Synthesis and biological evaluation of 3-aryl-quinoxaline-2- carbonitrile 1,4-Di-N-oxide derivatives as hypoxic selective anti-tumor agents

Article abstract of DOI:10.3390/molecules17089683

A series of 3-aryl-2-quinoxaline-carbonitrile 1,4-di-N-oxide derivatives were designed, synthesized and evaluated for hypoxic and normoxic cytotoxic activity against human SMMC-7721, K562, KB, A549 and PC-3 cell lines. Many of these new compounds displayed more potent hypoxic cytotoxic activity compared with TX-402 and TPZ in the tumor cells based evaluation, which confirmed our hypothesis that the replacement of the 3-amine with the substituted aryl ring of TX-402 increases the hypoxic anti-tumor activity. The preliminary SAR revealed that 3-chloro was a favorable substituent in the phenyl ring for hypoxic cytotoxicity and 7-methyl or 7-methoxy substituted derivatives exhibited better hypoxic selectivity against most of the tested cell lines. The most potent compound, 7-methyl-3-(3-chlorophenyl)-quinoxaline-2-carbonitrile 1,4-dioxide (9h) was selected for further anti-tumor evaluation and mechanistic study. It also exhibited significant cytotoxic activity against BEL-7402, HepG2, HL-60, NCI-H460, HCT-116 and CHP126 cell lines in hypoxia with IC₅₀ values ranging from 0.31 to 3.16 μM, and preliminary mechanism study revealed that 9h induced apoptosis in a caspase-dependent pathway.

Full text of DOI:10.3390/molecules17089683

Molecules 2012, 17, 9683-9696; doi:10.3390/molecules17089683
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of 3-Aryl-quinoxaline-2-
carbonitrile 1,4-Di-N-oxide Derivatives as Hypoxic Selective
Anti-tumor Agents
Yunzhen Hu 1,2, Qing Xia 1, Shihao Shangguan 1, Xiaowen Liu 3, Yongzhou Hu 1 and Rong Sheng 1,*
1
ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang University, Hangzhou 310058, China
2
Department of Pharmacy, the First Affiliated Hospital of college of Medicine, Zhejiang University,
Hangzhou 310006, China
3
Institute of Pharmacology & Toxicology, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou 310058, China
* Author to whom correspondence should be addressed; E-Mail: shengr@zju.edu.cn;
Tel./Fax: +86-571-8820-8458.
Received: 6 July 2012; in revised form: 1 August 2012 / Accepted: 2 August 2012 /
Published: 13 August 2012
Abstract: A series of 3-aryl-2-quinoxaline-carbonitrile 1,4-di-N-oxide derivatives were
designed, synthesized and evaluated for hypoxic and normoxic cytotoxic activity against
human SMMC-7721, K562, KB, A549 and PC-3 cell lines. Many of these new compounds
displayed more potent hypoxic cytotoxic activity compared with TX-402 and TPZ in the
tumor cells based evaluation, which confirmed our hypothesis that the replacement of
the 3-amine with the substituted aryl ring of TX-402 increases the hypoxic anti-tumor
activity. The preliminary SAR revealed that 3-chloro was a favorable substituent in the
phenyl ring for hypoxic cytotoxicity and 7-methyl or 7-methoxy substituted derivatives
exhibited better hypoxic selectivity against most of the tested cell lines. The most potent
compound, 7-methyl-3-(3-chlorophenyl)-quinoxaline-2-carbonitrile 1,4-dioxide (9h) was
selected for further anti-tumor evaluation and mechanistic study. It also exhibited significant
cytotoxic activity against BEL-7402, HepG2, HL-60, NCI-H460, HCT-116 and CHP126
cell lines in hypoxia with IC50 values ranging from 0.31 to 3.16 μM, and preliminary
mechanism study revealed that 9h induced apoptosis in a caspase-dependent pathway.
Keywords: 3-aryl-2-quinoxaline-carbonitrile 1,4-dioxide; hypoxic cytotoxic activity; SAR

Molecules 2012, 17
9684
1. Introduction
Hypoxia is an inevitable circumstance in most solid tumors resulting from rapid tumor growth with
an inefficient microvascular system. Tumor cells within these regions show resistance to radiotherapy
and chemotherapy and present a tremendous challenge to cancer therapy [1,2]. Hypoxia also
distinguishes solid tumor cells from physiologically normal cells and is marked as an attractive and
exploitable therapeutic target. Five classes of chemical moieties (quinones, nitroimidazoles, aromatic
N-oxides, aliphatic N-oxides and transition metal complexes) have been identified as hypoxic
cytotoxins in recent years. These compounds were selectively activated by reductive enzymes within
hypoxic environment and generated toxic metabolites causing cell death [3].
As classical aromatic N-oxides derivatives, tirapazamine (3-aminobenzotriazine-1,4-dioxide, 1,
TPZ, Figure 1) and 3-amino-2-quinoxalinecarbonitrile 1,4-di-N-oxide (TX-402, 2, Figure 1) were
extensively studied in the past years. TPZ was bioreductively activated through the one-electron
reduction of the benzotriazine-l,4-di-N-oxide moiety by reductase to form hydroxyl and benzotriazinyl
radicals that cause DNA damage. It had already been introduced into phase II and III clinical trials in
combination with radiotherapy and chemotherapy for advanced head and neck cancers [4,5]. TX-402 also
exhibited efficient hypoxic selective anti-tumor activities against various tumor cells with a similar
DNA damage mechanism [6]. Although both of them exhibited poor extravascular transport, the
unique one-electron reduction activation mechanism and encouraging antitumor profiles have
stimulated in recent years intense research efforts in the design and synthesis of a variety of TPZ and
TX-402 derivatives [7–10]. For example, benzotriazine-1,4-dioxide derivatives SN 29751 (3, Figure 1)
and SN 30000 (4, Figure 1) were identified as the promising secondary generation TPZ analogues by
using a spatially resolved pharmacokinetic/pharmacodynamic (SR-PKPD) model that considers tissue
penetration explicitly during lead optimization [7,10]. Beatriz reported the synthesis and the biological
evaluation of a series of 2-arylcarbonyl-3-trifluoromethylquinoxaline-1,4-di-N-oxide derivatives. The
most potent compound 2-(thiophene-2-carbonyl)-3-trifluoromethylquinoxaline 1,4-di-N-oxide (5, Figure 1)
not only exhibited good cytotoxic activity against NCI 60 cell lines with mean GI50 value of 0.07 μM,
but also showed positive activity in an in vivo hollow fiber assay [8].
To overcome the poor extravascular transport of TPZ, our lab have synthesized and evaluated 3-aryl
amino and 3-(alkoxymethylamino) benzotriazine-1,4-dioxide derivatives 6 and 7 (Figure 1) through
introduction of lipophilic groups into the C-3 amino of TPZ. Most of these compounds were more
potent than TPZ in the tumor cell lines assay and some of them exhibited higher hypoxia selectivity.
The preliminary SAR study revealed that the introduction of an aromatic group at the C-3 amino was
favorable for hypoxic cytotoxic activity and the physico-chemical study showed a positive correlation
between hypoxic activity and lipophilicity within a certain range [11–13].
With a similar drug design strategy, we also synthesized a series of 3-phenyl-2-ethylthio- (or
2-ethylsulfonyl)-quinoxaline-1,4-dioxide derivatives through replacement of the 2-cyano and 3-amine
moieties with 2-ethylthio (or 2-ethysulfonyl) and the 3-aryl of TX-402. The 2-ethylsulfonyl derivatives
displayed moderate to good antiproliferative activity in hypoxia, while the 2-ethylthio derivatives
showed almost no activity in the cell-based test. These results implicated that the electron-withdrawing
2-ethylsulfonyl moiety was necessary for hypoxic activity probably due to its modulation of the
one-electron reduction potential of molecules. Among all the synthesized compounds,

