Merrick R. Almond

5-Ethynyl-2(1H)-pyrimidinone: Aldehyde oxidase-activation to 5-ethynyluracil, a mechanism-based inactivator of dihydropyrimidine dehydrogenase

5-Ethynyluracil is a potent mechanism-based inactivator of dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) in vitro (Porter et al., J Biol Chem 267: 5236-5242, 1992) and in vivo (Spector et al., Biochem Pharmacol, 46: 2243-2248, 1993. 5-Ethynyl-2(1H)-pyrimidinone was rapidly oxidized to 5-ethynyluracil by aldehyde oxidase. The substrate efficiency (kcat/Km) was 60-fold greater than that for N-methylnicotinamide. In contrast, xanthine oxidase oxidized 5-ethynyl-2(1H)-pyrimidinone to 5-ethynyluracil with a substrate efficiency that was only 0.02% that of xanthine. Because 5-ethynyl-2(1H)-pyrimidinone did not itself inactivate purified DPD in vitro and aldehyde oxidase is predominately found in liver, we hypothesized that 5-ethynyl-2(1H)-pyrimidinone could be a liver-specific inactivator of DPD. We found that 5-ethynyl-2(1H)-pyrimidinone administered orally to rats at 2 micrograms/kg inactivated DPD in all tissues studied. Although 5-ethynyl-2(1H)-pyrimidinone produced slightly less inactivation than 5-ethynyluracil, the two compounds showed fairly similar patterns of inactivation of DPD in these tissues. At doses of 20 micrograms/kg, however, 5-ethynyl-2-pyrimidinone and 5-ethynyluracil produced equivalent inactivation of DPD. Thus, 5-ethynyl-2(1H)-pyrimidinone appeared to be an efficient, but not highly liver-selective prodrug of 5-ethynyluracil.

Enzymatic elimination of fluoride from α-fluoro-β-alanine

Abstract Rat liver homogenates catalyzed the elimination of fluoride from ( R , S )- α -fluoro- β -alanine. The substrate specificity and physical properties of the defluorinating enzyme were similar to those of mitochondrial l -alanine-glyoxylate aminotransferase II (EC 2.6.1.44, AlaAT-II). Furthermore, AlaAT-II activity, measured with l -alanine and glyoxylate as substrates, copurified with the α-fluoro-β-alanine-defluorinating enzyme. The NH 2 -terminal sequence (18 residues) of the enzyme did not show significant sequence similarity with any of the proteins currently listed in GenBank. The purified enzyme catalyzed the transamination of l -alanine (Ala) and glyoxylate (glyx) at pH 8.5 by a ping-pong mechanism with kinetic parameters of k cat = 17 sec −1 , K L -Ala = 3.2 mM, and K glyx = 0.3 mM, respectively. The kinetic parameters for the defluorination of ( R )- α -fluoro- β -alanine and ( S )- α -fluoro- β -alanine were k cat = 6.2 and 2.6 min −1 , respectively, and K m = 2.7 and 0.88 mM, respectively. l -Alanine potently inhibited the defluorination reaction with an apparent K i of 0.024 mM. ( R , S )- α -fluoro- β -alanine converted the optical spectrum of the enzyme-bound cofactor from the pyridoxal form to the pyridox-amino form, which indicated that this cofactor may participate in the defluorination reaction. The product of the enzymatic reaction, malonic semialdehyde, reacted nonenzymatically with ( R , S )- α -fluoro- β -alanine to form an adduct that was detected spectrally. AlaAT-II was not inactivated during dehalogenation of ( R , S )- α -fluoro- β -alanine but was inactivated completely during dehalogenation of β-chloro- l -alanine.

NEW SYNTHESIS OF L-FMAU FROM L-ARABINOSE

ABSTRACT A new synthesis of 2′-deoxy-2′-fluoro-5-methyl-β-L-arabinofuranosyl uracil (13, L-FMAU) was achieved in 10 steps from L-arabinose.

Synthesis of 2-amino-9-(3'-azido-2',3'-dideoxy-beta-D-erythro-pentofuranosyl)-6-methoxy-9H-purine (AzddMAP) and AzddGuo

Abstract A novel synthesis of AzddMAP and AzddGuo is described starting from 1, 2-O-isopropylidene-alpha-D-xylofuranose. The key step in this synthesis involves the replacement of a diphenyl carbamoyl protecting group in the purine 6-position by methanol.

(R)-5-Fluoro-5,6-dihydrouracil : kinetics of oxidation by dihydropyrimidine dehydrogenase and hydrolysis by dihydropyrimidine aminohydrolase

The biologically active isomer of 5-fluoro-5,6-dihydrouracil [(R)-5-fluoro-5,6-dihydrouracil, R-FUH2] was synthesized to study the kinetics of its enzymatic oxidation and hydrolysis by homogeneous dihydropyrimidine dehydrogenase (DPDase) and dihydropyrimidine aminohydrolase (DPHase), respectively. DPDase catalyzed the slow oxidation of R-FUH2 at pH 8 and 37 degrees with a Km of 210 microM and a kcat of 0.026 sec-1 at a saturating concentration of NADP+. The catalytic efficiency (kcat/Km) of DPDase for R-FUH2 was 1/14th of that for 5,6-dihydrouracil (UH2). In the opposite direction, DPDase catalyzed the reduction of 5-fluorouracil (FU) with a Km of 0.70 microM and a kcat of 3 sec-1 at a saturating concentration of NADPH. Thus, DPDase catalyzed the reduction of FU 30,000-fold more efficiently than the oxidation of R-FUH2. In contrast to the slow oxidation of R-FUH2 by DPDase, R-FUH2 was hydrolyzed very efficiently by DPHase with a Km of 130 microM and a kcat of 126 sec-1. The catalytic efficiency of DPHase for the hydrolysis of R-FUH2 was approximately twice that for the hydrolysis of UH2. Because R-FUH2 is hydrolysis of R-FUH2 was approximately twice that for the hydrolysis of UH2. Because R-FUH2 is hydrolyzed considerably more efficiently than it is oxidized and because the activity of DPHase was 250- to 500-fold greater than that of DPDase in bovine and rat liver, the hydrolytic pathway should predominate in vivo.

Novel Synthesis of (+/−)-cis-4-Amino-2-cyclopentene-1-methanol, a Key Intermediate in the Preparation of Carbocyclic 2′,3′-Didehydro-2′,3′-dideoxy Nucleosides

Abstract Novel synthetic routes directed toward the preparation of (+/−)-cis -4-amino-2-cyclopentene-1-methanol are described. The routes investigated involve azide openings of chiral and non-chiral cyclopentyl epoxides.

Synthesis of 1-(3-Azido-2,3-Dideoxy-Beta-D-Ribo-Hexofuranosyl)Thymine

Abstract The nucleoside derivative 1-(3-azido-2,3-dideoxy-beta-D-ribo-hexofuranosyl)thymine has been synthesized from 3-0-benzyl-1,2-0-isopropylidene-alpha-D-glucofuranose-5,6-carbonate in an overall yield of 16%. The key step in the synthesis involves the selective deacetylation of a nucleoside derivative having a cyclic carbonate moiety.