Synthesis of Platinum(IV) Anticancer Prodrugs Bearing Carboxylate, Carbamate, and Carbonate Axial Ligands and Exploration of the Activation of Platinum(IV) Prodrugs in Live Cells

Abstract

Platinum(IV) prodrug is attracting increasing attention as the next-generation platinum-based antineoplastic drug candidate. Numerous efforts have been made to design Pt(IV) complexes endowed with promising properties. However, the linkage used to obtain most of these Pt(IV) complexes is limited to carboxylate, which speeds down the expansion of the library of Pt(IV) prodrugs. The conjugations of novel Pt(IV) complexes via carbamate or carbonate linkage are promising strategies to alleviate the problem of the narrow scope. In addition, the available methods applied to monitor the reduction of Pt(IV) complexes heavily depend on either test tube assays or cell lysate studies, leading to a lack of knowledge about how Pt(IV) prodrugs are processed at the cellular level. To compensate for the unavailability of cellular-based tools, designing a sensitive probe that enables monitoring of the reduction of Pt(IV) complexes in live cells is warranted. The former part of the thesis aims to conjugate a series of ligands to Pt(IV) complexes via different linkages and enrich the family of Pt(IV) complexes. The latter section aims to develop a Pt(IV)-based probe featuring a live cell imaging property to determine the fate of Pt(IV) complexes at the cellular level.

Platinum (IV) complexes containing carboxylate and carbamate ligands at the axial position have been reported previously. A better understanding of the similarity and differences between the two types of ligands will provide us with new insights and more choices for designing novel Pt(IV) complexes. We systematically investigated and compared the properties of Pt(IV) complexes bearing the two types of ligands. Ten pairs of unsymmetric Pt(IV) complexes bearing axial carbamate or carboxylate ligands were synthesized and characterized. The stability of these Pt(IV) complexes in a PBS buffer with or without a reducing agent was investigated, and most of these complexes exhibited good stability. Besides, most Pt(IV) prodrugs with carbamate axial ligands were reduced faster than the corresponding ones with carboxylate ligands. Furthermore, the aqueous solubilities and lipophilicities of these Pt(IV) complexes were tested. All the carbamate complexes showed better aqueous solubility and decreased lipophilicity as compared to those of the corresponding carboxylate complexes due to the increased polarity of carbamate ligands. The biological properties of these complexes were also evaluated. Many carbamate complexes showed cytotoxicity similar to that of the carboxylate complexes, which may derive from the lower cellular accumulation but a faster reduction of the former. Our research highlights the differences between the Pt(IV) prodrugs containing carbamate and carboxylate axial ligands, and may contribute to the future rational design of Pt-based anticancer prodrugs.

Pt(IV) complexes bearing axial carbonate linkages have attracted much attention recently. A synthetic method behind this allows the hydroxyl group of bioactive ligands to be attached to the available hydroxyl group of Pt(IV) complexes, and the rapid release of free drugs is achieved after the reduction of carbonate-linked Pt(IV) complexes. Further understanding of the properties of Pt(IV) carbonates, such as hydrolytic stability and reduction profiles, however, is hindered by limited research. Herein, six mono-carbonated Pt(IV) complexes in which the axial carbonate ligands possess various electron-withdrawing powers were synthesized, and the corresponding mono-carboxylated analogues were also prepared as references, to highlight the different properties. The influence of the coordination environment on the hydrolysis and reduction rate of Pt(IV) carbonates and carboxylates was explored. The mono-carbonated Pt(IV) complexes were both less stable and reduced faster than the corresponding mono-carboxylated ones. Moreover, the hydrolysis and reduction profiles were dependent not only on the electron-withdrawing ability of the carbonates but also on the nature of the opposite axial ligands. Besides, the exploration of the hydrolytic pathway for Pt(IV) carbonates suggested that the process proceeded by an attack of OH− on the carbonyl carbon, followed by elimination, which was different from that of Pt(IV) carboxylates. This study provides some information on the influence of axial carbonate ligands, with different electron-withdrawing abilities on the properties of the Pt(IV) center, which may inspire new thoughts on the design of “multi-action” Pt(IV) prodrugs.

Platinum(IV) prodrugs are the most promising candidates as novel anticancer agents. The reduction of Pt(IV) prodrugs in cells is the essential step in activating these complexes. However, the investigation for the reduction profile of Pt(IV) prodrugs is mainly limited to test tube assays or cell lysate-based studies. Herein, we reported a Pt(IV)-caged aminoluciferin, 33c, a bioluminescent reporter for the visualization of Pt(IV) complex in live cells and animals. 33c depends on a reduction-dependent cleavage reaction to release aminoluciferin. Concurrent with this reduction, the initially inactive aminoluciferin center of the complex can now be recognized and oxidized by luciferase to emit light, thus allowing the in-situ monitoring of the reduction process and distribution of Pt(IV) complexes in luciferase-expressing cells or tissues within an animal. The efficiency of releasing luciferin scaffolds among three different bioluminescent probes 33c, 34b, and 35c in live cells and the correlation between their reduction rate and cytotoxicity were tested. The roles of glutathione (GSH), glutathione peroxidase 4 (GPX4), and cysteine (Cys) in the reduction of Pt(IV) complex were also investigated. This work provides an example of monitoring the reduction of Pt(IV) complexes in a real-time, non-invasive, and live-cell manner, making a contribution to understanding the components that reduce Pt(IV) complexes in live cells, affording a starting point for interrogating the bioinorganic chemistry in disease pathology with molecular bioluminescence imaging.

Date of Award	29 Jun 2023
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Guangyu ZHU (Supervisor)