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5. Conclusion
6.1.3. Electrochemical results
The redox potentials of compounds 3-14 were measured by cyclic
voltammetry in acetonitrile as a solvent at room temperature, using
a platinum electrode and 0.1 M tetraethylamonium tetrafluoroborate
as the supporting electrolyte. The voltammograms were run in the
potential range 0.0e2.0 V versus non-aqueous Ag/Agþ.
In conclusion, we have shown that the cytotoxicity of phenyl-
aminonaphthoquinones depends upon the nature of the donor
phenyl and the acceptor quinone groups. The two selected quinones
(7 and 9) markedly influenced cellular metabolism and impaired
cell proliferation. They appeared relatively safe when assessed on
healthy fibroblasts. Given the high incidence of undesirable adverse
effects induced by the majority of currently available anticancer
drugs and considering the selective cytotoxic effects of quinones 7
and 9, these compounds appear to have promising and interesting
potential anticancer activities.
6.2. Pharmacology
6.2.1. Cell lines
Human cancer cell lines (T24, DU145, MCF7) were cultured in
high-glucose Dulbecco’s modified Eagle medium (Gibco, Grand
Island, NY, USA) supplemented with 10% fetal calf serum, penicillin
6. Experimental protocols
(100 U/mL), and streptomycin (100 mg/mL). Balb/3T3 cells (normal
mouse fibroblasts) were cultured in the same medium, except that
the fetal calf serum was replaced by 10% newborn calf serum. All
cultures were kept at 37 ꢁC in 95% air/5% CO2 at 100% humidity.
Phosphate-buffered saline (PBS) was purchased from Gibco. Cells
were incubated at the indicated times at 37 ꢁC with or without
quinones at various concentrations.
6.1. Chemistry
6.1.1. General remarks
All reagentswerecommerciallyavailablereagentgradesandwere
used without further purification. Melting points were determined
on a Stuart Scientific SMP3 apparatus and are uncorrected. 1H NMR
spectrawere recorded indeuterochloroform(CDCl3) ona Bruker AM-
200 instrument. 13C NMR spectra were obtained in CDCl3 at 50 MHz.
Chemical shifts are expressed in ppm downfield relative to tetra-
6.2.2. Cellular assays
The cytotoxicity of the quinones was assessed by following the
reduction of MTT to formazan blue [23]. Briefly, cells were seeded
into 96-well plates at a density of 10 000 cells/well for 24 h and then
incubated for 48 h with or without the compounds. Cells were then
washed twice with warm PBS and incubated with MTT (0.5 mg/mL)
for 2 h at 37 ꢁC. Blue formazan crystals were solubilized by adding
methylsilane (TMS,
d scale), and the coupling constants (J) are
reported in Hertz. The elemental analyses were performed in a Fison
SA, modelEA-1108apparatus. Silica gelMerck 60(70e230mesh)was
used for preparative column chromatography and thin layer chro-
matography (TLC) aluminum foil 60F254 for analytical TLC.
100 ml DMSO/well, and the colored solution was subsequently read
at 550 nm. Results are expressed as % of MTT reduction compared to
untreated control conditions. ATP content was determined after 6 h
of incubation using the ATPlite assay from Perkin Elmer (Waltham,
MA, USA) according to the procedures described by the suppliers,
and the results are expressed as nmol ATP/mg of protein. The
amount of protein was determined by the method of Lowry using
BSA as a reference [24]. Caspase-3 activity was monitored after 6 or
24 h incubation. Briefly, after incubation with the test compounds,
cells were washed twice with PBS, lysed and centrifuged. Cell
supernatants were then incubated with a specific peptide substrate,
Asp-Glu-Val-Asp-AFC (DEVD-AFC). Substrate cleavage was deter-
mined kinetically at room temperature using a Victor X2 spectro-
photometer (380 nm excitation, 500 nm emission) (Perkin Elmer,
Waltham, MA, USA). The results are expressed as Units/mg protein,
as originally described by Nicholson et al. [17]. Clonogenic assays
were performed by seeding cells (500) in six-well plates at a single-
cell density. Cells were allowed to adhere overnight, then treated
with quinones for 24 h, washed with warm PBS, given fresh
medium, and allowed to grow for 10 days. Clonogenic survival was
determined by staining colonies using crystal violet.
