Med Chem Res
cytotoxicity results, this modification might result in syn-
ergistic action against certain tumor cell lines. Further
biological evaluation is in progress to better define the
antineoplastic activity of these compounds and to clarify
whether spin-labeled 5-FU analogues might display
decreased side effects compared with 5-FU.
target compounds 3a–f were characterized by m.p., ESR, IR,
and HRMS spectroscopic analyses.
Cell culture and determination of cytotoxic activity
A-549 (non-small cell lung carcinoma), DU145 (androgen-
independent prostate cancer), and KB (nasopharyngeal car-
cinoma) cell lines were obtained from the Lineberger Com-
prehensive Cancer Center (UNC-CH). KBvin (vincristine-
resistant KB) cell line was a generous gift of Professor Y.-C.
Cheng, Yale University. Cells were cultured in RPMI 1640
medium containing 25 mM HEPES and 2 mM L-glutamine
(Mediatech), supplemented with 10 % heat-inactivated fetal
bovine serum (Hyclone), 100 IU penicillin, 100 lg/mL
streptomycin, and0.25 lg/mLamphotericinB (Mediatech)in
5 % CO2 and 95 % air at 37 °C.
Antiproliferative activity was determined by the sulfo-
rhodamine B (SRB) colorimetric assay as previously
described (Nakagawa-Goto et al., 2011). In brief, cells
(3–5 9 103 cells/well) were seeded in 96-well plates filled
with culture medium containing various concentrations of
sample and incubated for 72 h. At the end of the exposure
period, cells were fixed with cold 50 % trichloroacetic acid
followed by staining with 0.04 % SRB (Sigma Chemical
Co.). The absorbance of solubilized SRB was measured at
515 nm on a Microplate Reader ELx800 (Bio-Tek Instru-
ments, Winooski, VT) with Gen5 software. All results are
representative of three or more experiments.
Conclusion
We have synthesized novel spin-labeled derivatives of
5-FU and evaluated their cytotoxic effects against four
tumor cell lines by the SRB method. Among all tested
compounds, compounds 3d and 3f were more cytotoxic
than 5-FU against the A-549 lung cancer cell line and merit
further investigation for development into clinical trial
candidates against non-small cell lung cancer.
Experimental
Chemistry
Melting points were taken on a Kofler melting point
apparatus and uncorrected. IR spectra were obtained on
NIC-5DX spectra photometer, mass spectral analysis was
taken on ZAB-HS and Bruker Daltonics APEXII49e
instruments, and ESR spectra were obtained with a Bruker
ER-200D-SRC X-band spectrometer. The synthetic com-
pounds were purified by flash chromatography on Merck
silica gel (70–230 mesh). Thin-layer chromatography
(TLC) was performed on silica gel plates with a fluorescent
indicator (Merck Silica Gel 60 F2540.25 mm thick). The
N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-
amino acids (9a–f) (Hankovszky et al., 1979) and
1-hydroxymethyl-5-fluorouracil used for the experiments
were prepared by modifications of previous procedures
(Ahmad et al., 1987; Ouyang et al., 2011).
Acknowledgments This study was supported financially by the
National Natural Science Foundation of China (30800720); the
Young Scholars Science Foundation of Lanzhou Jiaotong University
(2011011); and the Fundamental Research Funds for the Central
Universities (lzujbky-2013-69). This study was also supported in part
by the Cancer Research Center of Excellence, Taiwan (DOH-100-
TD-C-111-005), as well as NIH Grant CA177584 from the National
Cancer Institute awarded to K.H. Lee.
Conflict of interest The authors report no conflict of interests.
General procedure for the synthesis of target
References
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A mixture of a N-(1-oxyl-2,2,6,6-tetramethyl-4-piper-
idinyloxycarbonyl)-aminoacid(0.001 M), 1-hydroxymethyl-
5-fluorouracil (0.001 M), and dimethylaminopyridine
(DMAP, 0.1 g) was stirred in dichloromethane (10 mL) for
5 min at room temperature under nitrogen. N,N-Dicyclohex-
ylcarbodiimide (DCC, 0.21 g, 0.001 M) was added, and the
reaction mixture was stirred for 2 h. The reaction mixture was
filtered, and the filtrate was evaporated under reduced pres-
sure. The residue was separated by flash column chromatog-
raphy (gradient elution with mixtures of dichloromethane–
acetone) on silica gel and monitored by TLC. Synthesized
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