 2-provided by the invention (1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone is H22 tumor-bearing mice.Synthetic 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone compound all do not make significant difference to tumor-bearing mice thymus index and spleen index, former apigenin has remarkably influenced to the thymus index of tumor-bearing mice, show synthetic 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone compound do not affect mammiferous immunologic function, and former apigenin suppresses mammiferous immunologic function.
Anticancer experiment in vitro
(1) experiment material
Former apigenin is separated and obtains from common pin hair fern underground part by this laboratory early stage, extract to separate with Structural Identification method and be: the medicinal material rhizome 4kg (picking up from Lushan Mountain) of seasoning suitably pulverizes, with ethanol percolate extraction, 60 DEG C of decompression and solvent recoveries obtain medicinal extract, gained medicinal extract adds suitable quantity of water and suspends, and extracts and gets 4 positions successively with sherwood oil, ether, ethyl acetate, propyl carbinol.Ethyl acetate extract is through silica gel column chromatography, and trichloromethane-methyl alcohol gradient elution obtains F
1-F
55 parts, wherein F
1through silica gel column chromatography, trichloromethane-methanol-eluted fractions, then obtain yellow powder shape compound, ESI-MS (m/z): 286[M through Sephadex LH-20 purifying]
+,
1h-NMR (400MHz, in DMSO): δ 7.01 (2H, d, J=9.6Hz, H-2 ', 6 '), δ 6.35 (2H, d, J=9.6Hz, H-3 ', 5 '), δ 6.50 (1H, s, H-3), δ 6.19 (1H, s, H-8);
13c NMR (100MHz, inDMSO): δ 185.5 (C-4 '), δ 182.4 (C-4), δ 168.0 (C-2), δ 165.2 (C-7), δ 162.1 (C-5), δ 158.0 (C-9), δ 148.4 (C-2 ', 6 '), δ 129.4 (C-3 ', 5 '), δ 107.1 (C-3), δ 104.5 (C-10), δ 99.8 (C-6), δ 94.5 (C-8), δ 69.5 (C-1 ').
The former apigenin data consistent of above-mentioned experimental data and bibliographical information, therefore be judged as former apigenin [referring to Lin AS, et al.Newcytotoxic flavonoids from Thelypteris torresiana.Planta Med.2005,71:867-870];
2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone is synthetic and qualification by the inventive method.
Two kinds of test compound mother liquors of this of different concns are dissolved in DMSO.When experiment, add cultured cells with 1000 times of dilutions.
The gynecological tumor cell strain using in the present invention is as follows: humanized's breast carcinoma cell strain (MDA-MB-231 and MCF-7), humanized's Ovarian Cancer Cells (SKOV3) are preserved center (China Center for TypeCulture Collection purchased from Chinese Typical Representative bacterial strain,, and be incubated in the DMEM high glucose medium that has added 10% foetal calf serum CCTCC).
The humanized's prostate cancer cell (PC-3) using in the present invention is preserved center purchased from Chinese Typical Representative bacterial strain, is incubated in the DMEM high glucose medium that has added 10% foetal calf serum.
The digestive system tumor cell strain using in the present invention is as follows: humanized's hepatoma cell strain (HepG2), humanized's pancreas cancer cell strain (PANC-1) are preserved center purchased from Chinese Typical Representative bacterial strain, are incubated in the DMEM high glucose medium that has added 10% foetal calf serum.
The hematological system tumor cell strain using in the present invention is as follows: humanized's multiple myeloma cell (RPMI8226, U266) is preserved center purchased from Chinese Typical Representative bacterial strain, is incubated in 1640 substratum that added 10% foetal calf serum.
The blood system normal cell using in the present invention is provided by Central China University of Science and Technology's preclinical medicine institute immunization experiment chamber for human peripheral blood single nucleus cell (PBMC), and is incubated in 1640 substratum that added 10% foetal calf serum.
The metabolic system normal cell behaviour embryonic kidney cell line (HEK293) using in the present invention is preserved center purchased from Chinese Typical Representative bacterial strain, and is incubated in 1640 substratum that added 10% foetal calf serum.