Molecules 2012, 17
9685
3-(4-bromophenyl)-2-(ethylsulfonyl)-6-methylquinoxaline-1,4-dioxide (Q39, 8, Figure 1) not only
exhibited good antiproliferative activity in extensive cell lines in hypoxia, but also inhibited SMMC-7721
tumor growth in a dose-dependent manner in a human tumor xenograft mice model [14–16].
Based on these research results, we have envisioned that replacement of the 3-amino moiety of
TX-402 with a substituted aryl would be favorable for hypoxic anti-tumor activity. To find new lead
compounds with enhanced potency and hypoxic selectivity, we report here the design, synthesis and
evaluation of a series of 3-aryl-2-quinoxalinecarbonitrile-1,4-di-N-oxide derivatives 9a–t (Figure 1) as
hypoxic selective anti-tumor agents. Although compounds containing this skeleton have been reported
as antimalarial agents [17–19], their hypoxic anti-tumor characteristic has never been disclosed. The
main objective of present study was to investigate the effect of the replacement of the 3-amine moiety
with a substituted 3-aryl moiety and the modification of substituent at the 7-position of TX-402 on
anti-tumor activity and hypoxic selectivity. The study also has led to the identification of several new
potent hypoxic selective anti-tumor compounds.
Figure 1. The structures of aromatic N-oxides with hypoxic cytotoxic activity.
O-
N+ CN
O-
N+
O-
N+
N
N
N+ NH2
O-
N
O
N+
O-
N+ NH2
O-
O
2, TX-402
3, SN 29751
1, TPZ
O-
N+
O-
N+
O-
N+
O
R1
S
Cl
N
N
O
R2
N
N+
O-
N
N+
O-
N+ CF3
O-
H
4, SN 30000
6
5
O-
Br
O-
O-
N+
O
N+
O-
N+
R1
N+ CN
R1
H3C
N
N+
O-
R2
N+
O-
7
N
O
S
R2
CH2CH3
H
O
9a-9t
8, Q39
2. Results and Discussion
2.1. Chemistry
The synthetic route of 3-aryl-2-quinoxalinecarbonitrile-1,4-di-N-oxides 9a–t is shown in
Scheme 1. Refluxing of substituted benzoates 10a–e with acetonitrile in the presence of sodium
methoxide provided arylacetonitriles 11a–e, followed by the classical Beirut reaction
with 5-substituted benzofuroxans 12a–d in ethanol with catalytic amount of potassium carbonate at
room temperature to yield target compounds 9a–t. The structures of all the newly synthesized
compounds were confirmed by IR, 1H-NMR and HRMS.

Molecules 2012, 17
9686
Scheme 1. The synthetic route to compounds 9a–t.
O
O
CN
Et
a
O
R2
O-
R2
R1
N+ CN
11a-e
10a-e
b
N+
O-
O
N
R2
R1
O
9a-9t
N
12a-d
Reagents and Conditions: (a) NaOMe, CH3CN, reflux, 3–5 h; (b) C2H5OH, K2CO3, r.t., 3–8 h.
2.2. Pharmacology
2.2.1. In Vitro Cytotoxic Activity
All the newly synthesized compounds were assayed for in vitro cytotoxicity against five human
cancer cell lines, including SMMC-7721 (hepatoma), K562 (chronic myeloid leukemia), KB
(epidermoid carcinoma of the nasopharynx), A549 (nonsmall cell lung carcinoma) and PC-3 (prostate
cancer) under normoxic and hypoxic conditions. TX-402 and TPZ were employed as positive controls
and the antiproliferative activity results are summarized in Table 1.
As shown in Table 1, many of 3-aryl-2-quinoxalinecarbonitrile-1,4-di-N-oxide derivatives showed
higher or similar hypoxic cytotoxic activity and selectivity in comparison with those of TX-402 and
TPZ against most of the tested cell lines, in particular for the SMMC-7721, K562 and KB cell lines.
Obviously, the hypoxic cytotoxic potency of 9a–t was highly dependent on the 3-position and
7-position substitutents of the quinoxaline. Such as, compound 9a (3-phenylquinoxaline-2-carbonitrile
1,4-dioxide) exhibited weak to good cytotoxicity against the SMMC-7721, K562, KB, A549 and PC3
cell lines (IC50 = 1.58, 17.53, 1.53, 8.08 and 25.0 μM, respectively). Compound 9h (7-methyl-3-(3-
chlorophenyl)-quinoxaline-2-carbonitrile 1,4-dioxide) showed good hypoxic cytotoxic activity against
five cell lines, with IC50 values of 0.76, 0.92, 0.53, 4.91 and 2.25 μM, respectively. By comparison
with the IC50 values of the TX-402 (>50, 13.1, 0.98, >50 and 5.87 μM, respectively), our hypothesis
that the replacement of 3-amine with substituted aryl ring of TX-402 increased the hypoxic anti-tumor
activity was confirmed.
The substituents on the 3-phenyl moiety affect the anti-tumor activity by changing the electronic
and lipophilic properties of the entire molecule. Comparing the cytotoxic activity of 9c and 9h with
that of 9a and 9f suggested that an electron-withdrawing 3-chloro group in the 3-phenyl moiety
increased cytotoxicity against most tested cell lines, particularly for the SMMC-7721, K562 and KB
cell lines. The substituents on the 7-position of the quinoxaline ring also have a significant impact on
anti-tumor activity and hypoxia selectivity because of the disparity in the electronic properties of the
resulting molecules. As shown in Table 1, 7-chloro derivatives 9p and 9q exhibited better hypoxic
antiproliferative activity than the 7-unsubstituted derivative 9a and 9b in most tested cell lines.

