Phenazine N,N′-dioxide scaffold as selective hypoxic cytotoxin pharmacophore. Structural modifications looking for further DNA topoisomerase II-inhibition activity

Article abstract of DOI:10.1039/c3md00022b

Phenazine-5,10-dioxides have been identified as prodrugs for antitumour therapy that undergo hypoxic-selective bioreduction, in the solid tumour cells, to form cytotoxic species. We investigated structural modifications of the phenazine-5,10-dioxide scaff

Full text of DOI:10.1039/c3md00022b

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Phenazine N,N0-dioxide scaffold as selective hypoxic
cytotoxin pharmacophore. Structural modifications
looking for further DNA topoisomerase II-inhibition
activity†
Cite this: DOI: 10.1039/c3md00022b
a
a
b
c
´
´
´
Mariana Gonda,‡ Marcos Nieves,‡ Elia Nunes, Adela Lopez de Cerain,
c
a
a
*
*
´
Antonio Monge, Marıa Laura Lavaggi, Mercedes Gonzalez
and Hugo Cerecetto
a
*
Phenazine-5,10-dioxides have been identified as prodrugs for antitumour therapy that undergo hypoxic-
selective bioreduction, in the solid tumour cells, to form cytotoxic species. We investigated structural
modifications of the phenazine-5,10-dioxide scaffold attempting to find new selective hypoxic
cytotoxins with additional ability to inhibit DNA topoisomerase II. Four series of new phenazine-5,10-
dioxides aryl-substituted connected by different linkers were prepared. The clonogenic survivals of V79
cells on aerobic and anaerobic conditions were determined, and studies of oxic DNA-interaction and
hypoxic DNA topoisomerase II-inhibition, for the most relevant derivatives, were performed. Four new
hypoxic-selective cytotoxins were identified at the assayed doses. In some of them were operative the
DNA-interaction and/or the inhibition of DNA topoisomerase II. For one of the unselective cytotoxin
biotransformation studies were performed on aerobic and anaerobic conditions, explaining the lack of
selectivity.
Received 12th December 2012
Accepted 25th January 2013
DOI: 10.1039/c3md00022b
www.rsc.org/medchemcomm
development of a distinct therapy for treating cancer, the use of
bioreductive antitumour agents (BAA).5 BAA are prodrugs,
Introduction
It is well known that the existence of hypoxic and necrotic
regions in solid tumours1 is consequence of the rapid growth of
cancerous cells and their decient vascularisation that produces
molecular oxygen diffusion decreasing.2 The hypoxic regions are
associated to refractory radio- and chemotherapy.3 Additionally,
the hypoxia in tumours alters cellular metabolism tending to
select for a more malignant phenotype, increasing mutation
rates, and the expression of genes associated with angiogenesis
and tumour invasion, and it is associated with a more metastatic
phenotype of human cancers.4 By enhancing metastasis,
hypoxia can compromise curability of tumours by surgery. This
common feature of cancerous cells, hypoxia, is used for the
capable to be bioreduced under hypoxic conditions to further
drugs that produce cytotoxic events causing different degrees of
cancerous cells damage. N-oxide derivatives have been described
among the compounds classied as BAA.6,7 We have reported
phenazine N,N0-dioxide (PDO) as a hypoxia-activated prodrug
pharmacophore that potentially interacts with DNA aer the
corresponding bioreduction in hypoxic conditions.8 The PDO 1
(Scheme 1a) displayed excellent in vitro selective cytotoxic
property and in vivo antitumoral activity.9 For this, selective
anaerobic-reduction and its relation to bioreductive activity, and
the ability to release_OH in hypoxia were proved using enzymatic
mammal systems.9,10 Furthermore, we have tried to improve the
PDO DNA interaction by generating new derivatives with
increased planar p-conjugation8d or exible moieties that
increase DNA-stacking.11 PDO 2 (Scheme 1a) is an example of the
latter mentioned approach that partially improve, comparing to
PDO 1, the interaction with DNA (see UV results, Scheme 1a) but
with the concomitant loss of cytotoxic activity. On the other
hand, the PDOs 3–5 were toxic in both conditions, non-selective,
at the assayed dose (20 mM) (Scheme 1b).8a,11f Besides, some non-
selective PDO cytotoxins displayed in vitro aerobic-antitumor
activity against Caco-2 cells.12 Recently, we also described QSAR-
models that provided structural information for the design of
new PDO derivatives with improved potency.13
aGrupo de Quımica Medicinal-Laboratorio de Quımica Organica, Facultad de
´
´
´
´
´
´
Ciencias-Facultad de Quımica, Universidad de la Republica, Igua 4225, 11400
Montevideo, Uruguay. E-mail: megonzal@fq.edu.uy; hcerecet@fq.edu.uy; Fax: +598
2 5250749; Tel: +598 2 5258618 ext. 216
b
´
´
Laboratorio de Radiobiologıa, Dpto. de Biofısica, Facultad de Medicina, Universidad
´
de la Republica, Uruguay
c
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´
Centro de Investigacion en Farmacobiologıa Aplicada, Universidad de Navarra,
Pamplona, Spain
† Electronic supplementary information (ESI) available: Detailed experimental
procedures and spectroscopic characterization of benzofuroxan (IV), Fig. 1S, 2S,
3S, and 4S. See DOI: 10.1039/c3md00022b
‡ These authors contributed equally to this article.
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Scheme 1 Phenazine N,N0-dioxide (PDO) previously developed as hypoxia-activated prodrugs.
DNA topoisomerase II, an enzyme that manipulates DNA products, we have paid attention in the molecular structures of
topology, such as knots, tangles, and catenanes, remaining on berberrubine and resveratrol (Scheme 2). For the synthetic
DNA aer replication or transcription, has been described as an products, we have been interested in certain N-oxides which
excellent target for the development of anticancer drugs.14
A
combine in their structures pharmacophores moieties for
series of DNA topoisomerase II inhibitors are currently used in hypoxic-cytotoxicity and, aer bioreduction, become DNA
therapy, such as epipodophyllotoxins, antracyclines, antraqui- topoisomerase II inhibitors, i.e. DACA N-oxide and AQ4N
nones (i.e. mitoxantrone, mtx, Scheme 2), and acridines (i.e. (Scheme 2).15
amsacrine, m-AMSA, Scheme 2), for the treatment of different
Taking into account our developed PDOs, the structural
kind of cancers. The investigation in the DNA topoisomerase II exingencies from QSAR-models,13 and some DNA top-
inhibitors development is currently very wide ranging from oisomerase II inhibitors we planned PDOs aryl-substituted
natural products to synthetic compounds. Among the natural modifying the linker moiety (series a, b, c, and d, Scheme 3). The
Scheme 2 DNA topoisomerase II inhibitors used as template in the current design.
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rationality was to: (i) use PDOs 1 and 3–5 as parent compounds;
The syntheses of the new PDOs belonging to series c, 13–18,
(ii) include an extra p-interacting moiety, like in PDO 2, with were obtained via Beirut processes (Scheme 4b) using benzo-
certain exibility to improve the DNA-interaction capability or furoxans (I)–(III)17 and the nucleophiles p-aminophenol or
the DNA topoisomerase II inhibition, like in m-AMSA which p-hydroquinone in moderate yields. Derivatives from series d,
stacks the acridinyl ring into the AT-base sequences and 19–22, were prepared using as starting material 4,5-diuoro-2-
orientates the exible anilino side-chain in the DNA-minor nitroaniline (Scheme 4c) through a nucleophilic reaction fol-
groove11a,b or binds, via the methanesulfonylanilino moiety, at lowed by a transformation to the corresponding benzofuroxan,
the interface of DNA topoisomerase II and DNA11c–e (series a and (IV), which was expanded via Beirut processes to 19 or 20.
d); (iii) use sulfonamide moiety as linker like in the DNA top- Product 19, aer its purication, was obtained mixed with the
oisomerase II m-AMSA (series b); (iv) use ethenyl moiety as linker corresponding imine, as result of the secondary reaction
like in the DNA topoisomerase II resveratrol (series c); (v) include between the compound and p-aminophenol (see Fig. 1S in
in the aryl position a benzo[d]dioxolyl moiety like in the DNA ESI†). Then, in order to include in the lateral chain a moiety that
topoisomerase II berberrubine (series a and c); (vi) include in the strengthens the interaction with DNA at the acidic biological
aryl substituent a guanidinium moiety that could reinforce the pH,18 the aldehydes 19, mixed with the imine, and 20 were
interaction with DNA (series d).16 Additionally, the biological reacted with aminoguanidine producing, the aminoguanidones
effects of all the new compounds was analyzed by use of a clo- 21 and 22 in good yields.
nogenic assay with V79 cells in simulated oxic and hypoxic
Additionally, in order to conrm the relevance of the N-oxide
conditions. The PDOs DNA-interaction capability and DNA moiety in the biological response of these prodrugs, deoxygen-
topoisomerase II inhibition ability of some of these prodrugs ated derivatives were developed (23–25, Scheme 5). In order to
were also studied. For one of the unselective PDO bioreduction simulate the bioreductive process, chemical reductions were
studies, on aerobic and anaerobic conditions, were performed carried out with sodium dithionite, considered as an analogue
in order to explain the lack of selectivity.
of reductive enzymes (Scheme 5).19 Derivative 12 produced, in
the assayed conditions, the mono-reduced derivative 23, N10
-
oxide while derivative 20, in the assayed conditions, produced
the mono-oxygenated derivative 24, isolated as mixture of N5-
and N10-oxide isomers, and phenazine 25.
