Synthesis and antitumor evaluation of novel bis-triaziquone derivatives

Article abstract of DOI:10.3390/molecules14072306

Aziridine-containing compounds have been of interest as anticancer agents since late 1970s. The design, synthesis and study of triaziquone (TZQ) analogues with he aim of obtaining compounds with enhanced efficacy and reduced toxicity are an ongoing resear

Full text of DOI:10.3390/molecules14072306

Molecules 2009, 14, 2306-2316; doi:10.3390/molecules14072306
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antitumor Evaluation of Novel Bis-Triaziquone
Derivatives
Cheng Hua Huang 1,3, Hsien-Shou Kuo 2, Jia-Wen Liu 3 and Yuh-Ling Lin 3,*
1
Cathay General Hospital, 280 Renai Rd. Sec.4, Taipei, Taiwan
2
Department of Biochemistry, Taipei Medical University, 250 Wu-Hsin St. Taipei, Taiwan
3
College of Medicine, Fu-Jen Catholic University, 510 Chung Cheng Rd., Hsin-Chuang, Taipei
Hsien 24205, Taiwan
* Author to whom correspondence should be addressed; E-mail: med0018@mail.fju.edu.tw
Received: 30 April 2009; in revised form: 19 June 2009 / Accepted: 23 June 2009 /
Published: 29 June 2009
Abstract: Aziridine-containing compounds have been of interest as anticancer agents since
late 1970s. The design, synthesis and study of triaziquone (TZQ) analogues with the aim of
obtaining compounds with enhanced efficacy and reduced toxicity are an ongoing research
effort in our group. A series of bis-type TZQ derivatives has been prepared and their
cytotoxic activities were investigated. The cytotoxicity of these bis-type TZQ derivatives
were tested on three cancer lines, including breast cancer (BC-M1), oral cancer (OEC-M1),
larynx epidermal cancer (Hep2) and one normal skin fibroblast (SF). Most of these synthetic
derivatives displayed significant cytotoxic activities against human carcinoma cell lines, but
weak activities against SF. Among tested analogues the bis-type TZQ derivative 1a showed
lethal effects on larynx epidermal carcinoma cells (Hep2), with an LC50 value of 2.02 M,
and also weak cytotoxic activity against SF cells with an LC50 value over 10 M for 24 hr
treatment. Comparing the viability of normal fibroblast cells treated with compound 1a and
TZQ, the LC50 value of the latter was 2.52 M, indicating more toxicity than compound 1a.
This significantly decreased cytotoxicity of compound 1a towards normal SF cells, while
still maintaining the anticancer activity towards Hep2 cells is an interesting feature. Among
the seven compounds synthesized, compound 1c has similar toxicity effects on the three
cancer cell lines and SF normal cells as the TZQ monomer.

Molecules 2009, 14
Keywords: bioreductive compound; bis-triaziquone derivatives; cytotoxicity
2307
Introduction
The bioreductive aziridinylbenzoquinone drugs are a class of compounds designed to exploit one of
the features of solid tumor biology, namely tumor hypoxia, caused by an inadequate blood supply to
solid tumors; such regions generally are resistant to radiation, chemotherapeutic and other O2-requiring
treatments [1-4]. The ideal bioreductive drug should be administered as an inactive prodrug that is only
activated under low-oxygen conditions by one or two electron reductases [5]. The aziridine substituted
benzoquinones such as mitomycin C, triaziquone (TZQ), RH1, and tirapazamine (TPZ), are four of the
principal aziridinylquinone class of hypoxia-specific cytotoxins that are being developed for clinical
use [6-8]. These agents are composed of aziridinyl moieties on a quinone structure, and they are
converted by reductive metabolism into a bifunctional alkylating species that can cross-link major
groove DNA by interacting predominantly at guanine-N7 [9]. In case of di-aziridinyl substituted
quinones such as TPZ and CI-1010. the highly cytotoxic bifunctional alkylating agent can cross-link
DNA in cells, resulting in complex cellular mechanisms that lead to cell death by apoptosis or necrosis
[10-11]. These bioreductive drugs, as mentioned previously, have been developed to exploit the
oxygen deficiency in the hypoxic fraction of solid tumors on the premise that hypoxic cells should
show a greater propensity for reductive metabolism than well-oxygenated cells [12-16].
