X. Tang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 801–805
805
5. Liu, Z.; Gui, Q.; Fu, T.; Liu, G. Zhonghua Yi Xue Za Zhi 1996, 76, 214.
6. Liu, Z.; Liu, G.; Zhang, S. Cancer Lett. 1996, 108, 67.
7. Liu, J.; Waalkes, M. P. Toxicology 2005, 208, 289.
8. Huerta, S.; Chilka, S.; Bonavida, B. Int. J. Oncol. 2008, 33, 909.
9. Chen, L.; Zhang, Y.; Kong, X.; Lan, E.; Huang, Z.; Peng, S.; Kaufman, D. L.; Tian, J.
J. Med. Chem. 2008, 51, 4834.
10. Ling, Y.; Ye, X.; Ji, H.; Zhang, Y.; Lai, Y.; Peng, S.; Tian, J. Bioorg. Med. Chem. 2010,
18, 3448.
48 h, respectively. The IC50 of doxorubicin was determined by MTT assay. The
reversal fold (RF) value was calculated by dividing IC50 of doxorubicin alone by
IC50 of doxorubicin in combination with 7i or 7j. Experiments were conducted
in triplicates and repeated three times independently.
21. Intracellular doxorubicin accumulation assay: After cultured as described
above, MCF-7 cells and MCF-7/Adr cells were seeded into 24-well plates at
1.0 ꢀ 105 cells per well. The original medium was removed and the cells were
incubated in the 160 lL RPMI 1640 containing no serum. The cells were then
11. Lai, Y.; Shen, L.; Zhang, Z.; Liu, W.; Zhang, Y.; Ji, H.; Tian, J. Bioorg. Med. Chem.
Lett. 2010, 20, 6416.
treated with 7i, 7j or DDB at 25
were exposed to doxorubicin (5
l
M for 1.5 h. At the end of this step, the cells
lM) and the control group was treated with
12. Ling, Y.; Ye, X.; Zhang, Z.; Zhang, Y.; Lai, Y.; Ji, H.; Peng, S.; Tian, J. J. Med. Chem.
2011, 54, 3251.
13. Frederiksen, L. J.; Siemens, D. R.; Heaton, J. P.; Maxwell, L. R.; Adams, M. A.;
Graham, C. H. J. Urol. 2003, 170, 1003.
14. Matthews, N. E.; Adams, M. A.; Maxwell, L. R.; Gofton, T. E.; Graham, C. H. J.
Natl. Cancer Inst. 2001, 93, 1879.
doxorubicin (5 lM) alone. After incubation at 37 °C for 1.5 h, the cells were
washed three times with 4 °C Hank’s resolution. The cells were then lysed and
centrifuged, and the concentration of doxorubicin was determined by HPLC.
Fluorescence detection was operated at excitation and emission wavelengths
of 495 and 560 nm, respectively. The content of protein was measured by
Bradford assay. The final doxorubicin concentration was calibrated by
concentration of protein. Experiments were conducted in triplicates and
repeated three times independently.
15. Sullivan, R.; Graham, C. H. Curr. Pharm. Des. 2008, 14, 1113.
16. Fruttero, R.; Crosetti, M.; Chegaev, K.; Guglielmo, S.; Gasco, A.; Berardi, F.; Niso,
M.; Perrone, R.; Panaro, M. A.; Colabufo, N. A. J. Med. Chem. 2010, 53, 5467.
17. General procedure for the synthesis of intermediates 5a–j: A solution of 4a
(365 mg, 0.80 mmol) and CF3COOH (1 mL) in dry CH2Cl2 (4 mL) was stirred at
room temperature for 2 h. Then Et3N (2 mL) was slowly added, and the mixture
was stirred at room temperature for 30 min. The salts were removed by
filtration over a pad of Celite. The solvent was removed under reduced pressure
and the crude material was purified by column chromatography (PE/
EtOAc = 1:1) to give pure 5a (268 mg) in 94% yield. The compounds 5b–j
were similarly prepared as 5a.
18. General procedure for the synthesis of the target compounds 7a–j: To a solution of
5a (714 mg, 2.00 mmol) in dry CH2Cl2 (40 mL), 6 (808 mg, 2.00 mmol), EDCI
(573 mg, 3.00 mmol), and DMAP (268 mg, 2.20 mmol) were added and the
mixture was stirred at room temperature for 5 h. Then the reaction mixture
was poured into water and extracted with ethylacetate (3 ꢀ 25 mL). The
solvent was evaporated under reduced pressure and the crude material was
purified by column chromatography (PE/EtOAc = 3:1) to give pure 7a in 76%
yield. The target compounds 7b–j were similarly produced as 7a.
22. Gong, P.; Cederbaum, A. I.; Nieto, N. Mol. Pharmacol. 2004, 65, 130.
23. Intracellular accumulation of digoxin: Caco-2 cells were seeded into 24-well
plates at 1.0 ꢀ 105 cells per well. The cells were then treated with 7i, 7j or DDB
at 5, 10, 25 lM. After incubation for 1.5 h at 37 °C, digoxin (5 lM) was added to
each well, and the cells were incubated for another 1.5 h at 37 °C. Then, the
accumulation of digoxin was stopped by washing the cells three times with
4 °C Hank’s resolution. The cells were lysed and centrifuged, and the
concentration of digoxin was determined by liquid chromatography mass/
mass spectrometry (LC/MS/MS) and calibrated as described above.
Experiments were conducted in triplicates and repeated three times
independently.
