Study On The Differences In The Toxicity Of Chiral Oxindole Derivatives To Zebrafish Danio Rerio

Chiral is a ubiquitous feature in nature and it acts as a very important role in biology, stereoscopic chemistry and medicine. In this paper, we studied on the toxic activity and differences in toxicity between chiral compounds and its enantiomers and explored the mechanism of their toxicity by using the zebrafish.The chiral compounds that derived from phosphoric, acid and esters are newly and synthesized. It includes the structure of oxidation of indole and phosphoric acid ester. Previous studies reported that oxidation of indole is a heterocyclic compounds with biological activity, which can be the basic structure of a lot of pharmaceutically active molecules. Therefore the oxidation of indole containing the construction of 3-disubstituted oxidation of indole has important pharmacological activities. While some of the chiral organic phosphoric acid ester and its derivatives have insecticidal, weeding or bactericidal activities. In this study, the toxic activity of the oxidation of the derivatives of the phosphoric acid ester compound has not been reported. There may be a difference in the toxic activity of these compounds with two chiral centers. So the deep research on the enantiomeric level, can be more realistic and accurate in evaluating their impact on the environment and human health, and can be better guide the rational use of these chiral substances in our life.After the study of chiral compounds'toxic activity, and the toxicity difference between the chiral compounds and its enantiomers, the results showed that:racemate of LTR10(Tert-Butyl-6-Chloro-3-[-2-(diethoxyphosphoryl)-3-ethoxy-3-oxo-propyl]-2-oxo-3-phenylindoline-1-carboxylate), LTR11 (Tert-Buty1-6-Bromo-3-[-2-(diethoxyphos-phoryl)-3-ethoxy-3-oxo-propyl]-2-oxo-3-phenylindoline-l-carboxylate), LTR12 (Tert-Butyl-3-[-2-(diethoxyphosphoryl)-3-ethoxy-3-oxo-propyl]-7-fluoro-2-oxo-3-phenylindoline-1-carboxylate) and its enantiomers have teratogenic effects in the development of zebrafish embryos. The abnormal phenotype mainly includes:embryonic developmental delay, body axis bending, embryonic tail bending, peritoneopericardial edema, yolk sac below the heart depression, heart development into a tube, heart blood stasis, hypopigmentation, heart rhythm deceleration and so on. After the treatment of zebrafish embryos with LTR10 and LTR11, cardiac malformations were observed at 48 hpf, and the treatment with LTR12, cardiac malformations were observed at 72 hpf.At 48 hpf, the half lethal concentrations of raceme LTR10,7R,18S-isomer(10a), 7S,18R-isomer(10b) for Zebrafish were 511.476 ?M,271.194 ?M,634.840 ?M, respectively. The half lethal concentrations of raceme LTR11,7R,18S-isomer(11a), 7S,18R-isomer(llb) for Zebrafish were 469.842 ?M,145.879 ?M,41.345 ?M, respectively. The half lethal concentrations of raceme LTR12,7R,18S-isomer(12a), 7S,18R-isomer(12b) for Zebrafish were 175.372 ?M,674.314 ?M,304.78 ?M, respectively. There was a significant toxicological effects difference between the enantiomers, among them, the half lethal effect between the LTR11, 11a and 11b is significant difference. The joint toxicity of 7R,18S-isomer and 7S,18R-isomer, We found that there are antagonistic joint action between enantiomers of LTR10 and LTR11, and synergistic action between enantiomers of LTR12.According abnormal phenotypes of zebrafish embryos after chiral compounds treatment, we chose cmlcl and myl7(cmlc2) which associated with early embryonic heart development of zebrafish, and tbx20 concerned with heart looping to study the toxic mechanism of chiral compounds. With significant differences in toxicity, LTR11 isomers (11a and 11b) were used in this thesis to explore the mechanism of the retardant cardiac development and heart looping by using in situ hybridization. The results showed that under the same treatment concentration (30 ?M), lla promoted the expression of myl7, while 11b inhibited the expression of my17. At early stage of the zebrafish embryo (12 hpf), under the treatments of 15% lethal concentration or higher concentrations, 11a and 11b inhibited the expression of my17, cmlcl and tbx20 in heart. As the embryo development, 11a and 11b influenced the expression of my17, cmlcl and tbx20 gene as well as had significantly difference at its effect.High concentrations of 11a and 11b inhibited the expression of cmlcl, influenced the expression of my17 and promoted the expression of tbx20 during the development of the zebrafish heart, suggesting that the chiral oxindole derivatives affected the early development of the zebrafish heart by influencing expressions of cmlcl and my17, or influenced the process of heart cyclization by changing the expression pattern of tbx20. At the same time, we found that regardless of the same treatment concentration, or the same lethal concentration treatment, there are significant differences in the toxicological effects between 11a and 11b in the zebrafish embryonic development, suggesting that the toxicities of the enantiomers (11a and 11b) are differences.