The tumor tissue has a lower oxidative reduction (redox) potential relative to most normal tissues,
which could increase the reductive activation of these quinone derivatives in tumors [11]. Therefore,
the selectivity of bioreductive drugs is governed not only by differences in oxygen tension between
tumor and normal tissue, but also by levels of enzymes catalyzing bioreductive activation such as
DT-diaphorase [4,12,13]. This fact led to publication by Workman and Walton of the concept of
“enzyme-directed bioreductive development” in 1990 [14]. In many cases, the biological activity of
quinones is attributed to their ability to accept electrons to form the corresponding radical anion or
dianion species. A quinone moiety substituted with an aziridine has been shown as a potent alkylating
agent due to bioreduction either by one-electron reducing enzymes (e.g. NADPH cytochrome P450
reductase, cytochrome b5 reductase) or by two-electron reducing enzyme ((NADP)H oxidoreductase,
NQO1) to form the corresponding aziridinyl hydroquinones [15-17]. The hydroquinone in the
corresponding aziridinyl hydroquinone effectively changes the pK of the aziridine ring such that it is
protonated and become activated toward nucleophilic attack under physiological pH. For the purpose
of obtaining new more potent antitumor compounds that can improve the current chemotherapeutic
cancer treatments, in the past five years, a series of bis-type aziridinylnaphthoquinone biorereductive
compounds have been developed in our laboratory and their anticancer activities evaluated. These
bis-type aziridinylnaphthaquinone compounds have various lengths of spacer between the two
naphthoquinone core structures, as the relationship among polymethylene chain length, DNA
alkylation reactivity and cytotoxicity had been studied in our previous reports [18-19]. Cytotoxicity to
various tumor cells varies with the chain length for some analogues, but DNA alkylation reactivity is

Molecules 2009, 14
2308
related to the presence of aziridinyl groups at least one in each naphthoquinone structure [18-19]. The
aziridinyl moiety within the analogues served an important alkylation group [18], but the cytotoxic
effects of the synthetic analogues towards carcinoma cells might not solely be due to the aziridinyl
moiety, as the quinone structure is common in numerous natural products that are associated with
antitumor activities [20]. The cytotoxic mechanisms induced by quinones include redox cycling and
the production of superoxide and other reactive oxygen radicals, reactions with thiols and amines,
drug-uptake, and DNA alkylations [21-22]. Pharmacomodulation of biologically active compounds
through conjunctive approaches had become an area of very active research in different fields of
medicinal chemistry [23]. One of the aims in this study was to develop an efficient synthetic approach
to construct a series of bis-triaziridine substituted benzoquinone derivatives for evaluation of their
cytotoxic activities. The efficient two steps synthesis developed herein provides speedy creation of a
series of bis-triaziridinyl benzoquinone analogues.
Based on these reasons, we tried to find the bis-type quinone compounds with higher antitumor
activity herein. The clinical therapeutic TZQ might be the best candidate as a lead for bis-type core
structures linked by various chains length and spacer atoms, to evaluate the cytotoxicity on various
human cancer cell lines. The syntheses of 1a and its analogues were compelling due to their cytotoxic
activity and their status as potential new leads in antitumor drug discovery efforts toward hypoxic
fraction of solid tumors. In this study, we report the synthesis of bis-triaziridinylbenzoquinones 1b-g
and our investigation of the cytotoxic activities of 1a-g. We believe that the synthetic route disclosed
herein provides an efficient approach toward the preparation of 1b-g. We have also studied a part of
the cytotoxic mechanism holds promise for providing the development of a new generation of hypoxic
specific cytotoxins. The detailed mechanism study is currently under way in our laboratory.
Results and Discussion
We have synthesized a series of eight bis-triaziridinyl-benzoquinone derivatives 1a-g from TZQ (2)
by the route as describe in Scheme 1, and evaluated their activity. The bis-type TZQ was linked by
spacers with various length and different numbers of carbon and oxygen compositions.
