Study of benzo[a]phenazine 7,12-dioxide as selective hypoxic cytotoxin-scaffold. Identification of aerobic-antitumoral activity through DNA fragmentation

Article abstract of DOI:10.1016/j.bmc.2010.04.074

Phenazine 5,10-dioxides are prodrugs for antitumor therapy that undergo hypoxic-selective bioreduction to form cytotoxic species. Here we investigate the expanded system benzo[a]phenazine 7,12-dioxides as selective hypoxic cytotoxin-scaffold. The clonogenic survival of V79 cells on aerobic and anaerobic conditions, conduct us to study antiproliferative activity on Caco-2 tumoral cells in normoxia. Electrochemical, DNA-interaction and DNA-damage studies were performed to establish the mode of action. The results demonstrated the potential biological properties of the studied scaffold being derivatives 6-10 structural hits for further chemical-modifications to become into therapeutics for solid tumors. Compounds 6 and 8 with cytotoxicity against V79 cells in both conditions (aerobia and anaerobia) were also cytotoxic against Caco-2 tumoral cells in aerobiosis.

Full text of DOI:10.1016/j.bmc.2010.04.074

Bioorganic & Medicinal Chemistry 18 (2010) 4433–4440
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Study of benzo[a]phenazine 7,12-dioxide as selective hypoxic
cytotoxin-scaffold. Identification of aerobic-antitumoral activity through DNA
fragmentation
a
a
b
b
María Laura Lavaggi , Mauricio Cabrera , María de los Ángeles Aravena , Claudio Olea-Azar ,
c
c
d
d
e
Adela López de Ceráin , Antonio Monge , Gisela Pachón , Marta Cascante , Ana María Bruno ,
Lía I. Pietrasanta , Mercedes González , Hugo Cerecetto
f
a,
a,
*
*
a
Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, 11400 Montevideo, Uruguay
Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Chile
Centro de Investigación en Farmacobiología Aplicada, Universidad de Navarra, Pamplona, Spain
b
c
d
Department of Biochemistry and Molecular Biology, Faculty of Biology, Institute of Biomedicine of University of Barcelona (IBUB) and IDIBAPS, Unit Associated with CSIC,
Barcelona, Spain
e
Departamento de Química Orgánica, Facultad de Farmacia y Bioquímica, Argentina
Centro de Microscopías Avanzadas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
f
a r t i c l e i n f o
a b s t r a c t
Article history:
Phenazine 5,10-dioxides are prodrugs for antitumor therapy that undergo hypoxic-selective bioreduction
to form cytotoxic species. Here we investigate the expanded system benzo[a]phenazine 7,12-dioxides as
selective hypoxic cytotoxin-scaffold. The clonogenic survival of V79 cells on aerobic and anaerobic con-
ditions, conduct us to study antiproliferative activity on Caco-2 tumoral cells in normoxia. Electrochem-
ical, DNA-interaction and DNA-damage studies were performed to establish the mode of action. The
results demonstrated the potential biological properties of the studied scaffold being derivatives 6–10
structural hits for further chemical-modifications to become into therapeutics for solid tumors. Com-
pounds 6 and 8 with cytotoxicity against V79 cells in both conditions (aerobia and anaerobia) were also
cytotoxic against Caco-2 tumoral cells in aerobiosis.
Received 18 March 2010
Revised 18 April 2010
Accepted 22 April 2010
Available online 26 April 2010
Keywords:
Benzo[a]phenazine 7,12-dioxides
Bioreductive agents
DNA fragmentation
Ó 2010 Elsevier Ltd. All rights reserved.
1
. Introduction
Since the 1960’ it is well known the existence of hypoxic and
expression of genes associated with angiogenesis and tumor inva-
sion, and is associated with a more metastatic phenotype of human
4
cancers. By enhancing metastasis, hypoxia can compromise cur-
ability of tumors by surgery. This common feature of cancerous
cells is used for the development of a distinct therapy for treating
cancer. On the basis of using prodrugs, capable to be bioreduced
under hypoxic conditions (bioreductive antitumor agents,5 BAA)
to further drugs that produce cytotoxic events causing different
degrees of cancerous cells damage, hypoxic selective cytotoxins
have been developed. Molecular oxygen reverts the bioreductive
process in normal oxygenated tissues giving the selectivity to this
process the redox properties of the drugs and the reductases
1
necrotic regions in solid tumors representing the hypoxic regions
up to 20% of the total tumor mass. This situation is the result of the
cancerous cells isolation from the blood supply, as consequence of
their rapid growth and deficient vascularisation, decreasing the
diffusion of molecular oxygen.2 The hypoxic cells are associated
3
with increased resistance to radiation and chemotherapy. Con-
ventional anticancer drugs in clinical use are antiproliferative
agents that kill dividing cells, by attacking DNA (synthesis, replica-
tion or processing). These anticancer drugs could be ineffective be-
cause the hypoxic tumor cells are not dividing rapidly.
In addition, it has also been demonstrated that hypoxia in
tumors alters cellular metabolism tending to select for a more
malignant phenotype, increases mutation rates, increases
6
enzymes levels. Among the compounds classified as BAA N-oxide,
7
nitro and quinone derivatives have been described. This hypoxic
tissues special condition has been employed as a strategy for deliv-
ering traditional antineoplastic agents. Consequently a great num-
ber of hybrid compounds have been described where N-oxide,
nitro, azido, quinone, metal ion, 1,2-benzisoxazolyl and sulfoxide
8
moieties have been employed as redox-activated trigger entities.
*
Corresponding authors. Tel.: +598 2 5258618; fax: +598 2 5250749.