Molecules 2012, 17
Table 1. Cytotoxicity of 2-cyano-3-aryl-quinoxaline 1,4-dioxides against five cancer cell lines in hypoxia and in normoxia.
9687
Cytotoxicity(IC50, μM) and HCR
Comp.
R1
R2
SMMC-7721
N b
K562
N
KB
A549
N
PC3
N
H a
>50
4.75
1.58
5.07
1.09
1.82
1.43
0.63
2.98
0.76
0.93
1.6
HCR c
-
H
HCR
H
N
HCR
9.03
0.34
H
HCR
-
H
HCR
TX-402
TPZ
9a
-
-
-
>50
13.1
1.81
17.53
17.7
1.03
19.58
4.01
2.04
0.64
0.92
1.07
6.59
7.98
0.76
2.02
1.37
4.49
2.66
0.79
1.73
16.55
1.67
>50
-
0.98
8.85
>50
1.93
>50
7.43
52
5.87
>50
-
-
32.79
15.7
100
6.90
19.41
45.4
15.7
11.32
47.1
3.98
22.6
3.16
7.54
4.32
13.3
16.8
13.53
6.76
3.34
9.23
9.39
0.87
4.62
38.5
1.48
10.72 18.71 6.29
3.85
6.44
1.25
4.40
1.77
5.28
1.33
>2.30
2.27
0.90
1.47
0.71
>3.27
>5.71
2.51
1.06
5.36
1.50
1.00
0.12
0.94
1.17 20.97 17.92
H
H
9.94
2.59
0.89
1.53
7.9
16.46 10.76 8.08
14.75 1.87 21
4.59
13.61 2.68
25
18.2
3
24.6
7.56
15.03
>50
0.98
0.42
9b
9c
H
3-CH3
3-Cl
4-Br
4-NO2
H
19.72
4.59
26.3
H
5
10.99 0.76
6.04 10.61 46.71
11.2 19.8
8.02 42.31
5.01
9d
9e
H
32.9
8.28
100
18.08
5.79
2.41
0.99
5.08
2.83
16.4
>3.05
>4.83
6.37
H
12
4.24
7.69
10.36 >50
9f
CH3
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
OCH3
Cl
158.73
1.00
11.08 4.54
34.9
9.38
12.5
>50
3.8
24.2
>50
9g
3-CH3
3-Cl
4-Br
4-NO2
H
2.97
10.34
6.18
43
4.94
8.20
4.04
2.02
2.11
0.72
0.53
0.17
5.12
3.97
10.65 14.79 21.72
2.19 4.13
5.96 35.06 36.15 32.46
6.21
>8.05
5.57
9h
9i
13.61
6.65
4.91 11.13
2.25 12.55
5.54 20.26
3.66
9j
26.88
67.24
3.66
8.71
1.70
17
25
28.3
6.76
4.41
39
1.38
9k
9l
1.16
1.7
78
66.72 16.81 6.31
4.46
>50
>50
43.5
6.43
3-CH3
3-Cl
4-Br
4-NO2
H
6.23
4.83
13.84
3.92
4.27
1.73
1.73
23.7
23.6
17.80 1.16
6.23
4.87
6.63
3.54
3.25
5.37
3.43
15.3
8.75
>50 >11.34
9m
9n
9o
0.9
5.37
3.35
2.44
2.06
3.53
1.10
2.67
2.33
0.89
1.42
4.03
5.41
0.33
0.62
0.51
3.63
4.92
2.25 17.55
7.80
3.41
1.35
6.99
>5.04
3.92
0.26
0.86
0.12
1.62
0.46
0.37
0.8
115.33
2.42
1.65 11.32 28.43
8.4
28.61
25.2
0.65
6.17
5.75 30.84
18.5
3.76 11.77 11.74
6.53
18.7
9p
9q
9r
9.28
9.85
3.89 27.19
9.93 >50
3.06 11.98
Cl
3-CH3
3-Cl
4-Br
4-NO2
4.68
11.85 19.11 12.3
1.92
17.82 4.91 33.59
2.38 0.48 5.72
Cl
2.16
9s
Cl
2.08
3.4
11.39
6.94
4.1
5.4
50.9
16.1
13.3
13.8
9t
Cl
a H = Hypoxia: 3% percentage of oxygen. b N = Normoxia: 20% percentage of oxygen. c HCR, hypoxic cytotoxicity ratio.

Molecules 2012, 17
9688
On the other hand, the introduction of electron-donating methyl or methoxy groups into
the 7-position of the quinoxaline ring improved the hypoxic selectivity against most cell lines, in
particular for the SMMC-7721, K562 and KB cell lines. For example, 7-methyl and 7-methoxy-
substituted quinoxaline derivatives 9f and 9n showed very high hypoxic selectivity against SMMC-
7721 cell line, with HCR values of 159 and 115, respectively, which were 23- and 16.7-fold more
selective than TPZ (HCR = 6.90). The 7-methyl-substituted quinoxaline derivative 9i was the most
hypoxic selective cytotoxin against the KB cell line (HCR value of 35.1), which is an 11.8-fold
improvement compared with TPZ (HCR value of 2.97).
Among all the five tested cell lines, the SMMC-7721 was the most sensitive cell line to these newly
synthesized quinoxaline derivatives, with IC50 values in the 0.37–5.07 μM range and HCR values
between 1.0 and 158.73. The A549 one was the most resistant cell line to the hypoxic cytotoxic effect
of these derivatives, with IC50 values in the range of 5.72–36.15μM and HCR values between 0.12 and
5.71. This result was consistent with that of 2-arylcarbonyl-3-trifluoromethylquinoxaline-1,4-di-N-
oxide derivatives [8], suggesting that these two series of quinoxaline-1,4-di-N-oxide derivatives may
possess similar anti-tumor characteristics.
Compound 9h aroused our great interest because of its high hypoxic antiproliferative activity
against all the five cell lines with IC50 values range from 0.53 to 4.91 μM. It was further evaluated in
other six tumor cell lines in hypoxia and in normoxia, including Human hepatoma BEL-7402, HepG2,
Human promyelocytic leukemia HL-60, Human lung cancer NCI-H460, Human colon cancer HCT-116
and Human neuroblastoma CHP126. The results in Table 2 showed that 9h also exhibited significant
cytotoxicity against all six tested human tumor cell lines with IC50 values in the range of 0.31–3.16 μM
in hypoxia. It also showed moderate to good hypoxia selectivity with HCR values between 1.52 and
17.8. These results suggest that 9h might be a promising candidate for further development as hypoxic
selective anti-tumor agent.
Table 2. Cytotoxic activity of 9h against six human cancer cell lines in hypoxia and in normoxia.
IC50 (M)
Cell line
HCR c
H a
N b
Human hepatoma BEL-7402
Human hepatoma HepG2
2.23
1.76
3.16
2.64
1.71
14.7
13.1
4.80
5.96
4.92
5.51
6.60
7.44
1.52
2.26
2.88
17.8
Human promyelocytic leukemia HL-60
Human lung cancer NCI-H460
Human colon cancer HCT-116
Human neuroblastoma CHP126
0.31
b
a
H = Hypoxia: 3% percentage of oxygen. N = Normoxia: 20% percentage of oxygen.
c HCR, hypoxic cytotoxicity ratio.
2.2.2. Mechanism Studies
To investigate the mechanism of these newly synthesized quinoxaline derivatives, compound 9h
was further assayed for its effect on cell cycle progression and apoptosis-associated protein expression.
As shown in Figure 2A, spontaneous apoptosis (control) was seen in 8.42% of SMMC-7721 cells in
normoxia and 9.78% in hypoxia. In normoxia, 9h (20 μM) did not induce obvious apoptosis (13.4%)

