DIHYDROOROTATE ANALOGS AS ENZYME AND TUMOR INHIBITORS (DIHYDROPYRIMIDINE, PYRIMIDINE, ANTITUMOR)

DAVID PETER SAWICK, Purdue University

Abstract

The biosynthesis of ribo- and deoxyribonucleic acids by cells is essential for cell growth and replication. Many antitumor drugs in use today act by interfering with nucleic acid biosynthesis, but through a lack of selectivity for tumor cells, they may be toxic to normal cells at therapeutic doses. In contrast, this research involved targeting for inhibition a biochemical "bottleneck" specific to tumor cells: the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase which supplies precursors for nucleic acid biosynthesis. Effective inhibitors of this enzyme could potentially affect tumor cells selectively thereby lessening toxic side effects. Toward this end, several series of potential inhibitors were designed, synthesized, and tested against the enzyme. The enzyme was isolated from rat liver and assayed in a vesicle preparation using a coupled spectrophotometric assay which had been optimized (K(,m) for L-dihydroorotate was 12.5 (mu)M). Many of the structure activity relationships required for designing effective inhibitors of this key enzyme were established. Testing of derivatives synthesized such as 5-methyl-, 5-hydroxy-methyl-, and 5-ethylorotic acid indicated some bulk tolerance in the 5-position. Reductions of these derivatives to the cis and trans isomers indicated that trans stereochemistry was required for good enzyme inhibition in the 5,6-dihydro series. Replacement of the 6-carboxyl group by a hydroxy group or sulfonamide group was accommodated. However, the 6-phosphate, 6-cyanide, 6-azide, 6-tetrazole, and 6-diazo derivatives were not inhibitors. Other perturbations of the pyrimidine-2,4-dione ring, such as replacement of the amide nitrogen with carbon, led to inactive compounds. The most potent inhibitor tested was 5-aza-5,6-dihydroorotic acid (K(,i) of 9.2 (mu)M). A model for the enzyme active site was proposed which takes these findings into account as well as stereospecific mechanistic considerations. These findings enable the more rational design of potent and selective mechanism based inhibitors of dihydroorotate dehydrogenase. The compounds synthesized were tested against rat hepatoma cells and KB cells in vitro. The 5-aza-5,6-dihydroorotic acid and uracil-6-sulfonamide were as effective as phosphonacetyl-L-aspartate (PALA) at a concentration of 100 (mu)g/ml (100% inhibition of hepatoma cell growth). Against KB cells, these compounds were inactive.

Degree

Ph.D.

Subject Area

Organic chemistry

COinS