9
We have recently reported the first examples of a new group of
E-mail addresses: megonzal@fq.edu.uy (M. González), hcerecet@fq.edu.uy
(
H. Cerecetto).
N-oxide-hypoxia-activated prodrugs, compounds that display
0
968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.074

4
434
M. L. Lavaggi et al. / Bioorg. Med. Chem. 18 (2010) 4433–4440
toxic selectivity to cells at low oxygen concentrations. Phenazine
,10-dioxide derivatives (Scheme 1a), structurally related to other
N-oxide bioreductive agents, were selected for their DNA– -stack-
stage of the work we have selected one of the best described
5
synthetic methodology to prepare the BPDO system (6–13,
1
2
p
Scheme 2). This procedure allowed us to prepare, besides the de-
sired BPDO phenols after pH changes, 5-unsubstituted derivatives,
being six of them no described previously (Scheme 2).
ing structure that potentially interact with DNA after the corre-
sponding bioreduction in hypoxic conditions. Amines 2, and 4
displayed excellent selective cytotoxic properties while phenols,
except the lead compound 1, were non-cytotoxic in both of the
studied conditions (i.e., 3 and 5, Scheme 1b). Besides, some of the
phenazine 5,10-dioxides displayed in vitro aerobic-antitumor
The compounds resulted from the ortho (6–10) and para (11–
13) coupling procedures with the corresponding phenolate anion.
We were enable to isolate, after acidification and by precipitation,
3
the phenol derivatives when –R = –OCH and –(1,3-dioxolan-2-yl).
10
activity against Caco-2 cells. The selective anaerobic-reduction
and its relation to bioreductive activity were proved using enzy-
matic mammal systems.11 On the other hand, none of them were
able to interact with DNA,9 hence the proposed extra-mechanism
is not functional in this system.
The acidified mother liquors showed a muddy complex mixture of
products which after chromatographic resolution did not yield the
desired phenols. All the structures were elucidated on the basis of
assignment of the large chemical shift of the proton at 1-position in
the NMR spectra (signal near to 10.50 ppm, Figure S1a, Supporting
Information) and on the characteristic chemical shift of the peri-N-
oxide proton at 11-position, then the complete proton and carbon
assignments were performed in terms of NOE-diff, COSY, HSQC,
and HMBC experiments (Figure S1b, Supporting Information).
For that reason we investigated
benzo[a]phenazine 7,12-dioxide system (BPDO, Scheme 1c), that
potentially improves DNA- -stacking properties, through the ex-
a
new scaffold, the
p
panded chromophoric system, maintaining the bioreductive capa-
bility through the N-oxide pharmacophore. Therefore we have
prepared a selected series of BPDO derivatives, in vitro evaluated
its clonogenic capacity in normoxia and hypoxia and studied some
aspects related to mechanism of action.
1
2
The previous described BPDO, that is, 7 and 12, were completely
characterized in term of NMR spectroscopies. As we previously ob-
9
served, using 5-methoxybenzofuroxan as starting material only
one of the two possible products, 9- or 10-substituted BPDO, was
isolated by precipitation. According to the spectroscopic data
derivative 10 was assigned as the 10-methoxy isomer. The other
BPDOs were characterized as a non separable mixture of 9- and
2
2
. Methods and Results
1
0-isomers because they could not be isolated either by crystalli-
.1. Chemistry
zation or by chromatographic methods (preparative-TLC, column
chromatography, direct- and reverse-HPLC). The very close physi-
cochemical behaviors of each positional isomer did not allow us
to separate them by physicochemical methods. The purity of
BPDOs was established by TLC and microanalysis.
According to our previous biological results, we have prepared
BPDOs containing as -R substituents the most relevant ones from
the parent compound (1, 2, and 4, Scheme 1b). In this exploratory
a
O
selective bioreduction
N
X
N
N
X
in hypoxic conditions
Potential DNA- -stacking
π
R
R
pharmacophore
N
O
.-
O2
O2
b
O
N
O
N
O
OH
X
N
X
Br
H C
3
H CO
3
N
O
1
N
O
N
O
2, -X= -NH2
4, -X= -NH2
SFox = 80 %
SFhypox = 0 %
SFox = 61 %
SFhypox = 11 %
SFox = 87 %
SFhypox = 23 %
3
, -X= -OH
5, -X= -OH
SFox = 93 %
SFhypox = 100 %
SFox = 100 %
SFhypox = 87 %
c
O
N
8
6
7
5
9
X
BPDO
R
Expanded chromophoric
system
10
N
4
3
1
1
12
O
1
2
bioreductive pharmacophore
Scheme 1. (a) Expected mechanism of action of the first series of developed phenazine 5,10-dioxides. (b) Selected derivatives, to exemplify was selected the data SFox
survival fraction in oxic conditions at 20 M) and SFhypox (survival fraction in hypoxic conditions at 20 M). (c) Benzo[a]phenazine 7,12-dioxides studied in this work.
(
l
l

M. L. Lavaggi et al. / Bioorg. Med. Chem. 18 (2010) 4433–4440
4435
OH
O
N
O
N
O
O
OH
N
N
R
R
R
N
O
N
O
MeONa/MeOH
-
-
-
-
-
R=-5-Br,
bfx1
-R=-Br,
-R= -H,
-R=-CH3,
6
7
-R=-Br,
-R=-H,
11
12
R=-H,
bfx2
bfx3
R=-5-CH ,
8
-R= -CH , 13
3
3
R= -5-(1,3-dioxolan-2-yl), bfx4
-R=-(1,3-dioxolan-2-yl),
9
10
R= -5-OCH ,
bfx5
-R= -OCH ,
3
3
after acidification(AcOH)
Scheme 2. Synthetic procedure used to prepare the benzo[a]phenazine 7,12-dioxides.
