The chemotherapeutic agent 6-mercaptopurine (6-MP) has been shown to react covalently with the ultimate carcinogenic metabolite of benzo[a]pyrene, 7-r,8-t-dihydroxy-9-t,10-t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), in aqueous solution, forming a single adduct. NMR studies of the HPLC-purified product were consistent with its identification as 10(S)-(6'-mercaptopurinyl)-7,8,9-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene. Reaction kinetics were analyzed by using both HPLC separation of the products formed and a spectrophotometric assay for adduct formation. A simple model in which direct reaction between 6-MP and BPDE takes place without formation of a physical complex was found to adequately predict the dependence of product ratios on 6-MP concentration. Variations in the observed rate constant for this reaction with changes in temperature, pH, and buffer concentration were determined and compared to the effects of these variables on the observed rate constant for BPDE hydrolysis. In each case, the processes were affected quite differently, suggesting that different rate-determining steps are involved. The data suggest that the reaction mechanism involves SN2 attack of the anion of 6-MP, formed by ionization of the sulfhydryl group, on carbon 10 of BPDE, resulting in a trans-9,10 reaction product.
Dried B. officinalis was purchased from a local store and from the Indiana Botanical Gardens, Hammond, Indiana. Seed was purchased from Stokes Nursery, and plants were grown to maturity for identification (CSU Herbarium No. 53478; D. Wilken, Department of Botany). Alkaloid isolations from gardengrown leaves and stems indicated similar alkaloid content whether fresh or dried. Roots contained the alkaloids as the free base, while fresh leaves had mainly N-oxides. A purchased, dried, bulk sample of plant fragments (496 g) yielded 48 mg of a crude alkaloid mixture (Zn reduction procedure). Tic (with ochloranil-Ehrlich's reagent visualization (5)] showed two major and four trace alkaloids. One major alkaloid was identified as lycopsamine by 360 MHz pmr, ms, and tic comparison with a standard and with literature (6) values. The second was tentatively identified as supinidine viridiflorate (either cynaustine or amabiline) (6,7) by 360 MHz pmr and ms. A standard sample was not available for this identification.
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