Synthesis of 8–17
General: The 1H NMR (200 MHz) and 13C NMR (50 MHz) spectra were recorded on a Brucker AV 200 spectrometer. The mass spectra of pure compounds were obtained using a Finigan Mat 95 mass spectrometer.
Tri-n-butyl-n-hexadecylphosphonium bromide (8): Equimolar amounts of 1-bromo-n-hexadecane and tri-n-butylphosphine were heated to 65 °C for 3 d. Phosphonium bromide 8 was isolated as a white solid (60–70 %):36 mp: 56–58 °C; 1H NMR (200 MHz, CDCl3, 25 °C): δ=0.81–0.98 (m, 12 H, 4CH3), 1.22 (br s, 22 H, 11CH2), 1.31–1.53 (m, 18 H, 9CH2), 2.36–2.50 ppm (m, 8 H, 4CH2); 13C NMR (50 MHz, CDCl3, 25 °C): δ=12.6 (s, 3CH3), 13.0 (s, CH3), 18.6 (d, 1JC–P=47.3 Hz, P(CH2n-Pr)3), 18.8 (d, 1JC–P=47.0 Hz, PCH2(CH2)14CH3); 21.4 (d, 3JC–P=4.5 Hz, P(CH2)2CH2(CH2)12CH3), 22.1 (s, CH2), 23.3 (s, mCH2), 23.5 (d, 2JC–P=11.6 Hz, P(CH2CH2)3), 28.4 (s, CH2), 28.8 (s, nCH2), 29.0 (s, CH2), 29.1 (br s, rCH2), 30.3 (d, 2JC–P=14.6 Hz, PCH2CH2(CH2)13CH3), 31.3 ppm (s, CH2) (where m+n+r=11C); MS (FAB+, Cs+,13 keV): m/z (%): 427 (100, C16H33P-nBu3) [M]+; MS (FAB−, Cs+,13 keV): m/z (%): 585 (7) [M+2Br]−, 587 (3) [M+2Br]−, 589 (7, 13, 7) [M+2Br]−, 222 (85), 79 (100) [M+Br]−, 81 (100) [M+Br]−; HRMS (EI): m/z [M]+ calcd for C28H60P: 427.44327, found: 427.44344; Anal. calcd for C28H60BrP: C 66.27, H 11.83, P 6.11, found: C 66.32, H 11.98, P 6.17.
Phosphonium iodides 9 and 10 were synthesized accordingly from triphenyl phosphine and methyl iodide or n-pentyl iodide, respectively, as previously described.37, 38
1-n-Butyl-3-methoxymethylimidazolium chloride (13): Methoxymethyl chloride (0.16 g, 0.15 mL, 2.01 mmol) was added to a stirred solution of 1-n-butylimidazole (0.25 g, 0.26 mL, 2.01 mmol), and the resulting mixture was stirred for 1 h at RT. Salt 13 was obtained as a yellow oil (0.41 g, 100 %): 1H NMR (200 MHz, CDCl3, 25 °C): δ=0.80 (t, 3 H, 3JH–H=7.4 Hz, CH3), 1.23 (q, 2 H, 3JH–H=7.4 Hz, CH2CH3), 1.79 (dt, 2 H, 3JH–H=7.4 Hz, CH2CH2CH3), 3.30 (s, 3 H, OCH3), 4.24 (t, 2 H, 3JH–H=7.3 Hz, NCH2), 5.65 (s, 2 H, CH2O), 7.63 (s, 2 H, HCCH), 10.64 ppm (s, 1 H, N+CH); 13C NMR (50 MHz, CDCl3, 25 °C): δ=12.1 (s, CH3CH2), 18.0 (s, OCH3), 30.6 (s, CH3CH2), 48.5 (s, CH3CH2CH2), 56.2 (s, NCH2), 78.6 (s, N+CH2O), 120.7 (s, NCHCH), 121.6 (s, N+CHCH), 134.5 ppm (s, NCHN+); MS (FAB+,Cs+, 13 keV): m/z (%): 373 (7) [2M+Cl]+, 169 (100) [M]+; MS (FAB−, Cs+, 13 keV): m/z (%): 443 (15) [2M+3Cl]−, 339 (18), 241 (62), 239 (100) [M+2Cl]−, 135 (76), 133 (68), 127 (70); HRMS (EI): m/z [M]+ calcd for C9H17N2O: 169.13409, found: 169.13366; Anal. calcd for C9H17ClN2O: C 52.76, H 8.37, N 13.68, found: C 52.38, H 8.72, N 13.96.
The synthesis of compound 16 (below) is given as a representative example for the synthesis of compounds 11, 12, 14–17.