The cytotoxic activities of 1a-g against three human epithelium solid carcinoma cell lines, larynx
Hep2, oral OEC-M1 and breast BC-M1 cells were investigated by the MTT assay. Normal skin
fibroblast (SF) was used as a control normal cell, and all of their LC50 values were listed in Table 1.
The LC50 values were obtained by drawing the lines of best fit from the curves of Figures 1 and 2.

Molecules 2009, 14
Scheme 1. The chemical synthesis of bis-triaziquone derivatives 1a-g and structure of TZQ.
2309
O
O
O
N
N
F
N
S R S
N
Et3N
+
HS R SH
N
N
N
N
N
3
O
O
O
TZQ
R
O
2
1
N
N
1a CH2CH2
1b CH2 (CH2)2CH2
1c CH2 (CH2)4CH2
1d CH2 (CH2)6CH2
N
O
1e CH2CH2O CH2CH2
1f CH2CH2O
Table 1. The cytotoxicity of 1a-g (LC50, M) by MTT assay in three cancer cell lines and
normal skin fibroblast (SF). The MTT assay was used to determine the cell viability after
an additional 24 hr of culture. Data were from triplet wells and are representative of three
separate experiments.
Compound
Hep2
OEC-M1
5.02±0.34
0.85±0.05
1.52±0.12
1.14±0.07
0.11±0.04
0.21±0.03
0.22±0.02
1.02±0.11
BC-M1
SF
> 10
1a
1b
1c
1d
1e
1f
1g
TZQ
2.02±0.16
1.02±0.04
0.88±0.06
1.82±0.14
0.25±0.08
0.42±0.03
0.11±0.02
0.72±0.05
5.52±0.28
1.25±0.16
1.07±0.09
0.89±0.14
0.21±0.06
0.21±0.04
0.53±0.10
2.05±0.17
2.12±0.12
2.35±0.13
2.48±0.14
0.63±0.07
0.32±0.04
0.63±0.08
2.52±0.17
Compound 1a produced the least toxicity towards the three tumor cell lines and SF normal cells.
Compounds 1e-g produced more toxicity to normal SF cells (LC50= 0.32±0.04 to 0.63±0.08 M) than
that of compounds 1a-d (LC50 values were 2.12±0.12~2.48±0.14 M or >10 M). These results
indicated that 1a, with an LC50 over 10 M to SF cells, is the safest compound among 1a-g. For
comparison, the clinical drug TZQ was also tested in this study, showing the LC50 values towards the
four cell lines given in Table 1. The relationships between various concentrations of compound 1a and
TZQ and activity towards Hep 2 and SF cells are expressed individually in Figures 1A and 1B.
According these results, both these compounds act in a dose dependent manner. With 1a showing less
potency than TZQ towards Hep 2 cells (Figure 1A), but with more safety on normal cells (SF) as
shown in Figure 1B.

Molecules 2009, 14
Figure 1. Compound 1a and TZQ inhibited the proliferation of cell lines Hep 2 (A) and SF
2310
(B) that were seeded for 18 hr before the addition of two compounds with various
concentrations. The MTT assay was used to determine the cell viability after an additional
24 hr of culture. Data were from triplet wells and are representative of three separate
experiments.
A
110
Hep 2 cell
100
90
80
70
60
50
40
30
1a compound
20
TZQ
10
0
2
4
6
8
10
1a compound (M)
B
120
100
80
SF cell
1a compound
60
40
20
TZQ
0
2
4
6
8
10
1a compound (M)

Molecules 2009, 14
Figure 2. Compounds 1b to 1f inhibited the proliferation of cell lines Hep 2 (A) and SF
2311
(B), 1a to 1f compounds and TZQ inhibited the proliferation of cell lines OEC-M1 (C) and
BC-M1 (D) that were seeded for 18 hr before the addition of two compounds with various
concentrations. The MTT assay was used to determine the cell viability after an additional
24 hr of culture. Data were from triplet wells and are representative of three separate
experiments.
