Biomimetic Metabolism of Kaurenoic Acid Validated by Microsomal Reactions

Abstract

The cytochrome P450 is a remarkable family of enzymes with widespread occurrence in animals and plants. In humans, they catalyze phase I metabolism reactions, such as oxidations and hydroxylations, playing a significant role in xenobiotics detoxification. Although mechanistic details of the catalytic cycle of cytochrome P450 are known, and human isoforms have their crystal structure solved, mimicking cytochrome P450 reactions using in vitro systems is still challenging. For this reason, biological assays using isolated enzymes or microsomal fractions are typically required to study the oxidative metabolism of new drugs before approval by regulatory agencies. Here we demonstrate that a series of metalloporphyrin catalysts generate the same oxidative product in vitro as a liver microsomal assay. The bioactive diterpene kaurenoic acid was chosen as a substrate model because it is a component of widely used phytomedicines in Brazil, such as guaco syrups and copaiba waxes. We evaluated the influence of reaction parameters such as catalyst type, oxygen donor type, solvent, and catalyst concentration on substrate consumption. Three products had their structures proposed by mass spectrometry, and among them, a dihydroxylated oxidation product was detected as a major metabolite after liver microsomal biotransformation and using porphyrin catalysts. We envision that the use of metalloporphyrins could be a useful strategy for biomimetic oxidation of kaurenic terpenes.

Oxidation Products from the Neolignan Licarin A by Biomimetic Reactions and Assessment of in vivo Acute Toxicity

“…On the other hand, biomimetic models are simple, with fewer interferences in the reaction medium, allowing the detection of minority products. This model has been applied to understand the phase I metabolism of several natural products, such as lapachol [12], piplartine [24][25], grandisin [26][27][28], monensin [29], and kaurenoic acid [9,30], in which some products were also observed using microsomal models or in vivo models. In this work, it was demonstrated that the biomimetic oxidation of licarin A by Jacobsenʼs catalyst was able to reproduce the oxidation system with RLM and HLM.…”

“…drolysis, oxidation, or reduction reactions that increase the polarity of drugs to promote their excretion, mainly by the kidney, through urine [8]. These reactions can be reproduced in vitro by biomimetic chemical models based on synthetic metalloporphyrins and metal-salen complexes, such as the Jacobsen catalyst [9].…”

“…The metalloporphyrins ([Fe(TPP)Cl]) exhibit a unit of iron (III) porphyrins; they are able to mimic the CYP enzymes and have shown similar products compared to in vivo experiments. Biomimetic oxidation systems reveal advantages in relation to other in vitro models because they minimize the use of animals for metabolism experiments, and high amounts of the oxidized products can be obtained, which improve its isolation at acceptable costs and can assist to understand the products yielded in vivo [9][10]. Moreover, this alternative in vitro metabolism model allows comparison and identification of products generated in vivo in preclinical or clinical phases of drug development [11][12].…”

“…Moreover, this alternative in vitro metabolism model allows comparison and identification of products generated in vivo in preclinical or clinical phases of drug development [11][12]. The chemical structure determination of products can be assessed by the application of the mass spectrometry technique for systematic investigation based on gas-phase fragmentation reactions, such as the electrospray ionization tandem mass spectrometry (ESI-MS/MS) coupled to liquid chromatography, which is an useful tool to analyze in vitro metabolism reactions [9].…”