Synthesis of novel spin-labeled derivatives of 5-FU as potential antineoplastic agents

Article abstract of DOI:10.1007/s00044-013-0906-8

Chemotherapy is a general treatment option for various cancers, including lung cancer. In order to find compounds with superior bioactivity and less toxicity against lung cancer, novel spin-labeled 5-fluorouracil (5-FU) derivatives (3a-f) were synthesized and evaluated against four human tumor cell lines (A-549, DU-145, KB, and KBvin). Two promising compounds 3d and 3f exhibited IC₅₀ values of 2.76 and 2.38 μM, respectively, against non-small cell lung carcinoma cell line A-549. These compounds were twofold more cytotoxic than 5-FU and less toxic against other tested cell lines. Compound 3f exhibited seven times more selective cytotoxicity against A-549 than 5-FU. Our results suggest that compounds 3d and 3f merit further investigation for development into clinical trial candidates for non-small cell lung cancer.

Full text of DOI:10.1007/s00044-013-0906-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0906-8
ORIGINAL RESEARCH
Synthesis of novel spin-labeled derivatives of 5-FU as potential
antineoplastic agents
•
Liu Yang Mei-Juan Wang Zhi-Jun Zhang Susan L. Morris-Natschke
•
•
•
•
Masuo Goto Jing Tian Ying-Qian Liu Chih-Ya Wang Xuan Tian
•
•
•
•
•
Xiao-Ming Yang Kuo-Hsiung Lee
Received: 16 September 2013 / Accepted: 30 December 2013
Ó Springer Science+Business Media New York 2014
Abstract Chemotherapy is a general treatment option for
various cancers, including lung cancer. In order to find
compounds with superior bioactivity and less toxicity
against lung cancer, novel spin-labeled 5-fluorouracil (5-
FU) derivatives (3a–f) were synthesized and evaluated
against four human tumor cell lines (A-549, DU-145, KB,
and KBvin). Two promising compounds 3d and 3f exhib-
ited IC50 values of 2.76 and 2.38 lM, respectively, against
non-small cell lung carcinoma cell line A-549. These
compounds were twofold more cytotoxic than 5-FU and
less toxic against other tested cell lines. Compound 3f
exhibited seven times more selective cytotoxicity against
A-549 than 5-FU. Our results suggest that compounds 3d
and 3f merit further investigation for development into
clinical trial candidates for non-small cell lung cancer.
Keywords 5-Fluorouracil Á Spin-labeled Á Nitroxide Á
Cytotoxicity
Introduction
5-Fluorouracil (5-FU, 1) is a widely used clinical treatment
for solid tumors, such as colorectal, breast, liver, gastric, and
other cancers. 5-FU is used as a combination chemotherapy
for lung cancer (Ghoshal and Jacob, 1997; Huang et al.,
2007; Isanbor and O’Hagan, 2006). As an antimetabolite,
5-FU was first synthesized in 1957 (Heidelberger et al.,
1957). It is a pyrimidine analogue of DNA and RNA, with a
fluorine atom at the C-5 position in place of the hydrogen of
uracil (Zhang et al., 2008). 5-FU is an irreversible inhibitor
of thymidylate synthetase and consequently interferes with
the formation of thymidylic acid from deoxyuridylic acid to
inhibit DNA synthesis. As a uracil analogue, 5-FU may be
incorporated into RNA to give an aberrant form of RNA,
leading to cytotoxicity and cell death (Bosch et al., 1958;
Cohen et al., 1958; Goldberg et al., 1966; Madoc-Jones and
Bruce, 1968; Kufe and Major, 1981). However, 5-FU is
poorly selective toward tumors, and it causes some unde-
sirable side effects, such as gastrointestinal tract reaction,
bone marrow depression, dermatologic changes, alopecia,
leucopenia, thrombocytopenia, neurotoxicity, cardiotoxic-
ity, and even death (Kennedy, 1999). These side effects have
interfered significantly with 5-FU’s clinical application.