2
2
.2. Biological characterization
(Scheme 1a), by the reverse reaction with molecular oxygen of the
BPDO-free radical generated by the reductases (futile cycle) or the
BPDO damage as result of its DNA–p-stacking property. Some
.2.1. In vitro normoxic and hypoxic cytotoxicity
All the BPDO were examined for its selective hypoxic-cytotoxic-
experiments were performed in order to probe these hypotheses
(see below). When derivative 6 was studied at lower doses it dis-
played similar cytotoxicity profile of derivatives 11 and 13. The
methoxy-derivative 10 was the only one with some degree of
selective cytotoxicity in hypoxia having values of SF in both condi-
tions similar to the methoxy-parent compound 4 (Scheme 1b),
though it is not relevant.
ities, in a pre-established model of V-79 cells.9
,13,14
They were ana-
lyzed as a non separable mixture of 9- and 10-isomers. Previous
results for similar chemical systems (quinoxaline 1,4-dioxides)
demonstrated that no difference between both positional isomers
were observed in the selective hypoxic cytotoxicities against V-
1
3
7
9 cells. The percentages of survival fractions (Table 1) were
measured at 20 M while derivative 6 was also evaluated at
M. Except derivatives 10 and 12, the BPDOs showed high to
l
1
0
l
2.3. Normoxic cytotoxicity studies
medium cytotoxicity in normoxia maintaining the 5-un-substi-
tuted derivatives 6–9 its cytotoxicity in hypoxia. These derivatives
were classified as non-selective cytotoxins. The 5-hydroxy-substi-
tuted derivatives 11 and 13 showed very particular cytotoxic pro-
files having high oxic cytotoxicities they were not toxic in hypoxia.
This particular toxicity could be the result of at least two different
processes, the formation of toxic species, that is, superoxide anion
To gain insight into BPDO mechanism of toxicity in oxic condi-
tions, first of all we studied the BPDO electrochemical behavior in
term of cyclic voltammetry in organic aprotic solvent (DMSO).9
BPDO displayed comparable voltammetric behavior in this condi-
tion showing two to three reduction peaks and the anodic counter-
parts. Especially, we focus our attention in the first reduction
potential because it could be used as a descriptor of the BPDO fea-
sibility to be reduced in hypoxic conditions. Table 2 lists the values
of the first N-oxide cathodic peaks which correspond to quasire-
versible processes. As expected the electron-donor-substituted
derivatives, like the 5-hydroxy-substituted 11 and 13, have more
negative cathodic potential than the electron-withdrawing-substi-
tuted ones, that is, derivative 8 (Epc = À1.36 V in 13 vs À0.95 V in
8). However, the reduction potential does not seem to be corre-
lated to the oxic toxicity since some of the most aerobic-toxic com-
pounds, that is, derivatives 11 and 13, have the lowest reduction
potentials that are unfavorable to be reverted by molecular oxygen.
For this reason, secondly we analyze the BPDO capability to
interact to DNA. As a first approach, we studied the binding
capacity by measuring the hypochromic and bathochromic effect
Table 1
BPDO (6–13) cytotoxic effects in normoxia and hypoxia on V-79 cells and cytotoxic
effects on tumoral Caco-2 cells for the normoxic-toxic BPDO on V-79 cells (6, 8, 11,
and 13)
O
N
X
R
N
O
SFa,b,c,d
VC50e,f
(lM)
Compd
–X
–R
Norm
Hypox
6
7
8
9
–H
–H
–H
–H
–Br
–H
–CH
0 (2)
40
11
52
88
0
0 (48)
45
0
60
44
100
93
85
23.0
nd
290.0
nd
nd
53.0
nd
140.0
g
Table 2
Reduction potentials and DNA interacting capability by UV studies of BPDO
derivatives
3
–1,3-Dioxolan-2-yl
–OCH
–Br
–H
–CH
1
1
1
1
0
1
2
3
–H
3
pc versus SCEa (V)
E
Compd
a24/a
0
–OH
–OH
–OH
b,c
6
7
8
9
nd
nd
0.88
1.00
0.71
100
9
c
3
À0.95
À0.85
À0.92
À1.22
À1.36
—
—
—
—
a
b
c
c
SF norm = survival fraction in normoxia at 20
SF hypox = survival fraction in hypoxia at 20
Values are means of two different experiments. The assays were done by
l
M.
nd
lM.
10
11
13
m-AMSA
Ethidium bromide
NSC 322921 (bis-benzimide)
Mitoxantrone
1.00
0.94
0.47
0.30
0.50
0.57
0.00
duplicate and using at least three repetitions, standard errors were not greater than
% for most assays.
Values in parentheses are at 10
VC50 = concentration that causes 50% decrease of Caco-2 cell viability in nor-
moxia, after 24 h of exposition.
Values are means of three different experiments. The assays were done by
triplicate and using at least three repetitions, standard errors were not greater than
% for most assays.
nd: not determined.
2
d
lM.
e
f
a
b
c
Peaks potentials (ꢀ±0.01 V) measured at a scan rate of 2.00 V/s.
Not determined.
5
g
Solubility problems in the assay milieu do not allow to perform the study.

4
436
M. L. Lavaggi et al. / Bioorg. Med. Chem. 18 (2010) 4433–4440
of BPDO absorbance in the UV spectra, in a 20 nm band centred on
the maximal absorbance value of each compound, at 0 and
been seen for other antitumoral DNA interacting agents such as
1
9
some platinum complexes.
This observation conducted us to study the aerobic antitumoral
1
5,16
2
4 h.
tween the final absorbance area (a24) and the absorbance of the
compound (a ). Values of 1.00 or superior to 1.00 indicate a total
The degree of interaction was expressed by the ratio be-
2
0
activity on Caco-2 cells. Therefore, VC50s (concentrations that
caused 50% decrease of cell viability), for the oxic-cytotoxic BPDOs
on V-79 cells, that is, 6, 8, 11, and 13, were determined (Table 1).
The bromo-derivatives, 6 and 11, were more toxics on Caco-2 cells
than the methyl-ones, 8 and 11, with the same cytotoxic-profile
that observed on V-79 system (SF normoxia, Table 1).