(2) experimental technique
Anticancer experiment in vitro adopts tetrazolium reduction method (mtt assay): in 96 orifice plates, inoculate 5 × 10
4/ ml cell, cultivates former apigenin and 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone compound effect 24 hours with different concns (0.1,0.5,1.0,5.0,10.0,50.0,100.0 μ M) after 18 hours.Measure the restraining effect of test compound to tumor cell line with MTT cell proliferation detection method.With microplate reader detection, detect wavelength 570nm.Think survival rate 100% without pharmaceutically-active groups of cells.Survival rate=(dosing group OD value/control group OD value) × 100%.Every group of experiment repeats 4 times.
(3) experimental result
Referring to table 1, is former apigenin and 2-(1-hydroxyl-4-ketone-2,5-the cyclohexadiene)-restraining effect of pyrans-4-ketone compound to different tumor cell lines.In table 1,2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone compound is to breast cancer cell, ovarian cancer cell, the restraining effect of prostate cancer cell, liver cancer cell and pancreatic cancer cell is better than former apigenin, particularly breast cancer cell (MDA-MB-231, MCF-7) and ovarian cancer cell (SKOV3) are had to stronger restraining effect, show its potentiality in gynecological neoplasm.Simultaneously its to human peripheral blood single nucleus cell (PBMC) and human embryonic kidney cell (HEK 293) restraining effect a little less than, point out it can not cause the untoward reaction of strong blood system and metabolic system inhibition.Compare, former apigenin is better than 2-(1-hydroxyl-4-ketone-2 to multiple myeloma (RPMI8226, U266) and human embryonic kidney cell's (HEK 293) restraining effect, 5-cyclohexadiene)-pyrans-4-ketone compound, but it is obvious equally to the restraining effect of PBMC cell, may cause the untoward reaction such as blood system and metabolic system inhibition.
The former apigenin of table 1 and 2-(1-hydroxyl-4-ketone-2,5-the cyclohexadiene)-restraining effect of pyrans-4-ketone compound to different cell strains
Two, animal is tested at body
(1) experiment material
Former apigenin is separated and obtains from common pin hair fern underground part by this laboratory early stage, and 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone is synthesized by this laboratory.Positive control medicine endoxan (CTX) is purchased from hospital of Tongji University of the Central China University of Science and Technology.
KM mouse, female, 17-19g, is provided by Central China University of Science and Technology's experimentation on animals center.Mouse is provided by pharmaceutical college of the Central China University of Science and Technology pharmacological evaluation chamber H22 tumor-bearing mice kind.
(2) experimental technique
1, test compound is to antitumor action in H22 Mice Body
The modeling of H22 tumor-bearing mice and grouping administration: under aseptic condition, extract the ascites of the H22 liver cancer mouse going down to posterity the 7th day, in 1: 4 ratio normal saline dilution, repeatedly charge and attack, fully mix, counting under the microscope, adjusting cell concn with physiological saline is 1 × 10
7/ mL, gets 0.2mL subcutaneous vaccination after iodine tincture disinfection under the armpit of every mouse right side.After the success of experiment mice inoculated tumour, be divided at random 5 groups, 6 every group, i.e. normal group (NS, 20mL/kg, ig), lotus knurl control group (NS, 20mL/kg, ig), CTX group (10mg/kg, ip), 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone compound group (10mg/kg, ig), former apigenin group (10mg/kg, ig).Started administration in second day, be administered once every day, and administration volume ig is 20mL/kg, and ip is 10mL/kg, successive administration 7 days.Next day after last administration, after weighing, put to death mouse, strip knurl piece and weigh, calculate tumour inhibiting rate, the average knurl of tumour inhibiting rate=(the average knurl weight of the average knurl weight-medication of lotus knurl control group group)/lotus knurl control group heavy × 100%.
2, the impact of test compound on H22 mouse hemopoietic function
Grouping and administering mode are with 1, and next day after last administration, mouse is plucked eyeball blood sampling, get blood 20 μ L and join in 400 μ L2% glacial acetic acid solutions and shake up, counting peripheral blood leucocyte sum.After weighing, put to death mouse, peel off left side femur, go out marrow inner cell with 2% glacial acetic acid solution 5mL, conventionally count bone marrow nucleated cell in femur.Blood and marrow are made respectively blood smear and bone marrow smear, add Rui Shi dyeing, and counting Peripheral blood cells, lymphocyte and marrow granulocyte series, erythron, calculate respectively Peripheral blood cells and count lymphocyte number ratio and the red ratio of marrow grain.