A
B
120
SF
1b
1c
1d
1e
1f
Hep 2
100
80
60
40
20
0
1b
1c
1d
1e
1f
100
80
60
40
20
0
1g
1g
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
Compounds (M)
Compounds (M)
C
D
1a
1b
1c
1d
1e
1f
1a
1b
1c
1d
TZQ
1e
1f
BC-M1
OEC-M1
100
100
80
60
40
20
0
80
60
40
20
0
1g
TZQ
1g
0
2
4
6
8
10
0
2
4
6
8
10
Copmounds (M)
Compounds (M)
Our previous study on a series of bis-aziridinylnaphthoquinone compounds identified that these
compounds exhibit a more potent response toward the solid tumors than towards tumors with good
circulation [18] and these results are supported by some reports indicating that there are difference in
the reductive metabolism between the solid tumors and the well circulated tumors [2]. TZQ was first
synthesized in 1958, and introduced clinically for the treatment of a number of cancers in the 1960s
[24-25]. This agent presumably produces its antitumor effects by alkylation of cellular components,
and has been shown to inhibit DNA and RNA synthesis [25]. More recent studies suggest that this
agent is a substrate for both one and two-electron reducing agents and that the cytotoxic activity may
result from protein alkylation and oxidative stress [26]. Because of its toxicity to bone marrow and
blood vessel walls it has been replaced by more effective agents and has not been used clinically for
many years. However, a more recent study of this agent as an adjuvant to surgery in carcinoma of
cervix found that there was no difference in five-year survival rates between the TZQ treated patients
and patients receiving conventional therapy [27]. In this study of a series of synthesized compounds, in
which bis-type TZQ analogues were linked by spacers with various of chain lengths and atoms,

Molecules 2009, 14
2312
compound 1a produced with the best profile in tumor cell Hep2 and normal cell SF. As seen in Scheme
I, the number of carbon atoms (1a-1d) or carbon and oxygen (1e-g) in the spacer between the two of
triaziridinylbenzoquinone structure increased from 1a to 1d, and 1e to 1g. There seems to be no
correlation between the cytotoxic activities and the linker distance. The most interesting finding was
that 1a showed significantly lowered cytotoxicity to normal SF cells (LC50 > 10 M) and still
maintained anticancer activity (LC50= 2.02±0.16 M) towards Hep2 cells. The results from this study
suggest that compound 1a is a novel class of bis-triaziridinyl benzoquinone cytotoxin against tumor
cells. Oxygen atoms were present or absent in the linker structures of the different compounds, which
offered the chance to examine the effect of different hydrophobicity in bis-type TZQ derivatives. In
linker structures with oxygen atoms show increased hydrophilic properties than without, so the
bis-type TZQ derivatives 1a-d are less hydrophilic than 1e-g. From Table 1, it seems that high
hydrophobicity is correlated with less cytotoxicity to normal fibroblasts.
Experimental
General
RPMI 1640 medium, DMEM medium, fetal bovine serum (FBS), 2 mM L-glutamine, MEM
non-essential amino acid, trypsin-EDTA solution, PBS, Hanks balance salt solution (HBSS),
pencillin-streptomycine and fungizone were purchased from Gibico Laboratories (Grand Island, NY,
-
USA). The compounds of NaHCO3 , MTT, trypan blue, EDTA, propidium iodide (PI), Hoechst 33258,
triaziquone (TZQ) were purchased from Sigma. Proton and carbon NMR were obtained on a Bruker
AMX-500 spectrometer. Chemical shifts are reported in ppm relative to tetramethylsilane (δ units).
Elemental analyses were recorded repetitively on a Micromass ZAB spectrometer and Perkin-Elmer
2400 elemental analyzer at the Analytical Facility of the National Taiwan University. IR spectra were
obtained on a Perkin-Elmer Spectrum RXI FT-IR system. All of chemicals were purchased from Acros,
Aldrich or TCI and used without further purification. Triaziridinylfluoro-1,4-benzoquinone (2) was
prepared according to the procedure published by Martynov et al. [28].
