Journal of Medicinal Chemistry
Article
decomposes over 180 °C; IR (ATR) 3682, 3322, 1619, 1603, 1579,
1510, 1432, 1416, 1298, 1246, 1206, 1182, 1109, 1077, 1019, 922, 872,
799, 767 cm−1; 1H NMR (D2O) δ 3.96 (s, 2H, CH2), 7.16 (d, 4H, J =
8.0 Hz, Ar), 7.28 (d, 4H, J = 8.0 Hz, Ar); 13C NMR (400 MHz, D2O)
δ 39.7 (CH2), 125.8, 129.8, 131.1, 141 (Ar), 157.4 (CN); HRMS (m/
z, −ES) 315.1569 calcd [M+ + H]; found 315.1563. Anal.
(C15H20Cl2N6O2·2.75H2O) C, H, N.
base). A quartz cell with a 1 cm path length was filled with a 1 mL
solution of DNA polymer or DNA compound complex. The DNA
polymer (150 μM base) and the compound solution (15 μM) were
prepared in a phosphate buffer [0.01 M Na2HPO4/NaH2PO4],
adjusted to pH 7, so that a compound to DNA base ratio of 0.1
was obtained. The thermal melting temperatures of the duplex or
duplex-compound complex obtained from the first derivative of the
melting curves are reported.
4,4′-Dihydroxyguanidiniumdiphenylether Dichloride (4b):
Methods 1 and 2. Yellow solid (11% of total); mp, decomposes
over 110 °C; IR (ATR) 3630, 3311, 1655, 1621, 1221, 1102, 910,
Biochemistry. Cell Culture. Human Caucasian promyelocytic
leukemia HL-60 and MCF-7 human breast carcinoma breast cells were
obtained from the European Collection of Cell Culures (Porton
Down, Wiltshire, U.K.). Neuroblastoma Kelly cells were a kind gift
from the Royal College of Surgeons (St. Stephens Green, Dublin 2,
Ireland). HL-60 and Kelly cells were grown in Roswell Park Memorial
Institute (RPMI) 1640 medium with GlutaMax I supplemented with
10% (v/v) fetal bovine serum (FBS) and 50 μg mL−1 penicillin/
streptomycin (pen−strep). MCF-7 cells were grown in minimum
essential medium (MEM) with GlutaMax I supplemented with 10%
(v/v) FBS, 1% nonessential MEM amino acids, and 50 μg mL−1 pen−
strep. Cells were grown at 37 °C in a humidified environment
maintained at 95% O2 and 5% CO2 and passaged at least twice weekly
(HL-60) or once weekly (Kelly, MCF-7) depending on their levels of
confluency. HL-60 cells were maintained at a density of between 2 ×
105 and 2 × 106 cells/mL. Adherent cells (Kelly cells and MCF-7)
were subcultured by trypsinization upon reaching 90% confluency.
Compound Preparation. Stock solutions (10 mM) of the
compounds were prepared in sterile ddH2O and were then sterile
filtered (0.2 μM filters). Required concentration ranges (10−0.1 mM)
of each drug were prepared in sterile ddH2O and stored at −20 °C
until required.
Cell Viability (AlamarBlue Assays). HL-60 cells in the log phase
of growth were seeded in 96-well plates at a density of 200 000 cells
mL−1 (200 μL/well or 40 000 cells/well) in complete RPMI medium.
The cells were then treated with a 1:100 dilution of stock
concentrations of drugs in triplicate. Three blank wells containing
200 μL of RPMI were set up as blanks. MCF-7 cells were seeded at a
density of 50 000 cells mL−1 (200 μL/well or 10 000/well) and Kelly
cells at 30 000 cells mL−1 (200 μL/well or 6000/well) and incubated
at 37 °C for 24 h. The cells were then treated with the drugs as for the
HL-60 cells. After 72 h of incubation, 20 μL of AlamarBlue was added
to each well. The plates were incubated in darkness at 37 °C for 4.5 h.