Accordingly, it is necessary to continuously design and
synthesize 5-FU analogues that have broad-spectrum or
Liu Yang and Mei-Juan Wang have contributed equally to this work.
L. Yang
Environmental and Municipal Engineering School,
Lanzhou Jiaotong University, Lanzhou 730000,
People’s Republic of China
M.-J. Wang Á Z.-J. Zhang Á J. Tian Á Y.-Q. Liu (&)
School of Pharmacy, Lanzhou University, Lanzhou 730000,
People’s Republic of China
e-mail: yqliu@lzu.edu.cn
S. L. Morris-Natschke Á M. Goto Á C.-Y. Wang Á X.-M. Yang Á
K.-H. Lee (&)
Natural Products Research Laboratories, UNC Eshelman School
of Pharmacy, University of North Carolina, Chapel Hill,
NC 27599-7568, USA
e-mail: khlee@unc.edu
Y.-Q. Liu Á K.-H. Lee
Chinese Medicine Research and Development Center, China
Medical University and Hospital, Taichung, Taiwan
X. Tian
State Key Laboratory of Applied Organic Chemistry, Lanzhou
University, Lanzhou 730000, People’s Republic of China
123

Med Chem Res
O
O
Scheme 1 Synthesis of
compounds 9a–f. Reagents and
conditions: i Na2WO4/H2O2/
EDTA; ii N,N’-carbonyl-
diimidazole/THF; iii p-
toluenesulfonic acid
monohydrate; iv NaN3/H2O;
v amino acids/MgO, 24 h
TosO
N
N
OH
O
OH
O
N
NH
ii
iii
i
iv
N
H
N
O
N
O
N
O
4
7
6
5
R
O
O
OH
N 3
N
H
O
O
O
a
R=Me
v
b
c
d
e
f
R=CHMe2
R=CH2CHMe2
R=CH2Ph
R=CH2CH 2SCH3
R=Proline
N
O
N
O
9a-f
8
selective antineoplastic activities and can overcome the
clinical limitations.
this intermediate (6) was reacted with p-toluenesulfonic acid
monohydrate to give a highly reactive tosylate (7). When
dissolved in an aqueous solution of sodium azide, 7 was
converted instantaneously into the alkoxycarbonyl azide (8).
Compounds 9a–f were obtained in good yields by reaction of
8 with the appropriate amino acids in the presence of MgO
(Hankovszky et al., 1979).
Furthermore, the introduction of a stable nitroxyl radical
into a pharmaceutical molecule can, to some extent, reduce
toxicity and augment antineoplastic activities (Gadjeva,
2002; Jin et al., 2006; Zheleva and Gadjeva, 2001). In our
previous studies, we successfully prepared several series of
spin-labeled antineoplastic drugs by modifying different
bioactive molecules. When compared with the parent
compounds, many of the spin-labeled compounds showed
significant antineoplastic activity with markedly decreased
toxicity (Liu et al., 2007, 2012; Liu and Tian, 2005; Tian
et al., 2002; Wang et al., 1993). In addition, L-amino acids
are actively transplanted into mammalian tissue, have good
water solubility, and are often used as carrier vehicles for
some drugs. Based on the above facts, we introduced the
nitroxyl radical moiety into 5-FU via a hydrophilic amino
acid spacer, in an attempt to simultaneously circumvent
5-FU’s limitations and develop superior pharmacological
profiles. Herein, we report the synthesis and structure–
cytotoxicity relationships of a series of novel spin-labeled
derivatives of 5-FU as potential antitumor agents.
