High-Yielding Synthesis of Methyl Orthoformate-Protected Hydroxytyrosol and Its Use in Preparation of Hydroxytyrosyl Acetate

The new methyl orthoformate of the powerful antioxidant hydroxytyrosol (or 2-(3,4-dihydroxyphenyl)ethanol) has been synthesized by a two-step high yielding procedure. The protection stabilizes hydroxytyrosol against fast oxidation and allows both easy chromatographic purification and long term storage. The protective group is hydrolyzed over pH = 10 and below pH = 5, thus allowing the release of the active principle under physiological conditions. The use of the methyl orthoformate-protected hydroxytyrosol allows the preparation of protected hydroxytyrosyl esters, like the acetate herein reported, by selective esterification of the alcoholic function. The subsequent quantitative deprotection under non-aqueous and mild conditions affords the hydroxytyrosyl acetate in high yields.

In view of the above biological properties, it is not surprising that the preparation of pure hydroxytyrosol, to be used as a dietary supplement or as a stabilizer in foods and cosmetic preparations, has been the subject of many patents [8] and articles [9].However, manipulation of 1 suffers from two difficulties.First, hydroxytyrosol is such a good antioxidant that it undergoes quick oxidation in the air, particularly on silica gel and in alkaline medium, [9b] to afford a black polymeric material.This behaviour does not allow either long-term storage, chromatographic purification or easy recovery of 1 from the abundant natural glycoside 2.
The second problem is concerned with the selective esterification of the primary hydroxyl in 1. Preparation of hydroxytyrosyl esters is of interest because the introduction of the ester function results in higher solubility in lipophilic environments, without any loss of antioxidant activity [10].However, due to the competition between alcoholic and phenolic hydroxyls, the simple use of acyl chlorides results in mixtures of mono-di-and tri-esterified derivatives [9a], unless laborious protection of the catechol function as benzyl ethers [10c] or cerium-catalyzed conditions [11] are applied.Better results have been reported under patented transesterification conditions [10a,b] but experimental details are not given.
On this basis, we have been engaged in searching for suitable and easy removable protective groups able to stabilize the catechol function and to allow regioselective esterification.Recently, we reported the novel acetonide 3 as a protected form of 1 [12].The acetonide 3 can be directly obtained from natural oleuropein 2 in good yields (76%) and is stable both over silica and after two months exposition to the air and light.
However, the high intrinsic stability of the acetonide group can be regarded as troublesome if the aim is to deprotect the cathecol function under very mild conditions, as required if protected hydroxytyrosol was used as an antioxidant additive in foods and cosmetics or for the synthesis of hydroxytyrosyl esters.
Therefore, we chose the unprecedented methyl orthoformate moiety (see compound 4 in Figure 2) as an easy removable protective group to be tested for the ability both to stabilize hydroxytyrosol against oxidation and to allow a high yielding synthesis of acetate 8, regarded as a model molecule for other hydroxytyrosyl esters.We report here the synthesis, properties and conditions of protection and removal of methyl-orthoformate 4 together with its use in the preparation of the acetate 8.

Results and Discussion
Synthesis of methyl orthoformate 4 was performed, as depicted in Figure 2, following the same route successfully adopted for the acetonide 3 [12].First experiments to obtain compound 6 through the installation of the methyl orthoformate protection in methyl ester 5 were carried out under transesterification conditions identical to those described for the preparation of corresponding acetonide 3 [12].However, when the substrate 5 and trimethyl orthoformate (TMO) were reacted in chloroform as solvent and camphorsulfonic acid as a catalyst, only small amounts of the desired orthoformate were detected in the reaction mixture.In the hypothesis that this negative result could be the result on the unfavorable transesterification equilibrium, the reaction was forced to product by removing the produced methanol.The use of benzene as solvent, Amberlist ® 15 as a catalyst and a Dean-Stark apparatus filled with molecular sieves to adsorb methanol, resulted in the quantitative production of the methyl orthoformate 6. Subsequent reduction of the ester moiety to give alcohol 4 required only a careful control of the substrate/reagent stoichiometry in order to avoid the formation of overreduction products 9 and 10, not isolated and identified by their GC-MS spectra.Indeed, the use of 1:1 substrate/reagent molar ratio allowed a high yielding recovery of the desired protected hydroxytyrosol 4.
As expected, protection as an orthoformic ester stabilized hydroxytyrosol against oxidation but the orthoformate group proved to be less stable than the previously studied acetonide group.In fact, this protection did not survive on commercial silica.However, chromatographic purification of 4 was still possible if pre-washed silica was used (see Experimental Section).Moreover, quantitative protection removal under non aqueous conditions was easily obtained after a short treatment with the acid resin Amberlist ® 15 in methanol.
Therefore, in order to apply the orthoformate protection both to the recovery of 4 from the glycoside oleuropein and to the synthesis of hydroxytyrosyl esters, we studied the hydrolytic behaviour of 4 at 25°C at various pHs.The experiments were carried out measuring the decrease of the substrate and the contemporary formation of hydroxytyrosol 1 by HPLC.Since preliminary experiments, carried out at pH= 14 and 12, showed complete consumption of 4 after 0.5 h and 1 h, respectively, accurate measurements were made only for pHs ≤ 10. Results are resumed in Figure 3, where only the percent decrease of substrate is shown.As clearly evidenced in Figure 3 and in contrast to the high stability of the acetonide 3 [12], the range of stability of the orthoformate protection is very narrow, ranging between pH=10 (t 1/2 = 25.9 h) and pH= 6.5 (t 1/2 = 112.9h).This feature ruled out any possibility to obtain 4 from oleuropein by applying the same protocol successfully used to get the acetonide 3. On the contrary, the easy hydrolysis of 4 below pH = 5 seemed to be very useful for the selective esterification of hydroxytyrosol at the primary alcoholic function.On these bases, we choose the hydroxytyrosyl acetate 8 as a model molecule and experimented how to get it by using 4 as a precursor.
Quantitative acetylation of hydroxytyrosol methyl-orthoformate 4 was easily carried out, with routine methods, by simply reacting the substrate in tetrahydrofuran (THF) with one equivalent of a mixture of pyridine and acetyl chloride in 1:1 molar ratio.Subsequent chromatographic purification over pre-washed silica gave the desired protected hydroxytyrosyl acetate 7 in 94% yield.In spite of the above reported easy hydrolysis of the orthoformate moiety in 4, deprotection of 7 was unexpectedly troublesome.Trial experiments showed that, under acid catalyzed methanolic transesterification, deprotection took always place together with the concomitant and undesired formation of free hydroxytyrosol.In the hypothesis that the deprotected acidic catechol groups could catalyze the subsequent ester methanolysis, the reaction was carried out by adding a phosphate buffer (pH = 7.2).This resulted in selective deprotection with formation of hydroxytyrosyl acetate 8 in 87% overall yield, with respect to the starting methyl orthoformate 4. In order to avoid loss of product by easy oxidation, the lipofilic resin Sephadex ® LH-20 was used for chromatographic purification.In conclusion, in view of the high yield and the purity (99% via HPLC) of the obtained hydroxytyrosyl acetate 8, this procedure represents a new alternative route towards hydroxytyrosyl esters.