Bioreductively activatable prodrug conjugates of phenstatin designed to target tumor hypoxia.

A variety of solid tumor cancers contain significant regions of hypoxia, which provide unique challenges for targeting by potent anticancer agents. Bioreductively activatable prodrug conjugates (BAPCs) represent a promising strategy for therapeutic intervention. BAPCs are designed to be biologically inert until they come into contact with low oxygen tension, at which point reductase enzyme mediated cleavage releases the parent anticancer agent in a tumor-specific manner. Phenstatin is a potent inhibitor of tubulin polymerization, mimicking the chemical structure and biological activity of the natural product combretastatin A-4. Synthetic approaches have been established for nitrobenzyl, nitroimidazole, nitrofuranyl, and nitrothienyl prodrugs of phenstatin incorporating nor-methyl, mono-methyl, and gem-dimethyl variants of the attached nitro compounds. A series of BAPCs based on phenstatin have been prepared by chemical synthesis and evaluated against the tubulin-microtubule protein system. In a preliminary study using anaerobic conditions, the gem-dimethyl nitrothiophene and gem-dimethyl nitrofuran analogues were shown to undergo efficient enzymatic cleavage in the presence of NADPH cytochrome P450 oxidoreductase. Each of the eleven BAPCs evaluated in this study demonstrated significantly reduced inhibitory activity against tubulin in comparison to the parent anti-cancer agent phenstatin (IC50=1.0µM). In fact, the majority of the BAPCs (seven of the eleven analogues) were not inhibitors of tubulin polymerization (IC50>20µM), which represents an anticipated (and desirable) attribute for these prodrugs, since they are intended to be biologically inactive prior to enzyme-mediated cleavage to release phenstatin.

Synthesis of a 2-aryl-3-aroyl indole salt (OXi8007) resembling combretastatin A-4 with application as a vascular disrupting agent.

The natural products colchicine and combretastatin A-4 are potent inhibitors of tubulin assembly, and they have inspired the design and synthesis of a large number of small-molecule, potential anticancer agents. The indole-based molecular scaffold is prominent among these SAR modifications, leading to a rapidly increasing number of agents. The water-soluble phosphate prodrug 33 (OXi8007) of 2-aryl-3-aroylindole-based phenol 8 (OXi8006) was prepared by chemical synthesis and found to be strongly cytotoxic against selected human cancer cell lines (GI50 = 36 nM against DU-145 cells, for example). The free phenol, 8 (OXi8006), was a strong inhibitor (IC50 = 1.1 µM) of tubulin assembly. The corresponding phosphate prodrug 33 (OXi8007) also demonstrated pronounced interference with tumor vasculature in a preliminary in vivo study utilizing a SCID mouse model bearing an orthotopic PC-3 (prostate) tumor as imaged by color Doppler ultrasound. The combination of these results provides evidence that the indole-based phosphate prodrug 33 (OXi8007) functions as a vascular disrupting agent that may prove useful for the treatment of cancer.

Regioselective synthesis of water-soluble monophosphate derivatives of combretastatin A-1.

The natural products combretastatin A-4 (CA4) and combretastatin A-1 (CA1) are potent cancer vascular disrupting agents and inhibitors of tubulin assembly (IC50 = 1-2 µM). The phosphorylated prodrugs CA4P and CA1P are undergoing human clinical trials against cancer. CA1 is unique due to its incorporation of a vicinal phenol, which has afforded the opportunity to prepare both diphosphate and regioisomeric monophosphate derivatives. Here, we describe the first synthetic routes suitable for the regiospecific preparation of the CA1-monophosphates CA1MPA (8a/b) and CA1MPB (4a/b). The essential regiochemistry necessary to distinguish between the two vicinal phenolic groups was accomplished with a tosyl protecting group strategy. Each of the four monophosphate analogues (including Z and E isomers) demonstrated in vitro cytotoxicity against selected human cancer cell lines comparable to their corresponding diphosphate congeners. Furthermore, Z-CA1MPA (8a) and Z-CA1MPB (4a) were inactive as inhibitors of tubulin assembly (IC50 > 40 µM), as anticipated in this pure protein assay.

Development of synthetic methodology suitable for the radiosynthesis of combretastatin A-1 (CA1) and its corresponding prodrug CA1P.

Synthetic methodology has been established suitable for the preparation of combretastatin A-1 (CA1) and its corresponding phosphate prodrug salt (CA1P) in high specific activity radiolabeled form. Judicious selection of appropriate phenolic protecting groups to distinguish positions on the A-ring from the B-ring of the stilbenoid was paramount for the success of this project. Methylation of the C-4' phenolic moiety by removal of the tert-butyldimethylsilyl protecting group in the presence of methyl iodide was accomplished in excellent yield without significant Z to E isomerization. This step (carried out with (12)C-methyl iodide as proof of concept in this study) represents the process in which a (14)C radioisotope could be incorporated in an actual radiosynthesis. CA1 is a natural product isolated from the African bush willow tree (Combretum caffrum) that has important medicinal value due, in part, to its ability to inhibit tubulin assembly. As a prodrug, CA1P (OXi4503) is in human clinical trials as a vascular disrupting agent.