Results and discussion
Design and synthesis of new PDOs
The starting material for the synthesis of the new PDOs
belonging to series a and b, 6–12 (Scheme 4a), were the parent
compounds 1, 3, and 4 that were used as nucleophiles through
its phenol or amino groups, respectively. Nucleophilic substi-
tution reactions11f using phenol 1 and benzylic-derivatives with
traditional heating were incomplete being the main products
the corresponding de-oxygenated analogues of the parent
compounds, leading to very low yield of the desired ethers (see
examples in Table 1). However, we were able to improve slightly
the yields of these reactions using microwave irradiation, as
energy source, minimizing the presence of de-oxygenated
products (Table 1). In the case of amines 3–5, reacting with
benzylic-derivatives, only derivative 11, from parent compound
In all cases, the products 6–25 were characterized and eval-
uated as a non-separable mixture of 7- and 8-isomers because
we could not separate them neither by crystallization nor by
chromatographic methods. The isomeric proportion, deter-
mined by 1H NMR, is shown in Table S1 (see ESI†). All the
proposed structures were established by 1H-, and 13C NMR
spectroscopies (see examples in ESI†), using COSY, HMQC or
HSQC, and HMBC experiments, and MS. The purity was
analyzed and established by TLC and microanalysis,
respectively.
In vitro normoxic and hypoxic cytotoxicity
4, was generated in very low yield and under microwave irra- The PDOs and the reduced derivatives were examined for their
diation (Table 1). The amine 3 only was able to react with the selective hypoxic-cytotoxicities in a pre-established model of
good electrophile p-toluenesulfonyl chloride generating V79 cells.8,11f,20 They were analysed as a non-separable mixture of
sulfonamide 12 in a very low yield.
7- and 8-isomers. Previous results for similar chemical systems
Scheme 3 General structure of the four series of designed PDOs herein.
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Scheme 4 Synthetic procedure used for preparation of PDO 6–22.
(quinoxaline 1,4-dioxides) demonstrated that no difference better than that of parent compound 2. The remaining studied
between both positional isomers was observed in the selective compounds displayed not relevant biological activities, except
hypoxic cytotoxicities against V79 cells.20 The percentages of derivative 17, belonging to series c, and the intermediate selec-
survival fractions (SF, Table 2) were measured at 20.0 mM while tivity derivatives 18, from series c, 22, from series d, and 24, a
derivative 8 was also evaluated at 10.0 mM. This compound, N-oxide derivative. Additionally, the complete de-oxygenated
from series a, showed high cytotoxicity in both conditions, derivative, 25, showed some degree of cytotoxicity in both
normoxia and hypoxia, at 20 mM but when the dose was lowered conditions. This result conrmed that aer parent compound
the toxicity disappeared without selectivity. Furthermore, three 20 is metabolised to the metabolite, 25, could harm the cell via
of six compounds in series a displayed some degree of selec- its own mechanism. The best behaviours as selective hypoxic
tivity, i.e. 6, 7, and 9, lower than that of compound 1 but clearly cytotoxins were observed in compounds 7 and 17.
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Table 1 Assayed conditions in the synthesis of series a derivatives
Starting material
Producta
Conditions
T (ꢀC)
Time (h)
Yieldb (%)
Main product
1
6
K2CO3/KI/18-c-6c/DMF
25
40
50
120
50
120
40
50
120
40
50
120
48
24
—
2
No reaction
Deoxygenated product
No reaction
Deoxygenated product
Deoxygenated product
Deoxygenated product
Deoxygenated product
No reaction
K2CO3/KI/18-c-6/DMF
5 atm/300 wd,e
0.7
0.4
0.5
0.4
24
0.3
0.3
48
—
<1
<1
10
5
—
11
—
—
7
CH3CN/5 atm/300 wd
K2CO3/CH3CN/5 atm/300 wd
K2CO3/CH3CN/5 atm/300 wd
K2CO3/KI/18-c-6/DMF
1
4
7
K2CO3/CH3CN/5 atm/300 wd
K2CO3/CH3CN/18-c-6/TBAI 8 atm/700 wd,f
K2CO3/KI/18-c-6/DMF
Deoxygenated product
No reaction
No reaction
11
K2CO3/CH3CN/5 atm/300 wd
K2CO3/CH3CN/18-c-6/TBAI 8 atm/700 wd
0.3
0.3
Deoxygenated product
a
b
c
d
e
See Scheme 4 for structures. Aer isolation by chromatography column. 18-c-6: crown ether 18-crown-6. Microwave conditions. Without
solvent. f TBAI: tetrabutylammonium iodide.
Study of lack of selectivity of PDO 12
DNA interaction capability
PDO 12 was one of the non-selective derivative towards both We analyzed the capability to interact with DNA, in oxic
conditions, normoxia and hypoxia. For that reason, we selected conditions, of selected PDO prodrugs and some reduced
it to perform an in vitro bio-reduction study. We studied both derivatives. For that, we used the intrinsic changes in the uo-
the metabolism in hypoxia and in normoxia of derivative 12 rescence of phenazine while varying calf thymus DNA (CT DNA)
using S9 fraction and rat liver microsomal and cytosolic frac- concentrations aer 30 min of incubation.11f,21 Because it is the
tions.10 For the hypoxic bioreductions nitrogen atmosphere static quenching of DNA to compounds, the quenching
were used while normoxic conditions were simulated in air constant (Kq) is considered as the formation constant of
atmosphere. The processes were monitored by chromatography compounds and DNA complexes,22 i.e., the binding constant of
(TLC) using mono-deoxygenated derivative 23 as standard of compounds with DNA. The Stern–Volmer plots provided the Kq
one of the possible metabolites.
of studied compounds at pH 6.0 (see examples in Fig. 3S in ESI,†
Derivative 12 suffered bioreduction in both gassing condi- Table 3).23 For PDO 7, it was also evaluated its DNA-interaction
tions (see representative chromatograms in Fig. 1 and Fig. 2S in ability measuring the hypochromic and bathochromic effect of
ESI†) and similar bioreductive behaviour in the three studied compound-absorbance in the UV spectra, in a 20 nm band
protein fractions. The corresponding phenazine 23 was identi- centered on the maximal absorbance value of each compound,
ed as one of the main metabolic product. Also, other metabolic at 0 and 24 h,24 in order to compare with parent PDOs 1 and 2
products were evidenced and, according to their higher Rf (Table 3).
values, it is possible to establish that one of these could be the
Firstly, the studied PDOs were able in different degrees to
corresponding phenazine analogue. Efforts to elucidate the interact with DNA showing in some cases, derivatives 7, 14, 15,
chemical structures of these metabolic products were not done. and 16, similar interacting capabilities compared with the
These results are completely in agreement with the behav- reference compound, toluene blue (TB, Table 3). Secondly, two
iour of compound 12 in the cellular model (Table 2).
PDOs displayed better DNA-interacting properties than TB, they
Scheme 5 Synthetic procedure used for preparation of deoxygenated PDO 23–25.
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Table 2 PDO and deoxygenated PDO cytotoxic effects in normoxia and hypoxia on V79 cells
SFa,b,c,d
SFa,b,c,d
Norm
Series
Compd
Norm
Hypox
Series
Compd
Hypox
a
6
7
8
9
10
11
12
89 ꢁ 10
100 ꢁ 6
0(86 ꢁ 5)
100 ꢁ 6
100 ꢁ 7
85 ꢁ 5
58 ꢁ 4
d
19e
20
21
22
77 ꢁ 3
100 ꢁ 7
100 ꢁ 7
100 ꢁ 8
77 ꢁ 5
95 ꢁ 5
44 ꢁ 5
0(100 ꢁ 4)
55 ꢁ 3
100 ꢁ 10
73 ꢁ 4
100 ꢁ 8
85 ꢁ 4
Deoxygenated derivatives of 20
b
c
57 ꢁ 7
66 ꢁ 6
24
25
98 ꢁ 2
67 ꢁ 3
61 ꢁ 4
61 ꢁ 5
13f
14f
15f
16f
17
50 ꢁ 3
100 ꢁ 10
100 ꢁ 7
100 ꢁ 7
100 ꢁ 5
45 ꢁ 4
50 ꢁ 4
100 ꢁ 10
55 ꢁ 4
100 ꢁ 10
100 ꢁ 7
18
75 ꢁ 2
a
b
c
SF norm ¼ survival fraction in normoxia at 20 mM. SF hypox ¼ survival fraction in hypoxia at 20.0 mM. Values are means of two different
d
e
experiments. The assays were done by duplicate and using at least three repetitions. Values in parenthesis are at 10.0 mM. Evaluated as
mixture together with the imine by-product. Some compound solubility problems in the assay milieu, and both conditions, were observed.
f
Table 3 DNA interacting capability by fluorescence-studies of PDOs, phenazine
mono-oxide and phenazine
a
Series
Compound
Ratio Kq
a
6
7
12
13
14
15
16
17
18
20
21
22
24
25
TB
1
1.52 ꢁ 0.37b
0.92 ꢁ 0.37 (0.63)c
1.37 ꢁ 0.28
1.94 ꢁ 0.23
1.13 ꢁ 0.15
0.80 ꢁ 0.16
1.21 ꢁ 0.17
0.22 ꢁ 0.19
0.74 ꢁ 0.19
0.66 ꢁ 0.17
0.01 ꢁ 0.01
0.01 ꢁ 0.01
0.24 ꢁ 0.15
1.42 ꢁ 0.21
1.00 ꢁ 0.19
(1.00)
b
c
d
Fig. 1 TLC chromatograms (see Experimental section for experimental condi-
tions) taken after 30 min of incubation of PDO 12 with different protein fractions
and in different gasification conditions. Lanes: (1) PDO 23; (2) PDO 12; incuba-
tions in hypoxic conditions with cytosolic (3), and microsomal (4) fractions;
incubations in normoxic conditions with cytosolic (5), and microsomal (6)
fractions.
