Synthesis of Lipophilic Esters of Tyrosol, Homovanillyl Alcohol and Hydroxytyrosol

Low-molecular weight phenols such as tyrosol, homovanillyl alcohol and hydroxytyrosol are valuable compounds that exhibit a high number of health-promoting effects such as antioxidant, anti-inflammatory and anticancer activity. Despite these remarkable properties, their applications such as dietary supplements and stabilizers of foods and cosmetics in non-aqueous media are limited for the hydrophilic character. With the aim to overcome this limitation, the paper describes a simple and low-cost procedure for the synthesis of lipophilic esters of tyrosol, homovanillyl alcohol and hydroxytyrosol. The reactions were carried out under mild and green chemistry conditions, at room temperature, solubilizing the phenolic compounds in dimethyl carbonate, an eco-friendly solvent, and adding a little excess of the appropriate C2–C18 acyl chloride. The final products were isolated in good yields. Finally, according to the “circular economy” strategy, the procedure was applied to hydroxytyrosol-enriched extracts obtained by Olea europaea by-products to prepare a panel of lipophilic extracts that are useful for applications where solubility in lipid media is required.


Chemicals
Solvents, reagents, standards, silica gel 60 F254 plates, and silica gel 60 were purchased from Sigma-Aldrich (Milan, Italy). Hydroxytyrosol was prepared by the IBX oxidation of tyrosol as already reported [19]. A patented procedure based on membrane technology was applied to obtain hydroxytyrosol-enriched extracts from Olea europaea by-products [45]. The vegetal material was extracted with water and then subject to microfiltration (MF), nanofiltration (NF), and reverse osmosis (RO). Microfiltration was carried out with tubular ceramic membranes in titanium oxide; nanofiltration and reverse osmosis were carried out using spiral wound module membranes of poly(ether sulfone) [45]. The collected fraction was concentrated using a heat pump evaporator (Vacuum Evaporators-Scraper Series, C&G Depurazione Industriale Srl, Firenze, Italy) [3,4]. High Performance Liquid Chromatographic (HPLC) analysis of the resulting extracts was carried out at λ = 280 nm using a diode array detector (DAD) to identify the polyphenolic compounds found in the sample. The quantitative data were referred to pure standards (tyrosol, hydroxytyrosol, oleuropein, and caffeic acid).

Instruments
NMR spectra ( 1 H NMR, 13 C NMR) were recorded using a 400-MHz nuclear resonance spectrometer Advance-III Bruker (Munich, Germany). Each sample (20 mg) was dissolved in chloroform-d3 or methanol-d4 (0.5 mL). The chemical shifts were expressed in parts per million (δ scale) and referenced to either the residual protons or carbon of the solvent.
HPLC analysis of the hydroxytyrosol-enriched extracts and the corresponding lipophilic fractions were carried out using a HP 1200 liquid chromatograph (Agilent Technologies, Palo Alto, CA, USA). The detector was a diode array and the column was a LiChrosorb RP-18 (250 × 4.60 mm, 5 µm i.d.; Merck, Darmstadt, Germany). A flow rate of 0.8 mL min −1 was used for 88 min working from 100% of solvent A (H 2 O at pH = 3.2) to 100% of solvent B (CH 3 CN).

Synthesis of Esters: General Procedure
The appropriate phenethyl alcohol (0.5 mmol) was solubilized in dimethyl carbonate (1.5 mL) at 25 • C. Then the acyl chloride was added (0.6 mmol) and the mixture was kept under stirring for 24 h. The reaction was monitored by thin-layer chromatography on silica gel plates using mixtures of dichloromethane and methanol (9.8/0.2, 9.5/0.5 or 9.0/1.0) as eluents. At the end, the solvent was distilled under reduced pressure and the residue was solubilized with ethyl acetate (10 mL); then a saturated solution of NaCl was added (5.0 mL). After the extraction with ethyl acetate (3 × 10 mL), the combined organic phases were washed with NaCl s.s. (10 mL), dried over Na 2 SO 4 , and filtered. The solvent was distilled under reduced pressure and the residue was purified by a silica gel chromatographic column using mixtures of dichloromethane and methanol (9.8:0.2, 9.5:0.5 or 9.0/1.0) as eluents. All compounds were characterized by NMR analysis. Tyrosyl and homovanillyl alcohol were obtained in yields ranging from 90 to 98%, and hydroxytyrosyl esters from 60 to 68%, as detailed in Table 1. Table 1. Esterification reactions of tyrosol 1, homovanillyl alcohol 2 and hydroxytyrosol 3 (yields calculated after chromatographic purification).  [12,19].