Molecules 2012, 17
9689
relative to controls. However, in hypoxia, it caused apoptosis in 33.42% of SMMC-7721 cells at 48 h.
These data clearly demonstrated that 9h exhibited a hypoxic-selective anti-tumor activity. Given that
caspase signaling plays a critical role in stress induced apoptosis, we were thus encouraged to explore
its role in 9h-induced SMMC-7721 cells apoptosis. As illustrated in Figure 2A, when SMMC-7721
cells were pretreated with pan-caspase inhibitor z-VAD-fmk (10.0 μM), 9h-induced apoptosis was
significantly reduced from 33.42% to 16.83% at 48 h (Figure 2A). Collectively, these results indicated
that 9h serving as a potential hypoxic-selective compound and inducing apoptosis in a caspase-dependent
pathway. In order to further validate our results, some proteins related to activation of caspase cascade
were also detected. The expression of procaspase-3, and PARP and actin were measured in
SMMC-7721 cells treated with 9h (20.0 μM, 48 h). As shown in Figure 2B, 9h decreased the protein
levels of procaspase-3, and induce the cleavage of PARP in hypoxia. All these data further
demonstrate the apoptosis triggered by 9h in hypoxia is mediated by caspase signaling.
Figure 2. Pharmacological mechanism study of 9h. (A) SMMC-7721 cells were incubated
in normoxia and in hypoxia, and were treated with 9h (10 μM) for 48 h. After treatment,
cells were harvested and detected of apoptosis by flow cytometry using PI apoptosis
detection kit. (B) SMMC-7721cells were harvested after the same treatment as A, cell
extract were collected and immunoblotted with procaspase-3 and PARP antibodies.
3. Experimental
3.1. General
Melting points were obtained on a B-540 Büchi melting-point apparatus and are uncorrected. IR
spectra were performed on a Brüker VECTOR 22 FTIR spectrophotometer in KBr pellets
1
(400–4000 cm−1). H-NMR spectra were recorded on a Brüker AM 500 instrument at 500 MHz
(chemical shifts are expressed as δ values relative to TMS as internal standard). Mass spectra (MS),

Molecules 2012, 17
9690
ESI (positive) were recorded on an Esquire-LC-00075 spectrometer. HRMS spectra were measured
with an Agilent 6224 TOF LC/MS.
3.2. Chemistry
3.2.1. General Procedure for the Synthesis of Benzoylacetonitriles 11a–e
A mixture of ethyl benzoate 10a–e (11.7 mmol), sodium methoxide (1.08 g, 20 mmol) and
acetonitrile (15 mL) was refluxed for 3 h. After cooling to room temperature, the formed white
precipitate was filtered and dissolved in water (50 mL). Three mol/L HCl (10 mL) was added to the
solution and the mixture was extracted with CH2Cl2 (50 mL × 2). The combined organic layer was
washed with brine and dried over anhydrous Na2SO4. The solvent was removed under reduced
pressure to give the crude product, which was recrystallized from CH2C12-petroleum ether to provide
pure benzoylacetonitrile.
Benzoylacetonitrile (11a) [20]. White solid (83.1%), m.p.: 79–80 °C (lit. 80–81°C); ESI-MS:
m/z = 146.3 [M + H]+.
3-Methybenzoylacetonitrile (11b) [21]. White solid (82.9%), m.p.: 74–75 °C (lit. 74–75 °C); ESI-MS:
m/z = 160.6 [M + H]+.
3-Chlorobenzoylacetonitrile (11c) [22]. White solid (81.6%), m.p.: 72–73 °C (lit. 71–73 °C); ESI-MS:
m/z = 180.3 [M + H]+.
4-Bromobenzoylacetonitrile (11d) [23]. White solid (80.3%), m.p.: 160–161 °C (lit. 161–162 °C);
ESI-MS: m/z = 225.2 [M + H]+.
4-Nitrobenzoylacetonitrile (11e) [23]. White solid (78.6%), m.p.: 122–123 °C (lit. 121–122 °C);
ESI-MS: m/z = 191.3 [M + H]+.
3.2.2. General Procedure for the Synthesis of 3-Aryl-2-quinoxalinecarbonitrile-1,4-di-N-oxide
Derivatives 9a–t
The substituted benzofuroxans 12a–d were synthesized according to a literature method and
confirmed by melting point comparison [24]. To a solution of benzoylacetonitrile 11a–e (5.0 mmol)
and benzofuroxan 12a–d (5.0 mmol) in ethanol (40 mL), a 1% amount of potassium carbonate was
added and the mixture was stirred at room temperature for 3 h. The precipitate was filtered and washed
with ethanol to give a yellow solid, followed by recrystallization from ethanol to yield pure product.
3-Phenylquinoxaline-2-carbonitrile-1,4-dioxide (9a) [17]. Yellow solid (46.5%), m.p.: 208–210 °C;
1
(lit. 206–207 °C) IR (KBr): ν 3092, 2235, 1625, 1594, 1490, 1343, 1090, 971, 770 cm−1; H-NMR
(CDCl3) δ 8.69 (dd, 1H, J1 = 9.0 Hz, J2 = 1.0 Hz, H-5), 8.62 (dd, 1H, J1 = 9.0 Hz, J2 = 1.0 Hz, H-8),
7.99 (td, 1H, J1 = 7.8 Hz, J2 =1.5 Hz, H-6), 7.94 (td, 1H, J1 = 7.8 Hz, J2 =1.5 Hz, H-7), 7.72–7.74
(m, 2H, H-3′ and H-5′), 7.60–7.63 (m, 3H, H-2′, H-4′ and H-6′); ESI-MS: m/z = 264 [M + H]+.