2
(0.88)
m-AMSA
mtx
maphd
(0.30)
(0.00)
0.48 ꢁ 0.22e
were phenylethenyl-derivative 13 and sulfonamide-derivative
12. It is clear the relationship between their oxic-cytotoxicities
(see values of SFnorm, Table 2) and DNA-interaction abilities.
This observation, in general, is a rule for all the population of
studied compounds, i.e. the worst DNA-interacting PDOs, 21
and 22, did not display oxic-cytotoxicities (Fig. 2a). Thirdly, the
level of phenazine oxygenation and DNA-interaction capability
a
Ratio Kq ¼ ratios of Stern–Volmer uorescence quenching constants of
b
the compounds and the reference compound (toluene blue, TB). See
c
text for comments. Values in parenthesis are ratios of a24 and a0 (see
Scheme 1). maph: 6-methyl-2-aminophenazine. e From ref. 11f.
d
are noticeably related, i.e. the deoxygenated PDO 25 interacted aggregation in the assay milieu of compound 6 at low DNA
better than the PDO parent compound 20 (Table 3, Fig. 2b). concentration that was abolished at higher concentrations of
Finally, p-nitrophenyl derivative 6 showed some particular the biomolecule. The values of Kq for this compound (see
behavior. The uorescence of PDO increased when DNA Fig. 4Sb and c,† Table 3) was calculated.
concentrations was increased to 40 mM and aer this, for higher
From a structural point of view we could draw attention to: (i)
DNA concentration, the effect of the quencher was evident the inclusion in series a, b, and d of an extra p-interacting
promoting the decreasing of the PDO uorescence (see Fig. 4Sa moiety with some exibility, like in m-AMSA,11a–c improved the
in ESI†). This phenomenon could be explained in term of DNA-interaction capability (compare PDOs 6, 7, 12 and 20 with
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Fig. 2 (a) Ratio of Kq vs. survival fractions in normoxia. The dot-line is only indicative. (b) Fluorescence spectra of PDO 20 (left) and the corresponding deoxygenated
derivative 25 (right) in PBS (50 mM, pH 6.0) after addition of different amounts of CT DNA.
parent compound 1); (ii) the sulfonamide moiety, like in m- topoisomerase II inhibition. Additionally, the incorporation of
AMSA, improve DNA interaction more than the benzyl moiety the benzylic moiety, in derivative 6, maintained this property
(compare PDOs 12 and 7 or 12 and 20); (iii) the incorporation of but the sulfonamido group, in derivative 12, does not contribute
a trans-ethenylaryl moiety in series c, like resveratrol, contrib- to enzymatic inhibition.
uted to increase the DNA-interaction capacity producing the cis-
ethenylaryl substitution compounds with lower Kq than those
with trans-arrangement (compare PDOs 13–16 with 17–18); (iv)
Experimental section
the aryloxy moiety, series d, improved the DNA interaction when
it was substituted by a 4-formyl group (i.e. PDO 20, compare
deoxy-derivative 25 with maph, Table 3); (v) guanidinium
moiety did not reinforce the interaction with DNA (compare
PDO 20 with 21 and 22).
Chemistry
General methods. Some starting materials were commer-
cially available research-grade chemicals and used without
further purication. All solvents were dried and distilled prior
to use. All the reactions were carried out in a nitrogen atmo-
sphere. Starting materials 1, 3, 4, and (I)–(III) were prepared
following synthetic procedures previously reported.8a,17 The
DNA topoisomerase II inhibition ability
We analyzed the ability to inhibit DNA topoisomerase II, in microwave-assisted synthesis were performed using a reactor
microaerobic conditions,25 hypoxia, of selected PDO prodrugs. WX-4000 (EU Chemical instruments). Melting points were
For that, we used a yeast survival test where wild type strains, determined with an electrothermal melting point apparatus
SC7K(Lys2-3) or JN362a, and mutant, ts14-16 or JN362at2-1, (Electrothermal 9100) and are uncorrected. Proton and carbon
strains were employed as models due to the second ones are NMR spectra were recorded on a Bruker DPX-400 spectrometer
mutated at the top2-1 locus underexpressing DNA top- at 298 K. The chemical shis values are expressed in ppm (d)
oisomerase II.26 We selected one derivative belonging to each relative to tetramethylsilane as internal standard and the J in
series to study in these assays. However members of series c and Hertz. Mass spectra were determined on a MSD 5973 Hewlett–
d were completely unsoluble in the yeast culture. Consequently, Packard spectrometer using electronic impact ionization.
we included in the studies parent compound 1, the selective Microanalyses were performed on a Fisons EA 1108 CHNS-O
PDO 6 from series a, the unselective PDO 12 from series b and instrument and were within (0.4% of the calculated composi-
etoposide as reference compound. All the compounds were tions). Column chromatography was carried out using Merck
incubated with cells during 60 min and in doses of 1 mM. As silica gel (60–230 mesh).
previously described for the well known hypoxic-cytotoxin tira-
General procedure for the synthesis of PDOs 6, 7 and 11. To a
pazamine,25a DNA topoisomerase II inhibition could be one of solution of the parent PDO (1 or 4, 1 eq.) in acetonitrile (25 mL
the mechanisms of action of parent PDO 1 (Fig. 3a), due to its mmolꢂ1 of 23), the corresponding benzyl halide (1 eq.), and
high surviving fraction in the mutant yeast ts14-16 under- K2CO3 (1 eq.) were added. In the cases of 7 and 11 preparation,
expressing this enzyme. Similarly, although to a lesser extent, 18-crown-6 ether (1 eq.) and TBAI (0.1 eq.) were also added. The
ꢀ
PDO 6 showed, in the assayed conditions (Fig. 3a), a behaviour reaction mixture was heated at 120 C using microwave energy
like PDO 1. Conversely, sulfonamide 12 did not produce a at the potency, pressure and time indicated in Table 1. Then the
signicant difference in the percentages of survival on the wild crude reaction mixture was partitioned between EtOAc and
type JN362a and mutant JN362at2-1 (Fig. 3b) indicating that, in aqueous HCl (10%). Aer the work up the organic layer was
these conditions, the mechanism of DNA topoisomerase II evaporated in vacuo and the residue was puried by column
inhibition was not operative.
chromatography (SiO2, petroleum ether : EtOAc (4 : 6)).
These results demonstrated, at a rst time, that the excellent
7(8)-Bromo-2-(4-nitrobenzyloxy)phenazine-5,10-dioxide (6).
selective hypoxic cytotoxin PDO 1 acts, at least in part, via DNA Brown-orange solid (10%). 1H NMR (DMSO-d6, 400 MHz) d
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Fig. 3 (a) Survival percentages corresponding to strains SC7K(lys2-3) (gray) and ts14-16 (black) exposed 60 min to 1 mM of parent compound 1 (left) or benzylic-PDO
6 (right). (b) Survival percentages of strains JN362a (gray) and JN362at2-1 (black) exposed 60 min to 1 mM of sulfonamido-PDO 12. Behaviour of etoposide at 1 mM
was also showed.
(ppm): 7-isomer, 5.58 (s, 2H), 7.74 (d, 2H, J ¼ 7.9 Hz), 7.95 (s,
7(8)-Bromo-2-(4-bromobenzyloxy)phenazine-5,10-dioxide (8).
Red solid (29%). 1H NMR (DMSO-d6, 400 MHz) d (ppm): 7-
1H), 8.04 (m, 3H), 8.46 (m, 2H), 8.70 (m, 2H); 8-isomer, 5.58
(s, 2H), 7.72 (d, 2H, J ¼ 7.9 Hz), 7.96 (s, 1H), 8.06 (m, 3H), 8.53
isomer, 5.40 (s, 2H), 7.53 (m, 2H), 7.76 (s, 1H), 8.01 (m, 3H), 8.59
(m, 2H), 8.54 (m, 2H). 13C NMR (from HMQC and HMBC
experiments) (DMSO-d6, 100 MHz) d (ppm): 7- and 8-isomers,
(m, 2H), 8.63 (m, 2H); 8-isomer, 5.38 (s, 2H), 7.51 (m, 2H), 7.56
(m, 2H), 7.76 (s, 1H), 7.75–7.95 (m, 3H), 8.51 (m, 2H). 13C NMR
70.1, 99.3, 122.2, 122.8, 122.7, 124.6, 129.3, 131.6, 135.4, 137.7,
144.7, 148.1. EI-MS: m/z (abundance, %): 7- and 8-isomers: 443/
441 (M+_, 0.2), 426 (1), 410 (1). (Found: C, 51.5; H, 2.56; N, 9.2.
(from HMQC and HMBC experiments) (DMSO-d6, 100 MHz) d
(ppm): 7- and 8-isomers, 70.1, 99.3, 122.2, 122.8, 122.7, 124.6,
128.8, 129.0, 130.7, 131.3, 132.5, 133.4, 134.3, 135.7, 161.5,
161.7. EI-MS: m/z (abundance, %): 7- and 8-isomers: 478/476/474
(M+_, 0.5/1/0.5), 459 (1), 443 (6). (Found: C, 47.7; H, 2.33; N, 5.6.