Subsequently, commercially available 5-FU (1) was
heated with 37 % aqueous formaldehyde for about 50 min
at 60 °C to yield 1-hydroxymethyl-5-fluorouracil (2) (Ah-
mad et al., 1987; Ouyang et al., 2011). The target esters
3a–f were obtained in excellent yields by the straightfor-
ward acylation of intermediate 2 with the corresponding
N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-
amino acids 9a–f in the presence of N,N-dicyclohexylcar-
bodiimide (DCC) and 4-dimethylaminopyridine (DMAP)
(Scheme 2). The presence of the nitroxide group in the
target compounds 3a–f was confirmed from ESR and IR
spectra. In the ESR spectra, the nitroxide group showed a
triplet with hyperfine coupling constants (aN) in the range
of 15.90–16.28 G due to interaction between an unpaired
electron and nitrogen nucleus of the aminoxyl radical
(g value = 2.006). In the IR spectra, bands characteristic of
the nitroxyl moiety appeared at 1,370 7 cm-1 as shown
in Table 1. Furthermore, melting point and high-resolution
mass spectrometry (ESI) data also characterized the target
compounds 3a–f (Table 1).
Results and discussion
Design and synthesis of novel 5-FU analogues
The synthetic route to the N-(1-oxyl-2,2,6,6-tetramethyl-4-
piperidinyloxycarbonyl)-amino acids 9a–f is depicted in
Scheme 1. 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl
(5) was synthesized in 83 % yield by catalytic oxidation of
4-hydroxy-2,2,6,6-tetramethylpiperidine (4) with sodium
tungstate–hydrogen peroxide–EDTA (Rozantsev, 1964).
The reaction of 5 with N,N-carbonyldiimidazole gave N-(1-
oxyl-2,2,6,6-tetramethylpiperidinyloxycarbonyl)-imidazole
(6) (Staab and Mannschreck, 1962). Without purification,
Effects of novel 5-FU analogues on tumor cell growth
Target compounds 3a–f were evaluated for in vitro cyto-
toxicity against four tumor cell lines, human alveolar
adenocarcinoma (A-549), human prostate carcinoma (DU-
145), human nasopharyngeal carcinoma (KB), and human
vincristine-resistant nasopharyngeal carcinoma (KBvin).
The parent compound 5-FU (1) was included as a positive
control, and the obtained IC50 values are shown in Table 2.
123

Med Chem Res
O
N
O
N
O
F
F
HN
F
a
b
c
d
e
f
R=Me
HN
HN
i
ii
R=CHMe2
R=CH2CHMe2
R=CH2Ph
R=CH2CH2SCH3
R=Proline
O
O
H
N
O
O
N
H
O
O
OH
O
N
R
O
3a-f
2
1
Scheme 2 Synthesis of target compounds 3a–f. Reagents and conditions: i HCHO; ii 9a–f/DCC/DMAP, 2 h
Table 1 Physical and spectroscopic data of compounds 3a–f
Cmpd. Molecular formula mp (°C) HRMS ([M ? NH4]?)
IR (cm-1
)
ESR
aN (G)
Calcd.
Found
g
3a
3b
3c
3d
3e
3f
C18H26FN4O7
85–87
64–66
87–89
73–75
69–71
83–85
447.1792
475.2437
489.2593
523.2437
507.2157
473.2280
447.1786
475.2440
489.2603
523.2442
507.2162
473.2275
3,330 (N–H), 1,698 (C=O), 1,364 (N–O), 1,244 (C–F) 16.16
3,341 (N–H), 1,717 (C=O), 1,367 (N–O), 1,241 (C–F) 15.81
3,338 (N–H), 1,717 (C=O), 1368 (N–O), 1,263 (C–F) 16.04
3,341 (N–H), 1,715 (C=O), 1,365 (N–O), 1,259 (C–F) 16.10
3,333 (N–H), 1,716 (C=O), 1,365 (N–O), 1,246 (C–F) 15.88
3,440 (N–H), 1,715 (C=O), 1,370 (N–O), 1,245 (C–F) 16.02
2.0060
2.0058
2.0058
2.0061
2.0060
2.0060
C20H30FN4O7
C21H32FN4O7
C24H30FN4O7
C20H30FN4O7S
C20H28FN4O7
Table 2 Cytotoxic activity of 3a–f against four human cancer cell lines
Compounds
IC50 (lM)
SIa
A-549
DU-145
KB
KBvin
3a
3b
3c
12.39 0.91
13.10 1.81
11.14 0.77
2.76 0.02
10.28 0.62
2.38 0.12
5.09 0.46
19.58 0.64
[20
[20
[20
1.7
1.5
1.3
6.0
1.1
7.5
2.4
[20
[20
12.87 0.32
13.15 0.34
11.36 0.71
15.53 0.97
10.97 0.45
14.30 0.23
17.62 0.75
11.60 0.57
18.29 0.21
12.79 0.22
17.08 1.66
19.09 0.78
11.71 0.12
[20
3d
3e
3f
5-FU
13.70 0.46
a
Selectivity index (fold selective against A-549): (mean IC50 against DU-145, KB, and KBvin)/IC50 against A-549
The selectivity index (SI) against A-549 was calculated as
mean IC50 against DU-145, KB, and KBvin divided by
IC50 against A-549. Our results demonstrated that 3f
showed the best SI (7.5) against A-549.
