Date of Award

May 2024

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Xiaohua Peng

Committee Members

Alexander E Arnold, Arsenio A Pacheco, Shama P Mirza, David N Frick

Keywords

Boron, Cancer, Fluorescence, Hydrogen peroxide, Prodrugs, Theranostics

Abstract

Triple negative breast cancer (TNBC) is a notoriously difficult disease to treat, and many of the existing TNBC chemotherapeutics lack tumor selectivity and the capability for simultaneously visualizing and monitoring their own activity in the biological context. However, TNBC cells have been known to generate high levels of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2). To this end, four novel small molecule theranostics 35, 55, 58 and 76 consisting of both H2O2-responsive nitrogen mustard and profluorogenic functionalities were designed and synthesized. These H2O2-responsive theranostics have been evaluated as targeted cancer therapeutics and bioimaging agents. The theranostics 35, 55, and 58 comprise of boronate esters that deactivate nitrogen mustard functional groups and fluorophores; however, they are capable of selective activation through H2O2-specific oxidative deboronation to release the active drug and fluorophore. The three theranostics demonstrated H2O2-inducible DNA-alkylating capability and fluorescence turn-on properties in addition to selective anticancer activity. They are particularly effective in killing TNBC MDA-MB-468 cells with high H2O2 level while safe to normal epithelial MCF-10A cell. This anticancer efficacy has translated well into xenograft mice, as 55 induced 8-fold tumor growth inhibition relative to the vehicle. At a dose of 5 mg/kg a day for 5-days per week over the course of an 8-week treatment, no obvious weight loss nor toxicity in the organs were observed. Moreover, the conjugated boron-masked fluorophores in 55 and 58 are highly responsive towards H2O2, which enabled tracking of the theranostics in live cells’ mitochondria and nucleus. The theranostics’ profluorescence character also enabled detection of the activated 55 derivate in mice blood plasma. The three theranostics 35, 55, and 58 are capable of both selective release of the active drug to take effect in H2O2-rich cancer sites and simultaneously monitoring its activity. This single molecule system is of utmost importance to understand the function, efficacy, and mechanism of the H2O2-activated prodrugs within the living recipient. In addition to theranostics, novel phenylboronic nitrogen mustard prodrugs 88a (d0) and 88b (d5) have been designed, synthesized, and evaluated for the purpose of identifying metabolites from in vitro MDA-MB-468 cell cultures. Both molecules are structurally identical except 88a’s ethyl functionality comprises of hydrogens, whereas isotope labeled 88b is composed of deuterium. Six metabolites were identified from TNBC cells treated with equimolar combinations of 88a and 88b, which suggested two separate metabolic pathways, either protodeboronation or deboronation. Protodeboronation was found in both abiotic environment and cellular matrix, while oxidative deboronation only occurs in the presence of cancer cells. Furthermore, prodrug 88a demonstrated modest physicochemical properties along with DNA-alkylating capability to produce cytotoxic interstrand-crosslinked (ICL) adducts. The therapeutic properties translated well into in vitro efficacy by inhibiting MDA-MB-468 cancer cell growth compared to normal, epithelial MCF-10A. This anticancer efficacy has translated well into xenograft mice, as 88a inhibited tumor growth by 9-fold relative to the vehicle. At a dose of 5 mg/kg a day for 5-days per week over the course of an 8-week treatment, no apparent toxicity was observed similar to theranostic 55. The data suggests that d0 and isotope labeled d5 are effective anticancer boron derivatives capable of distinguishing activity from normal, healthy cells.

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