K. H. Lam et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2266–2269
2269
[M(Br79)(Br81)+H]+ and 299.89 [M(Br81)(Br81)+H]+. 3-Bromo-2,6-dimethoxy-4-
diphenylphosphinoylpyridine (5a): 1H NMR (500 MHz, CDCl3): d 3.85 (s, 3H,
OCH3), 3.93 (s, 3H, OCH3), 6.34 (d, J = 15 Hz, 1H, PyH), 7.35–7.32 (m, 4H, PhH),
7.45–7.42 (m, 2H, PhH), 7.56–7.52 (m, 4H, PhH); 13C NMR (125 MHz, CDCl3): d
54.3, 55.1, 98.9, 108.4, 128.9, 130.4, 131.3, 132.1, 132.2, 146.0, 146.8, 160.0,
161.9; 31P NMR (125 MHz, CDCl3): d 31.6; mass spectrum (ESI): 441.8 [M+Na]+.
cells were re-suspended in complete cell culture medium at a concentration of
approximately 1 Â 105/mL and counted manually using a haematocytometer
under an inverted microscope. Cancer cells seeded in the 96 wells microtitre
plates for 24 h were prepared for the phosphine oxides screening. The
compounds were dissolved in molecular biology grade dimethylsulfoxide
(DMSO). Cisplatinum was used as the positive reference and added at
starting concentration of 167 M. Compounds were added at starting
concentration of 100 M and followed by a serial of twofold dilutions and
a
3,5-Dibromo-2,6-dimethoxy-4-diphenylphosphinoylpyridine
(5b):
1H
NMR
l
a
(500 MHz, CDCl3): d 3.99 (s, 6H, OCH3), 7.44–7.47 (m, 4H, PhH), 7.54–7.56 (m,
2H, PhH), 7.70–7.74 (m, 4H, PhH); 13C NMR (125 MHz, CDCl3): d 55.1, 101.2,
128.5, 128.9, 132.1, 132.3, 132.5, 132.6, 133.3, 144.1, 144.8, 158.9, 159.0; 31P
NMR (125 MHz, CDCl3): d 32.9; mass spectrum (ESI): 495.93 [M(Br79)(Br79)+H]+,
497.93 [M(Br79)(Br81)+H]+ and 499.93 [M(Br81)(Br81)+H]+. 2,6-Dimethoxy-3-
phenyl-4-diphenylphosphinoylpyridine (6a): 1H NMR (500 MHz, CDCl3): d 3.85
(s, 3H, OCH3), 3.93 (s, 3H, OCH3), 6.34 (d, J = 15 Hz, 1H, PyH), 7.08–6.95 (m, 6H,
PhH) 7.35–7.32 (m, 4H, PhH), 7.45–7.42 (m, 2H, PhH), 7.56–7.52 (m, 4H, PhH);
13C NMR (125 MHz, CDCl3): d 54.0, 54.3, 105.5, 105.6, 105.7, 119.6, 119.7, 127.3,
128.4, 128.5, 128.6, 131.7, 131.8, 132.5, 133.8, 133.9, 145.7, 146.4, 161.4, 161.5,
161.8, 161.9; 31P NMR (125 MHz, CDCl3): d 27.6; mass spectrum (ESI): 416.17
[M+H]+; 438.15 [M+Na]+. 2,6-Dimethoxy-4-diphenylphosphinoylpyridine (6b): 1H
NMR (500 MHz, CDCl3): d 3.88 (s, 6H, OCH3), 6.52 (d, J = 12 Hz, 2H, PyH), 7.51–
7.53 (m, 4H, PhH) 7.61–7.63 (m, 2H, PhH), 7.64–7.66 (m, 4H, PhH); 13C NMR
(125 MHz, CDCl3): d 54.4, 104.4, 129.2, 132.5, 132.6, 132.9, 133.0, 163.9, 164.0;
31P NMR (125 MHz, CDCl3): d 28.9; mass spectrum (ESI): 340.11 [M+H]+.
6. (a) Cell lines and cell culture. Human breast cancer MDAMB-231 and hepatoma
SKHep-1 cell lines were used for preliminary anti-cancer screening of these
compounds. Both cancer cell lines were routinely maintained in RPMI cell
culture medium supplemented with 5% heat inactivated fetal bovine serum and
penicillin/streptomycin antibiotics (complete cell culture medium). The cells
were incubated in a humidified cell culture incubator at 37 °C with 5% CO2.; (b)
Antiproliferation of phosphosine oxides. Cancer cells were removed from 75 cm3
sterile cell culture flasks with onefold trypsin and neutralized with fetal bovine
serum. After washing with phosphate buffered saline and centrifugation, cancer
l
incubated for a further 48 h. The effective concentration of DMSO in each case
was approximately 0.1% by volume. Untreated control received either total
complete cell culture medium or 0.1% DMSO. Afterwards, the evaluation of
possible anti-proliferative potential of our synthesized compounds was
evaluated by the one step ATP lite assay cell proliferation kit purchased from
PerkinElmer (Netherlands). The one step ATP lite assay is a homogeneous
experiment of determining the number of viable cells in culture based on
quantitation of the ATP present, which is an indicator of metabolically active
cells. The homogeneous assay procedure involves adding the single reagent
directly to cells cultured in serum-supplemented medium. This results in cell
lysis and generation of a luminescent signal proportional to the amount of ATP
present. The amount of ATP is directly proportional to the number of cells
present in culture.; (c) Morphological investigation of cancer cells. MDAMB-231
and SKHep-1 human carcinoma cells were seeded at a concentration of 1 Â 105/
mL. After 24 h, phosphine oxides were added (compound 5b at 60 lM) and
incubated for 48 h. Any morphological changes were compared with the vehicle
control under the inverted microscope.
7. Kok, S. H. L.; Chui, C. H.; Lam, W. S.; Chen, J.; Lau, F. Y.; Wong, R. S. M.; Cheng, G.
Y. M.; Lai, P. B. S.; Leung, T. W. T.; Tang, J. C. O.; Chan, A. S. C. Bioorg. Med. Chem.
Lett. 2007, 17, 1155.
8. Kok, S. H. L.; Gambari, R.; Chui, C. H.; Yuen, M. C. W.; Lin, E.; Wong, R. S. M.; Lau,
F. Y.; Cheng, G. Y. M.; Lam, W. S.; Chan, S. H.; Lam, K. H.; Cheng, C. H.; Lai, P. B. S.;
Yu, M. W. Y.; Cheung, . F.; Tang, J. C. O.; Chan, A. S. C. Bioorg. Med. Chem. 2008, 16,
3626.