Molecules 2012, 17
9691
3-(3-Methylphenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9b). Yellow solid (48.0%); m.p.:
208–209 °C; IR (KBr): ν 3101, 2235, 1634, 1493, 1340, 1276, 773 cm−1; 1H-NMR (CDCl3) δ 8.68 (dd,
1H, J1 = 8.5 Hz, J2 = 1.0 Hz, H-5), 8.61 (dd, 1H, J1 = 8.5 Hz, J2 = 1.0 Hz, H-8), 7.99 (td, 1H, J1 = 8.0 Hz,
J2 = 1.5 Hz, H-6), 7.93 (td, 1H, J1 = 8.0 Hz, J2 = 1.5 Hz, H-7), 7.49–7.53 (m, 3H, H-4′, H-5′ and H-6′),
7.41–7.43 (m, 1H, H-2´), 2.46 (s, 3H, CH3); HRMS (TOF) calc. for C16H12N3O2 [M + H]+: 278.0924,
found: 278.0927.
3-(3-Chlorophenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9c). Yellow solid (53.3%); m.p.:
1
195–197 °C; IR (KBr): ν 3085, 2232, 1647, 1593, 1490, 1436, 1336, 1086, 976, 773 cm−1; H-NMR
(CDCl3) δ 8.67 (d, 1H, J = 8.4 Hz, H-5), 8.61 (d, 1H, J = 8.4 Hz, H-8), 7.94–8.03 (m, 2H, H-6 and
H-7), 7.75 (s, 1H, H-2′), 7.55–7.61 (m, 3H, H-4′, H-5′ and H-6′); HRMS (TOF) calc. for C15H9ClN3O2
[M + H]+: 298.0378, found: 298.0377.
3-(4-Bromophenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9d). Yellow solid (47.2%); m.p.:
230–232 °C; IR (KBr): ν 3102, 2239, 1601, 1515, 1488, 1341, 1272, 1089, 978, 830, 772 cm−1;
1H-NMR (DMSO-d6) δ 8.51–8.55 (m, 2H, H-5 and H-8), 8.07–8.12 (m, 2H, H-6 and H-7), 7.80–7.83
(m, 2H, H-3′ and H-5′), 7.46–7.49 (m, 2H, H-2′ and H-6′); HRMS (TOF) calc. for C15H9BrN3O2
[M + H]+: 341.9873, found: 341.9868.
3-(4-Nitrophenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9e). Yellow solid (48.5% yield); m.p.:
1
240–241 °C; IR (KBr): ν 3105, 2238, 1598, 1515, 1344, 1275, 1089, 979, 855 cm−1; H-NMR
(DMSO-d6) δ 8.54–8.56 (m, 2H, H-5 and H-8), 8.47–8.49 (m, 1H, H-3′ and H-5′), 8.11–8.15 (m, 2H,
H-6 and H-7), 8.03–8.05 (m, 2H, H-2′ and H-6′); HRMS (TOF) calc. for C15H9N4O4 [M + H]+:
309.0618, found: 309.0621.
7-Methyl-3-phenylquinoxaline-2-carbonitrile-1,4-dioxide (9f). [17]. Yellow solid (48.3% yield); m.p.:
191–193 °C (lit. 190–191 °C); IR (KBr): ν 3089, 2237, 1650, 1612, 1332, 1276, 1093, 831, 699 cm–1;
1H-NMR (CDCl3) δ 8.48–8.58 (m, 1H, H-5), 8.41 (s, 1H, H-8), 7.79–7.81 (m, 1H, H-6), 7.71–7.73 (m,
2H, H-3′ and H-5′), 7.62 (m, 3H, H-2′, H-4′ and H-6′), 2.67 (s, 3H, CH3); ESI-MS: m/z = 278.4 [M + H]+.
7-Methyl-3-(3-methylphenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9g). Yellow solid (43.3% yield);
m.p.: 216–217 °C; IR (KBr): ν 3109, 2236, 1613, 1484, 1328, 1275, 1091, 983, 828, 785 cm−1;
1H-NMR (CDCl3) δ 8.40–8.58 (m, 2H, H-5 and H-8), 7.77–7.80 (m, 1H, H-6), 7.49–7.52 (m, 3H, H-4′,
H-5′ and H-6′), 7.42–7.43 (m, 1H, H-2′), 2.67 (s, 3H, CH3), 2.47 (s, 3H, CH3); HRMS (TOF) calc. for
C17H14N3O2 [M + H]+: 292.1080, found: 292.1083.
7-Methyl-3-(3-chlorophenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9h). Yellow solid (46.5% yield);
m.p.: 221–222 °C; IR (KBr): ν 3096, 2233, 1593, 1491, 1333, 1088, 982, 830 cm−1; 1H-NMR (CDCl3)
δ 8.50–8.57 (m, 1H, H-5), 8.40 (s, 1H, H-8), 7.80–7.82 (m, 1H, H-6), 7.75 (s, 1H, H-2′), 7.55–7.60 (m,
3H, H-4′, H-5′ and H-6′), 2.67 (s, 3H, CH3); HRMS (TOF) calc. for C16H11ClN3O2 [M + Na]+:
334.0354, found: 334.0352.
7-Methyl-3-(4-bromophenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9i). Yellow solid (48.7%); m.p.:
222–223 °C; IR (KBr): ν 3076, 2239, 1602, 1501, 1330, 1231, 1088, 982, 836 cm−1; 1H-NMR (CDCl3,

