R. Bernini et al. / European Journal of Medicinal Chemistry 46 (2011) 439e446
445
washed with a saturated solution of NaCl (5 ml) and dried over
Na2SO4. Following evaporation of the solvent, 3,4-dihydrox-
yphenethyl 5-(1,2-dithiolan-3-yl)pentanoate 8 was isolated in 62%
Following evaporation of the solvent and chromatographic purifi-
cation on silica gel (eluent: ethyl acetate/hexane 1:2),
¼
4-hydroxyphenethyl 5-(1,2-dithiolan-3-yl)pentanoate 12 was iso-
yield (21 mg, oil). 1H NMR (CDCl3, 200 MHz):
d
1.23e1.37 (m, 2H,
lated in 84% yield (27 mg, oil). 1H NMR (CDCl3, 200 MHz):
CH2),1.48e1.62 (m, 4H, 2 ꢂ CH2),1.78e1.91 (m,1H, CH2), 2.29 (t, 2H,
J ¼ 7.0 Hz, CH2), 2.33e2.48 (m, 2H, CH2), 2.80 (t, 2H, J ¼ 7.0 Hz, CH2),
3.00e3.15 (m, 2H, CH2), 3.43e3.51 (m, 1H, CH), 4.23 (t, 2H,
J ¼ 7.0 Hz, CH2), 6.51 (dd, 1H, J1 ¼ 8.0 Hz, J2 ¼ 2.1, Ph), 6.64 (d, 1H,
J ¼ 2.1 Hz, Ph), 6.69 (d, 1H, J ¼ 8.0 Hz, Ph). 13C NMR (CDCl3, 50 MHz):
d
1.30e1.43 (m, 2H, CH2), 1.50e1.67 (m, 4H, 2 ꢂ CH2), 1.79e1.92 (m,
1H, CH2), 2.25 (t, 2H, J ¼ 7.0 Hz, CH2), 2.32e2.48 (m, 1H, CH2), 2.80
(t, 2H, J ¼ 7.0 Hz, CH2), 3.05e3.14 (m, 2H, CH2), 3.42e3.56 (m, 1H,
CH), 4.19 (t, 2H, J ¼ 7.0 Hz, CH2), 6.71 (d, 2H, J ¼ 8.5 Hz, Ph), 7.00
(d, 2H, J ¼ 8.5 Hz, Ph). 13C NMR (CDCl3, 50 MHz):
d 24.6, 28.6, 29.6,
d
24.6, 28.6, 29.6, 34.4, 34.5, 38.4, 40.2, 56.3, 65.1, 115.0, 115.6, 120.6,
34.0, 34.2, 38.4, 40.2, 56.3, 65.2, 115.2, 115.3, 128.8, 129.8, 129.9,
155.2, 173.7. HR-MS m/z: 326.4761 (Mþ). Anal. Calcd. for
C16H22O3S2: C, 58.86; H, 6.79; O, 14.70; S, 19.64. Found: C, 58.75; H,
6.88; O, 14.65; S, 19.72.
129.8, 142.9, 144.2, 173.8. HR-MS m/z: 342.4755 (Mþ). Anal. Calcd.
for C16H22O4S2: C, 56.11; H, 6.47; O, 18.69; S, 18.73. Found: C, 56.20;
H, 6.55; O, 18.65; S, 18.60.
4.1.2. Synthesis depicted in Scheme 2
4.1.2.5. Step (e). Aromatic hydroxylation of 4-hydroxyphenethyl 5-
(1,2-dithiolan-3-yl)pentanoate 12 was performed according to the
procedure described in Section 4.1.1.2. Following chromatographic
purification of the reaction mixture on silica gel (eluent: ethyl
acetate/hexane ¼ 1:7), 3,4-dihydroxyphenethyl 5-(1,2-dithiolan-3-
yl)pentanoate 8 was recovered in 80% yield (27 mg).
4.1.2.1. Step (a). See the procedure described in Section 4.1.1.1.
Reaction time was 24 h. Following evaporation of the solvent, 4-
methoxyphenethyl methyl carbonate 9 was isolated as an oil in
quantitative yield. 1H NMR (CDCl3, 200 MHz):
d 2.89 (t, 2H,
J ¼ 7.1 Hz, CH2), 3.73 (s, 3H, COOCH3), 3.75 (s, 3H, OCH3), 4.27 (t, 2H,
J ¼ 7.1 Hz, CH2), 6.82 (d, 2H, J ¼ 8.6 Hz, Ph), 7.11 (d, 2H, J ¼ 8.6 Hz,
Ph). 13C NMR (CDCl3, 50 MHz):
d
34.3, 54.7, 55.2, 68.6, 114.0, 114.1,
4.2. Antiproliferative activity
129.2, 129.3, 129.9, 156.0, 158.4. HR-MS m/z: 210.2265 (Mþ). Anal.
Calcd. for C11H14O4: C, 62.85; H, 6.71; O, 30.44. Found: C, 62.92; H,
6.68; O, 30.40.
4.2.1. Test compounds
Hydroxytyrosol,
dimethyl sulphoxide (DMSO, Sigma) at final concentrations of 100,
150, and 300 M. For controls, the equivalent volume of 0.1% DMSO
a-lipoic acid, and ester 8 were dissolved in 0.1%
4.1.2.2. Step (b). 4-Methoxyphenethyl methyl carbonate 9 (0.5 mmol,
105 mg) was solubilised in THF (10 ml), then a 1 N solution of NaOH
(2 ml) was added. The mixture was stirred at room temperature and
monitored by thin layer chromatography. After 24 h, the solvent was
evaporated under vacuum. The residue was neutralised with a 1 N
solution of HCl and the aqueous phase was extracted with ethyl acetate
(3 ꢂ 10 ml). The organic phases were washed with a saturated solution
of NaCl (10 ml) and dried over Na2SO4. Following evaporation of the
solvent, 2-(4-methoxyphenyl)ethanol 10 was isolated in quantitative
m
alone was added.
