WO2015027669A2 - 苯基取代化合物及其药物组合物和应用 - Google Patents

苯基取代化合物及其药物组合物和应用 Download PDF

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WO2015027669A2
WO2015027669A2 PCT/CN2014/000808 CN2014000808W WO2015027669A2 WO 2015027669 A2 WO2015027669 A2 WO 2015027669A2 CN 2014000808 W CN2014000808 W CN 2014000808W WO 2015027669 A2 WO2015027669 A2 WO 2015027669A2
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dihydroxy
group
compound
hydrogen
cancer
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French (fr)
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WO2015027669A3 (zh
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贺小琼
詹华强
黄清龙
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香港科技大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C65/21—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing ether groups, groups, groups, or groups
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/11—Aldehydes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/01—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring by replacing functional groups bound to a six-membered aromatic ring by hydroxy groups, e.g. by hydrolysis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
    • C07C39/02—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring monocyclic with no unsaturation outside the aromatic ring
    • C07C39/08—Dihydroxy benzenes; Alkylated derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/27—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by oxidation
    • C07C45/29—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by oxidation of hydroxy groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C47/00—Compounds having —CHO groups
    • C07C47/52—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings
    • C07C47/56—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings containing hydroxy groups
    • C07C47/565—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings containing hydroxy groups all hydroxy groups bound to the ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/347—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups
    • C07C51/367—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups by introduction of functional groups containing oxygen only in singly bound form

Definitions

  • This application belongs to the field of chemical medicine, and in particular to phenyl substituted compounds and pharmaceutical compositions and uses thereof.
  • Cancer is a major disease that poses a serious threat to human health and life. It has become the first cause of death for human beings. According to the Global Cancer Statistics Report, in 2008, there were 12.661 million new cases of cancer and 7.546 million deaths. The number of new cancer cases in China was 2.816 million and the deaths were 1.958 million. In 2012, there were more than 14 million new cancer cases worldwide and more than 8.2 million deaths. The 2012 China Cancer Registration Annual Report disclosed that China had 3.12 million new cancer cases in 2012, with an average of 6 diagnosed as malignant tumors per minute. The incidence of cancer is still rising, and cancer has become a major health problem that seriously threatens people's health and life.
  • Cancer is still a refractory disease in the world.
  • cancer chemotherapy has a very important position in the treatment of cancer, and it is also a hope to completely solve the problem of cancer treatment in the future.
  • cancer chemotherapy drugs many drugs have toxic side effects, uncertain effects, poor selectivity and drug resistance of cancer cells, so that the clinical application and effect of cancer chemotherapy is greatly limited. Therefore, the development of chemotherapeutic drugs with clear curative effect, low toxicity and safety, especially selective anti-cancer effect, is an important direction and urgent task for the research of new anti-cancer drugs.
  • the present invention relates to 4-ethoxy-2-hydroxy-6-methylbenzoic acid having the structure of formula (1) or a pharmaceutically acceptable salt thereof,
  • the present invention relates to a phenyl-substituted compound having a structure of the formula (2), wherein R 1 is hydrogen, a C1-C4 alkyl group or a C1-C4 aldehyde group; and R 2 is hydrogen, or a pharmaceutically acceptable salt thereof a carboxyl group, a C1-C4 alkyl ester group, or a substituted or unsubstituted phenyl ester group, wherein the substituted phenyl group is one to three selected from the group consisting of a hydroxyl group, a C1-C4 alkyl group, and a C1-C4 alkyl ester. a substituent substituted with a phenyl group; R 3 is hydrogen or a C1-C4 alkyl group,
  • the compound is not 4-ethoxy-2-hydroxy-6-methylbenzoic acid, 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester, 2,4-dihydroxy-6 -ethyl methyl benzoate, 1,3-dihydroxy-5-methylbenzene, 2,6-dihydroxy-4-methylbenzaldehyde, methyl 2,4-dihydroxy-6-methylbenzoate And 3-aldehyde aldehyde-2,4-dihydroxy-6-methylbenzoic acid ethyl ester.
  • the present invention relates to an antitumor pharmaceutical composition
  • an active ingredient comprising 4-ethoxy-2-hydroxy-6-methylbenzoic acid having the structure of formula (1) or a structure of formula (2) a phenyl-substituted compound or a pharmaceutically acceptable salt thereof, wherein R 1 in the formula (2) is hydrogen, a C1-C4 alkyl group or a C1-C4 aldehyde group; and R 2 is hydrogen, a carboxyl group or a C1-C4 alkyl ester group.
  • a substituted or unsubstituted phenyl ester group which is a phenyl group substituted with 1 to 3 substituents selected from a hydroxyl group, a C1-C4 alkyl group and a C1-C4 alkyl ester group; 3 is hydrogen or a C1-C4 alkyl group.
  • the present invention relates to a pharmaceutical composition for combination antitumor, the active ingredient comprising 4-ethoxy-2-hydroxy-6-methylbenzoic acid having the structure of formula (1) or the structure of formula (2) a phenyl-substituted compound or a pharmaceutically acceptable salt thereof, and at least one of 5-fluorouracil, paclitaxel, cisplatin or cyclophosphamide, wherein R 1 in the formula (2) is hydrogen, C1-C4 alkyl or a C1-C4 aldehyde group; R 2 is hydrogen, a carboxyl group, a C1-C4 alkyl ester group, or a substituted or unsubstituted phenyl ester group, wherein the substituted phenyl group is 1-3 selected from a hydroxyl group, C1- a phenyl group substituted with a substituent of a C4 alkyl group and a C1-C4 alkyl ester group; and R 3 is
  • the present invention relates to a 4-phenyloxy-2-hydroxy-6-methylbenzoic acid having the structure of the formula (1) or a phenyl-substituted compound of the formula (2) or a pharmaceutically acceptable salt thereof
  • R 1 in the formula (2) is hydrogen, a C1-C4 alkyl group or a C1-C4 aldehyde group
  • R 2 is a hydrogen, a carboxyl group or a C1-C4 alkyl ester.
  • a phenyl group or a substituted or unsubstituted phenyl group, which is a phenyl group substituted with 1 to 3 substituents selected from the group consisting of a hydroxyl group, a C1-C4 alkyl group and a C1-C4 alkyl ester group;
  • R 3 is hydrogen or a C1-C4 alkyl group.
  • the present application relates to a method of inhibiting or treating tumor growth comprising inhibiting or therapeutically effective amount of 4-ethoxy-2-hydroxy-6-methylbenzoic acid having the structure of formula (1) or formula ( 2) a structural phenyl-substituted compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, administered to an individual in need of such treatment, wherein R 1 in formula (2) is hydrogen, C1-C4 alkyl or a C1-C4 aldehyde group; R 2 is hydrogen, a carboxyl group, a C1-C4 alkyl ester group, or a substituted or unsubstituted phenyl ester group, wherein the substituted phenyl group is 1-3 selected from a hydroxyl group, C1- a phenyl group substituted with a substituent of a C4 alkyl group and a C1-C4 alkyl ester group; and R 3 is hydrogen or a C1-C4 alkyl group
  • the compounds of the present invention having the structures of formula (1) and formula (2) can significantly kill and/or inhibit various tumor cells of humans and animals in vivo and in vitro, and can significantly promote apoptosis and necrosis of cancer cells, and regulate Cancer cell growth cycle, inhibit cancer cell proliferation. At the same dose, the cytotoxic effects of the compounds having the structures of formula (1) and formula (2) on normal human cells are significantly lower than those on human tumor cells.
  • Compounds having the structures of formula (1) and formula (2) have a significant synergistic antitumor effect in combination with clinical antitumor drugs such as cisplatin, 5-fluorouracil, paclitaxel or cyclophosphamide.
  • the compound having the structure of the formula (1) and the formula (2) or a pharmaceutically acceptable salt thereof can be used for the preparation of an antitumor drug, an antitumor drug composition, a combined antitumor drug composition or an auxiliary antitumor health product.
  • Figure 1 is a mass spectrum of the compound of formula (1)
  • Figure 2 is a nuclear magnetic resonance spectrum of the compound of the formula (1)
  • Figure 3 is a nuclear magnetic carbon spectrum and a DEPT spectrum of the compound of the formula (1);
  • Figure 4 is a graph showing the dose-effect curve of the compound of the formula (1) of the present invention against different tumor cell lines;
  • Figure 5 is a graph showing the time-effect, dose-effect relationship of the anti-tumor experiment of the compound of the formula (1) in vitro;
  • Figure 6 is a graph showing the relative tumor volume (RTV) at different time points in the in vivo anti-tumor (NCI-H460) test of the compound of the formula (1);
  • Figure 7 is a graph showing the in vivo anti-tumor (NCI-H460) test of the compound of the formula (1) in the present invention at different times Point relative tumor growth rate (%);
  • Figure 8 is a graph showing the weight of NCI-H460 tumor after administration of the compound of the formula (1) 10 times;
  • Figure 9 is a graph showing the relative tumor volume (RTV) at different time points in the in vivo antitumor (QGY-7703) test of the compound of the formula (1);
  • Figure 10 is a graph showing the relative proliferation rate (%) of tumors at different time points in the in vivo antitumor (QGY-7703) test of the compound of the formula (1);
  • Figure 11 is a graph showing the average tumor weight of each group at the end of the in vivo antitumor (QGY-7703) experiment of the compound of the formula (1);
  • Figure 12 is a photograph of a tumor of each group of animals at the end of the in vivo anti-tumor (QGY-7703) experiment of the compound of the formula (1);
  • Figure 13 is a photograph of the spleen of each group of animals at the end of the in vivo anti-tumor (QGY-7703) experiment of the compound of the formula (1);
  • Figure 14 is a flow cytometry test result of the solvent control of the compound of the formula (1) of the present invention (QGY-7703);
  • Figure 15 is a flow cytometry test result of the compound of the formula (1) of the present invention (QGY-7703);
  • Figure 16 is a Western Blot test of the effect of the compound of the formula (1) on the protein levels of QGY-7703, HCT-116, MCF-7 and A-549 cell lines; wherein: NC indicates a negative control and SP indicates a positive control star.
  • Staurosporine, A, B, D, L represent different analytes, where B represents the compound 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester, and D represents the compound 4- Ethoxy-2-hydroxy-6-methylbenzoic acid;
  • Figure 17 is a graph showing the anti-A-549 tumor effect of the compound of the formula (1) and 5-fluorouracil in vitro;
  • Figure 18 is a graph showing the anti-A-549 tumor effect of the compound of the formula (1) and cisplatin in vitro;
  • Figure 19 is a mass spectrum of the compound 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester
  • Figure 20 is a nuclear magnetic resonance spectrum of the compound 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester
  • Figure 21 is a nuclear magnetic carbon spectrum and DEPT spectrum of the compound 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester;
  • Figure 22 is a nuclear magnetic two-dimensional spectrum HMBC of the compound 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester;
  • Figure 23 is a nuclear magnetic two-dimensional spectrum HSQC of the compound 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester;
  • Figure 24 is a nuclear magnetic two-dimensional map Roesy of the compound 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester;
  • Figure 25 is a graph showing the dose-response effect of the compound methyl 2,4-dihydroxy-3,6-dimethylbenzoate on the inhibition of different human tumor cell lines and human normal lung epithelial cell line Beas-2b in vitro;
  • Figure 26 is a graph showing the dose-response relationship between the clinical anticancer drugs 5-fluorouracil and cisplatin on the inhibition of human normal lung epithelial cell line Beas-2b;
  • Figure 27 is a flow cytometric test result of the compound 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester of the present invention (QGY-7703);
  • Figure 28 is a Western Blot test of the effect of the compound 2,4-dihydroxy-3,6-dimethylbenzoate on the protein levels of QGY-7703, HCT-116, MCF-7 and A-549 cell lines.
  • NC indicates a negative control
  • SP indicates a positive control Staurosporine
  • A, B, D, and L indicate different analytes
  • B indicates compound 2,4-dihydroxy-3,6 Methyl dimethyl benzoate
  • D represents the compound 4-ethoxy-2-hydroxy-6-methylbenzoic acid.
  • Figure 29 is a view showing the action of the compound 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester and cisplatin in combination with anti-Hela tumor cells in vitro;
  • Figure 30 is a mass spectrum of the compound 2,4-dihydroxy-6-methylbenzoic acid ethyl ester
  • Figure 31 is a nuclear magnetic hydrogen spectrum of the compound 2,4-dihydroxy-6-methylbenzoic acid ethyl ester
  • Figure 32 is a nuclear magnetic carbon spectrum and DEPT spectrum of the compound 2,4-dihydroxy-6-methylbenzoic acid ethyl ester;
  • Figure 33 is a two-dimensional spectrum of the compound 2,4-dihydroxy-6-methylbenzoic acid ethyl ester HMBC;
  • Figure 34 is a two-dimensional spectrum HSQC of the compound 2,4-dihydroxy-6-methylbenzoic acid ethyl ester
  • Figure 35 is a two-dimensional spectrum of the compound 2,4-dihydroxy-6-methylbenzoic acid ethyl ester Roesy;
  • Figure 36 is a graph showing the amount-effect relationship of the in vitro inhibitory effect of the compound 2,4-dihydroxy-6-methylbenzoate on the different tumor cell lines and the human normal lung epithelial cell line Beas-2b;
  • Figure 37 is a diagram showing the effect of the compound 2,4-dihydroxy-6-methylbenzoic acid ethyl ester and cisplatin in the anti-Hela tumor cells in vitro;
  • Figure 38 is a mass spectrum of the compound 2,6-dihydroxy-4-methylbenzaldehyde
  • Figure 39 is a nuclear magnetic resonance spectrum of the compound 2,6-dihydroxy-4-methylbenzaldehyde
  • Figure 40 is a nuclear magnetic carbon spectrum and DEPT spectrum of the compound 2,6-dihydroxy-4-methylbenzaldehyde
  • Figure 41 is a graph showing the dose-response relationship of the anticancer effect of the compound 2,6-dihydroxy-4-methylbenzaldehyde in vitro;
  • Figure 42 is a mass spectrum of the compound 2,4-dihydroxy-6-methylbenzoic acid methyl ester
  • Figure 43 is a nuclear magnetic resonance spectrum of the compound 2,4-dihydroxy-6-methylbenzoic acid methyl ester
  • Figure 44 is a nuclear magnetic carbon spectrum and DEPT spectrum of the compound 2,4-dihydroxy-6-methylbenzoic acid methyl ester
  • Figure 45 is a mass spectrum of the compound 3-aldehyde-2,4-dihydroxy-6-methylbenzoic acid ethyl ester
  • Figure 46 is a nuclear magnetic hydrogen spectrum of the compound 3-aldehyde-2-(2-hydroxy-6-methylbenzoic acid ethyl ester);
  • Figure 47 is a nuclear magnetic carbon spectrum and DEPT spectrum of the compound 3-aldehyde-2-(2-hydroxy-6-methylbenzoic acid ethyl ester);
  • Figure 48 is a mass spectrum of the compound 3-aldehyde-2,4-dihydroxy-6-methylbenzoic acid 5-(ethoxycarbonyl)-3-hydroxy-2-methylphenyl ester;
  • Figure 49 is a nuclear magnetic hydrogen spectrum of the compound 3-aldehyde-2,4-dihydroxy-6-methylbenzoic acid 5-(ethoxycarbonyl)-3-hydroxy-2-methylphenyl ester;
  • Figure 50 is a nuclear magnetic carbon spectrum and DEPT spectrum of the compound 3-aldehyde-2,4-dihydroxy-6-methylbenzoic acid 5-(ethoxycarbonyl)-3-hydroxy-2-methylphenyl ester. .
  • C1-C4 alkyl means a straight or branched saturated hydrocarbon group having one to four carbon atoms.
  • Examples of the C1-C4 alkyl group include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, 1-methylpropyl, t-butyl and the like.
  • C1-C4 aldehyde group means a straight or branched aldehyde group having one to four carbon atoms.
  • Examples of the C1-C4 aldehyde group include, but are not limited to, formaldehyde group, acetaldehyde group, propionaldehyde group, isopropenyl group and the like.
  • C1-C4 alkyl ester group means "RCOO-", wherein R is a C1-C4 alkyl group as defined above.
  • pharmaceutical composition refers to a formulation of a compound of the present invention and a medium which is generally accepted in the art for delivery of a biologically active compound to a mammal such as a human.
  • Such media include all pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier is meant to include, but is not limited to, any adjuvant, excipient, glidant that has been approved by the U.S. Food and Drug Administration (FDA) for use in humans or animals. , sweeteners, thinners, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, disintegrating agents, solvents or emulsifiers The agent or the like has various forms of carriers which do not have side effects in constituting the pharmaceutical composition.