3, the impact of test compound on H22 immune function of mice
Modeling and medication are with 1, and after execution mouse, weighed thymus gland, spleen weight, calculate thymus index and spleen index.
(3) experimental result
Referring to table 2, is former apigenin and 2-(1-hydroxyl-4-ketone-2,5-the cyclohexadiene)-impact of pyrans-4-ketone compound on H22 tumor-bearing mice tumor growth.
The 2-of 10mg/kg (1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone compound and positive drug CTX are the most obvious to the tumor inhibition effect of H22 mouse as can be seen from Table 2, are respectively 34.12% and 41.47%.Relatively have utmost point significant difference with model group, and former apigenin restraining effect under same dose is not too obvious, with relatively there was no significant difference of model group.But CTX is larger on the body weight impact of mouse, make the weight loss of mouse, relatively there is significant difference with normal group, point out it to have certain toxic action to mouse.
Referring to Fig. 5, Fig. 6, Fig. 7 and Fig. 8, be tumor tissues HE dyeing, the visible model group of light Microscopic observation and former apigenin group tumour cell irregular arrangement, cut apart agglomerating distribution by interstitial composition, and chromatin is darker, and nuclear fission is more.CTX group and 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone group tumour cell queueing discipline, cell karyoplasmic ratio reduces, and dyes more shallow, and different in nature check figure order reduces.And visible a large amount of downright bad tumour cells and lymphocyte infiltration phenomenon in CTX group and 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-this tumor tissue section of two groups of pyrans-4-ketone.
Antitumor action in the former apigenin of table 2 and 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketoboidies (n=6,
)
With model group comparison
1): p < 0.01
With normal group comparison
*: p < 0.05
Referring to table 3, be former apigenin and 2-(1-hydroxyl-4-ketone-2,5-the cyclohexadiene)-impact of pyrans-4-ketone compound on H22 tumor-bearing mice blood system.CTX group and former apigenin group all have impact in various degree to mouse blood system as can be seen from Table 3.CTX group significantly reduces H22 mouse peripheral blood white corpuscle number, extremely significantly reduces granulocyte and lymphocytic ratio in peripheral blood leucocyte, the red ratio of bone marrow nucleated cell number and marrow grain.And former apigenin group significantly reduces granulocyte and lymphocytic ratio and the red ratio of medullary cell grain in peripheral blood leucocyte, extremely significantly reduce bone marrow nucleated cell number, point out these two kinds of compounds to suppress mouse hemopoietic system, particularly suppress granulocytic growth.And 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone compound is less to each Index Influence of mouse hemopoietic function, with relatively there was no significant difference of normal group.
The former apigenin of table 3 and 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone is to H22 tumor-bearing mice peripheral blood WBC, Peripheral blood cells lymphocyte ratio, and bone marrow nucleated cell number, the impact of the red ratio of medullary cell grain (n=6,
)
With normal group comparison
1)p < 0.05;
2)p < 0.01
Referring to table 4, be former apigenin and 2-(1-hydroxyl-4-ketone-2,5-the cyclohexadiene)-impact of pyrans-4-ketone compound on H22 tumor-bearing mice thymus index and spleen index.2-(1-hydroxyl-4-ketone-2 as can be seen from Table 4,5-cyclohexadiene)-restraining effect the minimum of pyrans-4-ketone to H22 mouse thymus and spleen, there was no significant difference compared with normal group, and former apigenin and CTX all have certain restraining effect to H22 mouse thymus and spleen.
The impact of the former apigenin of table 4 and 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone on H22 mouse thymus exponential sum spleen index (n=6,
)
With normal group comparison
1): p < 0.05;
2): p < 0.01
In sum, 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone compound is acted on breast cancer cell, ovarian cancer cell by the present invention, prostate cancer cell, liver cancer cell, pancreatic cancer cell, myeloma cell, this compound can effectively suppress cell proliferation.Wherein to breast cancer cell, ovarian cancer cell, prostate cancer cell, liver cancer cell, the inhibition of pancreatic cancer cell is better than former apigenin, and to blood system normal cell and the Normocellular toxicity of metabolic system lower than former apigenin.Confirm 2-(1-hydroxyl-4-ketone-2 by experimentation on animals, 5-cyclohexadiene)-pyrans-4-ketone compound has the effect of good inhibition tumor growth in vivo equally, and compare with former apigenin with positive drug CTX, have advantages of and do not affect the hemopoietic system of animal function and function of immune system.
