*2.2. Instruments*

NMR spectra (1H NMR, 13C 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−<sup>1</sup> was used for 88 min working from 100% of solvent A (H2O at pH = 3.2) to 100% of solvent B (CH3CN).

#### *2.3. 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 Na2SO4, 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).

*4-Hydroxyphenethyl acetate (Tyrosyl acetate)* **4**. Colorless oil. NMR spectra are in accordance with those reported in the literature [19,32,35].

*4-Hydroxyphenethyl butanoate (Tyrosyl butyrate)* **5**. Colorless oil. NMR spectra are in accordance with those reported in the literature [32].

*4-Hydroxyphenethyl hexanoate (Tyrosyl hexanoate)* **6**. Colorless oil. NMR spectra are in accordance with those reported in the literature [19,33].

*4-Hydroxyphenethyl octanoate (Tyrosyl caprylate)* **7**. Yellow oil. NMR spectra are in accordance with those reported in the literature [33,35].

*4-Hydroxyphenethyl decanoate (Tyrosyl capriate)* **8**. Yellow oil. NMR spectra are in accordance with those reported in the literature [33,35].

*4-Hydroxyphenethyl decanoate (Tyrosyl laurate)* **9**. Yellow oil. NMR spectra are in accordance with those reported in the literature [32,35].

*4-Hydroxyphenethyl tetradecanoate (Tyrosyl myristate)* **10**. Yellow oil. NMR spectra are in accordance with those reported in the literature [32,35].

*4-Hydroxyphenethyl palmitate (Tyrosyl palmitate)* **11**. Colorless oil. NMR spectra are in accordance with those reported in the literature [19,32,35].

*4-Hydroxyphenethyl stearate (Tyrosol stearate)* **12**. Colorless oil. NMR spectra are in accordance with those reported in the literature e [32,35].

*4-Hydroxyphenethyl oleate (Tyrosol oleate)* **13**. Yellow oil. NMR spectra are in accordance with those reported in the literature e [32,35].

*4-Hydroxyphenethyl linoleate (Tyrosol linoleate)* **14**. Yellow oil. NMR spectra are in accordance with those reported in the literature e [19,32,35].

*4-Hydroxy-3-methoxyphenethyl acetate (Homovanillyl acetate)* **15**. Colorless oil. NMR spectra are in accordance with those reported in the literature [12,19].

*4-Hydroxy-3-methoxyphenethyl butanoate (Homovanillyl butyrate)* **16**. Colorless oil. NMR spectra are in accordance with those reported in the literature [12].

*4-Hydroxy-3-methoxyphenethyl hexanoate (Homovanillyl hexanoate)* **17**. Yellow oil. NMR spectra are in accordance with those reported in the literature [19].

*4-Hydroxy-3-methoxyphenethyl octanoate (Homovanillyl caprylate)* **18**. Yellow oil. 1H-NMR (400 MHz, CDCl3) δ: 6.88–6.86 (1H, m, Ph-H), 6.74–6.72 (2H, m, Ph-H), 5.57 (1H, bs, OH), 4.27 (2H, t, J = 6.0 Hz, OCH2), 3.90 (3H, s, OCH3), 2.88 (2H, t, J = 6.0 Hz, Ph-CH2), 2.31 (2H, t, J = 8.0 Hz, COCH2), 1.60 (2H, m, CH2), 1.28 (8H, m, 4CH2), 0.90 (3H, m, CH3). 13C-NMR (100 MHz, CDCl3) δ: 173.4, 145.9, 143.8, 129.2, 121.1, 113.8, 110.9, 64.5, 55.4, 34.3, 33.9, 31.1, 28.6, 28.5, 24.5, 22.1, 13.5.

*4-Hydroxy-3-methoxyphenethyl decanoate (Homovanillyl capriate)* **19**. Yellow oil. NMR spectra are in accordance with those reported in the literature [12].

*4-Hydroxy-3-methoxyphenethyl dodecanoate (Homovanillyl laurate)* **20**. Yellow oil. 1H-NMR (400 MHz, CDCl3) δ: 6.97–6.95 (1H, m, Ph-H), 6.87–6.72 (2H, m, Ph-H), 5.40 (1H, bs, OH), 4.27 (2H, t, J = 8.0 Hz, OCH2), 3.89 (3H, s, OCH3), 2.88 (2H, t, J = 8.0 Hz, Ph-CH2), 2.31 (2H, t, J = 8.0 Hz, COCH2), 1.64–1.60 (2H, m, CH2), 1.28 (16H, m, 8CH2), 0.92–0.89 (3H, m, CH3). 13C-NMR (100 MHz, CDCl3) δ: 173.4, 145.9, 143.8, 129.1, 121.1, 113.9, 110.9, 64.5, 55.4, 34.3, 33.9, 31.4, 29.1, 29.0, 28.9, 28.8, 28.7, 28.5, 24.4, 22.1, 13.6.