By use of a Molecular Devices microplate reader, the fluorescence (F)
was then read at an excitation wavelength of 544 nm and an emission
wavelength of 590 nm. Cell viability was then determined by
subtracting the mean blank fluorescence (Fb) from the treated sample
fluorescence (Fs) and expressing this as a percentage of the
fluorescence of the blanked vehicle control (Fc), as shown in eq 1.
1
1499, 1406, 1286, 1167, 1077, 1014, 880, 862, 824 cm−1; H NMR
(D2O) δ 7.07 (d, 4H, J = 8.0 Hz, Ar), 7.25 (d, 4H, J = 8.0 Hz, Ar); 13
C
NMR (400 MHz, D2O) δ 119.7, 127.7, 128.5, 155.7 (Ar), 157.6 (CN);
HRMS (m/z, −ES) 315.1362 calcd [M+ + H]; found 317.1364. Anal.
(C14H19Cl3N6O3·2.5H2O) C, H, N.
4,4′-Dihydroxyguanidiniumdiphenylethylphenyl Dichloride
(4c): Methods 1 and 2. Orange solid (21%, 11% of total); mp,
decomposes over 165 °C; IR (ATR) 3671, 3414, 1658, 1590, 1565,
1514, 1496, 1451, 1420, 1404, 1371, 1304, 1243, 1096, 1051, 1018,
1
987, 824 cm−1; H NMR (D2O) δ 2.9 (s, 4H, CH2), 7.09 (d, 4H, J =
8.0 Hz, Ar), 7.21 (d, 4H, J = 8.0 Hz, Ar); 13C NMR (400 MHz, D2O)
δ 35.9 (CH2), 126.0, 130.1, 131.2, 141.9 (Ar), 158.0 (CN); HRMS
(m/z, −ES) 329.1726 calcd [M+ + H]; found 329.1732. Anal.
(C16H22Cl2N6O4·2H2O) C, H, N.
4,4′-Dihydroxyguanidiniumdiphenylthioether Dichloride
(4d): Methods 1 and 2. Brown solid (27%, 19% of total); mp,
decomposes over 247 °C; IR (ATR) 3692, 3301, 1651, 1611, 1591,
1568, 1515, 1490, 1405, 1344, 1302, 1274, 1244, 1179, 1108, 1080,
1014, 950, 904, 892, 862, 815, 736, 704, 672, cm−1; 1H NMR (D2O) δ
7.18 (d, 4H, J = 8.0 Hz, Ar), 7.36 (d, 4H, J = 8.0 Hz, Ar); 13C NMR
(400 MHz, D2O) δ 125.9, 131.9, 132.7, 133.8 (Ar), 156.9 (CN);
HRMS (m/z, −ES) 333.1119 calcd [M+ + H]; found 333.1134. Anal.
(C14H19Cl3N6O2S·3H2O) C, H, N.
4,4′-Dihydroxyguanidiniumdiphenylcarbonyl Dichloride
(4e): Methods 1 and 2. Yellow solid (23%, 8% of total); mp,
decomposes over 115 °C; IR (ATR) 3748, 3370, 1662, 1646, 1623,
1593, 1564, 1558, 1511, 1449, 1389, 1372, 1318, 1293, 1282, 1248,
1182, 1145, 1080, 1015, 987, 975, 959, 934, 865, 850, 763, 738, 686,
667 cm−1; 1H NMR (D2O) δ 7.34 (d, 4H, J = 8.0 Hz, Ar), 7.75 (d, 4H,
J = 8.0 Hz, Ar); 13C NMR (400 MHz, D2O) δ 123. 2, 131.3, 134.0,
138.1 (Ar) 156.1 (CN), 197.39 (CO); HRMS (m/z, −ES) 329.1362
calcd [M+ + H]; found 329.1361. Anal. (C15H18Cl2N6O3·2.4H2O) C,
H, N.