Brief description of the drawings
Fig. 1,2-of the present invention (1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone
1h-NMR collection of illustrative plates;
Fig. 2,2-of the present invention (1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone
1h-NMR partial enlarged drawing spectrum;
Fig. 3,2-of the present invention (1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone
13c-NMR collection of illustrative plates;
Fig. 4: the reacting flow chart of synthetic 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone;
Fig. 5: model group tumor tissue section (HE dyeing × 400);
Fig. 6: CTX group tumor tissue section (HE dyeing × 400);
Fig. 7: former apigenin group tumor tissue section (HE dyeing × 400);
Fig. 8: 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-Tong Zu tumor tissue section (HE dyeing × 400).
Embodiment
The present invention can be fully understood by following description of test, can be those skilled in the art reference is provided, but be only example, does not limit the scope of the present invention.
The preparation of 2-(1-hydroxyl-4-ketone-2,5-cyclohexadiene)-pyrans-4-ketone
(1) 2-(2-bromotrifluoromethane)-1,3-dioxolanes and Sodium Nitrite obtain yellow liquid material after nitration reaction purifying, its concrete operations are: by the 2-of 1 equivalent (2-bromotrifluoromethane)-1, Sodium Nitrite, the 0.5 equivalent Phloroglucinol of 3-dioxolanes, 1.3 equivalents are dissolved in DMSO, mix room temperature reaction 18 hours.By reaction mixture water-dispersion, add equal-volume dichloromethane extraction 2 times, get dichloromethane layer, 70 DEG C are reclaimed solvent.Product does taking sherwood oil/acetone volume ratio as the condition of 10: 1 in thin layer condition iodine cylinder and develops the color, and Rf value is 0.3.Doing purification on normal-phase silica gel by the condition that sherwood oil/acetone volume ratio is 10: 1 carries out column chromatography purification and obtains yellow liquid product, judge that with reference to pertinent literature this product is 2-(2-nitro-ethyl)-1,3-dioxolanes [referring to: Robyn L.Crumbie et al. α-Nitro ketones and esters from acylimidazoles.J.Org.Chem.1982,47:4040-4045.]
(2) 2-(2-nitro-ethyl)-1, under the condition that 3-dioxolanes and 4-acetylenylbenzene methyl ether exist at phenylcarbimide, carry out 1, after 3-Dipolar Cycloaddition purifying, obtain yellow liquid material, its concrete operations are: by 1 equivalent 2-(2-nitro-ethyl)-1,3-dioxolanes and 3 equivalent 4-acetylenylbenzene methyl ethers are dissolved in benzene, mix, add subsequently two triethylamines and 4 equivalent phenylcarbimides.90 DEG C of reflux of this mixture 18 hours, add water after being cooled to room temperature, stirring at room temperature 30 minutes.This mixture is with using equal-volume ethyl acetate extracting twice after diatomite filtration, and combined ethyl acetate extraction liquid, with saturated common salt washing 2 times, dried over mgso, recovery solvent.Product does taking cyclohexane/ethyl acetate volume ratio as the condition of 7: 3 in thin layer condition iodine cylinder and develops the color, and Rf value is 0.4.Product does purification on normal-phase silica gel column chromatography purification by the condition that cyclohexane/ethyl acetate volume ratio is 3: 1 and obtains yellow liquid product, judge that with reference to pertinent literature this product is 3-(1,3-dioxolanes-2-methylene radical) and-5-(4-p-methoxy-phenyl)-isoxzzole [referring to: Chun-Sing Li, Edith Lacasse.Synthesis of pyran-4-ones from isoxazoles.TetrahedronLetters.2002,43:3565-3568.].