General procedure for preparing bis-triazquones 1a-g
To a solution of triaziridinylfluoro-1,4-benzoquinone (1.0 mmol) in benzene (10 mL) was added a
solution of triethylamine (2.0 mmol) under stirring at 5-10Ԩ. The dimercaptan (0.5 mmol) was added
to the mixture dropwise over a period of 5 min. The mixture was stirred for 5 hours at room
temperature. Ethyl acetate and 1N HCl were added to the reaction mixture, and the organic layer was
separated. The aqueous layer was further extracted with ethyl acetate, and combined organic layers
were dried with MgSO4 and concentrated.
2,2’-[Ethane-1,2-diylbis(thio)]bis[3,5,6-tris(aziridin-1-yl)benzo-1,4-quinone](1a): The crude product
was purified by flash chromatography (CH2Cl2/MeOH, 96:4) to yield a black green solid (37% yield).
1
Mp＞300°C (DMF); IR (neat) νmax: 1643 cm-1; H-NMR (CDCl3): δ 2.28 (8H, s), 2.30 (8H, s), 2.42

Molecules 2009, 14
2313
(8H, s), 2.98 (4H, s); 13C-NMR (CDCl3): δ 30.12, 30.31, 31.04, 33.15, 120.9, 140.0, 143.1, 153.7,
171.5, 179.8; Anal. calcd. for C26H28N6O4S2: C, 56.50; H, 5.11; N, 15.21. Found: C, 56.44; H, 4.98; N, 15.02.
2,2’-[Butane-1,4-diylbis(thio)]bis[3,5,6-tris(aziridin-1-yl)benzo-1,4-quinone (1b): The crude product
was purified by flash chromatography (CH2Cl2/MeOH, 98:2) to yield a black green solid (31% yield).
1
Mp199-200°C (CH2Cl2); IR (neat) νmax: 1643 cm-1; H-NMR (CDCl3): δ 1.62 (4H, t, J=5.0 Hz), 2.27
13
(8H, s), 2.31 (8H, s), 2.40 (8H, s); C-NMR (CDCl3): δ 28.98, 30.09, 30.26, 30.72, 32.70, 122.12,
139.90, 143.03, 153.72, 177.69, 179.85; Anal. calcd. for C28H32N6O4S2: C, 57.91; H, 5.55, N, 14.47.
Found: C, 57.78; H, 5.73; N, 14.22.
2,2’-[Hexane-1,6-diylbis(thio)]bis[3,5,6-tris(aziridin-1-yl)benzo-1,4-quinone (1c): The crude product
was purified by flash chromatography (CH2Cl2/MeOH, 98:2) to yield a black green solid (30% yield).
1
Mp 154-156°C (CH2Cl2); IR (neat) νmax: 1643 cm-1; H-NMR (CDCl3): δ 1.25 (4H, m), 1.52 (4H, m),
13
2.23 (8H, s), 2.30 (8H, s), 2.41 (8H, s), 2.84 (4H, t, J=7.0 Hz); C-NMR (CDCl3): δ 28.26, 29.92,
30.11, 30.29, 30.77, 33.22, 122.68, 139.91, 143.04, 153.59, 177.77, 179.92; Anal. calcd. for
C30H36N6O4S2: C, 59.19; H, 5.96, N, 13.80. Found: C, 59.0; H, 5.98; N, 13.98.
2,2’-[Octane-1,8-diylbis(thio)]bis[3,5,6-tris(aziridin-1-yl)benzo-1,4-quinone (1d): The crude product
was purified by flash chromatography (CH2Cl2/MeOH, 98:2) to yield a black green solid (22% yield).
1
Mp 119-121°C (CH2Cl2); IR (neat) νmax: 1641 cm-1; H-NMR (CDCl3): δ 1.21 (4H, m), 1.31 (4H, m),
13
1.49 (4H, quin, J=7.5 Hz), 2.30 (8H, s), 2.39 (8H, s), 2.55 (8H, s), 2.83 (4H, t, J=7.5 Hz); C- NMR
(CDCl3): δ 28.58, 28.94, 29.98, 30.05, 30.22, 30.71, 30.26, 122.78, 142.97, 153.48, 177.72, 179.86;
Anal. calcd. for C32H40N6O4S2: C, 60.35; H, 6.33; N, 13.20. Found: C, 60.44; H, 6.51; N, 13.0.