  • FDA U.S. Food and Drug Administration
  • inhibitory effective amount and “therapeutically effective amount” are used interchangeably, meaning that the compound of the present application is sufficient to effectively inhibit tumor cells or treatment when administered to a human, preferably to a human, more preferably to a human.
  • the amount of tumor The amount of the compound of the present application constituting the "inhibitory effective amount” or the “therapeutically effective amount” will vary depending on the state of the selected compound or the subject to be administered, but those skilled in the art can, based on their own knowledge and the disclosure of the present application, An effective amount of a compound of the present application is determined according to common knowledge in the art.
  • the term "individual” refers to a mammal, preferably a human.
  • treating means administering a compound or formulation of the invention to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:
  • “Pharmaceutically acceptable salts” include “pharmaceutically acceptable acid addition salts” and “pharmaceutically acceptable base addition salts.”
  • “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free base, which are biologically or otherwise suitable and which are formed using inorganic or organic acids.
  • the inorganic acid is, for example but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, Aspartic acid, benzenesulfonic acid, benzenecarboxylic acid, 4-acetamidobenzenecarboxylic acid, camphoric acid, camphor-10-sulfonic acid, citric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexyl Alkyl sulfamic acid, dodecyl sulphate, ethane-1,2-d
  • “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acid, which are biologically or otherwise suitable. These salts are prepared by adding an inorganic base or an organic base to the free acid. In the present application, salts derived from inorganic bases are preferred, including but not limited to sodium, potassium, lithium salts and the like.
  • the present invention relates to 4-ethoxy-2-hydroxy-6-methylbenzoic acid having the structure of formula (1) or a pharmaceutically acceptable salt thereof,
  • the present invention relates to a phenyl-substituted compound having a structure of the formula (2), wherein R 1 is hydrogen, a C1-C4 alkyl group or a C1-C4 aldehyde group; and R 2 is hydrogen, or a pharmaceutically acceptable salt thereof a carboxyl group, a C1-C4 alkyl ester group, or a substituted or unsubstituted phenyl ester group, wherein the substituted phenyl group is one to three selected from the group consisting of a hydroxyl group, a C1-C4 alkyl group, and a C1-C4 alkyl ester. a substituent substituted with a phenyl group; R 3 is hydrogen or a C1-C4 alkyl group,
  • the compound is not 4-ethoxy-2-hydroxy-6-methylbenzoic acid, 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester, 2,4-dihydroxy-6 -ethyl methyl benzoate, 1,3-dihydroxy-5-methylbenzene, 2,6-dihydroxy-4-methylbenzaldehyde, methyl 2,4-dihydroxy-6-methylbenzoate And 3-aldehyde aldehyde-2,4-dihydroxy-6-methylbenzoic acid ethyl ester.
  • R 1 is hydrogen, methyl or formaldehyde.
  • R 2 is hydrogen, CH 3 COO-, C 2 H 5 COO-, HCOO-, or 5-(ethoxycarbonyl)-3-hydroxy-2-methylphenyl ester.
  • R 3 is hydrogen, methyl or ethyl.
  • the phenyl substituted compound having the structure of formula (2) is 3-aldehyde-2,4-dihydroxy-6-methylbenzoic acid 5-(ethoxycarbonyl)-3-hydroxy-2- Methyl phenyl ester.
  • the present invention relates to an antitumor pharmaceutical composition
  • an active ingredient comprising 4-ethoxy-2-hydroxy-6-methylbenzoic acid having the structure of formula (1) or a structure of formula (2) a phenyl-substituted compound or a pharmaceutically acceptable salt thereof, wherein R 1 in the formula (2) is hydrogen, a C1-C4 alkyl group or a C1-C4 aldehyde group; and R 2 is hydrogen, a carboxyl group or a C1-C4 alkyl ester group.
  • a substituted or unsubstituted phenyl ester group which is a phenyl group substituted with 1 to 3 substituents selected from a hydroxyl group, a C1-C4 alkyl group and a C1-C4 alkyl ester group; 3 is hydrogen or a C1-C4 alkyl group.
  • R 1 in formula (2) is hydrogen, methyl or formaldehyde.
  • R 2 in formula (2) is hydrogen, CH 3 COO-, C 2 H 5 COO-, HCOO-, or 5-(ethoxycarbonyl)-3-hydroxy-2-methylphenyl Ester group.
  • R 3 in formula (2) is hydrogen, methyl or ethyl.
  • the compound is selected from the group consisting of 4-ethoxy-2-hydroxy-6-methylbenzoic acid, methyl 2,4-dihydroxy-3,6-dimethylbenzoate, 2,4 - Dihydroxy-6-methylbenzoic acid ethyl ester, 1,3-dihydroxy-5-methylbenzene, 2,6-dihydroxy-4-methylbenzaldehyde, 2,4-dihydroxy-6-methyl Methyl benzoate, ethyl 3-aldehyde-2,4-dihydroxy-6-methylbenzoate or 3-aldehyde-2,4-dihydroxy-6-methylbenzoic acid 5-(ethoxy Carbonyl)-3-hydroxy-2-methylphenyl ester.
  • compositions may also include a pharmaceutically acceptable carrier.
  • a particular therapeutically effective amount of the composition can be determined by one of ordinary skill in the art based on the selected compound, the subject being administered.
  • the effective amount may range from 0.01 to 99.99%, preferably from 1 to 99%, more preferably from 10 to 90% by weight of the composition.
  • the pharmaceutical composition can be in the form of an injection, a powder, a tablet, an oral solution, a capsule, a granule, or a granule.
  • the present invention relates to a pharmaceutical composition for combination antitumor, the active ingredient comprising 4-ethoxy-2-hydroxy-6-methylbenzoic acid having the structure of formula (1) or the structure of formula (2) a phenyl-substituted compound or a pharmaceutically acceptable salt thereof, and at least one of 5-fluorouracil, paclitaxel, cisplatin or cyclophosphamide, wherein R 1 in the formula (2) is hydrogen, C1-C4 alkyl or C1-C4 aldehyde group; R 2 is hydrogen, carboxyl group, C1-C4 alkyl ester group, or substituted or unsubstituted phenyl ester group, the substituted phenyl group is 1-3 selected from hydroxyl group, C1- a phenyl group substituted with a substituent of a C4 alkyl group and a C1-C4 alkyl ester group; and R 3 is hydrogen or a C1-C4 alkyl
  • R 1 in formula (2) is hydrogen, methyl or formaldehyde.
  • R 2 in formula (2) is hydrogen, CH 3 COO-, C 2 H 5 COO-, HCOO-, or 5-(ethoxycarbonyl)-3-hydroxy-2-methylphenyl Ester group.
  • R 3 in formula (2) is hydrogen, methyl or ethyl.
  • the compound is selected from the group consisting of 4-ethoxy-2-hydroxy-6-methylbenzoic acid, methyl 2,4-dihydroxy-3,6-dimethylbenzoate, 2,4 - Dihydroxy-6-methylbenzoic acid ethyl ester, 1,3-dihydroxy-5-methylbenzene, 2,6-dihydroxy-4-methylbenzaldehyde, 2,4-dihydroxy-6-methyl Methyl benzoate, ethyl 3-aldehyde-2,4-dihydroxy-6-methylbenzoate or 3-aldehyde-2,4-dihydroxy-6-methylbenzoic acid 5-(ethoxy Carbonyl)-3-hydroxy-2-methylphenyl ester.
  • the above-mentioned 4-ethoxy-2-hydroxy-6-methylbenzoic acid having the structure of formula (1) or a phenyl-substituted compound of the formula (2) or a pharmaceutically acceptable salt thereof
  • the ratio of the molar concentration of at least one of the 5-fluorouracil, paclitaxel, cisplatin or cyclophosphamide is calculated to be 1:10 to 100:1.
  • the present invention relates to a 4-phenyloxy-2-hydroxy-6-methylbenzoic acid having the structure of the formula (1) or a phenyl-substituted compound of the formula (2) or a pharmaceutically acceptable salt thereof
  • R 1 in the formula (2) is hydrogen, a C1-C4 alkyl group or a C1-C4 aldehyde group
  • R 2 is a hydrogen, a carboxyl group or a C1-C4 alkyl ester.
  • a phenyl group or a substituted or unsubstituted phenyl group, which is a phenyl group substituted with 1 to 3 substituents selected from the group consisting of a hydroxyl group, a C1-C4 alkyl group and a C1-C4 alkyl ester group;
  • R 3 is hydrogen or a C1-C4 alkyl group.
  • R 1 in formula (2) is hydrogen, methyl or formaldehyde.
  • R 2 in formula (2) is hydrogen, CH 3 COO-, C 2 H 5 COO-, HCOO-, or 5-(ethoxycarbonyl)-3-hydroxy-2-methylbenzene Base group.
  • R 3 in formula (2) is hydrogen, methyl or ethyl.
  • the compound is selected from the group consisting of 4-ethoxy-2-hydroxy-6-methylbenzoic acid, methyl 2,4-dihydroxy-3,6-dimethylbenzoate, 2,4 - Dihydroxy-6-methylbenzoic acid ethyl ester, 1,3-dihydroxy-5-methylbenzene, 2,6-dihydroxy-4-methylbenzaldehyde, 2,4-dihydroxy-6-methyl Methyl benzoate, ethyl 3-aldehyde-2,4-dihydroxy-6-methylbenzoate or 3-aldehyde-2,4-dihydroxy-6-methylbenzoic acid 5-(ethoxycarbonyl)-3-hydroxy-2-methylphenyl ester.
  • the tumor is any one of the following human tumors: human breast cancer, prostate cancer, lung cancer, gastric cancer, cervical cancer, colon cancer, liver cancer, ovarian cancer, leukemia, neuroma, sarcoma, nasopharynx cancer.
  • the auxiliary anti-tumor health care product refers to an auxiliary treatment for a tumor patient in the treatment process of a tumor patient, which may have a sensitizing effect on the treatment of a tumor patient, but is not a health care product for the purpose of treatment.
  • the present application relates to a method of inhibiting or treating tumor growth comprising inhibiting or therapeutically effective amount of 4-ethoxy-2-hydroxy-6-methylbenzoic acid having the structure of formula (1) or formula ( 2) a structural phenyl-substituted compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, administered to an individual in need of such treatment, wherein R 1 in formula (2) is hydrogen, C1-C4 alkyl or a C1-C4 aldehyde group; R 2 is hydrogen, a carboxyl group, a C1-C4 alkyl ester group, or a substituted or unsubstituted phenyl ester group, wherein the substituted phenyl group is 1-3 selected from a hydroxyl group, C1- a phenyl group substituted with a substituent of a C4 alkyl group and a C1-C4 alkyl ester group; and R 3 is hydrogen or a C1-C4 alkyl group
  • R 1 in formula (2) is hydrogen, methyl or formaldehyde.
  • R 2 in formula (2) is hydrogen, CH 3 COO-, C 2 H 5 COO-, HCOO-, or 5-(ethoxycarbonyl)-3-hydroxy-2-methylbenzene Base group.
  • R 3 in formula (2) is hydrogen, methyl or ethyl.
  • the compound is selected from the group consisting of 4-ethoxy-2-hydroxy-6-methylbenzoic acid, methyl 2,4-dihydroxy-3,6-dimethylbenzoate, 2,4 - Dihydroxy-6-methylbenzoic acid ethyl ester, 1,3-dihydroxy-5-methylbenzene, 2,6-dihydroxy-4-methylbenzaldehyde, 2,4-dihydroxy-6-methyl Methyl benzoate, ethyl 3-aldehyde-2,4-dihydroxy-6-methylbenzoate or 3-aldehyde-2,4-dihydroxy-6-methylbenzoic acid 5-(ethoxy Carbonyl)-3-hydroxy-2-methylphenyl ester.
  • the tumor is any one of the following human tumors: human breast cancer, prostate cancer, lung cancer, gastric cancer, cervical cancer, colon cancer, liver cancer, ovarian cancer, leukemia, neuroma, sarcoma, nasopharynx cancer.
  • a compound having the structure of formula (1) or formula (2) has an inhibitory effective amount of tumor cells greater than or equal to 5.0 ⁇ M.
  • the therapeutically effective amount of the compound having the structure of formula (1) or formula (2) is greater than or equal to 10 mg/kg.
  • the 4-ethoxy-2-hydroxy-6-methyl benzoic acid compound of the present invention is a novel structural substance which can be chemically synthesized by a phase transfer catalytic reaction.
  • the compound or a salt thereof can be used as an antitumor chemotherapeutic drug, or as an antitumor drug composition as an active ingredient of an antitumor drug, and as an auxiliary antitumor health care product.
  • 4-Ethoxy-2-hydroxy-6-methylbenzoic acid can significantly kill and/or inhibit various cancer cells in humans and animals in vitro, such as human breast cancer, prostate cancer, lung cancer, stomach cancer, cervical cancer, Colon cancer, liver cancer cells, and mouse sarcoma, liver cancer, lung cancer cells, etc.; the compound at the same dose, the cytotoxic effect on normal human cells is significantly lower than the cytotoxic effect on sensitive human tumor cells.
  • the compound in vivo transplants human lung cancer and human liver cancer into balb/c nude mice, mouse sarcoma and mouse liver cancer transplanted in Kunming mice and ICR mice, and mouse lung cancer transplanted in C57BL/6J mice. Significant growth inhibition.
  • the compound has a synergistic anti-tumor effect in vitro and/or in vivo with clinically used anti-tumor chemotherapeutic drugs.
  • the compound has a selective anticancer effect, and the sensitivity of different cancer cell lines is different, and the liver cancer cells are very sensitive to the compound.
  • the compound can significantly promote cancer cell apoptosis, regulate cancer cell cycle and differentiation, and inhibit cancer cell proliferation.
  • the compound has no significant toxicity to mouse weight gain and organ development at an effective anticancer dose, or the toxicity is significantly lower than clinical anticancer drugs such as cyclophosphamide and 5-fluorouracil.
  • the compound 2,4-dihydroxy-3,6-dimethyllbenzoate of the present invention can be obtained by chemical synthesis.
  • the compound can be used as an antitumor chemotherapeutic drug, or as an antitumor drug composition as an active ingredient of an antitumor drug, and as an auxiliary antitumor health care product.
  • the compound can significantly kill and/or inhibit cancer cells such as human liver cancer, breast cancer, cervical cancer, lung cancer, nasopharyngeal cancer and the like in vitro.
  • the compound has a targeted anticancer effect, and the sensitivity of different cancer cell lines is different. Human liver cancer cells and cervical cancer cells are very sensitive to the compound, and human colon cancer and Xuanwei lung cancer are not sensitive to the compound.
  • the cytotoxic effect of the compound on human normal cell lines such as HUVEC and Beas-2b in vitro is significantly lower than that on sensitive cancer cell lines, and has selective anticancer effect.
  • the toxic effect of the compound on human normal cell lines is significantly lower than that of the clinical anticancer drugs 5-fluorouracil, cisplatin and paclitaxel.
  • the compound has synergistic anti-tumor effect with clinically used anti-tumor chemotherapeutic drugs such as cisplatin, 5-fluorouracil, paclitaxel and the like in vitro.
  • the compound can significantly promote cancer cell apoptosis, regulate cancer cell cycle and differentiation, and inhibit cancer cell proliferation.
  • the compound of the present invention 2,4-dihydroxy-6-methylbenzoic acid ethyl ester (ethyl 2,4-dihydroxy-6-methylbenzoate) can be obtained by chemical synthesis.
  • the compound can be used as an antitumor chemotherapeutic drug, or as an antitumor drug composition as an active ingredient of an antitumor drug, and as an auxiliary antitumor health care product.
  • the compound can significantly kill and/or inhibit cancer cells of human breast cancer, lung cancer, nasopharyngeal cancer, cervical cancer, liver cancer, leukemia and the like in vitro.
  • the compound has a targeted anticancer effect, and the sensitivity of different cancer cell lines varies greatly.
  • Human liver cancer cells and human cervical cancer cells are very sensitive to the compound, and human cholangiocarcinoma, Xuanwei lung cancer, and human colon cancer do not. sensitive.
  • the compound in vivo transplants mouse sarcoma S-180 and mouse liver cancer H22, C57BL/6J mice transplanted mouse lung adenocarcinoma 3ll and other tumors in Kunming mice and ICR mice, and balb/c nude mice transplanted humans
  • Both lung cancer NCI-H460 and human liver cancer QGY-7703 have significant growth inhibition effects.
  • the compound has a synergistic anti-tumor effect in vitro and/or in vivo with clinically used anti-tumor chemotherapeutic drugs such as cisplatin, 5-fluorouracil, paclitaxel, cyclophosphamide and the like.