C19H12Br2N2O3 required C, 47.9; H, 2.54; N, 5.9%).
C
19H12BrN3O5 required C, 51.6; H, 2.74; N, 9.5%).
7(8)-Bromo-2-(4-chlorobenzyloxy)phenazine-5,10-dioxide (7).
Red solid (11%). 1H NMR (DMSO-d6, 400 MHz) d (ppm): 7-isomer,
5.42 (s, 2H), 7.50 (m, 1H), 7.52 (d, 2H, J ¼ 7.8 Hz), 7.55 (m, 3H),
7.75 (m, 2H), 8.03 (m, 2H); 8-isomer, 5.40 (s, 2H), 7.61 (m, 2H),
7.68 (d, 2H, J ¼ 7.7 Hz), 7.93 (s, 1H), 8.53 (m, 3H), 8.51 (m, 2H).
13C NMR (from HMQC and HMBC experiments) (DMSO-d6, 100
MHz) d (ppm): 7- and 8-isomers, 72.9, 99.1, 108.3, 122.2, 122.6,
126.2, 126.3, 128.8, 129.0, 130.7, 131.3, 132.5, 133.4, 134.3, 135.7,
161.5, 161.7. EI-MS: m/z (abundance, %): 7- and 8-isomers: 432/
430 (M+_, 1.3/1), 429 (4), 413 (7). (Found: C, 53.0; H, 2.66; N, 6.4.
C19H12BrClN2O3 required C, 52.9; H, 2.80; N, 6.5%).
7(8)-Bromo-2-(4-methylthiobenzyloxy)phenazine-5,10-dioxide
1
(9). Brown-orange solid (23%). H NMR (DMSO-d6, 400 MHz) d
(ppm): 7-isomer, 2.73 (s, 3H), 5.43 (s, 2H), 7.32 (d, 2H, J ¼ 8.9 Hz),
7.33 (s, 1H), 7.54 (m, 3H), 8.46 (m, 2H), 8.66 (m, 2H); 8-isomer,
2.70 (s, 3H), 5.40 (s, 2H), 7.29 (d, 2H, J ¼ 8.9 Hz), 7.35 (s, 1H), 7.64
(m, 3H), 8.52 (m, 2H), 8.80 (m, 2H). 13C NMR (from HMQC and
HMBC experiments) (DMSO-d6, 100 MHz) d (ppm): 7- and 8-
isomers, 15.2, 72.3, 109.4, 121.1, 121.2, 121.5, 126.7, 126.9, 127.1,
128.3, 129.6, 131.5, 132.2, 132.4, 132.6, 135.5, 135.9, 138.1, 147.9.
EI-MS: m/z (abundance, %): 7- and 8-isomers: 444/442 (M+_, 1), 430
(3), 414 (7). (Found: C, 53.9; H, 3.25; N, 6.0; S, 7.1. C20H15BrN2O3S
required C, 54.2; H, 3.41; N, 6.3; S, 7.2%).
7(8)-Chloro-2-(4-chlorobenzylamino)phenazine-5,10-dioxide
(11). Violet solid (7%). 1H NMR (CD3OD : D2O (9 : 1), 400 MHz)
d (ppm): 7-isomer, 5.07 (s, 2H), 7.54 (m, 1H), 7.57 (m, 1H), 7.93
(m, 3H), 8.08 (m, 2H), 8.27 (m, 1H), 8.54 (d, 1H, J ¼ 9.2 Hz), 8.69
(m, 2H); 8-isomer, 5.07 (s, 2H), 7.29 (m, 1H), 7.57 (m, 1H),
7.64 (m, 2H), 7.87 (dd, 1H, J1 ¼ 9.12, J2 ¼ 2.3 Hz), 8.08 (m, 2H),
8.27 (m, 1H), 8.69 (m, 1H). EI-MS: m/z (abundance, %): 7- and 8-
isomers: 371/369 (M+_ꢂ O, 1/1.6), 257 (1), 245 (3). (Found: C, 58.9;
H, 3.07; N, 11.2. C19H13Cl2N3O2 required C, 59.1; H, 3.39;
N, 10.9%).
7(8)-Bromo-2-(benzo[d][1,3]dioxol-5-ylmethyloxy)phenazine-
1
5,10-dioxide (10). Brown-orange solid (23%). H NMR (DMSO-
d6, 400 MHz) d (ppm): 7-isomer, 4.60 (s, 2H), 6.1 (s, 2H), 7.47 (d,
2H, J ¼ 8.9 Hz), 7.96 (s, 1H), 8.14 (m, 2H), 8.48 (m, 2H), 8.71 (m,
2H); 8-isomer, 4.52 (s, 2H), 6.0 (s, 2H), 7.45 (m, 2H, J ¼ 8.9 Hz),
7.35–7.64 (m, 3H), 8.52 (m, 2H), 8.80 (m, 2H); 13C NMR (from
HMQC and HMBC experiments) (DMSO-d6, 100 MHz) d (ppm):
7- and 8-isomers, 72.9, 99.1, 108.3, 122.2, 122.6, 123.6, 126.8,
130.0, 134.5, 136.8, 138.6, 138.8. EI-MS: m/z (abundance, %): 7-
and 8-isomers: 440 (M+_, 1), 426 (4), 410 (7). (Found: C, 54.1; H,
3.00; N, 5.9. C20H13BrN2O5 required C, 54.4; H, 2.97; N, 6.3%).
7(8)-Bromo-2-(4-methylphenylsulfonylamino)phenazine-5,10-
dioxide (12). A mixture of the parent PDO 1 (0.16 mmol) and p-
toluenesulfonyl chloride (0.16 mmol) in acetonitrile (3 mL) was
heated at 120 ꢀC using microwave energy at 700 watts of potency
and 8 atm of pression during 20 min. Then the crude reaction
General procedure for the synthesis of PDOs 8–10. To a
solution of 1 (1 eq.) in DMF (5 mL per 0.03 mmol of 1) was
added the corresponding benzyl chloride (1 eq.), K2CO3 (1 eq.),
18-crown-6 ether (1 eq.), and KI (0.5 eq.). The mixture was
ꢀ
heated at 40 C for 24 h. Then the crude reaction mixture was
partitioned between EtOAc and aqueous HCl (10%). Aer the
work up the organic layer was evaporated in vacuo and the
residue was puried by column chromatography (SiO2, petro-
leum ether : EtOAc (4 : 6)).
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mixture was puried by column chromatography (SiO2, petro- 330 (M+_, 1), 314 (14), 297 (100). (Found: C, 72.4; H, 4.11; N, 8.6.
leum ether : EtOAc (3 : 7)). Orange solid (15%). 1H NMR (DMSO-
C
20H14N2O3 required C, 72.7; H, 4.27; N, 8.5%).
d6 : D2O (1 : 1), 400 MHz) d (ppm): 7-isomer, 2.38 (s, 3H), 7.40
2-Amino-7(8)-[E-2-(4-chlorophenyl)ethenyl]phenazine-5,10-
(m, 2H), 7.53 (m, 2H), 7.71 (dd, 1H, J1 ¼ 9.1, J2 ¼ 2.0 Hz), 7.86 (d, dioxide (15). Black solid (79%). 1H NMR (DMSO-d6 : D2O
2H, J ¼ 8.4 Hz), 7.92 (m, 1H), 8.29 (m, 2H); 8-isomer, 2.35 (s, 3H), (9.5 : 0.5), 400 MHz) d (ppm): 7- and 8-isomers, 7.42 (dd, 1H, J1 ¼
7.40 (m, 4H), 7.92 (m, 4H), 8.29 (m, 1H), 8.54 (d, 1H, J ¼ 2.0 Hz). 9.6, J2 ¼ 2.9 Hz), 7.43 (dd, 1H, J1 ¼ 9.6, J2 ¼ 3.0 Hz), 7.48 (d, 2H,
13C NMR (from HSQC and HMBC experiments) (DMSO-d6 : D2O J ¼ 8.5 Hz), 7.49 (d, 2H, J ¼ 8.3 Hz), 7.56 (d, 1H, J ¼ 16.7 Hz), 7.58
(1 : 1), 100 MHz) d (ppm): 7- and 8-isomers, 21.5, 128.9, 129.0, (d, 1H, J ¼ 16.7 Hz), 7.60 (d, 1H, J ¼ 2.8 Hz), 7.64 (d, 1H, J ¼ 16.7
129.1 (four carbons), 122.2 (two carbons), 129.9 (two carbons), Hz), 7.66 (d, 1H, J ¼ 3.0 Hz), 7.67 (d, 1H, J ¼ 16.3 Hz), 7.73 (d,
130.0 (two carbons), 131.0, 131.1, 131.2, 131.3, 131.4, 131.5. EI- 2H, J ¼ 8.5 Hz), 7.74 (d, 2H, J ¼ 8.5 Hz), 8.16 (dd, 1H, J1 ¼ 9.3,
MS: m/z (abundance, %): 7- and 8-isomers: 461/459 (M+_, 5), 445 J2 ¼ 1.6 Hz), 8.23 (dd, 1H, J1 ¼ 9.5, J2 ¼ 1.4 Hz), 8.42 (d, 1H, J ¼
(1), 304 (100), 288 (32). (Found: C, 49.5; H, 3.17; N, 8.9; S, 6.9. 9.6 Hz), 8.43 (d, 1H, J ¼ 9.6 Hz), 8.47 (d, 1H, J ¼ 9.4 Hz), 8.49 (d,
C
19H14BrN3O4S required C, 49.6; H, 3.07; N, 9.1; S, 7.0%).