(3) 3-(1,3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole obtains yellow solid matter after the ring-opening reaction of hexacarbonylmolybdenum purifying, its concrete operations are: by 1 equivalent 3-(1,3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole is dissolved in and in acetonitrile/water mixed solvent, adds 0.5 equivalent hexacarbonylmolybdenum, 90 DEG C of reflux of mixture 3 hours.Reclaim solvent, product does taking cyclohexane/ethyl acetate volume ratio as the condition of 2: 1 in thin layer condition iodine cylinder and develops the color, and Rf value is 0.25 left and right.Product does purification on normal-phase silica gel column chromatography purification by the condition that cyclohexane/ethyl acetate volume ratio is 2: 1 and obtains yellow solid product, judge that with reference to pertinent literature this product is (2Z)-3-amino-4-(1,3-dioxolanes-2-) and-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone [referring to: Chun-Sing Li, Edith Lacasse.Synthesis of pyran-4-ones from isoxazoles.TetrahedronLetters.2002,43:3565-3568.].
(4) (2Z)-3-amino-4-(1, 3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone carries out cyclization at acidic conditions and obtains yellow solid matter, its concrete operations are: by (2Z)-3-amino-4-(1, 3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone is dissolved in 1 volumes of acetic acid, again with 4 volume 80% formic acid dilutions, 60 DEG C of heating of reaction system were cooled to room temperature after 12 hours, after adding water, be extracted with ethyl acetate 3 times, sodium hydroxide solution is washed till neutrality, again with saturated common salt washing 2 times, dried over mgso.Recovery solvent after product does taking cyclohexane/ethyl acetate volume ratio as the condition of 2: 1 in thin layer condition iodine cylinder and develops the color, and Rf value is 0.15.Do purification on normal-phase silica gel column chromatography purification by the condition that cyclohexane/ethyl acetate volume ratio is 2: 1, obtain yellow solid product, judge that with reference to pertinent literature this product is that 2-(4-p-methoxy-phenyl)-pyrans-4-ketone is [referring to Chun-Sing Li, Edith Lacasse.Synthesis of pyran-4-ones from isoxazoles.Tetrahedron Letters.2002,43:3565-3568.].
(5) 2-(4-p-methoxy-phenyl)-pyrans-4-ketone is at BBr
3effect under carry out demethylating reaction and obtain yellow solid matter, its concrete operations are: under-75 DEG C or 0 DEG C of condition, 1 equivalent 2-(4-p-methoxy-phenyl)-pyrans-4-ketone is dissolved in methylene dichloride, slowly adds 2 equivalent 1mol/L BBr
3dichloromethane solution.Under mixture room temperature, stir 6 hours.Reaction finishes rear system and adds frozen water, ethyl acetate extracting twice, merge organic layer, obtain yellow solid product, with reference to pertinent literature judge this product be 2-(4-hydroxy phenyl)-pyrans-4 ketone [referring to Song Yanling, wait .3, the synthetic method of 5-dihydroxyl bibenzyl is improved. Chinese pharmaceutical chemistry magazine .2008,18:129-130,134; Zhuo Guanglan, complete synthesis etc. natural product (E)-trans-resveratrol. Chinese pharmaceutical chemistry magazine .2002,12:152-154].
(6) 2-(4-hydroxy phenyl)-pyrans-4 ketone carries out oxidizing reaction and obtains yellow solid matter under the effect of oxygenant and catalyzer, its concrete operations are: it is in the mixed solvent of 9: 1 that 1 equivalent 2-(4-hydroxy phenyl)-pyrans-4 ketone is dissolved in to acetonitrile/water volume ratio, adds 2 equivalent oxygenants and 0.5 equivalent catalyzer.Stirring at room temperature 5 hours.Reclaim solvent after product and do thin layer condition taking methylene chloride/methanol volume ratio as the condition of 30: 1 in ultraviolet 254nm observation, Rf value is 0.2.Product does purification on normal-phase silica gel column purification by the condition that methylene chloride/methanol volume ratio is 30: 1, obtains yellow solid product.Described oxygenant is [two (triacetoxyl group) iodine] benzene (PIFA) or iodobenzene diethyl ester (PIA), and described catalyzer is 2,2,6,6-tetramethyl--1-piperidone (TEMPO).This product is yellow solid, UV
252.5nm, ESI-MS (m/z): 203[M-1]
-,
1h-NMR (400MHz, in DMSO): δ 8.10 (1H, d, J=6.0Hz, H-6), δ 6.96 (2H, d, J=10.0Hz, H-2 ', 6 '), δ 6.30 (2H, d, J=10.0Hz, H-3 ', 5 '), δ 6.54 (1H, d, J=2.4Hz, H-3), δ 6.29 (1H, dd, J=6.0,2.4Hz, H-5),
13c NMR (100MHz, in DMSO): δ 185.3 (C-4 '), δ 178.2 (C-4), δ 167.1 (C-2), δ 156.9 (C-6), δ 148.4 (C-2 ', 6 '), δ 129.1 (C-3 ', 5 '), δ 116.9 (C-3), δ 113.5 (C-5), δ 69.0 (C-1 ') (see Fig. 1, 2, 3), judge that with reference to pertinent literature this product is 2-(1-hydroxyl-4-ketone-2, 5-cyclohexadiene) and-pyrans-4-ketone [referring to: An-Shen Lin et al.First total synthesis of protoapigenoneand its analogues as potent cytotoxic agents.J.Med.Chem.2007, 50:3921-2927.]