  • the compound can significantly promote cancer cell apoptosis, regulate cancer cell cycle and differentiation, and inhibit cancer cell proliferation.
  • the cytotoxic effect of the compound on human normal cell lines such as HUVEC and Beas-2b in vitro is significantly lower than that on sensitive cancer cell lines, and has selective anticancer effect.
  • the toxic effect of the compound on human normal cell lines is significantly lower than that of the clinical anticancer drugs 5-fluorouracil, cisplatin and paclitaxel, and the cytotoxic effect is low.
  • the compound of the present invention 1,3-dihydroxy-5-methylbenzene (5-methylbenzene-1,3-diol), can be obtained by chemical synthesis.
  • the compound can be used as an antitumor chemotherapeutic drug, or as an antitumor drug composition as an active ingredient of an antitumor drug, and as an auxiliary antitumor health care product.
  • the compound can significantly kill and/or inhibit human human breast cancer, cervical cancer, and liver cancer cells in vitro.
  • the compound 2,6-dihydroxy-4-methylbenzaldehyde of the present invention can be obtained by chemical synthesis.
  • the compound can be used as an antitumor chemotherapeutic drug, or as an antitumor drug composition as an active ingredient of an antitumor drug, and as an auxiliary antitumor health care product.
  • the compound can significantly kill and/or inhibit human breast cancer, lung cancer, cervical cancer, and liver cancer cells in vitro.
  • the compound of the present invention methyl 2,4-dihydroxy-6-methylbenzoate, can be obtained by chemical synthesis.
  • the compound can be used as an antitumor chemotherapeutic drug, or as an antitumor drug composition as an active ingredient of an antitumor drug, and as an auxiliary antitumor health care product.
  • the compound can significantly kill and/or inhibit human breast cancer, lung cancer, cervical cancer, colon cancer, and liver cancer cells in vitro.
  • the compound 3-formyl-2,4-dihydroxy-6-methylbenzoate of the present invention can be obtained by chemical synthesis.
  • the compound can be used as an antitumor chemotherapeutic drug, or as an antitumor drug composition as an active ingredient of an antitumor drug, and as an auxiliary antitumor health care product.
  • the compound can significantly kill and/or inhibit human cervical cancer, breast cancer, liver cancer, colon cancer cells in vitro.
  • the compound of the present invention 3-aldehyde- 2,4-dihydroxy-6-methylbenzoic acid 5-(ethoxycarbonyl)-3-hydroxy-2-methylphenyl ester (5-(ethoxycrbonyl)-3- Hydroxy-2-methylphenyl 3-formyl-2,4-dihydroxy-6-methylbenzoate is a new structural substance which can be chemically synthesized by catalytic reaction.
  • the compound can be used as an antitumor chemotherapeutic drug, or as an antitumor drug composition as an active ingredient of an antitumor drug, and as an auxiliary antitumor health care product.
  • the compound can significantly kill and/or inhibit human breast cancer, lung cancer, cervical cancer, colon cancer, and liver cancer cells in vitro.
  • the compound of the formula (1) of the present invention 4-ethoxy-2-hydroxy-6-methylbenzoic acid, is illustrated by a part of specific experimental examples (hereinafter, it is represented by D6B2 or D in the following figures; the same applies hereinafter; Figs. 18 and 28, D1 is 200 ⁇ M, D2 is 150 ⁇ M, D3 is 100 ⁇ M, D4 is 50 ⁇ M, and D5 is 25 ⁇ M. It has significant and selective anti-tumor effects, combined anti-tumor effects with clinical anti-tumor drugs, and its toxicological safety. Sexual and anti-tumor mechanism.
  • Test method The compound of the formula (1) 4-ethoxy-2-hydroxy-6-methylbenzoic acid was dissolved in DMSO, and an in vitro anticancer test was carried out at a final concentration of 25, 50, 100, 150, 200 ⁇ M. Human or animal tumor cells in logarithmic growth phase were collected, suspended in complete medium, seeded in 6-well plates, and 2 ml of cell suspension was added to each well. After incubating for 24 hours at 37 ° C in a 5% CO 2 incubator, the number of cells in the 3 wells was counted as the number of cells at 0 hours; the culture solution of the remaining wells was aspirated, and then the complete culture medium containing the corresponding concentration of the compound of formula (1) was added as designed.
  • the compound of formula (1) 4-ethoxy-2-hydroxy-6-methylbenzoic acid was applied to human breast cancer cell line MCF-7, human lung cancer cell line A-549, human colon cancer cell line HCT-116, Human hepatoma cell line QGY-7703 and the like all have significant anticancer effects.
  • the compound has a significant difference in the inhibition rate of different cancer cells, and human liver cancer cell QGY-7703 is very sensitive to the compound.
  • the inhibitory rates of this compound against QGY-7703, MCF-7, A-549 and HCT-116 were 95.07%, 78.26%, 50.39% and 51.84%, respectively.
  • the half-inhibitory concentration (IC50) was respectively They were 70 ⁇ M, 135 ⁇ M, 190 ⁇ M and 200 ⁇ M; the in vitro anticancer effect of this compound has a significant dose-response relationship.
  • the 72-hour in vitro anticancer results are shown in Figure 4.
  • microscopic examination of human cervical cancer Hela, human gastric cancer cell SGC-7901, human prostate cancer cell PC-3, human ovary Cancer cell SKOV-3, human leukemia cells K-562 and HL-60, human glioma cell U-251 And so on has significant in vitro inhibition or killing effect.
  • the dose-response relationship and time-effect relationship diagram (MTT method) of the compound against human cancer cell line QGY-7703 in vitro at different doses are shown in Fig. 5.
  • Test method Male balb/c nude mice (body weight 16-18 g), each right side of the back was subcutaneously inoculated with human large cell lung cancer NCI-H460 cell suspension 0.1 ml (5 ⁇ 10 7 cells / ml). On the 12th day after inoculation of cancer cells, most of the mice developed macroscopic tumors under the skin. The tumor volume of each mouse was measured and calculated using a vernier caliper. The tumor-bearing mice were randomly divided into a DMSO control group and a compound administration group of the formula (1) according to the tumor volume, and 8 tumor-bearing mice in each group.
  • the compound of formula (1) was prepared in sterile mass spectrometry pure DMSO at a dose of 30 mg/kg; the control mice were given an equivalent volume of DMSO.
  • the mice were intraperitoneally administered from the second day after the grouping, and administered once every other day, and the tumor volume and body weight were measured once every four days, and the relative volume of the tumor and the relative rate of increase of the tumor were calculated.
  • the mice were sacrificed, and the tumor, liver, spleen, kidney and testis were completely excised, the tumor weight and the weight of each organ were measured, and the tumor weight inhibition rate and each organ coefficient were calculated.
  • mice Female Balb/c nude mice (body weight 16 to 18 g) that were adaptively reared for 1 week were each inoculated with 0.1 ml (1 ⁇ 10 8 cells/ml) of QGY-7703 cell suspension subcutaneously in the right side. Nude mice were housed in the SPF laboratory. On the 21st day after inoculation of the cancer cells, the mice showed macroscopic tumors under the skin. The long diameter and transverse diameter of the mouse tumor were measured with a vernier caliper to calculate the tumor volume. Tumor-bearing mice were randomly divided into DMSO solvent control group, 5-fluorouracil (5-Fu) positive drug control group and compound (1) compound administration group according to tumor volume, and each group contained 6 tumor-bearing mice.
  • mice 4-Ethoxy-2-hydroxy-6-methylbenzoic acid was dissolved in sterile mass spectrometry pure DMSO, and then diluted in an equal volume of sterile PBS at a dose of 20 mg/kg; Volume of DMSO/PBS (1:1); 5-Fu was diluted in an equal volume of DMSO, and 5-Fu was used at a dose of 30 mg/kg.
  • Mice were administered intraperitoneally from the second day after grouping, once every other day, and tumor volume and body weight were measured once every 4 days. After 12 administrations, the mice were sacrificed, and the tumor, liver, spleen, and kidney were completely excised, and the tumor weight, the body weight of the mice, and the weight of each organ were measured.
  • results The results of inhibition of QGY-7703 tumor by the compound of formula (1) 4-ethoxy-2-hydroxy-6-methylbenzoic acid are shown in Fig. 9, Fig. 10, Fig. 11.
  • the compound of formula (1) significantly inhibited the growth of human liver cancer QGY-7703 (p ⁇ 0.05).
  • the tumor weight inhibition rate was 69.83%, and the relative tumor growth rate was 30.84%.
  • the relative volume of tumors was significantly lower than that of the control group (p ⁇ 0.05).
  • the inhibitory effect of this compound on human liver cancer is significantly stronger than that of lung cancer.
  • Photographs of QGY-7703 tumors of each group at the end of the experiment are shown in Fig. 12.
  • mice In the anti-cancer test in nude mice, the body weight of the mice was measured at each time point of measuring the tumor volume; at the end of the experiment, the mice were sacrificed, and the liver, spleen, kidney, testis (male) of the mice were completely taken out. ) and weigh and calculate the coefficients of each organ.
  • the NC in the chart below indicates the Negative Control.
  • the compound of formula (1) 4-ethoxy-2-hydroxy-6-methylbenzoic acid can affect the liver and spleen development of male nude mice, but does not affect the kidney and testicular development of male mice.
  • mice in the #:5-fluorouracil group died on the 20th day, and another 3 were extremely thin and sudden death;
  • the anticancer drug 5-fluorouracil and the compound of formula (1) significantly affected the body weight development of mice.
  • the effect of 5-fluorouracil on the body weight of mice was significantly higher than that of the compound.
  • 2 animals in the 5-Fu group died due to failure, and another 3 mice died of extreme exhaustion.
  • the compound of the compound of formula (1) was healthy and active at the end of the test without death.
  • the compound of formula (1) significantly promoted apoptosis and necrosis of cancer cells.
  • doses of 100 ⁇ M, 150 ⁇ M and 200 ⁇ M the DNA fragment peaks (P2) formed by apoptosis and necrosis were 13.5%, respectively. 24.7% and 32.7%, there was a significant dose-effect relationship, which was significantly higher than 6.8% of the control group, and the compound significantly increased the G0/G1 and G2/M peaks and cell cycle distribution ratio of cancer cells, which significantly affected Differentiation and cycle of cancer cells.
  • liver cancer cells QGY-7703, colon cancer cells HCT-116, breast cancer cells MCF-7, lung cancer cells A-549 were treated with the compound of formula (1) for 24 hours, then the cells were collected, lysed, adjusted for protein concentration, and Western Blot technology determination.
  • the results are shown in Figure 16 (SP indicates the positive control Staurosporine, SP1 was 50 nM, and SP2 was 100 nM).
  • Test method In the anti-cancer test in vitro, a negative control group, a dose of positive anticancer drug group, three doses of D6B2 group and three doses of D6B2 plus positive anticancer drug group were set, and cells were collected 72 hours after dosing. Count, calculate inhibition rate and perform statistical analysis.
  • 4-ethoxy-2-hydroxy-6-methylbenzoic acid and cyclophosphamide have a combined inhibition of mouse liver cancer H-22 in vivo.
  • cyclophosphamide was 40 mg/kg
  • the inhibition rate of mouse liver cancer H-22 was 75.83%
  • the inhibition rate of D26B2 to 40-22/kg was 38.99%.
  • the two were used together, the combined group of mice No tumor growth was observed.
  • the compound of the formula (1) 4-ethoxy-2-hydroxy-6-methylbenzoic acid
  • the compound of formula (1) is neutralized with sodium hydrogencarbonate under heating, and then adjusted to a pH of 7.0-7.5 with a soda ash solution, heated, evaporated, Crystallization forms the sodium salt of the compound of formula (1) (Sodium 4-ethoxy-2-hydroxy-6-methylbenzoate) which is soluble in water.
  • the compound of formula (1) can also form salts with other basic ions or groups.
  • the compound of the formula (1) 4-ethoxy-2-hydroxy-6-methylbenzoic acid, is dissolved in a small amount of DMSO, and is usually prepared by adding water for injection, fine filtration, and potting to prepare an injection.
  • the compound of the formula (1) 4-ethoxy-2-hydroxy-6-methylbenzoic acid or a salt thereof, is dissolved in a small amount of ethanol, methanol or distilled water, filtered through a sterile suction funnel, and then sterilely filtered. It is divided into ampoules and sterilized by low temperature freeze-drying to obtain a powder injection.
  • the compound of the formula (1) 4-ethoxy-2-hydroxy-6-methylbenzoic acid or a salt thereof, is added to an excipient in a ratio required for the preparation to prepare a powder.
  • the compound of the formula (1) 4-ethoxy-2-hydroxy-6-methylbenzoic acid or a salt thereof, is added to an excipient in a ratio required for the preparation, and granulated and tableted to prepare a tablet.
  • the compound of the formula (1), 4-ethoxy-2-hydroxy-6-methylbenzoic acid or a salt thereof, is prepared into an oral liquid by a conventional oral liquid preparation method.
  • the compound of the formula (1) 4-ethoxy-2-hydroxy-6-methylbenzoic acid or a salt thereof, is added to the excipient in a ratio required for the preparation to prepare a capsule, a granule or a granule.
  • the compound of the formula (1) 4-ethoxy-2-hydroxy-6-methylbenzoic acid or a salt thereof, is added to a food or other carrier in a ratio required for the product to prepare a health care product or other functional product.
  • the compound of the formula (1) 4-ethoxy-2-hydroxy-6-methylbenzoic acid or a salt thereof, is mixed with other antitumor drugs according to the needs of the treatment, and is prepared into various preparations to form a combined antitumor drug. Combination Things.
  • the compound 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester is a known structural compound which can be chemically synthesized or purchased from the market.
  • D6B1 2,4-dihydroxy-3,6-dimethylbenzoic acid of the present invention
  • B1 is 200 ⁇ M
  • B2 is 150 ⁇ M
  • B3 is 100 ⁇ M
  • B4 is 50 ⁇ M
  • B5 is 25 ⁇ M. It has significant and selective anti-tumor effects, combined anti-tumor effects with clinical anti-tumor drugs, and anti-tumor mechanism.
  • Test method Methyl 2,4-dihydroxy-3,6-dimethylbenzoate was dissolved in DMSO, and an in vitro anticancer test was carried out at a final concentration of 25, 50, 100, 150, 200 ⁇ M. Human tumor cells in logarithmic growth phase were collected, suspended in complete medium, seeded in 6-well plates, and 2 ml of cell suspension was added to each well. After incubating for 24 hours at 37 ° C in a 5% CO 2 incubator, the cell count of 3 wells was counted as the number of cells at 0 hours; the culture solution of the remaining wells was aspirated, and then the corresponding concentration of the compound 2,4-dihydroxy-3,6 was added.
  • the inhibition rates of the compounds against QGY-7703, MCF-7, A-549, HCT-116, Hela, CNE, HepG2 and XWLC were 96.46%, 58.94%, 63.50%, and 39.66%, respectively, at a concentration of 200 ⁇ M for 72 hours. 84.75%, 78.89%, 81.82%, and 13.62%, there is a significant dose-response relationship. Inhibition of different cell lines by methyl 2,4-dihydroxy-3,6-dimethylbenzoate The dose-effect relationship is shown in Figure 25.
  • the cells were withered at doses of 100 ⁇ M, 150 ⁇ M and 200 ⁇ M.
  • liver cancer cells QGY-7703, colon cancer cells HCT-116, breast cancer cells MCF-7 and lung cancer cell A-549 were treated with compound 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester for 24 hours.
  • the cells were collected, lysed and adjusted for protein concentration. The results were determined by Western Blot. Figure 28.
  • Methyl 2,4-dihydroxy-3,6-dimethylbenzoate significantly promoted the formation of PARP cleavage by apoptosis marker primers in hepatoma cell line QGY-7703 and colon cancer cells HCT-116.
  • the formation of PARP cleavage in cancer cell MCF-7 and lung cancer cell A-549 had no effect. This compound also did not affect the cleaved caspase-3 levels and PTEN expression of these 4 cancer cells.
  • Test method In the anti-cancer test in vitro, a negative control group, a dose of positive anticancer drug group, 3 doses of D6B1 group and three doses of D6B1 plus positive anticancer drug group were set, and cells were collected 72 hours after dosing. Count, calculate inhibition rate and perform statistical analysis.
  • the compound 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester has significant synergistic anticancer effect with 5-fluorouracil, paclitaxel, cisplatin and the like in vitro.
  • the in vitro combined effects of the compound 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl ester with cisplatin on Hela are shown in Table 7 and Figure 29.
