Biomolecules 2019, 9, 446
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(2H, m, H-aromatic), 5.01 (1H, m, C30-H), 3.64 (2H, m, C2-H, C6-H), 2.85 (1 H, m, C50-H), 2.56 (1 H,
m, C50-H), 2.38 (1H, m, C40-H), 1.75 (1H, m, C40-H), 1.43 (3H, s, -CH3), 1.38 (3H, s, -CH3); 13C NMR
(DMSO, 75 MHz) δ
(ppm): 198.5 (C-10), 175.1 (2C-3, 5), 172.4 (C-60), 168.6 (C-20), 141.4 (C-8), 140.9 (C-9),
133.0 (2C-12, 120), 129.4 (4C-14, 140), 127.9 (2C-15, 150), 127.4 (4C-13, 130), 79.17 (C-30), 55.7 (2C-1, 7),
49.5 (2C-2, 6), 30.6 (C-50), 21.4 (C-40), 11.9 (2C-11, 110); HRMS (m/z): calculated value for [M + Na]+
100% = 491.1577; found 100% = 491.1576, 35% = 492.1584.
4-(2,6-dioxopiperidin-3-yl)-1,7-diethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-trione (8)
◦
1
M.W. = 496.1997; C30H28N2O5; Yield: 40%; white powder, m.p. 232–234 C; H-NMR (300 MHz,
DMSO-d6+ TMS, δ/ppm): 11.12 (1H, s, -NH), 7.17 (6H, m, H-aromatic), 7.00 (2H, m, H-aromatic), 6.92
(2H, m, H-aromatic), 5.04 (1H, m, C30-H), 3.78 (2H, m, C2-H, C6-H), 2.84 (1 H, m, C50-H), 2.80 (1 H, m,
C50-H), 2.50 (1H, m, C40-H), 2.05 (2H, m, -CH2-), 1.87 (2H, m, -CH2-), 1.71 ( 1H, m, C40-H), 0.88 (6H, t,
J = 6.9 Hz, -CH3); 13C NMR (DMSO, 75 MHz)
δ
(ppm): 198.1(C-10), 175.5 (2C-3, 5), 172.4 (C-60), 168.6
(C-20), 141.8 (2C-8, 9), 133.4 (2C-13, 130), 129.2 (4C-15, 150), 127.1 (2C-16, 160), 127.4 (4C-14, 140), 59.3
(2C-1, 7), 49.6 (C-30), 43.5 (2C-2, 6), 30.6 (C-50), 21.4 (C-40), 19.0 (2C-11, 110), 9.0 (2C-12, 120); HRMS
(m/z): calculated value for [M + Na]+ 100% = 519.1890; found 100% = 519.1891, 30% = 520.1899.
4-(2,6-dioxopiperidin-3-yl)-1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-trione (9)
◦
1
M.W. = 592.1998; C38H28N2O5; Yield: 30%; white powder, m.p. 230–232 C; H-NMR (300 MHz,
DMSO-d6+ TMS, /ppm): 11.16 (1H, s, -NH), 7.69 (4H, m, H-aromatic), 7.36 (6H, m, H-aromatic), 6.91
(6H, m, H-aromatic), 6.78 (4H, m, H-aromatic), 5.16 (1H, m, C30-H), 4.60 (2H, m, C2-H, C6-H), 2.85 (1 H,
m, C50-H), 2.81 (1 H, m, C50-H), 2.50 (1H, m, C40-H), 1.74 (1H, m, C40-H); 13C NMR (DMSO, 75 MHz)
δ
δ
(ppm): 194.8 (C-10), 174.6 (2C-3, 5), 172.4 (C-60), 168.5 (C-20), 141.6 (2C-8, 9), 132.9 (4C-11, 110,15,150),
132.7 (4C-16, 160), 130.5 (4C-17, 170), 129.8 (4C-13, 130), 127.7 (2C-18, 180), 127.4 (4C-12, 120), 127.3 (2C-14,
140), 79.16 (C-30), 65.2 (2C-1, 7), 50.0 (2C-2, 6) 30.5 (C-50), 21.3 (C-40); HRMS (m/z): calculated value for
[M + Na]+ 100% = 615.1890; found 100% = 615.1890, 30% = 616.1909, 6.25% = 617.1952.
2.2. Cell Culturing and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
Cytotoxicity Assay
Human umbilical vein endothelial cells (purchased from Life Technologies, Carlsbad, CA,
USA) were cultured according to the manufacturer’s instructions in Medium 200 supplemented
with Low Serum Growth Supplement. 1
×
104 cells were seeded on each well on a 96-well plate
(Nunc, Roskilde, Denmark). The HeLa (human cervix carcinoma, cat. #93021013), CFPAC (human
pancreatic adenocarcinoma, cat. #91112501), K562 (chronic myelogenous leukemia, cat. #89121407),
HL-60 (acute myelogenous leukemia, cat. #98070106), and MOLT-4 (Human acute T lymphoblastic
leukemia, cat. #85011413) cells were purchased from European Collection of Authenticated Cell
Cultures (ECACC). They were cultured in an RPMI 1640 medium supplemented with antibiotics
(streptomycin, penicillin) and 10% fetal calf serum (20% for HL60), in a 5% CO2-95% air atmosphere.
7
×
103 cells were seeded on each well on a 96-well plate (Nunc). 24 hours later the cells were exposed
to the test compounds. The aliquots of stock solutions of the test compounds (freshly prepared
in DMSO) were added to the cell cultures to yield final concentrations of 1, 10−1, 10−2, 10−3, 10−4
and 10−5 mM. The concentration of DMSO in the cell culture medium was 1%. The IC50 value (the
concentration of a test compound required to reduce the cell survival fraction to 50% of the control)
was calculated from a dose-response curve and used as a measure of cellular sensitivity to a given
,
treatment. The IC50 values are given as means (
±
standard deviation (SD)) from 2 independent
experiments with 4 technical replicates. The cytotoxicity of all compounds was determined by the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Sigma, St. Louis, MO, USA).
Briefly, after 24 h or 48 h of incubation with a given compound, the cells were treated with the MTT
reagent and incubation was continued for 2 h. MTT-formazan crystals were dissolved in 20% SDS and
50% DMF at pH 4.7 and VIS light absorption was measured at 570 and 650 nm on a microplate reader