0
lack of affinity and a value of 0 indicate binding of the entire com-
pound to DNA (Table 2, Fig. S2a, Supplementary data). Derivatives
with highest oxic-toxicity, that is, 6, 8 and 13, were the best DNA-
0
interacting agents with values of a24/a between 0.47 and 0.88. The
9
(10)-methyl-derivatives 13 and 8 have the highest capabilities of
Further the DNA damages, on Caco-2 cells, promoted by the less
toxic derivatives, 8 and 13, were evaluated. Gel electrophoresis re-
sults indicate that derivative 13 is able to induce DNA fragmenta-
DNA-interactions being the 5-hydroxy-analogue, 13, as interactive
as NSC 322921, ethidium bromide or m-AMSA in our experimental
conditions (Table 2).
tion in oxic conditions beginning at 50
than the corresponding VC80. Its effect is similar to the positive
control used in this assay (staurosporine, Stau, at 10 M). On the
lM (Fig. 2), a dose lower
To profoundly study the DNA-interaction processes derivatives
and 13 were selected to perform further interacting experiments.
8
l
The degree of interaction by DNA-melting point changes promoted
by the compounds was carried out.16 Whereas the free DNA melted
at 73.5 °C the systems DNA/8 and DNA/13 did at 75.5 and 78.5 °C,
respectively (Fig. S2b, Supplementary data). The melting tempera-
ture displacements are in agreement with the obtained values a24
(Table 2) showing that derivative 13 could interact with DNA by
intercalation but in a minor way than others well known intercala-
/
a
0
17
tors (i.e., proflavine,
DT = 12.0 °C ).
Moreover, we studied DNA structural parameters of 13-modi-
fied DNA and unmodified (untreated) DNA via tapping mode atom-
ic force microscopy (AFM).18 We analyzed the effects on DNA
structure of individual DNA molecules by increasing the amount
of 13 (molar ratio 13:DNA varying between 1:1 to 100:1). Mea-
surements of 13-modified DNA populations indicate significant
changes respect to unmodified DNA control, in the contour length
(
cl) and at the end-to-end distance (eed) (Fig. 1). End-to-end dis-
tance shows clearly DNA shortening and eed/cl ratios were similar
for the three studied reactants proportions, near to the unity, and
significantly greater than with control DNA (ꢀ0.94 vs 0.51). This
could be indicating that derivative 13 promotes DNA breakage
yielding little DNA fragments. Also it could be suggested that the
mechanism of interaction of derivative 13 with DNA is not by
intercalation because no lengthening of DNA molecules is observed
(
Fig. 1b). Also condensate structures of DNA such as spheres and
Figure 2. Genomic DNA analysis after exposure to BPDOs. Caco-2 cells were
exposed for 24 h in oxic conditions to variable doses of 8 and 13.
rods were observed (magnification Fig. 1b). Similar structures have
a
b
ratio BPDO 13:DNA contour length (nm) end-to-end distance (nm) end-to-end distance/contour length
0
1
:1
:1
165
96
84
90
0.51
0.94
0.95
0.93
1
0:1
118
67
112
62
1
00:1
Figure 1. Top: (a) atomic force microscopy of unmodified linear DNA. (b) 13-modified linear DNA (ratio 100:1). Up: length parameters of DNA in the different experimental
conditions. In each case, contour lengths and end-to-end distances were measured for at least 100 molecules that could unequivocally be traced.

M. L. Lavaggi et al. / Bioorg. Med. Chem. 18 (2010) 4433–4440
4437
a
b 1.0
c
-0.7
0.8
1
00
10
8
-
1
1
1
0.8
8
6
4
2
0
0
0
0
0
1
4
2
10
-0.9
-1.0
13
0.6
0.4
0.2
0.0
pc
-
-
-
-
1.1
1.2
1.3
1.4
E
4
2
8
10
13
1
-
1.4 -1.3 -1.2 -1.1 -1.0 -0.9 -0.8 -0.7
Epc (V)
-1.4 -1.3 -1.2 -1.1 -1.0 -0.9 -0.8 -0.7
Epc (V)
0
20
40
60
80
100
SFOXIA (%)
Figure 3. (a) Epc versus SFhypoxia for phenazine 5,10-dioxide parent compounds (Scheme 1b) and the herein studied BPDO. Dot-lines show tendency. (b) Epc versus selectivity
for phenazine 5,10-dioxide parent compounds (Scheme 1b) and the herein studied BPDO. Categories of selectivity values were obtained by the relation between SFhypoxia and
SFnormoxia (category 1, ratio >6; category 0.5, 6 P ratio P 2; category 0, ratio <2). Dot-lines show tendency. (c) SFnormoxia versus Epc for phenazine 5,10-dioxide parent
compounds (Scheme 1b) and the herein studied BPDO.
other hand, 5-un-substituted analogue, 8, showed a different
behavior at these doses accordingly to its lower DNA fragmenta-
knowledge this is the first time that this kind of compounds is also
studied as bioreductive cytotoxins. Further structural modifica-
tions on the benzo[a]phenazine 7,12-dioxide scaffold, QSAR stud-
ies, and in vivo activities are currently underway.
tion, like Stau at 1 and 2
lM doses. These results are in agreement
with the findings on the others DNA-studies.
3
. Discussion
5. Experimental
We report the study of benzo[a] phenazine 7,12-dioxides with
All starting materials were commercially available research-
grade chemicals and used without further purification. All solvents
were dried and distilled prior to use. All the reactions were carried
out in a nitrogen atmosphere. Benzofuroxan bfx1–5 were prepared
potential use as cytotoxic-agents in normoxia. These derivatives
were studied as bioreductive cytotoxins nevertheless only the
methoxy-derivative, 10, displayed some degree of hypoxic-selec-
tivity at the studied doses (Table 1).