  • Methyl 2,4-dihydroxy-3,6-dimethylbenzoate and 5-fluorouracil and paclitaxel also have significant synergistic anticancer effects on human breast cancer cell MCF-7 in vitro.
  • Ethyl 2,4-dihydroxy-6-methylbenzoate is a known structural compound which can be chemically synthesized using 2,4-dihydroxy-6-methylbenzoic acid and ethanol as a raw material, or commercially available.
  • Test method The compound 2,4-dihydroxy-6-methylbenzoic acid ethyl ester was dissolved in DMSO and tested at concentrations of 25, 50, 100, 150, and 200 ⁇ M. Tumor cells were suspended in complete culture and inoculated into 6-well plates. After incubating for 24 hours at 37 ° C in a 5% CO 2 incubator, the cell count of 3 wells was counted as the number of cells at 0 hours; the culture solution of the remaining wells was aspirated, and then the corresponding concentration of the compound 2,4-dihydroxy-6-A was added.
  • a complete medium of ethyl benzoate 2 ml was prepared, and a DMSO solvent control and a positive drug control were set at the same time, and 9 wells were paralleled for each concentration and the control group.
  • 3 well cell counts were collected for each concentration, and the mean and standard deviation of cell numbers were calculated.
  • the cell growth curve was drawn and statistical difference test was performed. Or use 96-well plate culture, by MTT method The OD values after 24, 48 and 72 hours of drug treatment were determined, and the dose-effect relationship and time-effect relationship of the drugs were observed.
  • RESULTS Compound 2,4-dihydroxy-6-methylbenzoic acid ethyl ester on human breast cancer cell line MCF-7, human lung cancer cell line A-549, human liver cancer cell line QGY-7703, human cervical cancer cell line Hela Human leukemia K-562, human hepatoma cell line HepG2, human nasopharyngeal carcinoma cell line CNE, human hepatoma cell line HepG2, etc. all have significant anticancer effects.
  • the compound has a large difference in the inhibition rate of different cancer cells.
  • Human liver cancer cell QGY-7703 and cervical cancer Hela are very sensitive to the compound, and human cholangiocarcinoma QBC-939 and human colon cancer HCT-116 are not sensitive to the compound.
  • the inhibition rates of the compounds against QGY-7703, CNE, Hela, HepG2, XWLC, A-549, and HCT-116 were 93.86%, 73.12%, 90.75%, 75.18%, 51.13%, and 52.23, respectively, at a concentration of 200 ⁇ M for 72 hours. %, 43.50%, had obvious dose-response relationship; the inhibition rate of the compound on human normal cell lines HUVEC and Beas-2b was 16.52% and 26.01%, respectively, which was significantly lower than the cytotoxic effect on the above cancer cell lines.
  • the compound 2,4-dihydroxy-6-methylbenzoic acid ethyl ester has a selective anticancer effect.
  • the dose-effect relationship between the inhibitory effect of the compound 2,4-dihydroxy-6-methylbenzoate on some cancer cell lines and normal cell lines in vitro is shown in Fig. 36.
  • mice transplanted with mouse 2,4-dihydroxy-6-methylbenzoate to mouse sarcoma S-180, mouse liver cancer H-22, C57BL/6J mice transplanted in Kunming mice and ICR mice Lung cancer 3ll, as well as balb/c nude mice transplanted human lung cancer NCI-H460 and human liver cancer QGY-7703 have significant anti-cancer effects in vivo (p ⁇ 0.05).
  • the compound 2,4-dihydroxy-6-methylbenzoic acid ethyl ester (D) has significant synergistic anti-tumor effect with the anticancer drugs 5-fluorouracil, paclitaxel and cisplatin in vitro.
  • the results of in vitro anti-cervical cancer Hela in combination with the compound 2,4-dihydroxy-6-methylbenzoic acid ethyl ester and cisplatin are shown in Table 8 and Figure 37.
  • Test method In vitro anticancer test, a negative control group, a dose of positive anticancer drug group, 3 doses of compound 2,4-dihydroxy-6-methylbenzoic acid ethyl ester group and three doses of compound The combination of 2,4-dihydroxy-6-methylbenzoic acid ethyl ester plus positive anticancer drug group was collected at 72 hours after dosing, and the inhibition rate was calculated and statistical analysis was performed.
  • Ethyl 2,4-dihydroxy-6-methylbenzoate and 5-fluorouracil and paclitaxel also have significant in vitro synergistic anticancer effects on colon cancer cells and human breast cancer cells.
  • the phenol potassium salt of the intermediate product 3,5-dihydroxytoluene can be obtained by high temperature dechlorination hydrolysis reaction, and then acidified to obtain the compound 1,3-dihydroxyl group. -5-methylbenzene.
  • Test method 1,3-dihydroxy-5-methylbenzene was dissolved in DMSO for detection of anticancer activity in vitro.
  • Human tumor cells in logarithmic growth phase were suspended in complete medium and inoculated into 6-well plates. After incubating for 24 hours at 37 ° C in a 5% CO 2 incubator, the culture solution was aspirated, and then 2 ml of a complete culture solution containing the corresponding concentration of 1,3-dihydroxy-5-methylbenzene compound was added as designed, and a DMSO solvent control was also set. The sample and the DMSO control group were each made of 3 wells in parallel. At 72 hours after dosing, cells were collected for counting, and the mean and standard deviation of the number of cells were counted, and statistical difference test was performed.
  • Test results After treatment for 72 hours, the compound 1,3-dihydroxy-5-methylbenzene was administered at a dose of 20 ⁇ g/ml (161.3 ⁇ M) to human breast cancer MCF-7, human lung cancer A-549, human colon cancer HCT.
  • the inhibition rates of -116 were 63.01%, 53.83%, and 92.93%, respectively, and there was a significant difference (p ⁇ 0.05) compared with the control group.
  • the compound of the present example 2,6-dihydroxy-4-methylbenzaldehyde, can be formed from the substrate 2-hydroxymethyl-5-methylbenzene-1,3-diol by oxidation.
  • Test method 2,6-dihydroxy-4-methylbenzaldehyde was dissolved in DMSO to a concentration of four doses of 12.5 ⁇ M, 25.0 ⁇ M, 50.0 ⁇ M and 100 ⁇ M.
  • Test method The same method as in vitro anticancer test in Example 15. Methyl 2,4-dihydroxy-6-methylbenzoate was tested in DMSO.
  • Test results Compound 2,4-dihydroxy-6-methylbenzoate after 72 hours of drug treatment
  • the inhibition rates of human breast cancer MCF-7, human lung cancer A-549, and human colon cancer HCT-116 at doses of 20 ⁇ g/ml (110.0 ⁇ M) were 99.18%, 82.15%, and 81.00%, respectively, which were significant compared with the control group.
  • Sexual differences p ⁇ 0.05).
  • Ethyl 3-aldehyde-2,4-dihydroxy-6-methylbenzoate can be chemically synthesized or purchased from the market.
  • Test method The same method as in vitro anticancer test in Example 15. Ethyl 3-aldehyde-2,4-dihydroxy-6-methylbenzoate was tested in DMSO.
  • Test results After 72 hours of drug treatment, the compound 3-aldehyde-2,4-dihydroxy-6-methylbenzoic acid ethyl ester was administered at a dose of 20 ⁇ g/ml (89.3 ⁇ M) to human breast cancer MCF-7, human lung cancer.
  • the inhibition rates of A-549 and human colon cancer HCT-116 were 29.32%, 24.58% and 54.60%, respectively.
  • the inhibition of HCT-116 was significantly different from that of the control group (p ⁇ 0.05).
  • Ethyl 3,5-dihydroxy-4-methylbenzoate and 3-aldehyde- 2,4-dihydroxy-6-methylbenzoic acid (3-formyl-) 2,4-dihydroxy-6-methylbenzoic acid was mixed at a molar concentration of 1:1, and concentrated sulfuric acid was used as a catalyst.
  • the esterification reaction was carried out by heating at 60 ° C for 1 to 3 hours. After cooling, it is thoroughly washed with distilled water, and the water-insoluble portion is collected, and then separated by silica gel column chromatography to obtain a compound 3-aldehyde-2,4-dihydroxy-6-methylbenzene having a purity greater than 99.8%.
  • 5-(ethoxycarbonyl)-3-hydroxy-2-methylphenyl formate 5-(ethoxycarbonyl)-3-hydroxy-2-methylphenyl formate.
  • Test method The same in vitro anticancer test method as in Example 15. Tris-aldehyde-2,4-dihydroxy-6-methylbenzoic acid 5-(ethoxycarbonyl)-3-hydroxy-2-methylphenyl ester was tested in DMSO.
  • the compound of the formula (2) is first dissolved in a small amount of a medicinal solvent, and then formulated into a corresponding concentration by conventional addition of water for injection, finely filtered, and potted and sterilized to prepare an injection.
  • the compound or the salt of the formula (2) is dissolved in a solvent for injection or distilled water, filtered through a sterile suction filter funnel, and then aseptically finely filtered, and dispensed in an ampoule, freeze-dried at a low temperature, and then aseptically melt-sealed to obtain a powder injection.
  • the compound of the formula (2) or a salt is added to the excipient in a ratio required for the preparation to prepare a powder.
  • the compound of the formula (2) or a salt thereof is added to the excipient in a ratio required for the preparation, and granulated and tableted to prepare a tablet.
