Diterpenes as models for the study of their effect on neural progenitor cells and on cytochrome p-450 enzymes

Resumen

Therapeutic strategies designed to promote endogenous neurogenesis in the adult brain are a new way to approach the treatment of traumatic and neurodegenerative brain damage. Protein kinase C (PKC) are a group of kinases involved in many cell events, and it has recently been proposed that the modulation of their activity may lead to treatments to promote endogenous neurogenesis. Our research group have shown in previous studies that non-tumor pharmacophores like 12- deoxyphorbols (DPA) and lathyranes with ingol skeleton (ELAC) promote proliferation process in adult neural progenitor cell (NPC) via PKC-activating, thus improving the brain's endogenous neurogenic response to traumatic injury. Occurrence of these compounds on minute amounts on the known sources is a major obstacle to consider them adequate as therapeutic agents. Therefore, the development of these compounds as therapeutic agents requires the exploration of their chemistry in order to be able to guarantee the availability of sufficient quantities of the active compounds and to establish the minimum structural requirement for its activity. In this context, Euphorbia resinifera was selected based on its well-known potential as a source of secondary metabolites, mainly diterpenes. Exhaustive studies of the spectroscopic and spectrometric data were explored following two basic axes: 1- Phytochemical investigation : Phytochemical investigation on latex of Euphorbia resinifera led to the ísolation and identification of eighteen diterpene compounds, including one 12,20-dideoxyphorbol 13-ester, eight 12-deoxyphorbol 13-ester, five 12-deoxy-16-hydroxyphorbol 13,16-diesters, two ingol tetraesters and one daphnane. 2- Development of analytical methods for identification of Euphorbia component. A Ultra-High performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC/HRMS) based targeted and untargeted identification workflow using MSE data-independent acquisition tandem mass spectrometry was developed to analyze and assist isolation of secondary metabolites from a complex phytochemical source. This approach was applied for a biased untargeted screening of new diterpene esters from latex of a Euphorbia resinifera extract. The analysis of E. resinifera fractions revealed that previously not described diterpene esters, including 12,20-dideoxy-phorbol, 12-deoxyphorbol and 12-deoxy-16-hydroxy-phorbol esters, were present in the samples. Consequently, with the aim of using these skeletons as models of new pharmacological agents, 12-deoxy-16-hydroxyphorbol 12,13-diesters (DPPI, DPPT and DPPBz) and phorbol 12,13-diesters described in this work, has been investigated by testing the their ability to release TGFa and promote NPC proliferation, in vitro. The results showing in this thesis provide that those compounds that facilitate the release of TFGα, promote the proliferation of NPC. Furthermore, in order to shed light on phase I metabolism of tigliane derivative, a library of phorbol esters and 12-deoxyphorbol derivatives were tested against microsomal cytochrome P-450 enzymes (CYP450). Based on a high-throughput fluorescence assay of CYP inhibition, many of the tested compounds inhibited two classes of CYPs: CYP3A4 and CYP19.