The Chemical Modification Of Banlangen Acid And The Anti-tumor Activities Of Its Derivatives And QSAR

According to "pharmacopoeia of China (2010)", "Banlangen" is the dry root of Isatis indigotica Fort. (Cruciferae), or Baphicacanthus cusia (Nees) Bremek.. And it is the most commonly used traditional Chinese medicines for Chinese people. The study on the pharmacology shows that Banlangen could anti virus, anti bacterial, anti endotoxin, improving defence of immunity, and so on.In our previous work, the higher unsaturated fatty acids from Isatis tinctoria L., Banlangen-acid, showed antitumor activities in vitro and the growth of tumor could be inhibited by Banlangen-acid in mice. On the basis of the procedure of research and development of new drug in west countries, Banlangen-acid was taken as lead compound to be modified for the research of antitumor.28 derivatives in total were synthesized and their anti-tumor activities were screened. The quantitative structural activity relationship (QSAR) was developed. Compound J, N,N-bis(2-chloroethyl)docos-13-enamide, was selected to be a anti-tumor drug candidate and its mechanism of reversal activities of multidrug resistance (MDR) was studied. By means of investigating the anti-tumor activities, the reversal activities of multidrug resistance (MDR), the anti-tumor mechanism and the mechanism of multidrug resistance of the compound J in vitro and vivo, we attempt to offer the basic experimental evidence for its clinical trials.Compound J shown its good drug candidate properties which was noval, antitumor active, unique mechanism of action, easy to be synthesized and cheap starting materials. It fulfils the requirements for a Drug Candidate and will be taken into further development. The contents of this study is Followed.1. New suggestion for extraction and isolation of Banlangen-acid from Banlangen and Fufang Banlangen KeliA single solvent petroleumether was selected to make extraction and isolation of Banlangen-acid from Banlangen and Fufang Banlangen Keli instead of dichloromethane-95% ethanol and petroleumether-ethyl acetate.2. Synthesis of 28 derivatives of Banlangen-acidOn the basis of bioguide of their toxicity and antitumor bioassays,28 derivatives in total were synthesized with cheap starting materials, simple and classic reaction. The lead compound optimization was developed.3. The nanoemulsion of 28 derivatives of Banlangen-acidThe nanoemulsions of 28 derivatives of Banlangen-acid were prepared. Their Z-average size and polydispersity index were determined.4. The toxicity and antitumor bioassays of the derivatives of Banlangen-acid from Banlangen in vitro——screening for an anti-tumor drug candidateThe toxicities of their derivatives of Banlangen acids were evaluated by their hemolytic effects on red blood cell. The concentration of erucic acid methyl ester, one of the derivatives, in human hepatocellular line BEL-7404 was determined by TLC-GC. The growth and morphologic changes of the TCA8113 tongue carcinoma cells were observed in different concentrations of derivatives in vitro. The inhibition and reversal index of tumor cells were determined by MTT colorimetric assay and FCM so that the activities of antitumor of derivatives were discussed and the anti-tumor drug candidate could be selected.Compound J, N,N-bis(2-chloroethyl)docos-13-enamide, was selected Finally.5. The anti-tumor QSAR of Banlangen-acid from BanlangenTow estimated linear model of QSAR on antitumor of higher fatty acids were developed with computational Chemistry.6. The anti-tumor effect of compound J as an anti-tumor drug candidate6.1 Test in vitroThe safety evaluation of compound J were investigated with its hemolytic effect on red blood cell and the cytotoxic effects in HUV-EC-C cells by MTT assay. For testing the antitumor effects of compound J, it is determined in vitro that the drug susceptibility testing (DST) on TCA8113 and SKOV3 and the expression of biomarkers of cancer stem cells in TCA/0.1μg/ml ADM reversed by compound J.It showed that compound J did not enhance red blood cell hemolysis at concentrations lower than 100 mM/L and at the concentration range of 0-400 μg/mL in vitro, compound J did not affect the viability of HUV-EC-C. Compound J could make down regulation of CD44 expression level. Compound J exhibited low intrinsic cytotoxicity and anti-tumor effect in vitro.6.2 Test in vivoThe acute drug toxicity was tested on mice. The model of transplanted ascites tumor was made by vaccination of S180 in BALB/c nude mice and the dose-effect relationship for antitumor of compound J was analyzed.Compound J showed us non-toxic side effects in kunming mice (i.p. administration,4 mg per mouse) after observation for 14 days. Compound J significantly extended the average survival time of ascites S180 transplanted tumor mice, life extension rate is between 17%～17%. Compared with saline control group, the ascites S180 transplanted tumor mice lung and liver from medial-dose(5.0mg/kg) group of compound J showed normal-appearing. Compound J exhibited low intrinsic cytotoxicity and anti-tumor effect in vivo.