  • the compound of the formula (2) or a salt thereof is prepared into an oral liquid by a conventional oral liquid preparation method.
  • the compound of the formula (2) or a salt thereof is added to the excipient in a ratio required for the preparation to prepare a capsule, or a granule, or a granule.
  • the compound of the formula (2) or a salt thereof is added to a food or other carrier in a ratio required by the product to prepare a health care product or other functional article.
  • the compound of the formula (2) or a salt thereof is mixed with other antitumor drugs in proportion to the needs of the treatment to prepare various preparations to form a combined antitumor pharmaceutical composition.

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Abstract

本发明公开了具有式(1)和式(2)结构的苯基取代化合物及其药用组合物和应用。该化合物在体内和体外均能显著性杀灭和/或抑制人类和动物的多种肿瘤细胞，能显著性促进癌细胞凋亡和坏死、调节癌细胞生长周期、抑制癌细胞增殖。在同等剂量下，该化合物对人体正常细胞的细胞毒性作用显著性低于对人类肿瘤细胞的细胞毒作用。该化合物与临床抗肿瘤药物联用具有显著性协同抗肿瘤作用。该化合物或其药学上可接受的盐可应用于制备抗肿瘤的药物、抗肿瘤药物组合物、联合抗肿瘤药物组合物或辅助抗肿瘤保健品。

Description

苯基取代化合物及其药物组合物和应用 技术领域
本申请属于化学医药领域，特别是涉及苯基取代化合物及其药物组合物和应用。
背景技术
癌症是严重威胁人类健康和生命安全的一个重大疾病，目前已成为人类的第一位死因。据全球癌症统计报告，2008年全球新发癌症病例为1266.1万例，死亡756.4万例；中国癌症新发病例为281.6万例，死亡195.8万例。2012年全球新发癌症病例为1400多万例，死亡820多万例；《2012中国肿瘤登记年报》披露，中国2012年新发癌症病例312万，平均每分钟有6人确诊为恶性肿瘤。癌症的发病率还在上升，癌症已成为严重威胁人民健康和生命安全的一个主要卫生问题。
癌症在世界上目前仍是一种难治之症。癌症的化学药物治疗作为全身性治疗措施，在癌症的治疗中占有极重要的地位，也是今后彻底解决癌症治疗问题的希望。
临床上目前使用的癌症化疗药物，很多药物存在毒副作用大、疗效不确定、选择性差以及癌细胞耐药等问题，使癌症化学药物治疗在临床上的应用和效果受到很大限制。因此，开发疗效明确、低毒安全、特别是具有选择性抗癌作用的化疗药物，是抗癌新药研究的一个重要方向和迫切任务。
发明概述
一方面，本发明涉及具有式(1)结构的4-乙氧基-2-羟基-6-甲基苯甲酸或其药学上可接受的盐，
Figure PCTCN2014000808-appb-000001
另一方面，本发明涉及具有式(2)结构的苯基取代化合物或其药学上可接受的盐，其中，R1为氢、C1-C4烷基或C1-C4醛基；R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基，
Figure PCTCN2014000808-appb-000002
其中所述化合物不为4-乙氧基-2-羟基-6-甲基苯甲酸、2,4-二羟基-3,6-二甲基苯甲酸甲酯、2,4-二羟基-6-甲基苯甲酸乙酯、1,3-二羟基-5-甲基苯、2,6-二羟基-4-甲基苯甲醛、2,4-二羟基-6-甲基苯甲酸甲酯和3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯。
另一方面，本发明涉及一种抗肿瘤的药物组合物，其活性成分包括具有式(1)结构的4-乙氧基-2-羟基-6-甲基苯甲酸或式(2)结构的苯基取代化合物或其药学上可接受的盐，其中式(2)中R1为氢、C1-C4烷基或C1-C4醛基；R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基。
再一方面，本发明涉及一种联合抗肿瘤的药物组合物，其活性成分包括具有式(1)结构的4-乙氧基-2-羟基-6-甲基苯甲酸或式(2)结构的苯基取代化合物或其药学上可接受的盐，和5-氟尿嘧啶、紫杉醇、顺铂或环磷酰胺中的至少一种，其中式(2)中R1为氢、C1-C4烷基或C1-C4醛基；R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基。
又一方面，本发明涉及具有式(1)结构的4-乙氧基-2-羟基-6-甲基苯甲酸或式(2)结构的苯基取代化合物或其药学上可接受的盐在制备抗肿瘤 药物或辅助抗肿瘤保健品中的应用，其中式(2)中R1为氢、C1-C4烷基或C1-C4醛基；R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基。
另一方面，本申请还涉及抑制或治疗肿瘤生长的方法，包括将抑制或治疗有效量的具有式(1)结构的4-乙氧基-2-羟基-6-甲基苯甲酸或式(2)结构的苯基取代化合物或其药学上可接受的盐，或者上文所述的药物组合物给予需要此治疗的个体，其中式(2)中R1为氢、C1-C4烷基或C1-C4醛基；R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基。
本申请具有式(1)和式(2)结构的化合物在体内和体外均能显著性杀灭和/或抑制人类和动物的多种肿瘤细胞，能显著性促进癌细胞凋亡和坏死、调节癌细胞生长周期、抑制癌细胞增殖。在同等剂量下，具有式(1)和式(2)结构的化合物对人体正常细胞的细胞毒性作用显著性低于对人类肿瘤细胞的细胞毒作用。具有式(1)和式(2)结构的化合物与临床抗肿瘤药物如顺铂、5-氟尿嘧啶、紫杉醇或环磷酰胺联用具有显著性协同抗肿瘤作用。具有式(1)和式(2)结构的化合物或其药学上可接受的盐可应用于制备抗肿瘤的药物、抗肿瘤药物组合物、联合抗肿瘤药物组合物或辅助抗肿瘤保健品。
附图说明
图1为式(1)化合物的质谱图；
图2为式(1)化合物的核磁氢谱图；
图3为式(1)化合物的核磁碳谱图和DEPT谱图；
图4为本发明式(1)化合物对不同肿瘤细胞株的剂量-效应曲线图；
图5为本发明式(1)化合物体外抗肿瘤实验的时间-效应、剂量-效应关系曲线图；
图6为本发明式(1)化合物体内抗肿瘤(NCI-H460)试验时，不同时间点的肿瘤相对体积(RTV)；
图7为本发明式(1)化合物体内抗肿瘤(NCI-H460)试验时，不同时间 点的肿瘤相对增值率(％)；
图8为本发明式(1)化合物给药10次后的NCI-H460肿瘤重量；
图9为本发明式(1)化合物体内抗肿瘤(QGY-7703)试验时，不同时间点的肿瘤相对体积(RTV)；
图10为本发明式(1)化合物体内抗肿瘤(QGY-7703)试验时，不同时间点的肿瘤相对增殖率(％)；
图11为本发明式(1)化合物体内抗肿瘤(QGY-7703)实验结束时，各组的肿瘤平均重量；
图12为本发明式(1)化合物体内抗肿瘤(QGY-7703)实验结束时，各组动物的肿瘤照片；
图13为本发明式(1)化合物体内抗肿瘤(QGY-7703)实验结束时，各组动物的脾脏照片；
图14为本发明式(1)化合物溶剂对照的流式细胞仪检测结果(QGY-7703)；
图15为本发明式(1)化合物的流式细胞仪检测结果(QGY-7703)；
图16为本发明式(1)化合物对QGY-7703、HCT-116、MCF-7和A-549细胞株蛋白水平影响的Western Blot检测结果；其中：NC表示阴性对照，SP表示阳性对照物星状孢菌素(Staurosporine)，A、B、D、L表示不同的待测物，其中B表示化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯，D表示化合物4-乙氧基-2-羟基-6-甲基苯甲酸；
图17为本发明式(1)化合物与5-氟尿嘧啶的体外联合抗A-549肿瘤作用；
图18为本发明式(1)化合物与顺铂的体外联合抗A-549肿瘤作用；
图19为化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯的质谱图；
图20为化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯的核磁氢谱图；
图21为化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯的核磁碳谱图和DEPT谱图；
图22为化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯的核磁二维图谱HMBC；
图23为化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯的核磁二维图谱HSQC；
图24为化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯的核磁二维图谱Roesy；
图25为化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯体外对人类不同肿瘤细胞株和人类正常肺上皮细胞株Beas-2b抑制作用的剂量-效应关系曲线图；
图26为临床抗癌药物5-氟尿嘧啶、顺铂对人类正常肺上皮细胞株Beas-2b抑制作用的剂量-效应关系曲线图；
图27为本发明化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯的流式细胞仪检测结果(QGY-7703)；
图28为本发明化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯对QGY-7703、HCT-116、MCF-7和A-549细胞株蛋白水平影响的Western Blot检测结果；注：NC表示阴性对照，SP表示阳性对照物星状孢菌素(Staurosporine)，A、B、D、L表示不同的待测物，其中B表示化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯，D表示化合物4-乙氧基-2-羟基-6-甲基苯甲酸。
图29为本发明化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯与顺铂体外联合抗Hela肿瘤细胞作用；
图30为化合物2,4-二羟基-6-甲基苯甲酸乙酯的质谱图；
图31为化合物2,4-二羟基-6-甲基苯甲酸乙酯的核磁氢谱图；
图32为化合物2,4-二羟基-6-甲基苯甲酸乙酯的核磁碳谱图和DEPT谱图；
图33为化合物2,4-二羟基-6-甲基苯甲酸乙酯的二维谱图HMBC；
图34为化合物2,4-二羟基-6-甲基苯甲酸乙酯的二维谱图HSQC；
图35为化合物2,4-二羟基-6-甲基苯甲酸乙酯的二维谱图Roesy；
图36为本发明化合物2,4-二羟基-6-甲基苯甲酸乙酯对不同肿瘤细胞株和人类正常肺上皮细胞株Beas-2b的体外抑制剂量-效应关系曲线图；
图37为本发明化合物2,4-二羟基-6-甲基苯甲酸乙酯与顺铂的体外联合抗Hela肿瘤细胞作用；
图38为化合物2,6-二羟基-4-甲基苯甲醛的质谱图；
图39为化合物2,6-二羟基-4-甲基苯甲醛的核磁氢谱图；
图40为化合物2,6-二羟基-4-甲基苯甲醛的核磁碳谱图和DEPT谱图；
图41为化合物2,6-二羟基-4-甲基苯甲醛体外抗癌作用的剂量-效应关系曲线图；
图42为化合物2,4-二羟基-6-甲基苯甲酸甲酯的质谱图；
图43为化合物2,4-二羟基-6-甲基苯甲酸甲酯的核磁氢谱图；
图44为化合物2,4-二羟基-6-甲基苯甲酸甲酯的核磁碳谱图和DEPT谱图；
图45为化合物3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯的质谱图；
图46为化合物3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯的核磁氢谱图；
图47为化合物3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯的核磁碳谱图和DEPT谱图；
图48为化合物3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯的质谱图；
图49为化合物3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯的核磁氢谱图；
图50为化合物3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯的核磁碳谱图和DEPT谱图。
发明详述
除非另有说明，本文中所用的术语具有如下含义：
术语“C1-C4烷基”意指具有一到四个碳原子的直链或支链的饱和烃基。C1-C4烷基的实例具体包括但不限于：甲基、乙基、丙基、异丙基、丁基、异丁基、1-甲基丙基、叔丁基等。
术语“C1-C4醛基”意指具有一到四个碳原子的直链或支链的醛基。C1-C4醛基的实例具体包括但不限于：甲醛基、乙醛基、丙醛基、异丙醛基等。
术语“C1-C4烷基酯基”意指“RCOO-”，其中R为如上所定义的C1-C4的烷基。
本申请中所述的“药物组合物”指本申请化合物与通常被本领域所接受的将生物活性化合物输送至诸如人类等哺乳动物的介质所形成的制剂。这 样的介质包括所有药物可接受的载体。
本申请中所述的“药物可接受的载体”意指包括但不限于已经被美国食品与药品管理局(FDA)认可的、可用于人类或动物的任何佐剂、赋形剂、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、香味增强剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗压剂、崩解剂、溶剂或乳化剂等对组成药物组合物无副作用的各种形式的载体。
本申请中所述的“抑制有效量”和“治疗有效量”可互换地使用，意指当对个体优选哺乳动物，较优选对人类给药时，本申请化合物足以有效抑制肿瘤细胞或治疗肿瘤的量。根据所选化合物、施药对象的状态，构成“抑制有效量”或“治疗有效量”的本申请化合物的量将会不同，但是本领域的技术人员根据其自身的知识以及本申请的公开可以依本领域常识确定本申请化合物的有效量。
术语“个体”是指哺乳动物，优选为人类。
术语“治疗”意为将本发明所述化合物或制剂进行给药以预防、改善或消除疾病或与所述疾病相关的一个或多个症状，且包括：
(i)预防疾病或疾病状态在哺乳动物中出现，特别是当这类哺乳动物易患有该疾病状态，但尚未被诊断为已患有该疾病状态时；
(ii)抑制疾病或疾病状态，即遏制其发展；
(iii)缓解疾病或疾病状态，即使该疾病或疾病状态消退。
“药物可接受的盐”包括“药物可接受的酸加合盐”和“药物可接受的碱加合盐”。
“药物可接受的酸加合盐”指保持游离碱的生物学有效性和性质的那些盐，所述酸加合盐是在生物学或其它方面合适的并且是使用无机酸或有机酸来形成的，所述无机酸例如但不限于盐酸、氢溴酸、硫酸、硝酸、磷酸等，所述有机酸例如但不限于乙酸、2,2-二氯乙酸、己二酸、褐藻酸、抗坏血酸、天冬氨酸、苯磺酸、苯羧酸、4-乙酰胺基苯羧酸、樟脑酸、樟脑-10-磺酸、癸酸、己酸、辛酸、碳酸、肉桂酸、柠檬酸、环己烷基氨基磺酸、十二烷基硫酸、乙烷-1,2-二磺酸、乙烷磺酸、2-羟基乙烷磺酸、甲酸、富马酸、粘酸、龙胆酸、葡庚糖酸、葡糖酸、葡糖醛酸、谷氨酸、戊二酸、2-氧代-戊二酸、甘油磷酸、乙醇酸、马尿酸、异丁酸、乳酸、乳糖醛酸、 月桂酸、马来酸、苹果酸、丙二酸、扁桃酸、甲烷磺酸、萘-1,5-二磺酸、萘-2-磺酸、1-羟基-2-萘甲酸、烟酸、油酸、乳清酸、棕榈酸、双羟萘酸、丙酸、焦谷氨酸、丙酮酸、水杨酸、4-氨基水杨酸、癸二酸、硬脂酸、丁二酸、酒石酸、硫氰酸、对甲苯磺酸、三氟乙酸、十一碳烯酸等。
“药物可接受的碱加合盐”指保持游离酸的生物学有效性和性质的那些盐，所述碱加成盐是在生物学或其它方面合适的。向游离酸中加入无机碱或有机碱来制备这些盐。本申请中，由无机碱衍生的盐是优选的，包括但不限于钠、钾、锂盐等。
一方面，本发明涉及具有式(1)结构的4-乙氧基-2-羟基-6-甲基苯甲酸或其药学上可接受的盐，
Figure PCTCN2014000808-appb-000003
另一方面，本发明涉及具有式(2)结构的苯基取代化合物或其药学上可接受的盐，其中，R1为氢、C1-C4烷基或C1-C4醛基；R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基，
Figure PCTCN2014000808-appb-000004
其中所述化合物不为4-乙氧基-2-羟基-6-甲基苯甲酸、2,4-二羟基-3,6-二甲基苯甲酸甲酯、2,4-二羟基-6-甲基苯甲酸乙酯、1,3-二羟基-5-甲基苯、2,6-二羟基-4-甲基苯甲醛、2,4-二羟基-6-甲基苯甲酸甲酯和3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯。
在一些实施方案中，R1为氢、甲基或甲醛基。在一些实施方案中，R2为氢、CH3COO-、C2H5COO-、HCOO-，或5-(乙氧羰基)-3-羟基-2-甲基苯基酯基。在一些实施方案中，R3为氢、甲基或乙基。
在一些实施方案中，具有式(2)结构的苯基取代化合物为3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯。
另一方面，本发明涉及一种抗肿瘤的药物组合物，其活性成分包括具有式(1)结构的4-乙氧基-2-羟基-6-甲基苯甲酸或式(2)结构的苯基取代化合物或其药学上可接受的盐，其中式(2)中R1为氢、C1-C4烷基或C1-C4醛基；R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基。
在一些实施方案中，式(2)中R1为氢、甲基或甲醛基。在一些实施方案中，式(2)中R2为氢、CH3COO-、C2H5COO-、HCOO-，或5-(乙氧羰基)-3-羟基-2-甲基苯基酯基。在一些实施方案中，式(2)中R3为氢、甲基或乙基。
在一些实施方案中，所述化合物选自4-乙氧基-2-羟基-6-甲基苯甲酸、2,4-二羟基-3,6-二甲基苯甲酸甲酯、2,4-二羟基-6-甲基苯甲酸乙酯、1,3-二羟基-5-甲基苯、2,6-二羟基-4-甲基苯甲醛、2,4-二羟基-6-甲基苯甲酸甲酯、3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯或3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯。
上述药物组合物还可包括药学上可接受的载体。
本领域所属技术人员根据所选化合物、给药对象的情况可以确定组合物中具体的治疗有效量。在一些实施方案中，有效量可以占组合物重量的0.01-99.99％，优选为1-99％，更优选为10-90％。
在一些实施方案中，该药物组合物的剂型可以为：注射液、粉针剂、片剂、口服液、胶囊、颗粒剂或冲剂。
再一方面，本发明涉及一种联合抗肿瘤的药物组合物，其活性成分包括具有式(1)结构的4-乙氧基-2-羟基-6-甲基苯甲酸或式(2)结构的苯基取代化合物或其药学上可接受的盐，和5-氟尿嘧啶、紫杉醇、顺铂或环磷酰胺中的至少一种，其中式(2)中R1为氢、C1-C4烷基或C1-C4醛基； R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基。
在一些实施方案中，式(2)中R1为氢、甲基或甲醛基。在一些实施方案中，式(2)中R2为氢、CH3COO-、C2H5COO-、HCOO-，或5-(乙氧羰基)-3-羟基-2-甲基苯基酯基。在一些实施方案中，式(2)中R3为氢、甲基或乙基。
在一些实施方案中，所述化合物选自4-乙氧基-2-羟基-6-甲基苯甲酸、2,4-二羟基-3,6-二甲基苯甲酸甲酯、2,4-二羟基-6-甲基苯甲酸乙酯、1,3-二羟基-5-甲基苯、2,6-二羟基-4-甲基苯甲醛、2,4-二羟基-6-甲基苯甲酸甲酯、3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯或3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯。