24. The efflux ratio of digoxin: Caco-2 cells were seeded in Millicell (12 mm,
0.4
l
m, Millicell-PCF, Millipore, USA) at 1.0 ꢀ 105 cells/ml and the medium
was changed daily. The cells were cultured for 25 days to prepare the fully
differentiated confluent Caco-2 cell monolayer. Then the TEER of cell
monolayer was measured using an EVOMTM epithelial volt ohmmeter
(World Precision Instrument, Sarasota, FL) and the TEER over 400
X
ꢀ cm2
19. The data of selected compounds: 7i: yield 80%, mp 153–155 °C. IR (KBr,
m
):
cell monolayer was chosen for experiment. Prior to experiment, Hank’s
resolution was added to the apical and basolateral membranes and allowed
to equilibrate at 37 °C for 30 min. The resolution was removed and the
3058, 1721, 1626, 1490, 1417, 1380, 1320, 1173, 1031, 865 cmꢁ1 1H NMR
;
(CDCl3, 300 MHz, d ppm): 1.32 (d, 3H, CH3, J = 7.26 Hz), 1.94–2.01 (m, 4H,
CH2CH2), 3.67 (s, 3H, COOCH3), 3.95 (s, 3H, ArOCH3), 4.00 (s, 3H, ArOCH3), 4.14
(t, 2H, OCH2, J = 6.0 Hz), 4.44 (t, 2H, OCH2, J = 6.0 Hz), 4.74–4.79 (m, 1H,
CHCOO), 5.96–6.02 (m, 4H, 2 ꢀ OCH2O), 7.03 (s, 1H, Ar-H), 7.37 (s, 1H, Ar-H),
7.56–7.62 (m, 2H, 2 ꢀ Ar-H), 7.71–7.76 (m, 1H, Ar-H), 8.02–8.07 (m, 2H,
2 ꢀ Ar-H), 8.38–8.39 (br d, 1H, CONH, J = 7.2 Hz); ESI-MS: m/z 772.1 ([M+H]+).
Anal. Calcd. for C34H33N3O16S: C 52.92, H 4.31, N 5.45; Found: C 53.08, H 4.23,
compound 7i, 7j or DDB was added at final concentration of 25
resolution. The cells were incubated at 37 °C for 90 min, the resolution was
removed and digoxin (5 M) was added to the one compartment as ‘donor
pool’, and the other compartment as ‘accept pool’. After incubation at 37 °C for
90 min, 500 l of sample was removed from ‘accept pool’ for analyzing the
lM in Hank’s
l
l
concentration of digoxin using liquid chromatography mass/mass
N 5.29. 7j: yield 82%, mp 175–177 °C. IR (KBr,
m
): 3062, 1728, 1630, 1490, 1415,
spectrometry (LC/MS/MS). The apparent permeability coefficient (Papp) was
1366, 1315, 1171, 1042, 868 cmꢁ1 1H NMR (CDCl3, 300 MHz, d ppm): 1.32 (d,
;
calculated by the formula: Papp (cm/s) = (
for drug throughout during certain time Dt (min); A is surface area of the
D
Q/
D
t)/(A ꢀ C0).
DQ (lmol) stands
3H, CH3, J = 7.28 Hz), 3.68 (s, 3H, COOCH3), 3.92 (t, 2H, OCH2, J = 4.5 Hz), 3.96 (s,
3H, ArOCH3), 4.02 (s, 3H, ArOCH3), 4.11 (t, 2H, OCH2, J = 4.5 Hz), 4.37 (t, 2H,
OCH2, J = 4.5 Hz), 4.57 (t, 2H, OCH2, J = 4.5 Hz), 4.73–4.77 (m, 1H, CH), 5.96–
6.02 (m, 4H, 2 ꢀ OCH2O), 7.02 (s, 1H, Ar-H), 7.35 (s, 1H, Ar-H), 7.62–7.66 (m,
2H, Ar-H), 7.73–7.77 (m, 1H, Ar-H), 8.05–8.08 (m, 2H, Ar-H), 8.39–8.40 (br d,
1H, CONH, J = 7.2 Hz); ESI-MS: m/z 810.0 ([M+Na]+). Anal. Calcd. for
monolayer; C0 (lmol) is the initial concentration in the ‘donor poor’.
25. The effect of 7i or 7j on inhibiting Pgp expression in MCF-7/Adr cells was
determined by Western blot assay. MCF-7/Adr cells pretreated with
doxorubicin at 1.5 ꢀ 105/mL were incubated with 25
l
M 7i or 7j at 37 °C for
8 h. After harvested and lysed, the cell lysates (50
lg/lane) were separated by
C
34H33N3O17S: C 55.62, H 4.32, N 4.86; Found: C 55.38, H 4.56, N 4.64.
SDS–PAGE (8% gel) and transferred onto nitrocellulose membranes (PVDF). The
PVDF membranes were then incubated in 5% skim milk solution at 37 °C for
1.5 h, and washed and incubated with primary antibodies (diluted 1:800) for
Pgp and b-actin. After that, the membranes were washed, and incubated with
HRP-labeled goat anti-mouse IgG (diluted 1:800) for Pgp and HRP-labeled goat
anti-rabbit IgG (diluted 1:800) for b-actin at 37 °C for 1 h. Finally, the
membranes were stained using the detection reagent in the ECL detection kit.
20. The reversal effect of 7i and 7j was determined by the following method: MCF-
7 and MCF-7/Adr cells were grown in RPMI 1640-containing 10% fetal calf
serum at 37 °C in a 5% CO2 humidified atmosphere. For the log phase, the cells
were seeded into 96-well plates at 2.0 ꢀ 104 cells per well, the original
medium was removed and the cells were incubated in the 160 lL RPMI 1640
containing no serum. The cells were then treated with various concentrations
of doxorubicin in the presence or absence of 7i, 7j at 0.1, 1.0 and 10 M for
l