1H, J ¼ 9.4 Hz), 8.53 (d, 1H, J ¼ 1.3 Hz), 8.55 (d, 1H, J ¼ 1.2 Hz).
General procedure for the preparation of the PDOs 13–20. To 13C NMR (from HSQC and HMBC experiments) (DMSO-d6 : D2O
a solution of 3.9 mmol of metallic sodium in anhydrous MeOH (9.5 : 0.5), 100 MHz) d (ppm): 7- and 8-isomers, 99.9, 100.0, 117.4,
(25.0 mL), at ꢂ5 ꢀC and under nitrogen atmosphere, was added 117.8, 120.1, 120.6, 121.9, 122.0, 126.2, 126.5, 127.5, 128.1,
a solution of 20.0 mmol of the corresponding phenol (p-ami- 128.2, 129.0, 129.1, 129.2, 129.3, 129.4, 130.9, 131.3, 131.4,
nophenol or p-hydroquinone) and 20.0 mmol of the corre- 131.9, 133.2, 133.3, 133.4, 134.3, 135.3, 135.9, 135.9, 136.3,
sponding benzofuroxan, (I), (II), (III) or (IV) (see ESI for detailed 137.4, 137.8, 138.6, 140.2, 162.8, 162.9. EI-MS: m/z (abundance,
experimental procedures and spectroscopic characterization of %): 7- and 8-isomers: 365/363 (M+_, 1.6/5), 347 (30), 331 (39).
benzofuroxan (IV)†), in 5.0 mL of anhydrous MeOH and 30.0 mL (Found: C, 65.8; H, 3.55; N, 11.2. C20H14ClN3O2 required C, 66.0;
of anhydrous THF. Aer stirring at room temperature for 24 h H, 3.88; N, 11.5%).
ꢀ
and maintaining at ꢂ20 C for 24 h, the resulting precipitate
7(8)-[E-2-(4-Chlorophenyl)ethenyl)-2-hydroxyphenazine-5,10-
dioxide (16). Black solid (77%). 1H NMR (DMSO-d6 : D2O
was ltered, washed with THF yielding the desired product.
2-Amino-7(8)-(E-2-phenylethenyl)phenazine-5,10-dioxide (13). (9.5 : 0.5), 400 MHz) d (ppm): 7- and 8-isomers, 7.42 (dd, 1H, J1 ¼
Black solid (47%). 1H NMR (DMSO-d6 : D2SO4 (9.5 : 0.5), 400 9.6, J2 ¼ 2.5 Hz), 7.43 (dd, 1H, J1 ¼ 9.6, J2 ¼ 2.5 Hz), 7.49 (d, 2H,
MHz) d (ppm): 7- and 8-isomers, 7.20 (d, 1H, J ¼ 7.5 Hz), 7.26 (d, J ¼ 8.5 Hz), 7.50 (d, 2H, J ¼ 8.6 Hz), 7.57 (d, 1H, J ¼ 16.4 Hz), 7.60
1H, J ¼ 8.1 Hz), 7.32 (m, 4H), 7.37 (d, 1H, J ¼ 15.1 Hz), 7.40 (d, 1H, J ¼ 16.8 Hz), 7.64 (d, 1H, J ¼ 2.8 Hz), 7.65 (d, 1H, J ¼ 16.5
(d, 1H, J ¼ 17.3 Hz), 7.46 (s, 2H), 7.47 (d, 1H, J ¼ 15.8 Hz), 7.49 Hz), 7.65 (d, 1H, J ¼ 2.5 Hz), 7.66 (d, 1H, J ¼ 16.4 Hz), 7.74 (d,
(d, 1H, J ¼ 17.5 Hz), 7.60 (d, 2H, J ¼ 6.9 Hz), 7.63 (d, 1H, J ¼ 7.2 2H, J ¼ 8.6 Hz), 7.75 (d, 2H, J ¼ 8.6 Hz), 8.17 (dd, 1H, J1 ¼ 9.3,
Hz), 8.06 (d, 1H, J ¼ 9.3 Hz), 8.14 (d, 1H, J ¼ 9.3 Hz), 8.15 (d, 2H, J2 ¼ 1.4 Hz), 8.24 (dd, 1H, J1 ¼ 9.2, J2 ¼ 1.5 Hz), 8.43 (d, 1H, J ¼
J ¼ 9.5 Hz), 8.25 (d, 1H, J ¼ 10.0 Hz), 8.34 (d, 1H, J ¼ 8.7 Hz), 9.6 Hz), 8.44 (d, 1H, J ¼ 9.6 Hz), 8.48 (d, 1H, J ¼ 9.8 Hz), 8.50 (d,
8.40 (s, 1H), 8.43 (s, 1H). 13C NMR (from HSQC and HMBC 1H, J ¼ 9.7 Hz), 8.54 (d, 1H, J ¼ 1.2 Hz), 8.56 (d, 1H, J ¼ 1.2 Hz).
experiments) (DMSO-d6, 100 MHz) d (ppm): 7- and 8-isomers, 13C NMR (from HSQC and HMBC experiments) (DMSO-d6 : D2O
100.3, 101.5, 113.0, 113.1, 116.9, 117.2, 120.8, 122.9, 123.0, (9.5 : 0.5), 100 MHz) d (ppm): 7- and 8-isomers, 98.1, 98.7, 116.2,
125.7, 125.9, 126.4, 126.5, 126.7, 127.1, 127.4, 127.6, 127.9, 116.6, 119.3, 120.2, 123.5, 123.8, 125.2, 125.3, 126.0, 126.4,
129.0, 129.2, 133.3, 133.4, 133.5, 134.2, 135.4, 134.5, 136.3, 127.6, 127.9, 128.0, 128.2, 129.0, 129.1, 129.2, 129.5, 130.1,
136.7, 137.3, 138.3, 139.0, 144.7, 150.0, 150.1, 157.2, 157.6. EI- 130.2, 131.3, 131.7, 132.2, 132.5, 133.3, 134.3, 134.6, 134.8,
MS: m/z (abundance, %): 7- and 8-isomers: 329 (M+_, 2), 313 (37), 136.1, 136.9, 139.5, 141.3, 161.2, 161.9. EI-MS: m/z (abundance,
297 (47). (Found: C, 73.0; H, 4.56; N, 12.8. C20H15N3O2 required %): 7- and 8-isomers: 366/364 (M+_, 1.3/4), 347 (17), 332 (64).
C, 72.9; H, 4.59; N, 12.8%).
(Found: C, 65.6; H, 3.48; N, 7.8. C20H13ClN2O3 required C, 65.8;
2-Hydroxy-7(8)-(E-2-phenylethenyl)phenazine-5,10-dioxide H, 3.59; N, 7.7%).