Molecules 2012, 17
9692
500 MHz) δ 8.39–8.56 (m, 2H, H-5 and H-8), 7.80 (dd, 1H, J1 = 8.5 Hz, J2 = 1.0 Hz, H-6), 7.74–7.77
(m, 2H, H-3′ and H-5′), 7.28–7.31 (m, 2H, H-2′ and H-6′), 2.67 (s, 3H, CH3); HRMS (TOF) calc. for
C16H11BrN3O2 [M + H]+: 356.0029, found: 356.0032.
7-Methyl-3-(4-nitrophenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9j). Yellow solid (46.2%); m.p.:
1
240–241 °C; IR (KBr): ν 3106, 2237, 1602, 1519, 1341, 1092, 981, 936, 828 cm−1; H-NMR (CDCl3,
500 MHz) δ 8.52–8.58 (m, 1H, H-5), 8.46 (d, 2H, J = 8.5 Hz, H-3′ and H-5′), 8.43 (s, 1H, H-8), 7.95
(d, 2H, J = 8.5 Hz, H-2′ and H-6′), 7.82–7.85 (m, 1H, H-6), 2.69 (s, 3H, CH3); HRMS (TOF) calc. for
C16H10N4NaO4 [M + Na]+: 345.0594, found: 345.0597.
7-Methoxy-3-phenylquinoxaline-2-carbonitrile 1,4-dioxide (9k). [17]. Yellow solid (45.1%); m.p.:
217–219 °C, (lit. 222–223 °C); IR (KBr): ν 3097, 2239, 1610, 1496, 1332, 1249, 1091, 845, 753 cm−1;
1H-NMR (CDCl3, 500 MHz) δ 8.58 (d, 1H, J = 9.6 Hz, H-5), 7.88 (d, 1H, J = 2.8 Hz, H-8), 7.70–7.72
(m, 2H, H-3′ and H-5′), 7.60–7.61 (m, 3H, H-2′, H-4′ and H-6′), 7.55 (dd, 1H, J1 = 9.6 Hz, J2 = 2.8 Hz,
H-6), 4.06 (s, 3H, OCH3); ESI-MS: m/z = 294.2 [M + H]+.
7-Methoxy-3-(3-methylphenyl)quinoxaline-2-carbonitrile 1,4-dioxide (9l). Yellow solid (44.5%); m.p.:
203–204 °C; IR (KBr): ν 3101, 2235, 1610, 1504, 1395, 1329, 1258, 1131, 1012, 945, 849 cm−1;
1H-NMR (DMSO-d6, 500 MHz) δ 8.43 (d, 1H, J = 9.0 Hz, H-5), 7.79 (d, 1H, J = 3.0 Hz, H-8), 7.71
(dd, 1H, J1 = 9.0 Hz, J2 = 3.0 Hz, H-6), 7.47–7.53 (m, 3H, H-4′, H-5′ and H-6′), 7.41–7.43 (m, 1H,
H-2′), 4.03 (s, 3H, OCH3), 2.40 (s, 3H, CH3); HRMS (TOF) calc. for C17H14N3O3 [M + H]+: 308.1030,
found: 308.1033
7-Methoxy-3-(3-chlorophenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9m). Yellow solid (45.7%);
m.p.: 160–162 °C; IR (KBr): ν 3106, 2238, 1616, 1591, 1535, 1494, 1422, 1333, 1250, 1001, 814 cm−1;
1H-NMR (CDCl3) δ 8.56 (d, 1H, J = 9.6 Hz, H-5), 7.87 (d, 1H, J = 2.4 Hz, H-8), 7.74 (s, 1H, H-2′),
7.54–7.59 (m, 4H, H-6, H-4′, H-5′ and H-6′), 4.06 (s, 3H, CH3); HRMS (TOF) calc. for C16H11ClN3O3
[M + H]+: 328.0483, found: 328.0485.
7-Methoxy-3-(4-bromophenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9n). Yellow solid (45.3%);
1
m.p.: 184–186 °C; IR (KBr): ν 3092, 2238, 1608, 1504, 1332, 1248, 1016, 933, 836 cm−1; H-NMR
(CDCl3) δ 8.56 (d, 1H, J = 9.5 Hz, H-8), 7.86 (d, 1H, J = 2.5 Hz, H-5), 7.73–7.76 (m, 2H, H-3′ and
H-5′), 7.55 (dd, 1H, J1 = 9.5 Hz, J2 = 3.0 Hz, H-6), 7.27–7.31 (m, 2H, H-2′ and H-6′), 4.05 (s, 3H,
OCH3); HRMS (TOF) calc. for C16H11BrN3O3 [M + H]+: 371.9978, found: 371.9979.
7-Methoxy-3-(4-nitrophenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9o). Yellow solid (45.6%); m.p.:
1
238–240 °C; IR (KBr): ν 3105, 2235, 1615, 1525, 1361, 1327, 1255, 1013, 939, 858 cm−1; H-NMR
(CDCl3) δ 8.57 (d, 1H, J = 9.0 Hz, H-5), 8.59 (d, 2H, J = 9.0 Hz, H-3′ and H-5′), 7.95 (d, 2H,
J = 9.0 Hz, H-2′ and H-6′) , 7.90 (d, 1H, J = 2.5 Hz, H-8), 7.59 (dd, 1H, J1 = 9.0 Hz, J2 = 2.5 Hz, H-6),
4.08 (s, 3H, OCH3); HRMS (TOF) calc. for C16H11N4O5 [M + H]+: 339.0724, found: 339.0729.
7-Chloro-3-phenylquinoxaline-2-carbonitrile-1,4-dioxide (9p). [17]. Yellow solid (47.6%); m.p.:
221–223 °C (lit. 224–225 °C); IR (KBr): ν 3095, 2238, 1599, 1488, 1333, 1260, 1092, 984, 842,
1
770 cm−1; H-NMR (DMSO-d6) δ 8.55 (d, 1H, J = 9.0 Hz, H-5), 8.53 (s, 1H, H-8), 8.15 (dd, 1H,

Molecules 2012, 17
9693
J1 = 9.0 Hz, J2 = 2.0 Hz, H-6), 7.72–7.73 (m, 2H, H-3′ and H-5′), 7.61–7.63 (m, 3H, H-2′, H-4′ and
H-6′); ESI-MS: m/z = 298.4 [M + H]+.
7-Chloro-3-(3-methylphenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9q). Yellow solid (46.0%); m.p.:
215–216 °C; IR (KBr): ν 3099, 2235, 1636, 1489, 1400, 1325, 1260, 1095, 893, 831, 787 cm−1;
1H-NMR (DMSO-d6) δ 8.51–8.53 (m, 2H, H-5 and H-8), 8.14 (dd, 1H, J1 = 9.5 Hz, J2 = 2.5 Hz, H-6),
7.50–7.52 (m, 3H, H-4′, H-5′ and H-6′), 7.44–7.45 (m, 1H, H-2′), 2.40 (s, 3H, CH3); HRMS (TOF)
calc. for C16H11ClN3O2 [M + H]+: 312.0534, found: 312.0536.
7-Chloro-3-(3-chlorophenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9r). Yellow solid (45.8%); m.p.:
225–226 °C; IR (KBr): ν 3103, 2234, 1595, 1487, 1401, 1332, 1088, 987, 829 cm−1; 1H-NMR (CDCl3)
δ 8.56–8.67 (m, 2H, H-5 and H-8), 7.88–7.94 (m, 1H, H-6), 7.74 (s, 1H, H-2′), 7.56–7.62 (m, 4H, H-6,
H-4′, H-5′ and H-6′); HRMS (TOF) calc. for C15H8Cl2N3O2 [M + H]+: 331.9988, found: 331.9993.
7-Chloro-3-(4-bromophenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9s). Yellow solid (49.4%); m.p.:
1
223–224 °C; IR (KBr): ν 3102, 2235, 1600, 1492, 1332, 1220, 1091, 989, 835 cm−1; H-NMR
(DMSO-d6) δ 8.52-8.55 (m, 2H, H-5 and H-8), 8.15 (dd, 1H, J1 = 9.5 Hz, J2 = 2.5 Hz, H-6), 7.79–7.82
(m, 2H, H-3′ and H-5′), 7.46–7.50 (m, 2H, H-2′ and H-6′); HRMS (TOF) calc. for C15H8BrClN3O2
[M + H]+: 385.9483, found: 385.9489.
7-Chloro-3-(4-nitrophenyl)quinoxaline-2-carbonitrile-1,4-dioxide (9t). Yellow solid (47.9%); m.p.:
1
238–239 °C; IR (KBr): ν 3107, 2237, 1599, 1519, 1488, 1340, 1094, 987, 830 cm−1; H-NMR
(DMSO-d6) δ 8.54–8.58 (m, 1H, H-5 and H-8), 8.48–8.50 (m, 2H, H-3′ and H-5′), 8.17–8.19 (m, 1H,
H-6), 8.02–8.04 (m, 2H, H-2′ and H-6′); HRMS (TOF) calc. for C15H8ClN4O4 [M + H]+: 343.0229,
found: 343.0234.
3.3. Pharmacology
The tested eleven human cancer cell lines (SMMC-7721, K562, KB, A549, PC-3, BEL-7402,
HepG2, HL-60, NCI-H460, HCT-116 and CHP126) were purchased from the Cell Bank of China
Science Academy (Shanghai, China). The above cells were cultured in RPMI-1640 (Invitrogen Corp.,
Carlsbad, CA, USA) medium with heat-inactivated 10% fetal bovine serum, penicillin (100 units/mL)
and streptomycin (100 μg/mL) and incubated in normoxic atmosphere with 20% O2, 5% CO2 at 37 °C
or in hypoxic atmosphere with 3% O2, 5% CO2 (established in a hypoxia incubator [Forma Scientific,
Inc., Marietta, OH, USA] where N2 was used to compensate for the reduced O2 level).
3.3.1. Cytotoxicity Assay [25]
Cancer cells were seeded in 96-well microtiter plates (4,000 cells/well), and were cultured in
normoxia and hypoxia. The hypoxic cells were allowed to attach 1 day prior to the addition of these
compounds (0–50 μM) in complete medium (to various final concentrations in 200 μL of complete
medium) in replicates of 4 wells per condition. Plates were assayed at 72 h after initiation of drug
exposure. Afterwards, 10 μL of stock 3-[4,5-dimethylthia-zol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT, Sigma) solution was added to each well (0.5 mg/mL) for another 4 h incubation (37 °C, 5% CO2).