Building-up reactions schema is shown in Fig. 4.
The present invention can capable field technique personnel make various modifications, but does not depart from protection scope of the present invention.
Claims (10)
1. there is-pyrans-4-ketone with the compound 2-shown in following formula I structure (1-hydroxyl-4-ketone-2,5-cyclohexadiene),
2. compound claimed in claim 1 is in the application of preparing in antitumor drug.
Compound claimed in claim 1 for the preparation for the treatment of gynaecology system tumor, tumor of prostate, digestive system tumor, metabolic system tumour or/and the application in hematological system tumor medicine.
4. the preparation method of compound claimed in claim 1, comprises the following steps:
(1) get 2-(2-bromotrifluoromethane)-1,3-dioxolanes and Sodium Nitrite and Phloroglucinol obtain 2-(2-nitro-ethyl)-1,3-dioxolanes after nitration reaction purifying;
(2) get 2-(2-nitro-ethyl)-1,3-dioxolanes and 4-acetylenylbenzene methyl ether, under the condition existing at phenylcarbimide, carry out 1, after 3-Dipolar Cycloaddition purifying, obtain 3-(1,3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole;
(3) get 3-(1,3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole, after the ring-opening reaction of hexacarbonylmolybdenum purifying, obtain (2Z)-3-amino-4-(1,3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone;
(4) get (2Z)-3-amino-4-(1,3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone, carries out cyclization at acidic conditions and obtains 2-(4-p-methoxy-phenyl)-pyrans-4-ketone;
(5) get 2-(4-p-methoxy-phenyl)-pyrans-4-ketone, under the effect of BBr3, carry out demethylating reaction and obtain 2-(4-hydroxy phenyl)-pyrans-4-ketone;
(6) get 2-(4-hydroxy phenyl)-pyrans-4-ketone carries out oxidizing reaction and obtains 2-(1-hydroxyl-4-ketone-2 under the effect of oxygenant and catalyzer, 5-cyclohexadiene)-pyrans-4-ketone, described oxygenant is [two (trifluoroacetyl oxygen base) iodine] benzene or iodobenzene diethyl ester, described catalyzer is 2,2,6,6-tetramethyl--1-piperidone.
5. preparation method according to claim 4, it is characterized in that, what step (1) was described gets 2-(2-bromotrifluoromethane)-1, 3-dioxolanes, Sodium Nitrite and Phloroglucinol, after nitration reaction purifying, obtain 2-(2-nitro-ethyl)-1, the concrete grammar of 3-dioxolanes is: get 2-(2-bromotrifluoromethane)-1, 3-dioxolanes, Sodium Nitrite and Phloroglucinol are dissolved in organic solvent, mix, stirring at room temperature, reclaim solvent, carry out column chromatography purification by sherwood oil/acetone purification on normal-phase silica gel and obtain 2-(2-nitro-ethyl)-1, 3-dioxolanes, described organic solvent is DMSO, DMF, benzene, toluene or ether.