2
2
according to procedures previously described. Melting points
were determined with an electrothermal melting point apparatus
(Electrothermal 9100) and are uncorrected. Proton and carbon
NMR spectra were recorded on a Bruker DPX-400 spectrometer
at 298 K. The chemical shifts values are expressed in ppm (d) rela-
tive to tetramethylsilane as internal standard and the J in hertz.
Mass spectra were determined on a MSD 5973 Hewlett–Packard
spectrometer using electronic impact ionization. Microanalyses
were performed on a Fisons EA 1108 CHNS-O instrument and were
within (0.4% of the calculated compositions). Column chromatog-
raphy was carried out using Merck silica gel (60–230 mesh). Most
chemicals and solvents were analytical grade and used without
further purification.
The profile of hypoxic cytotoxicity of these phenazine dioxides
seems to be governed for the bio-reduction process. This fact could
be realized in Figure 3a where hypoxia-cytotoxic compounds, that
is, parent compounds 1, 2 and 4,9 or BPDO derivatives 8 and 10,
have reduction potential (Epc) between À0.92 to À1.14 V with
the lowest hypoxic survival fractions (SFhypoxia). In Figure 3b is pos-
sible to observe that selectivity reached a maximum and then de-
clined with the increasing of Epc. This kind of relationship has been
previously described between air-to-nitrogen differential cytotox-
icity and E1/2 for benzotriazine di-N-oxides.21 On the other hand, in
reference to oxic-cytotoxicity compounds with Epc higher than
À1.14 V (value for the selective parent compound 1, Scheme 1b)
show SFnormoxia higher than 50% (Fig. 3c). This result shows the
enzymatic reduction processes for compounds with the least neg-
ative Epc could be reverted by molecular oxygen consequently
these dioxides could not be cytotoxic in oxic conditions. However
the reduction of derivatives with more negative Epc, that is, 11
and 13 (Table 2), could not be efficiently reverted by molecular
oxygen and therefore they are cytotoxic in oxia. A special behavior
is observed in BPDO 8, according to its Epc (À0.95 V, Table 2) it
should be non-cytotoxic in oxic conditions; however SFnormoxia
for BPDO 8 is 11%. This fact could be clearly understood taking into
account the capability of BPDO 8 to promote interaction to DNA
5.1. General procedure for the preparation of the
benzo[a]phenazine 7,12-dioxide derivatives 6–13
To a solution of 7.3 mmol of NaOMe and 1.1 mL of MeOH, at
À5 °C and under nitrogen atmosphere, was added a solution of
20.0 mmol of
a-naphthol and 7.3 mmol of the corresponding
benzofuroxan in 7.3 mL of MeOH. After stirring at room tempera-
ture for 24 h and maintaining at À4 °C for 24 h, the resulting pre-
cipitate was filtered to yield the corresponding 5-unsubstituted-
benzo[a]phenazine 7,12-dioxide (6–10, –X = –H, Scheme 2). The
mother liquor from the reaction mixture was acidified with AcOH
and cooled at À4 °C for 24 h, to yield the corresponding 5-hydroxy-
benzo[a]phenazine 7,12-dioxide (11–13, –X = –OH, Scheme 2).
0
(a24/a = 0.71, Table 2) and some degree of DNA-damage (Fig. 2)
in oxic conditions. Besides, derivatives 6, 8, 11, and 13, were toxic
in aerobic conditions against V-79 cells and showed antitumoral
activity against Caco-2 cells (Table 1). On this cell line the least-
toxic derivative 8 and the medium-toxic derivative 13 showed
clear DNA-fragmentation capability at a sub-toxic dose (lower than
the corresponding VC80, Fig. S3, Supplementary data).
5.2. 9(10)-Bromobenzo[a]phenazine 7,12-dioxide (6)
Yellow solid (15%). Proportion of 9 and 10-isomers 49:51. 1
NMR (DMSO-d ) d: 9-isomer, 7.80–7.90 (3H, m), 8.05 (1H, dd,
= 9.4, J = 2.2), 8.30 (1H, s), 8.40 (1H, d, J = 9.2), 8.60 (2H, m),
0.57 (1H, d, J = 9.5); 10-isomer, 7.80–7.90 (3H, m), 8.05 (1H, dd,
= 9.4, J = 2.2), 8.30 (1H, s), 8.40 (1H, m), 8.90 (2H, m), 10.57
(1H, d, J = 9.5). C NMR (from the HMQC and HMBC experiments)
(DMSO-d ) d: 9- and 10-isomers, 118.5, 119.7, 120.5, 121.6, 122.9,
H
6
4
. Conclusions
J
1
2
1
We have identified new benzo[a]phenazine 7,12-dioxides as
promising antitumoral agents in oxic conditions taking this to
defined a scaffolds of anticancer drugs not ever described. To our
J
1
2
1
3
6

4
438
M. L. Lavaggi et al. / Bioorg. Med. Chem. 18 (2010) 4433–4440
1
24.2, 125.0, 126.7, 128.7, 129.4, 130.2, 132.9, 134.0, 134.8. MS, m/
126.7, 128.3, 128.7, 130.4, 131.0, 132.6, 133.0, 135.0, 142.5. MS,
+Å
+Å
z (%): 9- and10-isomers, 340 (M , 28), 326 (47), 310 (62). (Found: C,
m/z (%): 9- and10-isomers, 356 (M , 24), 340 (67), 324 (16). (Found:
C, 53.9; H, 2.3. C16 BrN requires C, 53.8; H, 2.5).
5
6.0; H, 2.4. C16
H BrN
9
2
O
2
requires C, 56.3; H, 2.7).