6.1.2. General procedure for the synthesis of
phenylaminonaphthoquinones
A suspension of quinone 1 (500 mg, 3.16 mmol) or 2 (500 mg,
2.20 mmol), the required amine (2 equiv.), CeCl3.7H2O (5% mmol in
respect to 1 or 2), and ethanol (15 mL) was left at room temperature
with stirring until completion of the reaction as indicated by TLC.
The reaction mixture was evaporated under reduced pressure and
the residue was column chromatographed (1:1:2 AcOEt/CH2Cl2/
light petroleum) to yield the corresponding aminoquinone (Table
1). Aminonaphthoquinones 3, 4, 5, 6, 7, 8, 9, 10, 13 and 14 were
identified by comparing their spectral properties (Melting point, IR,
1H NMR and 13C NMR) to those reported for these compounds in
the literature [19e22].
6.1.2.1. 2-[(2,5-Dimethoxyphenyl)amino] ꢀ1,4-naphthoquinone (11).
Prepared from quinone 1 and 2,5-dimethoxyaniline (24 h, 85%):
Red crystal, mp 128.2 ꢁC-128.5 ꢁC; 1H NMR (200 MHz, CDCl3):
d 8.14
(t, J ¼ 3.8 Hz, 1H), 8.11 (t, J ¼ 3.8 Hz, 1H), 8.0 (bs,1H, NHPh), 7.78 (dd,
J ¼ 1.6 Hz,1H), 7.69 (dd, J ¼ 1.6 Hz,1H), 6.72 (s, 1H), 6.54 (s, 1H), 6.26
(d, J ¼ 3.0 Hz, 1H), 6.22 (d, J ¼ 3.0 Hz, 1H), 3.88 (s, Me, 3H), 3.73 (s,
Me, 3H). 13C NMR (50 MHZ, CDCl3):
d 183.9, 181.9, 153.7, 143.6, 141.8,
134.8, 133.2, 132.3, 130.5, 127.6, 126.5, 126.1, 111.7, 111.3, 109.0, 102.1,
56.2, 55.9. Anal. Calcd. for C18H15NO4: C, 69.89; H, 4.89; N, 4.53.
Found: C, 69.88; H, 4.92; N, 4.55.
6.3. Statistical analysis
Data were analyzed using a one-way analysis of variance
(ANOVA) followed by Bonferroni test for significant differences
between means. For statistical comparison of results at a given time
point, data were analyzed using a Student’s t test. The level of
significance was set at p < 0.05.
6.1.2.2. 2-Chloro-3-[(2,5-dimethoxyphenyl)amino] -1,4-naphthoquin-
one (12). Prepared from quinone 2 and 2,5-dimethoxyaniline (24 h,
93%): Dark purple crystal, mp 146.7 ꢁC-146.9 ꢁC; 1H NMR (200 MHz,
CDCl3):
d
8.22 (d, J ¼ 3.8 Hz, 1H), 8.14 (d, J ¼ 3.8 Hz, 1H), 7.80 (t,
J ¼ 3.7 Hz,1H), 7.72 (t, J ¼ 3.7 Hz, 1H), 7.65 (s, 1H), 6.86 (d, J ¼ 4.4 Hz,
1H), 6.75 (m, 1H), 6.6 (s,1H, NHPh), 3.85 (s, Me, 3H), 3.81 (s, Me, 3H).
Acknowledgements
13C NMR (50 MHZ, CDCl3):
d 180.4, 177.4, 152.9, 146.9, 141.7, 134.9,
132.9, 132.6, 130.1, 127.1, 127.0, 126.9, 115.1, 111.4, 111.3, 111.0, 56.1,
55.9. Anal. Calcd. for C18H14ClNO4: C, 62.89; H, 4.10; N, 4.07. Found:
C, 62.88; H, 4.12; N, 4.02.
The authors are grateful to Isabelle Blave for her excellent
technical assistance. Financial support by the Universidad Arturo
Prat (UNAP) is gratefully acknowledged.