4.2.2. Test system and culture conditions
The human colorectal adenocarcinoma HT-29 cell line was
obtained from the American Type Culture Collection (ATCC, Rock-
ville, MD, USA) and grown in Dulbecco’s medium supplemented
with 10% fetal calf serum, 100 IU/ml penicillin, 100 mg/ml strep-
tomycin, and 0.3 mg/ml
L-glutamine, in a humidified incubator at
yield (0.5 mmol, 76 mg). 1H NMR (CDCl3, 200 MHz):
d 2.79 (t, 2H,
37 ꢁC, 5% CO2.
J ¼ 6.5 Hz, CH2), 3.77 (s, 3H, OCH3), 3.80 (t, 2H, J ¼ 6.6 Hz, CH2), 6.84 (d,
2H, J ¼ 8.7 Hz, Ph), 7.13 (d, 2H, J ¼ 8.6 Hz, Ph).13C NMR (CDCl3, 50 MHz):
4.2.3. Viability assessment
Cell viability was assessed by the trypan blue dye exclusion
assay.
d
38.3, 55.3, 63.8, 114.0, 129.8, 129.9, 130.4, 158.3. HR-MS m/z: 152.1904
(Mþ). Anal. Calcd. for C9H12O2: C, 71.03; H, 7.95; O, 21.03. Found: C,
70.89; H, 8.01; O, 21.10.
4.2.4. Proliferation assay
4.1.2.3. Step (c). See the procedure described in Section 4.1.1.5.
Following chromatographic purification of the reaction mixture on
silica gel (eluent: ethyl acetate/hexane ¼ 1:7), 4-methoxyphenethyl
5-(1,2-dithiolan-3-yl)pentanoate 11 was isolated in 92% yield. 1H
Cells (2 ꢂ 103 cells/ml) were cultured in triplicates in 96-well
plates with the different compounds at the indicated concentra-
tions at 37 ꢁC for 24 and 48 h. Proliferation was assessed using
a BrdU-ELISA kit (Roche Diagnostics) according to the manufac-
turer’s instructions. Proliferative results are reported as the net
NMR (CDCl3, 200 MHz): d 1.24e1.36 (m, 2H, CH2), 1.43e1.73 (m, 4H,
2 ꢂ CH2), 1.76e1.82 (m, 1H, CH2), 2.23 (t, 2H, J ¼ 7.0 Hz, CH2),
2.32e2.38 (m, 1H, CH2), 2.76 (t, 2H, J ¼ 7.0 Hz, CH2), 3.01e3.07
(m, 2H, CH2), 3.42e3.49 (m, 1H, CH), 3.67 (s, 2H, OCH3), 4.13 (t, 2H,
J ¼ 7.0 Hz, CH2), 6.73 (d, 2H, J ¼ 8.6 Hz, Ph), 7.02 (d, 2H, J ¼ 8.6, Ph).
absorbance values: absorbance at
l
¼ 450 nm of pulse-labelled
cells e absorbance at
l
¼ 450 nm of unlabelled cells.
4.2.5. Cell cycle analysis
13C NMR (CDCl3, 50 MHz):
d
24.6, 28.6, 33.7, 34.0, 34.2, 38.4, 40.2,
Cell cycle was analysed by flow cytometry. Briefly, cells (2 ꢂ 105)
were seeded in 6-well culture plates and maintained in a humidi-
fied atmosphere at 37 ꢁC, 5% CO2 for 24 h. Cell cultures were then
incubated with native or synthetic compounds at the indicated
concentrations. After 24 h, the cells were harvested, washed,
55.2, 56.3, 65.0, 113.8, 113.9, 129.8, 129.9, 158.3, 173.5. HR-MS m/z:
340.5027 (Mþ). Anal. Calcd. for C17H24O3S2: C, 59.97; H, 7.10; O,
14.10; S, 18.83. Found: C, 60.05; H, 7.12; O, 14.05; S, 18.78.
4.1.2.4. Step (d). 4-Methoxyphenethyl 5-(1,2-dithiolan-3-yl)pen-
tanoate 11 (0.1 mmol, 34 mg) was dissolved in dry THF (2 ml), then
the mixture was cooled at ꢃ15 ꢁC and a solution of 1 M boron
tribromide in dichloromethane (0.2 mmol) was added. After 2 h,
the solvent was evaporated under vacuum, and the residue was
solubilised with ethyl acetate (5 ml) and treated with a saturated
solution of NaHCO3 (2 ml). The aqueous phase was extracted with
ethyl acetate (3 ꢂ 5 ml). The organic phases were washed with
a saturated solution of NaCl (5 ml) and dried over Na2SO4.
resuspended in a hypotonic buffer, and incubated with 50 mg/ml
propidium iodide at 4 ꢁC for 1 h in the dark. The cells were
subsequently analysed using a FACScalibur (Becton Dickinson, San
Jose, CA, USA); at least 5 ꢂ 104 events were acquired and analysed
using CELLQuest software.
4.2.6. Statistics
Results from the proliferation and cell cycle experiments were
evaluated by the one-way ANOVA test; differences between control