(14). Black solid (36%). 1H NMR (DMSO-d6 : D2O (9.5 : 0.5), 400
2-Amino-7(8)-[Z-2-(benzo[d][1,3]dioxol-5-yl)ethenyl]phena-
MHz) d (ppm): 7- and 8-isomers, 7.34 (d, 1H, J ¼ 7.2 Hz), 7.35 (d, zine-5,10-dioxide (17). Black solid (30%). 1H NMR (DMSO-
1H, J ¼ 7.2 Hz), 7.43 (m, 4H), 7.58 (d, 1H, J ¼ 16.4 Hz), 7.60 d6 : D2O (9.5 : 0.5), 400 MHz) d (ppm): 7- and 8-isomers, 6.01 (s,
(d, 1H, J ¼ 17.3 Hz), 7.63 (d, 1H, J ¼ 16.8 Hz), 7.68 (s, 1H), 7.69 (s, 1H), 6.02 (s, 1H), 6.77 (s, 2H), 6.78 (d, 2H, J ¼ 8.4 Hz), 6.79 (d,
1H), 7.64 (d, 1H, J ¼ 18.0 Hz), 7.72 (d, 2H, J ¼ 7.2 Hz), 7.73 (d, 2H, J ¼ 7.7 Hz), 6.80 (d, 2H, J ¼ 7.8 Hz), 6.85 (d, 2H, J ¼ 8.5 Hz),
2H, J ¼ 7.3 Hz), 8.18 (dd, 1H, J1 ¼ 9.6, J2 ¼ 1.4 Hz), 8.26 (dd, 1H, 7.33 (dd, 1H, J1 ¼ 9.5, J2 ¼ 2.2 Hz), 7.34 (dd, 1H, J1 ¼ 9.4, J2 ¼ 2.3
J1 ¼ 9.4, J2 ¼ 1.4 Hz), 8.44 (d, 1H, J ¼ 9.6 Hz), 8.45 (d, 1H, J ¼ 9.7 Hz), 7.36 (d, 1H, J ¼ 2.3 Hz), 7.38 (d, 1H, J ¼ 2.3 Hz), 7.50 (dd,
Hz), 8.47 (d, 1H, J ¼ 9.0 Hz), 8.49 (d, 1H, J ¼ 9.3 Hz), 8.54 (d, 1H, 1H, J1 ¼ 9.2, J2 ¼ 1.6 Hz), 7.61 (dd, 1H, J1 ¼ 9.3, J2 ¼ 1.7 Hz), 8.30
J ¼ 1.2 Hz), 8.55 (d, 1H, J ¼ 9.3 Hz). 13C NMR (from HSQC and (d, 1H, J ¼ 9.0 Hz), 8.31 (d, 1H, J ¼ 10.0 Hz), 8.32 (d, 1H, J ¼ 9.0
HMBC experiments) (DMSO-d6 : D2O (9.5 : 0.5), 100 MHz) d Hz), 8.33 (d, 1H, J ¼ 9.1 Hz), 8.36 (d, 1H, J ¼ 1.5 Hz), 8.38 (d, 1H,
(ppm): 7- and 8-isomers, 100.0, 100.1, 112.8, 116.7, 117.2, 117.6, J ¼ 1.5 Hz). 13C NMR (from HSQC and HMBC experiments)
120.1, 120.5, 122.0, 122.1, 127.5, 127.6, 129.2, 129.3, 131.0, (DMSO-d6 : D2O (9.5 : 0.5), 100 MHz) d (ppm): 7- and 8-isomers,
131.5, 131.8, 132.8, 133.3, 133.4, 133.8, 134.5, 135.3, 136.4, 94.1, 94.3, 98.5, 101.6, 109.0, 109.2, 118.5, 119.1, 119.3, 120.0,
136.7, 136.9, 137.0, 137.3, 137.7, 139.0, 140.5, 141.5, 149.1, 121.3, 121.4, 123.5, 123.6, 125.0, 125.2, 127.3, 127.6, 127.7,
150.0, 162.2, 162.3. EI-MS: m/z (abundance, %): 7- and 8-isomers: 129.8, 130.1, 130.4 (three carbons), 132.1, 132.2, 132.9, 133.2,
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133.4, 134.0, 134.9, 136.0, 137.6, 137.9, 138.0, 140.5, 147.4, J ¼ 9.1 Hz), 8.32 (d, 1H, J ¼ 9.8 Hz). 13C NMR (from HSQC and
147.5, 147.6, 147.8, 152.3, 152.4. EI-MS: m/z (abundance, %): 7- HMBC experiments) (DMSO-d6 : D2O (9.5 : 0.5), 100 MHz) d
and 8-isomers: 373 (M+_, 2), 357 (44), 341 (42). (Found: C, 67.5; H, (ppm): 7- and 8-isomers, 98.7, 99.1, 105.3, 106.1, 106.2, 106.4,
3.99; N, 10.9. C21H15N3O4 required C, 67.6; H, 4.05; N, 11.2%). 120.1, 120.5, 121.2, 121.9, 122.0, 123.8, 125.2, 125.3, 128.6,
7(8)-[Z-2-(Benzo[d][1,3]dioxol-5-yl)ethenyl]-2-hydroxyphena- 129.0, 129.3, 129.7, 129.9, 130.4, 131.2, 133.0, 135.9, 136.4,
zine-5,10-dioxide (18). Black solid (19%). 1H NMR (DMSO- 145.2, 148.5, 150.1, 150.2, 152.8, 154.0, 154.7, 155.1, 159.5,
d6 : D2O (9.5 : 0.5), 400 MHz) d (ppm): 7- and 8-isomers, 6.03 (s, 160.3. EI-MS: m/z (abundance, %): 7- and 8-isomers: 421 (M+_, 2),
1H), 6.04 (s, 1H), 6.68 (d, 1H, J ¼ 6.2 Hz), 6.71 (d, 2H, J ¼ 6.0 Hz), 373 (4), 363 (13). (Found: C, 56.8; H, 3.56; N, 23.0. C20H16FN7O3
6.75 (s, 2H), 6.79 (d, 1H, J ¼ 8.1 Hz), 6.80 (d, 1H, J ¼ 7.8 Hz), 6.81 required C, 57.0; H, 3.83; N, 23.3%).
(d, 1H, J ¼ 7.6 Hz), 6.82 (d, 1H, J ¼ 8.4 Hz), 6.85 (d, 1H, J ¼ 7.0
7(8)-Fluoro-8(7)-(4-guanidinoiminomethylphenyloxy)-2-
Hz), 6.87 (d, 1H, J ¼ 6.5 Hz), 7.31 (dd, 1H, J1 ¼ 9.6, J2 ¼ 1.9 Hz), hydroxyphenazine-5,10-dioxide (22). Orange solid (55%). 1H
7.33 (dd, 1H, J1 ¼ 9.6, J2 ¼ 2.0 Hz), 7.50 (d, 1H, J ¼ 2.0 Hz), 7.52 NMR (DMSO-d6 : D2O (9.5 : 0.5), 400 MHz) d (ppm): 7- and 8-
(d, 1H, J ¼ 2.0 Hz), 7.89 (dd, 2H, J1 ¼ 7.3, J2 ¼ 2.0 Hz), 8.14 (d, isomers, 7.40 (d, 2H, J ¼ 8.0 Hz), 7.43 (d, 2H, J ¼ 8.6 Hz), 7.50 (dd,
1H, J ¼ 7.6 Hz), 8.16 (d, 1H, J ¼ 8.1 Hz), 8.18 (d, 1H, J ¼ 9.3 Hz), 2H, J1 ¼ 9.3, J2 ¼ 1.5 Hz), 7.68 (d, 2H, J ¼ 2.3 Hz), 7.76 (d, 1H, J ¼
8.26 (d, 1H, J ¼ 9.1 Hz), 8.27 (d, 1H, J ¼ 7.9 Hz), 8.32 (s, 1H). 13
C
8.2 Hz), 7.83 (d, 1H, J ¼ 8.0 Hz), 8.05 (d, 2H, J ¼ 7.1 Hz), 8.07 (d,
NMR (from HSQC and HMBC experiments) (DMSO-d6 : D2O 2H, J ¼ 8.0 Hz), 8.24 (s, 1H), 8.25 (s, 1H), 8.38 (d, 1H, J ¼ 9.7 Hz),
(9.5 : 0.5), 100 MHz) d (ppm): 7- and 8-isomers, 94.6, 94.9, 98.0, 8.42 (d, 1H, J ¼ 9.9 Hz), 8.44 (d, 1H, J ¼ 9.3 Hz), 8.45 (d, 1H, J ¼
99.0, 109.5, 110.3, 117.0, 117.9, 118.5, 119.3, 120.2, 120.7, 121.8, 10.7 Hz). 13C NMR (from HSQC and HMBC experiments)
122.4, 125.2, 125.4, 125.9, 126.2, 127.0, 127.3, 128.5, 128.8, (DMSO-d6 : D2O (9.5 : 0.5), 100 MHz) d (ppm): 7- and 8-isomers,
129.2, 129.6, 130.7, 130.9, 131.5, 132.1, 132.3, 133.5, 134.1, 99.9, 100.0, 106.2, 106.9, 107.1, 107.3, 120.4, 120.7, 121.0, 122.0,
135.7, 136.1, 136.2, 136.9, 137.2, 146.5, 146.8, 147.8, 147.9, 122.1, 123.9, 125.3, 125.6, 130.5, 130.9, 131.0, 131.3, 131.5,
151.1, 151.3. EI-MS: m/z (abundance, %): 7- and 8-isomers: 374 131.7, 131.9, 133.7, 137.1, 137.3, 146.3, 148.7, 150.0, 150.1,
(M+_, 4), 357 (21), 341 (46). (Found: C, 67.1; H, 3.65; N, 7.3. 153.6, 155.8, 156.2, 156.5, 161.0, 161.2. EI-MS: m/z (abundance,
C
21H14N2O5 required C, 67.4; H, 3.77; N, 7.5%).
7(8)-Fluoro-8(7)-(4-formylphenyloxy)-2-hydroxyphenazine- 56.7; H, 3.43; N, 20.0. C20H15FN6O4 required C, 56.9; H, 3.58; N,
5,10-dioxide (20). Red solid (57%). 1H NMR (DMSO-d6 : D2O 19.9%).
(9.5 : 0.5), 400 MHz) d (ppm): 7- and 8-isomers, 7.45–7.50 (two
7(8)-Bromo-2-(4-methylphenylsulfonylamino)phenazine N10
%): 7- and 8-isomers: 420 (M+_-2H, 1), 364 (6), 348 (2). (Found: C,
-
dd, 6H, J1 ¼ 8.8, J2 ¼ 2.4 and J1 ¼ 7.2, J2 ¼ 2.4 Hz), 7.66 (d, 1H, oxide (23). PDO 12 (0.087 mmol) was dissolved in concentrated
J ¼ 2.4 Hz), 7.72 (d, 1H, J ¼ 2.4 Hz), 7.95 (d, 1H, J ¼ 7.6 Hz), 8.03 hydrochloric acid (0.06 mL) and methanol (2.7 mL). Then
(d, 1H, J ¼ 8.4 Hz), 8.05 (d, 2H, J ¼ 8.8 Hz), 8.07 (d, 2H, J ¼ 7.2 sodium dithionite (0.35 mmol) was added at room temperature.