H
9
2 3
O
5
.3. Benzo[a]phenazine 7,12-dioxide (7)
5.8. 5-Hydroxybenzo[a]phenazine 7,12-dioxide (12)
1
2
Yellow solid (35%), mp >240.0 °C (lit. 250.0–253.0 °C dec.).12 1H
Yellow solid (35%), mp = 178.5–181.5 °C (lit. 179.0–180.0 °C).
1
H NMR (DMSO-d
= 9.3, J = 2.1), 8.24 (1H, d, J = 9.2), 8.52 (1H, d, J = 9.2), 8.71–
.82 (2H, m), 10.71 (1H, d, J = 9.3). C NMR (from the HMQC and
HMBC experiments) (DMSO-d ) d: 119.8, 120.4, 122.4, 122.8,
25.2, 128.3, 128.4, 128.8, 130.4, 130.9, 132.1, 133.8, 135.0,
6
) d: 7.93 (2H, m), 8.06 (2H, m), 8.19 (1H, dd,
6
NMR (DMSO-d /D2 O) d: 7.40–7.85 (5H, m), 8.40–8.61 (3H, m), 10.67
1
3
J
1
2
(1H,, d, J = 9.3). C NMR (from the HMQC and HMBC experiments)
(DMSO-d ) d: 119.8, 120.4, 122.4, 122.8, 125.2, 128.3, 128.4, 128.8,
130.4, 130.9, 132.1, 133.8, 135.0, 135.2. MS, m/z (%): 278 (M , 28),
13
8
6
+Å
6
1
1
7
262 (47), 246 (62). (Found: C, 68.9; H, 3.3. C16
69.1; H, 3.6).
10 2 3
H N O requires C,
+Å
35.2. MS, m/z (%): 262 (M , 100), 246 (75), 230 (23). (Found: C,
3.2; H, 3.9. C16 requires C, 73.3; H, 3.8).
10 2 2
H N O
5
.9. 5-Hydroxy-9(10)-methylbenzo[a]phenazine 7,12-dioxide
5
.4. 9(10)-Methylbenzo[a]phenazine 7,12-dioxide (8)
(
13)
Yellow solid (35%). Proportion of 9 and 10-isomers 65:35. 1
H
Yellow solid (15%). Proportion of 9 and 10-isomers 53:47. 1
NMR (DMSO-d /D O) d: 9-isomer, 2.68 (3H, s), 7.82 (1H, s), 7.90
3H, m), 8.40–8.50 (3H, m), 10.73 (1H, d, J = 9.5); 10-isomer, 2.68
(3H, s), 7.82–7.90 (4H, m), 8.50–8.55 (3H, m), 10.73 (1H, d,
H
NMR (DMSO-d
m), 8.50 (1H, m), 8.58 (2H, m), 10.66 (1H, d, J = 9.5); 10-isomer,
.66 (3H, s), 7.88 (3H, m), 8.19 (2H, m), 8.50 (1H, m), 8.69 (2H,
6
) d: 9-isomer, 2.66 (3H, s), 7.88 (3H, m), 8.19 (2H,
6
2
(
2
13
m), 10.66 (1H, d, J = 9.5). C NMR (from the HMQC and HMBC
experiments) (DMSO-d ) d: 9- and 10-isomers, 23.0, 23.1, 110.4,
13.0, 118.6, 121.6, 122.9, 123.9, 125.9, 126.8, 128.9, 129.0,
31.5, 132.9, 134.4, 135.1, 145.0. MS, m/z (%): 9- and 10-isomers,
1
3
J = 9.5).
C NMR (from the HMQC and HMBC experiments)
) d: 9- and 10-isomers, 22.8, 23.0, 108.3, 116.0, 118.4,
20.2, 122.1, 124.4, 126.0, 127.4, 129.5, 129.5, 131.0, 133.7,
6
(
1
DMSO-d
6
1
1
2
C
+Å
+Å
134.0, 135.5, 144.2. MS, m/z (%): 9- and 10-isomers, 292 (M , 10),
76 (93), 260 (100). (Found: C, 70.0; H, 3.9. C17 requires
76 (M , 57), 260 (100), 244 (36). (Found: C, 73.7; H, 4.2.
requires C, 73.9; H, 4.4).
2
12 2 3
H N O
17
12 2 2
H N O
C, 69.9; H, 4.1).
5
.5. 9(10)-(1,3-Dioxolan-2-yl)benzo[a]phenazine 7,12-dioxide
(
9)
5.10. Biology
Yellow solid (12%). Proportion of 9 and 10-isomers 52:48. 1
NMR (DMSO-d ) d: 9-isomer, 4.15 (4H, m), 6.03 (1H, s), 7.74 (1H,
dd, J = 8.8, J = 1.6), 7.76–8.00 (4H, m), 8.51 (1H, d, J = 8.8), 8.62
1H, d, J = 1.6), 8.63 (1H, m), 10.59 (1H, m); 10-isomer, 4.15 (4H,
m), 6.05 (1H, s), 7.58 (1H, dd, J = 9.2, J = 1.2), 7.76–8.00 (4H, m),
.24 (1H, d, J = 8.0), 8.73 (1H, d, J = 9.2), 8.82 (1H, d, J = 2.1), 10.59
H
5.10.1. Bio-reductive activity9,14 cells
V79 cells (Chinese hamster lung fibroblasts) were obtained
from ECACC (European Collection of Animal Cell Cultures) and
maintained in logarithmic growth as subconfluent monolayer by
trypsinization and subculture to (1–2) Â 10 cells/cm twice
weekly. The growth medium was EMEM (Eagle’s Minimal Essential
Medium), containing 10% (v/v) fetal bovine serum (FBS) and
6
1
2
(
4
2
1
2
8
1
3
(
(
1
1
3
1H, m). C NMR (from the HMQC and HMBC experiments)
DMSO-d ) d: 9- and 10-isomers, 66.5, 68.3, 102.6, 103.7, 121.5,
23.4, 124.0, 125.4, 128.3, 128.8, 129.6, 130.4, 131.0, 131.5,
penicillin/streptomycin at 100 U/100 lg/mL. Aerobic and hypoxic
cytotoxicity: suspension cultures. Monolayers of V79 cells in expo-
6
+Å
Å
32.6, 134.3, 134.7. MS, m/z (%): 9- and 10-isomers, 335 (M + H ,
), 318 (5), 302 (5), 301 (1). (Found: C, 68.0; H, 4.4. C19 re-
nential growth were trypsinized, and suspension cultures were
4
H
14
N
2
O
4
set up in 50 mL glass flasks: 2 Â 10 cells/mL in 30 mL of EMEM
quires C, 68.3; H, 4.2).
