Synthesis and antiproliferative activity of threo-5-fluoro-L-dihydroorotate.

Abstract

Fluorinated compounds play an important role in enzymology as well as clinical medicine. Based on the stereochemical preferences of dihydroorotate oxidase and enzymes that use fluoroaspartate, it was anticipated that threo-5-fluoro-L-dihydroorotate (t-FDHO) would have the properties of an antimetabolite. Thus, t-FDHO was synthesized via the reduction of 5-fluoroorotate using NADH and dihydroorotate dehydrogenase that was free of dihydroorotase. When the product was purified and studied by high field proton and carbon 13 NMR, the fluorine, the five carbons, and all the nonexchangeable protons were readily observed. Confirmation of threo configuration was obtained by examining the vicinal coupling constants between the substituents on carbon 5 and carbon 6 of the newly synthesized compound. Additionally, t-FDHO could be reoxidized to 5-fluoroorotate in the presence of dihydroorotate dehydrogenase and NAD+. Treatment of t-FDHO with dihydroorotase generated N-carbamyl-threo-3-fluoro-L-aspartate (CTF-ASP) which was also purified and characterized by NMR. The antiproliferative activity of t-FDHO was determined against a diploid human fibrosarcoma cell line (HT-1080). Fifty microM t-FDHO caused 50% inhibition of HT-1080 cell proliferation. During the 48-h toxicity study, extracellular t-FDHO underwent significant hydrolysis to CTF-ASP. Further extracellular degradation to fluoroaspartate was not seen. The antiproliferative activity of t-FDHO was not due to extracellular degradation since CTF-ASP itself was essentially nontoxic.

• Authors: P K Rathod, A Khatri
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 265 Issue 24 Pg. 14242-9 (Aug 25 1990) ISSN: 0021-9258 [Print] United States
• PMID: 2167311 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antineoplastic Agents
• Indicators and Reagents
• 5-fluorodihydroorotate
• Orotic Acid
• Dihydroorotate Oxidase

Topics

• Antineoplastic Agents (chemical synthesis)
• Cell Line
• Chromatography, Ion Exchange
• Dihydroorotate Oxidase (metabolism)
• Fibrosarcoma
• Humans
• Indicators and Reagents
• Magnetic Resonance Spectroscopy
• Orotic Acid (analogs & derivatives, chemical synthesis, isolation & purification, pharmacology)
• Tumor Cells, Cultured (cytology, drug effects)