Esterification of Hydroxytyrosol Present in the Extracts: General Procedure
A total of 25 mg (0.16 mmol) of hydroxytyrosol-enriched extracts were dissolved in dimethyl carbonate (5.0 mL), and 0.19 mmol (40-200 µL) of the appropriate acyl chloride was introduced. The reaction was kept at 25 • C under magnetic stirring for 24 h; then, dimethyl carbonate was distillated under reduced pressure by using a rotary evaporator (Laborota 4000, Heidolph, Munich, Germany). The mixture was solubilized with ethyl acetate and washed with a saturated solution of NaCl; then, the combined organic phases were dried over Na 2 SO 4 . After filtration, the solution was recovered, and the solvent was evaporated under reduced pressure. The content of hydroxytyrosol and the corresponding alkyl ester present in each sample was determined by HPLC-DAD analysis at λ = 280 nm. The yields of hydroxytyrosyl esters range from 60 to 66%.

Results and Discussion
Tyrosol 1, homovanillyl alcohol 2 or hydroxytyrosol 3 (Scheme 1) was solubilized in dimethyl carbonate (DMC), an eco-friendly solvent [47], and then a little excess of the appropriate acyl chloride (1.2 equiv.) was added. The reactions were stirred at room temperature for 24 h. After the work-up and column chromatographic purification, the corresponding esters were isolated in good yields ( Table 1). The experimental results confirmed that the esterification reactions proceeded chemoselectively on the alcoholic group due the higher nucleophilicity compared to the phenolic moiety, emphasized by DMC, as already observed by us [19,47]. Even if hydroxytyrosol esters were isolated in lower yields compared to the enzymatic procedures reported in the literature [30][31][32][33][34][35], the simplicity of the operations and low cost of the reagents makes the described procedure attractive.
Most of the isolated esters exhibit a strong antioxidant activity in lipid media as oils and emulsions [33,35,41]. Tyrosol caprylate 7, capriate 8, and laurate 9 show remarkable antimicrobial activity against Leishmania major, Leishmania infantum, Staphylococcus aureus, Staphylococcus xylosus, Bacillus cereus and Brevibacterium flavum [35]. Hydroxytyrosol acetate 26, found in olive oil [46], exhibits antioxidant activity in oil and emulsions [48]; hydroxytyrosol oleate 35, recently found in olive oil by-products [49], is effective as an anti-inflammatory agent. Both derivatives 26 and 35 show antiproliferative activity on human cervical cells (HeLa) [50]. Hydroxytyrosol butanoate 27, decanoate 30, palmitate 33, stearate 34, oleate 35 and linoleate 36 are promising therapeutic agents for topical use in consideration to their cutaneous permeability [44]. Finally, hydroxytyrosol-enriched extracts were esterified under the same experimental conditions using the C2-C18 acyl chlorides. These samples were obtained by a selective extraction of Olea europaea by-products using a sustainable process based on membrane technologies [46]. The extracts contain 60.53 ± 0.41 mg/g of hydroxytyrosol (6.0 % w/w) on a total polyphenols content of 98.14 ± 2.43 mg/g [3,4]. After each esterification reaction, the hydroxytyrosyl ester found in the mixture was characterized and quantified by HPLC-DAD analysis. According to already observed using pure hydroxtytyrosol, the yields of the esterification reactions range from 60 to 66%.
Recently, we evaluated the antiproliferative activity of lipophilic fractions containing hydroxytyrosyl butanoate, octanoate and oleate on the human colon cancer cell line HCT8-β8, a model of colorectal cancer [4]. The experimental data has shown that all fractions exhibited antiproliferative activity. The relevant effect of hydroxytyrosol oleate was related to the high lipophilicity and bioavailability of the compound for the presence of the unsaturated C18 chain [4].

Conclusions
Lipophilic tyrosyl, homovanillyl and hydroxytyrosyl esters were prepared using a green and mild procedure. The products were obtained in good yields, solubilizing 1, 2 or 3 in dimethyl carbonate, an eco-friendly solvent, and adding a little excess of the appropriate C2-C18 acyl chloride. The procedure was then applied to hydroxytyrosol-enriched extracts obtained by olive oil by-products to afford a panel of lipophilic fractions containing hydroxytyrosyl esters of different chain lengths to use for applications where the solubility in lipid media is required.

Conflicts of Interest:
The authors declare no conflict of interest.