D. Cappoen et al. / European Journal of Medicinal Chemistry 63 (2013) 731e738
737
Fung [DMEM medium containing 0.1% penicillin and 0.8% Fungizone
but without gentamycin]. Compounds were solubilized in DMSO at
stock concentrations of 10 mM. Serial dilutions of the compounds
were made in DMEM-Pen/Fung at 2 times the concentration of each
incubation, the cells were trypsinized, washed and resuspended
with PBS, and 10 L of cell suspension was mixed with 300 L low
m
m
melting point agarose. The agarose/cell suspension mix dissolved
cell suspension was then placed onto a frosted microscope slide and
left on ice for 5 min. After removal of cover slip, the slide was
subsequently placed in a jar containing lysis buffer (10% DMSO, 89%
lysing stock solution, 1% Triton x-100) for 1 h. After lysis, the
agarose cell suspension was subjected to denaturation for 40 min
(denaturation buffer pH > 13) electrophoresis (same buffer) for
20 min at 300 mA (temperature þ/ꢀ17 ꢂC for both denaturation/
electrophoresis). The slides were washed 3 times with neutraliza-
tion buffer for 5 min and dried in ice cold ethanol for 10 min.
Staining of the DNA was done with gel red (Biotum-VWR). For the
quantification of the DNA migration a fluorescence microscope was
used and the percentage of DNA in the comet tail of the cells’ nuclei
core was calculated in proportion to the total DNA present in the
nuclei (comet head þ tail) by imager software from Metasystems
Altlussheim Germany.
compound to be tested. A volume of 100
sion in DMEM-Pen/Fung containing 4000 RLU of bacteria (multi-
plicity of infection of 0.1) and 100 L of the serial compound dilutions
mL of the bacterial suspen-
m
were added to the macrophage cultures. To measure the effects of the
compounds on intracellular growth of M. tb, the infected macro-
phages were washed three times on day 5 to remove all extracellular
bacteria, incubated 1 h with 1% gentamycin to kill residual extra-
cellular bacteria, lysed with 200
wells washed four times with 200
in a 2.5 mL tube together with the 4 PBS washings. One hundred
m
L 1% Triton X-100 (Sigma) and the
L PBS. The lysate was transferred
L of
m
m
1% n-decanal in ethanol were added to the tube and luminescence
measured. RLU values shown were obtained from six replicate cul-
tures. Cell viability of the J774 A1 macrophages was visually observed
by Trypan blue dye exclusion.
Assessment of Cytotoxicity. Inhibitory effect on C3A human
hepatocytic cells was determined for the derivatives using a neutral
red uptake assay as described before. The C3A cells were grown in
DMEM þ 10% FCS until a semi confluent monolayer of cells was
obtained. The cells were trypsinized, washed and 40 000 cells were
seeded per well of a 96 well plate and left for recovery at 37 ꢂC, 5%
CO2. The following days, the compounds were solubilized in DMSO
(SigmaeAldrich) to stock concentrations of 18 mM. A serial dilution
of each compound was made in DMEM þ 10% FCS. The C3A cells
were washed and exposed to the derivatives by adding the serial
dilutions of the compounds to the wells. The plates were left for
incubation at 37 ꢂC, 5% CO2 for 24 h. After exposure, the cells were
4.2. Chemistry
All compounds were prepared according to previously
described procedures [16,17]. Briefly,
a
phenol derivative
(0.015 mol) and a dibromoalkane (0.0075 mol) were dissolved in a
mixture of ethanol and water (9/1) containing an alkaline hy-
droxide (0.015 mol). The mixture was heated under microwave
irradiation in an InitiatorÒ Biotage oven for 20 min at 120 ꢂC. After
cooling, the precipitate was filtered and thoroughly washed with
water, ethanol and ether.
All compounds have been described in the literature [16,24]. The
synthesized products were identified by comparison of the spectral
data. The purity of the compounds was estimated at more than 98%
based on NMR spectra. An example of a complete characterization
(FTIR, HPLC, mass spectrometry, 1H and 13C NMR spectra) of two
samples is shown in the Supplementary material.
washed with 200
(Sigma) was added per well. Subsequently the plates were incu-
bated for 3 h at 37 ꢂC, 5% CO2. The wells were washed with 200
PBS and 200 L of an ethanol/acetic acid (50%/1%) mixture was
mL PBS and 200 mL neutral red working solution
mL
m
added. The plates were left on the shaker until the color became
homogenous purple and the optical density was measured at
540 nm (NR max) and 620 nM (reference wavelength) with the
Paradigm detection platform. OD of treated cells was compared to
OD of untreated cells (equals 100% viability).
Acknowledgments
This work was partially supported by The Flemish Research
Foundation (FWO-Vlaanderen) (Grant G.0020.10N) to D.C., L.V. and
K.H. D.F. and J.J.V.D.E. thank F.R.S.-FNRS for financial support. V.M.
was supported by funding from the European Community’s Sev-
enth Framework Programme (FP7/2007-2013) under grant agree-
ment FP7-223681 TB PAN-NET.
Vitotox assay. Possible genotoxicity of the compounds was
analyzed using the Gentaur VitotoxÔ kit and the included protocol
was followed. In brief, TA104 RecN2-4 (genox) and TA104 (cytox)
Salmonella typhimurium bacteria were cultivated shaking at 36 ꢂC
for 16 h in poor LB BROTH medium (20 g LB broth e 1 g glucose e
0.345 g CaCl2$2H2O þ antibiotics/liter) (1% LB broth, 0.5% glucose,
0.172% CaClH2O, 2.25% NaCl). The bacterial culture was diluted 125
times with poor LB broth MEDIUM (2 g LB BROTH, 1 g glucose,
0.375 g CaCl2$2H2O, 4.5 g NaCl/liter) times incubated shaking for
1 h at 36 ꢂC. Cultures were diluted 10 fold with poor LB medium, S9
(S9 liver fraction from aroclor treated rats) was added to the
designated þS9 cultures to test the genotoxic/cytotoxic effects of
the metabolites of the compounds. The bacterial suspensions were
then incubated at 30 ꢂC and the luminescent signal was measured
for 4 h with a 5 min interval.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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Comet assay. Possible DNA breakage effects of the derivatives on
C3A cells were examined in the alkaline comet assay. The C3A cells
were grown in DMEM þ 10% FCS until a semi confluent layer of cells
was obtained. The cells were trypsinized, washed and seeded at
40,000 cells per well of a 24 well plate and left for recovery at 37 ꢂC,
5% CO2. The following day, compound 12 was dissolved in DMSO as
a stock concentration of 1 mM. Serial dilutions of each compound
were made in DMEM þ 10% FCS to obtain the final concentrations
(10 mM, 3 mM, 1 mM, 0.3 mM, 0.1 mM). The C3A cells were washed and
exposed to the derivatives by adding 1 mL of the serial dilutions to
each well. The plates were incubated at 37 ꢂC, 5% CO2 for 24 h. After
Cappoen, Davie