Hz), 8.39 (d, 1H, J ¼ 9.6 Hz), 8.43 (d, 1H, J ¼ 10.4 Hz), 8.44 (d, The mixture was stirred at room temperature for 3 h. The
1H, J ¼ 9.6 Hz), 8.46 (d, 1H, J ¼ 10.8 Hz), 10.01 (s, 1H), 10.02 (s, solvent was evaporated in vacuo and the residue was partitioned
1H). 13C NMR (from HSQC and HMBC experiments) (DMSO- between EtOAc (30 mL) and saturated solution of sodium
d6: D2O (9.5 : 0.5), 100 MHz) d (ppm): 7- and 8-isomers, 99.9, bicarbonate (30 mL). The organic phase was dried and evapo-
100.0, 106.8, 107.0, 108.6, 109.8, 119.5, 119.9, 122.1, 122.1, rated in vacuo. The crude mixture was puried by column
125.5, 125.7, 131.5, 132.0, 132.7, 132.8, 133.4, 133.5, 133.6, chromatography (SiO2, petroleum ether : EtOAc (3 : 7)). Yellow-
133.8, 137.3 (two carbons), 147.0, 147.1, 148.4, 148.6, 154.9, orange solid (45%). 1H NMR (DMSO-d6 : D2O (1 : 1), 400 MHz) d
157.5, 160.0, 160.3, 161.1, 161.2, 192.2, 192.3. EI-MS: m/z (ppm): 7-isomer, 2.34 (s, 3H), 7.37 (m, 3H), 7.65 (m, 1H), 7.76 (m,
(abundance, %): 7- and 8-isomers: 366 (M+_, 7), 350 (100), 334 1H), 7.93 (m, 5H); 8-isomer, 2.37 (s, 3H), 7.38 (m, 3H), 7.65 (m,
(55). (Found: C, 62.5; H, 2.99; N, 7.7. C19H11FN2O5 required C, 1H), 7.93 (m, 5H), 8.28 (d, 1H, J ¼ 2.4 Hz). 13C NMR (from HSQC
62.3; H, 3.03; N, 7.6%).
and HMBC experiments) (DMSO-d6 : D2O (1 : 1), 100 MHz) d
General procedure for the preparation of the phenazine- (ppm): 7- and 8-isomers, 22.5, 126.0, 127.5, 127.8, 128.5, 129.2,
5,10-dioxide derivatives 21 and 22. To a solution of the corre- 129.5, 129.7, 129.9, 130.0, 130.3, 130.4, 131.3, 131.4, 131.5 (two
sponding aldehyde (19, mixed with the imine by-product, or 20) carbons), 133.8, 134.0, 134.3. EI-MS: m/z (abundance, %): 7- and
(1 eq.) and aminoguanidine bicarbonate (1 eq.) in methanol 8-isomers: 445/443 (M+_, 100), 289 (32), 124 (8). (Found: C, 51.5;
(5 mL per mmol of aldehyde), was added concentrated hydro- H, 3.16; N, 9.1; S, 6.9. C19H14BrN3O3S required C, 51.4; H, 3.18;
chloric acid, two drops. Aer stirring at room temperature for 8 N, 9.5; S, 7.2%).
h ethyl ether (2 mL per mmol of aldehyde) was added. The
precipitate was ltered in vacuo and washed with ethyl ether.
Synthesis of 7(8)-uoro-8(7)-(4-formylphenyloxy)-2-hydrox-
yphenazine N-oxide (24) and 7(8)-uoro-8(7)-(4-for-
2-Amino-7(8)-uoro-8(7)-(4-guanidinoiminomethylphenyl- mylphenyloxy)-2-hydroxyphenazine (25). PDO 20 (0.2 mmol)
oxy)phenazine-5,10-dioxide (21). Black solid (59%). 1H NMR was dissolved in concentrated hydrochloric acid (0.1 mL) and
(DMSO-d6 : D2O (9.5 : 0.5), 400 MHz) d (ppm): 7- and 8-isomers, methanol (7 mL). Then sodium dithionite (0.8 mmol) was
7.31 (dd, 1H, J1 ¼ 9.6, J2 ¼ 1.6 Hz), 7.35 (dd, 1H, J1 ¼ 9.4, J2 ¼ 1.7 added at room temperature. The mixture was stirred at 50 ꢀC for
Hz), 7.40 (d, 4H, J ¼ 8.2 Hz), 7.49 (d, 1H, J ¼ 1.8 Hz), 7.53 (d, 1H, 3 h. The solvent was evaporated in vacuo and the residue was
J ¼ 2.1 Hz), 7.65 (d, 1H, J ¼ 8.4 Hz), 7.72 (d, 1H, J ¼ 8.0 Hz), 8.02 partitioned between EtOAc (30 mL) and saturated solution of
(d, 2H, J ¼ 7.6 Hz), 8.04 (d, 2H, J ¼ 8.2 Hz), 8.19 (s, 1H), 8.21 (s, sodium bicarbonate (30 mL). The organic phase was dried and
1H), 8.25 (d, 1H, J ¼ 9.8 Hz), 8.27 (d, 1H, J ¼ 9.6 Hz), 8.29 (d, 1H, evaporated in vacuo. The crude was puried by column
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ꢀ
chromatography (SiO2, petroleum ether : EtOAc (4 : 6)). The a water bath at 37 C, where they were gassed with humidied
rst eluted product corresponded to derivative 25 and the air or pure nitrogen.
second to derivative 24.
Treatment. Compounds solutions, 6–22, 24, and 25, were
7(8)-Fluoro-8(7)-(4-formylphenyloxy)-2-hydroxyphenazine N- prepared just before dosing. Stock solutions, 150-fold more
oxide (24). Yellow solid (24%). 1H NMR (DMSO-d6 : D2O concentrated, were prepared in pure DMSO (Aldrich) or steril-
(9.5 : 0.5), 400 MHz) d (ppm): as a mixture of at least four isomers, ized distilled water. Thirty min aer the start of gassing, 0.2 mL
7.27 (s, 1H), 7.31 (s, 1H), 7.34–7.59 (m, 12H), 7.65 (s, 1H), 7.71 (s, of the stock compound solution was added to each ask,
1H), 7.78 (d, 1H, J ¼ 8.1 Hz), 7.95 (d, 1H, J ¼ 8.1 Hz), 7.97–8.13 two asks per dose. In every assay there was one ask with 0.2
(m, 12H), 8.19 (d, 1H, J ¼ 10.6 Hz), 8.24 (d, 1H, J ¼ 11.2 Hz), mL of DMSO (negative control) and another with 7-chloro-3-[3-
8.38–8.47 (m, 4H), 9.98–10.04 (m, 4H). 13C NMR (from HSQC (N,N-dimethylamino)propylamino]-2-quinoxalinecarbonitrile-
and HMBC experiments) (DMSO-d6 : D2O (9.5 : 0.5), 100 MHz) d 1,4-dioxide hydrochloride (positive control).
(ppm): as a mixture of at least four isomers, 98.3, 98.4, 102.7,
Cloning. Aer 2 h exposure to the compound, the cells were
103.9, 105.1, 105.3, 106.0, 107.3, 108.8, 108.9, 114.8, 115.5, centrifuged and resuspended in plating milieu (EMEM plus
115.8, 115.9, 118.5, 118.9, 119.2, 119.3, 119.7, 120.2, 120.3, 10% (v/v) FBS and penicillin/streptomycin). Cell numbers were
120.5, 120.6, 120.8, 120.9, 121.0, 125.7, 125.8, 126.5 (two determined with a haemocytometer and 102 to 103cells were
carbons), 126.9, 127.2, 129.3, 129.9, 131.0, 131.1, 132.4 (two plated in 6-well plates to give a nal volume of 2 mL per 30 mm
carbons), 132.6, 132.7 (two carbons), 133.0, 133.6, 133.9, 134.6, of well. Plates were incubated at 37 ꢀC in 5% CO2 for 7 days and
134.8 (two carbons), 135.5, 136.3, 136.7, 137.0 (two carbons), then stained with aqueous crystal violet. Colonies with more
140.5, 140.8 (two carbons), 141.2, 141.7, 141.8, 143.1, 143.2, than 64 cells were counted. The plating efficiency (PE) was
143.9, 144.0, 147.6, 147.8, 148.0, 148.1, 153.5, 160.4, 160.9, calculated by dividing the number of colonies by the number of
161.0, 161.2, 192.2, 192.3, 192.4. EI-MS: m/z (abundance, %): as cells seeded. The percent of control-cell survival for the
mixture of isomers: 350 (M+_, 1), 279 (9), 263 (1). (Found: C, 64.9; compound-treated cultures (SFnormoxia and SFhypoxia) was
H, 3.05; N, 8.0. C19H11FN2O4 required C, 65.1; H, 3.17; N, 8.0%) calculated as PEtreated/PEcontrol ꢃ 100. The compounds were
7(8)-uoro-8(7)-(4-formylphenyloxy)-2-hydroxyphenazine (25). tested at 20 mM in duplicate asks both in aerobic and hypoxic
White solid (19%). 1H NMR (DMSO-d6 : D2O (9.5 : 0.5), 400 MHz) conditions.