Molecules 2012, 17
9694
After 4 h incubation, 200 μL of DMSO was added to each well and optical density (OD) was read at
570 nm by Thermo Multiskan Spectrum (Thermo Electron Corporation). The IC50 values were calculated
using the PrismPad computer program (GraphPad Software, Inc., San Diego, CA, USA) and were
defined as concentration of drug causing 50% inhibition in absorbance compared with control
(vehicle) cells.
3.3.2. Flow Cytometry Analysis [25]
The SMMC-7721 cells were treated with 9h and/or the general caspase inhibitor, z-VAD-fmk
(R&D Systems, Inc., Minneapolis, MN, USA), 9h (10 μM) alone for 24–48 h in 1% O2 or 20% O2
respectively; 9h (10 μM) +z-VAD-fmk (10.0 μM) for 48 h in hypoxia. Detection of apoptosis by
FACS Calibur flow cytometer (Becton Dickinson, Lincoln Park, NJ, USA) was performed using the
Propidium iodide (PI) apoptosis detection kit (BioVision, Mountain View, CA, USA).
3.3.3. Western Blot Analysis [25]
Proteins of SMMC-7721 cells incubated with 10 μM 9h both in hypoxia and normoxia were
extracted in radio-immunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris, 2 mM EGTA, 2 mM
EDTA, 25 mM NaF, 25 mM glycerophosphate, 0.2% Triton X-100, 0.3% NONIDET P-40, 0.1 mM
PMSF). Total protein concentrations of whole cell lysates were determined using BioRad BCA
method (Pierce, Rockford, IL, USA). Equal amounts of protein sampled from whole cell lysates were
subjected to electrophoresis on 8%–12% Tris-Glycine pre-cast gels (Novex, San Diego, CA, USA) and
electroblottedonto Immobilon-P Transfer Membrane (Millipore Corporation, Billerica, MA, USA), and
probed with primary antibodies and then incubated with a horseradish peroxidase (HRP) conjugated
secondary antibodies. Proteins were visualized using enhanced chemiluminescent (ECL) Western
Blotting detection reagents (Amersham Biosciences, Piscataway, NJ, USA).
4. Conclusions
In summary, a series of 3-aryl-2-quinoxalinecarbonitrile-1,4-di-N-oxide derivatives 9a–t have been
synthesized and evaluated for their hypoxic and normoxic cytotoxic activity. Many of these 3-aryl-2-
quinoxalinecarbonitrile-1,4-di-N-oxide derivatives showed better hypoxic cytotoxic activity and
higher hypoxic selectivity than that of TPZCN and TPZ against most tested cancer cell lines, in
particular for the SMMC-7721, K562 and KB cell lines. The preliminary SAR study revealed that the
3-(3-chlorophenyl) moiety was favorable for hypoxic cytotoxicity and the 7-methyl or 7-methoxy
moiety improved the hypoxic selectivity. Compound 9h decreased the protein levels of procaspase-3
and induced the cleavage of PARP in hypoxia, which suggested it induces apoptosis in a
caspase-dependent pathway.
Acknowledgements
This study was financially supported by the National Natural Science Foundation of China
(21072171, 81072575) and the National Key Tech Project for Major Creation of New Drugs
(NO. 2009ZX09501-003).