Triethyl(methylthiomethyl)ammonium chloride (16): Under Ar, methylthiomethyl chloride (1.93 g, 20 mmol) was added to a stirred solution of triethylamine (2.02 g, 20 mmol), and the resulting mixture was stirred for 72 h at RT. Salt 16 was obtained as a beige solid (3.95 g, 100 %): mp: 101–102 °C; 1H NMR (200 MHz, CDCl3, 25 °C): δ=1.41 (t, 9 H, 3JH–H=7.1, CH2CH3), 2.51 (s, 3 H, SCH3), 3.53 (q, 6 H, 3JH–H=7.1, CH2CH3), 5.06 (s, 2 H, N+CH2S); 13C NMR (50 MHz, CDCl3, 25 °C): δ=8.2 (s, N+CH2CH3), 18.0 (s, SCH3), 52.6 (s, N+CH2CH3), 64.2 (s, N+CH2S); MS (CI): m/z (%): 162 (100) [Et3N+CH2SCH3]+; HRMS (CI): m/z [M]+ calcd for C8H20NS: 162.13165, found: 162.13213; Anal. calcd for C8H20ClNS: C 48.54, H 10.11, S 16.18, N 7.08, found: C 48.31, H 10.02, S 15.93, N 7.10.
MTT assay: The cytotoxicity of compounds 8–17 was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This assay relies on the reduction of MTT by succinate dehydrogenase, intervening in the respiratory mitochondrial chain of viable cells. The water-soluble yellow MTT is converted to water-insoluble purple formazan, which is then dissolved in organic solvents and measured using a spectrophotometer. The percentage of living cells is calculated from the corrected formazan absorbance. HeLa, K562 or HUVEC cells (5×103 per well) were seeded in a 96-well plate (Nunc, Penfield, NY, USA). After a 24 h incubation period, the cells were exposed to the test compounds for 24 or 48 h. The cytotoxicity of each compound was tested at four concentrations: 50 nM, 500 nM, 50 μM and 500 μM. Compounds 9 and 10 were tested at 1 nM, 100 nM, 10 μM and 1 mM. Cells grown in the presence of 2 % DMSO served as a control (100 % viability). After 24 or 48 h incubation with the test compounds, the MTT reagent was added and incubation was continued for 2 h. The formazan crystals were dissolved in 20 % sodium dodecyl sulfate (SDS) and 50 % DMF (pH 4.7).The absorbance was measured at 570 and 630 nm using an ELx800 microplate reader (BioTek, Winooski, VT, USA). Based on the dose–response curves, IC50 values were calculated and represent the mean ±SD of eight (for compound 9) or four (for compound 10) replicate cultures.
Transfection evaluation: HEK293T (human embryonic kidney) grown in Dulbecco′s modified Eagle′s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) were transferred to a 96-well plate in 100 μL aliquots 24 h before transfection (2×104 cells per well). The transfection procedure for a single 96-well plate was as follows. OPTI-MEM (25 μL) was mixed with Lipofectamine 2000 (0.1 μL, Invitrogen, CA, USA). In parallel, OPTI-MEM (25 μL) was mixed with 0.05 μg pmaxGFP vector DNA (50 ng per well; Lonza, Basel, Switzerland). After a 5 min incubation period at room temperature, both solutions were combined resulting in a Lipofectamine–DNA ratio of 2:1 (v/w) and incubated for an additional 20 min. The mixture was then added to a 96-well plate as a positive control. For tested compounds, the transfection procedure was performed in the same way as described for lipofectamine, except that the amount of tested compound was adjusted to achieve a charge ratio of 5:1 and 10:1 (compound/DNA). Transfection mixtures containing either DNA plasmid or lipofectamine only were used as negative controls. Each transfection experiment was repeated in four separate wells. After an additional 48 h incubation at 37 °C in a 5 % CO2 atmosphere, the culture medium was removed, and the cells were lysed for 30 min in phosphate-buffered saline (PBS) containing 1 % Triton X-100 (200 μL per well, pH 7.4). Fluorescence was measured with a FLUOstar Omega plate reader (BMG Labtech, Offenburg, Germany), with excitation and emission filters of 485/20 nm and 520/20 nm, respectively.
Caspase-3/7 assay: HeLa cells were cultured in RPMI 1640 medium supplemented with antibiotics and 10 % fetal calf serum (FCS) at 37 °C in a 5 % CO2 atmosphere. Cells (15×103) were seeded in each well of a 96-well plate. After 24 h, cells were exposed to test compound at a concentration of four-times the IC50 value and incubated for another 24 h. As controls, cells were exposed to DMSO only (2 % final concentration), staurosporine (1 μM, Sigma, St. Louis, MO, USA) and cisplatin (70 μM, Sigma, St. Louis, MO, USA). The induction of cell apoptosis was analyzed using the Apo-ONE homogeneous caspase-3/7 assay (Promega, Madison, WI, USA). After 24 h of incubation with the test compounds, cells were treated with the caspase-3/7 reagent (according to manufacturer’s instructions) and incubated for an additional hour at room temperature. The fluorescence intensity was measured using a FLUOstar Omega plate reader (BMG Labtech, Offenburg, Germany), with excitation and emission filters of 485/20 nm and 520/20 nm, respectively. Untreated cells and cells treated with staurosporine were used as negative and positive controls, respectively.