在一些实施方案中，上述具有式(1)结构的4-乙氧基-2-羟基-6-甲基苯甲酸或式(2)结构的苯基取代化合物或其药学上可接受的盐与所述5-氟尿嘧啶、紫杉醇、顺铂或环磷酰胺中的至少一种的比例按摩尔浓度计算为1:10～100:1。
又一方面，本发明涉及具有式(1)结构的4-乙氧基-2-羟基-6-甲基苯甲酸或式(2)结构的苯基取代化合物或其药学上可接受的盐在制备抗肿瘤药物或辅助抗肿瘤保健品中的应用，其中式(2)中R1为氢、C1-C4烷基或C1-C4醛基；R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基。
在一些实施方案中，式(2)中的R1为氢、甲基或甲醛基。在一些实施方案中，式(2)中的R2为氢、CH3COO-、C2H5COO-、HCOO-，或5-(乙氧羰基)-3-羟基-2-甲基苯基酯基。在一些实施方案中，式(2)中的R3为氢、甲基或乙基。
在一些实施方案中，所述化合物选自4-乙氧基-2-羟基-6-甲基苯甲酸、2,4-二羟基-3,6-二甲基苯甲酸甲酯、2,4-二羟基-6-甲基苯甲酸乙酯、1,3-二羟基-5-甲基苯、2,6-二羟基-4-甲基苯甲醛、2,4-二羟基-6-甲基苯甲酸甲酯、3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯或3-醛基-2,4-二羟基-6-甲基苯甲酸 5-(乙氧羰基)-3-羟基-2-甲基苯基酯。
在一些实施方案中，所述肿瘤为以下人类肿瘤中的任一种：人乳腺癌、前列腺癌、肺癌、胃癌、宫颈癌、结肠癌、肝癌、卵巢癌、白血病、神经瘤、肉瘤、鼻咽癌。
所述辅助抗肿瘤保健品，是指在肿瘤病人的治疗过程中，用于肿瘤病人的辅助治疗，可能对肿瘤病人的治疗具有增敏作用，但不以治疗为目的保健品。
另一方面，本申请还涉及抑制或治疗肿瘤生长的方法，包括将抑制或治疗有效量的具有式(1)结构的4-乙氧基-2-羟基-6-甲基苯甲酸或式(2)结构的苯基取代化合物或其药学上可接受的盐，或者上文所述的药物组合物给予需要此治疗的个体，其中式(2)中R1为氢、C1-C4烷基或C1-C4醛基；R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基。
在一些实施方案中，式(2)中的R1为氢、甲基或甲醛基。在一些实施方案中，式(2)中的R2为氢、CH3COO-、C2H5COO-、HCOO-，或5-(乙氧羰基)-3-羟基-2-甲基苯基酯基。在一些实施方案中，式(2)中的R3为氢、甲基或乙基。
在一些实施方案中，所述化合物选自4-乙氧基-2-羟基-6-甲基苯甲酸、2,4-二羟基-3,6-二甲基苯甲酸甲酯、2,4-二羟基-6-甲基苯甲酸乙酯、1,3-二羟基-5-甲基苯、2,6-二羟基-4-甲基苯甲醛、2,4-二羟基-6-甲基苯甲酸甲酯、3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯或3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯。
在一些实施方案中，所述肿瘤为以下人类肿瘤中的任一种：人乳腺癌、前列腺癌、肺癌、胃癌、宫颈癌、结肠癌、肝癌、卵巢癌、白血病、神经瘤、肉瘤、鼻咽癌。
在体外抗肿瘤试验中，具有式(1)或式(2)结构的化合物对肿瘤细胞的抑制有效量大于或等于5.0μM。
采用注射给药时，具有式(1)或式(2)结构的化合物治疗有效量大于或等于10mg/kg。
本发明4-乙氧基-2-羟基-6-甲基苯甲酸(4-ethoxy-2-hydroxy-6-methyl benzoic acid)化合物为新结构物质，可以通过相转移催化反应进行化学合成。该化合物或其盐类可作为抗肿瘤化学治疗药物，或作为抗肿瘤药物活性成分的抗肿瘤药物组合物，以及作为辅助抗肿瘤保健品。4-乙氧基-2-羟基-6-甲基苯甲酸体外能显著性杀灭和/或抑制人类和动物的多种癌细胞，如人乳腺癌、前列腺癌、肺癌、胃癌、宫颈癌、结肠癌、肝癌细胞，以及小鼠肉瘤、肝癌、肺癌细胞等；该化合物在同等剂量下，对人体正常细胞的细胞毒作用显著性低于对敏感人类肿瘤细胞的细胞毒作用。该化合物体内对balb/c裸鼠移植人类肺癌和人类肝癌，以及昆明种小鼠和ICR小鼠移植的小鼠肉瘤和小鼠肝癌，C57BL/6J小鼠移植的小鼠肺癌等肿瘤，均具有显著性的生长抑制作用。该化合物与临床使用的抗肿瘤化疗药物在体外和/或体内都具有联合协同抗肿瘤作用。该化合物具有选择性抗癌作用，不同癌细胞株的敏感性不同，肝癌细胞对该化合物非常敏感。该化合物能显著性促进癌细胞凋亡、调节癌细胞周期与分化、抑制癌细胞的增殖。该化合物在有效抗癌剂量下，对小鼠的体重增长和脏器发育无显著性毒性，或毒性作用显著性低于临床抗癌药物如环磷酰胺和5-氟尿嘧啶。
本发明化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯(methy2,4-dihydroxy-3,6-dimethyllbenzoate)可以由化学合成得到。该化合物可作为抗肿瘤化学治疗药物，或作为抗肿瘤药物活性成分的抗肿瘤药物组合物，以及作为辅助抗肿瘤保健品。该化合物体外能显著性杀灭和/或抑制人类肝癌、乳腺癌、宫颈癌、肺癌、鼻咽癌等癌细胞。该化合物具有靶向性抗癌作用，不同癌细胞株的敏感性不同，人类肝癌细胞和宫颈癌细胞对该化合物非常敏感，人结肠癌和宣威肺癌对该化合物不敏感。该化合物在体外对人类正常细胞株如HUVEC、Beas-2b的细胞毒作用显著性低于对敏感癌细胞株的细胞毒作用，具有选择性抗癌作用。该化合物对人类正常细胞株的毒性作用显著性低于临床抗癌药物5-氟尿嘧啶、顺铂、紫杉醇，毒性低。该化合物与临床使用的抗肿瘤化疗药物如顺铂、5-氟尿嘧啶、紫杉醇等在体外具有联合协同抗肿瘤作用。该化合物能显著性促进癌细胞凋亡、调节癌细胞周期与分化、抑制癌细胞的增殖。
本发明的化合物2,4-二羟基-6-甲基苯甲酸乙酯(ethyl 2,4-dihydroxy-6-methylbenzoate)可以由化学合成得到。该化合物可作为抗肿瘤化学治疗药物，或作为抗肿瘤药物活性成分的抗肿瘤药物组合物，以及作为辅助抗肿瘤保健品。该化合物体外能显著性杀灭和/或抑制人类乳腺癌、肺癌、鼻咽癌、宫颈癌、肝癌、白血病等癌细胞。该化合物具有靶向性抗癌作用，不同癌细胞株的敏感性差异很大，人肝癌细胞和人宫颈癌细胞对该化合物非常敏感，人胆管癌、宣威肺癌、人结肠癌对该化合物不敏感。该化合物体内对昆明种小鼠和ICR小鼠移植的小鼠肉瘤S-180和小鼠肝癌H22，C57BL/6J小鼠移植的小鼠肺腺癌3ll等肿瘤，以及balb/c裸鼠移植人类肺癌NCI-H460和人类肝癌QGY-7703均具有显著性的生长抑制作用。该化合物与临床使用的抗肿瘤化疗药物如顺铂、5-氟尿嘧啶、紫杉醇、环磷酰胺等在体外和/或体内具有联合协同抗肿瘤作用。该化合物能显著性促进癌细胞凋亡、调节癌细胞周期与分化、抑制癌细胞的增殖。该化合物在体外对人类正常细胞株如HUVEC、Beas-2b的细胞毒作用显著性低于对敏感癌细胞株的细胞毒作用，具有选择性抗癌作用。该化合物对人类正常细胞株的毒性作用显著性低于临床抗癌药物5-氟尿嘧啶、顺铂、紫杉醇，细胞毒作用低。
本发明的化合物1,3-二羟基-5-甲基苯(5-methylbenzene-1,3-diol)可以由化学合成得到。该化合物可作为抗肿瘤化学治疗药物，或作为抗肿瘤药物活性成分的抗肿瘤药物组合物，以及作为辅助抗肿瘤保健品。该化合物体外能显著性杀灭和/或抑制人类人乳腺癌、宫颈癌、肝癌细胞。
本发明的化合物2,6-二羟基-4-甲基苯甲醛(2,6-dihydroxy-4-methylbenzaldehyde)可以由化学合成得到。该化合物可作为抗肿瘤化学治疗药物，或作为抗肿瘤药物活性成分的抗肿瘤药物组合物，以及作为辅助抗肿瘤保健品。该化合物体外能显著性杀灭和/或抑制人类人乳腺癌、肺癌、宫颈癌、肝癌细胞。
本发明的化合物2,4-二羟基-6-甲基苯甲酸甲酯(methyl2,4-dihydroxy-6-methylbenzoate)可以由化学合成得到。该化合物可作为抗肿瘤化学治疗药物，或作为抗肿瘤药物活性成分的抗肿瘤药物组合物，以及作为辅助抗肿瘤保健品。该化合物体外能显著性杀灭和/或抑制人类人乳腺癌、肺癌、宫颈癌、结肠癌、肝癌细胞。
本发明的化合物3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯(ethyl3-formyl-2,4-dihydroxy-6-methylbenzoate)可以由化学合成得到。该化合物可作为抗肿瘤化学治疗药物，或作为抗肿瘤药物活性成分的抗肿瘤药物组合物，以及作为辅助抗肿瘤保健品。该化合物体外能显著性杀灭和/或抑制人类人宫颈癌、乳腺癌、肝癌、结肠癌细胞。
本发明的化合物3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯(5-(ethoxycrbonyl)-3-hydroxy-2-methylphenyl 3-formyl-2,4-dihydroxy-6-methylbenzoate)为新结构物质，该化合物可以通过催化反应进行化学合成得到。该化合物可作为抗肿瘤化学治疗药物，或作为抗肿瘤药物活性成分的抗肿瘤药物组合物，以及作为辅助抗肿瘤保健品。该化合物体外能显著性杀灭和/或抑制人类人乳腺癌、肺癌、宫颈癌、结肠癌、肝癌细胞。
以下通过实施例对本发明做进一步的阐述。
实施例1：式(1)所示化合物4-乙氧基-2-羟基-6-甲基苯甲酸的合成及结构鉴定
1、制备方法
相转移催化合成与柱层析分离
利用相转移催化反应，在氢氧化钠存在下，2,4-二羟基-6-甲基苯甲酸与硫酸二乙酯(摩尔比为0.2:0.18)，在催化剂HA-1(双十八烷基甲基胺氯化苄季铵盐)作用下于60-65℃反应3h。反应完毕后，冷却到25℃，倒入冰水中，用稀盐酸调PH值至中性，分出有机层；水层用甲苯萃取2次，合并有机层；水洗有机层3次，无水硫酸钠干燥。减压蒸去溶剂甲苯，产物再采用硅胶柱层析技术进行分离纯化，得到化合物4-乙氧基-2-羟基-6-甲基苯甲酸。
2、结构鉴定
化合物4-乙氧基-2-羟基-6-甲基苯甲酸的质谱图(如图1所示)；
化合物4-乙氧基-2-羟基-6-甲基苯甲酸的核磁氢谱图(如图2所示)；
化合物4-乙氧基-2-羟基-6-甲基苯甲酸的核磁碳谱图和DEPT谱图(如图3所示)。
实施例2：式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸的生物学实验结果
下面通过部分具体实验例证来说明本发明的式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸(以下图表中以D6B2或D表示，下同；图16、17、18及28中D1为200μM、D2为150μM、D3为100μM、D4为50μM、D5为25μM)具有显著性和选择性抗肿瘤作用、与临床抗肿瘤药物的联合抗肿瘤作用、以及其毒理学安全性、抗肿瘤作用机理。
1、4-乙氧基-2-羟基-6-甲基苯甲酸的体外抗癌作用：
试验方法：式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸以DMSO溶解，按最终浓度为25、50、100、150、200μM进行体外抗癌试验。收集对数生长期人类或动物肿瘤细胞，以完全培养基混悬后，接种于6孔板，每孔加入2ml细胞悬液。37℃、5％CO2培养箱培养24小时后，取3孔细胞计数作为0小时的细胞数；吸去其余孔的培养液，然后按设计分别加入含有相应浓度式(1)化合物的完全培养液2ml，同时设DMSO溶剂对照。每浓度及DMSO对照组做9孔平行，细胞在加药后于37℃、5％CO2培养箱继续培养。于加药后24h、48h、72h，每浓度分别收集3孔细胞进行计数，计算细胞数的平均值和标准差，绘制细胞生长曲线，进行统计学差异检验。同时采用96孔板培养，以MTT法测定药物处理24、48和72小时后的OD值，观察药物的时间-效应关系。
结果：式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸对人乳腺癌细胞株MCF-7、人肺癌细胞株A-549、人结肠癌细胞株HCT-116、人肝癌细胞株QGY-7703等均具有显著性抗癌作用。该化合物对不同癌细胞的抑制率有显著性差异，人类肝癌细胞QGY-7703对该化合物非常敏感。在200μM浓度、72小时，该化合物对QGY-7703、MCF-7、A-549和HCT-116的抑制率分别为95.07％、78.26％、50.39％和51.84％，其半数抑制浓度(IC50)分别为70μM、135μM、190μM和200μM；该化合物的体外抗癌作用具有明显的剂量-反应关系。72小时的体外抗癌结果见图4。在其它体外抗癌活性筛选试验中，式(1)化合物处理癌细胞72小时后，在显微镜下见对人宫颈癌细胞Hela、人胃癌细胞SGC-7901、人前列腺癌细胞PC-3、人卵巢癌细胞SKOV-3、人白血病细胞K-562和HL-60、人神经胶质瘤细胞U-251 等具有显著性体外抑制或杀灭作用。
该化合物在不同剂量下对人肝癌细胞QGY-7703体外抗癌作用的剂量-效应关系图和时间-效应关系图(MTT法)见图5。
2、4-乙氧基-2-羟基-6-甲基苯甲酸对balb/c裸鼠移植的人类大细胞肺癌NCI-H460的体内抗癌作用
试验方法：雄性balb/c裸鼠(体重16～18g)，每只右侧背部皮下接种人类大细胞肺癌NCI-H460细胞悬液0.1ml(5×107cells/ml)。接种癌细胞后的第12天，大部分小鼠皮下出现肉眼可见肿瘤。用游标卡尺测量、计算每只小鼠的肿瘤体积。将荷瘤小鼠按肿瘤体积大小随机分为DMSO对照组和式(1)化合物给药组，每组8只荷瘤小鼠。式(1)化合物以无菌的质谱纯DMSO配制，给药剂量为30mg/kg；对照组小鼠给予同等体积的DMSO。小鼠从分组后的第二天开始腹腔注射给药，隔天给药1次，每4天测定1次肿瘤体积和体重，计算肿瘤的相对体积、肿瘤的相对增值率。给药10次后，处死小鼠，完整剥离肿瘤、肝、脾、肾和睾丸，测定肿瘤重量以及各脏器重量，计算肿瘤重量抑制率及各脏器系数。
结果：4-乙氧基-2-羟基-6-甲基苯甲酸对NCI-H460肿瘤的抑制结果见图6、图7、图8。4-乙氧基-2-羟基-6-甲基苯甲酸能显著性抑制人类肺癌NCI-H460的生长(p<0.05)，试验结束时，肿瘤的重量抑制率为43.51％，肿瘤的相对增值率为51.96％，给药组的肿瘤重量、肿瘤相对体积与阴性对照组(以NC表示，下同)比较，呈显著性下降(p<0.05)。
3、4-乙氧基-2-羟基-6-甲基苯甲酸对裸鼠体内移植性人类肝癌QGY-7703的抗癌作用
试验方法：对经适应性饲养1周的雌性balb/c裸鼠(体重16～18g)，每只在右侧背部皮下接种QGY-7703细胞悬液0.1ml(1×108cells/ml)。裸鼠饲养在SPF实验室。接种癌细胞后的第21天，小鼠皮下出现肉眼可见肿瘤。用游标卡尺测量小鼠肿瘤的长径和横径，计算肿瘤体积。将荷瘤小鼠按照肿瘤体积大小随机分为DMSO溶剂对照组、5-氟尿嘧啶(5-Fu)阳性药物对照组和式(1)化合物给药组，每组共6只荷瘤小鼠。4-乙氧基-2- 羟基-6-甲基苯甲酸以无菌的质谱纯DMSO溶解后，再以等体积无菌PBS稀释，给药剂量为20mg/kg；溶剂对照组小鼠给予同等体积的DMSO/PBS(1:1)；5-Fu以等体积DMSO稀释，5-Fu使用剂量为30mg/kg。小鼠从分组后的第二天开始腹腔注射给药，隔天给药1次，每4天测定1次肿瘤体积和体重。给药12次后，处死小鼠，完整剥离肿瘤、肝、脾、肾，测定肿瘤重量、小鼠体重以及各脏器重量。
结果：式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸对QGY-7703肿瘤的抑制结果见图9、图10、图11。式(1)化合物能显著性抑制人类肝癌QGY-7703的生长(p<0.05)，试验结束时，肿瘤的重量抑制率为69.83％，肿瘤的相对增值率为30.84％，给药组的肿瘤重量、肿瘤相对体积与对照组比较，呈显著性下降(p<0.05)。该化合物对人类肝癌的抑制作用显著性强于对肺癌的抑制作用。实验结束时各组的QGY-7703肿瘤照片见图12。
4-乙氧基-2-羟基-6-甲基苯甲酸对昆明种小鼠和ICR小鼠移植的小鼠肉瘤S-180、小鼠肝癌H-22，以及C57BL/6J小鼠移植的小鼠肺癌3ll均具有显著性的体内抗癌作用(p<0.05)，并具有剂量-效应关系。
4、4-乙氧基-2-羟基-6-甲基苯甲酸对裸鼠的毒性作用
实验方法：裸鼠体内抗癌试验中，在测量肿瘤体积的各时间点，同时测量小鼠的体重；在试验结束时，处死小鼠，完整取出小鼠肝脏、脾脏、肾脏、睾丸(雄鼠)并称重，计算各脏器系数。以下图表中的NC表示阴性对照组(Negative Control)。
(1)对雄鼠体重和脏器发育的影响
表1.雄性balb/c小鼠不同时间点的体重(g)
Figure PCTCN2014000808-appb-000005
*：P＜0.05
结果：给药4次后，式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸对小鼠体重增长呈现抑制作用。
表2.雄性balb/c小鼠的脏器系数
Figure PCTCN2014000808-appb-000006
*：P＜0.05
结果：式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸能影响雄性裸鼠的肝脏和脾脏发育，但不影响雄性小鼠的肾脏和睾丸发育。
(2)对雌性小鼠死亡、体重和脏器发育的影响
表3.雌性balb/c小鼠不同时间点的体重(g)
Figure PCTCN2014000808-appb-000007
#：5-氟尿嘧啶组2只小鼠在第20天死亡，另有3只极度消瘦、濒死；
*：从第8天开始，5-氟尿嘧啶和D6B2组体重显著性低于溶剂对照组，P<0.05；
结果：抗癌药物5-氟尿嘧啶和式(1)化合物能显著性影响小鼠体重发育，5-氟尿嘧啶对小鼠体重影响显著性高于该化合物。在实验的第20天，5-Fu组有2只动物因衰竭死亡，另有3只小鼠极度衰竭而濒死。式(1)化合物组动物在试验结束时仍健康活泼、无死亡。
表4.雌性balb/c小鼠的脏器系数
Figure PCTCN2014000808-appb-000008
*：P＜0.05
结果：4-乙氧基-2-羟基-6-甲基苯甲酸能影响雌性小鼠肾脏发育，而5-氟尿嘧啶能显著性影响小鼠的脾脏和肾脏发育(各组小鼠的脾脏照片见图13)。该化合物对小鼠体重和脏器的发育影响显著性低于抗癌药物5-氟尿嘧啶。
5、4-乙氧基-2-羟基-6-甲基苯甲酸对肿瘤细胞凋亡和细胞周期的影响：
实验方法：肝癌细胞QGY-7703经式(1)化合物处理48h后，采用流式细胞仪(FACS)检测细胞周期和细胞凋亡情况。结果见图14和图15。
结果：式(1)化合物能显著性促进癌细胞的凋亡和坏死，药物处理组在剂量为100μM、150μM和200μM时，细胞凋亡和坏死形成的DNA碎片峰(P2)分别为13.5％、24.7％和32.7％，有明显的剂量-效应关系，显著性高于对照组的6.8％，并且该化合物显著性推后癌细胞的G0/G1和G2/M峰值和细胞周期分布比例，明显影响癌细胞的分化和周期。
6、4-乙氧基-2-羟基-6-甲基苯甲酸对细胞凋亡标志蛋白裂解的半胱天冬酶-3(Cleaved Caspase-3)和裂解的PARP(Cleaved PARP)水平、及肿瘤抑制基因PTEN表达的影响：
实验方法：肝癌细胞QGY-7703、结肠癌细胞HCT-116、乳腺癌细胞MCF-7、肺癌细胞A-549经式(1)化合物处理24小时后，收集细胞、裂解、调整蛋白浓度，采用Western Blot技术测定。结果见图16(SP表示阳性对照物Staurosporine,SP1为50nM,SP2为100nM)。
结果：4-乙氧基-2-羟基-6-甲基苯甲酸能显著性促进肝癌细胞 QGY-7703、结肠癌细胞HCT-116中细胞凋亡标志引物裂解的PARP(Cleaved PARP)的形成，对乳腺癌细胞MCF-7、肺癌细胞A-549中裂解的PARP的形成无作用。该化合物也不影响癌细胞的裂解的半胱天冬酶-3(Cleaved Caspase-3)水平和PTEN的表达。
7、4-乙氧基-2-羟基-6-甲基苯甲酸与其它抗癌药物的联合抗肿瘤作用：
在体外抗癌试验中，化合物4-乙氧基-2-羟基-6-甲基苯甲酸与抗癌药物5-氟尿嘧啶、紫杉醇和顺铂具有显著性体外联合协同抗肿瘤作用。结果见表5、表6和图17、图18。
试验方法：体外抗癌试验中，设置阴性对照组、一个剂量的阳性抗癌药物组、三个剂量的D6B2组和三个剂量的D6B2加阳性抗癌药物组，在加药后72小时收集细胞计数，计算抑制率和进行统计分析。
表5.D6B2联合5-Fu对人肺癌A-549细胞的体外抑制作用(n＝3，x±s)
Figure PCTCN2014000808-appb-000009
与对照组比较,*P＜0.05，**P＜0.01；与5-FU比较,△P＜0.05，△△P＜0.01.