4,4′-Dihydroxyguanidiniumdiphenylamide Dichloride (4f):
Methods 1 and 2. Off-white solid (14% of total); mp, decomposes
over 190 °C; IR (ATR) 3646, 3178, 1652, 1627, 1602, 1549, 1515,
1475, 1412, 1340, 1298, 1243, 1180, 1120, 1077, 1019, 985, 904, 891,
844, 831, 812, 708, 656 cm−1; 1H NMR (D2O) δ 7.32 (d, 4H, J = 8.0
Hz, Ar), 7.37 (d, 4H, J = 8.0 Hz, Ar) 7.57 (d, 4H, J = 8.0 Hz, Ar), 7.89
(d, 4H, J = 8.0 Hz, Ar); 13C NMR (400 MHz, D2O) δ 123.2, 124.3,
126.2, 128.8, 130.2, 132.0, 136.1, 137.3 (Ar), 156.7 (CN), 157.3 (CN),
168.3 (CO); HRMS (m/z, −ES) 344.1471 calcd [M+ + H]; found
344.1461. Anal. (C15H19Cl2N7O3·2H2O) C, H, N.
F − Fb
s
× 100 = % cell viability
F − Fb
(1)
c
The results were then plotted as nonlinear regression, sigmoidal dose−
response curves on Prism, from which the IC50 value for each drug was
determined.
N-Phenylhydroxyguanidinium Hydrochloride (4g):18 Meth-
ods 1 and 2. Yellow oil (49%, 27% of total); IR (film) 3654, 3417,
1655, 1611, 1590, 1567, 1499, 1461, 1449,1390, 1341, 1314, 1298,
1249, 1196, 1181, 1156, 1119, 1068, 1032, 1004, 983, 966, 914, 943,
Determination of DNA Content (Flow Cytometry) of HL-60
Cells. HL-60 cells were seeded at 200 000 cells mL−1 in T25 flasks and
treated with appropriate drug concentrations or ddH2O as vehicle
control for 24, 48, or 72 h. Cells were harvested at each time-point by
centrifugation at 300g for 8 min and the pellets resuspended in 200 μL
of nonsterile PBS. The cells were then fixed by a dropwise addition of
2 mL of ice-cold 70% EtOH/PBS while gently being vortexed.
Following overnight fixation at −20 °C, 10 μL of nonsterile FBS was
added to the cells and the solutions were centrifuged at 800g for 10
min. Ethanol was then drained, the cell pellet resuspended in PBS
containing 0.5 mg mL−1 RNase-A (to denature RNA) and 0.15 mg
mL−1 propidium iodide (PI, a fluorescent DNA binding dye) and then
incubated in the dark at 37 °C for 30 min. The PI fluorescence was
measured on a linear scale using a FACS Calibur flow cytometer
(Becton Dickinson, San Jose, CA). Data collections (10 000 events per
sample) were gated to exclude cell debris and cell aggregates. The
1
893, 842, 826, 795, 753, 694, 685 cm−1; H NMR (D2O) δ 7.23 (d,
2H, J = 8.0 Hz, Ar), 7.27 (t, 1H, J = 8.0 Hz, Ar), 7.44 (t, 2H, J = 8.0
Hz, Ar), 7.96 (br s, 2H), 9.92 (br s, 1H), 10.11 (br s, 1H), 10.88 (br s,
1H); HRMS (m/z, −ES) 152.0824 calcd [M+ + H]; found 152. 0823.
DNA Thermal Denaturation Assays. Thermal melting experi-
ments were conducted with a Varian Cary 300 Bio spectrophotometer
equipped with a 6 × 6 multicell temperature-controlled block.
Temperature was monitored with a thermistor inserted into a 1 mL
quartz cuvette containing the same volume of water as in the sample
cells. Absorbance changes at 260 nm were monitored from a range of
20 to 90 °C with a heating rate of 1 °C per min and a data collection
rate of five points per °C. The salmon sperm DNA was purchased
from Sigma Aldrich (extinction coefficient ε260 = 6600 cm−1 M−1
457
dx.doi.org/10.1021/jm301358s | J. Med. Chem. 2013, 56, 451−459