In order to investigate the structural features responsible for the cytotoxicity of the naturally occurring lignan nordihydroguaiaretic acid, the synthesis of four structural analogues of NDGA is proposed for the purpose of studying their oxidative metabolism. One analogue in particular (1), a mono-catechol analogue, is successfully synthesized employing a double Stobbe condensation approach. Following synthesis of this compound a series of oxidation experiments is performed consisting of: incubation in rat liver microsomes with and without the trapping agent glutathione (GSH), oxidation with mushroom tyrosinase, oxidation with silver oxide, and oxidation with horseradish peroxidase. Results are analyzed via HPLC and UPLC-MS. It is found that 1 does not autoxidize at pH 7.4 as NDGA does. Two products are produced during incubation of 1 in rat liver microsomes with UPLC-ESI(-)-MS results giving m/z of 879.2 and 574.18. This is consistent with 1 plus 2 GSH and 1 plus 1 GSH respectively; confirming 1 will oxidize to an electrophilic moiety. Oxidation with mushroom tyrosinase is found to produce high levels of product two with m/z 574.2. Oxidation with horseradish peroxidase is found to produce high levels of the m/z 879.2 product. Silver Oxide produced multiple products rather than the expected one major product, but most are found to be inconsistent with the products seen during rat liver microsomal incubation, and are not pursued.

In order to investigate the structural features responsible for the cytotoxicity of the naturally occurring lignan nordihydroguaiaretic acid, the synthesis of four structural analogues of NDGA is proposed for the purpose of studying their oxidative metabolism. One analogue in particular (1), a mono-catechol analogue, is successfully synthesized employing a double Stobbe condensation approach. Following synthesis of this compound a series of oxidation experiments is performed consisting of: incubation in rat liver microsomes with and without the trapping agent glutathione (GSH), oxidation with mushroom tyrosinase, oxidation with silver oxide, and oxidation with horseradish peroxidase. Results are analyzed via HPLC and UPLC-MS. It is found that 1 does not autoxidize at pH 7.4 as NDGA does. Two products are produced during incubation of 1 in rat liver microsomes with UPLC-ESI(-)-MS results giving m/z of 879.2 and 574.18. This is consistent with 1 plus 2 GSH and 1 plus 1 GSH respectively; confirming 1 will oxidize to an electrophilic moiety. Oxidation with mushroom tyrosinase is found to produce high levels of product two with m/z 574.2. Oxidation with horseradish peroxidase is found to produce high levels of the m/z 879.2 product. Silver Oxide produced multiple products rather than the expected one major product, but most are found to be inconsistent with the products seen during rat liver microsomal incubation, and are not pursued.

The rising use of natural products creates an imperative need for an enhanced awareness of the safety of current and new products making their way into the marketplace. An important example is natural products containing lignans as the principal active component. Despite their structural similarity the lignan of creosote bush can cause hepato- and renal toxicity while the lignans of flaxseed have no reported serious toxicity. This dissertation aimed to investigate the oxidative metabolism of such lignans to determine whether reversible, competitive interactions and/or bioactivation may explain the differences in their apparent toxicity.<p> The first objective was to study the metabolism and bioactivation of nordihydroguaiaretic acid (creosote bush) and secoisolariciresinol (flaxseed). Nordihydroguaiaretic acid metabolism in rat liver microsomes led to the production of three glutathione adducts formed via ortho¬-quinone reactive intermediates. This metabolism was independent of NADPH and thus attributed to autoxidation. Secoisolariciresinol metabolism yielded lariciresinol and no glutathione adducts suggesting an absence of bioactivation to reactive quinone intermediates.<p> The second objective was to study the autoxidation of nordihydroguaiaretic acid. The major autoxidation product was a unique, stable schisandrin-like cyclolignan which was the result of nordihydroguaiaretic acid cyclization. The half-life of nordihydroguaiaretic acid in aqueous solution, pH 7.4, 37ºC is 3.14 hours suggesting the cyclolignan may be responsible for some of the biological effects of nordihydroguaiaretic acid.<p> The third objective was to study the inhibition of cytochrome P450 isoforms 1A2, 2B, 2C11 and 3A by lignans derived from creosote bush and flaxseed. None of the lignans caused irreversible inhibition. Both creosote bush and flaxseed lignans caused reversible inhibition of P450 enzyme activity that involved competitive or mixed-type inhibition, however the inhibition was present at nonphysiologically relevant concentrations. Activation of cytochrome P450 isoforms was also observed at low lignan concentrations. The results suggest that P450-mediated bioactivation or reversible inhibition cannot explain the differences in toxicity noted between the lignans of creosote bush and flaxseed.<p> This work suggests a minimal risk for drug-lignan interactions at P450 enzymes. Further studies are warranted to determine the presence and biological and toxicological role of the nordihydroguaiaretic acid cyclolignan in herbal preparations.

The rising use of natural products creates an imperative need for an enhanced awareness of the safety of current and new products making their way into the marketplace. An important example is natural products containing lignans as the principal active component. Despite their structural similarity the lignan of creosote bush can cause hepato- and renal toxicity while the lignans of flaxseed have no reported serious toxicity. This dissertation aimed to investigate the oxidative metabolism of such lignans to determine whether reversible, competitive interactions and/or bioactivation may explain the differences in their apparent toxicity.<p> The first objective was to study the metabolism and bioactivation of nordihydroguaiaretic acid (creosote bush) and secoisolariciresinol (flaxseed). Nordihydroguaiaretic acid metabolism in rat liver microsomes led to the production of three glutathione adducts formed via ortho¬-quinone reactive intermediates. This metabolism was independent of NADPH and thus attributed to autoxidation. Secoisolariciresinol metabolism yielded lariciresinol and no glutathione adducts suggesting an absence of bioactivation to reactive quinone intermediates.<p> The second objective was to study the autoxidation of nordihydroguaiaretic acid. The major autoxidation product was a unique, stable schisandrin-like cyclolignan which was the result of nordihydroguaiaretic acid cyclization. The half-life of nordihydroguaiaretic acid in aqueous solution, pH 7.4, 37ºC is 3.14 hours suggesting the cyclolignan may be responsible for some of the biological effects of nordihydroguaiaretic acid.<p> The third objective was to study the inhibition of cytochrome P450 isoforms 1A2, 2B, 2C11 and 3A by lignans derived from creosote bush and flaxseed. None of the lignans caused irreversible inhibition. Both creosote bush and flaxseed lignans caused reversible inhibition of P450 enzyme activity that involved competitive or mixed-type inhibition, however the inhibition was present at nonphysiologically relevant concentrations. Activation of cytochrome P450 isoforms was also observed at low lignan concentrations. The results suggest that P450-mediated bioactivation or reversible inhibition cannot explain the differences in toxicity noted between the lignans of creosote bush and flaxseed.<p> This work suggests a minimal risk for drug-lignan interactions at P450 enzymes. Further studies are warranted to determine the presence and biological and toxicological role of the nordihydroguaiaretic acid cyclolignan in herbal preparations.