表6D6B2联合顺铂(DDP)对肺癌A-549细胞的抑制作用(n＝3，
Figure PCTCN2014000808-appb-000010
)
Figure PCTCN2014000808-appb-000011
Figure PCTCN2014000808-appb-000012
与对照组比较,*P＜0.05，**P＜0.01；与DDP比较,△P＜0.05，△△P＜0.01.
紫杉醇在1nM浓度时，体外对MCF-7的抑制率为28.85％，D6B2在浓度为100μM时对MCF-7的抑制率为15.38％，二者联合作用时对MCF-7的抑制率为52.16％，4-乙氧基-2-羟基-6-甲基苯甲酸与紫杉醇在体外对人乳腺癌细胞MCF-7具有显著性联合协同体外抗癌作用。
在体内抗癌试验中，4-乙氧基-2-羟基-6-甲基苯甲酸与环磷酰胺在体内具有联合抑制小鼠肝癌H-22。环磷酰胺在40mg/kg时，对小鼠肝癌H-22的抑制率为75.83％，D6B2在40mg/kg时对H-22的抑制率为38.99％，二者联合使用时，联合组小鼠未见有肿瘤生长。
实施例3：
式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸与碳酸氢钠在加热条件下进行中和反应，然后以纯碱溶液调PH值至7.0-7.5，加热、蒸发、结晶即形成式(1)化合物的钠盐(Sodium4-ethoxy-2-hydroxy-6-methylbenzoate)，其能溶于水。式(1)化合物也能与其它碱性离子或基团形成盐类。
实施例4：
式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸，以少量DMSO溶解，按常规加注射用水，精滤，灌封灭菌制成注射液。
实施例5：
式(1)化合物的钠盐(Sodium4-ethoxy-2-hydroxy-6-methylbenzoate)， 按常规加注射用水溶解，精滤，灌封灭菌制成注射液。
实施例6：
式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸或其盐类，以少量乙醇、甲醇或蒸馏水溶解后，用无菌抽滤漏斗过滤，再无菌精滤，分装于安瓿中，低温冷冻干燥后无菌熔封得粉针剂。
实施例7：
式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸或其盐类，按制剂需要的比例加入赋形剂，制成粉剂。
实施例8：
式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸或其盐类，按制剂需要的比例加入赋形剂，制粒压片制成片剂。
实施例9：
式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸或其盐类，按常规口服液制法制成口服液。
实施例10：
式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸或其盐类，按制剂需要的比例加入赋形剂，制成胶囊、或颗粒剂、或冲剂。
实施例11：
式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸或其盐类，按产品需要的比例加入食品或其它载体，制成保健品或其它功能性用品。
实施例12:
式(1)化合物4-乙氧基-2-羟基-6-甲基苯甲酸或其盐类，按治疗需要，与其它抗肿瘤药物按比例混合，制成各种制剂，形成联合抗肿瘤药物组合 物。
实施例13：
化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯(methyl2,4-dihydroxy-3,6-dimethyllbenzoate)的化学结构鉴定、制备方法、抗癌作用和作用机理。
1、化学结构鉴定
化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯的质谱图(图19)、核磁氢谱图(图20)、核磁碳谱图和DEPT谱图(图21)；
化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯的二维图谱HMBC(图22)、HSQC(图23)和Roesy(图24)；
2、制备方法
化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯为已知结构化合物，可化学合成，或从市场购买。
3、化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯的生物学实验结果
下面通过部分具体实验例证来说明本发明的化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯(以下图表及文中以“D6B1”或“B”表示，下同。图18和28中B1为200μM、B2为150μM、B3为100μM、B4为50μM、B5为25μM)具有显著性和选择性抗肿瘤作用、与临床抗肿瘤药物的联合抗肿瘤作用、以及抗肿瘤作用机理。
3.1体外抗癌作用
试验方法：2,4-二羟基-3,6-二甲基苯甲酸甲酯以DMSO溶解，按最终浓度为25、50、100、150、200μM进行体外抗癌试验。收集对数生长期人类肿瘤细胞，以完全培养基混悬后，接种于6孔板，每孔加入2ml细胞悬液。37℃、5％CO2培养箱培养24小时后，取3孔细胞计数作为0小时的细胞数；吸去其余孔的培养液，然后分别加入含有相应浓度化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯的完全培养液2ml，设DMSO溶剂对照和阳性药物对照。每浓度及对照组做9孔平行，细胞在加药后继续培养。于加药后24h、48h、72h，每浓度分别收集3孔细胞进行计数，计算细胞数的平均值和标准差，绘制细胞生长曲线，进行统计学差异检验。或者采用96孔板培养，以MTT法测定药物处理24、48和72小时后的OD值，观察药 物的时间-效应关系和时间-效应关系。
结果：2,4-二羟基-3,6-二甲基苯甲酸甲酯对人肝癌细胞株QGY-7703和HepG2、人宫颈癌细胞株Hela、人鼻咽癌细胞株CNE、人乳腺癌细胞株MCF-7、人肺癌细胞株A-549、人白血病细胞株K-562等均具有显著性抗癌作用。该化合物对不同癌细胞的抑制率差异大，肝癌细胞对该化合物非常敏感，结肠癌、宣威肺癌对该化合物不敏感。在200μM浓度、72小时，该化合物对QGY-7703、MCF-7、A-549、HCT-116、Hela、CNE、HepG2和XWLC的抑制率分别为96.46％、58.94％、63.50％、39.66％、84.75％、78.89％、81.82％和13.62％，存在明显的剂量-反应关系。2,4-二羟基-3,6-二甲基苯甲酸甲酯对不同细胞株的抑制作用剂量-效应关系见图25，化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯对人体正常细胞的细胞毒作用显著性低于对一些癌细胞的细胞毒作用，具有选择性抗癌作用。抗癌药物5-氟尿嘧啶和顺铂对人体正常肺上皮细胞Beas-2b抑制作用的剂量-效应关系见图26。比较图26与图25结果，可见化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯对人体正常细胞的毒性作用显著性低于抗癌药物5-氟尿嘧啶和顺铂。
3.2对肿瘤细胞凋亡和细胞周期的影响：
实验方法：QGY-7703经化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯处理48h后，采用流式细胞仪(FACS)检测细胞周期和细胞凋亡情况。结果见图27。
结果：化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯能显著性促进癌细胞的凋亡和/或坏死，药物处理组在剂量为100μM、150μM和200μM时，细胞凋亡和/或坏死形成的DNA碎片峰(P2)分别为6.9％、8.7％和28.3％，有明显的剂量-效应关系，显著性高于对照组的4.1％，并且该化合物显著性推后癌细胞的G0/G1和G2/M峰和细胞周期分布比例，明显影响癌细胞的分化和周期。
3.3对细胞凋亡通路裂解的半胱天冬酶-3和裂解的PARP水平、及肿瘤抑制基因PTEN表达的影响：
实验方法：肝癌细胞QGY-7703、结肠癌细胞HCT-116、乳腺癌细胞 MCF-7、肺癌细胞A-549经化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯处理24小时后，收集细胞、裂解、调整蛋白浓度，采用Western Blot技术测定结果见图28。
结果：2,4-二羟基-3,6-二甲基苯甲酸甲酯能显著性促进肝癌细胞QGY-7703、结肠癌细胞HCT-116中细胞凋亡标志引物裂解的PARP的形成，对乳腺癌细胞MCF-7、肺癌细胞A-549中裂解的PARP的形成无作用。该化合物也不影响这4个癌细胞的裂解的半胱天冬酶-3水平和PTEN的表达。
3.4与其它抗癌药物的体外联合抗肿瘤作用：
试验方法：体外抗癌试验中，设置阴性对照组、一个剂量的阳性抗癌药物组、3个剂量的D6B1组和三个剂量的D6B1加阳性抗癌药物组，在加药后72小时收集细胞计数，计算抑制率和进行统计分析。
化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯与5-氟尿嘧啶、紫杉醇、顺铂等药物具有显著性体外联合协同抗癌作用。化合物2,4-二羟基-3,6-二甲基苯甲酸甲酯与顺铂对Hela的体外联合作用结果见表7和图29。
表7D6B1联合顺铂(DDP)对Hela细胞的体外抑制作用(n＝3，
Figure PCTCN2014000808-appb-000013
)
Figure PCTCN2014000808-appb-000014
与DMSO对照组比较,*P＜0.05，**P＜0.01；与DDP比较,△P＜0.05.
2,4-二羟基-3,6-二甲基苯甲酸甲酯与5-氟尿嘧啶、紫杉醇在体外对人乳腺癌细胞MCF-7也具有显著性联合协同抗癌作用。
实施例14：
化合物2,4-二羟基-6-甲基苯甲酸乙酯(ethyl2,4-dihydroxy-6-methylbenzoate)的化学结构鉴定、体外与体内抗癌作用、作用机理。
1、化学结构鉴定
化合物2,4-二羟基-6-甲基苯甲酸乙酯的质谱图(图30)、核磁氢谱图(图31)和核磁碳谱图和DEPT谱图(图32)；
化合物2,4-二羟基-6-甲基苯甲酸乙酯的核磁二维图谱HMBC(图33)、HSQC(图34)、Roesy(图35)；
2、制备方法
2,4-二羟基-6-甲基苯甲酸乙酯为已知结构化合物，可以采用2,4-二羟基-6甲基苯甲酸和乙醇为原料进行化学合成，或从市场购买。
3、化合物2,4-二羟基-6-甲基苯甲酸乙酯的生物学实验结果
下面通过部分具体实验例证来说明本发明的化合物2,4-二羟基-6-甲基苯甲酸乙酯具有显著性和选择性体外体内抗肿瘤作用、与临床抗肿瘤药物的联合抗肿瘤作用、以及抗肿瘤作用机理。
3.1体外抗癌作用：
试验方法：化合物2,4-二羟基-6-甲基苯甲酸乙酯以DMSO溶解，按浓度为25、50、100、150、200μM进行试验。肿瘤细胞以完全培养液混悬后，接种于6孔板。37℃、5％CO2培养箱培养24小时后，取3孔细胞计数作为0小时的细胞数；吸去其余孔的培养液，然后分别加入含有相应浓度化合物2,4-二羟基-6-甲基苯甲酸乙酯的完全培养液2ml，同时设DMSO溶剂对照和阳性药物对照，每浓度及对照组做9孔平行。加药后24h、48h、72h，每浓度分别收集3孔细胞计数，计算细胞数的平均值和标准差，绘制细胞生长曲线，进行统计学差异检验。或者采用96孔板培养，以MTT法 测定药物处理24、48和72小时后的OD值，观察药物的剂量-效应关系和时间-效应关系。
结果：化合物2,4-二羟基-6-甲基苯甲酸乙酯对人乳腺癌细胞株MCF-7、人肺癌细胞株A-549、人肝癌细胞株QGY-7703、人宫颈癌细胞株Hela、人白血病K-562、人肝癌细胞株HepG2、人鼻咽癌细胞株CNE、人肝癌细胞株HepG2等均具有显著性抗癌作用。该化合物对不同癌细胞的抑制率差异大，人类肝癌细胞QGY-7703和宫颈癌Hela对该化合物非常敏感，人胆管癌QBC-939、人结肠癌HCT-116等对该化合物不敏感。在200μM浓度、72小时，该化合物对QGY-7703、CNE、Hela、HepG2、XWLC、A-549、HCT-116的抑制率分别为93.86％、73.12％、90.75％、75.18％、51.13％、52.23％、43.50％，具有明显的剂量-反应关系；该化合物对人体正常细胞株HUVEC和Beas-2b的抑制率分别为16.52％和26.01％，显著性低于对上述癌细胞株的细胞毒作用，化合物2,4-二羟基-6-甲基苯甲酸乙酯具有选择性抗癌作用。化合物2,4-二羟基-6-甲基苯甲酸乙酯对部分癌细胞株和正常细胞株体外抑制作用的剂量-效应关系结果见图36。
3.2体内抗癌作用：
化合物2,4-二羟基-6-甲基苯甲酸乙酯对昆明种小鼠和ICR小鼠移植的小鼠肉瘤S-180、小鼠肝癌H-22，C57BL/6J小鼠移植的小鼠肺癌3ll，以及balb/c裸鼠移植人肺癌NCI-H460和人肝癌QGY-7703均具有显著性的体内抗癌作用(p<0.05)。
3.3与其它抗癌药物的体外联合抗癌作用：
化合物2,4-二羟基-6-甲基苯甲酸乙酯(D)与抗癌药物5-氟尿嘧啶、紫杉醇和顺铂具有显著性体外联合协同抗肿瘤作用。化合物2,4-二羟基-6-甲基苯甲酸乙酯与顺铂体外联合抗宫颈癌Hela的结果见表8和图37。
试验方法：体外抗癌试验中，设阴性对照组、一个剂量的阳性抗癌药物组、3个剂量的化合物2,4-二羟基-6-甲基苯甲酸乙酯组和三个剂量的化合物2,4-二羟基-6-甲基苯甲酸乙酯加阳性抗癌药物联合用药组，在加药后72小时收集细胞计数，计算抑制率和进行统计分析。
表8.化合物2,4-二羟基-6-甲基苯甲酸乙酯(D)联合DDP对宫颈癌Hela的体外抑制作用(n＝3，
Figure PCTCN2014000808-appb-000015
)
Figure PCTCN2014000808-appb-000016
与阴性对照组比较,*P＜0.05，**P＜0.01；与DDP比较,△P＜0.05.