containing 10% (v/v) FBS and HEPES (10 mM). The glass flasks were
submerged and stirred in a water bath at 37 °C, where they were
gassed with humidified air or pure nitrogen. Treatment: Com-
pounds solutions were prepared just before dosing. Stock solu-
tions, 150-fold more concentrated, were prepared in pure DMSO
(Aldrich) or sterilized distilled water. Thirty minutes after the start
of gassing, 0.2 mL of the stock compound solution was added to
each flask, two flasks per dose. In every assay there was one flask
with 0.2 mL of DMSO (negative control) and another with 7-
chloro-3-[3-(N,N-dimethylamino)propylamino]-2-quinoxalinecar-
bonitrile 1,4-dioxide hydrochloride (positive control). Cloning:
After 2 h exposure to the compound, the cells were centrifuged
and resuspended in plating medium (EMEM plus 10% (v/v) FBS
and penicillin/streptomycin). Cell numbers were determined with
5
.6. 10-Methoxybenzo[a]phenazine 7,12-dioxide (10)
1
Yellow solid (6%); mp >240.0 °C. H NMR (DMSO-d
s), 7.69 (1H, dd, J = 9.6, J = 2.4), 7.92 (2H, m), 8.00 (1H, d, J = 2.4),
.19 (1H, dd, J = 7.6, J = 2.0), 8.26 (1H, d, J = 9.2), 8.55 (1H, d,
J = 9.6), 8.73 (1H, d, J = 9.6), 10.68 (1H, d, J = 9.3). C NMR (from
the HMQC and HMBC experiments) (DMSO-d ) d: 9- and 10-iso-
mers, 57.7, 106.3, 108.6, 119.4, 120.7, 122.3, 122.8, 124.7, 127.9,
28.5, 128.9, 130.7, 131.9, 133.6, 135.2, 135.5, 145.3. MS, m/z (%):
6
) d: 4.07 (3H,
1
2
8
1
2
13
6
1
9
+Å
- and 10-isomers, 292 (M , 98), 276 (54), 260 (15). (Found: C,
6
8.6; H, 3.9. C17
12 2 3
H N O requires C, 68.9; H, 4.1).
2
3
5
.7. 9(10)-Bromo-5-hydroxybenzo[a]phenazine 7,12-dioxide
a haemocytometer and 10 –10 cells were plated in 6-well plates
to give a final volume of 2 mL/30 mm of well. Plates were incu-
(
11)
2
bated at 37 °C in 5% CO for 7 days and then stained with aqueous
Proportion of 9 and 10-isomers 55:45. Yellow solid (12%). 1
H
crystal violet. Colonies with more than 64 cells were counted. The
plating efficiency (PE) was calculated by dividing the number of
colonies by the number of cells seeded. The percent of control-cell
survival for the compound-treated cultures (SFnormoxia and
SFhypoxia) was calculated as PEtreated/PEcontrol  100. The
NMR (DMSO-d
6
/D
= 9.3, J
2
O) d: 9-isomer, 6.78 (1H, s), 7.70–7.87 (2H, m),
= 2.1), 8.27 (1H, d, J = 9.3), 8.48 (2H, m),,
7
1
(
(
(
.86 (1H, dd, J
1
2
0.60 (1H, m); 10-isomer, 6.78 (1H, s), 7.70–7.87(2H, m), 7.60
= 9.3, J = 2.1), 8.48 (2H, m), 8.69 (1H, d, J = 9.3), 10.60
1H, m). C NMR (from the HMQC and HMBC experiments)
DMSO-d ) d: 9- and 10-isomers, 119.9, 120.8, 122.0, 122.9, 125.6,
1H, dd, J
1
1
2
3
compounds were tested at 20
bic and hypoxic conditions.
lM in duplicate flasks both in aero-
6

