Date of Award

August 2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Xiaohua peng

Committee Members

James Cook, Alexander Arnold, Arsenio Pacheco, Shama Mirza

Keywords

Alkylating agent, DNA cross-linking agent, Modified nitrogen mustard

Abstract

DNA is the carrier of genetic information, making its chemical reactivity and function extremelyimportant. DNA interstrand cross-links (ICL) leads to covalent bind of two strands, which prevents the separation of double helix. DNA ICLs are the sources of the cytotoxicity of antitumor and anticancer agents. This thesis focuses on investigating the reactivity of a varieties of aromatic compounds towards DNA. Some of these molecules can be activated by photo irradiation to induce DNA ICL formation via alkylation. Others undergo activation by H2O2 to release DNA cross-linking agents. We investigated the mechanism of function of these inducible DNA cross-linking agents as well as their biomedical application. The first part of this thesis is focusing on design, synthesis, and mechanism investigation of the anthracene analogues as photo-inducible DNA cross-linking agents. In an effort to increase the efficiency of photo-inducible DNA cross-linking formation by bifunctional aromatic compounds for biological applications, we used a well-known DNA intercalator anthracene for this study. We synthesized a series of bifunctional compounds containing the 9,10-anthryl core structure, and various benzylic leaving groups and investigated their photo reactivity towards DNA at 350 nm. The efficiency of DNA ICL formation strongly depends on the leaving groups and compound solubility. Compounds 1a-c with bromo, trimethyl ammonium salts or triphenyl phosphonium salts as leaving groups showed faster reaction rate and higher DNA cross-linking efficiency than the remaining compounds. Compounds containing amine (1d and 1e), acetate (1f), or ether groups (1g-j) as leaving groups showed very low DNA ICL efficiency and low reaction rate. DNA ICL formation induced by 1b was inhibited by both radical and cation trapping agents, indicating that both free radicals and cations are involved in DNA crosss-linking process. However, none of the trapping agents was able to inhibit ICL completely even with very high concentration, which might be due to the intercalating interaction between anthracene moiety and duplex DNA. The free radical trapping adducts y-z were detected in the photoreaction of 1b with TEMPO, suggesting the generation of free radicals x in photo-induced DNA cross-linking process of 1b-z. We also observed formation the cation trapping adducts n that provide direct evidence for the generation of carbocation m in the photo-induced DNA ICL formation induced by 1b-c. Collectively, these data suggested that both both free radicals and carbo cations may have been involved in photo-induced DNA ICL formation by these anthracene analogues. The second part of this thesis is focusing on in vivo study of H2O2-activated DNA alkylating agents. We investigated the in vivo efficacy and safety of H2O2-activated nitrogen mustard prodrug 11. The CD-1 mice and athymic nude mice were used for the safety and in vivo efficacy study, respectively. We tested the cytotoxicity of 11 against triple negative breast cancer cell line (MDA-MB-468) and a few renal cancer cell lines (UO-31, SN-12C, A498, TK-10 and CAKI-1). Repeated dose toxicity study was used to identify the safe doses for in-vivo efficacy study. The athymic nude mice were xenografted with MDA-MB-468 breast cancer cell line. We observed that 11 greatly reduced tumor size (>50%) without any obvious side effects such as no obvious weight loss and other unusual behaviors for mice. We did not observe the changes of normal tissues including lung, liver, spleen, brain etc. Pharmacokinetic study showed that duration time of 11 is double of the parent molecule x. In addition, we observed that 11 showed better efficacy but less toxic to mice than the clinically used DNA alkylating agent chlorambucil. Further study suggested that the breast cancer MDA-MB-468 cells showed high H2O2 level and quenching the H2O2 level decreased the anticancer activity of 11, which indicated that there is a correlation between the anticancer activity of 11 and ROS level in cancer cells. Metabolite extraction study was also conducted on cell culture in an effort to identify activated metabolite.

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