Molecules 2012, 17
9695
References
1. Martin, B.; Wilson, W.R. Exploiting tumour hypoxia in cancer treatment. Nat. Rev. Cancer 2004,
4, 437–447.
2. Wardman, P. The importance of radiation chemistry to radiation and free radical biology (The
2008 silvanus thompson memorial lecture). Br. J. Radiol. 2009, 82, 89–104.
3. Wilson, W.R.; Hay, M.P. Targeting hypoxia in cancer therapy. Nat. Rev. Cancer 2011, 11, 393–410.
4. Rischin, D.; Peters, L.; Fisher, R.; Macann, A.; Denham, J.; Poulsen, M.; Jackson, M.; Kenny, L.;
Penniment, M.; Corry, J.; et al. Tirapazamine, cisplatin, and radiation versus fluorouracil, cisplatin,
and radiation in patients with locally advanced head and neck cancer: A randomized phase II trial
of the trans-tasman radiation oncology group (TROG 98.02). J. Clin. Oncol. 2005, 23, 79–87.
5. Rischin, D.; Peters, L.; ÓSullivan, B.O.; Giralt, J.; Fisher, R.; Yuen, K.; Trotti, A.; Bernier, J.;
Bourhis, J.; Ringash, J.; et al. Tirapazamine, cisplatin, and radiation versus cisplatin and radiation
for advanced squamous cell carcinoma of the head and neck (TROG 02.02, HeadSTART): A
phase III trial of the trans-tasman radiation oncology group. J. Clin. Oncol. 2010, 28, 2989–2996.
6. Monge, A.; Martinez-Crespo, F.J.; Lopez, A.; Palop, J.A.; Narro, S.; Senador, V.; Marin, A.;
Sainz, Y.; Gonzalez, M. Hypoxia-selective agents derived from 2-Quinoxalinecarbonitrile 1,4-Di-N-
oxides. 2. J. Med. Chem. 1995, 38, 4488–4494.
7. Hay, M.P.; Hicks, K.O.; Pchalek, P.; Lee, H.H.; Blaser, A.; Pruijn, F.B.; Anderson, R.F.;
Shinde, S.S.; Wilson, W.R.; Denny, W.A. Tricyclic [1,2,4]triazine 1,4-dioxides as hypoxia
selective cytotoxins. J. Med. Chem. 2008, 51, 6853–6865.
8. Solano, B.; Junnotula, V.; Marín, A.; Villar, R.; Burguete A.; Vicente E.; Pérez-Silanes, S.;
Aldana, I.; Monge, A.; Dutta, S.; et al. Synthesis and biological evaluation of new 2-arylcarbonyl-
3-trifluoromethylquinoxaline 1,4-di-N-oxide derivatives and their reduced analogues. J. Med. Chem.
2007, 50, 5485–5492.
9. Magda, M.F.I.; Kamelia, M.A.; Eman, N.; Dalia, H.S.; Yousry, A.A. New quinoxaline 1, 4-di-N-
oxides: Anticancer and hypoxia-selective therapeutic agents. Eur. J. Med. Chem. 2010, 45,
2733–2738.
10. Hicks, K.O.; Siim, B.G.; Jaiswal, J.K.; Pruijn, F.B.; Fraser, A.M.; Patel, R.; Hogg, A.; Liyanage, S.;
Dorie, M.J.; Brown, M.; et al. Pharmacokinetic/Pharmacodynamic modeling identifies SN30000
and SN29751 as tirapazamine analogues with improved tissue penetration and hypoxic cell killing
in tumors. Clin. Cancer Res. 2010, 16, 4946–4957.
11. Jiang, F.Q.; Yang, B.; Fan, L.L.; He, Q.J.; Hu, Y.Z. Synthesis and hypoxic-cytotoxic activity of some
3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives. Bioorg. Med. Chem. Lett. 2006, 16, 4209–4213.
12. Xia, Q.; Zhang, L.; Zhang, J.; Sheng, R.; Yang, B.; He, Q.J.; Hu, Y.Z. Synthesis, hypoxia-
selective cytotoxicity of new 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives Euro. J. Med.
Chem. 2011, 46, 919–926.
13. Jiang, F.Q.; Weng, Q.J.; Sheng, R.; Xia, Q.; He, Q.J.; Yang, B.; Hu, Y.Z. Synthesis, structure and
hypoxic cytotoxicity of 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives Arch. Der. Pharm.
2007, 340, 258–263.
14. Sheng, R.; Xu, Y.; Weng, Q. J.; Xia, Q.; He, Q.J.; Yang, B.; Hu, Y.Z. Synthesis and cytotoxic
activity of 3-phenyl-2-thio-quinoxaline1,4-dioxide derivatives in hypoxia and in normoxia.
Drug. Discov. Ther. 2007, 1, 119–123.

Molecules 2012, 17
9696
15. Weng, Q.J.; Wang, D.D.; Guo, P.; Fang, L.; Hu, Y.Z. He, Q.J.; Yang, B. Q39, a novel synthetic
quinoxaline 1,4-di-N-oxide compound with anti-cancer activity in hypoxia. Eur. J. Pharmacol.
2008, 581, 262–269.
16. Weng, Q.J.; Zhang, J.; Cao, J.; Xia, Q.; Wang, D.D.; Hu, Y.Z.; Sheng, R.; Wu, H.H.; Zhu, D.F.;
Zhu, H.; et al. Q39, a quinoxaline 1,4-di-N-oxide derivative, inhibits hypoxia-inducible factor-1α
expression and the Akt/mTOR/4E-BP1 signaling pathway in human hepatoma cells. Invest New
Drug. 2011, 29, 1177–1187.
17. Zarranz, B.; Jaso, A.; Aldana, I.; Monge, A.; Maurel, S.; Deharo, E.; Jullian, V.; Sauvain, M.
Synthesis and antimalarial activity of new 3-arylquinoxaline-2-carbonitrile derivatives.
Arzneim. Forsch. 2005, 55, 754–761.
18. Vicente, E.; Duchowicz, P.R.; Benitez, D.; Castro, E.A.; Cerecetto, H.; Gonzalez, M.; Monge, A.
Anti-T. cruzi activities and QSAR studies of 3-arylquinoxaline-2-carbonitrile di-N-oxides.
Bioorg. Med. Chem. Lett. 2010, 20, 4831–4835.
19. Vicente, E.; Lima, L.M.; Bongard, E.; Charnaud, S.; Villar, R.; Solano, B.; Burguete, A.;
Perez-Silanes, S.; Aldana, I.; Vivas, L.; Monge, A. Synthesis and structure-activity relationship of
3-phenylquinoxaline 1,4-di-N-oxide derivatives as antimalarial agents. Eur. J. Med. Chem. 2008,
43, 1903–1910.
20. Puterová, Z.; Andicsová, A.; Végh, D. Synthesis of π-conjugated thiophenes starting from
substituted 3-oxopropanenitriles via gewald reaction. Tetrahedron 2008, 64, 11262–11269.
21. Dornow, A.; Kuhlcke, I.; Baxmann, F. Über einige derivate der benzoylessigsäure. Chem. Ber.
1949, 82, 254–257.
22. Alon, H.; Lena, L.; Michal, W.; Iris, O.G.; Abraham, N.; Amnon, H. De Novo parallel design,
synthesis and evaluation of inhibitors against the reverse transcriptase of human
immunodeficiency virus type-1 and drug-resistant variants. J. Med. Chem. 2007, 50, 2370–2383.
23. Ridge, D.N.; Hanifin, J.W.; Harten, L.A.; Johnson, B.D.; Menschik, J.; Nicolau, G.;
Sloboda, A.E.; Watts, D.E. Potential antiarthritic agents. 2. Benzoylacetonitriles and
.beta.-aminocinnamonitriles. J. Med. Chem. 1979, 22, 1385–1389.
24. Gaughran, R.J.; Picard, J.P.; Kaufman, J.V.R. Contribution to the chemistry of benzfuroxan and
benzfurazan derivatives. J. Am. Chem. Soc. 1954, 76, 2233–2236.
25. Yang, B.; Patrick, R.C. Tirapazamine cytotoxicity for neuroblastoma is p53 Dependent.
Clin. Cancer. Res. 2005, 11, 2774–2780.
Sample Availability: Samples of the compounds are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).