2,4-二羟基-6-甲基苯甲酸乙酯与5-氟尿嘧啶、紫杉醇对结肠癌细胞、人乳腺癌细胞也具有显著性体外联合协同抗癌作用。
实施例15：
化合物1,3-二羟基-5-甲基苯的制备方法和抗癌作用。
1、制备方法
(1)化学合成法
以3-甲基-4-氯苯酚为原料，通过高温脱氯水解反应，可得到中间产物3,5-二羟基甲苯的酚钾盐，再经酸化处理后可得到化合物1,3-二羟基-5-甲基苯。
Figure PCTCN2014000808-appb-000017
2、体外抗癌作用
试验方法：1,3-二羟基-5-甲基苯以DMSO溶解成进行体外抗癌活性检测。对数生长期人类肿瘤细胞，以完全培养基混悬后，接种于6孔板。37℃、5％CO2培养箱培养24小时后，吸去培养液，然后按设计加入含有相应浓度1,3-二羟基-5-甲基苯化合物的完全培养液2ml，同时设DMSO溶剂对照。样品及DMSO对照组各做3孔平行。于加药后72h，收集细胞进行计数，计算细胞数的平均值和标准差，进行统计学差异检验。
试验结果：药物处理72小时后，化合物1,3-二羟基-5-甲基苯在剂量为20μg/ml(161.3μM)对人乳腺癌MCF-7、人肺癌A-549、人结肠癌HCT-116的抑制率分别为63.01％、53.83％和92.93％，与对照组比较具有显著性差异(p<0.05)。
实施例16：
化合物2,6-二羟基-4-甲基苯甲醛的化学结构鉴定、制备方法和抗癌作用。
1、化学结构鉴定
化合物2,6-二羟基-4-甲基苯甲醛的质谱图(图38)、核磁氢谱图(图39)、核磁碳谱图和DEPT谱图(图40)；
2、制备方法
(1)化学合成法
氧化法：
由底物2-羟甲基-5-甲基苯-1,3-二醇通过氧化反应可生成本实施例化合物2,6-二羟基-4-甲基苯甲醛。
Figure PCTCN2014000808-appb-000018
甲醛反应法：
化合物1,3-二羟基-5甲基苯和甲醛反应，生成2-羟甲基-5-甲基苯-1,3-二醇,后者再按上面步骤氧化反应即生成化合物2,6-二羟基-4-甲基苯甲 醛，经柱层析进行分离纯化。
Figure PCTCN2014000808-appb-000019
3、体外抗癌作用
试验方法：2,6-二羟基-4-甲基苯甲醛以DMSO溶解成浓度为12.5μM、25.0μM、50.0μM和100μM四个剂量进行试验。
试验结果：药物处理72小时后，四个浓度的化合物2,6-二羟基-4-甲基苯甲醛对人乳腺癌MCF-7的抑制率分别为5.58％、8.81％、27.46％和53.98％，在剂量为50.0μM和100μM时与对照组比较具有显著性差异(p<0.05)，且具有明显的剂量-效应关系。体外对人不同肿瘤细胞株的抗肿瘤效果见图41(横坐标的1、2、4、8分别表示剂量为12.5μM、25.0μM、50.0μM和100μM)。
实施例17：
化合物2,4-二羟基-6-甲基苯甲酸甲酯(methyl2,4-dihydroxy-6-methylbenzoate)的化学结构鉴定、制备方法和抗癌作用。
1、化学结构鉴定
化合物2,4-二羟基-6-甲基苯甲酸甲酯的质谱图(图42)、核磁氢谱图(图43)、核磁碳谱图和DEPT谱图(图44)；
2、制备方法
(1)化学合成法：
以2,4-二羟基-6-甲基苯甲酸为原料，与甲醇进行酯化反应，可生成化合物2,4-二羟基-6-甲基苯甲酸甲酯。
3、体外抗癌作用
试验方法：同实施例15中的体外抗癌试验方法。2,4-二羟基-6-甲基苯甲酸甲酯以DMSO溶解进行试验。
试验结果：药物处理72小时后，化合物2,4-二羟基-6-甲基苯甲酸甲酯 在剂量为20μg/ml(110.0μM)对人乳腺癌MCF-7、人肺癌A-549、人结肠癌HCT-116的抑制率分别为99.18％、82.15％和81.00％，与对照组比较具有显著性差异(p<0.05)。
实施例18：
化合物3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯(ethyl3-formyl-2,4-dihydroxy-6-methylbenzoate)的化学结构鉴定、制备方法和抗癌作用。
1、化学结构鉴定
化合物3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯的质谱图(图45)、核磁氢谱图(图46)、核磁碳谱图和DEPT谱图(图47)；
2、制备方法
3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯可化学合成或从市场购买。
3、体外抗癌作用
试验方法：同实施例15中的体外抗癌试验方法。3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯以DMSO溶解进行试验。
试验结果：药物处理72小时后，化合物3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯在剂量为20μg/ml(89.3μM)对人乳腺癌MCF-7、人肺癌A-549、人结肠癌HCT-116的抑制率分别为29.32％、24.58％和54.60％，对HCT-116的抑制与对照组比较具有显著性差异(p<0.05)。
实施例19：
化合物3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯(5-(ethoxycrbonyl)-3-hydroxy-2-methylphenyl-3-formyl-2,4-dihydroxy-6methyl benzoate)的化学结构鉴定、制备方法和抗癌作用。
1、化学结构鉴定
化合物3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯的质谱图(图48)、核磁氢谱图(图49)、核磁碳谱图和DEPT谱图(图50)，该化合物为新结构化合物。
2、制备方法
(1)化学合成法
将3,5-二羟基-4-甲基苯甲酸乙酯(ethyl3,5-dihydroxy-4-methylbenzoate)和3-醛基-2,4-二羟基-6甲基苯甲酸(3-formyl-2,4-dihydroxy-6-methylbenzoic acid)按照摩尔浓度以1:1混合，以浓硫酸作为催化剂，在60℃下加热回流进行酯化反应1～3小时。冷却后，以蒸馏水进行充分洗涤，收集不溶于水的部分，再采用硅胶柱层析进行分离，可得到纯度大于99.8％的化合物3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯。
Figure PCTCN2014000808-appb-000020
3、体外抗癌作用
试验方法：同实施例15的体外抗癌试验方法。3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯以DMSO溶解进行试验。
试验结果：药物处理72小时后，化合物3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯在剂量为20μg/ml(53.5μM)时对人乳腺癌MCF-7、人肺癌A-549、人结肠癌HCT-116的抑制率分别为70.00％、69.07％和81.95％，与对照组比较具有显著性差异(p<0.05)。
实施例20：
2,4-二羟基-3,6-二甲基苯甲酸甲酯、2,4-二羟基-6-甲基苯甲酸乙酯、2,4-二羟基-6-甲基苯甲酸甲酯、3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯、3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯化合物与氢氧化钠、碳酸氢钠、氢氧化钾、碳酸氢钾等强碱在加热的情况下，发生水解化学反应，然后，以盐酸等调PH值至7.5，蒸馏后用蒸馏水进行洗涤、重结晶，可得到2,4-二羟基-3,6-二甲基苯甲酸钠(钾)、2,4-二羟基-6-甲基苯甲酸钠(钾)、3-醛基-2,4-二羟基-6-甲基苯甲酸钠(钾)等盐类。2,4-二羟基-3,6-二甲基苯甲酸甲酯、2,4-二羟基-6-甲基苯甲酸乙酯、2,4-二羟基-6-甲基苯甲酸甲酯、3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯、3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯化合物也能与其它碱性离子或基团形成盐类。
实施例21
2,4-二羟基-3,6-二甲基苯甲酸甲酯、2,4-二羟基-6-甲基苯甲酸乙酯、2,4-二羟基-6-甲基苯甲酸甲酯、3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯、3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯化合物与氢氧化钠、碳酸氢钠、氢氧化钾、碳酸氢钾等强碱在加热的情况下，发生水解化学反应，然后，以盐酸等调PH值至3-5，蒸馏后用蒸馏水进行洗涤、重结晶，可得到2,4-二羟基-3,6-二甲基苯甲酸、2,4-二羟基-6-甲基苯甲酸、3-醛基-2,4-二羟基-6-甲基苯甲酸。
实施例22：
式(2)化合物，先以少量药用溶媒溶解，然后按常规加注射用水配制成相应浓度，精滤，灌封灭菌制成注射液。
实施例23：
按实施例(20)反应生成的2,4-二羟基-3,6-二甲基苯甲酸、2,4-二羟基-6-甲基苯甲酸、3-醛基-2,4-二羟基-6-甲基苯甲酸的盐类物质，按常规加注射用水溶解，精滤，灌封灭菌制成注射液。
实施例24：
式(2)化合物或盐类，以注射用溶媒或蒸馏水溶解后，用无菌抽滤漏斗过滤，再无菌精滤，分装于安瓿中，低温冷冻干燥后无菌熔封得粉针剂。
实施例25：
式(2)化合物或盐类，按制剂需要的比例加入赋形剂，制成粉剂。
实施例26：
式(2)化合物或其盐类，按制剂需要的比例加入赋形剂，制粒压片制成片剂。
实施例27：
式(2)化合物或其盐类，按常规口服液制法制成口服液。
实施例28：
式(2)化合物或其盐类，按制剂需要的比例加入赋形剂，制成胶囊、或颗粒剂、或冲剂。
实施例29：
式(2)化合物或其盐类，按产品需要的比例加入食品或其它载体，制成保健品或其它功能性用品。
实施例30:
式(2)化合物或其盐类，按治疗需要，与其它抗肿瘤药物按比例混合，制成各种制剂，形成联合抗肿瘤药物组合物。
以上所述实施例仅部分表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明的保护范围应以所附权利要求为准。

Claims (36)

  1. 具有式(1)结构的4-乙氧基-2-羟基-6-甲基苯甲酸或其药学上可接受的盐，
    Figure PCTCN2014000808-appb-100001
  2. 一种抗肿瘤的药物组合物，其中，其活性成分包括权利要求1所述的4-乙氧基-2-羟基-6-甲基苯甲酸或其药学上可接受的盐。
  3. 根据权利要求2所述的药物组合物，其中，所述药物组合物的剂型为：注射液、粉针剂、片剂、口服液、胶囊、颗粒剂或冲剂。
  4. 一种联合抗肿瘤的药物组合物，其中，其活性成分包括权利要求1所述的4-乙氧基-2-羟基-6-甲基苯甲酸或其药学上可接受的盐，和5-氟尿嘧啶、紫杉醇、顺铂或环磷酰胺中的至少一种。
  5. 根据权利要求4所述的药物组合物，其中，权利要求1所述的4-乙氧基-2-羟基-6-甲基苯甲酸或其药学上可接受的盐与所述5-氟尿嘧啶、紫杉醇、顺铂或环磷酰胺中的至少一种的比例按摩尔浓度计算为1:10～100:1。
  6. 权利要求1所述的4-乙氧基-2-羟基-6-甲基苯甲酸或其药学上可接受的盐在制备抗肿瘤药物或辅助抗肿瘤保健品中的应用。
  7. 根据权利要求6所述的应用，其中所述肿瘤为以下人类肿瘤中的任一种：人乳腺癌、前列腺癌、肺癌、胃癌、宫颈癌、结肠癌、肝癌、卵巢癌、白血病、神经瘤、肉瘤、鼻咽癌。
  8. 具有式(2)结构的苯基取代化合物或其药学上可接受的盐，其中R1为氢、C1-C4烷基或C1-C4醛基；R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基，
    Figure PCTCN2014000808-appb-100002
    其中所述化合物不为4-乙氧基-2-羟基-6-甲基苯甲酸、2,4-二羟基-3,6-二甲基苯甲酸甲酯、2,4-二羟基-6-甲基苯甲酸乙酯、1,3-二羟基-5-甲基苯、2,6-二羟基-4-甲基苯甲醛、2,4-二羟基-6-甲基苯甲酸甲酯和3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯。
  9. 根据权利要求8所述的化合物，其中所述R1为氢、甲基或甲醛基。
  10. 根据权利要求8所述的化合物，其中所述R2为氢、CH3COO-、C2H5COO-、HCOO-，或5-(乙氧羰基)-3-羟基-2-甲基苯基酯基。
  11. 根据权利要求8所述的化合物，其中所述R3为氢、甲基或乙基。
  12. 根据权利要求8所述的化合物，其中所述化合物为3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯。
  13. 一种抗肿瘤的药物组合物，其中，其活性成分包括具有式(2)结构的苯基取代化合物或其药学上可接受的盐，其中R1为氢、C1-C4烷基或C1-C4醛基；R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基，
    Figure PCTCN2014000808-appb-100003
    其中所述化合物不为4-乙氧基-2-羟基-6-甲基苯甲酸。
  14. 根据权利要求13所述的药物组合物，其中所述R1为氢、甲基或 甲醛基。
  15. 根据权利要求13所述的药物组合物，其中所述R2为氢、CH3COO-、C2H5COO-、HCOO-，或5-(乙氧羰基)-3-羟基-2-甲基苯基酯基。
  16. 根据权利要求13所述的药物组合物，其中所述R3为氢、甲基或乙基。
  17. 根据权利要求13所述的药物组合物，其中所述化合物选自2,4-二羟基-3,6-二甲基苯甲酸甲酯、2,4-二羟基-6-甲基苯甲酸乙酯、1,3-二羟基-5-甲基苯、2,6-二羟基-4-甲基苯甲醛、2,4-二羟基-6-甲基苯甲酸甲酯、3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯或3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯。
  18. 根据权利要求13-17中任一项所述的药物组合物，其中，所述药物组合物的剂型为：注射液、粉针剂、片剂、口服液、胶囊、颗粒剂或冲剂。
  19. 一种联合抗肿瘤的药物组合物，其中，其活性成分包括具有式(2)结构的苯基取代化合物或其药学上可接受的盐，和5-氟尿嘧啶、紫杉醇、顺铂或环磷酰胺中的至少一种，其中式(2)中R1为氢、C1-C4烷基或C1-C4醛基；R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基，
    Figure PCTCN2014000808-appb-100004
    其中所述化合物不为4-乙氧基-2-羟基-6-甲基苯甲酸。
  20. 根据权利要求19所述的药物组合物，其中所述R1为氢、甲基或甲醛基。
  21. 根据权利要求19所述的药物组合物，其中所述R2为氢、 CH3COO-、C2H5COO-、HCOO-，或5-(乙氧羰基)-3-羟基-2-甲基苯基酯基。
  22. 根据权利要求19所述的药物组合物，其中所述R3为氢、甲基或乙基。
  23. 根据权利要求19所述的药物组合物，其中所述化合物选自2,4-二羟基-3,6-二甲基苯甲酸甲酯、2,4-二羟基-6-甲基苯甲酸乙酯、1,3-二羟基-5-甲基苯、2,6-二羟基-4-甲基苯甲醛、2,4-二羟基-6-甲基苯甲酸甲酯、3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯或3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯。
  24. 根据权利要求19-23中任一项所述的药物组合物，其中，所述的具有式(2)结构的苯基取代化合物或其药学上可接受的盐与所述5-氟尿嘧啶、紫杉醇、顺铂或环磷酰胺中的至少一种的比例按摩尔浓度计算为1:1～10:1。
  25. 具有式(2)结构的苯基取代化合物或其药学上可接受的盐在制备抗肿瘤药物或辅助抗肿瘤保健品中的应用，其中R1为氢、C1-C4烷基或C1-C4醛基；R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基，
    Figure PCTCN2014000808-appb-100005
    其中所述化合物不为4-乙氧基-2-羟基-6-甲基苯甲酸。
  26. 根据权利要求25所述的应用，其中所述R1为氢、甲基或甲醛基。
  27. 根据权利要求25所述的应用，其中所述R2为氢、CH3COO-、C2H5COO-、HCOO-，或5-(乙氧羰基)-3-羟基-2-甲基苯基酯基。
  28. 根据权利要求25所述的应用，其中所述R3为氢、甲基或乙基。
  29. 根据权利要求25所述的应用，其中所述化合物选自2,4-二羟基 -3,6-二甲基苯甲酸甲酯、2,4-二羟基-6-甲基苯甲酸乙酯、1,3-二羟基-5-甲基苯、2,6-二羟基-4-甲基苯甲醛、2,4-二羟基-6-甲基苯甲酸甲酯、3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯或3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯。
  30. 根据权利要求25-29中任一项所述的应用，其中所述肿瘤为以下人类肿瘤中的任一种：人乳腺癌、前列腺癌、肺癌、胃癌、宫颈癌、结肠癌、肝癌、卵巢癌、白血病、神经瘤、肉瘤、鼻咽癌。
  31. 抑制或治疗肿瘤生长的方法，包括将抑制或治疗有效量的具有式(2)结构的苯基取代化合物或其药学上可接受的盐，其中R1为氢、C1-C4烷基或C1-C4醛基；R2为氢、羧基、C1-C4烷基酯基，或取代或未取代的苯基酯基，所述取代的苯基为被1-3个选自羟基、C1-C4烷基和C1-C4烷基酯基的取代基取代的苯基；R3为氢或C1-C4烷基，
    Figure PCTCN2014000808-appb-100006
    或者权利要求2-5中任一项所述的药物组合物，或者权利要求13-24中任一项所述的药物组合物给予需要此治疗的个体。
  32. 根据权利要求31所述的方法，其中所述R1为氢、甲基或甲醛基。
  33. 根据权利要求31所述的方法，其中所述R2为氢、CH3COO-、C2H5COO-、HCOO-，或5-(乙氧羰基)-3-羟基-2-甲基苯基酯基。
  34. 根据权利要求31所述的方法，其中所述R3为氢、甲基或乙基。
  35. 根据权利要求31所述的方法，其中所述化合物选自4-乙氧基-2-羟基-6-甲基苯甲酸、2,4-二羟基-3,6-二甲基苯甲酸甲酯、2,4-二羟基-6-甲基苯甲酸乙酯、1,3-二羟基-5-甲基苯、2,6-二羟基-4-甲基苯甲醛、2,4-二羟基-6-甲基苯甲酸甲酯、3-醛基-2,4-二羟基-6-甲基苯甲酸乙酯或3-醛基-2,4-二羟基-6-甲基苯甲酸5-(乙氧羰基)-3-羟基-2-甲基苯基酯。
  36. 根据权利要求31-35中任一项所述的方法，其中所述肿瘤选自人乳腺癌、前列腺癌、肺癌、胃癌、宫颈癌、结肠癌、肝癌、卵巢癌、白血病、神经瘤、肉瘤和鼻咽癌。
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