6. preparation method according to claim 4, it is characterized in that, what step (2) was described gets 2-(2-nitro-ethyl)-1, 3-dioxolanes and 4-acetylenylbenzene methyl ether, under the condition existing at phenylcarbimide, carry out 1, after 3-Dipolar Cycloaddition purifying, obtain 3-(1, 3-dioxolanes-2-methylene radical) concrete grammar of-5-(4-p-methoxy-phenyl)-isoxzzole is: get 2-(2-nitro-ethyl)-1, 3-dioxolanes and 4-acetylenylbenzene methyl ether are dissolved in organic solvent, mix, add subsequently triethylamine and phenylcarbimide, this mixture reflux adds water after being cooled to room temperature after 12-18 hour, stir, diatomite filtration, then be extracted with ethyl acetate, combined ethyl acetate extraction liquid, reclaim solvent, product obtains 3-(1 with cyclohexane/ethyl acetate purification on normal-phase silica gel column chromatography purification, 3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole, described organic solvent is benzene, toluene, DMSO, DMF or ether.
7. preparation method according to claim 4, it is characterized in that, what step (3) was described gets 3-(1, 3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole, after the ring-opening reaction of hexacarbonylmolybdenum purifying, obtain (2Z)-3-amino-4-(1, 3-dioxolanes-2-) concrete grammar of-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone is: get 3-(1, 3-dioxolanes-2-methylene radical)-5-(4-p-methoxy-phenyl)-isoxzzole is dissolved in acetonitrile/water mixed solvent, then add hexacarbonylmolybdenum, mixture reflux 3-5 hour, reclaim solvent, cyclohexane/ethyl acetate purification on normal-phase silica gel column chromatography purification obtains (2Z)-3-amino-4-(1, 3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone.
8. preparation method according to claim 4, it is characterized in that, what step (4) was described gets (2Z)-3-amino-4-(1, 3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone, carrying out at acidic conditions the concrete grammar that cyclization obtains 2-(4-p-methoxy-phenyl)-pyrans-4-ketone is: get (2Z)-3-amino-4-(1, 3-dioxolanes-2-)-1-(4-p-methoxy-phenyl)-2-butylene-1-ketone is dissolved in the mixed organic solvents of formic acid/acetic acid, after reflux 12-18 hour, be cooled to room temperature, after adding water, be extracted with ethyl acetate, sodium hydroxide solution is washed till neutrality, after reclaiming solvent, use cyclohexane/ethyl acetate purification on normal-phase silica gel column chromatography purification, obtain 2-(4-p-methoxy-phenyl)-pyrans-4-ketone.
9. preparation method according to claim 4, is characterized in that, what step (5) was described gets 2-(4-p-methoxy-phenyl)-pyrans-4-ketone, at BBr
3effect under carry out the concrete grammar that demethylating reaction obtains 2-(4-hydroxy phenyl)-pyrans-4-ketone and be: under-75 DEG C or 0 DEG C of condition, get 2-(4-p-methoxy-phenyl)-pyrans-4-ketone and be dissolved in methylene dichloride, add BBr
3, under mixture room temperature, stir 6-8 hour, add frozen water, ethyl acetate extraction, merges organic layer, obtains 2-(4-hydroxy phenyl)-pyrans-4-ketone.
10. preparation method according to claim 4, it is characterized in that, what step (6) was described gets 2-(4-hydroxy phenyl)-pyrans-4-ketone at oxygenant [two (trifluoroacetyl oxygen base) iodine] benzene or iodobenzene diethyl ester and catalyzer 2, 2, 6, under the effect of 6-tetramethyl--1-piperidone, carry out oxidizing reaction and obtain 2-(1-hydroxyl-4-ketone-2, 5-cyclohexadiene)-pyrans-4-ketone concrete grammar is: gets 2-(4-hydroxy phenyl)-pyrans-4-ketone and is dissolved in the mixed solvent of acetonitrile and water, add oxygenant and catalyzer, stirring at room temperature 3-6 hour, methylene chloride/methanol purification on normal-phase silica gel column purification for product, obtain 2-(1-hydroxyl-4-ketone-2, 5-cyclohexadiene)-pyrans-4-ketone, described oxygenant is [two (trifluoroacetyl oxygen base) iodine] benzene or iodobenzene diethyl ester, described catalyzer is 2, 2, 6, 6-tetramethyl--1-piperidone, described acetonitrile and the mixed solvent of water are by volume for the ratio of 9:1 mixes by acetonitrile and water.
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