**3.2.1 Synthetic approaches**

Hydroxytyrosyl alkyl ethers have been obtained (Madrona et al., 2009) in a three-step procedure starting from hydroxytyrosol isolated from olive oil waste waters (Scheme 8). This procedure requires first the selective protection of the aromatic hydroxyl groups *via* benzylation with benzyl bromine in the presence of K2CO3, and then the addition of an alkyl iodide under basic conditions, and the subsequent deprotection by catalytic hydrogenation (Pd/C) to obtain the corresponding ethers.

The yield for the alkylation step varies depending on the length of the alkyl chain; as depicted in Scheme 8, the yields decrease as the length of the alkyl chain increases, due to the reduced solubility of the corresponding long chain alkyl iodides in the solvent (DMSO).

Biological Properties of Hydroxytyrosol and Its Derivatives 387

phenolic compounds on their antioxidant potential against cell damage: HT methyl and

Isochroman fragment is a ubiquitous scaffold that can be found in natural products, drugs and agrochemicals (Larghi & Kaufman, 2006). Access to dihydroxyisochromans derived from HT can be achieved by using the oxa-Pictet-Spengler reaction, by reaction of arylethanols with aldehydes, ketones or masked-carbonyl derivatives (Guiso et al. 2001). The reaction is highly regioselective, as intramolecular cyclization takes place mainly in the less hindered position, as it can be deduced from its reaction mechanism, shown in Scheme 9. Two of the synthetized isochromans (Fig. 4) have been detected in olive oil

ethyl ethers are less effective than HT propyl and butyl ethers.

**3.3 Hydroxytyrosol-derived isochromans** 

Fig. 4. Isochromans naturally present in olive oil

Scheme 9. Synthesis of hydroxytyrosol isochroman derivatives

interest to study these metabolites and their biological activities.

**3.4 Hydroxytyrosol glucoronide derivatives** 

Hydroxytyrosol isochroman derivatives shown in Fig. 4 were effective free radical scavengers able to inhibit platelet aggregation and thromboxane release (Togna, 2003).

One of the major metabolic pathways found *in vivo* for dietary phenolic compounds such as hydroxytyrosol is *O*-conjugation via glucuronidation and sulfation. Therefore, it is of

(Bianco et al., 2001).

Scheme 8. Synthesis of hydroxytyrosyl alkyl ethers by alkylation with alkyl iodides

The oxidative stability of lipid matrix in the presence of these compounds, measured by the Rancimat method, has shown that these derivatives retain the high protective capacity of free hydroxytyrosol and similar induction times, having higher induction times than butylhydroxytoluene (BHT) and α-tocopherol (Madrona et al., 2009). These results are in agreement with those obtained in the case of hydroxytyrosyl esters, covered in the previous section (Mateos et al., 2008). The antioxidant activity has been checked by the DPPH, FRAP and ABTS assays in a hydrophilic medium (Pereira-Caro et al., 2009). The antioxidant activity of the lipophilic hydroxytyrosyl ethers was slightly lower in bulk oils and higher in hydrophilic media in comparison with their reference HT, supporting the polar paradox. The length of the alkyl chain did have a positive influence in hydrophilic medium for ethers with a short alkyl chain (methyl, ethyl, propyl), while ethers with longer alkyl chains (from butyl to octadecyl) maintained or decreased their antioxidant activity, probably due to the steric effect of the hydrocarbon chains.

#### **3.2.2 Biological activity**

In order to evaluate the safety and potential biological activity of these ethers, studies of their transport, absorption and metabolism in cellular and animal models have been developed (Pereira-Caro et al., 2010a, 2010b) using a human hepatoma cell line (HepG2) as a model system of the human liver and human enterocyte-like Caco-2/TC7 cells, which are commonly used to characterize the intestinal absorption of a range of drugs, nutrients, and other xenobiotics.

The results showed a direct relationship between the lipophilic nature of each compound and the level of metabolization; as an example, hydroxytyrosyl butyl ether biotransformation was complete after 18 h, whereas small amounts of the others remained after the same time. Furthermore, an intestinal absorption increase was observed from methyl to *n*-butyl ethers.

Protective effects against oxidative stress have also been studied (Pereira-Caro et al., 2011) using HepG2 cells, the ones previously employed to assess the metabolism of the synthesized HT ethers. The results obtained show the potential to prevent cell damage induced by *tert*-butyl hydroperoxide (*t*-BuOOH) and the ability to maintain unaltered cellular redox status, partially after 2 hours of pretreatment and almost completely after 20 hours. These results are in accordance with those obtained with hydroxytyrosol (Martín et al., 2010), but they also show the relevance of the role of the lipophilic character of the phenolic compounds on their antioxidant potential against cell damage: HT methyl and ethyl ethers are less effective than HT propyl and butyl ethers.