M. L. Lavaggi et al. / Bioorg. Med. Chem. 18 (2010) 4433–4440
4439
5
.11. Antiproliferative activity on Caco-2 cells10,23 cells
5.15. AFM experiments18
Caco-2 cells (colorectal adenocarcinoma, ATCC/HTB-37), pro-
vided by the American Type Culture Collection (ATCC, Manassas,
VA, USA), were grown in Dulbecco’s Modified Eagle’s Medium
DMEM, Gibco, Prat de Llobregat, Barcelona, Spain) supplemented
with 10% fetal bovine serum (FBS, Gibco, Prat de Llobregat,
Barcelona, Spain) and 1% antibiotic (10.000 U/mL penicillin and
DNA was prepared from a pGEM-3-Zf(À)-derived plasmid con-
taining the duplicated multiple-cloning site of pBEND2 (pGB). The
linear 350 bp fragment was dissolved in 10 mM Tris–HCl (pH 7.5),
and aliquots were allowed to react with studied BPDO for 20 min at
37 °C. The BPDO was previously dissolved in DMSO and added to
the DNA solution to give a final DMSO concentration of 20% (v/
v). The ratio BPDO:DNA was varied between 100:1 and 1:1. The
solution was adjusted to 0.1 M NaCl, and hydrolysis products were
removed by multiple extractions with ethyl acetate and ether, fol-
lowed by ethanol precipitation and ether extraction of the precip-
itate. Measurements: DNA samples were diluted in HM1 buffer
[20 mM HEPES/KOH (pH 7.6) and 10 mM magnesium acetate] to
(
1
0.000
Spain). Cells were maintained as monolayer cultures at 37 °C in a
humidified atmosphere with 5% CO . Treatment: The assay was
performed using the method described in the literature slightly
modified. Samples containing 200
cell suspension (2 Â
0 cells/mL) were plated in 96 well-flat-bottomed microtiter
plates. After adherence of the cells within 24 h of incubation at
lg/mL streptomycin, Gibco, Prat de Llobregat, Barcelona,
2
2
3
lL
4
1
a concentration of 2–3 ng/lL. Approximately 8 ng of DNA was ad-
3
1
7 °C, studied derivatives at concentrations ranging from 1 to
000 M were added to different wells (3–4 per concentration).
sorbed to a freshly cleaved mica surface (glued to steel disks), al-
lowed to incubate at ambient temperature, then gently washed
with Milli-Q water, and dried under a gentle stream of filtered,
dry nitrogen. In some cases, the samples were further dried under
vacuum. AFM imaging was performed in tapping mode on a Vecco-
Digital Instruments microscope, model Multimode (MMAFM)
NanoScope IIIa-Quadrex, with a vertical J scanner having a maxi-
l
After additional incubation time (24 and 48 h) at 37 °C in a humid-
ified incubator with 5% CO , MTT dissolved in PBS and sterile fil-
tered was added to all the wells at a final concentration of 1 mg/
mL. Following 1 h of incubation, the generated formazan was dis-
2
solved with 100 lL DMSO per well. The optical density was mea-
sured using an ELISA plate reader (Merck ELISA system MIOS
mal lateral range of approximately 150
lm. Standard silicon canti-
version 3.2.) at 550 nm. The derivatives concentrations that caused
levers 125 m in length were used for all tapping in air images. The
l
5
0% decrease of cell viability (VC50) was calculated. The experi-
cantilever oscillation frequency was tuned to 280–320 nm, and
samples were scanned at 3–5 lines/s. Images were processed by
flattening (using NanoScope software) to remove background
slope. All images that are shown were analyzed by tapping in air.
ments were performed in triplicate.
5
.12. Cyclic voltammetric studies24
5
.16. Agarose gel electrophoresis studies10
DMSO (spectroscopy grade) was obtained from Aldrich. Tetra-
butylammonium perchlorate (TBAP), used as supporting electro-
lyte, was obtained from Fluka. Cyclic voltammetry was carried
out using a Metrohm 693 VA instrument with a 694 VA Stand con-
vertor and a 693 VA Processor, in DMSO (ca. 1.0 mM), under a
nitrogen atmosphere at room temperature (TBAP, ca. 0.1 mM) as
supporting electrolyte. A three-electrode cell configuration was
used, a mercury dropping working electrode, a platinum wire aux-
iliary electrode, and a saturated calomel reference electrode. Volt-
age scan rates ranged from 0.10 to 2.00 V/s.
DNA fragmentation was determined using ‘Real Pure Extraction
6
Kit’. In short, 2 Â 10 Caco-2 cells/well were cultured in 100-mm
plates and treated for 24 h with BPDO between 50 lM and the
corresponding VC50 doses. Chromosomal DNA was isolated (Real,
Durviz, Valencia, Spain) and analyzed in a 1% agarose gel visualiz-
ing with ethidium bromide staining. This assay was performed on
four separate occasions. Staurosporine was used as positive control
20
(
1–10 lM).
Acknowledgements
5
5
.13. DNA interaction studies
.13.1. UV–visible spectroscopy9
Financial supports from Comisión Honoraria de Lucha contra el
Cáncer (Uruguay), Project CTP 07/P11 (Government of Navarra,
Spain, within the program of the Work Community of Pyrenees),
Project SAF2008-00164 (Spanish Government, Ministerio de Cien-
cia e Innovación), Project 2009SGR1308 (Government of Catalonia),
networks ISCIII-RTICC (RD06/0020/0046 and RD06/0020/1037
(Red Temática de Investigación Cooperativa en Cáncer, Instituto
de Salud Carlos III), ANPCyT, CONICET and UBA (Argentine) are
acknowledged. We thank PEDECIBA-ANII for scholarship to MLL
and MC and AMSUD-PASTEUR network, PEDECIBA and CSIC-Uni-
versidad de la República for fellowships. L.I.P. is member of
CONICET.
DNA solution: calf thymus DNA (12.5 mg) was slowly magnet-
ically stirred in 5 mL Tris–HCl buffer (10 mM, pH 7.4) for 24 h at
4
°C. From this solution, 0.6 mL was diluted with the same buffer
À4
to 25 mL. Test compound solution: it was prepared at 10 M con-
centration using a minimal volume of adequate solvent, no more
À5
than 5%, and then diluted adding water to 2 Â 10 M. No effect
on DNA was observed by these concentrations of solvents. Study:
A 3.0 mL sample of this resulting solution was mixed with 3.0 mL
of DNA solution described above. The mixtures were slowly ro-
tated during 24 h and subsequently their UV spectra were re-
corded using a 1-cm cell at 20 °C on a Jasco V-570 UV/VIS/NIR
spectrophotometer. Areas were calculated automatically by the
apparatus.
Supplementary data
5
.14. Changes in DNA melting point17
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.074.
Thermal denaturation experiments were carried out by moni-
toring absorbance variations with temperature at 260 nm. The ra-
tio of DNA:BPDO was 1:1 for all experiments, where the DNA was
prepared as in the previous described experiment. The absorbance
of the mixture was using a 1-cm cell on a Jasco V-570 UV/VIS/NIR
spectrophotometer.
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