2,2’-[Oxybis(ethane-2,1-diylthio)]bis[3,5,6-tris(aziridin-1-yl)benzo-1,4-quinone (1e): The crude
product was purified by flash chromatography (CH2Cl2/MeOH, 99:1) to yield a black green solid (25%
1
yield). Mp 89-91°C (CH2Cl2); IR (neat) νmax: 1643 cm-1; H-NMR (CDCl3): δ 2.12 (8H, s), 2.23 (8H,
s), 2.40 (8H, s), 2.96 (4H, t, J=6.0 Hz), 3.50 (4H, t, J=6.0 Hz); 13C-NMR (CDCl3): δ29.19, 29.59,
30.04, 32.58, 70.37, 121.54, 139.85, 143.06, 153.59, 177.57, 179.71; Anal. calcd. for C28H32N6O5S2: C,
56.36; H, 5.41; N, 14.08. Found: C, 56.44; H, 5.24; N, 14.30.
2,2’-[Ethane-1,2-diylbis(oxyethane-2,1-diylthio)]bis[3,5,6-tris(aziridin-1-yl)benzo-1,4-quinone
(1f):
The crude product was purified by flash chromatography (CH2Cl2/MeOH, 99:1) to yield an opaque oil
1
(22% yield). IR (neat) νmax: 1643 cm-1; H-NMR (CDCl3): δ 2.27 (8H, s), 2.32 (8H, s), 2.43 (8H, s),
13
3.05 (4H, t, J=7.5 Hz), 3.51 (4H, s), 3.61 (4H, t, J=7.5 Hz); C-NMR (CDCl3): 29.31, 30.10, 31.88,
32.58, 70.17, 70.68, 121.59, 139.90, 143.16, 153.73, 177.67, 179.80; Anal. calcd. for C30H36N6O6S2: C,
56.23; H, 5.66; N, 13.12. Found: C, 56.18; H,5.86; N, 13.0.
2,2’-[Oxybis(ethane-2,1-diyloxyethane-2,1-diylthio)]bis[3,5,6-tris(aziridin-1-yl)benzo-1,4-quinone
(1g): The crude product was purified by flash chromatography (CH2Cl2/MeOH, 99:1) to yield an
opaque oil (26% yield). IR (neat) νmax: 1643 cm-1; 1H-NMR (CDCl3): δ 2.26 (8H, s), 2.31 (8H, s), 2.42
(8H, s), 3.06 (4H, t, J=7.5 Hz), 3.59 (12H, m); 13C-NMR (CDCl3): 29.36, 30.06, 31.84, 32.54, 70.18,

Molecules 2009, 14
2314
70.62, 70.94, 121.60, 139.85, 143.12, 153.66, 177.62, 179.76; Anal. calcd. for C32H40N6O7S2: C, 56.12;
H, 5.89; N, 12.27. Found: C, 56.32; H,5.71; N, 12.10.
Cell culture
The cell lines OEC-M1 (human oral epidermal carcinoma), Hep-2 (human larynx epidermal
carcinoma), BC-M1 (human breast adenocarcinoma) were cultured in the RPMI 1640 medium with 10
% fetal bovine serum (FBS), 2 mM L-glutamine, 25 mM Hepes. Skin fibroblasts (SF) were cultured in
DMEM medium with 10 % fetal bovine serum (FBS), 2 mM L-glutamine, MEM non-essential amino
acid. The cell culture medium for the four cell lines all contained pencillin-streptomycin and fungizone.
All the medium and supplements were purchased from Gibco Laboratories (Grand Island, NY, USA).
All cells were incubated in a humidified atmosphere of 5 % CO2 at 37°C. Numbers of cells were
counted after trypsinization by a Neubauer hemocytometer (VWR, Scientific Corp. Philadelphia, PA,
USA).