more potent than 5-FU, with IC50 values of 10.97, 12.79, and
13.70, respectively. Against the indicated cell lines, the rank
orders of activity based on the different amino acid linkages
were as follows: for A-549, L-proline [ L-phenylalanine [
L-methionine [ L-leucine [ L-alanine [ L-valine; for DU-
145, L-methionine [ L-leucine [ L-phenylalanine [ L-pro-
line [ L-alanine [ L-valine; for KB L-methionine [ L-leu-
cine[ L-phenylalanine[ L-proline[ L-valine C L-alanine;
and for KBvin, L-methionine [ L-leucine [ L-phenylala-
nine [ L-valine C L-proline C L-alanine. These results
showed that the structures of the L-amino acids can have
potential effects on the bioactivity of these compounds.
Thus, we have successfully introduced a stable nitroxyl
radical into 5-FU via an L-amino acid linkage. Based on the
5-FU and its spin-labeled derivatives showed the same
order of cell line sensitivity: A-549 [ DU-145 [ KB [
KBvin (decreasing potency of test compound). Against the
A-549 cell line, compounds 3d and 3f, with IC50 values of
2.762 and 2.38 lM, respectively, were twofold more potent
than 5-FU, with an IC50 value of 5.09 lM. Furthermore, these
compounds exhibited good selectivity against A-549, sug-
gesting less toxicity for normal cells. Against the DU-145,
KB, and KBvin cell lines, compound 3e, with IC50 values of
11.36, 11.60, and 11.71 lM, respectively, was as or slightly
123

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
compounds (3a–f)
Ahmad S, Ozaki S, Nagase T, Iigo M, Tokuzen R, Hoshi A (1987) A
facile method for synthesis of N-acyloxymethyl-5-fluorouracils,
as a class of antitumor agents. Chem Pharm Bull 35:4137–4143
Bosch L, Harbers E, Heidelberger C (1958) Studies on fluorinated
pyrimidines. V. Effects on nucleic acid metabolism in vitro.
Cancer Res 18:335–343
Cohen SS, Flaks JG, Barner HD, Loeb MR, Lichtenstein J (1958) The
mode of action of 5-fluorouracil and its derivatives. Proc Natl
Acad Sci USA 44:1004–1012
Gadjeva VG (2002) Two spin labeled triazenes: relationship between
biochemical and biological activities. Int J Pharm 247:39–45
Ghoshal K, Jacob ST (1997) An alternative molecular mechanism of
action of 5-fluorouracil, a potent anticancer drug. Biochem
Pharmacol 53:1569–1575
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
123

Med Chem Res
Goldberg AR, Machledt JH Jr, Pardee AB (1966) On the action of
fluorouracil on leukemia cells. Cancer Res 26:1611–1615
Hankovszky HO, Hideg K, Lex L, TIgyi J (1979) Nitroxyls; IV.
