Síntesis química y enzimática de biofenoles lipofilos del olivo con actividad antiinflamatoriaoptimización de la reacción de Krapcho

Résumé

In this PhD Thesis it is decribed the synthesis of a series of lipophilic compounds, from biophenols contained in both olive leaf and extra virgin olive oil (EVOO), under the general hypothesis that an increase in the lipophilic character results in an improvement in the biological activity of the compound, as in real biological systems absorption processes take place through lipophilic cell membranes. The fulfillment of this hypothesis would lead us to the conclusion that a greater lipophilicity of a biophenol leads to a better bioavailability and to a greater efficiency in the exercise of the therapeutic action. Specifically, the transformations described in this PhD Thesis aimed at this purpose have been carried out by chemical or enzymatic procedures, or by a combination of both methodologies. First, a novel method for the synthesis of phenol 3,4-dihydroxyphenylglycol (DHPG), a natural compound contained in EVOO, has been developed from phenacyl chloride in 3 steps and with an overall yield of 38% through a simple and easily scalable synthetic route. Second, the synthesis of two new families of lipophilic derivatives 10‒14 and 23‒27 was performed by esterification with carboxylic acids of 2 to 16 carbon atoms from O-benzyl protected catechols 4 and 17, derived from of DHPG 3 and HT 67, respectively. Third, it has been established the synthetic utility of new enzymatic extracts from microorganisms with marked activity in transesterification reactions on model phenols and carbohydrates compounds, concluding that the best results are obtained for deacylation reactions, which turns out to be very selective. As a result, the synthesis of HTAc 29 has been achieved from the triacetylated derivative of HT 142, and also a novel family of lipophilic compounds of DHPG 30-39 was obtained from peracetyl DHPG 2. This family is characterized by displaying the deprotected catechol group and by the substitution in benzylic position of an acetoxy group by an alkoxy group derived from the alcohol used as solvent in the deacylation step. Additionally, in the deacylation of peracetylated disaccharides such as trehalose and sucrose, a preference has been observed for the selective deacylation of one of the monosaccharides leaving the second unchanged, allowing the desymmetrization of trehalose and the access to new derivatives such as the penta-O-acetylated 42 and tetra-O-acetylated 43 in just two steps: peracylation followed by regioselective deacylation. Finally, selective deacylation of the peracetylated ligstroside 147 and oleuropein 148 with these enzyme extracts has allowed the regioselective synthesis of compounds 49 and 50, acetylated only in the glucose ring. The regioselective acylation with acceptable yield (56‒74%) of the O-6 position of the glucose moiety of oleuropein with acyl groups of 2, 4 and 8 carbons was achieved by using Thermomyces lanuginosus, a commercial lipase. Forth, some of the synthesized derivatives have been tested in different models of inflammation. Thus, glycol 3 and HTAc 29 have been shown to be efficient modulators of the inflammatory response of LPS-stimulated peritoneal macrophages, modulating the level of nitrites and the expression of proinflammatory enzymes into the cell. Moreover, derivative 29 has been shown to be an excellent therapeutic agent in inflammatory diseases such as ulcerative colitis, rheumatoid arthritis and systemic lupus erythematosus, combating inflammation and efficiently correcting the level of oxidative stress associated to these pathologies. Following this trend, preliminary studies with peritoneal macrophages indicate that the lipophilic derivatives of oleuropein have a similar effect to that observed for the HTAc 29. Both the peracetylated oleuropein 148 and the partially acylated derivatives obtained enzymatically, the tetraacetylated 50 and the monoacetylated 46, are better than the oleuropein, in terms of the modulation of the inflammatory response and the level of oxidative stress of peritoneal macrophages. Fifth, the Krapcho demethoxycarbonylation reaction applied to the oleuropein has been optimized to obtain, with acceptable yield, the acetylated oleacein or aceolein 51, together with the acetylated dehydrated oleuroside aglycone (DOA) 52, by heating at 150 °C in DMSO followed by acetylation, without prior purification. The same biomimetic strategy has been applied to phenolic extracts of olive oil containing the monoaldehyde aglycones of ligstroside and oleuropein 65 and 66, as well as to the same aglycones previously separated and purified. This methodology allows not only a new access to oleocanthal and oleacein, but also the possibility of enriching phenolic extracts, easy to obtain from EVOOs, in oleocantal and oleacein, two important highly active biophenols with application in the Pharmaceutical and Food industries. Different synthetic routes for stabilizing oleacein 53 have also been explored in order to enable their chromatographic purification, obtaining the aforementioned acetylated derivative 51 and the compounds derived from regioselective reductions 59 and 60, besides to monoacetals of oleacein and oleocanthal 61‒64. Compounds 51 and 52 resulting from the Krapcho reaction on oleuropein followed by acetylation have been tested, together with the lipophilic derivatives of ligstroside 49 and 147 and oleuropein 50-52 and 148 as antiproliferative agents against various human solid tumor cell lines. The results show a progressive improvement of the cellular growth inhibition data as the lipophilicity of the secoiridoid glycosides increases. In addition, both 51 and 52 present better results than the peracetylated HT 142 in all cell lines tested. The compounds with better activity show a higher selectivity to cells of cervix-uterine cancer (HeLa) with GI50 (8.6 ± 4.4) μM, (23 ± 4.3) μM and (15 ± 3.2) μM for 148, 51 and 52 respectively. Finally, a method of quantification by 1H-NMR of the aldehyde secoiridoid derivatives present in the phenolic fraction of the EVOOs has been proposed, allowing quantification of oleacein 53, oleocanthal 57, and monoaldehyde aglycones of ligstroside 65 and oleuropein 66 by integration of the signal corresponding to aldehyde protons above 9.0 ppm.