d (ppm): 7- and 8-isomers, 7.30 (d, 1H, J ¼ 2.8 Hz), 7.34 (d, 1H, J ¼
DNA interaction studies.21
DNA solutions. Calf thymus DNA (CT DNA, 12.5 mg, Sigma
2.4 Hz), 7.40 (d, 1H, J ¼ 8.4 Hz), 7.43 (m, 1H), 7.43 (d, 2H, J ¼ 8.8
Hz), 7.60 (dd, 1H, J1 ¼ 9.9, J2 ¼ 2.6 Hz), 7.80 (d, 1H, J ¼ 8.8 Hz), Chemical Co., USA) was slowly magnetically stirred in 5 mL
7.93 (d, 1H, J ¼ 8.8 Hz), 8.01 (d, 2H, J ¼ 8.8 Hz), 8.03 (d, 2H, J ¼ phosphate buffer solution (PBS) (50 mM, pH 6.0) for 24 h at
ꢀ
8.8 Hz), 8.08 (d, 1H, J ¼ 9.6 Hz), 8.12 (d, 1H, J ¼ 9.2 Hz), 8.15 (d, 4 C. From this solution, the further necessary dilutions were
1H, J ¼ 12.4 Hz), 8.20 (d, 1H, J ¼ 11.6 Hz), 9.99 (s, 1H), 10.01 (s, performed using the same buffer. Test compound solution: It
1H). 13C NMR (from HSQC and HMBC experiments) (DMSO- was prepared at 20 mM concentration using PBS (50 mM, pH 6.0)
d6 : D2O (9.5 : 0.5), 100 MHz) d (ppm): 7- and 8-isomers, 118.3, and 20% of DMSO. No effect on DNA was observed by these
118.8, 127.1 (two carbons), 131.0 (two carbons), 132.6 (two concentrations of solvents.
carbons), 132.8, 133.0, 138.2, 138.3, 139.1 (two carbons), 139.6,
Study. Fluorescence spectra were recorded on a Varioskan
139.7, 141.5 (two carbons), 145.0 (two carbons), 146.2, 147.1, Flash 2.4.1 spectrometer. The uorescence quenching titrations
147.2, 150.2, 153.5 (two carbons), 155.9, 156.0, 160.3 (two with CT DNA were performed by keeping the compounds
carbons), 160.9, 161.0, 192.1, 192.2. EI-MS: m/z (abundance, %): concentrations constants (20 mM) and varying the nucleic acid
7- and 8-isomers: 334 (M+_, 1), 317 (1), 279 (11). (Found: C, 68.0; H, concentrations (0–400 mM) maintaining the total volume of the
3.10; N, 8.2. C19H11FN2O3 required C, 68.3; H, 3.32; N, 8.4%).
solution constant. Fluorescence emission spectra were recorded
in the wavelength range of 320–840 nm by exciting the
compounds at the corresponding wavelength with the excita-
tion slit widths of 12 nm. The intrinsic binding constants of
compounds with CT DNA were determined by uorescence
Biology
Bio-reductive activity.8,9,11f
Cells. V79 cells (Chinese hamster lung broblasts) were titrations. The data were plotted according to the Stern–Volmer
obtained from ECACC (European Collection of Animal Cell equation:27 I0/I ¼ 1 + Kq [Q], where, I0 and I are the uorescence
Cultures) and maintained in logarithmic growth as subcon- intensities in the absence and presence of CT DNA, respectively.
uent monolayer by trypsinization and subculture to (1–2) ꢃ Kq is the Stern–Volmer uorescence-quenching constant. [Q] is
104 cells per cm2 twice weekly. The growth milieu was EMEM the concentration of quencher.
(Eagle's Minimal Essential Milieu), containing 10% (v/v) foetal
bovine serum (FBS) and penicillin/streptomycin at 100 U per Dawley rats pretreated with Aroclor 1254 was obtained from
In vitro bio-reduction study.9,10 The S9 fraction of Sprague-
100 mg mLꢂ1
Aerobic and hypoxic cytotoxicity. Suspension cultures. Mono-
.
Moltox (Boone, North Carolina, USA).
Preparation of the rat liver microsomal and cytosolic fractions.
layers of V79 cells in exponential growth were trypsinized, and Livers were obtained from male Sprague-Dawley rats (198–
suspension cultures were set up in 50 mL glass asks: 2 ꢃ 104 202 g) from “Centro de Investigaciones Nucleares” (UdelaR,
cells per mL in 30 mL of EMEM containing 10% (v/v) FBS and Montevideo, Uruguay). The animals were allowed for food and
HEPES (10 mM). The glass asks were submerged and stirred in water ad libitum. The experimental protocols with animals were
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adhered to the Principles of Laboratory Animal Care.28 The centrifuged and washed twice with sterilized distilled water and
animals were sacriced by cervical dislocation and the livers, resuspended in sterilized distilled water. Cells were plated in
maintained in a ice bath, were perfused in situ with an ice-cold Petri dishes at a number of 102 for negative control and 102 to
KCl (0.9%) solution and washed with 3 volumes of Tris–HCl 103 per plate for treated cells. Plates were incubated for 3–6 days
(0.05 M)-sucrose (0.25 M) pH ¼ 7.4 in a Potter-Elvehjem glass- and number of cell colonies was determinated. Negative control
Teon homogeniser. The homogenates were centrifuged for cell growth was 100% and treated cells surviving fraction was
30 min at 900 g at 4 ꢀC and the supernatant fraction was calculated as (surviving cellstreated/surviving cellscontrol) ꢃ 100.
centrifuged at 10000 g for 1 h at 4 ꢀC. The pellet was discarded Statistical analysis was performed with the method of binomial
and the supernatant fraction was further centrifuged at distribution, with a condence interval of 95%.
100 000g for 1 h at 4 ꢀC. Metabolic assays were carried out with
microsomes and cytosol either fresh or frozen in Tris–HCl
buffer and stored at ꢂ80 C.
Conclusions
ꢀ
Incubation of PDO 12 with rat microsomal and rat cytosolic In conclusion, we designed and prepared a new series of
fractions and S9 fraction in normoxia and hypoxia. Our procedure phenazine dioxide derivatives as potential selective hypoxic
was adapted from published methods.29 The standard incuba- cytotoxins. Different moieties were included in the phenazine
tion mixture, in nitrogen or air purged ask, contained MgCl2 scaffold trying to increase the DNA or DNA topoisomerase II
(1.3 mM), NADPH generating system (NADP+, 0.4 mM; glucose interaction capabilities. Four of the new derivatives, PDOs 6, 7,
6-phosphate, 3.5 mM; 0.5 U mLꢂ1 glucose 6-phosphate dehy- 9, and 17, showed promising in vitro proles. One of them, PDO
drogenase) in a 0.1 M potassium phosphate buffer (pH 7.4) 6, together with the parent compound, PDO 1, showed capa-
containing EDTA (1.5 mM) and the corresponding PDO (40 mM) bility to inhibit DNA topoisomerase II in hypoxia. The
dissolved in dimethylsulfoxide (DMSO). Nitrogen gas was compounds DNA interaction abilities, in oxia, were related to
passed through a silica gel trap. The experiments were per- the oxic cytotoxicities.
formed by triplicate. Aer pre-equilibration of the mixture at 37
According to our results, the incorporation of a benzyl-group,
ꢀC, gassing with nitrogen or air, appropriate volume of micro- i.e. in derivatives 6, 7, and 9, was related to the desired bio-
somal, cytosolic or S9 suspension was added to give a nal activity. In contrast, the arylethenyl-moieties were, in general,
protein concentration of 1 mg mLꢂ1. The mixtures were incu- responsible of normoxic cytotoxicity and the sulfonamido-
ꢀ
bated for 30–120 min at 37 C. Three control incubations were group produced non-selective compound.
used: (1) 0.1 M potassium phosphate buffer (pH 7.4) (C1); (2)
The present results can inspire the synthesis of new bio-
(C1) + NADPH-generating system (C2); 3) (C2) + heat-inactivated reductive agents.
cytosolic or microsomal fractions (C3).
TLC monitoring of metabolites. The incubated mixtures were
Acknowledgements
extracted with EtOAc (3 ꢃ 400 mL) and the organic layer was
´
evaporated to dryness in vacuo. The residue was treated twice Financial supports from Comision Honoraria de Lucha contra
´
with EtOAc, 500 mL each. TLC experiments were performed el Cancer (Uruguay) is acknowledged. We thank PEDECIBA-
using Al2O3 as solid phase and CH2Cl2 : MeOH (99 : 1). The ANII for scholarship to MLL, ANII for scholarships to MN and
´
spots were visualised using UV-light (240 nm), directly for its MG, and PEDECIBA and CSIC-Universidad de la Republica for
characteristic colours (PDO, orange; phenazine monoxides, fellowships. We thank J. Nitiss for the JN yeast strains.
yellow) or by spraying with
a solution of p-anisaldehy-
de : H2SO4(c) : EtOH (95 : 4 : 1) followed by heating.
Notes and references
DNA topoisomerase II inhibition studies. Yeast cells were
grown in liquid nutrient milieu YPD [1% yeast extract (USB,
Cleveland, OH), 2% bactopeptone (USB), and 2% dextrose
(Sigma, St Louis, MO)]. Cells were grown and maintained at 30
ꢀC for 1–7 days with aeration by shaking, without added nutri-
ents. Thereaer, samples of these cultures were grown in
nutrient YPD up to the exponential phase (2–5 ꢃ 107 cells per
mL). Solid used nutrient milieu was YPDA (YPD + 2% Agar,
USB).26a
Treatment. Compounds solutions, were prepared just before
dosing. Stock solutions, 20-fold more concentrated, were
prepared in pure DMSO (Aldrich) or in sterilized distilled water.
Cells (1.5 mL from exponential phase cultures) were incubated
with 0.1 mL of compounds solutions in closed asks, shaked for
sixty min, two asks per dose. In every assay there was one ask
with 0.1 mL of DMSO (negative control) and another with eto-
poside (positive control) at 30 ꢀC (SC7K(lys2-3), JN362a and
JN362at2-1) and at 22 ꢀC for ts14-16. Aer treatment, cells were
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