*4-Hydroxy-3-methoxyphenethyl tetradecanoate (Homovanillyl myristate)* **21**. Yellow oil. 1H-NMR (400 MHz, CDCl3) δ: 6.87–6.85 (1H, m, Ph-H), 6.74–6.72 (2H, m, Ph-H), 5.60 (1H, bs, OH), 4.27 (2H, t, J = 8.0 Hz, OCH2), 3.89 (3H, s, OCH3), 2.88 (2H, t, J = 8.0 Hz, Ph-CH2), 2.31 (2H, t, J = 8.0 Hz, COCH2), 1.64–1.60 (2H, m, CH2), 1.28 (20H, m, 10CH2), 0.92–0.89 (3H, m, CH3). 13C-NMR (100 MHz, CDCl3) δ: 173.4, 145.9, 143.8, 129.1, 121.1, 113.9, 110.9, 64.5, 55.4, 34.3, 33.9, 31.4, 29.1, 29.0, 28.9, 28.8, 28.7, 28.6, 28.5, 28.4, 24.5, 22.2, 13.6.

*4-Hydroxy-3-methoxyphenethyl palmitate (Homovanillyl palmitate)* **22**. Colorless oil. NMR spectra are in accordance with those reported in the literature [19,30].

*4-Hydroxy-3-methoxyphenethyl stearate (Homovanillyl stearate)* **23**. Yellow oil. NMR spectra are in accordance with those reported in the literature [12].

*4-Hydroxy-3-methoxyphenethyl oleate (Homovanillyl oleate)* **24**. Yellow oil. NMR spectra are in accordance with those reported in the literature [19].

*4-Hydroxy-3-methoxyphenethyl linoleate (Homovanillyl linoleate)* **25**. Yellow oil. NMR spectra are in accordance with those reported in the literature [19].

*3,4-Dihydroxyphenethyl acetate (Hydroxytyrosyl acetate)* **26**. Yellow oil. NMR spectra are in accordance with those reported in the literature [12,19,46].

*3,4-Dihydroxyphenethyl butanoate (Hydroxytyrosyl butyrate)* **27**. Colorless oil. NMR spectra are in accordance with those reported in the literature [4,12,44].

*3,4-Dihydroxyphenethyl hexanoate (Hydroxytyrosyl hexanoate)* **28**. Yellow oil. NMR spectra are in accordance with those reported in the literature [19].

*3,4-Dihydroxyphenethyl octanoate (Hydroxytyrosyl caprylate)* **29**. Colorless oil. NMR spectra are in accordance with those reported in the literature [34].

*3,4-Dihydroxyphenethyl decanoate (Hydroxytyrosyl capriate)* **30**. Yellow oil. NMR spectra are in accordance with those reported in the literature [12,44].

*3,4-Dihydroxyphenethyl dodecanoate (Hydroxytyrosyl laurate)* **31**. Yellow oil. NMR spectra are in accordance with those reported in the literature [31].

*3,4-Dihydroxyphenethyl tetradecanoate (Hydroxytyrosyl myristate)* **32**. Yellow oil. NMR spectra are in accordance with those reported in the literature [30].

*3,4-Dihydroxyphenethyl palmitate (Hydroxytyrosyl palmitate)* **33**. Yellow oil. NMR spectra are in accordance with the literature [19,31,44].

*3,4-Dihydroxyphenethyl stearate (Hydroxytyrosyl stearate)* **34**. Yellow oil. NMR spectra are in accordance with those reported in the literature [12,31,44].

*3,4-Dihydroxyphenethyl oleate (Hydroxytyrosyl oleate)* **35**. Colorless oil. NMR spectra are in accordance with those reported in the literature [19,31,44].

*3,4-Dihydroxyphenethyl linoleate (Hydroxytyrosyl linoleate)* **36**. Colorless oil. NMR spectra are in accordance with those reported in the literature [19,31,44].

#### *2.4. 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 Na2SO4. 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%.

#### **3. 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 [30–35], 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 e ffective 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].

**Scheme 1.** Esterification reactions of phenolic compounds **1**, **2** and **3**.

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].