Cytotoxicity assay (MTT assay)
The MTT assay was according to the method of Skehan et al. [29]. One day before drug application,
cells were seeded in 96-well flat-bottomed microtiter plates (3,000-5,000 cells/well). Epithelium
carcinoma cells were incubated for 24 hr with drugs, applied as serial 1:2 dilutions (100 μL/well)
ranging from 10 μM down to 0.05 μM. Twenty microliter of MTT (5 mg/mL) were added to each well
and incubated for 4 hr at 37°C. The formazan product was dissolved by adding dimethyl sulfoxide
(DMSO, 100 μL) to each well, and the plates were read at 550 nm. All measurements were performed
in triplicate and each experiment was repeated at least three times. The LC50 values were calculated
from the 50 % formazan formation compared with control without drugs addition.
Conclusion
The synthetic bis-triaziquone derivatives herein displayed various cytotoxic activities against three
tumor cell lines and normal skin fibroblasts (SF). Among all of these derivatives compound 1a
produced the most potent cytotoxicity to Hep2 cells and least toxicity to SF. This compound is a
possible future anticancer drug.
Acknowledgments
This work was supported by Cathy General Hospital (CGH-FJU-9505).
References and Notes
1. Brown, J.M. Tumor microenvironment and the response to anticancer therapy. Cancer Biol. Ther.
2002, 1, 453-458.

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2. Robert, G.B.; Stuart, T. Hypoxia: Targeting the tumour. Anticancer Agents Med. Chem. 2006, 6,
281-286.
3. Wouters, B.G.; Weppler, S.A.; Koritzinsky, M.; Landuyt, W.; Nuyts, S.; Theys, J.; Chiu, R.K.;
Lambin, P. Hypoxia as a target for combined modality treatments. Eur. J. Cancer 2002, 38,
240-257.
4. Shannon, A.M.; Bouchier-Hayes, D.J.; Condron, C.M.; Toomey, D. Tumor hypoxia,
chemotherapeutic resistance and hypoxia-related therapies. Cancer Treat. Rev. 2003, 29, 297-307.
5. McKeown, S.R.; Coweny, R.L.; Williamsy, K.J. Bioreductive Drugs: from Concept to Clinic.
Clin. Oncol. 2007, 19, 427-442.
6. Phillips, R.M.; Burger, A.M.; Loadman, P.M.; Jarrett, C.M.; Swaine, D.J.; Fiebig, H.H. Predicting
tumor responses to mitomycine C on the basis of DT-diaphorase activity or drug metabolism by
tumorhomogenares: implications for enzyme-directed bioreductive drug development. Cancer Res.
2000, 60, 6384-6390.
7. Kim, J.Y.; Kim, C.H.; Stratford, I.J.; Patterson, A.V.; Hendry, J.H. The bioreductive agents RH1
and gamma-irradition both cause G2/M cell cycle phase arrest and polyploidy in a p53-mutated
human breast cancer cell line. Int. J. Radiat. Oncol. Biol. Phys. 2004, 58, 376-358.
8. Loredana, M.; Ian, O. Tirapazamine: From Bench to Clinical Trials Cur. Clin. Pharmacol. 2006,
1, 71-79.
9. Mattes, W.B.; Hartly, J.A.; Kohn, K.W. Mechanism of DNA strands breakage by piperidine at site
of 7N-alkyguanines. Biochim. Biophys. Acta 1986, 868, 71-76.
10. Lin, P.S.; Ho, K.C.; Yang, S.J. Tirapazamine (SR4233) interrupts cell cycle progression and
induces apoptosis. Cancer Lett. 1996, 105, 249-255.
11. Lee, A.E.; Wilson, W.R. Hypoxia-dependent retinal toxicity of bioreductive anticancer prodrugs in
mice. Toxicol. Appl. Pharmacol. 2000, 163, 50-59.
12. Smitskamp-Wilms, E.; Giaccone, G.; Pinedo, H.M.; Laan, B.F.A.M.; Peters, G.J. DT-diaphorase
activity in normal and neoplastic human tissues; an indicator for sensitivity to bioreductive agents?
Br. J. Cancer 1995, 72, 917-921.
13. Danson, S.; Ward, T.H.; Butler, J.; Ranson, M. DT-diaphorase: a target for new anticancer drugs.
Cancer Treat. Rev. 2004, 30, 437-449.