Synthesis of spin-labeled N-(4-piperidinyloxycarbonyl)imida-
zoles and 4-piperidinyloxycarbonyl azides and their reaction
with amino acid derivatives. Synthesis 1:530–531
Heidelberger C, Chaudhuri NK, Danenberg P, Mooren D, Griesbach
L, Duschinsky R, Schnitzer RJ, Pleven E, Scheiner J (1957)
Fluorinated pyrimidines, a new class of tumour-inhibitory
compounds. Nature 179:663–666
Madoc-Jones H, Bruce WR (1968) On the mechanism of the lethal
action of 5-fluorouracil on mouse
28:1976–1981
Nakagawa-Goto K, Wu PC, Lai CY, Hamel E, Zhu H, Zhang L,
Kozaka T, Ohkoshi E, Goto M, Bastow KF, Lee KH (2011)
Antitumor agents. 284. New desmosdumotin B analogues with
bicyclic B-ring as cytotoxic and antitubulin agents. J Med Chem
54:1244–1255
Ouyang L, He DS, Zhang J, He G, Jiang B, Wang Q, Chen ZJ, Pan JZ,
Li YH, Guo L (2011) Selective bone targeting 5-fluorouracil
prodrugs: synthesis and preliminary biological evaluation. Bio-
org Med Chem 19:3750–3756
Rozantsev EG (1964) Organic free radicals with a hydroxy group. Izv
Akad Nauk SSSR Ser Kim 2:2187–2191
L cells. Cancer Res
Huang J, Wang JW, Gong T, Zhang ZR (2007) Synthesis and
characterization of insulin-5-Fu conjugate, enabling insulin as
multi-drug carrier via dendritic approach. Chin Chem Lett
18:247–250
Isanbor C, O’Hagan D (2006) Fluorine in medicinal chemistry: a
review of anti-cancer agents. J Fluor Chem 127:303–319
Jin Y, Chen SW, Tian X (2006) Synthesis and biological evaluation
of new spin-labelled derivatives of podophyllotoxin. Bioorg Med
Chem 14:3062–3068
Staab HA, Mannschreck A (1962) Synthesis of carboxylic acid esters
by the imidazolide method. Chem Ber 95:1284–1297
Tian X, Zhang FM, Li WG (2002) Antitumor and antioxidant activity
of spin labeled derivatives of podophyllotoxin (GP-1) and
congeners. Life Sci 70:2433–2443
Kennedy BJ (1999) 5-Fluorouracil toxicity: old or new? Cancer
86:1099–1100
Kufe DW, Major PP (1981) 5-Fluorouracil incorporation into human
breast carcinoma RNA correlates with cytotoxicity. J Biol Chem
256:9802–9805
Liu YQ, Tian X (2005) Synthesis of novel spin-labeled derivatives of
podophyllotoxin as potential antineoplastic agents. Synth Com-
mun 35:2749–2758
Liu YQ, Yang L, Tian X (2007) Podophyllotoxin: current perspec-
tives. Curr Bioact Compd 3:37–66
Wang JZ, Tian X, Tsumura H, Shimura K, Ito H (1993) Antitumor
activity of a new low immunosuppressive derivative of podo-
phyllotoxin (GP-11) and its mechanisms. Anticancer Drug Des
8:193–202
Zhang N, Yin Y, Xu SJ, Chen WS (2008) 5-Fluorouracil: mechanisms
of resistance and reversal strategies. Molecules 13:1551–1569
Zheleva AM, Gadjeva VG (2001) Spin labelled nitrosoureas and
triazenes and their non-labelled clinically used analogues—a
comparative study on their physicochemical properties and
antimelanomic effects. Int J Pharm 212:257–266
Liu YQ, Ohkoshi E, Li LH, Yang L, Lee KH (2012) Design, synthesis
and cytotoxic activity of novel spin-labeled rotenone derivatives.
Bioorg Med Chem Lett 22:920–923
123