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Isotope effects were determined for the oxidative demethylation of the substituted N-methyl-N-(trideuteriomethy1)anilines la-d, and the corresponding N,N-bis(dideuteriomethy1)anilines 2 a 4 , by microsomal cytochrome P-450. The pairs ofp-cyano-andp-nitro-N,N-dimethylanilines were found to have the same intramolecular isotope effects, while the unsubstituted and p-chloro derivatives had different isotope effects. It is concluded that, in general, intramolecular isotope effects measured for the enzymatic oxidations of N-methyl-N-(trideuteriomethy1)anilines are susceptible to masking. The isotope effect for the hydrogen (deuterium) atom abstractions from PhN(CH& vs PhN(CD3)z by the tert-butoxy radical was found to be 2.5. Interestingly, this is the same as the isotope effect measured for the cytochrome P-450 oxidation of N,N-bis(dideuteriomethy1)aniline (2a). These results are discussed with respect to the use of isotope effects for distinguishing the oxidative dealkylation mechanisms of amines by cytochrome P-450 and by related enzymes.

Three analogues of thiazole-4-carboxamide adenine dinucleotide (TAD) (1-3) containing a fluorine atom at the C2' of the adenine nucleoside (in the ribo and arabino configuration) and at the C3' (in the ribo configuration) were synthesized in high yield from the corresponding 5'-monophosphates of 2'-deoxy-2'-fluoroadenosine (9), 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (17), and 3'-deoxy-3'-fluoroadenosine (14), respectively. Pure 2',3'-O-isopropylidene-tiazofurin 5'-phosphorimidazolide (8) was obtained by phosphorylation of the protected tiazofurin followed by treatment with carbonyldiimidazole and HPLC purification. Reaction of 8 with 9 in DMF-d7 (monitored by 1H and 31P NMR) afforded the desired dinucleotide 12, which after deisopropylidenation gave 1 in 82% yield. Small amounts of symmetrical dinucleotides AppA (10, 7.2%) and TRppTR (11, 8.0%) were also isolated during HPLC purification of the major product 12. In a similar manner, compounds 2 and 3 were obtained by coupling of 8 with 14 and 17 in 80% and 76% yield, respectively. All newly prepared fluoro-substituted compounds as well as beta-CF2-TAD, earlier synthesized by us, showed good inhibitory activity against inosine monophosphate dehydrogenase type II, the isozyme which is predominant in neoplastic cells. Binding of 1 (Kis = 0.5 microM), 2 (Kis = 0.7 microM), and 3 (Kis = 2.9 microM) was comparable to that of TAD (Ki = 0.2 microM). The difluoromethylene bisphosphonate analogue, beta-CF2-TAD (Ki = 0.17 microM), was found to be equally effective as the best cofactor-type inhibitor, beta-CH2-TAD (Ki = 0.11 microM). Interestingly, the level of inhibition of horse liver alcohol dehydrogenase by these compounds was found to be much lower (0.1 mM for 1 and 2 and no inhibition up to 10 mM for 3). These findings show that inhibition of tumor-induced inosine monophosphate dehydrogenase type II is selective and may be of therapeutic interest.

This article is available online at http://dmd.aspetjournals.org ABSTRACT:Quinoline was used to probe the steric and electronic contributions to rates of aromatic oxidation of nitrogen-containing, multiring substrates by cytochrome P450 (P450) enzymes. The regioselectivity of the P450 oxidation of quinoline was determined experimentally by identifying and measuring the ratios of metabolites. The laboratory results were compared with those obtained computationally by modeling the electronic effects for aromatic hydroxylation of the substrate. Calculated values predict 8-hydroxyquinoline to have the lowest relative activation energy, whereas 3-hydroxyquinoline was calculated to have the highest relative activation energy. In contrast, 3-hydroxyquinoline was produced to a much greater extent relative to 8-hydroxyquinoline. The sharp contrast observed between the computationally obtained energies and the ratios of products identified experimentally indicates that steric factors play a role in determining the regioselectivity of P450 enzymes with quinoline. To further probe steric contributions to product formation, isoquinoline was used as a substrate and the results were compared with those obtained with quinoline. Isoquinoline N-oxide was determined to be the major metabolite of isoquinoline with all of the P450 enzymes used. These results provide further evidence for the steric influence on the regioselectivity of P450 enzymes with quinoline.The cytochrome P450 enzymes are a superfamily of monooxygenases that function to metabolize both endogenous and exogenous compounds (Ortiz de Montellano, 1995). These enzymes are large contributors to both the prevention and induction of chemical toxicities and carcinogenicity. Although many of these processes result in detoxification, occasionally a more toxic compound is formed (Jones et al., 2002). Since P450 1 enzymes play a central role in bioactivation and detoxification, the ability to predict the products of P450-mediated reactions is a valuable tool. This tool can assess potential risks associated with environmental exposures as well as assist in drug design (Jones et al., 2002).The cytochrome P450 enzymes accommodate a vast array of xenobiotics by metabolizing compounds of a broad chemical diversity. An individual isoform can metabolize multiple substrates and a single substrate at multiple positions. The P450 family catalyzes a wide variety of reactions, including aromatic oxidation, nitrogen and sulfur heteroatom oxidations, and aliphatic hydroxylation (Ortiz de Montellano, 1995). These processes are accomplished through the unique characteristics of the cytochrome P450 family, in that the transition states of reactions are not tightly bound and the enzyme catalyzes the activation of molecular oxygen (Jones and Korzekwa, 1996). Thus, for many reactions, ratios of metabolites produced reflect the intrinsic ease of oxidation at a given position in a molecule, whereas for other reactions oxidation is directed to a specific position by the binding orientation of the s...