14. Workman, P.; Walton, M.I. Enzyme directed bioreductive drug development. In Selective
Activation of Drug by Redox Processes; Adams, G.E., Breccia, A., Fielden, E.M., Wardman, P.,
Eds.; Kluwer Academic/ Plenum Press: New York, NY, USA, 1990; pp.173-191.
15. Szmigiero, L.; Kohn, K.W. Mechanism of DNA strand breakage and interstrand cross-linking by
diazirinylbenzoquinone (Diaziquone) in isolated nuclei from human cell. Cancer Res. 1984, 44,
4453-4457.
16. Begleiter, A. Clinical applications of quinone-containing alkylation agents. Front. Biosci. 2000, 5,
153-171.
17. Basu, S.; Brown, J.E.; Flannigan, G.M.; Gill, J.H.; Loadman, P.M.; Martin, S.W.; Naylor, B.;
Scally, A.J.; Seargent, J.M.; Shah, T.; Puri, R.; Phillips, R.M. Immunohistochemical analysis of
NAD(P)H: quinone oxidoreductase and NADPH cytochrome P450 reductase in human superficial

Molecules 2009, 14
2316
bladder tumors: relationship between tumour enzymology and clinical outcome following
intravesical mitomycin C therapy. Int. J. Cancer 2004, 109, 703-709.
18. Huang, S.T.; Kuo, H.S.; Lin, C.M.; Tsai, H.D.; Peng, Y.C.; and Lin, Y.L. Synthesis and biological
evaluation of novel bis-aziridinylnapthoquinone derivatives. Oncol. Res. 2003, 11, 199-204.
19. Huang, S.T.; Tsai, H.D.; Kuo, H.S.; Yang, Y.P.; Peng, Y.C.; Lin, Y.L. A novel
bis-aziridinylnaphthoquinone with anti-solid tumor activity in which induced apoptosis is
associated with altered expression of bcl-2 protein. ChemBioChem 2004, 5, 797-803.
20. O’Brien, P.J. Molecular mechanisms of quinone cytotoxicity. Chem. Biol. Interact. 1991, 80,
1-41.
21. Bulter, J.; Hoey, B.M. Redox cyling drugs and DNA damage In DNA and Free Radicals;
Halliwell, B., Aruoma, O.I., Eds.; Ellis Horwood: London, UK, 1993, pp. 243-265.
22. Powis, G. Free radical formation by antitumour quinines. Free Radic. Biol. Med. 1989, 6, 63-101.
23. Tietze, L.F.; Bell, H.P.; Chandrasekhar S. Natural Product Hybrides as New Leaders for Drugs
Discovery Angew. Chem. Int. Ed. 2003, 42, 3996-4028.
24. Gauss, W. Uber die umsetzung einiger alkoxy-p-benzochinone mit antylenimin. Chem. Ber. 1958,
91, 2216-2222.
25. Eckhardt, G.B.S.J.; Hall, T.C.; Winkler, A. Drug Therapy of Cancer. World Health Organization:
Geneva, Switzerland, 1973.
26. Silva, J.M.; Rao, D.N.; O’Brien, P.J. Modulation of trenimon-induced cytoxicity by
DT-disphorase in isolated rat hepatocytes under aerobic versus hypoxic conditions. Cancer Res.
1992, 52, 3015-3021.
27. Gasparri, F.; Periti, P.; Scarselli, T.; Mazzei, L.; Savino, F.; Branconi, F.; Dancygier R.G.
Perioperative chemotherapy in treatment of carcinoma of uterine cervix. Chemioterapia 1986, 5,
44-52.
28. Martynov, V.S.; Makarova, A.N.; Berlin, A.Y. Diaminobis(ethylenimino)benzoquinones.II.
Reactions of ethyleniminofluoro-1,4-benzoquinones with phenol and derivatives of tyrosin. Biol.
Akivn. Soedin. Akad. Nauk SSSR [Chem. Abstr. 1965, 63, 18252b].
29. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J.T.; Bokesch,
H.; Kenney, S.; Boyd, M.R. New colorimetric cytotoxicity assay for anticancer-drug screen. J.
Nat. Inst. 1990, 82, 1107-1112.
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